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

Abstract

Six analogues 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 described in the preceding paper in this journal was used as a model for the enzymatic inactivation of substance P. The synthetic analogues 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 analogues 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 analogue, described, an heptapeptide analogue 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 analogue was about a third as potent as substance P in competing for binding to receptor sites for this peptide in rat brain membranes and a tenth 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 α‐amino protecting group in conjunction with t‐butyloxycarbonyl as an ɛ‐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 ionexchanger. The peptides were finally purified on a semi‐preparative reverse‐phase column. Copyright © 1981, Wiley Blackwell. All rights reserved