An integrated conformational study is reported on the structurally modified nucleosides 4'-thiothymidine (1), an isoelectronic analogue of natural thymidine, and (E)-5-(2-bromovinyl)-2'-deoxy-4'-thiouridine (2). The conformation of 1 and 2 in D2O solution was inferred from the vicinal proton-proton NMR coupling constants and nuclear Overhauser (nOe) contacts. Significant adaptions of the conventional routines for J-coupling analysis in nucleos(t)ide structures were necessary due to the presence of sulfur instead of O4' in the thiofuranose ring. A pseudorotational equation of the form nu(j) = a(j)nu(m) cos (P + epsilon(j) + 144-degrees (j - 2)) was used to account for the nonequilateral nature of the 4'-thiofuranose ring. The parameter sets a0, ..., a4 and epsilon0, ..., epsilon4 were deduced from a set of ab initio (HF/3-21 G level) molecular orbital calculations. Analysis of the J-coupling constants measured for 1 and 2 revealed that (i) the C4'-C5' bond is primarily in the gamma+ or gamma(t) conformation and (ii) the 4'-thiofuranose ring has a preference for a South-type (C2'-endo/C3'-exo) puckered conformation. The preference for the South conformation is slightly larger for 1 (73% at 300 K) than for 2 (66% at 300 K). The pseudorotational parameters of the South conformer are as follows: P = 177-degrees, nu(m) = 43-degrees for 1, and P = 177-degrees, nu(m) = 44-degrees for 2. The results reveal that another conformer with a North-type puckered conformation of the 4'-thiofuranose ring is also present in solution, to an extent of almost-equal-to 27% for 1 and 34% for 2 at ambient temperature. The pseudorotational parameters describing the thiofuranose conformation of the minor conformer are as follows: P = 13-degrees, nu(m) = 45-degrees for 1, and P = 9-degrees, nu(m) = 45-degrees for 2. The characterization of the minor conformer must be regarded as an essential complement to the results of X-ray crystallographic analyses. One-dimensional nOe measurements indicated a predominant anti conformation of the thymine base in 1 and the modified uridine base in 2. The crystal structures of 1 and 2 were found to be grossly similar. The most important characteristics are as follows: compound 1, P = 177.7-degrees, nu(m) = 47.9-degrees, X = -144.7-degrees, gamma = 179.5-degrees; compound 2, P = 179.5-degrees, nu(m) = 48.6-degrees, chi = -140.0-degrees, and gamma = 174.1-degrees. Furthermore, it is concluded that the preferred conformation of 1 and 2 in solution is in close agreement with the X-ray crystal structure. The present results indicate that it is dangerous to rely solely on X-ray crystallographic information in attempts to explain or predict biological activity of modified nucleosides. The best basis for formulating structure-activity relationships for modified nucleosides is probably a combined interpretation of solid state and solution conformational data.
