CN107652302B - Compounds, conjugates, kits and uses thereof - Google Patents
Compounds, conjugates, kits and uses thereof Download PDFInfo
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- CN107652302B CN107652302B CN201610586821.7A CN201610586821A CN107652302B CN 107652302 B CN107652302 B CN 107652302B CN 201610586821 A CN201610586821 A CN 201610586821A CN 107652302 B CN107652302 B CN 107652302B
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- 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
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- 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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- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
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- 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 a compound, a conjugate, a kit for detecting vitamin D and application of the kit in detecting vitamin D. The compound has a structure represented by formula (1), wherein L represents a linker arm, and R represents1、R2And R3Are respectively and independently hydrogen radical, hydroxyl radical, C1～3Alkoxy radical, C1～3Alkyl radical, C2～3Alkenyl or C2～3Alkynyl. The compound of the invention can accurately detect vitamin D.
Description
Technical Field
The present invention relates to the field of analysis. In particular, the invention relates to compounds, conjugates, kits and uses thereof. More specifically, the invention relates to compounds, conjugates, kits for detecting vitamin D and the use of the kits for detecting vitamin D.
Background
Vitamin D refers to a substance having a parent nucleus structural unit, and 25-hydroxyvitamin D is commonly known.
At present, the principle of detecting vitamin D by using an immunological method is as follows: because the vitamin D has no functional group which can be directly connected with the labeled molecule, a structure (called a connecting arm which can be carboxyl, amino and the like) which can be directly connected with the labeled molecule is introduced by a chemical synthesis method to obtain the vitamin D derivative. Subsequently, the labeling molecule is attached to the linker arm of the vitamin D derivative via a chemical bond, resulting in a vitamin D labeled molecule that is capable of directly or indirectly generating a detectable signal. In the detection process, the vitamin D labeled molecules compete with the vitamin D in the sample to be detected to bind the antibody (the bound vitamin D labeled molecules and the concentration of the vitamin D in the sample have an inverse proportional function relationship). After the immune reaction process, an optical signal value is generated. And (4) obtaining the concentration value of the vitamin D to be detected through assignment and calibration, thereby completing the determination of the vitamin D content in the sample to be detected.
However, currently available vitamin D derivatives are still under development.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art.
The present invention has been completed based on the following findings of the inventors:
in a kit for detecting vitamin D by a competitive method, the performance of a vitamin D derivative is an important factor influencing the quality of the kit, and the selection of the position of a connecting arm on the vitamin D derivative is a difficult point, so that the synthesis feasibility is realized, and the obtained vitamin D derivative can be effectively combined with an antibody. Further, the present inventors have conducted extensive studies on the structure of vitamin D and found that a vitamin D derivative can be obtained by derivatizing a linker at the 9-, 11-or 12-carbon of the core structure of vitamin D. The introduced connecting arm can not damage the structure of the antigenic determinant, and the obtained vitamin D derivative can be effectively and specifically combined with a corresponding antibody, and has high combination rate. In addition, the vitamin D derivative can be connected with a labeling molecule, and a detectable signal (such as color development, light signal change and the like) is directly or indirectly generated by the connected labeling molecule, so that the vitamin D can be accurately detected.
In view of this, in a first aspect of the invention, a compound is provided. According to an embodiment of the present invention, the compound has a structure represented by the following formula, wherein L represents a linker arm, R1、R2And R3Are respectively and independently hydrogen radical, hydroxyl radical, C1～3Alkoxy radical, C1～3Alkyl radical, C2～3Alkenyl or C2～3Alkynyl. The inventor finds that the vitamin D has 9 and 1 in the parent nucleus structure through a large amount of experimentsThe vitamin D derivative of the invention, which is obtained by deriving the connecting arm from the carbon at the 1 or 12 position, can not damage the structure of an antigenic determinant by introducing the connecting arm, so that the compound can be effectively and specifically combined with a corresponding antibody. In addition, the compound is easy to obtain and popularize and apply.
