Microfluidic separation of (S)-ibuprofen using enzymatic reaction

This study was investigated for the enantioselective separation of (S)-ibuprofen using the ionic liquid in the microfluidic device. A stable and thin ionic liquid flow (ILF) was made by controlling the flow rate of the ILF in the microfluidic channel. In addition, coupling lipase as a biocatalyst with the ILF based on the microfluidic device showed the facilitative and selective transport of (S)-ibuprofen across the ILF, indicating successful optical resolution of a racemic mixture. Subsequently, the enantioselectivity was evaluated in the transport ratio (η) of (R)- and (S)-ibuprofen, the optical resolution ratio (α) and enantiomeric excess of (S)-ibuprofen (ee_S).     

Enzymatic formation and esterification of (S)-mandelonitrile

The (S)-selective hydroxynitrile lyase from Hevea brasiliensis (HbHNL) catalyzes the trans-cyanohydrin reaction (transcyanation). The equilibrium of this two-step reaction sequence is not favorable unless a large excess of acetone cyanohydrin (1) is used. Therefore, the coupling of this reaction with a follow-up reaction was investigated. It was established that the trans-cyanohydrin reaction could be performed in organic media, making it possible to couple it with a lipase-catalyzed acylation. Candida antarctica lipase B (CAL-B) shows a high selectivity (E=100) for (S)-mandelonitrile (4) and is, therefore, the ideal candidate for this type of multi-step one-pot reaction.     

Esterified, optical pure (3S, 3′S)-astaxanthin from flowers of Adonis annua

The quantitative carotenoid composition of the red flower petals of Adonis annua is reported. Optically pure (3S, 3′S)-astaxanthin occurs both as a diester (64% of total carotenoid) and as a monoester (11%). The optical purity was determined by hydrolysis of the natural esters in the absence of oxygen and subsequent HPLC analysis of the paren -ketol esterified with (−)-camphanic acid. All non-animal sources hitherto examined synthesize pure 3S,3′S- or 3R,3′R-isomers of astaxanthin, whereas marine animal sources contain mixtures of all three optical isomers, including the meso form.     

Preparation of (2S, 3R)-methyl-3-phenylglycidate using whole cells of Pseudomonas putida

Preparation of (2S, 3R)-methyl 3-phenylglycidate via enantioselective hydrolysis of racemic phenylglycidate was carried out using whole cells of Pseudomonas putida. Under optimal conditions (2S, 3R)-methyl-3-phenylglycidate could be got with ee value 99 and 48% chemical yield.     

Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotorula glutinis

A recombinant yeast Pichia pastoris carrying the gene encoding epoxide hydrolase (EH) of Rhodotorula glutinis was constructed and used for producing (S)-styrene oxide by enantioselective hydrolysis of racemic mixtures of styrene oxides. The EH gene was obtained by PCR amplification of cDNA of R. glutinis and integrated into the chromosomal DNA of P. pastoris to express EH under the control of AOX promoter. The recombinant yeast has a high hydrolytic activity toward (R)-styrene oxide as 358 nmol min⁻¹ (mg cell)⁻¹, which is about 10-fold higher than that of wild type R. glutinis. When kinetic resolution was conducted by the recombinant yeast at a high initial epoxides concentration of 526 mM that constitutes an epoxide–water two-liquid phase, chiral (S)-styrene oxide with an enantiomeric excess (e.e.) higher than 98% was obtained as 36% yield (theoretical, 50%) at 16 h.     

Lipase-catalyzed production of optically active (S)-flurbiprofen in aqueous phase reaction system containing chiral succinyl β-cyclodextrin

The lipase-catalyzed production of optically active (S)-flurbiprofen was carried out in a dispersion reaction-system induced by chiral succinyl β-cyclodextrin (suβ-CD). The optimal reaction conditions were 500 mM (R,S)-flurbiprofen ethyl ester ((R,S)-FEE), 600 units of Candida rugosa lipase per 1 mmol of (R,S)-FEE, and 1000 mM suβ-CD at 37 °C for 72 h. An extremely high enantiomeric excess of 0.98 and conversion yield of 0.48 were achieved in the dispersed aqueous phase reaction system containing chiral suβ-CD added as a dispenser and chiral selector. The inclusion complex formability of the immiscible substrate (S)- and (R)-form of FEE with suβ-CD was compared using a phase-solubility diagram, DSC, and ¹H NMR. (S)-Isomer formed a more stable and selective inclusion complex with chiral suβ-CD. It was hydrolyzed much more selectively by lipase from C. rugosa, due to the selective structural modification through inclusion complexation with chiral suβ-CD.     

