Chemical synthesis and receptor binding of catfish somatostatin: a disulfide-bridged beta-D-Galp-(1 -> 3)-alpha-D-GalpNAc O-glycopeptide

The glycopeptide hormone catfish somatostatin (somatostatin-22) has the ami no 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 connect ing the two Cys residues, and the major naturally occurring glycoform conta ins D-GalNAc and D-Gal O-glycosidically linked to Thr(5). The linear sequen ce was assembled smoothly starting with an Fmoc-Cys(Trt)-PAC-PEG-PS support , using stepwise Fmoc solid-phase chemistry. In addition to the nonglycosyl ated peptide, two glycosylated forms of somatostatin-22 were accessed by in corporating as building blocks, respectively, N*-Fmoc-Thr(Ac3-ccD-CalNAc)-O H and N-alpha-Fmoc-Thr(Ac-4-P-D-Gal-(1--3)-Ac-2-alpha-D-GalNAc)-OH. Acidoly tic deprotection/cleavage of these peptidyl-resins with trifluoroacetic aci d/scavenger cocktails gave the corresponding acetyl-protected glycopeptides with free sulfhydryl functions. Deacetylation, by methanolysis in the pres ence of catalytic sodium methoxide, was followed by mild oxidation at pH 7, mediated by N alpha-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 se veral cases, presence of the disaccharide moiety resulted in 2-fold tighter binding.