Analysis of O-sulfotyrosine-mediated bio-interactions using synthetic peptides

1.基于我们开发的简便高效的固相法，进行了大分子形式的酪氨酸(SO_3H)多肽的化学合成。i)人大胃泌素-II及其C端甘氨酸扩展肽的合成采用收敛段缩合法制备人CCK-58。在本合成中，2-氯三丁基树脂被广泛地用作固体载体来制备硫酸酯链段和部分保护的硫酸酯链段。CCK-58具有与CCK-33.2相当的葡萄糖依赖的胰岛素样生长活性。以Fmoc为基础的固相法合成了三种含有硫化酪氨酸残基的α螺杆菌毒素(PnIA、PnIB和EPI)。采用同时氧化法和两步选择性氧化法形成两个二硫键。在PnIA和PnIB的同时方法中，添加…反应介质中较多的二甲基亚砜是减少二硫键异构体生成的关键。在整个合成过程中，硫磺被保持在最低限度。利用天冬氨酸的α-羧基作为固相载体的锚定基团，采用一种新的固相方法合成了α-芋螺毒素EPI。对三种硫酸化的α-芋螺毒素进行了生物测定(抑制牛肾上腺嗜铬细胞分泌儿茶酚胺的作用)，并发现它们与非硫酸盐的同类毒素具有等效性。这意味着这些α-芋螺毒素上的硫酸盐基团不是其生物活性的决定因素。此外，我们还发现这些α-芋螺毒素的二硫键异构体对儿茶酚胺的分泌有微弱的抑制作用。3.采用固相法制备了不同大小的大鼠CCK多肽(CCK-33、CCK-22和CCK-12)，并将它们作为高效液相色谱分离前CCK蛋白水解物的标志物。4使用合成的硫酸化多肽，发现凝血因子XI(FXI)与血小板膜糖蛋白Ibα结合，而不是通过涉及三个硫化酪氨酸残基的阴离子灰尘，而是通过GplbαN-末端的富亮氨酸重复序列。较少

1 We carried out the chemical synthesis of large molecular forms of Tyr(SO_3H)-containing peptides based on the facile and efficient solid-phase method developed by us.i) Human big gastrin-II and its C-terminal Gly-extended peptide were prepared by the convergent segment condensation approach on the resin.ii) Human cholecystokinin (CCK)-58 was prepared by the silver-ion mediated thioester segment condensation approach. In this synthesis, the 2-chlorotrityl resin was extensively used as a solid support to prepare the sulfated segment and partially protected thioester segments. CCK-58 exhibited glucose-dependent insulinotropic activity at levels comparable to CCK-33.2 Three α-conotoxins (PnIA, PnIB, and EpI) that contain the sulfated tyrosine residue were synthesized by the Fmoc-based solid-phase method. Both a simultaneous oxidation approach and a two-step selective oxidation approach were employed to form the two disulfide linkages. In a simultaneous approach for PnIA and PnIB, additio … More n of DMSO in the reaction medium was critical to reduce the production of disulfide bond isomers. Desulfation was kept minimum throughout synthesis. α-Conotoxin EpI was synthesized using a novel solid-phase approach, in which α-carboxyl function of Asp was utilized as an anchoring group with a solid support. Three sulfated α-conotoxins were subjected to bioassay (Inhibitory effects of catecholeamine secretion from bovine adrenal chromaffin cells) and were found to be equipotent with their non-sulfate counterparts. This implies that the sulfate groups on these α-conotoxins are not determinants for their biological activities. Also we found that the disulfide bond isomers of these α-conotoxins had a weak but an apparent inhibitory effects of catecholeamine secretion.3 Various sized rat CCK-peptides (CCK-33, CCK-22, and CCK-12) were prepared by our solid-phase method and they were used as markers on the HPLC separations of the proteolytic products from proCCK. A model for the progression of pro-CCK processing in AtT 20-cells was proposed.4 Using the synthetic sulfated peptides, factor XI (FXI) was found to bind with the platelet glycoprotein Ibα not via an anionic duster in which three sulfated tyrosine residues were involved, but via the leucine-rich repeat sequences within the N-terminal domain of Gplbα. Less