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

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

Abstract

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

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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URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(25)64767-X

https://www.cjcatal.com/EN/Y2025/V78/I11/144

Figures/Tables 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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