CN117024332A - Near-infrared singlet oxygen luminescent probe with high signal-to-noise ratio and high stability and preparation method and application thereof - Google Patents
Near-infrared singlet oxygen luminescent probe with high signal-to-noise ratio and high stability and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
- C07D213/28—Radicals substituted by singly-bound oxygen or sulphur atoms
- C07D213/30—Oxygen atoms
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
- C07D209/12—Radicals substituted by oxygen atoms
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
- 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
- C07D215/14—Radicals substituted by oxygen atoms
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention belongs to the technical field of fine chemical engineering, and provides a near-infrared singlet oxygen luminescence probe with high signal-to-noise ratio and high stability, and a preparation method and application thereof. The luminescent probe is synthesized in a modularized mode, 4-methylquinoline is taken as a raw material, and the luminescent probe reacts with iodoethane to obtain a quinoline salt intermediate with active methyl; taking 2-bromo-5-hydroxybenzaldehyde as a raw material, obtaining a phosphate intermediate through an acetal reaction and a phosphoesterification reaction, and then reacting with 2-norbornone to obtain an enaldehyde intermediate; the quinoline salt intermediate and the enal intermediate are further reacted to obtain the luminescent probe. The luminescent probe prepared by the invention has long-wavelength luminescence, and can effectively penetrate biological tissues for living body imaging; the method also has excellent light stability, can avoid false signals caused by light in the PDT efficiency evaluation process to the greatest extent, and can rapidly and accurately acquire the singlet oxygen yield due to high sensitivity and high selectivity.
Description
Technical Field
The invention relates to the technical field of fine chemical engineering, in particular to a near-infrared singlet oxygen luminescence probe with high signal-to-noise ratio and high stability, and a preparation method and application thereof.
Background
Singlet oxygen [ ] 1 O 2 ) Is a high-activity oxygen generated in the biological metabolism process and plays an important role in a plurality of physiological processes such as cell signal transduction, immune response, gene expression and the like. In addition, photodynamic therapy (PDT) which has been attracting attention in recent years has been advancing in cancer therapy, and more new photosensitizers have been developed, which function to sensitize oxygen production under illumination 1 O 2 Thereby killing cancer cells. Thus, detection 1 O 2 To explore the biological and medical process the function of the Chinese medicine is of great significance.
However, due to 1 O 2 Has extremely high reactivity and extremely short life, and its living detection is challenging. The currently commonly used singlet oxygen detection method mainly comprises the following steps: (1) phosphorescence measurement at 1270 nm; (2) electron paramagnetic resonance method; (3) spectrophotometry represented by ABDA; (4) a fluorescence method represented by SOSG fluorescent probe; (5) chemiluminescence method. Among them, the first three methods are generally used for in vitro detection and are not suitable for in vivo detection scenarios. Commercial SOSG probes are short in wavelength and reported in numerous literature to have themselvesCertain photosensitivity, which may give rise to false signals under illumination, further limits their application in PDT evaluation. The chemiluminescence method effectively avoids the interference of background fluorescence and remarkably improves the signal to noise ratio as no external light source is needed for real-time excitation, and is a powerful tool in the detection field. However, the existing chemiluminescent probes face the problems of short wavelength and poor stability. Therefore, how to provide a chemiluminescent probe with long wavelength emission, high sensitivity and good light stability is a problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a near-infrared singlet oxygen luminescence probe with high signal-to-noise ratio and high stability, and a preparation method and application thereof. The method aims to solve the technical problems of short wavelength and poor stability of the existing chemiluminescent probe.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a near-infrared singlet oxygen luminescence probe with high signal-to-noise ratio and high stability, and the structural formula of the near-infrared singlet oxygen luminescence probe is shown as formula I:
wherein R is 1 The structural formula of (a) is any one of formulas II-IV;
R 2 the structural formula of (C) is formula V or formula VI;
the R is 3 、R 4 And R is 5 The structural formula of (a) is any one of formulas VII-IX independently;
the invention provides a preparation method of the near infrared singlet oxygen luminescent probe, which comprises the following steps:
s1, mixing 4-methylquinoline, ethyl iodide and an organic solvent A, and reacting to obtain a quinoline intermediate;
s2, mixing 2-bromo-5-hydroxybenzaldehyde, tetrabutylammonium tribromide, tert-butyldimethyl chlorosilane and an organic solvent B, and carrying out an acetal reaction to obtain an acetal intermediate; mixing an acetal intermediate, trimethoxy phosphorus and an organic solvent C, and performing a phospho esterification reaction to obtain a phosphate intermediate; mixing a phosphate intermediate, 2-norbornone, butyllithium and an organic solvent D, and performing HWE reaction to obtain an enal intermediate;
and S3, mixing the quinoline intermediate, the enal intermediate, the catalyst and the organic solvent E, and performing Knoevenagel condensation reaction to obtain the near-infrared singlet oxygen luminescent probe.
