CN106280533B - A kind of near infrared fluorescent dye and its synthetic method and for parasite fluorescence labeling - Google Patents

Abstract

The invention belongs to the field of bio-optical marking of parasitic diseases, and mainly relates to the synthesis of a near-infrared fluorescent dye, which is applied to the fluorescent marking of schistosomiasis adults. A near-infrared fluorescent dye with a conjugated diene bond structure, more specifically, the molecular structure is linked by three olefinic bonds, and the hydrophilic group has a sulfonic acid group (‑SO ₃ H) and a carboxyl group (‑COOH); and This near-infrared fluorescent dye can be used for fluorescent labeling imaging of Schistosoma adult worms. The design, synthesis and fluorescent labeling of adult worms of Schistosoma japonicum were realized. Near-infrared fluorescent dyes make the emission of fluorescent dyes in the near-infrared region through a conjugated system, and then combine with hydrophilic sulfonic acid groups and carboxyl groups for water-soluble modification. This type of near-infrared fluorescent dyes is used to fluorescently label Schistosoma adults, effectively The problem of poor fluorescent staining effect, background fluorescence interference and poor light penetration in fluorescence imaging of Schistosoma japonicum adults was solved.

Description

A kind of near-infrared fluorescent dye and its synthesis method and its use in parasite fluorescent labeling

technical field

The invention belongs to the field of bio-optical labeling for the prevention and treatment of parasitic diseases. Specifically, the invention relates to the synthesis of a near-infrared organic fluorescent dye and its application to the fluorescent labeling of schistosomiasis adults. According to a novel method, the near-infrared fluorescent dye has excellent fluorescent labeling effect on Schistosoma adult worms.

technical background

Schistosomiasis is an infectious disease that is prevalent in tropical and subtropical regions, seriously endangering people's health and affecting social and economic development. Schistosomiasis is endemic in 74 countries and regions. There are currently about 652 million people in the world at risk, 193 million infected people, and about 120 million symptomatic cases, of which 20 million are severe cases. my country is the main endemic area of schistosomiasis. Schistosomiasis is a typical zoonotic parasitic disease transmitted by water sources. Its life cycle can be divided into seven main stages: egg, miracidia, mother sporidia, sporozoites, cercariae, juveniles, and adults. After entering the human body, the cercariae of Schistosoma japonicum develop into juvenile worms, and then grow into adults after about 30 days. The adults cause great damage to the human body, causing hepatosplenomegaly, liver cirrhosis, etc., and severe cases can cause death in the late stage. Drugs (such as praziquantel) that have a good killing effect on adult flukes have been developed, but due to the long-term and extensive use of anti-schistosomiasis drugs, schistosomiasis has developed resistance to them. At the same time, the actual mechanism of action of this type of drug to kill adult worms is still unclear, so in-depth research on anti-schistosomiasis drugs is still needed. However, the traditional research method (mainly the detection of dead insects) to study the possible mechanism of action of drugs on adults cannot accurately and directly reflect the mode and site of action of drugs on living organisms, which affects the accuracy of research results. and credibility, so it is necessary to seek new research methods to explore its mechanism of action. At present, fluorescence imaging is a sensitive, non-invasive, and low-cost visualization detection technology with a resolution of hundreds of nanometers, which can realize in vivo imaging from cells to organisms. Fluorescence imaging technology has the advantages of sensitive monitoring, rapid imaging, and simultaneous observation of multiple molecular events, so it has broad application prospects in the study of the mechanism of action of anti-schistosome drugs.

Fluorescence imaging is used in the research of schistosomiasis. Andrea B. Kohn et al. used 4,5-diaminoXuorescein-2 fluorescent probe to detect the content of nitric oxide in schistosomiasis. The research results found that the release of nitric oxide in worms depends on nitric oxide synthase (NOS), indicating that nitric oxide enzyme plays an important role in the physiology of schistosomes. SATO H. et al. used fluorescence imaging to observe the excretory system of adults of Schistosoma mansoni. The fluorescent probe resorufin (resorufin) was used to observe the mode of action of the excretory system of the parasite. The research results will help to understand the physiological function of the pronephroid system of Schistosoma mansoni.

