Design,Synthesis And Application Of Enzyme-Responsive ESIPT Fluorescent Molecular Probes

Nitroreductase（NTR）is a flavin mononucleotide（FMN）cofactor-dependent protein expressed mainly in E.coli that reduces aromatic nitro compounds to amine compounds in the presence of reduced nicotinamide adenine dinucleotide（NADH）.Nitroreductase is highly expressed in hypoxic solid tumor environments and further promotes tumor proliferation and metastasis.Nitroreductase is one of the important biomarkers for the detection of tumor hypoxia microenvironment.Therefore,it is of great significance to develop a sensitive and efficient method for the detection of nitroreductase in tumor cells.Monoamine oxidase（MAO）is mainly composed of two subtypes of monoamine oxidase-A（MAO-A）and monoamine oxidase-B（MAO-B）,which can catalyze the oxidation of monoamines to the corresponding reaction products of aldehydes,hydrogen peroxide and ammonia or substituted amines.MAO-A and MAO-B show a high degree of homology,but they are associated with different symptoms.Among them,MAO-A mainly decomposes serotonin,norepinephrine and epinephrine,which is closely related to mental disorders.MAO-A content in tumor cell is also changed.Most assays are currently responsive to both MAO-A and MAO-B,necessitating the development of assays specific for MAO-A.To date,there have been a variety of detection methods for the above two enzymes,including fluorescence,colorimetric,and magnetic resonance,but these methods require the use of large-scale instruments and are expensive.Fluorescence imaging method has been used to detect various tumor markers due to its high sensitivity,small damage to the body,real-time imaging and many other advantages.Among them,the fluorescent probe based on ESIPT mechanism has the advantages of large Stokes shift,long wavelength,high signal-to-noise ratio,and is often used for the design of small molecule fluorescent probes.Therefore,in this paper,we constructed two fluorescent probes based on ESIPT mechanism for the detection of nitroreductase and monoamine oxidase-A,respectively.（1）A common 2-（benzo[d]thiazole-2-yl）-4-methylphenol（HBT）based on an ESIPT mechanism was selected as a fluorescent skeleton.An electron-withdrawing group was connected to prolong a conjugated system,and a fluorescent platform SNN was obtained.Then a recognition group of nitroreductase was connected,and the probe SNN-NO₂ was obtained.The probe SNN-NO₂ was used to detect nitroreductase in vitro and in vivo,and nitroreductase in cells and zebrafish were also be detected.（2）The probe SNN-NH₂ was obtained by connecting a recognition group of monoamine oxidase-A with the fluorescent platform SNN,and the probe SNN-NH₂ was used for detecting monoamine oxidase-A in vivo and in vitro respectively and detecting monoamine oxidase-A in cells and zebrafish.