高活性工程亚胺还原酶的创制及高效催化合成手性2-芳基吡咯烷

doi:10.1016/S1872-2067(25)64767-X

催化学报 ›› 2025, Vol. 78: 144-155.DOI: 10.1016/S1872-2067(25)64767-X

高活性工程亚胺还原酶的创制及高效催化合成手性2-芳基吡咯烷

陈馨茹^a^,¹, 金添^a^,¹, 张弛^a, 朱振宇^a, 沈昕元^a, 陈琦^a, 王静^b^,^*(), 许建和^a, 郑高伟^a^,^*()

^a华东理工大学生物反应器工程国家重点实验室, 上海生物制造协同创新中心, 上海 200237
^b复旦大学附属中山医院生殖医学中心, 上海 200237

收稿日期:2025-04-23 接受日期:2025-05-25 出版日期:2025-11-18 发布日期:2025-10-14
通讯作者: *电子信箱: wang.jing4@zs-hospital.sh.cn (王静), gaoweizheng@ecust.edu.cn (郑高伟).
作者简介:¹共同第一作者.
基金资助:
国家自然科学基金(32371547);国家自然科学基金(22008068);国家重点研发计划(2024YFA0917800);国家重点研发计划(2021YFA0911400);上海市科学技术委员会(24HC2810300)

Engineering an imine reductase for enhanced activity and reduced substrate inhibition: Asymmetric synthesis of chiral 2-aryl pyrrolidines

Xin-Ru Chen^a^,¹, Tian Jin^a^,¹, Chi Zhang^a, Zhen-Yu Zhu^a, Xin-Yuan Shen^a, Qi Chen^a, Jing Wang^b^,^*(), Jian-He Xu^a, Gao-Wei Zheng^a^,^*()

^aState Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China
^bReproductive Medicine Center, Zhongshan Hospital, Fudan University, Shanghai 200237, China

Received:2025-04-23 Accepted:2025-05-25 Online:2025-11-18 Published:2025-10-14
Contact: *E-mail: wang.jing4@zs-hospital.sh.cn (J. Wang), gaoweizheng@ecust.edu.cn (G.-W. Zheng).
About author:¹Contributed equally to this work.
Supported by:
National Natural Science Foundation of China(32371547);National Natural Science Foundation of China(22008068);National Key Research and Development Program of China(2024YFA0917800);National Key Research and Development Program of China(2021YFA0911400);Science and Technology Commission of Shanghai Municipality(24HC2810300)

摘要/Abstract

摘要：

手性胺作为一类重要的手性中间体, 在医药、化工、农业等领域具有广泛应用价值. 其中, 手性2-芳基取代吡咯烷结构单元是众多药物分子的关键药效团. 以阿替卡仑(Aticaprant)为例, 该药物作为强效、高亲和力的选择性Kappa阿片受体拮抗剂, 在抑郁症、躁郁症及创伤后应激障碍等情绪相关疾病的治疗中展现出重要临床价值. 亚胺还原酶作为NAD(P)H依赖型氧化还原酶, 能够高效催化亚胺的不对称还原反应, 为手性胺化合物的合成提供绿色途径. 然而, 目前文献报道的高活力工程化亚胺还原酶催化剂(> 50 U mg^‒1)仍较罕见. 本文通过定向进化成功获得一种催化活力显著提升的突变体, 并构建了原位树脂吸附反应体系, 有效缓解了底物及产物对酶的抑制效应, 最终实现了手性2-芳基吡咯烷的高效酶法合成.

