CN106010507A - Dye-molecule-marked tetrazine probe, and preparation method and application thereof - Google Patents

Dye-molecule-marked tetrazine probe, and preparation method and application thereof Download PDF

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CN106010507A
CN106010507A CN201610348541.2A CN201610348541A CN106010507A CN 106010507 A CN106010507 A CN 106010507A CN 201610348541 A CN201610348541 A CN 201610348541A CN 106010507 A CN106010507 A CN 106010507A
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molecule
dye molecule
tetrazine
dye
labelling
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蔡林涛
李文军
潘正银
张鹏飞
刘宏
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Shenzhen Institute of Advanced Technology of CAS
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • G01—MEASURING; TESTING
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    • 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
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • 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
    • C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
    • 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
    • C09K2211/1074—Heterocyclic compounds characterised by ligands containing more than three nitrogen atoms as heteroatoms
    • 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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Abstract

The invention provides choline analogs and a dye-molecule-marked tetrazine probe. The coupling reaction between the choline analogs and the dye-molecule-marked tetrazine probe can quickly occur in the normal-temperature physiological environment without any copper catalyst, thereby preventing the toxic influence of metal catalysis on the cells and living body. After the choline analogs and dye-molecule-marked tetrazine probe are subjected to coupling reaction, the detection indicates that the marked dye molecule can quickly perform positioning and tracing on the choline analogs on cytomembranes in a high-sensitivity high-resolution way. The invention also provides a preparation method of the choline analogs and dye-molecule-marked tetrazine probe. The method is simple and easy to implement, can implement large-scale production, and has low production cost. The invention also provides application of the choline analogs and dye-molecule-marked tetrazine probe.

Description

By tetrazine probe of dye molecule labelling and its preparation method and application
The application be aimed at December in 2013 within 31st, be submitted to Patent Office of the People's Republic of China, Application No. 201310752617.4, Invention entitled " cholinomimetic and its preparation method and application, by tetrazine probe of dye molecule labelling and preparation method thereof And application " the divisional application made of parent application, the content of above-mentioned parent application is incorporated herein in the way of introducing.
Technical field
The present invention relates to biological medicine Material Field, be specifically related to cholinomimetic and its preparation method and application, by contaminating Tetrazine probe of material molecular marker and its preparation method and application.
Background technology
Choline (Cho) is the important component part in membrane structure and lipoprotein composition.In eukaryote, phospholipid head Holding most common group is choline, is phospholipid the abundantest in cell membrane containing phosphatidyl choline, is also that the primary structure of cell membrane becomes Point, in cellular metabolism and signal conduct, play vital effect,
Adrian Salic seminar utilizes choline metabolic pathway in cell, by the end modified alkynes to choline Base, achieves its labeled in situ to cell on the premise of not affecting its function, and with the dyestuff of band nitrine by click copper The metabolism behavior to intracellular choline that is catalyzed carries out tracer analysis.But, use tradition nitrine-alkynyl in the method Husigen [3+3] cycloaddition reaction, the existence of catalyst copper can cause the toxicity of cell, thus at living cells or even live body Application is very restricted.
Hai-Yan Xie seminar uses novel click to be catalyzed without copper, uses nitrine instead and modifies choline, and with its with carefully Born of the same parents cultivate altogether, usually detect, in the case of without copper with without copper [3+2] click cycloaddition reagent benzo cyclooctyne DBCO-fluorescence Checking nitrine-choline is successfully marked on cell membrane.But the response speed of this reaction is relatively slow, thus easily the most in vivo by Remove and affect its efficiency combined.
Therefore, it is necessary to provide a kind of low to cytotoxicity, and react choline spike quick, highly sensitive, high-resolution Method.
Summary of the invention
For solving the problems referred to above, the invention provides a kind of cholinomimetic and its preparation method and application.Additionally, this Bright additionally provide a kind of by tetrazine probe of dye molecule labelling and its preparation method and application.
The present invention provide cholinomimetic and by the coupling reaction between the tetrazine probe of dye molecule labelling without copper Catalysis, it is to avoid metal catalytic is to cell and the toxic effect of live body, it is possible to high-resolution quick, highly sensitive ground is on cell membrane Cholinomimetic carry out positioning and spike.