Further, in the step S1, the molar volume ratio of the 4-methylquinoline to the iodoethane to the organic solvent A is 5-10 mmol: 20-30 mmol: 5-10 mL; the reaction temperature is 100-150 ℃ and the reaction time is 5-10 h.
Further, in the step S2, the molar volume ratio of the 2-bromo-5-hydroxybenzaldehyde, tetrabutylammonium tribromide, tertiary butyl dimethyl chlorosilane and the organic solvent B is 5-10 mmol:0.5 to 1mmol: 10-20 mmol: 20-40 mL; the temperature of the acetalization reaction is 20-30 ℃, and the time of the acetalization reaction is 6-8 h.
Further, in the step S2, the molar volume ratio of the acetal intermediate, trimethoxy phosphorus and the organic solvent C is 20 to 30mmol: 30-40 mmol: 10-20 mL; the temperature of the phosphating reaction is-5 ℃, and the time of the phosphating reaction is 8-12 h.
Further, in the step S2, the molar volume ratio of the phosphate intermediate, 2-norbornone, butyllithium and the organic solvent D is 10 to 20mmol: 15-30 mmol: 15-30 mmol: 10-20 mL; the temperature of the HWE reaction is-20-0 ℃, and the time of the HWE reaction is 4-8 h.
Further, in the step S3, the molar volume ratio of the quinoline intermediate, the enal intermediate, the catalyst and the organic solvent E is 0.1 to 1mmol:0.1 to 1mmol: 0.1-1 mL: 8-20 mL; the temperature of the Knoevenagel condensation reaction is 100-150 ℃, and the time of the Knoevenagel condensation reaction is 5-10 h.
Further, the catalyst is one or more of piperidine, triethylamine, morpholine and pyridine.
Further, the organic solvent A is toluene, o-dichlorobenzene or acetonitrile; the organic solvent B is methanol or acetonitrile; the organic solvent C is dichloromethane or 1, 4-dioxane; the organic solvent D is dichloromethane, tetrahydrofuran or diethyl ether; the organic solvent E is toluene or acetonitrile.
The invention also provides application of the near infrared singlet oxygen luminescent probe in detection of singlet oxygen.
Compared with the prior art, the invention has the following beneficial effects:
the luminous probe provided by the invention has a simple structure and is simpler and more convenient to synthesize. The invention expands the emission wavelength of the probe through the conjugated connection of the small molecular fluorophore and the Schaap chemiluminescent substrate precursor, and the electron-rich double bond precursor of the chemiluminescent substrate can react with singlet oxygen in a high specificity manner to generate a 1, 2-dioxetane structure, and then the 1, 2-dioxetane structure is decomposed under the induction of the phenoxy anions to generate a near infrared chemiluminescent signal. In addition, the probe has excellent light stability, can avoid false signals caused by light in the PDT efficiency evaluation process to the greatest extent, and can rapidly and accurately acquire the singlet oxygen yield due to high sensitivity and high selectivity.
