CN111675696A - Light affinity probe molecule based on triazole active molecule and preparation method and application thereof - Google Patents
Light affinity probe molecule based on triazole active molecule and preparation method and application thereof Download PDFInfo
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- 239000000523 sample Substances 0.000 title claims abstract description 88
- 150000003852 triazoles Chemical class 0.000 title claims abstract description 66
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 33
- 239000013067 intermediate product Substances 0.000 claims abstract description 21
- 150000002762 monocarboxylic acid derivatives Chemical class 0.000 claims abstract description 21
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 20
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 20
- 125000000304 alkynyl group Chemical group 0.000 claims abstract description 16
- 238000005516 engineering process Methods 0.000 claims abstract description 15
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229940014800 succinic anhydride Drugs 0.000 claims abstract description 12
- 230000000694 effects Effects 0.000 claims abstract description 9
- 238000009833 condensation Methods 0.000 claims abstract description 4
- 230000005494 condensation Effects 0.000 claims abstract description 4
- 238000012790 confirmation Methods 0.000 claims description 15
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 14
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 7
- DIXBSCZRIZDQGC-UHFFFAOYSA-N diaziridine Chemical compound C1NN1 DIXBSCZRIZDQGC-UHFFFAOYSA-N 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
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- 230000008569 process Effects 0.000 claims description 5
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- 238000002372 labelling Methods 0.000 claims description 3
- 238000006482 condensation reaction Methods 0.000 claims description 2
- 238000005222 photoaffinity labeling Methods 0.000 abstract description 4
- 150000003384 small molecules Chemical class 0.000 abstract description 2
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- 238000000635 electron micrograph Methods 0.000 description 5
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 4
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 4
- 229960000956 coumarin Drugs 0.000 description 4
- 235000001671 coumarin Nutrition 0.000 description 4
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- 239000003960 organic solvent Substances 0.000 description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 238000012650 click reaction Methods 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 3
- 230000002427 irreversible effect Effects 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
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- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical group C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- CYWSDGUZWKUALI-UHFFFAOYSA-N 3-azidocoumarin Chemical compound C1=CC=C2OC(=O)C(N=[N+]=[N-])=CC2=C1 CYWSDGUZWKUALI-UHFFFAOYSA-N 0.000 description 1
- NJACESZMZLMGBG-UHFFFAOYSA-N 4-[3-[1-[[4-(6-methoxypyridin-3-yl)phenyl]methyl]triazol-4-yl]anilino]-4-oxobutanoic acid Chemical compound COC1=CC=C(C=N1)C1=CC=C(CN2N=NC(=C2)C=2C=C(C=CC=2)NC(CCC(=O)O)=O)C=C1 NJACESZMZLMGBG-UHFFFAOYSA-N 0.000 description 1
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical class [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
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- C07—ORGANIC CHEMISTRY
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- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
A light affinity probe molecule based on triazole active molecule, a preparation method and application thereof, wherein the triazole active molecule and succinic anhydride are stirred in acetonitrile to obtain an intermediate product with monocarboxylic acid; and (3) obtaining the photoaffinity probe molecule based on the triazole active molecule under the condensation effect of a linker containing photoaffinity groups bisaziridine and alkynyl and an intermediate product with monocarboxylic acid under EDC & HCl. The preparation method of the photoaffinity probe molecule of the triazole active molecule is simple, easy to realize and high in yield. The photoaffinity probe of the triazole active molecule can be used for confirming target protein of the triazole active molecule and verifying feasibility of a photoaffinity labeling technology in confirming a small molecule target.
Description
Technical Field
The invention relates to a light affinity probe molecule based on triazole active molecules, a preparation method and application thereof.
Background
The light affinity labeling technology (PAL) combines the advantages of modern molecular biology, cell biology, medicinal chemistry, analytical chemistry and other subjects, and applies the synthesized light affinity probe molecules to generate high-activity intermediates under the irradiation of light with specific wavelength, which can be directly irreversibly covalently crosslinked with the protein specifically combined with the medicinal molecules to realize the capture of the medicinal target protein molecules. It is one of the core tools for researching the interaction between ligand and receptor on molecular level, and has great promoting effect on the elucidation of the interaction mechanism between ligand and receptor and the discovery of new target of medicine.