First aspect, the invention provides a kind of cholinomimetic, and structural formula is as shown in P1:
In formula, k is the natural number of 2～4.
Second aspect, present invention also offers the preparation method of a kind of cholinomimetic, comprises the steps:
S10, provide compound A and compound B that following structural formula represents:
Under conditions of catalyst exists, described compound A and compound B is dissolved in first by the mol ratio of 1:1～1.5 In organic solvent, obtain reactant liquor, after described reactant liquor reacts 0.5～1 hour at 20～30 DEG C, through extraction, dry, silica gel Column chromatographic isolation and purification obtains compound C, its structural formula as shown at c:
S20, provide the compound D that following structural formula represents:
In formula, k is the natural number of 2～4,
Under conditions of catalyst exists, by the mol ratio of 1:2～5, described compound C and compound D is dissolved in second has In machine solvent, obtain reactant liquor, after described reactant liquor reacts 0.5～1 hour at 20～30 DEG C, through extraction, dry, silicagel column Chromatography purification obtains compound E, and its structural formula is as shown in E:
In formula, k is the natural number of 2～4,
S30, described compound E and dimethylethanolamine are dissolved in the 3rd organic solvent by the mol ratio of 1:1～3, To reactant liquor, after described reactant liquor reacts 12～16 hours at reflux, concentrated, silica gel column chromatography separating purification obtains Compound P1, its structural formula is as shown in P1:
In formula, k is the natural number of 2～4.
It should be noted that in the structural formula that the present invention relates toSymbol for positive charge.
Preferably, in described step S10, described catalyst is pyridine.
Preferably, in described step S10, the molar concentration of described catalyst is 2～4 times of described compound A.
Preferably, in described step S10, the mode of described extraction is to add saturated chlorine in reactant liquor after the completion of reaction Change ammonium salt solution to extract.
Preferably, in described step S10, described dry mode is for do extraction gained organic facies anhydrous sodium sulfate Dry, it is evaporated.
Preferably, in described step S10, described first organic solvent is dichloromethane, oxolane, chloroform or N, N-bis- Methylformamide.
Preferably, in described step S20, described catalyst is triethylamine or DIPEA.
Preferably, in described step S20, the molar concentration of described catalyst is 2～5 times of described compound C.
Preferably, in described step S20, the mode of described purification is to add two after the reactant liquor after reaction being terminated concentrates Chloromethanes is diluted, and with NaOH solution and solution washing.
Preferably, in described step S20, described dry mode is for do extraction gained organic facies anhydrous sodium sulfate Dry, it is evaporated.
Preferably, in described step S20, described second organic solvent be DMF, dimethyl sulfoxide or Oxolane.
Preferably, in described step S30, described 3rd organic solvent be DMF, dimethyl sulfoxide or Oxolane.
The synthetic method of the cholinomimetic that the present invention provides, with divinyl macromer (1R, 8S, the 9r)-dicyclo of non-copper catalysis [6.1.0] nonyl-4-alkynyl-9-base methanol (BCN) is initial compounds, by the activation to its terminal hydroxyl, enters with bromo-amine class Row reaction obtains Bromo-intermediates, and finally reacts generation cholinomimetic with dimethylethanolamine.The method is easy and simple to handle, anti- Answer mild condition, favorable reproducibility.
The preferred steps of the method includes: the 1) activation (BCN-pNC) of BCN: exist with p-nitrophenyl chloro-formate (pNC) Under conditions of pyridine and dichloromethane, BCN is activated；2) synthesis of intermediate B CN-Br: BCN-pnc and 2-after activation Bromine ethamine generates intermediate bromo BCN-Br under the effect of triethylamine；3) the cholinomimetic synthesis (BCN-that BCN modifies Cho): BCN-Br obtains target compound BCN-Cho with dimethylethanolamine in oxolane (THF) backflow.
The compound A that the present invention provides is divinyl macromer (1R, 8S, 9r)-dicyclo [6.1.0] nonyl-4-alkynyl-9-base methanol (BCN), compound B be p-nitrophenyl chloro-formate (pNC), compound C be BCN-pNC, compound D is bromo-amine class, chemical combination Thing E is BCN-Br.
Preferably, described bromo-amine class is 2-bromine ethamine or 3-bromine propylamine.
