Method for preparing nitrogen-branched two-arm polyethylene glycol derivative

Abstract

Disclosed in the present invention is a method for preparing a nitrogen-branched two-arm polyethylene glycol derivative. The nitrogen-branched two-arm polyethylene glycol derivative is as represented by general formula (1), and the method comprises a reaction between P-amide and BSM. The P-amide is a linear polyethylene glycol derivative containing an amide bond, and the BSM is a bifunctionalized small molecule compound. The definition of each symbol is as described herein. The present invention overcomes, by means of the rational design of reaction routes and reaction conditions, problems such as difficulty in separation and purification during the synthesis of analogous target products. Moreover, the present invention has the advantages of simple operation, high product purity, and high functionalization modification rate.

Description

Preparation method of nitrogen-branching two-arm polyethylene glycol derivative TECHNICAL FIELD The present application relates to the field of polymer synthesis chemistry, in particular to a preparation method of nitrogen-branching two-arm polyethylene glycol derivative, and especially to a preparation method of nitrogen-branching two-arm polyethylene glycol derivative with simple process and high product purity. BACKGROUND Polyethylene glycol derivatives can be coupled with drug molecules (including protein drugs and small organic molecule drugs), peptides, sugars, lipids, oligonucleotides, affinity ligands, co-factors, liposomes and biomaterials, etc. through covalent bonds to realize the PEGylation modification of drugs and other biological related substances. The modified drug molecules can have many excellent properties of polyethylene glycol, such as hydrophilicity, flexibility, anticoagulant property, etc. Compared with linear structure, polyethylene glycol derivatives containing branched structure can realize unique "umbrella" protection effect and achieve better modification effect. The branched two-arm polyethylene glycol derivative has been widely used in the field of drug modification, including carboxylic acid derivatives with the structure type of The prior art CN108659227A discloses a method for preparing the derivative using two linear monofunctional polyethylene glycol derivatives as raw materials. The reaction is realized by the secondary amine group (-NH-) contained in one of the raw materials and the activated carboxyl group (such as -C(=O)NHS) contained in the other raw material, and after the reaction is completed, an acid anhydride is added for further reaction, and then an ion column is used for separation and purification. This preparation method must use an acid anhydride to reduce the interference of the raw material containing a secondary amine group in the separation and purification step, which not only increases the operation steps, but also cannot be directly applied to the preparation of compounds sensitive to acid anhydride. Therefore, it is necessary to develop a new preparation method to obtain nitrogen-branching two-arm polyethylene glycol derivative with simple process and easy separation of product. SUMMARY In order to achieve the above-mentioned purpose, the present application provides an embodiment as follows: The preparation method of the nitrogen-branching two-arm polyethylene glycol derivative, characterized in that the nitrogen-branching two-arm polyethylene glycol derivative is shown as general formula (1), and the method comprises the reaction as shown below: Wherein, P-amide is a linear polyethylene glycol derivative containing an amide bond, and BSM is a bifunctional small molecule compound; T1, T2 are each independently C 1-20 hydrocarbon oxy group; n1, n2 are the polymerization degree of polyethylene glycol chain, selected from integers from 8 to 1000; L1, L2, L3 are each independently an alkylene group or a divalent linking group formed by an alkylene group and a heteroatom-containing group in combination; W is a group capable of reacting with the amide bond in the P-amide and forming a trivalent nitrogen branching core; L0 is a linking bond or a divalent linking group formed after W reacts with the amide bond in the P-amide; F1 is -COOH, -COONa or -COOK; The reaction is carried out under strong alkaline conditions or moderate alkaline conditions; Both the P-amide and the compound represented by general formula (1) are monodisperse or polydisperse. The present application also provides another embodiment: A preparation method of a nitrogen-branched two-arm polyethylene glycol derivative, characterized in that the nitrogen-branched two-arm polyethylene glycol derivative is represented by general formula (2), and the method comprises the following two steps: Step one: obtaining the compound represented by general formula (1) by the preparation method in the foregoing embodiments; Step two: performing one or more functional modification on the compound represented by general formula (1) to obtain the compound represented by general formula (2): T1, T2, n1, n2, L1 and L2 in the compound represented by general formula (2) are the same as those in the compound represented by general formula (1); L4 is a linking group formed after the functional modification and connecting the trivalent nitrogen branching core and F2; k is an integer from 1 to 100; Each F2 is independently a hydrogen atom or a functional group capable of reacting with a biologically relevant substance or a protected form thereof. The present application also provides another embodiment: A preparation method of a nitrogen-branched two-arm polyethylene glycol derivative and a biologically relevant substance, characterized in that the preparation method of the combination comprises preparing the nitrogen-branched two-arm polyethylene glycol derivative by using the preparation method in any of the foregoing embodiments. Compared with the prior art, the present application has the following beneficial effects: Beneficial Effect 1: This application prepares a nitrogen-branched two-arm polyethylene glycol derivative as shown in general formula (1) by reacting a linear polyethylene glycol derivative P-amide, which contains no terminal reactive groups but has an intermediate amide bond, with a bifunctional small molecule compound BSM. The amide bond itself has low reactivity, and the intermediate amide bond of P-amide is to some extent embedded by two PEG segments, theoretically making it difficult to perform coupling reactions with other compounds. However, the preparation method of this application can overcome the aforementioned problems through reasonable design of reaction conditions, enabling the coupling reaction based on the amide bond to proceed smoothly. For example, Example 8 confirms that different synthetic routes for E1-1 can improve the yield by selecting specific solvents. Beneficial Effect 2: One of the raw materials used in this application is P-amide, a polyethylene glycol derivative without terminal polar groups, and the other is a small molecule compound BSM. Since BSM differs significantly in molecular weight from the compound shown in general formula (1), its diffusion rate in the ion exchange column differs significantly. Whether BSM contains polar functional groups (e.g., carboxyl groups) as shown in general formula (1) or not will not affect the separation and purification of the product. On the other hand, compared with existing technologies where polyethylene glycol raw materials and products contain the same polar functional groups (e.g., carboxyl groups), P-amide in this application does not contain terminal polar groups, thus avoiding interference with the final product during separation and purification. Therefore, the preparation method of this application does not require additional operations to overcome the adverse effects of polar functional groups in the raw materials on product separation and purification, and can more easily obtain products with high purity and high functionalization modification rate. For example, the final product of Example 1.1 is two-arm polyethylene glycol carboxylic acid. Since the raw material does not use polyethylene glycol carboxylic acid derivatives, the separation and purification of the final product can be achieved efficiently using an ion exchange chromatography column. Beneficial Effect 3: In this application, the coupling reaction between P-amide and BSM can be carried out not only under strongly alkaline conditions, but also under moderately alkaline conditions at relatively low temperatures and without the need for transition metal catalysis, thus expanding the selection range of BSM. Attached Figure Description Figure 1 shows the P-amide (S1-3) prepared in Example 1.1. 1 H NMR spectrum. Figure 2 shows the nitrogen-branched two-arm polyethylene glycol acetic acid derivative (E1-1) prepared in Example 1.1. 1 H NMR spectrum. Figure 3 shows the nitrogen-branched two-arm polyethylene glycol aldehyde derivative (E2-1) prepared in Example 2. 1 H NMR spectrum. Figure 4 shows the nitrogen-branched two-arm polyethylene glycol succinimide ester derivative (E3-1) prepared in Example 3. 1 H NMR spectrum. Figure 5 is a GPC profile of E1-1. Figure 6 is a GPC profile of E2-1. Figure 7 is a GPC profile of E3-1. Figure 8 is a HPLC profile of testing the functionalization modification rate of E1-1. Figure 9 is a HPLC profile of testing the functionalization modification rate of E2-1. Figure 10 is a HPLC profile of testing the functionalization modification rate of E3-1. DETAILED DESCRIPTION The present application is described in detail for specific embodiments, however, it is to be understood that the application is by no means limited to the specific embodiments described and that various alterations and modifications will occur to those skilled in the art upon reading the present description. The description in the cited documents differs from the description in the present application, the description in the present application is controlling. This principle applies to all cited documents throughout the specification. 1. Glossary Unless otherwise described, all technical and scientific terms used in the present application have the meanings commonly understood by one of ordinary skill in the art. The disclosures of all cited patents and other publications are incorporated herein by reference in their entirety. In the event that the description of a term in this document conflicts with the description of the same term in any document incorporated herein by reference, the description of the term as set forth in this document will control. In the present application, the term "independently" means that the described objects are not restricted by each other. For example, "T1, T2 are independently C 1-20 The description of "hydrocarbyloxy" means that the specific forms of T1, T2 can be the same or different, and are not restricted by each other, such as T1, T1 can both be methoxy, or T1 is methoxy and T2 is ethoxy. In the present application, the term "combination" refers to the combination of any two or more listed elements. Any element in the combination can be one or more of the same or different forms. For example, the divalent linking group satisfying the "combination of alkylene and heteroatom-containing group" can be -CH2C(=O)-, -CH2C(=O)CH2-, -CH2OCH2CH2-, etc. In the present application, unless otherwise specified, the terms "including", "containing", and the like have an open and inclusive meaning, and can be interpreted as "including but not limited to" or "non- limitingly including". In this application, numerical ranges are designated by the use of the terms "about," "approximately," "around," or "circa," followed by a number that has not been specified as an integer, a percentage, a fraction, or other type of numerical value. Unless specifically stated, numerical ranges include both endpoints and all the intervening values, as well as any and all subranges therebetween. For example, "1-3" means 1, 3, and any number in between, such as, 1.1, 1.2, 1.3, 2.8, 3. The endpoints of the ranges are provided as example of the broader range itself or an example of a smaller range falling within the broader range. Unless specifically stated, an average can be an integer or a non-integer. Unless specifically stated, the number of groups is an integer by default; for example, -(CH2) 1-4 - means -(CH2)-, -(CH2)2-, -(CH2)3-, -(CH2)4-, and the like. means and the like. In this application, "about," "approximately," "around," or "circa," followed by a number that has not been specified as an integer, a percentage, a fraction, or other type of numerical value, means ±10% of the value. In some cases, the range can be enlarged to ±15%, but not more than ±20%. For example, "about 10 kDa" means 9-11 kDa, and in some cases, 8.5-11.5 kDa, but not more than 8-12 kDa. For another example, when the molecular weight of a PEG component is about 5 kDa, the molecular weight is allowed to be selected from 5 kDa ±10%, i.e., 4500-5500 Da. In this application, "about," "approximately," "around," or "circa," followed by a number that has not been specified as an integer, a percentage, a fraction, or other type of numerical value, means ±10% of the value. In some cases, the range can be enlarged to ±15%, but not more than ±20%. For example, "about 10 kDa" means 9-11 kDa, and in some cases, 8.5-11.5 kDa, but not more than 8-12 kDa. For another example, when the molecular weight of a PEG component is about 5 kDa, the molecular weight is allowed to be selected from 5 kDa ±10%, i.e., 4500-5500 Da. In this application, "heteroatom" means an atom other than carbon and hydrogen in an organic compound, including but not limited to O, S, N, P, Si, F, Cl, Br, I, B, and the like. In this application, the term "residue" means a group formed by the loss of a part of an atom or a group from a compound. In the present application, the term "linker" refers to a group having two or more attachment points. Unless otherwise specified, a linker can be attached to other groups at either of its attachment points, as the context of the present application requires. For example, if L in -CH2-L-CH2CH2- is -C(=O)O-, the specific case can be -CH2-C(=O)O-CH2CH2- or -CH2-OC(=O)-CH2CH2-. For certain specific divalent linkers, the "er" in the name can be used interchangeably with "bond", e.g., divalent amide group and amide bond, divalent ether group and ether bond. In the present application, the term "linking bond" does not contain any atom and only serves as a connection. A linking bond in a chemical structure can be marked with , e.g., represents a monovalent group -(CH2)4CH3. In particular, when the definition of a group contains "linking bond", it means that the group can be absent and replaced by a linking bond. In the present application, the number of carbon atoms in a group can be marked at the subscript position of "C". Unless otherwise specified, the number of carbon atoms does not include the contribution of substituents. For example, C 1-12 represents "having 1 to 12 carbon atoms". For another example, C 1-10 Alkylene represents any one of alkylene having a number of carbon atoms in the range of 1-10, i.e., C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 Unless otherwise specified, any one of alkylene can be linear, branched or cyclic structure. In the present application, the term "substituted" is used to describe a group or a compound, which means that at least one hydrogen atom of the group or the compound is replaced by a substituent, and unless otherwise specified, the type, number and number of atoms of the substituent are not particularly limited. Relative to the "unsubstituted" structure, the "substituted" structure is also called "substituted form". Unless otherwise specified, the "substituent" can be a single atom or a polyatomic group, but does not include hydrogen atoms. For hydrocarbon groups (such as alkyl, alkenyl, alkynyl) or hydrocarbon derivative residues (such as heterohydrocarbon groups), the substituents in their substituted forms include but are not limited to =O, -OR", =NR", =N-OR", -NR"R", -SR", -F, -Cl, -Br, -I, -SiR"R"R", -C(=O)R", -OC(=O)R", -C(=O)OR", -OC(=O)OR", -C(=O)NR"R", -OC(=O)NR"R", -NR"C(=O)R", -NR"C(=O)NR"R", -NR"C(=O)OR", -CN, -NO2, etc.; wherein each R" is independently hydrogen, C 1-10 alkyl or C 1-10heteroalkyl; optionally, any two R" attached to the same nitrogen atom form a cyclic structure; for example, -NR"R" includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. "Optionally substituted" means that the chemical structure being described can be substituted or unsubstituted. In the present application, the term "modification" refers to a process of changing the structure of a group or a compound by a chemical reaction known in the art. In the present application, the term "functional group" is a substituent or moiety that determines the chemical properties of an organic compound and can initiate a characteristic chemical reaction of the molecule. A compound containing a functional group is referred to as a "functionalized compound". In the present application, "functional modification" refers to a process of obtaining a specific functional group by modification. A compound modified by functional modification is also referred to as a "functional derivative" of the compound, such as a carboxyl functional derivative, an aldehyde functional derivative, etc.; wherein the compound being modified can or can not contain a functional group. A compound containing a functional group is also referred to as a "functional compound", and can be classified as a mono-functional compound, a di-functional compound, and a multi-functional compound according to the number of functional groups. A di-functional compound contains two functional groups, which can be the same or different. A multi-functional compound contains at least three functional groups, and any two functional groups can be the same or different. In the present application, the term "amide" refers to a compound with the general formula R-C(=O)-N(R')2, wherein R is an organic group, each R' is independently H or an organic group, and R or R' is an organic group when it is connected to (C=O) or N in the general formula via its carbon atom. Depending on R', the amide can be a primary amide, a secondary amide, and a tertiary amide, wherein both R' of the primary amide are H, one R' of the secondary amide is H and the other R' is an organic group, and both R' of the tertiary amide are organic groups. The secondary amide is also referred to as an N-substituted amide, and the tertiary amide is also referred to as an N,N-disubstituted amide. In the present application, the term "amide group" refers to a monovalent, divalent, or trivalent group formed by losing part of the atoms or groups from an amide. For example, -C(=O)NH- is a divalent amide group, and -C(=O)N< is a trivalent amide group. Unless otherwise specified, the "amide bond" in the present application refers to -C(=O)NH- by default. In the present application, the term "sulfonate group" refers to a group with the general formula R-S(=O)2O-, wherein R is an organic group and is connected to S in the general formula via its carbon atom. The sulfonate group in the present application includes, but is not limited to, a methanesulfonate group, a benzenesulfonate group, a p-toluenesulfonate group, etc. In the present application, the term "active ester group" is an ester group with high reactivity, including but not limited to the active ester groups disclosed in CN110591079B

