Chemoenzymatic enantioselective synthesis of phenylglycine and phenylglycine amide by direct coupling of the Strecker synthesis with a nitrilase reaction

E. Eppinger, J. Gröning, A. Stolz
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引用次数: 3

Abstract

The conversion of rac-phenylglycinonitrile by different variants of the nitrilase from Pseudomonas fluorescens EBC191 (EC 3.5.5.1) was studied and the amounts and chiral composition of the formed phenylglycine and phenylglycine amide compared. Muteins that converted rac-phenylglycinonitrile to extraordinarily high amounts of phenylglycine or phenylglycine amide were tested for the chemoenzymatic enantioselective one-pot synthesis of (R)- and (S)-phenylglycine and (R)- and (S)-phenylglycine amide. The chemoenzymatic synthesis combined the initial step in the traditional chemical Strecker synthesis which results in the formation of rac-phenylglycinonitrile from benzaldehyde, cyanide, and ammonia with the enzymatic conversion of the formed nitrile by the nitrilase variants. The aminonitrile synthesis was optimized in order to obtain conditions which allowed under mildly alkaline conditions (pH 9.5) maximal yields of phenylglycinonitrile and the in-situ racemization of the compound. The racemic phenylglycinonitrile was directly converted under the alkaline conditions without any interposed purification step by cells of Escherichia coli overexpressing recombinant nitrilase variants. The application of a mutant of E. coli defect in a (S)-phenylglycine amide hydrolysing peptidase (E. coli JM109ΔpepA) expressing a highly reaction- and (R)-specific nitrilase variant allowed the synthesis of (R)-phenylglycine with ee-values ≥ 95% in yields up to 81% in relation to the initially added benzaldehyde. These yields indicated a dynamic kinetic resolution which involved the racemization of (S)- to (R)-phenylglycinonitrile under the used alkaline conditions with the concurrent hydrolysis of (R)-phenylglycinonitrile to (R)-phenylglycine. The addition of resting cells of E. coli JM109ΔpepA synthesizing an amide forming nitrilase variant to the final product of the Strecker synthesis and/or using E. coli strains with an intact aminopeptidase gene resulted in the preferred formation of (S)-phenylglycine amide, (R)-phenylglycine amide or (S)-phenylglycine.
Strecker合成和腈水解酶反应直接偶联化学酶对映选择性合成苯甘氨酸和苯甘氨酸酰胺
研究了荧光假单胞菌EBC191(EC 3.5.5.1)腈水解酶的不同变体对rac-苯基甘氨酸腈的转化,并比较了形成的苯基甘氨酸和苯基甘氨酸酰胺的量和手性组成。将rac-苯基甘氨酸腈转化为极高量的苯基甘氨酸或苯基甘氨酸酰胺的蛋白质被测试用于化学酶对映选择性一锅合成(R)-和(S)-苯基甘氨酸以及(R)和(S”-苯基甘氨酸酰胺。化学酶合成结合了传统化学Strecker合成中的初始步骤,该步骤导致由苯甲醛、氰化物和氨形成rac-苯基缩水甘油腈,并通过腈水解酶变体对形成的腈进行酶转化。对氨基腈的合成进行了优化,以获得在温和碱性条件(pH 9.5)下允许苯基缩水甘油腈的最大产率和化合物的原位外消旋化的条件。外消旋苯基缩水甘油腈在碱性条件下通过过表达重组腈水解酶变体的大肠杆菌的细胞直接转化,而不需要任何中间的纯化步骤。在表达高度反应性和(R)特异性腈水解酶变体的(S)-苯基甘氨酸酰胺水解肽酶(E.coli JM109ΔpepA)中应用大肠杆菌缺陷突变体,可以合成ee值≥95%的(R)-苯甘氨酸,相对于最初加入的苯甲醛,产率高达81%。这些产率表明了动态动力学拆分,其涉及在所用碱性条件下(S)-到(R)-苯基甘氨酸腈的外消旋,同时(R)–苯基甘氨酸腈水解到(R。将合成酰胺形成腈水解酶变体的大肠杆菌JM109ΔpepA的静息细胞添加到Strecker合成的最终产物中和/或使用具有完整氨基肽酶基因的大肠杆菌菌株,导致优选形成(S)-苯基甘氨酸酰胺、(R)-苯基甘氨酸酰胺或(S)–苯基甘氨酸。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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