MODIFICATION OF BIOLOGICALLY IMPORTANT NATURAL PRODUCT SCAFFOLDS THROUGH BIOCATALYSIS, USING TAXUS ACYLTRANSFERASES By Irosha Nayanthika Nawarathne A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chemistry 2011 ABSTRACT MODIFICATION OF BIOLOGICALLY IMPORTANT NATURAL PRODUCT SCAFFOLDS THROUGH BIOCATALYSIS, USING TAXUS ACYLTRANSFERASES By Irosha Nayanthika Nawarathne ® ® The antineoplastic agents paclitaxel (Taxol ) and docetaxel (Taxotere ) are currently supplied commercially by plant cell fermentations which rely on a biological source. The production of paclitaxel and related compounds can therefore be improved by understanding the biosynthesis of these metabolites in detail. Structure-activity-relationship studies have led to the development of highly promising paclitaxel analogues compared to the parent molecule through acyl group modifications, which are currently obtained by in-effective semisynthetic methods. Instead, catalysis of Taxus acyltransferases belonging to the BAHD plant superfamily can potentially be applied to developing the biotechnological production of paclitaxel and its analogues. Thus, the potential biocatalytic applications of a 2-O-benzoyltransferase (TBT) and a 13-O-3-amino-3phenylpropanoyltransferase (BAPT) involved in paclitaxel biosynthesis were studied. Two site-directed mutations within the wild-type 2-O-benzoyltransferase cDNA (tbt), from Taxus cuspidata plants, yielded an encoded protein containing replacement amino acids at Q19P and N23K that map to a solvent-exposed loop region. The likely significant changes in the biophysical properties invoked by these mutations caused the overexpressed, modified TBT (mTBT) to partition into the soluble enzyme fraction about 5-fold greater than the wild-type enzyme. Sufficient protein could now be acquired to examine the scope of the substrate specificity of mTBT by incubation with 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III that was mixed individually with various substituted benzoyls, alkanoyls, and (E)-butenoyl CoA donors. The mTBT catalyzed production of several 7,13-O,O-diacetyl-2-O-acyl-2-O-debenzoylbaccatin III analogues demonstrated the broad specificity of mTBT, suggesting that a plethora of 2-O-acyl variants of the antimitotic paclitaxel can be assembled through biocatalytic sequences. The scope of the taxane co-substrate specificity of mTBT hydrolysis was examined by incubating CoASH with various taxane co-substrates. The mTBT hydrolysis was highly regiospecific to the C-2 benzoyl moiety. However, the substrate scope was fairly limited. The 2O-benzoylated taxoids hydroxylated at C-7 and/or both C7 and C13 were not productive in mTBT hydrolytic reaction, suggesting that the hydroxylation at C-7 precludes the mTBT hydrolysis. This observation was verified by examining the taxane co-substrate specificity of mTBT acylation and further analysis allowed for pinpointing the relative timing of 2-Obenzoylation and the 7β-hydroxylation steps in the overall paclitaxel pathway. The wild-type 13-O-3-amino-3-phenylpropanoyltransferase cDNA (bapt), from Taxus cuspidata plants was recombinantly expressed to produce BAPT. Several arylpropanoyl CoA thioesters were incubated with baccatin III and purified BAPT to examine the substrate specificity; each formed the corresponding 13-O-arylpropanoylbaccatin III analogue. Also, the mechanistic studies of BAPT demonstrated the substrate-assisted catalysis of BAPT. The conducted substrate specificity studies of Taxus acyltransferases suggest the potential application of Taxus acyltransferases in developing biotechnological production of paclitaxel and its analogues. The fine details of paclitaxel biosynthesis are also advantageous in improving the production of paclitaxel through plant cell fermentations. Moreover, a variety of directed evolutionary analyses can be employed to potentially produce new catalyst derivatives that are able to transfer an even greater or more refined scope of novel acyl groups to the taxane core or other diterpene scaffolds. I dedicate this dissertation to my loving parents, Kaman Nawarathne and Murin Dias, my wonderful sister Dr. Iresha Meenuka Nawarathne, and my adorable nephew Aadithya Perera for their continuous love and encouragement, and my dearly husband Tharanga Wijetunge for his patience, sacrifices, and the support. iv ACKNOWLEDGMENTS For many students, years spent in graduate school are the most painful years of their entire life. To me, graduate life was truly a wonderful, memorable time. When I came to graduate school at MSU from the other side of the world in 2005, I never thought MSU would become my second home. Apparently, many wonderful people I met at MSU made it as comfortable as my first home. Thank you all for making my graduate experience very delightful. My sincere gratitude goes to the most wonderful person I met in graduate school, my research advisor Prof. Kevin D. Walker. Every time when I got lost in graduate school (before and after joining your group) and wanted to give up, you cheered me up and helped me regaining my confidence. By giving opportunities in the laboratory and elsewhere, you helped me immensely to grow as an independent researcher and a matured individual. I remember the way you made a special effort to appreciate every single small achievement I had throughout the graduate school. You are truly an outstanding advisor; you will always be my role model. I am truly grateful to my graduate advisory committee members, Prof. Robert P. Hausinger (Second reader), Prof. Gregory L. Baker, and Prof. Babak Borhan. I appreciate your invaluable guidance not only to be successful in the graduate school but even beyond that. Thank you so much for observing my strengths and weaknesses closely and advising me to overcome the weaknesses. I am also thankful to all of you for supporting me finding a suitable postdoctoral position and letting me use the resources from your research groups. Additionally, I am indebted to Prof. Baker for giving me a chance to come to MSU and enjoy the graduate school. I feel so blessed to have you all in my committee. v I am also thankful to Prof. A. Daniel Jones (Director, Mass Spectrometry Facility) for all his kindness and guidance. Although, you are not an official member of my advisory committee, your advice was invaluable. Thank you very much for directing me to the correct path all the time and helping me to explore suitable career options. I would also like to express my sincere gratitude to the organic faculty in the Department of Chemistry for clarifying thousands of questions I had about my research, seminars, and course work. You were always available in need. I specially thank Prof. Gary Blanchard (Associate Chair for Education) for helping me obtain funding from the College of Natural Sciences several times. I would also like to express my gratefulness to Dr. Chrysoula Vasileiou for her kindness, continuous advice, and invaluable discussions on biological details of my research and Dr. Ardeshir Azadnia for always trusting me and giving me an opportunity to teach which ever course I desired. My sincere appreciation goes to several wonderful people who work at Department of Chemistry making my graduate life very delightful. Thank you Dr. Daniel Holmes, Mr. Kermit Johnson (NMR facility), Dr. Thomas Carter, Mr. Paul Reed, Mr. Chris Preffer (Computer facility) for being patient with me. At times (rather many times), I asked you very trivial questions. Regardless, you were patient and very resourceful and never let me walk away without an answer. I would love to extend my immense thankfulness to Nancy, Debbie, Deann, Jeanne, Lisa, Joni, and Brenda. You always had a smile on your face seeing me. I will certainly miss you all. My gratitude extends to Ms. Lijun Chen and the MSU Mass Spectrometry Facility for the enormous support during the frequent usage of the facility, and Dr. Kaillathe (Pappan) Padmanabhan in the Macromolecular Computer Facility, Department of Biochemistry. The help vi I got from the MSU Research Technology Support Facility (RTSF) was remarkable in gaining biological research experience at MSU. Therefore, I would like to thank Mr. Joseph Leykam, Mr. Jeff Landgraf, Ms. Colleen Curry, and Ms. Stacy Trzos for helping me in various ways. I am obliged to thank the staff at the MSU Protein Expression Laboratory for their insightful discussions, sharing of the technical knowledge, and the various bacterial cell line donations. I would extend my sincere gratitude to the staff at the MSU writing center, MSU Simply Speaking Toastmasters Club, and the organizers of graduate school workshops which were enormously helpful in developing various professional skills. Without funding from the Department of Chemistry, the MSU College of Natural Sciences (CNS), the MSU graduate school, NSF, and the Michigan Agricultural Experiment Station, I would not be able to successfully complete my PhD program. Thank you very much for giving me various teaching and research assistantships and fellowships. I specially would like to thank Prof. Richard Schwartz and Ms. Teri Roache in CNS for their exceptional help in granting me several fellowships. Dr. Mark Ondari and (Dr.) Danielle Nevarez Mcbride, you two were truly inspirational. I am so glad that I got to be with you every single day in the lab throughout graduate school. Mark, you are a brother I never had till I came to the USA. Danielle, you filled me with my sister’s love which I missed coming to the USA. I can not expect better colleagues than you two. I will terribly miss you. The rest of the wonderful people I met in Walker lab are significant in making my graduate life truly a success. I would like to thank Dr. Catherine Loncaric, Dr. Brad Cox, Dr. Washington Mutatu, Dr. Lei Feng, Mr. Sanjit Sanyal, Ms. Karin Klettke, and Mr. Clifton Foster for teaching me various techniques, disciplines, and work ethics in the lab. My sincere appreciation goes to Sue, Yemane, Jelani, Udayanga, Getrude, Dennis, Behnaz, Ruth, Dilini, and vii Chelsea for making the lab very much fun and home like. Uday, despite the age difference, you will always be my twin brother. It is painful to miss you all. Aws, Josh, Farhiya, Yvonne, Amanda, Thomas, Ebony, Noelle, Jaime, and many more, thank you for all your technical support. You decorated the lab with love and fashion. Aws and Farhiya, you two were wonderful to work with. I will always be proud of your achievements. Josh, you make me laugh all the time. As I always tell you, “you are the best”. I would like to extend my gratitude to all current and past members of the Borhan, Wulff, Baker, Hausinger, Maleczka, and Spence groups for sharing their resources. I was always welcomed in your laboratories. I specially thank Calvin, Luiz Sanchez, Tarun, Glenn, Mariam, Greg, Parul, Aparajitha, and Chao for being wonderful friends since the very first months of the program. Calvin, you would never realize how much I appreciate your friendship; you made friends with me when I really needed one. I sincerely thank Anil, Munmun, Xiofei, Wenjing, Hong, Matt Nethercott, Rahman, Mercy, Toyin, Aman K., Aman D, Luiz (small), Hovig, and Allison for your warm friendship throughout. Anil and Munmun, I will impatiently wait to meet you in future ACS conferences. I thank Ms. Amber Cordell, Mr. Matt Fhaner, the YCC (MSU local section) group, and the International and Departmental Orientation Committees for taking my excess energy away and using it appropriately in volunteer work. I am thankful to all my teachers in school and college; specially, Prof. Dilip de Silva for giving me many opportunities to grow as a scientist; Mr. Kariyawasam (high-school Chemistry tutor) for inspiring me through the small-scale experiments we did in Organic Chemistry classes and believing in me even when I did not believe in myself; late Ms. Mahanama, although I can not see you any more, your enthusiasm still lives inside me. viii I am grateful to Dr. Chandana Summithrarachchi and his family for the care and love since the day I came to the USA. My gratitude extends to all the Srilankans live in Lansing for making it a small Srilanka here in the USA. I am also grateful to past and present Srilankan friends I met everyday in Chemistry; specially, Nishotha, Uday, Wathsala, Salinda, Dilini, and Damith for sharing my good and bad days patiently. You never forgot to save me a snack; never were so busy to accompany me when needed. You will be truly missed. Finally, I owe all my success to my close and extended family. Amma (mom), this was originally your dream; you helped me dream big and showed me the path to make it a reality. You are really the inspiration and the courage I had from the day I born. Thaththa (dad), you walked me through the woods, swam with me in the ocean, trained me to speak to plants, birds, and animals, and you showed me the beauty of the nature. If I have a fashion for science, it all came from you. My one and only Akka (sister), being the big sister and excelling everything you did, you took the lead and I just came along. Thank you for the overwhelming love and unlimited tolerance you always had for me. Ayya (my husband Tharanga), I owe you a lot. You sacrificed a number of your dreams to let my dream come true. Thank you for putting up with me even when I was so stressed out; I would be nothing without your love and protection. ix TABLE OF CONTENTS LIST OF TABLES……………………………………………………………………………… xv LIST OF FIGURES……………………………………………………………………………. xvi LIST OF ABBREVIATIONS………………………………………………………………… xxvi 1. INTRODUCTION ................................................................................................................. 1 1.1. Introduction ..................................................................................................................... 1 1.2. The Supply Crisis of Paclitaxel....................................................................................... 2 1.3. The Analogues of Paclitaxel ........................................................................................... 5 1.4. Biosynthesis of Paclitaxel ............................................................................................... 8 1.5. Taxus Acyltransferases: Members of BAHD Superfamily........................................... 13 1.6. Biocatalysis: Using Taxus Acyltransferases ................................................................. 15 References ………………………………………………………………………………………. 22 2. DESIGN AND SUBSTRATE SPECIFICITY STUDIES OF MODIFIED TAXUS 2-ODEBENZOYLBACCATIN III 2α-O-BENZOYLTRANSFERASE (mTBT) .................... 30 2.1. Introduction………………………………………………………………………… ... 30 2.2. Experimental ................................................................................................................. 34 2.2.1. Substrates, Reagents, and General Instrumentation .............................................. 34 2.2.2. Subcloning the Wild-Type TBT cDNA ................................................................ 35 2.2.3. Relative Soluble and Insoluble Expression of wtTBT.......................................... 36 2.2.4. Synthesis of 7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III ................................ 37 2.2.5. Synthesis of 7-O-Acetylbaccatin III ..................................................................... 39 2.2.6. Synthesis of 7-O-Acetyl-13-oxobaccatin III ......................................................... 40 3 2.2.7. Synthesis of [13- H]-7-O-Acetylbaccatin III ....................................................... 41 2.2.8. 3 Synthesis of [13- H]-7,13-O,O-Diacetylbaccatin III............................................ 41 3 2.2.9. Synthesis of [13- H]-7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III .................. 42 2.2.10. General Synthesis of Aroyl/Alkyl CoA Thioesters .............................................. 43 2.2.10.1. Quantification of Synthesized Acyl CoA Thioesters Using Ellman Assays 44 2.2.10.2. Synthesis of Benzoyl CoA ............................................................................ 44 2.2.10.3. Synthesis of 3-Chlorobenzoyl CoA .............................................................. 45 2.2.10.4. Synthesis of 4-Chlorobenzoyl CoA .............................................................. 45 2.2.10.5. Synthesis of 3-Cyanobenzoyl CoA ............................................................... 45 2.2.10.6. Synthesis of 3-Methoxybenzoyl CoA ........................................................... 46 2.2.10.7. Synthesis of 2-Fluorobenzoyl CoA ............................................................... 46 2.2.10.8. Synthesis of 3-Fluorobenzoyl CoA ............................................................... 47 2.2.10.9. Synthesis of 4-Fluorobenzoyl CoA ............................................................... 47 2.2.10.10. Synthesis of 2-Methylbenzoyl CoA .............................................................. 47 2.2.10.11. Synthesis of 3-Methylbenzoyl CoA .............................................................. 48 2.2.10.12. Synthesis of 4-Methylbenzoyl CoA .............................................................. 48 x 2.2.10.13. Synthesis of 2-Furancarbonyl CoA ............................................................... 48 2.2.10.14. Synthesis of 3-Furancarbonyl CoA ............................................................... 49 2.2.10.15. Synthesis of 2-Thiophenecarbonyl CoA ....................................................... 49 2.2.10.16. Synthesis of 3-Thiophenecarbonyl CoA ....................................................... 50 2.2.10.17. Synthesis of 5-Thiazolecarbonyl CoA .......................................................... 50 2.2.10.18. Synthesis of Phenylacetyl CoA ..................................................................... 50 2.2.10.19. Synthesis of Cyclohexanoyl CoA ................................................................. 51 2.2.11. Synthesis of Product Standards ............................................................................. 51 2.2.11.1. General Procedures ....................................................................................... 51 2.2.11.2. Synthesis of 7,13-O,O-Diacetylbaccatin III.................................................. 51 2.2.11.3. Synthesis of 7,13-O,O-Diacetyl-2-O-debenzoyl-2-O-(3fluorobenzoyl)baccatin III ........................................................................... 52 2.2.11.4. Synthesis of 2,7,13-O,O,O-Triacetyl-2-O-debenzoylbaccatin III ................ 53 2.2.11.5. Synthesis of 7,13-O,O-Diacetyl-2-O-debenzoyl-2-O-(3thiophenecarbonyl)baccatin III .................................................................... 53 2.2.12. wtTBT Activity Assay, Protein Purification, and Substrate Specificity Studies .. 54 2.2.13. Strategies Used to Improve the Solubility of wtTBT ........................................... 56 ™ 2.2.13.1. Application of pMAL Protein Fusion and Purification System ................ 56 2.2.13.2. Solubilization, Unfolding, and Refolding of Wild-type TBT ....................... 58 2.2.13.2.1. Method I ................................................................................................... 58 2.2.13.2.2. Method II ................................................................................................. 59 2.2.13.2.3. Method III ................................................................................................ 60 2.2.13.3. Coexpression of wtTBT and Chaperone Proteins ......................................... 61 2.2.13.4. Comparison of wtTBT and Codon-optimized wtTBT .................................. 63 2.2.13.5. Construction of a Homology Model of wtTBT ............................................ 63 2.2.13.6. Site-Directed Mutagenesis of wt-tbt cDNA.................................................. 64 2.2.13.7. Comparison of the Soluble Expression of wtTBT and mTBT ...................... 64 2.2.13.8. mTBT Activity Assay and Protein Purification ............................................ 64 2.2.14. Kinetic Evaluation of mTBT with 7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III and Benzoyl CoA Co-substrates ........................................................................... 66 2.2.15. Kinetic Evaluation of mTBT with 7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III and Competing CoA Substrates ............................................................................ 68 2.2.16. Relative kcat Values of mTBT with Various Acyl CoA Thioesters ...................... 70 2.3. Results and Discussion ................................................................................................. 71 2.3.1. Strategies Used to Improve the Solubility of wtTBT ........................................... 77 2.3.1.1. Optimization of Several Parameters in wtTBT Expression .......................... 77 2.3.1.2. Use of Fusion Proteins to Enhance Partitioning of wtTBT into the Soluble Fraction…………………………………………………………………… 77 2.3.1.3. Solubilization, Unfolding, and Refolding of wtTBT .................................... 78 2.3.1.4. Co-expression of wtTBT and Chaperone Proteins ....................................... 79 2.3.1.5. Strategies to Overcome the Biased Codon Usage of the Expression Host ... 80 2.3.1.6. Construction of a Mutant tbt and Functional Expression ............................. 82 2.3.2. Relative Substrate Specificity of mTBT with Various Aroyl CoA Thioesters and 7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III ..................................................... 87 xi 2.3.3. KM and kcat Values of mTBT with 7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III and Various Aroyl CoA Thioesters....................................................................... 95 2.4. Conclusion .................................................................................................................... 98 2.5. Future Directions .......................................................................................................... 99 2.5.1. Expansion of the Catalytic Site of mTBT ............................................................. 99 2.5.2. Coupling of mTBT to a Promiscuous Benzoyl CoA Ligase ............................... 100 2.5.3. In Vivo mTBT Catalysis in Producing Analogues of Paclitaxel ......................... 102 Appendix A ................................................................................................................................. 106 References…. .............................................................................................................................. 163 3. PINPOINTING THE TIMING OF 2-O-BENZOYLATION IN THE OVERALL PACLITAXEL BIOSYNTHETIC PATHWAY ............................................................... 169 3.1. Introduction ................................................................................................................. 169 3.2. Experimental ............................................................................................................... 177 3.2.1. Substrates, Reagents, and General Instrumentation ............................................ 177 3.2.2. Acquiring Crude Cell Lysate of modified TBT (mTBT), Activity Assay, and Protein Purification ............................................................................................. 178 3.2.3. General Note ....................................................................................................... 178 3.2.4. General Procedures ............................................................................................. 178 3.2.5. Synthesis of 13-O-Acetylbaccatin III ................................................................. 179 3.2.6. Synthesis of 7-O-Acetyl-13-O-triethylsilylbaccatin III ...................................... 179 3.2.7. Synthesis of 7-O-Acetyl-2-O-debenzoyl-13-O-triethylsilylbaccatin III ............. 180 3.2.8. Synthesis of 7-O-Acetyl-2-O-debenzoylbaccatin III .......................................... 181 3.2.8.1. First Trial .................................................................................................... 181 3.2.8.2. Second Trial ................................................................................................ 182 3.2.9. Synthesis of 7-O-Triethylsilylbaccatin III .......................................................... 183 3.2.10. Synthesis of 13-O-Acetyl-7-O-triethylsilylbaccatin III ...................................... 184 3.2.11. Synthesis of 13-O-Acetyl-2-O-debenzoyl-7-O-triethylsilylbaccatin III ............. 185 3.2.12. Synthesis of 13-O-Acetyl-2-O-debenzoylbaccatin III………………………… 186 3.2.12.1. First Trial .................................................................................................... 186 3.2.12.2. Second Trial ................................................................................................ 187 3.2.13. Synthesis of 2-O-Debenzoyl-7-O-triethylsilylbaccatin III ................................. 188 3.2.14. Synthesis of 2-O-Debenzoylbaccatin III ............................................................. 188 3.2.15. Characterization of the mTBT-Catalyzed Deacylation ....................................... 189 3.2.16. Kinetic Evaluation of mTBT Incubated with 7,13-O,O-Diacetylbaccatin III and CoASH ................................................................................................................ 190 3.2.17. Identification of Putative Taxane Co-substrates for the mTBT Deacylation ...... 191 3.2.18. Identification of Putative Taxane Co-substrates for the C2-Benzoylation Reaction Catalyzed by mTBT ............................................................................................ 192 3.3. Results and Discussion ............................................................................................... 193 3.3.1. Characterization and the Kinetic Evaluation of the mTBT Deacylation ............ 193 3.3.2. Assessing the Productive Taxanes in the Deacylating Reaction Catalyzed by mTBT .................................................................................................................. 196 3.3.3. Synthesis of 2-O-Debenzoylated Taxoids and the Identification of the Taxane Cosubstrate Scope of Benzoylation at C-2 Catalyzed by mTBT............................. 202 3.3.4. Plausible Explanation for the Differential Reactivity of mTBT ......................... 212 xii 3.3.5. Pinpointing the Timing of 2-O-Benzoylation in Overall Paclitaxel Biosynthetic Pathway ............................................................................................................... 214 3.4. Conclusion .................................................................................................................. 218 3.5. Future Directions ........................................................................................................ 219 3.5.1. Further Characterization of the mTBT Deacylation and Acylation and the Inhibition Studies ................................................................................................ 219 3.5.2. Designing Productive Taxane Co-substrates for the mTBT Catalysis by Disrupting the H-bonding Interactions ............................................................... 220 3.5.3. Alternative Hypothesis to Explain the Differential Reactivity of mTBT ........... 220 Appendix B……………………………………………………………………………………. 225 References…..…………………………………………………………………………………. 250 4. SUBSTRATE SPECIFICITY STUDIES AND SUBSTRATE-ASSISTED CATALYSIS OF TAXUS BACCATIN III 13α-O-3-AMINO-3-PHENYLPROPANOYLTRANSFERASE (BAPT) ............................................................................................................................... 255 4.1. Introduction ................................................................................................................. 255 4.1.1. Substrate Specificity Studies of BAPT ............................................................... 255 4.1.2. Substrate-Assisted Catalysis of BAPT................................................................ 262 4.2. Experimental ............................................................................................................... 267 4.2.1. Substrates, Reagents, and General Instrumentation ............................................ 267 4.2.2. Subcloning the Wild-Type bapt cDNA .............................................................. 268 4.2.3. Site-Directed Mutagenesis of wt-bapt ................................................................ 269 4.2.4. Expression and Purification of wtBAPT............................................................. 269 4.2.5. Expression and Purification of mBAPT .............................................................. 271 4.2.6. General Protocol for the Synthesis of Arylpropanoyl Coenzyme A Thioesters . 271 4.2.6.1. Synthesis of (R)-3-Amino-3-phenylpropanoyl CoA .................................. 272 4.2.6.2. Synthesis of (R)-3-Amino-3-(2-fluorophenyl)propanoyl CoA .................. 274 4.2.6.3. Synthesis of (R)-3-Amino-3-(2-methylphenyl)propanoyl CoA ................. 274 4.2.6.4. Synthesis of (R)-3-Amino-3-(2-thiophenyl)propanoyl CoA ...................... 275 4.2.6.5. Synthesis of (R)-3-Hydroxy-3-phenylpropanoyl CoA ............................... 275 4.2.6.6. Synthesis of 3-Phenylpropanoyl CoA ........................................................ 276 4.2.7. wtBAPT Activity Assay ..................................................................................... 276 4.2.8. mBAPT Activity Assay....................................................................................... 277 4.2.9. Substrate Specificity Studies of wtBAPT ........................................................... 278 4.2.10. Assessing Putative Substrate-Assisted Catalysis of BAPT ................................ 278 4.2.11. Relative Inhibition of wtBAPT catalysis by Other Phenylpropanoyl CoA Thioesters ............................................................................................................ 279 4.3. Results and Discussion ............................................................................................... 280 4.3.1. Expression and Activity Assay of the Taxus Baccatin III 13α-O-3-amino-3phenylpropanoyltransferase (BAPT) .................................................................. 280 4.3.2. Substrate Specificity Studies of wtBAPT with Synthetically-derived Phenyl/Heterolylpropanoyl CoA Thioesters and Baccatin III ............................ 286 4.3.3. Substrate-Assisted Catalysis of BAPT................................................................ 290 4.4. Conclusion .................................................................................................................. 293 4.5. Future Directions ........................................................................................................ 294 4.5.1. Expansive Substrate Specificity Studies of BAPT ............................................. 294 xiii 4.5.2. The Coupling of BAPT to a Promiscuous CoA Ligase ...................................... 294 4.5.3. A Detailed Understanding of the Catalytic Mechanism of BAPT ...................... 296 Appendix C ................................................................................................................................. 298 References. .................................................................................................................................. 309 xiv LIST OF TABLES Table 2.1 – Rarely used codons in E. coli..................................................................................... 81 Table 2.2 – Relative kinetics of mTBT with aroyl- and short-chain hydrocarbon-CoA’s and 7,13O,O-diacetyl-2-O-debenzoylbacctin III co-substrates ................................................ 91 Table 3.1 – Comparison of diagnostic proton NMR data on 45 and 46 with 36 ........................ 206 Table 3.2 – Comparison of diagnostic carbon NMR data on 45 and 46 with 36........................ 206 Table 3.3 – The diagnostic proton NMR data on 37, 38, and 39 ................................................ 208 Table 3.4 – The diagnostic carbon NMR data on 37, 38, and 39 ............................................... 208 xv LIST OF FIGURES ® ® Figure 1.1 – The structures of anticancer agent paclitaxel (Taxol ) and docetaxel (Taxotere )1 Figure 1.2 – Paclitaxel stabilize the microtubules leading to cell death ...................................... 2 Figure 1.3 – The structures of paclitaxel precursor baccatin III and 10-O-deacetylbaccatin III . 4 Figure 1.4 – The Structure-Activity Relationships (SAR) of paclitaxel ...................................... 6 Figure 1.5 – Paclitaxel analogues currently in phase I or phase II clinical trials ........................ 7 Figure 1.6 – The basic carbon frame configuration of most taxoids ........................................... 8 Figure 1.7 – Paclitaxel biosynthetic pathway starting from primary metabolism to the paclitaxel precursor baccatin III............................................................................ 10 Figure 1.8 – Last steps of paclitaxel biosynthetic pathway; N-benzoylphenylisoserine side chain construction at C13 of baccatin III .............................................................. 11 Figure 1.9 – A list of common “off-pathway metabolites” which are proposed to divert pathway flux away from paclitaxel production .................................................... 12 Figure 1.10 – Several improved paclitaxel analogues in preclinical studies with acyl group modifications......................................................................................................... 18 Figure 1.11 – Scheme of synthesis of improved paclitaxel analogue. ......................................... 19 Figure 1.12 – A potential route of semisynthesizing improved paclitaxel analogue incorporating biocatalysis of Taxus acyltransferases in both forward and reverse direction ...... 20 Figure 2.1 – A few biosynthetically derived and variously acylated metabolites formed by the function of BAHD plant superfamily acyltransferases ......................................... 31 Figure 2.2 – The C-2 acyl modified potential candidates for developing novel taxane agents . 32 Figure 2.3 – The reaction of the recombinant Taxus 2-O-benzoyltransferase (TBT) ............... 34 Figure 2.4 – Synthesis of 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III .................................. 72 3 Figure 2.5 – Synthesis of [13- H]-7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III ................... 73 Figure 2.6 – SDS-PAGE and Coomassie blue staining of recombinantly expressed, soluble and insoluble fractions of wtTBT isolated from E. coli BL21(DE3) .......................... 74 xvi Figure 2.7 – (A) A portion of the reverse-phase HPLC profile of authentic unlabeled 7,13O,O- diacetylbaccatin III with A228 monitoring of the effluent; (B) Partial 3 reverse-phase radio-HPLC profile of [ H]-labeled biosynthetic product derived by incubation of wtTBT crude soluble enzyme extract with benzoyl CoA and [133 H]-7,13-O,O-diacetyl-2-O-debenzoylbaccatin III with radioactivity monitoring of the effluent. ....................................................................................................... 75 3 Figure 2.8 – Partial reverse phase radio-HPLC profiles of [ H]-labeled biosynthetic products derived by separate incubations of wtTBT crude soluble enzyme extract and [133 H]-7,13-O,O-diacetyl-2-O-debenzoylbaccatin III with 2-furancarbonyl CoA or 2-thiophenecarbonyl CoA with radioactivity monitoring of the effluent. ............ 76 Figure 2.9 – SDS-PAGE and Coomassie blue staining of recombinantly expressed, chemically denatured, refolded, and purified wtTBT from insoluble inclusion bodies isolated from E. coli BL21(DE3) ....................................................................................... 79 Figure 2.10 – SDS-PAGE and Coomassie blue staining of resolved proteins of both soluble and insoluble fractions from E. coli BL21(DE3) transformed with the pET28a vector, separately expressing wild-type tbt cDNA insert and the codon-optimized tbt insert ...................................................................................................................... 82 Figure 2.11 – Partial amino acid sequence alignment of TBT orthologues used to design the site-directed mutant of the wild-type TBT from Taxus cuspidata. The sequences of two TBT orthologues from Taxus × media and T. wallichiana and other operationally soluble Taxus acyltransferases involved in paclitaxel biosynthesis are included in the alignment ................................................................................ 83 Figure 2.12 – SDS-PAGE and Coomassie blue staining of recombinantly expressed, soluble wtTBT and mTBT isolated from E. coli BL21(DE3) ........................................... 84 Figure 2.13 – Western blot analysis of the soluble expression of wtTBT and mTBT enzyme in crude extracts of transformed E. coli cells. ........................................................... 85 Figure 2.14 – The homology model of wtTBT based on the crystal structure of the related BAHD family vinorine synthase (VS, EC 2.3.1.160), in which both Q19 and N23 amino acid residues were found to be part of a solvent-exposed loop structure positioned on the enzyme surface. ........................................................................ 86 Figure 2.15 – A schematic diagram of mTBT and aroyl CoA ligase coupled system in producing analogues of paclitaxel through biocatalysis. ..................................................... 101 Figure 2.16 – The transformation of benzoate to benzoyl CoA by benzoate-CoA ligase……. 102 xvii Figure A 1 – The nucleotide sequence of wild-type tbt from Taxus cuspidata (accession no. AF297618) including the gene sequence for N-terminal His6-tag epitope and the corresponding amino acid sequence of wtTBT in pET28a vector. ..................... 107 Figure A 2 – The mutant tbt nucleotide sequence including the gene sequence for N-terminal His6-tag epitope and the corresponding amino acid sequence of mTBT in pET28a vector................................................................................................................... 109 Figure A 3 – Nucleotide sequence alignment of wild-type tbt cDNA (wt_tbt) from Taxus cuspidata and codon optimized synthetic tbt gene (opt_tbt) from GenScript. ... 112 Figure A 4 – Amino acid sequence alignment of TBT to the constructed double mutant, two different orthologs of TBT from different Taxus species, and other soluble acyl transferases involved in the paclitaxel biosynthetic pathway ............................. 115 Figure A 5 – Linear regression analysis of a concentration series of the product standard, 7,13O,O-diacetylbaccatin III calibration standard plotted against the total ion current + + of the [M + H] plus [M + Na] ions measured by LC-ESIMS ......................... 116 Figure A 6 – MS/MS fragment ion profiles of authentic 7,13-O,O-diacetylbaccatin III ........ 117 Figure A 7 – MS/MS fragment ion profiles of authentic 7,13-O,O-diacetyl-2-O-debenzoyl-2-O(3-fluorobenzoyl)baccatin III .............................................................................. 118 Figure A 8 – MS/MS fragment ion profiles of authentic 2,7,13-O,O,O-triacetyl-2-Odebenzoylbaccatin III .......................................................................................... 119 Figure A 9 – MS/MS fragment ion profiles of authentic 7,13-O,O-diacetyl-2-O-debenzoyl-2-O(3-thiophenecarbonyl)baccatin III ...................................................................... 120 Figure A 10 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetylbaccatin III derived by catalysis of mTBT ............................................................................. 121 Figure A 11 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(3-methylbenzoyl)baccatin III derived by catalysis of mTBT... 122 Figure A 12 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(4-methylbenzoyl)baccatin III derived by catalysis of mTBT... 123 Figure A 13 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(2-fluorobenzoyl)baccatin III derived by catalysis of mTBT .... 124 Figure A 14 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(3-fluorobenzoyl)baccatin III derived by catalysis of mTBT .... 125 xviii Figure A 15 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(4-fluorobenzoyl)baccatin III derived by catalysis of mTBT .... 126 Figure A 16 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(4-chlorobenzoyl)baccatin III derived by catalysis of mTBT. .. 127 Figure A 17 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(2-furancarbonyl)baccatin III derived by catalysis of mTBT .... 128 Figure A 18 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(3-furancarbonyl)baccatin III derived by catalysis of mTBT .... 129 Figure A 19 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(2-thiophenecarbonyl)baccatin III derived by catalysis of mTBT. ............................................................................................................................. 130 Figure A 20 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(3-thiophenecarbonyl)baccatin III derived by catalysis of mTBT1. ............................................................................................................................. 131 Figure A 21 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(5-thiazolecarbonyl)baccatin III derived by catalysis of mTBT 132 Figure A 22 – MS/MS fragment ion profile of the biosynthesized 2,7,13-O,O,O-triacetyl-2-Odebenzoylbaccatin III derived by catalysis of mTBT ......................................... 133 Figure A 23 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-propanoylbaccatin III derived by catalysis of mTBT ................ 134 Figure A 24 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-butyrylbaccatin III derived by catalysis of mTBT ..................... 135 Figure A 25 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(2-butenoyl)baccatin III derived by catalysis of mTBT ............ 136 Figure A 26 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-hexanoylbaccatin III derived by catalysis of mTBT .................. 137 Figure A 27 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-isobutyrylbaccatin III derived by catalysis of mTBT. ............... 138 Figure A 28 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-cyclohexanoylbaccatin III derived by catalysis of mTBT ......... 139 1 Figure A 29 – H NMR of 7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III ............................... 140 xix Figure A 30 – 13 C NMR of 7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III .............................. 141 1 Figure A 31 – H NMR of 7,13-O,O-Diacetylbaccatin III ......................................................... 142 Figure A 32 – 13 C NMR of 7,13-O,O-Diacetylbaccatin III ....................................................... 143 1 Figure A 33 – H NMR of Benzoyl CoA ................................................................................... 144 1 Figure A 34 – H NMR of 3-Chlorobenzoyl CoA ..................................................................... 145 1 Figure A 35 – H NMR of 4-Chlorobenzoyl CoA ..................................................................... 146 1 Figure A 36 – H NMR of 3-Cyanobenzoyl CoA ...................................................................... 147 1 Figure A 37 – H NMR of 3-Methoxybenzoyl CoA .................................................................. 148 1 Figure A 38 – H NMR of 2-Fluorobenzoyl CoA ...................................................................... 149 1 Figure A 39 – H NMR of 3-Fluorobenzoyl CoA ...................................................................... 150 1 Figure A 40 – H NMR of 4-Fluorobenzoyl CoA ...................................................................... 151 1 Figure A 41 – H NMR of 2-Methylbenzoyl CoA ..................................................................... 152 1 Figure A 42 – H NMR of 3-Methylbenzoyl CoA ..................................................................... 153 1 Figure A 43 – H NMR of 4-Methylbenzoyl CoA ..................................................................... 154 1 Figure A 44 – H NMR of 2-Furancarbonyl CoA ...................................................................... 155 1 Figure A 45 – H NMR of 3-Furancarbonyl CoA ...................................................................... 156 1 Figure A 46 – H NMR of 2-Thiophenecarbonyl CoA .............................................................. 157 1 Figure A 47 – H NMR of 3-Thiophenecarbonyl CoA .............................................................. 158 1 Figure A 48 – H NMR of 5-Thiazolecarbonyl CoA ................................................................. 159 1 Figure A 49 – H NMR of Phenyl acetyl CoA ........................................................................... 160 xx 1 Figure A 50 – H NMR of Cyclohexanoyl CoA ........................................................................ 161 Figure 3.1 – A potential semi-biocatalytic route to efficatious paclitaxel analogues with various acyl groups R1, R2, R3, and R4 at C-10, C-2, C-3', and C-3' amino group respectively incorporating Taxus acyltransferases in both forward (forward) and reverse (deacylation) direction ............................................................................ 170 Figure 3.2 – Several selected commercially available and semisynthesized taxane substrates to test the substrate specificity of mTBT hydrolytic reaction. ................................ 172 Figure 3.3 – Paclitaxel biosynthetic pathway starting from primary metabolism to the paclitaxel precursor baccatin III.......................................................................... 174 Figure 3.4 – Last steps of paclitaxel biosynthetic pathway; N-benzoylphenylisoserine side chain construction at C13 of baccatin III ............................................................ 176 Figure 3.5 – The characterization of mTBT-catalyzed deacylation. Shown are the steady-state kinetic parameters of mTBT catalysis for the CoASH co-substrate ................... 195 Figure 3.6 – LC-MS ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoylbaccatin III .......................................................................................... 196 Figure 3.7 – LC-MS ion profile of the biosynthesized 7-O-acetyl-2-O-debenzoylbaccatin III derived by mTBT deacylation ............................................................................. 198 Figure 3.8 – LC-MS ion profile of the biosynthesized 7-O-acetyl-2-O-debenzoyl-13oxobaccatin III derived by mTBT deacylation ................................................... 199 Figure 3.9 – Several identified productive and non-productive taxane substrates when tested for the substrate specificity of mTBT deacylation .............................................. 201 Figure 3.10 – The proposed 2-O-debenzoylated taxoids to test the effect of functional group at C7 on mTBT 2-O-benzoylation reaction. The functionality at C13 is also varied to see any effects on the mTBT catalysis ................................................................ 201 Figure 3.11 – First attempt of synthesis of 2-O-debenzoylated taxoids ................................... 205 Figure 3.12 – The proposed mechanism for the oxetane ring opening and the furan ring formation in the presence of HF/pyridine reagent during the first attempt of synthesis of 2-O-debenzoylated taxoids ............................................................. 206 Figure 3.13 – A ball and stick models of expected product (37) and the resulted and rearranged product (45) in the first attempt to synthesize 37………………………………207 Figure 3.14 – Second attempt of synthesis of 2-O-debenzoylated taxoids ............................... 