BIOCATALYSIS OF AROMATIC - HYDROXY - - AMINO ACIDS VIA REGIO - AND STEREOSELECTIVE AMINATION OF TRANS - 3 - ARYL GLYCIDATES U SING A PHENYLALANINE AMINOMUTASE By Prakash Kumar Shee A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemistry Doctor of Philosophy 20 20 ABSTRACT - HYDROXY - - AMINO ACIDS VIA REGIO - AND STEREOSELECTIVE AMINATION OF TRANS - 3 - ARYLGLYCIDATES USING A PHENYLALANINE AMINOMUTASE By Pra kash Kumar Shee Biocatalytic process - development continue s to advance to ward discovering alternative transformation reactions to synthesize medicinally important molecules such as, - h ydroxy - - amino acids . These bifunctional building blocks, a subclass of noncanonical amino acids , have two stereocenters and are v aluable in the natural product, pharmaceut ical, and agrochemical sectors . Here, a 5 - methylidene - 3,5 - dihydro - 4 H - imidazol - 4 - one (MIO) - dependent phenylalanine aminomutase from Taxus canadensis ( Tc PAM ) w as repurposed to ir reversibl y bio catalyze an intermolecular amino group transfer regioselectively from (2 S ) - styryl - - alanine to ring - substituted racemic trans - 3 - arylglycidates (i.e., cinnamate epoxide s) to make the corresponding arylserines. The reaction scope of MIO - aminomutases, which primarily catalyze the reversible interconversion - - amino acids, can also catalyze the hydroamination - - amino acids. Here, we explore glycidates as a new class of substrates for the MIO - amin omutase family of enzymes. Racemic trans - 3 - arylglycidates were regio - and stereoselectively aminat ed to produce a mixture of anti - arylserine enantiomers predominantly . 3 - Arylglycidates usually prefer nucleophilic attack at the benzylic C due to stabilizat ion of the partial cationic properties + ) at the C by the aryl ring, a stabilization that does not occur at C . Tc PAM catalysis , however, inverted this inherent nucleophilic regioselectivity by aminating at the C of trans - 3 - ar ylglycidates to make ar ylserine predominantly (97%) over aryl iso s erine ( 3 % ) . From among twelve substrates, t he aminomutase r ing - opened 3 ' - Cl - phenylglycidate to 3 ' - Cl - phenylserine 140 times faster than it opened the 4' - Cl - isomer , which was turned over slowest among all epoxides tested. GC/MS analysis of c hiral auxiliary derivatives of the biocatalyzed ar ylserine analogue s showed that each product mixture contained (2 S )+ ( 2 R ) - anti and (2 S )+(2 R ) - syn pairs with the anti - isomers predominating (~90:10 dr ). Integrating the vicinal proton signals in the 1 H - NMR spectrum of the biocatalyzed ar ylserines and calculating the chemical shift di anti and syn proton signals confirmed the diastereomeric ratios and relative stereochemistries . Application of a ( 2 S ) - threonine aldolase from E. coli further established the absolute stereoche mistry of the chiral derivatives of th e diastereomeric biocatalyzed products. The 2 R :2 S ratio for the biocatalyzed anti - isomers was highest (88:12) for 3 ' - NO 2 - phenylserine and lowest (66:34) for 4 ' - F - phenylserine . This showed that the stereospecificity of Tc PAM is , in part , directed by the sub stituent - type on the arylglycidate analogue. We also synthesized enantiopure 3 - phenylglycidates and incubated them separately with Tc PAM. The absolute configurations of the biocatalyzed a nti - phenylserine (major) and phenyl iso serine (minor) were evaluated t o gain insights on the substrate specificity and selectivity of Tc PAM for aminating 3 - phenylglycidate enantiomers. Tc PAM converted (2 S ,3 R ) - 3 - phenylglycidate to (2 S ) - anti - phenylserine predominantly ( 89 %) and (2 R ,3 S ) - 3 - phenylglycidate to (2 R ) - anti - phenylseri ne (88%) over their antipode s with inversion of configuration at C in each case . Both glycidate enantiomers formed a small amount (~10%) of the syn - phenylserine with retention of configuration at the C . Tc PAM had a slight preference toward (2 S ,3 R ) - 3 - phenylglycidate, which was turned over ( = 0.3 min - 1 ) 1.5 t imes faster than the (2 R ,3 S ) - glycidate ( = 0.2 min - 1 ). The k inetics data showed that the a mination of arylglycidate process follows a two - substrate ping - pong mechanism with competitive inhibition by the epoxide substrate at higher concentration. Copyright by PRAKASH KUMAR SHEE 2020 v I dedicate this dissertation to my parents for their countless sacrifices to fulfill my dream, my brother for taking all the blows up front and paving me a smoother road, and my s ister for su pporting me always . You all made it possible for me to become who I am today. vi ACKNOWLEDGMENTS Behind every doctorate, and every doctoral student, are people who played a vital role in guiding, inspiring, and supporting its recipient. While I cannot hope to thank everyone who contributed as professors, peers, or friends, I would, here, like to acknowledge those who have had among the most profound impacts upon my research and this dissertation. I would first like to thank my advisor, Dr. Kevin D. Walker, for the extensive training, guidance, encouragement and motivation, which extended far beyond the laboratory, helped me to grow as an independent researcher. T his dissertation and its associated publications would not have been irreplaceable role Dr. Walker played in my academic development. I am similarly thankfu l to the members of the graduate advisory committee: Prof. Xuefei Huang (second reader), Prof. James H. Geiger, Prof. John W. Frost, and Prof. Gary J. Blanchard. Prof. Huang mentored me throughout graduate school. I really appreciate his invaluable support during the final phase of my dissertation . Prof. Geiger, too, provided me with consistently engaging intellectual discussion and feedback, providing much - needed perspective on the structure and mechanism of our protein molecule. I am similarly indebted to Prof. Frost, whose inputs and guidance on the characterization of the enantiopure glycidols is greatly appreciated. I must also thank Prof. Blanchard, who offered me a doctoral position at Michigan State University. Prof. Blanchard has remained a stalwart ally in the many years since, and also wrote a number of recommendation letters that helped me obtain much - needed scholarships and funding. vii Furthermore, I would like to extend my sincere appreciation both to Prof. A. Daniel Jones and his entire team at Ma s s Spectrometry and Metabolomics Core. Prof. Jones taught me all of the minute details and fine parameters of using complex mass spectrometry instruments. Prof. Jones was always available for discussions , and helped me troubleshoot a great many of the prob lems I experienced with LCMS or GCMS in my graduate life. The expertise I gained in his laboratory played a pivotal role in my pursuit of a career. I would like to thank Lijun Chen, Dr. Tony Schilmiller, and Dr. Casey Johnny for all the extensiv e training and enormous support during the frequent usage of the Mass Spectrometry. At the MTR - NMR Facility, Dr. Daniel Holmes helped me develop a novel method to establish ce was cruc ial to the development of my dissertation. I cannot possibly overstate my thanks to Dr. Holmes for the multitude of suggestions and the guidance he offered in designing many key experiments. I would like to recognize and thank Prof. Honggao Yan (BMB, MSU ), whose collaboration helped deduce a kinetic equation for a two substrate ping - pong mechanism . This work provided I would like to extent my sincer e appreciat ion to Prof. Babak Borhan, who graciously shared his laboratory space and all the resources it encompasses. On countless occasions, I have set up moisture and temperature sensitive epoxidation reactions in the Borhan laboratory, and synthesized compounds o f interest. Prof. Robert E. Maleczka (Chair) was unerringly supportive. Near the end of my studies, he was actively involved in advocating for a safe return to research during the 2020 coronavirus is likely I would have never been able to finish my research timely. viii I would also, yet again, like to thank my mentors and peers in the Walker Laboratory. They are all, without exceptional, extremely resourceful professionals who made my doctoral candidacy a success. To Dr. Nishanka Dilini Ratnayake and Dr. Edith N. Onyeozili: I would like both of you to know that you are nothing short of amazing, and I am vastly grateful for your introduction to this research project. Furthermore, both of you made vital co ntributions to the initial stages of the project which undoubtedly laid the foundations for my dissertation. Dr. Ruth N jeri Muchiri: you treated me like an younger brother, and I am thankful for your mentorship and guidance. You were approachable and infor mative, and I truly enjoyed learning from and alongside you. Much of my time in the Walker Laboratory was spent with Dr. Tyler Walter, with whom I made many memories. I enjoyed my time at Michigan State with you, Dr. Walter, and must also thank you for the cultural e ducation you imparted. It was, like so many other things you have done, par excellence. I wish you the best in all of your future endeavors. I would also like to thank Jean - Bosco Shingiro, Gayanthi Attanayake, and Aimen Al - Hilfi for making my la st years at Michigan State even more memorable. You all made and fostered an accepting, friendly work environment. I hope for success for all of you in your continued research. Many thanks to all of the fantastic undergraduates who worked with me at the Wa lker Labora tory. Of special note: Olivia, Brendyn, Lawrence, Shahrazad, Jeshua, Ciara, and Arianna. Not only were you all great partners in the laboratory, but you provided phenomenal technical support, too. Olivia: your contribution with the aldolase enzy me was high ly significant to the shape and form of my dissertation. Thank you! Alongside the Walker Laboratory, I vastly appreciate the current and past members of the Wulff, Borhan, Maleczka, Jackson, Weliky, Geiger and Smith groups for sharing their reso urces. I wa s always welcomed in your laboratories; your courtesy meant much, both in the provision of ix resources and in the construction of my professional identity. Of the members of these groups, I would like to individually thank Wei, Xiaopeng, Yubai, Ru wanthi, Tay eb, Pengchao, Hadi Gholami, Ali Akbar, Debarshi, Soham, Ankush, James, Saeedeh, Dan, Arzoo, Badru - Dean, and Hadi Nayebi. I will, most certainly, miss sharing ideas with each of you, as well as your camaraderie. At the institutional level, I rema in indebted to the Department of Chemistry, the Department of Biochemistry and Molecular Biology, the Michigan State University College of Natural Sciences, and the Graduate School for providing me with assistantships and fellowships, all necessary for the continuati on of my research and graduate life at Michigan State and in the United States. To those of you overseas: I, too, remain as indebted to you as anyone else. My professors at the Indian Institute of Technology Bombay, and before that at Jadavpur U niversity, sparked and fanned my passions for sciences. They brought me to Chemistry, and they helped fulfil my ambition of finding a high - quality doctoral program. I must particularly offer my never - ending thanks to Prof. Sambasivarao Kotha, who patiently guided me throughout my M.Sc. research project. Prof. Swadesh R. Roy Chowdhury, and Prof. Sanjay Bhar at Jadavpur University, believed in me from the beginnings of my higher education. They were moral bulwarks and educational champions, who lent me the ve ry essentia l support I needed as an underprivileged student. And, to my friends: you made my eight - year - long stay at Michigan State and in East Lansing a pleasure. Perhaps more importantly, you helped keep me sane! I, like most every other graduate student , had my ha rd times. You all made it possible to retain some energy, and to power through with a happy, still - intact soul. I love each of you with all my heart, and I accord special thanks to Dr. Dhritabrata Mandal, Dr. Saurja Dasgupta, Dr. Kalyan Santra, Dr. Krishna ja Duvvuri, Dr. Ananya Rakshit, Dr. Sanhita Sinharay, Dr. Tamal K . Ghosh, Dr. Anup Adhikari, Dr. Raza Haque, x Dr. Zakia Alavi, Dr. Ali Haque, Beena Haque, Dr. Abhishek Dutta Chuwdhury, Dr. Tarang Chugh, Yashesh Dhebar, George Kapali, Ryan Farrick , and (Dr.) Supriya Ghosh, for their constant love and support, intellectual input, and suggestions in writing this dissertation. No amount of appreciation is enough for all the beautiful people who supported me selflessly during my undergraduate studies, when higher education seemed a scarcely - affordable dream. My sincere thanks and gratitude go to Mrs. Ajanta Sadhu (didi), Mr. Arindam Sadhu, Mr. Abhijit Sengupta (kaku), the Srijan Sujan family, and the late Mr. Gautam Sen. You all stood by me and believed in me when I desperately needed your love and support. You made me part of your families, and I could never have asked for me. I will always love each and every one of you, no matter the oceans now between us. In my youth, too, I was also exceptionally fo rtunate to have a friend, a philosophic mentor, and a guide in Dr. Souvagya Biswas (Vagyo Da). He inspired me from my time at Jadavpur, and has motivated me ever since. He mentored me, and he treated me like a younger brother. Vagyo Da prepared me for ever y hurdle I could have faced as a young student, from the IIT entrance exam to the infamous GRE. Whatever I needed, he was able to help, from applications to universities abroad, to my first flight, graduate frustrations, job hunt, and thesis defense. You h ave long be en my mentor, as well as a brother and there is nothing more I could ask of you that you did not offer. I will, most certainly, miss staying within an easy drive of your home. Lastly, but never least, there is not a single chance I could have go tten to whe re I am now without the unconditional, unwavering support of my family. I owe every shred of my success to you. Maa and Baba: you raised me right, even though it meant making what must have been millions of sacrifices. Maa, you gave me my passio n, the abil ity to dream beyond where I was. You showed me the power of education, and its rare ability to uplift the impoverished. And Baba, xi while your love language was always silent, I never once questioned its presence. You worked, relentlessly and tire lessly, to raise three children, give them food, and the best education we could afford. You were generous with your family, and with all around you; I know, even at work, you never stopped thinking of ways to our obstacles. Even now, writing this, I still cannot ima gine what strength it must have taken for you to do all that you did; you are my true hero, and my enduring inspiration. Maa and Baba, I cannot give enough words to appreciate your sacrifices, for myself, for my brother, and for my sister. And i t was my ol You are the one who first lit my interest, and you are the reason I chose to pursue chemistry. I may have followed in your footsteps blindly for a time, but your wisdom a nd directio n have given meaning to my professional life. I am forever grateful that you chose to take responsibility for my young life, to share your success, to help me grow in comfortable shade as you stood against and endured the worst of weather. My si ster, Mon, you supported me through my worst days when I was depressed and directionless. You never, not once, failed to brighten my day, to give me new resolve. Thank you for putting up with me and being my strongest supporter . I love you with all my hear t. Lastly, Boudi, my sister - in - law and Ryka, I am sorry that I lost so much time with our family while chasing my dream. Thank you for never making me question my path, for always making me smile whenever we talked. I hope, now that I am passing one stage of life for another, that I can make up for the years I have been gone. All of you, my family: I would be nothing without your love, your protection, and guidance. I will love you always, no matter where I may be, no matter our separation, no matter this d istance of necessity. Thank you. xii TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ....................... xiv LIST OF FIGURES ................................ ................................ ................................ ...................... xv LIST OF SCHEMES ................................ ................................ ................................ .................. xxvi KEY TO ABBREVIATIONS ................................ ................................ ................................ ... xxvii Chapter 1: Overview of Biocatalysis in Organic Chemistry and Pharmaceutical Industry ............ 1 REFERENCES ................................ ................................ ................................ ......................... 13 Chapter 2: Biocatalysis of Arylserines and Arylisoserines using Phenylalanine Aminomutase from Taxus canadensis ( Tc PAM) and 3 - Arylglycidate Racemates. ................................ ... 20 2.1 Introduction ................................ ................................ ................................ ......................... 20 2.1.1 Aminomutases: Enzyme Class (EC) of 1,2 - Amino Acid Isomerases ....................... 20 2.1.2 Mechanistic Diversity of Aminomutases ................................ ................................ .. 21 2.1.3 MIO - depe ndent Aminomutases ................................ ................................ ................. 21 2.1.4 Overview of Aryl Amino Acid Aminomutases ................................ ......................... 23 2.1.5 MIO Function ................................ ................................ ................................ ............ 24 2.1.6 Intramolecular and Intermolecular Mechanisms of MIO - Aminomutases ................. 27 2.1.7 Arylserines and Arylisoserines ................................ ................................ .................. 30 2.2 Experimental ................................ ................................ ................................ ....................... 36 2.2.1 Chemicals and Reagents ................................ ................................ ............................ 36 2.2.2 Instrumentation ................................ ................................ ................................ .......... 37 2.2.3 General Procedure for the Syntheses of trans - 3 - Arylglycidate Analogues .............. 37 2.2.4 Stereochemical Analysis of trans - 3 - Arylglycidates using GC/EI - MS. .................... 41 2.2.5 Stability of Racemic Arylglycidates in Assay Buffer ................................ ............... 42 2.2.6 Expression and Purification of Tc PAM. ................................ ................................ .... 43 2.2.7 Expression and Purification of ( 2 S ) - Threonine Aldolase ( ltaE ). .............................. 44 2.2.8 Control Assay Experiments: Activity of Tc PAM toward Arylglycidates ................. 45 2.2.9 Biocatalysis of Arylserine and Arylisoserine with Tc PAM. ................................ ..... 45 2.2.10 Measurement of Kinetic Parameters. ................................ ................................ ...... 45 2.2.11 General Procedure for the Syntheses of Arylserines. ................................ .............. 46 2.2.12 Establishing Relative Stereoconfiguration ( syn / anti ) of Arylserine Diastereomers using 1 H NMR Spectroscopy. ................................ ................................ ................ 48 2.2.13 General Method for Derivatizing Arylserines with a Chiral Auxiliary. .................. 48 2.2.14 (2 S ) - Threonine Aldolase catalyzed resolution of Arylserine Diastereomers .......... 50 2.2.15 Computation al Methods for Biomolecular Docking. ................................ .............. 50 2.2.16 Calculation of Covalent van der Waals Volumes. ................................ ................... 51 2.3 Results and Discussion ................................ ................................ ................................ ....... 52 2.3.1 Synthesis and Characterization of the Racemic 3 - Arylglycidate Substrates. ............ 52 2.3.2 Separation of trans - 3 - Arylglycidate Enantiomers using a Chiral - GC/EI - MS ......... 53 2.3.3 Hydrolysis of Arylglyc idates in Assay Buffer ................................ .......................... 54 xiii 2.3.4 Control - Assay Experiments ................................ ................................ ....................... 56 2.3.5 Biocatalysis of Aryl serines and Arylisoserines. ................................ ........................ 58 2.3.6 Synthesis of Arylserine Diastereomers. ................................ ................................ .... 60 2.3.7 Assessing the Regiochemistry of the Tc PAM - catalyzed Transamination Reaction. 61 2.3.8 Establishing Relative Stereoconfiguration of Arylserines by 1 H NMR Analysis. .... 63 2.3.9 Relative Stereochemistry of the Tc PAM Reaction by Chiral Auxiliary Derivatization. ................................ ................................ ................................ ................................ 66 2.3.10 Absolute Stereochemistry of the Tc PAM Reaction by Aldolase Resolution. ......... 67 2.3.11 Epoxide Subst rate Docking Model of Tc PAM . ................................ ....................... 69 2.3.12 Kinetics of Arylserine Biocatalysis. ................................ ................................ ........ 73 2.3.13 Kinetics of Aryl iso serine Biocatalysis. ................................ ................................ ... 75 2.4 Conclusion ................................ ................................ ................................ .......................... 77 APPENDIX ................................ ................................ ................................ ............................... 80 REFERENCES ................................ ................................ ................................ ....................... 127 Chapter 3: Insight into the Mechanism of Regio - and Stereoselective Amination of Enantiopure 3 - Phenylglycidate Isomers to Phenylserine by Tc PAM. ................................ .............. 141 3.1 Introduction ................................ ................................ ................................ ....................... 141 3.2 Experimental ................................ ................................ ................................ ..................... 144 3.2.1 Chemicals and Reagents ................................ ................................ .......................... 144 3.2.2 Synthesis of (2 R ,3 R ) - 3 - Phenylglycidol ( 21a ) 35 ................................ ....................... 144 3.2.3 Synthesis of (2 S ,3 S ) - 3 - Phenylglycidol ( 21b ). ................................ ......................... 145 3.2.4 General Procedure to Characterize the Enantiomeric Excess of Glycidols ( 21a and 21b ). ................................ ................................ ................................ ...................... 146 3.2.5 Synthesis of Potassium (2 S ,3 R ) - 3 - Phenylglycidate ( 14a a) from 21a . ..................... 146 3.2.6 Synthesis of Potassium (2 R ,3 S ) - 3 - Phenylglycida te ( 14a b) from 21b. .................... 147 3.2.7 Enantiopurity of the Synthesized 3 - Phenylglycidates. ................................ ............ 147 3.2.8 Production of Phenylserine by Tc PAM Biocatalysis. ................................ ............. 148 3.2.9 General Method for Chiral Auxiliary Derivatization of Phenylserines and Phenylisoserines. ................................ ................................ ................................ .. 148 3.2.10 K inetic Analysis. ................................ ................................ ................................ ... 150 3.3 Results and Discussion ................................ ................................ ................................ ..... 151 3.3.1 Synthesis of and Characterization of Enantioenriched 3 - Phenylglycidate Substrates ( 14aa and 14ab ). ................................ ................................ ................................ ... 151 3.3.2 Enantiopurity of the Synthesized 3 - Phenylglycidate isomers ( 14aa and 14ab ). ..... 152 3.3.3 Glycidate Ring - Opening by Tc PAM Catalysis and Analysis of its Stereoselectivity. ................................ ................................ ................................ .............................. 153 3.3.4 Probing the Formation of the Anti - a nd Syn - Phenylserines using Computational Docking. ................................ ................................ ................................ ............... 160 3.3.5 Proposed Mechanism and Derivation of the Kinetic Equation. .............................. 163 3.3.6 Kinetic Analyses. ................................ ................................ ................................ ..... 167 3.4 Conclusion ................................ ................................ ................................ ........................ 171 3.5 Future Studies. ................................ ................................ ................................ .................. 172 APPENDIX ................................ ................................ ................................ ............................. 176 REFERENCES ................................ ................................ ................................ ....................... 188 xiv LIST OF TABLES Table 1.1. Various Enzymatic Processes Involving Epoxides in Microorganisms. ....................... 9 Table 2.1. - Arylalanine Products Catalyzed b y MIO - Dependent Aminomutases and Their Corresponding Biosynthetic Products. ................................ ................ 26 Table 2.2. GC Oven Heating Parameters. ................................ ................................ .................... 42 Table 2.3. Synthesis of 3 - Arylglycidate Analogues a and Isolated Yields . ................................ .. 52 Table 2.4. Control Experiments. ................................ ................................ ................................ .. 56 Table 2. 5. 500 MHz 1 H NMR data for the synthetic and biocatalyzed arylserine analogues ( 15 a 15 l ) recorded in D 2 O at pH 1.5. ................................ ................................ ................................ .... 64 Table 2.6. 500 MHz 1 H NMR data for the chemically synthesized arylserine analogues ( 15 a 15 l ) recorded in D 2 O at a pH range of 0 - 4.0. ................................ ................................ ....................... 65 Table 2.7. Kinetics of Tc PAM for Turnover of Arylglycidates ( 14a 14l) to Arylserines ( 15 a 15 l ) and Arylisoserines ( 16a 16l ). ................................ ................................ ................................ ....... 74 Tabl e 3.1. Kinetics of Tc PAM for turnover of 3 - phenylglycidate enantiomers to phenylserine a . ................................ ................................ ................................ ................................ ..................... 171 xv LIST OF FIGURES Figure. 1.1. Comparison of chemocatalyti c and biocatalytic processes to sitagliptin. (A) Chemocatalytic synthesis of sitagliptin involves enamine ( 2 ) formation, followed by rhodium - catalyzed asymmetric hydrogenation at high pressure. (B) The biocatalytic route involves direct amination of prosit agliptin ketone ( 1 ), followed by phosphate salt formation to yield enantiopure 4 ). ................................ ................................ ............................. 5 Figure. 1.2. Different enzymatic routes toward the synthesis of the key side chain of Atorvastatin ( 8 ) (Lipitor ® ). These routes show a combination of KRED with a halohydrin dehalogenase (HHDH) ( Route I , developed by Codexis), a nitrilase ( Route II ) , 37 or an aldolase ( Route III ) . 38 ... 7 Figure. 1.3. Pathway to acetoacetate from 1 - propene in Xanthobacter sp. ................................ . 11 Figure. 1.4. Partial b iosynthesis of Taxol from (2 S ) - - phenylalanine in Taxus plants. .............. 12 Figure. 1.5. Biocatalysis of a rylserine and arylisoserine analogues from trans - 3 - arylglycidates and (2 S ) - styryl - - alanine ( 12 ) by using an MIO - aminomutase. The asterisks (*) identify a chiral center. ................................ ................................ ................................ ................................ ....................... 12 Figure. 2.1. 1,2 - Amino group isomerization catalyzed by aminomutases. ................................ .. 20 Figure. 2.2. A) The m echanism of how the MIO forms from a conserved triad of active site residues. B) Reactions of the MIO moiety with nucleophiles from NaBH 4 or KCN resulted in the loss of aminomutase activity due to MIO - inactivation. ................................ ............................... 23 Figure. 2.3. The mechanism of MIO - aminomutase catalyzed reaction via an amino - alkylation - amino acid. Route a : the (3 S ) - - arylalanine isom er is made via a pathway where the acrylate intermediate does not rotate before being re - aminated at the C . Route b : the (3 R ) - isomer via a pathway where the arylacrylate intermediate rotates inside the active site before being re - aminated at the C . ................................ ................................ ................................ .......... 24 Figure. 2.4. Transaminase activity of Tc PAM with A) its natural substrate; B) surrogate (2 S ) - styryl - - alanine. ................................ ................................ ................................ ............................ 27 Figure. 2.5. Tch PAM - catalyzed addition of ammonia to substituted arylacrylates. .................... 28 Figure. 2.6. Proposed covalent binding of a 3 - arylglycidate inhibitor in the Sg TAM active site A) Formation of a dihydroxy ether intermediate reported by Montanavon and co - workers. 17 B) Reinterpretation - hydroxy - - amino adduct formed by MIO - NH 2 as informed by the proposed amino - linked adducts made in the Pa PAM crystal structures. 29 ........ 30 Figure. 2.7. - phenyl - - hydroxy - - amino acid building blocks in bioactive compounds. Stereochemical designations listed in brackets [] are described in Figure. 2.8 . ................................ ................................ ................................ ............... 31 xvi Figure. 2.8. Stereoisomerism convention for A) Arylserine, and B) Arylisoserine used herein . The equivalent, archaic designations for arylserine diastereomers are listed in brackets []. ............... 32 Figure. 2.9. - hydroxy - - amino acid building blocks in bioactive compounds. ................................ ................................ ................................ ............... 33 Figure. 2.10. - hydroxy - - amino acids: a). aza - Claisen rearrangement; b). proteinogenic amino acid derivatization; c). asymmetric hydrogenation; d). Strecker reaction; e). Sharpless aminohydroxylation and dihydroxylation reaction; f). Electrophilic amination; g). Mannich type reaction; h). Oxy - Michael addition; i). Asymmetric aldol condensati on; and j). multicomponent reaction. ................................ ............. 34 Figure. 2.11. Biocatalysis of arylserine and arylisoserine analogues from trans - 3 - arylglycid ates and (2 S ) - styryl - - alanine by using an MIO - aminomutase. The asterisk ( * ) indicates a chiral center. ................................ ................................ ................................ ................................ ....................... 36 Figure. 2.12. E nantiopurity of the synthetically derived methyl esters of A) 3 - phenylglycidate ( 14a ) (49:51), extracted ion m / z 121; B) 3 - (3' - OCH 3 - phenyl)glycidate ( 14b ) (50:50), extra cted ion m / z 151; C) ( 14c ) 3 - (3' - CH 3 - phenyl)glycidate ( 14c ) (49:51), extracted ion m / z 135; D) 3 - (3' - F - phenyl)glycidate ( 14d ) (50:50), extracted ion m / z 139; E) 3 - (3' - Cl - phenyl)glycidate ( 14e ) (50:50), extracted ion m / z 155; and F) 3 - (3' - Br - phenyl)glycidat e ( 14f ) (50:50), extracted ion m / z 199 analyzed by chiral GC/EI - MS. The base peak ion was used for extracted - ion selection of the derivatives; partial chromatograms are shown. The ratio of each enantiomer is shown in parentheses. ................................ ................................ ................................ ................................ ... 53 Figure. 2.13. E nantiopurity of the synthetically derived methyl esters of G) 3 - (3' - NO 2 - phenyl)glycidate ( 14g ) (47:53), extracted ion m / z 166; H) 3 - (4' - NO 2 - p henyl)glycidate ( 14h ) (46:54), extracted ion m / z 166; I) 3 - (4' - CH 3 - phenyl)glycidate ( 14i ) (50:50), extracted ion m / z 135; J) 3 - (4' - F - phenyl)glycidate ( 14j ) (50:50), extracted ion m / z 139; K) 3 - (4' - Cl - phenyl)glycidate ( 14k ) (47:53), extracted ion m / z 155; a nd L) 3 - (4' - Br - phenyl)glycidate ( 14l ) (50:50), extracted ion m / z 199.analyzed by chiral GC/EI - MS. The base peak ion was used for extracted - ion selection of the derivatives; partial chromatograms are shown. The ratio of each enantiomer is shown in parenthes es. ................................ ................................ ................................ ................................ ... 54 Figure. 2.14. 1 H NMR of 3 - phenylglycidate ( 14a ) in deuterated Assay Buffer. A) NMR spectra were recorded at 15 min intervals s tarting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the ratio of 14a :dihydroxy product = 100:0, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14a :dihydroxy product = 100:4. See Appendix for the associated 1 H NMR data for other arylglycidate analogues ( 14b - 14l ). ................................ .. 55 Figure. 2.15. Relative abundances of arylisoserine ( 16b ) and arylserine ( 15 b ) products made from C - and C - amination, respectively, of 3 - (3 ' - OCH 3 - phenyl)glycidate ( 14b ) (1 mM) when incubated with 2M NH 4 OH and Tc PAM (100 µ g/mL) ( top trace ) and without Tc PAM ( bottom trac e ). ..... 58 Figure. 