A new enzymatic process for the enantioselective cleavage of N-benzyloxycarbonyl (Cbz) groups from protected amino acids and related compounds has been developed. The Cbz-deprotecting enzyme was isolated from cell extracts of Sphingomonas paucimobilis SC 16113 and purified to homogeneity. The purified protein has a molecular weight of 155,000 daltons and a subunit size of 44,000 daltons.

Baccatin III is a polycyclic diterpene which can be used for the semi-synthesis of paclitaxel and analogs. An enzymatic process was developed for the conversion of 10-deacetylbaccatin III (10-DAB) to baccatin III without requiring protection of the 7-hydroxyl group. A C-10 deacetylase from Nocardioides luteus SC 13912 was immobilized on diethylaminoethyl cellulose (DEAE-Cellulose) and the immobilized enzyme was used in the biotransformation process. The reaction was catalyzed using vinyl acetate as acyl donor at ambient temperature and at pH 7. A reaction yield of 51% was obtained.

[4S-(4I.7I,10aJ)]1-Octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01) is a key chiral intermediate for the synthesis of Omapatrilat (BMS-186716), a new vasopeptidease inhibitor under development. By using a selective enrichment culture technique we have isolated a strain of Sphingomonas paucimobilis SC 16113, which contains a novel L-lysine epsilon-aminotransferase. This enzyme catalyzed the oxidation of the epsilon-amino group of lysine in the dipeptide dimer N-2-[N[phenyl-methoxy)-carbonyl] L-homocysteinyl] L-lysine)1,1-disulphide (BMS-201391-01) to produce BMS-199541-01. The aminotransferase reaction required alpha-ketoglutarate as the amino acceptor. Glutamate formed during this reaction was recycled back to alpha-ketoglutarate by glutamate oxidase from Streptomyces noursei SC 6007. Fermentation processes were developed for growth of S. paucimobilis SC 16113 and S. noursei SC 6007 for the production of L-lysine epsilon-amino transferase and glutamate oxidase, respectively. L-lysine epsilon-aminotransferase was purified to homogeneity and N-terminal and internal peptides sequences of the purified protein were determined. The mot wt of L-lysine epsilon-aminotransferase is 81 000 Da and subunit size is 40 000 Da. L-lysine epsilon-aminotransferase gene (lat gene) from S. paucimobilis SC 16113 was cloned and overexpressed in Escherichia coli. Glutamate oxidase was purified to homogeneity from S. noursei SC 6003. The mol wt of glutamate oxidase is 125 000 Da and subunit size is 60 000 Da. The glutamate oxiadase gene from S. noursei SC 6003 was cloned and expressed in Streptomyces lividans. The biotransformation process was developed for the conversion of BMS-201391-01 to BMS-199541-01 by using L-lysine epsilon-aminotransferase expressed in E. coli. In the biotransformation process, for conversion of BMS-201391-01 (CBZ protecting group) to BMS-199541-01, a reaction yield of 65-70 65-70 M% was obtained depending upon reaction conditions used in the process. Phenylacetyl or phenoxyacetyl protected analogues of BMS-201391-01 also served as substrates for L-lysine epsilon-aminotransferase giving reaction yields of 70 M% for the corresponding BMS-199541-01 analogs. Two other dipeptides N-[N[(phenylmethoxy)carbonyl]-L-methionyl]-L-lysine (BMS-203528) and N,2-[S-acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine (BMS-204556) were also substrates for L-lysine epsilon-aminotransferase. N-alpha-protected (CBZ or BOC)-L-lysine were also oxidized by L-lysine E-aminotransferase. (C) 2000 Elsevier Science Inc. All rights reserved.

The 2-ketoreductase from Gluconobacter oxydans (SC 13851) catalyzes the reduction of 2-pentanone to (S)-(+)-2-pentanol. The 2-ketoreductase was purified 295-fold to homogeneity from G. oxydans cell extracts. The purified 2-ketoreductase had a molecular mass of 29 kDa with a specific activity of 17.7 U/mg. (S)-(+)-2-pentanol was prepared on a pilot scale (3.2 kg of 2-pentanone input) using Triton X-100-treated G. oxydans cells. After 46 h, 1.06 kg (32.3 M%) of (S)-(+)-2-pentanol of >99% enantiomeric excess (ee) was produced. Journal of Industrial Microbiology & Biotechnology (2000) 25, 171–175.

