Department of Chemistry, Indian Institute of Science Education and Research (IISER) Bhopal, Bhopal Bypass Road, Bhauri, Bhopal, 462066, Madhya Pradesh, India.
Nat Commun. 2024 Jan 29;15(1):859. doi: 10.1038/s41467-024-45124-2.
Proteins labelled site-specifically with small molecules are valuable assets for chemical biology and drug development. The unique reactivity profile of the 1,2-aminothiol moiety of N-terminal cysteines (N-Cys) of proteins renders it highly attractive for regioselective protein labelling. Herein, we report an ultrafast Z-selective reaction between isatin-derived Baylis Hillman adducts and 1,2-aminothiols to form a bis-heterocyclic scaffold, and employ it for stable protein bioconjugation under both in vitro and live-cell conditions. We refer to our protein bioconjugation technology as Baylis Hillman orchestrated protein aminothiol labelling (BHoPAL). Furthermore, we report a lipoic acid ligase-based technology for introducing the 1,2-aminothiol moiety at any desired site within proteins, rendering BHoPAL location-agnostic (not limited to N-Cys). By using this approach in tandem with BHoPAL, we generate dually labelled protein bioconjugates appended with different labels at two distinct specific sites on a single protein molecule. Taken together, the protein bioconjugation toolkit that we disclose herein will contribute towards the generation of both mono and multi-labelled protein-small molecule bioconjugates for applications as diverse as biophysical assays, cellular imaging, and the production of therapeutic protein-drug conjugates. In addition to protein bioconjugation, the bis-heterocyclic scaffold we report herein will find applications in synthetic and medicinal chemistry.
带有小分子的定点标记蛋白质是化学生物学和药物开发的宝贵资源。蛋白质 N 末端半胱氨酸(N-Cys)上 1,2-氨硫醇部分的独特反应性使其成为区域选择性蛋白质标记的理想选择。在此,我们报告了一种超快的 Z 选择性反应,涉及靛红衍生的 Baylis-Hillman 加合物和 1,2-氨硫醇,形成双杂环支架,并在体外和活细胞条件下将其用于稳定的蛋白质生物偶联。我们将我们的蛋白质生物偶联技术称为 Baylis-Hillman 调控的蛋白质氨硫醇标记(BHoPAL)。此外,我们还报告了一种基于硫辛酸连接酶的技术,用于在蛋白质的任何所需位置引入 1,2-氨硫醇部分,从而使 BHoPAL 位置不可知(不限于 N-Cys)。通过将这种方法与 BHoPAL 结合使用,我们在单个蛋白质分子上的两个不同特定位置生成带有不同标签的双重标记蛋白质生物偶联物。总之,我们在此公开的蛋白质生物偶联工具包将有助于生成单标记和多标记的蛋白质-小分子生物偶联物,用于生物物理测定、细胞成像和治疗性蛋白质-药物偶联物的生产等多种应用。除了蛋白质生物偶联之外,我们报告的双杂环支架还将在合成和药物化学中得到应用。