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 (Ntz AD+ ) are described. Enzymatic reactions, photophysically monitored in real time, show Ntz AD+ and Ntz ADH 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 Ntz AD+ is converted to Ntz ADH, reflecting a complementary photophysical behavior to that of the native NAD+ /NADH. Ntz AD+ and Ntz ADH serve as substrates for NADase, which selectively cleaves the nicotinamide's glycosidic bond yielding tz ADP-ribose. Ntz AD+ 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 tz 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+ .