CN116178349A - Golgi targeting near infrared fluorescent probe for detecting cysteine, preparation method and application thereof - Google Patents
Golgi targeting near infrared fluorescent probe for detecting cysteine, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a Golgi apparatus targeting near infrared fluorescent probe for detecting cysteine, a preparation method and application thereof, wherein the molecular formula of the fluorescent probe is C36H34N3O6S+, and the structural formula is as follows:
Description
Technical Field
The invention belongs to the technical field of organic synthesis, and particularly relates to a Golgi targeting near infrared fluorescent probe for detecting cysteine, a preparation method and application thereof.
Background
Biological thiols such as cysteine, homocysteine, glutathione play a vital role in maintaining redox balance in vivo. But the three are similar in structure and chemical property and are difficult to distinguish.
Fluorescent photoprobes have received much attention because of their relatively high sensitivity and ease of detection of biologically active molecules within biological cell systems. More importantly, compared with other technologies for detecting trace molecules, the fluorescent probe can detect trace small molecules in cells and organisms in situ in real time without damaging cells and organelles. By confocal microscopy fluorescence imaging and the like, researchers have studied biomolecules in organelles such as mitochondria, lysosomes, endoplasmic reticulum, and the like. The tracking detection of biological thiols in living organisms has been very mature in recent years. However, probes for in situ investigation of small molecules in the golgi apparatus are currently rare. To our knowledge, although many excellent cysteine-specific fluorescent probes have been reported, near infrared fluorescent probes for in situ detection of cysteines in the golgi apparatus are only in a few columns. Therefore, the Cys content in cells and organisms can be monitored in real time in a targeted manner, and the method has important significance for early diagnosis of diseases and evaluation of the diseases.
Since thiols have a very important role in biology, clinic and environment, designing and developing a method for detecting thiols will have a great impact on human life. Some common detection and analysis technologies such as high performance liquid chromatography, electrochemical methods, capillary electrophoresis methods and the like, but the methods have high equipment cost, poor selectivity and complicated operation steps. In contrast, fluorescent probes that are simple, efficient and capable of targeted localization are of interest.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a Golgi apparatus targeting fluorescent probe for detecting cysteine. The fluorescent probe has stable cysteine response, good linear relation and selectivity, and is successfully used for cysteine imaging in living cell golgi and in vivo fluorescence imaging of mice.
The invention also provides a preparation method and application of the fluorescent probe.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a Golgi apparatus targeting fluorescent probe for detecting cysteine, the molecular formula is C 36 H 34 N 3 O 6 S + The structural formula is as follows:
the preparation method of the targeted Golgi fluorescent probe for detecting cysteine comprises the following steps:
(1) Cyclohexanone, phosphorus tribromide and DMF are reacted in chloroform to obtain a compound 1;
(2) 2, 4-dihydroxybenzaldehyde and 3, 4-dihydro-2H-pyran react under the action of a catalyst of pyridinium tosylate to obtain a compound 2;
(3) Reacting the compound 2, the compound 1 and potassium carbonate in DMF to obtain a compound 3;
(4) Adding trifluoroacetic acid into dichloromethane in the compound 3, and stirring for reaction to obtain a compound 4;
(5) Reacting p-aminobenzenesulfonamide with chloroacetyl chloride to obtain a compound 5;
(6) Reacting the compound 5 with 2, 3-trimethyl-3H-indole to obtain a compound 6;
(7) Reacting the compound 6 with the compound 4 to obtain a compound 7;
(8) And (3) reacting the compound 7, the acryloyl chloride and the triethylamine in DMF to obtain the fluorescent probe.
Preferably, the preparation process of the compound 1 in the step (1) specifically comprises the following steps: under the ice bath condition, dripping phosphorus tribromide into a mixed solution of N, N-dimethylformamide and chloroform, stirring for 0.5-1.5 h, dripping cyclohexanone into the solution, stirring for complete reaction, neutralizing to neutrality, extracting a product with dichloromethane, and concentrating by rotary evaporation to obtain a target product.
Preferably, the preparation process of the compound 2 in the step (2) specifically comprises the following steps: weighing 2, 4-dihydroxybenzaldehyde and pyridine p-toluenesulfonate respectively, and adding into CH 2 Cl 2 And adding 3, 4-dihydro-2H-pyran, stirring to react completely, steaming, and performing column chromatography to obtain compound 2.
Preferably, the preparation process of the compound 3 in the step (3) specifically comprises the following steps: adding the prepared compound 2, the compound 1 and potassium carbonate into DMF, stirring at 35-45 ℃ for complete reaction, cooling to room temperature, filtering and concentrating to obtain concentrated solution of the compound 3.
Preferably, the preparation process of the compound 4 in the step (4) specifically comprises: and (3) adding trifluoroacetic acid into the concentrated solution obtained in the step (3), stirring at 35-45 ℃ for complete reaction, cooling to room temperature, carrying out suction filtration, washing with ethanol, and drying to obtain the compound 4.