208 xxi Figure 3.15 – LC-MS ion profile of the biosynthesized 7,13-O,O-diacetylbaccatin III derived by mTBT-catalyzed deacylation .............................................................................. 210 Figure 3.16 – LC-MS ion profile of the biosynthesized 7-O-acetylbaccatin III derived by mTBT-catalyzed deacylation .............................................................................. 211 Figure 3.17 – Several identified productive and non-productive taxane substrates when tested for the substrate specificity of mTBT 2-O-benzoylation reaction ...................... 212 Figure 3.18 – A ball and stick model in Chem & Bio 3D 12.0 (CambridgeSoft) and the corresponding molecular structure of 7-O-acetylbaccatin III ............................. 214 Figure 3.19 – The binding constants (Ks) reported for highly functionalized taxoids with Taxus 7β-hydroxylase ................................................................................................... 216 Figure 3.20 – Proposed order of several steps in the paclitaxel biosynthetic pathway starting from 25 to the paclitaxel precursor 1 considering that the 2-O-benzoylation occurs immediately after the 2α-hydroxylation ............................................................. 216 Figure 3.21 – The proposed 2-O-benzoyl-7-deoxytaxoids to understand the exact timing of 2-Obenzoylation and/or 7β-hydroxylation in the overall paclitaxel biosynthetic pathway ............................................................................................................... 217 Figure 3.22 – Proposed order of several steps in the paclitaxel biosynthetic pathway starting from 25 to the paclitaxel precursor 1 considering that the 2-O-benzoylation occurs further down in the pathway ............................................................................... 217 Figure 3.23 – Shown are several 2-O-benzoylated and 2-O-debenzoylated taxoids designed to purposely disrupt the interactions between 7-OH and 9-CO .............................. 221 Figure 3.24 – Shown are several naturally occurring advanced taxoids which are 7-O-acetylated and 2-O-benzoylated. .......................................................................................... 222 Figure 3.25 – The revised timing of 2-O-benzoylation in the overall paclitaxel biosynthetic pathway and the additional acetylation-deacetylation steps included in between 2O-benzoylation and the 7β-hydroxylation considering the alternative hypothesis in explaining the differential reactivity of mTBT ............................................... 223 Figure B 1 – MS/MS fragment ion profiles of authentic 7,13-O,O-diacetyl-2-Odebenzoylbaccatin III .......................................................................................... 225 Figure B 2 – MS/MS fragment ion profiles of authentic 7-O-acetyl-2-O-debenzoylbaccatin III ............................................................................................................................. 226 Figure B 3 – MS/MS fragment ion profiles of authentic 7,13-O,O-diacetylbaccatin III......... 227 xxii Figure B 4 – MS/MS fragment ion profiles of authentic 7-O-acetylbaccatin III...................... 228 Figure B 5 – MS/MS fragment ion profile of the biosynthesized 7-O-acetyl-2-Odebenzoylbaccatin III derived by catalysis of mTBT ......................................... 229 Figure B 6 – MS/MS fragment ion profile of the biosynthesized 7-O-acetyl-2-O-debenzoyl-13oxobaccatin III derived by catalysis of mTBT .................................................... 230 Figure B 7 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetylbaccatin III derived by catalysis of mTBT ............................................................................. 231 Figure B 8 – MS/MS fragment ion profile of the biosynthesized 7-O-acetylbaccatin III derived by catalysis of mTBT .......................................................................................... 232 Figure B 9 – Figure B 10 – 1 H NMR of rearranged product formed during the first attempt of 7-O-acetyl-2O-debenzoylbaccatin III synthesis ...................................................................... 233 13 C NMR of rearranged product formed during the first attempt of 7-O-acetyl-2O-debenzoylbaccatin III synthesis ...................................................................... 234 1 Figure B 11 – H NMR of rearranged product formed during the first attempt of 13-O-acetyl-2O-debenzoylbaccatin III synthesis ...................................................................... 235 Figure B 12 – 13 C NMR of rearranged product formed during the first attempt of 13-O-acetyl-2O-debenzoylbaccatin III synthesis ...................................................................... 236 1 Figure B 13 – H NMR of 7-O-Acetyl-2-O-debenzoylbaccatin III ........................................... 237 Figure B 14 – 13 C NMR of 7-O-Acetyl-2-O-debenzoylbaccatin III .......................................... 238 1 Figure B 15 – H NMR of 13-O-Acetyl-2-O-debenzoylbaccatin III ......................................... 239 Figure B 16 – 13 C NMR of 13-O-Acetyl-2-O-debenzoylbaccatin III ........................................ 240 1 Figure B 17 – H NMR of 2-O-Debenzoylbaccatin III .............................................................. 241 Figure B 18 – 13 C NMR of 2-O-Debenzoylbaccatin III............................................................. 242 Figure B 19 – HMQC of 7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III .................................. 243 Figure B 20 – HMQC of rearranged product formed during the first attempt of 7-O-acetyl-2-Odebenzoylbaccatin III synthesis .......................................................................... 244 xxiii Figure B 21 – HMQC of rearranged product formed during the first attempt of 13-O-acetyl-2-Odebenzoylbaccatin III synthesis .......................................................................... 245 Figure B 22 – HMQC of 7-O-Acetyl-2-O-debenzoylbaccatin III .............................................. 246 Figure B 23 – HMQC of 13-O-Acetyl-2-O-debenzoylbaccatin III ............................................ 247 Figure B 24 – HMQC of 2-O-Debenzoylbaccatin III ................................................................. 248 Figure 4.1 – Paclitaxel, Docetaxel, and several other efficacious paclitaxel analogues with modified C-13 side chain .................................................................................... 256 Figure 4.2 – Shown are some paclitaxel analogues with improved targeted drug delivery. DHA-linked paclitaxel is currently in phase III clinical studies ......................... 257 Figure 4.3 – Scheme of semisynthesis of paclitaxel and/or its analogues with various acyl groups .................................................................................................................. 260 Figure 4.4 – A potential biocatalytic route using Taxus acyltransferases to produce paclitaxel and/or its analogues with various acyl groups .................................................... 261 Figure 4.5 – The characterization of the recombinant Taxus baccatin III 13α-O-3-amino-3phenylpropanoyltransferase (BAPT) .................................................................. 262 Figure 4.6 – Proposed mechanism for SAC by H64A subtilisin BPN9ʹ with a peptide substrate containing a P2 histidine ..................................................................................... 264 Figure 4.7 – Proposed catalytic mechanism of vinorine synthase ........................................... 265 Figure 4.8 – Partial amino acid sequence alignment of BAPT to several other members of BAHD plant superfamily. The partial sequences of other Taxus acyltransferases involved in paclitaxel biosynthesis and several other BAHD family members outside the Taxus spp. are included in the alignment ......................................... 266 Figure 4.9 – Proposed substrate-assisted catalytic mechanism of BAPT. ............................... 266 Figure 4.10 – Several synthetically-derived phenylpropanoyl CoA thioesters that are proposed to be useful in investigating the substrate-assisted catalytic mechanism of BAPT ............................................................................................................................. 267 Figure 4.11 – SDS polyacrylamide gel electrophoresis and Coomassie blue staining of resolved proteins of partially purified, soluble fraction from E. coli BL21(DE3) transformed with the pET28a vector, expressing wild-type bapt cDNA insert .. 281 Figure 4.12 – Synthesis of phenyl/heterolyl propanoyl CoA thioesters .................................... 283 xxiv Figure 4.13 – The LC-MS chromatogram of extracted wtBAPT activity assay with baccatin III and (R)-3-amino-3-phenylpropanoyl CoA .......................................................... 284 Figure 4.14 – MS/MS fragment ion profile of the biosynthesized (R)-3'-amino-3phenylpropanoyl baccatin III derived by catalysis of wtBAPT .......................... 285 Figure 4.15 – MS/MS fragment ion profile of the biosynthesized (R)-3'-Amino-3-(2fluorophenyl)propanoyl baccatin III derived by catalysis of wtBAPT ............... 287 Figure 4.16 – MS/MS fragment ion profile of the biosynthesized (R)-3'-Amino-3-(2thiophenyl)propanoyl baccatin III derived by catalysis of wtBAPT .................. 288 Figure 4.17 – An array of proposed acyl CoA donors which are conceivably catalyzed by wtBAPT to transfer the acyl group to 13-hydroxyl of baccatin III .................... 289 + + Figure 4.18 – The plot of the total ion abundance of [M + H] and [M + Na] molecular ions of (3'R)-β-phenylalanoyl baccatin III vs the type of phenylpropanoyl CoA thioester used in the inhibition studies as the inhibitor...................................................... 292 Figure 4.19 – The general two-step mechanism of acyl CoA biosynthesis …………………. 296 Figure C 1 – The nucleotide sequence of wild-type bapt from Taxus cuspidata (accession no. AY082804) including the gene sequence for N-terminal His6-tag epitope and the corresponding amino acid sequence of wtBAPT in pET28a vector ................... 299 Figure C 2 – The mutant bapt nucleotide sequence including the gene sequence for N-terminal His6-tag epitope and the corresponding amino acid sequence of mBAPT in pET28a vector. .................................................................................................... 301 Figure C 3 – LC-MS ion profile of the biosynthesized (R)-3'-Amino-3-(2fluorophenyl)propanoyl baccatin III derived by catalysis of wtBAPT ............... 302 Figure C 4 – LC-MS ion profile of the biosynthesized (R)-3'-Amino-3-(2-thiophenyl)propanoyl baccatin III derived by catalysis of wtBAPT ...................................................... 303 Figure C 5 – Figure C 6 - 1 1 H NMR of (R)-3-amino-3-phenylpropanoyl CoA ............................................. 304 H NMR of (R)-3-amino-3-(2-fluorophenyl)propanoyl CoA .............................. 305 1 Figure C 7 – H NMR of (R)-3-amino-3-(2-methylphenyl)propanoyl CoA ............................. 306 1 Figure C 8 – H NMR of (R)-3-amino-3-(2-thiophenyl)propanoyl CoA .................................. 307 xxv LIST OF ABBREVIATIONS Ac2O, Acetic anhydride AIDS, Acquired immune deficiency syndrome AMP, Adenosine monophosphate ATP, Adenosine triphosphate BadA, Benzoate-coenzyme A ligase BAHD, Benzylalcohol-O-acetyltransferase; anthocyanin-O-hydroxycinnamoyltransferase; anthranilate hydroxycinnamoyl/benzoyltransferase; deacetylvindolidine 4-Oacetyltransferase BAPT, Taxus baccatin III 13α-O-3-amino-3-phenylpropanoyltransferase BEBT, Benzyl alcohol O-benzoyltransferase from Clarkia breweri Boc, tert-Butoxycarbonyl BPBT, Benzyl/phenylethyl alcohol O-benzoyltransferase from Petunia × hybrida BzOH, Benzoic acid CAN, Cerium(IV) ammonium nitrate CD, Circular dichroism CDCl3, Deuterated chloroform CDI, N,N'-Carbonyldiimidazole cDNA, Complementary deoxyribonucleic acid CoA, Coenzyme A CoASH, Free coenzyme A thiol D2O, Deuterated water DAB, Deacetylbaccatin III xxvi DBAT, 10-Deacetylbaccatin III-10β-O-acetyltransferase DFGWG, Aspartate; phenylalanine; glycine; tryptophan; glycine motif DHA, Docosahexaenoic acid Dm3MaT3, Anthocyanin malonyltransferase homolog from Chrysanthemum morifolium DMADP, Dimethylallyl diphosphate DMAP, 4-Dimethylaminopyridine DMF, Dimethylformamide DTNB, 5,5'-Dithiobis-2-nitrobenzoic acid DTT, Dithiothreitol E. coli, Escherichia coli EDTA, Ethylenediaminetetraacetic acid ESI-MS/MS, Electrospray ionization tandem mass spectrometer EtCOCl, Ethyl chloroformate EtOAc, Ethyl acetate FDA, U.S. Food and Drug Administration GGDP, Geranylgeranyl diphosphate GGDPS, Geranylgeranyl diphosphate synthase GST, Glutathione S-transferase GTP, Guanosine-5'-triphosphate HCBT, Anthranilate N-hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus HMBC, Heteronuclear multiple bond correlation 2-D NMR spectroscopic technique HMQC, Heteronuclear multiple quantum coherence 2-D NMR spectroscopic technique HOAc, Acetic acid xxvii HPLC, High performance liquid chromatography IDP, Isopentenyl diphosphate IDPI, Isopentenyl diphosphate isomerase IPTG, Isopropyl-β-D-1-thiogalactopyranoside LB, Luria-Bertani medium LC, Liquid chromatography LDA, Lithium diisopropylamide LiHMDS, Lithium bis(trimethylsilyl)amide MBP, Maltose binding protein MeCN, Acetonitrile MEP, Methylerythritol phosphate pathway MHz, Megahertz MS, Mass Spectrometer mTBT, modified wild-type 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III 2-O-benzoyltransferase from T. cuspidata MWCO, Molecular weight cut off NDBTBT, 3'-N-Debenzoyl-2'-deoxytaxol N-benzoyltransferase NDTBT, 3'-N-Debenzoyl-2'-deoxytaxol N-benzoyltransferase NMR, Nuclear Magnetic Resonance OD, Optical density PAM, Phenylalanine aminomutase PCF, Plant Cell Fermentation PCR, Polymerase chain reaction PMP, para-methoxyphenyl xxviii PMSF, Phenylmethanesulfonylfluoride PPi, Inorganic phosphate PTLC, Preparative thin layer chromatography Q-ToF, quadrupole time of flight Red-Al, Bis(2-methoxyethoxy)aluminum hydride RNA, Ribonucleic acid RT, Room temperature SAC, Substrate-assisted catalysis SAR, Structure-activity relationship SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis TAT, Taxadien-5α-ol O-acetyltransferase from T. cuspidata T. brevifolia, Taxus brevifolia T. cuspidata, Taxus cuspidata TBAF, Tetrabutylammonium fluoride TBT, 2-O-debenzoylbaccatin III 2-O-benzoyltransferase from T. cuspidata t-BuOH, tert-Butanol TES, Triethylsilyl TFA, Trifluoroacetic acid THF, Tetrahydrofuran TIPS, Triisopropylsilyl TLC, Thin layer chromatography TMS, Trimethylsilyl tRNA, Transfer ribonucleic acid xxix TRX, Thioredoxin TS, Taxadiene synthase UV, Ultraviolet VS, Vinorine synthase from Rauvolfia serpentina wtBAPT, wild-type Taxus baccatin III 13α-O-3-amino-3-phenylpropanoyltransferase from T. cuspidata wtTBT, wild-type 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III 2-O-benzoyltransferase from T. cuspidata xxx 1. INTRODUCTION 1.1. Introduction ® Paclitaxel (Taxol ) (1) (Figure 1.1), isolated from Taxus brevifolia (T. brevifolia, Pacific Yew) plant extracts in 1971, is arguably one of the most celebrated naturally occurring anticancer 1,2,3 agents in recent decades. It is also one of the few organic compounds, which, like benzene ®4 and aspirin, is more recognizable by its trade name Taxol . AcO O O OH NH O O O OH HO O O HO H OAc O O O OH NH O O OH O HO H OAc O O ® Paclitaxel (Taxol ) (1) ® Docetaxel (Taxotere ) (2) ® ® Figure 1.1 - The structures of anticancer agent paclitaxel (Taxol ) and docetaxel (Taxotere ) ® ® Unlike other anticancer agents, such as vinblastine (Velban ) and vincristine (Oncovin ) that act as inhibitors of tubulin polymerization, paclitaxel promotes and stabilizes microtubule assembly (Figure 1.2) and, ultimately, disrupts mitosis leading to cell cycle arrest and 5-7 apoptosis. Paclitaxel was approved by the U.S. Food and Drug Administration (FDA) for the 8 treatment of refractory ovarian cancer in 1992 1 and for the treatment of refractory or 9 anthracycline-resistant breast cancer in 1994, currently both paclitaxel and its semisynthetic ® analogue docetaxel (Taxotere ) (2) (Figure 1.1) are used for the treatments of ovarian, breast, and non-small-cell lung cancers and also in advanced forms of AIDS-related Kaposi’s 6,10,11 sarcoma. Heterodimer f ormation α-Tubulin Polymerization/ Elongation Initiation (αβ) β-Tubulin Paclitaxel Stabilized microtubule by paclitaxel interaction (12 protof ilaments with 22 nm diameter) Figure 1.2 – Paclitaxel stabilizes the microtubules leading to cell death. This figure is adapted 12 from the published work of Cragg and co-workers. For interpretation of the references to color in this and all other figures, the reader is referred to the electronic version of this dissertation These two taxane pharmaceuticals are also highly utilized in the management of heart disease and are receiving interest in newly developed regimens against Alzheimer’s disease and 13-16 tuberculosis. Consequently, the combined annual sales of these two drugs are well over $1 7 billion (USA dollars). 1.2. The Supply Crisis of Paclitaxel Despite the fact that paclitaxel is currently a widely used drug, it was almost not approved for anticancer therapy due to the solubility issues and the hypersensitivity reactions caused by the 2 2,6 chemicals used in drug formulations during clinical trials. However the demand of paclitaxel expanded dramatically, since FDA approved paclitaxel to be used in the treatment of ovarian 2,12 cancers in 1992 and breast cancers in 1994. High demand of paclitaxel was not possible to satisfy at that point due to the complications associated with its supply. The natural resources of paclitaxel are Taxus spp. which are slow-growing evergreen shrubs or small trees. T. brevifolia 1-3 was the plant in which paclitaxel was discovered. As the curiosity towards the development of paclitaxel increased for various clinical trials and black market sales, large-scale harvesting of T. brevifolia ensued. Unfortunately, the T. brevifolia grew most abundantly in old-growth forest inhabitated by the endangered spotted owl. The prospect of over-harvesting thus raised serious environmental concerns, and, more importantly, the amount of paclitaxel acquired by this means was insufficent to meet the demand.12,17 Further, the extraction process conducted by Hauser 12,17 Chemical Research of Boulder, Colorado, was costly and labor-intensive. All-together, the cost of production and the poor yields from the plant spelled a bleak future for paclitaxel as a new pharmaceutical. Fortunately, Potier and coworkers discovered substantial amounts of 10-Odeacetylbaccatin III (3) (Figure 1.3) and smaller amounts of baccatin III (4) (Figure 1.3) in 18 needles of Taxus baccata and then a semisynthetic process was developed by Holton and coworkers, where (3) was coupled to a synthetically derived phenylisoserine side chain precursor 19 to produce paclitaxel. This method was licensed to Bristol-Myers Squibb (BMS) who began 20 mass production and global sales of the drug. Although there are several successful approaches of total synthesis of paclitaxel reported, these were never intended to address the supply issue of 3 paclitaxel, and as anticipated were multistep and low-yielding. Instead, very elegant chemistry 21-28 was displayed wherein stereochemical and regiochemistry control were hallmarks. 10-O-Deacetylbaccatin III (3) Baccatin III (4) Figure 1.3 - The structures of paclitaxel precursor baccatin III and 10-O-deacetylbaccatin III While the semisynthetic method was sufficient to sustain the supply/demand cycle till 2002 for Bristol Myers Squibb, there were complications with the method. The yield of (3) isolated from Taxus spp. was unpredictable due to the fluctuations in weather and extensive damage was caused to the native Taxus populations in several countries from harvesting conditions for 29,30 paclitaxel precursors. Also the isolation and purification of precursors for the semisynthesis from plant tissue with abundant phenolics, lipids, and other contaminants required a substantial 31 effort making the process less effective. Therefore, the semisynthetic method was replaced by the current commercial supply of paclitaxel, plant cell fermentation technology developed by 32 Python Biotech. This methodology is environmentally sustainable and offers several advantages over the other methods such as not being subjected to weather, season, or contamination, it can be grown independently of its origin, and productivity can be increased by 2,29,30,33 optimizing the culture conditions, cell lines, and by adding precursors or elicitors Since this methodology relies on a biological source, the titers of paclitaxel, and related compounds, 34-36 can therefore be improved by understanding the biosynthesis of these metabolites in detail. 4 In addition to Taxus cell culture fermentation, a paclitaxel-producing endophytic fungus (Taxomyces andeanae) was discovered in 1993, and recent advancements in this area are 37,38 destined to sustain the supply of paclitaxel. 1.3. The Analogues of Paclitaxel Despite its potent biological activities, paclitaxel, as with most any drug, causes undesirable side 39 Also the low water-solubility, lack of tissue effects, which limits the dose that can be used. specific targeting, blood-brain barrier permeability, and acquired drug resistance towards 7,12,40 paclitaxel are important issues to be addressed. Therefore, it is essential to develop analogues of paclitaxel with better pharmacological properties and improved activities especially geared towards drug-resistant cell lines. The extensive Structure-Activity-Relationship (SAR) studies carried out by several researchers have been a great tool for identifying key functional 12 groupings necessary for drug efficacy (Figure 1.4). These studies have led to the development of efficacious analogues (Figure 1.5) (in some cases in a combinatorial approach), as 6,12,40-45 summarized in several reviews. 5 May removed without a signif icant loss of activity; some modif ications are more ef f ective N-acyl group is required May change without a huge decrease in activity; O OH some groups are more ef f ective AcO O NH O O Some alkenyl, substituted phenyls, and, heteroaromatic groups improve the activity OH Free hydroxyl or a hydrolyzable ester is required O HO H OAc O O Necessary f or activity Some changes are usef ul; but f ree hydroxyl isn't ef f ective Acyloxy group is essential; certain alkenyls and substituted aromatics improve activity Figure 1.4 - The Structure-Activity Relationships (SAR) of paclitaxel. This figure is adapted 12 from the published work of Cragg and co-workers In brief, each functional group of the parent drug has been modified separately or in combination, and the resulting molecule has been tested for cytotoxicity and tubulin binding affinity. Several paclitaxel analogues with improved efficacy have been identified as candidates 7,45-56 for phase I and phase II clinical studies (Figure 1.5). The active pharmacophore of paclitaxel was found to comprise most of the acyl groups around the tricyclic core of the molecule; therefore modifications of acyl functions were conspicuously targeted in developing 6,57 efficacious paclitaxel analogues (Figure 1.4). 6 * 48 Taxoprexin * 7-hexanoylpaclitaxel * * R: COCH2(CH2CH=CH)6CH2CH3 (5) 48 49 R: CO(CH2)5CH3 (6) BMS-184476 50 RPR-109881A (10) R1: Ph; R2: Ph (8) 56 BMS-275183 R: OCH2SCH3 (7) * 51 BMS-188797 R1: OC(CH3)3; R2: C(CH3)3 (9) * 52 TXD258 R1: Me; R2: Me; R3: Ph (11) 54 TL-00139 R1: COCH2CH3; R2: H, R3: 2-Furanyl (12) AcO O O O OH N NH O O H OH O O O O O OAc O O N NH O O F 53 O OH 55 O HO H OAc O O Ortataxel (13) DJ-927 (14) Figure 1.5 – Paclitaxel analogues currently in phase I or phase II clinical trials (analogues in * phase 2 clinical trials are indicated in an asterisk ) 7 1.4. Biosynthesis of Paclitaxel Interest in the biosynthesis of paclitaxel continues to be driven by the reliance on its biological 31,34,36 mode (plant cell fermentation (PCF)) of production. As mentioned previously, PCF is a 29,34 renewable, environmentally-friendly and cost-effective resource of this molecule. Foreseeably, PCF could potentially also be developed to biocatalyze efficacious analogues of paclitaxel. Paclitaxel is a diterpenoid found in a variety of Taxus plant species and was first 3 isolated from T. brevifolia. These species produce about 400 different taxoids, all of which are based upon the unique taxane core (15) (a pentamethyl[9.3.1.0]tricyclopentadecane, Figure 36,58-60 1.6). H 15 Figure 1.6 – The basic carbon frame configuration of most taxoids Up until 1990s, all diterpenoids were thought to arise from geranylgeranyl diphosphate (GGDP, 18) exclusively via the mevalonic acid pathway. However, a non-mevalonic acid pathway (i.e., the methylerythritol phosphate (MEP) pathway) to terpenes was relatively recently discovered and elucidated, showing the genesis of the precursors used in mono-, di-, and 61-64 tetraterpenoid biosynthesis. It has been shown that the taxane building blocks (IDP (16) and 61 DMADP (17)) are derived from the non-mevalonate pathway, and the sequential coupling of these primary metabolites produce 18, the universal precursor of diterpene biosynthesis. The first committed step on the paclitaxel pathway is the cyclization of 18 to taxa-4(5),11(12)-diene (19), 8 36 which is the parent of all taxoids with the skeleton of 15 . paclitaxel uses ~19 enzymatic steps. (Figure 1.7) 31,36,65-70 From 18, the biosynthesis of and includes a series of ~seven cytochrome P450-mediated oxygenations, two acetylations, a benzoylation, an oxidation at C9, and closure of the oxetane ring which leads to 4. Attachment of β-phenylalanine at C13 of 4 and 36,71- assembly of the N-benzoylphenylisoserine side chain completes the pathway (Figure 1.8). 91 9 IDP (16) a DMADP (17) GGDP (18) taxa-4(5),11(12)-diene (19) HO 1 5 H H 20 taxa-4(20),11(12)-dien-5α-ol (20) e OH 1 5 f OAc H H 20 taxa-4(20),11(12)-dien -5α-yl-acetate (21) 1 5 H 20 g OAc H 5α-acetoxytaxa-4(20),11(12)dien-10β-ol (22) 2-debenzoyltaxane (23) 3 4 Figure 1.7 – Paclitaxel biosynthetic pathway starting from primary metabolism to the paclitaxel precursor baccatin III (4). Step a: Isopentenyl diphosphate isomerase (IDPI); b: Geranylgeranyl diphosphate synthase (GGDPS); c: Taxadiene synthase (TS); d: Cytochrome P450 Taxadiene 5α-hydroxylase; e: Taxa-4-(20),11(12)-diene-5α-ol-O-acetyltransferase; f: Cytochrome P450 taxane 10β-hydroxylase; g: several steps; h: Taxane 2α-O-benzoyltransferase; i: 10Deacetylbaccatin III-10β-O-acetyltransferase. Abbreviations: MEP - Methylerythritol phosphate; IDP - Isopentenyl diphosphate; DMADP - Dimethylallyl diphosphate; GGDP - Geranylgeranyl diphosphate. 10 α-phenylalanine (24) AcO HO β-phenylalanine (25) β-phenylalanine CoA (26) O OH 26 13 HO O H OBz OAc β-phenylalanoyl baccatin III (27) 4 AcO NH2 O 3' O OH 13 O O HO H OAc O O AcO 1. m 2. n O OH NH O 2' O 13 O 3' HO H OAc O O OH O 27 1 Figure 1.8 - Last steps of paclitaxel biosynthetic pathway; N-benzoylphenylisoserine side chain construction at C13 of baccatin III (4). Step j: Phenylalanine aminomutase (PAM); k: Unknown CoA thioester ligase; l: Baccatin III 13-O-phenylpropanoyltransferase; m: Cytochrome P450 hydroxylase; n: Taxane N-benzoyltransferase 58 In planta, paclitaxel is one of about 400 taxoids in Taxus. A review of the characterized taxoids suggest that in addition to the acetylations at the C4 and C10 positions of paclitaxel, acetyl groups can be variously introduced at the C1, C2, C7, C9, and C13 positions of the taxane core, resulting in a diverse group of naturally occurring taxoids that may be involved in defense mechanisms of the plant (Figure 1.9). Notably, construction of these off-pathway metabolites likely divert carbon flux away from paclitaxel which could explain why the plant was a poor 11 source for isolating the target drug in the early days of paclitaxel discovery and 36,58,92,93 application. taxa-4(20)-11(12)diene-5α10β-14β-triol (28) AcO taxa-4(20)-11(12)diene-2α5α-10β-14β-tetra-acetate (29) OH AcO O OAc OAc OAc 7 AcO 13 HO O H OBz OAc O 13 HO 19 O H OBz OAc 1β-hydroxy-baccatin III (30) O AcO O OH NH O 2' O 13 O 3' HO H OAc O O OH Paclitaxel C (33) 19-hydroxy-13-oxo-baccatin III (32) Figure 1.9 – A list of common “off-pathway metabolites” which are proposed to divert pathway flux away from paclitaxel production baccatin VI (31) Although none of the acyltransferases involved in off–pathway biosynthesis are yet known, identification of these acyltransferases will be useful for improving the biological 34,36 production of paclitaxel and its precursors. Despite the immense work reported on paclitaxel biosynthesis, the order of some steps in the pathway remains unclear, and the full complement of extant paclitaxel biosynthetic genes has not yet been successfully expressed in a heterologous host. Thus, a thorough understanding of the biosynthesis of paclitaxel and that of other abundant 12 taxoids is essential for improving biological production techniques as well as for using Taxus 31,34,90,91,94-97 genes towards developing paclitaxel analogues through biocatalysis. 1.5. Taxus Acyltransferases: Members of BAHD Superfamily Most of the approximately 400 different naturally occurring taxoids produced by Taxus species have common structural and functional group motifs, while a few others deviate from the norm with regards to oxygenation pattern, carbon skeleton arrangement, and acyl group 58,60 regiochemistry. To date, only very few Taxus acyltransferases have been identified and/or 34,36,75,78-84 characterized. Five of the characterized Taxus acyltransferases are involved in paclitaxel biosynthesis and belong to a large superfamily of plant-derived acyltransferases (designated BAHD), an acronym derived from first four biochemically characterized enzymes of 98,99 this superfamily (BEAT, AHCT, HCBT, and DAT). Enzymes in this family utilize acyl 98,99 coenzyme A (acyl donor substrate) and amino or hydroxy group acceptors as cosubstrates. The members in clade V of this superfamily have been subdivided further into subgroups, and 99 the second subgroup consists of the Taxus acyltransferases involved paclitaxel biosynthesis. Understanding the biochemistry of the BAHD members is potentially useful because acyl transferases play crucial roles in modifying the characteristics of plant metabolites such as alterating the polarity, volatility, solubility, chemical stability, and biological activity, e.g., in 99 terms of plant signaling or defense. BAHD acyltransferases contain signature amino acid motifs such as an HXXXD, identified as catalytic (H) and structural (D) residues, and a DFGWG motif located near the carboxyl terminus and presumed to comprise a substrate access channel.99 13 Knowledge of the catalytic mechanism and the function of the shared motifs of the members of BAHD superfamily have been well-supported by the structural data obtained for vinorine 100-103 synthase and anthocyanin malonyltransferases. These structural data along with mutational studies show that members of this family can acylate a wide variety of substrates in 99 vitro; but the range of specificity among different enzymes varies extensively. For example, some BAHD members show a restricted range of substrate usage, while others have wide 88-90,99,104-107 substrate specificities in vitro and/or in vivo. It is important to note that the distribution and abundance of a particular acylated secondary metabolite (i.e., specialized compounds) 108,109 produced in planta may not reveal the promiscuity of a particular acyltransferase due to the limited array of naturally occurring substrates. Thus, it becomes important to evaluate whether novel acylated metabolites can be formed by a BAHD acyltransferase incubated in vitro with non-natural substrates. Furthermore, respective upregulation or downregulation of particular BAHD enzymes in vivo might directly increase the levels of pharmaceutically important compounds or commodity chemicals, or decrease the production of “off-pathway metabolites,” which could indirectly increase the levels of potentially 34,36,99,104,110,111 beneficial compounds made in planta. Despite the importance and the prevalence of BAHD acyltransferases, very few of these catalysts have been extensively analyzed in terms of their substrate specificity. Hence, understanding the Taxus acyltransferases in greater detail is vital not only towards the production of paclitaxel or its analogues, but also towards understanding the mechanism of the substrate selectivity of BAHD members. 14 1.6. Biocatalysis: Using Taxus Acyltransferases As mentioned earlier, the SAR of paclitaxel revealed that most of the acyl groups play a 12 principal role in drug efficacy (Figure 1.4). Also, several improved paclitaxel analogues with various acyl group modifications have been identified as highly promising candidates for further clinical and preclinical studies (cf. Figure 1.5 and Figure 1.10). 7,40,45,46,112-114 Currently, these paclitaxel analogues are obtained by semisynthetic methods that require, often times, redundant protection group manipulations of functional groups on the taxane core to direct the acyl group replacement chemistry. These added protecting group related steps generally affect 115 the overall product yield. As an example, one route to semisynthesize an efficacious analogue of paclitaxel (46) involves 13 total steps. Briefly, three reactive hydroxyl groups of (3) are silyl protected, followed by reductive ester cleavage to remove the benzoyl group at the C2 hydroxyl. Then the selective acylation at the C2 hydroxyl with activated 3-azidobenzoic acid is immediately followed by the deprotection of 7, 10, and 13 hydroxyl groups, resulting in 53. Selective re-protection of the hydroxyl group at C7 is achieved before the acylation of the hydroxyl group at C10 with propanoyl chloride, forming 55. Selective acylation at the C13 hydroxyl with enantiopure βlactam (54) followed by deprotection of C2 and C7 hydroxyls forms the desired paclitaxel 40,116-118 analogue (46) (Figure 1.11). Although not included in the reaction scheme, the synthesis of enantiopure β-lactam involves 5 additional steps, increasing the number of chemical 40 steps to synthesize analogue 46 . 15 In contrast, the application of biocatalytic acylation and deacylation in a semibiosynthetic procedure could potentially reduce the number of protection-deprotection steps required for the assembly of next generation paclitaxel compounds. With regards to acylation, the several identified Taxus acyltransferases deliver acyl groups from the corresponding acylcoenzyme A thioesters (acyl-CoA) to paclitaxel pathway intermediates to produce the drug, and they have been examined with a vast range of substrates in standard steady-state kinetic 78,81-84,88-91 evaluations in vitro. In terms of deacylation, BAHD family members are also found 119 to catalyze regiospecific CoA-dependent deacetylation reactions. Therefore, Taxus acyltransferases conceivably can be utilized in regioselective deacylation of different acyl moieties around the paclitaxel core. A shorter (5 steps) and potentially improved route to semisynthesize paclitaxel analogue 46 employs Taxus acyltransferase catalysis in both the forward (acylation) and reverse (deacylation) reactions. In this route (3) is selectively acylated at C10 without any prior protections of other free hydroxyls present in the molecule. The transfer of the propanoyl group to the hydroxyl group at C10 is catalyzed by the biocatalyst DBAT (10-O-deacetylbaccatin III-10β-O-acetyltransferase). Then through TBT (7,13-O,O-diacetyl-2-O-debenzoylbaccatin III-2α-O-benzoyltransferase) catalysis in both reverse and forward directions, the regiospecific removal of benzoyl group at C2 and the incorporation of 3-azidobenzoyl moiety at the free hydroxyl at C2 are achieved to form 58. The corresponding side chain of the paclitaxel analogue 46 is transferred to the hydroxyl at C13 of baccatin core using BAPT (baccatin III-13α-O-phenylpropanoyltransferase) to form biosynthetic product 59, which is immediately subjected to NDTBT (3'-N-debenzoyl-2'deoxytaxol N-benzoyltransferase) catalysis to form the desired paclitaxel analogue 46 (Figure 1.12). Thus, the biocatalytic approach to synthesize paclitaxel analogue 46 would include only 5 16 steps, while the semisynthetic route uses 13 steps. This reduction in the number of assembly steps by employing biocatalysis selectively could potentially increase the production yield of various paclitaxel analogues, including analogue 46. Thus, understanding the cryptic mechanistic aspects of the Taxus acyltransferases would facilitate the designing of new biocatalysts capable of modifying acyl groups of the taxane core or other diterpene scaffolds. 17 45 R: Bz (34) , R: 4-OMe(C6H4)CO (35) R: 4-OMe(C6H4)(CH2)2CO (36) R: Me2C=CH (39) 45 112 R: cyclopent-1-enyl (37) 45 112 R: cyclohex-1-enyl (38) 45 R: Me2C=CH (41) 45 R: Me2CHCH2- (40) RO O O F F OH O OH AcO O HO H OAc O O H N O O OH O O OH O H O OBz OAc X 113 113 R: Ac; X: MeO (43) , R: Ac; X: N3 (44) R: EtCO; X: MeO (45) R: Me2NCO; X: MeO (47) 113 113 113 40 R: Me2CHCH2- (42) NH O O 40 , R: EtCO; X: N3 (46) 113 , R: Me2NCO; X: N3 (48) 51 114 113 113 R: MeOCO; X: MeO (49) , R: MeOCO; X: N3 (50) Figure 1.10 - Several improved paclitaxel analogues in preclinical studies with acyl group modifications 18 3 TIPSO 52 5 steps F N O 53 O F t-Boc O O O NH O O 54 F O OH F OH O HO H OAc O O N3 Paclitaxel analogue (46) 55 Figure 1.11 – Scheme of synthesis of improved paclitaxel analogue 46. Reagents, conditions used in each step, and the stepwise yields: (i) TESCl, imidazole, DMF, RT, 96%; (ii) sodium bis(2-methoxyethoxy)aluminum hydride, THF, -10 ºC, 97%; (iii) 3-azidobenzoic acid, DIC, DMAP, CH2Cl2, 88%; (iv) HF/pyridine, pyridine/MeCN; (v) TESCl, imidazole, DMF, RT, 2 h, 83% for two steps; (vi) LiHMDS, EtCOCl, THF, 82%; (vii) LiHMDS, THF, -40 ºC, 30 min; (viii) HF/pyridine, pyridine/MeCN, 0 ºC – RT, 18 h, ~80% for two steps. Overall yield is ~0.4% without considering the 5 steps involved in synthesis of enantiopure β-lactam. Note - TIPS – triisopropyl silyl; PMP – para-methoxyphenyl group. 19 O HO O OH O O O OH O b a HO HO O H OBz OAc 3 O HO O H OBz OAc 56 HO HO HO O H OH OAc 57 O O O OH O c O HO H OAc O O O OH NH2 O d HO O OH O F F OH O HO H OAc O O N3 N3 58 59 46 Figure 1.12 - A potential route of semisynthesizing improved paclitaxel analogue 46 incorporating biocatalysis of Taxus acyltransferases in both forward and reverse direction. 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DESIGN AND SUBSTRATE SPECIFICITY STUDIES OF MODIFIED TAXUS 2-O-DEBENZOYLBACCATIN III 2α-O-BENZOYLTRANSFERASE (mTBT) 2.1. Introduction Plant-derived acyltransferases that utilize a coenzyme A acyl donor and an amino or hydroxy group acceptor as co-substrates are assigned to a large superfamily, designated BAHD. 1 Occurrences of the BAHD acyltransferases are seemingly ubiquitous within the plant kingdom, and despite their prevalence, the function of several of the acyltransferases remain unknown. Generally, the BAHD members acetylate and phenylpropanoylate amino and/or hydroxy groups of secondary metabolites. In addition, benzoylation of plant secondary metabolites also occurs to 2 invoke specific physiological effects in planta. The compendium of biosynthetic products made by these catalysts is a vast group of variously acylated metabolites found in different plant species with important biological activities (Figure 2.1). 1,2 A cladogram of functionally characterized BAHD family members has been constructed on the basis of phylogenetic analysis of the protein sequences, and the evolutionary tree was shown to branch into five major 1,3 clades. All six of the benzoyltransferases in this superfamily reside in clade V, which includes O-benzoyltransferase (BEBT) isozymes from Clarkia breweri (AAN09796) and Nicotiana tabacum (AAN09798), a functionally similar benzyl/phenylethyl alcohol O-benzoyltransferase (BPBT, AAU06226) from Petunia × hybrida, the 2-O-debenzoylbaccatin III 2-Obenzoyltransferase (TBT, Q9FPW3) from Taxus cuspidata (described herein), and two N30 acyltransferases, an N-debenzoylpaclitaxel N-benzoyltransferase (NDBTBT, AAM75818) from Taxus canadensis, and an anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT, 3 CAB06430) from Dianthus caryophyllus. Benzylbenzoate (1) Clarkia breweri Benzylacetate (2) Clarkia breweri O Chlorogenic acid (3) Nicotiana tabacum O O O OH OH O OH HO HO O O O O O OH OH O OH NH O O O O OH HO H O O O O O OH Anthocyanidin 3-O-3'',6''-O-dimalonylglucoside (4) Dendranthema morifolium Paclitaxel (5) Taxus spp. Vindoline (6) Dianthramide B (7) Catharanthus roseus Dianthus caryophyllus Figure 2.1 – A few biosynthetically derived and variously acylated metabolites formed by the function of BAHD plant superfamily acyltransferases. Acyl groups are highlighted in blue. However, the substrate specificity of most recombinantly expressed and/or native BAHD benzoyltransferases (outside of the Taxus family) has been examined with only a single or very 1 limited substrates in standard steady-state kinetic evaluations in vitro. 31 O O O O O O OH NH O OH N3 O HO H OAc O O O OH O HO H OAc O O MeO 4 4 SB-T-121304 (8) O O O O OH NH O O O O O SB-T-11033 (9) O OH NH O O OH O HO H OAc O O S 5 6 2-O-heteroaroyl taxanes (10) taxane-antibody immunoconjugate (11) O MeO O O O OH NH O O F O F OH O HO H OAc O O N3 8 SB-T-12855-3 (14) 7 SB-T-10104 , R: isobutyl at C3' (12) 7 SB-T-10204 , R: isobutenyl at C3' (13) Figure 2.2 – The C-2 acyl modified potential candidates for developing novel taxane agents The application of the 2α-O-benzoyltransferase (TBT) on the paclitaxel biosynthetic pathway 4-15 toward the production of potentially useful C-2 modified paclitaxel compounds 32 (Figure 2.2) can be greatly bolstered by knowledge of its substrate specificity with various taxane and acyl CoA thioester cosubstrates. The extended substrate specificity of only one Taxus Oacyltransferase (a 10-O-acetyltransferase (DBAT)) has been examined thus far. DBAT was shown to have modest selectivity for three short-chain alkanoyl CoA thioesters and to utilize 416,17 and 10-O-deacetyltaxanes as acyl group acceptors in vitro. Several taxane analogues with 18 different acyl groups at C-10 have been made in vivo in E. coli engineered to express DBAT. Encouraged by the modest flexibility of DBAT, the substrate scope of TBT was evaluated for potential application towards biosynthesizing an array of useful C-2 modified taxane analogues. TBT was first characterized as a necessary catalyst on the biosynthetic pathway to paclitaxel by incubating benzoyl CoA and TBT with 7,13-O-O-diacetyl-2-O-debenzoylbaccatin III (17); the transfer of the benzoyl moiety from the corresponding CoA thioester to 17 to form 7,13-O-O19 diacetylbaccatin III (18) was observed (Figure 2.3). The reported KM values for 7,13-O-O- diacetyl-2-O-debenzoylbaccatin III (17) and benzoyl CoA (15) are 0.64 mM and 0.30 mM, 19 respectively. In the same report, acetyl CoA (16), the only other CoA thioester tested at the time with TBT, was also identified as a productive cosubstrate with Vrel of 1.5% compared to 19 benzoyl CoA (Figure 2.3). Herein, the kinetic details of an extended array of substrates incubated with TBT are described, and the potential use of TBT as a biocatalyst in the development of novel compounds is also discussed. 33 O O O O O O R: Ph (15), R: Ac (16) O O HO H OH O O O O O O O O O HO H O O O O O R 7,13-O,O-diacetyl-2-O-acyl2-O-debenzoylbaccatin III R: Ph (18), R: Ac (19) Figure 2.3 – The reaction of the recombinant Taxus 2-O-benzoyltransferase (TBT) 7,13-O,O-diacetyl2-O-debenzoylbaccatin III (17) 2.2. Experimental 2.2.1. Substrates, Reagents, and General Instrumentation The Taxus cuspidata cDNA (tbt) (accession no. AF297618) was a generous donation of Washington State University Research Foundation (Pullman, WA). Baccatin III (20) was 3 purchased from Natland (Research Triangle Park, NC). Sodium [ H]borohydride and CoASH were purchased from American Radiolabeled Chemicals Inc. (Saint Louis, MO). The restriction endonucleases were purchased from New England Biolabs (Ipswich, MA). Material required for thin layer chromatography (TLC), preparative TLC, and flash column chromatography were purchased from EMD Chemicals Inc. (Gibbstown, NJ). Acyclic short-chain-hydrocarbon carbonyl CoA thioesters and all other reagents were obtained from Sigma-Aldrich and used without further purification, unless indicated otherwise. HPLC analyses of semi-synthetic and biosynthetic products were carried out using a reversed-phase column (Econosphere C18, 5 μm, 250 × 4.6 mm, Alltech, Mentor, OH) attached to an Agilent 1100 HPLC system (Agilent Technologies, Wilmington, DE). The HPLC was 34 connected in series with a UV detector and a Packard Radiomatic Flow-One Beta 150TR radioactivity detector (Perkin-Elmer, Shelton, CT), which mixed the effluent with 3a70B Complete Counting Cocktail (Research Products International, Mount Prospect, IL) in detecting radioactive products. A Varian Inova-300, Varian UnityPlus-300, Varian Inova-500, and Varian 1 UnityPlus-500 instruments were used to acquire H and 13 C NMR spectra. An electrospray ionization tandem mass spectrometer (ESI-MS/MS) (Q-ToF Ultima Global, Waters, Milford, MA) was used to acquire mass analysis of semi-synthetic compounds by direct injection. The biosynthetic compounds were separated and analyzed using a reversed-phase column (Betasil C18, 5 μm, 150 × 2.1 mm, Thermo Fisher Scientific Inc., Waltham, MA) attached to a capillary HPLC system (CapLC capillary HPLC, Waters, Milford, MA) connected to ESI-MS/MS (Q-ToF Ultima Global, Waters, Milford, MA). Other general information not included here is noted elsewhere in the text. 2.2.2. Subcloning the Wild-Type TBT cDNA Standard microbial and recombinant techniques used throughout this work were described by Sambrook. 20 Turbo Pfu DNA polymerase (Stratagene) was used in all the PCR reactions. A sticky-end PCR method 21 was conducted to amplify the wild-type tbt (accession no. AF297618) insertion from a pCWori+ vector with an appropriate primer set [pair 1: forward primer (5′TGGGCAGGTTCAATGTAGAT-3′) and reverse primer (5′- GATCCTTATAACTTAGAGTTACATATTTTAGCCAC-3′); pair 2: forward primer (5′TATGGGCAGGTTCAATGTAGAT-3′) and reverse primer (5′- CTTATAACTTAGAGTTACATATTTTAGCCAC-3′); the nucleotides of BamHI and NdeI restriction sites are italicized and underlined]. By this method, the tbt cDNA was transferred 35 21 from the previously used pCWori+ vector to pET28a (Novagen), designated p28wtTBT. The nucleotide sequence of wild-type tbt and the amino acid sequence of wtTBT are shown in Figure A 1. This exchange incorporated an N-terminal His6-tag epitope on the expressed TBT for immunoblot analysis of the expressed protein and purification by His-Select Nickel Affinity Gel (Sigma, St. Louis, MO) binding. The plasmid containing the tbt cDNA and a pET28a vector without an insert were used to separately transform E. coli BL21(DE3) following a described protocol (Stratagene). 