Synthesis And Application Of Coumarin-based Near-infrared Probes

Fluorescent probes are widely used in the detection of various heavy metals and amino acids due to their high sensitivity and good selectivity.The study of near-infrared functional fluorescent probes for in vivo and pathological applications has become a trend in recent years.Although many fluorescent probes are constantly being developed,it is not enough to reach the near-infrared and be applied to the detection of living organisms.Therefore,coumarin-based near-infrared fluorescent probes are widely used due to their superior fluorescence detection performance.Based on the advantages of coumarin and near-infrared,several different fluorescent probes with good permeability,large Stokes shift and high sensitivity were designed and synthesized.According to different coumarin precursors,two series of coumarin precursors,3-amino-7-hydroxycoumarin（HL）and6-amino-3-ethoxycarbonylcoumarin（CL）,were studied and synthesized.Among them,HL3and HL4 can detect amino acids,and CL4 and CL5 can detect metal ions and amino acids.This subject is based on the complexation mechanism and the coordination of N atoms,metal ions and amino acids contained in the coumarin precursor itself.The HL series of probes tested the fluorescence properties and specificity of HL3 and HL4.The probe HL3 is in the medium of MeCN/H₂O（6:4,v/v）,and the probe HL3 responds to His off-type fluorescence.After adding His to the probe HL3,with the excitation wavelength of 370 nm,the selectivity change of the probe HL3 to His cannot be observed in the ultraviolet,but the fluorescence intensity can be observed in the fluorescence emission spectrum at 638nm.After quenching,the fluorescence will stabilize within 15 s.It can be observed by fluorescence spectroscopy that the probe HL3 is a specific selection for His,and there is no obvious change after adding other competing amino acids.Through the curve determination of different concentrations of His,the minimum detection limit of His was calculated to be1.2μM.A possible detection mechanism was proposed by testing and characterizing the fluorescence properties of the probe HL3.The probe HL4 was introduced into the structure of isophor nitrile,so that a push-pull system was formed in the molecule.At the excitation wavelength of 422 nm,the probe HL4 was found to specifically detect Lys in the medium of DMF/H₂O（9:1,v/v）.In the emission fluorescence spectrum at 696 nm,the probe HL4 is a fluorescence-on-type probe to Lys,and the anti-interference effect is better after adding other competing amino acids.Through the titration experiments of different concentrations of Lys,the lowest detection limit was calculated to be 0.6μM,the complexation ratio of probe to Lys was 1:1,and the response time was 42 min.Through the stability test of the probe pH,it was found that the probe HL4 was relatively stable at pH 5-11.A possible mechanism is proposed by fluorescence testing and characterization methods.The CL series of probes tested the fluorescence properties and specificity of CL4 and CL5.The probe CL4 can be used to detect Arg and His in the medium of MeCN/H₂O（1:9,v/v）,and the fluorescence is reduced.Compared with other amino acids,the detection effect of Arg and His is poor,but the anti-interference is better.Through the titration experiments of different concentrations of Arg and His,the minimum detection limits were calculated to be 0.8μM and 0.9μM,respectively.Probe CL4 has a good detection effect for detecting Cu²⁺.It can specifically recognize Cu²⁺in the medium of MeCN/H₂O（1:9,v/v）,and the fluorescence is quenched after adding Cu²⁺.Because the structure contains C=S,it will make Ag⁺/Hg²⁺have some interference on the detection of Cu²⁺by probe CL4.Through the titration experiments of different concentrations of Cu²⁺,the lowest detection limit was calculated to be 0.9μM,and the complexation ratio of probe CL4 to Cu²⁺was 2:1.A possible detection mechanism is proposed by fluorescence testing and characterization methods.Probe CL5 can specifically detect Lys and Cu²⁺in the medium of DMF/H₂O（9:1,v/v）.At the excitation wavelength of410 nm,after adding Lys to the probe CL5,the fluorescence was enhanced at the emission wavelength of 699 nm,and the color changed from yellow to green,and the anti-interference effect was good.The detection limit was 0.3μM and the response time was 6 min.The complexation ratio of probe CL5 and Lys is 1:1.Adding Cu²⁺to probe CL5,the fluorescence is quenched,the color changes from yellow to red,and the anti-interference effect is good,the detection limit is 0.8μM,and the response time is 20 s.The complexation ratio is 1:1.Probe CL5 has a reversible effect,and the detection of Cu²⁺can further detect biological thiols.When biothiol was added to the probe CL5-Cu²⁺,the fluorescence enhancement returned to the fluorescence intensity of the original probe CL5,but the fluorescence intensity increased more obviously after the addition of GSH.The detection limits of Cys,Hcy,and GSH were 1.1μM,0.4μM,0.3μM.In addition,the probe can be used to detect Cu²⁺multiple times by its reversibility.At the same time,the probe CL5 was also successfully applied in Hela cells.