本文以绿产色链霉菌(Streptomyces viridochromogene)来源的亚胺还原酶SvIRED为研究对象, 以2-(3,5-二甲基苯基)吡咯啉为模式底物, 通过半理性设计策略对其进行分子改造以提高催化性能. 研究聚焦于底物结合位点与辅酶结合口袋的关键区域, 经过多轮迭代饱和突变, 成功获得性能显著提升的突变体SvIRED_M3. 该突变体的活力由初始的1.6 U mg^‒1大幅提升至136.8 U mg^‒1, 突破了亚胺还原酶活力100 U mg^‒1的阈值, 为目前文献报道中比活力最高的亚胺还原酶. 研究还测定了野生型SvIRED及最优突变体SvIRED_M3的底物谱, 结果显示, 相较于野生型SvIRED, 最优突变体SvIRED_M3对25种2-取代吡咯啉的亚胺还原反应及16种醛胺底物的还原胺化反应均展现出更优异的催化效率. 此外, 研究还通过引入弱酸型阳离子交换树脂D152构建反应-分离耦合体系, 有效缓解了底物和产物对酶的抑制作用. 该优化体系成功实现了多种手性2-芳基取代吡咯烷的高效合成, 并完成了抗抑郁药物阿替卡仑(Aticaprant)关键手性中间体(S)-2-(3,5-二甲基苯基)吡咯烷的克级规模制备, 转化率达98.6%, 分离收率90%, 时空产率高达438 g L^‒1 d^‒1. 这些研究成果充分展现了工程化亚胺还原酶在手性2-芳基吡咯烷合成中的巨大应用前景.

综上, 本文成功开发了高催化活性的亚胺还原酶突变体, 构建了原位树脂吸附反应体系, 有效解决了高浓度底物和产物对酶的抑制问题, 成功实现了吡咯烷类化合物的绿色高效生物催化合成, 为酶催化过程强化提供了重要参考.

关键词: 不对称还原, 亚胺还原酶, 酶定向进化, 原位树脂吸附, 2-芳基吡咯烷

Abstract:

Imine reductases (IREDs) have been extensively used for the imine reduction and reductive amination to access various amines. However, poor activity and severe substrate/product inhibition limit their widespread application in industry. Herein, an engineered IRED from Streptomyces viridochromogenes was developed through four rounds of directed evolution. The engineered SvIRED displayed a significant increase in specific activity to 136.8 U mg⁻¹, the highest reported for an IRED to date. Molecular dynamics simulations elucidated the surge in specific activity during mutations. The best mutant can also catalyse the reductive coupling of aldehyde homologs and primary amines with up to 66.9 U mg⁻¹. Additionally, we established an in-situ product adsorption system using resin, which significantly alleviated substrate/product inhibition and enhanced substrate loading to 100 g L⁻¹. Under optimal conditions, a wide range of chiral 2-aryl-pyrrolidines were successfully produced at high substrate loadings (50-100 g L⁻¹) with enantiomeric excess over 99%. The usefulness of this biocatalytic system was further demonstrated by preparation of pharmaceutically relevant chiral 2-aryl pyrrolidines, particularly the decagram-scale synthesis of the key chiral aticaprant intermediate with 90% isolated yield, >99% ee, and 438 g L⁻¹ d⁻¹ space-time yield.

Key words: Asymmetric reduction, Imine reductase, Enzyme evolution, In-situ resin adsorption, 2-Aryl pyrrolidines

陈馨茹, 金添, 张弛, 朱振宇, 沈昕元, 陈琦, 王静, 许建和, 郑高伟. 高活性工程亚胺还原酶的创制及高效催化合成手性2-芳基吡咯烷[J]. 催化学报, 2025, 78: 144-155.

Xin-Ru Chen, Tian Jin, Chi Zhang, Zhen-Yu Zhu, Xin-Yuan Shen, Qi Chen, Jing Wang, Jian-He Xu, Gao-Wei Zheng. Engineering an imine reductase for enhanced activity and reduced substrate inhibition: Asymmetric synthesis of chiral 2-aryl pyrrolidines[J]. Chinese Journal of Catalysis, 2025, 78: 144-155.

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图/表 10

Fig. 1. Representative bioactive molecules with chiral 2-aryl substituted pyrrolidines motifs.

Scheme 1. Synthetic strategies of chiral 2-aryl-substituted-pyrroli- dines.

Table 1 Performance of the best mutants from different rounds of evolution.

