ABSTRACT
Nicotinamides are ubiquitous cofactors used by many biological systems as redox agents. Stereospecifically labeled cofactors are useful in many studies of nicotinamide-dependent enzymes. Enzyme-directed synthesis of these cofactors is rather common but their stability imposes significant challenges on yield, purity, and preservation. This paper presents the stereospecific synthesis of reduced R- and S-[4-3H] beta-nicotinamide adenine dinucleotide 2' phosphate (NADPH). The method of Valera et al. [Biochem. Biophys. Res. Commun. 148 (1987) 515] was modified to a synthetic procedure that produces both isotopic diastereomers within 2h with an improved yield of 75-90% after purification and lyophilization. In the synthesis, [4-3H]NADP+ was generated as an intermediate (which can be isolated if desired). The specific radioactivities reported here are 2.7 and 1.1 Ci/mmol for the S and R diastereomers, respectively. Specific radioactivities ranging from carrier-free to trace labeling can be achieved with a minor change to the procedure.
