PLASMA DESORPTION MASS-SPECTROMETRY OF 2 SYNTHETIC SARAFOTOXINS - SIDE REACTIONS AND CHARACTERIZATION OF THE INTERMEDIATES

Sarafotoxins (SRTXs) form a family of toxic and potent vasoconstrictor peptides of 21 amino acids and two disulfide bonds. They are present in the venom of the burrowing asp Atractaspis engaddensis. We have mad e two derivatives of the amino acid sequence of SRTX-b, one of the mos t potent isotoxins, in the solid phase. First, we replaced Ser(2) by T hr, to investigate whether, as previously postulated, this change is r esponsible for the weak activities of SRTXs c and d. Secondly, we repl aced Ser(2), Asp(18) and Val(19) respectively by Thr, Gly and Ile, wit h a view to generating SRTX-e whose amino acid sequence was deduced fr om cDNA. Solid-phase peptide synthesis (SPPS) was performed according to the tert-butyloxycarbonyl strategy and the disulfides were paired s equentially using a selective chemistry. The disulfide 1-15 was formed by oxidation of cysteines(1,15) with ferricyanide, whereas disulfide 3-11 was made by iodine oxidation of Acm-blocked cysteines(3,11). By p lasma desorption mass spectrometry (PDMS), we monitored all possible s ide reactions that occurred during the synthesis. We thus observed a b enzyl shift in mass spectra when aspartic and glutamic acid side chain s were protected by a benzyl group during tile SPPS. This could be cir cumvented by using instead, a cyclohexyl protecting group. We also not ed the oxidation of the methionine and the tryptophan side chain (form ation of methionine sulfoxide and oxindole ring of tryptophan) to a sm all extent during the cleavage peptide/solid phase oxidation of the me thionine side chain during the formation of the disulfide 1-15 by ferr icyanide. These known side reactions could be avoided by using a mixtu re of scavengers (ethanedithiol, dimethylsulfide, thioanisole, m-creso l and skatole) and by shortening the ferricyanide oxidation time to on e hour. The sequential disulfide bond formation starting from the line ar peptide (mono-disulfide and bi-disulfide) was also monitored by PDM S. PDMS therefore proved to be a sensitive analytical method for the m easurement of the molecular weights of synthetic or natural peptides ( SRTX peptides in this application) in the field of peptide and protein chemistry.