CN114478363A - AIE molecule with double-end-group modified sites, multi-module probe, and preparation methods and applications thereof - Google Patents

AIE molecule with double-end-group modified sites, multi-module probe, and preparation methods and applications thereof Download PDF

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CN114478363A
CN114478363A CN202111586492.3A CN202111586492A CN114478363A CN 114478363 A CN114478363 A CN 114478363A CN 202111586492 A CN202111586492 A CN 202111586492A CN 114478363 A CN114478363 A CN 114478363A
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compound
aie
formula
probe
polypeptide
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娄筱叮
夏帆
段冲
胡晶晶
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China University of Geosciences
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Abstract

The invention discloses an AIE molecule with double-end-group modification sites, a multi-module probe, a preparation method and application thereof, wherein the AIE molecule with the double-end-group modification sites has a structure shown as a formula I:
Figure DDA0003428021960000011
the invention further controls the charge ratio of the AIE molecule and the polypeptide AB to prepare a unilateral modified AIE-AB probe; and the unilateral modified AIE-A is prepared by regulating the reaction charge ratio of the AIE molecule and the polypeptide A, and then the AIE-A and the polypeptide B are further reacted to prepare the bilateral modified A-AIE-B probe. The invention also provides two multi-module probes with the same composition and different arrangement modes and the cancer cell killing capacity, and the result shows that the double-side modified A-AIE-B probe has higher cancer cell selectivity, and the probe AIE-AB has higher toxicity.

Description

AIE molecule with double-end-group modified sites, multi-module probe, and preparation methods and applications thereof
Technical Field
The invention relates to the technical field of organic chemical synthesis, in particular to an AIE molecule with double-end-group modification sites and multi-module probes with different arrangement modes prepared by the AIE molecule, and particularly relates to a multi-module probe with the same composition and different arrangement modes and a preparation method of the multi-module probe.
Background
Since neoplastic diseases are seriously threatening human health, the development and improvement of efficient tumor drug probes have been far reaching. The common medicine generally needs to go through a plurality of processes such as blood circulation, tumor enrichment, tissue penetration, cell internalization, medicine release and the like in order to realize tumor treatment, and due to the complex microenvironment of organisms, the traditional diagnosis and treatment preparation has the problems of insufficient enrichment in tumor tissues, low cell internalization efficiency, weak subcellular organelle positioning capacity and the like, so that the exertion of the medicine effect is seriously influenced. Therefore, in order to overcome multiple obstacles in tumor diagnosis and treatment, it is important to modify the diagnosis and treatment preparation to have a series of functions to combat the complex microenvironment of organisms. The modular strategy is a strategy of constructing one or more multifunctional probes by modifying a plurality of functional modules such as a cell selective module, a cell membrane penetrating module, an organelle targeting module and the like on a drug molecule, thereby realizing better treatment effect.
The existing modular probe development strategies mainly include two types: developing a new module or optimizing the function of the module, for example, recognizing the sequence of the protein which acts with enzyme by learning the interaction mechanism of the enzyme and the protein, then artificially synthesizing, then introducing the functional peptide into cells by the aid of the existing functional peptide, and then generating competitive reaction with the enzyme so as to achieve the purpose of regulating the cell behavior. And b, connecting a plurality of functional modules into a molecule through covalent bonds so as to achieve the aim of improving the treatment effect. For example, cell-selective polypeptides are often modified onto drug molecules for increasing tumor cell selectivity; cell membrane penetrating polypeptides are often used to increase the tumor cell internalization efficiency of therapeutic drug probes; therapeutic polypeptides are often modified onto drug molecules for the promotion of mitochondrial dysfunction to enhance therapeutic efficacy. However, in the modified modular probe modified by the above method, whether the interaction between adjacent modules will affect the function of each module has been ignored by researchers, and is always an important consideration in designing multi-module probes. Therefore, the development of fluorescent molecules with double modification sites and therapeutic effects to promote the research and development of modular probes is a technical problem to be solved in the field.
Disclosure of Invention
In order to improve the technical problem, the invention provides a compound with a structure shown in formula I:
Figure BDA0003428021940000021
according to an embodiment of the invention, the compound of formula I is an AIE molecule with a double-ended modification site.
The invention also provides a preparation method of the compound with the structure shown in the formula I, which comprises the step of reacting the compound 4 with sodium azide to prepare the compound with the structure shown in the formula I;
the compound 4 has the following structure:
Figure BDA0003428021940000031
according to the embodiment of the invention, the mass ratio of the sodium azide to the compound 4 is (1-2): 1, illustratively 1:1, 1.5:1, 2: 1.
