Engineering imine reductases enables stereodivergent reductive amination of 4-substituted cyclohexanones

doi:10.1016/S1872-2067(26)65279-5

Abstract

Abstract:

Imine reductases (IREDs) enable sustainable routes to stereochemically defined amines. However, their synthetic application to 4-substituted cyclohexanones, important scaffolds in pharmaceutical chemistry, remains underexplored. Here, we identified a key residue in IREDs that acts as a stereochemical switch governing cis/trans selectivity, and established a feasible and efficient approach for stereodivergent synthesis of 4-substituted cyclohexylamines. Phylogeny-guided mining of IREDs homologues yielded a subset of ten enzymes capable of catalyzing reductive amination of 4-ethylcyclohexanone in high efficiency whereas distinct diastereoselectivity. MD simulations reveal that residue 127 of IRZ06 modulates substrate orientation through steric gating and hydrophobic interactions. Mutagenesis at I127 produced two complementary variants I127L and I127P. Using a range of ketones substrates and amine donors, I127L afforded cis-products up to 95% de, whereas I127P resulted trans-products with up to 92% de. Further assessment was conducted at equivalent position of residue 127 in IRZ06 across nine IREDs, demonstrating its essential role in diastereoselectivity. Specifically, residue Pro at 127 consistently induced trans-selectivity, and Leu favored cis-selectivity. This work reveals a key residue governing diastereoselectivity of IREDs, offering a generally applicable strategy for engineering IREDs to access stereochemically defined cyclohexylamine building blocks.

Key words: Imine reductases, Stereodivergent reductive amination, 4-Substituted cyclohexylamines, Diastereoselectivity, Biocatalysis

Shunyu Zhao, Xinping Zhang, Xiangyuan Gu, Jieyu Zhou, Ye Ni. Engineering imine reductases enables stereodivergent reductive amination of 4-substituted cyclohexanones[J]. Chinese Journal of Catalysis, 2027, 92: 442-451.

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URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(26)65279-5

https://www.cjcatal.com/EN/Y2027/V92/I1/442

Figures/Tables 9

Fig. 1. Preparation of 4-substituted cyclohexylamines. (a) Examples of bioactive molecules containing 4-substituted cyclohexylamines. (b) Strategies for the asymmetric synthesis of 4-substituted cyclohexylamines employing transaminases and imine reductases in previously reports. (c) Stereodivergent synthesis of cis/trans-products by IREDs via modulating a key residue developed in this study.

Fig. 2. A phylogenetic tree of imine reductases. The four distinct imine reductase clusters are indicated by different colors. Branches corresponding to literature-reported enzymes with excellent catalytic activity are marked in blue, and the candidate enzymes selected in this study are highlighted in red.

Table 1 Conversions and stereoselectivities of IREDs toward 1a.

Enzyme	Conv. (%)	d.e. (%)
IRZ01	85±3.2	1±0.8 (cis)
IRZ02	0	0
IRZ03	95±2.1	69±0.3 (trans)
IRZ04	96±2.4	64±3.1 (trans)
IRZ05	30±5.5	27±1.5 (cis)
IRZ06	61±3.0	48±1.1 (cis)
IRZ07	63±4.8	71±2.5 (trans)
IRZ08	>99	51±5.2 (trans)
IRZ09	83±2.5	>99 (trans)
IRZ10	>99	71±0.5 (trans)

Fig. 3. Model of wild-type IRZ06 with the docked ligands. Residues subjected to mutagenesis are represented as white sticks. NADPH and 1a are represented as green sticks.

Fig. 4. Synthesis of cis/trans 4-substituted cyclohexylamines with different amines catalyzed by IRZ06 and its variants. Standard conditions: ketone (5 mmol/L), amine (a‒d, 4 equiv.), NADP+ (1 mmol/L), D-glucose (30 mmol/L), BmGDH (0.1 mg/mL), IREDs (1 mg/mL), in PBS buffer (100 mmol/L, pH = 7.5) at 30 °C for 24 h.

Fig. 5. Mechanism analysis of stereodivergent-functionalization for I127P and I127L by MD simulations. (a) Approach direction of NADPH toward substrate is characterized by the sign of ϕC4P. (b) Proposed mechanism of reductive amination process. (c) Statistics of ϕCSP for the imine intermediate within the active site of the IRZ06(WT), I127P, and I127L variants. (d) Conformation maps of WT in pre-reaction state simulation. (e) Conformation maps of I127L in pre-reaction state simulation. (f) Conformation maps of I127P in pre-reaction state simulation. The X-axis shows the distance between NADPH-C4 and the imine carbon, and the Y-axis shows the dihedral angle of NADPH-C4 relative to the cyclohexane plane in active site.

Fig. 6. Intermolecular interactions of the imine intermediate in the active site of (a) WT, (b) I127P, and (c) I127L.

Fig. 7. Sequence conservation analysis logo of IREDs. The homologous residue 127 was indicated by a red dashed rectangle.

Table 2 Diastereoselectivities of IREDs toward 1a.

Enzyme	de (%)	Enzyme	de (%)	Enzyme	de (%)
IRZ01	1±0.8 (cis)	IRZ01-V119L	42±1.6 (cis)	IRZ01-V119P	87±1.5 (trans)
IRZ03	69±0.3 (trans)	IRZ03-T120L	56±1.1 (trans)	IRZ03-T120P	81±2.3 (trans)
IRZ04	64±3.1 (trans)	IRZ04-V125L	12±1.0 (trans)	IRZ04-V125P	92±2.1 (trans)
IRZ05	27±1.5 (cis)	IRZ05-V129L	23±0.8 (trans)	IRZ05-V129P	69±6.5 (trans)
IRZ07	71±2.5 (trans)	IRZ07-V120L	29±5.5 (trans)	IRZ07-V120P	96±0.5 (trans)
IRZ08	51±5.2 (trans)	IRZ08-V122L	49±6.3 (trans)	IRZ08-V122P	91±1.0 (trans)
IRZ09	>99 (trans)	IRZ09-P137L	73±2.5 (trans)
IRZ10	71±0.5 (trans)	IRZ10-V121L	69±3.2 (trans)	IRZ10-V121P	>99 (trans)

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