Biocatalytic Synthesis of Chiral Alcohols and Amino Acids for Development of Pharmaceuticals

Chirality is a key factor in the safety and efficacy of many drug products and thus the production of single enantiomers of drug intermediates and drugs has become increasingly important in the pharmaceutical industry. There has been an increasing awareness of the enormous potential of microorganisms and enzymes derived there from for the transformation of synthetic chemicals with high chemo-, regio- and enatioselectivities. In this article, biocatalytic processes are described for the synthesis of chiral alcohols and unntural aminoacids for pharmaceuticals.


Introduction
For preparation of drugs and their intermediates, the synthesis of single enantiomers has become increasingly important in the pharmaceutical industry [1]. Single enantiomers can be produced by either by chemical or biocatalytic routes. The advantages of biocatalysis over chemical synthesis are that enzyme-catalyzed reactions are often highly enantioselective and regioselective. They can be carried out under mild conditions at ambient temperature and atmospheric pressure, thus avoiding the use of more extreme reaction conditions which could cause problems with isomerization, racemization, epimerization, and rearrangement of compound. Microbial cells and wide variety and class of enzymes derived there from can be used for chiral synthesis. Enzymes can be immobilized and reused for many cycles. In addition, enzymes can be over expressed to make biocatalytic processes economically efficient, and enzymes with modified activity can be tailor-made. Directed evolution of biocatalysts can lead to increased enzyme activity, selectivity and stability [2][3][4][5][6][7][8][9][10][11][12][13][14][15]. A number of review articles [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] have been published on the use of enzymes in organic synthesis. This chapter provides The NAD-dependent (S)-reductase (SHBR) from Pseudomonas putida SC 16269 was also purified to homogeneity, its N-terminal and internal sequences were determined and cloned and expressed in Escherichia coli. To regenerate the cofactor NADH required for reduction we have also cloned and expressed the NAD + dependent formate dehydrogenase gene from Pichia pastoris in Escherichia coli. Recombinant Escherichia coli expressing (S)-reductase was used to catalyze the reduction of 6-ketobuspirone to (S)-6-hydroxybuspirone, in >98% yield and >99.8% e.e. at 50 g/L substrate input [37].
A mixture of ethyl 3-keto-5-hydroxy 9 (major) and 5-keto-3-hydroxy 10 (minor) was obtained from partial microbial reduction of ketoester 7. These two mixtures were subjected to microbial reduction by Acinetobacter sp SC13874 cells for 6 h (incomplete reduction). The reduction provided the dihydroxy esters with the isomeric composition. The results indicated that the second reduction of the monohydroxy compound by SC13874 cells was quite enantiospecific. Reduction of the 3-keto-5-hydroxy 9 provided predominantly the (3R)-hydroxy, while reduction of the 3-hydroxy-5-keto ester 10 provided predominantly the (5S)-hydroxy compound [43].
Cell extracts of A. calcoaceticus SC 13876 in the presence of NAD + , glucose, and glucose dehydrogenase reduced 7 to the corresponding monohydroxy compounds [3-hydroxy-5-oxo-6-(benzyloxy) hexanoic acid ethyl ester 9 and 5-hydroxy-3-oxo-6-(benzyloxy) hexanoic acid ethyl ester 10]. Both 9 and 10 were further reduced to the (3R,5S)-dihydroxy compound 4 in 92% yield and 99% e.e. by cell extracts. (3R,5S)-4 was converted to 11, a key chiral intermediate for the synthesis of compound 5 and Atorvastatin 6. Three different ketoreductases were purified to homogeneity from cell extracts of A. calcoaceticus SC 13876 and their biochemical properties were compared. Reductase I only catalyzes the reduction of ethyl diketoester 7 to its monohydroxy products whereas reductase II catalyzes the formation of dihydroxy products from monohydroxy substrates. A third reductase (III) was identified which catalyzes the reduction of diketoester 7 to syn-(3R,5S)-dihydroxy ester 4 [44], which now has been cloned and expressed in E. coli [44] and the reduction of diketoester 7 to syn-(3R,5S)-dihydroxy ester 4 was demonstrated by recombinant enzyme at 50 g/L substrate input with 10 g/L cell suspensions.
Using this method, an overall quantity of 23 kg of 18 was prepared. The key step was an aqueous enzymatic reduction using D-lactate dehydrogenase (D-LDH) and formate dehydrogenase (FDH). Mechanistically, the keto acid salt 20 is stereoselectively reduced to the corresponding R-hydroxy acid 18 in the presence of D-lactate dehydrogenase by NADH. The cofactor itself is oxidized to NAD + in the process. Subsequently, in the presence of formate dehydrogenase, NAD + is reduced back to NADH by ammonium formate, which was oxidized to CO 2 and NH 3 . In this fashion the expensive cofactor NAD + is regenerated by FDH, and only a catalytic amount of NAD + was required [52].