According to an embodiment of the present invention, the preparation method may be performed in the presence of a solvent such as an organic solvent. For example, the organic solvent may be selected from acetonitrile.
According to an embodiment of the present invention, the preparation method further comprises a step of separating a solid product from the reacted mixture after the reaction is completed. For example, a solid product is obtained by spin-drying the solvent. Further, the preparation method also comprises the step of purifying the product. For example, the purification may be performed using a column chromatography column. Preferably, the eluent for the column chromatography column separation is dichloromethane/methanol ═ (40-60): 1(v/v), illustratively 40:1, 50:1, 60: 1.
Preferably, the synthetic route for the compounds of formula I is as follows:
Figure BDA0003428021940000032
according to an embodiment of the present invention, said compound 4 is prepared by a process comprising: reacting the compound 3 with 1, 6-diiodobutane to obtain a compound 4;
the compound 3 has the following structure:
Figure BDA0003428021940000041
according to an embodiment of the present invention, the above reaction may be carried out in the presence of a solvent such as an organic solvent. For example, the organic solvent may be selected from acetonitrile.
According to an embodiment of the present invention, the reaction ratio of the compound 3 to 1, 6-diiodobutane is (400-600) mg:1mL, exemplary 400mg:1mL, 500mg:1mL, 600mg:1 mL.
According to an embodiment of the present invention, the preparation method further comprises a step of separating a solid product from the reacted mixture after the reaction is completed. For example, a solid product is obtained by spin-drying the solvent. Further, the preparation method also comprises the step of purifying the product. For example, the purification may be performed using a column chromatography column. Preferably, the eluent for the column chromatography column separation is dichloromethane/methanol (40-60): 1(v/v), exemplary 40:1, 50:1, 60: 1.
Preferably, the synthetic route of the compound 4 is as follows:
Figure BDA0003428021940000042
according to an embodiment of the present invention, said compound 3 is prepared by a process comprising: reacting the compound 2 with vinylpyridine to obtain the compound 3;
the compound 2 has the following structure:
Figure BDA0003428021940000051
according to an embodiment of the present invention, the above reaction may be carried out in the presence of a solvent such as an organic solvent. For example, the organic solvent may be selected from N-methylpyrrolidone.
In the reaction of tetraphenylethylene and vinylpyridine according to an embodiment of the present invention, it is preferred to add a base and a catalyst.
Preferably, the amount of the catalyst is 20-40% of the mass of tetraphenylethylene, and is exemplified by 20%, 30% and 40%.
Illustratively, the catalyst may be, for example, at least one of palladium acetate, tri-o-tolylphosphine, and palladium dichloride. Preferably palladium acetate and tri-o-tolylphosphine.
According to an exemplary embodiment of the invention, in the catalyst, the mass ratio of palladium acetate to tri-o-tolylphosphine is (1-2): 1, illustratively 1:1, 1.5:1, 2: 1.
Preferably, the amount of the base is 40-60% by mass of tetraphenylethylene, and is exemplified by 40%, 50% and 60%.
Preferably, the base is selected from one, two or more of potassium carbonate, sodium tert-butoxide, potassium phosphate and sodium acetate.
According to an embodiment of the present invention, the preparation method further comprises a step of separating a solid product from the reacted mixture after the reaction is completed. For example, a solid product is obtained by suction filtration with water. Further, the preparation method also comprises the step of purifying the product. For example, the purification may be performed using a column chromatography column. Preferably, the eluent for the column chromatography column separation is dichloromethane/methanol ═ (40-60): 1(v/v), illustratively 40:1, 50:1, 60: 1.
Preferably, the synthetic route of the compound 3 is as follows:
Figure BDA0003428021940000061
according to an embodiment of the present invention, the compound 2 is prepared by a process comprising: reacting the compound 1 with metal to obtain a compound 2;
the compound 1 has the following structure:
Figure BDA0003428021940000062
preferably, the mass ratio of the compound 1 to the metal is (1-2): 1, illustratively 1:1, 1.5:1, 2: 1.
Preferably, the metal is one or more of zinc, iron, aluminium and manganese.
Preferably, the reaction is carried out in the presence of a catalyst. Preferably, the reaction ratio of benzophenone to catalyst is (0.5-2) g:1mL, exemplary 0.5g:1mL, 1g:1mL, 2g:1 mL. For example, the catalyst is selected from titanium tetrachloride.
