Use of transaminases for the biosynthesis of enantiopure building blocks of two essential medicines: Ethambutol and Dolutegravir 2023 Josephine Tshegofatso Maboya Supervisors: Prof Dean Brady & Dr Daniel Pienaar A DISSERTATION SUBMITTED TO THE FACULTY OF SCIENCE, UNIVERSITY OF THE WITWATERSRAND, IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i Declaration I declare that the work presented in this dissertation is my own work and has not been submitted before for any degree or qualification in this or any other university. A dissertation submitted to the Faculty of Sciences at the University of the Witwatersrand, Johannesburg, for the Degree of Master of Sciences. Josephine Tshegofatso Maboya Date : 25-03-2024 ii Abstract (S)-2-Amino butan-1-ol and (R)-3–amino butan-1-ol play an important role as intermediates in the synthesis of the anti-tuberculosis drug ethambutol and HIV integrase inhibitor drug dolutegravir respectively. The current industrial preparation of these enantioenriched amino alcohols is quite a challenging process; it typically involves the use of harsh chemicals, results in low yields, and generates hazardous waste materials. Consequently, these methods tend to be expensive, and it has been demonstrated that the cost of these intermediates has a significant impact on the overall costs of the synthesis of the entire drug. Therefore, it is not surprising that the convenient, cost–effective, and environmentally benign production of these optically pure amino alcohols is still the subject of ongoing investigations. The chemo-enzymatic approach holds great potential to replace the conventional routes for the synthesis of enantiopure amines. Transaminase enzymes (ATAs), in particular, have gained much attention over time due to their remarkable capability to transform inexpensive ketone starting materials into valuable enantiopure amino alcohols. Through the utilization of the isopropyl amine donor system, pro-chiral ketone starting materials were effectively transformed into the desired (S)-isopropyl 2-aminobutanoate and (R)-isopropyl 3-aminobutanoate using transaminase biocatalysis. These reactions proceeded well under milder conditions such as ambient temperature and pressure conditions, and impressively under an aqueous environment. Three (S)-enantiomer selective “hit “enzymes were discovered (ATA-189, ATA-194, and ATA-254) for the biotransformation of alpha-keto ester substrate into an enantio-enriched amino ester product, with enantiomeric excess ranging between 95-99% and the yield was 15-73% depending on the enzyme and reaction conditions. However, when it came to dolutegravir intermediate, a different scenario unfolded. In this case, the majority of the ATA enzymes in our enzyme library fortuitously exhibited selectivity for the (R)-enantiomer. In particular, four highly enantioselective enzymes (ATA-254, ATA-261, ATA-262, and ATA-234) were discovered, demonstrating % e.e ranging from 93% to 99.99%, with corresponding yields from 38% to 45%. The successful biotransformation of an inexpensive pro-chiral starting material into highly valuable enantioenriched amino ester intermediates represents a significant achievement. Coupled with an effective reduction method to convert these intermediates into the corresponding amino alcohols, this biotransformation process holds immense potential for enabling the sustainable and cost- effective production of both of the valuable ethambutol and dolutegravir amine intermediates. iii Acknowledgments I extend my sincere gratitude to Prof. Dean and Dr Danie for their unwavering support and exceptional mentorship. Their guidance and wealth of knowledge have been pivotal in shaping my journey, and I am immensely thankful for the opportunities to learn and grow under their tutelage. I want to express my profound gratitude to Thapelo, Refilwe, and Dr. Eric for their invaluable support in utilizing GCMS and HPLC-MS techniques. Furthermore, I extend my deepest thanks to Professor Fernandes for conducting the single-crystal analysis and to Dr. Sam Gittings from Prozomix for generously providing us with enzyme samples. Your expertise and collaborative efforts have significantly elevated the quality of my research, far beyond what I could have accomplished on my own I extend my warmest thanks to Dr. Maphupha, Dr Thabo, and Dr. Charles for their significant contributions and guidance throughout this journey. Your insights have been invaluable in shaping the trajectory of my work. To my wonderful lab mates – Kabelo, Lebo, Khanya, Chris, Nduduzo, Mpumi, Cecilia and Bongi – your camaraderie and support have turned the laboratory into a place of both productivity and enjoyment. I am truly thankful for the shared experiences that made this journey memorable. To my beloved siblings, whose belief in me has been a constant source of inspiration, and to my mom, whose prayers and emotional support have been my pillars of strength – your unwavering faith has been instrumental in my success, and for that, I am truly grateful. I would also like to express my gratitude to my best friend, Dinah. The late nights we spent together in the laboratory, pushing through challenges, were made enjoyable by your presence. Your unwavering companionship has been a constant source of motivation. Lastly, I want to thank Shibo, Tshego, Mkhulule, Bongi and Saki for their unwavering emotional support. Your encouragement and understanding have been a guiding light, helping me navigate both the highs and lows of this journey. To everyone mentioned above including the whole of organic group, your support has been invaluable, and I am deeply thankful for your roles in my academic and personal growth. With heartfelt appreciation, iv Table of Contents Declaration .................................................................................................................................. i Abstract ...................................................................................................................................... ii Acknowledgments.................................................................................................................... iii List of Figures ......................................................................................................................... vii List of Schemes ......................................................................................................................... ix List of abbreviations ................................................................................................................. xi 1. INTRODUCTION .............................................................................................................. 1 1.1 Mechanism of action of ATAs .................................................................................... 2 1.1.1 The role of PLP-cofactor .................................................................................... 2 1.1.2 Transaminase reaction mechanism ...................................................................... 4 1.2 Chiral amines............................................................................................................... 5 1.2.1 Chemical synthesis of amines ............................................................................. 6 1.2.2 Biocatalytic Synthesis of chiral amines ............................................................... 8 1.3 Types of transaminases reaction ............................................................................... 12 1.3.1 Kinetic resolution transaminase reaction ........................................................... 13 1.3.2 Asymmetric synthesis of chiral amines using ATAs .............................................. 14 1.4 General developments and challenges of asymmetric ATAs reaction ...................... 16 1.4.1 Expansion of substrate scope and a choice of amino donor .............................. 16 1.4.2 Continous flow biotransformation ........................................................................... 17 1.5 Chiral amino alcohols................................................................................................ 19 1.5.1 Ethambutol ......................................................................................................... 20 1.5.2 Dolutegravir ................................................................................................................. 22 1.6 Aims and Objectives ................................................................................................. 25 1.6.1 Aims ......................................................................................................................... 25 1.6.2 Objectives ................................................................................................................ 25 v 2. Synthesis of an Ethambutol intermediate ......................................................................... 26 2.1 General information .................................................. Error! Bookmark not defined. 2.2 Substrate selection ..................................................................................................... 27 2.3 Substrate synthesis .................................................................................................... 29 2.4 Enzyme screening ..................................................................................................... 32 2.4.1 Enzyme Screening Method Development ......................................................... 32 2.4.2 Method development for product isolation and quantification .......................... 33 2.4.3 Enzyme screening results ................................................................................... 36 2.4.4 The quantitative analysis of the isopropyl amino ester 11 ................................. 44 2.4.5 Development of HPLC method for separation of R and S enantiomers. ........... 39 2.4.6 Determination of stereochemistry of the enzyme products. .............................. 42 2.4.7 Enzyme kinetics ................................................................................................. 44 2.3.7 Scale–up of ATAs reaction for ethambutol synthesis.............................................. 48 2.3 Reduction of amino ester to the amino alcohol .............................................................. 48 2.4 Conclusion and Future Work ....................................................................................... 51 3.Synthesis of a chiral Dolutegravir intermediate.................................................................... 54 3.1 General information ....................................................... Error! Bookmark not defined. 3.2 Substrate selection .......................................................................................................... 54 3.2 Substrate synthesis ......................................................................................................... 55 3.3 Quantification and stereochemistry determination ......................................................... 56 3.4 Enzyme screening ........................................................................................................... 62 3.5 Reduction of amino ester 18 into amino alcohol 5 ......................................................... 64 3.6 Conclusion and Future work .......................................................................................... 65 4. General conclusion and future work .................................................................................... 68 5. Experimental ........................................................................................................................ 70 5.1 General reagents and instrumentation ........................................................................... 