Enzymatic Reduction Process for Synthesis of Montelukast Intermediate
The discovery of the biological activity of the slow reacting substance of anaphylaxis (SRS-A) and its relation to the leukotrienes (LTC4, LTD4, and LTE4) and asthma, the search for leukotriene antagonists has been intensive. As part of an ongoing program for the development of specific LTD4 antagonists for the treatment of asthma and other associated diseases at Merck have identified Montelukast 25 ( Figure 7) as LTD4 antagonist [58][59][60]. Merck has described the synthetic route for the production of montelukast, using a stereoselective reduction of a ketone 26 to the (S)-alcohol 27 as the key step. The alcohol subsequently undergoes a Sn2 displacement with a thiol to give the R-configured final product [59,60]. The reduction of the ketone 26 to produce the chiral alcohol 27 requires stoichiometric amounts of the chiral reducing agent (−)-flchlorodiisopino campheylborane [(−)-DIP-chloride]. (−)-DIP-chloride is selective and avoids the side reactions but it is corrosive and moisture-sensitive, causing burns if it is allowed to contact the skin. The reaction must be carried out at −20 to −25 °C to achieve the best stereoselectivity. The quench and extractive work-up generate large volumes of waste solvent, due to the product's low solubility. The potential advantages of biocatalytic transformation of ketone to alcohol were recognized early on by researchers at Merck. However, only two microorganisms were identified as having activity on the bulky and hydrophobic substrate [61]. Due to several reasons, an enzymecatalyzed process for reduction of the ketone 26 was developed by Codexis. A ketoreductase was developed by directed evolution by high throughput screens using a slurry of the ketone substrate and high isopropanol concentration. Beneficial mutations among the various improved mutants were recombined in each round, and new mutations were made guided by ProSAR. The productivity of the final enzyme was improved 2,000-fold and stability was also substantially increased [62].
The final process was carried out as a slurry-to-slurry reaction at 45 °C , with the sparingly soluble ketone 26 being converted to an almost equally insoluble alcohol 27 at a concentration of 100 g/L substrate in aqueous isopropanol and toluene. A reaction yield of 99.3% and enantiomeric excess of 99.9% was obtained for alcohol 27 [62].

Anticancer Drug: Enzymatic Preparation of C-13 Paclitaxel Side-Chain Synthon
Among the antimitotic agents, paclitaxel (taxol ® ) 28 ( Figure 8), a complex, polycyclic diterpene, exhibits a unique mode of action on microtubule proteins responsible for the formation of the spindle during cell division. Various types of cancers have been treated with paclitaxel and it was approved for use by the FDA for treatment of ovarian cancer and metastatic breast cancer [63][64][65]. A key precursor for the paclitaxel semi-synthetic process is the chiral C-13 paclitaxel side-chain 29. An enzymatic enantioselective microbial reduction of 2-keto-3-(N-benzoylamino)-3-phenyl propionic acid ethyl ester 30 to yield (2R,3S)-N-benzoyl-3-phenyl isoserine ethyl ester 29 was demonstrated using two strains of Hansenula [66]. Preparative-scale bioreduction of ketone 30 was demonstrated using cell suspensions of Hansenula polymorpha SC 13865 and Hansenula fabianii SC 13894 in independent experiments. In both batches, a reaction yield of >80% and e.e.s of >94% were obtained for (2R,3S)-29. In a single-stage process, cells of H. fabianii were grown in a 15-L fermentor for 48 h, then the bioreduction process was initiated by addition of 30 g of substrate and 250 g of glucose and continued for 72 h. A reaction yield of 88% with an e.e. of 95% was obtained for (2R,3S)-29.

