Synthesis and biological evaluation of novel photo-clickable adenosine and cyclic ADP-ribose analogs: 8-N3-2′-O-propargyladenosine and 8-N3-2′-O-propargyl-cADPR

Abstract

A photo-clickable analog of adenosine was devised and synthesized in which the photoactive functional group (8-azidoadenosine) and the click moiety (2ʹ-O-propargyl-ether) were compactly combined within the structure of the adenosine nucleoside itself. We synthesized 8-N3-2ʹ-O-propargyl adenosine in four steps starting from adenosine. This photo-clickable adenosine was 5ʹ-phosphorylated and coupled to nicotinamide mononucleotide to form the NAD analog 8-N3-2ʹ-O-propargyl-NAD. This NAD analog was recognized by Aplysia californica ADP-ribosyl cyclase and enzymatically cyclized producing 8-N3-2ʹ-O-propargyl cyclic ADP-ribose. Photo-clickable cyclic-ADP-ribose analog was envisioned as a probe to label cyclic ADP-ribose binding proteins. The monofunctional 8-N3-cADPR has previously been shown to be an antagonist of cADPR-induced calcium release [T.F. Walseth et. al., J. Biol. Chem (1993) 268, 26686–26691]. 2ʹ-O-propargyl-cADPR was recognized as an agonist which elicited Ca2+ release when added at low concentration to sea urchin egg homogenates. The bifunctional 8-N3-2ʹ-O-propargyl cyclic ADP-ribose did not elicit Ca2+ release at low concentration or impact cyclic ADP-ribose mediated Ca2+ release either when added to sea urchin egg homogenates or when microinjected into cultured human U2OS cells. The photo-clickable adenosine will none-the-less be a useful scaffold for synthesizing photo-clickable probes for identifying proteins that interact with a variety of adenosine nucleotides.