CN105295896A - Specifically marked capsaicin fluorescent probe and synthetic method and application thereof - Google Patents
Specifically marked capsaicin fluorescent probe and synthetic method and application thereof Download PDFInfo
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- CN105295896A CN105295896A CN201510578824.1A CN201510578824A CN105295896A CN 105295896 A CN105295896 A CN 105295896A CN 201510578824 A CN201510578824 A CN 201510578824A CN 105295896 A CN105295896 A CN 105295896A
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- fluorescent probe
- synthetic method
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- 0 COc(cc(CNC(*Nc1ccc(*)c2n[o]nc12)=O)cc1)c1O Chemical compound COc(cc(CNC(*Nc1ccc(*)c2n[o]nc12)=O)cc1)c1O 0.000 description 1
Abstract
The invention relates to a specifically marked capsaicin fluorescent probe and a synthetic method and application thereof, which belong to the technical field of biology. Firstly, the invention discloses the specifically marked capsaicin fluorescent probe, and the structural formula (III) is as follows, wherein R=(CH2)2, (CH2)3, (CH2)4, (CH2)5, (CH2)6, (CH2)7, (CH2)8, (CH2)9, (CH2)10 or (CH2)11, the excitation wavelength is between 450 and 480 nm, and the emission wavelength is between 510 and 540 nm. The compound provided by the invention has the advantages of good lipid solubility, long excitation wavelength, simplicity and convenience in synthesis, mild markers and the like, and especially, a good tool is provided for the research on comformational change in a membrane protein gating process due to the difference of the fluorescence intensity of the compound in liposoluble and hydrosoluble environments.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of capsaicine fluorescent probe of specific marker and synthetic method thereof and application.
Background technology
The structure of protein, motion and the motion of function to life entity have very important meaning.The means of the artificial mixed forests of Study on Protein comprise fluorescence spectrum, nucleus magnetic resonance, electron microscope, X-ray crystalline diffraction etc.Wherein, the fluorescence detection of protein has the advantages such as marker site method is flexible, highly sensitive, detection method is easy and is used widely.But, current whole world Jin You minority mechanism possesses ability and adopts Fluorescein labeled ligand probe to study the dynamic process of ion channel structure function, its know-why is fluorescent probe on part specific site mark, and clearly this fluorescence labeling probe still has function.Subsequently, by the combination that detects between this probe and receptor membrane albumen and effect of dissociating, realize resolving this channel protein and activate and the dynamic process of inactivation.At present, this fluorescent labelling techniques is still in this forward position, field, is more and more subject to extensive concern.
TRPV1 passage is otherwise known as capsaicin receptor, it is the non-selective cation channel of a part gate, can by various exogen and endogenous physics and chemical stimulation excite, have been found that wide expression is in central nervous system and peripheral nervous system, relate to the sense of heat, the pain sensation transmission and modulation and integrate the important physiological function such as various pain information.Known main TRPV1 endogenic ligand is capsaicine, and itself does not possess fluorescent characteristic, and there is no capsaicine class fluorescent ligand at present, cannot carry out Visual Dynamic research to gating process such as the activation of TRPV1 passage and inactivations.
Summary of the invention
The problem of specificity fluorescent probe part is lacked in order to solve current TRPV1 channel protein, 7-nitro benzofuraxan (NBD) fluorophor is introduced the ligands specific capsaicin analog of TRPV1 by the present invention, successfully synthesizes a kind of capsaicine fluorescent probe of novel specific marker.
First, the invention discloses a kind of capsaicine fluorescent probe of specific marker, structure is as formula III:
Wherein, R=(CH
2)
2, (CH
2)
3, (CH
2)
4, (CH
2)
5, (CH
2)
6, (CH
2)
7, (CH
2)
8, (CH
2)
9, (CH
2)
10or (CH
2)
11.Its excitation wavelength is between 450-480nm, and emission wavelength is between 510-540nm.