Drawings
FIG. 1 is a hydrogen spectrum of 1-ethyl-4-methylquinoline salt prepared in example 1 of the present invention;
FIG. 2 is a carbon spectrum of a 1-ethyl-4-methylquinoline salt prepared in example 1 of the present invention;
FIG. 3 is a hydrogen spectrum of 2- (adamantan-2-ylidene (methoxy) methyl) -4-hydroxybenzaldehyde prepared in example 1 according to the present invention;
FIG. 4 is a carbon spectrum of 2- (adamantan-2-ylidene (methoxy) methyl) -4-hydroxybenzaldehyde prepared in example 1 according to the present invention;
FIG. 5 is a hydrogen spectrum of QMI prepared in example 1 of the present invention;
FIG. 6 is a carbon spectrum of QMI prepared in example 1 of the present invention;
FIG. 7 is a hydrogen spectrum of 1-ethyl-2, 3-trimethylindole salt prepared in example 2 of the present invention;
FIG. 8 is a carbon spectrum of 1-ethyl-2, 3-trimethylindole salt prepared in example 2 of the present invention;
FIG. 9 is a hydrogen spectrum of InCL prepared in example 2 of the present invention;
FIG. 10 is a graph of the carbon spectrum of InCL prepared in example 2 of the present invention;
FIG. 11 is a graph showing the ultraviolet absorption spectrum and fluorescence emission spectrum of QMI in PBS solution (containing 1% DMSO), wherein the concentration of QMI is 1.0X10 -5 mol/L；
FIG. 12 is a QMI in PBS buffer (1% DMSO, 1.0X10) -5 mol/L methylene blue) of the light-emitting image and intensity image;
FIG. 13 is a graph showing the intensity of chemiluminescent signal of QMI as a function of photosensitizer methylene blue concentration;
FIG. 14 is a graph showing signal enhancement comparison of QMI with commercial singlet oxygen probes ABDA and SOSG;
FIG. 15 is a graph showing the comparison of the photostability of QMI and conventional chemiluminescent probes DCM-OH-CF and QM-OH-CF;
fig. 16 is a diagram of the application of QMI in vivo imaging in HeLa tumor-bearing nude mice.
Detailed Description
The invention provides a near-infrared singlet oxygen luminescence probe with high signal-to-noise ratio and high stability, and the structural formula of the near-infrared singlet oxygen luminescence probe is shown as formula I:
wherein R is 1 The structural formula of (A) is any of formulas II-IVPreferably of formula III or formula IV, further preferably of formula III;
R 2 the structural formula of (a) is formula V or formula VI, preferably formula VI;
the R is 3 、R 4 And R is 5 The structural formula of (a) is independently any one of formulas VII-IX, preferably formula VII or formula IX, and further preferably formula VII;
in the invention, the curve marks in the formulas II to IX are all substitution positions.
The invention provides a preparation method of the near infrared singlet oxygen luminescent probe, which comprises the following steps:
s1, mixing 4-methylquinoline, ethyl iodide and an organic solvent A, and reacting to obtain a quinoline intermediate;
s2, mixing 2-bromo-5-hydroxybenzaldehyde, tetrabutylammonium tribromide, tert-butyldimethyl chlorosilane and an organic solvent B, and carrying out an acetal reaction to obtain an acetal intermediate; mixing an acetal intermediate, trimethoxy phosphorus and an organic solvent C, and performing a phospho esterification reaction to obtain a phosphate intermediate; mixing a phosphate intermediate, 2-norbornone, butyllithium and an organic solvent D, and performing HWE reaction to obtain an enal intermediate;
and S3, mixing the quinoline intermediate, the enal intermediate, the catalyst and the organic solvent E, and performing Knoevenagel condensation reaction to obtain the near-infrared singlet oxygen luminescent probe.
In the present invention, there is no precedence relationship between step S1 and step S2.
In the invention, in the step S1, the molar volume ratio of the 4-methylquinoline, the iodoethane and the organic solvent A is 5-10 mmol: 20-30 mmol: 5-10 mL, preferably 6-9 mmol: 21-27 mmol:6 to 9mL, more preferably 7 to 8mmol: 23-25 mmol: 7-8 mL; the temperature of the reaction is 100-150 ℃, preferably 110-140 ℃, and more preferably 120-130 ℃; the reaction time is 5 to 10 hours, preferably 6 to 9 hours, and more preferably 7 to 8 hours.
In the invention, in the step S2, the molar volume ratio of the 2-bromo-5-hydroxybenzaldehyde, tetrabutylammonium tribromide, tertiary butyl dimethyl chlorosilane and the organic solvent B is 5-10 mmol:0.5 to 1mmol: 10-20 mmol: 20-40 mL, preferably 6-9 mmol:0.6 to 0.9mmol: 12-18 mmol:24 to 36mL, more preferably 7 to 8mmol:0.7 to 0.8mmol: 14-16 mmol: 25-30 mL; the acetalization reaction temperature is 20 to 30 ℃, preferably 22 to 26 ℃, and more preferably 25 ℃; the time for the acetalization reaction is 6 to 8 hours, preferably 6.5 to 7.5 hours, and more preferably 7 hours.
