CN113912483A - Squaric acid derivative, maleic anhydride derivative, maleimide biological coupling material, preparation method and application thereof - Google Patents

Squaric acid derivative, maleic anhydride derivative, maleimide biological coupling material, preparation method and application thereof Download PDF

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CN113912483A
CN113912483A CN202110845665.2A CN202110845665A CN113912483A CN 113912483 A CN113912483 A CN 113912483A CN 202110845665 A CN202110845665 A CN 202110845665A CN 113912483 A CN113912483 A CN 113912483A
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aromatic hydrocarbon
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maleic anhydride
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唐本忠
王志明
李银
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South China University of Technology SCUT
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Abstract

The invention discloses a squaric acid derivative, a maleic anhydride derivative, a maleimide biological coupling material, and a preparation method and application thereof. The method comprises the steps of acylating and chlorinating by thionyl chloride and squaric acid, and then carrying out Friedel-crafts acylation reaction on the chlorinated thionyl chloride and an aromatic compound to obtain a squaric acid derivative; then maleic anhydride derivatives are generated under the induction of ultraviolet light, and the compounds can perform specific bioconjugation with aliphatic primary amine compounds to generate maleimide bioconjugation products with aggregation-induced emission properties. The method of the invention is a novel biological coupling method which can generate the aggregation-induced emission material with high efficiency. The method can modify natural biomolecules, drug molecules and natural proteins, endow the natural biomolecules, drug molecules and natural proteins with aggregation-induced emission properties, overcome the defect that the raw materials have no fluorescence or quenching caused by aggregation, and have wide prospects in biological applications.

Description

Squaric acid derivative, maleic anhydride derivative, maleimide biological coupling material, preparation method and application thereof
Technical Field
The invention belongs to the field of biomaterial synthesis, and particularly relates to a squaric acid derivative, a maleic anhydride derivative, a maleimide bioconjugate material, and a preparation method and application thereof.
Background
Bioconjugation refers to the attachment of functional groups or molecules (such as fluorescent groups, targeting groups, drug molecules, etc.) to biomolecules through covalent bonds, thereby imparting fluorescence, targeting, or therapeutic functions to the biomolecules, thereby promoting the rapid development of material science and biomedicine. There are many potential biological studies in nature that require bioconjugation, such as natural polysaccharides, proteins, lipids, nucleic acids, synthetic biofunctional polymers, biocompatible polyethylene glycols (PEGs), colloidal particles, even living organisms, etc., which typically have one or more functional groups including amino, carboxyl, sulfhydryl and hydroxyl groups for conjugation, with high efficiency of bioconjugation.
Researchers have also developed a number of bioconjugation reactions including (1) functional-COOH and primary amine biomolecules (-NH)2) Or has (-NH)2) The functional molecule and the biomolecule (-COOH) are coupled, and (2) the functional molecule and the biomolecule (-NH) with aldehyde group (-CHO)2) Coupling, (3) functional molecules and biomolecules with isothiocyanate (-NCS) (-NH)2) Coupling, (4) boric acid solution (-B (OH)2) And biomolecules containing glycol (- (OH)2) (5) coupling of functional molecules containing maleimide and biomolecules containing thiol groups (-SH), (6) azide-alkyne cycloaddition reaction, coupling reaction between azide-functionalized functional molecules and biomolecules with alkyne or functional molecules with alkyne moieties and biomolecules with azide, (7) metal-free catalyzed coupling reaction of functional molecules containing active alkyne groups with functional groups amine and thiol groups of biomolecules (Liu H, Xiong L H, Kwok R T K, et al]Advanced Optical Materials,2020,8(14): 2000162). However, the traditional coupling methods have the problems of low reaction activity, easy hydrolysis and instability of products, complex pre-modification, toxic metal catalysis or difficult synthesis of raw materials and the like, so that the vigorous development is limited to a certain extent, and therefore, the invention of a high-efficiency spontaneous and mild biological coupling reaction is necessary.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a squaric acid derivative, a maleic anhydride derivative, a maleimide biological coupling material, a preparation method and an application thereof.
The invention aims to provide a maleimide biological coupling fluorescent material with aggregation-induced emission performance, which is generated by specifically coupling a light-induced squaraine derivative and primary amine.
It is still another object of the present invention to provide a method for synthesizing the squaraine derivative having the property of enhancing photoinduced fluorescence.
The invention also aims to provide the preparation method of the maleimide bioconjugate material, which has the advantages of controllable time, reliable chemical selectivity, mild conditions, high reaction efficiency and stable product.
It is still another object of the present invention to provide a method for synthesizing the maleic anhydride derivative capable of performing efficient bioconjugation.
The invention also aims to provide application of the maleimide bioconjugate material.
The purpose of the invention is realized by at least one of the following technical solutions.
A kind of squaric acid derivatives, the structural general formula is shown as following formula I:
Figure BDA0003180452080000021
Figure BDA0003180452080000031
wherein R is2And R3Independently is one of aromatic hydrocarbon and aromatic hydrocarbon derivative groups.
Preferably, the squaraine derivative has a property of light-induced fluorescence enhancement.
