Enantioselective Synthesis of N‐Substituted Aspartic Acids Using an Engineered Variant of Methylaspartate Ammonia Lyase

Veetil, Vinod Puthan; Raj, Hans; Villiers, Marianne de; Tepper, Pieter G.; Dekker, Frank J.; Quax, Wim J.; Poelarends, Gerrit J.

doi:10.1002/cctc.201200906

Cited by 22 publications

(18 citation statements)

References 29 publications

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“…Enzymatic products were purified by two steps of ion-exchange chromatography. [20] The purified products were lyophilized and their identity was determined by using 1 H NMR, 13 C NMR and HRMS. The enantiomeric excess and absolute configuration of the product was determined by HPLC analysis on a chiral stationary phase.…”

Section: Communications Ascwiley-vchdementioning

confidence: 99%

“…[19] Using MAL-Q73A, a large variety of Nsubstituted L-aspartic acids were synthesized with high enantioselectivity (> 99% e.e.). [20] Structural analysis of MAL-Q73A showed that this mutant enzyme has an enlarged amine binding pocket, without changes in the orientation of active site residues, thus rationalizing its ability to convert the new amine substrates. [19] Recently, we reported another CÀ N lyase, ethylenediamine-N,N'-disuccinic acid (EDDS) lyase from Chelativorans sp.…”

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confidence: 99%

“…Previous work from our group demonstrated that the Q73A mutant of MAL exhibits an expanded amine scope, accepting various structurally distinct amines in hydroamination reactions. [19,20] This prompted us to first test the potential of MAL-Q73A for the asymmetric synthesis of N-cycloalkyl-substituted L-aspartic acids. Out of ten amines tested, MAL-Q73A only accepted amines 2 b, 2 e and 2 f as substrates (Table 1).…”

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confidence: 99%

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Biocatalytic Enantioselective Hydroaminations for Production of N‐Cycloalkyl‐Substituted L‐Aspartic Acids Using Two C−N Lyases

Zhang

Tepper

et al. 2019

Adv Synth Catal

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N-cycloalkyl-substituted amino acids have wide-ranging applications in pharma-and nutraceutical fields. Here we report the asymmetric synthesis of various N-cycloalkyl-substituted laspartic acids using ethylenediamine-N,N'-disuccinic acid lyase (EDDS lyase) and a previously engineered variant of methylaspartate ammonia lyase (MAL-Q73A) as biocatalysts. Particularly, EDDS lyase shows broad non-natural substrate promiscuity and excellent enantioselectivity, allowing the selective addition of homo-and heterocycloalkyl amines (comprising four-, five-and sixmembered rings) to fumarate, giving the corresponding N-cycloalkyl-substituted l-aspartic acids with > 99% e.e. This biocatalytic methodology offers an alternative synthetic choice to prepare difficult N-cycloalkyl-substituted amino acids. Given its very broad amine scope, EDDS lyase is an exceptionally powerful synthetic tool that nicely complements the rapidly expanding toolbox of biocatalysts for asymmetric synthesis of noncanonical amino acids.

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Section: Communications Ascwiley-vchdementioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Biocatalytic Enantioselective Hydroaminations for Production of N‐Cycloalkyl‐Substituted L‐Aspartic Acids Using Two C−N Lyases

Zhang

Tepper

et al. 2019

Adv Synth Catal

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“…Our group has previously reported that an engineered variant of 3-methylaspartate ammonia lyase (MAL-Q73A) accepts various amines, including butylamine (2 c, Table 1), for enantioselective hydroamination of fumarate (1). [21,22] Structurally, the amines 2 b and 2 a have, respectively, one and two extra methyl group(s) at C3 compared with 2 c. This difference prompted us to start our investigations by testing the branched amines 2 a and 2 b as unnatural substrates in the MAL-Q73A-catalyzed hydroamination of 1. Although 2 b was accepted by MAL-Q73A for slow hydroamination of 1 (see Figure S1 in the Supporting Information), yielding optically pure l-3 b (ee > 99 %), 2 a was unfortunately not accepted as a substrate by MAL-Q73A.…”

