CN113896700A

CN113896700A - Micromolecular formaldehyde fluorescent probe and application thereof

Info

Publication number: CN113896700A
Application number: CN202110751260.2A
Authority: CN
Inventors: 彭涛
Original assignee: Peking University Shenzhen Graduate School
Current assignee: Peking University Shenzhen Graduate School
Priority date: 2021-07-02
Filing date: 2021-07-02
Publication date: 2022-01-07
Anticipated expiration: 2041-07-02
Also published as: CN113896700B

Abstract

The invention discloses a micromolecular formaldehyde fluorescent probe and application thereof. The probe is based on an optimized probe construction strategy of 2-aza-coppu rearrangement reaction, the substituent of nitrogen atom is changed, and the sensitivity and response rate to formaldehyde are improved; changing the structure of a fluorophore to obtain a formaldehyde fluorescent probe with more fluorescence colors in the visible to near-infrared light region, and the formaldehyde fluorescent probe is used for imaging living cells, living tissues and living bodies; and formaldehyde fluorescent probes which target cell nucleuses, endoplasmic reticulum, mitochondria, lysosomes and other organelles are also constructed.

Description

Micromolecular formaldehyde fluorescent probe and application thereof

Technical Field

The invention relates to the technical field of organic small-molecule fluorescent probes, in particular to a small-molecule fluorescent probe for detecting formaldehyde in a biological sample and a preparation method and application thereof.

Background

As the simplest aldehyde molecule, formaldehyde is widely present in the natural and human living environment. Although there have been a number of reports on the harm of exogenous formaldehyde present in the environment to human health, endogenous formaldehyde produced in the body and the associated diseases caused by it have been relatively poorly studied. In recent years, research findings at home and abroad show that formaldehyde can be generated in mammals and human bodies, is a novel gas signal molecule in organisms, and participates in normal physiological processes and the occurrence process of a plurality of diseases. The production of formaldehyde (also referred to as endogenous formaldehyde relative to environmental formaldehyde contaminants) in the body is a normal physiological process mediated by an enzymatic system. For example, methanol can be oxidized intracellularly by Alcohol Dehydrogenase (ADH) to form formaldehyde; semicarbazide-sensitive amine oxidase (SSAO) is used for catalyzing the deamination of amine substances such as methylamine, histamine and polyamine in vivo to generate formaldehyde; lysine demethylase (LSD) and histone demethylase (JmjC domain-stabilizing histone demethylases, JHDM) also produce formaldehyde by catalyzing the demethylation of histone proteins. In addition, formaldehyde pollution factors in the environment, food, medicines and the like are also one of the sources of formaldehyde in the body. Meanwhile, there are corresponding molecular mechanisms in organisms to eliminate formaldehyde, wherein one of the main ways is to oxidize formaldehyde into formic acid by the action of aldehyde dehydrogenase (ALDH) or Alcohol Dehydrogenase (ADH) and then discharge the formic acid with urine, or finally convert the formic acid into carbon dioxide and discharge the carbon dioxide out of the body by respiratory movement.

Due to the balance of formaldehyde production and catabolic pathways, the concentration of formaldehyde endogenous to the organism is maintained in a moderate equilibrium state. In the blood of normal people, the concentration of formaldehyde is about 0.1mM, and the concentration of formaldehyde in urine of normal people is about 3.25 μ M; the content of formaldehyde in the brain can reach 0.2-0.4 mM. Under normal physiological conditions, endogenous formaldehyde is actively involved in the methylation and demethylation cycles of histones, and thus plays an important role in memory storage, retention and retrieval in humans.

However, excessive accumulation of endogenous formaldehyde can react with nucleic acids and proteins to form molecular cross-links, thereby affecting normal gene expression and protein structure and function. Thus, abnormal increases in endogenous formaldehyde concentrations have been linked to the development of a range of diseases, including malignancies, Alzheimer's disease, stroke, and diabetes, among others, and are considered likely to be the causative factors for these diseases. For example, studies at home and abroad over recent years have suggested that the accumulation of endogenous formaldehyde is closely linked to the production, proliferation and migration of tumor cells. The sample test of clinical patients shows that the formaldehyde content of the tumor tissues of the lung cancer patients is about 0.72 mM; the tumor tissue of breast cancer patients is present at about 0.75 mM. In contrast, the formaldehyde concentration in the paracancerous tissue is only 0.19 mM. The formaldehyde exposure of a certain dosage can cause canceration of different organs, and can obviously increase division and proliferation of cancer cells. In order to deeply understand the production and metabolic regulation mechanism of endogenous formaldehyde in the disease occurrence process and clarify the specific action of formaldehyde in the pathological process, the real-time detection and imaging of formaldehyde in biological samples such as living cells, living tissues and even living bodies are very necessary, and the method has important practical significance.

An ideal method for the detection of formaldehyde in biological samples would include several advantages: high sensitivity, high selectivity, nondestructive detection of samples, real-time imaging and the like. The current common methods for measuring formaldehyde comprise high performance liquid chromatography, gas chromatography, radioactive inspection, colorimetric analysis and the like. However, most of these methods have low sensitivity and require invasive disruption of the intact biological sample, thus presenting certain limitations. Compared with the prior art, the small-molecule fluorescent probe has the advantages of high sensitivity, low background, capability of being used for nondestructive detection and real-time visual imaging of biological samples and the like. As a typical organic functional molecule, the organic small molecule fluorescent probe can selectively bind or react with a target analyte to cause a change in fluorescence intensity or wavelength, thereby realizing detection of the analyte. In the detection of biological samples, the sensitivity of the fluorescent probe is high, and the background from the biological samples is low; and more importantly, fluorescent probes enable non-destructive testing of biological samples as well as visual fluorescence imaging analysis, which also enables real-time detection and imaging of analytes. Based on the above, the invention discloses a class of organic small-molecule fluorescent probes based on optimized aza-coppe (2-aza-Cope) rearrangement reaction, which are used for specific detection and imaging of formaldehyde in biological samples.

Disclosure of Invention

The invention aims to provide a small-molecule fluorescent probe for detecting formaldehyde in a biological sample, and a preparation method and application thereof, so as to solve the problems that the formaldehyde probe in the prior art is not high enough in sensitivity, not fast in response speed and the like.

In order to achieve the above purpose, the invention uses 2-aza-coppu rearrangement reaction as a trigger, introduces optimized sites on the nitrogen atom of homoallylamine, and connects variable fluorophores through 1, 2-ethylene on the other side, thereby developing a universal probe template. The probe reacts with formaldehyde to generate imine positive ions, and after 2-aza-coppu rearrangement and hydrolysis (beta-elimination), fluorophores are released to generate remarkable fluorescence, so that the probe can be used for specific detection and imaging of formaldehyde in biological samples. The principle is shown in fig. 32:

a micromolecular formaldehyde fluorescent probe has a structural general formula shown as the following (I):

wherein R is selected from H, alkyl, substituted alkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, hydroxyl, alkoxy, amino, substituted amino, -NHCOOEt, morpholine substituent;

wherein said substituted alkyl may be alkyl substituted with one or more substituents selected from the group consisting of hydroxy, amino, alkenyl, alkynyl, fluoro, chloro, bromo, iodo;

the substituted phenyl group may be phenyl substituted with one or more substituents selected from alkyl, alkoxy, hydroxy, amino, haloalkyl, hydroxyalkyl, hydroxyalkoxy, aminoalkyl, aminoalkoxy, alkenyl, alkynyl, fluoro, chloro, bromo, iodo;

the substituted benzyl group may be benzyl substituted with one or more substituents selected from alkyl, alkoxy, hydroxy, amino, haloalkyl, hydroxyalkyl, hydroxyalkoxy, aminoalkyl, aminoalkoxy, alkenyl, alkynyl, fluoro, chloro, bromo, iodo;

the substituted amino group may be an amino group substituted with one or more substituents selected from alkyl, alkoxy, hydroxy, haloalkyl, hydroxyalkyl, hydroxyalkoxy, alkenyl, alkynyl, fluoro, chloro, bromo, iodo;

a is selected from the group consisting of a fluorophore, a substituted fluorophore; a fluorophore refers to a molecule that is excited at a specific wavelength and emits fluorescence at the specific wavelength;

the substituted fluorophore may be a fluorophore substituted with an organelle targeting group;

z is selected from oxygen atoms

Carbamates, their preparation and their use

Wherein R is₁Selected from H, methyl, C_2-6Alkyl radical, C_2-6A substituted alkyl group.

Preferably, the first and second electrodes are formed of a metal,

r is selected from H and C_1-7Alkyl radical, C_1-7Substituted alkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, hydroxy, C_1-7Alkoxy, amino, substituted amino, -NHCOOEt, morpholine substituents,

wherein said C_1-7The substituted alkyl group may be C_1-7Alkyl is substituted with one or more substituents selected from hydroxy, amino, alkenyl, alkynyl, fluoro, chloro, bromo, iodo;

the substituted phenyl group may be phenyl substituted by one or more groups selected from C_1-6Alkyl radical, C_1-6Alkoxy, hydroxy, amino, halogeno C_1-6Alkyl, alkenyl, alkynyl, fluorine, chlorine, bromine and iodine;

the substituted benzyl group may be benzyl substituted by one or more groups selected from C_1-6Alkyl radical, C_1-6Alkoxy, hydroxy, halogeno C_1-6Alkyl radical, C_1-6Hydroxyalkyl radical, C_1-6Hydroxyalkoxy, C_1-6Substituted by amino alkoxy, alkenyl, alkynyl, fluorine, chlorine, bromine and iodine;

the substituted amino group may be an amino group substituted with one or more groups selected from C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Hydroxy, halogeno C_1-6Alkyl radical, C_1-6Hydroxyalkyl radical, C_1-6Hydroxyalkoxy, alkenyl, alkynyl, and substituent of fluorine, chlorine, bromine and iodine.

Preferably, the first and second electrodes are formed of a metal,

and R is selected from H, methyl, ethyl, n-butyl, isopropyl, propyl, isobutyl, cyclohexylmethyl, neopentyl, phenyl, p-methoxyphenyl, benzyl, o-methylbenzyl, 2, 5-dimethylbenzyl, p-methoxybenzyl, hydroxyl, methoxyl, amino, -NHCOOEt and morpholine substituent.

Preferably, R is p-methoxybenzyl.

Preferably, the first and second electrodes are formed of a metal,

the parent structure of the fluorophore is selected from coumarin, 2-methyl Tokyo green derivatives, fluorescein, 1, 8-naphthalimide derivatives, resorufin and amino hemicyanine; wherein the coumarin, the 2-methyl Tokyo green derivative, the fluorescein, the 1, 8-naphthalimide derivative, the resorufin and the amino cyanine can be respectively substituted by organelle targeting groups.

Preferably, the first and second electrodes are formed of a metal,

the organelle targeting group consists of an organelle positioning group and a connecting group for connecting the organelle positioning group and a fluorophore, wherein the organelle positioning group is selected from a nucleus positioning group, a mitochondrion positioning group, an endoplasmic reticulum positioning group and a lysosome positioning group.

Preferably, the first and second electrodes are formed of a metal,

the fluorophore parent structure is selected from 4-methyl umbelliferone, 2-methyl Tokyo green, ester group modified 2-methyl Tokyo green, 4-hydroxy-N-butyl-1, 8-naphthalimide, ester group modified 1, 8-naphthalimide, resorufin and amino hemicyanine pigment.

Preferably, the first and second electrodes are formed of a metal,

z is carbamate

In which R is₁Is H.

Preferably, the first and second electrodes are formed of a metal,

a structure comprising one of:

the invention also provides application of the micromolecular formaldehyde fluorescent probe in formaldehyde detection.

The invention also provides application of the micromolecular formaldehyde fluorescent probe in formaldehyde detection in living cells and tissues.

The invention also provides application of the micromolecular formaldehyde fluorescent probe in formaldehyde detection in living small animals.

The invention also provides application of the micromolecular formaldehyde fluorescent probe in preparation of a formaldehyde detection kit or test paper.

The invention has the advantages that:

(1) the micromolecular formaldehyde fluorescent probe only emits weak fluorescence before reacting with formaldehyde; under the condition of formaldehyde, the homoallylamine unit in the probe structure can effectively react with formaldehyde, a fluorophore matrix is generated through processes of imine intermediate, aza-copple rearrangement, beta-elimination and the like, and strong fluorescence enhancement is accompanied, so that the fluorescence detection of the formaldehyde is realized, and the steady state, signal conduction and function of the formaldehyde in a biological system can be researched;

(2) based on the optimized probe construction strategy of 2-aza-copro rearrangement reaction, the sensitivity and response rate of the probe obtained by changing the substituent of nitrogen atom to formaldehyde are improved, and the structure of a fluorophore can be changed to develop a formaldehyde fluorescent probe with more fluorescence colors;

(3) the probe with the wavelength from ultraviolet light to blue light region and the formaldehyde fluorescent probe with various colors from visible light to near infrared region are obtained, the formaldehyde selectivity of the probe is good, the sensitivity is good, and the formaldehyde detection limit can be as low as about 1 mu M;

(4) the near-infrared formaldehyde fluorescent probe can be used for living tissue and living body imaging;

(5) the formaldehyde fluorescent probe of the targeted organelle can be used for detecting organelles such as cell nucleus, endoplasmic reticulum, mitochondria, lysosome and the like, and has the advantages of good targeted positioning capability of the organelle, good formaldehyde selectivity and good sensitivity, wherein the formaldehyde detection limit is as low as about 1 mu M.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention:

FIG. 1 is a graph showing the experimental results of the degree of fluorescence response of a formaldehyde fluorescence probe molecule library in reaction with formaldehyde;

FIG. 2 is a graph showing the reaction rate of the fluorescent probe with formaldehyde and the reaction efficiency of formaldehyde molecular of the formaldehyde fluorescent probe; the ordinate is relative fluorescence intensity mean ± standard deviation, n is 3 replicates;

FIG. 3 is the effect of the number of side chain methyl groups on probe reactivity;

FIG. 4 shows the effect of the geminal dimethyl effect and the N-substituent effect on the probe reaction rate. The ordinate is relative fluorescence intensity mean ± standard deviation, n is 3 replicates;

FIG. 5 is a graph showing the results of the sensitive and selective detection of probe FP 445; wherein FIG. 5a is a graph showing the results of an experiment on the fluorescence response intensity of 10. mu.M probe FP445 to 0-2mM formaldehyde at 37 ℃ for 2 hours; FIG. 5b is a graph showing the results of fluorescence response intensity experiment of 10. mu.M probe FP445 to 500. mu.M formaldehyde at 37 ℃ for 0 to 180 minutes; FIG. 5c is a graph of the time dependence of 10. mu.M probe FP445 on 500. mu.M formaldehyde; FIG. 5d is a graph showing the results of fluorescence response intensity experiments for 10. mu.M probe FP445 to formaldehyde and other analytes at 0, 40, 80, 120 minutes;

FIG. 6 is a graph showing the results of the sensitivity and selectivity detection of probe FP 511; wherein FIG. 6a is a graph showing the results of an experiment on the fluorescence response intensity of 10. mu.M probe FP511 to 0-2mM formaldehyde at 37 ℃ for 2 hours; FIG. 6b is a graph showing the results of fluorescence response intensity experiment of 10. mu.M probe FP511 to 500. mu.M formaldehyde at 37 ℃ for 0 to 180 minutes; FIG. 6c is a graph showing the time dependence of 10. mu.M probe FP511 on 500. mu.M formaldehyde; FIG. 6d is a graph showing the results of fluorescence response intensity experiments for 10. mu.M probe FP511 to formaldehyde and other analytes at 0, 40, 80, 120 minutes;

FIG. 7 is a graph showing the results of the sensitive and selective detection of probe FP 511B; wherein, FIG. 7a is a graph showing the results of an experiment on the fluorescence response intensity of 10. mu.M probe FP511B to 0-2mM formaldehyde at 37 ℃ for 2 hours; FIG. 7b is a graph showing the results of fluorescence response intensity experiment of 10. mu.M probe FP511B to 500. mu.M formaldehyde at 37 ℃ for 0 to 180 minutes; FIG. 7c is a graph of the time dependence of 10. mu.M probe FP511B on 500. mu.M formaldehyde; FIG. 7d is a graph showing the results of fluorescence response intensity experiments for 10. mu.M probe FP511B to formaldehyde and other analytes at 0, 40, 80, 120 minutes;

FIG. 8 is a graph showing the results of the sensitive and selective detection of probe FP 511C; wherein, FIG. 8a is a graph showing the results of an experiment on the fluorescence response intensity of 10. mu.M probe FP511C to 0-2mM formaldehyde at 37 ℃ for 2 hours; FIG. 8b is a graph showing the results of fluorescence response intensity experiment of 10. mu.M probe FP511C to 500. mu.M formaldehyde at 37 ℃ for 0 to 180 minutes; FIG. 8c is a graph of the time dependence of 10. mu.M probe FP511C on 500. mu.M formaldehyde;

FIG. 8d is a graph showing the results of experiments on the fluorescence response intensity of 10. mu.M probe FP511C to formaldehyde and other analytes at 0, 40, 80, and 120 minutes;

FIG. 9 is a graph showing the results of the sensitivity and selectivity detection of probe FP 551; wherein, FIG. 9a is a graph showing the result of an experiment on the fluorescence response intensity of 0-2mM formaldehyde at 37 ℃ for 2 hours using 10. mu.M probe FP 551; FIG. 9b is a graph showing the result of an experiment on the fluorescence response intensity of 10. mu.M probe FP551 to 500. mu.M formaldehyde at 37 ℃ for 0 to 180 minutes; FIG. 9c is a graph showing the time dependence of 10. mu.M probe FP551 on 500. mu.M formaldehyde; FIG. 9d is a graph showing the results of fluorescence response intensity experiments for 10. mu.M probe FP551 to formaldehyde and other analytes at 0, 40, 80, 120 minutes;

FIG. 10 is a graph showing the results of the sensitivity and selectivity detection of probe FP 551B; wherein, FIG. 10a is a graph showing the result of an experiment on the fluorescence response intensity of 0-2mM formaldehyde at 37 ℃ for 2 hours with 10. mu.M probe FP 551B;

FIG. 10b is a graph showing the results of fluorescence response intensity experiment of 10. mu.M probe FP551B to 500. mu.M formaldehyde at 37 ℃ for 0 to 180 minutes; FIG. 10c is a graph of the time dependence of 10. mu.M probe FP551B on 500. mu.M formaldehyde; FIG. 10d is a graph showing the results of fluorescence response intensity experiments for 10. mu.M probe FP551B to formaldehyde and other analytes at 0, 40, 80, 120 minutes;

FIG. 11 is a graph showing the results of the sensitivity and selectivity detection of probe FP 551C; wherein, FIG. 11a is a graph showing the result of an experiment of the fluorescence response intensity of 10. mu.M probe FP551C to 0-2mM formaldehyde at 37 ℃ for 2 hours;

FIG. 11b is a graph showing the result of an experiment on the fluorescence response intensity of 10. mu.M probe FP551C to 500. mu.M formaldehyde at 37 ℃ for 0 to 180 minutes; FIG. 11C is a graph showing the time dependence of 10. mu.M probe FP551C on 500. mu.M formaldehyde; FIG. 11d is a graph showing the results of fluorescence response intensity experiments for 10. mu.M probe FP551C to formaldehyde and other analytes at 0, 40, 80, 120 minutes;

FIG. 12 is a graph showing the results of the sensitivity and selectivity detection of probe FP 585; wherein, FIG. 12a is a graph showing the experimental results of the fluorescence response intensity of 10. mu.M probe FP585 to 0-2mM formaldehyde at 37 ℃ for 2 hours; FIG. 12b is a graph showing the results of an experiment on the fluorescence response intensity of 10. mu.M probe FP585 to 500. mu.M formaldehyde at 37 ℃ for 0 to 180 minutes; FIG. 12c is a graph showing the time dependence of 10. mu.M probe FP585 on 500. mu.M formaldehyde; FIG. 12d is a graph showing the results of fluorescence response intensity experiments for 10. mu.M probe FP585 to formaldehyde and other analytes at 0, 40, 80, 120 min;

