CN110804009A - Chemiluminescent substrate with high chemiluminescent intensity, long wavelength and good stability, and preparation method and application thereof - Google Patents

Chemiluminescent substrate with high chemiluminescent intensity, long wavelength and good stability, and preparation method and application thereof Download PDF

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CN110804009A
CN110804009A CN201910956954.2A CN201910956954A CN110804009A CN 110804009 A CN110804009 A CN 110804009A CN 201910956954 A CN201910956954 A CN 201910956954A CN 110804009 A CN110804009 A CN 110804009A
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chemiluminescent
reaction
intensity
substrate
good stability
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郭志前
朱为宏
张玉涛
燕宸旭
徐清爽
李娟�
张辽
王婷
常茂菊
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East China University of Science and Technology
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    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/12—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
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Abstract

The invention provides a chemiluminescent substrate with high chemiluminescent intensity, long wavelength and good stability, and a preparation method and application thereof. The chemiluminescent substrate has a structure shown in formula I. In addition, the invention provides the autofluorescence wavelength and intensity of the chemiluminescent probe in a physiological environment, and application of the chemiluminescent probe in living body imaging. The test results show that the chemiluminescent probe provided by the invention has self-luminescence with long wavelengthFluoresce (600nm), can effectively penetrate skin tissues and has high autofluorescence intensity (>107p/s/cm2/sr), excellent thermal stability (no 1, 2-dioxetane structure contained) and diversified detection groups (applicable to different detection models).
Figure DDA0002227660030000011

Description

Chemiluminescent substrate with high chemiluminescent intensity, long wavelength and good stability, and preparation method and application thereof
Technical Field
The invention belongs to the field of fine chemical engineering, and particularly relates to a synthesis method and biological application of a novel chemiluminescence probe based on electron-rich methoxyl-alkenyl.
Background
Chemiluminescence is a light emission phenomenon that accompanies substances in carrying out chemical reactions. Because no external excitation light is needed, the chemiluminescence detection can effectively overcome the difficult problems of photobleaching, light scattering, autofluorescence and the like in the traditional fluorescence detection method (Talanta 2000,51, 415-439). In addition, chemiluminescence detection also has the remarkable advantages of low detection limit, high sensitivity, wide analyte detection concentration range and the like, so that chemiluminescence probes are concerned by chemists and biologists. In 1987, Paul Schaap first reported 1, 2-dioxyheterocycle as a high-energy state structure chemiluminescent substrate. Compared with the single defect of a detection object (active oxygen) of the traditional chemiluminescence substrate (such as luminol, acridinium ester and the like), the chemiluminescence probe suitable for various active substances can be constructed on the basis of the Schaap-type chemiluminescence substrate.
Although the Schaap-type chemiluminescent probe has the advantages of long luminescence half-life (hour scale) and stable signal intensity, the weak luminescence intensity is faced by the chemiluminescent probeThe main bottleneck. When applied to imaging biological samples, Schaap-type chemiluminescent probes often fail to meet the signal intensity requirements of the detector. Taking the phosphomonoesterase chemiluminescence probe (AMPPD) as an example (as shown in the following formula), the detection mechanism is as follows: (1) alkaline phosphatase induces AMPPD to generate hydrolysis reaction, and phosphate group leaves to generate unstable intermediate AMPPD-(ii) a (2) The high energy state 1, 2-dioxygen heterocyclic ring is cracked, and then a chemiluminescence signal is expressed. However, under physiological conditions, the 1, 2-dioxane structure is unstable, and therefore, the probe has low luminous intensity and is susceptible to environmental interference. In addition, the Schaap-type chemiluminescent probe has a short emission wavelength and is difficult to apply to in vivo imaging. Therefore, how to develop a novel chemiluminescent substrate with long luminescence wavelength, high intensity, good stability and a detection object capable of being popularized becomes a difficult problem to be solved urgently.