The above-mentioned use of traditional organic fluorescent dyes as fluorescent probes for the study of Schistosoma adult worms has the following disadvantages: (1) Biological spontaneous background fluorescence interference exists in short-wavelength excitation and emission. (2) Most of the fluorescent probes have poor photostability, which makes it impossible to observe fluorescence for a long time. (3) Most organic small molecule fluorescent dyes have poor hydrophilicity, which limits their biological applications. At the same time, the structure of Schistosoma adult worms has the following characteristics: (1) The body wall of Schistosoma adult worms is composed of body coat, basement membrane and body coat, and is connected to the body coat through cytoplasmic tubules, so that the body wall is separated by the surface layer without nuclei and cells body is covered. (2) Adult Schistosoma parasites in the blood vessels of mammals, and the body surface is in direct contact with the blood of the host. In order to avoid the immune attack of the host, the body is covered with a specific sugar that is the same as the host erythrocyte ab antigen, and the body surface of the adult worm appears Strong hydrophilicity. (3) The adult worms of Schistosoma japonicum are relatively large and have high requirements for the penetration of fluorescence. In view of the shortage of current fluorescent dyes and the contradiction between the physiological and structural characteristics of Schistosoma adult worms, it is a challenge to develop fluorescent dyes with good fluorescent labeling effects on Schistosoma adult worms.

The present invention combines the advantages of near-infrared fluorescent dyes, such as organisms rarely have autofluorescence in the near-infrared spectral region, so that the imaging of near-infrared fluorescent dyes is less disturbed by background fluorescence; secondly, because the intensity of scattered light is proportional to the fourth power of wavelength Inversely, near-infrared fluorescent dyes whose emission light is in the long-wave region are less disturbed by it, and have strong penetrating ability to biological tissues and less damage. Therefore, the inventors comprehensively considered factors such as the excitation and emission ranges of fluorescent dyes, water solubility, and physiological characteristics of schistosomiasis, and used near-infrared fluorescent dyes as fluorescent probes for fluorescent labeling of schistosomiasis adults, and successfully solved the fluorescence imaging process of schistosomiasis adults. The problems of poor fluorescence staining effect, large background fluorescence interference and poor fluorescence penetration.

Contents of the invention

The invention provides a near-infrared fluorescent dye, which has the performance of fluorescently marking schistosome adults and can be used as a fluorescent labeling reagent for schistosome adults.

The concrete structure of near-infrared fluorescent dye of the present invention is as follows:

Compounds of the present invention can be prepared according to the following synthetic routes:

The raw materials and reagents used in the preparation of the compounds of the present invention are known compounds, which can be obtained in the market, or can be prepared by methods known in the art.

Compound 1,1,2-trimethyl-1H-benzindole (1.0-2.0mmol) and 3-bromopropionic acid (1.2-2.4mmol) were heated in toluene at 110°C for 24 hours to obtain product M-1. The obtained intermediate product M-1 (1.0-2.0 mmol) and malondialdehyde diphenylamine (1.0-2.0 mmol) were heated in a solution of acetic anhydride at 110° C. for 2 hours to obtain the product M-2. Product M-2 (1.0-2.0mmol) and 3-(3-sulfonate-propyl)-1,1,2-trimethyl-1H-benzindole (1.0-2.0mmol) in potassium acetate and The target fluorescent dye NIR-COOH-SO ₃ H was synthesized by heating and reacting in the mixed solution of acetic anhydride at 70° C. for 2 hours.

The above-mentioned fluorescent dye is used as a fluorescent labeling probe for schistosomiasis adults, and the fluorescent labeling method includes the following steps:

(1) Obtain schistosome cercariae: cercariae are obtained from positive snails infected with miracidia. Take the positive snails and put them into clean water, and expose them to fresh cercariae under incandescent light for about 2 hours.

(2) Mice infection: Cut off the abdominal hair of six-week-old female mice, take warm water to moisten the abdominal skin of the mice, and stick the coverslip containing about 50 cercariae on the dehaired abdominal skin of the mice , kept for 20 minutes to allow the cercariae to penetrate the mouse skin for infection.

