Chemical synthesis and receptor binding of catfish somatostatin: A disulfide-bridged β-D-Galp-(1 → 3)-α-D-GalpNAc O-glycopeptide

The glycopeptide hormone catfish somatostatin (somatostatin-22) has the amino acid sequence H-Asp-Asn-Thr-VaL-Thr-Ser-Lys-Pro-Leu-Asn-Cys-Met-Asn- Tyr-Phe-Trp-Lys-Ser-Arg-Thr-Ala-Cys-OH; it includes a cyclic disulfide connecting the two Cys residues, and the major naturally occurring glycoform contains D-GalNAc and D-Gal O-glycosidically linked to Thr⁵. The linear sequence was assembled smoothly starting with an Fmoc-Cys(Trt)-PAC-PEG-PS support, using stepwise Fmoc solid-phase chemistry. In addition to the nonglycosylated peptide, two glycosylated forms of somatostatin-22 were accessed by incorporating as building blocks, respectively, N(α)-Fmoc- Thr(Ac₃-α-D-GalNAc)-OH and N(α)-Fmoc-Thr(Ac₄-β-D-Gal-(1→3)-Ac₂-α-D- GalNAc)-OH: Acidolytic deprotection/cleavage of these peptidyl-resins with trifluoroacetic acid/scavenger cocktails gave the corresponding acetyl- protected glycopeptides with free sulfhydryl functions. Deacetylation, by methanolysis in the presence of catalytic sodium methoxide, was followed by mild oxidation at pH 7, mediated by N(α)-dithiasuccinoyl (Dts)-glycine, to provide the desired monomeric cyclic disulfides. The purified peptides were tested for binding affinities to a panel of cloned human somatostatin receptor subtypes; in several cases, presence of the disaccharide moiety resulted in 2-fold tighter binding.