- A High-Throughput Screening Method for the Directed Evolution of Hydroxynitrile Lyase towards Cyanohydrin Synthesis
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Chiral cyanohydrins are useful intermediates in the pharmaceutical and agricultural industries. In nature, hydroxynitrile lyases (HNLs) are a kind of elegant tool for enantioselective hydrocyanation of carbonyl compounds. However, currently available methods for demonstrating hydrocyanation are still stalled at precise, but low-throughput, GC or HPLC analyses. Herein, we report a chromogenic high-throughput screening (HTS) method that is feasible for the cyanohydrin synthesis reaction. This method was highly anti-interference and sensitive, and could be used to directly profile the substrate scope of HNLs either in cell-free extract or fermentation clear broth. This HTS method was also validated by generating new variants of PcHNL5 that presented higher catalytic efficiency and stronger acidic tolerance in variant libraries.
- Zheng, Yu-Cong,Ding, Liang-Yi,Jia, Qiao,Lin, Zuming,Hong, Ran,Yu, Hui-Lei,Xu, Jian-He
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p. 996 - 1000
(2021/01/15)
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- Structure-Guided Tuning of a Hydroxynitrile Lyase to Accept Rigid Pharmaco Aldehydes
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The chiral vicinal C-O/C-N bifunctional groups generated from enzymatic hydrocyanation represents a useful methodology. However, construction of the pharmacophore of β2-adrenoreceptor agonists with this method remains a great challenge because of complete racemization of the benzylic alcohol during deprotection of the acetal groups. In this study, structure-guided redesign of a hydroxynitrile lyase originating from Prunus communis (PcHNL5) enables a highly enantioselective hydrocyanation of rigid benzo-ketal aldehyde which was proved to be resistant against racemization during the deprotection step, with dramatically improved productivity (>95% conversion vs 2-adrenoreceptor agonist, in an optically pure form (>99% ee) with an overall yield of 54%, which is the highest value reported.
- Hong, Ran,Li, Fu-Long,Lin, Guo-Qiang,Lin, Zuming,Xu, Jian-He,Yu, Hui-Lei,Zheng, Yu-Cong
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p. 5757 - 5763
(2020/06/09)
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- Immobilized Baliospermum montanum hydroxynitrile lyase catalyzed synthesis of chiral cyanohydrins
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Hydroxynitrile lyase (HNL) catalyzed enantioselective C–C bond formation is an efficient approach to synthesize chiral cyanohydrins which are important building blocks in the synthesis of a number of fine chemicals, agrochemicals and pharmaceuticals. Immobilization of HNL is known to provide robustness, reusability and in some cases also enhances activity and selectivity. We optimized the preparation of immobilization of Baliospermium montanum HNL (BmHNL) by cross linking enzyme aggregate (CLEA) method and characterized it by SEM. Optimization of biocatalytic parameters was performed to obtain highest % conversion and ee of (S)-mandelonitrile from benzaldehyde using CLEA-BmHNL. The optimized reaction parameters were: 20 min of reaction time, 7 U of CLEA-BmHNL, 1.2 mM substrate, and 300 mM citrate buffer pH 4.2, that synthesized (S)-mandelonitrile in ~99% ee and ~60% conversion. Addition of organic solvent in CLEA-BmHNL biocatalysis did not improve in % ee or conversion of product unlike other CLEA-HNLs. CLEA-BmHNL could be successfully reused for eight consecutive cycles without loss of conversion or product formation and five cycles with a little loss in enantioselectivity. Eleven different chiral cyanohydrins were synthesized under optimal biocatalytic conditions in up to 99% ee and 59% conversion, however the % conversion and ee varied for different products. CLEA-BmHNL has improved the enantioselectivity of (S)-mandelonitrile synthesis compared to the use of purified BmHNL. Nine aldehydes not tested earlier with BmHNL were converted into their corresponding (S)-cyanohydrins for the first time using CLEA-BmHNL. Among the eleven (S)-cyanohydrins syntheses reported here, eight of them have not been synthesized by any CLEA-HNL. Overall, this study showed preparation, characterization of a stable, robust and recyclable biocatalyst i.e. CLEA-BmHNL and its biocatalytic application in the synthesis of different (S)-aromatic cyanohydrins.