Enantioselective hydrolysis of (R,S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated solvents via lipases from Carica pentagona Heilborn and Carica papaya

A crude lipase prepared from Carica pentagona Heilborn latex was explored as an effective enantioselective biocatalyst for the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated organic solvents. Comparisons of the enzyme performance with that from Carica papaya lipase indicated that both lipases showed low tolerance to the hydrophilic solvent and were inhibited by (S)-naproxen and 2,2,2-trifluoroethanol. Improvements on the enzyme activity and enantioselectivty were demonstrated when both lipases in partially purified forms were employed. By using the thermodynamic analysis, the enantiomeric discrimination was mainly driven by the difference of activation enthalpy for all reaction systems except for employing Carica papaya lipase as the biocatalyst for (R,S)-fenoprofen 2,2,2-trifluoroethyl thioester.     

应用合成生物学策略构建全细胞生物催化剂合成(S)-乙偶姻

目的:在大肠杆菌宿主中过量表达丁二酮还原酶(DAR),同时构建辅酶NADH原位再生系统,利用全细胞高效催化丁二酮不对称还原合成(S)-乙偶姻。方法:PCR克隆多黏芽孢杆菌(Paenibacillus polymyxa) dar基因连到质粒pETDuet-1,转化至大肠杆菌(Escherichia coli) BL21(DE3),构建重组菌E. coli BL21(DE3)-DAR;通过Hi Trap TALON柱亲和层析纯化表达产物DAR酶蛋白,测定DAR的比酶活和分子动力学参数。在重组菌E. coli BL21(DE3)-DAR中构建辅酶NADH原位再生系统,协同表达枯草芽孢杆菌(Bacillus subtilis)的葡萄糖脱氢酶(GDH),构建重组菌E. coli BL21(DE3)-DAR/GDH,并以此重组菌为全细胞生物催化剂,优化催化条件,提高(S)-乙偶姻的产量和产率。结果:获得重组工程菌E. coli BL21(DE3)-DAR和E. coli BL21(DE3)-DAR/GDH。DAR以NADH为辅酶还原丁二酮的米氏常数Km、最大催化速率Vmax、催化常数Kcat分别为2. 59mmol/L、1. 64μmol/(L·min·mg)、12. 3/s,还原丁二酮生成(S)-乙偶姻光学的纯度为95. 86%,具有较好的催化效率和立体异构体选择性。构建辅酶NADH原位再生系统后,重组菌E. coli BL21(DE3)-DAR/GDH可高效催化丁二酮合成乙偶姻。在最优催化条件下分批补料,乙偶姻产量达51. 26g/L,转化率为81. 37%,生产速率为5. 13g/(L·h)。结论:使用非手性化合物原料丁二酮生产高附加值的手性化合物(S)-乙偶姻,以重组菌为全细胞生物催化剂合成(S)-乙偶姻,不需额外添加昂贵的辅酶,具有较高的生产应用价值。     

Fed-batch production of (S)-flurbiprofen in lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin and its extractive purification

Optically active (S)-flurbiprofen was produced fed-batch-wisely in a lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin (suβ-CD). A highly concentrated 480 mM (S)-flurbiprofen, corresponding to 117.0 g/l, with an enantiomeric excess of 0.98 and conversion yield of 0.48 was obtained. (S)-Flurbiprofen produced in an inclusion complex form with suβ-CD was extractively purified using three-step procedures: decomplexation of (S)-flurbiprofen and residual (R)-flurbiprofen ethyl ester ((R)-FEE) using the ethyl acetate, dissolution of (S)-flurbiprofen from (R)-FEE using a sodium bicarbonate solution, and selective precipitation of (S)-flurbiprofen using 2-propanol. Consequently, an extremely high concentration of 420 mM (S)-flurbiprofen with an optical purity higher than 98% was recovered after purification.     

Liquid chromatographic determination of total celiprolol or (S)-celiprolol and (R)-celiprolol simultaneously in human plasma

A method has been developed for the determination of total celiprolol (sum of enantiomers) or the enantiomers (R)-celiprolol and (S)-celiprolol in plasma by high-performance liquid chromatography with UV and fluorescence detection. After extraction from alkalinized plasma with methyl-tert-butyl ether and back-extraction into 0.01 M HCl (for total celiprolol determination) or after evaporation of the organic phase and derivatisation with R(−)-1-(1-naphthyl)ethyl isocyanate (enantiomer determination), total celiprolol or its diastereomeric derivatives were chromatographed on a reversed-phase HPLC column with a mixture of acetonitrile and phosphate buffer pH 3.5 (+0.05% triethylamine). Acebutolol was used as internal standard. Linearity was obtained in the range of 5 to 2000 ng/ml for total and 2.5 to 500 ng/ml for enantiomer determination. Intra-day and inter-day variation was lower than 10%. The method can be applied for analysis of plasma samples obtained from patients treated with oral racemic celiprolol doses.     