According to an embodiment of the present invention, the above reaction may be carried out in the presence of a solvent such as an organic solvent. For example, the organic solvent may be selected from tetrahydrofuran.
According to an embodiment of the present invention, the reaction may be carried out at a temperature of 20 ℃ to 100 ℃.
According to an embodiment of the present invention, the preparation method further comprises a step of separating a crude product after the reaction is completed. For example, the mixture after the reaction is extracted with an extractant, the filtrate is collected, and the solvent is dried by spinning to obtain the crude product. Preferably, the extractant may be a dichloromethane/water mixed solvent. Further, the preparation method also comprises the step of purifying the crude product to obtain the compound 2. For example, the purification can be performed using column chromatography to give compound 2. Preferably, the eluent for the column chromatography column separation is petroleum ether/ethyl acetate ═ (40-60): 1(v/v), illustratively 40:1, 50:1, 60: 1.
Preferably, the synthetic route of the compound 2 is as follows:
Figure BDA0003428021940000071
according to an embodiment of the present invention, the preparation method of the compound having the structure shown in formula I comprises the following steps:
s101, synthesizing a compound 2 from a compound 1, titanium tetrachloride and zinc powder by taking tetrahydrofuran as a solvent; the structural formula of the compound 2 is as follows:
Figure BDA0003428021940000072
s102, synthesizing a compound 3 by using potassium carbonate as an alkali, palladium acetate and tri-o-tolylphosphine as catalysts and N-methylpyrrolidone as a solvent in the compound 2 and vinyl pyridine; the structural formula of the compound 3 is as follows:
Figure BDA0003428021940000073
s103, synthesizing a compound 4 from a compound 3, 1, 6-diiodobutane by using acetonitrile as a solvent; the structural formula of the compound 4 is as follows:
s104, synthesizing a final product, namely a compound in a formula I from a compound 4, sodium azide and acetonitrile serving as a solvent, and separating and purifying by using column chromatography to obtain a pure final product, namely the compound in the formula I;
the reaction scheme of the compound of formula I is shown in FIG. 1.
The invention also provides application of the compound with the structure shown in the formula I in preparation of a fluorescent probe.
The invention also provides a compound with a structure shown in a formula II or a formula III:
Figure BDA0003428021940000081
the invention also provides a preparation method of the compound with the structure shown in the formula II or the formula III, which comprises the following steps:
reacting a compound with a structure shown in a formula I with a polypeptide A and a polypeptide B to prepare a compound with a structure shown in a formula II;
the structure of the polypeptide A is as follows:
Figure BDA0003428021940000082
the structure of polypeptide B is:
Figure BDA0003428021940000091
or reacting the compound with the structure shown in the formula I with the polypeptide AB to prepare a compound with the structure shown in the formula III;
the structure of the polypeptide AB is as follows:
Figure BDA0003428021940000092
according to an embodiment of the invention, the molar ratio of the compound of the structure represented by formula I to the polypeptide AB is (4-6): 1, illustratively 4:1, 5:1, 6: 1.
According to an embodiment of the invention, the molar ratio of the compound having the structure shown in formula I to the polypeptide A is (4-6): 1, and is exemplarily 4:1, 5:1, 6: 1.
According to an embodiment of the invention, the molar ratio of the compound of the structure represented by formula I to the polypeptide B is (0.5-2): 1, exemplary 0.5:1, 1:1, 2: 1.
According to an embodiment of the present invention, the reaction may be a click reaction through an azido-alkynyl group.
According to an embodiment of the present invention, the reaction may be performed in a solvent selected from one or both of DMSO, water; a mixed solvent of DMSO and water is preferable.
According to an embodiment of the invention, the mixing volume ratio of DMSO to water in the mixed solvent is (0.5-2): 1, and exemplary is 0.5:1, 1:1, 2: 1.
According to an embodiment of the present invention, the reaction may be carried out in the presence of a catalyst. For example, under the catalytic action of sodium ascorbate and cuprous bromide.
Preferably, the mass ratio of the compound with the structure shown in the formula I to the sodium ascorbate is (10-30): 1, and exemplary is 10:1, 20:1, 25:1 and 30: 1.
Preferably, the mass ratio of the compound with the structure shown in the formula I to the cuprous bromide is (40-60): 1, and is exemplarily 40:1, 50:1 or 60: 1.
According to an embodiment of the present invention, the preparation method further comprises a step of purifying the reaction product after the reaction is completed. For example, the purification may be performed by HPLC. Preferably, the separation is performed by reversed phase high performance liquid chromatography gradient elution.
