CN116903601A - Poly hydrogen sulfide near infrared chemiluminescence probe, preparation method and application thereof - Google Patents
Poly hydrogen sulfide near infrared chemiluminescence probe, preparation method and application thereof Download PDFInfo
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- CN116903601A CN116903601A CN202310662653.5A CN202310662653A CN116903601A CN 116903601 A CN116903601 A CN 116903601A CN 202310662653 A CN202310662653 A CN 202310662653A CN 116903601 A CN116903601 A CN 116903601A
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- hydrogen polysulfide
- near infrared
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- hydrogen
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- 239000000523 sample Substances 0.000 title claims abstract description 122
- 238000002360 preparation method Methods 0.000 title abstract description 11
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 title description 5
- 229910000037 hydrogen sulfide Inorganic materials 0.000 title description 5
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 68
- 239000001257 hydrogen Substances 0.000 claims abstract description 68
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 68
- 229920001021 polysulfide Polymers 0.000 claims abstract description 68
- 239000005077 polysulfide Substances 0.000 claims abstract description 68
- 150000008117 polysulfides Polymers 0.000 claims abstract description 67
- 238000001514 detection method Methods 0.000 claims abstract description 26
- 238000003384 imaging method Methods 0.000 claims abstract description 21
- 238000006243 chemical reaction Methods 0.000 claims description 20
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 12
- 150000001875 compounds Chemical class 0.000 claims description 12
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 8
- AYYGQBKLLWSKJB-UHFFFAOYSA-N 2-benzoylsulfanylbenzoic acid Chemical compound OC(=O)C1=CC=CC=C1SC(=O)C1=CC=CC=C1 AYYGQBKLLWSKJB-UHFFFAOYSA-N 0.000 claims description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 7
- 229910052760 oxygen Inorganic materials 0.000 claims description 7
- 239000001301 oxygen Substances 0.000 claims description 7
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 6
- JFOZKMSJYSPYLN-QHCPKHFHSA-N lifitegrast Chemical compound CS(=O)(=O)C1=CC=CC(C[C@H](NC(=O)C=2C(=C3CCN(CC3=CC=2Cl)C(=O)C=2C=C3OC=CC3=CC=2)Cl)C(O)=O)=C1 JFOZKMSJYSPYLN-QHCPKHFHSA-N 0.000 claims description 6
- 229960000907 methylthioninium chloride Drugs 0.000 claims description 6
- 238000006482 condensation reaction Methods 0.000 claims description 5
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- 238000006555 catalytic reaction Methods 0.000 claims description 3
- 238000004020 luminiscence type Methods 0.000 claims description 2
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims 2
- 241000699670 Mus sp. Species 0.000 abstract description 21
- 230000035945 sensitivity Effects 0.000 abstract description 6
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- 201000010099 disease Diseases 0.000 abstract 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 81
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- 239000011734 sodium Substances 0.000 description 50
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 26
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 11
- 239000011780 sodium chloride Substances 0.000 description 11
- 241001465754 Metazoa Species 0.000 description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 239000002504 physiological saline solution Substances 0.000 description 8
- 229910052708 sodium Inorganic materials 0.000 description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 6
- 229910002091 carbon monoxide Inorganic materials 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 5
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 5
- 239000007850 fluorescent dye Substances 0.000 description 5
- 241000894007 species Species 0.000 description 5
- DGYHPLMPMRKMPD-UHFFFAOYSA-N 2-azaniumylpent-4-ynoate Chemical compound OC(=O)C(N)CC#C DGYHPLMPMRKMPD-UHFFFAOYSA-N 0.000 description 4
- 230000003187 abdominal effect Effects 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 239000000377 silicon dioxide Substances 0.000 description 4
- -1 1, 2-dioxetane (1, 2-dioxetanone) Chemical compound 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
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- 230000035484 reaction time Effects 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- SLVIJGCNMLGLMH-UHFFFAOYSA-N 2h-pyran-2,3-dicarbonitrile Chemical group N#CC1OC=CC=C1C#N SLVIJGCNMLGLMH-UHFFFAOYSA-N 0.000 description 2
- BVTJGGGYKAMDBN-UHFFFAOYSA-N Dioxetane Chemical compound C1COO1 BVTJGGGYKAMDBN-UHFFFAOYSA-N 0.000 description 2
- 108010053070 Glutathione Disulfide Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 2
- 238000011503 in vivo imaging Methods 0.000 description 2
- 229920006008 lipopolysaccharide Polymers 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- SEEYREPSKCQBBF-UHFFFAOYSA-N n-methylmaleimide Chemical compound CN1C(=O)C=CC1=O SEEYREPSKCQBBF-UHFFFAOYSA-N 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- HYHCSLBZRBJJCH-UHFFFAOYSA-N sodium polysulfide Chemical compound [Na+].S HYHCSLBZRBJJCH-UHFFFAOYSA-N 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
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- 231100000419 toxicity Toxicity 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 125000000972 4,5-dimethylthiazol-2-yl group Chemical group [H]C([H])([H])C1=C(N=C(*)S1)C([H])([H])[H] 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- XUJNEKJLAYXESH-UWTATZPHSA-N D-Cysteine Chemical compound SC[C@@H](N)C(O)=O XUJNEKJLAYXESH-UWTATZPHSA-N 0.000 description 1
- 101100075747 Drosophila melanogaster Lztr1 gene Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
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- 239000013543 active substance Substances 0.000 description 1
- RPCBRIVIADRRGK-UHFFFAOYSA-N adamantane;dioxetane Chemical class C1COO1.C1C(C2)CC3CC1CC2C3 RPCBRIVIADRRGK-UHFFFAOYSA-N 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
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- 102000005936 beta-Galactosidase Human genes 0.000 description 1
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- 238000005415 bioluminescence Methods 0.000 description 1
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- 229940125904 compound 1 Drugs 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
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- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
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- 229940097275 indigo Drugs 0.000 description 1
- COHYTHOBJLSHDF-UHFFFAOYSA-N indigo powder Natural products N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 description 1
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- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
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- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
- C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
- C07D407/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a carbon chain containing aromatic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- 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"
-
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention relates to a near infrared chemiluminescence probe of hydrogen polysulfide, a preparation method and application thereof, wherein the structural formula of the chemiluminescence probe CL-SN is shown in a formula (I). The near-infrared chemiluminescent probe CL-SN has the advantages of high sensitivity, low detection limit, high signal-to-noise ratio, high selectivity, noninvasive real-time imaging and the like; the probe CL-SN realizes chemiluminescence imaging of the hydrogen polysulfide in H9c2 cells, can be further applied to the living body level of mice, and realizes detection of the hydrogen polysulfide in the living body level. The probe can be used for detecting the hydrogen polysulfide in a complex environment in a living body, and provides an effective detection tool for the change of the hydrogen polysulfide level in diseases.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a hydrogen polysulfide near-infrared chemiluminescent probe, a preparation method and application thereof.
