阅读说明：本技术 谷氨酰胺荧光探针及其制备方法和应用 (Glutamine fluorescent probe and preparation method and application thereof ) 是由 崔佳燕 黄瑾 马磊 黄维维 邱子言 于 2021-06-18 设计创作，主要内容包括：本发明公开了具有通式(I)的谷氨酰胺荧光探针及其制备方法和用途。该类探针的结构模块包括：谷氨酰胺、连接链和荧光报告基团。本发明的谷氨酰胺荧光探针可作为工具分子,对细胞内谷氨酰胺通量进行实时监测。更优地,此类结构的荧光探针可显示动物肿瘤模型病灶部位对谷氨酰胺的摄取情况,反映体内肿瘤的分布以及大小,丰富小动物活体成像及人类癌症筛查中对肿瘤的检测手段。(The invention discloses a glutamine fluorescent probe with a general formula (I) and a preparation method and application thereof. The structural module of the probe comprises: glutamine, a linker and a fluorescent reporter group. The glutamine fluorescent probe can be used as a tool molecule for monitoring the glutamine flux in cells in real time. Preferably, the fluorescent probe with the structure can display the glutamine uptake condition of the lesion site of the animal tumor model, reflect the distribution and the size of tumors in vivo and enrich the detection means of the tumors in small animal living body imaging and human cancer screening.)

1. A glutamine fluorescence probe is characterized in that it has a structure of general formula (1),

wherein the content of the first and second substances,

the Gln group has the structure

The Linker has the structure ofWherein n is an integer of 1-8, or the Linker does not exist;

f is a fluorescent reporter group with the structure of

2. The glutamine fluorescence probe according to claim 1, characterized in that, the glutamine fluorescence probe is one of the following compounds,

3. a method for preparing the glutamine fluorescent probe according to claim 1 or 2, characterized by comprising the steps of:

Fmoc-Gln (Trt) firstly carries out coupling reaction with 2-chlorotrityl chloride resin to generate Fmoc-Gln (Trt) -2-CTC resin, and the reaction solvent is DMF or DCM; after Fmoc group removal, the resin reacts with Fmoc-Linker to generate Fmoc-Linker-Gln (Trt) -2-CTC resin, and the reaction solvent is DMF or DCM; after Fmoc group is removed, the resin reacts with a fluorescence reporter group, and the reaction solvent is DMF or DCM; finally, the glutamine fluorescent probe is obtained by cracking the resin.

4. Use of the glutamine fluorescent probe of claim 1 or 2 for dynamic monitoring of intracellular glutamine flux.

5. Use of the glutamine fluorescence probe of claim 1 or 2 for imaging tumor distribution and size in an animal tumor model.

6. Use of the glutamine fluorescence probe of claim 1 or 2 for imaging tumor distribution and size in a human.

Technical Field

The invention relates to the technical field of compounds, in particular to the field of pharmaceutical chemistry, and specifically relates to a glutamine fluorescent probe and a preparation method and application thereof.

Background

Cancer cells are the source of cancer. Compared with normal cells, cancer cells have the characteristics of unlimited proliferation, transformation and easy metastasis. Cancer cells require a large intake of nutrients to meet their property of unlimited proliferation.

Cancer cell metabolism the well-known "Warburg effect" indicates that cancer cells are capable of high-rate consumption of glucose. FDG-PET (glucose radiotracer-positron emission tomography) which has been developed for human body can trace cancer cells according to their glucose metabolism, thereby precisely locating cancer cells and tissues. However, FDG-PET does not always provide a relatively accurate clinical diagnosis.

Glutamine is a non-essential amino acid and is the free amino acid with the highest content in the blood circulation of the body. The carbon of glutamine can be used for the synthesis of amino acids and fatty acids, and the nitrogen of glutamine can be used for the synthesis of purines and pyrimidines. High levels of glutamine in the blood provide a carbon source and a nitrogen source for cancer cells, support biosynthesis, energy metabolism and homeostatic balance in cancer cells, and promote tumor growth. On 7.4.2021, a research paper titled Cell-programmed nutrient characterization in the tissue micro environment was published in Nature journal by the research team of the university of Van der Pauw medical center, which overturns the development and perfection of cancer metabolic models and basic cancer cognition for 100 years.

At present, the detection means of glutamine in cancer cells mainly comprise the following methods: high performance liquid chromatography, enzyme linked immunosorbent assay, and glutamine/glutamic acid conversion assay. The high performance liquid chromatography needs a plurality of sample pretreatment steps, and the sample preparation is complex. The enzyme-linked immunoassay method relates to the use of antibodies, and has long time consumption and high requirement on temperature in the experimental process. The glutamine/glutamic acid conversion test method relates to the use of glutaminase, and has high requirements on the storage and transportation of a kit. Therefore, at present, no method which is simple in treatment, low in cost, easy to store and easy to detect is used for detecting the glutamine in the cancer cells.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a glutamine fluorescent probe which can be used for monitoring the glutamine flux in cells in real time and reflecting the size and distribution of tumors in an animal tumor model, and a preparation method and application thereof.

