Synthesis And Application Of Two-photon Fluorescent Probes Based On Naphthalimide

Biological imaging technology as the best noninvasive way to study living systems in a natural setting has obtained widespread attention in recent years. Two-photon fluorescence microscope has become the indispensable tool in the field of biological imaging due to the advantages of high signal-to-noise ratio, localized excitation, deeper tissue penetration, less photodamage, prolonged observation time, and better three-dimensional imaging. TP fluorescent probes as the render of fluorescent signals play key roles in two-photon imaging. So far, there are many of the two-photon imaging material have been reported, among which two-photon fluorescent probes based on organic small molecular have been development vigorous owning the good cell permeability, fast imaging, high specificity and easy modification.This paper, we have designed and synthesized reaction-based two-photon fluorescence probes:Z1-Z4. Experimental results showed that probe Z1 can ratio fluorescence imaging fluorine ions in cells and tissues, probe Z2 and Z3 can be applied to imaging thiol specificity, Z4 both can be used to measure the content of nitrite in practical samples, and quantitatively monitor nitric oxide in the mitochondria. Our works in concrete are mainly as follows: 1. First, a reaction-based two-photonratiometric fluorescence probe Z1 was developed and successfully applied to detect and image fluoride ion in living cells and tissues. The Z1 probe was designed to utilize an ICT mechanism, n-butylnaphthalimide as a fluorophore and tert-butyldiphenylsilane as a response group. Upon addition of fluoride ion, the Si-O bond in the Z1 would be cleaved, and then a stronger electron-donating group was released. The fluorescent changes at 450 and 540 nm, respectively, made it possible to achieve ratiometric fluorescence detection. The results indicated that the Z1 could ratiometrically detect and image fluoride ion in living cells and tissues in a depth of 250 μm by two-photon microscopy. 2. Then, a two-photon fluorescence imaging probe Z2 was designed to detect biothiols through a photoinduced electron transferpathway utilizing N-butyl-naphthalimide as the fluorophore and 2,4-dinitrobenzene sulfonyl as the responsive group, which were linked together by piperazine. The synthesized Z2 displayed high selectivity to biothiols, significant fluorescence off–on property. Moreover, Z2 showed good biocompatibility and insensitivity toward changes in the biologically relevant p H range(7.2–8.4), which enabled the utilization of Z2 to monitor biothiol levels not only in live cells but also in tissues at depths of 50–250 μm.3. Based-on the previous work, devised and synthesized thiol fluorescent probes ortho-Z3, meta-Z3 and para-Z3 based on photoinduced electron transfer(PET) pathway utilizing maleimide as the responsive group was reported here. The fluorescence intensity of these probes varied significantly depending on the position of the maleimide toward the naphthalimide fluorophore. Comparing to the meta and paraisomers, the ortho isomer displayed the best fluorescence response performances upon addition of thiols. The results indicated that relative position of the electron acceptor played key rolein the PET process, and probe ortho-Z3 could detect endogenous biothiols in serum samples and live cells successfully. 4. On the basis of the above works, a two-photon ratiometric fluorescence probe Z4 for detection of NO/NO2- and imaging of NO in mitochondria have been designed. Experiment results showed that, probe Z4 could be used to detect nitrite and nitric oxide. Probe Z4 has been successfully applied to quantitative detect nitrite in the actual samples and ratiometric imaging nitric oxide in mitochondria.