CN110551056B

CN110551056B - Cyanine compound, preparation method and application in detection of Golgi pH

Info

Publication number: CN110551056B
Application number: CN201910934847.XA
Authority: CN
Inventors: 唐波; 王慧; 李平; 杨玉云
Original assignee: Shandong Normal University
Current assignee: Shandong Normal University
Priority date: 2019-09-29
Filing date: 2019-09-29
Publication date: 2020-11-20
Anticipated expiration: 2039-09-29
Also published as: CN110551056A

Abstract

The invention discloses a cyanine compound, a preparation method and application in detecting Golgi pH, wherein the cyanine compound has the chemical structure as follows:

the cyanine compound provided by the disclosure is used as a fluorescent probe of pH in a Golgi body, not only has Golgi targeting capability, but also can realize photoacoustic-fluorescent bimodal imaging for sensitively detecting pH change of the Golgi body.

Description

Cyanine compound, preparation method and application in detection of Golgi pH

Technical Field

The disclosure relates to a cyanine compound, a preparation method thereof and application thereof in detecting the pH value of a Golgi apparatus.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

Intracellular pH plays an important role in many physiological processes, such as cell proliferation, regulation of enzymatic activity, and ion transport. The golgi apparatus is the main site of protein secretion, transport and assembly, and a number of protein glycosylation modifications occur in the golgi apparatus. As a weakly acidic organelle (pH 6.0-6.7), the Golgi apparatus undergoes proton exchange primarily by proton pumps or ion channels. Abnormal golgi pH can lead to cellular dysfunction, glycosylation disorders, and, in severe cases, disorganization and further disintegration of the golgi structure. Therefore, it is necessary to develop a detection means for in-situ real-time quantitative detection of golgi pH, which is a highly efficient target.

The fluorescence imaging technology has the advantages of simple operation, nondestructive in-situ imaging and the like, and is mostly used for diagnosis and detection of diseases in recent years. Among them, the near-infrared fluorescence imaging technology has the advantages of low background fluorescence and low phototoxicity, and is widely applied to cell imaging research. The photoacoustic imaging technology is a novel detection means in the field of biomedicine, and converts part of absorbed light energy into heat energy to send out ultrasonic signals. Compared with fluorescence imaging technology, photoacoustic imaging technology can achieve high-resolution nondestructive imaging detection at the living body level at a higher penetration depth, but cannot achieve cell imaging.

Disclosure of Invention

In order to solve the defects of the prior art, the purpose of the disclosure is to provide a cyanine compound, a preparation method and application in detection of the pH of a Golgi apparatus.

In order to achieve the purpose, the technical scheme of the disclosure is as follows:

in one aspect, the present disclosure provides a cyanine compound having a chemical structure as follows:

on the other hand, the disclosure provides a preparation method of the cyanine compound, which comprises the step of performing substitution reaction of aniline groups and chlorine by using Cy7-Cl and sulfanilamide as raw materials.

In a third aspect, the disclosure provides an application of the cyanine compound in detecting pH or detecting pH of golgi.

Under acidic conditions, the-NH-generated by the connection of the p-aminobenzenesulfonamide as a positioning group and a Cy7-Cl as a fluorophore can be protonated, so that the coplanar effect of the whole system is increased, and the fluorescence emission wavelength is shifted. With increasing acidity, the fluorescence emission of the probe is gradually red-shifted from 750nm to 810 nm.

In a fourth aspect, the disclosure provides an application of the cyanine compound in the preparation of a target golgi pH fluorescent probe.

In a fifth aspect, the disclosure provides an application of the cyanine compound in preparing a photoacoustic imaging in-situ pH detection probe.

The beneficial effect of this disclosure does:

1. the present disclosure provides fluorescent probes using cyanine compounds as efficient target golgi. Compared with the prior Golgi positioning group, the strategy for synthesizing the positionable fluorescent probe by using the benzenesulfonamide as the Golgi positioning group in the disclosure is easy to synthesize, low in cost and good in positioning effect.

2. The ratiometric fluorescent probe for detecting the pH of the Golgi apparatus, which is provided by the disclosure, shows good absorption and fluorescence spectrum response in buffer solutions with different pH values, and has good reversibility and photostability.

3. The cyanine compounds disclosed by the disclosure have good biocompatibility and very low toxicity to cells, and can be used for pH detection of a high-molar matrix in living cells.