According to an embodiment of the present invention, the method for preparing the compound having the structure represented by formula ii or formula iii comprises the steps of:
the first scheme is as follows:
s201, adding the polypeptide A and a compound with a structure shown in a formula I into a mixed solution of DMSO and water, adding sodium ascorbate and cuprous bromide, and stirring to react to obtain an intermediate product AIE-A;
s202, adding the polypeptide B and the intermediate product AIE-A into a mixed solution of DMSO and water, adding sodium ascorbate and cuprous bromide, and stirring to react to obtain a compound with a structure shown in a formula II; wherein, the structure of the polypeptide A is as follows:
Figure BDA0003428021940000101
the structure of polypeptide B is:
Figure BDA0003428021940000102
scheme II: adding the polypeptide AB and a compound with a structure shown in a formula I into a mixed solution of DMSO and water, adding sodium ascorbate and cuprous bromide, and stirring to react to obtain a compound with a structure shown in a formula III;
the structure of the polypeptide AB is as follows:
Figure BDA0003428021940000103
according to the invention, by selecting the polypeptide sequence RGDGPLGVRGRKKRKVRRR (polypeptide A) and the sequence HLAHLAHHLAHLAH (polypeptide B) with good cancer cell selective uptake and mitochondria interference capability, the polypeptide A and the polypeptide B are respectively modified at two ends of the AIE molecule, the selectivity and the killing capability of a probe to cancer cells can be greatly increased through the synergistic effect of the polypeptide A and the polypeptide B, and the damage of drug molecules to normal cells is reduced.
The invention also provides application of the compound with the structure shown as the formula II or the formula III as a fluorescent probe. Preferably, the fluorescent probe has a mitochondrial targeting effect and high-efficiency active oxygen generation capability, and can be used for tumor cell-specific targeting treatment. The invention also provides a compound with the structure shown in the formula II or the formula III for detecting integrin (alpha)vβ3) The use of (1).
The invention also provides a fluorescent probe which contains a compound with a structure shown as a formula II or a formula III.
The invention also provides a method for detecting the integrin by using the fluorescent probe, which comprises the step of mixing the integrin or a target object to be detected containing the integrin with the fluorescent probe.
According to an embodiment of the present invention, the method further comprises mixing a sample to be tested containing the integrin or a target substance to be tested containing the integrin with the fluorescent probe, measuring the luminous intensity of the mixed solution, and calculating the concentration of the integrin. Wherein the concentration of the fluorescent probe in the sample to be tested is 0.005-2mM, and 1mM is exemplified.
Preferably, the concentration of the target to be detected is calculated by substituting into a concentration-dependent standard curve of the target to be detected.
According to an embodiment of the present invention, the method further comprises preparing a solution of the fluorescent probe and a solution of integrin or a target to be tested containing integrin.
According to an embodiment of the invention, the method comprises in particular the steps of:
1) dissolving a compound with a structure shown as a formula II or a formula III in a mixed solvent of DMSO/water to obtain a solution of the fluorescent probe;
2) preparing integrin with different concentrations or a solution of a target object to be detected containing the integrin;
3) drawing a concentration-dependent standard curve of the integrin or the target object to be detected containing the integrin;
preferably, the step of plotting a concentration-dependent standard curve of said integrin is as follows: taking a fluorescent probe solution, taking one group of solutions as a blank sample, adding an integrin solution with a known concentration or a solution of a target object to be detected containing the integrin into the other groups of solutions respectively, mixing, incubating, measuring the luminous intensity of the mixed solution, taking the fluorescent intensity y of each group of mixed solution after adding the target object solution to be detected as a vertical coordinate, taking the concentration x of the target object solution to be detected as a horizontal coordinate, and making a concentration-dependent standard curve of the integrin or the target object to be detected containing the integrin;
4) detecting the concentration of the integrin or the target object to be detected containing the integrin;
preferably, the concentration of the target to be detected is specifically measured by the following steps: mixing the fluorescent probe solution with the integrin solution with unknown concentration or the solution of the target substance to be detected containing the integrin, incubating, measuring the luminous intensity of the mixed solution, and substituting the luminous intensity into the concentration-dependent standard curve of the integrin drawn in the step 3) to obtain the concentration of the integrin.
