CN113512022B - Multifunctional fluorescent link body based on pH response and preparation method and application thereof - Google Patents
Multifunctional fluorescent link body based on pH response and preparation method and application thereof Download PDFInfo
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- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
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Abstract
The pH response-based multifunctional fluorescent linker, the preparation method and the application thereof, wherein 9- (2-carboxyphenyl) -3, 6-bis (diethylamino) xanthene chloride and N-Boc-butanediamine are condensed under EDC and HCl to obtain an amino terminal intermediate with Boc protection; amino terminal intermediates with Boc protection in CF 3 Obtaining an intermediate with an amino terminal under the action of COOH; the intermediate with an amino terminal and dithio diacetic acid are condensed under EDC and HCl to obtain the multifunctional fluorescent linker based on pH response. The linker molecule can chemically modify the targeted anti-tumor drug molecule to construct a diagnosis and treatment integrated drug, and enters the body, fluorescence is started for marking and tracing under the catalysis of a tumor low-pH microenvironment, and simultaneously the targeted anti-tumor drug is released under the high-reduction tumor microenvironment, so that the purpose of diagnosis and treatment integration is realized.
Description
Technical Field
The invention belongs to the technical field of medicine preparation, and relates to a multifunctional fluorescent link body based on pH response, and a preparation method and application thereof.
Background
Cancer, also known as malignant tumor, is one of the major diseases that seriously endanger human health and life. According to the latest report of International journal of famous cancer, CA: A Cancer Journal for Clinicians, about 429.2 cases of new cancer and 281.4 cases of cancer death in China in 2015, the recent report of the condition of cancer in China accounts for 22% and 27% of the cases of new cancer and death worldwide respectively. Cancer is becoming an important factor in jeopardizing public health safety in China. Therefore, the timely, efficient and accurate diagnosis and treatment of the cancer is related to the life health and the quality of life of people and the sustainable development of economy and society. Traditional methods of tumor treatment mainly include surgical excision, chemotherapy (chemotherapy) and radiation therapy (radiotherapy). However, in the operation process, the tumor tissue is not easy to identify, and the proto-cancer cells are easy to transfer, so that the postoperative recurrence is easy to occur. Meanwhile, aiming at different individuals, the treatment effect is uneven, and the radiotherapy and chemotherapy are generally more expensive. Thus, screening of individual patients prior to treatment is a primary step.
"diagnosis and treatment integration" was first proposed by John Funkhouser in 1998, which defines diagnosis and treatment integration as "ability to intervene in a therapeutic means according to a disease state" (The Ability to Affect Therapy or Treatment of A Disease State). Along with rapid and vigorous development of diagnosis and treatment integration, the definition of the diagnosis and treatment integration is also widely expanded, and the diagnosis and treatment integration is currently widely considered to be a novel biomedical technology which organically combines diagnosis or monitoring and treatment of diseases. Because diagnosis and treatment functions are integrated into a whole, the diagnosis and treatment integrated device has obvious advantages compared with a single diagnosis or treatment means. Specifically, the diagnosis and treatment integration of cancer has great potential in the aspects of layering and personalized medicine treatment of patients, real-time monitoring of the medicine treatment process, feedback of the medicine treatment effect and the like.
Individual differences have led to the trend of personalized medicine because unified standardized treatment regimens do not achieve optimal efficacy for different cancer patients. According to the explanation given by the global leading personalized medical institution, andersen cancer center (MD Anderson Cancer Center), the core idea of personalized medicine as a cancer treatment strategy is to analyze the possibility of a patient responding to a specific treatment means based on various tumor markers and tumor cells, and combine different manifestations of drug metabolism, response and toxicity possibly caused by genetic factors of the patient, and simultaneously integrate tumor molecular spectra, tumor parts and other physiological characteristics of the patient to prepare a set of optimal treatment schemes aiming at individuals. From the above definition, in personalized medicine, various diagnosis and treatment means need to be organically combined in the whole treatment process, which is completely combined with the concept of cancer diagnosis and treatment integration, so that the development of the cancer diagnosis and treatment integration technology integrating diagnosis and treatment functions can strongly promote the development of personalized medicine. On the basis, the imaging and treatment functions are integrated, so that the dynamic distribution of the diagnosis and treatment integrated medicine in tumor tissues can be mastered in real time, and the optimal stimulation time can be mastered.