In a second aspect of the invention, the invention provides a conjugate. According to an embodiment of the invention, the conjugate comprises a compound as described above and a labeling molecule attached to the linker arm of the compound. The conjugate according to the embodiment of the invention can be effectively and specifically combined with the antibody, and the labeled molecule directly or indirectly generates a detectable signal through reaction with a detection substrate, so that the content of vitamin D in a sample can be accurately detected.
In a third aspect of the invention, a kit for detecting vitamin D is provided. According to an embodiment of the invention, the kit comprises the conjugate described above. The inventor finds that the kit can effectively and specifically bind to the antibody based on the conjugate, and the labeled molecule directly or indirectly generates a detectable signal through reaction with a detection substrate, so that the content of the vitamin D in a sample can be accurately detected.
In a fourth aspect of the invention, the invention proposes the use of a kit as described above for the detection of vitamin D. As described above, according to the embodiments of the present invention, vitamin D can be accurately and efficiently detected using the kit of the present invention.
Furthermore, according to embodiments of the present invention, the compound, the conjugate, the kit for detecting vitamin D, and the use of the kit for detecting vitamin D have at least one of the following advantages:
the compounds of the present invention are obtained by introducing a linker arm at the 9, 11 or 12 carbon of the vitamin D parent nucleus structure. The structure of the antigenic determinant is not damaged by introducing the connecting arm, the obtained compound can be effectively combined with the specificity of the corresponding antibody, and the compound is easy to obtain and popularize and apply. In addition, the compound can be connected to a marker molecule to obtain a conjugate, and the conjugate is applied to detection of vitamin D, so that the content of the vitamin D in a sample can be accurately detected based on an immunoassay principle (such as color development, optical signal change and the like).
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a schematic diagram of a process for detecting vitamin D content according to one embodiment of the present invention; and
FIG. 2 shows a standard graph according to one embodiment of the invention; and
fig. 3 shows a graph according to an embodiment of the invention.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
The present invention proposes compounds, conjugates, kits for detecting vitamin D and uses of the kits for detecting vitamin D in the detection of vitamin D, which will be described in detail below, respectively.
Compound (I)
In a first aspect of the invention, the invention features a compound. The compound has a structure shown in formula (1), wherein L represents a connecting arm, and R represents1、R2And R3Are respectively and independently hydrogen radical, hydroxyl radical, C1～3Alkoxy radical, C1～3Alkyl radical, C2～3Alkenyl or C2～3Alkynyl.
In addition, the inventors have conducted extensive experimental studies with respect to the determination of the site of derivation of the linker arm L of the compound represented by formula (1).
Specifically, first, as shown in the following formula:
the inventors found that the linker arm L can be easily introduced by using the carbon at position 9, 11 or 12 as a derivative site, the structure of the epitope is not destroyed, and the synthesized compound has high stability and L is not easily lost. The compound obtained by introducing L into the site can effectively and specifically bind with antigen, and further can accurately detect vitamin D. However, the compounds obtained by introducing the linker arm at other sites do not work well for vitamin D detection. Furthermore, the inventors introduced a linker arm using the carbon at position 9, 11 or 12 of the vitamin D parent nucleus as a crosslinking site, thereby obtaining a vitamin D derivative, which is a compound of the present application.
In addition, referring to the compound represented by formula (b), a ring system formed by a substituent L on a ring with a bond connected to the center represents that the substituent L substitutes for hydrogen on the carbon at the 9-position on the ring (the compound represented by formula (b 1)), hydrogen on the carbon at the 11-position (the compound represented by formula (b 2)), or hydrogen on the carbon at the 12-position (the compound represented by formula (b 3)).
According to an embodiment of the invention, the compound has the structure of one of the following. The compound can be effectively and specifically combined with an antibody, has high combination rate, and can further accurately detect vitamin D.