Synthesis of lobucavir prodrug, L-valine, [(1S,2R,3R)-3-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-2-(hydroxymethyl) cyclobutyl]methyl ester monohydrochloride (BMS 233866), requires regioselective coupling of one of the two hydroxyl groups of lobucavir (BMS 180194) with valine. Either hydroxyl group of lobucavir could be selectively aminoacylated with valine by using enzymatic reactions. N-[(Phenylmethoxy)carbonyl]-L-valine, [(1R,2R,4S)-2-(2-amino-6-oxo-1H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester (3, 82.5% yield), was obtained by selective hydrolysis of N,N'-bis[(phenylmethoxy)carbonyl]bis[L-valine], O,O'-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester (1) with lipase M, and L-valine, [(1R,2R,4S)-2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (4, 87% yield) was obtained by hydrolysis of bis[L-valine], O,O'-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester, dihydrochloride (2), with lipase from Candida cylindracea. The final intermediate for lobucavir prodrug, N-[(phenylmethoxy)carbonyl]-L-valine, [(1S,2R,4R)-3-(2-amino-6-oxo-1H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester (5), could be obtained by transesterification of lobucavir using ChiroCLEC BL (61% yield), or more selectively by using immobilized lipase from Pseudomonas cepacia (84% yield).

l-6-Hydroxynorleucine, a key chiral intermediate used for synthesis of a vasopeptidase inhibitor, was prepared in 89% yield and >99% optical purity by reductive amination of 2-keto-6-hydroxyhexanoic acid using glutamate dehydrogenase from beef liver. In an alternate process, racemic 6-hydroxynorleucine produced by hydrolysis of 5-(4-hydroxybutyl)hydantoin was treated with d-amino acid oxidase to prepare a mixture containing 2-keto-6-hydroxyhexanoic acid and l-6-hydroxynorleucine followed by the reductive amination procedure to convert the mixture entirely to l-6-hydroxynorleucine, with yields of 91 to 97% and optical purities of >99%.

The chiral intermediates (S)-3,4-dihydro-1,2(2H)-pyridinedicarboxylic acid, 1-(phenylmethyl)ester [BMS 202665-01] and (S)-3,4-dihydro-1,2(2H)-pyridinedicarboxylic acid, 1,1-dimethylethyl ester [BMS 264406-01] were prepared by oxidation of Nα-carbobenzoxy-l-lysine (Nα-CBZ-l-lysine) and Nα-t-butoxycarbonyl-l-lysine (Nα-t-BOC-l-lysine), respectively, by cell suspensions of Rhodotorula graminis SC 16005.

Chiral intermediate (-)-7-[N,N'-Bis(benzyloxycarbonyl)-N-(guanidino-heptanoyl)]-alpha-acetoxyglycine 2 was prepared for the total synthesis of a (-)-15 deoxyspergualin, an antitumor antibiotic and immunosuppressive agent. The stereoselective acetylation of racemic 7-[N,N'-Bis(benzyloxycarbonyl)-N-(guanidinoheptanoyl)]-alpha-hydroxyglycine 1 was carried out in methyl ethyl ketone (MEK) using lipase from Pseudomonas sp. (lipase AK). Vinyl acetate was used as an acylating agent. A reaction yield of 48% (theoretical max 50%) and an optical purity of 98% were obtained for S-(-)-acetate 2. The unreacted alcohol (+)-1 was obtained in 41% yield and 93.5% optical purity. (C) 1997 Elsevier Science Ltd.

The chiral intermediate (S) [1-(acetoxyl)-4-(3-phenyl)butyl]phosphonic acid, diethyl ester 2 was prepared for the total synthesis of a squalene synthase inhibitor, BMS-188494. The stereoselective acetylation of racemic [1-(hydroxy)-4-(3-phenyl)butyl] phosphonic acid, diethyl ester 1 was carried out in toluene using lipase from Geotrichum candidum. Isopropenyl acetate was used as an acylating agent. A reaction yield of 38% and an optical purity of 95% were obtained for chiral 2. (C) 1997 Elsevier Science Ltd.

