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The synthesis of optically active sulfoxides, compounds due to their unique properties, has been a main target for synthetic organic chemistry. Recent efforts in the field of biocatalysis have allowed the preparation of enantiopure sulfoxides starting from the corresponding sulfides while using relatively mild conditions. In fact, several different types of redox biocatalysts have been found that can catalyze enantio- and/or regioselective sulfoxidations. The most promising group of enzymes able to perform selective sulfoxidations is the flavin-containing monooxygenases (FMOs). Enzymes containing a flavin cofactor have already been widely studied and used in organic synthesis, especially in reduction and/or oxidation processes. This chapter highlights the recent efforts in the preparation of chiral sulfoxides catalyzed by different types of flavoenzymes, with special attention to the parameters that can improve their catalytic properties. Novel approaches that allow performing selective sulfoxidations in which modified flavin systems are used are also discussed.

Screening was carried out to identify strains useful for the preparation of (R)-1-cyclopropylethylamine and (R)-sec-butylamine by resolution of the racemic amines with an (S)-specific transaminase. Several Bacillus megaterium strains from our culture collection 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 E. coli SC16578. In contrast to the original B. 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 E. coli SC16578 was scaled up to give (R)-sec-butylamine⋅1/2 H2SO4 in 46.6% isolated yield with 99.2% ee. An alternative isolation procedure was also used to isolate (R)-sec-butylamine as the free base.

The non-proteinogenic amino acid 2-(3-hydroxy-1-adamantyl)-(2S)-aminoethanoic acid [2, (S)-3-hydroxyadamantylglycine], is a key intermediate required for the synthesis of Saxagliptin, a dipeptidyl peptidase IV inhibitor under development for treatment of type 2 diabetes mellitus. Keto acid 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid (1) was converted to (S)-3-hydroxyadamantylglycine by reductive amination using a phenylalanine dehydrogenase from Thermoactinomyces intermedius expressed in a modified form in Pichia pastoris or Escherichia coli. NAD (nicotinamide adenine dinucleotide) produced during the reaction was recycled to NADH (reduced form of nicotinamide adenine dinucleotide) using formate dehydrogenase. Pichia pastoris produces an endogenous formate dehydrogenase when grown on methanol, and the corresponding gene was cloned and expressed in E. coli. The modified phenylalanine dehydrogenase contains two amino acid changes at the C-terminus and a 12-amino acid extension of the C-terminus. The modified enzyme is more effective with keto acid 1 than the wild-type enzyme, but less effective with the natural substrate, phenylpyruvate. 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, and further scaled up using a preparation of the two enzymes expressed in E. coli.

Many of the familiar relations in the theory of linear viscoelasticity which relate the relaxation spectrum H(τ) to the experimentally measured quantities were extended to embrace the concept of shear dependent relaxation spectrum H(τ, ). The concept is based on the relation H(τ, ) = H(τ)h(θ)g(θ)3/2, where θ = c τ and c = 0.5. Here is the shear rate and τ is the relaxation time. The replacement of H(τ) by H(τ',) in the integral relations of the linear theory produced the corresponding nonlinear parameters, e.g., the shear rate dependent viscosity η(), the shear stress relaxation following cessation of steady-state flow σ(,t) and the dynamic parameters under parallel superimposed rotation η'(ω) and G'(ω, ). The concept was used successfully for several high-density polyethylene melts in interconversion between η(), c(,t) and the dynamic viscosity η'(ω) without involving coordinate shifts or empirical parameters. The failure of this approach for interconversion between η(ω) and η() for the highly branched polyethylenes was ascribed to the level of long-chain branching. For the high-density resins, the agreement between measured η'(ω,) and G'(ω,) and those calculated using H(τ,) was not quantitative since the parallel superimposed experiments require an additional term in ∂H(τ,)/∂ inside the integral.

