Efficient synthesis of d-branched-chain amino acids and their labeled compounds with stable isotopes using d-amino acid dehydrogenase

Hironaga Akita, Hirokazu Suzuki, Katsumi Doi, Toshihisa Ohshima

研究成果: ジャーナルへの寄稿記事

15 引用 (Scopus)

抄録

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-[1-13C,15N]leucine, d-[1-13C]leucine, d-[15N]leucine, d-[15N]isoleucine, and d-[15N]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.

元の言語英語
ページ(範囲)1135-1143
ページ数9
ジャーナルApplied Microbiology and Biotechnology
98
発行部数3
DOI
出版物ステータス出版済み - 1 1 2014

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Branched Chain Amino Acids
Isotopes
Oxidoreductases
Amino Acids
Leucine
Isoleucine
Valine
NADP
Glucose 1-Dehydrogenase
Keto Acids
Amination
NADPH Dehydrogenase
Enzymes
Regeneration
Mass Spectrometry
Magnetic Resonance Spectroscopy
Anti-Bacterial Agents
Glucose
Peptides

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Applied Microbiology and Biotechnology

これを引用

Efficient synthesis of d-branched-chain amino acids and their labeled compounds with stable isotopes using d-amino acid dehydrogenase. / Akita, Hironaga; Suzuki, Hirokazu; Doi, Katsumi; Ohshima, Toshihisa.

:: Applied Microbiology and Biotechnology, 巻 98, 番号 3, 01.01.2014, p. 1135-1143.

研究成果: ジャーナルへの寄稿記事

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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-[1-13C,15N]leucine, d-[1-13C]leucine, d-[15N]leucine, d-[15N]isoleucine, and d-[15N]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.",
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AB - 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-[1-13C,15N]leucine, d-[1-13C]leucine, d-[15N]leucine, d-[15N]isoleucine, and d-[15N]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.

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