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Cyclic adenosine 5'-diphosphate ribose (cADPR) is a ubiquitous Ca 2+ -releasing second messenger. Knowledge of its conformational landscape is an essential tool for unraveling the structure-activity relationship (SAR) in cADPR. Variable-temperature 1 H NMR spectroscopy, in conjunction with PSEUROT and population analyses, allowed us to determine the conformations and thermodynamic parameters of the furanose rings, γ-bonds (C4'-C5'), and β-bonds (C5'-O5') in the cADPR analogues 2'-deoxy-cADPR, 7-deaza-cADPR, and 8-bromo-cADPR. A significant finding was that, although the analogues are similar to each other and to cADPR itself in terms of overall conformation and population (ΔG°), there were subtle yet important differences in some of thermodynamic properties (ΔH°, ΔS°) associated with each of the conformational equilibria. These differences prompted us to propose a model for cADPR in which the interactions between the A2'-N3, A5″-N3, and H2-R5' atoms serve to fine-tune the N-glycosidic torsion angles (χ).

A series of 8-substituted analogues of cyclic ADP-4-thioribose (cADPtR, 3), which is a stable equivalent of Ca 2+ -mobilizing second messenger cyclic ADP-ribose (cADPR, 1), were designed as potential pharmacological tools for studies on cADPR-modulated Ca 2+ signaling pathways. These 8-amino analogue (8-NH 2 -cADPtR, 4), 8-azido analogue (8-N 3 -cADPtR, 5), and 8-chloro analogue (8-Cl-cADPtR, 6) were efficiently synthesized, where the stereoselective N1-β-thioribosyladenine ring closure reaction via an α/β-equilibrium of the 1-aminothioribose derivative and construction of the characteristic 18-membered pyrophosphate ring by Ag + -promoted activation of a phenyl phosphorothioate type substrate were the two key steps. Although 8-NH 2 -cADPR (2) is a well-known potent antagonist against cADPR-inducing Ca 2+ -release, the 4-thioribose congener 8-NH 2 -cADPtR turned out unexpectedly to be a full agonist in sea urchin egg homogenate evaluation system. This important finding suggested that the ring-oxygen in the N1-ribose of cADPR analogues is essential for the antagonistic activity in the Ca 2+ -signaling pathway, which can contribute to clarify the structure-agonist/antagonist activity relationship.

Cyclic adenosine diphosphate-carbocyclic-ribose (cADPcR, 2) is a stable equivalent of cyclic adenosine diphosphate-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. On the basis of the structure-activity relationship of cADPR-related compounds and three-dimensional structural modeling of cADPcR, we designed and synthesized cyclic-ADP-4″α-azidoethyl carbocyclic-ribose (N3-cADPcR, 3) to demonstrate that it has a highly potent Ca(2+)-mobilizing activity (EC50 = 24 nM). N3-cADPcR will be a useful precursor for the preparation of biological tools effective to investigate cADPR-mediated signaling pathways.

Two nicotinamide adenine dinucleotide (NAD(+)) analogues modified at the 6 position of the purine ring were synthesized, and their substrate properties toward Aplysia californica ADP-ribosyl cyclase were investigated. 6-N-Methyl NAD(+) (6-N-methyl nicotinamide adenosine 5'-dinucleotide 10) hydrolyzes to give the linear 6-N-methyl ADPR (adenosine 5'-diphosphoribose, 11), whereas 6-thio NHD(+) (nicotinamide 6-mercaptopurine 5'-dinucleotide, 17) generates a cyclic dinucleotide. Surprisingly, NMR correlation spectra confirm this compound to be the N1 cyclic product 6-thio N1-cIDPR (6-thio cyclic inosine 5'-diphosphoribose, 3), although the corresponding 6-oxo analogue is well-known to cyclize at N7. In Jurkat T cells, unlike the parent cyclic inosine 5'-diphosphoribose N1-cIDPR 2, 6-thio N1-cIDPR antagonizes both cADPR- and N1-cIDPR-induced Ca(2+) release but possesses weak agonist activity at higher concentration. 3 is thus identified as the first C-6 modified cADPR (cyclic adenosine 5'-diphosphoribose) analogue antagonist; it represents the first example of a fluorescent N1-cyclized cADPR analogue and is a new pharmacological tool for intervention in the cADPR pathway of cellular signaling.

