Amphiphilic multi-arm polyethylene glycol derivatives and uses thereof

Abstract

The application relates to the field of immunodiagnostic materials, in particular to an amphoteric multi-arm polyethylene glycol derivative of a compound shown in the following general formula and application thereof. The compound provided by the application can block the non-specific adsorption of interfering substances in a determination system, protects the surface of a solid-phase carrier, and can be used in practice as a low-cost substitute for a protein blocking agent.

Description

[0001] Priority Information

[0002] The present application claims priority to and the benefit of Chinese Patent Application No. 202211667523.2, filed December 23, 2022, and is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application relates to the field of protein detection technology, in particular to the field of immunodiagnostic materials. BACKGROUND

[0004] Blocking of protein binding surface (i.e. the surface of solid phase carrier in immunoassay reagent system) is very important in the field of immunoassay based on solid phase carrier. In immunoassay, non-specific adsorption of impurity proteins on the surface of solid phase carrier such as immunomagnetic beads, multi-well coated plates, fluorescent microspheres, latex microparticles, colloidal gold, glass, capillary, nylon and nitrocellulose membrane used in detection methods such as chemiluminescence, immunochromatography, Elisa, latex turbidity, etc. will lead to a significant decrease in signal-to-noise ratio, i.e. detection sensitivity, and may also cause false positives, resulting in errors in diagnosis of diseases and their progression and medical accidents.

[0005] Therefore, blocking of protein binding surface plays an important role in the entire diagnostic process. The commonly used blocking reagents currently mainly comprise protein reagents such as bovine serum albumin, casein, etc.; and serum such as fetal bovine serum, horse serum, etc.; and some small or large molecule compounds (hereinafter referred to as auxiliary reagents) such as gelatin, polyethylene glycol, triethanolamine, Tris, etc. The above protein components and the above auxiliary reagents can be used independently as blocking agents, or can be used as a whole as blocking agents. In some diagnostic processes, or in immunoblotting experiments, for example, there is a large demand for blocking agents, a low cost requirement and a high performance requirement.

[0006] Specifically, in the scenarios of chemiluminescence platform detection, Elisa platform detection, immunochromatography platform detection, latex turbidity platform detection, etc., a blocking agent is usually configured by adding bovine serum albumin (BSA) to the buffer, and some auxiliary reagents are also added to the buffer, i.e. a protein blocking agent is obtained. The characteristics of this kind of blocking agent are that it can better block the hydrophobic sites on the surface of solid phase carriers and other reagent molecules. However, BSA is difficult to purify and has high cost, and as a biological technology product, it has the problem of difficult control of batch-to-batch differences. At the same time, because proteins are attached to the surface of solid phase carriers, they can also interact physically and chemically with other protein components in the sample, reducing their ability to improve sensitivity. Finally, protein reagents can block the sites that should normally undergo physical and chemical binding, resulting in a decrease in detection signal value.

[0007] In addition, the above-mentioned auxiliary reagent can also be used alone as a blocking agent, i.e., a compound blocking agent. Currently, the main reagents used are polyethylene glycol, triethanolamine and some polysaccharides. The characteristics of such blocking agents are that the performance is good at times and poor at times, and various detection platforms (such as immunochromatographic detection platform, chemiluminescence detection platform, latex turbidimetric detection platform, etc.) and various solid-phase carriers cannot be uniformly used, which greatly limits the application scenarios of the blocking agent.

[0008] Therefore, it is urgent to develop a solid-phase carrier blocking reagent without protein components and capable of avoiding non-specific adsorption of a solid-phase carrier. SUMMARY

[0009] The present application aims to at least partially solve one of the technical problems in the related art. To this end, the purpose of the present application is to provide an amphoteric multi-arm polyethylene glycol derivative and its use in a solid-phase carrier non-specific adsorption blocking agent. The amphoteric multi-arm polyethylene glycol derivative provided by the present application has a hydrophobic group at the arm end, which can block the hydrophobic sites on the surface of the solid-phase carrier. The polyethylene glycol skeleton provides hydrophilic properties, prevents the non-specific adsorption of interfering substances in the sample or the reagents of the immunodetection system based on hydrophobic interaction, and improves the blocking effect of the solid-phase carrier. The blocking agent prepared from the amphoteric multi-arm polyethylene glycol derivative as the main component has a certain improvement in reagent sensitivity compared with the BSA blocking agent (protein blocking agent), and can be used as a lower-cost protein blocking agent substitute in practice.

[0010] To this end, the first aspect of the present application provides a compound, which is a compound represented by formula (I) or a tautomer, stereoisomer, hydrate or solvate of the compound represented by formula (I):

[0011]

[0012] wherein,

[0013] n and x are integers, 2≤n≤10; 1≤x≤10;

[0014] M is independently selected from an amide bond, an ether bond and a carbon-nitrogen single bond;

[0015] R in each monomer structure in the compound is independently selected from the following groups: C2-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, 3-8 membered heterocyclyl, C6-C8 aryl and 5-8 membered heteroaryl.