- Jangir, Nisha,Padhi, Santosh Kumar
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- Hydroxynitrile Lyase Isozymes from Prunus communis: Identification, Characterization and Synthetic Applications
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Biocatalysts originating from Badamu (Prunus communis) have been applied to catalyze the asymmetric synthesis of (R)-4-methylsulfanylmandelonitrile, a key building block of thiamphenicol and florfenicol. Here, four hydroxynitrile lyase (HNL) isozymes from Badamu were cloned and heterologously expressed in Pichia pastoris. The biochemical properties and catalytic performances of these isozymes were comprehensively explored to evaluate their efficiency and selectivity in asymmetric synthesis. Among then, PcHNL5 was identified with outstanding activity and enantioselectivity in asymmetric hydrocyanation. Under the optimized mild biphasic reaction conditions, seventeen prochiral aromatic aldehydes were converted to valuable chiral cyanohydrins with good yields (up to 94%) and excellent optical purities (up to >99.9% ee), which provide a facile access to numerous chiral amino alcohols, hypoglycemic agents, angiotension converting enzyme (ACE) inhibitors and β-blockers. This work therefore underlines the importance of discovering the most potent biocatalyst among a group of isozymes for converting unnatural substrates into value-added products. (Figure presented.).
- Zheng, Yu-Cong,Xu, Jian-He,Wang, Hui,Lin, Guo-Qiang,Hong, Ran,Yu, Hui-Lei
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supporting information
p. 1185 - 1193
(2017/04/13)
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- Asymmetric cyanohydrin formation from aldehydes catalyzed by manganese Schiff base complexes
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The catalyst generated in situ from Mn(OAc)2 and a chiral Schiff base ligand exhibited excellent catalytic abilities in asymmetric cyanohydrin formation from aldehydes with sodium cyanide in up to 99% enantioselectivity and good yield.
- Qu, Yanyang,Jing, Linhai,Wu, Zhiqing,Wu, Di,Zhou, Xiangge
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experimental part
p. 187 - 190
(2010/04/28)
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- Enzymatic kinetic resolution of racemic cyanohydrins via enantioselective acylation
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Enzymatic kinetic resolution of a series of aromatic and aliphatic cyanohydrins in organic media has been investigated. The behavior of potential lipases, molecular sieves, acyl reagent, reaction temperature, and organic solvents on the kinetic resolution was studied. The influence of substrate structure, steric, and electronic nature and position of the aryl substituent on the enantioselectivity was discussed. Under the optimized reaction conditions, good enantioselectivity could be achieved for most of the investigated compounds. Specifically, substrates 1a, 1c, 1d, 1f, 1u could be resolved with the kinetic enantiomer ratio (E) higher than 200.
- Xu, Qing,Xie, Yongli,Geng, Xiaohong,Chen, Peiran
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experimental part
p. 624 - 630
(2010/09/07)
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- Kinetic resolution of cyanohydrins via enantioselective acylation catalyzed by lipase PS-30
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By using lipase PS-30 as catalyst, the kinetic resolution of a series of racemic cyanohydrins has been achieved via enantioselective acylation. The values of kinetic enantiomeric ratio (E) reached up to 314. Substituent effect is also briefly discussed.
- Xu, Qing,Geng, Xiaohong,Chen, Peiran
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scheme or table
p. 6440 - 6441
(2009/04/06)
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- An R-selective hydroxynitrile lyase from Arabidopsis thaliana with an α/β-hydrolase fold
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Folding and selectivity: The noncyanogenic plant Arabidopsis thaliana contains a new hydroxynitrile lyase, which was cloned and characterized. This enzyme is readily available form a recombinant source, has a broad range of substrates, and enantioselectively transforms aliphatic and aromatic aldehydes as well as ketones into the corresponding R-cyanohydrins. (Chemical Equation Presented).
- Andexer, Jennifer,Von Langermann, Jan,Mell, Annett,Bocola, Marco,Kragl, Udo,Eggert, Thorsten,Pohl, Martina
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p. 8679 - 8681
(2008/09/18)
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- Highly enantioselective cyanosilylation of aldehydes catalyzed by novel β-amino alcohol-titanium complexes
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The β-amino alcohol 1b-Ti(Oi-Pr)4 complex has been shown to catalyze the enantioselective cyanosilylation of aldehydes efficiently. In the presence of 5 mol % of 1b-Ti(Oi-Pr)4 complex catalyst, the aromatic, conjugated, heteroaromatic, and aliphatic aldehydes were converted to their corresponding trimethylsilyl ethers of cyanohydrins in 90-99% yields with up to 94% ee under mild conditions.
