CN116283663A - Difunctional near infrared fluorescent probe for detecting monoamine oxidase A and viscosity as well as preparation and application thereof - Google Patents
Difunctional near infrared fluorescent probe for detecting monoamine oxidase A and viscosity as well as preparation and application thereof Download PDFInfo
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Abstract
The application discloses a near infrared fluorescent compound which is characterized by having a structural formula shown as a formula (I):
Description
Technical Field
The application relates to the field of fluorescence detection, in particular to a difunctional near infrared fluorescent probe for detecting monoamine oxidase A and viscosity, and preparation and application thereof.
Background
Monoamine oxidase (MAO) is a mammalian flavin enzyme, located mainly on the outer mitochondrial membrane, which catalyzes the oxidative degradation of monoamine neurotransmitters in biological systems. MAO-A expression levels are significantly increased in many physiological diseases, such as age-related diseases, heart diseases, kidney diseases and cancers. On the other hand, the cellular microenvironment index, viscosity, has been shown to be an important factor in the biological environment. Abnormal changes in these factors can lead to cell dysfunction as a marker of disease occurrence, as well as affect other small molecule indicators of activity in cells.
Therefore, the development of an effective fluorescent probe that simultaneously monitors monoamine oxidase a and viscosity to elucidate the crosstalk between these two parameters has become a focus of attention, having great significance in understanding the physiological and pathological roles of monoamine oxidase a and viscosity in the living system.
The small molecule fluorescent probe detection technology has the advantages of high sensitivity and strong visibility, and thus has been increasingly used in organisms. There is no report on the dual-function fluorescent probe of monoamine oxidase A and viscosity. Therefore, the design of a bifunctional fluorescent probe that synthesizes monoamine oxidase A and viscosity is very important for further understanding of its biological function.
Disclosure of Invention
The application provides a near infrared fluorescent compound with dual functions of detecting monoamine oxidase A and viscosity.
A near infrared fluorescent compound having a structural formula as shown in formula (I):
the application also provides a preparation method of the near infrared fluorescent compound, which comprises the following steps:
(1) Adding potassium carbonate into a solvent to react by taking a compound (II) and a compound (III) as reactants until the reaction is monitored, and distilling the solvent under reduced pressure to obtain a crude product;
(2) Adding a deprotection solvent into the obtained crude product for deprotection, and then separating and purifying to obtain a compound with a structural formula shown as (I):
the synthetic route is as follows:
optionally, in step (1): the ratio of the amounts of the compound (II), the compound (III) and the potassium carbonate substance is 1:1-1.2:2-4; further, the ratio of the amount of the compound (II), the compound (III) and the potassium carbonate is 1:1:2.
Optionally, the solvent is DMF or acetonitrile; further, the solvent is DMF.
Alternatively, the reaction conditions are: stirring and reacting for 10-14 h at 50-60 ℃.
Optionally, in step (2): the deprotection solvent is trifluoroacetic acid and anhydrous CH 2 Cl 2 Mixed solution of trifluoroacetic acid and anhydrous CH 2 Cl 2 The volume ratio of (2) is 1:4-1:5.
Alternatively, the separation and purification method may be as follows: concentrating the reaction solution under reduced pressure, separating and purifying by column chromatography, wherein the eluent is CH 2 Cl 2 :CH 3 OH (volume ratio 25:1) to give compound (I).
The compound (II) is disclosed in the specification, and the preparation method can be referred to as The synthesis and bioimaging of a biocompatible hydrogen sulfide fluorescent probe with high sensitivity and selection information analysis (Cambridge, united Kingdom) (2020), 145, (6), 2305-2310.
The compound (III) of the present invention is obtained by purchase.
The near infrared fluorescent compound has monoamine oxidase A and viscosity dual response functions, based on which:
the application also provides application of the near infrared fluorescent compound in preparation of a product for detecting monoamine oxidase A.
The application also provides application of the near infrared fluorescent compound in preparing a product for detecting viscosity.
The application also provides the use of the infrared fluorescent compound in preparing a product capable of simultaneously detecting monoamine oxidase A and viscosity.
Alternatively, the product may be in the form of a fluorescent probe, a test strip or a kit or other product that may be prepared.
Alternatively, the product is a product that detects monoamine oxidase a and/or viscosity in solution or within an organism's cell.