FIG. 13 is a graph showing the results of the sensitive and selective detection of probe FP 706; wherein, FIG. 13a is a graph showing the result of an experiment on the fluorescence response intensity of 10. mu.M probe FP706 to 0-2mM formaldehyde at 37 ℃ for 2 hours; FIG. 13b is a graph showing the results of an experiment on the fluorescence response intensity of 10. mu.M probe FP706 to 500. mu.M formaldehyde at 37 ℃ for 0 to 180 minutes; FIG. 13c is a graph showing the time dependence of 10. mu.M probe FP706 on 500. mu.M formaldehyde; FIG. 13d is a graph showing the results of fluorescence response intensity experiments for 10. mu.M probe FP706 to formaldehyde and other analytes at 0, 40, 80, and 120 minutes;

FIG. 14 is a graph showing the results of measuring the fluorescence response intensity of different probes at 10. mu.M for different concentrations of formaldehyde;

FIG. 15 is a graph showing the results of intracellular detection imaging of exogenous formaldehyde by probes FP511, FP511B, FP 511C;

FIG. 16 is a graph showing the results of intracellular detection imaging of exogenous formaldehyde by probes FP551, FP551B and FP 551C;

FIG. 17 is a photograph of confocal fluorescence imaging of probe FP511C against exogenous formaldehyde in HEK293T cells;

FIG. 18 is a photograph of confocal fluorescence imaging of probe FP551B against exogenous formaldehyde in HEK293T cells;

FIG. 19 is a confocal fluorescence imaging of probe FP585 to exogenous formaldehyde in HEK293T cells;

FIG. 20 is a photograph of confocal fluorescence imaging of probe FP706 to exogenous formaldehyde in HEK293T cells;

FIG. 21 is an image of probe FP585 vs. cellular endogenous formaldehyde;

FIG. 22 is a photograph of the endogenous formaldehyde of the cells imaged by probe FP 706;

FIG. 23 is an imaging plot of probe FP706 detecting endogenous formaldehyde in ex vivo brain tissue sections;

FIG. 24 is a graph showing the results of detection of the sensitivity, selectivity and detection limit of probe FP 551-Nuc; wherein, FIG. 24a is a graph showing the results of an experiment on the fluorescence response intensity of 0-2mM formaldehyde at 37 ℃ for 2 hours using 10. mu.M probe FP 551-Nuc; FIG. 24b is a graph showing the results of the fluorescence response intensity experiment of 10. mu.M probe FP551-Nuc to 500. mu.M formaldehyde at 37 ℃ for 0 to 180 minutes; FIG. 24c is a graph showing the time dependence of 10. mu.M probe FP551-Nuc on 500. mu.M formaldehyde; FIG. 24d is a graph showing the results of fluorescence response intensity experiments for 10. mu.M probe FP551-Nuc to formaldehyde and other analytes at 0, 40, 80, 120 minutes; FIG. 24e is a graph showing the results of fluorescence response intensity experiments for 10. mu.M probe FP551-Nuc against 0, 1 and 2.5. mu.M formaldehyde at 37 ℃ for 2 hours;

FIG. 25 is a photograph of fluorescent images of the probe FP551-Nuc for the detection of intracellular formaldehyde;

FIG. 26 is a graph showing the results of detection of the sensitivity, selectivity and detection limit of probe FP 551-ER; wherein, FIG. 26a is a graph showing the results of an experiment on the fluorescence response intensity of 0-2mM formaldehyde at 37 ℃ for 2 hours using 10. mu.M probe FP 551-ER; FIG. 26b is a graph showing the results of fluorescence response intensity experiment of 10. mu.M probe FP551-ER to 500. mu.M formaldehyde at 37 ℃ for 0 to 180 minutes; FIG. 26c is a graph showing the time dependence of 10. mu.M probe FP551-ER on 500. mu.M formaldehyde; FIG. 26d is a graph showing the experimental results of the fluorescence response intensity of 10. mu.M probe FP551-ER to formaldehyde and other analytes at 0, 40, 80, 120 minutes; FIG. 26e is a graph showing the results of fluorescence response intensity experiments for 10. mu.M probe FP551-ER to 0, 1, 2.5. mu.M formaldehyde at 37 ℃ for 2 hours;

FIG. 27 is a photograph of fluorescent image of the probe FP551-ER for detecting formaldehyde in cells;

FIG. 28 is a graph showing the results of detection of the sensitivity, selectivity and detection limit of probe FP 551-Mito; wherein, FIG. 28a is a graph showing the results of fluorescence response intensity experiment of 10. mu.M probe FP551-Mito to 0-2mM formaldehyde at 37 ℃ for 2 hours; FIG. 28b is a graph showing the results of fluorescence response intensity experiment of 10. mu.M probe FP551-Mito 500. mu.M formaldehyde at 37 ℃ for 0 to 180 minutes; FIG. 28c is a graph of the time dependence of 10. mu.M probe FP551-Mito on 500. mu.M formaldehyde; FIG. 28d is a graph showing the results of fluorescence response intensity experiments for 10. mu.M probe FP551-Mito formaldehyde and other analytes at 0, 40, 80, 120 minutes; FIG. 28e is a graph showing the results of fluorescence response intensity experiments for 10. mu.M probe FP551-

Mito

0, 1, 2.5. mu.M formaldehyde at 37 ℃ for 2 hours;

FIG. 29 is a photograph of fluorescent image of formaldehyde in cells detected by probe FP 551-Mito;

FIG. 30 is a graph showing the results of detection of the sensitivity, selectivity and detection limit of the probe FP 551-Lyso; wherein FIG. 30a is a graph showing the result of an experiment on the fluorescence response intensity of 0 to 2mM formaldehyde at 37 ℃ for 2 hours using 10. mu.M probe FP 551-Lyso; FIG. 30b is a graph showing the result of an experiment on the fluorescence response intensity of 10. mu.M probe FP551-Lyso to 500. mu.M formaldehyde at 37 ℃ for 0 to 180 minutes; FIG. 30c is a graph showing the time dependence of 10. mu.M probe FP551-Lyso on 500. mu.M formaldehyde; FIG. 30d is a graph showing the results of fluorescence response intensity experiments for 10. mu.M probe FP551-Lyso to formaldehyde and other analytes at 0, 40, 80, 120 minutes; FIG. 30e is a graph showing the results of the fluorescence response intensity experiment of 10. mu.M probe FP551-Lyso to 0, 1 and 2.5. mu.M formaldehyde at 37 ℃ for 2 hours;

FIG. 31 is a photograph of fluorescent image for detecting intracellular formaldehyde by the probe FP 551-Lyso;

FIG. 32 is a schematic diagram of the probe for specific detection and imaging of formaldehyde in a biological sample.

Detailed Description

The present invention will be described in detail with reference to the drawings and specific embodiments, which are illustrative of the present invention and are not to be construed as limiting the present invention.

The invention is based on the 2-aza-copal reaction of formaldehyde and homoallylamine, and the design strategy of a formaldehyde fluorescent probe is to introduce phenolic hydroxyl of a fluorophore to alpha-carbon atom of homoallylamine through a1, 2-ethylene connecting unit and introduce a reaction regulating group R to nitrogen atom; the formaldehyde reacts with the homoallylic amine derivative and then undergoes rearrangement and hydrolysis to finally generate a free fluorophore, thereby enhancing fluorescence.

Based on the design strategy, the invention synthesizes a series of fluorescent probes with different nitrogen atom substituents, optimizes the reaction rate and yield of formaldehyde and the homoallylamine derivative, and thus obtains the homoallylamine formaldehyde trigger with the best formaldehyde reactivity. Then, by utilizing the universality and modularization of a design strategy and changing the structure of a fluorophore, a series of novel formaldehyde fluorescent probes with various fluorescent colors are developed and further used for real-time imaging analysis of formaldehyde in living cells and living tissues, and a series of specific recognition probes targeting subcellular organelles are also designed and synthesized, and then the performance of the probes for detecting formaldehyde in the cells is characterized. The synthesis, application, etc. of each probe will be described in detail below.

All reagents were purchased from reagent companies and used without any purification treatment if not otherwise specified. All reactions were run under anhydrous conditions under argon. If not otherwise stated, the solvent is re-evaporated before use, THF is treated with the sodium-benzophenone system and methylene chloride with calcium hydride. Anhydrous DMF, DMSO, MeCN, MeOH, etc. were all purchased directly from the reagent company for use as solvents. The reaction was monitored by TLC using thin-layer silica gel plates developed with GF 254(60-F250, 0.2mm, Qingdao Seashore Seawa Seisakusho) using UV (254 nm and 365nm) or iodine or by immersion in acidic potassium permanganate, ninhydrin solution, followed by heat development. The silica gel used for flash column chromatography is from Qingdao marine silica gel 60 (300-400mesh ASTM). Generally, ethyl acetate and n-hexane are used as eluent, and the ratio adopted in the operation is the volume ratio of the ethyl acetate and the n-hexane.¹H NMR and¹³c NMR employs DRX-300(¹H：300MHz，¹³C: 75MHz) or Bruker Avance-400(¹H：400MHz，¹³C: 100MHz) or Bruker Avance-500(¹H：500MHz，¹³C: 125MHz) and the splitting of the nuclear magnetic peak is explained using abbreviations such as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and the like. The deuterated reagent is deuterated chloroform, deuterated dimethyl sulfoxide, deuterated methanol and the like, the solvent peaks of the hydrogen spectrum in the nuclear magnetic spectrum are respectively 7.26 (deuterated chloroform), 3.31 (deuterated methanol) or 2.50 (deuterated dimethyl sulfoxide), and the solvent peaks of the carbon spectrum are respectively 77.16 (deuterated chloroform), 49.00 (deuterated methanol) or 40.00 (deuterated dimethyl sulfoxide). High resolution mass spectra were determined on an ABI Q-star Elite high resolution mass spectrometer using ESI conditions.

General abbreviations in this specification

1. Formaldehyde fluorescent probe molecule library

1.1 Formaldehyde fluorescent Probe molecule Synthesis

Example 1 Formaldehyde fluorescent probe compound 2.7a-f scheme I:

singly protecting 1, 3-propylene glycol by using p-methoxybenzyl chloride (PMBCl), and oxidizing by using 2-iodoxybenzoic acid (IBX) to generate an aldehyde compound 2.1; then compound 2.1(1.0equiv.) in tetrahydrofuran (30mL) was added to the amine RNH at 0 deg.C₂(R is selected from H, propyl, benzyl, p-methoxybenzyl, o-methylbenzyl, 2, 5-dimethylbenzyl and neopentyl) solution (1.2equiv.), the reaction is carried out for 30min at room temperature to generate an imine intermediate, then the imine intermediate is reacted with allyl boronic acid pinacol ester (1.2equiv.) at room temperature for 8H, and the completion of the raw material reaction is monitored by thin layer chromatography. Concentrating, and separating by silica gel flash column chromatography (n-hexane: ethyl acetate ═ 10: 1) to obtain colorless oily liquid homoallylamine compound 2.2a-g, wherein R ═ H of 2.2a, R ═ propyl of 2.2b, R ═ benzyl of 2.2c, R ═ p-methoxybenzyl of 2.2d, R ═ o-methylbenzyl of 2.2e, R ═ 2, 5-dimethylbenzyl of 2.2f, and R ═ neopentyl of 2.2 g;

compounds 2.2a-g (1.0equiv.) of tetrahydroFuran solution (20mL) Boc was added at 0 deg.C₂O (1.2equiv.) protects amino, the reaction is carried out for 10h at room temperature, and the completion of the reaction of the raw materials is monitored by thin-layer chromatography. Concentrating, and separating by silica gel flash column chromatography (n-hexane: ethyl acetate: 20: 1) to obtain colorless oily liquid 2.3a-g, and obtaining compound 2.3 a-g;

2.3a-g (1.0equiv.) of compound (1.3 a) in dichloromethane and water (10: 1 by volume) was added 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ) at 0 deg.C, reacted at room temperature for 3h to remove the PMB protecting group, and the completion of the starting material reaction was monitored by thin layer chromatography. Then adding saturated sodium bicarbonate, dichloromethane and water (the volume ratio is 2: 2: 1) respectively, stirring vigorously at room temperature for 2 hours, extracting the organic phase for three times by using dichloromethane, washing the combined organic phase by using saturated saline solution, drying by using anhydrous sodium sulfate, filtering, concentrating, and separating by using silica gel flash column chromatography (normal hexane: ethyl acetate: 5: 1) to obtain colorless oily liquid alcohol 2.4 a-g;

a methylene chloride solution of alcohols 2.4a-b (1.0equiv.) was subjected to an Appel reaction at 0 ℃ for 12 hours with the addition of imidazole (1.1equiv.), triphenylphosphine (1.1equiv.), carbon tetrabromide (1.1equiv.), and the completion of the starting material reaction was monitored by thin layer chromatography. Then adding water, extracting the organic phase for three times by using dichloromethane, washing the combined organic phase by using saturated saline solution, drying the combined organic phase by using anhydrous sodium sulfate, filtering, concentrating, and separating by using silica gel flash column chromatography (normal hexane: ethyl acetate: 10: 1) to obtain colorless oily liquid 2.5 a-b;

a solution of alcohol 2.4d (742mg, 2.2mmol) in dichloromethane (15mL) was added imidazole (166mg, 2.44mmol), triphenylphosphine (639mg, 2.44mmol), iodine (616mg, 2.68mmol) at 0 deg.C for an Appel reaction at room temperature for 12h, and the completion of the starting material reaction was monitored by thin layer chromatography. Then adding water, extracting an organic phase by using dichloromethane (3X 30mL), washing the combined organic phases by using saturated saline solution, drying the combined organic phases by using anhydrous sodium sulfate, filtering, concentrating, and separating by using silica gel flash column chromatography (normal hexane: ethyl acetate: 10: 1) to obtain a colorless oily liquid for 2.5 d;

a solution of alcohols 2.4c-f (1.0equiv.) in methylene chloride was added p-toluenesulfonyl chloride (1.2equiv.), triethylamine (3.0equiv.), 4-dimethylaminopyridine (0.2equiv.) at 0 ℃. Reacting at room temperature for 12h to realize Ts protection of hydroxyl, and monitoring the completion of the reaction of the raw materials by thin-layer chromatography. Then adding water, extracting the organic phase for three times by using dichloromethane, washing the combined organic phase by using saturated saline solution, drying the combined organic phase by using anhydrous sodium sulfate, filtering, concentrating, and separating by using silica gel flash column chromatography (normal hexane: ethyl acetate is 10: 1) to obtain colorless oily liquid compounds 2.5c, 2.5e-f and 3.8;

4-Methylelendone (1.2equiv.), potassium carbonate (1.5equiv.) in N, N-dimethylformamide (5mL) was added to a solution of compounds 2.5a-b (1.0equiv.) in N, N-dimethylformamide (5mL) at 0 ℃ and reacted at room temperature for 12h, and the completion of the reaction was monitored by thin layer chromatography. Then adding water, extracting the organic phase for three times by using ethyl acetate, washing the combined organic phase for three times by using 1N hydrochloric acid solution, washing the combined organic phase by using saturated saline solution, drying the combined organic phase by using anhydrous sodium sulfate, filtering, concentrating, and separating by using silica gel flash column chromatography (normal hexane: ethyl acetate: 5: 1) to obtain white solid compounds 2.6 a-b;

a solution of 4-methylumbelliferone (48mg, 0.27mmol) and potassium carbonate (47mg, 0.34mmol) in N, N-dimethylformamide (5mL) was added to a solution of compound 2.5d (0.1g, 0.23mmol) in N, N-dimethylformamide (5mL) at room temperature, the reaction was carried out at room temperature for 12h, and the completion of the reaction of the starting materials was monitored by thin layer chromatography. Then adding water, extracting an organic phase by using ethyl acetate (3X 30mL), washing the combined organic phase for three times by using 1N hydrochloric acid solution, washing the organic phase by using saturated saline solution, drying the organic phase by using anhydrous sodium sulfate, filtering, concentrating, and separating by using silica gel flash column chromatography (normal hexane: ethyl acetate: 5: 1) to obtain a white solid compound 2.6 d;

a solution of 4-methylumbelliferone (1.2equiv.) and cesium carbonate (1.5equiv.) in N, N-dimethylformamide (5mL) was added at 0 ℃ to a solution of compounds 2.5c, e-f (1.0equiv.) in N, N-dimethylformamide (5mL), and the reaction was carried out at room temperature for 12 hours, followed by monitoring the completion of the reaction of the starting materials by thin layer chromatography. Then adding water, extracting the organic phase for three times by using ethyl acetate, washing the combined organic phase for three times by using 1N hydrochloric acid solution, washing the combined organic phase by using saturated saline solution, drying the combined organic phase by using anhydrous sodium sulfate, filtering, concentrating, and separating by using silica gel flash column chromatography (normal hexane: ethyl acetate: 5: 1) to obtain white solid compounds 2.6c and e-f;

finally, the compound 2.6a-f (0.1equiv.) was reacted with 4M hydrogen chloride in 1, 4-dioxane (3mL) at room temperature for 2h to remove the Boc protecting group and the completion of the reaction was monitored by thin layer chromatography. Direct concentration (three times with toluene addition) gave the target probes 2.7a-f as white solids. Synthetic routes are represented as route I:

2.7a(85mg,89％):R_f＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(300 MHz,MeOD)δ7.66(d,J＝8.8Hz,1H),7.06-6.78(m,2H),6.15(d,J＝1.2Hz, 1H),5.88(m,1H),5.38-5.15(m,2H),4.25(td,J＝6.4,1.8Hz,2H),3.54-3.35(m, 1H),2.57-2.31(m,5H),2.21-2.00(m,2H),1.90(s,1H).¹³C NMR(75MHz,MeOD) δ159.4,159.2,152.4,151.6,130.1,123.3,116.1,111.0,109.9,108.3,98.4,62.4, 45.9,35.6,29.9,14.6.HRMS calcd for C₁₆H₂₀NO₃[M+H]⁺274.1443,found 274.1438.

2.7b(35mg,79％):R_f＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(500 MHz,CDCl₃)δ7.50(d,J＝8.8Hz,1H),6.92-6.79(m,2H),6.14(d,J＝0.9Hz, 1H),5.88-5.76(m,1H),5.20-5.08(m,2H),4.15(m,2H),2.89-2.75(m,1H),2.71-2.51 (m,2H),2.41(d,J＝0.9Hz,3H),2.28(dt,J＝18.2,7.2Hz,2H),1.93(qd,J＝14.3, 7.6Hz,2H),1.50(m,2H),0.93(t,J＝7.4Hz,3H).¹³C NMR(126MHz,CDCl3)δ 162.1,161.3,155.3,152.5,135.1,125.4,117.8,113.5,112.5,111.9,101.5,66.2,54.3, 48.9,38.7,33.3,23.4,18.6,11.8.HRMS calcdfor C₁₉H₂₆NO₃[M+H]⁺316.1913, found 316.1910.

2.7c(12mg,73％):R_f＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(300 MHz,CDCl₃)δ7.49(d,J＝8.5Hz,1H),7.30(t,J＝6.1Hz,5H),6.83(t,J＝5.5 Hz,2H),6.15(d,J＝1.1Hz,1H),5.95-5.70(m,1H),5.27-4.94(m,2H),4.32-4.03(m, 2H),3.81(q,J＝13.0Hz,2H),3.03-2.71(m,1H),2.41(d,J＝1.1Hz,3H),2.39-2.21 (m,2H),2.04-1.79(m,2H).¹³C NMR(75MHz,CDCl3)δ162.1,161.5,155.4, 152.7,140.6,135.0,128.5,128.2,127.0,125.5,118.1,113.6,112.7,112.0,101.5,66.2, 53.5,51.2,38.7,33.4,18.8.HRMS calcd forC₂₃H₂₆NO₃[M+H]⁺364.1913,found 364.1905.