The synthesis of the leading candidate compound in an anticancer program required (S)-2-chloro-1-(3-chlorophenyl)-ethanol as an intermediate. Other possible candidate compounds used analogues of the S-alcohol. Of 119 microbial cultures screened for reduction of the corresponding ketone to the S-alcohol, Hansenula polymorpha ATCC 58401 (73.8% ee) and Rhodococcus globerulus ATCC 21505 (71.8% ee) had the highest enantioselectivity for producing the desired alcohol. A ketoreductase from Hansenula polymorpha, after purification to homogeneity, gave the S-alcohol with 100% ee. Amino acid sequences from the purified enzyme were used to design PCR primers for cloning the ketoreductase. The cloned ketoreductase required NADP(H), had a subunit molecular weight of 29,220 and a native molecular weight of 88,000. The cloned ketoreductase was expressed in E. coli together with a cloned glucose 6-phosphate dehydrogenase from Saccharomyces cerevisiae to allow regeneration of the NADPH required by the ketoreductase. An extract of E. coli containing the two recombinant enzymes was used to reduce 2-chloro-1-(3-chloro-4-fluorophenyl)-ethanone and two related ketones to the corresponding S-alcohols. Intact E. coli cells provided with glucose were used to prepare (S)-2-chloro-1-(3-chloro-4-fluorophenyl)-ethanol in 89% yield with 100% ee.

The synthesis of a new series of arylmethyltin chlorides is reported, ArnMeSnCl3−n (Ar = RC6H4, R = H, ethyl, i-propyl, t-butyl; hexyl, octyl). The synthesis involves initial formation of triarylmethyltin compounds, Ar3MeSn, via Grignard techniques followed by HCl–Et2O aryl group cleavage, preferably in a stepwise manner. Preliminary biological activity against Staphylococcus aureus illustrates the importance of the para-alkyl substituents and reinforces that an optimal hydrophobic character is needed for maximum efficacy. Copyright © 2005 John Wiley & Sons, Ltd.

The single-crystal structure of the hydrophobic actinide extractant di(p-tert-butylphenyl)-N,N-di-(iso-butyl)carbamoylmethylphosphine oxide (CMPO, 1) has been determined, as has that of its 1:1 chelate complex with uranyl nitrate (2), its 1:1 monodentate complexes with Ph3SnCl (3) and Me2SnCl2 (4) bonding via the PO group. A 1:1 bidentate complex with Ph2SnCl2 bonding via both PO and CO functionalities (5) was obtained which in solution exhibits a CO Sn bond dissociation to exist as the monodentate isomer coordinating only through the PO group. Copyright © 2005 John Wiley & Sons, Ltd.

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.

Chirality is a key factor in the efficacy of many drug products and thus the production of single enantiomers of drug intermediates has become increasingly important in the pharmaceutical industry. Biocatalysis is now accepted as a one of key methodologies for the preparation of chiral drug intermediates and fine chemicals. The biocatalytic production of several key intermediates in the synthesis of antihypertensive, anticholesterol, anti-Alzheimer’s, β3-receptor agonist, HIV-protease inhibitor, and other pharmaceuticals is described. These includes (1) the synthesis of L-6-hydroxynorleucine from racemic 6-hydroxynorleucine, (2) the enzymatic synthesis of (S)-allysine ethylene acetal by reductive deamination using phenylalanine dehydrogenase, (3) the synthesis of [4S-(4a,7a,10ab)]-1-octahydro-5-oxo-4-{[(phenylmethoxy)carbonyl]-amino}-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylic acid (BMS-199541–01) by enzymatic oxidation process using L-lysine-ϵ-aminotransferase, (4) the enzymatic synthesis of the lactol [3aS-(3aα,4α,7α,7aα)]-hexahydro-4,7-epoxyisobenzofuran-1(3H)-ol and corresponding lactone, (5) the microbial synthesis of (3R-cis)-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluoromethyl)-2H-1-benzazepin-2-one, (6) the microbial oxygenation of 6-cyano-2,2-dimethyl-2H-1-benzopyran to the corresponding chiral epoxide and (+)-trans diol, (7) the enantioselective microbial reductions of N-[4-(2-chloroacetyl)phenyl]methanesulfonamide and (4-benzyloxy-3-methanesulfonylamino)-2'-bromoacetophenone to the corresponding (R)-alcohols, (8) the enzymatic resolution of racemic α-methyl phenylalanine amides by amidase, (9) the enantioselective hydrolysis of diethyl methyl-(4-methoxyphenyl)-propanedioate by lipase PS-30, (10) the enantioselective microbial reduction of methyl 4-chloro-3-oxobutanoate, (11) the enzymatic synthesis of ethyl (3S,5R)-dihydroxy-6-(benzyloxy)hexanoate, (12) the enantioselective hydrolysis of racemic epoxide 1-{2',3'-dihydrobenzo[b]furan-4'-yl}-1,2-oxirane by epoxide hydrolase, (13) the biocatalytic dynamic kinetic resolution of R,S-1-{2',3'-dihydrobenzo[b]furan-4'-yl}-ethane-1,2-diol, and (13) the diastereoselective microbial reduction of (1S)-[3-chloro-2-oxo-1-(phenylmethyl)propyl]carbamic acid 1,1-dimethylethyl ester. 1 Introduction 2 Antihypertensive Drugs 3 Thromboxane A2 Antagonists 4 Calcium Channel Blocking Drugs 5 Potassium Channel Openers 6 Antiarrhythmic Agents 7 β3-Receptor Agonists 8 Anticholesterol Drugs 9 Microbial Resolution10 Anti-Alzheimer Drugs11 HIV Protease Inhibitors12 Conclusion