The application also provides a method for detecting monoamine oxidase A and viscosity for non-diagnostic and therapeutic purposes, comprising:
adding the near infrared fluorescent compound into a solution to be detected for reaction to obtain a reaction solution;
detecting the fluorescence value of the obtained reaction liquid under the conditions that the excitation wavelength is 450nm and the emission wavelength is 625nm, bringing the obtained fluorescence value into a standard curve, and calculating to obtain the concentration of monoamine oxidase A in the solution to be detected;
and detecting the fluorescence value of the obtained reaction liquid under the conditions of excitation wavelength of 420nm and emission wavelength of 560nm, bringing the obtained fluorescence value into a standard curve, and calculating to obtain the viscosity of the solution to be detected.
Optionally, when detecting the concentration of monoamine oxidase a, the near infrared fluorescent compound is added in an amount such that the ratio of the final concentration of the near infrared fluorescent compound to the concentration of monoamine oxidase a in the solution to be detected is 0.001m M: 1-50 mug/mL. Under the condition of the proportion, the compound (I) increases the fluorescence intensity at 625nm by nearly 60 times along with the increase of monoamine oxidase A in the buffer solution, and has good linear relation (R 2 = 0.9926). Has higher linear relation to monoamine oxidase A.
Alternatively, the near infrared fluorescent compound is added in an amount to detect the viscosityThe ratio of the final concentration to the viscosity of the solution to be measured was 0.001. 0.001m M:100cP to 100 cP. With the increase of the viscosity of the buffer solution, the fluorescence intensity at 560nm is improved by approximately 90 times, and the ratio has good linear relation (R 2 ＝0.9943)。
Compared with the prior art, the application has at least one of the following beneficial effects:
(1) The compound (I) can be used as a fluorescent probe with the functions of detecting monoamine oxidase A and viscosity at the same time.
(2) The specificity to monoamine oxidase A is good, and the monoamine oxidase A is not interfered by amino acid, active oxygen and various anions and cations.
(3) The sensitivity to viscosity is high.
(4) The fluorescence emission wavelength is longer, the near infrared fluorescence performance is realized, and the biological background interference is lower.
(5) The near infrared fluorescent probe synthesized by the application can be used for preparing a novel clinical diagnostic reagent for early warning of serious diseases, reduces the incidence rate of serious diseases of modern people, accords with the strategy of 'health China' and improves the health level of national people.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of the compound (I) prepared in example 1 in the present application.
FIG. 2 is a nuclear magnetic resonance spectrum of the compound (I) prepared in example 1 of the present application.
FIG. 3 is a fluorescence emission spectrum of the compound (I) prepared in example 2 of the present application, to which monoamine oxidase A/B was added at pH 7.4.
FIG. 4 is a graph showing the ultraviolet absorption spectrum of the compound (I) prepared in example 1 of the present application after various monoamine oxidase A concentrations are added at pH 7.4.
FIG. 5 is a graph showing fluorescence emission spectra of Compound (I) prepared in example 1 of the present application after various monoamine oxidase A concentrations are added at pH 7.4.
FIG. 6 is a graph showing the fluorescence emission spectrum of Compound (I) prepared in example 1 of the present application after various monoamine oxidase A concentrations were added at pH 7.4.
FIG. 7 shows the results of the selectivity test of the compound (I) prepared in example 1 of the present application.
FIG. 8 shows the results of the kinetic test of monoamine oxidase A of the compound (I) prepared in example 1 of the present application. FIG. 9 is a graph showing fluorescence emission spectra of the compound (I) prepared in example 1 of the present invention in Gly/PBS buffer (v/v=1:9 to 9:1), excitation wavelength 420nm.
FIG. 10 is a graph showing the linear relationship between the fluorescence intensity at 560nm of the compound (I) prepared in example 1 and Gly/PBS buffer (v/v=1:9 to 9:1) according to the present invention, and the emission wavelength of 420nm.
FIG. 11 is a plot of fluorescent spots of compound (I) prepared in example 1 of the present invention under different pH buffers (v/v=1/99).
FIG. 12 is a graph showing the effect of cell confocal fluorescence imaging of the compound (I) produced in example 1 of the present invention.
