CN102168104A - Coding molecule for marking biomolecule and preparing method thereof - Google Patents
Coding molecule for marking biomolecule and preparing method thereof Download PDFInfo
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- CN102168104A CN102168104A CN 201010592986 CN201010592986A CN102168104A CN 102168104 A CN102168104 A CN 102168104A CN 201010592986 CN201010592986 CN 201010592986 CN 201010592986 A CN201010592986 A CN 201010592986A CN 102168104 A CN102168104 A CN 102168104A
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Abstract
The invention relates to a coding molecule for marking biomolecules and a preparing method thereof. The coding molecule has a chain or dendritic shaped main skeleton which is provided with one or more than two luminescence groups along the length direction of the main skeleton at different positions, and the end of which is provided with a function group able to connect with bioactive molecules. The luminescence group consists of cyclometalated iridium coordination compound, cyclometalated ruthenium coordination compound, and rare earth metal coordination compound. The preparing method comprises: taking the luminescence group to be connected to the compound having more than two reaction sites to form a luminescence unit; taking a plurality of luminescence units to form the coding molecule having a main skeleton; or taking compound having more than two reaction sites for reacting to form the main skeleton of the coding molecule, and then connecting the luminescence group to the main skeleton to form coding molecule via the reacting of the active groups distributed upon the branched chain of the main skeleton with the corresponding active groups upon the luminescence groups. The invention allows selective coding over biomolecules.
Description
Technical field
The present invention be more particularly directed to a kind of functional groups such as carboxyl that contain, can carry out fluorescently-labeled coding molecule and its production and application in order to antagonist, antigen, enzyme, polypeptide, protein, amino modified biologically active substances such as nucleic acid.
Background technology
One peacekeeping two-dimensional bar technology is widely used in the article identification process based on computer management.When encoding and be used to recognize when reading the littler article of yardstick, the application of barcode just is restricted.Usually can only in outer packaging, print barcode.When " article " that need mark or coding are little when being biomacromolecule, traditional mode is to adopt the microballoon of the fluorescent substance of different emission that different biological molecules is encoded.This technology has been successfully applied to the high-throughput bio-analysis system of Luminex.Thereafter, people such as Nie also develop to utilize semiconductor-quantum-point etc. to carry out Methods for Coding (to consult " Quantum-dot-taggedmicrobeads for multiplexed optical coding of biomolecules ", NaturaBiotechnol.2001,19,631).This case contriver also once attempted adopting same quantum-dot coding method to develop a kind of encryption technology and (consulted " Cryptography based on the absorption/emissionfeatures of multicolor semiconductor nanocrystal quantum dots ", OpticsExpress, 2004,12,2925).But this type of method that adopts fluorescent microsphere and quantum-dot coding is all because of microballoon itself has certain volume, thereby is difficult to make the carrier of coded message further to narrow down to and can carry out fluorescence-encoded degree to single biomacromolecule.
At present, usually adopt fluorescence molecule that biomolecules is carried out the method for mark in the bioanalysis, this can be considered as a kind of coding of simple one digit number.Fluorescence molecule commonly used mainly contains organic fluorescence molecule and rare earth luminescence coordination compound etc.Wherein, organic fluorescent substance has the emmission spectrum of broad usually, also be difficult to a plurality of luminescence units of excitation light source excites, and the photochemical stability of organic fluorescent substance is not so good; And the rare earth luminescence coordination compound has very narrow emission peak, but different rare earth ion title complexs also needs different excitation light sources.
Recently, the investigator by on carbon nitrogen and nitrogen nitrogen ligand, introduce aldehyde radical, isosulfocyanate radical, iodo-acid amide and vitamin H etc. can with bioactive molecules link coupled group, some bioactive molecules has been carried out mark (has consulted " Biological labelling reagents and probes derived from luminescenttransition metal polypyridine com
Summary of the invention
The objective of the invention is to propose a kind of coding molecule that is used for the mark biomolecules and preparation method thereof, the molecule that has connected a plurality of different luminescence units in this coding molecule, each coding molecule has different luminosities, and the coding molecule end be provided with can with biologically active substance bonded active group, can encode to different biologically active substances thus, thereby overcome deficiency of the prior art.
