【作者】 张志平；

【导师】 郭庆祥；

【作者基本信息】 中国科学技术大学 ， 有机化学， 2011， 博士

【摘要】 蛋白质是化学生物学中重要的生理活性分子。在生物学研究中,利用修饰的蛋白质,可以更详细的揭示作用机理。但是这些分子难以用生物表达来获的,化学全合成却能够提供含有更种非天然氨基酸,特定位点同位素标记及成像标签的蛋白质,推动了在分子水平上理解蛋白质功能。化学选择性连接被发展用来有效的合成各种蛋白,其中自然化学连接方法应用最为广泛。该方法能够在温和的水溶液中高效地实现肽片段的连接,从而生成天然或者非天然的蛋白质。天然蛋白质半胱氨酸的丰度只有1.7%,且硫酯很难高效制备。近年来,很多化学家对其进行了大量的改进,以期突破半胱氨酸和硫酯的限制。进一步,用于可以用来进行连接的"Xaa-Cys"的位点就更加难以获得,最近发展的氨基酸巯基化,连接后进行脱硫的方法不失为一种很好的解决方案。但是,这种方案的要求是蛋白质中天然的半胱氨酸的巯基必须在脱硫时被有效的保护起来。别一个挑战是合成含有多个半胱氨酸的多肽或者蛋白质中一个经常遇到的困难是明确的几个二硫键形成的合成。为了解决这一困难,往往需要半胱氨酸残基正交保护,然后分步形成二硫键的。最后,多肽或者蛋白质的修饰缀合物化学还需要对活性半胱氨酸侧链进行选择性保护和在生物兼容的温和条件下脱除。目前在肽合成中常用几种保护基都不能满足蛋白质化学合成的需要,其要不脱除条件苛刻,要不与蛋白质化学合成不兼容。因此,必须发展一种能在温和条件下脱除的,并且与多肽蛋白质化学相融合的巯基保护基。本文中,作者在前人工作基础上,设计合成了一类与固相肽合成兼容新的半胱氨酸巯基保护基Hqm和Hgm。与之前报导的保护基相比,在多肽合成中脱除条件更为温和。经过测试和条件优化,发现两种保护基很容易在肼水溶液中高效脱除,并与自然化学连接的所有条件兼容。而且我们运用该保护基,成功合成了trifolitoxin和human neutrophil defensin hNP2,表明其适用于多肽和蛋白质化学合成。更多还原

【Abstract】 Proteins are important biological macromolecules that carry out most of the biochemical functions in chemical biology. The use of modified proteins can reveal more detailed biological mechanism. However, these molecules can’t be readily obtained through biological expression. The total chemical synthesis of proteins from unprotected peptide fragments has enabled the introduction of unnatural amino acids, site-specific isotopic labeling, and attachment of affinity tags or labels for imaging, and has great potential of increasing our understanding of the molecular basis of protein function.Chemoselective ligation methods have increased the efficiency of protein synthesis. In particular, currently native chemical ligation is most widely used, has led to higher reaction rates, higer reaction yields, and greater biocompatility. It can efficiently condense peptide segments in aqueous solutions under mild conditions to generate natural as well as non-natural proteins. While, the abundance of cysteine in natural proteins is only 1.7% and it is hard to prepare the thiol esters.In just few sho(?) years, various methods were developde to break through the limitations of cystein and thiol ester. For chemical synthesis of proteins without suitable Xaa-Cys ligation site(s), there is a need to use the recently developed "ligation-desulfurization" approach in which the desulfurization step is conducted in the presence of side chain protected Cys. Another challenge is the synthesis of multiple Cys-containing proteins, where an often encountered difficulty is unambiguous formation of several disulfide bridges. To solve this difficulty it often needs to conduct orthogonal protection of Cys residues for the stepwise formation of disulfide bonds. Finally, peptide-protein bioconjugation may also need selective protection and deprotection of active Cys side chains under biologically benign conditions.The popular thiol protecting groups reported before cannot meet the needs of the protein chemical synthesis, because of harsh deprotecting conditions compatibility. Thus, thiolprotecting groups which can be removed under mild conditions and compatible with the peptide and protein chemistry must be developed.In this paper, we designed and synthesis a new Cys side-chain protecting group which is compatible to the solid-phase peptide synthesis. They are readily deprotected by using aqueous hydrazine. To illustrate the method’s utility, trifolitoxin and human neutrophil defensin hNP2 were manually synthesized by using Hgm and Hqm. That shows they were suited for peptide and protein synthesis as useful protecting groups.更多还原