Background
Hydrogen sulfide (H) 2 S) is a third gas signaling molecule following Nitric Oxide (NO) and carbon monoxide (CO). H 2 S changes the structure and function of target protein mainly through sulfhydrylation modification of cysteine residue, and influences the activity of key receptor and signal path so as to play a physiological role. Polyhydrosulfide (H) 2 S n N.gtoreq.2) is H 2 Oxidation products of S. At H 2 In the S-related physiological process, the hydrogen polysulfide plays an important regulatory role. The hydrogen polysulfide can promote signal transduction between cells, is an important redox signal, and has the effects of cytoprotection, anti-inflammation, anticancer, antioxidation and the like.
The detection method of the hydrogen polysulfide mainly comprises the following steps: ultraviolet/visible light absorption spectrometry, mass spectrometry, chromatography, fluorescent probe methods. However, the first three methods require pretreatment of cells and tissues. The method has the defects of long detection time, high cost, complex operation and the like, and cannot detect the hydrogen polysulfide in real time and in situ. In order to explore the mechanism of action of hydrogen polysulfide in organisms, accurately detect the hydrogen polysulfide level, it is required to establish an effective real-time, dynamic and visual accurate detection technique.
The fluorescent probe has the advantages of noninvasive real-time monitoring, high sensitivity and selectivity, good biocompatibility and the like. Thus, many studies for detecting hydrogen polysulfide based on a fluorescent probe method have been reported. Although some prior hydrogen polysulfide fluorescent probes have advanced, noninvasive in-vivo imaging using these fluorescent probes is still affected by the light penetration depth of fluorescence of biological tissues under external light excitation, photobleaching and high tissue autofluorescence interference.
Unlike conventional fluorescence imaging, chemiluminescence (chemiluminesce) is a phenomenon in which light is generated by chemical excitation in a chemical reaction. Because no real-time excitation of an external light source is needed, chemiluminescence has little autofluorescence interference, and can realize higher signal-to-noise ratio and improve the sensitivity of in-vivo imaging. 1, 2-dioxetane (1, 2-dioxetanone) is the most common chemiluminescent substrate, and the hydroxyl groups in its structure facilitate structural modification, thereby developing a variety of chemiluminescent probes. A series of chemiluminescent probes have been successfully used to detect biologically active substances such as beta-galactosidase, hydrogen peroxide, and cysteine.
Near-infrared (NIR) imaging technology is a major trend in research in the field of detection of small bioactive molecules. However, the emission wavelength of the 1, 2-dioxetane is generally below 600nm, and the generated photons are easily absorbed by tissues, so that the detected optical signals are weakened. Dicyanopyran substituted 1, 2-dioxetane derivatives developed by Shbat et al have an emission that can red shift to 700nm. Therefore, the near infrared chemiluminescent probe is used for detecting the hydrogen polysulfide, which is beneficial to imaging deep tissues and further improves the detection sensitivity of the deep tissues.
Disclosure of Invention
The invention aims to provide a near infrared chemiluminescence probe for hydrogen polysulfide, and a preparation method and application thereof based on the prior art.
The aim of the invention can be achieved by the following measures:
a near infrared chemiluminescence probe of hydrogen polysulfide has a chemical structure shown in a formula (I),
a preparation method of a near infrared luminescent probe of hydrogen polysulfide comprises the following synthetic routes:
in the present invention, the reaction is carried out using 1g of the compound as a starting material, wherein the preparation method of 1g of the compound can be referred to as CN115417863A.
In the preparation method, the specific reaction steps comprise: the compound 1g and 2- (benzoylthio) benzoic acid are subjected to condensation reaction, and then the reaction product is catalyzed by methylene blue under the condition of introducing oxygen to obtain the compound of the formula (I).
