Single light source excitation for ratio-type fluorescent probe to distinguish biological thiols

Abstract

The present application relates to a single light source excitation ratio type fluorescent probe for simultaneously distinguishing and detecting Cys, GSH and Hcy, and belongs to the field of fluorescent probes. The molecular structure is as follows: the probe molecule itself emits red fluorescence, and when responding to Cys, emits yellow fluorescence and red fluorescence; when responding to GSH, emits green fluorescence and red fluorescence; when responding to Hcy, green fluorescence can be observed first, and then gradually red shifts to yellow fluorescence, and red fluorescence can be observed simultaneously. The probe molecule disclosed by the present application can not only single light source excitation ratio type simultaneously distinguish and detect three biological thiols, but also can realize rapid, qualitative and quantitative detection of three biological thiols, and has important application value in the field of biochemistry and the like.

Description

[0001] The present application relates to a single light source excitation ratio type fluorescent probe THQ-O-NBS for simultaneously distinguishing and detecting Cys, GSH and Hcy, including preparation, photophysical property research and application in the field of biological imaging, and belongs to the field of fluorescent probes. BACKGROUND

[0002] Small molecule biological thiols play an important role in many physiological processes and are closely related to many diseases. There are three representative biological thiols: cysteine (Cys), glutathione (GSH) and homocysteine (Hcy). Although the three have similar chemical structures and reactivity, the roles they play are completely different. Cys is an essential amino acid for protein synthesis, and its abnormal content can cause many diseases, such as edema, growth retardation, infantile lethargy, cardiovascular disease, liver damage, skin disease, hair discoloration, etc.; GSH is the highest content of biological thiols in the human body, with various cell functions, including maintaining intracellular redox activity, xenobiotic metabolism, intracellular signal transduction and gene regulation, and its abnormal content can cause cancer, leukemia, AIDS, etc.; the role of Hcy in the human body is still a highly controversial topic, and its normal concentration in the human body is 5-15mM, and its abnormal content can cause cardiovascular disease, osteoporosis, Alzheimer's disease, etc. Therefore, detecting the content of the three biological thiols in the human body may help early treatment of certain diseases. Compared with traditional detection techniques, fluorescence analysis has the advantages of high sensitivity, high selectivity, simple operation, etc., and can be applied to cell and in vivo imaging, so in recent years, the application of fluorescent probes to detect the content of biological thiols in the body has become more and more extensive. SUMMARY

[0003] The present application aims to provide a single light source excitation red ratio type fluorescent probe THQ-O-NBS for simultaneously distinguishing and detecting Cys, Hcy and GSH, which solves the defect that the existing fluorescent probes for simultaneously distinguishing and detecting three biological thiols are all switch type.

[0004] The technical solution of the present application is as follows:

[0005] The fluorescent probe THQ-O-NBS is designed based on tetrahydroquinoxaline fluoroborine dye, and the structure formula of the fluorescent probe THQ-O-NBS is as follows:

[0006]

[0007] The synthesis route of the probe THQ-O-NBS is as follows:

[0008] (a) 4-aminobenzoic acid and EDCI, DMAP were added to anhydrous DMF, stirred at room temperature for 30 min, then 4-hydroxyphenethylamine was added to the reaction solution, and reacted at room temperature for 24 h. The reaction was stopped, the reaction solution was poured into water, extracted with ethyl acetate three times, washed with saturated brine five times, dried over anhydrous sodium sulfate, and rotary evaporated to obtain a solid crude product. The white solid compound 1 was obtained by column chromatography, and the technical route was as follows,

[0009]

[0010] (b) Compound 1 and compound 2 obtained in the above step, and p-toluenesulfonic acid were added to anhydrous ethanol, and refluxed overnight. The reaction solution was poured into water, extracted with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, and rotary evaporated to obtain a solid crude product compound 3. The technical route was as follows,

[0011]

[0012] (c) Compound 3 obtained in the above step was dissolved in 1,2-dichloroethane, N,N-diisopropyl ethylamine was added, stirred for 10 min, then boron trifluoride ether was added, and the temperature was raised to 70°C for 3 h. The reaction was stopped, the reaction solution was poured into ice water, the pH was adjusted to weakly acidic with saturated sodium bicarbonate solution, extracted with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, and rotary evaporated to obtain a red oily liquid crude product. The red solid product THQ-OH was obtained by column chromatography. The technical route was as follows,

