Journal of Medicinal Chemistry J2′,3′ = 6.0 Hz and J2′,1′ = 2.7 Hz, H-2′), 5.04 (dd, 1H, J3′,2′ = 6.0 Hz and J3′,4′ = 2.4 Hz, H-3′), 4.55−4.54 (m, 1H, H-4′), 4.16−4.12 (m, 2H, H-5′), 2.03 (s, 3H, CH3), 1.63 (s, 3H, CH3), 1.44 (s, 9H, tBu), and 1.40 (s, 9H, tBu); 31P (decoupled, 109 MHz, CDCl3) δ −9.3 (s); HRMS (ES+) calcd for C27H36N6O11PS 683.1895 (MH+), found 683.1871; Rf = 0.14 (hexane/EtOAc, 6:4). 2′,3′-O-Isopropylidene-5′-O-(di-tert-butylphosphoramidite)- 6-thioinosine. 16 (80 mg, 0.116 mmol) was stirred in a solution of MeCN (10 mL) containing 10% (v/v) 2-mercaptoethanol (1 mL) and 1% DIPEA (0.1 mL) for 1 h. Water (15 mL) and DCM (30 mL) were then added. The organic layer was washed several times with water to remove the excess 2-mercaptoethanol. The organic phase was dried, filtered, and evaporated in vacuo to leave an oil which was purified by column chromatography on silica gel (DCM/acetone, 7:3) to give the title compound as a white waxy solid (45 mg, 75%). 1H (270 MHz, CDCl3) δ 8.38 (s, 1H, H-2), 8.36 (s, 1H, H-8), 6.14 (d, 1H, J1′,2′ = 2.2 Hz, H-1′), 5.52 (dd, 1H, J2′,3′ = 6.1 Hz and J2′,1′ = 2.1 Hz, H-2′), 5.04 (dd, 1H, J3′,2′ = 6.1 Hz and J3′,4′ = 2.5 Hz, H-3′), 4.56−4.54 (m, 1H, H-4′), 4.23−4.17 (m, 2H, H-5′), 1.63 (s, 3H, CH3), 1.44 (s, 18H, 2 × tBu), and 1.42 (s, 3H, CH3); 31P (decoupled, 109 MHz, CDCl3) δ −9.3 (s); HRMS (ES+) calcd for C21H34N4O7PS 517.1880 (MH+), found 517.1863. 6-Thioinosine 5′-Monophosphate (6-SH IMP, 14). 2′,3′-O- Isopropylidene-5′-O-(di-tert-butylphosphoramidite)-6-thioinosine (35 mg, 0.067 mmol) was stirred in a 50% aqueous TFA solution (2 mL) at room temperature for 24 h. The solvent was removed under reduced pressure and coevaporated several times with MeOH to remove any residual TFA. The residue was dissolved in water and worked up with EtOAc (2 × 10 mL). The aqueous layer was evaporated to dryness to produce the desired monophosphate 14 as a glassy solid (21 mg, 86%). HPLC, tR = 2.9 min at 320 nm; 1H (270 MHz, D2O) δ 8.94 (s, 1H, H-2), 8.23 (s, 1H, H-8), 6.05 (d, 1H, J1′,2′ = 3.9 Hz, H-1′), 4.60 (dd, 1H, J2′,3′ = 4.7 Hz and J2′,1′ = 3.9 Hz, H-2′), 4.41 (app t, 1H, J = 5.0 Hz, H-3′), 4.27 (dd, 1H, J4′,3′ = 5.0 Hz and J4′,5′a = 2.5 Hz, H-4′), 4.17 (ddd, 1H, J5′a,5′b = 11.8 Hz, J5′a,P = 4.4 Hz, and J5″a,4 = 2.5 Hz, H- 5′a), and 4.05 (ddd, 1H, J5′a,5′b = 11.8 Hz, J5′a,P = 5.5 Hz, and J5″a,4 = 2.8 Hz, H-5′b); 31P (decoupled, 109 MHz, D2O) δ 0.51 (s); HRMS (ES+) calcd for C10H14N4O7PS 365.0315 (MH+), found 365.0310. Nicotinamide 6-Mercaptopurine 5′-Dinucleotide (6-SH NHD+, 17). 14 (13 mg, 0.036 mmol) was dissolved in dry DMSO (1 mL) and coevaporated with dry DMF (5 × 3 mL). The residue was dissolved in DMSO (400 μL), to which were added morpholine (16 μL, 0.187 mmol), dipyridyl disulfide (19 mg, 0.089 mmol), and triphenylphosphine (24 mg, 0.089 mmol), at which point the solution became bright yellow. It was stirred for 1 h at room temperature, after which HPLC analysis showed completion of the reaction. Precipitation of the product occurred by dropwise addition of a solution of NaI in acetone (0.1M, 8 mL). The resulting precipitate was filtered, washed with acetone, and dried (δP 6.7 ppm and HPLC, tR = 5.9 min at 320 nm). It was then reacted with β-NMN+ (17 mg, 0.050 mmol) and MgSO4 (11 mg, 0.092 mmol) in a 0.2 M solution of MnCl2 in form- amide (0.35 mL) at room temperature overnight, after which HPLC analysis showed completion of the reaction (tR(β‑NMN) = 2.1 min and tR(6‑SH‑NHD) = 7 min). Precipitation occurred by dropwise addition of MeCN. The precipitate was filtered, dissolved in Milli-Q, and applied to a reverse-phase column, eluting with a gradient of MeCN in 0.05 M TEAB. Further treatment with Chelex 100 to remove any para- magnetic particles afforded the desired dinucleotide 17 as a glassy solid (10 mg, 15.2 μmol, 31% from 6-thio IMP). HPLC, tR = 6.9 min at 320 nm; 1H (270 MHz, D2O) δ 9.17 (s, 1H, HN2), 8.96 (d, 1H, J6,5 = 6.1 Hz, HN6), 8.65 (d, 1H, J4,5 = 7.7 Hz, HN4), 8.30 (s, 1H, H-2), 8.08 (s, 1H, H-8), 8.02−7.96 (m, 1H, HN5), 6.03 (d, 1H, J1″,2″ = 6.5 Hz, H-1″), 5.86 (d, 1H, J1′,2′ = 5.8 Hz, H-1′), 4.65 (app t, 1H, J2′,1′ = J2′,3′ = 5.5 Hz, H-2′), and 4.41−4.07 (m, 9H, Hsugar); 31P (decoupled, 109 MHz, D2O) δ −10.5 (d, J = 19.7) and −10.8 (d, J = 19.7); HRMS (ES+) calcd for C21H27N6O14P2S 681.0776 (MH+), found 681.0752; UV (H2O, pH 7.3) λmax 322 nm (ε/dm3 mol−1 cm−1 20 320). Cyclic 6-Thioinosine 5′-Diphosphate Ribose (6-Thio cIDPR, 3). 17 (13.5 μmol) was incubated with Aplysia cyclase (150 μL) in a 0.1 M NaHCO3 buffer (30 mL, pH 7.6) at room temperature. After 30 h, ■ ■ ■ Article HPLC analysis showed total consumption of starting material and formation of a new large peak at 11 min. The water was evaporated to a minimum, and the residue was applied on a RP-18 column, eluting with a gradient of MeCN in 0.05 M TEAB (0−65% over 300 mL). The product came through with 18% MeCN. The appropriate frac- tions were collected and evaporated under reduced pressure. The residue was passed through a small Chelex column previously washed with Milli-Q water to bring the pH down to 8. The product was washed off with Milli-Q water. It was then lyophilized to afford the desired cyclic dinucleotide as its sodium salt (7 μmol, 52%). HPLC, tR = 10.7 min at 320 nm; 1H (270 MHz, D2O) δ 9.32 (s, 1H, H-2), 8.29 (s, 1H, H-8), 6.65 (br s, 1H, H-1″), 5.99 (d, 1H, J1′,2′ = 6.3 Hz, H-1′), 5.20 (dd, 1H, J2′,1′ = 6.3 Hz and J2′,3′ = 5.0 Hz, H-2′), 4.64 (dd, 1H, J3′,2′ = 5.0 Hz and J3′,4′ = 2.2 Hz, H-3′), and 4.59−4.07 (m, 8H, H-4′, H-5′, H-2″, H-3″, H-4″, and H-5″); 31P (decoupled, 109 MHz, D2O) δ −9.2 (d, AB system, J = 11.8 Hz), −10.5 (d, AB system, J = 11.8 Hz); HRMS (ES−) calcd for C15H19N4O13P2S 557.0150 (MH−), found 557.0161; UV (H2O, pH 7.2). λmax 322 nm (ε/dm3 mol−1 cm−1 18 600). ■ ASSOCIATED CONTENT *S Supporting Information Full NMR characterization spectra, fluorescence spectrum, HPLC traces, computational data for target compound 3, and 13C NMR data for all compounds. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Phone: ++44-1225-386639. Fax: ++44-1225-386114. E-mail: B.V.L.Potter@bath.ac.uk. 1487 dx.doi.org/10.1021/jm201127y | J. Med. Chem. 2012, 55, 1478−1489 ACKNOWLEDGMENTS We thank the Wellcome Trust for Project Grant 084068 (to B.V.L.P. and A.H.G.) and Program Grant 082837 (to B.V.L.P.). Work in the Guse lab is supported by the Deutsche Forschungsgemeinschaft (DFG) and the Deutscher Akade- mischer Austauschdienst (DAAD). ■ ABBREVIATIONS USED cADPR, cyclic adenosine 5′-diphosphoribose; cIDPR, cyclic inosine 5′-diphosphoribose; cADPcR, cyclic adenosine 5′- diphosphocarbocyclic ribose; cIDPRE, N1‑[(2″-O-phosphoryl- ethoxy)methyl]-5′-O-phosphorylinosine 2″,5′-cyclic pyrophos- phate; NAD+, nicotinamide adenosine 5′-dinucleotide; 6-NMe + NAD , 6-N-Methyl nicotinamide adenosine 5′-dinucleotide; + + NGD , nicotinamide guanosine 5′-dinucleotide; NHD , nic- otinamide hypoxanthine 5′-dinucleotide; 6-thio NHD+, nicotina- mide 6-mercaptopurine 5′-dinucleotide; ADPRC, ADP-ribosyl cyclase; TEP, triethylphosphate; β-NMN + , β-nicotinamide 5′- mononucleotide; CDI, carbonyldiimidazole; TEAB, triethylammo- nium bicarbonate; IMP, inosine 5′-monophosphate; ribo-2′-F- NAD+, ribosyl-2′-fluoro-2′-deoxy-nicotinamide adenine dinucleotide REFERENCES (1) Clapper, D. L.; Walseth, T. F.; Dargie, P. J.; Lee, H. C. Pyridine- nucleotide metabolites stimulate calcium release from sea-urchin egg microsomes desensitized to inositol trisphosphate. J. Biol. Chem. 1987, 262, 9561−9568. (2) Dargie, P. J.; Agre, M. C.; Lee, H. C. Comparison of Ca2+ mobilizing activities of cyclic ADP-ribose and inositol trisphosphate. Cell Regul. 1990, 1, 279−290. (3) Guse, A. H. Biochemistry, biology, and pharmacology of cyclic adenosine diphosphoribose (cADPR). Curr. Med. Chem. 2004, 11, 847−855.