The Design, Synthesis And Applications Of Two-Photon Fluorescent Probe Based On Novel Fluorophores

Two-photon excitation fluorescence (TPEF) is a kind of anti-Stokes luminescence, which can be excited by near infrared light and give emission in visible region. In biological applications, using NIR light as excitation light source can effectively avoid interference from autofluorescence and scattered light, reduce photodamage and photobleaching, as well as improve penetration and spatial resolution. Two-photon excitation fluorescence has attracted increasing attention recent years and exhibits broad application prospects. Utilizing two-photon fluorescent probes for bioanalysis and bioimaging can overcome many disadvantages of one-photon fluorescent probes and is significant to achieve dynamic, real-time and in situ detection. Although various two-photon materials with favorable two-photon absorption cross sections have been developed, most of them are not appropriate for bioanalysis and bioimaging because of large structure, poor solubility and low quantum yield in water. Lack of two-photon fluorophores for biological applications has limited the development of two-photon probes. Thus, it is vital to design novel fluorophores and improve two-photon properties for further study.This dissertation focuses on developing various fluorophores which are appropriate for biological applications as well as possessing outstanding two-photon properties. Based on different luminous mechanisms and detection targets, a series of two-photon fluorescent probes are designed and synthesized. The main contents are as follows:(1) Two-photon calcium fluorescent probes TP-BAPTA and TP-CN-BAPTA are designed and synthesized utilizing stilbene derivatives as fluorophores. Both fluorophores have quadrupole configurations as D-π-D and D-π-A-π-D scaffolds, respectively (where D is an electron donator, A is an eclectron acceptor and π represents conjugate bridge). The fluorophore and receptor are connected through π-bond. Due to internal charge transfer (ICT), fluorescence of the probes quenches after binding to Ca2+. TP-BAPTA has a short conjugate chain which leads to its small two-photon absorption (TPA) cross section. The conjugate chain of TP-CN-BAPTA is longer than TP-BAPTA. In the meanwhile, cyanogroups are introduced in the core. Thus, TP-CN-BAPTA has a larger TPA cross section value and longer emission wavelength compared to TP-BAPTA. However, such a long conjugate structure is easy to cause molecular distortion especially in polar sovlents, resulting in a decrease of the quantum yield. Both probes featured high affinity, good selectivity, low cytotoxicity and pH-insensitivity in physiological range. TP-CN-BAPTA can be used for two-photon microscopy (TPM) imaging and also show responses to intracellular calcium.(2) Utilizing fluorene derivatives as fluorophores, two-photon calcium fluorescent probes FCal and FCa2are synthesized. Both fluorophores are D-π-A dipole scaffolds, which behave obvious solvatochromism. The fluorophore and receptor are connected through σ-bond according to photoinduced electron transfer (PET) principle. Both probes show20-22fold fluorescence enhancement in response to Ca2+high quantum yield in buffer solutions and maximum TP action cross section values of87-90GM under740nm excitation, which are rather larger than that of traditional probes. Besides, the probes have medium affinity and high selectivity toward Ca2+and are also pH-insensitivie in the biologically relevant range.(3) Fluorene derivatives are employed as fluorophores to synthesize a couple of two-photon magnesium fluorescent probes FMgl and FMg2. Photophysical properties of the probes are similar to the previous calcium probes. In response to Mg2+, FMgl and FMg2exhibit16-18fold fluorescence increase with quantum yields of0.12and0.18in buffer solutions and two-photon action cross section values of76-87GM, which demonstrate that this kind of fluorophores based on fluorene derivatives can be extensively used for synthesizing two-photon fluorescent probes due to the excellent photophysical properties. Both probes can also be applied for monitoring cytoplasmic Mg2+by TPM imaging. Combined with BCaM, a two-photon fluorescent probe for near-membrane Ca2+, FMg2allows dual-color imaging of Ca2+/Mg2+activities in live cells and [Mg2+]i/[Ca2+]m distributions in live tissues at a depth of100-200μm.(4) A two-photon nitric oxide probe QNO is synthesized based on quinoline fluorophore. The fluorophore possesses a D-π-A dipole scaffold with alkylamine group as the electron donor and benzothiazole group as the electron acceptor, which behaves obvious solvatochromism. o-phenylenediamine is used as receptor and shows high sensitivity and selectivity to nitric oxide. The fluorophore and receptor are connected through amide bond according to photoinduced electron transfer (PET) principle. QNO exhibits12-fold fluorescence enhancement in response to nitric oxide with quantum yield of0.2and two-photon action cross section value of52GM under810nm excitation, which demonstrate this quinoline fluorophore possesses favorable two-photon properties and tremendous application potential.