Ca<sup>2+</sup> is one of the most versatile and important intracellular messenger since it controls numerous cellular process. Nicotinic acid adenine dinucleotide phosphate (NAADP) has shown to be a very potent second messenger that triggers calcium release from intracellular stores at nanomolar concentration in a variety of biological systems. However, the NAADP binding protein has not yet been unambiguously identified. Previous work in our lab showed that a photosensitive probe, 5-azido-NAADP, was able to release calcium at low concentration in sea urchin egg homogenates and successfully label the NAADP binding proteins on irradiation. Based on this compound, we designed an effective bifunctional NAADP analog that contains a clickable alkyne at the 8-adenosyl position and a photoactive azide at the 5 position of nicotinic acid. In our chemo-enzymatic synthesis, a synthetic 8-substituted NAD is produced from NMN and an adenosine 5'-mono phosphate derivative. Next, NAD is phosphorylated by NAD kinase, followed by an enzyme catalyzed base exchange reaction, where ADP-ribosyl cyclase exchanges nicotinamide for a synthetic 5-substituted nicotinic acid. The clickable and photoactive NAADP analog is expected to be recognized as an NAADP agonist, to efficiently label the NAADP binding protein on irraditaion and to facilitate the isolation and purification of the protein by “click chemistry”.<br/>

Chemo-enzymatic synthesis of an effective bifunctional NAADP analog for isolation and purification of the NAADP receptor

253rd National Meeting (2017)

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