In recent years, the diagnosis and treatment integrated concept is mainly applied to the aspect of nano-delivery of medicines. Many research efforts have demonstrated that nanomaterials are not beneficial and harmless, and that they affect the normal physiological functions of organisms at both cellular, subcellular and protein levels.
Disclosure of Invention
The invention aims to provide a pH response-based multifunctional fluorescent linker, a preparation method and application thereof, wherein the pH response-based multifunctional fluorescent linker is used for chemically modifying targeted antitumor drug molecules and can be used for verifying the feasibility of realizing diagnosis and treatment integration of probes constructed by the pH response-based multifunctional fluorescent linker.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a multifunctional fluorescent linker based on pH response, the multifunctional fluorescent linker having the structural formula:
the preparation method of the multifunctional fluorescent linker based on pH response comprises the following steps:
a) Condensing 9- (2-carboxyphenyl) -3, 6-bis (diethylamino) xanthene chloride and N-Boc-butanediamine under EDC and HCl to obtain an amino terminal intermediate with Boc protection;
b) Amino terminal intermediates with Boc protection in CF 3 Obtaining an intermediate with an amino terminal under the action of COOH;
c) Condensing the intermediate with amino terminal with dithiodiacetic acid under EDC & HCl to obtain the multifunctional fluorescent linker based on pH response, wherein the structural formula is as follows:
the invention is further improved in that the specific process of step a) is as follows: 9- (2-carboxyphenyl) -3, 6-bis (diethylamino) xanthene chloride, EDC, HCl and HOBT are dissolved in anhydrous dichloromethane, and after being stirred uniformly at 0 ℃, DIPEA is added dropwise, after the dropwise addition, N-Boc-butanediamine is added for reaction, and an amino terminal intermediate with Boc protection is obtained.
The invention is further improved in that the specific process of the step a) is as follows: 1.13mmol of 9- (2-carboxyphenyl) -3, 6-bis (diethylamino) xanthene chloride, 1.69mmol of EDC.HCl and 1.35mmol of HOBT are dissolved in anhydrous dichloromethane, stirred uniformly at 0 ℃, 4.70mmol of DIPEA is added dropwise, the mixture is reacted after the dripping, 0.94mmol of N-Boc-butanediamine is added, and the reaction is carried out for 4 hours at room temperature to obtain an amino terminal intermediate with Boc protection.
The invention is further improved in that the specific process of the step b) is as follows: the amino-terminal intermediate with Boc protection was dissolved in anhydrous dichloromethane and CF was added dropwise at 0deg.C 3 COOH, stirring for 1h after dripping, monitoring by TLC until the reaction is completed, adding saturated sodium bicarbonate aqueous solution after the reaction is completed, titrating until the system is neutral, extracting, collecting an organic phase, and spin-drying to obtain an intermediate with an amino terminal.
The invention is further improved in that the specific process of the step b) is as follows: 0.37mmol of Boc-protected amino terminus intermediateThe body was dissolved in 20mL of anhydrous dichloromethane, and 2mL of CF was added dropwise at 0 ℃ 3 COOH, stirring for 1h at 0 ℃, then reacting for 24h at 25-30 ℃ to obtain an intermediate with an amino terminal.
The invention is further improved in that the specific process of step c) is as follows: dissolving dithio-diacetic acid in anhydrous dichloromethane, adding EDC, HCl and HOBT, stirring at 0 ℃ for 30min, then dripping triethylamine, and stirring at room temperature for 24h to obtain the multifunctional fluorescent linker based on pH response.