In a preferred embodiment, 1g of the compound is dissolved in a solvent together with 2- (benzoylthio) benzoic acid, EDC and DMAP are then added, and the mixture is stirred at 20-30 ℃ for condensation reaction for 0.2-3h; then under the condition of introducing oxygen and under the irradiation of yellow light, the reaction is catalyzed by methylene blue, and the compound shown in the formula (I) is obtained. The reaction temperature in the condensation reaction is room temperature, the reaction time is preferably 1h, and the reaction solvent is preferably dichloromethane. In the catalytic reaction, the reaction temperature is 20-30 ℃, preferably 25 ℃, and the reaction time is 0.2-3h, preferably 1h.
In a preferred embodiment, the molar ratio of compound 1g to 2- (benzoylthio) benzoic acid is 1:1 to 2, preferably 1:1.2 to 1.8, more preferably 1:1.5.
in another preferred embodiment, the molar ratio of DMAP to EDC is 1:2 to 4, preferably 1:2.8 to 3.2, more preferably 1:3.0.
in another preferred embodiment, the molar ratio of compound 1g to EDC is 1:0.5 to 2.5, preferably 1:1 to 2, more preferably 1:1.5.
in a preferred process for the preparation of a hydrogen polysulfide near infrared chemiluminescent probe, 1g is condensed with 2- (benzoylthio) benzoic acid, then EDC and DMAP are added in the presence of oxygen to react, and then the compound of formula (I) is obtained by methylene blue catalysis.
The near infrared chemiluminescence probe of the invention can be applied to chemiluminescence imaging for detecting the hydrogen polysulfide in cells or mice.
The near infrared chemiluminescence probe of the invention can be applied to the detection of endogenous hydrogen polysulfide in cells or mice.
The near infrared chemiluminescence probe of the invention can be applied to the aspect of chemiluminescence imaging detection of endogenous hydrogen polysulfide in cells or mice.
The near infrared chemiluminescence probe for the hydrogen polysulfide can be applied to quantitative detection of the hydrogen polysulfide.
The near infrared chemiluminescence probe of the invention can be applied to the aspect of detecting the physiological and pathological mechanism or signal transduction path of the hydrogen polysulfide in an organism.
The invention relates to a near infrared chemiluminescence probe of hydrogen polysulfide, which has a chemical structure shown in a formula (I), and is also called a probe CL-SN.
The invention has the beneficial effects that:
the invention designs and synthesizes a near infrared chemiluminescent probe CL-SN for identifying hydrogen polysulfide by taking dicyanopyran substituted adamantane-dioxetane as a near infrared chemiluminescent substrate and 2- (benzoyl mercapto) benzoate as an identification group. Probes CL-SN and Na 2 S 4 The reaction products of (2) are characterized by high resolution mass spectrometry to determine the probe reaction mechanism. The probe has good selectivity and high sensitivity (detection limit 39 nM). The probe CL-SN also realizes the chemiluminescent imaging detection of H9c2 cell-level hydrogen polysulfide. More importantly, the probe CL-SN realizes chemiluminescent imaging detection of the living body level hydrogen polysulfide. The method can provide a visual detection method for the physiological and pathological mechanism of the hydrogen sulfide in the organism and the signal transduction path, and has important significance for revealing the physiological and pathological mechanism of the hydrogen sulfide in the human body.
Drawings
FIG. 1 shows probes CL-SN and Na 2 S 4 Chemiluminescent kinetics measurements of the reaction;
in the figure, the upper line is CL-SN, and the lower line is CL-SN+Na 2 S 4 。
FIG. 2 shows the probe CL-SN and different concentrations of Na 2 S 4 A chemiluminescent response result map of (2);
the probe CL-SN and Na at different concentrations are shown 2 S 4 (0-500. Mu.M) in PBS buffer (20 mM, pH= 7.4,10% DMSO,10% FBS) at 37 ℃Chemiluminescent response after 1h incubation. Insert diagram: probe CL-SN chemiluminescence intensity and Na 2 S 4 Linear relationship of concentrations (0, 1,2, 4, 6, 8, 12, 14, 16, 18, 20 μm).