Antipsychotic Drug: Enzymatic Reduction of 1-(4-Fluorophenyl)4-[4-(5-Fluoro-2-Pyrimidinyl)1-Piperazinyl]-1-Butanone
The sigma receptor system in the brain and endocrine tissue has been target for development of new class of antipsychotic drugs [67,68]. Compound (R)-31 ( Figure 9) is a sigma ligand and has a high affinity for sigma binding site and antipsychotic efficacy. The enantioselective microbial reduction process was developed for the conversion of ketone 32 to both enantiomers of alcohol 31 [69]. Various microorganisms screened for the enatioselective reduction of 1-(4-fluorophenyl)4-[4-(5-fluoro-2pyrimidinyl)1-piperazinyl]-1butanone 32. From this screen, Mortierella ramanniana ATCC 38191 was identified to predominantly reduced compound 32 to (R)-31, while Pullularia pullulans ATCC 16623 was identified to predominantly reduced compound 32 to (S)-31. A single stage fermentation/biotransformation process was developed. Cells of M. ramanniana were grown in a 20-L fermentor and after 40 h growth period, the biotransformation process was initiated by addition of 40 g ketone 32 and 400 g glucose. The biotransformation process was completed in 24 h with a reaction yield of 100% and an e.e. of 98.9% for (R)-31. At the end of the biotransformation process, cells were removed by filtration and product was recovered from the filtrate in overall 80% yield [69].

Thrombin Inhibitor: Enzymatic Preparation of (R)-2-Hydroxy-3,3-Dimethylbutanoic Acid
Thrombin is a trypsin-like protease enzyme that plays a critical role in intrinsic and extrinsic blood coagulation. As a result of the enzymatic activation of numerous coagulation factors, thrombin is activated to cleave fibrinogen, producing fibrin, which is directly responsible for blood clotting. An imbalance between these factors and their endogenous activators and inhibitors can give rise to a number of disease states such as myocardial infarction, unstable angina, stroke, ischemia, restenosis following angioplasty, pulmonary embolism, deep vein thrombosis, and arterial thrombosis [82,83]. Consequently, the aggressive search for a potent, selective, and bioavailable thrombin inhibitor is widespread [84]. An intensive effort by Merck has led to the identification of thrombin inhibitor 53 [85]. The synthesis of 53 required a key chiral intermediate (R)-hydroxy ester 54. An enzymatic process was developed for the asymmetric reduction of ketoester 55 to (R)-54 using commercially available ketoreductase KRED1001 (Figure 14). The cofactor NADPH required for this reaction was regenerated using glucose dehydrogenase. The hydroxy ester (R)-54 was isolated as an oil and then saponified to the corresponding enantiomerically pure hydroxy acid (R)-56 without epimerization [86]. The enantiomerically pure (R)-56 was obtained in 82% isolated yield (>99.5% e.e.).

Endothelin Receptor Antagonist: Enantioselective Microbial Reduction of Keto Ester and Chloroketone
Endothelin is present in elevated levels in the blood of patients with hypertension, acute myocardial infarction and pulmonary hypertension. Two endothelin receptor sub-types have been identified which bind endothelin, thus causing vasoconstriction [87,88]. Endothelin receptor antagonists such as compound 57 (Figure 15) have potential therapeutic value. Synthesis of compound 57 required two key chiral intermediates (S)-alcohols 58 and 59. Enantioselective microbial reduction of a ketoester 60 and a chlorinated ketone 61 to their corresponding (S)-alcohols 58 and 59 was demonstrated using Pichia delftensis MY 1569 and Rhodotorula piliminae ATCC 32762 to afford desired products in >98% e.e. and >99% e.e, respectively [89]. Reductions were scaled up to 23 L to produce the desired (S)-alcohols in 88% and 97% yields, respectively.

