Ca2+ is a ubiquitous intracellular messenger that controls the most fundamental cellular processes. Nicotinic acid adenine dinucleotide phosphate (NAADP) is so far the most potent second messenger that can trigger calcium release from intracellular stores at nanomolar concentration in a variety of biological systems. The NAADP receptor, however, has not yet been unambiguously identified. With the objective to isolate and purify the NAADP binding protein(s), we have successfully synthesized three bi-functional NAADP analogs, which are 5-(3-azidopropyl)-8-azido-NAADP, 5-(prop-2-yn-1-yloxy)-8-azido-NAADP and 5-azido-8-ethynyl-NAADP. Each of the probes contains a photoactive aromatic azide which can covalently label the NAADP receptor, and a “clickable” terminal alkyne or alkyl azide that will enable us to attach the probes to an affinity tag. In the chemo-enzymatic synthesis, synthetic 8-substituted NAD was produced from coupling nicotinamide mononucleotide and an adenosine 5'-mono phosphate derivative. Next, the 8-substituted NAD was phosphorylated by recombinant human NAD kinase to give the corresponding NADP analog. Last, the nicotinamide in the NADP derivative was exchanged for a synthetic 5-substituted nicotinic acid by ADP-ribosyl cyclase to generate the bifunctional NAADP analog in reasonable yield. These compounds were characterized and assayed using sea urchin egg homogenates for their abilities to release Ca2+ from intracellular stores, to compete with [32P]NAADP in binding to the receptor, and to specifically photolabel the NAADP binding protein(s). Both 5-(3-azidopropyl)-8-azido-NAADP and 5-(prop-2-yn-1-yloxy)-8-azido-NAADP were shown to be high potency agonists of Ca2+ release and were able to label the NAADP binding protein(s) specifically at low concentration, while the 5-azido-8-ethynyl-NAADP is still under investigation.

Chemo-enzymatic synthesis of “clickable” and photoactive NAADP analogs for identification of the NAADP receptor

255th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.