Reactivity of Functional Groups on the Protein Surface:  Development of Epoxide Probes for Protein Labeling

• Published in: Journal of the American Chemical Society 2003-07, Vol.125 (27), p.8130-8133
• Main Authors: Chen, Gong, Heim, Alexander, Riether, Doris, Yee, Dominic, Milgrom, Yelena, Gawinowicz, Mary Ann, Sames, Dalibor
• Format: Article
• Language: English
• Subjects: Affinity Labels - chemistry; Biological and medical sciences; Carbonic Anhydrase II - chemistry; Carbonic Anhydrase II - metabolism; Epoxy Compounds - chemistry; Fundamental and applied biological sciences. Psychology; Humans; Molecular and cellular biology; Surface Properties
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja034287m

We present the development of new affinity probes for protein labeling based on an epoxide reactive group. Systematic screening revealed that an epoxide functionality possesses the special combination of stability and reactivity which renders it stable toward proteins in solution but reactive on the protein surface outside the active site (proximity-induced reactivity). Highly efficient and selective labeling of purified HCA II (human carbonic anhydrase II) was achieved. For instance, 2 equiv of epoxide probe 9 was sufficient for nearly quantitative labeling of HCA II (>90% yield, 20 h reaction time). MS analysis of the labeled protein revealed that 1 equiv of the probe was attached and that labeling occurred at a single residue (His 64) outside the active site. Importantly, epoxide probe 9 selectively labeled HCA II both in simple protein mixtures and in cellular extracts. In addition to the chemical insight and its relevance to many epoxide-containing natural products, this study generated a promising lead in the development of new affinity probes for protein labeling.