Artificial cascade biocatalysis for the synthesis of 2-aminocyclohexanols with contiguous stereocenters

doi:10.1016/S1872-2067(24)60158-0

Abstract

Abstract:

Chiral cyclic amino alcohols with contiguous stereocenters are key building blocks in the synthesis of bioactive molecules and pharmaceuticals. Artificial cascade biocatalysis represents an attractive method for the synthesis of chiral molecules bearing multiple stereocenters from readily available materials. Here we reported an artificial cascade biocatalysis comprising an epoxide hydrolase, an alcohol dehydrogenase, and a reductive aminase or an amine dehydrogenase. It can be utilized to access all four stereoisomers of 2-aminocyclohexanol with two contiguous stereocenters in high yields (up to 95%) and excellent stereoselectivity (up to 98% de) starting from readily available cyclohexene oxide without isolation of the intermediates. Additionally, the biocatalytic cascade has been successfully extended to the production of structurally diverse 2-(alkylamino)cyclohexanols by replacing ammonia with different organic amines.

Key words: Biocatalysis, Enzyme cascade, Amino alcohol, Reductive aminase, Amine dehydrogenase

Fei-Xiang Dong, Tian Jin, Xiaojuan Yu, Hong-Yue Wang, Qi Chen, Jian-He Xu, Gao-Wei Zheng. Artificial cascade biocatalysis for the synthesis of 2-aminocyclohexanols with contiguous stereocenters[J]. Chinese Journal of Catalysis, 2025, 68: 345-355.

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URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(24)60158-0

https://www.cjcatal.com/EN/Y2025/V68/I1/345

Figures/Tables 11

Fig. 1. Bioactive molecules and chiral ligands containing a cyclic amino alcohol with contiguous stereocenters motif.

Scheme 1. Design of artificial cascade biocatalysis for accessing all stereoisomers of 2-aminocyclohexanol 4. (A) Retrosynthetic analysis of 2-aminocyclohexanol 4 from cyclohexene oxide 1. (B) The artificial biocatalytic cascade comprising an epoxide hydrolase (EH), an alcohol dehydrogenase (ADH), a reductive aminase (RedAm) or an amine dehydrogenase (AmDH), an NADH oxidase (NOX) and a formate dehydrogenase (FDH). (C) Four different cascade routes for accessing all four 2-aminocyclohexanol stereoisomers from simple epoxide 1.

Fig. 2. Cascade biotransformation of module 1. (A) Recombinant E. coli strain coexpressing EH and ADH on plasmid pRSFDuet-1 used as whole-cell biocatalyst of module 1 for the synthesis of intermediate 3 from starting substrate 1. (B) Synthesis of (R)- and (S)-3 catalyzed by various E. coli strains coexpressing different EHs and ADHs. (C) GC chromatography of biotransformation mediated by E. coli (ReLEH-SZ529/BDHA) and E. coli (ReLEH-H178/BUDC).

Fig. 3. The screening of RedAms or AmDHs in module 2 through reductive amination of 3 yielded in module 1. Larger bars correspond to higher yield of 4 (red: (1R,2R)-4, green: (1R,2S)-4, orange: (1S,2R)-4, blue: (1S,2S)-4). Enzymes marked with asterisk are selected as the best catalysts for the construction of module 2.

Table 1 The synthesis of all four stereoisomers of 2-aminocyclohexanol 4 by different cascade routes under unoptimized reaction conditions.

Route	Module 1	Module 2	Product	Yield of 4 ^c (%)	de ^c (%)	ee ^c (%)
Route 1 ^a	E. coli (ReLEH-SZ529/BDHA)	AmtRedAm/BstFDH	(1R,2R)-4	90	97	>99
Route 2 ^b	E. coli (ReLEH-SZ529/BDHA)	LfAmDH_K68C/N261L/CbFDH	(1R,2S)-4	71	85	89
Route 3 ^a	E. coli (ReLEH-H178/BUDC)	RsRedAm/BstFDH	(1S,2R)-4	13	52	>99
Route 4 ^b	E. coli (ReLEH-H178/BUDC)	LfAmDH_K68C/N261L/CbFDH	(1S,2S)-4	67	87	98

Fig. 4. Directed evolution of wild-type (WT) RsRedAm to improve its properties. (A) Docking of (S)-3 and NADPH into the active site of WT RsRedAm. N92, W205, M235, and Q236 surrounding the substrate binding pocket were chosen for evolution. (B) Diastereomeric excess (de) of product (1S,2R)-4 generated by different RsRedAm mutants. (C) Analytic yield of product (1S,2R)-4 formed in biocatalytic cascades involving WT RsRedAm and RsRedAm-M3.

Fig. 5. Optimization of cascade modes. (A) Cascade mode A: module 1 and module 2 were cascaded using recombinant E. coli cells and cell-free extract of enzymes, respectively. (B) Cascade mode B: module 1 and module 2 were cascaded using two E. coli strains, respectively. (C) Cascade mode C: One single E. coli strain coexpressing EH, ADH, and AmDH was used for cascade of module 1 and module 2. Yield is referred to as analytic yield.

Fig. 6. Optimization of NH4+ concentration and pH of the cascade system. (A) Effect of NH4+ concentration on the yield of product 4. (1R,2R)-4 and (1S,2R)-4 were synthesized using RedAms of module 2. (1R,2S)-4 and (1S,2S)-4 were synthesized using AmDHs of module 2. (B) Biocatalytic reaction and spontaneous chemical ammonolysis occurred simultaneously in the artificial cascade system. (C) Effect of pH of cascade route 1 on optical purity of (1R,2R)-4. (D) Effect of pH of cascade route 2 on optical purity of (1R,2S)-4. (E) Effect of pH of cascade route 3 on optical purity of (1S,2R)-4. (F) Effect of pH of cascade route 4 on optical purity of (1S,2S)-4. Yield is referred to as analytic yield.

Table 2 Optimization of loading of RedAms or AmDHs in module 2.

Route	Cascade catalyst	RedAms or AmDHs loading (g L⁻¹)	Yield of 4 ^c (%)	de ^c (%)
Route 1^a	E coli (ReLEH-SZ529/ BDHA) + cell-free extract of AmtRedAm and BstFDH	5	92	97 (1R,2R)
		10	94	97 (1R,2R)
		15	95	97 (1R,2R)
		20	93	97 (1R,2R)
Route 2^b	E coli (ReLEH-SZ529/ BDHA) + cell-free extract of LfAmDH_K68C/N261L and CbFDH	5	72	87 (1R,2S)
		10	93	85 (1R,2S)
		15	93	85 (1R,2S)
		20	95	85 (1R,2S)
Route 3^a	E coli (ReLEH-H178/BUDC) + cell-free extract of RsRedAm-M3 and BstFDH	5	26	97 (1S,2R)
		10	40	98 (1S,2R)
		15	48	98 (1S,2R)
		20	50	98 (1S,2R)
Route 4^b	E coli (ReLEH-H178/BUDC) + cell-free extract of LfAmDH_K68C/N261L and CbFDH	5	61	91 (1S,2S)
		10	85	92 (1S,2S)
		15	94	92 (1S,2S)
		20	94	92 (1S,2S)

Fig. 7. Time course of cascade biocatalysis for the synthesis of (1R,2R)-4 (A), (1R,2S)-4 (B), (1S,2R)-4 (C), and (1S,2S)-4 (D) from starting substrate 1 in 100 mL preparative-scale under optimization reaction conditions.

Table 3 Synthesis of 2-(alkylamino)cyclohexanols using the artificial biocatalytic cascade with organic amines as amino donors.

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