2.16. Resonance and inductive stabilizations of the partial positive charge ( + ) (due to bond polarization) at the C of arylglycidate substrates 14a , 14b , and 14i assisted by the aryl - ring. For substrate 14h , the electron - withdrawing NO 2 group resonance destabi lizes the + at the xvii benzylic carbon (C ), and the regioselectivity is reversed, directing an incoming nucleophile to attack at C preferentially. ................................ ................................ ................................ ............ 59 Figure. 2.17. GC/EI - MS spectra of authentic A) (2 S )+(2 R ) - syn - phenylserine from Sigma - Aldrich, B) phenylserine biocatalyzed from 3 - phenylglycidate by Tc PAM catalysis (see Figure. 2.19 for GC profiles), and of C) (2 R ,3 S ) - syn - phenylisoserine ( see Figure. 2.72 for GC profile). Each hydroxy amino acid was derivatized to its O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. m / z 351) was not observed for either analyte. ................................ ...... 62 Figure. 2.18. An expanded 500 MHz 1 H NMR of A) synthesized phenylserine ( 15 a ), and B) of 15 a containing authentic ( 2S ) - syn - phenylserine recorded in D 2 O at pH 1.5. Chemical shift values are listed vertically above each peak, and the relative area under each peak is listed horizontally next to each peak. ................................ ................................ ................................ .......................... 63 Figu re. 2.19. Gas - chromatography/mass spectrometry e xtracted - ion ( m / z 179) chromatograms of A) synthesized phenylserine ( 15 a ), as shown in Scheme 2.1 B, was derivatized with a chiral auxiliary as per Scheme 2.1 A . Peaks at 7.95 min and 8.01 min correspond to (2 S )+(2 R ) - anti - phenylserine isomers, and those at 8.10 and 8.13 min correspond to (2 S )+(2 R ) - syn - phenylserine isomers; B) derivatized authentic (2 S )+(2 R ) - syn - phenylserine racemate (Sigma - Aldrich, contains 13% (2 S )+(2 R ) - anti - phenylserine as an impurity; C) chir al - auxiliary of biocatalyzed phenylserine made from the racemic 3 - phenylglycidate. Biocatalyzed (2 S )+(2 R ) - syn - phenylserine stereoisomers were also produced at ~9% (peaks at 8.10 and 8.13 min); and D) Extracted ion ( m / z 179) chromatogram of derivatized phen ylserine after treating with (2 S ) - TA for 15 min. ........... 67 Figure. 2.20. Distribution of enantiomers (2 R ) - anti (gray bars) and (2 S ) - anti (b lack bars) in biocatalyzed products for the substituted phenylserine analogues. ................................ .............. 68 Figure. 2.21. A) Lowest energy binding poses are shown of (2 S ,3 R ) - 3 - phenylglycidate ((2 S ,3 R ) - 14 a ) (orange sticks) and (2 R ,3 S ) - 3 - phenylglycidate ((2 R ,3 S ) - 14 a ) (light - gray sticks) at ce nter of the image. The conformations are consistent with the active site of Tc PAM consisting of residues (shown as dark gray and yellow sticks), the catalytic Tyr80, a putative catalytic Tyr322 (light blue sticks), binding contact Arg325 (golden - rod sticks ), and the methylidene imidazolone (MIO) moiety (green sticks). B) (2 S ,3 R ) - 14 a (orange sticks) and C) (2 R ,3 S ) - 14 a (light - gray sticks) are posed separately in the active site to highlight the nominal distances (<3.5 Å) that substituents on the aryl ring a re from active site residues. Printed on the aryl ring, ' m ' designates meta - positions (equivalent to the 3' - designation used in the text), and ' p ' designates the para - position (equivalent to the 4' - designation used in the text) per ligand. Heteroatoms are colored red for oxygen and blue for nitrogen. The images were produced with UCSF Chimera, 136 , and the docking conformations were generated with AutoDock Vina 127 from PDB code 3NZ4. Numbers are distances in Å. D) Tc PAM in complex with cinnamic acid, based o n PDB codes 3NZ4 and 4CQ5. ........................ 70 Figure. 2.22. a ) Rendering of the mechanism showing attack of the NH 2 - antibonding orbita l (gray lobe) at C for the C (2 S ,3 R ) - 14 a to produce (2 R ) - anti - phenylserine. b ) A double inversion - of - configuration mechanism is envisioned to access the minor syn - stereoisomer that proceeds through a putative lactone intermediate. ................................ ..... 71 xviii Figure. 2.23. Various viewing angles centering on the surfaces of A) Leu104, B) Leu108, and C) Lys427 deep in the aryl binding pocket of Tc PAM (PDB code 3NZ4) with poses of the docked (2 S ,3 R ) - 14 a (orange sticks) and (2 R ,3 S ) - 14 a (light - gray sticks). ................................ ................. 72 Figure. 2.24. SDS - PAGE gel of wild - type Tc PAM (82% pure) and (2 S ) - TA (99% pure) after Coomassie blue staining. Purity was estimated by a Kodak Gel Logic 100 Imaging System; lane 1: Tc PAM (10 µL of a 13.7 mg/mL solution); lane 2: Tc PAM (5 µL of a 13.7 mg/mL solution); lane 3: Pa geRuler © Prestained Ladder: MW (kDa) 170, 130, 100, 70, 55, 40, 35, 25, 15; lane 4: (2 S ) - TA (10 µL of a 8.8 mg/mL solution); and lane 5: (2 S ) - TA (5 µL of a 8.8 mg/mL solution). ................................ ................................ ................................ ................................ ....................... 81 Figure. 2.25. 1 H NMR of 3 - (3 - OCH 3 - phenyl)glycidate ( 14 b ) in deuterated Assay Buffer. A) NMR spectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the ratio of 14 b :dihydroxy product = 100:0, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 b :dihydroxy product = 100:3. . 82 Figure. 2.26. 1 H NMR of 3 - (3 - CH 3 - phenyl)glycidate ( 14 c ) in deuter ated Assay Buffer. A) NMR spectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the ratio of 14 c :dihydroxy product = 100:7, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 c :dihydroxy product = 100:12. .... 83 Figure. 2.27. 1 H NMR of 3 - (3 - F - phenyl)glycidate ( 14 d ) in deuterated Assay B uffer. A) NMR spectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the ratio of 14 d :dihydroxy product = 100:0, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 d :dihydroxy product = 100:2. ..... 84 Figure. 2.28. 1 H NMR of 3 - (3 - Cl - phenyl)glycidate ( 14 e ) in deuterated Assay Buffer. A) NMR spectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the ratio of 14 e :dihydroxy product = 100:0, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 e : dihydroxy product = 100:1. ...... 85 Figure. 2.29. 1 H NMR of 3 - (3 - Br - phenyl)glycidate ( 14 f ) in deuterated Assay Buffer. A) NMR spectra wer e recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the ratio of 14 f :dihydroxy product = 100:0, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 f :dihydroxy pr oduct = 100:1. ...... 86 Figure. 2.30. 1 H NMR of 3 - (3 - NO 2 - phenyl)glycidate ( 14 g ) in deuterated Assay Buffer. A) NMR spectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the ratio of 14 g :dihydroxy = 100:0, and B) 1 H NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of the ratio of 14 g :dihydroxy product = 100:2. ..... 87 Figure. 2.31. 1 H NMR of 3 - (4 - NO 2 - phenyl)glycidate ( 14 h ) in deuterated Assay Buffer. A) NMR spectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and t 7 = 90 min, the ratio of 14 h :dihydroxy product = 100:9, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 h :dihydroxy product = 100:9. ..... 88 Figure. 2.32. 1 H NMR of 3 - (4 - CH 3 - phenyl)glycidate ( 14 i ) in deuterated Assay Buffer. A) NMR spectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the xix ratio of 14 i :dihydroxy product = 100:1, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 i :dihydroxy product = 100:5. ...... 89 Figure. 2.33. 1 H NMR of 3 - (4 - F - phenyl)glycidate ( 14 j ) in deuterated Assay Buffer. A) NMR spectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and t 7 = 90 min, the ratio of 14 j :dihydroxy product = 100:4, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 j :dihydroxy product = 100:7. ...... 90 Figure. 2.34. 1 H NMR of 3 - (4 - Cl - phenyl)glycidate ( 14 k ) in deuterated Assay Buffer. A) NMR spectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and t 7 = 90 min, the ratio of 14 k :dihydroxy product = 100:0, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 k :dihydroxy product = 100:3. ..... 91 Figure. 2.35. 1 H NMR of 3 - (4 - Br - phenyl)glycidate ( 14 l ) in deuterated Assay Buffer. A) NMR spectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and t 7 = 90 min, the ratio of 14 l :dihydroxy produ ct = 100:0, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 l :dihydroxy product = 100:1. ...... 92 Figure. 2.36. A) Relative abundances of arylisoserine ( 16a ) and arylserine ( 15 a ) products made from C - and C - amination, respectively, of 3 - phenylglycidate ( 14a ) (1 mM) when incubated with 2M NH 4 OH and Tc PAM (100 µ g/mL) ( top trace ) and without Tc PAM ( bottom trace ). B) Relative abundances of arylisoserine ( 16i ) and arylserine ( 15 i ) products made from C - and C - amination, respectively, of 3 - (4 ' - CH 3 - phenyl)glycidate ( 14i ) (1 mM) when incubated with 2M NH 4 OH and Tc PAM (100 µ g/mL) ( top trac e ) and without Tc PAM ( bottom trace ). C) Relative abundances of arylisoserine ( 16h ) and arylserine ( 15 h ) products made from C - and C - amination, respectively, of 3 - ( 4 ' - NO 2 - phenyl)glycidate ( 14h ) (1 mM) when incubated with 2M NH 4 OH and Tc PAM (100 µ g/mL) ( to p trace ) and without Tc PAM ( bottom trace ). ................................ .............................. 93 Figure. 2.37. GC/EI - MS spectra of A) authentic ( 2 S /2 R ) - anti - - OCH 3 - phenylserine ( 15 b ) and B) 3 - OCH 3 - phenylserine biocatalyzed from 3 - - OCH 3 - phenyl)glycidate ( 14 b ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 381) was not observed f or either analyte. ................................ ................................ ................................ ................................ ....................... 94 Figure. 2.38. GC/EI - MS spectra of A) authentic ( 2 S /2 R ) - anti - - CH 3 - phenylserine ( 15 c ) and B) 3 - CH 3 - phenylseri ne biocatalyzed from 3 - - CH 3 - phenyl)glycidate ( 14 c ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 365) was not observed for either analyte. ................. 94 Figure. 2.39. GC/EI - MS spectra of A) authentic ( 2 S /2 R ) - anti - - F - phenylserine ( 15 d - F - phenylserine biocatalyzed from 3 - - F - phenyl)glycidate ( 14 d ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 369) was not observed for either analyte. ............................. 95 Figure. 2.40. GC/EI - MS spectra of A) authentic (2 S /2 R ) - anti - - Cl - phenylserine ( 15 e - Cl - phenylserine biocatalyzed from 3 - - Cl - phenyl)glycidate ( 14 e ) by Tc PA M catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 385) was not observed for either analyte. ............................. 95 xx Figure. 2.41. GC/EI - MS spectra of A) authentic (2 S /2 R ) - anti - - Br - phenylserine ( 15 f - Br - phenylserine biocatalyzed from 3 - - Br - phenyl)glycidate ( 14 f ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 429) was not observed for either analyte. ............................. 96 Figure. 2.42. GC/EI - MS spectra of A) authentic (2 S/ 2 R ) - anti - - NO 2 - phenylserine ( 15 g ) and B) - NO 2 - phenylserine biocatalyzed from 3 - - NO 2 - phenyl)glycidate ( 14 g ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as th eir O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 396) was not observed for either analyte. ................. 96 Figure. 2.43. GC/EI - MS spectra of A) authentic (2 S/ 2 R ) - anti - - NO 2 - phenylserine ( 15 h ) and B) - NO 2 - phenylserine biocatalyzed from 3 - - NO 2 - phenyl)glycidate ( 14 h ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [( 2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 396) was not observed for either analyte. ................. 97 Figure. 2.44. GC/EI - MS spectra of A) authentic ( 2 S /2 R ) - anti - 4 - CH 3 - phenylserine ( 15 i ) and B) 4 - CH 3 - phenylserine biocatalyzed from 3 - (4 - CH 3 - phenyl)glycidate ( 14 i ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbuty ryl] methyl ester. The molecular ion (M , m / z 365) was not observed for either analyte. ................. 97 Figure. 2.45. GC/EI - MS spectra of A) aut hentic ( 2 S /2 R ) - anti - - F - phenylserine ( 15 j - F - phenylserine biocatalyzed from 3 - - F - phenyl)glycidate ( 14 j ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 369) was not observed for either analyte. ............................. 98 Figure. 2.46. GC/EI - MS spectra of A) authentic (2 S /2 R ) - anti - - Cl - phenylse rine ( 15 k - Cl - phenylserine biocatalyzed from 3 - - Cl - phenyl)glycidate ( 14 k ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 385) was not obse rved for either analyte. ............................. 98 Figure. 2.47. GC/EI - MS spectra of A) authentic (2 S /2 R ) - anti - - Br - phenylserine ( 15 l - Br - phenyl serine biocatalyzed from 3 - - Br - phenyl)glycidate ( 14 l ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 429) was not observed for either analyte. ............................. 99 Figure. 2.48. Par tial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized phenylserine ( 15 a ) and B) biosynthetic phen ylserine produced from phenylglycidate ( 14a ) by Tc PAM. ................................ ................................ ................................ ................................ ....... 100 Figure. 2.49. Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemic ally synthesized 3 - OCH 3 - phenylserine ( 15 b ) and B) biosynthetic 3 - OCH 3 - phenylserine produced from 3 - (3 - OCH 3 - phenyl)glycidate ( 14 b ) by Tc PAM. ................................ ................................ ............................ 101 Figure. 2.50. Partial 1 H NMR (500 MHz, D 2 - CH 3 - phenylserine ( 15 c - CH 3 - phenylserine produced from 3 - (3 - CH 3 - phenyl)glycidate ( 14 c ) by Tc PAM. ................................ ................................ ............................. 102 xxi Figure. 2.51. Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized 3 - F - phenylserine ( 15 d - F - phenylserine produ ced from 3 - (3 - F - phenyl)glycidate ( 14d ) with Tc PAM. ................................ ................................ ......................... 103 Figure. 2.52. Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized 3 - Cl - phenylserine ( 15 e ) and B) biosynthetic 3 - Cl - phenylserine produced from 3 - (3 - Cl - phenyl)glycidate ( 14e ) by Tc PAM. ................................ ................................ ............................. 104 Figure. 2.53. Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized 3 - Br - phenylserine ( 15 f ) and B) biosynthetic 3 - Br - phenylserine produced from 3 - (3 - Br - phenyl)glycidate ( 14f ) by Tc PAM. ................................ ................................ ............................. 105 Figure. 2.54. Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized 3 - NO 2 - phenylserine ( 15 g ) and B) biosynthet ic 3 - NO 2 - phenylserine produced from 3 - (3 - NO 2 - phenyl)glycidate ( 14g ) by Tc PAM. ................................ ................................ ............................ 106 Figure. 2.55. Partial 1 H NMR (500 MHz, D 2 O at pH 1 .5) spectra of A) chemically synthesized 4 - NO 2 - phenylserine ( 15 h ) and B) biosynthetic 4 - NO 2 - phenylserine produced from 3 - (4 - NO 2 - phenyl)glycidate ( 14 h ) by Tc PAM. ................................ ................................ ............................ 107 Figure. 2.56. Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized 4 - CH 3 - phenylserine ( 15 i - CH 3 - phenylserine produced from 3 - (4 - CH 3 - phenyl)glycidate ( 14 i ) by Tc PAM. ................................ ................................ ............................. 108 Figure. 2.57. Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized 4 - F - phenylserine ( 15 j ) and B) biosynthetic - F - phenylserine produced from 3 - (4 - F - phenyl)glycidate ( 14 j ) by Tc PAM ................................ ................................ .............................. 109 Figure. 2.58. Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) sp ectra of A) chemically synthesized 4 - Cl - phenylserine ( 15 k - Cl - phenylserine produced from 3 - (4 - Cl - phenyl)glycidate ( 14 k ) by Tc PAM. ................................ ................................ ............................ 110 Figure. 2.59. Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized 4 - Br - phenylserine ( 15 l - Br - phenylserine produced from 3 - (4 - Br - phenyl)glycidate ( 14 l ) by Tc PAM. ................................ ................................ ............................. 111 Figure. 2.60. GC/EI - MS e xtracted - ion chromatograms of biosynthetic arylserines derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl esters A ) phenylserine ( 15 a ) (extracted ion m / z 179), B) 3 - OCH 3 - phenylserine ( 15 b ) (extracted ion m / z 209), C) 3 - methylphenylserine ( 15 c ) (extracted ion m / z 193), D) 3 - F - phenylserine ( 15 d ) (extracted ion m / z 197), E ) 3 - Cl - phenylserine ( 15 e ) (extracted ion m / z 213), and F) 3 - Br - phenylserine ( 15 f ) (extracted ion m / z 257). ........... 112 Figure. 2.61. GC/EI - MS e xtracted - ion chromatograms of biosynthetic arylserines derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl esters G) 3 - NO 2 - phenylserine ( 15 g ) (extracted ion m / z 224), H) 4 - NO 2 - phenylserine ( 15 h ) (extracted ion m / z 224), I) 4 - CH 3 - phenyl serine ( 15 i ) (extracted ion m / z 193), J) 4 - F - phenylserine ( 15 j ) (extracted ion m / z 197), K ) 4 - Cl - phenylserine ( 15 k ) (extracted ion m / z 213), and L) 4 - Br - phenylserine ( 15 l ) (extracted ion m / z 257) ................................ ................................ ................................ ................................ ....... 113 xxii Figure. 2.62. GC/EI - MS e xtracted - ion chromatograms of synthetic arylserines derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester before ( top trace ) and after 10 - mi n treatment with (2 S ) - TA ( bottom trace ): A) 3 - OCH 3 - phenylserine ( 15 b ) (extracted ion m / z 209), B) 3 - CH 3 - phenylserine ( 15 c ) (extracted ion m / z 193), C) 3 - F - phenylserine ( 15 d ) (extracted ion m / z 197), D) 3 - Cl - phenylserine ( 15 e ) (extracted ion m / z 213), E ) 3 - Br - phenylserine ( 15 f ) (extracted ion m / z 257), and F) 3 - NO 2 - phenylserine ( 15 g ) (extracted ion m / z 224). ................ 114 Figure. 2.63. GC/EI - MS e xtracted - ion chromatograms of synthetic arylserines derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester before ( top trace ) and after 10 - min treatment with (2 S ) - TA ( bottom trace ): G) 4 - NO 2 - phenylserine ( 15 h ) (extracted ion m / z 2 24); H) 4 - CH 3 - phenylserine ( 15 i ) (extracted ion m / z 193), I) 4 - F - phenylserine ( 15 j ) (extracted ion m / z 197), J ) 4 - Cl - phenylserine ( 15 k ) (extracted ion m / z 213) and K) 4 - Br - phenylserine ( 15 l ) (extracted ion m / z 257). ................................ ................................ ................................ ............... 115 Figure. 2.64. Michaelis - Menten kinetics for the turnover of arylglycidate analogues ( 14 a 14 f ) to their corresponding arylserines ( 15 a 15 f ). ................................ ................................ ................. 116 Figure. 2.65. Michaelis - Menten kinetics for the turnover of arylglycidate analogues ( 14 g 14l ) to their corresponding arylserines ( 15 g 15 l ). ................................ ................................ ................. 117 Figure. 2.66. Michaelis - Menten kinetics for the turnover of arylglycidate analogues ( 14 a 14 f ) to their corresponding aryl iso serines ( 1 6 a 16 f ). ................................ ................................ ............ 118 Figure. 2.67. Michaelis - Menten kinetics for the turnover of arylglycidate analogues ( 14 g 14l ) to their corresponding ary lisoserines ( 16 g 16l ). ................................ ................................ ............. 119 Figure. 2.68. The six - hour time course for the conversion of four representative 3 - arylglycidate racemates [3 - phenylglycidate ( 14a - F - ( 14d ) ( - Cl - ( 14e - Cl - ( 14k phenylglycidate (each at 400 µM)] to their corresponding arylserines in assays containing Tc PAM (100 µg/mL), and (2 S ) - styryl - - alanine (1 mM) in 50 mM NaH 2 PO 4 /Na 2 HPO 4 (p H 8.0), n = 3. Arylserine production is shown as a percentage relative to the initial epoxide substrate concentration. These example epoxides were turned over by Tc PAM from fastest (3' - Cl) to slowest (4' - Cl). ................................ ................................ ................................ ................................ ......... 120 Figure. 2.69. Lowest energy binding poses are shown of A) (2 R ,3 S ) - 14f ; B) (2 S ,3 R ) - 14f ; C) (2 R ,3 S ) - 14e ; D) (2 S ,3 R ) - 14e ; E) (2 R ,3 S ) - 14g ; F) (2 S ,3 R ) - 14g ; G) (2 R ,3 S ) - 14b ; and H) (2 S ,3 R ) - 14b . ................................ ................................ ................................ ................................ ............. 121 Figure. 2.70. Lowest energy binding poses are shown of A) (2 R ,3 S ) - 14l ; B) (2 S ,3 R ) - 14l ; C) (2 R ,3 S ) - 14k ; D) (2 S ,3 R ) - 14k ; E) (2 R ,3 S ) - 14h ; and F) (2 S ,3 R ) - 14h . ................................ ......... 122 Figure. 2.71. Putative binding pose of (2 S ,3 R ) - 3 - (3' - OCH 3 - phenyl)glycidate ( 14b ) in a confor mation aligned for NH 2 - MIO attack at C (labeled ' ') to make the arylisoserine ( 16b ). C is labeled with ' ' for reference. ................................ ................................ ................................ ...... 123 Figu re. 2.72. GC/EI - MS extracted ion ( m / z 106) chromatogram of authentic (2 R ,3 S ) - syn - phenylisoserine ( 16a ) derivatized as its O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester ( 18a ). ................................ ................................ ................................ ................................ ........... 123 xxiii Figure. 2.73. LC - ESI - MS/MS spectra of A) authentic ( 2 R ,3 S ) - syn - phenylisoserine ( 16 a ) (structures of diagnostic fragment ions are shown and used to characterize the fragment ions of the biocatalyzed arylisoserine analogues (below). B) phenylisoserine ( 16 a ); C) 3' - OCH 3 - phenylisoserine ( 16 b ); D) 3 ' - CH 3 - phenylisoserine ( 16 c ); and E) 3 ' - F - phen ylisoserine ( 16 d ) biocatalyzed by Tc PAM from the corresponding 3 - arylglycidates. ................................ ........... 124 Figure. 2.74. LC - ESI - MS/MS spectra of F) 3' - Cl - p henylisoserine ( 16 e ); G) 3 ' - Br - phenylisoserine ( 16 f ); H) 3' - NO 2 - phenylisoserine ( 16 g ); I) 4' - NO 2 - phenylisoserine ( 16h ); J) 4' - CH 3 - phenylisoserine ( 16 i ); and K) 4' - F - phenylisoserine ( 16 j ) biocatalyzed by Tc PAM from the corresponding 3 - arylglycidates. ................................ ................................ ................................ .. 125 Figure. 2.75. LC - ESI - MS/MS spectra of L) 4' - Cl - phenylisoserine ( 16 k ) and M) 4 ' - Br - phenylisoserine ( 16 l ) biocatalyzed by Tc PAM from the ir corresponding 3 - arylglycidates. ...... 126 Figure. 3.1. A) Reactions catalyzed by MIO - dependent ammonia lyases (ALs) and aminomutases (AM s), making either the acrylate or - amino acid from the corresponding - amino acid. The MIO and aminated - MIO (NH 2 - MIO) are shown. The asterisk (*) identifies an ( R / S ) - chiral center; B) - - arylalanine using engineere d PAL/PAM catalysis; C) PAM - mediated amine transfer from ( S ) - styryl - - alanine to acrylate acceptors. D) Addition of ammonia and primary amines to mesaconate analogues using engineered methylaspartate ammonia lyase (MAL) to produce (2 S ) - syn - aspartate analog ues. ................................ .............. 142 Figure. 3.2. Chiral HPLC separation of A). (2 R ,3 R ) - 3 - phenylglycidol ( 21a ), 92% e.e. ; B) .(2 S ,3 S ) - 3 - phenylglycidol ( 21b ), 90% e.e ; and C) a co - injection of enantioenriched samples of 21a and 21b . ................................ ................................ ................................ ................................ ............. 152 Figure. 3.3. Extracted ion ( m / z 121) chromatogram of (A) r acemic methyl 3 - phenylglycidate (51:49), (B) methyl (2 S ,3 R ) - 3 - Phenylglycidate (4:96, 12.56 min), and (C) methyl (2 R ,3 S ) - 3 - Phenylglycidate (95:5, 12.37 min) . ................................ ................................ ............................. 153 Figure. 3.4. Stereoisomerism convention for A) phenylserine, and B) phenylisoserine used herein. ................................ ................................ ................................ ................................ ..................... 154 Figure. 3.5. GC/EI - MS e xtracted - ion chromatograms with m / z 179 ion monitoring of A) phenylserine stereoisomers derivatized with a chiral auxiliary (cf. Scheme 3.2A ). An earlier study 29 involving enzymatic resolution with (2 S ) - threonine aldolase confirmed that peaks at 8.88 min and 8.93 min correspond to chiral derivatives of (2 S ) - anti - and (2 R ) - anti - phenylserine isomers, respectively, and peaks at 9.02 and 9.05 min correspond to the chi ral derivatives of (2 S ) - syn - and (2 R ) - syn - phenylserine isomers (cf. Figure. 2.19 ). Chiral derivatives of B) authentic (2 S ) - syn - phenylserine (Bachem); C) biocatalyzed phenylserine made from (2 S ,3 R ) - 3 - phenylglycidate (the (2 S ) - anti :(2 R ) - anti :(2 S ) - syn isomers are abundant at a relative ratio of 2:90:8); and D) biocatalyzed phenylserine made from (2 R ,3 S ) - 3 - phenylglycidate (the (2 S ) - anti :(2 R ) - anti :(2 R ) - syn isomers are abundant at a relative ratio of 76:15:9). ................................ ................................ ... 156 Figure. 3.6. GC/EI - MS e xtracted - ion chromatograms with m / z 106 ion monitoring of a chiral auxiliary derivative of A) authentic (2 R ,3 S ) - syn - phenylisoserine (Bachem); B) authentic (2 R , 3 R ) - anti - phenylisoserine (Chem Impex); C ) anti - phenylisoserine enantiomers synthesized from authentic racemic 3 - phenylglycidate; 42,43 D) (2 S ,3 S ) - anti - phenylisoserine synthesized from xxiv (2 S ,3 R ) - 3 - phenylglycidate ( 14aa ); E) (2 R ,3 R ) - anti - phenylisoserine made fr om (2 R ,3 S ) - 3 - phenylglycidate ( 14ab ); F) biocatalyzed (2 S ,3 S ) - anti - phenylisoserine made from (2 S ,3 R ) - 3 - phenylglycidate substrate ( 14aa ); and G) biocatalyzed (2 R ,3 R ) - anti - phenylisoserine made from (2 R ,3 S ) - 3 - phenylglycidate substrate ( 14ab ). Note, isoserines in Panels C , D , and E , were made from the corresponding glycidates by nucleophilic addition of NH 4 OH. ................................ ... 158 Figure. 3.7. Absolute stereo configuration of biocatalyzed phenylserine produced from A) (2 S ,3 R ) - 3 - phenylglycidate ( 14aa ); and B) (2 R ,3 S ) - 3 - phenylglycidate ( 14ab ). Proposed mechanism for the formation of C) (2 S ) - syn - phenylserine from 14aa ; and D) (2 R ) - syn - phenylserine from 14ab throug h intramolecular carboxylate assisted oxiranone formation followed by amination at C by MIO - NH 2 . ................................ ................................ ................................ ................................ ... 1 59 Figure. 3.8. A) Lowest energy bi nding conformations of A) (2 S ,3 R ) - 3 - phenylglycidate ( 14aa ) (light - grey sticks); and B) ( R ) - 3 - (( R ) - hydroxy(phenyl)methyl)oxiran - 2 - one ( 22aa ) (light grey sticks) are shown at the center of the image inside the Tc PAM active site. The residues shown here inclu de a catalytic Tyr80 (yellow sticks), a putative catalytic Tyr322 (yellow sticks), binding contact Arg325 (orange sticks), and the methylidene imidazolone (MIO) moiety (green sticks). Heteroatoms are colored red for oxygen and blue for nitrogen. The images were produced with UCSF Chimera, 45 and the docking conformations were generated with AutoDock Vina 44 from Tc PAM crystal structure (PDB code 3NZ4). ................................ ................................ .............. 162 Figure. 3.9. A) Lowest energy binding conformations of A) (2 R ,3 S ) - 3 - phenylglycidate ( 14ab ) (light - grey sticks); and B) ( R ) - 3 - (( R ) - hydroxy(phenyl)methyl)oxiran - 2 - one ( 22ab ) (light grey sticks) are shown at the center of the image inside the Tc PAM active site. The residues shown here include a catalytic Tyr80 (yellow sticks), a putative catalytic Tyr322 (yellow sticks), binding contact Arg325 (orange sticks), and the methylidene imidazolo ne (MIO) moiety (green sticks). Heteroatoms are colored red for oxygen and blue for nitrogen. The images were produced with UCSF Chimera, 45 and the docking conformations were generated with AutoDock Vina 44 from Tc PAM crystal structure (PDB code 3NZ4). ................................ ................................ .............. 163 Figure. 3.10. Modified Michaelis - Menten plots for the turnover of A) (2 S ,3 R ) - 3 - phenylglycidate ( 14aa ); and B) (2 R ,3 S ) - 3 - phenylglycidat e ( 14ab ) to their corresponding phenylserine at fixed concentration of the 3 - phenylglycidate enantiomer. Double reciprocal Lineweaver - Burk plot of C) (2 S ,3 R ) - 3 - phenylglycidate ( 14aa ); and D) (2 R ,3 S ) - 3 - phenylglycidate ( 14ab ) to their corresponding phenylser ine at fixed concentration of 3 - (calculated from each plot of Figure. 3.10 A vs [ 14aa ]; and F) Secondary plot of (calculated from each plot of Figure. 3.10 B vs [ 14ab ] to calculate the true K M of the glycidate substrates. ................ 170 Figure. 3.11. Par tial 1 H NMR (50 0 MHz, CDCl 3 ) spectra of (2 R ,3 R ) - 3 - Phenylglycidol ( 21a ) . 177 Figure. 3.12. Par tial 13 C NMR (126 MHz, CDCl 3 ) spectra of (2 R ,3 R ) - 3 - Phen ylglycidol ( 21a ) . ................................ ................................ ................................ ................................ ..................... 178 Figure. 3.13. Par tial 1 H NMR (500 MHz, CDCl 3 ) spectra of (2 S ,3 S ) - 3 - Phenylglycidol ( 21b ). 179 Figure. 3.14. Par tial 13 C NMR (126 MHz, CDCl 3 ) spectra of (2 S ,3 S ) - 3 - Phenylglycidol ( 21b ) . 180 xxv Figure. 3.15. Par tial 1 H NMR (500 MHz, D 2 O) spectra of Potassium (2 S ,3 R ) - 3 - Phenylglycidate ( 14aa ). ................................ ................................ ................................ ................................ ......... 181 Figure. 3.16. Par tial 13 C NMR (126 MHz, D 2 O) spectra of Potassium (2 S ,3 R ) - 3 - Phenylglycidate ( 14aa ). ................................ ................................ ................................ ................................ ......... 182 Figure. 3.17. Par tial 1 H NMR (500 MHz, D 2 O) spectra of Potassium (2 R ,3 S ) - 3 - Phenylglycidate ( 14ab ). ................................ ................................ ................................ ................................ ......... 183 Figure. 3.18. Par tial 13 C NMR (126 MHz, D 2 O) spectra of Potassium (2 R ,3 S ) - 3 - Phenylglycidate ( 14ab ). ................................ ................................ ................................ ................................ ......... 184 Figure. 3.19. GC/EI - MS spectra of A) authentic ( 2 R, 3 S ) - syn - phenylisoserine from Bachem ( 6 b ); B) authentic ( 2 R, 3 R ) - anti - phenylisoserine from ChemImpex C) derivatized anti - phenylisoserine enantiomers produced from au thentic racemic 3 - phenylglycidate; and D) . derivatized (2 S ,3 S ) - anti - phenylisoserine ( 16ac ) made from (2 S ,3 R ) - 3 - phenylglycidate ( 14aa ) and NH 4 OH. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The mol ecular ion (M , m / z 351) was not observed. ................................ ........................ 185 Figure. 3.20. GC/EI - MS spectra of A) derivatized (2 R ,3 R ) - anti - phenylisoserine ( 16ad ) m ade from (2 R ,3 S ) - 3 - phenylglycidate ( 14ab ) and NH 4 OH B) biocatalyzed (2 S ,3 S ) - anti - phenylisoserine ( 16ac ) made from (2 S ,3 R ) - 3 - phenylglycidate ( 14aa ) by Tc PAM; and C) biocatalyzed (2 R ,3 R ) - anti - phenylisoserine ( 16ad ) made from (2 R ,3 S ) - 3 - phenylglycidate ( 14ab ) by Tc PAM. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 351) was not observed. ................................ ........................ 186 Figure. 3.21. Gas - chromatography/mass spectrometry e xtracted - ion chromatograms with m / z 336 ion monitoring of derivatized anti - phenylserine and anti - phenylisoserine enantiomers produced from NH 4 OH assisted ring opening o f authentic racemic 3 - phenylglycidate. Peaks at 8.84 min and 8.90 min correspond to racemic anti - phenylserine (18%) produced from amination at the C , and peaks at 9.33 min and 9.41 min correspond to racemic anti - phenylisoserine produced from the amination at C . ................................ ................................ ................................ ........................... 187 xxvi LIST OF SCHEMES Scheme 2.1. A) Tc PAM was incubated with (2 S ) - styryl - - alanine ( 12 ) (1 mM) and separately with each trans - 3 - phenylglycidate racemate ( 14a 14l ) . Step a ) i) Tc PAM (1.7 mg/mL) in Assay Buffer, 2 min pre - equilibration , 14a 14l (10 mM), 29 ° C, 4 h. Step b ) Derivatization of putative phenylserines and phenylisoserines using a chiral auxiliary for stereoisomeric resolution. i ) (2 S ) - 2 - Methylbutyric anhydride, pyridine, rt, 20 min; ii ) 6 M HCl, pH 2; iii ) CH 2 N 2 , EtOAc/MeOH (3:1 v / v ), rt, 15 min; and iv ) chlorotrimethylsilane, pyridine, CH 2 Cl 2 , rt, 15 min. Insets : stereoisomerism o f (2 R ,3 S ) - syn - phenylisoserine. B) Synthesis of the stereoisomers of phenylserine analogues from glycine and a substituted benzaldehyde. a) step i ) triethylamine, n - BuOH and H 2 O, 12 h, rt; step ii ) 6 M HCl, pH 2; and step iii ) NaHCO 3 , pH 6 . ................................ ..................... 60 Scheme 3.1. Synthesis of (2 S ,3 R ) - 3 - phenylglycidate ( 14aa ) and (2 R ,3 S ) - 3 - phenylglycidate ( 14ab ). a) ( ) - DET, Ti(O i - Pr) 4 , t - BuOOH, 4 Å MS, CH 2 Cl 2 , 1 6 h, - DET, Ti(O i - Pr) 4 , t - BuOOH, 4 Å MS, CH 2 Cl 2 , 16 h, 30 °C; b) i ) RuCl 3 , NaIO 4 , NaHCO 3 , CH 3 CN/CCl 4 /H 2 O, rt, 72 h.; ii ) KHCO 3 , acetone/H 2 O, 0 °C, 1h. ................................ ................................ .............. 151 Scheme 3.2. Chiral auxiliary derivatization of A) authentic phenylserine diastereomers; and B) phenylisoserine diastereomers comprising (2 S ,3 S ) - anti - ( 16ac ), (2 R ,3 R ) - anti - ( 16ad ), and (2 R ,3 S ) - syn - phenylisoserine ( 16ab ). Derivatization was performed using (a) ( i ) (2 S ) - 2 - Methylbutyric anhydride, pyridine, rt, 15 min; ( ii ) 6 M HCl, pH 2; ( iii ) CH 2 N 2 , EtOAc/MeOH (3:1 v/v), rt, 10 min; and ( iv ) chlorotrimethylsilane, pyridine,CH 2 Cl 2 , rt, 15 min.; and (b) 2 M aq. NH 4 OH, rt, 1h. ................................ ................................ ................................ ................................ ..................... 155 Scheme 3.3. depicts the two - substrate ping - pong me chanism where (2 S ) - styryl - - alanine acts as the amino concentration (>1000 µM) of 3 - phenylglycidate. Inset 1 shows the scheme of a typical ping - pong mechanism. ................................ ................................ ................................ ................................ . 164 xxvii KEY TO ABBREVIATIONS n - BuOH: n - Butanol C Cl 4 : Carbon tetrachloride CDCl 3 : Deuterated chloroform CH 2 Cl 2 : Dichlo romethane CH 2 N 2 : Diazomethane CO 2 : Carbon dioxide D 2 O: Deuterium oxide DMSO: Dimethyl sulfoxide DNA: Deoxyribonucleic acid DOPS: ( 2 S , 3 R ) - 3,4 - D ihydroxyphenylserine E. coli : Escherichia coli EDTA: Ethylenediaminetetraacetic acid EPA: Environmental Protectio n Agency ESI: Electrospray ionization EtOAc: Ethyl acetate EtOH: Ethanol GC/EI - MS: Gas chromatography electron ionization mass spectrometry GDH: Glucose dehydrogenase H 2 O: Water HIV: H uman immunodeficiency virus HCl: Hydrochloric acid xxviii HHDH: H alohydrin deh a lo genase HRMS: High resolution mass spectrometry IPTG: Isopropyl - - D - thiogalactopyranoside KCN: Potassium cyanide KOH: Potassium hydroxide KRED: Ketored uctase LB: Lysogeny broth LC/ESI/MS: Liquid - chromatography electrospray ionization mass spectrometry LC /ESI/MRM: Liquid - chromatography electrospray ionization multiple reaction monitoring MgSO 4 : Magnesium Sulfate MIO: 4 - methylidene - 1 H - imidazol - 5(4 H ) - one Na 2 EDTA: Disodium ethylenediaminetetraacetate Na 2 HPO 4 : Disodium hydrogen phosphate NaBH 4 : Sodium borohydr ide NaCl: Sodium chloride NaHCO 3 : Sodium bicarbonate NaH 2 PO 4 : Sodium dihydrogen phosphate NCL: Native Chemical Ligation NH 4 OH: Ammonium hydroxide NMR: Nuclear Magnetic Resonance OD: Optical density PAL: Phenylalanine ammonia lyase PAM: Phenylalanine aminom utase xxix PCR: Polymerase chain reaction PDB: Protein data bank PLP : Pyridoxal - - phosphate SAM: S - adenosyl methionine SDS - PAGE: Sodium dodecyl sulfate - polyacrylamide gel electrophoresis TA: Threonine aldolase TAM: Tyrosine aminomutase TOF: Time of flight UCS F: University of California - San Francisco WWTP: Wastewater treatment plants 1 Chapter 1: Overview of Biocatalysis in Organic Chemistry and Pharmaceutical Industry Biocatalysis is defined as the use of living systems or their parts, such a s isolated enzymes and microbial - whole cells, to speed up t he chemical synthesis of organic compounds. 1 Biocatalytic processes are gradually appearing as an appealing technological alternative in the phar maceutical industry due to the excellent chemo - , regio - , an d stereoselectivity that biocatalysis offers over classical organic synthesis . Such catalysis often avoids using scarce metals, protecting group manipulations, and environmentally hazardous conditi ons, and involve milder conditions in water at room tempera ture, higher atom economy, and greener methods. 2 The Development of Biocatalysis : Its Beginning to Current State of the Art Humans have been using enzymes for centuries in fermentation to produce cheese, vinegar, and alcoholic beverages. In the modern era, it was the pioneering work of Louis Pasteur on s eparating a racemic mixture of ammonium tartrate u sing a culture of the fungus Penicillium glaucum that s et the first milestone in biocatalysis. 3 Pasteu r provided the first application of enzyme - catalyzed kinetic resolution, which is now a widely exercised technique in academia and industry. After this landmark research, biocatalysis advanced in the 20 th c entury, when scientists learned how to use whole - c ell cultures, cell extracts, or partially purified enzymes in various biocatalytic processes to make commodity chemicals. 4 - 8 Biocatalysis in Its First Wave . Techniq ues for the isolation and purification of enzymes were subsequently optimized i n the mid - 20 th century . These purified enzyme preparations were used in stereoselective transformations of non - natural substrates and high light the first wave of biocatalysis. 2,7 Some notable examples include employing a plant extract for the biocatalysis of ( R ) - (+) - mandelonitrile from benzaldehyde and hydrogen cyanide , 4 hydroxylating steroids within 2 microbial cell hosts, 8 converting glucose to the sweeter fructose isomer using a glucose isomerase, 9 - lacta m antibiotics on an industrial - scale using penicillin G acylase. 10 B iocatalysis in Its Second Wave . However, the major limitation in enzyme - catalyzed processes until the end of the 1970s was obtaining sufficient enzyme quantities for industrial purposes. 7 Conventional enzyme preparatio n methods relied on isolating native enzymes from the natural host, such as from microorganisms, insects, plants, or mammalian species. 11 Typically, these na tive enzymes were present in small amounts, making their application in biocatalysis intractable. With the advent of recombinant DNA technology, enzyme s could be overexpressed efficiently in non - native host organisms in a laboratory or industrial setting, marking the second wave of biocatalysis. 1 During this phase, structure - based protein engineering te chnologies expanded the substrate scope of enzymes and made it possible to biocatalyze a wide variety of non - natural products that could be used as intermediates in chemical synthesis or as final products. 7,11 Notable examples of these biocatalytic transformations include lipase - catalyzed resolution of 3 - phenylglycida te esters in the synthesis of the drug diltiazem (used to lo wer blood pressure), 12 carbonyl - reductase catalyzed synthesis of enantiomerically pure ethyl ( S ) - 4 - chloro - 3 - hydroxybutanoate to synthesize statins drugs to lower cho lesterol, 13 and nitrile hydratase catalyzed industr ial production of acrylic acid and methacrylic acid for the polymer industry. 14 Biocatal ysis in Its Third Wave . The third and the most significant w ave of biocatalysis accelerated the speed of biocatalytic optimizations through directed evolution approaches initiated in the mid and late 1990s and is still currently used. 15,16 The in vitro molecular biology methods were modeled on Darw inian evolution to rapidly and broadly modify the intrinsic structural and catalytic properties of an enzyme. This Darwinian approach includes random amino acid modificati on by altering the cDNA encoding the enzyme. 15,16 This technique uses error - prone PCR 3 or gene shuffling, followed by transforming the cDNA into an expression vector and subcloning in an expression host. The resulti ng cDNA libraries within these hosts are separately expressed and screened for mutant enzymes with improved thermal and operational stability, and broader substrate acceptance and selectivity, often uninformed by a crystal structure. 11 Industrial - scale biocatalysis primarily focused on ketoreductases (KREDs ), hydrolases, cofactor generation, and protein stability in organic solvents. 11,15,16 As a result of the biocatalysis advancements, remarkable new capabilities and properties were introduced into enzymes, such as accepting previously inert substrates. For example, a KRED from Microbacterium campoquemadoensis was engineered to turn over methyl ( E ) - 2 - (3 - (3 - (2 - (7 - chloroquinolin - 2 - yl)vinyl)phenyl) - 3 - oxopropyl)benzoate on the biosynthetic pathway of Montelukast, 17 and a transaminase from Art hrobacter sp. was modified to accept the substrate prositagliptin on the biosynthetic pathway of sitagliptin. 18,19 Another example includes changing the properties of a sesquite rpene synthase to biosynthesize diffe rent sesquiterpenes in Nicotiana tabacum 20 by diverting the amino acid metabolism pathway to produce branched - chain alcohols for biofuels. 21 Valuable biotechnological and bioinformatics tools emerged during t he third wave of biocatalysis that a d vanced gene synthesis and sequence analysis, protein engineering technologies (including directed evolution), and computer - based modeling and docking methods. Before this biotechnological windfall , enzym atic process es w ere designed to offset the limitation in stability and reactivity of the enzymes used, but currently the enzyme s are engineered and chosen from a smarter library of variants, suitable for the process specifications. 1,11,15 The third wave of biocatalys is significantly helped to generalize and widen the use of biocatalysis over the past few decades by complementing synthetic organic chemistry transformations in the pharmaceutical, agrochemical, materials, polymers, food, and fine - chemical 4 sectors. 7,11,22 - 24 The pharmaceutical industry has seen a remarkable application of biocatalytic processes. 25 The directed - evolution appro ach has been successfully commercialized by many companies like Codexis, Merck, Pfizer, and Arch Pharm Labs Limited. 2,11 A notable example includes the development o f a biocatalytic route for the commercial production of the antidiabetic compound sitagliptin, whi ch was previously synthesized chemically and marketed as Januvia ® ( 4 ), by Merck. Januvia ® was the first marketed antihyperglycemic drug for Type 2 diabetes me llitus and the most widely marketed dipeptidyl peptidase - 4 inhibitor worldwide, reaching sales of $6.36 billion in 2014 and expected to reach $7.53 billion in 2020. 26 - 28 It was only second to insulin glargine amongst the antidiabetics. 11,27 The first chemical synthesis of sitagliptin was achieved through asymmetric hydrogenation of an e namine ( 2 ) moiety, derived from prositagliptin ( 1 ), using a rhodium - based chiral catalyst (Rh - [Josiphos]) under high pressure (250 psi) ( Figure. 1 . 1 A ). This process suffered from inadequate stereoselectivity (97%) f or pharmaceutical standards, and the final product was contaminated with rhodium salts, requiring further purification steps at the expense of yield to increase the enantiomeric excess and purity. 29 5 Figure. 1 . 1 . Comparison of chemocatalytic and biocatalytic processes to sitagliptin. (A) Chemocatalytic synthesis of sitagliptin involves enamine ( 2 ) formation, followed by rhodium - catalyzed asymmetric hydrogenation at high pressure. (B) The biocatalytic route involves direct amination of prositagliptin ketone ( 1 ), follo wed by phosphate salt formation to yield enantiopure 4 ). Scientists at Merck and Codexis have recently used a bioengineering approach to rationally design an ( R ) - selective transamina se (ATA - 117) and optimize its properties for improved efficiency in si tagliptin manufacturing ( Figure. 1 . 1 B ). 18 ATA - 117, a homolog of an enzyme from Arthrobacter sp ., was initially active only toward methyl and other sma ll cyclic ketones. 30,31 A combination of computational modeling and site - saturation mutagenesis was used to cre ate a larger active site that could bind and turn over a truncated methyl ketone analogue similar to prositagliptin. These efforts resulted in marginal activity, only 4% turnover from 10 g/L enzyme and 2 g/L p rositagliptin substrate loading. After 11 round s of directed evolution and high throughput screening, the best ATA - 117 variant was found to catalyze the transaminase reaction with 99.95% e.e. After 27 mutations, the final ATA - 117 variant (6 g/L enzyme load ing) was capable of converting 200 g/L prositag liptin ketone to sitagliptin in 50% DMSO with a 92% assay yield. 1 8 In comparison with the Rh - catalyzed process, this biocatalytic route produced sitagliptin with a 13% increase in overall yield, a 53% increase in productivity, and a 19% reduction in total 6 waste resulting in the elimination of heavy metals and a reducti on in total manufacturing cost. 18 This process received the Presidential Green Chemistry Challenge Award (Green er Reaction Conditions Award) from the U.S. Env ironmental Protection Agency (EPA) in 2010. Another representative example is the multi - ton scale Codexis protocol of ethyl ( R ) - 4 - cyano - 3 - hydroxybutyrate ( 7 ), a key molecule constituting the side chain of ator vastatin (Lipitor ® ) ( 8 ). The cholesterol - lowering drug Lipitor ® had global sales of $11.9 billion in 2010. Due to a high demand of 7 for the synthesis of Atorvastatin (estimated to be m ore than 100 metric tons), it was desira ble to minimize the hazardous waste produced and cost involved while maintaining or improving the purity. 32 KREDs and other enzymes were thoroughly investigated to make the chiral intermediates involved in the sy nthesis of Atorvastatin and other statin analogs. Seven enzymatic processes were developed to vary the KRED properties to obtain the intermediate they were producing on the synthetic pathway ( Figure. 1 . 2 ). 7 The Codexis protocol ( Figure. 1 . 2 , Route I) employ ed a KRED - based reduction of 5 in combination with an NADP - dependent glucose dehydrogenase (GDH) for cofactor regeneration. This reduction produced 6 with 96% isolated yield and 99.5% e.e. The next step was catalyzed by a halohydrin dehalogenase (HHDH) tha t typically eliminates hal ides from a vicinal haloalcohol via an epoxide ring. 33,34 HHDH substrate selectivity was honed by directed evolution and could substitute the chloro functional group with a cyano to p roduc e 7 . Codexis was awarded the U.S. this process. 35 The three - enzyme biocatalytic protocol u sed milder conditions to produce 7 . The enzymatic process was more favorable to a previously reported chemical synthesis that involved an S N 2 replacement of the halogen with cyanide in alkaline medium (pH 10) at high temperature 7 (80 °C). These conditions gener ated many byproducts because both 6 and 7 were not stable in alkaline solution. The enzymes KRED and HHDH used in the commercial biocatalytic approach to 7 initial ly showed low activity, significant product inhibition, and poor stability under the operating condition. 27 After in vitro optimizati on through enzyme evolution using gene shuffling techniques resulted in an overall process where the volumetric productivity per mass catalyst load of the cyanation process was improved by ~2500 fold, a 14 - fold reduction in reaction time, a 7 - fold increase in substrate loading, a 25 - fold reduction in enzyme load, and a 50% increase in isolated yield compared to the synthetic route. 32,36 Figure. 1 . 2 . Different enzymatic routes toward the synthesis of the key side chain of Atorvastatin ( 8 ) (Lipitor ® ). These routes show a combination of KRED with a halohydrin dehalogenase (HHDH) ( Route I , develop ed by Codexis), a nitrilase ( Route II ) , 37 or an aldolase ( Route III ) . 38 Biocatalysis is no longer limited to the niches of small - molecule synthesis and application within the pharmaceutical industry. This field of bi ochemistry has evolved as a desirable 8 alternative for the ever - increasing demand for renewable energy sources and chemical feedstocks. 25 Recent advancements in laccase - mediated grafting of biopolymers, for example, lignocelluloses, cellulose, and chitosan have the potential to p rovide environmentally benign alternatives for the wood, textile, and paper industry. 39 Enzymes also have been used successfully as an enhancer in the biodegradation of synthetic polymers in the wastewater system. 40 Hydrolases from wastewater microorganisms were employed to breakdown poly(oxyethylene terephthalate) polymers in vitro and under realistic Waste Water Treatment Plants (WWTP) conditions. Biocatalysis in Its Fourth Wave . The fourth wave of biocatal ysis is ongoing . This phase includes a multidisciplinary approach of combining molecular genetics, metagenomics, and bioinformatics tools to discover and even synthesize novel enzymes that did not previously exist in nature . These techniques are combined w ith the development of multi - enzyme cascade reactions , immobiliza tion methods, and microreactor technolog ies . 41,42 In fourth - wave biocatalysis, t he target enzyme has been through iterative mutations and is often si gnificantly modified compared to the wild type, containing only 10 - 20% of the wild type residues. As an example, a protein with 300 amino acids has 2 0 300 possible sequence combinations of the 20 natural amino acids. Th e daunting scope of enzyme var iants ca nnot be achieved on ly using random muta genesis and high - throughput screening approach es . While t he field of de novo enzymes is emerging as an exci ting alternative in recent years, the resulting enzymes typically have much lower efficiency tha n what can be achieved from directed evolution techniques. 43,44 In the future, the advances of the fourth wave of biocatalysis need to merge the empirical random mutagenesis data with advanced computational tools to design opera tion and functional b iocatalysts. T hese two approaches will bode well for managing the large data set s gene rated across multiple protein evolution projects and will enhance the predictability of machine learning algorithms. 9 The Application of Epoxides in Biocatalysis This thesis uses glycidate (epoxide) substrates in an aminotransferase reaction to make stereo isomers of arylserines and arylisoserines; thus, it is fitting to provide a brief overview of the application of epoxides in biocatalysis. Epoxides p lay a vital role in the biocatalysis of value - added chemicals and pharmaceuticals due to the intrinsic reac tivity of the epoxide ring. 45,46 Enzymes that catalyze regio - and stereoselective ring - opening of epoxides include epoxide hydrolases (EHs), halohydrin dehalogenases (HHDHs), glutathio ne S - transferases, and CoM - transferases ( Table 1 . 1 ). 46 Epoxide hydrolases use water as the nucleophile to catalyze the hydrolysis of epoxides to the corresponding diols. The stereoselectivity of epoxide hydrolases can thus also be used for kine tic resolution of racemic epoxide substrates, producing a chiral 1,2 - dihydroxy compound while leaving the unreactive epoxide intact. Table 1 . 1 . Various Enzymatic Processes Involving Epoxides in Microorganisms. Enzyme Accepted nucleophile(s) Example of product(s) Epoxide h ydrolase H 2 O Halohydrin dehalogenase Cl , Br , I , CN , OCN , SCN , N 3 , NO 2 , HCO 2 Glutathione - S - transferase GSH, Cys CoM - transferase HHDHs are bacterial enzymes involved in the biodegradation of xenobiotic halogenated compounds. They catalyze the reversible dehalogenatio n of vicinal haloalcohols through intramolecular substitution of a halogen atom to yield an epoxide and halide. 33,46 HHDHs exhibit high regio - and enantioselectivity toward aliphatic and aromatic vicinal haloalcoho ls , and thus are employed in the production of enantiopure epoxides as well as their ring - open ed products . 34,47 10 HHDH from Agrobacterium radiobacter ( Ar HHDH) is the best - studied enzyme in this group and is reported to catalyze the epoxide ring - opening reaction with a range of ionic and non - ionic nucleophiles with high enantiomeric excess and high yields . 48,49 The non - halide nucleophiles accepted by the Ar HHDH are ambident ate anions with a linear sha pe that binds inside the tunnel - shaped active site. For example, while HHDHs generally do not accept NH 2 (or NH 3 ) with sp 3 (tetrahedral) hybridized orbital geometry as a nucleophile, HHDHs can tolerate linear azido nucleophiles to make an azido alcohol fr om a haloalcohol. The reduction of the azido group accessed the amino alcohols indirectly. 50 Several other enzymes involved in the microbial conversion of epoxides are also reporte d in the literature. Glu tathione S - transferases (GSH) are associated with the detoxification of compounds - Glu - Cys - Gly) that employs the catalytic thiol group of cysteine, followed by further metabolization. 51,52 Two distinct mechanisms are found for the metabolism of epoxides by glutathione; the most common one involves hydrogen bonding of the thiol group of glutathione to suitable groups of the enzyme, making it more nucleophilic. Alternatively, complexa tion of the epoxide substrate by met al ion like Mn (II) makes it more electrophilic, facilitating the nucleophilic attack by the glutathione thiol. The complete role of GSH in epoxide metabolism is not yet known. Epoxides are also common intermediates in t he biodegradation of alkenes in bacteria like Rhodococcus rhodochrous and Xanthobacter sp. where short - chain aliphatic alkenes such as ethylene, propylene, or 1 - butene are epoxidized using NADH - dependent monooxygenases . The epoxide then gets carboxylated b y CO 2 - keto acid as the final product by a process catalyzed by a four - component, multi - enzyme system ( Figure. 1 . 3 ). The epoxide is opened by a cofactor M (CoM, 2 - mercapto - 1 - ethyl sulfonate ) dependent transfe rase ( Table 1 . 1 ). 45,53 11 Figure. 1 . 3 . Pathway to acetoacetate from 1 - propene in Xanthobacter sp. Biocatalytic Transformations of Epoxide by an Aminotransfer Reaction ( Thesis Work ) . This thesis describes research done with variously substituted epoxides, 3 - arylgly cidates. These glycidates are regio - and stereoselectively aminated by employing a phenylalanine amino mutase (PAM) biocatalyst and (2 S ) - styryl - - alanine as an NH 2 donor to produce anti - arylserines as the major products. Phenylalanine aminomutases (PAMs) ar - - phenylalanine. 54 In Taxus plants, Tc PAM catalyzes the conversion of (2 S ) - - phenylalanine ( 9 ) to (3 R ) - - phenylalanine ( 10 ), which is the biosynthetic precursor of the phenylisoserinyl side chain of anticancer drug Taxol ( 11 ) ( Figure. 1 . 4 ). 55 - 57 The excellent product enantioselectivity (99.9 % e.e.) a nd broad substrate scope of Tc PAM make it an attractive - phenylalanine production. 58 In earlier work, (2 S ) - styryl - - alanine was used as a non - natural substrate to achieve aminotransferase activity using Tc PAM, transferring an amino group from ( 2 S ) - styryl - - alanine - - am ino acids. 59 12 Figure. 1 . 4 . Partial b iosynthesis of Taxol from (2 S ) - - phenylalanine in Taxus plants. In this work, a new class of acc eptor molecules, 3 - arylglycidates, were successfully transaminated using (2 S ) - styryl - - alanine ( 12 ) and Tc PAM to produce arylserines and arylisoserines ( Figure. 1 . 5 ). 60 Arylserine and arylisoserine scaffolds are found in clinically significant antibiotics, including vancom ycin and its analogues, 61 ,62 ristocetin from Amycolatopsis and teicoplanins from Actinoplanes , 63 chloramphenicol from Streptomyces , 64 katanosin depsipeptides from Cytophaga and Lysobacter bacteria, 65 and th e antineoplastic agent paclitaxel from Taxus sp p . and its analo gues. 57,66 Given the beneficial pharmacological, chemical, and physical properties of these aromatic hydroxy amino acids, there is considerable interes t in making these bifunctional molecules either synthetically, biocatalytically, or by a combination of both approaches. (2 S ) - Styryl - a - alanine ( 12 ) rac - 3 - Arylglycidate Arylserine Arylisoserine Figure. 1 . 5 . Biocatalysis of arylserine and arylisoserine analogues from trans - 3 - arylglycidates and (2 S ) - styryl - - alanine ( 12 ) by using an MIO - aminomutase. The asteris ks (*) identify a chiral center. 13 REFERENCES 14 R EFERENCES 1. Arnold, F. H. (2001) Combinatorial and Computational Challenges for Biocatalyst Design, Nature 409 , 253 - 257. 2. Truppo, M. D. (2017) Biocatalysis in the Pharmaceutical Industry: The Need for Speed, ACS Med. Chem. Lett. 8 , 476 - 480. 3. Pasteur, L. (1858) Mém C. R. Acad. Sci. 46 , 615 - 618. 4. Rosenthaler, L. (1908) Durch Enzyme Bewirkte Ssymmetrische Synthesen, Biochem. Z 14 , 238 - 253. 5. is a Comprehensive Handbook, Third, completely revised and enlarged edition / ed., pp 3 volumes (lii, 1985 pages), Wiley - VCH,, Weinheim, Germany. 6. Ortiz de Montellano, P. R . (2005) Cytochrome P450 : Structure, Mechanism, and Biochemistry , Third edition. ed., Kluwer Academic/Plenum Publishers, New York. 7. Bornscheuer, U. T., Huisman, G. W., Kazlauskas, R. 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Gueritte - Voegelein, F., Guenard, D., Lavelle, F., Le Goff, M. T., Mangatal, L., and Potier, P. (1991) Relationships Between the Structure of Taxol Analogs and Their Antimito tic Activity, J. Med. Chem. 34 , 992 - 998. 20 Chapter 2: Biocatalysis of Arylserines and Arylisoserines using Phenylalanine Aminomutase from Taxus canadensis ( Tc PAM ) and 3 - Arylglycidate Racemates. Reprinted (adapted) with permission from (Shee, P. K.; Ratnayake, N. D.; Walter, T.; Goethe, O.; - Aminomutase for the Amination of Cinnamate Epoxides to Arylserines and Arylisoserines. ACS Catal. 2019 , 9 (8), 7418 - 7430. ) Copyright ( 2019 ) American Chemical Society . 2.1 Introduction 2.1.1 Aminomutase s : Enzyme C lass (EC) of 1, 2 - Amino Acid Isomerases Isomerases (EC 5) are a general class of enzymes that convert a molecule from one isomer to another. 1 The intermolecular isomerase (EC 5.4) family has enzymes that promote the transfer of an acyl - , phospho - , amino - , hydroxy - or other functional group from one position of a molecule to another. 2 A minomutases (AMs) (EC 5.4.3) are a s ubclass of enzymes that catalyze reversible cross - exchange of an amino group and a proton on vicinal carbons of a substrate ( Figure. 2 . 1 ). 3 Figure. 2 . 1 . 1,2 - Amino group isomerization catalyzed by aminomutases. The AM family has gained much attention due to the application of the biocatalysts in the synthesis of medicinally important molecules. 4 This family is comprised of lysi ne 2,3 - (EC 5.4.3.2), 5,6 - lysine 5,6 - (EC 5.4.3.3), 7 - 9 D - lysine 5,6 - (EC 5.4.3.4), 10,11 D - ornithine 4,5 - (EC 5.4.3.5), 12,13 tyrosine 2,3 - (EC 5.4.3.6), 14 - 20 leucine 2,3 - (EC 5.4.3.7), 21,22 glutamate - 1 - semialdehyde 2,1 - (EC 5.4.3.8), 23,24 glutamate 2,3 - (EC 5.4.3.9), 25 and phenylalanine (EC 5.4.3.10 26,27 and 5.4.3.11 28,29 ) aminomutas es. A 2 - aza - L - tyrosine 2,3 - aminomutase was discovered, and based on amino acid sequence homology, like ly belongs to EC 5.4.3 - K21227. 30 The catalytic properties of these 21 aminomutases make them attractive for synthet ic ap plications because of their regio - and enantioselectivity, and potential for the preparation of valuable non - natural - amino acids. 3 2.1.2 Mechanistic Diversity of Aminomutases Aminomutases use various cofactors to catalyze the vicinal exchange of the amino group and proton in a molecule. The aminomutase reaction mechanism can be class ified into a homolytic or heterolytic pathway. The mechanisms of lysine 2,3 - , 5,6 - lysine 5,6 - , 7 - 9 D - lysine 5,6 - , 10,11 D - ornithine 4,5 - , 12,13 leucine 2,3 - , 21,22 and glutamat e 2,3 - aminomutases 25 invo lve radical intermediates, and thus follow a homolytic pathway. These enzymes use either S - adenosyl methionine (SAM), pyri doxal 5 - phosphate (PLP), and a [4Fe - 4S] + (iron - sulfur) cluster or adenosylcobalamin (vitamin B12) and PLP as cofactors. Tyrosine 2,3 - 14 - 20 and phenylalanine aminomutases 26 - 29 cat alyze their isomerization reaction using the heterolytic pathway to break and make bonds and require a 4 - methylidene - 1 H - imidazol - 5(4 H ) - one (MIO) catalytic gro up made by the backbone residues in the active site. 2.1.3 MIO - dependent Aminomutases The MIO - dependent aminomutases catalyze the isomerization / - amino acid via a heterolytic mechanism. 27,31 In 1998, Walker and coworkers reported on the activity of an aminomutase in the cell - free extracts from Taxus brevifolia plant tissue. 26 It was the first - ever reported aminomutase from a higher plant and also the first phenylalanine aminomutase from any source. It catalyzed the con version of (2 S ) - - phenylalanine to (3 R ) - - phenylalanine en route to the biosynthesis of anti - cancer drug Taxol. 26 It was shown that the intramolecular isom erization proceeded with retention of configuration at the C , which was different from single - electron transfer mechanisms used by microbial aminomutas es characterized at the time. However, n o claims were made that the AM from T. brevifolia was MIO - dependent. 22 Five years after the discovery of the T. brevifolia aminomutase activity, Christenson and coworkers discovered a novel MIO - dependent tyrosine amino mutase from Streptomyces globisporus ( Sg TAM) that showed high homology with a histidine a mmonia lyase from Streptomyces griseus (39% identity and 56% similarity) 32 and a phenylalanine ammonia lyase from Streptomyces maritimus (38% identity and 56% similarity). 33 Each of these MIO - dependent enzymes has the signature Ala - Ser - Gly motif that is the origin of this MIO - moiety in the active site. Sg TAM converts (2 S ) - - tyrosine to (3 S ) - - tyrosine during the biosynthesis of enediyne antitumor antibiotic C - 1027. 31 The MIO - moiety was previously identified by X - ray crystallographic analysis of a histidine ammonia lyase from Pseudomonas putida (EC 4.3.1.3) , 34 and the Ala - Ser - Gly residues were proposed to cyclize and form the MIO - group autocatalytically through sequential condensation reactions ( Figure. 2 . 2 A ). Based on previous studies that verified the function of the MIO, 35 - 37 Sg TAM was incuba ted with NaBH 4 ( 10 mM ) or KCN (2 mM). These nucleophilic reactants abrogated the aminomutase activity by fouling the reactivity of the MIO, as the nucleophile acceptor ( Figure. 2 . 2 B ). Additionally, mutation of the s erine residue (S153A) of the MIO triad decreased the Sg TAM activity by 340 - fold. This analogous mutation in other MIO - dependent enzymes also reduced the activity. 31 These results suggested that Sg TAM relied on a cata lytic MIO - moiety. 23 A B Figure. 2 . 2 . A) The m echanism of how the MIO forms from a conserved triad of active site residues. B) Reactions of the MIO moiety with nucleophiles from NaBH 4 or KCN resulted in the loss of aminomutase activity due to MIO - inactiva tion. In 2004, a random sequencing of a cDNA library derived from Taxus cuspidata cells that provided them several of the twelv e defined genes of Taxol biosynthesis, including a PAM. 38 A gene cloning method was used to acquire a phenylalanine ammonia lyase (PAL) - like sequence from the Taxus cuspidata cDNA library and expresse d in Escherichia coli . 27 The expressed enzyme was confirmed as an AM that was identical to the recombinant AM from Taxus chinensi s acquired earlier by rever se genetic approach. 39 This PAM from Taxus sp. ( Tc PAM ) contained the signature A SG motif in the active site, identifying the mutase as MIO - dependent. 27 Both plant Tc PAM and bacterial Sg TAM form their MIO by condensation of the Ala - Ser - Gly residues, 26,31 while the aminomutase from Pantoea agglomerans ( Pa PAM) uses a Thr - Ser - Gly sequence. 28 2.1.4 Overview of Aryl Amin o Acid Aminomutases MIO - dependent aminomutases have been discovered on various biosynthetic pathways ( Table 2 . 1 ). The mutases isomerize (2 S ) - - amino acids to non - proteinogenic - amino acids by intramolecular 2,3 - ami no group transfer. Tc PAM and Pa PAM catalyze the production of - phenylalanine on the pathways to Taxol and the antibiotic andrimid, respectively. 27,40 The bacterial Sg TAM is on the biosynthetic pathway to the antit umor antibiotic C - 1027, and a TAM from 24 Chondromyces cracatus ( Cc TAM) produce s - tyrosine on the pathway to cytotoxic chondramides. 18 Other instances of TAMs include Os TAM from rice ( Oryza sativa ) , 16 MdpC4 TAM from Actinomadura madurae on the pathway to the antitumor and antibiotic mad u ropeptin, 41 Mf TAM from Myxococcus fulvus and Mx TAM from Myxococcus sp. are involved in the bio s ynthesis of myxovalargin, 19 and KedY4 from Streptoalloteichus sp. Lies on the pathway to the antitumor and antibiotic kedarcidin. 30 KedY4 stereoselectively catalyzes the conversion of (2 S ) - - 2 - aza - tyrosine to (3 R ) - - 2 - aza - tyrosine, and it is also the first MIO - dependent aminomutase to accept a heteroaromatic amino acid as a substrate. 30 2.1.5 MIO Function The electrophilic methylidene group of MIO gets N - a - amino group of the sub strate ( Figure. 2 . 3 ). The resulting N - alkylated ammonium group is then displaced as an MIO - NH 2 adduct with simultaneous removal of H b y a catalytic Tyr residue. Figure. 2 . 3 . The mechanism of MIO - aminomutase catalyzed reaction via an amino - alkylation - amino acid. Route a : the (3 S ) - - arylalanine isomer is made via a pathway where the acrylate intermediate does not rotate before being re - aminated at the C . Route b : the (3 R ) - isomer via a pathway where the arylacrylate intermediate rotates inside the active site before being re - amina ted at the C . 25 This elimination reaction produces an arylacrylate intermediate, which is released in the lyase reaction, yet primarily serves as a scaffold for stereospecific amination at C - amino acids. For example, Tc PAM, Cc TAM, and Os TAM c atalyze the (3 R ) - - amino acid from the (2 S ) - - amino acid. On this pathway, the arylacrylate intermediate i s proposed to rotate in the active site - amination, resulting in the delivery of the amine group to the re - face of the arylacrylate to produce the (3 R ) - - isomer ( Figure. 2 . 3 ). The stereo configurations of the final biocatalyzed products and the migratory groups (NH 2 and H ) have been studied extensively for both phenylalanine - (PAMs) and tyrosine aminomutas es (TAMs) ( Table 2 . 