According to another embodiment of the invention, the compound has the structure of one of the following. The compound can be effectively and specifically combined with an antibody, has high combination rate, and can further accurately detect vitamin D.
Conjugates
In a second aspect of the invention, the invention provides a conjugate. The compounds of the present invention can be used in the form of conjugates of the compounds represented by formula (1). The term "conjugate" as used herein refers to a substance of the present invention formed by linking a compound represented by formula (1) to a labeling molecule via its linker arm. The conjugate according to the embodiment of the invention can be effectively and specifically combined with the antibody, and the labeled molecule directly or indirectly generates a detectable signal through reaction with a detection substrate, so that the content of vitamin D in a sample can be accurately detected.
According to the embodiment of the present invention, the kind of the labeling molecule is not strictly limited. According to some embodiments of the invention, the labeling molecule is an enzyme, an acridinium ester or an analogue thereof, ruthenium pyridine or an analogue thereof, luminol or an analogue thereof, isoluminol or an analogue thereof, fluorescein isothiocyanate or an analogue thereof. Thus, the labeling molecule directly or indirectly generates a detectable signal by reaction with the detection substrate to accurately determine the vitamin D content.
According to the embodiment of the present invention, the kind of the enzyme is not strictly limited. The horseradish peroxidase, alkaline phosphatase or acetylprotease has strong specific binding capacity with a catalytic substrate, and the horseradish peroxidase, alkaline phosphatase or acetylprotease has high efficiency and accurate and reliable detection results when being used for immunoassay. Thus, according to some embodiments of the invention, the enzyme is horseradish peroxidase, alkaline phosphatase, or acetylprotease.
It will be appreciated by those skilled in the art that the features and advantages described above for the compounds apply equally to the conjugates and will not be described in detail here.
Kit for detecting vitamin D
In a third aspect of the invention, a kit for detecting vitamin D is provided. According to an embodiment of the invention, the kit comprises the conjugate described above. The inventor finds that by using the kit of the invention, the labeled molecule can directly or indirectly generate a detectable signal through reaction with a detection substrate based on the fact that the conjugate can be effectively and specifically combined with the antibody, so that the content of the vitamin D in a sample can be accurately detected.
According to some embodiments of the invention, the vitamin D is 25-hydroxyvitamin D, such as 25-hydroxyvitamin D2 and D3.
It will be appreciated by the person skilled in the art that the features and advantages described above for the conjugates apply equally to the kit for the detection of vitamin D and will not be described in further detail here.
Application of kit for detecting vitamin D in detection of vitamin D
In a fourth aspect of the invention, the invention proposes the use of a kit as described above for the detection of vitamin D. As described above, according to the embodiments of the present invention, vitamin D can be accurately and efficiently detected using the kit of the present invention.
According to an embodiment of the present invention, the kit is for detecting vitamin D by competitive binding. Vitamin D derivatives in the kit have a structure similar to vitamin D and will competitively bind to the antibody. According to an embodiment of the present invention, a sample containing vitamin D can be reacted with the antibody coated on the solid phase carrier to obtain the solid phase carrier-antibody-vitamin D complex. Next, the solid support-antibody-vitamin D complex is reacted with the conjugate, and the vitamin D linked in the conjugate competitively displaces the vitamin D in the solid support-antibody-vitamin D complex, thereby forming a solid support-antibody-conjugate complex. Finally, the solid phase carrier-antibody-conjugate compound is mixed with a detection substrate, the labeled molecule connected with the conjugate reacts with the detection substrate to generate an optical signal, and the content of the vitamin D in the sample is determined based on the size of the optical signal. Specifically, the immobilization carrier may be a magnetic microparticle, colloidal gold, or cellulose membrane.