The third aspect, the invention provides a kind of by the tetrazine probe of dye molecule labelling, including dye molecule and tetrazine Molecule, described dye molecule forms covalent bond with the amino of tetrazine molecule, and described dye molecule and tetrazine molecule are by described common Valence link is connected, and described dye molecule is luminescent dye molecule or nir dye molecule, the structural formula such as P2 of described tetrazine molecule Shown in:
Wherein, R1For
R2For H orDescribedIn, X is CH or N.
Preferably, described luminescent dye molecule is rhodamine, Fluorescein isothiocyanate, o-phthalaldehyde(OPA), dimethylamino naphthalene Sulfonic acid chloride, fluorenes methoxy dicarbonyl chloride, 6-quinolin-2-ylamine base-N-hydroxy-succinamide carbamate or N-9-fluorenes methoxyl group phosphinylidyne- Amino acidic group-N-carboxylic acid anhydrides.
The luminescent dye molecule that the present invention uses is preferably but is not limited to described rhodamine, Fluorescein isothiocyanate, adjacent benzene Dicarbaldehyde, dansyl chloride, fluorenes methoxy dicarbonyl chloride, 6-quinolin-2-ylamine base-N-hydroxy-succinamide carbamate or N-9-fluorenes methoxyl group phosphinylidyne-amino acidic group-N-carboxylic acid anhydrides.
In the luminescent dye molecule that the present invention uses, described Fluorescein isothiocyanate is cyanate fluorescent dye, institute State o-phthalaldehyde(OPA) be the adjacent twain-aldehyde compound fluorescent dye of fragrance, described dansyl chloride be acyl chloride fluorescent dye, described Fluorenes methoxy dicarbonyl chloride is chloro-formate fluorochrome matter, described 6-quinolin-2-ylamine base-N-hydroxy-succinamide carbamate It is anhydrides fluorescent dye for carbonates fluorescent dye, described N-9-fluorenes methoxyl group phosphinylidyne-amino acidic group-N-carboxylic acid anhydrides, institute State cyanate, the adjacent twain-aldehyde compound of fragrance, acyl chloride, chloro-carbonic acid esters, carbonates or anhydrides fluorescent dye and there is energy and egg The active group of the amino reaction of white matter, can carry out fluorescent labeling to protein；And the tetrazine molecule that the present invention provides contain can For reaction amino, when using tetrazine molecule described in described luminescent dye molecule labelling, the active group of luminescent dye molecule with The amino reaction of described tetrazine molecule forms covalent bond, thus obtains described by the tetrazine probe of dye molecule labelling.
Preferably, when described luminescent dye molecule is Fluorescein isothiocyanate (FITC, structural formula is as shown in M), described Covalent bond is the sulfur carbon amino linkage that the free amino group of tetrazine molecule is formed through phosphinylidyne ammonification.
Preferably, when described luminescent dye molecule is rhodamine (structural formula is as shown in N), and described covalent bond is tetrazine molecule The amido link that formed through dehydrating condensation of free amino group,
Preferably, described nir dye molecule is indole cyanine dyes molecule or chlorin molecule.
It is further preferred that the structural formula of described indole cyanine dyes molecule is as shown in L:
Wherein, X is Cl, Br, I or ClO4, Y is O, N, S or CH (CH3)2, R1For alkyl or aromatic radical, R2For alkyl or virtue Perfume base, R3For H, CH3、SO3 —Or CH3O, R4For H, CH3、SO3 —Or CH3O, n are 0,1 or 2.
It is further preferred that described indole cyanine dyes molecule is Cy3 or Cy5.5.
Specifically, the structural formula of described Cy3 and Cy5.5 is as follows:
The nir dye molecule that the present invention uses is preferably but is not limited to described indole cyanine dyes molecule.
Fourth aspect, the invention provides a kind of preparation method by the tetrazine probe of dye molecule labelling, including following Step:
(1), providing tetrazine molecule, the structural formula of described tetrazine molecule is as shown in P2:
Wherein, R1For
R2For H orDescribedIn, X is CH or N；
(2), providing dye molecule, described dye molecule is luminescent dye molecule or nir dye molecule；
(3), described dye molecule is marked on the amino of described tetrazine molecule, obtains by the tetrazine of dye molecule labelling Probe, described is included dye molecule and tetrazine molecule by the tetrazine probe of dye molecule labelling, and described dye molecule divides with tetrazine The amino of son forms covalent bond, and described dye molecule is connected by described covalent bond with tetrazine molecule.