[0013]

[0014] (d) 3-chloro-o-phenylenediamine was dissolved in anhydrous ethanol, stirred to dissolve, then selenium dioxide was added, and the temperature was raised to 80°C for 2 h. The reaction was stopped, directly rotary evaporated, and purified by column chromatography to obtain a yellow solid product 4. The technical route was as follows,

[0015]

[0016] (e) Compound 4 prepared in the above step was added to concentrated sulfuric acid under ice bath, stirred for 5 min, then concentrated nitric acid was added dropwise, and the ice bath reaction was continued for 1 h. The reaction was stopped, the reaction solution was poured into ice water, a large amount of yellow solid was precipitated, and the solid was filtered and dried to obtain a yellow solid crude product 5. The technical route was as follows,

[0017]

[0018] (f) The compound 5 prepared in the above step was added into concentrated hydrochloric acid, and hydriodic acid was added drop by drop, and the reaction was carried out at room temperature for 1 h, and the reaction was stopped. The reaction solution was poured into saturated sodium sulfite solution, and 2 mol / L NaOH was added to adjust the pH of the reaction solution to 8, and dichloromethane was used for extraction, and the organic phase was filtered with diatomite, and dried with anhydrous sodium sulfate, and rotary evaporation was carried out to obtain yellow solid product 6, and the technical route was as follows,

[0019]

[0020] (g) The compound 6 prepared in the above step was dissolved in toluene, and triethylamine was added for continuous stirring for 10 min, and then dichlorosulfoxide was dissolved in toluene and added drop by drop into the reaction solution, and the temperature was raised to 100℃ for reaction for 2 h, and the reaction was stopped. The reaction solution was directly rotary evaporated and separated and purified by column chromatography to obtain white solid compound 7. The technical route was as follows,

[0021]

[0022] (h) Compound THQ-OH and compound 7 were dissolved in acetonitrile, and potassium carbonate was added, and the temperature was raised to 65℃ for reaction for 14 h, and the reaction was stopped. The reaction solution was poured into water, dichloromethane was used for extraction, and the organic phases were combined, dried with anhydrous sodium sulfate, and the solvent was removed by reduced pressure distillation, and separated and purified by column chromatography to obtain red solid product THQ-O-NBS, and the technical route was as follows,

[0023]

[0024] The molar ratio of p-aminobenzoic acid, p-hydroxyphenethylamine, EDCI and DMAP in step (a) is 1:1:1:2.5.

[0025] The molar ratio of compound 2 and compound 1 in step (b) is 1:1.2, and a small amount of p-toluenesulfonic acid.

[0026] The molar ratio of compound 3, boron trifluoride ether and N,N-diisopropylethylamine in step (c) is 1:1:1.5.

[0027] The molar ratio of 3-chloro-o-phenylenediamine and selenium dioxide in step (d) is 1:1.2.

[0028] The molar ratio of compound 5 and concentrated nitric acid in step (e) is 1:20.

[0029] The molar ratio of compound 6, concentrated hydrochloric acid and hydriodic acid in step (f) is 1:15:1.

[0030] The molar ratio of compound 7, dichlorosulfoxide and triethylamine in step (g) is 1:10:3.

[0031] The mass ratio of the compound 4, the compound 5 and the potassium carbonate in the step (h) is 1:2.5:1.

[0032] The eluent used in the column chromatography separation and purification in the steps (a), (c), (d), (g) and (h) is (V 二氯甲烷 :V 甲醇 = 100:1), (V 二氯甲烷 :V 甲醇 = 200:1), (V 石油醚 :V 乙酸乙酯 = 30:1), (V 二氯甲烷 :V 石油醚 = 1:1), (V 二氯甲烷 :V 石油醚 = 1:3), respectively.

[0033] The fluorescence probe testing method of the present application is as follows: the probe molecule is dissolved in DMSO / PBS (10.0 mM, v / v, 1 / 1, pH = 7.40), and the testing is carried out at room temperature (25℃). The biological thiols can be qualitatively and quantitatively detected, and the specific implementation method is described in detail in the implementation examples.

