Developing Fluorescent Probes To Trap Protein Vicinal Thiols And Sulfenylation In Living Cells

Redox signal transduction, especially the oxidative modification of protein thiols, correlates with many diseases and becomes an expanding research area. However, there was rare method for quick and specific detection, especially the in situ imaging of protein thiols and their oxidative modification in living cells, which hinder the further study of this field. In the present work, a series of fluorescent probes were designed and synthesized for specific detection of protein vicinal thiols and sulfenic acids. The photochemical properties and biocompatibility were also evaluated. Meanwhile, BSA and thioredoxin as the model protein were taken to test the selectivity, reaction kinetics and competition of these probes. Furthermore, the in situ imaging of vicinal thiol-containing proteins (VDPs) and protein sulfenylation were realized based on CPD, NPE, S2and S3.1. A series of dimedone-based probes were designed and synthesized to trap protein sulfenic acids. The fluorescent probe CPD was taken for the further assay and in situ imaging in living cells. Compared to the reported probe for protein sulfenic acids, CPD had the following advantages:(1) The first fluorescent probe used in micromolar scale, which had negligible influence on the cellular ROS level;(2) CPD was a cell-permeable probe that can be applied in physiological condition;(3) In situ imaging experiments show the changes of sulfenic acids upon the perturbation of redox environment could be quantified by CPD in live cell as well. The colocalization of CPD with commercial subcellular localization probes revealed that the cellular protein sulfenylation occurred mainly in endoplasmic reticulum (ER) and mitochondria.2. The stable acceptor (VTA2) was synthesized through the introduction of1,2-ethanedithiol (EDT) to phenylarsine oxide (PAO). The photo-stable1,8-naphthalimide was taken as the fluorophore. Meanwhile,4,5position of naphthalimide were modified with the biocompatible diglycol amine to obtain fluorescent probe NPE. NPE was the first fluorescent probes reported to selectively detect endogenous vicinal thiol-containing proteins (VDPs). NPE had negligible cell toxicity during the micromole range, it was a cell-permeable probe that can be applied in physiological condition. Fluorescence polarization and electrophoresis assay displayed the high selectivity of NPE for VDPs. In situ imaging experiments showed that the changes of VDPs could be quantified by NPE in live cell as well. The colocalization of NPE with commercial subcellular localization probes revealed cellular VDPs were mainly localized in mitochondria.3. Based on VTA2, three fluorescent probes (S2, S3and S5) were designed and synthesized respectively to trap extracellular vicinal thiol-containing proteins by conjugation of membrane-impermeable fluorophore (Cy3, Cy5or fluorescein).S2and S3were taken as for the further study of their interaction with extracellular VDPs. rBSA was taken as the model protein, and fluorescence polarization assay showed good selectivity and quick reaction time of S2and S3. Electrophoresis assay further confirmed the selectivity of the probes. Negligible cytotoxicity of the probes were observed during the concentration range of0-25μM.The in situ imaging assay showed S5was still cell permeable while S2, S3can be selectively stained on cell membrane. In contrast, no fluorescent signal was obtained by using control probe Cy3or Cy5. Hence, S2and S3were confirmed to selectively detect vicinal thiol-containing proteins on membrane. Especially, S3was an infrared-red probe that can be applicable to vivo imaging.4. A FRET based fluorescent probe was designed and synthesized by conjugation of fluorescent donor and acceptor with VTAF derivative. This probe can realize the ratio detection of vicinal thiol-containing proteins (VDPs). The in vitro assay showed the specific interaction of VTAF with VDPs, and the dual fluorescent signal can be obtained at the wavelength of470nm and540nm respectively. This probe can reduce the interference of the background and increase the signal-noise ratio.