Resolution and Synthesis of (S)-1-(2-Naphthyl)ethanol with Immobilized Pea Protein: As a New Biocatalyst

(S)-1-(2-Naphthyl)ethanol was yielded by immobilized pea (Pisum sativum L.) protein (IPP) from (R, S) 2-naphthyl ethanol (>99% ee, yield; about 50%), in which the (R)-enantiomer was selectively oxidized to 2-acetonaphthone. IPP could be reused consecutively at least three times without any decrease of yield and optical purity.     

Biochemical approaches to the synthesis of ethyl 5-(s)-hydroxyhexanoate and 5-(s)-hydroxyhexanenitrile

Three different biochemical approaches were used for the synthesis of ethyl 5-(S)-hydroxyhexanoate 1 and 5-(S)-hydroxyhexanenitrile 2. In the first approach, ethyl 5-oxo-hexanoate 3 and 5-oxo-hexanenitrile 4 were reduced by Pichia methanolica (SC 16116) to the corresponding (S)-alcohols, ethyl (S)-5-hydroxyhexanoate 1 and 5-(S)-hydroxyhexanenitrile 2, with an 80-90% yield and >95% enantiomeric excess (e.e). In the second approach, racemic 5-hydroxyhexanenitrile 5 was resolved by enzymatic succinylation, leading to the formation of (R)-5-hydroxyhexanenitrile hemisuccinate and leaving the desired alcohol 5-(S)-hydroxyhexanenitrile 2 with a yield of 34% (50% maximum yield) and >99% e.e. In the third approach, enzymatic hydrolysis of racemic 5-acetoxy hexanenitrile 6 resulted in the hydrolysis of the R-isomer to provide 5-(R)-hydroxyhexanenitrile, leaving 5-(S)-acetoxyhexanenitrile 7 with a 42% yield (50% maximum yield) and >99% e.e.     

羰基还原酶产生菌Candida ontarioensis制备(R)-2-氯-1-(3-氯苯基)乙醇

从实验室保藏的菌株中筛选获得Candida sp.PT2A,并通过18S rRNA鉴定为安大略假单胞菌Candida on-tarioensis。对C.ontarioensis不对称还原合成(R)-2-氯-1-(3-氯苯基)乙醇的发酵产酶条件和转化条件进行优化,确定了最适的发酵产酶条件和转化条件:温度30℃,初始pH 6.5,摇床转速180 r/min,菌体质量浓度200 g/L。采用2-氯-1-(3-氯苯基)乙酮质量浓度为10 g/L时,还原反应72 h,(R)-2-氯-1-(3-氯苯基)乙醇的e.e.值为99.9%,产率为99%;底物质量浓度提高至30 g/L时,产率下降为84.3%。采用十六烷基三甲基溴化铵(CTAB)对C.ontarioensis细胞进行通透性处理(CTAB g/L,4℃下处理20 min),在30 g/L底物下反应24 h,产物的e.e.和产率分别达到99.9%和97.5%。     

Biotransformation of (R)-(+)- and (S)-(-)-limonene by fungi and the use of solid phase microextraction for screening

The biotransformation of (R)-(-)- and (S)-(-)-limonene by fungi was investigated. More than 60 fungal cultures were screened for their ability to bioconvert the substrate, using solid phase microextraction as the monitoring technique. After screening, the best fungal strains were selected for further study and were grown as sporulated surface cultures in conical flasks and as submerged liquid cultures. It was found that (+)- and (-)-limonene were converted by Penicillium digitatum to alpha-terpineol (main metabolite), cis- and trans-p-menth-2-en-1-ol, neodihydrocarveol and limonene oxide (minor metabolites) using liquid cultures. The bioconversion of (R)-(-)- and (S)-(-)-limonene by Corwespora cassiicola yielded (1S,2S,4R)- and (1R,2R,4S)-limonene-1,2-diol respectively. The bioconversions by liquid cultures were also monitored by solid phase microextraction as a function of time. The optimum conversion of limonene to alpha-terpineol by Penicillium digitatum was obtained after 8 hours (yield up to 100%). Since an important pH-decrease was noticed in some liquid broths, the stability of limonene under acidic conditions was investigated. No acid catalysed conversion products were recovered after 8 days from control flasks at pH 3.5 containing limonene.     

