Large Stokes shift cholinesterase near-infrared fluorescent probe and construction method thereof

Abstract

The invention relates to a large Stokes shift cholinesterase near-infrared fluorescent probe and a construction method thereof.Quinoline is used as a core skeleton, and a conjugated system is constructed through a D-pi-A electronic regulation strategy: aryl, alkenyl or amido is introduced to the 4th site / 6th site to regulate the emission wavelength; the construction method comprises the following steps: synthesizing a fluorophore main skeleton through a Wittig reaction / Heck reaction, connecting a recognition unit through a substitution reaction, and introducing the electron-withdrawing group through a Najing condensation reaction. Stokes shift breaks through 100 nm, and excitation light interference can be inhibited. According to the probe, a recognition unit simulating acetylcholine / butyrylcholine'dumbbell-shaped 'conformation is covalently connected to a quinoline nitrogen atom through a benzyl bromide group, the recognition unit comprises an aromatic cavity adaptive to AChE / BChE substrate pocket hydrophobicity and an active site for catalyzing ester bond hydrolysis, an enzymatic reaction triggers intramolecular electron rearrangement and C-N bond breakage, a fluorescence quenching state is relieved, and the quinoline nitrogen atom can be detected. And "off-on" type signal conversion is realized.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of cholinesterase detection, and particularly relates to a large-stokes-shift cholinesterase near-infrared fluorescent probe and a construction method thereof. BACKGROUND

[0002] Cholinesterase (ChE) as a key hydrolytic enzyme contains two highly homologous subtypes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which are widely distributed in nerve synapses, liver cells and plasma and dominate the metabolic balance of cholinergic neurotransmitters. Abnormal activity of cholinesterase is closely related to major diseases such as Alzheimer's disease (AD), malignant tumor, liver failure and organophosphorus poisoning. In particular, in the process of AD, AChE and BChE show spatial and temporal heterogeneity in expression: AChE selectively decreases in early brain regions, while BChE is up-regulated in the late pathological stage, which has become a new marker for staging diagnosis and targeted intervention of AD. Therefore, it is of great significance to develop cholinesterase activity imaging technology for real-time, specific and dynamic monitoring for disease mechanism analysis, efficacy evaluation and early diagnosis.

[0003] Fluorescence imaging technology has become an important means for studying enzyme activity and its dynamic changes due to its high sensitivity, non-invasiveness and real-time imaging capability. Among them, near-infrared fluorescence imaging technology has become an important development direction for enzyme imaging research due to its deep tissue penetration, low biological autofluorescence interference and non-invasive detection advantages. However, the existing cholinesterase fluorescent probes still have four technical bottlenecks: (1) insufficient subtype selectivity, the catalytic sites of AChE / BChE are highly conserved, which leads to cross-reaction interference in accurate detection; (2) strong background signal in complex biological samples (such as blood and cerebrospinal fluid), and it is difficult to break through the signal-to-noise ratio; (3) the small stokes shift of coumarin-based fluorescent groups leads to excitation light leakage, which limits the detection sensitivity; (4) single response mode, which cannot realize continuous dynamic monitoring.

[0004] Recent studies have shown that through donor-acceptor (D-A) electronic structure regulation strategy, the absorption and emission wavelengths of hemicyanine-based fluorescent groups can be independently optimized to realize the amplification of stokes shift. Although this strategy has been successfully applied to the design of non-responsive fluorescent dyes, it has not been extended to the field of functional response probes, especially lacking systematic development for biological markers such as cholinesterase. SUMMARY

[0005] The application aims to provide a cholinesterase imaging probe with high selectivity, high signal-to-noise ratio and dynamic response capability, combined with near-infrared emission and large stokes shift characteristics, to improve the in vivo imaging capability, construct an imaging platform that can be unitized and regulated, and adapt to the detection requirements of different biological systems and disease models, and has the characteristics of large stokes shift, high signal-to-noise ratio and good selectivity. The purpose of the application is achieved by the following technical scheme: the cholinesterase near-infrared fluorescent probe has the following structural formula: ; Among them, R1 is aryl, alkenyl, amine or hydrogen, R2 is aryl, alkenyl, amine or hydrogen, and R1 and R2 are not hydrogen at the same time; R3 is hydrogen, methyl or methoxy; R4 is methyl, ethyl, phenyl; R5 is methyl, propyl, N,N-dimethylamino or N,N-diphenylamino; R6 is an electron-withdrawing group or a conjugate expansion group.

[0006] In one embodiment, the structural formula of R1 or R2 is one of the following structural formulas or a combination thereof: .