[0355] In the present application, the term "active ester group" is an ester group with high reactivity, including but not limited to the active ester groups disclosed in CN110591079B In this application, polyethylene glycol (PEG) and its derivatives refer to compounds containing -(CH2CH2O) n segments; wherein n is the degree of polymerization, which refers to the number of repeating units (-CH2CH2O-). The molecular weight of a polymer refers to the average molecular weight, and the index of molecular weight (Mn) is used by default. The molecular weight of a polyethylene glycol chain can also be measured by the degree of polymerization. Unless otherwise specified, the degree of polymerization refers to the average degree of polymerization, which is by default the number average degree of polymerization. n The size of the molecular weight of a polyethylene glycol chain can also be measured by the degree of polymerization. Unless otherwise specified, the degree of polymerization refers to the average degree of polymerization, which is by default the number average degree of polymerization. In this application, unless otherwise specified, any compound represented by a general formula includes its salts (preferably pharmaceutically acceptable salts), tautomers, stereoisomers, and solvates. In this application, "tautomer" refers to an isomer of a compound that results from tautomerism. Tautomerism refers to the process in which a hydrogen atom or proton is transferred within a molecule, resulting in a change in the structure of the compound, often involving the conversion of a single bond and a double bond. The tautomerism of this application includes but is not limited to keto-enol tautomerism, amide-imidic acid tautomerism, lactam-lactim tautomerism, enamine-imine tautomerism, enamine-enamine tautomerism (such as phosphopyridoxyl catalyzed enzymatic reaction), proton transfer tautomerism, valence tautomerism, etc. In the present application, the term "salt" is selected from any one, any two, or two or more combinations of acid addition salts with inorganic and / or organic acids and base addition salts with inorganic and / or organic bases. When a compound contains a basic moiety (such as, but not limited to, pyridine or imidazole) and an acidic moiety (such as, but not limited to, carboxylic acid), a zwitterion ("inner salt") can be formed and is included in the term "salt" as used. The "salt" can be a pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salt, or it can be another salt. Salts of compounds can be formed by reacting the compound with an amount of acid or base, such as an equivalent amount of acid or base, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization. Exemplary acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and the like. Exemplary base addition salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) and salts with amino acids such as arginine or lysine. Basic nitrogenous groups can be quaternized with such reagents as lower alkyl halides (for example, methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (for example, dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (for example, decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), aralkyl halides (for example, benzyl and phenethyl bromides), and others. In the present application, acid addition salts and base addition salts of the compounds of any of the general formulae are preferably pharmaceutically acceptable salts, and are considered equivalent to the free forms of the compounds for the purposes of the present disclosure. In the present application, "stereoisomer" refers to isomers that have the same sequence of atoms but differ in the arrangement of atoms in three-dimensional space. Stereoisomerism can be classified as enantiomeric or diastereomeric, where diastereomeric further includes geometric isomeric and conformational isomeric. Enantiomers are molecules that are mirror images of one another and cannot be superimposed on one another. In the present application, "solvate" refers to an aggregate that comprises one or more compounds molecules and one or more solvent molecules. The solvent can be water, and the corresponding solvate can be referred to as a hydrate, including mono-hydrate, di-hydrate, hemi-hydrate, sesqui-hydrate, tri-hydrate, tetra-hydrate, etc. The solvent can also be an organic solvent. In the present application, the source of amino acid is not particularly limited unless otherwise specified, which can be natural source, non-natural source, or mixture of both. In the present application, the structural type of amino acid is not particularly limited unless otherwise specified, which can refer to L-form, D-form, or mixture of both. In the present application, the definition and examples of amino acid and its derivative skeleton in the references CN104877127A, WO / 2016 / 206540A, CN106967213A, CN108530637A, CN108530617A and each cited reference are also incorporated herein by reference. Unless otherwise specified, the skeleton belongs to the residue. In the present application, the term "biologically relevant substance" includes but is not limited to the substances described, listed and cited in documents CN104877127A, WO / 2016 / 206540A, CN106967213A, CN108530637A, CN108530617A and the respective cited documents. In general, the biologically relevant substance includes but is not limited to the following substances: drugs, proteins, polypeptides, oligopeptides, protein mimics, fragments and analogs, enzymes, antigens, antibodies and fragments thereof, receptors, small molecule drugs, nucleosides, nucleotides, oligonucleotides, antisense oligonucleotides, polynucleotides, nucleic acids, aptamers, polysaccharides, proteoglycans, glycoproteins, steroids, lipid compounds, hormones, vitamins, phospholipids, glycolipids, dyes, fluorescent substances, targeting factors, targeting molecules, cytokines, neurotransmitters, extracellular matrix substances, plant or animal extracts, viruses, vaccines, cells, vesicles, liposomes, micelles, etc. The biologically relevant substance also includes its precursors, activated states, derivatives, isomers, mutants, analogs, mimics, polymorphs, pharmaceutically acceptable salts, fusion proteins, chemically modified substances, genetically recombined substances, etc., and can also be agonists, activators, activators, inhibitors, antagonists, modulators, receptors, ligands or ligands, antibodies and fragments thereof, enzymes (such as kinases, hydrolytic enzymes, cleavage enzymes, oxygen-reducing enzymes, isomerases, transferases, deaminases, deimineases, invertases, synthetases, etc.), enzyme substrates (such as blood coagulation cascade protease substrates, etc.), etc. The derivatives include but are not limited to glycosides, nucleosides, amino acids, polypeptide derivatives. Chemical modification products that form new reactive groups, including chemical modification products that modify the type of reactive groups, and chemical modification products that are generated after additional introduction of functional groups, reactive groups, amino acids or amino acid derivatives, polypeptides, etc. structures, are all chemical modification substances of biologically relevant substances. The biologically relevant substance is allowed to form a modified or complexed biologically relevant substance with the target molecule, the accessory or the delivery carrier before or after being combined with the functionalized compound (including polyethylene glycol derivatives). Among them, the pharmaceutically acceptable salt can be an inorganic salt such as hydrochloride, sulfate, phosphate, or an organic salt such as oxalate, malate, citrate, etc. In the present application, a "reactive group" refers to a group capable of participating in a reaction, including but not limited to various common functional groups in organic chemistry. It is often necessary to selectively react only the target reactive group during the preparation process. To prevent the unrelated reactive group from affecting the target reaction, the unrelated reactive group is usually protected. The "protection" of a reactive group refers to the strategy of reversibly converting the reactive group to be protected into an inert group (non-reactive group) by a specific reagent. The protected group, which is different from the unprotected form, is called a "protecting group". For example, -OTBS is a protected form of hydroxyl (-OH), in which the -TBS group is a protecting group for hydroxyl. The protecting group not only remains stable during the target reaction, but can also be removed by conventional techniques in the art as needed. In the present application, the term "deprotection" refers to the process of converting the protected group to the unprotected form. In the present application, the alternative range of the coupling reaction is not particularly limited, as long as the reactants can form the desired covalent linkage through the reaction. The covalent linkage can be a linkage based on a linkage or a linker. Exemplary coupling reactions include, but are not limited to, alkylation, condensation, amidation, esterification, thioesterification, ring-opening