Aggregation-induced emission stereoisomer, application and preparation method thereof

文档序号：335518 发布日期：2021-12-03 浏览：82次中文

阅读说明：本技术 聚集诱导发光的立体异构体及其用途和制备方法 (Aggregation-induced emission stereoisomer, application and preparation method thereof ) 是由唐本忠何学文于 2021-05-31 设计创作，主要内容包括：本发明涉及一系列在发光特性和生物医学活性方面具有明显差异的具有聚集诱导发光(AIE)特征的立体异构体及其制备方法。本发明还利用所述E/Z异构体对中的一种异构体在室温下具有高荧光量子产率并发射出强烈的荧光,而另一种异构体则几乎不发射荧光,以及所述E/Z异构体在光照下相互转化的能力,提供了所述异构体作为荧光探针在点亮型细胞成像中以及在可视化的药物筛选和开发中的应用。(The present invention relates to a series of stereoisomers with Aggregation Induced Emission (AIE) characteristics having significant differences in luminescent properties and biomedical activities, and a preparation method thereof. The invention also provides the application of the isomer as a fluorescent probe in lightening cell imaging and visual drug screening and development by utilizing the high fluorescence quantum yield of one isomer in the E/Z isomer pair at room temperature and strong fluorescence emission, almost no fluorescence emission of the other isomer and the capability of interconversion of the E/Z isomers under illumination.)

1. A stereoisomer having aggregation-induced emission characteristics, wherein said stereoisomer has the general structural formula E isomer:

or the R isomer, or a mixture of the R isomers,

wherein X is selected from any one of the following groups;

r and R' are one or more fluorescent chromophores of different structures coupled to one or more fluorophores, R being selected from the group consisting of hydrogen, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-N-chlorosuccinimide (NCS), alkyl-azide (-N₃) And alkyl-amino (-NH)₂) Any one of the above; r' is selected from the group consisting of hydrogen, halogen, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-N-chlorosuccinimide (NCS), alkyl-azide (-N)₃) And alkyl-amino (-NH)₂) Any one of the above; r is preferably hydrogen; r' is preferably hydrogen, more preferably a halogen atom; n is 1, 2, 3, 4, 5, 6, 7, 8 or 9.

2. The method of producing a stereoisomer with aggregation-induced emission characteristics according to claim 1, wherein the method comprises:

(1) in the presence of an organic solvent, dropwise adding a second reaction raw material into a mixture of the first reaction raw material and NaH, and stirring or heating and refluxing the reaction mixture in an inert gas environment to obtain a crude product;

wherein the first reaction raw material is 4-methoxybenzophenone or 4- (dimethylamino) benzophenone;

the chemical structural formula of the second reaction raw material is as follows:

wherein R' is selected from the group consisting of hydrogen, halogen, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-N-chlorosuccinimide (NCS), alkyl-azide (-N)₃) And alkyl-amino (-NH)₂) Preferably hydrogen, is added to the reaction mixture,more preferably a halogen atom;

(2) and separating and purifying the crude product to obtain a purified product, and further separating the E/Z isomer from the purified product by high performance liquid chromatography.

3. The process of claim 2, wherein the crude product is isolated and purified by a silica gel column.

4. The method of claim 2, wherein the E/Z isomers are separated by high performance liquid chromatography equipped with a C18 reverse phase column.

5. A fluorescent probe comprising the stereoisomer of claim 1 having aggregation-induced emission characteristics.

6. Use of a stereoisomer of claim 1 having aggregation-induced emission characteristics or a fluorescent probe of claim 5 in illuminated bioimaging.

7. Use according to claim 6, wherein the non-luminescent isomer is used for illuminated cell imaging by an isomerisation process under UV irradiation.

8. Use of a stereoisomer of claim 1 having aggregation-induced emission characteristics or a fluorescent probe of claim 6 in visual drug screening and development.

9. Use of a stereoisomer of claim 1 having aggregation-induced emission characteristics in the manufacture of a medicament for the treatment of cancer.

Technical Field

The present invention belongs to the field of biotechnology, and in particular relates to Aggregation Induced Emission (AIE) stereoisomers with great differences in luminescent properties and biomedical activities, uses thereof, and methods of preparation thereof.

Background

Exploring the relationship between molecular structure and properties is crucial to the development of material science. Isomers having the same composition but differing in structural structure, stereochemistry and chirality generally have a tremendous impact on molecular engineering and life sciences. Although scientists have made great efforts to study the important role of molecular chirality in determining its optical properties and pharmaceutical efficacy, little is known about how to modulate the properties of the E/Z stereoisomers. It has been reported that drugs with some geometric E/Z isomers exhibit significantly different biomedical activities. For example, tamoxifen (Z-tamoxifen) in the Z configuration is a weak estrogen agonist, but its E-configuration acts as a potent estrogen antagonist for the treatment of breast cancer. Cisplatin (Cis-platin) and carboplatin (trans-platin) show significant activity differences against cancer targets, with cisplatin showing high killing power against many types of cancer, while carboplatin shows little anti-cancer activity. Some recently reported fluorescent stereoisomer probes also have significantly different binding affinities for enzyme or nucleic acid (DNA) targets. However, tracking stereoisomers during endocytosis, biological target binding and responsive transformation, and ultimately visualizing their differences in biomedical activity, remains a significant challenge.

Efficient synthesis of E/Z stereoisomers with significant luminescent differences is important for elucidating their respective different optical properties and their transformations among each other, and may provide great opportunities for exploring their practical applications in molecular engineering and drug discovery. The stereochemistry of the carbon-carbon double bonds of Aggregation-Induced Emission luminescence (AIEgen), e.g. triphenylethylene, tetraphenylethylene and their derivatives, is very suitable for such studies, since their E/Z structures can be easily prepared by McMurry coupling to obtain benzophenones with asymmetric functional groups. In addition, they have aggregation-induced emission (AIE) properties through the restriction of intramolecular movement, and have greater absorption, greater luminosity and photobleaching resistance. These excellent luminescent properties overcome the disadvantages of quenching and photobleaching caused by aggregation caused by traditional fluorophores, and enable a wide range of practical applications in optoelectronic devices, chemical/biological analyte sensing, molecular imaging, dynamic process tracking and photoresponsive therapeutic applications based on functionalized aiegens.

However, the study of pure isomers with AIE properties is still in the initiative, due to the difficulty of separation due to the small difference in polarity. Although palladium-catalyzed coupling reactions can theoretically synthesize the pure isomers directly, it was found that the expected Z isomer is usually accompanied by the E isomer as a side product. Separation of some AIE stereoisomers has been successfully achieved by applying strategies such as enlarging the molecular size and increasing the polarity difference by attaching larger groups to the AIE core or stabilizing them through multiple hydrogen bonds. However, the complexity of the molecular design described above will limit the practical application of this strategy. Therefore, most applications use mixtures of directly prepared E/Z isomers, resulting in compromises to the properties and functions of these molecules.

Another arduous task is to identify the E/Z geometry after separation. Current methods rely on growing single crystals for X-ray diffraction to confirm the geometric configuration of isomers, but this is a cumbersome task and is only applicable to those isomers that can be easily deposited as single crystals. Although some of the separated isomers have different optical properties, such as variations in emission wavelength and small fluctuations in fluorescence intensity, it is still difficult to accurately distinguish the individual isomer types therein, let alone to follow up their potentially unique biomedical activities. Since isomerization is closely related to molecular motion, stereoisomers are often unstable in solution and result in changes in luminescent properties.

Therefore, there is a need in the art for a stereoisomer that has a simple structure, is stable, and is easy to separate, and provides a new tool for fluorescence imaging and visualized drug screening and development through the difference between its luminescent properties and biomedical activities.

Disclosure of Invention

The object of the present invention is to overcome the drawbacks of the prior art, to provide a pair of stereoisomers that is structurally simple, stable and easy to separate, and to provide their use in illuminated cell imaging and drug screening and development, based on their differences in luminescent properties and biomedical activity.

