Isomerization and Racemization of Menthols

Menthols were converted to Δ³-menthone enol acetate (VII) via menthones having only one asymmetric carbon atom. It was shown that the optical rotation of menthone enol acetate was proportional to the optical purity of starting menthols. Optical purity of original menthol could, therefore, be determined by optical rotation of menthone enol acetate derived from.     

Enantioselectivity-promoting factor in enzyme-mediated asymmetric hydrolysis of enol esters

Enantioselectivity-promoting factor enhances enantioselectivity of protonation in lipase AP-catalyzed asymmetric hydrolysis of enol esters. The factor was partially purified by chromatography using Phenyl-TOYOPEARL 650M and Sephacryl S-200HR. The hydrolysis of 2-benzyl-l-cyclohexenyl acetate by PLE in the presence of the purified factor produced (R)-2-benzylcyclo-hexanone in 92% ee, while the reaction without the factor gave the racemate.     

Asymmetric bioreduction of a bisaryl ketone to its corresponding (S)-bisaryl alcohol, by the yeast Rhodotorula pilimanae ATCC 32762

The screening of 310 microbial strains yielded eight as suitable biocatalysts for the asymmetric bioreduction of a highly hindered bisaryl ketone to its corresponding alcohols. The production of both enantiomers with elevated optical purity (ee>96%) was achieved by different microorganisms. When scaling up the asymmetric bioreduction process in laboratory bioreactors (23 l scale), the production of preparative amounts (1.5 g) of the (S) enantiomer with elevated optically purity (ee>96%) was achieved when employing the yeast Rhodotorula pilimanae ATCC 32762. Achieving this asymmetric bioreduction with enantiocomplementarity in employing such a hindered substrate is remarkable and highlights the potential of such biological approach.     

Process for the Stereoselective Biotransformation of Acetoacetic Acid Esters Using Yeasts

A process for the stereospecific reduction of acetoacetic acid esters to the 3-(S)-hydroxy-butanoic acid esters by the yeasts Saccharomyces cerevisiae and Candida utilis grown on glucose and ethanol media was developed. A continuous single stage steady state production system was found to be superior to pulse-, batch- and fed-batch systems in terms of optical product purity, biomass concentration and production rates.

Optical purity of 3-(S)-hydroxybutanoic acid esters produced with Saccharomyces cerevisiae and Candida utilis was dependent on pH. A maximal optical purity was obtained at pH2.2 from S. cerevisiae growing on ethanol medium. The specific product formation rate of the chemostat cultures was 0.02…0.05 gg^?1 h^?1. C. utilis was more productive than S. cerevisiae but it reconsumed the product under carbon limited growth conditions.     

Some Interesterification Reactions Involving Mucor Miehei Lipase

An enzyme-catalysed inter-esterification strategy is described which allows the preparation of esters of the chiral secondary alcohols (3) and (9) in states of high optical purity.     

Synthetic libraries of tyrosine-derived bacterial metabolites

The preparation of a collection of 131 small molecules, reminiscent of families of long chain N-acyl tyrosines, enamides and enol esters that have been isolated from heterologous expression of environmental DNA (eDNA) in Escherichia coli, is reported. The synthetic libraries of N-acyl tyrosines and their 3-keto counterparts were prepared via solid-phase routes, whereas the enamides and enol esters were synthesized in solution-phase.     

Total Synthesis of (+)-(2S,3R)-Piscidic Acid via Catalytic Asymmetric Dihydroxylation of a Trisubstituted Olefin

(+)-(2S,3R)-Piscidic acid was efficiently synthesized with high optical purity (90% e.e.) via Sharpless catalytic asymmetric dihydroxylation of a trisubstituted olefin in only 6 steps from commercially available 4-hydroxyphenylpyruvic acid as the starting material. The reaction proceeded with high optical purity by using the chiral ligands, dihydroquinidine 2,5-diphenyl-4,6-pyrimidinediyl diether or dihydroquinidine 1,4-anthraquinonediyl diether.     

Chirally selective synthesis of sugar moiety of nucleosides by chemicoenzymatic approach: L- and D-riboses, showdomycin, and cordycepin

The symmetric dimethyl esters derived from furan and dimethyl acetylenedicarboxylate was efficiently hydrolysed with pig liver esterase to yield half esters with high optical purity. Following chemical transformations afforded precursors with L-configuration, and chirality transfer through ester exchange was achieved to afford precursors with D-configuration of the sugar moiety of nucleosides. Thus, an efficient approach to L- and D-riboses, showdomycin, and cordycepin (3'-deoxy-adenosine) has been demonstrated.U     

Biocatalytic properties of a recombinant aldo-keto reductase with broad substrate spectrum and excellent stereoselectivity

