Expanding the Imine Reductase Toolbox by Exploring the Bacterial Protein‐Sequence Space

Abstract

Recent investigations on imine reductases (IREDs) have enriched the toolbox of potential catalysts for accessing chiral amines, which are important building blocks for the pharmaceutical industry. Herein, we describe the characterization of 20 new IREDs. A C-terminal domain clustering of the bacterial protein-sequence space was performed to identify the novel IRED candidates. Each of the identified enzymes was characterized against a set of nine cyclic imine model substrates. A refined clustering towards putative active-site residues was performed and was consistent both with our screening and previously reported results. Finally, preparative scale experiments on a 100 mg scale with two purified IREDs, IR_20 from Streptomyces tsukubaensis and IR_23 from Streptomyces vidiochromogenes, were carried out to provide (R)-2-methylpiperidine in 98% ee (71% yield) and (R)-1-methyl-1,2,3,4-tetrahydroisoquinoline in >98% ee (82% yield).

Asymmetric Ketone Reduction by Imine Reductases

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The rapidly growing area of asymmetrici mine reduction by imine reductases (IREDs)h as provided alternative routes to chiral amines. [2][3][4] In addition, IREDs were recently utilized as chiral catalysts forr eductive aminations, and showedh igh chemoselectivity for reducing the imine intermediate formed in situ while leaving the C=Ob ond unaffected. Assisted by an in silico analysis of energy barriers, we evaluated asymmetric hydrogenationso f carbonyls and iminesw hile considering the influence of substrate reactivity on the chemoselectivity of this novel class of reductases.W er eport the asymmetric reduction of C=Na s well as C=Ob onds catalysed by members of the IRED enzyme family.

The biocatalytic, stereocontrolled addition of hydrogen from NAD(P)H to a,b-unsaturatedc arbonyl compounds, cyclic/acyclic imines and aldehydes/ketones during asymmetric catalytic transformationi sahighlye fficient and competitive alternative for the synthesis of chiral products.

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Biocatalytic Reduction of 2‐Monosubstituted 3‐Thiazolines Using Imine Reductases

“…With the starting materials and reference compounds in hand, analytical methods for determining the conversion (by achiral GC) and enantioselectivity (by chiral SFC‐HPLC) were established (see Experimental section). Attracted by the recent successful utilization of the IRED from Mycobacterium smegmatis for the reduction of a range of prochiral 3‐thiazolines , we next conducted synthetic experiments at substrate concentrations of 20 mM rac ‐ 1a and rac ‐ 1b in order to get an insight into the suitability of this IRED to form the desired products. Towards this end, we used a tailor‐made recombinant whole‐cell biocatalyst , which contains the IRED from M. smegmatis and the GDH from Bacillus subtilis for in situ recycling of the cofactor NADPH (utilized in catalytic amount) using D ‐glucose as a co‐substrate (Table ).…”

“…The IREDs from M. smegmatis is recombinantly expressed in Escherichia coli ( E. coli ) BL21(DE3), according to a literature‐known procedure . A preculture (10 mL LB medium, containing 100 μg/mL of carbenicillin) of E. coli BL21(DE3) carrying the recombinant plasmid is cultivated overnight at 37°C.…”

The Amaryllidaceae alkaloids: biosynthesis and methods for enzyme discovery

“…An SDR in the ADH family, tetrahydroalstonine synthase, from Catharanthus roseus has been shown to reduce the imine bond on strictosidine to form tetrahydroalstonine (Stavrinides et al, 2015). Several more NADPH dependent imine reductases have been characterized in bacteria (Wetzl et al, 2015). After this reduction, norbelladine has been shown to be methylated by the class I methyltransferase N4OMT in Narcissus sp.…”