REPURPOSING A NON - RIBOSOMAL PEPTIDE SYNTHETASE ( TYROCIDINE SYNTHETASE A ) FOR AMINO ACYL COA BIOSYNTHESIS By Ruth Njeri Muchiri A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemistry Doctor o f Philo sophy 2015 ABSTRACT REPURPOSING A NON - RIBOSOMAL PEPTIDE SYNTHETASE ( TYROCIDINE SYNTHETASE A ) FOR AMINOACYL COA BIOSYNTHESIS By Ruth Njeri Muchiri In Taxus - amino - - phenylpropanoyls from coenzyme A to the diterpenoid baccatin III by an acyl CoA - - amino - - phenyl propanoyl CoAs in Taxus plants has not yet been isolated or characterized. In a screen for an alternative catalyst, a multienzyme, nonribosomal peptidyl synthetase on the pathway that produces tyrocidines was identified as a surrogate CoA ligase . The trido main starter module ( Phe AT E ) of the tyrocidine synthetase A normally activates ( S ) - - phenylalanine to an adenylate phosphate anhydride in the adenylation domain. The activated phenylalanine intermediate is then thioesterified by the pendent (i.e. covalent) pantetheine attached to the adjacent thiolation domain. In the current project, the adenylation and thiolation domains were found to function as - - phenylalanyl, and , more importantly, phenylisoserinyl CoA. The latte r two are known substrates of a phenylpropanoyltransferase (BAPT) on the biosynthetic pathway of the antimitotic paclitaxel . Tyc( Phe AT ) was used in additional specifi city studies with a focus on ar ylisoserine s, since the corresponding CoA thioester s are important for biosynthesizing precursors of the paclitaxel analog s (such as the prostate cancer drug carbazitaxel) . The stereospecificity of Tyc( Phe AT ) for the various stereoisomers o f phenylisoserine was explored, showing reduced turnover for the (2 R ,3 R ) isomer, and no turnover with (2 S , 3 R ) - p henylisoserine relative to the benchmark (2 R ,3 S ) - isomer . The latter (2 R , 3 S ) - diastereoisomer of phenylisoserine matches the stereochemistry of the natural side chain of paclitaxel. Thus, our preliminary work evaluated the substrate specificity of Tyc( Phe AT ) (taking advantage of its enantiospecificity) for racemates of ( 2 S , 3 R ) - and (2 R , 3 S ) - arylisoserine . Structure - a ctivity relationship analyses in earlier, independent studies show ed that ar ylisoserine is necessary for the effective anticancer activity of paclit axel . To access these activated CoA intermediates biosynthetically for use in a coupled enzyme assay with the paclitaxel pathway - specific 13 - O - phenylpropanoyltransferase (BAPT), the CoA ligase function of Tyc( Phe AT ) was employed to convert various aryl - and non - aryl isoserine analogs to their CoA thioesters. We propose the products of the Tyc( Phe AT ) reaction can be transferred to baccatin III by a permissive BAPT. The isoserine substrates were synthesized by the Staudinger reaction that formed the S chiff base between benzaldehyde analogs and p - anisidine . This base was then reacted with acetoxy acetyl chloride in the presence of triethylamine. The amino and hydroxyl groups of the lactam product of this reaction were deprotected. Finally, h ydrolysis of the lactam produced the isoserine analog. Tyc( Phe AT ) catalysis converted these analogs to their corresponding isoserinyl CoA s. All s ubstrates in which the phenyl ring was substituted at ortho - (F, Cl, NO 2 ), para - (F, Cl, Br, Me, OH, and NO 2 ), or meta - (F, Cl, Br, Me, OH, CH 3 O and NO 2 ) were converted to their CoA thioesters . Activity was also observed w ith the non - - ( cyclohexyl - ( thiophenyl ) isoserine analogs , but not with the aliphatic alkanyl groups (isopropyl - , and tert - butyl isoserine). This work provides a stepping stone towards novel biosynthesis of paclitaxel analogs with better efficacy than the parent drug. iv ( Dan Millman, Way of the Peaceful Warrior ) . v ACKNOWLEDGEMENTS It is with immense gratitude that I acknowledge the support and help of my graduate research advisor Dr. Walker, without whom this thesis would have remained a dream. I also thank my thesis second reader Dr. Borhan for putting in extra effort in proofreadi ng my thesis and providing great suggestions. Additionally, I appreciate my research committee members Dr. Tepe and Dr. Jones for sharing their pearls of wisdom with me during my early years in graduate school. I would also like to thank the former member s of Walker lab: Irosha for taking time to train me, Danielle for being a great friend, Getrude for being the big sister I needed when I was trying to find my way in graduate school, Mark, Washington, Sean, Dennis, Uday and Dilini. I also want to extend my gratitude to the current Walker lab members: Chelsea for fruitful discussions, Prakash and Tyler who create a friendly environment in the lab. I consider it an honor for the opportunity to mentor undergraduate students, Ashley and Aaron, and high school s tudent Asha. Their hard work and dedication was evident and I am greatly indebted by their contribution towards my research project. I thank Dr. Jones and Lijun for their assistance with mass spectrometry, and also Dr. Holmes for his assistance with the NM R. I owe my deepest gratitude to the Chemistry department at MSU for their financial support and the opportunity to shape my teaching skills. I wish to acknowledge the Forum for African Women Educationalists (FAWE) who saw the potential in me and put in th eir resources into my high school education. Without them, I may not have made it to the University of Nairobi. I cannot find words to express my gratitude to my dear mum, Mary Mukami. She instilled academic discipline, curiosity and spirit of hard work vi in me at a very tender age. I remember when she would challenge me to be the best student in my class with a promise of a gift at the end of the school year. Though at the time the drive was to receive the gift, I look back with a smile at how such childhood bait shaped my academic life. I can never forget how mum struggled to see me through my high school education and secured a full scholarship for me. I also extend my thanks to my dad, Muchiri, for his encouragement through - out the graduate school. I would like to also thank my siblings Lucas, Martha, Francis, Ben, Milly and Simon. They have been a great source of strength throughout my academic life. It gives me a great pleasure in acknowledging my primary school head teacher, Mr. Karanu, who has been a gr eat motivation. He saw the great potential in me and challenged me to aim higher. I am glad he was present to witness the great achievement during my advanced degree commencement. Last but not least, I would like to thank my friends; Eric for his help in m y thesis edits and comments, Waithira, my little sister for her love and support, Eunice, and the MSU - University Bible Fellowship church members for their constant prayers and encouragement. vii TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ....................... xi LIST OF FIGURES ................................ ................................ ................................ .................... x ii KEY TO ABBREVIATIONS ................................ ................................ ................................ .... x i x 1. INTRODUCTION ................................ ................................ ................................ ............. 1 1.1. Chemotherapeutic Agents f rom Natural Products ................................ ....................... 1 1.2. Paclitaxel Discovery and Supply Shortage ................................ ................................ .. 4 1.3. Paclitaxel Biosynthesis ................................ ................................ ................................ 8 1.4. In Search for an Aminoacyl CoA Ligase ................................ ................................ ... 10 1.4.1. ANL Superfamily ................................ ................................ ................................ .. 12 1.4.2. T yrocidine Synthetase A : Member of NRPS Enzyme Family .............................. 14 1.4.2.1. Adenylation (A) Domain ................................ ................................ ................ 16 1.4.2.2. Thiolation (T) Domain ................................ ................................ ................... 16 1.4.2.3. Epimerization (E) Domain ................................ ................................ ............. 17 REFERENCES ................................ ................................ ................................ ........................ 21 2. TYROCIDINE SYNTHETASE A (TycA) CATALYSIS IN THE BIOSYNTH ESIS OF AMINOACYL COA AND AMINO ACYL - N - ACETYLCYSTEAMINE ...................... 30 2.1. Introduction ................................ ................................ ................................ ................ 30 2.2. Experimental ................................ ................................ ................................ .............. 3 5 2.2.1. Substrates, Reagents, and General Instrumentation ................................ ............... 3 5 2.2.2. Expression of wild - type TycA cDNA ................................ ................................ .... 3 6 2.2.3. Construction and Expression of the TycA S563A Mutant ................................ .... 3 6 2.2.4. Pu ri fication and Characterization of TycA and the TycA S563A Mutant ............ 3 7 2.2.5. Synthesis of Phenylalan yl AMP ................................ ................................ ............. 3 8 2.2.6. Activity of wtTycA or m Phenylalanine, and (2 R ,3 S ) Phenylisoserine ................................ ................................ ................................ ....... 3 9 2.2.7. Synthesis of Product Standards ................................ ................................ .............. 41 2.2.7.1. Synthesis of [S Phenylalanyl) N acetyl]cysteamine ................... 41 2.2.7.2. N - deprotection of [S Phenylalanyl)]SNAC ................................ .. 43 2.2.7.3. Synthesis of [(2 R ,3 S ) - Phenylalanyl] N acetylcysteamine ............................. 4 5 2.2.8. Activity and Kinetic Evaluation of wtTycA or m TycA with N Acetylcysteamine and Aminophenylpropanoates ................................ ................................ ................ 4 6 2.2.9. Kinetic Analysis of the N acetylcysteamine Ligase Reaction Catalyzed by TycA or TycA S563A ................................ ................................ ................................ .......... 4 7 2.2.10. Activity and Kinetic Evaluation of wtTycA or m TycA as Ligases for Catalysis of Aminophenylpropanoyl CoA Thioesters ................................ ................ 4 9 2.2.11. Kinetic Analysis of the CoA Ligase Reaction Catalyzed by TycA and TycA S563A ................................ ................................ ................................ ..................... 50 viii 2.3. Results and Discussion ................................ ................................ ............................... 52 2.3.1. Expression and Purifi cation of the ATE Tridomain of wild - type and m utant tycA ................................ ................................ ................................ ......................... 52 2.3.2. Strategies used to Test the Activity of TycA with the Amino Acid Substrates ..... 53 2.3.3. Incubation of TycA and TycA S563A w ith N - Acetylcysteamine and Phenyl - propanoates ................................ ................................ ................................ ............. 57 2.3.4. Kinetic Analyses of TycA and TycA S563A w ith NAC ................................ ...... 61 2.3.5. Assessment of TycA and TycA S563A for CoA Ligase Activity ........................ 62 2.3.6. Kinetic Analyses of TycA and TycA S563A w ith Phenylpropanoates and CoA .. 6 6 2.3.7. Kinetic Anal yses of TycA and TycA S563A with Phenylpropanoates and ATP .. 6 9 2.3.8. Comparing the Kinetic Parameters of TycA/TycA S563A with Other CoA Ligases ................................ ................................ ................................ .................... 71 2.4. Conclusion ................................ ................................ ................................ ................. 72 APPENDIX ................................ ................................ ................................ ............................ 7 4 REFERENCES ................................ ................................ ................................ ...................... 8 9 3. TRUNCATION OF TYROCIDINE SYNTHETASE A (TycA( Phe ATE) to Tyc( Phe A ) and Tyc( Phe AT ) , ACTIVITY EVALUATION , AND STEREOSPECIFICITY STUDIES IN THE BIOSYNTHESIS OF AMINO ACYL COA THIOESTERS ............ 94 3.1. Introduction ................................ ................................ ................................ ................ 94 3.2. Experimental ................................ ................................ ................................ .............. 9 8 3.2.1. Substrates, Reagents, and General Instrumentation ................................ .............. 9 8 3.2.2. Tr uncation and Subcloning of wt tycA cDNA to Obtain tyc ( phe a ) cDNA ......... 9 8 3.2.3. Tyc (Phe A) Protein Expression and Purification ................................ ................. 9 9 3.2.4. Evaluation of Tyc(Phe A) Activity with CoA and Aminophenylpropanoids ..... 100 3.2.5. Assessing Tyc(Phe A) Substrate Binding by Tryptophan Fluorescence Quenching Studies ................................ ................................ ................................ .................. 101 3.2.6. Truncation of wt tycA or tycA S563A cDNA to Obtain tyc ( phe at) and tyc(phe at (S563A)) Constructs ................................ ................................ .......................... 101 3.2.7. Expression, Purification, and Characterization of Tyc(Phe AT) Proteins .......... 102 3.2.8. Activity Evaluation of Tyc(Phe AT) and Tyc(Phe AT (S563A)) with CoA and Amino phenylpropanoids ................................ ................................ ...................... 104 3.2.9. Kinetic Evaluation of Tyc(Phe AT) and Tyc(Phe AT (S563A)) for CoA and Aminophenylpropanoids ................................ ................................ ...................... 105 3.2.10. Stereospecificity of Tyc(Phe AT) for Phenylisoserine Stereoisomers ................ 106 3.2.10.1. Preparation of (2 R ,3 R ) Phenylisoserine ................................ ....................... 106 3.2.10.2. Preparation of (2 S ,3 R ) Phenylisoserine ................................ ....................... 10 7 3.2.10.3. Evaluation of Tyc(Phe AT) Activity for Phenylisoserine Stereoisomers .... 10 8 3.2.10.4. Kinetic Evaluation of Tyc(Phe AT) for CoA and Phenylisoserine Stereo - isomers ................................ ................................ ................................ .......... 10 8 3.2.10.5. Inhibition Studies of (2 R ,3 S ) Phenylisoserine by the Enantiomer (2 S ,3 R ) Phenylisoserine ................................ ................................ ............................... 10 9 3.3. Results and discussion ................................ ................................ ............................. 10 9 3.3.1. Cons truction and Expression of Tyc(Phe A) ................................ ....................... 10 9 3.3.2. Activity of Tyc(Phe A) with ATP, CoA, and Aminophenylpropanoids ............. 1 10 ix 3.3.3. Assessing Tyc(Phe A) Substrate Binding by Tryptophan Fluorescence Quenching Studies ................................ ................................ ................................ .................. 1 11 3.3.4. Construction and Expression of Tyc(Phe AT) and Tyc(Phe AT(S563A)) ........ 1 14 3.3.5. Activity and Kinetic Analysis of Tyc(Phe AT) with ATP, CoA, and Aminophenylpropanoids ................................ ................................ ...................... 1 15 3.3.6. Tyc(Phe AT) Secondary Structure Model and Comparison with CoA Ligases .. 11 7 3.3.6.1. Contrasting S tructural Features of Tyc(Phe AT) Model and 4 Chloro - benzoate:CoA Ligase ................................ ................................ .................... 11 9 3.3.6.2. Tyc (Phe AT) Structure Model Alignment with Acetate:CoA Ligase .......... 11 9 3.3.7. Sub strate Stereospecificity of Tyc(Phe AT) ................................ ........................ 1 21 3.3.7.1. In hibition Studies of (2 R ,3 S ) Phenylisoserine by the Enantiomer (2 S ,3 R ) Phenylisoserine ................................ ................................ ............................. 1 25 3.4. Conclusion ................................ ................................ ................................ ............... 1 26 APPENDIX ................................ ................................ ................................ .......................... 128 REFERENCE ................................ ................................ ................................ ....................... 142 4. SUBSTRATE SPECIFICITY STUDIES OF TYC(PHE AT) WITH ISOSERINE ANALOGS IN THE BIOSYNTHESIS OF ISOSERINYL COA THIOESTERS ........ 1 4 7 4.1. Introduction ................................ ................................ ................................ .............. 1 4 7 4.1.1. Paclitaxel Analog s and their Importance ................................ ............................. 1 4 7 4.1.2. Sources of Paclitaxel: An Historical Time Line ................................ ................... 1 4 9 4.1.3. Catalytic Activity of Baccatin III 13 O 3 Amino 3 Phenylpropanoyltransferase (BAPT) with Aminoacyl CoA Thioesters ................................ ............................ 1 5 1 4.1.4. Tyrocidine Synthetase A Tyc(Phe AT) Catalysis in Biosynthesis of Isoserinyl CoA Analogs ................................ ................................ ................................ ................. 1 5 3 4.2. Experimental ................................ ................................ ................................ ............ 1 5 6 4.2.1. Substrates, Reagents, and General Instrumentation ................................ ............. 1 5 6 4.2.2. Synthesis of Isoserine Analog s ................................ ................................ ............ 1 5 7 4.2.2.1. General Procedure 1: Synthesis of N ( p - Methoxyphenyl)benzylimines ...... 1 5 7 4.2.2.2. General Procedure 2: Synthesis of 2 - Azetidinones ................................ ....... 1 6 1 4.2.2.3. General Procedure 3: Deprotection of Azetidinones ................................ .... 1 7 2 4.2.2.4. General Procedure 4: Hydroxyl Deprotection of the 2 - Azetidinones ........... 1 8 2 4.2.2.5. General Procedure 5: Hydrolysis of Azetidinone Ring ................................ . 1 8 6 4.2.3. Synthesis of Pyridinyl Isoserine Analogs ................................ ............................. 1 9 5 4.2.3.1. General Procedure 6: Synthesis of Pyridinyl - N - benzhydryl Imine ............... 1 9 5 4.2.3.2. General Procedure 7: Synthesis of N - benzhydryl - 3 - acetoxy - 4 - (pyridinyl)azetidin - 2 - one ................................ ................................ .............. 1 9 7 4.2.3.3. General Procedure 8: Lactam Ring Opening Along with Amine and Hydroxyl Deprotectio n of N - benzhydryl - 3 - acetoxy - 4 - pyridinylazetidin - 2 - one ........... 1 9 9 4.2.4. Activity Assay for the Determination of the Substrate Specificity of Tyc(Phe AT) ................................ ................................ ................................ ........ 20 0 4.2.5. Apparent Rates of Tyc(Phe AT) with the Isoserine Substrates ........................... 20 1 4.2.6. Kinetic Evaluation of Tyc(Phe AT), CoA, and Racemic (2 R ,3 S ) - Phenyl - isoserine ................................ ................................ ................................ ................ 20 2 4.3. Results and Discussion ................................ ................................ ............................ 20 2 x 4.3.1. Synthesis of Isoserine Analogs via Staudinger Cycloaddition Reaction ............. 20 2 4.3.2. Relative Rates of Tyc(Phe AT) for Isoserine Substrate Analogs ........................ 20 5 4.3.3. Docking (2 R ,3 S ) Phenylisoserine into Grs1(Phe A) Domain ............................ 20 8 4.4. Conclusion ................................ ................................ ................................ ............... 2 1 1 4.5. Future Direction ................................ ................................ ................................ ....... 2 1 2 APPENDIX ................................ ................................ ................................ .......................... 215 REFERENCES ................................ ................................ ................................ .................... 2 5 8 5. PRELIMINARY SITE DIRECTED MUTAGENESIS STUDIES AND FUTURE DIRECTION IN THE BIOSYNTHESIS OF AMINOACYL COA THIOESTERS .... 2 64 5.1. Introduction ................................ ................................ ................................ .............. 2 64 5.1.1. Application of Bioengineering to Enhance Enzymatic Catalysis ........................ 2 64 5.1.2. Tyc(Phe AT) Point Mutation Sites to Expand the Aryl Ring Binding Site ........ 2 65 5.2. Experimental ................................ ................................ ................................ ............ 2 67 5.2.1. Substrates, Reagents, and General Instrumentation ................................ ............. 2 67 5.2.2. Tandem Site Directed Mutagenesis of tyc ( phe at ) cDNA ................................ .. 2 68 5.2.2.1. Mutation of Tyc(Phe AT) to Tyc(Phe AT(W227A, FFF Tyc(Phe AT (W227A)) ................................ ................................ ................. 2 68 5.2.2.2. Mutation o f Tyc(Phe AT) to Tyc(Phe AT(W227S, FFF Tyc(Phe AT(W227S)) ................................ ................................ .................. 2 69 5.2.3. Protein Expression and Activity Assays of Tyc(Phe AT) Mutants with Phenylisoserine Analogs and CoA ................................ ................................ ....... 2 70 5.2.4. Protein Purification of Tyc(Phe AT) Mutants ................................ ..................... 2 72 5.2.5. Activity of Tyc(Phe AT) Mutants with Isoserine Analogs ................................ . 2 73 5.3. Results and Discussion ................................ ................................ ............................ 2 74 5.3.1. Protein Expression and Purification of Tyc(Phe AT) Mutants ........................... 2 74 5.3.2. Activity Assays of Tyc(Phe AT) Mutants with Phenylisoserine Analogs .......... 2 75 5.3.2.1. Activity Evaluation of Tyc(Phe AT(W227S)) in the Screen for Isoserine Analogs in Isoserinyl CoA Biosynthesis ................................ ....................... 2 76 5.4. Tyc (Phe AT) and BAPT Coupled Assays Towards the Biosynthesis of Precursors of Paclitaxel Analogs ................................ ................................ ................................ ... 2 78 REFERENCES ................................ ................................ ................................ .................... 2 80 xi LIST OF TABLES Table 2.1. Steady state kinetic analysis of TycA and TycA S563A with phenylpropanoids, CoA and N acetylcysteamine ................................ ................................ ............................. 6 7 Table 2.2. Steady state kinetic analysis of TycA and TycA S563A with apparent saturation of phenylpropanoids and varying concentrations of ATP ................................ .............. 70 Table 2 . 3 . Ratio of k cat of phenylpropanoids in steady state biosynthesis of aminoacyl AMP or aminoacyl CoA catalyzed by TycA or TycA S563A ................................ ................ 71 Table 2 . 4 . Kinetic parameters of acyl CoA ligases from various microorganisms ..................... 72 Table 3. 1 . The dissociation constants for binding of (2 S ) phenylalanine ((2 S ) Phe) to Tyc(Phe A), Tyc(Phe AT), and Grs1(Phe A ) ................................ ....................... 1 13 Table 3. 2 . Steady state kinetic analysis of Tyc(Phe AT) and Tyc(Phe AT(S563A)) with CoA in comparison to Tyc(Phe ATE) and Tyc(Phe ATE(S563A)) ................................ .... 1 16 Table 3.3 . Kinetic parameters of Tyc(Phe AT) with phe nylisoserine stereoisomers ............... 1 23 Table 4.1 . Cytotoxic activity of paclitaxel analog s in microtubule assembly and against B16 melanoma cells ................................ ................................ ................................ ......... 1 4 8 Table 4.2 . Relative rates of Tyc(Phe - AT) with aryl - and non - aryl isoserine and CoA ............. 20 7 Table 5 . 1 . The proposed mutations of F201, F202 , and F206 of Tyc(Phe - AT) in tandem with W227 replacement residues ................................ ................................ ..................... 2 67 Table 5. 2 . The rates ( v app ) of Tyc(Phe AT(W227S)) mutant in comparison to Tyc(Phe AT) in the biosynthesis of isoserinyl CoA analogs ................................ ............................. 2 77 xii LIST OF FIGURES Figure 1. 1 . Podophyllotoxin and its derivatives ................................ ................................ ............ 2 Figure 1.2 . The vinca alkaloids; vinblastine and vincristine ................................ ......................... 2 Figure 1.3 . Camptothecin and the water soluble derivatives ................................ ......................... 3 Figure 1.4 . The structures of pacl itaxel (Taxol®) and its analog , docetaxel (Taxotere®) ............ 4 Figure 1.5 . The Holton methodology for the semis - lactam (A) and 10 - DAB precursor (B) ................................ ................................ ................................ ...... 6 Figure 1.6 . Biosynthetic pathway to paclitaxel starting from simple primary metabolite precursors to the baccatin III ................................ ................................ ....................... 9 Figure 1.7 . Biosynthesis of paclitaxel C 13 side chain (A) and th e enzyme catalyzed transfer to the baccatin III (B) ................................ ................................ ................................ ..... 10 Figure 1.8 . Synthesis of (2 R , 3 S ) phenylisoserinyl CoA via the mixed anhydride intermediate ... 11 Figure 1.9 . The tyrocidine synthetase cluster showing the three polypeptides; TycA, TycB, and TycC ................................ ................................ ................................ .......................... 15 Figure 1.10 . Scheme showing the conversion of apo - PCP (or T domain) to holo - PCP ............... 17 Figure 1.1 1 . Schematic representation of the reactions catalyzed by various domains within a module in tyrocidine synthetase ................................ ................................ ............. 18 Figure 2.1 . Scheme showing reaction s catalyzed by TycA (Phe - ATE) domains ....................... 31 Figure 2.2 . The stabilization of thiol - carboxylate intermediate formed during epimerization reaction in NRPS enzymes ................................ ................................ ....................... 32 Figure 2.3 . Possible pathways for the epimerization at the alpha carbon ................................ .... 33 Figure 2.4 . Synthesis of (2 R ,3 S ) phenylisoserinyl CoA via the mixed anhydride intermediate (panel A, see Figure 1.8 in Chapter 1 for full scheme) and the proposed biosynthetic route in the current study (panel B). ................................ ................................ ........... 3 5 Figure 2.5 . ( N Boc) ( S ) phenylalanyl SNAC ( B - 2 ) and ( N Boc) ( R / S ) phenylalanyl SNAC ( B - 3 ) ................................ ................................ ................................ .............. 41 xiii Figure 2.6 . Synthesis of (2 S ) phenylalanyl SNAC ................................ ................................ 42 Figure 2.7 . ( S ) Phenylalanyl SNAC ( B - 7 ) Phenylalanyl SNAC · HCl salt ( B - 8 ) .......... 43 Figure 2.8 . (2 R ,3 S ) Phenylisoserinyl SNAC ................................ ................................ ............... 4 5 Figure 2.9 . SDS - PAGE gel of fractions eluted from a Ni - affinity column containing TycA constructs ................................ ................................ ................................ ................... 53 Figure 2.10 . Radioactivity - guided assay of the phenylalanine adenylation domain of TycA ..... 5 4 Figure 2.11 . The exchange reaction, performed in the absence of thiolation activity, measures - 18 O 4 - ATP with 16 O 4 - pyrophosphate ............................. 5 5 Figure 2.12 . The proposed biosynthesis of aminoacyl CoAs using TycA ................................ ... 5 7 Figure 2.13 . The LC E SI MS MS spectr a of aminoacyl SNACs ................................ .............. 5 9 Figure 2.14 . Molecular fragment ions profile resulting from LC ESI MS/MS of biosynthetic aminoacyl CoAs ................................ ................................ ................................ ...... 63 Figure 2.15 . The LC ESI MS MS spectr a of bio synthetic aminoacyl CoAs ............................ 6 4 Figure 2.16 . Kinetic model for CoA thioesterification reaction catalyzed by TycA and TycA S563A ................................ ................................ ................................ ..................... 71 Figure I 1. MRM profile obtained by LC ESI MS/MS of biosyntheti phenylpropanoyl SNAC ................................ ................................ ........................... 7 5 Figure I 2. MRM profile obtained by LC ESI MS/MS of biosyntheti phenylpropanoyl SNAC ................................ ................................ ........................... 7 5 Figure I 3. MRM profile obtained by LC ESI MS/MS of biosynthetically derived (2 S ,3 R ) phenylisoserinyl SNAC ................................ ................................ ............................ 7 6 Figure I 4. MRM profile obtained by LC ESI phenylalanyl CoA ................................ ................................ ................................ ..... 7 6 Figure I 5. MRM profile obtained by LC ESI MS/MS of biosynthetica phenylalanyl CoA ................................ ................................ ................................ ..... 7 7 Figure I 6. MRM profile obtained by LC ESI MS/MS of biosynthetically derived (2 R ,3 S ) phenylisoserinyl CoA ................................ ................................ ............................... 7 7 Figure I 7 . The LC ESI MS analyses of authentic aminoacyl SNACs ................................ ..... 7 8 xiv Figure I 8. Total ion chromatograms obt ained by LC ESI MS analysis (scan mode: m / z 100 to 1200) of the compounds in TycA assays ................................ ................................ .. 7 9 Figure I 9 . Determination of kinetic parameters for A) phenylalanine, B) phenylalanine, C) (2 R ,3 S ) phenylisoserine and D) CoA with wtTycA using Hannes Wolf plots . 81 Figure I 10 . Determination of kinetic parameters for A) phenylalanine, B) phenylalanine, C) (2 R ,3 S ) phenylisoserine and D) N acetylcysteamine with wtTycA using Hannes Wolf plots ................................ ................................ ................................ ................. 82 Figure I 11. 1 H - NMR spectrum of ( S ) phenylalanyl SNAC standard ................................ ... 83 Figure I 12. 13 C - NMR spectrum of ( S ) phenyl alanyl SNAC standard ................................ .. 8 4 Figure I 13. 1 H - NMR spectrum phenylalanyl SNAC · HCl salt standard ............................ 8 5 Figure I 14. 13 C - NMR spectrum phenylalanyl SNAC · HCl salt standard .......................... 8 6 Figure I 15. 1 H - NMR spectrum of (2 R ,3 S ) phenylisoserinyl SNAC · standard .......................... 8 7 Figure I 16. 13 C - NMR spectrum of (2 R ,3 S ) phenylisoserinyl SNAC standard ......................... 8 8 Figure I 17. Trypsin digestion sequence analysis of TycA purified by Ni - affinity chromatog raphy ................................ ................................ ................................ ...... 8 8 Figure 3.1. Representation of reactions catalyzed by different adenylate forming enzymes ...... 9 7 Figure 3.2. Deprotection of (2 R ,3 R ) phenylisoserine ................................ ............................... 106 Figure 3.3. Deprotection of (2 S ,3 R ) phenylisoserine ................................ ................................ 10 7 Figure 3. 4 . SDS polyacrylamide gel electrophoresis (12% acrylamide) and Coomassie Blue staining of recombinantly expressed Tyc(Phe A) ................................ ................. 1 10 Figure 3. 5 . Plot of observed fluorescence change with increasing concentration of (2 S ) phenylalan ine ................................ ................................ ................................ .......... 1 13 Figure 3. 6 . SDS - polyacrylamide gel electrophoresis (12% acrylamide) and Coomassie blue staining of recombinantly ex pressed Tyc(Phe AT) ................................ ............... 1 15 Figure 3. 7 . Comparison of 4 ClBzCL with Tyc(Phe AT) model ................................ ............. 11 8 Figure 3. 8 . Comparison of active site of 4 ClBzCL with Tyc(Phe AT) model ....................... 11 9 Figure 3. 9 . Comparison of active site of AcCL with Tyc(Phe AT) model ............................... 1 21 xv Figure 3. 1 0 . Structure of paclitaxel ................................ ................................ ............................ 1 22 Figure 3. 1 1 . Grs1(Phe A) active site (cyan) in complex with aminophenylpropanoids ........... 1 24 Figure 3.1 2 . Lineweaver Burk plots for the inhibition of Tyc(Phe AT) by (2 S ,3 R ) phenyl - isoserine ................................ ................................ ................................ ................. 1 26 Figure II 1. Amino acid sequence variation in the highly conserved motifs in the acyl adenylate enzyme family ................................ ................................ ................................ ........ 12 9 Figure II 2. Amino acid sequence alignment of cl ose adenylation domain homolog s of NRPS family ................................ ................................ ................................ ...................... 1 30 Figure II 3. The amino acid sequence a lignment of Tyc(Phe AT), acetyl CoA and 4 - chloro - benzoate:CoA ligase ................................ ................................ ............................... 1 32 Figure II 4. Spectra obtained by equilibrium fluorescence titration of (2 S ) phenylalanine to K + HEPES ................................ ................................ ................................ ............... 1 34 Figure II 5. Spectra obtained by equilibrium fluorescence titration of (2 S ) phenylalanine at increasing concentrations (0 A) ( ) ............................ 1 35 Figure II 6 . Plot of observed fluorescence change with varying concentrations of AMP at fixed concentration of Tyc(Phe A) ................................ ................................ .................. 1 35 Figure II 7 . Plot of observed fluorescence change with varying concentrations of (2 S ) phenyl - alanine at fixed concentr ation of Tyc(Phe AT) ................................ ..................... 1 36 Figure II 8 . Plot of observed fluorescence change with varying concent rations of AMP at fixed concentration of Tyc(Phe AT) ................................ ................................ ............... 1 36 Figure II 9 . 1 H - NMR spectrum of (2 R ,3 R ) p henylisoserine ................................ .................... 13 7 Figure II 10 . 13 C NMR spectrum of (2 R ,3 R ) phenylisoserine ................................ ................. 13 8 Figure II 11 . 1 H - NMR spectrum of (2 S ,3 R ) phe nylisoserine ................................ ................... 13 9 Figure II 12 . 13 C - NMR spectrum of (2 S ,3 R ) phenylisoserine ................................ ................. 1 40 Figure II 13 . Lineweaver - Burk plots for the inhibition of Tyc(Phe AT) in the presence of (2 S ,3 R ) phenylisoserine ................................ ................................ ...................... 1 41 Figure 4.1. The paclitaxel structure framework ................................ ................................ ......... 1 4 7 Figure 4.2. Some of the paclitaxel analogs with modification at C 13 ................................ ...... 1 4 9 xvi Figure 4.3. The biosynthesis of paclitaxel in plants starting from geranylgeranyl diphosphate (GGDP) ................................ ................................ ................................ .................. 1 5 0 Figure 4.4. The proposed biosynthesis of paclitaxel ................................ ................................ . 1 5 1 Figure 4.5. A representative 11 step semisynthesis of paclitaxel analogs ................................ 1 5 3 Figure 4. 6 . Synthesis of (2 R ,3 S ) - phenylisoserinyl CoA via the mixed anhydride inter - media te ................................ ................................ ................................ ................... 1 5 4 Figure 4. 7 . The reversible adenylation of ( R / S ) phenylalanine by Tyc(Phe A) domain through ATP hydrolysis ................................ ................................ ................................ ....... 1 5 5 Figure 4. 8 . Synthesis of N - protected imines from benzaldehyde analogs and p - anisidine ....... 1 5 7 Figure 4. 9 . Scheme showing the general synthesis of 2 - azetidinones ................................ ....... 1 6 1 Figure 4. 10 . Scheme showing general method for deprotection of amine ................................ 1 7 2 Figure 4.1 1 . Scheme showing the general method for hydroxyl deprotection .......................... 1 8 2 Figure 4.1 2 . Scheme showing the general method for the lactam ring hydrolysis .................... 1 8 6 Figure 4.1 3 . Scheme showing general synthesis of 2 - pyridin yl imines ................................ ..... 1 9 5 Figure 4.1 4 . Scheme showing the synthesis of pyridin yl - 2 - azetidinones ................................ .. 1 9 7 Figure 4.1 5 . A scheme showing the synthesis of isoserine analogs ................................ .......... 20 3 Figure 4.1 6 . Synthesis of pyridinylisoserine isomers ................................ ................................ 20 4 Figure 4.1 7. Reaction mechanism for the formation of benzhydrylacetamide .......................... 20 5 Figure 4.1 8 . (2 R ,3 S ) phenylisoserine (green) docked into Grs1(Phe - A) active site ................. 2 1 0 Figure 4.1 9 . Synthesis of cabazetaxel (Jevtana®) ................................ ................................ ..... 21 3 Figure III 1. MS/MS fragment ion profile of the biosynthes ized 3 - F phenylisoserinyl CoA ................................ ................................ ................................ ..................... 2 1 6 Figure III 2. MS/MS fragment ion profile of the biosynthesized 3 - Cl phenylisoserinyl CoA ................................ ................................ ................................ ..................... 2 1 7 Figure III 3. MS/MS fragment ion profile of the biosynthesized 3 - Br phenylisoserinyl CoA ................................ ................................ ................................ ..................... 2 1 8 xvii Figure III 4. MS/MS fragment ion profile of the bio synthesized 3 - Me phenylisoserinyl CoA ................................ ................................ ................................ ..................... 2 1 9 Figure III 5. MS/MS fragment ion profile of the biosynthesized 3 - NO 2 phenylisoserinyl CoA ................................ ................................ ................................ ..................... 2 2 0 Figure III 6. MS/MS fragment ion profile of the biosynthesized 4 - OMe phenylisoserinyl CoA ................................ ................................ ................................ ..................... 2 2 1 Figure III 7. MS/MS fragment ion profile of the biosynthesized 4 - OH Phenylisoserinyl CoA ................................ ................................ ................................ ..................... 2 2 2 Figure III 8. MS/MS fragment ion profile of the biosynthesized thiophenylisoserinyl CoA ................................ ................................ ................................ ..................... 2 2 3 Figure III 9. MS/MS fragment ion profile of the biosynthesized cyclohexylisoserinyl CoA ... 2 2 4 Figure III 10. 1 H Homodecoupling NMR spectrum of 3 - Hydroxy - 4 - (4 - fluoro phenyl)azetidin - 2 - one ................................ ................................ ................................ ....................... 2 2 5 Figure III 11. H 2 O/D 2 O exchange NMR spectrum of 3 - Hydroxy - 4 - (4 - fluoro phenyl)azetidin - 2 - one ................................ ................................ ................................ ....................... 2 2 6 Figure III 12. 1 H - NMR spectrum of phenylisoserine ................................ ............................... 2 2 7 Figure III 13. 1 H - NMR spectrum of p - F phenylisoserine ................................ ........................ 2 2 8 Figure III 14. 1 H - NMR spectrum of p - Cl phenylisoserine ................................ ...................... 2 2 9 Figure III 15. 1 H - NMR spectrum of p - Br phenylisoserine ................................ ...................... 2 3 0 Figure III 16. 1 H - NMR spectrum of p - Me phenylisoserine ................................ ..................... 2 3 1 Figure III 17. 1 H - NMR spectrum of p - OMe phenylisoserine ................................ .................. 2 3 2 Figure III 18. 1 H - NMR spectrum of p - OH phenylisoserine ................................ .................... 2 3 3 Figure III 19. 1 H - NMR spectrum of p - NO 2 phenylisoserine ................................ ................... 2 3 4 Figure III 20. 1 H - NMR spectrum of m - F phenylisoserine ................................ ....................... 2 3 5 Figure III 21. 1 H - NMR spectrum of m - Cl phenylisoserine ................................ ..................... 2 3 6 Figure III 22. 1 H - NMR spectrum of m - Br phenylisoserine ................................ ..................... 2 3 7 Figure III 23. 1 H - NMR spectrum of m - Me phenylisoserine ................................ .................... 2 3 8 xviii Figure III 24. 1 H - NMR spectrum of m - OMe phenylisoserine ................................ ................. 2 3 9 Figure III 25. 1 H - NMR spectrum of m - OH phenylisoserine ................................ ................... 2 4 0 Figure III 26. 1 H - NMR spectrum of m - NO 2 phenylisoserine ................................ .................. 2 4 1 Figure III 27. 1 H - NMR spectrum of o - F phenylisoserine ................................ ........................ 2 4 2 Figure III 28. 1 H - NMR spectrum of o - Cl phenylisoserine ................................ ...................... 2 4 3 Figure III 29. 1 H - NMR spectrum of o - Br phenylisoserine ................................ ...................... 2 4 4 Figure III 30. 1 H - NMR spectrum of o - Me phenylisoserine ................................ ..................... 2 4 5 Figure III 31. 1 H - NMR spectrum of o - OMe phenylisoserine ................................ .................. 2 4 6 Figure III 32. 1 H - NMR spectrum of o - NO 2 phenylisoserine ................................ ................... 2 4 7 Figure III 33. 1 H - NMR spectrum of trimethylisoserine ................................ ............................ 2 4 8 Figure III 34. 1 H - NMR spectrum of cyclohexylisoserine ................................ ......................... 2 4 9 Figure III 35. 1 H - NMR spectrum of thiopheneisoserine ................................ .......................... 2 5 0 Figure III 36. 1 H - NMR spectrum of isopropylisoserine ................................ ........................... 2 5 1 Figure III 37. 1 H - NMR spectrum of 2 - pyridinylisoserine ................................ ........................ 2 5 2 Figure III 38. 13 C - NMR spectrum of 2 - pyridinelisoserine ................................ ....................... 2 5 3 Figure III 39. 1 H - NMR spectrum of N - benzhydrylacetamide ................................ .................. 2 5 4 Figure III 40. 13 C - NMR spectrum of N - benzhydrylacetamide ................................ ................. 2 5 5 Figure III 41 . 1 H - NMR spectrum of 3 - pyridineisoserine ................................ .......................... 2 5 6 Figure III 4 2. LC - ESI - MS of intermediates and products obtained from the hydrolysis and deprotection of N - benzhydryl - 3 - acetoxy - 4 - (2 - pyridinyl)azetidin - 2 - one ........... 2 5 7 Figure 5.1. Tyc(Phe AT) model (grey cartoon) in complex with phenylalanine ...................... 2 66 Figure 5 . 2 . SDS polyacrylamide gel electrophoresis (12% acrylamide) and Coomassie blue staining of recombinantly expressed Tyc(Phe AT) mutants ................................ . 2 75 Figure 5.3 . Proposed Tyc(Phe AT) and BAPT - coupled assays aimed at novel biosynthesis of paclitaxel analogs ................................ ................................ ................................ ... 2 79 xix KEY TO ABBREVIATIONS Ac 2 O, Acetic anhydride AcCl, Acetyl chloride AIDS, Acquired immune deficiency syndrome AMP, Adenosine monophosphate ANL, Acyl - CoA synthetase , n on - ribosomal peptide synthetase , and l uciferase enzymes ATP, Adenosine triphosphate BAPT, Taxus - O - 3 - amino - 3 - phenylpropanoyltransferase Boc, tert - Butoxycarbonyl n - BuLi, n - butyllithium CAN, Cerium (IV) ammonium nitrate CDCl 3 , Deuterated chloroform cDNA, Complementary deoxyribonucleic acid CoA, Coenzyme A CoASH, Free coenzyme A thiol D 2 O, Deuterated water DAB, Deacetylbaccatin III DBAT, 10 - Deacetylbaccatin III - - O - acetyltransferase DCC, Dicyclohexylcarbodiimide DIPEA , N , N - Diisopropylethylamine DMAP, 4 - Dimethylaminopyridine DMF, Dimethylformamide xx DMSO, Dimethyl sulfoxide E. coli , Escherichia coli EDTA, Ethylenediaminetetraacetic acid EE, Ethyl vinyl ether ESI - MS/MS, Electrospray ionization tandem mass spectrometer EtCOCl, Ethyl chloroformate Et 3 SiCl, C hlorotriethylsilane EtOAc, Ethyl acetate FDA, U.S. A Food and Drug Administration GGDP, Geranylgeranyl diphosphate GGDPS, Geranylgeranyl diphosphate synthase Grs1(Phe - A), Gramicidin synthetase 1 adenylation domain HEPES , 4 - (2 - hydroxyethyl) - 1 - piperazineethanesulfonic acid g H S QC, heteronuclear single quantum coherence 2 - D NMR spectroscopic technique HOAc, Acetic acid HOBt , Hydroxybenzotriazole HPLC, High performance liquid chromatography HRMS , High resolution mass spectromet ry IC 50 , 50% inhibitory concentration ED 50 , 50% Effective dosage I P P, Isopentenyl diphosphate IDPI, Isopentenyl diphosphate isomerase D 1 thiogalactopyranoside xxi k cat , catalytic turnover K M , Michaelis constant LB, Luria - Bertani medium LC, Liquid chromatography LDA, Lithium diisopropylamide LiHMDS, Lithium bis(trimethylsilyl)amide MEP, Methylerythritol phosphate pathway MHz, Megahertz MS, Mass Spectrometer MRM, Multiple reaction monitoring MWCO, Molecular weight cut off Na 2 SO 4 , Sodium sulpha te NaHCO 3 , Sodium bicarbonate NDTBT, 3' - N - Debenzoyl - 2' - deoxytaxol N - benzoyltransferase NMR, Nuclear Magnetic Resonance NRPS, Non - ribosomal peptide synthetase OD, Optical density PAM, Phenylalanine aminomutase PCF, Plant Cell Fermentation PCP, Peptidyl carrier protein PCR, Polymerase chain reaction Tyc( Phe ATE ) , Phenylalanine adenylation, thiolation and epimerization PMP, para - methoxyphenyl xxii PPi, Inorganic phosphate 4' Ppant , 4' Phosphopantetheinyl Q - ToF, quadrupole time of flight RT, Room temperature SAR , Structure - activity relationship SDS - PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis SNAC, N - acetylcysteamine T. brevifolia, Taxus brevifolia T. cuspidata, Taxus cuspidata BuOH, Butanol TES, Triethylsilyl TFA, Trifluoroacetic acid THF, Tetrahydrofuran TIPS, Triisopropylsilyl TLC, Thin layer chromatography TMS, Trimethylsilyl tRNA, Transfer ribonucleic acid UPLC , Ultra performance liquid chromatography UV, Ultraviolet YT, Yeast extract and bacto tryptone medium 1 1. INTRODUCTION 1.1. Chemotherapeutic Agents From Natural Products Natural products have historically been excellent leads for the discovery of biologically active compounds including therapeutics . 1 For instance , 40% of all anticancer drugs developed before 2002 are natural products, while an additional 20% are synthetic c ompounds, based on natural product pharmacophore. 2 Natural anticancer agents can be of microbial or plant origin. The microbial or mo re commonly referred to as antibiotics constitute the majority of these agents. 3 The anthracycline antibiotics in use currently include doxorubicin, 4 - 6 daunorubicin, 7 , 8 epirubicin , 9 and idarubicin. 10 - 13 The bleomycin derivatives are used as glycosylated oligopeptide antibiotics, 14 while other antibiotics such as pyrroloindole and phenoxazinone chromopeptide are comm only administered as mitomycin C , 15 , 16 and a c tinomycin D , 17 respectively in combination with other drugs . These antibiotics are important in the treatment of different cancers and have been extensively reviewed. 13 , 15 , 17 , 18 The plant - derived anticancer agents have attracted attention both in academia and commercial fields. 19 - 22 Their use as traditional medicine date s back to 1800s, 22 but the isolati on and identification of t heir constituent active ingredients was not pursued until mid - 1900. 23 , 24 Podophyllotoxin is probably the first documented anticancer agent of plant origin ( Figure 1 . 1 ) . 23 , 25 It is derived from the American mandrake or May apple ( Podophyllum peltat um L) and was identified as a potent drug for the treatment of benign tumor, C ondylomata acuminate in 1942. 24 Due to its toxicity, derivatives namely etoposide 26 and tenipos ide 27 were developed for therapeutic use. To overcome the solubility challenges associated with these derivatives, etoposide phosphate (Etopophos ® ) 27 was developed and approved for the treatment of K 2 sarcoma, lung, te sticular cancers, lymphoma, non - lymphocytic leukemia , and gliobla s toma multiforme. 28 , 29 Figure 1.1. Podophyllotoxin and its derivatives Additionally, early discoveri es included the vinca alkaloids vinblastine and vincristine in 1958 . 30 These drugs have continuously b een used alongside other combinations as chemotherapeutic agents against non - small cell lung cancer, leukemia, lymphoma, H disease, bladder, brain , and breast cancers ( Figure 1. 2 ) . 31 The vinca alkaloids prevent growth of ca ncerous cells by inhibiting microtubule formation during mitosis. 32 , 33 Figure 1. 2 . The vinca alkaloids; vinblastine and vincristine 3 Later in 1966, the search for more potent anticancer agent s from natural products led to the isolation of camptothecin by researchers, Wall and Wani. 34 This quinolone alkaloid was extracted from the bark and stem of camptotheca acuminate (the happy tree), a tree indigenous to China. 34 The seemingly common solubility challenge with the natural anti cancer ex tracts was also observed with camptothecin in addition to severe adverse reactions. 35 These drawbacks halted the early development of camptothecin a nd its clinical trials. Nevertheless, due to its unique mechanism of action, namely inhibition of topoisomerase 1, 36 - 39 new interest in camptothecin led to the development of analog s that overcame the solubility and toxicity issues 40 ( Figure 1.3 ). Currently, topotecan (Hycamtin ® ) and irinotecan (Camptosar ® ) are approved by FDA for use in ovarian, cervical, colon , and small cell lung cancers. 41 Figure 1.3 . Camptothecin and the water soluble derivatives Among the discoveries made from nature to date , the most interesting is perhaps paclitaxel (Taxol ® ). 42 It is a complex diterpenoid, with a phenylisoserine side cha i n and generally adorned with hydroxyl functionalities ( Figure 1.4 ). 42 It is not surprising then, that it too k ~30 4 years from discovery to commercialization. 43 - 45 The follow ing sections will highlight important studies from discov ery to the current status of paclitaxel and a proposal on a new approach towards its production. 1.2. Paclitaxel Discovery and Supply Shortage The antineoplastic drug, paclitaxel (Taxol ® ) is one of the most important drugs in chemotherapy following its FDA approval in 1992. 46 Initially, paclitaxel was used for the treatment of refractory ovarian cancer and refractory or anthracycline - resistant breast cancer. 47 , 48 Over the years , paclitaxel and its analog , docetaxel (Taxotere ® ) ( Figure 1. 4 ) ha ve found wide application in the treatment of various cancers including metastatic breast cancer, ovarian , and lung cancer , and also A ids - related K . 49 - 52 Whereas other chemotherapy drugs, for example vinblastine and vincristine inhibit tubulin polymerization, p aclitaxel has a unique mode of acti on where it promotes microtubule polymerization, thus arresting cell division and eventually lead ing to apoptosis of the target cells . 53 Its unique mechanism of action prompted curiosity among the scientific community and partly contributed to its early development . 46 Figure 1. 4 . The structure s of paclitaxel ( Taxol ® ) and its analog , docetaxel ( Taxotere ® ) 5 Initially, paclitaxel was extracted from the inner bark of the pacific yew tree, Taxus brevifolia at very low yields ( 0.02 % w/w). 54 This means of supply raised concern s about its sustainability in addition to the labor intensive and high c ost of production . 54 A supply shortage of paclitaxel in its early development prompted the need for alternative means o f production. The initial approach was a semisynthetic process from 10 - deacetylbaccatin III (10 - DAB) , which was discovered in European yew, Taxus baccata . 55 , 56 Several semisynthetic methods were developed including the coupling of a phenylisoserine precursor ( - lactam synthesized through six steps ) to 10 - DAB ( Figure 1. 5 ) . 57 , 58 Bristol - Myers Squibb ( BMS ) adopted the semisynthesis method for commercial production of paclitaxel . 59 The application of semisynthesis method required use of environmentally harmful solvents and redundant protection/deprotection steps and was therefore aba ndoned for commercial production of paclitaxel. 59 6 A) B) Figure 1. 5 . The Holton methodology for the - lactam ( A ) and 10 - DAB precursor (B ) . In the search for a renewable and environmentally friendly production of paclitaxel, a lot of effort was geared towards the development of a biosynthetic process. 60 U ltimately, a 7 sustainable and environmentally safer Taxus plant cell fermentation (PCF) was developed by Phyton Biotech . 60 The first patent for PCF was filed in 1991 and produced paclitaxel at 1 3 mg · L . 61 Currently, Phyton Biotech USA and SamyangGenex, South Korea a re the two companies producing paclitaxel using PCF on an industrial scale . 62 PCF ha s been able to overcome challenges associated with its competitor methods, for example, production is independent of geographical and seasonal variations, it also provides uniform quality paclitaxel , and it is a renewable and environmentally friendly method . 63 , 64 However, there are still pertinent challenges associated with PCF including genetic ins tability, heterogeneous culture, low growth rates compared to bacterial cultures, variable yields, susceptibility to shear stresses in bioreactors , and aggregation. 65 Numerous proposals on how to overcome these drawbacks have been laid out and they mainly focus on molecular biological studies on taxane biosynthesis and how its accumulation affects metabolic profiles and gene expression in Taxus species cell cultures. 66 New taxane sources such as endophytic fungi of T axus and C orylus avellana cell culture have also been identified and show promise in the biotechnological production of taxanes in the future. 67 While the semisynthetic method to produce the drug was considered environmentally harmful, it ho wever avoided the likely rate - limiting biosynthetic steps occurring in planta 68 and proceeded with higher yields . The slow enzymatic steps in Taxus plants and derived cell cultures 68 likely contribute to the low production yields of paclitaxel . Be sides the produc tion challenges, paclitaxel also showed low solubility in water. 69 The discovery of a suitable formulation in a mixture of ethanol and Cremophor EL ® (currently referred to as Koll ipher EL ® ) proved effective during clinical trial s and is still administered in this form. 54 , 70 8 1.3. Paclitaxel Biosynthesis The biosynthes is of paclitaxel in Taxus plants involves ~19 steps from geranylgeranyl diphosphate (GGDP), the u niversal diterpenoid precursor derived from deoxyxylulose phosphate pathway. 71 , 72 The cyclization of GGDP by taxadiene synthase to the tricyclic taxane skeleton 73 , 74 is followed by eight cytochrome P450 - mediated oxygenations, three acyl CoA - dependent acylations, an oxidation at C9, and oxetane (D - ring) fo rmation to provide baccatin III. 75 This diterpene intermediate is further modified in five steps via the attachmen - phenylalanine at C13 by a CoA - dependent acyltransferase, P450 hydroxylation at C2' and final N - benzoylation to produce paclitaxel ( Figure s 1. 6 and 1. 7 ) . 76 - 78 All the genes for the CoA thioester - dependent acyltransferases on the paclitaxel biosynthetic pathway were identified in previous studies . 79 - 81 Among them are genes that code for 13 - O - phenylpropanoyl transferase (BAPT) and N - benzoyltransferase enzymes , which have been characterized in the biosynthesis pathway . 80 , 81 Two benzoyltransferases and a 13 - O - phenylpropanoyltransferase from Taxus were characterized separately in assays with a suitable paclitaxel precursor. 80 The benzoyl - and p henylpropanoyl CoA thioester co - substrates were synthesized by reaction of a mixed anhydride with CoA . 80 - phenylalanine and phenylisoserine needed prote cting chemistry to cap the reactive amino or hydroxyl groups on the propanoid side chain be fore the CoA coupling step . 80 By application of these acyltransferases in the late - stage biosynthesis of paclitaxel, the biocatalytic method can be imagined to advance through direct side chain attachment to the baccatin III core (see Figure 1. 7 , bottom panel ). Additionally , th is biocatalytic conversion would also short - circuit the protecting group chemistries and hazardous solvents obligatory for the synthetic route. 9 Figure 1. 6 . Biosynthetic pathway to p aclitaxel starting from simple primary metabolite precursors to the baccatin III . The various enzyme catalyzed steps are shown : (a) geranylgeranyl diphosphate synthase, (b) GG D P cyclization by taxadiene synthase, (c) P450 oxygena se, (d) 5 - O - acetylation by a taxa - - ol acetyltransferase, (e) P450 oxygenases, (f) 2 - O - benzoylation by a 2 - O - debenzoylbaccatin III benzoyltransferase; (g) 10 - O - acetylation by a 10 - deacetylbaccatin III acyltransferase . 10 A) B) Figure 1. 7 . Biosynthesis of paclitaxel C - 13 side chain ( A ) and the enzyme catalyzed transfer to the baccatin III ( B ) ; (h) the conversion of (2 S ) - - phenylalanine to its (3 R ) - is catalyzed by an aminomutase (PAM) ; (i) phenylpropanoyl CoA is catalyzed by unidentified phenylalanyl CoA ligase in Taxus plants ; (j) 13 - O - acylation by a phenylpropanoyltransferase ; (k) C - 2' - oxidation by a P450 hydroxylase; (l) N - benzoylation by a taxane N - benzoyltransferase 1.4. In search for an Ami n oacyl CoA Ligase The protecting group chemistry used to synthesize the amino phenylpropano yl CoA thioesters in the earlier paclitaxel biosynthetic studies 80 ( Figure 1. 8 ) can f oreseeably be averted by employing a chemoselective car boxylate:CoA ligase during acyl CoA biosynthesis. Further, such a ligase 11 would avoid the solvent incompatibility of synthetic methods used to make acyl CoA thioesters when a hydrophobic acid anhydride is coupled with the hydrophilic CoA . Figure 1 . 8 . Synthesis of (2 R ,3 S ) phenylisoserinyl CoA via the mi xed anhydride intermediate . (i) CH 2 Cl 2 /THF, DMAP in CH 2 Cl 2 , benzyl chloroformate, rt, 1 h, 90% yield ; (ii) CH 3 CN , DMAP in CH 3 CN , Boc 2 O , rt, 24 h, 20% yield ; (iii) CH 3 OH, 6% Mg(OCH 3 ) 2 , rt, 1 h, 80% yield ; (iv) step (ii), 80% yield ; (v) 2 M NaOH, 12 h ; (vi) THF, ethyl chloroformate, rt, 1 h ; (vii) CoASH in 0.4 M NaHCO 3 , t - BuOH, rt, 0.5 h ; (viii) HCOOH, rt, 10% yield. Synt - - phenylalanyl CoA were carried out similarly except for the 2 - hydroxyl protection. Earlier studies showed that Taxus 13 - O - acyltransferase can attach a 3 - a mino - 3 - phenylpropanoyl by means of its synthetically - derived CoA thioester to the C13 - hydroxyl of baccatin III to form an advanced precursor of paclitaxel. 80 The need to synthesize the 3 - amino - 3 - phenylpropanoyl CoA thioesters is a major limitation towards the development of an in vitro biosynthetic approach to paclitaxel starting from bac catin III. To overcome this challenge, an alternative aminoacyl CoA ligase was proposed based on available literature as described in the following sections. 12 1.4.1. ANL Superfamily Adenylation or adenylate forming enzymes are diverse in nature. They are involved in unique biological processes, for example biosynthesis of secondary metabolites, 82 lipid metabolism, 83 protein synthesis 84 and degradation, 85 DNA synthesis , 86 and CoA biosynthesis. 87 , 88 Over the years, a lot of research has been dedicated to identifying adenylation enzymes through mechanistic and structural studies. 89 - 93 The term ANL was coined to designate the three main subfamilies of adenylate - forming enzymes, namely A cyl CoA synthetases, N on - ribosomal peptide synthetases (NRPSs), and the L uciferase enzymes. 94 The ANL enzymes catalyze two partial reactions; the initial adenylation half reaction starts with the binding of carboxylic acid and ATP to the enzyme active site, where the carboxylate attack on ATP - phosphate leads to the formation of acyl - adenylate intermediate. 95 In the second half reaction, the acyl - AMP is attacked by a nucleophilic oxygen, sulfur , or nitrogen atom of an acceptor molecule leading to formation of an ester, thioester , or amide respectively. 95 The acceptor molecule is either a small molecule such as CoA (in acyl CoA biosynthesis), a peptidyl carrier protein (PCP, also referred to as thiolation domain) in NRPSs or to the molecular oxygen in luciferase enzymes. 96 Besides the similar adenylation partial reaction mechanism in ANL superfamily, there is a high structural homology among many members. 96 The crystal structures of at least 16 proteins from ANL superfamily have been solved and used to identify core sequences that are highly conserved and the role they play in the catalysis. 94 Notably, the structures of these enzymes show a unique architecture; they have a large N - terminal and a small C - terminal domain . The roles of these two domains have been elucidated using gramicidin synthetase A (Grs 1( Phe A ) ) a denylation domain and acetyl CoA ligase structures. 92 , 97 It was shown that after the first half 13 reaction (shown with Grs 1 (Phe A) ), the enzyme adopts a different conformation for the second thiolation reaction through a 140 ° rotation of the C - terminal domain (shown with acetyl CoA ligase). 97 To validate this observation , a conserved Lys l ocated at the C - terminal domain in the ANL superfamily was separately mutated to different re sidues in propionyl CoA synthetase and firefly luciferase . 98 , 99 A drastic reduction in enzyme activity for the first ha lf acyl adenylation reaction was reported. 98 , 99 Howeve r, the Lys mutants in propionyl CoA synthetase converted propionyl AMP to propionyl CoA, hence further suggesting that the conserved Lys only catalyzes the first half reaction. 98 From the crystal structure , this Lys is located at 3 Å from the active site in Grs 1 (Phe A) . 92 Due to the close proximity to the carboxylate group of phenylalanine substrate, it was proposed that this residue is important in positioning the substrate at the right orientation for a nucleophilic attack on ATP - phosphate. 92 However, in acetyl CoA ligase structure, the Lys at similar position was located 25 Å from the active site and was not shown to participate in the thiolation reaction. 97 The biochemical and structural data implicating the conserved Lys was suggested to support a novel catalytic strategy for ANL enzymes. 94 It has been proposed that the adenylate - forming enzymes adopt a Grs 1 ( Phe A ) - like conformation in the first half adenylation reaction. Following the formation of the act ive acyl - adenylate intermediate, the enzyme adopts a different conformation for the second half reaction. 94 - 97 In NRPSs, it has been argued that the enzyme conformational change s also happen even though the thiol donor phosphopantetheine is covalently attached to the thiolation domain. 94 The crystal structure of surfactin A module (a NRPS enzyme) , showed that the position of a serine residue on which the phosphopantetheine group ( of PCP or thiolation domain) would attach was not position ed to allow shuttling of acyl group between domains in NRPS without a conformational change. 100 Therefore , the C - terminal domain would have to rotate to enable the 14 shuttling of the acyl moiety along th e epimeri zation domain template to complete the catalytic reaction. 94 , 100 1.4.2. Ty rocidine Synthetase A : Member of NRPS Enzyme F amily NRPSs are a family of enzymes involved in biosynthesis of important natural products, including antibiotics (e.g tyrocidine, gramici din S, penicillins, vancomycin , and bacitracin), 101 siderophores (e.g, enterobactin and mycobactin), 102 toxins (e.g syringomycin and HC - toxin) , 103 , 104 and immunosuppressive agents (e.g rapamycin and cyclosporin). 105 - 107 Due to their importance and divers ity , immense studies have bee n done to understand the biochemistry of this family of enzymes ranging from gene identity to structur e elucidation. 94 , 106 , 108 , 109 NRPSs have been shown to consist of modules that are organized in an order corresponding to the amino acid sequence of the final product in a co - lin ear fashion (the so called co - linearity rule). 108 In this rule, each module is responsible for recognition, activation, modification (where necessary) , and incorporation of a single amino acid into the growing peptide chain. 108 Tyrocidine synthetase is a member of NRPS family and is naturally found in Bacillus brevis strain, a soil bacterium. It consists of ten modules designated TycA (contains module 1 ) , TycB (contains module s 2 4) , and TycC (contains module s 5 10) . 110 The starter module , TycA incorporates a D - phenylalanine, TycB incorporates three residues (L - proline, L - phenylalanine and D - phenylalanine) and TycC incorporates the remaining six residues (L - asparagine, L - glutamine, L - tyrosine , L - valine, ornithine , and L - leucine). The tyrocidine synthetase modules are further subdivided into domains, each responsible for one catalytic function ( Figure 1. 9 ) . 110 - 112 15 Figure 1.9 . The tyrocidine synthetase cluster showing the three polypeptides; TycA, TycB , and TycC , that hosts one, three , and six modules respectively. Each of these modules contain necessary catalytic domains responsible for incorporation of a single amino acid residue into the growing pepti de chain: Adenylation domain ( A , grey), Thiolation domain ( T , black oval), Condensation domain ( C , yellow), Epimerization domain ( E , blue), Thioesteras e domain ( TE , green). The figure is redrawn from Benoit & Florian (2009 ) with permission from John Wiley & Sons, copyright 2009. 16 1.4.2.1. Adenylation (A) Domain The A domain (about 550 amino acids) bears the substrate recognition and activation site. A domain activates its cognate amino acid into its adenylate form at the expense of ATP with loss of pyrophosphate (P P i). 109 Due to its catalytic role and reaction mechanism, A domain is classified under the ANL superfamily described in S ection 1.4. 94 The A domain s are structurally and functionally independent and catalyze the amino acid incorporation with similar spec ificity as the wild type enzyme ( eg . TycA, consisting of a denylation, thiolation, and epimerization domains) . 113 , 114 Additionally, TycA adenylation domain exhibits broad substrate scope , a desirable feature in the synthesis of different aminoacyl AMP derivatives. 115 , 116 The aminoacyl adenylate intermediates formed are then covalently bound to the enzyme through a thioester linkage to phophopantetheine moiety in the adjacent thiolation domain. 117 1.4.2.2. Thiolation (T) Domain Thiolation domains, also referred to as peptidyl carrier domains (PCP) are located downstream of the respective A domain partner in tyrocidine synthetase cluster. 116 Each T domain contains about 100 amino acids and hosts a conserved serine where a 4' phosphopantetheine (4' Ppant ) cofactor is attached during post translational modification. 114 The conversion of T domain from apo to holo form enzyme is me diated by a 4' phosphopantetheine transferase that catalyzes the nucleophilic attack of a hydroxyl group of the highly conserved serine to the phosphate of CoA therefore transferring the Ppant moiety onto the T domain ( Figure 1 . 10 ). 108 The Ppant thiol attacks the aminoacyl - adenylate displacing the AMP to form a relatively stable aminoacyl - thioester interm ediate ( Figure 1.1 1 ) . 17 Figure 1. 10 . Scheme showing the conversion of apo - PCP (or T domain ) to holo PCP . The 4 ' Ppant T (4 ' phosphopantetheinyl transferase) catalyzes the nucleophilic attack of hydroxyl of a conserved serine residue ( - phosphate of CoA hence transferring the P pant moiety to the PCP ( T domain ) . 1.4.2.3. Epimerization (E) Domain The E domain belongs to a class of cofactor indep endent epimerases that catalyze the de - and reprotonation of C of an enzyme bound aminoacyl S Ppant (in TycA) or peptidyl S Ppant (e.g in TycB , module 4 ). 108 This domain is composed of ~450 amino acids and is embedded within each module that has been shown to convert an L to a D amino acid. 118 At equilibrium, both L and D isomers are formed, but only the D isomer is accepted by the highly enantioselective condensation domain located on the adjacent module ( Figure 1.9 ) . 119 As mentioned before, A and T domain s are independent catalytic domains. On the contrary, the activity of E domain relies on the presence of an upstream partner T domain ( Figure 1.1 1 ) . 120 18 Figure 1.1 1 . Schematic representation of the reactions catalyzed by various domains within a module in tyrocidine synthetase . Note: Epimerization (E) domain is present in modules 1 and 4 where the L amino acid is converted to the D isomer in tyrocidine synthetase . The 4' Ppant precursor is shown as a wiggly line. In NRPS enzymes, the cha in elongation occurs through amide bond formation catalyzed by the condensation (C) domain. 121 The growing aminoacyl (or peptidyl) chain is covalently attached to the T domain through a thioester bond and is transferred to the attacking aminoacyl residue whic h is in a thioester linkage to the downstream T domain in the next module. 121 In tyrocidine synthetase, each elongation module has a CAT triad ( Figure 1.9 ). 108 O nce the necessary a mino acid residue s have been incorporated , t he cyclization of the final product is catalyzed by the termination module referred to as thioesterase (TE) domain . The intramolecular cyclization of tyrosidine occurs when the amine group of D - phenylalanine, the first residue incorporated by TycA module attacks the thioester bon d of the last residue (Leucine) 122 (see Figure 1.9 ). 19 Tyrocidine synthetase A (TycA) comprises of AT E domain s that bind, activate and isomerize L phenylalanine. 116 Naturally, the A domain of TycA has similar adenylation partial reaction as CoA ligases. 96 The second partial thiolation reaction proceeds with different thiol donors (Ppant in TycA and CoA in acyl CoA ligases) that differentiate the two adenylases. 96 The biosynthesis of paclitaxel in T axus plants employs an aminoacyl CoA ligase in the pathway that remains unidentified. 79 To develop and implement a novel semi - biosynthetic pathway towards paclitaxel production, there is a need to identify an alternative aminoacyl CoA ligase. TycA was selected as an ideal candidate due to the numerous similarities it sh ares with the acyl CoA ligases . 94 , 96 Additionally, TycA activate s phenyl alanine which is structurally similar to the substrate needed for the biosynthesis of paclitaxel and its analog s. 116 Furthermore, the broad substrate scope displayed further qualifies this adenylase as an ideal aminoacyl CoA ligase for investigation in the current study . The availability of structural information within the ANL super family provides a clue on the binding site archite cture of close homolog s which is handy in designing new substrates for TycA . 92 , 94 , 123 The projects aimed at elucidating the activity of TycA as a CoA ligase will be highlighted in the following three chapters. Chapter two will focus on characterization, activity assessment , and kinetic parameters of TycA (Phe ATE) in the biosynthesis of aminophenylpropano yl CoA products. Firstly, the development of TycA activity assays using N acetylcysteamine as a thiol donor will be discussed. The comparison of TycA with other known aminoacyl CoA ligases will also be highlighted. The focus of c hapte r three is dissecting TycA domains to understand their role in CoA ligase reaction and also the domain s that are needed for maximum CoA ligase activity. The activity of TycA mutants (Phe A and Phe AT) with the aminophenylpropanoates will be 20 discussed . Additionally, the activity of these mutants will be analyzed using structural model s that are based on close homologs of Tyc( Phe A ) . Chapter four will focus on synthesis and characterization of isoserine analog s that are important motifs of paclitaxel de rivatives . TycA substrate scope studies with the isoserine analog s will also be discussed . Chapter five will focus on engineering Tyc( Phe AT ) with the aim of expand ing the substrate scope of this CoA ligase . P reliminary mutation studies will be discussed. 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Crystallogr. 2014 , 70 , 1442. (119 ) Stachelhaus, T.; Walsh, C. T. Biochemistry 2000 , 39 , 5775. (120) Linne, U.; Doekel, S.; Marahiel, M. A. Biochemistry 2001 , 40 , 15824. (121) Stachelhaus, T.; Mootz, H. D.; Bergendahl, V.; Marahiel, M. A. J. Biol. Chem. 1998 , 273 , 22773. (122) Trauger, J. W.; Kohli, R. M.; Mootz, H. D.; Marahiel, M. A.; Walsh, C. T. Nature 2000 , 407 , 215. (123) Wu, R.; Reger, A. S.; Lu, X.; Gulick, A. M.; Dunaway - Mariano, D. Biochemistry 2009 , 48 , 4115. 29 This Chapter is adapted from our published work in the Journal of Chemistry & Biology (Muchiri, R.; Walker, Kevin D. Chem. Biol. 2012 , 19 , 679). 30 2. TYROCIDINE SYNTHETASE A (TycA) CATALYSIS IN THE BIOSYNTHESIS OF AMINOACYL COA AND AMINO ACYL - N - ACETYL CYSTEAMINE 2.1. Introduction Tyrocidine synthetase A ( TycA or Phe ATE) is a member of the broad acyl - adenylate - forming enzyme family that includes numerous CoA ligases , including those that transfer CoA to acetate 1 , fatty acid, 2 coumarate , 3 O - succinylbenzoate, carnitine, bile acid , and b enzoa te . 4 , 5 During the CoA ligase catalysis, the acyl - adenylate intermediate, formed by nucleophilic atta ck of carboxylate subst rate - phosphate group of ATP, serves as the acyl donor to the thiol group of CoA. Though alkanoyl - , alkenyl - , and aroyl CoA ligases have been extensively studied , 6 - 10 only few studies have been reporte d for the amino acyl CoA ligases . 11 - 13 TycA belongs to a non - ribosomal peptide synthetase (NRPS) family of enzymes that are characterized by a unique set of modular proteins. 14 Each module is responsible for inco rpo rating a single amino acid substrate into a growing polypeptide chain through assistance of structurally independent domains that activate a nd condense the carboxyl substrate. 15 TycA consists of A denylation ( A ), T hiolation ( T ) , and E pimerization ( E ) domains (designated Phe ATE ) . The A domain recognizes and binds the amino acid, and then uses the cosubstrate ATP to adenylate the carboxylate of the substrate to form the aminoacyl adenylate. 16 The activated acyl moiety is then transferr ed to the sulfhydryl group of 4' - phosphopantetheine (Ppant) cofactor that is covalently bound to the adjacent thiolation domain . The latter cofactor is added during posttranslational modification of the apoprotein . 17 P revious studies showed that Ppant is cova lently tethered to a serine residue within a highly conserved motif within the thiolation domain, characterized by the CoreT residues 31 (GG[H,D]S[L,I]. 18 The posttranslational modification is catalyzed by a CoA dependent 4 ' - phosphopantetheinyl transferase. The Ppant cofactor is critical in tethering the acyl intermediate as a thioester in Tyc( Phe ATE ) . 17 The covalently attached thioester intermediate is epi merized by the epimerizatio n domain and acyl moiety is subsequently transferred to the downstream modules ( Figure 2. 1 ). 19 The E domains of NRPS catalyze isomerization reaction s that involve cat alytic transfer of protons that inverts stereochemistry. 18 This type of reaction traditionally follows the minimal number rule , which defines reaction classes that use or may be thought to use one catalytic residue where theoretically two or more could be employed . For this rule, there is a dual economy in the use of catalytic gr oups for the epimerization reaction . 20 Figure 2. 1 . Scheme showing reaction s catalyzed by TycA ( Phe ATE ) domains . D omains shown are: A denylation ( A ), Thiolation ( T ) [ phosphopantethein yl arm (squiggly line) ] and the E pimerization ( E ) domain s. The dipeptide intermediate is shown in the downstream TycB (module 2) com prised of the Condensation ( C ), Adenylation ( A ) and Thiolation ( T ) domains. In NRPS epimerization domains, the cleavage of the C - H bond yield an C - carbanion intermediate that is envisioned to be stabilized by resonance, forming a planar thio 32 carboxy late anion. R eprotonat ion on either face of the thio l carboxylate forms the L or D amino acid species ( Figure 2.2 ) . 18 , 21 Figure 2.2 . The stabilization of thiol carboxylate intermediate formed d uring epimerization reaction in NRPS enzymes . Two alternative enzyme catalysis mechanism are described to explain this proton transfer step. 18 One mechanism employs two bases where one removes the C - proton from the substrates and the conjugate acid of the second enzymatic base delivers a proton back to the opposite face ( Figure 2. 3 A ). By contrast, a one - base mechanism uses a single enzym at ic base to both deprotonate and reprotonate the aminoacyl - S Ppant ( Figure 2 . 3 B ) . To differentiate between these two mechanisms in g ramicidin synthetase A (Grs 1 ) epimerization domain, r adiolabeled L [2 3 H] phenyl alanine was used as the substrate. In this study, there could be no label retained after epimerization to the D phenylalanyl S phosphopantethei nyl - acyl enzyme product. The mechanistic evaluation of the epimerization step between L phenyl alanine to D phenyl alanine o n the gramicidin synthetase pathway therefore supported a two base mechanism . 18 33 Figure 2. 3 . Possible pathways for the epimerization at the alpha carbon : A ) Two base mechanism where one active site base abstracts the proton from the substrate and a second active site base donates a proton. B ) One base mechanism for epimerization where only one residue abstracts and donates the proton. On the t yrocidine NRPS pathway , the final product is formed through iterative process es on the three separate catalytic modules (TycA, TycB and TycC). Generally, the adenylation, thioesterification , and epimerization (where necessary) reaction cycle is repeated with the inserti on of a condensation step that forms an amide bond between amino acid residues . 22 Despite the wealth of mechanistic information on the chemistry of the adenylation domains on NRPS pathways, overviewed in the previous discussion, applic ation of this information towards making tyrocidine and gramicidin analogs is limited . 23 - 26 In earlier studies, the mechanistic similarities bet ween CoA ligases and the A domains of NRPSs were elucidated. 27 In one study, the enterobact in A domain on the enterobactin NRPS pathway in Escherichia coli was shown to use CoA and pantetheine in thio l dependent release of PPi in place of the pendent Ppant moiety of the thiolation domain. 28 It remained unclear in this study whether the thiol surrogates were binding the active site during the catalysis of the thioester or were non - enzymatically scaven ging the acyl - adenylate intermediate released from the active site. Encouraged by t h e earlier study where the A domain of an NRPS pathway could apparently utilize CoA to form activated A B 34 thioesters, TycA (Phe ATE) was examined to evaluate whether it could en zymatically catalyze the production of acyl CoAs under steady state conditions. - like mechanism of TycA prompted us to explore this phenylpropanoid a denylation enzyme towards adenylating phenylisoserine for the potential production of pheny lisoserinyl CoA that likely appears on the biosynthetic pathway of the anticancer drug paclitaxel (Taxol). 12 In earlier studies, the phenylpropanoyltransferase was characterized with amino phenylpropanoyl CoA substrates that were synthesized using a mixed anhydride and CoA. 29 This synthesis involve d eight steps that require d protection/deprotection of the amine group and wa s challenged by the solvent incompatibility of the hydrophobic acid anhydride int ermediate and the hydrophilic Co A in the synthesis method ( Figure 2.4 ). The protection/deprotection chemistry and the use of non - environmental friendly and expensive solvents and reagents can be avoided by emp loying a chemoselective carboxylate CoA ligase. To evaluate the substrate specificity and kinetics of the Tyc( Phe AT ) domains of the TycA module, the conserved serine residue on the T domain was mutated to prevent enzyme bound phosphopantetheinylation of the subs trate and decouple the activity of the Tyc(Phe AT) from downstream epimerization domain . In addition, the earlier study indirectly assessed the formation of a thioester using exchange of 32 PPi into ATP to measure the reversible formation of an acyl AMP . He rein, electrospray ionization mass spectrometry was employed to directly evaluate the formation of the amino acyl CoA and also evaluate th e release, if any, of the amino acyl AMP intermediate. 35 (A) Previous studies (B) This study Figure 2.4 . Synthesis of (2 R ,3 S ) phenyl isoserinyl CoA via the mixed anhydride intermediate ( panel A , see Figure 1.8 in C hapter 1 for full scheme ) and the proposed biosynthetic route in the current study (panel B) . 2.2. Experimental 2.2.1. Substrates, Reagents, and General Instrumentation B ovine serum albumin was obtained from Thermo Scientific (Rockford, IL), N Boc ( S ) phenylalanine and N acetylcysteamine were purchased from Sigma Aldrich, N Boc ( R , S ) phenylalanine was obtained from Alfa Aesar , (Ward Hill, MA), N Boc (2 R ,3 S ) phenylisoserine was obtained from PepTech Corp. ( Burlington, MA) , CoA was purchased from American R adiolabeled Chemicals Inc. (St. Louis, MO) . All other reagents were obtained from Sigma Aldrich and were used without further purification, unless noted otherwise. A Varian Inova - 300 or a V arian UnityPlus500 instrument was used to acquire 1 H - and 13 C - NMR . A Q - ToF Ultima electrospray ionization high resolution mass spectrometer ( ESI MS, Waters, Milford, M A) with a Waters 2795 HPLC and Quattro Premier ESI MS coupled with Acquity® UPLC system w ere used for mass spectral analysis. 36 2.2.2. Expression of wild - type TycA cDNA A cDNA clone of wild - type tyrocidine synthetase A (TycA) was obtained as a gift from Florian Hollfelder (University of Cambridge, UK). Cloned cDNA tycA was inserted into a pSU18 vector, and the plasmid was designated pSU18 Tyc A His encoding expression for a C terminal His 6 epitope. The plasmid was used to transform E . coli BL21 (DE3) that was grown in 2xYT medium (100 mL), containing Bacto Tryptone (1.6 g), Bacto Yeast Extract (1.0 g), NaCl (0.5 g), and chloramphenicol (20 mg · mL 1 ) at 37 °C for 12 h. A 10 m L aliquot of the seed culture was used to inoculate 2 × YT medium (5 × 1 L). The bacteria were grown at 37 °C to OD 600 ~0.6, at which time isopropyl D 1 thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, and the culture was grown for 4 h at 30 °C. The cells were pelleted by centrifugation (30 min, 4000 g ) at 4 °C, resuspended in Binding buffer (20 mM Tris HCl buffer containing 0.5 M NaCl and 5 mM imidazole at pH 7.8), lysed by sonication (Misonix XL 2020 sonicator, Misonix, Inc. Far mingdale, NY), and then centrifuged at 15,000 g for 0.5 h. The supernatant was decanted and centrifuged at 149,000 g for 2 h to remove cell wall debris and light membranes. 