reaction, ring-closing condensation, addition reaction, cycloaddition, α,β-unsaturated bond addition, alkyne addition, Schiff base reaction combined with reduction, click reaction, azide-alkyne addition reaction, 1,3-dipolar cycloaddition, Diels-Alder addition reaction, thiol-yne reaction, thiol-ene reaction, and thiol-vinyl reaction. The reaction conditions of the coupling reaction are related to the type of covalent linkage generated by the reaction, and the existing published techniques can be used. In the present application, for a polydisperse polymer, the molecular weight is allowed to be within ±10% of the given value, in some cases it can be extended to ±15% but not more than ±20%. For example, the molecular weight of a certain polydisperse polymer is 5 kDa, then the molecular weight of its individual molecules and macro-components is allowed to be within the range of 4500-5500 Da, and the content or purity of the polymer is calculated based on the components with an average molecular weight between 4500-5500 Da. In the present application, the molecular weight or the degree of polymerization of a monodisperse polymer is allowed to deviate by no more than ±5% when the degree of polymerization is greater than or equal to 10, and the degree of polymerization is allowed to deviate by no more than ±0.5 when the degree of polymerization is less than 10. When the content of a component that meets the foregoing requirements reaches a certain percentage (preferably greater than or equal to 90%, more preferably greater than 95%, more preferably greater than 96%, more preferably greater than 98%, and more preferably 99-100%), a monodisperse macro-product of the target component is considered to have been obtained. Even if the content ratio does not meet the foregoing requirements, as long as the preparation method of the present application or a similar method that is substantially the same in terms of preparation concept is used, the product obtained, which is insufficient in content, or the component that appears as a main product, a co-product, or a by-product, whether or not it is isolated and purified, is within the scope of the present application. In the present application, a polymer is considered to have monodispersity when the polydispersity index (PDI) thereof is no more than 1.005, which can be denoted as PDI = 1, and is considered to have polydispersity when the PDI thereof is more than 1.005. In the present application, the default unit of molecular weight is Dalton (Da). In the present application, a “small molecule” refers to a compound having a molecular weight of no more than 1000 Da. In the present application, the term “drug” includes any agent, compound, composition, or mixture that provides a physiological or pharmacological effect in vivo or in vitro, and often provides a beneficial effect. The type of the “drug” is not particularly limited, and includes, but is not limited to, a drug, a vaccine, an antibody, a vitamin, a food, a food additive, a nutritional agent, a nutraceutical, and other agents that provide a beneficial effect. The range of the “drug” in which the physiological or pharmacological effect is produced in vivo is not particularly limited, and can be systemic or local. The “drug” is not particularly limited in terms of activity, and is mainly an active substance that can interact with other substances, but can also be an inert substance that does not interact; wherein the inert drug can be converted into an active form by an in vivo action or a certain stimulus. Among them, a “small molecule drug” is a biologically relevant substance or a small molecule mimetic or active fragment thereof having a molecular weight of no more than 1000 Da. 2. A compound of general formula (1) In the present application, the preparation method of the compound represented by general formula (1) includes the following reaction: wherein P-amide is a linear polyethylene glycol derivative containing an amide bond, and BSM is a bifunctional small molecule compound; T1, T2 are each independently C 1-20 hydrocarbyloxy; n1, n2 are the degree of polymerization of the polyethylene glycol chain, and are selected from integers from 8 to 1000; L1, L2, L3 are each independently an alkylene group or a divalent linking group formed by combination of an alkylene group and a heteroatom-containing group; W is a group capable of reacting with the amide bond in the P-amide and forming a trivalent nitrogen branched core; L0 is a linking bond or a divalent linking group formed after W reacts with the amide bond in the P-amide; BSM and F1 in general formula (1) are each independently -COOH, -COONa or -COOK; The reaction is carried out under strong alkaline conditions or moderate alkaline conditions. The P-amide and the compound represented by general formula (1) are each monodisperse or polydisperse. In one specific embodiment of the present application, F1 in the BSM is -COONa or -COOK, and F1 in general formula (1) is -COOH. In one specific embodiment of the present application, F1 in the BSM is -COOH, and F1 in general formula (1) is -COONa or -COOK. In one specific embodiment of the present application, F1 in the BSM is the same as F1 in general formula (1), and is each -COOH, -COONa or -COOK. In one specific embodiment of the present application, T1, T2 are each independently C 1-10 hydroxyl, preferably any one of methoxy, ethoxy, propoxy, butoxy, pentoxy, hexyloxy and benzyloxy, more preferably methoxy. In one specific embodiment of the present application, n1, n2 are each independently an integer of 20-700, preferably an integer of 50-500. In one specific embodiment of the present application, L1, L2 are each independently a*-(CH2) p -Z-(CH2) q -b* or a*-Z-(CH2) q -b*, L3 is a*-(CH2) p -Z-(CH2) q- b*; wherein, a*, b* are each a bond; in P-amide, a* of L1 is connected with polyethylene glycol segment, b* of L1 is connected with -NH-, a* of L2 is connected with polyethylene glycol segment, b* of L2 is connected with -C(=O)-; in BSM, a* of L3 is connected with W, b* of L3 is connected with F1; wherein, p, q are each independently selected from an integer from 1 to 10; Z is a bond or a divalent linking group not participating in the reaction; when Z is not a bond, it is preferably selected from any one of -C(=O)-, -C(=S)-, -O-, -S-, -C(=O)O-, -OC(=O)-, -SC(=O)- and -C(=O)S-; preferably, L1 is -CH2CH2-; preferably, L2 is -CH2-; preferably, L3 is -CH2-, -CH2CH2- or -CH2CH2CH2-. In one specific embodiment of the present application, W is a halogen group or a sulfonate group, selected from any one of -F, -Cl, -Br, -I, alkyl sulfonate group and aryl sulfonate group, preferably -Br. In one specific embodiment of the present application, L0 is a bond or C 1-6 Alkylene, preferably a bond or methylene, more preferably a bond. In one specific embodiment of the present application, the base used in the aforementioned strong basic condition or moderate basic condition is an organic base or an inorganic base, selected from any one of sodium tert-butoxide, potassium tert-butoxide, triethylamine, pyridine, 4-dimethylaminopyridine, imidazole, diisopropylethylamine, sodium hydroxide, potassium hydroxide, sodium hydride, potassium hydride, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium acetate, potassium acetate, trisodium phosphate, tripotassium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, monosodium hydrogen phosphate and monopotassium hydrogen phosphate; wherein, sodium hydride, potassium hydride, sodium hydroxide or potassium hydroxide is preferably used in the strong basic condition, and triethylamine, trisodium phosphate or tripotassium phosphate is preferably used in the moderate basic condition. In one specific embodiment of the present application, the solvent used in the aforementioned reaction for preparing the compound of general formula (1) is selected from any one of water, methanol, ethanol, benzene, toluene, xylene, diethyl ether, petroleum ether, tetrahydrofuran, dioxane, dichloromethane, chloroform, dichloroethane, dimethylformamide, dimethylacetamide, 1,4-dioxane, acetonitrile, dimethyl sulfoxide, ethyl acetate, or a mixed solvent containing any two or three of the aforementioned solvents; When the reaction is carried out under strong basic condition, the solvent used is preferably toluene, dichloromethane or tetrahydrofuran, more preferably toluene or dichloromethane, most preferably toluene; When the reaction is carried out under moderate basic condition, the solvent used is preferably toluene, dichloromethane, tetrahydrofuran or acetonitrile, more preferably dichloromethane or tetrahydrofuran, most preferably tetrahydrofuran. In one embodiment of the present application, the reaction temperature for preparing the compound of general formula (1) is selected from 20-130℃; when the reaction is carried out under strong alkaline conditions, the reaction temperature is preferably 60-100℃, more preferably 80℃; when the reaction is carried out under moderate alkaline conditions, the reaction temperature is preferably 30-70℃, more preferably 50℃. In one embodiment of the present application, the