d-Amino Acids Indirectly Inhibit Biofilm Formation in Bacillus subtilis by Interfering with Protein Synthesis

The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine and was reported to inhibit biofilm formation via the incorporation of these d-amino acids into the cell wall. Here, we show that l-amino acids were able to specifically reverse the inhibitory effects of their cognate d-amino acids. We also show that d-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding d-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of d-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of d-amino acids without losing the ability to incorporate at least one noncanonical d-amino acid, d-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of d-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis.     

Amino Acid Isomerization in the Production of l-Phenylalanine from d-Phenylalanine by Bacteria

To establish an advantageous method for the production of l-amino acids, microbial isomerization of d- and dl-amino acids to l-amino acids was studied. Screening experiments on a number of microorganisms showed that cell suspensions of Pseudomonas fluorescens and P. miyamizu were capable of isomerizing d- and dl-phenylalanines to l-phenylalanine. Various conditions suitable for isomerization by these organisms were investigated. Cells grown in a medium containing d-phenylalanine showed highest isomerization activity, and almost completely converted d- or dl-phenylalanine into l-phenylalanine within 24 to 48 hr of incubation. Enzymatic studies on this isomerizing system suggested that the isomerization of d- or dl-phenylalanine is not catalyzed by a single enzyme, “amino acid isomerase,” but the conversion proceeds by a two step system as follows: d-pheylalanine is oxidized to phenylpyruvic acid by d-amino acid oxidase, and the acid is converted to l-phenylalanine by transamination or reductive amination.     

Probing the Sophisticated Synergistic Allosteric Regulation of Aromatic Amino Acid Biosynthesis in Mycobacterium tuberculosis Using ?-Amino Acids

Chirality plays a major role in recognition and interaction of biologically important molecules. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of the shikimate pathway, which is responsible for the synthesis of aromatic amino acids in bacteria and plants, and a potential target for the development of antibiotics and herbicides. DAH7PS from Mycobacterium tuberculosis (MtuDAH7PS) displays an unprecedented complexity of allosteric regulation, with three interdependent allosteric binding sites and a ternary allosteric response to combinations of the aromatic amino acids l-Trp, l-Phe and l-Tyr. In order to further investigate the intricacies of this system and identify key residues in the allosteric network of MtuDAH7PS, we studied the interaction of MtuDAH7PS with aromatic amino acids that bear the non-natural d-configuration, and showed that the d-amino acids do not elicit an allosteric response. We investigated the binding mode of d-amino acids using X-ray crystallography, site directed mutagenesis and isothermal titration calorimetry. Key differences in the binding mode were identified: in the Phe site, a hydrogen bond between the amino group of the allosteric ligands to the side chain of Asn175 is not established due to the inverted configuration of the ligands. In the Trp site, d-Trp forms no interaction with the main chain carbonyl group of Thr240 and less favourable interactions with Asn237 when compared to the l-Trp binding mode. Investigation of the MtuDAH7PS_N175A variant further supports the hypothesis that the lack of key interactions in the binding mode of the aromatic d-amino acids are responsible for the absence of an allosteric response, which gives further insight into which residues of MtuDAH7PS play a key role in the transduction of the allosteric signal.     

Amino Acid Racemization in Pseudomonas putida KT2440

d-Amino acids have been shown to play an increasingly diverse role in bacterial physiology, yet much remains to be learned about their synthesis and catabolism. Here we used the model soil- and rhizosphere-dwelling organism Pseudomonas putida KT2440 to elaborate on the genomics and enzymology of d-amino acid metabolism. P. putida KT2440 catabolized the d-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. With the exception of phenylalanine, each of these amino acids was racemized by P. putida KT2440 enzymes. Three amino acid racemases were identified from a genomic screen, and the enzymes were further characterized in vitro. The putative biosynthetic alanine racemase Alr showed broad substrate specificity, exhibiting measurable racemase activity with 9 of the 19 chiral amino acids. Among these amino acids, activity was the highest with lysine, and the k_cat/K_m values with l- and d-lysine were 3 orders of magnitude greater than the k_cat/K_m values with l- and d-alanine. Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. However, DadX did show 6- and 9-fold higher k_cat/K_m values than Alr with l- and d-alanine, respectively. The annotated proline racemase ProR of P. putida KT2440 showed negligible activity with either stereoisomer of the 19 chiral amino acids but exhibited strong epimerization activity with hydroxyproline as the substrate. Comparative genomic analysis revealed differences among pseudomonads with respect to alanine racemase genes that may point to different roles for these genes among closely related species.     

