SYNTHESIS OF TRIGGERABLE FUSOGENS FOR MEMBRANE BILAYERS

双层膜可触发融合剂的合成

• 批准号: 2629050
• 负责人: DAVID H THOMPSON
• 金额: $ 16.72万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2629050
• 关键词: biomaterial development /preparation; chemical structure function; chemical synthesis; confocal scanning microscopy; flow cytometry; high performance liquid chromatography; hydrolysis; lipid bilayer membrane; membrane fusion; membrane model; vinyl ether

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).

描述（改编自申请人摘要）：主要目的 拟议的研究是合成新的化合物， 控制脂质介导的膜融合。 一个跨学科的项目是 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， 加速这一至关重要的进程。 申报材料 将被掺入客体膜囊泡中，作为掩蔽的、非融合的 在局部化学触发后将变得融合的化合物， 暴露于低pH值或氧化环境-一个暴露过程， 类似于流感血凝素和其它病毒蛋白质基膜 融合途径 初步的计算实验已经被用来 设计一套合成目标分子，含有乙烯基醚键 在最佳战略位点的基序，这将有效地促进膜 在该连接的降解已经被触发之后的融合。 合成 由Run & Thompson开发的方法[J. Org. Chem. Eur.J. 1996 2 1505]将用于安装不稳定的乙烯基醚键 在所提出的一系列掩蔽的两亲性融合子中。 所得 然后将测试化合物促进膜融合的能力 在能够展开的实验条件下的模型膜系统中 通过乙烯基醚键断裂而形成融合剂。 HPLC分析和膜 融合荧光测定将用于监测融合剂的速率 暴露、囊泡脂质混合和化学后囊泡内容物混合 激活已经发生;这些结果将与预测进行比较 平均场单链理论。 的物理特性 膜结构，在触发之前和之后，也将执行 使用31 P 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 将测定它们促进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。 在不存在内体摄取的情况下，细胞质膜融合效率， 也将使用流式细胞术和激光共聚焦显微镜来确定 技术，以评估这些材料的细胞内的效用， 递送亲水性试剂通常经历缓慢的 膜易位（例如，肽，反义寡核苷酸，和 质粒）。