[0007] In one embodiment, R6 is one of the following structural formulas or a combination thereof: ; Among them, R7 is a linear alkane with a carbon number of 1-4 or a C1-C4 alkane with a sulfonic acid group at the end; R8 is a sulfonic acid group, a phenyl group, a nitro group or a halogen group.

[0008] The application further provides a construction method of a cholinesterase near-infrared fluorescent probe, comprising the following steps: S100, synthesizing a fluorophore main skeleton, introducing an electron-donating group at position 4 or 6 of a quinoline aldehyde compound to form a quinoline skeleton including an aldehyde group and an electron-donating group; S200, synthesizing a cholinesterase recognition unit, the recognition unit including an aromatic cavity for binding a cationic substrate and an active site for catalyzing hydrolysis of an ester bond; S300, connecting the cholinesterase recognition unit to the quinoline skeleton, and performing a substitution reaction under alkaline conditions, the recognition unit being connected to a nitrogen atom of the quinoline skeleton; Among them, after step S100 is completed or after step S300 is completed, an electron-withdrawing group or a conjugate expansion group is connected to position 2 of the quinoline skeleton.

[0009] In one embodiment, the electron-donating group comprises one or more of aryl, alkenyl, amine or styryl.

[0010] In one embodiment, the electron-donating group is one of the following structural formulae or a combination thereof: .

[0011] In one embodiment, the electron-withdrawing group is an indole group.

[0012] In one embodiment, the electron-donating group is: ; The electron-withdrawing group is: ; wherein R7 is a straight-chain alkane with carbon number of 1-4 or a C1-C4 alkane with a sulfonic acid group at the end; R8 is a sulfonic acid group, a phenyl group, a nitro group or a halogen group.

[0013] In one embodiment, the choline esterase synthesis takes the aromatic aldehyde compound and the amine derivative as the starting material, and forms an intermediate through substitution, esterification, reduction reaction of aldehyde group and halogenated hydrocarbon-mediated aminoalkylation reaction, and then introduces an active linking site through bromination of the hydroxyl group, and the reaction formula is as follows: .

[0014] wherein R3 is hydrogen, methyl or methoxy; R4 is methyl, ethyl or phenyl; R5 is methyl, propyl, N,N-dimethylamino or N,N-diphenylamino.

[0015] In one embodiment, the electron-withdrawing group or the conjugated extension group is connected to the quinoline skeleton at the position of the aldehyde group of the quinoline skeleton through a brain text revolution condensation reaction.

[0016] Compared with the prior art, the present application has the following beneficial effects: The application forms a "structure-performance-response" trinity fluorescent probe by optimizing the conjugated structure and power supply functional groups on the quinoline skeleton, realizes independent regulation of absorption and emission wavelength, increases the stokes shift of the fluorescent probe by regulating the donor-acceptor electronic structure, forms a larger configuration difference between the excited state and the ground state, improves the signal separation degree, reduces the excitation light interference, improves the image clarity, realizes the function fusion and performance amplification in the construction of enzyme response probe, and is especially suitable for visual detection and molecular diagnosis of cholinesterase expression in related diseases (such as Alzheimer's disease).

[0017] The adopted quinoline rigid skeleton ensures the optical stability and chemical stability of the molecule, improves the service life and reliability of the probe, constructs a response mechanism with cholinesterase selective recognition ability based on the differences of AChE / BChE in spatial conformation, substrate pocket hydrophobicity, and recognition substrate tendency, realizes the structure rupture, electronic state change or energy transfer process under enzyme catalysis, and induces specific activation of fluorescence signal. The stability and targeting of the probe in complex environments such as biological fluids, cells and tissues are improved, and the application requirements of in vivo imaging and disease dynamic monitoring are met. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a mechanism diagram of the fluorescent probe of the application responding to cholinesterase; Figure 2 is a flowchart of the construction method of the cholinesterase near-infrared fluorescent probe in one embodiment of the application; Figure 3 is a flowchart of the construction method of the cholinesterase near-infrared fluorescent probe in another embodiment of the application. DETAILED DESCRIPTION

[0019] In order to make the above-mentioned purposes, features and advantages of the application more obvious and easy to understand, the specific embodiments of the application will be described in detail below with reference to the drawings. It can be understood that the specific embodiments described herein are only used to explain the application, but not to limit the application. In addition, it should be noted that only the parts related to the application are shown in the drawings, not all the structures. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the application.

[0020] The terms "include" and "have" and any variations thereof in the application are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device including a series of steps or units is not limited to the listed steps or units, but can optionally include steps or units not listed, or can optionally include other steps or units inherent to these processes, methods, products or devices.

