CN107652302A - Compound, conjugate, kit and application thereof - Google Patents
Compound, conjugate, kit and application thereof Download PDFInfo
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- CN107652302A CN107652302A CN201610586821.7A CN201610586821A CN107652302A CN 107652302 A CN107652302 A CN 107652302A CN 201610586821 A CN201610586821 A CN 201610586821A CN 107652302 A CN107652302 A CN 107652302A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03001—Alkaline phosphatase (3.1.3.1)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
Abstract
The invention discloses compound, conjugate, detect the purposes of the kit and kit of vitamin D in vitamin D is detected.The compound has the structure shown in formula (1), wherein, L represents linking arm, R1、R2And R3It is separately hydrogen-based, hydroxyl, C1~3Alkoxy, C1~3Alkyl, C2~3Alkenyl or C2~3Alkynyl.The compound of the present invention can detect to vitamin D exactly.
Description
Technical field
The present invention relates to analysis field.In particular it relates to compound, conjugate, kit and application thereof.More
Body, the present invention relates to compound, conjugate, detect the use of the kit and kit of vitamin D in vitamin D is detected
On the way.
Background technology
Vitamin D refers to the material with following mother nucleus structure unit, common are 25-hydroxy-vitamin D.
At present, it is using the principle of immunization method detection vitamin D：It can be directly connected to due to no on vitamin D
The functional group of mark molecule is, it is necessary to which the structure that the method introducing being chemically synthesized can be directly connected to mark molecule (is referred to as
Linking arm, can be carboxyl, amino etc.), obtain vitamin D derivative.Then, mark molecule is connected to by dimension by chemical bond
On the linking arm of raw plain D derivative, vitamin D mark molecule is obtained, the mark molecule can directly or indirectly be produced and can examined
Survey signal.In detection process, vitamin D competition binding antibody in vitamin D mark molecule and sample to be tested (with reference to dimension
There is inverse proportion function relation in raw plain D mark molecules) with vitamin D concentrations in sample.By immunoreaction process, light letter is produced
Number value.Calibrated by assignment, obtain the concentration value of vitamin D to be measured, so as to complete the survey to vitamin D content in sample to be tested
It is fixed.
However, vitamin D derivative applicable at present still have it is to be developed.
The content of the invention
It is contemplated that at least solves one of technical problem present in prior art.
The present invention is the following discovery based on inventor and completed：
In the kit of competition law detection vitamin D, the performance of vitamin D derivative is to influence the weight of kit quality
Factor is wanted, and the position selection of linking arm is a difficult point in vitamin D derivative, should have the feasibility of synthesis, ensure again
Obtained vitamin D derivative can be effectively combined with antibody.And then the present inventor passes through the knot to vitamin D
Structure carries out further investigation discovery, by deriving linking arm on 9,11 or 12 carbon of vitamin D mother nucleus structure, so as to obtain
Vitamin D derivative.The linking arm of introducing will not destroy the structure of antigenic determinant, and obtained vitamin D derivative can have
Effect ground is specifically bound with corresponding antibodies, and Percentage bound is higher.In addition, vitamin D derivative can be connected with mark molecule, enter
And by the mark molecule of connection directly or indirectly caused detectable signal (such as colour developing, change in optical signal), so as to
To be detected exactly to vitamin D.
In view of this, in the first aspect of the present invention, the present invention proposes a kind of compound.According to the implementation of the present invention
Example, the compound have the structure shown in following formula, wherein, L represents linking arm, R1、R2And R3It is separately hydrogen-based, hydroxyl
Base, C1~3Alkoxy, C1~3Alkyl, C2~3Alkenyl or C2~3Alkynyl.Inventor has found by many experiments, by female in vitamin D
Compound of the invention --- the vitamin D derivative that linking arm obtains is derived on 9,11 or 12 carbon of nuclear structure, is introduced
Linking arm will not destroy the structure of antigenic determinant, therefore the compound effectively can be specifically bound with corresponding antibodies.This
Outside, the compound is readily available, application easy to spread.
