EPR distance measurement in a doubly nitroxide-labelled helical beta-peptide

A terminally protected β-hexapeptide, based on trans-(3R,4S)-β-TOAC and trans-(1S,2S)-ACHC, synthesized using classical solution methods, was found by FT-IR absorption and CD techniques to adopt the 3-14-helical conformation. EPR measurements of 1 in MeOH and HFIP (at 1.0 and 0.1 mM concentrations) were performed in the temperature range 120 K - 318 K. The spectra show three sharp lines with separations of about 1.5 mT (the same at all temperatures) superimposed on two broad signals, the separation of which increases as the temperature is lowered. The solvent and concentration effects are of minor significance. The spectra of the polycrystalline solid samples at low temperature extend over about 30 mT and their shape is mainly governed by the electron dipolar interaction. The intramolecular distance (6.1 ± 0.1 Å) between the nitroxide labels of the two β-TOAC residues at positions i and i+3, obtained from the analysis of the low temperature spectra, allowed us to confirm the expected ternary helical structure.