Chiral intermediates S-(+)-2-pentanol and S-(+)-2-heptanol were prepared by a lipase-catalyzed enzymatic resolution proces. Among various lipases evaluated for the stereoselective acylation of racemic alcohols, lipase B from Candida antarctica catalyzed the acylation of the undesired enantiomer of racemic alcohols leaving the desired S-(+)-alcohols unreacted. A reaction yield of 43–45% and an enantiomeric excess (e.e.) of >99% were obtained for S-(+)-2-pentanol or S-(+)-2-heptanol when the reaction was carried out using vinyl acetate or succnic anhydride as acylating agent. In an alternative process, an enantioselective hydrolysis of 2-pentyl acetate was demonstrated using lipase B giving S-(+)-2-pentyl acetate and R-(−)-2-pentanol. A reaction yield of 45% and an e.e. of 98.6% were obtained for S-(+)-2-pentyl acetate.

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 a human immunodeficiency virus protease inhibitor, BMS-186318. The stereoselective reduction of (1S) [3-chloro-2-oxo-1(phenylmethyl)propyl] carbamic acid, 1,1-dimethylethyl ester 1 was carried out using microbial cultures, among which Streptomyces nodosus SC 13149 efficiently reduced 1 to 2a. A reaction yield of 80%, enantiomeric excess (e.e.) of 99.8%, and diastereomeric purity of 99% were obtained for chiral alcohol 2a. Chiral l-6-hydroxy norleucine 3, an intermediate in the synthesis of antihypertensive drug, was prepared by reductive amination of 2-keto-6-hydroxyhexanoic acid 4 using beef liver glutamate dehydrogenase. The cofactor NADH required for this reaction was regenerated using glucose dehydrogenase from Bacillus sp. A reaction yield of 80% and e.e. of 99.5% were obtained for l-6-hydroxynorleucine 3. To avoid the lengthy chemical synthesis of the ketoacid, a second route was developed in which racemic 6-hydroxynorleucine [readily available from hydrolysis of 5-(4-hydroxybutyl) hydantoin 5] was treated with d-amino acid oxidase from Trigonopsis variabilis to selectively convert the d-isomer of racemic 6-hydroxynorleucine to 2-keto-6-hydroxyhexanoic acid 4 and l-6-hydroxynorleucine 3. Subsequently, the 2-keto-6-hydroxyhexanoic acid 4 was converted to l-6-hydroxynorleucine by reductive amination using glutamate dehydrogenase. A reaction yield of 98% and an e.e. of 99.5% were obtained.