3-Receptor Agonist: Reduction of 4-Benzyloxy-3-Methanesulfonylamino-2'-Bromo-Acetophenone
3-Adrenergic receptors are found on the cell surfaces of both white and brown adipocytes and are responsible for lipolysis, thermogenesis, and relaxation of intestinal smooth muscle [95]. Consequently, several research groups are engaged in developing selective 3 agonists for the treatment of gastrointestinal disorders, type II diabetes, and obesity [96,97]. Biocatalytic syntheses of chiral intermediates required for the total synthesis of 3 receptor agonists 66 ( Figure 17) has been demonstrated [98]. The microbial reduction of 4-benzyloxy-3-methanesulfonylamino-2'-bromo-acetophenone 67 to the corresponding (R)-alcohol 68 has been demonstrated [98] using Sphingomonas. paucimobilis SC 16113. The growth of S. paucimobilis SC 16113 was carried out in a 750-L fermentor and harvested cells (60 kg) were used to conduct the biotransformation in 10-L and 200-L preparative batches using 20% (wt/vol, wet cells). In some batches, the fermentation broth was concentrated 3-fold by microfilteration and subsequently washed with buffer by diafilteration and used directly in the bioreduction process. In all the batches, reaction yields of >85% and e.e.s. of >98% were obtained. The isolation of alcohol 68 from the 200-L batch gave 320 g (80% yield) of product with an e.e. of 99.5%.
In an alternate process, frozen cells of S. paucimobilis SC 16113 were used with XAD-16 hydrophobic resin (50 g/L) adsorbed substrate at 10 g/L concentration. In this process, an average reaction yield of 85% and an e.e. of >99% were obtained for alcohol 68. At the end of the biotransformation, the reaction mixture was filtered on a 100 mesh (150 μm) stainless steel screen, and the resin retained by the screen was washed with water. The product was then desorbed from the resin with acetonitrile and crystallized in 75% overall yield with a 99.8% e.e. [98].

Integrin Receptor Agonist: Enzymatic Preparation of (R)-Allylic Alcohol
(R)-allylic alcohol 76 ( Figure 19) was required as an intermediate for the synthesis of a desired monanoic derivate useful as an integrin receptor antagonist for the inhibition of bone desorption and treatment of osteoporosis [102]. A pilot scale whole cell process was developed for the enantioselective 1,2-reduction of prochiral alpha,beta-unsaturated ketone 77 to (R) allylic alcohol, (R)-76 using Candida chilensis [103]. Initial development showed high enantiomeric excess (>95%) but low product yield (10%). Further process development, using a combination of statistically designed screening and optimization experiments, improved the desired alcohol yield to 90%. The fermentation growth stage, particularly medium composition and growth pH, had a significant impact on the bioconversion while process characterization identified diverse challenges including the presence of multiple enzymes, substrate/product toxicity, and biphasic cellular morphology. Manipulating the fermentation media allowed control of the whole cell morphology to a predominantly unicellular broth, away from the viscous pseudohyphae, which were detrimental to the bioconversion. The activity of a competing enzyme, which produced the undesired saturated ketone 78 and (R)-saturated alcohol 79, was minimized to < or =5% by controlling the reaction pH, temperature, substrate concentration, and biomass level. Despite the toxicity effects limiting the volumetric productivity, a reproducible and saleable process was demonstrated at pilot scale with high enantioselectivity (e.e. > 95%) and overall yield greater than 80% [104]. The whole cell approach proved to be a valuable alternative to chemical reduction routes. Figure 19. Integrin receptor agonist: Enzymatic preparation of (R)-allylic alcohol.

NK1 Receptor Antagonists: Enzymatic Synthesis of (S)-3,5-Bistrifluoromethylphenyl Ethanol
The synthesis of (S)-3,5-bistrifluoromethylphenyl ethanol, (S)-80, (Figure 20), an intermediate for the synthesis of NK-1 receptor antagonists 81 [104] was demonstrated from a ketone 82 via asymmetric enzymatic reduction process [105]. The isolated enzyme alcohol dehydrogenase from Rhodococcus erythropolis reduced the poorly water soluble substrate with an excellent enantiomeric excess (>99.9%) and good conversion (>98%). The optimized process was demonstrated up to pilot scale using concentration (390 mM) using a easy isolation process achieving overall isolation yields (>90%). Process improvements at preparative scale, demonstrated increase in the substrate input to 580 mM achieving a space time yield of 260 g/L/day [105].

