Chemoselective Staudinger-induced Michael-additions to antibodies to analyze protein homeostasis in C. elegans

在该提案中，一种新的无金属化学选择性缀合策略将被应用于修饰抗体，用于分析在维持蛋白质稳态中具有关键相关性的蛋白质-蛋白质相互作用。在这项共同努力中，Hackenberger实验室在生物正交反应工程和Kirstein实验室在蛋白质稳态方面的专业知识将被结合起来，以确定新的伴侣蛋白复合物参与蛋白质解聚及其在C中的蛋白质底物。优雅此外，我们的目标是采用抗体药物缀合物在体内将淀粉样蛋白形成的修饰剂靶向A β形成的原纤维。随着施陶丁格诱导的迈克尔加成用于抗体中半胱氨酸残基的化学选择性缀合的发展，将引入蛋白质修饰的新概念。这种新技术使得两个复杂的功能性构建块的模块化和无金属偶联成为可能，而不需要中间保护基团的操作。此外，通过该方法引入膦酰胺部分，其允许在蛋白质缀合物中直接掺入光或酶可裂解的键。这种新的合成工具将被应用于获得功能性抗体，用于鉴定未知的蛋白质底物以及特定分子伴侣的相互作用，并将进一步用于设计光可切割的抗体药物缀合物，以靶向表达Abeta 1 -42肽的阿尔茨海默病活动物模型中的淀粉样蛋白原纤维。

In this proposal a novel metal-free chemoselective conjugation strategy will be applied to modify antibodies for the analysis of protein-protein interactions of key relevance in the maintenance of protein homeostasis. In this joint effort the expertise of the Hackenberger laboratory in the engineering of bioorthogonal reactions and the Kirstein laboratory in protein homeostasis will be combined to identify novel chaperone complexes involved in protein disaggregation and their protein substrates in C. elegans. In addition, we aim to employ antibody drug conjugates to target modifiers of amyloid formation to Abeta formed fibrils in vivo. With the development of a Staudinger-induced Michael-addition for the chemoselective conjugation of cysteine residues in antibodies a new concept for the modification of proteins will be introduced. This novel technique enables the modular and metal-free coupling of two complex functional building blocks without the need of intermediate protecting group manipulations. Additionally, a phosphonamidate moiety is introduced by this method, which allows the direct incorporation of a light- or enzymatically cleavable bond in protein conjugates. This new synthetic tool will be applied to obtain functional antibodies for the identification of unknown protein substrates as well as interaction partners of specific chaperones and will furthermore be used in the design of light-cleavable antibody drug conjugates to target amyloid fibrils in living animal models for Alzheimer's disease that express Abeta1-42 peptides.