For achieving the above object, the present invention has adopted following technical scheme:
A kind of coding molecule that is used for the mark biomolecules, it is characterized in that, described coding molecule has chain or dendroid main body framework, along its length different positions place has one or more luminophores on this main body framework, and the terminal setting of this main body framework functional group that can be connected with bioactive molecules;
Described luminescence unit comprises Cyclometalated iridium coordination compound, ring metal Ru coordination compound and rare earth metal coordination compound.
In a preferred embodiment, described coding molecule is main body framework with the peptide chain, and this main body framework art end be provided with can with the carboxyl of amino coupled in the bioactive molecules.This coding molecule can be under EDC and NHS effect, and with the biomolecular reaction that has amido, thereby realization is to the mark of biomolecules.
Order between distance between the aforementioned luminophore and the different luminophore can change.
Be connected with one or more water soluble groups on described main body framework and/or the luminophore, described water soluble group comprises sodium group.
The preparation method of coding molecule is characterized in that as mentioned above, and this method is:
Get luminophore and be connected on the compound with two above reaction site and form luminescence unit, get a plurality of luminescence units reactions again and form coding molecule with main body framework;
Or, get the main body framework of compound reaction formation coding molecule with two above reaction site, by being distributed in the corresponding active group reaction on active group on the main body framework side chain and the luminophore, luminophore being connected to forming coding molecule on the main body framework again.
The preparation method of coding molecule is characterized in that as mentioned above, and this method comprises the steps:
Get the luminophor and the amino acid reaction that have reaction site and obtain luminescence unit; To the amido and the carboxyl deprotection of aforementioned luminescence unit, obtain free amido and carboxyl respectively, utilizing polypeptid synthesising process that luminescence unit is connected to form with the peptide chain by amido linkage again is the target product of main body framework.
The present invention has prepared a kind of novel coding molecule, along with the number and the kind of luminophor on every kind of coding molecule changes, makes that the luminosity of every kind of coding molecule is all inequality, thereby can carry out the selective coding to biomolecules.The present invention also utilizes the carboxyl isoreactivity group of the terminal band of coding molecule, by biological linked reaction protein, polypeptide, antibody and amino modified nucleic acid etc. is contained amino bioactive molecules and is connected, thereby realize coding to bioactive molecules.
Embodiment
At the defective of existing biomarker method, this case contriver proposes a kind of synthetic method that reaches with this coding molecule mark biologically active substance of novel coding molecule through studying for a long period of time and putting into practice.
Following is the technical process of a kind of novel coding molecule and mark biologically active substance thereof among the present invention, and it comprises the steps:
(1) luminescence unit is synthetic
This luminescence unit obtains luminescence unit by luminophor and the amino acid reaction that has reaction site.Its reaction expression is as follows:
(2) connection of luminescence unit
Respectively to the amido and the carboxyl deprotection of luminescence unit; obtain free amido and carboxyl; utilize peptide synthesis technology that luminescence unit (identical or different) is connected by amido linkage, form, repeat aforesaid operations and can obtain final coding molecule as connecting more luminescence unit.Its reaction expression is as follows:
(3) biomarker
This technological process can adopt following two kinds of methods: a kind of method is to utilize carboxylic acid on the coding molecule and the amino on the biomolecules at EDC[1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride] and NHS (N-hydroxy-succinamide) effect formation peptide bond down, its reaction expression is as follows:
Another kind method is that the coding molecule that has carboxyl is converted into succinimide ester by reacting with NHS under DCC (dicyclohexylcarbodiimide) effect, and the latter forms peptide bond with amino again in buffered soln, and its reaction expression is as follows:
Below in conjunction with some preferred embodiments technical scheme of the present invention is elaborated.
Embodiment 1: main body framework partly is the synthetic of linear coding molecule
The synthesis step of compound 3: get compound 1 (1mmol), HOSu (1mmol) adds 20 milliliters of exsiccant DMF in one 50 ml flasks, add DCC (1mmol), stirring at room 20 hours, add Boc-Methionin (2mmol), DIEA (1mmol), stirring at room 24 hours, reaction finishes, revolve and desolvate, cross post, get product.Ultimate analysis: C, 49.42%; H, 3.82%; N, 7.04% (measured value); C, 49.39%; H, 3.8/4%; N, 7.02% (C
41H
38F
4IrN
5O
8Theoretical value).
