CN110511245B - Near-infrared fluorescent probe SHCy-P based on thiohemicyanine dye and preparation method and application thereof - Google Patents
Near-infrared fluorescent probe SHCy-P based on thiohemicyanine dye and preparation method and application thereof Download PDFInfo
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- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
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- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/10—The polymethine chain containing an even number of >CH- groups
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The invention provides a near-infrared fluorescent probe SHCy-P based on thiohemicyanine dye and a preparation method and application thereof. Adding heptamethine indocyanine dye solution dropwise into acetonitrile of potassium carbonate and 3-hydroxythiophenol to react to obtain a product SHCy, and adding the SHCy into dry CH2Cl2Dissolving and dropping POCl3And pyridine, and obtaining a solid probe SHCy-P after reaction. The probe synthesis process is simple in operation process and mild in reaction condition. The prepared probe has high selectivity and sensitivity, can react with acid phosphatase in a wider pH range to cause obvious change of a fluorescence emission spectrum in a near infrared region, thereby realizing the specific detection of the acid phosphatase. Meanwhile, cell imaging experiments show that the probe has good cell membrane permeability, low cytotoxicity and lysosome targeting positioning function, and the fluorescence imaging detection of acid phosphatase in human prostate cancer (PC-3) cells is successfully realized.
Description
Technical Field
The invention relates to the technical field of molecular fluorescent probes, in particular to a near-infrared fluorescent probe for detecting acid phosphatase based on thiohemicyanine dye, and a preparation method and application thereof.
Background
In recent years, thanks to the development of fluorescence imaging technology, the fluorescence probe technology can realize non-invasive and high-time-space resolution visual detection at the level of cells and organelles due to the advantages of selectivity, sensitivity, quick response and the like, and therefore, the fluorescence probe technology has great application potential in the field of detecting and monitoring bioactive molecules. Compared with a fluorescent probe with an emission spectrum positioned in an ultraviolet-visible light region, the near-infrared fluorescent probe with the emission spectrum positioned in a light transmission region (650-900nm) of the biological tissue shows better tissue penetrability and smaller light damage, can effectively avoid interference caused by background fluorescence of the biological tissue, and is favored by researchers.
Acid phosphatases are a class of hydrolases widely distributed in mammals, whose catalytic activity is closely related to human health. Clinically, acid phosphatase is used as a tumor marker of metastatic prostate cancer, and can be used for diagnosing prostate diseases and prostate cancer by detecting the concentration and activity of acid phosphatase. At present, the work of detecting the acid phosphatase by using a fluorescent probe technology is relatively less, and the fluorescent-on imaging detection of the acid phosphatase in cells cannot be realized. Therefore, the development of the fluorescence-activated near-infrared fluorescent probe with high selectivity and sensitivity for detecting the acid phosphatase in the living cells has very important scientific significance and practical value.
Disclosure of Invention
The invention aims to provide a near-infrared fluorescent probe capable of specifically detecting acid phosphatase in-vitro and in-vivo environments, a preparation method and application thereof aiming at the problems of the existing fluorescent probe for detecting the acid phosphatase.
The technical scheme of the invention is as follows:
the designed and synthesized near-infrared fluorescent probe SHCy-P based on thiohemicyanine dye has the following structural general formula:
a preparation method of the near-infrared fluorescent probe SHCy-P based on the thiohemicyanine dye comprises the following preparation processes:
the preparation method comprises the following specific steps:
1) the heptamethine indole cyanine dye solution is slowly dripped into acetonitrile solution of potassium carbonate and 3-hydroxythiophenol, and then the mixture reacts for 3 hours at 50 ℃, and after the reaction is finished, the mixture is cooled to room temperature to obtain reaction liquid. And (3) carrying out suction filtration on the reaction solution, and carrying out reduced pressure distillation and concentration on the filtrate to obtain a crude product. Separating and purifying the crude product by using column chromatography silica gel, wherein the volume ratio of eluent is 100: 1-2 in dichloromethane-methanol to obtain a blue intermediate SHCy. The molar ratio of the heptamethine indole cyanine dye to the potassium carbonate to the 3-hydroxythiophenol is 1:2: 2; the weight ratio of the heptamethine indocyanine dye to the solvent acetonitrile is 1: 6-7; the weight ratio of the total weight of the potassium carbonate and the 3-hydroxythiophenol to the solvent acetonitrile is 1: 14-15.
