Journal of Medicinal Chemistry Article Figure 5. Two views of a model of the position of the adenine of the intermediate from ribo-2′-F-NAD+·ADPRC complex immediately prior ring closure. The hydrogen bond from the base to Glu-98 is shown. The model was built by rotating individual bonds in the PDB 3I9O structure (ribo-2′- F-NAD+·ADPRC) using the Schrödinger software running under Maestro, version 9.0.111. The color scheme for the ligand is as follows: pink, carbon; blue, nitrogen; red, oxygen; white, hydrogen; light blue, fluorine; orange, phosphorus. The color scheme for the residues is the same as for the ligand except the carbons are green. N9/C-1′ bond to adopt the syn configuration. It can then approach the C-1″ by stacking with Trp-140 and having the 6-amino group forming at least one hydrogen bond to Glu-98 to further orient and hold the adenine in position. This pro- posed mechanism has some support from the kinetic data for Glu-98 mutants, as indeed mutation of this residue was shown to reduce cyclase activity.65 NHD+ (or NGD+) would be unable to form the hydrogen bonds to Glu-98, and the lack of stabili- zation may partly explain the lack of cyclization at N-1. Exactly why 6-thio NHD+ cyclizes at N1 is unknown at present. It is intuitively clear that in order for 6-thio NHD+ to cyclize, the enzyme should be capable of stabilizing it in its thiol form, although the mercaptopurine base normally exists pre- dominantly in its thione form in solution.66 In the case where the protonation state plays a role in the reaction mechanism, the pKa values of N1, N7, and X1 (NH2, OH, SH) and X2 (O, NH, S) were calculated computationally (Figure 6; see Figure 6. Tautomeric form of purines. also Supporting Information). The calculations were done on the nucleoside (adenosine, inosine, and 6-thioinosine) and each respective tautomer; we presume that it is reasonable to pro- pose that these values would follow the same trend for the corresponding NAD/NHD analogue. For three of the possible ionization states, the program was unable to calculate a pKa, presumably because they are too extreme. At physiological pH, most of these ionizable groups are going to be either fully protonated or deprotonated, the exception being for the oxygen-containing compound (see Supporting Information). In the A tautomer the X1 ionization − (OH → O ) has a pKa of 6.56, and in the B tautomer the N1 − ionization (NH → N ) has a pKa of 8.67. These two values are within the range of physiological pH, which means that at this pH there will be a mixture of four tautomeric forms for the oxygen-containing compound (Figure 7) while there will Figure 7. Presumed different forms of the oxygen containing com- pound that may be present at physiological pH. be only two forms of the nitrogen- and sulfur-containing compound. Previously, we argued that the deciding interaction through the adenine ring was via hydrogen bond donation from the base, since the hypoxanthine base has only hydrogen acceptor character in its keto form. However, the 6-thiopurine base has virtually no hydrogen bond acceptor or donor character, which may imply that hydrogen bonding is unimportant at C-6 and that cyclization is influenced by other factors such as the size of the substituent (the sulfur atom is larger than the nitrogen and oxygen atoms; see Supporting Information), the conformation about the glycosidic bond (for the oxygen containing nucleo- tides), electronic properties, or a combination of these. It may be that an H-bonding interaction with the enzyme locks the purine of NHD+ in the anti form, leading to N7 cyclization. However, this interaction could be very weak or nonexistent with the 6-thiopurine, allowing it to rotate to facilitate N1 cyclization. In the case of 8-Br NHD+ the strong orienting effect of the bromo group may be enough to overcome such H-bonding. Protein crystallography with such a ligand may reveal those interactions as important for catalysis to occur. Pharmacology. The Ca2+ release activity of the newly synthesized 6-thio cIDPR 3 was evaluated fluorimetrically in 1483 dx.doi.org/10.1021/jm201127y | J. Med. Chem. 2012, 55, 1478−1489