2.2.3. Construction and Expr ession of the TycA S563A Mutant A S563A amino acid mutation was incorporated into the wild - type TycA clone by site directed mutagenesis. The oligonucleotide primer pair used to incorporate the point mutation (underlined) was as follows: Forward primer S563A For: 5' TTA CTC GCT CGG CGG AGA T GC G AT CCA AGC GAT CCA G GT CG 3'; Reverse primer S563A Rev: 5' CGA CCT GGA TCG CTT GGA T CG C AT CTC CGC CGA GCG AGT AA 3'. The correct synthesis of the mutant cDNA was verified by DNA sequencing. The resultant plasmid encoding a C terminal His tag (designated pSU18 TycA S563A His) was used to transform E. coli BL21 (DE3) cells. A 10 37 mL culture of E . coli transformed with the PSU18 vector was grown in 2xYT medium at 37 °C with chloramphenicol (20 mg · mL 1 ) selection for 12 h. T he 10 mL inoculum was transferred to a new batch of 2 × YT medium (1 L), as described previously for the expression of the wild type t ycA clone. The bacteria were grown at large scale at 37 °C to OD 600 ~0.6, and the cDNA expression was induced by IPTG , and the culture was grown for 4 h at 30 °C. The cells were pelleted by centrifugation (30 min, 4000 g ) at 4 °C, resuspended in Binding buffer , lysed by sonication, and the corresponding soluble protein fraction was clarified by centrifugation as described earlier to remove cell - wall debris and light membranes. 2.2.4. Pur ification and C haracterization of TycA and th e TycA S563A Mutant Crude soluble enzyme was separately isolated fr om bacteria expressing the wild - type tycA or tycA S563A. Each fraction contained ~15 mg total protein as estimate d by the Bradford protein assay . 30 These fractions were independently loaded onto a nickel nitrilotriacetic acid affinity column (Qiagen, Valencia, CA) and eluted according to the protocol described by the manufacturer. The column was washed w ith increasing concentration of imidazole (20 250 mM) in Binding buffer . SDS PAGE slabs were loaded with aliquots from each fraction that eluted off the nickel - affinity column and stained with Coomassie Blue. Fractions that contained >95% pure protein co rresponding to a molecular weight consistent with that of TycA and TycA S563A at 123 kDa were combined. The enzymes eluted in ~50 mM imidazole (100 mL) were separately loaded into a Centriprep size selective (100,000 MWCO) centrifugal filtration unit (Mill ipore, Billerica, MA). The protein solutions were concentrated to 1 mL, and the buffer was exchanged with the Assay b uffer (50 mM HEPES containing 100 mM NaCl and 1 mM EDTA at pH 8.0) over several dilution/concentration cycles. The final purity of the enzy me was estimated 38 by SDS PAGE with Coomassie Blue staining. The final p rotein concentration was determined by Beer's Law and measuring the absorbance of the protein solution at A 280 on a NanoDrop ND1000 Spectrophotometer ( Thermo Scientific, Wilmington, DE ). The extinction coefficient ( 280 = 142685 M 1 cm 1 ) and the molecular weight of T ycA and TycA S563A were 122675 g · mol 1 and 122,6 75 g · mol 1 , respectively). The purified protein was stored at 5 mg/mL at 80 °C. The protein sequence of the isolated TycA recombinant protein was confirmed by LC/ electrospray ionization tandem mass spectrometry analysis at the Michigan State University Proteomics Facility ( Appendix I , Figure I 11 ) . 2.2.5. Synthesis of Phenylalanyl AMP Authentic phenylalanyl Amp was synthesized by a previously described procedure . 31 Briefly, to ( 2 S ) phenyl alanine ( 330.4 mg, 2.0 mmol , 1.04 equiv ) and adenosine monophosphate ( 666.6 mg, 1.92 mmol , 1 equiv ) were added distilled water (3.2 mL) and pyridine (10.4 mL) in a glass - stoppered round bottom flask. The solution was acidified with 8 N HCl (0.25 mL) and stirred at 0 °C for 15 min. A solution of N,N' - dicyclohexylcarbodiimide (10.3 g, 50 mmol) dissolved in pyridine (12 mL) was then added, and the reaction was stirred for 4 h. The reaction was terminated by the addition o f ice - cold acetone (150 mL), and after 1 min, the precipitate of crude ( 2 S ) phenyl alanyl adenylate was rapidly sunction filtered through 55 mm grade filter paper (Whatman, England ) on a Büchner funnel. The filter cake was washed with ice - cold acetone/ethyl alcohol (60:40, 3 × 5 mL), then with ice - cold diethyl ether (5 mL), and air - dried on the filter. The crude product was suspended in ice - cold water (10 mL), rapidly vacuum filtered, and the insoluble particulates were washed with ice - cold water (3 × 5 mL). All of the filtrates were combined, and 8N HCl was added to adjust the pH to 3. Cold ethanol (100 mL) was then 39 added and the solution was stored at 4 °C for 18 h. The resultant precipitate was separated by centrifugation, washed with ethanol, and the samp le was lyophilized to a residue (309 mg). The sample was judged to be 32% pure by LC MS and was dissolved in D 2 O and analyzed by NMR. 1 H - NMR - H), 4.18 (m, 2H, ribose 5' - H), 4.27 (m, 1H, ribose 4' - H), 4.43 (m, 2H, PheCH 2 ), 6.04 (d, 1H, 1' - H), 7.09 (s, 5H, aromatic H), 8.20 (s, 1H, 3' - H), 8.42 (s, 1H, 8' - H). 13 C - 149.0 , 147.0, 144.0, 131.0, 130.0, 129.0 , 120 , 89.0, 75.0, 65.0, 56.0, 35.0. 2.2.6. Activity of wtTycA or m TycA with ATP and Phenylalanine, and (2 R ,3 S ) Phenylisoserine Substrates ( S ) phenylalanine, ( R ) phenylalanine, and (2 R ,3 S ) phenylisoserine (each at 1 mM) were separately incubated at 31 °C in single stopped time (1 h) reactions containing 100 mM HEPES (pH 8.0), ATP (1 mM), MgCl 2 (3 mM) and TycA or TycA S563A control reactions were carried out in parallel under the same conditions used for assays containing enzyme, where TycA, TycA S563A , ATP, or amino acid was omitted from the assay. The reactions were quenched by acidi fication to pH ~2 (10% formic acid in distilled water) and lyophilized to dryness. The resultant samples were separately dissolved in aqueous 0.01 M f Premier XE Mass Spectrometer coupled with Acquity® UPLC s 30 eluted with a solvent gradient of a cetonitrile (Solvent A) and 0.05 % triethylamine in distilled water (Solvent B) (held at 2.5 % Solvent A for 3.17 min, increased to 100 % Solvent A over 5 sec with a 2 min hold, and then returned to 2.5 % Solvent A over 5 sec with a 50 sec hold) at a flow 40 rate of 0.4 mL/min. T he effluent from the column was di rected to the mass spectrometer set to negative ion mode electrospray ionization with a scan range of m / z 50 1000 . After identifying productive substrates for TycA and TycA S563A in the screen for AMP adenylases function, substrates ( S ) phenylalanine and ( R ) phenylalanine (each at 1 8.0), MgCl 2 (3 mM), and TycA or TycA S563A (20 reactions were acid quenched (10 % f ormic acid) at various time points (1, 5, 10, 15, 20, 30, 60, 90 and 120 min) to establish steady state conditions. At the end of each reaction and prior to sample to correct for variations of the analyte. The products of the enzyme - catalyzed reaction were quantified by a liquid chromatography multiple reaction monitoring (MRM) mass spectrometry technique 32 on the Quattro Premier XE Mass Spectrometer coupled with Acquity® UPLC system fitted with a C18 Ascentis Express column (2.5 × 50 mm, 2.7 described before, with a solvent gradient of a cetonitrile (Solvent A) and 0.05 % triethylamine in distilled water (Solven t B) (held at 2.5 % Solvent A for 3.17 min, increased to 100 % Solvent A over 5 sec with a 2 min hold, and then returned to 2.5 % Solvent A over 5 sec with a 50 sec hold) at a flow rate of 0.4 mL/min. The effluent from the chromatography column was directed to the mass spectrometer where the first quadrupole mass analyzer (in negative ion mode) was set to select for the [M H] - ion of a biosynthesized acyl AMP product. The selected ion was then directed to a collision gas chamber where the collision energy was optimized to maximize the a bundance of a single signature fragment ion ( m / z 134, the adenine fragment of the adenosine moiety ) in negative ion 41 mode. This ion was resolved in the second quadrupole mass analyzer by MRM of the adenine transition ion fragment ( m / z 134). The peak area of ion m / z 134 for each biosynthetic phenylpropionyl AMP ester was converted to concentration units using linear regression of a dilution series of authentic adenine (at six intervals from 0.01 to 20 M) plotted against the correspondi ng ion abundance ( m / z 134, in negative ion mode). The initial velocity ( v o ) of phenylpropionyl AMP production was used to calculate K M and k cat according to the Michaelis Menten equation ( R 2 was typically between 0.90 and 0.99). 2.2.7. Synthesis of Product Standards 2.2.7.1. Synthesis of [ S Phenylalanyl) N acetyl]cysteamine Figure 2.5 . ( N Boc) ( S ) phenylalanyl SNAC ( B - 2 ) and ( N Boc) ( R / S ) phenylalanyl SNAC ( B - 3 ) . [ S ((2 S ) and (3 R / S ) phenylalanyl) N acetyl]cysteamine (i.e., (2 S ) and (3 R / S ) phenyl alanyl SNAC ) were synthesized according to a reported procedure, with some modifications ( Figure 2.6 ) . 33 42 Figure 2.6 . Synthesis of (2 S ) phenyl alanyl SNAC . (i) a) DCC, HOBt, DIPEA, THF, rt. 45 min, b) N - acetylcysteamine, rt. 12 h. (ii) TFA/DCM, 0 °C, 4 h. Generally, to N Boc ( 2S ) or N Boc (3 R/S ) phenylalanine (530 mg, 2 mmol) dissolved in tetrahydrofuran (15 18 mL) were added N , N dicyclohexylcarbodiimide (372.2 mg, 2 mmol ), 1 hydroxybenzotriazole monohydrate (255.2 mg, 2 mmol), and N , N ' diisopropylethylamine (258.5 , 8 mmol) at 24 °C. After 45 min, N acetylcysteamine (NAC) (238 mg, 2 mmol) was added to the reaction, and the solution was stirred for ~12 h. The contents of th e reaction mixture were gravity filtered (42.5 mm filter paper, Whatman, Stockton, NJ), and the filtrate was concentrated under vacuum. The resultant resi due was then dissolved in ethyl acetate (8 mL), and extracted with an equal volume of 10% aqueous NaHCO 3 . The aqueous layer was separated, and the organic layer was extracted twice more with 10% aqueous NaHCO 3 . The organic fraction was dried (Na 2 SO 4 ), filtered, and the solvent was removed under vacuum. The resultant crude product was purified by silica gel flash chromatography (3 5% gradient of methanol in chloroform). The fractions containing the product, as judged by the thin layer chromatography (TLC) ( R f = 0.15 and 0.12 for N Boc phenyl alanyl SNAC and N Boc ( R / S ) phenyl alanyl SNAC , respectively) were combined separately and concentrated to afford N Boc phenyl alanyl SNAC (552 mg, 75% isolated yield). 1 H - NMR (500 MHz, DMSO d 6 1.32 (s, 9H, methyl H of Boc), 1.80 (s, 3H, H 1 ), 2.79 (dd, J = 13.7, 10.0, Hz, 1H, H b 7 ), 2.87 (t, J = 6 .0 Hz, 2H, H a 4 , H b 4 ), 3.05 (dd, J = 13.9, 5.0 Hz, 1H, H a 7 ), 3.15 (q, J = 6.0, 2H, H a 3 , H b 3 ), 4.23 (dd, J = 7.2, 5.0 Hz, 1H, H 6 ), 7.20 7.27 (m, 5H, aromatic protons ), 7.67 (d, J = 43 8.30 Hz, 1H, OC(O)NH); 8.04 (t, J = 5.0 Hz, 1H, C(O)NH). 13 C - NMR (125 MHz, DMSO d 6 202.33, 169.91, 155.97, 138.20, 129.78 , 128.86, 127.10 , 79.34, 62.97, 42.85, 38.79, 28.83, 24.20, 23.22. N Boc ( R / S ) phenyl alanyl SNAC (487 mg, 66% isolated yield). 1 H - NMR (500 MHz, DMSO d 6 H of Boc), 2.07 (s, 3H , H 1 ), 2.84 (ddd, J = 12.0, 6.0, 6.0Hz, 2H, H a 4 , H b 4 ), 3.08 (q, J = 6.0, 2H, H a 3 , H b 3 ), 3.29 (dd, J = 15.0, 9.0 Hz, 1H, H a 6 ), 3.40 (dd, J = 15.0, 5.5, 1 H, H b 6 ), 4.64 (m, 1H, H 7 ), 7.37 7.51 (m, 5H, aromatic protons ), 8.02 (t, J = 5.5 Hz, 1H, C(O)NH); 8.63 (bs, 1H, OC(O)NH). 13 C - NMR (125 MHz, DMSO d 6 : 196.49 , 169.89, 155.35, 14 3 . 11 , 128.99, 127.75, 12 7.04 , 78.67, 51.93 , 50 .72 , 38.86, 28 .89 , 28.68, 23.22. 2.2.7.2. N - deprotection of [S Phenylalanyl)] SNAC Figure 2. 7 . ( S ) Phenylalanyl SNAC ( B - 7 Phenylalanyl SNAC·HCl salt ( B - 8 ) To remove the Boc groups, N Boc ( S ) and N Boc ( R / S ) phenyl alanyl SNAC were separately dissolved in dichloromethane (4 mL), and trifluoroacetic acid was added dropwise over 4 h at 0 °C. The reaction progress was monitored by normal phase thin layer chromatography (5% MeOH in CHCl 3 ) until complete. Excess trifluoroacetic acid was removed prior to isolating the ( S ) phenyl alanyl SNAC by concentrating the reaction volume to 2 mL under vacuum, diluting 2 fold in dichloromethane, and then concentrating to 1 2 mL. This 44 dilution/concentration cycle was repeated three times, after which, the solvent was removed completely. To the residue containing ( S ) ph enyl alanyl SNAC was added ethyl acetate and dilute aqueous NaOH at 0 °C to partition the ( S ) phenyl alanyl SNAC and aqueous soluble contaminants, respectively. The organic layer was decanted and then removed under vacuum. Water (2 mL) was added to the remaining residue to which 1 M HCl (2 mL) was added at 0 °C. Ethyl acetate was added (2 × 2 mL) to extract any remaining t - butanol and SNAC, and the organic layer was decanted. The water fraction was lyophilized to yield ( S ) phenyl alanyl SNAC as the hyd rochloride salt isolated at ~31 % yield ( 90 mg), based on the N Boc protected starting material . 1 H - NMR (500 MHz, DMSO d 6 : 1.78 (s, 3H, CH 3 ), 2.69 (dd, J = 13.6, 10.0 Hz, 1H, H b 7 ), 2.80 (t, J = 6.0 Hz, 2H, H a 4 , H b 4 ), 2.96 (dd, J = 13.6, 5.0 Hz, 1H, H a 7 ), 3.12 (q, J = 6.0, 2H, H a 3 , H b 3 ), 3.62 (dd, J = 8.7, 5.0 Hz, 1H, H 6 ), 7.17 7.29 (m, 5H, aromatic protons ), 8.02 (t, J = 5.0 Hz, 1H, C(O)NH). 13 C - NMR (125 MHz, DMSO d 6 168. 15, 136. 78 , 128.3 2, 127.16, 125.30 , 61.74, 39.51, 37.22, 26.64, 21.53. The exact mass was determined in the positive ion mode on a Quadrupole Time of Flight Tandem Mass Spectrom eter : observed m / z = 267.1164; calculated m / z = 267.1167 for C 13 H 19 N 2 O 2 S. After deprotection of ( N Boc) ( R / S ) phenyl alanyl SNAC , excess trifluoroacetic acid was removed prior to isolating the product, as described above, except the residue was dissolved in 1 M HCl (2 mL, at 0 °C) to exchange the trifluoroacetate salt for the hydrochloride salt of the product. The sample was lyophilized to dryness, resulting in the hydrochloride salt of ( R / S ) phenyl alanyl SNAC (48 mg, ~88.1% yield). 1 H - NMR (500 MHz, DMSO d 6 CH 3 ), 2.84 (ddd, J = 12.0, 6.0, 6.0 Hz, 2H, H a 4 , H b 4 ), 3.08 (q, J = 6.0 Hz, 2H, H a 3 , H b 3 ), 3.31 (dd, J = 15.0, 9.0 Hz, 1H, H a 6 ), 3.45 (dd, J = 15.0, 5.5 Hz, 1H, H b 6 ), 4.63 (m, 1H, H 7 ), 7.35 7.60 (m, 5H, aromatic protons ), 8.06 (t, J = 5.5 Hz, 1H, C(O)NH), 8.73 (bs, 3 H, H 3 N). 45 13 C - NMR (125 MHz, DMSO d 6 196.47 , 169.98 , 137.06 , 129.58 , 129.37, 128.40 , 51.64, 48.07, 38.57, 28.92, 23.18. The exact mass was determined in the positive ion mode on a Quadrupole Time of Flight Tandem Mass Spectrometer : observed m / z = 267.1173; calculated m / z = 267.1167 for C 13 H 19 N 2 O 2 S. 2.2.7.3. Synthesis of [(2 R ,3 S ) - Phenylalanyl] N acetylcysteamine Figure 2. 8 . (2 R ,3 S ) Phenylisoserinyl SNAC . N Boc ( 2 R , 3 S ) Phenylisoserinyl SNAC was synthesized according to the procedure described phenyl alanyl SNAC thioesters. To N Boc ( 2 R , 3 S ) phenylisoserine (100 mg, 0.36 mmol) dissolved in tetrahydrofuran (~6 mL) were added N , N dicyclohexylca rbodiimide (74.27 mg, 0.36 mmol ), 1 hydroxybenzotriazole monohydrate (48.64 mg, 0.36 mmol), and N , N ' diisopropylethylamine (23.29 mg, 0.72 mmol) and s tirred at 24 °C. After 45 min, NAC (85.61 mg, 0.72 mmol) was added to the reaction, and the solution was s tirred for ~12 h. The reaction work up and purification were done as described above, resulting in crude N Boc ( 2 R , 3 S ) p henylisoserinyl SNAC (40 mg, 29% yield), which was used for the N Boc deprotection without further purification . To remove the Boc groups, N Boc ( 2 R , 3 S ) p henylisoserinyl was dissolved in dichloromethane (4 mL), and trifluoroacetic acid was added dropwise over 3 h at 0 °C. The reaction progress was monitored by normal phase TLC (5% methanol in chloroform) until comple te. The work up was done according to the procedure 46 described for phenyl alanyl SNAC resulting in ( 2 R , 3 S ) phenylisoserinyl SNAC (20 mg, ~24% isolated yield). 1 H - NMR (500 MHz, DMSO d 6 CH 3 ), 2.81 (m, 2H, H a 4 , H b 4 ), 3.15 (m , 2H, H a 3 , H b 3 ), 4.39 (d, J = 5.0 Hz, 1H, H 6 ), 4.45 (m, 1H, H 7 ), 7.40 7.47 (m, 5H, aromatic protons ), 8.09 (t, J = 5.6 Hz, 1H, C(O)NH), 8.52 (br.d, J = 5.0 Hz , 2 H, N H 2 ). 13 C - NMR (125 MHz, DMSO d 6 , 129.51, 129.16, 128.77, 78.24, 57.09, 38.3 8, 28.33, 23.24 . The exact mass was determined in the positive ion mode on a Quadrupole Time of Flight Tandem Mass Spectrometer : observed m / z = 283.1108; calculated m / z = 283.1116 for C 13 H 19 N 2 O 3 S . 2.2.8. Activity and Kinetic Evaluation of wtTycA or m TycA with N Acetylcysteamine and Aminophenylpropanoates Substrates ( S ) phenylalanine, ( R ) phenylalanine, and (2 R ,3 S ) phenylisoserine (each at 1 mM) were separately incubated at 31 °C in single stopped time (1 h) reactions containing 100 mM HEPES (pH 8.0), ATP (1 mM), MgCl 2 (3 mM), N acetylcysteamine (1 mM), and TycA or TycA Various control reactions were carried out in parallel under the same conditions used for the enzyme assay, where TycA or TycA S563A, ATP, or N acetylcysteamine was omitted from separate assays. The reactions were quenched by acidifying to pH ~2 (6 M HCl) and lyophilized to dryness. The resultant residues were separately dissolved Quadrupole Time of Flight Tandem Mass Spectromet er coupled with 2795 HPLC system fitted with a reverse phase Halo C18 column ( 30 °C) and the analytes were eluted with a solvent gradient of 0 15% of acetonitrile (Solvent A ) and 0.1% formic acid in distilled water (Solvent B) at a flow rate of 0.2 mL/min. 47 2.2.9. Kinetic Analysis of the N acetylcysteamine Ligase Reaction C atalyzed by TycA or TycA S563A Substrates ( S ) phenylalanine, ( R ) phenylalanine, and (2 R ,3 S ) phenylisoserine (each at 1 mM) were separately incubated with TycA S563A (20 µg) in the presence of N acetylcysteamine (1 mM), ATP (1 mM), and Mg 2+ (3 mM) to establish steady state conditions with respect to protein concentration and time at 31 °C. Under steady state cond itions, either ( S ) phenylalanine, ( R ) phenylalanine, or (2 R ,3 S ) phenylisoserine at 5, 10, 20, 40, 80, 160, 250, 500, 1000 , and 2000 µM was separately incubated with TycA or TycA S563A (20 µg), ATP (1 mM), Mg 2+ (3 mM), and N acetylcysteamine (1 mM) in dup licate, single stopped time in distilled water), and ( N Boc) p henyl alanyl SNAC or ( N Boc) p henyl alanyl SNAC (1 µM) wer e added as an internal standard when ( S ) phenylalanine or ( R ) phenylalanine was used as the substrate, respectively. The samples were analyzed on a Quattro Premier XE Mass Spectrometer coupled with Acquity® UPLC system fitted with a C18 Ascentis ® Express column (2.5 × 50 mm, 2.7 µm) at 30 °C. and the analytes were eluted with a solvent gradient of acetonitrile ( Solvent A) and 0.05% triethylamine in distilled water (Solvent B) (held at 2.5 % Solvent A for 3 .17 min , increased to 100 % Sol vent A over 5 sec with a 2 min hold, and then returned to 2.5 % Solvent A over 5 sec with a 50 - sec hold) at a flow rate of 0.4 mL/min. In brief, the effluent from the chromatography column was directed to the Quattro Premier ESI mass spectrometer, in multiple reaction monitoring ( MRM ) scan mode, to quantify the biosynthetic acyl SNAC products. The monitored fragment ions were m / z 120.06, 131.10 , and 105.95 for ( S ) phenyl alanyl , ( R ) phenyl alanyl , and ( 2 R, 3 S ) phenylisoserinyl SNAC , respectively . A st andard curve was used to convert the 48 peak area under the curve of the monitored fragment ion to concentration for each biosynthetic phenylpropionyl SNAC . Authentic ( S ) phenyl alanyl , ( R / S ) phenyl alanyl and (2 R ,3 S ) phenylisoserinyl SNAC were used to cons truct the standard curves by correlating the peak area under the curve of the monitored ion to co ncentration of the standard (at 0.16, 0.32, 0.63, 1.3, 2.5, 5, 10, 20, 40, 80, 160 and 320 M) using linear regression analysis. The initial velocity ( v o ) production of ( S ) phenyl alanyl , ( R / S ) phenyl alanyl , and (2 R ,3 S ) phenylisoserinyl SNAC was plotted against substrate concentration and fit by non linear regression to the Michaelis Menten equation ( R 2 was typically 0.99) to calculate the Michaelis param eters ( K M and k cat ). The K M values of TycA and TycA S563A for N acetylcysteamine was assessed by incubating each enzyme separately with ( S ) phenylalanine (1 mM), MgCl 2 (3 mM), ATP (1 mM), and N acetylcysteamine at 5, 10, 20, 40, 80, 160, 250, 500, 1000 , and 2000 µM at 31 °C for 20 min. The reactions were quenched by acidifying to pH ~2 (10% formic acid in distilled water), and internal standard ( N Boc) p henyl alanyl SNAC (1 µM) was added to each sample to correct for variations of the analyte. The SNAC thioester products of the enzyme - catalyzed reaction were quantified by a liquid chromatography multiple reaction monitoring (MRM) mass spectrometry technique, and the monitored fragment ion ( m / z 120.06) derived from the SNAC thioester analytes in the efflu ent were quantified identically to the procedure described earlier. The initial velocity ( v o ) production of ( R ) phenyl alanyl SNAC made in separate assays was plotted against substrate concentration and fit by non linear regression to the Michaelis Menten equation ( R 2 was typically between 0.97 and 0.99 ) to calculate the Michaelis constant ( K M ). 49 2.2.10. Activity and Kinetic Evaluation of wtTycA or m TycA as Ligases for and Aminop henyl propanoyl Co A Thioesters Similar experiments were done as described above for the N acetylcysteamine ligase screen study, except CoA (1 mM) was used in place of N acetylcysteamine with the substrates ( S ) phenylalanine, ( R ) phenylalanine and (2 R ,3 S ) phenylisoserine in different 1 mL assays. The enzyme assays for this study contained 100 mM HEPES (pH 8.0), ( S ) phenylalanine (1 mM), ATP (1 mM), MgCl 2 (3 mM), CoA ( 1 mM), and TycA or TycA incubated at 31 °C for 1 h. Various control reactions were carried out in parallel under the same conditions used for assays containing enzyme, where TycA, TycA S563A, ATP, or CoA was omitted from the assay. The reactions were quenched by acidifying to pH ~2 (6 M HCl) and lyophilized to dryness. The resultant samples were se parately dissolved in aqueous 0.01 M HCl Quadrupole Time of Flight Tandem Mass Spectrometer coupled with a 2795 HPLC system fitted with a reverse phase Halo C18 column ( 5 cm × 2.1 mm) at 30 e was loaded onto the column and the analytes were eluted with a solvent gradient of 0 15% of acetonitrile (Solvent A) and 0.1% formic acid in distilled water (Solvent B) at a flow rate of 0.2 mL/min. The effluent from the column was directed to the mass spectrometer set to negative ion mode with a scan range of m / z 200 1000 atomic mass units. Authentic phenylalanyl CoA was used as a model to identify the diagnostic ion cleavage transitions of the three acyl CoA products. 50 2.2.11. Kinetic Analysis of the CoA Ligase Reaction C atalyzed by TycA and TycA S563A After identifying productive substrates for TycA and TycA S563A in the screen for CoA ligase function, substrates ( S ) phenylalanine, ( R ) phenylalanine, and (2 R ,3 S ) phenylisoserine (each at 1 mM) wer (pH 8.0), ATP (1 mM), MgCl 2 (3 mM), CoA (1 mM), and TycA or TycA S563A (50 µg or 20 µg ) to establish steady state conditions with respect to protein concentration and time at 31 °C. Under steady state conditions, ( S ) phenylalanine, ( R ) phenylalanine, and (2 R ,3 S ) phenylisoserine at 5, 10, 20, 40, 80, 160, 250, 500, 1000 , and 2000 µM were separately incubated with TycA or TycA S563A (20 µg) for 30 min. At the end of each reaction and prior to mass spectrometry analysis, acetyl CoA (1 µM) was added as the internal standard to each sample to correct for variations of the analyte. The products of the enzyme - catalyzed reaction were quantified by a liquid chromatography MRM mass spectrometry tech nique on the Quattro Premier XE Mass Spectrometer coupled with Acquity® UPLC system fitted with a C18 Ascentis o nto the column and the analytes were eluted with a solvent gradien t of acetonitrile (Solvent A) and 0.05% triethylamine in distilled water (Solvent B) (held at 2.5 % Solvent A for 3.17 min, increased to 100 % Solvent A over 5 sec with a 2 min hold, and then returned to 2.5% Solvent A over 5 sec with a 50 - sec hold) at a flow rate of 0.4 mL/min. The effluent from the chromatography column was directed to the mass spectrometer where the first quadrupole mass analyzer (in negative ion mode) was set to select for the molecul ar ion of a biosynthesized acyl CoA product. The selected ion was then directed to a collision gas chamber wherein the collision energy was optimized to maximize the abundance of a single signature fragment ion ( m / z 51 408.31) monitored in the seco nd quadrupole mass analyzer (in negative ion mode). The monitored ion was derived by a fragmentation reaction in the CoA moiety that is characteristic of acyl CoA thioesters analyzed by this MRM method. The peak area under the curve of the monitored fragme nt ion m / z 408.31 corresponding to each biosynthetic phenylpropionyl CoA thioester was converted to concentration by comparing the peak area of the same ion produced by authentic CoA (at 0.0 48 , 0.0 98, 0.20, 0.39, 0.78, 1.6, 3.1, 6.3, 12.5, 25, 50, 100 M) using linear regression analysis. The initial velocity ( v o ) production of ( S ) phenyl alanyl , ( R ) phenyl alanyl and (2 R ,3 S ) phenylisoserinyl CoA made in separate assays was plotted against substrate concentration and fit by non linear regression to the Mi chaelis Menten equation ( R 2 was between 0.85 and 0.99) to calculate the Michaelis parameters ( K M and k cat ). The K M values of TycA and TycA S563A for CoA w ere assessed by incubating each enzyme separately with ( S ) phenylalanine (1 mM), MgCl 2 (3 mM), ATP (1 mM), and CoA at 0.05 , 0.1 , 0.2 , 0.4 , 0.8 , 1 . 6, 3 . 2 , 6 . 4 , 12 . 8 , and 25 . 6 µ M at 31 °C for 20 min. At the end of each reaction and prior to mass spectrometry analysis, acetyl CoA (1 µM) was added as the internal standard to each sample to correct for v ariations of the analyte. The products of the enzyme - catalyzed reaction were quantified by a liquid chromatography MRM mass spectrometry technique, and the monitored fragment ion ( m / z 408.31) derived from the CoA thioester analytes in the effluent were qua ntified identically to the procedure described earlier herein. The initial velocity ( v o ) production of ( S ) phenylalanyl CoA made in separate assays was plotted against substrate concentration and fit by non linear regression to the Michaelis Menten equation ( R 2 was typically between 0.92 and 0.98) to calculate the Michaelis constant ( K M ). 52 2.3. Results and Discussion 2.3.1. Expression and Purifi cation of the ATE Tridomain of W ild - type and Mutant t ycA To test the TycA tridomain module as a potential CoA ligase, the wild - type tycA cDNA encoding the A , T , and E domains was subcloned into a pSU18 vector and heterologously expressed as a His6 fusion in E . coli BL21(DE3). In addition, Ser563 of TycA was changed to Ala563 (TycA S563A) since the T - domain of a functional TycA module req uires covalent attachment of 4' Ppant to a conserved serine by the 4' Ppant transferase enzyme. This mutation was envisaged to prevent phosphopantetheinylation of the conserved serine residue by E. coli BL21, known to contain a phosphopantetheinyl transferase gene. The T - domain mutant TycA S563A was expressed similarly. The isolated soluble enzymes were Ni - affinity purified to ~95%, based on SDS - PAGE analysis with Coomassie Blue staining, and the apparent molecular mass (123 kDa) was consistent with the theoretical value ( Figure 2. 9 ) . Wild - type TycA and the S563A mutant (TycA S563A) were expressed, i solated, and purified from a 5 - L bacterial culture, resulting in ~15 mg of protein used for activity assays. In this preliminary study, t he stereochemistry of the amino phenylpropanoyl side chain of the biosynthetic CoA and SNAC thioesters was not evaluated . Thus, it is unknown if the E domain of TycA or TycA S563A racemized a chiral center(s) of the CoA thioester products. 53 Figure 2. 9 . SDS - PAGE gel of fractions eluted from a Ni - affinity column containing TycA constructs . A ) TycA S563A mutant. Lane L : Molecular weight standards. A profile of the protein eluted in Binding buffer cont ained the following: Lane 1 : 50 mM imidazole (fraction 1); Lane 2 : 50 mM imidazole (fraction 2); Lane 3 : 100 mM imidazole (fraction 3); and Lane 4 : 200 mM imidazole (fraction 4). B ) SDS - PAGE gel (7% acrylamide) of fractions eluted from a Ni - affinity column that contained the wild type TycA. Lane L : Molecular weight standards; Lanes 1 and 2 are profiles of protein eluted under the same conditions desc ribed previously for m TycA . Binding buffer : (0.5 M NaCl, 20 m M Tris - HCl, 5 m M imidazole, pH 7.8) . 2.3.2. Strategies u sed to Test the Activity of TycA w ith the Amino Acid Substrates Numerous different assays have been developed to explore the a denylation step of ATP - dependent a denylation and thioesterification of aminoacyl substrates by TycA domain and the analogous Grs 1 domain on the pathway s to tyrocidines A D and gramicidin. Radioactive assays were principally employed for members of this a cyl - adenylation family, including assays for NRPS domains and CoA ligases. Since a bonafide end product is not derived from the amino acyl substrate for these partial biosynthetic reaction s comprising a subset of the complete module matrix on NRPS pathways , creative cofactor/byproduct exchange assays were developed to assess the catalysis of the NRPS candidates. In an earlier study, the amino acid specificity of the adenylation domain of TycA was studied in a dynamic exchange assay using radioactive 32 PPi A B 225 150 100 75 50 35 25 225 150 100 75 L 1 2 3 4 1 2 L kDa kDa 54 a nd unlabeled ATP. 34 , 35 This assay relies on the dynamic reversibility of the amino acid adenylation (amino acyl AMP ), inorganic diphosphate ( PPi ) and ATP formed during catalysis . As the reaction progresses towards equilibrium, 32 PPi hydrolyzes the acyl AMP intermediate, forming AT 32 P. T he specific activity of this end product was measured to assess the activity of TycA ( Figure 2.10 ). This classical radiolabeled 32 PPi / ATP exchange assay has limitations : the cost s of the 32 PPi and disposal of the radioactive material are high an d t 1/2 of 32 PPi is short (~14 days). Radiolabeled assay was also used in a benzoyl CoA ligase assay. 36 In this study, radiolabeled benzoic acid, putative CoA ligase, and CoA were incubated together. At the completion of the re action, the unreacted radiolabe led ben zoic acid was separated by solvent extraction; the remaining radioactivity in the aqueous phase suggested the amount of biosynthesized benzoyl CoA. Figure 2 . 10 . Radioactivity - guided assay of the phenylalanine adenylation domain of TycA . * The reverse reaction forces the formation of radioactive ATP by addition of excess 32 PPi . This radioactive ATP is measured in the ATP - PPi exchange assay. A nother PPi / ATP exchange assay to characteriz e an - 18 O 4 - ATP pyrophosphate exchange as outlined in Figure 2.11 . 37 In this assay, mass sensitive observation of PP i exchange was key in the detection of adenylation domain catalyzed mass shifts on either side of the exchange equation. As back exchange is favored by using PPi in - 18 O 4 - ATP. T he 55 time - - 16 O 4 - - 18 O 4 - ATP with an adenylase was measured by MALDI - T o F MS. Figure 2.11 . The exchange reaction, performed in the absence of thiolation activity, measures equilibrium - 18 O 4 - ATP with 16 O 4 - pyrophosphate - 16 O 4 - ATP is measured using MALDI To F to indirectly analyze and quantify the adenylation domain - catalyzed acyl adenylate product. Another assay employed acyl CoA ligase 11 to characterize the acyl CoA formation by measuring the amount of AMP formed. The AMP analysis wa s done using a spectrophotometric HPLC method a nd authentic AMP standard. In addition, studies on the gramicidin synthetase pathway analyzed the phenylalanyl adenylate intermediate by UV HPLC. 38 The chromatographically isolated product was analyzed by MALDI T o F MS . In the same study, the first evidence was provided for a covalently bound thioester (L [ 1 4 C] phenylalanyl S Ppant ) and the isomer (D [ 1 4 C] phenylalanyl S Ppant ) adducts on the gramicidin pathway. L [ 14 C]Phe nylalanine su bstrate was incubated with wild - type holo Grs1 , containing the 4 ' phosphopantetheinyl group, and the H753A mutant (with an inactive epimerization domain), in two separate assays. The time - course analysis for the phenylalanyl adenylate showed that about 75% of the initial L [ 14 C]Phe ( 450 mCi/mmol ) was 56 converted to L [ 14 C]Phe AMP over 500 ms . The [ 14 C] phenylalanyl S Ppant enzyme complexes were hydrolyzed by incubation with potassium hydroxide and extracted with methanol, followed by centrifug ation to get rid of the enzyme. This product was t hen analyzed after the reaction reached equilibrium; ~ 85% of substrate L [ 14 C]Phe was converted to enzyme bound forms . 38 These previ ous studies with acyl adenylase enzyme family heavily relied on indirect assay methods to test for the formation of acyl adenylate intermediates or acyl CoA products. The PPi /ATP exchange assay provide d information about the reversible formation of acyl adenylate intermediates. The assa y wa s uninformative about the second half reaction catalyzed by adenylation domain. Secondly, direct analysis of acyl AMP formation is challenging due to slow release of this intermediate in to the solution. As mentioned previously, studies on TycA showed t hat phenylalanyl AMP is formed when a TycA mutant, l acking a functional Ppant group was incubated with ( S ) phenylalanine. This intermediate is proposed to be labile yet remains tightly bound in the TycA active site. 38 It is th erefore proposed that the acyl AMP intermediate is susceptible to attack by a nucleophile added to the assay. The formation of the aminoacyl adenylate intermediate could be determined indirectly . Therefore, based on these initial studies, a hypothesis was proposed stating that the adenylation domain could potentially function as a potential amino phenylpropanoid:CoA ligase ( Figure 2. 12 ). In the current study, mass spectrome try was used to assess if TycA could m ake CoA thioesters of phenylalanine and (2 R ,3 S ) phenylisoserine via their corresponding adenylate intermediate. To test this hypothesis, assays were developed for non - radiative detection of phenylpropanoyl AMP intermediates of TycA catalysis and for the CoA ligase fun ction of TycA. 57 Figure 2. 12 . The proposed biosynthesis of aminoacyl CoAs using TycA . The TycA domains shown are: adenylation (A), thiolation domain (T) (without the Ppant group) and the epimerization (E) domain. CoA serves as a surrogate thiol donor to form the acyl CoA products. 2.3.3. Incubation of TycA and TycA S563A w ith N - Acetylcysteamine and Phenylpropanoates 4' Phosphopantetheinyl (i.e., the cysteamine amide of pantothenic acid) is attached posttranslationally to the T domain of a functional TycA module. 15 On the natural pathway, t he thiol group of the pho sphopantetheinyl serves as a nucleophile that reacts with the phenylalanyl AMP to form an intermediary pendent thioester on the tyrocidine pathway. In the present study, N acetylcysteamine (NAC, the terminal fragment of CoA) was explored as a surrogate of TycA and TycA S563A when incubated with ( S ) phenylalanine, ( R ) phenylalanine, and (2 R ,3 S ) phenylisoserine . N acetylcysteamine was chosen for the initial activity development to conserve the expensive CoA, which was th e ultimate thiol donor of interest. 58 A mixture of TycA or TycA S563A and N acetylcysteamine (1 mM) w as incubated with ( S ) phenylalanine, ( R ) phenylalanine, or (2 R ,3 S ) phenylisoserine for 1.5 h . The products of each assay were analyzed by LC ESI MS in scan mode. The first - stage mass spectrometer (positive ion mode) was set to select for the [M + H] + ions phenyl alanyl ( m / z 267, eluting at 4.49 and 3.01 min, respectively) and (2 R ,3 S ) phenylisoserinyl N acetylcysteamine (i.e., phenylisoserinyl SNAC ) ( m / z 283, elut ing at 2.32 min) ( Appendix , Figure s I 1 , I 2 , and I 3 ). These ions were directed to an inert gas collision chamber, and the resulting fragment ions were analyzed by the second - stage mass spectrometer set to positive ion scan mode ( Figure 2. 13 ). The sample in which TycA or TycA S563A and NAC w ere incubated separately with ( S ) phenylalanine , ( R ) phenylalanine , and (2 R ,3 S ) phenylisoserine contained an analyte that generated fragment ions consistent with the respective phenylpropanoyl SNAC ( Figure 2. 1 3 ). The MS and MS/MS data for each biosynthetic SNAC matched those of authentic standards (see Appendix I , Figure I 7 ). Control assays that lacked the enzyme catalyst from the assay mixture, but includ ed the necessary cofactors and phenylpropanoate substrat e did phenyl alanyl SNAC nor phenylisoserinyl SNAC . 59 A) B ) Figure 2.13 . The LC ESI MS MS spectra of aminoacyl SNACs . m / z 267, 267, and 283 ([M + H] + ) correspond to molecula r ion of biosynthetic ( S ) phenylalanyl SNAC ( B ), ( R ) phenylalanyl SNAC ( C ), and (2 R ,3 S ) phenylisoserinyl SNAC ( D ). The fragments corresponding to the ions in the spectrum are s hown in top panel (A) . 60 Figure 2.13 . C ) D ) 61 2.3.4. Kinetic A nalyses of TycA and TycA S563A w ith NAC The k cat and K M values w ere calculated for TycA or TycA S563A by incubating each catalyst separately with a dilution series of ( S ) phenylalanine , ( R ) phenylalanine , or (2 R ,3 S ) phenylisoserine substrates in the presence of N acetylcysteamine (1 mM) and the necessary reagents and cofactors. The reaction products were analyzed by a quantitative LC ESI MRM method, as before. In brief, the relative amount of each biosynthetic acyl SNAC was determined by the ion abunda nce of the transition ions [M + H] + m / z 120, [M + H] + m / z 131, and [M + H] + m / z 106 for ( S ) p henyl alanyl , ( R ) phenylalanyl , and (2 R ,3 S ) phenylisoserinyl SNAC thioesters, respectively. The transition ion abundance was converted to concentration units by linear regression analysis by charting the relationship between ion abundance and concentration of authentic phenylpropan oyl SNAC (the synthesis procedure is described in the experimental section) that matched the biosynthesized product. Analysis of the catalytic parameters for TycA and TycA S563A with varying concentrations of NAC foll owed a similar trend as when incubated with CoA and the phenylpropanoates (described below) . TycA was more catalyti cally efficient (6 - fold higher ) when NAC was incubated with ( R ) phenylalanine compared with the other phenylpropanoates ( Table 2. 1 ). The K M values of TycA S563A were higher for all the phenylpropanoate and NAC cosubstrates compared with the K M values of TycA . The 15 - fold slower turnover of ( R ) phenylalanine by TycA S563A to its SNAC thioester caused the catalytic efficiency of the mutant en zyme to fall 30 - fold lower compared with that of TycA for the same reaction. In addition, the efficiency of TycA S563A for ( S ) phenylalanine was increased 2.5 - fold over that of TycA for the same substrate, due to its superior k cat ( Table 2. 1 ). Despite th e higher k cat of TycA S563A compared with that of TycA for the conversion of (2 R ,3 S ) pheny lisoserine to its acyl SNAC, the K M of TycA S563A was 3.9 - fold 62 greater than that of TycA for the same reaction. Thus, these latter parameters reduced the k cat / K M of T ycA S563A by 3.3 - fold compared with that of TycA for the conversion of (2 R ,3 S ) phenylisoserine to its SNAC thioester. The K M for TycA and TycA S563A with NAC were calculated by incubating each catalyst separately with a dilution series of NAC in the presence of ( S ) phenylalanine at apparent saturation, Mg 2+ , and ATP. The catalytic efficiency of TycA was 2.5 - fold higher than that of TycA S563A incubated under the same conditions with varying concentrations of NAC. 2.3.5. Assessment of TycA and TycA S563A f or CoA L igase A ctivity ( S ) Phenylalanine, ( R ) phenylalanine, and (2 R ,3 S ) phenylisoserine were added separately to reaction mixtures containing heterologously expressed TycA or TycA S563A with ATP, MgCl 2 , and CoA. The product mixtures were screened by LC ESI mass spectrometry in scan mode. The ESI MS ion profiles in negative ion mode contained a diagnostic, negative molecular i on [M H] phenyl alanyl ( m / z 913, phenyl alanyl ( m / z 913, eluting at 2.47 min), and (2 R ,3 S ) phenyl isoserinyl CoA ( m / z 929, eluting at 2.26 min) ( Appendix , Figure s I 4, I 5 and I 7 ). To confirm the identity of the phenylpropano yl CoA thioesters, the molecular ions were evaluated further by tandem MS/MS analysis by collision - induced dissociation of the [M H] ion. The analytes in each of the samples incubated separately with ( S ) phenylalanine , ( R ) phenylalanine , and (2 R ,3 S ) phenylisoserine produced fragment ions consistent with the corresponding CoA thioesters ( Figure s 2. 1 4 and 2. 1 5 ). Control assays lacking TycA or TycA S563A, ATP, or CoA from the appropriate enzyme assay mixtur e did not yield a detectable [M 63 H] ion consistent with the m phenyl alanyl phenyl alanyl , nor phenylisoserinyl CoA, as expected for a ATP/Mg 2+ dependent CoA ligase (see Appendix , Figure I 8 ) . Figure 2. 1 4 . Molecular fragment ions profile resulting fro m LC ESI MS/MS of biosynthetic aminoacyl CoAs : ( S ) phenyl alanyl CoA ( m / z 913), ( R ) phenylalanyl CoA ( m / z 913), and (2 R ,3 S ) phenyl isoserinyl CoA ( m / z 929). 64 Figure 2.1 4 . A ) Figure 2. 1 5 . The LC ESI MS MS spectra of biosynthetic aminoacyl CoAs . m / z 913, 913, and 929 are ions corresponding to the biosynthetic ( S ) phenyl alanyl CoA ( A ), ( R ) phenylalanyl CoA ( B ), and (2 R ,3 S ) phenyl isoserinyl CoA ( C ). The molecular fragments corresponding to the ions in the spectrums are shown in Figure 2.14 above. 65 Figure 2.1 5 . B ) C ) 66 2.3.6. Kinetic A nalyses of TycA and TycA S563A w ith Phenylpropanoates and CoA The kinetic parameters of TycA and TycA S563A were calculated by separately incubating each catalyst with a varying concentration of ( S ) phenylalanine , ( R ) phenylalanine , or (2 R ,3 S ) phenylisoserine substrates in the presence of necessary cofactors and Mg 2+ . Aliquots from reactions containing TycA or TycA S563A were quenched by acidification to pH 2. The resulting CoA thioester products were analyz ed by a quantitative LC ESI MRM method to detect the [M H] m / z 408 transition, common to each phenylpropanoyl CoA used in this study. The abundance of the transition ion was converted to concentration units by linear regression (using CoA as the quantitation standard). The catalytic efficiencies of TycA with CoA and each amino acid were within the same order of magnitude as when SNAC was used. The higher k cat of TycA for ( R ) phenylalanine over the other amino acids tested coupled with a K M value similar to that for the other phenylpropanoate substrates resulted in a 5 - and 10 - fold higher catalytic preference of TycA for ( R ) phenylalanine than for ( S ) phenylalanine and (2 R ,3 S ) phenylisoserine, respectively. The K M of TycA S563A followed a similar trend as that of TycA and was slightly lower when incubated with the natural substrate ( S ) phenylalanine compared to the non - natural ( R ) phenylalanine, yet had a nearly 6 - fold higher K M for (2 R ,3 S ) phenylisoserine ( Table 2.1 ) . The ca talytic efficiency of TycA S563A with ( R ) phenylalanine was highest among the amino phenylpropanoates tested. Similarly , the kinetic parameters for TycA and TycA S563A with a dilution series of CoA were calculated by incubating each catalyst separately i n the presence of ( S ) phenylalanine at 1 mM. The resulting CoA thioester product was analyzed by a quantitative LC ESI MRM method, as before. The K M value of 67 TycA was 2 - fold higher compared to the mutant, hence resulting in a 2.5 - fold lower catalytic ef ficiency of TycA for CoA compared to TycA S563A. Table 2. 