Design And Synthesis Of Fluorescent Probes Based On Quinoxaline-Xanthene Fluorophores For Imaging

Owing to the high sensitivity,simple operation and non-destructive detection of small molecule fluorescent probes,which have been widely used in imaging and detection of active molecules in organisms.For example,for reactive oxygen species such as HOCl or biological macromolecules such as nitroreductase,scientists have developed a large number of detection tools and revealed their functions in physiological and pathological processes.However,most of the reported probes still suffer from insufficient sensitivity,small Stokes shift and short wavelength.In response to the above problems,we starts with the regulation of dye structure.This paper has developed an enhanced highly sensitive hypochlorous acid（HOCl）fluorescent probe and a ratiometric nitroreductase（NTR）fluorescent probe based on quinoxaline-xanthene dyes.The specific work are as follows:Firstly,for the low sensitivity and small Stokes shift of current HOCl fluorescent probes.With the help of theoretical calculations,We proposed a general strategy for constructing high-sensitivity enhanced fluorescent probes by regulating the electrophilicity of the 9 carbon atoms of Rhodamine Pyronine.As a proof of concept,we synthesized three HOC1 fluorescent probes RB-S,PZQ-S and DQF-S based on rhodamine B and its derivatives.The experimental results showed that the electron-donating group stabilizes the charge of the 9-position carbon atom of Rhodamine Pyronine.Compared with the other two probes,the sensitivity of DQF-S to HOCl is significantly improved（detection limit:0.2 n M）.The response time is also faster（<10 s）.In addition,due to the enhanced electron donation performance of Pyronin and the breaking of the symmetry of the dye electron distribution,DQF-S has the advantages of far infrared emission（>650 nm）and large Stokes shift（81 nm）.Biological imaging studies showed that DQF-S can not only effectively detect endogenous HOCl in cells,but also achieve the detection of HOCl in mouse tumor in vivo.Second,the ratio fluorescent probes take the advantages of multi-channel signal output and accurate imaging results.However,for rhodamine dyes,the design of ratio fluorescent probes is more complicated and difficult to synthesize.We designed and synthesized rhodamine derivatives suitable for the development of ratio fluorescent probes.By further regulating the structure of the large Stokes shift dye DQF-RB,we synthesized a novel rhodamine dye RDQF-RB with a secondary amine group at the 3-position of xanthene and its acetylated derivatives.Spectral experimental studies showed that RDQF-RB has the characteristics of long-wavelength emission（>650 nm）and large Stokes shift（85 nm）,and the optical properties of its acetylated product are close to traditional rhodamine dyes(λ_ab/λ_em=548 nm/581 nm),the above results indicated that RDQF-RB has a good potential in the construction of ratio fluorescent probes.Besides,we successfully applied this structure to other types of dyes and tested their spectral properties.Thirdly,In view of the problem that most of the current NTR probes have a short emission wavelength or can only be imaged through a single channel signal.Based on the dye RDQF-RB developed in the previous chapter,we synthesized a ratiometric nitroreductase（NTR）probe RDQF-NTR.The experimental results showed that in the presence of NTR,the maximum emission wavelength of the probe RDQF-NTR is gradually red-shifted from 588 nm to 655 nm,which has a good ratio response.Furthermore,in vitro experiments showed that the probe RDQF-NTR has the advantages of rapid response（<10 min）and low Michaelis constant（K_m=5.41μM）to NTR.RDQF-NTR has good selectivity which can not be interfered by other enzymes and biologically active substances.Bioimaging results showed that RDQF-NTR can detect endogenous NTR in cells and in cirrhotic tissues,indicating that RDQF-NTR has a good application prospect in NTR detection in physiological and pathological processes.