Advances in Enzymatic Synthesis of D-Amino Acids

Abstract

In nature, the D-enantiomers of amino acids (D-AAs) are not used for protein synthesis and during evolution acquired specific and relevant physiological functions in different organisms. This is the reason for the surge in interest and investigations on these "unnatural" molecules observed in recent years. D-AAs are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. In past years, a number of methods have been devised to produce D-AAs based on enantioselective enzymes. With the aim to increase the D-AA derivatives generated, to improve the intrinsic atomic economy and cost-effectiveness, and to generate processes at low environmental impact, recent studies focused on identification, engineering and application of enzymes in novel biocatalytic processes. The aim of this review is to report the advances in synthesis of D-AAs gathered in the past few years based on five main classes of enzymes. These enzymes have been combined and thus applied to multi-enzymatic processes representing in vitro pathways of alternative/exchangeable enzymes that allow the generation of an artificial metabolism for D-AAs synthetic purposes.

Keywords: D-amino acids; biocatalysis; cascade reactions; protein engineering; stereoselective reactions.

Figure 1

Use of aminotransferases in production of D-AAs. Synthesis of D-AAs from the corresponding α-keto acids and ammonia by coupling: (A) four enzymes, namely D-amino acid aminotransferase, glutamate racemase, glutamate dehydrogenase and formate dehydrogenase [11]; (B) tryptophan synthase from S. enterica, L-amino acid deaminase from P. myxofaciens and T242G variant of D-aminotransferase variant from Bacillus sp. YM-1 for the synthesis of D-tryptophan derivatives [12]; (C) L-methionine γ-lyase from F. nucleatum and D-amino acid aminotransferase from Bacillus sp. to convert L-methionine into D-homoalanine [13].

Figure 2

Stereoinversion and deracemization of: (A) phenylalanine derivatives by means of L-amino acid deaminase and a D-selective transaminase [18]; (B) alanine derivatives by alanine dehydrogenase from B. subtilis, NADH oxidase and the engineered D-selective ω-transaminase from Arthrobacter sp. [19].

Figure 3

Use of engineered D-amino acid dehydrogenases in production of D-AAs. Synthesis of D-AAs by: (A) direct conversion by engineered D-amino acid dehydrogenase of 4-Br-phenylpyruvic acid into D-4-Br-phenylalanine, subsequently coupled with a panel of arylboronic acids to give D-biarylalanine derivates [21]; (B) the multi-enzymatic system made of E. coli L-threonine ammonia lyase, D-amino acid dehydrogenase and formate dehydrogenase converted L-threonine into D-2-aminobutyric acid [25].

Figure 4

One-pot stereoinversion of L-AAs by P. mirabilis L-amino acid deaminase and recombinant meso-diaminopimelate D-dehydrogenase from S. thermophilum; formate dehydrogenase was used to regenerate NADPH.

Figure 5

The application of the hydantoinase process to the synthesis of D-tryptophan. In this case, hydantoin racemase from A. aurescens was coupled to D-hydantoinase from A. tumefaciens and D-carbamoylase from A. crystallopoietes [34].

Figure 6

The application of PcPAL in D-AAs synthesis. (A) Resolution of substituted D-phenylglycines by the I460V PcPAL variant [39]. (B) Preparation of enantiopure D-propargylglycine by enantioselective deamination of L-propargylglycine catalyzed by PcPAL immobilized on magnetic nanoparticles [43].

Figure 7

The application of AvPAL in D-AAs synthesis. (A) Production of substituted D-phenylalanines by enantioselective hydroamination of cinnamic acid derivatives using AvPAL [44]. (B) Production of pNO2-phenylalanine from the corresponding racemate employing AvPAL, P. mirabilis D-amino acid deaminase, and amine borane [45]. (C) Chemo-enzymatic pathway for the synthesis of optically pure D-(2,4,5-trifluorophenyl)alanine from 2,4,5-trifluoro-cinnamic acid generated from trifluorobenzaldehyde using the DAAT T242G variant from Bacillus sp. YM-1, PmLAAD and AvPAL [47].

Figure 8

Production of D-AAs by L-amino acid oxidase. (A) Two-step biocatalytic synthesis of D-5-Br-tryptophan using a one-pot approach by combining tryptophan 5-halogenase from Streptomyces rugosporus with RebO, without intermediary purification. Iteration of enantioselective oxidation of the L-enantiomer by RebO and subsequent chemical reduction into the racemate induced accumulation of the D-AA [53]. (B) Application of ancestral variant of LAAO (AncLAAO) in the deracemization of substituted phenylalanines and phenylglycine [55].

Figure 9

Biocatalytic application of PmaLAAD. (A) Stereoinversion of 12.5 mM L-4-NO₂-phenylalanine, using 62.5 mM borane tert-butylamine, 0.1 mg PmaLAAD/mL, at 25 °C and pH 7.5 [61]; (B) schematic structure of the active site of the triple variant F318A/V412A/V438P PmaLAAD with L-1-naphthylalanine modelled as substrate [62].