Imine Reductase Research Articles

One-Pot Biocatalytic Cascade Reduction of Cyclic Enimines for the Preparation of Diastereomerically Enriched N-Heterocycles.

Ene-reductases (EREDs) catalyze the reduction of electron-deficient C═C bonds. Herein, we report the first example of ERED-catalyzed net reduction of C═C bonds of enimines (α,β-unsaturated imines). Preliminary studies suggest their hydrolyzed ring-open ω-amino enones are the likely substrates for this step. When combined with imine reductase (IRED)-mediated C═N reduction, the result is an efficient telescoped sequence for the preparation of diastereomerically enriched 2-substituted saturated amine heterocycles.

Imine Reductase Based All-Enzyme Hydrogel with Intrinsic Cofactor Regeneration for Flow Biocatalysis.

All-enzyme hydrogels are biocatalytic materials, with which various enzymes can be immobilized in microreactors in a simple, mild, and efficient manner to be used for continuous flow processes. Here we present the construction and application of a cofactor regenerating hydrogel based on the imine reductase GF3546 from Streptomyces sp. combined with the cofactor regenerating glucose-1-dehydrogenase from Bacillus subtilis. The resulting hydrogel materials were characterized in terms of binding kinetics and viscoelastic properties. The materials were formed by rapid covalent crosslinking in less than 5 min, and they showed a typical mesh size of 67 ± 2 nm. The gels were applied for continuous flow biocatalysis. In a microfluidic reactor setup, the hydrogels showed excellent conversions of imines to amines for up to 40 h in continuous flow mode. Variation of flow rates led to a process where the gels showed a maximum space-time-yield of 150 g·(L·day)−1 at 100 μL/min.

Engineering imine reductases for industrial applications

Imine reductases are promising catalysts, facilitating a direct stereoselective route to secondary amines. Now, protein engineering has created stable and efficient variants that allow their application in kilogram-scale synthesis.

Fermentative Production of N-Alkylated Glycine Derivatives by Recombinant Corynebacterium glutamicum Using a Mutant of Imine Reductase DpkA From Pseudomonas putida.

Sarcosine, an N-methylated amino acid, shows potential as antipsychotic, and serves as building block for peptide-based drugs, and acts as detergent when acetylated. N-methylated amino acids are mainly produced chemically or by biocatalysis, with either low yields or high costs for co-factor regeneration. Corynebacterium glutamicum, which is used for the industrial production of amino acids for decades, has recently been engineered for production of N-methyl-L-alanine and sarcosine. Heterologous expression of dpkA in a C. glutamicum strain engineered for glyoxylate overproduction enabled fermentative production of sarcosine from sugars and monomethylamine. Here, mutation of an amino acyl residue in the substrate binding site of DpkA (DpkAF117L) led to an increased specific activity for reductive alkylamination of glyoxylate using monomethylamine and monoethylamine as substrates. Introduction of DpkAF117L into the production strain accelerated the production of sarcosine and a volumetric productivity of 0.16 g L−1 h−1 could be attained. Using monoethylamine as substrate, we demonstrated N-ethylglycine production with a volumetric productivity of 0.11 g L−1 h−1, which to the best of our knowledge is the first report of its fermentative production. Subsequently, the feasibility of using rice straw hydrolysate as alternative carbon source was tested and production of N-ethylglycine to a titer of 1.6 g L−1 after 60 h of fed-batch bioreactor cultivation could be attained.

Evolved enzyme improves route to drug candidate

Chemists at the pharmaceutical company GlaxoSmithKline had some problems with the route originally devised to install a chiral secondary amine in the drug candidate GSK2879552. To make a key intermediate of the molecule, which is in Phase II clinical trials for the treatment of small-cell lung cancer and acute leukemia, they first had to separate enantiomers of the secondary amine. Then, following reductive amination, they had to use a complex extraction process that required several environmentally unfriendly solvents and generated boron waste. Looking to make their synthesis greener, simpler, and less expensive, the chemists turned to imine reductase enzymes. Such enzymes are known to perform reductive amination reactions, but until now they have been used only with simple amines and carbonyl compounds and in conditions that aren’t amenable to industrial syntheses. The GSK team, led by Gheorghe-Doru Roiban, put an imine reductase enzyme from the bacterium Saccharothrix espanaensis through three