1 ). T hese enzymes can be categorized based on the absolute configuration of the biocatalyzed - amino acid. Sg TAM 31 and Pa PAM 40 pr oduce (3 S ) - - arylalanines , while Tc PAM 42 and Cc TAM 18 catalyze the formation of (3 R ) - - enantiomer. However, TAMs exhibit less en a ntioselectiv ity compared to the PAMs. TAMs produce one enantiomer of the - amino acid under kinetic control, but the opposite enantiomer is also produced when equilibrium is reached after prolonged incubation of the enzyme with the substrate . 19,31 As an example, Sg TAM produces (3 S ) - - tyrosine as the major product i nitially. As the reaction proceeds, the 3 R :3 S ratio a pproaches 1:1 after prolonged incubation likely due to the partial inversion and retention of stereoc onfiguration pathways used by TAMs. 3,18,31,43 By comparison, Cc TAM produces a mixture of (3 R ) - - tyrosine (90%) and (3 S ) - - tyrosine (10%) initially, and the 3 R :3 S ratio of the mixture reached 85:15 as the reaction r eached equilibrium . 14 The product distribution of Cc TAM also v aries with pH, At pH 7, the 3 R :3 S ratio was at 83:17, and then changed to 73:27 at pH 9. Os TAM from rice plants, however, has better enantioselectivity among the TAMs and makes (3 R ) - - tyrosine predominantly (97%) with 3% (3 S ) - - enantiomer, the 97:3 R : S was kept over a pH range from 7 - 10 over 24 h incubation. 26 Table 2 . 1 . - Arylalanine Products Catalyzed by MIO - Dependent Aminomutases and Their Corresponding Biosynthetic Products. Enzyme Substra te Product Configuration at C and C Biosynthetic product Phenylalanine Aminomutase ( Taxus sp. ) Retention Anti - cancer Taxol Phenylalanine Aminomutase ( Pantoea agglomerans ) Unknown Antibiotic andrimid Tyrosine Aminomutase ( Streptomyces globisporus ) Inversion Antitumor antibiotic C - 1027 1:1 (3 S ):(3 R ) - - tyrosine at equilibrium Inversion and retention Tyrosine Aminomutase ( Chondromyces cracatu s) Retention Cytotoxic chondramides 6:1 (3 R ):(3 S ) - - tyrosine at equilibrium Inversion and retention Tyrosine Aminomutase ( Oryza sativa ) Retention Unknown Tyrosine Aminomutase ( Actinomadura madurae ) Unknown Antitumor antibiotic maduropeptin Tyrosine Aminomutase ( Myxococcus sp. ) Bacterial protein biosynthesis inhibitor myxovalargin 2 - Aza - tyrosine Aminomutase ( Streptoalloteichus sp. ) Unk nown Antitumor antibiotic kedarcidin Thus, TAMs catalyze the isomerization reaction with both inversion and retention of configuration - - carbons giving mixtures of enantiomers for the product . By contrast, PAMs are highly enantioselective (> 99.9%), and thus are projected to be more suitable for applica tions in 27 scalable biocatalytic production of enantiopure - arylalanines as synthetic building block for pharmaceuticals. 44 - 49 2.1.6 Intramolecular and Intermo lecular Mechanisms of MIO - Aminomutases 2.1.6.1 Transaminase Studies of Tc PAM Using (2 S ) - Styryl - - alanine as Amine Donor Tc PAM catalyzes t he formation of (3 R ) - - phenylalanine with intramolecular exchange of the - amino group and the pro - (3 S ) - proton with retention - o f - configuration at both the reaction termini. 42,50 Tc PAM was co - incubated with both [ 15 N] - ( S ) - - phenylalanine and [ring,3 - 2 H 6 ] - ( E ) - cinnamate , and the r e was 97% incorporation of the [ 15 N]amino group intramolecularly into ( R ) - - phenylalanine. However, a small amount (3%) of [ 15 N, 2 H 6 ] - - phenylalanine also formed due to intermolecular amine transfer from the crossover reaction ( Figure. 2 . 4 A ). 50 This discovery set up the platform for exploring further transaminase studies with Tc PAM. Various non - n atural amino acids were tested as the initial amine donor substrate and (2 S ) - styryl - - alanine was found to be 1.5 times faster than the natural su bstrate (2 S ) - - phenylalanine, and Tc PAM predominantly catalyzed the formation of (2 E ,4 E ) - styrylacrylate (99%) with only 1% - aminated product. A B Figure. 2 . 4 . Transaminase activity of Tc PAM with A) its natural substrate; B) su rrogate (2 S ) - styryl - - alanine. 28 Later, in a burst phase kinetics study, Tc PAM was shown to transfer the amino group from ( 2 S ) - styryl - - alanine, employed as a surrogate s ubstrate, to exogenously s upplied arylacrylates via an MIO - NH 2 adduct. 51 Th e transaminase function of Tc PAM catalyz ed efficient intermolecul ar exchange of the amino group to make - - amino acids ( Figure. 2 . 4 B ) . 50,51 2.1.6.2 Transaminase Studies employing Ammonium Hydroxide as the Amine Donor In 2009, an alternate route to make enantiopure - and - amino acids from the amination of arylacrylates, catalyzed by a PAM from Taxus chinensis ( Tch PAM). 49 An aqueous solution of ammonia (NH 4 OH, 6 M, pH 10) was used as the amino group source, in which a series of arylacrylates with electron - donating and electron - withdrawing substituents on the aryl rin g were incubated with Tch PAM. The arylacrylates were converted to their corresponding - or - amino acids ( Figure. 2 . 5 ). The regioselectivity of the : - amination ratios varied from 99:1 (for 2 - Br) to 9:91 (for 4 - i - Pr); however, typically more evenly distributed mixtures of - and - amino acids were formed from most of the other acrylate substrates. Active site mutagenesis (Q319M) on Tch PAM improved the regioselectivity toward - amination from 35:65 to 9:91 - to - amination (for 4 - fluoro - cinnamate), and thus, enantioenriched - amin o acids were biocatalyzed from arylacrylates through one - step ammonia addition ( Figure. 2 . 5 ). 52,53 Figure. 2 . 5 . Tch PAM - catalyzed addition of ammonia to substituted arylacrylates. 29 2.1.6.3 Employing Epoxides (Glycidates) as Amino Group Acceptors in MIO - Catalysis In an earlier study, a c ovalently trapped 3 - phenylglycidate ( i.e., cinnamate epoxide) was seen in the active site of Sg TAM crystal (PDB: 2RJR) , which helped dissect its mechanism. 17 The trapped glycidate formed a presumed phenylpropan - 3 - o l covalently attached to the MIO via an ether linkage. 17 The authors assumed the crystallization m edia contained no exogenous amino group resources, and thus, the MIO was armed as HO - MIO by the addition of water. T his modified MIO was deemed the source of the ether - linked enzyme/substrate adduct. The ability of Sg TAM to trap a glycidate hinted that the reaction pathway for any MIO - enzyme should be aborted theoretically by covalent inhibition by a glycidate substrate ( Figure. 2 . 6 A). It is interesting t o note that the crystal structures of an MIO - dependent Pa PAM solved in a subsequent study showed dual - - phenylalanines (the natural substrate and product, resp ectively) covalently linked to the MIO moiety. 29 The latter study demonstrated that residual ammonia remained in the buffer after enzyme purification through affinity chromatography, likely coming from the Lysogen y Buffer (LB) used to grow the bacteria in which Tc PAM was overexpressed. Dialyzed Tc PAM en zyme preparations were incubated in assays buffers containing cinnamate with the trace concentration of ammonia (NH 4 OH) found in commercial LB media remaining after enzyme affinity chromatography. The trace ammonia was sufficient for Tc PAM to convert cinna mate to - - phenylalanine. 29 Thus, we reinterpreted the covalently - linked structures of Sg TAM to be - hydroxy - - amino a dduct ( Figure. 2 . 6 B ), rather than a dihydroxy e ther intermediate attached to the MIO ( Figure. 2 . 6 A ). 17 30 A B Figure. 2 . 6 . Proposed covalent binding of a 3 - arylglycidate inhibitor in the Sg TAM active site A) Formation of a dihydroxy ether intermediat e reported by Montanavon and co - workers. 17 B) Reinterpretation of bound - hydroxy - - amino adduct formed by MIO - NH 2 as informed by the proposed amino - linked adducts made in the Pa PAM crystal structures. 29 Encouraged by the intermolecular mechanism of Tc PAM and our rein terpretation of the chemistry of MIO enzymes with epoxides , we hypothesized that 3 - arylglycidates could be potential amine acceptors during the transamination reaction catalyzed by Tc PAM. H ere, w e describe a new application for the Tc PAM catalyst to transf er an amino group to various ring - substituted 3 - arylglycidat es to make ar ylserine s (major) and arylisoserines (minor). 2.1.7 Arylserines and Arylisoserines 2.1.7.1 Importance and Occurrence in Medicinally Active Compounds Nonproteinogenic amino acids are a specialized c lass of organic compounds that often have intrinsic biological activity . 3,54 - 56 More frequently, t hese vital amino acids are found in peptides with antiviral, 57 antitumor, 58 anti - inflammatory , 59 or immunosuppressive 60 activities. - H ydroxy - - amino acids reside in an important subclass of nonproteinogenic amino acids, including arylserines , hydroxyaspartic acid, and hydroxyleucine . These bifunctional compounds introduce two chiral centers when used as biomolecular building blocks. I ncorporation of unique stereochemistry into th e residues of peptide chains can change their properties by, for example, increasing the stability against peptidases and prolonging bioavailability. 61 - 63 - hy droxy - - amino acid scaffolds ( Figure. 2 . 7 ) are found in clinically signific ant antibiotics, including glycopeptide vancomycin and its analogues, ristocetin from Amycolatopsis 64,65 and teico planins 31 from Actinoplanes , 66 the phenylpropanoid chloramphenicol from Streptomyces , 67 and katanosin depsipeptides from Cytophaga and Lysobacter bacteria. 68,69 The pentapeptide g ymnangiamide from marine hydroid Gymnangium regae shows anticancer activity. 70 ( 2 S , 3 R ) - 3,4 - D ihydroxyphenylserine ( DOPS , Droxidopa ) is used for hypotension and as a Parkinsonian therapeutic, 71 - 73 while N - arylsulfonyl derivatives of phenylserine ethyl esters function as non - steroidal anti - inflammat ory drugs. 7 4 Gymnangiamide [(2 S ) - syn ] (anticancer) Vancomycin [ ( 2 R ) - anti ] (antibiotic) Katanosin [(2 S ) - syn ] (antibiotic) D ihydroxyphenylserine ( DOPS ) [(2 S ) - syn ] ( neurotransmitter prodrug) Chloramphenicol [(2 S ) - syn ] (antibiotic) N - ( p - Br - Benzenesulfonyl ) - syn - 4' - nitrophenylserine racemate (analgesic) Figure. 2 . 7 . - phenyl - - hydroxy - - amino acid building blocks in bioactive compounds. Stereochemical designations listed in brackets [] are described in Figure. 2 . 8 . H acids have also been used recent ly in protein synthesis by chemical ligation at N - terminal serine and threonine sites , and this technique can likely be expanded to incorpora te other . 75 Addition ally, an attractive subclass of non - - h ydroxy - - amino acid ami des are drug leads for their analgesic and immunostimulant activities. 76 - 79 32 A B (2 S ) - syn [ L - threo ] (2 R ) - syn [ D - threo ] (2 S ) - anti [ L - erythro ] (2 R ) - anti [ D - erythro ] (2 S ,3 R ) - syn (2 R ,3 S ) - syn (2 S ,3 S ) - anti (2 R ,3 R ) - anti Figure. 2 . 8 . Stereoisomerism convention for A) Arylserine, and B) Arylisoserine used herein . The equivalent, archaic designations for arylserine diastereomers are listed in brackets []. Sim ilarly, the interest in - h ydroxy - - amino acid s has grown significantly in recent years, stemming partly from their abundance in biologically active molecules such as bestatin, 80 edeine, 81 tatumine, 82 and microginin, 83 and also due to the use of these iso - amino acids to synthesize protease inhibitors ( Figure. 2 . 9 ). 84 Prominent members of this class of compounds include isoserine, 85 isothreonine, 86 3 - amino - 2 - hydroxydecanoic acid, 87 3 - amino - 2 - hydroxy - 4 - phenylbut yric acid 88 and 3 - phenylisoserine. 89 Paclitaxel (Taxol ® ), has been a widely used drug in taxane chemotherapy since its FDA approval in 1992, contains a (2 R ,3 S ) - 3 - phenylisoserine side chain ( Figure. 2 . 9 ). It is the drug of choice for certain types of ovarian and breast cancers as it blocks the microtubule disassembly. 90 - 96 An analogue, docetaxel (Taxotere ® ), is used in metastatic breast cancer, ovarian and lung cancer, and also AIDS r . 97 - 100 Cabazitaxel (Jevtana ® ) is potent newer generation taxane used against castration resistant prostate cancer. 101 - 103 Other instances of molecules containing a phenyli soserine moiety include ( ) - cytoxazone, which is a potent, synthetic chemotherapeutic agent for atopic der matitis and asthma, 104,105 and a lactarius sesqui terpene, modified synthetically with a phenylisoserine side chain, has antiviral, cytotoxic, and anti - proliferative properties ( Figure. 2 . 9 ). 106,107 33 Paclitaxel: R 1 = Ph, R 2 = Ac, R 3 = H Docetaxel:R 1 = t - BuO, R 2 = H, R 3 = H Cabazitaxel:R 1 = t - BuO, R 2 = CH 3 , R 3 = CH 3 (Anticancer) Bestatin (Protease inhibitor) ( ) - Cytoxazone (Antibiotic) Lactarius se squiterpene (Antiviral, Cytotoxic) Figure. 2 . 9 . - hydroxy - - amino acid building blocks in bioactive compounds. 2.1.7.2 Current Methods for the Chemical Synthesis of Arylser ine Given the beneficial pharmacological , chemical, and physical properties of a compound - h ydroxy - - amino acid s, t here is considerable interest in making these bifunctional scaffolds by synt hetic approaches or in combination with biocatalytic routes. 79,108,109 Various synthetic st rategies have been used to control the regio - - h ydroxy - - amino acids , including asymmetric aldol condensation, oxy - Michael addition, electrophilic amina tion, aminohydroxylation of alkenes, and aza - Claisen rearrangement( Figure. 2 . 10 ). 71,110 - 113 34 Figure. 2 . 10 . - hydroxy - - amino acids: a). aza - Claisen rearrangement; b). proteinogenic amino acid derivatization; c). asymmetric hydrogenation; d). Strecker reaction; e). Sharpless aminohydroxylation and dihydroxylati on reaction; f). Electrophilic amination; g). Mannich type reaction; h). Oxy - Michael addition; i). Asymmetric aldol condensation; and j). multicomponent reaction. Several of these methods incorporate protecting group manipulations to direct regiochemistry, add a functionalized chiral auxiliary to incorporate stereogenic centers, or apply heavy - metal catalysts to promote the reactions. These s ynthetic approaches are often highly efficient and s tereoselective, but chemical manufacturers observe that these rou tes often violate green chemistry principles through the generation of hea vy - metal waste, poor atom economy built on the synthesis of intricate chiral - ligand catalysts, and frequently fall short as sustainable methods. 114 - 116 2.1.7.3 Current methods for the Biocatalysis of Arylserines Biocatalysis is emerging as a valuable complementary tool for organic chemists to acces s regio - and stereocontrolled chemical transformations that otherwise use complex synthetic chiral ligand s and often proceed through multiple steps in conventional methods. 115,117 - 119 Biocatalytic routes towards (2 S )+(2 R ) - ( syn / anti ) - - hydroxy - - amino acids ( Figure. 2 . 8 ) mixture s regularly use threonine aldolases (TAs). 120 TAs are divided into two groups depending on the ( R ) - or ( S ) - stereochemistry 35 at the C of threonine , where the amino group is attached. While the substrate specificity of TAs depends on the glycine donor substrat e , they have broad specificity for the aldehyde, including non - natural aryl aldehydes when used to produce arylserines. 121 TAs are divided further into four subgroups, depending on the stereochemistry at the C of threonine where the hydroxyl group is attached . High - s p ecificity (2 S ) - syn - TAs make only ( 2 S ) - syn - threonin es , (2 S ) - anti - TAs are stereoselective for (2 S ) - anti - threonine s , while low - specificity ( 2 S ) - TAs make a mixture of (2 S ) - syn - and (2 S ) - anti - threonine s . Only low - specificity ( 2 R ) - TAs are known and make mixtures of (2 R ) - syn - and (2 R ) - anti - threonine s . 109 TA specificity is also used i n industrial chemical processes for enantiospecific enzyme resolution to cleave (via a retro - aldol reaction) one enantiomer of a syn - or anti - - hydroxy - - amino acid pair. 109 This process stereospecifically enriches one stereoisomer over the other in a mixture. For example, a low specificity (2 R ) - TA stereospecifically catalyzed the retro - aldol cleavage o f the (2 R ) - isomer in a (2 S )+(2 R ) - syn - racemate to resolve the intact (2 S ) - syn - (3',4' - methylenedioxy)phenylserine enantiomer, a precursor of the therapeutic drug (2 S ) - syn - DOPS ( L - DOPS). 122 2.1.7.4 Biocatalysis of Arylserine and Arylisoserine using a MIO - Aminomutase In t his work , we used a n irreversible , TA - independent biocatalytic method employing a repurposed 5 - methylidene - 3,5 - dihydro - 4 H - imidazol - 4 - one (MIO) - dependent phenylalanine aminomutase ( Tc PAM) 27,46,123 to make various ring - substituted phenylserines . Tc PAM converts ( 2S ) - - phenylalanine to ( 3R ) - - phenyl alanine on the biosynthetic pathway to paclitaxel (Taxol). The inherent transaminase activity - amino acid isomerase , 50,124 was repurposed to transfer an amino group from a surrogate donor substrate ( 2S ) - styryl - - alanine to various ring - 36 substituted trans - 3 - arylglycidates to make different arylserine and arylisoserine analogues ( Figur e. 2 . 11 ). Figur e. 2 . 11 . Biocatalysis of arylserine and arylisoserine analogues from trans - 3 - arylglycidates and (2 S ) - styryl - - alanine by using an MIO - aminomutase. The asterisk ( * ) indicates a chiral center. To our surprise, in an initial pilot study, when the unsubstituted trans - 3 - phenylglycidate was incubated with Tc PAM, the amino group was transferred at the C - hydroxy - - amino acid phenylserine. Encouraged by this preliminary data, we hypothesized that the electroni c effects of the substituents on the aromatic ring of 3 - phenylglycidates would impact the regioselectivity of the amino group transfer. This exploration is the first instance of using an aminomutase to biocatalyze pharmaceutically and industrially importan t hydroxy amino acids. 2.2 Experimental 2.2.1 Chemicals and Rea gents trans - Cinnamic acid, trans - 3 - methoxycinnamic acid , trans - 3 - meth yl cinnamic acid , trans - 3 - fluoro cinnamic acid , trans - 3 - chloro cinnamic acid , trans - 3 - bromo cinnamic acid , trans - 3 - nitro cinnamic acid , tra ns - 4 - fluoro cinnamic acid , trans - 4 - chloro cinnamic acid , trans - 4 - bromo cinnamic acid , trans - 4 - nitro cinnamic acid , oxone monopersulfate, sodium phosphate monobasic, sodium phosphate dibasic heptahydrate, benzaldehyde, 3 - methylbenzaldehyde, 3 - methoxybenzaldehyd e, 3 - fluorobenzaldehyde, 3 - chlorobenzaldehyde, 3 - bromobenzaldehyde, 3 - nitrobenzaldehyde, 4 - methylbenzaldehyde, 4 - fluorobenzaldehyde, 4 - chlorobenzaldehyde, 4 - bromobenzaldehyde, 4 - nitrobenzaldehyde, rac - (2 S +2 R ) - syn - phenylserine, pyridoxal - - phosphate, chlor otrimethylsilane, and (2 S ) - (+) - methylbutyric anhydride were purchased from 37 Millipore - Sigma (Burlington, MA). trans - 4 - M eth yl cinnamic a cid and 1,1,1 - trifluoroacetone were purchased from Oakwood Chemical (Estill, SC). (2 S ) - Styryl - - alanine and L - 3 - bromo - - phe nylalanine was purchased from Chem Impex (Wood Dale, IL). (2 S ) - syn - Phenylserine and (2 R ,3 S ) - p henylisoserine hydrochloride was purchas ed from Bachem (Torrance, CA). All chemicals were used without further purification unless noted . 2.2.2 Instrumentation A gas chr omatograph (6890N, Agilent) coupled with a mass selective detector (5973 inert , Agilent) operated in electron impact mode (70 eV ionization voltage) was used for the analysis of derivatized amino acids. The instrument was equipped with a capillary GC colum n (30 m × 0.25 mm × 0.25 uM; HP - 5MS; J & W Scientific) with He as the carrier gas (flow rate, 1 mL/min). The injector port (at 250 °C) was set to splitless injection mode. A 1 - uL aliquot of each sample was injected using an Agilent 7683 aut o - sampl er (Agile nt, Atlanta, GA). A capillary GC column (25 m × 0.25 mm × 0.39 mm; CP - Chirasil - Dex CB, thickness 0.25 µm; Agilent Technologies, Santa Clara, CA) was used for the analysis of methyl - 3 - arylglycidates. All 1 H (500 MHz) and 13 C NMR (126 MHz) spectra w ere recor ded using a Varian superconducting NMR - spectrometer using standard acquisition parameters. LC/ESI/MSMS analyses were recorded with a Xevo G2 - XS QTof (Waters, Milford, MA), and LC/ESI by MRM spectral data was acquired on a Xevo TQ - S (Waters, Milfor d, MA) in strument. 2.2.3 General Procedure for the Syntheses of trans - 3 - Arylglycidate Analogues Th e racemic arylglycidates were synthesized according to a procedure described previously. 125 In a 50 - mL single - necked round - bottomed flask, a stirred slurry of a trans - aryl acrylic acid analogue ( 13 a - 13 l ) (0.75 mmol) in acetone (515 µL, 7.5 mmol) was treated first wit h sodium bicarbonate (3. 3 mmol) and then with dropwise addition o f water (515 µL). To the resulting thick 38 mixture, a solution of oxone monopersulfate (1. 4 mmol, contains 1. 8 equiv of KHSO 5 ) in 0.4 mM Na 2 EDTA solu ti on (1.6 mL) was added dropwise for 1 h while the temperature was kept at ~25 °C and the pH at 7.5. T he mixt ure was then stirred an additional 6 h and cooled to 5 °C. The reaction was acidified to pH 2 (12 M HCl ) and mixed with ethyl acetate (5 mL) with rapi d stirring. The mixture was then filtered and extract ed with et hyl acetate (3 × 50 mL). The combined orga nic fractions were washed with saturated NaCl, dried over anhydrous MgSO 4 , filtered, and concentrated under vacuum . After all the solvent was removed, each trans - aryl glycidic acid analog was isolated as an oily residue. The oily residue was dissolved in e thanol ( EtOH ) (1 mL), cooled on ice, and treated with a solution of KOH (3. 6 mmol) dissolved in EtOH (1 mL). The resulting thick slurry was filtered, a nd the residue was washed with EtOH and then dried under vacuum to provide the potassium 3 - arylglycidate s as a racemic mixture. Potassium 3 - phenyl glycidate ( 14 a ). By f ollowing the general procedure , 420 mg (88% yield ) of 14 a was made . 1 H NMR (500 MHz, D 2 J = 7.8, 5.7 Hz, 3H), 7.37 7.33 (m, 2H), 3.97 (d, J = 2.1 Hz, 1 H), 3.56 (d, J = 2.2 Hz, 1 H). 13 C NMR (126 MHz, CD 3 129.14, 128.93, 126.20, 59.04, 57.66. HRMS (ESI - TOF) m / z : [M K] calcd for C 9 H 7 O 3 163.0395; Found 163. 0398. Potassium 3 - (3' - OCH 3 - phenyl) glycidate ( 14 b ). Conversion of the 3' - OCH 3 - cinnamate starting material was incomplete, yielding a mixture of glycidate and cinnamat e at 8 8 :1 2 ( 14 b : 13 b ). Following the general procedure yielded 264 mg of 14 b ( 41 %). 1 H NMR (500 MHz, DMSO - d 6 ) 7.23 (t, J = 7.9 Hz, 1H), 6.86 6.76 (m, 3H), 3.73 (s, 3H), 3.68 (d, J = 2.0 Hz, 1H), 3.00 (d, J = 2.0 Hz, 1H). 13 C NMR (126 M Hz, DMSO - d 6 60.99, 55.58, 55.04. HRMS (ESI - TOF) m / z : [M K] calcd for C 10 H 9 O 4 193.0501 ; Found 193.0504 . 39 Potassium 3 - (3' - CH 3 - phenyl) glycidate ( 14 c ). By f ollowing the general procedure , 240 mg (78% yield ) of 14 c was made . 1 H NMR (500 MHz, DMSO - d 6 J = 7.5 Hz, 1 H), 7.09 (d, J = 7.5 Hz, 1 H), 7.02 (d, J = 8.3 Hz, 2H), 3.70 (d, J = 2.0 Hz, 1 H), 3.09 (d, J = 2.0 Hz, 1 H), 2.25 (s, 3H). 13 C NMR (126 MHz, DMSO - d 6 60.45, 56.60, 21.57. HRMS (ESI - TOF) m / z : [M K] calcd f or C 10 H 9 O 3 177.0552; Found 177.0551. Potassium 3 - (3' - F - phenyl) glycidate ( 14 d ). By f ollowing the general procedure , 300 mg (63% yield ) of 14 d was made ; 1 H NMR (500 MHz, DMSO - d 6 7.27 (m, 1 H), 7.14 6.98 (m, 3H), 3.79 (d, J = 1.7 Hz, 1 H), 3.14 (d, J = 2.0 Hz, 1 H). 13 C NMR (126 MHz, DMSO - d 6 163.80, 141.40, 131.03, 122.54, 115.26, 112.88, 60.70, 55.68. HRMS (ESI - TOF) m / z : [M K] calcd for C 9 H 6 O 3 F 181.0301; F o und 181.0302. Potassium 3 - (3' - Cl - phenyl) glycidate ( 14 e ). By f ollowing the general procedure , 760 mg (86% yield ) of 14 e was made ; 1 H NMR (500 MHz, DMSO - d 6 7.31 (m, 2H), 7.29 (s, 1 H), 7.22 (d, J = 6.8 Hz, 1 H), 3.75 (d, J = 2.1 Hz, 1 H), 3.01 (d, J = 2.1 Hz, 1 H). 13 C NMR (126 MHz, DMSO - d 6 HRMS (ESI - TOF) m / z : [M K] calcd for C 9 H 6 O 3 Cl 197.0005; Found 197.0009. Potassium 3 - (3' - Br - phenyl) glycidate ( 14 f ). By f ollowing the general procedure , 960 mg (91% yield ) of 14 f was made ; 1 H NMR (500 MHz, DMSO - d 6 7.40 (m, 2H), 7.27 (m, 2H), 3.74 (d, J = 6.6 Hz, 1 H), 3.01 (d, J = 6.5 Hz, 1 H). 13 C NMR (126 MHz, DMSO - d 6 130.95, 130.93, 128.71, 125.21, 122.14, 61.74, 55.14. HRMS (ESI - TOF) m / z : [M K] calcd for C 9 H 6 O 3 Br 240.9500; Found 24 0.9501. Potassium 3 - (3' - NO 2 - phenyl) glycidate ( 14 g ). By f ollowing the general procedure , 262 mg (57% yield ) of 14 g was made ; 1 H NMR (500 MHz, DMSO - d 6 8.10 (m, 1 H), 8.06 (t, J = 2.0 Hz, 40 1 H), 7.76 7.70 (m, 1 H), 7.63 (t, J = 7.9 Hz, 1 H), 3.93 (d, J = 2.0 Hz, 1 H), 3.06 (d, J = 2.1 Hz, 1 H). 13 C NMR (126 MHz, DMSO - d 6 54.49. HRMS (E SI - TOF) m / z : [M K] calcd for C 9 H 6 NO 5 208.0246; Found 208.0247. Potassium 3 - (4' - NO 2 - phenyl) glycidate ( 14 h ). 1,1,1 - Trifluoroacetone (1.8 equiv) was used instead of a cetone and the reaction was stirred for 24 h after the addition of oxone. Yield: 154 mg of 14 h (28%); 1 H NMR (500 MHz, DMSO - d 6 J = 8.7 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 3.90 (d, J = 2.0 Hz, 1 H), 3.04 (d, J = 2.0 Hz, 1 H). 13 C NMR (1 26 MHz, DMSO - d 6 146.95, 146.72, 126.81, 123.54, 61.91, 54.66. HRMS (ESI - TOF) m / z : [M K] calcd for C 9 H 6 NO 5 208.0246; Found 20 8.0249. Potassium 3 - (4' - CH 3 - phenyl) glycidate ( 14 i ). Following the general procedure yielded 320 mg of 14 i (68%). 1 H NMR (500 MHz, DMSO - d 6 7.09 (m, 4H), 3.71 (d, J = 2.0 Hz, 1 H), 3.15 (d, J = 1.7 Hz, 1 H), 2.23 (s, 3H). 13 C NMR (126 MHz, DMSO - d 6 126.88, 60.33, 57.18, 21.69. HRMS (ESI - TOF) m / z : [M K] calcd for C 10 H 9 O 3 177.0552; Found 177.0549. Po tassium 3 - (4' - F - phenyl) glycidate ( 14 j ). Following the general procedure yielded 426 mg of 14 j (91%); 1 H NMR (500 MHz, DMSO - d 6 J = 8.7, 5.5 Hz, 2H), 7.13 (t, J = 8.9 Hz, 2H), 3.76 (d, J = 2.0 Hz, 1 H), 3.14 (d, J = 2.1 Hz, 1 H). 13 C NMR (126 MHz, DMSO - d 6 161.82, 134.24, 128.77, 128.70, 116.24, 116.07, 60.45, 56.22. HRMS (ESI - TOF) m / z : [M K] calcd for C 9 H 6 O 3 F 181.0301; F ound 181.0301. Potassium 3 - (4' - Cl - phenyl) glycidate ( 14 k ). Following the general procedure yielded 137 mg of 14 k (86%); 1 H NMR (500 MHz, DMSO - d 6 J = 8.5 Hz, 2H), 7.39 (d, J = 8.5 Hz, 2H), 4.16 (d, J = 1.8 Hz, 1 H), 3.66 (d, J = 1.8 Hz, 1 H). HRMS (ESI - TOF) m / z : [M K] calcd for C 9 H 6 O 3 Cl 197.0005; Found 197.0007. 41 Potassium 3 - (4' - Br - phenyl) glycidate ( 14 l ). Following the general procedure yielded 1.2 g of 14 l (64%); 1 H NMR (500 MHz, DMSO - d 6 J = 8.1 Hz, 2H), 7.19 (d, J = 8.0 Hz, 2H), 3.76 (d, J = 2.0 Hz, 1 H ), 3.17 (d, J = 2.0 Hz, 1 H ). 13 C NMR ( 126 MHz, DMSO - d 6 137.42, 132.53, 129.12, 122.52, 60.46, 56.71. HRMS (ESI - TOF) m / z : [M K] calcd for C 9 H 6 O 3 Br 240.9500; Found 240.9503. 2.2.4 Stereochemical A nalysis of trans - 3 - Arylglycidates using GC/EI - MS. Each potassium 3 - arylglycidate (0.05 mmol), suspended in 1 mL H 2 O , was titrated with 6 N HCl (pH 3). The resulting aryl glycidic acids were extracted into ethyl acetate (2 mL) , and the organic solution was treated with diazomethane (0.9 equiv) dissolved in ether. The org anic solvent gas chromatography/electron - impact mass spectrometry (GC/EI - MS). Chiral GC/EI - MS analysis was performed on an Agilent 6890N gas chromatograph equipped with a capillary GC colu mn (25 m × 0.25 mm × 0.39 mm; CP - Chirasil - Dex CB, thickness 0.25 µm; Agilent Technologies, Santa Clara, CA) with He as the carrier gas (flow rate, 1 mL/min). The injector port (at 250 °C) was set to splitless injection mode. A 1 - uL aliquot of each sample w as injected using an Agilent 7683 auto - sampler (Agilent, Atlanta, GA). Various GC heating gradients were used to elute each of the epoxide racemates from the column ( Table 2 . 2 ). Mass spectra were recorded in the mas s range of 50 400 m / z to analyze the analogues of methyl 3 - arylglycidate ( Figure. 2 . 12 and Figure. 2 . 13 ). 42 Table 2 . 2 . GC Oven Heating Parameters. Glycidate GC/EI - MS conditions 14 a , 14 i I nitial column temperatur e started at 70 °C, then increased at 40 °C/min to 95 °C with a 7 min hold , ramp ed at 10 °C/min to 150 °C, then increased by 30 °C/min to 175 °C, and returned to 70 °C over 3 min. 14 b Initial column temperature started at 70 °C , then increased at 40 °C/min to 90 °C with a 10 min h old, ramp at 8 °C/min to 150 °C and held for 8.5 min, then increased by 20 °C/min to 180 °C, and returned to 70 °C over 3 min. 14 c Initial column temperature started at 70 °C, then incr eased at 40 °C/min to 90 °C with a 7 min hold, ramp at 10 °C/min to 140 °C, then increased by 30 °C/min to 170 °C, and returned to 70 °C over 3 min. 14 d , 14 j Initial column temperat ure started at 70 °C , then increased at 40 °C/min to 95 °C with an 8 min hold, ramp at 10 °C/min to 140 °C, then increased by 30 °C/min to 180 °C, and returned t o 70 °C over 3 min. 14 e Initial column tempera ture started at 70 °C, then increased at 40 °C/min to 95 °C with a 10 min hold, then ramp at 8 °C/min to 150 °C , then increased by 30 °C/min to 180 °C, and returned to 70 °C over 3 min. 14 f , 14 l Initial column temperature started at 70 °C , then increased at 40 °C/min to 90 °C with a 5 min hold, ramp at 10 °C/min to 150 °C and held for 7 min, then increased by 20 °C/min to 180 °C, and returned to 70 °C over 3 min. 14 g , 14 h Initial column temperature started at 70 °C , then increased at 40 °C/min to 95 °C with a 15 min hold, then ramp at 8 °C/min to 150 °C with a 10 min h old, then increased by 10 °C/min to 185 °C, and ret urned to 70 °C over 3 min. 14 k Initial column temperature started at 70 °C , then increased at 40 °C/min to 90 °C with a 2 min hold, then ramp at 10 °C/min t o 150 °C with a 7 min hold, then increased by 20 °C/min to 180 °C, and returned to 70 °C over 3 min. 2.2.5 Stability of Racemic Arylglycidates in Assay Buffer The water in A ssay Buffer ( 20 mL, 50 mM NaH 2 PO 4 /Na 2 HPO 4 , pH 8.0, 0 % glycero l) was evaporated under a s tream of nitrogen gas, D 2 O (20 mL) was added and then evaporated to replace the exchangeable protons with deuterons. This deuteron - for - proton exchange was repeated to depress the HDO signal in the 1 H NMR spectrum, and the concentrated buffer was dissolved in D 2 O to achieve a final concentration was 50 mM phosphates. Each glycidate substrate ( 14 a - 14 l ) was dissolved separately in the deuterated Assay Buffer and analyzed in a time course 1 H NMR experiment. Spectra were recorded every 15 min, starting from t 1 = 0 min to t 7 = 90 min to monitor the formation of dihydroxy products resulting fr om ring - opening of the epoxides. After this 90 min 43 time course, the samples were then stored inside the NMR tube at ~25 ° C for 7 days, and the quality of the sample was assessed by 1 H NMR ( Figure. 2 . 14 ) . 2.2.6 Expression and Purification of Tc PAM. The tcpam cDNA (codon - optimized for expression in bacteria) was previously ligated into the expression vector pET28a(+), 27 and the recombinant - plasmid encoded an N - terminal His 6 tag. Esch erichia coli BL21(DE3) cells, transformed to express Tc PAM, were grown at 37 °C for 12 h in 150 mL of Lysogeny brot h (LB). Separate aliquots (25 mL) of this inoculum culture were then added to each of six 1 - L cultures of LB supplemented with kanamycin (50 incubated at 37 °C until OD 600 = 0.7. Isopropyl - - D - thiogalactopyranoside ( IPTG, added to the cultures with expression conducted at 16 °C for 16 h. The cells were harvested by centrifugation at 4,650 g (15 min), and the pe llets were diluted in resuspension buffer (100 mL of 50 mM sodium phosphate containing 5% (v/v) glycerol and 300 mM NaCl, pH 8.0). The cells were then lysed by brief sonication [one 10 - s burst at 60% power with a 20 - s rest interval for 20 cycles on a Mison ix Sonicator (Danbury, CT)]. The cellular debris was removed by centrifugation at 27,200 g (20 min) followed by high - speed centrifugation at 142,000 g (90 min) to remove light membrane debris. The resultant crude aminomutase in the solub le fraction was purif ied by nickel - nitrilotriacetic acid affinity chromatography according to the protocol described by the manufacturer (Invitrogen, Carlsbad, CA); Tc PAM eluted in 50 mL of 250 mM imidazole dissolved in resuspension buffer. Fractions conta ining active soluble Tc PAM (76.5 kDa) were combined and loaded onto a size - selective centrifugal filtration unit (30,000 NMWL, Millipore Sigma, Burlington, MA). The protein solution was concentrated and diluted over several cycles until the imidazole and s alt concentrations we re <1 µM, and the final volume was 1 mL (~14 mg of Tc PAM ) . The quantity of Tc PAM was measured using a Nanodrop spectrophotometer 44 (ThermoFisher Scientific, Waltham, MA) , and the purity (82%) was assessed by SDS - PAGE with Coomassie Blue staining using Kodak Gel Logic 100 Imaging System (version 3.6.3) to integrate the relative intensities of the scanned protein bands. 2.2.7 Expression and Purification of ( 2 S ) - Threonine Aldolase ( lta E ). A cDNA (from E. coli , accession number: P75823) encoding a low specificity (2 S ) - threonine aldolase ( l ta E ) was ligated into a pET28a(+) vector, encoding an appended N - terminal His 6 - tag, to make a recombinant plasmid (designated as pKDW 014 _lsTA) was purchased from GenScript (Pi scataway, NJ). Escherichia coli BL21(DE3) cells were then tr ansformed with pKDW 014 _lsTA to overexpress the l ta E gene by standard protocols ( Millipore Sigma, Burlington , MA ). Transformed bacteria were used to inoculate LB (100 mL) and grown at 37 °C for 12 h. Separate aliquots (15 mL) of this inoculum culture were a dded to each of three 1 - L cultures of LB 600 = 0.6, and IPTG to induce expression at 16 °C fo r 16 h . The cells were harvested by centrifugation at 4,650 g (1 5 min), and the pellets were diluted in resuspension buffer (at pH 7.0 ) containing 10 µM PLP and 300 mM NaCl. The cells were then lysed by brief sonication [one 10 - s burst at 6 0% power with a 2 0 - s rest interval for 20 cycles on a Misonix Sonicator ], and th e cellular debris was removed by centrifugation at 27,200 g (20 min) followed by high - speed centrifugation at 142,000 g (90 min) to remove light membrane debris. The clarified lysate containing crude (2 S ) - TA in the soluble fraction was purified by nickel - nit rilotriacetic acid affinity chromatography according to the protocol described by the manufacturer ( Invitro gen, Carlsb ad , CA ); (2 S ) - TA was eluted in 50 mL of 250 mM imidazole. Fractions containing active soluble (2 S ) - TA (36. 