Chiral intermediates were prepared by biocatalytic processes with oxidoreductases for the chemical synthesis of some pharmaceutical drug candidates. These include: (i) the microbial reduction of 1-(4-fluorophenyl)-4-[4-(5-fluoro-2-pyrimidinyl)-1-piperazinyl]-1-butanone (1) to R-(+)-1-(4-fluorophenyl)-4-[4-(5-fluoro-2-pyrimidinyl)-1-piperazinyl]-1-butanol (2) [R-(+)-BMY 14802], an antipsychotic agent; (ii) the reduction of N-4-(1-oxo-2-chloroacetyl ethyl) phenyl methane sulfonamide (3) to the corresponding chiral alcohol (4), an intermediate for d-(+)-N-4-{1-hydroxy-2-[(-methylethyl)amino]ethyl}phenyl methanesulfonamide [d-(+) sotalol], a β-blocker with class III antiarrhythmic properties; (iii) biotransformation of Nɛ-carbobenzoxy (CBZ)-l-lysine (7) to Nɛ-CBZ-l-oxylysine (5), an intermediate needed for synthesis of (S)-1-[6-amino-2-{[hydroxy(4-phenylbutyl)phosphinyl]oxy}1-oxohexyl]-l-proline (ceronapril), a new angiotensin converting enzyme inhibitor (6) and (iv) enzymatic synthesis of l-β-hydroxyvaline (9) from α-keto-β-hydroxyisovalerate (16). l-β-Hydroxyvaline (9) is a key chiral intermediate needed for the synthesis of S-(Z)-{[1-(2-amino-4-thiazolyl)-2-{[2,2-dimethyl-4-oxo-1-(sulfooxy)-3-azetidinyl] amino}-2-oxoethylidene]amino}oxyacetic acid (tigemonam) (10), an orally active monobactam.

The chiral intermediate (1S,2R) [3-chloro-2-hydroxy-1-(phenylmethyl)propyl] carbamic acid, 1,1-dimethylethyl ester 2a was prepared for the total synthesis of an HIV protease inhibitor, BMS-186318. The stereoselective reduction of (1S) [3-chloro-2-oxo-1-(phenylmethyl)propyl] carbamic acid, 1,1-dimethyl-ethyl ester 1 was carried out using microbial cultures among which Streptomyces nodosus SC 13149 efficiently reduced 1 to 2a. A reaction yield of 80% was obtained. The optical purity of 99.8% and the diastereomeric purity of 99% were obtained for chiral alcohol 2a. (C) 1997 Elsevier Science Ltd.

alpha-(3-Chloropropyl)-4-fluorobenzenemethanol, a possible intermediate for synthesis of a potential anti-psychotic agent alpha-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazine butanol (BMS 181100), was resolved by acetylation using isopropenyl acetate and lipase PS-30 in heptane. S-alcohol was obtained in 42% yield with >99% optical purity. R-acetate was obtained with 92.6% optical purity by stopping the reaction after 46% conversion. The enzymatically produced acetate was hydrolyzed by lipase PS-30 to give R-alcohol with >99% optical purity after 62-72% conversion. BMS 181100 acetate ester was treated with lipase GC-20 in buffer containing 10% toluene to give the R-alcohol with 97.9% optical purity after 47.6% conversion. The rate and enantioselectivity of hydrolysis by lipase GC-20 were very dependent on the organic solvent. E values ranged from 1 in the absence of organic solvent to >100 with dichloromethane and toluene.