The invention is further improved in that the specific process of the step c) is that 3.991mmol of dithio diacetic acid, 1.915mmol of EDC and 1.915mmol of HOBT are dissolved in anhydrous methylene dichloride at the temperature of 0 ℃, 1.915mmol of triethylamine is added dropwise after stirring for 30min at the temperature of 0 ℃, then 0.798mmol of intermediate with an amino terminal is added, and stirring is carried out for 24h at the temperature of 25-30 ℃ to obtain the multifunctional fluorescent linker based on pH response.
The application of the multifunctional fluorescent linker based on pH response in preparing diagnosis and treatment integrated medicines for treating tumors is provided.
The invention is further improved in that the pH responsive multifunctional fluorescent linker and PyBop are placed in anhydrous dichloromethane at the temperature of 0 ℃, triethylamine is added dropwise after being stirred uniformly at the temperature of 0 ℃, then an anti-tumor drug is added, and the mixture is stirred for 24 hours at the temperature of 25 ℃ to obtain the diagnosis and treatment integrated drug.
The invention is further improved in that 0.074mmol of pH responsive multifunctional fluorescent linker and 0.148mmol of PyBop are added into anhydrous methylene dichloride at 0 ℃, triethylamine 0.295mmol is added dropwise after stirring for 30min at 0 ℃, then 0.074mmol of antitumor drug is added, and stirring is carried out at room temperature for 24h, thus obtaining the diagnosis and treatment integrated drug.
The invention is further improved in that the antitumor drug is Li Nifa Ni, sorafenib, bevacizumab or atilizumab; the tumor is human umbilical vein cells, cervical cancer cells or breast cancer cells.
Use of a multifunctional fluorescent linker based on pH response as described above for the construction of a drug probe for labelling.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, 9- (2-carboxyphenyl) -3, 6-bis (diethylamino) xanthene chloride, N-Boc-butanediamine and dithiodiacetic acid are used for synthesizing a pH response-based multifunctional fluorescent linker, and the linker molecule can chemically modify targeted anti-tumor drug molecules so as to construct a diagnosis and treatment integrated drug which enters the body, and fluorescence is started for marking and tracing under the catalysis of a tumor low pH microenvironment, and simultaneously targeted anti-tumor drug release is carried out under a high-reduction tumor microenvironment, so that the purpose of diagnosis and treatment integration is realized. The pH response multifunctional fluorescent link preparation method is simple, easy to implement and wide in application range.
The pH responsive multifunctional fluorescent linker can chemically and covalently modify targeted anti-tumor drug molecules to construct a diagnosis and treatment integrated drug, and further trigger fluorescent lighting to perform fluorescent marking of tumor parts through stimulation of microenvironment of tumors (liver cancer, lung cancer, breast cancer, cervical cancer and the like), and simultaneously release the targeted anti-tumor drug molecules, so that marking tracing and treatment of tumors are achieved. The probe molecules constructed by the linker can improve the biotoxicity of the nano delivery drug system in the aspect of application of diagnosis and treatment integrated concepts, and simultaneously expand the application pluripotency of the diagnosis and treatment integrated concepts. The pH responsive multifunctional fluorescent link body can be used for constructing a diagnosis and treatment integrated drug and verifying the feasibility of the drug in realizing diagnosis and treatment integration.
Drawings
FIG. 1 is a synthetic route diagram of a pH responsive multifunctional fluorescent linker provided by the present invention;
FIG. 2 shows the results of cell imaging.
FIG. 3 shows the results of inhibition of proliferation of A549 cells.
FIG. 4 shows the results of inhibition of EA.hy926 cell proliferation.
FIG. 5 shows the results of inhibition of Hela cell proliferation.
FIG. 6 shows the results of MDA-MB-231 cell proliferation inhibition.
FIG. 7 is a fluorescence spectrum of a pH sensitive fluorescent linker.
FIG. 8 is a graph of pH sensitive fluorescent linker-pH quantification.