[0016] Further, 2≤n≤6; further, n is 2, 4 or 6; further, n is 2.

[0017] Further, 1≤x≤8; further, 1≤x≤4; further, x is 2.

[0018] Further, M is an amide bond.

[0019] Further, R in each monomer structure of the compound is independently selected from the group consisting of C4-C6 alkyl, C4-C6 alkenyl, C4-C6 alkynyl, C4-C6 cycloalkyl, 4-6 membered heterocyclyl, aryl;

[0020] Further, R in each monomer structure of the compound is independently selected from C4-C6 alkyl;

[0021] Further, R in each monomer structure is C6 alkyl.

[0022] Compared with protein blocking reagents which are difficult to purify, high in cost, and easy to interact with other protein components in samples, and small molecule blocking reagents which are poor in blocking effect and easy to cause over-blocking, the compound as shown in formula (I) has a hydrophobic group at the arm end, can block the hydrophobic sites on the surface of a solid carrier, and at the same time, the polyethylene glycol skeleton provides hydrophilic properties, blocks the interference with protein adsorption, and at the same time, as little as possible affects the normal binding of antibodies and antigens, and improves the detection sensitivity.

[0023] The second aspect of the present application provides a blocking agent comprising the compound according to the first aspect of the present application.

[0024] Further, the blocking agent further comprises BSA or casein.

[0025] Further, the blocking agent further comprises at least one buffer selected from the group consisting of tris-hydroxymethyl aminomethane buffer, phosphoric acid buffer, ammonium sulfate buffer, boric acid buffer, 2-(N-morpholine) ethanesulfonic acid buffer, 3-morpholine propyl sulfonic acid buffer, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer, and triethanolamine buffer.

[0026] The second aspect of the present application also provides a diluent comprising the compound according to the present application; preferably, the diluent is at least one selected from the group consisting of magnetic bead diluent, colloidal gold diluent, latex diluent, AE marker diluent, luminol marker diluent, isoluminol marker diluent, AP marker diluent, HRP marker diluent, fluorescent material marker diluent, quantum dot material marker diluent, up-conversion luminescent material marker diluent, electrochemiluminescent material marker diluent, and sample diluent. Further, the diluent further comprises an antibody or an antigen.

[0027] The second aspect of the present application also provides a detection reagent comprising the compound according to the present application, and further comprising at least one of a magnetic bead, colloidal gold, latex, AE marker (acridinium ester, referred to as AE), luminol marker, isoluminol marker, AP marker (alkaline phosphatase, referred to as AP), HRP marker (horseradish peroxidase, referred to as HRP), fluorescent material marker, quantum dot material marker, upconversion luminescent material marker, and electrochemiluminescent material marker.

[0028] The fluorescent material mainly includes fluorescein isothiocyanate, tetraethenyl rhodamine, tetramethyl rhodamine isothiocyanate, a substance that generates fluorescence after acting on an enzyme, and lanthanides. The quantum dot material is mainly selected from cadmium selenide colloidal quantum dots, cadmium telluride colloidal quantum dots, cadmium selenide / zinc sulfide core-shell structure colloidal quantum dots, cesium lead bromide perovskite quantum dots, cesium lead iodide perovskite quantum dots, and the like. The cadmium selenide / zinc sulfide core-shell structure colloidal quantum dots are core-shell structure colloidal quantum dots with cadmium selenide as the core and zinc sulfide as the shell. The upconversion luminescent material is a special material composed of rare earth elements doped in the crystal lattice of the crystal. Under the excitation of near-infrared light, the absorbed low-energy near-infrared photons can be converted into high-energy visible light through multi-photon absorption or energy transfer. The electrochemiluminescent agent includes trispyridine ruthenium and the like.

[0029] The “×× marker” named in the present application refers to a reagent component connected with a material with a prefix “××”. The “×× diluent” named in the present application refers to a reagent solution used for dissolving the material with the prefix “××” in the process of immunodetection or the process of preparing a detection reagent.

[0030] The AE marker includes materials in acridinium ester-labeled antibodies, acridinium ester-labeled antigens, and acridinium ester-labeled other detection reagents, which may be, for example, carrier proteins, carrier polymers, carrier compounds, and the like. Similarly, the luminol marker, isoluminol marker, AP marker, HRP marker, fluorescent material marker, quantum dot material marker, upconversion luminescent material marker, and electrochemiluminescent material marker are also understood accordingly.

[0031] The present application also provides a kit comprising the compound according to the present application.