Annals of the New York Academy of SciencesVolume 750, Issue 1 p. 166-174 Stereoselective Microbial Reduction of 2-Keto-3-(N-Benzoylamino)-3-Phenyl Propionic Acid Ethyl Ester Synthesis of Taxol Side-chain Synthon RAMESH N. PATEL, RAMESH N. PATEL Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this authorAMIT BANERJEE, AMIT BANERJEE Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this authorJEFFREY M. HOWELL, JEFFREY M. HOWELL Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this authorCLYDE G. McNAMEE, CLYDE G. McNAMEE Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this authorDAVID BRZOZOWSKI, DAVID BRZOZOWSKI Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this authorVENKAT NANDURI, VENKAT NANDURI Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this authorJOHN K. THOTTATHIL, JOHN K. THOTTATHIL Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this authorLASZLO J. SZARKA, LASZLO J. SZARKA Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this author RAMESH N. PATEL, RAMESH N. PATEL Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this authorAMIT BANERJEE, AMIT BANERJEE Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this authorJEFFREY M. HOWELL, JEFFREY M. HOWELL Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this authorCLYDE G. McNAMEE, CLYDE G. McNAMEE Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this authorDAVID BRZOZOWSKI, DAVID BRZOZOWSKI Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this authorVENKAT NANDURI, VENKAT NANDURI Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this authorJOHN K. THOTTATHIL, JOHN K. THOTTATHIL Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this authorLASZLO J. SZARKA, LASZLO J. SZARKA Department of Microbial Technology and Chemical Process Research Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 191 New Brunswick, New Jersey 08903Search for more papers by this author First published: March 1995 https://doi.org/10.1111/j.1749-6632.1995.tb19946.xCitations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 Wani, M. C., H. L. Taylor, M. E. Wall, P. Coggon & A. T. McPhail. 1971. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J. Am. Chem. Soc. 93: 2325–2327. 2 Kingston, D. G. I. 1991. The chemistry of taxol. Pharm. Ther. 52: 1–34. 3 Lythgoe, B. The taxus alkaloids. In The alkaloids. Chemistry and physiology. R. H. F. Manske & Ed.: vol. X: 597–626. Academic Press. New York . 4 Schiff, P. B., J. Fant & S. B. Horowitz. 1979. Promotion of microtubule assembly in vitro by taxol. Nature 277: 665–667. 5 Miller, R. W., R. G. Powell, C. R. Smith, E. Arnold & J. Clardy. 1981. Antileukemic alkaloids from Taxus wallichiana Zucc. J. Org. Chem. 46: 1469–1471. 6 Senith, V., S. Blecherr, M. Colin, D. Guenard, F. Plcot, P. Potier & P. J. Varenne. 1981. New derivatives of taxol. J. Nat. Prod. (Lloydia) 47: 131–137. 7 Holton, R. A., R. R. Juo, H. B. Kim, A. D. Williams, S. Harusawa, R. E. Lowenthal & S. Yogai. 1988. A synthesis of tauxsin. J. Am. Chem. Soc. 110: 6558–6560. 8 Denis, J-N., A. E. Greene, A. Aarao Serre & M-J. Luche. 1986. An efficient enantioselective synthesis of the taxol side chain. J. Org. Chem. 51: 46–50. 9 Christen, A. A., J. Bland & D. M. Gibson. 1989. Cell culture as a means to produce taxol. Proc. Am. Assoc. Cancer Res. 30: 566–568. 10 Ojima, I., I. Habus, M. Zhao, M. Zucco, Y. H. Park, C. M. Sun & T. Brigaud. 1992. New and efficient approaches to the semisynthesis of taxol and its C-13 side chain analogs by means of β-lactam synthon method. Tetrahedron 48: 6985–7012. 11 Deng, L. & E. N. Jacobson. 1992. A practical, highly enantioselective synthesis of the taxol side chain via asymmetric catalysis. J. Org. Chem. 57: 4320–4323. 12 Palomo, C., A. Arrieta, F. Cossio, J. H. Aizpuruna, A. Mielgo & N. Aurrekoetxea. 1990. Highly stereoselective synthesis of α-hydroxy β-amino acids through β-lactams: application to the synthesis of the taxol and bestatin side chains and related systems. Tetrahedron Lett. 31: 6429–6432. 13 Denis, J-N., A. Correa & A. E. Greene. 1990. An improved synthesis of the taxol side chain and of RP 56976. J. Org. Chem. 51: 46–50. 14 Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein dye binding. Anal. Biochem. 72: 248–254. 15 Barlett, P. A. 1980. Stereocontrol in the synthesis of acyclic systems: Applications to natural product synthesis. Tetrahedron 36: 2–72. 16 Ward, O. P. & C. S. Young. 1990. Reductive biotransformations of organic compounds by cells and enzymes of yeast. Enzyme Microb. Technol. 12: 482–492. 17 Csuk, R. & B. I. Glanzer. 1991. Baker's yeast mediated transformations in organic chemistry. Chem. Rev. 91: 49–97. 18 Jones, J. B. & J. F. Back. 1976. In Application of biochemical systems in organic synthesis. J. B. Jones, C. J. Sih & D. Perlman & Eds.: 248–376. John Wiley & Sons. New York . 19 Jones, J. B. 1986. Mechanisms of enzymatic reaction stereochemistry. P. A. Frey, Eds.: 3–14. Elsevier Science. Amsterdam . 20 Keinen, E., E. K. Hafeli, K. K. Seth & R. Lamed. 1981. Thermostable enzymes in organic synthesis. 2. Assymetric reduction of ketones with alcohol dehydrogenase from Thermoanaerobium brockii. J. Am. Chem. Soc. 108: 162–168. 21 Patel, R. N., C. T. Hou, A. I. Laskin & P. Derelanko. 1981. Microbial production of methylketones: Properties of a purified secondary alcohol dehydrogenase from Pichia sp. J. Appl. Biochem. 3: 218–232. 22 Bradshaw, C. W., W. Hummel & C-H. Wong. 1992. Lactobacillus kefir alcohol dehydrogenase: A useful catalyst for synthesis. J. Org. Chem. 57: 1532–1536. 23 Bradshaw, C. W., H. Fu, G-J Shen & C-H. Wong. 1992. A Pseudomonas sp. alcohol dehydrogenase with broad substrate specificity and unusual stereoselectivity for organic synthesis. J. Org. Chem. 57: 1526–1532. 24 Holland, H. L., E. J. Bergen, P. C. Chenchaliah, S. H. Khan, B. Munoz, R. W. Ninnis & D. Richards. 1957. Microbial reduction by Morteirella isaballina. Can. J. Chem. 65: 502–507. 25 Patel, R. N., A. Banerjee, M. Liu, R. L. Hanson, R. Ko, J. Howell & L. J. Szarka. 1992. Microbial reduction of 1-(4-fluorophenyl)-4-[4-(5-fluoro-2-pyrimidinyl)-1-piperazinyl]butan-1-one. Biotechnol. Appl. Biochem. 17: 139–153. 26 Brieva, R., J. Z. Crich & C. J. Sih. 1993. Chemoenzymatic synthesis of the C-13 side chain of taxol: Optically-active 3-hydroxy-4-phenyl β-lactam derivatives. J. Org. Chem. 58: 1068–1075. 27 Gou, D-M., Y-C. Liu & C-S. Chen. 1993. A practical chemoenzymatic synthesis of the taxol C-13 side chain N-benzoyl-(2R,3S)-3-phenylisoserine. J. Org. Chem. 58: 1287–1289. 28 McGuire, W. P., E. K. Rowinsky, N. B. Rosenhain, F. C. Grumbine, D. S. Effinger, D. K. Armstrong & R. C. DoneHower. 1989. Ann. Intern. Med. 111: 273–280. 29 Denis, J-N., A. E. Greene, D. Guenard, F. Gueritte-Voegelein, L. Mangatal & P. Potier. 1988. Highly efficient, practical approach to natural taxol. J. Am. Chem. Soc. 110: 5917–5919. Citing Literature Volume750, Issue1Enzyme Engineering XIIMarch 1995Pages 166-174 ReferencesRelatedInformation

