亚胺还原酶催化动态动力学拆分还原胺化合成双手性中心N-取代氨基酰胺和氨基酯

doi:10.1016/S1872-2067(25)64769-3

催化学报 ›› 2025, Vol. 77: 144-152.DOI: 10.1016/S1872-2067(25)64769-3

亚胺还原酶催化动态动力学拆分还原胺化合成双手性中心N-取代氨基酰胺和氨基酯

徐泽菲^a, 冯进辉^a, 刘祥涛^a, 李倩^a, 刘卫东^a, 姚培圆^a^,^b^,^*(), 吴洽庆^a^,^*(), 朱敦明^a^,^b^,^*()

^a中国科学院天津工业生物技术研究所, 工业酶国家工程研究中心, 国家合成生物技术创新中心, 天津市生物催化技术工程研究中心, 天津 300308
^b低碳合成工程生物学全国重点实验室, 天津 300308

收稿日期:2025-05-05 接受日期:2025-07-01 出版日期:2025-10-18 发布日期:2025-10-05
通讯作者: *电子信箱: zhu_dm@tib.cas.cn (朱敦明),wu_qq@tib.cas.cn (吴洽庆),yao_py@tib.cas.cn (姚培圆).
基金资助:
国家重点研发计划(2021YFC2102000);国家自然科学基金(32401273);国家自然科学基金(22177130);天津市合成生物技术创新能力提升行动项目(TSBICIP-CXRC-035);天津市合成生物技术创新能力提升行动项目(TSBICIP-KJGG-009);中国博士后科学基金(2021M703437)

Asymmetric synthesis of chiral N-substituted amino amides and esters with two chiral centers by imine reductase-catalyzed dynamic kinetic resolution via reductive amination

Zefei Xu^a, Jinhui Feng^a, Xiangtao Liu^a, Qian Li^a, Weidong Liu^a, Peiyuan Yao^a^,^b^,^*(), Qiaqing Wu^a^,^*(), Dunming Zhu^a^,^b^,^*()

^aNational Center of Technology Innovation for Synthetic Biology, National Engineering Research Center of Industrial Enzymes and Tianjin Engineering Research Center of Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
^bState Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin 300308, China

Received:2025-05-05 Accepted:2025-07-01 Online:2025-10-18 Published:2025-10-05
Contact: *E-mail: zhu_dm@tib.cas.cn (D. Zhu), wu_qq@tib.cas.cn (Q. Wu), yao_py@tib.cas.cn (P. Yao).
Supported by:
National Key R&D Program of China(2021YFC2102000);National Natural Science Foundation of China(32401273);National Natural Science Foundation of China(22177130);Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-CXRC-035);Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-009);China Postdoctoral Science Foundation(2021M703437)

摘要/Abstract

摘要：

手性N-取代氨基酰胺和氨基酯是农药与医药领域的重要结构单元, 广泛存在于具有生物活性的化合物中, 如抗高血压药物贝那普利、依那普利以及抗癫痫药物布瓦西坦等. 然而, 这类化合物的不对称合成仍面临挑战, 尤其是具有多个手性中心的N-取代氨基酰胺/氨基酯. 传统化学合成方法常需要苛刻的反应条件或贵金属催化剂,且难以控制α-取代醛与氨基酰胺/氨基酯还原胺化反应中的手性. 因此, 开发高效、绿色的合成多手性N-取代氨基酰胺和氨基酯的方法具有重要意义.