Optimization of (R,S)-1-phenylethanol kinetic resolution over Candida antarctica lipase B in ionic liquids

The kinetic resolution of racemates constitutes one major route to manufacture optically pure compounds. The enzymatic kinetic resolution of (R,S)-1-phenylethanol over Candida antarctica lipase B (CALB) by using vinyl acetate as the acyl donor in the acylation reaction was chosen as model reaction. A systematic screening and optimization of the reaction parameters, such as enzyme, ionic liquid and substrates concentrations with respect to the final product concentration, were performed. The enantioselectivity of immobilized CALB commercial preparation, Novozym 435, was assayed in several ionic liquids as reaction media. In particular, three different ionic liquids: (i) 1-butyl-3-methylimidazolium hexafluorophosphate [bmim][PF₆], (ii) 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][BF₄] and (iii) 1-ethyl-3-methylimidazolium triflimide [emim][NTf₂] were tested. At 6.6% (w/w) of Novozym 435, dispersed in 9.520 M of [bmim][PF₆] at 313.15 K, using an equimolar ratio of vinyl acetate/(R,S)-1-phenylethanol after 3 h of bioconversion, the highest possible conversion (50%) was reached with enantiomeric excess for substrate higher than 99%.     

Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets

Several lichen compounds, i.e. lobaric acid (1), a β-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic -methylene-γ-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a β-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 μg/ml: 1 93.4±6.62%, 2 98,5±1.19%, 5 14.7±2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose–response relationship in the range of 3.33–100 μg/ml. According to the calculated IC₅₀ values the highest inhibitory activity was observed for the depsidone 1 (IC₅₀=28.5 μM) followed by 2 (IC₅₀=77.0 μM). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (IC₅₀=24.6 μM).     

酵母脂肪酶分子进化及催化合成(S)-1,4-苯并二口恶烷*

Objective: 1,4-Benzodioxane is an important chiral intermediate for antihypertensive (Proroxan and Doxazosin), antidepressant (MCK-242) and other drugs, and it displays a broad spectrum of applications in the pharmaceutical field. Currently, in spite of high-yield advantage of chemical synthesis, there are some problems of environmental pollution and low production safety. Using lipase to catalyze synthesis of 1,4-benzodioxane provides a new pathway of green synthesis of 1,4-benzodioxane. However, natural enzymes face the dilemma of poor enantioselectivity. Therefore, molecular evolution was performed on Candida antarctica lipase B, and a technical route for the catalytic synthesis of 1,4-benzodioxane was established. Methods: Firstly, the key amino acid residues involved in substrate binding and conversion in the active center of Candida antarctica lipase B were analyzed, and saturation mutagenesis libraries on the interaction sites were constructed. Improved mutants with high efficiency and high enantioselectivity were then obtained using HPLC detection. Furthermore, catalytic synthesis conditions of mutant D223N/A225K were systematically optimized. Results: The results indicated that the mutants mainly derived from the pairwise site D223/A225 (such as D223N/A225K and D223G/A225W) were biased towards the synthesis of (S)-isoforms, while most of the mutants derived from the pairwise site E188/I189 (such as E188D/I189M) showed a bias for the synthesis of (R)-isoforms. Compared with WT, the ee_s value of the best mutant D223N/A225K to synthesize (S)-1,4-benzodioxane was increased from 11.9% to 29.3%. After systematic optimization of the reaction conditions, an ee_s value of (93.9±0.16)% and a conversion rate of (47.5±2.33)% were achieved using mutant D223N/A225K to catalyze kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate in n-butanol/phosphate buffered saline (20∶80, V/V) biphasic solvent at 37℃ for 50 min. Conclusion: An efficient kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate was successfully achieved by molecular evolution and optimization of conditions, which provides a new example for the creation of new enzymes by protein engineering technology, and also provides a theoretical and technical foundation for the efficient synthesis of (S)-1,4-benzodioxane molecules by enzymatic methods.     

Enantioselective analysis of (R)- and (S)-atenolol in urine samples by a high-performance liquid chromatography column-switching setup

An HPLC column-switching method for the enantioselective determination of (R,S)-atenolol in human urine was developed and validated. Diluted urine samples were injected onto a LiChrospher ADS restricted access column and atenolol was separated from most of the matrix components using 0.01 M Tris buffer. The atenolol peak was sharpened by a step gradient of 30% acetonitrile and the atenolol-containing fraction was switched onto an enantioselective column. Separation of the atenolol enantiomers was carried out on a Chirobiotic T (Teicoplanin) column using acetonitrile–methanol–acetic acid–triethylamine (55:45:0.3:0.2, v/v/v/v) as eluent. Detection of the effluent was performed by fluorescence measurement. Several experiments were carried out to suppress the high blank reading, which was efficiently achieved using Tris buffer in the first dimension. For the enantioselective analysis of (R)- and (S)-atenolol in plasma under the same conditions the sample capacity of the ADS column is considerably lower.     