70 5.2 Experimental procedures ............................................................................................... 70 vi 5.2.1 Synthesis of diisopropyl oxalate ............................................................................. 70 5.2.2 Grignard reaction for the synthesis of α-Keto-butyric-isopropyl ester substrate and α-Keto-butyric-ethyl ester ................................................................................................. 72 5.2.3 Transesterification reaction for the synthesis of isopropyl acetoacetate . ............... 73 5.2.4 Synthesis of reference rac and (S) -2-aminobutyric –isopropyl ester and reference rac & (S) -2-aminobutyric –ethyl ester. ............................................................................ 75 5.2.5 Synthesis of reference rac and (S) -3-aminobutyric–isopropyl ester. ..................... 76 5.2.6 Derivatization of 2-aminobutyric–isopropyl ester into para-nitro benzoic-isopropyl ester for HPLC method development. .............................................................................. 79 5.2.7 Derivatization of 3-aminobutyric–isopropyl ester into para-nitro benzoic-isopropyl ester for HPLC method development. .............................................................................. 80 5.2.8 Reduction of 2-aminobutyric–isopropyl ester into 2-aminobutanol ....................... 80 5.2.9 Reduction of 3-aminobutyric–isopropyl ester into 3-aminobutanol ....................... 81 5.3 Enzyme reaction ............................................................................................................ 82 5.3.1 General enzyme screening procedure ..................................................................... 82 5.4 X-Ray diffraction crystal structure method. .................................................................. 83 5.5 LCMS data: Ethambutol ................................................................................................. 84 5.5.1 Achiral: HPLC analysis ........................................................................................... 87 5.5.2 Chiral: HPLC analysis ............................................................................................. 89 5.6 LCMS Data: Dolutegravir ......................................................................................... 93 5.6.1 Achiral: HPLC analysis ........................................................................................... 93 5.6.2 Chiral : HPLC analysis ............................................................................................ 95 References .............................................................................................................................. 100 NMR DATA .......................................................................................................................... 109 Cost estimation tables ................................................................................................................ 1 vii List of Figures Figure 1: The collective alanine transaminase function in the human liver and skeletal muscle cells.3 .......................................................................................................................................... 1 Figure 2:The significance of PLP functional groups on its overall function, redrawn from reference.12 ................................................................................................................................. 3 Figure 3 :The examples of chiral amine-containing pharmaceutical drugs.18 .......................... 6 Figure 4: An example of chiral resolution using the crystallization method. ............................ 7 Figure 5: Biocatalytic application of ATAs for the synthesis of pharmaceutically active compounds. .............................................................................................................................. 16 Figure 6: Immobilization of E. coli cells and PLP in methacrylate polymeric resin followed by flow chemistry transformation40 ......................................................................................... 18 Figure 7: Amino alcohol-containing drugs. ............................................................................. 19 Figure 8: First-line Anti-TB drug combination . ..................................................................... 22 Figure 9:The population of new HIV infections in South Africa categorized by sex and gender from 2010 to 2021.53 ................................................................................................................ 24 Figure 10: Docking of keto butyric acid in the ATA active site.64Error! Bookmark not defined. Figure 11: The standard curve of concentration of amino ester versus peak area by LCMS (number of variables, (n)= 8). .................................................................................................. 36 Figure 12: Comparison between LCMS results of ATA-230 and the standard amino ester products. ................................................................................................................................... 37 Figure 13: The graph illustrating the comparison between the activities of the enzymes on substrate 10. ............................................................................................................................. 38 Figure 14: The graph illustrating the comparison between the activities of the enzymes on substrate 11. ............................................................................................................................. 38 Figure 15: The graph illustrating the comparison between the activities of the enzymes, Series 1 (blue) represents the activity of the enzymes in a repeat experiment, and Series 2 (orange), represents the activity of the first screen.................................................................................. 45 Figure 16: The LCMS chromatography peaks of the derivatized (rac)-17 (above) and (S)- standard 17 (below)................................................................................................................. 41 Figure 17: The crystal structure of the derivative 17 of the ATA-189 enzyme product. ...... 44 Figure 18:Peak area versus time graph of the ATA-194 (blue), ATA-254 (grey) and ATA-189 (orange) catalysed reaction at 40°C. ........................................................................................ 47 viii Figure 19:Mass spectrum of ATA-254 catalysed amino alcohol product. .............................. 51 Figure 20: In the 1H NMR spectrum of the enzyme product (above), the stereogenic proton has shifted from 3.5 ppm to 1.8 ppm in comparison to the standard spectrum (below). ............... 53 Figure 22: 1H NMR data of an enamino ester 21 illustrating the two distinct peaks that distinguish a product from the starting material. ..................................................................... 57 Figure 23: 1H NMR showing the indication of newly formed stereogenic center when utilizing sodium triacetoxyborohydride reagent. ................................................................................... 58 Figure 24: Liquid chromatogram of the standard amino ester 18 obtained when using an achiral reverse phase column. .............................................................................................................. 59 Figure 25: Standard curve of peak area of the amine reference product 18 versus the concentration (ppm) (n=6). ...................................................................................................... 60 Figure 26: LCMS chromatography peaks of the derivatized rac-25 and enantiopure (S)-25 (below). .................................................................................................................................... 61 Figure 27: Comparison between the activities of active enzymes towards the biotransformation of pro-chiral 20 into the corresponding amino-ester 21. .......................................................... 62 Figure 28: Chiral liquid chromatogram showing the (R)-selectivity of ATA-261 and ATA-241. .................................................................................................................................................. 64 Figure 29: In the 1H NMR spectrum of the enzyme product (above), the stereogenic proton has shifted from 3.5 ppm to 1.8 ppm in comparison to the standard spectrum (below). ............... 67 ix List of Schemes Scheme 1: The summary of the overall ATA reaction mechanism .......................................... 4 Scheme 2: The transamination reaction mechanism.14 .............................................................. 5 Scheme 3: Different schematic routes for the synthesis of primary racemic amines . .............. 7 Scheme 4: Different schematic routes for the synthesis of optically pure amine.20 .................. 9 Scheme 5: An example of the application of dynamic kinetic resolution in improved hydrolases resolution % yield of the enantiopure products ....................................................................... 10 Scheme 6: Kinetic resolution of racemic amine using MAO followed by deracemization ..... 10 Scheme 7: Asymmetric synthesis of optically pure amines using imine reductases. .............. 11 Scheme 8: Example of AmDHs enzyme catalysed reaction.27 ................................................ 12 Scheme 9: ATA kinetic resolution coupled with amine oxidases.32 ........................................ 13 Scheme 10: The application of sequential stereoselective ATA catalysed reactions to synthesise mexiletine.33 ............................................................................................................................. 14 Scheme 12: Facile synthesis of Ethambutol from unnatural amino acid 3 ............................. 20 Scheme 14: Industrial synthesis of Ethambutol 1 .61,62 ............................................................ 26 Scheme 15: Our proposed synthesis route to produce Ethambutol 1 ...................................... 27 Scheme 16: Different routes for biotransformation of pro-chiral ketones into amines using ATAs. ....................................................................................................................................... 29 Scheme 28: Enzyme route for the synthesis of (S)-amino alcohol 4. ..................................... 50 Scheme 29: The current industrial method for the production of (R)-3-amino-1-butanol (5) intermediate.............................................................................................................................. 54 Scheme 30: Proposed ATA biotransformation for the synthesis of the dolutegravir intermediate (5) ............................................................................................................................................. 55 Scheme 31: Transesterification of ethyl acetoacetate 22 with isopropanol to synthesize the isopropyl acetoacetate 20. ........................................................................................................ 56 Scheme 32: Transesterification of methyl acetoacetate 18 with isopropanol to synthesize the isopropyl acetoacetate 20. ........................................................................................................ 56 Scheme 33: Reductive amination of isopropyl acetoacetate 2 0 into a (rac) - amino ester 21. .................................................................................................................................................. 57 Scheme 34: An alternative synthesis of racemic and (S)-amino ester reference compound 21. .................................................................................................................................................. 