Preferably, R is2And R3The aromatic hydrocarbon and aromatic hydrocarbon derivative group of (a) is a phenyl group, naphthyl group, tetraphenylvinyl group, thiophene ring, pyridine ring, furan ring, 4-methoxyphenyl group, diethylaminophenyl group, dimethylaminophenyl group, carbazolylphenyl group, carbazolyl group, phenothiazinyl group, phenoxazinyl group, bithiophenyl group, thienocyclopentadienyl group, 9, 10-dihydro-9, 9-dimethylazinyl group, 9, 10-dihydro-9, 9-diphenylacridinyl group, 10-H-spiro [ acridine-9, 9' -fluorene]One of a phenyl group, a diphenylamino group, a triphenylamino group, a diphenylaminothiophene group, a bithiophene group, a fused thiophene group, a thienocyclopentadienyl group, a naphthylaminophenyl group, or a bipyridine amino group.
The structural general formula of the maleic anhydride derivative is shown as the following formula II:
Figure BDA0003180452080000032
wherein R is2And R3Independently is one of aromatic hydrocarbon and aromatic hydrocarbon derivative groups.
Preferably, the maleic anhydride derivative has high-efficiency bioconjugation properties.
Preferably, R is2And R3The aromatic hydrocarbon and aromatic hydrocarbon derivative group of (a) is a phenyl group, naphthyl group, tetraphenylvinyl group, thiophene ring, pyridine ring, furan ring, 4-methoxyphenyl group, diethylaminophenyl group, dimethylaminophenyl group, carbazolylphenyl group, carbazolyl group, phenothiazinyl group, phenoxazinyl group, bithiophenyl group, thienocyclopentadienyl group, 9, 10-dihydro-9, 9-dimethylazinyl group, 9, 10-dihydro-9, 9-diphenylacridinyl group, 10-H-spiro [ acridine-9, 9' -fluorene]One of a phenyl group, a diphenylamino group, a triphenylamino group, a diphenylaminothiophene group, a bithiophene group, a fused thiophene group, a thienocyclopentadienyl group, a naphthylaminophenyl group, or a bipyridine amino group.
The structural general formula of the maleimide biological coupling material is shown as the following formula III:
Figure BDA0003180452080000041
wherein R is1Independently is one of aliphatic hydrocarbon, aromatic hydrocarbon, aliphatic hydrocarbon derivative group and aromatic hydrocarbon derivative group; r2And R3Independently is one of aromatic hydrocarbon and aromatic hydrocarbon derivative groups.
Preferably, the maleimide bioconjugate material has a fluorescence material with aggregation-induced emission properties.
Preferably, said R is1The aliphatic hydrocarbon of (a) is a linear or branched alkyl group; the R is1The aromatic hydrocarbon group and aromatic hydrocarbon derivative group of (a) is phenyl, naphthyl, tetraphenylvinyl, thiophene ring, pyridine ring, furan ring, 4-methoxyphenyl, carbazolylphenyl, dimethylaminophenyl, triphenylamine group, diphenylamino, phenothiazine phenyl, phenoxazinylphenyl, bithiophene group, thienocyclopentadienyl;
further preferably, R is1The aliphatic hydrocarbon and aliphatic hydrocarbon derivative groups of (a) are methyl, ethyl, propyl, butyl, isobutyl, tert-butyl, cyclohexyl, lysine derivatives;
more preferably, R is1Is ethyl, butyl, hexyl, phenyl, lysine derivatives and4-methoxyphenyl group.
Preferably, R is2And R3The aromatic hydrocarbon and aromatic hydrocarbon derivative group of (a) is a phenyl group, naphthyl group, tetraphenylvinyl group, thiophene ring, pyridine ring, furan ring, 4-methoxyphenyl group, diethylaminophenyl group, dimethylaminophenyl group, carbazolylphenyl group, carbazolyl group, phenothiazinyl group, phenoxazinyl group, bithiophenyl group, thienocyclopentadienyl group, 9, 10-dihydro-9, 9-dimethylazinyl group, 9, 10-dihydro-9, 9-diphenylacridinyl group, 10-H-spiro [ acridine-9, 9' -fluorene]One of a phenyl group, a diphenylamino group, a triphenylamino group, a diphenylaminothiophene group, a bithiophene group, a fused thiophene group, a thienocyclopentadienyl group, a naphthylaminophenyl group, or a bipyridine amino group.
Further preferably, R is2And R3Is phenyl, 4-methoxyphenyl, carbazole phenyl, triphenylamine, phenothiazine phenyl, phenoxazine phenyl.
The preparation method of the squaric acid derivative comprises the following steps:
(1) heating 3, 4-dihydroxy-3-cyclobutene-1, 2-diketone (squaric acid) and thionyl chloride to 30-80 ℃, and then adding N, N-dimethylformamide as a catalyst to react to obtain 3, 4-dichloro-3-cyclobutene-1, 2-diketone;
(2) carrying out Friedel-crafts acylation reaction on an aromatic compound and the 3, 4-dichloro-3-cyclobutene-1, 2-diketone in the step (1) to obtain a kind of squaric acid derivatives of which the 3,4 positions are substituted by aromatic groups;
the molar ratio of the 3, 4-dihydroxy-3-cyclobutene-1, 2-dione, thionyl chloride and N, N-dimethyl formamide in the step (1) is 1: 2-20: 0.1 to 1; the reaction time in the step (1) is 3-8 hours.