reaction time for preparing the compound of general formula (1) is 4-24 hours, preferably 10-24 hours. In one embodiment of the present application, the reaction for preparing the compound of general formula (1) further uses a phase transfer catalyst; the phase transfer catalyst is a quaternary ammonium salt, a quaternary phosphonium salt, a sulfonium salt or an arsenic salt, preferably a quaternary ammonium salt, more preferably tetrabutylammonium fluoride or tetrabutylammonium bromide. In a more specific embodiment of the present application, the reaction is carried out under strong alkaline conditions, and the molar ratio of P-amide, BSM, base and phase transfer catalyst is 1:(10-30):(20-70):(5-15), preferably 1:20:(40-50):10. In a more specific embodiment of the present application, the reaction is carried out under moderate alkaline conditions, and the molar ratio of P-amide, BSM, base and phase transfer catalyst is 1:(10-30):(20-70):(30-50), preferably 1:20:(40-50):40. In one embodiment of the present application, the method for preparing the compound of general formula (1) further comprises reacting linear polyethylene glycol amine derivative LP-1 and linear polyethylene glycol carboxylic acid derivative LP-2 to obtain P-amide, and the reaction is as follows: wherein, LP-1 and LP-2 are monodisperse or polydisperse; T1, T2, n1, n2, L1 and L2 in LP-1 and LP-2 are the same as in general formula (1). In one embodiment of the present application, the number average molecular weight of the compound of general formula (1) is 1-100kDa, preferably 2-50kDa, more preferably 10-50kDa, and most preferably 10kDa, 20kDa, 30kDa, 40kDa or 50kDa. In one embodiment of the present application, the structure of the compound of general formula (1) is selected from any one of the following: 3. A compound of general formula (2) In the present application, the method for preparing the compound of general formula (2) comprises the following two steps: Step 1: obtaining the compound of general formula (1) by any of the aforementioned methods; Step two: the compound of general formula (1) is subjected to one or more functional modification to obtain a compound of general formula (2): T1, T2, n1, n2, L1and L2in general formula (2) are the same as those in general formula (1); L4is a linker formed after the functional modification, which connects the trivalent nitrogen branched core with F2; k is an integer from 1 to 100; each F2independently is a hydrogen atom or a functional group capable of reacting with a biorelevant substance or a protected form thereof. In one specific embodiment of the present application, L4in general formula (2) can be the same as or different from -L0-L3-in general formula (1). In one specific embodiment of the present application, F2is any one of an epoxy group, a hydroxyl group, a thiol group, a carboxyl group, an amino group, an aldehyde group, an active ester group, an active carbonate group, a carbamate group, an isocyanate group, an isothiocyanate group, a succinimidyl group, a maleimidyl group, an alkenyl group, an alkynyl group, an alkenoate group, an azido group, a cyano group, a dithiopyridyl group, an alpha-haloacetylalkynyl group, a folate group, a rhodamine group, a biotin group, a monosaccharide group and a polysaccharide group, or a protected form thereof; F2is preferably any one of the following structures: In one specific embodiment of the present application, L4is a divalent linker or a trivalent linker; preferably, L4is selected from any one of -CH2C(=O)-Y-, -CH2C(=O)-Y-(CH2) j -CH2C(=O)-Y-(CH2) j -M-, -CH2C(=O)-Y-(CH2) j -M-(CH2) j - and -CH2C(=O)-NH-A, and the left end thereof is connected with the trivalent nitrogen branched core in general formula (2); wherein, Y is -O- or -NH-; M is any one of -O-, -S-, -S-S-, -C(=O)O-, -OC(=O)- and -OC(=O)O-; each j independently is an integer from 1 to 10, preferably 1, 2, 3, 4 or 5; A is a trivalent residue of an amino acid, which is a residue obtained after all amino groups, carboxyl groups, hydroxyl groups and mercapto groups of an amino acid are removed; Preferably, L4is -CH2-, -CH2C(=O)O-, -CH2C(=O)OCH2CH2-, -CH2C(=O)NHCH2CH2-, -CH2C(=O)OCH2CH2OC(=O)CH2CH2- and any one of the foregoing, and the left end thereof is connected to the trivalent nitrogen branched core in general formula (2). In one embodiment of the present application, the aforementioned k is an integer from 1 to 8, preferably 1, 2 or 3, more preferably 1. In one embodiment of the present application, the aforementioned functional modification is performed once, twice or thrice in succession. In one embodiment of the present application, each functional modification is a micro-modification or a small molecule modification; the micro-modification is selected from any one of protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation, acidification and basification; the small molecule modification is selected from a coupling reaction, preferably an alkylation reaction, an amidation reaction or an esterification reaction; the small molecule is a bifunctional or multifunctional small molecule. The alkylation reaction refers to a coupling reaction that introduces an optionally substituted alkyl group to the substrate. The optionally substituted alkyl group is -CH2R0, wherein R0is a hydrogen atom, an optionally substituted hydrocarbyl group or an optionally substituted heterohydrocarbyl group. The amidation reaction and the esterification reaction refer to coupling reactions that form amide bonds and ester bonds, respectively. In one embodiment of the present application, the compound represented by general formula (1) is subjected to a functional modification to obtain a compound represented by general formula (2). In one embodiment of the present application, the compound represented by general formula (1) is subjected to a micro-modification to obtain a compound represented by general formula (2); preferably, the micro-modification is protection, deprotection or acidification. In one embodiment of the present application, the compound represented by general formula (1) is subjected to a small molecule modification to obtain a compound represented by general formula (2). In one embodiment of the present application, F1of the compound represented by general formula (1) is -COONa or -COOK, and F2of the compound represented by general formula (2) is -COOH. In one embodiment of the present application, F1of the compound represented by general formula (1) is -COOH, and F2of the compound represented by general formula (2) is selected from any one of the foregoing. In one embodiment of the present application, the structure of the compound represented by general formula (2) is selected from any one of the following: 4. A conjugate of a nitrogen branched two-arm polyethylene glycol derivative and a biologically relevant species In the present application, the method for preparing the conjugate of the nitrogen- branched two-armed polyethylene glycol derivative and the biologically relevant substance comprises using any of the aforementioned methods to prepare the nitrogen-branched two-armed polyethylene glycol derivative. In one specific embodiment of the present application, the biologically relevant substance is selected from any one of the following: a drug, a protein, a polypeptide, an oligopeptide, a protein mimetic, a fragment, an enzyme, an antigen, an antibody and fragments thereof, a receptor, a gene-related substance aptamer, a polysaccharide, a proteoglycan, a glycoprotein, a lipid compound, a hormone, a vitamin, a vesicle, a liposome, a dye, a fluorescent substance, a targeting factor, a cytokine, a neurotransmitter, an extracellular matrix substance, a plant or animal extract, a virus, a vaccine, a micelle; preferably, the biologically relevant substance is selected from any one of the following: a small molecule drug, a nucleic acid, a phospholipid, a glycolipid; more preferably, the biologically relevant substance is selected from any one of the following: a small molecule drug, a nucleoside, a nucleotide, an oligonucleotide, an antisense oligonucleotide, a polynucleotide; wherein the small molecule drug is preferably selected from any one of the following: a flavonoid, a terpenoid, a carotenoid, a saponin, a steroid, a quinone, an anthraquinone, a fluoroquinone, a coumarin, an alkaloid, a porphyrin, a polyphenol, a macrolide, a monobactam, a phenylpropanoid, an anthracycline, an aminoglycoside. In one specific embodiment of the present application, the biologically relevant substance is a genetically engineered drug selected from any one of the following: an antibody, an antibody fragment, an interleukin, a lysozyme, an interferon, a growth hormone, an erythropoietin, and a granulocyte colony-stimulating factor; and the interferon is preferably an alpha-, beta- or gamma-interferon. In one specific embodiment of the present application, the method for preparing the conjugate of the nitrogen-branched two-armed polyethylene glycol derivative and the biologically relevant substance comprises the step of generating a covalent linkage by reacting the reactive group of the nitrogen-branched two-armed polyethylene