Preferably, the preparation process of the compound 5 in the step (5) specifically comprises: suspending the sulfanilamide in acetone, dropwise adding chloracetyl chloride at room temperature, stirring the reaction mixture at 70-80 ℃ to react completely, cooling to room temperature, adding ice water, stirring the obtained mixture, filtering, washing the collected solid with ice water, recrystallizing with ethanol, filtering, and drying the obtained solid to obtain the compound 5.
Preferably, the preparation process of the compound 6 in the step (6) specifically comprises: and (3) dissolving the compound 5 obtained in the step (5) in acetonitrile under the nitrogen atmosphere, adding 2, 3-trimethyl-3H-indole by a syringe, stirring, refluxing, completely reacting, performing vacuum rotary evaporation on the reacted liquid, adding dichloromethane, and performing vacuum suction filtration to obtain a solid, namely the compound 6.
Preferably, the preparation process of the compound 7 in the step (7) specifically comprises: dissolving the compound 6 and the compound 4 in ethanol, stirring, refluxing and reacting until solid precipitate is generated, and filtering to obtain a solid, namely the compound 7.
Preferably, the preparation process of the compound 8 in the step (8) specifically comprises the following steps: and (3) dissolving the compound 7 in DMF under nitrogen atmosphere, adding triethylamine, stirring uniformly, adding acryloyl chloride under ice bath condition, stirring for a period of time in ice bath, stirring at room temperature, reacting completely, removing DMF by vacuum rotary evaporation, adding distilled water, stirring, vacuum filtering, and vacuum drying the obtained solid to obtain the fluorescent probe.
Preferably, in step (1), PBr 3 The mole ratio of DMF to cyclohexanone is 2.5-2.6 to 2.3:1.
Preferably, in step (2), the molar ratio of 2, 4-dihydroxybenzaldehyde, 3, 4-dihydro-2H-pyran and pyridine p-toluenesulfonate is 1 (1-1.2): 0.05.
preferably, in step (3), the molar ratio of compound 1, compound 2 to potassium carbonate is 2:1:3.
Preferably, in step (4), the molar ratio of compound 3 to trifluoroacetic acid is 10 (1-2).
Preferably, in the step (5), the molar ratio of the sulfanilic acid amide to the chloroacetyl chloride is 1 (0.9-1.5).
Preferably, in step (6), the molar ratio of compound 5 to 2, 3-trimethyl-3H-indole is 1 (1-1.5).
Preferably, in step (7), the molar ratio of compound 4 to compound 6 is 1 (1 to 1.5).
Preferably, in the step (8), the molar ratio of the compound 7, triethylamine and the acryloyl chloride is 1 (1-4): 1-4.
The fluorescent probe is applied to cysteine detection for the purpose of diagnosis of non-diseases.
The application of the upper fluorescent probe in preparing a cell fluorescent imaging agent.
The application of the fluorescent probe in preparing the targeting positioning Golgi fluorescent probe.
Compared with the prior art, the invention has the beneficial effects that:
1. the acryl of the fluorescent probe of the invention is cleaved under the induction of cysteine, accompanied by a remarkable enhancement of NIR fluorescence emission, while the probe can rapidly distinguish cysteine from other bioactive small molecules such as thiol and amino acid according to different kinetic rates thereof. And the probe is visible to the naked eye with a significant change in fluorescence intensity and a concomitant significant color change.
2. The fluorescent probe provided by the invention is simple to synthesize, has high molar absorption coefficient in an NIR region and good biocompatibility, can successfully perform fluorescent imaging in living cells and mice, and can successfully verify the Golgi localization capability. The probe can sensitively and selectively identify the change of the cysteine content in cells and mice, and has a strong application prospect in biomedicine.
Drawings
FIG. 1 is a synthetic route of a near infrared fluorescent probe prepared in example 1;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of the near infrared fluorescent probe prepared in example 1;
FIG. 3 is a nuclear magnetic resonance spectrum of the near infrared fluorescent probe prepared in example 1;
FIG. 4 is an ultraviolet absorption spectrum of the near infrared fluorescent probe prepared in example 1 in relation to cysteine response over time;
FIG. 5 is a fluorescence spectrum of the near infrared fluorescent probe prepared in example 1 showing the time response of cysteine;
FIG. 6 is a graph showing the change of the ultraviolet absorption spectrum of the near infrared fluorescent probe prepared in example 1 according to the concentration of cysteine;
FIG. 7 shows the fluorescence spectrum of the near infrared fluorescent probe prepared in example 1 according to the concentration of cysteine;
FIG. 8 is a graph showing the linear relationship between the fluorescence emission wavelength and the intensity at 705nm of the near infrared fluorescent probe prepared in example 1;
FIG. 9 is an ultraviolet absorption spectrum of a near infrared fluorescent probe prepared in example 1 with different amino acids and ions;
FIG. 10 shows different ions (1) probe, (2) NO, in order from left to right 2 - 、(3)AcO - 、(4)H 2 PO 4 - 、(5)S - 、(6)F - 、(7)Cl - 、(8)Br - 、(9)Zn 2+ 、(10)Cu 2+ 、(11)Fe 3+ A bar chart of fluorescence intensity at 705.0nm of (12) Phe, (13) Glu, (14) Mel, (15) gin, (16) Flr, (17) Ser, (18) Leu, (19) GSH, (20) Hcy, (21) Cys, and the near infrared fluorescent probe prepared in example 1;
FIG. 11 is a chart showing the ability of near infrared fluorescent probe prepared in example 1 to recognize cysteine as interference test: from left to right, in order, (1) probe, (2) NO 2 - 、(3)AcO - 、(4)H 2 PO 4 - 、(5)S - 、(6)F - 、(7)Cl - 、(8)Br - 、(9)Zn 2+ 、(10)Cu 2+ 、(11)Fe 3+ 、(12)Phe、(13)Glu、(14)Mel、(15)Gln、(16)Flr、(17)Ser、(18)Leu、(19)GSH、(20)Hcy、(21)Cys。
FIG. 12 is a graph showing the color comparison of cysteine and near infrared fluorescence probe prepared in example 1;
FIG. 13 is a biological imaging of near infrared fluorescent probes for cysteine detection in living cells;
FIG. 14 is a biological imaging of oxidative stress of near infrared fluorescent probes in living cells;
FIG. 15 is a diagram of a near infrared fluorescent probe localization capability test biological imaging in living cells;
FIG. 16 is a biological imaging of near infrared fluorescent probes for cysteine detection in mice;
FIG. 17 is a biological imaging of near infrared fluorescent probe human liver cancer HepG-2 cell line nude mouse engrafting tumor.