Because the fluorescence intensity of NBD fluorophor in fat-soluble bad border is significantly better than aqueous environment, this feature can significantly strengthen target compound and membrane protein receptor TRPV1 in conjunction with time fluorescence intensity.
Secondly, present invention also offers the synthetic method of the capsaicine fluorescent probe of specific marker.Synthetic route is shown below, by Compound I and Compound II per under HOBT, DCC, DMAP catalysis in organic solvent condensation form.
Wherein, R=(CH
2)
2, (CH
2)
3, (CH
2)
4, (CH
2)
5, (CH
2)
6, (CH
2)
7, (CH
2)
8, (CH
2)
9, (CH
2)
10or (CH
2)
11.
Described organic solvent is one or more of DMF, acetonitrile, dioxane, acetone, chloroform, methylene dichloride, methyl alcohol and ethanol, or the arbitrary proportion mixing of above-mentioned solvent, is preferably DMF.
Preferably, concrete grammar is: get 1 equivalents of compound I and 1 equivalents of compound II, be dissolved in DMF, add the HOBT of 1 equivalent, the DCC of 1 equivalent, the DMAP of 0.1 equivalent, stirring at room temperature 24 hours; Removing organic solvent, column chromatography, with methylene dichloride: methyl alcohol=10:1 wash-out, obtains corresponding compound III.
The purposes that the capsaicine fluorescent probe that present invention also offers specific marker in addition marks for the specificity fluorescent of TRPV1 channel protein.
The present invention relates to a kind of novel fat-soluble fluorescent probe applying to the activation of transient receptor potential vanilloid receptor1 (transientreceptorpotentialvanilloid1, TRPV1) channel protein and synthetic method thereof.This compounds modifies fluorophor 7-nitro benzofuraxan (NBD) with the ligands specific capsaicine analog of TRPV1 to form, have good fat-soluble, longer excitation wavelength, there is the advantages such as synthesis is easy, mark is gentle simultaneously, particularly this compounds is in the fat-soluble difference with the fluorescence intensity in aqueous environment, in membranin gating process, the research of conformational change provides good instrument.As the effective fluorescent probe of one, this compounds may be used for the specificity fluorescent mark of ligand binding TRPV1 channel protein, has important help to the dynamic process of resolving the gates such as the activation of TRPV1 passage and inactivation.The excitation wavelength of such probe is between 450-480nm, and emission wavelength, between 510-540nm, by the use to this serial different chain length capsaicin analog, can describe the structure of protein, dynamics and biological function more accurately.
Accompanying drawing explanation
The HEK293 cell of Fig. 1 transfection TRPV1 protein expression, gives compound III, the map of current that TRPV1 passage is activated on the 10th second in record; In lavation in the 28th second removing compound III, pathway closure current vanishes;
The HEK293 cell of Fig. 2 transfection TRPV1 protein expression, gives compound III on the 10th second in record, and this compound TRPV1 passage on film is combined, and cell fluorescence intensity increases; In lavation in the 28th second removing compound III, cell fluorescence intensity weakens until disappear gradually;
The HEK293 cell of Fig. 3 transfection TRPV1 protein expression, gives compound III on the 10th second in record, and this compound TRPV1 passage on film is combined, and single-cell fluorescence imaging showed cell fluorescence intensity strengthens gradually; In lavation in the 28th second removing compound III, single-cell fluorescence imaging showed cell fluorescence intensity weakens gradually until disappear.
Embodiment
Set forth the present invention further below in conjunction with specific embodiment, should be understood that following examples are only not used in for illustration of the present invention and limit the scope of the invention.
In the following example, method therefor if no special instructions, is ordinary method.Material required in following examples or reagent, be market if no special instructions and buy.
Instrument:
Nuclear magnetic resonance analyser (BrukerAM400 type nuclear magnetic resonance analyser)
Spectrophotofluorometer (Japanese Shimadzu Corporation, RF-5301PC)
Ultraviolet analysis instrument for three purposed (physico-optical instrument factory of upper Nereid section, ZF-1 ultraviolet analysis instrument for three purposed)
AL204 electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.
