Design,synthesis And Biological Application Of Infrared Rosamine Fluorescent Probes

Fluorescent probe is composed of a fluorophore(host),a recognition group(guest)and a linking group.So far,the design principles of fluorescent molecular probes based on the mechanism of fluorescence quenching mainly include the following: Photoinduced Electron Transfer(PET),Intramolecular Charge Transfer(ICT),Fluorescence Resonance Energy Transfer(FRET),Excited-State Intramolecular Proton Transfer(ESIPT)and Aggregation-Induced Emission(AIE),etc.Among them,PET mechanism is the most commonly used for fluorescence quenching mechanism in the field of molecular design.When non-radiative electron transfer occurs between fluorophore and the guest,fluorescence quenches.There are two types of PET,both a-PET(acceptor-excited PET)and d-PET(donor-excited PET)can cause fluorescence quenching.The a-PET refers that photoinduced electrons can transfer from the highest occupied molecular orbital(HOMO)of the guest to the excited host moiety.In contrast,d-PET refers that electrons transfer from the excited host moiety to the lowest unoccupied molecular orbital(LUMO)of the guest.At present,the main method for verifying and distinguishing these two mechanisms is(Time-Dependent)Density Functional Theory((TD)DFT)calculations,and the process is cumbersome and complicated.Therefore,it is of great theoretical significance and value to develop a rapid method for judging PET in fluorescent molecular probes.Due to the differences in the relative electron density on the fluorophore and guest groups,different fluorophores are linked to the same guest to achieve different detection purposes.Similarly,the same fluorophores are linked to different guest groups,often resulting in inconsistent results.So a simple method based on the relative electron density between the fluorophore and guest groups can be established to determine the PET effect.Actually,the use of the PET mechanism to design various types of probes for the detection of small molecules and enzyme is also a recent hot topic.In this paper,three types of molecules based on rosamine were synthesized and designed,and their optical properties and biological applications were studied in depth.There were mainly divided into three parts.The first part was based on the classical organic bond theory to establish a simple method for PET fluorescent probes designing.And the latter two parts mainly studied the detection of biomolecules from the perspective of organelle-targeting function.The detail contents are as follows.1.Five different rosamine derivatives(probes 3a-3e)were obtained by linking nitrogen-containing heterocycles with different electron donating abilities and pyronine,and their p H responses were studied.When pyronine was connected to an electron-donating indole ring,the a-PET process was turned on,but when the indole was protonated,PET was prohibited.When pyronine was linked to an electron-withdrawing quinoline or pyridine,d-PET was inhibited,fluorescence was turned on.While nitrogen was protonated to form a strong electron-withdrawing pyridinium under strong acidic conditions,and d-PET was allowed,fluorescence was quenched.Five kinds of infrared fluorescence p H probes,with high selectivity,fast response and good reversibility,could be used for p H fluorescence titration in solution.Among them,the relative fluorescence intensity of probe 3d was enhanced by 263-fold,and probe 3e with improved biocompatibility had a suitable p Ka,so that it had lysosome-targeting ability in V79 and He La cells,and probe 3e with ON-OFF-type exhibited strong and weak signals in normal cells and cancer cells,respectively.And it could be used for the monitoring of intracellular p H under acidic conditions.The results showed that the PET effects was more likely to happen if the bigger difference of the electronic density between the host and the guest,thereby we provided a convenient method for designing ON-OFF fluorescent probes according to classical organic bond theory.2.The probe 3c designed in the previous work was stained with different cancer cells and normal cells.The results showed that this molecule was the mitochondrial targeting probe for most cells.The novel open fluorescent probes 5 and 9 were designed and synthesized for detecting important small molecules in mitochondria.There was electron transfer between pyridinium and pyronine,d-PET was allowed,and fluorescence was closed.When H2O2 or H2 S was added exogenously,the reaction site leaved,free pyronine-pyridine was released,and fluorescence was turned on.The probes had the advantages of high fluorescence quantum yield,red light emission,good stability and good water solubility,and they could be used for exogenous detection of H2O2 and H2 S in solution and cells.The detection limits of probes 5 and 9 were 57 n M and 110 n M,were all in the medium level of the reported probes,respectively.This work provides a basis for the development of organelle-targeted probes using the PET mechanism and is of great significance for targeting functions and drug delivery in biomedicine.3.The mitochondrial targeting function of the pyronine-pyridinium skeleton in the previous work was continued,and a novel ON-OFF type fluorescent probe 11 was designed to detect biological enzymes in the mitochondria.In the solution,the probe 11 had a low detection limit of 2.2 ng/m L for nitroreductase.Under normoxic conditions,there is mainly a type I NTR with two-electron-reduced;under hypoxic conditions,there is a type II NTR with single electron-reduced.The type I and type II nitroreductases could be distinguished by cell imaging,and the whole detection process was also performed in mitochondria.