2.7d(50mg,79％):R_f＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(300 MHz,MeOD)δ7.69(d,J＝8.8Hz,1H),7.47(d,J＝8.6Hz,2H),6.96(dd,J＝ 13.2,5.5Hz,3H),6.87(d,J＝2.2Hz,1H),6.17(s,1H),5.90(m,1H),5.48-5.17(m, 2H),4.27(dd,J＝11.9,4.9Hz,4H),3.80(s,3H),3.68-3.51(m,1H),2.79-2.57(m, 2H),2.44(d,J＝0.6Hz,3H),

2.40-2.21(m,2H).¹³C NMR(75MHz,MeOD)δ162.0,161.6,160.8,155.0, 154.3,131.6,131.3,126.1,122.8,119.7,114.3,113.9,112.4,111.1,101.2,64.6,54.7, 54.5,48.0,34.3,29.3,17.4.HRMS calcd for C₂₄H₂₈NO₄[M+H]⁺394.2018,found 394.2010.

2.7e(60mg,67％):R_f＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(400 MHz,CDCl₃)δ9.70(d,J＝58.4Hz,2H),7.67(s,1H),7.43(d,J＝8.2Hz,1H), 7.11(s,3H),6.87-6.54(m,2H),6.08(s,1H),5.76(s,1H),5.19(t,J＝11.3Hz,2H), 4.22(s,1H),4.05(s,3H),3.28(s,1H),2.67(d,J＝34.8Hz,2H),2.40(s,3H),2.35(s, 3H),2.24(s,2H).¹³CNMR(101MHz,CDCl₃)δ161.3,155.1,152.6,137.6,132.1, 131.2,131.0,129.5,128.8,126.8,125.7,120.2,114.0,112.2,102.0,64.7,54.0,45.2, 35.2,30.1,19.9,18.7.HRMScalcd for C₂₄H₂₈NO₃[M+H]⁺378.2069,found 378.2062.

2.7f(15mg,65％):R_f＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(500 MHz,CDCl₃)δ7.22(d,J＝8.6Hz,1H),6.79(t,J＝7.4Hz,1H),6.70(d,J＝7.2 Hz,2H),6.53(d,J＝8.0Hz,1H),6.46(s,1H),5.73(s,1H),5.50(d,J＝7.1Hz,1H), 4.95(dd,J＝40.3,13.4Hz,2H),3.91(dd,J＝32.0,18.4Hz,4H),2.89(d,J＝1.3Hz, 1H),2.33(s,2H),2.03(s,6H),2.01(s,3H),1.97(d,J＝7.6Hz,2H).¹³C NMR(126 MHz,CDCl₃)δ161.6,161.0,154.5,153.5,137.8,131.2,129.4,128.6,127.4,125.7, 119.7,113.7,112.0,111.0,101.1,64.7,56.7,42.8,34.2,29.4,18.7,17.3.HRMS calcd for C₂₅H₃₀NO₃[M+H]⁺392.2226,found 392.2223.

example 2 formaldehyde fluorescent probe compound 2.7g synthetic route II:

synthesis of Compound 2.4g referring to the synthesis of Compounds 2.4a-g of example 1 wherein the imine intermediate is formed using an amine RNH2, R being selected from neopentyl, to give compound 2.4g, the Mitsunobu reaction is used and 2.4g (0.77g, 2.7mmol), 4-methylumbelliferone (713mg, 4.05mmol) and triphenylphosphine (1.06g, 4.05mmol) in tetrahydrofuran (20mL) is slowly added diethyl azodicarboxylate (0.64mL, 4.05mmol) at 0 deg.C and heated to 95 deg.C for reflux reaction for 12h and thin layer chromatography monitors the completion of the starting reaction. Cooling to room temperature, adding water, extracting the organic phase with ethyl acetate (3 × 30mL), washing the combined organic phases with 0.1N hydrochloric acid solution three times, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating, separating by silica gel flash column chromatography (N-hexane: ethyl acetate 15: 1) to obtain 2.6g of white solid compound, finally adding 4M hydrogen chloride in 1, 4-dioxane solution (3mL) to react at room temperature for 2h to remove Boc protecting group, and monitoring by thin layer chromatography that the raw materials are completely reacted. Direct concentration (three additional toluene bands) yielded 2.7g of the target probe as a white solid. The synthetic route is shown as route II:

2.7g(35mg,64％):R_f＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(400 MHz,MeOD)δ7.67(d,J＝8.8Hz,1H),6.96(dd,J＝8.9,2.5Hz,1H),6.89(d,J＝ 2.5Hz,1H),6.15(d,J＝1.1Hz,1H),5.87(m,1H),5.30-5.15(m,2H),4.32-4.14(m, 2H),2.76(d,J＝12.0Hz,1H),2.67(d,J＝12.0Hz,1H),2.49(t,J＝6.7Hz,2H), 2.43(d,J＝1.1Hz,3H),2.20-2.06(m,2H),1.01(s,10H).¹³C NMR(101MHz, MeOD)δ162.1,162.0,155.1,154.4,133.5,126.1,118.3,113.7,112.6,111.0, 101.1,65.8,57.0,56.3,36.1,30.9,30.6,26.6,17.4.HRMS calcd for C₂₁H₃₀NO₃ [M+H]⁺344.2226,found 344.2221.

EXAMPLE 3 Formaldehyde fluorescent Probe Compounds 2.7h and 2.7i Synthesis route III

Nucleophilic addition was achieved by adding 15mL of allyl Grignard (allyl magnesium bromide) (1.5N) to a tetrahydrofuran solution (30mL) of the aldehyde 2.1(1.94g, 10mmol) at-70 deg.C for 2h at room temperature and monitoring the completion of the starting material reaction by thin layer chromatography. Then quenching the reaction with water, extracting the organic phase with diethyl ether (3 × 30mL), washing the combined organic phases with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating, and separating by silica gel flash column chromatography (n-hexane: ethyl acetate 10: 1) to obtain a colorless oily liquid compound 2.8;

after the compound 2.8(1.08g, 4.6mmol) in dichloromethane (30mL) and water (3mL) was added 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone at 0 deg.C and reacted at room temperature for 3h to remove the PMB protecting group, the starting material was monitored by thin layer chromatography for completion of the reaction. Then, saturated sodium bicarbonate (40mL), dichloromethane (40mL) and water (20mL) were added, and the mixture was stirred vigorously at room temperature for 2 hours, and the organic phases were extracted with dichloromethane (3X 40mL), and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The concentrated product was dissolved in diethyl ether (25mL), and lithium aluminum hydride (0.26g, 6.9mmol) was slowly added thereto at 0 ℃ to conduct reduction reaction at room temperature for 1 hour. Quench with water and continue the reaction for 15 minutes. Wet sodium sulfate was added, followed by filtration, concentration, and silica gel flash column chromatography (n-hexane: ethyl acetate ═ 2: 1) to separate a colorless oily liquid diol compound 2.9.

A solution of compound 2.9(425mg, 3.7mmol) in dichloromethane (15mL) was reacted at 0 deg.C with the addition of p-toluenesulfonyl chloride (744mg, 3.9mmol), triethylamine (1.03mL, 7.4mmol), 4-dimethylaminopyridine (73mg, 0.6mmol) at room temperature for 12h to selectively protect the primary alcohol with Ts to give compound 2.10; the completion of the reaction of the starting materials was monitored by thin layer chromatography. Then, water was added, the organic phase was extracted with dichloromethane (3 × 30mL), the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel flash column chromatography (n-hexane: ethyl acetate 4: 1) to give 2.10 as a colorless oily liquid compound;

4-Methyleumbelliferone (1.24g, 3.8mmol) and cesium carbonate (0.67g, 3.8mmol) in N, N-dimethylformamide (20mL) A solution of compound 2.10(854mg, 3.2mmol) in N, N-dimethylformamide (5mL) was added at 40 deg.C and the reaction was allowed to proceed for 12h at 40 deg.C, and the completion of the starting material reaction was monitored by thin layer chromatography. Then, water was added, the organic phases were extracted with ethyl acetate (3 × 30mL), the combined organic phases were washed with 0.1N hydrochloric acid solution (3 × 30mL), washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel flash column chromatography (N-hexane: ethyl acetate 2: 1) to give compound 2.11 as a white solid;

compound 2.11(726mg, 2.7mmol) in dichloromethane (20mL) was oxidized at 0 deg.C for 12 hours with dessimutan oxidant (1.35g, 3.2mmol) and the starting material was monitored by thin layer chromatography for completion. Then adding water, extracting the organic phase by using ethyl acetate (3 × 30mL), washing the combined organic phases by using 0.1N sodium hydroxide (3 × 30mL), washing the combined organic phases by using saturated saline solution, drying the combined organic phases by using anhydrous sodium sulfate, filtering, concentrating, and separating by using silica gel flash column chromatography (normal hexane: ethyl acetate: 3: 1) to obtain a white solid compound 2.12;

the synthetic route is shown as follows:

compound 2.12(1.0equiv.) in dichloromethane was added hydroxylamine hydrochloride or methoxylamine hydrochloride (1.2equiv.) at 0 deg.C, added sodium acetate solid (2.0equiv.), reacted at room temperature for 12h, and the completion of the starting material reaction was monitored by thin layer chromatography. Then water was added, the organic phase was extracted three times with dichloromethane, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The formation of the imine product was confirmed by nuclear magnetic resonance and LC-MS, the concentrated product was dissolved in methanol, followed by addition of sodium cyanoborohydride (3.0equiv.) and a 1: 1 to about 5, and the reaction is continued at room temperature for 5 hours to reduce the imine. After concentration, 1N hydrochloric acid solution was added to adjust the pH to about 1, then ammonia was used to carefully adjust the pH to 8-10, and the organic phase was extracted with ether. The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated and separated by flash column chromatography on silica gel (n-hexane: ethyl acetate 4: 1) to give a white solid 2.7h-i, the synthetic route is shown below:

2.7h(43mg,77％):R_f＝0.3(silica gel,hexanes:ethyl acetate＝3:1)；¹H NMR (400MHz,CDCl₃)δ7.46(d,J＝8.8Hz,1H),6.92-6.70(m,2H),6.10(d,J＝1.0Hz, 1H),5.83-5.70(m,1H),5.13(dd,J＝12.1,6.0Hz,2H),4.20-4.02(m,2H),3.51(s, 3H),3.19-3.00(m,1H),2.36(dd,J＝6.0,1.1Hz,3H),2.34-2.18(m,2H),1.94(m, 2H).¹³C NMR(101MHz,CDCl₃)δ162.0,161.4,155.3,152.7,134.8,125.6,118.2, 113.6,112.7,112.0,101.5,66.3,62.3,57.2,36.6,31.3,18.7.HRMS calcd for C₁₇H₂₂NO₄[M+H]⁺304.1549,found 304.1541.

2.7i(47mg,77％):R_f＝0.3(silica gel,hexanes:ethyl acetate＝3:1)；¹H NMR (300MHz,CDCl₃)δ7.47(d,J＝8.8Hz,1H),6.94-6.67(m,2H),6.12(d,J＝0.8Hz, 1H),5.80(m,1H),5.33-5.01(m,4H),4.15(td,J＝6.3,2.5Hz,2H),3.28-3.03(m,1H), 2.47-2.20(m,5H),2.16-1.88(m,3H).¹³C NMR(126MHz,CDCl3)δ161.7,161.3, 155.2,152.5,134.0,125.5,118.7,113.7,112.6,111.9,101.4,66.0,58.3,35.8,30.4, 18.6.HRMS calcd forC₁₆H₂₀NO₄[M+H]⁺290.1392,found 290.1387.

EXAMPLE 4 Formaldehyde fluorescent Probe Compound 2.7j-n Synthesis route IV

Acetic acid was added to a methanol solution of compound 2.12(1.0equiv.) to adjust the pH of the reaction solution to 4 to 5, followed by addition of a sodium cyanoborohydride (3.0equiv.) reducing agent, followed by slow dropwise addition of a methanol solution of aniline (3.0equiv.) at ordinary temperature to react with compound 2.12 for 12 hours, and the resulting imine was immediately reduced without occurrence of other side reactions. The completion of the reaction of the starting materials was monitored by thin layer chromatography. Then, water was added thereto, the organic phase was extracted three times with dichloromethane, and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by flash column chromatography on silica gel (n-hexane: ethyl acetate 4: 1) to obtain compound 2.7j having a phenyl substituent as a white solid.

Referring to the reaction mode, the target product 2.7k-m with a urethane structure, p-methoxyphenyl and morpholine substituent is synthesized, and the specific process is as follows:

the intermediate product 2.13 with-NHBoc structure can be synthesized by taking the compound 2.12 as a raw material and referring to the synthesis method of the target product 2.7k-M, and finally, the target product 2.7n with amino substituent can be obtained by reacting with 4M hydrogen chloride solution of 1, 4-dioxane at room temperature for 2h to remove Boc protecting group. The specific process is as follows:

2.7j(51mg,74％):R_f＝0.3(silica gel,hexanes:ethyl acetate＝3:1)；¹H NMR (300MHz,CDCl3)δ7.49(d,J＝8.8Hz,1H),7.16(dd,J＝8.4,7.5Hz,2H),6.86(dd, J＝8.8,2.5Hz,1H),6.80(d,J＝2.4Hz,1H),6.74-6.58(m,3H),6.14(d,J＝1.0Hz, 1H),5.96-5.72(m,1H),5.24-5.04(m,2H),4.25-4.07(m,2H),3.87-3.68(m,1H), 2.45-2.32(m,5H),2.15(m,1H),2.02-1.86(m,1H),1.27(s,1H).¹³C NMR(75MHz, CDCl₃)δ161.9,161.4,155.3,152.6,147.5,134.3,129.5,125.6,118.2,117.6,113.7, 113.5,112.6,112.0,101.6,65.8,49.9,39.1,34.0,18.8.HRMS calcd for C₂₂H₂₄NO₃[M+H]⁺350.1756,found 350.1750.

2.7k(42mg,65％):R_f＝0.3(silica gel,hexanes:ethyl acetate＝3:1)；¹H NMR (300MHz,CDCl3)δ7.47(d,J＝8.8Hz,1H),6.98-6.60(m,2H),6.31(s,1H),6.12 (d,J＝1.1Hz,1H),5.98-5.71(m,1H),5.27-5.05(m,2H),4.27-4.03(m,4H), 3.31-3.09(m,1H),2.38(d,J＝1.1Hz,3H),2.32-2.10(m,2H),1.94(q,J＝6.2Hz, 2H),1.24(t,J＝7.1Hz,4H).¹³CNMR(75MHz,CDCl₃)δ161.9,161.4,157.7,155.3, 152.6,134.8,125.6,118.1,113.7,112.6,112.0,101.6,66.1,61.5,57.0,37.4,31.5, 18.7,14.7.HRMS calcd for C₁₉H₂₅N₂O₅[M+H]⁺361.1763,found 361.1759.

2.7l(45mg,63％):R_f＝0.3(silica gel,hexanes:ethyl acetate＝3:1)；¹H NMR (500MHz,CDCl3)δ7.50(d,J＝8.8Hz,1H),6.87(dd,J＝8.8,2.4Hz,1H),6.82(d, J＝2.4Hz,1H),6.79-6.71(m,2H),6.64-6.56(m,2H),6.15(d,J＝1.0Hz,1H),5.85 (m,1H),5.15(dd,J＝13.2,6.3Hz,2H),4.19(m,2H),3.76(d,J＝7.7Hz,3H),3.67 (dd,J＝8.1,5.1Hz,1H),2.43-2.35(m,5H),2.13(m,1H),1.93(m,1H).¹³C NMR (126MHz,CDCl₃)δ161.9,161.2,155.3,152.4,152.2,141.6,134.3,125.5,118.2, 115.1,115.0,113.6,112.4,112.0,101.6,65.9,55.8,51.0,39.0,34.0,18.6.HRMS calcd for C₂₃H₂₆NO₄[M+H]⁺380.1862,found380.1863.

2.7m(34mg,65％):R_f＝0.3(silica gel,hexanes:ethyl acetate＝3:1)；¹H NMR (500MHz,CDCl3)δ7.51(d,J＝8.8Hz,1H),6.90-6.81(m,2H),6.15(d,J＝1.1Hz, 1H),5.92-5.68(m,1H),5.20-5.09(m,2H),4.19(tq,J＝9.4,6.5Hz,2H),3.67(dd,J ＝9.3,6.0Hz,4H),3.13-3.07(m,1H),2.69(s,2H),2.58(s,2H),2.42(d,J＝1.1Hz, 3H),2.32(dd,J＝12.9,6.9Hz,1H),2.22-2.15(m,1H),2.02-1.95(m,2H).¹³C NMR (126MHz,CDCl₃)δ162.1,161.2,155.3,152.4,135.3,125.4,117.8,113.5,112.6, 111.9,101.4,67.1,66.5,56.8,52.9,38.2,33.3,18.6.HRMS calcd for C₂₀H₂₇N₂O [M+H]⁺359.1971,found 359.1959.

2.13(53mg,62％):R_f＝0.3(silica gel,hexanes:ethyl acetate＝5:1)；¹H NMR (300MHz,CDCl3)δ7.47(d,J＝8.8Hz,1H),6.89-6.76(m,2H),6.12(d,J＝1.1Hz, 2H),5.94-5.74(m,1H),5.22-5.07(m,2H),4.25-4.06(m,3H),3.26-3.09(m,1H), 2.38(d,J＝1.1Hz,3H),2.26(dd,J＝12.7,6.4Hz,2H),1.93(dd,J＝12.8,6.5Hz, 2H),1.44(s,9H).¹³CNMR(101MHz,CDCl₃)δ162.0,161.4,155.4,152.6,141.7, 134.9,125.6,118.0,113.7,112.7,112.0,101.6,66.0,56.9,37.4,31.8,29.8,28.4,18.7. HRMS calcd for C₂₁H₂₈N₂NaO₅[M+Na]⁺411.1896,found 411.1890.

2.7n(26mg,67％):R_f＝0.3(silica gel,hexanes:ethyl acetate＝3:1)；¹H NMR (300MHz,MeOD)δ7.64(d,J＝8.8Hz,1H),7.04-6.77(m,2H),6.12(s,1H),5.90 (m,1H),5.27(dd,J＝17.6,13.7Hz,2H),4.25(td,J＝9.6,3.8Hz,2H),3.53-3.38(m, 1H),2.56(m,2H),2.41(s,3H),2.19(dd,J＝5.6,2.4Hz,2H).¹³C NMR(75MHz, MeOD)δ162.1,161.9,154.9,154.3,132.5,126.0,118.8,113.7,112.6,111.0,101.2, 65.0,57.7,34.5,29.1,17.4.HRMS calcd for C₁₆H₂₁N₂O₃[M+H]⁺289.1552,found 289.1548.

EXAMPLE 5 Formaldehyde fluorescent Probe Compound 2.7o-t synthetic route V

A solution of compound 2.7a (1.0equiv.) in N, N-dimethylformamide is added with potassium carbonate (2.0equiv.), iodide (1.2equiv.), or bromide (2.0equiv.), sodium iodide (1.0equiv.), reacted at room temperature or 70 ℃ for 10-20h, preferably 12h, with SN2 of a different haloalkane to introduce a nitrogen atom alkyl substituent, and the completion of the reaction of the starting materials is monitored by thin layer chromatography. Then adding water, extracting the organic phase for three times by using ethyl acetate, washing the combined organic phase for three times by using 1N hydrochloric acid solution, washing the combined organic phase by using saturated saline solution, drying the combined organic phase by using anhydrous sodium sulfate, filtering, concentrating, and separating by using silica gel flash column chromatography (normal hexane: ethyl acetate: 5: 1) to obtain a white solid compound 2.7 o-t. The synthetic route is represented as follows:

2.7o(30mg,65％):R_f＝0.3(silica gel,hexanes:ethyl acetate＝6:1)；¹H NMR (500MHz,CDCl₃)δ7.49(d,J＝8.8Hz,1H),6.95-6.75(m,2H),6.13(d,J＝1.0Hz, 1H),5.93-5.64(m,1H),5.16(dd,J＝11.6,6.2Hz,2H),4.24-4.04(m,2H),3.31(s, 2H),2.98-2.76(m,1H),2.47(s,3H),2.40(d,J＝1.1Hz,3H),2.36-2.29(m,2H), 2.01-1.95(m,2H).¹³C NMR(126MHz,CDCl₃)δ161.9,161.2,155.3,152.4,134.4, 125.5,118.1,113.6,112.5,111.9,101.6,65.9,56.0,37.6,33.0,32.4,18.5.HRMS calcd for C₁₇H₂₂NO₃[M+H]⁺288.1600,found 288.1594.

