CN105153191A - Biotin active ester, biotin active ester synthesis method and usages of biotin active ester - Google Patents
Biotin active ester, biotin active ester synthesis method and usages of biotin active ester Download PDFInfo
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- CN105153191A CN105153191A CN201510545346.4A CN201510545346A CN105153191A CN 105153191 A CN105153191 A CN 105153191A CN 201510545346 A CN201510545346 A CN 201510545346A CN 105153191 A CN105153191 A CN 105153191A
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- active ester
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
- C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
- C07D495/04—Ortho-condensed systems
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/13—Labelling of peptides
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Abstract
The invention provides a biotin active ester, a biotin active ester synthesis method and usages of the biotin active ester. A chemical formula (I) of the biotin active ester is shown in the specification, and a chemical name of the biotin active ester is biotin-N-hydroxy-5-norbornene-2,3-dicarboximide ester (Biotin-ONB). The biotin active ester synthesis method includes that biotin is reacted with N-hydroxy-5-norbornene-2,3-dicarboximide in a solvent to generate the biotin active ester as shown in the chemical formula (I). The biotin active ester is high in stability and capable of serving as a replacement reagent of Biotin-OSU to be widely applied to the fields of biological detection, separation and the like, the synthesis method is simple and convenient, and high yield and high purity are achieved.
Description
Technical field
the present invention relates to the field of chemical synthesis, relate more specifically to the active ester of a kind of vitamin H and synthetic method, purposes.
Background technology
vitamin H (Biotin) side chain has a carboxyl, carboxyl can react with amino, hydroxyl etc. and generate new compound, thus by changing the pendant moiety in biotin molecule, can synthesize different types of biotin derivative.Such as; vitamin H is converted into the derivative containing active ester by the reaction of Biotin end carboxyl; the active ester compound of vitamin H is widely used in the nonradioactive labeling of the biomolecules such as nucleic acid, protein; its marker not only can be used as probe or the signaling molecule of various normal, aberrant gene and protein detection, and is also used to specific nucleic acid in recent years and is separated with the affinity chromatography of protein or analyzes.
vitamin H-N-succinimide ester (Biotin-OSU) is the active ester of a kind of vitamin H generally adopted at present, but due to its leaving away property of succinimide ester structure very strong, be not suitable for steady in a long-term preservation, generally all need now-making-now-using, use very inconvenient.In addition, succinimide ester structure-activity is higher, does not possess selectivity during amino in labelled protein, and these have all limited to its application.Thus, research and development are badly in need of a kind of more stable, the active ester of the vitamin H that selectivity is stronger.
Summary of the invention
for overcoming the problems referred to above of the prior art, the invention provides the active ester of a kind of new vitamin H and synthetic method thereof, the active ester of this vitamin H has satisfactory stability and selectivity, can amino better in labelled protein, be applied to bioseparation, detection etc., and its simple synthetic method, productive rate is higher.
the technical solution used in the present invention is:
on the one hand, the invention provides the active ester of a kind of vitamin H, it has following chemical formula:
（I）。
the chemical name of the active ester of this vitamin H is: vitamin H-N-hydroxyl-5-norbornylene-2,3-dicarboximide ester (Biotin-ONB), can be used as the active ester of a kind of novel vitamin H and replace the application of vitamin H-N-succinimide ester, there is better selectivity and stability, can steady in a long-termly preserve.
on the other hand, present invention also offers the synthetic method of the active ester of above-mentioned vitamin H, comprise the following steps: vitamin H and N-hydroxyl-5-norbornylene-2,3-dicarboximide (HONB) react in a solvent, the active ester of production (I) compound biotin.
the reaction scheme of this synthetic method is as follows:
further, in organic solvent, there is condensation reaction, the active ester of production (I) compound biotin in vitamin H and N-hydroxyl-5-norbornylene-2,3-dicarboximide under the effect of condensing agent.
preferably, above-mentioned condensing agent is N, N '-dicyclohexylcarbodiimide (DCC), N, N '-DIC (DIC) or EDC hydrochloride.
particularly, above-mentioned organic solvent is selected from one or more in DMF, dimethyl sulfoxide (DMSO), methylene dichloride, acetonitrile, dioxane.
further, vitamin H and sulfur oxychloride or oxalyl chloride are obtained by reacting intermediate, and then this intermediate and N-hydroxyl-5-norbornylene-2,3-dicarboximide (HONB) react in organic solvent, obtain the active ester of formula (I) compound biotin.