Rotary Evaporators (Nanjing Cole's plant and instrument company limited, RE-1302BV-G2100 type rotatory evaporator)
Heat-collecting magnetic stirring device (Shanghai Yu Ying Instrument Ltd., DF-101S (digital display automatic constant-temperature agitator)
Reagent:
Vanillin (Aladdin reagent, AR, >=98%)
Oxammonium hydrochloride (Chemical Reagent Co., Ltd., Sinopharm Group, AR, >=98%)
6-aminocaprolc acid (Aladdin reagent, AR, 98%)
4-chloro-7-nitro-2,1,3-benzofuraxan (NBD-Cl) (Sigma, AR, >=99%)
I-hydroxybenzotriazole (HOBT) (Aladdin reagent, AR, >=97%)
Dicyclohexylcarbodiimide (DCC) (Chemical Reagent Co., Ltd., Sinopharm Group, AR, >=95%)
DMAP (DMAP) (Aladdin reagent, AR, >=97%)
DMF (DMF) (Chemical Reagent Co., Ltd., Sinopharm Group, AR, >=95%)
Acetonitrile (Chemical Reagent Co., Ltd., Sinopharm Group, AR, >=99.0%)
Dioxane (Chemical Reagent Co., Ltd., Sinopharm Group, AR, >=99.5%)
Acetone (Chemical Reagent Co., Ltd., Sinopharm Group, AR, >=99.5%)
Chloroform (Chemical Reagent Co., Ltd., Sinopharm Group, AR, >=99.0%)
Methylene dichloride (Chemical Reagent Co., Ltd., Sinopharm Group, AR, >=99.5%)
Methyl alcohol (Chemical Reagent Co., Ltd., Sinopharm Group, AR, >=99.7%)
Ethanol (Chemical Reagent Co., Ltd., Sinopharm Group, AR, >=95.0%)
The capsaicine fluorescent probe of specific marker shown in structure formula III is obtained by two kinds of chemical compounds Is, II condensation under the condition of HOBT, DCC, DMAP.
Embodiment 1:
Get 1 equivalents of compound I and 1 equivalent R=(CH
2)
5compound ii, is dissolved in DMF, adds 1 equivalent HOBT, 1 equivalent DCC, 0.1 equivalent DMAP, stirring at room temperature 24 hours.Removing organic solvent, column chromatography, with methylene dichloride: methyl alcohol=10:1 wash-out, obtains corresponding compound III.Confirm to coincide with target product through nuclear-magnetism spectrum, its proton nmr spectra ownership is as follows:
1hNMR (500MHz, DMSO-d
6): δ 9.54 (s, 1H), 8.81 (s, 1H), 8.50 (t, 1H, J=6Hz), 8.16 (t, 1H, J=6Hz), 6.79 (d, 1H, J=1.5Hz), 6.68 (d, 1H, J=8Hz), 6.62 (dd, 1H, J=8Hz, J=2Hz), 6.39 (d, 1H, J=9Hz), 4.13 (d, 2H, J=5.5Hz), 3.72 (s, 3H), 3.44 (s, 2H), 2.13 (t, 2H, J=7.5Hz), 1.68 (dt, 2H), 1.57 (dt, 2H), 1.36 (d, 2H, J=6Hz).The fluorescence exciting wavelength of this compound in methanol solvate is 466nm, and emission wavelength is 528nm.In biological activity assay, this compound fails to activate TRPV1 channel protein generation current.