Wherein compound 1 is 9- (2-carboxyphenyl) -3, 6-bis (diethylamino) xanthene chloride, compound 2 is N-Boc-butanediamine, compound 3 is N- (9- (2- ((4- ((tert-butoxycarbonyl) amino) butyl) carbamoyl) phenyl) -6 (diethylamino) -8a,10 a-dihydro-3H-xanthen-3-ylidene) -N-ethyl ethylamine, compound 4 is N- (9- (2- ((4-aminobutyl) carbamoyl) phenyl) -6- (diethylamino) -8a,10 a-dihydro-3H-xanthen-3-ylidene) -N-ethyl ethylamine, and compound 5 is dithiodiacetic acid. Reagents and conditions: (a) EDC HCl, HOBT, DIPEA, DCM,0 ℃ to rt,24h; (b) TFA, DCM, 0deg.C to rt,24h; (c) EDC HCl, HOBT, TEA, DCM,0℃to rt,24h.
Detailed Description
The invention will now be described in further detail with reference to the drawings and to specific examples, which are given by way of illustration and not limitation.
The invention constructs diagnosis and treatment integrated molecules on the basis of small molecules, and uses the functions of realizing marking and treatment so as to expand the realization means of diagnosis and treatment integration in the aspect of cancer treatment.
The invention synthesizes pH response multifunctional fluorescent linker molecules by using 9- (2-carboxyphenyl) -3, 6-bis (diethylamino) xanthene chloride, N-Boc-butanediamine and dithiodiacetic acid, and the linker can be used for constructing 'diagnosis and treatment integrated' medicines by chemically modifying targeted antitumor drug molecules.
The pH response multifunctional fluorescent linker provided by the invention has the following structure:
the name of the pH responsive multifunctional fluorescent linker is as follows:
n- (9- (2- ((4- (2- ((carboxymethyl) disulfanyl) acetamido) butyl) carbamoyl) phenyl) -6- (diethylamino) -3H-xanthen-3-ylidene) -N-ethyl ethylamine.
The preparation method of the multifunctional fluorescent linker with pH response for constructing the diagnosis and treatment integrated drug provided by the invention is described in detail below with reference to the synthetic route and specific synthetic examples shown in FIG. 1.
Referring to fig. 1, a synthetic route for a pH-responsive multifunctional fluorescent linker comprises the steps of:
a) Condensing 9- (2-carboxyphenyl) -3, 6-bis (diethylamino) xanthene chloride and N-Boc-butanediamine under EDC and HCl to obtain an amino terminal intermediate with Boc protection;
the specific operation of the step a) is as follows:
dissolving 9- (2-carboxyphenyl) -3, 6-bis (diethylamino) xanthene chloride, EDC, HCl and HOBT in anhydrous dichloromethane, stirring uniformly at 0 ℃, dropwise adding DIPEA, reacting for 3min, adding N-Boc-butanediamine, reacting at room temperature for 4h to obtain an amino terminal intermediate crude product with Boc protection, washing with saturated sodium bicarbonate for three times, washing with saturated sodium chloride, drying an organic phase with anhydrous sodium sulfate, and separating by column chromatography to obtain the amino terminal intermediate with Boc protection;
b) Amino terminal intermediates with Boc protection in CF 3 Obtaining an intermediate with an amino terminal under the action of COOH;
the specific operation of the step b) is as follows:
the amino-terminal intermediate with Boc protection was dissolved in anhydrous dichloromethane and CF was added dropwise at 0deg.C 3 And (3) after stirring for 1h, monitoring by TLC until the reaction is completed, thus obtaining a crude product containing an amino terminal intermediate, regulating the pH value of the system to be neutral by saturated sodium bicarbonate, extracting and collecting an organic phase, respectively drying and washing the organic phase by saturated sodium chloride and anhydrous sodium sulfate, and carrying out decompression spin-drying to obtain the intermediate with the amino terminal.
c) The intermediate with an amino terminal and dithio diacetic acid are condensed under EDC and HCl to obtain the multifunctional fluorescent linker based on pH response.