In the second aspect of the present invention, the present invention proposes a kind of conjugate.According to an embodiment of the invention, it is described conjugated
Thing includes compound described above and mark molecule, and the mark molecule is connected with the linking arm of the compound.Root
Effectively it can be combined according to the conjugate of the embodiment of the present invention with antibody specificity, by being reacted with detection substrate, mark molecule
Detectable signal is directly or indirectly produced, so as to detect the content of vitamin D in sample exactly.
In the third aspect of the present invention, the present invention proposes a kind of kit for detecting vitamin D.According to the reality of the present invention
Example is applied, the kit includes conjugate described above.Inventor has found, using the kit of the present invention, based on conjugated
Thing can be combined effectively with antibody specificity, and by being reacted with detection substrate, mark molecule directly or indirectly produces detectable
Signal, so as to detect the content of vitamin D in sample exactly.
In the fourth aspect of the present invention, the present invention proposes a kind of kit described above in vitamin D is detected
Purposes.As it was previously stated, according to an embodiment of the invention, can be accurately and effectively to vitamin D using the kit of the present invention
Detected.
In addition, according to an embodiment of the invention, compound, conjugate, the kit and the kit for detecting vitamin D
Purposes in vitamin D is detected has at least one of following advantages：
By introducing linking arm on 9,11 or 12 carbon of vitamin D mother nucleus structure, so as to obtain the compound of the present invention.
The structure of antigenic determinant will not be destroyed by introducing linking arm, and resulting compound can be tied effectively with corresponding antibodies specificity
Close, and the compound is readily available, application easy to spread.In addition, the compound is connectable in mark molecule, it is conjugated
Thing, and it is applied to the detection of vitamin D, immune detection principle (such as colour developing, change in optical signal) can be based on exactly
Detect the content of vitamin D in sample.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Brief description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become in the description from combination accompanying drawings below to embodiment
Substantially and it is readily appreciated that, wherein：
Fig. 1 shows the process schematic of detection vitamin D content according to an embodiment of the invention；And
Fig. 2 shows canonical plotting according to an embodiment of the invention；And
Fig. 3 shows curve map according to an embodiment of the invention.
Embodiment
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
The kit that the present invention proposes compound, conjugate, the kit for detecting vitamin D and detection vitamin D exists
The purposes in vitamin D is detected, will be described in greater detail respectively below.
Compound
In the first aspect of the present invention, the present invention proposes a kind of compound.The compound has the knot shown in formula (1)
Structure, wherein, L represents linking arm, R1、R2And R3It is separately hydrogen-based, hydroxyl, C1~3Alkoxy, C1~3Alkyl, C2~3Alkenyl
Or C2~3Alkynyl.
It should be noted that the linking arm L on compound shown in formula (1) derives the determination in site, inventor is carried out
Lot of experiments.
Specifically, first, it is shown below：
Inventor has found, using 9,11 or 12 carbon as derivative site, can relatively easily introduce linking arm L, will not break
The structure of bad antigenic determinant, and the compound stability synthesized is strong, L is not easy to be lost.Chemical combination obtained by introducing L in the site
Thing effectively can be specifically bound with antigen, and then vitamin D can be detected exactly.However, with other
Compound obtained by site introducing linking arm detects ineffective to vitamin D.And then inventor is with vitamin D parent nucleus
9,11 or 12 carbon as crosslink sites, linking arm is introduced, so as to obtain compound-vitamin D derivative of the application.
It should be noted that referring to compound shown in formula (b), substituent L draws a key connection to institute's shape on the ring at center
Into member ring systems represent the hydrogen (compound as shown in formula (b1)) on 9 carbon, the hydrogen on 11 carbon above substituent L substitution rings
Hydrogen (compound as shown in formula (b3)) in (compound as shown in formula (b2)) or 12 carbon.
According to an embodiment of the invention, the compound has the structure of one below.The compound can be effectively with resisting
Body is specifically bound, and Percentage bound is higher, and then vitamin D can be detected exactly.
According to another embodiment of the invention, the compound has the structure of one below.The compound can have
Effect ground is specifically bound with antibody, and Percentage bound is higher, and then vitamin D can be detected exactly.