Figure BDA0002227660010000011
Disclosure of Invention
Aiming at the bottlenecks of short wavelength, weak intensity and poor stability of the existing 1, 2-dioxetane type chemiluminescent probe, the invention aims to provide a chemiluminescent probe with long chemiluminescent wavelength, high intensity and excellent stability. The chemical modifiability of the substances is fully utilized: the chemical emission wavelength of the system is expanded to a near infrared region by introducing an electron-withdrawing fluorescent unit to expand a pi system; the fluorescent unit is optimized through molecular engineering, so that the energy conversion efficiency is improved, and the chemiluminescence signal intensity is improved; the stability of the chemiluminescent probe is improved by replacing the 1, 2-dioxa ring structure with an electron rich double bond.
The invention provides a general preparation method of the chemiluminescence probe with long wavelength chemiluminescence, high intensity and excellent stability, a partial absorption, fluorescence and self-luminescence fluorescence spectrum and application thereof in biological detection. The chemical composition and function of the chemiluminescent probe are as follows: (1) a fluorophore unit for extending the chemiluminescence wavelength and enhancing the chemiluminescence intensity; (2) an electron-rich double bond unit can be controlled to generate a high energy state 1, 2-dioxetane structure; (3) a response unit that specifically recognizes the detection substance.
Under the condition of a detection object, the probe generates specific response, and the detection unit leaves to generate a chemiluminescent precursor with phenolic hydroxyl anions. And then, under the excitation of white light, the electricity-rich double bond part in the chemiluminescent precursor and oxygen generate addition reaction to generate a 1, 2-dioxetane structure, and the structure is cracked under the excitation of phenolic hydroxyl anions to show a remarkable chemiluminescent signal.
The invention is realized by the following scheme:
in one aspect, the chemiluminescent substrate of the invention has a structure represented by formula I
In the formula I, R1Independently selected from any one of small molecule fluorophores shown in formulas II-V (wherein the mark position of the curve is a substitution position, the same below); in the formula II R3Is one of hydrogen atom, bromine atom, amino group and carboxyl group, in the formulas II and III, R4Is one of ethyl or propyl sodium sulfonate.
Figure BDA0002227660010000022
R2Independently selected from any one of the detection groups shown in the formulas VI-IX.
Figure BDA0002227660010000031
The general preparation method comprises the following steps:
the synthesis of the compound adopts a modular preparation mode, 2-bromo-5-hydroxybenzaldehyde is taken as an initial raw material, and a phosphate intermediate is obtained through an acetal reaction, a hydroxyl protection reaction and a phosphitylation reaction in sequence; the phosphate intermediate is further reacted with an adamantanone compound (horner-Watts-Eimers reaction) to prepare an olefin intermediate; activating the olefin intermediate by a metal organic reagent, and reacting the olefin intermediate with N, N-dimethylformamide to prepare an olefine aldehyde intermediate; the olefine aldehyde intermediate further has a micromolecular fluorophore reaction (Knoevenagel condensation reaction) of active methyl, and a chemiluminescent substrate is prepared; the substrate is further connected with various detection units to obtain the final chemiluminescent probe.
Figure BDA0002227660010000032
Universal detection method
The chemiluminescent probe firstly reacts with a detected object (such as enzyme or active micromolecule) to detect group leaving, and a chemiluminescent precursor with phenolic hydroxyl negative ions is released; then, under the excitation of white light, the chemiluminescent precursor rapidly generates addition reaction with oxygen to generate a 1, 2-dioxetane structure; the structure is cleaved under the action of phenolic hydroxyl anions to release a chemiluminescent signal.
Figure BDA0002227660010000033
Drawings
FIG. 1 UV absorption of F-QM-OH (see example 1 for details) in PBS solution (containing 1% DMSO) (10)-5mol·L-1) Fluorescence and chemiluminescence spectra (10)-4mol·L-1)；
Wherein the abscissa is wavelength (nm), the left ordinate is absorbance, and the right ordinate is relative intensity of fluorescence and chemiluminescence.
FIG. 2. chemiluminescence patterns and intensities (5 x 10) of F-QM-OH (see example 1 for details) with increasing water content in a mixture of water and DMSO-5mol·L-1)；
Wherein, 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 is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 99% from left to right in sequence, and the three groups are parallel; the percentage of DMSO (%) is plotted on the abscissa of the quantitative bar graph, and the number of photons per unit area and per unit time (p/s/cm2/sr) is plotted on the ordinate of the quantitative bar graph.
