TOTAL CHEMICAL SYNTHESIS OF CADPR AND CADPR ANALOGS

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Cyclic adenosine diphosphate ribose (cADPR) appears to be a new and important second messenger that mediates Ca2+ release. The large majority of cADPR preparations in the literature are enzymatic syntheses. in vivo and in vitro, ADP-ribosyl cyclase catalyzes the conversion of nAD+ to cADPR. A biomimetic chemical synthesis of cADPR from NAD+ has recently been reported. Herein the first non-enzymatic total chemical synthesis of cADPR is proposed. Such a synthesis is a prerequisite to the eventual total chemical synthesis of cADPR photoaffinity labels (PALs) because it is unlikely that the available methods for the synthesis of cADPR can be sufficiently modified. cADPR acyclic precursors will be prepared from nucleotide and glycoside building blocks. These acyclic precursors can be "armed" and activated towards oxocarbenium ion formation and ring closure using the methodology of Fraser-Reid. This analysis is based on the common intermediacy of oxocarbenium ions intermediates in the biomimetic and enzymatic cyclizations of NAD+ and in Fraser-Reid's work. The proposed synthesis has the advantage that it is easily adapted to construct cADPR analogues, including cADPR PALs and cADPR affinity chromatography (AC) ligands. With a method for the chemical synthesis of cADPR and analogues in hand we will turn our attention to the biochemical evaluation of the cADPR analogues. Specifically constructed cADPR PALs and AC ligands are expected to be invaluable tools for identifying, purifying, and isolating cADPR receptor, effector, and binding proteins. Assays for biological activity and photoaffinity labeling experiments will use the original sea urchin egg systems, with the long term goal being the development of a set of general tools to study this new Ca2+-mediated second messenger system. The knowledge and methodology gained in the course of this work are expected to make important contributions to our understanding of this and potentially other second messenger pathways. Should the proposed experiments prove successful we would then be equipped to significantly expand the scope of the project to include systems more complex than the sea urchin.