Conjugate
In the second aspect of the present invention, the present invention proposes a kind of conjugate.The compounds of this invention can be used as shown in formula (1)
The form of the conjugate of compound uses." conjugate " used herein refers to that the present invention is led to as compound shown in formula (1)
Cross the material that its linking arm is connected and formed with mark molecule.Conjugate according to embodiments of the present invention can effectively with antibody
Specific binding, by being reacted with detection substrate, mark molecule directly or indirectly produces detectable signal, so as to examine exactly
The content of vitamin D in test sample sheet.
According to an embodiment of the invention, considered critical is not made to the species of mark molecule.According to some implementations of the present invention
Example, the mark molecule are enzyme, acridinium ester or its analog, pyridine ruthenium or its analog, luminol or its analog, different Rumi
Promise or its analog, fluorescein isothiocynate or its analog.Thus, by being reacted with detection substrate, mark molecule directly or
Detectable signal is produced indirectly, to accurately determine the content of vitamin D.
According to an embodiment of the invention, considered critical is not made to the species of enzyme.Wherein, horseradish peroxidase, alkaline phosphorus
Sour enzyme or acetyl protease and catalysis substrate specificity binding ability are strong, efficiency high during for immune detection, and testing result is accurate
Reliably.Thus, according to some embodiments of the present invention, the enzyme is horseradish peroxidase, alkaline phosphatase or acetyl albumen
Enzyme.
It will be appreciated to those of skill in the art that it is equally applicable to above for the feature and advantage described by compound
The conjugate, will not be repeated here.
Detect the kit of vitamin D
In the third aspect of the present invention, the present invention proposes a kind of kit for detecting vitamin D.According to the reality of the present invention
Example is applied, the kit includes conjugate described above.Inventor has found, using the kit of the present invention, based on conjugated
Thing can be combined effectively with antibody specificity, and by being reacted with detection substrate, mark molecule can be produced directly or indirectly can
Detection signal, so as to detect the content of vitamin D in sample exactly.
According to some embodiments of the present invention, vitamin D is 25-hydroxy-vitamin D, for example, 25-OH Vintamin D2 and
D3。
It will be appreciated to those of skill in the art that it is equally applicable to above for the feature and advantage described by conjugate
The kit of the detection vitamin D, will not be repeated here.
Detect purposes of the kit of vitamin D in vitamin D is detected
In the fourth aspect of the present invention, the present invention proposes a kind of kit described above in vitamin D is detected
Purposes.As it was previously stated, according to an embodiment of the invention, can be accurately and effectively to vitamin D using the kit of the present invention
Detected.
According to an embodiment of the invention, kit is that vitamin D is detected using competition binding method.In kit
Vitamin D derivative structure is similar to vitamin D, will competitively binding antibody.According to a particular embodiment of the invention, can be with
First coated antibody in sample and solid phase carrier containing vitamin D is reacted, obtains solid phase carrier-antibody-vitamin D
Compound.Then, solid phase carrier-antibody-vitamin D compound is reacted with conjugate, the dimension connected in conjugate
Raw plain D is by the competitive vitamin D substituted in solid phase carrier-antibody-vitamin D compound, so as to form solid phase carrier-anti-
Body-conjugate complex.Finally, solid phase carrier-antibody-conjugate complex is mixed with detection substrate, connects conjugate
The mark molecule connect reacts with detection substrate and produces optical signal, and determines vitamin D in sample based on optical signal size
Content.Specifically, immobilization carrier can be magnetic particle, collaurum or cellulose membrane.
It will be appreciated to those of skill in the art that above for detection vitamin D kit described by feature and
Advantage, purposes of the kit of the detection vitamin D in vitamin D is detected is equally applicable to, will not be repeated here.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or bar in embodiment
Part, carried out according to the technology described by document in the art or condition or according to product description.Agents useful for same or instrument
The unreceipted production firm person of device, being can be by the conventional products of acquisition purchased in market.
