Solvation of nitroxides

Abstract

The e.s.r. spectra of nitroxide radicals are widely used to study environmental effects, especially for labelled biomolecules. However, e.s.r. spectra are, in general, time-averaged records of the environmental constraints experienced by the NO˙ species. This averaging often shows up as a contribution to the linewidths, but the hyperfine coupling, A(¹⁴N), and g values are time-averaged values.

We have used non-averaging spectroscopic techniques (infrared, Raman and electronic spectroscopies) to obtain further information about the structures of nitroxide solvates. Unfortunately, the environmental sensitivity of the N—O stretching frequency proved to be so low that definitive results were difficult to obtain without careful computer analyses. Since the visible absorption band at ca. 23 000 cm^–1 can be measured with great accuracy, it was used in the major part of this study.

Systematic variations in v_max for the 23000 cm^–1 band for pure solvents followed a similar trend to that for the CO stretching frequency of acetone. This suggests that the mode of solvation for the CO and NO units is similar. For mixed solvents, in contrast to previous work on acetone systems, we were not generally able to resolve the experimental spectra into two or more components because the shift-width ratios were too small. However, by experimenting with a series of computer-constructed curves, we have established that skew bands comprising two Gaussian bands of very different intensities can be uniquely analysed to give the position of the weak band. Hence, we have shown that for water + aprotic-solvent systems nitroxides form three distinct solvates, whilst methanol + aprotic-solvent systems form only two. Since the intermediate band of the aqueous systems is close to that for the nitroxide in methanol, we postulate that water forms two hydrogen bonds, falling to one as the aprotic solvent is added, whereas methanol only forms a monosolvate. These conclusions are identical with those deduced for acetone solutions, using the CO stretching band as a gauge. Detailed comparison shows that the nitroxides behave as slightly stronger bases than the ketones. These results are used to reinterpret certain e.s.r. results, and it is suggested that nitroxide spin labels used in the study of membranes are often mono- rather than di-hydrated.