FIG. 3 is the selectivity of probe CL-SN to hydrogen polysulfide;
in the figure, probe CL-SN (10 μm) was incubated in PBS buffer (20 mm, ph= 7.4,10% DMSO,10% FBS) for 1h at 37 ℃, chemiluminescent intensity for each thiol, 1.blank; gsh (1 mM); gsh (10 mM); gssg (1 mM); 5.CH 3 SSSCH 3 (1mM)；6.Hcy(100μM)；7.S 2 O 3 2- (1mM)；8.SO 4 2- (1mM)；9.S 2 O 4 2- (1mM)；10.HSO 3 - (1mM)；11.Cys(1mM)；12.L-Cys(1mM)；13.D-Cys(1mM)；14.Na 2 S(100μM)；15.S 8 (1mM)；16.Na 2 S 4 (1mM))。
FIG. 4 is a sample of probe CL-SN vs. Na 2 S 4 Selectivity of (2);
it was incubated in PBS buffer (20 mm, ph= 7.4,10% DMSO,10% FBS) at 37 ℃ for 1h, the chemiluminescent intensity of probe CL-SN (10 μm) on active oxygen and active nitrogen, 1.blank;2.H 2 O 2 (100μM)；3.ClO - (100μM)；4. t BuOOH(100μM)；5.·OH(100μM)；6. 1 O 2 (100μM)；7.O 2- (100μM)；8.NO 3 2- (100μM)；9.NO 2- (100μM)；10.ONOO - (100μM)；11.NO(100μM)；12.Na 2 S 4 (500μM))。
FIG. 5 shows the probe CL-SN vs. Na 2 S 4 Selectivity of (2);
it was incubated in PBS buffer (20 mM, pH= 7.4,10% DMSO,10% FBS) at 37℃for 1h, and the chemiluminescent intensity of probe CL-SN (10. Mu.M) on inorganic salt ions was 1.blank; zn (Zn) 2+ (1mM)；3.Na + (1mM)；4.K + (1mM)；5.Mg 2+ (1mM)；6.Cu 2+ (1mM)；7.Ca 2+ (1mM)；8.Fe 3+ (1mM)；9.Fe 2+ (1mM)；10.Al 3+ (1mM)；11.Cl - (1mM)；12.I - (1mM)；13.F - (1mM)；14.HPO 4 2- (1mM)；15.H 2 PO 4 - (1mM)；16.CO 3 2- (1mM)；17.HCO 3 - (1mM)；18.Na 2 S 4 (500μM))。
FIG. 6 is a sample of probe CL-SN vs. Na 2 S 4 Selectivity of (2);
it was incubated in PBS buffer (20 mM, pH= 7.4,10% DMSO,10% FBS) at 37℃for 1h, probe CL-SN (10. Mu.M) was 1.blank for chemiluminescent intensity of amino acids; glu (1 mM); trp (1 mM); tyr (1 mM); ser (1 mM); arg (1 mM); phe (1 mM); his (1 mM); ala (1 mM); val (1 mM); pro (1 mM); gly (1 mM); na. Na 2 S 4 (500μM))。
FIG. 7 pH vs. probes CL-SN and Na 2 S 4 Influence of the reaction;
it was incubated at 37℃for 7h, probe CL-SN (10. Mu.M) and Na 2 S 4 (500. Mu.M)) in different pH buffers (20 mM, pH 4.0, 4.5, 5.0, 6.0, 6.5, 7.0, 7.4, 7.5, 8.0, 8.5 and 9.0,10% DMSO,10% FBS). The upper line in the figure is CL-SN+Na 2 S 4 The lower line is CL-SN.
FIG. 8 is a stability test result of the probe CL-SN;
it was incubated at 37℃for 1h and probe CL-SN (10. Mu.M) was chemiluminescent at 37℃in PBS buffer (20 mM, pH=7.4, 10% DMSO,10% FBS). The upper dot in the figure shows CL-SN in PBS buffer, and the lower dot shows PBS buffer.
FIG. 9 is a graph showing the effect of probe CL-SN on H9c2 cell viability;
in the diagram a: survival of H9c2 cells incubated with CL-SN (0, 5,10,20, 50. Mu.M) for 24H cells; in the diagram B: viability of H9c2 cells incubated with CL-SN (10. Mu.M) for various times (0, 6,12,18, 24H).
FIG. 10 is a cytoluminescent image of H9c2 cell exogenous hydrogen polysulfide;
in the figure, a is: cells were incubated with CL-SN (10. Mu.M) for 30min at 37 ℃. In the figure, B is: cells were incubated with CL-SN (10. Mu.M) for 30min at 37℃and then with Na 2 S 4 (25. Mu.M) incubating for 10min. In the figure, C is: cells were incubated with CL-SN (10. Mu.M) for 30min at 37℃and then with Na 2 S 4 (50. Mu.M) for 10min. In the figure, D is: cells were pretreated with NMM (1 mmol) for 1h, then incubated with CL-SN (10. Mu.M) for 30min, and then with Na 2 S 4 (50. Mu.M) for 10min. In the figure, E is: the total photon flux of chemiluminescence was quantified for each set of cell areas. Data are expressed as mean ± SD (n=3). *** Comparison with group a P<0.001。 ### Comparison with group C P<0.001。
FIG. 11 is a cytochemiluminescent imaging of H9c2 cells endogenous to the hydrogen polysulfide;
in the figure, a is: cells were incubated with CL-SN (10. Mu.M) for 30min at 37 ℃; in the figure, B is: cells were incubated with LPS (1. Mu.g/mL) at 37℃for 16h, then with CL-SN (10. Mu.M) for 30min; in panel C, cells were incubated with DL-propylglycine (PAG, 200. Mu.M) at 37℃for 30min, followed by addition of LPS (1. Mu.g/mL) for 16h and then CL-SN (10. Mu.M) for 30min; in the figure, D is: quantitatively measuring the total photon flux of chemiluminescence of each group of cell areas; *** comparison with group a P<0.001。 ### Comparison with group C P<0.001。
FIG. 12 is a sample of the detection of in vivo horizontal hydrogen polysulfide by probe CL-SN;
in the figure, a is: mice were injected intraperitoneally with probe CL-SN (100 μΜ,100 μl, DMSO: physiological saline = 1:9); in the figure, B is: abdominal injection of Na into mice 2 S 4 (2 mM, 100. Mu.L of physiological saline) for 10min, and then intraperitoneally injecting probe CL-SN (100. Mu.M, 100. Mu.L, DMSO: physiological saline=1:9); in the figure, C is: abdominal injection of Na into mice 2 S 4 (4 mm,100 μl of physiological saline) for 10min, and then intraperitoneally injecting probe CL-SN (100 μΜ,100 μl, DMSO: physiological saline=1:9); in the figure, D is: mice were intraperitoneally injected with LPS (10 μg/mL,100 μl of saline) for 24h, followed by CL-SN (100 μΜ,100 μl, DMSO: saline=1:9); in the figure, E is: mice were pre-treated with DL-propylglycine (2 mm,100 μl of physiological saline) for 30min and then were intraperitoneally injected with LPS (10 μg/mL,100 μl of physiological saline) for 24h, and CL-SN (100 μl, DMSO: physiological saline=1:9) was intraperitoneally injected; f in the figure is: the total photon flux of the abdominal chemiluminescence of each group of mice was quantitatively determined. Data as mean ± SD (n=3); *** comparison with group a P<0.001。 ### Comparison with group D P<0.001。
FIG. 13 is a probe CL-SN+Na 2 S 4 HRMS of the reaction product; wherein, (M-H) - :403.0486,found,403.0486。
Fig. 14 is probe CL-SN: 1 H NMR(400MHz,CDCl 3 )。
fig. 15 is probe CL-SN: 13 C NMR(100MHz,CDCl 3 )。
FIG. 16 is HRMS of probe CL-SN; wherein (M+Na) - C 46 H 35 ClN 2 O 7 S,817.1746；found817.1702。
Detailed Description
The invention will be better understood from the following examples. However, it will be readily appreciated by those skilled in the art that the description of the embodiments is provided for illustration only and should not limit the invention as described in detail in the claims.