According to an embodiment of the present invention, the integrin or the target solution to be tested containing the integrin in step 2) is obtained by mixing the integrin or the target solution to be tested containing the integrin with a buffer solution;
preferably, the buffer solution can be selected from buffer solutions with pH values of 7-11; for example, the buffer solution may be selected from the group consisting of HEPES aqueous solution, MES buffer solution, Tris-HCl buffer, NaOH-H3BO3Buffer solution, NaCO3-NaHCO3Buffer solutions, phosphate buffer solutions, and the like;
according to an exemplary embodiment of the invention, the buffer solution is selected from Phosphate Buffered Saline (PBS) at pH 7.4;
according to an exemplary embodiment of the present invention, the integrin solution is obtained by mixing integrin with PBS solution; the concentration of the integrin is more than 0 and not more than 40. mu.g/mL, preferably 1 to 40. mu.g/mL.
According to an embodiment of the present invention, the temperature of the incubation in step 4) may be selected from 30 to 50 ℃, preferably the temperature of the incubation is selected from 35 to 40 ℃, according to an exemplary embodiment of the present invention, the temperature of the incubation is 37 ℃;
the incubation time can be 1-10 min, and preferably, the incubation time is 20-40 min; according to an exemplary embodiment of the invention, the incubation time is 30 min;
preferably, the concentrations and volumes of the fluorescent probe solution and the integrin solution of different concentrations may be mixed in any ratio.
The invention also provides a kit which comprises the fluorescent probe.
The invention also provides a biosensor which comprises the fluorescent probe.
The invention also provides the application of the fluorescent probe, the kit and/or the biosensor in detecting the integrin.
The invention has the beneficial effects that:
existing modular probes do not completely overcome the complex environment of the organism and are thus directed to clinical applications, and the proximity interaction and arrangement of the modules has been overlooked. In view of the above, the present invention explores the influence of the arrangement mode on the intracellular performance of the multi-module probe based on the existing modular probe development strategy to develop the multi-module probe with the same composition but different arrangement modes. And by comparing the intracellular performance of probes in different arrangements, it was investigated whether the function of the responding module was affected by the proximity module. Meanwhile, the reaction activity of the double-end-group modification sites often directly influences the synthesis yield of target molecules, and only if the end-group modification groups are properly selected, the generation of target products can be controlled by controlling the reaction feeding ratio, so that the generation of byproducts is prevented. Specifically, the method comprises the following steps:
(1) the AIE molecule synthesized by the invention has the advantages of fluorescence emission in a near infrared region, high-efficiency active oxygen generation capability (figure 5) and the like.
(2) The AIE molecule with the double-end-group modification site can prevent the generation of byproducts by controlling the reaction charge ratio and the reaction time so that the polypeptide is modified at only one end of the AIE molecule and the other end is exposed.
(3) The multi-modular probe A-AIE-B of the invention has good affinity to integrin, potential for tumor cell specific targeting and good biosafety (FIG. 11, FIG. 14), while the AIE-AB probe has a higher helix (FIG. 13), thus showing good cancer cell killing effect (FIG. 15). The probe A-AIE-B of the invention has higher selectivity on tumor cells, and the probe AIE-AB has higher cytotoxicity. Therefore, the arrangement mode of the modules can be adjusted to obtain multi-module probes with different functional requirements.
Drawings
FIG. 1 is a flow chart of the preparation of AIE molecules with double-ended modification sites.
FIG. 2 is a nuclear magnetic hydrogen spectrum characterization of AIE molecules with double-terminal modification sites.
FIG. 3 is a nuclear magnetic carbon spectrum characterization of AIE molecules with double-ended modification sites.
Figure 4 is a mass spectral characterization of AIE molecules with double-ended modification sites.
FIG. 5 is a graph showing the active oxygen generating capacity of AIE molecules having double-terminal modification sites.
FIG. 6 is a flow chart of the preparation of AIE-AB.
FIG. 7 is a mass spectrum characterization of AIE-AB.
FIG. 8 is a flow chart of the preparation of AIE-A.
FIG. 9 is a flow chart of the preparation of A-AIE-B.
FIG. 10 is a mass spectrum characterization of A-AIE-B.
FIG. 11 (A) and (B) are the fluorescence change curve and affinity quantification of the double-sided modified multi-modular probe A-AIE-B bound to integrin, respectively.
FIG. 12 (A) and (B) show fluorescence change curves and affinity quantification of the single-edge modified multi-modular probe AIE-AB bound to integrin, respectively.
FIG. 13 is a circular dichromatic representation of multi-modular probes in different arrangements.
FIG. 14 (A) and (B) show the cytotoxicity of probes AIE-AB and A-AIE-B, respectively, in the absence of light.
