Chemiluminescence substrate with high chemiluminescence intensity, long wavelength and good stability, and preparation method and application thereof

Abstract

The application provides a chemical luminescence substrate with high chemical luminescence intensity, long wavelength and good stability, and a preparation method and application thereof. The chemical luminescence substrate has a structure shown in formula I. In addition, the application provides spontaneous fluorescence wavelength and intensity of the chemical luminescence probe in a physiological environment, and application of the chemical luminescence probe in in-vivo imaging. Test results show that the chemical luminescence probe provided in the application has long-wavelength spontaneous fluorescence (600 nm), can effectively penetrate skin tissue, has high spontaneous fluorescence intensity (> 10 7 p / s / cm 2 / sr), excellent thermal stability (without 1,2-dioxetane structure) and diversified detection groups (suitable for different detection models).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fine chemical industry, and particularly relates to a synthesis method and biological application of a novel chemical luminescence probe based on electron-rich methoxy-alkyl. BACKGROUND

[0002] Chemiluminescence is a kind of light radiation phenomenon accompanied by the chemical reaction of a substance. Since no external excitation light is needed, chemiluminescence detection can effectively overcome the problems such as light bleaching, light scattering and autofluorescence that are difficult to solve in traditional fluorescence detection (Talanta 2000, 51, 415-439). In addition, chemiluminescence detection has the advantages of low detection limit, high sensitivity, wide range of analyte detection concentration, etc., so chemiluminescence probes are concerned by chemists and biologists. In 1987, Paul Schaap first reported a chemical luminescence substrate with 1,2-dioxetane as a high-energy state structure. Compared with the traditional chemical luminescence substrate (such as luminol, acridinium ester, etc.), the defect of single detection object (reactive oxygen species), the chemical luminescence probe based on Schaap-type chemical luminescence substrate can be constructed for a variety of active substances.

[0003] Although the Schaap-type chemical luminescence probe has the advantages of long luminescence half-life (hour level) and stable signal intensity, the main bottleneck of this kind of chemical luminescence probe is weak luminescence intensity. When applied to biological sample imaging, the Schaap-type chemical luminescence probe often cannot meet the signal intensity requirements of the detector. Take the alkaline phosphatase chemiluminescence probe (AMPPD) as an example (as shown below), its detection mechanism is as follows: (1) alkaline phosphatase induces AMPPD to undergo hydrolysis reaction, and the phosphate group is removed to generate unstable intermediate AMPPD - ; (2) the high-energy state 1,2-dioxetane is cleaved, and then the chemiluminescence signal is generated. However, under physiological conditions, the 1,2-dioxetane structure is not stable, so the luminescence intensity of the probe is low and easy to be disturbed by the environment. In addition, the luminescence wavelength of the Schaap-type chemical luminescence probe is short, which is difficult to apply to in vivo imaging. Therefore, how to develop a new type of chemical luminescence substrate with long luminescence wavelength, high intensity, good stability and wide detection range has become a difficult problem to be solved.

[0004] SUMMARY

[0005] In view of the bottleneck of short wavelength, weak intensity and poor stability of the existing 1,2-dioxetane type chemiluminescence probe, the application aims to provide a kind of chemiluminescence probe with long wavelength, high intensity and excellent stability. The chemical modifiability of the substance is fully utilized: the chemical emission wavelength is extended to the near-infrared region by introducing electron-withdrawing fluorescent units to expand the pi system; the energy conversion efficiency is improved by optimizing the fluorescent unit through molecular engineering, and the chemiluminescence signal intensity is improved; the stability of the chemiluminescence probe is improved by using electron-rich double bonds instead of 1,2-dioxetane structure.

[0006] The application provides a general preparation method of the above-mentioned chemiluminescence probe with long wavelength, high intensity and excellent stability, and partial absorption, fluorescence and self-luminescence fluorescence spectrum, and application thereof in biological detection. The chemical composition and function of the chemiluminescence probe are as follows: (1) fluorescent unit, which extends the chemiluminescence wavelength and enhances the chemiluminescence intensity; (2) electron-rich double bond unit, which can controllably generate high-energy state 1,2-dioxetane structure; and (3) response unit, which specifically recognizes and detects substances.