preferably, intermediate and N-hydroxyl-5-norbornylene-2,3-dicarboximide (HONB) react one or more that the organic solvent used is selected from DMF, dimethyl sulfoxide (DMSO), methylene dichloride, acetonitrile, dioxane.
preferably, vitamin H and N-hydroxyl-5-norbornylene-2,3-dicarboximide (HONB) at room temperature react in a solvent, more preferably, react at 20-50 DEG C.
preferably, the time that vitamin H and N-hydroxyl-5-norbornylene-2,3-dicarboximide (HONB) react in a solvent is 12-24 hour, preferred 16-20 hour.
further, the synthetic method of the active ester of this vitamin H also comprises the step of the thick product generated after reaction being carried out to recrystallization, the solvent that recrystallization uses is Virahol, but is not limited to this, also can use and can carry out recrystallization with other solvents of further purifying crude product to this thick product.
in another, present invention also offers the purposes of the active ester of a kind of above-mentioned vitamin H as protein labeling reagent.
in addition, the reagent such as the organic solvent used in the present invention are not limited to described kind, and other reagent that can play suitable effect also should be included in protection scope of the present invention.
compared with prior art, the present invention has the following advantages: the invention provides a kind of new active ester of protein labeling reagent vitamin H " vitamin H-N-hydroxyl-5-norbornylene-2; 3-dicarboximide ester (Biotin-ONB) ", and synthesizes and method of purification.The method route is simple, and productive rate is higher, and purity reaches more than 98%.The stability of this vitamin H active ester Biotin-ONB is high, stablely under room temperature condition can preserve half a year more than, the Biontin-OSU of alternative widely used less stable is at present used in the field such as biological detection, separation as protein labeling reagent, and there is good amino selectivity, this has important reference value to the synthesis of the active ester of other biological element and application, and then enlightenment can be provided to synthesize a lot of derivative containing vitamin H, expand the range of application of vitamin H.
Embodiment
below in conjunction with specific embodiment, the present invention is further elaborated.
embodiment 1
24.4g vitamin H and 18.8gN-hydroxyl-5-norbornylene-2 is added in 500ml there-necked flask, 3-dicarboximide (HONB) wherein, add 300mlDMF again, 22.7gDCC is dripped under ice-water bath cooling, there is solid DCU to generate in system, dropwise, remove ice bath, room temperature reaction 16 hours, suction filtration, a small amount of DMF of filter cake washs, filtrate evaporate to dryness, with recrystallisation from isopropanol, obtain the active ester of 31.2g vitamin H, i.e. vitamin H-N-hydroxyl-5-norbornylene-2,3-dicarboximide ester, molar yield is 77%, HPLC purity 98.5%.
the nuclear magnetic data of this vitamin H-N-hydroxyl-5-norbornylene-2,3-dicarboximide ester prepared by embodiment 1 is as follows:
HNMR(400MHz，DMSO)：δ6.43–6.33(2H,m)，6.1(2H,br)，4.37–4.27(1H,m)，4.18–4.12(1H,m)，3.5(2H,br)，3.12–3.07(1H,m)，2.88–2.81(1H,m)，2.68–2.52(3H,m)，1.69–1.23(9H,m)，1.69–1.23(9H,m)。
embodiment 2
in 500ml there-necked flask, add 24.4g vitamin H and 18.8gHONB, then add 300mlDMF, under ice-water bath cooling, drip 13.9gDIC, dropwise, remove ice bath, room temperature reaction 24 hours, suction filtration, a small amount of DMF of filter cake washs, and filtrate is blended into 1L Virahol, have a large amount of solid to separate out, suction filtration, filter cake uses recrystallisation from isopropanol again, obtain the active ester of 28.1g vitamin H, i.e. vitamin H-N-hydroxyl-5-norbornylene-2,3-dicarboximide ester, molar yield is 69%, HPLC purity 98.1%.
the nuclear magnetic data of this vitamin H-N-hydroxyl-5-norbornylene-2,3-dicarboximide ester prepared by embodiment 2 is as follows: HNMR (400MHz, DMSO): δ 6.43 – 6.33 (2H, m), 6.1 (2H, br), 4.37 – 4.27 (1H, m), 4.18 – 4.12 (1H, m), 3.5 (2H, br), 3.12 – 3.07 (1H, m), 2.88 – 2.81 (1H, m), 2.68 – 2.52 (3H, m), 1.69 – 1.23 (9H, m), 1.69 – 1.23 (9H, m).
