Reversed-phase high-performance liquid chromatography studies of α-MSH fragments

α-Melanotropin (α-MSH) is a linear tridecapeptide (Ac-Ser-Tyr-Syr-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂), that is primarily known for its ability to stimulate melanosome dispersion within integumental melanocytes (F. J. H. Tilders, D. F. Swaab and T. B. van Wimersma Greidanus (Editors), Frontiers of Hormone Research, Vol. 4, Karger, Basel, 1977; J. Ramachandran, S. W. Farmer, S. Liles and C. H. Li, Biochem. Biophys. Acta, 428 (1976) 347). In our efforts to understand the relationship of structure and conformation to the biological activities of α-MSH, we have prepared a series of diastereoisomeric analogues based on the highly potent analogue Ac-[Nle⁴, d-Phe⁷]-αMSH_4-11-NH₂ (T. K. Sawyer, V. J. Hruby, B. C. Wilkes, M. T. Draelos, M. E. Hadley and J. Bergsneider, J. Med. Chem., 25 (1982) 1022). These analogues differed only in the amino acid substituted in the seven position, which was thought to be a critical residue for the biological activity of α-MSH. The chromatographic behavior of these analogues was examined on a C₁₈ Vydac (16-μm) reversed-phase column with five different mobile phases. The selectivity (α) for the analogues was compared in 0.10% trifluoroacetic acid (TFA), 0.10% heptafluorobutyric acid (HFBA) and 0.25 M triethylammonium phosphate (TEAP) using either acetonitrile or methanol as the organic modifier. With only one exception all analogues substituted with a d-amino acid in the seven position were eluted prior to their l-amino acid counterparts. As expected due to the greater ionic strength, the TEAP buffer allowed the greatest selectivity for the separation of these-α-MSH analogues, but it was surprising to find that the TFA buffer had a greater influence on selectivity that the HFBA buffer with either organic modifier. The probable mechanism of retention for the α-MSH analogues in perfluoroalkanoic buffers was also investigated. In addition, the relatioship between the retention time and the hydrophobicity of the seven position substitution was examined. Although the data were somewhat limited, a lack of correlation between the hydrophobicity of the seven position residue and retention time was observed.