Gerrit Jan Poelarends

C─N lyases from the aspartase/fumarase superfamily are attractive biocatalysts for the production of noncanonical amino acids via selective hydroamination of α , β ‐unsaturated carboxylic acids. However, the substrate scope of these enzymes remains largely restricted to primary amines, as secondary amines are usually poorly accepted. Here, we report the engineering of ethylenediamine‐ N , N’ ‐disuccinic acid (EDDS) lyase for enantioselective hydroaminations with secondary amines as non‐native substrates. This engineered C─N lyase variant (Y26A/Q159V/R294E) not only displays a 20‐fold increase in activity for dimethylamine addition to fumarate, but also processes secondary amines that show no reactivity with the wild‐type enzyme. Expanding the substrate scope of this C─N lyase to include secondary amines establishes a viable enzymatic platform for the enantioselective synthesis of structurally diverse tertiary amines.

The significance of L‐aspartic acid derivatives lies in their multifaceted functionalities and their extensive utility as ‘green’ surfactants, tools for neurobiological research, and synthons for pharmaceuticals and food additives. This makes these noncanonical amino acids important molecules in scientific research and industrial applications. C─N lyases are attractive biocatalysts for the production of L‐aspartic acid derivatives via asymmetric hydroamination of α,β ‐unsaturated carboxylic acids. However, the substrate scope of these enzymes is largely limited to small amines, as bulky aliphatic amines are generally poorly accepted. Here, we report the structure‐based engineering of ethylenediamine‐ N,N’ ‐disuccinic acid (EDDS) lyase enabling efficient and selective hydroaminations with large aliphatic amines as non‐native substrates. This engineered C─N lyase shows up to a 600‐fold increase in activity toward sterically demanding amines and even efficiently converts large non‐native amines that are entirely unreactive with the wild‐type enzyme. Molecular docking revealed that the mutations widened and enhanced the hydrophobicity of the amine‐binding pocket, strengthening interactions with bulky aliphatic amines. The results underscore that structure‐inspired protein engineering can be an efficient approach to enlarge the substrate scope of a C─N lyase, enabling the efficient enzymatic synthesis of various enantiopure L‐aspartic acid derivatives (>99% ee), including compounds with potential applications as biodegradable surfactants.

Macrophage migration inhibitory factor (MIF) is a promising therapeutic target for cancer and inflammatory diseases. Nevertheless, the identification of MIF inhibitors with druglike properties remains a challenge. In this study, we aimed to identify MIF inhibitors with a novel pharmacophore. Screening of a focused compound library led to the identification of compound 26, which inhibited MIF and D-Dopachrome Tautomerase (D-DT) with with micromolar potency. Enzyme kinetic characterization revealed that compound 26 acts as a reversible, mixed-type inhibitor of MIF. NMR titration analysis indicated that the interaction interface of compound 26 is adjacent to the MIF catalytic pocket, and extends across the allosteric site and CD74 activation motif. In subsequent cell-based assays, compound 26 moderately inhibited proliferation and suppressed MIF-induced ERK phosphorylation in A549 cells. Together, these findings establish compound 26 with the 4-(3-(trifluoromethyl)phenyl)-1H-pyrazole scaffold as a new MIF inhibitor class with a novel pharmacophore as an inspiration source for MIF-directed drug discovery.

D-dopachrome tautomerase (D-DT), also known as macrophage migration inhibitory factor-2, is a member of the MIF cytokine family and plays a key role in cancer and inflammation. Molecules that bind to the D-DT or MIF-1 tautomerase site could block their biological activity. However, relatively few D-DT inhibitors have been reported. In this study, we designed, synthesized, and screened a focused compound library. This led to the identification of 4h, a furan-2-carboxylic acid derivative with IC₅₀ values of 2.4 μM for D-DT and 9.8 μM for MIF-1. Subsequent SAR optimization yielded the more potent inhibitor 10b, exhibiting IC₅₀ values of 1.5 μM for D-DT and 1.0 μM for MIF-1. The specific interactions of 4h with D-DT and MIF-1 were explored using ¹H-¹⁵N NMR endpoint titrations. 4h also inhibited D-DT-induced ERK phosphorylation in A549 cells. Thus, 4h and 10b represent a new class of inhibitors that can be utilized as tools to investigate the biological functions of D-DT and MIF-1.

The thia‐Michael addition is one of the most versatile and practical reactions for CS bond formation in organic synthesis, but remains a continuing challenge for biocatalysis. Here we report engineered cofactor‐independent enzymes, based on the promiscuous enzyme 4‐oxalocrotonate tautomerase, for the enantioselective synthesis of various ( R )‐β‐thioenals via the nucleophilic addition of aliphatic thiols to cinnamaldehyde. These engineered enzymes efficiently promote the formation of the carbon–sulfur bond with good stereocontrol, affording the desired ( R )‐β‐thioenal products with excellent conversions (up to 99%) and with moderate‐to‐high enantiopurity (e.r. up to 95:5). Our results highlight the power of catalytic promiscuity for expanding the biocatalytic repertoire for non‐natural reactions.

