Stereoselective preparation of deuterated reduced nicotinamide adenine nucleotides and substrates by enzymatic synthesis

✍ Scribed by Ronald E. Viola; Paul F. Cook; W.W. Cleland

• Publisher: Elsevier Science
• Year: 1979
• Tongue: English
• Weight: 597 KB
• Volume: 96
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(79)90590-6

✦ Synopsis

A-Side (4-R)-(4-2H)-reduced nicotinamide adenine dinucleotide (NADD) was prepared by a stepwise oxidation of ethanol-d, to acetate in the presence of NAD, alcohol dehydrogenase, and aldehyde dehydrogenase. The B-side (4-S) isomer of NADD was prepared using the glucose dehydrogenase activity of glucose-6-phosphate dehydrogenase to oxidize glucosel-d in 40% dimethyl s&oxide. Subsequent purification of the reduced nucleotides was achieved using a column of strongly basic polystyrene macroporous resin (AG MP-1) eluted with 0.2 M LiCl, pH 10, and applying the pooled NADD peak to a polyacrylamide gel (Bio-Gel P-2) column. The final A26,,/A340 ratio obtained for these preparations was below 2.3. Preparation of the deuterated reduced nucleotides in this manner allows production of specifically deuterated substrates by coupled enzymatic synthesis. L-Malate-2-d was prepared by coupled synthesis of A-side NADD to the reduction of oxaloacetate by the A-side enzyme malate dehydrogenase.