2) After the intermediate SHCy was reacted with phosphorus oxychloride in dry dichloromethane added with pyridine at room temperature for 4 hours, the reaction solution was poured into ice water and stirred overnight, followed by extraction with dichloromethane, the organic phase was collected and dried over anhydrous sodium sulfate, and concentrated by distillation under reduced pressure to obtain a crude product. Separating and purifying the crude product by using column chromatography silica gel, wherein the volume ratio of eluent is 100: 2-5 of dichloromethane-methanol to obtain a blue powder probe SHCy-P. The molar ratio of the intermediate SHCy to the phosphorus oxychloride and the pyridine is 1: 1.5: 4.5; the weight ratio of the intermediate SHCy to the dichloromethane is 1: 60-70; the volume ratio of the pyridine to the dichloromethane is 1: 60-70; the volume ratio of the ice water to the reaction liquid is 4-5: 1; the volume ratio of the dichloromethane to the ice water in the extraction process is 2-2.5: 1.
The application of the near-infrared fluorescent probe SHCy-P based on the thiohemicyanine dye is as follows:
the near-infrared fluorescent probe SHCy-P is used for selectively identifying the acid phosphatase from different enzyme species, the acid phosphatase can cause the remarkable fluorescence enhancement of a probe solution, and the probe can be used for carrying out fluorescence imaging detection on the acid phosphatase in cells.
The invention has the advantages and beneficial effects that:
the invention designs and synthesizes a near-infrared fluorescent probe SHCy-P based on thiohemicyanine dye. The probe has simple synthesis process and mild reaction condition. The prepared probe has high selectivity and sensitivity, can react with acid phosphatase in a wider pH range to cause obvious change of a fluorescence emission spectrum in a near infrared region, thereby realizing the specific detection of the acid phosphatase. Meanwhile, the probe has good cell membrane permeability, low cytotoxicity and a lysosome targeting positioning function, and successfully realizes the fluorescent imaging detection of acid phosphatase in human prostate cancer (PC-3) cells.
Drawings
FIG. 1 shows the structure of fluorescent probe SHCy-P.
FIG. 2 shows the fluorescence enhancement at 765nm of the fluorescent probe SHCy-P caused by different enzymes.
FIG. 3 is a photograph showing fluorescence images of PC-3 intracellular ACP with the fluorescent probe SHCy-P.
Detailed Description
Example 1, intermediate SHCy was prepared as follows:
the preparation method comprises the following specific steps:
1) stirring at room temperature under the protection of argon, slowly dropwise adding a heptamethine indocyanine dye (570mg, 1mmol) solution into acetonitrile (10mL) of potassium carbonate (272mg, 2mmol) and 3-hydroxythiophenol (252mg, 2mmol), reacting at 50 ℃ for 3 hours, and cooling to room temperature after the reaction is finished to obtain a reaction solution;
2) and (3) carrying out suction filtration on the reaction solution, and carrying out reduced pressure distillation and concentration on the filtrate to obtain a crude product. Separating and purifying the crude product by using column chromatography silica gel, wherein the volume ratio of eluent is 100: 1-2 to obtain a target product SHCy which is a blue solid and has a yield of 81.6 percent; melting point: is more than 300 ℃.
1H NMR(DMSO-d6,400MHz,ppm)8.23(1H),7.78(1H),7.70(1H),7.54(1H),7.48(2H),7.34(1H),7.08(1H),6.91(1H),6.68(1H),3.89(3H),2.73(2H),2.67(2H),1.84(2H),1.74(6H)；13C NMR(DMSO-d6,100MHz,ppm)178.02,159.94,152.10,144.50,142.76,142.68,135.89,135.24,133.16,131.38,129.33,127.82,126.38,123.15,121.84,117.45,113.90,111.17,107.07,50.88,33.26,32.12,27.81,26.70,20.54。
Example 2, preparation route of probe SHCy-P was as follows:
the preparation method comprises the following specific steps:
1) intermediate SHCy (100mg, 0.2mmol) was weighed into a 50mL round bottom flask and 5mL dry CH was added2Cl2Dissolving, and dropwise adding 1.5 equivalent of POCl3(35. mu.L, 0.3mmol) and 75. mu.L pyridine, under argon atmosphere, and stirred at room temperature for 4 hours.
2) The reaction solution was poured into 20mL of ice water and stirred overnight, extracted with 3 × 15mL of dichloromethane, the organic phase was spin-dried, and purified by silica gel column chromatography (developing solvent: CH (CH)2Cl2MeOH 50/1-20/1) yielded 40mg of blue solid probe SHCy-P in 35% yield. M.p. 208-.