2.2.3. Relative Soluble and Insoluble Expression of wtTBT The wild-type tbt was expressed in E. coli, and the respective recombinant enzyme, wtTBT was 19 harvested according to the previously reported protocol with some modifications. In brief, BL21(DE3) bacterial cultures transformed to express the wild-type tbt clone were grown overnight at 37 °C in 5 mL of Luria-Bertani medium (Neogen, Lansing, MI) supplemented with 50 μg/mL kanamycin (Roche). The 5-mL inoculum was added to 1 L of Luria-Bertani medium supplemented with the appropriate antibiotic, the cells were grown at 37 °C to OD600 ≈ 1, gene expression was induced with 50 μM isopropyl-β-D-1-thiogalactopyranoside (IPTG) (Gold Biotechnology, St Louis, MO), and the cultures were incubated at 18 °C. After 16 h, the density of cultures was assessed by OD600 monitoring. The cells were harvested by centrifugation at 4000g for 20 min at 4 °C and resuspended in assay buffer [25 mM 3-(N-morpholino)-2hydroxypropanesulfonic acid (Research Products International Corp., Mt. Prospect, IL), 5% glycerol (v/v), 3 mM dithiothreitol (Roche), pH 7.4] to a concentration of 0.3 g of cells/mL. The cells were lysed by sonication at 4 °C [5 × 1-min bursts at 50% power with 1-min intervals, and then 2 × 15 s-bursts at 70% power with 2-min intervals, using a Misonix XL-2020 sonicator 36 (Farmingdale, NY)]. The homogenate was centrifuged at 15000g to pellet the debris; the pellet was resuspended in assay buffer and saved for further analysis; the supernatant was clarified by centrifugation at 190,000g to provide the soluble enzyme fraction. In order to assess the relative concentration of recombinantly expressed wtTBT in the crude soluble and insoluble fractions, first, the total protein concentrations of each sample were determined by comparison against BSA standards ranging from 2 to 10 mg/mL. Equal amounts of protein from both the soluble and insoluble fractions were subjected to SDS PAGE, and the separated proteins were visualized by Coomassie blue staining. Kodak 1D Image Analysis Software (Version 3.6.3) was used to integrate the intensity of the staining of the overexpressed TBT enzyme bands (at ~50 kDa) on the gel. The relative amounts of overexpressed protein were also verified by Western immunoblot analysis, according to a method based on the 1-Step TMB-Blotting Kit (Pierce, Rockford, IL) with the following antibodies: monoclonal anti-polyhistidine and anti-mouse IgG peroxidase conjugate (Sigma, St. Louis, MO). 2.2.4. Synthesis of 7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III (17) 19 To a stirred solution of 7,13-O,O-diacetylbaccatin III (spectral data below) (18) (100 mg, 149 μmol) in dry THF (7 mL) at 0 °C under N2 was added (dropwise) bis(2methoxyethoxy)aluminum hydride (>65 wt % in toluene, 3 equiv). After stirring for 2.5 h at 0 °C, the reaction was quenched by dropwise addition of saturated NH4Cl, and the mixture was stirred for 10 min, then warmed to room temperature and diluted with EtOAc (50 mL), followed by the addition of H2O (10 mL). The aqueous phase was separated and extracted again with EtOAc (2 × 25 mL). The combined organic fractions were washed with brine and H2O and then 37 dried over anhydrous Na2SO4. The solvent was evaporated, and the crude product was purified by silica gel flash column chromatography (10:90 to 60:40 (v/v), linear gradient of EtOAc in hexanes) to yield pure 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (17) (54.8 mg, 65% yield): 1 H NMR (300 MHz, CDCl3) δ: 1.01 (s, H-16), 1.19 (s, H-17), 1.73 (s, H-19), 1.80 (ddd, J ) 2.1, 3.6, 12.5 Hz, H-6β), 1.87 (d, J = 1.5 Hz, H-18) 1.99 (s, C(O)CH3 at C-10), 2.11 (s, C(O)CH3 at C-7), 2.12 (s, C(O)CH3 at C-13), 2.18 (s, C(O)CH3 at C-4), 2.44 (br s, OH at C-2), 2.55 (ddd, J = 2.4, 7.2, 14.3 Hz, H-6α), 2.67 (d, J = 4.8, H-14), 3.55 (d, J = 6.6 Hz, H-3), 3.87 (dd, J = 6.3, 11.4 Hz, H-2), 4.57 (d, J = 10.8 Hz, H-20α), 4.59 (d, J = 10.8 Hz, H-20β), 4.94 (d, J = 7.8 Hz, H5), 5.22 (dd, J = 3.6, 8.7 Hz, H-7), 6.12 (t, J = 8.8 Hz, H-13), 6.16 (s, H-10) (Figure A 29) (cf. Figure 2.4 for proton numbering); 13 C NMR (75 MHz, CDCl3) δ: 202.84, 170.67, 170.49, 169.60, 169.09, 141.19, 132.83, 83.90, 82.16, 78.48, 77.96, 75.68, 74.22, 71.78, 69.92, 56.25, 47.26, 42.89, 36.12, 33.64, 26.28, 22.65, 21.36, 21.30, 20.91, 20.81, 14.90, 11.15 (Figure A 30); + HRMS (ESI-TOF) m/z 567.2465 [M + H] ; calculated for C28H39O12: 567.2442. 1 The NMR data for 7,13-O,O-diacetylbaccatin III (18). H NMR (300 MHz, CDCl3) δ: 1.14 (s, H-16), 1.19 (s, H-17), 1.78 (s, H-19), 1.82 (ddd, J = 1.5, 3.0, 12.5 Hz, H-6β), 1.94 (s, H18) 2.01 (s, C(O)CH3 at C7), 2.15 (s, C(O)CH3 at C10), 2.18 (s, C(O)CH3 at C13), 2.23 (d, J = 9 Hz, H-14), 2.32(s, C(O)CH3 at C4), 2.58 (ddd, J = 7.5, 8.0, 15.0 Hz, H-6α), 3.93 (d, J = 7 Hz, H-3), 4.13 (d, J = 8.5 Hz, H-20α), 4.30 (d, J = 8.5 Hz, H-20β), 4.95 (d, J = 9.5 Hz, H-5), 5.57 (dd, J = 7.5, 10.5 Hz, H-7), 5.64 (d, J = 7.0 Hz, H-2), 6.14 (t, J = 9.0 Hz, H-13), 6.23 (s, H-10), 7.46–8.05 (aromatic protons) (Figure A 31) (cf. Figure 2.4 for proton numbering); 38 13 C NMR (75 MHz, CDCl3) δ: 202.26, 170.60, 170.43, 169.73, 169.10, 167.15, 141.61, 133.96, 132.68, 130.26, 129.41, 128.88, 84.19, 81.11, 78.98, 76.52, 75.62, 74.76, 71.62, 69.77, 56.29, 47.45, 43.35, 35.76, 33.56, 26.60, 22.67, 21.42, 21.30, 20.93, 20.86, 14.96, 10.97 (Figure A 32); + HRMS (ESI-TOF) m/z 671.2692 [M + H] ; calculated for C35H43O13: 671.2704. 2.2.5. Synthesis of 7-O-Acetylbaccatin III (21) To a stirred solution of baccatin III (20) (100 mg, 170 μmol) in dry THF (3 mL) at 23 °C under N2 were added Ac2O (5 equiv), 4-(N,N-dimethylamino)pyridine (4-DMAP) (5 equiv), and Et3N (24 μL, 172 μmol). The reaction was monitored by TLC; upon completion, the reaction was diluted with EtOAc (50 mL) and quenched with H2O (10 mL). The mixture was stirred for 15 min, and the aqueous fraction was separated and extracted with EtOAc (2 × 25 mL). The combined organic fractions were washed with saturated CuSO4, brine, 0.1 M HCl, and H2O and dried over anhydrous Na2SO4. The organic solvent was evaporated, and the crude product was purified by silica gel flash column chromatography (40:60 to 60:40 (v/v), linear gradient of 1 EtOAc in hexanes) to yield pure 7-O-acetylbaccatin III (21) (104 mg, 97% yield): H NMR (500 MHz, CDCl3) δ: 1.06 (s, H-16), 1.11 (s, H-17), 1.77 (s, H-19), 1.80 (m, H-6β), 2.01 (s, C(O)CH3 at C-7), 2.08 (s, H-18), 2.15 (s, C(O)CH3 at C-10), 2.26 (s, C(O)CH3 at C-4), 2.59 (ddd, J = 2.5, 8.5, 14.2 Hz, H-6α), 3.98 (d, J = 6.5 Hz, H-3), 4.13 (d, J = 8.5 Hz, H-20α), 4.29 (d, J = 8.5 Hz, H-20β), 4.83 (br t, H-13), 4.95 (d, J = 8.5 Hz, H-5), 5.58 (dd, J = 3.5, 7.0 Hz, H-7), 5.60 (d, J = 3.5 Hz, H-2), 6.25 (s, H-10), 7.46-8.08 (aromatic protons) (cf. Figure 2.5 for proton numbering); 13 C NMR (125 MHz, CDCl3) δ: 202.60, 170.92, 170.66, 169.16, 167.23, 144.77, 133.91, 39 131.93, 130.33, 129.54, 128.86, 84.27, 80.97, 78.84, 76.59, 76.12, 74.62, 71.81, 68.19, 56.42, 47.70, 43.04, 38.72, 33.62, 26.90, 22.79, 21.33, 21.0, 20.28, 15.40, 10.94; HRMS (ESI-TOF) m/z + 629.2592 [M + H] ; calculated for C33H41O12: 629.2598. 2.2.6. Synthesis of 7-O-Acetyl-13-oxobaccatin III (22) To a stirred solution of 7-O-acetylbaccatin III (21) (80 mg, 127 μmol) in dry CH2Cl2 (10 mL) at 23 °C under N2 was added activated MnO2 powder (1 g). The reaction was monitored by TLC, and upon completion of the reaction, the mixture was filtered to remove excess MnO2. The filtrate was diluted with EtOAc (15 mL) and quenched with H2O (10 mL). The aqueous fraction was separated and extracted with EtOAc (2 × 25 mL). The combined organic fractions were washed with brine and H2O and dried over anhydrous Na2SO4. The organic solvent was evaporated, and the crude product was purified by silica gel flash column chromatography (30:70 to 60:40 (v/v), linear gradient of EtOAc in hexanes) to yield pure 7-O-acetyl-13-oxobaccatin III 1 (22) (79 mg, 100% yield): H NMR (500 MHz, CDCl3) δ: 1.18 (s, H-16), 1.19 (s, H-17), 1.74 (s, H-19), 1.78 (ddd, J ) 1.5, 3.0, 12.5 Hz, H-6β), 2.01 (s, H-18), 2.09 (s, C(O)CH3 at C-7), 2.17 (s, C(O)CH3 at C-10), 2.19 (s, C(O)CH3 at C-4), 2.58 (ddd, J = 7.5, 9.5, 14.5 Hz, H-6α), 2.64 (d, J = 19.5 Hz, H-14β), 2.92 (d, J = 19.5 Hz, H-14α), 4.00 (d, J = 6.5 Hz, H-3), 4.08 (d, J = 7.0 Hz, H-20α), 4.30 (d, J = 8.5 Hz, H-20β), 4.90 (d, J = 8.5 Hz, H-5), 5.56 (dd, J = 7.5, 10.5 Hz, H-7), 5.63 (d, J = 7.0 Hz, H-2), 6.33 (s, H-10), 7.46-8.03 (aromatic protons) (cf. Figure 2.5 for proton numbering); 13 C NMR (125 MHz, CDCl3) δ: 200.64, 198.14, 170.63, 170.22, 168.73, 167.00, 151.96, 141.06, 134.25, 130.27, 129.00, 128.94, 84.00, 80.57, 78.61, 76.69, 76.23, 72.77, 71.56, 40 57.08, 46.72, 43.70, 42.80, 33.59, 33.13, 21.83, 21.25, 20.91, 18.32, 13.93, 10.60; HRMS (ESI+ TOF) m/z 627.2440 [M + H] ; calculated for C33H39O12: 627.2442. 2.2.7. 3 Synthesis of [13- H]-7-O-Acetylbaccatin III (23) To a stirred solution of 7-O-acetyl-13-oxobaccatin III (22) (7 mg, 11 μmol) in dry THF (5 mL) at 3 0 °C under N2 were added sodium [ H]borohydride (333 μmol, specific activity of 150 Ci/mol) dissolved in a minimum amount of 0.01 M NaOH. The reaction mixture was warmed to room temperature, and the progress of the reaction was monitored by TLC. After 3 h, more 7-O-acetyl13-oxobaccatin III (22) (14.0 mg, 22 μmol) was added to the reaction, which was stirred for 3 h, diluted with EtOAc (5 mL), and quenched with H2O (5 mL). The mixture was stirred for 15 min, and the aqueous fraction was separated and extracted with EtOAc (2 × 5 mL). The combined organic fractions were dried over anhydrous Na2SO4, the solution was filtered, the filtrate was concentrated in vacuo, and the crude product was purified on silica gel PTLC (60:40 (v/v), 3 EtOAc/hexanes) to yield pure [13- H]-7-O-acetylbaccatin III (23) (20 mg, 0.61 mCi, 95% radiochemical purity by radio-HPLC). 2.2.8. 3 Synthesis of [13- H]-7,13-O,O-Diacetylbaccatin III 3 To a stirred solution of [13- H]-7-O-acetylbaccatin III (23) (20 mg, 0.61 mCi) in dry THF (5 mL) at 23 °C under N2 were added Ac2O (100 equiv), 4-DMAP (40 equiv), and Et3N (24 μL, 172 μmol), and the starting material was depleted after 28 h, as determined by TLC monitoring of the reaction progress. An aliquot of unlabeled 7-O-acetylbaccatin III (21) (12 mg, 19 μmol) 41 was added to the reaction and stirred in the solution for 3 h. The mixture was then diluted with EtOAc (5 mL), quenched with H2O (5 mL), and stirred for 15 min; the aqueous fraction was separated and extracted with EtOAc (2 × 5 mL). The combined organic fractions were washed with saturated CuSO4, brine, 0.1 M HCl, and H2O and dried over anhydrous Na2SO4. The organic solvent was evaporated, and the crude product was purified on silica gel PTLC (60:40 3 (v/v), EtOAc/hexanes) to yield pure [13- H]-7,13-O,O-diacetylbaccatin III (21 mg, 0.59 mCi, 97% radiochemical purity by radio-HPLC). 2.2.9. 3 Synthesis of [13- H]-7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III (24) 3 To a stirred solution of [13- H]-7,13-O,O-diacetylbaccatin III (21 mg, 0.59 mCi, 30 Ci/mol) in dry THF (5 mL) at 0 °C under N2 was added (dropwise) bis(2-methoxyethoxy)aluminum hydride (Red-Al) (>65 wt % in toluene, 3 equiv). After stirring for 2.5 h, the reaction was quenched by dropwise addition of saturated NH4Cl at 0 °C, and the mixture was stirred for 10 min, warmed to room temperature, and diluted with EtOAc (5 mL) and H2O (5 mL). The aqueous phase was separated and extracted again with EtOAc (2 × 5 mL). The combined organic fractions were washed with brine and H2O and then dried over anhydrous Na2SO4. The solvent was evaporated, and the crude product was purified by silica gel PTLC (60:40 (v/v), 3 EtOAc/hexanes) to yield pure [13- H]-7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (24) (6.6 mg, 0.35 mCi, 99% radiochemical purity by radio-HPLC, 30 Ci/mol specific activity). The LCESI-MS (positive ion mode) fragmentation profile and the HPLC profile of authentic unlabeled 42 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (17), described earlier in section 2.2.4 were 3 identical to those obtained for the [ H]-labeled material. 2.2.10. General Synthesis of Aroyl/Alkyl CoA Thioesters Several aroyl CoA donors (heteroaroyls, variously substituted benzoyls, cyclohexanoyl, phenylacetyl) were synthesized via a previously described method that proceeds through a mixed 16 ethyl carbonate anhydride. Briefly, Et3N (3.0 μL, 30 μmol) was added to a solution of the carboxylic acid (27 μmol) in 5:2 CH2Cl2/THF (v/v, 1.4 mL) under N2 gas. The mixture was stirred for 10 min at 23 °C, ethyl chloroformate (2.9 μL, 30 μmol) was added in one portion, and the reaction was stirred for 1 h at 23 °C. The solvents were evaporated under reduced pressure, and the residue was dissolved in 0.5 mL of t-BuOH. Coenzyme A as the sodium salt (23 mg, 30 μmol dissolved in 0.5 mL of 0.4 M NaHCO3) was added to the solution, and the mixture was stirred for 0.5 h at 23 °C and then quenched with dropwise addition of 1 M HCl, to adjust the pH between 3 and 5. The solvents were evaporated under vacuum, and the residue was dissolved in H2O (5 mL, pH 5) and loaded onto a miniature C18 silica gel preparatory column (10% capped with TMS) that was washed with 100% MeOH (50 mL) and pre-equilibrated with distilled H2O (100 mL, pH 5). The sample was washed with H2O (100 mL, pH 5) and then eluted with 15% MeOH in H2O (50 mL, pH 5). The fractions containing aroyl CoA, as determined by TLC, were combined, and the solvent was evaporated to yield pure product (90-99% yield, except phenylacetyl CoA and cyclohexanoyl CoA which were obtained at 56.4% and 38.7% yield, respectively). 43 2.2.10.1. Quantification of Synthesized Acyl CoA Thioesters Using Ellman Assays The quantification of each acyl CoA thioester was achieved by measuring the CoA content using Ellman assay conditions, before and after cleavage of the thioester bond with alkaline hydrolysis 22 following an established procedure. For this hydrolysis, an aliquot of each of the acyl CoA sample was mixed with 2 M NaOH (final concentration 0.5 M) and heated at 50 °C for 30 min. One molar HCl was added to lower the pH, which was then adjusted to 6–7 with 0.5 M NaHCO3 buffer. The content of CoA was determined in an aliquot of this neutralized sample using 10 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB or Ellman reagent) in 100 mM K2PO4, pH 8.0, by measuring the absorbance at 412 nm and then using a molar extinction coefficient of 13700 M-1 cm-1. The difference in absorbance between hydrolyzed and non hydrolyzed samples was used to calculate the acyl-CoA concentrations. The purity of each sample was verified this way by Ellman assaying and found that there was 10-20% inorganic salts present in each synthesized CoA thioester sample. 2.2.10.2. Synthesis of Benzoyl CoA 1 The product was isolated in >99% yield. H NMR (300 MHz, D2O) δ: 0.58 (s, H-10'), 0.71 (s, H-11'), 2.27 (t, J = 7.2 Hz, H-4'), 2.98 (t, J = 7.2 Hz, H-1'), 3.27 (m, H-2' and H-5'), 3.39 (m, H5''), 3.66 (m, H-5''), 3.84 (s, H-7'), 4.08 (br s, H-9'), 4.41 (br s, H-4''), 5.92 (d, J = 5.7 Hz, H-1''), 7.26 (dd, J = 9.6, 7.8 Hz, H-4 and H-6) 7.43 (t, J = 8.7 Hz, H-5), 7.62 (d, J = 8.7 Hz, H-3 and H7), 7.92 (s, adenine-CH), 8.31 (s, adenine-CH) (cf. Figure A 33 for proton numbering); LC− 2− 3− ESIMS (negative ion mode), m/z: 870.1 (M-H) , 434.5 (M-2H) , 289.3 (M-3H) . 44 2.2.10.3. Synthesis of 3-Chlorobenzoyl CoA 1 The product was isolated in >99% yield. H NMR (500 MHz, D2O) δ: 0.78 (s, H-10'), 0.91 (s, H-11'), 2.45 (t, J = 6.5 Hz, H-4'), 3.18 (m, H-1'), 3.45 (m, H-2' and H-5'), 3.58 (m, H-5''), 3.85 (m, H-5''), 4.03 (s, H-7'), 4.26 (br s, CH2-9'), 4.57 (br s, H-4''), 6.06 (d, J = 7.5 Hz, H-1''), 7.37 (dd, J = 9.0, 7.5 Hz, H-6), 7.54 (d, J = 8.5 Hz, H-5), 7.68 (d, J = 7.5 Hz, H-7), 7.72 (br.m, H-3), 8.08 (s, adenine-CH), 8.45 (s, adenine-CH) (cf. Figure A 34 for proton numbering); LC-ESIMS − 2− 3− (negative ion mode), m/z 926.0 (M+Na-2H) , 451.5 (M-2H) , 300.6 (M-3H) . 2.2.10.4. Synthesis of 4-Chlorobenzoyl CoA 1 The product was isolated in 95% yield. H NMR (500 MHz, D2O) δ: 0.63 (s, H-10'), 0.77 (s, H11'), 2.32 (m, H-4'), 3.05 (t, J = 8.0 Hz, H-1'), 3.32 (m, H-2' and H-5'), 3.43 (m, H-5''), 3.70 (m, H-5''), 3.88 (s, H-7'), 4.11 (br s, H-9'), 4.44 (br s, H-4''), 5.94 (d, J = 8.5 Hz, H-1''), 7.27 (d, J = 10.5 Hz, H-4 and H-6), 7.60 (d, J = 10.5 Hz, H-3 and H-7), 7.98 (s, adenine-CH), 8.34 (s, adenine-CH) (cf. Figure A 35 for proton numbering); LC-ESIMS (negative ion mode), m/z − 2− 3− 904.1 (M-H) , 451.5 (M-2H) , 300.7 (M-3H) . 2.2.10.5. Synthesis of 3-Cyanobenzoyl CoA 1 The product was isolated in >99% yield. H NMR (500 MHz, D2O) δ: 0.60 (s, H-10'), 0.73 (s, H-11'), 2.28 (t, J = 8.7 Hz, H-4'), 3.04 (t, J = 7.2 Hz, H-1'), 3.29 (m, H-2' and H-5'), 3.39 (m, H5''), 3.65 (m, H-5''), 3.85 (s, H-7'), 4.07 (br s, H-9'), 4.40 (br s, H-4''), 5.89 (d, J = 7.5 Hz, H-1''), 7.42 (dd, J = 8.4, 6.0 Hz, H-6), 7.46 (d, J = 7.8 Hz, H-7), 7.70 (ddd, J = 7.8, 2.0,1.1 Hz, H-5), 7.93 (s, H-3), 8.09 (s, adenine-CH), 8.30 (s, adenine-CH) (cf. Figure A 36 for proton 45 − 2− numbering); LC-ESIMS (negative ion mode), m/z 895.2 (M-H) , 447.0 (M-2H) , 297.6 (M3− 3H) . 2.2.10.6. Synthesis of 3-Methoxybenzoyl CoA 1 The product was isolated in >99% yield. H NMR (300 MHz, D2O) δ: 0.63 (s, H-10'), 0.76 (s, H-11'), 2.31 (t, J = 6.0 Hz, H-4'), 3.04 (t, J = 6.9 Hz, H-1'), 3.31 (m, H-2' and H-5'), 3.43 (m, H5''), 3.70 (s, H-8 and H-5''), 3.87 (s, H-7'), 4.12 (br s, H-9'), 4.41 (br s, H-4''), 5.93 (d, J = 7.2 Hz, H-1''), 7.01 (dd, J = 8.7, 2.8 Hz, H-5), 7.14 (dd, J = 2.8, 1.4 Hz, H-3), 7.22 (dd, J = 10.5, 6.9 Hz, H-6), 7.30 (dd, 8.4, 1.6 Hz, H-7), 7.96 (s, adenine-CH), 8.32 (s, adenine-CH) (cf. Figure A 37 − 2− for proton numbering); LC-ESIMS (negative ion mode), m/z 900.2 (M-H) , 449.5 (M-2H) , 3− 299.3 (M-3H) . 2.2.10.7. Synthesis of 2-Fluorobenzoyl CoA 1 The product was isolated in 97% yield. H NMR (300 MHz, D2O) δ: 0.57 (s, H-10'), 0.72 (s, H11'), 2.26 (t, J = 6.6 Hz, H-4'), 3.10 (m, H-1'), 3.26 (m, H-2' and H-5'), 3.37 (m, H-5''), 3.66 (m, H-5''), 3.83 (s, H-7'), 4.05 (br s, H-9'), 4.39 (br s, H-4''), 5.90 (d, J = 6.9 Hz, H-1''), 7.02 (m, H-6), 7.39 (m, H-4), 7.56 (t, J = 6.9 Hz, H-7), 7.85 (t, J = 5.7 Hz, H-5), 7.94 (s, adenine-CH), 8.31 (s, adenine-CH) (cf. Figure A 38 for proton numbering); LC-ESIMS (negative ion mode), m/z − 2− 3− 888.0 (M-H) , 443.5 (M-2H) , 295.3 (M-3H) . 46 2.2.10.8. Synthesis of 3-Fluorobenzoyl CoA 1 The product was isolated in 98% yield. H NMR (300 MHz, D2O) δ: 0.60 (s, H-10'), 0.73 (s, H11'), 2.27 (t, J = 6.6 Hz, H-4'), 3.01 (t, J = 6.9 Hz, H-1'), 3.27 (m, H-2' and H-5'), 3.40 (m, H-5''), 3.68 (m, H-5''), 3.86 (s, H-7'), 4.07 (br s, H-9'), 4.41 (br s, H-4''), 5.90 (d, J = 6.9 Hz, H-1''), 7.16 (t, J = 6.9 Hz, H-6) 7.27 (d, J = 6.9 Hz, H-5), 7.33 (d, J = 9.6 Hz, H-3), 7.45 (d, J = 9.6 Hz, H-7), 7.94 (s, adenine-CH), 8.31 (s, adenine-CH) (cf. Figure A 39 for proton numbering); LC-ESIMS − 2− 3− (negative ion mode), m/z 888.0 (M-H) , 443.5 (M-2H) , 295.4 (M-3H) . 2.2.10.9. Synthesis of 4-Fluorobenzoyl CoA 1 The product was isolated in >99% yield. H NMR (300 MHz, D2O) δ: 0.59 (s, H-10'), 0.72 (s, H-11'), 2.26 (t, J = 6.6 Hz, H-4'), 2.98 (t, J = 6.6 Hz, H-1'), 3.25 (m, H-2' and H-5'), 3.37 (m, H5''), 3.65 (m, H-5''), 3.85 (s, H-7'), 4.07 (br s, H-9'), 4.40 (br s, H-4''), 5.91 (d, J = 4.5 Hz, H-1''), 6.98 (t, J = 8.7 Hz, H-4 and H-6) 7.68 (t, J = 8.7 Hz, H-5 and H-7), 7.93 (s, adenine-CH), 8.31 (s, adenine-CH) (cf. Figure A 40 for proton numbering); LC-ESIMS (negative ion mode), m/z − 2− 3− 888.0 (M-H) , 443.5 (M-2H) , 295.4 (M-3H) . 2.2.10.10. Synthesis of 2-Methylbenzoyl CoA 1 The product was isolated in >99% yield. H NMR (300 MHz, D2O) δ: 0.54 (s, H-10'), 0.68 (s, H-11'), 2.14 (s, H-8), 2.27 (t, J = 6.6 Hz, H-4'), 2.97 (t, J = 6.6 Hz, H-1'), 3.27 (m, H-2' and H-5'), 3.35 (m, H-5''), 3.62 (m, H-5''), 3.81 (s, H-7'), 4.05 (br s, H-9'), 4.37 (br s, H-4''), 5.92 (d, J = 5.7 Hz, H-1''), 7.06 (m, H-4 and H-6) 7.22 (t, J = 5.7 Hz, H-5), 7.43 (d, J = 8.1 Hz, H-7), 7.94 (s, 47 adenine-CH), 8.31 (s, adenine-CH) (cf. Figure A 41 for proton numbering); LC-ESIMS − 2− 3− (negative ion mode), m/z 884.1 (M-H) , 441.6 (M-2H) , 294.1 (M-3H) . 2.2.10.11. Synthesis of 3-Methylbenzoyl CoA 1 The product was isolated in >99% yield. H NMR (300 MHz, D2O) δ: 0.57 (s, H-10'), 0.71 (s, H-11'), 2.15 (s, H-8), 2.26 (t, J = 6.6 Hz, H-4'), 2.97 (t, J = 6.6 Hz, H-1'), 3.26 (m, H-2' and H-5'), 3.37 (m, H-5''), 3.65 (m, H-5''), 3.83 (s, H-7'), 4.06 (br s, H-9'), 4.38 (br s, H-4''), 5.88 (d, J = 7.2 Hz, H-1''), 7.12 (dd, J = 9.6, 7.2 Hz, H-6), 7.22 (m, H-5), 7.42 (m, H-3 and H-7), 7.90 (s, adenine-CH), 8.29 (s, adenine-CH) (cf. Figure A 42 for proton numbering); LC-ESIMS − 2− 3− (negative ion mode), m/z 884.2 (M-H) , 441.6 (M-2H) , 294.1 (M-3H) . 2.2.10.12. Synthesis of 4-Methylbenzoyl CoA 1 The product was isolated in >99% yield. H NMR (300 MHz, D2O) δ: 0.57 (s, H-10'), 0.71 (s, H-11'), 2.17 (s, H-8), 2.26 (m, H-4'), 2.98 (t, J = 6.6 Hz, H-1'), 3.26 (m, H-2' and H-5'), 3.37 (m, H-5''), 3.65 (m, H-5''), 3.83 (s, H-7'), 4.06 (br s, H-9'), 4.39 (br s, H-4''), 5.88 (d, J = 6.6 Hz, H1''), 7.50 (d, J = 9.0 Hz, H-3 and H-7) 7.04 (d, J = 9.0 Hz, H-4 and H-6), 7.90 (s, adenine-CH), 8.29 (s, adenine-CH) (cf. Figure A 43 for proton numbering); LC-ESIMS (negative ion mode), − 2− 3− m/z 884.2 (M-H) , 441.6 (M-2H) , 294.1 (M-3H) . 2.2.10.13. Synthesis of 2-Furancarbonyl CoA 1 The product was isolated in 90% yield. H NMR (500 MHz, D2O) δ: 0.57 (s, H-10'), 0.70 (s, H11'), 2.30 (t, J = 7.2 Hz, H-4'), 2.44 (t, J = 7.2 Hz, H-1'), 2.57 (t, J = 7.2 Hz, H-1'), 3.15 (t, J = 7.2 Hz, H-2'), 3.28 (m, H-2' and H-5'), 3.38 (m, H-5''), 3.65 (m, H-5''), 3.83 (s, H-7'), 4.08 (br s, H48 9'), 4.42 (br s, H-4''), 6.00 (d, J = 6.9 Hz, H-1''), 6.38 (dd, J = 3.3, 1.7 Hz, H-4), 6.83 (dd, J = 3.9, 0.9 Hz, H-3), 7.42 (dd, J = 1.9, 0.8 Hz, H-5), 8.08 (s, adenine-CH), 8.37 (s, adenine-CH) (cf. − Figure A 44 for proton numbering); LC-ESIMS (negative ion mode), m/z 860.0 (M-H) , 429.5 2− 3− (M-2H) , 286.0 (M-3H) . 2.2.10.14. Synthesis of 3-Furancarbonyl CoA 1 The product was isolated in 90% yield. H NMR (300 MHz, D2O) δ: 0.64 (s, H-10'), 0.77 (s, H11'), 2.31 (t, J = 6.3 Hz, H-4'), 2.98 (t, J = 7.8 Hz, H-1'), 3.27 (m, H-2'), 3.32 (m, H-5'), 3.43 (m, H-5''), 3.72 (m, H-5''), 3.90 (s, H-7'), 4.12 (br s, H-9'), 4.46 (br s, H-4''), 6.01 (d, J = 5.7 Hz, H1''), 6.59 (d, J = 2.9 Hz, H-5), 7.43 (dd, J = 2.9, 2.3 Hz, H-4), 8.06 (s, adenine-CH), 8.40 (s, adenine-CH) (cf. Figure A 45 for proton numbering); LC-ESIMS (negative ion mode), m/z − 2− 3− 860.0 (M-H) , 429.5 (M-2H) , 286.0 (M-3H) . 2.2.10.15. Synthesis of 2-Thiophenecarbonyl CoA 1 The product was isolated in 95% yield. H NMR (500 MHz, D2O) δ: 0.57 (s, H-10'), 0.70 (s, H11'), 2.27 (t, J = 7.8 Hz, H-4'), 2.41 (t, J = 7.8 Hz, H-1'), 2.56 (t, J = 7.8 Hz, H-1'), 3.13 (t, J = 7.8 Hz, H-2'), 3.23 (t, J = 7.8 Hz, H-2'), 3.28 (t, J = 7.8 Hz, H-5'), 3.37 (m, H-5''), 3.64 (m, H-5''), 3.84 (s, H-7'), 4.06 (br s, H-9'), 4.42 (br s, H-4''), 6.00 (d, J = 6.3 Hz, H-1''), 6.94 (dd, J = 4.5, 4.2 Hz, H-4), 7.40 (d, J = 4.5 Hz, H-3), 8.04 (s, H-5), 8.10 (s, adenine-CH), 8.38 (s, adenine-CH) (cf. − Figure A 46 for proton numbering); LC-ESIMS (negative ion mode), m/z 875.9 (M-H) , 437.5 2− 3− (M-2H) , 291.2 (M-3H) . 49 2.2.10.16. Synthesis of 3-Thiophenecarbonyl CoA 1 The product was isolated in 95% yield. H NMR (300 MHz, D2O) δ: 0.56 (s, H-10'), 0.70 (s, H11'), 2.25 (t, J = 6.9 Hz, H-4'), 2.94 (t, J = 6.9 Hz, H-1'), 3.24 (m, H-2' and H-5'), 3.37 (m, H-5''), 3.65 (m, H-5''), 3.82 (s, H-7'), 4.07 (br s, H-9'), 4.39 (br s, H-4''), 5.93 (d, J = 5.4 Hz, H-1''), 7.22 (m, H-5), 7.27 (m, H-4), 7.95 (s, H-3), 8.00 (s, adenine-CH), 8.32 (s, adenine-CH) (cf. Figure A − 2− 47 for proton numbering); LC-ESIMS (negative ion mode), m/z 875.9 (M-H) , 437.5 (M-2H) , 3− 291.3 (M-3H) . 2.2.10.17. Synthesis of 5-Thiazolecarbonyl CoA 1 The product was isolated in 95% yield. H NMR (300 MHz, D2O) δ: 0.61 (s, H-10'), 0.74 (s, H11'), 2.32 (m, H-4'), 3.06 (t, J = 7.8 Hz, H-1'), 3.31 (m, H-2' and H-5'), 3.42 (m, H-5''), 3.69 (m, H-5''), 3.87 (s, H-7'), 4.12 (br s, H-9'), 4.44 (br s, H-4''), 6.03 (d, J = 6.0 Hz, H-1''), 8.08 (s, H-4), 8.39 (s, H-3), 8.88 (s, adenine-CH), 9.06 (s, adenine-CH) (cf. Figure A 48 for proton − 2− numbering); LC-ESIMS (negative ion mode), m/z 877.1 (M-H) , 438.1 (M-2H) , 291.7 (M3− 3H) . 2.2.10.18. Synthesis of Phenylacetyl CoA 1 The product was isolated in 56.4% yield. H NMR (300 MHz, D2O) δ: 0.55 (s, H-10'), 0.69 (s, H-11'), 2.05 (t, J = 6.6 Hz, H-4'), 2.79 (t, J = 6.6 Hz, H-1'), 3.11 (m, H-2' and H-5'), 3.34 (s, H-2), 3.37 (m, H-5''), 3.64 (m, H-5''), 3.68 (s, H-2), 3.82 (s, H-7'), 4.05 (br s, CH2-9'), 4.40 (br s, H-4''), 5.96 (d, J = 5.7 Hz, H-1''), 7.03 – 7.21 (m, H-4 to H-8 ), 8.02 (s, adenine-CH), 8.35 (s, adenine- 50 CH) (cf. Figure A 49 for proton numbering); LC-ESIMS (negative ion mode), m/z 906.0 [M + − − 2− Na - 2H] , 884.0 [M - H] , 441.5 [M - 2H] . 2.2.10.19. Synthesis of Cyclohexanoyl CoA 1 The product was isolated in 38.7% yield. H NMR (300 MHz, D2O) δ: 0.56 (s, H-10'), 0.69 (s, H-11'), 1.55 (m, H-5), 2.26 (t, J = 6.9 Hz, H-4'), 2.40 (t, J = 6.9, H-6), 2.55 (t, J = 8.1 Hz, H-4), 2.75 (t, J = 6.0 Hz, H-1'), 3.11 (t, J = 6.0, H-3 and H-7), 3.27 (m, H-2' and H-5'), 3.35 (m, H-5''), 3.64 (m, H-5''), 3.82 (s, H-7'), 4.04 (br s, CH2-9'), 4.39 (br s, H-4''), 5.98 (d, J = 7.5 Hz, H-1''), 8.08 (s, adenine-CH), 8.35 (s, adenine-CH) (cf. Figure A 50 for proton numbering); LC- ESIMS − 2− (negative ion mode), m/z 876.0 [M - H] , 437.5 [M - 2H] . 2.2.11. Synthesis of Product Standards 2.2.11.1. General Procedures The reactions were generally monitored by TLC and, upon completion, were diluted with EtOAc (5 mL) and quenched with H2O (3 mL). The mixture was stirred for 5 min, and the aqueous fraction was separated and extracted with EtOAc (2 × 5 to 20 mL, depending on the extent of portioning of the product to the organic fraction). The combined organic fractions were washed with brine, 0.1 M HCl, and H2O, and dried over anhydrous Na2SO4. 2.2.11.2. Synthesis of 7,13-O,O-Diacetylbaccatin III (18) 7,13-O,O-diacetylbaccatin III (18) was prepared according to an established procedure and the 1 H and 13 C NMR data are reported in section 2.2.4. 51 2.2.11.3. Synthesis of 7,13-O,O-Diacetyl-2-O-debenzoyl-2-O-(3-fluorobenzoyl)baccatin III The method to synthesize 7,13-O,O-diacetyl-2-O-debenzoyl-2-O-(3-fluorobenzoyl)baccatin III 13 was adapted from a previously described procedure. To a stirred solution of 7,13-O,O- diacetyl-2-O-debenzoylbaccatin III (17) (20 mg, 35 μmol) in dissolve in a mixture of dry THF (0.2 mL) and dry toluene (0.2 mL) at 50 °C under N2 were added dicyclohexylcarbodiimide (158 mg, 22 equiv), 4-DMAP (5 mg, 1 equiv), and 3-fluorobenzoic acid (108 mg, 22 eq) under refluxing conditions. The reaction temperature was increased gradually up to 80 °C over 40 h. After monitoring the reaction progress by TLC and isolating the product by general work-up procedures (after ~80% conversion), the organic solvent was evaporated, and the crude product was purified by silica gel PTLC (40:60 (v/v) EtOAc:hexanes) to yield 7,13-O,O-diacetyl-2-O1 debenzoyl-2-O-(3-fluorobenzoyl)baccatin III (17 mg, 71% yield). H NMR (300 MHz, CDCl3) δ: 1.13 (s, H-16), 1.19 (s, H-17), 1.78 (s, H-19), 1.82 (m, H-6β), 1.94 (s, H-18), 2.01 (s, C(O)CH3 at C-7), 2.15 (s, C(O)CH3 at C-10), 2.19 (s, C(O)CH3 at C-13), 2.32 (s, C(O)CH3 at C-4), 2.58 (ddd, J = 2.6, 6.6, 14.4 Hz, H-6α), 3.93 (d, J = 6.9 Hz, H-3), 4.12 (d, J = 8.4 Hz, H20α), 4.28 (d, J = 8.4 Hz, H-20β), 4.96 (d, J = 5.7 Hz, H-5), 5.57 (dd, J = 9.9, 6.3 Hz, H-7), 5.61 (d, J = 6.9 Hz, H-2), 6.14 (t, J = 8.4 Hz, H-13), 6.24 (s, H-10), 7.30–7.85 (aromatic protons; + HRMS (ESI-TOF) m/z 689.2610 [M + H] ; calculated for C35H42O13: 689.2609. 52 2.2.11.4. Synthesis of 2,7,13-O,O,O-Triacetyl-2-O-debenzoylbaccatin III To a stirred solution of 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (17) (13 mg, 23 μmol) in dry CH2Cl2 (0.4 mL) at 25 °C under N2 were added Ac2O (83 μL, 35 equiv), 4-DMAP (5 mg, 1.8 equiv), and Et3N (4.5 μL, 2 equiv). After monitoring the reaction progress by TLC and isolating the product by general work-up procedures (at reaction completion after ~2 h), the organic solvent was evaporated, and the pure product was characterized as 2,7,13-O,O,O1 triacetyl-2-O-debenzoylbaccatin III without purification (14 mg, 99% yield). H NMR (300 MHz, CDCl3) δ: 1.06 (s, H-16), 1.16 (s, H-17), 1.71 (s, H-19), 1.80 (m, H-6β), 1.90 (s, H-18), 2.00 (s, C(O)CH3 at C-7), 2.02 (s, H-14), 2.06 (s, C(O)CH3 at C-2), 2.13 (s, C(O)CH3 at C10), 2.15 (s, C(O)CH3 at C-13), 2.21 (s, C(O)CH3 at C-4), 2.57 (ddd, J = 2.8, 8.4, 13.8 Hz, H-6α), 3.80 (d, J = 6.9 Hz, H-3), 4.16 (d, J = 7.8 Hz, H-20α), 4.47 (d, J = 7.8 Hz, H-20β), 4.95 (d, J = 9.0 Hz, H-5), 5.34 (d, J = 6.6 Hz, H-2), 5.53 (dd, J = 7.8, 12.0 Hz, H-7), 6.10 (t, J = 9.6 Hz, H+ 13), 6.18 (s, H-10); HRMS (ESI-TOF) m/z 609.2552 [M + H] ; calculated for C30H41O13: 609.2547. 2.2.11.5. Synthesis of 7,13-O,O-Diacetyl-2-O-debenzoyl-2-O-(3thiophenecarbonyl)baccatin III The method to synthesize 7,13-O,O-diacetyl-2-O-debenzoyl-2-O-(3-thiophenecarbonyl)baccatin 13 III was adapted from a previously described procedure. To a stirred solution of 7,13-O,O- diacetyl-2-O-debenzoylbaccatin III (17) (10 mg, 18 μmol) in dry THF (0.2 mL) at 80 °C under N2 were added dicyclohexylcarbodiimide (67 mg, 22 equiv), 4-DMAP (3 mg, 1 equiv), and 353 thiophenecarboxylic acid (50 mg, 22 equiv) under refluxing conditions. After monitoring the reaction progress by TLC and isolating the product by general work-up procedures (at reaction completion after ~36 h), the organic solvent was evaporated, and the product was purified by loading onto a C-18 silica gel column (10% capped with TMS) (0:100 (v/v) distilled H2O:MeOH) to yield 7,13-O,O-diacetyl-2-O-debenzoyl-2-O-(3-thiophenecarbonyl)baccatin III 1 (12 mg, 98% yield). H NMR (300 MHz, CDCl3) δ: 1.09 (s, H-16), 1.14 (s, H-17), 1.81 (s, H19), 1.85 (s, H-18), 1.96 (s, C(O)CH3 at C-7), 2.07 (s, H-14), 2.10 (s, C(O)CH3 at C-10), 2.13 (s, C(O)CH3 at C-13), 2.26 (s, C(O)CH3 at C-4), 2.53 (m, H-6α), 3.86 (d, J = 6.3 Hz, H-3), 4.16 (d, J = 8.1 Hz, H-20α), 4.29 (d, J = 8.1 Hz, H-20β), 4.90 (d, J = 9.6 Hz, H-5), 5.50 (d, J = 8.1 Hz, H2), 5.53 (d, J = 8.1 Hz, H-7), 6.09 (t, J = 8.1 Hz, H-13), 6.17 (s, H-10), 7.34 – 8.24 (aromatic + protons); HRMS (ESI-TOF) m/z 677.2278 [M + H] ; calculated for C33H41O13: 677.2268. 2.2.12. wtTBT Activity Assay, Protein Purification, and Substrate Specificity Studies An aliquot (0.9 mL) of the soluble wtTBT preparation as a crude mixture, described in section 3 2.2.3, was incubated at 31 °C with [13- H]-7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (24) (33 μM, 1 μCi) and 1 mM benzoyl CoA (15), and the total volume was adjusted to 1 mL with assay buffer [25 mM 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 5% glycerol (v/v), 3 mM dithiothreitol, pH 7.4]. After 4 h, the reaction was quenched and extracted with EtOAc (4 × 2 mL), the respective organic fractions were combined, the solvent was evaporated, and the residue was dissolved in 50 μL of MeCN. A 25 μL aliquot was loaded onto a reversed-phase column (Econosphere C18, 5 μm, 250 × 4.6 mm, Alltech, Mentor, OH) and eluted at 1 mL/min 54 with a linear gradient of solvent A:solvent B [solvent A: 97.99% H2O with 2% MeCN and 0.01% H3PO4 (v/v); solvent B: 99.99% MeCN with 0.01% H3PO4 (v/v)] from 70:30 (v/v) to 40:60 (v/v) over 31 min, then to 0:100 (v/v) over another 2 min, and returning to the initial conditions over 7 min with a 5 min hold on an Agilent 1100 HPLC system. The HPLC was connected in series with a UV detector and a Packard Radiomatic Flow-One Beta 150TR radioactivity detector, which mixed the effluent with 3a70B Complete Counting Cocktail. The UV absorbance and radioactivity profiles of the biosynthetic product isolated from the assays containing crude enzyme extracts of cells expressing wild-type tbt were compared to those of the control assays containing extracts of cells transformed with vector without an insert. Once active wtTBT was verified by radiochemically-guided assays, plasmid p28wtTBT encoding the wildtype tbt was heterologously expressed in large-scale (4 L) E. coli cell cultures. After 16 h, the cells were harvested and processed as before except that, instead of 3-(N-morpholino)-2hydroxypropanesulfonic acid buffer, the cell pellet was resuspended in 50 mM Na3PO4 buffer (pH 8.0), which was compatible with the affinity resin used in a later purification step. The soluble fraction of each sample was separately loaded onto a Whatman DE-52 anion-exchange column (2.5 × 6 cm, 15 g resin) to remove small molecules and cell debris. The flow-through was discarded, wtTBT eluted from the ion-exchange resin in 100 mM NaCl (200 mL), and the latter fraction was subjected to consecutive concentration/dilution cycles in assay buffer by centrifugation (30 000 MWCO, YM30 membrane, Millipore, Billerica, MA). An aliquot (0.9 mL) of this relatively purified, concentrated recombinant protein, was incubated at 31 °C for 4 h 3 with [13- H]-7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (24) (33 μM, 1 μCi) and separately with either benzoyl CoA (15), 2-thiophenecarbonyl CoA, or 2-furancarbonyl CoA, each at 1 55 mM. The total volume was adjusted to 1 mL with assay buffer. Each assay was processed and analyzed following the methodology used for activity assay of wtTBT. That is, the UV absorbance and radioactivity profiles of the biosynthetic products isolated from the assays containing relatively purified and concentrated wtTBT soluble enzyme extracts were compared to those of the control assays containing extracts of cells transformed with vector without an insert. 2.2.13. Strategies Used to Improve the Solubility of wtTBT 2.2.13.1. Application of pMAL ™ Protein Fusion and Purification System Turbo Pfu DNA polymerase (Stratagene) was used in the following PCR reactions. A sticky-end PCR method 21 was conducted to amplify wild-type tbt with an appropriate primer set [pair 1: forward primer (5′- GATCCATGGGCAGGTTCAATGTAG-3′) and reverse primer (5′TTTATAACTTAGAGTTACATATTTTAGCCACATATTTG-3′); pair 2: forward primer (5′CATGGGCAGGTTCAATGTAGATATG-3′) and reverse primer (5′- AGCTTTTATAACTTAGAGTTACATATTTTAGCCAC-3′); the nucleotides of BamHI and HindIII restriction sites are italicized and underlined]. By this method, wild-type tbt cDNA was 21 transferred from the pET28a vector ™ to the pMAL -c2X vector (New England Biolabs, Ipswich) designated pMALwtTBT. This exchange incorporated the up-stream malE gene, which encoded the maltose binding protein (MBP) in fusion with the expressed TBT for improved solubility, immunoblot analysis, and purification by amylose affinity column chromatography 23 (New England Biolabs, Ipswich). The plasmid pMALwtTBT was used to transform BL21(DE3) E. coli cells, and the cells were grown overnight at 37 °C in 50 mL of Luria-Bertani 56 medium supplemented with 2 mg/mL glucose and 50 μg/mL kanamycin. Then 5 mL of inoculum was separately added to each of 6 L of Luria-Bertani medium supplemented with the appropriate antibiotic and glucose. The cells were grown at 37 °C to an OD600 ≈ 1, gene expression was induced with 50 μM isopropyl-β-D-1-thiogalactopyranoside (IPTG), and the cultures were incubated at 18 °C for 16 h. The cells were harvested and lysed as described in section 2.2.3, except the cell pellet was diluted in column buffer [20 mM Tris-HCl, 200 mM NaCl, 1 mM dithiothreitol, pH 7.4] to a concentration of 0.1 g cells/mL (wet weight) prior to sonication. After pelleting the cell debris after sonication, the supernatant was decanted, diluted five-fold with column buffer, and incubated with Amylose resin (3 g, New England Biolabs) for 30 min at 4 °C. The mixture was poured into an Econo column (Bio-Rad, 20 cm × 2.5 cm), and the column buffer was drained at a flow rate of 1 mL/min. The eluent was reloaded to the column and drained; this cycle was repeated twice more to maximize binding. The resin was washed with 12 column volumes of column buffer, and the bound protein was eluted with 2 column volumes of column buffer containing 10 mM maltose, followed by two column volumes of column buffer containing 20 mM maltose. Each elution solution in the maltose gradient was separately collected, concentrated by ultracentrifugation (30 000 MWCO, YM30 membrane), and analyzed further by SDS-PAGE with Coomassie blue staining of the proteins. The recombinant protein eluted in column buffer containing 20 mM maltose as evidenced by migration on SDS-PAGE at an Rf similar to that of the calculated molecular weight of MBP-TBT fusion (~94 kDa). The cells transformed with the vector without an insert were processed and purified similarly, and by comparison, no bands were detectable by the detection methods described; however, MBP (42.5 kDa) was highly expressed and visible by Coomassie blue staining on SDS-PAGE. The low stability of the produced MBP-TBT fusion was observed in Western immunoblot analysis, 57 according to a method based on the 1-Step TMB-Blotting Kit (Pierce, Rockford, IL) with the following antibodies: monoclonal anti-MBP (New England Biolabs, Ipswich) and anti-rabbit IgG peroxidase conjugate (Sigma, Saint Louis). 2.2.13.2. Solubilization, Unfolding, and Refolding of Wild-type TBT 2.2.13.2.1. Method I The wtTBT was expressed and harvested according to the methodology used in section 2.2.3 in the large-scale (2 L) study. The method of solubilizing inclusion bodies under denaturing 20,24 conditions and refolding of the denatured protein was adapted from a previous report. The wet cell pellet was resuspended in Buffer D (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA at pH 8.0 in 1 g/3 mL), the cells were lysed by the addition of 300 µg/mL of lysozyme (Roche), and the suspension was stirred for 20 min at 4 °C in the presence of 0.1 mM PMSF (Roche). The cell debris was pelleted by centrifugation at 15000g for 15 min according to section 2.2.3. The pellet was resuspended to 1 g/3 mL in Buffer D and the suspension was incubated at 37 °C for 30 min after the addition of 1 mg/mL of RNase free recombinant DNase I (Roche). The suspension was washed at 1 g cell suspension in 10 mL of Buffer E (100 mM Tris-HCl, 2 M urea at pH 8.5) and the inclusion bodies were collected by centrifugation at 15000g for 15 min at 4 °C. These inclusion bodies were solubilized for 1 h at a dilution of 1 g/3 mL of Buffer F (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, 8 M urea, 1 mM DTT at pH 8.0) at 25 °C, and then the suspension was diluted to 500 mL with Buffer G (50 mM K2HPO4, 1 mM EDTA, 50 mM NaCl, 5 mM DTT). This mixture was kept at 25 °C for 30 min prior to adjusting the pH to 8.0 and incubating another 30 min at 25 °C. This suspension was concentrated to 2 mL using centrifugation (30,000 MWCO, YM30 membrane, Millipore, Billerica, MA) and analyzed 58 further using the visual detection methods described in section 2.2.3. The activity of putatively refolded protein was also analyzed using the assay described in section 2.2.3. 2.2.13.2.2. Method II The wtTBT was expressed and harvested according to the methodology used in section 2.2.3 in the large-scale (4 L) cell preparation. The cell pellet was resuspended in Binding Buffer-A (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl at pH 7.9), the cells were lysed by sonication according to section 2.2.3, and then centrifuged at 15000g for 30 min to clarify the soluble and insoluble fractions. The insoluble fraction was processed further by a previously described method for solubilizing inclusion bodies under denaturing conditions; essentially, denatured 25 inclusion bodies are refolded while immobilized on-column. The pellet containing inclusion bodies was resuspended successively in 1 g/5 mL of Pellet Washing Buffer-I and then Buffer-II (i.e., Binding Buffer-A including 2 M and 4 M urea, respectively, plus 1% Triton X-100 at pH 8.0), mixed on an Lab-Line 3520 orbital lab shaker (Lab-Line Instruments Inc, Melrose Park, IL) at 50 rpm for 1 h for each buffer, and then centrifuged at 15000g for 30 min to clarify the pellet. The clarified pellet was resuspended in Denaturing Buffer (Binding Buffer-A containing 8 M urea plus 1% Triton X-100) to 1 g/5 mL, mixed on the orbital shaker for 30 min, and then centrifuged at 15000g for 30 min. The soluble fraction was applied to a His-Select Nickel Affinity Gel (3 g) that was pre-equilibrated with Equilibration Buffer ( Binding Buffer-A containing 8 M urea, 20 mM β-mercaptoethanol, 5 mM DTT, and 1% Triton X-100 at pH 8.0) in an Econo column (20 cm × 2.5 cm, Bio-Rad) at 4 °C and drained at 0.5 mL/min. This was repeated once more. Then the successive washings were carried out using Wash Buffers-I, -II, and -III (Binding Buffer-A containing 20 mM β-mercaptoethanol, 5 mM DTT, 1% Triton X-100, 5 mM imidazole, and 6 M, 4 M, and 2 M urea, respectively, at pH 8.0). Then Wash Buffer-IV 59 (Binding Buffer-A containing 20 mM β-mercaptoethanol, 5 mM DTT, 1% Triton X-100, and 5 mM imidazole at pH 8.0) was used to wash the resin 4 times, and during the last wash, the resin was stored in the buffer overnight to facilitate refolding. On the following day, His-tagged TBT bound to the resin was eluted (5 × 5 mL) with Elution Buffer-I (Binding Buffer-A containing 20 mM β-mercaptoethanol, 5 mM DTT, and 250 mM imidazole at pH 8.0). Each fraction eluting from the column was concentrated separately to 1 mL using centrifugation (30,000 MWCO, YM30 membrane) and analyzed further using the visual detection methods described in section 2.2.3. The activity of putatively refolded protein was also analyzed using the assay described in section 2.2.12. 2.2.13.2.3. Method III The wtTBT was expressed and harvested according to the methodology used in section 2.2.3 in the large-scale (1 L) cell preparation. The cell pellet was resuspended in Binding Buffer-B (10 mM Tris-HCl at pH 8.0), the cells were lysed by sonication according to section 2.2.3, and then centrifuged at 15000g for 30 min to obtain the soluble and insoluble fractions. The insoluble fraction was cleaned further by resuspending it in 1 g/3 mL of Pellet Washing Buffer-III (50 mM Tris-HCl including 2 M urea, 500 mM NaCl, and 2% Triton X-100 at pH 8.0) and centrifuged at 15000g for 20 min. This was repeated twice to obtain a cleaner pellet. Then the pellet was solubilized in 1 g/2 mL of Solubilizing Buffer (70 mM NaH2PO4 including 6 M guanidine HCl, 14 mM β-mercaptoethanol, and 80 mM NaCl at pH 8.0) and centrifuged for 20 min at 15000g. The supernatant from that centrifugation was applied to a His-Select Nickel Affinity Gel (3 g) under denaturing conditions. The resin was pre-equilibrated with the Binding Buffer-C (20 mM NaH2PO4 including 500 mM NaCl and 8 M urea at pH 8.0) in an Econo column (Bio-Rad, 20 60 cm × 2.5 cm) at 4 °C and incubated for 1 h. Then the filtrate was eluted off at 0.5 mL/min rate. Protein bound to the resin was washed with three column volumes of Wash Buffer-IV (20 mM NaH2PO4 including 500 mM NaCl and 8 M urea at pH 6.0) and eluted at the same rate. The bound His-tagged protein was eluted from the column using two column volumes of Elution Buffer-II (20 mM NaH2PO4 including 500 mM NaCl and 8 M urea at pH 4.0). The elution was added drop wise to a 20 times (v/v) of cold Refolding Buffer (2 mM Tris-HCl including 1 mM EDTA and 0.2 mM DTT at pH 8.0) at 4 °C, while mechanically stirring vigorously. Then the mixture was incubated without shaking overnight allowing the refolding of wtTBT. The mixture was concentrated by ultracentrifugation (30,000 MWCO, YM30 membrane), exchanged the buffer to Assay Buffer (25 mM 3-(N-morpholino)-2-hydroxypropanesulfonic acid containing 5% glycerol and 3 mM DTT at pH 8.3), and analyzed further using the visual detection methods described in section 2.2.3. The activity of putatively refolded protein was also analyzed using the assay described in section 2.2.12. 2.2.13.3. Coexpression of wtTBT and Chaperone Proteins Several chaperones (GroEL, GroES, DnaK, DnaJ, Tf, GrpE) obtained from cDNA supplied in a Chaperone Plasmid Set (TaKaRa Bio Inc.) were co-expressed wtTBT in E. coli following an 26 established protocol. There were five different plasmids with chaperone "teams" as follows: pG-KJE8 (DnaK-DnaJ-GrpE-GroES-GroEL), pGro7 (GroES-GroEL), pKJE7 (DnaK-DnaJGrpE), pG-Tf2 (GroES-GroEL-Tf), pTf16 (Tf). In brief, each chaperone plasmid was used separately to transform chemically competent cells E. coli BL21(DE3) cells, and the transformants were selected on Luria-Bertani agarose plates containing 20 µg/mL chloramphenicol. The transformed cells containing chaperone plasmids were made chemically 61 competent by standard calcium chloride treatment in liquid Luria-Bertani media containing 20 µg/mL chloramphenicol to facilitate attachment of plasmid DNA to the competent cell membrane. The plasmid p28wtTBT was used to retransform each of the prepared competent cells, and the transformants were selected on Luria-Bertani agarose plates containing 20 µg/mL chloramphenicol and 50 µg/mL kanamycin. Each transformant containing p28wtTBT and one of the chaperone teams were coexpressed in 1 L of LB medium containing 20 μg/mL chloramphenicol, 50 µg/mL kanamycin, 0.5 mg/mL L-arabinose, and/or 5 ng/mL tetracycline at 30 °C. Both L-arabinose and tetracycline were needed to induce the expression of chaperones when plasmid pG-KJE8 was expressed; L-arabinose only was used to express pGro7, pKJE7, or pTF16; tetracycline only was used to express pG-Tf2. When the culture densities reached OD600 ~ 0.6, IPTG (0.5 mM final concentration) was added to each culture and they were incubated at 30 °C for 6 h. Then the cultures were harvested, processed, and partially purified using the same method in section 2.2.12. For each sample, the fractions containing wtTBT were combined and subjected to consecutive concentration by centrifugation (30,000 MWCO, YM30 membrane) and dilution in Assay Buffer (at pH 7.4). Each concentrated sample was analyzed further using the visual detection methods described in section 2.2.3 and the activity was analyzed using the assay described in section 2.2.12. As a trial, 4 L of transformants co-expressing pG-KJE8 and p28wtTBT were grown at same conditions described, harvested, processed, and partially purified using anion-exchange chromatography. The fractions containing wtTBT enzyme were combined, and additional NaCl was added to a final concentration of 300 mM and incubated with His-Select Nickel Affinity Gel (2 g, Sigma-Aldrich) in batch mode at 4 °C. After 1 h, the mixture was poured into an Econo column (20 cm × 2.5 cm, Bio-Rad), and the Lysis Buffer (50 mM Na3PO4, 300 mM NaCl, 10 62 mM imidazole) was drained. The resin was washed with 7 column volumes of Wash Buffer-V (Lysis Buffer containing 20 mM imidazole, pH 8.0), and the bound protein was eluted with a step gradient of one column volume of Elution Buffer-III (Lysis Buffer containing 100 mM imidazole), followed by 1 column volume of Elution Buffer-IV (Lysis Buffer containing 200 mM imidazole). The imidazole was removed from the purified protein solution by consecutive concentration/dilution cycles in Assay Buffer (at pH 7.4) by centrifugation (30,000 MWCO, YM30 membrane). These concentrated fractions were analyzed further using the visual detection methods described in section 2.2.3 and the activity was analyzed using the assay described in section 2.2.12. 2.2.13.4. Comparison of wtTBT and Codon-optimized wtTBT The tbt gene was codon-optimized for expression in E. coli by GenScript Corp. (Piscataway, NJ), restriction digested with NdeI and BamHI, and then subcloned from the pUC57 cloning vector and ligated into the pET28a expression vector cut with the same restriction enzymes. The resulting plasmid was designated p28optTBT. The p28optTBT and p28wtTBT plasmid were separately expressed in E. coli, and the enzymes were isolated and partially purified, and analyzed separately by the methods described in section 2.2.3 and the activity was analyzed using the assay described in section 2.2.12. The sequence alignment of wild-type tbt cDNA and the codon-optimized tbt cDNA are shown in Figure A 3. 2.2.13.5. Construction of a Homology Model of wtTBT A homology model of wtTBT was constructed using the Meta-Server software and other 27,28 computational biology tools. This model was based on the sequence homology and 63 available structural data of vinorine synthase (EC 2.3.1.160), an acetyltransferase included in BAHD superfamily. 2.2.13.6. 