Embodiment 1
In this embodiment, compound shown in formula (6) in following manner：
Synthetic route is as follows：
Wherein, compound 7, the structure of R group are as follows：
Synthesize compound 2：
120mg compounds 1 (0.46mmol) are dissolved in containing 2mL CH3CN/H2O(10：1) 20mL of mixed solution is former
In the flask of bottom and constant temperature is to 8-10 DEG C, sequentially adds 1,1,1 trifluoroacetone (0.4mL, 4.6mmol), ammonium persulfate-sodium bisulfate
(Oxone) (848mg, 1.38mmol) and NaHCO3(232mg, 2.76mmol), addition 10mL water is used in combination after fully reacting 6h
20mL CH2Cl2Extraction.Anhydrous sodium sulfate drying is used after extract brine It, revolving removes solvent after filtering.It is resulting
Solids by chromatographing column separating purification (ethyl acetate/petroleum ether=1：1) 79mg is obtained after, 0.29mmol water white transparencies
Oily compound 2, yield 62%.
1H NMR(CDCl3, 300MHz) and 2.25 (2H, m), 2.10-1.10 (17H, m), 1.06 (6H, s), 0.75 (3H,
D), 0.48 (3H, s)；HRMS(EI/CI)C18H32O2(M-H2O)+Estimate peak：262.2276 actual peak：262.2275.
Synthesize compound 3：
By 1.4g, 5.0mmol compound 2 is dissolved in 400mL CH2Cl2In, sequentially add 1.6g, 1.74mL, 15mmol
Lutidines and 1.58mL, 7mmol TESOTf, 25min is stirred at -80 DEG C, adds 30mL water to stop reacting.Take out
Organic layer and with anhydrous sodium sulfate drying, revolving removes CH after filtering2Cl2, resulting residue with 15% ethyl acetate/
Petroleum ether solution isolates and purifies to obtain 1.96g, 4.96mmol colorless oils compound 3, yield 99% in chromatographic column.
1H NMR(CDCl3, 300MHz) and 2.35 (2H, dd), 2.24-1.12 (25H, m), 1.12 (6H, s), 0.85 (4H,
T), 0.52 (3H, s), 0.43 (6H, q)；HRMS(EI/CI)C24H46O2Si (M+H) estimates peak：395.3345 actual peak
395.3348。
Synthesize compound 4：
By 2.68mL, 1.94g, 19.2mmol diisopropylamine are dissolved in 64mLTHF, and are added dropwise in ice-water bath
12mL 1.6M n-BuLi THF solution.After 15min, mixed liquor is placed in -78 DEG C, and is added dropwise containing compound 3
The THF solution 30mL of (6.30g, 16.0mmol).After 1h, it is added dropwise into mixed liquor containing 2.78g, 19.2mmol PhSeCl
THF solution 20mL, and add 4：The mixed solution of 1 saturated aqueous common salt and 10% hydrochloric acid, with 2 × 100mL dichloromethane
Organic matter in extractive reaction liquid simultaneously dries organic layer with anhydrous magnesium sulfate, is removed after filtering with Rotary Evaporators organic molten
Agent, and be redissolved in after residue is dissolved in 200mL dichloromethane and be cooled to 0 DEG C, adds 6.82g several times, and 80%,
32mmol MCPBA.After reacting 15min, with 1：1 saturated aqueous common salt and the mixed solution of saturated sodium bicarbonate stop reacting,
And organic layer is obtained by extraction with dichloroethanes, with being spin-dried for after anhydrous magnesium sulfate dry filter.By obtained organic matter with 5% second
Acetoacetic ester/petroleum ether solution obtains 3.11g, 7.89mmol colourless oil liquid compound 4, yield after purification on chromatographic column
For 49%.
1H NMR(CDCl3, 300MHz) and 6.48 (1H, dt), 6.03 (1H, d), 2.32-0.94 (29H, m), 0.83 (4H,
T, J=8Hz), 0.53 (3H, s), 0.41 (6H, q, J=7Hz)；HRMS(EI/CI)C24H44O2Si (M+H) estimates peak：
393.3144 actual peak 393.3248.