4. The cyanine compounds disclosed by the disclosure have near-infrared fluorescence and photoacoustic response properties, and can realize the positioning of subcellular organelles and the imaging of deep tissues in a living body with high spatial resolution. And is successfully applied to the detection of in-situ pH in an inflammation mouse model.

5. The cyanine compound disclosed by the disclosure is simple to synthesize, has obvious reversible color change in acidic and alkaline environments, and is expected to be developed into a commercial pH test paper.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1 is a spectrum of CPH prepared in example 1 of the present disclosure in different pH buffers, where a is an ultraviolet absorption spectrum and b is a fluorescence spectrum;

FIG. 2 is a graph showing reversibility of CPH, a probe prepared in example 1 of the present disclosure, in an environment of acidic and basic reciprocating changes;

FIG. 3 is a diagram of confocal fluorescence images of cells after the probe CPH prepared in example 1 of the present disclosure is co-stained with different subcellular organelle localization dyes in human normal hepatocytes HL-7702, wherein a is Golgi dyes (the scales in the left, middle and right panels are consistent), b is mitochondrial dyes (the scales in the left, middle and right panels are consistent), c is lysosomal dyes (the scales in the left, middle and right panels are consistent), d is a trend graph of fluorescence intensities of the probe CPH and the Golgi dyes, e is a trend graph of fluorescence intensities of the probe CPH and the mitochondrial dyes, and f is a trend graph of fluorescence intensities of the probe CPH and the lysosomal dyes;

fig. 4 is a representation of confocal fluorescence imaging of probe CPH prepared in example 1 of the present disclosure in human hepatoma cells SMCC-7721 in buffer media of different pH as a function of pH, which is the superposition of cellular confocal fluorescence imaging and bright field, where a is pH 6.0, b is pH 6.5, c is pH 7.0, d is pH 7.5, and e is pH 8.0;

FIG. 5 is a graph showing in situ photoacoustic imaging characterization of probe CPH prepared in example 1 of the present disclosure in detecting pH changes of Golgi apparatus at normal and lesion sites in a mouse model of inflammation;

fig. 6 is a graph of photoacoustic imaging intensity data output of the probe CPH prepared in example 1 of the present disclosure for detecting pH changes of golgi at normal and lesion sites in a mouse model of inflammation.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the 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 disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In order to realize the purpose of photoacoustic-fluorescent bimodal imaging for sensitively detecting the change of the pH value of the Golgi apparatus, the disclosure provides a cyanine compound, a preparation method and application in detecting the pH value of the Golgi apparatus.

In one exemplary embodiment of the present disclosure, a cyanine compound is provided, which has a chemical structure as follows:

in another embodiment of the present disclosure, a preparation method of the cyanine compound includes a step of performing a substitution reaction of an aniline group and chlorine using Cy7-Cl and sulfanilamide as raw materials.

The chemical structural formula of the raw material Cy7-Cl is shown as

The chemical structure of the raw material sulfanilamide is

The synthetic route is as follows:

in one or more embodiments of this embodiment, the substitution reaction conditions are: heating to not less than 90 ℃ under alkaline condition to carry out reaction.

In this series of examples, sodium hydroxide was used to provide alkaline conditions. When the molar ratio of Cy7-Cl to sodium hydroxide is 1: 1-2, an alkaline environment can be better provided for the substitution reaction.

In the series of embodiments, the reaction temperature is 90-95 ℃ and the reaction time is 12-16 h. The reaction efficiency of the compound can be increased.

In one or more embodiments of the embodiments, the charge ratio of Cy7-Cl to sulfanilamide is 1: 2-5. The Cy7-Cl reaction can be completed, and the yield of the cyanine compound is improved.

In one or more embodiments of this embodiment, the solvent of the reaction system is N, N-dimethylformamide.

In one or more embodiments of this embodiment, the material after the reaction is purified by column chromatography after the solvent is removed in order to increase the purity of the cyanine compound.

In this series of examples, the developing solvent for column chromatography was ethyl acetate.

In a third embodiment of the present disclosure, there is provided an application of the cyanine compound in detecting pH or detecting golgi pH.

In a fourth embodiment of the disclosure, an application of the cyanine compound in the preparation of a target golgi pH fluorescent probe is provided.

In a fifth embodiment of the disclosure, an application of the cyanine compound in preparing a photoacoustic imaging in-situ pH detection probe is provided.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific embodiments.

