FTIR AND EPR STUDY OF RADICALS OF AROMATIC-AMINO-ACIDS 4-METHYLIMIDAZOLE AND PHENOL GENERATED BY UV IRRADIATION

The oxidized radicals of aromatic amino acids, 4-methylimidazole and p henol were generated by UV irradiation at low temperature. The radical s were monitored by EPR spectroscopy. For the first time, infrared (IR ) modes characteristic of the tyrosine, tryptophan, phenylalanine, and histidine radicals were obtained by FTIR difference spectroscopy betw een the ground state and the radical state. The effects of D- (on Tyr and phenol) or N-15- (on His) labeling on the IR modes of the radicals were studied, as were the influence of hydrogen bonding and of pH. Th ese parameters were studied to model the possible radical structures a nd environments in proteins. The radicals obtained with tyrosine, phen ol, and 4-ethylphenol present six main IR modes: a combination mode at similar to 2110-2106 cm(-1); the nu 8b(CC) mode at 1550-1556 cm(-1), the nu 7a(CO) and nu 19b(CC) modes both at 1515-1500 cm(-1), which are distinctly affected by D-labeling of the phenol ring; the 14(nu CC delta CH) mode at 1290-1288 cm(-1), which is strongly modified when th e radical is hydrogen bonded; and the 9a(CC) mode at 1163-1159 cm(-1). These IR modes partly confirm the assignments made by resonance Raman (RR) spectroscopy and should help to obtain precise structure and for ce field calculations for the radicals. The deprotonated 4-methylimida zole radical (4-MeIm(.)) is obtained at pH 12. It has characteristic I R modes at 1593 nu(CC), 1425 delta CH3, 1376 delta CH3, 1315 cm(-1), 1 213 cm(-1) and 1098 cm(-1) delta(CH). The methyl modes seem strongly d ownshifted upon radical formation, while the ring modes appear less af fected. In particular, the C4C5 double-bond character is conserved. Th e protonated 4-methylimidazole radical formed at pH less than or equal to 6 is characterized by signals at 1433 cm(-1), 1380 cm(-1), 1310 cm (-1), 1227 cm(-1), and 1172 cm(-1). The histidine and tyrosine radical s present similar IR modes as the corresponding model of their sidecha in. For all the amino acids, the nu(as)(COO-) and nu(s)(COO-) modes of the terminal carboxylate were respectively up- and downshifted by sim ilar to 20 cm(-1) upon the radical formation. This effect suggests tha t, in a protein, the amide bond of the amino acid could also be influe nced by the radical formation. (C) 1995 John Wiley & Sons, Inc.