1,29,30 Site-Directed Mutagenesis of wt-tbt cDNA Two point-mutations were simultaneously incorporated into the wild-type tbt cDNA, while ligated in the pET28a vector, using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) to change codon 19 from CAA to CCA, and codon 23 from AAT to AAA, with the following oligonucleotide sets CGCCATGCCTTCCATCGCCCAAAAAAATCC-3′) GGTGCAGGATTTTTTTGGGCGATGGAAGGC-3′); [forward and the primer reverse nucleotide (5′- primer mutation sites (5′are italicized and underlined]. The amino acid replacements Q19P and N23K were encoded by these mutations in the protein sequence of the wt-tbt, respectively; the resultant plasmid was designated p28PK-TBT and used to transform E. coli BL21(DE3) cells. The nucleotide sequence of mutant tbt and the encoded amino acid sequence (designated mTBT) are shown in Figure A 2. 2.2.13.7. Comparison of the Soluble Expression of wtTBT and mTBT The wild-type tbt and a cognate of tbt containing site-directed point mutations were expressed in E. coli, and the respective recombinant enzymes were harvested and processed according to the method described in the previous section 2.2.3, except that the relative concentration of recombinantly expressed wild-type (wtTBT) or mutant (mTBT) enzyme in the crude soluble fraction was assessed. 2.2.13.8. mTBT Activity Assay and Protein Purification An assessment of the relative quantity of solubly expressed wtTBT and mTBT in E. coli (described in section 2.2.13.7) revealed that the recombinant mTBT enzyme partitioned into the 64 soluble enzyme fraction approximately 5-fold better than the wild-type form. Therefore, an aliquot (0.9 mL) of the soluble mTBT preparation as a crude mixture, described in section 3 2.2.13.7, was incubated at 31 °C for 4 hours with [13- H]-7,13-O,O-diacetyl-2- Odebenzoylbaccatin III (24) (33 μM, 1 μCi) and 1 mM benzoyl CoA (15), and the total volume was adjusted to 1 mL with assay buffer. The assay was processed and analyzed as described in section 2.2.12. The UV absorbance and radioactivity profiles of the biosynthetic product isolated from the assays containing crude enzyme extracts of cells expressing tbt mutant were compared to those of the control assays containing extracts of cells transformed with vector without an insert. Once active mTBT was verified by radiochemically guided assays, plasmid p28PK-TBT encoding the mutant tbt was expressed in large scale (4 L), transformed E. coli cell cultures. After 16 h, the cells were harvested and processed as described in section 2.2.3 except that, instead of 3-(N-morpholino)-2-hydroxypropanesulfonic acid buffer, the cell pellet was resuspended in 50 mM Na3PO4 buffer (pH 8.0), which was compatible with the affinity resin used in the latter purification step. The soluble fraction of each sample was separately loaded onto a Whatman DE-52 anion-exchange column (2.5 × 6 cm, 15 g resin) to remove small molecules and cell debris. A small fraction (~15%) of the total mTBT protein eluted in the flowthrough and the remainder eluted from the ion-exchange resin in 100 mM NaCl (200 mL). The fractions containing mTBT enzyme were combined, and additional NaCl was added to a final concentration of 300 mM and incubated with His-Select Nickel Affinity Gel (3 g) in batch mode at 4 °C. After 1 h, the mixture was poured into an Econo column (Bio-Rad, 20 cm × 2.5 cm), and the lysis buffer was drained. The resin was washed with seven column volumes of wash buffer (50 mM Na3PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), and the bound protein was 65 eluted with a step gradient of one column volume of elution buffer (50 mM Na3PO4, 300 mM sodium chloride, pH 8.0) containing 100 mM imidazole, followed by one column volume of elution buffer containing 200 mM imidazole. The imidazole was removed from the purified protein solution by consecutive concentration by centrifugation (30 000 MWCO, YM30 membrane) and dilution in assay buffer (25 mM 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 5% glycerol (v/v), 3 mM dithiothreitol, pH 7.4). The recombinant protein migrated to an Rf by SDS-PAGE similar to that of the calculated molecular weight of mTBT (~50 kDa), which was visualized by Coomassie blue staining according to section 2.2.3. Kodak ID Image Analysis Software (version 3.6.3) was used to integrate the relative intensity of each type of enzyme band against BSA standards ranging from 2 to 10 mg/mL. The cells transformed with the vector without any insert were processed and purified similarly, and by comparison, mTBT was not detectable by any of the detection methods described above. 2.2.14. Kinetic Evaluation of mTBT with 7,13-O,O-Diacetyl-2-O- debenzoylbaccatin III and Benzoyl CoA Co-substrates Linearity of the rate of the mTBT catalyzed reaction with respect to protein concentration and time was established with the natural benzoyl CoA (15) at 50 μM, while the co-substrate 7,13O,O-diacetyl-2-O-debenzoylbaccatin III (17) was maintained at apparent saturation (1 mM) in 10 mL of assay buffer. Aliquots (1 mL) were collected, and the biosynthetic reaction was stopped by the addition of 500 μL of EtOAc at 5, 10, 20, 30, and 40 min and at 1, 2, 3, 4, and 5 h. Baccatin III (5 μg) was added as the internal standard to correct for the loss of analyte during the extraction of product with organic solvent. Each sample was extracted with EtOAc (2 × 4 mL), and the organic fractions were combined and then evaporated in vacuo. The resultant residue was 66 dissolved in 200 μL of MeCN, and a 10-μL aliquot of the sample was loaded onto a reversedphase column (Betasil C18, 5 μm, 150 × 2.1 mm, Thermo Fisher Scientific Inc., Waltham, MA), eluting at 0.3 mL/min with a linear gradient of solvent A:solvent B [solvent A: 99.50% H2O with 0.5% H3PO4 (v/v); solvent B: 99.50% MeCN with 0.5% H3PO4 (v/v)] from 70:30 (v/v) to 0:100 (v/v) over 7 min, holding at 100% solvent B for 2 min, and returning to the initial conditions over 1.10 min, with a 50 s hold on a capillary HPLC system (CapLC capillary HPLC, Waters, Milford, MA). The effluent from the HPLC column was directed to a Q-ToF Ultima Global ESI+ + MS/MS. The peak areas of the selected molecular ions [M + H] and [M + Na] derived by electrospray ionization of the biosynthetic product that was directed to and detected by the first stage mass spectrometer were converted to concentration by comparison to the peak areas of the + + [M + H] and [M + Na] ions generated by authentic 7,13-O,O-diacetylbaccatin III (18) ranging from 0 to 5 μM at 1.2 μM intervals (Figure A 5). Samples containing high concentrations of biosynthetic product in the assays were diluted 10- to 20-fold so that the peak areas fell within the linear range of the MS detector. Evaluation of the steady-state parameters was determined by incubating 50 μg of purified mTBT protein in 1 mL assays for 5 min. The concentration of benzoyl CoA (15) was independently varied from 0 to 1000 μM in separate assays, while the 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (17) was maintained at apparent saturation (1 mM). The initial velocity (vo) was plotted against the co-substrate concentration, and the 2 equation of the best-fit nonlinear regression curve (R > 0.99) was determined (Microsoft Excel 2003, Microsoft Corp., Redmond, WA) to calculate kcat and KM. 67 2.2.15. Kinetic Evaluation of mTBT with 7,13-O,O-Diacetyl-2-O- debenzoylbaccatin III and Competing CoA Substrates To identify productive co-substrates in pilot assays, 100 μg of purified mTBT in 1 mL of buffer, 3 [13- H]-7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (24) (33 μM, 1 μCi), and an acyl CoA (26) at 1 mM were incubated in the same reaction flask. The assays were incubated for 4 h at 31 °C, quenched, and extracted with EtOAc (2 × 4 mL). The organic fractions were evaporated in vacuo, and the resultant residues were separately dissolved in 50 μL of MeCN, loaded onto a reversed-phase column (Econosphere C18, 5 μm, 250 × 4.6 mm, Alltech, Mentor, OH), and eluted at 1 mL/min with a linear gradient of solvent A:solvent B [solvent A: 97.99% H2O with 2% MeCN and 0.01% H3PO4 (v/v); solvent B: 99.99% MeCN with 0.01% H3PO4 (v/v)] on an Agilent 1100 HPLC system equipped for UV detection and radioactivity monitoring of the effluent. To confirm the identity of the products obtained from productive co-substrates, 100 μg of purified mTBT in 1 mL of assay buffer, 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (17), and an acyl CoA (26), each at 1 mM, were incubated in a mixture. After 4 h at 31 °C, the assays were quenched and extracted with EtOAc (2 × 4 mL). The organic fractions were evaporated in vacuo, and the resultant residue was dissolved in 200 μL of MeCN. A 10 μL aliquot was + + subjected to electrospray ionization, and the molecular ions ([M + H] and [M + Na] ) of the de novo biosynthetic 7,13-O,O-diacetyl-2-O-acyl-2-O-debenzoylbaccatin III (25) analyte were detected in single-stage and tandem mass spectrometer modes (Figure A 10 to Figure A 28). The relative abundances, detected by the mass spectrometer, of selected ions between the various biosynthetic 2-O-acyl-2-O-debenzoylated samples at the same concentrations were shown to be identical among the following exemplary authentic standards (Figure A 6 to Figure A 9). The 68 + + abundance of [M + H] and [M + Na] ions generated by identical electrospray ionization of authentic 7,13-O,O-diacetylbaccatin III (18) (Figure A 6), 7,13-O,O-diacetyl-2-O-debenzoyl-2O-(3-fluorobenzoyl)baccatin III (Figure A 7), and 2,7,13-O,O,O-triacetyl-2-O- debenzoylbaccatin III (Figure A 8) was separately quantified over a range from 0 to 5 μM at 1.2 μM intervals and then compared. Control assays in which one co-substrate and/or enzyme was omitted were processed and analyzed identically to the methods described above. After productive CoA substrates were identified, the relative catalytic efficiency of mTBT with each productive acyl CoA thioester substrate identified above was assessed (based on a previous 31 procedure). Each CoA thioester (50 μM) was separately incubated with 50 μM benzoyl CoA (15), purified mTBT (50 μg), and 1 mM 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (17) in a 1 mL assay for 5 min at 31 °C. The assays were processed similarly to the method described earlier in this report, including the addition of baccatin III (20) (5 μg) as the internal standard to correct for losses during the extraction. The relative amount of each biosynthetic product made in each assay was resolved by LC-ESI-MS. Samples containing product at concentrations above the linear range of detection of the mass spectrometer were diluted 20-fold prior to analysis. The + peak areas in the following diagnoses were corrected with respect to the area under the [M + H] + and [M + Na] ion peaks of the baccatin III internal standard. The sum of the corrected areas + + under the [M + H] and [M + Na] ion peaks of each biosynthetic 7,13-O,O-diacetyl-2-O-acyl-2O-debenzoylbaccatin III analogues (25) was divided by the sum of the total area under the peaks of the same ions of the biosynthetically derived 7,13-O,O-diacetylbaccatin III (18) present in the same mixed-substrate assay. To assess the relative specificity constants for mTBT and each acyl CoA (26), the acquired ratios were each multiplied by the specificity constant (kcat/KM) of 69 mTBT when benzoyl CoA (15) and 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (17) were used as substrates earlier. Productive acyl CoA substrates of mTBT that did not produce detectable product when assayed at 50 μM for 5 min under standard reaction conditions were incubated instead at 250 μM in the competitive substrate assays. In the latter case, the sum of the areas + + under the [M + H] and [M + Na] ion peaks of each 2-O-acylated biosynthetic product was reduced 5-fold and used as the numerator in the quotient, as described above, to calculate the relative kcat/KM of the less competitive CoA substrates. 2.2.16. Relative kcat Values of mTBT with Various Acyl CoA Thioesters The apparent maximal turnover rates (kcat) of mTBT for the productive acyl CoA thioesters, identified above, were assessed by incubating each CoA thioester (1 mM) separately with 50 μg of purified mTBT enzyme and 1 mM 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (17) in a 1 mL assay for 5 min at 31 °C. The assays were extracted as described above, after the addition of baccatin III (5 μg) to each assay as the internal standard to correct for losses during extraction of the product. The amount of each biosynthetic product was determined by LC-ESI-MS, as + + described previously. The sum of the peak areas of the [M + H] and [M + Na] ions of each Oacylated biosynthetic product (7,13-O,O-diacetyl-2-O-acyl-2-O-debenzoylbaccatin III) (25) was + converted to concentration by comparing the sum of the areas of the [M + H] and [M + Na] + ions of the concentration standards comprised of 7,13-O,O-diacetylbaccatin III (18), and the apparent kcat of mTBT at saturating concentrations of each acyl CoA (26) and 7,13-O,Odiacetyl-2-O-debenzoylbaccatin III (17) was reported. 70 2.3. Results and Discussion In a previous study, the tbt cDNA, the expression and the recombinantly expressed TBT enzyme 19 was identified and was characterized. In the earlier study, the soluble wild-type TBT enzyme + was expressed in E. coli JM109 host cells from pCWori in native form without an affinity tag. The overexpressed enzyme was marginally purified by only anion exchange chromatography, and its activity was characterized in a crude milieu of other enzymes. Assays were conducted 14 with 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (17) and [7- C]benzoyl CoA as co19,32 substrates. In the current study, a preliminary screen was carried out for the functional expression of a 2-O-benzoyltransferase and its substrate specificity was examined by co3 incubating the enzyme with several different acyl CoA thioesters and [13- H]-7,13-O,Odiacetyl-2-O-debenzoylbaccatin III (24) obtained via a synthetic route described in a previous 33-35 report. The radioactive substrate had the same retention time as authentic 7,13-O,O- diacetyl-2-O-debenzoylbaccatin III (17), synthesized from commercially available baccatin III (20) (Figure 2.4), thus verifying the stereoselectivity of the borotritide reduction step (Figure 36 2.5), as per earlier studies. 71 O O O OH O 7 13 HO HO H O O O O O a O O O O O 7 O O 13 HO H O O O b O O O O 7 O O 7,13-O,O-Diacetylbaccatin III (18) Figure 2.4 – Synthesis of 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (17) a Ac2O, 4-DMAP, Et3N, 97% yield Baccatin III (20) b O O 2 HO H OH O O O 17 Red-Al, 0 °C, 65% yield In the current study, a different enzyme expression system that added an epitope tag to the expressed TBT was explored for selective purification. The wild-type tbt cDNA was transferred + 21 from pCWori (a low copy number, low expression vector) system) 37 into pET28a (a high expression developed for expression of recombinant proteins under control of the strong bacteriophage T7 promoter sequence and contained fusion tags useful in protein purification and detection. The resultant plasmid was used to transform E. coli BL21(DE3) host cells to provide the source of T7 RNA polymerase (on the genome) under the expression control of the lacUV5 37 promotor, which is IPTG inducible. The plasmid carrying the tbt gene was designated as p28wtTBT and expressed in vivo. More than 95% of wtTBT partitioned into the insoluble fraction, likely promoted by aggregation of low-solubility TBT during overexpression (Figure 38 2.6). 72 O O O OH 7 HO 13 HO H O O O a O O 20 7-O-Acetylbaccatin III (21) 3 3 [13- H]-7-O-Acetylbaccatin III (23) 7-O-Acetyl-13-oxo baccatin III (22) [13- H]-7,13-O,O-Diacetyl2-O-debenzoylbaccatin III (24) 3 Figure 2.5 – Synthesis of [13- H]-7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III (24) a b c d Ac2O, 4-DMAP, Et3N, 97% yield MnO2, 100% yield 3 Sodium [ H]borohydride, 0 ºC, 0.61 mCi, 95% radiochemical purity (i) Ac2O, 4-DMAP, Et3N, 0.59% mCi, 97% radiochemical purity (ii) Red-Al, 0 ºC, 0.35 mCi, 99% radiochemical purity 73 L1 L2 L3 100 kDa 75 kDa 50 kDa TBT 35 kDa 25 kDa Figure 2.6 – SDS polyacrylamide gel electrophoresis and Coomassie blue staining of recombinantly expressed, soluble and insoluble fractions of wtTBT (arrowed) isolated from E. coli BL21(DE3). L1 shows the resolved proteins from 10 µg of total soluble proteins from E coli transformed with the p28wtTBT vector expressing the wild-type tbt cDNA insert; L2 contains molecular weight standards (Lonza); L3 shows the resolved proteins from 10 µg of total insoluble proteins from E. coli transformed with the p28wtTBT vector expressing the wild-type tbt cDNA insert The fraction with over 95% insoluble TBT and the supernatant of the crude cell lysate containing 3 minimal amounts of soluble TBT were separately tested for activity with synthesized [13- H]7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (24) and benzoyl CoA (15). TBT in the insoluble fraction was inactive while the minimal amounts of TBT present in the soluble fraction showed a signal in the radio-HPLC detector corresponding to the authentic 7,13-O,O-diacetylbaccatin III (18) (Figure 2.7). The significant proportion of expressed wtTBT partitioning to the insoluble fraction coupled with its inactivity created a challenge to acquire sufficient protein for the substrate specificity and kinetics analyses. 74 400 300 200 100 0 120 100 80 60 40 20 0 16 18 20 22 24 26 Figure 2.7 – (A) A portion of the reverse-phase HPLC profile of authentic unlabeled 7,13-O,Odiacetylbaccatin III (18) with A228 monitoring of the effluent (Rt = 22.6 min, dotted line); (B) 3 Partial reverse-phase radio-HPLC profile of [ H]-labeled biosynthetic product (Rt = 22.6 min, solid line) derived by incubation of wtTBT crude soluble enzyme extract with benzoyl CoA (15) 3 and [13- H]-7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (24) with radioactivity monitoring of the effluent. Despite the low soluble expression of wtTBT, there was sufficient enzyme to conduct very preliminary specificity assays. The soluble crude enzyme lysate was used in assays containing 75 3 [13- H]-7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (24) and heterole carbonyl CoA thioesters in separate assays. New signals in the radio-HPLC detector profile corresponding to the 3 formation of [13- H]-7,13-O,O-diacetyl-2-O-debenzoyl-2-O-heterolecarbonylbaccatin III were detected in each assay (cf. Figure 2.8 for examples). 500 400 300 200 100 0 14 16 18 20 22 24 3 Figure 2.8 – Partial reverse phase radio-HPLC profiles of [ H]-labeled biosynthetic products 3 derived by separate incubations of wtTBT crude soluble enzyme extract and [13- H]-7,13-O,Odiacetyl-2-O-debenzoylbaccatin III (24) with 2-furancarbonyl CoA (Rt = 17.3 min, dotted red line) or 2-thiophenecarbonyl CoA (Rt = 20.7 min, solid black line) with radioactivity monitoring of the effluent. Encouraged by these findings, the purification of soluble active wtTBT from the crude enzyme extract utilizing an N-terminal His6-tag was attempted in order to increase the specific activity. Ni-affinity chromatography of the His6-tag fusion protein yielded insufficient amounts of protein to cover the scope of substrates for testing. Therefore, attempts were made to minimize or 76 eliminate the possible cause(s) of insoluble expression of inactive aggregated enzyme and thus increase soluble expression. 2.3.1. Strategies Used to Improve the Solubility of wtTBT 2.3.1.1. Optimization of Several Parameters in wtTBT Expression The strategies employed to enhance the solubility of wtTBT are discussed herein. Several attempts were made to increase the soluble overexpression of wtTBT by optimizing parameters such as the: 1) growth temperature, 2) growth media, 3) optimal growth density of E. coli prior to IPTG induction, 4) concentration of IPTG, 5) induction temperature, 6) induction time, 7) lysis buffer used, 8) additives included in lysis buffer (e.g., mild detergents, protease inhibitors, low percentages of stronger detergents, lysozyme), 9) the method of cell lysis, and 10) parameters related to each method of cell lysis. Regrettably, significant improvements in the soluble protein expression were not observed after application of the optimization steps (data not shown). 2.3.1.2. Use of Fusion Proteins to Enhance Partitioning of wtTBT into the Soluble Fraction Fusion proteins have a wide range of applications; some facilitate the detection, stability, and/or the purification of the fused partner, while others increase the yield of the recombinant protein by 39 producing it in the form of a fusion. Highly soluble proteins such as maltose-binding protein (MBP), glutathione S-transferase (GST), ubiquitin, and thioredoxin (TRX) have been shown to 40,41 increase the soluble partitioning of their aggregation-prone fusion partners in many cases. ™ Therefore, the pMAL protein fusion and purification system (New England Biolabs, Ipswich, 23,42 MA) was tested for improving the soluble expression of wtTBT. 77 The tbt cDNA was inserted into the pMAL-c2X vector downstream of the malE gene encoding MBP. This expression system produced a MBP-TBT fusion that was purified by amylose affinity chromatography in 23 batch mode specific for MBP binding. This fusion system, however, did not significantly improve the partitioning of wtTBT into the soluble fraction nor did it enhance the purification of the expressed fusion protein. Also, low stability of the produced MBP-TBT fusion was observed in Western immunoblot analysis using monoclonal anti-MBP (New England Biolabs) and antirabbit IgG peroxidase conjugate (Sigma, Saint Louis) (data not shown). Several strategies reported to prevent the instability of the fusion due to activities of E. coli proteases were attempted but failed. 2.3.1.3. Solubilization, Unfolding, and Refolding of wtTBT 43 Recombinant proteins produced in E. coli are often expressed as inclusion bodies. Despite the potential high-yield and convenient isolation of the produced protein, this method of biological protein synthesis can produce misfolded insoluble protein. 43 Commonly, unfolding/refolding inclusion bodies in an attempt to solubilize these aggregates can be achieved through the addition of denaturants such as urea or guanidinium chloride and reducing agents such as dithiothreitol (DTT) or 2-mercaptoethanol (BME). After unfolding the protein, refolding is typically achieved by removal of the denaturant through dilution, diafiltration, or dialysis, often under optimized 44 redox conditions. Several protocols which were previously successful in denaturing and refolding other enzymes were examined for solubilizing and refolding the wtTBT from low24,25,45,46 soluble aggregates. Although, wtTBT could be isolated and purified in the soluble 78 fraction in many cases (Figure 2.9; only one example is shown), regaining the activity was never possible due likely to the inability of finding the exact conditions for the refolding process. L1 L2 L3 L4 100 kDa 75 kDa TBT 50 kDa 35 kDa 25 kDa Figure 2.9 – SDS-PAGE and Coomassie blue staining of recombinantly expressed, chemically denatured, refolded, and purified wtTBT from insoluble inclusion bodies (arrowed) isolated from E. coli BL21(DE3) according to the Method III described in section 2.2.13.2.3. L1 shows the resolved proteins from 10 μg of total soluble protein of concentrated effluent from the His-select nickel affinity column chromatography after the denaturing, solubilizing, and refolding of the inclusion bodies; L2 shows the resolved proteins from 15 μg of total soluble protein of concentrated effluent from the His-select nickel affinity column chromatography after the denaturing, solubilizing, and refolding of the inclusion bodies; L3 contains molecular weight standards (Lonza); L4 shows the resolved proteins from 10 μg of total soluble protein from E. coli transformed with the p28wtTBT vector expressing the wild-type tbt cDNA insert before any chemical denaturing or solubilizing was performed. 2.3.1.4. Co-expression of wtTBT and Chaperone Proteins In vivo folding of proteins is an energy-dependent process assisted by molecular chaperones and folding catalysts (foldases).47,48 While folding catalysts accelerate rate-limiting steps along the folding pathway (e.g., the isomerization of peptidyl-prolyl bonds and the formation and reshuffling of disulfide bridges), molecular chaperones facilitate proper folding by binding partially folded proteins and maintaining them in a soluble or translocation-competent 47,48 conformation reducing protein aggregation. Several reports have demonstrated that the co- 79 overexpression of folding modulators can improve the soluble recovery yields of many, but not 49-51 all, aggregation-prone recombinant polypeptides. Co-expression of several chaperones (GroEL, GroES, DnaK, DnaJ, Tf, GrpE) of a Chaperone Plasmid Kit (TaKaRa Bio Inc.) along with expression of wtTBT was tested, while optimizing conditions to improve the soluble yield of wtTBT. Isolation of the resultant soluble wtTBT from highly abundant chaperones was a challenge as a consequence of this strategy (data not shown). The presence of abundant chaperone proteins in the soluble fraction reduced the specific activity of wtTBT significantly. 2.3.1.5. Strategies to Overcome the Biased Codon Usage of the Expression Host Synonymous codons are used at different frequencies in different genomes, as well as in the same genome. The codon usage preference in each organism correlates with the composition of the tRNA pool. 52 Although E. coli is a preferred host for heterologous protein production, the process can be challenging due to the biased codon usage. When the codon bias of the gene to be expressed differs significantly from that used by E. coli, the concentrations of the E. coli tRNAs for the lesser-used codons are insufficient to optimally translate the RNA. The outcome of rare codons includes translational errors such as stalling, termination, amino acid substitution and possibly frameshifting; competition for rare tRNAs may also adversely affect the expression of 53 host genes or elicit a stringent response. This could be an issue in effectively expressing the tbt cDNA from Taxus plants in E. coli. There are 102 rare codons present in the wild-type tbt cDNA sequence among 441 total codons (Table 2.1). 80 53 Table 2.1 – Rarely used codons in E. coli Amino Acid Rare Codon(s) Arginine Leucine Isoleucine Serine Glycine Proline Threonine AGG, AGA, CGG, CGA CUA, CUC AUA UCG, UCA, AGU, UCC GGA, GGG CCC, CCU, CCA ACA The general strategies to overcome the biased codon usage include targeted mutagenesis to remove the rare codons, addition of rare codon tRNAs in specific cell lines, or costly but effective production of synthetic genes replacing the rare codons. 53,54 Improvement of the heterologous expression of tbt was attempted by increasing the cognate tRNA in the host by inserting the cDNA encoding wild type tRNA used by E. coli on a multiple copy plasmid. The BL21-CodonPlus(DE3)-RIPL strain (Stratagene) (Novagen) 56 55 and several different Rosetta strains were used as expression hosts. The improvement of soluble partitioning of wtTBT was not obtained using these host strains transformed to express tRNAs for rare codons (data not shown). Often the synthetic production of codon-optimized genes improves the protein 57 expression level significantly in case of biased codon-usage of E. coli. Therefore, the production of a synthetically-derived codon-optimized tbt gene was accomplished utilizing ® 58 Gene-On-Demand technology. The so-derived tbt contained only 18 different rare codons according to Table 2.1 with a total of 102 occurrences. The expression of codon-optimized synthetic tbt was accomplished in the pET28a vector in BL21(DE3) E. coli, resulting in higher 81 overall protein production but did not improve the partitioning ratio of wtTBT to the soluble fraction (Figure 2.10). L1 L2 L3 L4 L5 100 kDa 75 kDa TBT 50 kDa 35 kDa 25 kDa Figure 2.10 – SDS-PAGE and Coomassie blue staining of resolved proteins of both soluble and insoluble fractions from E. coli BL21(DE3) transformed with the pET28a vector, separately expressing wild-type tbt cDNA insert and the codon-optimized tbt insert. L1 shows the resolved proteins from 10 μg of total soluble protein from E. coli transformed with the p28wtTBT vector expressing the wild-type tbt cDNA insert; L2 shows the resolved proteins from 10 μg of total soluble protein from E. coli transformed with the pET28a vector expressing the codon-optimized tbt insert; L3 contains molecular weight standards (Lonza); L4 shows the resolved proteins from 10 μg of total insoluble protein from E. coli transformed with the p28wtTBT vector expressing the wild-type tbt cDNA insert; L5 shows the resolved proteins from 10 μg of total insoluble protein from E. coli transformed with the pET28a vector expressing the codon-optimized tbt insert. 2.3.1.6. Construction of a Mutant tbt and Functional Expression As an alternative to in vitro chemical manipulations and the other described strategies to overcome the hydrophobic interactions that promote the formation of inclusion bodies, the amino acid sequence alignment of TBT orthologues from other Taxus spp. was reevaluated for potential differences in sequence that may affect differential expression across species. General strategies for increasing protein solubility include site-directed mutagenesis, which aims to enhance 82 solubility by replacing hydrophilic amino acids with others that contribute more favorably to interactions with bulk H2O. 59 TBT_T. cuspidata_wt : TBT_T. cuspidata_mut: TBT_T. media : TBT_T. wallichiana : DBAT_T. cuspidata : TAT_T. cuspidata : NDTBT_T. canadensis : BPPT_T. cuspidata : ------MGRFNVDMIERVIVAPCLQSPKNILHLSPI… ------MGRFNVDMIERVIVAPCLPSPKKILHLSPI… ------MGRFNVDMIERVIVAPCLPSPKKILRLSPI… ------MGRFNVDMIERVIVAPCLPSPKKILHLSPI… MAGS---TEFVVRSLERVMVAPSQPSPKAFLQLSTL… MEKT----DLHVNLIEKVMVGPSPPLPKTTLQLSSI… MEKAG-STDFHVKKFDPVMVAPSLPSPKATVQLSVV… MKKTGSFAEFHVNMIERVMVRPCLPSPKTILPLSAI… Figure 2.11 – Partial amino acid sequence alignment of TBT (taxane 2α-O-benzoyltransferase) orthologues used to design the site-directed mutant of the wild-type TBT from Taxus cuspidata (T. cuspidata). The sequences of two TBT orthologues from Taxus × media and T. wallichiana and other operationally soluble Taxus acyltransferases involved in paclitaxel biosynthesis are included in the alignment. TBT_T. cuspidata_wt, wild-type TBT from T. cuspidata (accession no. AF297618); TBT_T. cuspidata_mut, site-directed mutant TBT with Q19P and N23K replacements; TBT_T. media, TBT from Taxus × media (accession no. AY675557); TBT_T. wallichiana, TBT from Taxus wallichiana (accession no. AY970522); DBAT_T. cuspidata, 10deacetylbaccatin III 10-O-acetyltransferase from T. cuspidata (accession no. AF193765); TAT_T. cuspidata, taxadien-5α-ol O-acetyltransferase from T. cuspidata (accession no. AF190130); NDTBT_T. canadensis, 3′N-debenzoyl-2′-deoxytaxol N-benzoyltransferase from T. canadensis (accession no. AF466397); BPPT_T. cuspidata, 3-amino-3phenylpropanoyltransferase from T. cuspidata (accession no. AY082804). The sequence analysis revealed that TBT orthologues, isolated from Taxus x media and T. wallichiana, and other operationally soluble, functionally defined acyltransferases from T. candensis and T. cuspidata deposited in the GenBank database, contain a highly conserved proline (P) residue instead of a glutamine (Q) at position 19 of wtTBT (Figure 2.11). Additionally, the two TBT orthologues contain tandem lysine residues (K22, K23). Guided by these parameters, the tbt cDNA obtained from T. cuspidata was mutated at codons CAA19CCA and AAT23AAA. The mutant tbt cDNA from the pET28a vector was expressed in E. coli to yield functional, recombinant mutant TBT (mTBT) protein (1 mg mTBT/L cells) containing two 83 point-mutations (Q19P and N23K), compared to 5-fold less of the identically expressed wtTBT (200 μg of wtTBT/L cells), as determined by SDS-PAGE analysis and Coomassie blue staining (Figure 2.12) of the soluble proteins in the respective E. coli lysates. L1 L2 L3 100 kDa 75 kDa TBT 50 kDa 35 kDa 25 kDa Figure 2.12 – SDS-PAGE and Coomassie blue staining of recombinantly expressed, soluble wtTBT and mTBT (arrowed) isolated from E. coli BL21(DE3). L1 contains molecular weight standards (Lonza); L2 shows the resolved proteins from 10 μg of total soluble protein from E. coli transformed with the p28wtTBT vector expressing the wild-type tbt cDNA insert; L3 shows the resolved proteins from 10 μg of total soluble protein from E. coli transformed with the p28PK-TBT vector expressing the CAA19CCA and AAT23AAA mutant tbt cDNA insert. Western blot analysis of cell extracts from E. coli expressing p28wtTBT and p28PK-TBT further showed that the level of soluble mTBT was significantly increased over the expression of soluble wtTBT (Figure 2.13). Furthermore, when the structure of wtTBT was modeled on the crystal structure of the related BAHD family vinorine synthase, K23 was found to be part of a solvent29,60 exposed loop structure positioned on the enzyme surface (Figure 2.14). 61 pH of E. coli is approximated between 7.4 and 7.8, 84 Since the internal the lysine side chain amino group (pKa ~10) would also carry a partial net positive charge shared between the residues and would likely increase the protein solubility in bulk H2O. In addition, the juxtaposition of K23 and K22 conceivably creates a dipole moment in the loop domain that could likely further facilitate soluble expression. This significant increase in soluble expression fortuitously overcame the bottleneck to efficiently express TBT in operationally soluble form and, therefore, the production of functional catalyst was scaled-up to conduct the present substrate specificity investigation. MW L1 L2 197 kDa 125 kDa 83 kDa * 37 kDa 17 kDa Figure 2.13 – Western blot analysis of the soluble expression of wtTBT and mTBT enzyme in crude extracts of transformed E. coli cells. Comparison of the mTBT-His6 fusion (L1) and the wtTBT-His6 fusion (L2) by western-immunoblot analysis with the monoclonal anti polyhistidine and anti-mouse IgG peroxidase conjugate. The band corresponding to the expressed mTBT protein is indicated by an asterisk. The locations of the molecular weight standards (Kaleidoscope Prestained Standards, Bio-Rad Laboratories) are shown. Moreover, the mutant tbt cDNA was expressed with an N-terminal His6-tag epitope for immunoblot verification and purification to ~70% purity by nickel-affinity resin chromatography. SDS-PAGE analysis of the partially purified mTBT revealed a predominant 85 pair of proteins at Rf values consistent with those of chaperones GroEL and DnaK (57.4 and 69.1 62 kDa, respectively), each comprising about 10% of the total protein (data not shown). The catalytic activity and quantity of mTBT in the presence of the presumed chaperones was judged sufficient for the purpose of the present investigation. Q19 N23 Figure 2.14 – The homology model of wtTBT based on the crystal structure of the related BAHD family vinorine synthase (VS, EC 2.3.1.160), in which both Q19 and N23 amino acid residues were found to be part of a solvent-exposed loop structure positioned on the enzyme surface. Frequently, in vivo insolubility can result from low protein stability, where the hydrophobic sites normally buried within a protein can be exposed and interact with partially or completely 59 unfolded protein in the expression host, rendering the protein inactive. Therefore, protein solubility problems during overexpression might also be facilitated by approaches to increase protein stability. Proline is well known to define, in part, the conformation of secondary structural elements of proteins that provide structural rigidity, which often translates into 63 structural stability when these pyrrolidine structures are situated proximally. 86 Proline residues are well conserved throughout the primary amino acid sequences (at 11 positions) of members of the BAHD acyltransferase superfamily involved in paclitaxel biosynthesis (Figure A 4). The frequent occurrence of proline in the Taxus acyltransferases may contribute an important role in enzyme function and in defining the biophysical behavior of these catalysts. Although the inclusion of Q19P and N23K in mTBT represents modest mutations in terms of the primary amino acid sequence, these exchanges significantly influenced the physical properties that increased the soluble expression. Individual point mutants at each site were not constructed to separately dissect the effects of the individual mutations on the differential soluble expression of mTBT; this aspect will be probed in future investigations. 3 Radiolabeled [13- H]-7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (24) and benzoyl 19 CoA (15) were used in pilot-scale assays, similar to a previously described method, and the appearance of a de novo biosynthetic product with similar retention time to that of authentic 18 demonstrated enzyme activity. A larger-scale assay to produce sufficient de novo biosynthetic product for LC-ESI-MS characterization was conducted with mTBT (50 µg) in 1-mL assays along with benzoyl CoA and the taxane acceptor, each at 1 mM. Direct injection on the LC-ESIMS confirmed the identity of the product as 7,13-O,O-diacetylbaccatin III (Figure A 6 and Figure A 10), thus verifying the mutant enzyme was functional. 2.3.2. Relative Substrate Specificity of mTBT with Various Aroyl CoA Thioesters and 7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III Pilot studies were initially conducted to determine the array of productive acyl CoA substrates from a small library of these activated thioesters that were synthesized by a previously described 64 process or obtained from commercial sources. After using 24 and a single acyl CoA substrate 87 in separate assays, 18 CoA thioesters, in addition to benzoyl CoA (15), were found to be utilized by mTBT. Each biosynthetic product was further characterized by incubating mTBT and 17 at 1 mM separately with different acyl CoA substrates (1 mM). EtOAc was added to the assays to partition the acylated products into the organic phase. The extracted products were verified as 2O-acyl taxane analogues (25) by LC-ESI-MS/MS. The first-stage mass spectrometer was set to + select for the [M + H] ion of the product, which was directed into a fragmentation chamber, and the resulting fragment ions were analyzed by the second-stage mass spectrometer set to scan mode. Typical diagnostic fragment ions of 2-O-acyl-2-O-debenzoylbaccatin III analogues (25) were evaluated using authentic 18 as a model to identify the various ion cleavage points. + + + Diagnostic ions are m/z 671.2 [M + H] , 551.2 [m/z 671.2 - 2HOAc] , 491.2 [m/z 551 - HOAc] , + + 429.2 [m/z 551.2 - RCOOH at C-2] , 369.2 [m/z 429.2 - HOAc] , and 309.2 [m/z 369.2 + HOAc] (Figure A 10 to Figure A 28). Analogous assays conducted with enzyme isolated from E. coli harboring the empty vector did not show detectable product derived from any of the acyl CoA’s (26). Most of the aroyl CoA thioesters examined in this study were productive with mTBT and 17, when both substrates were assayed at 1 mM. The specificity constant (kcat/ KM) of mTBT for each functional acyl CoA was estimated from the amount of the 2-O-acylated taxane made from the corresponding thioester in a competitive substrate reaction under typical 31 assay conditions. The compounds in the resulting product mixture were separated by reversed- phase HPLC with electrospray ionization of the effluent, and the abundance of the putative biosynthetic analyte ion was quantified by LC-ESI-MS/MS. When compared to the total number of ionizable heteroatoms in the entire biosynthetic molecules of, for example, 7,13-O,O-diacetyl- 88 2-O-debenzoyl-2-O-(3-thiophenecarbonyl) baccatin III (molecular formula C33H40O13S), the ionizable sites on the heterole side chains at the C-2 hydroxy group are about ~8% (1-sulfur in the aryl side chain compared to 13-oxygen sites in the remainder of the molecule). Therefore, the mode of electrospray ionization of the 7,13-O,O-diacetylbaccatin III (the 2-O-benzoyl analogue) standard (18) was deemed essentially similar to the ionization mechanism for each biosynthetic 2-O-acyl-2-Odebenzoyl analogue derived by mTBT catalysis, regardless of the C-2 side chain. Therefore, instead of synthesizing and characterizing the several different authentic standards corresponding to each proposed de novo 2-O-acyl biosynthetic product to calculate the concentrations of biosynthetic products, authentic 7,13-O,O-diacetylbaccatin III (18) was used as the general standard for all of the 2-O-acyl analogues with quantitation of ion abundance by MS (Figure A 5). Exemplary 7,13-O,O-diacetyl-2-O-acyl-2-O-debenzoylbaccatin III analogues (Figure A 7 to Figure A 9) were synthesized and analyzed identically by LC-ESIMS at concentrations similar to those of 18. A correlation of 1:1 was observed between concentration and ion abundance among the 2-O-acyl analogues tested (data not shown), which supported the use of 18 as the all-purpose calibration standard. Ion abundance was converted to analyte concentration by a linear regression equation that correlated the peak areas under the signals + + generated for selected ions [M + H] and [M + Na] detected by the mass spectrometer with a series of 7,13-O,O-diacetylbaccatin III standards at various concentrations (Figure A 5). From the analysis of the ion abundances, the relative specificity constants (Table 2.2) were calculated on the basis of the specificity constant (kcat/KM) = 1.8 min -1 -1 mM ) calculated for mTBT with benzoyl coenzyme A (Entry 1A). The catalytic efficiency for 4-methylbenzoyl CoA (Entry 4A) was equal to that of the natural substrate; the efficiency of mTBT for the other coenzyme A 89 -1 thioesters of substituted-aroyls or heterole carbonyls ranged from 0.28 to 3.9 min 2.2, column A). 90 -1 mM (Table Table 2.2 – Relative Kinetics of mTBT with aroyl- and short-chain hydrocarbon-CoA’s and a,b 7,13-O,O-diacetyl-2-O-debenzoylbacctin III (17) co-substrates O O O mTBT HO H OR O O O R-SCoA 26 B A C kcat/KM -1 -1 (min mM ) kcat -1 (min ) KM (μM) 1 1.8 0.19 110 2 --- --- --- 3 b O H3C a --- 2.4 × 10 -3 --- 4 1.8 0.021 12 3.9 0.65 170 0.28 0.075 270 0.62 0.076 120 5 6 O O O 7,13-O,O-diacetyl-2-O-acyl2-O-debenzoylbaccatin III 25 7,13-O,O-diacetyl-2-Odebenzoylbaccatin III 17 R Derived from CoA O O 7 F 91 Table 2.2 (Cont’d) R Derived from CoA A B C kcat/KM -1 -1 (min mM ) kcat -1 (min ) KM (μM) --- --- --- --- --- --- 10 --- --- --- 11 0.38 0.016 42 12 0.92 0.11 120 13 2.2 0.014 6.4 14 2.9 0.13 45 15 2.9 0.51 180 16 0.35 0.13 370 8 9 O Cl O S O N S 92 Table 2.2 (Cont’d) A R Derived from CoA B C 0.25 510 3.0 0.35 120 21 1.2 57 20 35 1.7 49 21 0.24 0.010 42 22 0.42 0.023 55 23 --- --- --- 24 O 0.49 19 O KM (μM) 18 O kcat -1 (min ) 17 O kcat/KM -1 -1 (min mM ) b a --- 2.6 × 10 -4 --- a The rate of product formed by the transfer of the corresponding acyl group from CoA by mTBT catalysis was considered to be first-order (i.e., significantly below saturation); the value -1 b reported is Vo (nmol min ). The catalytic efficiency was not calculated for these entries, since the corresponding acyl CoA’s were not strong competitive inhibitors of benzoyl CoA in mixed substrate assays, and consequently, the associated products were not detectable. kcat/KM values -1 -1 -1 are listed as min mM , kcat values are listed as min , and KM values are listed as μM. The standard error for each of these parameters measured from triplicate assays is ~5%. The dashed line (---) indicates that the corresponding biosynthetic products were below the detection limits of the mass spectrometer 93 The p-substituted aroyls within a regioisomeric series had the highest catalytic efficiency, while the biosynthetic product in assays with the m- or o-substituted aroyl CoA thioesters (Table 2.2, Entries 2, 8, 9, and 10) was below the detection limits of the mass spectrometer. The o- and mfluorobenzoyl CoA analogues and m-methylbenzoyl CoA were the exception; the catalytic -1 efficiency with the o-fluoro substrate was 3.9 min mM -1 mM -1 -1 (Entry 5A) compared to 0.62 min for the p-fluoro isomer (Entry 7A). While the m-methylbenzoyl CoA was productive, it was nevertheless transferred by mTBT at comparatively lower efficiency than the other productive acyl CoA thioesters (Entry 3). Intriguingly, mTBT showed higher catalytic efficiency with several of the acyclic and some heterocarbonyl thioesters than with benzoyl CoA. For shorthydrocarbon-chain (C3 and C4) thioesters (Entries 18A, 19A, and 20A) the efficiencies were 1.7, 12, and 19 times greater, respectively, than with benzoyl CoA, with the branched C4 isobutyryl CoA (Entry 22A) as the exception, being 4 times slower. Compared to the specificity constant of mTBT for benzoyl CoA, other alkanoyl CoA thioesters (acetyl and hexanoyl, Entries 17A and -1 21A) were utilized by the mutant benzoyltransferase at lower efficiency (0.49 and 0.24 min -1 mM , respectively). Cyclohexanoyl CoA was also utilized by mTBT to form a 2-O-acyl-2-Odebenzoyl analogue, but the reaction was orders of magnitude slower. The efficiency of mTBT with heterole carbonyl thioesters 3-furanyl- and 2- and 3-thiophene-carbonyls (Table 2.2, Entries 13A, 14A, and 15A) were 1.2, 1.6, and 1.6 times greater, respectively, than with benzoyl CoA, while 2-furanyl- and 5-thiazole-carbonyls were 2.0 and 5.1 times less efficient than the natural substrate (Entries 12A and 16A, respectively). 94 2.3.3. KM and kcat Values of mTBT with 7,13-O,O-Diacetyl-2-O- debenzoylbaccatin III and Various Aroyl CoA Thioesters Still guided by efforts to conserve the stocks of the synthetically acquired acyl CoAs and the semisynthetic 2-O-debenzoyl taxane substrate, kcat values were separately calculated at apparent saturation (1 mM) for the taxane substrate and each of the 19 productive aroyl CoAs at apparent saturation (estimated at 1 mM) for 5 min, in triplicate runs. Each sample was analyzed by LCESI-MS/MS to verify the identity of the 2-O-acylated product and to quantify the relative rate at which the biosynthesized products were formed. The KM values were calculated by multiplying the kcat values by the reciprocal of the corresponding catalytic efficiency values (KM/kcat) (Table 2.2). For mTBT assays with 3-methylbenzoyl- and cyclohexanoyl-CoA, the turnovers of substrate to product were comparatively 70- and 700- fold slower, respectively, than the other CoA thioesters used. In the assays conducted earlier, the relative kcat/KM values for these substrates could not be calculated since they were not strong competitive inhibitors of benzoyl CoA at 50 µM when incubated for 5 min in the mixed substrate assays; within this period, their derived taxane products could not be detected, even at 1 mM substrate. The rates at which the product was formed from 3-methylbenzoyl- and cyclohexanoyl-CoA by mTBT catalysis were therefore considered first-order (i.e., significantly below enzyme saturation, i.e., kcat) at 1 mM after incubating for 4 h and are reported as Vo (cf. Table 2.2, Entries 3B and 24B). The other CoA substrates (Entries 2, 8, 9, 10, and 23, Table 2.2) were incubated similarly at 1 mM for 4 h, and the corresponding biosynthetic products derived by mTBT catalysis were not detectable. mTBT showed similar to superior catalytic efficiency with several acyl coenzyme A thioesters 95 (Table 2.2, Entries 4, 5, 13-15, and 18-20, in column A) compared to benzoyl CoA. Calculating the kcat values of mTBT for various acyl CoA’s, however, revealed which kinetic parameter (kcat or KM) most influenced the magnitude of the efficiency parameter. Butenoyl CoA (Table 2.2, Entry 20) was ~19-fold more efficient with mTBT than was benzoyl CoA. This difference was predominantly dictated by the near 9-fold greater turnover of butenoyl CoA over benzoyl CoA by mTBT, coupled with the ~2-fold decrease in KM for butenoyl CoA over the natural CoA substrate. Conversely, the magnitude of the catalytic efficiency of mTBT for 3-furanoyl CoA -1 (Entry 13A, 2.2 min -1 -1 mM ), which was nearly equal to the efficiency of mTBT for benzoyl -1 CoA (1.8 min mM ), was predominantly influenced by KM. While the turnover number (kcat) for the 3-furanoyl donor was 14-fold slower, the KM was 17-fold lower (6.4 µM) for this substrate compared to that calculated for benzoyl CoA (110 µM), indicating that the catalytic efficiency was balanced by superior binding of the former acyl CoA. Compared to the KM for benzoyl CoA, mTBT displayed lower KM values for eight aroyl CoA substrates (Table 2.2, Entries 4, 11, 13, 14, 19, 20, 21, and 22; column C). There are only three instances (Entries 4, 13, and 14), however, where a lower KM offset the deficient turnover rate that reciprocally equalized (or increased) the magnitude of the specificity constant (Entries 4A, 13A, and 14A) of mTBT compared to that for benzoyl CoA. The results of this study demonstrated the expansive substrate specificity of the recombinantly expressed mutant of the wtTBT (mTBT) in purified form when incubated with several acyl-CoA donor substrates and 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (17). mTBT transferred aroyl 4-substituted benzoyl analogues and 2- and 3-heterole carbonyl 96 groups, including 5-thiazole carbonyl, which is a similar structural motif found in the antimitotic epothilone drug family. 