In recent years, the technology is mainly applied to the confirmation of drug molecular target protein, and the capture and confirmation of the drug molecular target are mainly realized based on a photo-crosslinking technology, a bio-orthogonal technology, a related biological analysis technology and the like. The light affinity probe molecule is designed and synthesized by directly carrying out structural modification on the basis of not influencing the medicinal activity of a target, respectively introducing a light reaction active group and designing and synthesizing the light affinity probe molecule by a bioorthogonal handle, and the light affinity probe molecule is subjected to specific irreversible covalent capture of a target protein of a target compound under the irradiation of light with specific wavelength. And then the identification confirmation of the captured target protein is realized through a bio-orthogonal reaction.
The probe molecules in the existing target confirmation technology cannot be stably combined with the drug target molecules, and false positive results are easily caused. In addition, the existing technology usually needs to connect a large volume of fluorescent group on the drug molecule for analysis, which can cause the defects of reduced activity, poor solubility, poor cell permeability and the like of the probe molecule.
Disclosure of Invention
The invention aims to provide a light affinity probe molecule based on triazole active molecules, a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a molecular structure formula of a photoaffinity probe based on triazole active molecules is as follows:
a preparation method of a photoaffinity probe molecule based on triazole active molecules comprises the following steps:
1) stirring triazole active molecules and succinic anhydride in acetonitrile to obtain an intermediate product with monocarboxylic acid;
2) under the condensation effect of EDC & HCl, a linker containing photoaffinity groups, namely diaziridine and alkynyl and an intermediate product with monocarboxylic acid are subjected to condensation reaction to obtain a photoaffinity probe molecule based on a triazole active molecule, wherein the structural formula is as follows:
the further improvement of the invention is that the specific process of the step 1) is as follows: and dissolving triazole active molecules and succinic anhydride in acetonitrile, and reacting for 8h at 60 ℃ to obtain an intermediate product with monocarboxylic acid.
The further improvement of the invention is that 1.40mmol of triazole active molecules and 2.10mmol of succinic anhydride are dissolved in acetonitrile and react for 8h at 60 ℃ to obtain an intermediate product with monocarboxylic acid.
The invention has the further improvement that the triazole active molecule is 3- (1- (4- (6-methoxypyridine-3-yl) benzyl) -1H-1,2, 3-triazole-4-yl) aniline.
The further improvement of the invention is that the specific process of the step 2) is as follows: dissolving the intermediate product with the monocarboxylic acid obtained in the step 1) in anhydrous tetrahydrofuran, then adding EDC & HCl and HOBt, then dropwise adding DIPEA, stirring for 1h at 0 ℃, then adding a linker containing photoaffinity groups bisaziridine and alkynyl, and stirring for 24h at room temperature to obtain the photoaffinity probe molecule based on the triazole active molecule.
The further improvement of the invention is that 0.17mmol of intermediate product with monocarboxylic acid is dissolved in anhydrous tetrahydrofuran, then 0.225mmol of EDC HCl and 0.17mmol of HOBt are added, the mixture is stirred evenly at 0 ℃, then 0.75mmol of DIPEA is added drop by drop, the mixture is stirred for 1h, 0.15mmol of linker containing photo-affinity group biaziridine and alkynyl is added, the mixture is stirred for 24h at room temperature, and the photo-affinity probe molecule based on triazole active molecule is obtained.
A further development of the invention is that the linker containing the photoaffinity group diaziridine and the alkynyl group is 2- (3- (-3-butyn-1-yl) -3H-diaziridine-3-yl) ethan-1-amine.
The application of the light affinity probe molecule based on the triazole active molecule in the light affinity labeling technology is provided.