The specific operation of the step c) is as follows:
dissolving dithiodiacetic acid, EDC, HCl and HOBT in anhydrous dichloromethane at 0 ℃, stirring for 30min at 0 ℃, then dripping triethylamine, adding an amino terminal intermediate, stirring for 24h at room temperature to obtain a multifunctional fluorescent linker crude product based on pH response, washing the crude product with saturated sodium bicarbonate for three times, washing with saturated sodium chloride, drying an organic phase with anhydrous sodium sulfate, and separating by column chromatography to obtain the multifunctional fluorescent linker based on pH response.
The multifunctional fluorescent linker containing the pH response is applied to the construction of 'diagnosis and treatment integrated' medicines.
Example 1
The preparation process of the multifunctional fluorescent linker with pH response is shown in figure 1,
a) The condensation of 9- (2-carboxyphenyl) -3, 6-bis (diethylamino) xanthene chloride and N-Boc-butanediamine under EDC and HCl gives an amino terminal intermediate with N-Boc protection, which comprises the following steps:
9- (2-carboxyphenyl) -3, 6-bis (diethylamino) xanthene chloride (1.13 mmol,0.50 g), EDC and HCl (1.69 mmol,0.32 g) and HOBT (1.35 mmol,0.18 g) are dissolved in anhydrous dichloromethane, after stirring uniformly at 0 ℃, DIPEA (4.70 mmol,0.61 g) is added dropwise, after reaction for 3min, N-Boc-butanediamine (0.94 mmol,0.18 g) is added for reaction for 4h at room temperature to obtain a crude product with Boc protected amino end intermediate, the crude product is washed three times by saturated sodium bicarbonate, washed by saturated sodium chloride and dried by anhydrous sodium sulfate, the solvent is distilled off under reduced pressure, the crude product is separated and purified by a chromatographic column, the target compound is obtained by eluting with ethyl acetate, and the target compound is obtained by 0.23g and the yield is 39.70%;
LC-MS(ESI,m/z):380.14[M+H] + ，378.10[M-H] -- 。
b) N-Boc protected amino terminal intermediate in CF 3 Under the action of COOH, an intermediate with an amino terminal is obtained, and the specific process is as follows:
the Boc-protected amino-terminal intermediate (0.37 mmol,0.23 g) was dissolved in 20mL dry dichloromethane and 2mL CF was added dropwise at 0deg.C 3 COOH, reacting at 0 deg.C for 1 hr, reacting at 25-30 deg.C for 24 hr to obtain crude product of photoaffinity linker containing biodegradable disulfide bond, and saturated carbonic acidThe pH of the system is regulated to be neutral (pH is approximately equal to 7-8) by sodium hydrogen, the organic phase is collected by extraction of methylene dichloride, the organic phase is respectively dried and washed by saturated sodium chloride and anhydrous sodium sulfate, the solvent is distilled off under reduced pressure, and the intermediate with the amino terminal is obtained, and the weight is 0.16g, and the yield is 84.20%.
LC-MS(ESI,m/z):514.71[M+H] + ，512.65[M-H] - 。
c) Condensing the intermediate with amino terminal with dithiodiacetic acid under EDC & HCl to obtain the belt, the specific process is as follows:
dithiodiacetic acid (3.991 mmol,0.77 g), EDC & HCl (1.915 mmol,0.39 g) and HOBT (1.915 mmol,0.27 g) are dissolved in anhydrous dichloromethane at 0 ℃, triethylamine (1.915 mmol,0.20 g) is added dropwise after stirring for 30min at 0 ℃, an intermediate with an amino end (0.798 mmol,0.43 g) is added, stirring is carried out for 24h at 25-30 ℃ to obtain a pH-response-based multifunctional fluorescent linker crude product, the crude product is obtained by washing with saturated sodium bicarbonate three times, washing with saturated sodium chloride and drying the organic phase with anhydrous sodium sulfate, the solvent is distilled off under reduced pressure, the crude product is separated and purified by a chromatographic column, and ethyl acetate/methanol (V/V=10/1) is used for eluting to obtain the target compound, the target compound is 0.46g, and the yield is 85.15%.
