CN102219791A - Color biotin used for large biological molecule marking, as well as applications in preparing large biological molecule marking reagent - Google Patents
Color biotin used for large biological molecule marking, as well as applications in preparing large biological molecule marking reagent Download PDFInfo
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- CN102219791A CN102219791A CN2011100876553A CN201110087655A CN102219791A CN 102219791 A CN102219791 A CN 102219791A CN 2011100876553 A CN2011100876553 A CN 2011100876553A CN 201110087655 A CN201110087655 A CN 201110087655A CN 102219791 A CN102219791 A CN 102219791A
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- biotin
- biomacromolecule
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- biological element
- color
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- 0 *C(c1ccc(C(c(cc2)ccc2O)=C(C=C2)C=CC2=O)c(C(O)=O)c1)=O Chemical compound *C(c1ccc(C(c(cc2)ccc2O)=C(C=C2)C=CC2=O)c(C(O)=O)c1)=O 0.000 description 1
- BMAYSFOJWCKKPU-UHFFFAOYSA-O CCC(c1cc(OCC[NH3+])cc(OCCN)c1)=O Chemical compound CCC(c1cc(OCC[NH3+])cc(OCCN)c1)=O BMAYSFOJWCKKPU-UHFFFAOYSA-O 0.000 description 1
- UMRBNESUAJXRDG-UHFFFAOYSA-N CCOC(C(CCCCN)NC(c1ccc(C(c(cc2)ccc2OC(C)=O)(c(cc2)ccc2OC(C)=O)OC2=O)c2c1)=O)=O Chemical compound CCOC(C(CCCCN)NC(c1ccc(C(c(cc2)ccc2OC(C)=O)(c(cc2)ccc2OC(C)=O)OC2=O)c2c1)=O)=O UMRBNESUAJXRDG-UHFFFAOYSA-N 0.000 description 1
- WBDPEHUAZGWQLE-UHFFFAOYSA-N CCOC(C(CCCCNC(OC(C)(C)C)=O)N)=O Chemical compound CCOC(C(CCCCNC(OC(C)(C)C)=O)N)=O WBDPEHUAZGWQLE-UHFFFAOYSA-N 0.000 description 1
- XPJDXILQMZXKJT-UHFFFAOYSA-N CCOC(C(CCCCNC(OC(C)(C)C)=O)NC(c1ccc(C(c(cc2)ccc2OC(C)=O)(c(cc2)ccc2OC(C)=O)OC2=O)c2c1)=O)=O Chemical compound CCOC(C(CCCCNC(OC(C)(C)C)=O)NC(c1ccc(C(c(cc2)ccc2OC(C)=O)(c(cc2)ccc2OC(C)=O)OC2=O)c2c1)=O)=O XPJDXILQMZXKJT-UHFFFAOYSA-N 0.000 description 1
- KKXQYHBPRRISRZ-UHFFFAOYSA-O [NH3+]c1ccc(C(c(cc2)ccc2O)=C(C=C2)C=CC2=O)c(C(O)=O)c1 Chemical compound [NH3+]c1ccc(C(c(cc2)ccc2O)=C(C=C2)C=CC2=O)c(C(O)=O)c1 KKXQYHBPRRISRZ-UHFFFAOYSA-O 0.000 description 1
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Abstract
The invention relates to a color biotin used for large biological molecule marking, as well as preparation method thereof. The indicator can be used for trace antigen, antibody determination, enzyme immunity determination, immunofluorescence assay, quantitive detection and new researched positioning observation technology, such as tumor development diagnosis, targeted treatment, and the like. After the color biotin is conjugated with large molecular active material, the conjugate is not developed under physiology conditions, thus having no interference on application of biotin and avidin under normal conditions. Amaranth can be developed after pH is adjusted to certain value, and the absorbance is relative to the content of biotin, thus being capable of calculating the mol number of biotin contained in each molecular active material according to the absorbance. The biotin conjugate has similar function after being combined with the avidin specifically, thus realizing tracer analysis process with naked eyes, greatly improving efficiency, sensitivity and repeatability, and filling blank of international technology.