Example 1:

synthesis of fluorescent probe:

cy7-Cl (0.51g,1mmol) and p-aminobensulfonamide (0.86g,5mmol) were weighed accurately into a two-necked flask under nitrogen and 8mL of DMF with water removed was added as solvent. Additional sodium hydroxide (0.04g,1mmol) was added to provide a basic environment for the reaction. Slowly heating to 90 ℃ and reacting for 12 hours. After the reaction is finished, the product is subjected to reduced pressure distillation to remove a solvent DMF, and then is subjected to separation and purification by silica gel column chromatography, and ethyl acetate is used as a developing agent. Purification gave a blue solid (0.1g, 15%) which was designated CPH.¹H NMR(400MHz,CDCl₃)8.23(d,J＝13.2Hz,1H),7.83(d,J＝8.5Hz,1H),7.20(t,J＝7.6Hz,2H),6.96(t,J＝7.4Hz,1H),6.75(d,J＝7.8Hz,1H),6.66(d,J＝8.6Hz,1H),5.59(d,J＝13.2Hz,1H),5.29(s,1H),3.79(d,J＝6.9Hz,2H),2.55(t,J＝6.0Hz,2H),2.04(s,1H),1.86–1.79(m,1H),1.55(s,6H),0.88(dd,J＝8.6,4.8Hz,3H).¹³C NMR(101MHz,CDCl₃)：147.52,142.11,139.20,128.97,128.74,126.68,124.96,120.94,120.49,113.05,106.20,93.10,52.41,46.30,36.48,34.83,30.89,29.46–27.97,27.15,26.18,24.80,24.52,21.66,20.83,13.10,10.35。

Effect experiment of CPH:

generally, the dye molecules can be dissolved in physiological saline, buffer solution or water-soluble organic solvent such as acetonitrile, dimethylsulfoxide, etc., and then added with appropriate buffer solution and other organic reagents for the test. The absorption and fluorescence spectral response of the probe CPH in PBS buffer solutions with different pH values along with the change of the pH value are respectively researched and used for imaging experiments of living cells and inflammatory mice. The living cell staining method is to incubate the cultured cells in a culture solution containing probe molecules, remove the incubation solution after incubation for a certain time, and perform a confocal imaging experiment. The mouse staining method is to inject the probe into the normal part and inflammation part of the mouse in situ, and after a period of time, perform photoacoustic imaging on the part of the mouse injected with the probe.

The probe CPH is used for ultraviolet absorption, fluorescence emission and reversible selection experiments in buffers with different pH values:

the UV absorption and fluorescence response properties of probe CPH in PBS (0.1M) at different pH were explored. Buffers of different pH were prepared by adding as little sodium hydroxide or hydrochloric acid solution as possible to PBS at pH 7.4. The absorption spectrum of the probe CPH (50 mu M) shows a change with high sensitivity with the increase of the pH value (5.0-8.7). As shown in FIG. 1a, in a buffer medium with a pH of 5.0, the maximum absorption peak of the probe is around 800nm, and the maximum absorption blue of the probe shifts to around 610nm as the pH increases. And since the light absorption intensity (detectable by PA signal) of the probe at 800nm gradually decreases with the increase of pH, and the light absorption at 690nm is basically unchanged, the probe has the potential to detect pH compared with photoacoustic imaging. FIG. 1b shows the fluorescence spectral response of probe CPH (2. mu.M) in buffers with different pH values (5.0-8.7). Under the excitation of light at 675nm, the fluorescence intensity of the probe at 810nm is gradually reduced along with the gradual increase of the pH, and the fluorescence intensity at 750nm is gradually increased, so that the fluorescence response of the probe with high sensitivity to the pH is shown, and the pH can be quantitatively detected through ratiometric fluorescence.

FIG. 2 shows the reversibility of probe CPH in an acidic and basic reciprocating environment. The probe (2. mu.M) was added to PBS at pH 4.5 and fluorescence detected, after which the pH was continuously adjusted to shift between 4.5 and 9.5 and the spectral response was recorded. Ordinate FI_750nm/FI_810nmRepresents the ratio of the fluorescence intensities of the probe at 750nm and 810 nm. The results show that CPH consistently exhibits good, transient fluorescence response with good reversibility in the buffer at pH 4.5 and 9.5.

