Imine Reductase Research Articles

An Engineered Imine Reductase for Highly Diastereo‐ and Enantioselective Synthesis of β‐Branched Amines with Contiguous Stereocenters

Abstractβ‐Branched chiral amines with contiguous stereocenters are valuable building blocks for preparing various biologically active molecules. However, their asymmetric synthesis remains challenging. Herein, we report a highly diastereo‐ and enantioselective biocatalytic approach for preparing a broad range of β‐branched chiral amines starting from their corresponding racemic ketones. This involves a dynamic kinetic resolution‐asymmetric reductive amination process catalyzed using only an imine reductase. Four rounds of protein engineering endowed wild‐type PocIRED with higher reactivity, better stereoselectivity, and a broader substrate scope. Using the engineered enzyme, various chiral amine products were synthesized with up to >99.9 % ee, >99 : 1 dr, and >99 % conversion. The practicability of the developed biocatalytic method was confirmed by producing a key intermediate of tofacitinib in 74 % yield, >99.9 % ee, and 98 : 2 dr at a challenging substrate loading of 110 g L−1. Our study provides a highly capable imine reductase and a protocol for developing an efficient biocatalytic dynamic kinetic resolution‐asymmetric reductive amination reaction system.

A Quantum Mechanical Approach to The Mechanism of Asymmetric Synthesis of Chiral Amine by Imine Reductase from Stackebrandtia Nassauensis.

The asymmetric synthesis of tetrahydroisoquinolines (THIQs) has gained importance in recent years due to their significant potential in drug development studies. In this study, the conversion of 1-methyl-3,4-dihydroisoquinoline substrate to a chiral amine, 1-methyl-1,2,3,4-tetrahydroisoquinoline, under the catalysis of the stereoselective imine reductase enzyme from Stackebrandtia nassauensis (SnIR) was investigated in detail to elucidate the mechanism and explain the experimental enantioselectivity. The results were found to be in agreement with the experimental data. To elucidate the reaction mechanism, quantum mechanical calculations were performed by considering a large cluster of the active site of the enzyme. In this regard, possible reaction pathways leading to both R- and S-products with the corresponding intermediates and the transition states for the hydride transfer from the cofactor to the substrate were considered by density functional theory (DFT) calculations, and the factors contributing to the observed stereoselectivity were sought. The calculations supported a stepwise mechanism rather than the concerted protonation and the hydride transfer steps. The stereoselectivity in the hydride transfer was found to be due not only to the stability of the enzyme-subtrate complex but also to the corresponding reaction barriers. The calculations were performed at the wB97XD/6-311+G(2df,2p)//B3LYP/6-31G(d,p) level of theory using the PCM approach.

Expanding the repertoire of imine reductases by mining divergent biosynthetic pathways for promiscuous reactivity

Expanding the repertoire of imine reductases by mining divergent biosynthetic pathways for promiscuous reactivity

Biosynthesis of the α-D-Mannosidase Inhibitor (-)-Swainsonine.

(-)-Swainsonine is a polyhydroxylated indolizidine alkaloid with potent inhibitory activity against α-D-mannosidases. In this work, we successfully reconstituted swainsonine biosynthetic pathway both in vivo and in vitro. Our study unveiled a surprising epimerization mechanism involving an imine reductase (SwnN) and two non-heme iron and α-ketoglutarate-dependent oxygenases (SwnH2 and SwnH1). SwnH2 is a multifunctional enzyme responsible for both C2-hydroxylation and amine desaturation, while SwnN completes the oxidation-reduction sequence to achieve the net redox neutral epimerization. Notably, SwnN exhibits substrate-dependent stereospecificity and the stereochemical outcome of SwnN-catalyzed iminium reduction is determined by SwnH1-catalyzed C8-hydroxylation. We also discovered that an O -acetyl ester can serve as a detachable protecting/directing group, altering the site-selectivity of SwnH2-catalyzed hydroxylation while maintaining the stereoselectivity. Insights gained from the biochemical characterization of these tailoring enzymes enabled biocatalytic synthesis of a new polyhydroxylated indolizidine alkaloid, opening doors to the biosynthesis of diverse natural product-based glycosidase inhibitors.

Bifunctional Imine Reductase Cascades for the Synthesis of Saturated N-Heterocycles.

