Total synthesis of cyclic ADP-carbocyclic-ribose, a stable mimic of Ca2+-mobilizing second messenger cyclic ADP-ribose

The synthesis of cyclic ADP-carbocyclic-ribose (cADPcR, 4) designed as a stable mimic of cyclic ADP-ribose (cADPR, 1), a Ca2+-mobilizing second messenger, was achieved using as the key step a condensation reaction with the phenylthiophosphate-type substrate 14 to form an intramolecular pyrophosphate linkage. The N-1-carbocyclic-ribosyladenosine derivative 16 was prepared via the condensation between the imidazole nucleoside derivative 17, prepared from AICA-riboside (19), and the readily available optically active carbocyclic amine 18. Compound 16 was then converted to the corresponding 5' '-phosphoryl-5'-phenylthiophosphate derivatives 14. Treatment of 14 with AgNO3 in the presence of molecular sieves (3 A) in pyridine at room temperature gave the desired cyclization product 32 in 93% yield, and subsequent acidic treatment provided the target cADPcR (4). This represents a general method for synthesizing biologically important cyclic nucleotides of this type. 1H NMR analysis of cADPcR suggested that its conformation in aqueous medium is similar to that of cADPR. cADPcR, unlike cADPR, was stable under neutral and acidic conditions, where under basic conditions, it formed the Dimroth-rearranged N6-cyclized product 34. cADPcR was also stable in rat brain membrane homogenate which has cADPR degradation activity. Furthermore, cADPcR was resistant to the hydrolysis by CD38 cADPR hydrolase, while cADPR was rapidly hydrolyzed under the same conditions. When cADPcR was injected into sea urchin eggs, it caused a significant release of Ca2+ in the cells, an effect considerably stronger than that of cADPR. Thus, cADPcR was identified as a stable mimic of cADPR.     

Design and synthesis of 4″,6″-unsaturated cyclic ADP-carbocyclic-ribose, a Ca-mobilizing agent selectively active in T cells

We previously developed cyclic ADP-carbocyclic-ribose (cADPcR, 3a) as a stable mimic of cyclic ADP-ribose (cADPR, 1), a Ca²⁺-mobilizing second messenger. The unsaturated carbocyclic-ribose analogs of cADPR, i.e., 4″,6″-didehydro-cADPcR (8a) and its inosine congener 4″,6″-didehydro-cIDPcR (8b) were newly designed and successfully synthesized using the key intramolecular condensation reaction with S-phenyl phosphorothioate-type substrates. The Ca²⁺-mobilizing potency of the compounds was examined in sea urchin egg homogenates, NG108-15 neuronal cells, and permeabilized Jurkat T-lymphocytes, which may indicate that 4″,6″-didehydro-cADPcR is the first cADPR analog selectively active in T cells. Acid-base behavior and conformation of 8a were also investigated and compared with those of cADPcR.     

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.     

High throughput fluorescence-based assays for cyclic ADP-ribose,NAADP, and their metabolic enzymes

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are two novel Ca(2+) messengers derived respectively from NAD and NADP. Since their discovery in sea urchin eggs, both have now been shown to serve messenger functions in a wide range of cells from plant to human. In this article, a series of fluorimetric assays for cADPR, NAADP and their metabolic enzymes is compiled. The enzyme assay makes use of an analog of NAD, nicotinamide guanine dinucleotide, which is non-fluorescent but is cyclized by the enzymes to a fluorescent analog of cADPR, cyclic GDP-ribose. Other NAD utilizing enzymes are not capable of catalyzing the cyclization and thus produce no interference. The fluorimetric assays for cADPR and NAADP make use of coupled-enzyme amplification and can readily detect nanomolar concentrations of either messenger. All the assays described can be performed in multi-well format, allowing ready automation and use in high throughput screening. An added advantage of these assays is that all the required reagents are commercially available, facilitating general adoption of the techniques by all those who are interested in the physiology and enzymology of the novel Ca(2+) signaling pathways mediated by cADPR and NAADP.     

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.     

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.     

Discovery of a small-molecule inhibitor for kidney ADP-ribosyl cyclase: Implication for intracellular calcium signal mediated by cyclic ADP-ribose

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca2+-mobilizing second messenger, cyclic ADP- ribose (cADPR), from beta-NAD+. A prototype of mammalian ADPR-cyclases is a lymphocyte antigen CD38. Accumulating evidence indicates that ADPR-cyclases other than CD38 are expressed in various cells and organs. In this study, we discovered a small molecule inhibitor of kidney ADPR-cyclase. This compound inhibited kidney ADPR-cyclase activity but not CD38, spleen, heart or brain ADPR-cyclase activity in vitro. Characterization of the compound in a cell-based system revealed that an extracellular calcium-sensing receptor (CaSR)- mediated cADPR production and a later long-lasting increase in intracellular Ca2+ concentration ([Ca2+]i) in mouse mesangial cells were inhibited by the pre-treatment with this compound. In contrast, the compound did not block CD3/TCR-induced cADPR production and the increase of [Ca2+]i in Jurkat T cells, which express CD38 exclusively. The long-lasting Ca2+ signal generated by both receptors was inhibited by pre-treatment with an antagonistic cADPR derivative, 8-Br-cADPR, indicating that the Ca2+ signal is mediated by the ADPR-cyclase metabolite, cADPR. Moreover, among structurally similar compounds tested, the compound inhibited most potently the cADPR production and Ca2+ signal induced by CaSR. These findings provide evidence for existence of a distinct ADPR-cyclase in the kidney and basis for the development of tissue specific inhibitors.     