(3) Obtain schistosomiasis adult worms: feed the infected mice for 6 weeks, dislocate their necks and kill them. Strictly follow the requirements of aseptic operation, cut open the abdominal skin and peritoneum in sequence, and infuse them into the hepatic porta-mesenteric vein Collect adults. The collected adult worms were rinsed three times in normal saline and distributed to petri dishes. After adding RPMI-1640 (Roswell Park Memorial Institute) cell culture medium and culturing for 2 hours, the worms with good activity were screened under a microscope for fluorescence imaging.

(4) Fluorescence imaging of adults of Schistosoma japonicum: the fluorescent dye was dissolved in dimethyl sulfoxide (DMSO) to accurately prepare a 1 mmol stock solution, and then diluted with PBS buffer to a 5 μM dilution. Pipette 200 μL of the diluted solution into a petri dish, pick the adults with tweezers and add them to the fluorescent dye dilution, incubate at room temperature for 5 minutes to 6 hours, transfer the adults to a fluorescence imaging vessel, and use a confocal fluorescence microscope under 635nm excitation The fluorescent labeling of adults was observed, and fluorescent signals at 650-750 nm were collected for fluorescent imaging.

The inventors have designed and synthesized near-infrared organic fluorescent dyes for the study of fluorescent labeling of schistosome adult worms, which can successfully solve the problems of poor fluorescent labeling effect, large background fluorescence interference and poor light penetration in the process of adult fluorescent imaging.

Description of drawings

Fig.1 Absorption spectrum and fluorescence emission spectrum of fluorescent dye NIR-COOH-SO ₃ H

Figure 2 Laser confocal fluorescence imaging of fluorescent dye NIR-COOH-SO ₃ H cells

Figure 3 Laser confocal fluorescence imaging of Schistosoma japonicum adult worms labeled with fluorescent dye NIR-COOH-SO ₃ H

Figure 4 Laser confocal fluorescence imaging of adult head organs of Schistosoma japonicum labeled with fluorescent dye NIR-COOH-SO ₃ H

Fig.5 Signal-to-noise ratio of fluorescent imaging of Schistosoma japonicum labeled with fluorescent dye NIR-COOH-SO ₃ H

Detailed ways

Specific examples of the compounds of the present invention are given below, which illustrate the present invention by way of examples, but do not constitute any limitation thereto. The raw materials used in this example are all known compounds, which can be obtained from commercial sources, or can be synthesized according to the methods designed in relevant literature.

In the following examples, the physical and chemical parameters involved are measured by the following instruments: ¹ H NMR spectrum is measured at 400 MHz on BrukerMercuryplus, with TMS as internal standard; mass spectral data MALDI-TOF-MS is on AB SCIEX 5800 mass spectrometer Obtained; UV-visible absorption spectrum was completed on Shimadzu UV-2700 UV-visible absorption spectrometer; fluorescence emission spectrum was measured by PerkinElmer LS-55 fluorescence spectrometer; fluorescence imaging of schistosomiasis adults was collected on OLYMPUSFV1000 laser confocal fluorescence microscope, laser provided 635nm excitation light source, collect emission fluorescence signals in the range of 650nm-750nm.

Example 1

Synthesis of fluorescent dye NIR-COOH-SO ₃ H:

(1) Synthesis of M-1: Add 1mmol of 1,1,2-trimethyl-1H-benzindole and 1.2mmol of 3-bromopropionic acid into a three-necked flask, then add 30mL of toluene as solvent. Under the protection of nitrogen, the reaction system was reacted at 110° C. for 24 hours. After cooling to room temperature, the reaction solution was extracted with dichloromethane (10 mL×3), the organic layers were combined, dried over anhydrous sodium sulfate, and separated by column chromatography to obtain M-1. NMR data: ¹ H NMR (400MHz, CDCl ₃ ) δ8.07(dd, J＝13.6,8.3Hz, 5H), 7.80–7.54(m, 2H), 4.05(d, J＝7.0Hz, 2H), 3.26 (s, 5H), 1.87 (s, 6H), 1.19 (t, J=6.9Hz, 3H).