Detailed Description
The invention is further illustrated, but not limited, by the following examples and figures.
Example 1
The preparation process of the fluorescent probe specifically comprises the following steps:
(1) Preparation of Compound 1
The synthetic route is as follows:
separately measuring CaH 2 Dried DMF (11.2 mL,146 mmol), anhydrous CHCl 3 50mL was added to a 100mL two-necked flask and stirred with a magnetic stirrer. Measuring phosphorus tribromide (PBr) 3 ) (12.4 mL,130.7 mmol) was placed in a constant pressure dropping funnel, and then slowly dropped into the flask under ice-water bath (0 ℃ C.) to slowly turn the solution into cream yellow. After about 50 minutes of reaction, the water bath was removed, cyclohexanone (5 mL,56.5 mmol) was weighed into the flask and stirred at room temperature. The reaction was monitored by TLC, after TLC plates were dried with phosphomolybdic acid, the reaction was stopped after no starting points for cyclohexanone were shown, and the reaction was completed for about 16 hours. The solution in the bottle was added to a large beaker containing 100mL of ice water with stirring, and then the anhydrous sodium carbonate solid was added with stirring for neutralization. 100mL of CH is added 2 Cl 2 Extracting, separating, transferring the lower organic phase into clean conical flask, na 2 SO 4 Drying, followed by filtration and rotary evaporation gave compound 1 (liquid) having a mass of 6.5280g and a yield of about 67.6%.
(2) Preparation of Compound 2
The synthetic route is as follows:
2, 4-dihydroxybenzaldehyde (2.76 g,20 mmol) and pyridine p-toluenesulfonate (PPTS, catalyst, 0.25 g,1 mmol) were weighed into a 50mL round bottom flask (cover at any time, catalyst was easy to absorb water, slightly more in the experiment), and 30mL CH was measured 2 Cl 2 Added to the flask and stirring was started. Thereafter, 3, 4-dihydro-2H-pyran (4.15 mL,45.5 mmol) was weighed into the flask, the metal sand bath temperature was set at 30℃and stirring was continued. The reaction was monitored by spot TLC plates and stopped for about 8 h. Rotary steaming after the reaction is completed, and separating by column chromatography (eluent petroleum ether: ethyl acetate volume ratio =15:1) to give compound 2 as white crystals, 3.81g in mass, about 86.1% in yield.
(3) Preparation of Compound 3
The synthetic route is as follows:
compound 2 (2.00 g,9 mmol), anhydrous potassium carbonate (3.73 g,27 mmol) and DMF 10mL were added sequentially to a 250mL round bottom flask and stirred. Then, weighed amount of Compound 1 (3.41 g,18 mmol) was added to the flask, and the reaction was stirred at 40 ℃. The reaction was monitored by spot TLC plate, and the reaction was stopped after about 16h of completion, and cooled to room temperature. Insoluble solids were removed by suction filtration. The organic phase was transferred to a clean round bottom flask and spin evaporated to give an orange solution (part of the solvent was retained) which was used directly in the next reaction without work-up.
(4) Preparation of Compound 4
The synthetic route is as follows:
crude product 3 (1.76 g,5.6 mmol) and 10ml of dichloromethane were taken in a round-bottomed flask, and 0.5mmol of trifluoroacetic acid was added thereto and stirred in a sealed condition at 40 ℃. The reaction was monitored by spot TLC, after the reaction was substantially completed, the heating and stirring were stopped, cooled to room temperature, suction filtration was performed, and then an orange-yellow solid was obtained by washing with ethanol, and compound 4 was obtained after vacuum drying, the mass of the solid was 1.0480g, and the yield was about 70.8%.