1.1 preparation of solution:
(1) Preparing a probe CL-SN (namely a compound of a formula (I)) solution; under the dark environment, the probe CL-SN is dissolved in DMSO to prepare a 1mM probe solution.
(2)Na 2 S 4 Preparation of solutions (as a source of hydrogen polysulfide): na (Na) 2 S 4 A solution of 20mM sodium polysulfide was prepared in PBS buffer (20 mM, pH=7.4, 10% DMSO).
(3) Solutions of other biological analytes, including active sulfur species (RSS, including HSO 3 - ；SO 3 2- ；S 2 O 3 2- ；SO 4 2- ；S 2 O 4 2- ；GSSG；S 8 ；CH 3 SSSCH 3 The method comprises the steps of carrying out a first treatment on the surface of the Cys; hcy; GSH), reactive oxygen species (ROS, including H 2 O 2 ；ClO - ；O 2 - ；·OH； t BuOOH； 1 O 2 ) Reactive nitrogen (RNS, including NO; NO (NO) 2 - ；ONOO - ；NO 3 - ) Inorganic salt ion (K) + ；Ca 2+ ；Na + ；Mg 2+ ；Fe 3+ ；Zn 2+ ；Cu 2+ ；CO 3 2- ；HCO 3 - ；Cl - ；I - ；HPO 4 2- ；H 2 PO 4 - ) Amino acids (Glu; trp; tyr; ser; arg; phe; his; ala; met; leu; val; pro; gly), and the like.
1.2 cells:
species and strain: rat cardiomyocyte H9c2 cell line. The source is as follows: cell bank of China academy of sciences.
1.3 animals:
species and strain; healthy male Kunming mice, weighing 20-25g. The source is as follows: xuzhou university of medical science laboratory animal center.
EXAMPLE 1 Synthesis of Compound of formula (I) (i.e., compound 1 CL-SN)
Compound 1g (52.2 mg,0.1 mmol), 2- (benzoylthio) benzoic acid (39 mg,0.15 mmol) was dissolved in 10mL of dichloromethane, EDC (29 mg,0.15 mmol) and DMAP (6.1 mg,0.05 mmol) were then added and the reaction stirred at room temperature for 1h. After completion of the reaction, the reaction mixture was washed with distilled water (100 mL) and extracted with methylene chloride (3X 100 mL). The organic layer was treated with anhydrous Na 2 SO 4 Drying, concentrating under reduced pressure to remove the solvent. The crude product was purified by column chromatography on silica (silica, PE: EA,20:1 v/v) to give 50mg of a yellow solid. The yellow solid was dissolved in 20mL of methylene chloride, methylene blue (3 mg,0.1 mmol) was added thereto, oxygen was introduced, and the reaction was performed for 1 hour under reduced pressure to remove the solvent. The crude product was purified by column chromatography on silica gel (silica, PE: EA,17:3 v/v) to give 90mg of a white solid. The yield thereof was found to be 30%.
TLC(silica,PE:EA,17:3v/v)：Rf＝0.45。 1 H NMR(400MHz,CDCl 3 ):δ8.87-8.80(m,
1H),8.50-8.31(m,2H),8.16-8.11(m,1H),7.88-7.58(m,8H),7.49-7.37(m,2H),7.24-7.20(m,2H),6.98-6.80(m,2H),3.27-3.24(m,3H),3.03(s,1H),2.27-2.24(m,1H),2.07-1.63(m,12H). 13 C NMR(100MHz,CDCl 3 ):δ164.01,161.85,156.15,153.36,152.99,152.55,152.14,135.26,134.93,134.71,134.63,131.26,130.95,130.72,130.60,129.10,128.16,127.44,126.24,126.14,125.87,124.93,123.18,118.75,118.66,117.72,117.62,116.69,116.43,115.57,115.29,111.70,108.32,105.57,96.56,62.36,49.94,36.65,33.99,33.73,32.31,31.65,29.78,26.24,25.87,20.58.HRMS(ESI + ):(M+Na) - C 46 H 35 ClN 2 O 7 S,817.1746；found 817.1702.
