CN113354627B - Near-infrared fluorescent compound for detecting viscosity and preparation and application thereof - Google Patents
Near-infrared fluorescent compound for detecting viscosity and preparation and application thereof Download PDFInfo
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- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
- C07D409/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N11/00—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N11/00—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties
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Abstract
The invention provides a high-sensitivity near-infrared fluorescent compound for detecting viscosity, and a preparation method and application thereof, wherein the structure of the near-infrared fluorescent compound is shown as a formula (I). The invention has the following beneficial effects: the compound (I) can be used as a fluorescent probe for detecting viscosity, has the fluorescence excitation of ex-480 nm and em-670 nm, has larger Stokes shift, has the advantages of low background interference, small light damage to biological samples and the like, has high viscosity sensitivity, and provides an effective research tool for researching the physiological action of viscosity in cells.
Description
(I) technical field
The invention relates to a near-infrared fluorescent compound for detecting viscosity, a preparation method thereof and application thereof in preparing a fluorescent probe for detecting viscosity.
(II) background of the invention
A stable intracellular microenvironment is an important condition for maintaining the normal operation of a series of vital activities (such as proliferation, differentiation, metabolism, etc.) of cells. In which intracellular viscosity is involved in processes such as material exchange, intermolecular interaction between biomolecules, chemical signaling, and metabolic transfer, and plays an especially important role. Abnormalities in viscosity are associated with a number of diseases and cellular disorders, such as atherosclerosis, diabetes, Alzheimer's disease, malignancies, etc., and thus by monitoring viscosity, early diagnosis of these conditions can be made. Therefore, viscosity can be used as a reference index for measuring the state of living cells. At present, the traditional method for detecting viscosity is mainly based on in vitro viscosity, and cannot realize viscosity detection on a cell level, so that a novel method for detecting intracellular viscosity is urgently needed to be developed.
In recent years, small molecule fluorescent probes have become powerful tools for detecting biomolecules and biological parameters in living systems due to their advantages of high sensitivity, noninvasive detection, high selectivity, easy operation, real-time monitoring, good biocompatibility and the like. Therefore, the novel viscosity fluorescent probe with the near infrared emission performance has potential application value. The high-sensitivity viscosity fluorescent probe synthesized by the method can be used as a novel clinical diagnosis technology to early warn serious diseases, reduce the incidence rate of the serious diseases of modern people, accord with the strategy of 'healthy China', and improve the national health level.
Disclosure of the invention
The invention aims to provide a high-sensitivity near-infrared fluorescent compound for detecting viscosity, and a preparation method and application thereof.
The technical scheme adopted by the invention is as follows:
a near infrared fluorescent compound for detecting viscosity has a structure shown in formula (I):
the invention also relates to a method for preparing the near-infrared fluorescent compound, which comprises the following steps: adding the compound (II), the compound (III) and piperidine into absolute ethyl alcohol, stirring and reacting at 80-90 ℃ for 12-14 h, and separating and purifying reaction liquid after the reaction is finished to obtain a compound (I);
the amount ratio of the compound (II), the compound (III) and the piperidine is 1: 1-1.2: 0.2-0.4.
Preferably, the amount ratio of the compound (II), the compound (III) and the piperidine is 1:1.2: 0.2.
The separation and purification method comprises the following steps: the reaction solution was concentrated under reduced pressure and purified by silica column chromatography with a volume ratio of dichloromethane/methanol of 1: 10 as eluent, collecting eluent, and drying to obtain compound (I).
The compound (II) of the present invention is a compound disclosed therein, and its production method can be referred to in the literature (M.Riome, K.Port, A.Wijkhuisen, D.Audisc, F.Taran, Fluorogenic immunological reactions: bioorganic alcohols for double tube-on-chip-and-release reactions, Chemical Communications, (2020) 183-.
The compound (III) of the present invention is a disclosed compound, and its preparation method can be referred to in the literature (Y.Ando, Y.Homma, Y.Hiruta, D.Citterio, K.Suzuki, Structural characteristics and optical properties of a series of solvents of solvent fluorescent substances displaying and displacement-height estimation, Dyes and Pigments, (2009) 198-206).
The invention also relates to application of the near-infrared fluorescent compound in preparing a fluorescent probe for viscosity detection.
Specifically, the fluorescent probe is used for measuring the intracellular viscosity value, and the viscosity value is about 20-80 cp.
Preferably, the fluorescent probe is used for measuring the intracellular viscosity value of Hela of human cervical carcinoma cells. The experimental results show that the compound (I) can detect the change of the viscosity in Hela cells, the average fluorescence intensity is 18.039, and after 2 mu M nystatin is added, the average fluorescence intensity is 41.566. The mean fluorescence intensity after changing the intracellular viscosity by the addition of 5. mu.M nystatin was 56.669.
