Research for novel fluorescent probe and unnatural amino acid labeling for live cell imaging

Abstract

Visualization of specific protein in live system is crucial for understanding biological system, such as clarification of important cellular mechanisms, function of a specific protein, and protein-protein interactions in a living cell. In addition, development of high resolution microscopy such as nanoscopy and super-resolution microscopy (SRM) further promotes the development of protein labeling techniques. To satisfy these needs, a number of protein labeling techniques such as fusing fluorescent protein (FP) or self-labelling enzyme, and tagging using peptide tags or unnatural amino acids were developed. Each technique has its own strong points, but also has some critical limitations. Especially, in case of FP and self-labelling enzyme, because of their relatively big size, it could perturb some protein properties. Hence while devising the labeling techniques with small tagging unit, we focused on the UAA labeling which seemed more potential to selective labeling compared to peptide tags that have high background signal. Also, we chose the SuFEx click chemistry which is remarkably good to be used under physiological condition without interfering biological system and used BODIPY-based probe which is known for its high photostability and quantum yield. To find the best SuFEx target, we screened several candidates including natural amino acids and analogs with several functional groups by using LC-MS. Among them we found fluorotyrosine is a great SuFEx target which forms high amount of stable adduct even in the presence of free thiols. Also, we found that by adjusting the number and site of fluorides the SuFEx efficiency could be enhanced. Based on these result, we devised the new UAA whose structure has the fluorophenol group linked to Lys. With well-designed target UAA combined with terrific fluorescent sulfonyl fluoride probe, we tried to find an ideal protein labeling technique.