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An efficient biocatalytic process has been developed for the resolution of methyl (1 RS )- N - t Boc-6-hydroxy-3,4-dihydro-1 H -isoquinoline-1-carboxylate rac - 1 by means of Seaprose S-mediated enantioselective hydrolysis to afford (1 S )- 2 and (1 R )- 1 in 87% and 93% isolated yield, 101% and 96% potency, and ee >99.8% and >99.5%, respectively.

An efficient biocatalytic process has been developed for the resolution of 1-(3′-bromophenyl)ethylamine ( RS )- 1 by way of enantioselective lipase-mediated ( R )-selective acylation with ethyl 2-methoxyacetate to afford ( S )-amine ( S )- 1 and ( R )-2″-methoxyacetamide (( R )- 2 ) in 91–95% and 90–92% isolated yield, respectively, and both with >99% ee.

Microbial reduction of ethyl 1-benzyl-3-oxo-piperidine-4-carboxylate by the majority of evaluated microorganisms gave the ethyl cis-(3R,4R)-1-benzyl-3R-hydroxy-piperidine-4R-carboxylate as the major product in high diastereo- and enantioselectivities. The 3R,4R-hydroxy ester was produced in 97.4% diastereomeric excess (de) and 99.8% enantiomeric excess (ee) by Candida parapsilosis SC16347, while 99.5% de and 98.2% ee were obtained from reduction by Pichia methanolica SC16415. A few of the evaluated microorganisms gave 10–40% of the ethyl trans-(3R,4S)-1-benzyl-3R-hydroxy-piperidine-4S-carboxylate as the minor product.

Previously we have demonstrated the reduction of ethyl diketoester 4 to the corresponding dihydroxy ester 6a by Acinetobacter sp. SC13874. Recently we screened more than 100 cultures for microbial reduction of both the ethyl and t-butyl diketoesters 4 and 5. Most yeast cultures showed a preference for reduction at the C-3 with low enantioselectivity. Among the three Acinetobacter strains screened, Acinetobacter sp. SC13874 reduced both compounds 4 and 5 to the corresponding (3R)- and (5S)-monohydroxy compounds. Monohydroxy compounds were isolated and their absolute configurations determined. (3R)- and (5S)-Monohydroxy compounds were reduced further to the corresponding dihydroxy esters 6a and 8a to provide alternate routes for the synthesis of compounds 14a and 16a, potential intermediates for the synthesis of HMG-CoA reductase inhibitors. Cell suspensions of Acinetobacter sp. SC13874 reduced the ethyl diketoester 4 to a mixture of desired syn and undesired anti diastereomers. The desired syn-(3R,5S)-dihydroxy ester 6a was obtained with an enantiomeric excess (ee) of 99% and a diastereomeric excess (de) of 63%. Cell suspensions reduced the t-butyl diketoester 5 to a mixture of mono- and dihydroxy esters with the dihydroxy ester showing an ee of 87% and de of 51% for the desired syn-(3R,5S)-dihydroxy ester 8a. Three different ketoreductases were purified to homogeneity, and their biochemical properties compared. Reductase I only catalyzes the reduction of ethyl diketoester 4 to its monohydroxy products 10 and 11, whereas reductase II catalyzes the formation of dihydroxy products 6 and 7 from monohydroxy substrates 10 and 11. A third reductase (III) was identified, which catalyzes the reduction of diketoester 4 to syn-(3R,5S)-dihydroxy ester 6a.

Enantioselective asymmetric hydrolysis of (1α,2β,3α)-2-(benzyloxymethyl)-cyclopent-4-ene-1,3-diol diacetate 1 to the corresponding (+)-monoacetate 2 was carried out using lipase PS-30 from Pseudomonas cepacia or pancreatin. A reaction yield of 85 M % with an enantiomeric excess (ee) of 98% was obtained. Using pancreatin, a reaction yield of 75 M % with an ee of 98.5% was obtained. Asymmetric acetylation of (1α,2β,3α)-2-(benzyloxymethyl)-cyclopent-4-ene-1,3-diol 3 to the corresponding (−)-monoacetate 4 was carried out using lipase PS-30 with isopropenyl acetate as the acylating agent. A reaction yield of 80 M % with an ee of 98% was obtained for (−)-monoacetate 4 .

The enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione 1 to either of the corresponding (R)- or (S)-6-hydroxy-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-diones 2 and 3 is described.

