JACS: azulene derived two-photon fluorescence probe for biological imaging of ROS / RNs

ROS and RNS are important intermediates in many physiological and pathological processes High hydrogen peroxide (H 2O 2) production is closely related to the pathogenesis of many diseases such as cancer and neurodegenerative diseases Similarly, peroxynitrite (ONOO -) can react uncontrollably with various biomolecules due to its strong oxidation and nitrification, which is closely related to various cardiovascular diseases, neurodegenerative diseases, inflammation and cancer Based on this, it is very urgent to use reasonable technology to clarify the role of ROS / RNs in biological system The two-photon fluorescence probe has the excitation wavelength of near-infrared region and deeper tissue penetration, which is a good choice to solve the above problems Recently, Hwan Myung Kim of Asian University in Korea and Tony D James, Simon E Lewis of Bath University in the UK reported a new bioimaging fluorescence group azulene, which has become an effective two-photon fluorescence probe after being connected with the borate receptor motif, and can be used for the detection of active oxygen Good cell penetration, excellent peroxynitrite selectivity, photostability and very low cytotoxicity Relevant achievements were published on J am Chem SOC (DOI: 10.1021 / JACS 9b09813) under the title of "azulene derived fluorescent probe for bioimaging: detection of reactive oxygen and nitrogen specifications by two photon microscopy" (source: J am Chem SOC.) azulene is a kind of non substituted bicyclic aromatic hydrocarbon, which is isomerized with naphthalene, but has different properties It has a very high intrinsic dipole moment (1.08d) Because of the resonance structure, the smaller ring has a higher electronic density and the two rings are both discrete 6 π aromatic system (scheme 1a) In the presence of ROS / RNs, the C-B bond of the borate group linked to the seven membered ring is oxidized to form a C-O bond, which shows the change of fluorescence (scheme 1b) This is because the borate is scheme 1c and the hydroxyl is scheme 1D The electronic effect of borate weakens the intrinsic polarization of azulene, which leads to the decrease of the intramolecular charge transfer (ICT) effect in az-6-bpin system, while the hydroxyl group enhances the intrinsic polarization of azulene, which makes the ICT effect and fluorescence in az-6-oh system stronger (source: J am Chem SOC.) 6-hydroxyazulene has poor stability due to the existence of keto tautomers Therefore, the author obtained the final probe 1 (scheme 2a) by introducing the ester group at position 1 and 3 of azulene and the electron donor amino group at position 2 The substitution of amino group enhanced the resonance effect (scheme 2b) which was in contradiction with the inherent polarity The ester group not only increased the stability of product 2, but also enhanced the resonance effect, thus enhancing the inherent polarity (scheme 2C) of azulene (source: j.am Chem SOC.) calculated the DFT of probe molecule 1 and oxidation product 2, and obtained the molecular orbitals (Homo, LUMO and LUMO + 1) of each compound (Figure 1) In addition, the author was lucky to get the crystal structure of compound 2 (Figure 2) in THF / n-hexane (source: J am Chem SOC.) then, the fluorescence emission spectrum of the probe was measured in PBS buffer / methanol system (Figure 3) Under the excitation of 350 nm light, probe 1 showed no fluorescence, while oxidation product 2 showed a bright fluorescence signal at 483 nm (source: J am Chem SOC.) pH titration experiment showed that the fluorescence intensity of compound 2 decreased significantly at lower pH or pH higher than 8, and it showed the maximum fluorescence intensity (Figure 4) at pH 7-8, which made the probe feasible for cell imaging (source: J am Chem SOC.) then, the author tested the selectivity of probe 1 to ROS / RNs Many literatures have confirmed that borate receptor has response to ONOO - and H 2O 2, but the high nucleophilic of ONOO - makes its reaction rate much higher than H 2O 2 The results showed that probe 1 showed high selectivity to ONOO - at 5 and 30 minutes, but almost no response to H 2O 2 (Figure 5) (source: J am Chem SOC.) in order to further explore the ability of probe 1 to detect ROS in living cells, the authors measured the fluorescence intensity of 1 in raw 264.7 macrophages pretreated with H 2O 2 (2 mm) and ONOO - (100 μ m) respectively Compared with the negative control group stained with probe 1 only, the cells pretreated with ROS showed three times fluorescence enhancement (Figure 6B, c) In addition, PMA, LPS / IFN - γ were used to stimulate macrophages to release ROS, or SIN-1 was used to pretreat cells, then probe 1 was used to dye cells, the fluorescence intensity increased significantly (Figure 6D, e, g) The fluorescence intensity induced by ROS decreased significantly (Figure 6F, H) All the above results indicate that probe 1 can be used to detect ONOO - and H 2O 2 in vivo (source: J am Chem SOC.) finally, the authors explored the ability of probe 1 to visualize ONOO - and H 2O 2 in vivo The hippocampal sections of rats were stained with probe 1 In accordance with the results of cell experiments, TPM images showed that it showed weak fluorescence signal (Figure 7a, e), while PMA and SIN-1 processing enhanced fluorescence signal (Figure 7b, c), and ebselen processing weakened fluorescence signal (Figure 7d, f) This confirmed that probe 1 can be used to detect ROS in tissues (source: J am Chem SOC.) in a word, the author designed and synthesized a new two-photon fluorescence probe based on azulene fluorophore, which has better cell permeability, photostability and lower cytotoxicity, and can be used for the detection of peroxynitrite and hydrogen peroxide in cells and living tissues.