1. Steady state kinetic analys i s of TycA and TycA S563A with phenylpropanoids , CoA , and N acetylcysteamine . TycA Substrate K M k cat (min - 1 ) k cat / K M (s - 1 · M - 1 ) ( S ) Phenylalanine 41.9 ± 2.0 0.25 ± 0.01 99.4 ± 6.2 ( R ) Phenylalanine 50.6 ± 7.9 1.6 ± 0.3 527 ± 129 (2 R ,3 S ) Phenylisoserine 89.3 ± 15.0 0.25 ± 0.02 46.7 ± 8.7 CoA a 1976 ± 175 0.75 ± 0.05 6.3 ± 0.7 TycA S563A ( S ) Phenylalanine 33.9 ± 4.0 0.69 ± 0.08 339 ± 56 ( R ) Phenylalanine 62.3 ± 1.0 3.00 ± 0.04 803 ± 17 (2 R ,3 S ) Phenylisoserine 191 ± 10 0.43 ± 0.01 37.5 ± 2.1 CoA a 804 ± 26 0.90 ± 0.08 18.7 ± 1.8 TycA with NAC Substrate K M k cat (min - 1 ) k cat / K M (s - 1 · M - 1 ) ( S ) Phenylalanine 27.0 ± 6.5 0.05 ± <0.01 30.8 ± 9.7 ( R ) Phenylalanine 30.4 ± 3.9 0.36 ± 0.06 197 ± 42 (2 R ,3 S ) Phenylisoserine 132 ± 56 0.19 ± 0.01 24.0 ± 10.3 NAC a 153 ± 22 0.09 ± <0.01 9.8 ± 1.8 TycA S563A with NAC ( S ) Phenylalanine 37.8 ± 1.6 0.20 ± 0.002 88.1 ± 3.8 ( R ) Phenylalanine 58.9 ± 6.4 0.024 ± 0.001 6.8 ± 0.8 (2 R ,3 S ) Phenylisoserine 512 ± 43 0.31 ± 0.05 10.1 ± 1.8 NAC a 268 ± 4 0.10 ± <0.01 6.2 ± 0.6 a Kinetic parameters for CoA or NAC were determined with ( S ) phenylalanine as cosubstrate . The kinetic parameters calculated for TycA show different behavior with the three phenylpropanoate substrates with CoA. Based on the K M of TycA, the enzyme binds the natural substrate ( S ) phenylalanine and surrogate substrate ( R ) phenylalanine nearly t he same, but ~2 - fold better than (2 R ,3 S ) phenylisoserine ( Table 2.1 ). These data suggested that TycA prefers substrates with an amino group at C or C , but not phenylisoserine, containing both a C - hydroxyl and C - amino group. For (2 R ,3 S ) phenylisoserine , the stereochemistry of the C amino group is oriented identical to the amino group of ( R ) phenylalanine , while the C 68 hydroxyl group is oriented opposite to that of the C amino group of ( S ) phenylalanine . Thus, the hydroxyl group li kely affects binding either sterically, electrostatically, or through unfavorable H bonding that alters the trajectory of the substrate. Initial studies had showed that E . c oli contained a 4' Ppant acyltransferase that catalyzed the posttranslational modi fication of peptide synthetases with the cofactor 4' Ppant . Therefore, the TycA catalyzed amino acyl CoA biosynthesis reaction would likely compete with the internal thioesterification by the pantetheine thiol donor. Therefore, to remove any residual competi ti ve thioesterification by the native Ppant thiol , an S563A mutation was made to prevent 4' phosphopantetheinylation. T he turnover ( k cat ) of TycA S563A was only slightly greater than TycA with each phenylpropanoate substrate and CoA as the thiol don or, suggesting that CoA access was unimpeded by the 4' Ppant arm at the reaction center of Tyc( Phe A ) . The increase in k cat of TycA S563A phenyl alanine and (2 R ,3 S ) phenylisoserine was offset by a nearly equal increase in K M (suggesting poorer binding) compared with TycA, resulting in similar catalytic efficiency of the catalysts with these substrates. Considering the catalytic efficiency of TycA did not vary by more than 3 - fold over TycA S563A suggested that CoA at 1 mM was perhaps higher than the slight amount, if any, TycA holoenzyme containing the covalent Ppant in the T domain. Thus, CoA likely attacks the phenylpropanoate phosphoric acid anhydride in the A domain of TycA without much competition from the pendent thiol pr osthetic group. TycA was therefore considered operationally similar to TycA S563A and principally in its apo enzyme form. The K M of TycA for each phenylpropanoate substrate with NAC at apparent saturation was different, but approximately the same order of m agnitude as when CoA was used. This suggested that NAC (a smaller, structural mimic of CoA) did not affect binding of the 69 phenylpropanoate to the Tyc( Phe A ) ( Table 2. 1 ). The K M phenyl alanine bound the best to TycA and (2 R ,3 S ) phenylisoserine the worst in the presence of NAC, similar to when CoA was the cosubstrate. Further, the k cat values of TycA were faster for CoA turnover than the rates for NAC under steady state reaction conditions ( Table 2. 1 ). The k cat / K M of TycA with ea ch phenylpropanoate and CoA ranged between 1.5 - and 3 - fold higher than when NAC was the thiol donor, suggesting that CoA is more catalytically competent than NAC. However, the efficiency of TycA (with NAC) was highest for ( R ) phenylalanine , followed by ( S ) phenylalanine , and then by (2 R ,3 S ) phenylisoserine . This trend was similar to (but the values were lower than) that for the phenylpropanoids when CoA was used in place of NAC ( Table 2. 1 ). This further suggested the two stereogenic functional groups of phenylisoserine are somehow affecting the thioesterification catalysis . 2.3.7. Kinetic A nalyses of TycA and TycA S563A w ith Phenylpropanoates and ATP The chemical syntheses of authentic amino phenylpropano yl AMP s for use as quantitation standards were low yielding; t herefore, the biosynthetic acyl AMP s were quantified by LC ESI MRM for kinetic analyses and adenosine was used as the standard (the abundant molecular ion fragment of amino phenylpropanoyl AMP s quantified using the MRM method corresponds to aden ine) . The catalytic efficiency value of TycA for the conversion of ( S ) phenylalanine and ( R ) phenylalanine (ATP in the assays was at apparent saturation ) to ( S ) phenyl alan yl and ( R ) phenylalanyl AMP was ~ 2 - and 15 - fold lower, respectively, than the catalytic efficiency values for the conversion of the same substrates to the ir corresponding amino phenylpropanoyl CoAs ( Table 2.2 ). A similar trend was observed for TycA S563A , where the catalytic efficiency 70 was ~ 7 - and 46 - fold lowe r, respectively, for the conversion of the same substrates to their AMP anhydrides than the values for the conversion to the corresponding CoA thioesters. Phenylisoserinyl AMP was below the limits of detection in similar assays. Table 2. 2 . Steady state kinetic analysis of TycA and TycA S563A with apparent saturation of phenylpropanoids and varying concentrations of ATP TycA Substrate k cat (min - 1 ) k cat / K M (s - 1 · M - 1 ) ( S ) Phenylalanine 0.13 ± 0.01 51.7 ± 1.7 ( R ) Phenylalanine 0.10 ± 0.01 32.9 ± 0.14 (2 R ,3 S ) Phenylisoserine a a TycA S563A Substrate ( S ) Phenylalanine 0.09 ± 0.01 44.2 ± 0.2 0 ( R ) Phenylalanine 0.06 ± 0.01 16.1 ± 1.2 (2 R ,3 S ) Phenylisoserine a a a Phenylisoserinyl AMP was below limits of detection in the LC ESI MS . The slower steady - state production rate of the amino phenylpropanoate AMP s detected in solution did not account for the production rate of the corresponding amino phenyl propano yl CoAs ( Table 2.1 ). The greater catalytic efficiency for CoA thioester producti on over amino phenylpropanoyl AMP biosynthesis confirmed that CoA displaced AMP from the amino phenylpropanoyl AMP in complex with TycA or TycA S563A , at steady state, and not from the acyl AMP intermediate in solution to form the thioesters. Previously, amino acyl AMP formation was studied using Grs 1 under single turn over reaction conditions. It was reported that about 74 84% of the amino acids were enzyme bound, but in the absence of t he T domain the increase of aminoacyl AMP in solution w as only 20%. This suggested that the acyl AMP s were labile and were rapidly transferred to the covalently - linked Ppant group. However, in the absence of Ppant, the acyl AMP s are tightly bound to the active site and the leak rate into the solution is slow . 21 , 38 In the current study, t he ratio of k cat (CoA) / k cat(AMP) was greater than one for all the substrates tested 71 which shows that the formation of acyl CoA did not arise from non catalytic reaction of CoA with the acyl AMP in solution, but rather that CoA binds to the TycA active site ( Table 2. 3 , Figure 2.1 6 ) . Tabl e 2.3 . Ratio of k cat of phenylpropanoids in steady state biosynthesis of amino acyl AMP or amino acyl CoA catalyzed by TycA or TycA S563A . TycA Substrate k cat(CoA) (min - 1 ) k cat(ATP) (min - 1 ) k cat(CoA) / k cat(ATP) ( S ) Phenylalanine 0.25 ± 0.01 0.13 ± 0.01 1.92 ( R ) Phenylalanine 1.6 ± 0.3 0.10 ± 0.01 16.0 (2 R ,3 S ) Phenylisoserine 0.75 ± 0.05 a >>1 TycA S563A Substrate ( S ) Phenylalanine 0.69 ± 0.08 0.09 ± 0.01 7.67 ( R ) Phenylalanine 3.00 ± 0.04 0.06 ± 0.01 50.0 (2 R ,3 S ) Phenylisoserine 0.43 ± 0.01 a >>1 a Phenylisoserinyl AMP was below limits of detection in the LC ESI MS. Figure 2.1 6 . Kinetic model for CoA thioesterification reaction catalyzed by TycA and TycA S563A . E is TycA, E · AmPheprop - yl AMP is amino phenylpropanoyl AMP anhydride TycA complex. 2.3.8. Comparing the Kinetic Parameters of TycA/ TycA S563A w ith Other CoA Ligases Compared to the K M values of bacterial CoA ligases for CoA ( 100 ) and acyl substrates (10 , those of TycA and TycA S563A are 72 about same order of magnitude, whereas the carboxylate substrates (34 variable (re fer to Tables 2. 1 and 2 . 4 ). CoA ligases on catabolic pathways in various bacteria convert, for exam ple, propionate, 39 benzoate , 40 and 4 chlorobenzoate 41 to the ir corresponding CoA thioesters with superior catalytic efficiency (1.65 × 10 6 , 3 .96 × 10 7 , 1.02 × 10 7 s - 1 · M - 1 , respectively ) compared to those on secondary metabolic pathways ( < 2,500 s 1 · 1 ) such as for TycA (used in tyrocidines A D biosynthesis, described here as CoA ligase), phenylacetate: CoA ligase from Penicillium chrysogenum , 11 and cinnamate: CoA ligase from Streptomyces coelicolor . 42 The role of cinnamoyl CoA is as yet un defined ; however, it may play a role in biosynthesis because Streptomyces sp . are known to produce a v ariety of secondary prod ucts . 42 Table 2. 4 . Kinetic p arameters of a cyl CoA ligases from v arious m icro organisms. CoA Ligase (CL) (Organism) Substrate K M k cat (min - 1 ) k cat / K M (s - 1 · M - 1 ) Ref Propionyl - CL ( Salmonella enterica) propionate 20 1980 1.65 × 10 6 40 CoA 215 2520 1.98 × 10 5 Benzoate - CL ( Pseudomonas st. KB 740 ) benzoate 11 26,000 3.94 × 10 7 43 CoA 100 NL NL Phenylacetate - CL ( Penicillium chrysogenum ) phenylacetate 6100 84 230 44 CoA 940 NL NL Cinnamate - CL ( Streptomyces coelicolor) cinnamate 190 28.5 2500 45 CoA NL NL NL 4 - Chlorobenzoate - CL ( Alcaligenes sp. AL3007) 4 - Cl - benzoate 0.9 552 1.02 × 10 7 41 CoA 310 NL 3.00 × 10 4 NL: no listing 2.4. Conclusion The importance of acyl CoA substrates has been demonstrated in the bio synthesis of key therapeutic compounds such as tetracyclin, macrolides, actinorhodin , 46 erythromycin, rifamycin, 47 rapamycin , 48 and p aclitaxel . 43 , 49 , 50 Though most of the known CoA ligases utilize an 73 alkyl/alkenyl carboxylate substrate, less information is known for the amino phenylpropanoid CoA ligases. One stud y explored a phenylpropanoate:CoA ligase and found that it could transfer CoA to phenyl alanine. 11 This limited knowledge on amino acid CoA ligases for use in biocatalytically modifying bioactive secondary metabolites, warrants further discovery of new routes to access these acyl CoA substrates. This study highlighted the acy l CoA ligase activity of an NRPS adenylation domain; whereas in earlier studies, the catalytic transfer of CoA was suspected as fortuitous, non - catalyzed event. The ability of TycA to catalyze the synthesis of the amino acyl CoA intermediates on the paclita xel biosynthetic pathway opens exploration towards designing novel paclitaxel analogs . phenyl alanyl , phenyl alanyl , and (2 R ,3 S ) phenyl isoserinyl CoA under similar assay conditions. This biosynthetic method has an advantage over the conventional methods that have been challenged by reactions involving multiple steps that require protection/deprotection of the amine group and is challenged by the solvent incompatibility of the hydrophobic acid anhydride intermediate and the hydrophilic CoA in the synthesis method . Also, for the first time, the bio synthesis of (2 R ,3 S ) phenyl isoserinyl CoA has been demonstrated in this study. In addition, this study showed that besides CoA or a pendent Ppant group, TycA can also use the small thiol donor N a cetylcysteamine (NAC) in the ligase reaction to make acyl SNACs. The a cyl SNACs have been used extensively as surrogate substrates to interrogate the substrate specificity of NPRS catalysts, 51 , 52 PKS - catalyzed pathways , 53 - 56 and transformations on the mitomycin C pathway , 57 and a l so in interrogating proofreading mechanism that releases stalled intermediates from PKs. 58 Biocatalytic production of acyl SNACs would thus bypass the protection/deprotection chemistry required in some thioester syntheses. 74 APPENDIX 75 Figure I 1 . MRM profile obtained by LC ESI MS/MS of biosynthetically derived phenyl propanoyl SNAC [M + H] + m / z 120 . Figure I 2 . MRM profile obtained by LC ESI MS/MS of biosynthetically derived phenyl propanoyl SNAC [M + H] + m / z 131 . 76 Figure I 3 . MRM profile obtained by LC ESI MS/MS of biosynthetically derived (2 S ,3 R ) phenyl isoserinyl SNAC [M + H] + m / z 131 . Figure I 4 . M RM profile obtained by LC ESI MS/ M S of biosynthetically derived phenylalanyl CoA [M H] - m / z 408 . 77 Figure I 5 . MRM profile obtained by LC ESI MS/MS of biosynthetically derived phenylalanyl CoA [M H] - m / z 408 . Figure I 6 . MRM profile obtained by LC ESI MS/MS of biosynthetically derived (2 R ,3 S ) phenylisoserinyl CoA [M H] - m / z 408 . 78 A B C Figure I 7 . The LC ESI MS analyses of authentic aminoacyl SNACs . The t otal ion profile of the LC ESI MS analyses (left column) and the fragment ions derived by MS/MS of the corresponding [M + H] + molecular ion (right column) of authentic A) ( S ) phenyl alanyl SNAC , molecular ion ([ M + H] + , m / z 267.1), B ) ( R ) phenyl alanyl SNAC , molecular ion ([ M + H] + , m / z 267.1), and C) ( 2 R ,3 S ) phenyl isoserinyl SNAC ([ M + H] + , m / z 283.1) are shown . 79 A C B D Figure I 8 . Total ion chromatograms obtained by LC ESI MS analysis (scan mode: m / z 100 to 1200) of the compounds in TycA assays (incubated for 30 min at 31 °C) containing the enzyme ATP (1 mM, 100 nmol in 0.1 mL), CoA (1 mM), Mg 2+ (3 mM) and A) Phenylalanine, D ) Phenylalanine or G ) (2 R ,3 S ) Phe nyl isoserine (each at 1 mM). Control assays: B , E , and H lacked ATP, C , F , and I lacked CoA, and J lacked phenylpropanoid substrate. The identity of the individual peaks was determined by selected - ion monitoring (post - run) of the [M H] - ions of each analyte and comparison to the retention time and fragment ions of authentic standards. Retention times : Hepes buffer (0.3 min); AMP and ADP (0.3 to 0.6 min); ATP and (2 S ,3 R ) phenylisoserine phenyl phenyl alanine (1.0 min); CoA (1.6 min); unknown contaminant an adenylate phosphate compound that lacked the pantetheine side chain b y LC MS/MS analysis phenylalanyl CoA (2.0 phenylalanyl CoA (2.2 min); phenylisoserinyl CoA (2.4 min extracted peak ion). Proportions of ATP, ADP, and AMP , respectively, in product mixture of reaction represented in A ) 79, 18, and 3 nmol; C ) 82, 17, and 1 nmol; D ) 82, 9, and 10 nmol; F ) 75, 21, and 3 nmol; G ) 85, 14, and 1 nmol; I ) 87, 13, and below limits of detection; J ) 89, 10, and below limits of detect ion . 80 Figure I 8 . E H F I G J 2.4 81 A B C D Figure I 9 . Determination of kinetic parameters for A) phenyl alanine, B) phenyl alanine, C) (2 R ,3 S ) phenylisoserine , and D) CoA with wt TycA using Hannes Wolf plot s . Conditions: 20 µg wtTycA, Hepes buffer, 100 mM, pH 8.0, 1 mM ATP, 3 mM MgCl 2 and 1 mM CoA (except when varying phenyl alanine was used at 1 mM concentration). 82 A B C D Figure I 10 . Determination of kinetic parameters for A) phenyl alanine, B) phenyl alanine, C) (2 R ,3 S ) phenylisoserine , and D) N acetylcysteamine with wtTycA using Hannes Wolf plot s . Conditions: 20 µg wtTycA, Hepes buffer ( 100 mM, pH 8.0 ) , 1 mM ATP, 3 mM MgCl 2 and 1 mM N acetylcysteamine (except when varying N phenyl alanine was used at 1 mM concentration). 83 Figure I 11 . 1 H - NMR spectrum of ( S ) p henyl alanyl SNAC standard . DMSO d 6 was used as solvent. 1 4 - a,b 3 - a,b 7 - b 9 - 13 NH 7 - a 6 NH 2 * 84 Figure I 12 . 13 C - NMR spectrum of ( S ) p henyl alanyl SNAC standard . DMSO d 6 was used as solvent. 1 4 11 8 3,7 6 10&12 9&13 2 5 85 Figure I 13 . 1 H - NMR spectrum of p henyl alanyl SNAC · HCl salt standard . DMSO d 6 was used as solvent. 1 6 b 4 - a,b 6 a 3 - a,b 7 9 - 13 NH NH 3 86 Figure I 14 . 13 C - NMR spectrum of p henyl alanyl SNAC · HCl salt standard . DMSO d 6 was used as solvent. 4 8 7 6 9 - 12 2 5 3 1 87 Figure I 15 . 1 H - NMR spectrum of (2 R ,3 S ) p henylisoserinyl SNAC standard . The inset shows H/D exchange NMR spectr um . 1 4 - a,b 6 3 - a,b 7 NH 2 9 - 13 D 2 O DMSO d 6 DMSO d 6 * NH 88 Figure I 16 . 13 C - NMR spectrum of (2 R ,3 S ) p henylisoserinyl SNAC standard . DMSO d 6 was used as solvent. Figure I 17 . Trypsin digestion sequence analysis of TycA purified by Ni - affinity chromatography . Assembly of the peptide fragment sequences and comparison to the sequence of the wtTycA (Accession No. AAC45928) deposited in GenBank shows 59 % coverage (sequenced residues highlighted in yellow). 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Introduction Non - ribosomal peptide synthetases ( NRPSs ) are multimodular pe ptide synthetases comprising structurally and functionally independent domains on a single poly peptide chain . 1 These enzymes are expressed sequentially from the contiguous DNA template . 2 Previous NRPSs studies revealed that the catalytic module s withi n t he polypeptide chain contain adenylation (A) domain , thiolation (T) domain , 3 and a modifying domain which c an be an epimerization (gramicidin and tyrocidine) , 3 N methylation (in cyclosporine A) , 4 N formylation (in anabaenopeptilide 90 A), 5 oxidation (in bleomycin and epothiline), 6 and reduction (yersiniabactin and pyochelin) . 7 The adenylation and thiolation domains are responsible for amino acyl adenylation and thioesterification on a specific 4' phosphopantetheine prosthetic group , respectively . 8 The thioester intermediate is then modified as needed by the corr esponding modif ying domain. For example , in tyrocidine synthetase A (TycA) module, the C of the activated phenylalanyl pho sphopantetheine intermediate is epimerized by the epimerization ( E ) domain prior to the transfer of the activated phenylalanine to the first domain of the adjacent module (also referred to as the chain elongation module ). 9 The elongation modules studied so far contain domains that participate in substrate activation (A domain), thioesterification ( T domain ), modification (where necessary), and condensation (C domains). 10 95 I ndividual domains that make up the modules in NRPSs have be en isolated as stable enzymes, with some of them retaining activity independently of the partner domains. 11 - 13 Generally, t he catalytic independence of the various d omains within a module has been demonstrated through deletion studies. For e xample, gramicidin synthetase 1 ( Grs1 ) module o n the gramicidin biosynthesis pathway was mutated to leave the ade nylation and thiolation domains (designated herein as Grs1 (Phe AT ) ) . 12 The del etion in Grs1 caused no reduction in the adeny lation of the natural substrate ( S ) phenylalanine compared to the wild type reactivity based on ATP/PP i exchange assay . 12 Also, the Grs1(Phe AT) was able to form a T domain linked 4' phospho pantetheinyl (4' Ppant ) thioester. A further deletion of 100 amino acid residu es at the C terminal end of Grs1 ( Grs1( Phe AT ) to Grs1( Phe A ) ) could only activate the ( S ) phenylalanine to its adenylate form, which was not covalently bound to the enzyme, hence suggesting loss of T domain activity. 12 The NRPSs domain independence has also been studied using tyrocidine synthetase A ( TycA ) . Firstly, the ( S ) phenyl alanyl - AMP intermediate was generated by addition of TycA A domai n ( Tyc( Phe A )) to an assay containing ( S ) phenylalanine and ATP . 11 When the thiolation domain was added into this reaction mixture , a covalently bound ( S ) phenylalanine - S Ppant thioester on the T domain was observed albeit in small amounts. 11 This study suggested that independent domains can perform their respective catalytic activity without a need for a covalent peptide bond joining them . In the current study, it was hypothesized that the catalytic efficiency of TycA, which already uses aminophenylpropanoate substrates, would improve by eliminating thiolation and epimerization domains through mutagenes is. It was expected that CoA would serve as a thiol donor in place of 4' Ppant to access amino phenylpropanoyl CoA s biosynthetically. Additionally, 96 deletion of E domain would prevent any possibility of epimerization of amino acyl CoA products . CoA has been described as a Ppant surro gate in the TycA catalyzed amino acyl CoA biosynthesis (Chapter 2) . 14 Additionally, CoA is not a natural sub strate for TycA, and hence to rationalize how CoA is able to serve as a thiol donor in a reaction catalyzed by Tyc( Phe A ) and /or Tyc( Phe AT ) described herein , homology modeling of Tyc( Phe AT ) on the structure of Grs 1 ( Phe A ) and comparison of the resulting model to acyl CoA structures will be discussed. Based on their similar reaction mechanism and presence of conserved peptide motifs, the acyl CoA s and NRPSs have been classified into a superfamily of adenylate forming enzymes 15 ( Figure 3. 1 ). Specifically, there is an AMP binding motif that is conserved in all members of this family (SGXTGKPKG) 16 , 17 (See appendix , Figure II 1 ). The amino acid seque nce identity between these acyl adenylate forming enzymes is low (20 30%). However, many members of this family share high structural homology. 15 Crystal structures of different enzymes in this family have been solved, including oxi do reductase luciferase from Photinus pyralis ( PDB code: 1LCI ), 18 Grs 1 ( Phe A domain) from Bacillus brevis ( PDB code: 1AMU), 19 DhbE from Bacillus subtilis ( PDB code: 1MDF ), 20 acetate: CoA ligase from Salmone l la enterica ( PDB code: 1PG4 ), 21 4 - c hlorobenzoate:CoA ligase from Alcaligenes sp. ( PDB code: 3CW9 ), 22 benzoate:CoA ligase from Burkholderia x enovorans ( PDB code: 2V7B ), 23 and malonyl CoA ligase from Streptomyces coelicolor ( PDB code: 3NY R ). 24 Analysis of th e crystal structures of adenylate forming family shows high similarities, for example, the overall folds of firefly luciferase and acetate: CoA ligase were shown to be v ery similar to those of NRPSs A domains. 15 , 20 97 Acyl CoA ligases tRNA synthetases Thiol template synthetases Firefly luciferase Figure 3. 1 . Representation of reactions catalyzed by different adenylate forming enzyme s . Generally, the A domains of NRPSs share sequence identities of ~30 60%, thus making the Grs1 ( Phe A ) domain structure an archetype for most amino acid activating A domai ns in NRPSs. 20 With the wealth of information on amino acid sequence, structure , and function of acyl adenylate enzyme family, it is possible to understand the CoA thioesterification reaction catalyzed by TycA domains . In the study described herein, Tyc( Phe AT ) w as modeled on Grs1( Phe A ) structure and compared to acetate: CoA and 4 c hlorobenzoate:CoA ligase in complex with CoA. 98 3.2. Experimental 3.2.1. Substrates, Reagents, and General Instrumentation N Boc (2 R ,3 R ) phenyl isoserine and (2 S ,3 R ) N benzoyl 3 phenyl isoserine were obtained from Peptech (Bedford, MA). The DNA oligo primers were purchased from IDT (Commercial Park Coralville, Iowa). The DNA polymerase was obtained from New England BioLabs (Ipswich, MA). A V arian Inova - 300 or a V arian UnityPlus500 instrument was used to acquire 1 H - and 13 C - NMR . A Q ToF Ultima electrospray ionization high resolution mass spectrometer ( ESI MS , Waters, Milford, MA) with a Waters 2795 HPLC and Quattro Premier XE coupled with Acquity ® UPLC system w ere used for mass spectr ometry analysis . 3.2.2. Truncation and S ubcloning of wt tyc A cDNA to Obtain tyc ( phe a ) cDNA T he tyc A cDNA e ncoding the adenylation domain Tyc( Phe A ) was amplified by PCR from the original expression vector pSU18 to install terminal restriction sites for subcloning into pET28a (Novagen) , designated pET28a Tyc( Phe A ) His . The PCR was performed using Pfu turbo ® DNA P olymera se and 10 × reaction buffer (New England Biolabs, Ipswich, MA) following the manufacturer s protocol. The oligonucleotides used are as follows (bold, restriction sites): 5' Nco I Forward primer GAG AAA TTA A CC ATG GTA GCA AAT CAG GCC - 3' and 5' Sal I reverse primer TGT GTC GAC GCC CAG CTT GAC GAA ATA AGA TGG 3'. The tyc ( p he a ) cDNA was digested using Nco I and Sal I restriction enzymes , and T4 ligase (N ew England Biolabs, Ipswich, MA) was used to insert the cDNA into pET28a vector that was digested similarly . The fusion sites between the vector and the tyc ( p he a ) gene were confirmed by DNA sequencing (MSU Research Technology Support Facility: G enomics , East La n sing, MI). 99 3.2.3. Tyc( Phe A ) Protein Expression and Purification The plasmid Tyc( Phe A ) His encoding a C terminal His tag was used to transform E. coli BL21 (DE3) cells. A 10 mL culture of E. coli transformed with the pET28a vector was grown in LB medium at 37 °C supplemented with kanamycin (50 mg · mL 1 ) for 12 h. The 10 mL inoculum was transferred to a new batch of LB medium (1 L). The bacteria were grown at 37 °C to OD 600 ~0.6, and the cDNA expression was induced by isopropyl D 1 thiogalactopyranoside (IPTG) , and the culture was grown further for 18 h at 16 °C. The cells were pelleted by centrifugation (30 min, 4000 g ) at 4 °C, resuspended in Binding buffer (20 mM Tris HCl buffer containing 0.5 M NaCl and 5 mM imidazole at pH 7.8) , lysed by sonication (Misonix sonicator , Farmingdale, NY), and the corresponding soluble protein fraction was clarified by cen trifugation at 15,000 g for 0.5 h. The supernatant was then decanted and centrifuged at 135,000 g for 1.5 h to remove cell - wall debris and light membranes. The crude Tyc( Phe A ) His in the soluble fraction was purified by nickel nitrilotriacetic acid (Ni - NTA) affinity chromatography (Qiagen, Valencia, CA) and eluted according to the protocol described by the manufacturer. The column was eluted with increasing concentration of imi dazole (20 250 mM) in Binding buffer . Fractions containing Tyc( Phe A ) His were identified by SDS PAGE analysis and Coomassie Blue staining . The Tyc( Phe A ) fractions eluting at 50 and 100 mM imidazole were combined (75 mL) and concentrated ( 1 mL ) by size - selective centrifuga tion ( Centriprep 30,000 MWCO unit ; Mi l lipore , Billerica, MA ) . T he buffer was exchanged with the Assay buffer (50 mM H EPES containing 100 mM NaCl and 1 mM EDTA at pH 8.0) over five dilution/concentration cycles. E nzyme purity was estimated by SDS PAGE with Coomassie Blue staining. The protein concentration ( 35 mg/mL ) was determined by measuring the A 280 absorbance on a NanoDrop ND1000 Spectrophotometer (Thermo Scientific, 100 Wilmington, DE). The calculated extinction coeffi cient and molecular weight of Tyc( Phe A ) were 280 = 60405 M 1 cm 1 and 5 8.06 kDa , 25 respectively . The purified protein was store d at - 80 °C. 3.2.4. Evaluation of Tyc( Phe A ) Activity w ith CoA and Aminophenylpropanoids Substrates ( S ) phenylalanine, ( R ) phenylalanine, and (2 R ,3 S ) phenylisoserine (each at 1 mM) were separately incubated at 31 °C in single stopped time (1 h) reactions containing 100 mM HEPES (pH 8.0) , ATP (1 mM), MgCl 2 (3 mM), CoA or N acetylcysteamine (1 mM), and Tyc( Phe A ) ( done in parallel under the same conditions used for assays containing enzyme, except Tyc( Phe A ) , ATP, or CoA was omitted. The reactions were quenched by acidifying to pH ~2 ( 10 % formic acid ) and lyophilized to dryness. The resultant samples were separately dissolved in 2 O (pH 4.0) and analyzed using a Quattro Premier XE Mass Spectrometer coupled to a n Acquity® UPLC system fitted with a C18 Ascentis Express column (2.5 × sample was loaded onto the column , and the analytes were eluted with a solvent gradient of acetonitrile (Solvent A) and 0.05% triethylamine in distilled water (Solvent B) (held at 2.5 % Solvent A for 3 .17 m in, increased to 100 % solvent A over 5 sec with a 2 min hold, and then returned to 2.5 % Solvent A over 5 sec with a 50 - sec hold) at a flow rate of 0.3 mL/min. The effluen t from the column was directed to the mass spectrometer set to negative ion mode with a scan range of m / z 200 1000 atomic mass units. During the development of an LC ESI MS MS method for the analysis of the aminophenylpropanoyl CoA products, a common dia gnostic fragme nt ion ( m / z 408) was identified phenyl alanyl CoA was used to validate the observed LC ESI MS MS profile of the biosynthetic products. 101 3.2.5. Assessing Tyc( Phe A ) Substrate Binding by Tryptophan Fluorescence Quenching Studies A liquots of (2 S ) phenylalanine , between 0 were added to a solution containing Tyc( Phe A ) at . The excitation wavelength was set at 280 nm and the emission spectra were recorded from 300 420 nm. The fluorescence intensity was recorded after every addition of ( S ) phenylalanine as an average of three readings. In the control experiment, K + HEPES was titrated to 2.0 mL Tyc( Phe A ) ligand. The equilibrium fluorescence titration experiments and control , where Tyc(Phe A) was omitted , were repeated using a fixed excitation wavelength at 280 nm and fixed emission wavelength at 340 nm. Similar fluorescence titration experiments were done using a fixed concentration of Tyc( Phe A ) ( 2 S ) phenylalanine con centration of AMP (0 + HEPES (pH 7.5). The fluorescence intensity obtained for the control experiment in the absence of Tyc(Phe A) was subtracted from that of the test samples containing the enzyme and ligand (( S ) phenylalanine) . The fluorescen ce change observed at 340 nm was plotted vs the lig and concentration and equation 1 was applied in the curve analysis using KaleidaGraph program to obtain the binding constants ( K d ) ( Figure 3. 5 and Appendix, Figures II 7 II 9 ). 3.2.6. Truncation of wt tyc A or tyc A S563A cDNA to Obtain tyc ( p he at ) and tyc ( p he at ( S563A ) ) Constructs In C hapter two, the full length tycA (also identified as tyc ( phe ate ) and single point mutant tycA S563A are described. The latter encoded Tyc( Phe AT E( S563A )) that removed the serine residue needed for covalent 4' phosphopantetheinylation in the native reaction. The full - length tyc ( phe 102 ate ) cDNA encoding Tyc( Phe AT E) and tyc ( phe ate ( S563A) ) encoding Tyc( Phe AT E(S563A)) were truncated to encode Tyc( Phe AT ) and Tyc( Phe AT (S563A)). Briefly, tycA or tycA (S563A) was amplified by PCR with the following oligonucleotides ( bold, restriction sites): 5' Nco I Forward primer GAG AAA TTA A CC ATG GTA GCA AAT CAG GCC 3' and Sal I reverse primer 5' CGC AAG CTT GTC GAC GCC GCT TTT TCT CGT CGT GCT CTT GAC 3' to generate the fragment Tyc( Phe AT ) and install terminal restriction sites for subcloning from the original expression vector pSU18 into pET28a (Novagen). The PCR was performed using Pfu turbo® DNA polymerase and 10 × reaction buffer from New England Biolabs (Ipswich, MA) following the manufacturer The resultant t yc ( p he at ) and t yc ( p he at ( S563A ) ) fragment s w ere individually digested using Nco I and Sal I restriction enzymes . T4 ligase (New England Biolabs, Ipswich, MA) was used to separately insert the cDNA into pET28a vector that was digested similarly. The fusion sites between the vector and the tyc ( phe at ) or tyc ( phe at ( S563A ) ) gene were confirmed by DNA sequencing (MSU Research Technology Support Facility: G enomics, East Lansing, MI ). 3.2.7. Expression , Purification, and Characterization of Tyc( Phe AT ) Proteins The resultant t yc ( p he at ) and t yc ( phe at ( S563A ) ) plasmid s encoding a C terminal His tag (designated Tyc( Phe AT ) His and Tyc( Phe AT ( S563A ) ) His , respectively ) were separately used to transform E . coli BL21 (DE3) cells. Five 10 mL culture s of the two E . coli transform ant were separately grown in LB · mL kanamycin at 37 °C for 12 h as described above . A 10 mL aliquot of each seed culture expressing Tyc( Phe AT ) His and Tyc( Phe AT (S563A)) His was used to inoculate LB medium (5 × 1 L for each transformant ). The bacteria were grown at 37 °C to OD 600 ~0.6, then IPTG was added to a final concentration of 103 0.5 mM, and the culture was grown for 18 h at 16 °C. The cells were pelleted by centrifugation (30 min, 4000 g ) at 4 °C, resuspended in Binding buffer (20 mM Tris HCl buffer containing 0.5 M NaCl and 5 mM imidazole at p H 7.8), lysed by sonication , and then centrifuged at 15,000 g for 0.5 h. The supernatant was decanted and centrifuged at 135,000 g for 1.5 h to remove cell wall debris and light membranes. Crude soluble protein isolated from bacteria expressing the Tyc( Phe AT ) and Tyc( Phe AT (S563A) ) was estimated by the Bradford protein assay at ~50 mg and ~75 mg total protein , respectively. 26 These fraction s w ere independently loaded onto a Ni - NTA affinity column (Qiagen, Valencia, CA) and eluted according to the protocol described by the manufacturer. The column was eluted with increasing concentration of imidazole (20 250 mM) in Binding buffer . Fractions containing the His - tagged enzymes were i dentified by SDS PAGE analysis and Coomassie Blue staining . Fractions eluting in 50 100 mM imidazole were combined and showed >95% pure protein corresponding to a molecular weight consistent with that of the calculated molecular weight for Tyc( Phe AT ) or Tyc( Phe AT (S563A) ) at 69.57 kDa. The enzyme solutions (100 mL) for each enzyme were s eparately concentrated to 1 mL by size - selective centrifugation (Centriprep 30,000 MWCO unit; Millipore, Billerica, MA). The buffer was exchanged with the Assay buffer (50 mM HEPES containing 100 mM NaCl and 1 mM EDTA at pH 8.0) over five dilution/concentration cycles. Enzyme purity was estimated by SDS PAGE with Coomassie Blue sta ining. The concentration of each protein (35 and 28 mg/mL for Tyc( Phe AT ) and Tyc( Phe AT (S 563A) ), respectively ) was determined by measuring the A 280 absorbance on a NanoDrop ND1000 Spectrophotometer (Thermo Scientific, Wilmington, DE). The calculated extinction coefficient and molecular weight of either Tyc( Phe AT ) or Tyc( Phe 104 AT (S563A) w as 280 = 60405 M 1 cm 1 27 and 69.57 kDa, respectively. The purified protein s w ere stored at - 80 °C. 3.2.8. Activity Evaluation of Tyc( Phe AT ) and Tyc( Phe AT (S563A) ) w ith CoA and Amino phenylpropanoids phenylalanine, phenylalanine , and (2 R , 3 S ) phenylisoserine (each at 1 mM) were separately incubated at 31 °C in s ingle stopped time (1 h) assays containing 100 mM HEPES (pH 8.0) , ATP (1 mM), MgCl 2 (3 mM), Co A (1 mM), and Tyc( Phe AT ) or Tyc( Phe AT (S563A) ) Various control reactions were done in parallel under the same conditions used for assays containing enzyme, w here Tyc( Phe AT ) or Tyc( Phe AT (S563A)) , ATP, or amino acid was omitted from the assay. The reactions were quenched by acidification to pH ~2 (10% formic acid in dH 2 O ) and lyophilized to dryness. The resultant samp les were separately dissolved 2 O (pH 4.0) and analyzed using a Quattro Premier XE Mass Spectrometer coupled to an Acquity ® UPLC system fitted with a C18 Ascentis Express column and the analytes were eluted with a solvent gradient of acetonitrile (Solvent A) and 0.05 % triethylamine in distilled water (Solvent B ) (held at 2.5 % Solvent A for 3.17 min, increased to 100 % Solvent A over 5 sec with a 2 min hold, and then returned to 2.5 % Solvent A over 5 sec with a 50 sec hold) at a flow rate of 0.4 mL/min. The effluent from the column was directed to the mass spectro meter set to negative ion mode with a scan range of m / z 50 1000 atomic mass units. 105 3.2.9. Kinetic Eva luation of Tyc( Phe AT ) and Tyc( Phe AT (S563A) ) for CoA and Aminophenylpropanoids After identifying productive substrates for Tyc( Phe AT ) or Tyc( Phe AT (S563A) ) , ( R ) phenylalanine, and (2 R ,3 S ) phenylisoserine (each at 1 mM) we re separately incubated in 1 m L reactions containing 100 mM HEPES (pH 8.0) , ATP (1 mM), MgCl 2 (3 mM), Co A (1 mM), and Tyc( Phe AT ) or Tyc( Phe AT (S563A)) (100 µg) to establish steady state conditions with respect to protein concentration and time at 31 °C. Under steady state conditions, ( R ) phenylalanine and (2 R ,3 S ) phenylisoserine at 5 2000 µM were separately incubated with Tyc( Phe AT ) or Tyc( Phe AT (S 563A)) (20 µg) for 15 min. At the end of each reaction and prior to mass spectrometry analysis, acetyl CoA (1 µM) was added as the internal standard to each sample to correct for losses during workup . The biosynthetic products were quantified by a LC ESI MRM (liquid chromatography electro spray multiple reaction monitoring) mass spectrometry on the Quattro Premier XE Mass Spectrometer coupled to an Acquity ® UPLC system fitted with a C18 Ascentis Express column (2.5 × 50 mm, 2.7 µm) at 30 °C. An aliquot (5 uted with a solvent gradient as described in Section 3.2.8. The effluent from the column was directed to the mass spectrometer where the first quadrupole mass analyzer (in negative ion m ode) was set to select for the molecular ion of a biosynthesized acyl CoA product. The selected ion was then directed to a collision gas chamber wherein the collision energy was optimized to maximize the abundance of a signature fragment ion ( m / z 408.31 , d erived by a fragmentation reaction in the CoA moiety of the acyl CoA ) monitored in the s econd quadrupole mass analyzer in negative ion mode . The peak area under the curve of the monitored fragment ion m / z 408.31 corresponding to each biosynthetic phenylpro pionyl CoA thioester was converted to concentration by comparison 106 to a standard curve of authentic CoA ( 0.048 The initial velocity ( v o ) production of ( R ) phenylalanyl , and (2 R ,3 S ) phenylisoserinyl CoA made in separate assays was plotted against substrate concentration and fit by non linear regression to the Michaelis Menten equation ( R 2 was typically between 0.90 and 0.99 ) to calculate the K M and k cat . The K M values of Tyc( Phe AT ) and Tyc( Phe AT (S563A) ) for CoA was assessed by incubating each enzyme separately with ( R ) phenylalanine, MgCl 2 (3 mM), ATP (1 mM), and Co A varied between 5 at 31 °C for 15 min. At the end of each reaction and prior to mass spectrometry analysis, acetyl CoA (1 µM) was added as the internal standard to each sample. The products of the enzyme - catalyzed reaction s were quantified by a LC MRM mass spectrometry, and the monitored fragment ion ( m / z 408.31) derived from the CoA thioester analytes in the effluent wer e quantified identically to the procedure described earlier in this Section . The initial velocity ( v o ) production of ( R ) pheny lalanyl CoA made in separate assays was plotted against substrate concentration and fit by non linear regression to the Michaelis Menten equation (R 2 was 0.9 ) to calculate K M . 3.2.10. S tereos pecificity of Tyc( Phe AT ) for Phenylisoserine Stereoisomers 3.2.10.1. Preparation of (2 R ,3 R ) - Phenylisoserine Figure 3.2 . Deprotection of (2 R ,3 R ) phenylisoserine The N Boc (2 R ,3 R ) phenylisoserine was deprotec ted as previously reported . 2 8 Briefly, to 5 mg 107 ( 0.018 mmol) of the N Boc (2 R ,3 R ) phenylisoserine in a 25 - mL round bottom flask was added dichloromethane (1 mL) . T o the solu tion stirred at 0 °C was added dropwise TFA: DCM (1:1 v/v, 1 mL total) until all the starting material was deprotected (monitored by TLC; 1:3:6 acetic acid: dH 2 O: t - butanol). The reaction was concentrated under vacuum to 1 mL, then diluted 2 - fold in dichloromethane, and concentrated to 1 mL. This dilution/concentration cycle was repeated three times, and then the solvent was removed completely. The residue was dried under vacuum t o obtain a white solid ( 3.2 mg, 0.018 mmol, 100 % yield) which was judged to be 100 % pure by 1 H - NMR analysis. 1 H - NMR (500 MHz, D 2 O) : 4.78 4.82 (m, H - 2 & H - 3 ) , 7.41 7 .42 (m, aromatic protons). 13 C - NMR (126 MHz, D 2 O) C1), 131.50 (C4 ), 129.70 ( C7 ), 128.90 (C5 & C9), 127.89 (C6 & C8 ), 70.38 (C2), 55.87 ( C 3 ). 3.2.10.2. Preparation of (2 S ,3 R ) Phenylisoserine Figure 3. 3 . Deprotection of (2 S ,3 R ) phenylisoserine To a 25 mL round - bottom flask, 5 mg (0.018 mmol) of (2 S ,3 R ) N benzoyl 3 phenylisoserine and 7 N HCl (1 mL) were added and the reaction stirred under reflux for 24 h. The reaction was then cooled down to room temperature and diethyl ether was added to remove benzoic acid, leaving the product in the aqueous layer, which was lyophilized to obtain (2 S ,3 R ) phenylisoserine ( 2.2 mg, 0.012 mmol, 57 % yield at >98 % purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O ) 4.56 (d, J = 6.36 Hz, H - 2 ) , 4.64 (d, J = 6.36 Hz, H - 3 ) , 7.41 7.53 (m, 108 aromatic protons). 13 C - NMR (126 MHz, D 2 O) 176.30 (C1), 133.44 (C4 ), 129.27 (C7 ), 128.98 (C5 & C9 ), 127.17 (C 6 & C8 ), 7 3 . 35 (C 2), 57.42 (C 3). 3.2.10.3. Evaluation of Tyc( Phe AT ) Activity for Phenylisoserine Stereoisomers (2 R, 3 S ) , (2 R, 3 R ) , and (2 S ,3 R ) P henylisoserine (each at 1 mM) were separately incubated at 31 °C in single stopped time (1 h) assays, containing 100 mM HEPES (pH 8.0) , ATP (1 mM), MgCl 2 (3 mM), Co A (1 mM), and Tyc( Phe AT ) acidifying to pH ~2 (10 % formic acid) and lyophilized to dryness. The resultant samples were separately dissolved in dH 2 O ( pH 4.0 ) and analyzed by LC ESI MRM as described in Section 3.2.9 . 3.2.10.4. Kinetic Evaluation of Tyc( Phe AT ) for CoA an d Phenylisoserine S tereoisomers The kinetic parameters of Tyc( Phe AT ) for (2 R, 3 R ) phenylisoserine were determined to provide a basis of comparison with the (2 R, 3 S ) phenylisoserine isomer ( described in Section 3.2.9 ). (2 R, 3 R ) P henylisoserine (at 1 mM) was incubated in a 1 mL reaction containing 100 mM HEPES (pH 8.0) , ATP (1 mM), MgCl 2 (3 mM), Co A (1 mM), and Tyc( Phe AT ) (10 0 µg ·mL ) to establish steady - state conditions with respect to protein concentration and time at 31 °C. Aliquots ( ) were taken at various time points between 0 2 h and quenched with 10 % Acetyl CoA (1 µM) was added as the internal standard and the samples were analyzed as described in Section 3.2.8 . The kinetic assays with (2 R, 3 R ) phenylisoserine were done similarly to the procedure described for (2 R, 3 S ) phenylisoserine in Section 3.2. 