Rhodamine-methylene blue derivative fluorescent probe, and preparation method therefor and use thereof

Abstract

The present invention belongs to the technical field of organic synthesis. Provided are a rhodamine-methylene blue derivative fluorescent probe (I), and a preparation method therefor and the use thereof. In the preparation method for the fluorescent probe, raw materials are easily available, and a synthesis method is simple and convenient. The fluorescent probe can achieve synchronous double-channel fluorescence recognition of intracellular ClO- and ATP, and has a detection limit regarding ClO- of 0.90 nM and a detection limit regarding ATP of 23.60 nM; therefore, the fluorescent probe can be used for fluorescence quantitative detection of ClO- and ATP, can also be used in synchronous fluorescence imaging of ClO- and ATP in cells under the stimulation of a drug, and has a wide potential application value.

Description

A rhodamine-methylene blue derivative fluorescent probe and its preparation method and application Technical Field

[0001] The present invention relates to the technical field of organic synthesis, in particular to a rhodamine-methylene blue derivative fluorescent probe and a preparation method and application thereof. Background Art

[0002] Hypochlorous acid (HClO) / hypochlorite (ClO - ) is widely used as a disinfectant in daily life. - It is also an important reactive oxygen species (ROS) produced during cellular aerobic respiration and is involved in many physiological processes. While cells are undergoing aerobic respiration, they synthesize adenosine triphosphate (ATP) through oxidative phosphorylation to provide energy for cell activities. Its level is also a sensitive signal of cell health. - It has an extremely close relationship with ATP. The dynamic balance and growth and decline of the two play a key role in the life processes of organisms such as survival, development, aging and death. Dysfunctions such as abnormal oxidative stress and energy metabolism disorders in cells are important inducers of lesion formation and metastasis of many malignant diseases (such as Alzheimer's disease, cardiovascular disease, epilepsy and cancer). Therefore, it is necessary to develop effective tools to monitor cellular ClO - and ATP levels are particularly important, which helps to elucidate the physiological relationship between the two and the pathogenesis of related diseases.

[0003] In recent years, fluorescent molecular probe technology has become the mainstream method of biological detection due to its high sensitivity, simple operation and low cost. However, most of the existing fluorescent probes can only detect ClO - or ATP, but cannot detect ClO simultaneously - and ATP, limiting its further practical application.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide a rhodamine-methylene blue derivative fluorescent probe and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a rhodamine-methylene blue derivative fluorescent probe, the structural formula of which is shown below:

[0008] The present invention also provides a preparation method of the rhodamine-methylene blue derivative fluorescent probe, comprising dissolving methylene blue phosgene and Na2CO3 in an organic solvent, then adding an organic solution of rhodamine B aminoethylpiperazine, stirring for reaction, pouring into ice water, extracting, separating the organic phase, removing the solvent by rotary evaporation, and purifying to obtain the rhodamine-methylene blue derivative fluorescent probe.

[0009] Preferably, the molar ratio of methylene blue phosgene to Na2CO3 is 1:3.

[0010] Preferably, the mass volume ratio of the methylene blue phosgene to the organic solvent is 1 g:10 mL; and the organic solvent comprises dichloromethane.

[0011] Preferably, the molar ratio of the rhodamine B aminoethylpiperazine to the methylene blue phosgene is 4:1; the solvent of the organic solution of rhodamine B aminoethylpiperazine includes dichloromethane with a concentration of 2.296 mol·L -1 .

[0012] Preferably, the stirring reaction is carried out at room temperature for 5 hours with a stirring frequency of 600 rpm.

[0013] Preferably, the extraction is performed using ethyl acetate.

[0014] Preferably, the rotary evaporation conditions are: 50° C., 0.1 MPa; and the purification is silica gel column chromatography purification.

[0015] Preferably, the eluent for the silica gel column chromatography is ethyl acetate:petroleum ether=1:300 (v:v).

[0016] The present invention also provides the rhodamine-methylene blue derivative fluorescent probe in ClO - and ATP detection.

[0017] The present invention also provides the rhodamine-methylene blue derivative fluorescent probe as ClO - and the application of ATP fluorescent probes in cell imaging.