LC-MS(ESI,m/z):678.10[M+H] + ，676.80[M-H] -- 。
The multifunctional fluorescent link body containing the pH response can be applied to diagnosis and treatment integration. The application range is wider, and the multifunctional fluorescent linker based on pH response is a universal type, can be used for modifying active biological small molecules with anti-tumor activity, such as Li Nifa Ni, sorafenib and the like, and can also be used for modifying monoclonal antibody drug molecules with therapeutic effect, such as anti-angiogenesis monoclonal antibody drug bevacizumab and the like, PD1/PDL1 monoclonal antibody drug actizumab and the like.
Preferably, the invention utilizes pH responsive multifunctional fluorescent linker chemical modification targeting anti-tumor drug molecule Li Nifa Ni to construct Li Nifa Ni diagnosis and treatment integrated drug molecule, and utilizes the drug molecule to perform tracing, positioning, imaging analysis and activity screening on some tumor cells.
Construction of Li Nifa Ni "diagnosis and treatment integrated" drug molecules: the pH response multifunctional fluorescent linker (0.074 mmol,0.05 g) and PyBop (0.148 mmol,0.08 g) are dissolved in anhydrous dichloromethane at 0 ℃, triethylamine (0.025 mmol,0.03 g) is added dropwise after stirring for 30min at 0 ℃, li Nifa Ni (0.074 mmol,0.03 g) is added next, stirring is carried out for 24h at room temperature, li Nifa Ni medical molecule crude product is obtained, saturated sodium bicarbonate is used for washing three times, saturated sodium chloride is used for washing, anhydrous sodium sulfate is used for drying organic phase, the solvent is removed by reduced pressure, the crude product is separated and purified by a chromatographic column, the target compound is obtained by eluting with ethyl acetate/methanol (V/V=10/1), the weight is 0.02g, and the yield is 27.6%, thus Li Nifa Ni medical molecule is obtained.
The application of the 'diagnosis and treatment integrated' targeting anti-tumor drug molecules constructed by the multifunctional fluorescent linker containing the pH response in-situ tumor imaging.
The Li Nifa Ni diagnosis and treatment integrated drug molecule synthesized by the design is utilized to image the EA.hy926 cell.
Specific cell imaging steps:
1) Cell seed plates: cell density per well was 4.3 x 105 cells/mL, incubated overnight at 37 ℃;
2) Cell administration: the concentration of each hole of probe is 4 mu mol/L, and the probe is incubated for 2 hours at 37 ℃;
3) Washing: washing the cells three times with PBS to remove excess probe molecules;
4) Cell fixation: after washing was completed, 1mL of 3.7% paraformaldehyde (925 ul 4% paraformaldehyde+75 ul PBS) was added to the cells at room temperature, fixed for 30 minutes, washed twice with PBS (slightly stirred for 1-2 minutes), and permeabilized with PBS solution containing 0.1% Triton X-100 at room temperature for 10 minutes. Cells were then washed twice with PBS, stirred slowly for 1 min, blocked with PBS containing 2% BSA (containing 0.05% Tween-20) for 30min at room temperature, and washed twice with PBS (containing 0.05% Tween-20). Each time for 5 minutes, gently shake.
5) Sealing piece: 5ul of anti-fluorescence quenching sealing tablets were added to each group, and nail oil sealing sheets were added around.
6) And (3) detecting by a fluorescence microscope: the cell imaging result is shown in figure 2, the probe concentration is 4 mu mol/L, and as can be seen from figure 2, tumor cells are fluorescently labeled, so that the constructed probe can realize the labeling in diagnosis and treatment integration, namely one step of diagnosis.
The application of the 'diagnosis and treatment integrated' targeting antitumor drug molecule constructed by the multifunctional fluorescent linker containing the pH response in the aspect of antitumor activity.
The Li Nifa Ni diagnosis and treatment integrated drug molecule synthesized by the design is used for carrying out proliferation inhibition analysis and evaluation on EA.hy926 cells (human umbilical vein cells), hela (cervical cancer cells) and MDA-MB-231 (breast cancer cells).