2.7p(60mg,50％):R_f＝0.3(silica gel,hexanes:ethyl acetate＝6:1)；¹H NMR(400MHz,MeOD)δ7.62(d,J＝8.8Hz,1H),6.94(dd,J＝8.8,2.5Hz,1H), 6.85(d,J＝2.5Hz,1H),6.11(d,J＝1.1Hz,1H),5.88(m,1H),5.33-5.16(m,2H), 4.29-4.13(m,2H),3.30(dd,J＝3.3,1.6Hz,1H),3.02(qd,J＝7.2,2.8Hz, 2H),2.58-2.42(m,2H),2.39(d,J＝1.1Hz,3H),2.15(dt,J＝8.8,2.7Hz,2H),1.28(t, J＝7.2Hz,3H).¹³C NMR(101MHz,MeOD)δ162.0,161.9,155.0,154.2,132.8, 126.0,118.7,113.7,112.5,111.0,101.1,65.1,54.8,40.5,35.6,30.4,17.4,11.6. HRMS calcd for C₁₈H₂₄NO₃[M+H]⁺302.1756,found 302.1745.

2.7q(98mg,35％):R_f＝0.3(silica gel,hexanes:ethyl acetate＝6:1)；¹H NMR (300MHz,CDCl3)δ7.48(d,J＝8.8Hz,1H),6.86(dd,J＝8.8,2.5Hz,1H),6.80(d, J＝2.4Hz,1H),6.12(d,J＝1.0Hz,1H),5.87(m,1H),5.25(dd,J＝25.4,7.8Hz, 2H),4.41-4.24(m,1H),4.16(dt,J＝10.3,6.4Hz,1H),3.45(dq,J＝13.3,6.5Hz, 2H),2.79(dd,J＝12.6,6.8Hz,1H),2.67(dd,J＝15.1,7.5Hz,1H),2.51-2.21(m, 5H),1.49(dd,J＝13.1,6.4Hz,6H).¹³C NMR(75MHz,CDCl3)δ162.5,161.5, 161.3,155.2,152.7,132.4,125.8,119.9,114.0,112.2,101.9,64.7,52.5,48.7,35.8, 30.6,19.8,19.5,18.7.HRMS calcd for C₁₉H₂₆NO₃[M+H]⁺316.1913,found 316.1908.

2.7r(58mg,34％):R_f＝0.3(silica gel,hexanes:ethyl acetate＝6:1)；¹H NMR (400MHz,MeOD)δ7.66(d,J＝8.8Hz,1H),7.02-6.80(m,2H),6.14(d,J＝1.1Hz, 1H),5.87(m,1H),5.34-5.12(m,2H),4.30-4.11(m,2H),3.29-3.23(m,1H), 3.01-2.81(m,2H),2.48(dd,J＝9.9,3.9Hz,2H),2.42(d,J＝1.1Hz,3H),2.13(qd,J ＝6.1,3.4Hz,2H),1.61(dd,J＝15.5,7.9Hz,2H),1.41(dd,J＝15.1,7.5Hz,2H), 0.96(t,J＝7.4Hz,3H).¹³C NMR(101MHz,MeOD)δ162.2,161.9,155.0,154.4, 133.0,126.1,118.5,113.7,112.5,111.0,101.1,65.3,55.1,45.5,35.9,30.6,29.5,19.9, 17.3,12.8.HRMS calcd for C₂₀H₂₈NO₃[M+H]⁺330.2069,found 330.2063.

2.7s(47mg,21％):R_f＝0.3(silica gel,hexanes:ethyl acetate＝6:1)；¹H NMR (400MHz,CDCl3)δ7.44(d,J＝8.8Hz,1H),6.82(dd,J＝8.8,2.5Hz,1H),6.76(d, J＝2.4Hz,1H),6.07(d,J＝1.1Hz,1H),5.82(m,1H),5.28-5.08(m,2H),4.20(m, 2H),3.45-3.21(m,1H),2.80-2.69(m,2H),2.66(dd,J＝11.0,4.5Hz,1H),2.53(dd,J ＝14.7,7.3Hz,1H),2.34(d,J＝1.1Hz,3H),2.33-2.26(m,1H),2.25-2.03(m,2H), 1.02(dd,J＝6.6,2.1Hz,6H).¹³CNMR(101MHz,CDCl3)δ161.5,161.3,155.2, 152.7,133.1,125.7,119.5,113.9,112.4,112.1,101.9,65.5,55.3,52.7,36.2,30.7, 26.6,20.9,20.8,18.7.HRMS calcd for C₂₀H₂₈NO₃[M+H]⁺330.2069,found 330.2061.

2.7t(48mg,33％):R_f＝0.3(silica gel,hexanes:ethyl acetate＝6:1)；¹H NMR (500MHz,MeOD)δ7.68(d,J＝8.8Hz,1H),7.07-6.80(m,2H),6.15(s,1H),5.90 (m,1H),5.41-5.20(m,2H),4.36-4.15(m,2H),3.53-3.39(m,1H),2.91(dd,J＝11.4, 5.8Hz,2H),2.60(dd,J＝13.2,6.4Hz,2H),2.44(s,3H),2.33-2.13(m,2H),1.91-1.65 (m,6H),1.42-1.16(m,4H),1.08(td,J＝12.1,2.8Hz,2H).¹³C NMR(126MHz, MeOD)δ161.9,161.7,154.9,154.1,132.4,126.0,118.9,113.7,112.4,111.0,101.2, 65.1,55.7,51.1,35.6,35.0,30.4,29.8,25.7,25.3,17.2.HRMS calcd forC₂₃H₃₂NO₃ [M+H]⁺370.2382,found370.2378.

1.2 reactivity of Formaldehyde fluorescent Probe library with Formaldehyde

20 fluorescent probes with different nitrogen atom substituents and coumarin as model fluorophores were obtained by the synthesis methods of examples 1 to 5, and a molecular library of formaldehyde probes was constructed. In order to screen the probes for optimal reactivity, the present invention measures their response to formaldehyde in vitro. These probes were first dissolved in PBS buffer (10. mu.M), reacted with 100 equivalents of formaldehyde at 37 ℃ for 2 hours, respectively, and then the fluorescence intensity at 445nm was measured, and the results are shown in FIG. 1.

20 formaldehyde fluorescent probe molecules can realize response to formaldehyde. Among the fluorescent probes having an alkyl substituent, the probe having a cyclohexylmethyl substituent has the best fluorescent response. The probe with the benzyl substituent group also has better fluorescence response, and after a methoxy group is introduced into a benzyl para position, the response of the corresponding probe to formaldehyde is greatly improved. Probes with phenyl substituents have a relatively poor fluorescence response. While probes with nitrogen or oxygen heteroatom substituents have a general fluorescence response only for the methoxy substituent, the other responses to formaldehyde are poor.

For some probes with better formaldehyde reactivity, their response fluorescence intensity to 50 equivalents of formaldehyde at 37 ℃ was measured within 0-180 minutes at 20 minute intervals, and the results are shown in FIG. 2. The response rate of the p-methoxybenzyl probe with the best fluorescent response is far ahead of the other probes. Among the 20 kinds of probes synthesized with various types of substituents, the probe with p-methoxybenzyl substituent had the best formaldehyde fluorescence response efficiency and rate.

Regarding the influence factors of the difference of the fluorescence response of different probes to formaldehyde, the invention firstly analyzes that the substituent influences the nucleophilicity of nitrogen atoms, and further influences the speed of generating imine positive ions by amino and formaldehyde; and steric hindrance of substituents, a larger steric hindrance may be detrimental to the addition of formaldehyde, and thus may affect the fluorescent response to formaldehyde. The steric hindrance of the nitrogen atom substituent has influence on the 2-aza-copro rearrangement process, thereby influencing the generation of the product. The probe with p-methoxybenzyl group has great advantages in both nucleophilicity and steric hindrance of nitrogen atom, and the result also proves that the probe indeed has the best fluorescence response.

The invention also makes a comparative experiment aiming at the gem-dimethyl effect and the influence of the N-substituent on the response of the probe, and synthesizes the probe with different numbers of methyl on the side chain based on the coumarin fluorophore.

As shown in the following synthetic route of products 2.17a and 2.17b with propyl substituent, products 2.14a and 2.14b with Ts protected hydroxyl group can be obtained according to the prior art, and the detailed synthetic process of products 2.14a and 2.14b is not repeated herein. A solution of 4-methylumbelliferone (1.2equiv.) and cesium carbonate (1.2equiv.) in N, N-dimethylformamide was added to a solution of compounds 2.14a-b (1.0equiv.) in N, N-dimethylformamide at 40 ℃. The reaction was then carried out at 40 ℃ for 12 hours and the completion of the reaction of the starting materials was monitored by thin layer chromatography. Then adding water, extracting the organic phase for three times by using ethyl acetate, washing the combined organic phase for three times by using 0.1N hydrochloric acid solution, washing the combined organic phase by using saturated saline solution, drying the combined organic phase by using anhydrous sodium sulfate, filtering, concentrating, and separating by using silica gel flash column chromatography (normal hexane: ethyl acetate: 15: 1) to obtain 2.15a and 2.15 b; a solution of tin dichloride (1.5equiv.) and thiophenol (4.4equiv.) in acetonitrile was added with triethylamine (4.4equiv.) at 0 ℃ and reacted at room temperature for 15 minutes; then 2.15a-b (1.0equiv.) is added for reaction at room temperature for 12 hours, azide groups are reduced, and the completion of the reaction of the raw materials is monitored by thin-layer chromatography. After concentration, the resulting product was dissolved in methylene chloride, washed three times with 2N sodium hydroxide solution, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to flash column chromatography on silica gel (methylene chloride: methanol 10: 1) to obtain products 2.16a and 2.16b each having 1 or 3 methyl groups in the side chain. Referring to scheme V, compounds 2.16a-b (1.0equiv.) and potassium carbonate (1.0equiv.) in N, N-dimethyl sulfoxide were reacted at room temperature with iodopropane (1.0equiv.) for 12h or 30h at 50 deg.C and monitored by thin layer chromatography for completion of the starting material reaction. Then water was added, the organic phase was extracted three times with ethyl acetate, the combined organic phases were washed three times with 1N hydrochloric acid solution, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel flash column chromatography (dichloromethane: methanol 10: 1) to give products 2.17a and 2.17b having 1 or 3 methyl groups in the side chain and a propyl substituent. The specific synthetic route is as follows:

as shown in the synthetic route of products 2.16c, 2.17c having propyl substituent, a methanol solution (5mL) of aldehyde compound 2.1 (262mg, 1.35mmol) was added with a methanol solution (1mL) of 7N ammonia at 0 ℃ to react at room temperature for 30min to form imine, then 3, 3-dimethylallylboronic acid pinacol ester (0.36mL, 1.62mmol) was added to continue the reaction at room temperature for 10h, and the completion of the starting material reaction was monitored by thin layer chromatography. Concentrating, and separating by silica gel flash column chromatography (dichloromethane: methanol 10: 1) to obtain colorless oily liquid 2.18(158mg, 45%) which is homoallylic amine compound with gem-dimethyl 2.18; tetrahydrofuran solution of Compound 2.18 was added Boc₂Reacting O at room temperature for 10h to protect amino with Boc group to obtain compound 2.19; adding 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone into dichloromethane and water solution of the compound 2.19 at 0 ℃ to react for 3 hours at room temperature, and removing a PMB protecting group to obtain a compound 2.20; taking a compound 2.20 as a raw material, adding p-toluenesulfonyl chloride, triethylamine and 4-dimethylaminopyridine into a dichloromethane solution of the compound 2.20 at 0 ℃ to react for 12 hours at room temperature according to a synthesis method of the compound 2.5e-f, and protecting a hydroxyl group with Ts to obtain a compound 2.21; taking a compound 2.21 as a raw material, and referring to a synthesis method of a compound 2.6e-f, obtaining a compound 2.22, except that the reaction temperature is 45 ℃; taking a compound 2.22 as a raw material, and obtaining a compound 2.16c by referring to the synthesis method of the compounds 2.7 a-f; the compound 2.16c is used as a raw material, and the synthesis method of the compounds 2.17a-b is referred, except that the reaction is carried out for 24h at room temperature, and the product 2.17c with 2 methyl groups on the side chain and propyl substituents is obtained.

The present invention examined the response of probes 2.16a-c and 2.17a-c to formaldehyde. These probes were reacted with 50 equivalents of formaldehyde at 37 ℃ for 2 hours, and then the fluorescence intensity at 445nm was measured, and the results are shown in FIG. 3. As can be seen from the figure, when the substituent R of the nitrogen atom is a hydrogen atom, the fluorescence response of the probe with three methyl groups on the side chain skeleton is best, and the reactivity of the probe can be increased by the geminal dimethyl effect. When the nitrogen substituent R is propyl, the probe without any side chain methyl has the best fluorescence response. The reason for this is presumably that the presence of both a side chain skeleton substituent and a nitrogen atom substituent increases steric hindrance and affects the rearrangement reaction.

In order to further compare the gem-dimethyl effect and the N-substituent effect, the response rates of the probe 2.7d with the p-methoxybenzyl substituent, the probe 2.7b with the propyl substituent and the probe 2.16b with the side chain having 3 methyl groups to formaldehyde were simultaneously detected, and the results are shown in FIG. 4. As is apparent from FIG. 4, under the same conditions, the probe with p-methoxybenzyl substituent has higher fluorescence response intensity and the reaction rate is much higher than that of the other two probes, which further proves that the probe with p-methoxybenzyl group screened by the invention is superior to the probe with three methyl groups on the side chain in the in vitro detection of formaldehyde. As the parent fluorophore structures adopted by the probes for comparison are the same, the p-methoxybenzyl substituent-based homoallylamine group obtained by screening has better formaldehyde reactivity than the geminal dimethyl homoallylamine group.

In summary, there are two main factors affecting the reactivity of probe formaldehyde: the first factor is the nucleophilicity of the nitrogen atom substituent, and the size of the nucleophilicity of the substituent can influence the stability of an imine product generated by the reaction of the probe and formaldehyde; the second factor is the steric hindrance of the nitrogen substituent, and the size of the steric hindrance of the substituent may affect the stability during the rearrangement reaction.

2. Development of formaldehyde fluorescent probe with various wavelengths

2.1 Formaldehyde fluorescent Probe Synthesis based on fluorophores of different wavelengths

The maximum fluorescence emission wavelength of the target probe 2.7d is 445nm, named FP445, and the excitation and emission wavelengths thereof belong to the region from ultraviolet light to blue light. The invention also replaces coumarin fluorophores with a plurality of fluorophores with different excitation and emission wavelengths, develops a series of formaldehyde fluorescent probes with different wavelengths, and is used for detecting and imaging formaldehyde in biological samples. See in particular examples 6-13 below.

Example 6 design and Synthesis of Formaldehyde fluorescent Probe FP511 based on 2-methyl Tokyo Green

The 2-methyl Tokyo green can be excited at a wavelength of about 500nm in an aqueous medium and emits green light with a wavelength of about 515 nm. This example first synthesizes 2-methyl tokyo green fluorophore with a fluorescein backbone. Reacting 2, 2', 4, 4' -tetrahydroxybenzophenone for 24 hours at 170 ℃ to obtain a precursor 3, 6-dihydroxy-xanthene-9-ketone of fluorescein; after two phenolic hydroxyl groups are protected by TBS, the TBS is reacted with 2-bromotoluene under the condition of n-butyllithium, and finally the TBS is removed by acidolysis to obtain a target product 2-methyl Tokyo green. The synthetic route is represented as follows:

a solution of 2-methylTokyo green (45mg, 0.18mmol) and potassium carbonate (38mg, 0.27mmol) in N, N-dimethylformamide (5mL) was added at room temperature to a solution of compound 2.5d (80mg, 0.18mmol) in N, N-dimethylformamide (2 mL). The reaction is carried out for 12h at 50 ℃, and the completion of the reaction of the raw materials is monitored by thin layer chromatography. Then, water was added, the organic phase was extracted with ethyl acetate (20mL), the combined organic phases were washed with 0.1N hydrochloric acid solution (3 × 20mL), washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel flash column chromatography (dichloromethane: methanol ═ 20: 1) to give a yellow solid compound 3.1, followed by reaction with 4M hydrogen chloride in 1, 4-dioxane at room temperature for 2h to remove the Boc protecting group, to give a target probe compound 3.2, which emits 511nm and is therefore named probe FP511, and its synthetic route is as follows:

3.2(15mg,58％):R_f＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(500 MHz,MeOD)δ7.59-7.49(m,2H),7.47(t,J＝7.4Hz,1H),7.25(dd,J＝12.2,4.8 Hz,3H),7.15(t,J＝2.3Hz,1H),7.12(d,J＝1.2Hz,1H),7.11-7.09(m,1H),6.91(dt, J＝9.0,2.2Hz,1H),6.86-6.77(m,2H),6.63(dd,J＝9.6,2.0Hz,1H),6.52(d,J＝1.9 Hz,1H),5.86(m,1H),5.18(dd,J＝23.9,6.1Hz,2H),4.34-4.12(m,2H),3.80(dd,J ＝35.1,13.0Hz,2H),3.71(d,J＝2.9Hz,3H),3.01-2.89(m,1H),2.47-2.30(m,2H), 2.08(t,J＝5.4Hz,3H),2.04-1.92(m,2H).¹³C NMR(126MHz,MeOD)δ185.9, 164.9,160.0,159.1,155.2,153.6,135.9,134.7,132.1,131.3,130.6,130.4,129.7, 129.7,129.6,128.8,128.3,125.9,117.2,117.1,114.6,114.2,113.5,104.2,100.8,66.3, 54.2,52.9,49.5,37.7,32.1,18.2.HRMS calcd forC₃₄H₃₄NO₄[M+H]⁺520.2488, found 520.2483.

example 7 design and Synthesis of Formaldehyde fluorescent Probe FP511B based on 2-methyl Tokyo Green

Tetrahydrofuran is used as a solvent, a fluorescein precursor 3, 6-dihydroxy-xanthene-9-ketone is used as an initial raw material, 2-methoxyethoxymethyl chloride and sodium hydride are added at 0 ℃, the reaction is carried out for 10 hours at room temperature, and 2-methoxyethoxymethyl is used for protecting hydroxyl; then adding isopropyl magnesium chloride and 4-iodine-3-methyl benzoate at the temperature of 20 ℃ below zero, and reacting for 12 hours at room temperature; adding trifluoroacetic acid, reacting at room temperature for 30min, and removing the MEM protecting group by acidolysis to obtain 18, 4-carboxyl-Tokyo green methyl ester fluorophore containing an ester group;

compound 2.4d (208mg, 0.62mmol), fluorophore 18, 4-carboxy-Tokyo Green methyl ester (111mg, 0.31mmol), tri-n-butylphosphine (0.15mL, 0.62mmol) in tetrahydrofuran (15mL) was added slowly diisopropyl azodicarboxylate (0.12mL, 0.62mmol) at 0 deg.C, and at 50 deg.C, Mitsunobu reaction occurred for 12h, and thin layer chromatography monitored that the starting material reaction was complete. After cooling to room temperature, water was added, the organic phase was extracted with ethyl acetate (3 × 30mL), the combined organic phases were washed three times with 0.1N hydrochloric acid solution, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel flash column chromatography (dichloromethane: ethyl acetate ═ 20: 1) to give compound 3.3 as a yellow solid, and finally reacted with 4M hydrogen chloride in 1, 4-dioxane at room temperature for 2h to remove the Boc protecting group to give compound 3.4, which is the target product FP 511B. The synthetic route is represented as follows:

3.4(32mg,35％):R_f＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(400 MHz,MeOD)δ8.11(s,1H),8.04(d,J＝8.0Hz,1H),7.34(d,J＝7.9Hz,1H),7.28 (d,J＝8.5Hz,2H),7.14-7.06(m,1H),7.01(t,J＝9.1Hz,2H),6.88(dd,J＝9.0,2.3 Hz,1H),6.80(dd,J＝8.7,2.2Hz,2H),6.57(dd,J＝9.6,2.0Hz,1H),6.42(d,J＝2.0 Hz,1H),5.84(m,1H),5.27-5.09(m,2H),4.23(td,J＝10.1,4.0Hz,2H),4.01-3.77 (m,5H),3.68(d,J＝1.3Hz,3H),3.10(d,J＝5.9Hz,1H),2.44(dd,J＝13.6,7.0Hz, 2H),2.19-1.97(m,5H).¹³C NMR(101MHz,MeOD)δ186.0,166.5,164.9,159.8, 159.6,155.1,151.8,137.0,136.9,133.8,131.5,131.4,131.0,130.2,129.5,129.4, 128.8,127.0,118.0,117.2,114.8,113.8,113.8,105.1,104.6,101.1,66.1,54.4,53.4, 51.7,49.2,36.7,31.3,18.4.HRMS calcd forC₃₆H₃₆NO₆[M+H]⁺578.2543,found 578.2514.

example 8 design and Synthesis of Formaldehyde fluorescent Probe FP511C based on 2-methyl Tokyo Green

After a solution of compound 3.3(118mg, 0.17mmol) in tetrahydrofuran (5mL) was added with a solution of lithium hydroxide (15mg, 0.61mmol) in water (2mL) at 0 ℃ for 30min to hydrolyze the methyl ester, the pH was adjusted to 1 with 1N hydrochloric acid solution, then isopropyl alcohol (10mL) was added, the organic phase was extracted with dichloromethane (20mL), the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel flash column chromatography (N-hexane: ethyl acetate: dichloromethane: 1: 0.2) to separate and obtain compound 3.5 as a yellow solid;

compound 3.5(162mg, 0.247mmol), dimethyl 2, 2' -diacetic acid ester (60mg, 0.3mmol), 1-hydroxybenzotriazole (52mg, 0.37mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (72mg, 0.37mmol) in N, N-dimethylformamide (10mL) was added with N, N-diisopropylethylamine (60. mu.L, 0.35mmol), reacted at 60 ℃ for 12 hours to effect condensation reaction to give an amide product, and the completion of the starting material reaction was monitored by thin layer chromatography. Then, water was added, the organic phase was extracted with ethyl acetate (30mL), the combined organic phases were washed with 0.1N hydrochloric acid solution (3 × 15mL), washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel flash column chromatography (dichloromethane: methanol ═ 20: 1) to give a yellow solid 3.6, and finally reacted with a1, 4-dioxane solution of 4M hydrogen chloride at room temperature for 2 hours to remove the Boc protecting group to give compound 3.7 having a diester structure, i.e., the target product FP 511C. The synthetic route is represented as follows:

3.7(30mg,93％):R_f＝0.3(silica gel,DCM:MeOH＝10:1)；¹H NMR(500 MHz,MeOD)δ7.54(s,1H),7.48(d,J＝7.8Hz,1H),7.35(dd,J＝7.8,1.9Hz,1H), 7.26(d,J＝8.1Hz,2H),7.13(s,1H),7.09(d,J＝9.4Hz,2H),6.90(d,J＝8.9Hz, 1H),6.81(dd,J＝8.6,3.1Hz,2H),6.61(dd,J＝9.6,1.9Hz,1H),6.47(d,J＝1.9Hz, 1H),5.85(m,1H),5.18(dd,J＝17.8,13.7Hz,2H),4.38(s,2H),4.33(s,2H), 4.30–4.17(m,2H),3.94–3.78(m,5H),3.77(s,3H),3.70(d,J＝2.2Hz,3H), 3.09–2.99(m,1H),2.41(dd,J＝15.3,7.3Hz,2H),2.09(t,J＝5.8Hz,3H),2.04(dd, J＝11.7,6.0Hz,2H).¹³C NMR(126MHz,MeOD)δ186.0,172.5,169.9,169.5, 164.7,159.8,159.6,155.1,151.5,137.1,136.3,134.4,133.6,131.0,130.1,129.5, 129.4,129.4,128.7,128.7,124.2,117.9,117.4,114.5,114.0,113.2,104.5,101.1, 66.3,54.3,53.2,51.6,49.6,48.9,36.5,31.6,29.8,26.2,18.5.HRMS calcd forC₄₁H₄₂N₂O₉[M+H]⁺707.2969,found 707.2965.。

example 9 design and Synthesis of 1, 8-naphthalimide-based Formaldehyde fluorescent Probe FP551

The 1, 8-naphthalimide derivative can be excited at the wavelength of about 440nm and can emit yellow light with the wavelength of about 550 nm.

First, a fluorophore 4-hydroxy-N-butyl-1, 8-naphthalimide having a1, 8-naphthalimide backbone was prepared. Carrying out reflux reaction on 4-bromo-1, 8-naphthalic anhydride and butylamine in ethanol for 24 hours to obtain 4-bromo-N-butyl-1, 8-naphthalimide; then using methanol as a solvent, adding sodium methoxide and copper sulfate pentahydrate, and reacting for 8 hours at 80 ℃ to obtain 4-methoxy-N-butyl-1, 8-naphthalimide; then reacting for 12h at 120 ℃ under the action of 57% hydriodic acid aqueous solution, and removing the methyl to obtain the fluorophore 4-hydroxy-N-butyl-1, 8-naphthalimide. The synthetic route is represented as follows:

then, adding p-toluenesulfonyl chloride, triethylamine and 4-dimethylaminopyridine into a dichloromethane solution of the compound 2.4d at 0 ℃ to react for 12 hours at room temperature, and protecting hydroxyl of the compound 2.4d by Ts to obtain a product 3.8; then adding the compound 3.8 into a 4-hydroxy-N-butyl-1, 8-naphthalimide fluorophore and a solution of cesium carbonate in N, N-dimethylformamide at 50 ℃, reacting at 50 ℃ for 12h to obtain a compound 3.9, then reacting with a solution of 4M hydrogen chloride in 1, 4-dioxane at room temperature for 2h, and removing a Boc protecting group to obtain a compound 3.10, namely a target probe FP551, wherein the synthetic route is shown as follows:

3.10(35mg,77％):R_f＝0.3(silica gel,DCM:MeOH＝15:1)；¹H NMR(500 MHz,MeOD)δ8.28(d,J＝7.1Hz,1H),8.19(dd,J＝16.9,8.2Hz,2H),7.53(t,J＝ 7.7Hz,1H),7.48(d,J＝8.2Hz,2H),7.02(d,J＝8.2Hz,1H),6.86(d,J＝8.3Hz, 2H),5.98(dd,J＝16.9,9.8Hz,1H),5.38(dd,J＝41.7,13.6Hz,2H),4.47-4.24(m, 4H),4.07-3.94(m,2H),3.70(s,3H),3.64(s,1H),2.87-2.67(m,2H),2.61-2.35(m, 2H),1.69-1.52(m,2H),1.38(dd,J＝14.9,7.4Hz,2H),0.96(t,J＝7.3Hz,3H).¹³C NMR(126MHz,MeOD)δ164.1,163.6,160.7,159.3,132.9,131.6,131.2,130.9, 128.7,128.1,125.7,122.9,122.6,121.8,119.8,114.6,114.2,106.3,66.7,64.9,54.4, 54.0,39.5,34.2,29.9,29.6,19.9,12.8.HRMS calcd forC₃₀H₃₅N₂O4[M+H]⁺ 487.2597,found 487.2591.

example 10 design and Synthesis of 1, 8-naphthalimide-based Formaldehyde fluorescent Probe FP551B

Refluxing 4-bromo-1, 8-naphthalic anhydride and 4-aminobutyric acid in ethanol for 8h to obtain a product, and adding K₂CO₃And methanol is used as a solvent, and the compound 3.11 is obtained after the reflux reaction for 10 hours. The compound 3.11(3.08g, 10.88mmol) and 57% hydriodic acid aqueous solution (100mL) are mixed at room temperature and reacted for 12h at 120 ℃, the methyl group is removed to obtain the compound 3.12 with a carboxyl structure, and the thin-layer chromatography monitors that the raw materials are completely reacted. Then, the mixture was filtered, washed with water, and separated to obtain compound 3.12 as a yellow solid. Compound 3.12(270mg, 0.9mmol) in methanol (5mL) was reacted with concentrated sulfuric acid (0.2mL) at 90 ℃ under reflux for 12h, and the completion of the reaction was monitored by thin layer chromatography. Then cooling to room temperature, and concentrating to obtain a yellow solid fluorophore compound 3.13 with a monoester structure; slowly adding diisopropyl azodicarboxylate into tetrahydrofuran solution of the compound 2.4d, the compound 3.13 and tri-n-butylphosphine at 0 ℃, carrying out Mitsunobu reaction for 12h at 50 ℃ to obtain a product 3.14, finally carrying out reaction for 2h at room temperature by using 1, 4-dioxane solution of 4M hydrogen chloride, and removing Boc protecting groups to obtain a compound 3.15 with a monomethyl ester structure, namely a target product FP 551B. The synthetic route is represented as follows:

3.15(47mg,70％):R_f＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(500 MHz,CDCl3)δ8.60(d,J＝7.1Hz,1H),8.54(d,J＝8.3Hz,1H),8.41(d,J＝8.3 Hz,1H),7.66(t,J＝7.8Hz,1H),7.22(d,J＝8.4Hz,2H),7.04(d,J＝8.3Hz,1H), 6.74(d,J＝8.4Hz,2H),5.92-5.78(m,1H),5.30-5.05(m,2H),4.44(dd,J＝14.2,7.9 Hz,1H),4.37(dt,J＝9.3,5.9Hz,1H),4.26(t,J＝7.1Hz,2H),3.89-3.74(m,2H), 3.72(s,3H),3.67(s,3H),3.08-2.98(m,1H),2.47(t,J＝7.6Hz,2H),2.42(s,2H), 2.25(d,J＝7.2Hz,1H),2.20-2.06(m,4H).¹³CNMR(126MHz,CDCl₃)δ173.4, 164.6,163.9,160.2,158.7,134.7,133.6,131.5,129.4,128.7,125.8,123.5,122.3, 118.2,114.7,113.7,108.5,105.9,66.4,55.1,53.0,51.6,50.3,39.3,38.3,33.2,31.7, 23.5.HRMS calcd for C₃₁H₃₅N₂O₆[M+H]⁺531.2495,found531.2492.

example 11 design and Synthesis of 1, 8-naphthalimide-based Formaldehyde fluorescent Probe FP551C

Referring to the method for synthesizing the compound 3.5 from the compound 3.3, the compound 3.16 is synthesized by using the compound 3.14 as a raw material; referring to compound 3.5, compound 3.6 was synthesized by starting with compound 3.16 except that the reaction temperature was 50 ℃; synthesis of Compound 2.7a-g with reference to Compound 2.6a-g Compound 3.18 was synthesized starting from Compound 3.17 to give the target product FP551C having a diester structure. The synthetic route is represented as follows:

example 12 design and Synthesis of resorufin-based Formaldehyde fluorescent Probe FP585

Resorufin can be excited at a wavelength of about 573nm and emits orange light at a wavelength of about 585 nm.

Tetrahydrofuran is used as a solvent, triphenylphosphine and a diethyl azodicarboxylate reagent are used, a Mitsunobu reaction is carried out, a resorufin fluorophore and a compound 2.4d are directly reacted for 12 hours at room temperature to be connected together to obtain a product 3.19, finally, a1, 4-dioxane solution of 4M hydrogen chloride is used for reaction for 2 hours at room temperature, a compound 3.20 and a target probe FP585 are obtained after Boc protecting groups are removed, and the synthetic route is shown as follows:

3.20(8mg,49％):R_f＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(300 MHz,MeOD)δ7.74(d,J＝8.9Hz,1H),7.52(d,J＝9.8Hz,1H),7.34(d,J＝8.6 Hz,2H),7.00(dd,J＝8.9,2.6Hz,1H),6.94(d,J＝2.6Hz,1H),6.88(dd,J＝9.1,2.5 Hz,2H),6.83(dd,J＝9.8,2.1Hz,1H),6.30(d,J＝2.1Hz,1H),5.95-5.76(m,1H), 5.26(dd,J＝21.3,5.8Hz,2H),4.31-4.14(m,2H),4.00(d,J＝7.1Hz,1H),3.75(d,J ＝3.6Hz,3H),2.60-2.41(m,2H),2.20-2.03(m,2H).¹³C NMR(75MHz,MeOD)δ 187.0,163.3,159.9,150.6,145.8,144.9,135.2,133.4,133.2,131.5,130.4,128.8, 127.5,118.3,114.3,113.9,105.5,100.5,65.8,54.4,53.6,48.9,36.2,30.9.HRMS calcd for C₂₄H₂₈NO₄[M+H]⁺431.1971,found 431.1953.

example 13 design and Synthesis of Formaldehyde fluorescent Probe FP706 based on amino hemicyanine

The excitation wavelength of the amino-bearing hemicyanine is 670nm, and the emission wavelength is 706 nm.

Firstly synthesizing near-infrared emission fluorophore amino hemicyanine dye AXPI, taking a compound with the same functional group as the amino hemicyanine dye and o-nitrophenol as raw materials, taking acetonitrile as a solvent, reacting for 4h at room temperature under the condition of potassium carbonate, then taking methanol as a solvent, and reacting in SnCl₂And carrying out reflux reaction for 12h under the condition of HCl to obtain the fluorophore amino cyanine AXPI. The synthetic route is represented as follows:

n, N-diisopropylethylamine (0.24mL, 1.47mmol) was added to a solution of compound AXPI (134mg, 0.33mmol) in acetonitrile (10mL), triphosgene (233mg, 0.78mmol) in acetonitrile was added slowly, the reaction was carried out in an ice bath for 2 hours, the ice bath was removed, the mixture was heated to reflux for 3 hours, cooled to room temperature, diluted with dichloromethane (15mL), filtered, and then compound 2.4d (298mg, 0.89mmol) was added to the filtrate, the reaction was carried out at room temperature for 3 hours, and the completion of the reaction of the starting materials was monitored by thin layer chromatography. Concentration and flash column chromatography on silica gel (dichloromethane: methanol ═ 20: 1) afforded 3.21(105mg, 36%) as a yellow solid. Referring to the method for synthesizing 2.7a-g from 2.6a-g, compound 3.22 was synthesized from compound 3.21 to obtain the objective fluorescent probe FP 706.

The synthetic route is shown as follows:

3.22(35mg,73％):R_f＝0.25(silica gel,DCM:MeOH＝10:1)；¹H NMR(500 MHz,MeOD)δ8.76(d,J＝14.8Hz,1H),7.86(s,1H),7.67(d,J＝7.4Hz,1H), 7.59(dd,J＝17.3,7.6Hz,2H),7.56-7.51(m,2H),7.49(dd,J＝7.8,4.4Hz,2H), 7.41-7.30(m,2H),6.99(d,J＝8.5Hz,2H),6.56(d,J＝14.9Hz,1H),5.98-5.83(m, 1H),5.36(dd,J＝32.5,13.5Hz,2H),4.38(t,J＝6.4Hz,3H),4.32(dd,J＝14.9,8.5 Hz,3H),3.77(s,3H),3.51(d,J＝3.9Hz,1H),2.80(d,J＝5.4Hz,2H),2.73(d,J＝ 5.7Hz,3H),2.69-2.62(m,1H),2.27(dt,J＝18.9,9.3Hz,1H),2.23-2.14(m,1H), 1.98(d,J＝7.0Hz,4H),1.84(s,6H),1.11(t,J＝7.3Hz,3H).¹³C NMR(126MHz, MeOD)δ178.0,161.4,160.8,153.7,153.5,145.7,142.8,142.0,141.6,133.1,131.4, 131.4,128.9,128.1,128.1,127.1,122.6,122.4,119.7,117.2,115.7,114.4,114.2, 112.7,104.1,103.8,61.0,54.4,54.4,50.7,48.5,46.3,34.2,29.3,28.7,27.0,23.6, 20.9,20.2,10.2.HRMS calcd for C₄₃H₅₀N₃O₄[M]⁺672.3796,found 672.3814.

2.2 Probe sensitivity, Selective detection

The invention detects the sensitivity of the nine probes FP445, FP511, FP511B, FP511C, FP551, FP551B, FP551C, FP585 and FP706 with different excitation and emission wavelengths to formaldehyde response.

The excitation and emission wavelengths of the fluorophore and the probe are measured by ultraviolet spectrophotometer Shanghai Tianmei UV-1000, the fluorescence intensity value is measured by fluorescence spectrophotometer Hitachi F-4600, and the fluorescence intensity value of a 96-pore plate is measured by enzyme labeling instrument BIOTEKSynergy H1.

If not otherwise stated, all probes were configured as 20mM DMSO stock solutions when detecting the reaction of the probes with formaldehyde. To examine the sensitivity of the probe to formaldehyde, 50. mu.L of the probe stock solution was diluted to 100mL with 2 XPBS buffer solution having a pH of 7.4 to prepare a 10. mu.M solution for use. When the selectivity of the probe to formaldehyde and the detection limit were determined, 21.7. mu.L of the probe stock solution was diluted to 40mL with 2 XPBS buffer solution having a pH of 7.4 to prepare 11.1. mu.M solution for use. The equivalent ratio of probe to formaldehyde as used herein refers to the ratio of the amounts of the substances. The formaldehyde is aqueous solution of formaldehyde. The volume of formaldehyde solution added in all reactions did not exceed 5% of the volume of probe solution. The reactions were all carried out in a thermostat at 37 ℃. The fluorescence response multiple of the detection probe to formaldehyde response refers to calculating the ratio of the fluorescence intensity of the maximum emission peak position of an experimental group to the fluorescence intensity of the maximum emission peak position of a control group under the condition of 50 equivalent formaldehyde.

The fluorescence intensity for detecting the sensitivity and selectivity of the probe was measured using a fluorescence spectrophotometer Hitachi F-4600.

In the aspect of sensitivity detection, the invention examines the response degree of a 10 mu M probe after reacting with formaldehyde with different concentrations for two hours at 37 ℃ and the fluorescence response of the 10 mu M probe after reacting with 500 mu M formaldehyde at 37 ℃ for different times.

When the invention detects the response of the probe to the formaldehyde concentration, the concentration of the adopted probe is 10 mu M, the volume is 5mL, the equivalent ratio of the formaldehyde to the probe is respectively 0, 5, 10, 20, 50, 100, 150 and 200, the total number is 8 gradients, after the formaldehyde with the corresponding equivalent is added, the mixture is shaken and shaken evenly, then the mixture is kept stand in a constant temperature box at 37 ℃ for 2 hours, and then the fluorescence is detected by a fluorescence spectrophotometer. The value of the histogram in the present invention is the ratio of the fluorescence intensity of the probe at the maximum emission wavelength at which the formaldehyde equivalent is 50 to the fluorescence intensity at the maximum emission wavelength at which the formaldehyde equivalent is 0. The same applies hereinafter.

When the response of the probe to the formaldehyde reaction time is detected, the concentration of the adopted probe is 10 mu M, the volume of the adopted probe is 5mL, the equivalent ratio of the formaldehyde to the probe is 50, the formaldehyde is added, the probe is shaken and shaken up, then the obtained product is placed in a constant temperature box at 37 ℃ and stands still, the reaction time is respectively 0, 20, 40, 60, 80, 100, 120, 140, 160 and 180 minutes, and 10 time points are counted, and then the fluorescence of each time point is detected by a fluorescence spectrophotometer. In the graph obtained when the reaction rate of the probe for formaldehyde was detected in this paper, the data used was the ratio of the fluorescence intensity of the probe at the maximum wavelength at each time point to the fluorescence intensity at the maximum wavelength at 0 minute. The same applies hereinafter.