[0032] The present application also provides the use of the compound according to the present application, or the blocking agent, or the diluent, or the kit in blocking a solid phase carrier. Preferably, the solid phase carrier is selected from at least one of a microsphere, a plate, and a membrane. More preferably, the solid phase carrier is selected from at least one of a magnetic microsphere, a plastic microsphere, a latex microparticle, a fluorescent microsphere, colloidal gold, a microwell plate, glass, a capillary, nylon, and nitrocellulose membrane.

[0033] The present application has the following beneficial effects over the prior art:

[0034] The compounds of the present application and blocking reagents comprising the same can be used as a universal material in the practice of immunoassay blocking solid phase carriers to improve the performance of reagents in the overall assay platform.

[0035] Additional aspects and advantages of the present application will be in part apparent and in part pointed out below in the description. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The chromatography-mass spectrometry profile of the compound obtained in Example 1 of the present application is shown, which proves that the amphoteric four-arm polyethylene glycol derivative PEG2-CONH-C6H 13 . DETAILED DESCRIPTION

[0037] Embodiments of the present application are described in detail below. The embodiments described below are examples only and are not intended to limit the present application, as interpreted in the broadest light possible.

[0038] It should be noted that the terms "first", "second" are used only for descriptive purposes and are not to be construed as indicating or implying relative importance or an indicated number of technical features. Thus, features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise stated, the meaning of "a plurality" is two or more.

[0039] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and any values are provided as approximate descriptions of the ranges and are accomplished by the values within each range. For numeric ranges, the endpoints of each range, the endpoints of each range and individual point values, and individual point values can be combined with each other to generate one or more new numeric ranges, which should be considered as specifically disclosed herein.

[0040] In order that the present application can be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in this document concur in meaning with the general usage of those terms by those skilled in the art to which the present application belongs.

[0041] In this document, the terms "comprising" or "including" are open-ended, that is, they include the stated subject-matter, but not to the exclusion of additional subject-matter.

[0042] In this document, the terms "optionally", "optional" or "optional" generally mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0043] Definitions and explanations of terms

[0044] Unless otherwise indicated, the definitions of groups and terms used in the present application and the claims, including definitions of examples, illustrative examples, preferred definitions, definitions recited in tables, definitions of specific compounds in examples, etc., can be combined and combined with each other in any manner. The group definitions and compound structures after such combination should be within the scope of the present application.

[0045] The term "stereoisomer" refers to isomers that have the same structure except for the arrangement of atoms in space, including cis-trans isomers, enantiomers, diastereomers and conformers.

[0046] The term "tautomers" refers to isomers of functional groups due to the rapid movement of an atom in two positions in the molecule. The compounds of the present application can exhibit tautomerism. Tautomeric compounds can exist in two or more interconvertible forms. Proton-shift tautomers result from the migration of a hydrogen atom covalently bonded between two atoms. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually result in a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates; in phenols, the enol form predominates. The present application includes all tautomeric forms of the compounds.

[0047] The term "solvate" refers to a compound of the present application or a salt thereof, including stoichiometric or non-stoichiometric amounts of a solvent, bound by non-covalent intermolecular forces, which is water when the solvent is water, a hydrate.

[0048] The term "C2-C8alkyl" is to be understood as meaning a straight-chain or branched saturated monovalent hydrocarbon group having 2 to 8 carbon atoms. Said alkyl group is, for example, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, and the like. "C4-C6alkyl" is to be understood as meaning a straight-chain or branched saturated monovalent hydrocarbon group having 4 to 6 carbon atoms. Other related terms are to be construed likewise.

[0049] The term "C2-C8alkenyl" is to be understood as preferably meaning a straight-chain or branched monovalent hydrocarbon group, which contains one or more double bonds and which has 2, 3, 4, 5, 6, 7, 8 carbon atoms. "C4-C6alkenyl" is to be understood as preferably meaning a straight-chain or branched monovalent hydrocarbon group, which contains one or more double bonds and which has 4 to 6 carbon atoms. Other related terms are to be construed likewise.

[0050] The term "C2-C8alkynyl" is to be understood as preferably meaning a straight-chain or branched monovalent hydrocarbon group, which contains one or more triple bonds and which has 2, 3, 4, 5, 6, 7, 8 carbon atoms. "C4-C6alkynyl" is to be understood as preferably meaning a straight-chain or branched monovalent hydrocarbon group, which contains one or more triple bonds and which has 4 to 6 carbon atoms. Other related terms are to be construed likewise.

[0051] The term "C3-C8cycloalkyl" is to be understood as meaning a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 8 carbon atoms. Such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. "C4-C6cycloalkyl" is to be understood as meaning a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 4 to 6 carbon atoms. Other related terms are to be construed likewise.