The synthesis step of compound 4: get compound 2 (1mmol), Boc-lysine methyl ester (2mmol), DMAP (1mmol) is in one 100 ml flasks, add 20 milliliters of toluene, add DCC (1mmol), stirring at room 2 hours, reaction finishes, revolve and desolvate, add 60 milliliters of methylene dichloride, add trifluoroacetic acid (6mmol), stirring at room 24 hours, reaction finishes, and revolves to desolvate, and crosses post and gets product.Ultimate analysis: C, 59.53%; H, 4.25%; N, 7.11% (measured value); C, 59.50%; H, 4.28%; N, 7.08% (C
49H
42IrN
5O
6Theoretical value).
The synthesis step of compound 5: get compound 3 (1mmol), be dissolved in DMF, add HOAt (1.5mmol), compound 2 (1mmol) is cooled to-20 degree, adds EDC (1.5mmol), stirring reaction 16 hours, reaction finishes, and adds 1N hydrochloric acid, use ethyl acetate extraction, organic phase is used saturated aqueous common salt respectively, sodium hydrogen carbonate solution, saturated common salt washing, revolve and desolvate, cross post and get product.This product is dissolved among the 20mL THF, adds 10mL aqueous sodium hydroxide solution (1N), stirring at room 4.5 hours, reaction finishes, and drips dilute hydrochloric acid solution in reaction solution, transfers slightly acidic, has solid to separate out suction filtration, and water, normal hexane washing get product respectively.Ultimate analysis: C, 54.56%; H, 3.87%; N, 7.04% (measured value); C, 54.54%; H, 3.86%; N, 7.07% (C
90H
76F
4Ir
2N
10O
14Theoretical value).
Embodiment 2: main body framework partly is coding molecule synthetic of branch type
Compound 10
The synthesis step of compound 9: get compound 6 (1mmol), DMAP (1mmol) is in one 100 ml flasks, add 20 milliliters of DMF, get compound 8 (1mmol) and be made into DMF solution, under magnetic agitation, the drips of solution that is made into is added in the reaction flask, dropwise, stirring at room 24 hours, reaction finishes, and revolves to desolvate, and crosses post and gets product.Ultimate analysis: C, 63.86%; H, 4.95%; N, 6.05% (measured value); C, 63.84%; H, 4.92%; N, 6.08% (C
49H
45EuN
4O
5Theoretical value).
The synthesis step of compound 10: get compound 7 (1mmol) in one 100 ml flasks, add 20 milliliters of DMA, get compound 9 (1mmol) and add 10 milliliters of DMA wiring solution-formings, under agitation this drips of solution is added in the reaction flask, dropwise, stirring at room 30 minutes, reaction finishes, and reaction solution is poured in the water, there is solid to separate out, suction filtration, drying gets product.This product is dissolved among the 20mL THF, adds 10mL aqueous sodium hydroxide solution (1N), stirring at room 5 hours, reaction finishes, and drips dilute hydrochloric acid solution in reaction solution, transfers slightly acidic, has solid to separate out suction filtration, and water, normal hexane washing get the finished product respectively.Ultimate analysis: C, 50.65%; H, 3.15%; N, 7.61% (measured value); C, 50.67%; H, 3.10%; N, 7.65% (C
85H
62EuN
11Na
4O
17RuS
5Theoretical value).
Embodiment 3 compounds 5 mark BSA (bovine serum albumin) under EDC and NHS effect
In the PBS buffered soln (pH=7.4) of compound 5 (1mmol), add NHS (1.05mmol) and EDC (1mmol), at room temperature reacted 30 minutes, add BSA then, stirred overnight at room temperature.The mixture molecular weight cut-off of gained is that the dialysis tubing of the 10000Da purifying of dialysing in PBS solution was removed uncrosslinked compound in three days, promptly obtains the BSA of crosslinked underlined compound.