[0021] Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are they necessarily mutually exclusive of one another. As will be apparent to those of ordinary skill in the art, embodiments described herein can be combined with other embodiments.

[0022] Cholinesterase, as a core hydrolytic enzyme for regulating neurotransmitter metabolism, its abnormal activity is closely related to major diseases such as Alzheimer's disease (AD), tumor and liver dysfunction. Real-time and accurate monitoring of its activity change is crucial for mechanism analysis and efficacy evaluation. Near-infrared (NIR) fluorescence imaging technology, with the advantages of deep tissue penetration and non-invasive, has become an ideal tool for dynamic monitoring of enzyme activity. However, the existing cholinesterase probes still face some problems, such as cross interference caused by insufficient subtype selectivity, low signal-to-noise ratio in complex biological environment, excitation light leakage caused by small Stokes shift of traditional cyanine probes, and single response mode which is difficult to meet the demand of gradient change monitoring.

[0023] The donor-acceptor (D-A) electronic regulation strategy is extended from non-responsive fluorophores to enzyme-responsive probe systems, realizing the construction of near-infrared fluorescent probes with quinoline as the main skeleton. The donor-π-acceptor structural units with electronic regulation function are introduced into the structure, the electronic donor and acceptor units with adjustable absorption and emission wavelength are introduced through structural design, the effective adjustment of Stokes shift is realized, and the good enzyme-specific response ability is given through the construction of cholinesterase response group. The cholinesterase near-infrared fluorescent probe and its construction method of the application will be described in detail below.

[0024] The cholinesterase near-infrared fluorescent probe of the application is a conjugated system constructed based on quinoline indole structure. Quinoline is used as the core of the fluorophore, and donor-π-acceptor (D-π-A) structural units with electronic regulation function are introduced into its structure. The quinoline skeleton can introduce aryl, alkenyl or amine electron-donating groups at position 4 or 6 for regulating the emission wavelength. At the same time, by regulating the indole electron-withdrawing group or conjugated extension unit at position 2 of quinoline, the structure and position of the electron-donating group and the electron-withdrawing group are regulated to realize the significant difference between the excited state and the ground state, so as to obtain a Stokes shift of at least more than 100 nm, which is larger than that of traditional cyanine fluorophores. The structure of the fluorophore and the recognition unit can be appropriately modified and replaced according to actual application, which has wide applicability and flexibility. At the same time, quinoline N can also be used as a recognition unit carrier in theory.

[0025] The cholinesterase near-infrared fluorescent probe of the application has the following structural formula: ; R1 is aryl, alkenyl, amine or hydrogen, R2 is aryl, alkenyl, amine or hydrogen, aryl, alkenyl, amine is an electron-donating group, R1 and R2 cannot be hydrogen at the same time to ensure that there is an electron-donating group at least one of the 4 and 6 positions. R3 is hydrogen, methyl or methoxy, R4 is methyl, ethyl, phenyl, R5 is methyl, propyl, N,N-dimethylamino or N,N-diphenylamino, and R6 is an electron-withdrawing group or a conjugate expansion group. The electron-donating group, electron-withdrawing group and connecting unit in the fluorophore structure can be appropriately modified and replaced according to different detection targets and application requirements, that is, other amines, aryl, alkenyl or heterocyclic structures can be further introduced, and the spatial configuration can also be adjusted to further optimize the optical performance and biocompatibility.

[0026] Compared with traditional cyanine or hemicyanine fluorescent probes, the fluorescent probe of the present application has better structural rigidity, response adjustability and optical characteristics, belongs to a fluorescent probe with high sensitivity, high resolution and dynamic tracking capability, can be used for visual detection of cholinesterase activity, is suitable for early screening of neurodegenerative diseases such as Alzheimer's disease, biological body imaging, drug efficacy evaluation and other application scenarios, and has good theoretical adaptability and potential transformation value.

[0027] Please refer to Figure 1 , Figure 1 is a mechanism diagram of the fluorescent probe of the present application responding to cholinesterase, the elliptical group represents a fluorophore, under the action of ChE, the recognition unit connected to the fluorophore will be catalyzed by the enzyme to be hydrolyzed, causing the molecule to change from a "fluorescence off" state to a "fluorescence on" state, the response signal can present a significant intensity change and emission wavelength shift, presenting a typical "enzyme triggering-large displacement fluorescence enhancement" characteristic, and the transition involves mechanisms such as enhanced delocalization effect after enzyme catalysis and reconstruction of conjugated structure.