Chiral intermediates were prepared by biocatalytic processes for the chemical synthesis of three pharmaceutical drug candidates. These include (i) the synthesis of [(3R-cis)-3-(acetyloxy)-4-phenyl-2-azetidinone2 for the semi-synthesis of paclitaxel (taxol)5, an anticancer compound; (ii) synthesis of chiral (exo,exo)-7-oxabicyclo [2.2.1] heptane-2,3-dimenthanol monoacetate ester9 for the chemoenzymatic preparation of a thromboxane A2 antagonist; (iii) the enzymatic synthesis ofS-(−) 3-benzylthio-2-methylpropanoic acid, a key chiral intermediate for the synthesis of antihypertensive drugs captopril10 or zofenopril13.

10-Deacetylabaccatin III (10 DAB), an important precursor for paclitaxel semisynthesis, is enhanced in yew extracts using C10-deacetylase and C13-deacylase enzymes.(4) C10-deacetylase is an intracellular enzyme produced by the fermentation of a soil microorganism, Nocardioides luteus (SC 13912). During the fermentation of Nocardioides luteus, the growth of cells reaches a maximum growth at 28 h. C10-deacetylase enzyme activity starts at 26 h and peaks at 38 h of the fermentation. The cells are recovered by centrifugation. The C10-deacetylase enzyme was purified from the Nocardioides luteus cells. The enzyme was purified 190-fold to near homogeneity. The purified enzyme appeared as a single band on 12.5% SDS-PAGE analysis with a molecular weight of 40,000 daltons. (c) 1995 John WileySons, Inc.

Chiral intermediates were prepared by biocatalytic processes for the chemical synthesis of four pharmaceutical drug candidates. These include: (i) the microbial reduction of 3,5-dioxo-6-(benzyloxy) hexanoic ethyl ester to (3S,5R)-dihydroxy-6-(benzyloxy) hexanoic acid ethyl ester, an intermediate for a new anticholesterol drug; (ii) synthesis of (2R,3S)-(-)-N-benzoyl-3-phenyl isoserine ethyl ester, a taxol side-chain synthon; (iii) the microbial oxygenation of 2,2-dimethyl-2H-1-benzopyran-6-carbonitrile to the corresponding (3S,4S) epoxide and (3S,4R)-trans diol, intermediates for synthesis of potassium channel opener; (iv) the biotransformation of (exo,exo)-7-oxabicyclo [2.2.1] heptane-2,3-dimethanol to the corresponding chiral lactol and lactone, intermediates for thromboxane A2 antagonist.

A class of doubly stochastic Poisson processes, which is termed a Markov-modulated Poisson process, is studied. The maximum likelihood method is used to make inferences about the Markov-modulated Poisson process. Expressions are derived for the likelihood function and for second-order properties of both counts and intervals. A simple two-state model is applied to a set of exposure data and to simulated data. Bivariate generalization of this process is also studied.

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.

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.