In the screening of 11 E. coli strains overexpressing recombinant oxidoreductases from Bacillus sp. ECU0013, an NADPH-dependent aldo-keto reductase (YtbE) was identified with capability of producing chiral alcohols. The protein (YtbE) was overexpressed, purified to homogeneity, and characterized of biocatalytic properties. The purified enzyme exhibited the highest activity at 50°C and optimal pH at 6.5. YtbE served as a versatile reductase showing a broad substrate spectrum towards different aromatic ketones and keto esters. Furthermore, a variety of carbonyl substrates were asymmetrically reduced by the purified enzyme with an additionally coupled NADPH regeneration system. The reduction system exhibited excellent enantioselectivity (>99% ee) in the reduction of all the aromatic ketones and high to moderate enantioselectivity in the reduction of α- and β-keto esters. Among the ketones tested, ethyl 4,4,4-trifluoroacetoacetate was found to be reduced to ethyl (R)-4,4,4-trifluoro-3-hydroxy butanoate, an important pharmaceutical intermediate, in excellent optical purity. To the best of our knowledge, this is the first report of ytbE gene-encoding recombinant aldo-keto reductase from Bacillus sp. used as biocatalyst for stereoselective reduction of carbonyl compounds. This study provides a useful guidance for further application of this enzyme in the asymmetric synthesis of chiral alcohol enantiomers.     

Enantioselective hydrolysis of esters of secondary alcohols using lyophilized Bakers'' yeast

A number of acetoxycarboxylic acid esters were hydrolysed enantioselectively by Saccharomyces cerevisiae Hansen leading to chiral hydroxycarboxylic acid esters of high optical purity. The scope and limitations of this method with respect to the substitutional pattern of substrates were investigated. Subcellular localization of the hydrolytic activity on the plasma membrane led to the assumption that unspecific carboxyl esterases are responsible for hydrolysis of this type of substrate. Comparative experiments using viable cells and lyophilized cells as source of enzyme revealed the latter to be superior with respect to enantioselection and ease of handling.     

Large-scale preparation of (+)-disparlure,the gypsy moth pheromone,by a practical chemico-enzymatic procedure

The Sharpless asymmetric epoxidation of 8-methyl-2-nonen-1-ol performed on a large scale (over 5 moles) at room temperature gave (2S, 3R)-2, 3-epoxy-8-methyl-1-nonanol with 52%ee. The produced epoxy alcohol of low optical purity was subjected to lipase-catalyzed enatioselective acylation in order to increase the optical purity up to 85%ee. Recrystalyzation of the corresponding 3, 5-dinitrobenzoate gave optically pure epoxy alcohol. (+)-Disparlure, the gypsy moth pheromone, was synthesized in two steps from the thus obtained optically pure epoxy alcohol.     

南海深海微生物酯酶EST12-7在制备(R)-2-氯丙酸乙酯中的应用研究

利用来源南海深海的微生物酯酶EST12-7不对称水解反应拆分制备（R）-2-氯丙酸乙酯。并探寻了温度、pH、底物浓度、有机溶剂和反应时间等因素对酯酶EST12-7催化制备（R）-2-氯丙酸乙酯的影响。结果表明,深海微生物酯酶EST12-7催化制备（R）-2-氯丙酸乙酯的最佳反应条件为：13.8 μg/ml酯酶EST12-7,50 mmol/L（±）-2-氯丙酸乙酯,2%正癸醇,pH8.5,30℃,0.05mol/L Tris-HCl,反应60 min。在最佳反应条件下,（±）-2-氯丙酸乙酯的转化率可达49%,所制备的（R）-2-氯丙酸乙酯的光学纯度为98%。通过对酯酶EST12-7拆分制备（R）-2-氯丙酸甲酯和（R）-2-氯丙酸乙酯进行比较,2-氯丙酸酯中的链长对酯酶EST12-7拆分反应有极大的影响。     

Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase

Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.     

Asymmetric Bioreduction of an Allylic Ketosulfone to its Corresponding (R)-Alcohol

The screening of 144 microbial strains yielded several suitable biocatalysts for the asymmetric bioreduction of an allylic ketosulfone to its corresponding (R)-alcohol. The production of the desired enantiomer with elevated optical purity (ee > 90%) was achieved by the yeast strain Candida magnoliae MY 1785. The enantiocomplementary chiral reduction, the production of the (5)-alcohol, was also achieved when employing other microbial strains. The production of preparative amounts (about 2.5 g) of the (R)-enantiomer with elevated optical purity (ee > 90%) was completed when employing the yeast strain Candida magnoliae.     

Diastereoselective synthesis, binding affinity for vitamin D receptor, and chiral stationary phase chromatography of hydroxy analogs of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol.

A series of analogs of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol were obtained with an additional hydroxyl in the aliphatic side chain at carbon atom C-24. These analogs were synthesized by direct and diastereo-selective alpha-hydroxylation of enolates derived from respective vitamin D esters using Davies chiral oxaziridines. The use of (+)-(2R,8aS)-(8, 8-dichlorocamphoryl)sulfonyl oxaziridine resulted in (R) stereochemistry of the new asymmetric center for both series of analogs. Similarly, (-)-(2S,8aR) oxaziridine gave (S) analogs. The diastereomeric purity of hydroxy analogs was determined by high-performance liquid chromatography on a chiral stationary phase. High diastereopurity of hydroxylation of vitamin D esters was obtained without the use of any chiral auxiliary. The binding affinity of (24R)-1,24,25-trihydroxycholecalciferol for the calf thymus intracellular vitamin D receptor was one order of magnitude higher than that of the respective (24S)-diastereomer.     