FIG. 3 self-luminescent substrate F-QM-B (concentration 5 x 10)-5mol·L-1) Chemiluminescence images were generated in Tris buffer (10% DMSO in water) with hydrogen peroxide and with or without light.
FIG. 4 in vivo imaging application of the self-luminescent substrate F-QM-B on A549 subcutaneous tumor model mice.
Detailed Description
In a preferred embodiment of the present invention:
R1independently selected from any one of small molecule fluorophores shown in II-VII;
R2any one of detection groups independently selected from formulas VIII-XI;
R3is one of hydrogen atom, bromine atom, amido and carboxyl;
R4is one of ethyl or propyl sodium sulfonate;
in a further preferred embodiment, R1Independently selected from any one of the groups shown in II, R2Is a borate group;
further preferred R3Independently selected from hydrogen atom, R4Is one of ethyl and propyl sodium sulfonate;
further preferred R4Is ethyl;
the invention is further illustrated by the following examples, which are intended only for a better understanding of the contents of the invention. The examples given therefore do not limit the scope of protection of the invention:
example 1
Taking a quinoline nitrile chemiluminescence substrate as an example, the specific synthetic route is as follows:
Figure BDA0002227660010000051
synthesis of 4-bromo-3- (dimethoxymethyl) phenol
Figure BDA0002227660010000052
Adding 4-bromo-3-hydroxy-phenol (1g,4.55mmol) and 20mL of methanol into a 50mL dry single-neck bottle, and stirring to dissolve; then p-toluenesulfonic acid (171.3mg,0.91mmol) was added and refluxed for 6 h; after the reaction was completed, the reaction solution was poured into 200mL of a dilute sodium carbonate solution (2g), and extracted with ethyl acetate (200mL × 2), and the extracts were combined, dried over anhydrous sodium sulfate, spin-dried, and purified by column chromatography to obtain 0.7g of a pale pink liquid, yield 57%.
1H NMR(400MHz,CDCl3-d1,ppm):δ＝7.40-7.38(d,J＝8.4Hz,1H,Ph-H),δ＝7.12-7.11(d, J＝3.2Hz,1H,Ph-H),δ＝6.73-6.70(d-d,J1＝8.8Hz,J2＝3.2Hz,1H,Ph-H),δ＝5.84(s,1H, Ph-OH),δ＝5.50(s,1H,-CH-O-),δ＝3.41(s,6H,-O-CH3).13C NMR(100MHz,CDCl3-d1,ppm): 155.51,133.74,117.82,115.34,112.71,103.37,54.35
Synthesis of (4-bromo-3- (dimethoxymethyl) phenoxy) (tert-butyl) dimethylsilane
Figure BDA0002227660010000053
4-bromo-3- (dimethoxymethyl) phenol (500mg,2.02mmol), imidazole (368mg, 4.04mmol) and 15mL of dichloromethane were added to a 50mL round-bottomed flask and dissolved with stirring; dropwise adding a mixed solution of TBSCl (411mg,6.06mmol) and dichloromethane (5ml) in an ice bath, and stirring at normal temperature after dropwise adding; the reaction progress was monitored by a dot plate, and after completion of the reaction, 50ml of methylene chloride was added, and the organic layer was dried with water (100 ml. times.5) and anhydrous sodium sulfate and spin-dried to obtain 0.6g of a pale pink liquid with a yield of 82%.
1H NMR(400MHz,CDCl3-d1,ppm):δ＝7.40-7.37(d,J＝8.6Hz,1H,Ph-H),δ＝7.10-7.09(d, J＝3.0Hz,1H,Ph-H),δ＝6.71-6.68(d-d,J1＝8.6Hz,J2＝3.0Hz,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).13C NMR(100MHz,CDCl3-d1,ppm):155.02,137.71,133.47,121.91,120.17,114.02,102.79,53.86, 25.64,18.20,-4.48.
