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

1. 基于对Tyr （SO_3Na）在TFA中脱硫和保护基团Arg （Pbf）和Ser （^tBu）脱保护的动力学研究，我们提出在低温下90%的TFA水处理是直接合成含Tyr （SO_3H）肽的合适脱保护/裂解方案。建立了一种基于fmoc的高效固相合成含Tyr （SO_3H）肽的方法。该方法包括两个关键特征：(1)使用2-氯三烷基树脂作为固体载体；(2)基于tfa介导的低温酸解的s_n1型脱保护/裂解协议。用这种方法制备了各种分子形式的胃泌素- ii和胆囊收缩素（CCK），其中包括大胃泌素- ii和CCK-39，没有明显的困难。研究了基于fmoc的固相段缩合法在合成人大胃泌素ii及其c端gly延伸形式（G34-Gly sulfate）中的应用。在这些合成过程中，2-氯三烷基树脂被专门用作锚定树脂来制备含有c端Pro残基的三个肽段和含有Tyr （SO_3H）的树脂结合段。基于各种含Tyr （SO_3H）肽的质谱行为，我们发现阴离子Tyr （SO_3H）残基倾向于在水溶液和非极性条件下与阳离子官能团形成稳定的共轭酸碱对，特别是Arg残基的胍功能。采用硫酯段缩合法制备了大分子型胆囊收缩素（CCK）肽CCK-39和CCK-58，其中含有c端Tyr （SO_3H）的硫酯段和含有c端Pro残基的部分保护硫酯段为缩合段。对最终缩合产物进行简短的TFA处理，分别得到CCK-39和CCK-58 6。以硫酸催产素为模型肽，研究了对Cys保护基团的脱保护方法和对Tyr （SO_3H）残基不进行脱硫的二硫键形成方法。采用Cys （Trt）保护基团，经tfa介导低温酸解，再经空气氧化形成2个二硫键，合成了玉米螺毒素a-conotoxin Epl。少

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