glycol derivative with the biologically relevant substance. DETAILED DESCRIPTION The method for preparing the present application comprises any protection and deprotection process required by the reaction. The intermediates and final products prepared in the present application can be purified by purification methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, anti-precipitation, membrane dialysis, supercritical extraction, column chromatography, etc. The characterization of the structure and molecular weight of the final product can be confirmed by characterization methods including but not limited to nuclear magnetic resonance, electrophoresis, ultraviolet-visible spectrophotometer, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism, etc. In the present application, it is preferred to use H-NMR for structure confirmation, gel permeation chromatography (GPC) for measuring the number average molecular weight (Mn) and the polydispersity index (PDI), and high performance liquid chromatography (HPLC) for testing the functional modification rate. 1 H-NMR for structure confirmation, gel permeation chromatography (GPC) for measuring the number average molecular weight (Mn) and the polydispersity index (PDI), and high performance liquid chromatography (HPLC) for testing the functional modification rate. n The preparation of the nitrogen-branched two-armed polyethylene glycol derivative is described below in conjunction with some specific examples. The following examples are further to illustrate the present application, but not to limit the scope of protection of the present application. Example 1: Preparation of nitrogen branched two-armed polyethylene glycol carboxylic acid derivative Example 1.1: Preparation of nitrogen branched two-armed polyethylene glycol acetic acid derivative (40 kDa) E1-1 Method 1: Nitrogen branched two-armed polyethylene glycol sodium acetate derivative was obtained by coupling reaction, and the nitrogen branched two-armed polyethylene glycol acetic acid derivative E1-1 was obtained by acidification without purification. Step a: Under nitrogen atmosphere, methoxy polyethylene glycol ethylamine derivative S1-1 (20.00 g, 1.0 mmol, Mw= 20 kDa, PDI = 1.01), methoxy polyethylene glycol acetic acid derivative S1-2 (20.00 g, 1.0 mmol, Mw= 20 kDa, PDI = 1.01), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCI, 0.96 g, 5.0 mmol), 1-hydroxybenzotriazole (HOBt, 0.68 g, 5.0 mmol) and triethylamine (TEA, 0.81 g, 8.0 mmol) were sequentially dissolved in 200 mL dichloromethane (DCM) under ice bath, then raised to room temperature, and stirred for 24 h. After the reaction was completed, it was concentrated, 200 mL water was added, and it was washed with ethyl acetate (100 mL*2). The aqueous phase was extracted with dichloromethane (100 mL*2). The organic phases were combined, concentrated, and recrystallized to obtain S1-3 (30.88 g). n = 20 kDa, PDI = 1.01), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCI, 0.96 g, 5.0 mmol), 1-hydroxybenzotriazole (HOBt, 0.68 g, 5.0 mmol) and triethylamine (TEA, 0.81 g, 8.0 mmol) were sequentially dissolved in 200 mL dichloromethane (DCM) under ice bath, then raised to room temperature, and stirred for 24 h. After the reaction was completed, it was concentrated, 200 mL water was added, and it was washed with ethyl acetate (100 mL*2). The aqueous phase was extracted with dichloromethane (100 mL*2). The organic phases were combined, concentrated, and recrystallized to obtain S1-3 (30.88 g). n = 20 kDa, PDI = 1.01), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCI, 0.96 g, 5.0 mmol), 1-hydroxybenzotriazole (HOBt, 0.68 g, 5.0 mmol) and triethylamine (TEA, 0.81 g, 8.0 mmol) were sequentially dissolved in 200 mL dichloromethane (DCM) under ice bath, then raised to room temperature, and stirred for 24 h. After the reaction was completed, it was concentrated, 200 mL water was added, and it was washed with ethyl acetate (100 mL*2). The aqueous phase was extracted with dichloromethane (100 mL*2). The organic phases were combined, concentrated, and recrystallized to obtain S1-3 (30.88 g). 1 H NMR (400 MHz, (CD3)2SO) δ: 7.63 (t, 1H, -C(=O)NH-), 3.87 (s, 2H, -OCH2C(=O)NH-), 3.74-3.37 (m, PEG, -O(CH2)2NHC(=O)-), 3.24 (s, 6H, -OCH3). Step b: The above S1-3 (20.00 g, 0.5 mmol), sodium bromoacetate (S1-4, 1.61 g, 10.0 mmol), sodium hydride (NaH, 60%; 1.00 g, 25.0 mmol), tetrabutylammonium bromide (TBAB, 1.61 g, 5.0 mmol) were added to a three-necked flask containing 200 mL toluene (Tol). The three-necked flask was equipped with mechanical stirring, a thermometer and a reflux condenser. It was heated to 80°C, stirred and refluxed, and the reaction was carried out. After the reaction overnight, the reaction liquid was cooled to room temperature, acidified with hydrochloric acid (6N) to pH = 3, and extracted twice with dichloromethane (100 mL*2). The organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by ion exchange chromatography column to obtain E1-1 (6.91 g). 1H NMR (400 MHz, (CD3)2SO) d: 4.26-3.94 (m, 4H, -OCH2C(=0)N<,>NCH2COOH), 3.70-3.36 (m, PEG, -OCH2CH2N<), 3.24 (s, 6H, -OCH3). GPC measured molecular weight M n = 40.1 kDa, PDI = 1.01. The functionalization modification rate is 100%. Method two: directly obtain the nitrogen branched two-arm polyethylene glycol acetic acid derivative E1-1 through coupling reaction. Into a reaction flask equipped with a magnetic stirring bar and 200 mL of tetrahydrofuran (THF), S1-3 (20.00 g, 0.5 mmol, M n = 40.0 kDa, PDI = 1.01), bromoacetic acid (S1-5, 1.39 g, 10.0 mmol), potassium phosphate (K3PO4, 4.24 g, 20.0 mmol) and tetrabutylammonium bromide (TBAB, 6.44 g, 20.0 mmol) were added successively. The reaction flask was capped and wrapped with sealing film, and the reaction was stirred at 50 °C for 24 hours. After the reaction was completed, 200 mL of water was added, and ethyl acetate was used for washing (100 mL*2). The water phase was extracted with dichloromethane (100 mL*2). The organic phase was combined and concentrated under reduced pressure, and ion exchange chromatography column purification was performed to obtain the nitrogen branched two-arm polyethylene glycol acetic acid derivative E1-1 (6.17 g). 1 H NMR (400 MHz, (CD3)2SO) d: 4.26-3.94 (m, 4H, -OCH2C(=0)N<,>NCH2COOH), 3.70-3.36 (m, PEG, -OCH2CH2N<), 3.24 (s, 6H, -OCH3). GPC measured molecular weight M n = 40.1 kDa, PDI = 1.01. The functionalization modification rate is 100%. Example 1.2: Preparation of nitrogen branched two-arm polyethylene glycol acetate derivative E1-2 Using the method one of Example 1.1, the acidification treatment in step b is changed to extraction under alkaline conditions to obtain the sodium salt form E1-2 of E1-1, the structure is as follows: Example 1.3: Preparation of nitrogen branched two-arm polyethylene glycol acetic acid derivative (5 kDa, 10 kDa) The linear mono-functionalized polyethylene glycol derivative starting materials S1-1 and S1-2 were simultaneously replaced with corresponding structures having a number average molecular weight of about 5 kDa or 10 kDa, respectively, using Method 1 or Method 2 of Example 1.1, and otherwise without change, to produce nitrogen-branched, two-armed polyethylene glycol carboxylic acid derivatives having the same structure as E1-1 and a number average molecular weight of 10.1 kDa and 20.1 kDa, respectively, and a functionalization modification rate of 100% in each case. Example 2: Preparation of nitrogen-branched, two-armed polyethylene glycol aldehyde derivatives The compound of general formula (1) can be functionally modified to obtain a compound of general formula (2) containing an aldehyde group functional group. The functional modification can be achieved by a bifunctional small molecule, such as S2-1 containing an amino group and an acetal group (a protected form of an aldehyde group). The method for preparing the nitrogen-branched, two-armed polyethylene glycol propionaldehyde derivative E2-1 is as follows: Under a nitrogen atmosphere, E1-1 (20.05 g, 0.5 mmol), 3-aminopropanal diethyl acetal (S2-1, 1.47 g, 10.0 mmol), EDC HCI (5.76 g, 30.0 mmol), HOBt (4.05 g, 30.0 mmol), and TEA (4.55 g, 45.0 mmol) were sequentially dissolved in 200 mL of dichloromethane in an ice bath, then raised to room temperature, and stirred for 24 h. After the reaction was completed, it was concentrated, 200 mL of water was added, and it was washed with ethyl acetate (100 mL*2). The aqueous phase was adjusted to pH 1.0 with 1 mol / L HCI under an ice bath. After 4 h of reaction at room temperature, it was extracted twice with dichloromethane (100 mL*2). The organic phases were combined, washed with saturated brine, dried, filtered, concentrated, and recrystallized from anhydrous isopropyl alcohol to obtain E2-1 (5.73 g). 