N1-[(5' '-O-Phosphorylethoxy)methyl]-5'-O-phosphorylinosine 5',5''-cyclicpyrophosphate (cIDPRE 2a) and the 8-substituted derivatives 8-bromo-, 8-azido-, 8-amino-, and 8-Cl-cIDPRE (2b-e) were synthesized from N1-[(5''-acetoxyethoxy)methyl]-2',3'-O-isopropylideneinosine (5) in good yields. The pharmacological activities of cIDPRE and the 8-substituted derivatives (2a-e) were analyzed in intact and permeabilized human Jurkat T-lymphocytes. The results indicate that cIDPRE permeates the plasma membrane, releases Ca2+ from an intracellular, cADPR-sensitive Ca2+ store, and subsequently initiates Ca2+ release-activated Ca2+ entry. The Ca(2+)-releasing activity of cIDPRE was confirmed directly in permeabilized cells. Using time-resolved confocal Ca2+ imaging at the single cell level, the development of global Ca2+ signals starting from local small Ca2+ signals evoked by cIDPRE was observed. 8-N3-cIDPRE 2c and 8-NH2-cIDPRE 2d were similarly effective in their agonistic activity as compared to cIDPRE 2a, showing almost indistinguishable concentration-response curves for 2a, 2c, and 2d and very similar kinetics of Ca2+ signaling. In contrast, the halogenated derivatives 8-Br- and 8-Cl-cIDPRE (2b and 2e) did not significantly elevate [Ca2+]i. Therefore, cIDPRE 2a, 8-N3-cIDPRE 2c, and 8-NH2-cIDPRE 2d are novel membrane permeant cADPR mimic and may provide important novel tools to study cADPR-mediated Ca2+ signaling in intact cells.

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In Jurkat T cells, the type 3 ryanodine receptor (RyR) was knocked-down by stable integration of plasmid expressing type 3 ryanodine receptor antisense RNA. Ca 2ϩ signaling by ligation of the T cell receptor (TCR)/CD31 complex is a complicated process involving the formation and breakdown of the second messengers D-myo-inositol 1,4,5-trisphosphate (IP 3 ) and cyclic adenosine diphosphoribose (cADPR) in a temporally coordinated fashion (1-3). In addition to IP 3 and cADPR, nicotinic acid adenine dinucleotide phosphate (NAADP) is an essential regulator of T cell Ca 2ϩ signaling (4), although the exact role of this nucleotide has not yet been clarified. Jurkat T cells preincubated with the specific, membrane-permeant cADPR antagonist 7-deaza-8-Br-cADPR (5) showed characteristic defects in the onset and in the longlasting phase of TCR/CD3-and ␤ 1 -integrin-mediated Ca 2ϩ signaling (2, 6). In addition, in peripheral human T cells, 7-deaza-8-Br-cADPR efficiently blocked expression of the activation antigens CD25 and MHCII and blocked proliferation upon CD3

We previously developed cyclic ADP-carbocyclic ribose (cADPcR, 2) as a stable mimic of cyclic ADP-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. A series of the N1-ribose modified cADPcR analogues, designed as novel stable mimics of cADPR, which were the 2"-deoxy analogue 3, the 3"-deoxy analogue 4, the 3"-deoxy-2"-O-(methoxymethyl) analogue 5, the 3"-O-methyl analogue 6, the 2",3"-dideoxy analogue 7, and the 2",3"-dideoxydidehydro analogue 8, were successfully synthesized using the key intramolecular condensation reaction with phenylthiophosphate-type substrates. We investigated the conformations of these analogues and of cADPR and found that steric repulsion between both the adenine and N9-ribose moieties and between the adenine and N1-ribose moieties was a determinant of the conformation. The Ca(2+)-mobilizing effects were evaluated systematically using three different biological systems, i.e., sea urchin eggs, NG108-15 neuronal cells, and Jurkat T-lymphocytes. The relative potency of Ca(2+)-mobilization by these cADPR analogues varies depending on the cell-type used: e.g., 3"-deoxy-cADPcR (4) > cADPcR (2) > cADPR (1) in sea urchin eggs; cADPR (1) >> cADPcR (2) approximately 3"-deoxy-cADPcR (4) in T-cells; and cADPcR (2) > cADPR (1) > 3"-deoxy-cADPcR (4) in neuronal cells, respectively. These indicated that the target proteins and/or the mechanism of action of cADPR in sea urchin eggs, T-cells, and neuronal cells are different. Thus, this study represents an entry to cell-type selective cADPR analogues, which can be used as biological tools and/or novel drug leads.