CN113185559B - Glycosyl calixarene fluorescent probe for detecting tumors - Google Patents
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Abstract
The invention discloses a glycosyl calixarene fluorescent probe for detecting tumors, which is characterized by having a structure shown in a formula (I):
Description
Technical Field
The invention belongs to the field of biological analysis and detection, and particularly relates to a glycosyl calixarene fluorescent probe for detecting tumors and application thereof.
Background
At present, tumors become one of the major diseases seriously threatening human health in the world, and along with the development of research on anti-tumor drugs, the research on early diagnosis and targeted diagnosis and treatment of tumors also draws more and more attention.
The basic principle of the method is to utilize tumor biomarkers which exist specifically in tumor cells or tumor tissues or have obvious difference with normal cells or normal tissues, and achieve the purpose of detecting or treating tumors in a targeted manner by selectively recognizing and inhibiting the biomarkers. Research has shown that malignant tumors can proliferate, spread and metastasize continuously, and one of the important physiological mechanisms is to continuously absorb and take up the special nutrients it needs. These nutrients include sugar molecules, amino acids, vitamins, nucleosides, and the like. As early as 1924, the german biochemical, otto woberg, found that healthy cells could efficiently utilize aerobic metabolism to produce sufficient energy from a limited carbohydrate nutrient source, while most tumor cells, due to their hypoxic environment, provided their own energy through massive sugar absorption and metabolism. This phenomenon is known as the warburg effect. Woberg thus acquired The physiological or medical Nobel Prize in 1931 (ref: Warburg, O.science,123:309 (1956); "The Nobel Prize in Physiology or Medicine 1931". Nobel prize.org. < http:// www.nobelprize.org/Nobel _ prizes/medium/laurates/1931/>).
In summary, the following steps: based on tumor Woberg effect, because tumor cells specifically and highly express the sugar transport channel protein GLUT compared with healthy cells, the GLUT is expected to realize the targeted identification of tumors by taking the GLUT as a tumor specific marker.
The synthesis method of glycosyl calixarene and the effect of the glycosyl calixarene and agglutinin are reported in documents (reference: Sansone, F.Chem.Soc.Rev.,2013,42,4623), however, the glycosyl calixarene fluorescent probe and the application are not reported at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a glycosyl calixarene fluorescent probe for detecting tumors.
The second purpose of the invention is to provide the application of the glycosyl calixarene fluorescent probe for detecting the tumor in preparing a tumor detection reagent.
The technical scheme of the invention is summarized as follows:
the glycosyl calixarene fluorescent probe for detecting the tumor has a structure shown in a formula I:
wherein the content of the first and second substances,
R1selected from (A-1) or (A-2);
the structural formula of the (A-1) is as follows:
the structural formula of the (A-2) is as follows:
R2selected from:
the glycosyl calixarene fluorescent probe for detecting the tumor is applied to preparing a tumor detection reagent.
Advantageous effects
The glycosyl calixarene fluorescent probe for detecting the tumor can be directly used in a cell system to analyze and detect high-expression GLUT tumor cells, so that the screening and the identification of the tumor cells are completed. The glycosyl calixarene fluorescent probe for detecting the tumor targeting the tumor Wobbe effect can be directly used in a cell line to analyze and detect the expression degree of GLUT protein of a tumor cell, so that the screening of the tumor cell is completed. Provides effective means and useful tools for early screening and diagnosis of tumors, development of new antitumor drugs and the like.