9 . 109 3.2.10.5. Inhibition Studies of (2 R ,3 S ) Phenylisoserine by the Enantiomer (2 S ,3 R ) Phenylisoserine The inhibition of Tyc(Phe AT) by the (2 S ,3 R ) phenylisoserine was determined by incubating (2 R , 3 S ) phenylisoserine at varying concentrations (0.01 2 mM), ATP (1 mM), CoA (1 mM), and MgCl 2 (3 mM). A fixed concentration of (2 S ,3 R ) phenylisoserine (0.25 mM) and Tyc(Phe AT) (20 µg) were added to each of the assay and the reaction incubated at 31 ° C for 30 min. The assays were quenched by acidifying to pH ~2 (10% formic acid) and analyzed by LC ESI MRM as described in Section 3.2.9. 3.3. R esults and D iscussion 3.3.1. Construction and Expression of Tyc( Phe A ) The t yc ( p he a ) cDNA en coding the Tyc( Phe A ) domain was heterologously expressed as a C - terminal His6 - fusion in E . coli BL21(DE3). The solubly - expressed enzyme was isolated and Ni - affinity purified to 98 % for use in the activity assays. The Tyc( Phe A ) protein from both the soluble and insoluble fractions was analyzed by SDS PAGE and Coomasie blue staining ( Figure 3. 4 ). 110 Figure 3. 4 . SDS polyacrylamide gel electrophoresis (12% acrylamide) and Coomassie B lue staining of recombinantly expressed Tyc( Phe A ) isolated from E. coli BL21(DE3). Lane 1 : whole cell contents before IPTG induction ; Lane 2 : whole cell contents after IPTG induction . The profile of the soluble protein eluted from Ni - affinity resin chromatography with Binding buffer containe d the following ; Lane 3 : flow through; Lane 5 : 50 mM imidazole (fraction 1); Lane 6 : 100 mM imidazole (fraction 2); L: Molecular weight standard; Lane 7 : 200 mM imidazole (fraction 3) . 3.3.2. Activity of Tyc( Phe A ) w ith ATP, CoA , and Aminop henylpropanoids In a previous study, the Tyc ( Phe A ) domain was reported as active when its stand - alone thiolation (T) domain Tyc ( Phe T) complement was added to the reaction mixture that included ATP and [ 14 C] phenylalanine . 11 The phenylalanine was thioesterified by the pendent pantetheinyl thiol donor on the holo Tyc( Phe T ) domain . This study confirmed the formation of a phenylalanyl AMP intermediate. This earlier stud y suggest ed that Tyc( Phe A ) is an active stand - alone domain that catalyzed phenylalanine adenylation. In the current stud y, it was hypothesized that CoA would serve as a thiol donor based on similarities to the Tyc(Phe T) bound 4' Ppant moiety. T he enzyme activity of Tyc( Phe A ) was tested in separate assays containing ATP ( 1 mM ) , MgCl 2 ( 3 mM ) , CoA ( 1 mM ) , , phenylalanine , or (2 R , 3 S ) phenylisoserine . N acetylcysteamine was also used as a thiol donor in place of CoA in separate assays under similar conditions . C ontrol reactions were done in 1 2 3 5 6 L 7 225 150 100 50 35 25 kDa 70 Lane: 111 parallel under the same conditions except Tyc( Phe A ) , ATP, or CoA was omitted . No amino acyl CoA n or aminoacyl N acetylcysteamine was detected by ESI mass spectrometer for phenylalanine phenylalanine , or (2 R , 3 S ) phenylisoserine . Thus , CoA binding was either affected by the absence of thiolation domain , or t he expressed Tyc( Phe A ) was inactive. To address the se arguments, a tryptophan fluorescence quenching technique was used to assess indirectly the functional expression of the Tyc( Phe A ) truncation. In addition, Tyc( Phe AT ) and Tyc( Phe AT (S563A)) were expressed to assess the dependence of the CoA thioesterification on the thiolation domain. The S563A mutant was construc ted to eliminate 4' phosphopantetheinylation of the T domain, and thus eliminate competition between the pendent thiol and the diffusible CoA added to the activity assay. 3.3.3. Assessing Tyc( Phe A ) Substrate Binding by Tryptophan Fluorescence Quenching Studies In principle, fluorescen ce quenching is observed when molecular contact between a fluorophore and a quencher molecule occurs. The contact can be as a result of diffusive encounter (dynamic quenching) or due to complex formation (static quenching). 29 When a contact between the quencher and the fluorophore (which is in the excited state ) is established, its excited electron in the LUMO (lowest unoccupied molecular orbital) returns to ground state. The fluorophore is unable to emit light and the energy is dissipated as heat. 29 The fluorescence quenching technique has been exploited to study protein conformation changes and interactions with other molecules. 30 , 31 Tryptophan fluorescence is highly sensitive to its local environment and is affected by any changes to the protein conformation as a result of ligand binding. Therefore, 112 tryptophan has been used as a probe to study prot ein dynamics and intermolecular interactions including p rotein ligand binding . 30 , 31 Previously, a tryptophan quenching titration experiment was used to determine the binding constants of amino acids to Grs 1 , a homolog of TycA. 32 , 33 Increase in tryptophan quenching with in Grs 1 ( Phe A domain) in the presence of varying concentrations of acrylamide showed a linear correlation. 33 Th is observation led to the conclusion that a single tryptophan residue was involved in the quenching, which was presumed to be W 239 . 33 This previous study prompted the application of equilibrium tryptophan fl uorescence quenching to determine the binding outcome of (2 S ) phenylalanine to the Tyc( Phe A ) domain examined herein. Equation 1 shows the relationship between the change in the observed fluorescence ( F obs ), the ligand dissociation constant K d , and the maximum observed difference in tryptophan fluorescence quenching when the ligand is saturating ( F max ). F obs = F 0 F [S] ; where F 0 is the fluorescence emission without the ligand and F [S] is the quenched fluorescence reading of Tyc( Phe A ) at ligand concentration [S]. The K d of Tyc( Phe A ) (0. 1 µ M) for (2 S ) phenylalanine was 4.0 ± 1 .0 with a F max o f 24.3 . Equation 1 S imilar equilibrium titration s were repeated with Tyc( Phe AT ), (a functional amino acyl CoA ligas e ) . The similar K d value of 4 .0 ± 1 .0 for Tyc( Phe AT ) agreed with that for Tyc( Phe A ) suggesting that (2 S ) phenylalanine could bind the Tyc( Phe A ) active site . 113 Figure 3. 5 . Plot of observ ed fluorescence change with increasing concentr ation of (2 S ) phenylalanine (0 96 µM) titrated to 0.1 µM Tyc( Phe A ) (in K + HEPES, pH 7.5, 25 °C). The curve was fit by the non - linear regression curve defined by equation 1. In an earlier s t udy , analysis of the Grs1( Phe A ) domain ( 62 % sequence similarity with Tyc ( Phe A )) reported a dissociation constant for (2 S ) phenylalanine of 6 .0 ± 1 .0 32 which is similar to that determined in the current stud y ( Table 3.1 ). These results suggest that the inability of Tyc(Phe A) to catalyze aminoacyl CoA biosynthesis stem from its failure to bind CoA in a catalytically competent orientation for nucleophilic displacement of AMP from the adenylate intermediate as discussed in Se ction 3.3.6. Table 3. 1 . The dissociation constants fo r binding of (2 S ) phenylalanine ((2 S ) Phe) to Tyc( Phe A ) , Tyc ( Phe AT ) , and Grs1( Phe A) . Substrate K d Tyc( Phe A ) ) T yc ( Phe AT ) Grs1( Phe A) 32 (2 S ) Phe 4 .0 ± 1 .0 4 .0 ± 1 .0 6 .0 ± 1 .0 (2 S ) Phe + AMP 8 .0 ± 1 .0 7 .0 ± 1 .0 *ND *Not determined 114 In a previous study , a standalone A domain (EntE) , that belongs to the NRPS enzyme family was shown to activate dihydroxybenzoate to its adenylate anhydride. 34 The earlier study evaluate whether CoA would act as a s urrogate of the native 4 ' Ppant . The formation of dihydroxybenzoyl CoA was analyzed indirectly by measuring the formation of PPi using a continuous spectrophotometric assa y . A dearth of PPi (that confirmed the production of dihydroxybenzoyl CoA ) was detected , suggesting that the product formed fortuitously and non - enzymatically in solution. 34 A similar conclusion was derived from earlier studie s in which Tyc( Phe A ) and Tyc(Phe T) domains were expressed separately and mixed as untethered standalone domains with phenylalanine and ATP. Similar to the results for the EntE study, only a small amount of Tyc(Phe T) was thioesterified. Based on the re sults from the previous EntE and Tyc(Phe T) thioesterification studies, it could be imagined that in the current study, Tyc( Phe A ) catalyzes the adenylation of phenylalanine . Thereafter, CoA merely non - enzymatically thioesterifies (2 S ) phenylalanine by reaction with the AMP anhydride intermediate when it dissociates from the enzyme. Since no observable CoA thioester product was obtained in reactions with Tyc( Phe A ) and the needed cofactors, even with detection by an LC ESI MRM method , it is evident t hat CoA binds to the Tyc( Phe AT E) or Tyc( Phe AT ) active site, where it forms acyl thioesters, and this binding is interrupted in the absence of Tyc(Phe T) domain, as discussed in the next Section (3.3.4). 3.3.4. Construction and Expression of Tyc( Phe AT ) and Tyc( Phe AT (S563A) ) T he wild - type tyc A cDNA encoding the A , T , and E domains was truncated t o test the activity of Tyc( Phe AT ) d idomain for amino acyl CoA biosynthesis . In addition, a mutant Tyc( Phe AT (S563A)) derived from Tyc( Phe AT ) was expressed to eliminate the native pantetheinylation 115 of S563; this way, the only thiol/nucleophile would be the one added exogenously. The apparent molecular mass ( 69.5 kDa) of the expressed Tyc( Phe AT ) and Tyc( Phe AT (S563A)) on SDS PAGE was consisten t with the theoretical value ( Figure 3. 6 ) . A ) B ) Figure 3. 6 . SDS - polyacrylamide gel electrophoresis (12% acrylamide) and Coomassie blue staining of recombinantly expressed Tyc( Phe AT ) ( A ) and Tyc( Phe AT (S563A) ) ( B ) which were separately isolated from E. coli BL21(DE3). A ) Lane 1 shows the soluble cell crude lysate before purification. The profile of the soluble protein eluted from nickel affinity resin chromatography with Binding buffer contained the following; Lane 3 : 20 mM imidazole wash; Lane 4 : 50 mM imidazole (fraction 1); Lane 5 : 100 mM imidazole (fraction 2); L: Molecular weight standard; Lane 6: 200 mM imidazole (fraction 3). B ) Lane 1 : 1 0 mM imidazole wash; Lane 2 : 2 0 mM imidazole wash; Lane 3 : 50 mM imidazole (fraction 1); Lane 5 : 100 mM imidazole (fraction 2); Lane 6 : 200 mM imidazole (fraction 3) ; Lane 7 : 250 mM imidazole (fraction 4 ) . 3.3.5. Activity and Kinetic Analysis of Tyc( Phe AT ) w ith ATP, CoA , and Amino p henylpropanoid s Tyc( Phe AT ) and Tyc( Phe AT (S563A) ) were incubated with CoA, cofactors, and separately with ( S ) , ( R ) phenylalanine , or (2 R , 3 S ) phenylisoserine . Analysis of the assay products by LC MS method showed a mass profile matching that of authentic phenylalanyl CoA . The results reveal that truncation of Tyc( Phe AT ) and Tyc( Phe AT ( S563A )) enhanced turn over rates for the three substrates examined compa red to Tyc( Phe AT E) . The k cat of ( R ) phenylalanine was 1 2 3 5 6 L 7 1 3 4 5 L 6 225 100 50 35 25 kDa 69.5 116 the most improved with either catalyst by 7 - fold compared to the wild - type Tyc( Phe AT E) , whereas the K M was within the same range for both wild type Tyc ( Phe AT E) and the truncated version Tyc( Phe AT ). Table 3.2 . Steady state kinetic analysis of Tyc( Phe AT ) and Tyc( Phe AT (S563A) ) with CoA in comparison to Tyc( Phe AT E) and Tyc( Phe AT E(S563A)) Tyc( Phe AT ) Substrate K M k cat (min - 1 ) k cat / K M (s - 1 · M - 1 ) ( R ) phenylalanine 45.9 ± 2.6 0 14.6 ± 0.5 00 4018 ± 119 (2 R ,3 S ) Phenylisoserine 440 ± 62.1 1.51 ± 0. 17 0 33.5 ± 1.7 0 CoA a 208 ± 57.2 10.4 ± 2 .00 838 ± 74 .0 Tyc( Phe AT (S563A) ) ( R ) phenylalanine 32.6 ± 4.7 0 22 ± 4. 1 0 11244 ± 485 (2 R ,3 S ) Phenylisoserine 348 ± 140 1.06 ± 0.5 7 0 50.6 ± 8.2 0 CoA a 687 ± 250 31.4 ± 8.3 0 762 ± 87 .0 Tyc( Phe AT E) ( R ) phenylalanine 50.6 ± 7.9 0 1. 6 ± 0.3 00 527 ± 129 (2 R ,3 S ) Phenylisoserine 89.3 ± 15.0 0.25 ± 0.02 0 46.7 ± 8.7 0 CoA b 1976 ± 175 0.75 ± 0.05 0 6.3 ± 0.7 00 Tyc( Phe AT E(S563A)) ( R ) phenylalanine 62.3 ± 1.0 0 3.00 ± 0. 04 803 ± 17 .0 (2 R ,3 S ) Phenylisoserine 191 ± 10 .0 0.43 ± 0.01 37.5 ± 2.1 0 CoA b 804 ± 26 .0 0.90 ± 0.08 18.7 ± 1.8 0 a Kinetic measurements for CoA were done in the presence of ( R ) phenylalanine , ATP , and MgCl 2 . b Kinetic measurements for CoA were performed in the presence of ( S ) phenylalanine , ATP , and MgCl 2 . All values are expressed as means ± standard deviations of triplicates. The kinetic values of Tyc( Phe AT E) and Tyc( Phe AT E(S563A)) are listed for comparison to those of Tyc( Phe AT ) and Tyc( Phe AT (S563A)). Similarly, (2 R ,3 S ) phenyl isoserine showed a gain in turn over rate with both Tyc( Phe AT ) (6 - fold increase) and Tyc( Phe AT (S563A) ) (2.5 - fold increase ) compared to the Tyc( Phe AT E) and Tyc( Phe AT E(S563A)) respectively . Tyc( Phe AT (S563A)) displayed the highest k cat value when incubated with CoA (35 - fold increase) compared to the Tyc( Phe AT E(S563A)) counterpar t. Additionally, Tyc( Phe AT ) showed a 4 - fold improvement in k cat with CoA compared to the w ild type Tyc ( Phe AT E) . The catalytic efficiency ( k cat / K M ) of Tyc( Phe AT ) for CoA improved 10 orders - of - magnitude over that of Tyc( Phe AT E) due mainly to lower K M 117 increase in catalytic efficiency of Tyc( Phe AT ) and Tyc( Phe AT (S563A)) at 8 - fold and 14 - fold, respectively due mainly to a 7 - fold increase in k cat for ( R ) phenylalanine wa s also observed . The catalytic efficiency of Tyc( Phe AT ) for (2 R ,3 S ) phenylisoserine was lower compared to that of Tyc ( Phe AT E ) due to a higher K M value despite the 6 - fold greater k cat ( Table 3.2 ) . Overall, the catalytic efficiencies of Tyc( Phe AT ) and Tyc( Phe AT (S563A)) were superior to those for the wild - type Tyc( Phe AT E) and Tyc( Phe AT E(S563A)) counterparts, respectively for the three substrates. These results suggest that eliminating the epimerization (E) domain likely improved produ ct dissociation from the multidomain enzyme as evidenced by the improved turn over rates ( Table 3.2 ) . In a ddition, the possibility of epimerization of amino acyl CoA product s was eliminated . 3.3.6. Tyc( Phe AT ) Secondary S t ructure Model and C omparison w ith CoA L igases Previous studies showed that the secondary structures of acetate: CoA ligase ( AcCL, from Salmonella enterica , PDB code: 1PG4 ) , Grs1( Phe A PDB code: 1AMU ), and DhbE (a free standing adenylation domain from Bacillus subtilis , PDB code: 1MDF ) are similar, 15 while their amino acid sequence homology is only moderate (19 25% similar) . 15 It is well known that Tyc ( Phe AT ) normally thioesterify phenylalanine with a pantetheinyl side chain covalently linked to the T domain through a phosphate ester bond . By contrast , CoA ligases use diffusible CoA in the bulk medium to thioesterify the acyl substrates . 35 Phosphopantetheine is a precursor of CoA and, by comparison lacks the adenine moiety at the 4' phosphate terminus. In the current study, Tyc( Phe A ) did not biosynthesize amino acyl CoA , when CoA was used as a cosubstrate with the three amino acid substrates described herein , whereas Tyc( Phe AT ) was active. To help explain how CoA serve d as a thiol donor in the reaction catalyzed by Ty c( Phe AT ) and not 118 Tyc( Phe A ), homology models of Tyc( Phe A ) and Tyc( Phe AT ) ( SWISS model program ) 36 were built on the structure of Grs1( Phe A ) ( 65% amino acid sequence identity). It should be noted that the amino acid residues of the phenylalanine binding pockets of Tyc( Phe AT ) and Grs1( Phe A ) are 100% identical . The resulting Ty c(Phe AT) model was compared to 4 chlorobenzoate:CoA (4 ClBzCL ; PDB code: 3CW9 ) and acetate: CoA (AcCL , PDB code: 1PG4 ) ligases ( Figure 3. 7 ) . Tyc( Phe AT ) model was superimposed on the structure of 4 ClBzCL and AcCL separately ( Figure 3. 7 ) . There are key features common to Tyc( Phe AT ) and both ligases used herein. The residues used to bind the carboxylate substrate, ATP, and Mg 2+ and those used to catalyze the reaction are highly conserved. In addition, both Tyc( Phe AT ) model and 4 ClBzCL have a channel that accommodates pantetheine chain which is mainly surrounded by hydrophobic and a few hydrophilic residues ( Figure 3. 8 ) . A) B) Figure 3. 7 . Comparison of 4 ClBzCL with Tyc(Phe AT) model . A) Overlay of Tyc( Phe AT ) model (cyan ribbon ) and 4 ClBzCL (green ribbon ) in complex with CoA ( carbon skeleton shown as yellow spheres) . The amino acid sequence alignment between the two proteins show 21% sequence identity. B) Overlay of Tyc( Phe AT ) model (cyan ribbon ) and AcCL (magenta ribbon) in complex with CoA (hot pink spheres). Tyc( Phe AT ) shows 19% amino acid sequence identity to AcCL . 119 3.3.6.1. Contrasting Structural Features of Tyc( Phe AT ) Model and 4 C hlorobenzoate:CoA Ligase Key differences are seen between Tyc( Phe AT ) and the CoA ligases, for instance the binding site of the s tructures of Tyc( Phe AT ) and 4 ClBzCL are noticed . Specifically, the key residues that form favorable interactions with the adenosyl moiety in 4 ClBzCL ( S407, W440, K477, R475, D472, F473 , and R87 ) align the CoA binding pocket around the adenosyl group and also form favorable electrostatic contact s with the 5 ' diphosphate bridge. 22 Tyc( Phe AT ) residues at the same position (I424, A457, S491, L489, A486, Y487 , and E110) are largely hydrophobic that likely do not form favorable interaction s with the polar diphosphatidyl adenosyl moiety . A) B) Figure 3. 8 . Comparison of active site of 4 ClBzCL with Tyc(Phe AT) model . A ) Shown are residues (green sticks) that constitute the binding pocket of CoA (yellow) in 4 ClBzCL . B ) Shown are residues (cyan) that surround the proposed binding channel for CoA (yellow) in Tyc( Phe AT ) model. 3.3.6.2. Tyc( Phe AT ) Structure Model Alignment w ith Acet ate: CoA Ligase Comparison of Tyc( Phe AT ) model to that of AcCL structure show s similar residues oriented around the adenosyl group of CoA . In AcCL, the adenine ring of CoA forms stacking W440 K477 R475 D472 F473 R87 L489 A457 A486 Y487 E110 I424 120 interaction s with the peptide backbone between G164 and G165 ( Figure 3 . 9 A ). The other residues that contribute to the hydrophobic environment are F163 and I196 ( Figure 3. 9 A ) . Additionally, the 5 ' di phosphate on the adenosyl of CoA forms favorable interactions with the side chain of R191 whereas 3 ' phosphate interacts with R584 and R194 ( Figure 3. 9 A ) . The pantetheine chain is supported by interaction between the carbon skeleton and hydrophobic residues ( G524 and A357 ) and also the hydrogen bonding of the two amines with polar residues ( S523 and H525 ). In Tyc( Phe AT ) , the adenine binding site is surrounded by hydrophilic residues D108, E110 and R428 which are not positioned for a favorable stacking interaction with the adenine . Also, the 3 ' phosphate binds at the surface of Tyc(Phe AT) and lacks important H - bonding interactions. Thus, the differences in binding site architecture around the adenosyl moiety most likely contribute to unfavorable binding of CoA to Tyc(Phe - AT). However, the pantetheine binding channel consists of residues similar to those of the AcCL and the 4 ClBzCL . These observations s hed light on how the acyl adenylate enzymes are tailored to suite their respective catalytic reactions ; the acyl CoA ligases need a framework to support the binding site of adenine moiety of CoA , whereas Tyc( Phe AT ) requires a channel that binds only the pantetheinyl moiety . 121 A) B) Figure 3. 9 . Comparison of active site of AcCL with Tyc(Phe AT) model . A ) Shown are residues (magenta) that contribute to CoA (yellow) binding interactions in AcCL . B ) Shown are residues (cyan) that constitute the proposed CoA binding pocket in Tyc( Phe AT ) . In AcCL and the 4 ClBzCL, the adenine of CoA is bound on the surface of the protein , and the pantethein yl moiety is directed into the active site through a hydrophobic channel ( Figure s 3. 8 A and 3. 9 A ). The T - domain of the Tyc( Phe AT ) model was excluded by the SWISS modeling program, since the Grs1( Phe A ) structure upon which it was modeled also lacked the conjugate T domain. These jettisoned residues likely have an added role in binding CoA ( see Appendix , Figure II 3 ) . The T domain residues must help CoA to bind Tyc( Phe AT ), as evidenced by the lack of amino acyl CoA biosynthetic activity by Tyc( Phe A ), which lacked the T domain as described herein (Section 3.3.1). 3.3.7. Substrate Stereospecificity of Tyc( Phe AT ) The A domai n of Tyc( Phe AT E) has broad substrate specificity for both S and R amino acids. 37 Specifically, both S and R phenylalanine are converted with similar efficiency to a pendent R584 I196 G165 G164 F163 R191 H525 R194 E110 D108 K244 R425 R428 G424 A357 S423 122 thioester via an AMP anhydride. Thereafter, the E domain epimerizes the S phenylalanine to the R isomer at a ratio of 1:2 in favor of R phenylalanine . However, only the R isomer proceeds along the catalytic chain to form the downstream dipeptide bond with proline on the biosynthetic pathway to tyrocidines A D in Bacillus brevis ( Chapter 2, Figure 2.1 ) . 37 , 38 Generally, for NRPS family members, the A domai n s are known to be highly promiscuous, 1 , 37 , 39 , 40 and the E domain s are gat e keepers and play a major role in substrate se lectivity and stereospecificity . 41 - 43 Howev er, the interest of using Tyc ( Phe AT E) in the studies described herein was to identify phenylisoserine: CoA ligase that could be used in the bio synthesis of phenylisoserinyl CoA , an important substrate for a 13 O 3 amino 3 phenyl propano yl transferase (BAPT) on the paclitaxel biosynthetic pathway. 44 The (2 R ,3 S ) stereochemistry of the phenylisoserinyl moiety at C 13 of paclitaxel is important for its biological activity ( Figure 3. 10 ). 45 It was t hus necessary to assess the stereospecificity of Tyc( Phe AT ) for the phenylisoserine diastereoiso mers before thi biosynthesis of paclitaxel or its analogs . Figure 3. 1 0 . Structure of p aclitaxel Tyc( Phe AT ) turn ed over (2 R ,3 R ) isomer at a 2 - fold slower rate ( k cat = 0.78 min - 1 ) than the (2 R ,3 S ) - isomer ( k cat = 1.5 min - 1 ) ; the latter has the stereochemistry of the natural side chain 123 of paclitaxel . The CoA thioester of (2 S ,3 R ) phenylisoserine was below the limits of detection ( Table 3.3 ). In order to elucidate the differences observed in the Tyc( Phe AT ) stereospecificity studies , the (2 R ,3 S ) , (2 R ,3 R ) , and (2 S ,3 R ) phenylisoserine isomers were docked into the Grs1( Phe A ) crysta l structure using Autodock V ina program 46 ( Figure 3. 11 B D ). The residues for phenylpropanoyl binding in Grs1( Phe A ) are identical to those used by Tyc( Phe A ), thus the ligand coordinates established by the Grs1( Phe A ) chassis were deemed representative of Tyc( Phe A ). Table 3.3 . Kinetic parameters of Tyc( Phe AT ) with phenylisoserine stereoisomers Substrate K M (µM) k cat (min - 1 ) (2 R ,3 S ) Phenylisoserine 440 ± 62.1 1.51 ± 0.17 (2 R ,3 R ) Phenylisoserine 375 ± 68. 8 0.78 0 ± 0.1 3 *(2 S ,3 R ) Phenylisoserine --- --- *The kinetic constants of Tyc(Phe AT) for (2 S ,3 R ) phenylisoserine were not obtained as the CoA product was below the detection limit of the LC ESI MS. The docking conformation s of the phenylisoserine isomer s w ere compared to that of (2 S ) phenylalanine in complex with Grs 1( Phe A ) structure (PDB: 1AMU) ( Figure 3.1 1 A D ). T he trajectory of ( 2 S ) phenylalanine is established in the active site through a hydrogen bondi ng interaction between the amino group of the substrate and D 235 , and a salt bridge between the carboxylate and K 517 ( Figure 3.1 1 A ). The phenyl ring is surrounded by common hydrophobic residues ( A236, W239, I299, A322, I330, and C 331 ) in the structures. However, a major difference between phenylalanine and the docked phenylisoserine isomers is the carbon skeleton around the rotatable bond between ipso carbon of phenyl ring and C 3. This rotation o rients the isoserine moiety in a conformation that enables favorable interactions in the active site. In (2 R ,3 S ) phenylisoserine ( Figure 3.1 1 B ) , H bonding interaction between C2 OH and active site D235 is observed. The C3 NH 2 forms polar interaction through hydrogen bonding with D235, 124 while the carboxylate group continues to interact with K517 but gains an additional interaction with G324 . A) B) C) D) Figure 3.1 1 . Grs1(Phe A) active site (cyan) in complex with aminophenylpropanoids : A ) (2 S ) phenylalanine (yellow). B ) (2 R ,3 S ) phenylisoserine (green), C ) (2 R ,3 R ) phenylisoserine (magenta) and (2 R ,3 S ) phenylisoserine (green) superimposed, and D ) (2 S ,3 R ) p henylisoserine (orange) and (2 R ,3 S ) phenylisoserine (green) superimposed. K517 D235 W239 A236 I330 T278 I299 C331 A322 K517 D235 W239 A236 I330 T278 I299 C331 A322 K517 D235 W239 A236 I330 T278 I299 C331 A322 K517 D235 W239 A236 I330 T278 I299 C331 A322 G324 G324 G324 125 Similar hydrogen bonding interaction between C2 OH and active site D235 is seen for (2 R ,3 R ) phenylisoserine ( Figure 3.1 1 C ) . However, t he C3 NH 2 is oriented in the opposite direction and the H bonding contact with D235 is lost. Interestingly, t his difference does not affect the binding of (2 R ,3 R ) isomer since the K M ( 375 ± 68. 8 µM ) is similar to that of ( 2 R ,3 S ) phenylisoserine ( 440 ± 62.1 µM ). The catalytic rate of Tyc( Phe AT ) however , is 2 - fold lower for (2 R ,3 R ) phenylisoserine , compared to that for ( 2 R ,3 S ) phenylisoserine . The binding of (2 S ,3 R ) phenylisoserine to the active site shows a different orientation of the phenyl ring compared to ( 2 R ,3 S ) phenylisoserine ( Figure 3.1 1 D ) . Also , the C3 NH 2 binds in a different site, away from D235 ( the ligand that contributes to the H bonding ) . The loss of Tyc( Phe AT ) activity with (2 S ,3 R ) phenylisoserine could be due to the different orientation of the phenyl ring in the bi nding pocket in addition to the lack of H bonding between the C3 NH 2 and active site D235. 3.3.7.1. Inhibition Studies of (2 R ,3 S ) P henylisoserine by the E nantiomer (2 S ,3 R ) P henylisoserine In the presence of (2 S ,3 R ) phenylisoserine at 250 µM , the K M of Tyc( Phe AT ) for (2 R ,3 S ) phenylisoserine was 500 µM which was nearly identical to the K M ( 436 µM ) of Tyc( Phe AT ) in the absence of the inhibitor . The k cat however , decreased by 1 5 - fold ( Figure 3.1 2 ) and the K I (92.0 µ M) was calculated using E quation 3.2 . 47 ' = (1+[I]/ K I ) Equation 3.2 126 Figure 3.1 2 . Lineweaver Burk plots for the inhibition of Tyc( Phe AT ) by (2 S ,3 R ) phenylisoserine . The K M value of (2 R ,3 S ) phenylisoserine without inhibitor wa s 436 µM , and k cat was 1. 5 min - 1 . In the presence of ( 2 S ,3 R ) phenylisoserine ( 25 µM ), the K M was 500 µM and the k cat was 0.1 0 min - 1 . The results indicate a non - competitive inhibition of Tyc( Phe AT ) by (2 S ,3 R ) phenylisoserine suggesting that this inhibitor binds allosterically to a site other than t he active site and affects the catalytic turnover. 3.4. Conclusion Numerous studies on NRPS modules have dissect ed substrate recognition and the mechanisms of acylation, thioesterification , 3 , 5 , 9 , 12 , 48 - 53 and epimerization catalyzed by the domains . 32 , 39 , 54 - 58 This information was useful for identifying an NRPS module Tyc( Phe AT E) (i.e TycA) that could function as a CoA ligase and thioesterify phenylisoserine, a paclitaxel pathway intermediate, to 0.25 m M (2 S ,3 R ) 0 m M (2 S ,3 R ) 127 its biologically relevant CoA thioester. The design and biochemical validation of Tyc( Phe AT ) mutants gave us access to catalysts that biosynthesized amino acyl CoA s at a superior rate (~7 - fold for ( R ) phenylalanine and (2 R ,3 S ) phenylisoserine ) compared to the wild - type Tyc( Phe AT E) enzymes. The ability of Tyc( Phe AT ) to catalyze the CoA ligase - like reaction traces back to the structura l and sequence similarities with acyl CoA ligases. The alignment of Tyc( Phe AT ) model on either acetate: CoA or 4 c hlorobenzoate:CoA ligase showed some similarities in the residues lining the active sites where carboxylate, ATP, Mg 2+ , an d pantetheine arm of CoA binds. However, striking differences were observed with the composition of r esidues around adenosyl binding cavity CoA ligases have residues that support the binding of adenosyl moiety (for example W440, K447, R475, D472, F473 a nd R87 in 4 c l oro benzoate:CoA ligas e) whereas Tyc( Phe AT ) consists of residues that are not suitable for favorable stacking with the adenosyl moiety o r electrostatic interaction s with the phosphate groups. The binding affinity and catalytic efficiency of Tyc( Phe AT ) c oul d likely improve upon changing the residues near the adenosyl terminus of CoA (A457, S491, L498, A486, Y487 and E110) to those found in CoA ligases. Tyc( Phe AT ) was stereospecific for phenylisoserine diastereomers (2 R ,3 S ) phenylisoserine ( k rel = 1.0), followed by (2 R ,3 R ) ( k rel = 0.5) , and then (2 S ,3 R ) ( k rel = 0). Molecular docking of (2 R ,3 S ) phenylisoserine into the active site of Tyc( Phe AT ) homolog (Grs1 ( Phe A ) ) showed that the C 2 OH and C 3 NH 2 substituents form ed favorable polar contacts through H - bonding with D23 4 , while the phenyl ring of the substrate formed hydrophobic interactions in the aryl ring binding pocket . The findings from this study will enable Tyc( Phe AT ) to be used in the development of a biosynthe tic route towards paclitaxel and its analogs containing (2 R ,3 S ) arylisoserines. 128 APPENDIX 129 Protein Motif I Motif II Motif III CoA ligases 4 C hloro b enzo ate CL 161 T SG T TG LP K G 170 302 YGT TE 306 376 Y R TS D 380 Coumarate CL T SG T TG PP K G YGS TE YRT GD 4 H ydroxy be nzo ate CL S SG S TG RP K G IGS TE TKS GD O Succinatebenzoate CL T SG T TG PQ K A FGM TE FNT GD Acetate CL T SG S TG KP K G YWQ TE YFT GD Ac et yl CL T SG T TG NP K G WGM TE FST GD Thiol template acyl adenylate forming enzymes Grs1 1 90 T SG S TG N P K G 1 99 YGP TE YRT GD TycA 178 T SG T TG KP K G 187 YGP TE 376 YRT GD 380 EntE SG G T TG TP K L FGMA E YCS GD Luciferase enzymes Luciferase a 198 S SG S TG LP K G 207 340 YGL TE 344 418 LHS GD 422 Luciferase b S SG T TG LP K G FGL TE LHS GD Figure II 1 . A mino acid sequence variation in the highly conserved motifs in the a cyl adenylate enzyme family . CL is CoA ligase, 4 chloro benzoate CL is from Pseudomonas sp. , c oumarate CL is from Mycobacterium leprae , 4 hydroxy benzoate CL is from Rhodopseudomas palustris , O s uccinatebenzoate CL is from Staphylococcus aureus , acetate CL is from Neurospora crassa , acetyl CL is from Pseudomonas olevorns , TycA and Grs 1 are from B acillus brevis , E ntE is adenylation domain from enterobactin synthetase from E. coli . a Luciferase is from Photinus pyralis and b Luciferase is from the green - emitting strain of the click beetle. 130 Figure II 2 . Amino acid sequence alignment of close adenylation domain homolog s of NRPS family that show 16 61% amino acid sequence identity. The residues that are conserved across all sequences are highlighted in black, whereas the residues conserved in some of the sequences are highlighted in grey. 131 Figure II 2 . 132 Figure II 3 . The amino acid sequence alignment of Tyc( Phe AT ) , ac et yl CoA and 4 - chlorobenzoate:CoA ligase are shown. The overall sequence identity of Tyc( Phe AT ) to ac et yl CoA and 4 chlorobenzoate:CoA ligase is 19 and 21% respectively. The Tyc( Phe AT ) residues that are not aligned to either CoA ligase (residues numbered 639 to 740) belong to t hiolation domain. 133 Figure II 3 . 134 Figure II 3 . Figure II 4 . Spectra obtained by equili brium fluorescence titration of (2 S ) phenylalanine to K + HEPES , pH 7.5 , 25 °C . This assay lacked the enzyme and was used as the control experiment. The emission wavelength was scanned from 300 to 420 nm using a fixed excitation wavelength of 280 nm. 0 2 4 6 8 10 12 14 16 300 320 340 360 380 400 420 Intensity Wavelength (nm) M M M M M M M M M M M 135 Figure II 5 . Spectra obtained by equilibrium fluorescence titrat ion of (2 S ) phenylalanine at increasing concentrations ( 0 Tyc( Phe A ) (in 50 mM K + Hepes buffer, pH 7.5 at 25 °C ) ). The emission wavelength was scanned from 300 to 420 nm using a fixed excitation wavelength of 280 nm. F igure II 6. Plot of observed fluorescence change with varying concentrations of AMP at fixed concentration of Tyc(Phe A) (in K + HEPES, pH 7.5, 25 °C) containing 96 µM (2 S ) phenylalanine. The curve was fitted using equation 1 in KaleidaGraph program. 0 10 20 30 40 50 60 70 80 300 320 340 360 380 400 420 Intensity Wavelength (nm) M M M M M M M M M M 136 Figure II 7. Plot of observed fluorescence change with varying concentrations of (2 S ) phenylalanine at fixed concentration of T yc(Phe AT) (in K + HEPES, pH 7.5, 25 °C). The curve was fitted using equation 1 in KaleidaGraph program. Figur e II 8 . Plot of observed fluorescence change with varying concentration s of AMP at fixed concentration of Tyc( Phe AT ) (in K + HEPES , pH 7.5, 25 ° C ) containing 96 µM (2 S ) phenylalanine . The curve was fitted using equation 1 in KaleidaGraph program. 137 Figure II 9 . 1 H - NMR spectrum of (2 R ,3 R ) phenylisoserine D 2 O H - 2& 3 H - 5 - 9 138 Figure II 10 . 13 C - NMR spectrum of (2 R ,3 R ) phenylisoserine C - 3 C - 2 C - 6&8 C - 5&9 C - 7 C - 4 C - 1 139 Figure II 1 1 . 1 H - NMR spectrum of (2 S ,3 R ) phenylisoserine H - 3 H - 2 H - 5 - 9 140 F igure II 12 . 13 C - NMR spectrum of (2 S ,3 R ) phenylisoserine C - 3 C - 2 C - 6&8 C - 5&9 C - 7 C - 4 C - 1 141 Figure II 1 3 . Lineweaver - Burk plots for the inhibition of Tyc( Phe AT ) in the presence of (2 S ,3 R ) phenylisoserine at 0 mM , 0.01 mM , 0.04 mM , and 0.25 mM. (2 R ,3 S ) phenylisoserine was used as the substrate at varying concentrations (0.001 2 mM). 1 42 REFERENCES 143 REFERENCES (1) Finking, R.; Marahiel, M. A. Annu. Rev. Microbiol. 2004 , 58 , 453. (2) Schwarzer, D.; Finking, R.; Marahiel, M. A. Nat. Prod. Rep. 2003 , 20 , 275. (3) Marahiel, M. A.; Stachelhaus, T.; Mootz, H. D. Chem. 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So far, different paclitaxel analog s with improved IC 50 and ED 50 over the parent drug against multidrug resistant ovarian carcinoma, pancreatic cancer, B16 melanoma, human lung cancer, and colon cancer cell lines have been developed. 3 , 5 - 12 Among them are analog s modified at the C 13 side chain ( Figure 4.1 ) . 3 , 5 , 11 Figure 4.1 . The paclitaxel structure framework showing C 13 (2 R ,3 S ) phenylisoserine side chain Structure - activity relationship studies have shown that the isoserine side chain at C 13 of paclitaxe l is important for it s activity 13 , 14 with maximum activity seen for (2 R ,3 S ) phenylisoserine and lesser activity for the other stereoisomers. 15 Moreover, the 2' hydroxy l is necessary fo r activity ; when O acetylat ed, the drug is inactivated. 14 R eplacement of 2' OH with 148 fluorin e or thiol decreases binding of the paclitaxel analog to the cellular microtubule by two fold. 16 , 17 Q uantitative assessment and c omputational analysis showed that the 2' hydroxyl accounts for 80% of the binding free energy of the side chain . 18 Removal of both 2' hydroxyl and 3' benzoyl group s rendered the resultant N debenzoyl 2' deoxy paclitaxel inactive. 18 Following this , various with modifications at the phenyl ring have been synthesized and shown to have comparable or improved activity to the parent drug ( Table 4. 1 ). 3 , 11 , 19 - 22 Most of these analog s have sho wn promise in treatment of drug - resistant tumor cells . For example, O rtataxel (Spectrum Pharmaceuticals), 23 currently in phase 2 clinical trials is active agai n st breast and non small cell lung cancer resist ant to paclitaxel and docetaxel . 23 Tesetaxel, an oral taxane , currently in phase 1 clinical trials is used in combination with the antineoplastic pharmaceutical C apecitabine in patients with solid tum ors 24 ( Figure 4. 2 ) . Table 4.1 . Cytotoxic activity of paclitaxel analogs in microtubule assembly and against B16 melanoma cells. Microtubule assembly ED 50 /ED 50 (Paclitaxel) Inhibition of B16 melanoma cells proliferation ED 50 /ED 50 (Paclitaxel) Microtubule assembly ED 50 /ED 50 (Paclitaxel) Inhibition of B16 melanoma cells proliferation ED 50 /ED 50 (Paclitaxel) D 2 1.0 1.0 D 6 0.7 0.8 D 3 0.5 1.0 D 7 0.9 0.3 D 4 0.4 1.3 D 8 0.9 3.3 D 5 0.4 27 D 9 0.2 - D 10 ED 50 /ED 50 (Taxol ® ) cytotoxicity against HCTVM46 human colon carcinoma cells (0.2) D 11 microtubule disassembly ED 50 /ED 50 (Taxol ® ) (0.8) 149 Figure 4. 2 . Some of the p aclitaxel analogs with modification at C 13 . D 1 2 , D 1 3 , D 1 5 and D 1 7 are currently in clinical trials. 4.1.2. Sources of Paclitaxel: A n Historical Time Line P aclitaxel was isolated initially from the bark of Pacific Y ew ( Taxus brevifolia ) at low yield ( 0.02% w/w ) . 25 This unsustainable meth od was succeeded by semi synthesis 26 that supplied paclitaxel for commercial distrib ution by Bristol Myers Squibb . 11 The semi synthetic method employed a phenylisoserine precursor ( a lactam synthesized through six steps), which was coupled to the C 13 hydroxy l of deacetylbaccatin III. 27 This strategy needed large volumes of hazardous solvents and reagents , and implemented redundant protection/deprotection steps. 28 Bristol Myers Squibb abandoned the semisynthetic method and adopted an environmentally conscience Taxus pla nt cell fermentation process. 29 While this greener biological method of pro duction produces paclitaxel at 500 kg/ year by Phyton Pharmaceutical, the plant cells h ave competing pathways that divert part of the metabolic flux from paclitaxel ( Figure 4. 3 ) . 30 To address these challenges, a sustainable biolo gical method can be envisioned that directs carbon 150 flux from an abundant pathway intermediate to one desired end product, such as paclitaxel or an an a log ( Figure 4. 4 ) . Figure 4. 3 . The biosynthesis of paclitaxel in plants starting from geranylgeranyl diphosphate (GGDP) . The different competing pathways are also shown. 151 Figure 4.4. The proposed biosynthesis of paclitaxel through coupling (2 R ,3 S ) phenylisoserine to baccatin III by 13 O phenylpropanoyltransferase (BAPT) , an acyl CoA dependent enzyme. 4.1.3. Cataly tic Activity of Baccatin III 13 O 3 Amino 3 Phenylpropanoyltransferase (BAPT) w ith Amino acyl CoA Thioesters To develop a biosynthetic approach, enzymes on the paclitaxel pathway have been identified and characterized . 2 In partic ular, acyl CoA - dependent 13 O phenyl propanoyltransferase (BAPT) regioselectively phenylpropanoylates the C 13 hydroxy l of baccatin III . 31 An N benzoyl transferase (NDTBT) N benzoylates the phenylisoserin yl side chain using the appropriate acyl CoA subst rates ( Figure 4. 4 ) . In an earlier study, the molecular cloning and heterologous expression of BAPT was described and found to be regioselective . 32 Moreover, the apparent K M values of BAPT were 2.4 phenylalanyl CoA , respectively. 32 Also, t he catalytic turn over of BAPT with 3 phenylisoserinyl CoA was ~ 2.5 times s lower compared to phe nylalanyl CoA , and no activity was observed with phenylalanyl CoA or N b enzoylp henylisoserinyl CoA. 32 The synthesis of the aminoacyl CoA thioesters reported in the earlier study involve d eight steps requir ing protection/deprotection of the amine and hydroxy l 152 functional group s (see Figure 1.8 in Chapter 1). Additionally, this method wa s complicated by solvent incompatibility of the hydrophobic acid anhydride inte rmediate and the hydrophilic CoA reactant . 32 The broad substrate scope of the acyltransferases has been reported in earlier studies. 33 - 35 For example, both BAPT and NDTBT showed specificity for various aroyl, heteroaroyl, alkanoyl, and alkenoyl CoA thioesters. 33 , 35 B APT is hypothesized to catalyz e the transfer of various isoserinyl analog s to the C 13 hydroxyl of baccatin III . This catalysis provides a direct route to a precursor of a paclitaxel analog. In addition, it replaces the multistep semisynthesis of paclitaxel analog s ( Figure 4. 5 ) and thus eliminate s the use of environmental harmful solvents and reagents . 27 153 Figure 4. 5 . A representative 11 step semisynthe sis of paclitaxel analog s (i) LDA, - 78 ° C, (ii) a) Bu 4 N - F + , b) ethyl vinyl ether , H + , c) BzCl, Et 3 N, DMAP, (iii) pyridine, DMAP, (iv) 0.5 M HCl, EtOH . 4.1.4. Tyrocidine Synthetase A Tyc( Phe AT ) C atalysis in B iosynthesis of Isoserinyl CoA Analogs Chapter 3 describes the use of the Tyc(Phe AT) didomain as a CoA ligase that biocatalyzed (2 S ) , (3 R ) phenylalanyl CoA, and (2 R ,3 S ) phenylisoserinyl CoA from the corresponding amino acids. Thus, Tyc(Phe AT) could help by - pass lengthy chemical synthesis of arylisoserinyl CoA thioesters for use by acyl CoA dependent acyltransferases ( Figure 4.6 ) . 154 A ) B ) Figure 4.6 . A) Synthesis of (2 R ,3 S ) - phenylisoserinyl CoA via the mixed anhydride intermediate . (i) CH 2 Cl 2 /THF, DMAP in CH 2 Cl 2 , benzyl chloroformate, rt, 1 h, 90% yield; (ii) CH 3 CN, DMAP in CH 3 CN, Boc 2 O, rt, 24 h, 20% yield; (iii) CH 3 OH, 6% Mg(OCH 3 ) 2 , rt, 1 h, 80% yield; (iv) step (ii), 80% yield; (v) 2 M NaOH, 12 h; (vi) THF, ethyl chloroformate, rt, 1 h; (vii) CoASH in 0.4 M NaHCO 3 , t - BuOH, rt, 0.5 h; (viii) HCOOH, rt, 10% yield. Synthe - - phenylalanyl CoA were carried out similarly except for the 2 - hydroxyl protection. B ) The proposed biosynthesis of isoserinyl CoA thioesters. An earlier study described the wide range of adenylation domain of Tyc( Phe ATE ) for natural and unnatural amino acid substrates, which differed in their electronic properties and size. 36 The earlier study, however, only evaluated the ability of Tyc( Phe A ) to form an adenylate 155 anhydride intermediate through an indirect ATP/[ 32 P]PPi exchange experiment . This earlier work, showing adenylation of various substrates together with our discovery of the CoA ligase function of Tyc(Phe AT) prompted us to investigate the didomain catalyst for the production of isoserinyl CoA thioesters 36 , 37 ( Figure 4. 7 ). A ) B ) Other amino acid substrates Figure 4. 7 . A ) The reversible adenylation of ( R / S ) phenyl alanine by Tyc( Phe A ) domain through ATP hydrolysis forming the ( R / S ) phenyl alanyl adenylate. B ) Natural and unnatural amino acids that are substrates of TycA. The rates of adenylation ( k cat ) by Tyc(Phe A) are shown in parentheses. The substrates whose rates of adenylation are not indicated had less than 20% activity of the ( R / S ) phenylalanine substrate . 156 Figure 4. 7 . 