Enzymatic Preparation of Chiral Amino Acids
The reductive amination of -keto acids using amino acid dehydrogenases to be one of the most useful methods because the enzymes have good stability, broad substrate specificity and very high enantioselectivity and can be used at high substrate concentrations as keto acids are soluble in aqueous system. The reductive aminations process coupled to an enzymatic cofactor regeneration system are most prominent method for preparation of chiral amino acids. For most enzymes, the required cofactor is NADH but NADPH is required in some cases. Yeast formate dehydrogenase is commonly used for NADH regeneration and glucose dehydrogenase usually from Bacillus species may be used for either NADH or NADPH regeneration. There are excellent reviews on the amino acid dehydrogenases and examples of their synthetic utilities [106][107][108][109].

Tigemonam: Enzymatic Synthesis of (S)-β-Hydroxyvaline
(S)-β-hydroxyvaline 83 (Figure 21), is a key chiral intermediate required for the total synthesis of orally active monobactam [110], Tigemonam 84. Chiral amino acids have been made from corresponding keto acids by reductive amination process [111]. The synthesis of (S)-β-hydroxyvaline 83 from -keto-β-hydroxyisovalerate 85 by reductive amination using leucine dehydrogenase from Bacillus sphaericus ATCC 4525 has been demonstrated [112]. The NADH required for this reaction was regenerated by either formate dehydrogenase from Candida boidinii or glucose dehydrogenase from Bacillus megaterium. The required substrate 85 was generated either from -keto-βbromoisovalerate or its ethyl esters by hydrolysis with sodium hydroxide in situ. In this process, an overall reaction yield of 98% and an enantiomeric excess of 99.8% were obtained for the L-βhydroxyvaline 83. Figure 21. Tigemonam: Enzymatic synthesis of (S)-β-hydroxyvaline.

Atazanavir: Enzymatic Synthesis of (S)-Tertiary-Leucine
Atazanavir 86 is an acyclic aza-peptidomimetic, a potent HIV protease inhibitor [53,54]. Synthesis of atazanavir required (S)-tertiary leucine 87 (Figure 22). An enzymatic reductive amination of ketoacid 88 to amino acid 87 by recombinant Escherichia coli expressing leucine dehydrogenase from Thermoactinimyces intermedius has been demonstrated. The reaction required ammonia and NADH as a cofactor. NAD produced during the reaction was converted back to NADH using recombinant Escherichia coli expressing formate dehydrogenase from Pichia pastoris. A reaction yield of >95% with an e.e. of >99.5% was obtained for 87 at 100 g/L substrate [113]. Leucine dehydrogenase from Bacillus strain has also been cloned and expressed and used in reductive amination process [114,115].