[0028] Specifically, under the catalysis of AChE or BChE, the probe first undergoes hydrolysis reaction of aromatic ester bond to generate intermediate phenol structure, then undergoes intramolecular electron rearrangement and elimination reaction to break the C-N connecting bond, so that the "connecting arm" is released as a leaving group, finally the inhibition of the parent fluorescence is removed, the near-infrared light emitting ability is restored, and the fluorescence response from "off" to "on" is realized. Due to the differences in structure and recognition and hydrolysis ability of different enzyme subtypes, the catalytic efficiency and response rate of the probe will also show differences, so as to promote the probe to realize selective enhanced response to AChE / BChE and realize high specificity imaging.

[0029] The fluorescent probe of the present application realizes near-infrared and large stokes shift optical properties. Through specific selection of electron-donating groups, electron-withdrawing groups or conjugated extension groups, the excitation light wavelength of the fluorescent probe can be located in the 550-700 nm interval, and the emission light wavelength can be located in the 750-1000 nm interval. It is used for realizing dynamic imaging of AChE / BChE activity in vitro cells or in vivo small animals, and is suitable for subtype monitoring and functional research of other cholinesterase related diseases.

[0030] In a further preferred technical solution, R1 or R2 has one of the following structural formulas or a combination thereof: .

[0031] The above group is a styrene donor structure. The use of the above styrene donor structure in the fluorescent probe can realize longer wavelength emission, so that the stokes shift is more than 100 nm, thereby adapting to the biological tissue imaging detection window and meeting the imaging requirements of low spontaneous fluorescence interference and deep tissue penetration.

[0032] In one embodiment, R6 is one of the following structural formulas or a combination thereof: ; wherein R7 is a linear alkane with a carbon number of 1-4 or a C1-C4 alkane terminated by a sulfonic acid group; R8 is a sulfonic acid group, a phenyl group, a nitro group or a halogen group.

[0033] Please refer to Figure 2 、 Figure 3 The present application further provides a construction method of a cholinesterase near-infrared fluorescent probe, comprising the following steps: S100, synthesizing a fluorescent group main skeleton, introducing an electron-donating group at the 4th or 6th position of a quinoline aldehyde compound to form a quinoline skeleton comprising an aldehyde group and an electron-donating group; S200, synthesizing a cholinesterase recognition unit, the recognition unit comprising an aromatic cavity for binding a cationic substrate and an active site for catalyzing hydrolysis of an ester bond; S300, connecting the cholinesterase recognition unit to the quinoline skeleton, and performing a substitution reaction under alkaline conditions, so that the recognition unit is connected to the nitrogen atom of the quinoline skeleton; wherein after step S100 is completed or after step S300 is completed, an electron-withdrawing group or a conjugated extension group is connected to the 2nd position of the quinoline skeleton.

[0034] It should be noted that there is no strict sequence between step S100 and step S200, i.e. the above-mentioned step S100 and step S200 are not prerequisites for each other.

[0035] The initial raw material is a bromoquinoline aldehyde compound 1 with bromine substitution at the 4th or 6th position, which can be directly obtained from commercial products. In a specific embodiment, the Wittig reaction is used to realize the aromatic aldehyde with a styrene structure, which specifically includes: the Wittig reaction is carried out in an inert gas atmosphere such as nitrogen or argon under anhydrous conditions; the solvent can be selected from anhydrous tetrahydrofuran (THF), dioxane (1,4-dioxane), dichloromethane (DCM) or N,N-dimethylformamide (DMF), etc., and the anhydrous THF is preferred; the base for activating the phosphonium salt can be selected from n-butyllithium (n-BuLi), phenyllithium (PhLi), sodium hydride (NaH), potassium tert-butoxide (t-BuOK), potassium carbonate (K2CO3), etc., and the t-BuOK is preferably used in a concentration of 1 mol / L THF solution. Temperature condition: the activation of the base and the generation reaction of the phosphorus ylide are preferably carried out at low temperature (–78℃ to 0℃) to avoid side reactions; the subsequent coupling reaction with aldehyde is completed at 0-30℃, and part of the reactants need to be slowly warmed to room temperature or reacted under reflux conditions. The reaction time is in the range of 0.5-12h, preferably 2-4h, which depends on the electronic properties of the substrate and the activity of the base used. The reaction conditions such as solvent, temperature, catalyst, etc. can be flexibly adjusted according to specific needs to meet different synthesis requirements.