Accordingly, in a first aspect, the present invention provides a stereoisomer having aggregation-induced emission characteristics, wherein the stereoisomer has the general structural formula E isomer:

or the R isomer, or a mixture of the R isomers,

wherein X is selected from any one of the following groups:

r and R' are one or more fluorescent chromophores of different structures coupled to one or more fluorophores, R being selected from the group consisting of hydrogen, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-N-chlorosuccinimide (NCS), alkyl-azide (-N₃) And alkyl-amino (-NH)₂) Any one of the above; r' is selected from the group consisting of hydrogen, halogen, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-N-chlorosuccinimide (NCS), alkyl-azide (-N)₃) And alkyl-amino (-NH)₂) (ii) a R is preferably hydrogen; r' is preferably hydrogen, more preferablyIs selected as a halogen atom; n is 1, 2, 3, 4, 5, 6, 7, 8 or 9.

In a second aspect, the present invention provides a method for producing a stereoisomer having aggregation-induced emission characteristics according to the first aspect, wherein the method comprises:

wherein the first reaction raw material is 4-methoxybenzophenone or 4- (dimethylamino) benzophenone;

the chemical structural formula of the second reaction raw material is as follows:

(2) and separating and purifying the crude product to obtain a purified product, and further separating the E/Z isomer from the purified product by high performance liquid chromatography.

In some embodiments, the crude product is isolated and purified by silica gel column.

In some embodiments, the E/Z isomers are separated by high performance liquid chromatography equipped with a C18 reverse phase column

In a third aspect, the present invention provides a fluorescent probe, wherein the fluorescent probe comprises the stereoisomer having aggregation-induced emission characteristics according to the first aspect of the present invention.

In a fourth aspect, the present invention provides a stereoisomer of the first aspect having aggregation-induced emission characteristics or a fluorescent probe of the third aspect for use in illuminated bioimaging.

In some embodiments, the invention uses the non-luminescent isomer for illuminated cell imaging by an isomerization process under uv irradiation.

In a fourth aspect, the present invention provides the use of a stereoisomer of the first aspect having aggregation-induced emission characteristics or a fluorescent probe of the third aspect in drug screening and development for visualization.

In a fifth aspect, the present invention provides the use of a stereoisomer of the first aspect having aggregation-induced emission characteristics for the preparation of an anti-cancer medicament.

The invention has the beneficial effects that:

1) provides a series of stable Aggregation Induced Emission (AIE) stereoisomers with simple molecular structure, wherein the E/Z isomers can be easily separated by HPLC;

2) each pair of E/Z isomers shows great difference in luminescence property and biomedical activity, and has mutual isomerization ability under illumination;

3) by utilizing the difference of the luminescence characteristics and the biomedical activity of each pair of E/Z isomers, a new tool is provided for the luminous bioluminescence imaging, and a new possibility is provided for the development and screening of visual medicines.

Drawings

Figure 1 shows a synthetic roadmap for a series of stereoisomers with aggregation-induced emission characteristics.

FIG. 2 shows the elution separation profile of E/Z-TPAN-OM by HPLC.

FIG. 3 shows deuterated chloroform (CDCl)₃) Hydrogen of the E/Z-TPAN-OM mixture and purified E-TPAN-OM and Z-TPAN-OM alone ((¹H) Spectra.

FIG. 4 shows normalized absorption spectra of E/Z-TPAN-OM measured in Dimethylsulfoxide (DMSO) solvent.

FIG. 5 shows the characterization of aggregation-induced emission characteristics of E/Z-TPAN-OM, wherein a is a fluorescence spectrum of E-TPAN-OM under constant change of water volume fraction in a mixed solvent of DMSO and water; panel b is a graphical representation of the intensity of the fluorescence emission peak of E/Z-TPAN-OM as a function of water content.

FIG. 6 shows the optical characterization of E/Z-TPAN-OM, wherein a is the absorption spectrum of solid E/Z-TPAN-OM, respectively labeled with absorption peaks; FIG. b is a fluorescent photograph of E/Z-TPAN-OM powder under 365nm UV light at Room Temperature (RT) and liquid nitrogen (CT, 77K), respectively, and absolute quantum yield (. PHI.) of E-TPAN-OM_F) Is marked; FIG. c is a plot of the fluorescence spectra of solid E/Z-TPAN-OM at Room Temperature (RT) or under liquid nitrogen (CT, 77K), respectively, indicating the emission wavelength (. lamda.) and decay lifetime (. tau.); and d is a graph of the temperature-dependent E/Z-TPAN-OM fluorescence emission intensity.

FIG. 7 is a graph showing the results of isomer transformation experiments of E/Z-TPAN-OM under 365 nanometer (nm) UV lamp for 150 min, wherein a is E/Z-TPAN-OM in deuterated chloroform (CDCl)₃) Nuclear magnetic hydrogen spectrum of (1)¹H NMR), proton resonances in the aromatic rings in zone 1 and zone 3 belong to Z-TPAN-OM and E-TPAN-OM, respectively, and proton resonances for methyl groups in zone 2 and zone 4 belong to Z-TPAN-OM and E-TPAN-OM, respectively; panel b is a graph of the results of an isomer conversion experiment monitored by High Performance Liquid Chromatography (HPLC) of E/Z-TPAN-OM at 280 nanometers (nm), with elution peaks in zones 5 and 6 belonging to Z-TPAN-OM and E-TPAN-OM, respectively, and elution peaks in zone 7 from their photo-cyclized products.

Fig. 8 shows a graph of the results of an illuminated cell imaging experiment.

FIG. 9 shows optical characterization of E/Z-TPAN-OH; wherein, the figure a shows a fluorescence spectrum diagram of E/Z-TPAN-OH under the condition that the volume fraction of water in a mixed solvent of DMSO and water is changed continuously; FIG. b is a graph showing the fluorescence spectrum of solid E/Z-TPAN-OH at Room Temperature (RT) in which the absolute quantum yield (. PHI.) of Z-TPAN-OH_F) Is plotted, and the absolute quantum yield (. PHI.) of E-TPAN-OH_F) Then below the instrument detectable range.

FIG. 10 is a graph showing the results of an enzymatic reaction experiment of E/Z-TPAN-POH; wherein, Panel a shows the scheme for the enzymatic reaction of E/Z-TPAN-POH to E/Z-TPAN-OH at room temperature, the absolute yield (. PHI.) of solid Z-TPAN-POH_F) Is marked out; FIG. b is a high performance liquid chromatogram of a monitored alkaline phosphatase (ALP) -catalyzed hydrolysis of Z-TPAN-POH; FIG. c is a graph showing the hydrolytic conversion of E/Z-TPAN-POH; E/Z-The concentration of TPAN-POH was 100 micromole per liter and ALP was 1.05 nanomole per liter.

FIG. 11 is a graph showing the results of an affinity study of E/Z-TPAN-POH with ALP enzyme; FIG. a is an overlay structure of the Z-TPAN-POH binding to the active site of ALP; panel b is an overlay structure of E-TPAN-POH binding to ALP active site; FIG. c shows the binding energy of E/Z-TPAN-POH to ALP enzyme, respectively.

FIG. 12 shows a graph of cell activity of E/Z-TPAN-POH after action in various types of cancer cell lines; FIG. a is a graph showing the activity of E/Z-TPAN-POH at various concentrations after 2 hours of incubation with human cervical cancer cells (HeLa); FIG. b is a graph of cell activity after incubation of various concentrations of E/Z-TPAN-POH with human renal carcinoma cells (ACHN) for 12 hours; FIG. c is a graph of cell activity after 6 hours of incubation of various concentrations of E/Z-TPAN-POH with human osteosarcoma cells (U2R); FIG. d is a graph of cell viability of E/Z-TPAN-POH at various concentrations after 12 hours incubation with human osteosarcoma cells (HOS); (e) a cell activity graph of E/Z-TPAN-POH with different concentrations after 6 hours of incubation with human osteosarcoma cells (SJSA-1); (f) cell activity plots of different concentrations of E/Z-TPAN-POH after 2 hours incubation with human hepatoma cells (HepG 2).

FIG. 13 shows a HeLa cell profile after incubation with Z-TPAN-POH; panel a is a cell map after 2 hours incubation with Z-TPAN-POH; panel b is a cell map after 6 hours incubation with Z-TPAN-POH; panel c is a cell map after 12 hours incubation with Z-TPAN-POH.