The compound (I) can be used as a fluorescent probe for detecting viscosity, the fluorescence excitation of the fluorescent probe is ex-480 nm and em-670 nm, the fluorescent probe has large Stokes shift, and the fluorescent probe has the advantages of low background interference, small light damage to biological samples and the like. The quantum yield of the fluorescent probe in Gly/PBS buffer (v/v ═ 9:1) is 0.25, the fluorescence is enhanced by about 130 times, and the fluorescent probe can be applied to the fluorescence quantitative detection of viscosity. The fluorescence detection principle for quantifying the viscosity concentration is as follows: the compound (I) can limit the rotation of a carbon-carbon single bond of a structure in a solution with certain viscosity, so that the non-radiation energy of the probe is reduced, the fluorescent light is turned-on, and the change of the fluorescent intensity of the probe at 670nm is detected when the excitation is 480nm, so that the viscosity concentration is obtained.
In contrast to the prior art, compound (1) as described in the reference (T.Chen, Z.K.Chen, R.Y.Liu, A NIR fluorescent probe for detection of vision and physiology imaging in live cells, Organic Biomolecular Chemistry, 2019,17, 6398-:
compound (1) as a fluorescent probe, with a fluorescence excitation reported as ex＝505nm，em685nm allows viscosity detection in cells but is less sensitive to viscosity response, with only about a 5.4 fold increase in fluorescence intensity with increasing viscosity and 685nm fluorescence emission in Gly/PBS buffer (v/v-1: 9 to 9:1), not meeting the requirement for near-infrared viscosity probes. The compound (I) has obviously increased fluorescence intensity along with the increase of the viscosity of the buffer solution, the fluorescence intensity at 680nm is improved by nearly 130 times, and a good linear relation is achieved, so that the compound (I) provided by the invention can be used as a fluorescence probe for detecting the intracellular viscosity, and an effective research tool is provided for researching the physiological action of the viscosity in cells.
The invention has the following beneficial effects: the compound (I) can be used as a fluorescent probe for detecting viscosity, has the fluorescence excitation of ex-480 nm and em-670 nm, has larger Stokes shift, has the advantages of low background interference, small light damage to biological samples and the like, has high viscosity sensitivity, and provides an effective research tool for researching the physiological action of viscosity in cells.
(IV) description of the drawings
FIG. 1 is a nuclear magnetic hydrogen spectrum of compound (I) prepared in example 1 of the present invention.
FIG. 2 shows a nuclear magnetic carbon spectrum of Compound (I) prepared in example 1 of the present invention.
Fig. 3 is a graph showing fluorescence absorption spectra of compound (I) prepared in example 1 of the present invention added to PBS buffer and Gly/PBS buffer (v/v ═ 9: 1).
Fig. 4 shows fluorescence emission spectra (pH 7.4) of compound (I) prepared in example 1 in Gly/PBS buffer (v/v ═ 1:9 to 9:1) according to the present invention. Excitation wavelength 480nm, emission wavelength 670 nm.
Fig. 5 is a graph of the linear relationship (pH 7.4) of compound (I) prepared in example 1 of the present invention in Gly/PBS buffer (v/v ═ 1:9 to 9: 1). Excitation wavelength 480nm, emission wavelength 670 nm.
Fig. 6 is a fluorescence diagram of the selectivity results of compound (I) prepared in example 1 of the present invention in DMSO/PBS buffer (pH 7.4, v/v 1/99). 1-20 are respectively PBS and Mn2+、Ser、Phe、Arg、BSA、CO3 2-、OAc-、NO3 -、HSO3 -、PO4 3-、H2S、Cl-、Fe3+、K+、ClO-、H2O2、ONOO-GSH, Cys, glycerol. FIG. 6 excitation wavelength 480nm, emission wavelength 670 nm.
Fig. 7 is a fluorescence diagram of the selectivity results of compound (I) prepared in example 1 of the present invention in DMSO/PBS buffer (pH 7.4, v/v 1/99). 1-9 are PBS, dichloromethane, chloroform, 1,4 dioxane, DMF, acetone, tetrahydrofuran, ethylene glycol, and glycerol, respectively. FIG. 7 excitation wavelength 480nm, emission wavelength 670 nm.
Fig. 8 is a fluorescence diagram of compound (I) prepared in example 1 according to the present invention under different pH buffer conditions (v/v-1/99).
FIG. 9 is an image of a cell image of Compound (I) prepared in example 1 of the present invention.