It will be appreciated by those skilled in the art that the features and advantages described above in relation to the kit for the detection of vitamin D are equally applicable to the use of the kit for the detection of vitamin D in the detection of vitamin D and will not be described in further detail herein.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
In this example, a compound represented by the formula (6) was prepared as follows:
the synthetic route is as follows:
wherein, the structures of the compound 7 and the R group are shown as follows:
synthesis of Compound 2:
120mg of Compound 1(0.46mmol) was dissolved in a solution containing 2mL of CH3CN/H2Adding O (10: 1) mixed solution into a 20mL original bottom flask, keeping the temperature to be 8-10 ℃, and sequentially adding 1,1Trifluoroacetone (0.4mL, 4.6mmol), Oxone (Oxone) (848mg, 1.38mmol) and NaHCO3(232mg, 2.76mmol), reacted well for 6h, then 10mL water was added and 20mL CH was used2Cl2And (4) extracting. The extract was washed with brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The obtained solid was separated and purified by a chromatography column (ethyl acetate/petroleum ether ═ 1: 1) to obtain 79mg, 0.29mmol of colorless transparent oily compound 2, and the yield was 62%.
1H NMR(CDCl3，300MHz)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)+Peak estimation: 262.2276, actual peak: 262.2275.
synthesis of compound 3:
dissolve 1.4g, 5.0mmol of Compound 2 in 400mL CH2Cl21.6g, 1.74mL, 15mmol lutidine and 1.58mL, 7mmol TESOTF were added sequentially, stirred at-80 ℃ for 25min, and 30mL water was added to stop the reaction. The organic layer was taken out and dried over anhydrous sodium sulfate, filtered, and then CH was removed by rotary evaporation2Cl2The residue obtained was purified by separation on a column chromatography using 15% ethyl acetate/petroleum ether to give 1.96g, 4.96mmol of compound 3 as a colorless oil in 99% yield.
1H NMR(CDCl3，300MHz)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)C24H46O2Estimated Si (M + H) peak: 395.3345, actual peak 395.3348.
Synthesis of compound 4:
2.68mL, 1.94g, 19.2mmol of diisopropylamine were dissolved in 64mL THF and 12mL of a 1.6M n-BuLi solution in THF was added dropwise in an ice-water bath. After 15min, the mixture was placed at-78 ℃ and 30mL of THF solution containing Compound 3(6.30g, 16.0mmol) was added dropwise. After 1h, 20mL of a THF solution containing 2.78g, 19.2mmol of PhSeCl was added dropwise to the mixture, and 4: 1, extracting the organic matter in the reaction solution with 2X 100mL of dichloromethane, drying the organic layer with anhydrous magnesium sulfate, filtering, removing the organic solvent with a rotary evaporator, dissolving the residue, redissolving in 200mL of dichloromethane, cooling to 0 deg.C, and adding 6.82g, 80%, 32mmol of MCPBA in portions. After 15min of reaction, the reaction mixture was quenched with 1: the reaction was stopped with a mixed solution of saturated brine and saturated sodium hydrogencarbonate of 1, and the resulting mixture was extracted with dichloroethane to obtain an organic layer, which was dried over anhydrous magnesium sulfate, filtered and then spin-dried. The obtained organic substance was purified by a column chromatography using 5% ethyl acetate/petroleum ether solution to obtain 3.11g, 7.89mmol of compound 4 as a colorless oily liquid with a yield of 49%.
1H NMR(CDCl3，300MHz)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)C24H44O2Estimated Si (M + H) peak: 393.3144, actual peak 393.3248.
