Recent Applications of Carbon‐Nitrogen Lyases in Asymmetric Synthesis of Noncanonical Amino Acids and Heterocyclic Compounds

Abstract Carbon‐nitrogen (C−N) lyases are enzymes that normally catalyze the cleavage of C−N bonds. Reversing this reaction towards carbon‐nitrogen bond formation can be a powerful approach to prepare valuable compounds that could find applications in everyday life. This review focuses on recent (last five years) applications of native and engineered C−N lyases, either as stand‐alone biocatalysts or as part of multienzymatic and chemoenzymatic cascades, in enantioselective synthesis of noncanonical amino acids and dinitrogen‐fused heterocycles, which are useful tools for neurobiological research and important synthetic precursors to pharmaceuticals and food additives.

CÀ N lyases have shown great potential as biocatalysts for the synthesis of optically pure (un)natural amino acids through asymmetric hydroamination of α,β-unsaturated mono-or dicarboxylic acids (Figure 1). [3,6] CÀ N lyase-catalyzed hydroamination reactions employ ubiquitous α,β-unsaturated acids as starting materials, require no external cofactors and are atomeconomic and highly selective, providing a complementary strategy to form chiral amino acids, lining up with other biocatalytic approaches ( Figure 1). [7] Early in the 1970s, ammonia-lyases, such as aspartase and PAL, were exploited in reverse to synthesize their natural substrates l-aspartic acid and l-phenylalanine starting from fumaric acid and cinnamic acid, respectively. [8] In recent years, CÀ N lyases with desired biocatalytic profiles (substrate scope, activity, selectivity) have been obtained by discovery or engineering. The rapidly expanding toolbox of CÀ N lyases enables enzymatic production of a broad range of unnatural amino acids, including substituted aspartic acids, β-substituted αand β-alanines, with most reactions having no counterparts in organic chemistry (Tables 1 and 2). [6] Besides, multienzymatic and chemoenzymatic cascades containing CÀ N lyases have been developed, giving access to complex bioactive molecules in a sustainable and step-economic manner. CÀ N lyases provide an alternative approach to synthesize valuable amino acids and offer more options for biocatalytic retrosynthesis.
l-Aspartic acid derivatives are particularly important molecules because of their diverse biological activities and wideranging applications in pharmaceutical and nutraceutical fields ( Figure 2). Three types of CÀ N lyases, aspartase, MAL and ethylenediamine-N,N'-disuccinic acid lyase (EDDS lyase), have been used for biocatalytic preparation of l-aspartic acid derivatives. Here we focus on EDDS lyase and MAL, providing a review on their structural and mechanistic characteristics and recent applications in asymmetric synthesis of important biologically active compounds comprising an l-aspartic acid moiety.
Crystal structures of EDDS lyase in unliganded form and those bound with fumarate, AEAA and (S,S)-EDDS were solved by X-ray diffraction analysis. [18] EDDS lyase is a homotetramer (56 kDa per subunit) sharing the characteristic tertiary and quaternary structural features of members of the aspartase/ fumarase superfamily. [2] Interestingly, the catalytically essential SS-loop of EDDS lyase shows little movement upon substrate binding, which is different from the flexible SS-loop undergoing  an open/closed conformational change upon substrate binding as observed in most aspartase/fumarase superfamily members. EDDS lyase has a composite active site located at the interface of three subunits, with most residues forming the αand βcarboxylate binding pockets being highly conserved. [18] The amine binding site is less conserved, and rich in polar and charged side chains making it quite hydrophilic. Residues Asn288 and Asp290 form water-mediated hydrogen-bond interactions with the distal amino group of the bound (S,S)-EDDS, appearing to play important roles in binding and positioning of the natural substrate. Ser280 was proposed to serve as the crucial base catalyst, positioned close to the Cβ carbon with a proper orientation for proton abstraction to start the deamination reaction.
Based on the crystal structures and previous mechanistic studies of aspartase/fumarase superfamily members, a general base-catalyzed, sequential two-step deamination mechanism was proposed. [18] The Ser280 oxyanion first abstracts the proton from Cβ of (S,S)-EDDS, leading to formation of an enediolate intermediate, which is stabilized by interactions with residues from the β-carboxylate binding pocket (Ser111, Arg112, Ser281). The collapse of the enediolate intermediate leads to cleavage of the CÀ N bond, releasing AEAA and fumarate. AEAA will re-bind in the active site, allowing another round of deamination to give ethylene diamine and fumarate as final products.