Stereoselective synthesis of chirally deuterated (S)-D-(6-(2)H(1))glucose

Chirally deuterated (S)-D-(6-(2)H(1))glucose has been prepared in good overall yield from d-(6,6'-(2)H(2))glucose by a short, five-step synthesis from D-(6,6-(2)H(2))glucose utilizing (R)-(+)-Alpine-Borane [(R)-9-[(6,6-dimethylbicyclo[3.1.1]hept-2-yl)methyl]-9-borabicyclo[3.3.1]nonane]. Suitably protected methyl 2,3,4-tri-O-benzyl-D-(6,6-(2)H(2))glucopyranoside was prepared and the deuterated O-6 primary alcohol was oxidized to an aldehyde by Swern oxidation. Stereoselective reduction with nondeuterated (R)-(+)-Alpine-Borane gave methyl 2,3,4-tri-O-benzyl-(6S)-D-(6-(2)H(1))glucopyranoside, which was deprotected under standard conditions to afford the title compound. The key stereoselective reduction step was achieved in 90% yield. The preparation uses economical, commercially available starting materials and will be useful for elucidating biosynthetic mechanisms.     

Synthesis and preliminary evaluation of (R,R)(S,S) 5-(2-(2-[4-(2-[(18)F]fluoroethoxy)phenyl]-1-methylethylamino)-1-hydroxyethyl)-benzene-1,3-diol ([(18)F]FEFE) for the in vivo visualisation and quantification of the beta2-adrenergic receptor status in lung

The (18)F-labeled beta2-adrenergic receptor ligand (R,R)(S,S) 5-(2-(2-[4-(2-[(18)F]fluoroethoxy)phenyl]-1-methylethylamino)-1-hydroxyethyl)-benzene-1,3-diol, a derivative of the original highly selective racemic fenoterol, was synthesized in an overall radiochemical yield of 20% after 65 min with a radiochemical purity higher than 98%. The specific activity was in the range of 50-60 GBq/micromol. In vitro testing of the non-radioactive fluorinated fenoterol derivative with isolated guinea pig trachea was conducted to obtain an IC(50) value of 60 nM. Preliminary ex vivo organ distribution and in vivo experiments with positron emission tomography (PET) on guinea pigs were performed to study the biodistribution as well as the displacement of the radiotracer to prove specific binding to the beta2-receptor.     

Computational study of Simultaneous synthesis of optically active (RS)-1,2,4-butanetriol trinitrate (BTTN)

In respective water or ethanol polarizable continuum cavity environments, simultaneous aldol condensation was performed using density functional theory (DFT) computational method to model the synthesis of optically active (RS)-1,2,4-butanetriol trinitrate (BTTN). The results of reaction energy barrier analysis suggested feasible routes with lower activation energies to obtain either the (R)- or (S)-configuration product in ethanolic solution. In addition, local analysis of average inter-particulate distances of reaction species revealed that a stronger inter-particulate interaction accompanied a shorter average distance in the ethanol system. The stabilization effect also indicated that related syntheses would be able to proceed in ethanol. Furthermore, relative to the production of (R)-BTTN, a lower overall energy of 425.3 kJ/mol was required for the synthesis of (S)-BTTN. Through analysis of the effects of temperature on the reaction rates of individual parallel stages of (R)- and (S)-species synthesis, it was simple to adjust the reaction temperature accordingly to differentiate between relative rates in order to obtain a product of a specific configuration.

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Biomimetic conversion of (3S)-(-)-neodictyoprolenol to optically pure (1S,2R)-(-)-dictyopterene B,marine algal sex pheromone

Both enantiomers of (3S)-(-)- and (3R)-(+)-Neodictyoprolenol [(3S,5Z,8Z)-(-)-1,5,8-undecatrien-3-ol] were successfully converted to the algal sex pheromone, (1S,2R)-(-)-dictyopterene B and (1R,2S)-(+)-dictyopterene B in high enantiomeric purities (e. e. > 99%), respectively, by the biomimetic reaction involving phosphorylation and elimination under a mild condition.     

