Studies on the Sulfated Tyrosine Containing Peptides Using a Novel Solid-Phase Synthetic Method

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1. Based on the kinetic studies concerning the desulfation of Tyr (SO_3Na) and the deprotection of the protecting groups, Arg (Pbf) and Ser (^tBu), in TFA, we proposed 90% aqueous TFA treatment at low temperature is a suitable deprotection/cleavage protocol for the direct synthesis of Tyr (SO_3H)-containing peptides.2. An efficient Fmoc-based solid-phase method for the synthesis of Tyr (SO_3H)-containing peptides was developed. This approach involves two key features : (1) use of the 2-chlorotrityl resin as a solid support, and (2) a S_N1-type deprotection/cleavage protocol based on the TFA-mediated acidolysis at low temperature. Various molecular forms of gastrin-II and cholecystokinin (CCK) involving big gastrin-II and CCK-39 were prepared by this approach without notable difficulty.3. Application of the Fmoc-based solid-phase segment condensation approach to the synthesis of human big gastrin-II and its C-terminal Gly-extended form (G34-Gly sulfate) was investigated. In these synthe … More ses, 2-chlorotrityl resin was exclusively employed for an anchor resin to prepare the three peptide segments having the C-terminal Pro residue and the Tyr (SO_3H)-containing resin-bound segment.4. Based on the mass spectrometric behaviors of the various Tyr (SO_3H)-containing peptides, we indicated that an anionic Tyr (SO_3H) residue tends to form a stable conjugate acid-base pair with cationin functional groups, especially the guanidine function of the Arg residues, in aqueous solutions and under nonpolar conditions.5. The big-molecular-form cholecystokinin (CCK)-peptides, CCK-39 and CCK-58, were prepared by the thioester segment condensation method using two or three peptide segments (the C-terminal Tyr (SO_3H)-containing segment and the partially protected thioester segments having C-terminal Pro residues). A brief TFA treatment of the final condensation product afforded objective CCK-39 and CCK-58, respectively6. Deprotection methods of the protecting groups for Cys and disulfide bond formation methods, in which the desulfation from the Tyr (SO_3H) residue was not associated, were examined using oxytocin sulfate as a model peptide. Corn-snail toxin, a-conotoxin Epl, was synthesized using Cys (Trt) protecting group and TFA-mediated acidolysis at low temperature followed by the air oxidation to form two disulfide bonds. Less