Saturated N-heterocycles constitute a vital scaffold for pharmaceutical chemistry but are challenging to access synthetically, particularly asymmetrically. Here, we demonstrate how imine reductases can achieve annulation through tandem inter- and intramolecular reductive amination processes. Imine reductases were used in combination with further enzymes to access unsubstituted, α-substituted, and α,α'-disubstituted N-heterocycles from simple starting materials in one pot and under benign conditions. This work shows the remarkable flexibility of these enzymes to have broad activity against numerous substrates derived from singlular starting materials.

Biocatalytic Desymmetrization for the Atroposelective Synthesis of Axially Chiral Biaryls Using an Engineered Imine Reductase.

The enzymatic atroposelective synthesis of biaryl compounds is relatively rare, despite considerable attention received by biocatalysis in academic and industrial sectors. Imine reductases (IREDs) are an important class of enzymes that have been applied in the asymmetric synthesis of chiral amine building blocks. In this work, two IREDs (IR140 and IR189) were identified to catalyze the efficient desymmetrization of biaryls utilizing various amine donors. Further protein engineering enabled the identification of variants (IR189 M8-M9 and IR189 M13-M14) that are able to catalyze the formation of both (R) and (S) atropisomers in excellent yields and atroposelectivities for up to 24 examples (up to 99% ee and yield). The absolute configuration and rotational barriers were confirmed, and the reactions were readily enlarged to allow isolation of the atropisomeric products in 99% ee and 82% isolated yields. The optically pure biaryl amines were further derivatized into various synthetically useful atropisomers. To shed light on the molecular recognition mechanisms, molecular dynamics (MD) simulations were performed, offering plausible explanations for the improved atroposelectivities and enzymatic activities. The current strategy expands the scope of IRED-catalyzed synthesis of axially chiral biaryl amines, contributing significantly to the field of atroposelective biocatalysis.

Engineered Imine Reductase Catalyzed Enantiodivergent Synthesis of Alkylated Amphetamines.

Less steric ketones exhibited low stereoselectivity toward M5 due to their difficulty in restricting the free rotation of the imine intermediate. An engineered enantio-complementary imine reductase from M5 was obtained with catalytic activity. We identified four key residues that play essential roles in controlling stereoselectivity. Two mutants, I149Y-W234L (up to 99%S ee) and L200M-F260M (up to 99%R ee), were achieved, showing excellent stereoselectivity toward the tested substrates, offering valuable biocatalysts for synthesizing alkylated amphetamines.

Biocatalytic Desymmetrization for the Atroposelective Synthesis of Axially Chiral Biaryls Using an Engineered Imine Reductase

The enzymatic atroposelective synthesis of biaryl compounds is relatively rare, despite considerable attention received by biocatalysis in academic and industrial sectors. Imine reductases (IREDs) are an important class of enzymes that have been applied in the asymmetric synthesis of chiral amine building blocks. In this work, two IREDs (IR140 and IR189) were identified to catalyze the efficient desymmetrization of biaryls utilizing various amine donors. Further protein engineering enabled the identification of variants (IR189 M8‐M9 and IR189 M13‐M14) that are able to catalyze the formation of both (R) and (S) atropisomers in excellent yields and atroposelectivities for up to 24 examples (up to 99% ee and yield). The absolute configuration and rotational barriers were confirmed, and the reactions were readily enlarged to allow isolation of the atropisomeric products in 99% ee and 82% isolated yields. The optically pure biaryl amines were further derivatized into various synthetically useful atropisomers. To shed light on the molecular recognition mechanisms, molecular dynamics (MD) simulations were performed, offering plausible explanations for the improved atroposelectivities and enzymatic activities. The current strategy expands the scope of IRED‐catalyzed synthesis of axially chiral biaryl amines, contributing significantly to the field of atroposelective biocatalysis.

Synthesis of Substituted Acyclic and Cyclic N-Alkylhydrazines by Enzymatic Reductive Hydrazinations.

