Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD Analogue.

The synthesis, photophysics, and biochemical utility of a fluorescent NAD analogue based on an isothiazolo[4,3-d]pyrimidine core (NAD) are described. Enzymatic reactions, photophysically monitored in real time, show NAD and NADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as NAD is converted to NADH, reflecting a complementary photophysical behavior to that of the native NAD/NADH. NAD and NADH serve as substrates for NADase, which selectively cleaves the nicotinamide's glycosidic bond yielding ADP-ribose. NAD also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer ADP-ribose to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD.