125971-93-9Relevant articles and documents
Enzymatic preparation of t-butyl-6-cyano-(3R, 5R)-dihydroxyhexanoate by a whole-cell biocatalyst co-expressing carbonyl reductase and glucose dehydrogenase
Wu, Xuri,Gou, Xudong,Chen, Yijun
, p. 104 - 110 (2015)
Statins are the most effective drugs for hyperlipidemia-related diseases by competitively inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase. Because of the difficulty and environmental concerns associated with chemical preparation of the chiral diols of statin side chains, different biocatalytic approaches have been explored and the two-step bio-reduction process for the introduction of two chiral hydroxyl groups has been industrialized. However, the high costs and poor stability of nicotinamide cofactors in the process was a major limiting factor. In the present study, a whole-cell biocatalyst simultaneously expressing carbonyl reductase and glucose dehydrogenase was constructed. This biocatalyst was then used to synthesize t-butyl-6-cyano-(3R, 5R)-dihydroxyhexanoate via enzymatic reduction of t-butyl-6-cyano-(5R)-hydroxy-3-carboxylhexanoate, which involves in the self-recycling of endogenous cofactors. After systematic optimization, the bioconversion was complete with a productivity of 120 g l-1 day-1 without exogenous addition of cofactors after 7 h at 35 g/L substrate concentration. Thus, the present system has simplified the process and improved the overall efficiency for the preparation of statin side chains.
Tailoring an aldo-keto reductase KmAKR for robust thermostability and catalytic efficiency by stepwise evolution and structure-guided consensus engineering
Li, Shu-Fang,Xie, Jian-Yong,Qiu, Shuai,Zhou, Sheng-Yi,Wang, Ya-Jun,Zheng, Yu-Guo
, (2021)
t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an advanced chiral diol intermediate of the cholesterol-lowering drug atorvastatin. KmAKRM5 (W297H/Y296W/K29H/Y28A/T63M) constructed in our previous work, displayed good biocatalytic performance on (3R,5R)-2. In the present work, stepwise evolution was applied to further enhance the thermostability and activity of KmAKRM5. For thermostability enhancement, N109 and S196 located far from the active site were picked out by structure-guided consensus engineering, and mutated by site-directed mutagenesis (SDM). For catalytic efficiency improvement, the residues A30 and T302 adjacent to the substrate-binding pocket were subjected to site-saturation mutagenesis (SSM). As a result, the “best” mutant KmAKRM9 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C) was developed, of which T5015 and Tm were 5.0 °C and 8.2 °C higher than those of KmAKRM5. Moreover, compared to KmAKRM5, KmAKRM9 displayed a 1.9-fold (846 vs 2436 min) and 6.7-fold (126 vs 972 min) longer half-lives at 40 and 50 °C, respectively. Structural analysis suggested that beneficial mutations introduced additional hydrophobic interactions and hydrogen bonds, contributing rigidification of the flexible loops and the increase of internal forces, hence increasing the thermostability and activity. 5 g DCW (dry cell weight) L?1 KmAKRM9 completely reduced 350 g L?1 t-butyl 6-cyano-(5R)-hydroxy-3-oxo-hexanoate ((5R)-1), within 3.7 h at 40 °C, yielding optically pure (3R,5R)-2 (d.e.p > 99.5%) with a space-time yield (STY) of 1.82 kg L?1 d?1. Hence, KmAKRM9 is a robust biocatalyst for the synthesis of (3R,5R)-2.