Detailed Description
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all, of the embodiments of the present application. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
Example 1: preparation of Compound (I)
Compound II (1.14 g,5 mmol), compound III (1.19 g,5 mmol), K 2 CO 3 (1.38 g,10 mmol) was added to dry 5mL DMF and stirred at 50deg.C for 12h to give a mixture; the mixture was then diluted with ethyl acetate (40 mL) and washed three times with 40mL of water; the organic layer was separated with anhydrous Na 2 SO 4 Drying;the solvent was removed under reduced pressure to give the intermediate as an orange oil.
To the resulting orange oil was added trifluoroacetic acid (0.5 mL) and anhydrous CH 2 Cl 2 (2 mL) was placed at 0deg.C, stirred for 1h, and then the solvent was removed by evaporation under reduced pressure. By CH 2 Cl 2 :CH 3 Silica gel chromatography with OH (25:1) as eluent gave compound (I) as an orange solid (0.063 g, 75% yield).
1 H NMR(500MHz,Chloroform-d)δ7.92(s,1H),7.75(s,1H),7.62(s,1H),7.47(s,2H),7.37(s,1H),7.22–7.09(m,2H),6.99(d,J＝4.4Hz,2H),6.90–6.79(m,2H),6.35(s,1H),5.49–5.36(m,2H),4.08–3.87(m,2H),2.76–2.62(m,2H),2.51–2.43(m,2H),2.35–2.24(m,2H),2.09–1.95(m,2H),1.37–1.30(m,2H),1.21–1.05(m,6H).
13 C NMR(151MHz,CDCl3)δ169.05,155.74,153.35,152.27,141.87,139.91,136.21,134.59,133.87,131.59,131.54,130.14,128.84,128.52,128.44,125.05,124.10,122.19,120.46,119.14,117.22,113.36,112.64,79.97,79.28,68.85,66.74,43.02,42.08,39.33,35.14,32.08,28.06.
The nuclear magnetic hydrogen spectrum is shown in figure 1, and the nuclear magnetic carbon spectrum is shown in figure 2.
Example 2: fluorescence emission spectra of compound (I) (1 μm) with monoamine oxidase a/B (10 μg/M L) in DMSO/PBS buffer (v/v=1/199) at pH 7.4.
A100. Mu. Mol/mL mother liquor of compound (I) (prepared in example 1) was prepared in advance, 4. Mu.L of PBS buffer added to 0.396mL monoamine oxidase A/B (10. Mu.g/m L) was pipetted by a pipette, and the mixture was shaken at 37℃for two hours, added to a 96-well plate, and the fluorescence spectrum of compound (I) was measured by a multifunctional microplate reader, as shown in FIG. 3, and the excitation wavelength was 450nm.
The data in fig. 3 show that compound (I) has significant specificity for monoamine oxidase a.
Example 3: compound (I) (1 μm) was tested by uv absorbance spectroscopy at 7.4 pH in DMSO/PBS buffer (v/v=1/199) at different monoamine oxidase a concentrations.
A100. Mu. Mol/mL stock solution of compound (I) (prepared in example 1) was prepared in advance, 4. Mu.L of PBS buffer (final monoamine oxidase A concentration was 1. Mu.g/mL, 2. Mu.g/mL, 3. Mu.g/mL, 4. Mu.g/mL, 5. Mu.g/mL, 6. Mu.g/mL, 7. Mu.g/mL, 8. Mu.g/mL, 9. Mu.g/mL, 10. Mu.g/mL) added to 0.396mL of the different monoamine oxidase A concentration was pipetted by a pipette, and the ultraviolet absorption spectrum of compound (I) was measured by a multifunctional microplate reader at 37℃for two hours, see FIG. 4.
The experimental result shows that when monoamine oxidase A does not exist in the reaction system, the compound (I) only has an absorption peak at 420 nm; when the monoamine oxidase A concentration is higher, the absorption peak of the compound (I) gradually red shifts to 450nm.
Example 4: compound (I) (1 μm) was detected by fluorescence spectroscopy of selective results in DMSO/PBS buffer (ph=7.4, v/v=1/199).
A100. Mu. Mol/mL stock solution of compound (I) (prepared in example 1) was prepared in advance, 4. Mu.L of PBS buffer (final monoamine oxidase A concentration was 5. Mu.g/mL, 10. Mu.g/mL, 15. Mu.g/mL, 20. Mu.g/mL, 25. Mu.g/mL, 30. Mu.g/mL, 35. Mu.g/mL, 40. Mu.g/mL, 45. Mu.g/mL, 50. Mu.g/mL) added to 0.396mL of the different monoamine oxidase A concentration was pipetted by a pipette, and the fluorescence spectrum of compound (I) was measured by a multifunctional microplate reader at 37℃for two hours, see FIG. 5. A linear fit of the fluorescence intensity of compound (I) at 625nm with monoamine oxidase A concentration is shown in FIG. 6, with an excitation wavelength of 450nm.