(2) Synthesis of M-2: 1 mmol of M-1 and 1 mmol of malondialdehyde diphenylamine were dissolved in 25 mL of acetic anhydride, and reacted at 110° C. for 2 hours. Cool to room temperature, neutralize by adding aqueous sodium bicarbonate solution, extract the reaction solution with dichloromethane (10 mL×3), combine the organic layers, dry over anhydrous sodium sulfate, and separate by column chromatography to obtain M-2. Characterization data of high-resolution mass spectrometry: MALDI-TOF-MS: m/z 453.2175.

(3) Synthesis of NIR-COOH-SO ₃ H: 1.0 mmol of M-2 compound, 1.0 mmol of 3-(3-sulfonic acid-propyl)-1,1,2-trimethyl-1H- Add benzoindole and 1.0 mmol of potassium acetate to the round bottom flask, then add 30 mL of acetic anhydride. Under the protection of nitrogen, the reaction system was reacted at 70° C. for 2 hours. Cool to room temperature, neutralize by adding aqueous sodium bicarbonate solution, extract the reaction solution with dichloromethane (10 mL×3), and separate by column chromatography to obtain NIR-COOH-SO ₃ H. NMR characterization data: ¹ H NMR (400MHz, MeOD) δ8.38(s, 2H), 8.22(s, 2H), 7.98(d, J＝8.1Hz, 4H), 7.71(d, J＝8.8Hz, 1H ),7.62(d,J=8.6Hz,3H),7.46(s,2H),6.71(t,J=12.3Hz,1H),6.49(d,J=13.5Hz,1H),6.35(d,J ＝11.1Hz, 1H), 4.50(s, 4H), 3.05(t, J＝6.6Hz, 2H), 2.84(t, J＝6.9Hz, 2H), 2.40–2.22(m, 2H), 1.99(s ,12H). Characterization data of high-resolution mass spectrometry: MALDI-TOF-MS: m/z 649.2711.

Example 2

Absorption spectrum and fluorescence spectrum test of fluorescent dye NIR-COOH-SO ₃ H:

The fluorescent dye NIR-COOH-SO ₃ H was made into a ^{1 mmol·L -1} ethanol mother solution, and then diluted for testing. Ultraviolet Spectrum Measurement: The scanning range is 500nm-850nm, and the ultraviolet absorption spectrum characteristics are recorded. The solution determined above was then used to measure the fluorescence spectrum. Fluorescence spectrum measurement: the excitation wavelength is 620nm, and the emission spectrum range is 640nm-850nm. The absorption spectrum and fluorescence spectrum are shown in Figure 1, and the test results show that the emission peak of the fluorescent dye is located in the near-infrared region of 650-850nm.

Example 3

Cell fluorescence imaging of fluorescent dye NIR-COOH-SO ₃ H:

(1) Reagents and materials

Dimethyl sulfoxide (AR) was purchased from Aladdin Chemical Reagent Co., Ltd., RPMI 1640 culture medium was purchased from Thermo Fisher Scientific, and human cervical cancer cell HeLa was purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.

(2) Cell fluorescence imaging

Human cervical cancer cells HeLa were cultured in RPMI 1640 medium containing 10% fetal bovine serum and grown adherently at 37°C, 5% CO ₂ and 95% air atmosphere. The cell fluorescence imaging of the fluorescent dye NIR-COOH-SO ₃ H was tested on an OLYMPUSFV1000 laser confocal fluorescence microscope, the excitation wavelength was 635nm, and the fluorescence signal at 650-750nm was collected. The results of the cell fluorescence imaging are shown in Figure 2, and the experimental results show that the dye NIR-COOH-SO ₃ H can well fluorescently label the cells.

Example 4

Fluorescence labeling imaging of Schistosoma adult worms with fluorescent dye NIR-COOH-SO ₃ H:

(1) Reagents and materials

Reagents: Dimethyl sulfoxide (AR) was purchased from Aladdin Chemical Reagent Co., Ltd., RPMI 1640 culture solution was purchased from Thermo Fisher Scientific, and phosphate buffer (PBS, pH=7.4) was made by ourselves.

Cercariae: obtained from positive Oncomelania snails provided by the Jiangsu Institute of Schistosomiasis Control.