Rational design and characterization of D-Phe-Pro-D-Arg-derived direct thrombin inhibitors

The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both l- and d-amino acids, with the general sequence d-Phe(P3)-Pro(P2)-d-Arg(P1)-P1′-CONH₂. The P1′ position was scanned with l- and d-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1′ position. The lead tetrapeptide, d-Phe-Pro-d-Arg-d-Thr-CONH₂, competitively inhibits α-thrombin''s cleavage of the S2238 chromogenic substrate with a K_i of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with P1′ l-isoleucine (fPrI), l-cysteine (fPrC) or d-threonine (fPrt)) in complex with human α-thrombin were determined by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of the enzyme. The contacts established between the d-Arg residue in position P1 and thrombin are similar to those observed for the l-isomer in other substrates and inhibitors. However, fPrC and fPrt disrupt the active site His57-Ser195 hydrogen bond, while the combination of a P1 d-Arg and a bulkier P1′ residue in fPrI induce an unfavorable geometry for the nucleophilic attack of the scissile bond by the catalytic serine. The experimental models explain the observed relative potency of the inhibitors, as well as their stability to proteolysis. Moreover, the newly identified direct thrombin inhibitors provide a novel pharmacophore platform for developing antithrombotic agents by exploring the conformational constrains imposed by the d-stereochemistry of the residues at positions P1 and P1′.     

Regulation of sulfate uptake by amino acids in cultured tobacco cells

The sulfur requirements of tobacco (Nicotiana tabacum L. var. Xanthi) XD cells grown in chemically defined liquid media can be satisfied by sulfate, thiosulfate, l-cyst(e)ine, l-methionine or glutathione, and somewhat less effectively by d-cyst (e) ine, d-methionine or dl-homocyst (e)ine. Sulfate uptake is inhibited after a 2 hr lag by l-cyst (e)ine, l-methionine, l-homocyst(e)ine or l-isoleucine, but not by any of the other protein amino acids, nor by d-cyst(e)ine. l-cyst(e)ine is neither a competitive nor a non-competitive inhibitor of sulfate uptake. Its action most closely resembles apparent uncompetitive inhibition. Inhibition of sulfate uptake by l-cyst(e)ine can be partially prevented by equimolar l-arginine, l-lysine, l-leucine, l-phenylalanine, l-tyrosine or l-tryptophan, but is little affected by any of the other protein amino acids. The effective amino acids are apparent competitive inhibitors of l-cyst(e)ine uptake after a 2 hr lag. Inhibition of sulfate uptake by l-methionine cannot be prevented, nor can uptake of l-methionine be inhibited by any single protein amino acid. The results suggest the occurrence of negative feedback control of sulfate assimilation by the end products, the sulfur amino acids, in cultured tobacco cells.     

Detection of d-amino acids in purified proteins synthesized in Escherichia coli

It has long been believed that amino acids comprising proteins of all living organisms are only of the l-configuration, except for Gly. However, peptidyl d-amino acids were observed in hydrolysates of soluble high molecular weight fractions extracted from cells or tissues of various organisms. This strongly suggests that significant amounts of d-amino acids are naturally present in usual proteins. Thus we analyzed the d-amino acid contents of His-tag-purified β-galactosidase and human urocortin, which were synthesized by Escherichia coli grown in controlled synthetic media. After acidic hydrolysis for various times at 110°C, samples were derivatized with 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole (NBD-F) and separated on a reverse-phase column followed by a chiral column into d- and l-enantiomers. The contents of d-enantiomers of Ala, Leu, Phe, Val, Asp, and Glu were determined by plotting index d/(d + l) against the incubation time for hydrolysis and extrapolating the linear regression line to 0 h to eliminate the effect of racemization of amino acids during the incubation. Significant contents of d-amino acids were reproducibly detected, the d-amino acid profile being specific to an individual protein. This finding indicated the likelihood that d-amino acids are in fact present in the purified proteins. On the other hand, the d-amino acid contents of proteins were hardly influenced by the addition of d- or l-amino acids to the cultivation medium, whereas intracellular free d-amino acids sensitively varied according to the extracellular conditions. The origin of these d-amino acids detected in proteins was discussed.     