65 A benefit of chemoselective biocatalytic attachment of heterole carbonyls onto the taxane core is that the diene functional group can potentially be further modified, for example, by Diels-Alder chemistry to form novel heterocyclic taxanes with biological activity. Notably, a previous investigation showed that the Taxus N-benzoyltransferase on the paclitaxel biosynthetic pathway indiscriminately transferred o-, p-, or m-substituted 64 benzoyl groups from CoA, with a slight bias for the p-isomer. In contrast, mTBT showed singular preference for the p-substituted regioisomers within a homologous series and only marginally recognized 3-methylbenzoyl CoA as a substrate. Therefore, the positioning of the substituent on the aromatic ring of aroyl CoA substrates may reveal that occluding sterics are present in the active site of mTBT. It was hypothesized that mTBT, characterized as a benzoyltransferase, would transfer aroyl groups more efficiently to the 2-O-debenzoyl substrate; therefore, the superior catalytic constants of the catalyst for C3 and C4 hydrocarbonoyl CoA’s and butenoyl CoA’s were phenomenological. Short-chain alkanoyl and alkenoyl groups and a cyclohexanoyl group from the CoA donor to the diterpene co-substrate were also transferred to the taxane acceptor substrate by mTBT (cf. Table 2.2). Likely, the active site of mTBT can accommodate the conformational flexibility and smaller size of the acyclic CoA substrates, including the various conformational isomers of the cyclohexanoyl moiety. The transfer of these latter groups by mTBT indicates that the aromatic ring of the acyl CoA is not obligatory for catalysis. 97 2.4. Conclusion By understanding the scope of the mTBT specificity, the current biological sources of the antimitotic drug paclitaxel (cell cultures derived from Taxus spp. 66 and several fungal 67,68 cultures that biosynthesize paclitaxel) can potentially be utilized to produce novel paclitaxel molecules. To date, only acetyl, benzoyl, 2-methylbutenoyl, and 2-methylbutanoyl have been 69 identified at the C-2 hydroxy group of naturally occurring taxanes. The array of C-2-O adducts in vivo can be conceivably expanded by separately feeding one of the several carboxylic acids as a substrate to a paclitaxel producing organism engineered to produce any of the acyl CoA thioesters (described herein) for the construction of an unnatural paclitaxel pathway intermediate modified at C-2 by mTBT catalysis. As mentioned previously, TBT is included in a distinct 1,3 phylogenetic clade containing other benzoyltransferases. The wide specificity of mTBT includes several non-natural CoA substrates and thus supports the “patchwork model” of metabolic pathway evolution. This model hypothesizes that an organism uses a minimal gene content to provide a biocatalyst with maximum biochemical flexibility to provide product 70 diversity, a model envisioned for the survival of an ancestral organism. The broad specificity of mTBT suggests that this catalyst has likely maintained the flexibility it possessed during the early stages of evolution, according to the patchwork model. The several different kinds of acyl groups transferred in assays containing mTBT in vitro are remarkable, especially considering the limited, naturally occurring acyl CoA thioester substrates [acetyl, benzoyl, 2-methylbutenoyl, and 2-methylbutanoyl, which are variable with regard to sterics and electronics] available in the native host. Obviously, the acyl groups attached to the various taxane acceptors in planta at the C-2 hydroxy group of naturally occurring taxanes are likely sufficient to confer the necessary 98 fitness to the host. While it is reasonable to speculate that the specificity of mTBT was already honed by the few acyl CoA substrates based on metabolite occurrence, the latent extended specificity of the benzoyltransferase suggests a relatively tolerant active site of mTBT on the taxane biosynthetic pathways. Obviously, the range of alkanoyl, alkenoyl, and aroyl CoA thioesters accommodated by the catalyst in this study has been established over the course of evolution by the modest pool of dissimilar natural CoA substrates in the original hosts. Other 1,64,71 BAHD family acyltransferases also display broad substrate specificity profiles with varying acyl CoA donors and a range of alcohol and amine acceptor substrates. The current knowledge base of the BAHD acyltransferases, however, does not provide sufficient mechanistic information that enables speculation on what moderates the kinetic parameters of mTBT in terms of substituent regiochemistry in the aroyl CoA substrate, regarding sterics and electronic effects, and preferential catalytic efficiency for acyclic hydrocarbon carbonyls. Foreseeably, as more structural information becomes available for these BAHD acyltransferases, valuable insight into the mechanism of substrate specificity can be dissected. Moreover, directed evolutionary analyses can be employed to potentially produce new catalyst derivatives that are able to transfer an even greater or more refined scope of novel acyl groups to the taxane core or other diterpene scaffolds. 2.5. Future Directions 2.5.1. Expansion of the Catalytic Site of mTBT In contrast to a previous investigation showing that the Taxus N-benzoyltransferase on the paclitaxel biosynthetic pathway indiscriminately transferred o-, p-, or m-substituted benzoyl groups from CoA, 64 mTBT showed significantly higher preference for the p-substituted 99 regioisomers within each homologous series compared to o- or m- substituted isomers. This strongly suggests the presence of occluding sterics in the active site of mTBT. However, the biocatalytic transferring of m-substituted aroyl groups to the C2 hydroxyl of paclitaxel core producing novel analogues with C2 modifications are quite important in drug development. There are many reported C2 modified paclitaxel analogues with m-substituted benzoyl derivatives in clinical and preclinical stages such as SB-T-121304 (8), SB-T-11033 (9), SB-T7,9,10,12,15 12855-3 (14), and etc. (Figure 2.2). Therefore, improved substrate specificity of mTBT by assisting the catalysis of m-substituted aroyl transfers by changing the active site sterics is needed. We propose to increase the active site volume and reduce the occluding steric interactions between the substrates and the active site through rationally designed site-directed mutagenesis based on structural data of mTBT. However, the available structural data in the BAHD superfamily is minimal and limited to only four crystal structures; with and without ligands of heavy atom labeled or non-labeled enzymes. 29,60,72,73 Hence, acquiring the structural data of pure mTBT with and without bound substrates will be attempted. 2.5.2. Coupling of mTBT to a Promiscuous Benzoyl CoA Ligase As described in this study, mTBT demonstrated expansive substrate specificity with similar to superior catalytic efficiencies with various coenzyme A thioesters compared to benzoyl CoA. However, the overall turnover of substrate to the corresponding product was not very effective in any of the above transformations supplied with both cosubstrates at saturating concentrations. The recovery of remaining taxane and acyl coenzyme A thioester cosubstrates from the assay medium was not feasible. Therefore, it is intriguing to think of utilizing relatively cheap and more prevalent material as the cosubstrates in the transformations and also recycling the 100 substrates in the process. As a starting point, one could consider coupling mTBT catalysis with enzymatic synthesis of acyl CoA thioesters (26). The coupled system would utilize ubiquitous carboxylic acids (27) as cosubstrates and recycle the coenzyme A thiol (CoASH) (28), an expensive substrate, in the process (Figure 2.15). Aroyl CoA ligase Carboxylic acids (27) Mg2+, ATP Free CoA thiol (28) AcO AcO Acyl Coenzyme A thioesters (26) O OAc mTBT 2 HO O H OR OAc 25 17 Figure 2.15 – A schematic diagram of mTBT and aroyl CoA ligase coupled system in producing analogues of paclitaxel through biocatalysis. Benzoyl CoA (15) is the most common central intermediate of the anaerobic catabolism of a variety of aromatic compounds, which are then reduced to alicyclic compounds through the 74,75 “biological Birch reduction mechanism”. Hence, enzymatic synthesis of benzoyl CoA through bacterial benzoate:coenzyme A ligase (EC 6.2.1.25) is extensively studied (Figure 76-83 2.16). 82 There are several reported benzoyl CoA ligases with broad substrate specificities. 76 The benzoate-coenzyme A ligase, encoded by badA from Rhodopseudomonas palustris 80- has been recombinantly expressed in E. coli, purified by His-select nickel affinity chromatography, 101 tested with a range of aromatic carboxylic acids so far, and found to be highly promiscuous (unpublished data). Benzoylate (29) 28 Benzoate-CoA Ligase 15 Figure 2.16 – The transformation of benzoate to benzoyl CoA (15) by benzoate-CoA ligase. Also, the in vitro coupling of this promiscuous aroyl CoA ligase with mTBT has demonstrated highly efficient production of C2 acyl group modified taxane derivatives (25) (unpublished data), once the taxane cosubstrate (17), respective carboxylic acid (27), and CoASH (28) are provided. The goal is to co-express badA and the mutant tbt clone in E. coli using a similar approach 18 previously developed in our laboratory to develop biochemical production of precursors of paclitaxel analogues with the exogenous supply of the corresponding carboxylic acid and the taxane cosubstrate. 2.5.3. In Vivo mTBT Catalysis in Producing Analogues of Paclitaxel The overproduction of natural products with complex architecture and biological significance in heterologous hosts has been achieved through the recent developments in metabolic engineering 84-93 and synthetic biology. The production of paclitaxel in engineered genetically tractable microbial strains such as E. coli, Saccharomyces cerevisiae, Arabidopsis thaliana, transgenic 102 tomato fruits, and Physcomitrella patens (transgenic moss) has been approached through the overproduction of taxadiene, the first committed intermediate of paclitaxel biosynthesis, by isoprenoid pathway optimizations. 94-99 The production level of ~1 g/L of taxadiene has been 96 achieved in a recent report using E. coli as the heterologous host. A continuation of this work has demonstrated the formation of taxadiene-5α-ol from taxadiene to constitute the very first P450-mediated oxidation reaction in the paclitaxel biosynthetic pathway through genetic 96 engineering. The complete synthesis of paclitaxel in a heterologous host is certainly a daunting task which requires the constitution of several more hydroxylation and acylation reactions (some 100-102 are yet to be identified) downstream of taxadiene-5α-ol. However, the full understanding of the metabolism of each step in the paclitaxel biosynthetic pathway would essentially facilitate the bioengineering of Taxus cell cultures for higher and sustainable production of paclitaxel (5) and/or its analogues either by overexpression of genes controlling the limiting steps or by suppressing the “off pathway” metabolism. 100,102,103 Therefore, an understanding of the in vivo production of paclitaxel (5), its precursors, or its analogues utilizing a single or a combination of Taxus acyltransferases is profitable. As a pilot study, the in vivo production of baccatin III (20), an obligatory intermediate, and analogues has been demonstrated by functional expression of dbat encoding DBAT (10-O-deacetylbaccatin III 10β-O-acetyltransferase), derived from Taxus, 16,18 and acyl-CoA synthases/transferases, derived from the E. coli genome. Encouraged by the previous work, the in vivo production of 7,13-O,O-diacetylbaccatin III (18) through mTBT catalysis was tested in an E. coli BL21(DE3) strain by exogenously supplying the corresponding taxane and acyl coenzyme A cosubstrates. However, the catalytic production of 18 was not 103 detectable mainly due to the lack of intake of the taxane cosubstrate into the host strain. The in vivo production of corresponding products through coupled mTBT- BadA (benzoate-coenzyme A ligase) system has to be revisited utilizing more technically amenable microbial hosts. Also, the production of biologically significant analogues of paclitaxel with acyl group modifications (Figure 2.2) through metabolic engineering of combinations of Taxus acyltransferases needs to be examined in detail. 104 APPENDIX A 105 Appendix A atgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggcagccat 60 M G S S H H H H H H S S G L V P R G S H atgggcaggttcaatgtagatatgattgagcgagtgatcgtggcgccatgccttcaatcg 120 M G R F N V D M I E R V I V A P C L Q S cccaaaaatatcctgcacctctcccccattgacaacaaaactagaggactaaccaacata 180 P K N I L H L S P I D N K T R G L T N I ttatcagtctacaatgcctcccagagagtttctgtttctgcagatcctgcaaaaacaatt 240 L S V Y N A S Q R V S V S A D P A K T I cgagaggctctctccaaggtgctggtttattatcccccttttgctggaaggctgagaaac 300 R E A L S K V L V Y Y P P F A G R L R N acagaaaatggggatcttgaagtggagtgcacaggggagggtgccgtctttgtggaagcc 360 T E N G D L E V E C T G E G A V F V E A atggcggacaacgacctttcagtattacaagatttcaatgagtacgatccatcatttcag 420 M A D N D L S V L Q D F N E Y D P S F Q cagctagtttttaatcttcgagaggatgtcaatattgaggacctccatcttctaactgtt 480 Q L V F N L R E D V N I E D L H L L T V caggtaactcgttttacatgtggaggatttgttgtgggcacaagattccaccatagtgta 540 Q V T R F T C G G F V V G T R F H H S V tctgatggaaaaggaatcggccagttacttaaaggcatgggagagatggcaaggggggag 600 S D G K G I G Q L L K G M G E M A R G E tttaagccctcgttagaaccaatatggaatagagaaatggtgaagcctgaagacattatg 660 F K P S L E P I W N R E M V K P E D I M tacctccagtttgatcactttgatttcatacacccacctcttaatcttgagaagtctatt 720 Y L Q F D H F D F I H P P L N L E K S I caagcatctatggtaataagctttgagagaataaattatatcaaacgatgcatgatggaa 780 Q A S M V I S F E R I N Y I K R C M M E gaatgcaaagaatttttttctgcatttgaagttgtagtagcattgatttggctggcaagg 840 E C K E F F S A F E V V V A L I W L A R acaaagtcttttcgaattccacccaatgagtatgtgaaaattatctttccaatcgacatg 900 T K S F R I P P N E Y V K I I F P I D M aggaattcatttgactcccctcttccaaagggatactatggtaatgctattggtaatgca 960 R N S F D S P L P K G Y Y G N A I G N A 106 tgtgcaatggataatgtcaaagacctcttaaatggatctcttttatatgctctaatgctt 1020 C A M D N V K D L L N G S L L Y A L M L Ataaagaaatcaaagtttgctttaaatgagaatttcaaatcaagaatcttgacaaaacca 1080 I K K S K F A L N E N F K S R I L T K P tctacattagatgcgaatatgaagcatgaaaatgtagtcggatgtggcgattggaggaat 1140 S T L D A N M K H E N V V G C G D W R N ttgggattttatgaagcagattttggatggggaaatgcagtgaatgtaagccccatgcag 1200 L G F Y E A D F G W G N A V N V S P M Q caacaaagagagcatgaattagctatgcaaaattattttctttttctccgatcagctaag 1260 Q Q R E H E L A M Q N Y F L F L R S A K aacatgattgatggaatcaagatactaatgttcatgcctgcatcaatggtgaaaccattc 1320 N M I D G I K I L M F M P A S M V K P F aaaattgaaatggaagtcacaataaacaaatatgtggctaaaatatgtaactctaagtta 1380 K I E M E V T I N K Y V A K I C N S K L taa - 1440 Figure A 1 – The nucleotide sequence of wild-type tbt from T. cuspidata (accession no. AF297618) including the gene sequence for N-terminal His6-tag epitope and the corresponding amino acid sequence of wtTBT in pET28a vector (Novagen). The highlighted region shows the incorporated N-terminal His6-tag epitope on wtTBT which is used for immunoblot analysis of the expressed protein and purification by His-Select Nickel Affinity column chromatography. 107 atgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggcagccat 60 M G S S H H H H H H S S G L V P R G S H atgggcaggttcaatgtagatatgattgagcgagtgatcgtggcgccatgccttccatcg 120 M G R F N V D M I E R V I V A P C L P S cccaaaaaaatcctgcacctctcccccattgacaacaaaactagaggactaaccaacata 180 P K K I L H L S P I D N K T R G L T N I ttatcagtctacaatgcctcccagagagtttctgtttctgcagatcctgcaaaaacaatt 240 L S V Y N A S Q R V S V S A D P A K T I cgagaggctctctccaaggtgctggtttattatcccccttttgctggaaggctgagaaac 300 R E A L S K V L V Y Y P P F A G R L R N acagaaaatggggatcttgaagtggagtgcacaggggagggtgccgtctttgtggaagcc 360 T E N G D L E V E C T G E G A V F V E A atggcggacaacgacctttcagtattacaagatttcaatgagtacgatccatcatttcag 420 M A D N D L S V L Q D F N E Y D P S F Q cagctagtttttaatcttcgagaggatgtcaatattgaggacctccatcttctaactgtt 480 Q L V F N L R E D V N I E D L H L L T V caggtaactcgttttacatgtggaggatttgttgtgggcacaagattccaccatagtgta 540 Q V T R F T C G G F V V G T R F H H S V tctgatggaaaaggaatcggccagttacttaaaggcatgggagagatggcaaggggggag 600 S D G K G I G Q L L K G M G E M A R G E tttaagccctcgttagaaccaatatggaatagagaaatggtgaagcctgaagacattatg 660 F K P S L E P I W N R E M V K P E D I M tacctccagtttgatcactttgatttcatacacccacctcttaatcttgagaagtctatt 720 Y L Q F D H F D F I H P P L N L E K S I caagcatctatggtaataagctttgagagaataaattatatcaaacgatgcatgatggaa 780 Q A S M V I S F E R I N Y I K R C M M E gaatgcaaagaatttttttctgcatttgaagttgtagtagcattgatttggctggcaagg 840 E C K E F F S A F E V V V A L I W L A R acaaagtcttttcgaattccacccaatgagtatgtgaaaattatctttccaatcgacatg 900 T K S F R I P P N E Y V K I I F P I D M aggaattcatttgactcccctcttccaaagggatactatggtaatgctattggtaatgca 960 R N S F D S P L P K G Y Y G N A I G N A tgtgcaatggataatgtcaaagacctcttaaatggatctcttttatatgctctaatgctt 1020 C A M D N V K D L L N G S L L Y A L M L ataaagaaatcaaagtttgctttaaatgagaatttcaaatcaagaatcttgacaaaacca 1080 I K K S K F A L N E N F K S R I L T K P 108 tctacattagatgcgaatatgaagcatgaaaatgtagtcggatgtggcgattggaggaat 1140 S T L D A N M K H E N V V G C G D W R N ttgggattttatgaagcagattttggatggggaaatgcagtgaatgtaagccccatgcag 1200 L G F Y E A D F G W G N A V N V S P M Q caacaaagagagcatgaattagctatgcaaaattattttctttttctccgatcagctaag 1260 Q Q R E H E L A M Q N Y F L F L R S A K aacatgattgatggaatcaagatactaatgttcatgcctgcatcaatggtgaaaccattc 1320 N M I D G I K I L M F M P A S M V K P F aaaattgaaatggaagtcacaataaacaaatatgtggctaaaatatgtaactctaagtta 1380 K I E M E V T I N K Y V A K I C N S K L taa - 1440 Figure A 2 – The mutant tbt nucleotide sequence including the gene sequence for N-terminal His6-tag epitope and the corresponding amino acid sequence of mTBT in pET28a vector (Novagen). The highlighted regions show the modified amino acid residues, Q19P and N23K of wtTBT to mTBT through site-directed mutagenesis, modifications of nucleotide sequence to form mutant tbt, and the incorporated N-terminal His6-tag epitope on wtTBT which is used for immunoblot analysis of the expressed protein and purification by His-Select Nickel Affinity column chromatography. 109 * 20 * 40 wt_tbt : ATGGGCAGGTTCAATGTAGATATGATTGAGCGAGTGATCGTGGC : opt_tbt : ATGGGCCGTTTTAATGTAGACATGATCGAACGTGTTATCGTGGC : 44 44 * 60 * 80 wt_tbt : GCCATGCCTTCAATCGCCCAAAAATATCCTGCACCTCTCCCCCA : opt_tbt : CCCGTGTCTGCAGTCCCCGAAGAACATCCTGCATCTGTCCCCGA : 88 88 * 100 * 120 * wt_tbt : TTGACAACAAAACTAGAGGACTAACCAACATATTATCAGTCTAC : opt_tbt : TCGACAACAAAACCCGCGGCCTGACTAACATTCTGTCTGTTTAC : 132 132 140 * 160 * wt_tbt : AATGCCTCCCAGAGAGTTTCTGTTTCTGCAGATCCTGCAAAAAC : opt_tbt : AACGCGAGCCAGCGCGTATCTGTCTCCGCCGATCCAGCAAAAAC : 176 176 180 * 200 * 220 wt_tbt : AATTCGAGAGGCTCTCTCCAAGGTGCTGGTTTATTATCCCCCTT : opt_tbt : TATTCGTGAGGCGCTGTCTAAGGTGCTGGTGTACTATCCGCCAT : 220 220 * 240 * 260 wt_tbt : TTGCTGGAAGGCTGAGAAACACAGAAAATGGGGATCTTGAAGTG : opt_tbt : TTGCAGGCCGCCTGCGTAACACCGAAAATGGTGATCTGGAAGTT : 264 264 * 280 * 300 wt_tbt : GAGTGCACAGGGGAGGGTGCCGTCTTTGTGGAAGCCATGGCGGA : opt_tbt : GAATGTACCGGTGAAGGCGCCGTTTTTGTGGAGGCGATGGCCGA : 308 308 * 320 * 340 * wt_tbt : CAACGACCTTTCAGTATTACAAGATTTCAATGAGTACGATCCAT : opt_tbt : TAATGACCTGAGCGTTCTGCAGGATTTTAACGAGTATGATCCTA : 352 352 360 * 380 * wt_tbt : CATTTCAGCAGCTAGTTTTTAATCTTCGAGAGGATGTCAATATT : opt_tbt : GCTTCCAGCAACTGGTGTTCAATCTGCGTGAGGATGTAAATATT : 396 396 400 * 420 * 440 wt_tbt : GAGGACCTCCATCTTCTAACTGTTCAGGTAACTCGTTTTACATG : opt_tbt : GAAGATTTGCATCTGCTGACCGTGCAGGTGACCCGTTTTACCTG : 440 440 * 460 * 480 wt_tbt : TGGAGGATTTGTTGTGGGCACAAGATTCCACCATAGTGTATCTG : opt_tbt : CGGTGGCTTTGTTGTGGGTACGCGTTTCCACCACTCCGTGAGCG : 484 484 110 * 500 * 520 wt_tbt : ATGGAAAAGGAATCGGCCAGTTACTTAAAGGCATGGGAGAGATG : opt_tbt : ATGGCAAAGGTATTGGTCAGCTCCTGAAGGGCATGGGTGAGATG : 528 528 * 540 * 560 * wt_tbt : GCAAGGGGGGAGTTTAAGCCCTCGTTAGAACCAATATGGAATAG : opt_tbt : GCGCGTGGCGAGTTTAAGCCGTCGCTGGAACCGATCTGGAATCG : 572 572 580 * 600 * wt_tbt : AGAAATGGTGAAGCCTGAAGACATTATGTACCTCCAGTTTGATC : opt_tbt : CGAAATGGTTAAGCCGGAGGATATCATGTATCTTCAGTTCGATC : 616 616 620 * 640 * 660 wt_tbt : ACTTTGATTTCATACACCCACCTCTTAATCTTGAGAAGTCTATT : opt_tbt : ACTTTGACTTCATTCATCCGCCGCTCAATCTGGAAAAAAGCATT : 660 660 * 680 * 700 wt_tbt : CAAGCATCTATGGTAATAAGCTTTGAGAGAATAAATTATATCAA : opt_tbt : CAAGCCTCGATGGTTATCTCTTTCGAGCGTATCAATTATATTAA : 704 704 * 720 * 740 wt_tbt : ACGATGCATGATGGAAGAATGCAAAGAATTTTTTTCTGCATTTG : opt_tbt : GCGCTGCATGATGGAGGAATGTAAAGAATTCTTTTCGGCGTTCG : 748 748 * 760 * 780 * wt_tbt : AAGTTGTAGTAGCATTGATTTGGCTGGCAAGGACAAAGTCTTTT : opt_tbt : AAGTTGTGGTTGCCTTAATCTGGCTGGCGCGCACGAAGAGCTTT : 792 792 800 * 820 * wt_tbt : CGAATTCCACCCAATGAGTATGTGAAAATTATCTTTCCAATCGA : opt_tbt : CGTATTCCGCCAAATGAGTATGTGAAAATCATCTTCCCGATTGA : 836 836 840 * 860 * 880 wt_tbt : CATGAGGAATTCATTTGACTCCCCTCTTCCAAAGGGATACTATG : opt_tbt : CATGCGCAATTCGTTTGATAGCCCGCTTCCTAAAGGCTATTACG : 880 880 * 900 * 920 wt_tbt : GTAATGCTATTGGTAATGCATGTGCAATGGATAATGTCAAAGAC : opt_tbt : GTAATGCAATTGGCAACGCGTGTGCTATGGACAATGTGAAAGAC : 924 924 * 940 * 960 : CTCTTAAATGGATCTCTTTTATATGCTCTAATGCTTATAAAGAA : 968 wt_tbt 111 opt_tbt : CTGTTAAATGGCAGCCTGCTGTATGCTCTGATGCTGATTAAAAA : 968 * 980 * 1000 * wt_tbt : ATCAAAGTTTGCTTTAAATGAGAATTTCAAATCAAGAATCTTGA : 1012 opt_tbt : GAGCAAATTTGCTCTGAATGAAAACTTCAAGTCGCGCATTCTGA : 1012 1020 * 1040 * wt_tbt : CAAAACCATCTACATTAGATGCGAATATGAAGCATGAAAATGTA : 1056 opt_tbt : CCAAGCCGAGCACGTTGGACGCGAACATGAAACACGAAAATGTT : 1056 1060 * 1080 * 1100 wt_tbt : GTCGGATGTGGCGATTGGAGGAATTTGGGATTTTATGAAGCAGA : 1100 opt_tbt : GTGGGCTGCGGCGATTGGCGCAATCTGGGGTTCTATGAAGCGGA : 1100 * 1120 * 1140 wt_tbt : TTTTGGATGGGGAAATGCAGTGAATGTAAGCCCCATGCAGCAAC : 1144 opt_tbt : TTTTGGCTGGGGCAACGCGGTGAACGTAAGTCCGATGCAACAGC : 1144 * 1160 * 1180 wt_tbt : AAAGAGAGCATGAATTAGCTATGCAAAATTATTTTCTTTTTCTC : 1188 opt_tbt : AACGCGAACATGAACTGGCAATGCAGAACTATTTCCTGTTTCTG : 1188 * 1200 * 1220 * wt_tbt : CGATCAGCTAAGAACATGATTGATGGAATCAAGATACTAATGTT : 1232 opt_tbt : CGCTCCGCGAAAAACATGATTGACGGTATCAAAATCCTGATGTT : 1232 1240 * 1260 * wt_tbt : CATGCCTGCATCAATGGTGAAACCATTCAAAATTGAAATGGAAG : 1276 opt_tbt : TATGCCGGCGAGCATGGTGAAACCGTTTAAAATCGAAATGGAAG : 1276 1280 * 1300 * 1320 wt_tbt : TCACAATAAACAAATATGTGGCTAAAATATGTAACTCTAAGTTA : 1320 opt_tbt : TTACCATTAACAAATACGTCGCGAAAATCTGCAACTCCAAACTG : 1320 wt_tbt : TAA--- : 1323 opt_tbt : TAATAA : 1326 Figure A 3 – Nucleotide sequence alignment of wild-type tbt cDNA (wt_tbt) from Taxus cuspidata and codon optimized synthetic tbt gene (opt_tbt) from GenScript (Piscataway). An additional stop codon was added to the opt_tbt in designing the synthetic gene sequence. Both amino acid sequences remained the same otherwise. 112 AF297618 Mutant_TBT AY675557 AY970522 AF193765 AF190130 AF466397 AY082804 : : : : : : : : * 20 * 40 ------MGRFNVDMIERVIVAPCLQSPKNILHLSPIDNKT-------MGRFNVDMIERVIVAPCLPSPKKILHLSPIDNKT-------MGRFNVDMIERVIVAPCLPSPKKILRLSPIDNKT-------MGRFNVDMIERVIVAPCLPSPKKILHLSPIDNKT-MAGS---TEFVVRSLERVMVAPSQPSPKAFLQLSTLDNLPGV MEKT----DLHVNLIEKVMVGPSPPLPKTTLQLSSIDNLPGV MEKAG-STDFHVKKFDPVMVAPSLPSPKATVQLSVVDSLT-I MKKTGSFAEFHVNMIERVMVRPCLPSPKTILPLSAIDNMA-- : : : : : : : : 34 34 34 34 39 38 40 40 AF297618 Mutant_TBT AY675557 AY970522 AF193765 AF190130 AF466397 AY082804 : : : : : : : : * 60 * 80 -RGLTNILSVYNAS-QRVSVSADPAKTIREALSKVLVYYPPF -RGLTNILSVYNAS-QRVSVSADPAKTIREALSKVLVYYPPF -RALTNILSVYNAS-QRVSVSADPAETIREALSKVLVYYPPF -RALTNTLSVYNAS-QRVSVSADPAETIREALSKVLVYYPPF RENIFNTLLVYNAS--DR-VSVDPAKVIRQALSKVLVYYSPF RGSIFNALLIYNASPSPTMISADPAKPIREALAKILVYYPPF CRGIFNTLLVFNAP--DN-ISADPVKIIREALSKVLVYYFPL -RAFSNVLLVYAAN-MDR-VSADPAKVIREALSKVLVYYYPF : : : : : : : : 74 74 74 74 78 80 79 79 AF297618 Mutant_TBT AY675557 AY970522 AF193765 AF190130 AF466397 AY082804 : : : : : : : : * 100 * 120 AGRLRNTENGDLEVECTGEGAVFVEAMADNDLSVLQDFNEYD AGRLRNTENGDLEVECTGEGAVFVEAMADNDLSVLQDFNEYD AGRLRNTENGDLEVECTGEGAVFVEAMADNDLSVLQDFNEYD AGRLRSTENGKLEVECTGEGAVFVEAMADNDLSVLQDFNEYD AGRLRKKENGDLEVECTGEGALFVEAMADTDLSVLGDLDDYS AGRLRETENGDLEVECTGEGAMFLEAMADNELSVLGDFDDSN AGRLRSKEIGELEVECTGDGALFVEAMVEDTISVLRDLDDLN AGRLRNKENGELEVECTGQGVLFLEAMADSDLSVLTDLDNYN : : : : : : : : 116 116 116 116 120 122 121 121 AF297618 Mutant_TBT AY675557 AY970522 AF193765 AF190130 AF466397 AY082804 : : : : : : : : * 140 * 160 PSFQQLVFNLREDVNIEDLHLLTVQVTRFTCGGFVVGTRFHH PSFQQLVFNLREDVNIEDLHLLTVQVTRFTCGGFVVGTRFHH PSFQQLVFYLPEDVNIEDLHLLTVQVTRFTCGGFVVGTRFHH PSFQQLVFNLPEDVNIEDLHLLTVQVTRFTCGGFVVGTRFHH PSLEQLLFCLPPDTDIEDIHPLVVQVTRFTCGGFVVGVSFCH PSFQQLLFSLPLDTNFKDLSLLVVQVTRFTCGGFVVGVSFHH PSFQQLVFWHPLDTAIEDLHLVIVQVTRFTCGGIAVGVTLPH PSFQQLIFSLPQDTDIEDLHLLIVQVTRFTCGGFVVGANVYG : : : : : : : : 158 158 158 158 162 164 163 163 AF297618 Mutant_TBT AY675557 AY970522 AF193765 AF190130 AF466397 AY082804 : : : : : : : : * 180 * 200 * SVSDGKGIGQLLKGMGEMARGEFKPSLEPIWNREMVKPEDIM SVSDGKGIGQLLKGMGEMARGEFKPSLEPIWNREMVKPEDIM SVSDGKGIGQLLKGMGEMARGEFKPSLEPIWNREMVKPEDIM SVSDGKGIGELLKGMGDMARGEFKPSLEPIWSREMVKPEDIM GICDGLGAGQFLIAMGEMARGEIKPSSEPIWKRELLKPEDPL GVCDGRGAAQFLKGLAEMARGEVKLSLEPIWNRELVKLDDPK SVCDGRGAAQFVTALAEMARGEVKPSLEPIWNRELLNPEDPL SACDAKGFGQFLQSMAEMARGEVKPSIEPIWNRELVKLEHCM : : : : : : : : 200 200 200 200 204 206 205 205 113 AF297618 Mutant_TBT AY675557 AY970522 AF193765 AF190130 AF466397 AY082804 : : : : : : : : 220 * 240 * Y-LQFDHFDFIHPPLNLEKSIQASMVISFERINYIKRCMMEE Y-LQFDHFDFIHPPLNLEKSIQASMVISFERINYIKRCMMEE Y-LQFDHFDFIHPPLNLEKSIQASMVISFERINYIKRCMMEE Y-LQFDQFDFIRPPLNLEKSIQASMVISFEKINYIKRCMMEE YRFQYYHFQLICPPSTFGKIVQGSLVITSETINCIKQCLREE Y-LQFFHFEFLRAPSIVEKIVQTYFIIDFETINYIKQSVMEE H-LQLNQFDSICPPPMLEELGQASFVINVDTIEYMKQCVMEE P-FRMSHLQIIHAPVIEEKFVQTSLVINFEIINHIRRRIMEE : : : : : : : : 241 241 241 241 246 247 246 246 AF297618 Mutant_TBT AY675557 AY970522 AF193765 AF190130 AF466397 AY082804 : : : : : : : : 260 * 280 * CKEFFSAFEVVVALIWLARTKSFRIPPNEYVKIIFPIDMRNS CKEFFSAFEVVVALIWLARTKSFRIPPNEYVKIIFPIDMRNS CKEFFSAFEVVVALIWLARTKSFRIPPNEYVKIIFPIDMRNS CNEFFSAFEVVVALIWLARTKSFRIPPNEYVKIIFPMDMRNS SKEFCSAFEVVSALAWIARTRALQIPHSENVKLIFAMDMRKL CKEFCSSFEVASAMTWIARTRAFQIPESEYVKILFGMDMRNS CNEFCSSFEVVAALVWIARTKALQIPHTENVKLLFAMDLRKL RKESLSSFEIVAALVWLAKIKAFQIPHSENVKLLFAMDLRRS : : : : : : : : 283 283 283 283 288 289 288 288 AF297618 Mutant_TBT AY675557 AY970522 AF193765 AF190130 AF466397 AY082804 : : : : : : : : 300 * 320 * FDSPLPKGYYGNAIGNACAMDNVKDLLNGSLLYALMLIKKSK FDSPLPKGYYGNAIGNACAMDNVKDLLNGSLLYALMLIKKSK FDSPLPKGYYGNAIGNACAMDNVKDLLNGSLLYALMLIKKSK FDPPLPKGYYGNAIGNACAMDNVKYLLNGSLLYALMLIKKSK FNPPLSKGYYGNFVGTVCAMDNVKDLLSGSLLRVVRIIKKAK FNPPLPSGYYGNSIGTACAVDNVQDLLSGSLLRAIMIIKKSK FNPPLPNGYYGNAIGTAYAMDNVQDLLNGSLLRAIMIIKKAK FNPPLPHGYYGNAFGIACAMDNVHDLLSGSLLRTIMIIKKSK : : : : : : : : 325 325 325 325 330 331 330 330 AF297618 Mutant_TBT AY675557 AY970522 AF193765 AF190130 AF466397 AY082804 : : : : : : : : 340 * 360 * 3 FALNENFK-SRILTKPSTLDANMKHENVVGCGDWRNLGFYEA FALNENFK-SRILTKPSTLDANMKHENVVGCGDWRNLGFYEA FALNENFK-SRILTKPSALDANMKHENVVGCGDWRNLGFYEA FALYENFK-SRILTKPSTLDANMKHENVVGCGDWRNLGFYEA VSLNEHFT-STIVTPRSGSDESINYENIVGFGDRRRLGFDEV VSLNDNFK-SRAVVKPSELDVNMNHENVVAFADWSRLGFDEV ADLKDNYSRSRVVTNPYSLDVNKKSDNILALSDWRRLGFYEA FSLHKELN-SKTVMSSSVVDVNTKFEDVVSISDWRHSIYYEV : : : : : : : : 366 366 366 366 371 372 372 371 AF297618 Mutant_TBT AY675557 AY970522 AF193765 AF190130 AF466397 AY082804 : : : : : : : : 80 * 400 * 420 DFGWGNAVNVSPMQQQREHELAMQNYFLFLRSAKNMIDGIKI DFGWGNAVNVSPMQQQREHELAMQNYFLFLRSAKNMIDGIKI DFGWGNAVNVSPMQQQREHELAMQNYFLFLRSTKNMVDGIKI DFGWGNAVNVSPMQQQREHELAMQNYFLFLRSAKNMIDGIKI DFGWGHADNVSLVQHGLKDVSVVQSYFLFIRPPKNNPDGIKI DFGWGNAVSVSPVQQQS--ALAMQNYFLFLKPSKNKPDGIKI DFGWGGPLNVSSLQR-LENGLPMFSTFLYLLPAKNKSDGIKL DFGWGDAMNVSTMLQQQEHEKSLPTYFSFLQSTKNMPDGIKM : : : : : : : : 408 408 408 408 413 412 413 413 114 AF297618 Mutant_TBT AY675557 AY970522 AF193765 AF190130 AF466397 AY082804 : : : : : : : : * 440 * LM-FMPASMVKPFKIEMEVTINKYVAKICNSKL LM-FMPASMVKPFKIEMEVTINKYVAKICNSKL LM-FMPASMVKPFKIEMEVIINKYVAKICNSKL LM-FMPSSMVKPFKIEMEVTINKYVAKICNSNL LS-FMPPSIVKSFKFEMETMTNKYVTKP----LM-FLPLSKMKSFKIEMEAMMKKYVAKV----LLSCMPPTTLKSFKIVMEAMIEKYVSKV----LM-FMPPSKLKKFKIEIEAMIKKYVTKVCPSKL : : : : : : : : 440 440 440 440 440 439 441 445 Figure A 4 – Amino acid sequence alignment of TBT to the constructed double mutant, two different orthologs of TBT from different Taxus species, and other soluble acyl transferases involved in the paclitaxel biosynthetic pathway. AF297618, taxane 2α-O-benzoyltransferase from Taxus cuspidata (Accession no AF297618); Mutant TBT, double site-directed mutant of TBT replacing Gln19 and Asn23 to Pro19 and Lys23 respectively using site directed mutagenesis; AY675557, taxane 2α-O-benzoyltransferase from Taxus x media (Accession no AY675557); AY970522, taxane 2α-O-benzoyltransferase from Taxus wallichiana (Accession no AY970522); AF193765, 10-deacetylbaccatin III 10-O-acetyltransferase from Taxus cuspidata (Accession no AF193765); AF190130, taxadien-5α-ol O-acetyltransferase from Taxus cuspidata (Accession no AF190130); AF466397, 3′-N-debenzoyl-2′-deoxytaxol-N-benzoyltransferase from Taxus canadensis (Accession no AF466397); AY082804, 3-amino-3-phenylpropanoyltransferase from Taxus cuspidata (Accession no AY082804). Highly conserved proline residues are highlighted. 115 Total Current Detected for [M+H]+ and [M+Na]+ Ions 1800 1600 1400 1200 1000 800 600 400 200 0 0 1 2 3 4 5 Concentration of 7,13-O,O-Diacetylbaccatin III (pmol/mL) Figure A 5 – Linear regression analysis of a concentration series of the product standard, 7,13+ O,O-diacetylbaccatin III calibration standard plotted against the total ion current of the [M + H] + plus [M + Na] ions measured by LC-ESIMS; measured in duplicate (R2 = 0.98). The standard error was calculated to be at ~10%. 116 429.2 100 7,13-O,O-diacetylbaccatin III Exact Mass : 670.2 369.2 AcO % Relative Abundance 80 AcO 60 O OAc O HO H OAc O O 309.2 40 20 671.2 491.2 551.2 0 100 200 300 400 500 600 700 m/z Figure A 6 – MS/MS fragment ion profiles of authentic 7,13-O,O-diacetylbaccatin III is shown. + + Diagnostic ions are m/z: 671.2 [M + H] , 551.2 [m/z 671.2 – 2HOAc] , 491.2 [m/z 551 – + + + HOAc] , 429.2 [m/z 551.2 – RCOOH at C-2] , 369.2 [m/z 429.2 – HOAc] , and 309.2 [m/z + 369.2 – HOAc] , where R = benzoyl. 117 309.2 100 7,13-O,O-diacetyl-2-O-debenzoyl2-O-(3-fluorobenzoyl)baccatin III Exact mass - 688.2 AcO % Relative Abundance 80 60 O OAc O HO H OAc O O AcO 40 369.2 F 20 429.2 509.2 689.2 0 0 100 200 300 400 500 600 700 800 m/z Figure A 7 – MS/MS fragment ion profiles of authentic 7,13-O,O-diacetyl-2-O-debenzoyl-2-O+ (3-fluorobenzoyl)baccatin III is shown. Diagnostic ions are m/z: 689.2 [M + H] , 509.2 [m/z + + + 689.2 – 3HOAc] , 429.2 [689.2 – 2HOAc – RCOOH at C-2] , 369.2 [m/z 429.2 - HOAc] , and + the 309.2 [m/z 369.2 – HOAc] , where R = 3-fluorophenyl. 118 100 429.2 2,7,13-O,O,O-triacetyl2-O-debenzoylbaccatin III Exact Mass : 608.2 % Relative Abundance 80 AcO 369.2 60 AcO 40 O OAc O HO H OAc O O 609.2 309.2 20 489.2 549.2 0 100 200 300 400 500 600 700 800 m/z Figure A 8 – MS/MS fragment ion profiles of authentic 2,7,13-O,O,O-triacetyl-2-O+ debenzoylbaccatin III is shown. Diagnostic ions are m/z: 609.2 [M + H] , 549.2 [m/z 609.2 – + + + + HOAc] , 489.2 [m/z 549.2 – HOAc] , 429.2 [m/z 489.2 – RCOOH at C-2]+, 369.2 [m/z 429.2 – HOAc] , 309.2 [m/z 369.2 – HOAc] , where R = acetyl. 119 369.2 100 309.2 7,13-O,O-diacetyl-2-O-debenzoyl2-O-(3-thiophenecarbonyl)baccatin III Exact Mass : 676.2 % Relative Abundance 80 AcO 60 AcO 429.2 40 O OAc O HO H OAc O O S 20 497.2 557.2 677.2 0 100 200 300 400 500 600 700 800 m/z Figure A 9 – MS/MS fragment ion profiles of authentic 7,13-O,O-diacetyl-2-O-debenzoyl-2-O+ (3-thiophenecarbonyl)baccatin III is shown. Diagnostic ions are m/z: 677.2 [M + H] , 557.2 [m/z + + + 677.2 – 2HOAc] , 497.2 [m/z 557.2 – HOAc] , 429.2 [m/z 557.2 – RCOOH at C-2] , 369.2 [m/z + + 429.2 – HOAc] , 309.2 [m/z 369.2 – HOAc] , where R = 3-thiophenecarbonyl. 120 369.2 100 7,13-O,O-diacetylbaccatin III Exact Mass : 670.2 AcO % Relative Abundance 80 309.2 60 AcO 429.2 O OAc O HO H OAc O O 40 20 491.2 0 0 100 200 300 400 500 551.2 671.2 600 700 800 m/z Figure A 10 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetylbaccatin III + derived by catalysis of mTBT is shown. Diagnostic ions are m/z: 671.2 [M + H] , 551.2 [m/z + + + 671.2 – 2HOAc] , 491.2 [m/z 551.2 – HOAc] , 429.2 [m/z 551.2 – RCOOH at C-2] , 369.2 [m/z + + 429.2 – HOAc] , 309.2 [m/z 369.2 – HOAc] , where R = benzoyl. 121 309.2 100 369.2 80 % Relative Abundance 7,13-O,O-diacetyl-2-O-debenzoyl2-O-(3-methylbenzoyl)baccatin III Exact Mass : 684.2 AcO 60 AcO O OAc O HO H OAc O O 40 CH3 429.2 20 505.2 565.2 685.2 0 100 200 300 400 500 600 700 800 m/z Figure A 11 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(3-methylbenzoyl)baccatin III derived by catalysis of mTBT is shown. + + Diagnostic ions are m/z: 685.2 [M + H] , 565.2 [m/z 685.2 – 2HOAc] , 505.2 [m/z 565.2 – + + + HOAc] , 429.2 [m/z 565.2 – RCOOH at C-2] , 369.2 [m/z 429.2 – HOAc] , 309.2 [m/z 369.2 – + HOAc] , where R = 3-methylbenzoyl. 122 AcO O AcO 80 % Relative Abundance 685.2 7,13-O,O-diacetyl-2-O-debenzoyl2-O-(4-methylbenzoyl)baccatin III Exact Mass : 684.2 100 AcO 60 AcO OAc O OAc OH AcO O H O HO O O O O OAc 40 429.2 H3 C CH3 20 369.2 565.2 505.2 309.2 0 100 200 300 400 500 600 700 800 m/z Figure A 12 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(4-methylbenzoyl)baccatin III derived by catalysis of mTBT is shown. + + Diagnostic ions are m/z: 685.2 [M + H] , 565.2 [m/z 685.2 – 2HOAc] , 505.2 [m/z 565.2 – + + + HOAc] , 429.2 [m/z 565.2 – RCOOH at C-2] , 369.2 [m/z 429.2 – HOAc] , 309.2 [m/z 369.2 – + HOAc] , where R = 4-methylbenzoyl. 123 369.2 100 429.2 7,13-O,O-diacetyl-2-O-debenzoyl2-O-(3-fluorobenzoyl)baccatin III Exact Mass : 688.2 AcO % Relative Abundance 80 60 O HO H OAc O O AcO 309.2 O OAc F 40 509.2 20 569.2 689.2 629.2 0 100 200 300 400 500 600 700 800 m/z Figure A 13 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(2-fluorobenzoyl)baccatin III derived by catalysis of mTBT is shown. Diagnostic + + ions are m/z: 569.2 [m/z 689.2 – 2HOAc] , 509.2 [m/z 569.2 – HOAc] , 429.2 [m/z 569.2 – + + + RCOOH at C-2] , 369.2 [m/z 429.2 – HOAc] , 309.2 [m/z 369.2 – HOAc] , where R = 2fluorobenzoyl. 124 369.2 100 429.2 7,13-O,O-diacetyl-2-O-debenzoyl2-O-(3-fluorobenzoyl)baccatin III Exact Mass : 688.2 % Relative Abundance 80 AcO 60 O HO H OAc O O AcO 309.2 O OAc 40 F 509.2 20 569.2 689.2 629.2 0 100 200 300 400 500 600 700 800 m/z Figure A 14 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(3-fluorobenzoyl)baccatin III derived by catalysis of mTBT is shown. Diagnostic + + + ions are m/z: 689.2 [M + H] , 629.2 [m/z 689.2 – HOAc] , 569.2 [m/z 689.2 – 2HOAc] , 509.2 + + + [m/z 569.2 – HOAc] , 429.2 [m/z 569.2 – RCOOH at C-2] , 369.2 [m/z 429.2 – HOAc] , 309.2 + [m/z 369.2 – HOAc] , where R = 3-fluorobenzoyl. 125 369.2 100 % Relative Abundance 80 7,13-O,O-diacetyl-2-O-debenzoyl2-O-(4-fluorobenzoyl)baccatin III Exact Mass : 688.2 AcO 429.2 60 O HO H OAc O O AcO 309.2 O OAc 40 F 20 509.2 0 100 200 300 400 500 569.2 600 689.2 700 800 m/z Figure A 15 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(4-fluorobenzoyl)baccatin III derived by catalysis of mTBT is shown. Diagnostic + + + ions are m/z: 689.2 [M + H] , 569.2 [m/z 689.2 – 2HOAc] , 509.2 [m/z 569.2 – HOAc] , 429.2 + + + [m/z 569.2 – RCOOH at C-2] , 369.2 [m/z 429.2 – HOAc] , 309.2 [m/z 369.2 – HOAc] , where R = 4-fluorobenzoyl. 126 369.2 100 7,13-O,O-diacetyl-2-O-debenzoyl2-O-(4-chlorobenzoyl)baccatin III Exact Mass : 704.2 AcO % Relative Abundance 80 O OAc 309.2 60 429.2 O HO H OAc O O AcO 40 Cl 20 527.2 587.2 705.2 0 100 200 300 400 500 600 700 800 m/z Figure A 16 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(4-chlorobenzoyl)baccatin III derived by catalysis of mTBT is shown. Diagnostic + + + ions are m/z: 705.2 [M + H] , 587.2 [m/z 705.2 – 2HOAc] , 527.2 [m/z 587.2 – HOAc] , 429.2 + + + [m/z 587.2 – RCOOH at C-2] , 369.2 [m/z 429.2 – HOAc] , 309.2 [m/z 369.2 – HOAc] , where R = 4-chlorobenzoyl. 127 309.2 100 7,13-O,O-diacetyl-2-O-debenzoyl2-O-(2-furancarbonyl)baccatin III Exact Mass : 660.2 % Relative Abundance 80 AcO 60 AcO 369.2 40 O OAc O HO H OAc O O O 429.2 481.2 20 541.2 661.2 0 100 200 300 400 500 600 700 800 m/z Figure A 17 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(2-furancarbonyl)baccatin III derived by catalysis of mTBT is shown. Diagnostic + + + ions are m/z: 661.2 [M + H] , 541.2 [m/z 661.2 – 2HOAc] , 481.2 [m/z 541.2 – HOAc] , 429.2 + + + [m/z 481.2 – RCOOH at C-2] , 369.2 [m/z 429.2 – HOAc] , 309.2 [m/z 369.2 – HOAc] , where R = 2-furancarbonyl. 128 309.2 100 7,13-O,O-diacetyl-2-O-debenzoyl2-O-(3-furancarbonyl)baccatin III Exact Mass : 660.2 369.2 % Relative Abundance 80 AcO O OAc 429.2 60 AcO HO O O H O OAc 40 O 20 481.2 541.2 661.2 0 100 200 300 400 500 600 700 800 m/z Figure A 18 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(3-furancarbonyl)baccatin III derived by catalysis of mTBT is shown. Diagnostic + + + ions are m/z: 661.2 [M + H] , 541.2 [m/z 661.2 – 2HOAc] , 481.2 [m/z 541.2 – HOAc] , 429.2 + + + [m/z 481.2 – RCOOH at C-2] , 369.2 [m/z 429.2 – HOAc] , 309.2 [m/z 369.2 – HOAc] , where R = 3-furancarbonyl. 129 309.2 100 7,13-O,O-diacetyl-2-O-debenzoyl369.2 2-O-(2-thiophenecarbonyl)baccatin III Exact Mass : 676.2 AcO % Relative Abundance 80 60 429.2 AcO O OAc O HO H OAc O O 40 S 20 497.2 557.2 677.2 0 100 200 300 400 500 600 700 800 m/z Figure A 19 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(2-thiophenecarbonyl)baccatin III derived by catalysis of mTBT is shown. Diagnostic ions are m/z: 677.2 [M + H]+, 557.2 [m/z 677.2 – 2HOAc]+, 497.2 [m/z 557.2 – HOAc]+, 429.2 [m/z 497.2 – RCOOH at C-2]+, 369.2 [m/z 429.2 – HOAc]+, 309.2 [m/z 369.2 – HOAc]+, where R = 2-thiophenecarbonyl. 130 369.2 100 309.2 7,13-O,O-diacetyl-2-O-debenzoyl2-O-(3-thiophenecarbonyl)baccatin III Exact Mass : 676.2 % Relative Abundance 80 AcO 60 AcO 429.2 40 O OAc O HO H OAc O O S 20 497.2 557.2 677.2 0 100 200 300 400 500 600 700 800 m/z Figure A 20 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(3-thiophenecarbonyl)baccatin III derived by catalysis of mTBT is shown. Diagnostic ions are m/z: 677.2 [M + H]+, 557.2 [m/z 677.2 – 2HOAc]+, 497.2 [m/z 557.2 – HOAc]+, 429.2 [m/z 497.2 – RCOOH at C-2]+, 369.2 [m/z 429.2 – HOAc]+, 309.2 [m/z 369.2 – HOAc]+, where R = 3-thiophenecarbonyl. 131 AcO 80 % Relative Abundance 678.2 7,13-O,O-diacetyl-2-O-debenzoyl2-O-(5-thiazolecarbonyl)baccatin III Exact Mass : 678.2 100 60 AcO O OAc O HO H OAc O O 40 S N 20 309.2 369.2 498.2558.2 0 100 200 300 400 500 600 700 800 m/z Figure A 21 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(5-thiazolecarbonyl)baccatin III derived by catalysis of mTBT is shown. Diagnostic ions are m/z: 678.2 [M + H]+, 558.2 [m/z 678.2 – 2HOAc]+, 498.2 [m/z 558.2 – HOAc]+, 369.2 [m/z 498.2 – RCOOH at C-2]+, 309.2 [m/z 369.2 – HOAc]+, where R = 5thiazolecarbonyl. 132 429.2 100 % Relative Abundance 80 2,7,13-O,O,O-triacetyl2-O-debenzoylbaccatin III Exact Mass - 609.2 309.2 60 AcO 369.2 O OAc O HO H OAc O O AcO 40 489.2 20 549.2 609.2 0 100 200 300 400 500 600 700 800 m/z Figure A 22 – MS/MS fragment ion profile of the biosynthesized 2,7,13-O,O,O-triacetyl-2-O+ debenzoylbaccatin III is shown. Diagnostic ions are m/z: 609.2 [M + H] , 549.2 [m/z 609.2 – + + + + HOAc] , 489.2 [m/z 549.2 – HOAc] , 429.2 [m/z 489.2 – RCOOH at C-2]+, 369.2 [m/z 429.2 – HOAc] , 309.2 [m/z 369.2 – HOAc] , where R = acetyl. 133 309.2 100 7,13-O,O-diacetyl-2-O-debenzoyl2-O-propionylbaccatin III Exact Mass : 622.2 369.2 % Relative Abundance 80 AcO 60 429.2 AcO O OAc O HO H OAc O O 40 20 503.2 0 100 200 300 400 500 623.2 600 700 800 m/z Figure A 23 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-propanoylbaccatin III derived by catalysis of mTBT is shown. Diagnostic ions are m/z: 623.2 [M + H]+, 503.2 [m/z 623.2 – 2HOAc]+, 429.2 [m/z 503.2 – RCOOH at C-2]+, 369.2 [m/z 429.2 – HOAc], 309.2 [m/z 369.2 – HOAc]+, where R = propanoyl. 134 309.2 100 7,13-O,O-diacetyl-2-O-debenzoyl2-O-butyrylbaccatin III Exact Mass : 636.2 % Relative Abundance 80 AcO O OAc 369.2 60 AcO HO O O H O OAc 40 20 429.2 457.2 517.2 0 0 100 200 300 400 500 600 700 m/z Figure A 24 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-butyrylbaccatin III derived by catalysis of mTBT is shown. Diagnostic ions are m/z: 517.2 [m/z 637.2 – 2HOAc]+, 457.2 [m/z 517.2 – HOAc], 429.2 [m/z 517.2 – RCOOH at C2]+, 369.2 [m/z 429.2 – HOAc], 309.2 [m/z 369.2 – HOAc]+, where R = butyryl. 135 369.2 100 AcO 429.2 80 % Relative Abundance 7,13-O,O-diacetyl-2-O-debenzoyl2-O-(2-butenoyl)baccatin III Exact Mass : 634.2 O OAc 309.2 AcO 60 HO O O H O OAc 40 20 515.2 635.2 0 100 200 300 400 500 600 700 800 m/z Figure A 25 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-(2-butenoyl)baccatin III derived by catalysis of mTBT is shown. Diagnostic ions + are m/z: 635.2 [M + H] , 515.2 [m/z 635.2 – 2HOAc]+, 429.2 [m/z 515.2 – RCOOH at C-2]+, 369.2 [m/z 429.2 – HOAc], 309.2 [m/z 369.2 – HOAc]+, where R = 2-butenoyl. 136 309.2 100 7,13-O,O-diacetyl-2-O-debenzoyl2-O-hexanoylbaccatin III Exact Mass : 664.2 AcO % Relative Abundance 80 60 AcO 369.2 O OAc O HO H OAc O O 40 20 429.2 665.2 545.2 0 100 200 300 400 500 600 700 800 m/z Figure A 26 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-hexanoylbaccatin III derived by catalysis of mTBT is shown. Diagnostic ions are + m/z: 665.2 [M + H] , 545.2 [m/z 665.2 – 2HOAc]+, 429.2 [m/z 545.2 – RCOOH at C-2]+, 369.2 [m/z 429.2 – HOAc], 309.2 [m/z 369.2 – HOAc]+, where R = hexanoyl. 137 369.2 100 7,13-O,O-diacetyl-2-O-debenzoyl2-O-isobutyrylbaccatin III Exact Mass : 636.2 309.1 309.2 AcO % Relative Abundance 80 60 AcO 429.2 429.2 O OAc O HO H OAc O O 40 20 517.2 517.2 457.2 577.2 637.2 577.3 637.2 0 100 200 300 400 500 600 700 800 m/z Figure A 27 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-isobutyrylbaccatin III derived by catalysis of mTBT is shown. Diagnostic ions + + are m/z: 637.2 [M + H] , 577.2 [m/z 637.2 – HOAc] , 517.2 [m/z 577.2 – HOAc]+, 457.2 [m/z + 517.2 – HOAc] , 429.2 [m/z 545.2 – RCOOH at C-2]+, 369.2 [m/z 429.2 – HOAc], 309.2 [m/z 369.2 – HOAc]+, where R = isobutyryl. 138 429.2 100 369.2 7,13-O,O-diacetyl-2-O-debenzoyl2-O-cyclohexanoylbaccatin III Exact Mass : 676.2 AcO % Relative Abundance 80 AcO 309.2 60 40 O OAc O HO H OAc O O 677.2 557.2 20 497.2 617.2 0 100 200 300 400 500 600 700 800 m/z Figure A 28 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoyl-2-O-cyclohexanoylbaccatin III derived by catalysis of mTBT is shown. Diagnostic + + ions are m/z: 677.2 [M + H] , 617.2 [m/z 677.2 – HOAc]+, 557.2 [m/z 617.2 - HOAc] , 497.2 + [m/z 557.2 – HOAc , 429.2 [m/z 557.2 – RCOOH at C-2]+, 369.2 [m/z 429.2 – HOAc], 309.2 [m/z 369.2 – HOAc]+, where R = cyclohexanoyl. 139 O O O O O 7 O O 13 HO H OH O O O 1 Figure A 29 – H NMR of 7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III 140 O O O O O 7 O O 13 HO H OH O O O Figure A 30 – 13 C NMR of 7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III 141 O O O O O 7 O O 13 HO H O O O O O 1 Figure A 31 – H NMR of 7,13-O,O-Diacetylbaccatin III 142 O O O O O 7 O O 13 HO H O O O O O Figure A 32 – 13 C NMR of 7,13-O,O-Diacetylbaccatin III 143 H 4 O 1 S 7 O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 33 – H NMR of Benzoyl CoA 144 H Cl 5 O 1 S 7 O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 34 – H NMR of 3-Chlorobenzoyl CoA 145 Cl H 4 O 1 S 7 O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 35 – H NMR of 4-Chlorobenzoyl CoA 146 H CN 5 O 1 S 7 O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 36 – H NMR of 3-Cyanobenzoyl CoA 147 8 CH3 O H 5 O 1 S 7 O 1′ N H N NH2 OH OH O N O O O N N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 37 – H NMR of 3-Methoxybenzoyl CoA 148 4 H F O 1 S 7 O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 38 – H NMR of 2-Fluorobenzoyl CoA 149 H F 5 O 1 S 7 O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 39 – H NMR of 3-Fluorobenzoyl CoA 150 F H 4 O 1 S 7 O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 40 – H NMR of 4-Fluorobenzoyl CoA 151 4 H 8 CH3 O 1 S 7 O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 41 – H NMR of 2-Methylbenzoyl CoA 152 8 CH3 H 5 O 1 S 7 O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 42 – H NMR of 3-Methylbenzoyl CoA 153 8 H3C H 4 O 1 S 7 O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 43 – H NMR of 4-Methylbenzoyl CoA 154 H 3 5 O O 1 S O 1′ N H N NH2 N OH OH O N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 44 – H NMR of 2-Furancarbonyl CoA 155 H O 3 O 1 S 5 O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 45 – H NMR of 3-Furancarbonyl CoA 156 H 3 5 S O 1 S O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 46 – H NMR of 2-Thiophenecarbonyl CoA 157 H S 3 O 1 S 5 O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 47 – H NMR of 3-Thiophenecarbonyl CoA 158 H 3 N 4 S O 1 S O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 48 – H NMR of 5-Thiazolecarbonyl CoA 159 H 4 2 O 1 S O 7 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure A 49 – H NMR of Phenyl acetyl CoA 160 H 4 O 1 S 7 O 1′ N H N NH2 OH OH O N N O O O N N P P 5′′ 9′ H O O H O OH HO H P OH O OH O 4′ 1 Figure A 50 – H NMR of Cyclohexanoyl CoA 161 REFERENCES 162 REFERENCES (1) D'Auria, J. 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Therefore, the acyl group modifications at C-10, C-2, C-4, and C-13 hydroxy-, and C-3' amino- positions are targets for developing efficacious paclitaxel analogues; some are currently in clinical usage and in clinical trial studies. 1,3-6 To date, biotechnological production is the most sustainable and cost-effective way of producing 7,8 analogues of paclitaxel in addition to the alternative, semisynthetic approach. Potentially, biotechnological production of efficacious paclitaxel analogues can be achieved through the 7,9,10 metabolic engineering of Taxus plant cell cultures, 11,12 Escherichia coli, 14-16 species, Saccharomyces cerevisiae, 13 and/or other recombinant hosts such as and alternative tractable transgenic As an example, a potential semi-biosynthetic route of producing efficacious paclitaxel analogues with various acyl groups at C-10, C-2, C-13, and C-3' (7) can be substituted into the paclitaxel structure via a semi-biosynthetic route producing efficacious analogues (Figure 3.1). Due to the high regiospecificity of the Taxus acyltransferases, in general, their application as biocatalysts in this route would eliminate the many protection-deprotection 169 manipulations involved in semisynthesis, thus significantly improving the overall efficiency of the process. 