FIG. 15 (A) and (B) show the cytotoxicity of probes AIE-AB and A-AIE-B, respectively, under light irradiation.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
In the following examples of the present invention, polypeptide A, polypeptide B and polypeptide AB were purchased from Gill Biochemical (Shanghai) Co., Ltd.
Example 1
Referring to fig. 1, a method for synthesizing an AIE molecule with double modification sites comprises the following steps:
step S101, weighing 2g of benzophenone, 2mL of titanium tetrachloride and 2g of zinc powder, dissolving the benzophenone, the titanium tetrachloride and the zinc powder in 100mL of tetrahydrofuran, stirring at 70 ℃, condensing and refluxing for 24h, extracting with a dichloromethane/water (volume ratio is 1: 1) mixed solvent after the reaction is finished, collecting an organic phase, spin-drying the solvent with a rotary evaporator, purifying a crude product with a silica gel column by using petroleum ether/ethyl acetate 50/1 as an eluent to obtain tetraphenyl ethylene, wherein the yield is 34.3% (wherein: the yield is (actual yield/theoretical yield) × 100%);
step S102, weighing 1g of tetraphenylethylene, 200mg of palladium acetate, 500mg of potassium carbonate, 100mg of tri-o-tolylphosphine and 1mL of vinylpyridine, dissolving the mixture in 100mL of N-methylpyrrolidone, stirring, condensing and refluxing for 24 hours, cooling after the reaction is finished, adding 300mL of water, and performing suction filtration to obtain a solid crude product, wherein the crude product is subjected to silica gel column by using dichloromethane/ethyl acetate-50/1 as an eluent to obtain TPE-Py, and the yield is 79.4%;
step S103, weighing 500mg of TPE-Py and 1mL of 1, 6-diiodobutane, dissolving the TPE-Py and 1mL of 1, 6-diiodobutane in 100mL of acetonitrile, stirring, condensing and refluxing for 24h, and then carrying out rotary evaporation on a solvent by using a rotary evaporator to obtain a solid crude product, wherein the crude product is subjected to silica gel column by using dichloromethane/ethanol-50/1 as an eluent to obtain TPE-I, and the yield is 58.4%;
step S104, weighing 300mg of TPE-I and 200mg of sodium azide, dissolving in 100mL of acetonitrile, stirring, condensing, refluxing for 24h, and then carrying out rotary evaporation on the solvent by using a rotary evaporator to obtain a solid crude product, wherein the crude product is subjected to silica gel column by using dichloromethane/methanol-50/1 as an eluent to obtain AIE, and the yield is 77.9%.
The AIE molecule obtained in this example has a hydrogen spectrum characterization as shown in fig. 2, a carbon spectrum characterization as shown in fig. 3, and a mass spectrum characterization as shown in fig. 4. The above characterization demonstrated the success of the preparation of the AIE molecule.
Weighing 1.0mol of AIE molecular solid prepared in the embodiment, and dissolving the AIE molecular solid in 1mL of DMSO to obtain 1mmol/L AIE mother solution; weighing 5.0mol of active oxygen detection reagent 9, 10-anthryl-bis (methylene) dipropanedioic acid (ABDA) and dissolving in 1mL of DMSO to obtain 5mmol/L ABDA mother liquor; adding 10 μ L of the above ABDA mother liquor into 1mL of deionized water, adding 5 μ L of the above AIE mother liquor, mixing, recording the ultraviolet absorption spectrum of ABDA at 300-500nm with an ultraviolet spectrophotometer, and recording the ultraviolet absorption spectrum once every 30s of illumination.
As a result, as shown in FIG. 5, the UV absorption of ABDA under white light irradiation hardly changed without adding AIE molecules; whereas, after the addition of AIE molecules, the absorption of ABDA rapidly decreased within 4min under white light illumination. This indicates that: the AIE molecule has excellent active oxygen generating ability.
Example 2
Referring to fig. 6, the method for preparing the multi-modular probe AIE-AB includes the following steps:
weighing 50mg of AIE molecule and polypeptide AB in a molar ratio of 5:1, adding into DMSO and water at a volume ratio of 1:1, adding 2mg of sodium ascorbate and 1mg of cuprous bromide, fully stirring at room temperature for 24h, after the reaction is finished, carrying out reverse high performance liquid chromatography gradient elution on the reaction solution (the method is that a sample is dissolved in aqueous solution or acetonitrile, applied to a Kromasil C18 column (10 mu m, 250 multiplied by 4.6mm) from Teknokroma, and eluted at the speed of 2mL/min, the gradient is from 20% to 100% (0.01min 20%, 20min 40%, 25min 60%, 30min 90%, 50min 100% of solvent B, wherein the solvent A is water (0.1% TFA solution), the solvent B is acetonitrile (0.1% TFA solution)), separating and purifying to obtain the AIE-AB, and the yield is 27.4%.