The invention further improves the application of the photoaffinity probe molecule based on the triazole active molecule in the aspect of confirming the protein target.
Compared with the prior art, the invention has the following beneficial effects:
the invention uses the linker simultaneously containing photoaffinity groups of diaziridine and alkynyl to connect with triazole active molecules to obtain the photoaffinity probe molecules of the triazole active molecules. The photoaffinity probe molecule can be specifically and irreversibly combined with a target protein of a triazole active molecule, and a bioorthogonal handle in the probe molecule and another bioorthogonal handle containing fluorescein or biotin carry out click reaction to confirm the target protein. The preparation method of the photoaffinity probe molecule of the triazole active molecule is simple, easy to realize and high in yield.
The photoaffinity probe molecule of the triazole active molecule can carry out specific covalent binding on the target protein of the active molecule, and further realize the subsequent confirmation analysis of the captured target protein through click reaction. The method utilizes a photoaffinity labeling technology to realize the confirmation of the target protein of the drug action and can overcome the defects that the probe molecule can not stably bind the drug and the activity of the probe molecule is reduced, the solubility is poor and the cell permeability is poor in the existing confirmation target technology. The photoaffinity labeling technology of the invention is used for target confirmation by combining the synthetic small molecule photoaffinity probe with the bioorthogonal technology, and makes up for the defects of the prior art. The micromolecular photoaffinity probe based on the triazole active molecule can be used for confirming the target protein of the triazole active molecule and verifying the feasibility of a photoaffinity labeling technology in the aspect of confirming the micromolecular target.
Drawings
FIG. 1 is a synthesis route diagram of a photoaffinity probe molecule based on triazole active molecules provided by the invention;
wherein, the compound 1 is 3- (1- (4- (6-methoxypyridin-3-yl) benzyl) -1H-1,2, 3-triazol-4-yl) aniline (triazole active molecule), the compound 2 is succinic anhydride, the compound 3 is 4- ((3- (1- (4- (6-methoxypyridin-3-yl) benzyl) -1H-1,2, 3-triazol-4-yl) phenyl) amino) -4-oxobutyric acid, the compound 4 is linker 2- (3- (-3-butyn-1-yl) -3H-bisaziridin-3-yl) ethane-1-amine containing photoaffinity group bisaziridine and alkynyl, the compound X is triazole active molecule photoaffinity probe molecule.
Fig. 2 is a graph of the localization of a detection probe (blue) by click reaction in ea.hy926 cells using azidocoumarin. Wherein, (a) is the fluorescence localization of the probe of the control group, (b) is the nuclear localization of PI in the same field of view of the control group, (C) is the fluorescence localization and nuclear localization of the probe of the control group, (d) is the electron micrograph of the same group of cells of the control group, (e) is the fluorescence localization of the probe of the P1 group, (f) is the nuclear localization of PI in the same field of view of the P1 group, (g) is the fluorescence localization and nuclear localization of the probe of the P1 group, (h) is the electron micrograph of the same group of cells of the P1 group, (i) is the nuclear localization of PI in the same field of view of the fluorescence localization of the probe of the P1-C group, (j) is the nuclear localization of PI in the same field of view of the P1-C group, (k) is the fluorescence localization and nuclear localization of the electron micrograph of the probe of the P1-C group, and (l) is the electron micrograph of the same group of cells of the P1-C group.
Fig. 3 is an enlarged view of fig. 2, where (m) is an enlarged view of fig. (a), (n) is an enlarged view of fig. (b), (o) is an enlarged view of fig. (c), (p) is an enlarged view of fig. (e), (q) is an enlarged view of fig. (f), (r) is an enlarged view of fig. (g),(s) is an enlarged view of fig. (i), (t) is an enlarged view of fig. (j), and (u) is an enlarged view of fig. (k).
Detailed Description
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
The invention obtains the photoaffinity probe molecule by connecting triazole active molecules by using a linker containing photoaffinity groups, namely, diaziridine and alkynyl. The photoaffinity probe molecule can be used for confirming target protein of triazole active molecule.