59 Scheme 35: Derivatization of the standard amine 21 using para- nitro benzoyl chloride to form amide 25. .................................................................................................................................. 61 x Scheme 36:Proposed biotransformation towards the synthesis of (R)-amino ester 21. ........... 62 xi List of abbreviations AIDS Acquired Immune Deficiency Syndrome API Active pharmaceutical ingredients α-TAms Alpha transaminases ART Antiretroviral therapy ATAs Amino transaminases BF3.(OEt)2 Boron trifluoride diethyletherate DCC N, N'-Dicyclohexylcarbodiimide DCM Dichloromethane DKR Dynamic kinetic resolution DMAP 4-dimethylamino pyridine DNA Deoxyribonucleic acid DIPEA N, N-Diisopropylethylamine e.e Enantiomeric excess FDA Food and Drug Administration HILIC Hydrophilic interaction chromatography HIV Human immunodeficiency virus InSTI Integrase strand transfer inhibitor IREDs Imine Reductases IPA Isopropyl amine KR Kinetic resolution K+Pi Potassium monophosphate buffer LCMS liquid chromatograph mass spectrometer MAO Monoamine oxidases NADP Nicotinamide adenine dinucleotide phosphate (NADP) NRTIs Nucleoside reverse transcriptase inhibitors PLP Pyridoxal-5-phosphate PMP Pyridoxamine-5-phosphate PPM Part per million THF Tetrahydrofuran ω-TAms Omega transaminases WHO World Health Organization 1 CHAPTER 1: 1. INTRODUCTION Aminotransferases (ATAs), also known as transaminases (EC 2.6.1.-), are groups of enzymes that catalyse the interconversion of amine and keto groups in the presence of pyridoxal-5- phosphate (PLP) enzyme co-factor. These enzymes play a major role in central metabolism in both animals and plants.1 ATAs are commonly found in all parts of the plants, and they are responsible for the catalysis of the reversible transaminase between glutamate and pyruvate into oxoglutarate and alanine, which plays a major role in the protein synthesis.1–3 ATAs have a tissue-specific distribution in humans, some are found in the kidneys, cardiac and skeletal muscle but the major distribution is found in the liver.2,3 The importance of transaminase in glucose formation is illustrated in Fig.1. In the muscle cell, pyruvate and glutamate are converted to alanine and 𝛼-ketoglutarate. The alanine product then enters the blood circulation and is taken up by the liver where alanine transaminase hepatocytes convert it back to pyruvate, which plays a major role in glucose synthesis (Fig. 1). This function is essential for glucose regulation, particularly during stressful conditions like vigorous exercise and food deprivation.1,2,4 Generally, ATAs play a crucial role in cell viability, metabolism, and excretion of toxic substances in the body. Figure 1: The collective alanine transaminase function in the human liver and skeletal muscle cells.3 In bacterial cells (e.g. E. coli), there are several overlapping ATAs enzyme activities that collectively participate in the biosynthesis of alanine, which is a major building block of the bacterial cell wall, while they also catalyse alanine degradation depending on the metabolic and environmental conditions. The enzymatic function of ATAs and their biological significance was discovered and recognized by Braunstein and his colleagues in the late 1930s.5 Their discoveries resolved several biochemical puzzles despite working with limited resources and this team continued 2 over decades advancing the study of ATAs and other PLP-dependant enzymes. Cooper and Meister published a historical review paper in 1988 as a tribute to Braunstein; the paper gave a detailed explanation of the events and pioneers involved in the discovery of ATAs, as well as enzyme characteristics.5 Their findings involve: • The mechanism of action of aspartate transaminase enzymes. • Biological significance of ATAs, wherein they observed the conversion of glutamate and aspartate to succinate when using muscle tissue extracts and concluded that a mammalian cell should at least contain two different ATAs, a glutamate-pyruvate ATAs and aspartate-pyruvate ATAs. • The importance of PLP as an enzyme co-factor was also highlighted. • The stereospecificity of the ATAs reaction was observed as they found only one enantiomeric configuration between the reversible interconversion of alanine and glutamate. Since the first ATA demonstration by Braunstein in the 1930s, various ATAs were subsequently discovered. ATAs are now classified into two different groups, the α- transaminases (α-TAms) or ω-transaminases (ω-TAms) depending on their substrate specificity. 6In the case of α-TAms, the presence of a carboxylic group in the α-position to the carbonyl functionality is exclusively required, hence α-TAms only allow for the formation of α-amino acids and the corresponding α- keto acids. 7,8 𝜔-TAms are technically more useful than α-TAms as they allow amination of non-alpha positioned substrates thus having a wide range of substrates including ketones, aldehydes, and keto acids. ω-TAms exhibit enantio-preferences, being either R or S–selective. Over decades the majority of ω-TAms discovered in nature have been (S)-selective; it is only recently that (R)-selective ω-TAms have been identified or engineered. 9ω-TAms biocatalysis is currently applied in pharmaceuticals, chemical and agricultural industries for the interconversion of pro-chiral ketones into high-value enantiopure intermediates. Mechanism of action of ATAs 1.1.1 The role of PLP-cofactor 3 Pyridoxal-5-phosphate (PLP), also known as an active form of vitamin B6, represents the most versatile organic co-factor utilized in biochemistry. PLP-dependent enzymes are involved in the catalysis of numerous chemical reactions, including those catalysed by transaminases, racemases, aldolases, decarboxylases, lyases and others.10 Besides this broad functional diversity, all PLP-dependent enzymes are structurally grouped into seven-fold types based on different evolutionary lineages. Of all 7-fold types, fold type 1 is the most populated and is more structurally and functionally diverse compared to the others.11 The family of aminotransferases are a part of this widely diverse fold-type I PLP–dependent enzymes. Fold type I enzymes share certain mechanistic characteristics with other fold types such as a covalent binding of PLP with lysine residue to form a Schiff base (Fig. 2).10 Figure2: The significance of PLP functional groups on its overall function, redrawn from reference.12 PLP acts as a co-factor because of its specific structural properties. The structure of PLP contains a phosphate group and a hetero aromatic pyridine ring bearing a hydroxyl and an aldehyde group (Fig. 2). All functional groups in PLP are vital in the mode of action and binding interaction of PLP–dependent enzymatic reactions. The aldehyde group is responsible for imine formation (Schiff base) with the free unprotected amine, i.e. the internal aldimine is formed with the lysine residue of the enzyme, and the external aldimine formed with the amine of the amino donor or a substrate (Fig. 2).10 The interconversion between internal and external 4 aldimine is a reversible process due to its thermodynamic properties, which makes it possible for the binding of the substrate, product release, and the regeneration of the co-factor (PLP).10 The phenolic oxygen of the PLP is responsible for maintaining the planar conformation of the Schiff base, which is essential for electron delocalization. The oxygen also interacts with the imine nitrogen through keto-enol tautomerization which favours the keto-enamine tautomer and the planer conformation of imine and the pyridine ring (Fig. 2). The pyridine ring is important for the stabilization of the carbaionic intermediate that is formed in the reaction, this is done through resonance stabilization and delocalization of electrons by the electron-sink character of PLP (further discussed below). The PLP binds in the active site through the phosphate group, however, exceptions are observed in some fold types. The phosphate group was also proposed to function as an acid-base catalyst, which promotes proton transfer during external aldimine formation.10 1.1.2 Transaminase reaction mechanism The reaction mechanism of transaminases was extensively studied by crystallographic investigation of enzyme and substrate complexes. Although there are different sub-classes of transaminases (2.6.1.1-2.6.1.6), they all follow the same mechanism but differ significantly in substrate specificity. ATAs catalyse the oxidative deamination of amine donor and reductive amination of amine acceptor, thus the whole mechanism is divided into two distinct half- reactions (Scheme 1).7,13,14 The first half-reaction involves the conversion of the amine donor to its corresponding ketone, while the PLP cofactor is interconverted into its amine version, pyridoxamine-5-phosphate (PMP).14 In the second half-reaction, the amine acceptor (ketone) is converted into a final amine product and PLP is regenerated.14 Scheme 1: The summary of the overall ATAs reaction mechanism. 5 The first step in transamination involves the formation of the internal aldimine (Schiff base).10,14 This is followed by a nucleophilic attack of the amino donor at the iminium carbon of PLP thus undergoing transaldimination transformation to form external aldimine.7,14 The proton on Cα is abstracted by the lysine residue resulting in a planar quinoid intermediate, which is followed by the carbanion resonance stabilization over the entire compound (electron sink effect) resulting in new bond formation.7,14,15 Catalytic lysine then donates a proton at C4 resulting in a ketimine intermediate. Finally, ketimine is hydrolyzed to a keto acid and PMP, thus one-half reaction is complete followed by a second half which constitutes the same intermediates but in the reversed order resulting in a corresponding new amine product as well as PLP regeneration.14 Scheme 2: The transamination reaction mechanism.14 Chiral amines Chiral amines play an increasingly important role as building blocks in the pharmaceutical and agrochemical industries.16 In the case of a racemic compound, both R and S enantiomers may have the same structural and physical characteristics, however, they often have different biological responses. The most famous example of the importance of chirality is thalidomide, which is the drug that was used in the early 1960s for the treatment of nausea and alleviation of morning sickness in pregnant women.17 The use of racemic thalidomide resulted in a lot of infants being born with deformities of limbs and only half of these children survived. This effect was due to the biological response from one of the enantiomers, the R enantiomer was a potent drug with multiple therapeutic applications, while the S enantiomer had severe 6 teratogenic side effects.17 The US Food and Drug Administration (FDA) and the European Committee for Proprietary Medicinal Products introduced strict regulation requiring the synthesis of single stereoisomer drugs, and this came into effect in 1992.18. More than 90% of Top selling drugs originate from enantiopure chiral amines, Fig.3 shows examples of drugs containing a single amine enantiomer.18 Figure 3 :The examples of chiral amine-containing pharmaceutical drugs.18 1.1.3 Chemical synthesis of amines The are numerous chemical reaction techniques that can be used to synthesize amines. The most facile route includes the direct substitution of an alkyl halide in a simple SN2 reaction (Scheme 3). The problem with the substitution reaction is the requirement of an excessive amount of ammonia in order to drive the reaction to a desired product. The primary amine product is also reactive towards the alkyl halide; therefore this results in an unfavourable over- alkylation if minimum amount of ammonia is used. Alternative synthesis of amines includes the reductive amination reaction, which is the most commonly used and versatile way of forming C-N bonds (Scheme 3). The are two steps involved in this reaction, this includes the imine / the enamine formation