FIG. 7 is a representation of the mass spectrum of the AIE-AB molecule prepared in this example, showing the success of the preparation of the AIE-AB molecule.
Example 3
Referring to fig. 8, the method for preparing the multi-module probe AIE-a includes the steps of:
weighing 50mg of AIE molecule and polypeptide A in a molar charge ratio of 5:1, adding into DMSO and water at a volume ratio of 1:1, adding 2mg of sodium ascorbate and 1mg of cuprous bromide, fully stirring at room temperature for 24h, after the reaction is finished, carrying out reverse high performance liquid chromatography gradient elution on the reaction solution (the method is that a sample is dissolved in aqueous solution or acetonitrile, applied to a Kromasil C18 column (10 mu m, 250 multiplied by 4.6mm) from Teknokroma, and eluted at the speed of 2mL/min, the gradient is from 20% to 100% (0.01min 20%, 20min 40%, 25min 60%, 30min 90%, 50min 100% of solvent B, wherein the solvent A is water (0.1% TFA solution), and the solvent B is acetonitrile (0.1% TFA solution)), separating and purifying to obtain the AIE-A, and the yield is 34.7%.
Example 4
The preparation method of the multi-module probe A-AIE-B comprises the following steps:
referring to FIG. 9,10 mg of AIE-A prepared in example 3 and polypeptide B were mixed at a molar feed ratio of 1:1, adding the mixture into a reactor with a volume ratio of 1:1, adding 2mg of sodium ascorbate and 1mg of cuprous bromide into a mixed solvent of DMSO and water, stirring at room temperature for 24h, and performing reverse phase high performance liquid chromatography gradient elution on the reaction solution after the reaction is finished (the method is that a sample is dissolved in aqueous solution or acetonitrile, applied to a Kromasil C18 column (10 mu m, 250 multiplied by 4.6mm) from Teknokroma and eluted at the speed of 2mL/min, wherein the gradient is from 20% to 100% (0.01min 20%, 20min 40%, 25min 60%, 30min 90%, 50min 100% of solvent B, wherein the solvent A is water (0.1% TFA solution) and the solvent B is acetonitrile (0.1% TFA solution)), separating and purifying to obtain A-AIE-B, and the yield is 48.3%.
FIG. 10 is a mass spectrum characterization chart of A-AIE-B prepared in this example, and the results show the success of the preparation of A-AIE-B molecules.
Example 5
Detection of affinity of the probe to integrin: with DMSO/water ═ 1:1 (A-AIE-B) to obtain 1mM of a probe stock solution (AIE, AIE-AB, A-AIE-B) which was stored in a refrigerator at-20 ℃.
mu.L of each probe stock solution (AIE-AB, A-AIE-B) was added to 1mL of PBS (50mM, pH 7.4), and integrin (. alpha.) was added to the PBS at different concentrationsνβ3) (0, 10. mu.g/mL, 20. mu.g/mL, 30. mu.g/mL, 40. mu.g/mL), incubated at 37 ℃ for 0.5h, and the fluorescence of the mixed solution was recorded by detection with a fluorescence spectrometer.
As shown in FIGS. 11 and 12, the affinities of the two probes for the integrin were calculated from the slopes of the fitted curves, respectively. Wherein: the affinity of the AIE-AB probe for integrin was 0.020 and the affinity of the A-AIE-B probe for integrin was 0.057. This indicates that: the A-AIE-AB probe has higher potential tumor selectivity compared with the AIE-AB probe.
And (3) detecting the circular dichroism spectrum of the probe: 50 μ L of each of the above probe stocks (AIE, AIE-AB, a-AIE-B) was added to a 1mL PBS solution (50mM, pH 7.4)/trifluoroethanol 1: the spectrum of the mixed solution was recorded in 1 (volume ratio) of the mixed solvent by a circular dichroism spectroscopy, and the result is shown in fig. 13. As can be seen from the figure, probe AIE-AB showed higher negative absorptions at 208nm and 222nm than probe A-AIE-B. This indicates that: probe AIE-AB has a higher alpha-helix ratio than probe A-AIE-B, and thus has greater therapeutic potential for cancer cells.
