A method of preparation and purification of (4R)-deuterated-reduced nicotinamide adenine dinucleotide phosphate.
(4R)-Deuterated-reduced nicotinamide adenine dinucleotide phosphate, (4R)-[2H]NADPH, was prepared by reduction of NADP+ using an NADP(+)-dependent alcohol dehydrogenase (EC 1.1.1.2) from Thermoanaerobium brockii and isopropanol-d8 as substrate at 43 degrees C, pH 9. More than 80% of the product was identified as reduced cofactor by reverse-phase (ODS) HPLC, and a 1H NMR study showed that all of the reduced cofactor was (4R)-deuterated. Less than 10% of the product was oxidized cofactor, the remainder being impurities from the breakdown of the dinucleotide compound. Subsequent purification carried out by semipreparative reverse-phase HPLC with 0.1 M NaCl at pH 8.5 gave a compound of more than 96% purity. Separated (4R)-[2H]NADPH fractions were freeze-dried and the white solid was stored at 5 degrees C with desiccant.
Jeong
S S
SS
Department of Biochemistry, University of Sydney, New South Wales, Australia.
Gready
J E
JE
D000433:1-Propanol; D000426:Alcohol Dehydrogenase; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003903:Deuterium; D007553:Isotope Labeling / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009249:NADP / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D010084:Oxidation-Reduction
D009249:NADP; D000433:1-Propanol; D003903:Deuterium; D000426:Alcohol Dehydrogenase