Co-evolution of activity and thermostability of an aldo-keto reductase KmAKR for asymmetric synthesis of statin precursor dichiral diols
Chen, Yi,Cheng, Feng,Jin, Ling-Jun,Li, Shu-Fang,Qiu, Shuai,Wang, Ya-Jun,Zheng, Yu-Guo
, (2020)
Aldo-keto reductase KmAKR-catalyzed asymmetric reduction offers a green approach to produce dichiral diol tert-butyl 6-substituted-(3R,5R/S)-dihydroxyhexanoates, which are important building blocks of statins. In our previous work, we cloned a novel gene of NADPH-specific aldo-keto reductase KmAKR (WT) from a thermotolerant yeast Kluyveromyces marxianus ZJB14056 and a mutant KmAKR-W297H/Y296W/K29H (Variant III) has been constructed and displayed strict diastereoselectivity towards tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1) but moderate activity and stability. Herein, to further co-evolve its activity and thermostability, we performed semi-rational engineering of Variant III by using a combinational screening strategy, consisting of tertiary structure analysis, loop engineering, and alanine scanning. As results, the “best” variant KmAKR-W297H/Y296W/K29H/Y28A/T63M (Variant VI) was acquired, whose Km, kcat/Km towards (5R)-1 was 0.66 mM and 210.77 s?1 mM?1, respectively, with improved thermostability (half-life of 14.13 h at 40 °C). Combined with 1.5 g dry cell weight (DCW) L-1 Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for NADPH regeneration, 4.5 g DCW L-1 Variant VI completely reduced (5R)-1 of up to 450 g L?1 within 7.0 h at 40 °C, yielding the corresponding optically pure tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-3, >99.5% d.e.p) with a space–time yield (STY) of 1.24 kg L?1 day?1, and this was the highest level documented in literatures so far on substrate loading and STY of producing (3R,5R)-3. Besides (5R)-1, Variant VI displayed strong activity on tert-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-2). 4.5 g DCW L-1 Variant VI completely reduced 400 g L?1 (5S)-2, within 5.0 h at 40 °C, yielding optically pure tert-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-4, >99.5% d.e.p) with a STY of 1.34 kg L?1 day?1. In summary, Variant VI displayed industrial application potential in statins biomanufacturing.
Improving the catalytic efficiency of aldo-keto reductase KmAKR towards t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate via semi-rational design
Yu, Han,Qiu, Shuai,Cheng, Feng,Cheng, Ying-Nan,Wang, Ya-Jun,Zheng, Yu-Guo
, (2019)
t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an important chiral diol synthon of atorvastatin calcium. Previously, we constructed a variant KmAKR-W297H (M1) of Kluyveromyces marxianus aldo-keto reductase (KmAKR, designated as M0), possessing excellent diastereoselectivity but moderate activity towards t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). In this work, KmAKR-W297H/Y296W/K29H (M3) was developed via semi-rational design. It exhibited much improved catalytic efficiency towards (5R)-1. The Km values of M3 for NADPH and (5R)-1 were 0.15 mmol/L and 1.41 mmol/L, and the maximal reaction rate vmax was 55.56 μmol/min/mg. Compared with M1, the catalytic efficiency kcat/Km of M3 was increased 2.64-fold. Coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration, M3 took 3.5 h to completely reduce (5R)-1 at up to 100.0 g/L, producing 237.4 mmol/L (3R,5R)-2 in d.e.P value above 99.5%. The space-time yield (STY) of M3-catalyzed (3R,5R)-2 synthesis was 372.8 g/L/d.
Atorvastatin calcium intermediate as well as preparation method and application thereof
-
, (2019/04/04)
The invention discloses an atorvastatin calcium intermediate as well as a preparation method and application thereof. A synthesis process of the intermediate is environmentally-friendly, simple to operate and low in EHS risk; raw materials are easy to obtain; a used chemical reagent is small in toxicity and low in cost; and the synthesis process is a green synthesis process suitable for the industrial production. Moreover, the intermediate provided by the invention is applied to the synthesis of atorvastatin calcium and a key intermediate thereof, the route is relatively short, the yield is high, the industrial production cost of the atorvastatin calcium is effectively reduced, and the atorvastatin calcium intermediate has a relatively high industrial application prospect.
Preparation method of atorvastatin calcium intermediate
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Paragraph 0044-0048; 0050-0054; 0056-0060; 0062-0066, (2018/03/24)
The invention belongs to the technical field of pharmaceutical chemistry and in particular relates to a preparation method of an atorvastatin calcium intermediate. According to the preparation methodprovided by the invention, an intermediate compound II is used as a starting material and the starting material is transformed into a lithium reagent compound III and an aldehyde group compound IV step by step; then the lithium reagent compound III and the aldehyde group compound IV react with hydroxylamine to generate an oxime compound V; an oxime group is dehydrated to obtain cyano I-A; and thenprotection is carried out to obtain a target compound I. Compared with an existing report, the route effectively avoids the utilization of highly toxic substances including sodium cyanide and hydrocyanic acid; and reactions in the whole route are common, conditions are moderate and the yield is relatively good. The preparation method provided by the invention is more beneficial to large-scale industrial production of the intermediate I.