In the invention, in the step S2, the molar volume ratio of the acetal intermediate, trimethoxy phosphorus and the organic solvent C is 20-30 mmol: 30-40 mmol: 10-20 mL, preferably 22-28 mmol: 32-38 mmol:12 to 18mL, more preferably 24 to 26mmol: 34-36 mmol: 14-16 mL; the temperature of the phosphotidation reaction is-5 ℃, preferably-3-2 ℃, and more preferably 0 ℃; the time for the phosphating reaction is 8 to 12 hours, preferably 9 to 11 hours, more preferably 10 hours.
In the present invention, in the step S2, the molar volume ratio of the phosphate intermediate, 2-norbornone, butyllithium and the organic solvent D is 10 to 20mmol: 15-30 mmol: 15-30 mmol: 10-20 mL, preferably 12-18 mmol: 18-26 mmol: 18-26 mmol:12 to 18mL, more preferably 14 to 16mmol: 20-25 mmol: 20-25 mmol: 14-16 mL; the HWE reaction temperature is-20-0 ℃, preferably-15-5 ℃, and more preferably-10 ℃; the time for the HWE reaction is 4 to 8 hours, preferably 5 to 7 hours, more preferably 6 hours.
In the invention, in the step S3, the molar volume ratio of the quinoline intermediate, the enal intermediate, the catalyst and the organic solvent E is 0.1-1 mmol:0.1 to 1mmol: 0.1-1 mL: 8-20 mL, preferably 0.3-0.7 mmol:0.3 to 0.8mmol: 0.2-0.8 mL:10 to 16mL, more preferably 0.4 to 0.5mmol:0.4 to 0.6mmol: 0.4-0.6 mL: 12-15 mL; the temperature of the Knoevenagel condensation reaction is 100-150 ℃, preferably 110-140 ℃, and further preferably 120-130 ℃; the Knoevenagel condensation reaction time is 5 to 10 hours, preferably 6 to 9 hours, more preferably 7 to 8 hours.
In the present invention, the catalyst is one or more of piperidine, triethylamine, morpholine and pyridine, preferably piperidine, triethylamine or morpholine, and more preferably piperidine.
In the present invention, the organic solvent a is toluene, o-dichlorobenzene or acetonitrile, preferably toluene or o-dichlorobenzene, and more preferably toluene; the organic solvent B is methanol or acetonitrile, preferably methanol; the organic solvent C is dichloromethane or 1, 4-dioxane, preferably dichloromethane; the organic solvent D is dichloromethane, tetrahydrofuran or diethyl ether, preferably dichloromethane or tetrahydrofuran, and more preferably dichloromethane; the organic solvent E is toluene or acetonitrile, preferably toluene.
The invention also provides application of the near infrared singlet oxygen luminescent probe in detection of singlet oxygen.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A25 mL single-necked flask was charged with 4-methylquinoline (1.0 g,7.0 mmol), ethyl iodide (3.3 g,21.0 mmol) and 5mL toluene, and the reaction was carried out at 110℃for 6 hours. After the reaction was completed, the mixture was cooled to room temperature, suction filtration was performed, and the cake was washed with toluene and dried to obtain 1.97g of 1-ethyl-4-methylquinoline salt as a yellow solid in 94% yield.
FIG. 1 is a hydrogen spectrum of 1-ethyl-4-methylquinoline salt: 1 HNMR(400MHz,CDCl 3 ,ppm):δ1.81(t,J＝7.6Hz,3H,NCH 2 CH 3 ),3.02(s,3H,pyridine-CH 3 ),5.38(q,J＝7.2Hz,2H,NCH 2 CH 3 ),7.98-8.03(m,2H,phenyl-H),8.19-8.24(m,1H,phenyl-H),8.36-8.41(m,2H,phenyl-H),10.31(d,J＝6.0Hz,1H,phenyl-H).
