Chemo-enzymatic synthesis of adenine substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs

Abstract

Nicotinamide adenine dinucleotide phosphate (NADP) is an indispensable metabolic co-substrate and nicotinic acid adenine dinucleotide phosphate (NAADP) is an important Ca²⁺ releasing intracellular second messenger. Exploration of the NADP and NAADP interactome often requires the synthesis of NADP derivatives substituted on the adenosine nucleoside. The introduction of the 2'-phosphate of NADP makes the synthesis of substituted NADP derivatives difficult. We have employed recombinant human NAD kinase expressed in E. coli as an enzymatic reagent to convert readily available synthetic NAD derivatives to NADP analogs, which were subsequently transformed into NAADP derivatives using enzyme catalyzed pyridine base exchange. 8-Ethynyl-NADP, 8-ethynyl-NAADP and 5-N₃-8-ethynyl-NAADP were synthesized starting from a protected 8-ethynyladenosine using a combination of chemical and enzymatic steps and the NAADP derivatives shown to be recognized by the sea urchin NAADP receptor at low concentration. Our methodology will enable researchers to produce mono- and bi-substituted NADP and NAADP analogs that can be applied in proteomic studies to identify NADP and NAADP binding proteins.