Synthesize compound 6：
In N2Under protection, by 7.62mmol, 0.85M tert-butyl lithium is dissolved in 4.5mL heptane, at a temperature of -85 DEG C
The anhydrous ether solution 20mL of compound containing 8mL 5 (3.81mmol, 1.52g) is slowly added to, and stirs 30min.Meanwhile another
By 0.48mL, 1.9mmol n-Bu in one reaction vessel3P and 362mg, 1.9mmol CuI adds the anhydrous of 15mL dryings
In ether, CuI/n-Bu is obtained3P complex.At -85 DEG C, by CuI/n-Bu3P solution adds the previous tert-butyl lithium containing n-
Anhydrous ether solution, and obtained mixed solution is placed in -50 DEG C and reacts 1h, then it is re-cooled to -78 DEG C.It is added dropwise
Anhydrous ether solution containing compound 4 (373mg, 0.95mmol) and after stirring 30min, adds the water of 2.5mL saturated ammonium chlorides
Solution, stop reacting and adding 25mL absolute ethers.Saturated ammonium chloride solution, water and saturated common salt water washing are used successively, and
Dried with anhydrous magnesium sulfate.Obtained liquid organic matter is spin-dried for after filtering with 8：1 petrol ether/ethyl acetate is crossed post separation and obtained
To 535mg, 0.57mmol colourless transparent liquids compound 6, yield 60%.
1H NMR(CDCl3, 300MHz) and 8.0-7.5 (2H, s), 6.2-6.0 (2H, s), 4.59 (2H, s), 3.7-3.0
(23H, m), 2.8-2.0 (35H, m), 1.5-0.2 (26H, m)；HRMS(EI/CI)C49H90N4O9SSi (M+H) estimates peak：
939.6321 actual peak：939.8851.
Synthesize compound 8：
-78℃N2Under protection, by 0.18mL, the tetrahydrofuran solution of the positive tert-butyl lithiums of 1.6M (0.28mmol) adds 1mL
Containing 123.6mg, in the tetrahydrofuran of 0.28mmol compounds 7, after stirring 40min, it is added dropwise dissolved with 65mg,
The tetrahydrofuran of the drying of 0.07mmol compounds 6.After stirring 2h in -78 DEG C, 0.4mL methanol is added to stop
Reaction, then diluted with 50mL absolute ethers.Mixed liquor adds after using saturated ammonium chloride solution and saturated common salt water washing successively
Enter the drying of anhydrous slufuric acid ammonium, concentration.Obtained residue is with 50：1 ethyl acetate/petroleum ether solution crosses post and obtains 64.8mg,
0.055mmol compounds 8, yield 79%.
1H NMR(CDCl3, 300MHz) and 8.0-7.5 (2H, s), 6.2-6.0 (4H, m), 5.0-4.5 (4H, m), 3.7-3.0
(24H, m), 2.8-2.0 (39H, m), 1.5-0.2 (43H, m)；HRMS(EI/CI)C64H116N4O9SSi2(M+H) peak is estimated：
1173.8772, actual peak：1173.6587.
Compound shown in synthesis type (6)：
0.0514mmol 60.3mg compounds 8 are dissolved in 6mL 9：In the mixed liquor of 1 acetone/water, 4.6mg is added,
Cooled down after 0.0184mmolPPTs, the 3h that flows back, use saturated sodium bicarbonate solution, water and saturation after being diluted with ethyl acetate successively
Brine It, filter, concentrate after being dried with anhydrous magnesium sulfate.Residue is with 2：1 petrol ether/ethyl acetate mixed liquor crosses post
Obtain 48.0mg, compound shown in 0.0509mmol formulas (6), yield 98%.
1H NMR(CDCl3, 300MHz) and 8.0-7.5 (2H, s), 6.2-6.0 (6H, m), 5.0-4.5 (4H, m), 3.7-3.0
(24H, m), 2.8-2.0 (39H, m), 1.5-0.2 (13H, m)；HRMS(EI/CI)C52H88N4O9S (M+H) estimates peak：
945.6852 actual peak：945.7658.