1 HNMR (400 MHz, (CD3)2SO) δ: 4.26-3.85 (m, 4H, -OCH2C(=O)N<,>NCH2C(=O)NH-), 3.74-3.35 (m, PEG, -OCH2CH2N<,-C(=O)NHCH2CH2CHO), 3.23 (s, 6H, -OCH3), 2.69-2.65 (m, 2H, -C(=O)NHCH2CH2CHO). The molecular weight M n = 40.2 kDa, PDI = 1.01. The functionalization modification rate was 99.1%. Example 3: Preparation of nitrogen-branched, two-armed polyethylene glycol succinimidyl ester derivatives The compound of general formula (1) can be functionally modified once to obtain a compound of general formula (2) containing an active ester group functional group. The functional modification can be achieved by a carboxyl activating agent, such as N-hydroxysuccinimide (NHS). The preparation method of nitrogen branched two-arm polyethylene glycol succinimidyl ester derivative E3-1 is as follows: E1-1 (4.01 g, 0.1 mmol) was dissolved in 40 mL of dichloromethane, and NHS (0.35 g, 3.0 mmol) and N, N'-dicyclohexyl carbodiimide (DCC, 0.62 g, 3.0 mmol) were added, and the reaction was stirred at room temperature for 24 hours. After the reaction was completed, the insoluble matter was removed by filtration, concentrated under reduced pressure, and recrystallized and purified to obtain E3-1 (3.65 g). 1 H NMR (400 MHz, (CD3)2SO) δ: 4.78-4.13 (m, 4H, -OCH2C(=O)N<,>NCH2CONHS), 3.70-3.38 (m, PEG, -OCH2CH2N<), 3.24 (s, 6H, -OCH3), 2.86-2.78 (m, 4H, NHS). The molecular weight M n = 40.2 kDa, PDI = 1.01. The functional modification rate was 99.5%. Example 4: Preparation of nitrogen branched two-arm polyethylene glycol p-nitrophenyl ester derivative The compound represented by general formula (1) can be subjected to two consecutive functional modifications to obtain two compounds represented by general formula (2), for example, two consecutive coupling reactions are performed using a bifunctional small molecule. The preparation method of nitrogen branched two-arm polyethylene glycol p-nitrophenyl ester derivative E4-2 is as follows: Step a: Under a nitrogen atmosphere, E1-1 (8.02 g, 0.2 mmol), ethylene glycol (S4-1, 0.62 g, 10.0 mmol), 4-dimethylaminopyridine (DMAP, 0.24 g, 2.0 mmol), and TEA (0.81 g, 8.0 mmol) were sequentially dissolved in 80 mL of dichloromethane, and a dichloromethane solution (20 mL) of EDC HCl (1.54 g, 8.0 mmol) was slowly added dropwise under ice bath, and then the temperature was raised to room temperature, and the reaction was stirred for 24 h. After the reaction was completed, it was concentrated, 200 mL of water was added, and it was washed with ethyl acetate (100 mL*2). The aqueous phase was extracted with dichloromethane (100 mL*2). The organic phases were combined, concentrated, and recrystallized and purified to obtain a nitrogen branched two-arm polyethylene glycol derivative E4-1 (7.51 g) also satisfying general formula (2). The molecular weight M n = 40.2 kDa, PDI = 1.01. The functional modification rate was 99.9%. Step b: To a solution of E4-1 (4.02 g, 0.1 mmol) and TEA (0.51 g, 5.0 mmol) in 40 mL of dichloromethane, a solution of p-nitrophenyl chloroformate (S4-2, 1.01 g, 5.0 mmol) in 10 mL of dichloromethane was added dropwise under ice bath. The mixture was stirred for one hour and then allowed to warm to room temperature. The reaction was continued for 24 hours. After the reaction was completed, the mixture was extracted, concentrated, and purified by recrystallization to give E4-2 (3.49 g). 1 H NMR (400 MHz, (CD3)2SO) δ: 8.28 (d, 4H, Ar), 7.38 (d, 4H, Ar), 4.47-4.38 (m, 4H, -C(=O)OCH2CH2OC(=O)O-), 4.27-3.92 (m, 4H, -OCH2C(=O)N<,>NCH2COO-), 3.74-3.36 (m, PEG, -OCH2CH2N<), 3.24 (s, 6H, -OCH3). The molecular weight was measured by GPC to be M n = 40.3 kDa, PDI = 1.01. The functionalization modification rate was 99.8%. Example 5: Preparation of nitrogen-branched two-arm polyethylene glycol amine derivative The preparation method of nitrogen-branched two-arm polyethylene glycol propylamine derivative E5-1 is as follows: First, a nitrogen-branched two-arm polyethylene glycol carboxylic acid derivative is synthesized, and then a nitrogen-branched two-arm polyethylene glycol amine derivative containing another functional group is obtained by functionalization modification, which corresponds to the preparation method of the compound represented by general formula (2). E1-1 (4.01 g, 0.1 mmol) and ethylenediamine containing a Boc-protected amino group (S5-1, 0.48 g, 3.0 mmol) were dissolved in 40 mL of dichloromethane, and N,N-diisopropylethylamine (DIPEA, 0.65 g, 5.0 mmol), HOBt (0.41 g, 3.0 mmol), and EDC HCl (0.58 g, 3.0 mmol) were added in sequence under ice bath. The mixture was allowed to warm to room temperature and stirred overnight. After the reaction was completed, the mixture was washed with saturated sodium bicarbonate solution (10 mL*2), water (10 mL*2), and brine (10 mL*2) in sequence. The mixture was concentrated under reduced pressure, and the Boc protection was removed using a TFA / DCM mixed solution (1:1 v / v), followed by washing with purified water and dichloromethane extraction. The extraction was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by recrystallization to give E5-1 (3.33 g). 1H NMR (400 MHz, (CD3)2SO) δ: 4.30-3.86 (m, 4H, -OCH2C(=0)N<,>NCH2C(=0)NH-), 3.74-3.33 (m, PEG, -OCH2CH2N<,-C(=0)CH2CH2NH2), 3.24 (s, 6H, -OCH3), 2.84 (t, 2H, -C(=0)CH2CH2NH2). The molecular weight M n = 40.2 kDa, PDI = 1.01. The functionalization modification rate is 99.9%. Example 6: Preparation of nitrogen branched two-arm polyethylene glycol maleimide derivative The preparation method of nitrogen branched two-arm polyethylene glycol maleimide derivative E6-1 is as follows: E4-1 (4.02 g, 0.1 mmol) was dissolved in 40 mL of anhydrous dichloromethane, and EDC HCI (0.77 g, 4.0 mmol) and TEA (0.40 g, 4.0 mmol) were added. DMAP (0.12 g, 1.0 mmol) was added to a 15 mL dichloromethane solution of 3-maleimide propionic acid containing furan protecting group (S6-1, 1.19 g, 5.0 mmol). After mixing the two solutions, the reaction was stirred at room temperature for 24 hours. After the reaction was completed, 40 mL of water was added, and washed with ethyl acetate (20 mL*2). The aqueous phase was extracted with dichloromethane (20 mL*2). The organic phase was combined and concentrated under reduced pressure, 40 mL of toluene was added, and 0.40 g of 2,6-di-tert-butyl-4-methylphenol (BHT) was added, heated to 125°C, and stirred for 5 hours to remove the furan protecting group. Precipitated with anhydrous diethyl ether, recrystallized with anhydrous isopropanol to obtain E6-1 (3.76 g). 1 H NMR (400 MHz, (CD3)2SO) δ: 6.70 (s, 2H, Mal), 4.24-3.93 (m, 4H, -OCH2C(=0)N<,>NCH2COO-), 3.85 (t, 2H, -O(C=0)CH2CH2-Mal), 3.76-3.36 (m, PEG, -OCH2CH2N<), 3.24 (s, 6H, -OCH3), 2.69 (t, 4H, -C(=0)OCH2CH2OC(=0)-), 2.54 (t, 2H, -O(C=0)CH2CH2-Mal). The molecular weight M n = 40.3 kDa, PDI = 1.01. The functionalization modification rate is 100%. Example 7: Preparation of nitrogen branched two-arm polyethylene glycol dicarboxylic acid derivative The trifunctional small molecule is used for functional modification, and a bifunctional compound represented by general formula (2) can be obtained. The preparation method of the nitrogen branched two-arm polyethylene glycol dicarboxylic acid derivative E7-1 is as follows: E1-1 (4.01 g, 0.1 mmol) and glutamic acid containing two tBu-protected carboxyl groups (S7-1, 0.78 g, 3.0 mmol) were dissolved in 40 mL of dichloromethane, and DIPEA (0.65 g, 5.0 mmol) and DMAP (0.12 g, 1.0 mmol) were added. After stirring at room temperature for 5 min, EDC HCl (0.58 g, 3.0 mmol) was added, and the reaction was stirred at room temperature overnight. After the reaction was completed, it was washed successively with saturated sodium bicarbonate solution (10 mL*2), water (10 mL*2), and brine (10 mL*2). After concentration under reduced pressure, the tBu protection was removed with a TFA / DCM mixed solution (1:1 v / v), and then washed with purified water and extracted with dichloromethane. The extract was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by recrystallization to obtain E7-1 (3.69 g). 1 H NMR (400MHz, (CD3)2SO) δ: 4.45-4.41 (m, 1H, -C(=O)NHCH<), 4.25-3.86 (m, 4H, -OCH2C(=O)N<,>NCH2C(=O)NH-), 3.75-3.35 (m, PEG, -OCH2CH2N<), 3.23 (s, 6H, -OCH3), 2.41 (t, 2H, >CHCH2CH2COOH), 2.20-1.95 (m, 2H, >CHCH2CH2COOH). The molecular weight M n = 40.2 kDa, PDI = 1.01. The functional modification rate was 99.4%. Example 8: Investigation of the effect of different reaction solvents on the yield The effect of the solvent used on the yield of E1-1 was investigated for the reaction of S1-3 and S1-4 in step 2 of the method of Example 1, and the reaction of S1-3 and S1-5 in Method Two, and the results are shown in Table 1. The experimental results show that the preparation method of the present application can achieve a functional modification rate of more than 90% in various conventional solvents, and can also achieve a high reaction yield in a specific solvent. Table 1 Effect of solvent on yield of E1-1 The above only describes the embodiments of the present application and does not limit the patent scope of the present application. Any equivalent structure or equivalent flow transformation using the content of the present application specification, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application. It will be apparent to those skilled in the art that various modifications and variations can be made in the present application without departing from the spirit or scope of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. It is intended that the present application cover modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

