Journal of Medicinal Chemistry tert-butyl and isopropylidene protecting groups could then be cleaved under acidic conditions. However, in our hands the phosphorylation step could not be repeated, as the starting material is insoluble in most organic solvents except DMF and DMSO, and therefore only the starting material was recovered after 24 h with no sign of product. Thio-substituted nucleotides are particularly useful in molec- ular biology, as they have been incorporated into oligonucleo- tides by chemical methods and used for postsynthetic modi- fication.43−46 During the former process, the sulfur is always protected to avoid unnecessary side reactions during phosphi- tylation and oxidation of the phosphite.47 Since the synthesis of 6-thio IMP is hampered by the lack of solubility of 6-thioino- sine, protecting the sulfur should further solve the solubility issue encountered. Various protecting groups have been used such as cyanoethyl, which can be removed by treatment with DBU. Although the cyanoethyl group has been used extensively, we decided to utilize the 2,4-dinitrophenyl (DNP) group, as previous studies have shown that it can easily be removed with mercaptoethanol under very mild alkaline conditions.48 The synthesis of 6-thio NHD+ 10 is outlined in Scheme 2. 2′,3′-O-Isopropylidene-6-thioinosine 13 was prepared in a very high yield (86% over five steps) using published methods start- ing from inosine 12.41,49 Protection of the sulfur was achieved by treatment of 2′,3′-O-isopropylidene-6-thioinosine with tri- ethylamine and 2,4-dinitrofluorobenzene. The yellow product 15 obtained after flash chromatography could then be selec- tively phosphitylated at the 5′-hydroxyl using the tert-butyl protected phosphitylating reagent in high yield to give 16. Removal of the DNP protecting group was accomplished by treatment with mercaptoethanol under mild alkaline conditions. Careful purification by flash chromatography led to a very clean product in 75% yield. Next, both the isopropylidene and tert- butyl protecting groups were removed very cleanly by treat- ment with 50% aqueous TFA to generate the desired 6-thio IMP 14 in 86% yield, without further purification. This clearly is the advantage of the DNP group, as an ion-exchange purifi- Article cation step is avoided during which, from our experience, some product often gets unavoidably lost. 14 was thus obtained in a satisfying 48% yield after a four-step procedure from 2′,3′-O- isopropylidene-6-thioinosine. With 6-thio IMP in hand, we proceeded to synthesize the key intermediate 6-thio NHD+ 17. The synthesis of the pyrophos- phate was achieved using a procedure initially reported by Moffatt50 and later improved by Lee et al.51 that relies on the coupling of a sugar phosphate with a nucleotide phosphoro- morpholidate in the presence of a Lewis acid. We have previously used this method to successfully generate various NAD+ and NHD+ analogues in relatively high yield.30,52 6-Thio IMP was first activated using a combination of morpholine/dipyridyl disulfide and triphenylphosphine to yield the morpholidate that was then condensed with β-NMN+ with MnCl2 as Lewis acid. A 31P NMR shift of around −10 ppm clearly indicated the formation of a pyrophosphate linkage. Purification on reverse-phase chromatog- raphy afforded the desired 6-thio NHD+ 17 as the sole product. 6-Thio NHD+ was then incubated with Aplysia californica cyclase. There are several possible outcomes for this reaction (Figure 3), i.e., formation of 6-thio N1-cIDPR, 6-thio N7-cIDPR as for the oxo congener, 6-thio IDPR (6-thioinosine 5′-diphos- phate ribose), and it is possible to envisage cyclization on to the sulfur to give the 6-thio-S-cIDPR. The new product displayed a molecular ion of 557 in the ES− mass spectrum, which ruled out the formation of both the hydrolyzed product 6-thio IDPR and surprisingly also the N7 cyclized product 6-thio N7-cIDPR, for which a mass of 575 and 558, respectively, would be expected. 1H NMR spectroscopy showed a chemical shift of 5.2 ppm for H-2′, a typical value for a nucleotide in the syn conformation, which also ruled out the formation of 6-thio N7-cIDPR. Addi- tionally, a C-6 13C chemical shift of 175 ppm is typical of a thione form (thiol, ∼155 ppm), therefore ruling out the putative sulfur- cyclized product 6-thio S-cIDPR as a possibility. Crucially, a gHMBC spectrum showed cross-peaks between the H-2 proton of the purine ring (δ 9.32 ppm) and the anomeric carbon C-1″ of the “northern” ribose (δ 94.9 ppm) and between the anomeric Figure 3. Four possible outcomes from the incubation of 6-thio NHD+ with ADPRC. 1481 dx.doi.org/10.1021/jm201127y | J. Med. Chem. 2012, 55, 1478−1489