10-O-Deacetylbaccatin III (1) O R1 O O O OH O R1 c O HO H OAc O O O OH NH2 O d HO 3 Baccatin III R1: Me (2) R3 O OH O HO H OAc O O R2 R2 4 5 Paclitaxel R1: Me; R2: Ph; R3: Ph; R4: Ph (6) Various R1, R2, R3, R4 combinations (7) Figure 3.1 – A potential semi-biocatalytic route to efficatious paclitaxel analogues with various acyl groups R1, R2, R3, and R4 at C-10, C-2, C-3', and C-3' amino group respectively (7) incorporating Taxus acyltransferases in both forward (acylation) and reverse (deacylation) direction. Step a: 10-Deacetylbaccatin III-10β-O-acetyltransferase (DBAT); b: Deacylation of modified 2-O-debenzoyl-7,13-O,O-diacetylbaccatin III-2α-O-benzoyltransferase (mTBT); c: Acylation of mTBT; d: Baccatin III-13α-O-phenylpropanoyltransferase (BAPT); e: 3'-NDebenzoyl-2'-deoxytaxol-N-benzoyltransferase (NDTBT) 170 The acylation reactions of these Taxus acyltransferases have been well studied and proven to 17-26 show broad substrate specificities in vitro and/or in vivo, while the deacylation reactions of Taxus acyltransferases and/or other related enzymes have been reported only sporadically. 24,27 The potential application of the acylation reaction catalyzed by a mutant 2-O-debenzoyl-7,13O,O-diacetylbaccatin III 2α-O-benzoyltransferase (mTBT) in producing C-2 modified taxane analogues (Figure 3.1, step c) was discussed in Chapter 2. However, the enzymatic deacylation of mTBT, which is a key step in the semi-biosynthetic route of producing paclitaxel analogues (Figure 3.1, step b), has not yet been investigated to date. A related enzyme of Taxus acyltransferases, vinorine synthase (EC 2.3.1.160), was shown to catalyze the regiospecific deacetylation of vinorine in the presence of CoASH (35); this reverse reaction of vinorine synthase shows higher catalytic efficiency compared to the forward reaction of the same 27 enzyme. 27 Also, the reported deacetylation reaction is very substrate specific. Likewise, mTBT may be able to debenzoylate the C-2 benzoyl group of various taxane substrate/s in the presence of 35. Several commercially available and semisynthesized taxane substrates were selected to analyze the substrate specificity of mTBT deacylating reaction (Figure 3.2). 171 AcO O H OBz OH 4-O-Deacetylbaccatin III R: H (9) 13-O-Acetyl-4-Odeacetylbaccatin III R: Ac (10) AcO O OAc RO 10-O-Deacetylbaccatin III R: H (1) Baccatin III R: Ac (2) 7,13-O,O-Diacetylbaccatin III (8) 13-O-Acetylbaccatin III (12) HO O H OBz OAc 7-O-Acetyl-13-oxobaccatin III (13) O 7-O-Acetylbaccatin III (11) O OH HO Paclitaxel R1: Me; R2: Ph (6), Docetaxel R1: H; R2: OC(CH3)3 (14) Figure 3.2 – Several selected commercially available and semisynthesized taxane substrates to test the substrate specificity of mTBT deacylating reaction. In addition, examination of range of taxane substrates that mTBT can deacylate may also aid in pinpointing the exact time in which the 2-O-benzoylation occurs on the overall paclitaxel pathway. Some of the genes on the highly complex paclitaxel biosynthetic pathway have been 5,7,9,17-26,28-48 expressed and their function has been characterized (Figure 3.3 and Figure 3.4). The first committed step on the paclitaxel pathway is the cyclization of the primary metabolite geranylgeranyl diphosphate (GGDP, 17) produced through the primary metabolism, to taxa29,30 4(5),11(12)-diene (18). Then several oxygenations, two acetylations, a benzoylation, 172 oxidation at C-9, oxetane ring formation, and the final side chain assembly and attachment at C17-26,34-47 13, complete the paclitaxel biosynthesis. IDP (15) GGDP (17) DMADP (16) 1 H 5 H 20 Taxa-4(5),11(12)-diene (18) 5 1 OAc H 20 H Taxa-4(20),11(12)-dien-5α-yl-acetate (19) OH Taxa-4(20),11(12)-dien-5αol (20) Taxa-4(20),11(12)-dien-5α,13α-diol (21) HO OH j 9 1 5α-Acetoxytaxa-4(20),11(12)-dien-10β-ol (22) 5 OAc H 20 H 5α-Acetoxytaxa-4(20),11(12)dien-9α/β,10β-diol (23) 19 1 2 h 5 OAc H 20 H OH 5α-Acetoxytaxa-4(20),11(12)-dien-2α-ol (24) Figure continues… 173 R = H or Ac Taxusin R: Ac (25) R = H or Ac 7β-Hydroxytaxusin R: Ac (26) RO OR k RO 26 RO h 27 k 2 OR H OH 2α-Hydroxytaxusin R: Ac (27) H OR OH 7 l 2 OR H OH 2α,7β-Dihydroxytaxusin R: Ac (28) RO 13 H 2-Debenzoyl-10-deacetylbaccatin III (29) AcO O OH 10 29 7 O H OBz OAc (2) (1) Figure 3.3 – Paclitaxel biosynthetic pathway starting from primary metabolism to the paclitaxel precursor baccatin III (2). Step a: Isopentenyl diphosphate isomerase (IDPI); b: Geranylgeranyl diphosphate synthase (GGDPS); c: Taxadiene synthase (TS); d: Cytochrome P450 Taxadiene 5α-hydroxylase; e: Taxa-4-(20),11(12)-diene-5α-ol-O-acetyltransferase (TAT); f: Cytochrome P450 taxane 13α-hydroxylase; g: Cytochrome P450 taxane 10β-hydroxylase; h: Cytochrome P450 taxane 2α-hydroxylase; i: Cytochrome P450 taxane 9α/β-hydroxylase; j: Several unknown steps; k: Cytochrome P450 taxane 7β-hydroxylase; l: Unknown step/s; m: Modified 2-Odebenzoyl-7,13-O,O-diacetylbaccatin III-2α-O-benzoyltransferase (mTBT); n: 10-ODeacetylbaccatin III-10β-O-acetyltransferase. Abbreviations: MEP - Methylerythritol phosphate; IDP - Isopentenyl diphosphate; DMADP - Dimethylallyl diphosphate; GGDP - Geranylgeranyl diphosphate. HO 174 HO Despite the presumed order of oxygenations based on a survey of known taxoids,28,47 more recent findings and observations suggest that paclitaxel biosynthesis is not a linear pathway and that there are several branch points in the mid-pathway that likely lead to other related taxoids 7 that are not on the paclitaxel pathway (Figure 3.3). Consequently, the continuity of the pathway gets disrupted by several unknown or identified, but divergent steps occur in the mid-pathway (for example Figure 3.3 steps e, f, g, h, i, j, k, and l). The apparent dead end routes assembled by arranging the taxane structures, known to date, on a linear catabolic path, suggest that the route to paclitaxel requires the use of perceived dead-end product. The complexity of the oxygenation and acylation patterns seen in the taxoids is likely due to the promiscuity of the enzymes in 24,41 planta Therefore, the interpretations of in vitro assays employing a limited set of test substrate(s) and expressed protein catalysts from Taxus spp. can be misleading. Isolation and identification of a true substrate(s) needed to characterize each mid-pathway step may remain elusive, if the metabolite is at a low concentration in its natural resource. In addition, without theses key substrates, it is difficult to ascertain whether the cDNA encoding the enzymes are 9,39,40,45,46,49 currently in-hand. Moreover, some of these genes already identified and characterized in vitro as on-pathway enzymes may actually be involved exclusively or in part in 9 “off-pathway” steps. As a consequence of the complexity of paclitaxel biosynthesis, several important steps and intermediates have not yet been identified. Therefore, to shed light on one of the putative pathway steps, the deacylating reaction of mTBT will be assayed with 2-O-benzoyl taxanes in attempt to evaluate the organization, and possibly the regulation of the paclitaxel pathway. The scope of this chapter entails designing a 175 rational set of taxane substrates that may elucidate the timing of 2-O-benzoylation in the paclitaxel biosynthetic scheme. α-phenylalanine (30) β-phenylalanine (31) β-phenylalanine CoA (32) 32 β-phenylalanoyl baccatin III (33) 2 N-Debenzoylpaclitaxel (34) 6 Figure 3.4 – Last steps of paclitaxel biosynthetic pathway; N-benzoylphenylisoserine side chain construction at C13 of baccatin III (2). Step o: Phenylalanine aminomutase (PAM); p: Unknown CoA thioester ligase; q: Baccatin III-13α-O-phenylpropanoyltransferase (BAPT); r: Cytochrome P450 hydroxylase; s: 3'-N-Debenzoyl-2'-deoxytaxol-N-benzoyltransferase (NDTBT) 176 3.2. Experimental 3.2.1. Substrates, Reagents, and General Instrumentation The Taxus cuspidata cDNA (tbt) (accession no. AF297618) was a generous donation of Washington State University Research Foundation (Pullman, WA). 10-O-Deacetylbaccatin III (1) and baccatin III (2) were purchased from Natland (Research Triangle Park, NC) while paclitaxel (6) and docetaxel (14) were purchased from OChem Inc. (Des Plaines, IL). CoASH (35) was purchased from American Radiolabeled Chemicals Inc. (Saint Louis, MO). Material required for thin layer chromatography (TLC), preparative TLC, and flash column chromatography were purchased from EMD Chemicals Inc. (Gibbstown, NJ). All other reagents were obtained from Sigma-Aldrich and used without further purification, unless indicated otherwise. A Varian Inova-300, Varian UnityPlus-300, Varian Inova-500, Varian UnityPlus-500, 1 and Varian Inova-600 instruments were used to acquire H, 13 C, HMQC, HMBC NMR spectra. An electrospray ionization tandem mass spectrometer (ESI-MS/MS) (Q-ToF Ultima Global, Waters, Milford, MA) was used to acquire mass analysis of semi-synthetic compounds by direct injection. The biosynthetic compounds were separated and analyzed using a reversed-phase column (Betasil C18, 5 μm, 150 × 2.1 mm, Thermo Fisher Scientific Inc., Waltham, MA) attached to a capillary HPLC system (CapLC capillary HPLC, Waters, Milford, MA) connected to the ESI-MS/MS (Q-ToF Ultima Global, Waters, Milford, MA). Other general information not included here is noted elsewhere in the text. 177 3.2.2. Acquiring Crude Cell Lysate of modified TBT (mTBT), Activity Assay, and Protein Purification The plasmid p28PK-TBT in E. coli BL21(DE3) cells prepared according to section 2.2.13.6 was used in acquiring crude mTBT cell lysate using the method described in section 2.2.13.7. The activity assay and the further purification of mTBT were achieved following a protocol described in section 2.2.13.8. 3.2.3. General Note 19 7,13-O,O-Diacetylbaccatin III (8) was prepared according to an established procedure and the spectral data are shown in section 2.2.4. The synthesis and the spectral data of 7-Oacetylbaccatin III (11) were described in section 2.2.5. The synthesis and the spectral data of 7O-acetyl-13-oxobaccatin III (13) were described in section 2.2.6. The synthesis and the spectral data of 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III were described in section 2.2.4. 4-ODeacetylbaccatin III (9) and 13-O-acetyl-4-O-deacetylbaccatin III (10) were generous donations; the synthesis and the spectral data of these compounds were reported in a previous 24 communication. The synthesis, quantification, and the spectral data of benzoyl CoA were described in sections 2.2.10, 2.2.10.1, and 2.2.10.2 respectively. 3.2.4. General Procedures The reactions were generally monitored by TLC and, upon completion, were diluted with EtOAc (10 mL) and quenched with H2O (5 mL). The mixture was stirred for 5 min, and the aqueous fraction was separated and extracted with EtOAc (2 × 5 to 20 mL, depending on the extent of 178 portioning of the product to the organic fraction). The combined organic fractions were washed with saturated CuSO4, brine, 0.1 M HCl, and H2O, and dried over anhydrous Na2SO4. 3.2.5. Synthesis of 13-O-Acetylbaccatin III (12) The compound 12 was synthesized similarly to a procedure described for the synthesis of 13-O50 butyrylbaccatin III, except acetic anhydride was used in place of butyryl chloride as the acyl 1 donor. H NMR (500 MHz, CDCl3) δ: 1.12 (s, H-16), 1.23 (s, H-17), 1.66 (s, H-19), 1.89 (s, H18), 2.19 (s, C(O)CH3 at C-13), 2.22 (s, C(O)CH3 at C-10), 2.23 (s, H-14), 2.31 (s, C(O)CH3 at C-4), 2.54 (m, H-6β), 3.81 (d, J = 9.0 Hz, H-3), 4.14 (d, J = 8.5 Hz, H-20α), 4.29 (d, J = 8.5 Hz, H-20β), 4.42 (dd, J = 7.0, 10.5 Hz, H-7), 4.95 (d, J = 9.5 Hz, H-5), 5.64 (d, J = 6.0 Hz, H-2), 6.16 (t, J = 9.5 Hz, H-13), 6.29 (s, H-10), 7.46-8.05 (aromatic protons); 13 C NMR (125 MHz, CDCl3) δ: 204.03, 171.55, 170.41, 169.99, 167.24, 143.24, 133.99, 133.01, 130.29, 129.45, 128.91, 84.64, 81.31, 79.46, 76.64, 75.97, 75.23, 72.45, 69.960, 58.86, 46.04, 43.29, 35.95, 29.94, 26.93, 22.77, 21.74, 21.46, 21.10, 15.35, 9.73; HRMS (ESI-TOF) m/z 629.2570 [M + + H] ; calculated for C33H41O12: 629.2598. 3.2.6. Synthesis of 7-O-Acetyl-13-O-triethylsilylbaccatin III (41) To a stirred solution of 11 (100 mg, 159 µmol) in dry DMF (5 mL) at 50 °C under N2 were added triethylsilyl chloride (20 equiv) and imidazole (20 equiv). The reaction was monitored by TLC. Upon completion, the product was isolated by general work-up procedures, the organic solvent was evaporated, and the crude product was purified by silica gel flash column 179 chromatography (30:70 to 60:40 (v/v), linear gradient of EtOAc in hexane (115 mg, 98% yield, 1 1 99% purity by H NMR): H NMR (600 MHz, CDCl3) δ: 0.65 ( m, OSi(CH2CH3)3 at C13), 0.99 (t, J = 7.2 Hz, OSi(CH2CH3)3 at C13),1.11 (s, H-16), 1.23 (s, H-17), 1.76 (s, H-19), 2.00 (s, C(O)CH3 at C-7), 2.03 (s, H-18), 2.12 (m, H-6β), 2.14 (s, C(O)CH3 at C-4), 2.22 (m, H-14), 2.27 (s, C(O)CH3 at C-10), 2.58 (m, H-6α), 3.92 (d, J = 7.2 Hz, H-3), 4.11 (d, J = 8.4 Hz, H20α), 4.28 (d, J = 8.4 Hz, H-20β), 4.92 (t, J = 8.4 Hz, H-13), 4.95 (d, J = 9.6 Hz, H-5), 5.58 (dd, J = 7.2, 10.8 Hz, H-7), 5.60 (d, J = 7.8 Hz, H-2), 6.25 (s, H-10), 7.45-8.06 (aromatic protons) (cf. Figure 3.11 for proton numbering); 13 C NMR (125 MHz, CDCl3) δ: 202.53, 170.26, 169.67, 168.92, 167.03, 146.24, 133.63, 130.50, 130.03, 129.32, 128.59, 83.99, 80.49, 79.33, 76.26, 75.90, 74.88, 71.44, 68.41, 55.82, 47.12, 42.99, 39.94, 33.24, 26.34, 22.18, 21.13, 21.08, 20.76, + 15.07, 10.82, 6.89, 4.81; LC-ESIMS (positive ion mode), m/z 743.30 [M + H] , 765.30 [M + + Na] . 3.2.7. Synthesis of 7-O-Acetyl-2-O-debenzoyl-13-O-triethylsilylbaccatin III (43) To a stirred solution of 41 (71 mg, 96 µmol) in dry THF (5 mL) at 0 °C under N2 was added (dropwise) bis(2-methoxyethoxy)aluminum hydride (> 65 wt % in toluene, 3 equiv). After stirring for 2.5 h, the reaction was quenched by dropwise addition of saturated NH4Cl at 0 °C, and the mixture was stirred for 10 min, warmed to room temperature, and processed according to the general work-up procedures. The organic solvent was evaporated, and the crude product was purified by silica gel flash column chromatography (10:90 to 60:40 (v/v), linear gradient of 180 1 1 EtOAc in hexane (46 mg, 54% yield, 99% purity by H NMR); H NMR (600 MHz, CDCl3) δ: 0.63 ( m, OSi(CH2CH3)3 at C13), 0.98 (t, J = 7.8 Hz, OSi(CH2CH3)3 at C13), 1.00 (s, H-16), 1.15 (s, H-17), 1.73 (s, H-19), 1.81 (m, H-6β), 1.89 (dd, J = 8.4, 15.6 Hz, H-14), 1.97 (s, H-18), 1.99 (s, C(O)CH3 at C7), 2.07 (dd, J = 8.4, 15.6 Hz, H-14), 2.12 (s, C(O)CH3 at C4), 2.16 (s, C(O)CH3 at C10), 2.57 (m, H-6α), 3.53 (d, J = 6.6 Hz, H-3), 3.85 (d, J = 6.6 Hz, H-2), 4.58 (t, J = 9.6 Hz, H-20α), 4.59 (d, J = 9.6 Hz, H-20β), 4.94 (t, J = 8.4 Hz, H-13), 4.96 (d, J = 9.6 Hz, H5), 5.52 (dd, J = 3.8, 10.2 Hz, H-7), 6.18 (s, H-10) (cf. Figure 3.11 for proton numbering); 13 C NMR (125 MHz, CDCl3) δ: 203.03, 170.31, 169.33, 168.93, 145.82, 130.75, 83.59, 81.77, 78.51, 77.62, 75.99, 74.66, 71.63, 68.46, 55.82, 47.07, 42.48, 40.58, 33.36, 26.03, 22.21, 21.09, + 20.76, 15.04, 10.97, 6.91, 4.82; LC-ESIMS (positive ion mode), m/z 639.30 [M + H] , 661.30 + [M + Na] . 3.2.8. Synthesis of 7-O-Acetyl-2-O-debenzoylbaccatin III (37) 3.2.8.1. First Trial To a stirred solution of 43 (32 mg, 50 µmol) in dry THF (2 mL) and pyridine (1 ml) at 0 °C under N2 was added 0.5 ml HF/pyridine (70% as HF and 30% as pyridine). After stirring for 4 h, 0.3 ml of HF/pyridine (70% as HF and 30% as pyridine) was added. After following the reaction by TLC for 16 h, it was quenched by addition of EtOAc (10 ml) and H2O (5 ml), stirred for 10 min, and warmed to room temperature. The crude compound was dried under high vacuum, and the product was purified by silica gel flash column chromatography (30:70 to 100:00 (v/v), linear 181 gradient of EtOAc in hexane followed by a (5:95 to 10:90 (v/v), linear gradient of MeOH in 1 EtOAc (21 mg, 81% yield, 97% purity by H NMR). The purified compound (45) was subjected 1 to a complete spectral characterization; H NMR (600 MHz, CDCl3) δ: 1.01 (s, H-16), 1.11 (s, H-17), 1.31 (s, H-19), 1.87 (m, H-6β), 1.98 (s, C(O)CH3 at C7), 2.05 (s, H-18), 2.13 (s, C(O)CH3 at C10), 2.16 (s, C(O)CH3 at C4), 2.21 (m, H-6α), 2.28 (dd, J = 9.0, 15.0 Hz, H-14), 2.44 (dd, J = 6.6, 15.0 Hz, H-14), 3.51 (d, J = 7.2 Hz, H-3), 3.67 (d, J = 12.0 Hz, H-20α), 4.06 (d, J = 6.6 Hz, H-2), 4.32 (t, J = 9.0 Hz, H-5), 4.35 (d, J = 12.0 Hz, H-20β), 4.87 (t, J = 7.2 Hz, H-13), 5.11 (dd, J = 4.3, 12.6 Hz, H-7), 6.28 (s, H-10) (Figure B 9) (cf. Figure 3.11 for proton numbering); 13 C NMR (125 MHz, CDCl3) δ: 203.36, 172.62, 170.25, 169.02, 144.68, 134.41, 94.95, 85.12, 76.55, 76.26, 71.10, 71.06, 69.61, 68.14, 53.60, 51.27, 42.79, 38.68, 32.36, 25.43, 22.13, 20.75, + 20.73, 20.57, 16.44, 15.24 (Figure B 10); HRMS (ESI-TOF) m/z 525.2311 [M + H] ; calculated for C26H37O11: 525.2336. 3.2.8.2. Second Trial To a stirred solution of 43 (40 mg, 63 µmol) in dry THF (4 mL) at -78 °C under N2 was added (dropwise) tetrabutylammonium fluoride (TBAF) (1.0 M in THF, 2 equiv). The reaction was warmed to 4 °C and more TBAF (2 equiv) was added (dropwise). After following the reaction by TLC for 36 h (~ 90% conversion), the reaction was quenched by addition of EtOAc (10 ml) and H2O (5 ml), stirred for 10 min, and warmed to room temperature. The crude compound was dried under high vacuum, and the product was purified by silica gel flash column chromatography (30:70 to 100:00 (v/v), linear gradient of EtOAc in hexane (30 mg, 92% yield, 182 1 1 97% purity by H NMR); H NMR (600 MHz, CDCl3) δ: 1.00 (s, H-16), 1.23 (s, H-17), 1.74 (s, H-19), 1.82 (m, H-6β), 1.97 (m, H-14), 2.00 (s, C(O)CH3 at C7), 2.02 (s, H-18), 2.12 (s, C(O)CH3 at C4), 2.14 (s, C(O)CH3 at C10), 2.22 (dd, J = 10.2, 16.2 Hz, H-14), 2.43 (br.s, OH), 2.58 (m, H-6α), 3.60 (d, J = 7.2 Hz, H-3), 3.84 (br.d, J = 7.2 Hz, H-2), 4.57 (d, J = 10.2 Hz, H20α), 4.61 (d, J = 10.2 Hz, H-20β), 4.83 (t, J = 7.8 Hz, H-13), 4.95 (d, J = 9.0 Hz, H-5), 5.53 (dd, J = 7.2, 10.8 Hz, H-7), 6.18 (s, H-10) (Figure B 13) (cf. Figure 3.14 for proton numbering); 13 C NMR (500 MHz, CDCl3) δ: 202.90, 170.51, 170.41, 168.93, 144.22, 131.82, 83.64, 81.87, 77.91, 77.71, 75.96, 74.05, 71.78, 67.92, 56.16, 47.36, 42.30, 38.94, 33.47, 29.67, 22.51, 21.11, + 20.76, 19.99, 15.15, 10.84 (Figure B 14); HRMS (ESI-TOF) m/z 525.2357 [M + H] ; calculated for C26H37O11: 525.2336. 3.2.9. Synthesis of 7-O-Triethylsilylbaccatin III (40) To a stirred solution of baccatin III (2) (100 mg, 170 µmol) in dry DMF (5 mL) at 40 °C under N2 were added triethylsilyl chloride (5 equiv), and imidazole (4 equiv). The reaction was monitored by TLC. Upon completion, the product was isolated using general work-up procedures. Then the organic solvent was evaporated, and the crude product was purified by silica gel flash column chromatography (30:70 to 60:40 (v/v), linear gradient of EtOAc in hexane 1 1 ( 113 mg, 95% yield, 99% purity by H NMR); H NMR (300 MHz, CDCl3) δ: 0.54 ( q, J = 8.1 Hz, OSi(CH2CH3)3 at C7), 0.89 (t, J = 8.1 Hz, OSi(CH2CH3)3 at C7), 0.99 (s, H-16), 1.15 (s, H17), 1.64 (s, H-19), 1.83 (m, H-6β), 2.14 (s, H-18), 2.14 (s, C(O)CH3 at C4), 2.21 (m, H-14), 2.24 (s, C(O)CH3 at C10), 2.48 (m, H-6α), 3.84 (d, J = 7.2 Hz, H-3), 4.10 (d, J = 9.0 Hz, H-20α), 183 4.26 (d, J = 9.0 Hz, H-20β), 4.45 (dd, J = 6.6, 10.5 Hz, H-7), 4.78 (t, J = 7.8 Hz, H-13), 4.92 (d, J = 8.4 Hz, H-5), 5.59 (d, J = 6.9 Hz, H-2), 6.42 (s, H-10) (cf. Figure 3.11 for proton numbering), 7.43 – 8.06 (aromatic protons); 13 C NMR (125 MHz, CDCl3) δ: 202.28, 170.64, 169.34, 167.02, 144.11, 133.54, 132.50, 130.02, 129.35, 128.53, 84.17, 80.74, 78.65, 76.45, 75.76, 74.71, 72.29, 67.78, 58.58, 47.20, 42.70, 38.30, 37.16, 26.72, 22.57, 20.87, 20.05, 14.87, + 9.88, 6.68, 5.21; HRMS (ESI-TOF) m/z 701.3334 [M + H] ; calculated for C37H53O11Si: 701.3357. 3.2.10. Synthesis of 13-O-Acetyl-7-O-triethylsilylbaccatin III (42) To a stirred solution of 40 (100 mg, 143 µmol) in dry THF (5 mL) at 23 °C under N2 were added acetic anhydride (20 equiv), dimethylaminopyridine (20 equiv), and triethylamine (48 μL, 86 μmol). The reaction was monitored by TLC, and upon completion, it was processed using general work-up procedures. The organic solvent was evaporated, and the crude product was purified by silica gel flash column chromatography (30:70 to 60:40 (v/v), linear gradient of 1 1 EtOAc in hexane ( 105 mg, 98% yield, 99% purity by H NMR); H NMR (300 MHz, CDCl3) δ: 0.54 ( q, J = 7.8 Hz, OSi(CH2CH3)3 at C7), 0.88 (t, J = 7.8 Hz, OSi(CH2CH3)3 at C7), 1.13 (s, H-16), 1.18 (s, H-17), 1.64 (s, H-19), 1.85 (m, H-6β), 2.00 (s, H-18), 2.14 (s, C(O)CH3 at C4), 2.16 (s, C(O)CH3 at C13), 2.21 (m, H-14), 2.29 (s, C(O)CH3 at C10), 2.47 (m, H-6α), 3.79 (d, J = 7.2 Hz, H-3), 4.11 (d, J = 8.4 Hz, H-20α), 4.26 (d, J = 8.4 Hz, H-20β), 4.44 (dd, J = 6.6, 10.5 Hz, H-7), 4.91 (d, J = 9.6 Hz, H-5), 5.63 (d, J = 7.5 Hz, H-2), 6.11 (t, J = 8.7 Hz, H-13), 6.42 (s, H-10), 7.43 – 8.03 (aromatic protons) (cf. Figure 3.11 for proton numbering); 184 13 C NMR (125 MHz, CDCl3) δ: 201.81, 170.15, 169.69, 169.20, 166.98, 140.64, 133.63, 133.51, 130.01, 129.27, 128.58, 84.13, 81.06, 78.84, 76.45, 75.15, 74.79, 72.24, 69.65, 58.49, 46.92, 43.09, 37.18, 35.45, 26.46, 22.54, 21.20, 20.83, 20.68, 14.49, 9.97, 6.70, 5.24; LC-ESIMS (positive ion + + mode), m/z 743.30 [M + H] , 765.30 [M + Na] . 3.2.11. Synthesis of 13-O-Acetyl-2-O-debenzoyl-7-O-triethylsilylbaccatin III (44) ° To a stirred solution of 42 (54 mg, 72 µmol) in dry THF (5 mL) at 0 C under N2 was added (dropwise) bis(2-methoxyethoxy)aluminum hydride (> 65 wt % in toluene, 3 equiv). After stirring for 3 h, the reaction was quenched by dropwise addition of saturated NH4Cl at 0 ºC, and the mixture was stirred for 10 min, warmed to room temperature, and processed following the general work-up procedures. The organic solvent was evaporated, and the crude product was purified by silica gel flash column chromatography (10:90 to 60:40 (v/v), linear gradient of 1 1 EtOAc in hexane (30 mg, 65% yield, 99% purity by H NMR); H NMR (300 MHz, CDCl3) δ: 0.53 ( q, J = 8.1 Hz, OSi(CH2CH3)3 at C7), 0.88 (t, J = 8.1 Hz, OSi(CH2CH3)3 at C7), 1.07 (s, H-16), 1.16 (s, H-17), 1.62 (s, H-19), 1.87 (m, H-6β), 1.95 (s, H-18), 2.12 (s, C(O)CH3 at C4), 2.13 (s, C(O)CH3 at C13), 2.14 (s, C(O)CH3 at C10), 2.41 (br s, OH), 2.47 (m, H-6α), 2.61 (d, J = 5.1 Hz, H-14), 3.43 (d, J = 6.3 Hz, H-3), 3.89 (dt, J = 6.3, 8.1 Hz, H-2), 4.39 (dd, J = 6.6, 10.8 Hz, H-7), 4.56 (t, J = 9.3 Hz, H-20α), 4.59 (d, J = 9.3 Hz, H-20β), 4.92 (d, J = 9.3 Hz, H-5), 6.10 (t, J = 8.7 Hz, H-13), 6.35 (s, H-10) (cf. Figure 3.11 for proton numbering); 13 C NMR (125 MHz, CDCl3) δ: 202.43, 170.22, 169.54, 169.22, 140.26, 133.57, 83.86, 82.18, 78.34, 77.90, 185 75.20, 74.24, 72.27, 69.78, 58.48, 46.76, 42.61, 37.27, 35.88, 26.14, 25.01, 22.59, 21.19, 20.62, + 14.49, 10.20, 6.73, 5.22; LC-ESIMS (positive ion mode), m/z 639.30 [M + H] , 661.30 [M + + Na] . 3.2.12. Synthesis of 13-O-Acetyl-2-O-debenzoylbaccatin III (38) 3.2.12.1. First Trial ° To a stirred solution of 44 (26 mg, 40 µmol) in dry THF (2 mL) and pyridine (1 ml) at 0 C under N2 was added 0.5 ml HF/pyridine (70% as HF and 30% as pyridine). After following the reaction by TLC for 3 h, it was quenched by addition of EtOAc (10 ml) and H2O (5 ml), stirred for 10 min, and warmed to room temperature. The crude compound was dried under high vacuum, and the product was purified by silica gel flash column chromatography (30:70 to 100:00 (v/v), linear gradient of EtOAc in hexane followed by a (5:95 to 10:90 (v/v), linear 1 1 gradient of MeOH in EtOAc (21 mg, 99% yield, 96% purity by H NMR); H NMR (600 MHz, CDCl3) δ: 1.04 (s, H-16), 1.25 (s, H-17), 1.27 (s, H-19), 1.73 (m, H-6β), 1.83 (s, H-18), 2.07 (s, C(O)CH3 at C13), 2.16 (s, C(O)CH3 at C10), 2.18 (s, C(O)CH3 at C4), 2.21 (dd, J = 8.4, 15.6 Hz, H-14), 2.28 (m, H-6α), 2.45 (dd, J = 8.4, 15.6 Hz, H-14), 3.29 (d, J = 7.2 Hz, H-3), 3.66 (d, J = 12.0 Hz, H-20α), 3.78 (dd, J = 4.2, 12.6 Hz, H-7), 4.13 (d, J = 6.6 Hz, H-2), 4.24 (t, J = 9.6 Hz, H-5), 4.34 (d, J = 12.0 Hz, H-20β), 6.16 (t, J = 10.2 Hz, H-13), 6.20 (s, H-10) (Figure B 11) (cf. Figure 3.11 for proton numbering); 13 C NMR (125 MHz, CDCl3) δ: 206.67, 171.99, 170.13, 170.03, 141.54, 135.58, 95.30, 85.78, 76.60, 76.06, 71.14, 71.03, 70.18, 69.78, 55.12, 49.96, 186 43.09, 35.54, 34.06, 25.03, 22.14, 21.51, 20.98, 20.74, 15.66, 15.01 (Figure B 12); HRMS (ESI+ TOF) m/z 525.2359 [M + H] ; calculated for C26H37O11: 525.2336. 3.2.12.2. Second Trial To a stirred solution of 44 (30 mg, 47 µmol) in dry THF (4 mL) at -78 °C under N2 was added (dropwise) tetrabutylammonium fluoride (TBAF) (1.0 M in THF, 2 equiv). The reaction was warmed to 4 °C and added (dropwise) more TBAF (3 equiv). After following the reaction by TLC for 32 h (~ 80% conversion), the reaction was quenched by addition of EtOAc (10 ml) and H2O (5 ml), stirred for 10 min, warmed to room temperature. The crude compound was dried under high vacuum, and the product was purified by silica gel flash column chromatography 1 (30:70 to 100:00 (v/v), linear gradient of EtOAc in hexane (21 mg, 87% yield, 98% purity by H 1 NMR); H NMR (600 MHz, CDCl3) δ: 1.01 (s, H-16), 1.24 (s, H-17), 1.62 (s, H-19), 1.83 (s, H18), 1.88 (m, H-6β), 2.00 (dd, J = 7.8, 15.0 Hz, H-14), 2.13 (s, C(O)CH3 at C4), 2.17 (s, C(O)CH3 at C13), 2.20 (s, C(O)CH3 at C10), 2.39 (br.s, OH), 2.46 (dd, J = 8.4, 15.0 Hz, H-14), 2.53 (m, H-6α), 3.43 (d, J = 7.8 Hz, H-3), 3.90 (dd, J = 5.2, 8.4 Hz, H-2), 4.37 (dd, J = 7.2, 10.2 Hz, H-7), 4.57 (d, J = 9.6 Hz, H-20α), 4.62 (d, J = 9.6 Hz, H-20β), 4.96 (d, J = 9.6 Hz, H-5), 6.15 (t, J = 7.8 Hz, H-13), 6.21 (s, H-10) (Figure B 15) (cf. Figure 3.14 for proton numbering); 13 C NMR (125 MHz, CDCl3) δ: 204.33, 171.22, 170.13, 169.56, 142.58, 132.90, 84.04, 82.18, 78.70, 77.82, 75.76, 74.67, 72.24, 69.82, 58.57, 45.59, 42.54, 36.16, 35.62, 26.34, 22.50, 21.43, + 21.12, 20.84, 15.04, 9.68 (Figure B 16); HRMS (ESI-TOF) m/z 525.2360 [M + H] ; calculated for C26H37O11: 525.2336. 187 3.2.13. Synthesis of 2-O-Debenzoyl-7-O-triethylsilylbaccatin III (47) To a stirred solution of 42 (24 mg, 32 µmol) in dry THF (5 mL) at 0 °C under N2 was added (dropwise) bis(2-methoxyethoxy)aluminum hydride (> 65 wt % in toluene, 5 equiv). After stirring for 3 h, the reaction was quenched by dropwise addition of saturated NH4Cl at 0 ºC, and the mixture was stirred for 10 min, warmed to room temperature, and processed following the general work-up procedures. The organic solvent was evaporated, and the crude product was purified by silica gel flash column chromatography (10:90 to 60:40 (v/v), linear gradient of 1 1 EtOAc in hexane (11 mg, 60% yield, 99% purity by H NMR); H NMR (600 MHz, CDCl3) δ: 0.54 ( m, OSi(CH2CH3)3 at C7), 0.89 (t, J = 7.8 Hz, OSi(CH2CH3)3 at C7), 1.04 (s, H-16), 1.05 (s, H-17), 1.62 (s, H-19), 1.98 (m, H-6β), 2.01 (s, H-18), 2.10 (s, C(O)CH3 at C4), 2.13 (s, C(O)CH3 at C10), 2.40 (br.s, H-14), 2.48 (m, H-6α), 2.52 (d, J = 6.0 Hz, OH), 3.47 (d, J = 6.6 Hz, H-3), 3.85 (dd, J = 6.6, 7.8 Hz, H-2), 4.40 (dd, J = 6.6, 10.8 Hz, H-7), 4.54 (d, J = 8.4 Hz, H-20α), 4.60 (d, J = 8.4 Hz, H-20β), 4.80 (br.m, H-13), 4.93 (d, J = 9.6 Hz, H-5), 6.35 (s, H-10) (cf. Figure 3.14 for proton numbering); 13 C NMR (125 MHz, CDCl3) δ: 202.81, 170.40, 169.34, 143.59, 132.82, 83.85, 82.05, 77.92, 77.87, 75.86, 74.30, 72.42, 67.90, 58.66, 47.15, 42.25, 38.80, 37.32, 26.57, 22.60, 20.88, 20.00, 14.87, 10.09, 6.70, 5.24; LC-ESIMS (positive ion + + mode), m/z 597.40 [M + H] , 619.40 [M + Na] . 3.2.14. Synthesis of 2-O-Debenzoylbaccatin III (39) To a stirred solution of 47 (22 mg, 37 µmol) in dry THF (4 mL) at -78 °C under N2 was added (dropwise) tetrabutylammonium fluoride (TBAF) (1.0 M in THF, 2 equiv). The reaction was 188 warmed to 4 °C and added (dropwise) more TBAF (3 equiv). After following the reaction by TLC for 32 h (~ 80% conversion), the reaction was quenched by addition of EtOAc (10 ml) and H2O (5 ml), stirred for 10 min, warmed to room temperature. The crude compound was dried under high vacuum, and the product was purified by silica gel flash column chromatography 1 (30:70 to 100:00 (v/v), linear gradient of EtOAc in hexane (15 mg, 84% yield, 97% purity by H 1 NMR); H NMR (600 MHz, CDCl3) δ: 0.98 (s, H-16), 1.23 (s, H-17), 1.62 (s, H-19), 1.87 (m, H-6β), 1.97 (s, H-18), 2.14 (s, C(O)CH3 at C4), 2.17 (dd, J = 6.6, 12.6 Hz, H-14), 2.20 (s, C(O)CH3 at C10), 2.40 (br.s, OH), 2.47 (d, J = 6.6 Hz, H-14), 2.54 (m, H-6α), 3.47 (d, J = 7.2 Hz, H-3), 3.86 (dd, J = 6.6, 7.2 Hz, H-2), 4.38 (m, H-7), 4.57 (d, J = 9.0 Hz, H-20α), 4.62 (d, J = 9.0 Hz, H-20β), 4.88 (t, J = 9.0 Hz, H-13), 4.97 (d, J = 9.0 Hz, H-5), 6.23 (s, H-10) (Figure B 17) (cf. Figure 3.14 for proton numbering); 13 C NMR (125 MHz, CDCl3) δ: 204.63, 171.24, 170.30, 145.88, 132.14, 84.01, 82.06, 78.28, 77.77, 76.25, 74.77, 72.42, 67.99, 58.71, 46.01, 42.17, 39.15, 35.70, 29.69, 22.52, 20.88, 20.82, 15.52, 9.56 (Figure B 18); HRMS (ESI-TOF) + m/z 483.2243 [M + H] ; calculated for C24H35O10: 483.2230. 3.2.15. Characterization of the mTBT-Catalyzed Deacylation Purified mTBT (50 μg) was incubated in assay buffer with 1 mM 7,13-O,O-diacetylbaccatin III (8), and 1 mM CoASH (35). After 3 h at 31 °C, the assay was quenched and extracted with EtOAc (2 × 4 mL). The organic fractions were evaporated in vacuo, and the resultant residue was dissolved in MeCN (200 μL). A 10-μL aliquot of the sample was loaded onto a reversed-phase column (Betasil C18, 5 μm, 150 × 2.1 mm, Thermo Fisher Scientific Inc., Waltham, MA) on a 189 capillary HPLC system (CapLC capillary HPLC, Waters, Milford, MA). The column was eluted at 0.3 mL/min with a linear gradient of solvent A: solvent B [solvent A: 99.50% H2O with 0.5% H3PO4 (v/v); solvent B: 99.50% MeCN with 0.5% H3PO4 (v/v)] from 70:30 (v/v) to 0:100 (v/v) over 7 min, holding at 100% solvent B for 2 min, and returning to the initial conditions over 1.10 min, with a 50 s hold. The effluent from the HPLC column was directed to an ESI-MS/MS (Q+ + ToF Ultima Global, Waters, Milford, MA), and the molecular ions ([M + H] and [M + Na] ) of the de novo biosynthetic 2-O-debenzoyltaxane analyte were detected in single-stage and tandem mass spectrometer modes (Figure 3.6). The mass spectral data of biosynthetic 2-Odebenzoylated product was shown to be identical to the exemplary authentic standards (Figure B 1). Control assays in which one co-substrate and/or enzyme was omitted, and a control assay with only 8 in assay buffer were processed and analyzed identically to the methods described above. 3.2.16. Kinetic Evaluation of mTBT Incubated with 7,13-O,O-Diacetylbaccatin III and CoASH Linearity of the rate of the mTBT catalyzed reaction with respect to protein concentration and time was established with 7,13-O,O-diacetylbaccatin III (8) at 50 μM, while co-substrate 35 was maintained at apparent saturation (1 mM) in 10 mL of assay buffer. Aliquots (1 mL) were collected, and the biosynthetic reaction was stopped by the addition of 500 μL of EtOAc at 5, 10, 20, 30, and 40 min and at 1, 2, 3, 4, and 5 h. Compound 2 (5 μg) was added as the internal standard to correct for the loss of analyte during the extraction of product with organic solvent. Each sample was extracted with EtOAc (2 × 4 mL), and the organic fractions were combined and 190 then evaporated in vacuo. The resultant residue was dissolved in 200 μL of MeCN, and a 10-μL aliquot of the sample was subjected to electrospray ionization. The peak areas of the selected + + molecular ions [M + H] and [M + Na] derived by electrospray ionization of the biosynthetic product that was directed to and detected by the first stage mass spectrometer were converted to + + concentration by comparison to the peak areas of the [M + H] and [M + Na] ions generated by authentic 7,13-O,O-diacetylbaccatin III (8) ranging from 0 to 5 μM at 1.2 μM intervals. Samples containing high concentrations of biosynthetic product in the assays were diluted 10- to 20-fold so that the peak areas fell within the linear range of the MS detector. Evaluation of the steadystate parameters was determined by incubating 30 μg of purified mTBT protein in 1 mL assays for 5 min. The concentration of 8 was independently varied from 0 to 1000 μM in separate assays, while 35 was maintained at apparent saturation (1 mM). The initial velocity (vo) was plotted against the co-substrate concentration, and the equation of the best-fit nonlinear 2 regression curve (R > 0.99) was determined (Microsoft Excel 2003, Microsoft Corp., Redmond, WA) to calculate kcat and KM. 3.2.17. Identification of Putative Taxane Co-substrates for the mTBT Deacylation To identify productive taxane co-substrates in pilot assays, 50 μg of purified mTBT in 1 mL of assay buffer, 35, and taxane co-substrate (all the taxane substrates shown in Figure 3.2), each at 1 mM, were incubated in separate assay mixtures. After 3 h at 31 °C, the assays were quenched and extracted with EtOAc (2 × 4 mL). The organic fractions were evaporated in vacuo, and the resultant residue was dissolved in 200 μL of MeCN. A 10 μL aliquot was subjected to 191 + + electrospray ionization, and the molecular ions ([M + H] and [M + Na] ) of the de novo biosynthetic 2-O-debenzoyltaxane analytes were detected in single-stage and tandem mass spectrometer modes (Figure 3.7, Figure 3.8, Figure B 5, and Figure B 6). The mass spectral data of biosynthetic 2-O-debenzoylated samples were shown to be identical to the exemplary authentic standards (Figure B 1 and Figure B 2). Control assays in which one co-substrate and/or enzyme was omitted, and control assays with each of the taxane co-substrate in assay buffer were processed and analyzed identically to the methods described above. 3.2.18. Identification of Putative Taxane Co-substrates for the C2- Benzoylation Reaction Catalyzed by mTBT To identify productive taxane co-substrates in pilot assays, 50 μg of purified mTBT in 1 mL of assay buffer, benzoyl CoA, and taxane co-substrate (all the taxane substrates shown in (Figure 3.10), each at 1 mM, were incubated in a mixture. After 3 h at 31 °C, the assays were quenched and extracted with EtOAc (2 × 4 mL). The organic fractions were evaporated in vacuo, and the resultant residue was dissolved in 200 μL of MeCN. A 10 μL aliquot was subjected to + + electrospray ionization, and the molecular ions ([M + H] and [M + Na] ) of the de novo biosynthetic 2-O-benzoylated taxane analytes were detected in single-stage and tandem mass spectrometer modes (Figure 3.15, Figure 3.16, Figure B 7, and Figure B 8). The mass spectral data of biosynthetic 2-O-benzoylated samples were shown to be identical to the exemplary authentic standards (Figure B 3 and Figure B 4). Control assays in which one co-substrate and/or enzyme was omitted were processed and analyzed identically to the methods described above. 192 3.3. Results and Discussion 3.3.1. Characterization and the Kinetic Evaluation of the mTBT Deacylation A detailed study of regiospecific acylation and deacylation of Taxus acyltransferases is essential to understand the potential application of Taxus acyltransferases to produce 6 or its analogues. In chapter 2, the expansive scope of productive acyl CoA thioester substrates in the mTBT reaction 26 as well as the potential biocatalytic application of the catalyst were discussed in detail. mTBT can catalyze the regiospecific transfer of the benzoyl/acyl moiety from the corresponding CoA thioester to the C2 hydroxyl group of 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III (36) to produce 7,13-O,O-diacetylbaccatin III (8) or 7,13-O,O-diacetyl-2-O-acyl-2-O- debenzoylbaccatin III as demonstrated in the previous chapter. In the previous study, evaluating 3 the substrate specificity of mTBT, only either [ H]-labeled or unlabeled 36 were used as the 26 taxane co-substrates, due to synthetic challenges encountered while attempting to acquire 51-58 various 2-O-debenzoylated taxoids by established procedures. Comparatively, the synthesis of variously 2-O-aroylated taxoids is less challenging and, more importantly, some of the 2-Obenzoylated taxoids are commercially available (Figure 3.2). However, the enzymatic deacylation of 2-O-benzoylated taxoids by mTBT has not been investigated to date. Therefore, purified mTBT was incubated with CoASH and 2-O-benzoylated taxane co-substrate 8, each at 1 mM, and the product(s) were analyzed (Figure 3.5). A de novo biosynthetic product formation was detected by LC-MS and verified by tandem mass spectroscopy by identifying the diagnostic + + fragment ions m/z: 429.2 [m/z 567.3 – 2HOAc –H2O] , 387.2 [m/z 567.3 – 3HOAc] , 327.2 [m/z 387.2 – HOAc]+, 309.2 [m/z 429.2 – 2HOAc]; [M + H] is 567.3 (Figure 3.6). The detected 193 biosynthetic product showed similar characteristics to that of authentic 36 (Figure B 1) demonstrating the mTBT-catalyzed deacylation of the 2-O-benzoyl group (Figure 3.5). None of the control assays showed any analogous product, confirming that the 2-O-debenzoylation was enzymatic and not a result of a chemical degradation under the assay conditions used. Also, no other mTBT catalyzed deacylated products were observed in addition to 8, when screened by LC-MS/MS for all combinations of singly and multiply-deacylated products. Therefore, mTBTcatalyzed deacylation was confirmed to be highly chemoselective for 2-O-debenzoylation. A set of steady-state kinetic parameters for mTBT-catalyzed deacylation were determined by incubating 30 µg of purified mTBT with varying concentration of 35 in separate assays, while 8 was maintained at apparent saturation. The ion abundance of biosynthetic product (36) formed in each assay was converted to analyte concentration by a linear regression equation that + + correlated the peak areas under the signals generated for selected ions [M + H] and [M + Na] detected by the mass spectrometer with a series of standards at various concentrations. The -1 specificity constant of mTBT-catalyzed deacylation was calculated to be 0.15 min mM -1 for 35 by plotting the initial velocity (vo) calculated from above data against the concentration of 35. 194 O O O CoASH O O O O O O O HO H OH O 35 O O O HO H O O O O O O O O 7,13-O,O-diacetyl-2-O7,13-O,O-diacetylbaccatin III debenzoylbaccatin III (36) (8) Figure 3.5 – The characterization of mTBT-catalyzed deacylation. Shown are the steady-state kinetic parameters of mTBT catalysis for the CoASH co-substrate (35) 195 309.2 % Relative Abundance 100 80 60 327.2 40 387.2 429.2 20 0 100 200 300 400 500 600 m/z Figure 3.6 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetyl-2-Odebenzoylbaccatin III (36) derived by mTBT-catalyzed deacylation is shown. Diagnostic ions + + were found as m/z: 429.2 [m/z 567.3 – 2HOAc –H2O] , 387.2 [m/z 567.3 – 3HOAc] , 327.2 [m/z 387.2 – HOAc]+, 309.2 [m/z 429.2 – 2HOAc]; [M + H] is 567.3. The MS/MS fragment ion profile of the authentic 36 is shown in Error! Reference source not found. 3.3.2. Assessing the Productive Taxanes in the Deacylating Reaction Catalyzed by mTBT Pilot studies were conducted to determine the array of productive taxane co-substrates (obtained from semi-synthesis as described in 3.2 or commercial sources) that could be productively hydrolyzed by mTBT (Figure 3.2) After incubating 35 and a single taxane co-substrate each at 1 mM concentration in separate assays with purified mTBT, EtOAc was added to the assays to partition any 2-O-debenzoylated products into the organic phase. The extracted products were 196 subjected to LC-MS to detect any de novo product formation by selecting for the corresponding + + + [M + H] and [M + Na] ions of the plausible products. The [M + H] ion of the product was further subjected to MS/MS analysis. Typical diagnostic fragment ions of 2-O-debenzoylated taxoids were evaluated using authentic 36 and 7-O-acetyl-2-O-debenzoylbaccatin III (37) as a model to identify the various ion fragmentation patterns (Figure B 1 and Figure B 2). 7-Oacetylbaccatin III (11) and 7-O-acetyl-13-oxobaccatin III (13) co-substrates formed 7-O-acetyl2-O-debenzoylbaccatin III and 7-O-acetyl-2-O-debenzoyl-13-oxobaccatin III respectively (Figure 3.7 and Figure 3.8), while no deacylated product was detected in assays containing the other taxoids. Typical diagnostic fragment ions of 2-O-debenzoylated taxane analogues + + + possessed m/z of [M + H – HOAc – H2O] , [M + H – HOAc – 2H2O] , [M + H– 2HOAc] , [M + + + + H– 2HOAc – H2O ] , [M + H– 2HOAc – 2H2O ] , [M + H – 3HOAc] , [M + H – 3HOAc – + + H2O] , and [M + H – 3HOAc – 2H2O] (Figure B 5 and Figure B 6). The control assays did not show detectable products derived by chemical hydrolysis of the taxoids. 197 525.3 100 % Relative Abundance 80 60 40 465.2 547.3 20 387.2 0 0 100 200 300 400 500 600 m/z Figure 3.7 – LC-MS ion profile of the biosynthesized 7-O-acetyl-2-O-debenzoylbaccatin III (37) + derived by mTBT deacylation is shown. Diagnostic ions were found as m/z: 547.2 [M + Na] , + + 525.2 [M + H] , 465.2 [m/z 525.2 –HOAc] , 387.2 [m/z 465.2 – HOAc – H2O]+. The MS/MS fragment ion profiles of the authentic and the biosynthesized 37 are shown in Figure B 2 and Figure B 5 respectively 198 523.3 100 % Relative Abundance 80 60 40 20 545.3 385.2 445.2 0 0 100 200 300 400 500 600 m/z Figure 3.8 – LC-MS ion profile of the biosynthesized 7-O-acetyl-2-O-debenzoyl-13-oxobaccatin + III derived by mTBT deacylation is shown. Diagnostic ions were found as m/z: 545.2 [M + Na] , + + 523.2 [M + H] , 445.2 [m/z 523.2 –HOAc - H2O] , 385.2 [m/z 445.2 – HOAc]+. The MS/MS fragment ion profile of the biosynthesized 13 is shown in Figure B 6 From all the tested taxane co-substrates, only 8, 11, and 13 (Figure 3.9) were found to form any detectable products in the mTBT-catalyzed deacylating reaction. Comparison of these productive substrates to the other taxanes tested showed that an acetoxy group is present at C-7 in all the productive substrates, while the non-productive substrates had a hydroxyl group at this position (Figure 3.9). This observation suggested that the acetoxy group at C-7 may play a role in the mTBT catalyzed 2-O-debenzoylation while the hydroxyl group at C-7, by contrast, may hinder 199 the same reaction. Up until now, the 2-O-debenzoylation reaction catalyzed by mTBT has served as a diagnostic tool to predict productive 2-hydroxytaxane substrates for mTBT in the forward direction, catalyzing the 2-O-benzoylation. The precision of using this diagnostic tool was further evaluated by analyzing the structural characteristics of 36, the only taxane substrate tested for the mTBT acylation and found to be productive. Consequently, 36 does contain acetoxy group at C-7 further suggesting that the acetoxy group at C-7 may play a role in the mTBT catalysis while the hydroxyl group at C-7, by contrast, may hinder the catalysis. Thus, to further test whether varying functional group at C-7 affects the 2-O-benzoylation reaction of mTBT, a select few of synthetically derived 2-O-debenzoylated taxane substrates were examined (Figure 3.10). In Addition, the functional group at C-13 was also varied (either an acetyl or hydroxyl) to test whether this exchange affected mTBT catalysis. 200 AcO O OAc 7 O 8 HO 11 AcO O OH O H OBz OAc 13 7 O H OBz OH R: H - 9 and R: Ac - 10 RO R: H - 1 and R: Ac - 2 HO 12 R1: Me; R2: Ph - 6 and R1: H; R2: OC(CH3)3 - 14 Figure 3.9 – Several identified productive (8, 11, 13) and non-productive (1, 2, 6, 9, 10, 12, 14) taxane substrates when tested for the substrate specificity of mTBT deacylation. All the productive taxane substrates have O-acetyl group at C7 (shown in red) while all the nonproductive substrates have hydroxy- group at C7 (shown in blue) 7-O-Acetyl-2-O-debenzoylbaccatin III (37) R1: Ac; R2: H 13-O-Acetyl-2-O-debenzoylbaccatin III (38) R1: H; R2: Ac 7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III (36) R1: Ac; R2: Ac 2-O-Debenzoylbaccatin III (39) R1: H; R2: H Figure 3.10 – The proposed 2-O-debenzoylated taxoids to test the effect of functional group at C7 on mTBT 2-O-benzoylation reaction. The functionality at C13 is also varied to see any effects on the mTBT catalysis 201 3.3.3. Synthesis of 2-O-Debenzoylated Taxoids and the Identification of the Taxane Co-substrate Scope of Benzoylation at C-2 Catalyzed by mTBT As mentioned previously, the synthesis of 2-O-debenzoylated taxoids are highly challenging due to intramolecular rearrangements involving the liberated hydroxyl at C-2 and the oxetane ring 51-58 under various reaction conditions during the debenzoylation step. (Figure 3.10) used to test the function of mTBT 19,26 The default substrate 36 was derived in a straightforward synthesis from 2 (Figure 3.1), thus, 2 was also used to synthesize modified taxoids 37, 38, 39 (Figure 3.10). The first attempt to synthesize these 2-O-debenzoylated taxoids (Figure 3.11) in parallel resulted in products with exact masses as determined by HRMS analysis identical to those 1 calculated for the desired products. Upon H-NMR analysis, however, the spectra revealed that the expected chemical shifts at C-2, C-5, and C-20 (Figure 3.10 for oxetane taxoid numbering) were inconsistent with a structurally similar grouping found in the authentic 2-O-debenzoylated analogue, 36. In particular, the different proton chemical shifts of the H-2, H-5, and H-20 signals and the increased geminal coupling constants of the H-20 protons suggested that the oxetane ring was no longer intact (Table 3.1). This skeletal rearrangement was confirmed by complete 1 spectral characterization ( H- and 13 C-NMR and 2D-NMR HMQC, HMBC techniques). Considering only the proton chemical shifts of H-2 (δ: 3.87 ppm), H-5 (δ: 4.95 ppm), H-20α (δ: 4.57 ppm), and H-20β (δ: 4.59 ppm) that are contained in or proximate to the oxetane functional grouping of 36, it was found that the analogously positioned protons of the synthetically-derived 2-O-debenzoylated product (45) were at δ: 4.06, 4.32, 3.67, and 4.35 for H-2, H-5, H-20α and H20β, respectively. Moreover, the geminal coupling constant for the H-20 proton of 36 was J = 10.8 Hz, while at J = 12.0 Hz for the analogous protons in the product isolated in this synthesis 202 of 45. The HMQC data (Figure B 19 to Figure B 21) further supported that the oxetane ring was rearranged to a cyclic ether at C-2 while the ether bond at C-5 was cleaved. This was clearly shown by the relative downfield shift of the C-2 signal in the 13 C-NMR spectra due to the deshielding effects caused by the formation of the cyclic ether bond. Also, an upfield shift of the C-5 signal was observed in the 13 C-NMR spectra, likely due to the cleavage of the oxetane ether bond. (Table 3.2). In general, the characteristics of a rearranged oxetane ring were also observed for the product 46, synthesized in parallel with 45. Briefly, protons H-2 (δ: 4.13 ppm), H-5 (δ: 4.24 ppm), H-20α (δ: 3.66 ppm), and H-20β (δ: 4.34 ppm) of the synthetically-derived 2-Odebenzoylated product (46) showed the same trend in its 1H-NMR spectrum as compound 45, with J = 12.0 Hz for the H-20 geminal coupling constant. As mentioned, oxetane ring opening can occur in the presence of either Lewis acid or base (electrophilic or nucleophilic, respectively) reagents, by facilitating neighboring group effects, particularly when attempting to remove the benzoyl group at C-2 or a silyl protecting group from 51-58 2-O-debenzoyl taxoids. The hydroxyl group at C-2 is known to intramolecularly attack the C-20 of the oxetane ring in the presence of Lewis acids or bases forming rearranged 51,57,58 taxoids Analogously, when HF-pyridine is used alternatively in the final deprotection step of the synthesis to access 2-O-debenzoyltaxanes, described herein, the protonation of the oxygen atom of the oxetane ring likely promoted ring opening with plausible nucleophilic attack by the hydroxyl group at C-2 (Figure 3.12). The absolute stereochemistry at C-2 and C-5 of both 45 and 46 remained unchanged during the rearrangement according to the proposed mechanism. The molecular topology of 45 was very similar to that of expected product, 37 as elaborated using molecular models of each in Figure 3.13 despite the formation of the furan ring in between 203 C-2α and C-4β. Analogously, the created molecular models of 46 and 38 were compared and identified to be very similar. Also, the absolute structural configurations were further verified by comparing the various NMR spectra of 45 and 46 to the other reported isobaccatin III 51,57,58 derivatives. In the next attempt, 2-O-debenzoylated taxoids 37, 38, and 39 were successfully synthesized, when TBAF was used in place of HF/pyridine as the silyl group deprotection agent in the last step (Figure 3.14). TBAF presents an aprotic and bulkier alkyl ammonium counter ion of fluoride that likely precludes coordination with the oxetane ring (Figure 3.12); therefore, preventing structural rearrangements, as substantiated by HMQC data (cf. Figure B 22 to Error! Reference source not found.). It is worth noting that during the synthesis of 44, the debenzoylating reagent (Red-Al) was used in slight excess, which led to the removal of acyl groups at both C-2 and C-13, forming 47. Compound 47 was then conveniently converted to 2O-debenzoylated taxoid 39. After a complete spectral characterization of synthesized 2-Odebenzoylated taxoids (Table 3.3 and Table 3.4), each (at 1 mM) was incubated with purified mTBT (50 µg) and benzoyl CoA (1 mM) to evaluate the scope of taxanes that mTBT could benzoylate at the C-2 hydroxyl. 