WO2024041225A1 - 一种聚乙二醇醛衍生物的制备方法 - Google Patents

一种聚乙二醇醛衍生物的制备方法 Download PDF

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WO2024041225A1
WO2024041225A1 PCT/CN2023/105409 CN2023105409W WO2024041225A1 WO 2024041225 A1 WO2024041225 A1 WO 2024041225A1 CN 2023105409 W CN2023105409 W CN 2023105409W WO 2024041225 A1 WO2024041225 A1 WO 2024041225A1
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polyethylene glycol
derivative
preparation
aldehyde
acetal
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PCT/CN2023/105409
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English (en)
French (fr)
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林昇
王爱兰
朱琦
翁文桂
刘超
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厦门赛诺邦格生物科技股份有限公司
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Publication of WO2024041225A1 publication Critical patent/WO2024041225A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331—Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/321—Polymers modified by chemical after-treatment with inorganic compounds
    • C08G65/323—Polymers modified by chemical after-treatment with inorganic compounds containing halogens
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331—Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C08G65/332—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof
    • C08G65/3322—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof acyclic
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/337—Polymers modified by chemical after-treatment with organic compounds containing other elements
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2650/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G2650/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule characterized by the type of post-polymerisation functionalisation
    • C08G2650/04—End-capping
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the field of polymer synthesis chemistry, and in particular to a method for preparing polyethylene glycol acetal or aldehyde derivatives.
  • PEGylation is one of the important methods for modifying drugs or biologically related substances.
  • the modified drug molecules will possess many of the excellent properties of polyethylene glycol (such as hydrophilicity, flexibility, anticoagulant properties, etc.).
  • polyethylene glycol aldehyde modifiers with aldehyde end groups are very important modifiers in the protein field and have the advantages of high selectivity and high activity retention.
  • the aldehyde group has a certain selectivity for the N-terminus of the protein, because the N-terminal amino group of the protein has a lower PKa than the side chain amine group. When the side chain amino group is protonated at a certain pH, it loses the ability to nucleophilically attack the aldehyde group.
  • the N-terminal amino group is not yet protonated, and also has the ability to nucleophilic attack; the aldehyde group can also form a Schiff base with the N-terminal amino group of the protein. After reduction, a stable imine connection can be obtained, and the positive charge of the amino group is maintained, which is important for maintenance It plays an important role in the structure and activity of proteins.
  • polyglycol aldehydes can be obtained by oxidizing the terminal hydroxyl groups of polyethylene glycol, adding oxygen to the mixture of PEG and catalyst to oxidize -CH2OH groups to -CHO. However, under most oxidative conditions, the PEG chain will decompose, and the end group conversion rate of the PEG chain is not high. Polyglycol aldehydes can also be prepared by the acetal method, which is obtained by introducing linear acetal groups at the end of the PEG chain and then hydrolyzing them.
  • CN1763122A discloses that the base-catalyzed reaction of 3-hydroxypropionaldehyde diethyl acetal and PEG mesylate will produce a large amount of unstable PEG vinyl ether side reactions, and the reaction yield is lower than 85%-90% , intermolecular coupling will also cause the purity of the prepared polyethylene glycol aldehyde product to be low, thus affecting the modification efficiency of PEG aldehyde to proteins and other drugs.
  • CN102037056A discloses a method for preparing high-purity polyglycol aldehyde, but it is not suitable for commercialization because the reaction steps are complicated.
  • the present invention provides a method for preparing polyethylene glycol acetal derivatives represented by formula (1) or formula (2).