Imine reductases (IREDs) provide promising opportunities for the synthesis of various chiral amines. Initially, asymmetric imine reduction was reported, followed by reductive aminations of aldehydes and ketones via imines. Herein we present the reductive amination of structurally diverse carbonyls and dicarbonyls with hydrazines (reductive hydrazination), catalyzed by the IRED from Myxococcus stipitatus. In analogy to IRED-catalyzed reductive aminations, various carbonyls and dicarbonyls could react with simple hydrazines to produce substituted acyclic and cyclic N-alkylhydrazines. By incorporating and scaling up hydrogenase cofactor regeneration system, we demonstrated the scalability and atom-efficiency of an H2-driven double reductive hydrazination, highlightling the potential of IREDs in biocatalysis.

Atroposelective Synthesis of Axial Biaryls by Dynamic Kinetic Resolution Using Engineered Imine Reductases

AbstractAxially chiral biaryl compounds are ubiquitous scaffolds in natural products, bioactive molecules, chiral ligands and catalysts, but biocatalytic methods for their asymmetric synthesis are limited. Herein, we report a highly efficient biocatalytic route for the atroposelective synthesis of biaryls by dynamic kinetic resolution (DKR). This DKR approach features a transient six‐membered aza‐acetal‐bridge‐promoted racemization followed by an imine reductase (IRED)‐catalyzed stereoselective reduction to construct the axial chirality under ambient conditions. Directed evolution of an IRED from Streptomyces sp. GF3546 provided a variant (S‐IRED‐Ss‐M11) capable of catalyzing the DKR process to access a variety of biaryl aminoalcohols in high yields and excellent enantioselectivities (up to 98 % yield and >99 : 1 enantiomeric ratio). Molecular dynamics simulation studies on the S‐IRED‐Ss‐M11 variant revealed the origin of its improved activity and atroposelectivity. By exploiting the substrate promiscuity of IREDs and the power of directed evolution, our work further extends the biocatalysts’ toolbox to construct challenging axially chiral molecules.

ThermoLink: Bridging disulfide bonds and enzyme thermostability through database construction and machine learning prediction.

Disulfide bonds, covalently formed by sulfur atoms in cysteine residues, play a crucial role in protein folding and structure stability. Considering their significance, artificial disulfide bonds are often introduced to enhance protein thermostability. Although an increasing number of tools can assist with this task, significant amounts of time and resources are often wasted owing to inadequate consideration. To enhance the accuracy and efficiency of designing disulfide bonds for protein thermostability improvement, we initially collected disulfide bond and protein thermostability data from extensive literature sources. Thereafter, we extracted various sequence- and structure-based features and constructed machine-learning models to predict whether disulfide bonds can improve protein thermostability. Among all models, the neighborhood context model based on the Adaboost-DT algorithm performed the best, yielding "area under the receiver operating characteristic curve" and accuracy scores of 0.773 and 0.714, respectively. Furthermore, we also found AlphaFold2 to exhibit high superiority in predicting disulfide bonds, and to some extent, the coevolutionary relationship between residue pairs potentially guided artificial disulfide bond design. Moreover, several mutants of imine reductase 89 (IR89) with artificially designed thermostable disulfide bonds were experimentally proven to be considerably efficient for substrate catalysis. The SS-bond data have been integrated into an online server, namely, ThermoLink, available at guolab.mpu.edu.mo/thermoLink.

One-Pot Multienzyme Cascades for Stereodivergent Synthesis of Tetrahydroquinolines.

The tetrahydroquinoline (THQ) framework is commonly found in natural products and pharmaceutically relevant molecules. Apart from the use of transition metal catalysts and chiral phosphoric acids, the chiral 2-substituted 1,2,3,4-THQs are synthesized using amine oxidase biocatalysts. However, the use of imine reductases (IREDs) in their asymmetric synthesis remained unexplored. In the current work, IREDs are employed in telescopic multienzyme cascades to catalyze the intramolecular reductive amination leading to chiral 2-alkyl and 2-aryl substituted-1,2,3,4-tetrahydroquinolines starting from inexpensive nitroalkenones. The cascades containing NtDBR (an ene reductase), NfsB (a nitro reductase) with either Na2S2O4 or V2O5, various IREDs, and glucose dehydrogenase (for NADPH regeneration) are used to synthesize a broad range of (R)/(S)-2-alkyl-substituted (THQs) (26 examples) with high yield (up to 93%) and excellent ee (up to 99%) in one-pot. The method further facilitates the one-pot biocatalytic synthesis of chiral 2-aryl substituted THQs (26 examples) from amino chalcones. Lastly, the asymmetric synthesis of several (R)- and (S)-THQ based intermediates of Hancock alkaloids showed the practical application of the newly developed biocatalytic cascades.