(5) Preparation of Compound 5
The synthetic route is as follows:
paramamide (1.0 g,5.81 mmol) was suspended in acetone and chloroacetyl chloride (0.45 ml,5.53 mmol) was added dropwise at room temperature. The reaction mixture was stirred at 75 ℃ for 1 hour and then cooled to room temperature. Ice water was added and the resulting mixture was stirred for a period of time and filtered. The collected solid was washed with ice water and then recrystallized from ethanol. The solid was collected by filtration and dried in vacuo to give compound 5 as a white solid (0.88 g) and the filtrate was concentrated to give more product 0.34g in about 89.2% yield. Compound 5 profile information is as follows:
1 H NMR(400MHz,DMSO)δ10.64(s,1H),7.77(q,J＝8.9Hz,4H),7.29(s,2H),4.31(s,2H).
13 C NMR(101MHz,DMSO)δ165.65(s),141.82(s),139.44(s),127.28(s),119.46(s),44.02(s).
(6) Preparation of Compound 6
The synthetic route is as follows:
compound 5 (2.48 g,10 mmol) was dissolved in acetonitrile and 2, 3-trimethyl-3H-indole (1.9 g,12 mmol) was injected into a solution of compound 5 in acetonitrile and reacted under reflux stirring at 120℃for 12 hours under nitrogen protection. The reaction solution after the reaction is steamed in a rotary way in vacuum, after the rotary steaming is finished, dichloromethane is added, the mixture is vibrated, kept stand, decompressed and filtered to obtain the compound 6 (3.5 g,9.4 mmol), and the yield is about 94.2%. The nuclear magnetic information of compound 6 is as follows:
1 H NMR(400MHz,DMSO)δ12.19(s,1H),8.03(d,J＝4.1Hz,1H),7.90–7.78(m,5H),7.63(d,J＝3.0Hz,2H),7.34(s,2H),5.80(s,2H),2.89(s,3H),1.60(s,6H).
13 C NMR(101MHz,DMSO)δ200.26(s),162.79(s),141.86(s),141.57–141.47(m),139.76(s),130.05(s),129.61(s),127.25(s),124.09(s),119.45(s),115.37(s),54.92(s),50.74(s),22.59(s),14.93(s).
(7) Preparation of Compound 7
The synthetic route is as follows:
under the protection of nitrogen, compound 4 (228 mg,1 mmol), compound 6 (372 mg,1 mmol) and 10mL of ethanol are added into a 25mL round bottom flask, then magnetons are added for reflux reaction for 12h at 80 ℃, the mixture is placed in a refrigerator after the reaction is finished and cooled for 30min, and the precipitated solid is decompressed and filtered to obtain solid product 510mg, namely compound 7, and the yield is about 90.3%. The nuclear magnetic information of compound 7 is as follows:
1 H NMR(400MHz,DMSO)δ11.65(s,1H),11.02(s,1H),8.62(d,J＝14.6Hz,2H),7.80(dt,J＝11.5,8.2Hz,5H),7.64(s,1H),7.52(dt,J＝24.7,7.8Hz,3H),7.41(t,J＝7.4Hz,1H),7.31(s,2H),7.01(d,J＝1.8Hz,1H),6.93(dd,J＝8.5,1.8Hz,1H),6.55(d,J＝14.6Hz,1H),5.49(s,2H),2.75–2.59(m,4H),1.80(s,12H).
13 C NMR(101MHz,DMSO)δ178.25(s),164.60(s),162.64(s),162.55(s),154.83(s),142.59(s),141.73(s),141.68(s),139.49(s),136.27(s),129.91(s),127.28(s),126.83(s),126.39(s),123.18(s),119.35(s),115.61(s),115.08(s),114.99(s),112.61(s),103.86(s),102.51(s),56.49(s),50.50(s),48.22(s),28.70(s),28.29(s),20.41(s),19.03(s).
(8) Preparation of probes
The synthetic route is as follows:
compound 7 (56.9 mg,0.1 mmol) was added to 5mL of DMF under nitrogen, and triethylamine (40 mg,0.4 mmol) was injected into the solution of compound 7 using a syringe. Acryloyl chloride (36 mg,0.4 mmol) was poured into the reaction flask under ice-bath conditions, and stirred for five minutes in an ice bath followed by stirring at room temperature for 1 hour. After the reaction is finished, DMF is removed by vacuum rotary evaporation, distilled water is added for stirring, then vacuum filtration is carried out again, and the obtained solid powder is dried in vacuum, thus obtaining the final near infrared probe 55mg, and the yield is about 87.4%. The nuclear magnetic hydrogen spectrum and the carbon spectrum of the fluorescent probe prepared in this example are shown in fig. 2 and 3, respectively, and specific nuclear magnetic data are as follows:
1 H NMR(400MHz,DMSO)δ11.68(s,1H),8.66(d,J＝15.0Hz,1H),7.81(dt,J＝7.7,5.1Hz,5H),7.66(dd,J＝11.1,8.2Hz,2H),7.60–7.45(m,4H),7.31(s,2H),7.22(dd,J＝8.4,2.2Hz,1H),6.73(d,J＝15.1Hz,1H),6.62(dd,J＝17.3,1.1Hz,1H),6.48(dd,J＝17.2,10.3Hz,1H),6.25(dd,J＝10.3,1.1Hz,1H),5.61(s,2H),2.77–2.61(m,4H),1.81(s,6H).
