手性侧链订书肽的合成及性能分析

发布时间：2018-06-12 12:12

本文选题：烯烃复分解反应(RCM) + staple　；参考：《华侨大学》2017年硕士论文

【摘要】：多肽的生物活性与构型密切相关,在多数情况下,由于灵活多变的结构使得它容易受蛋白酶水解作用的影响以及带来低效的细胞穿透性;所以稳定的多肽构象对于多肽药物的发展有重要意义。固定构型后的多肽穿膜机制虽然还未揭示,但通过报道的200多个装订肽的摄取方式的统计显示,影响多肽领域最有价值的性质—细胞穿透性能的因素主要是肽的电荷以及装订的位置与类型。在许多情况下,装订肽的穿膜性超过未修饰的多肽。目前已经出现了很多修饰多肽的方法来使得螺旋构象稳定。螺旋稳定化的策略包括盐桥、金属螯合物和共价环化方法,例如二硫化物和内酰胺桥。由于连接位置i,i+4,i+7和i+11处于α-螺旋的同一侧(i代表肽链上某氨基酸所在位数),所以这些侧链的共价连接可以促进体系的螺旋构象。Grubbs和Blackwell通过与O-烯丙基丝氨酸残基的闭环复分解反应(RCM)开创了非天然碳—碳键成环。该反应在Verdine及其同事发展下,将非天然α,α-二取代氨基酸与烯烃系链引入肽序列中,这种新的碳氢化合物骨架稳定方法显示出最好的稳定性与穿膜性能,为后期研究提供了理论依据。结合近期我们课题组研究发现,在多肽侧链特定位置引入一个R构型的手性中心,该手性中心有助于多肽形成α-螺旋结构。引入手性中心通过RCM关环构建环肽以及RCM关环本身均会出现异构体问题,出现每种异构体对螺旋度是否具有一致性的问题,摆在了我们面前。这促使我们应用RCM修饰多肽的方法研究其所形成的构型对多肽二级结构的影响,并寻找最优构型。基于实验室前期工作,这次我们研究的重点放在是手性中心对结构的影响。建立短肽模型(X_1-AAA-X_2)来验证侧链手性中心对Stapled Peptides的二级构象影响。选取HIV(FITC-Beta-Ala-ITF-X_1-DLL-X_2-YYGP-NH_2)多肽作为靶点,验证体系通过RCM方法添加手性中心之后该体系穿膜性的变化。(X_1,X_2为S_5、mS_5、mS_5(2-Ph)的一种;mS_5(2-Ph)代表手性中心为S构型,其侧链为5个碳键长度的的非天然氨基酸,且其α位无支链(m),在侧链2位是苯基)。通过结构鉴定、穿膜性能鉴定以及多肽稳定性实验我们得出了Z/R-装订肽具有最好α螺旋含量,并且还具有最佳的穿透细胞膜能力及最佳的抗水解能力。
[Abstract]:The bioactivity of polypeptide is closely related to its configuration. In most cases, it is easy to be affected by protease hydrolysis and bring low cell penetration due to its flexible and changeable structure. Therefore, stable polypeptide conformation is of great significance for the development of polypeptide drugs. Although the mechanism of polypeptide transmembrane after fixed configuration has not been revealed yet, the statistics of the uptake patterns of more than 200 binding peptides reported, The most valuable properties in the field of peptides-cell penetration are mainly the charge of peptides, binding position and type. In many cases, binding peptides are more permeable than unmodified peptides. There have been many methods of modifying peptides to stabilize the helix conformation. Spiral stabilization strategies include salt bridges, metal chelates, and covalent cyclization methods, such as disulfide and lactam bridges. Because the junction positions iGrubbs and i11 are located on the same side of the 伪 -helix to represent the number of amino acids on the peptide chain, the covalent bonding of these side chains can promote the helical conformation of the system. Grubbs and Blackwell promote the helical conformation of the system by interacting with O- allyl sericin. The closed-loop double decomposition reaction of acid residues (RCM) creates unnatural carbon-carbon bond ring formation. With the development of Verdine and his colleagues, the unnatural 伪, 伪 -disubstituted amino acids and alkenes were introduced into the peptide sequence. It provides a theoretical basis for the later study. In combination with our recent research, we found that a chiral center of R configuration was introduced at the specific position of the peptide side chain, which was helpful to the formation of 伪 -helix structure of the peptide. The introduction of chiral centers to construct cyclic peptides via RCM ring and the problem of isomers appearing in RCM ring itself, and the question of whether each isomer has consistency on helicity is in front of us. This prompted us to use RCM modified polypeptide method to study the influence of its configuration on the secondary structure of polypeptide and to find the optimal configuration. Based on prelab work, our focus this time is on the effects of chiral centers on structures. Short peptide model X _ 1-AAA-X _ 2) was established to test the effect of chiral center of side chain on the secondary conformation of Stapled peptides. Using the HIVFITC-Beta-Ala-ITF-X1-DLL-X2-YYGP-NH2) peptide as the target, the verification system verifies the change of the system's membrane penetration after the chiral center is added by the RCM method. Moreover, the 伪 -site has no branched chain, and the second position in the side chain is phenyl. Through the structure identification, membrane performance identification and polypeptide stability test, we obtained that the Z / R binding peptide has the best 伪 helix content, and also has the best ability to penetrate the cell membrane and the best ability to resist hydrolysis.
【学位授予单位】：华侨大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O629.72

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