[0007] Under the condition of the detected substance, the probe specifically responds, the detection unit is removed, and a chemiluminescence precursor with a phenolic hydroxyl negative ion is generated. Then, under white light excitation, the electron-rich double bond part of the chemiluminescence precursor reacts with oxygen to generate 1,2-dioxetane structure, which is stimulated by the phenolic hydroxyl negative ion to crack, and a significant chemiluminescence signal is generated.

[0008] The application is realized by the following scheme:

[0009] In one aspect, the chemiluminescence intensity of the chemiluminescence substrate disclosed by the application is high, the wavelength is long, and the chemiluminescence substrate is oxidation-free, and the structure is shown as formula I

[0010]

[0011] In formula I, R1 is independently selected from any one of the small molecule fluorophores shown in formulae II-V (wherein the curved mark is a substitution site, and the same below); in formula II, R3 is one of a hydrogen atom, a bromine atom, an amine group and a carboxyl group; in formulae II and III, R4 is one of ethyl or propyl sulfonate sodium.

[0012]

[0013] R2 is independently selected from any one of the detection groups shown in formulae VI-IX.

[0014]

[0015] General preparation method:

[0016] The synthesis of the compound adopts a modular preparation method. 2-bromo-5-hydroxybenzaldehyde is used as a starting material, and an acetal reaction, hydroxyl protection and phosphatidation reaction are sequentially performed to obtain a phosphonate intermediate. The phosphonate intermediate is further reacted with an adamantyl ketone compound (Hunig-Wozniewski-Emmons reaction) to prepare an olefin intermediate. The olefin intermediate is activated by a metal organic reagent, and is reacted with N,N-dimethylformamide to prepare an enal intermediate. The enal intermediate is further reacted with a small-molecule fluorescent group having a reactive methyl group (Knoevenagel condensation reaction) to prepare a chemiluminescent substrate. The substrate is further connected with various detection units to obtain the final chemiluminescent probe.

[0017]

[0018] General detection method

[0019] The chemiluminescent probe is first reacted with a detected substance (such as an enzyme or an active small molecule), and a detection group is removed to release a chemiluminescent precursor having a phenolic hydroxyl anion. Subsequently, under white light excitation, the chemiluminescent precursor rapidly undergoes an addition reaction with oxygen to generate a 1,2-dioxetane structure. The structure is cleaved to release a chemiluminescent signal under the action of the phenolic hydroxyl anion.

[0020] BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 UV absorption (10 -5 mol·L -1 ), fluorescence and chemiluminescence spectra (10 -4 mol·L -1 ) of.F-QM-OH (see Example 1) in a PBS solution (containing 1% DMSO);

[0022] The abscissa is the wavelength (nm), and the left ordinate is the absorbance, and the right ordinate is the relative intensity of fluorescence and chemiluminescence.

[0023] Figure 2 Chemiluminescence images and intensity (5*10 -5 mol·L -1 ) of.F-QM-OH (see Example 1) in a water and DMSO mixed solvent with increasing water content;

[0024] The upper part is the image collected by the Imaging Quant 4000 system, and the lower part is the quantification of the light intensity in the image. The water content in the image from left to right is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 99%, and three groups are in parallel; in the quantitative column chart, the horizontal coordinate is the percentage content of DMSO (%), and the vertical coordinate is the number of photons per unit area and unit time (p / s / cm2 / sr).

[0025] Figure 3 . The self-luminescent substrate F-QM-B (concentration of 5*10 -5 mol·L -1 ) in Tris buffer solution (containing 10% DMSO) with hydrogen peroxide and whether light is chemiluminescence image.

[0026] Figure 4 . The application of the self-luminescent substrate F-QM-B in the in vivo imaging of A549 subcutaneous tumor model mice. DETAILED DESCRIPTION

[0027] In a preferred technical solution of the present application:

[0028] R1 is independently selected from any one of the small molecule fluorophores shown in Ⅱ-Ⅶ;

[0029] R2 is independently selected from any one of the detection groups shown in formula Ⅷ-Ⅺ;

[0030] R3 is one of hydrogen atom, bromine atom, amine group and carboxyl group;

[0031] R4 is one of ethyl or propyl sulfonic acid sodium;

[0032] In a further preferred technical solution, R1 is independently selected from any one of the groups shown in Ⅱ, and R2 is a borate group;

[0033] More preferably, R3 is independently selected from hydrogen atom, and R4 is one of ethyl and propyl sulfonic acid sodium;

[0034] More preferably, R4 is ethyl;