Preferably, in described step (2), described luminescent dye molecule is rhodamine, Fluorescein isothiocyanate, O-phthalic Aldehyde, dansyl chloride, fluorenes methoxy dicarbonyl chloride, 6-quinolin-2-ylamine base-N-hydroxy-succinamide carbamate or N-9- Fluorenes methoxyl group phosphinylidyne-amino acidic group-N-carboxylic acid anhydrides.
The luminescent dye molecule that the present invention uses is preferably but is not limited to described rhodamine, Fluorescein isothiocyanate, adjacent benzene Dicarbaldehyde, dansyl chloride, fluorenes methoxy dicarbonyl chloride, 6-quinolin-2-ylamine base-N-hydroxy-succinamide carbamate or N-9-fluorenes methoxyl group phosphinylidyne-amino acidic group-N-carboxylic acid anhydrides.
Preferably, in described step (2), described nir dye molecule is indoles Molecule of Cyanine Dyes.
It is further preferred that the structural formula of described indole cyanine dyes molecule is as shown in L:
Wherein, X is Cl, Br, I or ClO4, Y is O, N, S or CH (CH3)2, R1For alkyl or aromatic radical, R2For alkyl or virtue Perfume base, R3For H, CH3、SO3 —Or CH3O, R4For H, CH3、SO3 —Or CH3O, n are 0,1 or 2.
It is further preferred that described indole cyanine dyes molecule is Cy3 or Cy5.
The nir dye molecule that the present invention uses is preferably but is not limited to described indole cyanine dyes molecule.
Preferably, in described step (3), when described luminescent dye molecule is that (FITC, structural formula is such as Fluorescein isothiocyanate Shown in M) time, described covalent bond is the sulfur carbon amino linkage that the free amino group of tetrazine molecule is formed through phosphinylidyne ammonification.
Preferably, in described step (3), when described luminescent dye molecule is rhodamine (structural formula is as shown in N), described common Valence link is the amido link that the free amino group of tetrazine molecule is formed through dehydrating condensation,
Preferably, in described step (3), on the described amino that dye molecule is marked at tetrazine molecule, obtain by dyestuff The operation of the tetrazine probe of molecular marker is:
Under conditions of N, N-dicyclohexylcarbodiimide and 4-dimethylamino pyridine exist, by dye molecule and compound P2 is dissolved in organic solvent by the mol ratio of 1:1～1.5, obtains reactant liquor, described reactant liquor react at 20～30 DEG C 0.5～ After 1 hour, through extraction, be dried, silica gel column chromatography separating purification obtains described by the tetrazine probe of dye molecule labelling；Wherein, The molar concentration of described N, N-dicyclohexylcarbodiimide and 4-dimethylamino pyridine be respectively described dye molecule 0.9～ 2.5 times and 0.1～0.5 times.
It is further preferred that described dye molecule is rhodamine, the structural formula of described rhodamine is as shown in N:
It is further preferred that described by the structural formula of the tetrazine probe of dye molecule labelling as shown in P3；
Under this optimum condition, the present invention uses 4-[1,2,4,5] tetrazine-3-base-benzylamine (Tz) to enter rhodamine B (Rb) Line flag: Tz and Rb is at condensing agent N, N-dicyclohexylcarbodiimide (DCC) and catalyst 4-dimethylamino pyridine (DMAP) The lower condensation of effect, generates tetrazine-rhodamine (Tz-Rb).Tz is the dienophile of non-copper catalysis, it is possible to divinyl macromer BCN often Temperature is lower there is quick coupling reaction such that it is able to quickly whether contain BCN-Cho on inspection cell membrane.
The preparation method that fourth aspect present invention provides uses ligand groups (1R, 8S, the 9r)-dicyclo of non-copper catalysis [6.1.0] nonyl-4-alkynyl-9-base methanol (BCN) modifies choline, it is to avoid metal catalytic is to cell and the toxic effect of live body； The highly effective reaction speed of the non-copper catalysis click chemistry of tetrazine group and BCN make choline be marked with higher sensitivity and Resolution.