In the selective detection, the invention examines the fluorescence response intensity of 10 μ M probe at 37 ℃ to 400M formaldehyde and other analytes at 0, 40, 80 and 120 minutes respectively. Other analytes include acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, pyruvaldehyde, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde, chloral, and fifteen potentially interfering active small molecules.

The concentration of the probe selectively adopted by the detection of the invention is 11.1 MuM, the volume is 1.8mL, and the final concentration is 10 MuM. PBS buffer concentration 10mM, pH 7.4, volume 200 u L. The concentrations of formaldehyde, acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde and chloral are all 4mM, the volumes are all 200 muL, and the final concentrations are all 400 muM. Methylglyoxal concentration was 1mM, volume was 200. mu.L, final concentration was 100. mu.M. The data obtained when the selectivity of the probe for formaldehyde is detected in the present invention is the ratio of the fluorescence intensity at the maximum wavelength after 0, 40, 80, 120 minutes of reaction at 37 ℃ after the probe is added with the analyte to the fluorescence intensity at the maximum wavelength at 0 minutes. The same applies hereinafter.

The response intensity of 10. mu.M probe FP445 to 0-2mM formaldehyde at 37 ℃ for 2 hours was measured, and the result is shown in FIG. 5a, and the degree of response of probe FP445 to formaldehyde increases with the increase of formaldehyde concentration within 50 equivalents. Through calculation of fluorescence intensity, the fluorescence response multiple of the probe FP445 to formaldehyde is 24.5 times under the condition of 50 equivalent formaldehyde.

The response intensity of 10. mu.M probe FP445 to 500. mu.M formaldehyde at 37 ℃ within 0-180min was measured, and the result is shown in FIG. 5 b. The response degree of the probe to formaldehyde increases along with the increase of time within 180 minutes, and the fluorescence intensity reaches a maximum basically when the reaction time is 120 minutes.

As can be seen from the 10. mu.M probe FP445 response to formaldehyde versus reaction time curve in FIG. 5c, the fluorescence response of the probe to formaldehyde increased linearly over 120 minutes.

The fluorescence response intensity of 10. mu.M probe FP445 to formaldehyde and other analytes at 0, 40, 80, 120 minutes was measured, and the results are shown in FIG. 5d, with the ordinate being the relative luminescence intensity and the abscissa being 1-17: PBS, formaldehyde, acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, pyruvaldehyde, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde, chloral, as can be seen from FIG. 5d, probe FP445 has good selectivity for formaldehyde.

The results of measuring the fluorescence response intensity of 10. mu.M probe FP511 reacted with 0-2mM formaldehyde at 37 ℃ for 2 hours are shown in FIG. 6 a. Within 50 equivalents of formaldehyde, the degree of response of probe FP511 to formaldehyde increases with increasing formaldehyde concentration. Through calculation of fluorescence intensity, the fluorescence response multiple of the probe FP511 to formaldehyde is 7.3 times under the condition of 50 equivalent formaldehyde.

The degree of response of 10. mu.M probe FP511 to formaldehyde at 37 ℃ with 500. mu.M formaldehyde within 0-180min was examined, and the results are shown in FIG. 6 b. The response degree of the probe to formaldehyde increases along with the increase of time within 180 minutes, and the fluorescence intensity reaches a maximum basically when the reaction time is 120 minutes. As can be seen from the curve of the response of probe FP511 to formaldehyde versus reaction time in FIG. 6c, the fluorescence response of the probe to formaldehyde increases linearly within 120 minutes.

The fluorescence response intensity of 10. mu.M probe FP511 to formaldehyde and other analytes at 0, 40, 80 and 120 minutes was measured, and the results are shown in FIG. 6d, where the ordinate represents the relative luminescence intensity, and the abscissa 1 to 17 represent: PBS, formaldehyde, acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, pyruvaldehyde, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde, chloral, as can be seen from FIG. 12, probe FP511 has good selectivity to formaldehyde.

The results of detecting the fluorescence response intensity of 10. mu.M probe FP511B to 0-2mM formaldehyde at 37 ℃ for 2 hours are shown in FIG. 7a, and the degree of response of probe FP511B to formaldehyde increases with the increase of the formaldehyde concentration within 50 equivalents. Through calculation of fluorescence intensity, the fluorescence response multiple of the probe FP511B to formaldehyde is 9.7 times under the condition of 50 equivalent formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP511B to 500. mu.M formaldehyde at 37 ℃ for 0-180 minutes was measured, and the result is shown in FIG. 7b, wherein the response degree of the probe to formaldehyde increases with the time within 180 minutes, and the fluorescence intensity reaches a maximum substantially when the reaction time is 120 minutes. As can be seen from the curve of the response of probe FP511B to formaldehyde versus reaction time in FIG. 7c, the fluorescence response of the probe to formaldehyde increased linearly over 120 minutes.

The fluorescence response intensity of 10. mu.M probe FP511B to formaldehyde and other analytes at 0, 40, 80 and 120 minutes was measured, and the results are shown in FIG. 7d, where the ordinate represents the relative luminescence intensity, and the abscissa represents 1 to 17: PBS, formaldehyde, acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, pyruvaldehyde, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde, chloral, it can be seen from FIG. 7d that probe FP511B has good selectivity for formaldehyde.

The results of measuring the response of 10. mu.M probe FP511C to 0-2mM formaldehyde at 37 ℃ for 2 hours are shown in FIG. 8a, and the degree of response of the probe to formaldehyde increases with the increase of the formaldehyde concentration within 50 equivalents. Through calculation of fluorescence intensity, the fluorescence response multiple of the probe FP511C to formaldehyde is 7.9 times under the condition of 50 equivalent formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP511C to 500. mu.M formaldehyde at 37 ℃ for 0-180 minutes was measured, and the result is shown in FIG. 8b, wherein the response degree of the probe to formaldehyde increases with the time within 180 minutes, and the fluorescence intensity reaches a maximum substantially when the reaction time is 120 minutes.

As can be seen from the 10. mu.M probe FP511C response to formaldehyde versus reaction time in FIG. 8c, the fluorescence response of the probe to formaldehyde increased linearly over 100 minutes.

The fluorescence response intensity of 10. mu.M probe FP511C to formaldehyde and other analytes at 0, 40, 80 and 120 minutes was measured, and the results are shown in FIG. 8d, where the ordinate represents the relative luminescence intensity, and the abscissa represents 1 to 17: PBS, formaldehyde, acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, pyruvaldehyde, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde, chloral, it can be seen from FIG. 8d that probe FP511C has good selectivity for formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP551 to 0-2mM formaldehyde at 37 ℃ for 2 hours was measured, and as shown in FIG. 9a, the degree of response of probe FP551 to formaldehyde increased with the increase in formaldehyde concentration within 50 equivalents. By calculation of fluorescence intensity, the fluorescence response multiple of the probe FP551 to formaldehyde was 33.7 times under the condition of 50 equivalents of formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP551 to 500. mu.M formaldehyde at 37 ℃ for 0-180 minutes was measured, and the result is shown in FIG. 9b, in 180 minutes, the degree of response of the probe to formaldehyde increases with time, and the fluorescence intensity reaches a maximum substantially when the reaction time is 120 minutes.

As can be seen from the curve of 10. mu.M probe FP551 response to formaldehyde versus reaction time in FIG. 9c, the fluorescence response of the probe to formaldehyde increased linearly over 180 minutes.

The fluorescence response intensity of 10. mu.M probe FP551 to formaldehyde and other analytes at 0, 40, 80, and 120 minutes was measured, and the results are shown in FIG. 9d, where the ordinate represents relative luminescence intensity, and 1 to 17 on the abscissa represent: PBS, formaldehyde, acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, pyruvaldehyde, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde, chloral, as can be seen from FIG. 9d, probe FP551 has good selectivity to formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP551B to 0-2mM formaldehyde at 37 ℃ for 2 hours was measured, and as shown in FIG. 10a, the degree of response of the probe to formaldehyde increased with the increase of formaldehyde concentration within 50 equivalents. Through calculation of fluorescence intensity, the fluorescence response multiple of the probe FP551B to formaldehyde is 138.6 times under the condition of 50 equivalent formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP551B to 500. mu.M formaldehyde at 37 ℃ for 0-180 minutes was examined, and the result is shown in FIG. 10 b. The response degree of the probe to formaldehyde increases along with the increase of time within 180 minutes, and the fluorescence intensity reaches a maximum basically when the reaction time is 120 minutes.

As can be seen from the curve of 10. mu.M probe FP551B in FIG. 10c for formaldehyde response versus reaction time, the fluorescence response of the probe to formaldehyde increased linearly over 100 minutes.

The fluorescence response intensity of 10. mu.M probe FP551B to formaldehyde and other analytes at 0, 40, 80, and 120 minutes was measured, and the results are shown in FIG. 10d, where the ordinate represents relative luminescence intensity, and the abscissa represents 1 to 17: PBS, formaldehyde, acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, pyruvaldehyde, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde, chloral, as can be seen from FIG. 10d, probe FP551B has good selectivity for formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP551C to 0-2mM formaldehyde at 37 ℃ for 2 hours was measured, and as shown in FIG. 11a, the degree of response of the probe to formaldehyde increased with the increase of formaldehyde concentration within 50 equivalents. Through calculation of fluorescence intensity, the fluorescence response multiple of the probe FP551C to formaldehyde is 115.7 times under the condition of 50 equivalent formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP551C to 500. mu.M formaldehyde at 37 ℃ for 0-180 minutes was measured. As shown in FIG. 11b, the response of the probe to formaldehyde increased with time within 180 minutes, and the fluorescence intensity was substantially maximized at a reaction time of 120 minutes.

As can be seen from the curve of the response of probe FP551C to formaldehyde versus reaction time in FIG. 11c, the fluorescence response of the probe to formaldehyde increased linearly over 120 minutes.

The fluorescence response intensity of 10. mu.M probe FP551C to formaldehyde and other analytes at 0, 40, 80, and 120 minutes was measured, and the results are shown in FIG. 11d, where the ordinate represents relative luminescence intensity, and the abscissa represents 1 to 17: PBS, formaldehyde, acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, pyruvaldehyde, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde, chloral, as can be seen from FIG. 11d, probe FP551C has good selectivity for formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP585 to 0-2mM formaldehyde at 37 ℃ for 2 hours was measured. As shown in FIG. 12a, the degree of response of the probe to formaldehyde increases with the increase in the concentration of formaldehyde within 50 equivalents. Through calculation of fluorescence intensity, the fluorescence response time of the probe FP585 to formaldehyde is 5.6 times under the condition of 50 equivalent formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP585 to 500. mu.M formaldehyde at 37 ℃ for 0-180min was measured. As a result, as shown in FIG. 12b, the probe response to formaldehyde increased with time within 180 minutes, and the fluorescence intensity was substantially maximized at a reaction time of 120 minutes.

As can be seen from the curve of the response of probe FP585 to formaldehyde versus reaction time in FIG. 12c, the fluorescence response of the probe to formaldehyde increased linearly over 80 minutes.

The fluorescence response intensity of 10. mu.M probe FP585 to formaldehyde and other analytes at 0, 40, 80, and 120 minutes was measured, and the results are shown in FIG. 12d, where the ordinate is relative luminescence intensity, and 1-17 on the abscissa are: PBS, formaldehyde, acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, pyruvaldehyde, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde, chloral, as can be seen from FIG. 12d, probe FP585 has good selectivity for formaldehyde.

The fluorescence response intensity of probe FP706 at 10. mu.M to 0-2mM formaldehyde at 37 ℃ for 2 hours was measured, and the result is shown in FIG. 13a, in which the response degree of probe to formaldehyde increased with the increase of formaldehyde concentration within 50 equivalents. Through calculation of fluorescence intensity, the fluorescence response multiple of the probe FP706 to formaldehyde is 30.1 times under the condition of 50 equivalent formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP706 to 500. mu.M formaldehyde at 37 ℃ for 0-180 minutes was measured, and the result is shown in FIG. 13b, wherein the degree of response of the probe to formaldehyde increases with time within 180 minutes, and the fluorescence intensity reaches a maximum substantially when the reaction time is 120 minutes.

As can be seen from the 10. mu.M probe FP706 versus formaldehyde response versus reaction time in FIG. 13c, the fluorescence response of the probe to formaldehyde increased linearly over 80 minutes.

The fluorescence response intensity of 10. mu.M probe FP706 to formaldehyde and other analytes at 0, 40, 80 and 120 minutes was measured, and the results are shown in FIG. 13d, where the ordinate represents the relative luminescence intensity, and the abscissa 1 to 17 represent: PBS, formaldehyde, acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, pyruvaldehyde, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde, chloral. As can be seen in FIG. 13d, probe FP706 is very selective for formaldehyde.

2.3 limits of detection of Probe

The fluorescence intensity of the detection limit of the detection probe is measured by a microplate reader BIOTEK Synergy H1.

The concentration of the probe selectively adopted by the detection of the invention is 11.1 MuM, the volume is 90 MuL, and the final concentration is 10 MuM. The formaldehyde concentrations were 0, 10, 25. mu.M, respectively, the volume was 10. mu.L, and the final concentrations were 0, 1, 2.5. mu.M, respectively. Three groups of parallel experiments are simultaneously carried out by adopting a 96-well plate, formaldehyde with different concentrations is added into different holes, probe solution is added, and then reaction is carried out for two hours at the temperature of 37 ℃, and then a value is detected by adopting an enzyme-labeling instrument. The same applies hereinafter.

The detection limit of the nine probes FP445, FP511, FP511B, FP511C, FP551, FP551B, FP551C, FP585 and FP706 with different excitation and emission wavelengths on formaldehyde is evaluated in a buffer solution system. 10 μ M of the probe (a) FP445, (b) FP511, (c) FP511B, (d) FP511C, (e) FP551, (f) FP551B, (g) FP551C, (h) FP585, (i) fluorescence response intensity of each FP706 to formaldehyde at a concentration of 0, 1, 2.5 μ M, respectively, was measured at 37 ℃ for 2 hours. The results are shown in fig. 14, where the ordinate represents the mean value of relative fluorescence intensity ± standard deviation, n is 3 replicates, and the abscissa represents the concentration of formaldehyde. Statistical analysis was performed using one-way anova with P less than 0.05, P less than 0.01, P less than 0.001, P less than 0.0001. The detection capability of the 10 mu M probe on formaldehyde basically reaches 1 mu M.

2.4 intracellular detection and imaging of exogenous Formaldehyde with Probe

The invention evaluates the fluorescence detection and imaging capability of the 9 probes on formaldehyde in living cells.

First, the influence of a probe having an emission wavelength of 511nm on the sensitivity of the probe after the introduction of an ester group was evaluated. Under the same conditions, HEK293T cells were incubated with a balanced salt solution containing 20. mu.M of the FP511, FP511B, FP511C probes for 60 minutes, followed by addition of 0 or 1mM formaldehyde and incubation at 37 ℃ for 60 minutes, and the fluorescence results obtained under a confocal microscope are shown in FIG. 15. As can be seen from the figure, the background fluorescence signal of the probe is substantially the same without the addition of formaldehyde, while the probe FP511C has a more significant fluorescence signal enhancement after the addition of formaldehyde. Therefore, the FP511C probe with a diester structure is the best structure at this wavelength.

Next, the influence of a fluorophore having an emission wavelength of 551nm on the sensitivity of the probe after the introduction of an ester group was evaluated. Under the same conditions, HEK293T cells were incubated with buffer solutions containing 10. mu.M of FP551, FP551B, FP551C probes, respectively, for 30 minutes, then 0 or 1mM formaldehyde was added and incubated at 37 ℃ for 60 minutes, and the fluorescence results obtained under a confocal microscope are shown in FIG. 16. As can be seen from the figure, the fluorescence signal of FP551B background is higher than that of the other two probes without adding formaldehyde, and after adding formaldehyde, the fluorescence signal of probe FP551B is more obviously enhanced, and the other two probes have no signal enhancement basically. Therefore, the FP551B probe having a monoester structure is an optimum structure at this wavelength.

The effect of confocal fluorescence imaging of probe FP511C on exogenous formaldehyde in HEK293T cells was evaluated. HEK293T cells were incubated in DEME medium containing 10% bovine embryo serum and CO at 37 deg.C₂Culturing under the condition of 5% content. HEK293T cells were seeded into polylysine-coated confocal dishes and incubated overnight before cell imaging. The following day, the cells were washed twice with buffer and cells were added to buffer containing 20. mu.M of Probe FP511C with 5% CO at 37 ℃₂HEK293T cells were incubated for an additional 60 minutes under conditions before being processed into groups. Then, formaldehyde was added to the cells at final concentrations of 0, 0.1, 0.2, 0.3, 0.5, and 1mM, respectively, and incubated at 37 ℃ for 60 minutes. Washing with buffer solution for three times, imaging the cells with a Nikon A1R laser confocal microscope, wherein the channel excitation wavelength of the probe FP511C is 488nm, and the collection wave band is 500-560 nm; the results are shown in FIGS. 17A-F; a formaldehyde experimental group with the concentration of 1mM is subjected to living cell staining by using a Hoechst dye, the channel excitation wavelength of a cell nucleus dye Hoechst33342 is 405nm, and the collection wave band is 425-475 nm. The results are shown in FIG. 17G. The fluorescence intensity of the Image was quantified by Image J, and the fluorescence images of different formaldehyde concentrations were quantified, and as a result, as shown in fig. 17H, the ordinate represents the relative fluorescence intensity average ± standard deviation, n is 3 repetitions, and the abscissa represents the formaldehyde concentration. Statistical analysis was performed using one-way analysis of variance in GraphPad Prism, P < 0.05, P < 0.01, P < 0.0001.

Evaluation of Probe FP551B against exogenous Formaldehyde in HEK293T cellsConfocal fluorescence imaging effect. HEK293T cells were incubated in DEME medium containing 10% bovine embryo serum and CO at 37 deg.C₂Culturing under the condition of 5% content. HEK293T cells were seeded into polylysine-coated confocal dishes and incubated overnight before cell imaging. The following day, the cells were washed twice with buffer and cells were added to buffer containing 10. mu.M of probe FP551B with 5% CO at 37 ℃₂HEK293T cells were incubated for an additional 30 minutes under conditions before being processed into groups. Then, formaldehyde was added at concentrations of 0, 0.1, 0.2, 0.3, 0.5, 1mM, respectively, and incubated at 37 ℃ for 60 minutes. Washing with buffer solution three times, imaging the cells with Nikon A1R confocal laser microscopy, the channel excitation wavelength of probe FP551B being 488nm, the collection band being 510-590nm, the results are shown in A-F of FIG. 18; the result of staining live cells with Hoechst dye on a formaldehyde test group at a concentration of 1mM, wherein the channel excitation wavelength of the nuclear dye Hoechst33342 was 405nm, and the collection wavelength was 425 and 475nm is shown in FIG. 18G. The fluorescence intensity of the Image was quantified by Image J, and the fluorescence images of different formaldehyde concentrations were quantified, and as a result, as shown in fig. 18H, the ordinate represents the relative fluorescence intensity average ± standard deviation, n is 3 repetitions, and the abscissa represents the formaldehyde concentration. Statistical analysis was performed using one-way anova with P less than 0.05 and P less than 0.0001.