[0034] The response mechanism of the fluorescence probe of the present application is as follows: the probe THQ-O-NBS is red fluorescence itself, and NBS is the recognition group. As shown in the following figure, after the nucleophilic substitution reaction of the probe THQ-O-NBS and the biological thiol and the Smiles rearrangement, the NBS part is separated from the probe molecule and releases the red dye THQ-OH. The probe and Cys produce yellow fluorescence and red fluorescence after response; the probe and Hcy produce green fluorescence after response, and with the passage of time, the green fluorescence disappears, and this process is accompanied by red fluorescence; the probe and GSH produce green fluorescence and red fluorescence after response.

[0035] The fluorescence probe THQ-O-NBS of the present application is red fluorescence itself, and after response with Cys, red fluorescence with a maximum emission wavelength of 650nm and yellow fluorescence with a maximum emission wavelength of 550nm are obtained; after response with GSH, red fluorescence with a maximum emission wavelength of 650nm and green fluorescence with a maximum emission wavelength of 525nm are obtained; after response with Hcy, red light with a maximum emission wavelength of 650nm is obtained, and green fluorescence with a maximum emission wavelength of 525nm appears first, and with the passage of time, the green fluorescence at 525nm gradually red shifts to yellow fluorescence with a maximum emission wavelength of 550nm.

[0036] The probe molecule described in the present application has a simple synthesis route, low cost, good biocompatibility, and can realize rapid and simultaneous differential detection of biological thiols. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The figure is the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe THQ-OH-NBS of the application in deuterated DMSO, the horizontal coordinate is chemical shift, and the vertical coordinate is intensity.

[0038] Figure 2 The figure is the nuclear magnetic resonance hydrogen spectrum of the fluorescent dye THQ-OH of the application in deuterated DMSO, the horizontal coordinate is chemical shift, and the vertical coordinate is intensity.

[0039] Figure 3 The figure is the response of the fluorescent probe (10 μM) of the application to related analytes (Glu, Arg, Lys, Tyr, Leu, Pro, Trp, Ser, Thr, Asp, Val, lie, His, Ala, Phe, Met and Gly, with a concentration of 10.0 μM) in DMSO / PBS buffer (10.0 mM, v / v, 1 / 1, pH = 7.40), the horizontal coordinate is wavelength, and the vertical coordinate is fluorescence intensity.

[0040] Figure 4 The figure is the sensitivity detection diagram of the fluorescent probe THQ-O-NBS of the application to Cys, GSH and Hcy.

[0041] Figure 5 The figure is the response process mechanism diagram of the fluorescent probe of the application.

[0042] DETAILED DESCRIPTION

[0043] Example 1: Synthesis of compound 1

[0044] Under the protection of argon, p-aminobenzoic acid (200.0 mg, 1.46 mmol), EDCI (280.0 mg, 1.46 mmol) and DMAP (446.0 mg, 3.65 mmol) were dissolved in 4 mL of anhydrous DMF, stirred at room temperature for 30 min, then p-hydroxyphenethylamine was dissolved in 1 mL of anhydrous DMF, added dropwise to the reaction solution, and continued to stir at room temperature for 24 h. The reaction was stopped, the reaction solution was poured into saturated brine, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate for 30 min, and rotary evaporated to obtain the crude product. Finally, column chromatography (silica gel 200-300 mesh, eluent V 二氯甲烷 :V 甲醇 = 100:1) to obtain compound 1, a milky white solid, 252.2 mg, with a yield of 68.4%.

[0045] Example 2: Synthesis of compound 3

[0046] In a 25 mL round bottom flask, compound 2 (50.0 mg, 0.21 mmol), compound 1 (66.3 mg, 0.26 mmol), a trace of p-toluenesulfonic acid, 5 mL of absolute ethanol were added. The solution changed from red yellow green to dark red, the reaction was carried out at 80 °C overnight, and the reaction was stopped. The reaction solution was poured into saturated brine solution, extracted with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate for 30 min, and rotary evaporated to get a red viscous liquid, 130.9 mg was weighed and directly used in the next step.

