Chemically Induced Vinylphosphonothiolate Electrophiles for Thiol-Thiol Bioconjugations
Chemically Induced Vinylphosphonothiolate Electrophiles for Thiol-Thiol Bioconjugations
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DOI：
10.1021/jacs.0c03426
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发表时间：
2020-05-20
影响因子：
15
通讯作者：
Hackenberger, Christian P. R.
中科院分区：
文献类型：
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作者：
Baumann, Alice L.;Schwagerus, Sergej;Hackenberger, Christian P. R.
在这里，我们介绍了乙烯基硫代膦酸盐作为一类新型的半胱氨酸选择性亲电试剂，用于蛋白质标记和形成稳定的蛋白质-蛋白质偶联物。我们开发了一种直接的合成路线，将亲核的硫醇转化为亲电的、硫醇选择性的乙烯基硫代硫酸酯：在这个方案中，中间形成的二硫化物可以在酸性条件下被乙烯基亚膦化学选择性地取代，得到所需的乙烯基硫代膦。值得注意的是，该反应序列能够将乙烯基硫代膦亲电体直接安装在多肽和蛋白质中的半胱氨酸侧链上。除了标记具有良好半胱氨酸选择性的单抗曲妥珠单抗外，我们还应用我们的方法将两种蛋白质与独特的半胱氨酸残基进行位点特异性连接，产生不可水解性硫代磷酸连接的二泛素和泛素-α-突触核蛋白结合物。后者被认为是随后的酶泛素化反应的底物。
Herein we introduce vinylphosphonothiolates as a new class of cysteine-selective electrophiles for protein labeling and the formation of stable protein-protein conjugates. We developed a straightforward synthetic route to convert nucleophilic thiols into electrophilic, thiol-selective vinylphosphonothiolates: In this protocol, intermediately formed disulfides can be chemoselectively substituted with vinylphosphonites under acidic conditions to yield the desired vinylphosphonothiolates. Notably, this reaction sequence enables the installation of vinylphosphonothiolate electrophiles directly on cysteine side chains within peptides and proteins. In addition to labeling the monoclonal antibody trastuzumab with excellent cysteine-selectivity, we applied our protocol for the site-specific conjugation of two proteins with unique cysteine residues yielding a nonhydrolyzable phosphonothiolate-linked diubiquitin and an ubiquitin-a-synuclein conjugate. The latter was recognized as a substrate in a subsequent enzymatic ubiquitination reaction.