Vanlev: Enzymatic Synthesis of (S)-6-Hydroxynorleucine
Vanlev 89 ( Figure 23) is an antihypertensive drug which acts by inhibiting angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP) [116]. (S)-6-Hydroxynorleucine 90 is a key intermediate in the synthesis of Vanlev. The synthesis and complete conversion of 2-keto-6-hydroxyhexanoic acid 91 to (S)-6-hydroxynorleucine 90 was demonstrated by reductive amination using beef liver glutamate dehydrogenase [117]. As depicted, compound 91, in equilibrium with 2-hydroxytetrahydropyran-2carboxylic acid sodium salt 92, was converted to 90. The reaction requires ammonia and NADH. NAD produced during the reaction was recycled to NADH by the oxidation of glucose to gluconic acid using glucose dehydrogenase from Bacillus megaterium. The reaction was complete in about 3 h at 100 g/L substrate input with a reaction yields of 92% and e.e. of 99.8% for (S)-6-hydroxynorleucine. The synthesis and isolation of keto acid 91 required several steps. In a second, more convenient process the ketoacid was prepared by treatment of racemic 6-hydroxy norleucine 90 [produced by hydrolysis of 5-(4-hydroxybutyl) hydantoin 93] with (R)-amino acid oxidase (Figure 24) After the e.e. of the unreacted (S)-6-hydroxynorleucine had risen to 99.8%, the reductive amination procedure was used to convert the mixture containing the 2-keto-6-hydroxyhexanoic acid entirely to (S)-6-hydroxynorleucine in 97% yield with 99.8% e.e. from racemic 6-hydroxynorleucine at 100 g/L substrate input [117]. The (S)-6-hydroxynorleucine prepared by the enzymatic process was converted chemically to Valev 89 [118].   from Thermoactinomyces intermedius [119]. The reaction required ammonia and NADH; NAD produced during the reaction was recyled to NADH by the oxidation of formate to CO 2 using formate dehydrogenase [FDH]. T. intermedius PDH was cloned and expressed in Escherichia coli and recombinant culture was used as a source of PDH. Expression of T. intermedius PDH in P. pastoris, inducible by methanol, allowed generation of both enzymes in a single fermentation as methanol grown cells of P. pastoris also contained formate dehydrogease. A total of 197 kg of 94 was produced in three 1,600-L batches using a 5% concentration of substrate 95 with an average yield of 91 M % and e.e. >98% [119]. (S)-allysine ethylene acetal was converted to Vanlev 89 [118].
Saxagliptin 96 [121,122] (Figure 26), a DPP-IV inhibitor developed by Bristol-Myers Squibb and now approved for type 2 diabetic treatment by Food and Drug administration, requires (S)-N-boc-3hydroxyadamantylglycine 97 as an intermediate. A process for conversion of the keto acids 98 to the corresponding amino acid 99 using (S)-amino acid dehydrogenases was developed. A modified form of a recombinant phenylalanine dehydrogenase cloned from Thermoactinomyces intermedius and expressed in Pichia pastoris or Escherichia coli was used for this process. NAD produced during the reaction was recycled to NADH using formate dehydrogenase. The modified phenylalanine dehydrogenase contains two amino acid changes at the C-terminus and a 12 amino acid extension of the C-terminus [124].
Production of multi-kg batches was originally carried out with extracts of Pichia pastoris expressing the modified phenylalanine dehydrogenase from Thermoactinomyces intermedius and endogenous formate dehydrogenase. The reductive amination process was further scaled up using a preparation of the two enzymes expressed in single recombinant E. coli. The amino acid 99 was directly protected as its boc derivative without isolation to afford intermediate. Yields before isolation were close to 98% with 100% e.e. [124]. Reductive amination was also conducted using cell extracts from E.coli strain SC16496 expressing PDHmod and cloned FDH from Pichia pastoris. Cell extracts after polyethyleneamine treatment, clarification and concentration were used to complete the reaction in 30 h with >96% yield and >99.9% e.e. of product 99. This process has now been used to prepare several hundred kg of boc-protected amino acid 97 to support the development of Saxagliptin [124].

Enzymatic Synthesis of (S)-Neopentylglycine
The enantioselective synthesis of (S)-neopentylglycine 100 ( Figure 27) has been developed by Groeger et al. [125]. Recombinant whole cell containing leucine dehydrogenase and formate dehydrogenase was used in the reductive amination of the corresponding -keto acid 101. The desired (S)-neopentylglycine was obtained with >95% conversion and a high enantioselectivity of >99% e.e. at substrate concentrations of up to 88 g/L. Spiroheterocyclic compounds [morpholine-4-carboxylic acid amides of heterocyclic cyclohexylalanine and neopentylglycine derivatives and their analogs] are useful as reversible inhibitors of cysteine proteases such as cathepsin S useful in the treatment of variety of autoimmune diseases [126].

