Institute of Organic Chemistry III, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany.
Bioconjug Chem. 2012 Mar 21;23(3):500-8. doi: 10.1021/bc200556n. Epub 2012 Feb 29.
The site-selective modification of the proteins RNase A, lysozyme C, and the peptide hormone somatostatin is presented via a kinetically controlled labeling approach. A single lysine residue on the surface of these biomolecules reacts with an activated biotinylation reagent at mild conditions, physiological pH, and at RT in a high yield of over 90%. In addition, fast reaction speed, quick and easy purification, as well as low reaction temperatures are particularly attractive for labeling sensitive peptides and proteins. Furthermore, the multifunctional bioorthogonal bioconjugation reagent (19) has been achieved allowing the site-selective incorporation of a single ethynyl group. The introduced ethynyl group is accessible for, e.g., click chemistry as demonstrated by the reaction of RNase A with azidocoumarin. The approach reported herein is fast, less labor-intensive and minimizes the risk for protein misfolding. Kinetically controlled labeling offers a high potential for addressing a broad range of native proteins and peptides in a site-selective fashion and complements the portfolio of recombinant techniques or chemoenzymatic approaches.
通过动力学控制标记方法，实现了 RNase A、溶菌酶 C 和肽激素生长抑素等蛋白质的位点选择性修饰。这些生物分子表面的单个赖氨酸残基在温和条件、生理 pH 值和室温下与活化的生物素化试剂反应，产率超过 90%。此外，快速的反应速度、快速简便的纯化以及低温反应对于标记敏感的肽和蛋白质特别有吸引力。此外，还实现了多功能生物正交生物偶联试剂（19），允许选择性地引入单个乙炔基。引入的乙炔基可用于点击化学等反应，如 RNase A 与叠氮香豆素的反应。本文报道的方法快速、劳动强度低，最大限度地减少了蛋白质错误折叠的风险。动力学控制标记为以位点选择性方式处理广泛的天然蛋白质和肽提供了巨大的潜力，并补充了重组技术或化学酶方法的组合。