Synthesis of compound 6:
in N27.62mmol, 0.85M tert-butyllithium were dissolved in 4.5mL heptane with protection, and 20mL of a solution of 8mL compound 5(3.81mmol, 1.52g) in dry ether was added slowly at-85 ℃ and stirred for 30 min. Meanwhile, in another reaction vessel, 0.48mL of 1.9mmol of n-Bu3P and 362mg, 1.9mmol of CuI were added to 15mL of dry, anhydrous ether to give CuI/n-Bu3A complex of P. At-85 deg.C, adding CuI/n-Bu3Adding the solution P into the previous anhydrous ether solution containing n-tert-butyl lithium, placing the obtained mixed solution at-50 ℃ for reacting for 1h, and then cooling to-78 ℃ again. After an anhydrous ether solution containing compound 4(373mg, 0.95mmol) was added dropwise and stirred for 30min, 2.5mL of an aqueous solution of saturated ammonium chloride was added, the reaction was stopped and 25mL of anhydrous ether was added. The extract was washed with saturated ammonium chloride solution, water and saturated brine in this order, and dried over anhydrous magnesium sulfate. The liquid organic matter obtained by spin-drying after filtration was treated with 8: 1 was subjected to column chromatography using petroleum ether/ethyl acetate to give 535mg, 0.57mmol of colorless transparent liquid compound 6 in a yield of 60%.
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(35H，m)，1.5-0.2(26H，m)；HRMS(EI/CI)C49H90N4O9Predicted peak for SSi (M + H): 939.6321, actual peak: 939.8851.
synthesis of compound 8:
-78℃N20.18mL of 1.6M n-tert-butyllithium (0.28mmol) in tetrahydrofuran was added to 1mL of tetrahydrofuran containing 123.6mg, 0.28mmol of Compound 7 under protection, and after stirring for 40min, 65mg was dissolved by dropwise addition,
0.07mmol of Compound 6 in dry tetrahydrofuran. After stirring at-78 ℃ for 2h, 0.4mL of methanol was added to stop the reaction, which was then diluted with 50mL of anhydrous ether. The mixture was washed with a saturated ammonium chloride solution and a saturated brine in this order, and then dried with anhydrous ammonium sulfate and concentrated. The residue obtained was purified by a 50: the ethyl acetate/petroleum ether solution of 1 was passed through the column to give 64.8mg, 0.055mmol of Compound 8 in 79% yield.
1H NMR(CDCl3，300MHz)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) predicted peak: 1173.8772, actual peak: 1173.6587.
synthesizing a compound represented by the formula (6):
0.0514mmol 60.3mg of compound 8 was dissolved in 6mL 9: 1, 4.6mg of 0.0184mmol of PPTs are added to the acetone/water mixture, the mixture is refluxed for 3 hours, cooled, diluted with ethyl acetate, washed with saturated sodium bicarbonate solution, water and saturated brine in sequence, dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was purified by 2: the petroleum ether/ethyl acetate mixture of 1 was subjected to column chromatography to give 48.0mg, 0.0509mmol, 98% yield of the compound represented by formula (6).
1H NMR(CDCl3，300MHz)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)C52H88N4O9Predicted peak of S (M + H): 945.6852, actual peak: 945.7658.
example 2
In this example, a compound represented by formula (5) was synthesized according to the following procedure:
the synthetic route is as follows:
wherein the structures of the compound 5, the compound 7 and the R group are shown as follows:
the specific synthesis steps are as follows:
compounds 2 and 3 were synthesized according to the procedure of example 1.
Synthesis of compound 10:
1.34mL, 0.97g, 9.6mmol of diisopropylamine were dissolved in 32mL THF and 6mL of a 0.8M solution of n-BuLi in THF was added dropwise to the ice-water bath. After 15min, at N2Under protection, the mixture was placed at-78 ℃ and 20mL of THF solution containing compound 3(3.15g, 8.0mmol) was added dropwise, after 1h, 24mL of THF solution containing compound 5(16.0mmol, 6.58g) was added slowly, stirred for 1h, and then the mixture was stirred with a 1: the reaction was stopped with a mixed solution of saturated brine and saturated sodium hydrogencarbonate of 1, and the resulting mixture was extracted with dichloroethane to obtain an organic layer, which was dried over anhydrous magnesium sulfate, filtered and then spin-dried. The obtained organic matter was treated with 1: the ethyl acetate/petroleum ether solution of 8 was purified on a column chromatography to give 4.42g, 4.48mmol of the colorless oily liquid compound 10 in 56% yield.
