Cyclic peptide substrates of pp60c-src. Synthesis and evaluation.

To study the effects of constrained conformation and amino acid sequence on their kinetic parameters, a series of cyclic peptides were synthesized and each was tested as both a substrate and an inhibitor of pp60c-src, the product of the src proto-oncogene. The amino acid sequences were derived from Glu-Leu-Pro-Tyr-Ala-Gly and from the autophosphorylation site of pp60c-src (Ile-Glu-Asp-Asn-Glu-Tyr-Ala-Ala-Arg-Gln-Gly). Linear precursor peptides were synthesized by SPPS on aminomethylated polystyrene resin using the Fmoc-tert-butyl protection scheme with 4-hydroxymethyl-3-methoxyphenoxyacetic acid as the linkage agent. The peptides were cleaved from the support with 1% TFA in dichloromethane with the N-terminal Fmoc and the side-chain protecting groups in place. Removal of the Fmoc group with diethylamine and cyclization with BOP afforded cyclic peptides in 55-78% yield. Side-chain deprotection and further purification gave the final products in 25-48% yields based on their linear precursors. Based on the activities of the linear analogues, cyclization had little effect on the binding (Ki and Km) and rate of phosphorylation (Vmax) of cyclo(Glu-Leu-Pro-Tyr-Ala-Gly) and cyclo(Ile-Glu-Asp-Asn-Glu-Tyr-Ala-Ala-Arg-Gln). A series of cyclic decapeptides that contained the dipeptide D-Phe-Pro inserted in various positions in the autophosphorylation sequence showed marked differences in Ki, Km and Vmax. Compared to the well characterized linear substrate Val-5 angiotensin II, the D-Phe-Pro-containing cyclic peptides have higher Vmax values but differ little in Km, with values in the millimolar range.