d-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling

Peptidoglycan is an essential and highly conserved mesh structure that surrounds bacterial cells. It plays a critical role in retaining a defined cell shape, and, in the case of pathogenic Gram-positive bacteria, it lies at the interface between bacterial cells and the host organism. Intriguingly, bacteria can metabolically incorporate unnatural d-amino acids into the peptidoglycan stem peptide directly from the surrounding medium, a process mediated by penicillin binding proteins (PBPs). Metabolic peptidoglycan remodeling via unnatural d-amino acids has provided unique insights into peptidoglycan biosynthesis of live bacteria and has also served as the basis of a synthetic immunology strategy with potential therapeutic implications. A striking feature of this process is the vast promiscuity displayed by PBPs in tolerating entirely unnatural side chains. However, the chemical space and physical features of this side chain promiscuity have not been determined systematically. In this report, we designed and synthesized a library of variants displaying diverse side chains to comprehensively establish the tolerability of unnatural d-amino acids by PBPs in both Gram-positive and Gram-negative organisms. In addition, nine Bacillus subtilis PBP-null mutants were evaluated with the goal of identifying a potential primary PBP responsible for unnatural d-amino acid incorporation and gaining insights into the temporal control of PBP activity. We empirically established the scope of physical parameters that govern the metabolic incorporation of unnatural d-amino acids into bacterial peptidoglycan.     

A Highly Stable d-Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

d-Amino acid oxidase (DAO) is a biotechnologically attractive enzyme that can be used in a variety of applications, but its utility is limited by its relatively poor stability. A search of a bacterial genome database revealed a gene encoding a protein homologous to DAO in the thermophilic bacterium Rubrobacter xylanophilus (RxDAO). The recombinant protein expressed in Escherichia coli was a monomeric protein containing noncovalently bound flavin adenine dinucleotide as a cofactor. This protein exhibited oxidase activity against neutral and basic d-amino acids and was significantly inhibited by a DAO inhibitor, benzoate, but not by any of the tested d-aspartate oxidase (DDO) inhibitors, thus indicating that the protein is DAO. RxDAO exhibited higher activities and affinities toward branched-chain d-amino acids, with the highest specific activity toward d-valine and catalytic efficiency (k_cat/K_m) toward d-leucine. Substrate inhibition was observed in the case of d-tyrosine. The enzyme had an optimum pH range and temperature of pH 7.5 to 10 and 65°C, respectively, and was stable between pH 5.0 and pH 8.0, with a T₅₀ (the temperature at which 50% of the initial enzymatic activity is lost) of 64°C. No loss of enzyme activity was observed after a 1-week incubation period at 30°C. This enzyme was markedly inactivated by phenylmethylsulfonyl fluoride but not by thiol-modifying reagents and diethyl pyrocarbonate, which are known to inhibit certain DAOs. These results demonstrated that RxDAO is a highly stable DAO and suggested that this enzyme may be valuable for practical applications, such as the determination and quantification of branched-chain d-amino acids, and as a scaffold to generate a novel DAO via protein engineering.     