13 the fluorescent probes prepared in this example were tested using C NMR (101 MHz, DMSO). Delta. 180.64(s), 164.25(s), 164.18(s), 160.33(s), 153.12(s), 152.71(s), 142.34(s), 142.25(s), 141.68(s), 139.58(s), 134.89(s), 132.15(s), 130.33(s), 129.44(s), 128.87(s), 127.94(s), 127.78(s), 127.28(s), 123.28(s), 120.12(s), 119.88(s), 119.39(s), 115.33(s), 113.50(s), 110.45(s), 106.74(s), 51.29(s), 29.08(s), 27.98(s), 24.19(s), 20.16(s):
1) Preparation of stock solution for detection
a. Sample solution of fluorescent probe for detecting cysteine (1.00X 10) -3 mol/L) of the following components: accurately weighing 0.063g (M=636) fluorescent probe, dissolving in 10mL dimethyl sulfoxide to obtain a solution with a concentration of 1.00×10 -3 mol/L solution.
b. Deionized water is used to prepare the amino acids and ions with concentration of 1.0X10 -2 mol/L solution.
Preparation of hepes buffer solution (10 mm, ph=7.4):
1.19g of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES, molecular weight 238) was accurately weighed, dissolved in 400mL of distilled water at a concentration of 1.0X10 -2 The pH of the above solution was adjusted to 7.4 with a mol/L aqueous sodium hydroxide solution, and then the volume was adjusted to 500mL with distilled water.
The buffer solutions used in the following assays were all mixed solutions of HEPES (10 mm, ph=7.4) and methanol at a volume ratio of 5:1, and the experimental water was deionized water.
2) Detection analysis
3mL of the buffer solution was removed by a pipette, and 30. Mu.L of probe stock solution (1.00X 10) -3 mol/L) was used as a blank. Then 30. Mu.L of 1.00X 10 was added -2 The change of the probe in 5min was detected by using a mol/L cysteine aqueous solution. Scanning parameter setting for detecting ultraviolet absorption spectrum, wherein the starting point is 400nm, and the end point is800nm, fast, 1nm spacing, results are shown in FIG. 4. From fig. 4, it can be seen that the absorbance of the system increases with time after the addition of the cysteine solution, and is accompanied by a remarkable red shift phenomenon; a new peak type appears at 675nm, the increasing trend of absorbance of the system gradually slows down with the increase of time, and the strongest value can be reached in five minutes.
3mL of the buffer was removed by a pipette, and 30. Mu.L (1.00X 10) -3 mol/L) probe stock solution, mixing well, scanning out the contrast line of only probe, then adding 30 mu L of 1.00×10 -2 And (3) detecting the change of fluorescence intensity after adding the cysteine aqueous solution in mol/L. The excitation wavelength of fluorescence was 640nm, the excitation slit width was 5.0nm, and the emission slit width was 2.5nm, and the results are shown in FIG. 5. As can be seen from FIG. 5, the fluorescence intensity of the probe system was gradually increased with time after the addition of cysteine, and it can be seen from the figure that the trend of increase in fluorescence intensity of the system was gradually slowed down with time, stopping after about 50 minutes, and the fluorescence intensity of the system reached an equilibrium state.
3mL of the buffer was removed by a pipette, and 30. Mu.L (1.00X 10) -3 mol/L) of the probe stock solution, then, different equivalents of cysteine aqueous solutions were added, and the change trend of the absorbance of the system with the concentration of cysteine was detected, and the result is shown in FIG. 6. As can be seen from fig. 6, the absorbance of the system changes with increasing concentration after adding different concentrations of cysteine to the probe, and it can be seen that the absorbance of the system gradually decreases with increasing concentration of cysteine at 600nm, accompanied by a red shift phenomenon, a new absorption peak appears at 675nm, and gradually increases with increasing concentration of cysteine. Saturation was reached when 6Equiv was added; the color of the solution changed from deep blue to light blue.
3mL of the buffer was removed by a pipette, and 30. Mu.L (1.00X 10) -3 mol/L) of the probe stock solution into a fluorescence cell, then adding different equivalents of cysteine aqueous solutions, fully and uniformly mixing, and detecting the fluorescence spectrum of the probe system, wherein the result is shown in figure 7. As can be seen in FIG. 7, the probe dissolvesAfter adding different concentrations of cysteine solutions, the fluorescence intensity of the system is gradually enhanced along with the increase of the concentration of the cysteine. FIG. 8 is a graph showing the relationship between the fluorescence intensity values (705 nm) detected at different concentrations; the linear correlation degree of the probe is 0.990 when the probe is subjected to linear fitting under the concentration of 1:6 equivalent of cysteine, and the fitting equation is y=33.61x+45.41, so that the fluorescence intensity at 705.0nm is in positive correlation in a certain cysteine concentration range. The limit of detection of cysteine (LOD) by the probe is calculated by the formula: the formula lod=3σ/k, where σ is the standard deviation of 5 blank replicates and k is the slope of the linear equation of the different concentrations of cysteine versus the corresponding fluorescence intensity at 705.0 nm. The limit of detection of Cys by the probe was calculated to be 0.134. Mu.M.