The cholinomimetic that the present invention provides is formed containing choline by the phosphorylation of choline kinase at intracellular energy The phospholipid of analog, this phospholipid is incorporated on cell membrane by a series of intracellular metabolism, thus carries out former to cell Position labelling.Then being combined on surface of cell membrane with described by the tetrazine probe of dye molecule labelling of present invention offer be provided Cholinomimetic generation non-copper catalysis click chemical reaction, then by detecting the dye molecule on described tetrazine probe, can be right The cholinomimetic of surface of cell membrane carries out spike.
The present invention provide cholinomimetic and by the coupling reaction between the tetrazine probe of dye molecule labelling room temperature, Physiological environment just can quickly occur, and be catalyzed without copper, it is to avoid metal catalytic is to cell and the toxic effect of live body；Its Secondary, can high-resolution quick, highly sensitive the cholinomimetic on cell membrane be entered by detecting the dye molecule of described labelling Row location and spike.
5th aspect, the invention provides the gallbladder described in a kind of cholinomimetic as described in relation to the first aspect or the third aspect The application in preparing cell in-situ detection kit of the preparation method of alkali analog.
6th aspect, the invention provides a kind of as described in second aspect by the tetrazine probe or of dye molecule labelling Described in four aspects by the preparation method of the tetrazine probe of dye molecule labelling in preparing cell in-situ detection kit should With.
7th aspect, the invention provides a kind of cell in-situ detection compound, and described cell in-situ detection compound is Cholinomimetic as described in relation to the first aspect and carried out coupling by the tetrazine probe of dye molecule labelling as described in second aspect The compound obtained after reaction.
It is further preferred that the structural formula of described compound is as shown in P4:
In formula, k is the natural number of 2～4.
Cholinomimetic and its preparation method and application that the present invention provides, by dye molecule labelling tetrazine probe and Preparation method and application have the advantages that
1) present invention provide cholinomimetic and by the coupling reaction between the tetrazine probe of dye molecule labelling often Temperature, physiological environment just can quickly occur, and without copper catalysis, it is to avoid metal catalytic is to cell and the toxic effect of live body；
2) present invention provides cholinomimetic and by the coupling reaction between the tetrazine probe of dye molecule labelling after, logical Cross and detect the dye molecule of described labelling and can high-resolution quick, highly sensitive the cholinomimetic on cell membrane be positioned And spike；
3) cholinomimetic that the present invention provides is simple with by the preparation method of the tetrazine probe of dye molecule labelling, Large-scale production can be carried out, and production cost is low；
4) present invention provide cholinomimetic be can be used for preparing cell in-situ by the tetrazine probe of dye molecule labelling Detection kit.
Accompanying drawing explanation
The preparation flow figure of the cholinomimetic that Fig. 1 provides for the embodiment of the present invention 1～3；
The mass spectrum of the cholinomimetic that Fig. 2 provides for the embodiment of the present invention 1；
Fig. 3 for the embodiment of the present invention 4～6 provide by the preparation flow figure of the tetrazine probe of dye molecule labelling；
The fluorescence spectrum figure of the sample that Fig. 4 provides for the embodiment of the present invention 4.
Detailed description of the invention
The following stated is the preferred embodiment of the present invention, it is noted that for those skilled in the art For, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications are also considered as Protection scope of the present invention.
The preparation flow figure of a kind of cholinomimetic that Fig. 1 provides for the present invention, in conjunction with Fig. 1, the invention provides enforcement Example 1～3 is as follows:
Embodiment 1
The synthesis of a kind of cholinomimetic (BCN-Cho), comprises the steps:
1) activation (BCN-pNC) of BCN
In 100mL anhydrous methylene chloride, dissolve the BCN of 5mmol, wherein add the pyridine of 10mmol, after stirred for several minute Add the p-nitrophenyl chloro-formate (pNC) to 5mmol, at 20 DEG C, react 30min, add saturated ammonium chloride after terminating molten Liquid, extraction, organic facies anhydrous sodium sulfate is dried, and is evaporated, silica gel column chromatography separating purification, obtains white solid BCN-pNC and (produces Rate is 70%)；
2) synthesis of intermediate B CN-Br
The BCN-pNC of 4.4mmol is dissolved in the DMF (DMF) of 10mL, adds the 2-of 10mmol Bromine ethylamine hydrochloride and the triethylamine of 10mmol, react 30min at 20 DEG C.Mixture adds a certain amount of dichloromethane after concentrating Alkane, and with the NaOH solution of 1N and solution washing, organic facies anhydrous sodium sulfate is dried, and is evaporated, and silica gel column chromatography separates pure Change, obtain white solid BCN-Br (productivity is 70%)；
3) cholinomimetic synthesis (BCN-Cho) that BCN modifies
The BCN-Br of 5mmol is dissolved in the oxolane (THF) of 20mL, adds the dimethylethanolamine of 10mmol, After being sufficiently stirred for, react 12 hours under reflux state.Reactant liquor concentrates, silica gel column chromatography separating purification to white solid BCN- Cho (productivity is 60%).
