Synthesis and Biological Properties of Enzyme‐Resistant Analogues of Substance P

Abstract

Six analogs of substance P were synthesized with the aim of developing a metabolically stable peptide that would retain the biological activity of substance P. A recently isolated and characterized substance-P-degrading enzyme from human brain with a high specificity for substance P was used as a model for the enzymatic inactivation of substance P. The synthetic analogs were designed to protect the peptide bonds on the carboxyl side of residues 6, 7 and 8 of substance P, which represent the sites of cleavage by substance-P-degrading enzyme. To test for increased enzymatic resistance, the analogs were incubated with the enzyme, the digests were separated on a high-performance liquid chromatography reverse-phase column and the peptide fragments were collected and identified by amino acid analysis. Of the analogs described, an heptapeptide analog of residues 5-11, < Glu-Gln-Phe-MePhe-MeGly-Leu-MetNH2, showed almost complete resistance both towards substance-P-degrading enzyme and to degradation on exposure to rat hypothalamic slices. This analog was .apprx. 1/3 as potent as substance P in competing for binding to receptor sites for this peptide in rat brain membranes and 1/10 as potent in eliciting contractions of the guinea pig ileum. The peptides were synthesized using the solid-phase technique with polydimethylacrylamide as a solid support and the coupling was achieved with pre-formed symmetrical anhydrides in dimethylacetamide. Fluorenylmethyloxycarbonyl was used as an .alpha.-amino protecting group in conjunction with t-butyloxycarbonyl as on .epsilon.-amino protecting group. Ammoniolytic cleavage from the resin was followed by stepwise elution from an SP-Sephadex column, deprotection with trifluoroacetic acid and chromatography on a Bio-Rex 70 ion-exchanger. The peptides were finally purified on a semi-preparative reverse-phase column.