Drawings
FIG. 1 is the excitation and emission fluorescence spectra of the probe of example 1;
FIG. 2 is the excitation and emission fluorescence spectra of the probe of example 2;
FIG. 3 is the excitation and emission fluorescence spectra of the probe of example 3;
FIG. 4 shows the excitation and emission fluorescence spectra of the probe of example 4;
FIG. 5 is the excitation and emission fluorescence spectra of the probe of example 5;
FIG. 6 is the excitation and emission fluorescence spectra of the probe of example 6;
FIG. 7 is a graph showing the effect of the sugar-based calixarene fluorescent probe for detecting tumor (prepared in example 1) of the present invention on detecting and diagnosing tumor;
FIG. 8A is a comparison of the enrichment effect of the sugar-based calixarene fluorescent probe for detecting tumors of example 1 in GLUT high-expressing cells and GLUT normal-expressing cells; b is the comparison of the enrichment effect of the glycosyl calixarene fluorescent probe for detecting tumors in the GLUT high expression cells and the GLUT normal expression cells in the embodiment 2; c is the comparison of the enrichment effect of the glycosyl calixarene fluorescent probe for detecting tumors in GLUT high expression cells and GLUT normal expression cells in the embodiment 3.
Detailed Description
The present invention is further illustrated by the following specific examples, which are intended to be illustrative, but not limiting, of the present invention.
Example 1
Preparation of glycosyl calixarene fluorescent probe I-a for detecting tumors
1) Preparation of intermediate compound 3:
compound 1 (commercially available) (500mg) and ethyl bromoacetate (Compound 2, 170. mu.L) were dissolved in 10mL of dry N, N-Dimethylformamide (DMF), 140mg of cesium fluoride was added, and the mixture was heated to 40 ℃ and stirred for 16 hours. The reaction solution was diluted with 300mL of ethyl acetate, washed with 150mL of a saturated aqueous solution of ammonium chloride in a separatory funnel, and the organic phase was washed with 150mL of water and 150mL of a saturated aqueous solution of sodium chloride, respectively. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the crude product was purified by silica gel column separation to give the desired intermediate compound 3(328mg, 58%, white solid)
1H NMR(600MHz,CDCl3)δ7.02(s,4H),6.81(s,4H),4.72(s,2H),4.45(d,J＝13.2Hz,4H),4.30(q,J＝7.1Hz,4H),3.32(d,J＝13.2Hz,4H),1.34(t,J＝7.1Hz,3H),1.26(s,18H),0.97(s,18H).
2) Preparation of intermediate compound 4:
dissolving a compound 3(328mg) in 9mL of absolute ethyl alcohol (EtOH) to obtain an ethanol solution of the compound 3, dissolving 89mg of sodium hydroxide in 6mL of water, dropwise adding the solution into the ethanol solution of the compound 3, uniformly mixing, heating to 80 ℃, stirring for 4 hours, carrying out reduced pressure rotary evaporation to remove most of the solvent after the reaction is finished, adjusting the pH of the concentrated reaction solution to 1 by using 5% dilute hydrochloric acid, then precipitating a solid, carrying out reduced pressure suction filtration, and drying to obtain a target product, namely an intermediate compound 4(300mg, 95.2% and white solid).
1H NMR(400MHz,DMSO-d6)δ14.72(s,2H),7.08(d,J＝2.3Hz,2H),6.90(s,2H),6.86–6.74(m,4H),4.32(d,J＝11.8Hz,2H),4.08(d,J＝12.5Hz,2H),3.74(s,2H),3.14(d,J＝12.2Hz,4H),1.16(d,J＝13.7Hz,27H),1.04(s,9H).
3) Synthesis of intermediate compound 6:
compound 4(100mg) was dissolved in 410. mu.L of thionyl chloride (SOCl)2) Then, the mixture was heated to 80 ℃ and stirred for 4 hours. After the reaction is finished, performing reduced pressure rotary evaporation to remove thionyl chloride, directly dropwise adding the obtained crude product into a solution formed by dissolving the raw material compound 5(166mg) and 12 mu L of Pyridine (Pyridine) in 2mL of dry Dichloromethane (DCM) without treatment, and stirring at room temperature under the protection of argon overnight; most of the organic solvent was removed by rotary evaporation under reduced pressure, and the residue was diluted with 20mL of ethyl acetate and washed with 10mL of water and 10mL of a saturated aqueous solution of sodium chloride, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed by rotary evaporation under reduced pressure, and then the crude product was subjected to silica gel column separation and purification to obtain the objective compound 6(60mg, 49%, yellow solid).