embodiment 3
24.4g vitamin H and 18.8gHONB is added in 500ml there-necked flask, add 200ml dioxane and 21.1gEDC hydrochloride again, room temperature reaction 12 hours, suction filtration, a small amount of acetonitrile wash of filter cake, use recrystallisation from isopropanol again, obtain the active ester of 26.1g vitamin H, i.e. vitamin H-N-hydroxyl-5-norbornylene-2,3-dicarboximide ester, molar yield is 64%, HPLC purity 98.1%.
the nuclear magnetic data of this vitamin H-N-hydroxyl-5-norbornylene-2,3-dicarboximide ester prepared by embodiment 3 is as follows:
HNMR(400MHz，DMSO)：δ6.43–6.33(2H,m)，6.1(2H,br)，4.37–4.27(1H,m)，4.18–4.12(1H,m)，3.5(2H,br)，3.12–3.07(1H,m)，2.88–2.81(1H,m)，2.68–2.52(3H,m)，1.69–1.23(9H,m)，1.69–1.23(9H,m)。
embodiment 4
to in 100ml there-necked flask, add 24.4g vitamin H and 50ml sulfur oxychloride, room temperature reaction 24 hours, is spin-dried for excess thionyl chloride, then adds toluene band acid twice, and the intermediate obtained is for subsequent use.
in 250ml there-necked flask, add 18.8gHONB, then add 150ml methylene dichloride, ice-water bath cooling is lower drips above-mentioned intermediate, dropwise, remove ice bath, room temperature reaction 20 hours, suction filtration, the a small amount of ethyl acetate of filter cake is washed, then uses recrystallisation from isopropanol, obtains the active ester of 30.1g vitamin H, i.e. vitamin H-N-hydroxyl-5-norbornylene-2,3-dicarboximide ester, molar yield is 74%, HPLC purity 98.1%.
the nuclear magnetic data of this vitamin H-N-hydroxyl-5-norbornylene-2,3-dicarboximide ester prepared by embodiment 4 is as follows:
HNMR(400MHz，DMSO)：δ6.43–6.33(2H,m)，6.1(2H,br)，4.37–4.27(1H,m)，4.18–4.12(1H,m)，3.5(2H,br)，3.12–3.07(1H,m)，2.88–2.81(1H,m)，2.68–2.52(3H,m)，1.69–1.23(9H,m)，1.69–1.23(9H,m)。
embodiment 5
to in 100ml there-necked flask, add 24.4g vitamin H and 60ml oxalyl chloride, room temperature reaction 24 hours, is spin-dried for excessive oxalul chloride, then adds toluene band acid twice, and the intermediate obtained is for subsequent use.
in 250ml there-necked flask, add 18.8gHONB, then add 100ml acetonitrile, ice-water bath cooling is lower drips above-mentioned intermediate, dropwise, remove ice bath, room temperature reaction 20 hours, suction filtration, the a small amount of ethyl acetate of filter cake is washed, then uses recrystallisation from isopropanol, obtains the active ester of 29.1g vitamin H, i.e. vitamin H-N-hydroxyl-5-norbornylene-2,3-dicarboximide ester, molar yield is 71.5%, HPLC purity 99.4%.
the nuclear magnetic data of this vitamin H-N-hydroxyl-5-norbornylene-2,3-dicarboximide ester prepared by embodiment 5 is as follows:
HNMR(400MHz，DMSO)：δ6.43–6.33(2H,m)，6.1(2H,br)，4.37–4.27(1H,m)，4.18–4.12(1H,m)，3.5(2H,br)，3.12–3.07(1H,m)，2.88–2.81(1H,m)，2.68–2.52(3H,m)，1.69–1.23(9H,m)，1.69–1.23(9H,m)。
the various embodiments described above specifically describe the building-up process of the active ester biological element of vitamin H of the present invention-N-hydroxyl-5-norbornylene-2,3-dicarboximide ester, and its structured data, confirm its structure further.It is more stable that vitamin H-N-hydroxyl-5-norbornylene-2,3-dicarboximide ester of the present invention can be used as one, and the Biotin-OSU of alternative widely used less stable is at present used in the field such as biological detection, separation as protein labeling reagent.Show through stability test, vitamin H-N-hydroxyl-5-norbornylene-2 of the present invention, 3-dicarboximide ester can be stablized at ambient temperature and preserves more than half a year, can solve the active ester Biotin-OUS of existing vitamin H unstable, the problem that cannot preserve for a long time.
above specific embodiment of the present invention is illustrated; but protection content of the present invention is not only limited to above embodiment; in art of the present invention, the usual knowledge of a GPRS, just can carry out diversified change within the scope of its technology main idea.