[0035] The following examples are further described to better understand the content of the present application. Therefore, the examples do not limit the protection scope of the present application:

[0036] Example 1

[0037] Taking quinoline cyanide chemiluminescent substrate as an example, the specific synthesis route is as follows:

[0038]

[0039] 1. Synthesis of 4-bromo-3-(dimethoxymethyl)phenol

[0040]

[0041] In 50 mL dry single neck flask, 4-bromo-3-hydroxy-phenol (1 g, 4.55 mmol) was dissolved in 20 mL methanol, then p-toluenesulfonic acid (171.3 mg, 0.91 mmol) was added, refluxed for 6 h. After the reaction was completed, the reaction solution was poured into 200 ml dilute sodium carbonate solution (2 g), and extracted with ethyl acetate (200 mL x 2), dried with anhydrous sodium sulfate, rotary evaporation, column chromatography purification, light pink liquid 0.7 g, yield 57%.

[0042] 1 H NMR (400 MHz, CDC13-dl, ppm): δ = 7.40-7.38 (d, J = 8.4 Hz, 1H, Ph-H), δ = 7.12-7.11 (d, J = 3.2 Hz, 1H, Ph-H), δ = 6.73-6.70 (d-d, J1= 8.8 Hz, J2= 3.2 Hz, 1H, Ph-H), δ = 5.84 (s, 1H, Ph-OH), δ = 5.50 (s, 1H, -CH-O-), δ = 3.41 (s, 6H, -O-CH3). 13 C NMR (100 MHz, CDC13-dl, ppm): 155.51, 133.74, 117.82, 115.34, 112.71, 103.37, 54.35

[0043] 2. Synthesis of (4-bromo-3-(dimethoxymethyl)phenoxy)(tert-butyl)dimethylsilane

[0044]

[0045] In 50 mL round bottom flask, 4-bromo-3-(dimethoxymethyl)phenol (500 mg, 2.02 mmol) was dissolved in 15 mL dichloromethane, then imidazole (368 mg, 4.04 mmol) was added, under ice bath, TBSCl (411 mg, 6.06 mmol) and dichloromethane (5 ml) were added dropwise, after the addition was completed, the reaction was stirred at room temperature, TLC monitoring reaction progress, after the reaction was completed, 50 ml dichloromethane was added, washed with water (100 ml x 5), the organic layer was dried with anhydrous sodium sulfate, rotary evaporation, light pink liquid 0.6 g, yield 82%.

[0046] 1H NMR (400 MHz, CDC13-dl, ppm): δ = 7.40-7.37 (d, J = 8.6 Hz, 1H, Ph-H), δ = 7.10-7.09 (d, J = 3.0 Hz, 1H, Ph-H), δ = 6.71-6.68 (d-d, Ji = 8.6 Hz, J2= 3.0 Hz, 1H, Ph-H), δ = 5.48 (s, 1H, -CH-O-), δ = 3.38 (s, 6H, -O-CH3), δ = 0.97 (s, 9H, -Si-C-(CH3)3), δ = 0.20 (s, 6H, -Si-CH3). 13 C NMR (100 MHz, CDC13-dl, ppm): 155.02, 137.71, 133.47, 121.91, 120.17, 114.02, 102.79, 53.86, 25.64, 18.20, -4.48.

[0047] 3. Synthesis of dimethyl ((2-bromo-5-((tert-butyldimethylsilyl)oxy)phenyl)(methyloxy)methyl)phosphonate

[0048]

[0049] In a 25 mL reaction tube, add the product of the previous step (2.5 g, 6.29 mmol) and 3 mL of N,N-dimethylformamide, stir to mix well, then add trimethyl phosphite (1.03 g, 7.55 mmol) and a solution of boron trifluoride etherate in dichloromethane (8.3 mL, 7.55 mmol), dropwise. After the dropwise addition is complete, allow the reaction to proceed at room temperature for 16 h. After the reaction is complete, add an appropriate amount of saturated aqueous sodium bicarbonate solution to the reaction mixture and wash with ethyl acetate (150 mL x 3). Combine the extracts, dry over anhydrous sodium sulfate, and rotary evaporate to obtain a yellowish liquid crude product. This product is a reactive intermediate and is used in the next step without further purification.

