Department of Chemistry, University College London, 20 Gordon Street, WC1H OAJ, London, United Kingdom.
LifeArc, Accelerator Building, SBC Open Innovation Campus, SG1 2FX, Stevenage, United Kingdom.
Bioconjug Chem. 2021 Apr 21;32(4):672-679. doi: 10.1021/acs.bioconjchem.1c00057. Epub 2021 Mar 12.
Linkers that enable the site-selective synthesis of chemically modified proteins are of great interest to the field of chemical biology. Homogenous bioconjugates often show advantageous pharmacokinetic profiles and consequently increased efficacy . Cysteine residues have been exploited as a route to site-selectively modify proteins, and many successfully approved therapeutics make use of cysteine directed conjugation reagents. However, commonly used linkers, including maleimide-thiol conjugates, are not stable to the low concentrations of thiol present in blood. Furthermore, only a few cysteine-targeting reagents enable the site-selective attachment of multiple functionalities: a useful tool in the fields of theranostics and therapeutic blood half-life extension. Herein, we demonstrate the application of the pyridazinedione motif to enable site-selective attachment of three functionalities to a protein bearing a single cysteine residue. Extending upon previously documented dual modification work, here we demonstrate that by exploiting a bromide leaving group as an additional reactive point on the pyridazinedione scaffold, a thiol or aniline derivative can be added to a protein, post-conjugation. Thiol cleavability appraisal of the resultant C-S and C-N linked thio-bioconjugates demonstrated C-S functionalized linkers to be cleavable and C-N functionalized linkers to be noncleavable when incubated in an excess of glutathione. The plug-and-play trifunctional platform was exemplified by attaching clinically relevant motifs: biotin, fluorescein, a polyethylene glycol chain, and a model peptide. This platform provides a rare opportunity to combine up to three functionalities on a protein in a site-selective fashion. Furthermore, by selecting the use of a thiol or an amine for functionalization, we provide unique control over linker cleavability toward thiols, allowing this novel linker to be applied in a range of physiological environments.
能够实现化学修饰蛋白质的位点选择性合成的连接子在化学生物学领域具有重要意义。均相生物缀合物通常表现出有利的药代动力学特征，因此具有更高的疗效。半胱氨酸残基已被用作位点选择性修饰蛋白质的途径，许多成功批准的治疗药物都利用半胱氨酸定向缀合试剂。然而，常用的连接子，包括马来酰亚胺-巯基缀合物，在血液中低浓度的巯基存在下不稳定。此外，只有少数几种针对半胱氨酸的试剂能够实现多个功能的位点选择性连接：这是治疗诊断学和治疗血液半衰期延长领域的有用工具。在此，我们展示了使用哒嗪二酮基序来实现带有单个半胱氨酸残基的蛋白质的三个功能的位点选择性连接。在以前记录的双重修饰工作的基础上，我们证明了通过利用溴化物离去基团作为哒嗪二酮骨架上的另一个反应点，可以在缀合后将巯基或苯胺衍生物添加到蛋白质中。对所得的 C-S 和 C-N 连接硫代生物缀合物的硫醇可裂解性评估表明，当在过量谷胱甘肽中孵育时，C-S 功能化的连接子是可裂解的，而 C-N 功能化的连接子是不可裂解的。该即用型三功能平台通过连接临床相关的基序（如生物素、荧光素、聚乙二醇链和模型肽）得到了例证。该平台为在蛋白质上以位点选择性方式结合多达三个功能提供了难得的机会。此外，通过选择使用巯基或胺进行功能化，我们可以对硫醇的连接子可裂解性进行独特的控制，从而使这种新型连接子能够应用于一系列生理环境中。