5 kDa) were combined and loaded o nto a size - selective centrifugal filtration unit ( 15,000 NMWL, Millipore Sigma ). The protein solution was concentrate d and diluted over several cycles 45 until the imidazole and salt concentrations were <1 µM , and the final volume was 1 mL. The concentration of (2 S ) - TA was measured (8.8 mg/mL) using a Nanodrop spectrophotometer ( ThermoFisher Scientific). T he purity ( 99 %) was assessed by SDS - PAGE with Coomassie Blue staining using a Kodak Gel Logic 100 Imaging System (version 3.6.3) to integrate the relative i ntensities of the scanned protein bands . 2.2.8 Control Assay Experiments: Activity of Tc PAM toward Arylglycidates 3 - (3' - M ethoxyphenyl)glycidate ( 14 b ) was used as a represe ntative substrate in the control assays that contained all the necessary components for an operational assay except either the amine group donor [ (2 S ) - styryl - - alanine ( 1 mM ) or NH 4 OH (2 M)] or the enzyme was omitted. Each assay was analyzed using a liquid - chromatography electrospray - ionization and analyzed by a multiple reaction monitoring (LC/ESI - MRM ) method . 2.2.9 Biocatalysis of Ar ylserine and Arylisoserine with Tc PAM . A solution of ( 2 S ) - styryl - ( 12 ) (1 mM) in Assay Buffer ( 50 mM Na H 2 PO 4 / Na 2 HPO 4 buffer , pH 8. 0, 5% glycerol) was preincubated with Tc PAM ( 100 µg/mL) for 2 min . 3 - Arylglycidate ( 14 a - 14 l ) (1 mM) was added to the solution, and the assay was mixed at 31 ° C on a rocking shaker for 2.5 h. The reaction was then stopped with 10 % formic acid to adjust the pH to 3 .0 , and 3' - bromo - - phenylalanine (50 n M) was added as an internal standard. This reaction mixture was analyzed by LC/ESI - MRM method . 2.2.10 Measurement of Kinetic Parameters . The steady - state enzyme kinetic constants were calculated by varying each arylglycidate substrat ing the concentration in assays containing Tc PAM (100 µg/mL) and ( 2 S ) - styryl - ( 12 ) (1 mM) . The reactions were terminated with 10% formic acid (pH 3.0) , and 3' - br omo - - phenylalanine (50 n M) was added as 46 an internal standard . T he resultant ring - opened biocatalyzed products wit hout, derivatization, were quantified by LC/ESI - MRM. The apparent kinetic parameters for the production of arylserine and arylisoserine ( , , ) were calculated by non - linear regression with Origin Pro 9.0 software (Northampton, MA) , using the Michaelis - Menten equation: v o = [E o ] k cat /( K M + [S]) . 2.2.11 General Procedure for the Syntheses of Ary lserines . The aryl serine analogue s were synthesized according to a procedure described previously. 126 T riethylamine (22 mmol) was added to a solution of glycine (5 mmol) in water (4 mL). To this solution, an aryl aldehyde ( 19 a - 19 l ) (10 mmol) was added dropwise over 15 min , and the mixture was stirre d for 12 h at ~25 °C . The color of the reaction mixture gradually changed from clear and colorless to yellow - brown. n - B utanol (3 mL) was added , and the triethylamine was evaporated under vacuum . The butanolic solution was diluted with w ater (3 mL) , and the mixture was acidified to pH 2 with HCl ( 6 M). The acidified solution was stirred at ~25 °C for 3 h and partitioned against ethyl acetate (2 × 5 mL) to remove the unreacted aryl aldehyde . The aqueous layer was separated and neutralized to pH 6.0 with a sat urated Na H CO 3 solution to precipitate the aryl serine. The mixture was stirred for 1 h at 0 °C , and the arylserine product was washed with water (3 mL) and dried under vacuum to yield the mixture of aryl serine diastereomers. Phenylserine ( 15 a ). 1 H NMR (500 MHz, D 2 O, pH 1.5) anti - 7.36 (m, 5H), 5.37 (d, J = 4.2 Hz, 1 H), 4.33 (d, J = 4.2 Hz, 1 H). syn - 7.36 (m, 5H), 5.41 (d, J = 4.0 Hz, 1 H), 4.24 (d, J = 4.0 Hz, 1 H). anti : syn = 75:25 . - OCH 3 - phenylserine ( 15 b ). 1 H NMR (500 MHz, D 2 O, pH 1.5) anti - 6.88 (m, 4H), 5.36 (d, J = 4.0 Hz, 1 H), 4.36 (d, J = 4.0 Hz, 1 H). syn - 6.88 (m, 4H), 5.41 (d, J = 3.7 Hz, 1 H), 4.27 (d, J = 3.7 Hz, 1 H). anti : syn = 71:29 . 47 - CH 3 - phenylserine ( 15 c ). 1 H NMR (500 MHz, D 2 O, pH 1.5) anti - 7.16 (m, 4H), 5.33 (d, J = 4.2 Hz, 1 H), 4.33 (d, J = 4.2 Hz, 1 H). syn - 7.16 (m, 4H ), 5.37 (d, J = 4.0 Hz, 1 H), 4.23 (d, J = 4.0 Hz, 1 H). anti : syn = 68:32 . - F - phenylserine ( 15 d ). 1 H NMR (500 MHz, D 2 O, pH 1.5) anti - 7.09 (m, 4 H), 5.39 (d, J = 4.0 Hz, 1 H), 4.38 (d, J = 3.9 Hz, 1 H), syn - 7.09 (m, 4H), 5.45 (d, J = 3.7 Hz, 1 H), 4.29 (d, J = 3 .7 Hz, 1 H). anti : syn = 71:29 . - Cl - phenylserine ( 15 e ). 1 H NMR (500 MHz, D 2 O, pH 1.5) anti - 7.26 (m, 4H), 5.35 (d, J = 4.0 Hz, 1 H), 4.32 (d, J = 3.9 Hz, 1 H), syn - 7.26 (m, 4H), 5.39 (d, J = 3.9 Hz, 1 H), 4.22 (d, J = 3.9 Hz, 1 H). anti : syn = 97:3 . - Br - phenylserine ( 15 f ). 1 H NMR (500 MHz, D 2 O, pH 1.5) anti - 7.30 (m, 4H), 5.36 (d, J = 4.0 Hz, 1 H), 4.33 (d, J = 4.0 Hz, 1 H). syn - 7.30 (m, 4H), 5.40 (d, J = 3.9 Hz, 1 H), 4.23 (d, J = 3.9 Hz, 1 H). anti : syn = 58:42 . - NO 2 - phenylserine ( 15 g ). 1 H NMR (500 MHz, D 2 O, pH 1.5) anti - isomer 8.31 7.65 (m, 4H), 5.50 (d, J = 3.7 Hz, 1 H), 4. 42 (d, J = 3.7 Hz, 1 H). syn - 7.68 (m, 4H), 5.54 (d, J = 3.8 Hz, 1 H), 4.32 (d, J = 3.9 Hz, 1 H). anti : syn = 96:4 . - NO 2 - phenylserine ( 15 h ). 1 H NMR (500 MHz, D 2 O, pH 1.5) anti - isomer 1 H NMR 8.26 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 9.0 Hz, 2H), 5.49 (d, J = 3.6 Hz, 1 H), 4.43 (d, J = 3.6 Hz, 1 H). syn - isomer J = 8.8 Hz, 2H), 7.70 (d, J = 9.0 Hz, 2H), 5.55 (d, J = 3.9 Hz, 1 H) 4.33 (d, J = 3.8 Hz, 1 H). anti : syn = 83:17 . - CH 3 - phenylserine ( 15 i ). 1 H NMR (500 MHz, D 2 O, pH 1.5) anti - 7.24 (m, 4H), 5.33 (d, J = 4.2 Hz, 1 H), 4.34 (d, J = 4.3 Hz, 1 H). syn - 7.34 7.2 4 (m, 4H), 5.37 (d, J = 4.1 Hz, 1 H), 4.25 (d, J = 4.1 Hz, 1 H). anti : syn = 72:28 . 48 - F - phenylserine ( 15 j ). 1 H NMR (500 MHz, D 2 O, pH 1.5) anti - 7.38 ( m, 4H), 7.18 (qd, J = 8.9, 2.1 Hz, 4H), 5.41 (d, J = 4.0 Hz, 1 H), 5.37 (d, J = 4.2 Hz, 1 H), 4.35 (d, J = 4.2 Hz, 1 H), 4.26 (d, J = 4.0 Hz, 1 H). anti : syn = 52:48 . - Cl - phenylserine ( 15 k ). 1 H NMR (500 MHz, D 2 O, pH 1.5) anti - 7.37 (m, 4H), 5.36 (d, J = 4.0 Hz, 1 H), 4.34 (d, J = 4.1 Hz, 1 H). syn - 7.37 (m, 4H), 5.40 (d, J = 4.0 Hz, 1 H), 4.24 (d, J = 4.0 Hz, 1 H). anti : syn = 45:55 . - Br - phenylserine ( 15 l ). 1 H NMR (500 MHz, D 2 O, pH 1.5 ) anti - 7.31 (m, 4H), 5.38 (d, J = 4.0 Hz, 1 H ), 4.38 (d, J = 4.0 Hz, 1 H ). syn - 7.31 (m, 4H), 5.42 (d, J = 4 .0 Hz, 1 H ), 4.28 (d, J = 4.0 Hz, 1 H ). anti : syn = 58:42 . 2.2.12 Establishing Relative Stereoc onfiguration ( syn / anti ) of Arylserine Di astereomers using 1 H NMR Spectroscopy. A 1 H NMR - based method was developed to characterize the relative stereoconfiguration ( syn vs anti ) of arylserine diastereomers without further chemical derivatization. A mixture of chemically synthesized arylserine diastereomers ( 15 a - 15 l ) (0.17 mmol) was dissolved in D 2 O (700 µ L). 1 H NMR spectra were recorded in triplicate studies for a range of pH varying from 4.0 - 1.0 at every 0.5 pH interval. HCl (6 M) was added dropwise to adjust th e pH, which was measured using Taylor colorpHast ® pH test strips 0 - 6 (Millipore - Sigma (Burlington, MA). Chemical shifts ( ) for H and H , coupling constant between H and H ( 3 J H - H ), and the chemical shift difference between H and H 1 H NMR analyses. 2.2.13 General Method for Derivatizing Ar ylserines with a Chiral Auxiliary. A mixture of all fou r dia stereomers of aryl serine ( 15 a - 15 l ) (0.21 mmol) was dissolved in Assay Buffer (1 mL). T o this solution were added pyridine (50 µL, 0.62 mmol) and ( 2S ) - 2 - methylbutyric anhydride (60 µL, 0.30 mmol ), and the reaction mixture was stirred for 20 min at ~25 °C . The 49 solution wa s adjusted to pH 2 (6 M HCl ) to quench the reaction, and the N - protected ary lserine was extracted with ethyl acetate (2 mL). The organic layer was separated and evaporated under a stream of nitrogen gas, and t he resultant residue was dissolved in 3:1 EtOAc/MeOH ( v/v) (1 mL) . D iazomethane in diethyl ether was added dropw ise to obtain the methyl ester, and the solvent was removed under a stream of nitrogen gas. T he resulting methyl ester was diss olved in dichloromethane (1 mL) to which pyridine (100 µL, 1.24 mmol) and chlorotrimethylsilane (150 µL, 1.18 mmol ) were added, and the solution was stir red for 15 min at ~25 °C. The reaction was quenched with water (1 mL ), and the organic fraction was separated and analyzed by gas chromatography coupled with elec tron - impact mass spectrometry (GC/EI - MS) . GC/EI - MS analysis was performed on an Agilent 6890 N gas chromatograph equipped with a capillary GC column ( 30 m × 0. 25 mm × 0. 25 uM; HP - 5MS; J & W Scientific) with He as the carrier gas (flow rate, 1 mL/min). The i njector port (at 25 0 °C) was set to splitless injection mode. A 1 - uL aliquot of each sample was injected using an Agilent 7683 aut o sampler (Agilent, Atlanta, GA). Initial column temperature started at 50 °C , and it was increased at 50 °C/min to 150 °C , then increased by 20 °C/min to 200 °C. It was then ramped at 10 °C/min to 225 °C , with a 5 - min hold , and finally increased by 25 °C/min to 250 °C. - NO 2 - ( 15 g - NO 2 - phenylserines ( 15 h ) i nitial column temperature started a t 50 °C , and it was increased at 50 °C/min to 225 °C , with a 9 - min hold. It was again ramped at 1 °C/min to 22 8 °C , and finally increased by 60 °C/min to 250 °C. The gas chromatograph was coupled to a mass - selective detector (Agilent, 5973 inert ) operated in electron impact mode ( 70 eV ionization voltage). All spectra were recorded in the mass range of 50 40 0 m / z (except 50 450 m / z for 15 f and 15 l ) to analyze the hydroxy amino acid s derivatized as their O - trimethylsilyl N - [( 2 S ) - 2 - methylbutyryl] methyl esters . 50 2.2.14 (2 S ) - Threonine Aldolase catalyzed resolution of Arylserine Diastereomers A low sp ecificity (2 S ) - threonine aldolase (TA) expressed from Escherichia coli was used to catalyze the retro - aldol cleavage of arylserine diastereomers and help assign the absolute stereoc onfigurations . This enzyme catalyzes reversible diastereoselective retro al dol cleavage o f (2 S ) - syn - and ( 2 S ) - anti - arylserine, selectively from a mixture of all four diastereomers, to produce aryl aldehyde and glycine . A mixture of all four dia stereomers of aryl serine ( 15 a - 15 l ) (0.21 mmol) and pyridoxal - - phosphate (PLP, 1 mM) was dissolved in Assay Buffer (910 µL). To this solution (2 S ) - TA ( 1 mg, 8.72 mg/mL ) was added, and the reaction mixture was incubated at 3 1 °C . Aliquots were withdrawn at 0, 10, 20, and 40 min for 15 a - 15 l to assess the timeframe of the steady - state turnover. Each aliquot was derivatized according to the pr ocedure described in Scheme 2 . 1 A to convert the m to their corresponding O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl esters, and analyzed by GC/EI - MS to determine that (2 S ) - TA was at steady state during the 10 - min incubation . 2.2.15 C omputational Methods for Biomolecular Docking. Docking of the trans - 3 - arylglycidate enantiomers was performed using AutoDock Vina (version 1.1.2) 127 and AutoDock Tools (version 4.2.6). 128 The trans - cinnam ate ligands and water molecules were first removed from the crystal structure of Taxus canadensis phenylalanine aminomutase ( Tc PAM; PDB code: 3NZ4). Then the (2 R ,3 S ) - and (2 S ,3 R ) - 3 - arylglycidate ligands ( 14 a 14 l ) and enzyme files were converted to the .pdbqt file format using the AutoDock Tools software to make them compatible with AutoDock Vina. An empty grid was created to define the binding site wh ere the ligands would be docked in the Tc PAM active site . AutoDock Vina provided 9 ori entations for each of the docked epoxides into the active site of Tc PAM. The lowest energy 51 (i.e., apparent highest affinity) and logically docked orientation for each epo xide enantiomer was chosen . 2.2.16 Calculation of Covalent van der Waals Volumes. The molecul ar volumes of the substituents on the aryl carboxylate substrates were estimated as Connolly solvent - excluded volume 129 using Chem 3D Ultra software (ver . 6 3.0, Perkin Elmer) and a probe radius of 0.2 Å. The volum e of a phenyl radical was subtracted from the estimated volume of a phenyl attached to a substituent. The geometries of polyatomic substituents such as CH 3 , OCH 3 , N O 2 attached to phenyl were MM2 energy minimized using the d efault parameters of the Chem 3D U ltra software. 52 2.3 Results and Discussion 2.3.1 Synthesis and Characterization of the Racemic 3 - Arylglycidate Substrates. Various commercially available aryl acrylic acids ( 13 a 13 l ) were oxidized with Murray's reagent to synthesize the corresponding ring - substituted 3 - arylglycidates ( 14 a 14 l ) as their racemates ( Table 2 . 3 ). 125 The enantiomeric ratios of each racemate methyl ester w ere ~1:1 by chiral GC/EI - MS analysis ( Figure. 2 . 12 and Figure. 2 . 13 ) . Trifluoroacetone was used instead of acetone to modify Murray's reagent and oxidize the less reactive NO 2 - cinnamate analogues to epoxides 14 g and 14 h . The 3 - ( 4' - OCH 3 - phenyl)glycidate rapidly hydrolyzed to the dihydroxy compound under these reaction conditions and thus could not be tested in this study. Table 2 . 3 . Synthesis of 3 - Arylglycidate Analogues a and Isolated Yields . 13 ( a l ) a 14 ( a l ) Entry R Time (h) Yield (%) Entry R Time (h) Yield (%) 14a H 2 88 14 g - NO 2 24 30 14b - OCH 3 24 41 b 14 h - NO 2 24 28 14c - CH 3 2 78 14 i - CH 3 24 68 14d - F 12 63 14j - F 16 91 14e - Cl 2 86 14k - Cl 16 86 14f - Br 2 91 14l - Br 6 64 a Step i) NaHCO 3 , acetone (or 1,1,1 - trifluoroacetone), H 2 O ( ) (or NaHCO 3 , CF 3 C(O)CH 3 , H 2 O when R = NO 2 ); step ii) o xone in 0.4 mM EDTA, 2 - 24 h, 25 ° C ; and step iii) KOH in EtOH. b The product was an 88:12 mixture of compounds 14 b and 13b , respectively. 53 2.3.2 Separation of trans - 3 - Arylglycidate Enantiomers using a Chiral - GC/EI - MS The synthesized arylglycidate analogues were found to be racemic ( Figure. 2 . 12 and Figure. 2 . 13 ). A B C D E F Figure. 2 . 12 . E nantiopurity of the synthetically derived methyl esters of A) 3 - phenylglycidate ( 14 a ) (49:51), extracted io n m / z 121; B) 3 - (3' - OCH 3 - phenyl)glycidate ( 14 b ) (50:50), extracted ion m / z 151; C) ( 14c ) 3 - (3' - CH 3 - phenyl)glycidate ( 14 c ) (49:51), extracted ion m / z 135; D) 3 - (3' - F - phenyl)glycidate ( 14 d ) (50:50), extracted ion m / z 139; E) 3 - (3' - Cl - phenyl)glyc idate ( 14 e ) (50:50), extracted ion m / z 155; and F) 3 - (3' - Br - phenyl)glycidate ( 14 f ) (50:50), extracted ion m / z 199 analyzed by chiral GC/EI - MS. The base peak ion w as used for extracted - ion selection of the derivatives; partial chromatograms are shown. The ratio of each enantiomer is shown in parentheses. 54 G H I J K L Figure. 2 . 13 . E nantiopurity of the synthetically derived met hyl esters of G) 3 - (3' - NO 2 - phenyl)glycidate ( 14 g ) (47:53), extracted ion m / z 166; H) 3 - (4' - NO 2 - phenyl)glycidate ( 14 h ) (46:54), extracted ion m / z 166; I) 3 - (4' - CH 3 - phenyl)glycidate ( 14 i ) (50:50), extracted ion m / z 135; J) 3 - (4' - F - phenyl)glycid ate ( 14 j ) (50:50), extracted ion m / z 139; K) 3 - (4' - Cl - phenyl)glycidate ( 14 k ) (4 7:53), extracted ion m / z 155; and L) 3 - (4' - Br - phenyl)glycidate ( 14 l ) (50:50), extracted ion m / z 199.analyzed by chiral GC/EI - MS. The base peak ion was used for extract ed - ion selection of the derivatives; partial chromatograms are shown. The ratio of each enantiomer is shown in parentheses. 2.3.3 Hydrolysis of Arylglycidates in Assay Buffer Racemic 3 - arylg lycidates ( 14 a - 14 l ) were stable in Assay Buffer up to 90 min, and there was n o significant hydrolysis of the epoxide ring after 1 week in Assay Buffer fo r most of the glycidates ( Figure. 2 . 14 ). However, 14 a , 14 c , and 14 i showed a slight (~5%) conversion to the dihydroxy product after one week of incubation. These results suggested that the 3 - arylglycidate substrates are stable in the Assay Buffer during the a mination reaction catalyzed by Tc PAM. 55 14 a dihyd roxy product from 14 a A t 7 t 6 t 5 t 4 t 3 t 2 t 1 B (ppm) Figure. 2 . 14 . 1 H NMR of 3 - phenylglycidate ( 14 a ) in deuterated Assay Buffer. A) NMR s pectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the ratio of 14 a :dihydroxy product = 100:0, and B) NMR spectrum after storing the sample in deuterated A ssay Buffer at ~25 ° C for 1 week, the ratio of 14 a :dihydroxy product = 100:4. See Appendix for the associated 1 H NMR data for other arylglycidate analogues ( 14 b - 14 l ). 56 2.3.4 Control - Assay Experiments Table 2 . 4 . Control Experiments. Experiment (Exp) NH 4 OH (1 mM) NH 4 OH (2 M) (2 S ) - styryl - - alanine ( 12 ) (1 mM) 3 - (3' - OCH 3 - phenyl)glycidate ( 14 b ) (1mM) Tc PAM (100 µg/mL) 3' - OCH 3 - phenylserine ( 15 b ) produced (µM) 3' - OCH 3 - phenylisoserine ( 16 b ) produced (µM) 1 + + 0.05 0.0 2 2 + + + 31 11 3 + + + 0.06 0.02 4 + + 0.03 0.0 1 5 + + + 1 7 2 30 6 + + 16 2 50 In a pilot study that helped guide our kinetic analyses in our earlier published work 48 and later in this Chapter 2: fo r the substrate 3 - (3' - OCH 3 - phenyl)glycidate ( 14 b ) , we observed that Tc PAM turned over 14 b to the highest proportion of isoserine (37%) relative to the serine isomer (63%) compared to the other glycidate substr ates used in the study. We theorized that this higher isoseri ne proportioning likely resulted from nonenzymatic amination of the glycidate at the more electropositive C . Thus, 14 b was used as the model substrate in the control experiments to assess the contribution of nonenzymatic amination of the glycidates to the isoserine /serine distribution compared to that made from Tc PAM - catalyzed amination. Experiment 1 (Exp - 1 ), containing Tc PAM and the glycidate substrate 14 b , was incubated without an amine source, and a small amount of the hydroxy amino acids (~0.07 µM) was detected. Ex p - 3 was identical to Exp - 1, except it contained 1 mM NH 4 OH as the amine source, yet made a similarly low quantity of hydroxy amino acids (~0.08 µ M) as in Exp - 1. These experiments 57 suggested that the supplemental 1 mM NH 4 OH did not improve enzyme turnover of the glycidate to its hydroxy amino acids compared to when the assay only had residual ammonia likely coming from the buffer containing the purified enzyme. 29 Control Exp - 4, containing 1 mM NH 4 OH yet no Tc PAM bioca talyst, contained a similar quantity of hydroxy amino acids (~0.04 µ M), as in Exp - 1 and Exp - 3. The small amount of hydroxy amino acids from 14 b purportedly developed from occasional nonenzymatic epoxide - ring opening by t he NH 4 OH. Control Exp - 5 (with biocatalyst) and 6 (without biocatalyst) were incubated with 14 b and 2 M NH 4 OH a s the amine source ( Table 2 . 4 ). A mixture of serine 15 b and isoserine 16 b products, at 247 µM were measured in Exp - 5 and at 266 µM in Exp - 6, were simila r and showed that at an elevated concentration of NH 4 OH in the buffer, nonenzymatic ammonia - assisted ring - opening of the epoxide occurs predominantly. The condi tions for control Exp - 5 and Exp - 6 were repeated for other glycidate substrates ( 14 a , 14 i , and 14 h ), and their product distributions were similar ( Figure. 2 . 36 in Appendix ) To summarize, Exp - 3 and Exp - 4 are identical to Exp - 5 and Exp - 6, respectively, except that the former two assays contain 1 mM NH 4 OH, making trace amount s of products and the latter two 2 M NH 4 OH, making >200 uM of products. Therefore, from these control experiments, we concluded that when the NH 4 + salts are over 1 mM, the arylglycidates are aminated nonenzymatically. These results also informed us that the typical 6 M NH 4 + salts used in earlier studies by other groups needed to stimulate an MIO - dependent enzyme to aminate arylacrylates 46,49,52 should be avoided while introducing glycidates as substrates. The results of the preceding control assays informed us on Exp - 2 that contained all the necessary components yet included 1 mM (2 S ) - styryl - - alanine as the amine source to ensure the NH 4 + salts in solution was <1 mM. In control Exp - 2, 14 b was converted to serine 15 b and isoserine 16 b (~42 µM combined, 74% serine; 26% isoserine) by Tc PAM - catalyzed amination. The result 58 of the control experime nts using 14 b helped us design an operational transamination assay that was driven by enzyme catalysis and not b y an uncatalyzed side reaction to convert arylglycida tes to hydroxy/amino - 3 - arylpropanoates. Figure. 2 . 15 . Relative abundances of arylisoserine ( 16 b ) and arylserine ( 15 b ) products made from C - and C - aminati on, respectively, of 3 - (3 ' - OCH 3 - phenyl)glycidate ( 14 b ) (1 mM) when incubated with 2M NH 4 OH and Tc PAM (100 µ g/mL) ( top trace ) and w ithout Tc PAM ( bott om trace ). 2.3.5 Biocatalysis of Arylserines and Arylisoserines . Each arylglycidate substrate was incubated separately with the amine donor (2 S ) - styry l - - alanine ( 12 ) and Tc PAM 51 at pH 8.0 to make the ring - opened hydroxy amino aci ds, arylserines, and arylisoserines. (2 S ) - S tyryl - - alanine ( 12 ) was used to aminate the MIO moiety of Tc PAM instead of 6 M ammonium salts at pH 9 used in several previous studies to convert various cinnamates to - - amino acids. 46,49,53 Further, as we showed in this thesis through control experiments, an earlier study also demonstrated that high concentrations of ammonia non enzymatically conv ert arylglycidates by nearly exclusive amination at the benzylic carbon (C ) to aryl isoserine. 130 Th e selective synthetic amination of arylglycidates at C is directed by the 59 ability of the benzylic functional group to resonance stabi lize the + formed in the transition state ( Figure. 2 . 16 ) . This electronic - based regioselectivity would therefore be sensitive to the e lectronic effects of substituents on the aryl ring. Control experiments (with or without the Tc PAM catalyst) in th is study showed that after incubating 2 M NH 4 OH in 50 mM phosphate buffer, pH 8 for 2.5 h with the 3 - arylglycidate substrate bearing a mesomer ically electron - withdrawing 4' - NO 2 ( 14 h ), the amination reaction yielded a ~40:60 mixture of isoserine to serine, with the latter predominating ( Figure. 2 . 15 A ). The 4' - NO 2 4' - NO 2 substituent constant) 131 is positioned to pull electron density of the epoxide oxygen toward C , polarize the C O bond, and thus encourage nucleophilic amination at C to form mostly ar ylserines as observed ( Figure. 2 . 16 ) . By contrast, 3 - phenylglycidate ( 14 a [H] = 0.0) and its 3' - OCH 3 ( 14 b [3 ' - OCH 3 ] = 0.12) and 4' - CH 3 ( 14 i [4' - CH 3 ] = - 0.17) analogues have similar electronic influence on the epoxide ring opening. Substrates 14 a , 14 b , and 14 i favored intrinsic amination at C to form the corresponding 93% compared to the lesser amount of aryl serine when incubated with 2 M NH 4 OH ( Figure. 2 . 15 ), as observed in earlier synthetic studies. 130,132 Thus, (2 S ) - s tyry l - - alanine ( 12 ) was chosen as a milder amine source to dissect the regiochemistry and stereoselectivity of Tc PAM transamination catalysis. Figure. 2 . 16 . Resonance and inductive stabilizations of the partial positive charge ( + ) (due to bond polarization) at the C of arylglycidate substrates 14 a , 14 b , and 14 i assisted by the aryl - ring. For substrate 14 h , the electron - withdrawing NO 2 group resonance destabilizes the + at the benzylic carbon (C ), and the regioselectivity is reversed, directing an incoming nucleophile to attack at C preferentially. 60 2.3.6 Synthe sis of Aryl serine Diastereomer s. Various ring - substituted arylserine analogues ( 15 a 15 l ) were synthesized using the aldol condensation reaction between the correspo nding aryl aldehyde ( 19 a 19 l ) and glycine ( Scheme 2 . 1 B ). anti - Arylserine was formed predominantly over their syn - isomers. The ant i : syn ratio varied from 97:3 (for 3 - Cl) to 45:55 (for 4 - Cl). This gave access to all possible stereoisomers of arylserines ( Figure. 2 . 8 A ) which were later used as synthetic standards to characterize the biocatalyz ed arylserines formed from 3 - arylglycidates and Tc PAM. A B 14 12 a 15 16 b b 17 18 19 a (2 S ) - syn - 15 (2 S ) - anti - 15 (2 R ) - syn - 15 (2 R ) - anti - 15 Entry R anti : syn Entry R anti : syn 15 a H 75:25 15 g - NO 2 96:4 15 b - OCH 3 71:29 15 h - NO 2 83:17 15 c - CH 3 68:32 15 i - CH 3 72:28 15 d - F 71:29 15 j - F 52:48 15 e - Cl 97:3 15 k - Cl 45:55 15 f - Br 58:42 15 l - Br 58:42 Scheme 2 . 1 . A) Tc PAM was incubated with (2 S ) - styryl - - alanine ( 12 ) (1 mM) and separately with each trans - 3 - phenylglycidate racemate ( 14 a 14 l ) . Step a ) i) Tc PAM (1.7 mg/mL) in Assay Buffer, 2 min pre - equilibration , 14 a 14 l (10 mM), 29 ° C, 4 h. Step b ) Deriv atization of putative phenylserines and phenylisoserines using a chiral auxiliary for stereoisomeric resolution. i ) (2 S ) - 2 - Methylbutyric anhydride, pyridine, rt, 20 min; ii ) 6 M HCl, pH 2; iii ) CH 2 N 2 , EtOAc/MeOH (3:1 v / v ), rt, 15 min; and iv ) chlorotrimeth ylsilane, pyridine, CH 2 Cl 2 , rt, 15 min. Insets : stereoisomerism of (2 R ,3 S ) - syn - phenylisoserine. B) Synthesis of the stereoisomers of phenylserine analogues from glycine and a substituted benzaldehyde. a) step i ) triethylamine, n - BuOH and H 2 O, 12 h, rt; ste p ii ) 6 M HCl, pH 2; and step iii ) NaHCO 3 , pH 6 . 61 2.3.7 Assessing the Regiochemistry of the Tc PAM - cataly zed Transamination Reaction. The regiochemistry and relative stereochemistry of the mutase amination reaction were assigned by derivatizing the products made from biocatalysis. For example, authentic racemic (2 S )+(2 R ) - syn - phenylserine [(2 S )+(2 R ) - syn - 15 a ] and ( 2 R, 3 S ) - syn - phenylisoserine [( 2 R, 3 S ) - syn - 16 a ] standards ( Figure. 2 . 8 ) were converted to their O - trimethylsilyl N - [( 2 S ) - 2 - methylbutyryl] methyl esters ( Scheme 2 . 1 A ). The diagnostic fragment ions shown in the GC/EI - MS profile for the derivatized authentic phenylserine racemate were identical to those of the biocatalyzed phenylserine diastereomers yet distinct from those of the ( 2 R, 3 S ) - syn - phen ylisoserine (( 2 R, 3 S ) - syn - 16 a ) standard derivatized identically ( Figure. 2 . 17 ). GC/EI - MS fragmentation of the other biocatalytically made phenylserine analogues ( 15 a 15 l ) was identical to the ion profiles of the correspondi ng synthetic phenylserine analogues derivatized identically ( See Appendix ). 62 A B Relative Ion Abundance (%) m / z Relative Ion Abundance (%) m / z C Relative Ion Abundance (%) m / z m / z 336 Panels A + B m / z 336 Panel C m / z 245 Panels A + B m / z 235 Panel C m / z 190 Panel C m / z 179 Panels A + B m / z 106 Panel C m / z 73 Figure. 2 . 17 . GC/EI - MS spectra of aut hentic A) (2 S )+(2 R ) - syn - phenylserine from Sigma - Aldrich, B) phenylserine biocatalyzed from 3 - phenylglycidate by Tc PAM catalysis (see Figure. 2 . 19 for GC profiles), and of C) (2 R ,3 S ) - syn - phenylisoserine (see Figure. 2 . 72 for GC profile ). Each hydroxy amino acid was derivatized to its O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. m / z 351) was not observed for either analyte. Tc PAM could theoreti cally transfer the amino group to either C or C of the glycidate substrate to make phenylserine or phenylisoserine , respectively . We hypothesized that the aryl ring alone and the electron ics of the substituents attached to it would differentially affect the reactivity at C toward nucleophilic a minatio n, as they did in our control experiments. However, t he regiochemical analysis showed that regardless of the electronic nature of the substituent attached to the aryl ring , Tc PAM aminated predominantly at C to open the 3 - arylglycidates . 63 2.3.8 Establishin g Relative Stereoc onfiguration of Arylserines by 1 H NMR Analysis. A 1 H NMR method was developed to establish the relative syn / anti - stereoc onfigurations of the four synthesized arylserine diastereomers. The coupling constants for the vicinal protons (H and H ) were similar ( 3 J H - H ( anti ) and 3 J H - H ( syn ) for the underivatized synthetic anti - and syn - phenylserine diastereomers ( 15 a ) . Thus, differences in H and H coupli ng constants could not be used to distinguish the an ti - and syn - diastereomers, as done in another study. 133 Thus, a small amount of authentic (2 S )+( 2 R ) ) - syn - phenylserine racemate was added to the synthesized mixture of anti - and syn - phenylserine diastereomers ( Figure. 2 . 18 A ) . The 1 H NMR of the "spiked" sample showed a relative increase of the ( Figure. 2 . 18 B ). A B (2 S ) - anti (2 S ) - syn Figure. 2 . 18 . An expanded 500 MHz 1 H NMR of A) synthesized phenylserine ( 15 a ), and B) of 15 a containing authentic ( 2S ) - syn - phenylserine recorded in D 2 O at pH 1.5. Chemical sh ift values are listed vertically above each peak, and the relative area under each peak is listed horizontally next to each peak. The signals to the syn - phenylserine enantiom ers. The "inner doublets" at 4.33 and anti = 1.04 ppm, pH 1.5) were thus assigned to the anti - phenylserine enantiomers. We used the trends in 64 anti / syn - stereochemistries and anti : syn rat ios of the remaining s ynthetic and biocatalyzed arylserine analogues without derivatization . Each chemically synthesized and biocatalyzed arylserine analogue was dissolved separately in D 2 O at pH 1.5 and analyzed by 1 H NMR. syn (~1.15 ppm, pH 1.5) of the "outer doublets" anti ) (~1.05 ppm, pH 1.5) for the "inner doublets" ( Tab le 2 . 5 and Figure. 2 . 48 Figure. 2 . 59 ) . Tab le 2 . 5 . 500 MHz 1 H NMR data for the synthetic and biocatalyzed arylserine analogues ( 15 a 15 l ) recorded in D 2 O at pH 1.5. R 3 J H - H anti (Hz) 3 J H - H syn (Hz) anti b (ppm) syn b (ppm) 3 J H H biocatalyzed (Hz) biocatalyzed (ppm) anti : syn c (NMR) a anti : syn c (GC - MS) 15 a H 4.0 4.0 1.04 1.17 4.0 1.05 93:7 91:9 15 b - OCH 3 4.0 4.0 1.02 1.15 4.0 1.02 100:0 95:5 15 c - CH 3 4.0 4.0 1.0 0 1.14 4.0 1.01 100:0 96:4 15 d - F 4.0 4.0 1.02 1.16 4.0 1.04 98:2 95:5 15 e - Cl 4.0 4.0 1.03 1.18 4.0 1.01 91:9 94:6 15 f - Br 4.0 4.0 1.02 1.17 4.0 1.02 93:7 92:8 15 g 3 - NO 2 4.0 4.0 1.08 1.21 3.5 1.08 93:7 92:8 15 h - NO 2 3.5 4.0 1.06 1.22 4.0 1.09 97:3 96:4 15 i - CH 3 4.0 4.0 1.00 1.13 4.0 0.99 100:0 95:5 15 j - F 4.0 4.0 1.03 1.16 4.0 1.03 93:7 97:3 15 k - Cl 4.0 4.0 1.03 1.16 4.0 1.00 100:0 91:9 15 l - Br 4.0 4.0 1.00 1.13 4.0 0.98 100:0 95:5 a "0" indicates that the syn - isomer was below the limits of detection of the NMR (at ~100 nmol). b anti and syn values for the synthetic phenylserine analogues . c anti : syn ratio for the biocatalyzed ar ylserine analogues. n = 3. Standard error < 1%. 65 The NMR analysis substantiated that Tc PAM aminated the epoxide substrates predominantly at C to produce anti - arylserine analogues as the major product, regardless of the substituent on syn - phenylserines was also made ( See Appendix Figure. 2 . 48 Figure. 2 . 59 ) . Table 2 . 6 . 500 MHz 1 H NMR data for the chemically synthesized arylserine analogues ( 15 a 15 l ) recorded in D 2 O at a pH range of 0 - 4.0. Entry R pH 3 J H - H ( anti ) (Hz) 3 J H - H ( syn ) (Hz) anti b (ppm) syn b (ppm) 15 a H 4.0 4.0 4.0 1.26 1.37 3.0 4.0 4.0 1.23 1.34 1.5 4.0 4.0 1.05 1.18 1.0 4.0 4.0 0.99 1.13 15 b - OCH 3 2.0 4.0 4.0 1.03 1.16 1.5 4.0 4.0 1.01 1.14 15 c - CH 3 2.0 4.0 4.0 1.02 1.16 1.5 4.0 4.0 1.00 1.14 15 d - F 3.0 4.0 4.0 1.22 1.34 2.5 4.0 4.0 1.22 1.34 1.5 4.0 4.0 1.02 1.16 15 e - Cl 3.5 4. 0 4. 5 1.24 1.36 3.0 4. 0 4. 5 1.23 1.36 2.0 4. 0 4. 5 1.08 1.22 1.5 4.0 4. 0 1.03 1.18 15 f - Br 3.5 4.0 4.5 1.23 1.36 3.0 4.0 4 .5 1.23 1.35 2.0 4.0 4.0 1.12 1.24 1.5 4.0 4.0 1.02 1.17 1.0 4.0 4.0 0.97 1.12 15 g - NO 2 3.5 3.5 4.5 1.31 1.40 3.0 3.5 4.5 1.30 1.40 1.5 4.0 4.0 1.08 1.21 15 h - NO 2 3.0 3.5 4.0 1.27 1.40 2.5 3.5 4.0 1.26 1.39 1.5 3.5 4.0 1.06 1.22 15 i - CH 3 2.5 4.0 4.0 1.05 1.17 1.5 4.0 4.0 1.00 1.13 15 j - F 2.0 4.0 4.0 1.10 1.22 1.5 4.0 4.0 1.03 1.16 15 k - Cl 3.5 4.0 4.5 1.25 1.36 3.0 4.0 4.5 1.25 1.36 1.5 4.0 4.0 1.03 1.16 15 l - Br 1.5 4.0 4.0 1.00 1.13 1.0 4.0 4.0 0.94 1.09 66 2.3.9 Rela tive Stereochemistry of the Tc PAM Reaction by Chiral Auxiliary Derivatization. An orthogonal GC/MS separation method was used to verify the relative stereoc onfiguration of the biocatalyzed arylserines assigned earlier in this study by a 1 H NMR method. The chiral N - ( S ) - methylbutyryl - O - trimethylsilyl methyl ester derivatization of the arylserines described herein were extended to a diastereomeric mixture of synthesized (2 S )+(2 R ) - syn - 15 a and (2 S )+(2 R ) - anti - 15 a ( Figure. 2 . 19 A ), commercial - grade (2 S )+(2 R ) - syn - 15 a ( Figure. 2 . 19 B ), and 15 a bioc atalyzed by Tc PAM from the racemic 3 - phenylglycidate ( 14 a ) ( Figure. 2 . 19 C ) ( Scheme 2 . 1 A ). GC/MS profiles for the N , O - derivatives of the c ommercial (2 S )+(2 R ) - syn - phenylserine had m ajor peaks at retention times 8.10 min and 8.13 min ( Figure. 2 . 19 B ) and thus established the stereochemistry of the later - eluting isomeric pair in the mixture. The retention times of these most abundant peaks in the commercial sample matched those of the low er abundant peaks for the derivatized isomers in the synthetic 15 a sample ( Figure. 2 . 19 A ). The derivatized enantiomers in the phenylserine diastereomeric mixtures eluting at 7.95 min and 8.01 min were therefore assigned as (2 S )+(2 R ) - anti - phenylserine ( Figure. 2 . 19 ) . In summary, the derivatives of the anti - phenylserine enantiomers eluted first, and the syn - phenylserine enantiomers eluted later from the GC/MS. In the biocatalyzed sample, Tc PAM diastereoselectively turned over the racemate of p henylglycidate to a mixture of (2 R )+(2 S ) - anti - phenylserines with o ne enantiomer predominating ( Figure. 2 . 19 C ) . The arylserines made from glycidate s 14 b 14 l by Tc PAM catalysis were derivatized to 17 b 17 l and analyzed by GC/MS to assign the relative stereochemistries ( Figure. 2 . 60 and Figure. 2 . 61 ). The bio catalyzed products contained (2 R )+(2 S ) - anti - arylserines as the major stereoisomers, with one enantiomer predominating , while the (2 R )+(2 S ) - syn - Tab le 2 . 