Glucogen like Peptide: Enzymatic Deracemization Racemic Amino Acid to (S)-Amino Acid
The (S)-amino-3-[3-{6-(2-methylphenyl)}pyridyl]-propionic acid 102 ( Figure 28) is a key intermediate required for synthesis of GLP-1 mimics or GLP-1 receptor modulators. Such receptor modulators are potentially useful for the treatment of type II diabetes treatment [127,128].  [129] in 72% isolated yield with >99.4% e.e. from racemic amino acid 103 using combination of two enzymes (R)-amino acid oxidase from Trigonopsis variabilis expressed in Escherichia coli and (S)-aminotransferase from Sporosarcina ureae cloned and expressed in Escherichia coli. (S)-aspartate was used as amino donor. A (S)-aminotransferase was also purified from a soil organism identified as Burkholderia sp. and cloned and expressed in Escherichia coli and used in this process [131]. In enzymatic process racemic amino acid was first treated with (R)-amino acid oxidase for 4 h to convert racemic amino acid to mixture of (S)-amino acid and keto acid 104. Subsequently in the same reaction mixture (S)-aminotransferase was charged to convert keto acid 104 to (S)-amino acid 102 to get 85% yield at the end of the biotransformation process. This process was scaled up to 100 L scale at a substrate input of 1.5 kg.
In an alternate process, the enzymatic dynamic resolution of racemic amino acid 103 was also demonstrated. (R)-selective oxidation with celite-immobilized (R)-amino acid oxidase from Trigonopsis variabilis expressed in Escherichia coli in combination with chemical imine reduction with borane-ammonia gave a 75% in process yield and 100 e.e. of (S)-amino acid 102 [129].

Preparation of (R)-Amino Acid
(R)-Amino acids are increasingly becoming important building blocks in the production of pharmaceuticals and fine chemicals, and as chiral directing auxiliaries and chiral synthons in organic synthesis [130,131]. Using both rational and random mutagenesis, Rozzell and Novick [132] have created the broad substrate range, nicotinamide cofactor dependent, and highly stereoselective (R)-amino acid dehydrogenase. This new enzyme is capable of producing (R)-amino acids via the reductive amination of the corresponding 2-keto acid with ammonia. This biocatalyst was the result of three rounds of mutagenesis and screening performed on the enzyme meso-diaminopimelate (R)-dehydrogenase from Corynebacterium glutamicum. The first round targeted the active site of the wild-type enzyme and produced mutants that were no longer strictly dependent on the native substrate.
The second and third rounds produced mutants that had an increased substrate range including straightand branched-aliphatic amino acids and aromatic amino acids. The very high selectivity toward the (R)-enantiomer (95% to >99% e.e.) was shown to be preserved three rounds of mutagenesis and screening [132]. This new enzyme was active against variety of amino acids could complement and improve upon current methods for (R)-amino acid synthesis. The synthesis of (R)-cyclohexylalanine 105 ( Figure 29) was developed by reductive amination of cyclohexylpyruvate 106 to yield (R)-105 in 98% yield and >99% e.e. (R)-105 is a potential chiral intermediate for the synthesis of thrombin inhibitor Inogatran 107 [133]. The deracemisation of DL-amino acids using L-amino acid oxidase from Proteus myxofaciens and amine-boranes as chemical reducing agents has been investigated. Amine-boranes were found to be of particular interest in terms of reactivity and chemoselectivity compared to sodium borohydride and cyanoborohydride. Starting from the racemic amino acids, a range of D-amino acids were prepared in yields of up to 90% and e.e. >99% [134].
(R)-Amino acid 108 was prepared in 68% isolated yield with >99% e.e. from racemic amino acid 110 using (S)-amino acid oxidase from Proteus mirabilis expressed in Escherichia coli in combination with a commercially available (R)-transaminase using (R)-alanine as amino donor [137]. The (R)-enantiomer was also prepared in 79% isolated yield with >99% e.e. from the corresponding keto acid 111 using the (R)-transaminase with racemic alanine as the amino donor. The rate and yield of this reaction could be accelerated by addition of lactate dehydrogenase (with NAD + , formate and formate dehydrogenase to regenerate NADH) to remove the inhibitory pyruvate produced during the reaction. A (R)-transaminase was identified and purified from a soil organism identified as Bacillus thuringiensis and cloned and expressed in Escherichia coli. The recombinant (R)-transaminase was very effective for the preparation of (R)-108 and gave a nearly complete conversion of 111 to (R)-108 without the need for additional enzymes for pyruvate removal [137]. Figure 30. Calcitonin gene-related peptide receptors (antimigraine drugs): Enzymatic preparation of (R)-2-amino-3-(7-methyl-1H-indazol-5-yl)propanoic acid.

