EPR studies of N-15- and H-2-substituted pH-sensitive spin probes of imidazoline and imidazolidine types

The isotopically substituted analogs of pH-sensitive imidazoline and imidaz olidine radicals have been synthesized and investigated with electron param agnetic resonance (EPR) spectroscopy. The introduction of H-2 and N-15 into the structure of the radical is a useful approach to enhance the informati on obtained from spin-labeling experiments. The spectra of the radicals hav e been analyzed with 9.8 (X-band) and 130 GHz (D-band) EPR spectroscopy. Th e substitution of H-1 for H-2 leads to significant narrowing of Gaussian li ne width, while the substitution of N-14 for N-15 in the nitroxyl fragment decreases both the number of spectral lines and Lorentzian line width. Thes e effects result in a significant increase in the peak intensities up to 5- 7 times for X-band EPR spectra of one of the imidazoline radicals (R4). The increase in spectral resolution allowed us to reveal the hyperfine interac tion splitting with the attached proton (0.36 G) in the protonated form of the radical R4. The influence of proton exchange of the radicals with phosp hate and acetate buffers on their EPR spectra has been studied in H2O and D 2O. The corresponding rate constants of the proton exchange have been calcu lated from fitting of the simulated EPR spectra line shapes to experimental spectra. The data obtained demonstrated the advantages of the isotopically substituted spin pH probes in spectral resolution and sensitivity which ca n be an important factor particularly for applications in vivo where the fu ndamental sensitivity is much lower. The sensitivity of EPR spectra of thes e spin probes to the buffer capacity could be of practical importance takin g into account the biological relevance of monitoring this parameter in som e pathological states.