Description
Technical field
The present invention relates to the vitamin H indicator, especially a kind of band colour biological that is used for the biomacromolecule mark is plain and in the application of preparation biomacromolecule labelled reagent, this indicator can be used for that micro-antigen, antibody are qualitative, the new technology of enzyme immunoassay, fluorescence immunoassay, detection by quantitative and position observation research, as carries out tumor imaging diagnosis and targeted therapy etc.
Background technology
Vitamin H is a kind of small molecules somatomedin that extensively distributes in the animal and plant body, biotin-avidin system (Biotin-Avidin System, BAS) be the strongest up to now non-covalent coupling system, in biomedical technology, obtained using widely, almost can bind and close with the various marking objects of present research success.This system both can be used for a series of macromole active substances such as coupling antibody, can be used for the mark of multiple materials such as enzyme again, had the characteristics of highly sensitive, high specific, high stability.The mortise of biotin-avidin and labelled reagent high affinity and multistage scale effect make the immune labeled and relevant tracer analysis of BAS sensitive more.It become be widely used at present that active protein extracts, antigen-antibody is qualitative, the new technology of enzyme-linked immunoassay, fluorescence immunoassay, detection by quantitative and position observation research, as carry out tumor imaging diagnosis and targeted therapy, but because therefore the no charateristic avsorption band of biotin molecule itself is unfavorable for that spike detects and observation.
Traditional to the hydroxyphenyl azobenzoic acid (4 '-hydroxyazobenzene-2-carboxylic acid, HABA) method is earlier HABA to be mixed with avidin, HABA and avidin combine a little less than forming one, in conjunction with after complex compound at the 500nm place one maximum absorption band is arranged, to need quantitative biotin labeling thing to join in the mixed solution of HABA and avidin then, vitamin H is competed with avidin with HABA and is combined, thereby HABA is replaced the absorbancy that has reduced the 500nm place by vitamin H.Because the competitive effect of vitamin H and HABA to absorbancy to influence diversity ratio less, so this method sensitivity is very low, accuracy of measurement is not enough and can consume substrate.The HABA method only is suitable for the bigger protein molecule of concentration at present,
The SureLINK vitamin H method of existing report has broken through the restriction of traditional HABA method, can detect optical density under the 354nm wavelength, but still the need ultraviolet spectrophotometer is auxiliary and sensitivity is relatively low.
Still there is not at present the indicator that can carry out tracer analysis macromole labeling process in the world by naked eyes.Domestic use be the import like product mostly, but imported product can only be measured labeling process under ultraviolet, and costs an arm and a leg.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art part, provide the band colour biological that is used for the biomacromolecule mark of a kind of efficient height, sensitivity and favorable reproducibility plain and in the application of preparation biomacromolecule labelled reagent.
The objective of the invention is to be achieved through the following technical solutions:
A kind of band colour biological element that is used for the biomacromolecule mark, structural formula is as follows:
Wherein, Y has the functional group of at least three linkage function groups, and wherein three linkage function groups are connected with A, B and vitamin H respectively, and B represents the triphenylmethane indicator, the A representative
And described A is
And described Y is
And described B comprises
And described B comprises
R is CH
3
And described B comprises
R is Br or Cl.
And described B is
And the structural formula of described band colour biological element is
Be used for of the application of the band colour biological element of biomacromolecule mark at preparation biomacromolecule labelled reagent.
Advantage of the present invention and positively effect are as follows:
1, the present invention modify to introduce chromophore by biotin derivative being carried out side chain, not only makes biomacromolecule after by biotin labeling the colour developing ability be arranged promptly, and make vitamin H with also can in specific pH scope, develop the color after avidin combines.This band colour biological element is after with the coupling of macromole active substance, and conjugate does not develop the color under physiological condition, thereby can interference not arranged to the application of vitamin H and avidin under normal operation.But when regulating pH displaing amaranth after the certain numerical value, its absorbancy is relevant with the content of vitamin H, thereby can calculate the mole number of the contained vitamin H of per molecule active substance from its absorbancy.The plain conjugate of this band colour biological with the avidin specific combination after similar functions is also arranged, therefore realized under naked eyes, carrying out the tracer analysis process, improved efficient, sensitivity and circulation ratio greatly, filled up the international technology blank
2, the present invention finishes the design of multiple terminal reactive group by polystep reaction, unique three-dimensional frame design is with vitamin H, chromophore and be connected the end group perfect adaptation of biomacromolecule, this indicator can be combined with amino, carboxyl, sulfydryl, aldehyde radical etc., a series of macromole active substances such as coupling antibody have increased the diversity of product.