[0050] 4. Synthesis of (3-(adamantan-2-ylidene(methyloxy)methyl)-4-bromophenoxy)(tert-butyl)dimethylsilane

[0051]

[0052] In a 100 mL reaction tube, add the product of the previous step (500 mg, 1.14 mmol), sodium hydride (82 mg, 3.42 mmol), and 30 mL of THF. Add adamantanone (170 mg, 1.14 mmol) dropwise. Stir at room temperature for 1 h. Quench the reaction by adding deionized water and extract with ethyl acetate (30 mL x 5). Dry over anhydrous sodium sulfate and rotary evaporate to obtain a yellowish liquid product (300 mg, 57% yield).

[0053] 1 H NMR (400MHz, CDCl3-d1, ppm): δ = 7.44-7.42 (d, J 3 =8.6Hz, 1H, Ph-H), δ = 6.74-6.73 (d, J = 2.9Hz, 1H, Ph-H), δ = 6.70-6.68 (dd, J1 = 8.6Hz, J2 = 3.0Hz, 1H, Ph-H), δ = 3.32 (s, 3H, -O-CH3), δ = 3.26 (s, 1H, -Adamantane-H), δ=2.09(s,1H,-Adamantane-H), δ=2.00-1.79(m,12H,-Adamantane-H), δ=0.97(s,9H,-Si-C-(CH3)3), δ=0.19(s,6H,-Si-CH3).

[0054] 5. Synthesis of 2-(adamantane-2-methylene(methoxy)methyl)-4-hydroxybenzene

[0055]

[0056] Add 500 mg (1.08 mmol) of the product from the previous step and 5 mL of tetrahydrofuran to a 25 mL reaction tube, stir until homogeneous, and freeze-dry three times in ethanol at -78 °C. While stirring at -78 °C, add dropwise n-butyllithium solution (1 mL, 2.4 mmol). After the addition is complete, continue the reaction at -78 °C for 2 h. Then add N,N-dimethylformamide (0.4 mL) dropwise and stir continuously for 1 h. Then, transfer to room temperature and react for 0.5 h. After the reaction is complete, quench the reaction with 1 mL of deionized water. Add ethyl acetate (50 mL), wash with saturated brine (50 mL × 3), dry the organic layer with anhydrous sodium sulfate, and rotary evaporate to obtain a yellow liquid crude product. Purify the crude product using a flash column chromatography to obtain 280 mg of a white solid product, with a yield of 87%.

[0057] 1H NMR (400 MHz, CDC13-dl, ppm): δ = 10.13 (s, 1H, -CHO), δ = 7.95-7.93 (d, J = 8.4 Hz, 1H, Ph-H), δ = 6.92-6.90 (d-d, J1= 8.8 Hz, J2= 2.4 Hz, 1H, Ph-H), δ = 6.80-6.79 (d, J = 2.4 Hz, 1H, Ph-H), δ = 3.32 (s, 6H, -0-CH3), δ = 3.32 (s, 1H, -Adamantane-H), δ = 1.97 (s, 1H, -Adamantane-H), δ = 1.94-1.66 (m, 12H, -Adamantane-H).

[0058] Mass spectrometry (ESI-MS, m / z): [M-H + ] calcd. for [C 19 H 22 O3-H + ] 297.1791; found 297.1490.

[0059] 6. Synthesis of F-QM-OH

[0060]

[0061] In a 100 mL round bottom flask, add A (222 mg, 0.74 mmol), quinoline (208 mg, 0.89 mmol) and 50 ml acetonitrile, stir to mix well; then add sodium acetate (73 mg, 0.89 mmol); heat to reflux for 10 h, monitor the reaction progress by TLC. After the reaction is completed, rotary evaporation to get brown yellow crude product, purify the crude product by flash column to get light red solid product 80 mg, yield 18%.