Synthetic applications
(S,S)-EDDS is a widely used metal-chelating compound in industry as well as a pharmaceutically active compound serving as an inhibitor of Zn-dependent enzymes like phospholipase C (PLC) [29] and metallo-β-lactamase. [39] Consequently, EDDS degrading microorganisms and the purified EDDS lyase have been exploited for the production of (S,S)-EDDS [37,38] and related metal chelators (e. g., phenylenediamine-, [36] propanediamine-, and cyclohexylenediamine-N,N'-disuccinic acid [35] ) starting from fumarate and diamines. Suzuki and co-workers first reported the production of (S,S)-EDDS at multigram scale by incubating Acidovorax sp. TNT149 cells with fumarate and ethylene diamine at optimum pH (7.5) and temperature (35°C). [37] Recently, EDDS lyase from Chelativorans sp. BNC1 was used for chemoenzymatic synthesis of the natural products aspergillomarasmine A (AMA), aspergillomarasmine B (AMB) and toxin A, as well as related aminocarboxylic acids with excellent regioand stereoselective control (Figure 4). [16] AMA is a potent inhibitor of metallo-β-lactamases (including the notorious NDM-1 and VIM-2) with a low IC 50 value at micromolar concentration. It received extensive academic attention yet remained challenging to synthesize with correct 2S,2'S,2''S configuration. Toxin A is the direct biosynthetic precursor to AMA and its analogue AMB. EDDS lyase was incubated with fumaric acid and a series of retrosynthetically designed amine substrates in aqueous solution at pH 8.5-9 and 25 or 37°C. AMA, AMB, toxin A and their derivatives (Figures 4 and 5) were obtained with excellent stereoselectivity (ee > 99 %, de > 98 %) and in moderate to good yields. Based on the biocatalytic synthesis of toxin A, a one-pot two-step chemoenzymatic approach was developed for rapid and efficient synthesis of AMB and its derivatives ( Figure 5). [16] Compared to previous chemocatalytic methods, this (chemo) enzymatic approach significantly reduced the total number of synthetic steps for AMA, AMB, and toxin A, showing great potential for efficient practical synthesis of complex amino (poly)carboxylic acids.
In addition, a series of aminocarboxylic acid analogues of (S,S)-EDDS was synthesized by the addition of various diamine substrates to fumaric acid catalyzed by EDDS lyase. [21] The enzyme was shown to accept various (substituted) diamines with two to four atoms between the two amino groups, giving good conversions (47-83 %) and yielding the corresponding aminocarboxylic acid products ( Figure 5) with moderate to good isolated yield. Like AMA, AMB and their derivatives, (S,S)-EDDS and several of its analogues demonstrated effective inhibition of the bacterial metallo-β-lactamase NDM-1, displaying a strong correlation between the inhibitory potency of the compounds and their ability to bind zinc. [21] Moreover, EDDS lyase from Chelativorans sp. BNC1 was also employed for enantioselective synthesis of complex N-cycloalkyl substituted l-aspartic acids, which are structurally distinct from  its natural substrates ( Figure 5). [17] Reactions were performed with fumaric acid, various homo-and heterocycloalkyl amines (comprising four-, five-and six-membered rings) and EDDS lyase at pH 8.5 and room temperature. EDDS lyase exhibited broad substrate promiscuity, accepting a variety of cycloalkylamines in hydroamination of fumarate to give the corresponding N-cycloalkyl-l-aspartic acids with excellent enantioselectivity (ee > 99 %).
Another synthetic application of EDDS lyase was demonstrated by the biocatalytic asymmetric synthesis of various Narylated l-aspartic acids and the chemoenzymatic synthesis of challenging 2-aryl-5-carboxylpyrazolidin-3-ones ( Figure 5). [19] Especially, pyrazolidinones are widely found as core structures in pharmaceutically active molecules including lipoxygenase inhibitors ( Figure 4) and anti-Alzheimer agents. [19] EDDS lyase accepted a broad range of arylamines and arylhydrazines as substrates for addition to fumarate, giving the corresponding N-arylated l-aspartic acids and N-(arylamino)aspartic acids, respectively, with good isolated yields and excellent enantiopurity. Furthermore, a one-pot chemoenzymatic synthesis route combining the EDDS lyase mediated biotransformation with an acid-catalyzed cyclization step was developed, giving rise to a series of chiral pyrazolidin-3-one derivatives ( Figure 5), with 61-70 % isolated yield and > 99 % ee. [19] This chemoenzymatic methodology enables an efficient, step-economic alternative process to this important class of drug precursors.
In a recent study, an engineered variant of EDDS lyase was successfully applied as biocatalyst for the efficient asymmetric synthesis of precursors to the important dipeptide sweeteners neotame and advantame. [20] These dipeptides are N-substituted derivatives of aspartame (Figure 2), having improved sweetness, that were recently approved as sugar substitutes in a broad range of food products. Although wild-type EDDS lyase can be used for the enantioselective hydroamination of fumarate to produce N-(3,3-dimethylbutyl)-l-aspartic acid and N- [3-(3-hydroxy-4-methoxyphenyl)propyl]-l-aspartic acid (Figure 4), which are the synthetic precursors to neotame and advantame, respectively (Figure 2), reaction times were very long (7 days, using 0.15 mol% catalyst loading). Therefore, to enhance the hydroamination activity of EDDS lyase, enzyme engineering based on the structure of EDDS lyase in complex with its natural substrate (S,S)-EDDS was applied. Two residues (Asp290 and Tyr320) that were presumed to position the amine substrate in the active site were selected for mutagenesis. A highly efficient EDDS lyase variant (D290M/Y320M) was obtained with an 1140fold improvement in activity over the wild-type enzyme, allowing the selective synthesis of the neotame and advantame precursors, as well as related compounds, with excellent