Claims (6)
1. coding molecule that is used for the mark biomolecules, it is characterized in that, described coding molecule has chain or dendroid main body framework, along its length different positions place has one or more luminophores on this main body framework, and the terminal setting of this main body framework functional group that can be connected with bioactive molecules;
Described luminophore comprises Cyclometalated iridium coordination compound, ring metal Ru coordination compound and rare earth metal coordination compound.
2. the fluorescently-labeled coding molecule of biologically active substance that is used for according to claim 1 is characterized in that described coding molecule is main body framework with the peptide chain, and this main body framework end be provided with can with the carboxyl of amino coupled in the bioactive molecules.
3. the fluorescently-labeled coding molecule of biologically active substance that is used for according to claim 1 is characterized in that be connected with one or more water soluble groups on described main body framework and/or the luminophore, described water soluble group comprises sodium group.
4. the fluorescently-labeled coding molecule of biologically active substance that is used for according to claim 2 is characterized in that described coding molecule is under EDC and NHS effect, with the biomolecular reaction that has amido, thereby realizes biomolecules is carried out mark.
5. be used for the preparation method of the fluorescently-labeled coding molecule of biologically active substance according to claim 1, it is characterized in that, this method is:
Get luminophore and be connected on the compound with two above reaction site and form luminescence unit, get a plurality of luminescence units reactions again and form coding molecule with main body framework;
Or, get the main body framework of compound reaction formation coding molecule with two above reaction site, by being distributed in the corresponding active group reaction on active group on the main body framework side chain and the luminophore, luminophore being connected to forming coding molecule on the main body framework again.
6. the preparation method who is used for the fluorescently-labeled coding molecule of biologically active substance according to claim 5 is characterized in that this method comprises the steps:
Get the luminophor and the amino acid reaction that have reaction site and obtain luminescence unit;
To the amido and the carboxyl deprotection of aforementioned luminescence unit, obtain free amido and carboxyl respectively, utilizing polypeptid synthesising process that luminescence unit is connected to form with the peptide chain by amido linkage again is the target product of main body framework.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015527338A (en) * | 2012-08-02 | 2015-09-17 | エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ | New bis-iridium complexes for ECL production |
| JP2015530977A (en) * | 2012-08-02 | 2015-10-29 | エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ | New iridium-based complexes for ECL |
| JP2015530978A (en) * | 2012-08-02 | 2015-10-29 | エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ | New iridium-based complexes for ECL |
| CN109682967A (en) * | 2018-12-17 | 2019-04-26 | 暨南大学 | Application of the PAMAM in the reagent that preparation is used for immune detection |
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| CN1772729A (en) * | 2005-11-14 | 2006-05-17 | 武汉大学 | Polymerisable fluorescent functional monomer and its prepn and use |
| CN101747381A (en) * | 2008-12-19 | 2010-06-23 | 苏州纳凯科技有限公司 | Cyclometalated iridium coordination compound used as biomarker |
| CN101750486A (en) * | 2008-12-19 | 2010-06-23 | 苏州纳凯科技有限公司 | Method for labeling antibody by fluorophore generated by combination of iridium coordination compound and histidine |
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| CN101747381A (en) * | 2008-12-19 | 2010-06-23 | 苏州纳凯科技有限公司 | Cyclometalated iridium coordination compound used as biomarker |
| CN101750486A (en) * | 2008-12-19 | 2010-06-23 | 苏州纳凯科技有限公司 | Method for labeling antibody by fluorophore generated by combination of iridium coordination compound and histidine |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015527338A (en) * | 2012-08-02 | 2015-09-17 | エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ | New bis-iridium complexes for ECL production |
| JP2015530977A (en) * | 2012-08-02 | 2015-10-29 | エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ | New iridium-based complexes for ECL |
| JP2015530978A (en) * | 2012-08-02 | 2015-10-29 | エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ | New iridium-based complexes for ECL |
| US10227366B2 (en) | 2012-08-02 | 2019-03-12 | Roche Diagnostics Operations, Inc. | Bis-iridium-complexes for manufacturing of ECL-labels |
| CN109682967A (en) * | 2018-12-17 | 2019-04-26 | 暨南大学 | Application of the PAMAM in the reagent that preparation is used for immune detection |
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Application publication date: 20110831 |