1H NMR(DMSO-d6,400MHz,ppm):δ＝8.26(1H),7.76(1H),7.72(1H),7.62(s,1H),7.53(3H),7.31(1H),7.11(1H),6.74(1H),3.92(3H),2.73(2H),2.66(2H),1.84(2H),1.76(6H)；13C NMR(DMSO-d6,100MHz,ppm):δ＝178.57,151.49,144.92,142.91,142.71,135.05,133.72,132.28,132.14,129.33,128.08,126.68,124.03,123.35,123.11,115.16,114.14,107.79,99.66,52.60,52.54,51.11,33.46,32.15,27.81,20.46；31PNMR(DMSO-d6,160MHz,ppm):δ＝-4.81。
Fluorescent detection application of the probe SHCy-P:
the probe was prepared at a concentration of 5.0X 10-3And storing the solution in mol/L of DMF in dark for later use. The detection method comprises the following steps:
1) selective detection of Probe SHCy-P
The probes were prepared at 1X 10 in Tris-HCl buffer solution, respectively-53mL of the solution to be detected in mol/L, then respectively adding 2U/mL of different enzymes (1-7: acid phosphatase, alkaline phosphatase, beta-galactosidase, lysozyme chloride, thrombin, glucose oxidase and trypsin), keeping the temperature at 37 ℃ for 30 minutes, exciting at the wavelength of 700nm, testing the fluorescence emission spectrum of each solution, and then calculating the fluorescence intensity enhancement at 765 nm. As shown in FIG. 2, only the acid phosphatase caused a significant fluorescence enhancement.
2) Probe SHCy-P for fluorescent imaging detection of acid phosphatase in PC-3 cells
The above description is provided for further details of the present invention with reference to specific embodiments, and it should not be understood that the present invention is not limited to these descriptions, and it should be understood that those skilled in the art can make several simple deductions or substitutions without departing from the spirit of the present invention, and all shall be considered as belonging to the protection scope of the present invention.
Claims (5)
1. A near-infrared fluorescent probe SHCy-P based on thiohemicyanine dye is characterized in that the probe can selectively identify acid phosphatase to cause the change of a fluorescence emission spectrum in a near-infrared region, and the structural general formula is as follows:
2. the preparation method of the near-infrared fluorescent probe SHCy-P based on thiohemicyanine dye as claimed in claim 1, is characterized in that the preparation process is as follows:
the method comprises the following steps:
1) slowly dripping the heptamethine indocyanine dye solution into acetonitrile solution of potassium carbonate and 3-hydroxythiophenol, then reacting for 3 hours at 50 ℃, and cooling to room temperature after the reaction is finished to obtain reaction liquid;
2) and (3) carrying out suction filtration on the reaction liquid, carrying out reduced pressure distillation and concentration on the filtrate to obtain a crude product, separating and purifying the crude product by using column chromatography silica gel, wherein the volume ratio of the eluent is 100: 1-2 to obtain a blue intermediate SHCy with near-infrared emission;
3) adding SHCy obtained in the step 2), phosphorus oxychloride and pyridine into a round-bottom flask, and adding into a dry dichloromethane chamber for reaction for 4 hours;
4) pouring the reaction liquid obtained in the step 3) into ice water, stirring overnight, extracting with dichloromethane, collecting an organic phase, drying with anhydrous sodium sulfate, carrying out reduced pressure distillation and concentration to obtain a crude product, and separating and purifying the crude product by using column chromatography silica gel, wherein the volume ratio of the eluent is 100: 2-5 of dichloromethane-methanol to obtain a blue powder probe SHCy-P.
3. The method for preparing the near-infrared fluorescent probe SHCy-P based on the hemicyanine dye as claimed in claim 2, wherein: the molar ratio of the heptamethine indole cyanine dye to the potassium carbonate to the 3-hydroxythiophenol is 1:2: 2; the weight ratio of the heptamethine indocyanine dye to the solvent acetonitrile is 1: 6-7; the weight ratio of the total weight of the potassium carbonate and the 3-hydroxythiophenol to the solvent acetonitrile is 1: 14-15.
4. The method for preparing the near-infrared fluorescent probe SHCy-P based on the hemicyanine dye as claimed in claim 2, wherein: the molar ratio of the intermediate SHCy to the phosphorus oxychloride and the pyridine is 1: 1.5: 4.5; the weight ratio of the intermediate SHCy to the dichloromethane is 1: 60-70; the volume ratio of the pyridine to the dichloromethane is 1: 60-70; the volume ratio of the ice water to the reaction liquid is 4-5: 1; the volume ratio of the dichloromethane to the ice water in the extraction process is 2-2.5: 1.
5. The use of a near-infrared fluorescent probe SHCy-P for detecting acid phosphatase based on a thiohemicyanine dye according to claim 1, wherein the probe is used for detecting acid phosphatase in a solution.
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