Self-Immolative Difluorophenyl Ester Linker for Affinity-Based Fluorescence Turn-on Protein Detection

Abstract

Currently most fluorogenic probes are developed for the analysis of enzymes, where a bond breaking or rearrangement reaction is required to transform a nonfluorescent enzymatic substrate into a fluorescent product. However, this approach cannot be used for proteins that do not possess enzymatic activities. In this article, we show that fluorogenic probes with a self-immolative difluorophenyl ester linker can mimic the bond disassembly processes of fluorogenic enzyme substrates for the rapid analysis of nonenzymatic proteins. Although numerous self-immolative reagents have shown promising applications in sensors, drug delivery systems, and material chemistry, all of them are triggered by either enzymes or small reactive molecules. In our strategy, the probe binds to the protein via a specific protein–ligand interaction, inducing a chemical reaction between the self-immolative linker and an amino acid of the protein, thereby triggering a cascade reaction that leads to the activation and release of the fluorogenic reporter. In contrast, a phenyl ester linker without the difluoro substituent cannot be triggered to release the fluorogenic reporter. With this probe design, live-cell imaging of extracellular and intracellular endogenous tumor marker proteins can be achieved with high selectivity and sensitivity.

Imaging of Carbonic Anhydrase Level in Epilepsy with an Environment-Sensitive Fluorescent Probe

“…To date, fluorescent probes are considered to be one of the most powerful tools for monitoring biological species in biological systems. – On account of their desirable selectivity and sensitivity, excellent biocompatibility, and favorable spatial and temporal resolution, they have got a wide application in the CA detection. – Up to now, dozens of different types of CA fluorescent probes have been reported, – e.g., direct-conjugated recognition group on fluorophores; , one- or two-step photoaffinity probes; and one- or two-step electrophilic reaction probes. , However, in view of the availability, stability, and structural characterization clarity of CAs, most researchers regard CAs as model proteins for highlighting more modular research in probe designs. – In addition, the emission wavelength of the existing CAs environment-sensitive probes is short, which limits the in-depth application of probes in living tissues. ,, A red or near-infrared fluorescent probe (>600 nm) allows penetration into deep tissues with little autofluorescence interference, which is suitable for imaging CAs in living cells or tissues and is an ideal choice for exploring biological systems. – …”