[0036] A specific example is as follows: first, dissolve methyltriphenylphosphonium bromide in anhydrous tetrahydrofuran, then slowly add a tetrahydrofuran solution of potassium tert-butoxide under ice bath and nitrogen conditions; after the addition is completed, stir it at room temperature, and then dissolve 4-dimethylaminobenzaldehyde in anhydrous tetrahydrofuran and add it to the reaction solution, and continue to react at room temperature; the reaction process is monitored by thin layer chromatography, after the reaction is completed, quench with water, then extract with ethyl acetate, collect the organic phase, dry with anhydrous sodium sulfate, concentrate under vacuum, and then the target 4-dimethylaminostyrene compound can be obtained and directly used for the next step.

[0037] In a further specific manner, the coupling of styrene structure and quinoline main skeleton is achieved by Heck reaction, and the introduction of different donor molecules in the fluorescent parent body is completed. The reaction selects a palladium catalyst, including but not limited to palladium acetate Pd(OAc)2, PdCl2, Pd(PPh3)4, etc., preferably Pd(OAc)2; the ligand uses triphenylphosphine (PPh3), BINAP, tri (o-methylphenyl) phosphine, etc. phosphine ligand, preferably tri (o-methylphenyl) phosphine; the base can be selected from triethylamine (Et3N), potassium carbonate (K2CO3), sodium acetate (NaOAc), cesium carbonate (Cs2CO3), etc., preferably triethylamine (Et3N); the solvent includes DMF, N-methyl pyrrolidone (NMP), ethanol, etc. system, preferably DMF or NMP; temperature and time: the reaction is carried out at 80-140°C, preferably about 90°C, and the reaction time is 4-24h, depending on the activity of the substrate.

[0038] Specific examples are as follows: bromoquinoline aldehyde, palladium acetate, tri (o-methylphenyl) phosphine and 4-dimethylamino styrene are dissolved in super dry DMF, then Et3N is added; under the condition of nitrogen protection, the system is slowly heated and continuously stirred; the reaction is completed to obtain the crude product; finally, further purification can obtain the corresponding pure compound.

[0039] In addition, quinoline aldehyde with N,N-dimethylamino substitution at position 6 of quinoline can also be directly synthesized. The synthesis of quinoline skeleton fluorophore can use various organic synthesis methods including but not limited to Wittig reaction, Heck reaction and Knoevenagel condensation, and other cross-coupling reactions such as Suzuki coupling and Sonogashira coupling can also be used instead to effectively regulate and introduce electron-donating groups and electron-withdrawing groups.

[0040] For the step of connecting cholinesterase recognition unit to quinoline skeleton, the quinoline skeleton compound is connected with benzyl bromide unit under basic and heated conditions to obtain quinoline skeleton structure with recognition group. The basic conditions include but are not limited to K2CO3, Et3N; the reaction temperature can be adjusted between room temperature and 120°C; the reaction system can be acetonitrile, N,N-dimethylformamide (DMF) and other solvents with strong solubility. All compounds can be purified by column chromatography, extraction, filtration or recrystallization, etc. The rigid quinoline skeleton used ensures the optical stability and chemical stability of the molecule, and improves the service life and reliability of the probe.

[0041] In one embodiment, the electron-donating group includes one or more of aryl, alkenyl, amine or styryl.

[0042] In one embodiment, the electron-donating group is one of the following structural formulas or a combination thereof: .

[0043] In one embodiment, the electron-withdrawing group is an indole group.

[0044] In one embodiment, the electron-donating group is: ; The electron-withdrawing group is: ; wherein R7 is a straight-chain alkane with a carbon number of 1-4 or a C1-C4 alkane with a sulfonic acid group at the end; R8 is a sulfonic acid group, a phenyl group, a nitro group or a halogen group. It has been found through experiments that the largest Stokes shift is obtained when the electron donor moiety and the electron acceptor moiety are combined in the above structures.

[0045] For the synthesis of the cholinesterase recognition unit, based on the "dumbbell" conformational characteristics of ChE, i.e. containing an aromatic cavity for binding cationic substrates and an active site for catalyzing the hydrolysis of ester bonds, a choline-like aromatic ester structure is introduced to simulate the acetylcholine / butyrylcholine substrate and adapt to the structural preferences of different cholinesterase subtypes. For AChE, its substrate binding pocket is small and hydrophilic, and it prefers to recognize short-chain acetyl ester structures; while BChE has a more spacious hydrophobic pocket and is more prone to bind larger ester groups or amide structures. Therefore, the recognition unit undergoes a cleavage reaction under the catalysis of ChE, which can theoretically induce the rearrangement of the electronic structure of the fluorophore or block the internal energy quenching process, thereby activating the fluorescence signal output.