FIG. 14 shows a HeLa cell profile after incubation with E-TPAN-POH; panel a is a cell map after 2 hours incubation with E-TPAN-POH; panel b is a cell map after 6 hours incubation with E-TPAN-POH; panel c is a cell map after 12 hours incubation with E-TPAN-POH.

FIG. 15 shows a HeLa cell profile after 12 hours incubation with Z-TPAN-POH (500. mu.M); FIG. a is an image of HeLa cells at 0 min in a bright field; panel b is an image of HeLa cells in a fluorescence field at 0 min.

FIG. 16 shows a HeLa cell profile after 12 hours incubation with E-TPAN-POH (500. mu.M); FIG. a is an image of HeLa cells at 0 min in a bright field; panel b is an image of HeLa cells at 0 min in the fluorescence field; FIG. c is an image of HeLa cells in the fluorescent field after 10 minutes of irradiation with a mercury lamp.

FIG. 17 shows Raman spectra of cell extracts after incubation of HeLa cells with Z/E-TPAN-POH, respectively; wherein characteristic peaks of the silicon and nitrile groups are marked.

FIG. 18 shows HPLC elution profiles of HeLa cell extracts alone and after co-incubation with 500. mu.M of Z/E-TPAN-POH, respectively, peaks 1 to 5 can be judged as cell substrate, E-TPAN-POH precursor, Z-TPAN-POH precursor, E-TPAN-OH hydrolysate and Z-TPAN-POH hydrolysate, respectively, based on their respective elution times.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The described embodiments are only a few embodiments of the invention, not all embodiments. All other embodiments are available to the person skilled in the art based on the embodiments of the invention and are within the scope of protection of the invention.

Throughout this application, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently described subject matter belongs.

The invention provides a stereoisomer with aggregation-induced emission characteristics, which is characterized in that the structural general formula of the stereoisomer is an E isomer:

or the R isomer, or a mixture of the R isomers,

wherein, X is selected from any one of methoxyl, alkoxyl, hydroxyl, ethyl phosphoric acid, alkyl phosphoric acid, amino, dimethylamino, diethylamino, pyridine, imidazole and sulfydryl;

As used herein, "halogen" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

As used herein, "alkyl" refers to a straight or branched chain saturated hydrocarbon group. Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, sec-butyl, tert-butyl), pentyl, hexyl, and the like. In various embodiments, the alkyl group can have 1 to 40 carbon atoms (i.e., C1-40 alkyl), for example, 1 to 30 carbon atoms (i.e., C1-30 alkyl). In some embodiments, alkyl groups may have 1 to 6 carbon atoms, and may be referred to as "lower alkyl". Examples of lower alkyl groups include methyl, ethyl, propyl (e.g., n-propyl and isopropyl), and butyl (e.g., n-butyl, isobutyl, sec-butyl, tert-butyl). In some embodiments, alkyl groups may be substituted as described herein. An alkyl group is typically not substituted with another alkyl, alkenyl, or alkynyl group.

As used herein, "heteroatom" refers to an atom of any element other than carbon or hydrogen, including, for example, nitrogen, oxygen, silicon, sulfur, phosphorus, and selenium.

As used herein, "aryl" refers to an aromatic monocyclic hydrocarbon ring system or a polycyclic ring system in which two or more aromatic hydrocarbon rings are fused together (i.e., have a common bond), or at least one aromatic monocyclic hydrocarbon ring is fused to one or more cycloalkyl and/or cycloheteroalkyl rings. The aryl group may have 6 to 24 carbon atoms in its ring system (e.g., a C6-24 aryl group), which may include multiple fused rings. In some embodiments, the polycyclic aryl group can have 8 to 24 carbon atoms. Any suitable ring position of the aryl group can be covalently linked to the defined chemical structure. Examples of the aryl group having only an aromatic carbocyclic ring include phenyl, 1-naphthyl (bicyclic), 2-naphthyl (bicyclic), anthracenyl (tricyclic), phenanthrenyl (tricyclic), pentacenyl (pentacyclic), and the like. Examples of polycyclic ring systems in which at least one aromatic carbocyclic ring is fused to one or more cycloalkyl and/or cycloheteroalkyl rings include benzo derivatives of cyclopentane (i.e., indanyl, which is a 5, 6-bicyclic cycloalkyl/aryl ring system), cyclohexane (i.e., tetrahydronaphthyl, which is a 6, 6-bicyclic cycloalkyl/aryl ring system), imidazoline (i.e., benzimidazolyl, which is a 5, 6-bicyclic cycloheteroalkyl/aryl ring system), and pyran (i.e., other examples of aryl include benzodioxolyl, chromanyl, indolyl, etc., hi some embodiments, in some embodiments, the aryl group can have one or more halogen substituents, and may be referred to as "haloaryl". Perhaloaryl, i.e., all hydrogen atoms are replaced with halogen atoms (e.g., -C).₆F₅) Substituted aryl groups are included in the definition of "haloaryl". In certain embodiments, an aryl group is substituted with another aryl group, and may be referred to as a biaryl group. Each aryl group in the biaryl group may be substituted as disclosed herein.

As used herein, "heteroaryl" refers to an aromatic monocyclic ring system containing at least one ring heteroatom selected from oxygen (O), nitrogen (N), sulfur (S), silicon (Si), and selenium (Se), or a polycyclic ring system in which at least one ring present in the ring system is aromatic and contains at least one ring heteroatom. Polycyclic heteroaryls include those having two or more heteroaryl rings fused together, and having at least one fused to one or moreThose of a monocyclic heteroaryl ring on multiple aromatic carbocyclic, non-aromatic carbocyclic and/or non-aromatic cycloheteroalkyl rings. The heteroaryl group as a whole may have, for example, 5 to 24 ring atoms, and contain 1 to 5 ring heteroatoms (i.e., a 5-to 20-membered heteroaryl group). The heteroaryl group may be attached to the defined chemical structure at any heteroatom or carbon atom that results in a stable structure. Typically, the heteroaryl ring does not contain oxygen, sulfur, or sulfur linkages. However, one or more of the nitrogen or sulfur atoms in the heteroaryl group can be oxidized (e.g., pyridine nitroxide thiophenesulfoxide, thiophenesulfur, sulfur dioxide). Examples of heteroaryl groups include, for example, 5 or 6 membered monocyclic and 5-6 membered bicyclic ring systems as shown below, wherein T is O, S, NH, N-alkyl, N-aryl, N- (arylalkyl) (e.g., N-benzyl), SiH₂SiH (alkyl), Si (alkyl)₂SiH (arylalkyl), Si (arylalkyl)₂Or Si (alkyl) (arylalkyl). Examples of such heteroaryl rings include pyrrolyl, furanyl, thienyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl, benzofuranyl, benzothienyl, quinolinyl, 2-methylquinolinyl, isoquinolinyl, quinoxalinyl, benzotriazolyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl heteroaryl further examples include 4, 5, 6, 7-tetrahydroindolyl, tetrahydroquinolinyl, benzothienopyridyl, benzofuropyridyl, and the like. In some embodiments, heteroaryl groups may be substituted as described herein.

In another aspect, the present invention provides a method for preparing a stereoisomer having aggregation-induced emission characteristics, comprising: in the presence of an organic solvent, dropwise adding a second reaction raw material into a mixture of the first reaction raw material and NaH, and stirring or heating and refluxing the reaction mixture in an inert gas environment to obtain a crude product; wherein the first reaction raw material is 4-methoxybenzophenone or 4- (dimethylamino) benzophenone, and the chemical structural formula of the second reaction raw material is as follows:

(2) separating and purifying the crude product to obtain a purified product, and further separating the stereoisomer from the purified product by high performance liquid chromatography.

The E/Z-TPAN-OM obtained by the preparation method can be synthesized into E/Z-TPAN-OH by further adding boron tribromide, the E/Z-TPAN-OH can be obtained by further adding dichloromethane containing triethylamine, and finally the E/Z-TPAN-POH can be obtained by adding trimethyl bromosilane to the E/Z-TPAN-POE. A series of E/Z isomers constructed based on triphenylethylene motifs can be prepared on a large scale by the simple synthetic method.