Identification of a Robust Carbonyl Reductase for Diastereoselectively Building syn-3,5-Dihydroxy Hexanoate: A Bulky Side Chain of Atorvastatin
Gong, Xu-Min,Zheng, Gao-Wei,Liu, You-Yan,Xu, Jian-He
supporting information, p. 1349 - 1354 (2017/09/23)
t-Butyl-6-cyano-(3R,5R)-dihydroxyhexanoate is an advanced chiral precursor for the synthesis of the side chain pharmacophore of cholesterol-lowering drug atorvastatin. Herein, a robust carbonyl reductase (LbCR) was newly identified from Lactobacillus brevis, which displays high activity and excellent diastereoselectivity toward bulky t-butyl 6-cyano-(5R)-hydroxy-3-oxo-hexanoate (7). The engineered Escherichia coli cells harboring LbCR and glucose dehydrogenase (for cofactor regeneration) were employed as biocatalysts for the asymmetric reduction of substrate 7. As a result, as much as 300 g L-1 of water-insoluble substrate was completely converted to the corresponding chiral diol with >99.5% de in a space-time yield of 351 g L-1 d-1, indicating a great potential of LbCR for practical synthesis of the very bulky and bi-chiral 3,5-dihydroxy carboxylate side chain of best-selling statin drugs.
Process for the enantioselective enzymatic reduction of hydroxy keto compounds
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Paragraph 0060-0064, (2016/02/03)
In a process for the enantioselective enzymatic reduction of a hydroxy ketone of general formula I wherein R1═C1-C6 alkyl and R2═—Cl, —CN, —OH, —H or C1-C6 alkyl, into a chiral diol of general formula II wherein R1 and R2 have the same meaning as in formula I, the hydroxy ketone is reduced with an oxidoreductase in the presence of NADH or NADPH as a cofactor, wherein a) the hydroxy ketone is provided in the reaction at a concentration of ≧50 g/l,b) the oxidized cofactor NAD or NADP having formed is regenerated continuously by oxidation of a secondary alcohol of general formula RXRYCHOH, wherein RX, RY independently represent hydrogen, branched or unbranched C1-C8-alkyl and Ctotal≧3, andc) the reduction of the hydroxy ketone and the oxidation of the secondary alcohol are catalyzed by the same oxidoreductase.
Asymmetric synthesis of optically active methyl-2-benzamido-methyl-3-hydroxy-butyrate by robust short-chain alcohol dehydrogenases from Burkholderia gladioli
Chen, Xiang,Liu, Zhi-Qiang,Huang, Jian-Feng,Lin, Chao-Ping,Zheng, Yu-Guo
, p. 12328 - 12331 (2015/07/27)
Three short-chain alcohol dehydrogenases from Burkholderia gladioli were discovered for their great potential in the dynamic kinetic asymmetric transformation of methyl 2-benzamido-methyl-3-oxobutanoate, and their screening against varied organic solvents and substrates. This is the first report of recombinant enzymes capable of achieving this reaction with the highest enantio- and diastereo-selectivity.
Cloning, expression and enzymatic characterization of an aldo-keto reductase from Candida albicans XP1463
Wang, Ya-Jun,Liu, Xiao-Qing,Luo, Xi,Liu, Zhi-Qiang,Zheng, Yu-Guo
, p. 44 - 50 (2015/09/15)
An aldo-keto reductase encoding gene caakr was cloned from Candida albicans XP1463 (CCTCC M 2014382), and heterologously expressed in Escherichia coli. The aldo-keto reductase CaAKR is NADH-dependent with a molecular weight of approximately 38.6 kDal including a His6-Tag. It is active and stable at 30°C and pH 7.0. The maximal reaction rate (Vmax), apparent Michaelis-Menten constant (Km) and catalytic constant (kcat) for t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((R)-1a) were 11.50 mmol/L min, 1.91 mmol/L and 218.50 min-1. Besides atorvastatin's chiral synthon t-butyl 6-cyano-(3R,5R)- dihydroxy hexanoate ((R,R)-1b), it can synthesize N,N-2-dimethyl-(3S)-hydroxy-3-(2-thienyl)-1-propanine ((S)-9b) and methyl 1-[E]-2-[3-[3-[2-(7-chloro-2-quinoliny) ethenyl] phenyl]-(3S)-hydroxy propy] benzoate ((S)-10b), the chiral intermediates of duloxetine and montelukast, displaying potential applications in pharmaceutical industry. 2015 Elsevier B.V. All rights reserved.