Embodiment 2
In this embodiment, according to compound shown in following process synthesis type (5)：
Synthetic route is as follows：
Wherein, the structure of compound 5, compound 7 and R group is as follows：
Specific synthesis step is as follows：
Compound 2 and 3 is synthesized according to the method for embodiment 1.
Synthesize compound 10：
By 1.34mL, 0.97g, 9.6mmol diisopropylamine are dissolved in 32mLTHF, and are added dropwise in ice-water bath
6mL0.8M n-BuLi THF solution.After 15min, in N2Under protection, mixed liquor is placed in -78 DEG C, and is added dropwise containing change
After the THF solution 20mL, 1h of compound 3 (3.15g, 8.0mmol), the THF containing compound 5 (16.0mmol, 6.58g) is slowly added to
Solution 24mL, after stirring 1h, with 1：1 saturated aqueous common salt and the mixed solution of saturated sodium bicarbonate stop reacting, and use dichloro
Organic layer is obtained by extraction in ethane, with being spin-dried for after anhydrous magnesium sulfate dry filter.By obtained organic matter with 1：8 ethyl acetate/
Petroleum ether solution obtains 4.42g, 4.48mmol colourless oil liquids compound 10, yield 56% after purification on chromatographic column.
1H NMR (CDCl3,300MHz) 8.0-7.5 (2H, s), 6.2-6.0 (2H, s), 4.59 (2H, s), 3.7-3.0
(23H, m), 2.8-2.0 (34H, m), 1.5-0.2 (27H, m)；HRMS(EI/CI)C49H90N4O9SSi (M+H) estimates peak：
939.6426 actual peak：939.8651.
Synthesize compound 11：
-78℃N2Under protection, by 0.18mL, the tetrahydrofuran solution of the positive tert-butyl lithiums of 1.6M (0.28mmol) adds 1mL
Containing 123.4mg, in the tetrahydrofuran of 0.28mmol compounds 7, after stirring 40min, it is added dropwise dissolved with 65.7mg,
The tetrahydrofuran of the drying of 0.07mmol compounds 10.After stirring 2h in -78 DEG C, 0.4mL methanol is added to stop reacting, so
Diluted afterwards with 50mL absolute ethers.Mixed liquor after saturated ammonium chloride solution and saturated common salt water washing successively with adding anhydrous sulphur
Sour ammonium is dried, concentration.Obtained residue is with 50：1 ethyl acetate/petroleum ether solution crosses post and obtains 62.4mg,
Compound 11 pure 0.0532mmol, yield 76%.
1H NMR(CDCl3, 300MHz) and 8.0-7.5 (2H, s), 6.2-6.0 (4H, m), 5.0-4.5 (4H, m), 3.7-
3.0 (24H, m), 2.8-2.0 (38H, m), 1.5-0.2 (44H, m), HRMS (EI/CI) C64H116N4O9SSi2(M+H) peak is estimated：
1173.8659, actual peak：1173.6798.
Compound shown in synthesis type (5)：
0.0514mmol 60.2mg compounds 11 are dissolved in 6mL 9：In the mixed liquor of 1 acetone/water, add
Cooled down after 4.7mg, 0.0185mmol PPTs, the 3h that flows back, successively with saturated sodium bicarbonate solution, water after being diluted with ethyl acetate
And saturated common salt water washing, filter, concentrate after being dried with anhydrous magnesium sulfate.Residue is with 2：1 petrol ether/ethyl acetate is mixed
Conjunction liquid crosses post and obtains 47.6mg, the compound shown in 0.0504mmol formulas (5), yield 98%.
1H NMR(CDCl3, 300MHz) and 8.0-7.5 (2H, s), 6.2-6.0 (6H, m), 5.0-4.5 (4H, m), 3.7-3.0
(24H, m), 2.8-2.0 (38H, m), 1.5-0.2 (14H, m) HRMS (EI/CI) C52H88N4O9S (M+H) estimates peak：
945.6745 actual peak：945.7632.
Comparative example 1
In the comparative example, compound shown in formula (8) in following manner.