EXAMPLE 2 detection Performance study of chemiluminescent Probe CL-SN on Hydrogen polysulfide
Under a dark environment, the probe CL-SN is dissolved in DMSO to prepare a mother solution concentration (1 mM). Na (Na) 2 S 4 (as a source of hydrogen polysulfide) in PBS buffer (20 mM, pH= 7.4,10% DMSO) to make up a 20mM sodium polysulfide solution. Probe CL-SN (final concentration 10. Mu.M) and Na 2 S 4 (final concentration 500. Mu.M) chemiluminescent signal was detected in PBS (10 mM,10% FBS, pH 7.4) at 37 ℃. Each set of data was run at least three times in parallel and the results were expressed as mean±sd.
Test conditions: chemiluminescent in vitro experiments were performed using a Varioskan LUX multi-function microplate reader for chemiluminescent kinetics measurements. Observation Probe CL-SN (10. Mu.M) and CL-SN (10. Mu.M) +Na 2 S 4 (500. Mu.M) chemiluminescent response was measured over several hours. As can be seen from fig. 1, as the reaction time increases, the chemiluminescent intensity increases and decreases, and at 15min, the chemiluminescent intensity peaks; around 2h, there is still a strong chemiluminescent signal.
Example 3 chemiluminescent probe CL-SN detection limit determination of Hydrogen polysulfide
The calculation method comprises the following steps: the standard deviation of the blank, i.e., the chemiluminescent intensity of the detection probe CL-SN in the HEPES buffer system, was measured and calculated after 10 repetitions. Then the probe CL-SN and Na are measured 2 S 4 Linear equation of reaction in the range of 0-20. Mu.M. The calculation formula of the detection limit is as follows: 3 sigma/k. k represents probe and Na 2 S 4 Slope of the linear equation of the reaction, σ, represents standard deviation of the blank.
As a result, as shown in FIG. 2, probe CL-SN was reacted with Na at various concentrations 2 S 4 After incubation (0-500. Mu.M), the chemiluminescent intensity follows Na 2 S 4 The increase in concentration was gradually increased (28-fold). And the chemiluminescent intensity shows good linear relation in the range of 0-20 mu M, and the detection limit of the probe CL-SN is 39nM. Experimental results show that the probe CL-SN has good detection sensitivity, and can quantitatively detect the hydrogen polysulfide.
Solutions of other biological analytes were also tested for selectivity of the probe for hydrogen polysulfide.
As shown in FIGS. 3 to 6, the probes CL-SN and Na 2 S 4 Incubation produces a distinct chemiluminescent signal, but with other active sulfur species (RSS, including HSO 3 - ；SO 3 2- ；S 2 O 3 2- ；SO 4 2- ；S 2 O 4 2- ；GSSG；S 8 ；CH 3 SSSCH 3 The method comprises the steps of carrying out a first treatment on the surface of the Cys; hcy; GSH), reactive oxygen species (ROS, including H 2 O 2 ；ClO - ；O 2 - ；·OH； t BuOOH； 1 O 2 ) Reactive nitrogen (RNS, including NO; NO (NO) 2 - ；ONOO - ；NO 3 - ) Inorganic salt ion (K) + ；Ca 2+ ；Na + ；Mg 2+ ；Fe 3+ ；Zn 2+ ；Cu 2+ ；CO 3 2- ；HCO 3 - ；Cl - ；I - ；HPO 4 2- ；H 2 PO 4 - ) Amino acids (Glu; trp; tyr; ser; arg; phe; his; ala; met; leu; val; pro; gly) will hardly generate chemiluminescent signals. Therefore, the probe CL-SN has the advantage of good selectivity to hydrogen polysulfide.
Example 4 influence of pH on the reaction of chemiluminescent Probe CL-SN with Hydrogen polysulfide and stability experiment of Probe CL-SN
(1) Effect of PH on the reaction of chemiluminescent Probe CL-SN with Hydrogen polysulfide
To not be provided withDetection of Na with pH vs. Probe CL-SN 2 S 4 Is to probe CL-SN and Na 2 S 4 Incubation was performed at different pH conditions. As shown in FIG. 7, the probe is relatively stable under different pH conditions and does not decompose. At Na (Na) 2 S 4 In the presence of the solution, when the pH value of the solution is 4.0 to 8.0, the concentration of phenoxy anions in the solution system is increased, and the chemiluminescence intensity is gradually enhanced. With the enhancement of the alkalinity of the solution, the enhancement of the protonation effect of the phenoxy anions gradually reduces the chemiluminescence intensity [8 2]. The greater intensity of chemiluminescence in the pH range of 7.0 to 9.0 suggests that probe CL-SN can sensitively detect hydrogen polysulfide under physiological conditions (ph=7.4).
(2) Stability test of Probe CL-SN
Probes were incubated in PBS (10 mM,10% FBS, pH 7.4) buffer for 7h at 37 ℃. As shown in the results of FIG. 8, although probe CL-SN produces a weak chemiluminescent signal in the buffer, it does not affect the detection of hydrogen polysulfide. The trace amount of probe CL-SN is decomposed because FBS in the system has esterase activity and reacts with the ester bond of the probe, thereby causing a weak response signal. Therefore, the probe can be used for detecting the hydrogen polysulfide sensitively even though being partially decomposed in a PBS buffer solution system containing 10% FBS, and has better stability.