204 O O O O OH O 7 HO 13 HO H O O O 2 O OR1 7 a O O R2O R1: Ac (11), R1: TES (40) 13 HO H O O O O O R1: Ac; R2: TES (41) R1: TES; R2: Ac (42) R1: Ac; R2: TES (43), R1: TES; R2: Ac (44) R1: Ac; R2: H (45), R1: H; R2: Ac (46) Figure 3.11 – First attempt of synthesis of 2-O-debenzoylated taxoids, 37, 38 a Ac2O, 4-DMAP, Et3N, 97% yield for 11; TESCl, imidazole, 40 °C, 95% yield for 40 b c d TESCl, imidazole, 50 °C, 98% yield for 41; Ac2O, 4-DMAP, Et3N, 98% yield for 42 Red-Al, 0 °C, 54% yield for 43; Red-Al, 0 °C, 65% yield for 44 HF/pyridine, pyridine, 0 °C, 81% yield for 45, HF/pyridine, pyridine, 0 °C, 99% yield for 46 The compound names; (40): 13-O-triethylsilylbaccatin III; (41): 7-O-Acetyl-13-Otriethylsilylbaccatin III; (42): 13-O-Acetyl-7-O-triethylsilylbaccatin III; (43): 7-O-Acetyl—2-Odebenzoyl-13-O-triethylsilylbaccatin III; (44): 13-O-Acetyl-2-O-debenzoyl-7-Otriethylsilylbaccatin III; (45): Rearranged 7-O-acetyltaxoid; (46): Rearranged 13-O-acetyltaxoid 205 1 Table 3.1 – Comparison of diagnostic proton NMR data on 45 and 46 with 36 Proton Chemical Shifts (ppm), Splitting Patterns, and Coupling Constants (Hz) H NMR 36 45 46 signal H-10 H-5 H-20β H-20α H-2 H-3 6.16 (s) 4.95 (d, J = 7.8) 4.59 (d, J = 10.8) 4.57 (d, J = 10.8) 3.87 (dd, J = 6.3, 11.4) 3.55 (d, J = 7.8) 6.28 (s) 4.32 (t, J = 9.0) 4.35 (d, J = 12.0) 3.67 (d, J = 12.0) 4.06 (d, J = 6.6) 3.51 (d, J = 7.2) 6.20 (s) 4.24 (d, J = 9.6) 4.34 (d, J = 12.0) 3.66 (d, J = 12.0) 4.13 (d, J = 6.6) 3.29 (d, J = 7.2) Table 3.2 – Comparison of diagnostic carbon NMR data on 45 and 46 with 36 Chemical Shifts (ppm) C NMR 36 45 46 signal 13 C-5 C-4 C-1 C-20 C-10 C-2 C-3 83.90 82.16 78.48 77.96 75.68 74.22 47.26 71.10 94.95 76.26 71.06 76.55 85.12 51.27 71.14 95.30 76.06 71.03 76.60 85.78 49.96 Figure 3.12 – The proposed mechanism for the oxetane ring opening and the furan ring formation in the presence of HF/pyridine reagent during the first attempt to synthesize of 2-Odebenzoylated taxoids 206 Figure 3.13 – A ball and stick models of expected product (37, shown to the left) and the resulted and rearranged product (45, shown to the right) in the first attempt to synthesize 37. The subtle changes in the overall topology of 45 (mainly the positioning of C-2) facilitate the ether linkage in between C-2 and C-20 without affecting the stereochemistry at C-2 and C-4. Thus, the C2-O bond (equatorial, down) and the C4-C20 bond (axial, up) in 45 remain the same in the furan ring system as compared to the oxetane ring system in 37. The color coding for each atom and several groups are described: carbon – black, oxygen – red, hydrogen – white, methyl group – yellow, acetoxy group – brown. Carbons, C-1 to C-5 and C-20 are labeled for further understanding. The bond in between C5-O and C-20 in 37 which was cleaved and the bond in between C2-O and C-20 in 45 which was formed during the rearrangement are shown in purple. The photography was by Irosha Nawarathne 207 O O O O OR1 7 R2O 13 HO H O O O O O O OR1 7 c O O R2O 13 HO H O OH O O OR1 7 d O O R2O 13 HO H OH O R1: Ac; R2: TES (41) R1: Ac; R2: TES (43) R1: Ac; R2: H (37) R1: TES; R2: Ac (42) R1: TES; R2: Ac (44) O O R1: H; R2: Ac (38) R1: TES; R2: H (47) R1: H; R2: H (39) Figure 3.14 – Second attempt of synthesis of 2-O-debenzoylated taxoids, 37, 38, 39 c Red-Al (3 equiv), 0 °C, 54% yield for 43; Red-Al (3 equiv), 0 °C, 65% yield for 44; Red-Al (5 equiv), 0 °C, 60% yield for 47 d TBAF, -78 0 °C to 0 °C, 92% yield for 37; TBAF, -78 0 °C to 0 °C, 87% yield for 38; TBAF, 78 0 °C to 0 °C, 84% yield for 39 The compound (47) is 2-O-debenzoyl-7-O-triethylsilylbaccatin III 1 H NMR signal H-10 H-5 H-20β H-20α H-2 H-3 13 C NMR signal C-5 C-4 C-1 C-20 C-10 C-2 C-3 Table 3.3 – The diagnostic proton NMR data on 37, 38, and 39 Proton Chemical Shifts (ppm), Splitting Patterns, and Coupling Constants (Hz) 37 38 39 6.18 (s) 4.95 (d, J = 9.0) 4.61 (d, J = 10.2) 4.57 (d, J = 10.2) 3.84 (br d, J = 7.2) 3.60 (d, J = 7.2) 6.21 (s) 4.96 (t, J = 9.6) 4.62 (d, J = 9.6) 4.57 (d, J = 9.6) 3.90 (dd, J = 5.2, 8.4) 3.43 (d, J = 7.8) 6.23 (s) 4.97 (d, J = 9.0) 4.62 (d, J = 9.0) 4.57(d, J = 9.0) 3.86 (dd, J = 6.6, 7.2) 3.47 (d, J = 7.2) Table 3.4 – The diagnostic carbon NMR data on 37, 38, and 39 Chemical Shifts (ppm) 37 38 39 83.64 81.87 77.91 77.71 75.96 74.05 47.36 84.04 82.18 78.70 77.82 75.76 74.67 45.59 208 83.01 82.06 78.28 77.77 76.25 74.77 46.01 After incubating benzoyl CoA and a single 2-O-debenzoylated taxane co-substrate, each at 1 mM, in separate assays with purified mTBT, EtOAc was added to the assays to partition any 2O-benzoylated products into the organic phase. The extracted products were subjected to LCMS(/MS). Typical diagnostic fragment ions of 2-O-benzoylated taxoids were evaluated using authentic 8, 11, 12, and 2 as models to identify the various ion cleavage points (Figure B 3 and Figure B 4; MS data of 12, and 2 are not shown). Taxane co-substrates 36 and 37 were converted to their 2-O-benzoylated products 8 and 11, respectively (Figure 3.15 and Figure 3.16), while no anticipated product was detected for the other tested 2-O-debenzoylated taxoids. Typical diagnostic fragment ions of 2-O-benzoylated taxane analogues showed were m/z of [M + + + + H– 2HOAc] , [M + H– 3HOAc] , [M + H– HOAc – BzOH – H2O] , [M + H– 2HOAc – + + + BzOH] , [M + H – 3HOAc - BzOH] , [M + H – 3HOAc – BzOH – H2O] , [M + H – 4HOAc + + BzOH] , [M + H – 4HOAc – BzOH – H2O] (Figure B 5 and Figure B 6). The control assays did not show detectable products for any of the taxoids. 209 671.3 100 % Relative Abundance 80 60 693.3 40 20 611.3 309.2 369.2 429.2 551.2 0 0 100 200 300 400 500 600 700 800 m/z Figure 3.15 – LC-MS ion profile of the biosynthesized 7,13-O,O-diacetylbaccatin III (8) derived + by mTBT-catalyzed deacylation is shown. Diagnostic ions were found as m/z: 693.3 [M + Na] , + + + 671.3 [M + H] , 611.3 [m/z 671.3 - HOAc] , 551.2 [m/z 611.3 – HOAc] , 429.2 [m/z 551.2 – + BzOH] , 369.2 [m/z 429.2 – HOAc]+, 309.2 [m/z 369.2 – HOAc]+. The MS/MS fragment ion profiles of the authentic and the biosynthesized 8 are shown in Figure B 3 and Figure B 7 respectively 210 651.3 100 % Relative Abundance 80 60 569.2 629.3 40 20 327.2 387.2 509.2 0 0 100 200 300 400 500 600 700 800 m/z Figure 3.16 – LC-MS ion profile of the biosynthesized 7-O-acetylbaccatin III (11) derived by + mTBT-catalyzed deacylation is shown. Diagnostic ions were found as m/z: 651.3 [M + Na] , + + + 629.3 [M + H] , 569.2 [m/z 629.3 - HOAc] , 509.2 [m/z 569.2 – HOAc] , 387.2 [m/z 509.2 – + BzOH] , 327.2 [m/z 387.2 – HOAc]+. The MS/MS fragment ion profiles of the authentic and the biosynthesized 11 are shown in Figure B 4 and Figure B 8 respectively Similar to the mTBT-catalyzed deacylation, the results of taxane specificity studies on mTBT for the 2-O-benzoylation also shows that taxoids with an acetoxy group at C-7 were productive, while those with a hydroxyl group at C-7 were non-productive (Figure 3.17). The similar pattern of both mTBT-catalyzed deacylation and acylation rejecting C-7 hydroxylated and accepting C-7 acetylated taxoid substrates further indicate that the hydroxyl group somehow hinders mTBT 211 catalysis or the acetyl group assists the same catalysis. The underlying possibilities were further analyzed. AcO O OAc 7 HO 8 O H OH OAc 37 AcO O OH HO 7 O H OH OAc 39 38 Figure 3.17 – Several identified productive (8, 37) and non-productive (38, 39) taxane substrates when tested for the substrate specificity of mTBT 2-O-benzoylation reaction. All the productive taxane substrates have O-acetyl group at C7 (shown in red) while all the non-productive substrates have a hydroxy- group at C7 (shown in blue) HO 3.3.4. HO Plausible Explanation for the Differential Reactivity of mTBT In a previous study, a Taxus 10-O-acetyltransferase (DBAT) was found to also have 4-Oacetyltransferase activity, converting 4-O-deacetylbaccatin III (9) to 2 (cf. Figure 3.2 for 24 structures). However, when 9 was acetylated at the hydroxyls at C-7, C13, or both C-7 and C- 13, DBAT did not form any detectable products, suggesting that catalytic acetylation of the C-7 hydroxyl precluded acetylation at the C-4 hydroxyl. Consequently, a putative biosynthetic pathway to paclitaxel was formulated wherein acetylation of the C-4 hydroxyl occurred after the 24 Current dogma suggests that C-4 acetoxy of advance taxanes bearing oxetane ring was formed. a 4-acetoxy-4(20),5-oxetane functional grouping (including paclitaxel) are derived from 4(20)24 oxirane-5-acetoxy taxanes. This latter tenet was brought into question by the 4-O212 acetyltransferase activity observed in the earlier study. During the earlier investigation where 13O-acetyl-4-O-deacetylbaccatin III (10) was synthesized an unexpected reactivity was noted for 24,59 The chemical reactivity of C-13 hydroxyl was both the C-7 and C-13 hydroxyl groups of 9. enhanced while that of the C-7 hydroxyl was reduced in 9, compared to the reciprocal rates of reactivity for the analogous hydroxyl groups in 10-deacetyl baccatin III (1) baccatin III (2) (both have a 4-acetoxy group) (cf. Figure 3.2). 24,59 The C-13 hydroxyl of 9 could be acetylated without needing the typical protection of the hydroxyl at C-7, as needed for 1 and 2 to direct the chemoselectivity. The unusual reactivity of 9 was likely due to the disruption of the regular intramolecular H-bonding network of the 4-O-acetylated taxoids (9) compared to 4-hydroxy taxoids (1 and 2). The 7-hydroxy-4-O-deacetylbaccatins (9, 10) may also engage in a significantly modified intramolecular H-bonding network that is absent in the C-7 acetylated 4O-deacetylbaccatins. As a result, the effective binding conformation of the substrates in the 24 DBAT active site may likely change, leading to the observed differential reactivity of DBAT. By analogy, the different reactivity of mTBT with C-7-acetoxy versus C-7-hydroxyl taxanes may be a consequence of an altered intramolecular H-bonding network. To explore the effect of functional group at C-7 to the intramolecular interactions of the corresponding molecule, 60 preliminary molecular modeling studies (MM2) were conducted. According to the MM2 studies of various 2-O-benzoylated and 2-O-debenzoylated taxane analogues, the distance between the atoms of hydroxyl group at C-7 (7-OH) and the carbonyl oxygen at C-9 (9-CO) is 1.9 Å thus intramolecular H-bonding is plausible (Figure 3.18; only the model of 11 is shown). This H-bonding interaction could initiate a conceivable conformational change of the substrate may occur precluding the mTBT catalysis. 213 Figure 3.18 – A ball and stick model in Chem & Bio 3D 12.0 (CambridgeSoft) and the corresponding molecular structure of 7-O-acetylbaccatin III (11). Shown is the distance between the 7-OH and the 9-CO in A° 3.3.5. Pinpointing the Timing of 2-O-Benzoylation in Overall Paclitaxel Biosynthetic Pathway Several characterized steps of the paclitaxel biosynthetic pathway show that taxusin (10) and 7β40 hydroxytaxusin (26) are productive substrates of P450 taxane 2α-hydroxylase (Figure 3.3). Also, P450 taxane 2α-hydroxylase resembles the P450 taxoid 7β-hydroxylase, which also uses 10 as a substrate. Both 2α- and 7β-hydroxylases are capable of the reciprocal conversion of their 39,40 respective pentaol tetraacetate products to the common hexaol tetraacetate (Figure 3.3). However, if 7β-hydroxylation occurs prior to/immediately after the 2α-hydroxylation in the paclitaxel biosynthetic pathway, a key step in the pathway, mTBT-catalyzed 2-O-benzoylation will likely be prevented, according to the previously devised hypothesis. Consequently, the order of these three steps in the overall paclitaxel biosynthetic pathway is postulated to be 2α214 hydroxylation, 2-O-benzoylation, and 7β-hydroxylation respectively (Figure 3.20 and Figure 3.22). In addition, the binding studies of Taxus 7β-hydroxylase with several highly substituted 39 taxoids including 2-O-acyltaxoids were analyzed in evaluating the proposed order of reactions (Figure 3.19). The tested 2-O-acyltaxoids showed poor binding to the enzyme compared to the other tested substrates suggesting that the 7β-hydroxylation does not immediately follow the 2O-benzoylation step (formation of 2-O-benzoyltaxusin, 50), but likely occurs further down in the pathway with more advanced 2-O-acyltaxoids containing an intact oxetane ring (Figure 3.20). Several 2-O-benzoyl-7-deoxytaxoids (51) were designed to further analyze the exact timing of 7β-hydroxylation in future investigations (Figure 3.22). Alternatively, the 2-O-benzoylation may occur further down in the paclitaxel pathway to form an advanced 2-O-benzoyltaxoid which will subsequently get oxidized by 7β-hydroxylase (Figure 3.22). 2-Hydroxytaxusin (27) can be tested with mTBT to indirectly rationalize this idea. Although the exact timing of the 2-O-benzoylation in the overall paclitaxel biosynthetic pathway can not be predicted at this point, continued analysis of these preliminary findings will pinpoint the exact timing of the 2-O-benzoylation. 215 AcO OAc AcO OAc 7 AcO 2 H H K s = 0.14 M AcO OAc 7 OAc 7 2 AcO H OAc H OAc AcO K s = 13.3 M 2 H H OAc OAc K s = 555.0 M 2-Acetoxytaxusin (48) 2-Acetoxy-5-Odeacetyltaxusin (49) Figure 3.19 – The binding constants (Ks) reported for highly functionalized taxoids with Taxus 39 7β-hydroxylase. The 2-acetoxytaxoids showed poor Ks compared to that of 25 25 R = H or Ac R: Ac (27) R = H or Ac R: Ac (25) R = H or Ac 2-O-Benzoyltaxusin R: Ac (50) RO O OH d 7 2 Pathway continued... O H OBz OAc R = H or Ac R = H or Ac R: H (1) 2-O-benzoyl-7-deoxytaxoids (51) Figure 3.20 – Proposed order of several steps in the paclitaxel biosynthetic pathway starting from 25 to the paclitaxel precursor 1 considering that the 2-O-benzoylation occurs immediately after the 2α-hydroxylation. Step a: Cytochrome P450 taxane 2α-hydroxylase; b: mTBT; c: Several steps in the pathway; d: Cytochrome P450 taxane 7β-hydroxylase RO 216 HO 10-O-Acetyl-7-deoxybaccatin III (52) R1: Ac; R2: H 13-O-Acetyl-7-deoxybaccatin III (53) R1: H; R2: Ac 10,13-O,O-Diacetyl-7-deoxybaccatin III (54) R1: Ac; R2: Ac 7-Deoxybaccatin III (55) R1: H; R2: H Figure 3.21 – The proposed 2-O-benzoyl-7-deoxytaxoids (51) to understand the exact timing of 2-O-benzoylation and/or 7β-hydroxylation in the overall paclitaxel biosynthetic pathway R = H or Ac R: Ac (25) R = H or Ac R: Ac (27) R = H or Ac 7-Deoxy-2hydroxytaxoids (56) RO O OH d 7 2 Pathway continued... O H OBz OAc R = H or Ac R = H or Ac 7-Deoxy-2-O-benzoyltaxoids (51) R: OH (1) Figure 3.22 – Proposed order of several steps in the paclitaxel biosynthetic pathway starting from 25 to the paclitaxel precursor 1 considering that the 2-O-benzoylation occurs further down in the pathway. Step a: Cytochrome P450 taxane 2α-hydroxylase; b: Several steps in the pathway; c: mTBT; d: Cytochrome P450 taxane 7β-hydroxylase RO 217 HO 3.4. Conclusion Potentially, biotechnological production of efficacious paclitaxel analogues can be achieved 7,9,10 through the metabolic engineering of Taxus plant cell cultures, hosts such as E. coli, 11,12 13 S. cerevisiae, and/or other recombinant 14-16 and alternative tractable transgenic species. 17-26 acylation reactions of Taxus acyltransferases have been well studied, The and proven to show broad substrate specificities in vitro and/or in vivo, while the deacylation reactions of Taxus 24,27 acyltransferases and/or other related enzymes have been reported sporadically. In this study, the mTBT-catalyzed deacylation was studied in detail to understand its potential biocatalytic applications. Interestingly, the mTBT-catalyzed deacylation was highly chemoselective for 2-Odebenzoylation. More surprisingly, the scope of taxane substrates for mTBT-catalyzed deacylation was very limited. The results of the taxane specificity studies on mTBT-catalyzed deacylation showed that the taxane substrates hydroxylated at C-7 and/or both C-7 and C-13 were non-productive, suggesting that the hydroxylation at C-7 precluded the mTBT deacylation. Similarly, the taxane substrate specificity studies on mTBT-catalyzed acylation reaction verified also showed that the taxanes with C-7 and/or both C-7 and C-13 were non-productive. Further evidence from molecular modeling studies (MM2) demonstrated the plausible H-bonding interactions of 7-OH and 9-CO, which may hinder the mTBT catalysis in both forward and reverse directions due to the changes in effective binding confirmation of the substrate to the enzyme. Further analysis of these findings allowed the pinpointing of the relative timing of 2-Obenzoylation and the 7β-hydroxylation steps in the overall paclitaxel pathway. A complete understanding of the organization and/or the regulation of this pathway is undoubtedly advantageous in crafting more efficacious biotechnological production of 6. 218 3.5. Future Directions 3.5.1. Further Characterization of the mTBT Deacylation and Acylation and the Inhibition Studies The limited taxane substrate scope of mTBT-catalyzed deacylation and acylation are demonstrated in this study. However, a detailed kinetic evaluation of each productive taxane substrate is necessary to understand the potential biocatalytic application of these reactions in producing analogues of 6. Therefore, the steady-state kinetic parameters of mTBT-catalyzed 2O-benzoylation of 36 and mTBT-catalyzed 2-O-debenzoylation of 8 will be measured. Thereafter, the specificity constants of the mTBT-catalyzed deacylation and acylation reactions for each of the productive taxane co-substrate will be estimated from the amount of the taxoid made from the corresponding taxane substrate in a competitive substrate reaction under typical 61 assay conditions in future studies. The 2-O-benzoylated taxoids with hydroxyl group at C-7 and/or both C7 and C13 were non-productive substrates of mTBT in either the forward or reverse directions, suggesting that the hydroxylation at C-7 precludes the mTBT catalysis. This idea was further supported by the molecular modeling studies demonstrating the plausible H-bonding interactions in between 7OH and 9-CO. An inhibition study of mTBT-catalyzed deacylation with 8 and a seperate inhibition study of mTBT-catalyzed acylation with 36 with taxanes bearing C7-hydroxyl group will be conducted to assess if enzymatic reaction was inhibited by the presence of hydroxyl group at C-7 in future investigations. 219 3.5.2. Designing Productive Taxane Co-substrates for the mTBT Catalysis by Disrupting the H-bonding Interactions If the hydroxyl group at C-7 is merely responsible for hindering the mTBT catalysis in both forward and reverse directions due to the H-bonding interactions with 9-CO, the disruption of the respective interactions by varying the functionality or the stereochemistry at C-7 would likely restore the catalytic activity of mTBT with each substrate. Several 2-O-benzoylated and 2-Odebenzoylated taxoids are designed to purposely disrupt the interactions between 7-OH and 9CO (Figure 3.23). Testing of these semi-synthetically derived taxane co-substrates with mTBT in the respective catalytic reaction will likely support/reject the formulated hypothesis. However, as some of the designed substrates may yet hinder the reaction due to occluding sterics, the data interpretation needs caution. 3.5.3. Alternative Hypothesis to Explain the Differential Reactivity of mTBT Due to the evident intermolecular interactions between 7-OH and 9-CO, the mTBT catalysis was precluded by the presence of hydroxyl group at C-7 of the tested substrates. However, all the productive substrates possessed an acetyl group at the C-7 position (co-incidentally, the productive substrates did not have the free hydroxyl group). Thus, one could propose an alternative hypothesis to explain the different reactivity of mTBT with tested taxane cosubstrates. The acetoxy group at C-7 may assist the mTBT catalysis in both the forward and reverse directions. Consequently, the 2-O-benzoylation in the paclitaxel biosynthetic pathway catalyzed by mTBT would selectively/preferably occur in 7-O-acetyltaxoids. Although none of the prevalent advanced taxoids such as 1, 2, 6, or 14, (cf. Figure 3.2 for structures) hold an acetoxy group at C-7 there are several naturally occurring 7-O-acetyltaxoids benzoylated at C-2 supporting this alternative hypothesis (Figure 3.24). The acetyl group at C-7 may assist the 220 mTBT catalysis, requiring that a couple of steps be added to the paclitaxel biosynthetic pathway (Figure 3.25). The proposed 7-O-acetyltransferase may catalyze both acetylation and the deacetylation at C-7. The probable occurrence of a 7-O-acetyltransfrease can be investigated utilizing various 7-hydroxytaxoids as the taxane co-substrates to validate the alternative 9,38-40 hypothesis. The findings will likely be important in discovering an “off-pathway” 9 acyltransferase if the alternative hypothesis is rejected based on the gathered data. 7-Deoxybaccatin III (55) R: Bz 2-O-Debenzoyl-7-deoxybaccatin III (57) R: H AcO O OCH3 7-Epibaccatin III (58) R: Bz 2-O-Debenzoyl-7-epibaccatin III (59) R: H AcO O OTES 7 7 2 O O AcO HO H OAc HO H OAc OR OR 7-O-Triethylsilylbaccatin III (40) R: Bz 7-O-Methylbaccatin III (60) R: Bz 2-O-Debenzoyl-7-O-methylbaccatin III 2-O-Debenzoyl-7-O-triethylsilylbaccatin III (61) R: H (62) R: H Figure 3.23 – Shown are several 2-O-benzoylated and 2-O-debenzoylated taxoids designed to purposely disrupt the interactions between 7-OH and 9-CO AcO 2 221 OH HO OAc 7 2 O H OBz OAc Taxuspine E (64) AcO OAc OAc HO Baccatin VI (63) HO OAc OAc HO 7 AcO 9-O-Deacetylbaccatin VI (65) 7 2 O HO H OAc OBz HO 10-O-Deacetylbaccatin VI (66) 2 HO O H OBz OAc 13-O-Deacetylbaccatin VI (67) O O 10-O-Hydroxyacetylbaccatin VI (68) O OAc 7 NH O 2 O H OH OBz OAc 10-O-Deacetyl-10-dehydro-7-O-acetyltaxol A (69) Figure 3.24 – Shown are several naturally occurring advanced taxoids which are 7-O-acetylated and 2-O-benzoylated. O 222 HO R = H or Ac R: H (29), R: Ac (38) RO O OAc 7 2 R = H or Ac R: H (70), R: Ac (36) RO O OH c 7 2 O H OBz OAc R = H or Ac R = H or Ac R: H (71), R: Ac (8) R: H (1), R: Ac (12) Figure 3.25 – The revised timing of 2-O-benzoylation in the overall paclitaxel biosynthetic pathway and the additional acetylation-deacetylation steps included in between 2-O-benzoylation and the 7β-hydroxylation considering the alternative hypothesis in explaining the differential reactivity of mTBT. Step a: Unknown 7-O-acetyltransferase; b: mTBT c: Unknown 7-Odeacetyltransferase or the deacetylation by a RO O HO H OAc OBz RO 223 HO APPENDIX B 224 APPENDIX B 309.2 100 7,13-O,O-Diacetyl2-O-debenzoylbaccatin IIII Exact Mass : 567.3 % Relative Abundance AcO O OAc 80 AcO 327.2 60 HO O H OH OAc 40 369.2 387.2 20 0 100 200 300 400 500 600 m/z Figure B 1 – MS/MS fragment ion profiles of authentic 7,13-O,O-diacetyl-2-Odebenzoylbaccatin III is shown. Diagnostic ions were found as m/z: 387.2 [m/z 567.3 – + + + 3HOAc] , 369.2 [m/z 387.2 – H2O] , 327.2 [m/z 387.2 – HOAc] , and 309.2 [m/z 369.2 – + HOAc] . 225 309.2 100 7-O-Acetyl2-O-debenzoylbaccatin III Exact Mass : 525.2 AcO 80 O OAc % Relative Abundance 327.2 387.2 60 HO HO 369.2 O H OH OAc 40 429.2 20 525.2 0 0 100 200 300 400 500 600 m/z Figure B 2 – MS/MS fragment ion profiles of authentic 7-O-acetyl-2-O-debenzoylbaccatin III is + shown. Diagnostic ions were found as m/z: 525.2 [M + H] , 429.2 [m/z 525.2 – HOAc – + + + 2H2O] , 387.2 [m/z 525.2 – 2HOAc – 2H2O] , 369.2 [m/z 429.2 – HOAc] , 327.2 [m/z 387.2 – + + HOAc] , and 309.2 [m/z 369.2 – HOAc] . 226 369.2 100 7,13-O,O-diacetylbaccatin III Exact Mass : 670.2 AcO O OAc % Relative Abundance 80 AcO 309.2 60 O HO H OAc O O 429.2 40 20 551.2 671.2 491.2 0 0 100 200 300 400 500 600 700 800 m/z Figure B 3 – MS/MS fragment ion profiles of authentic 7,13-O,O-diacetylbaccatin III is shown. + + Diagnostic ions were found as m/z: 671.2 [M + H] , 551.2 [m/z 671.2 – 2HOAc] , 491.2 [m/z + + + 551 – HOAc] , 429.2 [m/z 551.2 – BzOH] , 369.2 [m/z 429.2 – HOAc] , and 309.2 [m/z 369.2 – + HOAc] . 227 387.2 100 7-O-Acetylbaccatin IIII Exact Mass : 629.3 AcO O OAc % Relative Abundance 80 HO 60 40 O HO H OAc O O 309.2 327.2 369.2 20 429.2 491.2 629.3 0 0 100 200 300 400 500 600 700 m/z Figure B 4 – MS/MS fragment ion profiles of authentic 7-O-acetylbaccatin III is shown. + + Diagnostic ions were found as m/z: 629.3 [M + H] , 491.2 [m/z 629.3 – 2HOAc – H2O] , 429.2 + + [m/z 629.3 – HOAc – BzOH – H2O] , 387.2 [m/z 629.3 – 2HOAc – BzOH] , 369.2 [m/z 429.2 – + + + HOAc] , 327.2 [m/z 387.2 – HOAc] , and 309.2 [m/z 369.2 – HOAc] . 228 309.2 100 7-O-Acetyl2-O-debenzoylbaccatin III Exact Mass : 525.2 327.2 % Relative Abundance 80 AcO 387.2 HO 369.2 60 HO O OAc O H OH OAc 40 429.2 20 525.2 0 100 200 300 400 500 600 m/z Figure B 5 – MS/MS fragment ion profile of the biosynthesized 7-O-acetyl-2-Odebenzoylbaccatin III derived by catalysis of mTBT is shown. Diagnostic ions were found as + + m/z: 525.2 [M + H] , 429.2 [m/z 525.2 – HOAc – 2H2O] , 387.2 [m/z 525.2 – 2HOAc – + + + 2H2O] , 369.2 [m/z 429.2 – HOAc] , 327.2 [m/z 387.2 – HOAc] , and 309.2 [m/z 369.2 – + HOAc] . 229 385.2 7-O-Acetyl-2-O-debenzoyl13-oxobaccatin III Exact Mass : 523.2 100 AcO % Relative Abundance 80 O 60 367.2 O OAc HO O H OH OAc 40 307.2 20 343.2 403.2 445.2 523.2 0 100 200 300 400 500 600 m/z Figure B 6 – MS/MS fragment ion profile of the biosynthesized 7-O-acetyl-2-O-debenzoyl-13oxobaccatin III derived by catalysis of mTBT is shown. Diagnostic ions were found as m/z: + + + 523.2 [M + H] , 445.2 [m/z 523.2 – HOAc – H2O] , 385.2 [m/z 445.2 – HOAc] , 367.2 [m/z + + + 385.2 – H2O] , 343.2 [m/z 523.2 – 3HOAc] , and 307.2 [m/z 367.2 – HOAc] . 230 369.2 100 7,13-O,O-Diacetylbaccatin III Exact Mass : 671.3 309.2 AcO % Relative Abundance 80 429.2 AcO 60 O OAc O HO H OAc O O 40 20 491.2 551.3 671.3 0 100 200 300 400 500 600 700 800 m/z Figure B 7 – MS/MS fragment ion profile of the biosynthesized 7,13-O,O-diacetylbaccatin III + derived by catalysis of mTBT is shown. Diagnostic ions were found as m/z: 671.3 [M + H] , + + + 551.3 [m/z 671.3 – 2HOAc] , 491.2 [m/z 551.3 – HOAc] , 429.2 [m/z 551.3 – BzOH] , 369.2 + + [m/z 429.2 – HOAc] , and 309.2 [m/z 369.2 – HOAc] . 231 387.2 100 7-O-Acetylbaccatin III Exact Mass : 629.3 AcO % Relative Abundance 80 HO 60 309.2 40 20 O OAc O HO H OAc O O 327.2 369.2 429.2 509.2 0 0 100 200 300 400 500 600 700 m/z Figure B 8 – MS/MS fragment ion profile of the biosynthesized 7-O-acetylbaccatin III derived + by catalysis of mTBT is shown. Diagnostic ions were found as m/z: 509.2 [629.3 – 2HOAc] , + + 429.2 [m/z 629.3 – HOAc – BzOH – H2O] , 387.2 [m/z 509.2 – BzOH] , 369.2 [m/z 429.2 – + + + + HOAc] , 327.2 [m/z 387.2 – HOAc] , and 309.2 [m/z 369.2 – HOAc] ; [M + H] is 629.3. 232 O O O O O 7 HO 13 HO OH H O O O 1 Figure B 9 – H NMR of rearranged product formed during the first attempt of 7-O-acetyl-2-O-debenzoylbaccatin III synthesis 233 O O O O O 7 HO 13 HO OH H O O O Figure B 10 – 13 C NMR of rearranged product formed during the first attempt of 7-O-acetyl-2-O-debenzoylbaccatin III synthesis 234 O O O OH 7 O O 13 HO H O OH O O 1 Figure B 11 – H NMR of rearranged product formed during the first attempt of 13-O-acetyl-2-O-debenzoylbaccatin III synthesis 235 O O O OH 7 O O 13 HO H O OH O O Figure B 12 – 13 C NMR of rearranged product formed during the first attempt of 13-O-acetyl-2-O-debenzoylbaccatin III synthesis 236 O O O O O 7 HO 13 HO H OH O O O 1 Figure B 13 – H NMR of 7-O-Acetyl-2-O-debenzoylbaccatin III 237 O O O O O 7 HO 13 HO H OH O O O Figure B 14 – 13 C NMR of 7-O-Acetyl-2-O-debenzoylbaccatin III 238 O O O OH 7 O O 13 HO H OH O O O 1 Figure B 15 – H NMR of 13-O-Acetyl-2-O-debenzoylbaccatin III 239 O O O OH 7 O O 13 HO H OH O O O Figure B 16 – 13 C NMR of 13-O-Acetyl-2-O-debenzoylbaccatin III 240 O O O OH 7 HO 13 HO H OH O O O 1 Figure B 17 – H NMR of 2-O-Debenzoylbaccatin III 241 O O O OH 7 HO 13 HO H OH O O O Figure B 18 – 13 C NMR of 2-O-Debenzoylbaccatin III 242 O O O O O 7 O O 13 HO H OH O O O Figure B 19 – HMQC of 7,13-O,O-Diacetyl-2-O-debenzoylbaccatin III 243 O O O O O 7 HO 13 HO H O OH O O Figure B 20 – HMQC of rearranged product formed during the first attempt of 7-O-acetyl-2-O-debenzoylbaccatin III synthesis 244 O O O OH 7 O O 13 HO H O OH O O Figure B 21 – HMQC of rearranged product formed during the first attempt of 13-O-acetyl-2-O-debenzoylbaccatin III synthesis 245 O O O O O 7 HO 13 HO H O OH O O Figure B 22 – HMQC of 7-O-Acetyl-2-O-debenzoylbaccatin III 246 O O O OH 7 O O 13 HO H OH O O O Figure B 23 – HMQC of 13-O-Acetyl-2-O-debenzoylbaccatin III 247 O O O OH 7 HO 13 HO H OH O O O Figure B 24 – HMQC of 2-O-Debenzoylbaccatin III 248 REFERENCES 249 REFERENCES (1) Kingston, D. 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Mass Spectrom. 2004, 15, 233-243. 253 The Subcloning and the site-directed mutagenesis of bapt cDNA were performed by Dr. Brad Cox. The synthesis of (R)-3-hydroxy-3-phenylpropanoyl CoA and 3phenylpropanoyl CoA were conducted by Yemane Mengistu. 254 4. SUBSTRATE SPECIFICITY SUBSTRATE-ASSISTED BACCATIN STUDIES CATALYSIS III OF AND TAXUS 13α-O-3-AMINO-3- PHENYLPROPANOYLTRANSFERASE (BAPT) 4.1. Introduction 4.1.1. Substrate Specificity Studies of BAPT Structure-Activity Relationship (SAR) studies on paclitaxel (1) and its analogues bearing semisynthetically derived phenylpropanoid side chains revealed the dependence on the side chain 1-6 functional groups and their regio- and stereochemistry for biological activity of 1. Many efficacious paclitaxel analogues have been reported that vary primarily at the 3'-phenyl, Nbenzoyl, and 2'-hydroxy functional groups; these are typically coupled with and without other acyl group modifications elsewhere in the molecule (e.g. see Figure 4.1 and Figure 4.2). The ® best-known non-natural analogue of paclitaxel is docetaxel (Taxotere ) (2), currently in clinical 2 use, which has a N-butoxy carbonyl for N-benzoyl replacement in the C-13 side chain. Other analogues (Figure 4.1) also show improved activity 12-14 delivery (Figure 4.2). 255 7-11 while others facilitate the targeted drug O HO O NH O O O HO H OAc O O O O OH * ® Paclitaxel (Taxol ) R1: Ac; R2: Ph (1) ®2 * Docetaxel (Taxotere ) R1: H; R2: C(CH3)3 (2) * * O 8 * DJ-927 (4) O O O O OH 7 TL-00139 (3) 9 BMS-275183 (5) O OH O O NH O O S 10 O O OH NH O O O HO H OAc O O O O OH * O HO H OAc O O 11 Bis cyclopropanated analog (6) Simotaxel (7) Figure 4.1 – Paclitaxel (1), Docetaxel (2), and several other efficacious paclitaxel analogues with modified C-13 side chains. The analogues currently in clinical usage or clinical trials are * indicated by an asterisk . 256 O S O O O S O OH NH O O OH O HO H OAc O O Cl 12 13,14 DHA-linked paclitaxel (8) mAb-taxoid conjugates (9) Figure 4.2 – Shown are some paclitaxel analogues with improved targeted drug delivery. DHAlinked paclitaxel (8) is currently in phase III clinical studies. Note, DHA is docosahexaenoic acid. Due to the low yields of naturally occurring paclitaxel from Taxus trees, Bristol-Myers 15 Squibb licensed a semisynthetic approach developed by Holton and coworkers. This method was a long-standing means of producing 1 and 2 in large-scale, and relied on an abundant natural precursor 10-O-deacetylbaccatin III (10) used as the starting material. This approach involved a series of redundant protecting group manipulations that affected the overall yield (Figure 16-18 4.3), and in addition, the multitude of large-scale synthetic steps employed several different petroleum based solvents and reagents that were environmentally hazardous. Therefore, BristolMyers Squibb adopted an alternative means of commercial scale production of 1 via Taxus Plant 19 Cell Fermentation (PCF), licensed from Phyton. Both the semi-synthetic approach (in part) and the PCF method rely on the knowledge of biosynthesis of paclitaxel (Figure 4.4). Many of the 20,21 ~19 biosynthetic steps on the paclitaxel pathway have been identified. 257 Of these, five Taxus acyltransferases have been characterized, including a CoA thioester-dependent Taxus baccatin III 22 13α-O-3-amino-3-phenylpropanoyltransferase (BAPT). In an earlier study, the recombinantly expressed BAPT was shown to transfer (3RS)-β-phenylalanoyl group from its corresponding CoA thioester to baccatin III (19) to form (3'RS)-β-phenylalanoyl baccatin III (29). The kinetic parameters were reported as KM = 2.4 µM for baccatin III (19) and 4.9 µM for (3RS)-β22 phenylalanoyl CoA (25) (Figure 4.5). The biosynthetic product (29) was chemically N- 1 benzoylated, and then demonstrated by H NMR analysis to contain a single 3'R-epimer of 2'22 deoxypaclitaxel by comparison with an authentic (3'RS)-2'-deoxypaclitaxel standard. The stereochemistry of this epimer was consistent with that of 1 at the 3'-position. In the same report, (2R,3S)-phenylisoserinoyl CoA (27) was also identified as a productive CoA substrate, however, with a 2.5-fold slower catalytic turn-over compared to (3RS)-β-phenylalanoyl CoA (25), while (S)-α-phenylalanoyl CoA (26) and N-benzoyl-(2R,3S)-phenylisoserinoyl CoA (28) were non22 productive (Figure 4.5). Thus, considering the phenylpropanoyltransferase reaction, by analogy to the semisynthetic method described earlier, BAPT can potentially be applied in aqueous biocatalytic, in vitro coupling of the abundant natural product 10 or 19 to an intact 3-aminophenylpropanoid side chain. This enzyme-based reaction can potentially replace the multistep semisynthesis of 1 and its isoserinyl analogues, thus avoiding the deleterious effects of the solvents and reagents used in the previous synthetic approach. BAPT presumably catalyzes one of the last five steps on the paclitaxel pathway in Taxus sp., which includes a phenylalanine aminomutase (PAM) that converts α-phenylalanine (20) to β-phenylalanine (21), 21 is ultimately converted to its CoA thioester by a ligase, and the resultant CoA is transferred to the C-13 hydroxyl of 10 or 19 by 258 22 BAPT catalysis. Understanding the flux of the various recombinantly expressed catalysts involved in constructing the last portions of 1 is advantageous towards producing 1 and its analogues via biocatalytic routes (Figure 4.4) that can potentially substitute for several steps in the semisynthetic pathway (Figure 4.3). 259 10-O-Deacetylbaccatin III R1: Bz (10) 10-O-Deacetyl-7-Otriethylsilylbaccatin III R1: Bz (11) 7-O-triethylsilylbaccatin IIII R1: Bz; R2: Ac (12) (13) Ph 14 15 R3 R N PMP PMP aldimine (17) 16 Paclitaxel R1: Bz; R2: Ac; R3: Ph; (18) R4: Ph (1) Figure 4.3 – Scheme of semisynthesis of paclitaxel (1) and/or its analogues with various acyl groups. Reagents, conditions used in each step, and the stepwise yields: (i) TESCl, imidazole, DMF, RT, 96%; (ii) LiHMDS, R2COCl, THF, 82%; (iii) LDA, THF; (iv) 17, 96% for two steps; (v) CAN, CH3CN, H2O, 88%; (vi) R4COCl, TEA, DMAP, CH2Cl2, RT, 97%; (vii) LiHMDS, THF, -40 ºC, 30 min; (viii) HF/pyridine, pyridine/MeCN, 0 ºC – RT, 18 h, ~80% for two steps. Overall yield is ~0.5% without considering the syntheses of 13 and 17. Note, TIPS is triisopropyl silyl; PMP is para-methoxyphenyl group. 260 O HO O OH R2 O O OH a HO O HO H OAc O O HO R1 R1 10-O-Deacetylbaccatin III R1: Bz (10) O R2 α-phenylalanine R3: Ph (20) O Baccatin III R1: Bz; R2: Ac (19) β-phenylalanine R3: Ph (21) β-phenylalanine CoA R3: Ph (22) O O OH R2 NH2 O HO O HO H OAc O O O HO H OAc O O R1 Baccatin III R1: Bz; R2: Ac (19) R3 S-CoA O OH NH2 O d β-phenylalanine CoA R3: Ph (22) O R3 O O HO H OAc O O R1 β-phenylalanoyl baccatin III R1: Bz; R2: Ac; R3: Ph (23) 3'-N-Debenzoylpacitaxel Paclitaxel R1: Bz; R2: Ac; R3: Ph; R4: Ph R1: Bz; R2: Ac; R3: Ph (24) (1) Figure 4.4 – A potential biocatalytic route using Taxus acyltransferases to produce paclitaxel (1) and/or its analogues with various acyl groups. Step a: 10-Deacetylbaccatin III-10β-Oacetyltransferase (DBAT); b: Phenylalanine aminomutase (PAM); c: Phenylalanine CoA ligase; d: Baccatin III-13α-O-phenylpropanoyltransferase (BAPT); e: Cytochrome P450 hydroxylase; f: 3'-N-Debenzoyl-2'-deoxytaxol N-benzoyltransferase (NDTBT) 261 O O HO O OH O HO H OAc O O R3 R 4 SCoA R 1 R2 O Crude soluble BAPT (25), (26), (27), (28) (3'RS)-β-phenylalanoyl baccatin III (29) 19 Figure 4.5 – The characterization of the recombinant Taxus baccatin III 13α-O-3-amino-3phenylpropanoyltransferase (BAPT). A single epimer of (3'RS)-β-phenylalanoyl baccatin III was formed but the stereochemistry of the epimer has not been investigated yet. The descriptions of the utilized CoA thioesters are shown. (25): (3RS)-β-phenylalanoyl CoA R1: NH2; R2, R3, R4: H and R2: NH2; R1, R3, R4: H (26): (S)-α-phenylalanoyl CoA R3: NH2; R1, R2, R4: H (27): (2R,3S)-phenylisoserinoyl CoA R2: NH2; R4: OH; R1, R3: H (28): N-benzoyl-(2R,3S)-phenylisoserinoyl CoA R2: BzNH; R4: OH; R1, R3: H 4.1.2. Substrate-Assisted Catalysis of BAPT A substrate-assisted catalysis (SAC) process is feasible, particularly, when the substrate of the corresponding enzyme contains a functional group which may be involved in the proposed 23,24 reaction mechanism. SAC has been convincingly demonstrated in the context of several engineered enzymes including representatives of two major classes, serine proteases and 23,25-28 GTPases, as well as lysozyme and hexose-1-phosphate uridylyl transferase. SAC also contributes to the activity of naturally occurring enzymes as evidenced by members of two large 23,29-32 classes, GTPases and type II restriction endonucleases, and additionally lysozyme. As an example for SAC, mutated serine protease, subtilisin BPN' with alanine in place of catalytic histidine (H64A subtilisin) has been shown to decrease the catalytic efficiency by approximately 262 a million-fold; 33 however, a P2 substrate histidine can substitute functionally for the missing catalytic histidine in the H64A subtilisin converting it from a broadly specific to a histidine34 specific protease (Figure 4.6). BAPT and the four other characterized Taxus acyltransferases on the paclitaxel pathway belong to a large superfamily of plant-derived acyltransferases, designated BAHD; the members 35,36 of this family generally contain a signature HXXXD catalytic amino acid motif. Biochemical and mutagenesis studies on vinorine synthase showed that the highly conserved His-160 serves as a catalytic base in the general acid/base reaction (Figure 4.7), while the 35,37-41 conserved Asp residue likely serves a structural role. 263 D32 N155 His P2 O O HN N H O X H HN O R Enzyme-Substrate Complex (E.S) R NH O H O N N H S221 Enzyme-Intermediate Complex (E.I) Enzyme-Product Complex (E.Ac) Figure 4.6 – Proposed mechanism for SAC by H64A subtilisin BPN9' with a peptide substrate containing a P2 histidine. Shown are enzyme-substrate (E.S) and enzyme-intermediate (E.I) complexes, and an acyl enzyme intermediate (E.Ac); peptide substrate containing His P2 is shown in red. The position of the mutated catalytic residue emulated by the substrate has been omitted for clarity. Subsequent deacylation is the reverse direction of the shown acylation 23,34 reaction where water occupies the position of the leaving group amine. 264 Figure 4.7 – Proposed catalytic mechanism of vinorine synthase. ROH is 17-deacetylvinorine. His-160 residue acts as the catalytic base in the deprotonation of the oxygen atom of the acceptor substrate (ROH), allowing a nucleophilic attack on the carbonyl carbon of acetyl CoA, which in turn is proposed to form a tetrahedral intermediate. This intermediate then decomposes by eliminating CoA which gets reprotonated to produce the free CoASH and the acetylated product, vinorine (ROAc). In contrast to vinorine synthase, BAPT contains a natural mutation of this conserved His to a Gly residue (GXXXD) (Figure 4.8). Therefore, a postulated mechanism of BAPT catalysis utilizes the 3-amino functional group of the (3RS)-β-phenylalanoyl CoA (25) or, in general, the 3-amino functional group of potentially other propanoyl co-substrates as the general base (Figure 4.9). If SAC is indeed occurring, then removal of the amino functional group from the substrate should 23,24,34 impair catalysis. Hence, a series of synthetically-derived phenylpropanoyl CoA thioesters (Figure 4.10) are proposed to be tested with BAPT along with some mutagenesis studies to investigate the plausible SAC mechanism. The details of substrate specificity studies and a conceivable SAC mechanism for BAPT are discussed in this chapter. 265 BAPT_T. cuspidata_wt : DBAT_T. cuspidata : TBT_T. cuspidata : NDTBT_T. canadensis : TAT_T. cuspidata : VS_R. serpentina : Dm3MaT3_C. morifolium: ...IVQVTRFTCGGFVVGANVYGSACDAKGFGQ... ...VVQVTRFTCGGFVVGVSFCHGICDGLGAGQ... ...TVQVTRFTCGGFVVGTRFHHSVSDGKGIGQ... ...IVQVTRFTCGGIAVGVTLPHSVCDGRGAAQ... ...VVQVTRFTCGGFVVGVSFHHGVCDGRGAAQ... ...AVKISFFECGGTAIGVNLSHKIADVLSLAT... ...SVQVTLFPNQGIAIGITNHHCLGDASTRFC... Figure 4.8 – Partial amino acid sequence alignment of BAPT to several other members of the BAHD plant superfamily. The partial sequences of other Taxus acyltransferases involved in paclitaxel biosynthesis and several other BAHD family members outside the Taxus spp. are included in the alignment. BAPT_T. cuspidata_wt, wild-type BAPT from T. cuspidata (accession no. AY082804); DBAT_T. cuspidata, 10-O-deacetylbaccatin III 10-Oacetyltransferase from T. cuspidata (accession no. AF193765); TBT_T. cuspidata, 7,13-O,Odiacetyl-2-O-debenzoylbaccatin III 2-O-benzoyltransferase from T. cuspidata (accession no. AF297618); NDTBT_T. canadensis, 3'N-debenzoyl-2'-deoxytaxol N-benzoyltransferase from Taxus canadensis (accession no. AF466397); TAT_T. cuspidata, taxadien-5α-ol Oacetyltransferase from T. cuspidata (accession no. AF190130); VS_R. serpentina, vinorine synthase from Rauvolfia serpentina (accession no: AJ556780); Dm3MaT3_C. morifolium, anthocyanin malonyltransferase homolog from Chrysanthemum morifolium. Figure 4.9 – Proposed substrate-assisted catalytic mechanism of BAPT. ROH is baccatin III (19). The amino functional group of (3RS)-β-phenylalanoyl CoA (25) acts as the catalytic base in the deprotonation of 13-hydroxyl group of 19, allowing a nucleophilic attack on the carbonyl carbon of 25 which in turn is proposed to form a tetrahedral intermediate between the CoA thioester and baccatin III (19). This intermediate then gets reprotonated to produce the free CoASH and the acylated product, (3'RS)-β-phenylalanoyl baccatin III (29). 266 3-Phenylpropanoyl CoA (30) (R)-3-Hydroxy-3-phenylpropanoyl CoA (31) 3-Keto-3-phenylpropanoyl CoA 3-amino-3-phenyl-2-propenoyl (32) CoA (33) Figure 4.10 – Several synthetically-derived phenylpropanoyl CoA thioesters that are proposed to be useful in investigating the substrate-assisted catalytic mechanism of BAPT 4.2. Experimental 4.2.1. Substrates, Reagents, and General Instrumentation The T. cuspidata bapt cDNA (accession no. AY082804) was a generous donation of Washington State University Research Foundation (Pullman, WA). Baccatin III was purchased from Natland (Research Triangle Park, NC). CoASH was purchased from American Radiolabeled Chemicals Inc. (Saint Louis, MO). Phenylpropanoic acid and (R)-3-amino-3-phenylpropanoic acid were purchased from Alfa Aesar (Ward Hill, MA) while all the other utilized (R)-β-amino acids were purchased from PepTech (Burlington, MA). The restriction endonucleases were purchased from New England Biolabs (Ipswich, MA). C-18 (carbon 11%) reversed phase silica gel was purchased from Silicycle, (Quebec City, Canada). Other material required for thin layer chromatography (TLC), preparative TLC, and flash column chromatography were purchased from EMD Chemicals Inc. (Gibbstown, NJ). All other reagents were obtained from SigmaAldrich and used without further purification, unless indicated otherwise. 267 A Varian Inova-300, Varian UnityPlus-300, Varian Inova-500, and Varian UnityPlus-500 instruments were used to acquire 1 13 H and C NMR spectra. An ESI-MS tandem mass spectrometer (Q-ToF Ultima Global, Waters, Milford, MA) was used to acquire mass analysis of semi-synthetic compounds by direct injection. The biosynthetic compounds were separated and analyzed using a reversed-phase column (Betasil C18, 5 μm, 150 × 2.1 mm, Thermo Fisher Scientific Inc., Waltham, MA) in a capillary HPLC system (CapLC capillary HPLC, Waters, Milford, MA) connected to an electrospray ionization tandem mass spectrometer (ESI-MS/MS) (Q-ToF Ultima Global, Waters, Milford, MA). Other general information not included here is noted elsewhere in the text. 4.2.2. Subcloning the Wild-Type bapt cDNA Microbial and recombinant techniques used for the bapt cDNA are described by Sambrook. 42 Turbo Pfu DNA polymerase (Stratagene) was used in all the PCR reactions. A sticky-end PCR 43 method was conducted to amplify a wild-type T. cuspidata bapt (accession no. AY082804) insertion from a pCWori+ vector with the following primer set [forward primer (5'AATCCATATGAAGAAGACAGGTTCGT-3') and reverse primer (5'- CGCGGATCCTCATAACTTTGACGGA-3', the nucleotides of BamHI and NdeI restriction sites are italicized and underlined]. By this method, the T. cuspidata bapt cDNA was transferred from the previously used pSBET vector 43 to pET28a (Novagen), designated p28wtBAPT. The nucleotide sequence of wild-type bapt and the amino acid sequence of wtBAPT are shown in Figure C 1. This exchange incorporated an N-terminal His6-tag epitope on the expressed BAPT for immunoblot analysis of the expressed protein and purification by His-Select Nickel Affinity 268 Gel (Sigma, St. Louis, MO) binding. The plasmid containing the bapt cDNA and a pET28a vector without an insert were used to separately transform E. coli BL21(DE3) following a described protocol (Stratagene). 4.2.3. Site-Directed Mutagenesis of wt-bapt A single point-mutation G183H was incorporated into the wild-type bapt cDNA using the standard site-directed mutagenesis techniques by changing two nucleotides with an appropriate primer sets [pair 1: forward primer (5'-AGCGAATGTGTATCATAGTGCATGCGAT-3') and reverse primer (5'-CGCGGATCCTCATAACTTTGACGGA-3'); pair 2: forward primer (5'AATCCATATGAAGAAGACAGGTTCGT-3') and reverse primer (5'- ATCGCATGCACTATGATACACATTCGCT -3'); the nucleotide mutation sites are italicized, bolded, and underlined; the nucleotides of BamHI and NdeI restriction sites are italicized and underlined]. The amino acid replacements G183H was made by these mutations in the encoded protein sequence of the wt-bapt; the resultant plasmid was designated p28G-BAPT and used to transform E. coli BL21(DE3) cells. The nucleotide sequence of mutant bapt and the amino acid sequence of mBAPT are shown in Figure C 2. 