  • the embodiments are as follows:
  • a method for preparing polyethylene glycol acetal derivatives characterized in that its structure is as shown in formula (1) or formula (2):
  • n is an integer from 20 to 1000;
  • t is independently an integer from 1 to 6;
  • k is an integer from 1 to 8; when k is 1, R is selected from H, -CH 3 , -CH 2 CH 3 , -TBS, -Bn, -(CH 2 ) tp -COOH, -(CH 2 ) tp - Any one of COOtBu, -(CH 2 ) tp -N 3 , tp is an integer from 1 to 3; when k is an integer from 2 to 8, R is a multivalent branched structure, selected from divalent to octavalent;
  • the preparation method is as follows:
  • polyethylene glycol derivative I- 1 or I-3 containing the functional group React at 20-90°C for 2-24 hours to obtain the polyethylene glycol acetal derivative represented by formula (1) or formula (2);
  • the X 1 Each time the X 1 appears, it is independently any one of H, p-toluenesulfonyl, and methanesulfonyl; the X 2 each time it appears, it is independently any one of -OH, -SH, -NH 2 , - Any one of Cl, -Br, p-toluenesulfonyl, methanesulfonyl, trifluorosulfonyl, and trifluoroethylsulfonyl;
  • the invention also provides a method for preparing polyethylene glycol aldehyde derivatives, which can be obtained by subjecting the acetal derivatives represented by formula (1) or formula (2) to acid treatment, and formula (1) or The acetal derivative represented by formula (2) is prepared according to the aforementioned method.
  • the present invention also provides a polyglycol aldehyde derivative-modified biologically relevant substance obtained according to the aforementioned preparation method, which is characterized in that the biologically related substance is selected from the group consisting of peptides, polypeptides, proteins, polysaccharides, steroids, and nucleosides. Any of acids, oligonucleotides, polynucleotides, and fats.
  • the invention provides a method for preparing polyethylene glycol acetal and corresponding polyethylene glycol aldehyde derivatives.
  • small molecule cyclic acetal derivatives and polyethylene glycol are used as raw materials.
  • a series of linear and nonlinear polyethylene glycol aldehyde derivatives functionalized with a single aldehyde group and multiple aldehyde groups were prepared.
  • the preparation method of the present invention has fewer reaction steps and does not require column chromatography separation and purification operations.
  • the obtained product has high purity and high terminal functional group substitution rate, and can provide more high-quality polyglycol aldehyde modifiers in the field of polyethylene glycol modification.
  • Figure 1 is the nuclear magnetic resonance spectrum ( 1 HNMR) of mPEG-propionaldehyde-20k (P1-2) prepared in Example 1 of the present invention
  • Figure 2 is a nuclear magnetic resonance spectrum ( 1 HNMR) of mPEG-butyraldehyde-20k (P2-2) prepared in Example 2 of the present invention
  • Figure 3 is a nuclear magnetic resonance spectrum ( 1 HNMR) of PEG-dibutyraldehyde-30k (P3-2) prepared in Example 3 of the present invention
  • Figure 4 is a nuclear magnetic resonance spectrum ( 1 HNMR) of PEG-dipropionaldehyde-3.4k (P4-2) prepared in Example 4 of the present invention
  • Figure 5 is the nuclear magnetic resonance spectrum ( 1 HNMR) of the four-arm PEG-propionaldehyde-10k (P5-2) prepared in Example 5 of the present invention
  • Figure 6 is the NMR spectrum of HO-PEG-propionaldehyde-20k (P6-2) prepared in Example 6 of the present invention.
  • Figure 7 is the nuclear magnetic spectrum of N 3 -PEG-propionaldehyde-20k (P7-2) prepared in Example 7 of the present invention.
  • Figure 8 is a GPC spectrum of mPEG-propionaldehyde-20k (P1-2) prepared in Example 1 of the present invention.
  • Figure 9 is a GPC spectrum of mPEG-butyraldehyde-20k (P2-2) prepared in Example 2 of the present invention.
  • Figure 10 is the GPC spectrum of PEG-dibutyraldehyde-30k (P3-2) prepared in Example 3 of the present invention.
  • FIG 11 is the GPC spectrum of PEG-dipropionaldehyde-3.4k (P4-2) prepared in Example 4 of the present invention.
  • Figure 12 is a GPC spectrum of four-arm PEG-propionaldehyde-10k (P5-2) prepared in Example 5 of the present invention.