Outwitting EF-Tu and the ribosome: translation with d-amino acids

Key components of the translational apparatus, i.e. ribosomes, elongation factor EF-Tu and most aminoacyl-tRNA synthetases, are stereoselective and prevent incorporation of d-amino acids (d-aa) into polypeptides. The rare appearance of d-aa in natural polypeptides arises from post-translational modifications or non-ribosomal synthesis. We introduce an in vitro translation system that enables single incorporation of 17 out of 18 tested d-aa into a polypeptide; incorporation of two or three successive d-aa was also observed in several cases. The system consists of wild-type components and d-aa are introduced via artificially charged, unmodified tRNA^Gly that was selected according to the rules of ‘thermodynamic compensation’. The results reveal an unexpected plasticity of the ribosomal peptidyltransferase center and thus shed new light on the mechanism of chiral discrimination during translation. Furthermore, ribosomal incorporation of d-aa into polypeptides may greatly expand the armamentarium of in vitro translation towards the identification of peptides and proteins with new properties and functions.     

Abiotic Racemization Kinetics of Amino Acids in Marine Sediments

The ratios of d- versus l-amino acids can be used to infer the sources and composition of sedimentary organic matter. Such inferences, however, rely on knowing the rates at which amino acids in sedimentary organic matter racemize abiotically between the d- and the l-forms. Based on a heating experiment, we report kinetic parameters for racemization of aspartic acid, glutamic acid, serine, and alanine in bulk sediment from Aarhus Bay, Denmark, taken from the surface, 30 cm, and 340 cm depth below seafloor. Extrapolation to a typical cold deep sea sediment temperature of 3°C suggests racemization rate constants of 0.50×10⁻⁵–11×10⁻⁵ yr⁻¹. These results can be used in conjunction with measurements of sediment age to predict the ratio of d:l amino acids due solely to abiotic racemization of the source material, deviations from which can indicate the abundance and turnover of active microbial populations.     

Physiology and Substrate Specificity of Two Closely Related Amino Acid Transporters,SerP1 and SerP2, of Lactococcus lactis

The serP1 and serP2 genes found adjacently on the chromosome of Lactococcus lactis strains encode two members of the amino acid-polyamine-organocation (APC) superfamily of secondary transporters that share 61% sequence identity. SerP1 transports l-serine, l-threonine, and l-cysteine with high affinity. Affinity constants (K_m) are in the 20 to 40 μM range. SerP2 is a dl-alanine/dl-serine/glycine transporter. The preferred substrate appears to be dl-alanine for which the affinities were found to be 38 and 20 μM for the d and l isomers, respectively. The common substrate l-serine is a high-affinity substrate of SerP1 and a low-affinity substrate of SerP2 with affinity constants of 18 and 356 μM, respectively. Growth experiments demonstrate that SerP1 is the main l-serine transporter responsible for optimal growth in media containing free amino acids as the sole source of amino acids. SerP2 is able to replace SerP1 in this role only in medium lacking the high-affinity substrates l-alanine and glycine. SerP2 plays an adverse role for the cell by being solely responsible for the uptake of toxic d-serine. The main function of SerP2 is in cell wall biosynthesis through the uptake of d-alanine, an essential precursor in peptidoglycan synthesis. SerP2 has overlapping substrate specificity and shares 42% sequence identity with CycA of Escherichia coli, a transporter whose involvement in peptidoglycan synthesis is well established. No evidence was obtained for a role of SerP1 and SerP2 in the excretion of excess amino acids during growth of L. lactis on protein/peptide-rich media.     

Cyclic tetrapeptides with –SS– bridging between amino acid side chains for potent histone deacetylases’ inhibition

Cyclic depsipeptide FK228 with an intramolecular disulfide bond is a potent inhibitor of histone deacetylases (HDAC). FK228 is stable in blood because of its prodrug function, whose –SS– bond is reduced within the cell. Here, cyclic peptides with –SS– bridges between a variety of amino acids were synthesized and assayed for HDAC inhibition. Cyclic peptide 3, cyclo(-l-amino acid-l-amino acid-l-Val-d-Pro-), with an –SS– bridge between the first and second amino acids, was found to be a potent HDAC inhibitor. Cyclic peptide 7, cyclo(-l-amino acid-d-amino acid-l-Val-d-Pro-), with an –SS– bridge between the first and second amino acids, was also a potent HDAC inhibitor.     

Properties of an Aminotransferase of Pea (Pisum sativum L.)