本文设计利用亚胺还原酶催化外消旋α-取代醛基酯与手性氨基酰胺或酯的动态动力学拆分还原胺化反应, 实现手性N-取代氨基酰胺及氨基酸酯的不对称合成. 这要求亚胺还原酶能够接受手性氨基酰胺作为氨基供体, 并控制醛官能团的β-手性中心, 同时须避免反应过程中手性氨基酰胺的外消旋化, 因此极具挑战性. 本文首先通过筛选157种野生型亚胺还原酶, 发现来自Streptomyces aureocirculatus的IR104可高非对映选择性催化合成布瓦西坦(d.r.=96:4), 但其比活力仅有3 mU/mg. 为提高IR104的催化效率, 对其进行了蛋白质工程改造, 针对活性口袋内共38个残基进行了两轮定点饱和突变和迭代组合突变, 获得了三突变体D191E/L195I/E253S, 其催化效率较野生型提高204倍, 但非对映选择性为90:10. 随后对催化活性中心底物结合位点周围残基进行定点突变, 找到显著影响立体选择性的M258, 最终确认四突变体D191E/L195I/E253S/M258A(M3), 其催化效率为4.09 L/(mmol min)(野生型的102倍), 产物的非对映选择性达到98:2. 晶体结构分析和分子动力学模拟表明, 突变体活性提升源于酶与辅酶NADPH及底物相互作用的优化, E191的羧酸基团与NADPH的羟基及I195分别形成了氢键, 且I195与亚胺中间体形成疏水相互作用, 这些作用稳定氢转移过程并缩短亚胺与NADPH的距离. 进一步分析显示, M3中的A258虽不与亚胺直接作用, 但相邻的E259与S253形成额外氢键, 推测A258改变亚胺周围氢键网络, 使酶构象更紧凑、底物定位更精确, 这虽提高了立体选择性, 却降低了活性中心结构的柔性, 导致催化活性下降. 为拓展该突变酶的应用, 分别测试了5种醛和17种氨基供体, 突变体M3的催化活性均有所提升, 并对脂肪族取代的底物表现出高立体选择性. 最终成功实现布瓦西坦的克级规模制备, 并合成了17种具有双手性中心的N-取代氨基酰胺/氨基酯, 非对映选择性最高达99:1, 产率达96%, 这些产物进一步转化为γ-和δ-内酰胺.

综上, 本研究为双手性中心N-取代氨基酰胺/氨基酯及其内酰胺的不对称合成提供了绿色、高效的生物合成方法, 扩展了亚胺还原酶在动态动力学拆分还原胺化中的应用, 促进了医药和农药领域的相关化合物合成技术的进步.

关键词: 亚胺还原酶, 动态动力学拆分还原胺化, 定向进化, N-取代氨基酰胺, N-取代氨基酯

Abstract:

Chiral N-substituted amino amides and esters are ubiquitous scaffolds in pesticides and pharmaceutical chemicals, but their asymmetric synthesis remains challenging especially for those with multiple chiral centers. In this study, IR104 from Streptomyces aureocirculatus was identified from 157 wild-type imine reductases for the synthesis of (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl) butanamide (antiepileptic drug Brivaracetam) via dynamic kinetic resolution reductive amination from ethyl 3-formylhexanoate and (S)-2-aminobutylamide with high diastereoselectivity. To further improve the catalytic efficiency of IR104, its mutant D191E/L195I/E253S/M258A (M3) was identified by saturation mutagenesis and iterative combinatorial mutagenesis, which exhibited a 102-fold increase in the catalytic efficiency relative to that of wild-type enzyme and high diastereoselectivity (98:2 d.r.). Crystal structural analysis and molecular dynamics simulations provided some insights into the molecular basis for the improved activity of the mutant enzyme. The imine reductase identified in this study could accept chiral amino amides/esters as amino donors for the dynamic kinetic resolution reductive amination of racemic α-substituted aldehydo-esters, expanding the substrate scope of imine reductases in the dynamic kinetic resolution-reductive amination. Finally, IR104-M3 was successfully used for the preparation of Brivaracetam at gram scale. Using this mutant, various N-substituted amino amides/esters with two chiral centers were also synthesized with up to 99:1 d.r. and 96% yields and subsequently converted into γ- and δ-lactams, providing an efficient protocol for the synthesis of these important compounds via enzymatic dynamic kinetic resolution-reductive amination from simple building blocks.

Key words: Imine reductase, Dynamic kinetic resolution-reductive amination, Directed evolution, N-substituted amino amide, N-substituted amino ester

徐泽菲, 冯进辉, 刘祥涛, 李倩, 刘卫东, 姚培圆, 吴洽庆, 朱敦明. 亚胺还原酶催化动态动力学拆分还原胺化合成双手性中心N-取代氨基酰胺和氨基酯[J]. 催化学报, 2025, 77: 144-152.