3, the present invention is an end product with the plain molecule of band colour biological, and new synthesis route and processing parameter are finished in the design research and development, reaction conditions gentleness, safety, and product purity can reach more than 99.0%.
Description of drawings: please with in all nuclear-magnetism figure stickups.
Fig. 1 is the nuclear magnetic spectrogram of intermediate 2 among the concrete preparation method 1 of the present invention;
Fig. 2 is the nuclear magnetic spectrogram of intermediate 3 among the concrete preparation method 1 of the present invention;
Fig. 3 is the nuclear magnetic spectrogram of intermediate 7 among the concrete preparation method 1 of the present invention;
Fig. 4 is the nuclear magnetic spectrogram of intermediate 8 among the concrete preparation method 1 of the present invention;
Fig. 5 is the nuclear magnetic spectrogram of intermediate 9 among the concrete preparation method 1 of the present invention;
Fig. 6 is the nuclear magnetic spectrogram of intermediate 11 among the concrete preparation method 1 of the present invention;
Fig. 7 is the nuclear magnetic spectrogram of intermediate 13 among the concrete preparation method 1 of the present invention;
Fig. 8 is the nuclear magnetic spectrogram of intermediate 14 among the concrete preparation method 1 of the present invention;
Fig. 9 does not show the photo of color when neutrality for vitamin H of the present invention;
Figure 10 is the photo of vitamin H of the present invention displaing amaranth when pH changes.
Embodiment
Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
A kind of band colour biological element that is used for the biomacromolecule mark, structural formula is as follows:
Wherein, Y has the functional group of at least three linkage function groups, and wherein three linkage function groups are connected with A, B and vitamin H respectively, and B represents the triphenylmethane indicator, the A representative
Described Y is
Described B comprises
Wherein R is halogen, alkane, NO
2, SO
3H, CN, CHO or COOH.
Described B comprises that R is CH
3
Described B comprises that R is Br or Cl.
Described B is
The structural formula of band colour biological element is
Also comprise as the various combinations of the molecular formula of Y, A, B representative, just do not enumerate one by one.
Be used for of the application of the band colour biological element of biomacromolecule mark at preparation biomacromolecule labelled reagent.
The present invention finishes the design of multiple terminal reactive group by polystep reaction, unique three-dimensional frame design is with vitamin H, chromophore and be connected the end group perfect adaptation of biomacromolecule, this indicator can be combined with amino, carboxyl, sulfydryl, histidine residues etc., can coupling antibody etc. a series of macromole active substances.
A kind of preparation method who is used for the band colour biological element of biomacromolecule mark, present method is an example with a kind of concrete structure, same present method of synthetic method of the band colour biological element of all the other structures, concrete steps are as follows:
The concrete steps of the reaction of each step are as follows:
Synthesizing of intermediate 2
Weighing 1 (70g, 0.36mol) and phenol (68.6g 0.73mol) puts into the 500mL there-necked flask, is heated to 80 ℃, treats that phenol all melts, and is warming up to 120 ℃ then, the reaction 18h.Add the 500mL methylene dichloride, with the saturated sodium bicarbonate solution washing repeatedly, merge water, transfer pH to 2 with the hydrochloric acid of 1mol/L, a lot of thick solids are separated out, and filter, and oven dry is carried out recrystallization with chloroform, obtain light brown solid 25g, productive rate 18.94%, figure is as follows for its nuclear-magnetism:
1H-NMR(DMSO，300MHz)δ＝9.711(s，1H)，δ＝7.656-8.316(m，3H)，δ＝7.023-7.051(m，4H)，δ＝6.719-6.756(m，4H)
Synthesizing of intermediate 3
Weighing intermediate 2 (20g, 0.055mol) in the there-necked flask of 1000mL, the diacetyl oxide of measuring 400mL adds in the there-necked flask, is heated to 80 ℃, reaction 10h is spin-dried for diacetyl oxide, carries out column chromatography for separation and purifies, obtain 10g intermediate 3 at last, productive rate 40.58%, figure is as follows for its nuclear-magnetism:
1H-NMR(CDCl
3，300MHz)δ＝7.726-8.219(m，3H)，δ＝7.327-7.438(m，4H)，δ＝7.158-7.216(m，4H)，δ＝2.248(s，6H)
Synthesizing of intermediate 4
(10g 0.0224mol) is dissolved in the 40mL sulfur oxychloride weighing intermediate 3, is heated to backflow, and reaction 20h, TLC follow the tracks of reaction to complete, and stopped reaction is spin-dried for sulfur oxychloride, and purifying does not directly drop into next step reaction, weighing crude product 10g.