4.2.4. Expression and Purification of wtBAPT The wild-type bapt was expressed in E. coli, and the respective recombinant enzyme, wtBAPT was harvested according to the previously reported protocol with some modifications in large 22 scale (4 L). In brief, BL21(DE3) bacterial cultures transformed to express the wild-type tbt clone were grown overnight at 37 °C in 5 mL of Luria-Bertani medium (Neogen, Lansing, MI) supplemented with 50 μg/mL kanamycin (Roche). The 5 mL inoculum was separately added to 1 L of Luria-Bertani medium supplemented with the appropriate antibiotic, the cells were grown at 269 37 °C to an OD600 ≈ 1, gene expression was induced with 50 μM isopropyl-β-D-1thiogalactopyranoside (Gold Biotechnology, St Louis, MO), and the cultures were incubated at 18 °C. After 16 h, the OD600 was assessed. The cells were harvested by centrifugation at 4000g for 20 min at 4 °C and resuspended in lysis buffer (50 mM Na3PO4, 300 mM NaCl, pH 8.0) containing 10 mM imidazole to a concentration of 0.3 g cells/mL. The cells were lysed by sonication at 4 °C [5 × 1 min bursts at 50% power with 1 min intervals, and then 2 × 30 s bursts at 70% power with 2 min intervals, using a Misonix XL-2020 sonicator (Farmingdale, NY)]. The homogenate was centrifuged at 15000g to pellet the debris. The soluble fraction was incubated with His-Select Nickel Affinity Gel (3 g, Sigma-Aldrich) in batch mode at 4 °C. After 1 h, the mixture was poured into an Econo column (Bio-Rad, 20 cm × 2.5 cm), and the lysis buffer was drained. The resin was washed with ten column volumes of wash buffer (lysis buffer containing 20 mM imidazole, pH 8.0), and the bound protein was eluted with a step gradient of two column volumes of elution buffer-1 (lysis buffer containing 100 mM imidazole), followed by two column volumes of elution buffer-2 (lysis buffer containing 200 mM imidazole). The imidazole was removed from the purified protein solution by consecutive concentration by centrifugation (30,000 MWCO, YM30 membrane, Millipore, Billerica, MA) and dilution in assay buffer (50 mM Na3PO4, 5% glycerol (v/v), pH 7.4). The recombinant protein migrated to an Rf by SDSPAGE similar to that of the calculated molecular weight of BAPT (~50 kDa), which was visualized by Coomassie blue staining. Kodak ID Image Analysis Software (version 3.6.3) was used to integrate the relative intensity of each type of enzyme band against BSA standards ranging from 2 to 10 mg/mL. The cells transformed with the vector lacking an insert were 270 processed and purified similarly, and by comparison, TBT was not detectable by any of the detection methods described above. 4.2.5. Expression and Purification of mBAPT The mutant bapt was expressed in E. coli, in large-scale (4 L), and the respective recombinant enzyme mBAPT was harvested, processed, purified, and analyzed according to the protocol described in section 4.2.4. 4.2.6. General Protocol for the Synthesis of Arylpropanoyl Coenzyme A Thioesters (38) 3-Phenylpropanoic acid (48), (R)-3-hydroxy-3-phenylpropanoic acid (47), and several other Nprotected arylpropanoic acids (35) were either purchased or synthesized and utilized in syntheses of arylpropanoyl CoA donors via a previously described method that proceeds through a mixed 22,44 ethyl carbonate anhydride. Note: the protecting group manipulations are provided under the corresponding CoA synthesis where necessary. Briefly, Et3N (3.0 μL, 30 μmol) was added to a solution of each arylpropanoic acid (27 μmol) in 5:2 CH2Cl2/THF (v/v, 1.4 mL) under N2 gas. The mixture was stirred for 10 min at 23 °C, ethyl chloroformate (5.8 μL, 60 μmol) was added in one portion, and the reaction was stirred for 1 h at 23 °C. The solvents were evaporated under reduced pressure, and the residue was dissolved in 0.5 mL of t-BuOH. Coenzyme A as the sodium salt (23 mg, 30 μmol dissolved in 0.5 mL of 0.4 M NaHCO3) was added to the solution, and the mixture was stirred for 0.5 h at 23 °C and then quenched with dropwise addition of 1 M HCl, to adjust the pH to 3-5. The solvents were evaporated under vacuum, and the residue was dissolved in H2O (5 mL, pH 5) and loaded onto a C18 silica gel column (10% capped with TMS) 271 that was washed with 100% MeOH (50 mL) and pre-equilibrated with distilled H2O (100 mL, pH 5). The samples were washed with H2O (100 mL, pH 5) and then eluted with 15% MeOH in H2O (50 mL, pH 5). The fractions containing the product, as determined by TLC, were combined, and the solvent was evaporated to yield pure product. If N-protected, the product was deprotected to the desired crude CoA thioester using strategies described earlier. The crude product was then purified using the same purification protocol described above to yield pure arylpropanoyl CoA thioesters (25 – 82% yield). Each arylpropanoyl CoA was quantified using Ellman assay conditions, before and after cleavage of the thioester bond with alkaline hydrolysis 44 following an established procedure. In brief, an aliquot of each of the arylpropanoyl CoA sample was mixed with 2 M NaOH (final concentration 0.5 M) and heated at 50 °C for 30 min. Hydrochloric acid (1 M) was added to lower the pH to 6–7 with 0.5 M NaHCO3 buffer. The content of CoASH was determined in an aliquot of the sample using 10 mM 5,5'-dithiobis(2nitrobenzoic acid) (DTNB or Ellman reagent) in 100 mM K2PO4, pH 8.0 and measuring the absorbance at 412 nm of 13700 M -1 -1 45 cm . The difference in absorbance between the hydrolyzed and non-hydrolyzed samples was used to calculate the arylpropanoyl CoA concentrations. Each synthesized CoA thioester sample was estimated by the Ellman assay to contain 10-30% inorganic salts. 4.2.6.1. Synthesis of (R)-3-Amino-3-phenylpropanoyl CoA (40) To a stirred solution of (R)-3-amino-3-phenylpropanoic acid (39) in 3:2 0.5 M NaHCO3/t-BuOH (v/v, 5 mL) at 23 °C was added di-t-butyl dicarbonate (2 equiv). Upon the completion of the 272 reaction as demonstrated by TLC, it was quenched with 1 M HCl (pH ~ 1.0), diluted with H2O (15 mL). The aqueous fraction was separated and extracted with EtOAc (3 × 25 mL). The combined organic fractions were washed with saturated brine and dried over anhydrous Na2SO4. The organic solvent was evaporated to yield (R)-N-Boc-3-amino-3-phenylpropanoic acid (quantitative). Without any further purification, the product was used for the synthesis of (R)-NBoc-3-amino-3-phenylpropanoyl CoA following the general protocol described in section 4.2.6. (R)-N-Boc-3-amino-3-phenylpropanoyl CoA was then deprotected as follows. To a stirred solution of (R)-N-Boc-3-amino-3-phenylpropanoyl CoA in H2O (pH ~ 5) at 0 °C was added (dropwise) trifluoroacetic acid (99%), and the reaction was monitored by TLC. Upon completion, the reaction was diluted with H2O (5 mL), and then the solvents were evaporated under vacuum. The residue was dissolved in H2O (3 mL) and purified by reverse-phase flash column chromatography as described in section 4.2.6. The product (R)-3-amino-31 phenylpropanoyl CoA (40) was isolated in 62% yield. H NMR (300 MHz, D2O) δ: 0.69 (s, H10'), 0.82 (s, H-11'), 2.20 (m, H-4'), 2.36 (br.m, 1'), 2.91 (d, J = 8.0 Hz, H-2), 2.96 (d, J = 8.0 Hz, H-2), 3.13 (m, H-2'), 3.30 (m, H-2' and H-5'), 3.49 (m, H-5''), 3.75 (m, H-5''), 3.92 (s, H-7'), 4.14 (m, H-9'), 4.46 (br.s, H-4''), 4.71 (s, H-3), 4.98 (br.s, NH2), 6.04 (d, J = 5.0 Hz, H-1''), 7.31 (m, aromatic-H), 8.25 (s, adenine-CH), 8.51 (s, adenine-CH) (cf. Figure C 5 for position of proton and the number of protons at a particular position); LC-ESIMS (negative ion mode), m/z: 913.2 − 2− (M-H) , 456.1 (M-2H) . 273 4.2.6.2. Synthesis of (R)-3-Amino-3-(2-fluorophenyl)propanoyl CoA (42) (R)-N-Boc-3-amino-3-(2-fluorophenyl)propanoic acid was prepared using (R)-3-amino-3-(2fluorophenyl)propanoic acid (41) in quantitative yield following the procedure described in section 4.2.6.1. Then the general protocol described in section 4.2.6 was used to synthesize (R)N-Boc-3-amino-3-(2-fluorophenyl)propanoyl CoA. Finally, (R)-3-amino-3-(2- fluorophenyl)propanoyl CoA (42) was prepared following the deprotection step described in section 4.2.6.1, then purified by the general procedure in section 4.2.6 to form the product (34% 1 yield). H NMR (300 MHz, D2O) δ: 0.69 (s, H-10'), 0.81 (s, H-11'), 2.22 (m, H-4'), 2.35 (m, H2), 2.48 (m, H-2), 2.85 (d, J = 9.0 Hz, H-1'), 3.29 (t, J = 3.9 Hz, H-2'), 3.35 (t, J = 3.9 Hz, H-5'), 3.50 (m, H-5''), 3.74 (m, H-5''), 3.91 (s, H-7'), 4.15 (m, H-9'), 4.48 (s, H-4''), 4.77 (s, H-3), 4.93 (br.m, NH2), 6.06 (d, J = 5.5 Hz, H-1''), 7.33 (m, aromatic-H), 8.29 (s, adenine-CH), 8.52 (s, adenine-CH) (cf. Figure C 6 for position of proton and the number of protons at a particular − − position); LC-ESIMS (negative ion mode), m/z: 931.2 (M-H) , 953.2 (M-2H+Na) . 4.2.6.3. Synthesis of (R)-3-Amino-3-(2-methylphenyl)propanoyl CoA (44) The same protocol used to synthesize 42 was utilized in synthesizing (R)-3-amino-3-(2methylphenyl)propanoyl CoA (44) except that the starting material was (R)-3-amino-3-(21 methylphenyl)propanoic acid (43). The final product yield was 70%. H NMR (300 MHz, D2O) δ: 0.63 (s, H-10'), 0.76 (s, H-11'), 1.16 (s, H-10), 2.17 (d, J = 6.9 Hz, H-4'), 2.27 (t, J = 6.9 Hz, H-2), 2.54 (t, J = 6.9 Hz, H-2), 2.80 (m, H-1'), 3.08 (m, H-2'), 3.24 (m, H-2' and H-5'), 3.43 (m, H-5''), 3.69 (m, H-5''), 3.85 (t, J = 6.6 Hz, H-7'), 4.08 (br.s, H-9'), 4.42 (br.s, NH2), 4.73 (m, H3), 4.89 (t, J = 8.7 Hz, H-4''), 5.02 (t, J = 8.7 Hz, H-3''), 6.01 (d, J = 4.8 Hz, H-1''), 7.00 (m, H-7), 274 7.08 (m, H-6 and H-8), 7.23 (m, H-9), 8.21 (s, adenine-CH), 8.47 (s, adenine-CH) (cf. Figure C 7 for position of proton and the number of protons at a particular position); LC-ESIMS (negative − 2− ion mode), m/z: 927.1 (M-H) , 463.0 (M-2H) . 4.2.6.4. Synthesis of (R)-3-Amino-3-(2-thiophenyl)propanoyl CoA (46) The same protocol used to synthesize 42 was utilized to synthesize (R)-3-amino-3-(2thiophenyl)propanoyl CoA (46), except that the starting material was (R)-3-amino-3-(21 thiophenyl)propanoic acid (45). The final product yield was 82%. H NMR (300 MHz, D2O) δ: 0.70 (s, H-10'), 0.82 (s, H-11'), 2.24 (t, J = 4.2 Hz, H-4'), 2.90 (t, J = 4.2 Hz, H-1'), 3.19 (t, J = 3.9 Hz, H-2'), 3.30 (m, H-2), 3.37 (t, J = 3.9 Hz, H-5'), 3.49 (m, H-5''), 3.75 (m, H-5''), 3.94 (s, H-7'), 4.14 (m, H-9'), 4.59 (s, NH2), 4.89 (t, J = 4.5 Hz, H-4''), 5.03 (t, J = 4.8 Hz, H-3''), 6.08 (d, J = 3.3 Hz, H-1''), 6.97 (m, H-6), 7.13 (t, J = 2.1 Hz, H-7), 7.38 (m, H-5), 8.25 (s, adenine-CH), 8.51 (s, adenine-CH) (cf. Figure C 8 for position of proton and the number of protons at a − 2− particular position); LC-ESIMS (negative ion mode), m/z: 919.1 (M-H) , 459.0 (M-2H) , 941.1 − (M-2H+Na) . 4.2.6.5. Synthesis of (R)-3-Hydroxy-3-phenylpropanoyl CoA (31) (R)-3-Hydroxy-3-phenylpropanoyl CoA (31) was synthesized using (R)-3-hydroxy-3- phenylpropanoic acid (47) as the starting material based on the general protocol described in section 4.2.6, except the solvent mixture (5:1 CH2Cl2: THF (v/v, 1.8 mL) for the step b of the 1 reaction scheme (Figure 4.12) was varied slightly to yield 25% product. H NMR (300 MHz, D2O) δ: 0.66 (s, H-10'), 0.78 (s, H-11'), 2.39 (m, H-4'), 2.80 (m, H-2), 3.20 (m, H-1'), 3.27 (m, 275 H-2' and H-5'), 3.46 (dd, J = 4.8 Hz and J = 9.6 Hz, H-5''), 3.80 (dd, J = 4.8 Hz and J = 9.6 Hz, H-5''), 3.96 (s, H-7'), 4.06 (s, H-9'), 4.46 (dd, J = 2.7 Hz and J = 5.3 Hz, H-4''), 4.60 – 4.82 (m, H-2'' and H-3''), 5.80 (m, H-3), 6.40 (d, J = 6.9 Hz, H-1''), 7.21 – 7.27 (m, aromatic-H), 8.20 (s, − adenine-CH), 8.43 (s, adenine-CH); LC-ESIMS (negative ion mode), m/z: 914.0 (M-H) , 456.5 2− − (M-2H) , 936.0 (M-2H+Na) . 4.2.6.6. Synthesis of 3-Phenylpropanoyl CoA (30) 3-Phenylpropanoyl CoA (30) was synthesized by the general protocol described in section 4.2.6, 1 starting from phenylpropanoic acid (48) to yield 51% product. H NMR (300 MHz, D2O) δ: 0.67 (s, H-10'), 0.81 (s, H-11'), 2.26 (t, J = 6.0 Hz, H-4'), 2.44 (m, H-1'), 3.16 (d, J = 5.5 Hz, H-2'), 3.25 (m, H-3), 3.31 (d, J = 6.5 Hz, H-5'), 3.48 (m, H-5''), 3.75 (m, H-5''), 3.94 (s, H-7'), 4.14 (s, H-9'), 4.50 (br.s, H-4''), 4.68 (m, H-2'' and H-3''), 6.05 (d, J = 5.0 Hz, H-1''), 7.08 – 7.26 (m, aromatic-H), 8.12 (s, adenine-CH), 8.45 (s, adenine-CH); LC-ESIMS (negative ion mode), m/z: − 2− − 898.0 (M-H) , 448.5 (M-2H) , 920.0 (M-2H+Na) . 4.2.7. wtBAPT Activity Assay Purified wtBAPT (100 μg) (purified according to section 4.2.4), was incubated in assay buffer (1 mL of 50 mM Na3PO4, 5% glycerol (v/v), pH 7.4), with 1 mM baccatin III (19), and 1 mM (R)3-amino-3-phenylpropanoyl CoA (40). After 4 h at 31 °C, the assay was quenched and extracted with EtOAc (2 × 4 mL). The organic fractions were evaporated in vacuo, and the resultant residue was dissolved in MeCN (200 μL). A 10-μL aliquot of the sample was loaded onto a reversed-phase column (Betasil C18, 5 μm, 150 × 2.1 mm, Thermo Fisher Scientific Inc., Waltham, MA) connected to a capillary HPLC system (CapLC capillary HPLC, Waters, Milford, 276 MA). The column was eluted at 0.3 mL/min with a linear gradient of solvent A: solvent B [solvent A: 99.50% H2O with 0.5% H3PO4 (v/v); solvent B: 99.50% MeCN with 0.5% H3PO4 (v/v)] from 70:30 (v/v) to 0:100 (v/v) over 7 min, holding at 100% solvent B for 2 min, and returning to the initial conditions over 1.10 min, with a 50 s hold. The effluent from the HPLC column was directed to an ESI-MS/MS (Q-ToF Ultima Global, Waters, Milford, MA), and the + + molecular ions ([M + H] and [M + Na] ) of the de novo biosynthetic (R)-β-phenylalanoyl baccatin III analyte were detected in single-stage and tandem mass spectrometer modes (Figure 4.13 and Figure 4.14). Control assays in which the co-substrate and/or enzyme was omitted were processed and analyzed identically to the methods described above. 4.2.8. mBAPT Activity Assay Purified mBAPT (100 μg) (purified according to section 4.2.5), was incubated in assay buffer (1 mL of 50 mM Na3PO4, 5% glycerol (v/v), pH 7.4), with 1 mM baccatin III (19), and 1 mM (R)3-amino-3-phenylpropanoyl CoA (40). After 4 h at 31 °C, the assays were quenched and extracted with EtOAc (2 × 4 mL). The organic fractions were evaporated in vacuo, and the resultant residue was dissolved in 200 μL of MeCN. A 10 μL aliquot was subjected to + electrospray ionization as described in section 4.2.7, and the molecular ions ([M + H] and [M + + Na] ) of the de novo biosynthetic (R)-β-phenylalanoyl baccatin III were analyzed in both singlestage and tandem mass spectrometer modes. Control assays in which the co-substrate and/or enzyme was omitted were processed and analyzed identically to the methods described above. 277 4.2.9. Substrate Specificity Studies of wtBAPT To identify other productive co-substrates, purified wtBAPT (100 μg) was incubated in assay buffer (1 mL of 50 mM Na3PO4, 5% glycerol (v/v), pH 7.4), with 1 mM baccatin III (19), and 1 mM (R)-3-amino-3-(2-fluorophenyl)propanoyl CoA (42) or (R)-3-amino-3-(2- thiophenyl)propanoyl CoA (46). After 4 h at 31 °C, the assays were quenched and extracted with EtOAc (2 × 4 mL). The organic fractions were evaporated in vacuo, and the resultant residue was dissolved in MeCN (200 μL). A 10-μL aliquot was subjected to electrospray ionization as + + described in section 4.2.7, and the molecular ions ([M + H] and [M + Na] ) of the de novo biosynthetic products were were analyzed in both single-stage and tandem mass spectrometer modes (Figure 4.15, Figure 4.16, Figure C 3, and Figure C 4). Control assays, in which the cosubstrate and/or enzyme were omitted, were processed and analyzed identically to the methods described above. 4.2.10. Assessing Putative Substrate-Assisted Catalysis of BAPT Purified wtBAPT and mBAPT (100 μg of each) were separately incubated 4 h at 31 °C in assay buffer (1 mL of 50 mM Na3PO4, 5% glycerol (v/v), pH 7.4), with 1 mM baccatin III (19), and separately with each CoA thioester substrate (R)-3-amino-3-phenylpropanoyl CoA (40), (R)-3hydroxy-3-phenylpropanoyl CoA (31), and phenylpropanoyl CoA (30), each at 1 mM. The assays were quenched and extracted with EtOAc (2 × 4 mL). The organic fractions were evaporated in vacuo, and the resultant residue was dissolved in MeCN (200 μL). A 10-μL aliquot was subjected to electrospray ionization as described in section 4.2.7, and the molecular ions ([M + + + H] and [M + Na] ) of the de novo biosynthetic products were analyzed in single-stage and 278 tandem mass spectrometer modes. Control assays in which the co-substrate and/or enzyme was omitted were processed and analyzed identically to the methods described above. 4.2.11. Relative Inhibition of wtBAPT catalysis by Other Phenylpropanoyl CoA Thioesters As a preliminary method to check whether wtBAPT catalysis was inhibited by other phenylpropanoyl CoA thioesters, (1 mM (R)-3-hydroxy-3-phenylpropanoyl CoA (31) and phenylpropanoyl CoA (30)) were separately incubated in the presence of (R)-3-amino-3phenylpropanoyl CoA (40) (200 μM), purified wtBAPT (100 μg), and 1 mM baccatin III (19) in a 1-mL assay for 3 h at 31 °C. The assays were quenched and docetaxel (2) (5 μg) was added as the internal standard to correct for losses during the extraction. The assays were extracted with EtOAc (2 × 4 mL), the organic fractions were evaporated in vacuo, and the resultant residue was dissolved in 200 μL of MeCN. A 10-μL aliquot was analyzed by electrospray ionization tandem + mass spectrometry as described in section 4.2.7, and the molecular ions ([M + H] and [M + + Na] ) of the biosynthetic product, (3'R)-β-phenylalanoyl baccatin III were detected in singlestage mass spectrometer mode. The peak areas corresponding to the relative abundance of [M + + + H] and [M + Na] of (3'R)-β-phenylalanoyl baccatin III in each assay were reported. The detected abundance of biosynthetic products were within the linear range of detection of the + + mass spectrometer, so that the peak areas for [M + H] and [M + Na] were comparable among the assays. Also the diagnosed peak areas of (3'R)-β-phenylalanoyl baccatin III in each assay + + were corrected with respect to the area under the [M + H] and [M + Na] ion peaks of the internal standard docetaxel (2). The relative inhibition of wtBAPT catalysis by other 279 phenylpropanoyl CoA thioesters was determined by comparing the normalized peak areas for [M + + + H] and [M + Na] of (3'R)-β-phenylalanoyl baccatin III in each assay. A control assay in which the enzyme was omitted was processed and analyzed identically to the methods described above. 4.3. Results and Discussion 4.3.1. Expression and Activity Assay of the Taxus Baccatin III 13α-O-3amino-3-phenylpropanoyltransferase (BAPT) In a previous study wild-type BAPT (wtBAPT) was recombinantly expressed from pSBET in E. 22 coli. The overexpressed wtBAPT partitioned primarily to the insoluble fraction as inclusion bodies. The attempts of partial purification of the expressed enzyme using anion exchange chromatography resulted in a significant loss of the activity, likely due to the instability of the enzyme. Therefore, the previous characterization of wtBAPT was conducted using crude soluble 22 enzyme lysate. In this study, the wild-type bapt cDNA was transferred from the pSBET vector into pET28a which encoded a His6-tag epitope; the resultant plasmid was designated as p28wtBAPT and was used to transform E. coli BL21(DE3) cells. The N-terminal His6-tag epitope encoded in the plasmid allowed for affinity purification of wtBAPT by nickel-affinity chromatography and verification by western immunoblotting, using an anti-His6 antibody. SDSPAGE analysis of the partially purified wtBAPT (Figure 4.11) revealed three dominant proteins (each ~15% of the total protein) at Rf values consistent with those of chaperones GroEL, GroES, 46,47 and Dnak (at ~ 80, 70 and 69.0 kDa, respectively). 280 However, there was sufficient partially purified, active wtBAPT to conduct further studies. Further purification of wtBAPT will be attempted in future investigations. L1 L2 100 kDa 75 kDa BAPT 50 kDa 35 kDa 25 kDa Figure 4.11 – SDS polyacrylamide gel electrophoresis and Coomassie blue staining of resolved proteins of partially purified, soluble fraction from E. coli BL21(DE3) transformed with the pET28a vector, expressing wild-type bapt cDNA insert. L1 contains molecular weight standards (Lonza); L2 shows the resolved proteins from 10 μg of total soluble protein from E. coli transformed with the p28wtBAPT plasmid expressing the wild-type bapt cDNA insert after purification by nickel-affinity resin chromatography. The other dominant proteins in the soluble enzyme extract pointed using arrows are likely GroEL, GroES, and Dnak (~ 80, 70 and 69.0 46,47 kDa, respectively). In the earlier report on the characterization the recombinantly expressed wtBAPT, the transfer of the (3RS)-β-phenylalanoyl moiety from the corresponding CoA thioester to baccatin III (19) was 22 investigated (Figure 4.5). 22 deoxypaclitaxel, The resultant biosynthetic product (29) was a single 3'-epimer of 2'- but the absolute stereochemistry was not confirmed. It was suggested, however, that the 3'-amino was the same as that of paclitaxel. In this study, the single enantiomer (R)-3-amino-3-phenylpropanoyl CoA (40) was used to screen the function of wtBAPT. (R)-3- 281 22,44 Amino-3-phenylpropanoyl CoA (40) was synthesized by adapting reported methods and implementing modifications as needed (Figure 4.12). The activity assay of wtBAPT with baccatin III (19) and the single enantiomer, (R)-3-amino-3-phenylpropanoyl CoA (40) was productive and the product formation was detected by LC-MS (Figure 4.13) and verified by tandem mass spectroscopy (Figure 4.14) by identifying the diagnostic fragment ions (m/z: 734.4 + + + [M + H] , 569.3 [m/z 734.4 – RCOOH at C-13] , 509.3 [m/z 569.3 – HOAc] , 387.2 [m/z 509.3 + + + – BzOH] , 327.2 [m/z 387.2 – HOAc] , and 166.1 [RCOOH at C-13 + H] ; R is (R)-3-amino-3phenylacetyl). Although the product stereochemistry could not be verified by mass spectrometry techniques, the transfer of the acyl group from (R)-3-amino-3-phenylpropanoyl CoA (40) to C13 hydroxyl group of baccatin III (19) suggested that the (R)-3-amino-3-phenylpropanoyl baccatin III diastereoisomer was formed. This idea was further supported by the absence of any racemase/mutase activity of the wtBAPT and the well characterized 3'-stereochemistry of (1). However, the stereochemistry of the wtBAPT reaction and the evaluation of the steady-state kinetic parameters will be attempted in future investigations. 282 Phenyl/heterolylpropanoic acids (34) N-Bocphenyl/heterolyl propanoic acids (35) Corresponding N-protected/other mixed anhydrides (36) N-Protected/ other phenyl/heterolyl propanoyl CoA thioesters (37) Phenyl/heterolyl propanoyl CoA thioesters (38) Figure 4.12 – Synthesis of phenyl/heterolyl propanoyl CoA thioesters (40, 42, 44, 46, 31, a,b,c,d a b 30) ( di-t-butyl dicarbonate, NaHCO3, t-BuOH, quantitative; Et3N, ethylchloroformate, c d º CH2Cl2, THF; CoASH, NaHCO3, t-BuOH, 25 – 82% yield; Trifluoroacetic acid, H2O, 0 C, 25 – 82% yield. The descriptions of the utilized phenyl/heterolylpropanoic acids and synthesized phenyl/heterolyl propanoyl CoA thioesters are shown. The amino functionality of all the 3amino-3-phenylpropanoic acids was protected using t-butoxy group (Boc); if protected, the deprotection was performed to gain the corresponding 3-amino-3-phenyl/heterolylpropanoyl CoA thioester. (R)-3-Amino-3-phenylpropanoic acid (39) was used to synthesize (R)-3-Amino-3phenylpropanoyl CoA (40) R2: NH2; R1, R3, R4: H; X: phenyl. (R)-3-Amino-3-(2fluorophenyl)propanoic acid (41) was used to synthesize (R)-3-Amino-3-(2fluorophenyl)propanoyl CoA (42) R2: NH2; R1, R3, R4: H; X: 2-fluorophenyl. (R)-3-Amino-3(2-methylphenyl)propanoic acid (43) was used to synthesize (R)-3-Amino-3-(2methylphenyl)propanoyl CoA (44) R2: NH2; R1, R3, R4: H; X: 2-methylphenyl. (R)-3-Amino-3(2-thiophenyl)propanoic acid (45) was used to synthesize (R)-3-Amino-3-(2thiophenyl)propanoyl CoA (46) R2: NH2; R1, R3, R4: H; X: 2-thiophenecarbonyl. (R)-3Hydroxy-3-phenylpropanoic acid (47) was used to synthesize (R)-3-hydroxy-3-phenylpropanoyl 283 CoA (31) R4: OH; R1, R2, R3: H; X: phenyl. 3-Phenylpropanoic acid (48) was used to synthesize 3-phenylpropanoyl CoA (30) R1, R2, R3, R4: H; X: phenyl Product 100 Substrate % Relative Abundance 80 60 40 20 0 0 2 4 6 8 10 12 14 Retention Time (min) Figure 4.13 – The LC-MS chromatogram of extracted wtBAPT activity assay with baccatin III (19) and (R)-3-amino-3-phenylpropanoyl CoA (49). The corresponding peaks of biosynthetic product ((R)-3'-amino-3-phenylpropanoyl baccatin III) and the taxane substrate (19) are labeled. 284 166.1 100 % Relative Abundance 80 509.3 60 40 569.3 20 327.2 387.2 734.4 0 100 200 300 400 500 600 700 800 m/z Figure 4.14 – MS/MS fragment ion profile of the biosynthesized (R)-3'-amino-3phenylpropanoyl baccatin III derived by catalysis of wtBAPT is shown. Diagnostic ions are + + found at m/z: 734.4 [M + H] , 569.3 [m/z 734.4 – RCOOH at C-13] , 509.3 [m/z 569.3 – + HOAc]+, 387.2 [m/z 509.3 – BzOH] , 327.2 [m/z 387.2 – HOAc]+, 166.1 [RCOOH at C-13 + H]+, where R = (R)- 3-amino-3-phenylacetyl. 285 4.3.2. Substrate Specificity Studies of wtBAPT with Synthetically-derived Phenyl/Heterolylpropanoyl CoA Thioesters and Baccatin III (19) Preliminary studies on the substrate specificity of wtBAPT were conducted by incubating synthetically-derived phenyl/heterolylpropanoyl CoA thioesters, (R)-3-amino-3-(2- fluorophenyl)propanoyl CoA (42) and (R)-3-amino-3-(2-thiophenyl)propanoyl CoA (46) (each at 1 mM) (Figure 4.12) with wtBAPT and baccatin III (19) in separate assays, and products were verified as 13-O-acylbaccatin III analogues by LC-MS. The first-stage mass spectrometer was + set to select for the [M + H] ion of the corresponding product, which was directed into a fragmentation chamber, and the resulting fragment ions were analyzed by the second-stage mass spectrometer set to the scan mode. Typical diagnostic fragment ions of 13-O-acylbaccatin III + + analogues possessed m/z of ([M + H – RCOOH] , [M + H– RCOOH – HOAc] , [M + H– + + RCOOH – HOAc – BzOH] , [M + H– RCOOH – BzOH – 2HOAc] , [M + H– RCOOH – + + BzOH – 2HOAc – H2O] , and [RCOOH + H] ; R is the 13-O-acyl group transferred from the CoA donor to (19) by wtBAPT catalysis (Figure 4.15 and Figure 4.16 ) 286 184.1 100 % Relative Abundance 80 60 509.3 40 20 327.2 309.2 387.2 569.3 0 0 100 200 300 400 500 600 700 800 m/z Figure 4.15 – MS/MS fragment ion profile of the biosynthesized (R)-3'-Amino-3-(2fluorophenyl)propanoyl baccatin III derived by catalysis of wtBAPT is shown. Diagnostic ions + are found at m/z: 569.3 [m/z 752.4 ([M + H] +) – RCOOH at C-13] , 509.3 [m/z 569.3 – HOAc]+, + + 387.2 [m/z 509.3 – BzOH] , 327.2 [m/z 387.2 – HOAc]+, 309.2 [m/z 327.2 – H2O] , 184.1 [RCOOH at C-13 + H]+, where R = (R)- 3-amino-3-(2-fluorophenyl)acetyl. 287 172.1 100 155.1 509.3 % Relative Abundance 80 60 327.2 40 569.3 309.2 387.2 20 449.2 0 100 200 300 400 500 600 700 800 m/z Figure 4.16 – MS/MS fragment ion profile of the biosynthesized (R)-3'-Amino-3-(2thiophenyl)propanoyl baccatin III derived by catalysis of wtBAPT is shown. Diagnostic ions are + found at m/z: 569.3 [m/z 740.4 ([M + H] +) – RCOOH at C-13] , 509.3 [m/z 569.3 – HOAc]+, + + 449.2 [m/z 509.3 – HOAc] , 387.2 [m/z 509.3 – BzOH] , 327.2 [m/z 387.2 – HOAc]+, 309.2 + [m/z 327.2 – H2O] , 172.1 [RCOOH at C-13 + H]+, 155.1 [RCOOH at C-13 + H – NH3]+, where R = (R)- 3-amino-3-(2-thiophenyl)acetyl. Both the 3-amino-phenyl- and -heterolylpropanoyl CoA thioester at apparent saturation (1 mM) were productive in assays containing wtBAPT and baccatin III (19) to form C-13 modified taxane analogues. The results also demonstrated the regiospecificity of wtBAPT with different, yet analogous substrates. However, more acyl CoA donors need to be tested to fully evaluate the substrate specificity of wtBAPT as a biocatalyst for producing paclitaxel analogues with modified C-13 side chains. An array of proposed acyl CoA donors is shown in Figure 4.17. 288 These co-substrates are designed to examine the effect of the substituent/s of the phenyl group on BAPT catalysis, the possibility of catalyzing the transfer of heterolyl/alkanyl/alkenylpropanoyl side chains to the hydroxyl at C-13 of baccatin III (19). More importantly, the substrate will allow us to explore the prospect of developing efficacious paclitaxel analogues with modified C13 side chains (Figure 4.1 and Figure 4.2) through the biocatalytic route (Figure 4.4). If BAPT efficiently catalyses the transfer of the proposed phenylisoserionoyl CoA thioesters (Figure 4.17) to the 13-hydroxyl of baccatin III, the previously suggested biocatalytic route can potentially be simplified by eliminating the cytochrome P450 hydroxylase (Figure 4.4, step e). Once the array of productive co-substrates is identified, the specificity constant (kcat/KM) of wtBAPT for each functional CoA donor will be examined in future investigations. X: m-/p-F series (50) X: o-/m-/p-CH3 series (51) X: o-/m-/p-C(CH3)3 series (52) X: o-/m-/p-OH series (53) X: o-/m-/p-NO2 series (54) R: 3-thiophenyl (55), R: 2/3-furanyl series (56) R: 5-thiazolyl (57), R: 2/3-pyrrolyl series (58) R: 2/3/4-pyridinyl series (59) R: 2-(3-fluoropyridinyl) (60) R: 2-methylpropyl (61), R: 2-methylpropenyl (62) R: 2,2-dimethylcyclopropyl (63), R: t-butyl(64) R: o-/m-/p-fluorophenyl series (65), R: o-/m-/p-methylphenyl series (66) R: 2/3-thiophenyl series (67), R: 2/3-furanyl series (68) R: 2/3/4-pyridinyl series (69), R: 2-(3-fluoropyridinyl) (70), R: 2-methylpropyl (71) R: 2-methylpropenyl (72), R: 2,2-dimethylcyclopropyl (73), R: t-butyl(74) Figure 4.17 – An array of proposed acyl CoA donors which are conceivably catalyzed by wtBAPT to transfer the acyl group to 13-hydroxyl of baccatin III (19) 289 4.3.3. Substrate-Assisted Catalysis of BAPT wtBAPT is hypothesized to utilize the 3-amino group of the acyl side chain of productive CoA thioester substrates as the general base in the catalytic mechanism (Figure 4.9). To assess whether wtBAPT reaction proceeds through substrate-assisted catalysis (SAC), a series of, phenylpropanoyl CoA thioesters (Figure 4.10) were synthesized according to reported methods 22,44 (Figure 4.12). wtBAPT (100 µg) was incubated for 4 h in 1 mL of assay buffer with 1 mM baccatin III (19), and each CoA substrate ((R)-3-amino-3-phenylpropanoyl CoA (40), (R)-3-hydroxy-3-phenylpropanoyl CoA (31), and 3-phenylpropanoyl CoA (30)) at 1 mM. The products were isolated and analyzed by LC-tandem mass spectroscopy to detect the molecular + + ions ([M + H] and [M + Na] ) of the de novo biosynthetic products. Assuming the (R)stereochemistry at 3' position, (R)-β-phenylalanoyl baccatin III was observed in the sample incubated with the aminated substrate 40; however, biosynthetic products of 3-hydroxy- (31) and 3-dihydro- (30) phenylpropanoyl CoA thioester were not detected. Encouraged by the results of this pilot study that suggested a substrate-assisted catalytic pathway for BAPT, a preliminary inhibition study of wtBAPT catalysis was conducted to assess if the non-aminated 30 and 31 CoA thioesters competitively inhibited the reaction with productive (R)-3-amino-3phenylpropanoyl CoA (40). Both 30 and 31 (at 1 mM) were incubated separately for 4 h with baccatin III (1 mM), wtBAPT (100 μg), and 40 (200 μM), the products were extracted, and + + analyzed by LC-MS while monitoring for molecular ions ([M + H] and [M + Na] ) of the biosynthetic product, (R)-β-phenylalanoyl baccatin III. After correcting for losses during sample workup by employing an internal standard and subtracting background signals, the total ion + + abundance contribution from [M + H] and [M + Na] molecular ions of (3'R)-β-phenylalanoyl 290 baccatin III was tabulated (cf. Figure 4.18). These data demonstrated that the rate at which wtBAPT catalyzed the transfer of the acyl group of 40 to 13-hydroxyl of (19) to form (3'R)-βphenylalanoylbaccatin III was reduced by 2- and 7-fold by co-incubation of 30 and 31, respectively. Understandably, the type of inhibition cannot be interpreted from this single-point datum; however, it is anticipated that the inhibition is likely competitive as demonstrated for other Taxus acyltransferases tested with structurally similar substrates. In future studies, to assess the exact mode of inhibition of wtBAPT by each impaired 48,49 CoA thioester, standard Michaelis-Menten inhibition kinetic studies will be conducted. Irreversible inhibition will be examined by the dilution effects and/or time dependence of the 48 inhibition effect. If necessary, other proposed 3-amino phenylpropanoyl CoA thioesters will be synthesized and examined with the inhibitor CoA thioesters. 291 [M + H]+ and [M + Na]+ molecular ions of (R)-β-phenylalanoyl baccatin III The total ion abundance of 12000 10000 8000 6000 4000 2000 0 1 2 3 The type of phenylpropanoyl CoA thioester Figure 4.18 – A plot of the total ion abundance of [M + H]+ and [M + Na]+ molecular ions of (3'R)-β-phenylalanoyl baccatin III vs the type of phenylpropanoyl CoA thioester used in the inhibition studies as the inhibitor; performed in duplicate. 1: (R)-3-amino-3-phenylpropanoyl CoA (40), 3: 3-phenylpropanoyl CoA (30), 2: (R)-3-hydroxy-3-phenylpropanoyl CoA (31) The glycine residue of the GXXXD motif in the BAPT amino acid sequence was reconstituted by site-directed mutagenesis to the conserved HXXXD motif found in other Taxus acyltransferases and other member of the large BAHD acyltransferase superfamily., It was imagined that the G H modified BAPT could catalyze the transfer of impaired CoA thioesters (lacking the 3-amino functionality) (Figure 4.10) to 13-hydroxyl of corresponding taxane substrate(s). In this scenario, the histidine would serve as the general base in the catalysis rather than the 3'-amino group of the substrate. However, none of the tested phenylpropanoyl CoA thioesters (with or without 3-amino functionality, 40, 30, 31) were productive with the modified 292 BAPT. There are several, as yet unidentified, reasons why this modified BAPT enzyme was not functional, including incorrect folding or reduced structural integrity of the enzyme caused by the site-mutation. To verify this postulation, purified G H modified BAPT will be scanned by circular dichroism (CD) spectroscopy and compared to the CD specturm of the purified wtBAPT in its native, active state. 4.4. Conclusion To date, only acetyl and phenylisoserinoyl have been identified at the C-13 hydroxy group of 50 naturally occurring taxanes, without considering the N-substituents of the C-13 side chain. However, many efficacious paclitaxel analogues with modified C-13 side chains have been reported (Figure 4.1 and Figure 4.2). An understanding of the substrate scope of the recombinant BAPT enzyme can potentially be advantageous in developing paclitaxel (1) and/or its analogues through the proposed biocatalytic route (Figure 4.4) and/or to substitute several steps in the semisynthetic route (Figure 4.3). In this study, several arylpropanoyl CoA thioesters were utilized in identifying the substrate scope of BAPT and found to be productive. Therefore, BAPT can foreseeably be utilized as a biocatalyst in developing C-13 modified paclitaxel analogues. Also, the catalytic mechanism of BAPT was analyzed in this study by rationally designing 3-amino impaired phenylpropanoyl CoA thioesters, by designing inhibition assays, and through mutagenesis studies. The results verified that the 3-amino functionality of the cosubstrate likely assists the BAPT catalysis acting as the general base in enzymatic reaction. Once structural data of BAPT and/or other Taxus acyltransferases become available, further insight into the catalytic mechanism of BAPT can be delineated. 293 4.5. Future Directions 4.5.1. Expansive Substrate Specificity Studies of BAPT The current study showed the utility of (R)-3-amino-3-phenylpropanoyl CoA, (R)-3-amino-3-(2fluorophenyl)propanoyl CoA, and (R)-3-amino-3-(2-thiophenyl)propanoyl CoA as substrates by wtBAPT hinting at the expansive substrate scope of BAPT. However, to fully understand the potential use of BAPT as a biocatalyst in developing paclitaxel analogues with variously modified C-13 side chains, further examination of wtBAPT with proposed aryl/alkanylpropanoyl and phenylisoserinoyl CoA thioesters (Figure 4.17) is necessary. The additional potential advancements of the future study are described in the results and discussion (section 4.3.2). 4.5.2. The Coupling of BAPT to a Promiscuous CoA Ligase Although the synthetically-derived aroylpropanoyl CoA thioesters were used in identifying the substrate scope of BAPT enzyme in this study, the proposed biocatalytic route to produce paclitaxel and/or its analogues (Figure 4.4) utilizes the biosynthetically-derived β-phenylalanine CoA (22) as the co-substrate in BAPT reaction (step d). The chemical synthesis of arylpropanoyl CoA thioesters is a multi-step strategy including two protection-deprotection manipulations (when functional groups are on the second and/or third carbons), several extractions, and a couple of flash column chromatographic steps (Figure 4.12). More importantly, the reported overall product yields for the synthesized arylpropanoyl CoA thioesters are not that high (25 – 82%). Therefore, the investigation of a biocatalytic route to produce arylpropanoyl CoA thioesters will be useful in developing paclitaxel and/or its analogues. According to the proposed biocatalytic route, the abundant α-phenylalanine (20) gets converted to β-phenylalanine (21) through the Taxus phenylalanine aminomutase (PAM) catalysis. In the next step β-phenylalanine 294 (21) is converted to β-phenylalanine CoA (22) by a Taxus phenylalanine CoA ligase (step b and c in Figure 4.4). Although PAM has been extensively studied and verified to act as a 51-53 biocatalyst, the Taxus β-phenylalanine CoA ligase is not yet discovered. There are numerous reports on the aroyl CoA ligases and the CoA ligases involved in phenylpropanoyl biosynthesis. 54-64 Unfortunately, none of these reported CoA ligases have been shown to utilize β-phenylalanines as substrates. According to the general mechanism of acyl CoA biosynthesis, first the substrate (75) reacts with ATP to form an activated acyl-adenylate intermediate (76) with the simultaneous release of pyrophosphate (PPi). Then the adenylate group is replaced by CoA and AMP is released forming acyl CoA (77) (Figure 4.19). Likewise, β-phenylalanine (21) needs to be activated in a similar mechanism to form β-phenylalanine CoA thioester (22). How are activated amino acids including β-phenylalanine (21) formed in biological systems? The amino acid activation does not occur by CoA ligases; instead, it is generally achieved by aminoacyl tRNA-synthetases and the adenylation domains of non-ribosomal peptide synthetases. The adenylating activation step (Figure 4.19) occurs similar to the acyl CoA ligases in members 65,66 of these two enzyme classes, while the second step varies. However, some amino-acyl CoA ligase activity of these enzyme classes has been reported including the formation of β67,68 phenylalanines, suggesting that there might also be amino-acyl CoA ligases which catalyze the formation of amino-acyl CoA thioesters. In fact, a CoA ligase from Penicillium chrysogenum has found recently to synthesize amino-acyl CoA thioesters including both (R) and (S)-β69 phenylalanine CoA thioesters. The highest catalytic activity was reported for (R)-β- 295 phenylalanine forming (R)-β-phenylalanine CoA thioester with the wild-type enzyme; a modification of the enzyme further enhanced the catalytic efficiency for the same substrate. O R SCoA Carboxylic acid (75) Acyl-adenylate intermediate (76) Acyl CoA (77) Figure 4.19 – The general two-step mechanism of acyl CoA biosynthesis. Note – ATP is adenosine 5'-triphosphate; AMP is adenosine 5'-monophosphate Conceivably, a coupled system of this β-phenylalanine CoA ligase and BAPT can be applied in developing an efficient biocatalytic route to produce paclitaxel and/or its analogues. Also there are several reported adenylation domains of non-ribosomal peptide synthetases, which are 70-74 capable of forming adenylated-aroylpropanoyl intermediates with higher efficiency. Coupling of these enzyme domains to BAPT in the presence of CoASH is also being investigated (unpublished data). 4.5.3. A Detailed Understanding of the Catalytic Mechanism of BAPT A complete understanding of the catalytic mechanism of BAPT is vital in developing it as a biocatalyst. Therefore, the studies of substrate-assisted catalytic mechanism of BAPT can be further developed as described in the results and discussion (section 4.3.3). Also, once the structural data of purified wtBAPT or other Taxus acyltransferases become available, the designing of mutants based on the active site morphology for probing the reaction mechanism will be less complicated. 296 APPENDIX C 297 APPENDIX C atgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggcagccat 60 M G S S H H H H H H S S G L V P R G S H atgaagaagacaggttcgtttgcagagttccatgtgaatatgattgagcgagtcatggtg 120 M K K T G S F A E F H V N M I E R V M V agaccgtgcctgccttcgcccaaaacaatcctccctctctccgccattgacaacatggca 180 R P C L P S P K T I L P L S A I D N M A agagctttttctaacgtattgctggtctacgctgccaacatggacagagtctctgcagat 240 R A F S N V L L V Y A A N M D R V S A D cctgcaaaagtgattcgagaggctctctccaaggtgctggtttattattacccttttgct 300 P A K V I R E A L S K V L V Y Y Y P F A gggcggctcagaaataaagaaaatggggaacttgaagtggagtgcacagggcagggtgtt 360 G R L R N K E N G E L E V E C T G Q G V ctgtttctggaagccatggctgacagcgacctttcagtcttaacagatctggataactac 420 L F L E A M A D S D L S V L T D L D N Y aatccatcgtttcagcagttgattttttctctaccacaggatacagatattgaggacctc 480 N P S F Q Q L I F S L P Q D T D I E D L catctcttgattgttcaggtaactcgttttacatgtgggggttttgttgtgggagcgaat 540 H L L I V Q V T R F T C G G F V V G A N gtgtatggtagtgcatgcgatgcaaaaggatttggccagtttcttcaaagtatggcagag 600 V Y G S A C D A K G F G Q F L Q S M A E atggcgagaggagaggttaagccctcgattgaaccgatatggaatagagaactggtgaag 660 M A R G E V K P S I E P I W N R E L V K ctagaacattgtatgcccttccggatgagtcatcttcaaattatacatgcacctgtaatt 720 L E H C M P F R M S H L Q I I H A P V I gaggagaaatttgttcaaacatctcttgttataaactttgagataataaatcatatcaga 780 E E K F V Q T S L V I N F E I I N H I R cgacgcatcatggaagaacgcaaagaaagtttatcttcatttgaaattgtagcagcattg 840 R R I M E E R K E S L S S F E I V A A L 298 gtttggctagcaaagataaaggcttttcaaattccacatagtgagaatgtgaagcttctt 900 V W L A K I K A F Q I P H S E N V K L L tttgcaatggacttgaggagatcatttaatccccctcttccacatggatactatggcaat 960 F A M D L R R S F N P P L P H G Y Y G N gcctttggtattgcatgtgcaatggataatgtccatgaccttctaagtggatctcttttg 1020 A F G I A C A M D N V H D L L S G S L L cgcactataatgatcataaagaaatcaaagttctctttacacaaagaactcaactcaaaa 1080 R T I M I I K K S K F S L H K E L N S K accgtgatgagctcatctgtagtagatgtcaatacgaagtttgaagatgtagtttcaatt 1140 T V M S S S V V D V N T K F E D V V S I agtgattggaggcattctatatattatgaagtggactttgggtggggagatgcaatgaac 1200 S D W R H S I Y Y E V D F G W G D A M N gtgagcactatgctacaacaacaggagcacgagaaatctctgccaacttatttttctttc 1260 V S T M L Q Q Q E H E K S L P T Y F S F ctacaatctactaagaacatgccagatggaatcaagatgctaatgtttatgcctccatca 1320 L Q S T K N M P D G I K M L M F M P P S aaactgaaaaaattcaaaattgaaatagaagctatgataaaaaaatatgtgactaaagtg 1380 K L K K F K I E I E A tgtccgtcaaagttatga C P S K L M I K K Y V T K V 1440 - Figure C 1 – The nucleotide sequence of wild-type bapt from T. cuspidata (accession no. AY082804) including the gene sequence for N-terminal His6-tag epitope and the corresponding amino acid sequence of wtBAPT in pET28a vector (Novagen). The highlighted region shows the incorporated N-terminal His6-tag epitope on wtBAPT which is used for immunoblot analysis of the expressed protein and purification by His-Select Nickel Affinity column chromatography. 299 atgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggcagccat 60 M G S S H H H H H H S S G L V P R G S H atgaagaagacaggttcgtttgcagagttccatgtgaatatgattgagcgagtcatggtg 120 M K K T G S F A E F H V N M I E R V M V agaccgtgcctgccttcgcccaaaacaatcctccctctctccgccattgacaacatggca 180 R P C L P S P K T I L P L S A I D N M A agagctttttctaacgtattgctggtctacgctgccaacatggacagagtctctgcagat 240 R A F S N V L L V Y A A N M D R V S A D cctgcaaaagtgattcgagaggctctctccaaggtgctggtttattattacccttttgct 300 P A K V I R E A L S K V L V Y Y Y P F A gggcggctcagaaataaagaaaatggggaacttgaagtggagtgcacagggcagggtgtt 360 G R L R N K E N G E L E V E C T G Q G V ctgtttctggaagccatggctgacagcgacctttcagtcttaacagatctggataactac 420 L F L E A M A D S D L S V L T D L D N Y aatccatcgtttcagcagttgattttttctctaccacaggatacagatattgaggacctc 480 N P S F Q Q L I F S L P Q D T D I E D L catctcttgattgttcaggtaactcgttttacatgtgggggttttgttgtgggagcgaat 540 H L L I V Q V T R F T C G G F V V G A N gtgtatcatagtgcatgcgatgcaaaaggatttggccagtttcttcaaagtatggcagag 600 V Y H S A C D A K G F G Q F L Q S M A E atggcgagaggagaggttaagccctcgattgaaccgatatggaatagagaactggtgaag 660 M A R G E V K P S I E P I W N R E L V K ctagaacattgtatgcccttccggatgagtcatcttcaaattatacatgcacctgtaatt 720 L E H C M P F R M S H L Q I I H A P V I gaggagaaatttgttcaaacatctcttgttataaactttgagataataaatcatatcaga 780 E E K F V Q T S L V I N F E I I N H I R cgacgcatcatggaagaacgcaaagaaagtttatcttcatttgaaattgtagcagcattg 840 R R I M E E R K E S L S S F E I V A A L gtttggctagcaaagataaaggcttttcaaattccacatagtgagaatgtgaagcttctt 900 V W L A K I K A F Q I P H S E N V K L L tttgcaatggacttgaggagatcatttaatccccctcttccacatggatactatggcaat 960 F A M D L R R S F N P P L P H G Y Y G N 300 gcctttggtattgcatgtgcaatggataatgtccatgaccttctaagtggatctcttttg 1020 A F G I A C A M D N V H D L L S G S L L cgcactataatgatcataaagaaatcaaagttctctttacacaaagaactcaactcaaaa 1080 R T I M I I K K S K F S L H K E L N S K accgtgatgagctcatctgtagtagatgtcaatacgaagtttgaagatgtagtttcaatt 1140 T V M S S S V V D V N T K F E D V V S I agtgattggaggcattctatatattatgaagtggactttgggtggggagatgcaatgaac 1200 S D W R H S I Y Y E V D F G W G D A M N gtgagcactatgctacaacaacaggagcacgagaaatctctgccaacttatttttctttc 1260 V S T M L Q Q Q E H E K S L P T Y F S F ctacaatctactaagaacatgccagatggaatcaagatgctaatgtttatgcctccatca 1320 L Q S T K N M P D G I K M L M F M P P S aaactgaaaaaattcaaaattgaaatagaagctatgataaaaaaatatgtgactaaagtg 1380 K L K K F K I E I E A M I K K Y V T K V tgtccgtcaaagttatga 1440 C P S K L Figure C 2 – The mutant bapt nucleotide sequence including the gene sequence for N-terminal His6-tag epitope and the corresponding amino acid sequence of mBAPT in pET28a vector (Novagen). The highlighted regions show the modified amino acid residue, G183H of wtBAPT to mBAPT through site-directed mutagenesis, modifications of nucleotide sequence to form mutant bapt, and the incorporated N-terminal His6-tag epitope on wtBAPT which is used for immunoblot analysis of the expressed protein and purification by His-Select Nickel Affinity column chromatography. 301 752.4 100 Exact Mass - 751.3 AcO % Relative Abundance 80 F O OH NH2 O O 60 O HO H OAc O O 40 20 793.5 569.3 387.2 0 0 100 200 300 400 500 600 700 800 m/z Figure C 3 – LC-MS ion profile of the biosynthesized (R)-3'-Amino-3-(2fluorophenyl)propanoyl baccatin III derived by catalysis of wtBAPT is shown. Diagnostic ions + + + are found at m/z: 793.5 [M + CH3CN + H] , 752.4 [M + H , 569.3 [m/z 752.4 ([M + H] ) – + + RCOOH at C-13] , 387.2 [m/z 509.3 – BzOH] , where R = (R)- 3-amino-3-(2fluorophenyl)acetyl. 302 740.4 100 Exact Mass - 740.3 AcO % Relative Abundance 80 O OH NH2 O O 60 S O HO H OAc O O 40 20 509.3 781.4 569.3 0 0 100 200 300 400 500 600 700 800 m/z Figure C 4 – LC-MS ion profile of the biosynthesized (R)-3'-Amino-3-(2-thiophenyl)propanoyl baccatin III derived by catalysis of wtBAPT is shown. Diagnostic ions are found at m/z: 781.4 + + + [M + CH3CN + H] , 740.4 [M + H] , 569.3 [m/z 740.4 ([M + H] +) – RCOOH at C-13] , 509.3 [m/z 569.3 – HOAc]+, where R = (R)- 3-amino-3-(2-thiophenyl)acetyl. 303 H 6 O 3 9 1 S NH2 O 1′ N H N NH2 OH OH O N O O O N N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure C 5 – H NMR of (R)-3-amino-3-phenylpropanoyl CoA 304 6 H F 3 9 O 1 S NH2 O 1′ N H N NH2 OH OH O N O O O N N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure C 6 - H NMR of (R)-3-amino-3-(2-fluorophenyl)propanoyl CoA 305 H 8 3 6 H3C 10 O O 1 S NH2 O 1′ N H N NH2 OH OH O N O O O N N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure C 7 – H NMR of (R)-3-amino-3-(2-methylphenyl)propanoyl CoA 306 H 5 7 S O 3 1 S NH2 O 1′ N H N NH2 OH OH O N O O O N N N P P 5′′ 9′ H O O H O OH HO H O P OH OH O 4′ 1 Figure C 8 – H NMR of (R)-3-amino-3-(2-thiophenyl)propanoyl CoA 307 REFERENCES 308 REFERENCES (1) Cragg, G. M. L.; Kingston, D.; Newman, D. J. 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