Chiral synthesis of LSD1 inhibitor GSK2879552 enabled by directed evolution of an imine reductase

Imine reductases catalyse the reductive amination of aldehydes or ketones with amines to produce chiral amines—a key transformation in the preparation of fine chemicals and active pharmaceutical ingredients. Although significant progress has been recently made in the field, their industrial application has not been demonstrated. Herein, we describe a wild-type imine reductase that was engineered to perform reductive amination with concomitant substrate amine resolution to give a commercially relevant manufacturing process to lysine-specific demethylase-1 inhibitor GSK2879552. Three rounds of evolution resulted in an enzyme variant showing a >38,000-fold improvement over wild type. The engineering of a more stable and active enzyme variant enabled process optimization to an economic, high quality and sustainable operating space. Using the evolved enzyme, kilogram quantities of a key intermediate to GSK2879552 were produced in 84% yield, at 99.9% purity and >99.7% enantiomeric excess, with improved process mass intensity. Imine reductases have been regarded as one of the most promising enzymes by the pharmaceutical industry—but their industrial application is still lacking. This work reports the successful industrial application of an imine reductase, enabled by directed evolution.

Synthesis of β‐Chiral Amines by Dynamic Kinetic Resolution of α‐Branched Aldehydes Applying Imine Reductases

AbstractImine reductases (IREDs) allow the one‐step preparation of optically active secondary and tertiary amines by reductive amination of ketones. Until now, mainly α‐chiral amines have been prepared by this route. In this study, we explored the possibility of synthesizing β‐chiral amines, a class of compounds which is also frequently found as structural motif in pharmaceuticals but much more challenging to prepare due to the following reasons: (i) The aldehyde substrate already contains the chiral center and needs to be racemized to enable full conversion. (ii) Because the intermediate imine bears the stereo center two carbon atoms remote to the imine nitrogen, it is more challenging to achieve high enantioselectivity compared to α‐chiral amine synthesis. For investigating the proof of concept, we first confirmed that different IREDs are able to convert a variety of α‐branched aldehydes when combined with five different amine substrates. The IRED from Streptomyces ipomoeae was a suitable enzyme facilitating the dynamic kinetic resolution of 2‐phenylpropanal and a substituted 2‐methyl‐3‐phenylpropanal: the corresponding N‐methylated β‐chiral amines were obtained with >95 % conversion and 78 and 95 %ee. Other amines were formed with low to moderate enantiomeric excess. This exemplifies the potential of IREDs for the one‐step synthesis of secondary β‐chiral amines, but also the challenge to identify highly selective enzymes for a desired amine product.

An Asymmetric Reductase That Intercepts Acyclic Imino Acids Produced in Situ by a Partner Oxidase.

Acyclic imines are unstable in aqueous conditions. For this reason, known imine reductases, which enable the synthesis of chiral amines, mainly intercept stable cyclic imines. Here we report the detailed biochemical and structural characterization of Bsp5, an imino acid reductase from the d-2-hydroxyacid dehydrogenase family that reduces acyclic imino acids produced in situ by a partner oxidase. We determine a 1.6 Å resolution structure of Bsp5 in complex with d-arginine and coenzyme NADPH. Combined with mutagenesis work, our study reveals the minimal structural constraints for its biosynthetic activity. Furthermore, we demonstrate that Bsp5 can intercept more complex products from an alternate oxidase partner, suggesting that this oxidase-imino acid reductase pair could be evolved for biocatalytic conversion of l-amino acids to d-amino acids.

Cascade Biotransformation to Access 3‐Methylpiperidine in Whole Cells

AbstractSynthesis of 3‐methylpiperidine from 1,5‐diamino‐2‐methylpentane in preparative scale is reported by using recombinant Escherichia coli cells expressing a variant of the diamine oxidase from Rhodococcus erythroprolis and an imine reductase from Streptosporangium roseum. Optimization of process parameters for cultivation and bioconversion led to substantial improvements in the initial laboratory procedure. The transformation of the methyl‐substituted diamine substrate to the N‐heterocyclic product was successfully scaled‐up from shake‐flask to a 20 L bioreactor with increased substrate concentrations. Remarkably, we obtained 67 % of 3‐methylpiperidine product from 140 g substrate within 52 h.