[0052] The term "3-8 membered heterocyclyl" means a saturated, monovalent, monocyclic or bicyclic hydrocarbon ring which contains 1-5, preferably 1-3, heteroatoms selected from N, O and S. In particular, the heterocyclyl group can include, but is not limited to: a 4-membered ring, such as azetidinyl, oxetanyl; a 5-membered ring, such as tetrahydrofuranyl, dioxolanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a 6-membered ring, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl; or a 7-membered ring, such as diazepanyl. Optionally, the heterocyclyl group can be benzo-fused. The heterocyclyl group can be bicyclic, for example, but not limited to, a 5,5 membered ring, such as hexahydrocyclopenta[c]pyrrol-2(lH)-yl ring, or a 5,6 membered bicyclic ring, such as hexahydropyrrolo[l,2-a]pyrazin-2(lH)-yl ring. The ring containing a nitrogen atom can be partially unsaturated, i.e. it can contain one or more double bonds, for example, but not limited to, 2,5-dihydro-lH-pyrrolyl, 4H-[l,3,4]thiadiazinyl, 4,5-dihydrooxazolyl or 4H-[l,4]thiazinyl, or it can be benzo-fused, for example, but not limited to, dihydroisoquinolinyl. Other related terms follow by analogy.

[0053] The term "C6-C8aryl" is to be understood as preferably meaning a monovalent aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring having 6-8 carbon atoms. In particular a ring having 6 carbon atoms ("C6aryl"), for example phenyl; or a ring having 8 carbon atoms ("C8aryl"), for example indanyl or indenyl. Other related terms follow by analogy.

[0054] The term "5-8 membered heteroaryl" is to be understood as including a monovalent monocyclic, bicyclic or tricyclic aromatic ring system having 5-10 ring atoms and containing 1-5 heteroatoms independently selected from N, O and S. In particular, the heteroaryl group is selected from thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thio-4H-pyrazolyl and the like as well as benzo derivatives thereof, for example benzofuranyl, benzothienyl, benzoxazolyl, benzoisoxazolyl, benzoimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and the like as well as benzo derivatives thereof, for example quinolinyl, quinazolinyl, isoquinolinyl and the like; or azocinyl, indolizinyl, purinyl and the like as well as benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenoxazinyl, phenothiazinyl, phenoxazinyl and the like. Other related terms follow by analogy.

[0055] The term "PEG" shall be understood as polyethylene glycol. The term "amide bond" shall be understood as a double bond between carbon and oxygen and a single bond between nitrogen and hydrogen, i.e. -CO-NH-. The term "ether bond" shall be understood as the functional group of an ether, i.e. an oxygen atom connected to two hydrocarbon groups, which can be the same or different, i.e. -C-O-C-. The term "carbon-nitrogen single bond" shall be understood as a covalent bond between a carbon atom and a nitrogen atom, i.e. -CN-. Other related terms are to be understood analogously.

[0056] According to an embodiment of the present application, the present application provides a compound represented by formula (I) or a tautomer, a stereoisomer, a hydrate, a solvate of the compound represented by formula (I):

[0057]

[0058] wherein,

[0059] n and x are integers, 2≤n≤10; 1≤x≤10;

[0060] M is independently selected from an amide bond, an ether bond, a carbon-nitrogen single bond;

[0061] R in each monomer structure in the compound is independently selected from the following groups: C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C3-C 10 cycloalkyl, 3-10 membered heterocyclyl, C6-C 10 aryl, 5-10 membered heteroaryl.

[0062] The above "n and x are integers, 2≤n≤10; 1≤x≤10" shall be understood as n can be 2, 3, 4, 5, 6, 7, 8, 9, 10; x can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0063] According to an embodiment of the present application, in the compound represented by formula (I):

[0064] n is 2, 4 or 6; x is 1, 2, 3 or 4;

[0065] M is independently selected from an amide bond, an ether bond, a carbon-nitrogen single bond;

[0066] According to an embodiment of the present application, in the compound represented by formula (I):

[0067] n is 2, x is 2; M is an amide bond;

[0068] R in each monomer structure in the compound is C6alkyl;

[0069] According to a specific embodiment of the present application, the present application provides a method for preparing the compound represented by formula (I), specifically as follows:

[0070] Preparation of amphoteric multi-arm polyethylene glycol derivatives (PEG x Preparation of amphoteric multi-arm polyethylene glycol derivatives (PEG

[0071] Preparation of amphoteric multi-arm polyethylene glycol derivatives (PEG

[0072] Multi-arm polyethylene glycol-amino or multi-arm polyethylene glycol-carboxyl is dissolved in a solvent, a catalyst is added, and the multi-arm polyethylene glycol-amino or multi-arm polyethylene glycol-carboxyl is stirred at room temperature for 15 minutes to 1 hour at a molar ratio of 1:(1.1-1.5) to the compound Y1-R. After stirring at room temperature for 12-24 hours, the solution is filtered and column chromatography is performed using a 10-100% methanol-ethyl acetate mixed solvent to obtain the product.