FIG. 2 is a carbon spectrum of 1-ethyl-4-methylquinoline salt: 13 C NMR(100MHz,CDCl 3 ,ppm):δ158.16,148.23,136.89,135.80,130.14,129.49,126.98,123.43,119.18,53.65,20.74,15.96.Mass spectrometry(ESI-MS,m/z):calcd.for C 12 H 14 N + ,172.1126；found,172.1122.
to a 25mL Schlenk tube was added dimethyl ((2-bromo-5- ((tert-butyldimethylsilyloxy) phenyl) (methoxy) methyl) phosphonate (1.0 g,2.28 mmol), 2-adamantanone (411 mg,2.74 mmol), butyllithium (1 mL,2.74 mmol) and 5mL tetrahydrofuran, and reacted at-20℃for 6h. After the completion of the reaction, 100mL of ethyl acetate was added, and the mixture was washed 3 times with 60mL of water, and the organic phase was dried over anhydrous sodium sulfate, filtered and dried by spin. Column chromatography separation, developing solvent is petroleum ether and ethyl acetate (ethyl acetate accounts for 10%), and the white solid 2- (adamantane-2-subunit (methoxy) methyl) -4-hydroxy benzaldehyde 380.7mg is obtained with a yield of 56%.
FIG. 3 is a hydrogen spectrum of 2- (adamantan-2-ylidene (methoxy) methyl) -4-hydroxybenzaldehyde: 1 H NMR(400MHz,CDCl 3 ,ppm):δ1.69-1.97(m,12H,adamantane-H),2.24(s,1H,adamantane-H),3.31(s,4H,-O-CH 3 ,adamantane-H),6.78(d,1H,J＝2.52Hz,phenyl-H),6.89(dd,1H,J 1 ＝2.48Hz,J 2 ＝8.52Hz,phenyl-H),7.93(d,1H,J＝8.52Hz,phenyl-H),10.14(s,1H,-CHO).
FIG. 4 is a carbon spectrum of 2- (adamantan-2-ylidene (methoxy) methyl) -4-hydroxybenzaldehyde: 13 C NMR(100MHz,CDCl 3 ,ppm):δ191.37,160.41,142.03,138.32,134.55,129.81,128.64,115.82,57.33,38.91,36.94,29.95,28.14.Mass spectrometry(ESI-MS,m/z):[M-H] - calcd.for C 19 H 21 O 3 - ,297.1491；found,297.1490.
in a 50mL one-necked flask, 1-ethyl-4-methylquinoline salt (100 mg,0.33 mmol), 2- (adamantan-2-ylidene (methoxy) methyl) -4-hydroxybenzaldehyde (90 mg,0.30 mmol), 0.5mL piperidine and 10mL toluene were charged, and the temperature was raised to 110℃for reaction for 6 hours. After the reaction, cooling to room temperature, suction filtering to obtain a filter cake, separating by column chromatography, wherein the developing agent is dichloromethane and methanol (the methanol accounts for 1%), and obtaining 89mg of red solid which is named QMI and has the yield of 51%.
FIG. 5 is a hydrogen spectrum of QMI: 1 H NMR(400MHz,DMSO-d 6 ,ppm):δ1.58(t,J＝7.2Hz,3H,NCH 2 CH 3 ),1.72-1.95(m,12H,adamantane-H),2.21(s,1H,adamantane-H),3.24(s,3H,-O-CH 3 ),3.30(s,1H,adamantane-H),4.98(q,J＝7.2Hz,2H,NCH 2 CH 3 ),6.73(d,J＝2.4Hz,1H,phenyl-H),6.94(dd,J 1 ＝2.4Hz,J 2 ＝8.4Hz,1H,phenyl-H),7.97(d,J＝16Hz,1H,alkene-H),8.02(t,J＝8Hz,1H,phenyl-H),8.08(d,J＝6.8Hz,1H,phenyl-H),8.16(d,J＝16Hz,1H,alkene-H),8.21-8.27(m,2H,phenyl-H),8.54(d,J＝8.8Hz,1H,phenyl-H),8.98(d,J＝8Hz,1H,phenyl-H),9.30(d,J＝6.8Hz,1H,phenyl-H),10.34(s,1H,-OH).
FIG. 6 is a carbon spectrum of QMI: 13 C NMR(100MHz,DMSO-d 6 ,ppm):δ159.70,153.00,147.29,140.49,137.99,137.65,135.10,130.67,129.64,129.06,126.69,126.55,125.62,119.09,117.48,117.30,116.16,115.38,56.46,52.09,38.49,38.44,36.38,32.19,29.14,27.57,15.01.Mass spectrometry(ESI-MS,m/z):[M-I] + calcd.for C 31 H 34 NO 2 + ,452.2590；found,452.2584.
example 2
Into a 25mL one-necked flask, 2, 3-trimethylindole (1.0 g,6.3 mmol), iodoethane (2.9 g,18.8 mmol) and 5mL toluene were charged, and the temperature was raised to 110℃for reaction for 6 hours. After the reaction was completed, the mixture was cooled to room temperature, suction filtration was performed, and the cake was washed with toluene and dried to obtain 1.79g of 1-ethyl-2, 3-trimethylindole salt as a pale pink solid, with a yield of 90%.