The existing aggregation-induced emission molecules cannot directly separate the E/Z isomers through a common C18 reverse column due to small polarity differences, and the use of directly prepared mixtures of E/Z isomers in applications results in compromises of properties and functions of these molecules. The E/Z isomer in the present invention can be easily separated by High Performance Liquid Chromatography (HPLC) equipped with C18 reverse phase column, the crude product prepared according to the above method is filtered and the solvent is evaporated, and then separated and purified by silica gel column using n-hexane-dichloromethane M as eluent to obtain a purified product, which is dissolved in acetonitrile, and the E/Z isomer can be further separated by HPLC equipped with C18 reverse phase column using acetonitrile/water 85:15 (volume ratio) as eluent.

Aggregation-induced emission (AIE) refers to a phenomenon exhibited by compounds that exhibit a significant increase in luminescence when aggregated in an amorphous or crystalline (solid) state, whereas they exhibit little or no emission in dilute solutions. The AIE material has the advantages of strong luminescence property, high sensitivity, strong photobleaching resistance and the like in a solid state or a high-concentration state. The stereoisomers of the present invention all exhibit specific Aggregation Induced Emission (AIE) properties, and one isomer of each pair of E/Z isomers exhibits strong fluorescence emission, while the other isomer emits little fluorescence. The inventors have found that in some embodiments, when the substituent X is selected from methoxy or alkoxy, the Z isomer does not emit light, while the E isomer emits light, as shown by the results in the examples. In other embodiments, when the substituent X is selected from the group consisting of hydroxy, ethylphosphoric acid, alkylphosphoric acid, phosphoric acid, amino, dimethylamino, diethylamino, pyridine, imidazole, and mercapto, the Z isomer emits light, while the E isomer does not. This is because crystals are more closely arranged and intermolecular pores are smaller in the luminescent isomer, thereby causing the molecules to be restricted in motion and thus emitting fluorescence. In contrast, in the non-luminescent isomers, the crystals are more loosely arranged and the intermolecular pores are larger, resulting in a smaller degree of restriction of molecular motion and thus no fluorescence is emitted.

Further, the E/Z isomer may undergo interconversion under UV irradiation, and this conversion is bidirectional, i.e., the E isomer may be converted to the Z isomer, and the Z isomer may also be converted to the E isomer. In order to control the direction of the conversion, the starting material must be pure E or Z, and it is important to be able to obtain stable pure E or Z isomers.

In terms of detection sensing, fluorescent "lit-up" type ("turn-on" or "light-up") biological and chemical sensing systems can be successfully implemented using the above-described properties of the E/Z isomers. The object of the illuminated bioimaging includes various organelles, cells (e.g., cancer cells and normal cells), microorganisms (e.g., bacteria and fungi), and tissues, and enables long-term tracking. Thus, the stereoisomers provided by the present invention can be used alone or in combination with other ingredients in a variety of fluorescent probes, fluorescent dyes, pharmaceutical compositions or kits, and the like.

On the other hand, the E/Z isomers also exhibit different biomedical activities, such as enzymatic reactions and cytotoxicity.

Since the uptake of E/Z-TPAN-OM and E/Z-TPAN-OH by cells is relatively limited and difficult to verify the biomedical activity, the inventor further designs E/Z-TPAN-POH modified by phosphate group to improve the water solubility and endow the enzyme catalytic responsiveness. Phosphate groups are substrates of alkaline phosphatase (ALP), play an important role in cell signaling pathways, and have become important biomarkers in cancer diagnosis. In some embodiments, enzymatic recognition and reaction of the E isomer can be found to be faster and with higher affinity to ALP by HPLC detection of ALP catalyzed E/Z-TPAN-POH hydrolysis. In some embodiments, the E/Z isomer may react with proteases (including trypsin, pepsin, trypsin, cathepsin, papain, subtilisin, and the like) to hydrolyze peptide bonds between amino acid residues in polypeptide chains, hydrolases (phosphatases, lipases, glycosylases, and the like), and other responsive biomolecules present in biological systems.

Inspired by the great difference in enzymatic reactions of ALP by E/Z-TPAN-POH, the inventors further studied them in cancer cytotoxicity. Because the expression level of ALP is generally up-regulated in cancer cells in the case of biochemical and metabolic process abnormalities. In some embodiments, the inventors have found that the E/Z isomers exhibit a large difference in cytotoxicity, e.g., one isomer exhibits strong toxicity to cancer cells while the other isomer exhibits better biocompatibility for these cancer cells, e.g., Z isomer exhibits significant toxicity when substituent X is selected from ethyl phosphate, alkyl phosphate or phosphate, while E isomer is relatively more compatible with cells. This difference in toxicity was further demonstrated in various types of cancer cells, including human cervical cancer cells (HeLa), human renal Adenocarcinoma Cells (ACHN), human osteosarcoma cells (HOS, SJSA-1, and drug-resistant U2R), and human hepatoma cells (HepG2), mainly due to different rates of cellular uptake and accumulation in cancer cells.

Differences in toxicity of the E/Z isomers can be distinguished by contrasting strong fluorescent signals and thus the stereoisomers provided by the invention also provide potential tools for drug screening and therapeutic evaluation.

Examples

Experimental Material

Dimethyl sulfoxide (DMSO, 99.7%), alkaline phosphatase (ALP, from calf intestine), ethylene bischlorophosphite (97%), sodium hydride (NaH, 60% dispersion in mineral oil), trimethylbromosilane (TMSBr, 97%) and phenylacetonitrile (98%) were purchased from Sigma-Aldrich. 4-bromophenylacetonitrile (98%), triethylamine (TEA, 99.8%), diethanolamine (DEA, 98%), 4-methoxybenzophenone (97%), 4-methylbenzophenone (97%), toluene, 4- (dimethylamino) benzophenone (98%), chloroform (CHCl3), toluene, Dichloromethane (DCM), Tetrahydrofuran (THF), N-Dimethylformamide (DMF), methanol, acetonitrile (CH)₃CN), n-hexane, boron tribromide (BBr)₃1.0 mol/l in dichloromethane) and ethyl acetate from J&K. Phosphate buffered saline (10 × PBS) was purchased from Thermo Scientific (HyClone). Other compounds were purchased from AIEgen Biotech. All reagents and solvents were of analytical grade. The solvents, including Dichloromethane (DCM), toluene and Tetrahydrofuran (THF), were all distilled. Cancer cell lines include human cervical cancer cells (HeLa), human renal Adenocarcinoma Cells (ACHN), human osteosarcoma cells (HOS, SJSA-1 and drug-resistant U2R), and human hepatoma cells (HepG2) purchased from China center for type culture (CCTCC). Ultrapure water (18.2 M.OMEGA.cm) was prepared by a Milli-Q Direct-8 purification system (Millipore Corp.).

Experimental methods

1.1 characterization of the Properties of the stereoisomers

Fluorescence (FL) spectra and absolute solid quantum yields were measured using a Horiba FluoroLog-3 fluorescence spectrophotometer. Time resolved transient luminescence spectroscopy (TRPL): the sample was excited using a femtosecond (fs) laser and TRPL decay kinetic signals were measured with a time response within 100 picoseconds (ps) using a time-correlated single photon counting (TCSPC) module (picohard 300) and a SPAD detector (IDQ, id 100). Fluorescence emission spectra were recorded as a function of temperature using an FLS-980 fluorescence spectrometer (Edinburgh). Absolute fluorescence quantum yield was measured using a Hamamatsu spectrometer (C11347 Quantaurus). Shimadzu UV was used2550 Spectrophotometer (Shimadzu Co, Kyoto, Japan) measuring UV-visible spectra. The aggregation-induced emission fluorescence curves of TVP-S were measured in mixed solvents of dimethyl sulfoxide and chloroform at different volume ratios, each group containing 10. mu. mol/L of AIEgen, and fluorescence spectra were collected under excitation light at 460 nm. Collected at 1 square centimeter (cm) using a MicroRaman spectrometer (InVia, Renishaw) under 632nm laser excitation²) Raman spectrum of dried cell extract on silicon wafer, signal acquisition exposure time was 10 seconds(s). After 20 microliters of the samples were respectively dispersed on 3.0 mm-sized copper mesh covered with a continuous carbon film and dried at room temperature, Transmission Electron Microscope (TEM) imaging observation was performed on a Tecnai G220 (FEI, usa) transmission electron microscope at 80 kv voltage.

