Retention behaviour of paracelsin peptides on reversed-phase silicas with varying n-alkyl chain length and ligand density.

As part of further investigations on the characterization of the ligand-induced conformational stabilization of peptides, two series of n-alkyldimethylsilyl bonded silicas have been prepared. In series A the n-alkyl chain length, n, of the bonded phase was varied between 1 and 20 carbon atoms at a constant ligand density. In series B the ligand density, alpha exp, was gradually changed from 0 to 4.1 mumol/m2 on a C1, C4, C6, C8 and C18 bonded phase. The retention behaviour of four peptides of the paracelsin family were examined under isocratic conditions, using a ternary mobile phase of water-methanol-acetonitrile (22:39:39, v/v/v). Plots of k' versus n showed pronounced maxima between n = 2 and 4 carbon atoms, followed by a decrease by a factor of 3 at n = 5, whereas above 5 carbon atoms only a slight increase in k' was observed. The selectivity behaviour of the paracelsins A-D can be mainly rationalized by interaction of the amphiphathic polypeptide 3.6(13) (alpha)-helix with the hydrophobic ligand and protrusion of the key amino acid residues at positions 6 and 9 in the sequence. However, from experiments with a polystyrene stationary phase it is evident that hydrophobic interactions and different partition coefficients also contribute to the resolution of the paracelsin peptides. Furthermore, Van 't Hoff plots confirm significant free energy changes associated with retention. These observations provide the basis for evaluating the enthalpic and entropic changes associated with peptide interactions with n-alkyl silicas.