5 ). 67 A B C D Figure. 2 . 19 . Gas - chromatography/mass spectrometry e xtracted - ion ( m / z 179) chromatograms of A) synthesized phenylserine ( 15 a ), as shown in Scheme 2 . 1 B, was derivatized with a chiral auxiliary as per Scheme 2 . 1 A . Peaks at 7.95 min and 8.01 min correspond to (2 S )+(2 R ) - anti - phenylserine isomers, and th ose at 8.10 and 8.13 min correspond to (2 S )+(2 R ) - syn - phenylserine isomers; B) derivatized authentic (2 S )+(2 R ) - syn - phenylserine racemate (Sigma - Aldrich, contains 13% (2 S )+(2 R ) - anti - phenylserine as an impurity; C) chiral - auxiliary of biocatalyzed phenylserin e made from the racemic 3 - phenylglycidate. Biocataly zed (2 S )+(2 R ) - syn - phenylserine stereoisomers were also produced at ~9% (peaks at 8.10 and 8.13 min); and D) Extracted ion ( m / z 179) chromatogram of derivatized phenylserine after treating with (2 S ) - TA for 15 min. 2.3.10 Absolute Stereochemistry of the Tc PAM Reaction by Aldolase Resolution . To assess which enantiomer was biocatalyzed more abundantly among the (2 R )+(2 S ) - anti - arylserines , we used a low specificity (2 S ) - threonine aldolase (TA) from E. coli . 134 This enzyme resolution step cleaved the (2 S ) - anti diastereomers in to a benzaldehyde moiety and glycine, resolving the synthesized (2 R ) - anti - hydroxy amino ac id diastereomers. Each stereoisomeric mixture of synthesized arylserine (e ntries 15 a 15 l ) was treated with (2 S ) - TA. Aliquots were withdrawn at 15 min un der steady - state enzymatic turnover to measure the relative abundances of 68 the arylserine diastereomers. The remaining arylserine stereoisomers were treat ed sequentially with ( 2 S ) - 2 - methylbutyric anhydride, diazomethane, and chlorotrimethylsilane. Figure. 2 . 20 . Distribution of enantiomers (2 R ) - anti (gray bars) and (2 S ) - anti (black bar s) in biocatalyzed products for the substituted phenylserine analogues . GC/EI - MS analysis of each derivatized arylserine showed that the peak correspo nding to the (2 S ) - anti isomer diminished relative to that for the (2 R ) - enantiomer (see Figure. 2 . 19 D and Figure. 2 . 62 Figure. 2 . 63 ). We could then assign the 2 R - anti and 2 S - anti - isomers of each arylserine and calculate the 2 R : 2 S - anti ratio s of the biocatalyzed arylserines ( Figure. 2 . 20 ). The 2 R :2 S - anti ratios for the biocatalyzed arylserines ranged from 66:34 (4' - F - phenylserine, 15 j ) to 88:12 (3' - NO 2 - p henylserine, 15 g ) with the (2 R ) - anti isomer predominating ( Figure. 2 . 20 ). The 2 R :2 S ratios for e ach syn - biocatalyzed product could not be calculated accurately because of their ~10 - fold lower abundance than the anti - isomers and poorer resolution on the GC/MS (see Figure. 2 . 19 B as an example) . The anti : syn rati os calculated from the abundance of derivatized biocatalyzed ar ylserines determined by GC/MS analysis agreed with those calculated by the 1 H NMR method for the underivatized biocatalytically made arylserines ( Tab le 2 . 5 ). There was no clear trend that explained how a combination of substitue nt electronic effects, position, and sterics influenced the R : S ratios of the arylserines made. 69 2.3.11 Epoxide Substrate Docking Model of Tc PAM . We used solved structures of Tc PAM in com plex with cinnamic acid (PDB code: 3NZ4 and 4CQ5) to help interpret whether the proposed dockings of 3 - arylglycidate analogues in the Tc PAM active site by the AutoDock program were reasonable. The 3 - phenylglycidate enantiomers, (2 S ,3 R ) - 14 a and ( 2 R ,3 S ) - 14 a , docked in the crystal structure of t he TcPAM active site ( Figure. 2 . 21 A C ) were consistent with the conformation of the naturally occurring cinnamate intermediate in the crystal structure ( Figure. 2 . 21 D ); the carboxylate group pointed toward Arg325, and the aryl binding region compris ed key residues Ile431, Leu108, Leu104, and Leu227 ( Figure. 2 . 21 B and C ). Docking poses suggest that (2 S ,3 R ) - 14 a yields the (2 R ) - anti - phenylserine ( Figure. 2 . 22 ). The anti - stereochemistry of the arylserine products follows a mechanism proceeding through backside nucleophilic (S N 2) attack by the NH 2 group at C of the epoxide ring. The docked conformation of (2 S ,3 R ) - 14 a places the epoxide oxygen 2.7 Å from the catalytic Tyr80 of Tc PAM. Tyr80 normally functions as a general aci - - phenylalanine isomerase reaction catalyzed by Tc PAM. Here, we believe Tyr80 also serves as a general acid to promote protonation - initiated epox ide opening during amination by the NH 2 - MIO . Conversely, the docked ( 2 R ,3 S ) - 14 a enantiomer is poised for making the (2 S ) - anti - arylserine . The oxirane oxygen of ( 2 R ,3 S ) - 14 a sits 2.9 Å from Tyr322 that has been identified as a key residue involved in forming the MIO moiety and keeping the NH 2 - MIO adduct deprotonated so that it functions as a nucleophile during the no rmal Tc PAM - catalyzed isomerase reaction ( Figure. 2 . 21 A ). 135 Tc PAM likely uses Tyr322 effectively as a surrogate general acid for protonation - initiated epoxide opening during amination by the NH 2 - MIO of ( 2 R ,3 S ) - 14 a . 70 A B C D Figure. 2 . 21 . A) Lowest energy binding poses are shown of (2 S ,3 R ) - 3 - phenylglycidate ((2 S ,3 R ) - 14 a ) (orange sticks) and (2 R ,3 S ) - 3 - phenylglycidate ((2 R ,3 S ) - 14 a ) (light - gray sticks) at center of the image. The conformations are consistent with the active site of Tc PAM consisting of residues (shown as dark gray and yellow sticks), the catalytic Tyr80, a putative catalytic Tyr322 (light blue sticks), binding contact Arg325 (golden - rod sticks), and the methylidene imidazolone (MIO) moiety (green s ticks). B) (2 S ,3 R ) - 14 a (orange sticks) and C) (2 R ,3 S ) - 14 a (light - gray sticks) are posed separately in the active site to highlight the nominal distances (<3.5 Å) that substituents on the aryl ring are from active site residues. Printed on the aryl ring, ' m ' designates meta - positions (equivalent to the 3' - designation used in the text), a nd ' p ' designates the para - position (equivalent to the 4' - designation used in the text) per ligand. Heteroatoms are colored red for oxygen and blue for nitrogen. The images were produced with UCSF Chimera, 136 , and the docking conformations w ere g enerated with AutoDock Vina 127 from PDB code 3NZ4. Numbers are distances in Å. D) Tc PAM in complex with cinnam ic ac id, based on PDB codes 3NZ4 and 4CQ5. 71 In this study, the syn - arylserine isomers were made at low levels compared to the anti - isomers for each arylglycidate analogues , and their resolution as chiral derivatives on GC/MS was poor; thus , accurate measur ement s of the ratio of the syn - enantiomers could not be made. The (2 R ) - and (2 S ) - anti - arylserine isomers are made through S N antibonding orbital of C O bond of the (2 S ,3 R ) - and (2 R ,3 S ) - glycidate racemates , respectivel y ( Figure. 2 . 22 , route a ) . However, the 2 S - and 2 R - syn - arylserine diastereomers cannot be accessed through a similar S N 2 - type mechanism unless Tc PAM can rotate the substrate into a cisoid conformation that aligns C in a proper orientation for nucleophilic attack. b a Figure. 2 . 22 . a ) Rendering of the mechanism showing attack of the NH 2 - antibonding orbital (gray lobe) at C for the C (2 S ,3 R ) - 14 a to produce (2 R ) - anti - phenylserine. b ) A double inversion - of - configuration mechanism is envisioned to access the minor syn - stereoisomer that proceeds through a putative lactone intermediate. Earlier mechanistic studies suggest that during its natural reaction, Tc PAM rotates the cinnamate intermediate about a central axis through a bicycle - pedal motion to invert the faces of the - and - carbons 180 ° from their original positions. 124,135 This rotation enables the NH 2 group of the MIO adduct to add to the opposite face of C from which it was remo ved from the phenylalanine substrate (see Figure. 2 . 3 ). The oxirane ring prevents rotations about the axis between C and C . Thus, a double inversion - of - configuration mechanism is envisioned to access the minor syn - stereoisomers (<10% relative a bundance) tha t proceeds through a putative oxirano ne intermediate ( Figure. 2 . 22 , route b ). 137 Through the proposed oxiran one intermediate pathway, the (2 S ,3 R ) - 14 a that produces the more abundant (2 R ) - anti - phenylserine at 8.01 min 72 ( Figure. 2 . 19 C ) likely also makes the 2 S - syn - phenyl serine isomer ( Figure. 2 . 22 ) (see peak at 8.10 min) . T he (2 R ) - syn - phenylserine at 8.13 min ( Figure. 2 . 19 C ) is likely derived from the (2 R ,3 S ) - 14 a . The other ring - substituted 3 - arylglycidates behaved similarly ( Figure. 2 . 60 Figure. 2 . 61 ). A B C Figure. 2 . 23 . Various viewing angles centering on the surfaces of A) Leu104, B) Leu108, and C) Lys427 deep in the aryl binding p ocket of Tc PAM (PDB code 3NZ4) with poses of the docked (2 S ,3 R ) - 14 a (orange sticks) and (2 R ,3 S ) - 14 a (light - gray sticks). 73 2.3.12 Kinetic s of Arylserine Bio catalysis . The Michaelis parameters were measured by quantifying the underivatized biocatalyzed products by LC/MS - MRM analysis. The apparent K M and k cat of Tc PAM were calculated under steady - st ate conditions for the analogues of 3 - arylglycidate with the amino donor ( 12 ) at 1 mM ( Table 2 . 7 and Figure. 2 . 68 showing the conversion of four epoxi de example s h ). The catalytic efficiency ( k cat / K M ) of Tc PAM for 3' - CH 3 - phenylglycidate ( 14 c ) was the best among all subst rates tested; largely influe nced by its relatively lower K M compared to those for the 3' - halo ( 14 d , 14 e , an d 14 f ), 3' - NO 2 ( 14 g ), 4' - NO 2 ( 14 h ), and 4' - CH 3 ( 14 i ) substrates. Also , the catalytic efficiencies of Tc PAM for 14 c and two other 3' - substituted phenylglycidate s (3' - F ( 14 d ) and 3' - Cl ( 14 e )) were superior to those of their 4' - substituted counterparts; k cat / K M values fo r the latter two were principally influenced by their higher k cat compared to those of their 4' - substituted isomers ( 14 j and 14 k ). - - amino acid isomerization reaction at 3.0 min 1 , 124 which is 10 - fold faster than the average turnover rate ( 0.3 min 1) of TcPAM for the 3 - substituted arylglycidate substrates used in this study. However, the latter rate is similar to the average rate at which other MIO - dependent aminomutases catalyze the ir natural - to - amino acid isomerization reactions involved in specialized metabolism. 138 Thus, the turnove r of the 3 - substituted arylglycidates by TcPAM is within the same order of magnitude as mutases that make - amino acids in their natural hosts to confer an evolutionary advantage . Because the calculated docking conformations of the arylglycidates agreed w ith those of other MIO - enzymes in complex with their natural phenylpropanoid substrates, 17,29,53,124 we , therefore, used the models to help interpret how the position of the substituents could potentially affect ca talysis due to steric interac tions. Substrate docking models show that for each ligand enantiomer , 74 one of the two 3' - (" meta " - ) carbons of the aryl ring of the substrate can position its substituent in steric relief when pointed toward Lys427 ( Figure. 2 . 21 B ) and Ile431 ( Figure. 2 . 21 C ). This , in part , suggests that substrates 14 c , 14 d , and 14 e with relatively smaller 3' - substituents (CH 3 , F, and Cl, respectively) are more able to adopt a catalytically competent conformation compared to their 4' - isomers. The latter place substituents on the 4' - (" para " - ) carbon of each ligand in greater steric conflict with Leu104 ( Figure. 2 . 21 B and Figure. 2 . 23 A ), Leu108, and Leu227 ( Figure. 2 . 21 C , Figure. 2 . 23 A and B ), likely preventing epoxides 14 i , 14 j , and 14 k from being turned over as efficiently ( Table 2 . 7 ) ( Figure. 2 . 69 and Figure. 2 . 70 ). Table 2 . 7 . Kinetics of Tc PAM for Turnover of Arylglycidates ( 14 a 14 l ) to Arylse rines ( 15 a 15 l ) and Arylisoserines ( 16 a 16 l ). Entry R (min - 1 ) (min - 1 ) K M (µM) k cat / K M (M - 1 s - 1 ) Entry R (min - 1 ) (min - 1 ) K M (µM) k cat / K M (M - 1 s - 1 ) 14 a H 0.39 (0.10) a 0.003 (<0.001) 340 19 14 g 3' - NO 2 0.43 (0.07) 0.01 (0.001) 760 10 14 b - OCH 3 0.46 (0.14) 0.27 (0.15) 2000 6.5 14 h 4' - NO 2 0.75 (0.11) 0.004 (<0.001) 610 21 14 c - CH 3 0.31 (0.01) 0.03 (0.001) 50 113 14 i - CH 3 0.17 (0.03) 0.11 (0.01) 450 10 14 d 3' - F 0.62 (0.03) 0.009 (<0.001) 170 61 14 j 4' - F 0.04 (<0.01) 0.005 (<0.001) 40 17 14 e 3' - Cl 1.3 (0.1) 0.10 (0.01) 600 41 14 k 4' - Cl 0.01 (<0.01) 0.002 (<0.001) 50 4.5 14 f 3' - Br 0.02 (<0.01) 0.002 (<0.001) 140 2.5 14 l 4' - Br ~0.04 (<0.01) 0.01 (<0.0 01) 50 15 a Standard deviation in parenthesis ( n = 3). The "<" symbol indicates that the actual value, estimated to one significant figure, is shown. is the apparent k cat of arylserine production, is the apparent k cat of arylisoserine production, and k cat is the turnover rate for the production of both arylserine and arylisoserine by Tc PAM. The K M of Tc PAM for each substrate is calcul ated from the production of arylserine and arylisoserine combined. Furthermore , the 3 ' - Cl - Phenylglycidate ( 14 e ) was turned over ( k cat ) by Tc PAM faster than the other e poxides in this study and interestingly much more superior (>130 - fo ld ) than the 4' - Cl isomer ( 14 k ) ( Table 2 . 7 ). The other 3' - s ubstituted analogues turned over ~16 - fold and ~2 - fold faster than their 4' - substituted counterparts ( 14 j and 14 i ) , which were the 3' - F - ( 14 d ) and 3' - CH 3 - ( 14 c ) 75 phenylglycidate s , respec tively. The larger st eric 3 ' - Br - ( 14 f ) and 4' - Br - ( 14 l ) substrates were turned over with poorer catalytic efficiency, due mainly to their equally poor k cat values . The K M values of Tc PAM for both Br - substituted substrates were similar yet lower (estimating tighter binding) than for other substrates such a s 14 d , 14 e , 14 g , and 14 h that were turned over faster ( Table 2 . 7 ). This suggests that a yet unknown phenomenon res ulting in part from tighter binding and steric misalignment likely disrupted the turnover of the bulkier Br - substituted arylglycidates by Tc PAM ( Figure. 2 . 69 A and B , and Figure. 2 . 70 A and B ) . It was interesting to find that the 3 - ( 3 - OCH 3 - phenyl)glycidate ( 14 b ) with the largest estimated steric volume 129,139 (26.4 Å3) was turned over similar to most 3 - s ubstituted substrates with smaller substituents, such as H (4.5 Å3), F (7.7 Å3), or Cl (18.8 Å3) (see 14 a , 14 c , and 14 d as examples, Table 2 . 7 ). The significantly lower catalytic efficiency, yet similar turn over by TcPAM of 14 b as compared to those of 14 a , 14 c , and 14 d with smaller substituents, suggested that the larger - size OCH 3 group, through a curious pathwa y, affected the K M but not the turnover, like the Br group. The catalytic efficiency ( k cat / K M ) of Tc PAM for the 4' - NO 2 - substituted arylglycidate ( 14 h ) was ~2 - fold hig her than for the 3' - NO 2 - isomer ( 14 g ), which parallels the difference in their turnover ( k cat ). This regiospecificity trend was similar to that described earlier for s ubstrates 3' - Br - ( 14 f ) and 4' - Br - ( 14 l ), where the 4' - isomer was turn ed over s lightly faster than the 3' - isomer. 2.3.13 Kinetic s of Aryl iso serine Biocatalysis . To make the aryl isoserines, the NH 2 - MIO adduct needs to align the phenylpropanoid of the p henylglycidate for occasional amino group attack at C instead of at C ( See Figure. 2 . 71 for a representative AutoDock modeled conformation of 14 b ). We found by LC/ESI - MS/MS an alysis that Tc PAM converted each substituted Phenylglycidate to an aryl iso serine ( Figure. 2 . 73 Figure. 2 . 75 ). Among the substituted arylisoserines, the 3' - OCH 3 - phenyl isoserin e isomer 16 b was made 76 most abundantly at a 37:63 ratio with its corresponding 3' - OCH 3 - phenylserine 15 b , with the latter predominating ( Table 2 . 7 ). The next most abundant arylisoserines made by Tc PAM were the 3' - Cl ( 16 e ) and 4' - CH 3 ( 16 i ) at ~10:90 and 40:60 ratios with their cognate arylserines 15 e and 15 i , respectively. The remaining Phenylglycidate 03 min - 1 ) of their aryl isoserines ( Table 2 . 7 ). The sterically demanding 3' - OCH 3 substituent likely "nudged" the substrate enough to place C close to and in alignment with the NH 2 - MIO moiety to make the 3' - OCH 3 - phenylisoserine ( 16 b ) more abundantly. However, the other epoxide substrates bearing 3' - or 4' - substituents with steric volumes [NO 2 (18.2 Å 3 ), Cl (14.3 Å 3 ), CH 3 (21.3 Å 3 ) and Br (24.5 Å 3 )] equivalent to that of OCH 3 (26.4 Å 3 ) were turned over to their corresponding aryl isoseri nes less efficiently. Thus, a combination of the different steric volumes, electronics, atom geometries, and the 3' - and 4' - positions of the substituents must place the carbon skeleton of epoxides into various conformations that unpredictably promote or pr eclude NH 2 - MIO attack at C . Based on the putative docked conformation of 14 b ( Figure. 2 . 71 ) , the anti - stereochemistry between the hydroxyl and amino groups is expe cted for aryl isoserines when the epoxide is opened by S N 2 amination at C . The proposed anti - stereoisomerism of the aryl isoserines biocatalyzed in this study is unlike the syn - stereoch emistry of the phenylisoserine moiety found in the antineoplastic drug paclitaxel (see Figure. 1 . 4 ). 140 - phenylalanyl taxane, which is hydroxylated to a penultimate pheny lisoserinyl taxane intermediate, 141 but further evidence is needed to confirm this. Another report claims isolation of methyl phenylisoserinate by methanol extraction of Taxus plants as evidence of a putative, phenyli soserine preassembly pathway. 142 It is unclear, however, whether the phenyli soserinate ester was obtained b y methanolysis of phenylisoserinyl taxanes, such as an advanced paclitaxel precursor, present in the 77 plant. Nonetheless, if syn - phenylisoserine indeed occurs as a metabolite in plants, its biosynthetic pathway in Taxus plants remains unknown. While the tra ns - 3 - arylglycidates were biocatalytically converted likely to anti - aryl isoserines in this study, the results highlight 3 - arylglycidate s as potential naturally occurring precursors for direct amination to aryl isoserines. We imagine that naturally occurring cinnamate, derived from phenylalanine by ammonia lyase activity in plants, 143 can be converted to 3 - phenylglycidate by a - ketoglutarate - dependent hydroxylase. This family of no nheme Fe(II) hydroxylases convert alkenes to epoxides, 144 and specifically cinnamate to 3 - phenylglycidate in vitro. 145 As noted earlier, Tc PAM catalyzes the - - phenylalanine in its n atural reaction by rotating the arylacrylate intermediate inside the active site before being re - aminated at the C ( Figure. 2 . 3 ). 124 This rotation pathway allowed by Tc PAM m ay play a role in torqueing the ring - opened trans - epoxide intermediates into a conformation needed for making syn - isoserine. In future studies, we look to dissect the stereoselectivity of the Tc PAM reaction for arylisoserine production. 2.4 Conclusion The sur rogate activity of TcPAM catalyzed the asymmetric and regioselective transfer of an NH 2 group from a sacrificial amine group donor (2 S ) - styryl - - alanine to racemates of 3 - arylglycidate analogues in one step. Tc PAM made underivatized anti - stereoisomers of arylserines predominantly while producing significantly less of the syn - stereoisomer, and these observations provided valuable insights into an extended - - phenylalanine isomerization reaction. The electronic effects or steric volumes of the substituents on the ring of the arylglycidate substrates did not predictably change the regioselectivity of the nucleop hilic 78 attack by NH 2 . Docked poses of the arylglycidate racemate in the active site of Tc PAM suggested that the conserved Tyr80 , a general acid present in all other MIO enzymes 146 and proximate to the (2 S ,3 R ) - glycid ate, purportedly helped catalyze the more abundant (2 R ) - anti - arylserines. Docking analysis also identified Tyr322 as a putative general acid that likely facilitated protonation - initiated amination of the (2 R ,3 S ) - glycidate antipode to make the lesser abunda nt (2 S ) - anti - arylserines. In addition, (2 S ) - syn - - hydroxy amino acid stere oisomers are commonly found in specialized glycopeptide and depsipeptides natural products. Application of an MIO - aminomutase described here to biocatalyze enantiodivergent aryl serin es with anti - stereoisomerism between the two chiral centers has industrial and pharmaceutical relevance, allowing the possibility to perform stereochemical structure/activity studies. For example, replacement of a naturally occurring syn - arylserine stereoi somer for rarer anti - arylserine stereoisomers in a bioactive drug candidat e could increase drug bioavailability by slowing first - pass metabolism pathways or inhibiting bacterial resistance mechanisms of bioactive compounds built on this scaffold. 147,148 Future studies will assess the stereoselectivity and stereospecificity of Tc PAM with enantiopure glycidate su bstrates for making hydroxy amino acids. While the amino nucleophile of the loaded NH 2 - MIO adduct of Tc PAM primarily added the NH 2 to C to open the epoxide - ring forming arylserines, the epoxides were also converted to their arylisoserines via NH 2 attack a t C , likely yielding the anti - hydroxy amino acid isomers. This work provides an alternative biocatalytic route to access phenylserine and phenylisoserine analogues starting from 3 - phenylglycidates. This work extends the transaminase activity of MIO - aminom utases to a new class of acceptor molecules to produce hydroxy amino acids. These biocatalyzed and underivatized bifunctional aryl isose rines can be used as direct entry points into the synthesis of value added compounds. For example, isoserines are bioacti ve structural motifs 79 of drugs such as the anticancer pharmaceutical paclitaxel and its analogues, 149 - 151 and a series of inhibitors. 152 Foreseeably, the capacity of Tc PAM to produce aryl isoserines from glycidates , in part, provides a starting point for us to design novel fu nction into the enzyme. 80 APPENDIX 81 A PPENDIX MW (kDa) lane 1 lane 2 lane 3 lane 4 lane 5 170 130 100 70 55 40 35 25 15 Figure. 2 . 24 . SDS - PAGE gel of wild - type Tc PAM (82% pure) and (2 S ) - TA (99% pure) after Coomassie blue staining. Purity was estimated by a Kodak Gel Logic 10 0 Imaging System; lane 1: Tc PAM (10 µL of a 13.7 mg/mL solution); lane 2: Tc PAM (5 µL of a 13.7 mg/mL solution); lane 3: PageRuler © Prestained Ladder: MW (kDa) 170, 130, 100, 70, 55, 40, 35, 25, 15; lane 4: (2 S ) - TA (10 µL of a 8.8 mg/mL solution); and l ane 5: (2 S ) - TA (5 µL of a 8.8 mg/mL solution). 82 14 b dihydroxy product from 14 b A t 7 t 6 t 5 t 4 t 3 t 2 t 1 B (ppm) Figure. 2 . 25 . 1 H NMR of 3 - (3 - OCH 3 - phenyl)glycidate ( 14 b ) in deuterated Assay Buffer. A) NMR spectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, th e ratio of 14 b :dihydroxy product = 100:0, and B) NMR spectrum after storing the sam ple in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 b :dihydroxy product = 100:3. 83 14 c dihydroxy product from 14 c A t 7 t 6 t 5 t 4 t 3 t 2 t 1 B (ppm) Figure. 2 . 26 . 1 H NMR of 3 - (3 - CH 3 - phenyl)glycidate ( 14 c ) in deuterated Assay Buffer. A) NMR spectra were record ed at 15 min intervals s tarting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the ratio of 14 c :dihydroxy product = 100:7, and B) NMR spectrum after storing the sam ple in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 c :dihydroxy product = 100:12. 84 14 d dihydroxy product from 14 d A t 7 t 6 t 5 t 4 t 3 t 2 t 1 B (ppm) Figure. 2 . 27 . 1 H NMR of 3 - (3 - F - phenyl)glycidate ( 14 d ) in deuterated Assay Buffer. A) NMR spectra were recorded a t 15 min intervals start ing from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the ratio of 14 d :dihydroxy product = 100:0, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 d :dihydroxy product = 100:2. 85 14 e dihydroxy product from 14 e A t 7 t 6 t 5 t 4 t 3 t 2 t 1 B (ppm) Figure. 2 . 28 . 1 H NMR of 3 - (3 - Cl - phenyl)glycidate ( 14 e ) in deuterated Assay Buffer. A ) NMR spectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the ratio of 14 e :dihydroxy product = 100:0, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 e :dihydroxy product = 100:1. 86 14 f dihydroxy product from 14 f A t 7 t 6 t 5 t 4 t 3 t 2 t 1 B (ppm) Figure. 2 . 29 . 1 H NMR of 3 - (3 - Br - phenyl)glycidate ( 14 f ) in deuterated Assay Buffer. A) NMR spectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the ratio of 14 f :dihydroxy product = 100:0, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 f :dihydroxy product = 100:1. 87 14 g dihydroxy product from 14 g A t 7 t 6 t 5 t 4 t 3 t 2 t 1 B (ppm) Figure. 2 . 30 . 1 H NMR of 3 - (3 - NO 2 - phenyl)glycidate ( 14 g ) in deuterated Assay Buffer. A) NMR spectra were record ed at 15 min intervals s tarting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the ratio of 14 g :dihydroxy = 100:0, and B) 1 H NMR spectrum after storing the sample i n deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of the ratio of 14 g :dihydroxy product = 100:2. 88 14 h dihydroxy product from 14 h A t 7 t 6 t 5 t 4 t 3 t 2 t 1 B (ppm) Figure. 2 . 31 . 1 H NMR of 3 - (4 - NO 2 - phenyl)glycidate ( 14 h ) in deuterated Assay Buffer. A) NMR spectra were record ed at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and t 7 = 90 min, the ratio of 14 h :dihydroxy product = 100:9, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 h :dihydroxy product = 100:9. 89 14 i dihydroxy product from 14 i A t 7 t 6 t 5 t 4 t 3 t 2 t 1 B (ppm) Figure. 2 . 32 . 1 H NMR of 3 - (4 - CH 3 - phenyl)glycidate ( 14 i ) in deuterated Assay Buffer. A) NMR spectra were record ed at 15 min intervals s tarting from t 1 to t 7 . At t 1 = 0 min and at t 7 = 90 min, the ratio of 14 i :dihydroxy product = 100:1, and B) NMR spectrum after storing the sam ple in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 i :dihydroxy product = 100:5. 90 14 j dihydroxy product from 14 j A t 7 t 6 t 5 t 4 t 3 t 2 t 1 B (ppm) Figure. 2 . 33 . 1 H NMR of 3 - (4 - F - phenyl)glycidate ( 14 j ) in deuterated Assay Buffer. A) NMR spectra were recorded a t 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and t 7 = 90 min, the ratio of 14 j :dihydroxy product = 100:4, and B) NMR spectrum after storing the sample in de uter ated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 j :dihydroxy product = 100:7. 91 14 k dihydroxy product from 14 k A t 7 t 6 t 5 t 4 t 3 t 2 t 1 B (ppm) Figure. 2 . 34 . 1 H NMR of 3 - (4 - Cl - phenyl)glycidate ( 14 k ) in deuterated Assay Buffer. A) NMR spectra were recorded at 15 min intervals st arting from t 1 to t 7 . At t 1 = 0 min and t 7 = 90 min, the ratio of 14 k :dihydroxy product = 100:0, and B) NMR spectrum after storing the sample in deuterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 k :dihydroxy product = 100:3. 92 14 l dihydroxy product from 14 l A t 7 t 6 t 5 t 4 t 3 t 2 t 1 B (ppm) Figure. 2 . 35 . 1 H NMR of 3 - (4 - Br - phenyl)glycidate ( 14 l ) in deuterated Assay Buffer. A) NMR spectra were recorded at 15 min intervals starting from t 1 to t 7 . At t 1 = 0 min and t 7 = 90 min, the ratio of 14 l :dihydroxy product = 100:0, and B) NMR spectrum after storing the sample in d euterated Assay Buffer at ~25 ° C for 1 week, the ratio of 14 l :dihydroxy product = 100:1. 93 A B C Figure. 2 . 36 . A) Relative abundances of arylisoserine ( 16 a ) and arylserine ( 15 a ) products made from C - and C - amination, respectively, of 3 - phenylglycidate ( 14 a ) (1 mM) when incubated with 2M NH 4 OH and Tc PAM (100 µ g/mL) ( top trace ) and without Tc PAM ( bottom trace ). B) R elative abundances of arylisoserine ( 16 i ) and arylserine ( 15 i ) products made from C - and C - amination, respectively, of 3 - (4 ' - CH 3 - phenyl)glycidate ( 14 i ) (1 mM) when incubated with 2 M NH 4 OH and Tc PAM (100 µ g/mL) ( top trace ) and without Tc PAM ( bottom trace ). C) Relative abundances of arylisoserine ( 16 h ) and arylserine ( 15 h ) product s made from C - and C - amination, respectively, of 3 - ( 4 ' - NO 2 - phenyl)glycidate ( 14 h ) (1 mM) when incubated with 2M NH 4 OH and Tc PAM (100 µ g/mL) ( top trace ) and without Tc PAM ( bottom trace ). 94 GC/EI - MS Analysis of Phenylserine Analogues Derivatized as O - Trimethylsilyl N - [( 2S ) - 2 - Methylbutyryl] Methyl Esters A B Figure. 2 . 37 . GC/EI - MS s pectra of A) authentic ( 2 S /2 R ) - anti - - OCH 3 - phenylserine ( 15 b ) and B) 3 - OCH 3 - phenylserine biocatalyzed from 3 - - OCH 3 - phenyl)glycidate ( 14 b ) by Tc PA M catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 381) was not observed for either analyte. A B Figure. 2 . 38 . GC/EI - MS spectra of A) authentic ( 2 S /2 R ) - anti - - CH 3 - phenylserine ( 15 c ) and B) 3 - CH 3 - phenylserine biocatalyzed from 3 - - CH 3 - phenyl)glycidate ( 14 c ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethy lsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 365) was not observed for either analyte. 95 A B Figure. 2 . 39 . GC/EI - MS spectra of A) authentic ( 2 S /2 R ) - anti - - F - phenylserine ( 15 d ) - F - phenylserine biocatalyzed from 3 - - F - phenyl)glycidate ( 14 d ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 369) was not observed for either analyte. A B Figure. 2 . 40 . GC/EI - MS spectra of A) authentic (2 S /2 R ) - anti - - Cl - phenylserine ( 15 e ) - Cl - phenylserine biocatalyzed from 3 - - Cl - phenyl)glycidate ( 14 e ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethyl silyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 385) was not observed for either analyte. 96 A B Figure. 2 . 41 . GC/EI - MS spectra of A) authentic (2 S /2 R ) - anti - - Br - phenylserine ( 15 f ) - Br - phenylserine biocatalyzed from 3 - - Br - phenyl)glycidate ( 14 f ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 429) was not observed for either analyte. A B Figure. 2 . 42 . GC/EI - MS spectra of A) authentic (2 S/ 2 R ) - anti - - NO 2 - phenylserine ( 15 g ) and B) - NO 2 - phenylserine biocatalyzed from 3 - - NO 2 - phenyl)glycidate ( 14 g ) by Tc PAM c atalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 396) was not observed for either analyte. 97 A B Figure. 2 . 43 . GC/EI - MS spectra of A) authentic (2 S/ 2 R ) - anti - - NO 2 - phenylserine ( 15 h ) and B) - NO 2 - phenylserine biocatalyzed from 3 - - NO 2 - phenyl)glycidate ( 14 h ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethy lsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 396) was not observed for either analyte. A B Figure. 2 . 44 . GC/EI - MS spectra of A) authentic ( 2 S /2 R ) - anti - 4 - CH 3 - phenylserine ( 15 i ) and B) 4 - CH 3 - phenylserine biocatalyzed from 3 - (4 - CH 3 - phenyl)glycidate ( 14 i ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethy lsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 365) was not observed for either analyte. 98 A B Figure. 2 . 45 . GC/EI - MS spectra of A) authentic ( 2 S /2 R ) - anti - - F - phenylserine ( 15 j ) - F - phenylserine biocatalyzed from 3 - - F - phenyl)glycidate ( 14 j ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 369) was not observed for either analyte. A B Figure. 2 . 46 . GC/EI - MS spectra of A) authentic (2 S /2 R ) - anti - - Cl - phenylserine ( 15 k ) - Cl - phenylserine biocatalyzed from 3 - - Cl - phenyl)glycidate ( 14 k ) by Tc PAM catalysis. Each hydr oxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 385) was not observed for either analyte. 99 A B Figure. 2 . 47 . GC/EI - MS spectra of A) authentic (2 S /2 R ) - anti - - Br - phenylserine ( 15 l ) - Br - phenylserine biocatalyzed from 3 - - Br - phenyl)glycidate ( 14 l ) by Tc PAM catalysis. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 429) was not observed for either analyte. 100 syn - 15 a anti - 15 a A (ppm) B (ppm) Figure. 2 . 48 . Par tial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized phenylserine ( 15 a ) and B) biosynthetic phenylserine produced from phenylglycidate ( 14 a ) by Tc PAM. 101 syn - 15 b anti - 15 b A (ppm) B (ppm) Figure. 2 . 49 . Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized 3 - OCH 3 - phenylserine ( 15 b ) and B) biosynthetic 3 - OCH 3 - phenylserine produced from 3 - (3 - OCH 3 - phenyl)glycidate ( 14 b ) by Tc PA M. 102 syn - 15 c anti - 15 c A (ppm) B (ppm) Figure. 2 . 50 . Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemi cally synthesized - CH 3 - phenylserine ( 15 c - CH 3 - phenylserine produced from 3 - (3 - CH 3 - phenyl)glycidate ( 14 c ) by Tc PAM. 103 syn - 15 d anti - 15 d A (ppm) B (ppm) Figure. 2 . 51 . Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra o f A) chemically synthesized 3 - F - phenylserine ( 15 d - F - phenylserine produced from 3 - (3 - F - phenyl)glycidate ( 14 d ) with Tc PAM. 104 syn - 15 e anti - 15 e A (ppm) B (ppm) Figure. 2 . 52 . Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectr a of A) chemically synthesized 3 - Cl - phenylserine ( 15 e ) and B) biosynthetic 3 - Cl - phenylserine produced from 3 - (3 - Cl - phenyl)glycidate ( 14 e ) by Tc PAM. 105 syn - 15 f anti - 15 f A (ppm) B (ppm) Figure. 2 . 53 . Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized 3 - Br - phenylserine ( 15 f ) and B) bi osynthetic 3 - Br - phenylserine produced from 3 - (3 - Br - phenyl)glycidate ( 14 f ) by Tc PAM. 106 syn - 15 14 g anti - 15 14 g A (ppm) B (ppm) Figure. 2 . 54 . Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized 3 - NO 2 - phenylserine ( 15 g ) and B) biosynthetic 3 - NO 2 - phenylserine produced from 3 - (3 - NO 2 - phenyl)glycidate ( 14 g ) by Tc PAM. 107 syn - 15 h anti - 15 h A (ppm) B (ppm) Figure. 2 . 55 . Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized 4 - NO 2 - phenylserine ( 15 h ) and B) biosynthetic 4 - NO 2 - phenylserine produced from 3 - (4 - NO 2 - phenyl)glycidate ( 14 h ) by Tc PAM. 108 syn - 15 i anti - 15 i A (ppm) B (ppm) Figure. 2 . 56 . Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized 4 - CH 3 - phenylserine ( 15 i - CH 3 - phenylserine produced from 3 - (4 - CH 3 - phenyl)glycidate ( 14 i ) by Tc PAM. 109 syn - 15 j anti - 15 j A (ppm) B (ppm) Figure. 2 . 57 . Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) ch emically synthesized 4 - F - phenylserine ( 15 j - F - phenylserine produced from 3 - (4 - F - phenyl)glycidate ( 14 j ) by Tc PAM 110 syn - 15 k anti - 15 k A (ppm) B (ppm) Figure. 2 . 58 . Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) ch emically synthesized 4 - Cl - phenylserine ( 15 k ) and B) bi - Cl - phenylserine produced from 3 - (4 - Cl - phenyl)glycidate ( 14 k ) by Tc PAM. 111 syn - 15 l anti - 15 l A (ppm) B (ppm) Figure. 2 . 59 . Partial 1 H NMR (500 MHz, D 2 O at pH 1.5) spectra of A) chemically synthesized 4 - Br - phenylserine ( 15 l - Br - phenylserine produced from 3 - (4 - Br - phenyl)glycidate ( 14 l ) by Tc PAM. 112 A B C D E F Figure. 2 . 60 . GC/EI - MS e xtracted - ion chromatograms of biosynthetic arylserines derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl esters A ) phenylserine ( 15 a ) (extracted ion m / z 179), B) 3 - OCH 3 - phenylserine ( 15 b ) (extracted ion m / z 209), C) 3 - methylphenylserine ( 15 c ) (extracted ion m / z 193), D) 3 - F - phenylserine ( 15 d ) (extracted ion m / z 197), E ) 3 - Cl - phenylserine ( 15 e ) (extracte d ion m / z 213), and F) 3 - Br - phenylserine ( 15 f ) (extracted ion m / z 257). 113 G H I J K L Figure. 2 . 61 . GC/EI - MS e xtracted - ion chromatograms of biosynthetic arylserines derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl esters G) 3 - NO 2 - phenylserine ( 15 g ) (extracted ion m / z 224), H) 4 - NO 2 - phenylserine ( 15 h ) (extrac ted ion m / z 224), I) 4 - CH 3 - phenylserine ( 15 i ) (extracted ion m / z 193), J) 4 - F - phenylserine ( 15 j ) (extracted ion m / z 197), K ) 4 - Cl - phenylse rine ( 15 k ) (extracted ion m / z 213), and L) 4 - Br - phenylserine ( 15 l ) (extrac ted ion m / z 257) 114 A B C D E F Figure. 2 . 62 . GC/EI - MS e xtracted - ion c hromatograms of synthetic arylserines derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester before ( top trace ) and after 10 - min treatment with (2 S ) - TA ( bottom trace ): A) 3 - OCH 3 - phenylserine ( 15 b ) (extracted ion m / z 209), B) 3 - CH 3 - phenylserine ( 15 c ) (extracted ion m / z 193), C) 3 - F - phenylserine ( 15 d ) (extracted ion m / z 197), D) 3 - Cl - phenylserine ( 15 e ) (extracted ion m / z 213), E) 3 - Br - phenylserine ( 15 f ) (extracted ion m / z 257), and F) 3 - NO 2 - phenylserine ( 15 g ) (extracted ion m / z 224). 115 G H I J K Figure. 2 . 63 . GC/EI - MS e xtracted - ion chromatograms of synthetic arylserines derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester before ( top trace ) and after 10 - min treatment with (2 S ) - TA ( bottom trace ): G) 4 - NO 2 - phenylserine ( 15 h ) (extracted ion m / z 224); H) 4 - CH 3 - phenylserine ( 15 i ) (extrac ted ion m / z 193), I) 4 - F - phenylserine ( 15 j ) (extracted ion m / z 197), J ) 4 - Cl - phenylserine ( 15 k ) (extracted ion m / z 213) and K) 4 - Br - phen ylserine ( 15 l ) (extracted ion m / z 257). 116 Michaelis - Menten Plots of Tc PAM Biocatalytic Conversion of Ary lserine Analogues. 14 a 14 b Phenylserine Production (nM s - 1 ) 3 - Phenylgl ycidate (µM) 3' - OCH 3 - Phenylserine Production (nM s - 1 ) 3 - (3' - OCH 3 - phenyl)glycidate (µM) 14 c 14 d 3' - CH 3 - Phenylserine Production (nM s - 1 ) 3 - ( 3' - CH 3 - phenyl)glycidate (µM) 3' - F - Phenylserine Production (nM s - 1 ) 3 - (3' - F - phenyl)glycidate (µM) 14 e 14 f 3' - Cl - Phenylserine Production (nM s - 1 ) 3 - (3' - Cl - phenyl)glycidate (µM) 3' - Br - Phenyl serine Production (nM s - 1 ) 3 - (3' - Br - phenyl)glycidate (µM) Figure. 2 . 64 . Michaelis - Menten kinetics for the turnover of arylglycidate analogues ( 14 a 14 f ) to their corresponding arylserines ( 15 a 15 f ). 117 14 g 14 h 3' - NO 2 - Phenylserine Production (nM s - 1 ) 3 - (3' - NO 2 - phenyl)glycidate (µM) 4' - NO 2 - Phenylserine Production (nM s - 1 ) 3 - (4' - NO 2 - phenyl)glycidate (µM) 14 i 14 j 4 ' - CH 3 - Phenylserine Production (nM s - 1 ) 3 - (4 ' - CH 3 - phenyl)glycida te (µM) 4' - F - Phenylserine Production (nM s - 1 ) 3 - (4' - F - phenyl)glycidate (µM) 14 k 14 l 4' - Cl - Phenylserine Production (nM s - 1 ) 3 - (4' - Cl - phenyl)glycidate (µM) 4' - Br - Phenylserine Production (nM s - 1 ) 3 - (4' - Br - phenyl)glycidate (µM) Figure. 2 . 65 . Michaelis - Menten kinetics for the turnover of arylglycidate analogues ( 14 g 14 l ) to their corresponding arylse rines ( 15 g 15 l ). 118 Mic haelis - Menten Plots of Tc PAM Biocatalytic Conversion of Ary l iso serine Analogues. 14 a 14 b Phenylisoserine Production (nM s - 1 ) 3 - Phenylglycidate (µM) 3' - OCH 3 - Phenylisoserine Production (nM s - 1 ) 3 - ( 3' - OCH 3 - phenyl)glycidate (µM) 14 c 14 d 3' - CH 3 - Phenylisoserine Production (nM s - 1 ) 3 - ( 3' - CH 3 - phenyl)glycidate (µM) 3' - F - Phenylisoserine Production (nM s - 1 ) 3 - (3' - F - phenyl)glycidate (µM) 14 e 14 f 3' - Cl - Phenylisoserine Production (nM s - 1 ) 3 - (3' - Cl - phenyl)glycidate (µM) 3' - Br - Phenylisoserine Production (nM s - 1 ) 3 - (3' - Br - phenyl)glycidate (µM) Figure. 2 . 66 . Michaelis - Menten kinetics for the turnover of arylglycidate analogues ( 14 a 14 f ) to their corresponding aryl iso serines ( 16 a 16 f ). 119 14 g 14 h 3' - NO 2 - Phenylisoserine Production (nM s - 1 ) 3 - (3' - NO 2 - phenyl)gl ycidate (µM) 4' - NO 2 - Phenylisoserine Production (nM s - 1 ) 3 - (4' - NO 2 - phenyl)glycidate (µM) 14 i 14 j 4 ' - CH 3 - Phenylisoserine Production (nM s - 1 ) 3 - (4 ' - CH 3 - phenyl)glycidate (µM) 4' - F - Phenylisoserine Production (nM s - 1 ) 3 - (4' - F - phenyl)glycidate (µM) 14 k 14 l 4' - Cl - Phenylisoserine Production (nM s - 1 ) 3 - (4' - C l - phenyl)glycidate (µM) 4' - Br - Phenylisoserine Production (nM s - 1 ) 3 - (4' - Br - phenyl)glycidate (µM) Figure. 2 . 67 . Michaelis - Menten kinetics for the turnover of arylglycidate analogues ( 14 g 14 l ) to their corresponding arylis oserines ( 16 g 16 l ). 120 Figure. 2 . 68 . The six - hou r time course for the conversion of four representative 3 - arylglycidate racemates [3 - phenylglycidate ( 14 a - F - ( 14 d ) ( - Cl - ( 14 e - Cl - ( 14 k phenylglycidate (each at 400 µM)] to their corresponding arylserines in ass ays containing Tc PAM (100 µg/mL), and (2 S ) - styryl - - alanine (1 mM) in 50 mM NaH 2 PO 4 /Na 2 HPO 4 (pH 8.0), n = 3. Arylserine production is shown as a percentage relative to the initial epoxide substrate concentration. These example epoxides were turned over by Tc PAM from fastest (3' - Cl) to slowest (4' - Cl). 121 A B C D E F G H Figure. 2 . 69 . Lowest energy b inding poses are shown of A) (2 R ,3 S ) - 14 f ; B) (2 S ,3 R ) - 14 f ; C) (2 R ,3 S ) - 14 e ; D) (2 S ,3 R ) - 14 e ; E) (2 R ,3 S ) - 14 g ; F) (2 S ,3 R ) - 14 g ; G) (2 R ,3 S ) - 14 b ; and H) (2 S ,3 R ) - 14 b . 122 A B C D E F Figure. 2 . 70 . Lowest energy binding poses are shown of A) (2 R ,3 S ) - 14 l ; B) (2 S ,3 R ) - 14 l ; C) (2 R ,3 S ) - 14 k ; D) (2 S ,3 R ) - 14 k ; E) (2 R ,3 S ) - 14 h ; and F) (2 S ,3 R ) - 14 h . 123 Figure. 2 . 71 . Putative b inding pose of (2 S ,3 R ) - 3 - (3' - OCH 3 - phenyl)glycidate ( 14 b ) in a conformation aligned for NH 2 - MIO attack at C (labeled ' ') to make the arylisoserine ( 16 b ). C is labeled with ' ' for reference. Figure. 2 . 72 . GC/EI - MS extracted ion ( m / z 106) chromatogram of authentic (2 R ,3 S ) - syn - phenylisoserine ( 16 a ) derivatized as its O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester ( 18 a ). 124 A (R = H) [M+H] + [M+H 17] + [M+H 18] + [M+H 63] + [M+H 76] + [M+H 91] + B (R = H) C (R = 3' - OCH 3 ) m / z m / z D (R = 3' - CH 3 ) E (R = 3' - F) Figure. 2 . 73 . LC - ESI - MS/MS spectra of A) authentic ( 2 R ,3 S ) - syn - phenylisoserine ( 16 a ) (structures of diagnostic fragment ions are shown and used to characterize the fragment ions of the biocatalyzed arylisoserine a nalogues (below) . B) phenylisoserine ( 16 a ) ; C) 3' - OCH 3 - phenylisoserine ( 16 b ); D) 3 ' - CH 3 - phenylisoserine ( 16 c ); and E) 3 ' - F - phenylisoserine ( 16 d ) biocatalyzed by Tc PA M from the corresponding 3 - arylglycidates. 125 F (R = 3' - Cl) G (R = 3' - Br) H (R = 3' - NO 2 ) I (R = 4' - NO 2 ) J (R = 4' - CH 3 ) K (R = 4' - F) Figure. 2 . 74 . LC - ESI - MS/MS spectra of F) 3' - Cl - phenylisoserine ( 16 e ); G) 3 ' - Br - phenylisoser ine ( 16 f ); H) 3' - NO 2 - phenylisoserine ( 16 g ); I) 4' - NO 2 - phenylisoserine ( 16 h ); J) 4' - CH 3 - phenylisoserine ( 16 i ); and K) 4' - F - phenylisoserine ( 16 j ) biocatalyzed by Tc PAM from the corresponding 3 - arylglycidates. 126 L (R = 4' - Cl) M (R = 4' - Br) Figure. 2 . 75 . 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(2016) New Directions for Protease Inhibitors Directed Drug Discovery, Peptide Science 106 , 563 - 579. 141 Chapter 3: Insight into the Mechanism of Regio - and Stereoselective Amination of Enantiopure 3 - Phenylglycidate Isomers to Phenylserine by Tc PAM. 3.1 Introduction The 5 - methylidene - 3,5 - dihydro - 4 H - imidazol - 4 - one (MIO) family (EC 4.3.1.0 and 5.4.3.0) contains aminomutases ( AMs) and ammonia lyases (ALs). MIO - AMs catalyze the reversible cro ss exchange of an amine group and a hydrogen atom present on vicinal carbon of an amino acid, thus, - - amino acid is omers, 1 - 7 while the ALs - amino acid and its dehydroaminated analogue, the acrylates ( Figure. 3 . 1 A). 8 - 14 Several MIO - AMs have been engineered to conv - - amino acids with higher enantiosel ectivity. 15 - 17 AMs were also engineered by random mutagenesis within the active site ( Figure. 3 . 1 B ) 18 - 20 or repurposed using a non - natural amine donor substrate ( Figure. 3 . 1 C) 21 to undergo amination of various ring - substituted ar - - amino acids. The applic - heteroaryl - - amino acids - amino acids. 22 Similarly, ALs have been repurposed to achieve a broader substrate scope to convert acrylates, with electron - donating or - withdrawing substituents - amino acids through regioselective amination. Enantioselective - amination of arylacrylates was ach ieved through active site mutagenesis of ALs from Petroselinum crispum ( Pc PAL) and Anabaena variabilis ( Av PAL), whereas mutated AL from Streptomyces maritimus (EncP) and AM from Taxus chinensis ( Tch PAM) resulted in enantioselective - amination of arylacryl ates ( Figure. 3 . 1 B). 11,14,23,24 An engineered methylaspartate ammonia lyase (MAL) from Clostridium tetanomorphum exhibited significant expansion of the substrate scope for b oth the amine donor and substituted mesaconate. Several 142 combinations of aliphatic and primary aromatic amines were coupled regio - and stereo - selectively to many different mesaconate analogues to make (2 S ) - syn - aspartate analogues ( Figure. 3 . 1 D ). 25,26 B esides employing MIO - enzymes to make amino a cids, other efforts looked to explore mutants - amino acids to their acrylates. In another study, propargylglyc ine, an acyclic amino acid, was converted to ( E - AL from Petroselinum crispum. 27 Active site mutagenesis on a PAM from Taxus chinensis of the PAL activity in the enantioselective deamination of ( R 2 8 A B C D Figure. 3 . 1 . A) Reactions catalyzed by MIO - dependent ammonia lyases (ALs) and aminomutases (AMs), making either the acrylate or - amino acid from the corresponding - amino acid. The MIO and aminated - MIO (NH 2 - MIO) are shown. The asterisk (*) identifies an ( R / S ) - chiral c enter; B) - - arylalanine using engineered PAL/PAM catalysis; C) PAM - mediated amine transfer from ( S ) - styryl - - a lanine to acrylate acceptors. D) Addition of ammonia and primary amines to mesaconate analogues using en gineered methylaspartate ammonia lyase (MAL) to produce (2 S ) - syn - aspartate analogues. While much of the mechanistic analysis involving MIO - enzymes has c entered either on - - amino aci ds, our earlier work explored a new application of MIO - enzyme chemistry where we expanded the transaminase activity of MIO - aminomutases to a new class o f substrates, the epoxides, producing amino 143 alcohols. We repurposed an MIO - dependent phenylalanine amino mutase from Taxus canadensis ( Tc PAM) to convert trans - 3 - arylglycidates (i.e., trans - arylacrylate epoxides) to anti - arylserines. 29 Tc PAM normally isomerizes (2 S ) - - phenylalanine to (3 R ) - - phenylalanine. 30,31 and was shown to have broad substrate specificity for non - - arylalanines including the chain extended ( 2 S ) - styryl - - alanine. 21 In a kinetic study on T c PAM , the residence time of the NH 2 - MIO adduct was measured, after (2 S ) - styryl - - alanine donated its amino group to the enzyme, and the (2 E ,4 E ) - styrylacrylate product was released . 32 The burst phase study revealed that the aminat ed Tc PAM (NH 2 - MIO) lifetime was long enough to transfer the amino group intermolecularly from (2 S ) - styryl - - - - arylamino acids. 21 Encouraged by the intrinsic intermolecular transaminase activities exhibited by MIO - PAMs, 30,33 we used Tc PAM and (2 S ) - styryl - - alanine to convert trans - 3 - arylglycidate racemates to arylserines and arylisoserines. In our previous substrate specificity study (described in Chapter 2 of this thesis), we showed that Tc PAM has broad substrat e specificity and stereoselectivity toward forming (2 R ) - anti - arylserin es over their (2 S ) - anti - isomers, ranging from - F - - NO 2 - phenylserine. 29 We also used c rystal structures of Tc PAM in our earlier study to visualize the Tc PAM active site and infer the stereocontrol observed for the conversion of trans - 3 - arylglycidates to their correspondin g anti - arylserines. 18,34 From these data, we hypothesized that two active site tyrosine residues acted separately as general acids to facilitate stereoselective ring - opening of the glycidate racemates. In this stud y, we looked to verify that the isomeric ratio of the (2 R ) - anti and (2 S ) - anti - phenylserine products catalyzed by Tc PAM resulted from stereoselectivity for each isomer in the racemic mixture and not from bond rotation within one of the glycidate substrate i somers during catalysis. We also established the correlation between the glycidate enantiomer and the small amount of syn - phenylserine formed, which 144 provided valuable insight into the mechanism of amine group transfer from NH 2 - MIO to the glycidate backbone during catalysis. To test this hypothesis, here, we synthesized enantiop ure 3 - phenylglycidates from cinnamyl alcohol using the asymmetric Sharpless epoxidation reaction, 35 followed by RuCl 3 - catalyzed oxidation of the primary alcohol to the carboxylate. 36 Tc PAM was incubated with each 3 - phenylglycidate enantiomer, and the absolute configurations of the biocatalyzed anti - phenylserine (major) and phenyl i so serine (minor) were evaluated to gain further insights on the substrate specificity and selectivity of Tc PAM for aminating 3 - phenylglycidates. 29 The apparent Michaelis - Menten kinetic parameters of Tc PAM in a competitive inhibition ping - pong mechanism for each 3 - phenylglycidate enantiomer were measured. Here we summarize the details of these studies. 3.2 Experimental 3.2.1 Chemicals an d Reagents trans - Cinnamyl alcohol, (+) - diethyl tartrate (DET), ( ) - DET, sodium meta periodate, tert - butyl hydroperoxide solution (5.0 6.0 M) in nonane, chlorotrimethylsilane, and (2 S ) - (+) - methylbutyric anhydride were purchased from Millipore - Sigma (Burlin g ton, MA). Titanium(IV) isopropoxide and ruthenium (III) chloride hydrate were purchased from Oakwood Chemical (Estill, SC). (2 S ) - Styryl - - alanine and (2 R ,3 R ) - anti - phenylisoserine were purchased from Chem Impex (Wood Dale, IL). (2 S ) - syn - Phenylserine and (2 R ,3 S ) - phenylisoserine hydrochloride was purchased from Bachem (Torrance, CA). All chemicals were used without further purification unless noted. 3.2.2 Synthesis of (2 R ,3 R ) - 3 - Phenyl glycidol ( 2 1a ) 35 A mixture of Ti(O i - Pr) 4 (288 mg, 1.01 mmol), ( ) - DET (241 mg, 1.2 mmol), and activated , powdered 4 Å MS (50 0 mg) were stirred in anhydrous dichloromethane (CH 2 Cl 2 ) (20 mL) at 30 °C for 30 min. The reaction mixture was incubated over 30 min intervals before adding the next 145 reagent so the chiral Ti - DET complex could form and coordinate with each subsequent react ant at low temperature. Afterward, cinnamyl alcohol ( 20 ) (1.05 g, 7.8 mmol) dissolved in anhydrous CH 2 Cl 2 (2 mL) was then added, and the resulting mixture was stirred at - 30 °C for 30 min. The mixture was treated with 5.5 M t - BuOOH in nonane (3.6 mL, 19.5 mmol), stirred for 16 h at - 30 °C, warmed to 0 °C over 1 h, and poured into a freshly prepared solution of FeSO 4 (1.2 g) and tartaric acid (350 mg) in deionized H 2 O (2 mL) precooled to 0 °C. The two - phase solution was stirred for 30 mi n, and the aqueous ph ase was separated and extracted with diethyl ether (Et 2 O) (2 × 10 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered, and concentrated under vacuum to give a pale - yellow oil. The product was dissolved in Et 2 O (50 mL), cooled to 0 ° C, and treated with a 30% (w/v) solution of NaOH in saturated brine (1 mL) precooled to 0 °C. The two - phase mixture was stirred for 1 h at rt, and the aqueous layer was separated and treated with Et 2 O (2 × 10 mL). The combined organic extracts were dried ( Na 2 SO 4 ) and concentrated under vacuum. The resulting crude product was purified by flash column chromatography (silica gel, EtOAc/hexanes, 1:4) to yield (2 R ,3 R ) - 3 - phenylglycidol ( 2 1a ) as a clear liquid. I solated yield: 440 mg (38%); 9 2 % ee (based on HPLC separation). +49° ( c 1.00, CHCl 3 ), reported +45.9° ( c 1.00, CHCl 3 ). 37 R f = 0.5 of silica gel TLC (EtOAc/hexanes, 40:60). 1 H NMR (500 MHz, CDC l 3 7.27 (m, 5H), 4.06 (ddd, J = 12.7, 5.2, 2.4 Hz, 1H), 3.94 (d, J = 2.2 Hz, 1H), 3.81 (ddd, J = 12.7, 7.9, 3.8 H z, 1H), 3.23 (dt, J = 3.7, 2.3 Hz, 1H), 1.79 (dd, J = 7.8, 5.2 Hz, 1H). 13 C NMR (126 MHz, CDCl 3 61.3, 55.7. 3.2.3 Synthesis of (2 S ,3 S ) - 3 - Phenylglycidol ( 21b ). The following synthesis is based on a reported procedure, 35 and is analogous to that used to convert cinnamyl alcohol 20 to 2 1a , except (+) - DE T was used instead of ( ) - DET. The final 146 glycidol product ( 21b ) wa s purified by flash column chromatography (silica gel, EtOAc/hexanes, 1:4) to yield (2 S ,3 S ) - 3 - phenylglycidol ( 21b ) as a clear liquid. Isolated yield: 379 mg (33%); 9 0 % ee based on measured specific optical rotation: 48° ( c 1.00, CHCl 3 ), reported 50.1° ( c 1.00, CHCl 3 ). 37 R f = 0.5 on silica gel TLC (EtOAc/hexanes, 40:60). 1 H NMR (500 MHz, CDCl 3 ): 7.23 (m, 5H), 4.04 (dd, J = 12.8, 2.4 Hz, 1H), 3.94 (d, J = 2.2 Hz, 1H), 3.78 (dd, J = 12.8, 4.2 Hz, 1H), 3.31 3.16 (m, 2H). 13 C NMR (126 MHz, CDCl 3 1 25.7, 62.7, 61.4, 55.7. 3.2.4 General Procedure to Characterize the Enantiome ric Excess of Glycidols ( 2 1a and 21b ). To measure the enantiomeric excess ( ee ) of the 3 - phenylglycidols, s ynthesized and purified 3 - phenylglycidol (2 mg) ( 2 1a or 21b ) was dissolved in Et 2 O (2 mL), and an aliquot (10 µ L) of this solut ion was analyzed in HPLC (Agilent 1260) equipped with a chiral column (Chiralcel OD - H, 5 µ m, 4.6 mm x 150 mm) with i - PrOH/Hex (90:10) as the solvent at a flow rate of 1 mL/min . 3.2.5 Synthesis of Potassium (2 S ,3 R ) - 3 - Phenylglycidate ( 14 a a) from 2 1a . The following synthesis is based on a reported method. 36 RuCl 3 2 O (6 mg, 29 µM) was added to a biphasic mixture of solvents (CCl 4 (2 mL), acetonitrile (2 mL), and water (3 mL)) containing 2 1a (0.2 g, 1.32 mmol), sodium meta periodate 0.85 g, 3.96 mmol), and so dium bicarbonate (0.55 g, 6.6 mmol). The mixture was stirred for 72 h, then additional RuCl 3 2 O (6 mg, 29 µM) and sodium meta periodate (0.32 g, 1.5 mmol) were added, and the reaction was stirred for 24 h. CH 2 Cl 2 (8 mL) and then water (2 mL) were added to the sodium carboxylate solution. The product mixture was cooled to 0 °C, and the water layer was adjusted to pH 4.0 (6 M HCl). The aque ous layer was then extracted with Et 2 O (2 × 15 mL). The combined organic layers were dried (Na 2 SO 4 ), and the solvent was removed under vacuum to yield the carboxylic acid as a yellow oil. The carboxylic 147 acid was dissolved in acetone (5 mL) and water (0.5 m L) and (100 mg, 1 mmol) and treated with KHCO 3 to convert to its potassium salt ( 14 a a - decarboxylation. The final epoxides contained unreacted salts. Dioxane (10 µmol) was the inter nal standard used to quantify the 3 - phenylgly cidate stock solutions in Assay Buffer (50 mM NaH 2 PO 4 /Na 2 HPO 4 buffer (pH 8.0)) . Isolated yield: 171 mg (63%). 1 H NMR (500 MHz, D 2 7.35 (m, 5H), 3.97 (d, J = 2.5 Hz, 1H), 3.56 (d, J = 2.0 Hz, 1H). 13 C NMR (126 MHz, D 2 126.0, 58.8, 57.4. HRMS (ESI - TOF) m / z : [M - K] for C 9 H 7 O 3 : found 163.0403; calcd 163.0395. 3.2.6 Synthesis of Potassium (2 R ,3 S ) - 3 - Phenylglycidate ( 14 a b) from 21b . The potassium salt of (2 R ,3 S ) - 3 - phenylglycidate ( 14 a b ) was prepared following the same procedure to convert 2 1a to 14 a a . 36 Isolated yield: 185 mg (68%). 1 H NMR (500 MHz, D 2 7.42 7.35 (m, 5H), 3.97 (d, J = 2.5 Hz, 1H), 3.56 (d, J = 2.0 Hz, 1H). 13 C NMR (126 MHz, D 2 O) - TOF) m / z : [M K] for C 9 H 7 O 3 : found 163.0402; calcd 163.0395. 3.2.7 Enantiopurity o f the Synthesized 3 - Phenylglycidates. T he glycidates were suspended in H 2 O (1 mL) at 0 °C and titrated with 6 N HCl to pH 3. The resulting 3 - phenylglycidic acids were extracted into EtOAc (1 × 2 mL), and the organic fractions were combined, separated, and treated with diazo methane (0.9 equiv) dissolved in Et 2 O t o measure the enantiomeric excess ( ee ) of e ach synthetic potassium 3 - phenylglycidate (0.05 mmol) enantiomer. A stock of racemic potassium 3 - phenylglycidate, synthesized previously, 29 was also converted and enantio - enriched methyl 3 - phenylglyci dates was analyzed by gas chromatography/electron - impact mass spectrometry (GC/EI - MS) using an Agilent 6890N gas chromatograph equipped with 148 a capillary chiral GC column (25 m × 0.25 mm × 0.39 mm; CP Chirasil - Dex CB, th ickness 0.25 , Santa Clara, CA) with He as the carrier gas (flow rate, 1 mL/min). The injector port (at 250 °C) was set to splitless injection mode. A 1 - uL aliquot of each sample was injected using an Agilent 7683 auto - sampler (Agil ent, Atlanta, GA). Initial column tem perature started at 70 °C, then increased at 40 °C/min to 95 °C with a 7 min hold, ramped at 10 °C/min to 150 °C, then increased by 30 °C/min to 175 °C, and returned to 70 °C over 3 min. The gas chromatograph was couple d to a mass - selective detector (Agile nt, 5973 inert) operated in electron impact mode (70 eV ionization voltage). All spectra were recorded in the mass range of 50 375 m / z to analyze the methyl phenylglycidates. 3.2.8 Production of Phenylserine by Tc PAM Biocat alysis. A solution of ( 2 S ) - styryl - - a lanine (1 mM) in Assay Buffer was preincubated with Tc PAM (100 µg/mL) for 2 min, and then a potassium 3 - phenylglycidate enantiomer ( 14 a a or 14 a b ) (1 mM) was added. The assay was incubated at 31 °C and gently mixed on a rocking shaker (100 rpm) for 2.5 h. The reaction was quenched with 10% formic acid (pH 3.0). 3.2.9 General Meth od for Chiral Auxiliary Derivatization of Phenylserines and Phenylis oserines. A mixture of all four stereoisomers of phenylserine (0.2 mmol), synthesized previously, 29 was dissolved in Assay Buffer. To this solution were added pyridine (50 µL, 0.62 mmol) and ( 2S ) - 2 - methylbutyric anhydride (50 µL, 0.25 mmol), and the reaction mixture was stirred for 15 min at ~25 °C. The pH was a djusted to 3.0 (6 M HCl), and the N - protected phenylserine was extracted with EtOAc (1 × 2 mL). The organic layer was evaporated under a stream of nitrogen gas, and the resultant residue was dissolved in 3:1 EtOAc/MeOH (v/v) (1 mL). Diazomethane dissolved in Et 2 O was added dropwise until the yellow color persisted to obtain the methyl esters, and the 149 solvent was removed under a stream of nitrogen gas. The resulting methyl ester was dissolved in CH 2 Cl 2 (1 mL), to which pyridine (70 µL, 0.86 mmol) and chloro t rimethylsilane (100 µL, 0.79 mmol) were added. The solution was stirred for 15 min at ~25 °C, and the reaction was quenched with water (1 mL), and the organic layer was separated and analyzed by GC/EI - MS. To access authentic standards of anti - phenylisoser i ne enantiomers, enantiopure potassium 3 - phenylglycidates, 14 a a and 14 a b , and a stock of racemic potassium 3 - phenylglycidates, characterized and synt hesized previously, 29 were treated separately with NH 4 OH (2 M) in Assay buffer (1 mL) for 1 h. The hydroxy amino acids derived from the synthetic amination of the glycid a tes and authentic (2 R ,3 S ) - syn - phenylisoserine were then derivatized to their ( 2S ) - 2 - methylbutyramide chiral auxiliaries, followed by methyl esterification and O - silyl etherification, as done with the phenylserines. Each derivatized phenylisoserine sample w as analyzed by GC/EI - MS on an Agilent 6890N gas chromatograph equipped with a capillary GC column (30 m × 0.25 mm × 0.25 uM + 5 m EZ - Guard; VF - 5MS; Agilent Technologies, Santa Clara, CA) with He as the carrier gas (flow rate, 1 mL/min). The injector port ( at 250 °C) was set to splitless injection mode. A 1 - uL aliquot of each sample was injected using an Agilent 7683 aut o - sampler (Agilent, Atlanta, GA). Initial column temperature started at 50 °C, then increased at 50 °C/min to 150 °C, followed by 20 °C/min to 200 °C, then ramped at 10 °C/min to 225 °C with a 5 - min hold, and finally increased by 25 °C/min to 250 °C. The gas chromatograph was coupled to a mass - selective detector (Agilent, 5973 inert) operated in electron impact mode (70 eV ionization voltage) . All spectra were recorded in the mass range of 50 375 m / z to analyze the hydroxy amino acids derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl esters. 150 3.2.10 Kinetic Analysis. (2 S ) - styryl - - o 1 mM, at fixed assays containing Tc PAM (100 µg/mL) t o calculate the steady - state enzyme kinetic constan ts . The reactions were terminated with 10% formic acid (pH 3.0), and 3' - bromo - - phenylalanine (50 nM) was added as an internal standard. This reaction mixture was analyzed using a liquid chromatography - electrospray ionization - multiple reaction monitoring ( LC /ESI - MRM) method without further chemical derivatization. The apparent kinetic parameters ( and ) were calculated by non - linear regression with Origin Pro 9.0 software (Northampton, MA), using a modified Michaelis - Menten equati on (Eq. (1 ), See Supporting Information) . A secondary plot of vs the concentration of 3 - phenylglycidate enantiomers (Eq. (4) See Supporting Information ) was used to calculate the true kinetic parameters ( K M and k cat ) of each enantiomer. 151 3.3 R esults and Discussion 3.3.1 Synthesis of and Characterization of Enantioenriched 3 - Phenylglycidate Substrates ( 14 a a and 14 a b ). The enantiomeric potassi um 3 - phenylglycidates were made by oxidizing cinnamyl alcohol with the Sharpless asymmetric epoxidation catal yst using a ( ) DET or (+) - DET ligand to make (2 R ,3 R ) - 3 - phenylglycidol ( 2 1a ) or the (2 S ,3 S ) - iso mer, respectively ( Scheme 3 . 1 ). The absolute configuration of each 3 - phenylglycidol enantiomer was confirm ed by measuring the corresponding specific rotation in chloroform and comparing that with the reported litera ture. 37 Asymmetric Sharpless epoxidation employing ( ) - DET resulted in (2 R ,3 R ) - 3 - phenylglycidol ( 2 1a ), whereas the epoxidation with (+) - DET produced (2 S ,3 S ) - 3 - phe nylglycidol ( 21b ). Enantiopurity of each glycidol enantiomer w as calculated by measuring the relative peak areas of the glycidols separated on a HPLC fitted with a chiral column. Glycidol 21b eluted at 7.42 min (9 0 % ee ) followed by its enantiomer 2 1a (8.42 min, 9 2 % ee ) ( F igure. 3 . 2 ). Scheme 3 . 1 . Synthesis of (2 S ,3 R ) - 3 - phenylglycidate ( 14 a a ) and (2 R ,3 S ) - 3 - phenylglycidate ( 14 a b ). a) ( ) - DET, Ti(O i - Pr) 4 , t - BuOOH, 4 Å MS, CH 2 Cl 2 , 16 h, - DET, Ti(O i - Pr) 4 , t - BuOOH, 4 Å MS, CH 2 Cl 2 , 16 h, 30 °C; b) i ) RuCl 3 , NaIO 4 , NaHCO 3 , CH 3 CN/CCl 4 /H 2 O, rt, 72 h.; ii ) KHCO 3 , acetone/H 2 O, 0 °C, 1h. Glycidols 2 1a and 21b were was oxidized to their carboxylic acids using a RuCl 3 /NaIO 4 - based oxidation protocol ( Scheme 3 . 1 ) 36 under basic ( NaHCO 3 ) conditions to facilitate the deprotonation of the carboxylic acid and prevent any po tential decomposition due to spontaneous decarboxylation. Oxidation (2 R ,3 R ) - 3 - phenylglycidol ( 2 1a ) gave potassium (2 S ,3 R ) - 3 - phenylglycidate ( 14 a a ) whi le the (2 S ,3 S ) - 3 - phenylglycidol ( 21b ) produced potassium (2 R ,3 S ) - 3 - 152 phenylglycidate ( 14 a b ). In contrast to 43 h reaction time reported in the literatur e, 36 the oxidation reaction was run for 72 h to achieve complete oxidation to enantioenriched 3 - phenylglycidates. A B C F igure. 3 . 2 . Chiral HPLC separation of A). (2 R ,3 R ) - 3 - phenylglycidol ( 2 1a ), 92% e.e. ; B) .(2 S ,3 S ) - 3 - phenylglycidol ( 21b ), 90% e.e ; and C) a co - injection of enantioenriched samples of 2 1a and 21b . 3.3.2 Enantiopurity of the Synthesized 3 - Phenylglycidate isomers ( 14 a a and 14 a b ). T he enantiomeric excess ( ee ) of both (2 S ,3 R ) - and (2 R ,3 S ) - 3 - phenylglycidate potassium salts were measured by converting them to their co rresponding methyl esters using CH 2 N 2 and characterized by GC/EI - MS fitted with a chiral column. The retention times of the eluted peaks for the enantiopure me thyl 3 - phenylglycidate isomers were verified by comparing to the retention times of authentic rac emic methyl 3 - phenylglycidate standards characterized in my earlier work ( Chapter 2: , section 2.3.2 , Figure. 3 . 3 and Figure. 2 . 12 A). 29 Comparing the relative peak areas of the glycidate methyl esters analyzed by GC/EI - MS verif ied that the (2 S ,3 R ) - isomer was made at 153 92% ee and its (2 R ,3 S ) - antipode was prepared at 90% ee . The ee values of the glycidates tracked those of the corresponding glycidols from which they were synthesized . A B C Figure. 3 . 3 . Extracted ion ( m / z 121) chromatogram of (A) racemic methyl 3 - phenylglycidate (51:49), (B) methyl (2 S ,3 R ) - 3 - Phenylglycidate (4:96, 12.56 min), and (C) methyl (2 R ,3 S ) - 3 - Phenylglycidate (95:5, 12.37 min) . 3.3.3 Glycid ate Ring - Opening by Tc PAM Catalysis and Analysis of its Stereoselectivity. In our previous work, 29 we reported that Tc PAM co uld be repurposed to irreversibly biocatalyze an intermolecular amine transfer reaction from (2 S ) - styryl - - alanine to racemic 3 - arylglycidates to access arylserines and arylisoserines. Racemic glycidates were used to explore the enantioselectivity of Tc PAM for the conversion of the glycidates to their corresponding hydroxy amino acid products. Surpri singly, Tc PAM turned over the arylglycidate racemates to a mixture of anti - arylserine enantiomers as the major biocataly zed products, with a modest stereoprefer ence for the (2 R ) - anti isomer over its (2 S ) - anti antipode ( Figure. 3 . 4 ). T hese results inspired us to examine whether the isomeric ratio of the (2 R ) - anti - and (2 S ) - anti - arylserine products resulted 154 from the stereosp ecificity Tc PAM for a particular arylglycidate in the racemic mixture and not from bond rotation within one of the glycidate substrate isomers during catalysis . A (2 S ) - syn ( 15 a a ) (2 R ) - syn ( 15 a b ) (2 S ) - anti ( 15 a c ) (2 R ) - anti ( 15 a d ) B (2 S ,3 R ) - syn ( 16 a a ) (2 R ,3 S ) - syn ( 16 a b ) (2 S ,3 S ) - anti ( 16 a c ) (2 R ,3 R ) - anti ( 16 a d ) Figure. 3 . 4 . Stereoisomerism convention for A) phenylserine, and B) phenylisoserine used herein. The turnover ( ) of the trans - 3 - arylglycidate racemates ranged from 0.02 min - 1 for 3 - - chlorophenyl)glycidate to 1.3 min - 1 for 3 - - chlorophenyl)glycidate, and the turnover of the 3 - phenylglycidate was in between at 0.4 min - 1 . Thus, we chose to examin e the turnover of each trans - 3 - phenylglycidate enantiomer by Tc PAM to gain further insight into its stereospecificity and stereoselectivity. Tc PAM was incubated separately with each enantioenriched 3 - phenylglycidate ( 14 a a or 14 a b ) along with the amino group donor substra te (2 S ) - styryl - - alanine. Noticeably, Tc PAM turned over 14 a a ~1.6 - times faster than 14 a b to their anti - phenylserines ( 15 a c and 15 a d , respectively) preferentially over the syn - i somers ( 15 a a and 15 a b ). Tc PAM - catalyzed the ring - opening of each 3 - phenylglycidate preferentially at the C to produce phenylserine as the primary product. A small amount of anti - phenylisoserine (3%), resulting from amination at the C , was also made. 3 - + ) at the benzylic carbon, and this usuall y promotes nucleophilic attack at the C . For example, nucleophilic epoxide ring - opening by InCl 3 - catalyzed thiolysis, 38 bromolysis and iodolysis, 39 copper - catalyzed azidolysis, 40 and N - alkyl - (hydroxylamines) 41 occurred at C of 3 - phenylglycidates. In our study, 155 we observed a reversal of regioselectivi ty with Tc PAM as the biocatalyst and 12 as the amine - nucleophile donor where the 3 - phenylglycidates were ring - opened by nucleophilic cleavage of the C - O bond to produce phenylserine as the major biocatalyzed product . The relative and absolute stereoconfigurations of O - trimethylsilyl N - [( 2 S ) - 2 - methylbutyryl] methyl esters derivatives of phenylserine and phenylisoserine biocatalyzed by Tc PAM were assessed. Authentic standards of phenylserine chiral derivatives ( Scheme 3 . 2 A) were synthesized and characterized ( Scheme 2 . 1 B) and analyzed by GC/EI - MS (Section 2.3.9 and Figure. 3 . 5 A), as described in Chapt er 2 of this thesis. 29 A B Scheme 3 . 