[0062] 1H NMR (400 MHz, CDC13-dl, ppm): δ = 9.14 (d, J = 0.8 Hz, 1H, =CH-), δ = 7.77-7.73 (m, 1H, Ph-H), δ = 7.61-7.59 (d, J = 8 Hz, 1H, Ph-H), δ = 7.54-7.52 (d, J = 8 Hz, 1H, Ph-H), δ = 7.48-7.44 (t, J = 8 Hz, 1H, Ph-H), δ = 7.45-7.41 (d, J = 16 Hz, 1H, Alkene-H), δ = 7.09 (s, 1H, Ph-H), δ = 7.02-6.98 (d, J = 16 Hz, 1H, Alkene-H), δ = 6.90-6.87 (d-d, J1= 8.4 Hz, J2= 2.8 Hz, 1H, Ph-H), δ = 6.78-6.77 (d, J = 2.8 Hz, 1H, Ph-H), δ = 4.40-4.34 (q, J = 6.8 Hz, 2H, -N-CH2-CH3), δ = 3.30 (s, 3H, -0-CH3), δ = 3.30 (s, 1H, -Adamantane-H), δ = 2.22 (s, 1H, -Adamantane-H), δ = 1.96-1.75 (m, 12H, -Adamantane-H), δ = 1.55-1.51 (t, J = 7.2 Hz, 2H, -N-CH2-CH3). 13 C NMR (100 MHz, CDC13-dl, ppm): 164.03, 157.37, 154.93, 145.77, 143.13, 142.90, 142.38, 135.84, 130.51, 125.85, 122.79, 122.49, 121.19, 61.52, 48.96, 43.75, 41.70, 37.44, 34.26, 18.88

[0063] Mass spectrometry (ESI-MS, m / z): [M-H + ] calcd. for [C 34 H 33 N3O2-H + ] 514.2495; found 514.2491.

[0064] 7. Synthesis of F-QM-B

[0065]

[0066] In a 100 mg round bottom flask, add the self-luminescent quinoline substrate (150 mg, 0.29 mmol), cesium carbonate (472 mg, 1.45 mmol) and 30 ml of acetonitrile, stir the mixture until uniform, then add p-benzylbromoborate (255 mg, 0.87 mmol), react at room temperature for 3 hours; after the reaction is complete, wash with saturated ammonium chloride solution (100 mL x 3), dry the organic with anhydrous sodium sulfate, separate by column chromatography, and obtain 130 mg of red product, yield 61%.

[0067] 1 H NMR (400 MHz, CDC13-dl, ppm): δ = 9.14-9.17 (d-d, J1= 8.8 Hz, J2= 1.2 Hz, 1H, =CH-), δ = 7.85-7.83 (d, J = 8 Hz, 2H, Ph-H), δ = 7.77-7.72 (m, 1H, Ph-H), δ = 7.60-7.56 (t, J = 8 Hz, 2H, Ph-H), δ = 7.47-7.38 (m, 4H, Ph-H), δ = 7.09 (s, 1H, Ph-H), δ = 7.01-6.97 (m, 2H, Alkene-H), δ = 6.87-6.86 (d, J = 2.8 Hz, 1H, Alkene-H), δ = 5.15 (s, 2H, -0-CH2-Ph), δ = 4.39-4.33 (q, J = 7.2 Hz, 2H, -N-CH2-CH3), δ = 3.28 (s, 3H, -0-CH3), δ = 3.28 (s, 1H, -Adamantane-H), δ = 2.16 (s, 1H, -Adamantane-H), δ = 1.95-1.73 (m, 12H, -Adamantane-H), δ = 1.55-1.51 (t, J = 7.2 Hz, 2H, -N-CH2-CH3), δ = 1.35 (s, 12H, -C-(CH3)2).

[0068] Example 2

[0069] Other chemiluminescent hydroxyl substrates, specific synthesis routes are as follows:

[0070] 1. Synthesis of self-luminescent indole substrate

[0071]

[0072] In a 100 mL round bottom flask, add sulfonic acid-based indole salt (322 mg, 1.08 mmol), quinoline (300 mg, 0.90 mmol) and 50 ml acetonitrile, stir to mix well; then add sodium acetate (74 mg, 0.90 mmol); heat to reflux for 10 h, monitor the reaction progress by TLC. After the reaction is completed, rotary evaporation to get red crude product, the crude product is purified by flash column to get red solid product 270 mg, yield 53%.

[0073] 1 H NMR (400 MHz, CDC13-dl, ppm): δ = 7.82-7.79 (m, 1H, Ph-H), δ = 7.59-7.57 (d, J = 8 Hz, 1H, Ph-H), δ = 7.53-7.49 (m, 3H, Ph-H), δ = 7.48-7.44 (d, J = 16 Hz, 1H, Alkene-H), δ = 7.12 (s, 1H, Ph-H), δ = 7.02-6.98 (d, J = 16 Hz, 1H, Alkene-H), δ = 6.92-6.89 (d-d, J1= 8.4 Hz, J2= 2.8 Hz, 1H, Ph-H), δ = 6.81-6.80 (d, J = 2.8 Hz, 1H, Ph-H), δ = 4.42-4.36 (q, J = 6.8 Hz, 2H, -N-CH2-CH2-), δ = 3.30 (s, 3H, -O-CH3), δ = 3.30 (s, 1H, -Adamantane-H), δ = 3.10-3.04 (m, 2H, -CH2-SO3 - ) δ = 2.22 (s, 1H, -Adamantane-H), δ = 1.96-1.75 (m, 12H, -Adamantane-H), δ = 1.62-1.58 (m, J = 7.2 Hz, 2H, -N-CH2-CH2-CH3).