from the corresponding ketone or aldehyde followed by the reduction reaction. The most common reducing reagent for this purpose is sodium triacetoxyborohydride (NaBH(OAc)3) which can selectively reduce imines in the presence of aldehydes into amines. Other reducing agents such as sodium borohydride (NaBH4) and sodium cyanoborohydride (NaBH4CN) can also be used. Amines can also be formed from the reduction of other nitrogen-containing functional groups, amides, nitriles, and azides (Scheme 3). https://www.masterorganicchemistry.com/glossary/nabh4/ https://www.masterorganicchemistry.com/glossary/reducing-agent/ https://www.masterorganicchemistry.com/glossary/nabh4/ https://www.masterorganicchemistry.com/glossary/nabh4/ 7 Scheme 3: Different schematic routes for the synthesis of primary racemic amines. All the reactions mentioned above result in the racemic mixtures of amine products. To achieve a single enantiomer, a chiral resolution step needs to be applied. The most commonly used resolution step for large-scale production includes recrystallization of the desired enantiomer by forming a diastereoisomeric salt.19 Formation of diastereoisomers from different resolution agents (mostly carboxylic chiral agents) results in two compounds with different physical properties, thus making them easy to be separated via distillation, crystallization, etc.19 During chiral resolution steps, only 50% of the initial quantity of the reactants can be converted to the desired product whilst the other 50% can be recovered as an unwanted precusor.20 However, if the undesired enantiomer can be racemized, it is possible to undergo the whole optical resolution repeatedly until all the products are in a single enantiomer form (Fig. 4). Figure 4: An example of chiral resolution using the crystallization method. Chemical synthesis strategies for stereospecific synthesis of chiral amines is still a challenging process, it requires time-consuming resolution steps, uses harsh chemicals, results in low 8 product yield, and generates toxic waste material.21,22 Even the recent synthesis approaches, which involve high temperature and pressure conditions, harsh chiral auxiliaries, and transition metals (like expensive ruthenium catalyst, which are non-reusable), are far from perfect. A more convenient and greener route for the production of optically pure amines is needed and is still an ongoing investigation. Biocatalysis, which is the use of biological substances to catalyse a chemical reaction, bears great potential for the synthesis of optically pure amines and thus has gained much attention in the past years.20 1.1.4 Biocatalytic Synthesis of chiral amines An efficient and sustainable synthesis of enantiopure amines is of great importance in the pharmaceutical industries and now there is a shift to the application of biocatalysis in the production of enantiopure amine-containing drugs. Biocatalytic synthesis of chiral amines has emerged as a green alternative route to chemical synthesis, they often work under milder conditions, operate in aqueous media and they are insensitive.23 The most appealing aspect of biocatalysis is that many enzymes are extremely stereoselective, often leading to enantioenriched amine compounds (sometimes achieving 99.9% e.e).20 In theory, various enzymatic pathways can be used to synthesize optically active amines, originating from different enzyme classes including transferases, hydrolases, and oxidoreductases (Scheme 4). Some enzymes, such as amine oxidases, transaminases, lipases, and other hydrolases, can distinguish between enantiomers of racemic substrates and thus can be used for a chiral resolution step. Others are capable of the more atom economical enantiospecific synthesis (imine reductases, amine dehydrogenases and again transaminases). 9 Scheme 4: Different schematic routes for the synthesis of optically pure amine.20 1.1.4.1 The use of hydrolases for resolution of racemic amines. Hydrolase enzymes can be used for the kinetic resolution of racemic amines to obtain optically pure compounds. The protease family of hydrolases, including penG acylase, lipases, and amidases, inherently catalyses the stereoselective hydrolysis of amides.20 These enzymes work by preferentially attacking one enantiomer, and the final product contains a mixture of a single amine enantiomer and the amide form of the other enantiomer. BASF agricultural solution industry in Germany uses Burkholderia plantarii lipase at a large-scale for the production of optically pure aliphatic amines and amino alcohols.20 Although the method has been highly optimized to be performed on a large scale for the resolution of a wide variety of amines, kinetic resolution yields are still limited to less than 50% unless racemization is performed to facilitate the dynamic kinetic resolution (DKR) step. The kinetic dynamic resolution was applied to the synthesis of the anti-depressant pharmaceutical norsetraline (1R, 4S) by Bäckvall’s group.24 Initially, the racemic 1- aminotetralin underwent the dynamic resolution step using Candida antarctica lipase B (CALB) followed by the racemization using Ru-Shvo catalyst and this resulted in a 70% yield of the intermediate (Scheme 5). Five more steps were followed to afford 28% norsertraline.24 10 This example highlights how the lipase kinetic resolution step can be improved by incorporating racemization to facilitate dynamic kinetic resolution. The drawback is that most of the racemization catalysts are transition metal catalysts, so they are expensive and some produce toxic waste. To solve this problem, enzymes that are able to promote racemization must be incorporated. Scheme 5: An example of the application of dynamic kinetic resolution in improved hydrolases resolution % yield of the enantiopure products. 1.1.4.2 The use of monoamine oxidases (MAO) for the synthesis of enantiopure amines Monoamine oxidases (MAO) are members of the oxidoreductase class of enzymes and they play a major role in the catalysis of redox reactions, particularly oxidation.16,18 MAO catalyses the enantioselective oxidation of a racemic amine to form an imine and a single enantiomer (Scheme 6). The most efficient approach that harnesses the catalytic power of MAO is deracemization, in which the enantioselective oxidation is coupled with the concurrent non- selective chemical reduction. The process can occur recursively until a 100% yield of the single enantiomer is reached. This resolution step is more advantageous compared to lipase DKR because no additional organometallic catalyst is required for the deracemization step. Scheme 6: Kinetic resolution of racemic amine using MAO followed by deracemization. 11 The chiral resolution of MAO reveals a rather impressive technique to synthesize a single enantiomer in 100% yield. A disadvantage of this reaction is that it requires a non-selective chemical reducing agent to facilitate the deracemization step, and ammonium borane (NH3.BH3) is most commonly used for this purpose. Boron-containing reducing agents are inconvenient to use because they often lead to undesirable boron-amine complex formation. An alternative non-boron containing reducing agent such as lithium aluminium hydride must be employed for the reduction of imines. 1.1.4.3 The use of imine reductase (IREDs) for the synthesis of enantiopure amines. Imine reductases are NADPH–dependent enzymes that are able to catalyse the stereoselective reduction of imine to their corresponding amines.25 The most interesting part of the IRED resolution step is that it allows for the asymmetric synthesis of optically pure amines, both reductive amination reactions and imine reductions are possible (Scheme 7). Due to the unfavourable equilibrium of imine formation in aqueous media, IRED reactions have received little attention in the past few years. For this reaction to be successful, the enzyme must be chemo selective enough to avoid reduction of the carbonyl to the corresponding alcohol. Scheme 7: Asymmetric synthesis of optically pure amines using imine reductases. In 2010, Mitsukura’s group discovered two different stereoselective IREDs in filamentous bacteria. Streptomyces species GF3587 and 3546 exhibited a highly enantioselective reduction of 2-methyl-1-pyrroline to 2-methyl-1-pyrrolidine, yielding ~99.2% e.e (R) and 99.3% e.e (S) enantiomers with excellent conversion .25 Since then IREDs have become highly recognised and now it is used in the industrial synthesis of suvorexant (a drug used for the treatment of insomnia).26 Although this reaction has excellent stereoselectivity, it also has limited substrate specificity as the reductive amination of aldehydes with amines has proven difficult. It is clear that enzymatic reduction occurs when prochiral imines are formed, however, there has been 12 little evidence for the role of enzymes in imine formation. Understanding the substrate specificity of IRED requires further understanding of the overall mechanistic reaction. 1.1.4.4 The use of amine dehydrogenase (AmDHs) for the synthesis of enantiopure amines. Amine dehydrogenases catalyse the stereoselective reductive amination of ketones and aldehydes to enantiopure chiral amines. AmDHs allow the asymmetric synthesis of chiral amines, initializing from a prochiral substrate (Scheme 8). This reaction is different from other NADPH- dependent enzymes (such as IRED) in the sense that it accepts ammonia as an amino donor, however, other primary amine donors such as methylamine, ethylamine, and cyclopropyl amines and have been reported when operating under high concentrations.27 Mayol’s group successfully catalysed the stereoselective synthesis of (S)-4-aminopentanoic acid from the corresponding ketone substrate using the AmDHs enzyme derived from the Clostridium sticklandii strain (Scheme 8).28 Only a single enantiomer was obtained (99.5% e.e) with an excellent 90% conversion. AmDHs are extremely enantioselective thus there are no additional racemization or DKR step required. The problem with this reaction is that AmDHs have a limited substrate scope and are only often involved in the formation of primary amines. More amino donors need to be discovered for the expansion of the substrate range in order to allow the synthesis of secondary and tertiary amines, and further engineering still needs to be investigated to improve the substrate specificity of the enzyme. Scheme 8: Example of AmDHs enzyme catalysed reaction.27 Types of transaminases reaction 13 1.1.5 Kinetic resolution transaminase reaction KR transaminase reactions involve the resolution of a racemic mixture of amines to generate one enantiomer. In this case, a single enantiomer of a racemic mixture donates its amino group to form a corresponding ketone and the other enantiomer is then isolated from the reaction mixture (Scheme 9). Pyruvate is the most commonly used amino acceptor for this reaction, and only a half equivalence is required for the resolution of a single enantiomer, this behaviour favours the forward reaction thus making the KR reaction more thermodynamically favourable compared to the asymmetric synthesis. As in any other KR step, the maximum yield that can be obtained is 50%. To overcome this limitation, ATAs are typically coupled to oxidizing enzymes such as amine oxidases to facilitate DKR or the alanine by-product is deracemized in a single pot reaction. Rozzell and Turner’s groups demonstrated an efficient chiral resolution method utilizing ATAs in tandem with amino acid oxidases, and a product in 99.99% e.e with a substrate conversion of 99% was obtained.32 Scheme 9: ATA kinetic resolution coupled with amine oxidases.32 Kroutil’s group also applied ATAs to kinetic resolution in the synthesis of mexiletine, a drug used for the treatment of cardiac diseases and muscle stiffness.33 They successfully isolated 99% yield of a single enantiomer using a single pot reaction which consists of two different transaminase