BCN-Cho is carried out Mass Spectrometer Method, obtains mass spectrum (MS m/z): 309.2160 (M+).For absolutely proving the present invention Beneficial effect, the present embodiment additionally provides the mass spectrum of BCN-Cho, as shown in Figure 3.
Embodiment 2
The synthesis of a kind of cholinomimetic (BCN-Cho), comprises the steps:
1) activation (BCN-pNC) of BCN
In 100mL anhydrous methylene chloride, dissolve the BCN of 5mmol, wherein add the pyridine of 15mmol, after stirred for several minute Add the p-nitrophenyl chloro-formate (pNC) to 6mmol, at 25 DEG C, react 40min, add saturated ammonium chloride after terminating molten Liquid, extraction, organic facies anhydrous sodium sulfate is dried, and is evaporated, silica gel column chromatography separating purification, obtains white solid BCN-pNC and (produces Rate is 74%)；
2) synthesis of intermediate B CN-Br
The BCN-pNC of 4.4mmol is dissolved in the DMF (DMF) of 10mL, adds the 3-of 20mmol Bromine propylamin hydrochloride and the triethylamine of 15mmol, react 40min at 25 DEG C.Mixture adds a certain amount of dichloromethane after concentrating Alkane, and with the NaOH solution of 1N and solution washing, organic facies anhydrous sodium sulfate is dried, and is evaporated, and silica gel column chromatography separates pure Change, obtain white solid BCN-Br (productivity is 78%)；
3) cholinomimetic synthesis (BCN-Cho) that BCN modifies
The BCN-Br of 5mmol is dissolved in the oxolane (THF) of 20mL, adds the dimethylethanolamine of 15mmol, After being sufficiently stirred for, react 14 hours under reflux state.Reactant liquor concentrates, silica gel column chromatography separating purification to white solid BCN- Cho (productivity is 70%).
BCN-Cho is carried out Mass Spectrometer Method, obtains mass spectrum (MS m/z): 323.2327 (M+).
Embodiment 3
The synthesis of a kind of cholinomimetic (BCN-Cho), comprises the steps:
1) activation (BCN-pNC) of BCN
In 100mL anhydrous methylene chloride, dissolve the BCN of 5mmol, wherein add the pyridine of 20mmol, after stirred for several minute Add the p-nitrophenyl chloro-formate (pNC) to 7.5mmol, at 30 DEG C, react 60min, add saturated ammonium chloride after terminating molten Liquid, extraction, organic facies anhydrous sodium sulfate is dried, and is evaporated, silica gel column chromatography separating purification, obtains white solid BCN-pNC and (produces Rate is 69%)；
2) synthesis of intermediate B CN-Br
The BCN-pNC of 4.4mmol is dissolved in the DMF (DMF) of 10mL, adds the 4-of 15mmol Bromine butylamine hydrochloride and the triethylamine of 20mmol, react 60min at 30 DEG C.Mixture adds a certain amount of dichloromethane after concentrating Alkane, and with the NaOH solution of 1N and solution washing, organic facies anhydrous sodium sulfate is dried, and is evaporated, and silica gel column chromatography separates pure Change, obtain white solid BCN-Br (productivity is 80%)；
3) cholinomimetic synthesis (BCN-Cho) that BCN modifies
The BCN-Br of 5mmol is dissolved in the oxolane (THF) of 20mL, adds the dimethylethanolamine of 10mmol, After being sufficiently stirred for, react 16 hours under reflux state.Reactant liquor concentrates, silica gel column chromatography separating purification to white solid BCN- Cho (productivity is 75%).