[0074] 2. Synthesis of self-luminescent TCM substrate

[0075]

[0076] In a 100 ml dry flask, add single-sided TCM substrate (150 mg, 0.24 mmol) and self-luminescent aldehyde (100 mg, 1.5 eq), and add 35 ml acetonitrile to stir and dissolve, then add sodium acetate (36 mg, 0.26 mmol), reflux for about 6 h; monitor the reaction progress by TLC, after the reaction is completed, rotary evaporation to get red crude product, the crude product is purified by flash column to get red solid product 70 mg, yield 32%.

[0077] 1H NMR (400 MHz, CDC13-dl, ppm): δ = 8.09-8.08 (d, J = 8.0 Hz, 1H, Ph-H), δ = 7.67-7.41 (m, 14H, Ph-H), δ = 7.35-7.39 (m, 4H, Ph-H), δ = 7.23-7.22 (d, J = 4 Hz, 1H, Ph-H), δ = 7.13-7.12 (d, J = 4 Hz, 1H, Ph-H), δ = 6.97-6.95 (d, J = 8.4 Hz, 1H, Ph-H), δ = 6.16-6.12 (d, J = 16 Hz, 1H, Alkene-H), δ = 5.99-5.95 (d, J = 16 Hz, 1H, Alkene-H), δ = 3.23 (s, 1H, -Adamantane-H), δ = 3.16 (s, 3H, -O-CH3), δ = 2.08 (s, 1H, -Adamantane-H), δ = 1.93-1.68 (m, 12H, -Adamantane-H).

[0078] 3. Synthesis of self-luminescent BF2substrate

[0079]

[0080] In a 100 mL round bottom flask, add fluoroborane compound (200 mg, 0.95 mmol), self-luminescent aldehyde (341 mg, 1.14 mmol) and 50 ml acetonitrile, stir to mix well; then add n-butylamine (0.5 mL); heat to reflux for 10 h, monitor the reaction progress by TLC. After the reaction is completed, rotary evaporation to get red crude product, purify the crude product by flash column to get light red solid product 120 mg, yield 26%. 1 H NMR (400 MHz, CDC13-dl, ppm): δ = 8.09-8.08 (d, J = 8.0 Hz, 1H, Ph-H), δ = 7.67-7.41 (m, 14H, Ph-H), δ = 7.35-7.39 (m, 4H, Ph-H), δ = 7.23-7.22 (d, J = 4 Hz, 1H, Ph-H), δ = 7.13-7.12 (d, J = 4 Hz, 1H, Ph-H), δ = 6.97-6.95 (d, J = 8.4 Hz, 1H, Ph-H), δ = 6.16-6.12 (d, J = 16 Hz, 1H, Alkene-H), δ = 5.99-5.95 (d, J = 16 Hz, 1H, Alkene-H), δ = 3.23 (s, 1H, -Adamantane-H), δ = 3.16 (s, 3H, -O-CH3), δ = 2.08 (s, 1H, -Adamantane-H), δ = 1.93-1.68 (m, 12H, -Adamantane-H).

[0081] Example 3

[0082] Absorption, fluorescence and chemiluminescence spectra of F-QM-OH in aggregate state

[0083] F-QM-OH prepared in Example 1 was dissolved in analytical pure dimethyl sulfoxide to prepare a stock solution of 1.0 x 10 -2 M. Then 2 mL of DMSO / H2O mixed solvent with a water (H2O) content of 99% was prepared. 20 μL of the above stock solution was added to the prepared DMSO / H2O mixed solvent, and after mixing, it was transferred to an optical quartz cuvette (10 x 10 mm) for testing of the fluorescence spectrum. As shown in Figure 1 , with 480 nm as the excitation wavelength, the maximum emission peak of the F-QM-OH substrate was about 600 nm in the near-infrared region, with a Stokes shift of 120 nm; the chemiluminescence spectrum of F-QM-OH was basically consistent with the fluorescence spectrum.