A transaminase (aminotransferase, EC 2.6.1) fraction was partially purified from shoot tips of pea (Pisum sativum L. cv. Alaska) seedlings. With α-ketoglutarate as co-substrate, the enzyme transaminated the following aromatic amino acids: d,l-tryptophan, d,l-tyrosine, and d,l-phenylalanine, as well as the following aliphatic amino acids: d,l-alanine, d,l-methionine, and d,l-leucine. Of other α-keto acids tested, pyruvate and oxalacetate were more active than α-ketoglutarate with d,l-tryptophan. Stoichiometric yields of indolepyruvate and glutamate were obtained with d,l-tryptophan and α-ketoglutarate as co-substrates. The specific activity was three times higher with d-tryptophan than with l-tryptophan.     

Specificity Determinants for Lysine Incorporation in Staphylococcus aureus Peptidoglycan as Revealed by the Structure of a MurE Enzyme Ternary Complex

Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.     

Utilization of d-Lactate as an Energy Source Supports the Growth of Gluconobacter oxydans

d-Lactate was identified as one of the few available organic acids that supported the growth of Gluconobacter oxydans 621H in this study. Interestingly, the strain used d-lactate as an energy source but not as a carbon source, unlike other lactate-utilizing bacteria. The enzymatic basis for the growth of G. oxydans 621H on d-lactate was therefore investigated. Although two putative NAD-independent d-lactate dehydrogenases, GOX1253 and GOX2071, were capable of oxidizing d-lactate, GOX1253 was the only enzyme able to support the d-lactate-driven growth of the strain. GOX1253 was characterized as a membrane-bound dehydrogenase with high activity toward d-lactate, while GOX2071 was characterized as a soluble oxidase with broad substrate specificity toward d-2-hydroxy acids. The latter used molecular oxygen as a direct electron acceptor, a feature that has not been reported previously in d-lactate-oxidizing enzymes. This study not only clarifies the mechanism for the growth of G. oxydans on d-lactate, but also provides new insights for applications of the important industrial microbe and the novel d-lactate oxidase.     

A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily: PRODUCTION,FUNCTIONAL CHARACTERIZATION,AND STRUCTURE MODELING*

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.     

Active site model of (R)-selective ω-transaminase and its application to the production of d-amino acids

ω-Transaminase (ω-TA) is one of the important biocatalytic toolkits owing to its unique enzyme property which enables the transfer of an amino group between primary amines and carbonyl compounds. In addition to preparation of chiral amines, ω-TA reactions have been exploited for the asymmetric synthesis of l-amino acids using (S)-selective ω-TAs. However, despite the availability of (R)-selective ω-TAs, catalytic utility of the ω-TAs has not been explored for the production of d-amino acids. Here, we investigated the substrate specificity of (R)-selective ω-TAs from Aspergillus terreus and Aspergillus fumigatus and demonstrated the asymmetric synthesis of d-amino acids from α-keto acids. Substrate specificity toward d-amino acids and α-keto acids revealed that the two (R)-selective ω-TAs possess strict steric constraints in the small binding pocket that precludes the entry of a substituent larger than an ethyl group, which is reminiscent of (S)-selective ω-TAs. Molecular models of the active site bound to an external aldimine were constructed and used to explain the observed substrate specificity and stereoselectivity. α-Methylbenzylamine (α-MBA) showed the highest amino donor reactivity among five primary amines (benzylamine, α-MBA, α-ethylbenzylamine, 1-aminoindan, and isopropylamine), leading us to employ α-MBA as an amino donor for the amination of 5 reactive α-keto acids (pyruvate, 2-oxobutyrate, fluoropyruvate, hydroxypyruvate, and 2-oxopentanoate) among 17 ones tested. Unlike the previously characterized (S)-selective ω-TAs, the enzyme activity of the (R)-selective ω-TAs was not inhibited by acetophenone (i.e., a deamination product of α-MBA). Using racemic α-MBA as an amino donor, five d-amino acids (d-alanine, d-homoalanine, d-fluoroalanine, d-serine, and d-norvaline) were synthesized with excellent product enantiopurity (enantiomeric excess >99.7 %).