Claims

1. A process for the preparation of a nitrogen branched two-armed polyethylene glycol derivative, characterized in that, The nitrogen branched two-arm polyethylene glycol derivative is represented by the general formula (1) and the method comprises the reactions as shown below: wherein P-amide is a linear polyethylene glycol derivative containing an amide bond, and BSM is a bifunctional small molecule compound; T1, T2are each independently C 1-20 hydrocarbyloxy; n1 and n2 are the polymerization degree of the polyethylene glycol chain, and are an integer selected from 8-1000; L1, L2 and L3 are each independently an alkylene group or a divalent linking group formed by combination of an alkylene group and a heteroatom-containing group; W is a group capable of reacting with the amide bond in P-amide and forming a trivalent nitrogen branched core; L0 is a linking bond or a divalent linking group formed after W reacts with the amide bond in P-amide; F1 is -COOH, -COONa or -COOK; The reaction is carried out under strong alkaline conditions or moderate alkaline conditions; Both P-amide and the compound represented by general formula (1) are monodisperse or polydisperse.

2. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized by, T1, T2are each independently C 1-10 hydroxy, preferably any one of hydroxyl, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy and benzyloxy, more preferably hydroxyl.

3. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized by, n1 and n2 are each independently an integer selected from 20-700, preferably an integer selected from 50-500.

4. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized by, L1, L2are each independently a*-(CH2) p -Z-(CH2) q -b* or a*-Z-(CH2) q -b*, L3is a*-(CH2) p -Z-(CH2) q -b*; wherein a* and b* are each a linking bond; in P-amide, a* of L1 is connected to the polyethylene glycol segment, and b* of L1 is connected to -NH-; a* of L2 is connected to the polyethylene glycol segment, and b* of L2 is connected to -C(=O)-; in BSM, a* of L3 is connected to W, and b* of L3 is connected to F1; wherein p and q are each independently an integer selected from 1-10; Z is a linking bond or a divalent linking group that does not participate in the reaction; when Z is not a linking bond, it is preferably selected from any one of -C(=O)-, -C(=S)-, -O-, -S-, -C(=O)O-, -OC(=O)-, -SC(=O)- and -C(=O)S-; Preferably, L1 is -CH2CH2-; preferably, L2 is -CH2-; preferably, L3 is -CH2-, -CH2CH2- or -CH2CH2CH2-.

5. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized by, W is a halogen group or a sulfonate group, and is selected from any one of -F, -Cl, -Br, -I, alkyl sulfonate groups and aryl sulfonate groups, and is preferably -Br.

6. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, wherein, L0is a bond or C 1-6 alkylene, preferably a bond or methylene, more preferably a bond.

7. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized by, The base used in the strong alkaline conditions or the moderate alkaline conditions is an organic base or an inorganic base, and is selected from any one of sodium tert-butoxide, potassium tert-butoxide, triethylamine, pyridine, 4-dimethylaminopyridine, imidazole, diisopropylethylamine, sodium hydroxide, potassium hydroxide, sodium hydride, potassium hydride, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium acetate, potassium acetate, trisodium phosphate, tripotassium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, monosodium hydrogen phosphate and monopotassium hydrogen phosphate; wherein the strong alkaline conditions preferably use sodium hydride, potassium hydride, sodium hydroxide or potassium hydroxide, and the moderate alkaline conditions preferably use triethylamine, trisodium phosphate or tripotassium phosphate.

