人工Cu-TM1459金属酶催化不对称迈克尔加成反应

doi:10.11949/0438-1157.20240302

摘要/Abstract

摘要：

不对称迈克尔加成反应是合成手性化合物的重要反应，手性的构建一般由传统手性金属络合物催化完成，催化剂结构复杂，制备困难。人工金属酶可以利用生物大分子替代过渡金属手性催化剂，成为研究的热点。研究使用TM1459蛋白质支架，在原有的金属结合基序上，理性引入两个组氨酸和一个羧酸盐面部三联基序，配位Cu²⁺，制备了人工Cu-TM1459金属酶。将其用于催化不对称迈克尔加成反应研究，Cu-H52A/H58E变体金属酶具有中等反应活性和较高对映选择性（e.e.值达58%）。进一步通过分子对接和催化机理研究，对金属结合位点附近关键残基进行定点突变，I108A/C106V/K24E突变体催化该反应，产率99%，e.e.值93%。

关键词: 人工金属酶, 不对称迈克尔加成反应, 理性设计, 蛋白质, 生物催化, 催化剂

Abstract:

The asymmetric Michael addition reaction is an important reaction for synthesizing chiral compounds. Traditionally, chiral metal complexes are used as catalysts for chiral induction, but they are complex in structure and difficult to prepare. Artificial metalloenzymes, which utilize biomacromolecules to replace transition metal chiral catalysts, have become a research hotspot. In this study, the TM1459 protein scaffold was used to rationalize the introduction of two histidine residues and one carboxylic acid salt facial triad on the original metal binding motif, coordinating Cu²⁺ to prepare artificial Cu-TM1459 metalloenzyme. It was applied to catalyze the asymmetric Michael addition reaction, and the Cu-H52A/H58E variant metalloenzyme exhibited moderate reaction activity and high enantioselectivity (e.e. value up to 58%). Further studies on molecular docking and catalytic mechanism led to site-directed mutations of key residues near the metal binding site. The I108A/C106V/K24E mutant catalyzed the reaction with a yield of 99% and an e.e. value of 93%.

Key words: artificial metalloenzymes, asymmetric Michael addition reactions, rational design, protein, biocatalysis, catalyst

中图分类号:

TQ 028.8

张梦婷, 王书林, 桑熙, 元兴昊, 徐刚. 人工Cu-TM1459金属酶催化不对称迈克尔加成反应[J]. 化工学报, DOI: 10.11949/0438-1157.20240302.

Mengting ZHANG, Shulin WANG, Xi SANG, Xinghao YUAN, Gang XU. Artificial Cu-TM1459 metalloenzyme catalyzes the asymmetric Michael addition reaction[J]. CIESC Journal, DOI: 10.11949/0438-1157.20240302.

图/表 14

图1 人工金属酶应用于不对称迈克尔加成反应

Fig.1 Artificial metalloenzymes applied to asymmetric Michael addition reactions

图2 [fac-[M(His2)(O2CR)]三联体基序突变体库用于不对称迈克尔加成反应

Fig.2 [fac-[M(His2)(O2CR)] triplet mutant library for asymmetric Michael Addition reaction

表1 PCR反应体系

Table 1 The components of PCR system

试剂	体积/µL
PrimerStar^® MAX DNA聚合酶	25
DNA模板	0.5
正向引物	1
反向引物	1
dd H₂O	22.5

表2 PCR扩增步骤

Table 2 PCR steps

步骤	温度/℃	时间	循环/次
预变性	98	2.5 min	1
循环阶段	98	15 s	30
	55~65	15 s
	72	1 min 30 s
后延伸	72	5 min	1
保存	12	∞	－

图3 氮杂查尔酮化合物1a-1b

Fig.3 Nitrogen-containing chalcone compounds 1a-1b

表 3 fac-［Cu（His2）（O2CR）］三联体突变体库筛选

Table 3 Screening of fac-［Cu（His2）（O2CR）］ trinuclear mutant library

序号	TM1459突变体	产率/%	e.e./%
1	－	12	－
2	Cu²⁺	28	－
3	apo-H52A/H58E	13	-14
4	H52A	31	8
5	H52A/H58E	45	58
6	H52A/H54E	40	-5
7	H52A/H92E	55	-12
8	H58A/H52E	49	-13
9	H58A/H54E	51	-30
10	H58A/H92E	43	-4

图4 人工Cu-TM1459金属酶催化反应体系优化

Fig.4 Optimization of the catalytic reaction system of artificial Cu-TM1459 metalloenzyme

图5 配体1a的分子对接结果

Fig. 5 Molecular docking results of ligand 1a

图6 TM1459金属活性位点附近残基

Fig. 6 Residues near the active site of TM1459 metalloenzyme

图7 底物硝基甲烷与金属活性位点的相互作用

Fig. 7 The interaction between substrate nitromethane and metal active sites

图8 人工Zn-TM1459金属酶催化不对称迈克尔加成反应的可能机理

Fig. 8 Possible mechanism of asymmetric michael addition catalyzed by artificial Zn-TM1459 metalloenzyme

表4 关键氨基酸丙氨酸扫描

Table 4 Alanine scanning of key amino acids

进攻方向	突变体	产率/%	e.e./%
Re面	W56A	80	44
Re面	I108A	96	93
Si面	K24A	75	31
	R39A	88	61
	I49A	88	-8
	F94A	67	19
Re面或Si面	C106A	80	62

图9 定点突变改善金属酶催化性能

Fig. 9 Site-directed mutagenesis to enhance the catalytic performance of metalloenzymes

表5 组合突变优化人工Cu-TM1459的催化性能

Table 5 Optimizing catalytic performance of artificial Cu-TM1459 through combinatorial mutations

突变数/个	突变体	产率/%	e.e./%
2	I108A/C106V	79	92
3	I108A/C106V/W56Y	26	1
3	I108A/C106V/K24E	>99	93
4	I108A/C106V/K24E/W56Y	57	92

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