1.2 hydrolysis of Z/E-TPAN-POH catalyzed by ALP

100 μ M/L of Z/E-TPAN-POH was hydrolyzed with ALP (1.05nM) in diethanolamine buffer (DEA, 10 mmol/L, pH 9.8, containing 0.1 mmol/L magnesium chloride) and the mixture was incubated at 37 ℃ for various periods of time. The hydrolysis was monitored in real time by semi-preparative High Performance Liquid Chromatography (HPLC) equipped with a C18 reverse phase column (Agilent 1260infinite II) eluting at 10ml/min and 85:15 acetonitrile/water (vol/vol).

1.3 culture of cancer cells

25 square centimeter (cm) with vent cover (Corning)²) Cancer cells were cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS) on cell culture plates. All cells were at 37 ℃ and contained carbon dioxide (CO)₂5% concentration) in a moist incubator. Cells were dissociated from the bottom of the dish using a 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA) solution. Aliquots of cells were then seeded into 96-well plates and continued to grow for the required time in cell culture medium containing fetal bovine serum.

1.4 bacterial culture, imaging and real-time tracking

In fluorescence imaging of cells, 100 microliters of cancer cells (1.0 × 10 per well) were examined⁴Individual cells) were seeded in 96-well plates (Corning). At 37 ℃ and 5% CO₂After an overnight incubation under the conditions of (1),cells were washed once with 1 fold phosphate buffered saline (1 × PBS). 100 microliters of DMEM medium containing different amounts of the compounds was added to each well and at 37 ℃ and containing carbon dioxide (CO)₂5% concentration) was added. After washing twice with 1 × PBS, fluorescence images were taken on an Olympus IX 71 inverted fluorescence microscope with a 40-fold (40 ×) objective lens.

1.5 in vitro antibacterial assay

Cell activity was measured by the thiazole bromide blue tetrazolium (MTT) assay. Cancer cells were treated at 5.0X 10³The density of cells/well was seeded into 96-well plates and incubated with different concentrations of E/Z-TPAN-POH and E/Z-TPAN-OH for the indicated time. MTT (5.0mg/mL, 20. mu.L) was then added and the cells were incubated at 37 ℃ and 5% CO₂And incubated for an additional 4 hours. Then 150 μ L DMSO was added, gently shaken for 10 minutes to dissolve the precipitate. The absorbance was measured at a wavelength of 570nm with a microplate reader (Bio-Rad). Cell activity was obtained by normalizing the absorbance of the sample wells relative to the absorbance of the control wells and expressed as a percentage, and the activity of untreated cells was assigned as 100%. After incubation with the different isoforms, bright field pictures of the cells were taken on an Olympus IX 71 inverted fluorescence microscope with a 20-fold objective. For HPLC quantification of cellular uptake of the isoforms, 4X 10 fractions were collected⁶Cells were digested with 2.0mL of 10% nitric acid for 4 hours. The cell contents were then extracted with 5.0mL of chloroform. After evaporation, the sample was calibrated to 500 μ L by dissolution in a mixed solvent of acetonitrile/water (volume ratio 50/50). HPLC elution was performed at an elution ratio of 10mL/min and acetonitrile/water of 85/15 (vol), and a signal was detected at 310 nm.

EXAMPLE 1 Synthesis of stereoisomers

Synthesis of Z/E-TPAN-M

To a mixture of 4-methylbenzophenone (500mg, 2.55mmol) and NaH (122.4mg, 5.1mmol) was added toluene (60 mL). The mixture was then cooled to 0 ℃ with an ice water bath. Phenylacetonitrile (320 μ L, 2.78mmol) in THF (20mL) was added dropwise at 0 deg.C. The reaction mixture was brought to room temperature and stirred under nitrogen for 2 hours. By passingThe reaction was quenched by the addition of 10mL of anhydrous methanol. The residue resulting from the evaporation of the solvent was dissolved in diethyl ether. After filtration and evaporation of the solvent, the crude product was purified by silica gel column chromatography using a mixed solvent of n-hexane-dichloromethane as an eluent to give a white powder. Attempts were made to separate the products by high performance liquid chromatography by varying the elution conditions. However, HPLC using a C18 reverse phase column configuration failed to separate the isomers. For the mixture:¹H NMR(Bruker Avance，400MHz，CDCl₃)：7.46-7.43(m，5H)，7.38-7.36(m，2H)，7.30-7.17(m，16H)，7.03-6.99(m，4H)，6.91-6.89(m，2H)，2.42(m，3H)，2.31(m，3H)；¹³C NMR(Bruker Avance，100MHz，CDCl₃)，δ(ppm)：140.7，140.2，139.2，137.6，136.1，135.1，135.0，130.9，130.0，129.9，129.8，129.7，129.2，128.9，128.5，128.4，128.2，120.4，120.3，111.0，110.9，21.5，21.3。

synthesis of Z/E-TPAN-OM

After 30 min of vacuum treatment, THF (60mL) was added to a mixture of 4-methoxybenzophenone (500mg, 2.36mmol) and NaH (114mg, 4.44 mmol). Then phenylacetonitrile (300. mu.L, 2.61mmol) dissolved in THF (20mL) was added dropwise at 0 ℃. The reaction mixture was warmed to room temperature and heated to reflux under nitrogen for 12 hours. The reaction was quenched by the addition of 10mL of anhydrous methanol. After filtration and evaporation of the solvent, the crude product obtained was purified by silica gel column chromatography using 1:1 n-hexane-dichloromethane as eluent to give a yellow crude product. Finally, the crude product was dissolved in acetonitrile and the Z/E-TPAN-OM isomer was further separated by HPLC using a C18 reverse phase column under conditions of elution rate 10mL/min and acetonitrile/water 85:15 (volume ratio). The collection time of Z-TPAN-OM and E-TPAN-OM is set to 10.15 minutes to 10.60 minutes and 9.50 minutes to 9.90 minutes, respectively. For Z-TPAN-OM, the structure is determined by single crystal measurement.¹H NMR(Bruker Avance，400MHz，CDCl₃)：7.41-7.38(m，2H)，7.24-7.19(m，3H)，7.15-7.12(m，5H)，6.99-6.98(m，2H)，6.91-6.88(m，2H)，3.78(s，3H)；¹³C NMR(Bruker Avance，100MHz，CDCl₃)，δ(ppm)：161.1，157.6，139.4，135.2，132.7，131.8，131.0, 129.8, 129.1, 128.5, 128.3, 128.2, 120.8, 113.9, 110.0, 55.4; hrms (ei): for C₂₂H₁₇NO[M+H]⁺Calculated values: 312.1383, respectively; measured value: 312.1383. the structure of the E-TPAN-OM is then determined by single crystal measurement.¹H NMR(Bruker Avance，400MHz，CDCl₃)：7.44-7.43(m，5H)，7.31-7.29(m，2H)，7.26-7.23(m，2H)，6.94-6.91(m，2H)，6.71-6.69(m，2H)，3.77(s，3H)；¹³C NMR(Bruker Avance，100MHz，CDCl₃) δ (ppm): 160.2, 157.5, 140.7, 135.2, 132.6, 131.2, 130.1, 129.8, 129.7, 128.6, 128.4, 128.2, 120.5, 113.6, 110.3, 55.2; hrms (ei): for C₂₂H₁₇NO[M+H]⁺Calculated values: 312.1383, respectively; measured value: 312.1383.

synthesis of Z-TPAN-OH

Boron tribromide (1.92mL, 1.92mmol) was slowly added to a solution of compound Z-TPAN-OM (300mg, 0.96mmol) in dry dichloromethane (40mL) at 0 ℃ in an ice bath. The mixture was stirred at room temperature for 12 hours. The reaction was quenched with methanol and the mixture was extracted with dichloromethane. The collected organic layer was dried over anhydrous sodium sulfate. The crude product was then concentrated and purified on a silica gel column using dichloromethane as eluent. The Z-TPAN-OH obtained was a grey solid (248mg, 0.83mmol), the yield was 86.5% and its structure was determined by single crystal measurement.¹H NMR(400MHz，CD₃OD)，δ(ppm)：7.30-7.24(m，3H)，7.21-7.18(m，7H)，7.02-6.99(m，2H)，6.84-6.82(m，2H)；¹³C NMR(100MHz，CD₃OD), δ (ppm): 159.3, 158.7, 139.4, 135.4, 131.4, 131.3, 130.7, 129.4, 128.6, 128.1, 127.8, 120.2, 114.8, 108.9; HRMS (EI, Bruker Apex IV FTMS): for C₂₁H₁₅NO[M+H]⁺Calculated values: 298.1226, respectively; measured value: 298.1226.