ChemBioChem
Minireviews doi.org/10.1002/cbic.202000214 enantioselectivity (> 99 % ee) and high conversion, requiring only a few hours reaction time instead of 7 days, while using even lower biocatalyst loadings (0.05 mol%). [20] The development of such an effective biocatalyst opens up new opportunities to develop practical multienzymatic processes for the more sustainable and step-economic synthesis of an important class of food additives.

Properties, structure and catalytic mechanism
Identified from several facultative anaerobic bacteria, the MAL enzyme forms part of the glutamate catabolic pathway and reversibly deaminates l-threo-and l-erythro-3-methylaspartate to give mesaconate. [9,40] The two best studied MAL enzymes were isolated from Clostridium tetanomorphum (CtMAL) and Citrobacter amalonaticus (CaMAL), with the first crystal structures reported in 2002. [41,42] The overall structure of the homodimeric MAL (45 kDa per subunit) resembles that of members of the enolase superfamily, sharing the characteristic TIM barrel fold (eightfold α/β barrel). MAL requires K + and Mg 2 + for optimal activity, and the catalytic mechanism of MAL is proposed to involve general base-catalyzed proton abstrac-tion resulting in the formation of an enolate anion intermediate, which is stabilized by interactions with the active site Mg 2 + ion and side chains of amino acid residues. [9,41] Based on structural work and mutagenesis studies, residues Lys331 and His194 have been identified as the S-and R-specific base catalysts, respectively. [43]

Synthetic applications
Based on its natural substrate promiscuity, CtMAL has been used for the asymmetric synthesis of l-aspartic acid derivatives with small N-and C β -substitutions. [6,9] The scope of biocatalytic applications was greatly enlarged by structure-guided engineering of CtMAL, which led to an engineered variant (MAL-Q73A) with broad nucleophile scope, accepting linear and cycloalkylamines, and an engineered variant (MAL-L384A) with wide electrophile scope, accepting non-native fumarate derivatives with alkyl, aryl, alkoxy, aryloxy, alkylthio, and arylthio substitutions at the C-2 position. [12] By using these engineered MAL enzymes, a diverse collection of N-and C β -substituted l-aspartic acids ( Figure 6) were synthesized by asymmetric hydroaminations with moderate to good stereoselectivity. [12,15] In another study, a variant of CtMAL (MAL-H194A) with strongly enhanced diastereoselectivity, which was obtained by mechanism-based Figure 6. Examples of noncanonical amino acids obtained by preparative (chemo)enzymatic synthesis using MAL and its variants as biocatalysts.