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The key chiral intermediate 3,5-dihydroxy-6-(benzyloxy) hexanoic acid, ethyl ester 2a, was made by the stereoselective microbial reduction of 3,5-dioxo-6-(benzyloxy) hexanoic acid, ethyl ester 1. Among various microbial cultures evaluated, cell suspensions of Acinetobacter calcoaceticus SC 13876 reduced 1 to 2a. The reaction yield of 85% and optical purity of 97% was obtained using glycerol-grown cells. The substrate was used at 2 g l−1 and cells were used at 20% (w/v, wet cells) concentrations. The optimum pH for the reduction of 1 to 2a was 5.5 and the optimum temperature was 32°C. Cell extracts of A. calcoaceticus SC 13876 in the presence of NAD+, glucose, and glucose dehydrogenase reduced 1 to the corresponding monohydroxy compounds 3 and 4 [3-hydroxy-5-oxo-6-(benzyloxy) hexanoic acid ethyl ester 3, and 5-hydroxy-3-oxo-6-(benzyloxy) hexanoic acid ethyl ester 4]. Both 3 and 4 were further reduced to 2a by cell extracts. Reaction yield of 92% and optical purity of 99% were obtained when the reaction was carried out in a 1-l batch using cell extracts. The substrate was used at 10 g l−1. Product 2a was isolated from the reaction mixture in 72% overall yield. The GC and HPLC area % purity of the isolated product was 99% and the optical purity was 99.5%. The reductase which converted 1 to 2a was purified about 200-fold from cell extracts of A. calcoaceticus SC 13876. The purified enzyme gave a single protein band on SDS-PAGE corresponding to 35,000 daltons.