[0047] Example 3: Synthesis of compound THQ-OH

[0048] Compound 3 was dissolved in 6 mL of 1,2-dichloroethane under argon protection, 240 μL of N,N-diisopropylethylamine was added at room temperature, stirred for 10 min, then 250 μL of boron trifluoride etherate was added, the temperature was raised to 70 °C and the reaction was carried out for 3 h, and the reaction was stopped. The reaction solution was poured into saturated sodium bicarbonate solution, extracted with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate for 30 min, and rotary evaporated to get the crude product, which was finally separated by column chromatography (silica gel 200-300 mesh, eluent: V 二氯甲烷 / V 甲醇 = 200 / 1) to get the red solid product 47.2 mg, yield 45.9%. 1 H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 8.60 (d, J = 5.8 Hz, 1H), 8.57 (s, 1H), 7.90 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 8.2 Hz, 2H), 7.03 (d, J = 8.0 Hz, 2H), 6.69 (s, 1H), 6.67 (s, 1H), 6.66 (s, 1H), 6.14 (s, 1H), 3.57 (d, J = 5.2 Hz, 2H), 3.49 (d, J = 7.3 Hz, 2H), 3.42 (d, J = 7.3 Hz, 2H), 3.25 (d, J = 7.1 Hz, 2H), 3.13 (t, J = 5.0 Hz, 2H), 2.73 (t, J = 7.6 Hz, 2H), 1.14 (dt, J = 13.5, 6.9 Hz, 6H).

[0049] Example 4: Synthesis of compound 4

[0050] 3-Chloro-o-phenylenediamine (1.02 g, 7.04 mmol) was dissolved in 40 mL of absolute ethanol, after stirring to dissolve, selenium dioxide (959.0 mg, 8.56 mmol) was added, the temperature was raised to 80 °C and the reaction was carried out for 2 h, the reaction was stopped, and the crude product was directly rotary evaporated, which was finally separated by column chromatography (silica gel 200-300 mesh, eluent: V 石油醚 :V 乙酸乙酯= 30: 1) to give yellow solid product 1.3 g, yield 86.6%.

[0051] Example 5: Synthesis of compound 5

[0052] The above compound was added to a round bottom flask under ice bath, 30 mL concentrated sulfuric acid was added, after stirring for 5 min, 7 mL concentrated nitric acid was added drop by drop, the reaction was continued for 1 h under ice bath until the reaction was completed, and the reaction was stopped. Pour the reaction solution into ice water to precipitate a large amount of yellow solid, filter and dry to obtain yellow solid product 1.4 g, yield 90.6%.

[0053] Example 6: Synthesis of compound 6

[0054] Compound 5 (263.2 mg, 1.0 mmol) was added to a round bottom flask, 10 mL concentrated hydrochloric acid was added, and 2.5 mL hydroiodic acid was added drop by drop. The solution changed from yellow to black purple solution, and the reaction was complete after 1 h at room temperature. Stop the reaction, add 10 mL saturated sodium sulfite, and then add 2 mol / L NaOH to adjust the pH of the reaction solution to 8. The color of the solution gradually lightened, and DCM was extracted (3x50 mL). The organic phase was filtered with diatomite, and the yellow solid product was obtained by rotary evaporation, with a yield of 177.5 mg and a yield of 94.5%.

[0055] Example 7: Synthesis of compound 7

[0056] The compound 6 (150.0 mg, 0.8 mmol) obtained in the above step was dissolved in 6 mL of toluene and stirred to dissolve, then 540.1 mg of triethylamine was added and stirred for 10 min. 800.0 mg of SOCl2 was dissolved in 2 mL of toluene and added drop by drop to the reaction solution. The temperature was raised to 100°C and the reaction was continued for 2 h until the reaction was complete. The reaction was stopped. Finally, column chromatography (silica gel 200-300 mesh, eluent: V 石油醚 :V 二氯甲烷 = 1: 1) to give white solid product 100.2 mg, yield 57.8%.