Entry	Enzyme	Residue												Substrate loading (g L⁻¹)	Conv.^a (%)	ee^b (%)	Specific activity^c (U mg⁻¹)	Fold improvement of activity
Entry	Enzyme	214	221	137	130	131	135	217	97	40	73	127	126	Substrate loading (g L⁻¹)	Conv.^a (%)	ee^b (%)	Specific activity^c (U mg⁻¹)	Fold improvement of activity
1	SvIRED_WT	M	Y	V	D	I	A	V	L	T	T	I	A	1	97.6	>99 (S)	1.6	1.0
2	SvIRED_WT	M	Y	V	D	I	A	V	L	T	T	I	A	10	15.4	>99 (S)	1.6	1.0
3	SvIRED_M1	L	R	V	D	I	A	V	L	T	T	I	A	10	70.6	>99 (S)	3.5	2.2
4	SvIRED_M1-1	L	R	I	D	I	A	V	L	T	T	I	A	20	46.9	>99 (S)	5.2	3.3
5	SvIRED_M1-2	L	R	I	W	I	A	V	L	T	T	I	A	20	91.8	>99 (S)	7.4	4.6
6	SvIRED_M1-3	L	R	I	W	L	A	V	L	T	T	I	A	30	49.3	>99 (S)	8.3	5.2
7	SvIRED_M1-4	L	R	I	W	L	R	V	L	T	T	I	A	30	85.3	>99 (S)	9.8	6.1
8	SvIRED_M1-5	L	R	I	W	L	R	L	L	T	T	I	A	30	99.5	>99 (S)	7.8	4.9
9	SvIRED_M2	L	R	I	W	L	R	L	Y	T	T	I	A	50	64.8	>99 (S)	8.0	5.0
10	SvIRED_M2-1	L	R	I	W	L	R	L	Y	R	T	I	A	50	68.6	>99 (S)	12.9	8.1
11	SvIRED_M2-2	L	R	I	W	L	R	L	Y	R	S	I	A	50	74.7	>99 (S)	16.4	10.3
12	SvIRED_M2-3	L	R	I	W	L	R	L	Y	R	S	P	A	50	99.6	>99 (S)	76.8	48.0
13	SvIRED_M3	L	R	I	W	L	R	L	Y	R	S	P	G	50	99.5	>99 (S)	136.8	85.5

Table 2 Kinetic parameters and thermostability (Tm) of SvIRED and key mutants a.

Entry	Enzyme	K_i (mmol L⁻¹)	K_m (mmol L⁻¹)	k_cat (s⁻¹)	k_cat/K_m (s⁻¹ mL mol⁻¹)	T_m^b (°C)
1	SvIRED_WT	2.14 ± 0.31	0.02 ± 0.00	1.04 ± 0.04	52.00	53.4
2	SvIRED_M1	1.25 ± 0.34	0.26 ± 0.06	3.97 ± 0.55	15.27	52.7
3	SvIRED_M2	8.89 ± 4.08	0.02 ± 0.00	4.10 ± 0.20	205.00	54.1
4	SvIRED_M3	7.22 ± 1.94	0.12 ± 0.01	98.33 ± 4.57	819.42	53.6

Fig. 2. Structural analysis of SvIREDWT and SvIREDM3. (A) Distribution of all mutated sites. (B) Interaction differences between mutated sites 40, 73, and NADPH in SvIREDWT (left) and SvIREDM3 (right). (C) Interaction differences between mutated sites 97 and Y140 in SvIREDWT (left) and SvIREDM3 (right). (D) Substrate binding mode analysis of 1a in SvIREDWT (left) and SvIREDM3 (right). Substrate 1a and NADPH are indicated using green and magenta sticks, respectively. Hydrogen bonds are indicated using broken lines. Mutated sites on A chain and B chain are coloured violet and yellow, respectively. The distance of hydride transfer is described as “d”. “d” is 5.7 ± 0.6 ? for SvIREDWT and 4.9 ± 0.6 ? for SvIREDM3 (average of three parallel simulations).

