Qualitative and quantitative aspects of the degradation of several tripeptides derived from the antitumour peptide antagonist [Arg(6), D-Trp(7,9), MePhe(8)] substance P[6-11].

The tripeptides Arg-Trp-Phe, Arg-Trp-Phe-NH2, Phe-Trp-Arg and Phe-Trp-Arg-NH2 were subjected to a degradation study to get a more detailed insight into the degradation processes of the antitumor hexapeptide antagonist [Arg(6), D-Trp(7,9), MePhe(8)] substance P¿6-11¿ which was investigated in earlier research. Degradation kinetics as well as identities of degradation products of the tripeptides emerging in alkaline and acidic media were studied. The amidated forms (Arg-Trp-Phe-NH2, Phe-Trp-Arg-NH2) appear to be less stable than the carboxylic forms (Arg-Trp-Phe, Phe-Trp-Arg). Deamidation of the amide C-terminus, racemization of the Phe and Arg residues, ornithine formation, hydrolysis of the peptide backbone and diketopiperazine formation with elimination of the N-terminal fragments were the major degradative processes. Comparing these reactions with the reactions of antagonist [Arg(6), D-Trp(7,9), MePhe(8)] substance P¿6-11¿ it appeared that racemization of Phe and Arg, hydrolysis of the peptide backbone and diketopiperazine formation did not occur in detectable amounts in the hexapeptide. probably due to lower reaction rates of these reactions compared to the overall degradation rate of antagonist [Arg(6), D-Trp(7,9) MePhe(8)] substance P¿6-11¿.

Structural identification of the degradation products of the antitumor peptide antagonist [Arg6,D-Trp7,9,MePhe8]substance P (6-11).

The basic hexapeptide antagonist [Arg6,D-Trp7,9,MePhe8]-substance P (6-11) was degraded in acid and alkaline media. In acid solution, only one degradation product is found whereas in alkaline solution at least six products are formed. These compounds were analytically characterized and structurally identified by reversed-phase high-performance liquid chromatography, capillary electrophoresis, liquid chromatography/mass spectrometry, fast atom bombardment tandem mass spectrometry, optical rotation analysis, and chiral gas chromatography. The product formed in acidic solution is the terminally deamidated antagonist [Arg6,D-Trp7,9,MePhe8]substance P (6-11); this product was also found in alkaline degradation mixtures. Other important degradation products originate from racemization of the amino acid residue L-Met, formation of ornithine from Arg, and the oxidation of Met to its sulfoxide form.