A validated enantioselective assay for the simultaneous quantitation of (R)-, (S)-fluoxetine and (R)-, (S)-norfluoxetine in ovine plasma using liquid chromatography with tandem mass spectrometry (LC/MS/MS)

A liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantitation of (R)-, (S)-fluoxetine, and (R)-, (S)-norfluoxetine in ovine plasma. The analytes were extracted from ovine plasma at a basic pH using a single-step liquid-liquid extraction with methyl-tert-butyl ether. Chromatographic separation of all enantiomers was achieved using an AGP-chiral column with a run time of 10 min. (R)-, (S)-fluoxetine, and (R)-, (S)-norfluoxetine were quantitated at the total ion current (TIC) of multiple reaction monitoring (MRM) transitions of m/z 310.2→44.1, m/z 310.2→147.7 for (R)-, (S)-fluoxetine, and m/z 296.2→30.3, m/z 296.2→133.9 for (R)-, (S)-norfluoxetine. This method was validated for accuracy, precision, linearity, range, limit of quantitation (LOQ), selectivity, recovery, dilution integrity, matrix effect, and evaluation of carry-over. Observed accuracy ranges were as follows: (R)-fluoxetine -8.82 to 3.75%; (S)-fluoxetine -10.8 to 1.46%; (R)-norfluoxetine -7.50 to 0.37% and (S)-norfluoxetine -8.77% to -1.33%. Observed precision ranges were as follows: (R)-fluoxetine 5.29-11.5%; (S)-fluoxetine 3.91-11.1%; (R)-norfluoxetine 4.32-7.67% and (S)-norfluoxetine -8.77% to -1.33%. The calibration curves were weighted (1/X(2), n=4) and observed to be linear for all analytes with the following r(2) values: (R)-fluoxetine ≥ 0.997; (S)-fluoxetine ≥ 0.996; (R)-norfluoxetine ≥ 0.989 and (S)-norfluoxetine ≥ 0.994. The analytical range of the method was 1-500 ng/ml with an LOQ of 1 ng/ml for all analytes, using a sample volume of 300 μL.     

Intramolecular reductive cyclization strategy to the synthesis of (-)-6-methyl-3-hydroxy-piperidine-2-carboxylic acid, (+)-6-methyl-(2-hydroxymethyl)-piperidine-3-ol and their glycosidase inhibitory activity

The first stereoselective synthesis of (2S,3R,6S)-6-methyl-3-hydroxy-piperidine-2-carboxylic acid (-)-6 and (2R,3R,6S)-6-methyl-(2-hydroxymethyl)-piperidine-3-ol (+)-7 was achieved starting from readily available d-glucose in 14 steps with 17% overall yield for both the compounds. The key feature of the present strategy includes the Wittig-olefination for the preparation of required conjugated keto-azide 9 and construction of 2,3,6-trisubstituted piperidine skeleton 11 by applying intramolecular reductive cyclization of conjugated keto-azide intermediate. The glycosidase inhibitory activity of compounds 6 and 7 towards several glycosidases has been evaluated.     

Enzymatic resolution of (+/-)-gamma-cyclohomogeraniol and conversion of its (S)-isomer to (S)-gamma-coronal, the ambergris odorant.

Enzymatic acetylation of (+/-)-gamma-cyclohomogeraniol[2-(2',2'-dimethyl-6'-methylenecyc lohexyl)ethanol] with vinyl acetate in the presence of lipase AK yielded the acetate of its (R)-isomer, leaving its (S)-isomer intact. The (S)-isomer was chemically converted to (S)-gamma-coronal[2-methylene-4-(2',2'-dimethyl-6'-methylenecyclohexy l)butanal], the ambergris odorant.     

Microbial Asymmetric Reduction. Preparation of Optically Active Methyl trans-3-(4-Methoxyphenyl)Glycidates

As a result of screening of microorganisms, Mucor ambiguus IFO 6742 was found to reduce methyl 2-chloro-3-(4-methoxyphenyl)-3-oxopropionate (2) to give methyl (2S,3R)-2-chloro-3-hydroxy-3-(4-methoxyphenyl)propionate [(2S,3R)-3] in good yield with high enantioselectivity. The resulting (2S, 3R)-3 was converted into methyl (2S,3R)-3-(4-methoxyphenyl)glycidate [(2S,3R)-4] by treatment with sodium methoxide. On the other hand, its enantiomer, (2R,3S)-4 was obtained by the Mitsunobu esterification of (2S,3R)-3 and subsequent treatment with sodium methoxide. Also (2R,3S)-4 was obtained by the treatment of (2RS,3S)-3, which was obtained from 2 by Trichoderma viride OUT 4642, with sodium methoxide.     

