Synthetic cADPR analogues may form only one of two possible conformational diastereoisomers.

Cyclic adenosine 5'-diphosphate ribose (cADPR) is an emerging Ca2+-mobilising second messenger. cADPR analogues have been generated as chemical biology tools via both chemo-enzymatic and total synthetic routes. Both routes rely on the cyclisation of a linear precursor to close an 18-membered macrocyclic ring. We show here that, after cyclisation, there are two possible macrocyclic product conformers that may be formed, depending on whether cyclisation occurs to the "right" or the "left" of the adenine base (as viewed along the H-8 → C-8 base axis). Molecular modelling demonstrates that these two conformers are distinct and cannot interconvert. The two conformers would present a different spatial layout of binding partners to the cADPR receptor/binding site. For chemo-enzymatically generated analogues Aplysia californica ADP-ribosyl cyclase acts as a template to generate solely the "right-handed" conformer and this corresponds to that of the natural messenger, as originally explored using crystallography. However, for a total synthetic analogue it is theoretically possible to generate either product, or a mixture, from a given linear precursor. Cyclisation on either face of the adenine base is broadly illustrated by the first chemical synthesis of the two enantiomers of a "southern" ribose-simplified cIDPR analogue 8-Br-N9-butyl-cIDPR, a cADPR analogue containing only one chiral sugar in the "northern" ribose, i.e. 8-Br-D- and its mirror image 8-Br-L-N9-butyl-cIDPR. By replacing the D-ribose with the unnatural L-ribose sugar, cyclisation of the linear precursor with pyrophosphate closure generates a cyclised product spectroscopically identical, but displaying equal and opposite specific rotation. These findings have implications for cADPR analogue design, synthesis and activity.

Calcium-Mobilizing Behaviors of Neutral Cyclic ADP-Ribose Mimics that Integrate Modifications to the Nucleobase, Northern Ribose and Pyrophosphate.

Cyclic adenosine diphosphate ribose (cADPR) is an endogenous Ca2+ mobilizer involved in diverse cellular processes. Mimics of cADPR play a crucial role in investigating the molecular mechanism(s) of cADPR-mediated signaling. Here, compound 3, a mimic of cADPR in which a neutral triazole moiety and an ether linkage were introduced to substitute the pyrophosphate and "northern" ribose components, respectively, was synthesized for the first time. The pharmacological activities in Jurkat cells indicated that this mimic is capable of penetrating plasma membrane and inciting Ca2+ release from the endoplasmic reticulum (ER) through the action of ryanodine receptors (RyRs) and triggering Ca2+ influx. Furthermore, a uridine moiety was introduced in place of adenine and the new cADPR mimics 4 and 5 were synthesized. The results of biological investigation showed that these mimics also targeted RyRs and retained moderate Ca2+ agonistic activities. The results indicated that the neutral cADPR mimics had the same targets for inducing Ca2+ signaling.

Cyclic ADP-Carbocyclic-Ribose and -4-Thioribose, as Stable Mimics of Cyclic ADP-Ribose, a Ca2+-Mobilizing Second Messenger.

Cyclic ADP-ribose (cADPR), a general mediator involved in Ca2+ signaling, has the characteristic 18-membered ring consisting of an adenine, two riboses and a pyrophosphate, in which the two primary hydroxy groups of the riboses are linked by a pyrophosphate unit. This review focuses on chemical synthetic studies of cADPR analogues of biological importance. Although cADPR analogues can be synthesized by enzymatic and chemo-enzymatic methods using ADP-ribosyl cyclase, the analogues obtained by these methods are limited due to the substrate-specificity of the enzymes. Consequently, chemical synthetic methods providing a greater variety of cADPR analogues are required. Although early chemical synthetic studies demonstrated that construction of the large 18-membered ring structure is difficult, the construction was achieved using the phenylthiophosphate-type substrates by treating with AgNO3 or I2. This is now a general method for synthesizing these types of biologically important cyclic nucleotides. Using this method as the key step, the chemically and biologically stable cADPR mimic, cADP-carbocyclic-ribose (cADPcR) and -4-thioribose (cADPtR), were synthesized.

Design, Synthesis, and Identification of 4″α-Azidoethyl-cyclic ADP-Carbocyclic-ribose as a Highly Potent Analogue of Cyclic ADP-Ribose, a Ca(2+)-Mobilizing Second Messenger.

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.

Cyclic adenosine 5'-diphosphoribose (cADPR) mimics used as molecular probes in cell signaling.

