Boc Solid-Phase Synthesis of Conjugable Cyclic Depsipeptides for Use as Biological Probes

对药物、天然产物和毒素等生物活性化学物质作用机理的研究，可以通过将合成化学探针应用于生物系统，通常是细胞，而获得很大的信息。探针通常是活性分子与荧光基团形式的“示踪剂”的结合，使其在细胞中的位置得以可视化，但更重要的是，有待开发的分析方法可以显示其何时与细胞中的目标(通常是蛋白质)结合(例如，生物发光共振能量转移(BRET)分析)。为了将生物活性分子转化为偶联物，它们必须有一个反应性的‘手柄’。本项目的目的是开发快速合成具有生物活性的环多肽(含酯键的多肽)的方法，这些多肽以赖氨酸(或类赖氨酸)残基的氨基的形式具有这样的手柄。多肽的合成在固相上进行得最快：第一个残基通过其羧酸连接到不溶于水的聚合物树脂上，随后的迭代去保护和偶联反应建立了剩余的多肽，而不需要在每一步后进行纯化-只需从树脂上洗掉多余的试剂。在这个项目中，我们将利用较老的“BOC”方法，因为它不涉及碱性步骤(用哌啶去保护Fmoc)，这会导致脱脂肽合成的问题(酯键的水解)。然而，BOC方法通常被避免，因为它需要危险的氢氟酸来最终从树脂中去除产品。我们将开发与树脂的替代连接，以避免使用这种试剂，并对要使用的保护基团进行全面调查，因为脱脂肽的构建将允许在树脂上循环，然后从树脂中移除和纯化，然后转化为化学探针。

Research into the mechanism of action of bioactive chemicals, such as drugs, natural products and toxins, can be greatly informed by the application of synthetic chemical probes to biological systems, usually cells. Probes are often conjugates of the active molecule with a 'tracer' in the form of a fluorescent group that allows its location in a cell to be visualised but, more importantly, assays to be developed that show when it is binding to its target (usually a protein) in the cell (e.g. a bioluminescence resonance energy transfer (BRET) assay). In order to convert bioactive molecules into conjugates, they must have a reactive 'handle' incorporated. The aim of this project is to develop methods to rapidly synthesise bioactive cyclic depsipeptides (peptides containing an ester bond) bearing such a handle in the form of the amino group of a lysine (or lysine-like) residue. The synthesis of peptides is most rapidly performed on solid-phase: the first residue is attached, by its carboxylic acid, to an insoluble polymer resin and subsequent iterative deprotection and coupling reactions build up the rest of the peptide without the need to purify after each step - only wash off excess reagents from the resin. In this project, we will take advantage of the older "Boc"-method as it does not involve an alkaline step (Fmoc deprotection by piperidine) which leads to problems in depsipeptide synthesis (hydrolysis of the ester bond). However, the Boc-method is usually avoided due to it requiring dangerous hydrofluoric acid for the final removal of the product from the resin. We will develop alternative linkages to the resin to circumvent the use of this reagent as well as carry out a full investigation of protecting groups to be used as the depsipeptide is built that will allow on-resin cyclisation prior to removal from resin and purification, followed by conversion into chemical probes.