Embodiment 2:
Get 1 equivalents of compound I and 1 equivalent R=(CH
2)
11compound ii, is dissolved in DMF, adds 1 equivalent HOBT, 1 equivalent DCC, 0.1 equivalent DMAP, stirring at room temperature 24 hours.Removing organic solvent, column chromatography, with methylene dichloride: methyl alcohol=10:1 wash-out, obtains corresponding compound III.Confirm to coincide with target product through nuclear-magnetism spectrum, its proton nmr spectra ownership is as follows:
1hNMR (500MHz, DMSO-d
6) δ 9.54 (s, 1H), 8.80 (s, 1H), 8.51 (d, 1H, J=8.5Hz), 8.14 (t, 1H, J=6Hz), 6.79 (d, 1H, J=2Hz), 6.68 (d, 1H, J=8Hz), 6.62 (dd, 1H, J=8Hz, J=2Hz), 6.40 (d, 1H, J=9Hz), 4.13 (d, 2H, J=6Hz), 3.72 (s, 3H), 3.45 (s, 2H), 2.09 (t, 2H, J=7Hz), 1.67 (dt, 2H), 1.49 (m, 2H), 1.36 (m, 2H), 1.26 (m, 12H).The fluorescence exciting wavelength of this compound in methanol solvate is 467nm, and emission wavelength is 528nm.In biological activity test, this compound can activate TRPV1 passage generation current, and can synchronous detection strengthen to being combined with cytolemma TRPV1 passage the fluorescence intensity caused; After this compound is by wash-out, the fluorescence intensity of TRPV1 channel current and cytolemma all progressively disappears.
Wherein, chemical compounds I and compound ii are known compound.
The synthetic method of chemical compounds I is as follows:
Get 1 equivalent Vanillin and be dissolved in dehydrated alcohol, 1 equivalent oxammonium hydrochloride is water-soluble, and 1.1 equivalent of sodium hydroxide are water-soluble.Join in the ethanolic soln of Vanillin by the oxammonium hydrochloride aqueous solution and aqueous sodium hydroxide solution, 65 DEG C are refluxed 2.5 hours.Off-white solid is obtained except after desolventizing, water/ethyl acetate system extraction, collect ethyl acetate layer, drying is except desolventizing, gained compound reduces with Pd/C, after reaction terminates through filtering, water-soluble after concentrated, column chromatography purification, add NaOH and be stirred to and occur a large amount of white solid, suction filtration, vacuum-drying obtain chemical compounds I.
The synthetic method of compound ii is following (with R=(CH
2)
5for example):
1 equivalent 6-aminocaprolc acid, 3 equivalent NaHCO
3water-soluble, drip 1 equivalent NBD-Cl under ice bath, stirring at room temperature after 1.5 hours 50 DEG C reaction 12 hours.Separate out solid with the HCl aqueous solution after reacting completely, after filtration drying, obtain compound ii.
Claims (7)
1. the capsaicine fluorescent probe of the specific marker shown in structure formula III
Wherein, R=(CH
2)
2, (CH
2)
3, (CH
2)
4, (CH
2)
5, (CH
2)
6, (CH
2)
7, (CH
2)
8, (CH
2)
9, (CH
2)
10or (CH
2)
11.
2. the capsaicine fluorescent probe of specific marker according to claim 1, is characterized in that: its excitation wavelength is between 450-480nm, and emission wavelength is between 510-540nm.
3. the synthetic method of the capsaicine fluorescent probe of the specific marker described in claim 1 or 2, is characterized in that: by Compound I and Compound II per under HOBT, DCC, DMAP catalysis in organic solvent condensation form, its synthetic route is:
Wherein, R=(CH
2)
2, (CH
2)
3, (CH
2)
4, (CH
2)
5, (CH
2)
6, (CH
2)
7, (CH
2)
8, (CH
2)
9, (CH
2)
10or (CH
2)
11.
4. synthetic method according to claim 3, is characterized in that: described organic solvent is one or more the mixing in DMF, acetonitrile, dioxane, acetone, chloroform, methylene dichloride, methyl alcohol and ethanol.
5. synthetic method according to claim 4, is characterized in that: described organic solvent is DMF.
6. synthetic method according to claim 3, is characterized in that: concrete grammar is: get 1 equivalents of compound I and 1 equivalents of compound II, be dissolved in DMF, add the HOBT of 1 equivalent, the DCC of 1 equivalent, the DMAP of 0.1 equivalent, stirring at room temperature 24 hours; Removing organic solvent, column chromatography, with methylene dichloride: methyl alcohol=10:1 wash-out, obtains corresponding compound III.