Cyclic adenosine 5'-diphosphate ribose (cADPR) is a second messenger in the Ca(2+) signaling pathway. To elucidate its molecular mechanism in calcium release, a series of cADPR analogues with modification on ribose, nucleobase, and pyrophosphate have been investigated. Among them, the analogue with the modification of the northern ribose by ether linkage substitution (cIDPRE) exhibits membrane-permeate Ca(2+) agonistic activity in intact HeLa cells, human T cells, mouse cardiac myocytes and neurosecretory PC12 cell lines; thus, cIDPRE and coumarin-caged cIDPRE are valuable probes to investigate the cADPR-mediated Ca(2+) signal pathway.

A cell permeable NPE caged ADP-ribose for studying TRPM2.

Transient potential receptor melastatin-2 (TRPM2) is a non-selective Ca(2+)-permeable cation channel of the TRPM channel subfamily and is mainly activated by intracellular adenosine diphosphate ribose (ADPR). Here we synthesized a 1-(2-nitrophenyl)ethyl caged ADPR (NPE-ADPR) and found that uncaging of NPE-ADPR efficiently stimulated Ca(2+), Mg(2+), and Zn(2+) influx in a concentration-dependent manner in intact human Jurkat T-lymphocytes. The cation influx was inhibited by inhibitors or knockdown of TRPM2. Likewise, uncaging of NPE-ADPR markedly induced cation entry in HEK 293 cells that overexpress TRPM2. As expected, high temperature increased the ability of the photolyzed NPE-ADPR to induce cation entry, whereas acidic pH inhibited. Moreover, the absence of extracellular Ca(2+) significantly inhibited Mg(2+) and Zn(2+) influx after uncaging NPE-ADPR. On the other hand, the absence of extracellular Na(+) or Mg(2+) had no effect on photolyzed NPE-ADPR induced Ca(2+) entry. Taken together, our results indicated that NPE-ADPR is a cell permeable ADPR analogue that is useful for studying TRPM2-mediated cation entry in intact cells.

Synthesis and calcium mobilization activity of cADPR analogues which integrate nucleobase, northern and southern ribose modifications.

Novel cADPR mimics, which integrate nucleobase, northern and southern ribose modifications were synthesized. The key steps of the synthesis were a Cu(I)-catalyzed Hüisgen [3+2] cycloaddition and a microwave-assisted intramolecular pyrophosphorylation. Preliminary biological investigations showed that these cADPR mimics are membrane-permeating agonists of the calcium signaling pathway. The introduction of chlorine or fluorine at the 2'-position of the southern riboses led to a decrease of activity. The existence of a hydrophobic group on the 3'-OH of the southern riboses does not obviously alter the agonistic activity.

Total synthesis of a cyclic adenosine 5'-diphosphate ribose receptor agonist.

Stable cyclic adenosine 5'-diphosphate ribose (cADPR) analogues are chemical biology tools that can probe the Ca(2+) release mechanism and structure-activity relationships of this emerging potent second messenger. However, analogues with an intact "northern" ribose have been inaccessible due to the difficulty of generating the sensitive N1-ribosyl link. We report the first total synthesis of the membrane permeant, hydrolytically stable, cADPR receptor agonist 8-Br-N1-cIDPR via regio- and stereoselective N1-ribosylation of protected 8-bromoinosine.

Synthesis and use of cell-permeant cyclic ADP-ribose.

Cyclic ADP-ribose (cADPR) is a second messenger that acts on ryanodine receptors to mobilize Ca(2+). cADPR has a net negative charge at physiological pH making it not passively membrane permeant thereby requiring it to be injected, electroporated or loaded via liposomes. Such membrane impermeance of other charged intracellular messengers (including cyclic AMP, inositol 1,4,5-trisphosphate and nicotinic acid adenine dinucleotide phosphate) and fluorescent dyes (including fura-2 and fluorescein) has been overcome by synthesizing masked analogs (prodrugs), which are passively permeant and hydrolyzed to the parent compound inside cells. We now report the synthesis and biological activity of acetoxymethyl (AM) and butoxymethyl (BM) analogs of cADPR. Extracellular addition of cADPR-AM or cADPR-BM to neuronal cells in primary culture or PC12 neuroblastoma cells induced increases in cytosolic Ca(2+). Pre-incubation of PC12 cells with thapsigargin, ryanodine or caffeine eliminated the response to cADPR-AM, whereas the response still occurred in the absence of extracellular Ca(2+). Combined, these data demonstrate that masked cADPR analogs are cell-permeant and biologically active. We hope these cell-permeant tools will facilitate cADPR research and reveal its diverse physiological functions.

Aberrant cyclization affords a C-6 modified cyclic adenosine 5'-diphosphoribose analogue with biological activity in Jurkat T cells.

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.

Solid-phase synthesis of a new diphosphate 5-aminoimidazole-4-carboxamide riboside (AICAR) derivative and studies toward cyclic AICAR diphosphate ribose.