Synthetic route is as follows：
The synthesis of compound shown in formula (8)：
In N2Under protection, by 0.728mmol, 101mg K2CO3And 0.073mmol, 30mg compound 5 is dissolved in 5mL
In DMF, stirred at 80 DEG C, add 0.073mmol, 36mg compounds 13, add 10mL water stopping reaction after reacting 3h and add
15mL absolute ethers.Saturated ammonium chloride, water and saturated common salt water washing are used successively, and are dried with anhydrous magnesium sulfate.After filtering
Obtained liquid organic matter is spin-dried for 8：1 petrol ether/ethyl acetate crosses post separation and obtains 535mg, 0.037mmol 34mg
Compound shown in the formula (8) of colourless transparent liquid, yield 51%.
1H NMR(CDCl3, 300MHz) and 8.0-7.5 (2H, s), 6.2-6.0 (6H, m), 5.0-4.5 (4H, m), 3.7-3.0
(24H, m), 2.8-2.0 (38H, m), 1.5-0.2 (20H, m)；HRMS(EI/CI)C44H71NO12Estimate peak：It is 1013.3524 real
Border peak：1013.3841.
Embodiment 3
In this embodiment, respectively by the compound shown in formula (5), formula (6) and formula (8) with being connected with streptavidin
Alkaline phosphatase (ALP) mixes, and is incubated under 37 degrees Celsius, obtains conjugate 1,2 and 3.
Embodiment 4
In this embodiment, 1~3 pair of various concentrations vitamin D calibration object of conjugate being utilized respectively obtained by embodiment 3
Detected, main process schematic diagram is as shown in Figure 1.Comprise the following steps that：
(1) the vitamin D calibration object solution of 3 parts of various concentrations is taken to be mixed respectively with sample treatment liquid after under 37 degrees Celsius
It is incubated 12.5 minutes, so as to which vitamin D antigen be discharged from sample, obtains free vitamin D；
(2) the vitamin D calibration object after every kind of processing is subjected to operations described below respectively：
(2-1) adds the magnetic-particle for being coated with antibody into vitamin D calibration object, makes antibody and dimension of dissociating in calibration object
Raw plain D is combined, and forms magnetic-particle-antibody-vitamin D compound；
(2-2) is added obtained by embodiment 3 into the mixed liquor containing magnetic-particle-antibody-vitamin D compound respectively
Conjugate 1~3, be incubated 12.5 minutes under 37 degrees Celsius, the vitamin D that conjugate is connected will with calibration object dimension give birth to
Plain D competitive bindings antibody, the vitamin D being already integrated under displacement on magnetic-particle, formation magnetic-particle-antibody-conjugated
Thing compound, magnetic-particle-antibody-conjugate complex is then cleaned to remove unnecessary free vitamin D, antibody and alkalescence
Phosphatase；
(2-3) adds 3- (2- into three kinds of magnetic-particle-antibody-conjugate complex obtained by step (2-2) respectively
Spiral adamantane) -4- methoxyl groups -4- (3- phosphorus oxygens acyl)-phenyl -1,2- dioxane, 12.5 points are incubated under 37 degrees Celsius
Clock, record luminous reading.
Fig. 2, which is shown, is utilized respectively the song that conjugate 1,2 and 3 determines vitamin D concentrations in various concentrations calibration object solution
Line chart, specific data are as shown in table 1.It can be seen that it is relatively good detect resulting curve noise using conjugate 1 and 2,
Illustrate that compound can specifically bind antibody, so as to the vitamin D antigenic competition in calibration object, and then can survey exactly
Determine vitamin D concentrations in calibration object.And the curve luminous value obtained by conjugate 3 is very low, also sew without obvious gradient, explanation
Compound 3 can not be with vitamin D antibody binding.Thus, illustrate to be formed using 2 carbon of vitamin D mother nucleus structure as derivative site
Compound can not specifically binding antibody, so as to be detected to vitamin D.
The standard curve of table 1
Embodiment 5
Following two ways is respectively adopted and determines vitamin D content in 48 samples：
Mode (1)：Using conjugate 2, exempted from according to the step of embodiment 4 using auspicious CL-2000i Full-automatic chemiluminescences advanced in years
Epidemic disease analysis system is detected.