[0073] The above solvent is selected from ultrapure water, dichloromethane, tetrahydrofuran, acetonitrile;

[0074] The above catalyst is selected from carbodiimide compounds, active ester compounds, carbonium salt compounds, organophosphorus compounds, s-triazine derivatives, carbonyl diimidazole;

[0075] The above Y1 is selected from amino, carboxyl, acyl halide.

[0076] Preparation of amphoteric multi-arm polyethylene glycol derivatives (PEG

[0077] Multi-arm polyethylene glycol-hydroxyl is dissolved in a dilute alkali solution with a pH of 7.5-9.5, equilibrated to 0-4°C, and the compound CHO-R is added at a molar ratio of 1:(1.1-1.5) to the multi-arm polyethylene glycol. After stirring at 0-4°C for 12-24 hours, the solution is column chromatographed using a 10-100% methanol-ethyl acetate mixed solvent to obtain the product.

[0078] The above dilute alkali solution is selected from sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate.

[0079] Preparation of amphoteric multi-arm polyethylene glycol derivatives (PEG

[0080] Multi-arm polyethylene glycol-amino is dissolved in ultrapure water, a small amount of glacial acetic acid is added dropwise, and the compound CHO-R is added at a molar ratio of 1:(1.1-1.5) to the multi-arm polyethylene glycol. After stirring at room temperature for 0.5-2 hours, a reducing agent is added at a molar ratio of 1:(1.1-1.5) to the multi-arm polyethylene glycol. After stirring at room temperature for 4-8 hours, the solution is column chromatographed using a 10-100% methanol-ethyl acetate mixed solvent to obtain the product.

[0081] The above reducing agent is selected from sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride.

[0082] Formulation of amphoteric multi-arm polyethylene glycol derivative blocking agent

[0083] According to one specific embodiment of the present application, the present application provides a method for preparing a blocking agent, wherein the blocking agent comprises at least one of the above-mentioned end products.

[0084] An amphoteric multi-arm polyethylene glycol derivative 0.01-1 g is weighed into 10-100 mL of a buffer solution, 0.001-0.01 M sodium chloride, 0.001-0.005 M polyvinylpyrrolidone, and 0.01-0.1% polyvinyl alcohol are added respectively, stirred and dissolved, vacuum filtered using a 0.22 μm microporous filter membrane, and 0.01-0.05% sodium azide is added for storage.

[0085] The above-mentioned buffer solution is selected from the group consisting of tris-hydroxymethyl aminomethane buffer, phosphoric acid buffer, ammonium sulfate buffer, boric acid buffer, 2-(N-morpholine)ethanesulfonic acid buffer, 3-morpholinepropanesulfonic acid buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, and triethanolamine buffer.

[0086] The solutions of the present disclosure will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustration of the present disclosure and should not be considered as limiting the scope of the present disclosure. If a specific technique or condition is not specified in the examples, it is performed according to the technique or condition described in the literature in the art or according to the product manual. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained on the market.

[0087] Example 1: Preparation of amphoteric tetra-arm polyethylene glycol derivative PEG2-CONH-C6H 13 (n=2)

[0088] 0.05 mol of tetra-arm polyethylene glycol-carboxyl (x=2) is dissolved in ultrapure water, 0.06 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added, stirred at room temperature for 15 minutes, then 0.06 mol of Sulfo-NHS active ester and 0.06 mol of n-hexylamine are added, stirred at room temperature for 16 hours, filtered, and the filtrate is subjected to column chromatography using a 10-100% methanol-ethyl acetate mixed solvent to obtain the product, and the results of the chromatography-mass spectrometry are shown in Figure 1 .

[0089] Example 2: Preparation of amphoteric hexa-arm polyethylene glycol derivative PEG2-CONH-C6H 13 (n=4)

[0090] 0.05 mol of eight-armed polyethylene glycol-carboxyl (x = 2) was dissolved in ultrapure water, 0.06 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, stirred at room temperature for 15 minutes, then 0.06 mol of Sulfo-NHS active ester and 0.06 mol of n-hexylamine were added, stirred at room temperature for 16 hours, then filtered, and the filtrate was column chromatographed with 10-100% methanol-ethyl acetate mixed solvent to obtain the product.

[0091] Example 3: Preparation of amphoteric eight-armed polyethylene glycol derivative PEG2-CONH-C6H 13 (n = 6)

[0092] 0.05 mol of eight-armed polyethylene glycol-carboxyl (x = 2) was dissolved in ultrapure water, 0.06 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, stirred at room temperature for 15 minutes, then 0.06 mol of Sulfo-NHS active ester and 0.06 mol of n-hexylamine were added, stirred at room temperature for 16 hours, then filtered, and the filtrate was column chromatographed with 10-100% methanol-ethyl acetate mixed solvent to obtain the product.