4, synthesizing E-TPAN-OH,

the starting material is changed to E-TPAN-OM, and the synthesis method and reaction conditions are the same as those of the synthesis of Z-TPAN-OH. A pale yellow solid of E-TPAN-OH (260mg, 0.87mmol) was obtained in 90.6% yield and determined by single crystal measurementDetermining the structure of E-TPAN-OH.¹H NMR(400MHz，CD₃OD)，δ(ppm)：7.43-7.42(m，5H)，7.30-7.26(m，5H)；6.82-6.79(m，2H)，6.60-6.57(m，2H)；¹³C NMR(100MHz，CD₃OD), δ (ppm): 158.6, 140.9, 135.5, 132.4, 129.7, 129.6, 129.4, 129.3, 128.3, 128.1, 127.9, 120.0, 114.6, 109.2; HRMS (EI, Bruker Apex IV FTMS): for C₂₁H₁₅NO[M+H]⁺Calculated values: 298.1226, respectively; measured value: 298.1226

Synthesis of Z-TPAN-POE

To a mixture of Z-TPAN-OH (200mg, 0.67mmol) was added anhydrous dichloromethane (15mL) including triethylamine (TEA, 150. mu.L). The mixture was then cooled to 0 ℃ with an ice water bath. Diethyl chlorophosphite (138mg, 0.80mmol) was then added dropwise. The reaction mixture was allowed to warm to room temperature and stirred under nitrogen for 2 hours. The reaction was quenched by the addition of 10mL of anhydrous methanol. After filtration and evaporation of the solvent, the crude product obtained was isolated and purified by silica gel column using n-hexane-ethyl acetate as eluent to give a grey powder with a yield of 67.2% (196mg, 0.45mmol), and the structure of the Z-TPAN-POE obtained was determined by single crystal measurement.¹H NMR(Bruker Avance，400MHz，CDCl₃)：7.7.46-7.43(m，2H)，7.28-7.19(m，10H)，7.00-6.98(m，2H)，4.29-4.21(m，4H)，1.40-1.36(m，6H)；¹³C NMR(Bruker Avance，100MHz，CDCl₃)，δ(ppm)：156.0，151.3，151.1，138.2，136.4，134.1，131.0，130.2，129.0，128.5，127.9，127.8，127.7，119.4，119.3，110.9，64.2，64.1，15.5，15.4；³¹P NMR(200MHz，CDCl₃): δ -6.80; HRMS (EI, Bruker Apex IV FTMS): for C₂₅H₂₄NO₄P[M+H]⁺Calculated values: 434.1516, respectively; measured value: 434.1516.

synthesis of E-TPAN-POE

The starting material is changed into E-TPAN-OH, and the synthesis method and the reaction conditions are the same as those of the synthesis of the Z-TPAN-POE. E-TPAN-POE was obtained as a white solid (178mg, 0.41mmol) in a yield of 61.2% and its structure was determined by single crystal measurement.¹H NMR(400MHz，CDCl₃)，δ(ppm)：7.45-7.43(m，5H)，7.28-7.22(m，5H)；7.06-7.04(m，2H)，6.99-6.97(m，2H)，4.23-4.15(m，4H)，1.35-1.31(m，6H)；¹³C NMR(100MHz，CDCl₃)，δ(ppm)：156.0，150.5，139.5，135.0，134.1，131.8，129.4，129.3，129.0，128.0，127.9，119.4，119.2，119.1，111.1，64.1，15.5，15.4；³¹P NMR(200MHz，CDCl₃): δ -6.81; HRMS (EI, Bruker Apex IV FTMS): for C₂₅H₂₄NO₄P[M^±]Calculated values: 433.1443, respectively; measured value: 433.1443.

synthesis of Z-TPAN-POH

To a solution of Z-TPAN-POE (100mg, 0.23mmol) in dry dichloromethane (10mL) was added dropwise trimethylsilyl bromide (80. mu.L, 0.61mmol) at room temperature. The reaction mixture was stirred at room temperature under nitrogen for 24 hours, and the reaction was quenched with methanol (2 mL). After the reaction mixture was stirred at room temperature for 30 minutes, the solvent was distilled off under reduced pressure. The reaction mixture was dispersed in ethyl acetate (1 mL). The precipitated product was filtered and collected to give a grey solid in 78.3% yield (67.9mg, 0.18mmol) and the structure of Z-TPAN-POH was determined by single crystal measurement.¹H NMR(Bruker Avance，400MHz，CD₃OD)，δ(ppm)：7.45-7.43(m，2H)，7.32-7.19(m，10H)，7.02-7.00(m，2H)；¹³C NMR(Bruker Avance，100MHz，CD₃OD)，δ(ppm)：157.0，130.4，129.8，128.7，128.2，127.6，127.5，127.3，119.3，118.9，110.3；³¹P NMR(200MHz，CD₃OD): δ -5.37; HRMS (EI, Bruker Apex IV FTMS): for C₂₁H₁₆NO₄P[M+Na]⁺Calculated values: 400.0709, respectively; measured value: 400.0709.

synthesis of E-TPAN-POH

The starting material is changed into E-TPAN-POE, and the synthesis method and the reaction conditions are the same as those of the synthesis of the Z-TPAN-POH. The E-TPAN-POH obtained was a grey solid (60.4mg, 0.16mmol), the yield was 69.6%, and the structure was determined by single crystal measurement.¹H NMR(400MHz，CD₃OD)，δ(ppm)：7.47-7.45(m，5H)，7.28-7.24(m，5H)，7.06-6.97(m，4H)；¹³C NMR(100MHz，CD₃OD)，δ(ppm)：159.1，142.0，136.4，135.9，133.4，131.0，130.8，129.8，129.6，121.1，112.5；³¹P NMR(200MHz，CD₃OD): δ -6.01; HRMS (EI, Bruker Apex IV FTMS): for C₂₁H₁₆NO₄P[M+Na]⁺Calculated values: 400.0709, respectively; measured value: 400.0709.

synthesis of Z/E-TPAN-NM

After 30 minutes of vacuum treatment, toluene (60mL) was added to a mixture of 4- (dimethylamino) benzophenone (500mg, 2.22mmol) and NaH (107mg, 4.44 mmol). Phenylacetonitrile (280. mu.L, 2.44mmol) in toluene (20mL) was then added dropwise at 0 ℃. The reaction mixture was warmed to room temperature and heated to reflux under nitrogen for 12 hours. The reaction was quenched by the addition of 10mL of anhydrous methanol. After filtration and evaporation of the solvent, the crude product obtained was isolated and purified by silica gel column using a 1:1 n-hexane-dichloromethane M as eluent, a yellow crude product was obtained. Finally the crude product was dissolved in acetonitrile and the Z/E-TPAN-NM isomer was further separated by HPLC with C18 reverse phase column at an elution rate of 10mL/min and acetonitrile/water of 85: 15. The collection times for Z-TPAN-NM and E-TPAN-NM were set to 12.6-13.2 minutes and 11.5-12.1 minutes, respectively. For Z-TPAN-NM, the structure was determined by single crystal measurement.¹H NMR(Bruker Avance，400MHz，CDCl₃)：7.37-7.35(m，2H)，7.23-7.14(m，8H)，7.06-7.03(m，2H)，6.69-6.67(m，2H)，3.02(s，6H)；¹³C NMR(Bruker Avance，100MHz，CDCl₃) δ (ppm): 158.3, 151.5, 139.8, 135.8, 131.7, 131.2, 129.8, 128.7, 128.3, 128.0, 127.6, 127.4, 121.5, 111.1, 107.3, 40.1; hrms (ei): for C₂₃H₂₀N₂[M+H]⁺Calculated values: 325.1699, respectively; measured value: 325.1699. for E-TPAN-NM:¹H NMR(Bruker Avance，400MHz，CDCl₃)：7.41-7.34(m，7H)，7.27-7.21(m，3H)，6.85-6.82(m，2H)，6.44-6.42(m，2H)，2.93(s，6H)；¹³C NMR(Bruker Avance，100MHz，CDCl₃)，δ(ppm)：158.1，150.6，141.3，132.7，130.3, 129.6, 128.5, 128.3, 127.8, 125.8, 121.2, 110.9, 107.9, 40.0; hrms (ei): for C₂₃H₂₀N₂[M+H]⁺Calculated values: 325.1699, respectively; measured value: 325.1699.