1H NMR(600MHz,CDCl3)δ11.22(s,1H),9.77(s,1H),9.21(s,2H),8.25(d,J＝7.8Hz,1H),7.47(d,J＝7.8Hz,1H),7.08(d,J＝3.5Hz,6H),7.04(d,J＝2.1Hz,2H),4.80(s,2H),4.36(d,J＝13.9Hz,2H),4.22(d,J＝13.3Hz,2H),3.51(t,J＝13.0Hz,4H),1.22(d,J＝2.8Hz,27H),1.17(s,9H).
4) Synthesis of intermediate Compound 8-1:
compound 7-1(2,3,4, 6-tetra-O-acetyl-1- (2-bromoethoxy) -mannose 187mg, prepared according to The synthetic method reported in The Journal of Antibiotics (2015)68, 302-acetonitrile 312) was dissolved in 10mL of anhydrous acetonitrile, a catalytic amount of potassium iodide was added, heating was performed, reflux stirring was performed for 1 hour, 300mg of Compound 6 and 58mg of potassium carbonate were added, heating was continued under reflux, stirring was performed for 48 hours, most of The organic solvent was removed by rotary evaporation under reduced pressure, The residue was diluted with 100mL of ethyl acetate, and then washed with 50mL of water, 50mL of saturated aqueous sodium chloride solution. The final organic phase was dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed by rotary evaporation under reduced pressure, and then the crude product was subjected to silica gel column separation and purification to obtain the objective compound 8-1(200mg, 46%, yellow solid).
1H NMR(600MHz,CDCl3)δ10.75(s,1H),8.48(d,J＝7.9Hz,1H),7.44(d,J＝7.9Hz,1H),7.14–7.04(m,4H),6.77(s,3H),6.74(d,J＝4.5Hz,2H),6.68(s,1H),5.28–5.19(m,3H),4.86–4.77(m,2H),4.63(d,J＝15.8Hz,1H),4.54–4.45(m,2H),4.39–4.15(m,8H),3.95(d,J＝12.1Hz,1H),3.87(s,1H),3.47(d,J＝13.6Hz,2H),3.39–3.29(m,2H),2.10(s,3H),2.04(s,3H),1.90(s,3H),1.75(s,3H),1.29(s,18H),0.90(d,J＝8.9Hz,18H).
5) Preparing a glycosyl calixarene fluorescent probe I-a for detecting tumors:
compound 8-1(200mg) was dissolved in 6mL of anhydrous methanol (MeOH), cooled to 0 ℃ and then 2mL of an aqueous sodium hydroxide solution (0.4mmol/mL) was added dropwise to the reaction mixture, the temperature was raised to 65 ℃ and the mixture was stirred for 1 hour, most of the solvent was removed by rotary evaporation under reduced pressure, the concentrated reaction solution was diluted with 100mL of ethyl acetate and washed with 50mL of water in a separatory funnel, and the organic phase was washed with 50mL of a saturated aqueous sodium chloride solution. Separating organic phase, drying with anhydrous sodium sulfate, filtering, separating and purifying the crude product with silica gel column to obtain glycosyl calixarene fluorescent probe I-a (80mg, yield 47%, yellow solid) for detecting tumor as target product
1H NMR(600MHz,CDCl3)δ10.79(s,1H),8.42(d,J＝7.9Hz,1H),7.39(d,J＝7.9Hz,1H),7.08(d,J＝3.4Hz,4H),6.84–6.76(m,4H),6.69(d,J＝2.1Hz,2H),4.91(s,1H),4.73(d,J＝15.6Hz,1H),4.63(d,J＝15.6Hz,1H),4.37(dd,J＝29.8,13.6Hz,4H),4.21(dd,J＝24.2,13.0Hz,2H),4.04(s,1H),3.89(s,1H),3.82–3.64(m,4H),3.55(d,J＝8.4Hz,1H),3.49–3.38(m,3H),3.31(dd,J＝25.1,13.1Hz,2H),1.29(d,J＝2.6Hz,18H),0.94(s,9H),0.87(s,9H).