Among various micro-organisms screened for the stereoselective reduction of 4-chloro-1-(4-fluorophenyl)butan-1-one (1), Hansenula polymorpha [American Type Culture Collection (A.T.C.C.) 26012 and 86014], Nocardia salmonicolor [Squibb Culture (S.C.) 6370], Arthobacter simplex (A.T.C.C. 6949), Mycobacterium vaccae (A.T.C.C. 29678), Candida boidinii (A.T.C.C. 13821) and Saccharomyces cerevisiae (A.T.C.C. 13792) reduced compound 1 to the corresponding (R)-(+)-alcohol (2). In contrast, Lactobacillus kefir (A.T.C.C. 35411), Pullularia pullulans (A.T.C.C. 16623), Trigonopsis variabilis (A.T.C.C. 10679) and Cunninghamella echinulata (A.T.C.C. 26269) reduced compound 1 to the (S)-(-)-alcohol (2). When 1-(4-fluorophenyl)-4-(1-piperazinyl)butan-1-one (3) was used as substrate for the reduction, only Nocardia globerula (A.T.C.C. 12505) and Saccharomyces cerevisiae (A.T.C.C. 13792) converted compound 3 into the corresponding (R)-(+)-alcohol (4). Organisms which reduced compound 1 were inactive for the reduction of compound 3. 1-(4-Fluorophenyl)-4-[4-(5-fluoro-2- pyrimidinyl)butan-1-one (5) was reduced to the corresponding (R)-(+)-alcohol (6) by Mortierella ramanniana (A.T.C.C. 38191) and to the (S)-(-)-alcohol (6) by Pullularia pullulans (A.T.C.C. 16623). (R)-(+)-compound 2 and compound 4 are key chiral intermediates in the total chemical synthesis of (R)-(+)-compound 6, an effective antipsychotic agent under development at Bristol-Myers Squibb. A single-stage (fermentation/biotransformation) process and two-stage (fermentation and subsequent biotransformation by cell suspensions) process were developed for the stereoselective reduction of compound 5 to (R)-(+)-compound 6 by Mortierella ramanniana (A.T.C.C. 38191). In both processes, the reaction yield of 98% and the optical purity of 99.4% were obtained for (R)-(+)-compound 6. The enzyme which catalysed the reduction of compound 5 to (R)-(+)-compound 6 was purified to homogeneity. The purified protein consisted of a single polypeptide of 29 kDa.

Among various micro-organisms screened for the stereoselective reduction of 4-chloro-1-(4-fluorophenyl)butan-1-one (1), Hansenula polymorpha [American Type Culture Collection (A.T.C.C.) 26012 and 86014], Nocardia salmonicolor [Squibb Culture (S.C.) 6370], Arthobacter simplex (A.T.C.C. 6949), Mycobacterium vaccae (A.T.C.C. 29678), Candida boidinii (A.T.C.C. 13821) and Saccharomyces cerevisiae (A.T.C.C. 13792) reduced compound 1 to the corresponding (R)-(+)-alcohol (2). In contrast, Lactobacillus kefir (A.T.C.C. 35411), Pullularia pullulans (A.T.C.C. 16623), Trigonopsis variabilis (A.T.C.C. 10679) and Cunninghamella echinulata (A.T.C.C. 26269) reduced compound 1 to the (S)-(-)-alcohol (2). When 1-(4-fluorophenyl)-4-(1-piperazinyl)butan-1-one (3) was used as substrate for the reduction, only Nocardia globerula (A.T.C.C. 12505) and Saccharomyces cerevisiae (A.T.C.C. 13792) converted compound 3 into the corresponding (R)-(+)-alcohol (4). Organisms which reduced compound 1 were inactive for the reduction of compound 3. 1-(4-Fluorophenyl)-4-[4-(5-fluoro-2- pyrimidinyl)butan-1-one (5) was reduced to the corresponding (R)-(+)-alcohol (6) by Mortierella ramanniana (A.T.C.C. 38191) and to the (S)-(-)-alcohol (6) by Pullularia pullulans (A.T.C.C. 16623). (R)-(+)-compound 2 and compound 4 are key chiral intermediates in the total chemical synthesis of (R)-(+)-compound 6, an effective antipsychotic agent under development at Bristol-Myers Squibb. A single-stage (fermentation/biotransformation) process and two-stage (fermentation and subsequent biotransformation by cell suspensions) process were developed for the stereoselective reduction of compound 5 to (R)-(+)-compound 6 by Mortierella ramanniana (A.T.C.C. 38191). In both processes, the reaction yield of 98% and the optical purity of 99.4% were obtained for (R)-(+)-compound 6. The enzyme which catalysed the reduction of compound 5 to (R)-(+)-compound 6 was purified to homogeneity. The purified protein consisted of a single polypeptide of 29 kDa.

