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

Abstract

The synthesis, photophysics, and biochemical utility of a fluorescent NAD⁺ analogue based on an isothiazolo[4,3-d]pyrimidine core (N^tzAD⁺) are described. Enzymatic reactions, photophysically monitored in real time, show N^tzAD⁺ and N^tzADH 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 N^tzAD⁺ is converted to N^tzADH, reflecting a complementary photophysical behavior to that of the native NAD⁺/NADH. N^tzAD⁺ and N^tzADH serve as substrates for NADase, which selectively cleaves the nicotinamide's glycosidic bond yielding ^tzADP-ribose. N^tzAD⁺ 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 ^tzADP-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⁺.