13C NMR(150MHz,CDCl3)δ169.35,150.22,149.99,149.92,149.18,148.78,147.71,147.14,142.24,142.17,132.14,132.05,132.00,128.15,127.96,127.24,127.17,126.00,125.86,125.82,125.78,125.28,125.18,125.14,118.38,115.61,100.42,75.78,74.06,72.34,71.48,70.66,66.29,65.79,60.89,33.98,33.89,33.88,31.90,31.86,31.70,31.40,31.31,30.97,30.93.
HRMS:Calcd.For C60H74ClN3O12 +[M+Na]+:1086.4961,found:1086.4856.
Example 2
Preparing a glycosyl calixarene fluorescent probe I-b for detecting tumors:
steps 1) -3) were the same as Steps 1) -3) of example 1;
4) synthesis of intermediate Compound 8-2:
the intermediate 7-2(2,3,4, 6-tetra-O-acetyl-1- (2-bromoethoxy) -glucose 187mg, prepared according to The synthetic method reported in The Journal of Antibiotics (2015)68,302-; to obtain the intermediate compound 8-2.
5) Preparing a glycosyl calixarene fluorescent probe I-b for detecting tumors:
the compound 8-2 is used for replacing the compound 8-1 in the step 5) of the example 1, and the step 5) of the example 1 is otherwise the same as the step 5) of the example 1, so as to obtain the glycosyl calixarene fluorescent probe I-b (75mg, yield 43%) of a yellow solid target product for detecting tumors.
1H NMR(600MHz,CDCl3)δ10.67(s,1H),8.50(d,J＝7.9Hz,1H),7.68(s,1H),7.48(d,J＝7.9Hz,1H),7.15(d,J＝2.1Hz,1H),7.08(d,J＝5.3Hz,3H),7.05(d,J＝2.1Hz,1H),6.93(s,2H),6.69(d,J＝6.0Hz,2H),4.90(d,J＝15.4Hz,1H),4.40–4.32(m,4H),4.27(t,J＝13.0Hz,3H),4.16(d,J＝10.4Hz,1H),4.07(dd,J＝10.4,2.4Hz,1H),3.85(d,J＝7.6Hz,1H),3.81(dd,J＝11.9,3.5Hz,1H),3.72(dd,J＝11.9,4.7Hz,1H),3.54(d,J＝13.8Hz,1H),3.49–3.44(m,2H),3.35–3.26(m,3H),3.21(t,J＝9.1Hz,1H),3.03(dd,J＝9.0,4.4Hz,1H),1.29(d,J＝13.4Hz,18H),1.03(s,9H),0.86(s,9H).
13C NMR(150MHz,CDCl3)δ170.18,150.75,149.99,149.01,148.82,148.72,147.73,146.59,145.45,142.83,142.28,132.59,132.48,132.27,132.23,131.79,129.07,128.50,126.35,126.27,126.09,126.05,125.85,125.76,125.65,125.55,125.40,124.95,124.81,118.20,115.44,103.38,76.06,75.55,74.09,73.40,69.77,67.84,62.04,34.15,33.93,33.90,33.84,32.21,31.68,31.60,31.52,31.33,31.00,30.92,14.14.
Example 3
Preparation of glycosyl calixarene fluorescent probe I-c for detecting tumors:
steps 1) -3) were the same as Steps 1) -3) of example 1;
4) synthesis of intermediate Compound 8-3: 187mg of intermediate 7-3(2,3,4, 6-tetra-O-acetyl-1- (2-bromoethoxy) -galactose, prepared according to The synthetic method reported in The Journal of Antibiotics (2015)68,302-; to obtain the intermediate compound 8-3.
5) Preparation of glycosyl calixarene fluorescent probe I-c for detecting tumors:
the compound 8-3 is used for replacing the compound 8-1 in the step 5) of the example 1, and the step 5) of the example 1 is otherwise the same as the step 5) of the example 1, so as to obtain the glycosyl calixarene fluorescent probe I-c (60mg, yield 35%) of a yellow solid target product for detecting tumors.
