Contribution of Phosphates and Adenine to the Potency of Adenophostins at the IP₃ Receptor: Synthesis of All Possible Bisphosphates of Adenophostin A

Although adenophostin A (AdA), the most potent agonist of d-<i>myo-</i>inositol 1,4,5-trisphosphate receptors (IP₃R), is thought to mimic IP₃, the relative roles of the different phosphate groups and the adenosine motif have not been established. We synthesized all three possible bisphosphate analogues of AdA and glucose 3,4-bisphosphate (<b>7</b>, AdA lacking the 2′-AMP). 2′-Dephospho-AdA (<b>6</b>) was prepared via a novel regioselective dephosphorylation strategy. Assessment of the abilities of these bisphosphates to stimulate intracellular Ca²⁺ release using recombinant rat type 1 IP₃R (IP₃R1) revealed that <b>6</b>, a mimic of Ins­(4,5)­P₂, is only 4-fold less potent than IP₃, while <b>7</b> is some 400-fold weaker and even 3″-dephospho-AdA (<b>5</b>) is measurably active, despite missing one of the vicinal bisphosphate groups normally thought to be crucial for IP₃-like activity. Compound <b>6</b> is the most potent bisphosphate yet discovered with activity at IP₃R. Thus, adenosine has a direct role independent of the 2′-phosphate group in contributing toward the potency of adenophostins, the vicinal bisphosphate motif is not essential for activity at the IP₃R, as always thought, and it is possible to design potent agonists with just two of the three phosphates. A model with a possible adenine–R504 interaction supports the activity of <b>5</b> and <b>6</b> and also allows a reappraisal of the unexpected activity previously reported for the AdA regioisomer 2″-phospho-3″-dephospho-AdA <b>40