The photoaffinity probe molecule with the target confirmation function comprises the following components: n is a radical of1- (2- (3- (3-butyn-1-yl) -3H-bis-pyhdin-3-yl) ethyl) -N4- (3- (1- (4- (6-methoxypyridin-3-yl) benzyl) -1H-1,2, 3-triazol-4-yl) phenyl) succinamide.
The following will explain the preparation method of the photoaffinity probe molecule with target confirmation provided by the present invention in detail by combining the synthetic route and the specific synthetic example shown in fig. 1.
Referring to fig. 1, a preparation method of a photoaffinity probe molecule based on triazole active molecules comprises the following steps:
1) reacting 3- (1- (4- (6-methoxypyridine-3-yl) benzyl) -1H-1,2, 3-triazole-4-yl) aniline (namely triazole active molecules) with succinic anhydride in an acetonitrile solution to obtain an intermediate product with monocarboxylic acid;
the specific operation of the step 1) is as follows: dissolving 1.40mmol of triazole active molecules and 2.10mmol of succinic anhydride in 10mL of acetonitrile solution, reacting for 8h at 60 ℃, removing the organic solvent at low pressure after the reaction is finished, adding a proper amount of water, extracting with ethyl acetate, washing the extracted organic phase with saturated saline solution, drying with anhydrous sodium sulfate, evaporating the solvent at reduced pressure to obtain a crude product, and separating the crude product by using a chromatographic column to obtain an intermediate product with monocarboxylic acid.
2) Under the condensation action of the intermediate product with monocarboxylic acid and a linker containing photoaffinity groups of diaziridine and alkynyl with EDC & HCl as a condensing agent, obtaining a photoaffinity probe molecule based on triazole active molecules, namely a compound X;
the specific operation of the step 2) is as follows: dissolving 0.17mmol of intermediate product with monocarboxylic acid in 3mL of anhydrous tetrahydrofuran solution, adding 0.225mmol of EDC HCl (EDC HCl is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride) and 0.17mmol of HOBt (HOBt is 1-hydroxybenzotriazole), stirring uniformly at 0 ℃, dropwise adding 0.75mmol of DIPEA (DIPEA is diisopropylethylamine) in ice bath, stirring for 1h, adding 0.15mmol of linker containing photoaffinity group biaziridine and alkynyl, stirring for 24h at room temperature, after the reaction is finished, removing the organic solvent by low pressure rotation, adding a proper amount of ethyl acetate, respectively washing with water and saturated sodium bicarbonate, drying the organic phase obtained after washing with saturated sodium sulfate, removing the solvent by reduced pressure distillation to obtain a crude product, separating the crude product by a chromatographic column to obtain a photoaffinity probe based on a triazole active molecule, i.e. compound X.
The linker containing the photoaffinity group bisaziridine and alkynyl is 2- (3- (-3-butyn-1-yl) -3H-bisaziridin-3-yl) ethyl-1-amine, and the structural formula is as follows:
the chemical structural formula of the photoaffinity probe molecule with the confirmed drug molecule target provided by the invention is as follows:
the application of the photoaffinity probe molecule based on the triazole active molecule in the confirmation of the active molecule target protein.
Example 1
Referring to fig. 1, the structural formula of the triazole active molecule-based photoaffinity probe molecule with target confirmation effect is prepared by the following steps:
dissolving triazole active molecules and succinic anhydride in acetonitrile, and reacting at 60 ℃ for 8h for treatment to obtain an intermediate product with monocarboxylic acid; the specific process is as follows:
dissolving 1.40mmol of triazole active molecules and 2.10mmol of succinic anhydride in 10mL of acetonitrile solution, reacting for 8h at 60 ℃, and finishing the reaction; removing the organic solvent at low pressure, adding a proper amount of water, extracting with ethyl acetate, washing the extracted organic phase with saturated saline, drying with anhydrous sodium sulfate, evaporating the solvent under reduced pressure to obtain a crude product, separating the crude product by using a chromatographic column, eluting with petroleum ether/ethyl acetate (V/V-1/1) to obtain the target compound, weighing 0.4g, and obtaining an intermediate product with monocarboxylic acid with a yield of 62.5%.