8. The method for preparing nitrogen-branched two-arm polyethylene glycol derivatives according to claim 1, characterized by, The solvent used in the reaction is selected from any one of water, methanol, ethanol, benzene, toluene, xylene, diethyl ether, petroleum ether, tetrahydrofuran, dioxane, dichloromethane, chloroform, dichloroethane, dimethylformamide, dimethylacetamide, 1,4-dioxane, acetonitrile, dimethyl sulfoxide and ethyl acetate, or a mixed solvent containing any two or three of the aforementioned solvents is used. When the reaction is carried out under strong alkaline conditions, the solvent used is preferably toluene, dichloromethane or tetrahydrofuran, more preferably toluene or dichloromethane, and most preferably toluene; When the reaction is carried out under moderate alkaline conditions, the solvent used is preferably toluene, dichloromethane, tetrahydrofuran or acetonitrile, more preferably dichloromethane or tetrahydrofuran, and most preferably tetrahydrofuran.

9. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, wherein, The reaction temperature is selected from 20-130℃; when the reaction is carried out under strong alkaline conditions, the reaction temperature is preferably 60-100℃, and more preferably 80℃; when the reaction is carried out under moderate alkaline conditions, the reaction temperature is preferably 30-70℃, and more preferably 50℃.

10. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized by, The reaction time is 4-24 hours, and preferably 10-24 hours.

11. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized in that, The reaction also uses a phase transfer catalyst; the phase transfer catalyst is a quaternary ammonium salt, a quaternary phosphonium salt, a sulfonium salt or an arsenic salt, preferably a quaternary ammonium salt, and more preferably tetrabutylammonium fluoride or tetrabutylammonium bromide.

12. The method for preparing a nitrogen-branched two-arm polyethylene glycol derivative according to claim 11, characterized by, When the reaction is carried out under strong alkaline conditions, the molar ratio of P-amide, BSM, base and phase transfer catalyst is 1:(10-30):(20-70):(5-15), and preferably 1:20:(40-50):10; when the reaction is carried out under moderate alkaline conditions, the molar ratio of P-amide, BSM, base and phase transfer catalyst is 1:(10-30):(20-70):(30-50), and preferably 1:20:(40-50):

40.

13. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized in that, The method also includes reacting linear polyethylene glycol amine derivative LP-1 and linear polyethylene glycol carboxylic acid derivative LP-2 to obtain P-amide, the reaction being as follows: In the formula, LP-1 and LP-2 are both monodisperse or polydisperse; T1, T2, n1, n2, L1 and L2 in LP-1 and LP-2 are the same as those in the general formula (1).

14. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized in that, The number average molecular weight of the compound represented by the general formula (1) is 1-100 kDa, preferably 2-50 kDa, more preferably 10-50 kDa, and most preferably 10 kDa, 20 kDa, 30 kDa, 40 kDa or 50 kDa.

15. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized in that, F1 in the BSM is -COONa or -COOK, and F1 in the general formula (1) is -COOH; Alternatively, F1 in the BSM is -COOH, and F1 in the general formula (1) is -COONa or -COOK; Alternatively, F1 in the BSM is the same as F1 in the general formula (1), and both are -COOH, -COONa or -COOK.

16. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized in that, the structure of the compound of general formula (1) is selected from any one of the following:

17. A method for preparing a nitrogen branched two-armed polyethylene glycol derivative, characterized by, The nitrogen-branching two-arm polyethylene glycol derivative is represented by the general formula (2), and the method comprises the following two steps: Step one: obtaining the compound represented by the general formula (1) by the preparation method of any one of claims 1-15; Step two: the compound of general formula (1) is subjected to one or more functional modification to obtain the compound of general formula (2): T1, T2, n1, n2, L1 and L2 in the general formula (2) are the same as those in the general formula (1); L4 is a linking group formed after the functional modification, which links the trivalent nitrogen-branching core and F2; k is an integer of 1-100; each F2 is independently a hydrogen atom or a functional group capable of reacting with a biologically relevant substance or a protected form thereof.

18. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 17, characterized in that, F2 is any one of an epoxy group, a hydroxyl group, a thiol group, a carboxyl group, an amino group, an aldehyde group, an active ester group, an active carbonate group, a carbamate group, an isocyanate group, an isothiocyanate group, a succinimidyl group, a maleimido group, an alkenyl group, an alkynyl group, an alkenoate group, an azido group, a cyano group, a dithiopyridyl group, an alpha-haloacetyl alkynyl group, a folate group, a rhodamine group, a biotin group, a monosaccharide group, and a polysaccharide group, or a protected form thereof; F2 is preferably any one of the following structures:

19. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 17, characterized in that, L4 is a divalent or trivalent linker; preferably, L4 is selected from -CH2C(=O)-Y- or -CH2C(=O)-Y-(CH2). j -、-CH2C(=O)-Y-(CH2) j -M-、-CH2C(=O)-Y-(CH2) j -M-(CH2) j It is either - or -CH2C(=O)-NH-A<, and its left end is connected to the trivalent nitrogen branched nucleus in general formula (2); wherein, Y is -O- or -NH-; M is any one of -O-, -S-, -S-S-, -C(=O)O-, -OC(=O)- and -OC(=O)O-; each j is independently an integer of 1-10, preferably 1, 2, 3, 4 or 5; A is a trivalent residue of an amino acid, which is a residue obtained after all amino groups, carboxyl groups, hydroxyl groups and thiol groups of the amino acid are removed; Preferably, L4 is -CH2-, -CH2C(=0)0-, -CH2C(=0)0CH2CH2-, -CH2C(=0)NHCH2CH2-, -CH2C(=0)0CH2CH2OC(=0)CH2CH2-, and is any one of the following compounds, and the left end thereof is connected to the trivalent nitrogen branched core in general formula (2).

20. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 17, characterized in that, The k is an integer from 1 to 8, preferably 1, 2 or 3, more preferably 1.

21. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 17, characterized in that, The number of functional modifications is one, two or three times in succession.

22. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 17, characterized in that, Each functional modification is a micro-modification or a small molecule modification; the micro-modification is selected from any one of protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation, acidification and basification; the small molecule modification is selected from a coupling reaction, preferably an alkylation reaction, an amidation reaction or an esterification reaction; the small molecule is a bifunctional or multifunctional small molecule.

23. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 17, characterized in that, F1 of the compound represented by general formula (1) is -COONa or -COOK, and F2 of the compound represented by general formula (2) is -COOH.

24. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 17, characterized in that, F1 of the compound of Formula (1) is -COOH, F2 of the compound of Formula (2) is selected from any one of the following:

25. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 17, characterized in that, the structure of the compound of Formula (2) is selected from any one of the following:

26. A method for producing a conjugate of a nitrogen- branched two-armed polyethylene glycol derivative and a biorelated substance, characterized by, The preparation method of the conjugate comprises using the preparation method of any one of claims 1-25 to prepare the nitrogen branched two-arm polyethylene glycol derivative.

27. A process for the preparation of a conjugate of a nitrogen-branched two-armed polyethylene glycol derivative according to claim 26 and a biorelated substance, characterized in that, The biologically relevant substance is selected from any one of a drug, a protein, a polypeptide, an oligopeptide, a protein mimetic, a fragment, an enzyme, an antigen, an antibody and fragments thereof, a receptor, a gene-related substance aptamer, a polysaccharide, a proteoglycan, a glycoprotein, a lipid compound, a hormone, a vitamin, a vesicle, a liposome, a dye, a fluorescent substance, a targeting factor, a cytokine, a neurotransmitter, an extracellular matrix substance, a plant or animal extract, a virus, a vaccine, a micelle; Preferably, the biologically relevant substance is selected from any one of a small molecule drug, a nucleic acid, a phospholipid and a glycolipid; More preferably, the biologically relevant substance is selected from any one of a small molecule drug, a nucleoside, a nucleotide, an oligonucleotide, an antisense oligonucleotide and a polynucleotide; wherein the small molecule drug is preferably selected from any one of a flavone, a terpenoid, a carotenoid, a saponin, a steroid, a quinone, an anthraquinone, a fluoroquinone, a coumarin, an alkaloid, a porphyrin, a polyphenol, a macrolide, a monobactam, a phenylpropanoid, an anthracycline and an aminoglycoside.

28. The method of producing a conjugate of a nitrogen-branched two-arm polyethylene glycol derivative and a biologically-relevant substance according to claim 26, wherein The biologically relevant substance is a genetically engineered drug selected from any one of an antibody, an antibody fragment, an interleukin, a lysozyme, an interferon, a growth hormone, an erythropoietin and a granulocyte colony-stimulating factor; the interferon is preferably an α-, β- or γ-interferon.

29. A process for preparing a conjugate of a nitrogen-branched two-arm polyethylene glycol derivative of claim 26 with a biorelated substance, characterized by, The method comprises the step of generating a covalent linkage by reacting the reactive groups of the nitrogen branched two-arm polyethylene glycol derivative with the biologically relevant substance.

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