Journal of Medicinal Chemistry proton H-1″ (δ 6.65 ppm) with the carbon C-2 (δ 144.9 ppm) and C-6 (δ 175.5 ppm) of the nucleobase (Figure 3). Finally, the 31P−31P coupling constant for the pyrophosphate linkage in 3 showed J = 11.8 Hz, similar to that of cADPR and cIDPR (13.5 53 and 12.5 30 Hz, respectively), whereas the 31P−31P cou- pling of the linear IDPR generally has a much higher frequency (∼20 Hz). These analytical data thus provide evidence of a successful and surprising cyclization of 6-thio NHD+ into the corresponding 6-thio N1 cIDPR 3 (Figure 4). Figure 4. 1H/13C correlations based on gHMBC spectrum supporting the formation of the N1−C1″ bond. The photochemical properties of 6-thio N1-cIDPR 3 were also examined. While adenine and guanosine based nucleotides have an absorbance maximum of around 260 nm, replacement of the oxygen by a sulfur atom shifts the UV absorbance spec- trum to 320−340 nm. 6-Thio cIDPR seems to be no exception. The UV spectrum in water exhibited intense absorption at 320 nm (λmax) with an extinction coefficient (ε) of 18 600 mol−1 dm3 cm−1 (data not shown). A smaller peak at 267 nm was also observed. More interestingly, 6-thio cIDPR displays fluorescent properties in water at room temperature. When it is excited at 335 nm (just above the maximum UV absorption), an emission spectrum with a single peak at 415 nm was ob- served (see Supporting Information). In contrast, no fluores- cence was observed by the related N1-cIDPR (data not shown). Thus far, only N7-cyclized dinucleotides such as N7-cIDPR, N7-cGDPR, and etheno cyclic ADP diphosphoribose have been demonstrated to be fluorescent, and this property was used to rule out the formation of N7-cyclic product when no fluores- cence was observed.33 To the best of our knowledge, 6-thio N1-cIDPR is therefore the first fluorescent N1-cyclized cADPR analogue and this property may find applications in cADPR binding protein biochemistry. Conformational Analysis. There have been reports that the Ca2+-release activity and antagonism54,55 of cADPR ana- logues may be linked to their conformation in solution. The major puckering mode of cADPR is C2′ endo in the N9 ribose moiety with a syn conformation about the N9 glycosidic bond.6 The furanose ring is in equilibrium between C2′ endo/C3′ endo forms, and the ratio can be calculated from 1H NMR data following the equation [C2′ endo] = [J1′,2′/(J1′,2′ + J3′,4′)] × 100.56 Also, the H-2′ chemical shift can be used as an indicator of glycosidic bond conformation.57,58 Therefore, from our NMR data, 6-thio cIDPR exhibits a 74% C2′ endo puckering with a syn conformation about the glycosidic bond (Table 1), which is consistent with that observed by both cADPR and N1-cIDPR. Mechanistic Study. ADP-ribosyl cyclase can use different substrates to produce structurally distinct products. The cyclization of NAD+ analogues usually takes place at the N1 position of the adenine ring. However, NAD+ analogues, which have their N1 position blocked (e.g., etheno NAD+), cyclize at N7.32 If N1 is free but is electronically deactivated (e.g., in 2- fluoro NAD+62) and the purine is still adenine, then this seems Article C2′ endo (%) 64 30 61 30 74 Table 1. Conformational Analysis of NAD+ Analogues and Their Respective Cyclic Dinucleotides H-1′a H-2′a cADPR N7-cIDPR N1-cIDPR N7-cGDPR 6-thio N1-cIDPR 5.80 6.19 5.89 6.07 6.0 5.20 3.90 5.18 4.63 5.2 Δ1′‑2′b conf c J1′,2′a 0.6 syn 5.6 2.29 anti 3.0 0.71 syn 6.1 1.44 anti 2.8 0.8 syn 6.3 J3′,4′a 3.2 nd nd nd 2.2 aData obtained from various sources: cADPR;59 N7-cIDPR and N7- cGDPR;59−61 N1-cIDPR.30 bDifference in chemical shifts between H-1′ and H-2′. cPreferred glycosidic bond conformation. to be correctly aligned in the active site but the substrate is hydrolyzed rather than cyclized at N7. The enzyme can also cyclize NHD+, NGD+, and NXD+ to their respective N7 cyclized dinucleotide N7-cIDPR, N7-cGDPR, and N7- cXDPR.33 In those cases, the most nucleophilic N7 nitrogen in guanine/hypoxanthine is often invoked to rationalize cycliza- tion at this position.33 However, nucleophilicity is not the only factor to consider. We previously made correlations showing that the conforma- tion about the glycosidic bond of NHD+ analogues appears to play a role in their cyclization.52 We argued that a syn confor- mation about the glycosidic bond is key for a successful cyclization at N1, i.e., that the enzyme seems to utilize the linear precursor in its prearranged conformation (at least in the case of the hypoxanthine series and presumably the guanosine series as well). Indeed, NHD+ (anti) cyclizes at N7 (product also in anti conformation), 8-X-NHD+ (syn) cyclizes at N1 (products in syn conformation), and 7-deaza NHD+ (anti and no possibility to cyclize at N7) hydrolyzes to the linear 7-deaza IDPR.52 However, the NAD+ analogues cyclize at N1 regardless of their conformation; NAD+ (anti), 8-X NAD+ (syn), and 7-deaza NAD+ (anti) cyclize at N1, all forming products in their syn conformation. When 6-thio NHD+ is used as a substrate, the enzyme converts it into 6-thio N1-cIDPR, therefore sug- gesting that the enzyme utilizes this substrate in the same way as NAD+ but clearly differently from NHD+, although NHD+ and 6-thio NHD+ are structurally very similar. Lee et al. have intensively investigated the structures of both Aplysia ADP-ribosyl cyclase and human CD38 to unravel the catalytic mechanism of the NAD+ cyclization and cADPR hy- drolysis reactions. Recently, they suggested that the cyclization reaction of NAD+ analogues occurs through a four-step sequence where both residues Tyr-81 and Phe-174 play an instrumental role by stabilizing the nucleobase through π-stacking interac- tions in a folded conformation so that cyclization can occur.63,64 Mutagenesis confirmed that Phe-174 was likely to be respon- sible for folding the linear substrate in the correct confor- mation. However, this is only true for adenine-based substrates; mutagenesis did not affect the cyclization of NGD+ to N7- cGDPR. Therefore, it is likely that two residues are responsible for the different base cyclization sites (N1 versus N7) and that residue Phe-174 may also be involved in the cyclization of 6-thio NHD+ to 6-thio N1-cIDPR. With the help of molecular modeling, we investigated further how NAD+ cyclization to cADPR may occur. The ligand in the ribo-2′-F-NAD+·ADPRC structure (PDB code 3I9O)63 was manually manipulated by rotating individual bonds to approxi- mate the position the adenine would have to be in to attack C-1″ and form the N1-cyclized product (Figure 5). In order for NAD+ to cyclize at N1, the adenine must rotate around the 1482 dx.doi.org/10.1021/jm201127y | J. Med. Chem. 2012, 55, 1478−1489