reactions (Scheme 10). The first step involves the deamination of a single enantiomer (R–enantiomer in Scheme 10) to a ketone intermediate, this was followed by the recycling of pyruvate by amino acid oxidases which convert alanine back to pyruvate in the presence of oxygen. R-ATAs are then deactivated by heat followed by the addition of a second stereoselective ATAs to afford the conversion of the ketone intermediate to the amine, so using this step both R and S enantiomers can be isolated in a good yield. KR using ATA is only 14 efficient if the racemic starting material is inexpensive, when dealing with an expensive racemic compound asymmetric synthesis of amines using ATAs can be employed. Scheme 10: The application of sequential stereoselective ATA catalysed reactions to synthesise mexiletine.33 1.3.2 Asymmetric synthesis of chiral amines using ATAs. ATAs together with AmDHs have emerged as important tools in biocatalysis because they are both capable of catalyzing direct conversion of a prochiral carbonyl group into an optically pure amine.18 These two enzymatic routes have several advantages compared to other routes (routes that include KR and DKR steps), the most obvious advantage of this route is initializing the reaction with the cost-effective starting material (prochiral ketone) which is capable of reducing the overall cost of the entire synthetic route. Asymmetric synthesis often results in a 100% theoretical yield and also has less waste generation, these properties are highly desired in both pharmaceutical and agrochemical manufacturing companies.8 AmDHs rely on nicotinamide cofactors (NAD(P)+ /NAD(P)H) for the reaction to take place, however, these cofactors are expensive to use in vitro. To solve this problem whole cells can be used to eliminate the need to supply the expensive co-factor. Although these enzymes are extremely stereoselective, their application is not necessarily economically attractive because it involves high process costs. ATAs on the contrary do not belong to the family of nicotinamide–dependent enzymes, they use PLP co-factors that regenerate themselves during the synthetic reaction. The application of asymmetric transaminase reaction has only gained much attention after 20 centuries their discovery, primarily due to constraints such as unfavourable equilibrium, enzyme instability, and limited substrate specificity profile.34 The first asymmetric synthesis of amines was demonstrated by Shim and Kim, they used ATAs originating from Vibrio fluvialis to convert acetophenone to (S)-α-methyl benzylamine using L-alanine amino donor 15 (Scheme 11).35 To overcome the thermodynamic bias towards the starting material they used an excessive amount of amino donors and removed the pyruvate co-product by converting it to lactate to afford 90% yield of the product with 99 % e.e (Scheme 11).35 Scheme 11: The conversion of acetophenone to (S)-α-methyl benzylamine reaction performed by Shin and Kim.30 In recent years, asymmetric synthesis of achiral amines is widely used in both academia and industries to synthesize a different range of pharmaceutically active compounds or intermediates. Researchers from Merck and Codexis did several rounds of directed evolution and structurally guided protein engineering to form an R-selective ωTAms variant derived from Arthrobacter sp, this was applied for the asymmetric synthesis of sitagliptin (an anti-diabetic drug) from prositagliptin.7,29 Most notably, the use of ATAs to biosynthesize the blockbuster sitagliptin was shown to be more economically competitive than using a traditional chemical method, i.e. a chiral ruthenium-based catalyst to reductively aminate the ketone intermediate. The same mutant Arthmut11-ATA which allows bulky substrates was later applied for the asymmetric synthesis of the intermediate of ramastroban, which is a thromboxane receptor antagonist currently used for the treatment of asthma and coronary disease.36 Rivastigmine (Fig. 5), the most potent drug used in the treatment of early stages of Alzheimer’s disease as well as Parkinson’s disease was synthesized using ATA (Vfl-ATA) to form enantiopure chiral amine in 71% yield and 99% e.e.37 Mutti and colleagues also published a paper using different enzyme mutants to form amine products, a few of these products formed shows potential in API precursor production.37 This includes the production of Dilevalol (a hypertension drug) and formoterol (β-2 agonist). All of the examples mentioned above emphasize the value of biocatalysis and how pharmaceuticals are continuously moving toward a greener and a sustainable approach to the synthesis of APIs. 16 Figure 5: Biocatalytic application of ATAs for the synthesis of pharmaceutically active compounds. General developments and challenges of asymmetric ATAs reaction 1.1.6 Expansion of substrate scope and a choice of amino donor Over the years, significant improvements have been made to overcome the previously mentioned limitations (section 1.3.2) of ATAs in asymmetric synthesis. Through different enzyme engineering and evolution techniques, the enzyme variants that allow bulky substrates have been identified (i.e. Arthrobacter sp for the synthesis of Sitagliptin), which resulted in the 17 expansion of the substrate scope. Transamination can now be carried out in a wide range of substrates varying in size and constituents. Another major constraint was the unfavourable equilibrium due to the reversible nature of the reaction. To achieve a good conversion, the carbonyl co-product should be removed to prevent the reverse reaction. Shin and Kim investigated the transamination reaction and indicated that the choice of the amine donor and amine acceptor together with the possible product and coproduct is crucial in an ATA-catalysed reaction.35 In most circumstances, the equilibrium was far on the reactant side therefore there was a major need for equilibrium displacement.14 A natural amino acid, alanine has proven to be the most commonly used amine donor in ATA- catalysed reactions, however using it results in an unfavourable equilibrium (only a small amount of the desired amine products is formed, often less than 5%).14 Increasing the quantity of the alanine amine donor also did not lead to any improvement in the yield for most substrates; however, the was an exception when aminating the 4-methoxyphenyl acetone into a desired amine, 94% conversion was isolated with 16–fold alanine amino donor.11 The equilibrium can be displaced when using alanine by removing the pyruvate co-product. Lactate dehydrogenase is usually coupled in the reaction in order to convert the pyruvate by-product into lactic acid in the presence of NADH (nicotinamide adenine dinucleotide).11 This reaction requires an additional regeneration enzyme to recycle NADH and in most cases, glucose dehydrogenase and glucose are usually used.6 The are many other reactions that can be performed in tandem with the ATAs reaction to remove the pyruvate, however, it is clear that they are many biological compounds associated with this reaction thus using alanine as an amino donor in the ATAs reaction presents a relatively expensive synthetic route. This has driven a search for other suitable amino donors. Isopropyl amine (IPA) is the most convenient and promising amino donor, it is readily obtainable and cost-effective and the co-product acetone can be easily removed to shift the equilibrium.30,38 From an economic point of view, IPA is the most preferred amino donor in industries for ATA-mediated reactions (i.e. biosynthesis of sitagliptin).7,29 Although IPA performs better than alanine, an excessive amount is still required to drive the forward reaction to completion. 1.4.2 Continuous flow biotransformation Substrate insolubility and enzyme instability are one of the challenges that were causing a major restriction on the application of ATAs in asymmetric reductive amination reactions. This problem can be improved by incorporating ATAs catalysed reactions into optimized flow 18 chemistry.39,40 The application of this technique is not a recent development; however, only limited examples regarding its applications on ATAs have been published. Flow chemistry is rapidly developing in both industry and academia and it was proposed that this technique has the ability to improve half of biocatalytic processes.39,40 Continuous flow chemistry is mostly used in large-scale production, the system can be run for longer periods to increase productivity.44 Another advantage of continuous flow biotransformation is that it allows for substrates with low solubility and it is typically more reproducible and economically attractive compared to the comparable batch processes.39,41 Enzyme immobilization is often a vital component for successful flow chemistry, although enzymes can be used on their own, attachment of a cell to a protein structure is always advantageous. Furthermore, some enzymes exist in small quantities, and attachment of the enzyme to a reactor reduces the quantities required for processing.40 Enzyme immobilization has been shown to improve the selectivity, stability of the enzymes and reaction rates. During the process, the enzyme is retained and this behaviour results in the simplification of the purification process.39 Many different immobilization techniques have been applied to the ATAs flow chemistry reactions. Kroutil et al. applied continuous flow chemistry by first immobilizing an enzyme using methacrylate polymeric resin (Fig. 6).40 The immobilized enzyme was then loaded into a packed bed –reactor and the necessary reactant (transferred through syringes) were passed through the reactor to allow continuous flow asymmetric amination of ketone substrate, this resulted in an excellent conversion (70-94%) and greater than 99% e.e .40 Figure 6: Immobilization of E. coli cells and PLP in methacrylate polymeric resin followed by flow chemistry transformation40 Major improvements have been made in transaminase biocatalysis, and now the use of enzymes in pharmaceutical industries is expanding. Enzymes are easily accessible, and they are produced from inexpensive renewable resources. The application of biocatalysis in industries 19 does not only reduce the cost of production but also shifts the synthesis into a greener approach and sustainable industrial processes. Chiral amino alcohols Chiral amino alcohols are alcohol and amine–containing class of compounds with diverse applications, abundantly featured in many natural products, pharmaceuticals, and other bioactive molecules. These compounds can be derived from natural molecules, particularly amino acids, and they are widely involved in the synthesis of several bioactive compounds. Chiral amino alcohols also serve as a cost–effective precursors for the synthesis of organic and inorganic chiral ligands and chiral auxiliaries.411,2-Amino alcohol and 1,3-amino alcohol motifs, which are the primary focus of this project, are present in pharmaceutical active compounds.42 The are several amino-alcohol-containing drugs on the market, this includes ethambutol, raltegravir, embamide, tembamide, dolutegravir, arbutamine, and antibiotics such as lincomycin and levomycetin37 (Fig. 7). Figure 7: Amino alcohol-containing drugs. In this study, we focus on two of these active pharmaceutical ingredients (APIs), ethambutol and dolutegravir. The reason why both APIs are included here is that we plan to use one key enabling chemistry that will be applicable to both APIs. The key step in the preparation of both enantiopure amino-alcohols (2-(S)-aminobutan-1-ol for ethambutol and 3-(R)-aminobutan-1- 20 ol for dolutegravir) is the conversion of cheap achiral starting material into enantiopure amino- alcohols using transaminase enzymes. 