(2) Obtain cercariae of Schistosoma japonicum

The cercariae are obtained from positive snails infected with miracidia. Take the positive snails and put them in clear water, and expose them to fresh cercariae under incandescent light for about 2 hours.

(3) Mouse infection

Cut off the abdominal body hair of six-week-old female mice, take warm water to moisten the abdominal skin of the mice, and stick a cover glass containing about 50 cercariae on the mouse’s dehaired abdominal skin, and keep it for 20 minutes to allow the cercariae to penetrate. Infection was performed through the mouse skin.

(4) Obtain adults of schistosomiasis

After the infected mice were fed for 6 weeks, they were killed by neck dislocation. The abdominal skin and peritoneum were cut open successively, and the adult worms were collected in the hepatic portal-mesenteric vein by perfusion. The collected adult worms were rinsed three times in normal saline and distributed to petri dishes. After adding RPMI-1640 cell culture medium and culturing for 2 hours, the worms with good activity were screened under a microscope for fluorescence imaging.

(5) Schistosoma adult worm fluorescent labeling imaging method

Fluorescent dye NIR-COOH-SO ₃ H was dissolved in dimethyl sulfoxide (DMSO) to accurately prepare a 1 mmol stock solution, and then diluted with PBS buffer to a 5 μM dilution. Pipette 200 μL of the diluted solution into a petri dish, pick the adults with tweezers and add them to the organic fluorescent dye dilution, incubate at room temperature for 5 minutes to 6 hours, transfer the adults to a special vessel for fluorescence imaging, and use them under excitation at 635nm Laser confocal fluorescence microscopy was used to observe the fluorescent labeling of adults, and the fluorescent signals at 650-750 nm were collected for fluorescence imaging.

(6) Fluorescent labeling results

Refer to Figure 3, Figure 4 and Figure 5 for specific labeling results. From the fluorescence imaging images, it can be concluded that the fluorescent probe NIR-COOH-SO ₃ H can perform fluorescent labeling and imaging on Schistosoma adult worms. At the same time, we quantitatively analyzed the fluorescence intensity of Schistosoma adult worms, as shown in Figure 5, the fluorescence signal signal-to-noise ratio of the background area 1 (reference) and the worm body area 2 on the straight line in the figure is S/N>10, indicating that the Near-infrared fluorescent dyes are used for fluorescence imaging of Schistosoma adult worms with high signal-to-noise ratio.

Claims (1)

1. an application of a near-infrared fluorescent dye with the following formula structure, characterized in that: the near-infrared fluorescent dye can be used for schistosome adult fluorescent marker imaging; The fluorescent labeling method comprises the following steps: (1) Obtain schistosomiasis cercariae: the cercariae are obtained from positive snails infected with miracidia, take the positive snails and put them into clear water, and expose them to fresh cercariae under incandescent light for 2 hours; (2) Mice infection: Cut off the abdominal hair of six-week-old female mice, take warm water to moisten the abdominal skin of the mice, and stick a cover glass containing 50 cercariae on the dehaired abdominal skin of the mice. Keep for 20 minutes to allow the cercariae to penetrate the mouse skin to infect; (3) Obtain schistosomiasis adult worms: feed the infected mice for 6 weeks, dislocate their necks and kill them. Strictly follow the requirements of aseptic operation, cut open the abdominal skin and peritoneum in sequence, and infuse them into the hepatic porta-mesenteric vein Collect adults; rinse the collected adults in saline for 3 times and distribute them to petri dishes, add RPMI-1640 cell culture medium to culture for 2 hours, and screen the worms with good activity under microscope for fluorescence imaging; (4) Fluorescent imaging of schistosomiasis adults: the fluorescent dye was dissolved in dimethyl sulfoxide to accurately prepare 1 mmol of the mother solution, and then diluted with PBS buffer to a 5 μM dilution; pipette 200 μL of the dilution into a petri dish, and use tweezers to Pick the adults and add them to the fluorescent dye diluent, incubate at room temperature for 5 minutes to 6 hours, then transfer the adults to a fluorescence imaging vessel, observe the fluorescent labeling of the adults under a confocal fluorescence microscope under 635nm excitation, and collect the fluorescence at 650-750nm The signal was imaged fluorescently.