synthesis of Z/E-TPAN-NMBr

After 30 minutes of vacuum treatment, toluene (60mL) was added to a mixture of 4- (dimethylamino) benzophenone (500mg, 2.22mmol) and NaH (107mg, 4.44 mmol). 4-bromophenylacetonitrile (478mg, 2.44mmol) dissolved in toluene (20mL) was then added dropwise at 0 ℃. The reaction mixture was warmed to room temperature and heated to reflux under nitrogen for 12 hours. The reaction was then quenched by the addition of 10mL of anhydrous methanol. After filtration and evaporation of the solvent, the crude product obtained was isolated and purified by silica gel column using a 1:1 n-hexane-dichloromethane as eluent, a yellow crude product was obtained. The crude product was dissolved in acetonitrile and the Z/E-TPAN-NMBr isomer was further separated by HPLC with a C18 reverse phase column at an elution rate of 10mL/min and acetonitrile/water of 85: 15. The collection time of Z-TPAN-NMBr and E-TPAN-NMBr is set to 16.8-18.0 minutes and 15.0-16.2 minutes respectively. For Z-TPAN-NMBr, the structure was determined by single crystal measurement.¹H NMR(Bruker Avance，400MHz，CDCl₃)：7.36-7.34(m，2H)，7.30-7.26(m，2H)，7.23-7.19(m，2H)，7.09-7.02(m，4H)，6.68-6.66(m，2H)，3.03(s，6H)；¹³C NMR(Bruker Avance，100MHz，CDCl₃) δ (ppm): 158.9, 151.6, 139.5, 134.8, 131.7, 131.4, 131.3, 131.1, 129.0, 128.2, 127.0, 121.6, 121.1, 111.1, 105.8, 40.1; hrms (ei): for C₂₃H₁₉BrN₂[M+H]⁺Calculated values: 403.0804, respectively; measured value: 403.0804. for E-TPAN-NMBr:¹H NMR(Bruker Avance，400MHz，CDCl₃)：7.38-7.37(m，7H)，7.26-7.22(m，2H)，6.84-6.82(m，2H)，6.47-6.45(m，2H)，2.96(s，6H)；¹³C NMR(Bruker Avance，100MHz，CDCl₃) δ (ppm): 158.8, 150.8, 141.0, 135.0, 132.7, 131.7, 131.2, 130.3, 129.8, 128.3, 125.4, 121.7, 120.8, 111.0, 106.5, 40.0; hrms (ei): for C₂₃H₁₉BrN₂[M+H]⁺Calculated values: 403.0804, respectively; measured value: 403.0804.

EXAMPLE 2 separation of stereoisomers

To obtain a purified stereoisomer that can be used for property studies, the E/Z-TPAN-M mixture, which has two distinct methyl proton Nuclear Magnetic Resonance (NMR) peaks (2.41ppm and 2.28ppm), was first subjected to isomer separation. However, no matter what ratio of elution solvent is used in High Performance Liquid Chromatography (HPLC), the common C18 reverse phase column cannot separate the two isomers. After adding electron donor groups in the molecular structure, a significant difference in elution time occurred. The isomer pair containing the methoxy group (E/Z-TPAN-OM) was therefore eluted at a rate of 10 milliliters per minute (10mL/min) using acetonitrile/water (volume ratio v/v) 15/85 and the signal was detected at 280 nanometers (nm), and the E/Z-TPAN-OM could be easily isolated (fig. 2). The E/Z-TPAN-OM isomer having a simple structure would benefit from its simple and efficient synthetic method and diversified post-structural modification, compared to the existing isomers that need to expand their molecular structure to a large extent for separation. Purified E/Z-TPAN-OM isomer structure by nuclear magnetic hydrogen spectrum (¹H NMR and nuclear magnetic carbon Spectroscopy: (¹³C NMR), High Resolution Mass Spectrometry (HRMS), and X-ray single crystal diffraction. Notably, in Z and E-TPAN-OM¹In the H NMR spectrum, proton resonances in the methyl group appeared at 3.86ppm and 3.77ppm, respectively. The Z and E isomer aromatic proton chemical shifts also show significant differences, at 7.02ppm to 7.00ppm (zone 1) and 6.71ppm to 6.68ppm (zone 2), respectively (fig. 3). These significant differences can be used for structural confirmation and observation of the isomer transformation process. Since they can be synthesized under very mild conditions, the isomer derivatives including E/Z-TPAN-OH, E/Z-TPAN-POE and E/Z-TPAN-POH, etc. inherit the stereogeometric configuration of their precursor isomers (FIG. 1). Thus, purified isomers can be obtained without further separation. Their purity and geometry were also confirmed by the above measurements.

Example 3 characterization of stereoisomers

Successful purification of stereoisomers makes it possible to study their respective optical properties. In the solution state, the E/Z-TPAN-POH isomer has similar absorption wavelength in the ultraviolet band, and the maximum absorption is 332nm (figure 4). Surprisingly, the AIE curves of the stereoisomers in DMSO or water show a large difference, with the emission intensity of Z-TPAN-OM and E-TPAN-OM increased by 0.5-fold and 30-fold, respectively, at 99.5% water content (FIG. 5). TEM images clearly show their difference in aggregate morphology, where E-TPAN-OM tends to form regular rod-like shapes, even arranged in two-dimensional planes at a water content of 99.5%. In contrast, Z-TPAN-OM cannot form a regular morphology. The properties of stereoisomers in the solid state were further investigated due to the significant difference in solution state. As shown in fig. 6a, both isomers show red-shifted absorption spectra in the solid state, indicating the formation of a more planar conformation, compared to the E/Z-TPAN-OM isomer in solution. In addition, the E isomer undergoes a large red shift, probably due to its relatively strong donor-acceptor (D-A) structure. Again, there was a significant difference in fluorescence quantum yield for the E/Z-TPAN-OM isomer, with E-TPAN-OM quantum yields as high as 21.8%, while the Z isomer was barely luminescent (FIG. 6b, FIG. 6 c). The fluorescence lifetime of the E isomer is up to 938 picoseconds (ps), which is nearly 4 times that of the Z isomer, further demonstrating higher radiative transition efficiency in E-TPAN-OM. When E/Z-TPAN-OM is cooled to low temperature (CT, 77K), they both emit strong fluorescence, but the colors of the emitted light are green and blue, respectively. The significant enhancement of the fluorescence intensity of E/Z-TPAN-OM at low temperature (CT, 77K) can be attributed to a higher degree of intramolecular motion confinement, thus demonstrating that E/Z-TPAN-OM is a typical luminescent molecule with AIE characteristics (FIG. 6 d).