[0057] Example 8: Synthesis of compound THQ-O-NBS

[0058] Compound THQ-OH (35.0 mg, 0.07 mmol), compound 7 (22.5 mg, 0.1 mmol), K2CO3 (25.3 mg, 0.1 mmol) were added to a 25 mL round bottom flask, 5 mL acetonitrile, 65°C overnight reaction, stop the reaction. Pour the reaction solution into saturated brine, extract with dichloromethane, combine the organic phase, dry with anhydrous sodium sulfate for 30 min, and finally column chromatography (silica gel 200-300 mesh, eluent: V 二氯甲烷 :V 石油醚=1:3), yielding 12.2 mg of red solid product, yield 26.0%. 1 H NMR (400MHz, DMSO-d6) δ8.68 (d, J=8.1Hz, 2H), 8.57 (s, 1H), 7.92 (d, J=8.2Hz, 2H), 7.59 (d, J=8.2Hz, 2H), 7.45 (d,J=8.0Hz,2H),7.30(d,J=8.0Hz,2H),6.84(d,J=8.7Hz,1H),6.65(s,1H),6.14 (s,1H),3.58(d,J=7.3Hz,4H),3.49(d,J=7.2Hz,2H),3.25(q,J=6.8Hz,2H) ,3.14(d,J=5.2Hz,2H),2.95(t,J=7.3Hz,2H),1.14(dt,J=13.6,7.0Hz,6H).

[0059] Implementation Case 9: Selectivity Experiment of the Fluorescent Probe THQ-O-NBS of the Present Invention

[0060] The substances used in the selectivity assay included (Glu, Arg, Lys, Tyr, Leu, Pro, Trp, Ser, Thr, Asp, Val, Ille, His, Ala, Phe, Met, and Gly, at a concentration of 10.0 μM). The fluorescent probe (10 μM) was reacted in DMSO / PBS buffer (10.0 mM, v / v, 1 / 1, pH = 7.40) for 40 minutes. (See attached...) Figure 3 As shown, the probe molecule exhibits red fluorescence in solution. Upon addition of Cys, a gradual increase in fluorescence intensity at the maximum emission wavelength of 550 nm was observed, eventually reaching its maximum. Upon addition of GSH, a gradual increase in fluorescence intensity at the maximum emission wavelength of 525 nm was observed, also eventually reaching its maximum. Upon addition of Hcy, an initial increase in fluorescence intensity at 525 nm was observed, followed by a gradual redshift of the maximum emission wavelength to 550 nm after 40 minutes. No other fluorescence was observed upon addition of other biological components. Therefore, based on the different fluorescence signals, Cys, GSH, and Hcy can be successfully distinguished from other biological components, and these three biothiols (Cys, GSH, and Hcy) can be simultaneously distinguished based on their signal differences.

[0061] Implementation Case 10: Sensitivity Experiment of the Fluorescent Probe THQ-O-NBS of the Present Invention

[0062] To investigate the sensitivity of the probe THQ-O-NBS to Cys, GSH, and Hcy, we studied the fluorescence changes of THQ-O-NBS with varying concentrations of Cys, GSH, and Hcy. (See attached image.) Figure 4As shown, with the gradual increase of Cys concentration, the fluorescence intensity at 550 nm also gradually increased, when the Cys concentration reached 10 equiv., the fluorescence intensity reached a maximum, the reaction equilibrium; when adding GSH, with the increase of GSH concentration, the fluorescence intensity at 525 nm constantly strengthened, in 6 equiv. reached equilibrium; when adding Hcy after 40 min, the fluorescence intensity at 550 nm can be observed to enhance, when adding 9.0 equiv. Hcy, the fluorescence intensity at 550 nm reached a maximum. From F 550 / F 650 With the change of Cys concentration, it can be concluded that the fluorescence intensity and Cys concentration between 0-2.0 equiv. have a good linear relationship, the linear correlation coefficient is Cys linear equation: y = 0.0885x + 0.2216, R = 0.9209. From F 525 / F 650 With the change of GSH concentration, it can be concluded that the fluorescence intensity and concentration between 0-3.5 equiv. have a good linear relationship, the linear correlation coefficient is GSH linear equation: y = 0.05x + 0.3113, R = 0.9973. From F 550 / F 650 With the change of Hcy concentration, it can be concluded that the fluorescence intensity and concentration between 0-3.5 equiv. have a good linear relationship, the linear correlation coefficient is Hcy linear equation: y = 0.0992x + 0.1499, R = 0.9620. According to the signal-to-noise ratio S / N = 3, it can be calculated that the detection limit of probe THQ-O-NBS for Cys, GSH, Hcy is 0.24 nM, 3.65 nM, 0.7 nM, respectively. The experimental results show that the probe THQ-O-NBS can qualitatively and quantitatively detect Cys, GSH and Hcy within a certain concentration, and the detection limit shows that its sensitivity is high.

Claims

1. A single light source excited ratio fluorescent probe for distinguishing Cys, Hcy and GSH, which has the structure of:

Images