BCN-Cho is carried out Mass Spectrometer Method, obtains mass spectrum (MS m/z): 337.2607 (M+).
Fig. 3 for the present invention provide a kind of by the preparation flow figure of the tetrazine probe of dye molecule labelling；In conjunction with Fig. 3, this Invention provides embodiment 4～6 as follows:
Embodiment 4
A kind of by the preparation method of the tetrazine probe of dye molecule labelling, comprise the following steps:
The rhodamine Rb of 5mmol is dissolved in the middle of the DMF of 5mL, add N, the N-dicyclohexylcarbodiimide of 4.5mmol (DCC), the 4-dimethylamino pyridine (DMAP) of 0.5mmol and 5mmol tetrazine (Tz), stirring reaction 12 hours at 20 DEG C, instead Should add dichloromethane after terminating, wash with saturated aqueous common salt, organic facies anhydrous sodium sulfate is dried, and concentrates, silica gel column chromatography Isolated and purified obtain red powder Tz-Rb (productivity is 75%).
Tz-Rb is carried out Mass Spectrometer Method, obtains mass spectrum (MS m/z): 613.3159 (M+).
Embodiment 5
A kind of by the preparation method of the tetrazine probe of dye molecule labelling, comprise the following steps:
Being dissolved by the rhodamine Rb of 5mmol in the middle of a certain amount of DMF, N, the N-dicyclohexyl carbon two adding 7.5mmol is sub- Amine (DCC), the 4-dimethylamino pyridine (DMAP) of 0.25mmol and 7mmol tetrazine (Tz), stirring reaction 14 hours at 25 DEG C, Reaction adds dichloromethane after terminating, and washs with saturated aqueous common salt, and organic facies anhydrous sodium sulfate is dried, and concentrates, silica gel column layer Analyse and isolated and purified obtain red powder Tz-Rb (productivity is 80%), and use thin layer chromatography (TLC) check analysis the present embodiment system Standby Tz-Rb.
Embodiment 6
A kind of by the preparation method of the tetrazine probe of dye molecule labelling, comprise the following steps:
The rhodamine (Rb) of 5mmol is dissolved in the middle of a certain amount of DMF, adds N, the N-dicyclohexyl carbon two of 12.5mmol Imines (DCC), the 4-dimethylamino pyridine (DMAP) of 0.25mmol and 7.5mmol tetrazine (Tz), stirring reaction 16 at 30 DEG C Hour, reaction adds dichloromethane after terminating, and washs with saturated aqueous common salt, and organic facies anhydrous sodium sulfate is dried, and concentrates, silica gel Column chromatographic isolation and purification obtains red powder Tz-Rb (productivity is 85%), and uses thin layer chromatography (TLC) this enforcement of check analysis Tz-Rb prepared by example.
Embodiment 7
For absolutely proving beneficial effects of the present invention, the present embodiment uses the cholinomimetic BCN-Cho of embodiment 1 preparation With being reacted by the tetrazine probe Tz-Rb of dye molecule labelling of embodiment 4 preparation, reaction equation is as follows:
For absolutely proving beneficial effects of the present invention, the present embodiment additionally provides described BCN-Cho to be carried out with described Tz-Rb The spectrofluorimetry of products therefrom after reaction, as comparison, has the most also carried out spectrofluorimetry to described Tz-Rb, knot As shown in Figure 4, wherein, curve 1 is the fluorescence spectrum of the cholinomimetic Tz-Rb of embodiment 1 preparation to fruit, and curve 2 is described BCN-Cho and described Tz-Rb react after the fluorescence spectrum of products therefrom.Having Fig. 4 to understand, the fluorescence intensity of curve 1 is less than Curve 2, the tetrazine ring of the most described Tz-Rb improves the fluorescence intensity of Tz-Rb with BCN-cho after reacting.
The present invention provide cholinomimetic and by the coupling reaction between the tetrazine probe of dye molecule labelling room temperature, Physiological environment just can quickly occur, and be catalyzed without copper, it is to avoid metal catalytic is to cell and the toxic effect of live body.