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)C49H90N4O9Predicted peak for SSi (M + H): 939.6426, actual peak: 939.8651.
synthesis of compound 11:
-78℃N2under protection, 0.18mL of 1.6M n-tert-butyllithium (0) was added.28mmol) was added to 1mL of tetrahydrofuran containing 123.4mg, 0.28mmol of compound 7, and after stirring for 40min, dried tetrahydrofuran in which 65.7mg, 0.07mmol of compound 10 was dissolved was added dropwise. After stirring at-78 ℃ for 2h, 0.4mL of methanol was added to stop the reaction, which was then diluted with 50mL of anhydrous ether. The mixture was washed with a saturated ammonium chloride solution and a saturated brine in this order, and then dried with anhydrous ammonium sulfate and concentrated. The residue obtained was purified by a 50: the ethyl acetate/petroleum ether solution of 1 was subjected to column chromatography to give 62.4mg, 0.0532mmol of pure compound 11, 76% yield.
1H NMR(CDCl3，300MHz)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) predicted peak: 1173.8659, actual peak: 1173.6798.
synthesizing a compound represented by the formula (5):
0.0514mmol 60.2mg of compound 11 were dissolved in 6mL 9: 1, 4.7mg, 0.0185mmol of PPTs was added to the acetone/water mixture, refluxed for 3 hours, cooled, diluted with ethyl acetate, washed with a saturated sodium bicarbonate solution, water and saturated brine in this order, dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was purified by 2: the petroleum ether/ethyl acetate mixture of 1 was subjected to column chromatography to give 47.6mg, 0.0504mmol of the compound represented by the formula (5), and the yield was 98%.
1H NMR(CDCl3，300MHz)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)C52H88N4O9Predicted peak of S (M + H): 945.6745, actual peak: 945.7632.
comparative example 1
In this comparative example, the compound represented by the formula (8) was prepared in accordance with the following method.
The synthetic route is as follows:
synthesis of a compound represented by formula (8):
in N2Under protection, 0.728mmol of K with the concentration of 101mg2CO3And 0.073mmol, 30mg of Compound 5 dissolved in 5mL of DMF, stirring at 80 deg.C, adding 0.073mmol, 36mg of Compound 13, reacting for 3h, adding 10mL of water to stop the reaction, and adding 15mL of anhydrous ether. The extract was washed with saturated ammonium chloride, water and saturated brine in this order, and dried over anhydrous magnesium sulfate. The liquid organic matter obtained by spin-drying after filtration was treated with 8: 1 was subjected to column chromatography using petroleum ether/ethyl acetate to give 535mg, 0.037mmol, 34mg of a colorless transparent liquid, which was the compound represented by the formula (8), and the yield was 51%.
1H NMR(CDCl3,300MHz)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)C44H71NO12Peak estimation: 1013.3524, actual peak: 1013.3841.
example 3
In this example, the compounds represented by formula (5), formula (6) and formula (8) were mixed with streptavidin-linked alkaline phosphatase (ALP), respectively, and incubated at 37 degrees celsius to give conjugates 1,2 and 3.