[0018] The present invention discloses the following technical effects:

[0019] The present invention prepares a new type of rhodamine-methylene blue derivative fluorescent probe, the raw materials are easily available, and the synthesis method is simple; it can synchronously identify intracellular ClO with dual-channel fluorescence. - and ATP, ClO - The detection limit is 0.90nM and the detection limit for ATP is 23.60nM, so it can be used for ClO - and ATP fluorescence quantitative detection, which can be used to stimulate ClO in cells- Fluorescence imaging synchronized with ATP has a wide range of potential applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 is a rhodamine-methylene blue derivative fluorescent probe prepared in Example 1 of the present invention 1 H NMR spectrum;

[0022] FIG2 is a rhodamine-methylene blue derivative fluorescent probe prepared in Example 1 of the present invention 13 C NMR spectrum;

[0023] FIG3 is a mass spectrum of the rhodamine-methylene blue derivative fluorescent probe prepared in Example 1 of the present invention;

[0024] Figure 4 a is a rhodamine-methylene blue derivative fluorescent probe (1×10 -5 1×10 -4 mol / L analyte F - ,Cl - ,Br - ,I - ,ClO4 - ,PO4 3- ,H2PO4 - ,HPO4 2- , pyrophosphate (ppi), SO4 2- ,HSO3 - ,S 2- , Ca 2+ , Mg 2+ , K + , L-cysteine ​​(L-Cys), glutathione (GSH), ClO - ,H2O2,ONOO - , 1 O2,ROO - a is the fluorescence spectrum of the rhodamine-methylene blue derivative fluorescent probe (1×10 -51×10 -4 mol / L analyte PO4 3- ,H2PO4 - ,HPO4 2- , fluorescence spectra of ppi, L-Cys, GSH, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UDP) (excitation wavelength is 360 nm);

[0025] Figure 5 a is the fluorescence of the rhodamine-methylene blue derivative prepared in Example 1 of the present invention (1×10 -5 mol / L) EtOH:PBS (0.02 mol / L, pH = 7.4, 5:95, v:v) solution was titrated with different concentrations of ClO - The fluorescence spectrum of the graph shows that the fluorescence intensity at 680 nm changes with the ClO - Concentration linear variation trend diagram (excitation wavelength is 630nm); b is the fluorescence of the rhodamine-methylene blue derivative prepared in Example 1 of the present invention (1×10 -5 Figure 2. Fluorescence spectra of ATP titrated with EtOH:PBS (0.02 mol / L, pH = 7.4, 5:95, v:v) solution containing 0.1 mol / L ATP. The inset shows the linear variation trend of fluorescence intensity at 590 nm with ATP concentration (excitation wavelength is 360 nm).

[0026] Figure 6 shows images of Raw246.7 cells, where a is a yellow channel fluorescence image; b is a NIR channel fluorescence image; c is a bright field image; d is a superposition of yellow channel and NIR channel (Yellow+NIR) imaging; and e is a merged image of yellow channel, NIR channel, and bright field imaging.

[0027] Figure 7 is an image of Raw246.7 cells, where: a is a fluorescence image of the yellow channel; b is a fluorescence image of the NIR channel; c is a bright field image; d is an overlay of the yellow channel and NIR channel (Yellow+NIR) images; and e is an overlay of the yellow channel, NIR channel, and bright field images (Merge). DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0034] Example 1

[0035] The preparation method of rhodamine-methylene blue derivative fluorescent probe is as follows:

[0036] Methylene blue phosgene (MB-COCl, 1 g, 2.87 mmol) and Na₂CO₃ (0.91 g, 8.61 mmol) were added to dichloromethane (10 mL). A dichloromethane solution (5 mL) containing rhodamine B aminoethylpiperazine (RhB-AP, 6.357 g, 11.48 mmol) was then added. The mixture was stirred at 600 rpm at room temperature for 5 hours, then poured into ice water (200 mL) and extracted with ethyl acetate (3 × 150 mL). The organic phase was separated, and the solvent was removed by rotary evaporation at 50°C and 0.1 MPa. Purification by silica gel column chromatography (eluent: ethyl acetate:petroleum ether = 1:300, v:v) afforded the rhodamine-methylene blue derivative fluorescent probe in a 23% yield.

[0037] The obtained rhodamine-methylene blue derivative was subjected to nuclear magnetic resonance analysis using a nuclear magnetic resonance instrument, and the results were as follows:

[0038] 1 H NMR(400MHz,DMSO-d6)δ7.80-7.82(m,1H,Ar-H),7.52-7.57(m,2H,Ar-H),7.46(s,1H,Ar-H),7.44(s, 1H,Ar-H),6.71-6.72(d,2H,Ar-H),6.64-6.67(dd,2H,Ar-H),6.31-6.41(m,6H,Ar-H),3.32-3.37(q, 8H,4CH2-CH3), 3.07-3.10 (t, 2H, CH2), 3.07-3.10 (t, 2H, CH2), 3.00-3.03 (t, 4H, 2CH2), 2.91 (s, 12H, 4CH3), 1.94-1.97 (t, 4H, 3CH2), 1.86-1.89 (t, 2H, CH2), 1.09-1.12 (t, 12H, 4CH3-CH2). The specific H NMR spectrum is shown in Figure 1;