The solid-phase synthesis of the first example of a new diphosphate AICAR derivative is reported. The new substance is characterized by the presence of a 5'-phosphate group while a second phosphate moiety is installed on a 5-hydroxypentyl chain attached to the 4-N-position of AICAR. Cyclization of the diphosphate derivative by pyrophosphate bond formation allowed for the formation of a novel AICAR-based cyclic ADP-ribose (cADPR) mimic.

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.

Concise syntheses of trifluoromethylated cyclic and acyclic analogues of cADPR.

A novel trifluoromethylated analogue of cADPR, 8-CF3-cIDPDE (5) was designed and synthesized via construction of N1,N9-disubstituted hypoxanthine, trifluoromethylation and intramolecular condensation. A series of acyclic analogues of cADPR were also designed and synthesized. These compounds could be useful molecules for studying the structure-activity relationship of cADPR analogues and exploring the cADPR/RyR Ca2+ signalling system.

Trifluoromethylated cyclic-ADP-ribose mimic: synthesis of 8-trifluoromethyl-N(1)-[(5''-O-phosphorylethoxy)methyl]-5'-O-phosphorylinosine-5',5''-cyclic pyrophosphate (8-CF(3)-cIDPRE) and its calcium release activity in T cells.

A convenient trifluoromethylation method was firstly applied to the synthesis of 8- CF(3)-purine nucleosides. On the basis of this method, new protection and deprotection strategies were developed for the successful synthesis of the trifluoromethylated cyclic-ADP-ribose mimic, 8-CF(3)-cIDPRE 1. Using intact, fura-2-loaded Jurkat T cells compound 1 and 2',3'-O-isopropylidene 8-CF(3)-cIDPRE 14 were characterized as membrane-permeant cADPR agonists. Contrary to the 8-substituted cADPR analogues that mainly act as antagonists of cADPR in cells, 8-substituted cIDPRE derivatives were shown to be Ca(2+) mobilizing agonists. Here we report that even compound 1, the 8-substituted cIDPRE with the strong electron withdrawing CF(3) group, behaves as an agonist in T cells. Interestingly, also the partially protected 2',3'-O-isopropylidene 8-CF(3)-cIDPRE activated Ca(2+) signaling indicating only a minor role for the hydroxyl groups of the southern ribose of cADPR for its biological activity. To our knowledge 8-CF(3)-cIDPRE 1 is the first reported fluoro substituted cADPR mimic and 8-CF(3)-cIDPRE 1 and compound 14 are promising molecular probes for elucidating the mode of action of cADPR.

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.

Design, synthesis, and biological activity of carbocyclic analogues of cyclic ADP-ribose, a Ca2+-mobilizing second messenger.

An efficient method for the total synthesis of cyclic ADP-ribose (cADPR, 1) analogues was established. In this procedure, formation of the characteristic 18membered ring was key step, which was achieved by the AgNO3-or I2-promoted condensation with the phenylthiophosphate-type substrate forming an intramolecular pyrophosphate linkage. Using this method, a variety of carbocyclic analogues of cADPR have been synthesized to investigate the structure-activity-relationship, where cyclic ADP-carbocyclic-ribose (2) was identified as a stable and cell-type selective cADPR agonist.

Substitution at the 8-position of 3''-deoxy-cyclic ADP-carbocyclic-ribose, a highly potent Ca2+-mobilizing agent, provides partial agonists.

We previously showed that 3''-deoxy-cyclic ADP-carbocyclic-ribose (3''-deoxy-cADPcR, 4) is a stable and highly potent analogue of cyclic ADP-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. From these results, we designed and synthesized other 3''-modified analogues of cADPcR having a substituent at the 8-position and found that this modification at the 8-position made them partial agonists. Among these compounds, 8-NH(2)-3''-deoxy-cADPcR (10) was identified as a potent partial agonist with an EC(50) value of 17 nM.

Synthesis and agonist activity of cyclic ADP-ribose analogues with substitution of the northern ribose by ether or alkane chains.

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.

Synthesis of cyclic ADP-carbocylcic-xylose and its 3"-O-methyl analogue as stable and potent Ca2+ -mobilizing agents.

We previously showed that 3"-deoxy-cyclic ADP-carbocyclic-ribose (3"-deoxy-cADPcR, 3) is a stable and highly potent analogue of cyclic ADP-ribose (cADPR, 1), a Ca2+ -mobilizing second messenger. From these results, we newly designed another 3"-modified analogues of cADPcR and identified the N1-"xylo"-type carbocyclic analogue, i.e., cADPcX (4), as one of the most potent cADPR-related compounds reported so far.