[0093] Example 4: Preparation of amphoteric four-armed polyethylene glycol derivative PEG2-CN-C6H 13 (n = 2)

[0094] 0.05 mol of four-armed polyethylene glycol-amino (x = 2) was dissolved in ultrapure water, 0.1 mL of glacial acetic acid was added dropwise, 0.06 mol of n-hexyl aldehyde was added, stirred at room temperature for 1 hour, then 0.06 mol of sodium cyanoborohydride was added, stirred at room temperature for 4 hours, then the solution was column chromatographed with 10-100% methanol-ethyl acetate mixed solvent to obtain the product.

[0095] Example 5: Preparation of amphoteric four-armed polyethylene glycol derivative PEG3-CONH-C6H 13 (n = 2)

[0096] 0.05 mol of four-armed polyethylene glycol-carboxyl (x = 3) was dissolved in ultrapure water, 0.06 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, stirred at room temperature for 15 minutes, then 0.06 mol of Sulfo-NHS active ester and 0.06 mol of n-hexylamine were added, stirred at room temperature for 16 hours, then filtered, and the filtrate was column chromatographed with 10-100% methanol-ethyl acetate mixed solvent to obtain the product.

[0097] Example 6: Preparation of amphoteric four-armed polyethylene glycol derivative PEG4-CONH-C6H 13 (n = 2)

[0098] 0.05 mol of tetra-PEG-carboxyl (x = 4) was dissolved in ultrapure water, 0.06 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, stirred at room temperature for 15 minutes, then 0.06 mol of Sulfo-NHS active ester and 0.06 mol of n-hexylamine were added, stirred at room temperature for 16 hours, then filtered, and the filtrate was column chromatographed with 10-100% methanol-ethyl acetate mixed solvent to obtain the product.

[0099] Example 7: Preparation of amphoteric tetra-PEG derivative PEG2-CONH-CH3 (n = 2)

[0100] 0.05 mol of tetra-PEG-carboxyl (x = 2) was dissolved in ultrapure water, 0.06 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, stirred at room temperature for 15 minutes, then 0.06 mol of Sulfo-NHS active ester and 0.06 mol of aminomethane were added, stirred at room temperature for 16 hours, then filtered, and the filtrate was column chromatographed with 10-100% methanol-ethyl acetate mixed solvent to obtain the product.

[0101] Example 8: Preparation of amphoteric tetra-PEG derivative PEG2-CONH-C2H5 (n = 2)

[0102] 0.05 mol of tetra-PEG-carboxyl (x = 2) was dissolved in ultrapure water, 0.06 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, stirred at room temperature for 15 minutes, then 0.06 mol of Sulfo-NHS active ester and 0.06 mol of ethylamine were added, stirred at room temperature for 16 hours, then filtered, and the filtrate was column chromatographed with 10-100% methanol-ethyl acetate mixed solvent to obtain the product.

[0103] Example 9: Preparation of amphoteric tetra-PEG derivative PEG2-CONH-C4H9 (n = 2)

[0104] 0.05 mol of tetra-PEG-carboxyl (x = 2) was dissolved in ultrapure water, 0.06 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, stirred at room temperature for 15 minutes, then 0.06 mol of Sulfo-NHS active ester and 0.06 mol of n-butylamine were added, stirred at room temperature for 16 hours, then filtered, and the filtrate was column chromatographed with 10-100% methanol-ethyl acetate mixed solvent to obtain the product.

[0105] Example 10: Preparation of amphoteric tetra-PEG derivative PEG2-CONH-C8H 17 (n = 2)

[0106] 0.05 mol of tetra-PEG-carboxyl (x = 2) was dissolved in ultrapure water, 0.06 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, stirred at room temperature for 15 minutes, 0.06 mol of Sulfo-NHS active ester was added, and 0.06 mol of n-octylamine was added, stirred at room temperature for 16 hours, filtered, and the filtrate was column chromatographed with 10-100% methanol-ethyl acetate mixed solvent to obtain the product.

[0107] Example 11: Preparation of amphoteric tetra-PEG derivative PEG2-CONH-C 10 H 21 (n = 2)

[0108] 0.05 mol of tetra-PEG-carboxyl (x = 2) was dissolved in ultrapure water, 0.06 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, stirred at room temperature for 15 minutes, 0.06 mol of Sulfo-NHS active ester was added, and 0.06 mol of n-decylamine was added, stirred at room temperature for 16 hours, filtered, and the filtrate was column chromatographed with 10-100% methanol-ethyl acetate mixed solvent to obtain the product.