Example 5 cell level luminescence imaging experiments
(1) Cell culture
HT-22 cells, H9c2 cells were inoculated in DMEM high-sugar medium containing 10% fetal bovine serum and 100. Mu.g/mL penicillin-streptomycin, and the cells were placed at 37℃with 5% CO 2 Culturing in an incubator. When the cells grow to the logarithmic phase, the cells are passaged and inoculated in a black 96-well plate, and after the cells are grown by adherence, chemiluminescence imaging experiments at the cell level can be performed.
(2) Cytotoxic culture
The cytotoxicity of probe CL-SN was detected using the MTT method. HT-22 cells and H9c2 cells are inoculated on a 96-well cell culture plate with the density of 3-5 multiplied by 10 4 And/or holes. After cell adhesion, the cell is adhered with probe CL-SN with different concentrations0,5,10,20,50. Mu.M) was incubated for 24h, then 20. Mu.L of MTT dye (3- [4, 5-dimethylthiazol-2-yl) was added to each well]-2, 5-diphenyltetrazolium bromide, 5mg/mL in PBS buffer), after 4h incubation the medium was removed, and 150 μl DMSO was added to dissolve formazan crystals, shaking up and absorbance at 570nm was measured using an microplate reader. Incubation was performed with no added probe as control wells and with medium added MTT as blank wells, at least three determinations for each sample. The calculation formula is as follows: cell relative viability = (experimental well OD value-blank well OD value)/(control well OD value-blank well OD value) ×100%. After further incubation with the probe CL-SN (10. Mu.M) for various times (0, 6,12,18,24 h) the procedure was repeated.
As shown in FIG. 10-A, when the concentration of probe CL-SN was less than 10. Mu.M, the cell viability was 90% or more, indicating that the probe CL-SN was very low in toxicity, and at a concentration of 10. Mu.M or less, there was no effect on the cell activity.
As shown in FIG. 10-B, the cell viability of the probe CL-SN (10. Mu.M) incubated with the cells for 0h,6h,12h,18h,24h was further examined, and the probe CL-SN incubated with the cells for 0-24h at a concentration of 10. Mu.M did not cause toxicity to the cells, and the viability reached more than 90%.
Example 6, cell level chemiluminescent imaging experiments
(1) Exogenous hydrogen polysulfide cell chemiluminescence imaging
The cells were divided into the following groups: (1) Probe CL-SN (final concentration 10. Mu.M, in 1. Mu.L DMSO) was incubated with H9c2 cells for 30min. (2) After incubation of probe CL-SN (final concentration 10. Mu.M, in 1. Mu.L DMSO) with H9c2 cells for 30min, different concentrations of Na were added 2 S 4 (25, 50. Mu.M, in 10. Mu.L of sample) for 10min. (3) H9c2 cells were pre-incubated with N-methylmaleimide (NMM, 1mM, in 10. Mu.L of saline) for 1H, then probe CL-SN (final concentration 10. Mu.M, in 1. Mu.L of DMSO) was added for 30min, and Na was added 2 S 4 (50. Mu.M, in 10. Mu.L of sample) for 10min. Prior to imaging, the images were gently washed three times with phosphate buffer.
The experimental results are shown in FIG. 10, in which almost no chemiluminescent signal was observed after incubation of probe CL-SN with H9c2 cells for 30min (FIG. 10-A). After incubation of H9c2 cells with probe CL-SN for 30min, different concentrations were addedNa of (2) 2 S 4 After incubation (25. Mu.M, 50. Mu.M), the chemiluminescent intensity was significantly enhanced; and with addition of Na 2 S 4 The increase in concentration was further enhanced (675-fold, 1923-fold; FIG. 10-B; FIG. 10-C). After 1h pretreatment of cells with N-methylmaleimide (NMM as a hydrogen polysulfide scavenger), the cells were treated with probes CL-SN and Na 2 S 4 Incubation was followed with a significant decrease in chemiluminescent intensity (fig. 10D). The chemiluminescence in the figure is caused by exogenous hydrogen polysulfide, and the probe CL-SN can detect H9c2 cell exogenous hydrogen polysulfide.
(2) Cell chemiluminescence imaging of endogenous hydrogen polysulfide
The cells were divided into the following groups: (1) Probe CL-SN (final concentration 10. Mu.M, in 1. Mu.L DMSO) was incubated with H9c2 cells for 30min. (2) After incubation of H9c2 cells with lipopolysaccharide (LPS, 1. Mu.g/mL, in 10. Mu.L of sample) for 16H, probe CL-SN (final concentration 10. Mu.M, in 1. Mu.L of DMSO) was added and incubated for 30min. (3) H9c2 cells were pre-incubated with DL-propargylglycine (PAG, 200. Mu.M, in 10. Mu.L of saline) for 30min, then lipopolysaccharide (LPS, 1. Mu.g/mL, in 10. Mu.L of saline) for 16H, and then incubated with probe CL-SN (final concentration 10. Mu.M, 1. Mu.L of DMSO) for 30min. Prior to imaging, the images were gently washed three times with phosphate buffer.
No chemiluminescent signal was observed after incubation of probe CL-SN with the cells (fig. 11-a). Cells incubated with LPS for 16h and then with probe CL-SN showed a significant chemiluminescent signal (2340-fold; FIG. 11-B). Subsequently, cells were pretreated with DL-propargylglycine (PAG, CSE enzyme inhibitor) and incubated with LPS, probes in sequence, with a significant decrease in chemiluminescent intensity (fig. 11-C). The result shows that the probe CL-SN can carry out chemiluminescence imaging detection on endogenous hydrogen polysulfide in cells.