Synthesis of dimethyl ((2-bromo-5- ((tert-butyldimethylsilyl) oxy) phenyl) (methoxy) methyl) phosphonate
Adding the product of the last step (2.5g,6.29mmol) and 3mL of N, N-dimethylformamide into a 25mL reaction tube, stirring and mixing uniformly, then adding trimethyl phosphite (1.03g,7.55mmol) and a dichloromethane solution of boron trifluoride diethyl etherate (8.3mL, 7.55mmol), and reacting at room temperature for 16h after dropwise addition; after the reaction, adding a proper amount of saturated aqueous solution of sodium bicarbonate into the reaction solution for washing, extracting with ethyl acetate (150mL multiplied by 3), combining the extract liquor, drying with anhydrous sodium sulfate, and carrying out rotary evaporation to obtain a light yellow liquid crude product, wherein the product is an active intermediate and is put into the next reaction without further purification.
Synthesis of (3- (adamantan-2-ylidene (methoxy) methyl) -4-bromophenoxy) (tert-butyl) dimethylsilane
Figure BDA0002227660010000062
The product of the previous step (500mg,1.14mmol), sodium hydride (82mg,3.42mmol) and 30mL THF were added to a 100mL reaction tube; adamantanone (170mg,1.14mmol) was added dropwise, and stirred at room temperature for 1 hour; the reaction was quenched by addition of deionized water, extracted with ethyl acetate (30 mL. times.5), dried over anhydrous sodium sulfate, and rotary evaporated to give 300mg of a pale yellow liquid product in 57% yield.
1H NMR(400MHz,CDCl3-d1,ppm):δ＝7.44-7.42(d,J3＝8.6Hz,1H,Ph-H),δ＝6.74-6.73(d, J＝2.9Hz,1H,Ph-H),δ＝6.70-6.68(d-d,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).
Synthesis of 2- (adamantan-2-ylidene (methoxy) methyl) -4-hydroxybenzene
Figure BDA0002227660010000063
Adding the product of the last step (500mg,1.08mmol) and 5mL of tetrahydrofuran into a 25mL reaction tube, stirring and mixing uniformly, and freezing and pumping in ethanol at-78 ℃ for 3 times; under the condition of stirring at-78 ℃, dropwise adding n-butyllithium solution (1mL,2.4mmol), and continuing to react for 2h at-78 ℃ after dropwise adding; then dropwise adding N, N-dimethylformamide (0.4ml), and continuously stirring for 1 h; then the mixture is transferred to room temperature for reaction for 0.5 h; after the reaction, 1mL of deionized water is injected into the reaction solution to quench the reaction; then, ethyl acetate (50mL) was added, the mixture was washed with saturated brine (50 mL. times.3), the organic layer was dried over anhydrous sodium sulfate, and the mixture was rotary-evaporated to obtain a crude product as a yellow liquid; the crude product was purified by flash column to obtain 280mg of a white solid product with a yield of 87%.
1H NMR(400MHz,CDCl3-d1,ppm):δ＝10.13(s,1H,-CHO),δ＝7.95-7.93(d,J＝8.4Hz,1H, Ph-H),δ＝6.92-6.90(d-d,J1＝8.8Hz,J2＝2.4Hz,1H,Ph-H),δ＝6.80-6.79(d,J＝2.4Hz,1H, Ph-H),δ＝3.32(s,6H,-O-CH3),δ＝3.32(s,1H,-Adamantane-H),δ＝1.97(s,1H,-Adamantane-H), δ＝1.94-1.66(m,12H,-Adamantane-H).
Mass spectrometry(ESI-MS,m/z):[M-H+]calcd.for[C19H22O3-H+]297.1791；found 297.1490.
Synthesis of F-QM-OH
Figure BDA0002227660010000071
A (222mg,0.74mmol), quinolinecarbonitrile (208mg,0.89mmol) and 50mL acetonitrile are added into a 100mL round-bottom flask and stirred and mixed uniformly; then sodium acetate (73mg,0.89mmol) was added; the mixture was heated to reflux for 10h and the reaction progress was monitored by spotting plates. After the reaction is finished, a brown yellow crude product is obtained by rotary evaporation, and the crude product is purified by a flash column to obtain a light red solid product 80mg with the yield of 18%.