Preferably, the molar ratio of the 3, 4-dihydroxy-3-cyclobutene-1, 2-dione, thionyl chloride and N, N-dimethylformamide in the step (1) is in the range of 1: 10: 0.2; the reaction time in the step (1) is within the range of 5 hours.
The preparation method of the maleic anhydride derivative comprises the following steps:
dissolving the squaric acid derivative with an organic solvent in an atmospheric atmosphere, and irradiating the squaric acid derivative solution by using an ultraviolet lamp or a blue light lamp to obtain a maleic anhydride derivative;
the organic solvent is dichloromethane, tetrahydrofuran or N, N-dimethylformamide; the wavelength range of the ultraviolet lamp or the blue light lamp is 365nm-460 nm; the irradiation time period ranges from 2 to 12 hours.
Preferably, the wavelength range of the ultraviolet lamp or the blue light lamp is 410 nm; the irradiation time period ranged from 6 hours.
The preparation method of the maleimide biological coupling material comprises the following steps:
carrying out biological coupling on the maleic anhydride derivative and a primary amine compound at normal temperature to obtain a maleimide biological coupling material with aggregation-induced emission properties;
the molar ratio of the maleic anhydride derivative to the primary amine compound is 1: 1-10.
Preferably, the molar ratio of the maleic anhydride derivative to the primary amine compound is in the range of 1: 2.
The maleimide biological coupling material is applied to cell imaging.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the bioconjugation method provided by the invention is characterized in that the acid derivatives are converted into maleic anhydride derivatives under the light induction, and then the maleic anhydride derivatives are efficiently subjected to specific bioconjugation with primary amine compounds, so that the functionalization of various small molecules and drugs or biologically related molecules is quickly realized.
(2) The bioconjugation method provided by the invention can be used for lysine specific modification of in vitro unprotected peptides and natural proteins.
(3) The biological coupling method provided by the invention can be used for modifying the polymer and endowing the polymer with aggregation-induced luminescence property.
(4) The maleimide biological coupling material provided by the invention has aggregation-induced emission property, overcomes the defect that biomolecules do not have fluorescence, and can be used for cell imaging.
Drawings
FIG. 1a shows SQ-PhOCH in the preparation of example 43Ultraviolet visible absorption spectrum of molecules in tetrahydrofuran solution at different illumination time.
FIG. 1b is SQ-PhOCH3Fluorescence emission spectra of molecules in tetrahydrofuran solution at different illumination times.
FIG. 2a PEG prepared in example 92000-MM-PhOCH3Ultraviolet and visible absorption spectrum of (1).
FIG. 2b shows PEG prepared in example 92000-MM-PhOCH3Fluorescence emission spectrum of (1).
FIG. 3a is a graph of BA-MM-PhOCH in THF/water mixtures of varying water content3Change in relative fluorescence intensity.
FIG. 3b shows Lys1-MM-PhOCH in THF/water mixtures of different water contents3Change in relative fluorescence intensity.
FIG. 4 shows BSA-MM-PhOCH3SDS-PAGE of (5) results.
FIG. 5 shows Lys1-MM-PhOCH3And (3) evaluating the toxicity of the Hela cervical cancer cells.
FIG. 6 shows Lys1-MM-PhOCH3Imaging of the cells.
Detailed Description
The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the following processes, if not described in particular detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer and are considered to be conventional products available by commercial purchase.
Example 1
Squaric acid derivative (SQ-PhOCH)3) Preparation of the Material
Figure BDA0003180452080000071
The synthetic route is as follows:
Figure BDA0003180452080000072
a)3, 4-dichloro-cyclo-3-ene-1, 2-dione (2): in a 100mL two-necked flask, squaric acid (1g,8.78 mmol) and thionyl chloride (10mL,137mmol) were added, the mixture was heated to 60 ℃, 1mL of N, N-dimethylformamide was added, and the reaction was completed for 6 hours, and the excess thionyl chloride was removed by a rotary evaporator, and the product was collected and directly subjected to the next reaction.
b) Synthesis of 3, 4-bis (4-methoxyphenyl) cyclo-3-ene-1, 2-dione (3) (SQ-PhOCH)3): aluminum trichloride (2.58g,19.32mmol) was charged into a 100ml two-necked flask, and nitrogen-suction-nitrogen was circulated 3 times (10 minutes each), and then the compound (2) obtained in step (a) was dissolved in 20ml of dichloromethane and injected into the reaction flask. Then anisole (2.09g, 13.93mmol) was added and heated to 55 ℃ for 5 hours. After the reaction, the mixture was extracted with dichloromethane/water (the volume ratio of dichloromethane to water was 1/1), dried over anhydrous magnesium sulfate for 2 hours, and separated and purified by column chromatography to obtain a yellow pink color (1.94g) with a yield of 75%.1H NMR(400MHz,CDCl3)δ8.11(d,J＝9.0Hz,2H),7.04(d,J＝9.0Hz,2H),3.92 (s,3H).
