Temperature Dependence of Peptide NH Chemical Shifts in Benzene: Delineation of Solvent-Shielded and Exposed Amide Protons

1H-nmr is one of the most widely used techniques for the study of intramolecular hydrogen-bonded conformations of peptides in solution. The parameters used to delineate solvent-shielded amide NH groups include rates of hydrogen-deuterium exchange: solvent shifts paramagnetic radical-induced broadening: transfer of saturation from exchangeable solvent protons, and temperature coefficients (d\delta / dT) of NH chemical shifts, in strongly hydrogen-bond-accepting solvents like ${({CD}_3)}_2SO$. Of these, the use of temperature coefficients is probably the most widespread. In general, values <0.003 ppm/ deg C in ${({CD}_3)}_2SO$ have been taken as indicative of solvent-shielded, and presumably hydrogen-bonded, NH groups, while values >0.004 ppm/deg C have been assigned to exposed groups. The model compound, N-methylacetamide ${CH}_3CONH{CH}_3$ yields a value of 0.006 ppm/deg C in ${({CD}_3)}_2SO$. The large temperature coefficients for exposed NH protons presumably arise by the breaking of solute-solvent hydrogen bonds on increasing the temperature. Peptides dissolved in $CD{Cl}_3$ show low temperature coefficients for both solvent-shielded and exposed amide hydrogens, a reflection of the poor hydrogen-bonding capability of this solvent. We describe in this report the use of temperature coefficients of peptide NH groups in benzene $(C_6D_6)$, as a sensitive parameter for the determination of the degree of solvent exposure