Example 4 isomerization Process for E/Z-TPAN-OM and application in Lighting-type bioimaging

Since the protons in the methyl and aromatic regions show significant chemical shift differences, the E/Z-TPAN-OM isomerization process was first monitored using nuclear magnetic methods. And (3) placing the quartz tube filled with the deuterated chloroform solution of the E/Z-TPAN-OM under the irradiation of 365nm ultraviolet light at room temperature. As shown in fig. 7a, resonance of characteristic proton of E-TPAN-OM appears in the irradiated Z-TPAN-OM sample (4 region represented as methyl and 3 region represented as aromatic) indicating the occurrence of structural transformation. With its corresponding isomer, a conversion of E-TPAN-OM to the Z isomer is also achieved under uv radiation, where the characteristic proton resonance of the Z isomer in zone 1 and zone 2 occurs. In contrast, the nuclear magnetic spectrum of the sample stored under natural light did not substantially change even after 7 days, indicating the stability of E/Z-TPAN-OM in the state of solution. The isomerization process can also be monitored by HPLC due to the different elution times of E/Z-TPAN-OM. As shown in FIG. 7b, the peaks appearing in zone 5 and zone 6 after UV irradiation can be respectively identified as newly generated E/Z-TPAN-OM because they are located at the same elution position as the original isomer. Prolonged irradiation time results in a sharp decrease in the peak intensity of the E isomer while the peak intensity of the Z isomer increases and vice versa. After 150 minutes of irradiation, the ratio of Z to E isomers was about 1.14, approaching equilibrium. In addition, the fresh peak eluting longer (zone 7) may refer to the halo by-product which is believed to be less polar. Theoretical calculations indicate that the photochemistry of the E/Z-TPAN-OM isomer in solution goes through two decay pathways, intramolecular cyclization and E/Z isomerization. Consistent with the experimental data above, the cyclization pathway in solution is more favorable for the E isomer than for Z. This process is similar to previously reported photocyclization, which typically occurs in AIE systems composed of aromatic rings.

Since E/Z-TPAN-OM can be isomerized to each other, Z-TPAN-OM that does not emit light is expected to be applied to lighting type bio-imaging by being converted into E-isomer that emits light. Human cervical cancer cells (HeLa) were co-incubated with Z-TPAN-OM and fluorescence imaged at 0 min and 2 min after mercury lamp irradiation, respectively. As shown in fig. 8, no significant aggregation occurred outside the cell membrane after incubation with Z isomer. Since Z-TPAN-OM has inherent non-luminescent properties, the fluorescence signal cannot be detected under a microscope. Surprisingly, after 2 minutes of mercury lamp irradiation, HeLa cells were lit by green fluorescent signal. Blue fluorescence may be derived from the photocyclization product accompanying the Z-TPAN-OM isomerization process. If the control cells were not incubated with Z-TPAN-OM, no fluorescence signal was consistently present, indicating that the luminescent fluorescence signal did originate from the isomerization process. On the other hand, when E-TPAN-OM is incubated with living cells, rod-shaped aggregates are rapidly formed in the medium and hardly enter the cells, and green fluorescence comes only from the outer membrane of the cells. The fast aggregation mode of the E-TPAN-OM is very consistent with the phenomenon of TEM picture recording. The dynamic course of illuminated cell imaging using Z-TPAN-OM showed that the green fluorescence intensity reached a plateau after only 2 minutes of irradiation, indicating its ability to respond rapidly in living cells. Furthermore, luminescence imaging can be accurately manipulated on a spatial scale, while fluorescence signals are only generated in the illuminated area. Complementary to previously reported photocyclization activated turn-on fluorescence, the photoisomerization process provides another strategy to achieve super-resolution bioimaging.

EXAMPLE 5 enzymatic reaction of stereoisomers

Since the uptake of E/Z-TPAN-OM and E/Z-TPAN-OH by cells is relatively limited, the inventors further designed E/Z-TPAN-POH modified with phosphate groups to improve its water solubility and to impart its enzymatic responsiveness. The E/Z-TPAN-POH isomers retain the stereo structure of their respective precursors, and the absolute fluorescence quantum yields of E/Z-TPAN-OM, E/Z-TPAN-OH, E/Z-TPAN-POE, Z-TPAN-NM, and Z-TPAN-NMBr are collected by measuring the crystals, respectively. The fluorescence quantum yields of the other isomers in the amorphous state were collected separately by measuring the solid powder and they showed very different fluorescence quantum yields (table 1). Since E/Z-TPAN-POH and its hydrolysate E/Z-TPAN-OH (FIG. 10a) elute on different time scales, ALP catalyzed hydrolysis of E/Z-TPAN-POH can be quantitatively monitored by HPLC. As shown in FIG. 10b, as the reaction proceeded, the peak intensity of Z-TPAN-POH continuously decreased, and the peak intensity of the product accompanied by the same elution time as that of Z-TPAN-OH increased. Also, hydrolysis of E-TPAN-POH resulted in a significant decrease in peak intensity and an increase in E-TPAN-OH production. Importantly, the hydrolysis rate of E-TPAN-POH was nearly 4.2 times faster than that of Z, indicating that structural nuances can lead to large differences in enzyme recognition and reaction (fig. 10 c). In order to understand the binding pattern of E/Z-TPAN-POH to ALP, the inventors conducted molecular affinity studies on the structure of ALP (FIG. 11). The difference in binding energy over three-fold demonstrates that ALP binds with E-TPAN-POH with much greater affinity at its active site than with the Z isomer. The large differences in the enzymatic reactions of stereoisomers are similar to known drugs, for example, cisplatin exhibits significant activity against cancer targets compared to the trans counterpart.

TABLE 1 void volume (V) in crystal packing of solid stereoisomers_V) And fluorescence quantum yield (. PHI.)_F)

ND: there is no detection signal.

Example 6 cytotoxicity of stereoisomers

Inspired by the great difference in enzymatic reactions of ALP by E/Z-TPAN-POH, the inventors further studied them in cancer cytotoxicity. As shown in FIG. 12, E/Z-TPAN-POH showed significant toxicity after 2 hours of incubation with HeLa cells. When the concentration reaches 300. mu.M, the cell survival rate of Z-TPAN-POH is rapidly reduced to 0. In sharp contrast, cellular activity remained nearly 100% for the E isoform. This cytotoxicity is also related to the incubation time. After an extended period of time up to 12 hours, the toxicity of the Z isomer became more pronounced, as 200. mu.M of the Z isomer could completely kill the cells. However, the cellular activity of the E isomer remained above 70% at a concentration of 800. mu.M. The disrupted cell morphology after incubation with Z-TPAN-POH and the intact cell morphology corresponding to E-TPAN-POH also demonstrated strong differences in toxicity, further confirming the large difference in cytotoxicity of the isoforms (FIGS. 13 and 14). This correlation between cytotoxicity and E/Z configuration was demonstrated not only in HeLa cells, but also in a number of other cancer cell lines, including human renal Adenocarcinoma Cells (ACHN), human osteosarcoma cells (HOS, SJSA-1 and drug resistant U2R), and human hepatoma cells (HepG 2). As shown in fig. 12 b-12 f, the cells all showed surprising toxicity after incubation with Z-TPAN-POH, but good biocompatibility with the E isomer, indicating the same general trend for E/Z-TPAN-POH to have different toxicity to cancer cells. The apparent morphological changes of cells that occur ubiquitously in all cell lines after incubation with Z isomers further confirm the fate of cell death.

To investigate the origin of the difference in toxicity, the inventors first tested the toxicity of the enzymatic hydrolysate E/Z-TPAN-OH. After incubation with HeLa cells, the toxicity of E/Z-TPAN-OH is not obviously different. Next, the amount of cell uptake was evaluated, and as shown in FIG. 15, intense fluorescent signals from Z-TPAN-OH incubated HeLa cells appeared almost in every cell unit. However, for E-TPAN-OH, almost no fluorescence signal was observed even after 10 minutes of irradiation under a mercury lamp (FIG. 16). Cells incubated with Z-TPAN-POH at 2220cm^-1The unique raman peak at (which can be attributed to nitrile bond (C ≡ N)) showed stronger intensity than E-TPAN-POH incubated cells (fig. 17). The stronger signals of the Z-TPAN-POH precursor and Z-TPAN-OH product in the HPLC elution profile of the extracted contents of HeLa cells cultured with Z-TPAN-POH further confirm that Z isomer exhibits faster absorption rate and significant anticancer effect compared to E isomer (fig. 18). Also, since Z-TPAN-POH has a slow rate in enzymatic hydrolysis reaction of ALP, its accumulation in cancer cells in a large amount will further cause severe cytotoxicity. The difference in cytotoxicity due to stereoisomers indicates that the toxicity of Z-TPAN-POH results from its faster cellular uptake rate and large accumulation in cancer cells.

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Aggregation-induced emission stereoisomer, application and preparation method thereof

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