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

• Published in: Journal of the American Chemical Society Vol. 139; no. 44; pp. 15556 - 15559
• Main Authors: Rovira, Alexander R, Fin, Andrea, Tor, Yitzhak
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 08.11.2017; Amer Chemical Soc
• Subjects: ADP Ribose Transferases - metabolism; Alcohol Dehydrogenase - metabolism; Animals; Chemistry; Chemistry, Multidisciplinary; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Humans; NAD - analogs & derivatives; NAD - chemical synthesis; NAD - metabolism; NAD+ Nucleosidase - metabolism; Physical Sciences; Pyridines - chemical synthesis; Pyridines - chemistry; Pyridines - metabolism; Science & Technology; Substrate Specificity; Swine; Thiazoles - chemical synthesis; Thiazoles - chemistry; Thiazoles - metabolism; RIBOSYLATION; NICOTINAMIDE 1,N6-ETHENOADENINE DINUCLEOTIDE; NADH; ENZYME; ACID; ADP-RIBOSYLTRANSFERASE; FLUORESCENCE; ADENINE-DINUCLEOTIDE; RNA ALPHABET; DEHYDROGENASE
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.7b05852

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