FIG. 10 fluorescence quantification map for cellular imaging of Compound (I) prepared in example 1 of the present invention.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1: preparation of Compound (I)
Combining 1mmol of the compound (II)1.2mmol of the substance (III) and 0.2mmol of piperidine are added to a sufficient amount of an ethanol solvent (5ml) and stirred at 85 ℃ for 12 hours; concentrating the reactant at 45 deg.C under reduced pressure to 500uL, purifying with silica column chromatography, eluting with dichloromethane/methanol (v/v, 1: 10), collecting eluate, and drying to obtain compound (I) (yield 53%), whose nuclear magnetic hydrogen spectrum is shown in FIG. 1 and nuclear magnetic carbon spectrum is shown in FIG. 2.1H NMR(500MHz，CDCl3)。
Example 2: fluorescence absorption spectrum of Compound (I) (5. mu.M) in PBS and 90% by volume of glycerol (excitation wavelength: 480nm, emission wavelength: 670nm)
An amount of the compound (I) prepared in example 1 was accurately weighed, a probe stock solution was prepared with dimethyl sulfoxide at a concentration of 0.1mM, 2. mu.L of the solution was pipetted into 0.398mL of glycerol/PBS buffer (glycerol/PBS, v: v, 1:9) and then into a 96-well plate, and then the fluorescence emission spectrum of the compound (I) was measured.
The fluorescence spectrum is shown in FIG. 3, and the Stokes shift is calculated to be 190 nm. The experimental results show that compound (I) has a weak absorption at 480nm in PBS buffer when excited at 480nm wavelength; when the viscosity of the buffer is high, the absorption of the compound (I) at 480nm is strong, indicating that the probe is sensitive to the viscosity.
Example 3: compound (I), the fluorescence intensity of the probe varied with the viscosity in DMSO/PBS buffer (v/v-1/99), pH 7.4
An amount of compound (I) prepared in example 1 was weighed accurately, prepared into a probe stock solution with a concentration of 1mM using dimethyl sulfoxide, pipetted at 2 μ L into 0.398mL of PBS buffer solutions with different viscosity values (final viscosity values of 100cp, 200cp, 300cp, 400cp, 500cp, 600cp, 700cp, 800cp, 950cp, respectively), added to a 96-well plate at 37 ℃, and then the fluorescence spectrum of compound (I) was measured and plotted as a correlation linear curve.
The fluorescence spectra are shown in FIGS. 4 and 5. Data show thatThe fluorescence intensity of compound (I) increases significantly with increasing buffer viscosity. When the viscosity value is 950cp, the fluorescence intensity is improved by nearly 130 times, and a good linear relation (R) is realized2＝0.9985)。
Example 4: the change of fluorescence intensity of the probe with viscosity of compound (I) in DMSO/PBS buffer (v/v-1/99), pH 7.4
The fluorescence spectrum of compound (I) (5 μ M) of the present invention was detected under the conditions of DMSO/PBS buffer (pH 7.4, v/v 1/99) with selective results.
Accurately weighing a certain amount of compound (I), preparing 1mM probe mother liquor by using dimethyl sulfoxide, sucking 2 mu L of the probe mother liquor by a pipette, adding the probe mother liquor into 0.394mL, and then respectively adding 4 mu L of biologically-related active small molecule aqueous solutions (1-20 are respectively PBS and Mn)2+、Ser、Phe、Arg、BSA、CO3 2-、OAc-、NO3 -、HSO3 -、PO4 3-、H2S、Cl-、Fe3+、K+、ClO-、H2O2、ONOO-GSH, Cys and glycerol at a final concentration of 1mM), and the fluorescence value thereof was measured at 37 ℃. The fluorescence excitation wavelength is 480nm, and the emission wavelength is 670 nm.
The fluorescence spectrum is shown in FIG. 6. The experimental result shows that except glycerol, the fluorescence intensity of the compound (I) basically has no obvious change in the presence of other related bioactive molecules, and the anti-interference capability of the compound (I) is very good.
Example 5: fluorescence spectrum detection of selective results of compound (I) (5 μ M) in DMSO/PBS buffer (pH 7.4, v/v 1/99) in the present invention
An amount of compound (I) was accurately weighed, prepared into a 1mM probe stock solution using dimethyl sulfoxide, and 2. mu.L of the solution was pipetted into 0.394mL of the solution, followed by 4. mu.L of each solution (1-9 each of PBS, dichloromethane, chloroform, 1,4 dioxane, DMF, acetone, tetrahydrofuran, ethylene glycol, and glycerol, at a final concentration of 1mM), and the fluorescence value was measured at 37 ℃. The fluorescence excitation wavelength is 480nm, and the emission wavelength is 670 nm.