3mL of the buffer was removed by a pipette, and 30. Mu.L (1.00X 10) -3 mol/L) probe stock solution, the probes were scanned individually, and then the test substances (30. Mu.L, 1.00X 10) were added sequentially, respectively -2 mol/L)(NO 2 - 、AcO - 、H 2 PO 4 - 、S 2- 、F - 、Cl - 、Br - 、Zn 2+ 、Cu 2+ 、Fe 3+ Phe, glu, mel, gln, flr, ser, leu, GSH, hcy, cys), after 1 hour, the ultraviolet spectrum and fluorescence spectrum were measured, and the results are shown in FIG. 9, respectively, in which the maximum absorption wavelength at 600nm was significantly reduced and the ultraviolet absorption at 675nm was significantly enhanced to give new ultraviolet absorption, and different ions (NO 2 - 、AcO - 、H 2 PO 4 - 、S 2- 、F - 、Cl - 、Br - 、Zn 2+ 、Cu 2+ 、Fe 3+ Phe, glu, mel, gln, flr, ser, leu, GSH, hcy) the uv absorption after which no significant change occurs. FIG. 10 is a bar graph of fluorescence intensity at 705nm of maximum emission wavelength for probe and cysteine with other different ions and amino acids, and we can see that the fluorescence emission of probe solution is significantly enhanced after adding cysteine with weaker fluorescence emission at 705 nm. When it is addedIt has different ions (2-20 are NO in turn) 2 - 、AcO - 、H 2 PO 4 - 、S 2- 、F - 、Cl - 、Br - 、Zn 2+ 、Cu 2+ 、Fe 3+ Phe, glu, mel, gln, flr, ser, leu, GSH, hcy) the fluorescence intensity after which no significant change occurred.
3mL of the buffer was removed by a pipette, and 30. Mu.L (1.00X 10) -3 mol/L) stock solution of the probe, the fluorescence intensity of the probe was scanned alone, and then 30. Mu.L (1.00X 10) was added, respectively -2 The mol/L interferents (2-20 are NO in turn) 2 - 、AcO - 、H 2 PO 4 - 、S 2- 、F - 、Cl - 、Br - 、Zn 2+ 、Cu 2+ 、Fe 3+ Phe, glu, mel, gln, flr, ser, leu, GSH, hcy), and uniformly mixing, and performing fluorescence spectrum scanning; then 30. Mu.L of cysteine solution (1.00X 10) -2 mol/L), and after uniformly mixing for 40 minutes, fluorescence spectrum scanning is carried out, and the fluorescence intensity at 705nm is taken as a histogram. As can be seen from FIG. 11, the rest of the ions and amino acids have little interference with the probe and do not substantially react, and after cysteine is added, the fluorescence intensity is changed strongly, and the solution changes from the original deep blue to the light blue (FIG. 12). Therefore, the probe has good response effect on cysteine under the condition that the ion and the amino acid interference exist, has strong anti-interference capability on various amino acids, and shows macroscopic color change, thus representing the excellent performance of the near infrared probe.
Taking HeLa cells with logarithmic growth cycle, counting, regulating cell concentration, and culturing in 96-well plate according to 6×10 3 The cells/wells were inoculated, placed in an incubator containing 5% carbon dioxide and incubated overnight at 37 ℃. The bioamaging pattern of near infrared fluorescent probe molecules for cysteine detection in living cells is shown in FIG. 13. As can be seen from FIG. 13, group a, cells that were not incubated with the probe did not fluoresce. When HeLa cells were incubated with 10. Mu.M Probe at 37℃for 40min, weak fluorescence was observed, and this change in fluorescence was observedIt was shown that the probe is sensitive to the natural level of Cys in living cells and can be used to monitor the endogenous Cys of the cells (panel b in fig. 13). To further confirm that the probes specifically recognize Cys in living cells, group c was pretreated with Cys (100 μm) for 40min and then incubated with 10 μm probe for 40min for confocal imaging. Fluorescence was significantly enhanced compared to group b treated with probe alone (group c in fig. 13). To verify intracellular cysteine specificity, live cells were then incubated with an aqueous solution of thiol scavenger N-ethylmaleimide (NEM) (200. Mu.M) at 37℃for 40min, followed by incubation with 10. Mu.M Probe for 40min at 37 ℃. As expected, the fluorescence intensity was significantly reduced (group d in fig. 13). The experimental result shows that the near infrared fluorescent Probe has good responsiveness and selectivity to cysteine in the cell body and has good application prospect in the aspect of biological test.
