Improved Yield for the Enzymatic Synthesis of Radiolabeled Nicotinamide Adenine Dinucleotide

Labeled β-nicotinamide adenine dinucleotide (NAD) analogues have been critical for uncovering new biochemical connections and quantitating enzymatic activity. They function as tracers for enzymology, flux analyses, and in situ measurements. Nevertheless, there is limited availability of specific types of analogues, especially radiolabeled NAD isotopologues. Here, we describe an improved enzymatic synthesis reaction for ³²P- NAD⁺ with a yield of 98% ± 1%, using lowered concentrations of reactants and standard equipment. This represents the highest reported yield for the enzymatic synthesis of NAD⁺ to date. With the high yield we were able to directly use the reaction product to generate derivatives, such as ³²P-NADP. The high-yield enzymatic synthesis is versatile for a broad variety of labels and NAD derivatives. Its advantages include lowered concentrations of reactants, providing sufficient amounts of product for downstream applications, and minimizing intermediate purification steps.