Example 4
In this example, the conjugates 1-3 obtained in example 3 were used to detect vitamin D calibrators of different concentrations, and the main process is schematically shown in FIG. 1. The method comprises the following specific steps:
(1) respectively mixing 3 parts of vitamin D calibrator solutions with different concentrations with the sample treatment solution, and incubating at 37 ℃ for 12.5 minutes, so that vitamin D antigen is released from the sample, and free vitamin D is obtained;
(2) each treated vitamin D calibrator was subjected to the following procedure:
(2-1) adding the magnetic particles coated with the antibody into a vitamin D calibrator to combine the antibody with free vitamin D in the calibrator to form a magnetic particle-antibody-vitamin D compound;
(2-2) adding conjugates 1 to 3 obtained in example 3 to the mixture solution containing the magnetic particle-antibody-vitamin D complex, respectively, incubating the mixture solution at 37 ℃ for 12.5 minutes, wherein the vitamin D bound to the conjugates competitively binds to the antibody with the vitamin D in the calibrator, displacing the vitamin D bound to the magnetic particles to form a magnetic particle-antibody-conjugate complex, and washing the magnetic particle-antibody-conjugate complex to remove excess free vitamin D, antibody and alkaline phosphatase;
(2-3) to the three magnetic particle-antibody-conjugate complexes obtained in step (2-2) was added 3- (2-helical adamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxetane, respectively, and incubated at 37 ℃ for 12.5 minutes, and luminescence readings were recorded.
Fig. 2 shows a graph of the vitamin D concentration in calibrant solutions of different concentrations determined using conjugates 1,2 and 3, respectively, with specific data as shown in table 1. It can be seen that the signal-to-noise ratio of the curves obtained by using conjugates 1 and 2 for detection is good, indicating that the compound can specifically bind to the antibody, thereby competing with the vitamin D antigen in the calibrator and further accurately determining the vitamin D concentration in the calibrator. The curve obtained with conjugate 3 showed very low luminescence values and no significant gradient, indicating that conjugate 3 failed to bind to vitamin D antibodies. This suggests that a compound having the carbon at position 2 of the vitamin D parent nucleus structure as a derivative site cannot specifically bind to an antibody, and thus cannot detect vitamin D.
TABLE 1 Standard Curve
Example 5
The vitamin D content in 48 samples was determined in the following two ways:
mode (1): using conjugate 2, detection was performed using a Merrill CL-2000i full-automatic chemiluminescence immunoassay system according to the procedure of example 4.
Mode (2): the detection was carried out using a commercially available vitamin D kit (purchased from DiaSorin) according to the instructions of the kit using the lision analyzer detection system of DiaSorin.
The results are shown in fig. 3, where the correlation coefficient R can be determined2And 0.986, indicating that conjugate 2 of the present invention is effective in determining the vitamin D content in a sample, and the results are consistent with those of an art-recognized kit.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (8)
1. A compound having the structure of one of:
2. a conjugate, characterized in that the conjugate comprises: a compound of claim 1; and a labeling molecule attached to the linker arm of the compound.
3. The conjugate of claim 2, wherein the labeling molecule is an enzyme, acridinium ester, ruthenium pyridine, luminol, isoluminol, fluorescein isothiocyanate.
4. The conjugate of claim 3, wherein the enzyme is horseradish peroxidase, alkaline phosphatase, or acetylprotease.
5. A kit for the detection of vitamin D, comprising a conjugate according to any one of claims 2 to 4.
6. The kit of claim 5, wherein said vitamin D is 25-hydroxyvitamin D.
7. Use of the kit of claim 5 or 6 for the detection of vitamin D.
8. The use according to claim 7, wherein the kit is for the detection of vitamin D by competitive binding.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5232836A (en) * | 1988-05-04 | 1993-08-03 | Ire-Medgenix S.A. | Vitamin D derivatives: therapeutic applications and applications to assays of metabolites of vitamin D |
| 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 |
| WO2016103722A1 (en) * | 2014-12-24 | 2016-06-30 | Kyoto University | Vitamin d3 derivatives and pharmaceutical use thereof |
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|---|---|---|---|---|
| US5232836A (en) * | 1988-05-04 | 1993-08-03 | Ire-Medgenix S.A. | Vitamin D derivatives: therapeutic applications and applications to assays of metabolites of vitamin D |
| 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 |
| WO2016103722A1 (en) * | 2014-12-24 | 2016-06-30 | Kyoto University | Vitamin d3 derivatives and pharmaceutical use 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 |