Design,Synthesis And Study Of Near Infrared Fluorescent Probes For Detection Of Bioactive Small Molecules

There are many kinds of active small molecules distribute in living organisms,such as reactive oxygen species,reactive nitrogen species,amino acids,nucleotides,and various enzymes involved in life activities.Since these small molecules are involved in many physiological and pathological processes in the organism,this plays a vital role in maintaining normal physiological activities of life.Therefore,monitoring their level fluctuations can be used as an important indicator to measure the proliferation of cells and whether the metabolic process is normal.Based on the benzopyranonitrile structure-based fluorophore,three similar near-infrared probes Dicpl,Dicp2,and Dicp3 for detecting hydrogen peroxide were synthesized.Compared with Dicpl and Dicp2,Dicp3 shows excellent light properties.After Dicp3 reacted with hydrogen peroxide,the fluorescence intensity was significantly increased by 22 times and the color changed from yellow to red.The probe Dicp3 was also successfully applied to exogenous and endogenous hydrogen peroxide fluorescence imaging in Hi-5 cells and live nematode fluorescence imaging.More importantly,through live zebrafish imaging experiments,we found that Dicp3 was able to trace the excessive accumulation of hydrogen peroxide in zebrafish caused by copper-induced neuroma damage.The results indicate that Dicp3 is a near-infrared fluorescent probe capable of directly indirecting tracer inflammation by detecting fluctuations in intracellular hydrogen peroxide levels.The near-infrared probe Miyp for detecting hypochlorous acid is synthesized based on the fluorescene 1-indole formaldehyde,the ethylene bridge structure as the recognition group,and the positive charge of the ruthenium moiety as the mitochondrial targeting group.The probe itself is red fluorescent.When the hypochlorite attacks the ethylene bridge,its double bond is broken and the fluorescence changes from red to blue.Spectroscopic measurements under EtOH:PBS = 3:7(pH = 7.4)showed that Miyp is a highly selective photoinduced electron transfer fluorescent probe.Through fluorescence imaging and colocalization experiments of Hela cells,we also found that the overlap coefficient of the probe Miyp with commercial mitochondrial dyes is high.The results indicate that Miyp is a near-infrared fluorescent probe that targets mitochondria and detects fluctuations in hypochlorous acid levels in cells.Based on the near-infrared fluorophores 3-1 and 3-2 which consisted of xanthene and cyanine,the snail structure as the identification part of pH,four similar structures of near-infrared fluorescent probe Np-1,Np-2,Np-3,Np-4 for pH detection were synthesized.Spectroscopic tests showed that the probes Np-1,Np-2 and Np-4 were opened by proton attack,and the fluorescence intensity was significantly increased by 45,44,and 30 times,respectively.The color of Np-1 and Np-2 changed distinctly from colorless to blue,and the color of Np-4 changed from colorless to green.More importantly,the fluorescence imaging and colocalization experiments of Hela cells showed that,due to the introduction of the morpholine ring,the probes Np-2 and Np-4's superposition coefficient with the commercial lysosomal special dye is 0.9855 and 0.9733.The results show that Np-1,Np-2,and Np-4 are near-infrared fluorescent probes that are capable of detecting fluctuations in intracellular pH levels and targeting lysosomes.Based on the dephosphorylation of alkaline phosphatase,the introduction of phosphate as a recognition group,a near-infrared fluorescent probe Niap for the detection of alkaline phosphatase was designed and synthesized.Spectroscopic tests conducted under the conditions of MeOH:PBS = 1:9(pH = 8.0)showed that the fluorescence of Niap reacted with alkaline phosphatase to achieve the process from"off" to "on".It was an ICT fluorescent probe with excellent selectivity.In addition,the probe Niap was successfully applied to the fluorescence imaging of Hela cells,demonstrating its excellent biocompatibility and membrane permeability.The results show that Niap is a non-invasive near-infrared fluorescent probe for the detection of alkaline phosphatase in cells.In summary,this paper synthesizes fluorescent probes for detecting hydrogen peroxide,hypochlorous acid,pH and alkaline phosphatase in living organisms,which will provide a favorable platform for studying some key characteristics of cells under physiological or pathological conditions.And provide important information on the prevention and treatment of related diseases.