4.2. Experimental 4.2.1. Substrates, Reagents, and General Instrumentation The aryl and non aryl carboxaldehydes, acetoxyacetyl chloride, and p - methoxyaniline (also known as p - anisidine ) were purchased from Sigma Aldrich and were used without further purification, unless noted otherwise . Triethylamine was obtained from J. T. Baker chemicals, Phillipsburg, NJ. A V arian Inova - 300 or a V arian UnityPlus500 instrument was used to acquire 1 H - and 13 C - NMR . A Q - Tof Ultima electrospray ionization tandem mass spectrometer (ESI MS /MS , Waters, Milford, MA) with a Waters 2795 HPLC was used for mass spectr um analysis. The biosynthetic products were separated a nd analyzed using Acquity ® UPLC system fitted with a C18 Ascentis Express column (2.5 × 50 mm, 2.7 µm) on a Quattro Premier XE Mass 157 Spectrometer . Thin layer chromatography (TLC) plates and flash column chromatography Silica gel were purchased from EMD Chemicals Inc. (Gibbstown, NJ). 4.2.2. Synthesis of Isoserine Analogs 4.2.2.1. General Procedure 1 : Synthesis of N ( p - Methoxyphenyl)benzyli mines Figure 4. 8 . Synthesis of N - protected imines from benzaldehyde analogs and p - anisidine . To a n aldehyde solution ( 1 equiv) dissolved in benzene was added p - methoxyaniline (2.5 equiv ). Oven dried molecular sieves (~1.5 g) were added to remove water formed during the reaction. The reaction was sti rred at room temperature for 12 h , then filtered, dried (MgSO 4 ), and concentrated under vacuum. The crude mixture was purified by silica gel column 158 chromatography (1:4 EtOAc/hexane , v/v), and the fract ions containing the produ ct were combined and dried u nder vacuum to obtain the imine . The non - aryl imines (3 - trimethyl, 3 - cyclohexyl, 3 - thiophenyl, and 3 - dimethyl - N - ( p - Methoxyphenyl) - imine ) were not stable and hence not isolated. The lactams of these imine s were synthesized separately in one pot as described in general P rocedure 2, M ethod B ( Section 4.2.2.2) . p - Methoxyphenyl is abbreviated as PMP in the procedures described herein. N - ( p - Methoxyphenyl) - 3 - benzylimine (D 49a). B enzaldehyde (0.5 g, 4.72 mmol) and p - methoxyaniline ( 0.87 g, 7.08 mmol, 1.5 equiv) were treated according to Procedure 1 ( Section 4.2.2.1) to afford the imine (897 mg, 83 % yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 3.84 (s, PMP O CH 3 ), 6.91 7.93 ( aromatic protons), 8.49 ( s, imine CH ). N - ( p - Methoxyphenyl) - 3 - (4 - F luoro) - benzylimine (D 49b). p - F luorobenzaldehyde (0.5 g, 4.06 mmol, 1 equiv) and p - methoxyaniline (1.25 g, 10.16 mmol, 2.5 equiv) were treated according to Procedure 1 ( Section 4.2.2.1) to afford the imine (464 mg, 50% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 3.84 (s, PMP O CH 3 ), 6.92 7.89 ( aromatic protons), 8.45 ( s, imine CH ). 13 C - NMR (126 MHz, CDCl 3 - d , 156.78, 144.20, 130.53, 130.47, 122.13, 115.94, 115.76, 114.40, 55.48 . N - ( p - Methoxyphenyl) - 3 - (4 - C hloro) - benzylimine (D 49c). p - C hlorobenzaldehyde (0.45 g, 3.25 mmol, 1 equiv) and p - methoxyaniline (1 g, 8.13 mmol, 2.5 equiv) were treated according to Procedure 1 ( Section 4.2.2.1) to afford the imine (670 mg, 84% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 3.84 (s, PMP O CH 3 ), 6.94 7.82 ( aromatic protons ), 8.44 ( s, imine CH ). 159 N - ( p - Methoxyphenyl) - 3 - (4 - B romo) - benzylimine (D 49d) . p - B romobenzaldehyde (0.60 g, 3.25 mmol, 1 equiv) and p - methoxyaniline (1 g, 8.13 mmol, 2.5 equiv) were treated according to Procedure 1 ( Section 4.2.2.1) to afford the imine (500 mg, 53% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) : 3.85 (s, PMP O CH 3 ) , 6.95 7.77 ( aromatic protons ) , 8.45 ( s, imine CH ). 13 C - NMR (126 MHz, CDCl 3 - d 144.42, 135.39, 131.96, 129.91, 125.41, 122.24, 114.44, 55.50 . N - ( p - Methoxyphenyl) - 3 - (4 - Me thyl ) - benzylimine (D 49e) . p - T olualdehyde (1 g, 8.3 mmol, 1 equiv) and p - methoxyaniline (2.6 g, 20.8 mmol, 2.5 equiv) were treated according to Procedure 1 ( Section 4.2.2.1) to afford the imine (1.1 g, 57% yield at >98% purity by 1 H - NMR analysis ). 1 H - NMR (CDCl 3 - d , 500 MHz) 2.41 (s, p - CH 3 ) 3.83 (s, PMP O CH 3 ) , 6.94 7.81 (aromatic protons ) , 8.44 ( s, imine CH ). 13 C - NMR (126 MHz, CDCl 3 - d , 158.10, 145.12 , 141.47 , 133.88, 129.48, 128.58, 122.15, 114.35, 55.50, 21.63. N - ( p - Methoxyphenyl) - 3 - (4 - Methoxy) - benzylimine (D 49f). p - M ethoxybenzaldehyde ( 0.44 g, 3.25 mmol, 1 equiv) and p - methoxyaniline (1 g, 8.13 mmol, 2.5 equiv) were treated according to Procedure 1 ( Section 4.2.2.1) to afford the imine (310 mg, 40% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 3.83 (s, p - OCH 3 ) , 3.87 (s, PMP O CH 3 ) , 6.90 7.84 (aromatic protons ) , 8.41 ( s, imine CH ). 13 C - NMR (126 MHz, CDCl 3 - d 161.99 , 157.93 , 152.78, 145.27, 130.26, 129.48, 122.08, 116.41, 114.80, 55.50, 55.42 . N - ( p - Methoxyphenyl) - 3 - (4 - Acetoxy) - benzylimine (D 49g). p - A cetoxybenzaldehyde (0.5 g, 3.05 mmol, 1 equiv) and p - methoxyaniline ( 0.93 g, 7.6 mmol, 2.5 equiv) were treated according to Procedure 1 ( Section 4.2.2.1) to afford the imine (540 mg, 66% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 2.33 (s, OA c CH 3 ) , 3.84 (s, PMP O CH 3 ) , 6.94 7.92 ( aromatic protons ) , 8.47 ( s, imine CH ). 13 C - NMR (126 MHz, CDCl 3 - 160 d , 1 58.32, 157.05 , 152.69, 144.71, 134.16, 129.71, 122.17, 121.96, 114.37, 55.50, 21.18. N - ( p - Methoxyphenyl) - 3 - (4 - Nitro) - benzylimine (D 49h). p - N itrobenzaldehyde (1 g, 6.62 mmol, 1 equiv) and p - methoxyaniline (2.04 g, 16.56 mmol, 2.5 equiv) were treated according to Procedure 1 ( Section 4.2.2.1) to afford the imine (930 mg, 55% yield, at >98% puri ty by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 3.87 (s, PMP O CH 3 ) , 6.98 8.33 ( aromatic protons) , 8.60 ( s, imine CH ). 13 C - NMR (126 MHz, CDCl 3 - d , 149.01 , 143.60, 141.95 , 129.06, 123.97, 122.59, 114.54, 55.52. N - ( p - Methoxyphenyl) - 3 - (3 - Bromo) - benzylimine (D 49i). m - B romobenzaldehyde (1 g, 7.14 mmol, 1 equiv) and p - methoxyaniline (2.2 g, 17.85 mmol, 2.5 equiv) were treated according to Procedure 1 ( Section 4.2.2.1) to afford the imine (1.1 g, 54% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 3.84 (s, PMP O CH 3 ) , 6.93 8.09 (aromatic protons ) , 8.42 (s, imine C H). 13 C - NMR (126 MHz, CDCl 3 - d , 156.31, 144.2 , 138.44 , 133.77, 131.03, 130.22, 127.34, 123.02, 122.30, 114.41, 55.50. N - ( p - Methoxyphenyl) - 3 - (3 - A cetyl) - benzylimine (D 49j). m - A cetoxybenzaldehyde (1 g, 6.10 mmol, 1 equiv) and p - methoxyaniline (1.88 g, 15.25 mmol, 2.5 equiv) were treated according to Procedure 1 ( Section 4.2.2.1) to afford the imine (965 mg, 59 % yield, at > 98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 2.34 (s, p OAc CH 3 ), 3.85 (s, PMP O CH 3 ), 6.93 7.73 ( aromatic protons ), 8.47 (s, imine C H). 13 C - NMR (126 MHz, CDCl 3 - d 158.47 , 157.02, 151.08, 144.44, 138.06, 129.72, 126.38, 124.19, 122.26, 121.10 , 114.40 , 55.51 , 21.14. N - ( p - Methoxyphenyl) - 3 - (2 - Methyl) - benzylimine (D 49k). o - M ethylbenzaldehyde (1 g, 8.3 mmol, 1 equiv) and p - methoxyaniline (2.56 g, 20.75 mmol, 2.5 equiv) were treated according 161 to Procedure 1 ( Section 4.2.2.1) to afford the imine (1.3 g, 69 % yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 2.59 (s, o - CH 3 ), 3.84 (s, PMP O CH 3 ), 6.92 8.08 ( aromatic protons ), 8.77 (s, imine C H). 13 C - NMR (126 MHz, CDCl 3 - d , 157.17, 134.31, 130.92, 130.69, 127.49, 126.33 , 122.17, 114.35, 55.52 , 19.40 . N - ( p - Methoxyphenyl) - 3 - (2 - Methoxy) - benzylimine (D 49l). o - M ethoxybenzaldehyde (1 g, 7.35 mmol, 1 equiv) and p - methoxyaniline (2.71 g, 18.38 mmol, 2.5 equiv) were treated according to Procedure 1 ( Section 4.2.2.1) to afford the imine (1.14 g, 64 % yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 3.83 (s, PMP O CH 3 ), 3.90 (s, o - OCH 3 ), 6.91 8.16 (aromatic protons ), 8.94 (s, imine C H). 13 C - NMR (126 MHz, CDCl 3 - d 159.33 , 158.09 , 154.52, 145.68, 132.37, 127.33, 124.97, 122.39, 120.88, 114.30, 111.08, 55.55, 55.49. 4.2.2.2. General P rocedure 2: Synthesis of 2 - A zetidinones Figure 4. 9 . Scheme showing the general synthesi s of 2 - azetidinones . TEA is tri ethylamine. 162 Figure 4.9 . Method A: To a solution of imine (1 equiv) dissolved in dichloromethane (18 mL) at 0 ° C was added triethylamine (3 equiv). A solution of acetoxyacetyl chloride (2 equiv) in dichloromethane (8 mL) was then added dropwise and the reaction stirred at 0 ° C for an additional 5 min. The 163 mixture was warmed to room temperature and stirred for 2 5 h to complete the reaction. The solution was washe d successively with 5% (w/v) NaHCO 3 (15 mL), 5% v/v HCl (15 mL), and water (3 × 15 mL). The organic fraction was dried (MgSO 4 ) and concentrated under vacuum. The crude mixture was then purified by silica gel chromatography (1:4 EtOAc/hexane, v/v) to yield the desired 2 - azetidinones. Method B: The aldehyde (1 equiv) was dissolved in dichloromethane and p - methoxyaniline (1.1 equiv) was then added. The reaction was stirred at room temperature until the disappearance of starting material (the reaction progress was followed by TLC ( 1:4 EtOAc/hexane, v/v ). The molecular sieves were removed by filtration and the filtrate transferred to a clean , oven dried round bottomed flask, which was then sealed using a rubber septum. To this crude imine mixture, triethylamine was added (3 equiv) and stirred at 0 ° C. Acetoxyacetylchloride (2 equiv ) was separately dissolved in dichloromethane and the solution was added slowly to the reaction mixture. T he reaction was stirred at 0 ° C for additional 5 min, and then warmed up to room temperature. Once the reaction was complete (2 3 h), the solution was washed successively wi th 5% (w/v) NaHCO 3 (15 mL), 5% v/v HCl (15 mL), and water (3 × 15 mL). The organic fraction was dried (MgSO 4 ) and concentrated under vac uum. The crude mixture was purified by silica gel chromatography (1:4 EtOAc/hexane, v/v) to yield the r espective 2 - azetidinone. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 phenyl azetidin - 2 - one (D 50a). N - PMP - 3 - benzylimine ( 0.2 g, 0.95 mmol, 1 equiv) , triethylamine (2.85 mmol, 3 equiv), and acetoxyacetyl chloride (1.9 mmol, 2 equiv) were treated according to Procedure 2, Method A ( Section 4.2.2.2) to afford the lactam (108 mg, 37% yield, at >95% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.68 (s, acetyl CH 3 ), 3.76 (s, PMP OCH 3 ), 5.35 (d, J = 4.89 Hz, H - 3), 5.95 164 (d, J = 4.89 Hz, H - 2), 6.80 7.37 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 161.29, 156.62, 132.27, 130.28, 128.77, 128.46, 127.90, 118.81, 114.41, 76.36, 61.47, 55.43, 19.77. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (4 - fluorophenyl)azetidin - 2 - one (D 50b). N - PMP - 3(4 - F) - benzylimine (0.26 g, 1.15 mmol, 1 equiv), triethylamine (3.45 mmol, 3 equiv), and acetoxyacetyl chloride (2.3 mmol, 2 equiv) were treated according to Procedure 2, Method A (Section 4.2.2.2) to afford the lactam (115 mg, 40% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.73 (s, acetyl CH 3 ), 3.76 (s, PMP OCH 3 ), 5.33 (d, J = 4.89 Hz, H - 3), 5.91 (d, J = 4.89 Hz, H - 2), 6.80 7.31 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 76.84, 60.90, 55.55, 19.91. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (4 - chlorophenyl)azetidin - 2 - one (D 50c). N - PMP - 3(4 - Cl) - benzylimine (0.5 g, 2.04 mmol, 1 equiv), triethylamine (6.12 mmol, 3 equiv), and acetoxyacetyl chloride (4.08 mmol, 2 equiv) were treated according to Proce dure 2, Method A (Section 4.2.2.2) to afford the lactam (629 mg, 84% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.74 (s, acetyl CH 3 ), 3.76 (s, PMP OCH 3 ), 5.31 (d, J = 4.90 Hz, H - 3), 5.92 (d, J = 4.89 Hz, H - 2), 6.79 7.35 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 76.32, 60.88, 55.44, 19.88. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (4 - bromophenyl)azetidin - 2 - one (D 50d ). N - PMP - 3(4 - Br) - benzylimine (0.59 g, 2.06 mmol, 1 equiv), triethylamine (6.18 mmol, 3 equiv), and acetoxyacetyl chloride (4.12 mmol, 2 equiv) were treated according to Procedure 2, Method A (Section 4.2.2.2) to afford the lactam (640 mg, 95% yield, at >9 8% purity by 1 H - NMR analysis). 165 1 H - NMR (500 MHz, CDCl 3 - d ) 1.75 (s, acetyl CH 3 ), 3.76 (s, PMP OCH 3 ), 5.31 (d, J = 4.89 Hz, H - 3), 5.93 (d, J = 4.89 Hz, H - 2), 6.82 7.51 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 131.50, 130.00, 129.57, 122.93, 118.73, 114.49, 76.26, 60.95, 55.44, 19.89. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (4 - methylphenyl)azetidin - 2 - one (D 50e). N - PMP - 3(4 - Me) - benzylimine (0.5 g, 2.2 mmol, 1 equiv), triethylamine (6.6 mmol, 3 equiv), and acetoxyacetyl c hloride (4.4 mmol, 2 equiv) were treated according to Procedure 2, Method A (Section 4.2.2.2) to afford the lactam (369 mg, 51% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.71 (s, acetyl CH 3 ), 2.35 (s, p - CH 3 ), 3.76 (s, PMP OCH 3 ), 5.31 (d, J = 4.90 Hz, H - 3), 5.92 (d, J = 4.89 Hz, H - 2), 6.79 7.31 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 114.37, 76.38, 61.38, 55.44, 21.23, 19.92. N - ( p - Methoxyphen yl) - 3 - acetoxy - 4 - (4 - methoxyphenyl)azetidin - 2 - one (D 50f). N - PMP - 3(4 - OMe) - benzylimine (0.25 g, 1.04 mmol, 1 equiv), triethylamine (3.12 mmol, 3 equiv) and acetoxyacetyl chloride (2.08 mmol, 2 equiv) were treated according to Procedure 2, Method A (Section 4.2.2.2) to afford the lactam (279 mg, 79% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.73 (s, acetyl CH 3 ), 3.75 (s, PMP OCH 3 ), 3.80 (s, p - OCH 3 ), 5.29 (d, J = 4.90 Hz, H - 3), 5.89 (d, J = 4.89 Hz, H - 2), 6.78 7.30 (aromatic p rotons). 13 C - NMR (126 MHz, CDCl 3 - d 118.85, 114.37, 113.90, 76.50, 61.15, 55.44, 55.27, 19.94. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (4 - acetoxy phenyl)azetidin - 2 - one (D 50g). N - PMP - 3(4 - OAc) - benzylimine (0.54 g, 2.0 mmol, 1 equiv), triethylamine (5.96 mmol, 3 equiv), and acetoxyacetyl chloride (3.98 mmol, 2 equiv) were treated according to Procedure 2, Method A 166 (Section 4.2.2.2) to afford the lactam (692 mg, 94% yield, at >95% pu rity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.71 (s, acetyl CH 3 ) 2.29 (s, p - OAC CH 3 ), 3.76 (s, PMP OCH 3 ), 5.35 (d, J = 4.89 Hz, H - 3), 5.93 (d, J = 4.89 Hz, H - 2), 6.82 7.33 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 161.15, 156.89, 150.98, 130.09, 129.78, 128.94, 121.77, 118.81, 114.47, 76.36, 60.98, 55.45, 21.11, 19.80. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (4 - nitro phenyl)azetidin - 2 - one (D 50h). N - PMP - 3(4 - nitro) - benzylimine (0.93 g, 3.63 mmol, 1 equiv), triethylamine (10.89 mmol, 3 equiv) and acetoxyacetyl chloride (7.29 mmol, 2 equiv) were treated according to Procedure 2, Method A (Section 4.2.2.2) to afford the lactam ( 1.1 g, 82% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.74 (s, acetyl CH 3 ), 3.76 (s, PMP OCH 3 ), 5.45 (d, J = 4.89 Hz, H - 3), 6.00 (d, J = 4.89 Hz, H - 2), 6.81 8.25 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 161.41, 156.94, 148.19, 140.01, 129.66, 128.88, 123.72, 118.62, 114.61, 76.41, 60.66, 55.47, 19.90. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (3 - fluoro phenyl)azetidin - 2 - one (D 50i). 3 - Fluoro - benzaldehyde (1 g, 8.07 mmol, 1 equiv), p - methoxyaniline (1.49 g, 12.10 mmol, 1.5 equiv), triethylamine (24.2 mmol, 3 equiv) and acetoxyacetyl chloride (16.13 mmol, 2 equiv) were treated according to Procedure 2, Method B (Section 4.2.2.2) to afford the lactam (1.3 g, 51% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.74 (s, acetyl CH 3 ), 3.76 (s, PMP OCH 3 ), 5.33 (d, J = 4.89 Hz, H - 3), 5.95 (d, J = 4.89 Hz, H - 2), 6.80 7.33 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 130.24, 130.00, 123.67, 118.72, 115.96, 114.95, 114.48, 76.26, 60.84, 55.44, 19.85. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (3 - chloro phenyl)azet idin - 2 - one (D 50j). 3 - Chloro - benzaldehyde (1 g, 7.1 mmol, 1 equiv), p - methoxyaniline (1 .31 g, 10.67 mmol, 1.5 equiv), 167 triethylamine (21.34 mmol, 3 equiv) and acetoxyacetyl chloride (14.23 mmol, 2 equiv) were treated according to Procedure 2, Method B (Section 4.2.2.2) to afford the lactam (1.1 g, 47% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.74 (s, acetyl CH 3 ), 3.76 (s, PMP OCH 3 ), 5.30 (d, J = 4.89 Hz, H - 3), 5.94 (d, J = 4.89 Hz, H - 2), 6.80 7.34 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 129.83, 129.10, 127.93, 126.12, 118.72, 114.50, 76.25, 60.85, 55.45, 19.87. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (3 - bromo phenyl)azetidin - 2 - one (D 50k). N - PMP - 3(3 - bromo) - benzylimine (1.11 g, 3.85 mmol, 1 equiv), triethylamine (11.54 mmol, 3 equiv), and acetoxyacetyl chloride (7.7 mmol, 2 equiv) were treated according to Procedure 2, Method A (Section 4.2.2.2) to afford the lactam (500 mg, 33% yield, at >97% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.76 (s, acetyl CH 3 ), 3.77 (s, PMP OCH 3 ), 5.30 (d, J = 4.89 Hz, H - 3), 5.95 (d, J = 4.89 Hz, H - 2), 6.82 7.51 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 134.86, 132.06, 130.85, 130.10, 129.98, 126.61, 122.61, 118.73, 114.52, 76.27, 60.81, 55.47, 19.88. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (3 - methyl phenyl)azetidin - 2 - one (D 50 l ). 3 - Methylbenzaldehyde (0.5 g, 4.17 mmol, 1 equiv), p - methoxyaniline (0.77 g, 6.26 mmol, 1.5 equiv), triethylamine (12.51 mmol, 3 equiv) and acetoxyacety l chloride (8.34 mmol, 2 equiv) were treated according to Procedure 2, Method B (Section 4.2.2.2) to afford the lactam (760 mg, 81% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.70 (s, acetyl CH 3 ), 2.34 (s, m - CH 3 ), 3.77 (s, PMP OCH 3 ), 5.30 (d, J = 4.89 Hz, H - 3), 5.94 (d, J = 4.89 Hz, H - 2), 6.80 7.32 (aromatic protons). N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (3 - methoxy phenyl)azetidin - 2 - one (D 50m). 3 - Methoxybenzaldehyde (1 g, 7.35 mmol, 1 equiv), p - methoxyaniline (1.36 g, 11.03 mmol, 1.5 168 equiv), triethylamine (22.05 mmol, 3 equiv) and acetoxyacetyl chloride (14.7 mmol, 2 equiv) were treated according to Procedure 2, Method B (Section 4.2.2.2) to afford the lactam (1.6 g, 66% yield, at >95% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.73 (s, acetyl CH 3 ), 3.76 (s, m - OCH 3 ), 3.77 (s, PMP OCH 3 ), 5.30 (d, J = 4.89 Hz, H - 3), 5.95 (d, J = 4.89 Hz, H - 2), 6.79 7.30 (aromatic protons). N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (3 - acetoxy phenyl)azetidin - 2 - one (D 50n). N - PMP - 3 - (3 - OAc) - benzylimine (0.97 g, 3.59 mmol, 1 equiv), triethylamine (10.77 mmol, 3 equiv), and acetoxyacetyl chloride (7.18 mmol, 2 equiv) were treated according to Procedure 2, Metho d A (Section 4.2.2.2) to afford the lactam (1.05 g, 79% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.74 (s, acetyl CH 3 ), 2.27 (s, m - OAc CH 3 ), 3.76 (s, PMP OCH 3 ), 5.33 (d, J = 4.89 Hz, H - 3), 5.94 (d, J = 4.89 Hz, H - 2), 6.80 7.38 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 129.56, 125.41, 122.29, 121.09, 118.77, 114.45, 76.27, 61.00, 55.44, 21.07, 19.78. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (3 - nitro phenyl)azetidin - 2 - one (D 50o). 3 - Nitro - benzaldehyde (1 g, 6.62 mmol, 1 equiv), p - methoxyaniline (1.22 g, 9.93 mmol, 1.5 equiv), triethylamine (19.86 mmol, 3 equiv) and acetoxyacetyl chloride (13.24 mmol, 2 equiv) were treated according to Procedure 2 , Method B (Section 4.2.2.2) to afford the lactam (588 mg, 25% yield, at >95% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.73 (s, acetyl CH 3 ), 3.76 (s, PMP OCH 3 ), 5.45 (d, J = 4.89 Hz, H - 3), 5.99 (d, J = 4.89 Hz, H - 2), 6.80 8.25 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 133.84, 129.67, 129.64, 123.96, 123.01, 118.68, 114.64, 76.37, 60.62, 55.49 19.87. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (2 - fluoro phenyl)azet idin - 2 - one (D 50p). 2 - Fluoro - benzaldehyde (1 g, 8.07 mmol, 1 equiv), p - methoxyaniline (1.49 g, 12.1 mmol, 1.5 equiv), 169 triethylamine (24.2 mmol, 3 equiv), and acetoxyacetyl chloride (16.13 mmol, 2 equiv) were treated according to Procedure 2, Method B (Section 4.2.2.2) to afford the lactam ( 1.1 g, 41% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.76 (s, acetyl CH 3 ), 3.76 (s, PMP OCH 3 ), 5.67 (d, J = 4.89 Hz, H - 3), 6.02 (d, J = 4.89 Hz, H - 2), 6.80 7.36 (aromatic protons). 1 3 C - NMR (126 MHz, CDCl 3 - d 130.06, 129.05, 124.09, 119.80, 118.54, 115.77, 114.41, 75.98, 63.00, 55.32, 19.72. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (2 - chloro phenyl)azetidin - 2 - one (D 50q). 2 - Chlorobenzaldehyde (1 g, 7.11 mmol, 1 equiv), p - methoxyaniline (1.31 g, 10.67 mmol, 1.5 equiv), triethylamine (21.34 mmol, 3 equiv), and acetoxyacetyl chloride (14.23 mmol, 2 equiv) were treated according to Procedure 2, Method B (Section 4.2.2.2) to affor d the lactam (2.0 g, 80% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.76 (s, acetyl CH 3 ), 3.77 (s, PMP OCH 3 ), 5.79 (d, J = 4.89 Hz, H - 3), 6.17 (d, J = 4.89 Hz, H - 2), 6.81 7.45 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 130.16, 130.04, 129.84, 128.72, 126.77, 118.66, 114.49, 75.45, 58.22, 55.45, 19.94. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (2 - bromo phenyl)azetidin - 2 - one (D 50r). 2 - Bromobenzaldehyde (1 g, 5.41 mmol, 1 equiv), p - methoxyaniline (1.0 g, mmol, 1.5 equiv), triethylamine (16.22 mmol, 3 equiv) and acetoxyacetyl chloride (10.81 mmol, 2 equiv) were treated according to Procedure 2, Method B (Section 4.2.2.2) to afford the lactam (1.2 g, 59% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.77 (s, acetyl CH 3 ), 3.76 (s, PMP OCH 3 ), 5.76 (d, J = 4.89 Hz, H - 3), 6.19 (d, J = 4.89 Hz, H - 2), 6.81 7.64 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 130.11, 128.88, 127.37, 122 .55, 118.67, 114.50, 75.35, 60.64, 55.45 19.98. 170 N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (2 - methyl phenyl)azetidin - 2 - one (D 50s). N - PMP - 3(2 - Me) - benzylimine (1.28 g, 5.69 mmol, 1 equiv), triethylamine (17.08 mmol, 3 equiv) and acetoxyacetyl chloride (11.39 mmol, 2 equiv) were treated according to Procedure 2, Method A (Section 4.2.2.2) to afford the lactam (1.4 g, 75% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.68 (s, acetyl CH 3 ), 2.43 (s, o - CH 3 ), 3.77 (s, PMP OCH 3 ), 5.55 (d, J = 4.89 Hz, H - 3), 6.01 (d, J = 4.89 Hz, H - 2), 6.81 7.27 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d , 156.54, 136.74, 130.71, 130.29, 129.97, 128.46, 126.98, 125.87, 118.76, 114.39, 76.05, 58.26, 55.38, 19.74, 19.06. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (2 - methoxy phenyl)azetidin - 2 - one (D 50t). N - PMP - 3 - (2 - OMe) - benzylimine (1.14 g, 4.72 mmol, 1 equiv), triethylamine (14.16 mmol, 3 equiv) and acetoxyacetyl chloride (9.44 mmol, 2 equiv) were treated according to Procedure 2, Method A (Section 4.2.2.2) to afford the lactam (1.3 g, 84% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.71 (s, acetyl CH 3 ), 3.75 (s, PMP OCH 3 ), 3.85 (s, o - OCH 3 ), 5.73 (d, J = 4.89 Hz, H - 3), 6.06 (d, J = 4.89 Hz, H - 2), 6.79 7.32 (aromatic protons). N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - (2 - nitro phenyl)azetidin - 2 - one (D 50u) . 2 - Nitro - benzaldehyde (1 g, 6.62 mmol, 1 equiv), p - methoxyaniline (0.82 g, 6.62 mmol, 1.5 equiv), triethylamine (19.87 mmol, 3 equiv) and acetoxyacetyl chloride (13.25 mmol, 2 equiv) were treated according to Procedure 2, Method B (Section 4.2.2.2) to afford the lactam (1.1 g, 47% yield, at >95% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.77 (s, acetyl CH 3 ), 3.78 (s, PMP OCH 3 ), 6.06 (d, J = 5.38 Hz, H - 3), 6.36 (d, J = 5.38 Hz, H - 2), 6.84 8.22 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 130.04, 129.50, 129.32, 129.25, 125.44, 118.58, 114.64, 75.89, 60.18, 55.51, 19.97. 171 N - ( p - Methoxyphenyl) - 3 - ac etoxy - 4 - trimethylazetidin - 2 - one (D 50v). Trimethyl - acetaldehyde (1 g, 11.61 mmol, 1 equiv), p - methoxyaniline (1.43 g, 11.61 mmol, 1.0 equiv), triethylamine (26.13 mmol, 3 equiv), and acetoxyacetyl chloride (17.42 mmol, 2 equiv) were tre ated according to Procedure 2, Method B (Section 4.2.2.2) to afford the lactam (0.9 g, 35% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.00 (s, t - butyl (CH 3 ) 3 ), 2.18 (s, acetyl CH 3 ), 3.79 (s, PMP OCH 3 ), 4.23 (d, J = 5.38 Hz, H - 3), 6.15 (d, J = 5.38 Hz, H - 2), 6.88 7.29 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 157.15, 129.94, 121.99, 114.24, 73.46, 66.89, 55.47, 34.77, 27.02, 20.92. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - cyclohexylazetidin - 2 - one ( D 50w). Cyclohexyl - acetaldehyde (1 g, 8.92 mmol, 1 equiv), p - methoxyaniline (2.74 g, 22.29 mmol, 1.5 equiv), triethylamine (13.8 mmol, 3 equiv) and acetoxyacetyl chloride (9.2 mmol, 2 equiv) were treated according to Procedure 2, Method B (Section 4.2.2.2) to afford the lactam (223 mg, 70% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 0.96 1.78 (cyclohexyl protons), 1.85 (dd, J = 6.36, 2.93 Hz, cyclohexyl CH), 2.20 (s, acetyl CH 3 ), 3.81 (s, PMP OCH 3 ), 4.20 (t, J = 5.87 Hz, H - 3) , 6.05 (d, J = 5.38 Hz, H - 2), 6.87 7.37 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 55.47, 38.59, 29.78, 29.64, 26.13, 20.72. N - ( p - Methoxyphenyl) - 3 - acetoxy - 4 - thiophenylazetidin - 2 - one (D 50x). Thiophene - carboxaldehyde (1 g, 8.93 mmol, 1 equiv), p - methoxyaniline (1.1 g, 8.93 mmol, 1.5 equiv), triethylamine (26.78 mmol, 3 equiv) and acetoxyacetyl chloride (17.86 mmol, 2 equiv) were treated according to Procedure 2, Method B (Section 4.2.2.2) to afford the lactam (322 mg, 8% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.86 (s, acetyl CH 3 ), 3.76 (s, PMP OCH 3 ), 5.61 (d, J = 4.89 Hz, H - 3), 5.95 (d, J = 4.89 Hz, H - 2), 6.80 7.34 172 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 128.16, 127.08, 126.87, 118.85, 114.40, 76.52, 57.66, 55.45, 20.05. N - ( p - Methoxyphenyl) - 3 - a cetoxy - 4 - dimethylazetidin - 2 - one (D 50y). Isopropyl - aldeh yde (1 g, 13.89 mmol, 1 equiv), p - methoxyaniline (2.56 g, 20.84 mmol, 1.5 equiv), triethylamine (41.67 mmol, 3 equiv) and acetoxyacetyl chloride (27.78 mmol, 2 equiv) were treated according to Procedure 2, Method B (Section 4.2.2.2) to afford the lactam (327 mg, 9% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 0.96 1.01 (m, (CH 3 ) 2 ) 2.18 (s, acetyl CH 3 ), 2.23 (dd, J = 12.72, 6.85 Hz, H - 1), 3.79 (s, PMP OCH 3 ), 4.22 (t, J = 5.38 Hz, H - 3), 6.04 (d, J = 5.38 Hz, H - 2), 6.86 7.37 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 162.87, 156.71, 130.56, 119.78, 114.32, 73.82, 62.64, 55.48, 28.43, 20.74, 18.84. 4.2.2.3. General P rocedure 3 : Deprotection of A zetidinones Figure 4. 10 . Scheme showing general method for deprotection of amine . CAN is ceric ammonium nitrate . 173 Figure 4.10 . The deprotection of azetidinones was carried out similarly to a reported procedure. 38 Briefly, t o the solution of 2 - azetidinone (1 equiv) in CH 3 CN was added drop wise a solution of ceric ammonium nitrate ( (NH 4 ) 2 Ce(NO 3 ) 6 ) (3 equiv) in water at 0 °C. The mixture was stirred at 0 °C u ntil the disappearance of starting material and then diluted with water (20 mL). The mixture was then extracted with EtOAc ( 3 × 20 mL ). The organic layer was washed with 5% (w/v) NaHCO 3 (15 mL) and the aqueous extracts w ere washed with EtOAc (20 mL ). The combined organic extracts were washed with 10% (w/v) Na 2 SO 3 (15 mL), 5% (w/v) NaHCO 3 (15 mL) , and brine (15 mL) successively. The combined extracts were dried over MgSO 4 and 174 concentrated under vacuum. The crude mixture was purified by silica gel chr omatography (1:3 EtOAc/hexane, v/v) to yield the desired 2 - azetidinones. 3 - Acetoxy - 4 - phenylazetidin - 2 - one (D 51a) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - phenylazetidin - 2 - one (0.14 g, 0.43 mmol) in CH 3 CN (8.5 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (0.71 g, 1.3 mmo l , 3 equiv) in water (9 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (59.6 mg, 80% yield, at >95% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.68 (s, acetyl CH 3 ), 5.04 (d, J = 4.89 Hz, H - 3), 5.89 (dd, J = 4.89, 2.70 Hz, H - 2), 6.16 6.21 (m, amide NH), 7.30 7. 39 (aromatic protons). 3 - Acetoxy - 4 - (4 - fluoro phenyl)azetidin - 2 - one (D 51b) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (4 - fluorophenyl)azetidin - 2 - one (0.23 g, 0.7 mmol) in CH 3 CN (9 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (1.15 g, 2.1 mmol, 3 equiv) in water (10 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (63.1 mg, 78% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.73 (s, acetyl CH 3 ), 5.03 (d, J = 4.89 Hz, H - 3), 5.86 (dd, J = 4.89, 2.69 Hz, H - 2), 6.18 (br. s, amide NH), 7.04 7.32 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 19.89. 3 - Acetoxy - 4 - (4 - chloro phenyl)azetidin - 2 - one (D 51 c) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (4 - chlorophenyl)azetidin - 2 - one (0.40 g, 1.2 mmol) in CH 3 CN (14 mL) and (NH 4 ) 2 Ce(NO 3 ) 6 (1.91 g, 3.48 mmol, 3 equiv) in water (16 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (177 m g, 69% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.74 (s, acetyl CH 3 ), 5.02 (d, J = 4.89 Hz, H - 3), 5.87 (dd, J = 4.89, 2.93 Hz, H - 2), 6.34 (br. s., amide NH), 7.26 7.36 (aromatic protons). 175 13 C - NMR (126 MHz, CDCl 3 - d 169.01, 165.16, 134.60, 133.06, 128.90, 128.55, 78.28, 57.39, 19.91. 3 - Acetoxy - 4 - (4 - bromo phenyl)azetidin - 2 - one (D 51d) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (4 - bromophenyl)azetidin - 2 - one (0.25 g, 0.64 mmol) in CH 3 CN (9 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (1.04 g, 4.94 mmol , 3 equiv) in water (9 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (440 mg, 95% yield, at >95% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.74 (s, acetyl CH 3 ), 5.00 (d, J = 4.89 Hz, H - 3), 5.86 (dd, J = 4.89, 2.93 Hz, H - 2), 6.40 (br. s., amide NH), 7.19 7.50 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 19.93. 3 - Acetoxy - 4 - (4 - methyl phenyl)azetidin - 2 - one (D 51e) . N - ( p - methoxyphenyl) - 3 - ac etoxy - 4 - (4 - methylphenyl)azetidin - 2 - one (0.25 g, 0.8 mmol) in CH 3 CN (10 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (1.27 g, 2.31 mmol 3 equiv) in water (10 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (146 mg, 87% yield, at > 98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.70 (s, acetyl CH 3 ), 2.33 (s, p - CH 3 ), 4.99 (d, J = 4.89 Hz, H - 3), 5.85 (dd, J = 4.89, 2.69 Hz, H - 2), 6.33 (br. s., amide NH), 7.13 7.21 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d , 165.43, 138.50, 131.31, 129.01, 127.52, 78.26, 57.79, 31.19, 19.97. 3 - Acetoxy - 4 - (4 - methoxy phenyl)azetidin - 2 - one (D 51f) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (4 - methoxyphenyl)azetidin - 2 - one (0.20 g, 0.59 mmol) in CH 3 CN (7 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (0.96 g, 1.76 mmol, 3 equiv) in water (8 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (130.4 mg, 95% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.72 (s, acetyl CH 3 ), 3.81 (s, p - OCH 3 ), 176 4 .98 (d, J = 4.40 Hz, H - 3), 5.83 (dd, J = 4.40, 2.69 Hz, H - 2), 6.31 (br. s., amide NH), 6.87 7.25 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 126.29, 78.33, 57.56, 55.28, 19.93. 3 - Acetoxy - 4 - (4 - acetoxy phenyl)azetidin - 2 - one (D 51g) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (4 - acetoxyphenyl)azetidin - 2 - one (690 mg, 1.87 mmol) in CH 3 CN (30 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (3.08 g, 5.61 mmol, 3 equiv) in water (32 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (0.26 g, 54% yield, at >95% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.68 (s, acetyl CH 3 ), 2.29 (s, p - OAC CH 3 ), 5.02 (d, J = 4.89 Hz, H - 3), 5.84 (dd, J = 4.89, 2.69 Hz, H - 2), 6.56 (br. s., amide NH), 7.08 7.31 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 128.65, 121.61, 78.28, 57.40, 21.09, 19.87. 3 - Acetoxy - 4 - (4 - nitro phenyl)azetidin - 2 - one (D 51h) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (4 - nitrophenyl)azetidin - 2 - one (0. 72 g, 2.01 mmol) in CH 3 CN (25 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (3.31 g, 6.04 mmol, 3 equiv) in water (28 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (412 mg, 55% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.75 (s, acetyl CH 3 ), 5.18 (d, J = 4.89 Hz, H - 3), 5.97 (dd, J = 4.89, 2.93 Hz, H - 2), 6.47 (br. s., amide NH), 7.51 8.28 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 3 - A cetoxy - 4 - (3 - fluoro phenyl)azetidin - 2 - one (D 51i) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (3 - fluorophenyl)azetidin - 2 - one (1.35 g, 4.1 mmol) in CH 3 CN (54 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (6.74 g, 12.3 mmol, 3 equiv) in water (56 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (610 mg, 67% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.75 (s, acetyl CH 3 ), 5.06 (d, J = 4.90 Hz, H - 3), 5.91 (dd, J = 4.89, 177 2.93 Hz, H - 2), 6.33 (br. s., amide NH), 7.03 7.40 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 19.91. 3 - Acetoxy - 4 - (3 - chloro phenyl)azetidin - 2 - one (D 51j) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (3 - chlorophenyl)azetidin - 2 - one (1.15 g, 3.34 mmol) in CH 3 CN (47 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (5.49 g, 10.02 mmol, 3 equiv) in water (44 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one ( 647 mg, 81% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.76 (s, acetyl CH 3 ), 5.03 (d, J = 4.40 Hz, H - 3), 5.91 (dd, J = 4.89, 2.93 Hz, H - 2), 6.28 (br. s., amide NH), 7.19 7.36 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d , 128.86, 127.56, 125.82, 78.27, 57.41, 19.93. 3 - Acetoxy - 4 - (3 - bromo phenyl)azetidin - 2 - one (D 51k) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (3 - bromophenyl)azetidin - 2 - one (0.5 g, 1.29 mmol) in CH 3 CN (17 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (2.11 g, 3.86 mmol, 3 equiv) in water (20 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (255 mg, 70% yield, at >90% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.75 (s, acetyl CH 3 ), 5.01 (d, J = 4.89 Hz , H - 3), 5.90 (dd, J = 4.90, 2.90 Hz, H - 2), 6.24 (br. s., amide NH), 7.22 7.51 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 122.49, 78.25, 57.34, 19.92. 3 - Acetoxy - 4 - (3 - methyl phenyl)azetidin - 2 - one (D 51l) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (3 - methylphenyl)azetidin - 2 - one (0.76 g, 2.34 mmol) in CH 3 CN (30 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (3.85 g, 7.02 mmol, 3 equiv) in water (33 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (210 mg, 41% yield, at >99% purity by 178 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.70 (s, acetyl CH 3 ), 2.35 (s, m - CH 3 ), 5.00 (d, J = 4.89 Hz, H - 3), 5.88 (dd, J = 4.65, 2.69 Hz, H - 2), 6.27 (br. s., amide NH), 7.07 7.25 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 128.22, 128.09, 124.66, 78.19, 57.89, 21.36, 19.93. 3 - Acetoxy - 4 - (3 - methoxy phenyl)azetidin - 2 - one (D 51m) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (3 - methoxyphenyl)azetidin - 2 - one (1.05 g, 2.84 mmol) in CH 3 CN (35 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (4.67 g, 8.52 mmol, 3 equiv) in water (37 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (900 mg , 81% yield, at >85% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.72 (s, acetyl CH 3 ), 3.80 (s, m - OCH 3 ), 5.01 (d, J = 4.89 Hz, H - 3), 5.89 (dd, J = 4.89, 2.93 Hz, H - 2), 6.44 (br. s., amide NH), 6.82 7.29 (aromatic protons). 3 - Acetoxy - 4 - (3 - acetoxy phenyl)azetidin - 2 - one (D 51n) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (3 - acetoxyphenyl)azetidin - 2 - one (1.6 g, 4.83 mmol) in CH 3 CN (60 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (7.95 g, 17.5 mmol, 3 equiv) in water (62 mL) were treated according to Procedure 3 (Section 4.2.2 .3) to afford the azetidin one ( 570.7 mg, 50% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.74 (s, acetyl CH 3 ), 2.30 (s, m - OAc CH 3 ), 5.04 (d, J = 4.89 Hz, H - 3), 5.88 (dd, J = 4.89, 2.93 Hz, H - 2), 6.29 (br. s., amide NH), 7.03 7.40 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 129.40, 125.13, 121.92, 120.86, 78.19, 57.45, 21.08, 19.86. 3 - Acetoxy - 4 - (3 - nitro phenyl)azetidin - 2 - one (D 51o) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (3 - nitrophenyl)azetidin - 2 - one (0.59 g, 1.65 mmol) in CH 3 CN (20 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (2.72 g, 4.96 mmol, 3 equiv) in water (20 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one ( 231 mg, 56% yield, at >95% purity by 1 H - NMR analysis). 1 H - 179 NMR (500 MHz, CDCl 3 - d ) 1.73 (s, acetyl CH 3 ), 5.18 (d, J = 4.89 Hz, H - 3), 5.95 (dd, J = 4.40, 2.93 Hz, H - 2), 6.63 (br. s., amide NH), 7.55 8.26 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 168.93, 164.82, 148.28, 137.09, 133.65, 129.46, 123.71, 122.44, 78.42, 57.25, 19.91. 3 - Acetoxy - 4 - (2 - fluoro phenyl)azetidin - 2 - one (D 51p) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (2 - fluorophenyl)azetidin - 2 - one (1.08 g, 3.28 mmol) in CH 3 CN (43 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (5.4 g, 9.85 mmol, 3 equiv) in water (48 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (592 mg, 81% yield, at >95% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) s, acetyl CH 3 ), 5.34 (d, J = 4.90 Hz, H - 3), 5.99 (dd, J = 4.90, 2.69 Hz, H - 2), 6.53 (br. s., amide NH), 7.02 7.45 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 128.52, 123.97, 121.98, 115.35, 77.81, 51.99, 19.89. 3 - Acetoxy - 4 - (2 - chloro phenyl)azetidin - 2 - one (D 51q) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (2 - chlorophenyl)azetidin - 2 - one (1.97 g, 5.71 mmol) in CH 3 CN (75 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (9.39 g, 17.13 mmol, 3 equiv) in water (80 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (853 mg, 63% yield, at >95% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.76 (s, acetyl CH 3 ), 5.41 (d, J = 4.89 Hz, H - 3), 6.16 (dd, J = 4.89, 2.93 Hz, H - 2), 6.76 (br. s., amide NH), 7.27 7.48 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 116.13, 76.80, 55.29, 20.02. 3 - Acetoxy - 4 - (2 - bromo phenyl)azetidin - 2 - one (D 51r) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (2 - bromophenyl)azetidin - 2 - one (1.24 g, 3.17 mmol) in CH 3 CN ( 42 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (5.22 g, 9.52 mmol, 3 equiv) in water (44 mL) were treated according to 180 Procedure 3 (Section 4.2.2.3) to afford the azetidin one (381 mg, 42% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.77 (s, acetyl CH 3 ), 5.37 (d, J = 4.89 Hz, H - 3), 6.19 (dd, J = 4.89, 2.93 Hz, H - 2), 6.58 (br. s., amide NH), 7.20 7.58 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 123.20, 77.19 57.50 20.06. 3 - Acetoxy - 4 - (2 - me thyl phenyl)azetidin - 2 - one (D 51s) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (2 - methylphenyl)azetidin - 2 - one (1.39 g, 4.29 mmol) in CH 3 CN (50 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (7.05 g, 12.86 mmol, 3 equiv) in water (55 mL) were treated according to Procedure 3 (Section 4.2.2.3 ) to afford the azetidin one (487 mg, 52% yield, at >95% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.67 (s, acetyl CH 3 ), 2.29 (s, o - CH 3 ), 5.24 (d, J = 4.60 Hz, H - 3), 5.96 (dd, J = 4.60, 3.18 Hz, H - 2), 6.19 (br. s, amide NH), 7.13 7.43 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 128.36, 126.44, 125.80, 77.95, 54.92, 19.84, 18.88. 3 - Acetoxy - 4 - (2 - methoxy phenyl)azetidin - 2 - one (D 51t) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (2 - methoxyphenyl)azetidin - 2 - one (1.35 g, 3.96 mmol) in CH 3 CN (47 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (6.51 g, 11.88 mmol, 3 equiv) in water (52 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (815 mg, 88% yield, at >94% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.73 (s, acetyl CH 3 ), 3.78 (s, o - OCH 3 ), 5.37 (d, J = 4.89 Hz, H - 3), 6.06 (dd, J = 4.89, 2.93 Hz, H - 2), 6.26 (br. s., amide NH), 6.84 7.37 (aromatic protons). 13 C - NMR (12 6 MHz, CDCl 3 - d 123.03, 120.29, 110.08, 77.38, 55.35, 53.07, 20.06. 3 - Acetoxy - 4 - (2 - nitro phenyl)azetidin - 2 - one (D 51u) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - (2 - nitrophenyl)azetidin - 2 - one (0.21 g, 0.59 mmol) in CH 3 CN (7 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 181 (0.97 g, 1.77 mmol, 3 equiv) in water (8 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (90 mg, 61% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.79 (s, acetyl CH 3 ), 5.71 (d, J = 5.38 Hz, H - 3), 6.33 (dd, J = 5.38, 3.42 Hz, H - 2), 6.54 (br. s., amide NH), 7.55 8.20 (aromatic protons). 3 - Acetoxy - 4 - trimethylazetidin - 2 - one (D 51v) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - trimethylazetidin - 2 - one (0.36 g, 1.25 mmol) in CH 3 CN (17 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (2.05 g, 3.75 mmol, 3 equiv) in water (17 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one (167 mg, 72% yield, at >95% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 0.96 1.00 (m, t - b utyl CH 3 ), 2.16 (s, acetyl CH 3 ), 3.62 (d, J = 4.89 Hz, H - 3), 6.00 (d, J = 4.89 Hz, H - 2), 6.30 (br. s, amide NH). 13 C - NMR (126 MHz, CDCl 3 - d 166.85, 74.76, 63.17, 32.82, 26.00, 20.82. 3 - Acetoxy - 4 - cyclohexylazetidin - 2 - one (D 51w) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - cyclohexylazetidin - 2 - one (0.22 g, 0.7 mmol) in CH 3 CN (10 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (1.16 g, 2.11 mmol, 3 equiv) in water (11 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one ( 127 mg, 85% yield, at >95% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 0.84 1.81 (cyclohexyl protons), 2.15 (s, acetyl CH 3 ), 3.52 (dd, J = 9.29, 4.89 Hz, H - 3), 5.95 (dd, J = 4.89, 2.45 Hz, H - 2), 6.24 (br. s., amide NH). 3 - Acetoxy - 4 - thiophenylazetidin - 2 - one (D 51x) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - thiopheneazetidin - 2 - one (0.22 g, 0.7 mmol) in CH 3 CN (10 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (1.15 g, 2.09 mmol, 3 equiv) in water (12 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one ( 210 mg, 98% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.81 (s, acetyl CH 3 ), 5.25 (d, J = 4.89 Hz, H - 3), 5.83 (dd, J = 4.89, 2.69 Hz, 182 H - 2), 6.30 (br. s., amide NH), 6.95 7.35 (aromatic protons). 13 C - NMR (126 MHz, CDCl 3 - d 169.30, 165.48, 138.30, 127.16, 127.08, 126.28, 78.27, 54.07, 20.03. 3 - Acetoxy - 4 - dimethylazetidin - 2 - one (D 51y) . N - ( p - methoxyphenyl) - 3 - acetoxy - 4 - dimethylazetidin - 2 - one (0.33 g, 1.18 mmol) in CH 3 CN (17 mL), and (NH 4 ) 2 Ce(NO 3 ) 6 (1.94 g, 3.54 mmol, 3 equiv) in water ( 18 mL) were treated according to Procedure 3 (Section 4.2.2.3) to afford the azetidin one ( 133 mg, 66% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, CDCl 3 - d ) 0.84 (d, J = 6.85 Hz, CH 3 ), 0.97 (d, J = 6.85 Hz, CH 3 ), 1.83 1.93 (m, H - 1), 2.15 (s, acetyl CH 3 ), 3.50 (dd, J = 9.29, 4.89 Hz, H - 3), 5.94 (dd, J = 4.89, 2.45 Hz, H - 2), 6.28 (br. s., amide NH). 13 C - NMR (126 MHz, CDCl 3 - d 169.51, 166.16, 75.23, 60.67, 28.42, 20.61, 18.96, 18.74. 