Enzymatic resolution for the preparation of enantiomerically enriched D‐β‐heterocyclic alanine derivatives using Escherichia coli aromatic L‐amino acid transaminase

Abstract

An enzymatic resolution was carried out for the preparation of enriched beta-heterocyclic D-alanine derivatives using Escherichia coli aromatic L-amino acid transaminase. The excess of pyrazole, imidazole, or 1,2,4-triazole reacted with methyl-2-acetamidoacrylate in acetonitrile in the presence of potassium carbonate at 60 degrees C, directly leading to make the potassium salt of the corresponding N-acetyl-beta-heterocyclic alanine derivatives. After the acidic deprotection of the N-acetyl group, 10 mM of racemic pyrazolylalanine, triazolylalanine, and imidazolylalanine were resolved to D-pyrazolylalanine, D-triazolylalanine, and D-imidazolylalanine with 46% (85% ee), 42% (72% ee), and 48% (95% ee) conversion yield in 18 h, respectively, using E. coli aromatic L-amino acid transaminase (EC 2.6.1.5). Although the three beta-heterocyclic L-alanine derivatives have similar molecular structures, they showed different reaction rates and enantioselectivities. The relative reactivities of the transaminase toward the beta-heterocyclic L-alanine derivatives could be explained by the relationship between the substrate binding energy (E, kcal/mol) to the enzyme active site and the distance (delta, A) from the nitrogen of alpha-amino group of the substrates to the C4' carbon of PLP-Lys258 Schiff base. As the ratio of the substrate binding energy (E) to the distance (delta) becomes indicative value of k(cat)/K(M) of the enzyme to the substrate, the relative reactivities of the beta-heterocyclic L-alanine derivatives were successfully correlated with E/delta, and the relationship was confirmed by our experiments.

Cloning and Characterization of a Novel β-Transaminase fromMesorhizobiumsp. Strain LUK: a New Biocatalyst for the Synthesis of Enantiomerically Pure β-Amino Acids

“…A possible explanation would be the substrate recognition mechanism of the transaminase subgroup II enzymes. In general, the substrate in the active site is recognized by the two substrate binding pockets around the PLPlysine Schiff base (9,23). The main roles of the binding pockets are to recognize the side chain of the substrate and to anchor the carboxylate groups of the substrate (9, 39).…”

Engineering aromaticL-amino acid transaminase for the asymmetric synthesis of constrained analogs ofL-phenylalanine

“…Validation of the generated model was done with the programs PROSAII (Sippl, 1993) and PROCHECK (Laskowski et al, 1993). Substrate docking into the active site was carried out using FlexX program (St. Louis, MO) as described before (Cho et al, 2004). All structural analyses such as superimposition of structures and calculation of distance were carried out using SwissPdb viewer.…”

“…Separation of enantiomers was achieved with an isocratic elution of water (pH 2, perchloric acid) at a flow rate of 0.7 mL/min at 58C and analyzed using a UV detector at 210 nm. The values of enantiomeric excess (ee L ) were calculated as described before (Cho et al, 2004).…”

“…We have described the asymmetric synthesis of unnatural Lamino acids using AroATEs (Cho et al, 2003), and recently reported the active site model of the AroATEc (Cho et al, 2004). The AroATEs showed unique broad substrate specificity toward unnatural L-amino acids such as L-homophenylalanine and high enantioselectivity toward L-configuration, which make the AroATEs an attractive biocatalyst for preparing optically pure unnatural L-amino acids.…”

“…The alteration of substrate specificity of enzyme without changing its stereoselectivity requires elucidating key determinants of amino acid residues in the active site. For example, the active site of the AroAT from Escherichia coli (E. coli) (AroATEc) is consisted of a PLP-anchoring site and two substrate-binding pockets around PLP-Lys Schiff base (Cho et al, 2004). In the substrate-binding pockets, two arginine residues play important roles to recognize acarboxylate group and distal carboxylate group of the substrate, respectively.…”