RESUMO
The utilities of an α-methylene-ß-lactone (MeLac) moiety as a warhead composed of multiple electrophilic sites are reported. We demonstrate that a MeLac-alkyne not only reacts with diverse proteins as a broadly reactive measurement probe, but also recruits reduced endogenous glutathione (GSH) to assemble a selective chemical probe of GSH-ß-lactone (GSH-Lac)-alkyne in live cells. Tandem mass spectrometry reveals that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael or acyl addition. A peptide-centric proteomics platform demonstrates that the proteomic selectivity profiles of orlistat and parthenolide, which have distinct reactivities, are measurable by MeLac-alkyne as a high-coverage probe. The GSH-Lac-alkyne selectively probes the glutathione S-transferase P responsible for multidrug resistance. The assembly of the GSH-Lac probe exemplifies a modular and scalable route to develop selective probes with different recognizing moieties.
Assuntos
Lactonas/síntese química , Sondas Moleculares/síntese química , Humanos , Lactonas/química , Sondas Moleculares/química , Estrutura Molecular , Orlistate/análise , Proteômica , Sesquiterpenos/análise , Espectrometria de Massas em TandemRESUMO
In the present study, during removal of minor impurities by twin-column recycling chromatography with a solvent gradient, a compressing band effect was generated to offset band spreading and retain the principal component band within one column. For real-time monitoring, a detector was mounted on-line after the upstream column to monitor when the tail of the principal component was eluted from this column. When the conditions fluctuated, the column were switched to ensure successful separation without the need to determine the adsorption equilibrium in advance. Optimization of the operating conditions revealed that increasing the solvent gradient improved pre-impurity separation but impeded post-impurity removal. Changing the feed volume within a certain range hardly affected separation of the impurities, and increasing the number of cycles enhanced separation of the impurities.