Cys plays an important role in the Golgi apparatus, and some cancer cells or diseased cells will cause over-expression of Cys in the cells with increased oxidative stress, so it is necessary to study the concentration level of Cys under stress conditions. Taking HeLa cells with logarithmic growth cycle, counting, regulating cell concentration, and culturing in 96-well plate according to 6×10 3 The cells/wells were inoculated, placed in an incubator containing 5% carbon dioxide and incubated overnight at 37 ℃. HeLa cells were pretreated with different concentrations of monensin (none), respectively, and Cys produced during the process was observed by adding 10. Mu.M probe. As shown in fig. 14, the cells showed weak fluorescence after incubation of the probes with the cells (group a). After pretreatment with different concentrations of none (0.5. Mu.M, 1. Mu.M, 2. Mu.M), the mixture was incubated with a probe and confocal imaging was performed. As expected, the red fluorescent signal was progressively increased for the cells of groups b, c, d compared to the cells of group a. This is probably due to endogenous Cys production after the none stimulated golgi, and the fluorescence intensity gradually increased with increasing none concentration. It follows that the probe is able to track the change in Cys content during oxidative stress in the golgi apparatus.
To study the function of the probe in locating the golgi apparatus, heLa cells with logarithmic growth cycle were countedPost-adjustment of cell concentration in 96-well plates according to 6X 10 3 The cells/wells were inoculated, placed in an incubator containing 5% carbon dioxide and incubated overnight at 37 ℃. After pretreatment of HeLa cells with 100. Mu.M Cys for 40min, the cells were imaged and mapped with 10. Mu.M probe and commercial green Golgi targeting dye (BODIPYTR ceramide (5. Mu.M) for 2h, respectively, as shown in FIG. 15, the probe had strong tissue penetration to detect cysteines in the cells, and the red channel treated with the probe and the green channel treated with the Golgi targeting dye overlapped well, and the probe exhibited good co-labeling properties with the Golgi targeting dye, with a Pearson correlation coefficient of 0.88, indicating that the probe could target the Golgi.
To further investigate the use of probes, the present application performed in vivo imaging experiments on mice. As shown in FIG. 16, probe solutions (50.0. Mu.L, 10. Mu.M) and Cys (20.0. Mu.L, 20 mM) were injected into the peritoneal cavity of the nude mice. The fluorescence intensity of mice in group a gradually increased over time, reaching a maximum after 40 minutes, consistent with the time effect of the previous test. In control experiments, mice were injected with NEM (20.0. Mu.L, 100 mM) and Cys (20.0. Mu.L, 20 mM), pre-treated for 20min, and then injected with probe solution (50.0. Mu.L, 10. Mu.M). From group b, it can be seen that the fluorescence intensity of group a is much stronger than that of group b, since NEM reacts with cysteine in mice resulting in the elimination of cysteine. The results indicate that the probe can be used for in vivo cysteine detection.
In addition, cys concentrations were overexpressed in tumors, and probes were used herein to image Cys in HepG-2 cell line nude mouse transplants. As shown in fig. 17, a control group was established, and nude mice were injected with PBS (50.0 μl) in vivo, and it can be seen that the mice injection site did not have any fluorescence. In the experimental group, probe solutions (50.0. Mu.L, 10. Mu.M) were injected into the tumor site. The fluorescence intensity at the site of injection probe gradually increased over time and reached a maximum at 40 minutes, indicating a good fluorescent response of the probe to Cys in the tumor, due to the apparent fluorescent signal exhibited by the tumor region, which would aid in early diagnosis of the tumor. The result shows that the probe can be used for detecting Cys in vivo and in vitro and has potential application prospect in the aspect of biomedicine.
Claims (7)
1. A Golgi apparatus targeting near infrared fluorescent probe for detecting cysteine is characterized in that the molecular formula of the fluorescent probe is C 36 H 34 N 3 O 6 S + The structural formula is as follows:
2. the method for preparing the golgi targeted near infrared fluorescent probe for detecting cysteine according to claim 1, which is characterized by comprising the following steps:
(1) Cyclohexanone, phosphorus tribromide and DMF are reacted in chloroform to obtain a compound 1;
(2) 2, 4-dihydroxybenzaldehyde and 3, 4-dihydro-2H-pyran react under the action of a catalyst of pyridinium tosylate to obtain a compound 2;
(3) Reacting the compound 2, the compound 1 and potassium carbonate in DMF to obtain a compound 3;
(4) Adding trifluoroacetic acid into dichloromethane in the compound 3, and stirring for reaction to obtain a compound 4;
(5) Reacting p-aminobenzenesulfonamide with chloroacetyl chloride to obtain a compound 5;
(6) Reacting the compound 5 with 2, 3-trimethyl-3H-indole to obtain a compound 6;
(7) Reacting the compound 6 with the compound 4 to obtain a compound 7;
(8) And (3) reacting the compound 7, the acryloyl chloride and the triethylamine in DMF to obtain the fluorescent probe.