FIG. 7 is a hydrogen spectrum of 1-ethyl-2, 3-trimethylindole salt: 1 HNMR(400MHz,CDCl 3 ,ppm):δ1.63(t,J＝7.6Hz,3H,NCH 2 CH 3 ),1.67(s,6H,Indole-(CH 3 ) 2 ),3.17(s,3H,Indole-CH 3 ),4.79(q,J＝7.2Hz,2H,NCH 2 CH 3 ),7.58-7.61(m,3H,phenyl-H),7.70-7.72(m,1H,phenyl-H).
FIG. 8 is a carbon of a 1-ethyl-2, 3-trimethylindole saltSpectrogram of the graph: 13 C NMR(100MHz,CDCl 3 ,ppm):δ195.40,141.68,140.55,130.18,129.59,123.47,115.38,54.68,45.44,23.12,17.01,13.67.Mass spectrometry(ESI-MS,m/z):calcd.for C 13 H 18 N + ,188.1434；found,188.1430.
to a 50mL one-necked flask was added 1-ethyl-2, 3-trimethylindole salt (100 mg,0.32 mmol), 2- (adamantan-2-ylidene (methoxy) methyl) -4-hydroxybenzaldehyde (90 mg,0.30 mmol), 0.5mL piperidine and 10mL toluene, and the mixture was reacted at 110℃for 6 hours. After the reaction, cooling to room temperature, suction filtering to obtain a filter cake, separating by column chromatography, wherein the developing agent is methylene dichloride and methanol (the methanol accounts for 1 percent), and obtaining 112.5mg of red solid which is named as InCL, and the yield is 63 percent.
The preparation method of 2- (adamantan-2-ylidene (methoxy) methyl) -4-hydroxybenzaldehyde used in this example was the same as that of example 1.
Fig. 9 is a hydrogen spectrum of InCL: 1 H NMR(400MHz,DMSO-d 6 ,ppm):δ1.44(t,J＝7.2Hz,3H,NCH 2 CH 3 ),1.67(d,J＝12.8Hz,6H,Indole-(CH 3 ) 2 ),1.79-2.00(m,12H,adamantane-H),2.49(s,1H,adamantane-H),3.32(s,4H,-O-CH 3 ,adamantane-H),4.10(q,J＝7.2Hz,2H,NCH 2 CH 3 ),6.45(d,J＝14.4Hz,1H,alkene-H),6.49(d,J＝2Hz,1H,phenyl-H),6.72(d,J＝9.2Hz,1H,phenyl-H),7.04(d,J＝8Hz,1H,phenyl-H),7.20(t,J＝7.6Hz,1H,phenyl-H),7.32-7.38(m,2H,phenyl-H),7.89(d,J＝9.2Hz,1H,phenyl-H),8.19(d,J＝14.4Hz,1H,alkene-H).
fig. 10 is a carbon spectrum of incal: 13 C NMR(100MHz,DMSO-d 6 ,ppm):δ171.90,148.40,144.53,141.89,141.20,140.85,133.14,132.34,128.58,125.66,124.59,124.05,122.24,120.81,109.80,97.28,57.04,48.95,39.74,39.34,39.13,37.13,32.65,31.59,29.63,28.42,28.38,28.28,27.80,22.66,14.13,12.34.Mass spectrometry(ESI-MS,m/z):calcd for C 32 H 38 NO 2 + 468.2897; found 468.2891 characterization of Performance
1. Ultraviolet absorption spectrum and fluorescence spectrum of probe QMI:
the QMI prepared in example 1 was taken and dissolved in analytically pureDimethyl sulfoxide to 1.0X10 -3 mol/L mother liquor. 20. Mu.L of the above mother solution was added to PBS buffer to form 2mL of a mixed solution (DMSO 1%, QMI concentration 1.0X10) - 5 mol/L), and after being uniformly mixed, the mixture was transferred into an optical quartz cuvette (10X 10 mm) for testing the absorption spectrum and the fluorescence spectrum, and the result is shown in FIG. 11. As shown in fig. 11, in the mixed solvent with 99% water content, the maximum absorption of QMI is located at about 520nm; the maximum emission of QMI is at 650nm with 520nm as excitation wavelength.
