A Method of Preparation and Purification of (4R)-Deuterated-Reduced Nicotinamide Adenine Dinucleotide Phosphate

✍ Scribed by S.S. Jeong; J.E. Gready

• Publisher: Elsevier Science
• Year: 1994
• Tongue: English
• Weight: 372 KB
• Volume: 221
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1994.1411

✦ Synopsis

(4R)-Deuterated-reduced nicotinamide adenine dinucleotide phosphate, ((4 R)-\left[{ }^{2} H\right] N A D P H), was prepared by reduction of NADP ({ }^{+})using an NADP ({ }^{+})-dependent alcohol dehydrogenase (EC 1.1 .1 .2 ) from Thermoanaerobium brockii and isopropanol- (d_{8}) as substrate at (43^{\circ} \mathrm{C}), pH 9. More than (80 %) of the product was identified as reduced cofactor by reverse-phase (ODS) HPLC, and a ({ }^{1}) H NMR study showed that all of the reduced cofactor was ((4 R))-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 \mathrm{M} \mathbf{N a C l}) at (\mathrm{pH} 8.5) gave a compound of more than (96 %) purity. Separated (4R)(\left[^{2} H\right] N A D P H) fractions were freeze-dried and the white solid was stored at (5^{\circ} \mathrm{C}) with desiccant. (o 1994 Academic Press, Inc.