Synthesizing of intermediate 6
The weighing lysine hydrochloride (50g 0.228mol) puts into the there-necked flask of 2000mL, adds 1200mL ethanol, and the 10mL concentrated hydrochloric acid is heated to backflow, reaction 20h, and stopped reaction is spin-dried for ethanol and concentrated hydrochloric acid, obtains the mixture 60g of 5 and 6 hydrochloride.
Synthesizing of intermediate 7
(40g 0.16mol) joins in the there-necked flask of 1000mL the mixture of weighing intermediate 6, and the saturated sodium bicarbonate solution that slowly adds 500mL is with its whole dissolvings.Weighing tert-Butyl dicarbonate (34.9g then, 0.16mol) be dissolved in the 100mL methyl alcohol, then it being splashed in the there-necked flask with slower speed, the 5h afterreaction is complete, stopped reaction, with methylene dichloride aqueous phase extracted repeatedly, the combined dichloromethane layer is used anhydrous sodium sulfate drying, column chromatography for separation is purified after being spin-dried for solvent, obtain 12g intermediate 7, productive rate 19.05%, figure is as follows for its nuclear-magnetism:
1H-NMR(CDCl
3，300MHz)δ＝4.095-4.200(m，3H)，δ＝2.670-2.716(m，2H)，δ＝1.444-1.743(m，5H)，δ＝1.379(s，10H)，δ＝1.194-1.217(t，3H)。
Synthesizing of intermediate 8
Weighing intermediate 7 (2.95g, 10.76mmol) and triethylamine (2.2g 21.52mmol) adds in the there-necked flask of 250mL, and the methylene dichloride that adds 20mL is with its whole dissolvings.Then weighing intermediate 4 (5g 10.76mmol), is dissolved in it in methylene dichloride of 100mL, slowly splash in the there-necked flask, normal-temperature reaction, TLC follows the tracks of reaction to fully.Then methylene dichloride is spin-dried for, column chromatography for separation obtains 3g intermediate 8, productive rate 39.59%, and figure is as follows for its nuclear-magnetism:
1H-NMR(CDCl
3，300MHz)δ＝7.933-8.255(m，3H)，δ＝7.295-7.348(m，4H)，δ＝7.040-7.077(m，4H)，δ＝4.129-4.294(m，3H)，δ＝3.404-3.485(t，2H)，δ＝2.284(s，6H)，δ＝1.758-1.896(m，1H)，δ＝1.592-1.722(m，3H)，δ＝1.380(s，9H)，δ＝1.303-1.316(m，2H)，δ＝1.221-1.284(m，5H)。
Synthesizing of intermediate 9
(2g 2.84mmol) is dissolved in the 10mL methylene dichloride weighing intermediate 8, adds trifluoroacetic acid 10mL then, normal-temperature reaction 2h, the TLC monitoring reaction finishes, and methylene dichloride and trifluoroacetic acid are spin-dried for, add saturated sodium bicarbonate solution, transfer pH to 8～9, separate out a lot of solids, dissolve with methylene dichloride, anhydrous sodium sulfate drying, be spin-dried for solvent and get 1.5g intermediate 9, productive rate 87.42%, figure is as follows for its nuclear-magnetism:
1H-NMR(CDCl
3，300MHz)δ＝7.577-7.897(m，3H)，δ＝7.259-7.323(m，4H)，δ＝7.016-7.092(m，4H)，δ＝4.189-4.271(m，2H)，δ＝4.003-4.052(m，1H)，δ＝3.404-3.454(m，2H)，δ＝2.285(s，6H)，δ＝1.879-2.075(m，2H)，δ＝1.595-1.643(m，2H)，δ＝1.394-1.516(m，2H)，δ＝1.222-1.269(t，3H)。