Corrigendum: Corrigendum: Sequence‐Based In‐silico Discovery, Characterisation, and Biocatalytic Application of a Set of Imine Reductases

After publication, the authors noticed a mistake in the description of the analytical methods in the Supporting Information. For the chiral gas chromatography separation of compounds 2a–c, a derivatisation with acetic anhydride was used, but this information is missing in the original Supporting Information. We add the derivatisation step now and update the Supporting Information for clarity (see the section on ‘Gas chromatography (chiral stationary phase)’ on page S29). The authors apologize for the oversight.

Technical Considerations for Scale-Up of Imine-Reductase-Catalyzed Reductive Amination: A Case Study

Imine reductases (IREDs) have attracted increasing attention as novel biocatalysts for the synthesis of various cyclic and acyclic amines. Herein a number of guidelines and considerations toward th...

Synthesis of N-heterocycles from diamines via H2-driven NADPH recycling in the presence of O2

Herein, we report an enzymatic cascade involving an oxidase, an imine reductase and a hydrogenase for the H2-driven synthesis of N-heterocycles. Variants of putrescine oxidase from Rhodococcus erythropolis with improved activity were identified. Substituted pyrrolidines and piperidines were obtained with up to 97% product formation in a one-pot reaction directly from the corresponding diamine substrates. The formation of up to 93% ee gave insights into the specificity and selectivity of the putrescine oxidase.

Xylose as preferred substrate for sarcosine production by recombinant Corynebacterium glutamicum

The aim of this work was to study the fermentative production of the N-methylated amino acid sarcosine by C. glutamicum. Characterization of the imine reductase DpkA from Pseudomonas putida revealed that it catalyses N-methylamination of glyoxylate to sarcosine. Heterologous expression of dpkA in a C. glutamicum strain engineered for glyoxylate overproduction enabled fermentative production of sarcosine from sugars and monomethylamine. Glucose-based fermentation reached sarcosine production titers of 2.4 ± 0.1 g L−1. Sarcosine production based on the second generation feedstocks xylose and arabinose led to higher product titers of 2.7 ± 0.1 g L−1 and 3.4 ± 0.3 g L−1, respectively, than glucose-based production. Optimization of production conditions with xylose and potassium acetate blends increased sarcosine titers to 8.7 ± 0.2 g L−1 with a yield of 0.25 g g−1. This is the first example in which a C. glutamicum process using lignocellulosic pentoses is superior to glucose-based production.

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

The application of a straightforward biocatalytic technology for the reduction of racemic 2‐monosubstituted 3‐thiazolines, which are easily prepared via Asinger‐multicomponent reaction, is reported. The biocatalytic reduction yields racemic 2‐monosubstituted 3‐thiazolidines, which are difficult to be prepared by means of classic chemical routes, in moderate to high yields. Moreover, our study clarifies the stereochemical reaction course of the biocatalytic reduction. Furthermore, the efficiency of this biocatalytic technology is demonstrated in an experiment at an elevated substrate concentration of 60 mM leading to 96% conversion.

One-pot synthesis of a 3-thiazolidine through combination of an Asinger-type multi-component-condensation reaction with an enzymatic imine reduction

Addressing the combination of heterocyclic chemistry and biocatalysis, a one-pot synthesis of a 3-thiazolidine by means of an initial Asinger-type multi-component-condensation reaction under formation of a 3-thiazoline and a subsequent enzymatic reduction of this cyclic imine is reported. The one-pot process is conducted in a sequential mode and without the need for a spatial compartmentalization of the single reaction steps. For the biocatalytic reduction an imine reductase was used in combination with a glucose dehydrogenase and D-glucose for in situ-cofactor recycling. This process has been applied successfully towards the synthesis of (S)-2,2,3-trimethyl-1-thia-4-azaspiro[4.4]nonane on a 4.5 mmol scale leading to moderate conversion and excellent enantioselectivity of 99% enantiomeric excess.

Enantioselective Biocatalytic Reduction of 2 H-1,4-Benzoxazines Using Imine Reductases.

A biocatalytic reduction of 2 H-1,4-benzoxazines using imine reductases is reported. This process enables a smooth and enantioselective synthesis of the resulting cyclic amines under mild conditions in aqueous media by means of a catalytic amount of the cofactor NADPH as hydride source as well as glucose as the reducing agent used in stoichiometric amounts for in situ cofactor recycling. Several substrates were studied, and the 3,4-dihydro-2 H-1,4-benzoxazines were obtained with up to 99% ee. In addition, the efficiency of this reduction process based on imine reductases as catalysts has been demonstrated for one 2 H-1,4-benzoxazine on an elevated laboratory scale running at a substrate loading of 10 g L-1 in the presence of a tailor-made whole-cell catalyst.