Golgi targeting experiments with CPH:

human normal hepatocyte HL-7702 was cultured in DMEM medium containing polyclonal antibody and fetal bovine serum albumin. After incubating the cells with 500nM probes and commercial dyes (including Golgi, mitochondria, lysosomes) that localize different subcellular organelles for 30min, co-localization imaging experiments were performed using confocal laser microscopy. Co-localization cell imaging experiments As shown in FIG. 3, probe CPH in FIGS. 3a, b, c only overlaps well with the fluorescence of the commercial Golgi localization dye. FIG. 3d, e, f are graphs showing the trend of fluorescence intensity of probe CPH and commercial dye in a certain linear region. The better the fluorescence intensity overlap, the more consistent the trend, indicating the better the co-localization effect, and thus the probe exhibits the ability to efficiently target golgi.

Confocal fluorescence imaging experiments of probes in living cells at different pH:

the human liver cancer cell SMMC-7721 is cultured by DMEM culture solution containing double antibody and fetal bovine serum albumin. Cells were then co-incubated with CPH (500nM) for 10 min at 37 deg.C, washed free of probe and incubated with PBS buffer (0.01M) containing 10. mu.M nigericin, an ionogen, at various pH's (6.0,6.5,7.0,7.5,8.0) for an additional 10 min before confocal imaging. Due to the intervention of nigericin, the intracellular pH value will change according to the change of the PBS buffer pH. Limited by the instrument, the present disclosure collects only the fluorescence at 710-760 nm. As shown in FIG. 4, the probe showed weak fluorescence in the cells after the cells were incubated with PBS at pH 6.00, and the fluorescence of CPH in the cells gradually increased as the pH of the cell incubation solution increased from 6.00 to 8.00. The above results indicate that the probe CPH can be used with high sensitivity for monitoring pH changes in living cells.

Probe in situ photoacoustic imaging experiments on normal and inflammatory sites in a mouse model of inflammation:

mice were treated with saline intraperitoneally on the left side as a control, and LPS (2mg/mL, 400. mu.L) was injected at the same position on the right side to induce the production of abdominal inflammation. After 4 hours, the mice were anesthetized with chloral hydrate and the experiment was performed. Probe CPH (30 μ M, 50 μ L) was intraperitoneally injected at the site where LPS (right) and saline (left) were injected, and photoacoustic imaging detection was performed at 690nm and 800nm wavelengths, as shown in fig. 5. As a result, it was found that the photoacoustic signal intensity was significantly stronger on the LPS-stimulated side of the mouse at the wavelength of 800nm than on the side injected with physiological saline, and that the photoacoustic signal had a lower PA level_690nm/PA_800nmThe ratio shows that the LPS stimulation site has weak acidity and the pH value is lower, as shown in FIG. 6. Indicating that CPH can be used as a fluorescence and photoacoustic bimodal imaging probe to detect pH in a living body.

The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. A cyanine compound is characterized by having the following chemical structure:

2. a process for producing a cyanine compound according to claim 1, which comprises the step of carrying out a substitution reaction of an aniline group with chlorine using Cy7-Cl and sulfanilamide as raw materials; the structure of the Cy7-Cl is:

3. a process for the preparation of cyanine compounds according to claim 2, wherein the substitution reaction conditions are: heating to not less than 90 ℃ under alkaline condition to carry out reaction.

4. A process for the preparation of cyanine compounds according to claim 3, wherein sodium hydroxide is used to provide the basic conditions.

5. A method for producing a cyanine compound according to claim 4, wherein the molar ratio of Cy7-Cl to sodium hydroxide is 1:1 to 2.

6. A process for preparing cyanine compounds as claimed in claim 3, wherein the reaction temperature is 90-95 ℃ and the reaction time is 12-16 h.

7. A method for producing cyanine compounds according to claim 2, in which the charge ratio of Cy7-Cl to sulfanilamide is 1: 2-5.

8. A process for preparing cyanine compounds according to claim 2, in which the solvent of the reaction system is N, N-dimethylformamide.

9. A process for preparing cyanine compounds as claimed in claim 2, in which the reacted material is purified by column chromatography after the solvent is removed.

10. The method for preparing a cyanine compound according to claim 9, wherein the developing agent for column chromatography is ethyl acetate.

11. Use of a cyanine compound of claim 1 in the detection of golgi pH, which does not include the diagnosis or treatment of diseases.

12. The use of a cyanine compound of claim 1 in the preparation of a target golgi pH fluorescent probe.

13. The application of the cyanine compound of claim 1 in the preparation of photoacoustic imaging in-situ pH detection probes.