[0109] Example 12: Preparation of amphoteric tetra-PEG derivative PEG2-CONH-C 12 H 25 (n = 2)

[0110] 0.05 mol of tetra-PEG-carboxyl (x = 2) was dissolved in ultrapure water, 0.06 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, stirred at room temperature for 15 minutes, 0.06 mol of Sulfo-NHS active ester was added, and 0.06 mol of n-decylamine was added, stirred at room temperature for 16 hours, filtered, and the filtrate was column chromatographed with 10-100% methanol-ethyl acetate mixed solvent to obtain the product.

[0111] Example 13: Preparation of blocking agents A1-A6

[0112] For the preparation of blocking agents A1, A2, A3, A4, A5, A6, 0.05 g of the product of Examples 1, 2, 3, 4, 5, and 6, respectively, was dissolved in 10 mL of tris-hydroxymethyl aminomethane buffer, 0.005 M sodium chloride, 0.002 M polyvinylpyrrolidone, and 0.05% polyvinyl alcohol were added, respectively, stirred and dissolved, vacuum filtered using a 0.22 μm microporous filter, and 0.02% sodium azide was added for storage.

[0113] Example 14: Preparation of blocking agents D1-D6

[0114] For the preparation of blocking agents D1, D2, D3, D4, D5, D6, 0.1 g of the product of Examples 7, 8, 9, 10, 11, 12 respectively was dissolved in 10 mL of Tris buffer, 0.005 M sodium chloride, 0.002 M polyvinylpyrrolidone, 0.05% polyvinyl alcohol were added respectively, stirred and dissolved, vacuum filtered using a 0.22 μm microporous filter, and 0.02% sodium azide was added for storage.

[0115] Example 15: Blocking effect comparison

[0116] 5 mg of surface-coated antibody magnetic beads were respectively dispersed in 500 μL of BSA blocking agent, blocking agents A1-A6, D1-D6, washed once, and then re-dispersed in 500 μL of BSA blocking agent, blocking agents A1-A6, D1-D6, and incubated at 25°C for 16 hours. The above-mentioned BSA blocking agent was prepared by dissolving 1% BSA in Tris buffer and adding 0.005 M sodium chloride. The blocked magnetic beads were re-dispersed in 500 μL of working solution, and the noise RLU0and luminescence signal (RLU 20 , RLU 600 , RLU 10000 , the subscript number represents the relative content of the sample to be tested, and the greater the value, the higher the content) were detected, and the signal-to-noise ratio (P / N 20 , P / N 600 , P / N 10000 ) was calculated, and the results are shown in Table 1.

[0117] Table 1 Blocking effect comparison of two-sex multi-arm polyethylene glycol derivatives with different R groups

[0118]

[0119] The experimental results show that blocking agents A1 (prepared from PEG2-CONH-C6H 13 (n=2) of Example 1) and A2 (prepared from PEG2-CONH-C6H 13 (n=4) of Example 2) have lower noise and better signal-to-noise ratio compared to the control group. Under other conditions that are completely consistent, the BSA blocking agent is used as a control group, and blocking agent A1 has better blocking effect than A2 and A3 (prepared from PEG2-CONH-C6H 13 (n=6) of Example 3), the noise is reduced by 33% compared to the control group, the luminescence signal is slightly reduced, the overall signal-to-noise ratio is better than the control group, and the low value sensitivity (P / N 20 ) is improved by about 14%, and the performance of the reagent for detection is optimized. Through analysis, the four-arm polyethylene glycol derivative PEG2-CONH-C6H 13(n = 2) have hydrophobic groups at the end of the arms, which can block the hydrophobic sites on the surface of the magnetic beads. The polyethylene glycol skeleton provides hydrophilic properties, blocks the adsorption of interfering proteins, and at the same time minimizes the impact on the normal binding of antibody antigens. However, as the number of arms increases, a part of the arm end cannot be adsorbed on the surface of the magnetic beads due to steric hindrance, causing it to be free on the surface of the magnetic beads, which may cause weak non-specific adsorption, resulting in a slight decrease in blocking effect (Example 2). When the number of arms increases to 8, a large number of arm ends may be free on the surface of the magnetic beads, causing non-specific adsorption while blocking the binding of antibody antigens, so the noise of Example 3 increases and the luminescence signal decreases.

[0120] Table 2M is a comparison of the blocking effect of amide and carbon-nitrogen single bond amphoteric four-arm polyethylene glycol derivatives

[0121]

[0122] The experimental results show that under the condition that other conditions are completely consistent, the blocking effect of blocking agents A1 and A4 (prepared from Example 4 PEG2-CN-C6H 13 (n2) preparation) is not much different, and their noise values, luminescence signal values and signal-to-noise ratios are similar, so the type of M has little effect on the blocking effect.

