Emissive Synthetic Cofactors: A Highly Responsive NAD Analogue Reveals Biomolecular Recognition Features.

Apart from its vital function as a redox cofactor, nicotinamide adenine dinucleotide (NAD ) has emerged as a crucial substrate for NAD -consuming enzymes, including poly(ADP-ribosyl)transferase 1 (PARP1) and CD38/CD157. Their association with severe diseases, such as cancer, Alzheimer's disease, and depressions, necessitates the development of new analytical tools based on traceable NAD surrogates. Here, the synthesis, photophysics and biochemical utilization of an emissive, thieno[3,4-d]pyrimidine-based NAD surrogate, termed N AD , are described. Its preparation was accomplished by enzymatic conversion of synthetic ATP by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1). The new NAD analogue possesses useful photophysical features including redshifted absorption and emission maxima as well as a relatively high quantum yield. Serving as a versatile substrate, N AD was reduced by alcohol dehydrogenase (ADH) to N ADH and afforded ADP-ribose ( ADPr) upon hydrolysis by NAD -nucleosidase (NADase). Furthermore, N AD was engaged in cholera toxin A (CTA)-catalyzed mono( ADP-ribosyl)ation, but was found incapable in promoting PARP1-mediated poly( ADP-ribosyl)ation. Due to its high photophysical responsiveness, N AD is suited for spectroscopic real-time monitoring. Intriguingly, and as an N7-lacking NAD surrogate, the thieno-based cofactor showed reduced compatibility (i.e., functional similarity compared to native NAD ) relative to its isothiazolo-based analogue. The distinct tolerance, displayed by diverse NAD producing and consuming enzymes, suggests unique biological recognition features and dependency on the purine N7 moiety, which is found to be of importance, if not essential, for PARP1-mediated reactions.