Journal of Medicinal Chemistry Scheme 1. Synthesis of 6-NMe NAD+a Article aReagents and conditions: (i) TMSOTf, DBU, MeCN, 60 °C, 1 h; (ii) methylamine hydrochloride, DCM/EtOH/Et3N, 60 °C, overnight; (iii) NH3/MeOH, 0 °C, 3 h; (iv) POCl3, TEP, H2O, 0 °C, 3 h; (v) PPh3, dipyridyl disulfide, morpholine, room temp, 2 h; (vi) β-NMN+, MnCl2 in formamide, room temp, 48 h; (vii) Aplysia californica, 25 mM HEPES (pH 7.4), room temp. Scheme 2. Synthesis of 6-Thio NHD+ a aReagents and conditions: (i) dimethoxypropane, acetone, HClO4, 20 min, room temp; (ii) POCl3, TEP, H2O, 0 °C, 1 h; (iii) dinitrofluorobenzene, Et3N, MeCN, room temp, 1 h; (iv) diisopropyl-di-tert-butylphosphoramidite, tetrazole, DCM, room temp, 1 h, then mCPBA, −78 °C, 10 min; (v) 10% mercaptoethanol in MeCN + 1% DIPEA, room temp, 1 h; (vi) 50% aq TFA, room temp, overnight; (vii) morpholine, PPh3, dipyridyl disulfide, DMSO, room temp, 1 h; (viii) β-NMN+, 0.2 M MnCl2 in formamide, room temp, overnight. yield and lack of structural data in the initial report prompted us to investigate a new, reliable and more modern, route to 6-thio NHD+. The key compound is 14, which was previously synthesized mostly in the 1960s more or less successfully.38,39 We initially reasoned that the synthesis of 14 should be easily accomplished by selective phosphorylation of 6-thioinosine 12 by adaptation of a published method40 that we have used very successfully on a wide range of nucleosides. However, treat- ment of 6-thioinosine with POCl3 in TEP was unsuccessful because of the poor solubility of the starting nucleoside in TEP (Scheme 2). An isopropylidene protecting group at the 2′,3′- hydroxyls was then considered to have the double advantage of solving the solubility issue and being removable during the phosphorylation procedure, which is carried out under acidic conditions. Phosphorylation at the 5′-position proved to be very difficult. Indeed, HPLC analysis of the quenched reaction showed a complex mixture of products that could not be iso- lated by reverse-phase chromatography. We reasoned that there was a mixture of protected and deprotected material as well as products phosphorylated at the 5′-OH, the sulfur atom, or both, since the sulfur is more nucleophilic than the oxygen. Phosphoramidite chemistry has been reported on 2′,3′-O- isopropylidene-6-thioinosine 1341 using N,N-diisopropyl-di- tert-butylphosphoramidite as a phosphitylating reagent.42 Both 1480 dx.doi.org/10.1021/jm201127y | J. Med. Chem. 2012, 55, 1478−1489