两相体系中固定化黏红酵母CCZU-G5催化合成 (R)-2-羟基-4-苯基丁酸乙酯

通过筛选得到一株高立体选择性还原2-氧代-4-苯基丁酸乙酯（OPBE）合成(R)-2-羟基-4-苯基丁酸乙酯[(R)-HPBE]的菌株并鉴定为黏红酵母。研究了在异辛烷/水两相体系中固定化黏红酵母CCZU-G5不对称还原OPBE合成(R)-HPBE的反应条件。结果表明最适的反应条件为：在异辛烷比例为10%条件下,底物浓度为100 mmol/L,催化剂用量为0.45 g/mL,辅底物为40 g/L的葡萄糖。在建立的反应体系中反应16 h,(R)-HPBE产率最高,达83.5%,e.e.值99%以上。固定化酵母经7次重复使用后,产率和e.e.值分别维持在70%和99%以上。     

全细胞生物催化合成(R)-2-羟基-4-苯基 丁酸乙酯

利用全细胞生物催化合成(R)-2-羟基-4-苯基丁酸乙酯[(R)-HPBE],并利用氢核磁共振（1HNMR）、气相色谱-质谱联用(GC-MS)、液相色谱(LC)对产物结构进行表征。以2-羰基-4-苯基丁酸乙酯（OPBE）为唯一碳源,从葡萄园土壤中筛选分离得到一株高选择性不对称还原OPBE为(R)-HPBE的菌株酵母G5,通过形态和18S rDNA分析鉴定为粘质红酵母(Rhodotorula mucilaginosa CCZU- G5)。构建Rhodotorula mucilaginosa CCZU- G5全细胞催化还原OPBE合成(R)-HPBE的反应体系并进行了优化。结果表明最适催化反应条件为：在20 mL PBS反应体系中,反应温度为35 ℃,pH 7.5,菌体质量浓度0.2 g/mL,葡萄糖质量浓度3.0%,金属离子Ca2+ 浓度3 mmol/L,底物OPBE浓度为20 mmol/L,反应时间12 h,(R)-HPBE产率达82%,对映体过量值(e.e.)值为99.9%。     

全细胞生物催化合成(R)-2-羟基-4-苯基 丁酸乙酯

以2-羰基-4-苯基丁酸乙酯(OPBE)为碳源,从葡萄园土壤中筛选分离得到一株高选择性不对称还原OPBE为(R)-2-羟基-4-苯基丁酸乙酯[(R)-HPBE]的粘质红酵母(Rhodotorula mucilaginosa CCZU-G5),利用1HNMR、GC-MS、液相色谱(LC)对产物结构进行了表征。构建了Rhodotorula mucilaginosa CCZU-G5全细胞催化还原OPBE合成(R)-HPBE的反应体系并对反应条件进行了优化。结果表明,最适催化反应条件为:在20 mL磷酸盐缓冲液(PBS)反应体系中,反应温度为35℃,pH=7.5,菌体质量浓度200 g/L,葡萄糖质量浓度30.0 g/L,金属离子Ca~(2+)浓度3 mmol/L,底物OPBE浓度为20 mmol/L,反应时间12h,在此条件下,(R)-HPBE产率达82%,对映体过量值(e.e.)为99.9%。     

固定化沙雷氏菌脂肪酶拆分(R,S)-2-羟基-4-苯基丁酸乙酯

利用海藻酸钙-戊二醛交联法对本实验室筛选到的一株能选择性拆分(R,S)-2-羟基-4-苯基丁酸乙酯〔(R,S)-HPBE〕的沙雷氏菌脂肪酶进行固定化,并对固定化脂肪酶的酶活和拆分条件进行了研究。结果表明,最适反应温度为50℃,pH=7.0,底物浓度40 mmol/L,ρ(酶液)=200 g/L,在该条件下,反应10 h后,(R)-2-羟基-4-苯基丁酸乙酯〔(R)-HPBE〕产率达93.4%,光学纯度(e.e.)为96.2%。连续反应12批次,固定化脂肪酶仍可使(R)-HPBE产率维持在80%以上。     

酵母细胞催化4-氯-乙酰乙酸乙酯的不对称还原反应

研究了酵母细胞催化4-氯-乙酰乙酸乙酯还原为手性化合物4-氯-(S)-3-羟基丁酸乙酯反应的工艺条件,讨论反应条件对产物得率及对映体过量(e.e.值)的影响。将质量分数5%的酵母加入培养基中静置培养12h后,加入20%质量分数的蔗糖、2.5%质量分数的底物,在30℃、pH值7.2、200r/min振荡培养8h,最大产率和产物e.e.值分别可达91.7%和97.8%。而手性助剂L-谷氨酰胺和L-半胱氨酸并未对实验结果有明显影响。     