1.1.7 Ethambutol Ethambutol is listed as an essential World Health Organization (WHO) drug for use in combination drug therapy against tuberculosis (TB), which is considered the most infectious disease worldwide, resulting in the most human fatalities annually.43 Although ethambutol was discovered more than 50 years ago44, many decades of synthesizing and screening libraries of similar compounds have not led to the discovery of new compounds with improved potency and pharmacokinetic properties.45 Therefore, ethambutol remains a mainstay of TB treatment to this day and is on South Africa’s essential medicines list. Ethambutol plays a crucial role in lowering the incidence of the development of mycobacterial resistance against the other treatments (isoniazid, rifampin and pyrazinamide) which are used in combination with ethambutol in standard TB treatment.46 (S)-2-Amino butanoic acid 3 is the most important chiral building block for the chemical synthesis of ethambutol 1. According to Wilkinson and his colleagues, the (S, S)-configured diastereomer of ethambutol is the most effective agent against almost all the strains of mycobacterium.46–48 It has the highest biological activity compared to other derivatives and it is approximately 500 times more potent than the (R, R)-diastereomer.47,48 Scheme 12: Facile synthesis of Ethambutol from unnatural amino acid 3 Since the discovery of ethambutol in the 1960s, (S)-2-amino-1-butanol 4 has been used as a main precursor for its industrial-scale production.29 This amino alcohol can be prepared from amino butanoic acid or its ester 3. (S)-2-Amino butanoic acid can be easily reduced into amino- alcohol 4, followed by a reaction with 1,2-dichloroethane resulting in desired enantiomer of ethambutol 1 (Scheme 12). Since the synthesis initializes with an unnatural amino acid 3, this represents a relatively expensive starting material. Our aim is to reduce the cost of the starting material by using transaminase biocatalysis for its preparation, thus making the route more economically attractive. 21 1.1.7.1 How will it improve the manufacturing process? This asymmetric synthetic route would have several advantages over the existing industrial synthesis of ethambutol. A well-known, industrial synthesis of ethambutol contains a critical chiral resolution step of amino alcohol 4, by first converting the intermediate racemic (50/50 R and S) mixture of 4 into the corresponding (+)-tartrate salts. The required 2-(S) salt of 4 is selectively precipitated and only this component can be used subsequently. This creates waste, as more than 50% of the racemic compound 4 (the R component) is effectively discarded. It also results in a low yield, as only a maximum yield of 50% of the desired (S)-4 can be obtained in this way. 1.1.7.2 Tuberculosis disease and its prevalence. Tuberculosis (TB) is an infectious disease caused by a bacterium known as Mycobacterium tuberculosis.49 TB primarily targets the respiratory system; however, it can also spread to other parts of the body, including muscles and bones.50 The disease is transmitted when an infected individual expels the bacteria into the air through actions like sneezing or coughing, and another person inhales these airborne particles. Although this bacterium was discovered more than a century ago, it still remains the major cause of morbidity and mortality worldwide.51 According to the World Health Organization (WHO), TB claimed the lives of 1.6 million people in 2021, ranking it as the 13th leading cause of death and the second most prevalent infectious disease after COVID-19.52 South Africa continues to be the epicentre of the TB epidemic (172,200 TB cases were reported in 2021) and this nation still faces challenges in managing the treatment process.53 These challenges are exacerbated by human resource shortages and the disruptions from the impact of the COVID-19 pandemic.53 TB is curable and preventable. The current treatment regimen involves using first-line anti-TB drugs for new TB cases, and second-line anti-TB drugs are employed for cases of multi-drug resistance. Ethambutol, pyrazinamide, isoniazid, and rifampin are combination drug regimens that are used to treat TB bacterial infection in the initial intensive phase of therapy (Fig.8). 22 Figure 8: First-line Anti-TB drug combination. 1.5.2 Dolutegravir Dolutegravir is a potent, new-generation integrase strand transfer inhibitor drug against HIV that was fast-tracked for approval by the FDA in 2013.54 In June 2020, it was approved for the treatment of children born with HIV, from as young as 4 weeks old.45 As of the end of 2019, dolutegravir is one of the drugs recommended for first-line antiretroviral treatment (ART) for treatment-naïve individuals in South Africa.54 It is also recommended that people experiencing side effects with other treatment regimens switch to the new regimen of dolutegravir, tenofovir disoproxil fumarate and lamivudine, known as TLD. This is a fixed-dose combination including one integrase inhibitor (dolutegravir) and two NRTIs. With about 4.8 million people currently on antiretroviral treatment in South Africa,54 local demand for antiretroviral drugs such as dolutegravir is high, and dolutegravir is considered an essential medicine in South Africa. The new regimen that includes dolutegravir has a number of advantages, including the fact that it has fewer interactions with other medicines, such as those used to treat tuberculosis, which is a common co-morbidity in South Africa.43 The most efficient industrial synthesis of dolutegravir is dependent on the economic access to chiral amine building block (R)-3-amino-1-butanol (5) (see step 8 in red, Scheme 13). This is quite an expensive building block because it cannot be prepared from naturally occurring amino acids. Our aim is to develop an improved and convenient selective route for the synthesis of 3- aminobutanol using economical, cheap, and easily available starting materials. 23 Scheme 13: The most economical industrial synthesis of dolutegravir developed to date.55 Based on a materials requirement calculation for the route shown in Scheme 13, for every kilogram of dolutegravir produced, 463 g of (R)-3-aminobutan-1-ol (5) are required.55Thus, a cost reduction in this key building block will lead to a cost reduction in the reported synthesis. We plan to demonstrate the cost-effective preparation of 5 from an achiral ketone using transaminase. 1.1.7.3 1.5.2.1 Human Immunodeficiency Virus and its prevalence. Human Immunodeficiency Virus (HIV) is a pathogen that specifically targets and affects the individual’s CD4 T lymphocytes, resulting in a compromised immune system. This weakened immune system makes the body susceptible to other infectious diseases, including tuberculosis (TB). If left untreated, HIV infection can progress to an extremely serious condition known as Acquired Immune Deficiency Syndrome (AIDS). HIV has evolved into one of the most critical global public health challenges since it was initially reported in 1981.56 Since the beginning of this epidemic, over 40.4 million lives were tragically lost.57 Currently, there are approximately 39 million individuals living with HIV and a significant majority of them, roughly two-thirds 24 (25.6 million) reside in African countries.56 Over the past two decades, significant global efforts have been made to combat the HIV epidemic, and this resulted in a substantial progress on the number of infected individuals. For instance, in South Africa, there has been a notable decline in the number of new infections. In 2010, there were 401,608 new infections, whereas in 2021, that number decreased to 198,311, marking a 51% reduction (Fig.9).53 Figure 9:The population of new HIV infections in South Africa categorized by sex and gender from 2010 to 2021.53. The decline in HIV cases can primarily be attributed to improved HIV awareness and prevention efforts, as well as enhanced HIV treatment programs. Since the development of antiretroviral therapy, there has been a notable decline in the mortality rates among HIV- infected patients. Antiretroviral therapy is categorized into six distinct classes based on the drug's molecular mechanism and resistance profiles.57 The recommended optimal initial regimens for most patients typically include a combination of two nucleoside reverse transcriptase inhibitors (NRTIs) along with an integrase strand transfer inhibitor (InSTIs).58 InSTIs represent the newest class of antiretroviral drugs and they function by blocking the viral intergrase enzymes which play a major role in the insertion of viral’s DNA into DNA of a host’s T-lymphocytes.59 There are currently four FDA-approved InSTI drugs, these include the first-generation raltegravir and elvitegravir, as well as the second-generation dolutegravir and 25 bictegravir drugs.60 Dolutegravir is mostly preferred in first-line HIV therapy, and it is used in combination with either abacavir-lamivudine or tenofovir-emtricitabine NRTIs.60 Aims and Objectives 1.6.1 Aims The aim of this project is to use transaminase enzymes to convert cheap achiral ketone starting materials into high value enantiopure amino alcohols required as key building blocks in the synthesis of dolutegravir and ethambutol. 1.6.2 Objectives • Introduction of new keto ester enzyme substrates with improved enzyme- substrate binding properties to improve enzyme efficiency. • The screening of the substrates with different ω-Transaminases to discover new active enzymes with greater activity and stereoselectivity. • Identification of stereoselective enzymes from a given library of enzymes. • Biocatalytic synthesis of chiral amino alcohols for the industrial synthesis of ethambutol and dolutegravir. 26 Chapter 2: 2. Synthesis of an Ethambutol intermediate Ethambutol 1 is manufactured on an industrial scale through the process outlined in Scheme 14.61,62 During the process, nitropropane undergoes oxymethylation with formaldehyde resulting in 2-nitrobutanol which is subsequently reduced to form racemic amino butanol (Scheme14).61 The desired (S)-amino butanol is resolved using tartaric acid followed by the reaction with 1,2-dichloroethane to form (S,S)ethambutol 1. When assessing the overall expenses of the entire reaction scheme, it is evident that steps 1 and 2 account for approximately 75% of the total reaction costs (Table 8). Furthermore, only 50% of the desired enantiomer can be theoretically obtained, so this scheme is expensive and generates a substantial amount of waste. Due to unattractive environmental and economic consequences associated with a significant amount of side products in an industrial process, it is evident that there is a pressing need for the development of new efficient and sustainable synthetic routes to (S)-2-amino alcohol intermediate. Scheme 14: Industrial synthesis of Ethambutol 1 .61,62 27 In this chapter, we are exploring an alternative greener and enzymatic approach to the synthesis of (S)-2-amino butanol 4 intermediate. In this case, we entail to use transaminase enzymes to directly convert an affordable prochiral ketone 7 into (S)-amino ester 8, which can be subsequently reduced to the desired (S)-2-amino butanol 4(Scheme 15). This schematic route has the potential of decreasing the costs of synthesis of the intermediate which can decrease the overall cost of the production of the drug. As mentioned previously, South Africa still experience challenges with the TB-treatment, with one of the prominent obstacles being the high cost of medications. Reducing the cost of this drug could enhance affordability and potentially stimulate price-based competition within the local pharmaceutical industries. Scheme 15: Our proposed synthesis route to produce Ethambutol 1 Substrate selection Transaminases allow the biotransformation of carbonyl groups (ketones, aldehydes, and keto acids) into the corresponding amine. There are various aspects that have to be taken into consideration before choosing an ATAs substrate. This includes the solubility of reactants in water, the extraction of the amine product from the aqueous media and the binding interaction between the enzyme and the substrate. ATAs catalysis often requires organic co-solvents to enhance the solubility of reactants in aqueous media; however, the utilization of high concentrations of water-miscible organic solvents decreases the stability of ATAs enzymes. To prevent this the substrate should be soluble enough in an aqueous solvent in order to allow catalysis to take place. 