Experimental results show that the fluorescence intensity of the compound (I) is improved by nearly 60 times at 625nm along with the increase of monoamine oxidase A in the buffer solution, and meanwhile, the compound (I) has good linear relation (R 2 = 0.9926). Has higher linear relation to monoamine oxidase A.
Example 5: fluorescence spectroscopic detection of the selective results of compound (I) (1 μm) in DMSO/PBS buffer (ph=7.4, v/v=1/199)
Compound (I) (prepared in example 1) mother liquor of 100. Mu. Mol/mL was prepared in advance and pipetted by a pipetteAdding 4 μL into 0.392mL PBS buffer, and adding 4 μL of biological related active small molecule water solution (1-20 are (1) PBS and (2) Mn respectively) 2+ ；(3)Ser；(4)Phe；(5)Arg；(6)BSA；(7)CO 3 2- ；(8)OAc - ；(9)NO 3 - ；(10)HSO 3 - ；(11)PO 4 3- ；(12)H 2 S；Cl - ；(13)Fe 3+ ；(14)K + ；(15)ClO - ；(16)H 2 O 2 ；(17)ONOO - The method comprises the steps of carrying out a first treatment on the surface of the (18) GSH; (19) Mao-B; (20) Mao-a; final concentration was 1 mM), and the mixture was shaken at 37℃for two hours, and then added to a 96-well plate, and the fluorescence value of Compound (I) was measured by a multifunctional microplate reader. The fluorescence excitation wavelength was 450nm and the emission wavelength was 625nm.
The fluorescence spectrum is shown in FIG. 7. The experimental result shows that the fluorescent intensity of the compound (I) is not substantially changed obviously except monoamine oxidase A in the presence of other related bioactive molecules, and the anti-interference capability of the compound (I) is very good.
Example 6: results of kinetic test of compound (I) on monoamine oxidase a.
A100. Mu. Mol/mL mother liquor of compound (I) (prepared in example 1) was prepared in advance, 4. Mu.L of PBS buffer containing a monoamine oxidase A concentration of 50. Mu.g/mL was added to a pipette tip, and the mixture was shaken for two hours, and then added to a 96-well plate, and the kinetic curve of compound (I) to monoamine oxidase A was measured at 37℃using a multifunctional microplate reader, as shown in FIG. 8. The experimental result shows that the fluorescence intensity of the compound (I) at 625nm gradually and steadily increases along with time, and the reaction is basically complete after 60 minutes.
Example 7: the fluorescence intensity of the compound (I) varies with viscosity.
A100. Mu. Mol/mL mother liquor of compound (I) (prepared in example 1) was prepared in advance, a pipette aspirates 4. Mu.L of PBS buffer (final viscosity values 100cp,200cp,300cp,400cp,500cp,600cp,700cp,800cp,950 cp) added to 0.396mL of different viscosity values, and then added to a 96-well plate at 37℃and then the fluorescence spectrum of compound (I) was measured (see FIG. 9) and a relevant linear curve was made (FIG. 10).
DataIt was shown that compound (I) was excited at 420nm and emitted at 560 nm. With increasing viscosity of the buffer, fluorescence intensity at 560nm was increased by approximately 90 times, while having a good linear relationship (R 2 ＝0.9943)。
Example 8: point-plot of changes in pH versus fluorescence intensity for Compound (I) (1. Mu.M) prepared in example 1 of the present invention under PBS buffer and PBS: glycerol (v/v=1/1)
100. Mu. Mol/mL of compound (I) stock solution was prepared in advance, 4. Mu.L of the stock solution was pipetted into a pipette, added to 0.396mL of PBS buffer of different pH values and PBS: glycerol (v/v=1/9) solution (the final pH was adjusted to a value of 4 to 9 in the buffer, respectively), added to a 96-well plate, counted, and spotted. The fluorescence excitation wavelength is 510nm, and the emission wavelength is 680nm.
The dot pattern is shown in FIG. 11. The data indicate that compound (I) has no sensitivity to pH.
Example 9 cytogram of Compound (I).