不对称催化合成(R)-(+)-3′-氯-苯丙醇

[目的](R)-(+)-3′-氯-苯丙醇是一种手性农药中间体和应用于预防治疗葡萄孢菌所致的灰霉病的有效植物抗菌素,旨在研究一条产品光学纯度高的工艺路线。[方法]以间氯苯丙酮为原料、以铱催化剂和双胺膦手性配体对3′-氯-苯丙酮进行不对称催化加氢还原制备(R)-(+)-3′-氯-苯丙醇。[结果]通过正交试验优化,反应转化率95%以上,产品光学纯度93%e.e.值。[结论]该路线反应条件温和、转化率和光学纯度高,可用于工业生产。     

动态动力学拆分高效制备(R)-1,2,3,4-四氢异喹啉-1-羧酸

利用固定化的南极假丝酵母脂肪酶B(CAL-B)实现外消旋底物1,2,3,4-四氢异喹啉-1-羧酸酯[(±)-1]的动态动力学拆分高效制备(R)-1,2,3,4-四氢异喹啉-1-羧酸[(R)-1-TIC]。考察了不同来源CAL-B制剂的催化效果,以及关键反应条件对底物稳定性和酶催化速率的双重影响。结果表明:商品化脂肪酶制剂QLlip-9提供了最佳的催化效果;反应温度提高至30℃、醋酸铵缓冲液的初始pH值提高至8.0时,虽然底物稳定性略微下降,但完全转化所需时间大幅缩短,整体催化效率明显提高;pH值恒定控制策略则加快了反应后期无效底物的消旋化速率,从而进一步提高催化效率。当(±)-1浓度为20 g/L时,QLlip-9浓度降至10 g/L基本不影响催化效果,最佳酶/底物比率(酶与底物的质量比)达到1:2。在上述优化条件下,反应时间缩短至6h,转化率不低于99%,产物的对映体过量值(enantiomeric excess of product, e.e.p)为96%,生产速率达到0.24 g/(h·g酶),为目前文献报道最高水平的8倍。在此基础上,实现了8批次的重复利用,并在300 mL,1 000 mL和20 L体系中实现了逐级规模放大,最终通过浓缩结晶法提取获得e.e.p≥99%的(R)-1-TIC晶体(分离收率不低于80%)。以上研究不仅大幅提高了该反应的整体催化效率,并且降低了酶的使用量。     

烯丙基化合物对面包酵母立体选择性的影响机理

添加烯丙基化合物可以控制酵母催化不对称还原反应的立体选择性,以4-氯-3-羰基丁酸乙酯(COBE)为模型底物,用丙烯酰胺和烯丙基醇对酵母进行预处理,产物(S)-4-氯-3-羟基丁酸乙酯(S-CHBE)的对映体过量值(e.e.)可以增加至94%;而用烯丙基溴预处理,则可以得到R型产物. 通过DNS法和API ZYM试验条证明烯丙基类化合物对细胞及胞内酶系存在毒害作用,且作用大小取决于烯丙基所带基团. 预处理后的酵母细胞经PBS缓冲液多次洗涤后,其活性可以逐渐得到恢复. 随着底物浓度的增加,经烯丙基醇预处理的酵母细胞,其S型产物的e.e.值维持在90%左右,而经烯丙基溴处理后,反应得到的R型产物的e.e.值则从90%下降到45%左右. 说明烯丙基类化合物竞争性地吸附于酶的活性区域,且其所带基团决定了被吸附酶的类型. 以抑制剂类似物处理酵母细胞,其反应结果说明酵母体内催化不对称还原反应的两类酶存在着空间位阻效应.     

(R)-(+)-2,2’-双[二(3,5-二甲苯基)膦]-1,1’-联萘的合成

三溴化磷为原料,与二(3,5-二甲基苯基)氧化膦反应,得到二(3,5-二甲基苯基)溴化膦,通过催化偶联反应,得到(R)-(+)-2,2’-双[二(3,5-二甲苯基)膦]-1,1’-联萘。通过考察各步的反应条件,得出最佳的工艺条件,(R)-(+)-2,2’-双[二(3,5-二甲苯基)膦]-1,1’-联萘的收率可达67%,e.e.值为99.2%。     