Chirality is a key factor in the efficacy of many drug products and thus the production of single enantiomers of drug intermediates has become increasingly important in the pharmaceutical industry. Biocatalysis is now accepted as a one of key methodologies for the preparation of chiral drug intermediates and fine chemicals. The biocatalytic production of several key intermediates in the synthesis of antihypertensive, anticholesterol, anti-Alzheimer’s, β3-receptor agonist, HIV-protease inhibitor, and other pharmaceuticals is described. These includes (1) the synthesis of L-6-hydroxynorleucine from racemic 6-hydroxynorleucine, (2) the enzymatic synthesis of (S)-allysine ethylene acetal by reductive deamination using phenylalanine dehydrogenase, (3) the synthesis of [4S-(4a,7a,10ab)]-1-octahydro-5-oxo-4-{[(phenylmethoxy)carbonyl]-amino}-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylic acid (BMS-199541–01) by enzymatic oxidation process using L-lysine-ϵ-aminotransferase, (4) the enzymatic synthesis of the lactol [3aS-(3aα,4α,7α,7aα)]-hexahydro-4,7-epoxyisobenzofuran-1(3H)-ol and corresponding lactone, (5) the microbial synthesis of (3R-cis)-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluoromethyl)-2H-1-benzazepin-2-one, (6) the microbial oxygenation of 6-cyano-2,2-dimethyl-2H-1-benzopyran to the corresponding chiral epoxide and (+)-trans diol, (7) the enantioselective microbial reductions of N-[4-(2-chloroacetyl)phenyl]methanesulfonamide and (4-benzyloxy-3-methanesulfonylamino)-2'-bromoacetophenone to the corresponding (R)-alcohols, (8) the enzymatic resolution of racemic α-methyl phenylalanine amides by amidase, (9) the enantioselective hydrolysis of diethyl methyl-(4-methoxyphenyl)-propanedioate by lipase PS-30, (10) the enantioselective microbial reduction of methyl 4-chloro-3-oxobutanoate, (11) the enzymatic synthesis of ethyl (3S,5R)-dihydroxy-6-(benzyloxy)hexanoate, (12) the enantioselective hydrolysis of racemic epoxide 1-{2',3'-dihydrobenzo[b]furan-4'-yl}-1,2-oxirane by epoxide hydrolase, (13) the biocatalytic dynamic kinetic resolution of R,S-1-{2',3'-dihydrobenzo[b]furan-4'-yl}-ethane-1,2-diol, and (13) the diastereoselective microbial reduction of (1S)-[3-chloro-2-oxo-1-(phenylmethyl)propyl]carbamic acid 1,1-dimethylethyl ester. 1 Introduction 2 Antihypertensive Drugs 3 Thromboxane A2 Antagonists 4 Calcium Channel Blocking Drugs 5 Potassium Channel Openers 6 Antiarrhythmic Agents 7 β3-Receptor Agonists 8 Anticholesterol Drugs 9 Microbial Resolution10 Anti-Alzheimer Drugs11 HIV Protease Inhibitors12 Conclusion

A key intermediate, S-(−)-3-benzoylthio-2-methylpropanoic acid (1) was made in high optical purity by the lipase-catalyzed stereoselective esterification of racemic 1 with methanol in an organic solvent system. Among various lipases evaluated, Amano P-30 lipase from Pseudomonas sp. efficiently catalyzed the esterification of 1 to yield R-(+) methyl ester and unreacted S-(−) 1. A reaction yield of 40 mol% and an optical purity of 97.2% were obtained for compound 1 at a substrate concentration of 0.1 m (22 mg/ml). Lipase P-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (23 cycles) in the esterification reaction without loss of enzyme acitivity, productivity or optical purity. Among various solvents evaluated, toluene was found to be the most suitable organic solvent and methanol was the best alcohol for the esterification of racemic 1 by immobilized lipase. Substrate concentrations as high as 1.0 m were used in the esterification reaction. When the temperature was increased from 28° C to 60° C, the reaction time required for the esterification of 0.1 m substrate decreased from 16 h to 2 h. On increasing the methanol to substrate molar ratio from 1:1 to 4:1, the rate of esterification decreased. A lipase fermentation using Pseudomonas sp. ATCC 21 808 was developed. In the batch-fermentation process, 56 units/ml of extracellular lipase activity was obtained. A fed-batch process using soybean oil gave a significant increase in the lipase activity (126 units/ml). Crude lipase recovered from the filtrate by ethanol precipitation and immobilized on Accurel PP was also effective: S-(−) compound 1 was obtained in 35 mol% yield and 95% optical purity.

The production of single enantiomers of drug intermediates has become increasingly important in the pharmaceutical industry. Chiral intermediates and fine chemicals are in high demand from both the pharmaceutical and agrochemical industries for the preparation of bulk drug substances and agricultural products. The enormous potential of microorganisms and enzymes for the transformation of synthetic chemicals with high chemo-, regio- and enantioselectivities has been demonstrated. In this article, biocatalytic processes are described for the synthesis of chiral pharmaceutical intermediates.