Design and synthesis of 4″,6″-unsaturated cyclic ADP-carbocyclic ribose as a Ca-mobilizing agent

4″,6″-Didehydro-cADPcR (3), an unsaturated carbocyclic ribose analog of a Ca²⁺-mobilizing second messenger cyclic ADP-ribose (cADPR), was designed and successfully synthesized using a key intramolecular condensation reaction forming the 18-membered pyrophosphate ring structure with a S-phenyl phosphorothioate-type substrate. Biological evaluation showed that 4″,6″-didehydro-cADPcR is a potent Ca²⁺-mobilizing agent in T cells.     

环腺苷二磷酸核糖类似物的合成、表征及性质

通过次黄嘌呤N1-位取代及分子内环合等反应, 合成了由带芳基支链的含氮链替代天然北区核糖结构的环腺苷二磷酸核糖(cADPR)类似物cIDPRN. 该化合物与Jurkat T淋巴细胞在37 ℃下孵育18 h后, 经毛细管电泳分析, 结果表明该化合物具有良好的稳定性. 荧光分光光度计测定, 结果表明, 在有钙离子和无钙离子环境下, 该化合物胞外给药后均能引起浓度依赖性的钙离子释放. 由以上结果确定该化合物为具有膜透性的促细胞内钙释放激动剂.     

Synthesis of Modified Di- and Trisaccharide Fragments of N-Glycoproteins

Abstract

The syntheses of several analogues of disaccharide Manot(1→6)Mana-OCH₃ (1) and of trisaccharide Mana(1→6)[Mana(1→3)]Mana-OCH₃ (2) are reported. The syntheses are described of the diastereomeric 6-methyl derivatives 9a and 9b, which are representatives of fixed conformations of disaccharide 1. The syntheses of the 2-amino-2-deoxy analogues 15 and 17 and the synthesis of the 2-fluoro-2-deoxy analogue 28 are also reported.     

Small Molecule CD38 Inhibitors: Synthesis of 8-Amino-N1-inosine 5′-monophosphate,Analogues and Early Structure-Activity Relationship

Although a monoclonal antibody targeting the multifunctional ectoenzyme CD38 is an FDA-approved drug, few small molecule inhibitors exist for this enzyme that catalyzes inter alia the formation and metabolism of the N1-ribosylated, Ca²⁺-mobilizing, second messenger cyclic adenosine 5′-diphosphoribose (cADPR). N1-Inosine 5′-monophosphate (N1-IMP) is a fragment directly related to cADPR. 8-Substituted-N1-IMP derivatives, prepared by degradation of cyclic parent compounds, inhibit CD38-mediated cADPR hydrolysis more efficiently than related cyclic analogues, making them attractive for inhibitor development. We report a total synthesis of the N1-IMP scaffold from adenine and a small initial compound series that facilitated early delineation of structure-activity parameters, with analogues evaluated for inhibition of CD38-mediated hydrolysis of cADPR. The 5′-phosphate group proved essential for useful activity, but substitution of this group by a sulfonamide bioisostere was not fruitful. 8-NH₂-N1-IMP is the most potent inhibitor (IC₅₀ = 7.6 μM) and importantly HPLC studies showed this ligand to be cleaved at high CD38 concentrations, confirming its access to the CD38 catalytic machinery and demonstrating the potential of our fragment approach.     

Synthetic studies of furanosesquiterpenoid tetrahydrolinderazulenes. Total synthesis of (±)-echinofuran

Echinofuran (1) was isolated in 1992 and was reported to inhibit cell division of fertilized sea urchin eggs. The synthesis of 1 was achieved by a Ring A→Ring AC→Ring ABC approach employing 3-methyl-4-(trimethylsilyl)furan (2) as a precursor. A Suzuki coupling reaction and a Lewis acid mediated Friedel-Crafts cyclization were the other key steps in the construction of the ring systems. In other preliminary model studies, two furan-containing 5,7,5-fused tricyclic molecules were also realized.     

Robust synthesis of enantiopure cyclohexenyl analogues of 2/3-deoxyribose sugars as carbocyclic nucleoside precursors

An expedient synthesis of 2-deoxy (10) and 3-deoxy (11) cyclohexenyl analogues of 2-deoxy and 3-deoxy-d-ribose sugar from commercially available starting materials is reported. Highly efficient enzymatic resolution of the key compound 10 is described using lipase under hydrolytic conditions. The robust methodology applied here will be useful to synthesize cyclohexenyl nucleosides, which possess potent antiviral activity and are capable of gene silencing via RNAi or antisense applications.     