Modulation of Mucor miehei lipase properties via directed immobilization on different hetero-functional epoxy resins: Hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid

Purified lipase from Mucor miehei (MML) has been covalently immobilized on different epoxy resins (standard hydrophobic epoxy resins, epoxy-ethylenediamine, epoxy-iminodiacetic acid, epoxy-copper chelates) and adsorbed via interfacial activation on octadecyl-Sepabeads support (fully coated with very hydrophobic octadecyl groups). These immobilized enzyme preparations were used under slightly different conditions (temperature ranging from 4 to 25 °C and pH values from 5 to 7) in the hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid.

Different catalytic properties (activity, specificity, enantioselectivity) were found depending on the particular support used. For example, the epoxy-iminodiacetic acid-Sepabeads gave the most active preparation at pH 7 while, at pH 5, the ethylenediamine-Sepabeads was superior.

More interestingly, the enantiomeric ratio (E) also depends strongly on the immobilized preparation and the conditions employed. Thus, the octadecyl-MML preparation was the only immobilized enzyme derivative which exhibited enantioselectivity towards R isomer (with E values ranging from 5 at 4 °C and pH 7 to 1.2 at pH 5 and 25 °C).

The other immobilized preparations, in contrast, were S selective. Immobilization on iminodiacetic acid-Sepabeads afforded the catalyst with the highest enantioselectivity (E=59 under optimum conditions).     

Characterization of the biological conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene in direct micellar systems

The whole cell biological conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by the E. coli JM109(pPS1778) recombinant strain carrying the naphthalene dioxygenase and regulatory genes cloned from Pseudomonas fluorescens N3 in micellar systems has been investigated using biochemical and chemico-physical techniques. Reverse and direct micellar systems have been tested. Non-ionic surfactants (Tween and Triton X series) were found not to inhibit either the growth of the bacteria and the expression of the hydroxylating dioxygenase enzyme in such systems and were utilized in order to speed up the naphthalene conversion by increasing its solubility and also its bioavailability. The phase behavior of the direct micellar system was characterized through light scattering and other chemico-physical techniques. Further addition of isopropyl-palmitate 1–2% v/v to the micellar systems resulted in an increase of the apparent substrate concentration in solution and particularly its bioavailability thus allowing faster catalytic conversions resulting in an increase in productivity for the process. Since the cis-dihydrodiols are acquiring considerable potential as chiral pool synthons in asymmetric synthesis for a variety of industrial processes, possible applications for efficient small and large-scale production of such compounds are discussed.     

Experiment and simulation on kinetic resolution of (R,S)-2-chloromandelic acid by enzymatic transesterification

Optically pure 2-chloromandelic acid (ClMA) is a very important chiral drug intermediate for synthesis of (S)-clopidogrel, belonging to the platelet aggregation inhibitor. Enantioselective resolution of (R,S)-2-chloromandelic acid was carried out in organic solvent through irreversible transesterification catalyzed by lipase AK with vinyl acetate acting as the acyl donor. Effects of various conditions on enantioselectivity and activity of lipase were investigated, including organic solvents, temperature, water content, substrate ratio, enzyme loading, and reaction time. Based on homogeneous reaction and Ping-Pong bi-bi mechanism, a quantitative model was constructed to simulate and optimize the reaction process. Under the optimal conditions, excellent results were obtained with high conversion of (R)-2-ClMA (c _R, ≥98.85%) and large enantiomeric excess of substrate (ee _s, ≥98.15%). There is a good agreement between predicted values and experiment data, which indicates that the established method is a powerful tool for optimization of the enantioselective transesterification process for enantiomers separation.     

Chiral separation of rac-Ornidazole and detection of the impurity of (R)-Ornidazole in (S)-Ornidazole injection and raw material

(S)-Ornidazole is a subject of research as an antifertility agent in male animals at present. However, there seems to be no relative report on chiral separation for rac-Ornidazole, which has been used as an effective medicine for more than 30 years. In this article, the chiral separation of rac-Ornidazole on a Chiralcel OB-H column based on normal-phase high-performance liquid chromatography (NP-HPLC) is investigated and the methodology for detection of impurity of (R)-Ornidazole in (S)-Ornidazole injection and raw material is established. The novel mobile phase is utilized by mixing n-hexane, methanol and isopropyl alcohol (95:4:1, v/v/v) instead of the typical mobile phase of n-hexane and isopropyl alcohol, although the methanol, which offers a good resolution factor for the enantiomeric separation in this system, is not recommended on the Chiralcel OB-H column according to the instruction supplied by Daicel Chemical Ind., LTD (Japan).