Result above has absolutely proved that the cholinomimetic using the present invention to provide is visited with by the tetrazine of dye molecule labelling Pin can quickly occur coupling reaction under conditions of being catalyzed without copper, can be used for the cholinomimetic on cell membrane is carried out location and Spike, has low toxicity, advantage quick, highly sensitive, high-resolution.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention Any amendment, equivalent and the improvement etc. made within god and principle, should be included within the scope of the present invention.

Claims (7)

1. one kind by the tetrazine probe of dye molecule labelling, it is characterised in that include dye molecule and tetrazine molecule, described dyestuff Molecule forms covalent bond with the amino of tetrazine molecule, and described dye molecule is connected by described covalent bond with tetrazine molecule, described Dye molecule is luminescent dye molecule or nir dye molecule, and the structural formula of described tetrazine molecule is as shown in P2:
Wherein, R2For
R3For H orDescribedIn, X is CH or N.
2. as claimed in claim 1 by the tetrazine probe of dye molecule labelling, it is characterised in that
Described luminescent dye molecule is rhodamine, Fluorescein isothiocyanate, o-phthalaldehyde(OPA), dansyl chloride, fluorenes first Oxygen dicarbonyl chloride, 6-quinolin-2-ylamine base-N-hydroxy-succinamide carbamate or N-9-fluorenes methoxyl group phosphinylidyne-amino acidic group-N- Carboxylic acid anhydrides；Described nir dye molecule is indole cyanine dyes molecule or chlorin molecule.
3. one kind by the preparation method of the tetrazine probe of dye molecule labelling, it is characterised in that comprise the following steps:
(1), providing tetrazine molecule, the structural formula of described tetrazine molecule is as shown in P2:
Wherein, R1For
R2For H orDescribedIn, X is CH or N；
(2), providing dye molecule, described dye molecule is luminescent dye molecule or nir dye molecule；
(3), described dye molecule is marked on the amino of described tetrazine molecule, obtains being visited by the tetrazine of dye molecule labelling Pin, described is included dye molecule and tetrazine molecule by the tetrazine probe of dye molecule labelling, described dye molecule and tetrazine molecule Amino formed covalent bond, described dye molecule is connected by described covalent bond with tetrazine molecule.
4. as claimed in claim 3 by the preparation method of the tetrazine probe of dye molecule labelling, it is characterised in that described step (2), in, described luminescent dye molecule is rhodamine, Fluorescein isothiocyanate, o-phthalaldehyde(OPA), dansyl chloride, fluorenes Methoxy dicarbonyl chloride, 6-quinolin-2-ylamine base-N-hydroxy-succinamide carbamate or N-9-fluorenes methoxyl group phosphinylidyne-amino acidic group- N-carboxylic acid anhydrides；Described nir dye molecule is indole cyanine dyes molecule.
5. one kind as claimed in claim 3 by the preparation method of the tetrazine probe of dye molecule labelling, it is characterised in that described Described dye molecule is marked on the amino of tetrazine molecule in (3) by step, obtains by the tetrazine probe of dye molecule labelling Operation is:
Under conditions of N, N-dicyclohexylcarbodiimide and 4-dimethylamino pyridine exist, dye molecule is pressed with compound P2 The mol ratio of 1:1～1.5 is dissolved in organic solvent, obtains reactant liquor, and it is little that described reactant liquor reacts 0.5～1 at 20～30 DEG C Shi Hou, through extraction, be dried, silica gel column chromatography separating purification obtains described by the tetrazine probe of dye molecule labelling；Wherein, described The molar concentration of N, N-dicyclohexylcarbodiimide and 4-dimethylamino pyridine is respectively 0.9～2.5 times of described dye molecule With 0.1～0.5 times.
6. as claimed in claim 1 by the tetrazine probe of dye molecule labelling answering in preparing cell in-situ detection kit With.
7. a cell in-situ detection compound, it is characterised in that described cell in-situ detection compound is that a kind of choline is similar to Thing and the compound obtained after as claimed in claim 1 being carried out coupling reaction by the tetrazine probe of dye molecule labelling, wherein, The structural formula of described cholinomimetic is as shown in P1:
In formula, k is the natural number of 2～4.
CN201610348541.2A 2013-12-31 2013-12-31 Dye-molecule-marked tetrazine probe, and preparation method and application thereof Pending CN106010507A (en)

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