1H NMR(400MHz,CDCl3-d1,ppm):δ＝9.14(d,J＝0.8Hz,1H,＝CH-),δ＝7.77-7.73(m,1H, Ph-H),δ＝7.61-7.59(d,J＝8Hz,1H,Ph-H),δ＝7.54-7.52(d,J＝8Hz,1H,Ph-H),δ＝7.48-7.44(t,J ＝8Hz,1H,Ph-H),δ＝7.45-7.41(d,J＝16Hz,1H,Alkene-H),δ＝7.09(s,1H,Ph-H),δ＝7.02-6.98 (d,J＝16Hz,1H,Alkene-H),δ＝6.90-6.87(d-d,J1＝8.4Hz,J2＝2.8Hz,1H,Ph-H),δ＝6.78-6.77 (d,J＝2.8Hz,1H,Ph-H),δ＝4.40-4.34(q,J＝6.8Hz,2H,-N-CH2-CH3),δ＝3.30(s,3H,-O-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＝7.2Hz,2H,-N-CH2-CH3).13C NMR(100MHz,CDCl3-d1, 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
Mass spectrometry(ESI-MS,m/z):[M-H+]calcd.for[C34H33N3O2-H+]514.2495；found 514.2491.
Synthesis of F-QM-B
Figure BDA0002227660010000072
Adding a self-luminous quinoline substrate (150mg,0.29mmol), cesium carbonate (472mg,1.45mmol) and 30ml of acetonitrile into a 100mg round-bottom flask, uniformly stirring and mixing, then adding p-benzyl bromide borate (255mg,0.87mmol), and reacting at normal temperature for 3 hours; after the reaction, the red product 130mg was obtained by washing with saturated ammonium chloride solution (100 mL. times.3), drying with anhydrous sodium sulfate, and separating by column chromatography, with a yield of 61%.
1H NMR(400MHz,CDCl3-d1,ppm):δ＝9.14-9.17(d-d,J1＝8.8Hz,J2＝1.2Hz,1H,＝CH-), δ＝7.85-7.83(d,J＝8Hz,2H,Ph-H),δ＝7.77-7.72(m,1H,Ph-H),δ＝7.60-7.56(t,J＝8Hz,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＝2.8Hz,1H,Alkene-H),δ＝5.15(s,2H,-O-CH2-Ph),δ＝4.39-4.33(q,J＝7.2Hz, 2H,-N-CH2-CH3),δ＝3.28(s,3H,-O-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＝7.2Hz,2H, -N-CH2-CH3),δ＝1.35(s,12H,-C-(CH3)2).
Example 2
Other chemiluminescent hydroxyl substrates were synthesized by the following specific routes:
1. synthesis of self-luminescent indole substrates
Figure BDA0002227660010000081
Adding sulfonic indole salt (322mg,1.08mmol), quinolinecarbonitrile (300mg,0.90mmol) and 50mL acetonitrile into a 100mL round-bottom flask, and uniformly stirring and mixing; then sodium acetate (74mg,0.90mmol) was added; the mixture was heated to reflux for 10h and the reaction progress was monitored by spotting plates. After the reaction is finished, a red crude product is obtained by rotary evaporation, and the crude product is purified by a flash column to obtain a light red solid product 270mg with the yield of 53%.
1H NMR(400MHz,CDCl3-d1,ppm):δ＝7.82-7.79(m,1H,Ph-H),δ＝7.59-7.57(d,J＝8Hz,1H, Ph-H),δ＝7.53-7.49(m,3H,Ph-H),δ＝7.48-7.44(d,J＝16Hz,1H,Alkene-H),δ＝7.12(s,1H, Ph-H),δ＝7.02-6.98(d,J＝16Hz,1H,Alkene-H),δ＝6.92-6.89(d-d,J1＝8.4Hz,J2＝2.8Hz,1H, Ph-H),δ＝6.81-6.80(d,J＝2.8Hz,1H,Ph-H),δ＝4.42-4.36(q,J＝6.8Hz,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＝7.2Hz,2H, -N-CH2-CH2-CH3).
2. Synthesis of self-emissive TCM substrates
Figure BDA0002227660010000091
Adding a single-side TCM substrate (150mg,0.24mmol) and self-luminous aldehyde (100mg,1.5 eq) into a 100ml dry double-mouth bottle, adding 35ml acetonitrile, stirring for dissolving, adding sodium acetate (36mg,0.26mmol), and refluxing for about 6 hours; and (3) monitoring the reaction process by using a spot plate, after the reaction is finished, carrying out rotary evaporation to obtain a red crude product, and purifying the crude product by using a flash column to obtain a red solid product 70mg with the yield of 32%.
