SYNTHESIS OF TRIGGERABLE FUSOGENS FOR MEMBRANE BILAYERS

描述（改编自申请人的摘要）：主要目标 拟议的研究是合成可用于 控制脂质介导的膜融合。 一个跨学科项目是 提议涉及合成化学、理论、生物物理学和细胞生物学 旨在扩大目前可用的材料范围 加速这一根本性重要进程。 提议的材料 将被纳入客体膜囊泡中，作为掩蔽的、非融合的 在局部化学触发下将变得融合的化合物 暴露于低pH值或氧化环境——一个揭露过程 类似于流感血凝素和其他基于病毒蛋白的膜 融合途径。 初步计算实验已用于 设计一组包含乙烯基醚键的合成目标分子 最佳战略位点的基序，将有效促进膜 触发该连接退化后的融合。 合成的 Run & Thompson 开发的方法论 [J. 组织。化学。 1994 59 5758；化学。 欧洲J. 1996 2 1505]将用于安装不稳定的乙烯基醚键 在拟议的一系列掩蔽两亲性融合剂中。 由此产生的 然后将测试化合物促进膜融合的能力 在能够部署的实验条件下的模型膜系统中 通过乙烯基醚键断裂产生融合剂。 HPLC分析和膜 融合荧光测定将用于监测融合剂的速率 化学处理后揭开、囊泡脂质混合和囊泡内容物混合 已发生激活；这些结果将与预测进行比较 由平均场单链理论制成。 物理特性 膜结构在触发之前和之后也将被执行 使用 31P NMR、冷冻断裂电子显微镜和 X 射线散射 技术。 模型膜实验中最有效的融合剂 将测定其促进 KB 细胞质释放的功效 通过叶酸缀合的 DSPE-PEG 靶向细胞内体区室。 细胞质膜融合效率，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 评估这些材料在细胞内的效用的技术 递送通常经历缓慢速率的亲水性试剂 膜易位（例如肽、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).