Preparation Of Near-Infrared Fluorescent Probe And Its Application In The Detection Of Bioactive Molecules

There are a variety of active molecules involved in physiological activities in organisms,such as active oxygen free radicals,various biologically active enzymes,and nucleotides.The active molecules that are widely presented in mammalian tissues play an important role in physiological activities.The molecular level is closely related to the metabolism of the organism.Abnormal levels may cause a variety of diseases.Fluorescent probes can easily,effectively and accurately realize the tracking of target molecules by specifically binding to the recognition site,and optical detection has the advantage of visual observation.The molluscicide candidate PPU06 obtained in the previous research of our group could trigger the increase of alkaline phosphatase（ALP）activity in the snails.On this basis,we first designed and synthesized a near-infrared fluorescent probe for the detection of alkaline phosphatase.YOO1,HOO1 and Niap,after studying the spectral properties,try to use the probe Niap to study the mechanism of the prodrug of snail control.After the drug-incubated snails were frozen and sliced in vivo,and through the methods of incubating with probe Niap and observing and recording fluorescence changes,for the first time the monitoring results which presented the fluorescence of the ALP enzyme activity in the liver of the golden apple snails（Pomacea canaliculata）increased under the action of the prodrug PPU06.Based on the positive concentration-dependent relationship between the drug and the enzyme activity,it was inferred that the main site of action of the molluscicide candidate PPU06 on snails is the liver.After tissue homogenization,the enzyme activity increase results of the kit and the fluorescent probe detection results could be verified by each part of the golden apple snails.Therefore,the probe Niap could not only be used for the detection of ALP activity in organism,but also could be used to make auxiliary study in the mechanism of drug action.Based on the use of trimethoquinone as the group that recognizes diaphorase,five kinds of near-infrared fluorescent probes Milla 1,Milla 2,Milla 3,Milla 4 and Milla 5with different structures were designed and synthesized.Through test comparison,it was found that the probes Milla 1 and Milla 2 showed better test results.The two probes reacted with diaphorase in the presence of NADH to release fluorophores,and the fluorescence values of both increased nearly 5 times.Although the fluorescence intensity of the probe Milla 1 was much higher than that of Milla 2,it had the disadvantage that the consumption of enzyme detection is too high.In contrast,Milla 2showed better superiority,and the consumption of enzyme activity was only one-thirtieth of Milla 1.High sensitivity could solve the problem that the enzyme activity in organisms is weak and difficult to detect.Both had the advantages of high selectivity and detection of organisms.In this paper,four near-infrared fluorescent probes Nova 1,Nova 2,Nova 3 and Nova 4 were designed and synthesized using different recognition mechanisms to detect hypochlorous acid.On the basis of different fluorophores,probes Nova 1,Nova 2 and Nova 4 were obtained by modifying and linking different recognition groups that recognize hypochlorous acid.Among them,probe Nova 1 reacted with hypochlorous acid to realize the process from "off" to "on",the fluorescence value is increased by 18 times,showing the characteristics of high sensitivity and strong selectivity and could be used for biological detection.The probe Nova 3,a probe whose fluorescence was quenched by oxidative bond breaking through the strong oxidizing property of hypochlorous acid,has high-intensity fluorescence,and the fluorescence could be quenched completely after reacting with hypochlorous acid.It could be seen that the colorimetric changes before and after the probe reacts with hypochlorous acid,with high selectivity and sensitivity,could also be used for biological detection.In summary,this article designed and synthesized a series of near-infrared fluorescent probes to detect changes in the levels of alkaline phosphatase,diaphorase and hypochlorous acid in organisms.The mechanism of action of the molluscicide candidate was studied,which provided effective help for the mechanism research of the molluscicide candidate,and promoted the development of fluorescent probes for drug mechanism research.