The chiral intermediate (2R,3S)-(-)-N-benzoyl-3-phenyl isoserine ethyl ester 2a, a potential taxol 5 side-chain synthon, was prepared by microbial and enzymatic processes. Taxol 5, is an anticancer compound recently approved by FDA for the treatment of ovarian cancer. The stereoselective reduction of racemic 2-keto-3-(N-benzoylamino)-3-phenylpropionic acid ethyl ester 1 to the corresponding alcohol 2 was carried out using microbial cultures. Among microorganisms evaluated, Hansenula polymorpha SC 13865 and Hansenula fabianii SC 13894 effectively reduced compound 1 to the desired syn diastereomer 2a. Reaction yields of >80% and enantiomeric excesses of >98% were observed for these bioreduction process. About 10-20% of anti diastereomers (2c,2d) were produced during bioreduction.

A key chiral intermediate, lactol 2[3aS(3aα, 4α, 7α, 7aα)]-hexahydro-4,7-epoxy-isobenzofuran-1 (3H)-one, and the corresponding chiral lactone 3 were made in high optical purity by stereoselective enzymatic and microbial oxidation of the parent diol 1. Horse liver alcohol dehydrogenase (HLADH) in the presence of nicotinamide adenine dinucleotide (NAD+) and riboflavin oxidized diol 1 (exo, exo)-7-oxabicyclo [2.2.1] heptane-2,3-dimethanol to the corresponding lactol 2 and lactone 3. Regeneration of NAD+ required for the oxidation of diol 1 was carried out by the NADH-dependent alanine dehydrogenase and leucine dehydrogenase in the presence of alanine and leucine, respectively. Since expensive enzyme (HLADH) and cofactors (NAD+) were required for the oxidation reaction, various microorganisms were screened for the ability to catalyze the stereoselective oxidation of diol 1. Two organisms, Nocardia globerula ATCC 21505 and Rhodococcus sp. ATCC 15592, catalyzed the efficient oxidation of diol 1 to the corresponding chiral lactol 2 and lactone 3. The reaction yield of 70% and optical purity of 96% was obtained for lactone 3 prepared from the oxidation of diol 1 by cell suspensions (10% w/v, wet cells) of N. globerula ATCC 21505. Substrate was used at 5 gl−1 concentration. A 10% (w/v, wet cells) cell suspension of Rhodococcus sp. ATCC 15592, after 120 h reaction period, produced lactol 2 in 12% yield and lactone 3 in 34% yield. An overall 46% reaction yield was obtained based on diol 1 oxidation. Substrate was used at 5 gl−1 concentration. Optical purity of 96.7% and 98.4% was obtained for lactol 2 and lactone 3, respectively. Lactone 3 purified by slilica gel chromatography gave a specific rotation of +117° and enantiomeric excess of >99%.

A key chiral intermediate S(-)4-chloro-3-hydroxybutanoic acid, methyl ester 2 was made in high optical purity by the stereoselective reduction of 4-chloro-3-oxobutanoic acid methyl ester 1 by cell suspensions of Geotrichum candidum SC 5469. A reaction yield of 95% and optical purity of 96% was obtained for 2 by glucose-, acetate-, or glycerol-grown cells (10% w/v) of G. candidum SC 5469. Substrate was used at 10 mg ml-1 concentration. The optical purity of 2 was increased to 99% by heat treatment of cell suspensions (55-degrees-C for 30 min) prior to conducting bioreduction of 1 at 28-degrees-C.Glucose-grown cells of G. candidum SC 5469 have also catalyzed the stereoselective reduction of ethyl-, isopropyl-, and tertiary-butyl esters of 4-chloro-3-oxobutanoic acid and methyl- and ethyl esters of 4-bromo-3-oxobutanoic acid. A reaction yield of >85% and optical purity of >94% were obtained. NADP-dependent oxidoreductase responsible for the stereoselective reduction of beta-keto esters of 4-chloro- and 4-bromo-3-oxobutanoic acid was purified 100-fold. The molecular was eight of the purified enzyme is 950,000. The purified oxidoreductase was immobilized on Eupergit C and used to catalyze the reduction of 1 to 2. The cofactor NADP required for the reduction reaction was regenerated by glucose dehydrogenase.