The differences in conformation in solution of fluorosulfonylbenzoyl nucleosides were analyzed by fluorescence and proton nuclear magnetic resonance spectroscopy. The quantum yield of 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine (5'-FSB epsilon A) in aqueous solution is low (ø = 0.01) as compared to that of its parent nucleoside, ethenoadenosine (ø = 0.54), and increases approximately 5-fold when measured in a series of solvents of decreasing dielectric constant. The quantum yield of 5'-p-sulfonylbenzoyl-1,N6-ethenoadenosine covalently bound to glutamate dehydrogenase and pyruvate kinase is also 0.01, suggesting that the analogue may exist in the same conformation when enzyme-bound as when free in solution. In D2O, the resonances of the purine ring protons on 5'-FSB epsilon A, 5'-p-fluorosulfonylbenzoyl adenosine (5'-FSBA), and 5'-p-fluorosulfonylbenzoyl guanosine (5'-FSBG) are shifted upfield by about 0.1-0.3 ppm relative to the corresponding protons of their parent nucleosides. The calculated difference in chemical shift (delta delta) decreases as the dielectric constant of the solvent decreases. The delta delta decreases with increasing temperature. These data indicate that 5'-FSB epsilon A, 5'-FSBA, and 5'-FSBG exist in aqueous solution in a conformation in which the purine ring is intramolecularly stacked with the benzoyl moiety. From the magnitude of change in delta delta for 5'-FSB epsilon A, 5'-FSBA, and 5'-FSBG as a function of solvent, it appears that the three analogues differ in their sensitivity to disruption of stacking. The solution conformation of these three fluorosulfonylbenzoyl nucleoside analogues may be an important determinant of their reaction with various enzymes and may explain differences among the analogues in their reaction with a single enzyme.