(R)-(+)-2,2’-双[二(3,5-二甲苯基)膦]-1,1’-联萘的合成

三溴化磷为原料,与二(3,5-二甲基苯基)氧化膦反应,得到二(3,5-二甲基苯基)溴化膦,通过催化偶联反应,得到(R)-(+)-2,2’-双[二(3,5-二甲苯基)膦]-1,1’-联萘。通过考察各步的反应条件,得出最佳的工艺条件,(R)-(+)-2,2’-双[二(3,5-二甲苯基)膦]-1,1’-联萘的收率可达67%,e.e.值为99.2%。     

Reversal of Regioselectivity in Zinc-Dependent Medium-Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives

The zinc-dependent medium-chain alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is one of the most versatile biocatalysts for the stereoselective reduction of ketones to chiral alcohols. Despite its known broad substrate scope, ReADH only accepts carbonyl substrates with either a methyl or an ethyl group adjacent to the carbonyl moiety; this limits its use in the synthesis of the chiral alcohols that serve as a building blocks for pharmaceuticals. Protein engineering to expand the substrate scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2-oxo-4-phenylbutyrate) opens up new routes in the synthesis of ethyl-2-hydroxy-4-phenylbutanoate, an important intermediate for anti-hypertension drugs like enalaprilat and lisinopril. We have performed computer-aided engineering of ReADH toward ethyl 2-oxo-4-phenylbutyrate and octanone derivatives. W296, which is located in the small binding pocket of ReADH, sterically restricts the access of ethyl 2-oxo-4-phenylbutyrate, octan-3-one or octan-4-one toward the catalytic zinc ion and thereby limits ReADH activity. Computational analysis was used to identify position W296 and site-saturation mutagenesis (SSM) yielded an improved variant W296A with a 3.6-fold improved activity toward ethyl 2-oxo-4-phenylbutyrate when compared to WT ReADH (ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH W296A has a more than 16-fold increased activity toward octan-4-one (ReADH W296A: 0.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30-fold decreased activity toward octan-2-one (ReADH W296A: 0.23 U/mg and ReADH WT: 7.69 U/mg). Computational and experimental results revealed the role of position W296 in controlling the substrate scope and regiopreference of ReADH for a variety of carbonyl substrates.     

Enantioselective Hydrogenation of Ethyl-2-oxo-4-phenylbutyrate on Cinchonidine-Modified Pt/γ-Al2O3 Catalyst Using a Fixed-Bed Reactor

Enantioselective hydrogenation of ethyl-2-oxo-4-phenylbutyrate (EOPB) on a cinchonidine-modified Pt/-Al₂O₃ catalyst was carried out using a fixed-bed reactor to obtain the chiral product, (R)-(+)-ethyl-2-hydroxy-4-phenylbutyrate ((R)-(+)-EHPB). About 95% conversion and 68% ee of the (R)-(+)-EHPB were obtained for the cinchonidine-modified Pt/-Al₂O₃ catalyst in a continuous-flow reaction system with a fixed-bed reactor. It is observed that the ee of (R)-(+)-EHPB strongly depends on the different solvent and hydrogen pressure. The competitive adsorption between the solvent and reactant molecule plays an important role in determining the ee value of (R)-(+)-EHPB. This study also shows that a compact adsorption of chiral modifiers on the platinum surface is beneficial to the higher enantioselectivity.     

不对称催化合成(R)-2-羟基-4-苯基丁酸乙酯

用Pt—Cinchona生物碱体系催化加氢合成(R)-2-羟基-4-苯基丁酸乙酯。进行了催化剂的制备和比较研究,并考察了修饰剂用量、2-氧代-4-苯基丁酸乙酯的初始浓度、反应氢压和温度对转化率和(R)-2-羟基-4-苯基丁酸乙酯得率的影响,得到了适宜的反应条件,在此条件下产物中(R)-2-羟基-4-苯基丁酸乙酯得率可达70％以上。实验结果表明,本研究中不对称加氢速率与光学收率呈良好线性关系,对研究不对称合成反应的规律有一定的意义。     

负载型PVP-Pt催化剂催化2-氧-4-苯基丁酸乙酯的不对称加氢

分别采用乙醇、甲醛、甲酸钠、硼氢化钠为还原剂,聚乙烯吡咯烷酮（PVP）为稳定剂,制备了负载型PVP-Pt/γ-Al2O3催化剂,考察了催化剂对2-氧-4-苯基丁酸乙酯不对称加氢合成(R)-2-羟基-4-苯基丁酸乙酯的性能,其中硼氢化钠作为还原剂时性能最佳。研究了制备条件如还原温度、还原浓度、还原介质、PVP/Pt物质的量比以及反应溶剂、时间等因素对催化结果的影响。实验结果表明,在PVP/Pt=5∶1、还原温度0 ℃、NaBH4浓度为0.5 mol/L、乙醇作为还原介质制备的催化剂,对2-氧-4-苯基丁酸乙酯（EOPB）不对称加氢的转化率可达100%,对映选择性为76.8% e.e.。     