Example 2
Preparation of squaric acid derivative (SQ-TPA) material
Figure BDA0003180452080000081
The synthetic route is as follows:
Figure BDA0003180452080000091
synthesis of 3, 4-bis (4- (dianilino) phenyl) cyclo-3-ene-1, 2-dione (SQ-TPA): into a 100mL two-necked flask were charged aluminum trichloride (2.58g,19.32mmol) and triphenylamine (4.73g,19.32mmol), and nitrogen-suction-nitrogen was circulated 3 times (10 minutes each), and then the compound (2) obtained in step (a) in example 1 was dissolved in 20mL of dichloromethane, injected into a reaction flask, and then 4 portions were added0mL, heated to 55 ℃ and reacted for 5 hours. After the reaction is finished, dichloromethane/water (the volume ratio of dichloromethane to water is 1/1) is used for extraction, anhydrous magnesium sulfate is dried for 2 hours, and separation and purification are carried out by a column layer analysis method, so that red pink (3,49g) is obtained, and the yield is 70%.1H NMR(500MHz,CDCl3)δ7.98(d,J＝8.9Hz,2H),7.34(t,J＝7.9Hz, 4H),7.23–7.08(m,6H),7.03(d,J＝8.9Hz,2H).
Example 3
Preparation of squaric acid derivative (SQ-CZ) material
Figure BDA0003180452080000092
The synthetic route is as follows:
Figure BDA0003180452080000101
synthesis of 3, 4-bis (9-phenyl-9H-carbazol-3-yl) cyclobutene-1, 2-dione (SQ-CZ): aluminum trichloride (2.58g,19.32mmol) and triphenylamine (4.69g,19.32mmol) were charged in a 100mL two-necked flask, nitrogen-pump-nitrogen was circulated 3 times (10 minutes each), and then the compound (2) obtained in step (a) in embodiment 1 was dissolved in 20mL of dichloromethane, injected into a reaction flask, then 40mL was added, heated to 55 ℃ and reacted for 5 hours. After the reaction, the mixture was extracted with dichloromethane/water (the volume ratio of dichloromethane to water was 1/1), dried over anhydrous magnesium sulfate for 2 hours, and separated and purified by column chromatography to obtain yellow pink (3,52g) with a yield of 71%.1H NMR(400MHz,CDCl3)δ9.10,9.10,8.25,8.25,8.23,8.23,8.20, 8.18,7.68,7.66,7.64,7.60,7.60,7.58,7.56,7.54,7.52,7.50,7.48,7.46,7.44,7.42, 7.37,7.37,7.35,7.33,7.33,-3.85.
Example 4
Maleic anhydride derivative material (MAH-PhOCH)3) Preparation of
Figure BDA0003180452080000102
The synthetic route is as follows:
Figure BDA0003180452080000103
synthesis of the Compound 3, 4-bis (4-methoxyphenyl) furan-2, 5-dione (MAH-PhOCH)3): a100 mL dry round bottom flask was charged with 3, 4-bis (4-methoxyphenyl) cyclo-3-ene-1, 2-dione (SQ-PhOCH)3) (1g,3.40mmol), then 45mL of tetrahydrofuran was added to the two-necked flask and the mixture was irradiated with a 410nm blue light for 6 h. After the reaction was stopped, the solvent was removed by a rotary evaporator, and the reaction mixture was separated and purified by column chromatography to obtain 0.74g of a golden yellow solid with a yield of 70%.1H NMR(500MHz, CDCl3)δ7.56(d,J＝9.0Hz,2H),6.91(d,J＝9.0Hz,2H),3.85(s,3H).
We monitored the reaction dynamically using uv absorption spectroscopy and fluorescence emission spectroscopy. As can be seen from the UV absorption spectrum of FIG. 1a, the material SQ-PhOCH increases with the illumination time3The absorption peaks at 296nm and 362nm are obviously weakened; the absorption peak at 362nm is obviously broadened when the lamp is irradiated for 6min, the split peak at 12min is two absorption peaks of 342nm and 384nm, and when the illumination time is prolonged to 36 min, the short wavelength absorption finally shifts to 337nm in blue, the long wavelength absorption shifts to 397nm in red, and the product MAH-PhOCH3The ultraviolet absorption positions are consistent, and the reaction is dynamically monitored by using the change of the ultraviolet absorption spectrum.
At the same time, we also collected SQ-PhOCH3As shown in FIG. 1b, the fluorescence emission spectra at different illumination times show obvious enhancement along with the increase of the illumination time, and after 18min, the fluorescence intensity reaches the highest and is enhanced by about 200 times, thus showing an obvious light-induced fluorescence enhancement phenomenon.