1H NMR(400MHz,CDCl3-d1,ppm):δ＝8.09-8.08(d,J＝8.0Hz,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＝4Hz,1H,Ph-H),δ＝7.13-7.12(d,J＝ 4Hz,1H,Ph-H),δ＝6.97-6.95(d,J＝8.4Hz,1H,Ph-H),δ＝6.16-6.12(d,J＝16Hz,1H,Alkene-H), δ＝5.99-5.95(d,J＝16Hz,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).
3. Self-luminous BF2Synthesis of a substrate
Adding a fluoroboron compound (200mg,0.95mmol), a self-luminous aldehyde (341mg,1.14mmol) and 50mL of acetonitrile into a 100mL round-bottom flask, and uniformly stirring and mixing; then n-butylamine (0.5mL) was added; the mixture was heated to reflux for 10h and the reaction progress was monitored by spotting plates. After the reaction is finished, a red crude product is obtained by rotary evaporation, and the crude product is purified by a flash column to obtain a light red solid product of 120mg with the yield of 26%.1H NMR(400MHz,CDCl3-d1,ppm):δ＝7.77-7.73(m,1H,Ph-H),δ＝7.65-7.63(d,J＝8Hz,2H, Ph-H),δ＝7.59-7.57(d,J＝8Hz,2H,Ph-H),δ＝7.50-7.46(t,J＝8Hz,1H,Ph-H),δ＝7.38-7.34(d,J ＝16Hz,1H,Alkene-H),δ＝7.18-7.12(m,2H,Ph-H),δ＝6.58-6.54(d,J＝16Hz,1H,Alkene-H), δ＝6.12(s,1H,＝CH-CO-),δ＝3.30(s,3H,-O-CH3),δ＝3.30(s,1H,-Adamantane-H),δ＝2.22(s, 1H,-Adamantane-H),δ＝1.96-1.75(m,12H,-Adamantane-H).
Example 3
Absorption, fluorescence and chemiluminescence spectra of F-QM-OH in the aggregated state
The F-QM-OH prepared in example 1 was dissolved in analytically pure dimethyl sulfoxide to give a solution of 1.0X 10-2Stock solutions of M. Then preparing water (H)2O) DMSO/H with a content of 99%2O mixed solvent 2 mL. 20 μ L of the stock solution was added to the prepared DMSO/H2O mixed solvent, mixed evenly and transferred to an optical quartz cuvette (10X 10mm) to test the fluorescence spectrum. As shown in FIG. 1, with 480nm as the excitation wavelength, the maximum emission peak of the F-QM-OH substrate is located in the near infrared region at about 600nm, and the Stokes shift is 120 nm; the F-QM-OH chemiluminescence spectrum is essentially identical to the fluorescence spectrum.
Example 4
White light activated chemiluminescent precursor F-QM-OH
The F-QM-OH prepared in example 1 was dissolved in analytically pure dimethyl sulfoxide to give a solution of 1.0X 10-2Stock solutions of M, prepared simultaneously at 1.0X 10-2Adding 1 mu L of the stock solution into 10 mu L of trimethyl cyclodextrin solution, adding 189 mu L of Tris and DMSO solutions in different proportions, and finally obtaining DMSO solutions in different proportions, wherein the Tris solution contains 5.0 multiplied by 10 simultaneously-5F-QM-OH of M with 5.0X 10-4M trimethyl- β -cyclodextrin, white light (200 mW/cm) was used uniformly after mixing uniformly2) The light is irradiated for 2 minutes, and the chemiluminescence intensity is uniformly collected by adopting an Imaging Quant 4000 system. As shown in FIG. 2, F-QM-OH showed significant chemiluminescent signals in different ratios of Tris-DMSO buffer.
Example 5
Application of chemiluminescent probe F-QM-B in hydrogen peroxide detection
Dissolving F-QM-B (further prepared from F-QM-OH) in analytically pure dimethyl sulfoxide to obtain 1.0 × 10 solution-3Stock solutions of M. Then 10 mul of the stock solution is added into 180 mul of Tris solution and mixed evenly; two groups of the above solutions were taken, and one group was added with 10. mu.L of hydrogen peroxide solution (1.0X 10)-2M), another group was added with 10. mu.L Tris buffer as a control; uniformly mixing the white light and the white light(100mW/cm2) The light is irradiated for 2 minutes, and the chemiluminescence intensity is uniformly collected by adopting an Imaging Quant 4000 system. As a result, as shown in fig. 3, the hydrogen peroxide group showed a clear self-luminescence signal, while the control group showed almost no self-luminescence signal.
