Theoretical Investigation On Two-photon Fluorescent Probes For Toxic Heavy Metal Ions(Hg²⁺ And Pd²⁺) And Important Enzymes ??-Gal And CYP1A?

During the past decade,two-photon microscopy(TPM),which utilizes two near-infrared(NIR)photons as the excitation source,has been emerged as a new and indispensable imaging tool for biomedical research.Compared with the traditional one-photon fluorescence microscopy(OPFM),the two-photon fluorescence microscopy(TPFM)has a lot of outstanding advantages: strong penetration,accurate positioning,high resolution,little photobleaching and photodamage.Additionally,TPFM can get lower signal noise ratio(SNR)and dark field imaging.Considering the excellent properties,TPFM has great practical significance for the further development and application of biological detection.The combination of two-photon(TP)fluorescence probe and TPFM technology has become an imortant imaging-detection tool in biomedical application,meanwhile,organic TP fluorescent materials welcomed new opportunities and challenges.Up to now,TP fluorescent probes that can be effectively used in practical applications are still rare,which seriously limit their application in the biomedical imaging detection.So there is an urgent need to research and develop more excellent TP fluorescent probes.In this thesis,the chemical reaction type TP fluorescent probes for toxic heavy metal mercury ion(Hg2+),palladium ion(Pd2+)and important enzymes ?-galactosidase(?-Gal),cytochrome P450 1A(CYP1A)and its corresponding product molecules,are investigated from the theoretical chemistry view.The main purpose is to deeply reveal the internal relationship and regularity between the molecular structure and two-photon absorption(TPA)optical properties,and provide a valuable theoretical basis for designing more excellent TP fluorescent probes.The main contents are summarized as follows: 1.Theoretical study on two-photon fluorescent probe for toxic heavy metal mercury ionAs one kind of special heavy and enormous toxicity element,mercury is highly toxic.Once mercury enters the human body,trace amounts of mercury can cause severe mercury poisoning such as headache,Parkinson's disease and Minamata disease.Therefore,it is very important to effectively detect mercury ion(Hg2+)in vivo.In order to achieve good imaging detection of Hg2+,we designed a series of novel Hg2+ TP fluorescence probes DTA(1-9)and the corresponding products DMAA(1-9)based on the intramolecular charge transfer(ICT)mechanism.The geometrical and electronic structural characteristics of these fluorescent molecules,as well as the optical properties of one-photon absorption(OPA),emission and TPA have been studied theoretically by DFT and TDDFT methods.The results show that extending ? conjugation length results in red-shift of the OPA,emission and TPA spectra,lower excitation energy,and increased TPA cross-section(?max)values.The probes DTA6 and DTA8 and their corresponding reaction products DMAA6 and DMAA8,respectively,have excellent TPA properties and larger ?max values in the NIR,making them the most promising ratio TP Hg2+ fluorescence sensors in biological systems.We hope this study will provide useful information for synthesizing novel Hg2+ TP fluorescent molecules with good TPA properties in the NIR.2.Theoretical study on two-photon fluorescent probe for toxic heavy metal palladium ionsPalladium is emitted due to automobile catalytic converters,and with rapid growth in the number of cars,palladium ion(Pd2+)becomes a current threat to human health and environment.TP fluorescent probes are favorable and powerful molecular tools for Pd2+ detection due to localized excitation and reduced phototoxicity and photodamage.Basing on the ?-center expanded coumarin derivative,we designed a series of novel Pd2+ probes(R1-9)and the corresponding reaction products(P'1-9).The program systematically studied their electronic structures,OPA and emission properties.In addition,their two-photon absorption properties were calculated using the quadratic response theory combining DALTON program.The fluorescence of the synthesized P'1 molecule is enhanced because auxochromic and electron-donating –OH group of P'1 replacing a large –OCH2CCH group of probe R1,reduced the geometrical relaxation and vibrational relaxation energies in the excited states and increased intramolecular charge transfer.Introducing electron-withdrawing groups(–Cl or –CN)at the 4-position can effectively reduce the H?L energy gap,red shift the absorption and emission spectra,increase the transfer dipole moment,charge transfer and TPA cross-section value per molecular weight.The degined product molecule P'4 has a smaller internal conversion rate(KIC: 1.28×106 s-1 smaller than 9.09 × 1011 s-1)than the corresponding probe R4,and has the largest TPA cross-section value per molecular weight(3.91)and the longest fluorescence wavelength(590.3 nm).Thus R4 is the most promising probe among all the designed Pd2+ probes.It is hoped that this detailed study can provide guidance for the design and synthesis of new Pd2+ fluorescent probes.3.Theoretical study on two-photon fluorescent probe for biological ?-galactosidase enzymeAs a marker enzyme,biological ?-galactosidase(?-Gal)is well characterized and extensively applied in distinguishing cell types or specific haptens,especially in situ hybridizations,classification of mycobacteria,gene expression studies as well as senescent human cells in aging skin in vivo.However,the underlying biological mechanisms of ?-Gal and its role in senescence and aging still remain unknown.We investigated a series of novel ?-Gal ratio TP fluorescent probes by introducing different biological linkers(quinoline,benzo[d]thiophene,thiophene,diazine)and cyano groups into naphthalene fluorophore.The results show that the product molecules generally show longer absorption and emission wavelengths,stronger TP transition probability and larger net charge transfer than those of corresponding ?-Gal probes,due to their better molecular rigidity and larger transition dipole moment.Additionally,the net charge change dominated the TPA cross section(?max).Among the studied molecules,there is an optimal match for ?max and radiation rate(kr)following the increase of the net charge change to improve nonlinear optical activity.The substitution of benzo[d]thiazole with benzo[d]thiophene is an optimal approach for ?-Gal probe design with the largest fluorescence efficiency and ?max.So SG3 is the most promising biological TP ratio ?-gal fluorescent probe.4.Theoretical study on two-photon fluorescent probe for biological Cytochrome P4501A enzymeAs an enzyme with terminal oxygen,CYP1 A subfamily possesses the ability for catalyzing lots of environmental toxins,endogenous substrates and clinical drugs.Developing efficient methods for rapid detection and real-time monitoring of CYP1 A enzyme activity in complex biological system is of considerable significance to identify the potential abnormalities of cancer-involved enzyme.Toward this goal,we have firstly provided a series of 1,8-naphthalimide-based TP fluorescent chromophores with large TPA cross section(500 ~ 7000 GM)and remarkable changes in fluorescence spectra upon recognizing CYP1 A enzyme from theoretical aspect.Moreover,we have thoroughly studied the effects of heterocyclic acceptors(dichlorobenzene and benzothiadiazole)and donors(fluorene and carbazole)groups on the OPA,TPA,as well as the fluorescence properties of CYP1 A enzyme probes and the corresponding reaction products.The electron donating properties of the ?O? group in the ion product caused more charge transfer and longer charge transfer distance in the transition;secondly,the more 2Pz incorporation of N atom in NCON-1 contributing to ? bond(with adjacent C)and larger ? delocalized electronic structure on the whole ionic product NCON-1,resulted into smaller energy gap ?EL-H(3.20 e V),leading to the spectral redshift of product.The docking analysis suggested that the designed TP probes NCMN-3 and NCMN-5 can guarantee and achieve the excellent selectivity for CYP1 A enzyme,proving the rationality of this design strategy.