Ligand-Directed Acyl Imidazole Chemistry for Labeling of Membrane-Bound Proteins on Live Cells

Abstract

Chemistry-based protein labeling in living cells is undoubtedly useful for understanding natural protein functions and for biological/pharmaceutical applications. Here, we report a novel approach for endogenous membrane-bound protein labeling for both in vitro and live cell conditions. A moderately reactive alkyloxyacyl imidazole (AI) assisted by ligand-binding affinity (ligand-directed AI (LDAI)) chemistry allowed us to selectively modify natural proteins, such as dihydrofolate reductase (DHFR) and folate receptor (FR), neither of which could be efficiently labeled using the recently developed ligand-directed tosylate approach. It was clear that LDAI selectively labeled a single Lys(K32) in DHFR, proximal to the ligand-binding pocket. We also demonstrate that the fluorescein-labeled (endogenous, by LDAI) FR works as a fluorescent biosensor on the live KB cell surface, which allowed us to carry out unprecedented in situ kinetic analysis of ligand binding to FR.

Affinity purification in target identification: the specificity challenge

“…Parallel labeling experiments against the target protein carbonic anhydrase have shown that the moderately reactive epoxide crosslinker exhibits the optimal balance of selectivity and reactivity (Chen et al 2003). Hamachi, Fenical and their respective co-workers developed a series of novel traceless affinity labeling probes containing electrophilic organocatalysts such as acyl phenolate, (Hughes et al 2009) 4-dimethylaminopyridine (DMAP) (Koshi et al 2008;Sun et al 2011;Wang et al 2011), tosylate, (Tsukiji et al 2009a, b;Tamura et al 2012), and acyl imidazole (Fujishima et al 2012;Matsuo et al 2013;Miki et al 2014). By targeting specific nucleophilic groups in the ligand-binding pocket of the protein, these probes exhibited high target specificity (Fig.…”

Gold nanoprobes-based resonance Rayleigh scattering assay platform: Sensitive cytosensing of breast cancer cells and facile monitoring of folate receptor expression

“…These specific recognition-derived results suggested high expression of FA receptors on MCF-7 cells, moderate expression on HepG2 cells, and no expression on normal cells, which was in accord with flow cytometric analysis using fluorescence-labeling , confirming the validity of the proposed method. The signal for receptor expression was directly observed during RRS detection, which was more quantitative and facile than microscopic observation (Fujishima et al, 2012).…”

“…Current efforts for cells recognition and cell surface antibody analysis have been focused on the usage of biochips (Culha et al, 2004;Kwong et al, 2009;Ellis et al, 2012), flow cytometry , fluorescence-labeling imaging (Fujishima et al, 2012), and the theoretical calculation (Li et al, 2015). Although these methods are powerful for molecular details, they have shortcomings of cellular auto-fluorescence, time-consuming sample preparation, and sophisticated instrumentation.…”

Ligand-directed tosyl chemistry for in situ native protein labeling and engineering in living systems: from basic properties to applications

“…From a standpoint of organic chemistry, it is also crucial to expand the repertoire of organic reactions that can be used for traceless affinity labeling of proteins in biological conditions. For instance, we have recently developed two new techniques, the affinity-guided 4-dimethylaminopyridine (DMAP) (AGD) chemistry [43][44][45] and the ligand-directed acyl imidazole (LDAI) chemistry [46,47], both of which allow rapid selective native protein labeling. Other promising ligand-directed chemistries have also recently been emerging [48][49][50][51][52].…”