Mode (2)：Using commercially available vitamin D kit (being purchased from DiaSorin (Suo Lin) company), according to the kit
Specification detected using the LIAISON analyzer detecting systems of DiaSorin companies.
As a result show in figure 3, wherein it is possible to determine coefficient R2For 0.986, illustrate the energy of conjugate 2 of the present invention
Vitamin D content in enough effectively determination samples, the testing result of kit of the testing result with approving in the industry are consistent.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.Moreover, specific features, structure, material or the feature of description can be with office
Combined in an appropriate manner in one or more embodiments or example.In addition, in the case of not conflicting, the skill of this area
Art personnel can be tied the different embodiments or example and the feature of different embodiments or example described in this specification
Close and combine.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changed, replacing and modification.
Claims (10)
1. a kind of compound, it is characterised in that there is the structure shown in following formula：
Wherein, L represents linking arm,
R1、R2And R3It is separately hydrogen-based, hydroxyl, C1~3Alkoxy, C1~3Alkyl, C2~3Alkenyl or C2~3Alkynyl.
2. compound according to claim 1, it is characterised in that the compound has the structure of one below：
3. compound according to claim 1, it is characterised in that the compound has the structure of one below：
4. a kind of conjugate, it is characterised in that the conjugate includes：Compound described in any one of claims 1 to 3；With
And mark molecule, the mark molecule are connected with the linking arm of the compound.
5. conjugate according to claim 4, it is characterised in that the mark molecule be enzyme, acridinium ester or its analog,
Pyridine ruthenium or its analog, luminol or its analog, different luminol or its analog, fluorescein isothiocynate or its is similar
Thing.
6. conjugate according to claim 5, it is characterised in that the enzyme is horseradish peroxidase, alkaline phosphatase
Or acetyl protease.
7. a kind of kit for detecting vitamin D, it is characterised in that including the conjugate described in any one of claim 4~6.
8. kit according to claim 7, it is characterised in that the vitamin D is 25-hydroxy-vitamin D.
9. purposes of any one of claim 7 or 8 kit in vitamin D is detected.
10. purposes according to claim 9, it is characterised in that the kit is to vitamin D using competition binding method
Detected.
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| CN115850141A (en) * | 2019-02-01 | 2023-03-28 | 科美博阳诊断技术(上海)有限公司 | Intermediate compound and preparation method and application thereof |
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| WO1997024127A1 (en) * | 1995-12-29 | 1997-07-10 | A And D Assay, Incorporated | Labeled vitamin d compounds and the use thereof |
| CN101273062A (en) * | 2005-09-29 | 2008-09-24 | 霍夫曼-拉罗奇有限公司 | Antibodies against 25-hydroxyvitamin D |
| WO2015200180A1 (en) * | 2014-06-27 | 2015-12-30 | Siemens Healthcare Diagnostics Inc. | Compositions relating to vitamin d |
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| WO1997024127A1 (en) * | 1995-12-29 | 1997-07-10 | A And D Assay, Incorporated | Labeled vitamin d compounds and the use thereof |
| CN101273062A (en) * | 2005-09-29 | 2008-09-24 | 霍夫曼-拉罗奇有限公司 | Antibodies against 25-hydroxyvitamin D |
| WO2015200180A1 (en) * | 2014-06-27 | 2015-12-30 | Siemens Healthcare Diagnostics Inc. | Compositions relating to vitamin d |
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| CN115850141A (en) * | 2019-02-01 | 2023-03-28 | 科美博阳诊断技术(上海)有限公司 | Intermediate compound and preparation method and application thereof |
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Application publication date: 20180202 Assignee: Shenzhen Mindray Animal Medical Technology Co.,Ltd. Assignor: SHENZHEN MINDRAY BIO-MEDICAL ELECTRONICS Co.,Ltd. Contract record no.: X2022440020009 Denomination of invention: Compounds, conjugates, kits and uses thereof Granted publication date: 20200915 License type: Common License Record date: 20220804 |