1H NMR(600MHz,CDCl3)δ10.69(s,1H),8.48(d,J＝7.9Hz,1H),7.72(s,1H),7.46(d,J＝7.9Hz,1H),7.15(d,J＝14.7Hz,2H),7.10–7.03(m,5H),6.94(d,J＝3.6Hz,2H),6.69(d,J＝15.0Hz,2H),4.90(d,J＝15.5Hz,1H),4.35(dd,J＝14.0,8.5Hz,4H),4.30–4.21(m,4H),4.19(s,1H),4.11(d,J＝7.2Hz,2H),3.86(dd,J＝17.4,8.6Hz,3H),3.78(dd,J＝11.8,4.7Hz,2H),3.63–3.57(m,1H),3.54(d,J＝13.9Hz,1H),3.45(d,J＝13.8Hz,2H),3.34–3.25(m,3H),3.24–3.15(m,1H),2.86(d,J＝22.9Hz,2H),1.30(s,9H),1.28(s,9H),1.03(s,9H),0.86(s,9H).
13C NMR(151MHz,CDCl3)δ171.18,170.16,169.49,150.88,150.70,149.96,148.95,148.84,148.80,147.64,146.53,145.46,142.85,142.33,132.56,132.50,132.21,132.12,131.69,129.08,128.58,127.20,126.40,126.34,126.31,126.04,126.02,125.86,125.76,125.73,125.71,125.65,125.55,125.52,125.48,125.03,124.92,124.87,118.34,118.13,115.44,103.61,99.24,75.65,75.48,74.22,74.14,73.14,71.13,70.61,70.15,68.88,67.58,66.29,62.50,62.21,62.09,60.42,53.27,34.15,34.13,34.07,33.95,33.89,33.82,32.88,32.20,32.05,31.69,30.91,29.70,21.00,14.13.
Example 4
Preparation of fluorescent probes I-d for detecting or treating tumors:
1) preparation of intermediate compound 10:
compound 9(77mg, prepared according to the method reported in J.Phys.chem.A 2013,117, 1474-1482) was dissolved in 2mL of anhydrous acetone, a catalytic amount of potassium iodide was added, the mixture was stirred at 60 ℃ for 1 hour, 100mg of Compound 1 and 43mg of potassium carbonate were added, stirring was continued for 6 hours, the organic solvent was removed by rotary evaporation under reduced pressure, and the residue was diluted with 20mL of ethyl acetate and washed with 10mL of water and 10mL of saturated aqueous sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed by rotary evaporation under reduced pressure, and then the crude product was subjected to silica gel column separation and purification to obtain the objective product 10(50mg, 38%, white solid).
2) Preparation of fluorescent probes I-d for detecting or treating tumors:
synthesis of intermediate Compound 8-4: intermediate compound 10 is substituted for intermediate compound 6 in step 4) of example 1, and the same procedure as in step 4) of example 1 is followed; to obtain the intermediate compound 8-4.
The compound 8-4 is used for replacing the compound 8-1 in the step 5) of the example 1, and the other steps are the same as the step 5) of the example 1), so that the white solid target product, namely the glycosyl calixarene fluorescent probe I-d for detecting the tumor is obtained (75mg, the yield is 43%).
1H NMR(600MHz,CDCl3)δ10.72(s,1H),8.02(d,J＝8.7Hz,1H),7.76(s,1H),7.58(d,J＝8.7Hz,1H),7.21(d,J＝3.8Hz,2H),7.11–7.05(m,5H),6.86(d,J＝12.1Hz,4H),6.14(s,1H),4.77(s,1H),4.70(d,J＝15.3Hz,1H),4.58(d,J＝15.3Hz,1H),4.35(d,J＝13.2Hz,1H),4.28(d,J＝12.9Hz,2H),4.23–4.17(m,3H),3.90–3.70(m,12H),3.47–3.35(m,6H),2.37(s,3H),1.27(d,J＝5.3Hz,18H),0.99(d,J＝3.9Hz,18H).