Synthetic Nicotinamide Cofactors as Alternatives to NADPH in Imine Reductase-Catalyzed Reactions.

This study demonstrates the effectiveness of synthetic nicotinamide cofactors as cost-effective alternatives to NADPH in imine reductase (IRED) catalysis. The synthetic cofactors maintained catalytic activity and stereoselectivity, achieving high conversion rates. Molecular docking studies revealed key structural interactions influencing performance. Combining a glucose dehydrogenase (GDH) recycling system further enhanced the stability and efficiency. These findings highlight the potential of synthetic cofactors to reduce costs and improve the feasibility of IRED-catalyzed processes for industrial applications.

Biocatalytic Synthesis of Ruxolitinib Intermediate via Engineered Imine Reductase.

An enzyme catalyzed strategy for the synthesis of a chiral hydrazine from 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 is presented. An imine reductase (IRED) from Streptosporangium roseum was identified to catalyze the reaction between 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 to produce trace amounts of (R)-3-cyclopentyl-3-hydrazineylpropanenitrile 4. We employed a 2-fold approach to optimize the catalytic performance of this enzyme. First, a transition state analogue (TSA) model was constructed to illuminate the enzyme-substrate interactions. Subsequently, the Enzyme_design and Funclib methods were utilized to predict mutants for experimental evaluation. Through three rounds of site-directed mutagenesis, site saturation mutagenesis, and combinatorial mutagenesis, we obtained mutant M6 with a yield of 98% and an enantiomeric excess (ee) of 99%. This study presents an effective method for constructing a hydrazine derivative via IRED-catalyzed reductive amination of ketone and hydrazine. Furthermore, it provides a general approach for constructing suitable enzymes, starting from nonreactive enzymes and gradually enhancing their catalytic activity through active site modifications.

An Engineered Imine Reductase for Highly Diastereo- and Enantioselective Synthesis of β-Branched Amines with Contiguous Stereocenters.

β-Branched chiral amines with contiguous stereocenters are valuable building blocks for preparing various biologically active molecules. However, their asymmetric synthesis remains challenging. Herein, we report a highly diastereo- and enantioselective biocatalytic approach for preparing a broad range of β-branched chiral amines starting from their corresponding racemic ketones. This involves a dynamic kinetic resolution-asymmetric reductive amination process catalyzed using only an imine reductase. Four rounds of protein engineering endowed wild-type PocIRED with higher reactivity, better stereoselectivity, and a broader substrate scope. Using the engineered enzyme, various chiral amine products were synthesized with up to >99.9% ee, >99:1 dr, and >99% conversion. The practicability of the developed biocatalytic method was confirmed by producing a key intermediate of tofacitinib in 74% yield, >99.9% ee, and 98:2 dr at a challenging substrate loading of 110 g L-1. Our study provides a highly capable imine reductase and a protocol for developing an efficient biocatalytic dynamic kinetic resolution-asymmetric reductive amination reaction system.

Construction of Multi-Enzyme Integrated Catalysts for Deracemization of Cyclic Chiral Amines.

Multi-enzyme cascade catalysis has become an important technique for chemical reactions used in manufacturing and scientific study. In this research, we designed a four-enzyme integrated catalyst and used it to catalyse the deracemization reaction of cyclic chiral amines, where monoamine oxidase (MAO) catalyses the enantioselective oxidation of 1-methyl-1,2,3,4-tetrahydroisoquinoline (MTQ), imine reductase (IRED) catalyses the stereo selective reduction of 1-methyl-3,4-dihydroisoquinoline (MDQ), formate dehydrogenase (FDH) is used for the cyclic regeneration of cofactors, and catalase (CAT) is used for decomposition of oxidative reactions. The four enzymes were immobilized via polydopamine (PDA)-encapsulated dendritic organosilica nanoparticles (DONs) as carriers, resulting in the amphiphilic core-shell catalysts. The hydrophilic PDA shell ensures the dispersion of the catalyst in water, and the hydrophobic DON core creates a microenvironment with the spatial confinement effect of the organic substrate and the preconcentration effect to enhance the stability of the enzymes and the catalytic efficiency. The core-shell structure improves the stability and reusability of the catalyst and rationally arranges the position of different enzymes according to the reaction sequence to improve the cascade catalytic performance and cofactor recovery efficiency.