The activity was evaluated using MTT:
1) Cell seed plates: 96-well plate with cell density of 1 x 10 per well 5 cells/mL, 180 μl of cell suspension was added per well and incubated overnight at 37 ℃.
2) Cell administration: 6 concentration gradients were set up, 20. Mu. Mol/L, 4. Mu. Mol/L, 0.8. Mu. Mol/L, 0.16. Mu. Mol/L, 0.032. Mu. Mol/L, 0.0064. Mu. Mol/L, 20ml of each well was added and incubated at 37℃for 24h;
3) Cells were given MTT: 22 mu mol/L is added to each hole, and the mixture is incubated for 4 hours at 37 ℃;
4) Cell treatment: the liquid from each well was aspirated, 150. Mu. LDMSO was added and incubated on a shaker at room temperature for 10min.
5) Absorbance measurement: the absorbance at 490nm was measured by placing the 96-well plate on an enzyme-labeled instrument.
6) And (3) calculating inhibition rate: inhibition = negative well OD-donor well OD/negative well OD-void white OD
The cell proliferation inhibition results are shown in fig. 3-6, and it can be seen from fig. 3-6 that the constructed probe molecules have anti-cell proliferation activity similar to that of the positive drug molecules. The probe molecules constructed can be shown to realize one treatment step in the concept of 'diagnosis and treatment integration'.
1) Multifunctional fluorescent link fluorescence lighting characteristic investigation based on pH response: preparation of Britton-Robinson (B-R) buffer solution
Acid mixed solution of phosphoric acid, boric acid and acetic acid with the concentration of 0.04mol/L and 0.2mol/L NaOH solution are prepared into B-R buffer solutions with different pH values according to different proportions, and the specific preparation method is shown in the following table 1:
TABLE 1 configuration of B-R buffer solutions at different pH values
2) Preparation of stock solution
0.0678g of pH sensitive fluorescent linker was dissolved in 5mL of DMSO solvent to prepare a mother liquor having a concentration of 0.02 mol/L. Diluting 200 times to obtain a concentration of 1×10 -4 mol/L Li Nifa Ni fluorescent probe stock solution.
3) Preparation of linker solutions of different pH
And transferring 100 mu L of stock solution of the fluorescent linker into a 10mL volumetric flask, adding B-R buffer solutions with different pH values, and fixing the volume to 10mL to prepare a linker solution with the concentration of 1 mu mol/L under the corresponding pH condition.
4) Fluorescence measurement of probe solutions
And (3) measuring the fluorescence emission spectrum of lambda=500-700 nm when the excitation wavelength is lambda=365 nm by configuring the obtained fluorescent linker solutions with different pH values, obtaining the emission wavelength with the strongest fluorescence intensity, and making a pH sensitive fluorescent linker-pH value quantitative curve under the emission wavelength.
The scanned fluorescence spectra of the pH-sensitive fluorescent linker at different pH conditions are shown in FIG. 7:
as can be seen from fig. 7, the pH-sensitive fluorescent linker has peaks at emission wavelengths λ=580 nm and λ=645 nm under different pH conditions, wherein the fluorescence intensity is maximum at the emission wavelength λ=580 nm, so λ=580 nm is selected as the emission wavelength for pH quantification.
The fluorescence measurement results of the fluorescent linker with different pH values at the emission wavelength of lambda=580 nm are shown in FIG. 8, and it can be seen from FIG. 8 that the synthesized pH sensitive fluorescent linker has a significant trend of change in the pH=5.72-7.54 interval. The fluorescence intensity of the fluorescence link body increases along with the increase of the pH value in the pH=5.72-6.09 interval, and the variation amplitude of the fluorescence intensity is large; in the range of ph=6.09 to 7.54, the fluorescence intensity decreases with an increase in pH value, and the change range of the fluorescence intensity is relatively small. In addition, the pH change interval just accords with the difference between the tumor microenvironment and the normal human tissue microenvironment, namely, the pH sensitive fluorescent linker shows weaker fluorescence under the normal tissue pH condition (the pH is between 7.2 and 7.4), and the probe emits stronger fluorescence under the tumor tissue pH condition (the pH is between 6.2 and 6.9). Therefore, the change trend of the fluorescence of the pH sensitive fluorescent linker along with the pH accords with the difference of the pH of the tumor microenvironment and the pH of the normal human tissue microenvironment, and the purpose that the fluorescent probe linked with the linker generates fluorescence to perform fluorescent tracing after entering the tumor microenvironment can be achieved.
