1. Solid-phase synthesis of cionin, a protochordate-derived octapeptide related to the gastrin/cholecystokinin family of peptides, and its mono-tyrosine-sulfate-containing derivatives
K Kitagawa, S Futaki, T Yagami, S Sumi, K Inoue Int J Pept Protein Res. 1994 Feb;43(2):190-200. doi: 10.1111/j.1399-3011.1994.tb00522.x.
Cionin, a protochordate-derived octapeptide amide related to the gastrin/cholecystokinin family of peptides, contains two consecutive tyrosine sulfate residues. In order to gain insight into the role of the respective tyrosine sulfate residue in biological activity, cionin and its derivatives in which one of the two tyrosine sulfate residues was replaced by tyrosine, were prepared by two Fmoc-based solid-phase approaches. In approach (1) Fmoc-Tyr(SO3Na)-OH was employed as a building block to assemble the Tyr(SO3Na)-containing peptide-resin, and a global deprotection/cleavage was conducted with 90% aqueous TFA in the presence of m-cresol and 2-methylindole at 4 degrees C. In approach (2) the Tyr(Msib) [Msib = p-(methylsulfinyl)benzyl] derivative was used for the peptide-chain assembly to achieve sulfation on the selective Tyr residue. Partially protected peptide with the Msib/Msz protecting groups [Msz = p-(methylsulfinyl)benzyloxycarbonyl], obtained after peptide-resin cleavage, was treated with DMF-SO3 complex in the presence of ethanedithiol to achieve the sulfation of free Tyr residue and the reduction of the Msib/Msz groups to TFA-labile Mtb/Mtz groups [Mtb = p-(methylthio)benzyl, Mtz = p-(methylthio)benzyloxycarbonyl]. Final deprotection of the Mtb/Mtz groups with 90% aqueous TFA in the presence of m-cresol and 2-methylindole gave the desired cionin derivative, which contains the tyrosine sulfate residue at the selective position. Yields obtained with approach (2) were considerably higher than those obtained with approach (1). Cionin and mono-Tyr(SO3H)-containing derivatives were assayed on exocrine pancreas in dogs.
2. Facile solid-phase synthesis of sulfated tyrosine-containing peptides: Part II. Total synthesis of human big gastrin-II and its C-terminal glycine-extended peptide (G34-Gly sulfate) by the solid-phase segment condensation approach
K Kitagawa, C Aida, H Fujiwara, T Yagami, S Futaki Chem Pharm Bull (Tokyo). 2001 Aug;49(8):958-63. doi: 10.1248/cpb.49.958.
Application of the fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase segment condensation approach to the preparation of sulfated peptides was investigated through the synthesis of human big gastrin-II, a 34-residue sulfated tyrosine [Tyr(SO3H)]-containing peptide. Highly acid-sensitive 2-chlorotrityl resin (Clt resin) was exclusively employed as an anchor-resin for the preparation of the three peptide segments having the C-terminal Pro residue as well as of the Tyr(SO3H)-containing resin-bound segment. By using the PyBOP-mediated coupling protocol [PyBOP=benzotriazolyloxytris(pyrrolidino)phosphonium hexafluorophosphatel, we successively condensed each segment and constructed the 34-residue peptide-resin without any difficulty. The final acid treatment of the fully protected peptide-resin at low temperature (90% aqueous TFA, 0 degree C for 8 h), which can detach a Tyr(SO3H)-containing peptide from the resin and remove the protecting groups concurrently with minimum deterioration of the sulfate, afforded a crude sulfated peptide. After one-step HPLC purification, a highly homogeneous human big gastrin-II was easily obtained in 14% yield from the protected peptide-resin. The sulfate form of the C-terminal glycine-extended gastrin (G34-Gly sulfate), a posttranslational processing intermediate of gastrin-II, was also successfully prepared with the segment condensation approach (11% yield). These results demonstrated the usefulness of the segment condensation protocol for preparing large Tyr(SO3H)-containing peptides.
3. Evaluation of the final deprotection system for the solid-phase synthesis of Tyr(SO3H)-containing peptides with 9-fluorenylmethyloxycarbonyl (Fmoc)-strategy and its application to the synthesis of cholecystokinin (CCK)-12
T Yagami, S Shiwa, S Futaki, K Kitagawa Chem Pharm Bull (Tokyo). 1993 Feb;41(2):376-80. doi: 10.1248/cpb.41.376.
Acidolytic deprotection and cleavage conditions for an acid-labile Tyr(SO3H)-containing peptide were systematically examined with respect to acid, temperature, and scavenger. The 90% aqueous trifluoroacetic acid (TFA)-based reagent systems (90% aqueous TFA/m-cresol and 90% aqueous TFA/m-cresol/2-methylindole) at 4 degrees C were found to minimize the deterioration of Tyr(SO3Na) in the peptide. The latter deprotection/cleavage system was applied to the 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase synthesis of cholecystokinin (CCK)-12 on an acid-labile PAL-linked support (PAL = peptide amide linker), with Fmoc-Tyr(SO3Na)-OH as a building block.