9-Fluorenylmethyl chloroformate (Fmoc-Cl) as a useful reagent for the synthesis of pentafluorophenyl, 2,4,5-trichlorophenyl,pentachlorophenyl, p-nitrophenyl,o-nitrophenyl and succinimidyl esters of Nα-urethane protected amino acids

Summary 9-Fluorenylmethyl chloroformate has been demonstrated to be useful reagent for the synthesis of several commonly used active esters of Fmoc-/Boc-/Z-amino acids. These include pentafluorophenyl, 2,4,5-trichlorophenyl, pentachlorophenyl,p-nitrophenyl,o-nitrophenyl, and succinimidyl esters. The method is simple, rapid and efficient. All the compounds made have been isolated as crystaline solids in good yield and optical purity. They were fully characterized by IR, and¹H NMR.     

Dynamic kinetic resolutions and asymmetric transformations by enzymes coupled with metal catalysis

The combination of enzyme and metal catalysis is described as a useful method for the synthesis of optically active compounds. A key feature of this new methodology is the use of metal catalysts for the in situ racemization of enzymatically unreactive enantiomers in the enzymatic resolution of racemic substrates. So far, two combinations - lipase-ruthenium and lipase-palladium - have been developed for the efficient dynamic kinetic resolution of alcohols and amines. The use of these combinations has also been extended to catalysis of the asymmetric transformation of ketones, their enol acetates, and ketoximes. In most cases, enzyme-metal combination catalysis has provided good yields and high optical purities.     

Lipase-catalyzed asymmetric hydrolysis of 3-phenylglycidic acid ester,the key intermediate in the synthesis of diltiazem hydrochloride

Asymmetric hydrolytic enzymes for trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM], a key intermediate in the synthesis of diltiazem hydrochloride that is useful as a coronary vasodilator, were screened from 730 microorganisms. Among the microbial enzymes tested, Serratia marcescens lipase had the highest enantioselectivity (E=135) for hydrolysis of (±)-MPGM in a two-phase system using water (pH 8) and a water-immiscible solvent such as toluene. Resolution of (±)-MPGM by S. marcescens lipase gave (2R, 3S)-3-(4-methoxyphenyl)glycidic acid methyl ester [(−)-MPGM] with a reaction yield of 48% and optical purity of >99.9% e.e. After the reaction, the emulsion of toluene and water was separated into two clear layers by the addition of sodium dodecyl sulfate. The crystalline (−)-MPGM was isolated with a yield of over 43% and optical purity of 100% e.e. without column treatment. Diltiazem hydrochloride synthesis using asymmetric hydrolysis of (±)-MPGM was found to be a more efficient process compared to the conventional chemical synthetic process. Some enzymatic characterizations on asymmetric hydrolysis in two-phases of organic solvent-water by S. marcescens lipase were investigated.     

Analysis of the stereochemistry of lipoxygenase-derived hydroxypolyenoic fatty acids by means of chiral phase high-pressure liquid chromatography

A chiral phase HPLC method was developed for the simultaneous determination of the positional and optical isomers of the lipoxygenase-derived hydroxypolyenoic fatty acids. With a Bakerbond chiral phase HPLC column (dinitrobenzoyl phenylglycine as chiral phase) the positional and optical isomers of the reduced dioxygenation products (by triphenylphosphine or borohydride) of linoleic acid and arachidonic acid were separated after methylation of the carboxylic groups. No cumbersome chemical derivatization such as conversion to a diastereomer was necessary. As compared with the methods used up till now chiral phase HPLC proved to be simpler and more sensitive. About 10 pmol of hydroxy fatty acids suffice for an analysis. The chiral phase HPLC can be used for the preparative separation of the optical antipodes of the lipoxygenase products. An optical purity of more than 90% can be reached in one preparative run. The method was applied to the determination of the stereochemistry of the dioxygenation products of polyenoic fatty acids formed by the lipoxygenases from soybeans, reticulocytes, pea seeds (isoenzyme I and II), tomato fruits, by the quasilipoxygenase activity of hemoglobin, and by the methylene blue-mediated photooxidation of arachidonic acid.     

Chiral synthesis of alpha-phenylpyridylmethanols with Rhizopus arrhizus

The bioreduction of 2-benzoylpyridine (1a) with Rhizopus arrhizus afforded (S)-(+)-alpha-phenyl-2-pyridylmethanol (2a) in 82% enantiomeric excess (e.e.) while the asymmetric hydrolysis of its racemic acetate resulted in the antipode (R)-(-)-2a with 24% optical purity.