The effect of confocal fluorescence imaging of probe FP585 on exogenous formaldehyde in HEK293T cells was evaluated. HEK293T cells were incubated in DEME medium containing 10% bovine embryo serum and CO at 37 deg.C₂Culturing under the condition of 5% content. HEK293T cells were seeded into polylysine-coated confocal dishes and incubated overnight before cell imaging. The following day, the cells were washed twice with buffer and cells were added to buffer containing 10. mu.M probe FP585 in 5% CO at 37 ℃₂HEK293T cells were incubated for an additional 30 minutes under conditions before being processed into groups. Then, formaldehyde was added to the cells at final concentrations of 0, 0.1, 0.2, 0.3, 0.5, and 1mM, respectively, and incubated at 37 ℃ for 60 minutes. Washing three times with buffer, imaging the cells with a Nikon A1R confocal laser microscope, channel excitation of probe FP585The wavelength is 561nm, and the collection band is 570-670 nm. The results are shown in FIGS. 19A-F; a formaldehyde experimental group with the concentration of 1mM is subjected to living cell staining by using a Hoechst dye, the channel excitation wavelength of a cell nucleus dye Hoechst33342 is 405nm, and the collection wave band is 425-475 nm. The results are shown in FIG. 19G. The fluorescence intensity of the Image was quantified by Image J, and the fluorescence images of different formaldehyde concentrations were quantified, and as a result, as shown in fig. 19H, the ordinate represents the relative fluorescence intensity average ± standard deviation, n is 3 repetitions, and the abscissa represents the formaldehyde concentration. Statistical analysis was performed using one-way analysis of variance in GraphPad Prism, P < 0.05, P < 0.0001.

The effect of confocal fluorescence imaging of probe FP706 on exogenous formaldehyde in HEK293T cells was evaluated. HEK293T cells were incubated in DEME medium containing 10% bovine embryo serum and CO at 37 deg.C₂Culturing under the condition of 5% content. HEK293T cells were seeded into polylysine-coated confocal dishes and incubated overnight before cell imaging. The following day, the cells were washed twice with buffer and cells were added to buffer containing 2. mu.M probe FP706 at 37 ℃ with 5% CO₂HEK293T cells were incubated for an additional 30 minutes under conditions before being processed into groups. Then adding 0, 0.1, 0.2, 0.3, 0.5 and 1mM formaldehyde respectively, incubating for 60 minutes at 37 ℃, washing for three times by using buffer solution, imaging the cells by using a Nikon A1R laser confocal microscope, wherein the channel excitation wavelength of a probe FP706 is 640nm, and the collection wave band is 663-738nm, and the results are shown in A-F of figure 20; the result of staining live cells with Hoechst dye on a formaldehyde test group with a concentration of 1mM, wherein the channel excitation wavelength of the nuclear dye Hoechst33342 is 405nm, and the collection band is 425 and 475nm is shown in FIG. 20G. The fluorescence intensity of the Image was quantified by Image J, and the fluorescence images of different formaldehyde concentrations were quantified, and as a result, as shown in fig. 20H, the ordinate represents the relative fluorescence intensity average ± standard deviation, n is 3 repetitions, and the abscissa represents the formaldehyde concentration. Statistical analysis was performed using one-way analysis of variance in GraphPad Prism, P < 0.01, P < 0.001, P < 0.0001.

It can be seen from FIGS. 17-20 that the probe has a significant, dose-dependent fluorescence response to formaldehyde as the formaldehyde concentration increases. Cells were found to have good viability and integrity by staining live cells with Hoechst dye. They all had a significant, dose-dependent fluorescence response to formaldehyde and did not affect cell morphology and survival.

2.5 detection and imaging of cellular endogenous Formaldehyde with Probe

Human breast cancer cells, MCF-7 cells, highly express lysine-specific demethylase 1(LSD1), LSD1 removes the methyl group on N-methylated histone, generating formaldehyde as a byproduct, resulting in increased formaldehyde concentration in MCF-7 cells. The ability of probes FP585 and FP706 to detect and image endogenous formaldehyde in MCF-7 cells was examined.

The experimental groups were set up as four: control group (untreated cells), drug strong heart-in-mind (TCP) -treated cells, small molecule inhibitor GSK-LSD 1-treated cells, and formaldehyde scavenger NaHSO₃Treated cells. TCP and GSK-LSD1 are both LSD1 inhibitors, can weaken the activity of LSD1 and reduce the content of formaldehyde in cells; NaHSO₃Is a formaldehyde scavenger and reacts with formaldehyde to directly scavenge the formaldehyde.

MCF-7 cells were seeded in a confocal dish and incubated with DEME medium containing 10% bovine embryo serum at 37 ℃ in CO₂Culturing for 1 day under the condition of 5%. Endogenous formaldehyde cell imaging experiments were performed in three groups: (1) blank group cells were incubated with medium for 24 hours; (2) incubate cells with medium containing 20 μ M TCP for 24 hours; (3) cells were incubated with medium containing 1. mu.M GSK-LSD1 for 24 hours. Then three groups of cells were further incubated for 60 minutes with buffer containing the same treatment composition and containing 10 μ M FP585 probe, the cells were washed three times with buffer, confocal fluorescence imaging was performed, imaging parameters: the channel excitation wavelength of the probe FP585 is 561nm, and the collection band is 570-670 nm. The results are shown in FIGS. 21A, 21B, and 21C; the live cells of each experimental group were stained with Hoechst dye, and the results of confocal microscopy fluorescence imaging are shown in fig. 21D, E, F. Quantifying the fluorescence intensity of the Image by Image J, and quantifying the fluorescence imaging images of different experimental groups, such asFig. 21G shows the mean relative fluorescence intensity ± standard deviation as the ordinate, n is 3 replicates, and the abscissa is the experimental group. Statistical analysis was performed using one-way analysis of variance in GraphPad Prism, with P less than 0.0001, indicating that cells pretreated with the addition of two inhibitors had a reduction in fluorescence signal of about 25% compared to the control, which demonstrates that probe FP585 is able to detect endogenous formaldehyde in the cells.

MCF-7 cells were seeded in a confocal dish and incubated with DEME medium containing 10% bovine embryo serum at 37 ℃ in CO₂Culturing for 1 day under the condition of 5%. Endogenous formaldehyde cell imaging experiments were performed in four groups: (1) blank group cells were incubated with medium for 24 hours; (2) incubate cells with medium containing 20 μ M TCP for 24 hours; (3) incubating the cells with medium containing 1 μ M GSK-LSD1 for 24 hours; (4) with a solution containing 200. mu.M NaHSO₃The cells were incubated for 30 minutes with the buffer of (1), then the four groups of cells were further incubated for 30 minutes with a buffer containing the same treatment composition and containing a 2 μ MFP706 probe, the nuclei were stained with the nuclear dye Hoechst33342, the cells were washed with the buffer three times, confocal fluorescence imaging was performed, the channel excitation wavelength of the probe FP706 was 640nm, and the collection band was 663-738 nm. Fluorescence imaging results for control and experimental groups, as shown in FIGS. 22A-D; and (3) carrying out living cell staining on each experimental group by using a Hoechst dye, wherein the channel excitation wavelength of the Hoechst33342 dye is 405nm, and the collection wavelength band is 425-475 nm. As shown in FIGS. 22E-H; the fluorescence intensity of the Image was quantified by Image J, and the fluorescence images of the different experimental groups were quantified, as shown in fig. 22I, with the ordinate being the relative fluorescence intensity average ± standard deviation, n being 3 replicates, and the abscissa being the experimental group. And statistical analysis using one-way analysis of variance in GraphPad Prism, P less than 0.01, P less than 0.0001; it can also be seen from the figure that the fluorescence signal is reduced by about 25% compared with the control group after the two inhibitors are added, and the formaldehyde scavenger NaHSO is added₃The fluorescence signal of the experimental group (2) is only half of that of the control group. This result demonstrates that probe FP706 can also detect the endogenous formaldehyde in the cells.

2.6 detection and imaging of endogenous Formaldehyde in brain tissue sections with Probe

The invention selects a near-infrared fluorescent probe FP706 which has better tissue penetrability and lower tissue fluorescence background, selects a C57BL/6J mouse with the age of 4 months, and takes fresh brain tissue of the mouse to carry out section processing, wherein the section thickness is about 400 mu m. Three sets of experiments were set up in total, which were:

(1) incubating the tissue sections with artificial cerebrospinal fluid (ACSF) buffer for 30min, then incubating the tissue sections with ACSF buffer containing 2. mu.M probe FP706 for 60 min;

(2) with NaHSO containing 400. mu.M scavenger₃The tissue sections were incubated with ACSF buffer for 30 minutes and then replaced with NaHSO containing 400. mu.M₃And 2. mu.M ACSF buffer of Probe FP706 for 60 min;

(3) with NaHSO containing 400. mu.M scavenger₃The tissue sections were incubated for 90 minutes with ACSF buffer. During the treatment process, a cell nucleus dye Hoechst33342 is added to stain the cell nucleus for 1 hour, and finally, ACSF is used for washing three times to carry out confocal imaging. The channel excitation wavelength of the probe FP706 is 640nm, and the collection band is 663-738 nm. The channel excitation wavelength of the Hoechst33342 dye is 405nm, and the collection band is 425 and 475 nm.

As shown in FIG. 23, the fluorescence signal in the brain tissue section was significantly reduced in the experiment group (1) with only the probe, while the fluorescence signal in the brain tissue section was significantly reduced in the experiment group (2) with the formaldehyde scavenger and the probe, indicating that the formaldehyde content was lower than that in the experiment group without NaHSO₃The experimental group (1) had a significant reduction. This result demonstrates that probe FP706 is able to enter brain tissue and is able to detect endogenous formaldehyde in ex vivo brain tissue sections.

In conclusion, by replacing coumarin with other fluorophores with longer excitation and emission wavelengths, a series of probes with different fluorescence colors were developed, including FP511, FP551, FP585, FP706 probes, FP511B, FP551B probes with monoester structure, and FP511C, FP551C probes with diester structure, further expanding the types of formaldehyde fluorescence probes. Subsequently, the sensitivity and selectivity of probes FP445, FP511, FP511B, FP511C, FP551, FP551B, FP551C, FP585 and FP706 to formaldehyde were examined, and their detection limits were also evaluated. They all show good sensitivity, good formaldehyde selectivity, and a formaldehyde detection limit as low as about 1 μ M. The present study then evaluated the ability of probes FP511C, FFP551B, FP585, FP706 to detect intracellular exogenous formaldehyde, all of which had a significant, dose-dependent fluorescent response to formaldehyde and did not affect cell morphology and survival. The invention evaluates the response of probes FP585 and FP706 to the endogenous formaldehyde of cells, and the result proves that the probes can detect the endogenous formaldehyde of cells. And the near-infrared fluorescent probe FP706 successfully realizes the detection of endogenous formaldehyde in brain tissue slices due to the good tissue penetrability and the low tissue fluorescence background.

3. Development of organelle targeting formaldehyde fluorescent probe

3.1 development of Nuclear-Targeted Formaldehyde fluorescent probes

Formaldehyde is used as an important signal molecule in organisms and is directly connected with various organelles, and abnormal metabolism of formaldehyde can cause a series of abnormal tandem reactions and is related to various functional diseases. Therefore, the detection of formaldehyde in intracellular organelles is very important to reveal the more biological effects of formaldehyde. The development of formaldehyde fluorescent probes for subcellular targeting of organelles would help to better understand more physiological and pathological functions of formaldehyde. See examples 14-17 for details.

Example 14 Synthesis of FP551-Nuc

Using N, N-dimethylformamide as solvent, in K₂CO₃Under the condition of (1), Hoechst33258 and N-tert-butoxycarbonyl-4-bromobutylamine react for 12h at 60 ℃ to obtain a compound 4.1, and finally, the compound reacts for 2h at room temperature with a1, 4-dioxane solution of 4M hydrogen chloride, the Boc protecting group on an amino group is removed to obtain a compound 4.2, and the condensation reaction is carried out for 12h at room temperature under the condition that diisopropylethylamine is added into an N, N-dimethylformamide solution of the compound 4.2, N-tert-butoxycarbonyl-diglycol-carboxylic acid, 1-hydroxybenzotriazole and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to obtain an amide product 4.3, reacting again with 4M 1, 4-dioxane solution of hydrogen chloride at room temperature for 2h, removing Boc protecting group on amino to obtain amine product 4.4,

the synthetic route is represented as follows:

referring to the method for synthesizing compound 3.6 from compound 3.5, compound 4.5a was synthesized starting from compound 3.16 and compound 4.4, except that the reaction temperature was room temperature; then the compound 4.5a reacts with a1, 4-dioxane solution of 4M hydrogen chloride at room temperature for 2h to remove the Boc protecting group, and a compound 4.6a, FP551-Nuc is obtained.

The synthetic route is shown as follows:

4.6a(30mg,88％):R_f＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(400 MHz,MeOD)δ8.35(dd,J＝7.3,1.0Hz,1H),8.26(d,J＝8.3Hz,1H),8.12-8.03(m, 2H),7.91-7.81(m,3H),7.62-7.43(m,3H),7.15(dd,J＝15.1,5.2Hz,3H),7.03(dd,J ＝8.8,2.2Hz,1H),6.93(d,J＝8.9Hz,2H),6.87(d,J＝8.4Hz,1H),6.64(d,J＝8.7 Hz,2H),5.86-5.70(m,1H),5.32(t,J＝4.6Hz,1H),5.14(t,J＝12.8Hz,2H),4.11(d, J＝5.6Hz,2H),4.00(t,J＝6.1Hz,4H),3.81(dd,J＝15.0,9.9Hz,2H),3.72(t,J＝ 5.9Hz,2H),3.59(d,J＝4.1Hz,6H),3.50(t,J＝5.5Hz,2H),3.26-3.21(m,4H), 3.11-3.00(m,1H),2.82-2.70(m,4H),2.46-2.42(m,4H),2.39(d,J＝6.8Hz,2H), 2.28(t,J＝7.5Hz,2H),2.22-2.15(m,1H),2.00(dd,J＝10.5,4.8Hz,3H),1.96-1.88 (m,2H),1.87-1.75(m,2H),1.75-1.65(m,2H),1.58(d,J＝7.2Hz,1H),0.88(dd,J＝ 11.8,4.7Hz,2H).¹³C NMR(101MHz,MeOD)δ178.0,174.1,172.8,165.3,164.4, 163.8,161.1,160.7,159.9,159.4,153.8,152.3,150.6,148.1,133.7,133.3,131.0, 130.1,129.6,128.9,128.5,128.1,127.9,125.6,124.1,123.1,121.8,121.3,121.0, 118.2,115.0,114.8,114.2,113.6,106.0,100.6,69.9,69.4,67.5,67.0,65.6,54.7,53.0, 50.1,44.5,39.0,38.8,36.3,35.2,33.2,31.7,31.3,29.5,29.5,26.8,26.3,25.8,24.1, 22.5,13.1.HRMS calcd for C₆₆H₇₇N₁₀O₉[M+H]⁺1153.5875,found 1153.5873.

3.11 characterization of Probe sensitivity and selectivity

The fluorescence response intensity of 10. mu.M probe FP551-Nuc to 0-2mM formaldehyde at 37 ℃ for 2 hours was measured, and the result is shown in FIG. 24a, in which the response degree of the probe to formaldehyde increases with the increase of formaldehyde concentration within 50 equivalents. Through calculation of fluorescence intensity, the fluorescence response multiple of the FP551-Nuc probe to formaldehyde is 9.7 times under the condition of 50 equivalent formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP551-Nuc to 500. mu.M formaldehyde at 37 ℃ for 0-180 minutes was examined. As shown in FIG. 24b, the response of the probe to formaldehyde increased with time within 180 minutes, and the fluorescence intensity was substantially maximized at a reaction time of 120 minutes.

As can be seen from the time-dependent curve of 10. mu.M probe FP551-Nuc against 500. mu.M formaldehyde in FIG. 24c, the fluorescence response of the probe to formaldehyde increases linearly over 140 minutes.

The fluorescence response intensity of 10. mu.M probe FP551-Nuc to formaldehyde and other analytes at 0, 40, 80, 120 minutes was measured, and the results are shown in FIG. 24d, where the ordinate is relative luminescence intensity, and 1 to 17 on the abscissa are: PBS, formaldehyde, acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, pyruvaldehyde, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde, chloral, it can be seen from FIG. 24d that the probe FP551-Nuc has good selectivity to formaldehyde.

Detecting the fluorescence response intensity of 10 mu M probe FP551-Nuc to formaldehyde with the content of 0, 1 and 2.5 mu M under the reaction conditions of two hours and 37 ℃; the results are shown in fig. 24e, with the ordinate representing the mean value of relative fluorescence intensity ± standard deviation, and n being 3 replicates. Statistical analysis was performed by one-way anova with P less than 0.01 and the ability of probe FP551-Nuc to detect formaldehyde reached 1 μ M.

3.12 Living cell fluorescence imaging Studies with Targeted Nuclear Formaldehyde Probe

The response of the probe FP551-Nuc targeting the cell nucleus to formaldehyde in HeLa cells and the effect of cell nucleus localization were examined, and the results are shown in FIG. 25. HeLa cells were incubated in DEME medium containing 10% bovine embryo serum and CO at 37 deg.C₂Culturing under the condition of 5% content. HeLa cells were seeded onto a confocal dish and incubated overnight before cell imaging. The following day, the cells were washed twice with buffer, 40. mu.M probe FP551-Nuc was added to the cells, and then 5% CO was added at 37 ℃₂Incubation was continued for 30 minutes under the conditions. And then performing grouping processing. Adding formaldehyde with the final concentration of 0 or 1mM into the cells respectively, incubating for 60 minutes, then incubating with a cell nucleus stain (Hoechst 33342) for 15 minutes, washing with a buffer solution for three times to remove excess stain, and then carrying out confocal imaging, wherein the channel excitation wavelength of the probe FP551-Nuc is 488nm, and the collection wavelength band is 510-590 nm. The channel excitation wavelength of the Hoechst33342 dye is 405nm, and the collection band is 425 and 475 nm. As can be seen from FIG. 25, the fluorescence signal was very weak in the control group without formaldehyde, while the fluorescence signal was significantly enhanced in the experimental group with formaldehyde. The cell nucleus co-localization experiment of the hurst dye and the probe shows that the probe has good cell nucleus localization effect. This result also indicates that the probe FP551-Nuc targeting the nucleus can specifically detect formaldehyde in the nucleus.

3.2 development of Targeted endoplasmic reticulum Formaldehyde fluorescent probes

Example 15 Synthesis of FP551-ER

Using dichloromethane as a solvent, and reacting ethylenediamine, triethylamine and 4-toluenesulfonyl chloride for 12 hours at room temperature to obtain N- (2-aminoethyl) -4-methylbenzenesulfonamide, wherein the synthetic route is shown as follows:

referring to the method for synthesizing the compound 3.6 from the compound 3.5, the compound 4.5b is synthesized by using the compound 3.16 and N- (2-aminoethyl) -4-methylbenzenesulfonamide as raw materials, except that the reaction temperature is room temperature; referring to the method for synthesizing the compound 2.7a-g from the compound 2.6a-g, the compound 4.6b is synthesized by using the compound 4.5b as a raw material to obtain a probe FP551-ER, and the synthetic route is shown as follows:

4.6b(32mg,24％):Rf＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(500 MHz,MeOD)δ8.24(d,J＝7.2Hz,1H),8.18(d,J＝8.2Hz,1H),8.07(d,J＝8.3Hz, 1H),7.72(d,J＝8.2Hz,2H),7.46(t,J＝7.8Hz,1H),7.31(dd,J＝18.9,8.3Hz,4H), 6.93(d,J＝8.4Hz,1H),6.76(d,J＝8.5Hz,2H),5.93(m,1H),5.27(dd,J＝25.2, 13.7Hz,2H),4.38-4.25(m,2H),4.06-3.90(m,4H),3.67(s,3H),3.29-3.24(m,1H), 3.22(t,J＝6.1Hz,2H),2.97(t,J＝6.1Hz,2H),2.55(dd,J＝11.4,6.0Hz,2H),2.38 (s,3H),2.30-2.15(m,4H),2.00-1.86(m,2H).¹³C NMR(126MHz,MeOD)δ174.1, 164.3,163.7,159.8,159.5,143.3,137.5,133.7,133.2,130.9,130.0,129.4,128.7, 128.3,128.1,126.7,125.5,122.9,121.6,118.1,114.1,113.8,106.1,65.8,54.4,53.2, 49.0,42.0,39.1,39.2,36.5,33.0,31.4,23.8,20.1.HRMScalcd for C₃₉H₄₅N₄O₇S [M+H]⁺713.3009,found 713.2997.

