Peptide synthesis and its application to elucidate the functional aspects of sulfated peptides and proteins

1. 研究了在Tyr（SO3H）残渣上不造成硫酸盐酯损失的二硫键形成反应。采用两种方法制备了含有两个二硫键的α- conotoxin (PnIA和PnIB)；同时氧化法和两步选择性氧化法。两种方法在整个合成过程中都没有产生去硫肽。采用侧链锚定策略，将Asp残基的β-羧基用聚合物载体锚定，合成了另一种磺化a- concontoxin EpI。这三种硫酸盐酸化的α-conotoxin与非硫酸盐酸化的α-conotoxin相比，具有几乎相同的生物活性。这一结果表明，这些肽上的硫酸盐酯并不支配它们的生物活性。制备含Tyr（SO3H）残基的肽硫酯是化学合成大分子硫化肽的关键。用Fmoc-SPPS组装含Tyr（SO3H）肽后，保护肽进行硫代酯化反应，然后用TFA去除保护基团（4℃）。2 .利用含Tyr（SO3H）肽硫酯进行银离子介导的节段缩合，制备目的硫酸化肽。利用MALDI-TOF-MS对我们制备的各种磺化多肽进行分析，获得磺化多肽和蛋白质的质谱信息。此外，我们还研究了利用胰凝乳酶消化和质谱分析相结合的新方法来确定肽和蛋白质的硫酸化位点。合成了不同分子大小的大鼠胆囊收缩素（CCK）肽，并将其作为高效液相色谱（HPLC）检测前体蛋白水解产物的真实标记。这些研究表明，多种蛋白酶和激素原转化酶参与CCK的加工，其作用具有细胞和组织特异性。

1. We examined the disulfide-bond forming reaction which does not cause the loss of sulfate ester on Tyr(SO3H) residue. α-Conotoxins (PnIA and PnIB), which contain two disulfide-bonds, were prepared by two approaches; a simultaneous oxidation approach and a two-step selective oxidation approach. Both approaches did not produce the desulfated peptide throughout the synthesis. Another sulfated a-conotoxin, EpI, was also synthesized by the side-chain anchoring strategy in which β-carboxyl group of Asp residue was anchored with polymer-support. These three sulfated α-conotoxins were proved to be almost equipotent on biological activity compared with their non-sulfate counterparts. This result implies that the sulfate ester on these peptides does not govern their biological activities.2. Preparation of peptide thioester which contains the Tyr(SO3H) residue(s) is critical for the chemical synthesis of the large-molecular-form of sulfated peptide. After assembly of the Tyr(SO3H)-containing peptide by Fmoc-SPPS, the protected peptide was subjected to the thioesterification followed by the removal of protecting groups with TFA (4℃). The Tyr(SO3H)-containing peptide thioester was used for the Ag+-mediated segment condensation to prepare the objective sulfated peptide.3. Various sulfated peptides prepared by us were subjected to MALDI-TOF-MS analyses to obtain information on MS of sulfated peptides and proteins. Also we examined the novel approach to determine the sulfated site(s) of peptides and proteins using a combination of chymotrypsin digestion and MS analysis.4. Rat cholecystokinin (CCK)-peptides varying the molecular size, were synthesized and used as authentic markers on HPLC to determine the proteolytic products from the precursor protein. From these studies, it is suggested that various proteases and prohormone convertases are involved in CCK processing and their actions are cell and tissue specific.