Fig. 3. Asymmetric reduction of 2-substituted pyrrolines catalysed by SvIREDWT and SvIREDM3. Reaction conditions were 100 mmol L-1 substrate, KPi buffer (100 mmol L-1, pH = 6.0), 1 mmol L-1 NADP+, 150 mmol L-1 glucose, 5% (v/v) DMSO, 1 mg mL?1 purified enzyme, 1.5 mg BmGDH lyophilised cell-free extract, 0.5 mL reaction volume in a 2 mL tube, 30 °C, and shaking at 1000 rpm for 24 h. Conversion was determined by GC-FID. n.a., not available; n.d., not detected. Specific activity was determined at 0.2 mmol L-1 2-substituted pyrrolines (2 mmol L-1 for 27a), 0.1 mmol L-1 NADPH, KPi buffer (100 mmol L-1, pH = 6.0) and 30 °C using purified enzyme. Absolute configurations were determined by circular dichroism (CD) spectroscopy. a Reactions were not conducted due to low and undetected activity.

Fig. 4. Reductive amination of aldehydes catalysed by SvIREDWT and SvIREDM3. Reaction conditions were 100 mmol L-1 1-4, 1 mol L-1 A-D (10 equiv.), KPi buffer (100 mmol L-1, pH = 7.0), 1 mmol L-1 NADP+, 150 mmol L-1 glucose, 5% (v/v) DMSO, 1 mg mL?1 purified enzyme, 1.5 mg BmGDH lyophilised cell-free extract, 0.5 mL reaction volume in a 2 mL tube, 30 °C, and shaking at 1000 rpm for 24 h. Conversion was determined by GC-FID. Specific activity was determined at 10?mmol L-1 1-4, 100 mmol L-1 A-D, 0.1?mmol L-1 NADPH, KPi buffer (100 mmol L-1, pH = 7.0) and 30 °C using purified enzyme.

Table 3 Process development for efficient synthesis of (S)-1b using SvIREDM3 a.

Entry	Substrate loading (g L⁻¹)	Substrate addition	Feeding mode	Titration	Resin	Time (h)	Conv. (%)	ee (%)
1	50	batch	—	2 mol L⁻¹ HCl	—	1	99.0	>99
2	60	batch	—	2 mol L⁻¹ HCl	—	2	96.4	>99
3^[b]	60	batch	—	2 mol L⁻¹ HCl	—	24	96.3	>99
4	60	fed-batch	15/15/15/15 g L⁻¹ in 2 h	2 mol L⁻¹ HCl	—	3	99.1	>99
5	80	batch	—	2 mol L⁻¹ HCl	—	2	45.7	>99
6	80	fed-batch	20 g L⁻¹ per 1.5 h	2 mol L⁻¹ HCl	—	6	46.8	>99
7	80	fed-batch	45/25/10 g L⁻¹ in 3 h	1 mol L⁻¹ HCl/3 mol L⁻¹ K₂CO₃	—	6	75.4	>99
8^b	80	fed-batch	45/25/10 g L⁻¹ in 3 h	1 mol L⁻¹ HCl/3 mol L⁻¹ K₂CO₃	—	24	75.6	>99
9	80	batch	—	—	IRN150	6	94.1	>99
10	80	batch	—	—	HZ835	6	85.3	>99
11	80	batch	—	—	NKA-II	6	95.2	>99
12	80	batch	—	—	D152	6	99.0	>99
13	100	batch	—	—	D152	6	98.7	>99

Scheme 2. Preparative-scale synthesis of 2-aryl-substituted pyrrolidines using SvIREDM3 and resin D152. Reaction mixture (5 mL) containing 20 g L?1 SvIREDM3 wet cells, 50 g L?1 substrate (80 g L?1 for 4a), 1.5 eq. glucose, 50 mg BmGDH lyophilised cell-free extract, 1 mmol L?1 NADP+, 5% DMSO (v/v), 0.15 g mL?1 resin (dry mass), and KPi buffer (100 mmol L?1, pH = 6.0) was shaken at 30 °C for 20 h.

Fig. 5. Preparative-scale synthesis of (S)-1b by SvIREDM3 in a 100 mL in-situ resin adsorption system.

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高活性工程亚胺还原酶的创制及高效催化合成手性2-芳基吡咯烷

Engineering an imine reductase for enhanced activity and reduced substrate inhibition: Asymmetric synthesis of chiral 2-aryl pyrrolidines

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