Imine Reductase‐Catalyzed Enantioselective Reduction of Bulky α,β‐Unsaturated Imines en Route to a Pharmaceutically Important Morphinan Skeleton

AbstractThe morphinan skeleton is an important sub‐structure in many medicines such as dextromethorphan, and can be constructed from 1‐benzyl‐1,2,3,4,5,6,7,8‐octahydroisoquinoline (1‐benzyl‐OHIQ) derivatives. 1‐Benzyl‐3,4,5,6,7,8‐hexahydroisoquinolines (1‐benzyl‐HHIQs), the precursors of 1‐benzyl‐OHIQs, constitute a type of bulky α, β‐unsaturated imines. Until now, the application of imine reductases (IREDs) to α, β‐unsaturated imines has only rarely been reported. In this study, through evaluation of 48 IREDs, both enantiomers of 1‐(4‐methoxybenzyl)‐1,2,3,4,5,6,7,8‐octahydroisoquinoline (1‐(4‐methoxybenzyl)‐OHIQ) were obtained in high yield and excellent optical purity. Among the enzymes, the most steric hindrance‐tolerant IRED from Sandarearacinus amylolyticus (IR40) was able to convert various phenyl substituted 1‐benzyl‐HHIQ to the corresponding 1‐benzyl‐OHIQ derivatives with excellent enantiometric excess. These results provide an effective route to synthesize these important compounds via enantioselective reduction of bulky α, β‐unsaturated imine precursors, which can be readily prepared from 2‐(1‐cyclohexenyl)ethylamine and corresponding aryl acetic acids.magnified image

Chemoenzymatic Synthesis of Substituted Azepanes by Sequential Biocatalytic Reduction and Organolithium-Mediated Rearrangement.

Enantioenriched 2-aryl azepanes and 2-arylbenzazepines were generated biocatalytically by asymmetric reductive amination using imine reductases or by deracemization using monoamine oxidases. The amines were converted to the corresponding N'-aryl ureas, which rearranged on treatment with base with stereospecific transfer of the aryl substituent to the 2-position of the heterocycle via a configurationally stable benzyllithium intermediate. The products are previously inaccessible enantioenriched 2,2-disubstituted azepanes and benzazepines.

A Mechanism for Reductive Amination Catalyzed by Fungal Reductive Aminases

Reductive aminases (RedAms) catalyze the asymmetric reductive amination of ketones with primary amines to give secondary amine products. RedAms have great potential for the synthesis of bioactive chiral amines; however, insights into their mechanism are currently limited. Comparative studies on reductive amination of cyclohexanone with allylamine in the presence of RedAms, imine reductases (IREDs), or NaBH3CN support the distinctive activity of RedAms in catalyzing both imine formation and reduction in the reaction. Structures of AtRedAm from Aspergillus terreus, in complex with NADPH and ketone and amine substrates, along with kinetic analysis of active-site mutants, reveal modes of substrate binding, the basis for the specificity of RedAms for reduction of imines over ketones, and the importance of domain flexibility in bringing the reactive participants together for the reaction. This information is used to propose a mechanism for their action and also to expand the substrate specificity of RedAms usin...

Development of Biotransamination Reactions towards the 3,4-Dihydro-2H-1,5-benzoxathiepin-3-amine Enantiomers

The stereoselective synthesis of chiral amines is an appealing task nowadays. In this context, biocatalysis plays a crucial role due to the straightforward conversion of prochiral and racemic ketones into enantiopure amines by means of a series of enzyme classes such as amine dehydrogenases, imine reductases, reductive aminases and amine transaminases. In particular, the stereoselective synthesis of 1,5-benzoxathiepin-3-amines have attracted particular attention since they possess remarkable biological profiles; however, their access through biocatalytic methods is unexplored. Amine transaminases are applied herein in the biotransamination of 3,4-dihydro-2H-1,5-benzoxathiepin-3-one, finding suitable enzymes for accessing both target amine enantiomers in high conversion and enantiomeric excess values. Biotransamination experiments have been analysed, trying to optimise the reaction conditions in terms of enzyme loading, temperature and reaction times.