7. the purposes that marks for the specificity fluorescent of TRPV1 channel protein of the capsaicine fluorescent probe of specific marker according to claim 1.
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|---|---|---|---|
| CN201510578824.1A CN105295896B (en) | 2015-09-11 | 2015-09-11 | The capsaicine fluorescence probe and its synthetic method of a kind of specific marker and application |
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|---|---|---|---|
| CN201510578824.1A CN105295896B (en) | 2015-09-11 | 2015-09-11 | The capsaicine fluorescence probe and its synthetic method of a kind of specific marker and application |
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| CN105295896B CN105295896B (en) | 2017-12-29 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110940655A (en) * | 2019-12-30 | 2020-03-31 | 杭州柘大飞秒检测技术有限公司 | Method for detecting capsaicin |
| WO2021088209A1 (en) * | 2019-11-08 | 2021-05-14 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Capsaicin hapten and artificial antigen for detecting illegal cooking oil, preparation method therefor and application thereof |
| CN116217611A (en) * | 2023-05-10 | 2023-06-06 | 成都摩诃大龙医药科技有限公司 | Cyclobutanone derivative, preparation method and application |
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| CN1443170A (en) * | 2000-07-20 | 2003-09-17 | 神经原公司 | Capsaicin receptor ligands |
| CN101495469A (en) * | 2006-07-24 | 2009-07-29 | 赛诺菲-安万特 | N-(aminoheteroaryl)-1H-indole-2-carboxamide derivatives as antagonist of TRPV1 or VR1 receptor, preparation thereof and therapeutic use thereof |
| WO2014037394A1 (en) * | 2012-09-06 | 2014-03-13 | Sanofi | Fluorescence-labelled fatty acids and uses thereof |
| CN104327846A (en) * | 2014-11-18 | 2015-02-04 | 河南农业大学 | Hg<2+> ratio fluorescence probe containing rigid structure rhodamine and preparation method of Hg<2+> ratio fluorescence probe |
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2015
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Patent Citations (4)
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| CN1443170A (en) * | 2000-07-20 | 2003-09-17 | 神经原公司 | Capsaicin receptor ligands |
| CN101495469A (en) * | 2006-07-24 | 2009-07-29 | 赛诺菲-安万特 | N-(aminoheteroaryl)-1H-indole-2-carboxamide derivatives as antagonist of TRPV1 or VR1 receptor, preparation thereof and therapeutic use thereof |
| WO2014037394A1 (en) * | 2012-09-06 | 2014-03-13 | Sanofi | Fluorescence-labelled fatty acids and uses thereof |
| CN104327846A (en) * | 2014-11-18 | 2015-02-04 | 河南农业大学 | Hg<2+> ratio fluorescence probe containing rigid structure rhodamine and preparation method of Hg<2+> ratio fluorescence probe |
Non-Patent Citations (2)
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021088209A1 (en) * | 2019-11-08 | 2021-05-14 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Capsaicin hapten and artificial antigen for detecting illegal cooking oil, preparation method therefor and application thereof |
| CN110940655A (en) * | 2019-12-30 | 2020-03-31 | 杭州柘大飞秒检测技术有限公司 | Method for detecting capsaicin |
| CN110940655B (en) * | 2019-12-30 | 2022-02-08 | 杭州柘大飞秒检测技术有限公司 | Method for detecting capsaicin |
| CN116217611A (en) * | 2023-05-10 | 2023-06-06 | 成都摩诃大龙医药科技有限公司 | Cyclobutanone derivative, preparation method and application |
| CN116217611B (en) * | 2023-05-10 | 2023-07-11 | 成都摩诃大龙医药科技有限公司 | Cyclobutanone derivative, preparation method and application |
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| CN105295896B (en) | 2017-12-29 |
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