2. Chemiluminescent imaging of QMI probes in 96-well plates:
the QMI prepared in example 1 was dissolved in analytically pure dimethyl sulfoxide to prepare 5.0X10 -3 mol/L mother liquor. Simultaneous preparation of 1.0X10 -2 PBS buffer solution of mol/L surfactant (hexadecyl tributyl phosphine bromide) and 1.0X10 -3 Aqueous solutions of mol/L methylene blue. Adding 2 μl of QMI mother liquor, 10 μl of PBS buffer solution of surfactant and 2 μl of methylene blue solution into PBS/DMSO mixed solution to obtain 200 μl total volume of mixed solution, wherein the concentration of QMI is 5.0X10 -5 mol/L, methylene blue concentration of 1.0X10 -5 mol/L. After mixing uniformly, a laser (690 nm,200 mW/cm) 2 ) The illumination was performed for 1 minute and chemiluminescent intensities were collected using an Imaging Quant4000 system. As shown in fig. 12, the left side is the image collected by the Imaging Quant4000 system, the right side is the quantification of the light intensity in the image, the abscissa in the bar graph is the intensity of chemiluminescence before and after illumination of the mixed solution. It can be seen that there is little chemiluminescent signal before illumination and that there is about 492-fold enhancement in the signal generated after illumination.
3. Contrast of signal of QMI probe with photosensitizer concentration versus commercial probe detection effect:
mu.L of QMI mother liquor (5.0X10) -3 mol/L), 10. Mu.L of surfactant solution (hexadecyl tributyl phosphine bromide, 1.0X10) -2 mol/L) and different volumes of methylene blue solution (1.0X10 -3 mol/L) was added to PBS solutions to prepare respective mixed solutions having a total volume of 200. Mu.L and a water content of 99%, and the concentration of QMI in the mixed solutions was determinedIs 5.0X10 -5 mol/L, methylene blue concentration of 0.5X10 -5 ～4×10 -5 mol/L. Uniformly mixing, uniformly using laser (690 nm,200mW/cm 2 ) Illumination is carried out for 1 minute, and chemiluminescence intensity is collected uniformly by using an Imaging Quant4000 system. As shown in FIG. 13, the intensity of the collected chemiluminescent signal correlated well with the concentration of methylene blue, indicating that QMI can quantitatively detect singlet oxygen produced by methylene blue. As shown in fig. 14, the signal-to-noise ratio of QMI is significantly higher than that of commercial probes ABDA and SOSG under the same methylene blue concentration and light conditions.
4. QMI compares to stability of conventional chemiluminescent probes:
the conventional probes DCM-OH-CF, QM-OH-CF and the probe QMI of the invention were prepared as 1mmol/L solutions (PBS: DMSO=1:1), oxygen was bubbled in, and the samples were taken every 3min with continuous irradiation from a commercial white light source (5W), and the decomposition amounts of the respective probes were tested by High Performance Liquid Chromatography (HPLC). As shown in FIG. 15, compared to the rapid decomposition of conventional probes DCM-OH-CF and QM-OH-CF (both described in "Angew. Chem. Int. Ed.2020,59, 2-10"), QMI of the present invention only decomposed about 12.7% after 21min of continuous oxygen bubbling and light irradiation, indicating that QMI has significantly superior stability compared to conventional chemiluminescent probes.
5. Chemiluminescence imaging of QMI probes in mouse tumors:
HeLa tumor-bearing nude mice were used as imaging subjects, and left mice were injected with QMI solution in situ (QMI concentration 1.0X10 -4 mol/L), right mice were injected in situ with a mixed solution of QMI and photosensitizer (QMI concentration 1.0X10) -4 mol/L, photosensitizer concentration 1.0X10 -5 mol/L), after injection, the two mice were subjected to 1min of light (690 nm,200 mW/cm) 2 ) And mice were subjected to whole-body chemiluminescent imaging using PerkinElmer IVIS Spectrum Imaging System. Prior to the imaging experiments, nude mice were anesthetized with a gas containing 2.5% isoflurane.
