Solid phase peptide synthesis of human endothelin precursor peptides using two-step hard acid deprotection/cleavage...

Area of Science:

• Biochemistry
• Peptide Chemistry
• Molecular Biology

Background:

• Endothelin is a potent vasoconstrictor peptide.
• Understanding endothelin biosynthesis is crucial for cardiovascular research.
• Previous studies deduced endothelin precursor sequences from cDNA.

Purpose of the Study:

• To synthesize the putative human (hET-38) and porcine (pET-39) biosynthetic precursors of endothelin.
• To characterize the synthesized peptides and their biological activity.
• To explore alternative synthetic strategies and identify potential side products.

Main Methods:

• Solid-phase peptide synthesis (Boc-based) was employed.
• Cleavage from the resin was achieved using trimethylsilyl bromide and optimized with hydrogen fluoride.
• Disulfide bridges were formed via air-oxidation and analyzed using Endoproteinase Asp-N digestion and HPLC.

Main Results:

• Successful synthesis of hET-38 and pET-39.
• Characterization of disulfide bridge linkage modes.
• hET-38 exhibited lower in vitro activity compared to native endothelin.
• Five additional C-terminally elongated endothelin homologs were synthesized.
• An alternative synthesis of pET-39 using trimethylsilyl trifluoromethanesulfonate yielded a characterized major side product.

Conclusions:

• The study provides synthetic routes to key endothelin biosynthetic precursors.
• The synthesized analogs offer tools for studying endothelin structure-activity relationships.
• Comparative in vitro activity suggests structural determinants of endothelin's potent vasoconstriction.