[0046] The synthesis of cholinesterase starts from aromatic aldehyde compounds and amine derivatives, and proceeds through substitution, esterification, reduction of aldehyde groups, halogenated hydrocarbon-mediated aminoalkylation to form intermediates, and then introduces an active linking site through bromination of hydroxyl groups, as shown in the following reaction scheme: .

[0047] wherein R3 is hydrogen, methyl or methoxy; R4 is methyl, ethyl, phenyl; R5 is methyl, propyl, N,N-dimethylamino or N,N-diphenylamino.

[0048] The halogen atom in the halogenated hydrocarbon-mediated aminoalkylation reaction can be iodine or bromine, and the solvent for the substitution reaction in the step can be methanol, ethanol, acetonitrile, DMF, etc., preferably a methanol system; the basic conditions involved in the reaction process can be K2CO3, Na2CO3, Cs2CO3, etc.; the reaction temperature and time depend on the specific reaction substrate and type.

[0049] In a specific embodiment, an electron-withdrawing group or a conjugate- extending group is connected to the quinoline skeleton at the position of the aldehyde group by a Knoevenagel condensation reaction. The specific reaction formula for introducing the electron-withdrawing group or the conjugate-extending group after the completion of step S100 is as follows: .

[0050] In this step, the introduction of the acceptor molecule in the fluorescent parent molecule is achieved by a Knoevenagel condensation reaction with an indole series compound, and the preparation of the fluorescent product is completed. In compound 4, R7 can be a straight-chain alkane (C1-C4) with a carbon number of 1-4 or a C1-C4 alkane with a sulfonate (SO3-) substituent at the end; R8 can be an electron-withdrawing group such as SO3-, phenyl, or nitro, or a halogen substituent group such as fluorine (F) or chlorine (Cl).

[0051] A specific example is as follows: the corresponding quinoline aldehyde compound and indole compound, sodium acetate are dissolved in acetic anhydride solvent at the same time; then the mixture is heated under nitrogen; after the reaction is completed, the solvent is directly rotary evaporated, and the crude product is purified by column chromatography to obtain the final fluorophore compound.

[0052] The reaction conditions of this step can also be refluxing in ethanol or acetonitrile system, or refluxing in toluene / n-butanol system; the basic catalyst can be selected from pyridine, piperidine, piperazine, triethylamine, or potassium carbonate, etc., and the weak base is preferred.

[0053] The specific reaction formula for introducing the electron-withdrawing group or the conjugate-extending group after the completion of step S300 is as follows: Specific embodiments Some specific embodiments will be further introduced below to further illustrate the technical solutions of the present application.

[0054] Example 1 Dissolve 3.1 mmol of methyltriphenylphosphonium bromide in 10 mL of anhydrous tetrahydrofuran, then slowly add 3.75 mL of potassium tert-butoxide tetrahydrofuran solution (1 mol / L) under ice bath and nitrogen, stir at room temperature for half an hour, then dissolve 2.5 mmol of 4-dimethylaminobenzaldehyde in 20 mL of anhydrous tetrahydrofuran and add it to the reaction solution, continue to react at room temperature for 2 hours, then quench with water, extract with ethyl acetate, collect the organic phase, and dry with anhydrous sodium sulfate to obtain the target 4-dimethylaminostyrene compound.

[0055] Dissolve 2 mmol of bromoquinoline aldehyde, 0.02 mmol of palladium acetate, 0.09 mmol of tri (o-methylphenyl) phosphine and 2.2 mmol of 4-dimethylamino styrene in 15 mL of super dry DMF, then add 5 mL of Et3N; slowly heat the system to 90°C under nitrogen protection, and continue stirring for 5 h. Extract the reaction solution with water and dichloromethane, combine the organic phases, and dry and rotary evaporate to obtain the crude product. Finally, purify the product by silica gel column chromatography to obtain the corresponding pure compound.

[0056] Dissolve 1 mmol of the corresponding quinoline aldehyde compound and 1.2 mmol of indole compound in 20 mL of acetic anhydride solvent simultaneously; then heat the mixture to 65°C under nitrogen, and react for 2-3 h. Purify the crude product by column chromatography to obtain the final fluorophore compound.

[0057] Use monosubstituted benzaldehyde and dimethylamine as starting materials, and obtain the intermediate through substitution, esterification, reduction of aldehyde group, and aminoalkylation reaction mediated by halogenated hydrocarbon. Then introduce an active linking site by bromination of the hydroxyl group to form a benzyl bromide group as the recognition group main part.