3.21 characterization of Probe sensitivity and selectivity

The fluorescence response intensity of 10. mu.M probe FP551-ER to 0-2mM formaldehyde at 37 ℃ for 2 hours was measured, and as shown in FIG. 26a, the degree of response of the probe to formaldehyde increased with the increase of formaldehyde concentration within 50 equivalents. Through calculation of fluorescence intensity, the fluorescence response multiple of the probe FP551-ER to formaldehyde is 35.8 times under the condition of 50 equivalent formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP551-ER to 500. mu.M formaldehyde at 37 ℃ for 0-180 minutes was examined. As shown in FIG. 26b, the response of the probe to formaldehyde increased with time within 180 minutes, and the fluorescence intensity was substantially maximized at a reaction time of 120 minutes.

As can be seen from the time-dependent curve of 10. mu.M probe FP551-ER against 500. mu.M formaldehyde in FIG. 26c, the fluorescence response of the probe to formaldehyde increases linearly over 180 minutes.

The fluorescence response intensity of 10. mu.M probe FP551-ER to formaldehyde and other analytes at 0, 40, 80, 120 minutes was measured, and the results are shown in FIG. 26d, where the ordinate is relative luminescence intensity, and 1 to 17 on the abscissa are: PBS, formaldehyde, acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, pyruvaldehyde, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde, chloral, as can be seen from FIG. 26d, probe FP551-ER has good selectivity to formaldehyde.

The fluorescence response intensity of 10 μ M probe FP551-ER to formaldehyde with the content of 0, 1, 2.5 μ M at 37 ℃ for 2 hours was examined. The results are shown in fig. 26e, with the ordinate representing the mean of relative fluorescence intensity ± standard deviation, and n being 3 replicates. Statistical analysis was performed by one-way anova with P less than 0.001 and the ability of probe FP551-ER to detect formaldehyde reached 1 μ M.

3.22 Living cell fluorescence imaging Studies with Targeted endoplasmic reticulum Formaldehyde Probe

The response of the probe FP551-ER targeting the endoplasmic reticulum to formaldehyde in HeLa cells and the effect of localization of the endoplasmic reticulum were examined, and the results are shown in FIG. 27. HeLa cells were incubated in DEME medium containing 10% bovine embryo serum and CO at 37 deg.C₂Culturing under the condition of 5% content. HeLa cells were seeded onto a confocal dish and incubated overnight before cell imaging. The following day, the cells were washed twice with buffer, 40. mu.M probe FP551-ER was added to the cells, and then 5% CO was added at 37 ℃₂Incubation was continued for 30 minutes under the conditions. And then performing grouping processing. Adding formaldehyde with final concentration of 0 or 1mM into cells, incubating for 60 min, staining endoplasmic reticulum with ER-tracker Red, incubating for 15 min, washing with buffer solution for three times to remove excessive stain, and performing confocal imaging, wherein the channel excitation wavelength of probe FP551-ER488nm, and the collection band is 510-570 nm. The channel excitation wavelength of the ER-tracker Red dye is 587nm, and the collection band is 590-640 nm. As can be seen from the figure, the fluorescence signal was very weak in the control group without formaldehyde, while the fluorescence signal was significantly enhanced in the experimental group with formaldehyde. Through the endoplasmic reticulum co-localization experiment of the dye and the probe, the probe has good endoplasmic reticulum localization effect. This result also indicates that the probe FP551-ER targeting the endoplasmic reticulum can specifically detect formaldehyde in the endoplasmic reticulum.

3.3 development of a fluorescent Probe for Targeted mitochondrial Formaldehyde

Example 16 Synthesis of FP551-Mito

Taking (4-bromobutyl) -triphenyl phosphonium bromide as a starting material, and reacting the starting material with a methanol solution of ammonia for 12 hours at room temperature to obtain (4-aminobutyl) -triphenyl phosphonium bromide. The synthesis process is shown as follows:

referring to the method for synthesizing the compound 3.6 from the compound 3.5, the compound 4.5c is synthesized by taking the compound 3.16 and (4-aminobutyl) -triphenyl phosphine bromide as raw materials, except that the reaction temperature is room temperature; referring to the synthesis of 2.7a-g from 2.6a-g, compound 4.6c, FP551-Mito was synthesized starting from 4.5c, the synthetic route is shown below:

4.6c(100mg,77％):R_f＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(400 MHz,MeOD)δ8.33(dd,J＝7.3,1.0Hz,1H),8.26(dd,J＝13.2,4.7Hz,2H), 7.81-7.71(m,9H),7.68(td,J＝7.6,3.6Hz,6H),7.59(dd,J＝8.2,7.5Hz,1H),7.40 (d,J＝8.7Hz,2H),7.09(d,J＝8.4Hz,1H),6.78(d,J＝8.7Hz,2H),5.95(m,1H), 5.42-5.13(m,2H),4.42(dd,J＝11.6,6.0Hz,2H),4.21(q,J＝13.2Hz,2H),3.96(t, J＝7.2Hz,2H),3.66(s,3H),3.59-3.51(m,1H),3.46(td,J＝13.6,7.6Hz,2H),3.22 (t,J＝6.0Hz,2H),2.76-2.61(m,2H),2.50-2.30(m,2H),2.25-2.14(m,2H),1.88(dd, J＝14.5,7.3Hz,2H),1.81-1.64(m,4H).¹³C NMR(101MHz,MeOD)δ174.0,164.2, 163.6,160.2,159.8,134.9,133.6,133.4,132.6,131.0,131.0,130.3,128.8,128.4, 125.8,123.1,121.8,119.2,118.9,118.1,114.4,114.0,106.5,65.4,54.5,53.7,48.6, 39.2,37.5,35.1,33.4,30.3,24.3,21.4,20.9,19.5.HRMS calcd forC₅₂H₅₅N₃O₅P+ [M]⁺832.3874,found 832.3887.

3.31 characterization of Probe sensitivity and selectivity

The fluorescence response intensity of 10. mu.M probe FP551-Mito 0-2mM formaldehyde at 37 ℃ for 2 hours was measured, and the result is shown in FIG. 28a, wherein the response degree of the probe to formaldehyde increases with the increase of formaldehyde concentration within 50 equivalents. Through calculation of fluorescence intensity, the fluorescence response multiple of the probe FP551-Mito formaldehyde is 53.7 times under the condition of 50 equivalent formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP551-Mito 500. mu.M formaldehyde at 37 ℃ for 0-180 minutes was measured. As shown in FIG. 28b, the response of the probe to formaldehyde increased with time within 180 minutes. The fluorescence intensity was substantially maximized at a reaction time of 120 minutes.

As can be seen from the time-dependent curve of 10. mu.M probe FP551-Mito against 500. mu.M formaldehyde in FIG. 28c, the fluorescence response of the probe to formaldehyde increased linearly over 80 minutes.

The fluorescence response intensity of 10. mu.M probe FP551-Mito formaldehyde and other analytes at 0, 40, 80, 120 min was measured, and the results are shown in FIG. 28d, where the ordinate represents relative luminescence intensity, and the abscissa represents 1 to 17: PBS, formaldehyde, acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, pyruvaldehyde, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde, chloral, as can be seen from FIG. 28d, probe FP551-Mito has good selectivity to formaldehyde.

Fluorescence response intensity of 10. mu.M probe FP551-

Mito

0, 1, 2.5. mu.M formaldehyde at 37 ℃ for 2 hours. The results are shown in fig. 28e, with the ordinate representing the mean of relative fluorescence intensity ± standard deviation, and n being 3 replicates. Statistical analysis was performed by one-way anova with P less than 0.001 and the ability of probe FP551-Mito to detect formaldehyde reached 1 μ M.

3.32 Living cell fluorescence imaging Studies with Targeted mitochondrial Formaldehyde Probe

The response of the mitochondria-targeting probe FP551-Mito formaldehyde in HeLa cells and the mitochondrial localization effect were examined, and the results are shown in FIG. 29. HeLa cells were incubated in DEME medium containing 10% bovine embryo serum and CO at 37 deg.C₂Culturing under the condition of 5% content. HeLa cells were seeded onto a confocal dish and incubated overnight before cell imaging. The following day, the cells were washed twice with buffer, 40. mu.M probe FP551-Mito was added to the cells, and then 5% CO was added at 37 ℃₂Incubation was continued for 30 minutes under the conditions. And then performing grouping processing. Adding formaldehyde with the final concentration of 0 or 1mM into the cells respectively, incubating for 60 minutes, then incubating with a mitochondrial stain (Mito-tracker Red) for 15 minutes, washing with a buffer solution for three times to remove excess stain, and then carrying out confocal imaging, wherein the channel excitation wavelength of the probe FP551-Mito is 488nm, and the collection wavelength band is 510-570 nm. The channel excitation wavelength of the Mito-tracker Red dye was 578nm, and the collection band was 574-624 nm. As can be seen from the figure, the fluorescence signal was very weak in the control group without formaldehyde, while the fluorescence signal was significantly enhanced in the experimental group with formaldehyde. The mitochondria co-localization experiment of the dye and the probe shows that the probe has good mitochondria localization effect. This result also demonstrates that the mitochondrial targeting probe FP551-Mito is able to specifically detect formaldehyde in mitochondria.

3.4 development of lysosomal Formaldehyde-targeting fluorescent probes

Example 17 Synthesis of FP551-Lyso

Referring to the method for synthesizing the compound 3.6 from the compound 3.5, the compound 3.16 and N-aminoethyl morpholine are used as raw materials to synthesize the compound 4.5d, except that the reaction temperature is normal temperature; according to the method for synthesizing 2.7a-g from compound 2.6a-g, compound 4.6d was synthesized using compound 4.5d as a starting material to obtain probe FP551-Lyso having a lysosome-localizing group.

The synthetic route is shown as follows:

4.6d(50mg,89％):R_f＝0.2(silica gel,DCM:MeOH＝10:1)；¹H NMR(500 MHz,MeOD)δ8.38(d,J＝7.2Hz,1H),8.30(dd,J＝16.7,8.3Hz,2H),7.64(t,J＝ 7.8Hz,1H),7.52(d,J＝8.5Hz,2H),7.14(d,J＝8.3Hz,1H),6.90(d,J＝8.5Hz, 2H),6.00(td,J＝17.1,7.1Hz,1H),5.40(dd,J＝43.9,13.5Hz,2H),4.48(s,2H), 4.37(dd,J＝27.6,13.2Hz,2H),4.11(t,J＝6.9Hz,4H),3.89(s,2H),3.83-3.66(m, 6H),3.62(t,J＝5.6Hz,2H),3.41-3.36(m,2H),3.24(s,2H),2.81(m,2H),2.57(dd, J＝14.5,6.0Hz,1H),2.46(dd,J＝14.0,7.2Hz,1H),2.37(t,J＝7.1Hz,2H),2.04(p, J＝6.8Hz,2H).¹³C NMR(126MHz,MeOD)δ175.4,164.3,163.8,160.6,159.6, 133.3,131.6,131.3,131.1,128.8,128.4,125.8,122.9,122.6,121.7,119.8,114.5, 114.2,106.4,65.0,63.8,57.3,54.5,54.0,52.2,38.9,34.1,33.7,32.5,29.5,26.7, 23.4.HRMS calcd for C₃₆H₄₅N₄O₆[M+H]⁺629.3339,found 629.3326.

3.41 characterization of Probe sensitivity and selectivity

The fluorescence response intensity of 10. mu.M probe FP551-Lyso to 0-2mM formaldehyde at 37 ℃ for 2 hours was examined. As shown in FIG. 30a, the degree of response of the probe to formaldehyde increases with the increase in the concentration of formaldehyde within 50 equivalents. Through calculation of fluorescence intensity, the fluorescence response multiple of the probe FP551-Lyso to formaldehyde is 58.8 times under the condition of 50 equivalent formaldehyde.

The fluorescence response intensity of 10. mu.M probe FP551-Lyso to 500. mu.M formaldehyde at 37 ℃ for 0 to 180 minutes was examined. As shown in FIG. 30b, the response of the probe to formaldehyde increased with time within 180 minutes, and the fluorescence intensity was substantially maximized at a reaction time of 120 minutes.

As can be seen from the time-dependent curve of 10. mu.M probe FP551-Lyso against 500. mu.M formaldehyde in FIG. 30c, the fluorescence response of the probe to formaldehyde increased linearly within 100 minutes.

The fluorescence response intensity of 10. mu.M probe FP551-Lyso to formaldehyde and other analytes at 0, 40, 80, 120 minutes was measured, and the results are shown in FIG. 30d, with the ordinate being relative luminescence intensity and the abscissa being 1 to 17: PBS, formaldehyde, acetaldehyde, glucose, L-dehydroascorbic acid, glucurone, pyruvate, oxaloacetate, pyruvaldehyde, hydrogen peroxide, glutathione, glyoxal, benzaldehyde, 4-hydroxynonenal, p-nitrobenzaldehyde, p-methoxybenzaldehyde, chloral, as shown in FIG. 30d, the probe FP551-Lyso has good selectivity to formaldehyde.

Detecting the fluorescence response intensity of 10 mu M probe FP551-Lyso to formaldehyde with the content of 0, 1 and 2.5 mu M at the temperature of 37 ℃ for 2 hours; the results are shown in FIG. 30e, with the ordinate representing the relative fluorescence intensity. Mean ± standard deviation, n is 3 replicates. Statistical analysis was performed by one-way anova with P less than 0.0001 and FP551-Lyso probe reaching 1. mu.M detection of formaldehyde.

3.42 Living cell fluorescence imaging Studies with Targeted lysosomal Formaldehyde Probe

The response of the lysosome-targeting probe FP551-Lyso to formaldehyde in HeLa cells and the lysosome localization effect were examined, and the results are shown in FIG. 31. HeLa cells were incubated in DEME medium containing 10% bovine embryo serum and CO at 37 deg.C₂Culturing under the condition of 5% content. HeLa cells were seeded onto a confocal dish and incubated overnight before cell imaging. The following day, the cells were washed twice with buffer, 40. mu.M probe FP551-Lyso was added to the cells, and then 5% CO was added at 37 ℃₂Incubation was continued for 30 minutes under the conditions. And then performing grouping processing. Adding formaldehyde with the final concentration of 0 or 1mM into the cells respectively, incubating for 60 minutes, then incubating with a lysosome stain (Lyso-tracker Red) for 15 minutes, washing with a buffer solution for three times to remove excess stain, and then carrying out confocal imaging, wherein the channel excitation wavelength of a probe FP551-Lyso is 488nm, and the collection wavelength band is 510-570 nm. Of Lyso-tracker Red dyesThe channel excitation wavelength is 577nm, and the collection band is 565 and 615 nm. As can be seen from the figure, the fluorescence signal was very weak in the control group without formaldehyde, while the fluorescence signal was significantly enhanced in the experimental group with formaldehyde. Through a lysosome co-localization experiment of the dye and the probe, the probe has a good lysosome localization effect. This result also indicates that the lysosome-targeting probe FP551-Lyso can specifically detect formaldehyde in lysosomes.

In conclusion, on the basis of developing a series of formaldehyde fluorescent probes with different fluorescent colors, in order to further expand the diversity of the probes, four formaldehyde fluorescent probes FP551-Nuc, FP551-ER, FP551-Mito and FP551-Lyso which respectively target cell organelles such as cell nucleus, endoplasmic reticulum, mitochondria, lysosome and the like are developed, the sensitivity and the selectivity of the formaldehyde fluorescent probes to the formaldehyde are detected, and the detection limit of the formaldehyde fluorescent probes is evaluated. Likewise, they also exhibit good sensitivity, good formaldehyde selectivity, and a formaldehyde detection limit as low as about 1 μ M. The invention also preliminarily completes the characterization of the organelle targeted formaldehyde fluorescence probes FP551-Nuc, FP551-ER, FP551-Mito and FP551-Lyso in cells, and the organelle targeted positioning capability and the formaldehyde detection performance are all good.

The invention takes formaldehyde as a research object and takes a fluorescent probe as a tool to systematically research the construction of the formaldehyde fluorescent probe. In the design of the probe, the invention utilizes 2-aza-coppu rearrangement reaction as a trigger, develops a universal probe template, the probe reacts with formaldehyde to generate imine positive ions, and after 2-aza-coppu rearrangement and hydrolysis, fluorophores can be released to generate remarkable fluorescence. On the basis of the design strategy, the invention develops and designs the formaldehyde reactivity-based fluorescent probe, synthesizes more than 20 probes with different nitrogen atom substituent groups R, completes the optimization of a reaction site, inspects the influence of side chain methyl on the probe reactivity, successfully screens out the probe FP445 with the best reactivity, completes the design and synthesis of four formaldehyde probes with different colors by changing the structure of a parent fluorophore, and expands the diversity of the formaldehyde probes, wherein the fluorescent wavelength spans from visible light to a near infrared region. In vitro detection, the probes show good sensitivity, good formaldehyde selectivity and formaldehyde detection limit as low as about 1 mu M. The invention evaluates the detection capability of probes FP511C, FFP551B, FP585 and FP706 on exogenous formaldehyde in cells, and all the probes have obvious and dose-dependent fluorescence response on formaldehyde. The invention evaluates the response of probes FP585 and FP706 to the endogenous formaldehyde of cells, and the result shows that the probes can detect the endogenous formaldehyde of cells. And the near-infrared fluorescent probe FP706 successfully realizes the detection of endogenous formaldehyde in brain tissue slices due to the good tissue penetrability and the low tissue fluorescence background.

In order to research the effect of formaldehyde in specific organelles, the invention further expands the diversity of the probes, and designs and synthesizes four formaldehyde fluorescent probes FPA551-Nuc, FPA551-ER, FPA551-Lyso and FPA551-Mito respectively target the organelles such as nucleus, endoplasmic reticulum, mitochondria, lysosome and the like. These probes exhibit good formaldehyde detection sensitivity and good selectivity, as well as a detection limit as low as about 1 μ M. In cell experiments, they also exhibited good organelle targeting ability and formaldehyde detection performance.

The technical solutions provided by the embodiments of the present invention are described in detail above, and the principles and embodiments of the present invention are explained herein by using specific examples, and the descriptions of the embodiments are only used to help understanding the principles of the embodiments of the present invention; meanwhile, for a person skilled in the art, according to the embodiments of the present invention, there may be variations in the specific implementation manners and application ranges, and in summary, the content of the present description should not be construed as a limitation to the present invention.

Claims

1. A micromolecular formaldehyde fluorescent probe is characterized in that:

the structural general formula is shown as the following (I):

z is selected from oxygen atoms

Carbamates, their preparation and their use

2. The class of small molecule formaldehyde fluorescent probes of claim 1, characterized in that:

3. The class of small molecule formaldehyde fluorescent probes of claim 2, characterized in that:

4. The class of small molecule formaldehyde fluorescent probes of claim 2, characterized in that:

and R is p-methoxybenzyl.

5. The class of small molecule formaldehyde fluorescent probes of claim 1, characterized in that:

6. The class of small molecule formaldehyde fluorescent probes of claim 5, characterized in that:

7. The class of small molecule formaldehyde fluorescent probes of claim 5, characterized in that:

8. The class of small molecule formaldehyde fluorescent probes of claim 1, characterized in that:

z is carbamate

In which R is₁Is H.

9. The small molecule formaldehyde fluorescent probe according to any one of claims 1 to 8, characterized in that:

a structure comprising one of:

10. the use of the small molecule formaldehyde fluorescent probe of claim 1 in formaldehyde detection.

11. Use of a small molecule formaldehyde fluorescent probe according to claim 10 for formaldehyde detection in living cells, tissues and small living animals.

12. The use of the small molecule formaldehyde fluorescent probe of claim 10 in the preparation of a formaldehyde detection kit or test paper.