[0123] Table 3 Effect of arm length on blocking effect

[0124]

[0125] The experimental results show that relative to the control group, blocking agent A1 and A5 (prepared from Example 5 PEG3-CONH-C6H 13 (n = 2) preparation) have lower noise and better signal-to-noise ratio. Under the condition that other conditions are completely consistent, blocking agent A1 has better blocking effect than A5 and A6 (prepared from Example 6 PEG4-CONH-C6H 13 (n = 2) preparation). Analysis shows that in the structure of the multi-arm polyethylene glycol derivative, the longer the arm length, the weaker the rigidity and the stronger the flexibility of the arm. When the arm end is adsorbed on the hydrophobic surface of the magnetic beads, the range of available sites increases, and the polyethylene glycol skeleton deforms, causing different space effects, such as blocking or partially covering the coated antibodies, which affects their binding to antigens. This is particularly evident when the sample content is high. Therefore, it is particularly important to select the appropriate arm length to improve the performance of the reagent according to the sample content and detection conditions.

[0126] Comparison of blocking effects of amphoteric four-arm polyethylene glycol derivatives PEG2-CONHR (n = 2) with different 4R groups

[0127]

[0128] The experimental results show that the length of the alkyl carbon chain of the R group directly affects the blocking effect of the compound. When the R group contains 1 carbon atom, the blocking effect of the compound is not as good as that of the BSA blocking agent (the BSA test data in Table 3); when the R group contains 2 carbon atoms, the blocking effect of the compound is close to that of the BSA blocking agent; when the R group contains 2 carbon atoms, the blocking effect of the compound is close to that of the BSA blocking agent; when the R group contains 4 carbon atoms, the blocking effect of the compound is better than that of the BSA blocking agent; when the R group contains 6 carbon atoms, the blocking effect of the compound reaches a peak, and after that, the length of the carbon chain of the R group is continued to be extended, which leads to a decrease in the blocking effect. When the number of carbon atoms contained in the carbon chain of the R group increases to 10, the blocking effect decreases sharply, and the noise increases sharply, which can be because the too long carbon chain causes non-specific adsorption, and at the same time, the light emission signal is lost seriously, which can be because the too long carbon chain excessively covers the surface of the magnetic beads, which affects the binding of the antibody and the antigen. Therefore, the optimal selection of the R group is C 4-8 .

[0129] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and modified by those skilled in the art without contradiction.

[0130] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A compound, characterized in that, The compound is a compound of formula (I): （Ⅰ）； wherein, n, x are integers, 2≤n≤6; x is 2, 3 or 4; M is independently selected from an amide bond, a carbon-nitrogen single bond; R in each monomer structure of the compound is independently selected from the group consisting of C2-C8 alkyl.

2. The compound of claim 1, wherein R in each monomer structure of the compound is independently selected from the group consisting of C4-C6 alkyl.

3. The compound of claim 1, wherein R in each monomer structure is C6 alkyl.

4. The compound of claim 1, wherein M is an amide bond.

5. The compound of claim 1, wherein x is 2.

6. The compound of claim 1, wherein n is 2, 4 or 6.

7. The compound of claim 1, wherein n is 2.

8. A sealant, characterized by, The compound of any one of claims 1-7.

9. The sealant of claim 8, wherein, The BSA or casein is further included.

10. The sealant of claim 8, wherein The at least one buffer is further included and is selected from the group consisting of Tris buffer, phosphate buffer, ammonium sulfate buffer, boric acid buffer, 2-(N-morpholino)ethanesulfonic acid buffer, 3-morpholinopropanesulfonic acid buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, and triethanolamine buffer.

11. A diluent, characterized in that, The compound of any one of claims 1-7. The diluent is selected from the group consisting of at least one of magnetic bead diluent, colloidal gold diluent, latex diluent, AE label diluent, luminol label diluent, isoluminol label diluent, AP label diluent, HRP label diluent, fluorescent label diluent, quantum dot label diluent, up-conversion phosphor label diluent, electrochemiluminescent label diluent, and sample diluent.

12. A test reagent, characterized by, The compound of any one of claims 1-7, further comprising at least one of magnetic beads, colloidal gold, latex microparticles, AE label, luminol label, isoluminol label, AP label, HRP label, fluorescent material label, quantum dot material label, up-conversion phosphor material label, and electrochemiluminescent material label.

13. A kit characterized in that, The compound of any one of claims 1-7.

14. Use of the compound of any one of claims 1-7 or the blocking agent of any one of claims 8-10 or the diluent of claim 11 or the detection reagent of claim 12 or the kit of claim 13 for blocking a solid support.

15. Use according to claim 14, characterized in that, The solid support is selected from the group consisting of at least one of microspheres, plates, and membranes.

16. The use according to claim 14, characterized in that, The solid support is selected from the group consisting of at least one of magnetic beads, plastic microspheres, latex microparticles, fluorescent microspheres, colloidal gold, microwell plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

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