Example 5
Preparation of maleic anhydride derivative material (MAH-TPA)
Figure BDA0003180452080000111
The synthetic route is as follows:
Figure BDA0003180452080000121
synthesis of compound 3, 4-bis (9-phenyl-9H-carbazol-3-yl) furan-2, 5-dione (MAH-TPA): 3, 4-bis (4- (dianilino) phenyl) cyclo-3-ene-1, 2-dione (SQ-TPA) (1g,1,76mmol) was added to a 100mL dry round bottom flask, then 45mL tetrahydrofuran was added to the flask, and irradiation with a 410nm blue lamp was continued for 6 h. After the reaction was stopped, the solvent was removed by a rotary evaporator, and the reaction mixture was separated and purified by column chromatography to obtain 0.67g of a golden yellow solid with a yield of 65%.1H NMR(500MHz, CDCl3)δ7.51(d,J＝9.0Hz,1H),7.35–7.28(m,2H),7.14(dd,J＝16.7,8.0Hz, 3H),6.96(d,J＝8.9Hz,1H).
Example 6
Preparation of maleic anhydride derivative material (MAH-CZ)
Figure BDA0003180452080000122
The synthetic route is as follows:
Figure BDA0003180452080000131
synthesis of compound 3, 4-bis (9-phenyl-9H-carbazol-3-yl) furan-2, 5-dione (MAH-CZ): 3, 4-bis (9-phenyl-9H-carbazol-3-yl) cyclobutene-1, 2-dione (SQ-CZ) (1g,1,77mmol) was added to a 100mL dry round bottom flask, followed by addition of 45mL tetrahydrofuran in a two-necked flask and continuous irradiation with a 410nm blue lamp for 6H. After the reaction was stopped, the solvent was removed by a rotary evaporator, and the reaction mixture was separated and purified by column chromatography to obtain 0.70g of a golden yellow solid with a yield of 68%.1H NMR(500MHz,CDCl3)δ 8.57(s,1H),8.09(d,J＝7.7Hz,1H),7.63–7.56(m,3H),7.53(d,J＝7.4Hz,2H), 7.49–7.41(m,2H),7.39(d,J＝8.0Hz,1H),7.30(t,J＝10.1Hz,2H).
Example 7
Maleimide biological coupling material (BA-MM-PhOCH) with aggregation-induced emission performance3) Preparation of
Figure BDA0003180452080000132
The synthetic route is as follows:
Figure BDA0003180452080000141
synthesis of the Compound 1-benzyl-3, 4-bis (4-methoxyphenyl) -1H-pyrrole-2, 5-dione (BA-MM-PhOCH)3): adding 3, 4-bis (4-methoxyphenyl) furan-2, 5-dione (0.31g and 1mmol) into a 100mL dry round-bottom flask, then adding 30mL tetrahydrofuran, adding benzylamine (0.22g and 2mmol) after the raw materials are completely dissolved, stopping the reaction after reacting for 10 minutes at normal temperature, removing the solvent by using a rotary evaporator, and separating and purifying by column layer analysis to obtain 0.16g of yellow solid with the yield of 40%.1H NMR(400MHz,CDCl3)δ7.46(t,J＝8.5Hz,6H),7.32(dd,J＝14.9,7.5Hz, 4H),6.86(d,J＝8.9Hz,4H),4.78(s,2H),3.82(s,6H).
Example 8
Maleimide biological coupling material (Lys 1-MM-PhOCH) with aggregation-induced emission performance and good biocompatibility3) Preparation of
Figure BDA0003180452080000142
The synthetic route is as follows:
Figure BDA0003180452080000151
synthesis of the Compound methyl (S) -2- ((benzyloxy) carbonyl) amino) -6- (3, 4-bis (4-methoxyphenyl) -2, 5-dioxy-2, 5-dihydro-1H-pyrrol-1-yl) hexanoate (Lys 1-MM-PhOC)H3): in a 100mL dry round bottom flask was added 3, 4-bis (4-methoxyphenyl) furan-2, 5-dione (0.31g, 1mmol) followed by 30mL tetrahydrofuran to dissolve the starting material completely. Then, N-benzyloxycarbonyl-L-lysine methyl ester hydrochloride (0.60g, 2mmol) was dissolved in 10mL of methanol, 10mL of triethylamine was added thereto, the resulting solution of the treated N-benzyloxycarbonyl-L-lysine methyl ester hydrochloride in methanol was added to a solution of 3, 4-bis (4-methoxyphenyl) furan-2, 5-dione, the reaction was stopped after 30 minutes at room temperature, the solvent was removed by a rotary evaporator, and the mixture was separated and purified by column chromatography to obtain 0.21g of a yellow solid in a yield of 35%.1H NMR(400MHz, CDCl3)δ7.47(d,J＝8.7Hz,4H),7.41–7.27(m,5H),6.86(d,J＝8.6Hz,4H), 5.09(q,J＝12.2Hz,2H),4.37(d,J＝5.0Hz,1H),3.82(s,5H),3.73(s,2H),3.61 (t,J＝6.8Hz,2H),1.98–1.58(m,6H),1.43(s,2H).