产碱杆菌ECU0401催化拆分扁桃酸及其衍生物

从土壤中分离的1株产碱杆菌Alcaligenes sp.ECU0401具有扁桃酸脱氢酶活性,可以以扁桃酸、苯甲酰甲酸或苯甲酸为唯一C源生长,并且具有较高的脱氢酶活力。以外消旋扁桃酸为C源,采用分批补料策略培养（或反应）99h,扁桃酸累计投入量为30.4g/L,（S）-（＋）-扁桃酸被完全降解,（R）-（-）-扁桃酸回收产率为32.8%,对映体过量值（e.e.）〉99.9%。利用静息细胞作为催化剂不对称降解外消旋扁桃酸的氯代衍生物,制备获得光学活性的（R）-（-）-邻氯扁桃酸、（S）-（＋）-间氯扁桃酸和（S）-（＋）-对氯扁桃酸,光学纯度均超过99.9%e.e.。     

Production of (R)-3-amino-3-phenylpropionic acid and (S)-3-amino-3-phenylpropionic acid from (R,S)-N-acetyl-3-amino-3-phenylpropionic acid using microorganisms having enantiomer-specific amidohydrolyzing activity

(R)-3-Amino-3-phenylpropionic acid ((R)-beta-Phe) and (S)-3-amino-3-phenylpropionic acid ((S)-beta-Phe) are key compounds on account of their use as intermediates in synthesizing pharmaceuticals. Enantiomerically pure non-natural amino acids are generally prepared by enzymatic resolution of the racemic N-acetyl form, but despite the intense efforts this method could not be used for preparing enantiomerically pure beta-Phe, because the effective enzyme had not been found. Therefore, screening for microorganisms capable of amidohydrolyzing (R,S)-N-acetyl-3-amino-3-phenylpropionic acid ((R,S)-N-Ac-beta-Phe) in an enantiomer-specific manner was performed. A microorganism having (R)-enantiomer-specific amidohydrolyzing activity and another having both (R)-enantiomer- and (S)-enantiomer-specific amidohydrolyzing activities were obtained from soil samples. Using 16S rDNA analysis, the former organism was identified as Variovorax sp., and the latter as Burkholderia sp. Using these organisms, enantiomerically pure (R)-beta-Phe (>99.5% ee) and (S)-beta-Phe (>99.5% ee) with a high molar conversion yield (67%-96%) were obtained from the racemic substrate.     

Syntheses of quinolone hydrochloride enantiomers from synthons (R)- and (S)-2-methylpiperazine

A series of R and S enantiomers of 7-(3-methylpiperazin-1-yl) quinolone derivatives were synthesized from (R)- and (S)-tert-butyl 2-methylpiperazine-1-carboxylate and tested for their antibacterial activities on 14 kinds of bacteria. Although no distinct difference in in vitro antibacterial activities was observed, 2-64-fold difference between R and S enantiomers was observed in approximately 52% of cases.     

Metathesis-mediated synthesis of (R)-10-methyl-2-tridecanone, the southern corn rootworm pheromone

(R)-10-Methyl-2-tridecanone, the female sex pheromone of the southern corn rootworm (Diabrotica undecimpunctata howardi Barber), was synthesized in 9 steps from methyl (S)-3-hydroxy-2-methylpropanoate in a 15.7% overall yield. Olefin cross metathesis between (R)-6-methyl-1-nonene and 5-hexen-2-one employing Grubbs' first-generation catalyst was the key step of the synthesis.     

Efficient route to (S)-azetidine-2-carboxylic acid

A new and efficient route to (S)-azetidine-2-carboxylic acid (>99.9% ee) in five steps and total yield of 48% via malonic ester intermediates was established. As the key step, efficient four-membered ring formation (99%) was achieved from dimethyl (S)-(1'-methyl)benzylaminomalonate by treating with 1,2-dibromoethane (1.5 eq) and cesium carbonate (2 eq) in DMF. Krapcho dealkoxycarbonylation of dimethyl (1'S)-1-(1'-methyl)benzylazetidine-2,2-dicarboxylate, the product of this cyclization procedure, proceeded with preferential formation (2.7:1, 78% total yield) of the desired (2S,1'S)-monoester, with the help of a chiral auxiliary which was introduced on the nitrogen atom. The undesired (2R,1'S)-isomer could be converted to that with proper stereochemistry, by a deprotonation and subsequent re-protonation step. Finally, lipase-catalyzed preferential hydrolysis of the (2S,1'S)-monoester and subsequent deprotection provided enantiomerically pure (S)-azetidine-2-carboxylic acid in a 91% yield from the mixture of (2S,1'S)- and (2R,1'S)-isomers.     