反相高效液相色谱法测定农药中间体3-二氟甲基-1-甲基-1H-吡唑-4-甲酸及其主要杂质的含量

建立了反相高效液相色谱法测定农药中间体3-二氟甲基-1-甲基-1H-吡唑-4-甲酸和3-二氟甲基-1-甲基-1H-吡唑-4-羧酸酯含量方法.甲醇和20 mmol/L乙酸铵(pH 5.0)为流动相,采用HC-C18色谱柱,在波长240 nm处检测.该方法在12.387～1238.7 mg/L 3-二氟甲基-1-甲基-1H...     

HYDROLYSIS OF ETHYL-2-BROMOISOBUTYRATE ESTER IN AN ALKALINE SOLUTION

The hydrolysis of ethyl-2-bromoisobutyrate in an alkaline solution/organic solvent two-phase medium as well as the effect of quaternary ammonium salt (e.g., tetrabutylammonium bromide) on the hydrolysis of ethyl-2-bromoisobutyrate were investigated in this work. The proposed mechanism for the hydrolysis of ethyl-2-bromoisobutyrate was verified based on experimental analysis. Both the bromo-alkyl (Br-C bond) and ester (-COOR) functional groups of ethyl-2-bromo-isobutyrate hydrolyze into alcohol and acid. Several reaction steps for the hydrolysis of these bromo-alkyl and ester functional groups were developed and discussed. A kinetic model of each reaction step was developed in which the rate expression was derived. The intrinsic rate constants of the reactions were determined from the experimental hydrolyzing data of ethyl-2-bromoisobutyrate using curving fitting. The effects of the reaction conditions on the conversion of reactants were investigated in detail. The results indicated that the conversion of reactants is highly dependent on the concentration of alkaline compound in the aqueous solution. An optimum value of alkaline concentration to produce a maximum conversion of reactant was obtained. Moreover, the conditions for reducing the hydrolysis of halo ester by adding sodium carbonate are recommended.     

HYDROLYSIS OF ETHYL-2-BROMOISOBUTYRATE ESTER IN AN ALKALINE SOLUTION

The hydrolysis of ethyl-2-bromoisobutyrate in an alkaline solution/organic solvent two-phase medium as well as the effect of quaternary ammonium salt (e.g., tetrabutylammonium bromide) on the hydrolysis of ethyl-2-bromoisobutyrate were investigated in this work. The proposed mechanism for the hydrolysis of ethyl-2-bromoisobutyrate was verified based on experimental analysis. Both the bromo-alkyl (Br-C bond) and ester (-COOR) functional groups of ethyl-2-bromo-isobutyrate hydrolyze into alcohol and acid. Several reaction steps for the hydrolysis of these bromo-alkyl and ester functional groups were developed and discussed. A kinetic model of each reaction step was developed in which the rate expression was derived. The intrinsic rate constants of the reactions were determined from the experimental hydrolyzing data of ethyl-2-bromoisobutyrate using curving fitting. The effects of the reaction conditions on the conversion of reactants were investigated in detail. The results indicated that the conversion of reactants is highly dependent on the concentration of alkaline compound in the aqueous solution. An optimum value of alkaline concentration to produce a maximum conversion of reactant was obtained. Moreover, the conditions for reducing the hydrolysis of halo ester by adding sodium carbonate are recommended.     

Evolution of Glucose Dehydrogenase for Cofactor Regeneration in Bioredox Processes with Denaturing Agents

Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH (BmGDH_M0) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDH_M6 (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDH_M6 was also more stable than BmGDH_M0 when exposed to hydrophobic and enzyme-inactivating compounds such as acetophenone, ethyl 2-oxo-4-phenylbutyrate, and ethyl (R)-2-hydroxy-4-phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, BmGDH_M6 was successfully used for the asymmetric reduction of deactivating ethyl 2-oxo-4-phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.     

不对称合成(R)-2-羟基-4-苯基丁酸乙酯的动力学

采用10,11-二氢辛可尼定修饰的Pt/Al2O3催化体系,不对称加氢合成(R)-2-羟基-4-苯基丁酸乙酯,研究了各因素影响反应速率和光学收率的一般规律.根据推测的机理,建立了未修饰体系中加氢反应的本征动力学模型,结合实验数据选定了较佳的模型.假设不对称加氢反应总速率包括反应物在催化剂表面修饰位上和未修饰位上两部分的加氢速率,建立了不对称加氢的动力学模型,经拟合得到参数并进行了验证和讨论.