13C NMR(150MHz,CDCl3)δ168.03,161.32,154.09,152.64,149.76,149.59,149.34,149.14,147.80,142.70,141.36,132.41,132.26,132.17,127.98,127.72,126.12,126.04,125.98,125.55,125.47,125.43,125.36,116.55,116.26,113.53,107.52,100.47,75.30,74.12,72.50,71.62,70.72,66.67,65.92,61.18,34.01,33.90,32.19,32.10,31.91,31.70,31.65,31.64,30.96,30.95,18.52.
HRMS:Calcd.For C64H79NO13 +[M+Na]+:1092.5551,found:1092.5439.
Example 5
Preparation of fluorescent probes I-e for detecting or treating tumors:
1) synthesis of intermediate Compounds 8-5: intermediate 10 was used instead of intermediate 6 in step 4) of example 2, and step 4) of example 2 was otherwise the same; to obtain the intermediate compound 8-5.
2) Preparing a glycosyl calixarene fluorescent probe I-e for detecting tumors:
the compound 8-5 is used for replacing the compound 8-2 in the step 5) of the example 2, and the other steps are the same as the step 5) of the example 2), so that the white solid target product, namely the glycosyl calixarene fluorescent probe I-e (85mg, the yield is 64%) for detecting the tumor is obtained.
1H NMR(400MHz,CDCl3)δ10.74(s,1H),8.26(d,J＝10.6Hz,1H),7.85(s,1H),7.72–7.66(m,2H),7.62(s,1H),7.16(s,1H),7.11(s,2H),7.03(d,J＝3.5Hz,3H),6.85(d,J＝10.6Hz,2H),6.24(s,1H),4.96(d,J＝15.0Hz,1H),4.38–4.25(m,4H),4.21(d,J＝10.3Hz,2H),4.17–4.09(m,2H),3.89(d,J＝7.7Hz,1H),3.80(dd,J＝13.7,5.6Hz,2H),3.70(dd,J＝11.8,5.0Hz,1H),3.61(d,J＝13.7Hz,1H),3.51–3.29(m,6H),3.01(q,J＝8.8,7.1Hz,1H),2.87(d,J＝14.4Hz,1H),2.46(s,3H),1.28(d,J＝9.8Hz,18H),1.10(s,9H),0.99(s,9H).
13C NMR(150MHz,CDCl3)δ169.24,161.07,154.25,149.98,149.10,148.87,147.41,147.15,143.64,143.15,141.90,132.85,129.25,127.08,126.54,126.44,126.09,125.86,125.76,125.29,124.94,116.12,113.59,107.21,103.33,76.08,75.56,75.51,74.14,73.44,70.63,67.76,62.67,34.23,33.95,33.93,32.28,32.15,31.66,31.61,31.59,31.55,30.99,30.93,18.62.
Example 6
Preparation of fluorescent probes I-f for detecting or treating tumors:
1) synthesis of intermediate Compounds 8-6: intermediate 10 was used instead of intermediate 6 in step 4) of example 3, and step 4) of example 3 was otherwise the same; to obtain the intermediate compound 8-6.
2) Preparation of glycosyl calixarene fluorescent probe I-f for detecting tumors:
the compound 8-6 is used for replacing the compound 8-3 in the step 5) of the example 3, and the other steps are the same as the step 5) of the example 3), so that the white solid target product, namely the glycosyl calixarene fluorescent probe I-f (70mg, the yield is 40%) for detecting the tumor is obtained.