Biocatalysis contributes significantly to the development of more sustainable synthetic pathways by using mild reaction conditions and water as a solvent. However, many relevant classes of compounds, including privileged groups in drug design, are not yet accessible via enzymatic pathways. In this context, the development of an enzymatic route to indazoles remains an unmet challenge. Here, we present a nitroreductase-triggered indazole formation, in which 2-nitrobenzylamine derivatives are converted to reactive nitrosobenzylamine intermediates that spontaneously cyclize and aromatize to indazoles. Two nitroreductases accept a series of 2-nitrobenzylamine derivatives with excellent conversions (up to >99 %). In the case of N -substituted nitrosobenzylamines, 2 H -indazoles are formed, whereas other derivatives led to 1 H -indazoles. The synthetic value of the nitroreductase-triggered indazole formation is further demonstrated by successful coupling with an imine reductase in a sequential cascade reaction on a 50 mg scale. With this cascade, 2 H -indazoles are accessible from cheap 2-nitrobenzaldehyde and primary amines, resulting in up to 85 % conversion and 68 % isolated yield.

The enantioselective conjugate hydrocyanation of α , β ‐unsaturated aldehydes remains a long‐standing challenge in synthetic chemistry. Here, we report the redesign of 2‐deoxy‐D‐ribose‐5‐phosphate aldolase (DERA) into an efficient biocatalyst capable of promoting the asymmetric conjugate addition of hydrogen cyanide (generated in situ from trimethylsilyl cyanide) to aromatic enals via an iminium activation pathway. The evolved variant DERA‐CN enables the efficient formation of various C4‐nitriles with high conversions (up to 99%) and good enantioselectivity (up to 98% e.e.). Control experiments revealed a stepwise process involving enzyme‐catalyzed conjugate hydrocyanation followed by spontaneous 1,2‐addition of cyanide. Substrates with various electron‐donating and electron‐withdrawing groups are tolerated, providing access to various enantioenriched nitriles. This work expands the scope of DERA‐promoted iminium catalysis and provides a rare enzymatic platform for asymmetric conjugate hydrocyanation under mild aqueous conditions.

Optically pure N ‐functionalized α‐amino acids are valuable chiral building blocks for pharmaceuticals, nutraceuticals, and agrochemicals. Ethylenediamine‐ N,N ‐disuccinic acid lyase from Chelativorans sp. BNC1 catalyzes the addition of a wide range of aliphatic and aromatic primary amines to fumarate, producing the corresponding enantioenriched N ‐substituted L‐ aspartic acids. In this work, the enzyme was subjected to iterative cycles of site‐saturation mutagenesis and screened for increased activity for the addition of 2‐((methylamino)methyl)aniline to fumarate. The final variant displayed an activity of three orders of magnitude higher compared to the wild‐type enzyme. Unexpectedly, the enzyme catalyzed the hydroamination of fumarate with the aliphatic secondary amine of the starting substrate, rather than with the aromatic primary amine, leading to the formation of a tertiary amine. Exploring the substrate scope showed that the enzyme accepts various substituted N ‐methyl‐1‐phenylmethanamines for the hydroamination of fumarate, yielding N,N ‐disubstituted L ‐aspartic acids in high optical purity (up to >99% ee). Furthermore, we showed that the enzyme accepts several ortho ‐substituted anilines that were previously not accepted by the wild‐type enzyme, yielding the corresponding N ‐arylated L ‐aspartic acids in high enantiomeric excess (>99% ee). This serendipitous finding enables a new strategy for the biocatalytic synthesis of tertiary amines, unlocked within the C‐N lyase toolbox.

Optically pure N ‐functionalized α‐amino acids are valuable chiral building blocks for pharmaceuticals, nutraceuticals, and agrochemicals. Ethylenediamine‐ N,N ‐disuccinic acid lyase from Chelativorans sp. BNC1 catalyzes the addition of a wide range of aliphatic and aromatic primary amines to fumarate, producing the corresponding enantioenriched N ‐substituted L‐ aspartic acids. In this work, the enzyme was subjected to iterative cycles of site‐saturation mutagenesis and screened for increased activity for the addition of 2‐((methylamino)methyl)aniline to fumarate. The final variant displayed an activity of three orders of magnitude higher compared to the wild‐type enzyme. Unexpectedly, the enzyme catalyzed the hydroamination of fumarate with the aliphatic secondary amine of the starting substrate, rather than with the aromatic primary amine, leading to the formation of a tertiary amine. Exploring the substrate scope showed that the enzyme accepts various substituted N ‐methyl‐1‐phenylmethanamines for the hydroamination of fumarate, yielding N,N ‐disubstituted L ‐aspartic acids in high optical purity (up to >99% ee). Furthermore, we showed that the enzyme accepts several ortho ‐substituted anilines that were previously not accepted by the wild‐type enzyme, yielding the corresponding N ‐arylated L ‐aspartic acids in high enantiomeric excess (>99% ee). This serendipitous finding enables a new strategy for the biocatalytic synthesis of tertiary amines, unlocked within the C‐N lyase toolbox.

The aspartase/fumarase superfamily is a group of homologous enzymes that promote the reversible elimination of functional groups from succinyl‐containing compounds, typically yielding fumarate as the common product. Over the past 50 years, members of this superfamily have continuously demonstrated their power and significance as biocatalysts. This is exemplified by ethylenediamine‐N,N‐disuccinic acid (EDDS) lyase, which was shown to have an extraordinary amine scope, enabling the production of a wide variety of N‐substituted aspartic acids. In this work, we used this enzyme as a starting point for a homology‐based strategy to expand the biocatalytic toolbox of C−N bond‐forming enzymes. We selected 13 enzymes for biochemical characterization, and identified several EDDS‐lyase homologues that can accept L ‐amino acids as substrates in the hydroamination of fumarate to produce the corresponding aminopolycarboxylic acids. Lastly, we carried out a sequence similarity network analysis of the aspartase/fumarase superfamily, which suggests that EDDS lyase and its homologues may represent a distinct isofunctional subfamily, laying the foundations for future enzyme discovery and engineering campaigns.