Biological safety and tumor cytotoxicity detection of the probe: culturing HaLa cells for more than three generations in a cell culture medium (10% fetal calf serum, 1% penicillin, 1% streptomycin) in a 37 ℃ oven, digesting the cells with trypsin, centrifuging, discarding the supernatant, diluting the cells to 8000,10000/100. mu.L, adding into a 96-well plate, culturing for 24h, adding (0, 2.5. mu.M, 5. mu.M, 10. mu.M, 15. mu.M, 20. mu.M) probes with different concentrations into each well, culturing for 24h, removing the culture medium, adding (0, 2.5. mu.M, 5. mu.M, 10. mu.M, 15. mu.M, 20. mu.M) probes into each well, and culturing for 24hCulturing 10 μ L of 1mM MTT and 100 μ L of fresh culture medium for 4 hr, sucking out culture medium, adding 100 μ L DMSO into each well, shaking on shaker for 10min, detecting and recording absorbance value at 570nm of each well with microplate reader, and using formula (A)S/A0X 100%) the cell viability of each group of samples was calculated.
The results are shown in FIG. 14, where the cell viability was above 80% for both probes under non-light conditions. This indicates that: the probes of the invention have low toxicity and good biological safety. At the same time, as shown in FIG. 15, the concentration is 200mW/cm2Under white light illumination, the cell viability was 29% when 20. mu.M of AIE-AB probe was added, and 60% when 20. mu.M of A-AIE-B probe was added. This indicates that: the AIE-AB probe has higher potential tumor treatment effect than the A-AIE-B probe. Thus, it was demonstrated that probe A-AIE-B was more selective for tumor cells, while probe AIE-AB was more cytotoxic. Therefore, the arrangement mode of the modules can be adjusted to obtain multi-module probes with different functional requirements.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A compound having the structure of formula I:
Figure FDA0003428021930000011
2. the preparation method of the compound with the structure shown in the formula I in the claim 1 is characterized by comprising the steps of reacting a compound 4 with sodium azide to prepare the compound with the structure shown in the formula I;
the compound 4 has the following structure:
Figure FDA0003428021930000012
the mass ratio of the sodium azide to the compound 4 is (1-2): 1.
3. the method of claim 2, wherein compound 4 is prepared by a process comprising: reacting the compound 3 with 1, 6-diiodobutane to obtain a compound 4;
the compound 3 has the following structure:
Figure FDA0003428021930000021
the reaction ratio of the compound 3 to 1, 6-diiodobutane is (400-600) mg:1 mL;
the compound 3 is prepared by the following method, comprising the following steps: reacting the compound 2 with vinylpyridine to obtain the compound 3;
the compound 2 has the following structure:
Figure FDA0003428021930000022
the compound 2 is prepared by the following method, comprising the following steps: reacting the compound 1 with metal to obtain a compound 2;
the compound 1 has the following structure:
Figure FDA0003428021930000023
the mass ratio of the compound 1 to the metal is (1-2): 1.
4. use of a compound of formula I according to claim 1 and/or a compound of formula I prepared by the preparation method according to claim 2 or 3 for the preparation of a fluorescent probe.
5. A compound having the structure of formula II or III:
Figure FDA0003428021930000031
6. a process for preparing a compound of formula ii or iii according to claim 5, comprising:
reacting a compound with a structure shown in a formula I with a polypeptide A and a polypeptide B to prepare a compound with a structure shown in a formula II;
the structure of the polypeptide A is as follows:
Figure FDA0003428021930000032
the structure of polypeptide B is:
Figure FDA0003428021930000033
or reacting the compound with the structure shown in the formula I with the polypeptide AB to prepare a compound with the structure shown in the formula III;
the structure of the polypeptide AB is as follows:
Figure FDA0003428021930000041
the molar ratio of the compound with the structure shown in the formula I to the polypeptide AB is (4-6) to 1;
the molar ratio of the compound with the structure shown in the formula I to the polypeptide A is (4-6): 1;
the molar ratio of the compound with the structure shown in the formula I to the polypeptide B is (0.5-2): 1.
7. Use of a compound of formula II or III according to claim 5 and/or a compound of formula II or III prepared by the preparation method according to claim 6 as a fluorescent probe.
8. A fluorescent probe comprising a compound of formula ii or iii according to claim 5 and/or a compound of formula ii or iii prepared by the method of claim 6.
9. A kit comprising the fluorescent probe of claim 8.
10. A biosensor comprising the fluorescent probe according to claim 8.
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