Active‐Site‐Matched Fluorescent Probes for Rapid and Direct Detection of Vicinal‐Sulfydryl‐Containing Peptides/Proteins in Living Cells

Abstract

Vicinal-sulfydryl-containing peptides/proteins (VSPPs) play a crucial role in human pathologies. Fluorescent probes that are capable of detecting intracellular VSPPs in vivo would be useful tools to explore the mechanisms of some diseases. In this study, by regulating the spatial separation of two maleimide groups in a fluorescent dye to match that of two active cysteine residues contained in the conserved amino acid sequence (-CGPC-) of human thioredoxin, two active-site-matched fluorescent probes, o-Dm-Ac and m-Dm-Ac, were developed for real-time imaging of VSPPs in living cells. As a result, the two probes can rapidly respond to small peptide models and reduced proteins, such as WCGPCK (W-6), WCGGPCK (W-7), and WCGGGPCK (W-8), reduced bovine serum albumin (rBSA), and reduced thioredoxin (rTrx). Moreover, o-Dm-Ac displays a higher binding sensitivity with the above-mentioned peptides and proteins, especially with W-7 and rTrx. Furthermore, o-Dm-Ac was successfully used to rapidly and directly detect VSPPs both in vitro and in living cells. Thus, a novel probe-design strategy was proposed and the synthesized probe applied successfully in imaging of target proteins in situ.

Aminomaleimide fluorophores: a simple functional group with bright, solvent dependent emission

“…Bis-maleimides have been used to allow detection of bis-thiols (including reduced disulfides), 9 – 11 and maleimide-functional probes have been used for protein labelling, 12 and both single and two-photon imaging of cells where they allow detection of thiol functional peptides. 9 , 10 , 13 – 16 …”

“…[6][7][8] By this process, N-fluorophore maleimides have been used as probes for the detection of thiols such as cysteine and glutathione. Bis-maleimides have been used to allow detection of bis-thiols (including reduced disulfides), [9][10][11] and maleimidefunctional probes have been used for protein labelling, 12 and both single and two-photon imaging of cells where they allow detection of thiol functional peptides. 9,10,[13][14][15][16] Response to chemical stimuli can also be achieved where the reaction of two non-fluorescent species leads to the generation of a fluorophore.…”

“…Bis-maleimides have been used to allow detection of bis-thiols (including reduced disulfides), [9][10][11] and maleimidefunctional probes have been used for protein labelling, 12 and both single and two-photon imaging of cells where they allow detection of thiol functional peptides. 9,10,[13][14][15][16] Response to chemical stimuli can also be achieved where the reaction of two non-fluorescent species leads to the generation of a fluorophore. For example, the highly efficient tetrazolealkene/azirine-alkene cycloaddition results in an emissive pyrazoline product.…”

Engineered extracellular vesicles: potentials in cancer combination therapy

“…Click chemistry that utilizes copper-catalyzed azide-alkyne cyclo-addition reaction to attach specific molecules to EV membrane is one of the representatives [ 30 – 32 ]. Interlinkage between sulfhydryl and maleimide can also be employed to engineer EV membrane [ 33 – 35 ]. In these cases, chemical bonds contribute to the strong connection between target substances and EV membrane.…”

A water-soluble and incubate-free fluorescent environment-sensitive probe for ultrafast visualization of protein thiols within living cells

“…Probe DMTs-OCC and reference compound Ctrl were dissolved in dimethyl sulfoxide (DMSO) as storing solution. Preparation and preservation of BSA and other amino acids were based on the literature [ 23 ]. Hitachi F-7000 fluorescence spectrophotometer was used to record the fluorescence spectra.…”