Synthesizing of intermediate 11
(12.2g, 0.05mol), (6.5g, 0.056mol), (10.7g 0.056mol) joins in the DMF solution of 200mL stirred overnight at room temperature to EDCI to NHS to weighing vitamin H 10 together.Then reaction solution is revolved steaming, boil off most of DMF, cooling adds entry in this solution, a large amount of white solids occur, leaves standstill, and filters, and obtains white solid 12g, productive rate 70.39%, and figure is as follows for its nuclear-magnetism:
1H-NMR(DMSO，300MHz)δ＝6.400(s，1H)，δ＝6.341(s，1H)，δ＝4.269-4.311(m，1H)，δ＝4.110-4.151(m，1H)，δ＝3.084-3.098(m，1H)，δ＝2.791(s，4H)，δ＝2.628-2.677(t，2H)，δ＝1.422-1.655(m，8H)。
Synthesizing of intermediate 12
Weighing intermediate 9 (1.5g, 2.4mmol) and intermediate 11 (0.8g, 2.4mmol) put into the there-necked flask of 100mL, with its whole dissolvings, normal-temperature reaction 20h is spin-dried for DMF among the DMF of adding 20mL, column chromatography for separation is purified, but intermediate 11 can not remove totally when purifying, and gets the mixture of 1g intermediate 11 and 12, and productive rate is 48.83%.
Synthesizing of intermediate 13
(1g 1.2mmol) is dissolved in the tetrahydrofuran (THF) of 10mL weighing intermediate 12, adds the lithium hydroxide aqueous solution of the 10%g/ml of 10mL, stirring at normal temperature 18h, be spin-dried for solvent, add a spot of water dissolution again, transfer pH to 4 with dilute hydrochloric acid, separate out a lot of white solids, filter, oven dry gets 700mg intermediate 13, productive rate 80.92%, figure is as follows for its nuclear-magnetism:
1H-NMR(DMSO，300MHz)δ＝7.797-8.263(m，3H)，δ＝7.024-7.060(m，4H)，δ＝6.726-6.768(m，4H)，δ＝6.463(s，1H)，δ＝6.398(s，1H)，δ＝4.247-4.289(m，1H)，δ＝4.067-4.108(m，1H)，δ＝3.202-3.259(m，3H)，δ＝3.024-3.054(m，1H)，δ＝2.751-2.809(m，1H)，δ＝2.051-2.098(t，2H)，δ＝1.235-1.712(m，14H)。
Synthesizing of target compound 14
Weighing intermediate 13 (0.5g, 0.7mmol) N-hydroxy-succinamide (0.08g, 0.7mmol) and EDC hydrochloride (0.14g, 0.7mmol) be dissolved among the DMF of 5mL, stirring at normal temperature 20h is spin-dried for solvent, separate with thick silica-gel plate, final objective product 286mg, productive rate 50.39%, figure is as follows for its nuclear-magnetism:
1H-NMR(DM?SO，300MHz)δ＝8.274-8.299(2，1H)，δ＝8.114-8.133(2，1H)，δ＝7.935-8.7.962(2，1H)，δ＝7.022-7.067(m，4H)，δ＝6.751-6.789(m，4H)，δ＝6.443-6.418(d，2H)，δ＝4.074-4.297(m，3H)，δ＝3.557-3.571(d，2H)，δ＝3.216-3.257(m，2H)，δ＝3.135(s，4H)，δ＝3.026-3.062(m，2H)，δ＝2.076-2.123(t，2H)，δ＝1.184-1.668(m，12H)。
The colour developing experiment:
Solvent: DMSO 10ml, product 14:10mg transfers pH solution: 10% wet chemical;
Fig. 9 is dissolved among the 10ml DMSO for the 10mg product for neutrality;
Figure 10 is that alkalescence is to transfer the pH=9 of neutral solution with 10% solution of potassium carbonate, becomes red-purple.