28 Extraction of organic compounds is usually achieved by using the liquid-liquid extraction method, which is the distribution of the product between the aqueous and organic layers. In this case, the extraction efficiency is strongly dependent on the chemical structure and dissociation state of the amine or amino alcohol. Distribution of amines in an organic layer can only be achieved by changing the dissociation state of the amine through the adjustment of the pH using a specific base. Amino-alcohol has two polar groups, the amino group, and the alcohol group, that can undergo hydrogen bonding with the water molecule thus forming strong hydrogen bond interactions. The H-bonding properties of these types of compounds make the extraction difficult; this becomes a problem because it can result in a dramatic loss of product through poor recovery. To combat this issue, the substrate should be less polar enough in order to achieve a less polar product that can be easily extractable compared to amino alcohol. Previous structural studies have revealed that there are two pockets (P and O) in the active side of the ω-TAms (Fig.10). The P-pocket is located near the phosphate group of PLP, whereas the O-pocket is positioned in the proximity of the O3′ atom on PLP.63 The two pockets differ from each other in size, depending on the class of ω-TAms. The docking study that was done by Zhang and his colleagues on keto butyric acid indicates that the small pocket displays steric constraints, prohibiting the substituent with a larger group from binding while the larger pocket can accommodate the bulkier substituents (Fig. 10).64 The choice of the ATAs substrate is highly dependent on the enzyme's active site, the substrate should have small constituents (ethyl and methyl groups are recommended) on one end to fit in the small pocket of the active site, and it should also have a larger constituent at the other end to fit in the larger pocket of the active side. Figure 10: Docking of keto butyric acid in the ATA active site.64 29 In this study, the desired (S)-amino butanol 4 can be accessed by the transaminase biotransformation on pro-chiral hydroxyl ketone 9 or alpha-ketoseter (Scheme 16). The hydroxyl ketone (9) is a commercially available compound that provides the most facile single- step route for the synthesis of enantiopure amino alcohol 4. Hydroxyl ketones are quite soluble in water; therefore, no additional solvents like DMSO are required. Although this substrate allows a direct conversion into the desired product, amino alcohols are often difficult to extract from the aqueous layer. Furthermore, hydroxyl ketone 9 is relatively expensive (a gram of it costs R2472 from Sigma Aldrich), therefore using it is inconvenient in this project as the main objective is to decrease the cost of the production of ethambutol 1. The starting material, alpha keto esters are often cheap renewable compounds that can undergo biotransformation into amino esters 3 which are easily extractable in the aqueous media (Scheme 16). In this study, we introduce a cheap novel ATAs substrates (ethyl keto ester and isopropyl ketoesters) with the aim of decreasing the costs of synthesis of the high-valued (S)-amino alcohol 4 intermediate. Scheme 16: Different routes for biotransformation of pro-chiral ketones into amines using ATAs. Substrate synthesis Initially, there were two different substrates of interest in this part of the project, ethyl keto ester 10 and isopropyl keto ester 11 (Scheme 16). Our substrates were not commercially available; hence they were chemically synthesized in the lab. The Grignard reaction, which is 30 most commonly used in pharmaceuticals for the introduction of new-carbon-carbon bonding, was utilized. The starting material for ethyl keto ester substrate 10, diethyl oxalate, is a cheap, and commercially available compound. The diethyl oxalate was reacted with the Grignard reagent that was successfully synthesized using magnesium turnings and bromoethane (Scheme 17). The reaction was controlled at a low temperature (0°C) under nitrogen gas for 2 hours (Scheme 17). No over-substitution of an ester (to form 3, 4-hexadione) was observed, and only residual starting material (which can be recycled) and the desired product were isolated. The purification was achieved using silica-column chromatography and by using ethyl acetate- hexane mobile phase, only 15 % of the product was isolated. Substrate 10 is very volatile, so most of the product was lost during the solvent removal step. To avoid this issue, purification by distillation was attempted, however, it was unsuccessful as the product carried over together with the starting material. The purification solvent was then changed to 1% ether – hexane and only slight changes were observed as the yield increased from 15 to 25%. The product was characterised using a proton and a carbon NMR .1H NMR spectrum shows a peak integrating 2H at chemical shift of 2.80 ppm indicating a newly formed ethyl group adjacent to the carbonyl group whilst the deshielded ester CH2 group was observed at 4.22 ppm. The 13C NMR spectrum confirmed that the is no longer a symmetry in the compound as 6 carbon peaks were observed. Scheme17: Grignard reaction to prepare ATA substrate 10. In contrast to the synthesis of substrate 10, diisopropyl oxalate 12, is not commercially available. However, it can be readily prepared by performing a simple nucleophilic substitution reaction between oxalyl chloride and isopropanol in the presence of a base (Scheme 18). This method resulted in 67% yield of product 12, a 1H NMR spectrum shows a doublet peak at 1.37 ppm integrating for 12 protons (all the methyl groups are in the same chemical environment), and a heptet peak at 5.16 ppm with a coupling constant of 6.5 Hz for protons adjacent to methyl groups. Although the reaction was successful, most of the product was lost during the solvent 31 and pyridine removal. To solve this problem, Pyridine was replaced with DIPEA, and a more volatile solvent was used (DCM). With the new conditions, the reaction time was reduced to 5 hours and the yield was improved to 80%. Scheme 18: The synthesis of diisopropyl oxalate 12 Diisopropyl oxalate 12 undergone a Grignard reaction following the same procedure as in substrate 10 (Scheme 19). The same extraction procedure was used and the purification was modified by adding 1 % of toluene in the mobile phase (1-3% ether hexane). The product was isolated in the yield of 50% yield. The 1H NMR spectrum shows a quartet peak at 2.8 ppm with the coupling constant of 7.2 Hz representing the newly formed CH2 adjacent to the methyl and the carbonyl group. The 13C NMR spectrum also confirmed that the is no longer a symmetry in the compound as 6 carbon peaks were observed. Scheme 19: Grignard reaction to prepare ATA substrate 11. Grignard reaction resulted in an incomplete conversion of the starting material to the product and there were various challenges encountered during the purification procedure, this led to the exploration of different techniques to synthesis the substrates. The simple Fischer esterification reaction was investigated using a keto butyric acid starting material, this starting material is commercially available and in the presence of an alcohol and acid catalyst, it results in the formation of an ester (Scheme 20). In this reaction, no purification procedure was required, and the yield of the ethyl substrate 10 ranged from 79-82% whilst the isopropyl substrate 11 ranged from 80-95%. 32 Scheme 20: Fisher esterification for the synthesis of ATA substrates. Enzyme screening 2.1.1 Enzyme Screening Method Development In 2014, the Turner group at the Manchester Institute of Biotechnology published a very useful ATA screening method that can be applied to test for ATA enzyme activity on a specific ketone substrate, within any library of given ATA enzyme variants.65 The method entails using a smart donor amine that, upon substrate conversion to the desired amine product, will result in an easily detectable colour change, from colourless/yellowish to dark purple-black. Our group at Wits has acquired an extensive library of more than 30 ATA enzymes from UK enzyme producers Prozomix in Northumberland. Previous research in our lab applied the Turner method to screen for ATA enzyme activity on substrate 10, and it was found that almost all of the enzymes in our library were active in producing the desired amine product to varying degrees. The presence of the product was also confirmed by semi-quantitative HPLC-high- resolution mass spectrometry. These results motivated us to investigate this reaction further using the same substrate 10 and an additional substrate 11, which is a novel ATA substrate. Although the use of smart donors has proved to displace challenging reaction equilibria, applying this procedure in large-scale reactions can be a problem as it may lead to the introduction of many side reactions (diamine smart donors often result in amine side product, and amines are very reactive thus susceptible to side reactions).65 In this project, the isopropyl amino donor was utilized because it has proven to be convenient in large-scale transamination (isopropylamine is used as an amine donor in the production of sitagliptin ).7,29 Enzyme activity is broadly dependent on the defined conditions such as temperature, pH and the nature and strength of the ions, therefore the enzyme activity can only be accurately compared if such conditions are standardised. For our screening method development, we began by using pH 7.5 and a temperature of 30°C, these are typical optimal conditions that are used for enzymatic reactions. Monophosphate buffer (100 mM) was used to stabilize the pH; the more concentrated a buffer is, the higher the capacity to stabilize the pH. However, ATAs accept moderate ionic strength between 0.005 M to 0.1 M (only halophilic and thermophilic 33 enzymes prefer high concentrations). In this method, there is no observable colour change expected as in when using a diamine donor in the Turner method and the desired amine products are also UV inactive, so monitoring the reaction as time progresses is quite challenging.65 In our initial screening procedure, the reactions were left stirring (250 rpm) for a maximum of 24 hours to ensure completion, this is the duration that is normally used in the literature. Scheme 21 shows the route and the initial conditions that were used for our screening process. Scheme 21: Proposed ATA biotransformation towards the synthesis of (S)-amino butanol 2.1.2 Method development for product isolation and quantification Developing a method for quantitative and qualitative analysis of the enzyme amine products requires standard compounds, However, both, ethyl 2-aminobutanoate and isopropyl 2- aminobutanoate (whether enantio-enriched or racemates) are not commercially available. These standards were therefore prepared using a non-biocatalytic synthetic approach. Scheme 22 shows all attempted reductive amination reactions for the synthesis of rac-14, unfortunately all the reactions didn’t work and only traces of the product rac-14 was observed in proton NMR and mass spectrum. javascript: javascript: javascript: 34 Scheme 22: Reductive amination reaction to synthesis the racemic amino ester 14 standard These unsuccessful reactions emphasise how difficult the reductive amination is and how the emergence of the ATA biocatalysis reaction can overcome this. This synthesis