Section: Resultsmentioning

confidence: 99%

Engineered C–N Lyase: Enantioselective Synthesis of Chiral Synthons for Artificial Dipeptide Sweeteners

Zhang

Grandi

et al. 2019

Angewandte Chemie

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Aspartic acid derivatives with branched N‐alkyl or N‐arylalkyl substituents are valuable precursors to artificial dipeptide sweeteners such as neotame and advantame. The development of a biocatalyst to synthesize these compounds in a single asymmetric step is an as yet unmet challenge. Reported here is an enantioselective biocatalytic synthesis of various difficult N‐substituted aspartic acids, including N‐(3,3‐dimethylbutyl)‐l‐aspartic acid and N‐[3‐(3‐hydroxy‐4‐methoxyphenyl)propyl]‐l‐aspartic acid, precursors to neotame and advantame, respectively, using an engineered variant of ethylenediamine‐N,N′‐disuccinic acid (EDDS) lyase from Chelativorans sp. BNC1. This engineered C–N lyase (mutant D290M/Y320M) displayed a remarkable 1140‐fold increase in activity for the selective hydroamination of fumarate compared to that of the wild‐type enzyme. These results present new opportunities to develop practical multienzymatic processes for the more sustainable and step‐economic synthesis of an important class of food additives.

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“…MALs belong to the enolase superfamily and they catalyze a broader range of reactions than other ammonia lyases [1] They are potentially versatile and a promising target to design new variants with different substrate specificity or improved activity and stability. For example, the MAL variant isolated from Citrobacter amalonaticus (CaMAL) has been engineered for enantionselective synthesis of N-substituted aspartic acids, which are essential building blocks for pharmaceutical, artificial sweeteners, synthetic enzymes and peptidomimetics [10,11].…”

Section: Introductionmentioning

confidence: 99%

Conformational gating in ammonia lyases

Lambrughi

Maršić

Sáez-Jiménez

et al. 2019

Preprint

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14Ammonia lyases (AL) are enzymes of industrial and biomedical interest. Knowledge 15 of AL structure-dynamics-function relationship would be instrumental for making use 16 of the application potential of these enzymes. We investigated, using microsecond 17 molecular dynamics, the conformational changes in the proximity of the catalytic 18 pocket of a 3-methylaspartate ammonia lyase (MAL) as a model system. In 19 particular, we identified two regulatory elements in the MAL structure, i.e., the β 5-α2 20 loop, and the helix-hairpin-loop subdomain. We showed that they undergo 21 conformational changes switching from 'occluded' to 'open' states. We observed that 22 these rearrangements are coupled to changes in the accessibility of the active site. 23 The β 5-α2 loop and the helix-hairpin-loop subdomain modulate the formation of 24 tunnels from the protein surface to the substrate binding site, making the active site 25 more accessible to the substrate when they are in an open state. We pinpointed a 26 sequential mechanism, in which the helix-hairpin-loop subdomain needs to break a 27 subset of intramolecular interactions first, to then allow the opening of the β 5-α2 loop 28 and, as a consequence, make the AL catalytic pocket accessible for the substrate.29 2 Our data suggest that protein dynamics need to be considered in the design of new 1 AL variants for protein engineering and therapeutic purposes. 2 Keywords 3 molecular dynamics simulations, conformational changes, disordered regions, 4 conformational switches, correlated motions 5 6

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Enantioselective Synthesis of N‐Substituted Aspartic Acids Using an Engineered Variant of Methylaspartate Ammonia Lyase

Abstract: Scheme 1. Biocatalytic synthesis of a few N-substituted l-aspartic acids.[a] Dr.

Cited by 22 publications

References 29 publications

Biocatalytic Enantioselective Hydroaminations for Production of N‐Cycloalkyl‐Substituted L‐Aspartic Acids Using Two C−N Lyases

Biocatalytic Enantioselective Hydroaminations for Production of N‐Cycloalkyl‐Substituted L‐Aspartic Acids Using Two C−N Lyases

Engineered C–N Lyase: Enantioselective Synthesis of Chiral Synthons for Artificial Dipeptide Sweeteners

Conformational gating in ammonia lyases

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