Cell-permeant small-molecule modulators of NAADP-mediated Ca2+ release

Nicotinic acid adenine dinucleotide phosphate (NAADP, 1) is the most potent intracellular Ca2+ mobilizing agent in important mammalian cells and tissues, yet the identity of the NAADP receptor is elusive. Significantly, the coenzyme NADP is completely inactive in this respect. Current studies are restricted by the paucity of any chemical probes beyond NAADP itself, and importantly, none is cell permeant. We report simple nicotinic acid-derived pyridinium analogs as low molecular weight compounds that (1) inhibit Ca2+ release via the NAADP receptor (IC50 approximately 15 microM - 1 mM), (2) compete with NAADP binding, (3) cross the cell membrane of sea urchin eggs to inhibit NAADP-evoked Ca2+ release, and (4) selectively ablate NAADP-dependent Ca2+ oscillations induced by the external gastric peptide hormone agonist cholecystokinin (CCK) in murine pancreatic acinar cells.     

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.     

Rapid synthetic route toward structurally modified derivatives of cyclic adenosine 5'-diphosphate ribose

A concise synthesis of five new analogues of the second messenger cADPR (cyclic adenosine 5'-diphosphate ribose) is presented. The synthetic plan centered around the key derivative 8-Br-N1-cIDPR (cyclic 8-Br-inosine 5'-diphosphate ribose, 2), which was prepared in only three steps from IMP (inosine 5'-monophosphate) via an unusual enzymatic cyclization reaction. The enhanced stability of 2 allowed for the direct modification of this cyclic dinucleotide at the 8 position, providing the unsubstituted parent N1-cIDPR (4) as well as the 8-phenyl (5), 8-azido (6), and 8-amino (7) N1-cIDPR analogues. In Jurkat T-lymphocytes, N1-cIDPR 4 induced Ca2+ release with an almost identical profile as the natural agonist cADPR, illustrating the value of this approach.     

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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ signal pathway.     

Influence of calcium-induced aggregation on the sensitivity of aminobis(methylenephosphonate)-containing potential MRI contrast agents

A novel class of 1,4,7,10-tetraazacyclododecane-1,4,7-tris(methylenecarboxylic) acid (DO3A)-based lanthanide complexes with relaxometric response to Ca(2+) was synthesized, and their physicochemical properties were investigated. Four macrocyclic ligands containing an alkyl-aminobis(methylenephosphonate) side chain for Ca(2+)-chelation have been studied (alkyl is propyl, butyl, pentyl, and hexyl for L(1), L(2), L(3), and L(4), respectively). Upon addition of Ca(2+), the r(1) relaxivity of their Gd(3+) complexes decreased up to 61% of the initial value for the best compounds GdL(3) and GdL(4). The relaxivity of the complexes was concentration dependent (it decreases with increasing concentration). Diffusion NMR studies on the Y(3+) analogues evidenced the formation of agglomerates at higher concentrations; the aggregation becomes even more important in the presence of Ca(2+). (31)P NMR experiments on EuL(1) and EuL(4) indicated the coordination of a phosphonate to the Ln(3+) for the ligand with a propyl chain, while phosphonate coordination was not observed for the analogue bearing a hexyl linker. Potentiometric titrations yielded protonation constants of the Gd(3+) complexes. log K(H1) values for all complexes lie between 6.12 and 7.11 whereas log K(H2) values are between 4.61 and 5.87. Luminescence emission spectra recorded on the Eu(3+) complexes confirmed the coordination of a phosphonate group to the Ln(3+) center in EuL(1). Luminescence lifetime measurements showed that Ca-induced agglomeration reduces the hydration number which is the main cause for the change in r(1). Variable temperature (17)O NMR experiments evidenced high water exchange rates on GdL(1), GdL(2), and GdL(3) comparable to that of the aqua ion.     

Synthesis of unnatural 3′-phospha-2′-deoxyfuranose nucleoside analogues

This Letter describes the synthesis of racemic analogues of unnatural 2′-deoxy nucleoside with a phosphorus atom replacing the carbon atom in the 3′-position. A seven-step sequence was developed in racemic series to afford unnatural 3′-phospha-2′-deoxyfuranose nucleosides. The phospha nucleoside analogues were tested against HCV, but did not show any antiviral activity at a 10 μM maximum concentration used for the inhibition assays of analogues 2-T, 2-C and 4-Tα.     

Isolated cortical granules: a model system for studying membrane fusion and calcium-mediated exocytosis.

Cortical granules are secretory vesicles bound to the inner surface of the plasma membrane of sea urchin eggs. Intact granules can be isolated by shearing away the cytoplasm of eggs which have been bonded to a protamine-coated surface. When Ca2+ is added to preparations of isolated granules the granules fuse with each other and release their contents. It is believed that isolated cortical granules may be an excellent model system for the biochemical study of exocytosis.