Example 6
Chemiluminescence probe F-QM-B application in vivo detection
All in vivo experiments in the present invention followed the regulations for laboratory animal feeding and use and were approved by the animal feeding and use committee of university of eastern university of china. Experimental nude mice bearing tumors were purchased from shanghai slaike animal laboratories ltd, and were housed in sterile squirrel cages in a laminar flow hood in a sterile room and fed with food and water treated with high pressure steam.
To evaluate the in vivo performance of the chemiluminescent probe F-QM-B, tumor-bearing nude mice were used as imaging subjects and the conventional chemiluminescent dye luminol was used as a reference, as shown in FIG. 4, 3A 549 (human lung carcinoma cell) subcutaneous tumor mice were injected with F-QM-B trimethyl- β -cyclodextrin solution (F-QM-B concentration 1.0X 10) in situ from left to right-4M, trimethyl- β -Cyclodextrin concentration 1.0X 10-3M), trimethyl- β -cyclodextrin solution of F-QM-B and luminol solution after injection, left 1 mouse was illuminated for 2 minutes (white light, 400 mW/cm)2) And the mice were imaged for total chemiluminescence using a Perkin Elmer In-Vivo Professional Imaging System. Prior to imaging experiments, nude mice were anesthetized with 2.5% isoflurane gas.
As shown in fig. 4, the illuminated group (left 1) mice had a clear chemiluminescent signal, while the non-illuminated group (left 2) did not exhibit a clear chemiluminescent signal, indicating that F-QM-B exhibited a chemiluminescent signal only when it responded to hydrogen peroxide and was illuminated. In addition, the luminol group (left 3) also did not exhibit significant chemiluminescent signal, indicating that short wavelength chemiluminescent dyes did not meet in vivo imaging requirements. In conclusion, the chemiluminescent probe prepared by the invention has the remarkable advantages of long wavelength, controllable luminescence, good specificity and the like, and is successfully applied to the detection of hydrogen peroxide living bodies.

Claims (4)

1. A chemiluminescent substrate with high chemiluminescent intensity, long wavelength and good stability is disclosed, and the structure of the chemiluminescent substrate is shown as formula I:
in the formula I, R1Independently selected from any one of small molecule fluorophores shown in formulas II-VI (wherein the mark position of the curve is a substitution position, the same below); in the formula II R3Is one of hydrogen atom, bromine atom, amino group and carboxyl group, in the formulas II and III, R4Is one of ethyl or propyl sodium sulfonate;
Figure FDA0002227658000000012
R2is independently selected from any one of detection groups shown in formulas XII-XV;
Figure FDA0002227658000000013
2. a method for preparing the chemiluminescent substrate of claim 1 having high autofluorescence, long wavelength and good stability, comprising the steps of:
the synthesis of the compound adopts a modular preparation mode, 2-bromo-5-hydroxybenzaldehyde is taken as an initial raw material, and a phosphate intermediate is obtained through an acetal reaction, a hydroxyl protection reaction and a phosphitylation reaction in sequence; the phosphate intermediate is further subjected to an adamantanone reaction (a horner-Watts-Eimers reaction) to prepare an olefin intermediate; activating the olefin intermediate by a metal organic reagent, and reacting the olefin intermediate with N, N-dimethylformamide to prepare an olefine aldehyde intermediate; the olefine aldehyde intermediate is further subjected to a small molecular fluorophore reaction (Knoevenagel condensation reaction) with an active methyl group, so as to prepare the chemiluminescent substrate.
3. Use of the chemiluminescent substrate of claim 1 having high autofluorescence, long wavelength, and good stability for chemiluminescence detection and chemiluminescence enzyme immunoassay.
4. The use according to claim 3, wherein the chemiluminescent probe is obtained by linking the substrate prepared according to claim 2 to various types of detection units.
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