title: Efficient synthesis of D-branched-chain amino acids and their labeled compounds with stable isotopes using D-amino acid dehydrogenase.
abstract: D-Branched-chain amino acids (D-BCAAs) such as D-leucine, D-isoleucine, and D-valine are known to be peptide antibiotic intermediates and to exhibit a variety of bioactivities. Consequently, much effort is going into achieving simple stereospecific synthesis of D-BCAAs, especially analogs labeled with stable isotopes. Up to now, however, no effective method has been reported. Here, we report the establishment of an efficient system for enantioselective synthesis of D-BCAAs and production of D-BCAAs labeled with stable isotopes. This system is based on two thermostable enzymes: D-amino acid dehydrogenase, catalyzing NADPH-dependent enantioselective amination of 2-oxo acids to produce the corresponding D-amino acids, and glucose dehydrogenase, catalyzing NADPH regeneration from NADP(+) and D-glucose. After incubation with the enzymes for 2 h at 65°C and pH 10.5, 2-oxo-4-methylvaleric acid was converted to D-leucine with an excellent yield (>99 %) and optical purity (>99 %). Using this system, we produced five different D-BCAAs labeled with stable isotopes: D-[1-(13)C,(15)N]leucine, D-[1-(13)C]leucine, D-[(15)N]leucine, D-[(15)N]isoleucine, and D-[(15)N]valine. The structure of each labeled D-amino acid was confirmed using time-of-flight mass spectrometry and nuclear magnetic resonance analysis. These analyses confirmed that the developed system was highly useful for production of D-BCAAs labeled with stable isotopes, making this the first reported enzymatic production of D-BCAAs labeled with stable isotopes. Our findings facilitate tracer studies investigating D-BCAAs and their derivatives.
authors: Applied Molecular Microbiology and Biomass Chemistry, Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan.
authors: Hironaga
authors: H
authors: Akita
authors: Hirokazu
authors: H
authors: Suzuki
authors: Katsumi
authors: K
authors: Doi
authors: Toshihisa
authors: T
authors: Ohshima
mesh_terms: D000597:Amino Acids, Branched-Chain / Q000096:biosynthesis*; D003067:Coenzymes / Q000378:metabolism; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D042843:Glucose 1-Dehydrogenase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling / Q000379:methods*; D009249:NADP / Q000378:metabolism*; D013696:Temperature
chemical_list: D000597:Amino Acids, Branched-Chain; D003067:Coenzymes; D009249:NADP; D042843:Glucose 1-Dehydrogenase; D003605:D-Amino-Acid Oxidase; D005947:Glucose