The structural features needed for antagonism at the cyclic ADP-ribose (cADPR) receptor are unclear. Chemoenzymatic syntheses of novel 8-substituted 2'-deoxy-cADPR analogues, including 8-bromo-2'-deoxy-cADPR 7, 8-amino-2'-deoxy-cADPR 8, 8- O-methyl-2'-deoxy-cADPR 9, 8-phenyl-2'-deoxy-cADPR 10 and its ribose counterpart 8-phenyl-cADPR 5 are reported, including improved syntheses of established antagonists 8-amino-cADPR 2 and 8-bromo-cADPR 3. Aplysia californica ADP-ribosyl cyclase tolerates even the bulky 8-phenyl-nicotinamide adenine 5'-dinucleotide as a substrate. Structure-activity relationships of 8-substituted cADPR analogues in both Jurkat T-lymphocytes and sea urchin egg homogenate (SUH) were investigated. 2'-OH Deletion decreased antagonistic activity (at least for the 8-amino series), showing it to be an important motif. Some 8-substituted 2'-deoxy analogues showed agonist activity at higher concentrations, among which 8-bromo-2'-deoxy-cADPR 7 was, unexpectedly, a weak but almost full agonist in SUH and was membrane-permeant in whole eggs. Classical antagonists 2 and 3 also showed previously unobserved agonist activity at higher concentrations in both systems. The 2'-OH group, without effect on the Ca (2+)-mobilizing ability of cADPR itself, is an important motif for the antagonistic activities of 8-substituted cADPR analogues.

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 analogues of cADPR with adenine as base and ether (10a) or different alkane chain (10b-d) substitutions of the northern ribose were synthesized from protected imidazole nucleoside 1 in good yields. The pharmacological activities of cyclic inosine diphosphoribose ether (cIDPRE) and the compounds (10a-d) were analyzed in intact human Jurkat T-lymphocytes. The results indicate that the analogues 10a-d permeate the plasma membrane and are weak agonists of the cADPR/ryanodine receptor signaling system in intact human Jurkat T cells. They are the first membrane-permeant and biologically active cADPR analogues that contain ether or alkane bridges instead of the northern ribose and retain adenine as its base.

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.

Analogues of cyclic ADP-ribose (cADPR) incorporating a methylenebisphosphonate linkage in the place of the pyrophosphate have been synthesized from nicotinamide adenine dinucleotide analogues enzymatically using Aplysia californica ADP-ribosyl cyclase. Methylenebisphosphonate cyclic ADP-ribose (cADPR[CH(2)]) and methylenebisphosphonate cyclic 3-deaza-ADP-ribose (3-deaza-cADPR[CH(2)]) showed full agonist activity for release of Ca(2+) ions from sea urchin egg homogenates. The EC(50) for cADPR[CH(2)] was 856 nM and that for 3-deaza-cADPR[CH(2)] was 300 nM, about 15- and 5-fold less potent than cADPR, respectively.

Cyclic ADP-carbocyclic-ribose (cADPcR, 2) is a biologically and chemically stable equivalent of cyclic ADP-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. In this study, a series of 8-substituted analogues of cADPcR, namely the 8-chloro analogue 6 (8-Cl-cADPcR), the 8-azido analogue 7 (8-N(3)-cADPcR), the 8-amino analogue 8 (8-NH(2)-cADPcR), and the 8-phenylthio analogue 9 (8-SPh-cADPcR), were designed as effective pharmacological tools for studies on cADPR-modulated Ca(2+) signaling pathways. These target compounds were synthesized by a convergent route via 8-Cl-cADPcR bisacetonide (14) as the common intermediate, in which a method for forming the intramolecular pyrophosphate linkage by activation of the phenylthiophosphate type substrate 15 with AgNO(3) to produce 14 was used as the key step. The carbocyclic analogues were tested for activity in the sea urchin egg homogenate system. Compounds were assessed for their calcium-mobilizing effects and their ability to cross-desensitize with calcium release induced by a normally maximal concentration of cADPR, as well as cADPR antagonism of cADPR-evoked calcium release. While cADPcR was 3-4 times more potent than cADPR, the 8-substituted analogues were less efficacious, with 8-SPh-cADPcR largely acting as a competitive antagonist. Most surprisingly, given that 8-N(3)-cADPR and 8-NH(2)-cADPR are known as potent antagonists, 8-N(3)-cADPcR and 8-NH(2)-cADPcR were full agonists, but ca. 80 and 2 times less potent than cADPR, respectively. These data contribute to developing structure-activity relationships for the interaction of cADPR with its receptor.

Cyclic ADP-ribose (cADPR) is not only a potent endogenous calcium modulator but also a second messenger. However, studies on the mechanism of cADPR action were limited due to its instability and lack of available structural modifications in the N1-glyosyl unit of cADPR. In the present work, a series of N1-glycosyl mimics with different configurational glycosyls or an ether strand were designed and synthesized mimicking the furanose ring. S(N)2 substitutions were carried out between the protected inosine and glycosyl triflates to form the N1-glycosylinosine derivatives, accompanied with some O6-glycosyl-substituted as side products. The intramolecular cyclization was followed the strategy described by Matsuda et al. It was found that the 8-unsubstituted substrate could also be used to construct the intramolecular cyclic pyrophosphate. The activities of N1-glycosyl-substituted cADPR mimics were evaluated by induced Ca2+ release in rat brain microsomes and HeLa cells. It was found that the configuration of the N1-glycosyl moiety in cADPR is not a critical structural factor for retaining the activity of mobilizing Ca2+ release. More interestingly, the N1-acyclic analogue 6 exhibited strong activity by inducing Ca2+ release in both rat brain microsomes and HeLa cells. It constitutes a useful tool for further studies.