Atroposelective Synthesis of Axial Biaryls by Dynamic Kinetic Resolution Using Engineered Imine Reductases.

Axially chiral biaryl compounds are ubiquitous scaffolds in natural products, bioactive molecules, chiral ligands and catalysts, but biocatalytic methods for their asymmetric synthesis are limited. Herein, we report a highly efficient biocatalytic route for the atroposelective synthesis of biaryls by dynamic kinetic resolution (DKR). This DKR approach features a transient six-membered aza-acetal-bridge-promoted racemization followed by an imine reductase (IRED)-catalyzed stereoselective reduction to construct the axial chirality under ambient conditions. Directed evolution of an IRED from Streptomyces sp. GF3546 provided a variant (S-IRED-Ss-M11) capable of catalyzing the DKR process to access a variety of biaryl aminoalcohols in high yields and excellent enantioselectivities (up to 98 % yield and >99 : 1 enantiomeric ratio). Molecular dynamics simulation studies on the S-IRED-Ss-M11 variant revealed the origin of its improved activity and atroposelectivity. By exploiting the substrate promiscuity of IREDs and the power of directed evolution, our work further extends the biocatalysts' toolbox to construct challenging axially chiral molecules.

Enantioselective Synthesis of Secondary Amines by Combining Oxidative Rearrangement and Biocatalysis in a One-Pot Process.

This contribution describes the development of chemoenzymatic one-pot processes, which combine an oxidative rearrangement and a biotransformation catalyzed by an imine reductase (IRED), for the synthesis of highly enantiomerically enriched secondary amines, such as an aryl-substituted pyrrolidine and a benzazepine. The benefits of this chemoenzymatic one-pot approach include high overall conversions (up to >99%), high enantiomeric excesses (up to >99% ee), and a straightforward synthetic approach toward secondary amines without the need to isolate the formed intermediate. For the initial chemical reaction, namely, the oxidative rearrangement, PhI(OAc)2 in methanol is used as a non-natural reagent, whereas the enzymatic step requires only stoichiometric amounts of d-glucose along with catalytic amounts of IRED, glucose dehydrogenase (GDH), and the cofactor NADPH. This methodology, demonstrating the compatibility of a "classic" organic synthesis using a non-natural, highly reactive reagent and a subsequent biocatalytic step, can be applied for different amines as substrates, thus making this concept a versatile tool in synthetic organic chemistry in general and for enantioselective synthesis of heterocyclic secondary amines in particular.

Biocatalytic Oxidative Amination of para‐Substituted Phenols

AbstractBiocatalytic preparation of chiral amines is a large and burgeoning field in organic chemistry. Many enzymes and routes have been published, including transaminases, imine reductases, reductive aminases, amine dehydrogenases and others. However, all these routes rely on some sacrificial substrate, in the form of either amine donor or cofactor regeneration substrate. Herein, we report the direct oxidative amination of p‐substituted phenols catalyzed by an evolved flavoprotein oxidase, with the consumption of only substrate and O2, and release of H2O2. The substrate scope of the reaction is studied, and is tolerant of a diverse panel including ammonia, primary and secondary amines, and amino acids. The reaction is later employed at preparative scale to generate aminated products in 50–80 % yield. This report establishes flavoprotein oxidase as a new and economical member of the chemist's toolkit for biocatalytic generation of chiral amines, acting as oxidative aminase.

Engineered Imine Reductase for Asymmetric Synthesis of Dextromethorphan Key Intermediate.

(S)-1-(4-Methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline ((S)-1-(4-methoxybenzyl)-OHIQ) is the key intermediate of the nonopioid antitussive dextromethorphan. In this study, (S)-IR61-V69Y/P123A/W179G/F182I/L212V (M4) was identified with a 766-fold improvement in catalytic efficiency compared with wide-type IR61 through enzyme engineering. M4 could completely convert 200 mM of 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline into (S)-1-(4-methoxybenzyl)-OHIQ in 77% isolated yield, with >99% enantiomeric excess and a high space-time yield of 542 g L-1 day-1, demonstrating a great potential for the synthesis of dextromethorphan intermediate in industrial applications.