4.2.2.4. General P rocedure 4 : H ydroxyl D eprotection of the 2 - A zetidinones Figure 4. 1 1 . Scheme showing the general method for hydroxyl deprotection . 183 To a solution of 2 - azetidinone (1 equiv) in MeOH, saturated NaHCO 3 , and Na 2 CO 3 (0.1 equiv) were added at room temperature. After the disappearance of the starting material, the reaction mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by column chromatography (EtOAc:hexane 2:1 , v/v ) to yi eld the desired 2 - azetidinones. 3 - Hydroxy - 4 - (4 - fluoro phenyl)azetidin - 2 - one (D 52a) . 3 - A cetoxy - 4 - (4 - fluorophenyl)azetidin - 2 - one (108 mg, 0.48 mmol, 1 equiv) in MeOH (0.7 mL), saturated NaHCO 3 (1 mL) and Na 2 CO 3 (5 mg, 0.048 mmol, 0.1 equiv) were treated according to Procedure 4 ( Section 4.2.2.4) to afford the deprotected 2 - azetidinone ( 55.2 mg, 98% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, DMSO - d 6 ) 4.70 (d, J = 4.89 Hz, H - 3 ), 4.92 (ddd, J = 7.34, 4.89, 2.45 Hz, H - 2 ), 5.83 (d, J = 7.34 Hz, OH), 7.13 7.30 ( aromatic protons ), 8.46 (br. s., amide NH) (see Figure III 10 for a sample spectrum) . 13 C - NMR (126 MHz, DMSO - d 6 ) 170.44, 162.94, 134.56, 129.88, 115.17, 78.93, 57.22. 3 - Hydroxy - 4 - (4 - chloro phenyl)azetidin - 2 - one (D 52b) . 3 - A cetoxy - 4 - (4 - chloro - phenyl)azetidin - 2 - one (130 mg, 0.54 mmol, 1 equiv) in MeOH (0.8 mL), saturated NaHCO 3 (1.2 mL) and Na 2 CO 3 (5.7 mg, 0.05 mmol, 0.1 equiv) were treated according to Procedure 4 ( Section 4.2.2.4) to afford the deprotected 2 - azetidinone ( 100 mg, 93% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, DMSO - d 6 ) 4.71 (d, J = 4.90 Hz, H - 3 ), 4.92 4.95 (m, H - 2 ), 5.87 (br. s, OH), 7.23 7.43 ( aromatic protons ), 8.48 (br. s, amide NH). 3 - Hydroxy - 4 - (4 - bromo phenyl)azetidin - 2 - one (D 52c) . 3 - A cetoxy - 4 - (4 - bromo - phenyl)azetidin - 2 - one (440 mg, 1.6 mmol, 1 equiv) in MeOH (2.4 mL), saturated NaHCO 3 (3.45 mL) and Na 2 CO 3 (16.3 mg, 0.16 mmol, 0.1 equiv) were treated according to Procedure 4 ( Section 4.2.2.4) to afford the deprotected 2 - azetidinone (210 mg, 56% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, DMSO - d 6 ) 4.70 (d, J = 4.89 Hz, H - 3 ), 4.94 (ddd, J = 184 6.85, 4.89, 2.45 Hz, H - 2 ), 5.89 (d, J = 6.85 Hz, OH), 7.17 7.56 ( aromatic protons ), 8.51 (s, amide NH). 13 C - NMR (126 MHz, DMSO - d 6 ) 170.38, 138.00, 131.20, 130.16, 120.75, 79.04, 57.35. 3 - Hydroxy - 4 - (4 - methyl phenyl)azetidin - 2 - one (D 52d) . 3 - A cetoxy - 4 - (4 - methyl - phenyl)azetidin - 2 - one (146 mg, 0.7 mmol) in MeOH (1 mL), saturated NaHCO 3 (1.2 mL) and Na 2 CO 3 (7 mg, 0.07 mmo l) were treated according to Procedure 4 ( Section 4.2.2.4) to afford the deprotected 2 - a zetidinone (64.8 mg, 55% yield, at >95% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, DMSO - d 6 ) 2.29 (s, p - CH 3 ), 4.65 (d, J = 4.89 Hz, H - 3 ), 4.90 (ddd, J = 6.85, 4.89, 2.20 Hz, H - 2 ), 5.75 (d, J = 6.85 Hz, OH), 7.09 7.17 ( aromatic protons ), 8.40 (br. s., amide NH). 13 C - NMR (126 MHz, DMSO - d 6 ) 170.55, 136.67, 135.38, 128.86, 127.94, 78.97, 57.76 , 21.71. 3 - Hydroxy - 4 - (4 - methoxy phenyl)azetidin - 2 - one (D 52e) . 3 - A cetoxy - 4 - (4 - methoxy - phenyl)azetidin - 2 - one (120 mg, 0.51 mmol, 1 equiv) in MeOH (0.74 mL), saturated NaHCO 3 (1.1 mL) and Na 2 CO 3 (5.36 mg, 0.05 mmol, 0.1 equiv) were treated according to Procedure 4 ( Section 4.2.2.4) to afford the deprotected 2 - azetidinone ( 77.4 mg, 79% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, DMSO - d 6 ) 3.74 (s, p - OCH 3 ) , 4.63 (d, J = 4.89 Hz, H - 3) , 4.85 4.91 (m, H - 2 ) , 5.74 (d, J = 6.85 Hz, OH) , 6.88 7.19 ( aromatic protons ) , 8.38 (s, amide NH). 13 C - NMR (126 MHz, DMSO - d 6 57.47 55.52. 3 - Hydroxy - 4 - (4 - hydroxy phenyl)azetidin - 2 - one (D 52f) . 3 - A cetoxy - 4 - (4 - acetoxy - phenyl)azetidin - 2 - one (262 mg, 1.0 mmol, 1 equiv) in MeOH (3.2 mL), saturated NaHCO 3 (4.7 mL) and Na 2 CO 3 (10.45 mg, 0.1 mmol, 0.1 equiv) were treated according to Procedure 4 ( Section 4.2.2.4) to afford the deprotected 2 - azetidinone (112 mg, 63% yield, at >99% purity by 185 1 H - NMR analysis). 1 H - NMR (500 MHz, DMSO - d 6 ) 4.56 (d, J = 4.89 Hz, H - 3 ), 4.84 (ddd, J = 7.30, 4.89, 2.45 Hz, H - 2 ), 5.71 (d, J = 7.30 Hz, OH), 6.70 7.03 ( aromatic protons ), 8.33 (br. s, amide NH), 9.29 (s, p - OH). 13 C - NMR (126 MHz, DMSO - d 6 ) 170.61, 157.11, 129.22, 128.37, 115.10, 78.77, 57.59. 3 - Hydroxy - 4 - (2 - methyl phenyl)azetidin - 2 - one (D 52g) . 3 - A cetoxy - 4 - (2 - methyl - phenyl)azetidin - 2 - one (487 mg, 2.2 mmol, 1 equiv) in MeOH (3.0 mL), saturated NaHCO 3 (4.0 mL) and Na 2 CO 3 (23.35 mg, 0.22 mmol, 0.1 equiv) were treated according to Procedure 4 ( Section 4.2.2.4) to afford the deprotected 2 - azetidinone (60 mg, 15% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, DMSO - d 6 ) 2.19 (s, o - CH 3 ), 4.82 (d, J = 5.00 Hz, H - 3 ), 5.00 (ddd, J = 7.73, 5.00, 2.45 Hz, H - 2 ), 5.78 (d, J = 7.30 Hz, OH), 7.10 7.22 ( aromatic protons ), 8.47 (br. s., amide NH). 13 C - NMR (126 MHz, DMSO - d 6 ) 170.20, 136.68, 136.16, 129.94, 127.12, 127.12, 126.35, 125.84, 78.81, 56.47 , 19.16. 3 - Hydroxy - 4 - (2 - methoxy phenyl)azetidin - 2 - one (D 52h) . 3 - A cetoxy - 4 - (2 - methoxy - phenyl)azetidin - 2 - one (815 mg, 3.46 mmol, 1 equiv) in MeOH (5.0 mL), saturated NaHCO 3 (7.4 mL) and Na 2 CO 3 (36.4 mg, 0.35 mmol, 0.1 equiv) were treated according to Procedure 4 ( Section 4.2.2.4) to afford the deprotected 2 - azetidinone (214 mg, 32% yield, at >96% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, DMSO - d 6 ) 3.74 (s, o - CH 3 ), 4.88 (d, J = 4.90 Hz, 1H ) , 4.89 4.93 (m, 1 H) , 5.68 (d, J = 7.34 Hz, OH) , 6.91 7.26 ( aromatic protons ) , 8.33 (br. s., amide NH). 186 4.2.2.5. General P rocedure 5 : H ydrolysis of A zetidinone R ing Figure 4. 1 2 . Scheme showing the general method for the lactam ring hydrolysis . 187 Figure 4.12 . Method A: Hydrolysis of azetidinone ring to unveil the isoserine was carried out by addition of 7 N HCl. The reaction mixture was stirred at reflux conditions (85 ° C) for 24 h. The reaction was lyophilized yielding the hydrochloride salt of the isoserine products as racemic mixtures. Method B: The hydroxyl group deprotection and lactam ring hydrolysis were done in one pot reaction . Briefly, 7 N HCl was added to the 3 - acetoxy - azetidinones and the reaction was stirred at reflux conditions for 24 h. The reaction mixture was lyophilized to dryness to afford the isoserine products as racemic mixtures. (Refer to the Appendix for the 1 H - NMR spectrum assignment and numbering for the isoserine analog s). Phenylisoserine (D 53a) . 7 N HCl (1 mL) and 3 - hydroxy - 4 phenylazetidin - 2 - one (29 mg) were treated according to Procedure 5, Method A (Section 4.2.2.5) to afford the desired isoserine (30 mg, 78% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.36 (d, J = 6.40 Hz, 1H ), 4.55 (d, J = 6.40 Hz, 1H ), 7.35 7.42 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 176.30, 133.44, 129.27 , 128.98, 127.17, 73.35, 57.42. HRMS (ESI - TOF) m / z 182.0855 [M + H] + ; calculated for C 9 H 12 NO 3 : 182.0817. 4 - Fluorophenylisoserine (D 53b) . 7 N HCl (1 mL) and 3 - hydroxy - 4 - (4 - fluoro)phenylazetidin - 2 - one (13.3 mg, 0.07 mmol) were treated according to Procedure 5, 188 Method A (Section 4.2.2.5) to afford the desired isoserine (12.2 mg, 81% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.51 (d, J = 6.36 Hz, 1H ), 4.61 (d, J = 6.36 Hz, 1H ), 7.16 7.43 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.84, 163.96, 129.50, 128.71, 116.08, 71.70, 56.21. HRMS (ESI - TOF) m / z HRMS (ESI - TOF) m / z 200.0760 [M + H] + ; calculated for C 9 H 11 NO 3 F: 200.0723. 4 - Chlorophenylisoserine (D 53c) . 7 N HCl (2.0 mL) and 3 - hydroxy - 4 - (4 - chloro)phenylazetidin - 2 - one (40 mg, 0.20 mmol) were treated according to Procedure 5, Method A (Section 4.2.2.5) to afford the desired isoserine (42.6 mg, 84% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.53 (d, J = 6.40 Hz, 1H ), 4.65 (d, J = 6.40 Hz, 1H ), 7.44 7.51 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 174.02, 133.99, 131.53, 129.13, 128.87, 71.77, 56.29. HRMS (ESI - TOF) m / z 216.0464 [M + H] + ; calculated for C 9 H 11 NO 3 Cl: 216.0427. 4 - Bromophenylisoserine (D 53d) . 7 N HCl (12 mL) and 3 - hydroxy - 4 - (4 - bromo)phenylazetidin - 2 - one (186 mg, 0.77 mmol) were treated according to Procedure 5, Method A (Section 4.2.2.5) to afford the desired isoserine (220 mg, 100% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.58 (d, J = 6.40 Hz, 1H ), 4.66 (d, J = 6.40 Hz, 1H ), 7.38 7.65 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.43, 132.14, 131.86, 129.10, 123.26, 71.70, 56.21. HRMS (ES I - TOF) m / z 259.9960 [M + H] + ; calculated for C 9 H 12 NO 3 Br: 259.9922. 4 - Methylphenylisoserine (D 53e) . 7 N HCl (2 mL) and 3 - hydroxy - 4 - (4 - methyl)phenylazetidin - 2 - one (20 mg, 0.1 mmol) were treated according to Procedure 5, Method A (Section 4.2.2.5) to afford the desired isoserine (25.9 mg, 95% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 2.35 (s, p - CH 3 ), 4.49 (d, J = 6.40 Hz, 1H ), 4.57 (d, 189 J = 6.40 Hz, 1 H ), 7.29 7.37 ( benzylic protons). 13 C - NMR (126 MHz, D 2 O) 173.87, 140.24, 129.74, 129.62, 127.34, 71.87, 56.76, 20.19 . HRMS (ESI - TOF) m / z 196.1008 [M + H] + ; calculated for C 10 H 14 NO 3 : 196.0974. 4 - Methoxyphenylisoserine (D 53f) . 7 N HCl (2 mL) and 3 - hydroxy - 4 - (4 - methoxyl)phenylazetidin - 2 - one (25 mg, 0.13 mmol) were treated according to Procedure 5, Method A (Section 4.2.2.5) to afford the desired isoserine (27 mg, 99% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 3.84 (s, p - OCH 3 ), 4.57 4.62 (m, 2H ), 7.06 7.42 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 174.05, 159.65, 129.06, 128.8 9, 114.48, 71.91, 56.46, 55.24 . HRMS (ESI - TOF) m / z 212.0956 [M + H] + ; calculated for C 10 H 14 NO 4 : 212.0923. 4 - Hydroxyphenylisoserine (D 53g) . 7 N HCl (7 mL) and 3 - hydroxy - 4 - (4 - hydroxy)phenylazetidin - 2 - one (110 mg, 0.62 mmol) were treated according to Procedure 5, Method A (Section 4.2.2.5) to afford the desired isoserine (119 mg, 98% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.42 (d, J = 6.40 Hz, 1H ), 4.47 (d, J = 6.40 Hz, 1H ), 6.88 7 .27 (aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.95, 156.54, 129.10, 124.37, 115.84, 71.86, 56.50. HRMS (ESI - TOF) m / z 198.0798 [M + H] + ; calculated for C 9 H 12 NO 4 : 198.0766. 4 - Nitrophenylisoserine (D 53h) . 7 N HCl (3 mL) and 3 - acetoxy - 4 - (4 - nitro) - phenylazetidin - 2 - one (20 mg, 0.08 mmol) were treated according to Procedure 5, Method B (Section 4.2.2.5) to afford the desired isoserine ( 20 mg, 95% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.49 (d, J = 5.40 Hz, 1H ), 4.78 (d, J = 5.40 Hz, 1 H), 7.64 8.27 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.95, 148.14, 140.29, 128.62, 124.19, 71.54, 56.20. HRMS (ESI - TOF) m / z 227.0711 [M + H] + ; calculated for C 9 H 11 N 2 O 5 : 227.0668. 190 3 - Fluorophenylisoserine (D 53i) . 7 N HCl (20 mL) and 3 - acetoxy - 4 - (3 - fluoro) - phenylazetidin - 2 - one (600 mg, 2.69 mmol) were treated according to Procedure 5, Method B (Section 4.2.2.5) to afford the desired isoserine (507 mg, 98% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.52 (d, J = 5.87 Hz, 1H ), 4.63 (d, J = 5.87 Hz, 1 H), 7.12 7.50 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.78, 163.43, 13 5.15, 131.07, 123.15 , 116.55, 114.41, 71.59, 56.28. HRMS (ESI - TOF) m / z 200.0765 [M + H] + ; calculated for C 9 H 11 NO 3 F: 200.0723. 3 - Chlorophenylisoserine (D 53j) . 7 N HCl (20 mL) and 3 - acetoxy - 4 - (3 - chloro) - phenylazetidin - 2 - one (645 mg, 2.7 mmol) were treated according to Procedure 5, Method B (Section 4.2.2.5) to afford the desired isoserine (643 mg, 95% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.53 (d, J = 6.40 Hz, 1H ), 4.62 (d, J = 6.40 Hz, 1H), 7.30 7.46 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 17 3.32, 134.69, 134.25, 130.65, 129.70, 127.40, 125.66 , 71.29, 56.15. HRMS (ESI - TOF) m / z 216.0467 [M + H] + ; calculated for C 9 H 11 NO 3 Cl: 216.0427. 3 - Bromophenylisoserine (D 53k) . 7 N HCl (5 mL) and 3 - hydroxy - 4 - (3 - bromo) - phenylazetidin - 2 - one (112 mg, 0.47 mmol) were treated according to Procedure 5, Method A (Section 4.2.2.5) to afford the desired isoserine (134 mg, 98% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.50 (d, J = 5.87 Hz, 1H ), 4.60 (d, J = 5.87 Hz, 1H), 7.31 7.60 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.32, 134.97, 132.67, 13 0.86, 130.33, 126.10 , 122.32, 71.30, 56.09. HRMS (ESI - TOF) m / z 259.9964 [M + H] + ; calculated for C 9 H 11 NO 3 Br: 259.9922. 3 - Methylphenylisoserine (D 53l) . 7 N HCl (15 mL) and 3 - acetoxy - 4 - (3 - methyl) - phenylazetidin - 2 - one (405 mg, 1.8 mmol) were treated according to Procedure 5, Method A 191 (Section 4.2.2.5) to afford the desired isoserine (406 mg, 95% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 2.29 (s, m - CH 3 ), 4.49 (d, J = 6.80 Hz, 1H ), 4.53 (d, J = 6.80 Hz, 1H ), 7.16 7.31 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.94, 139. 42, 132.67, 130.21, 129.03, 127.81, 124.12, 71.90, 56.87, 20.29 . HRMS (ESI - TOF) m / z 196.1010 [M + H] + ; calculated for C 10 H 14 NO 3 : 196.0974. 3 - Methoxyphenylisoserine (D 53m) . 7 N HCl (25 mL) and 3 - acetoxy - 4 - (3 - methoxy) - phenylazetidin - 2 - one (774 mg, 3.3 mmol) were treated according to Procedure 5, Method B (Section 4.2.2.5) to afford the desired isoserine (798 mg, 98% yield, at >96% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 3.78 (s, m - O CH 3 ), 4.50 (d, J = 6.40 Hz, 1 H), 4.56 (d, J = 6.40 Hz, 1H), 6.96 7.40 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.56, 159.16, 13 4.26, 130.50, 119.81 , 115.16, 112.99, 71.62, 56.69 , 55.30. HRMS (ESI - TOF) m / z 212.0959 [M + H] + ; calculated for C 10 H 14 NO 4 : 212.0923. 3 - Hydroxyphenylisoserine (D 53n) . 7 N HCl (4 mL) and 3 - acetoxy - 4 - (3 - acetoxy ) - phenylazetidin - 2 - one (50 mg, 0.19 mmol) were treated according to Procedure 5, Method B (Section 4.2.2.5) to afford the desired isoserine (18 mg, 41% yield, at > 99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.48 (d, J = 6.40 Hz, 1H ), 4.52 (d, J = 6.40 Hz, 1 H), 6.86 7.32 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.96, 155.86, 134.49, 130.59, 119.13, 116.47, 114.20, 71.83, 56.68. HRMS (ESI - TOF) m / z 198.0806 [M + H] + ; calculated for C 9 H 12 NO 4 : 198.0766. 3 - Nitrophenylisoserine (D 53o) . 7 N HCl (8 mL) and 3 - acetoxy - 4 - (3 - nitro) - phenylazetidin - 2 - one (229 mg, 0.91 mmol) were treated according to Procedure 5, Method B (Section 4.2.2.5) to afford the desired isoserine (230 mg, 96% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.53 (d, J = 5.90 Hz, 1H ), 4.80 (d, J = 5.90 Hz, 1H), 7.64 192 8.35 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.29, 147.99, 134.83, 134.02, 130.47, 124.54 , 122.52, 71.09, 55.89. HRMS (ESI - TOF) m / z 227.0708 [M + H] + ; calculated for C 9 H 11 N 2 O 5 : 227.0668. 2 - Fluorophenylisoserine (D 53p) . 7 N HCl (18 mL) and 3 - acetoxy - 4 - (2 - fluoro) - phenylazetidin - 2 - one (502 mg, 2.24 mmol) were treated according to Procedure 5, Method B (Section 4.2.2.5) to afford the desired isoserine (518 mg, 98% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.57 (d, J = 6.80 Hz, 1H ) 4.82 (d, J = 6.80 Hz, 1H) 7.15 7.48 ( aromatic pro tons ). 13 C - NMR (126 MHz, D 2 O) 173.50, 161.02, 131.91, 128.76, 125.00, 119.61 , 116.08, 70.81, 51.61. HRMS (ESI - TOF) m / z 200.0762 [M + H] + ; calculated for C 9 H 11 NO 3 F: 200.0723. 2 - Chlorophenylisoserine (D 53q) . 7 N HCl (28 mL) and 3 - acetoxy - 4 - (2 - chloro) - phenylazetidin - 2 - one (870 mg, 3.64 mmol) were treated according to Procedure 5, Method B (Section 4.2.2.5) to afford the desired isoserine (896 mg, 98% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.57 (d, J = 5.87 Hz, 1H ), 5.15 (d, J = 5.87 Hz, 1H ), 7.37 7.52 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.59, 132.93, 130.90, 130.49, 130.14, 127.75, 127.72 , 70.52, 53.18. HRMS (ESI - TOF) m / z 216.0467 [M + H] + ; calculated for C 9 H 11 NO 3 Cl: 216.0427. 2 - Bromophenylisoserine (D 53r) . 7 N HCl (10 mL) and 3 - acetoxy - 4 - (2 - bromo) - phenylazetidin - 2 - one (395 mg, 1.4 mmol) were treated according to Procedure 5, Method B (Section 4.2.2.5) to afford the desired isoserine (396 mg, 96% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.55 (d, J = 5.87 Hz, 1H ), 5.15 (d, J = 5.87 Hz, 1H), 7.28 7.73 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.38, 133.53, 132.11, 13 1.12, 128.34, 193 127.78 , 123.20, 70.46, 55.47. HRMS (ESI - TOF) m / z 259.9962 [M + H] + ; calculated for C 9 H 11 NO 3 Br: 259.992 2. 2 - Methylphenylisoserine (D 53s) . 7 N HCl (6 mL) and 3 - hydroxy - 4 - (2 - methyl)phenylazetidin - 2 - one (54 mg, 0.31 mmol) were treated according to Procedure 5, Method A (Section 4.2.2.5) to afford the desired isoserine (71 mg, 95% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 2.32 (s, o - CH 3 ), 4.45 (d, J = 6.80 Hz, 1H ), 4.86 (d, J = 6.80 Hz, 1H ), 7.26 7.39 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.92, 136. 72, 131.25, 131.06, 129.39, 126.67, 125.77, 71.42, 52.62, 18.28 . HRMS (ESI - TOF) m / z 196.1011 [M + H] + ; calculated for C 10 H 14 NO 3 : 196.0974. 2 - Methoxyphenylisoserine (D 53t) . 7 N HCl (18 mL) and 3 - hydroxy - 4 - (2 - methoxy) - phenylazetidin - 2 - one (214 mg, 1.1 mmol) were treated according to Procedure 5, Method A (Section 4.2.2.5) to afford the desired isoserine (229 mg, 98% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 3.83 (s, o - OCH 3 ), 4.60 (d, J = 8.30 Hz, 1 H), 4.66 (d, J = 8.30 Hz, 1H), 6.99 7.44 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 174.04, 157 .06, 131.45, 129.64, 120.94 , 119.54 , 111.61, 70.53, 55.23, 55.15 . HRMS (ESI - TOF) m / z 212.0961 [M + H] + ; calculated for C 10 H 14 NO 4 : 212.0923. 2 - Nitrophenylisoserine (D 53u) . 7 N HCl (5 mL) and 3 - acetoxy - 4 - (2 - nitro) - phenylazetidin - 2 - one (92.6 mg, 0.37 mmol) were treated according to Procedure 5, Method B (Section 4.2.2.5) to afford the desired isoserine (94 mg, 97% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.64 (d, J = 5 .40 Hz, 1 H), 5.32 (d, J = 5.40 Hz, 1 H ), 7.62 8.10 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173. 40, 148.10, 134.45, 130.67 , 128.38, 128.01, 125.71, 70.59, 51.63. HRMS (ESI - TOF) m / z 227.0706 [M + H] + ; calculated for C 9 H 11 N 2 O 5 : 227.0668. 194 Trimethyl isoserine (D 53v) . 7 N HCl (5 mL) and 3 - acetoxy - 4 - trimethylazetidin - 2 - one (210 mg, 1.13 mmol) were treated according to Procedure 5, Method B (Section 4.2.2.5) to afford the desired isoserine (145 mg, 65% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR ( 500 MHz, D 2 O) 1.01 (s, t - butyl CH 3 ), 3.43 (d, J = 1.50 Hz, H - 3), 4.55 (d, J = 1.50 Hz, H - 2). 13 C - NMR (126 MHz, D 2 O) 175.92, 66.7 4, 60.51, 32.62, 25.47 . HRMS (ESI - TOF) m / z 162.1168 [M + H] + ; calculated for C 7 H 16 NO 3 : 162.1130. Cyclohexyl isoserine (D 53w) . 7 N HCl (7 mL) and 3 - acetoxy - 4 - cyclohexylazetidin - 2 - one (211 mg, 1.0 mmol) were treated according to Procedure 5, Method B (Section 4.2.2.5) to afford the desired isoserine (219 mg, 98% yield, at >98% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 1.00 1.15 (m, 2H), 1.16 1.26 (m, 4H), 1.57 1.64 (m, 1H ), 1.66 1.77 (m, 4 H ), 3.34 3.38 (m, H - 3 ), 4.49 (d, J = 2.93 Hz, H - 2). 13 C - NMR (126 MHz, D 2 O) 175.27, 67.64, 57.70, 37.05, 28.56, 28.03, 25.23, 25.11. HRMS (ESI - TOF) m / z 188.1324 [M + H] + ; calculated for C 9 H 18 NO 3 : 188.1287. Thiophenyl isoserine (D 53x) . 7 N HCl (5 mL) and 3 - hydroxy - 4 - thiopheneazetidin - 2 - one (144 mg, 0.68 mmol) were treated according to Procedure 5, Method B (Section 4.2.2.5) to afford the desired isoserine (145 mg, 65% yi eld, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 4.54 (d, J = 5.90 Hz, H - 2 ), 4.98 (d, J = 5.90 Hz, H - 3 ), 7.04 7.50 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.94, 134.05, 128.60, 127.89, 127.31, 71.66, 52.41. HRMS (ESI - TOF) m / z 188.0414 [M + H] + ; calculated for C 7 H 10 NO 3 S: 188.0381. Isopropyl isoserine (D 53y) . 7 N HCl (4 mL) and 3 - acetoxy - 4 - dimethylazetidin - 2 - one (139 mg, 0.81 mmol) were treated according to Procedure 5, Method B (Section 4.2.2.5) to afford the desired isoserine (142 mg, 95% yield, at >99% purity by 1 H - NMR analysis). 1 H - NMR (500 MHz, D 2 O) 0.99 (d, J = 6.85 Hz, CH 3 ), 1.97 2.07 (m, CH), 3.35 (dd, J = 3.42, 2.90 Hz, 195 H - 3), 4.46 (d, J = 3.42 Hz, H - 2). 13 C - NMR (126 MHz, D 2 O) 175.16, 67.91, 58.47, 27.83, 18.09, 1 7.38 . HRMS (ESI - TOF) m / z 148.1015 [M + H] + ; calculated for C 6 H 14 NO 3 : 148.0974. 4.2.3. Synthesis of P yridinyli soserine Analogs The method described for the isoserine analog s above was not successful with the synthesis of pyridinylisoserine. The oxidative removal of p - methoxy phenol group using cerium ammonium nitrate did not yield the expected product. This prompted a search for an alternative amine protecting group. Benzhydry l amine was chosen which could be easily cleaved off through hydrogenolysis. 4.2.3.1. General P rocedure 6 : S ynthesis of P yridinyl - N - benzhydryl I mine Figure 4. 1 3 . Sc heme showing synthesis of 2 - pyridinyl imines . To a solution of pyr idin yl carboxaldehyde ( 1 equ iv) dissolved in benzene was add ed benzhydrylamine ( 1.1 equiv) and oven dried molecular sieves (~1.5 g). The reaction was stirred 196 at room temperature for 12 h, then filtered, dried (MgSO 4 ), and concentrated under vacuum. The crude mixture was purified by silica gel column chromatography (1:4 EtOAc/hexane v/v), and the fractions containing the product were combined and dried under vacuum to obtain the imine. 2 - pyridinyl - N - benzhydryl imine (D 56a) was synthesized according to the G eneral P roced ure 6 ( Section 4.2.3.1) by addition of 2 - pyridin yl carboxaldehyde (21 mmo l, 2 mL, 1 equiv) to benzhydryl amine (23.4 mmol, 1.1 equiv) dissolved in benzene (20 mL) to yield the imine (5.7 g, 99.6 % yield, at >99% purity by 1 H - NMR analysis ) . 1 H - NMR (500 MHz, CDCl 3 - d ) 5.72 (s, benz hydryl CH ) , 7.24 8.66 ( aromatic protons ). 13 C - NMR (126 MHz, CDCl 3 - d 162.02, 154.75 , 149.40, 143.37, 136.54 , 128.58, 127.81, 127.22 , 124.91, 121.57 , 77.74 . 3 - pyridinyl - N - benzhydryl imine (D 56b) was synthesized according to the G eneral P rocedure 6 ( Section 4.2.3.1) by addition of 3 - pyridinyl carboxaldehyde (9.35 mmol, 0.9 mL, 1 equiv) to benzhydryl amine (10.3 mmol, 1.1 equiv) dissolved in benzene (10 mL) to yield the imine (2.5 g, 100% yield, at >99% purity by 1 H - NMR analysis ). 1 H - NMR (500 MHz, CDCl 3 - d ) 5.64 (s, benzhydryl CH), 7.23 8.25 ( aromatic protons ), 8.47 (s, imine C H), 8.66 8.93 ( aromatic protons ). 13 C - NMR (126 MHz, CDCl 3 - d , 151.65, 150.50, 143.43, 134.84, 131.78 , 128.54, 127.58 , 127.18 , 123.63, 78.14. 4 - pyridinyl - N - benzhydryl imine (D 56c) was synthesized according to the G eneral P rocedu re 6 ( Section 4.2.3.1) by addition of 4 - pyridin yl carboxaldehyde (9.35 mmol, 0.9 mL, 1 equiv) to benzhydryl amine (10.3 mmol, 1.1 equiv) dissolved in benzene (10 mL) to yield the imine (2.49 g, 98% yield, at >99% purity by 1 H - NMR analysis ). 1 H - NMR (500 MHz, CDCl 3 - d ) 5.68 (s, benzhydryl CH) , 7.26 7.73 ( aromatic protons ) , 8.43 (s, imine C H) , 8.71 8.74 ( aromatic protons ). 13 C - NMR (126 MHz, CDCl 3 - d 128.36, 127.59, 127.31, 122.22, 78.14. 197 4.2.3.2. General P rocedure 7: S ynthesis of N - benzhydryl - 3 - acetoxy - 4 - (pyridinyl)azetidin - 2 - one Figure 4.1 4 . Scheme showing the synthesis of pyridin yl - 2 - azetidinones . To a solution of imine ( D 56 , 1 equiv) dissolved in dichloromethane (38 mL) at 0 ° C was added triethylamine (11.04 mmol, 3 equiv). A solution of acetoxyacetyl chloride (7.36 mmol, 2 equiv) in dichloromethane (20 mL) was then added dropwise and the reaction stirred at 0 ° C for 2 h . The mixture was then warmed to room tempe rature and stirred for 1.5 h to complete the reaction. The solution was washed with water (3 × 15 mL) and the organic fraction dried (MgSO 4 ) and concentrated under vacuum. The crude mixture was then purified by silica gel chromatography (1:4 EtOAc/hexane, v/v) and the fractions containing product concentrated under vacuum to obtain the pure product. Synthesis of N - benzhydryl - 3 - acetoxy - 4 - (2 - pyridinyl)azetidin - 2 - one (D 57a) . 2 - P yridinyl - N - benzhydryl imine (1 g, 3.68 mmol, 1 equiv), triethylamine (11.04 mmol, 3 equiv) dissolved in dichloromethane (20 mL) at 0 ° C and acetoxyacetyl chloride (7.36 mmol, 2 equiv) 198 were treated according to Procedure 7 ( Section 4.2.3.2) to afford the N - benzhydryl - 3 - acetoxy - 4 - (2 - pyridinyl)azetidin - 2 - one (243 mg, 17.8 % yield, at >99% purity by 1 H - NMR analysis ). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.71 (s, acetyl CH 3 ) , 5.11 (d, J = 4.89 Hz, H - 3 ), 5.96 (s, benzhydryl CH) , 5.98 (d, J = 4.89 Hz, H - 2 ) , 6.97 8. 51 ( aromatic protons ). 13 C - NMR (126 MHz, CDCl 3 - d 127.96, 127.80, 123.36, 122 .82, 75.79, 63.11, 61.75, 19.94. Synthesis of N - benzhydryl - 3 - acetoxy - 4 - (3 - pyridinyl)azetidin - 2 - one (D 57b) . 3 - P yridinyl - N - benzhydryl imine (1 g, 3.68 mmol, 1 equiv), triethylamine (11.04 mmol, 3 equiv) dissolved in dichloromethane (20 mL) at 0 ° C and acetoxyacetyl chloride (7.36 mmol, 2 equiv) were treated according to Procedure 7 ( Section 4.2.3.2) to afford the N - benzhydryl - 3 - acetoxy - 4 - (3 - pyridinyl)azetidin - 2 - one ( 356 mg, 26% yield, at > 99% purity by 1 H - NMR analysis ). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.73 (s, acetyl CH 3 ), 4.98 (d, J = 4.89 Hz, H - 3 ), 5.92 (s, benzhydryl CH), 5.93 (d, J = 4.90 Hz, H - 2 ), 7.08 8.46 ( aromatic protons ). 13 C - NMR (126 MHz, CDCl 3 - d 168.93, 164.72, 150.00, 149.69, 137.47, 137.32, 136.02 , 128.78, 128.61 , 128.54, 76.06, 61.75, 60.29, 19.90. Synthesis of N - benzhydryl - 3 - acetoxy - 4 - (4 - pyridinyl)azetidin - 2 - one (D 57c) . 4 - P yridinyl - N - benzhydryl imine (1 g, 3.68 mmol, 1 equiv), triethylamine (11.04 mmol, 3 equiv) dissolved in dichloromethane (20 mL) at 0 ° C and acetoxyacetyl chloride (7.36 mmol, 2 equiv) were treated according to Procedure 7 ( Section 4.2.3.2) to afford the N - benzhydryl - 3 - acetoxy - 4 - (4 - py ridinyl)azetidin - 2 - one (164 mg, 12% yield, at >99 % purity by 1 H - NMR analysis ). 1 H - NMR (500 MHz, CDCl 3 - d ) 1.70 (s, acetyl CH 3 ), 4.93 (d, J = 4.89 Hz, H - 3 ), 5.90 (s, benzhydryl C H), 5.91 (d, J = 4.89 Hz, H - 2 ), 6.96 8.43 ( aromatic protons ). 13 C - NMR (126 MHz, CDCl 3 - d 199 169.01, 164.54, 149.40, 142.56, 137.42, 128.78, 128.63 , 127.93 , 123.23, 75.90, 61.95, 61.42, 19.90. 4.2.3.3. General P rocedure 8: L actam R ing O pening Along with A mine and H ydroxy l D eprotection of N - benzhydryl - 3 - acetoxy pyridinylazetidin - 2 - one Briefly, to the star t ing material, N - benzhydryl - 3 - acetoxy - 4 - (2 - pyridinyl)azetidin - 2 - one (100 mg, 0.27 mmol, 1 equ i v) dissolved in acetonitrile (2 mL), was added 6 N HCl (2 mL) . T he reaction mixture was stirred under reflux , monitored by TLC ( 4:1:1 buta nol/AcOH /H 2 O v/v ) and stopped after 12 h . The a cetonitrile was removed by evaporation under a stream of dry nitrogen and the resultant crude residue was dissolved in water (3 mL) and adjusted to pH 3 ( 3 N HCl ) . The solution was extracted with ethyl acetate (3 × 3 mL ) , t he organic layer was washed with dH 2 O (2 × 3 mL), and the aqueous fractions were combined and lyophilized . The resultant crude residue was loaded onto a C 18 reverse phase silica gel (SiliCycle, Quebec City, Canada) column ( diameter: 1.2 mm, height: 32 cm ) and eluted with 2.5 % acetonitrile in water. The fractions containing the product were pooled and d ried u nder vacuum to obtain 2 - pyridinylisoserine a s a white powder ( 41.1 mg, 84 % converted yield from the lactam, at >90% purity by 1 H - NMR analysis ). 1 H - NMR (500 MHz, D 2 O) 4.37 (d, J = 5.40 Hz, 1H ) , 4.69 (d, J = 5.40 Hz, 1 H) , 7.41 8.56 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 175.85, 152.11, 149.06, 138.14, 124.37, 122.94, 72.77, 57.20. HRMS (ESI - TOF) m / z 183.0777 [M + H] + ; calculated for C 8 H 11 N 2 O 3 : 183.0770. Removal of ethyl acetate from the organic layer under vacuum yielded benzhydrylacetamide as a white solid ( 45.6 mg , 75% yield ) . 1 H - NMR (500 MHz, CDCl 3 - d ) 2.04 (s, 3 H) , 6.18 (d, J = 7.34 Hz, 1 H) , 6.24 (d, J = 7.30 Hz, 1 H), 7.21 7.24 (m, 4 H) , 7.26 (t, 200 J = 2.20 Hz, 1 H) , 7.28 (d, J = 0.98 Hz, 1 H) , 7.30 7.35 (m, 4 H) . 13 C - NMR (126 MHz, CDCl 3 - d 141.49 , 128.67, 127.49, 127.42 , 57.01 , 23.36 (see Appendix Figure III 38 for numbering) . Synthesis of 3 - pyridin yl isoserine . 3 - pyridinylisoserine was syn thesized according to P rocedure 8 ( Section 4.2.3.3 ) except N - benzhydryl - 3 - acetoxy - 4 - ( 3 - pyridinyl)azetidin - 2 - one (350 mg, 0.94 mmol, 1 equ i v) was used as the azetidine precursor to make 3 - pyridinylisoserine as the HCl salt ( 161.5 mg, 94 % converted yield from the lactam , at >9 5 % purity by 1 H - NMR analysis ). 1 H - NMR (500 MHz, D 2 O) 4.59 (d, J = 4.40 Hz, 1H ), 5.07 (d, J = 4.40 Hz, 1H ), 8.14 8.96 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.31, 145.87, 142.27, 140.90, 134.19, 127.80, 70.35, 53.49. HRMS (ESI - TOF) m / z 183.0810 [M + H] + ; calculated for C 8 H 11 N 2 O 3 : 183.0770. Synthesis of 4 - pyridin yl isoserine . 4 - pyridinylisoserine was synthesized according to Procedure 8 ( Section 4.2.3.3) except N - benzhydryl - 3 - acetoxy - 4 - (4 - pyridinyl)azetidin - 2 - one ( 164 mg, 0.44 mmol, 1 equ i v) was used as the azetidine precursor to make the HCl salt of 4 - pyridin yl isoserine ( 57.9 mg, 72 % yield, at 8 0% purity by 1 H - NMR analysis ). 1 H - NMR (500 MHz, D 2 O) 4. 38 (d, J = 4.40 Hz, 1H ), 4.93 (d, J = 4.40 Hz, 1H ), 7.99 8.8 2 ( aromatic protons ). 13 C - NMR (126 MHz, D 2 O) 173.45, 153.88, 141.98, 126.21, 70.80, 55.68. HRMS (ESI - TOF) m / z 183.0809 [M + H] + ; calculated for C 8 H 11 N 2 O 3 : 183.0770. 4.2.4. Activity A ssay for the D etermination of the S ubstrate S pecificity of Tyc( Phe AT ) To identify productive isoserine substrates, each isoserine ( 2 mM ) was incubat ed separately with CoA (1 mM), ATP (1 mM), MgCl 2 (3 mM) and purified Tyc( Phe AT ) enzyme (1 mg) in 201 100 mM HEPES (pH 8.0 ) in a total volume of 1 m L . The reaction s w e re incubated at 31 °C. A liquots were withdrawn at 10 min, 20 min, 40 min, 1 h, 2 h, 4 h, 6 h and 8 h, and transferred to 96 well plates, quenched immediately with 10 % formic acid , and analyzed by LC - MS ( Quattro Premier XE Mass Spectrometer coupled with Acquity® UPLC system fitted with a C18 Ascentis Express column (2.5 × 50 mm, 2.7 µm) at 30 °C ) . A n aliquot ( ) of each sample was loaded onto the column , and the analytes were eluted with a solvent gradient of acetonitrile (Solvent A) and 0.05% triethylamine in distilled water (Solvent B) (held at 2.5% Solvent A for 3 .17 min, increased to 100% Solvent A over 5 sec with a 2 min hold, and then returned to 2.5% Solvent A over 5 sec with a 50 - sec hold) at a flow rate of 0.3 mL/min. 4.2.5. Apparent Rates of Tyc( Phe AT ) w ith the I soserine S ubstrates The conversion rate of Tyc(Phe AT) for thioesterification of the isoserine analog s to their isoserinyl CoA thioesters was determined by incubating each isoserine (2 mM) separately with ATP (1 mM), CoA (1 mM), MgCl 2 (3 mM) and purified Tyc( Phe AT ) enzyme (1 mg) i n 100 mM HEPES (pH 8.0 ) at 31 °C for 2 h in triplicate . Time course assays done using 1 mg Tyc(Phe AT) with the isoserine substrates provided a justification for the estimation of the relative rates to be at steady state at 2 h. At the end of each reaction , acetyl CoA (1 µM) was added as the internal standard to each sample to correct for losses during work - up . The biosynthetic thioester products were quantified by a liquid chromatography multiple reaction monitoring (M RM) mass spectrometry technique 39 on the Quattro Premier XE Mass Spectrometer with an elution gradient described in Section 4.2.4 . The effluent from the chromatography column was directed to a mass spectrometer where the first quadruple mass analyzer (in negative ion mode) was set to select for the molecul ar ion 202 of a biosynthesized acyl CoA . The selected ion was then directed to a collision gas chamber where the collision energy was optimized to maximize the abundance of a single signature fragment ion ( m / z 408.31) monitored in the second quadruple mass analyzer (in negative ion mode). The monitored ion was derived by a fragmentation reaction in the CoA moiety that is characteristic of acyl CoA thioesters analyzed by this MRM method. The peak area of the monitored fragment ion m / z 408.31 corresponding to each biosynthetic isoserinyl CoA thioester was converted to conce ntration by comparing the peak area of the same ion generated by authentic Co A using linear regression analysis. 4.2.6. Kinetic Evaluation of Tyc(Phe AT) , CoA , and Racemic (2 R ,3 S ) - Phenylisoserine Racemic (2 R ,3 S ) phenylisoserine at 5 2000 µM was incubated with ATP (1 mM), MgCl 2 (3 mM), CoA (1 mM), 100 mM HEPES (pH 8.0), and Tyc(Phe AT) ( 10 0 µg ·mL ) for 15 min. At the end of each reaction and prior to mass spectrometry analysis, acetyl CoA (1 µM) was added as the internal standard to each sample to correct for losses during workup. The biosynthetic products were quantified by a LC ESI MRM as described is Section 4.2.4. 4.3. Results and D iscussion 4.3.1. Synthesis of I soserine Analogs via Staudinger C ycloaddition R eaction The substituents on phenyl ring of the isoserine s used as substrates herein varied in their electronic properties and the size . The reported improved potency in paclitaxel analog s 203 containing modified isoserin yl moieties (see Table 4. 1 , Section 4.1.1 ) 11 , 19 , 40 guided the choice of some iso serines synthesized herein. Several methods for the synthesis of phenylisoserine analog s proceed through intermediary asymmetric epoxidation, 41 dihydroxylation, 42 chemoenzym ology , 43 aminohydroxylation , and Staudinger cycloaddition reactions. 44 Readily available starting materials make the la t ter method widely popular for - lactams . Also, unlike other methods, the Staudinger [2 + 2] cycloaddition reaction betwe en an imine and ketene is direct and facile for the synthesis of racemic isoserine analog s ( Figure 4.1 5 ) . Figure 4.1 5 . A scheme showing the synthesis of isoserine analog s . (i) (a) DCM , 4 Å molecular s ie ves , rt, 12 h, (b) TEA, acetoxyacetyl chloride , DCM, 0 °C, then rt, 2 5 h, (ii) ceric ammonium nitrate in H 2 O, CH 3 CN, 0 °C, 1 3 h, (iii) 7 N HCl, reflux, 24 h. The fi rst step involved the formation of a Schiff base between benzaldehyde analog s ( D 47 ) and p - anisidine ( D 48 ) . This base was then reacted with acetoxyacetyl chloride in the presence of triethylamine to form the cis lactam racemate via electrocyclic conrotatory ring lactam. 45 Oxidation by ceric ammonium nitrate removed the p - methoxy phenol group to afford the free amide . Ordinarily, treatm ent with base followed by acidic work up deprotected the hydroxyl group at C - lactam. However, reflux in 7 N HCl hydrolyzed D 51 to isoserine D 53 in a single step, shortening the route to isoserine racemates to three steps ( Figure 4.1 5 ) . 204 The pyridinylisoserines were synthesized with some modifications to the general method ( Figure 4.1 6 ). A benzhydryl amine intermediate was used as the amine source instead of p - anisidine. The synthesis of pyridin yl isoserine employed the [2 + 2] cycloaddit ion reaction step to form the lactam ring . A proposed deprotection sequence included acid - catalyzed hydrolysis of the lactam and O - acetyl group followed by hydrogenolysis to remove the benzhydryl moiety. Surprisingly, t he acidic reflux removed al l of the protecting groups, and the hydrogenolysis step was unnecessary ( Figure 4.1 6 ) . R 1 : 2 - P yridin yl ( D 54 a ), R: 3 - P yridin yl ( D 54 b ), R: 4 - P yridin yl ( D 54 c ) Figure 4.1 6 . S ynthesis of pyridi nyl i soserine isomers . (i) (a) DCM, 4 Å molecular si e ves , rt, 12 h, (b) TEA, acetoxyacetyl chloride , DCM, 0 °C, 3 h, ( ii) Acetonitrile, 7 N HCl, reflux, 24 h . The hydrolysis of the benzhydryl - protected pyridinyl lactams formed two products : t he expected aqueous soluble pyridin yl isoserine hydrochlorides and the benzhydrylacetamide whose identity was confirmed by 1 H - NMR . The H/D exchange NMR showed d isappearance of the doublet at 6.18 ppm, and loss of coupling to the proton resonance at 6.24 ppm upon addition of D 2 O ( Appen dix , Figure III 39 ). The gHSQC - NMR did not show evidence of a carbon correlating to the proton at 6.18 ppm. Using this information, the compound was identified as benzhydrylacetamide. This side product was formed through the amidation of benzhydryl carbocation intermediate by acetonitrile used to dissolve the starting material according to the 205 scheme below ( Figure 4.1 7 ). The reaction intermediate ( D 59 ) was identified by LC ESI MS anal ysis ( see Appendix, Figure III 42 ) . Figure 4.1 7 . Reaction mechanism for the formation of benzhydrylacetamide . 4.3.2. Relative Rates of Tyc(Phe AT) for Isoserine Substrate Analogs Racemic isoserine substrates synthesized by the Staudinger reaction (see Section 4.3.1 ) informed on the substrate scope of Tyc( Phe AT ). All substrates in which the phenyl ring had a substituent at ortho - (F, Cl, NO 2 ), para - (F, Cl, Br, Me, OH, and NO 2 ), or meta - (F, Cl, Br, Me, OH, CH 3 O and NO 2 ) position were converted to their acyl CoA by Tyc( Phe AT ) . The enzyme a ctivity was 206 also observed with the non - - - (thiophenyl) - isoserine analog s , but not with t he aliphatic groups (isopropyl - and tert - butyl isoserine) and pyridine analog s . The kinetic evaluation of racemic (2 R ,3 S ) - phenylisoserine showed a 12 - fold reduction in turnover rates of Tyc(Phe AT) (0.12 ± 0.010 min - 1 ) compared to that of the single isomer (2 R ,3 S ) - phenylisoserine (1.51 ± 0.17 min - 1 ). Also, t he data from initial analysis showed that the non - natural (2 S ,3 R ) - phenylisoserine inhibited Tyc(Phe AT) ( K I = 92.0 µM) in a (2 R ,3 S ) - phenylisoserine assay (see Chapter 3, Section 3.3.7.1) , thus confounding efforts to obtain accurate apparent Michaelis parameters for each racemic isoserine substrate tested herein. Therefore, the relative steady - state rate of Tyc( Phe AT ) for each racemic isoserine substrate was determined through analysis of the biosynthesized isoserinyl CoA by ESI MS MRM method . These assays, run at apparent steady - state gave an approximation of relative steady - state velocity of Tyc(Phe AT) for various isoserine analogs. The rate (7 nmol · h - 1 ) at which Tyc(Phe AT) converted (2 R ,3 S ) phenyl isoserine to (2 R ,3 S ) phenylisoserinyl CoA was set at 100 % and used for comparison against the apparent rates of Tyc( Phe AT ) for the other isoserine substrates at apparent saturation (also incubated at 2 mM) ( Table 4. 2 ). In general, the v app values of Tyc(Phe AT) for substrates with meta - substituents on the aryl ring w ere higher than for the para - and ortho - isomers . For example, the v app was ~1.5 - and 6 - fold lower for p - and o - fluoro isomers respectively compared to the m - fluoro isomer. Similarly, the v app for the methyl - substituent on phenyl ring decreased for ortho - methyl ( v app = below detection limit) and para - methyl ( v app = 1.13 nmol·h - 1 ) compared to meta - methyl substituent ( v app = 2.61 nmol·h - 1 ) ( Table 4. 2 ) . 207 Table 4. 2 . Relative rates of Tyc( Phe AT ) with ar yl - an d non - ar yl isoserine and CoA R v app (nmol · h - 1 ) v app R v app (nmol · h - 1 ) v app a 7.3 ± 0.91 100% l 0.22 ± 0.0 5 3.0% b 6.1 ± 0.41 84% m 1.1 ± 0.019 15% c 1.7 ± 0.27 24% n 0.04 ± 0.0 1 <1% d 1.4 ± 0.15 19% o 0.05 ± 0.0 1 <1% e 2.6 ± 0.15 36% p --- --- f 5.2 ± 0.10 72% q 0.98 ± 0.12 14% g 0.6 ± 0.07 8.2% r 0.11 ± 0.02 1.5% h 0.3 ± 0.03 3.9% s --- --- i 0.9 ± 0.09 12% t --- --- j 4.0 ± 1.0 55% u 0.18 ± 0.02 2.5% k 0.45 ± 0.12 6.2% v --- --- The dashed line ( --- ) indicates that the cor responding biosynthetic product was below the detection limit of the mass spectrometer. 208 Table 4. 2 . R v app (nmol · h - 1 ) v app R v app (nmol · h - 1 ) v app w 0.09 ± 0.005 1 .2 % z --- --- x --- --- ab --- --- y --- --- ac --- --- The v app values of Tyc(Phe AT) also decreased with size for the halogen substituents regardless of the position on the phenyl ring. For example, the fluoro substituent was converted to the CoA thioester the fastest in each category; o - F > o - Cl > o - Br ( v app = 0.98, 0.11, and 0.0 nmol·h - 1 respectively), p - F > p - Cl > p - Br ( v app = 4.0 , 0.45, and 0.22 nmol·h - 1 respectively), and m - F > m - Cl > m - Br ( v app = 6.1, 1.7, and 1.4 nmol·h - 1 respectively). The v app for the production of the acyl CoA analogs decreased also with the size of the para substituent (H > F > Cl > Br > OH ~ NO 2 > OMe), ranging from a v app = 3.98 nmol·h - 1 for p - fluoro to v app below detection limit for p - methoxy. Among the heteroaromatic analogs, only thiofuranylisoserine was converted to the CoA thioester b y Tyc(Phe AT) at v app = 0.89 nmol·h - 1 . There was no Tyc(Phe AT) activity observed for the pyridinylisoserine isomers. Similarly, the isopropyl - and trimethylisoserine analogs were not converted to their respective CoA thioesters in this study. 4.3.3. Docking (2 R ,3 S ) Phenylisoserine into Grs1(Phe A) Domain To gain further information about the binding interactions between the isoserine substrates and Tyc(Phe AT), (2 R ,3 S ) phenylisoserine was docked into the active site of Grs1(Phe A) which is 209 highly homologous to Tyc(Phe AT) ( Figure 4.1 8 ). In this model, the aryl ring of (2 R ,3 S ) phenylisoserine is docked according to that of the (2 S ) phenylalanine substrate in complex with Grs1(Phe A). The phenyl ring binding pocket is highly hydrophobic and consists of A236, W239, T278, I299, A322, I330, and C331 residues. The ortho - , meta - , and para - ring carbons point towards A236, T2 78 , and W239 , respectively (this is the residue numbering in Grs1(Phe - A) . These steric interactions may explain why the turnover of Tyc(Phe AT) for aromatic substrates with small substituents on phenyl ring is higher compared to the bulky substituents at similar positions on the phenyl ring . F or instance , the v app for m - fluoro was ~2.5 - fold higher compared to that for m - methylisoserine analog ( Table 4 . 2 ). It is also not surprising that the isoserine substrates with aliphatic side chains (namely isopropyl - , and trimethylisoserine analogs) were not converted to their CoA thioesters ( Table 4. 