- Li, Yan,He, Bin,Qin, Bo,Feng, Xiaoming,Zhang, Guolin
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p. 7910 - 7913
(2007/10/03)
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- Titanium and ruthenium binaphthyl Schiff base complexes as catalysts for asymmetric trimethylsilylcyanation of aldehydes
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Investigations on the catalytic behaviour of titanium complexes formed in situ from Ti(OPri)4 and a variety of Schiff bases, mainly the binaphthyl derivatives 2,2′-bis(3-R1-5-R 2-2-hydroxybenzylideneamino)-1,1′-binaphthyl, toward the asymmetric trimethylsilylcyanation of some aromatic and aliphatic aldehydes demonstrated that the titanium complex of the binaphthyl Schiff base with R1 = R2 = But is one of the best catalysts for such a process, with an enantiomeric excess (e.e.) as high as 96% obtained for m-tolualdehyde. Crystal structure determination of a nitrosylruthenium complex, [RuII(L)(NO)Cl] (L is the dianion of the binaphthyl Schiff base with R1 = R2 = Cl), revealed that the complex assumes a cis-β configuration with the binaphthyl moiety having a dihedral angle of 70.2°. After treatment with AgPF6, the ruthenium complex also exhibited a good catalytic property for the trimethylsilylcyanation of benzaldehyde albeit with a lower e.e. (24%). The Royal Society of Chemistry 1999.
- Zhou, Xiang-Ge,Huang, Jie-Sheng,Ko, Po-Hung,Cheung, Kung-Kai,Che, Chi-Ming
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p. 3303 - 3309
(2007/10/03)
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- Preparation of Chiral Cyanohydrins by an Oxynitrilase-Mediated Transcyanation
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The transcyanation of aromatic and aliphatic aldehydes 1 (RCHO) with acetone cyanohydrin is catalyzed by the enzyme D-oxynitrilase to afford (R)-cyanohydrins 2 (RCH(OH)CN).The biocatalytic method using acetone cyanohydrin gives products of high enantiomeric purity with better consistency than similar conditions using hydrogen cyanide as the cyanide source.The use of an ether-buffer biphasic solvent system is essential for producing products of optimum optical purity, but the solubility properties of the substrate have a pronounced effect on the enantiomeric purity of the final product.A discussion relating the solubility partition coefficient (log P) of a substrate to the enantiomeric purity of the product as a guide for predicting the outcome with new substrates is presented.Application of the method to the preparation of the following cyanohydrins is reported (R, ee): a, C6H5, 92percent ee; b, 3,4-(CH2O2)C6H3, 90percent ee; c, 2-(CH3O)C6H4, 96percent ee; d, C6H5CH2, 88percent ee; e, CH3SCH2CH2, 92percent ee; f, CH3(CH2)5CH2, 92percent ee; g, (CH3)3C, 92percent ee; h, c-C6H11, 96percent ee; i, CH3O2C(CH2)6CH2, 97percent ee; j, (E,E)-CH3CH=CHCH=CH, 96percent ee; k, (CH3)2C=CHCH2CH2C(CH3)=CH, 99percent ee.
- Ognyanov, Vassil I.,Datcheva, Violeta K.,Kyler, Keith S.
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p. 6992 - 6996
(2007/10/02)
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- A Convenient Route to (R)-α-Hydroxy Carboxylic Acids and (2R)-1-Amino-2-alkanols from (R)-Cyanohydrins
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(R)-Cyanohydrins, prepared in good to excellent yields with high optical purity by enzyme-catalyzed addition of hydrogen cyanide to aldehydes in organic solvents, are hydrolyzed with concentrated hydrochloric acid at ambient temperature, usually in very high yield, without any trace of racemization to give (R)-α-hydroxy carboxylic acids.Likewise, no racemization is observed by direct reduction of the (R)-cyanohydrins with lithium aluminium hydride to give (2R)-1-amino-2-alkanols.
- Ziegler, Thomas,Hoersch, Brigitte,Effenberger, Franz
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p. 575 - 578
(2007/10/02)
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- Asymmetric Hydrocyanation of Aldehydes Using Chiral Titanium Reagents
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Two highly enantioselective methods for hydrocyanation of aldehydes were developed by using chiral alkoxytitanium reagents.Treatment of benzaldehyde with cyanotrimethylsilane in the presence of a chiral alkoxytitanium affords mandelonitrile in good chemic
- Minamikawa, Hiroyuki,Hayakawa, Satoshi,Yamada, Tohru,Iwasawa, Nobuharu,Narasaka, Koichi
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p. 4379 - 4384
(2007/10/02)
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- The Asymmetric Hydrocyanation of Aldehydes with Cyanotrimethylsilane Promoted by a Chiral Titanium Reagent
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The asymmetric hydrocyanation of aldehydes with cyanotrimethylsilane proceeds by the use of a chiral alkoxy titanium(IV) to give the corresponding cyanohydrins with good enantioselectivity.
- Narasaka, Koichi,Yamada, Tohru,Minamikawa, Hiroyuki
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p. 2073 - 2076
(2007/10/02)
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