In order to achieve the above object, the present invention provides, in a first aspect, a glutamine fluorescent probe characterized by having a general formula (1) including glutamine (Gln), a linker and a fluorescent reporter group F:

wherein the content of the first and second substances,

the Gln group has the structure

The Linker has the structure ofWherein n is an integer of 1-8, or the Linker does not exist;

f is a fluorescent reporter group with the structure of

Preferably, the glutamine fluorescent probe is one of the following compounds,

the invention also provides a preparation method of the glutamine fluorescent probe, which comprises the following steps:

Fmoc-Gln (Trt) firstly carries out coupling reaction with 2-chlorotrityl chloride resin to generate Fmoc-Gln (Trt) -2-CTC resin, and the reaction solvent is DMF or DCM; after Fmoc group removal, the resin reacts with Fmoc-Linker to generate Fmoc-Linker-Gln (Trt) -2-CTC resin, and the reaction solvent is DMF or DCM; after Fmoc group is removed, the resin reacts with a fluorescence reporter group, and the reaction solvent is DMF or DCM; finally, the resin is cracked to obtain the corresponding glutamine fluorescent probe.

The glutamine fluorescent probe can be used as a tool molecule for monitoring the glutamine flux in cells in real time. Preferably, the fluorescent probe with the structure can display the ingestion condition of the glutamine at the lesion site of the animal tumor model, reflect the distribution and the size of the tumor in vivo and enrich the detection means of the tumor in the living body imaging of the small animal.

Drawings

FIG. 1 is a graph showing intracellular fluorescence after 4 hours without addition of the glutamine fluorescent probe and after addition of the glutamine fluorescent probe.

FIGS. 2a and 2b are contrast images of imaging before and after the use of a glutamine fluorescence probe, respectively.

Detailed Description

For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.

All of the starting materials of the present invention, without particular limitation as to their source, may be purchased commercially or prepared according to conventional methods well known to those skilled in the art.

To further illustrate the present invention, the glutamine fluorescent probe provided by the present invention is described in detail with reference to the following examples, and the scope of the present invention is not limited by the following examples.

EXAMPLE 1 preparation of Glutamine fluorescent Probe 1

Preparation of Fmoc-Gln (Trt) -2-CTC resin

The 2-chlorotrityl chloride resin (1g,0.4 mmol/g-3 mmol/g) was swollen in 10mL DCM for 1h, filtered and washed 3 times with DCM. Fmoc-Gln (Trt) (2g,3.4mmol), DIPEA (0.85g,6.6mmol) were added and reacted in DCM at room temperature for 2 h. After the reaction was complete, the coupling solution was removed by filtration and washed 3 times with DCM.

Preparation of Fmoc-Acp-Gln (Trt) -2-CTC resin

10mL of 20% piperidine/DMF solution was added to the resin obtained in the previous step and reacted for 30 min. When ninhydrin test is positive, the reaction is complete. After the reaction was completed, the mixture was filtered and washed 5 times with DMF. Fmoc-6-aminocaproic acid (1.7g,4.9mmol), HOBt (0.79g,5.9mmol), DIC (0.74g,5.9mmol) were added and reacted in DMF at room temperature for 2 h. When ninhydrin test is negative, the reaction is complete. After the reaction was completed, the coupling solution was removed by filtration and washed with DMF 3 times.

Preparation of FITC-Acp-Gln (Trt) -2-CTC resin

10mL of 20% piperidine/DMF solution was added to the resin obtained in the previous step and reacted for 30 min. When ninhydrin test is positive, the reaction is complete. After the reaction was completed, the mixture was filtered and washed 5 times with DMF. FITC (0.66g,1.7mmol) was added and the reaction was carried out in DMF for 2h at room temperature with the exclusion of light. When ninhydrin test is negative, the reaction is complete. After the reaction was completed, the coupling solution was removed by filtration and washed with DMF 3 times. Methanol washes were performed 2 times to shrink the resin.

Preparation of FITC-Acp-Gln

To the resin obtained in the previous step, 10mL of TFE/DCM ═ 1:3(V/V) solution was added, reaction was carried out at room temperature for 2H, filtration was carried out, DCM was used for washing 3 times, the obtained filtrate was concentrated, and 10mL of TFA/H was added₂And (3) reacting the solution with a 95:5(V/V) solution at room temperature for 2 hours, and recrystallizing with diethyl ether to obtain the fluorescent probe 1.

Compound FITC-Acp-Gln, C₃₂H₃₂N₄O₉S, yellow solid.¹H NMR(400MHz,CDCl₃)δ8.24(1H,s),7.73(1H,d,J＝8Hz),7.30(1H,d,J＝8Hz),7.19(2H,m),6.57(4H,m),4.18(1H,m),3.39(2H,m),2.14(4H,m),1.93(1H,m),1.74(1H,m),1.56(4H,m),1.34(2H,m).MS(ESI):m/z 649.20[M+H]⁺。

EXAMPLE 2 preparation of Glutamine fluorescent Probe 2

Preparation of Fmoc-Gln (Trt) -2-CTC resin

The 2-chlorotrityl chloride resin (1g,0.4 mmol/g-3 mmol/g) was swollen in 10mL DCM for 1h, filtered and washed 3 times with DCM. Fmoc-Gln (Trt) (2g,3.4mmol), DIPEA (0.85g,6.6mmol) were added and reacted in DCM at room temperature for 2 h. After the reaction was complete, the coupling solution was removed by filtration and washed 3 times with DCM.

