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 Ca2+ mobilizing second messenger cyclic adenosine diphosphoribose, termed 2‘ ‘-NH2-cyclic adenosine diphosphoribose, cyclic adenosine diphospho-carbocyclic-ribose, and 8-NH2-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‘ ‘-NH2-cyclic adenosine diphosphoribose mediated Ca2+ release was slightly more potent than that of the endogenous cyclic adenosine diphosphoribose in terms of the concentration−reponse relationship. Both compounds released Ca2+ from the same intracellular Ca2+ pool. In addition, the control compound 2‘ ‘-NH2-adenosine diphosphoribose was almost without effect. In contrast, only at much higher concentrations (≥50 μM) did the “northern” carbocyclic analogue, cyclic adenosine diphospho-carbocyclic-ribose, significantly release Ca2+ 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-NH2-cyclic adenosine diphospho-carbocyclic-ribose, expected to antagonize Ca2+ release as demonstrated previously for 8-NH2-cyclic adenosine diphosphoribose, did not inhibit cyclic adenosine diphosphoribose mediated Ca2+ release. This indicates that the 2‘ ‘-NH2-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 CH2 in a carbocyclic system obviously interferes with a crucial element of interaction between cyclic adenosine diphosphoribose and its receptor in T-lymphocytes.