1H NMR(600MHz,CDCl3)δ10.73(s,1H),8.11–8.02(m,1H),7.91–7.85(m,2H),7.62(d,J＝8.4Hz,2H),7.19–7.08(m,3H),7.04(d,J＝4.8Hz,3H),6.88–6.80(m,2H),6.24(s,1H),4.90(d,J＝14.9Hz,1H),4.38–4.29(m,3H),4.27–4.19(m,3H),4.14(t,J＝14.5Hz,2H),3.97(d,J＝3.0Hz,1H),3.91–3.79(m,3H),3.73(dd,J＝11.8,4.4Hz,1H),3.67–3.59(m,2H),3.56(dd,J＝9.4,3.3Hz,1H),3.46(d,J＝13.7Hz,1H),3.42–3.35(m,2H),3.21(t,J＝5.2Hz,1H),2.45(s,3H),1.28(d,J＝10.7Hz,18H),1.11(s,9H),0.99(s,9H).
13C NMR(150MHz,CDCl3)δ169.07,161.03,154.37,149.25,149.07,148.78,147.32,147.09,143.72,143.23,132.93,132.52,132.05,129.40,128.70,126.94,126.66,126.27,126.15,125.90,125.80,125.57,125.28,124.99,115.85,113.62,107.28,103.35,77.07,75.67,74.39,74.15,73.10,71.07,68.98,67.32,62.65,34.29,34.00,33.98,33.96,32.36,32.23,31.69,31.63,31.60,31.03,30.96,18.63.
Test example 1: measurement experiment of glycosyl calixarene fluorescent probe (short fluorescent probe) fluorescence spectrum for detecting tumor
1) An experimental instrument: thermo Scientific variosukan LUX.
2) Test compounds: the fluorescent probes I-a, I-b, I-c, I-d, I-e and I-f prepared in the above examples.
Testing the solvent: DMSO (dimethylsulfoxide)
3) Experimental procedure
(1) Each compound was weighed.
(2) Dissolve compound to 10mM in DMSO, vortex for 1min, dissolve compound well.
(3) Add 5. mu.l of the dissolved compound to 495. mu.l DMSO and vortex well. Taking 200ul of each of different compound solutions and test solvents, adding the different compound solutions and the test solvents into a black bottom-penetrating 96-hole culture plate, and detecting an absorption spectrum, an excitation spectrum and an emission spectrum by using a multifunctional microplate reader.
TABLE-1:
| EXAMPLES 1-6 fluorescent probes | |
| Absorption Spectrum (nm) | 300-800 |
| Excitation Spectrum (nm) | 200-500 |
| Emission Spectrum (nm) | 345-700 |
4) Data processing: the absorption spectrum, emission spectrum, excitation and emission fluorescence spectrum are derived by processing with originPro 8.5.
5) As a result: the excitation and emission fluorescence spectra of the fluorescent probes of the various embodiments of the present invention are shown in fig. 1-6.
In the fluorescent probe provided by the invention,
the excitation wavelength range of the fluorescent probes of I-a, I-b, I-c is as follows: 370nm to 390nm, emission wavelength range is: 520 nm.
The excitation wavelength range of the fluorescent probes of I-d, I-e and I-f is as follows: 320nm to 340nm, emission wavelength range is: 380 nm.
Test example 2: application of fluorescent probe in preparation of tumor detection reagent
1. 1mg of the fluorescent probe was weighed, prepared into a 10mM solution using analytically pure dimethyl sulfoxide (DMSO), and then diluted with PBS (pH 7) to 100. mu.M of the detection reagent. Is prepared before use or stored at-20 deg.c in dark place.
2. Tumor cell culture:
(triple negative breast cancer cells MDA-MB-231, human lung cancer cells A549, human normal lung epithelial cells BEAS-2B, and human embryonic kidney cells 293FT were purchased 5 months 2017 from Wuhan Punuoise Life technologies Ltd (https:// www.procell.com.cn /).
The cells were cultured under the corresponding cell culture conditions and used.