Example 9
Specific coupling of polymers with maleic anhydride derivatives to Primary amines (PEG)2000-MM-PhOCH3) Make a modification
Figure BDA0003180452080000152
Synthesis of the Compound 1-polyethylene glycol-3, 4-bis (4-methoxyphenyl) -1H-pyrrole-2, 5-dione (PEG)2000-MM-PhOCH3): 3, 4-bis (4-methoxyphenyl) furan-2, 5-dione (0.02g, 0.065mmol) was added to a 20mL polymerization tube, then 5mL dichloromethane was added, polyethylene glycol (130mg, 0.065mmol) having a molecular weight of 2k was added after the raw material was completely dissolved, the reaction was stopped after 30 minutes at room temperature, the solvent was removed by a rotary evaporator, and the mixture was separated and purified by column chromatography to obtain 0.05g of a yellow oily liquid with a yield of 35%.
In the hydrogen nuclear magnetic resonance spectrum, the aromatic region has obvious peaks at the positions of 7.40-7.43ppm and 6.80-6.83ppm, and has obvious difference with the positions of 7.56ppm and 6.91ppm of the raw material, thereby proving that the polymer is successfully modified. In addition, we are dealing with the compound PEG2000-MM-PhOCH3Is measured, as can be seen from FIG. 2a, this is achievedThe compound has a new absorption peak at 402nm and a fluorescence emission peak at 537nm from FIG. 2b, which again proves that PEG2000-MM-PhOCH3The coupling was successful. [ PEG2000-MM-PhOCH3]＝1mg/mL。
Example 10
Coupling product BA-MM-PhOCH3And Lys1-MM-PhOCH3Characterization of the AIE
FIGS. 3a and 3b show BA-MM-PhOCH based on the materials obtained in example 7 and example 8, respectively3And Lys1-MM-PhOCH3Fluorescence spectra under different water content conditions. As can be seen from the figure, the fluorescence of the two example materials is significantly reduced when the water content is increased from 0 to 70%, exhibiting a significant twisted intramolecular charge transfer effect; when the water content is further increased to 90%, the fluorescence is gradually enhanced, and the aggregation-induced emission phenomenon is shown. [ BA-MM-PhOCH ]3,Lys1-MM-PhOCH3]＝10μM。
Example 11
Modification of bovine serum albumin by specific coupling of maleic anhydride derivatives to primary amines (BSA-MM-PhOCH)3) And the modified bovine serum albumin is verified to be endowed with the fluorescence property by utilizing a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiment.
BSA-MM-PhOCH3The preparation of (1): 100mg of bovine serum albumin was added to a 50mL single-neck flask, and then 10mL of water was added to dissolve the bovine serum albumin completely, 10mg of 3, 4-bis (4-methoxyphenyl) furan-2, 5-dione dissolved in 2mL of tetrahydrofuran was added thereto, the reaction was performed under a closed condition at normal temperature for 30 minutes, and then the lid was opened and the mixture was stirred overnight to completely rinse the tetrahydrofuran, and the solvent water was removed by a lyophilizer to obtain 100mg of a yellow solid.
SDS-PAGE experiments and fluorescent gel imaging characterization
(1) Mounting plate
Overlapping a long piece of flat glass and a short piece of flat glass, standing to enable the bottom end to be in contact with the desktop, clamping the two pieces of glass on a rack by hands and preparing glue pouring.
(2) Polymerization of gels
According to the sequence and proportion of the solutions in the following table, 10% glue is prepared.
Figure BDA0003180452080000171
After the above solutions are added and mixed uniformly to prepare gel, the gel solution is slowly dripped along the inner surface of a long glass plate of a gel cavity by a dropper, and care is taken not to generate bubbles. Insert the sample mold (comb) and wait for the gel to set.
(3) Treatment of protein samples
0.2mg of standard bovine serum albumin, 0.2mg of BSA-MM-PhOCH and 0.5mg of BSA-MM-PhOCH were weighed out separately3Dissolved in 1mL of 0.5mol L-1pH6.8 Tris-hydrochloric acid buffer solution or distilled water, and then 200. mu.L of the sample treatment solution was added. Treating in 100 deg.C water bath for 2min, and cooling to room temperature.
(4) Sample application
And (3) clamping the two glass plates by hands, lifting the inserting plate to loosen the two glass plates, placing the two glass plates into an electrophoresis tank, adding a buffer solution into the position which is submerged in the glass plates, and adding the buffer solution into the position which is 3mm away from the upper edge of the plate glass in the outer tank, wherein the phenomenon that bubbles appear in the electrophoresis tank is avoided.
(5) Electrophoresis
After sample adding, covering the upper cover, connecting the electrophoresis apparatus, and after opening a switch of the electrophoresis apparatus, controlling the current to be 15-20 mA for about 15-20 min before feeding the sample into the gel; after the bromophenol blue indicator in the sample reaches the separation gel, the current rises to 30-45 mA, and the current is kept stable in the electrophoresis process. And stopping electrophoresis when the bromophenol blue indicator migrates to a position 1-2 cm away from the front edge for about 1-2 hours. If the room temperature is high, the electrophoresis tank is opened to circulate water, and the electrophoresis temperature is reduced.
