Construction And Application Of Bodipy-based Near-infrared Fluorescent Probe For Sensing Biological Thiols

Thiol-containing small molecules,such as cysteine(Cys),homocysteine(Hcy)and glutathione(GSH)are very important reactive sulfur species(RSS)in cells.They play crucial roles in the biochemical process of animals and plants,the metabolism of microscopic organisms,food processing and other aspects.However,the chemical structures and reactivity of Cys,Hcy and GSH are very similar,especially the side chains of Cys and Hcy differ by only one methylene.Therefore,it is a great challenge to distinguish the three or to identify a biothiol specifically.Among the common detection methods for bioactive small molecule compounds,fluorescent probe detection technology has attracted the attention of researchers due to its unique advantages.In particular,near-infrared(650-900 nm)fluorescent probes which have the advantages of minimal damage,less light scattering,deep tissue penetration and low background interference are more beneficial to the biological system to monitor small molecule compounds.In this paper,BODIPY fluorescent dye with favorable properties was selected as the parent nucleus structure through literature investigation and previous research of our group.By adjusting the conjugated structure of BODIPY and introducing various recognition groups to construct the probe,we expect the probe can achieve discrimination or specific detection of biological mercaptan compounds in the near infrared region,and explore the influence of the change of conjugation degree and recognition groups on the reactivity of a series of probes.In the second chapter,we modified the parent structure of the BODIPY dye,and used 1,3,3-trimethyl-2-methylene indolline acetaldehyde and BODIPY to form extended conjugation BODIPY through Knoevenagel condensation,which provided probe to reach near-infrared region.And at the same time,the 4-methoxyphenyl sulfide identified by our research group was introduced into the meso pisition of BODIPY as the detection group,which can be easily cleaved when the SNAr substitution reaction occurred.Based on the above,we constructed fluorescent probe(BDP-NIR),and expect that the probe would be extended to NIR region with conjugated olefinic bond so as to realize the discrimination or specific detection of Cys Hcy and GSH.Through in vitro investigations of the response to biothiol with BDP-NIR,the probe was found to respond to Cys in the near infrared region,and has a large Stokes shift(105 nm),high sensitivity and selectivity.The linear dynamic range from 0-500 ?M of the probe basically covers the physiological level of Cys in vivo,which is suitable for the quantitative detection of Cys in biological systems.Moreover,a novel quenching mechanism of fluorescence was supported by density functional theory(DFT)calculation.The reason for the fluorescence quenching of the probe may go through intersystem crossing(from singlet excited state to triplet excited state).More importantly,the probe has also achieved specific detection of Cys in the research of cell and live animal imaging applications.In the third chapter,in order to further explore the conjugation effect and the capability of the new detection group on the detecting wavelength region and activity for distinguishing biothiols(Cys,Hcy,and GSH)or specific detection,we introduced phenylselenyl ether into the meso site of BODIPY as recognition group.With gradually increased the conjugation degree of 3-and 5-substituents,we designed and synthesized probe BDP-Se-1,BDP-Se-2,BDP-Se-3 to compare the detecting behaviors and biological imaging applications.Through investigations of detecting of biothiols with these probes in solution,we found that the SNAr substitution reaction between the new recognition group and the biothiol was faster,and with the difference in the degree of conjugation,the selectivity and detecting wavelength region of the probe to Cys,Hcy and GSH presented significant difference: BDP-Se-1 without extended conjugation can detect Cys and Hcy/GSH in the green light region,but its selectivity was poor with potential response to ROS;on the contrary,the conjugated BDP-Se-2 can selectively detect Cys and Hcy in the yellow light region;fully extended conjugation in BDP-Se-3 allowed specifically response to Cys in the red light region.Both BDP-Se-2 and BDP-Se-3 did not respond to ROS.In addition,the reaction between BDP-Se-3 and Cys can be completed within 10 minutes at room temperature.The probe has been applied to the detection of endogenous Cys in Hela cells by confocal fluorescence imaging.