The specific culture conditions were as follows:
triple negative breast cancer cells MDA-MB-231: glutamine-containing DMEM solution, 10% serum, 1% diabody, 5% carbon dioxide, 37 ℃;
human lung cancer cell a 549: RPMI-1640 medium, 10% fetal bovine serum, 95% air, 5% carbon dioxide, 37 ℃;
human normal lung epithelial cells BEAS-2B: DMEM medium, 10% serum, 1% diabody, 5% carbon dioxide, 37 ℃;
culture references for human embryonic kidney cells 293 FT: biochemical and Biophysical Research Communications 487, (2017), 34-40;
preparation and culture of GLUT1-293FT cells (GLUT1 highly expressed human embryonic kidney cells) reference: biochemical and Biophysical Research Communications 487, (2017), 34-40.
3. Tumor detection:
the detection reagent prepared in step 1 of this test example was used to carry out a tumor detection test.
1) In a cell culture box, triple negative breast cancer cells MDA-MB-231, human lung cancer cells A549 and human normal lung epithelial cells BEAS-2B are respectively paved in different 96-well plates. After cells adhere to the wall overnight, the detection reagents prepared in step 1 (the fluorescent probe is prepared in example 1) are added respectively, the final concentration of the fluorescent probe is 500nM, and the cells are cultured for 60 minutes at 37 ℃. The plate was washed 3 times with ice-cold PBS (pH 7.0) and transferred to a microplate for photography, see FIG. 7.
As can be seen from fig. 7: the fluorescent probe I-a shows obvious green fluorescence in triple negative breast cancer cells MDA-MB-231 and human lung cancer cells A549, and shows punctate green fluorescence in human normal lung epithelial cells BEAS-2B, which proves that the fluorescent probe I-a can be used for detecting tumor cells.
2) In a cell culture box, human embryonic kidney cells 293FT and GLUT1-293FT cells were plated in different 96-well plates. After cells adhere to the wall overnight, the detection reagents (the fluorescent probes are prepared in examples 1, 2 and 3) are added respectively, the final concentration of the fluorescent probes is 3.125-500 mu M, and the cells are cultured for 60 minutes at 37 ℃. The plate was washed 3 times with ice-cold PBS (pH 7.0) and transferred to an microplate for photography. The results are shown in FIG. 8.
As can be seen from FIGS. 8A, 8B and 8C, the enrichment of the fluorescent probes I-a, I-B and I-C in the human embryonic kidney cells GLUT1-293FT with high GLUT1 is 1.5-3 times that of the human embryonic kidney cells 293 FT.
Experiments prove that the detection effects of the fluorescent probes I-B, I-c, I-d, I-e and I-f on triple negative breast cancer cells MDA-MB-231, human lung cancer cells A549 and human normal lung epithelial cells BEAS-2B are similar to those of I-a.
The enrichment effect of the fluorescent probe I-d in the human embryonic kidney cell GLUT1-293FT with high GLUT1 expression and the human embryonic kidney cell 293FT is similar to that of I-a.
The enrichment effect of the fluorescent probe I-e in human embryonic kidney cell GLUT1-293FT with high expression of GLUT1 and human embryonic kidney cell 293FT is similar to that of I-b.
The enrichment effect of the fluorescent probe I-f in the human embryonic kidney cell GLUT1-293FT with high GLUT1 expression and the human embryonic kidney cell 293FT is similar to that of I-c.
Claims (2)
1. The glycosyl calixarene fluorescent probe for detecting the tumor is characterized by having a structure shown in a formula (I):
wherein the content of the first and second substances,
R1selected from (A-1) or (A-2);
the structural formula of the (A-1) is as follows:
the structural formula of the (A-2) is as follows:
R2selected from:
2. the use of the glycosyl calixarene fluorescent probe for detecting tumor according to claim 1 in the preparation of a reagent for detecting tumor.
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| Gold nanoparticles decorated by clustered multivalent cone-glycocalixarenes actively improve the targeting efficiency toward cancer cells;Svetlana Avvakumova 等;《Chem. Commun》;20140731;第50卷;第11029-11032页 * |
| Molecular Architecture and Symmetry Properties of 1,3-Alternate Calix[4]arenes with Orientable Groups at the Para Position of the Phenolic Rings;Lucio Toma 等;《The Journal of Oraganic Chemistry》;20160921;第81卷;第9718-9727页 * |
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