(6) Fluorescence gel imaging
After electrophoresis is finished, a power supply is turned off, the glass plates are taken out, the rubber surface and one glass plate are separated by slightly prying the glass plate by a knife in a gap between the lower corner of the two long and short glass plates, then the film is slightly supported, the film is placed on a bottom plate of a fluorescence gel imager, and an optical filter 530nm combined channel with an excitation wavelength of 400nm and an emission wavelength of receiving is selected for imaging. And adjusting the focal length and then taking a picture.
(7) Dyeing and decoloring
And (3) placing the film which is photographed with the fluorescent gel image into a large culture dish for dyeing, and using 0.25% Coomassie brilliant blue dye solution for dyeing for 2-4 h.
Discarding the dyeing solution, rinsing the glue surface with distilled water for several times, then adding a decolorizing solution, performing diffusion decolorizing, and frequently changing the decolorizing solution until the protein band is clear.
Placing the fluorescent gel imaging device on a bottom plate of a fluorescent gel imaging device, and selecting a white light field. And adjusting the focal length and then taking a picture. As shown by SDS-PAGE result in FIG. 4, at the position of the band with molecular weight slightly lower than 70KDa, BSA-MM-PhOCH is observed under ultraviolet irradiation3A clear fluorescence band was seen, whereas standard BSA did not fluoresce, demonstrating the fluorescent material PhOCH3The coupling of MAH and bovine serum protein is successful, and the fluorescence property of the natural protein can be endowed through the coupling method.
Example 12
Toxicity evaluation of maleimide bioconjugate material with aggregation-induced emission performance and good biocompatibility
The invention researches the toxicity of photosensitizer by taking Hela cervical carcinoma cells as a model.
Hela cervical cancer cells are spread in a 96-well plate and cultured for 24 hours, and then Lys1-MM-PhOCH 78 with the concentration of 0. mu.M, 1. mu.M, 2. mu.M, 4. mu.M, 8. mu.M, 16. mu.M and 32. mu.M is added3Culturing in 100mL DMEM (10% fetal calf serum) medium for 24 hr, removing supernatant, adding 100mL MTT, culturing for 2 hr, stopping culturing, removing culture medium, adding 100 μ L dimethyl sulfoxide into each well, shaking on a shaker at low speed for 10min to dissolve the crystal, and detecting OD value with an enzyme-labeling instrument. FIG. 5 is a graph showing the toxicity evaluation result of Lys1-MM-PhOCH3 on MCF-7 breast cancer cells, and Lys1-MM-PhOCH3 has almost no biological dark toxicity on cells and shows good biocompatibility. Then, to investigate the phototoxicity of Lys1-MM-PhOCH3, cells were plated in 96-well plates with white light (50mW cm)-2) Culturing for 12 hr after 30 min irradiation, removing supernatant, adding 100mL MTT, culturing for 2 hr, terminating the culture, removing culture medium in the wells, adding 100 μ L dimethyl sulfoxide into each well, and standing and shakingOscillating on bed at low speed for 10min to make the crystal fully dissolve the OD value detected by enzyme-labeling instrument. At concentrations up to 64 μ M, the cells still had 80% survival, demonstrating that the molecule is not phototoxic.
Example 13
The coupling product Lys1-MM-PhOCH3Cell imaging experiments
Using Hela cervical carcinoma cell as model, laying Hela cell on a culture dish special for confocal culture, incubating for 24 hours for adherence, removing culture medium by suction, adding 10 μ M Lys1-MM-PhOCH after PBS washing3Cells were incubated for 2 hours, media aspirated, and then washed with PBS for characterization by confocal microscopy. As shown in FIG. 6, the coupling product Lys1-MM-PhOCH was used3Cells were successfully lightened at 2 h of staining, indicating Lys1-MM-PhOCH3Has good penetrability and biocompatibility. FIG. 6 shows Lys1-MM-PhOCH3And (3) imaging fluorescence of Hela cervical cancer cells. [ Lys1-MM-PhOCH3]＝10μM,Ex＝405nm， Em＝410–513nm，Laser 2％；Scale bar＝20μm。
The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should protect the present invention from changes, substitutions and modifications without departing from the spirit of the present invention.

Claims (10)

1. A kind of squaric acid derivatives is characterized in that the structural general formula is shown as the following formula I:
Figure FDA0003180452070000011
wherein R is2And R3Independently is one of aromatic hydrocarbon and aromatic hydrocarbon derivative groups.
2. The maleic anhydride derivative is characterized by having a structural general formula shown as the following formula II:
Figure FDA0003180452070000012
wherein R is2And R3Independently is one of aromatic hydrocarbon and aromatic hydrocarbon derivative groups.
3. The maleimide biological coupling material is characterized in that the structural general formula is shown as the following formula III:
Figure FDA0003180452070000013
wherein R is1Independently is one of aliphatic hydrocarbon, aromatic hydrocarbon, aliphatic hydrocarbon derivative group and aromatic hydrocarbon derivative group; r2And R3Independently is one of aromatic hydrocarbon and aromatic hydrocarbon derivative groups.
