Design Of A Fluorescent Small Molecule For Probing Sulfenic Acid Protein And In Situ Imaging Of Living Cells

Oxidative stress has been proved to be closely related to many diseases,especially neurodegenerative diseases like Alzheimer’s disease and Parkinson’s disease.During oxidative stress reaction,the sulfhydryl group of protein was first oxidized to sulfenic acid as a crucial intermediate which could be reduced to sulfhydryl group,or oxidized to sulfonic acid and finally sulfite as an irreversible product that results in persistent damage of protein.Sulfenic acid protein was a kind of importantly transient marker for oxidative stress in organisms and cells.The development of a method for the direct detection of sulfenic acid proteins,especially the degree of sulfenic acid of sensitive proteins involved in specific signaling pathways in vivo,was very important to study the mechanism of oxidative stress regulation and repair for oxidative damage.Nowadays,the immunological method was commonly used for the detection of sulfenic acid proteins.It suffers from the low sensitivity due to the steric hindrance around the sulfenic acid group.On the contrary,small molecule probes were insensitive to steric hindrance around the sulfenic acid group,and more versatile for probing different proteins.But the direct detection of sulfenic acid proteins by small molecules were unavailable.A series of fluorescent probes were designed using8-amino-1,3,6-naphthalene trisulfonate disodium as the fluorescent group,cyanoacetic acid,3,5-cyclohexanedione carboxylic acid and（phenylsulfonyl）acetic acid as reaction groups and bromoethylamine and p-aminobenzoic acid as link groups.Their fluorescence spectroscopic properties at different temperatures and pH were characterized;BSA after the treatment of H₂O₂ was used as the model for characterizing the FRET between probes and sulfenic acid BSA,and also the labeling selectivity in the presence of different metal ions and solvents;while the lung cancer epithelial cell A549 after the treatment of H₂O₂ as the model for imaging the sulfenic acid proteins in cells.After screening,the optimal probe8-（2-cyanoacetamide）-6-sulfonated naphthalene-1,3-disulfonate（Probe-I）was chosen,and its stability in the presence of oxidants and reductants as well as the lipid-water partition coefficient were analyzed.Lung cancer epithelial cell A549 and normal lung epithelial cell BEAS-2B were used as cell models for imaging of living cells by Probe-I,the toxicity to cells was detect by MTT assay.Under optimal staining conditions of probes for cells,the in-situ imaging in cells by Probe-I and intracellular locations were examined by fluorescence co-localization with commercialized intracellular probes.Compared with the reported fluorescent probes for the detection of sulfenic acid proteins,Probe-I showed the following advantages:1)The probe was insensitive to pH and temperature;the fluorescence spectra remained unchanged at pH 4¹0 and 0⁹0℃.2)H₂O₂ and DTT had little effect on the fluorescence spectra of the probe.3)The lipid-water partition coefficient LogP=0.1±0.03（n=3）guaranteed the permeability.4)FRET effect between the probe and sulfenic acid albumin was strong to support the sensitivity,and the negligible interference of different ions and solvents support the high selectivity of the probe.5)MTT assay indicated that the probe-I was nontoxic at 0²00μM to two types of cells.The optimized labeling conditions of the probe in lung cancer cell A549 and normal human lung epithelial cell BEAS-2B were the oxidation with H₂O₂ for 5min at 2 mM and 1 mM,respectively,the labeling with the probe at 100mM for 4 h.Intracellular co-localizated with commercialized probes showed the nucleolus was mainly imaged by the probe.In conclusion,the designed fluorescent probe-I could react directly with sulfenic acid group in the protein and there were strong FRET occured with tryptophan as the donor.thus probe-I with high stability at different pH,temperatures and redox reagents,and appropriate lipo-water partition coefficient for high permeability was effective for in situ imaging sulfenic acid proteins in cells,especially cell nucleolus.