LC-MS(ESI,m/z):458.49[M+H]+，456.49[M-H]-。
The specific process for preparing the photoaffinity probe molecule is as follows:
dissolving 0.17mmol of intermediate product with monocarboxylic acid in 3mL of anhydrous tetrahydrofuran solution, then adding 0.225mmol of EDC HCl and 0.17mmol of HOBt, stirring uniformly at 0 ℃, dropwise adding DIPEAA 0.75mmol under ice bath, stirring for 1h, then adding 0.15mmol of linker containing photoaffinity group biaziridine and alkynyl, stirring for 24h at room temperature, after the reaction is finished, removing the organic solvent by low-pressure rotation, adding a proper amount of ethyl acetate, then respectively carrying out water washing and saturated sodium bicarbonate washing, drying the organic phase obtained after the saturated sodium chloride washing by using anhydrous sodium sulfate, then evaporating the solvent under reduced pressure to obtain a crude product, separating the crude product by using a chromatographic column, eluting by using petroleum ether/ethyl acetate (V/V ═ 1/1) to obtain a target compound, weighing 0.03g, obtaining the yield of 34.69%, and obtaining a molecular photoaffinity probe based on triazole activity, namely a compound X, wherein the structure is as follows:
the hydrogen spectrum nuclear magnetic resonance data is as follows:1H NMR(400MHz,DMSO-d6)10.06(s,1H),8.62(s,1H),8.48(d,J＝2.3Hz,1H),8.17(s,1H),8.01(dd,J＝8.6,2.6Hz,1H),7.92(t,J＝5.4Hz,1H),7.69(d,J＝8.2Hz,2H),7.54(d,J＝8.2Hz,1H),7.47(d,J＝8.1Hz,3H),7.35(t,J＝7.9Hz,1H),6.91(d,J＝8.6Hz,1H),5.69(s,2H),3.89(s,3H),2.92(dd,J＝12.9,7.0Hz,2H),2.83(t,J＝2.6Hz,1H),2.57(t,J＝7.1Hz,2H),2.41(t,J＝7.1Hz,2H),1.99(m,J＝7.3,2.6Hz,2H),1.56(m,J＝25.7,7.3Hz,4H).13C NMR(101MHz,DMSO-d6)171.64,171.03,163.62,147.11,145.21,138.03,129.73,127.20,120.43,116.02,111.09,83.63,72.24,53.76,53.15,34.08,32.49,32.05,31.79,30.73,27.69,13.14.MS(EI)[M]+:m/z＝576.7.
example 2
The application of the photoaffinity probe molecule based on the triazole active molecule in the target protein confirmation.
After the triazole active molecule-based photoaffinity probe is incubated with cells, the probe and an action target thereof form an irreversible covalent bond through ultraviolet illumination, a fluorescent group (azido coumarin) is connected through click chemistry, the azido coumarin emits blue fluorescence, and the fluorescent localization is the action site of the probe. The experimental parameters were determined from preliminary experiments as follows: the concentration ratio of the fluorescein to the photoaffinity probe is 1:1, the concentration ratio of the competitor to the photoaffinity probe is 5:1, and the obtained result is relatively clear. For the target protein confirmation experiment, 4 mu M photoaffinity probe, 4 mu M azido coumarin, 20 mu M competitor, ultraviolet irradiation for 15min and other parameters are selected to treat the cells, and 1 mu g/mL fluorescent dye PI (propidium iodide) is used for counterstaining. The result proves that after the triazole active molecule-based photoaffinity probe is treated, the blue fluorescence is stronger in cell membranes and cytoplasm.
The blue fluorescence is weak because the triazole active molecule photoaffinity probe is not irradiated by ultraviolet light, after the probe is incubated with cells, the combination formed by the target recognition group which is not irradiated by the ultraviolet light and the target protein is a reversible non-covalent bond, and the probe molecule is easily washed away, so the fluorescence is weakened.
