Biocatalytic stereoselective synthesis of methyl mandelates by engineering a cytochrome P450 hydroxylase

Green Synthesis and Catalysis Pub Date : 2024-05-01 DOI:10.1016/j.gresc.2023.01.005

Lingzhi Xie , Yun Zhang , Ruyue Zhang , Haibo Cui , Baodong Cui , Wenyong Han , Nanwei Wan , Zhi Li , Yongzheng Chen

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Abstract

Chiral methyl mandelates are useful synthons in organic transformation and pharmaceutical synthesis. Green synthesis of these valuable compounds by direct C–H activating oxidative hydroxylation has attracted keen interest. Described herein is achieving the stereoselective and efficient bio-hydroxylation of methyl 2-phenylacetates to the chiral methyl mandelates by directed evolution of the cytochrome P450DA hydroxylase. In the present study, a new colorimetric high-throughput screening assay was successfully developed based on a dual-enzyme cascade for the engineering of the P450DA's hydroxylation activity. Several beneficial variants with enhanced bio-hydroxylation activity were created by combining random mutagenesis and site-saturated/directed mutagenesis strategies. Whole-cell bio-hydroxylation of various methyl 2-phenylacetates using the best septuplet-mutant P450DA-11 yielded the corresponding chiral methyl mandelates in up to 92% isolated yields and >99% ee.

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通过细胞色素 P450 羟化酶的生物催化立体选择性合成扁桃酸甲酯

手性甲基扁桃酸酯是有机转化和药物合成中的有用合成物。通过直接 C-H 激活氧化羟化来绿色合成这些有价值的化合物引起了人们的浓厚兴趣。本文介绍了通过细胞色素 P450DA 羟化酶的定向进化，实现 2-苯基乙酸甲酯到手性甲基扁桃酸甲酯的立体选择性和高效生物羟化。在本研究中，成功开发了一种基于双酶级联的新型比色高通量筛选测定法，用于 P450DA 的羟化活性工程。通过随机诱变和位点饱和/定向诱变相结合的策略，创造出了几种具有更强生物羟化活性的有益变体。使用最佳的七联突变体 P450DA-11 对各种 2-苯乙酸甲酯进行全细胞生物羟化反应，可获得相应的手性甲基扁桃酸酯，分离产率高达 92%，ee 为 99%。

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来源期刊

Green Synthesis and Catalysis

CiteScore

14.40

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0.00%

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