[0039] 13 C NMR (101 MHz, DMSO-d6) δ 167.10, 157.53, 153.61, 153.14, 148.77, 148.38, 131.21, 131.15, 130.60, 129.05, 128.73, 123.95, 111.71, 110.85, 108.54, 105.59, 97.61, 64.32, 55.41, 52.21, 45.78, 44.16, 37.08, 12.81. The specific C NMR spectrum is shown in Figure 2;

[0040] Mass spectrum ESI-MS: m / z = 865.4414 ([M+H] +,calc.865.4587),433.2263for([M+2H] 2+ ,calc.433.2333). The specific mass spectrum is shown in Figure 3.

[0041] Example 2

[0042] Rhodamine-methylene blue derivative fluorescent probe for ClO - and determination of optical properties of ATP

[0043] Rhodamine-methylene blue derivative fluorescent probe (1×10 -5 mol / L) EtOH:PBS (0.05mol / L, pH=7.4, 5:95, v:v) solution was added to 2.5×10 -4 mol / L analyte F - ,Cl - ,Br - ,I - ,ClO4 - ,PO4 3- ,H2PO4 - ,HPO4 2- ,ppi,SO4 2- ,HSO3 - ,S 2- , Ca 2+ , Mg 2+ , K + ,LL-Cys,GSH,ClO - ,H2O2,ONOO - , 1 O2 and ROO - , the fluorescence spectrometer was used for analysis, the excitation wavelength was 630nm, and the obtained fluorescence spectrum is shown in Figure 4a. Rhodamine-methylene blue derivative fluorescent probe (1×10 -5 1×10 -2 mol / L analyte, PO4 3- ,H2PO4 - ,HPO4 2- , ppi, L-Cys, GSH, ATP, ADP, AMP, GTP, CTP and UDP were analyzed by fluorescence spectrometer with an excitation wavelength of 360 nm. The obtained fluorescence spectrum is shown in FIG4 b. As can be seen from FIG4 a and b, the rhodamine-methylene blue derivative fluorescent probe prepared by the present invention is only sensitive to ClO - It has obvious response to ATP and the fluorescence signal can be used for ClO - Rapid identification of ATP and other analytes.

[0044] Rhodamine-methylene blue derivative fluorescent probe (1×10 -5 mol / L) EtOH:PBS (0.05 mol / L, pH=7.4, 5:95, v:v) solution was gradually added with ClO - and ATP solution, control ClO - The final concentrations of ATP in the detection system were 2.5×10 -4 mol / L and 1.5×10 -2 mol / L. As can be seen from Figure 5a, as ClO - As the concentration increases, the fluorescence intensity of the probe at 680nm gradually increases. As can be seen from Figure 5b, as the ATP concentration increases, the fluorescence intensity of the probe at 590nm gradually increases. By calculation, it can be concluded that the probe has a strong affinity for ClO - The detection limit is 0.90nM, and the detection limit for ATP is 23.60nM. Therefore, the rhodamine-methylene blue derivative fluorescent probe prepared by the present invention can be used for ClO - and fluorescent quantitative detection of ATP.

[0045] Example 3

[0046] Rhodamine-methylene blue derivative fluorescent probe for intracellular ClO - and ATP detection experiments

[0047] After the Raw246.7 cells were cultured through the relevant steps, fluorescence imaging was performed using an Olympus FV500-IX70 laser confocal microscope. The resulting cell imaging is shown in Figure 6. Control group (Control): Raw246.7 cells were cultured with 1×10 -5 mol / L fluorescent probe incubated for 30 min; Oligomycin A (Omy A) group: Raw246.7 cells were incubated with 2×10 -5 After incubation for 1 h, 1×10 -5 mol / L fluorescent probe incubated for 30 min; Omy A+ATP group: Raw246.7 cells were incubated with 2×10 -5 After incubation for 1 h, 1×10 -3 mol / L ATP for 1 h, and then 1×10 -5 mol / L fluorescent probe incubated for 30 min; N-acetylcysteine ​​(NAC) group: Raw246.7 cells were incubated with 5×10 -4 After incubation for 1 h with 1×10 -5 mol / L fluorescent probe incubation for 30 min; NAC+ClO -Group: Raw246.7 cells were treated with 5×10 -4 After incubation for 1 h with 2×10 -4 mol / L ClO - Incubate for 1 h, then use 1×10 -5 mol / L fluorescent probe was incubated for 30 min. After the control group cells were incubated with the probe, both the yellow channel and the NIR channel had certain fluorescence signals, indicating that ClO - The fluorescence of the yellow channel and NIR channel returned to the control level after incubation with Omy A and then with ATP, indicating that the probe can detect exogenous ATP in cells. - The fluorescence of the yellow channel was significantly enhanced, and the fluorescence of the NIR channel was significantly weakened. - After incubation, the fluorescence of the yellow channel and NIR channel returned to the level of the control group, indicating that the probe can detect exogenous ClO - .