Claims (10)
1. The multifunctional fluorescent linker based on pH response is characterized by comprising the following structural formula:
2. the preparation method of the multifunctional fluorescent linker based on pH response is characterized by comprising the following steps:
a) Condensing 9- (2-carboxyphenyl) -3, 6-bis (diethylamino) xanthene chloride and N-Boc-butanediamine under EDC and HCl to obtain an amino terminal intermediate with Boc protection;
b) Amino terminal intermediates with Boc protection in CF 3 Obtaining an intermediate with an amino terminal under the action of COOH;
c) Condensing the intermediate with amino terminal with dithiodiacetic acid under EDC & HCl to obtain the multifunctional fluorescent linker based on pH response, wherein the structural formula is as follows:
3. the method for preparing the multifunctional fluorescent linker based on pH response according to claim 2, wherein the specific process of step a) is as follows: 9- (2-carboxyphenyl) -3, 6-bis (diethylamino) xanthene chloride, EDC, HCl and HOBT are dissolved in anhydrous dichloromethane, and after being stirred uniformly at 0 ℃, DIPEA is added dropwise, after the dropwise addition, N-Boc-butanediamine is added for reaction, and an amino terminal intermediate with Boc protection is obtained.
4. The method for preparing the multifunctional fluorescent linker based on pH response according to claim 2, wherein the specific process of the step b) is as follows: the amino-terminal intermediate with Boc protection was dissolved in anhydrous dichloromethane and CF was added dropwise at 0deg.C 3 COOH, stirring for 1h after dripping, monitoring by TLC until the reaction is completed, adding saturated sodium bicarbonate aqueous solution after the reaction is completed, titrating until the system is neutral, extracting, collecting an organic phase, and spin-drying to obtain an intermediate with an amino terminal.
5. The method for preparing the multifunctional fluorescent linker based on pH response according to claim 2, wherein the specific process of step c) is as follows: dissolving dithio-diacetic acid in anhydrous dichloromethane, adding EDC, HCl and HOBT, stirring at 0 ℃ for 30min, then dripping triethylamine, and stirring at room temperature for 24h to obtain the multifunctional fluorescent linker based on pH response.
6. Use of the pH-response based multifunctional fluorescent linker of claim 1 for the preparation of a diagnostic integrated medicament for the treatment of tumors.
7. The use according to claim 6, wherein the pH-responsive multifunctional fluorescent linker and PyBop are mixed in anhydrous dichloromethane at 0 ℃, triethylamine is added dropwise after uniform stirring at 0 ℃, then an anti-tumor drug is added, and the mixture is stirred at 25 ℃ for 24 hours to obtain the diagnosis and treatment integrated drug.
8. The use according to claim 7, wherein the pH-responsive multifunctional fluorescent linker 0.074mmol and PyBop0.148mmol are stirred in anhydrous dichloromethane at 0 ℃ for 30min, and then triethylamine 0.295mmol is added dropwise, and then the antitumor drug 0.074mmol is added and stirred at room temperature for 24h to obtain the diagnosis and treatment integrated drug.
9. The use according to claim 6, wherein the anti-tumor drug is Li Nifa ni, sorafenib, bevacizumab or actizumab; the tumor is human umbilical vein cells, cervical cancer cells or breast cancer cells.
10. Use of the pH-response based multifunctional fluorescent linker of claim 1 for constructing a drug probe for labeling.
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