Microscale synthesis and kinetic isotope effect analysis of (4R)-[Ad-C-14,4-H-2] NADPH and (4R)-[Ad-H-3,4-H-2] NADPH

We present a one-pot chemo-enzymatic microscale synthesis of NADPH with two different patterns of isotopic labels: (4R)-[Ad-C-14,4-H-2] NADPH and (4R)-[Ad-H-3,4-H-2] NADPH. These co-factors are required by an enormous range of enzymes, and isotopically labeled nicotinamides are consequently of significant interest to researchers. In the current procedure, [Ad-C-14] NAD(+) and [Ad-H-3] NAD(+) were phosphorylated by NAD(+) kinase to produce [Ad-C-14] NADP(+) and [Ad-H-3] NADP(+), respectively. Thermoanaerobium brockii alcohol dehydrogenase was then used to stereospecifically transfer deuterium from C2 of isopropanol to the re face of C4 of NADP(+). After purification by HPLC, NMR analysis indicated that the deuterium content at the 411 position is more than 99.7%. The labeled cofactors were then used to successfully and sensitively measure kinetic isotope effects for R67 dihydrofolate reductase, providing strong evidence for the utility of this synthetic methodology.