[0084] Example 4

[0085] White light activated chemiluminescence precursor F-QM-OH

[0086] F-QM-OH prepared in Example 1 was dissolved in analytical pure dimethyl sulfoxide to prepare a stock solution of 1.0 x 10 -2 M, and a 1.0 x 10 -2 M solution of trimethyl-β-cyclodextrin in Tris buffer was prepared. Then 1 μL of the above stock solution was added to 10 μL of the trimethyl cyclodextrin solution, and 189 μL of different proportions of Tris and DMSO solution was added, finally obtaining different proportions of DMSO:Tris solution containing 5.0 x 10 -5 M F-QM-OH and 5.0 x 10 -4 M trimethyl-β-cyclodextrin. After mixing, white light (200 mW / cm 2 ) was uniformly irradiated for 2 minutes, and the chemiluminescence intensity was uniformly collected using the Imaging Quant 4000 system. The results are shown in Figure 2 , in different proportions of Tris-DMSO buffer, F-QM-OH showed significant chemiluminescence signal.

[0087] Example 5

[0088] Chemiluminescence probe F-QM-B for hydrogen peroxide detection

[0089] F-QM-B (further prepared from F-QM-OH) was dissolved in analytical pure dimethyl sulfoxide to prepare a stock solution of 1.0 x 10 -3M stock solution. Then 10 μL of the above stock solution was added into 180 μL of Tris solution and mixed well. Two groups of the above solution were prepared, one group was added with 10 μL of hydrogen peroxide solution (1.0 x 10 -2 M) and the other group was added with 10 μL of Tris buffer as control. After mixing well, white light (100 mW / cm 2 ) was used to irradiate for 2 minutes and the chemiluminescence intensity was collected by Imaging Quant 4000 system. As shown in Figure 3 , the hydrogen peroxide group showed obvious spontaneous light signal, while the control group had almost no spontaneous light signal.

[0090] Example 6

[0091] Application of chemiluminescence probe F-QM-B in vivo

[0092] All in vivo experiments in the present application comply with the regulations of laboratory animal feeding and use and are approved by the University Animal Feeding and Use Committee of East China University of Technology. The tumor-bearing nude mice used in the experiment were purchased from Shanghai Slek Animal Experiment Co., Ltd. and were fed in a sterile mouse cage in a laminar flow hood in a sterile room. The food and water used for feeding were treated with high-pressure steam.

[0093] To evaluate the in vivo application performance of chemiluminescence probe F-QM-B, tumor-bearing nude mice were used as imaging objects and traditional chemiluminescence dye luminol was used as a reference. As shown in Figure 4 , from left to right, three A549 (human lung cancer cells) subcutaneous tumor mice were injected with F-QM-B trimethyl-β-cyclodextrin solution (F-QM-B concentration 1.0 x 10 -4 M, trimethyl-β-cyclodextrin concentration 1.0 x 10 -3 M), F-QM-B trimethyl-β-cyclodextrin solution and luminol solution, respectively. After injection, the left 1 mouse was irradiated for 2 minutes (white light, 400 mW / cm 2 ) and the whole chemiluminescence imaging of the mouse was performed using Perkin Elmer In-Vivo Professional Imaging System. Before imaging experiment, the nude mice were anesthetized with 2.5% isoflurane gas.

[0094] As shown in Figure 4As shown, the light group (left 1) mice have obvious chemiluminescence signal, while the non-light group (left 2) does not have obvious chemiluminescence signal, which shows that F-QM-B only has chemiluminescence signal when responding to hydrogen peroxide and being irradiated. In addition, the luminol group (left 3) also does not have obvious chemiluminescence signal, which shows that the short-wavelength chemiluminescence dye does not meet the requirements of in vivo imaging. In summary, the prepared chemiluminescence probe has the advantages of long wavelength, controllable luminescence and good specificity, and is successfully applied to in vivo detection of hydrogen peroxide.

Claims

1. A preparation method of a chemiluminescent substrate with high chemiluminescent intensity, long wavelength and good stability, the structure of which is: The steps of the preparation method are as follows:

2. Application of a chemiluminescent substrate with high chemiluminescent intensity, long wavelength and good stability prepared according to claim 1 in hydrogen peroxide detection, wherein the application is non-disease diagnosis or treatment. ​

Images