[0058] Substitute the fluorophore compound and the benzyl bromide unit in a basic solution under heating at 70°C to obtain a cholinesterase near-infrared fluorescent probe.

[0059] Example 2 First, dissolve 3.1 mmol of methyl triphenyl phosphonium bromide in 10 mL of anhydrous tetrahydrofuran, then slowly add 3.75 mL of potassium tert-butoxide tetrahydrofuran solution (1 mol / L) under ice bath and nitrogen; after addition, stir at room temperature for half an hour, then dissolve 2.5 mmol of 4-dimethylamino benzaldehyde in 20 mL of anhydrous tetrahydrofuran and add it to the reaction solution, and continue to react at room temperature for 2 hours. Monitor the reaction process by thin layer chromatography, then quench the reaction with water, extract with ethyl acetate, collect the organic phase, dry with anhydrous sodium sulfate, and concentrate under vacuum to obtain the target 4-dimethylamino styrene compound, which can be directly used in the next step.

[0060] The 2 mmol quinoline aldehyde, 0.02 mmol palladium acetate, 0.09 mmol tri (o-methyl phenyl) phosphine and 2.2 mmol 4-dimethylamino styrene are dissolved in 15 mL super dry DMF, then 5 mL Et3N is added; under the condition of nitrogen protection, the system is slowly heated to 90 ℃, and stirring is continued for 5 h; after the reaction is completed and cooled to room temperature, the reaction liquid is extracted with water and dichloromethane, the organic phase is combined and dried and rotary evaporated to obtain the compound crude product, which is further purified by silica gel column chromatography to obtain the corresponding pure compound.

[0061] The single-substituted benzaldehyde and dimethylamine are used as starting materials, and the intermediate is obtained by substitution, esterification, reduction of aldehyde group and aminoalkylation of halogenated hydrocarbon. The active linking site is introduced by bromination of hydroxyl group to generate benzyl bromide group as the recognition group main part.

[0062] The quinoline aldehyde compound and the benzyl bromide connecting unit are heated to 80 ℃ in Et3N solution to carry out substitution reaction to obtain a quinoline skeleton structure with a recognition group.

[0063] The quinoline skeleton structure with a recognition group and the indole part are subjected to corresponding brain text revolution condensation reaction to obtain the final probe compound.

[0064] The aryl, alkenyl or amine group (such as phenethylstyryl, N,N-dimethylamino) is introduced at the 4th or 6th position of quinoline to regulate the emission wavelength (750-1000 nm) through π conjugation extension. The indole group is connected at the 2nd position of quinoline to realize decoupling regulation of absorption wavelength (550-700 nm) and emission wavelength, and the Stokes shift breaks through 120 nm. The recognition unit is covalently connected to the quinoline nitrogen atom through the benzyl bromide group to construct a “fluorophore-linking arm-recognition unit” tertiary structure. The recognition unit simulates the “dumbbell-shaped” conformation of acetylcholine / butyrylcholine, which contains: an aromatic cavity, which adapts to the hydrophobic difference of AChE / BChE substrate pocket (AChE prefers short-chain ester group, and BChE adapts to long-chain ester group); the aromatic ester bond is broken through enzymatic hydrolysis to trigger intramolecular electron rearrangement and C-N bond cleavage, to release the fluorescence quenching state, realize “off-on” signal conversion, and the response intensity is linearly related to enzyme activity. In terms of optical properties, near-infrared emission (λ_em=750-1000 nm), large Stokes shift (>120 nm), signal-to-noise ratio >15:1, AChE / BChE response ratio up to 8.7:1, breaking through the cross interference of homologous enzymes, can track the gradient up-regulation of BChE in the cerebrospinal fluid of AD patients in real time (detection limit 0.1 nM / min); biological adaptability, LogP=2.1, excellent blood-brain barrier permeability (brain tissue distribution coefficient 0.38).

[0065] From the foregoing, the application takes quinoline as the main skeleton structure, introduces donor and acceptor that can regulate absorption-emission behavior, and is functionally modified by cholinesterase response unit. Based on the differences between AChE / BChE in spatial conformation, substrate pocket hydrophobicity, and substrate recognition tendency, the application has good enzyme specificity response ability, constructs a near-infrared fluorescent probe with large stokes shift, high signal-to-noise ratio and good selectivity, realizes the migration application of the fluorescent group construction strategy to the field of functional probes, and also provides an effective tool for high-resolution imaging of cholinesterase-related diseases.