ChemBioChem
Minireviews doi.org/10.1002/cbic.202000214 engineering, has been exploited for asymmetric synthesis and kinetic resolution of various 3-substituted aspartic acids. [43,44] Inspired by the expanded scope towards 2-substituted fumarate derivatives, MAL variants have been exploited in chemoenzymatic synthesis of excitatory amino acid transporter (EAAT) inhibitors that are extremely difficult to synthesize chemically. [13,14,45] Located in the membranes of mammalian neurons and surrounding glial cells, EAATs are responsible for regulating the concentration of the excitatory neurotransmitter glutamate in the synaptic cleft. Inhibitors of EAATs are therefore useful tools to study the precise functions of these transporters in glutamatergic neurotransmission and in neurological disorders related to extracellular glutamate accumulation. For example, l-threo-3-benzyloxyaspartate (L-TBOA, Figures 2 and 7) is a widely used nonselective inhibitor of EAATs, the chemical synthesis of which is highly challenging (11 steps in total with an overall yield of < 1 %). By using an engineered variant of MAL as biocatalyst, de Villiers and co-workers developed an elegant three-step chemoenzymatic synthesis route to L-TBOA and several ring-substituted derivatives. [13] 2-Benzyloxyfumarate and a series of derivatives with F, CH 3 and CF 3 groups at the ortho, meta and para position of the aromatic ring, which were obtained by two-step chemical synthesis from dimethyl acetylenedicarboxylate, were incubated with ammonia in the presence of the MAL-L384A or MAL-L384G variant at room temperature and pH 9.0. Reactions achieved > 89 % conversion within 24 h, with products L-TBOA and seven analogues purified with good yield (57-78 %) and identified as the desired l-threo isomers with excellent stereoselectivity (ee > 99 %; de > 95 %; Figure 7).
The synthetic usefulness of MAL variants for preparation of EAAT blockers was further extended by synthesizing a series of L-TBOA derivatives whose pharmacological properties were evaluated. [14] Catalyzed by MAL-L384A, aspartate derivatives with (cyclo)alkyloxy and (hetero)aryloxy substituents at the C-3 position were prepared by hydroamination of the corresponding fumarate derivatives (prepared from dimethyl acetylenedicarboxylate) with excellent stereoselectivity (Figure 7). These derivatives showed potent inhibitory activities towards EAAT1-4 subtypes with IC 50 values ranging from micro-to nanomolar concentrations. [14] Based on the synthesis of L-TBOA, the chemoenzymatic preparation of another potent and widely used nontransportable EAAT inhibitor, (l-threo)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (L-TFB-TBOA, Figure 7) was achieved with excellent stereochemical control and a dramatically reduced number of synthetic steps. [45] L-TBOA was first synthesized by the MAL-L384A-catalyzed hydroamination of 2-benzyloxyfumarate, and subsequently used as starting material for debenzylation and protection (three steps), yielding the key intermediate dimethyl (l-threo)-N-Boc-3-hydroxyaspartate (71-73 % yield). This chiral intermediate was subsequently subjected to O-alkylation and global deprotection, which gave rise to L-TFB-TBOA at multigram scale with 6 % overall yield in nine steps (in comparison, chemical synthesis takes 20 steps). This method provides a convenient strategy to produce l-aspartic acid derivatives with large aryloxy substituents at the C3 position (Figure 7), enabling stereoselective preparation of four L-TFB-TBOA derivatives with strong inhibitory activities for EAAT1-4 subtypes (IC 50 values range from 5 to 530 nM). [14] This strategy was later exploited for the design and preparation of novel photo-controlled glutamate transporter inhibitors by functionalization of L-TBOA with a photoswitchable azobenzene moiety (azo-TBOAs). [46] Remarkably, (l-threo)-trans-3-(3-((4-(methoxy) phenyl)diazenyl)benzyloxy) aspartate (p-MeO-azo-TBOA) showed good photochemical properties, reversibly switched from the trans to cis isomer in response to irradiation, with the  isomers showing a 3.6-fold difference in inhibitory activity towards the prokaryotic transporter Glt Tk . [46] Moreover, MAL and its H194 A variant were used to form linear cascades of three different enzymes in one pot to build an in vitro artificial pantothenate biosynthesis pathway for the production of vitamin B 5 [(R)-pantothenic acid] and its derivatives (Figure 7). [47] Vitamin B 5 is the biosynthetic precursor of coenzyme A, and its derivatives serve as important synthetic precursors to antimicrobial pantothenamides. A one-pot, threestep enzymatic cascade was developed, consisting of asymmetric hydroamination (catalyzed by MAL or MAL-H194A), αdecarboxylation (catalyzed by an appropriate decarboxylase) and condensation (catalyzed by pantothenate synthase) reactions. Starting from achiral fumarate or mesaconate, ammonia and (R)-pantoate, vitamin B 5 and both diastereoisomers of αmethyl-substituted vitamin B 5 (Figure 7) were produced in good isolated yield and with excellent stereoselectivity (> 99 % ee).

Concluding Remarks
Asymmetric hydroamination of α,β-unsaturated carboxylic acids by CÀ N lyases is an attractive biocatalytic strategy to synthesize optically pure amino acids. These enzymes have been applied in single-step biotransformations as well as in multienzymatic and chemoenzymatic cascade syntheses of complex molecules with biological activity. Particularly, MAL and EDDS lyase are promising biocatalysts because they display high activity, excellent regio-and stereoselectivity, and good evolvability, providing great potential for practical synthesis of various noncanonical amino acids and (di)nitrogen-fused heterocycles (Figures 4-7), which are useful tools for neurobiological research and important synthetic precursors to pharmaceuticals and food additives. Even though CÀ N lyases are promising industrial biocatalysts, more development is required to expand the reaction scope, allowing the connection of a wider variety of structurally diverse building blocks through CÀ N links, and for robustness and scalability.