Coloured peptides: synthesis, properties and use in preparation of peptide sub-library kits

Abstract

Several methods were developed for the solid-phase synthesis (SPPS) of coloured peptides and peptide libraries. At first a bifunctional red compound, 4-(4-(N-ethyl-N-(3-(tert-butyloxycarbonyl)aminopropyl)amino)phenylazo)be nzoic acid (Boc-EPAB), was coupled with chloromethyl resin to obtain a new solid support suitable for SPPS using Boc chemistry. Peptides synthesized on this coloured resin had the chromophore at their C-termini. N-terminally coloured peptides were synthesized on a traditional solid support, coupled with chromophoric carboxylic acid before cleavage. A model pentapeptide, Phe-Ala-Val-Leu-Gly, and its ten derivatives were synthesized and their properties studied. It was found that the presence of chromophores decreases the water solubility of peptides. However, insertion of solubilizing tags (penta-lysine sequences or polyoxyethyl chains) into the molecule of any coloured derivative resulted in enhancement of the solubility. The RP-HPLC hydrophobicity indexes (phi0) of the coloured peptides were also determined because phi0 values are closely related to their water solubility. A coloured pentapeptide library was synthesized using the portioning-mixing method. Each component of this library contained the red azo dye (EPAB) and the penta-lysine tag. Before the last coupling step the samples were not mixed. All of the 19 sub-libraries obtained after cleavage were readily soluble in water, giving intense red solutions. The effect of chromophore (EPAB) and/or penta-lysine solubilizing tag on the biological activity was also studied. Potencies of the bovine neurotensin 8-13 fragment and its different coloured and penta-lysine derivatives were compared in isolated longitudinal muscle strips of guinea pig ileum. It was shown that the hexapeptide with penta-lysine tag had almost the same activity as the 8-13 fragment itself. The activity of the EPAB-derivative was found to be rather low. However, the presence of the solubilizing tag in the coloured hexapeptide compensated the negative effect of the chromophore.