The fluorescence spectrum is shown in FIG. 7. The experimental result shows that except glycerol, the fluorescence intensity of the compound (I) basically has no obvious change in the presence of other related bioactive molecules, and the anti-interference capability of the compound (I) is very good.
Example 6: the change in fluorescence intensity of compound (I) (5 μ M) prepared in example 1 of the present invention was plotted in the form of a dot plot of the change in pH and the change in fluorescence intensity in DMSO/PBS buffer (pH 7.4, v/v 1/99)
An amount of the compound (I) prepared in example 1 was accurately weighed, prepared into a probe stock solution with a concentration of 1mM using dimethyl sulfoxide, and 2. mu.L of the solution was pipetted into 0.398mL of PBS buffers with different pH values (so that the final pH values in the buffers were from 2 to 10.5, respectively), added to a 96-well plate, counted, and plotted. The fluorescence excitation wavelength is 480nm, and the emission wavelength is 670 nm.
The fluorescence spectrum is shown in FIG. 8. The data show that compound (I) is not pH sensitive.
Example 7: cytographic imaging of Compound (I) of the invention
A certain amount of the probe (I) was accurately weighed, prepared into a 10mM stock solution with dimethyl sulfoxide, and 2. mu.L of the stock solution was pipetted into 1.998 mM DMMEM medium. Adding 1mL of culture solution containing the compound (I) into Hela cells, incubating for 0.5h at 37 ℃, washing twice with DMEM medium, incubating for 30min at 37 ℃ with Nystatin (Nystatin) at concentrations of 2 μ M and 5 μ M, washing twice with PBS, and performing fluorescence imaging with Olympus Fluoview FV 1200 confocal microscope. FIG. 9 is a diagram of the effect of confocal fluorescence imaging of cells: the excitation wavelength of the compound (I) is 480nm, and the receiving wavelength range is 670-700 nm. FIG. 10 is a graph of fluorescence intensity: a is no nystatin added, b is 2 μ M nystatin added, and c is 5 μ M nystatin added.
The fluorescence spectrum is shown in FIG. 9. The experimental results show that the compound (I) can detect the change of the intracellular viscosity of Hela, the average fluorescence intensity is 18.039, and the average fluorescence intensity is 41.566 after the intracellular viscosity is changed by adding 2 mu M nystatin. The mean fluorescence intensity after changing the intracellular viscosity by the addition of 5. mu.M nystatin was 66.669.
Claims (7)
1. A near infrared fluorescent compound for detecting viscosity has a structure shown in formula (I):
2. a method of making the near-infrared fluorescent compound of claim 1, the method comprising: adding the compound (II), the compound (III) and piperidine into absolute ethyl alcohol, stirring and reacting at 80-90 ℃ for 12-14 h, and separating and purifying reaction liquid after the reaction is finished to obtain a compound (I);
3. the method according to claim 2, wherein the amount of the compound (II), the compound (III) and the piperidine is 1:1 to 1.2:0.2 to 0.4.
4. The method according to claim 3, wherein the amount of compound (II), compound (III) and piperidine is in a ratio of 1:1.2: 0.2.
5. The method according to claim 2, wherein the separation and purification method comprises: the reaction solution was concentrated under reduced pressure and purified by silica column chromatography with a volume ratio of dichloromethane/methanol of 1: 10 as eluent, collecting eluent, and drying to obtain compound (I).
6. Use of the near-infrared fluorescent compound of claim 1 for the preparation of a fluorescent probe for intracellular viscosity detection.
7. The use according to claim 6, wherein said fluorescent probe is used for the determination of the intracellular viscosity number of Hela, a human cervical cancer cell.
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| CA2129933A1 (en) * | 1993-08-12 | 1995-02-13 | Graham D. Darling | Viscosity probe |
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| CN112745258A (en) * | 2021-01-22 | 2021-05-04 | 齐鲁工业大学 | Mitochondrial fluorescent probe with high signal-to-noise ratio and preparation method and application thereof |
| CN112939957A (en) * | 2021-02-03 | 2021-06-11 | 山西大学 | Benzoindole derivative In-XY1, and synthesis method and application thereof |
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| CN109694361A (en) * | 2019-02-26 | 2019-04-30 | 南方医科大学 | A kind of benzothiazole 2- acetonitrile and its application |
| CN112745258A (en) * | 2021-01-22 | 2021-05-04 | 齐鲁工业大学 | Mitochondrial fluorescent probe with high signal-to-noise ratio and preparation method and application thereof |
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