6-Hydroxybuspirone is an active metabolite of the antianxiety drug buspirone. The (R)- and (S)-enantiomers of 6-hydroxybuspirone were prepared using an enzymatic resolution process. l -Amino acid acylase from Aspergillus melleus (Amano Acylase 30000) was used to hydrolyze racemic 6-acetoxybuspirone to (S)-6-hydroxybuspirone in 95% ee after 45% conversion. The remaining (R)-6-acetoxybuspirone with 88% ee was converted to (R)-6-hydroxybuspirone by acid hydrolysis. The ee of both enantiomers could be improved to 99% by crystallization as a metastable polymorph. (S)-6-Hydroxybuspirone was also obtained in 88% ee and 14.5% yield by hydroxylation of buspirone using Streptomyces antibioticus ATCC 14890.

Resolution of (±)- sec -butylamine by Candida antarctica lipase provided a very low enantiomeric excess of the residual amine when either ethyl or vinyl butyrate was used as the acylating agent. The enantiomeric excess was increased by using ethyl esters of long chain fatty acids. The rate of the reaction was increased by using methyl t -butyl ether as a solvent. ( S )- sec -Butylamine of very high enantiomeric excess was obtained by C. antarctica lipase catalyzed acylation with ethyl decanoate in methyl t -butyl ether.

The chiral intermediate ( S )-1-(2′-bromo-4′-fluoro phenyl)ethanol 2 was prepared by the enantioselective microbial reduction of 2-bromo-4-fluoro acetophenone 1 . Organisms from genus Candida , Hansenula , Pichia , Rhodotorula , Saccharomyces , Sphingomonas and Baker's yeast reduced 1 to 2 in >90% yield and 99% enantiomeric excess (ee). In an alternative approach, the enantioselective microbial reductions of methyl, ethyl, and tert -butyl 4-(2′-acetyl-5′-fluorophenyl) butanoates 3 , 5 , and 7 , respectively, were demonstrated using strains of Candida and Pichia . Reaction yields of 40–53% and ee's of 90–99% were obtained for the corresponding ( S )-hydroxy esters 4 , 6 , and 8 . The reductase, which catalyzed the enantioselective reduction of ketoesters was purified to homogeneity from cell extracts of Pichia methanolica SC 13825. It was cloned and expressed in Escherichia coli with recombinant cultures used for the enantioselective reduction of keto methyl ester 3 to the corresponding ( S )-hydroxy methyl ester 4 . On a preparative scale, a reaction yield of 98% and an ee of 99% was obtained.

The chiral intermediate (3R)-cis-3-acetyloxy-4-(1,1-dimethylethyl)-2-azetidinone 2 was prepared for the semi-synthesis of the new taxane 5, an orally active anticancer compound. The enantioselective enzymatic hydrolysis of cis-3-acetyloxy-4-(1,1-dimethylethyl)-2-azetidinone 1 to the corresponding undesired (S)-alcohol 3 and unreacted desired 2 was carried out using immobilized lipase PS-30 or BMS lipase. Reaction yields of >48% and enantiomeric excesses of >99% were obtained for the desired 2. Acetoxy β-lactam 2 was converted to hydroxy β-lactam 4 for use in the semisynthesis of 5.

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 the HIV protease inhibitor Atazanavir. The diastereoselective 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 Rhodococcus, Brevibacterium, and Hansenula strains reduced 1 to 2a. Three strains of Rhodococcus gave >90% yield. A diastereomeric purity of >98% and enantiomeric excess of >99.3% were obtained for alcohol 2a.

The chiral ester ethyl (2 R )-hydroxy-2-(1′,2′,3′,4′-tetrahydro-1′,1′,4′,4′-tetramethyl-6′-naphthalenyl) acetate 2 and the corresponding acid 4 were prepared as intermediates in the synthesis of the retinoic acid receptor gamma-specific agonist ( R )-3-fluoro-4-[[hydroxy(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)acetyl]amino]benzoic acid 7 . Enantioselective reduction of ethyl 2-oxo-2-(1′,2′,3′,4′-tetrahydro-1′,1′,4′,4′-tetramethyl-6′naphthalenyl)acetate 1 to alcohol 2 was carried out using Aureobasidium pullulans SC 13849 in 98% yield and with an enantiomeric excess (e.e.) of 96%. Among microorganisms screened for the reduction of 2-oxo-2-(1′,2′,3′,4′-tetrahydro-1′,1′,4′,4′-tetramethyl-6′-naphthalenyl)acetic acid 3 to hydroxy acid 4 , Candida maltosa SC 16112 and two strains of Candida utilis (SC 13983, SC 13984) gave reaction yields of >53% with e.e.s of >96%.

