W-BAND (95 GHZ) EPR SPECTROSCOPY OF NITROXIDE RADICALS WITH COMPLEX PROTON HYPERFINE-STRUCTURE - FAST MOTION

Many dynamic processes in Liquids fall into the rotational motion regi me with correlation times of 10(-11) to 10(-12) s, which are difficult to probe by conventional electron paramagnetic resonance (EPR) spectr oscopy (8.8-9.5 GHz, X-band). At 95 GHz (W-band), the range of rotatio nal correlation times (tau(R)) measured by EPR for the typical nitroxi de radicals is extended by a factor of 7 toward short times, producing more pronounced motional effects on the line width at the same tau(R) . However, for protonated nitroxide spin probes, the inhomogeneous bro adening caused by proton superhyperfine (shf) interactions still contr ibutes significantly to motionally narrowed 95 GHz spectra, and this m akes direct estimation of tau(R) inaccurate. A multifrequency approach to solve this problem is reported. Information on proton hyperfine in teractions can be obtained from X-band spectra. This significantly imp roves the accuracy of T-2(-1) determination from W-band data without a dditional NMR or ENDOR experiments. The utility of this approach is de monstrated by two examples of nitroxide probes with complex superhyper fine structure: (i) 3-doxyl-17 beta-hydroxy-5 alpha-androstane (probe #1) and (ii) 3-maleimido-PROXYL (probe #2). EPR spectra of these probe s at both X- and W-bands were studied. X-band EPR spectra from probe # 1 revealed a well-resolved proton hyperfine structure; hyperfine coupl ing constants were determined by least-squares computer simulation. Th ese hyperfine constants were used for successful simulations of the sp ectra at W-band, where proton hyperfine structure is not resolved. Ano ther way to correct for inhomogeneous broadening is to use experimenta l X-band spectra measured near the limit of complete motional narrowin g as an approximation of inhomogeneous envelope functions to fit exper imental spectra obtained at W-band. This methodology can be especially useful when proton hyperfine structure at X-band is poorly resolved, as it is for probe #2. The spectra at both frequencies were analyzed w ith a computer program for inhomogeneous line width simulation/fitting based on a fast convolution algorithm and a Levenberg-Marquardt optim ization. Microwave phase effects present in W-band spectra were correc ted directly by an adjustment of the microwave phase shift in the fitt ing algorithm. Results are analyzed in terms of anisotropic Brownian d iffusion theory.