（R）与（S）-羰基还原酶偶联一步法制备（S）-苯乙二醇

【目的】通过 (R) - 和(S) -羰基还原酶在大肠杆菌中偶联,实现了一步法制备（S）-苯乙二醇的生物转化过程。【方法】将来源于近平滑假丝酵母(Candida parapsilosis CCTCC M203011)的(R)- 羰基还原酶基因（rcr）和(S) -羰基还原酶基因（scr）串联于共表达载体pETDuetTM-1上。重组质粒pETDuet-rcr-scr转化稀有密码子优化型菌株Escherichia coli Rosetta,获得酶偶联重组菌株E. coli Rosetta / pETDuet-rcr-scr。当重组菌体培养至OD600 0.6-0.8时,添加终浓度1 mmol/L IPTG,30℃诱导蛋白表达10 h。【结果】SDS-PAGE结果表明(R)- 和(S) -羰基还原酶均明显表达,它们的相对分子质量分别为37 kDa和30 kDa。重组菌生物转化结果表明：在pH7.0的磷酸缓冲液中,添加5 mmol/L Zn2+时,获得产物(S)-苯乙二醇,产物光学纯度为91.3% e.e.,产率为75.9%。【讨论】采用分子重组技术成功整合了两种氧化还原酶的催化功能,实现了(S)- 苯乙二醇的一步法转化,为简化手性醇制备途径提供了一条崭新的思路。     

Diastereoselective reduction of 1-(4-fluorophenyl)-3(R)-[3-oxo-3-(4-fluorophenyl)-propyl]-4(S)-(4-hydroxyphenyl)azetidin-2-one to Ezetimibe by the whole cell catalyst Rhodococcus fascians MO22

The asymmetric microbial reduction of 1-(4-fluorophenyl)-3(R)-[3-oxo-3-(4-fluorophenyl)-propyl]-4(S)-(4-hydroxyphenyl)azetidin-2-one to 1-(4-fluorophenyl)-3(R)-[3(S)-hydroxy-3-(4-fluorophenyl)-propyl]-4(S)-(4-hydroxyphenyl)azetidin-2-one (Ezetimibe) by Rhodococcus fascians MO22 is described. The catalytic capability of the microorganism for reduction has been examined also with protected ketone, an intermediate from chemical synthesis of Ezetimibe. Various parameters of the bioreduction have been optimized: the strain converted 94.8% of ketone and 63% of protected ketone into Ezetimibe with the same de of 99.9%. In the later case, two chemical steps are replaced with a single biotransformation.     

Biocatalytic ammonolysis of (5S)-4,5-dihydro-1H-pyrrole-1,5-dicarboxylic acid, 1-(1,1-dimethylethyl)-5-ethyl ester: preparation of an intermediate to the dipeptidyl peptidase IV inhibitor Saxagliptin

An efficient biocatalytic method has been developed for the conversion of (5S)-4,5-dihydro-1H-pyrrole-1,5-dicarboxylic acid, 1-(1,1-dimethylethyl)-5-ethyl ester (1) into the corresponding amide (5S)-5-aminocarbonyl-4,5-dihydro-1H-pyrrole-1-carboxylic acid, 1-(1,1-dimethylethyl)ester (2), which is a critical intermediate in the synthesis of the dipeptidyl peptidase IV (DPP4) inhibitor Saxagliptin (3). Candida antartica lipase B mediates ammonolysis of the ester with ammonium carbamate as ammonia donor to yield up to 71% of the amide. The inclusion of Ascarite and calcium chloride as adsorbents for carbon dioxide and ethanol byproducts, respectively, increases the yield to 98%, thereby offering an efficient and practical alternative to chemical routes which yield 57-64%.