Zefei Xu, Jinhui Feng, Xiangtao Liu, Qian Li, Weidong Liu, Peiyuan Yao, Qiaqing Wu, Dunming Zhu. Asymmetric synthesis of chiral N-substituted amino amides and esters with two chiral centers by imine reductase-catalyzed dynamic kinetic resolution via reductive amination[J]. Chinese Journal of Catalysis, 2025, 77: 144-152.

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Enzymes	Toward 1a ^b				Toward (S)-2a ^c
Enzymes	K_m (mmol/L)	k_cat (min^-1)	k_cat/K_m (L/(mmol min))	Fold^d	K_m (mmol/L)	k_cat (min^-1)	k_cat/K_m ((L/m(mol min))	Fold ^e
IR104	7.13 ± 0.79	0.30 ± 0.00	0.04	1	23.3 ± 2.7	0.30 ± 0.00	0.01	1
D191E	1.96 ± 0.24	0.84 ± 0.24	0.43	11	12.6 ± 1.0	1.02 ± 0.00	0.08	8
L195I	1.91 ± 0.14	1.02± 0.00	0.53	13	11.1 ± 0.7	1.32 ± 0.00	0.12	12
M1	2.60 ± 0.19	23.76 ± 0.42	9.14	228	30.7 ± 1.7	28.08 ± 0.54	0.91	91
M2	3.55 ± 0.27	28.98 ± 0.01	8.16	204	42.3 ± 3.8	31.26 ± 1.02	0.74	74
M3	4.46 ± 0.37	18.24 ± 0.06	4.09	102	31.1 ± 3.7	19.56 ± 0.66	0.63	63

Enzymes	Toward 1a ^b				Toward (S)-2a ^c
Enzymes	K_m (mmol/L)	k_cat (min^-1)	k_cat/K_m (L/(mmol min))	Fold^d	K_m (mmol/L)	k_cat (min^-1)	k_cat/K_m ((L/m(mol min))	Fold ^e
IR104	7.13 ± 0.79	0.30 ± 0.00	0.04	1	23.3 ± 2.7	0.30 ± 0.00	0.01	1
D191E	1.96 ± 0.24	0.84 ± 0.24	0.43	11	12.6 ± 1.0	1.02 ± 0.00	0.08	8
L195I	1.91 ± 0.14	1.02± 0.00	0.53	13	11.1 ± 0.7	1.32 ± 0.00	0.12	12
M1	2.60 ± 0.19	23.76 ± 0.42	9.14	228	30.7 ± 1.7	28.08 ± 0.54	0.91	91
M2	3.55 ± 0.27	28.98 ± 0.01	8.16	204	42.3 ± 3.8	31.26 ± 1.02	0.74	74
M3	4.46 ± 0.37	18.24 ± 0.06	4.09	102	31.1 ± 3.7	19.56 ± 0.66	0.63	63

Substrate	Specific activities (mU/mg)^a		Improvement (fold) ^b
Substrate	WT	M3	Improvement (fold) ^b
1a+(S)-2a	3.1 ± 0.4	114.8 ± 12.5	37
1a+(S)-2b	8.2 ± 1.2	39.5 ± 4.1	5
1a+(S)-2c	ND^c	14.4 ± 4.2	—
1a+(S)-2d	ND^c	28.0 ± 2.8	—
1a+(S)-2e	ND^c	7.3 ± 0.6	—
1a+(S)-2f	ND^c	6.6 ± 0.4	—
1a+(S)-2g	ND^c	9.0 ± 1.5	—
1a+(S)-2h	ND^c	28.7 ± 5.3	—
1a+(S)-2i	2.7 ± 0.4	50.2 ± 6.4	19
1a+(S)-2j	2.1 ± 0.4	23.6 ± 3.1	11
1a+(S)-2k	3.4 ± 1.0	15.9 ± 2.0	5
1a+(S)-2l	1.6 ± 0.3	3.4 ± 0.0	2
1a+(S)-2m	ND^c	4.0 ± 0.2	--
1a+(S)-2n	1.2 ± 0.4	26.5 ± 2.8	22
1a+(S)-2o	1.9 ± 0.6	57.8 ± 3.5	30
1a+(S)-2p	ND^c	4.3 ± 1.5	—
1a+(R)-2q	NM^d	NM^d	—
1a+(R)-2a	ND^c	8.8 ± 0.7	—
1b+(S)-2a	6.0 ± 2.2	69.8 ± 12.5	12
1c+(S)-2a	2.9 ± 0.6	64.4 ± 5.6	22
1d+(S)-2a	ND^c	11.2 ± 0.9	—
1e+(S)-2a	6.7 ± 0.6	22.5 ± 2.2	2
1f+(S)-2a	4.6 ± 0.5	65.2 ± 2.7	16