The present invention modify to introduce chromophore by biotin derivative being carried out side chain, not only makes biomacromolecule after by biotin labeling the colour developing ability be arranged promptly, and make vitamin H with also can in specific pH scope, develop the color after avidin combines.This band colour biological element is after with the coupling of macromole active substance, and conjugate does not develop the color under physiological condition, thereby can interference not arranged to the application of vitamin H and avidin under normal operation.But when regulating pH displaing amaranth after the certain numerical value, its absorbancy is relevant with the content of vitamin H, thereby can calculate the mole number of the contained vitamin H of per molecule active substance from its absorbancy.The plain conjugate of this band colour biological with the avidin specific combination after similar functions is also arranged, therefore realized under naked eyes, carrying out the tracer analysis process, improved efficient, sensitivity and circulation ratio greatly, filled up the international technology blank.
Claims (9)
1. band colour biological element that is used for the biomacromolecule mark, it is characterized in that: structural formula is as follows:
Wherein, Y is the functional group with at least three linkage function groups, and three linkage function groups are connected with A, B and vitamin H respectively, and B represents the triphenylmethane indicator, and the A representative can be used for the active function group of macromole mark.
2. the band colour biological element that is used for the biomacromolecule mark according to claim 1, it is characterized in that: described A is
3. the band colour biological element that is used for the biomacromolecule mark according to claim 1, it is characterized in that: described Y is
4. the band colour biological element that is used for the biomacromolecule mark according to claim 1, it is characterized in that: described B comprises
Wherein R is halogen, alkane, NO
2, SO
3H, CN, CHO or COOH.
5. the band colour biological element that is used for the biomacromolecule mark according to claim 4 is characterized in that: described B comprises that R is CH
3
6. the band colour biological element that is used for the biomacromolecule mark according to claim 4 is characterized in that: described B comprises that R is Br or Cl.
7. the band colour biological element that is used for the biomacromolecule mark according to claim 4, it is characterized in that: described B is
8. the band colour biological element that is used for the biomacromolecule mark according to claim 1, it is characterized in that: the structural formula of described band colour biological element is
9. the arbitrary described application that is used for the band colour biological element of biomacromolecule mark at preparation biomacromolecule labelled reagent of claim 1 to 8.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011100876553A CN102219791A (en) | 2011-04-08 | 2011-04-08 | Color biotin used for large biological molecule marking, as well as applications in preparing large biological molecule marking reagent |
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| CN2011100876553A CN102219791A (en) | 2011-04-08 | 2011-04-08 | Color biotin used for large biological molecule marking, as well as applications in preparing large biological molecule marking reagent |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2019043946A (en) * | 2017-08-31 | 2019-03-22 | 国立大学法人埼玉大学 | Ligand for molecule purification, tag peptide for molecule purification, and molecule purification method using the same |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1375495A (en) * | 2001-03-15 | 2002-10-23 | 清华大学 | Light excisable biotinyted reagent and its synthesis and application |
| CN1377356A (en) * | 1999-09-30 | 2002-10-30 | 纽罗杰有限公司 | Certain alkylene diamine-substituted heterocycles |
| WO2010106347A2 (en) * | 2009-03-20 | 2010-09-23 | University Of Nottingham | Biomolecular labelling using multifunctional biotin analogues |
-
2011
- 2011-04-08 CN CN2011100876553A patent/CN102219791A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1377356A (en) * | 1999-09-30 | 2002-10-30 | 纽罗杰有限公司 | Certain alkylene diamine-substituted heterocycles |
| CN1375495A (en) * | 2001-03-15 | 2002-10-23 | 清华大学 | Light excisable biotinyted reagent and its synthesis and application |
| WO2010106347A2 (en) * | 2009-03-20 | 2010-09-23 | University Of Nottingham | Biomolecular labelling using multifunctional biotin analogues |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2019043946A (en) * | 2017-08-31 | 2019-03-22 | 国立大学法人埼玉大学 | Ligand for molecule purification, tag peptide for molecule purification, and molecule purification method using the same |
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Application publication date: 20111019 |