Design,Synthesis And Application Of Novel Near Infrared Fluorescent Probes For Detection Of Biothiols

The small biothiols in the living systems contain cysteine(Cys),homocysteine(Hcy),and glutathione(GSH).They play crucial roles in assisting protein synthesis and toxin elimination,maintaining the body's normal immune function,adjusting the stability of the intracellular redox balance and so on.The abnormal levels of these biothiols correlate with variety of diseases.Therefore,the detection of the concentration levels of those thiols in the living systems has scientific significance for the prevention and diagnosis of diseases,the study of physiological pathology,ect.Compared with traditional detection methods such as electrochemical analysis,surface-enhanced Raman scattering(SERS)and chromatographic analysis,fluorescent probes have drawn considerable attention because of their advantages such as good selectivity,high sensitivity and short response time.The near-infrared fluorescent(650-900 nm)probe with a longer emmision wavelength,stronger tissue penetrating ability,lower cell damage and so on,becomes one of the research hotspots in recent years.In this paper,a near-infrared fluorescent probe(NIR-GP)with good water-soluble ability was designed.NIR-GP was synthesized by taking Rhodamine as the basic structure and introducing the Fischer aldehyde to expanding conjugate system,which was based on the ring opening and closing to interrupt and restore the conjugate system to detect GSH.The UV-vis absorption and fluorescent spectra of NIR-GP with different amino acids such as GSH,Cys,Hcy,and HS-,S2-sulfur-containing ions indicated that the probe exhibited good detection ability for GSH.The fluorescence wavelength reached to 745 nm.The concentration,anti-interference and stability experiments demonstrated that NIR-GP had good detection capability for GSH,and the detection limit was 2.5 ?M.Simultaneously,NIR-GP could successfully discriminate and image the endogenous GSH with low cytotoxicity and good biocompatibility.From the above study,it is found that NIR-GP could discriminate GSH from other analytes in pure water,but the Stokes shift was small.In order to further improve the water solubility of the probe and increase the Stokes shift,we designed and synthesized a water-soluble Cys probe NIR-Py.We used xanthene derivative as the basic structure and acrylate as the recognition group,expanding conjugate system by introducing a water-soluble morpholine-pyridinium salt structure.The UV-vis absorption and fluorescent spectra of NIR-Py with different amino acids such as GSH,Cys,Hcy,and HS-,S2-sulfur-containing ions indicated that NIR-Py could selectively recognize Cys in PBS(10 mM)with a maximum emission peak at 675 nm,the Stokes shift was 149 nm.The concentration and anti-interference experiments demonstrated that NIR-Py exhibited good detection ability for Cys and the detection limit was 0.96 ?M.Through the analysis by HRMS,TLC,UV-vis and fluorescent spectra,the recognition mechanism of the fluorescent probe should be the Michael addition and cyclization reaction between Cys and probe that released fluorophore,followed by Intramolecular Charge Transfer(ICT)process to get the fluorescent signal at 675 nm.At the same time,NIR-Py has low cytotoxicity and good biocompatibility,enabling intracellular Cys fluorescence imaging.Based on excited-state intermolecular proton transfer(ESIPT),we designed and synthesized a fluorescent probe HBT-Py by combining morpholine-pyridine salt structure with 2-(2'-hydroxyphenyl)benzothiazole,which could recognize both glutathione and arginine(Arg)simultaneously.The UV-vis absorption and fluorescent spectra of HBT-Py with different amino acids such as GSH,Cys,Hcy,Arg and HS-,S2-sulfur-containing ions indicated that HBT-Py was able to uniquely recognize GSH and Arg under exactly the same test conditions.The emission wavelengths of GSH and Arg were 684 nm(near-infrared regions)and 469 nm(blue regions)excited by 400 nm,respectively,and the Stokes shift were 284 nm(GSH)and 69 nm(Arg).The concentration,anti-interference experiments demonstrated that HBT-Py existed good detection ability for GSH and Arg,the detection limits were 0.24 ?M(GSH)and 2.2 ?M(Arg),respectively.Through the analysis by HRMS,TLC,UV-vis and FL spectra,the recognition mechanism of the fluorescent probe for GSH should be the Michael addition and cyclization reaction between GSH and the probe that released fluorophore,followed by an Excited-state Intermolecular Proton Transfer(ESIPT)process to generate the fluorescence in near infrared region;the recognition mechanism for Arg should be that the oxyanion released by nucleophilic substitution between Arg and the ester group formed hydrogen bonds with Arg,which blocked the ESIPT(ESIPT-blocked)process and enhanced fluorescence in blue region.