2 ). This is possibly due to bulky methyl groups, which would not bind to the Tyc(Phe AT) active site in a conformation that would enable a catalytic reaction to occur. Additionally, since the residues lining the active site are mainly hydrophobic, the substrates containing hydrophilic substituents (for example hydroxyp henylisoserine isomers) would not be favored during catalysis as evident from their lower v app compared to the hydrophobic ones (for example v app for m - methyl was ~9 - fold higher (2.6 nmol·h - 1 ) compared to the m - hydroxy (0.29 nmol·h - 1 )). It can also be argu ed that before binding, the hydrophilic substituents would require desolvation to break the hydrogen bonding network with water in the medium, which would require an additional step and hence kinetically unfavorable. 210 Figure 4.1 8 . (2 R ,3 S ) phenyl isoserine (green) docked into Grs1(Phe - A) active site , a close homolog of Tyc( Phe AT ). Chapter 3 describes the enantiospecificity of Tyc(Phe AT) for (2 R ,3 S ) phenylisoserine over (2 S ,3 R ) phenylisoserine in aminoacyl CoA biosynthesis. Further inhibition studies showed the non - productive enantiomer inhibited Tyc(Phe AT) ( K I = 92 .0 µM). The isoserine substrates described here were synthesized as racemates. Considering that the (2 S ,3 R ) isomer is an inhibitor, the intrinsic K M and k cat of Tyc(Phe AT) for the isoserines could not be calculated. To overcome this challenge, there is need to develop a method to separate the isoserine racemates prior to their conversion to thioesters. A lipase - catalyzed enantioselective method is proposed based on previously reported studies. 46 - 51 In general, strategies aimed at separation of racemic amino acids mainly employ enzymatic reactions such as N phenylacetylation by penicillin acylase from E. coli , 46 amide bond cleavage of L - amino acids b y an aminoacylase from pig kidneys, formation of L anilide using papain as a chemoselective catalyst, 47 , 48 , 51 and separation of amino acids using lipases. Of all the chiral catalysts investigated in resolution of racemates, lipases are the most extensively studie d. 49 For example, the ability of lipase B from Candida K517 Cys 331 I330 A322 D235 A236 W239 I299 T278 G324 211 antarctica - amino esters through hydrolysis was investigated. 50 A 100% enantioselectivity was reported in good yields (42 48% ). Additionally, a lipase PS ( Pseudomonal cepacia - aryl - - amino acids with upto 100% enantioselectivity and 50% yields. 52 Even more encouraging, an enzymatic strategy for separation of (2 R ,3 S ) phenylisoserine from the enantiomer using a lipase PS (from Burkholderia cepacia ) was also reported in high yields and enantioselectivity. 53 From these initial studies, lipase PS from two different organisms ( Pseudomonal cepacia and Burkholderia cepacia ) is the most ideal in the separation of the isoserine racemates described here. The high enantioselectivity, yields , wide substrate scope, and application in gram scale separation of racemic aryl amino acids make this lipase a promising choice in the future investigations with the isoserine analogs. 4.4. Conclusion Earlier substrate specificity studies showed that compared to other NRPS adenylation enzymes , TycA could adenylate an array of - amino acids and a few - amino acids such as ( S ) - - homophenylalanine. 36 , 37 These data suggested indirectly that these amino acids could co ntinue along the polyketide pathway to tyrocidine analogs. No isoserine analogs were tested except the bifunctional phenylserine isomer that was nearly as active ( k cat = 2.5 ± 0.2 s - 1 ) as the natural substrate - phenylalanine ( k cat = 3.6 ± 0.1 s - 1 ) . 36 Tyc(Phe AT) is involved in transient thioesterification of variously acylated AMP intermediates on its native pathway, and this guided the study herein to test the activity of Tyc(Phe AT) with an isoserine and CoA substrates. In addition, the crystal stru cture of the orthologous gramicidin synthetase 1 (Grs1(Phe A)) helped to select potentially productive isoserine analogs based on the modeled docking conformation of 212 (2 R ,3 S ) phenylisoserine . It was interesting that Tyc(Phe AT) could catalyze the thioesteri fication of isoserines since the active site shown previously has high structural complementarity to the natural substrate ( S ) - - phenylalanine. 37 T he hydrophobicity of the binding pocket residues that bind the aryl ring of the isoserines likely caused arylisoserines with small hydrophobic substituents to bind in a catalyticall y favorable orientation in the active site, thus speeding their turnover to the thioester products better than substrates with more hydrophilic substituents. Tyc(Phe AT) has similar relaxed substrate specificity as observed for other biocatalysts on the b iosynthetic pathway of specialized metabolites. 54 - 62 This is a desirable feature in synthetic biology when selecting biocatalysts to engineer a biosynthetic pathway that can recognize surrogate substrates. Thus Tyc(Phe AT) catalyst has significant potential to biosynthesize an array of aminoacyl CoAs that ca n be used by different acyl CoA dependent acyltransferases to construct rare bioactive natural product analogs. For example, Tyc(Phe AT) has direct application towards designing a biosynthetic route to produce paclitaxel analogs that contain modified aryli soserine side chains. To the best of my knowledge, there is currently no isoserine CoA ligase reported besides Tyc(Phe AT). 4.5. Future Direction The synthesis of prostate cancer drug, cabazetaxel (Jevtana ® ) proceeds through an intermediate similar to that to paclitaxel ( Figure 4.19 ). Foreseeably, Tyc(Phe AT) can be used in the semi - biosynthetic process by providing biosynthetically derived precursors that can be further elaborated chemi cally to make the final product ( Figure 4.19 ) without the need for protection groups. 213 Figure 4.19 . Synthesis of cabazetaxel (Jevtana ® ) . The broken arrow shows processes yet to be demonstrated in the proposed biosynthe s is. Some of the paclitaxel analogs that have shown promise in clinical trials contain pyridinyl in place of the phenyl ring at C - 3 ' (see Table 4.1 and Figure 4. 2 ) . Therefore, to support 214 (see Figure 4.4 ) , en gineering Tyc(Phe AT) to turnover pyridinyl substrates (such as in Tesetaxel) is proposed. In the literature, various crystal structures of enzymes that bind pyridinyl containing substrates contain residues capable of forming H bonding interaction with the nitrogen of the pyridinyl moiety in the binding pocket . 63 - 67 For example, the nitrogen of pyrimidinedione form s H - bonding interaction with the side chain hydroxyl of S367 in hepatitis CV NS5B polymerase inhibitor . 68 Additionally, a pyridylacetic acid derivative in complex with human dipeptidyl peptidase - 4 binds close to Y5 47. 69 Also, the pyridinyl nitrogen at position 6 of naphthyridine forms a hydrogen b ond with K384 of a protein kinase C protein. 70 In a different study, the nitrogen of pyridyldiaminothiazole (an inhibitor of cyclin - dependent kinase 2) forms H - bonding interaction with K33, 71 whereas a Q726 side chain amine binds next to the nitrogen of pyrazoloquinoline derivative in the phosphodiesterase 10A active site . 72 This trend in H - bonding interaction was also observed with N - arylamidopyridinylacetamide and H163 in SARS coronavirus 3Cl protease. 73 Thus, mutation of the a ryl binding site in Tyc(Phe - AT) to parallel the residues capable of H - bonding in the highlighted examples may enhance the binding of pyridinylisoserine analogs in a conformation favorable for catalysis. At a starting point, the proposed mutations would involve the residues that interact wit h the phenyl ring carbons at the ortho - , meta - , and para - positions, namely A2 24 , T2 66 , and W2 27 , respectively (this is the residue numbering in Tyc(Phe AT)) . 215 APPENDIX 216 3 F - Phenylisoserinyl CoA ; m / z 946.9 [ M H ] - Figure III 1 . MS/MS fragment ion pro file of the biosynthesized 3 - F phenyl isoserinyl CoA derived by Tyc( Phe AT ) catalysis is shown. The diagnostic fragment ions were identified as m / z : 946.9 [ M H ] - , 600.0 [ m / z 946.9 AMP] - , 425.8 (fragment a), 408.0 [ m / z 425.8 H 2 O] - . 217 3Cl - Phenylisoserinyl CoA ; m / z 964.3 [ M H ] - Figure III 2 . MS/MS fragment ion profile of the biosynthesized 3 - Cl phenyl isoserinyl CoA derived by Tyc( Phe AT ) catalysis is shown. The diagnostic fragment ions were identified as m / z : 964.3 [ M H ] - , 823.9 [ m / z 964.3 3 Cl - benzylamine] - , 426.3 (fragment a), 408.1 [ m / z 426.3 H 2 O] - . 218 3Br - Phenylisoserinyl CoA; m / z 1009.4 [ M H ] - Figure III 3 . MS/MS fragment ion pr ofile of the biosynthesized 3 - Br phenyl isoserinyl CoA derived by Tyc( Phe AT ) catalysis is shown. The diagnostic fragment ions were identified as m / z : 1009.4 [ M H ] - , 824.3 [ m / z 1009.4 3 - Br - benzylamine] - , 651.5 [b H 2 PO4 H 2 O] - , 425.9 (fragment a), 407.9 [ m / z 425.9 H 2 O] - , 329.6 [ m / z 407.9 H 2 PO 3 ] - . 219 3Me - Phenylisoserinyl CoA ; m / z 943.1 [ M H ] - Figure III 4 . MS/MS fragment ion profile of the biosynthesized 3 - Me phenyl isoserinyl CoA derived by Tyc( Phe AT ) catalysis is shown. The diagnostic fragment ions were identified as m / z : 943.1 [ M H ] - , 596.3 [ m / z 943.1 AMP] - , 515.9 [ m / z 596.3 HPO 3 ] - , 426.4 (fragment a), 408.4 [ m / z 426.4 H 2 O] - . 220 3NO 2 - Phenylisoserinyl CoA ; m / z 974.3 [ M H ] - Figure III 5 . MS/MS fragment ion profile of the biosynthesized 3 - NO 2 phenyl isoserinyl CoA derived by Tyc( Phe AT ) catalysis is shown. The diagnostic fragment ions were identified as m / z : 974.3 [ M H ] - , 957.7 [ m / z 974.3 OH] - , 839.0 [ m / z 974.3 adenine] - , 732.5 [ m / z 974.3 3 - NO 2 phenyl isoserinyl H 2 O] - , 685.9 [b H 2 PO 3 ] - . 407.9 [a H 2 O] - . 221 3OMe - Phenylisoserinyl CoA ; m / z 959.2 [ M H ] - Figure III 6 . MS/MS fragment ion profile of the biosynthesized 4 - OMe phenyl isoserinyl CoA derived by Tyc( Phe AT ) catalysis is shown. The diagnostic fragment ions were identified as m / z : 879.7 [ m / z 959.2 HPO 3 ] - , 667.8 [ m / z 879.7 4 - OMe phenyl isoserinyl H 2 O] - , 426.6 (fragment a), 407.9 [ m / z 426.6 H 2 O] - . 222 4OH - Phenylisoserinyl CoA ; m / z 945.0 [ M H ] - Figure III 7 . MS/MS fragment ion profile of the biosynthesized 4 - OH Phenylisoserinyl CoA derived by Tyc( Phe AT ) catalysis is shown. The diagnostic fragment ions were identified as m / z : 765.9 [ m / z 945.0 4 - OH phenyl isoserinyl] - , 686.5 [ m / z 765.9 H 2 PO 3 ] - , 616.4 [ m / z 945.0 adenosine] - , 518.6 [ m / z 616.4 HPO 4 ] - , 425.8 (fragment a), 407.8 [ m / z 425.8 H 2 O] - , 346.4 [ m / z 425.8 HPO 3 ] - , 133.8 [adenine] - . 223 Thiophenylisoserinyl CoA ; m / z 935.5 [ M H ] - Figure III 8 . MS/MS fragment ion profile of the biosynthesized thiophen yl isoserinyl CoA derived by Tyc( Phe AT ) catalysis is shown. The diagnostic fragment ions were identified as m / z : 935.5 [ M H ] - , 726.5 [b H 2 PO 3 H 2 O] - , 588.6 [ m / z 935.5 AMP H 2 O] - , 425.8 (fragment a), 408.0 [ m / z 425.8 H 2 O] - 356.9 [fragment c m / z 408 ] - , 328.2 [ m / z 408.0 HPO 3 ] - . 224 Cyclohexylisoserinyl CoA ; m / z 935.8 [ M H ] - Figure III 9 . MS/MS fragment ion profile of the biosynthesized cyclohexylisoserinyl CoA derived by Tyc( Phe AT ) catalysis is shown. The diagnostic fragment ions were identified as m / z : 863.4 [M 4(H 2 O) ] - , 764.7 (fragment a), 408.0 [fragment b H 2 O] - . 225 Figure III 10. 1 H Homodecoupling NMR spectrum of 3 - Hydroxy - 4 - (4 - fluoro phenyl)azetidin - 2 - one . Panel 1 shows the 1 H - NMR before decoupling where H - 2 ' gave a ddd ( J = 7.34, 4.89, 2.45 Hz) as a result of long range coupling with NH as well as 3 ' - H and 2 ' - OH protons . Panel 2 shows loss of coupling for H - 2 ' when NH was decoupled resulting to a dd (coupling with 3 ' - H and 2 ' - OH , J = 7.34, 4.89 Hz ) . The inset shows the change in splitting pattern of H - 2 ' before (blue) and after (green) 1 H Homodecoupling NMR. H - 2 H - 3 H - 3 H - 2 OH NH Aromatic H 226 Figure III 1 1. H 2 O/D 2 O exchange NMR spectrum of 3 - Hydroxy - 4 - (4 - fluoro phenyl)azetidin - 2 - one . Panel 1 shows the H - NMR before D 2 O exchange where OH and NH proton peaks are observed. Panel 2 shows loss of a doublet at 5.89 corresponding to 2 ' - OH , and a broad single t at 8.46 ppm corresponding to NH proton . Also, there is loss of coupling to the H - 2 ' which changed from a ddd to a doublet. This confirms coupling of the 2 ' - H with OH as well as NH. H - 3 H - 2 NH OH H - 2 H - 3 1 2 Aromatic H Aromatic H 227 Figure III 1 2 . 1 H - NMR spectrum of phenylisoserine . 2 3 5 - 9 D 2 O 228 Figure III 1 3 . 1 H - NMR spectrum of p - F phenyl isoserine . 2, 3 6&8 5&9 D 2 O 229 Figure III 1 4 . 1 H - NMR spectrum of p - Cl phenyl isoserine . 2, 3 6&8 5&9 D 2 O 230 Figure III 1 5 . 1 H - NMR spectrum of p - Br phenyl isoserine . 2,3 6&8 5&9 D 2 O 231 Figure III 1 6 . 1 H - NMR spectrum of p - Me phenyl isoserine . 2,3 5,6,8,9 CH 3 D 2 O 232 Figure III 1 7 . 1 H - NMR spectrum of p - O Me phenyl isoserine . 2, 3 CH 3 D 2 O 5&9 6 &8 233 Figure III 1 8 . 1 H - NMR spectrum of p - OH phenyl isoserine . 2,3 5&9 6&8 D 2 O 234 Figure III 1 9 . 1 H - NMR spectrum of p - NO 2 phenyl isoserine . The asterisk (*) denotes an impurity. 2 3 6&8 5&9 * D 2 O 235 Figure III 2 0 . 1 H - NMR spectrum of m - F phenyl isoserine . 2,3 8 5,7,9 D 2 O 236 Figure III 2 1 . 1 H - NMR spectrum of m - Cl phenyl isoserine . 2,3 5,7 8 * D 2 O 9 237 Figure III 2 2 . 1 H - NMR spectrum of m - Br phenyl isoserine . 2, 3 8,9 5,7 D 2 O 238 Figure III 2 3 . 1 H - NMR spectrum of m - Me phenyl isoserine . 2,3 8 7 9 5 CH 3 239 Figure III 2 4 . 1 H - NMR spectrum of m - OMe phenyl isoserine . 2,3 8 5,7,9 CH 3 * * 240 Figure III 2 5 . 1 H - NMR spectrum of m - OH phenyl isoserine . 2,3 8 5,7 9 D 2 O 241 Figure III 2 6 . 1 H - NMR spectrum of m - NO 2 phenyl isoserine . 2 3 9 7 5 8 * D 2 O 242 Figure III 2 7 . 1 H - NMR spectrum of o - F phenyl isoserine . 2 3 6 - 9 D 2 O 243 Figure III 2 8 . 1 H - NMR spectrum of o - Cl phenyl isoserine . 2 3 D 2 O 6 - 9 244 Figure III 2 9 . 1 H - NMR spectrum of o - Br phenyl isoserine . 2 3 8& 9 6 7 D 2 O 245 Figure III 3 0 . 1 H - NMR spectrum of o - Me phenyl isoserine . 2 3 CH 3 D 2 O 6 - 9 246 Figure III 3 1 . 1 H - NMR spectrum of o - OMe phenyl isoserine . 2, 3 7 9 CH 3 8 6 D 2 O 247 Figure III 3 2 . 1 H - NMR spectrum of o - NO 2 phenyl isoserine . 2 3 8 9 6 7 D 2 O 248 Figure III 3 3 . 1 H - NMR spectrum of trimethylisoserine . 3 2 t - Bu - CH 3 D 2 O 249 Figure III 3 4 . 1 H - NMR spectrum of cyclohexylisoserine . 3 2 Cyclohexyl protons D 2 O 250 Figure III 3 5 . 1 H - NMR spectrum of thiopheneisoserine . 2 3 8 6 7 D 2 O 251 Figure III 3 6 . 1 H - NMR spectrum of isopropylisoserine . 3 2 CH 3 4 252 Figure III 3 7 . 1 H - NMR spectrum of 2 - pyridinylisoserine . 2 3 7 6 9 8 253 Figure III 3 8 . 13 C - NMR spectrum o f 2 - pyridin y lisoserine . 3 2 7,9 4 6 1 8 254 Figure III 3 9 . 1 H - NMR spectrum of N - benzhydrylacetamide (bottom panel). H 2 O/D 2 O exchange NMR shows the disappearance of NH coupling to CH and the disappearance of NH peak (top panel). CH 3 D 2 O H - 3 Aromatic protons NH H - 3 255 Figure III 40 . 13 C - NMR spectrum of N - benzhydrylacetamide . 1 Aromatic carbons 3 CH 3 256 Figure III 41 . 1 H - NMR spectrum of 3 - pyridine isoserine . 2 3 7 5 9 8 * 257 Figure III 4 2 . LC - ESI - MS of intermediates and products obtained from the hydrolysis and deprotection of N - benzhydryl - 3 - acetoxy - 4 - (2 - pyridinyl)azetidin - 2 - one . 258 REFERENCES 2 59 REFERENCES (1) Skeel, R. T. Handbook of cancer chemotherapy ; Lippincott Williams & Wilkins: Philadelphia [etc.], 1999. (2) Jennewein, S.; Croteau, R. Appl. Microbiol. Biotechnol. 2001 , 57 , 13. (3) Ojima, I.; Chen, J.; Sun, L.; Borella, C. P.; Wang, T.; Miller, M. L.; Lin, S.; Geng, X.; Kuznetsova, L.; Qu, C. J. Med. Chem. 2008 , 51 , 3203. (4) Rowinsky, M., Eric K Ann u. Rev. Med. 1997 , 48 , 353. (5) Ojima, I.; Duclos, O.; Kuduk, S. 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Application of Bioengineering to Enhance Enzym at ic Catal ysis Bioengineering is undoubtedly a powerful technique in enzymology. 1 Site directed mutagenesis has been employed in various studies aimed at improving enzyme catalysis, substrate specificity , and selectivity. 2 , 3 For example, a single A312G mutation in phenylacetate tRNA synthetase increased the activity for (3 R ) phenylalanine by 4 fold . 2 In a different study, an H322E mutation in the AspA adenylation domain of surfactin synthetase changed the selectivity from aspartate to asparagine . 3 Additionally, computational - guided mutations in the adenylation domain (Grs1(Phe A)) of gramicidin synthetase 1 shifted its selectivity from phenylalanine to leucine after incorporating a double mutation T278L/A301G into the Grs1(Phe A) domain . 4 This last study heavily relied on structure function information rather than sequence ba sed analysis which enabled accurate and rapid predictions on substrate selectivity for Grs1(Phe A) mutants . 4 The successes of the earlier mutational studies prompted us to explore the effects of point - mutations of Tyc(Phe AT) for binding and catalyzing various value - added arylisoserine su bstrates. Some of our preliminary proposed mutations (Section 5.1.2) were guided by sequence homology; however, computational methods could potentially reduce the anecdotal trial - and - error approach towards obtaining beneficial mutations. Coupling computati onal based selection of protein mutations with structural and sequence homology studies is advantageous over traditional sequence alignment methods alone. 4 265 5.1.2. Tyc( Phe AT ) Point Mutation Sites to Expand the Aryl Ring Binding Site The A - domain of Tyc(Phe AT) was modeled on the structure of the homologous Grs1(Phe A) solved in complex with AMP and (2 S ) phenylalanine at 1.9 Å resolution . 5 As mentioned earlier in Chapters 3 and 4, the residues that surround the phenyl ring are mainly hydrophobic (A224, W227, T266, A289, A310, I318 and C319) and favor binding of hydrophobic aryl substrates . 6 Although the bindi ng sit e of Tyc(Phe AT) adenylation domain is highly specific for the natural substrate ( 2 S ) phenylalanine), an earlier study showed its substrate tolerance for other natural and unnatural amino acids 6 - 8 (see Chapter 4 , Figure 4.7 ). However, the k cat of Tyc(Phe AT) is reduced for substrate s containing substituents on the aryl ring compared to phenylalanine . 6 , 7 Also, when several arylisoserine w ere used as substrates, Tyc( Phe AT ) was biased against para - substituted analogs , show ing reduced activity (see C hapter 4 , Section 4.3.3) . Thus, to increase Tyc(Phe AT) substrate specificity for the ring - substituted arylisoserines and heteroarylisoserines, mutation s of the residues lining the binding pocket are potentially necessary. The Tyc( Phe AT ) model ( Figure 5.1 ) revealed that the residue, W 227 forms stacking interaction s with the phenyl ring of the substrate and is oriented next to the para - carbon. T herefore , W227 plays a key role in substrate binding. We hypothesized that mutation of this residue could effect change in the substrate scope of Tyc( Phe AT ) for para - substituted isoserine analogs . The binding sites of 200 homologous adenylation enzymes were surveyed using the ConSurf Program , 9 and tryptophan (W) occurs frequently (43%) , at the position similar to Tyc(Phe AT) W227 ( Figure 5.1 ). Whe n tryptophan was not present in the various homologs , there were instead natural replacements including hydrophilic residues (C, E, K, M, S, T) , which occur in 19% of the 200 homologs surveyed and smaller hydropho bic residues (A, G, I, L) 266 occurring in 20% of the homologs . The mutatio ns of Tyc( Phe AT ) herein were based on the occurrence of these residues. Figure 5.1 . Tyc( Phe AT ) model (grey cartoon) in complex with p henylalanine (yellow sticks) showing the active site residues (cyan). The F201, F202 , and F206 residues that border the binding pocket closest to the active site W227 are shown in blue sticks . The survey of Tyc(Phe AT) homologs showed that second shell residues border W227 . In Tyc( Phe AT ) , t hree second shell Phe residues (F201, F202, and F206) surround W 227 and likely align W227 for favorable stacking with the substrate . Considering the function of these second - shell residues towards supporting the residue at 227 of Tyc(Phe AT), the mutation of W 227 was accompanied by tandem mutations of the second tier Phe triad to residues that normally accompany the replacement residues found in the Tyc(Phe AT) homologs. The aim here was to maintain the architecture of the binding site. The tandem mutations of interes t are proposed to replace W227 with smaller residues ( Table 5.1 ). As a proof - of - concept, a few mutations were designed and tested for activity as described in the following s ections. W227 F201 F202 F206 267 Table 5.1 . The proposed mutations of F201, F202 , and F206 of Tyc( Phe AT ) in tandem with W227 replacement residues. W227A a W227S W227G W227Q W227L ARY b A WF Y W RY A Y F A L F AQF F AQ S QF Q N AWF AWY AYY SWF WYH WHL WHY WQI WQT WHY WNF W227T WAY WCY WFA WFN WKY WSY YCY YFV W227M WRY W227E W227K WRY WYC W227C a The residues in homologs that are position ed similarly to W227 in Tyc( Phe AT ) are indicated as W227B . b Three letter sequences indicate residues occurring in tandem with W227B in at least one of the 200 homologs that align with F201, F202 , and F206 respectively in Tyc( Phe AT ) ; FFF XYZ indicates proposed mutations of F201, F202 , and F206 respectively. 5.2. Experimental 5.2.1. Substrates, Reagents, and General Instrumentation C o A was purchased from Lee BioSolutions (St. Louis, MO), DNA oligos were obtained from IDT Corporation (San Jose, CA) , and Phusion DNA polymerase was purchased from New England BioLabs (Ipswich, MA). All the other reagents were obtained from Sigma Aldrich and were used without further purification, unless noted otherwise. Quattro Premier ESI MS coupled with Acquity® UPLC system was used for mass spectral analysis. 268 5.2.2. Tandem Site Directed Mutagenesis of t yc ( p he at ) cDNA 5.2.2.1. Mutation of Tyc( Phe AT ) to Tyc( Phe AT (W227A, FFF ) ) and Tyc( Phe AT ( W227A )) Tyc(Phe AT) homologs that have Ala in the place of W227 are accompanied by several different residues that align with the Phe triad (F201, F202 , and F206 in Tyc( Phe AT ) ) in the second shell ( Table 5.1 ) . Hence , mutations F201A, F202R , and F206Y were proposed based on the frequency of occurrence among Tyc( Phe AT ) homologs (residues underlined under W227A column , Table 5.1 ) . In the following procedure, the resultant mutant cDNA plasmids were sequence verified ( MSU Research Technology Support Facility: Genomics, East Lansing, MI) . In Chapter 3, tyc ( phe at ) cDNA encoding Tyc(Phe AT) was obtained by truncating tyc ( phe ate ) that encodes Tyc(Phe - ATE) (Chapter 3, Section 3.2.6). T he tyc ( phe at ) cDNA was used herein as the template to incorporate F201A mutation. The mutagenic oligonucleotide primers used to incorporate F201 A into tyc ( phe at ) were as follows (modified bases are underlined) : 5' F201A Forward primer GCC AAT TTG CAA TCC GC T TTC CAA AAT TCG TTT GGC 3' and 5' F201A Reverse primer GCC AAA CGA ATT TTG GAA A GC GGA TTG CAA ATT GGC 3'. The resultant plasmid was designated Tyc ( Phe AT ( F201 A) ) His and was used as a template to incorporate the W227A mutation. The mutagenic primers used to incorporate th is mutation are as follows (modified bases are underlined): 5' W227A Forward primer TCG TTC GAC GCA TCC GTT GCG GAA ATG TTC ATG GCT TTG 3' and 5' W227A Reverse primer CAA AGC CAT GAA CAT TTC CGC AAC GGA TGC GTC GAA CGA 3'. The resultant plasmid was designated Tyc(Phe AT(F201A : W227A)) His and was used as a template to incorporate the last two mutations (F202R an d F206Y) in a single s tep . The mutagenic primers used to incorporate these mutations are as follows (modified bases are 269 underlined): 5' F202R / F206Y Forward primer AAT TTG CAA TCC GCT CG C CAA AAT TCG T A T GGC GTC ACC GAG 3' and 5' F202R / F206Y Reverse primer CTC GGT GAC GCC A T A CGA ATT TTG G CG A GC GGA TTG CAA ATT 3' . The resultant plasmid was designated Tyc( Phe AT ( W227A: F201A :F202R: F206Y ) ) His . In a separate experiment, tyc ( phe at ) cDNA that encodes Tyc(Phe AT) was used as the template to incorporate a single W227A mutation by site directed mutagenesis . The mutagenic primers used to incorporate th is mutation are as follows (modified bases are underlined): 5' W227A Forward primer TCG TTC GAC GCA TCC GTT GCG GAA ATG TTC ATG GCT TTG 3' and 5' W227A Reverse primer CAA AGC CAT GAA CAT TTC CGC AAC GGA TGC GTC GAA CGA 3'. T he plasmid was designated Tyc( Phe AT ( W227A ) ) His. 5.2.2.2. Mutation of Tyc( Phe AT ) to Tyc( Phe AT (W227S, FFF ) and Tyc( Phe AT ( W227S ) ) Tyc( Phe AT ) homologs that have Ser positioned similarly to W227 generally have second shell replacements of F201 A and F202Q, based on frequency of occurrence among the homologs . Mutation of F206 in Tyc( Phe AT ) was not necessary since the Phe was already present in homologs in which W227 was replaced by Ser (residues underlined under W227S column in Table 5.1 ). A s imilar procedure as described in Section 5.2.2 above was followed in the introduction of the tandem mutations . Briefly, the t yc ( p he at ( F201A ) ) plas mid generated above ( Section 5.2. 2 .1) was used as the template to incorporate W227S mutation . The mutagenic primers used to incorporate th is mutation are as follows (modified bases are underlined): 5' W227S Forward primer TCG TTC GAC GCA TCC GTT TCG GAA ATG TTC ATG GCT TTG 3' and 5' W227S Reverse primer CAA AGC CAT GAA CAT TTC CGA AAC GGA 270 TGC GTC GAA CGA 3'. The resultant plasmid was designated Tyc( Phe AT (W227S : F201A ) ) His and was used as a template to incorporate the F202Q mutation. The mutagenic primers used to incorporate th is mutation are as follows (modified bases are underlined): 5' F202Q Forward primer GCC AAT TTG CAA TCC GC T CAG CAA AAT TCG TTT GG C GTC 3' and 5' F202Q Reverse primer GAC GCC AAA CGA ATT TTG CTG A GC GGA TTG CAA ATT GGC 3'. The resultant plasmid was designated Tyc( Phe AT (W227S : F201A : F202Q) ) His . In a separate experiment, tyc ( phe at ) cDNA that encodes Tyc(Phe AT) was used as the template to incorporate a single W227S mutation. The mutagenic primers used to incorporate this mutation are as follows (modified bases are underlined): 5' W227S Forward primer TCG TTC GAC GCA TCC GTT TCG GAA ATG TTC ATG GCT TTG 3' and 5' W227S Reverse primer CAA AGC CAT GAA CAT TTC CGA AAC GGA TGC GTC GAA CGA 3'. The resu ltant plasmid was designated Tyc( Phe AT ( W227S ) ) His. 5.2.3. Protein Expression and Act ivity Assays of Tyc( Phe AT ) Mutants w ith Phenylisoserine Analogs and CoA The t yc ( phe at (W227A) and t yc ( p he at ( W227S ) ) plasmids encoding a C term inal His tag (designated Tyc( Phe AT (W227A) ) His and Tyc( Phe AT (W227S ) ) His , respectively) described in Section s 5.2. 2.1 and 5.2.2.2 , were separately used to transform E . coli BL21 (DE3) cells. C ultures of E. coli (10 mL) transformed with pET28a vector were separately grown in LB medium supplemented with kanamycin (50 ·mL 1 ) at 37 °C for 12 h. The seed culture of each mutant was used separately to inoculate LB medium (1 L ) , and the bacteria grown at 37 °C to OD 600 ~0.6 . I sopropyl D 1 thiogalactopyranoside (IPTG) was added (0.5 mM final concentration ), and the culture was grown for 18 h at 16 °C. The cells were pelleted by 271 centrifugation (30 min, 4000 g ) at 4 °C, resuspended in Binding buffer (20 mM Tris HCl ( pH 7.8 ) , 0.5 M NaCl , and 5 mM imidazole ), lysed by sonication , and then centrifuged at 15,000 g for 0.5 h. The supernatant was decanted and centrifuged at 135,000 g for 1.5 h to remove cell ular debris. To test for functional expression of either Tyc( Phe AT (W227A) ) or Tyc( Phe AT (W227S ) ) mutants, a 1 - mL aliquot of the crude soluble enzyme was assayed separately with ( R ) phenylalanine, (2 R ,3 S ) phenyl - , 4 - OH phenyl - , or 4 - OMe phenylisoserine each at 2 mM in assays containing ATP (1 mM), CoA (1 mM) , and MgCl 2 (3 mM) in 100 mM HEPES ( pH 8.0). The assays were incubated at 31 ° C for 2 h, and then quenched to pH 3.0 using 10% aqueous formic acid ( ~50 µL ). The assays were then lyophilized to dryness and dissolved in dH 2 O ( 200 µL ) . The biosynthetic aminoacyl CoA products were analyzed on a Quattro Premier Electro Spray Mass Spectrometer coupled to an Acquity ® UPLC system fitted with a C18 Ascentis onto the column and the analytes eluted with a solvent gradient of acetonitrile (Solvent A) a n d 0.05% triethylamine in distilled water (Solvent B) : held at 2.5% Solvent A for 3.17 min, increased to 100% Solvent A over 5 sec with a 2 min hold, and then returned to 2.5% Solvent A over 5 sec with a 50 sec hold at a flow rate of 0.4 mL/min. The effluent from the column was directed to the mass spectrometer set to negative ion mode with a scan range of m / z 10 0 1 2 00 atomic mass units. The tyc ( phe at (W227A : F201A : F202R : F206Y ) ) and t yc ( p he at (W227S : F201A : F202Q ) ) plasmids encoding C term inal His tag s (designated Tyc( Phe AT (W227A : F201A : F202R : F206Y ) ) His and Tyc( Phe AT (W227S :F201A: F202Q ) ) His , respectively) were used separately to transform E . coli BL21 (DE3) cells. C ulture s of E. coli 272 (10 mL) transformed with pET28a vector were grown separately in LB medium supplemented with kanamycin (50 · mL 1 ) at 37 °C for 12 h. T he seed culture of each mutant was separately used to inoculate LB medium ( 1 L) and grown at 37 °C to OD 600 ~0.6, at which time IPTG was added to a final concentration of 0.5 mM, and the culture grown for 18 h at 16 °C. The cells were harvested, lysed and pelleted down as described above . The crude lysate obtained from each soluble enzyme fraction was used to test the activity of Tyc( Phe AT (W227A : F201A : F202R : F206Y) ) or Tyc( Phe AT (W227S : F201A : F202Q ) ) mutant. T he crude soluble enzyme fraction was separately assayed with ( R ) phenylalanine, (2 R ,3 S ) phenyl - , 4 - OH phenyl - , or 4 - OMe phenylisoserine each at 2 mM in assays containing ATP (1 mM), CoA (1 mM) , and MgCl 2 (3 mM) in 100 mM HEPES ( pH 8.0). The assays were incubated at 31 ° C for 2 h, and then quenched to pH 3.0 using 10% aqueous formic acid ( ). The assays were lyophilized to dryness and dissolved in dH 2 O ( 200 µL ) . The reaction s were analyzed using Quattro P remier LC MS as described in this section. 5.2.4. Protein Purification of Tyc( Phe AT ) Mutants Crude soluble proteins isolated from bacteria expressing the Tyc( Phe AT ) mutant s were separately loaded into nickel nitri lotriacetic affinity column (Qiagen, Valencia, CA) and eluted according to the protocol described by the manufacturer. The column was eluted with increasing concentration of imidazole (20 300 mM) in Binding buffe r (20 mM Tris HCl buffer containing 0.5 M NaCl and 5 mM imidazole at pH 7.8) . Fractions containing the His tagged enzymes were identified by SDS PAGE and Coomassie Blue staining. The enzyme solution (100 mL) for each purified enzyme was concentrated to 1 mL by size selective centrifugation (Centriprep 30,000 MWCO unit; Millipore, Billerica, MA). The Binding buffer was exchanged with Assay b uffer 273 (50 mM HEPES containing 100 mM NaCl and 1 mM EDTA at pH 8.0) over five dilution/concentration cycles. The e nzyme purity was estimated using SDS P A GE and Coomassie Blue staining. The extinction coefficient and molecular weight of the mutants ( 280 = 60,405 M 1 cm 1 and 69.57 kDa, respectively), were calculated, and t he concentration of each protein was measur ed at A 280 absorbance on a NanoDrop ND1000 Spectrophotometer (Thermo Scientific, Wilmington, DE) : T yc( Phe AT (W227S) His ( 15 mg/mL) , Tyc(Phe AT(W227A) His ( 1.5 mg/mL), Tyc(Phe AT(W227A:F201A:F202R:F206Y) His ( 0. 25 mg/mL ), and Tyc( Phe AT (W227S : F201A : F202Q) His)) ( 0.5 mg/mL ) . The purified proteins were stored at 80 °C. 5.2.5. Activity of Tyc( Phe AT ) Mutants w ith Isoserine Analogs Separate assays with ( R ) phenylalanine, ( R ) phenylalanine, (2 R ,3 S ) phenyl - , 4 - OH phenyl - , or 4 - OMe phenylisoserine ( for s ynthesis, see Chapter 4 , Section 4.2.1 ) at 2 mM concentration were carried out by incubating ATP (1 mM), CoA (1 mM), and MgCl 2 (3 mM) with each purified Tyc( Phe AT ) mutant (1 mg /mL ) in 100 mM HEPES (pH 8.0). The reactions were incubated for 2 h at 31 °C, and transferred to 96 well plates, quenched with 10% formic acid (10 µL) after which acetyl CoA was added as an internal standard (1 µM) and the reactions analyzed by LC MS as described earlier (Section 5.2.3) . Tyc(Phe - AT (W22 7S ) ) mutant showed activity in the conversion of ( R ) phenylalanine, (2 R ,3 S ) phenyl - , 4 - OH phenyl - , and 4 - OMe phenylisoserine to their corresponding CoA thioesters. T o test the substrate specificity of Tyc(Phe - AT(W227S)) mutant, isoserine analogs (Refer to Table 5.2) were used at 2 mM in separate assays containing the enzyme (1 mg/mL) , ATP (1 mM), CoA (1 mM), and MgCl 2 (3 mM) in 100 mM HEPES (pH 8.0). The reactions were 274 incubated for 2 h at 3 1 °C and transferred to 96 well plates, qu enched with 10% formic acid (10 µL) after which acetyl CoA was a dded as an internal standard (1 µM) . T he reactions were analyzed by LC MS as described earlier (Section 5.2.3). 5.3. Results and Disc ussion 5.3.1. Protein Expression and Purification of Tyc( Phe AT ) Mutants Four Tyc( Phe AT ) mutants (MW 69. 5 kDa) [ Tyc( Phe AT (W227A) ) His, Tyc( Phe AT (W227S) ) His, Tyc( Phe AT (W227A : F201A : F202R : F206Y ) ) His , and Tyc( Phe AT (W227S : F201A : F202Q) ) His ] were heterologously expressed in E. coli BL21(DE3) . To test for the expression of these mutants, protein in the crude lysate s were analyzed by SDS PAGE and Coomassie Blue staining ( Figure 5. 2 ) . The crude enzymes were purified separately using Ni affinity column and concentrated to ~0.25 15 mg/ mL ( Figure 5. 2 ). The yields of recovered purified enzyme was variable amongst the heterologous ly express ed Tyc(Phe AT) mutants . Tyc(Phe AT(W227S)) ( 15 mg/mL ) expressed the best. Tyc(Phe AT(W227A), Tyc(Phe AT(W227A : F201A : F202R : F206Y)), and Tyc(Phe AT(W227S : F201A : F202Q)) were obtained at low yields ranging from 0. 2 5 1.5 mg/mL. This was not too surprising, since seemingly minor changes in the expressed protein may affect expression levels . 10 275 A A B C 1 2 D Figure 5.2 . SDS polyacrylamide gel electrophoresis (12% acrylamide) and Coomassie blue staining of recombinantly expressed Tyc(Phe AT) mutants which were separately isolated from E. coli BL21(DE3). A ) Tyc(Phe AT(W227A)) His; Lane 1 A : crude lysate. The profile of the soluble protein eluted from nickel affinity resin chromatography with Bindin g buffer contained the following; Lane 2 A : 300 mM imidazole (fraction 1); Lane 3 A : Tyc(Phe A) (positive marker , 70 µg ); Lane 4 A : flow through; L: Molecular weight standard. B ) Tyc(Phe AT(W227A:F201A:F202R: F206Y)) His; Lane 1 B : crude lysate; Lane 2 B : empty; Lane 3 B : 100 mM imidazole (fraction 1, 5 µ L (from 25 mL elution) ); Lane 4 B : 300 mM imidazole (fraction 2, 0.75 µg ); Lane 5 B : 100 mM imidazole (fraction 1, 10 µ L ); Lane 6 B : 300 mM imidazole (fraction 2, 0.75 µg ). C ) Tyc(Phe AT(S563A)) His; C1 ) Lane 1 C : Flow through; Lane 2 C : 10 mM imidazole (wash 1); Lane 3 C : 20 mM imidazole (wash 2); Lane 4 C : 300 mM imidazole (fraction 1, 75 µg ); Lane 5 C : 300 mM imidazole (fraction 1, 150 µg ); Lane 6 C : Tyc(Phe AT) (positive marker , 100 µg ). C2) Lane 1 : 300 mM imidazole (fraction 1, 15 µg ). D ) Tyc(Phe AT(W227S: F201A : F202Q)) His; Lane 1 : 50 mM imidazole (fraction 1); Lane 2 : 100 mM imidazole (fraction 2); Lane 3 : 200 mM imidazole (fraction 3); Lane 4 : 30 0 mM imidazole (fra ction 4); Lane 5 : Tyc(Phe AT) (positive marker , 10 µg ). 5.3.2. Activity Assays of Tyc(Phe AT) Mutants with Phenylisoserine Analogs The activity of four Tyc(Phe AT) mutants was determined in separate assays containing ATP , MgCl 2 , CoA , and ( R ) phenylalanine, (2 R ,3 S ) phenyl , 4 - OH phenyl , or 4 - OMe phenylisoserine substrate. Analysis of the assay products by LC ESI MS showed aminophenylpropanoyl CoA products only in assays containing Tyc(Phe AT (W227S). No kDa 100 225 150 70 50 35 1 L 2 3 4 L 1 2 3 4 5 6 L 1 2 3 4 5 6 1 L L 1 2 3 4 5 69. 5 kDa 69. 5 kDa Lane: Lane: 276 aminophenylpropanoyl CoA product s were detected in assays containing crude or purified Ty c(Phe AT(W227A), Tyc(Phe AT(W227A : F201A : F202R : F206Y) , or Tyc(Phe AT(W227S : F201A : F202Q) mutants. 5.3.2.1. Activity Evaluation of Tyc( Phe AT (W227S) ) in the S creen for Isoserine Analogs in Isoserinyl CoA Biosynthesis The Tyc(Phe AT(W227S)) activity screen with isoserine analogs (see Chapter 4, Section 4.2.2) was done in separate assays . Each assay c ontained an isoserine substrate ( 2 mM) , ATP (1 mM) , CoA (1 mM), and Tyc( Phe AT (W227S)) (1 mg) . The assays were analyzed using Quattro Premier LC ESI MS in negative ion mode. It w as envisioned that the mutation of W 227 to smaller residues (for example Ala and Ser) would reduce steric interactions and enable binding of phenylisoserine analogs with bulky substituents on the para - carbon (for example , p - meth yl , p - meth oxy , and p - nitro phenylisoserine ) . Further , change s in the electronic properties of the binding site around the para - position from hydrophobic to hydrophilic (W227S) would enhance the binding of hydrophilic substituents through H - bonding interaction . There was a drastic reduction or complete loss in the activity of Tyc(Phe AT(W227S) for most variously substitute d aryl isoserine analogs compared to that of Tyc(Phe AT) ( Table 5.2 ) . Notably, 2 - F , 3 - Me , 3 - F , and 3 - Cl substituted phenylisoserines decrease d the turnover of Tyc(Phe AT(W227S)) by two - orders of magnitude compared to that of Tyc(Phe AT). T his suggests that W 227 play s a major role in positioning the phenyl ring of the substrate in a catalytically competent conformation . Tyc(Phe AT) and Tyc(Phe AT(W227S)) similarly turned over the para - substituted analogs , with the exception of 4 - F ph e n ylisoserine (the v app was ~ 10 0 - fold lower) . Surprisingly , 277 this mu tant turned over 4 OMe phenylisoserine and 2 pyridinylisoserine, which were not productive substrates of Tyc( Phe AT ) . Table 5.2 . The rates ( v app ) of Tyc( Phe AT (W227S) ) mutant in comparison to Tyc( Phe AT ) in the biosynthesis of isoserinyl CoA analogs . S ubstrate Tyc( Phe AT (W227S) ) v app (nmol · h 1 ) (×10 2 ) Tyc( Phe AT ) v app (nmol · h 1 ) Phenylisoserine 4.4 7. 3 ± 0.91 3 - F Phenylisoserine 2.8 6.1 ± 0.41 3 - Cl Phenylisoserine 1.7 1.7 ± 0.27 3 - Br Phenylisoserine 1.8 1.4 ± 0.15 3 - Me Phenylisoserine 0. 6 2.6 ± 0.15 3 - NO 2 Phenylisoserine *ND 5.2 ± 0.10 3 - OMe Phenylisoserine 1.5 0.6 ± 0.07 3 - OH Phenylisoserine ND 0.3 ± 0.03 Thiophenylisoserine ND 0.9 ± 0.09 4 - F Phenylisoserine 4.6 4.0 ± 1.0 4 - Cl Phenylisoserine 40 0.45 ± 0.12 4 - Br Phenylisoserine 7.3 0.22 ± 0.05 4 - Me Phenylisoserine 42 1.1 ± 0.019 4 - OH Phenylisoserine ND 0.04 ± 0.01 4 - NO 2 Phenylisoserine ND 0.05 ± 0.01 4 - OMe Phenylisoserine 4 . 4 ND 2 - F Phenylisoserine 0.5 0.98 ± 0.12 2 - Cl Phenylisoserine ND 0.11 ± 0.02 2 - NO 2 Phenylisoserine ND 0.18 ± 0.02 Cyclohexylisoserine 0.2 0.09 ± 0.0 1 2 - Pyridinylisoserine 0.4 ND *ND indicates that the rates of either enzyme for these substrates could not be determined because their respective CoA thioester products were below detection limit of the LC ESI MS. Th e recovered catalytic activity of the Tyc( Phe AT (W227S)) mutant for 4 OMe phenylisoserine to its CoA thioester suggest that the steric barrier present in Tyc(Phe AT) was likely overcome by the W227S substitution , which supports our hypothesis. It is not immediately evident, however, why the Tyc( Phe AT (W227S) ) muta nt was able to catalyze 2 278 pyridinylisoserine to its CoA thio ester. O ne can imagine that the pyridinyl nitrogen is able to H - bond to the backbone amide of an active site residue placing the substrate in the correct trajectory for catalysis (See Chapter 4, Section 4.3.3) . In summary , t he Tyc( Phe AT (W227S) ) mutation showed new activity for 4 OMe phenylisoserine and 2 pyridinylisoserine as discussed above . As part of future direction , mutation of the residues around the aryl binding pocket in Tyc(Phe AT) would be necessary in improving the substrate specificity. The mutations outlined in Table 5.1 target W 227 and aim at improving the binding of para - substituted phenylisoserine substrates. As mentioned previously , computational guided predictions may provide information abou t favorable mutations that would likely improv e Tyc( Phe AT ) activity and substrate specificity with the isoserine analogs , and also reduce the work load involved in the biochemical characterization of the sequence guided mutations. One approach towards designing plausible mutations is through docking the isoserine substrates into the structure models of Tyc(Phe AT) mutants bearing different mutations around the phenyl ring. This approach was previously demonstrated with Grs1(Phe A) . 4 In this study, t he computationally predicted Grs1(Phe A) mutants showed increase in the enzyme catalytic efficiency ( k cat / K M ) for non - natural substrates compared to the wild - type enzyme. 4 For example, Grs1(Phe A( T278L / A301G)) mutant showed a 19 - fold increase in the k cat / K M with leucine in comparison to the wild - type Grs1(Phe A) . 4 5.4. Tyc( Phe AT ) and BAPT Coupled Assays Towards the Biosynthesis of Precursors of Paclitaxel Analogs The structure activity relationship studies of paclitaxel revealed the importance of C 13 side chain bearing the correct stereochemistry for biological activity. 11 - 13 The biosynthetic 279 incorporation of C 13 side chain is dependent on the cataly tic activity of a 13 O 3 aminophenylpropanoyltransferase (BAPT) that transfers aminoacyl groups from aminoacyl CoA thioesters to baccatin III ( C hapter 4 , Section 4.1.3 ) . From the studies described in C hapters 3 and 4, Tyc( Phe AT ) catalyzes the biosynthesis of various aminoacyl CoA thioesters. Thus , coupli ng the reactions catalyzed by Tyc( Phe AT ) and BAPT ( Figure 5.3 ) has the potential to overcome the challenges associated with the semisynthesis of paclitaxel, namely multiple reaction steps and hazardous reagents 14 , 15 (see Chapter 1, Section s 1. 2 and 1.3 ). Also, a biosynthetic method is environmental ly friendly and does not incur huge expenses required for solvent disposal. Figure 5.3. Proposed Tyc(Phe AT) and BAPT - coupled assays aimed at novel biosynthesis of paclitaxel analogs . 280 REFERENCES 281 REFERENCES (1) Brannigan, J. A.; Wilkinson, A. J. Nat. Rev. Mol. Cell Biol. 2002 , 3 , 964. (2) Koetsier, M. J.; Jekel, P. A.; Wijma, H. J.; Bovenberg, R. A. L.; Janssen, D. B. FEBS Lett. 2011 , 585 , 893. (3) Stevens, B. W.; Lilien, R. H.; Georgiev, I.; Donald, B. R.; Anderson, A. C. Biochemistry 2006 , 45 , 15495. 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