UC San Diego

A series of enzymatic transformations, which generate visibly emissive isofunctional cofactors based on an isothiazolo[4,3-d]pyrimidine analogue of adenosine (tz A), was developed. Nicotinamide adenylyl transferase condenses nicotinamide mononucleotide and tz ATP to yield Ntz AD+ , which can be enzymatically phosphorylated by NAD+ kinase and ATP or tz ATP to the corresponding Ntz ADP+ . The latter can be engaged in NADP-specific coupled enzymatic transformations involving conversion to Ntz ADPH by glucose-6-phosphate dehydrogenase and reoxidation to Ntz ADP+ by glutathione reductase. The Ntz ADP+ /Ntz ADPH cycle can be monitored in real time by fluorescence spectroscopy.