(3) In vivo horizontal polysulfide hydrogen chemiluminescence imaging
3.1 animal feeding
Adult male mice of the healthy Kunming species, weighing 20-25g, were supplied by the laboratory animal center of the Xuzhou medical university (animal license number: SYXK (Su) 2007-0037). The animal protocol is approved by the animal protection and use committee of Xuzhou medical university, and the animal experiment is conducted in accordance with the rule of using experimental animals by Chinese law. Animals should be familiar with the environment a week in advance before the experiment, and are put into cage group culture under the natural circadian rhythm illumination condition, the temperature is 22+/-2 ℃, the humidity is 50+/-10%, and the animals can eat drinking water freely.
3.2 in vivo horizontal chemiluminescent imaging
Chemiluminescent imaging detection was performed using an LB983 NightOWL II small animal in vivo imager, and the bioluminescence pattern was selected for an exposure time of 60 seconds. Image and data analysis was performed using indiGo software.
Mice were divided into the following groups: (1) Mice were intraperitoneally injected with probe CL-SN (100. Mu.M, 100. Mu.L, 10% DMSO's saline). (2) Abdominal injection of Na into mice 2 S 4 (2, 4mM, 100. Mu.L of sample) and then intraperitoneally injecting probe CL-SN (100. Mu.M, 100. Mu.L, 10% DMSO of sample). (3) Mice were first intraperitoneally injected with LPS (10. Mu.g/mL, 100. Mu.L of saline) for 24h and then with probe CL-SN (100. Mu.M, 100. Mu.L, 10% DMSO of saline). (4) The mice were first intraperitoneally injected with DL-propargylglycine (2 mM, 100. Mu.L of saline) for 30min, then intraperitoneally injected with LPS (10. Mu.g/mL, 100. Mu.L of saline) for 24h, and then injected with probe CL-SN (100. Mu.M, 100. Mu.L of saline, 10% DMSO).
As shown in FIG. 12, the mice injected with the probe alone served as a control group, and had weak chemiluminescent signals on the abdomen (FIG. 12A). Compared with the control group, na with different concentration is injected into the abdominal cavity 2 S 4 After that, the chemiluminescent intensity increases significantly and with Na 2 S 4 The concentration was increased to increase (34-fold, 91-fold; fig. 12B, fig. 12C). This demonstrates that probe CL-SN can detect exogenous hydrogen polysulfide at different concentrations in mice. LPS-injected mice also showed a pronounced chemiluminescent signal on their abdomen (125-fold; FIG. 12-D). When mice were intraperitoneally injected with DL-Propargylglycine (PAG), the chemiluminescent intensity was significantly reduced after reinjecting the probe (fig. 12-E). This illustrates that the chemiluminescent signal in FIG. 11D is caused by LPS-induced endogenous hydrogen polysulfide. From this, probe CL-SN can detect endogenous hydrogen polysulfide in mice.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments may be modified or some technical features may be replaced equivalently; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. A near infrared chemiluminescence probe of hydrogen polysulfide has a chemical structure shown in a formula (I),
2. the method for preparing the near infrared chemiluminescence probe of the hydrogen polysulfide, which is characterized by comprising the following synthetic route:
3. the method for preparing the near infrared luminescent probe for hydrogen polysulfide according to claim 2, characterized in that the reaction step comprises: the compound 1g and 2- (benzoylthio) benzoic acid are subjected to condensation reaction, and then the reaction product is catalyzed by methylene blue under the condition of introducing oxygen to obtain the compound of the formula (I).
4. The method for preparing a near infrared luminescent probe for hydrogen polysulfide according to claim 3, wherein 1g of the compound and 2- (benzoylthio) benzoic acid are dissolved in a solvent, EDC and DMAP are then added, and the reaction is stirred at room temperature for 1h; then under the condition of introducing oxygen and under the irradiation of yellow light, the compound of the formula (I) is obtained through the catalysis of methylene blue.
5. The method for preparing a near infrared luminescent probe for hydrogen polysulfide according to claim 4, wherein the molar ratio of 1g of compound to 2- (benzoylthio) benzoic acid in the reaction is 1:1 to 2, preferably 1:1.2 to 1.8; the solvent for the condensation reaction was methylene chloride.
6. The method for preparing the near infrared luminescence probe of hydrogen polysulfide according to claim 4, wherein the molar ratio of DMAP to EDC in the reaction is 1:2.8 to 3.2, preferably 1:3.0; the molar ratio of compound 1g to EDC was 1:0.5 to 2.5, preferably 1:1 to 2.
7. Use of the near infrared hydrogen polysulfide chemiluminescent probe of claim 1 for chemiluminescent imaging detection of endogenous hydrogen polysulfide in a cell or in a mouse.
8. The use of the near infrared chemiluminescent probe of claim 1 for quantitatively detecting hydrogen polysulfide.
9. Use of the near infrared chemiluminescent probe of hydrogen polysulfide of claim 1 for detecting the physiological and pathological mechanism of hydrogen polysulfide in an organism.
10. Use of the near infrared hydrogen polysulfide chemiluminescent probe of claim 1 for detecting the signal transduction pathway of hydrogen polysulfide in an organism.
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