2 . Chiral auxiliary derivatization of A) authentic phenylserine diastereomers; and B) phenylisoserine diastereomers comprising (2 S ,3 S ) - anti - ( 16 a c ), (2 R ,3 R ) - anti - ( 16 a d ), and (2 R ,3 S ) - syn - phenylisoserine ( 16 a b ). Derivatization was performed using (a) ( i ) (2 S ) - 2 - Methylbutyric anhydride, pyridine, rt, 15 min; ( ii ) 6 M HCl, pH 2; ( iii ) CH 2 N 2 , EtOAc/MeOH (3:1 v/v), rt, 10 min; and ( iv ) chlorotrimethylsilane, pyridine,CH 2 Cl 2 , rt, 15 min.; and (b) 2 M aq. NH 4 OH, rt, 1h. The peak elution order of (2 S ) - anti - ( 15 a c , 8.88 min) followed by (2 R ) - anti - phenylserine ( 15 a d ,8.93 min), and (2 S ) - syn - ( 15 a a , 9.02 min) followed b y (2 R ) - syn - phenylserine ( 15 a b , 9.05 min) has previously been established ( Figure. 2 . 19 and Figure. 3 . 5 ) . 29 156 A B C D Figure. 3 . 5 . GC/EI - MS e xtracted - ion chromatograms with m / z 179 ion monitoring of A) phenylserine stereoisomers derivatized with a chiral auxiliary (cf. Scheme 3 . 2 A ). An earlier study 29 involving enzymatic resolution with (2 S ) - threonine aldolase confirmed that peaks at 8.88 min and 8.93 min correspond to chiral derivatives of (2 S ) - anti - and (2 R ) - anti - phenylserine isomers, respectively, and peaks at 9.02 and 9.05 min correspond to the chiral derivatives of (2 S ) - syn - and (2 R ) - syn - phenylserine isomers (cf. Figure. 2 . 19 ). Chiral derivatives of B) authentic (2 S ) - syn - phenylserine (Bachem); C) biocatalyz ed phenylserine made from (2 S ,3 R ) - 3 - phenylglycidate (the (2 S ) - anti :(2 R ) - anti :(2 S ) - syn isomers are abundant at a relative ratio of 2:90:8); and D) biocatalyz ed phenylserine made from (2 R ,3 S ) - 3 - phenylglycidate (the (2 S ) - anti :(2 R ) - anti :(2 R ) - syn isomers are ab undant at a relative ratio of 76:15:9). Guided by accounts reported previously , 42,43 racemic 3 - phenylglycidate was first treated with 2 M NH 4 OH to make a mixture of anti - phenylisoserine enantiomers ( 16 a c and 16 a d ) in situ to establish the absolute stereoconfiguration of phenylisoserine diastereomers. Also, enantiopure 14 a a was treated with NH 4 OH to invert the configuration at the C to make (2 S ,3 S ) - anti - phenylisoserine ( 16 a c ) , 42,43 and enantioenric hed 14 a b was treated similarly to make the (2 R ,3 R ) - anti - phenyl isoserine ( 16 a d ) ( Scheme 3 . 2 B). These sy nthesized anti - phenylisoserine isomers and 157 comm ercial (2 R ,3 S ) - syn - ( 16 a b ) and (2 R ,3 R ) - anti - phenylisoserine ( 16 a d ) methyl esters were deriva tized with chiral auxiliaries and analyzed by GC/EI - MS ( Figure. 3 . 12 ). The GC/EI - MS fragmentation patterns for the biocatalyzed phenylisoserines were identical to those for the chiral auxiliary derivatives of the co mmercial (2 R ,3 S ) - syn - , (2 R ,3 R ) - anti - , and synthesized (2 S ,3 S ) - anti - and (2 R ,3 R ) - anti - phenylisoserine isomers ( Figure. 3 . 19 and Figure. 3 . 20 ). The derivati ve of the commercial (2 R ,3 S ) - syn - phenylisoserine ( 16 a b ) and (2 R ,3 R ) - anti - phenylisoserine ( 16 a d ) eluted at 9.05 min and 9.33 min, respectively ( Figure. 3 . 6 A and B ), whereas those of the anti - phenylisoserine racemate , prepared from 3 - phenylglycidate racemate , eluted at 9.33 and 9.41 min ( Figure. 3 . 6 C ). This data indicates that the pe ak eluting at 9.41 min corresponds to the derivatized (2 S ,3 S ) - anti - phenylisoserine ( 16 a c ). The retention times of the anti - phenylisoserines in the racemic mixture was identified by comparing them a gain st the retention times of enantioenriched (2 S ,3 S ) - anti - phenylisoserine ( 16 a c , 9.41 min) ( Figure. 3 . 6 D ), and (2 R ,3 R ) - anti - phenylisoserine ( 16 a d , 9.33 min) ( Figure. 3 . 6 E ) derivatized identically . GC/EI - MS analysis of the chiral derivat ives of the products biocatalyzed by Tc PAM from (2 S ,3 R ) - 3 - phenylglycidate ( 14 a a , 92% ee ) in the amine transfer reaction showed that (2 R ) - anti - phenylserine was the major product (90%) with (2 S ) - syn - phenylserine as the minor product (8%). The (2 S ) - syn - phenylserine is believed to be formed when the carboxylate group of the glycidate substrate nucleophilically and intramolecularly opens the epoxy ring and forms a three - membered oxiranone intermediate , ( R ) - 3 - (( R ) - hydroxy(phenyl)methyl)oxiran - 2 - one ( 22aa ), ( Figure. 3 . 7 C and Figure. 3 . 8 B ) before being aminated at the C by the MIO - NH 2 adduct in the Tc PAM active site . Also, t race amount of the (2 R ,3 S ) - 3 - phenylglycidate present at 4% in the substrate was converted to (2 S ) - anti - phenylserine at a relative abundance of 2% ( Figure. 3 . 5 C). 158 A B C D E F G Figure. 3 . 6 . GC/EI - MS e xtracted - ion chromatograms with m / z 106 ion monitoring of a chiral auxiliary derivative of A) authentic (2 R ,3 S ) - syn - phenylisoserine (Bachem) ; B) authentic (2 R ,3 R ) - anti - phenylisoserine (Chem Impex); C ) anti - phenylisoserine enantiomers synthesized from authentic racemic 3 - phenylglycidate; 42,43 D) (2 S ,3 S ) - anti - phenylisoserine synthesized from (2 S ,3 R ) - 3 - ph enylglycidate ( 14 a a ); E) (2 R ,3 R ) - anti - phenylisoserine made from (2 R ,3 S ) - 3 - phenylglycidate ( 14 a b ); F) biocatalyzed (2 S ,3 S ) - anti - phenylisoserine made from (2 S ,3 R ) - 3 - phenylglycidate substrate ( 14 a a ); and G) biocatalyzed (2 R ,3 R ) - anti - phenylisoserine made from (2 R ,3 S ) - 3 - phenylglycidate substrat e ( 14 a b ). Note, isoserines in Panels C , D , and E , were made from the corresponding glycidates by nucleophilic addition of NH 4 OH. By comparison, the chiral derivatives of the products biocatalyzed by Tc PAM from (2 R ,3 S ) - 3 - phenylglycidate ( 14 a b , 90% ee ) showed that (2 S ) - anti - phenylserine was the major product at only 76% relative abundance with the (2 R ) - syn - phenylserine present at 9% abundance ( Figure. 3 . 5 D). The (2 R ) - syn - phenylserine is formed from (2 R ,3 S ) - 3 - phenylglycidate ( 14 a b ) following a mechanism similar to that for the formation of the (2 S ) - syn - isomer via an oxiranone intermediate , ( S ) - 3 - (( S ) - hydroxy(phenyl)methy l)oxiran - 2 - one ( 22ab ) before being am inated at the C ( Figure. 159 3 . 7 D and Figure. 3 . 9 B ). It was interesting to find that Tc PAM converted the 5 % (2 S ,3 R ) - 3 - phenylglycidate impurity in the substrate to (2 R ) - anti - phenylserine at 15% relative abundance . The latter is compared to the 2% relative abundance of the (2 S ) - anti - phenylserine made from the 4% (2 R ,3 S ) - 3 - phenylglycidate impurity in the previous assay . These results suggested that Tc PAM has a stereospecific preference for (2 S ,3 R ) - 3 - phenylglycidate over the (2 R ,3 S ) - enantiomer. Figure. 3 . 7 . A bsolute stereoconfiguration of biocatalyzed phenylserine produced from A) (2 S ,3 R ) - 3 - phenylglycidate ( 14 a a ); and B) (2 R ,3 S ) - 3 - phenylglycidate ( 14 a b ). Proposed mechanism for the formation of C) (2 S ) - syn - phenylserine from 14 a a ; and D) (2 R ) - syn - phenylserine from 14 a b through intramolecular carboxyla te assisted oxiranone formation followed by amination a t C by MIO - NH 2 . Incubating the enantio enriched 3 - phenylglycidate s with 2 M NH 4 OH showed that nonenzymatic, regioselective ring - - carbon ( Figure. 3 . 21 ), producing phenylisoserine as the major product . This result demonstrated that nucleophilic attack on phenylglycidate occurs inherently at the more electropositive benzylic carbon (C ). In contrast, Tc PAM used (2 S ) - sty ryl - - alanine as an amine donor and catalyzed the amination of enantiopure 3 - phenylglycidates predominantly at C to produce phenylserine . It is A C B D 160 worth noting, in its natural reaction, Tc PAM transfers the amino group from the N H 2 - MIO adduct equally to both C and C - - amino acids. However, with 3 - phenylglycidates, amination is precluded at C , resulting in Tc PAM convert ing (2 S ,3 R ) - 3 - phenylglycidate ( 14 a a ) and (2 R ,3 S ) - 3 - phenylgl ycidate ( 14 a b ) to small amounts of (2 S ,3 S ) - anti - phenylisoserine (3%) ( Figure. 3 . 6 F ), and (2 R ,3 R ) - anti - phenylisoserine (1%) ( Figure. 3 . 6 G ), respectively. 3.3.4 Probing the Formation of the Anti - and Syn - Phenylserine s using Computational Docking. Protein - ligand computational docking studies were performed to explain the formation of syn - phenylserine isomers (minor product) biocatal yzed by Tc PAM . The crystal struct ure of Tc PAM in complex with cinnamate [PDB: 3NZ4] was used to model structures of (2 S ,3 R ) - ( 14 a a ) and (2 R ,3 S ) - 3 - phenylglycidate ( 14 a b ) and their ox iranone s ( R ) - 3 - (( R ) - hydroxy(phenyl)methyl)oxiran - 2 - one ( 22aa ) and ( S ) - 3 - (( S ) - hydroxy(phenyl)methyl)oxiran - 2 - one ( 22ab ) into the Tc PAM active site using AutoDock Vi na by the method described in Section 2.2.15 of this thesis. 44 3.3.4.1 Computati onal Docking of Intermediates Leading to Anti - phenylserines. Lowest energy docked conformation s of 3 - phenylglycidate enantiomers produc ing anti - phenylserines has been discussed in Section 2.3.11 of this thesis. T he lowest energy docked conformation of the (2 S ,3 R ) - 3 - phenylglycidate ( 14 a a ) inside the active site of Tc PAM hinted that the carboxylate oxygen c ould adopt a bidentate salt - bridge with Arg 325 ( Figure. 3 . 8 A). 29 The glycidate oxygen was within a hydrogen bonding distance from the catal ytic Tyr80 residue that usually - isomerization of phenylalanine catalyzed by Tc PAM . We hypothesize that Tyr80 acts as a general acid with the glycidates to prom ote 161 protonation - initiated ring opening of the epoxide during the amination at the C to produce the major biocatalyzed product, (2 R ) - anti - phenylserine. Notably, in the docking model, C of the glycidate substrate is positioned close to the NH 2 - MIO nitrogen ( Figure. 3 . 8 A ) , and this modeled geomet ry is spatially consistent for S N 2 nucleophilic attack by the amino group at C of the glycidate. Similarly, a low energy binding conformation of the (2 R ,3 S ) - 3 - phenyl glycidate ( 14 a b ) substrate was calcu lated by AutoDock Vina limited to the active site cavity of Tc PAM. 29,44 As shown for the antipode ( 14 a a ) of 14 a b modelled within Tc PAM , t he carboxylate purportedly can form a salt - bridge interaction with Arg325 , and the glycidate oxygen of 14 a b is placed proximate to Tyr322 residue in a projected hydrogen b onding contact , facilitating epoxide ring - opening during the S N 2 amination step to produce (2 S ) - anti - phenylserine ( Figure. 3 . 9 A ), in this case, as the major biocatalyzed product. 3.3.4.2 Computational Docking Intermediates on the Pathway to Syn - Phenylserines. W e hypothesized that the (2 S ) - syn - phenylserine was produced during Tc PAM catalysis through a ( R ) - 3 - (( R ) - hydroxy(phenyl)methyl)oxiran - 2 - one ( 22aa ) intermediate made by an intramolecular carb oxylate - assisted ring - opening of the glycidate. To provide some insight on this proposal, a low - energy docking conformation of the ( R ) - 3 - (( R ) - h ydroxy(phenyl)methyl)oxiran - 2 - one ( 22aa ) intermediate inside the Tc PAM active site was calculated using the AutoDock Vina program. 44 Similar to the other modelled phenylpropanoid structures shown her ein, the oxiranone (O=C - O) functional group is placed into a salt - bridge complex with Arg325 ( Figure. 3 . 8 B ). The calculated low - energy pose places the oxiranone oxygen and the hydroxyl group close to the Tyr80 resid ue ( Figure. 3 . 8 B ) and C of the oxiranone close to the NH 2 - MIO nitrogen. This docking pose suggests that the Tyr80 residue can potentially facilitate the transition of the hydroxy oxiranone 162 intermediate to ac cess the (2 S ) - syn - phenylserine isomer directly from intermediate 22aa through proton - transfer steps and S N 2 attack of the NH 2 - MIO at the C ( Figure. 3 . 8 B ) . Likewise , ( S ) - 3 - (( S ) - hydroxy(phenyl)methyl)oxiran - 2 - one ( 22ab ) is prop osed as an intermediate to the minor product (2 R ) - syn - phenylserine made analogous to 22aa . AutoDock Vina 44 calculated a low - energy binding pose for 22ab in complex with Tc PAM. While the antipode 22ab was modeled in the active site as the mirror image, it made binding intera c tions s imilar to those made by 22aa . The oxiranone formed a salt bridge with Arg325, an oxiranone oxygen is near Tyr80, and the hydroxyl group is positioned close to Tyr322, the latter two interactions likely indicating catalytic hyd rogen - bonding interactions. The docked conformation also placed C near the nitrogen atom of the NH 2 - MIO priming the mechanism for favorable S N 2 attack at the C thus forming the (2 R ) - syn - phenylserine ( Figure. 3 . 9 B ) . A B Figure. 3 . 8 . A) Lowest energy binding conformations of A) (2 S ,3 R ) - 3 - phenylglycidate ( 14 a a ) (light - grey sticks); and B) ( R ) - 3 - (( R ) - hydroxy(phenyl)methyl)oxiran - 2 - one ( 22aa ) (light grey sticks) are shown at the center of the image inside the Tc PAM active site. The residues shown here include a catalytic Tyr80 (yellow sticks), a putative catalytic Tyr322 (yellow sticks), binding contact Arg325 (orange sticks), and the m ethylidene imidazolone (MIO) moiety (green sticks). Heteroatoms are colored red for oxygen and blue for nitrogen. The images were produced with UCSF Chimera, 45 and the docking conformations were generated with AutoDock Vina 44 from Tc PAM crystal structure (PDB code 3NZ4). 163 A B Figure. 3 . 9 . A) Lowest energy binding conformations of A) (2 R ,3 S ) - 3 - phenylglycidate ( 14 a b ) (light - grey sticks); and B) ( R ) - 3 - (( R ) - hydroxy(phenyl)methyl)oxiran - 2 - one ( 22ab ) (light grey s ticks) are shown at the center of the image inside the Tc PAM active site. The residues shown here include a catalytic Tyr80 (yellow sticks), a putative catalytic Tyr322 (yellow sticks), binding contact Arg325 (orange sticks), a nd the methylidene imidazolon e (MIO) moiety (green sticks). Heteroatoms are colored red for oxygen and blue for nitrogen. The images were produced with UCSF Chimera, 45 and the docking conformations were generated with AutoDock Vina 44 from Tc PAM crystal structure (PDB code 3NZ4). 3.3.5 Proposed Mechanism and Derivation of the Kinetic Equation. A two - substrate ping - pong mechanism was proposed for Tc PAM to biocatalyze phenylserine from a glycidate and (2 S ) - styryl - - alanine. In a typical ping - pong mechanism ( Scheme 3 . 3 , inset 1 ), substrate 1 ( S 1 ) binds to the enzyme ( E ) forming an enzyme - substrate complex ( ES 1 ). This is followed by subsequent release of product 1 ( P 1 ), which is usually a fragment of S 1 . The remainder of S 1 stays covalently attached to the enzyme, resulting in a modified enzyme ( E * ). Next, substrate 2 ( S 2 ) binds and reacts with E * and forms a modified enzyme - substrate complex ( E * S 2 ) before releas ing the product 2 ( P 2 ). The r elease of P 2 restores E * to E . 46 This is a non - sequential mechanism, thus S 1 and S 2 do not have to bind E before releasing P 1 - to how the enzyme bounces back and forth from an E (*) S n state to its standard state ( E * or E ) . 47 164 Scheme 3 . 3 . depicts the two - substrate ping - pong mechanism where (2 S ) - styryl - - alanine acts as the amino higher concentration (>1000 µM) of 3 - phenylglycidate. Inset 1 shows the scheme of a typical ping - pong mechanism. B iocatalysis of phenylserine from 3 - phenylglycidate, similarly, involves two substrates, (2 S ) - styryl - - alanine ( S 1 ) and trans - 3 - phenylglycidate ( S 2 ) that bind the enzyme forming separate enzyme - substrate complexes to ultimately transfer an amino group from S 1 to S 2 . We hypothesized that the amino donor substrate, (2 S ) - styryl - - alanine ( S 1 ) transfers its amino group to the MIO of Tc PAM ( E ) to form a covalent N - alkylated adduct ( ES 1 ). Crystal structure s of analogous N - alkylated complexes of both - and - phenylalanine have been previously reported in a mechanistically similar Tch PAM (PDB:4C5 R ), 48 and a bacterial PAM (PDB:3UNV), 31 suggest ing a common covalent mechanism for other MIO - enzymes, including Tc PAM . The nucleophilic - or - amino group of the substrate is found to attack the electrophilic methylene carbon of MIO to form this N - alkylated adduct. The enzym e - substrate complex ( ES 1 ) is proposed to proceed through an elimination step, removing H and the NH 2 - MIO group ( E * ) to release th e first product, (2 E ,4 E ) - 165 styrylacrylate ( P 1 ). In an earlier burst - phase kinetic study, the formation of P 1 was monitored where the lifetime of the aminated Tc PAM (NH 2 - MIO, E * ) was found to be long enough to transfer the amine group to exogenously supplied acceptor substrates. 32 After the release of produ ct P 1 , S 2 ( trans - 3 - phenylglycidate) likely binds covalently to Tc PAM through an NH 2 - MIO ( E*S 2 ) adduct that ring - opens the glyc idate regioselective ly . This proposed covalent adduct is based on a previously reported crystal structure of a homologous MIO - AM ( Sg TAM from Streptomyces globisporus , PDB :2RJR ) . Sg TAM formed a covalent N - alkyl bond between the MIO moiety and an intermediat e derived from 3 - - fluorophenyl)glycidate analogous to that proposed for E * S 2 ( Figure. 2 . 6 A ). 49 To continue, our proposed mechanism proceeds through proton - transfer steps and deamination of the MIO releases the major biocatalyzed product anti - phenylserine ( P 2 ) to regenerate the apo - enzyme E ( Scheme 3 . 3 ) . Assembly of this rational mechanistic pathway led us to derive a modified Michae lis - Menten kinetic equation to support the hypothesis ( Eq. 3.2 ). The following rate equations were derived from the pi ng - pong mechanism. Eq (3.1) where and Substituting and in Eq (3.1), 166 Eq. (3.2) where, , . Under the condition of constant [ ], Eq. (3.3) Where Eq. (3.4) and Eq. (3.5) 167 3.3.6 Kinetic Analyses. The Michaelis - Menten kinetic parameters of Tc PAM for converting 3 - phenylglycidate stereoisomers to phenylserine isomers were facilitated by a quantitative LC - ESI - MRM method following the derived rate equations ( Eq. 3. 3 to Eq. 3. 5 ) . The rate of phenylserine production was measured against the concentration of the amino donor substrate, (2 S ) - styryl - - alanine , at different fixed co ncentrations of the 3 - phenylglycidate sub strate ( 14 a a or 14 a b ) . To dissect the mechanism of this sequential catalytic reaction pathway, the concentrations of both substrates, (2 S ) - styryl - - alanine and phenylglycidate, were independently vari ed to evaluate their effects on the turnover rate of Tc PAM to make phenylserine . The concentration of (2 S ) - styryl - - alanine was varied from 100 µM to 1mM for each of the four fixed concentrations of the 3 - phenylglycidate ( 14 a a or 14 a b ) (100 µM, 500 µM, 1 mM, and 2 mM). For each particular concentration of the (2 S ,3 R ) - 3 - phenylglycidate ( 14 a a ), the of Tc PAM was calculated at ~1 mM for (2 S ) - styryl - - alanine ( Figure. 3 . 10 A and Eq. 3.3 ). T he phenylserine production rate increased with increas ing (2 S ,3 R ) - 3 - phenylglycidate ( 14 a a ) up to 1 m M , following a traditional two substrate ping - pong mechanism . 50,51 The phenylserine production was significantly reduced when the glycidate 14 a a was at 2 .5 mM ( Figure. 3 . 10 A) , suggesting a competitive substrate inhibition. E vidence of 3 - phenylglycidate inhibition on Tc PAM catalysis was also supported by a Lineweaver - Burk plot s howing convergent Y - intercepts for c oncentrations of 14 a a at 0.5 mM, 1 mM and 2.5 mM that indicates an unchanged and increased ( Figure. 3 . 10 C ) , a hallmark of competitive inhibition . A similar kinetic trend was observed for substrate 14 a b , where the for Tc PAM was calculated at ~1 m M of (2 S ) - styryl - - alanine for each of the four diff erent concentrations of 14 a b ( Figure. 3 . 10 B ) . 14 a b also inhibited t he rate of phenyls erine production at 2 .5 mM ( Figure. 168 3 . 10 D). of Tc PAM for (2 S ) - styryl - - alanine ( S 2 ), calculated from each kinetics plot measured at various fixed glycidate ( S 1 ) concentrations , were plotted agains t the concentration of S 1 ( 14 a a or 14 a b ) to calculate the intrinsic K M and k cat of Tc PAM for 14 a a or 14 a b at steady state ( Eq. 3.4 ) ( Table 3 . 1 ) . T he catalytic efficiency ( k cat / K M ) of Tc PAM for 14 a a app eared to be 1.3 - fold higher than that for 14 a b largely due to the 1.5 - fold higher k cat for 14 a a over that for 14 a b . The inhibit ion constant ( K I ) for each of the glycidate substrates were calculated from Eq. 3.5 using a previously reported K M value of (2 S ) - styryl - - alanine (105 µM) . 32 (2 S ,3 R ) - 3 - phenylgl ycidate ( 14 a a ) was found to have a K I value lower than its (2 R ,3 S ) - enantiomer ( 14 a b ), indicating that 14 a a binds Tc PAM better than 14 a b during the phenylserine production , and likely affects the reaction turnover at higher concentrations by precluding the binding of the amino donor (2 S ) - styryl - - alanine ( S 1 ) to the active site . In our previous study with racemic 3 - phenylglycidate as the substrate, Tc PAM made a mixture of (2 R ) - anti - and (2 S ) - anti - phenylserine products, with the 2 R - isomer predominating (68:32, >2 times more abundant), sug gesting modest enantioselectivity. In this study, we wanted to assess whether the observed isomeric distribution of products resulted directly from enantioselectivity or was the stereoselectivity combined with enantiospecificity or other effects. Stereoiso meric phenylserines were made irreversibly by Tc PAM, which converted (2 S ,3 R ) - 3 - phenylglycidate to (2 R ) - anti - phenyls erine as the primary product 1.3 times faster than it converted (2 R ,3 S ) - 3 - phenylglycidate to (2 S ) - anti - phenylserine. This moderate substrate specificity did not account fully for the ~2 - fold enantioselectivity observed earlier for Tc PAM with the racemic 3 - phenylglycidate substrate. Access to each glycidate enantiomer enabled us to calculate that the competitive inhibition constant ( K I ) for (2 S , 3 R ) - 3 - phenylglycidate was 1.5 - fold lower than that for the (2 R ,3 S ) - isomer. In this study, we revealed that a combin ation of kinetic ( k cat ) and 169 thermodynamic ( K I ) differences likely account for the 2 - fold higher enantioselectivity for the (2 S ,3 R ) - glycidate substrate over its enantiomer in the racemate in the earlier study. 170 A B C D E F Figure. 3 . 10 . Modified Michaelis - Menten plots for the turnover of A) (2 S ,3 R ) - 3 - phenylglycidate ( 14 a a ); and B) (2 R ,3 S ) - 3 - phenylglycidate ( 14 a b ) to their corresponding phenylserine at fixed concentration of the 3 - phenylglycidate enantiomer. Double reciprocal Lineweaver - Burk plot of C) (2 S ,3 R ) - 3 - phenylglycidate ( 14 a a ); and D) (2 R ,3 S ) - 3 - phenylglycidate ( 14 a b ) to their corresponding phenylserine at fixed concentration of 3 - y plot of (cal culated from each plot of Figure. 3 . 10 A vs [ 14 a a ]; and F) Secondary plot of (calculated from each plot of Figure. 3 . 10 B vs [ 14 a b ] to calculate the true K M of the glycidate substrates. 171 Table 3 . 1 . Kinetics of Tc PAM for turnover of 3 - phenylglycidate enantiomers to phenylserine a . Entry K M (µM) k cat (min - 1 ) k cat / K M (M - 1 s - 1 ) K I c (µM) 14 a a 230 (127) b 0.31 (0.04) 22.8 350 14 a b 190 (54) 0.20 (0.01) 17.5 520 a In addition to phenylserine, 14 a a forms 3% phenylisoserine and 14 a b produces 1% phenylisoserine. b Standard deviation in parenthesis ( n = 3). c Inhibition was observed at 2500 µM of 14 a a or 14 a b . 3.4 C onclusion Tc PAM catalyzed the stereoselective amination of 3 - phenylglycidate enantiomers through a sequential ping - pong aminotransfer reaction using (2 S ) - styryl - - alanine as the amine donor and (2 S ,3 R ) - and (2 R ,3 S ) - 3 - phen ylglycidate substrates as the amino group acceptors. Under non - enzymatic conditions, amine nucleophiles intrinsically cleave 3 - phenylglycidates at the electropositive C - O bond to make ph enylisoserine more abundantly over the phenylserine isomer. 42,43 In this study, the constraints of the Tc PAM catalytic environment forced the amino nucleophile resourced from (2 S ) - styryl - - alanine to preferentially cleave the C - O bond of each trans - 3 - phenylgly cidate enantiomer to produce a single antipode of anti - phenylserine preferentially. Nucleophilic attack at the C - O bond of the glycidates was prevented by Tc PAM, and only between 1% and 3%, relative abundance , of anti - phenylisoserines from each of the trans - 3 - phenylglycidate enantiomers was made. Tc PAM also turned over each trans - enantiomers of phenylglycidate to a single antipode syn - phenylserine (~10% relative abundance), which were evident as a mixture in a n earlier study when racemic phenylglycidates were used as substrates. 29 Computational modeling of a proposed oxiranone intermediate, formed from an intramolecular carboxylate - assisted oxirane - cleavage, showed that the intermediate made binding contacts with active site residues similar to those made in other MIO enzymes with their natural substrates. The modeled structures were poised f or amination by the NH 2 - MIO to yield the syn - phenylserines. 172 This work provides an alternative biocatalytic route to access single antipode of anti - phenylserine starting from each enantiomer of trans - 3 - phenylglycidates. This work extends the transaminase a c tivity of MIO - aminomutases to a new class of acceptor molecules to produce bi - functionalized hydroxy amino acids. This biocatalytic approach provides an easier access to the (2 R ) - anti - enantiomer of - hydroxy - - amino acids, which is difficult to obtain in large quantities otherwise and is highly expensive. 52 It also lays the foundation for further exploration into active site mutagenesis of the enzyme to achie ve better enantio selectivity , and to accommodate structurally more complex aryl and aliphatic glycidates as well as expansion into other three membered heterocycles in the future. 3.5 Future Studies. Wild - type Tc PAM was used to biocatalyze the production of ar ylserines and arylisoserines from 3 - arylglycidate and (2 S ) - styryl - - alanine in Assay Buffer. Tc PAM efficiently utilized (2 S ) - styryl - - alanine as a sacrificial amino group donor to ring - open the arylglycidate substrate regioselectively at the C to produc e arylserine predominantly. After the amino group transfer, (2 S ) - sty ryl - - alanine is converted to (2 E ,4 E ) - styrylacrylate. Future studies will focus on these aspects . Active Site Mutagenesis : Low - energy docking conformations suggested that (2 S ,3 R ) - 3 - phenylg lycidate possibly forms a hydrogen bonding interaction with Tyr80 re sidue during the amination reaction. Similarly, computational docking conformations indicated that (2 R ,3 S ) - 3 - phenylglycidate is within the hydrogen bonding distance of Tyr322 residue that possibly plays a vital role during the protonation assisted aminatio n reaction. Tyr80 is also reported to be responsible for the H elimination of (2 S ) - styryl - - alanine to (2 E ,4 E ) - styrylacrylate, 32,34 thus , forming the essential NH 2 - MIO adduct for transaminase activity . We envision that using an Y322F 173 mutant of Tc PAM through a ctive site mutag enesis and employing it in phenylserine b iocatalysis would prevent the (2 R ,3 S ) - 3 - phenylglycidate from getting converted to (2 S ) - anti - phenylserine. Hence, (2 R ) - anti - phenylserine could be stereoselectively biocatalyzed from a racemic mixture 3 - phenylglycidat e by ring - opening only the (2 S ,3 R ) - isomer. In a n earlier report on a Taxus chinensis phenylalanine aminomutase ( Tch PAM) , 18,20 active site mutagenesis (Q319M) altered the regioselectivity of amination reaction of arylacrylates by destabilizing the binding contacts of the carboxylate group of the substrate. This mutagenesis helped in stabilizing the intermediate formed from - amination . T hus, enantiopure - amino acids were synthesized from a rylacrylates through one - step ammonia addition. We hypothesize that such mutagenesis would also affect the amination of 3 - arylglycidate moiety and promote more - amination reaction, producing arylisoserines. Expansion into cis - G lycidates to B iocatalyze syn - P henylserines and syn - Phenylisoserines : Due to the "backside" attack of the incoming nucleophile during an S N 2 reaction catalyzed by Tc PAM , the ring - opened, aminated produ ct generated from a trans - glycidate made the anti - stereoconfiguration of the hydroxy amino acid predominantly . However, the majority of the naturally occurring and medicinally important compounds with an arylserine motif ( Figure. 2 . 7 ) and those with an arylisoserine moiety have relative syn - stereoisomerism ( Figure. 2 . 9 ). Hence, future efforts will focus on engineer ing Tc PAM , using site directed mutagenesis and guided by low - energy docking conformations, to accommodate a cisoid glycidate substrate to produc e syn - hydroxyamino a cids. Accessing aliphatic - Hydroxy - - Amino acids : Non - proteinogenic amino acids containing an aliphatic side chain show a wide range of biological activities such as, cytotoxicity, anti - tumor, anti - fungal, and anti - HIV. 53 (2 R ) - anti - threonine, as an example, is present in callipeltins, 174 PCM1206, and plipastatin which showed potent anti - HIV activity and/or cytotoxicity. 52,54 (2 S ) - anti - 3 - h ydroxy - leucine is a key component of several natural peptide antibiotics that include telomycin, azinothricin, citropeptin, variapeptin, and A83586C. 53,55 These aliphatic hydroxy amino acids likely also be accessed via the Tc PAM catalyzed transamination reactions from the corresponding alkyl glycidates. Regenerating (2S) - Styryl - - alanine Enzymatically . (2 S ) - Styryl - - alanine acts as a sacrificial amine donor in this biocatalysis reaction and gets converted to (2 E ,4 E ) - styrylacrylate. The styrylacrylate byproduct can be easily extracted out of the aqueous reaction buffer with organic solvent. Recent descriptions in the literature show that a mutated phenylalanine ammonia lyase from Petroselinum crispum ( Pc PAL) can add a mmonia to (2 E ,4 E ) - styrylacrylate and regenerate (2 S ) - s tyryl - - alanine . 24 This recycling event will significantly increase the a pplicability and turnover efficiency of the Tc PAM - assisted amination of 3 - arylglycidates. Exploring Aziridines and Thiiranes as Amino Group Acceptors : This transaminase activity of Tc PAM employing (2 S ) - Styryl - - alanine as the amino group donor can likely be extended also to other acceptor molecules, such as aziridines and thiiranes. Amination of 3 - phenylaziridine - 2 - carboxylic acid will produce 2,3 - diamino - 3 - phenylpropanoic acid. This c lass of , - diamino acids are important structural units found in natural products, 56 peptide antibiotics, 57 and in medicinally valuable compounds. 58 Tc PAM assisted amination of 3 - phenylazi ridine - 2 - carboxylic acid a nalogues will provide an entry point to access these , - diamino acids. Similarly, Tc PAM catalyzed amination of 3 - phenylthiirane - 2 - carboxylic acid will produce 2 - amino - 3 - mercapto - 3 - phenylpropanoic acid (also known as phenylcystein e) or its regioisomer, phe nylisocysteine depending on the regioselectivity of the thiirane ring - opening. Phenylcysteines are known as metabolic sequester of acetaldehyde derived from ethanol oxidation in the blood 175 stream of chronic alcoholics and thus, red ucing acetaldehyde toxicit y by diverting it to urinary excretion pathways. 59 They have been found to lower the amount of ethanol - derived acetaldehyde by 40 - 60% in mice models. Phenylcysteines are also effectively used in Native Chemical Ligation (NCL) for the synthetic preparation of therapeutic peptides and protein targets. 60 176 APPENDIX 177 A PPENDIX Figure. 3 . 11 . Par tial 1 H NMR (500 MHz, CDCl 3 ) spectra of (2 R ,3 R ) - 3 - Phenylglycidol ( 2 1a ) . 178 Figure. 3 . 12 . Par tial 13 C NMR (126 MHz, CDCl 3 ) spectra of (2 R ,3 R ) - 3 - Phenylglycidol ( 2 1a ) . 179 F igure. 3 . 13 . Par tial 1 H NMR (500 MHz, CDCl 3 ) spectra of (2 S ,3 S ) - 3 - Phenylglycidol ( 21b ). 180 Figure. 3 . 14 . Par ti al 13 C NMR (126 MHz, CDCl 3 ) spectra of (2 S ,3 S ) - 3 - Phenylglycidol ( 21b ) . 181 Figure. 3 . 15 . Par tial 1 H NMR (500 MHz, D 2 O) spectra of Potassium (2 S ,3 R ) - 3 - Phenylgl ycidate ( 14 a a ). 182 Figure. 3 . 16 . Par tial 13 C NMR (126 MHz, D 2 O) spectra of Potassium (2 S ,3 R ) - 3 - Phenylglycidate ( 14 a a ). 183 Figure. 3 . 17 . Par tial 1 H NMR (500 MHz, D 2 O) spectra of Potassium (2 R ,3 S ) - 3 - Phenylglycidate ( 14 a b ). 184 Figure. 3 . 18 . Par tial 13 C NMR (126 MHz, D 2 O) spectra of Potassium (2 R ,3 S ) - 3 - Phenylglycidate ( 14 a b ). 185 GC/EI - MS Analysis of Phenylisoserine Diastereomers Derivatized as O - Trimethylsilyl N - [( 2S ) - 2 - Methylbutyryl] Methyl Esters A B C D Figure. 3 . 19 . GC/EI - MS spectra of A) authentic ( 2 R, 3 S ) - syn - phenylisoserine from Bachem ( 6 b ); B) authentic ( 2 R, 3 R ) - anti - phenylisoserine from ChemImpex C) derivatized anti - phenylisoserine enantiomers produced from authentic racemic 3 - phenylglycidate; and D) . derivatized (2 S ,3 S ) - anti - phenylisoserine ( 16 a c ) made from (2 S ,3 R ) - 3 - phenylglycidate ( 14 a a ) and NH 4 OH. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - me thylbutyryl] methy l ester. The molecular ion (M , m / z 351) was not observed. 186 A B C Figure. 3 . 20 . GC/EI - MS spectra of A) derivatized (2 R ,3 R ) - a nti - phenylisoserine ( 16 a d ) made from (2 R ,3 S ) - 3 - phenylglycidate ( 14 a b ) and NH 4 OH B) biocatalyzed (2 S ,3 S ) - anti - phenylisoserine ( 16 a c ) made from (2 S ,3 R ) - 3 - phenylglycidate ( 14 a a ) by Tc PAM; and C) biocatalyzed (2 R ,3 R ) - anti - phenylisoserine ( 16 a d ) made from (2 R ,3 S ) - 3 - phenylglycidate ( 14 a b ) by Tc PAM. Each hydroxyamino acid was derivatized as their O - trimethylsilyl N - [(2 S ) - 2 - methylbutyryl] methyl ester. The molecular ion (M , m / z 351 ) was not observed. 187 Figure. 3 . 21 . Gas - chromatography/mass spectrometry e xtracted - ion chromatograms with m / z 336 ion monitoring of derivatized anti - phenylserine and anti - phenylisose rine enantiomers produced from NH 4 OH assisted ring opening of authentic racemi c 3 - phenylglycidate. 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