10mM of compound (I) (prepared in example 1) stock solution was prepared in advance, and 2. Mu.L was pipetted into 1.998mL DMEM medium.
Blank group: 1mL of the culture solution containing the compound (I) was added to HeLa cells, incubated at 37℃for 0.5h, and washed twice with DMEM medium.
Experimental group (1): incubating with commercial chloroJilin (monoamine oxidase A inhibitor) at a concentration of 2. Mu.M/L at 37℃for 0.5h, washing twice with DMEM medium, then adding the culture solution containing Compound (I), incubating at 37℃for 0.5h, and washing twice with DMEM medium.
Experimental group (2): incubating with commercial nystatin at a concentration of 2. Mu.M/L at 37℃for 0.5h, washing twice with DMEM medium, then adding the culture broth containing Compound (I), incubating at 37℃for 0.5h, and washing twice with DMEM medium.
Blank and experimental groups (1) and (2) were each subjected to fluorescence imaging using a Olympus Fluoview FV 1200 confocal microscope. Yellow channel: the excitation wavelength is 405nm, and the receiving wavelength range is 520-580 nm. Red channel: the excitation wavelength is 488nm and the receiving wavelength is 580-650 nm.
Cell confocal fluorescence imaging effect is shown in fig. 12: the experimental result shows that the compound (I) can detect monoamine oxidase and viscosity change in Hela cells.
The above examples merely represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application is to be determined by the claims appended hereto.
Claims (10)
1. A near infrared fluorescent compound, characterized by having a structural formula as shown in formula (I):
2. the method for preparing a near infrared fluorescent compound according to claim 1, comprising:
(1) Adding potassium carbonate into a solvent to react by taking a compound (II) and a compound (III) as reactants until the reaction is monitored, and distilling the solvent under reduced pressure to obtain a crude product;
(2) Adding a deprotection solvent into the obtained crude product for deprotection, and then separating and purifying to obtain a compound with a structural formula shown as (I):
3. the method according to claim 2, wherein in step (1): the ratio of the amounts of the compound (II), the compound (III) and the potassium carbonate substance is 1:1-1.2:2-4; the solvent is DMF or acetonitrile; the reaction conditions are as follows: stirring and reacting for 10-14 h at 50-60 ℃.
4. The method according to claim 2, wherein in step (2): the deprotection solvent is trifluoroacetic acid and anhydrous CH 2 Cl 2 Mixed solution of trifluoroacetic acid and anhydrous CH 2 Cl 2 The volume ratio of (2) is 1:4-1:5;
the separation and purification are as follows: by CH 2 Cl 2 :CH 3 Silica gel chromatography was performed with OH (25:1) as eluent.
5. Use of the near infrared fluorescent compound of claim 1 for the preparation of a product for detecting monoamine oxidase a.
6. Use of the near infrared fluorescent compound of claim 1 for the preparation of a product for detecting viscosity.
7. Use of an infrared-fluorescent compound according to claim 1 for the preparation of a product in which monoamine oxidase a and viscosity can be detected simultaneously.
8. The use according to any one of claims 5 to 7, wherein the product is a fluorescent probe, a test strip or a kit.
9. A method for detecting monoamine oxidase a and viscosity for non-diagnostic and therapeutic purposes, comprising:
adding the near infrared fluorescent compound as claimed in claim 1 into a solution to be detected for reaction to obtain a reaction solution;
detecting the fluorescence value of the obtained reaction liquid under the conditions that the excitation wavelength is 450nm and the emission wavelength is 625nm, bringing the obtained fluorescence value into a standard curve, and calculating to obtain the concentration of monoamine oxidase A in the solution to be detected;
and detecting the fluorescence value of the obtained reaction liquid under the conditions of excitation wavelength of 420nm and emission wavelength of 560nm, bringing the obtained fluorescence value into a standard curve, and calculating to obtain the viscosity of the solution to be detected.
10. The method according to claim 9, wherein the near infrared fluorescent compound is added in an amount such that the ratio of the final concentration of the near infrared fluorescent compound to the concentration of monoamine oxidase a in the solution to be measured is 0.001 and m M: 1-50 mug/mL;
when the viscosity is detected, the addition amount of the near infrared fluorescent compound is 0.001m M in terms of the ratio of the final concentration of the near infrared fluorescent compound to the viscosity in the solution to be detected: 100cP to 100 cP.
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