Preparation of Rhodamine B-Gln (Trt) -2-CTC resin

10mL of 20% piperidine/DMF solution was added to the resin obtained in the previous step and reacted for 30 min. When ninhydrin test is positive, the reaction is complete. After the reaction was completed, the mixture was filtered and washed 5 times with DMF. Rhodamine B (2.4g,4.8mmol), PyAOP (2.5g,4.8mmol), HOAt (0.68g,4.8mmol), DIPEA (1.2g,4.8mmol) were dissolved in DMF, stirred at room temperature for 10min, added to the above resin and reacted at room temperature for 2 h. When ninhydrin test is negative, the reaction is complete. After the reaction was completed, the coupling solution was removed by filtration and washed with DMF 3 times. Methanol washes were performed 2 times to shrink the resin.

Preparation of Rhodamine B-Gln

To the resin obtained in the previous step, 10mL of TFE/DCM ═ 1:3(V/V) solution was added, reaction was carried out at room temperature for 2H, filtration was carried out, DCM was used for washing 3 times, the obtained filtrate was concentrated, and 10mL of TFA/H was added₂And (3) reacting the solution with a 95:5(V/V) solution at room temperature for 2 hours, and recrystallizing with diethyl ether to obtain the fluorescent probe 2.

Rhodamine B-Gln, C compound₃₃H₃₉N₄O₅ ⁺Dark red solid.¹H NMR(400MHz,CDCl₃)δ7.98(1H,m),7.49(1H,m),7.38(1H,m),7.29(1H,m),7.20(2H,m),6.87(2H,m),6.79(2H,m),4.33(1H,m),3.64(8H,m),2.34(2H,m),1.85(2H,m),1.27(6H,t),1.17(6H,t).MS(ESI):m/z 571.29[M]⁺。

Example 3

The prepared glutamine fluorescent probe is used for detecting the flux of glutamine in cancer cells in real time.

Before experiment, non-small cell lung cancer cell A549 was inoculated at 100000/well into a glass-bottom culture dish special for 35mm confocal microscope and cultured with DMEM complete medium containing fetal bovine serum (volume ratio: 10%), penicillin (100. mu.g/mL) and chainMycin (100. mu.g/mL). Placing the culture dish in a container containing 5% CO by volume₂And cultured in an incubator at 37 ℃ for 24 hours. A549 cells were washed three times with PBS (phosphate buffered saline, pH 7.4). Adding 1mL of Hoechst 33258 staining solution and cell membrane red fluorescent probe Dil, and placing in a container containing 5% CO by volume₂And culturing in an incubator at 37 ℃ for 20-30 minutes. The staining solution was discarded and a549 cells were washed three times with PBS. Adding DMEM medium containing glutamine fluorescent probe (FITC-Acp-Gln, 4mM) and placing in a medium containing 5% CO by volume₂Cultured in an incubator at 37 ℃. After 4 hours, the medium was discarded, and the A549 cells were washed three times with PBS and observed for intracellular glutamine fluorescence uptake under a rotary confocal microscope (Nikon CSU-W1 SoRa).

As shown in FIG. 1, when no glutamine fluorescent probe was added, no green fluorescence was evident in the cells; when 4 hours later, the glutamine fluorescent probe shows obvious green fluorescence in the cell, which indicates that the cell effectively takes up glutamine, and the probe can be used for detecting the glutamine flux in the cell.

Example 4

The prepared glutamine fluorescent probe is used for imaging the distribution and the size of the tumor in an animal tumor model.

A549-luc cells capable of stably expressing luciferase are used for constructing a nude mouse lung metastasis model. Selecting 6-week-old female nude mice, and mixing 100 μ L of the nude mice with 5 × 10⁶A549-luc cell suspension was injected into mice by tail vein injection. After 3 weeks of normal feeding, 0.5mg/kg glutamine fluorescence probe (Rhodamine B-Gln) was injected into the abdominal cavity of each mouse, and the circulation was in vivo for 6 hours. After 6 hours, 2mg of luciferase substrate D-luciferin is injected into the abdominal cavity of each mouse, and the transfer condition of the lung of the mouse can be detected by using bioluminescence in a small animal living body imaging system after 10min of reaction, and then the glutamine uptake condition of the lung transfer part is detected by using fluorescence.

The results are shown in FIGS. 2a and 2b, and the use of the luciferase substrate D-luciferin in FIG. 2a clearly shows the success of modeling the nude mouse lung metastasis model. The imaging display part of the glutamine fluorescent probe is highly overlapped with the imaging display part of the fluorescein in the graph in fig. 2b, which shows that the glutamine fluorescent probe can be used for detecting the distribution and the size of the tumor in the tumor model.

In this specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The description is thus to be regarded as illustrative instead of limiting.