Design And Synthesis Of Novel Near-infrared Light-excited Fluorescent Probes For Bioimaging

Fluorescence imaging has been widely applied for exploring bioactive species and biological processes in living systems owing to its substantial merits,such as high sensitivity,convenient preparation,and capability of real-time sensing.However,the applications of traditional fluorescence imaging techniques in biological areas are limited by excitation with short wavelengths,which are easily subjected to high cellular autofluorescence,photobleaching of dyes,and restricted tissue-penetration depth(<100 μM).In sharp contrast,the two-photon microscopy(TPM)approach,which excites a two-photon(TP)fluorophore with near-infrared(NIR)laser pulses,can efficiently address these issues and provide improved three-dimension spatial localization with reduced photodamage to bio-samples and increased tissue penetration depth.Thus,two-photon fluorescence imaging can be used as a promising tool for analyzing living cells and tissues.Additionally,near-infrared(NIR)fluorescence imaging is another powerful technique for monitoring of biomolecules in living systems.Absorption and emission wavelengths of the near-infrared fluorescent dyes are in the near-infrared(NIR)region,which can avoid the background absorption and fluorescence to insure the detection sensitivity and selectivity.Moreover,near-infrared fluorescence imaging has additional advantages including minimum light scattering and deep tissue penetration with less photodamage to the bio-samples,making it an attracted technique in biological research.To promote the development of bioimaging technology,the design and construction of the novel two-photon fluorescent probes and near-infrared fluorescent probes with excellent performance are the critical point.In this thesis,we designed and synthesized a series of fluorescent probes based on naphthalene derivatives and HuDa NIR dye for detecting of biological-related anions and molecules(nitroxyl(HNO),SO2 derivatives,hydrogen peroxide(H2O2)and cysteine(Cys)).The main contents are presented as follows:(1)In the secondary chapter,a FRET-based two-photon fluorescent turn-on probe,A-HCy,was proposed for specific detection of SO2 derivatives through the bisulfite/sulfite-promoted Michael addition reaction.In this FRET system,an acedan(2-acetyl-6-dialkylaminonaphthalene)moiety was selected as a two-photon donor and a hemicyanine derivative served as both the quencher and the recognition unit for bisulfite/sulfite.A-HCy itself exhibited a very weak fluorescent signal because of the quenching effect of the acceptor.However,when the conjugated structure in the acceptor was interrupted by bisulfite(HSO3-)through the Michael addition reaction,a significant fluorescent enhancement was observed.A-HCy exhibited excellent selectivity and rapid response to HSO3-.More importantly,probe A-HCy was first successfully applied in two-photon fluorescence imaging of biological SO2 derivatives in living cells and tissues,suggesting its great potential for practical application in SO2-related biological researches.(2)In the third chapter,we have developed a FRET-based TP fluorescent pro be P-Np-Rhod for detecting HNO for the first time.In this FRET system,a T P naphthalene derivative served as the energy donor and a rhodol fluorophore w as chosen as the energy acceptor which was modified with a(diphenylphosphino)-benzoate moiety as a recognition unit for HNO.In the absence of HNO,the r hodol existed in a non-fluorescent lactone form and the FRET is off.After the reaction between HNO and triphenylphosphine group,a corresponding aza-ylide which can attack the adjacent ester linker in an intramolecular manner to releas e hydroxyl groups was obtained.And then the closed spirolactone form was con verted to a conjugated fluorescent xanthene form to induce the occurrence of F RET.Probe P-Np-Rhod displayed rapid response for HNO and a large emission shift(93 nm).More importantly,P-Np-Rhod could be succesfully applied for TP fluorescence imaging of HNO in living cells and tissues with less cross-talk between dual emission channels.(3)In the fourth chapter,based on the ICT mechanism,we reported a novel two-photon fluorescent probe,Nap-OH-H2O2,for monitoring hydrogen peroxide.Probe Nap-OH-H2O2 consists of a two-photon naphthalene backbone and a boric acid ester which was used as a H2O2 reporter.The reaction of probe Nap-OH-H2O2 with H2O2 triggers the cleavage of a boronate-based protecting group,and as a result,restores the fluorescence of compound Nap-OH.This probe exhibits excellent selectivity and high sensitivity to H2O2 with low cytotoxicity.Furthermore,probe Nap-OH-H2O2 was successfully applied to cell imaging of hydrogen peroxide with two-photon microscopy in live cells.(4)In the fifth chapter,a novel fluorine substituted near-infrared fluorescent dye CCF-OH was designed and synthesized.Compared with CC33(pKa ＝7.5),CCF-OH displayed a lower pKa(6.3)and the fluorescence signal of CCF-OH becomes more steadily around rang of pH>7.In order to further verify the practicability of CCF-OH,we develop a mitochondria-targeted near-infrared fluorescent probe,N-Cys,for detecting Cys by using the trigger moiety acrylate group as the recognition unit for Cys.Probe N-Cys responded to Cys with a ratiometric NIR fluorescence signal and could be used to image endogenously produced Cys in living cells.Furthermore,Probe N-Cys was capable of detecting endogenous Cys in living mice.