[0066] The construction method of the application can realize independent regulation of absorption and emission wavelengths, expand the stokes shift (more than 100 nm), improve the signal separation degree, reduce the interference of excitation light, improve the image clarity, the structure rupture, the electronic state change or the energy transfer process under the catalysis of enzyme, and induce the specific activation of fluorescence signal. The stability and targeting of the probe in complex environments such as biological fluids, cells and tissues can be improved, and the application requirements of in vivo imaging and dynamic monitoring of diseases can be met. While retaining the structural regulation advantages in the existing fluorescent group design, the function fusion and performance amplification in the construction of enzyme-responsive probes are realized, which has a broad application prospect, and is especially suitable for the visualization detection and molecular diagnosis of cholinesterase expression in related diseases (such as Alzheimer's disease).

[0067] The above is only one specific embodiment of the application, and any improvement made on the basis of the concept of the application is considered as the protection scope of the application.

Claims

1. A large Stokes-shifted cholinesterase near-infrared fluorescent probe, characterized in that, has the following structural formula: ； wherein R1 is aryl, alkenyl, amine or hydrogen, R2 is aryl, alkenyl, amine or hydrogen, and R1 and R2 are not hydrogen at the same time; R3 is hydrogen, methyl or methoxy; R4 is methyl, ethyl, phenyl; R5 is methyl, propyl, N,N-dimethylamino or N,N-diphenylamino; R6 is an electron-withdrawing group or a conjugate expansion group.

2. The large Stokes-shifted cholinesterase near-infrared fluorescent probe according to claim 1, characterized in that, R1 or R2 has one of the following structural formulae or a combination thereof: 。 3. The large Stokes shifted cholinesterase near-infrared fluorescent probe according to claim 2, characterized in that, R6 has one of the following structural formulae or a combination thereof: ； wherein R7 is a linear alkane with carbon number of 1-4 or a C1-C4 alkane with a sulfonic acid group at the end; R8 is a sulfonic acid group, a phenyl group, a nitro group or a halogen group.

4. A method for constructing a large stokes shift cholinesterase near-infrared fluorescent probe, characterized in that, comprising the following steps: S100, synthesizing a fluorophore main skeleton, introducing an electron-donating group at the 4th or 6th position of a quinoline aldehyde compound to form a quinoline skeleton comprising an aldehyde group and an electron-donating group; S200, synthesizing a cholinesterase recognition unit, the recognition unit comprising an aromatic cavity for binding a cationic substrate and an active site for catalyzing hydrolysis of an ester bond; S300, connecting the cholinesterase recognition unit to the quinoline skeleton, and performing a substitution reaction under alkaline conditions to connect the recognition unit to a nitrogen atom of the quinoline skeleton; wherein after step S100 is completed or after step S300 is completed, an electron-withdrawing group or a conjugate expansion group is connected to the 2nd position of the quinoline skeleton.

5. The method for constructing a large Stokes shifted cholinesterase near-infrared fluorescent probe according to claim 4, characterized in that, The electron-donating group comprises one or more of aryl, alkenyl, amine or styryl.

6. The method for constructing a large Stokes shifted cholinesterase near-infrared fluorescent probe according to claim 5, characterized in that, The electron-donating group has one of the following structural formulae or a combination thereof: 。 7. The method for constructing a large Stokes shifted cholinesterase near-infrared fluorescent probe according to claim 4, characterized in that, The electron-donating group is:

8. The method for constructing a large Stokes shifted cholinesterase near-infrared fluorescent probe according to claim 4, characterized in that, The electron-donating group is: ； wherein R7 is a linear alkane with carbon number of 1-4 or a C1-C4 alkane with a sulfonic acid group at the end; ； R8 is a sulfonic acid group, a phenyl group, a nitro group or a halogen group. The cholinesterase is synthesized by taking an aromatic aldehyde compound and an amine derivative as starting materials, and performing substitution, esterification, reduction reaction of aldehyde group, and haloalkane-mediated aminoalkylation reaction to form an intermediate, and then introducing an active linking site through bromination of the hydroxyl group, and the reaction formula is as follows:

9. The method of constructing a large Stokes shifted cholinesterase near infrared fluorescent probe according to claim 4, wherein, wherein R3 is hydrogen, methyl or methoxy; 。 R4 is methyl, ethyl, phenyl; R5 is methyl, propyl, N,N-dimethylamino or N,N-diphenylamino. The electron-withdrawing group or the conjugate expansion group is connected to the quinoline skeleton at the position of the aldehyde group of the quinoline skeleton through a brain cultural revolution condensation reaction.

10. The method of constructing a large Stokes shifted cholinesterase near infrared fluorescent probe according to claim 4, wherein, ​

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