4. The squaric acid derivative according to claim 1, wherein R is2And R3The aromatic hydrocarbon and aromatic hydrocarbon derivative group of (a) is a phenyl group, naphthyl group, tetraphenylvinyl group, thiophene ring, pyridine ring, furan ring, 4-methoxyphenyl group, diethylaminophenyl group, dimethylaminophenyl group, carbazolylphenyl group, carbazolyl group, phenothiazinyl group, phenoxazinyl phenyl group, bithiophenyl group, thienocyclopentadienyl group, 9, 10-dihydro-9, 9-dimethylazinyl group, 9, 10-dihydro-9, 9-diphenylacridinyl group, 10-H-spiro [ acridine-9, 9' -fluorene]One of a phenyl group, a diphenylamino group, a triphenylamino group, a diphenylaminothiophene group, a bithiophene group, a fused thiophene group, a thienocyclopentadienyl group, a naphthylaminophenyl group, or a bipyridine amino group.
5. The maleic anhydride derivative according to claim 2, wherein R is2And R3The aromatic hydrocarbon and aromatic hydrocarbon derivative group is phenyl, naphthyl, tetraphenylvinyl, thiophene ring, pyridine ring, furan ring, 4-methoxyphenyl, diethylaminophenyl, dimethylaminophenyl, carbazolylphenyl, carbazolyl, phenothiazinyl, phenoxazinyl,Phenothiazinphenyl, phenoxazinylphenyl, bithienyl, thienocyclopentadienyl, 9, 10-dihydro-9, 9-dimethylazinyl, 9, 10-dihydro-9, 9-diphenylzinyl, 10-H-spiro [ acridine-9, 9' -fluorene]One of a phenyl group, a diphenylamino group, a triphenylamino group, a diphenylaminothiophene group, a bithiophene group, a fused thiophene group, a thienocyclopentadienyl group, a naphthylaminophenyl group, or a bipyridine amino group.
6. The maleimide bioconjugate material according to claim 3, wherein said R is1The aliphatic hydrocarbon of (a) is a linear or branched alkyl group; the aliphatic hydrocarbon and aliphatic hydrocarbon derivative groups are methyl, ethyl, propyl, butyl, isobutyl, tert-butyl, cyclohexane and lysine derivatives;
the R is1The aromatic hydrocarbon group and the aromatic hydrocarbon derivative group of (a) are phenyl, naphthyl, tetraphenylvinyl, thiophene ring, pyridine ring, furan ring, 4-methoxyphenyl, carbazolylphenyl, dimethylaminophenyl, trianilino, diphenylthiophene group, phenothiazine phenyl, phenoxazine phenyl, bithiophene group, thienocyclopentadienyl;
said R2And R3The aromatic hydrocarbon and aromatic hydrocarbon derivative group of (a) is a phenyl group, naphthyl group, tetraphenylvinyl group, thiophene ring, pyridine ring, furan ring, 4-methoxyphenyl group, diethylaminophenyl group, dimethylaminophenyl group, carbazolylphenyl group, carbazolyl group, phenothiazinyl group, phenoxazinyl phenyl group, bithiophenyl group, thienocyclopentadienyl group, 9, 10-dihydro-9, 9-dimethylazinyl group, 9, 10-dihydro-9, 9-diphenylacridinyl group, 10-H-spiro [ acridine-9, 9' -fluorene]One of a phenyl group, a diphenylamino group, a triphenylamino group, a diphenylaminothiophene group, a bithiophene group, a fused thiophene group, a thienocyclopentadienyl group, a naphthylaminophenyl group, or a bipyridine amino group.
7. The process for producing a squaric acid derivative according to claim 1, comprising the steps of:
(1) heating 3, 4-dihydroxy-3-cyclobutene-1, 2-diketone and thionyl chloride to 30-80 ℃, and then adding N, N-dimethylformamide as a catalyst to react to obtain 3, 4-dichloro-3-cyclobutene-1, 2-diketone;
(2) carrying out Friedel-crafts acylation reaction on an aromatic compound and the 3, 4-dichloro-3-cyclobutene-1, 2-diketone in the step (1) to obtain a kind of squaric acid derivatives with 3, 4-substituted aromatic groups;
the molar ratio of the 3, 4-dihydroxy-3-cyclobutene-1, 2-dione, thionyl chloride and N, N-dimethylformamide in the step (1) is 1: 2-20: 0.1 to 1; the reaction time in the step (1) is 3-8 hours.
8. The method for producing a maleic anhydride derivative according to claim 2, comprising the steps of:
dissolving the squaric acid derivative of claim 1 in an organic solvent under an atmospheric atmosphere, and irradiating the solution with an ultraviolet lamp or a blue lamp to obtain a maleic anhydride derivative;
the organic solvent is dichloromethane, tetrahydrofuran or N, N-dimethylformamide; the wavelength range of the ultraviolet lamp or the blue light lamp is 365nm-460 nm; the irradiation time period ranges from 2 to 12 hours.
9. The method for preparing a maleimide bioconjugate material according to claim 3, comprising the steps of:
biologically coupling the maleic anhydride derivative of claim 2 with a primary amine compound at normal temperature to obtain a maleimide biological coupling material with aggregation-induced emission properties;
the molar ratio of the maleic anhydride derivative to the primary amine compound is 1: 1-10.
10. Use of the maleimide-based bioconjugate material according to claim 3 for cellular imaging.
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