Referring to fig. 2, it can be seen that after the probe is incubated with the cell, the probe and its action target form an irreversible covalent bond by ultraviolet irradiation, and the fluorescent group (azido coumarin) is linked by click chemistry, the azido coumarin emits blue fluorescence, and the fluorescent localization is the action site of the photoaffinity probe. Probe columns (FIG. 2 (a), (e) and (i)) show the fluorescent localization of the Probe, PI columns (FIG. 2 (b), (f) and (j)) show the nuclear localization of PI in the same field of view, Merge columns (FIG. 2 (c), (g) and (k)) show the fluorescent localization and nuclear localization of the Probe, and DIC columns (FIG. 2 (d), (h) and (l)) show the electron micrographs of the same group of cells. Group D (FIG. 2 (a), (b) (c) and (D)) was a control group, treated with DMSO only, and irradiated with UV light. The P1 groups (in FIG. 2, (e), (f), (g) and (h)) were treated with photoaffinity probes. P1-C groups (i), (j), (k) and (l) are probe and competitor treatment groups.
FIG. 2 was observed using a high power objective lens, and the results are shown in FIG. 3. Group D (fig. 3 (m), (n), and (o)) was a control group, treated with DMSO, irradiated with UV light, and showed almost no fluorescence. The P1 group (P), (q) and (r) in FIG. 3) is treated by photoaffinity probe based on triazole active molecule, and the result shows that the blue fluorescence is stronger in cell membrane and cytoplasm; the results of the treatment of the photoaffinity probe with the competitor in the P1-C group ((s), (t) and (u) in FIG. 3) revealed that the fluorescence intensity was low, the binding sites of the probe decreased, and the fluorescence intensity of blue was reduced because the binding sites of fluorescein were decreased after washing away the unbound probe.
The results show that the target protein based on the triazole active molecule photoaffinity probe is positioned in the cell membrane and cytoplasm.
Claims (10)
1. A light affinity probe molecule based on triazole active molecules is characterized in that the light affinity probe molecule has the following structural formula:
2. a preparation method of a photoaffinity probe molecule based on triazole active molecules is characterized by comprising the following steps:
1) stirring triazole active molecules and succinic anhydride in acetonitrile to obtain an intermediate product with monocarboxylic acid;
2) under the condensation effect of EDC & HCl, a linker containing photoaffinity groups, namely diaziridine and alkynyl and an intermediate product with monocarboxylic acid are subjected to condensation reaction to obtain a photoaffinity probe molecule based on a triazole active molecule, wherein the structural formula is as follows:
3. the preparation method of the photoaffinity probe based on the triazole active molecules as claimed in claim 2, wherein the specific process of the step 1) is as follows: and dissolving triazole active molecules and succinic anhydride in acetonitrile, and reacting for 8h at 60 ℃ to obtain an intermediate product with monocarboxylic acid.
4. The preparation method of the photoaffinity probe molecule based on the triazole active molecule as claimed in claim 2, wherein 1.40mmol of triazole active molecule and 2.10mmol of succinic anhydride are dissolved in acetonitrile and react for 8h at 60 ℃ to obtain an intermediate product with monocarboxylic acid.
5. The preparation method of the photoaffinity probe molecule based on the triazole active molecule as claimed in any one of claims 2 to 4, wherein the triazole active molecule is 3- (1- (4- (6-methoxypyridin-3-yl) benzyl) -1H-1,2, 3-triazol-4-yl) aniline.
6. The preparation method of the photoaffinity probe molecule based on the triazole active molecule as claimed in claim 2, wherein the specific process of the step 2) is as follows: dissolving the intermediate product with the monocarboxylic acid obtained in the step 1) in anhydrous tetrahydrofuran, then adding EDC & HCl and HOBt, then dropwise adding DIPEA, stirring for 1h at 0 ℃, then adding a linker containing photoaffinity groups bisaziridine and alkynyl, and stirring for 24h at room temperature to obtain the photoaffinity probe molecule based on the triazole active molecule.