Substrate	Specific activities (mU/mg)^a		Improvement (fold) ^b
Substrate	WT	M3	Improvement (fold) ^b
1a+(S)-2a	3.1 ± 0.4	114.8 ± 12.5	37
1a+(S)-2b	8.2 ± 1.2	39.5 ± 4.1	5
1a+(S)-2c	ND^c	14.4 ± 4.2	—
1a+(S)-2d	ND^c	28.0 ± 2.8	—
1a+(S)-2e	ND^c	7.3 ± 0.6	—
1a+(S)-2f	ND^c	6.6 ± 0.4	—
1a+(S)-2g	ND^c	9.0 ± 1.5	—
1a+(S)-2h	ND^c	28.7 ± 5.3	—
1a+(S)-2i	2.7 ± 0.4	50.2 ± 6.4	19
1a+(S)-2j	2.1 ± 0.4	23.6 ± 3.1	11
1a+(S)-2k	3.4 ± 1.0	15.9 ± 2.0	5
1a+(S)-2l	1.6 ± 0.3	3.4 ± 0.0	2
1a+(S)-2m	ND^c	4.0 ± 0.2	--
1a+(S)-2n	1.2 ± 0.4	26.5 ± 2.8	22
1a+(S)-2o	1.9 ± 0.6	57.8 ± 3.5	30
1a+(S)-2p	ND^c	4.3 ± 1.5	—
1a+(R)-2q	NM^d	NM^d	—
1a+(R)-2a	ND^c	8.8 ± 0.7	—
1b+(S)-2a	6.0 ± 2.2	69.8 ± 12.5	12
1c+(S)-2a	2.9 ± 0.6	64.4 ± 5.6	22
1d+(S)-2a	ND^c	11.2 ± 0.9	—
1e+(S)-2a	6.7 ± 0.6	22.5 ± 2.2	2
1f+(S)-2a	4.6 ± 0.5	65.2 ± 2.7	16

Substrate	Analysis yields (%)	d.r.^a
1a+(S)-2b	78	92:8
1a+(S)-2c	55	90:10
1a+(S)-2d	74	91:9
1a+(S)-2e	76	65:35
1a+(S)-2f	51	99:1
1a+(S)-2g	24	88:12
1a+(S)-2h	70	93:7
1a+(S)-2i	95	95:5
1a+(S)-2j	76	94:6
1a+(S)-2k	71	96:4
1a+(S)-2l	55	86:14
1a+(S)-2m	50	85:15
1a+(S)-2n	86	94:6
1a+(S)-2o	85	95:5
1a+(S)-2p	34	94:6
1a+(R)-2q	30	3:97
1a+(R)-2a	31	89:11
1b+(S)-2a	89	97:3
1c+(S)-2a	76	88:12
1d+(S)-2a	55	94:6
1e+(S)-2a	33	51:49
1f+(S)-2a	98	85:15

亚胺还原酶催化动态动力学拆分还原胺化合成双手性中心N-取代氨基酰胺和氨基酯

Asymmetric synthesis of chiral N-substituted amino amides and esters with two chiral centers by imine reductase-catalyzed dynamic kinetic resolution via reductive amination

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