3. The method for preparing the golgi targeted near infrared fluorescent probe for detecting cysteine according to claim 2, which is characterized by comprising the following steps of;
(1) The preparation process of the compound 1 specifically comprises the following steps: dropwise adding phosphorus tribromide into a mixed solution of N, N-dimethylformamide and chloroform under ice bath condition, stirring for 0.5-1.5 h, dropwise adding cyclohexanone into the solution, stirring to completely react, neutralizing to neutrality, extracting a product with dichloromethane, and concentrating by rotary evaporation to obtain a target product;
(2) The preparation process of the compound 2 specifically comprises the following steps: weighing 2, 4-dihydroxybenzaldehyde and pyridine p-toluenesulfonate respectively, and adding into CH 2 Cl 2 Adding 3, 4-dihydro-2H-pyran, stirring, steaming, and performing column chromatography to obtain compound 2;
(3) The preparation process of the compound 3 specifically comprises the following steps: adding the prepared compound 2, the compound 1 and potassium carbonate into DMF, stirring at 35-45 ℃ for complete reaction, cooling to room temperature, filtering and concentrating to obtain a concentrated solution of the compound 3;
(4) The preparation process of the compound 4 specifically comprises the following steps: adding trifluoroacetic acid into the concentrated solution obtained in the step (3), stirring at 35-45 ℃ for complete reaction, cooling to room temperature, carrying out suction filtration, washing with ethanol, and drying to obtain a compound 4;
(5) The preparation process of the compound 5 specifically comprises the following steps: suspending the sulfanilamide in acetone, dropwise adding chloroacetyl chloride at room temperature, stirring the reaction mixture at 70-80 ℃ for complete reaction, cooling to room temperature, adding ice water, stirring the obtained mixture, filtering, washing the collected solid with ice water, recrystallizing with ethanol, filtering, and drying the obtained solid to obtain a compound 5;
(6) The preparation process of the compound 6 specifically comprises the following steps: dissolving the compound 5 obtained in the step (5) in acetonitrile under the nitrogen atmosphere, adding 2, 3-trimethyl-3H-indole by a syringe, stirring, carrying out reflux reaction completely, carrying out vacuum rotary evaporation on the reacted liquid, adding dichloromethane, carrying out vacuum suction filtration, and obtaining a solid, namely the compound 6;
(7) The preparation process of the compound 7 specifically comprises the following steps: dissolving the compound 6 and the compound 4 in ethanol, stirring, refluxing and reacting until solid precipitate is generated, and filtering to obtain a solid, namely the compound 7;
(8) The preparation process of the fluorescent probe specifically comprises the following steps: and (3) dissolving the compound 7 in DMF under nitrogen atmosphere, adding triethylamine, stirring uniformly, adding acryloyl chloride under ice bath condition, stirring for a period of time in ice bath, stirring at room temperature, reacting completely, removing DMF by vacuum rotary evaporation, adding distilled water, stirring, vacuum filtering, and vacuum drying the obtained solid to obtain the fluorescent probe.
4. The method for preparing a Golgi-targeted near infrared fluorescent probe for detecting cysteine according to claim 2 or 3, wherein in the step (1), PBr 3 The mole ratio of DMF to cyclohexanone is 2.5-2.6 to 2.3:1; in the step (2), the molar ratio of the 2, 4-dihydroxybenzaldehyde to the 3, 4-dihydro-2H-pyran to the pyridine p-toluenesulfonate is 1 (1-1.2): 0.05; in the step (3), the molar ratio of the compound 1 to the compound 2 to the potassium carbonate is 2:1:3; in the step (4), the molar ratio of the compound 3 to the trifluoroacetic acid is 10 (1-2); in the step (5), the molar ratio of the p-aminobenzene sulfonamide to the chloroacetyl chloride is 1 (0.9-1.5); in the step (6), the molar ratio of the compound 5 to the 2, 3-trimethyl-3H-indole is 1 (1-1.5); in the step (7), the molar ratio of the compound 4 to the compound 6 is 1 (1-1.5); in the step (8), the molar ratio of the compound 7 to the triethylamine to the acryloyl chloride is 1 (1-4), and the molar ratio of the compound 7 to the triethylamine to the acryloyl chloride is 1-4.
5. Use of a fluorescent probe according to claim 1 for cysteine detection for the purpose of diagnosis of non-diseases.
6. Use of the fluorescent probe of claim 1 for preparing a cell fluorescent imaging agent.
7. Use of the fluorescent probe according to claim 1 for preparing a targeted-positioning golgi fluorescent probe.
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| CN116925756A (en) * | 2023-07-21 | 2023-10-24 | 锦州医科大学 | Nano probe for specifically detecting cysteine in organism and application thereof |
| CN117586247A (en) * | 2023-11-16 | 2024-02-23 | 南华大学 | Mitochondrial targeting near infrared fluorescent probe with viscosity and cysteine specific response and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116925756A (en) * | 2023-07-21 | 2023-10-24 | 锦州医科大学 | Nano probe for specifically detecting cysteine in organism and application thereof |
| CN116925756B (en) * | 2023-07-21 | 2024-04-19 | 锦州医科大学 | Nano probe for specifically detecting cysteine in organism and application thereof |
| CN117586247A (en) * | 2023-11-16 | 2024-02-23 | 南华大学 | Mitochondrial targeting near infrared fluorescent probe with viscosity and cysteine specific response and preparation method and application thereof |
| CN117586247B (en) * | 2023-11-16 | 2024-05-10 | 南华大学 | Mitochondrial targeting near infrared fluorescent probe with viscosity and cysteine specific response and preparation method and application thereof |
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