Phenylpyruvate decarboxylase from Achromobacter eurydice was used to catalyse the asymmetric acyloin condensation of phenylpyruvate 1 with various aldehydes 2 to produce optically active acyloins PhCH2COCH(OH)R 3. The specific activity of the phenylpyruvate decarboxylase enzyme was increased by a factor of 332 after its purification. The molecular weight of the purified enzyme was shown to be 150 kDa by gel filtration chromatography, while SDS gel electrophoresis showed two sub-units with molecular weights of 90 and 40 kDa. The acyloin condensation yield decreased with increasing chain length for straight chain aliphatic aldehydes from 76% for acetaldehyde to 24% for valeraldehyde. The e.e.s of the acyloin products were 87–98%. Low yields of acyloin products were obtained with chloroacetaldehyde (13%) and glycoaldehyde (16%). Indole-3-pyruvate was a substrate of the enzyme and provided acyloin condensation product 3-hydroxy-1-(3-indolyl)-2-butanone 5 with acetaldehyde in 19% yield, while benzoylformate was not a substrate for the enzyme.

Cells obtained from growth of Achromobacter eurydice , Pseudomonas aromatica and Pseudomonas putida on l -phenylalanine containing medium catalyzed the enzymatic acyloin condensation of phenylpyruvic acid 1 and acetaldehyde 2 by phenylpyruvate decarboxylase to produce 3-hydroxy-1-phenyl-2-butanone 3 . The acyloin condensation by Achromobacter eurydice and P. aromatica was stereoselective, providing the 3 R enantiomer 3a with enantiomeric excess (ee) of 95% and 84%, respectively. A partially purified enzyme was prepared from the cell free extract of Achromobacter eurydice. The acyloin product 3a was obtained in 45% yield with an ee of 91% by using this partially purified preparation of phenylpyruvate decarboxylase.

Deracemization of racemic 1,2-diol, (RS)-1-{2′,3′-dihydrobenzo[b]furan-4′-yl}-ethane-1,2-diol (1), by stereoinversion by several microorganisms belonging to the genera Candida and Pichia provided the corresponding chiral (S)-diol, (S)-1-{2′,3′-dihydrobenzo[b]furan-4′-yl}-ethane-1,2-diol (2). (S)-Diol 2 was obtained in yields of 60–70% with 90–100% enantiomeric excess.

Among various microbial cultures evaluated, Rhodotorula glutinis SC 16293 and Aspergillus niger SC 16311 catalyzed the stereospecific hydrolysis of the racemic epoxide, RS -1-{2′,3′-dihydrobenzo[ b ]furan-4′-yl}-1,2-oxirane, 1 to the corresponding R -diol, R -1-{2′,3′-dihydrobenzo[ b ]furan-4′-yl}-ethane-1,2-diol, 3 . The S -epoxide, S -1-{2′,3′-dihydrobenzo[ b ]furan-4′-yl}-1,2-oxirane, 2 remained unreacted in the reaction mixture. A reaction yield of 45–50% (theoretical maximum yield is 50%) and an enantiomeric excess (ee) of >95% were obtained for unreacted S -epoxide 2 using each culture. Addition of 10% methyl tert -butyl ether to an aqueous reaction mixture during hydrolysis by R. glutinis improved the ee of the unreacted S -epoxide 2 to >99% (yield 48%) and that of the R -diol 3 to 79%. Unlike R. glutinis , hydrolysis of racemic epoxide 1 in the presence of 10% methyl tert -butyl ether by A. niger showed an adverse effect and gave S -epoxide 2 in 54% yield and 49% ee.

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.

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 .

Chiral intermediate (−)-7-[ N , N ′-Bis(benzyloxycarbonyl)- N -(guanidinoheptanoyl)]-α-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)]-α-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 98.5% optical purity.

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.

The chiral intermediate diol (3 S ,4 R )-trans-3,4-dihydro-3,4-dihydroxy-2,2-dimethyl-2 H -1-benzopyran-6-carbonitrile 2 . was prepared for the total synthesis of a potassium channel opener drug candidate. The stereoselective acetylation of racemic 1 was carried out with various lipases among which the lipases from Candida cylindraceae and Pseudomonas cepacia catalyzed the acetylation of the undesired enantiomer of racemic 1 to yield monoacetylated product and unreacted desired (+)-trans diol 2 . A reaction yield of >40% and an optical purity >90% were obtained using each lipase.

α-(3-Chloropropyl)-4-fluorobenzenemethanol, a possible intermediate for synthesis of a potential anti-psychotic agent α-(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.

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.