As shown in fig. 16, the left mice (no photosensitizer) did not collect chemiluminescent signals and the right mice (photosensitizer) exhibited significant chemiluminescent signals, approximately 90-fold different. The probe QMI was shown to be able to detect singlet oxygen produced by photosensitizers in vivo and to be applicable to assessing the effect of singlet oxygen production during PDT (all in vivo experiments in this invention were in compliance with the laboratory animal feeding and use regulations and were approved by the university of North university animal feeding and use Committee, laboratory tumor-bearing nude mice were purchased from Shanghai Meixuan Biotechnology Co., ltd., fed with high pressure steam treated foods and water in a sterile squirrel cage in a laminar flow hood in a sterile room).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The near-infrared singlet oxygen luminescence probe with high signal-to-noise ratio and high stability is characterized by having a structural formula shown in formula I:
wherein R is 1 The structural formula of (a) is any one of formulas II-IV;
R 2 the structural formula of (C) is formula V or formula VI;
the R is 3 、R 4 And R is 5 The structural formula of (a) is any one of formulas VII-IX independently;
2. the method for preparing the near infrared singlet oxygen luminescence probe according to claim 1, comprising the steps of:
s1, mixing 4-methylquinoline, ethyl iodide and an organic solvent A, and reacting to obtain a quinoline intermediate;
s2, mixing 2-bromo-5-hydroxybenzaldehyde, tetrabutylammonium tribromide, tert-butyldimethyl chlorosilane and an organic solvent B, and carrying out an acetal reaction to obtain an acetal intermediate; mixing an acetal intermediate, trimethoxy phosphorus and an organic solvent C, and performing a phospho esterification reaction to obtain a phosphate intermediate; mixing a phosphate intermediate, 2-norbornone, butyllithium and an organic solvent D, and performing HWE reaction to obtain an enal intermediate;
and S3, mixing the quinoline intermediate, the enal intermediate, the catalyst and the organic solvent E, and performing Knoevenagel condensation reaction to obtain the near-infrared singlet oxygen luminescent probe.
3. The preparation method according to claim 2, wherein in the step S1, the molar volume ratio of 4-methylquinoline, ethyl iodide and the organic solvent a is 5 to 10mmol: 20-30 mmol: 5-10 mL; the reaction temperature is 100-150 ℃ and the reaction time is 5-10 h.
4. The process according to claim 3, wherein in the step S2, the molar volume ratio of 2-bromo-5-hydroxybenzaldehyde, tetrabutylammonium tribromide, t-butyldimethylsilyl chloride and organic solvent B is 5 to 10mmol:0.5 to 1mmol: 10-20 mmol: 20-40 mL; the temperature of the acetalization reaction is 20-30 ℃, and the time of the acetalization reaction is 6-8 h.
5. The process according to claim 4, wherein in the step S2, the molar volume ratio of the acetal intermediate, trimethoxy phosphorus and the organic solvent C is 20 to 30mmol: 30-40 mmol: 10-20 mL; the temperature of the phosphating reaction is-5 ℃, and the time of the phosphating reaction is 8-12 h.
6. The process according to claim 3 or 5, wherein in step S2, the molar volume ratio of the phosphate intermediate, 2-norbornone, butyllithium and organic solvent D is 10 to 20mmol: 15-30 mmol: 15-30 mmol: 10-20 mL; the temperature of the HWE reaction is-20-0 ℃, and the time of the HWE reaction is 4-8 h.
7. The process according to claim 6, wherein in the step S3, the molar volume ratio of the quinoline intermediate, the enal intermediate, the catalyst and the organic solvent E is 0.1 to 1mmol:0.1 to 1mmol: 0.1-1 mL: 8-20 mL; the temperature of the Knoevenagel condensation reaction is 100-150 ℃, and the time of the Knoevenagel condensation reaction is 5-10 h.
8. The preparation method according to claim 2 or 7, wherein the catalyst is one or more of piperidine, triethylamine, morpholine and pyridine.
9. The preparation method according to claim 8, wherein the organic solvent a is toluene, o-dichlorobenzene or acetonitrile; the organic solvent B is methanol or acetonitrile; the organic solvent C is dichloromethane or 1, 4-dioxane; the organic solvent D is dichloromethane, tetrahydrofuran or diethyl ether; the organic solvent E is toluene or acetonitrile.
10. Use of the near infrared singlet oxygen luminescent probe of claim 1 for detecting singlet oxygen.
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