Synthesis of cyclic adenosine 5'-diphosphate ribose analogues: a C2'endo/syn "southern" ribose conformation underlies activity at the sea urchin cADPR receptor.

Novel 8-substituted base and sugar-modified analogues of the Ca(2+) mobilizing second messenger cyclic adenosine 5'-diphosphate ribose (cADPR) were synthesized using a chemoenzymatic approach and evaluated for activity in sea urchin egg homogenate (SUH) and in Jurkat T-lymphocytes; conformational analysis investigated by (1)H NMR spectroscopy revealed that a C2'endo/syn conformation of the "southern" ribose is crucial for agonist or antagonist activity at the SUH-, but not at the T cell-cADPR receptor.

Chemoenzymatic synthesis of 7-deaza cyclic adenosine 5'-diphosphate ribose analogues, membrane-permeant modulators of intracellular calcium release.

An optimized synthetic route to 7-deaza-8-bromo-cyclic adenosine 5'-diphosphate ribose (7-deaza-8-bromo-cADPR 3), an established cell-permeant, hydrolysis-resistant cyclic adenosine 5'-diphosphate ribose (cADPR) antagonist, is presented. Using NMR analysis, we found that 3 adopted a C-2' endo conformation in the N9-linked ribose and a syn conformation about the N9-glycosyl linkage, which are similar to that of cADPR. The synthetic route was also employed to produce 7-deaza-2'-deoxy-cADPR 4, a potential cell-permeant cADPR analogue. 3 and 4 were more stable to chemical hydrolysis, consistent with the observation that 7-deaza-cADPR analogues are more stable than their parent adenosine derivatives. 3 was also found to be stable to enzyme-mediated hydrolysis using CD38 ectoenzyme.

Novel hydrolysis-resistant analogues of cyclic ADP-ribose: modification of the "northern" ribose and calcium release activity.

Three novel analogues modified in the "northern" ribose (ribose linked to N1 of adenine) of the Ca(2+) mobilizing second messenger cyclic adenosine diphosphoribose, termed 2"-NH(2)-cyclic adenosine diphosphoribose, cyclic adenosine diphospho-carbocyclic-ribose, and 8-NH(2)-cyclic adenosine diphospho-carbocyclic-ribose, were synthesized (chemoenzymatically and by total synthesis) and spectroscopically characterized, and the pK(a) values for the 6-amino/imino transition were determined in two cases. The biological activity of these analogues was determined in permeabilized human Jurkat T-lymphocytes. 2"-NH(2)-cyclic adenosine diphosphoribose mediated Ca(2+) release was slightly more potent than that of the endogenous cyclic adenosine diphosphoribose in terms of the concentration-reponse relationship. Both compounds released Ca(2+) from the same intracellular Ca(2+) pool. In addition, the control compound 2"-NH(2)-adenosine diphosphoribose was almost without effect. In contrast, only at much higher concentrations (> or =50 microM) did the "northern" carbocyclic analogue, cyclic adenosine diphospho-carbocyclic-ribose, significantly release Ca(2+) from permeabilized T cells, whereas the previously reported "southern" carbocyclic analogue, cyclic aristeromycin diphosphoribose, was slightly more active than the endogenous cyclic adenosine diphosphoribose. Likewise, 8-NH(2)-cyclic adenosine diphospho-carbocyclic-ribose, expected to antagonize Ca(2+) release as demonstrated previously for 8-NH(2)-cyclic adenosine diphosphoribose, did not inhibit cyclic adenosine diphosphoribose mediated Ca(2+) release. This indicates that the 2"-NH(2)-group substitutes well for the 2"-OH-group it replaces; it may be oriented toward the outside of the putative cyclic adenosine diphosphoribose receptor binding domain and/or it can potentially also engage in H bonding interactions with residues of that domain. In sharp contrast to this, replacement of the endocyclic furanose oxygen atom by CH(2) in a carbocyclic system obviously interferes with a crucial element of interaction between cyclic adenosine diphosphoribose and its receptor in T-lymphocytes.