Corticotropin Releasing Factor (CRF)-1 Receptor Antagonist: Enzymatic Resolution by Transaminase
(R)-amines synthesis for Anxiety and depression are psychiatric disorders that constitute a major health concern worldwide. While numerous marketed treatments exits for both disorders, there continue to be need agents which may have increased efficacy and/or reduced side-effect profiles [138][139][140]. CRF-1 receptor antagonists have been proposed as novel pharmacological treatments for depression, anxiety and stress disorders [138][139][140][141]. (R)-sec-butylamine 112 and (R)-1-cyclopropylethylamine 113 ( Figure 31) are key chiral intermediates for the synthesis of CRF-1 receptor antagonists such as 114 [141,142]. We have developed enzymatic resolution process for the preparation of (R)-sec-butylamine and (R)-1-cyclopropylethylamine [143]. Screening was carried out to identify strains useful for the preparation of (R)-1-cyclopropylethylamine and (R)-sec-butylamine from the racemic amines with an (S)-specific transaminase. Several Bacillus megaterium strains as well as several soil isolates were found to have the desired activity for the resolution of the racemic amines to give the (R)-enantiomers. Using an extract of the best strain, Bacillus megaterium SC6394, the reaction was shown to be a transamination requiring pyruvate as amino acceptor and pyridoxal phosphate as a cofactor. Initial batches of both amines were produced using whole cells of Bacillus megaterium SC6394. The transaminase was purified to homogeneity to obtain N-terminal as well as internal amino acid sequences. The sequences were used to design polymerase chain reaction (PCR) primers to enable cloning and expression of the transaminase in Escherichia coli SC16578. In contrast to using Bacillus megaterium process, pH control and aeration were not required for the resolution of sec-butylamine and an excess of pyruvate was not consumed by the recombinant cells. The resolution of sec-butylamine (0.68 M) using whole cells of Escherichia coli SC16578 was scaled up to give (R)-sec-butylamine 1/2 H 2 SO 4 in 46.6% isolated yield with 99.2% e.e. An alternative isolation procedure was also used to isolate (R)-sec-butylamine as the free base. Using the same recombinant (S)-tansaminase, (R)-1cyclopropylethylamine was obtained in 42% isolated yield (theoretical max. 50%) and 99% e.e. [143].

Conclusions
The production of single enantiomers of drug intermediates is increasingly important in the pharmaceutical industry. Biocatalysis provides organic chemists an alternate opportunity to prepare pharmaceutically important chiral compounds. The examples presented in this review are only from a few selected articles for synthresis of chiral alcohols and unnatural amino acids. Different types of biocatalytic reactions are capable of generating a wide variety of chiral compounds useful in the development of drugs. The use of hydrolytic enzymes such as lipases, esterases, proteases, dehalogenases, acylases, amidases, nitrilases, epoxide hydrolases, and decarboxylases for the resolution of variety of racemic compounds and in the asymmetric synthesis of enantiomerically enriched chiral compounds. Dehydrogenases and aminotransferases has been successfully used along with cofactors and cofactor regenerating enzymes for the synthesis of chiral alcohols, aminoalcohols, amino acids and amines. Aldolases and decarboxylases have been effectively used in asymmetric synthesis by aldol condensation and acyloin condensation reactions. Monoxygenases have been used in enantioselective and regioselective hydroxylation, epoxidation, sulfoxidation and Baeyer-Villiger reactions. Dioxygenases have been used in the chemo-enzymatic synthesis of chiral diols. Enzymatic deracemization, dynamic resolution and stereoinversion, to achieve >50% yield and high e.e. by combination of chemo-and/or biocatalysts in sequential reactions or by a single biocatalyst. In the course of the last decade, progress in biochemistry, protein chemistry, molecular cloning, random and site-directed mutagenesis, directed evolution of biocatalysts under desired process conditions has opened up unlimited access to a variety of enzymes and microbial cultures as tools in organic synthesis. Future of bicatalysis for synthesis of chiral compounds looks very promising.