was only essential for method development and validation, therefore we compromised and decided to use some expensive commercially available (S) and (rac)-aminobutyric acid 15 to synthesis our standards. Simple acyl halide reaction was carried out in the presence of isopropanol to afford yellow oil product with an excellent yield of 78-86% for rac-14 and (S)-14 (Scheme23). The products were characterised using 1H and 13C NMR. As expected, the same NMR pattern was observed in both racemate and (S)-enantiomer .1H NMR spectrum shows a newly formed doublet of doublet peak with a coupling constant of 6.1 and 3.4 Hz integrating for six protons, these indicate the presence of two methyl groups that are in the same chemical environment. The proton adjacent to the methyl group was also observed as a multiplet at 5.11-5.00 ppm. These results indicate that the reaction was a success, and it was further confirmed by the presence of an additional methyl carbon at 20.9 ppm in 13C NMR spectrum. The ethyl 2-aminobutanoate rac-16 and (S)-16 standard were also successfully synthesised to afford a yellow oil product with the yield of 68-85 % and the product was also characterised using 1H and 13C NMR (Scheme 23). The 1H NMR spectrum shows a new quartet peak (J=6.5Hz) at 4.18 ppm indicating the presence of CH2 adjacent to the methyl group, the newly formed methyl group was also observed as a triplet (J=7.5 Hz) at 1.28 ppm. This serves as a confirmation of successful esterification formation. javascript: 35 Scheme 23: The synthesis of amino-ester standards using nucleophilic reaction. Our desired amine products are very small compounds and not UV active, the most convenient characterization technique that can be utilized in this project is gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Using the standard amine 14, we were able to develop a LC-MS reverse phase gradient method (5-95% H2O /ACN), retention time: 15 minutes, flow rate: 0.300 ml/min). The standard curve (Fig.11) was then developed using different concentrations of the standard amine (S)-14. Additionally, the excellent R2 value indicates that there is a perfect correlation between the variables thus the concentration of the enzyme product can be quantified accurately. 36 Figure 11: The standard curve of concentration of amino ester versus peak area by LCMS (number of variables, (n)= 8). 2.1.3 Enzyme screening results The ATA enzymes were screened using assays in order to discover their substrate specificity profile and their ability to form interesting stereo-selective amines. Qualitative and quantitative analysis of enzyme amine products are often difficult to achieve when working in a small scale. As a result, most ATA reactions are monitored via the detection of the ketone starting material. However, in this study, our keto substrates are quite unstable during analysis by LCMS, thus making them difficult to monitor, therefore the enzyme reaction progress was monitored using the amine products. Initially, 28 enzymes were selectively screened against substrate 10 and substrate 11 using the conditions illustrated in Scheme 21. The products were processed and extracted with HPLC- grade acetonitrile and directly injected into the LCMS vials for characterization. Fig. 12 shows a comparison between the amino-ester standard 14 and the enzyme product 14 LCMS results. Two peaks eluting at a retention time of 3.5 min ( [𝑀 + 𝐻]+ =146.1164 and [𝑀 + 𝐻]+ =104.0708 ) and 8.9 min( [𝑀 + 𝐻]+ =146.1164 and [𝑀 + 𝐻]+ =104.0708 ),slightly shifted in the standard compound were observed .The product existing in two different forms, the ionic and the neutral form, thus separating when using the HILIC column. Fragment [𝑀 + 𝐻]+=146.1164 represent the full mass of our desired product and [𝑀 + 𝐻]+=104.0708 corresponds to our product without the isopropyl ester group. From these results, we can y = 115286x R² = 0.9955 -2000000 -1000000 0 1000000 2000000 3000000 4000000 5000000 6000000 7000000 -20 -10 0 10 20 30 40 50 60 P ea k A re a Concentration ( PPM) 37 deduce that ATA-230 successfully catalysed the synthesis of amino ester 14 from a pro-chiral ketone 11. Figure 12: Comparison between LCMS results of ATA-230 and the standard amino ester products. From the LCMS data obtained, the peak of the neutral amine product 11 (8.9 min) was selected, it was then integrated, and the peak area of each enzyme product was compared. The graph in Figure 13 illustrates the comparison of the peak area of the amine product catalysed by different enzymes on substrate 10. Sixteen enzymes were active against our substrate and ATA-231, ATA-250 and ATA-247 showed the greatest activity while the activity of others, for example ATA-241 and ATA-239, were insignificant. Standard amino ester 14 ATA-230 amino-ester product 14 PRODUCT a b a b 38 Figure 13: A graph illustrating the comparison between the activities of the enzymes on substrate 10. Moving to substrate 11, only few enzymes from the selected assay were active in comparison to substrate 10, however, the activity was more than 100-fold greater than substrate 10 (Fig. 14). An in-depth investigation of the comparison of these two substrates is beyond the scope of this project; however, from the results presented in the graphs, it is clear that substrate 11 gives better activity than substrate 10. Due to this reason, we decided to eliminate substrate 10 and further investigate substrate 11. Figure 14: A graph illustrating the comparison between the activities of the enzymes on substrate 11. 0.0 20000.0 40000.0 60000.0 80000.0 100000.0 120000.0 140000.0 160000.0 2 3 0 2 3 1 2 3 2 2 3 3 2 3 4 2 3 5 2 3 6 2 3 7 2 3 8 2 3 9 2 4 0 2 4 1 2 4 2 2 4 3 2 4 4 2 4 5 2 4 6 2 4 7 2 4 8 2 4 9 2 5 0 2 5 1 2 5 2 2 5 3 2 5 4 2 5 5 2 6 1 2 6 2 am in e p ea k ar ea enzyme catalogue number 0 2000000 4000000 6000000 8000000 10000000 12000000 14000000 16000000 18000000 2 3 0 2 3 1 2 3 2 2 3 3 2 3 4 2 3 5 2 3 6 2 3 7 2 3 8 2 3 9 2 4 0 2 4 1 2 4 2 2 4 3 2 4 4 2 4 5 2 4 6 2 4 7 2 4 8 2 4 9 2 5 0 2 5 1 2 5 2 2 5 3 2 5 4 2 5 5 2 6 1 2 6 2 p ea k ar ea o f th e am in e enzyme catalogue number 39 2.1.4 Development of HPLC method for separation of R and S enantiomers. Determination of the presence of chiral compounds using chiral HPLC is becoming an essential tool in organic chemistry. This analytical technique makes it possible to efficiently obtain target enantiomers in high enantiomeric purities. Therefore, many of the detection methods have already been coupled to chiral HPLC to determine the enantiomeric excess of different enantiomers. In this project, direct chiral resolution of the racemic amino ester 14 was difficult to perform. Only a single peak eluted and the use of different solvents (methanol, water and acetonitrile) and solvent constituents (gradient and isocratic) proved to be unsuccessful. There are many techniques that can be used to improve the separation of the enantiomeric compounds, such as the increase in the number of theoretical plates or increasing the length of the column. In this case, two different columns that vary in column length and particle size were investigated; however, neither of them resulted in better separation. Derivatization is the most commonly used technique to improve the separation of enantiomers; this method works efficiently especially when using small analytes such as our isopropyl amino ester. The derivatization of amino ester was subsequently performed order to establish a method for the chiral HPLC detection of the derivative amide (Scheme 24). Primary amines are strong bases and in the presence of carboxylic acids they undergo a fast acid-base reaction to form a salt. In this case the amidation reaction is favoured if high-temperature conditions (< 100°C) are employed; however, this is inconvenient because our isopropyl amino ester (14) will not survive those conditions. To favour the amidation route, coupling reagents are normally used. Our first attempt involved the use of CDI (carbonyl imidazole) coupling reagent, unfortunately the reaction didn’t work, only traces of the product was observed (Scheme 24). Various solvents and temperatures were investigated, however, none of those optimizations resulted in an improved yield. CDI was then replaced with a more flexible and less expensive coupling reagent, dicyclohexylcarbodimine (DCC). DCC is one of the most widely used coupling reagents and it has been utilized since 1955 for peptide synthesis, it is also widely used along with DMAP in esterification reactions. The use of DCC reagent resulted in a good outcome compared to the CDI, the maximum yield of amide obtained was 60%, and the only problem encountered was the purification procedure as there were a lot of by-products and starting material spots on the TLC plate. The purification procedure was achieved using silica column and 10-25% ethyl acetate-hexane to afford a light-yellow powder product 17. The product was characterised using NMR and mass spectrometer. The 1H NMR spectrum shows the present of two aromatic peaks at 8.35 – 8.27 ppm and 8.03 – 7.94 ppm .The NH peak 40 was also observed at 6.90 ppm, this indicate that the coupling was a success and it was further confirmed by the presence of an amide carbon at 164.6 ppm in the 13C NMR spectrum .The mass spectrum also shown the fragment with the mass of [M+H]+= 295.1295 which correspond to the expected mass of our product 17. Using the same reaction procedure as in Scheme 24, the (S)-17 amide was synthesised from the (S)-14 amine. As expected, the NMR and the mass spec data of the (S)-17 corresponded to its racemate. Scheme 24: The derivatization of amino ester 14 into a corresponding amide 17 to improve chiral HPLC separation. 41 Figure 15: The LCMS chromatography peaks of the derivatized (rac)-17 (above) and (S)- standard 17 (below). The resolution of two enantiomers proved to be successful as a clear baseline was detected between the two enantiomers using a Chiralpak® AD column, a mobile phase of water/ acetonitrile (95:5) isocratic, UV detection at 254 and 324 nm and mass spec detection (Fig. 16). The S-derivative standard was also run on the instrument, using the same method and S R S 254 nm 324 nm 42 conditions. It eluted at a retention time of 9.8 minutes; these results indicate that the S- enantiomer elutes first followed by the R-enantiomer. Standards were run immediately before running samples in order to achieve accurate results or elution time. 2.1.5 Determination of stereochemistry of the enzyme products. Transaminase reactions were scaled up to 10 ml scale to provide a sufficient product for accurate analysis; this was followed by the derivatization to form the amide 17 (Scheme 25). Table 2 shows the summarised results obtained when using different enzymes, the % e.e. was derived from the enantiomer peak areas, and the value of the yields represents the % yield of the amino ester products. The yields of the amino ester products were relatively good; this was followed by the derivatization with the yield ranging from 25-45%. The NMR and the mass spec data of the derivatized compounds correspond well with the standard data. Scheme 25: The enzymatic synthesis of the amino ester 14 followed by the derivatization to form the UV active amide 17. ATA-230 gave an excellent conversion of starting material to the product (87% yield) with an excellent % e.e; however, the stereochemistry was undesired (96% R). As noted in the introductory chapter ethambutol exist as three diastereomers, the S, S being the therapeutically active while R, S is 16 times less potent and R, R is 500 less potent, this enzyme is not useful for our intended product, however, it can be applied in the synthesis of other products for example, (R)-amino alcohol can be used for chiral resolution of the optical isomers of 1,1′- binaphthalene-2,2′-diyl hydrogen phosphate which is a chiral ligand used for hydro carboxylation reactions. ATA– 262, 239, 251