Design,Synthesis And Properties Of Mitochondria-targeted Two-photon Fluorescent Sensors For The Detection Of Au³⁺

The recognition of biologically and environmentally important species in addition to imaging them have been an important research task in recent years.Compared to other analytical tools,small-molecular fluorescent sensors have several merits,including simplicity,low detection limit,high selectivity,nondestructive.And most importantly,with the aid of confocal microscopy,they are also powerful tools for monitoring biologically relevant species in vitro and/or in vivo.In comparison with one-photon microscopy?OPM?,two-photon microscopy?TPM?,which employs two near-infrared photons as the excitation source,offers several superiorities,including the capability of imaging deep inside a tissue,higher spacial resolution and longer observation time.Moreover,with the advent of TPM,recent developments on two-photon fluorescent sensors have enabled the visualization of biologically important analytes in living tissues and in the mouse.Furthermore,small-molecular fluorescent sensors have been flourished in the past decade,have been widely used in chemistry,biochemistry,medicine,environment and other fields.Mitochondria are membrane-bound organelles as well as the principal energy-producing compartments,play a pivotal role in diverse essential physiological processes in eukaryotic cells and in diseases.Because the gold lipophilic complex could be accumulated in mitochondria of cells,several Au?III?complexes are now considered as potential anticancer agents,which have been evidenced for involving mitochondrial cell death pathways.Nevertheless,gold ions will be highly toxic in certain biological systems by irreversible interaction with biomolecules,and finally will bring about serious damage to the liver,kidneys,and the peripheral nervous system.Especially Au³⁺ ions have the strong affinity to DNA,which will lead to the serious damage of DNA.Therefore,it's urgent for us to develop effective tools to specific,rapid,real-time monitoring Au³⁺ ions in the environment,agrochemical and potential living systems.By reviewing and discussing a great deal of literatures,on the basis of our previous work,via the structure modification of small-molecular fluorescent sensors,especially focusing on the coordination mode and type of coordination atoms in the receptor,we designed and synthesized several novel highly selective two-photon fluorescent sensors for Au³⁺.The structures of these two-photon fluorescent sensors were characterized by 1H NMR?13C NMR?MS and other methods.And their optical properties were investigated through UV-vis and fluorescent spectra etc.In addition,they were applied to cell imaging,mitochondrial co-localization imaging,tissue imaging,some of them were also carried out in vivo imaging,and made a series of meaningful results.This paper is mainly composed of three parts as follows:1.Brief introduction of the two-photon fluorescence,two-photon microscopy technology and mitochondria-targeted fluorescent sensors.2.Recently,our group presented a mitochondria-targeted two-photon fluorescent sensor?PyCM?for the detection of gold ions,which functioned by gold ions-mediated C=N bond hydrolysis mechanism.However,the sensor PyCM cannot make a distinction between Au³⁺ and Au+,probably due to the similar affinity of"N^N" combination to Au³⁺ and Au+ in the receptor of PyCM.According to the hard soft acid base?HSAB?principle,hard donor atoms such as N and O were often used for binding the hard metal ion Au³⁺,we envisioned,by modulating "N^N"combination in sensor PyCM to "N^O" combination in this work,that the compound 4 can be a good point of departure to develop an Au³⁺-selective two-photon fluorescent sensor.With the presence of Au³⁺,PyCM-1 exhibited a distinct "turn on"fluorescence response?16-fold enhancement?with a low detection limit?22 nM?and the large two-photon absorption cross sections?696 GM at 860 nm?.At the same time,the reaction mechanism of PyCM-1 to Au³⁺ was demonstrated in accordance with that of PyCM via UV-Vis spectra,1H NMR and Mass spectra.In addition,PyCM-1 could work well as a two-photon fluorescent sensor for specific detection of Au³⁺ in mitochondria,tissues and zebra fish,respectively.3.Based on PyCM-1,in virtue of the sulphur-affinity character of Au³⁺,we took the strategy to adjust the "N^O" combination in sensor PyCM-1 to "N^S"combination to design and fabricate sensors PyCM-2 and PyCM-3.We discovered that though the mechanism of "turn on" fluorescence response of Au³⁺ in aqueous medium by Schiff-base receptors has previously been proposed that the sensor rapidly coordinated with Au³⁺ firstly?i.e.,inhibited C=N isomerization mechanism?,then hydrolyzed to the corresponding products by the attack of water,but not be fully demonstrated.Here,we systematically demonstrate for the first time that the whole process of Au³⁺-promoted hydrolysis reaction.Compared with PyCM and PyCM-2,PyCM-3 exhibited the excellent selectivity for Au³⁺ without Hg²⁺ and Au+interference in aqueous medium.Confocal fluorescence imaging experiments indicated PyCM-3 could be applied for monitor Au³⁺ in mitochondria,tissues and zebrafish under the two-photon excitation with large two-photon absorption cross sections?685 GM at 860 nm?,little cytotoxicity and good biocompatibility.