7. The preparation method of the photoaffinity probe molecule based on the triazole active molecules as claimed in claim 6, wherein 0.17mmol of the intermediate product with the monocarboxylic acid is dissolved in anhydrous tetrahydrofuran, then 0.225mmol of EDC. HCl and 0.17mmol of HOBt are added, the mixture is stirred uniformly at 0 ℃, 0.75mmol of DIPEA is added dropwise, the mixture is stirred for 1h, 0.15mmol of a linker containing the bisaziridine and the alkynyl group is added, and the mixture is stirred for 24h at room temperature, so as to obtain the photoaffinity probe molecule based on the triazole active molecules.
8. The preparation method of the photoaffinity probe molecule based on the triazole active molecules as claimed in claim 2, wherein the linker containing the photoaffinity groups of diaziride and alkynyl is 2- (3- (-3-butyn-1-yl) -3H-diaziride-3-yl) ethan-1-amine.
9. An application of the light affinity probe molecule based on triazole active molecule as claimed in claim 1 in the light affinity labeling technology.
10. The use of claim 9, characterized by the use of photoaffinity probe molecules based on triazole active molecules for the confirmation of protein targets.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113149962A (en) * | 2021-03-16 | 2021-07-23 | 无锡海伦生物科技有限公司 | Preparation method and application of probe molecule containing photoaffinity group bisaziridine |
| CN114560847A (en) * | 2022-04-24 | 2022-05-31 | 中国中医科学院中药研究所 | Chloroquine photoaffinity molecular probe and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107382862A (en) * | 2017-07-17 | 2017-11-24 | 大连理工大学 | Sulphur/oxo naphthoyl imide compounds and its application |
| CN107540608A (en) * | 2017-07-17 | 2018-01-05 | 大连理工大学 | 4 substitution naphthoyl imide compounds and its application |
| CN108675988A (en) * | 2018-06-04 | 2018-10-19 | 西安交通大学 | A kind of pyridine compounds and their and application |
| CN109369622A (en) * | 2018-12-05 | 2019-02-22 | 西安交通大学 | A kind of light Affinity Probes molecule and preparation method thereof based on VEGFR-2 inhibitor BD7 |
-
2020
- 2020-06-08 CN CN202010513923.2A patent/CN111675696A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107382862A (en) * | 2017-07-17 | 2017-11-24 | 大连理工大学 | Sulphur/oxo naphthoyl imide compounds and its application |
| CN107540608A (en) * | 2017-07-17 | 2018-01-05 | 大连理工大学 | 4 substitution naphthoyl imide compounds and its application |
| CN108675988A (en) * | 2018-06-04 | 2018-10-19 | 西安交通大学 | A kind of pyridine compounds and their and application |
| CN109369622A (en) * | 2018-12-05 | 2019-02-22 | 西安交通大学 | A kind of light Affinity Probes molecule and preparation method thereof based on VEGFR-2 inhibitor BD7 |
Non-Patent Citations (2)
| Title |
|---|
| JIAQI FU等: "Accelerated cellular on- and off -target screening of bioactive compounds using microarrays", 《ORG. BIO MOL. CHEM.》 * |
| ZHENGQIU LI等: "Design and Synthesis of Minimalist Terminal Alkyne-Containing Diazirine Photo-Crosslinkers and Their Incorporation into Kinase Inhibitors for Cell- and Tissue-Based Proteome Profiling", 《ANGEW. CHEM. INT. ED.》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113149962A (en) * | 2021-03-16 | 2021-07-23 | 无锡海伦生物科技有限公司 | Preparation method and application of probe molecule containing photoaffinity group bisaziridine |
| CN114560847A (en) * | 2022-04-24 | 2022-05-31 | 中国中医科学院中药研究所 | Chloroquine photoaffinity molecular probe and preparation method and application thereof |
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