SYNTHESIS OF TRIGGERABLE FUSOGENS FOR MEMBRANE BILAYERS

描述(改编自申请人的摘要)：主要目标 这项拟议的研究是为了合成可用于 控制脂质介导的膜融合。一个跨学科的项目是 建议涉及合成化学、理论、生物物理学和细胞生物学 旨在扩大目前可用于 加速这一根本上重要的进程。建议使用的材料 将作为掩蔽的、非融合性的结合在客膜囊泡中 在局部化学触发时将成为融合性的化合物 暴露在低pH或氧化环境中--这是一种揭开面纱的过程 类似于流感血凝素和其他病毒蛋白基膜 融合途径。初步的计算实验已经被用于 设计一组含有乙烯基醚键的合成靶分子 在最佳战略位置的基序，这将有效地促进膜 这一链接退化后的聚变已被触发。合成的 方法论由Run&Thompson开发[J.Org.化学。1994年59 5758；化学。 Eur.J.1996 2 1505]将用于安装不稳定的乙烯基醚连接件 在提议的蒙面两亲引爆剂系列中。由此产生的 然后将测试化合物促进膜融合的能力。 在实验条件下的模型膜系统中能够展开 FusoGen通过乙烯基醚键断裂。高效液相分析与膜分离 融合荧光分析将用于监测FusoGen的比率 化学处理后的去掩蔽、囊泡脂肪混合和囊泡内容物混合 激活已发生；这些结果将与预测进行比较 由平均场单链理论制成。它的物理特征 还将执行膜结构，在触发之前和之后 使用31P核磁共振、冷冻断口电子显微镜和x射线散射 技巧。模型膜实验中最有效的融合剂 将检测它们在促进KB胞质释放方面的有效性 通过叶酸结合的DSPE-聚乙二醇靶向细胞内体隔间。 细胞膜融合效率，在没有内体摄取的情况下， 也将使用流式细胞术和激光共聚焦显微镜来确定 评估这些材料对细胞内的效用的技术 递送亲水试剂通常经历缓慢的速度 膜转位(例如，多肽、反义寡核苷酸和 质粒)。

DESCRIPTION (Adapted from applicant's abstract): The primary objective of the proposed research is to synthesize new compounds that can be used to control lipid-mediated membrane fusion. An interdisciplinary project is proposed involving synthetic chemistry, theory, biophysics, and cell biology aimed at expanding the range of materials that are currently available for accelerating this fundamentally important process. The proposed materials will be incorporated within guest membrane vesicles as masked, nonfusogenic compounds that will become fusogenic upon localized chemical triggering by exposure to low pH or oxidative environment--an unmasking process that is analogous to influenza hemagglutinin and other viral protein-based membrane fusion pathways. Preliminary computational experiments have been used to design a set of synthetic target molecules, containing vinyl ether bonding motifs at optimal strategic loci, that will efficiently promote membrane fusion after degradation of this linkage has been triggered. Synthetic methodology developed by Run & Thompson [J. Org. Chem. 1994 59 5758; Chem. Eur.J. 1996 2 1505] will be used to install the labile vinyl ether linkages in the proposed series of masked amphiphilic fusogens. The resulting compounds will then be tested for their ability to promote membrane fusion in model membrane systems under experimental conditions capable of deploying the fusogen via vinyl ether bond cleavage. HPLC analysis and membrane fusion fluorescence assays will be used to monitor the rates of fusogen unmasking, vesicle lipid mixing, and vesicle contents mixing after chemical activation has occurred; these results will be compared with the predictions made by mean-field single chain theory. Physical characterization of the membrane structures, before and after triggering, will also be performed using 31P NMR, freeze-fracture electron microscopy, and x-ray scattering techniques. The most efficient fusogens in the model membrane experiments will be assayed for their efficacy in promoting cytoplasmic release from KB cell endosomal compartments targeted via folate-conjugated DSPE-PEG. Cytoplasmic membrane fusion efficiency, in the absence of endosomal uptake, will also be determined using flow cytometry and laser confocal microscopy techniques to evaluate the utility of these materials for intracellular delivery hydrophilic reagents that typically experience slow rates of membrane translocation (e.g., peptides, antisense oligonucleotides, and plasmids).