Claims (10)
1. the active ester of vitamin H, is characterized in that, has following chemical formula:
（I）。
2. a synthetic method for the active ester of vitamin H as claimed in claim 1, is characterized in that, comprise the following steps: vitamin H and N-hydroxyl-5-norbornylene-2,3-dicarboximide react in a solvent, the active ester of production (I) compound biotin.
3. the synthetic method of the active ester of vitamin H according to claim 2, it is characterized in that: vitamin H and N-hydroxyl-5-norbornylene-2, in organic solvent, there is condensation reaction in 3-dicarboximide, the active ester of production (I) compound biotin under the effect of condensing agent.
4. the synthetic method of the active ester of vitamin H according to claim 3, is characterized in that: described condensing agent is N, N '-dicyclohexylcarbodiimide, N, N '-DIC or EDC hydrochloride.
5. the synthetic method of the active ester of vitamin H according to claim 3, is characterized in that: described organic solvent is selected from one or more in DMF, dimethyl sulfoxide (DMSO), methylene dichloride, acetonitrile, dioxane.
6. the synthetic method of the active ester of vitamin H according to claim 2, it is characterized in that: vitamin H and sulfur oxychloride or oxalyl chloride are obtained by reacting intermediate, then described intermediate and N-hydroxyl-5-norbornylene-2,3-dicarboximide reacts in organic solvent, obtains the active ester of formula (I) compound biotin.
7. the synthetic method of the active ester of vitamin H according to claim 6, is characterized in that: described organic solvent is selected from one or more in DMF, dimethyl sulfoxide (DMSO), methylene dichloride, acetonitrile, dioxane.
8. the synthetic method of the active ester of vitamin H according to claim 2, is characterized in that: the temperature of described reaction is 20-50 DEG C.
9. the synthetic method of the active ester of vitamin H according to claim 2, is characterized in that: the method also comprises the step to the thick product recrystallization generated after reaction, and the solvent that recrystallization uses is Virahol.
10. the active ester of vitamin H as claimed in claim 1 is as the purposes of protein labeling reagent.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DD227436A1 (en) * | 1984-10-04 | 1985-09-18 | Adw Ddr | PROCESS FOR THE PRODUCTION OF BIOTINYL ESTERS AND AMMUNES BZW. BIOTINYL-N-AMINO ALKANIC ACID ESTERS AND AMMUNES |
| JP2000256317A (en) * | 1999-03-02 | 2000-09-19 | Sagami Chem Res Center | Production of solid phase-carrying n-hydroxysuccinimide ester |
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2015
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DD227436A1 (en) * | 1984-10-04 | 1985-09-18 | Adw Ddr | PROCESS FOR THE PRODUCTION OF BIOTINYL ESTERS AND AMMUNES BZW. BIOTINYL-N-AMINO ALKANIC ACID ESTERS AND AMMUNES |
| JP2000256317A (en) * | 1999-03-02 | 2000-09-19 | Sagami Chem Res Center | Production of solid phase-carrying n-hydroxysuccinimide ester |
Non-Patent Citations (3)
| Title |
|---|
| FEI LIAO ET AL.: ""Homogeneous noncompetitive assay of protein via Förster-resonance-energy-transfer with tryptophan residue(s) as intrinsic donor(s) and fluorescent ligand as acceptor"", 《BIOSENSORS AND BIOELECTRONICS》 * |
| N. B. SANKARAN: ""Ring-Opening Metathesis Polymers for Biodetection and Signal Amplification: Synthesis and Self-Assembly"", 《MACROMOLECULES》 * |
| SUZANNE G.RZUCZEK ET AL: ""A Toxic RNA Catalyzes the In Cellulo Synthesis of Its Own Inhibitor"", 《ANGEW. CHEM. INT. ED.》 * |
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