[0048] After the Raw246.7 cells were cultured through the relevant steps, fluorescence imaging was performed using an Olympus FV500-IX70 laser confocal microscope. The resulting cell imaging is shown in Figure 7. Control group (Control): Raw246.7 cells were cultured with 1×10 -5 mol / L fluorescent probe was incubated for 30 min, and the glucose (Glu) concentration in the culture medium was 5.5×10 -3 mol / L; Lipopolysaccharide (LPS) group: Raw246.7 cells were incubated with 5 μg / mL LPS for 1 h, and then with 1×10 -5 mol / L fluorescent probe incubated for 30 min; No Glu group: Raw246.7 cells were incubated with 1×10 -5 mol / L fluorescent probe was incubated for 30 min, and Glu was not added to the culture medium; High Glu group: Raw246.7 cells were incubated with 1×10 -5 mol / L fluorescent probe was incubated for 30 min, and the Glu concentration in the culture medium was 2.5×10 -2 mol / L; acetaminophen (APAP) group: Raw246.7 cells were treated with 5×10 -5 After incubation with APAP for 1 h, 1×10 -5 mol / L fluorescent probe was incubated for 30 min. After the cells in the control group were incubated with the probe, both the yellow channel and the NIR channel had certain fluorescence signals, indicating that ClO -LPS is used to stimulate cells to produce ClO - The fluorescence of the yellow channel was significantly weakened, and the fluorescence of the NIR channel was significantly enhanced, indicating that the probe can detect endogenous ClO - When cells were incubated with the probe in a sugar-free medium, the yellow channel had almost no fluorescence, while the NIR channel fluorescence was significantly enhanced. Conversely, when cells were incubated with the probe in a high-glucose medium, the yellow channel fluorescence was significantly enhanced, while the NIR channel fluorescence was significantly weakened, indicating that the probe can detect endogenous ATP and that the impaired cellular ATP metabolism caused ClO - After APAP was used to stimulate the cells, there was almost no fluorescence in the yellow channel, and the fluorescence in the NIR channel was significantly enhanced, indicating that APAP induced ClO - The concentration of ATP increased, thereby inhibiting ATP production, indicating that the probe can be used for drug-stimulated ClO - Synchronous fluorescence imaging with ATP.

[0049] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A rhodamine-methylene blue derivative fluorescent probe, characterized in that: Its structural formula is as follows:

2. The method for preparing a rhodamine-methylene blue derivative fluorescent probe according to claim 1, characterized in that: Methylene blue phosgene and Na2CO3 are dissolved in an organic solvent, and then an organic solution of rhodamine B amineethylpiperazine is added, stirred for reaction, poured into ice water, extracted, the organic phase is separated, the solvent is removed by rotary evaporation, and purified to obtain the rhodamine-methylene blue derivative fluorescent probe.

3. The preparation method according to claim 2, characterized in that: The molar ratio of methylene blue phosgene to Na2CO3 is 1:

3.

4. The preparation method according to claim 2, characterized in that: The mass volume ratio of the methylene blue phosgene to the organic solvent is 1 g:10 mL; the organic solvent includes dichloromethane.

5. The preparation method according to claim 2, characterized in that: The molar ratio of the rhodamine B amine ethyl piperazine to the methylene blue phosgene is 4:1; the solvent of the organic solution of the rhodamine B amine ethyl piperazine includes dichloromethane with a concentration of 2.296 mol·L -1 .

6. The preparation method according to claim 2, characterized in that: The reaction conditions of the stirring reaction are stirring the reaction at room temperature for 5 hours and a stirring frequency of 600 rpm.

7. The preparation method according to claim 2, characterized in that: The extraction is performed by using ethyl acetate; the rotary evaporation conditions are: 50° C., 0.1 MPa; and the purification is performed by silica gel column chromatography.

8. A ClO - and a fluorescent quantitative detection method for ATP, characterized in that, Using the rhodamine-methylene blue derivative fluorescent probe of claim 1 to detect ClO in cells - and ATP were tested.

9. The rhodamine-methylene blue derivative fluorescent probe as claimed in claim 1 in ClO - and ATP detection.

10. The rhodamine-methylene blue derivative fluorescent probe as claimed in claim 1 as ClO - And the application of ATP fluorescent probes in cell imaging.

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