  • Figure 13 is an HPLC spectrum for testing the terminal substitution rate of mPEG-propionaldehyde-20k (P1-2) prepared in Example 1 of the present invention
  • Figure 14 is an HPLC spectrum for testing the terminal substitution rate of mPEG-butyraldehyde-20k (P2-2) prepared in Example 2 of the present invention
  • Figure 15 is an HPLC spectrum for testing the terminal substitution rate of PEG-dibutyraldehyde-30k (P3-2) prepared in Example 3 of the present invention.
  • Figure 16 is an HPLC spectrum for testing the terminal substitution rate of PEG-dipropionaldehyde-3.4k (P4-2) prepared in Example 4 of the present invention
  • Figure 17 is an HPLC spectrum for testing the terminal substitution rate of four-arm PEG-propionaldehyde-10k (P5-2) prepared in Example 5 of the present invention.
  • an integer interval marked in the form of an interval can represent a group composed of all integers within the interval range, and the range includes two endpoints.
  • the integer range 1-6 represents the group consisting of 1, 2, 3, 4, 5, and 6.
  • the numerical range in the present invention includes, but is not limited to, the numerical range expressed by integers, non-integers, percentages, and fractions. Unless otherwise specified, both endpoints are included.
  • the terminal group of the linking group and the substituent contained in the linking group are easily confused, use To mark the position of the linker to connect other groups, such as in the structural formula in, adopted To mark the two positions connecting other groups in the divalent linking group, the aforementioned two structural formulas respectively represent -CH(CH 2 CH 2 CH 3 ) 2 -, -CH 2 CH 2 CH(CH 3 ) 2 -CH 2 CH 2 -.
  • molecular weight represents the mass of a compound molecule
  • average molecular weight represents the mass of the general compound components in macroscopic substances. If there is no special provision, “average molecular weight” generally refers to "number average molecular weight” M n .
  • the number average molecular weight may be either the molecular weight of a polydisperse block or substance or the molecular weight of a monodisperse block or substance. Unless otherwise specified, the measurement unit of "molecular weight” and “average molecular weight” is Dalton, Da.
  • degree of polymerization can also be used to characterize the molecular weight of the polyethylene glycol chain, specifically referring to the number of repeating units (oxyethylene units, EO units) in a compound molecule.
  • average degree of polymerization preferably “number average degree of polymerization” is used to characterize the average value or number average value of the number of repeating units.
  • the molecular weight/degree of polymerization of a single molecule of a compound and the number average molecular weight/number average degree of polymerization of the compound components in the macroscopic substance are "equal” or “the same” or “equal to” or “approximately equal to ” (including other forms of equivalent expressions), unless otherwise specified, is not limited to being strictly equal in numerical value, but rather the index values are close or approximately equal, and the close or approximately equal preferred deviation does not exceed ⁇ 10%, more preferably partial The difference does not exceed ⁇ 5, usually based on the preset value.
  • the molecular weight of mPEG is 5kDa, which preferably means that the molecular weight value of a single molecule in the general formula is between 4500 and 5500Da, and the average molecular weight of the corresponding components of the corresponding prepared product is 5kDa, that is, when the average molecular weight value is between 4500 and 5500Da.
  • the product is the target product.
  • the "selected from"/"preferred" of any two objects are independent of each other.
  • the selected from/preferred of any two objects may be of the same level or Different levels.
  • LA and LB are each independently selected from A, B, and C. It can mean that both L A and LB are A, or it can be that L A is A and LB is B 1 (B 1 is B's a subordinate situation).
  • LA is preferably A (level 1 preference), more preferably A 1 to A 3 (level 2 preference), most preferably A 11 to A 13 (level 3 preference), and LB is preferably B (1 Preferred grade), more preferably B 1 to B 3 (preferred grade 2), most preferably B 11 to B 13 (preferred grade 3), preferably A is A 1 to A 3 (preferred grade 2) and B B 11 to B 13 (3rd level preference), or both A and B may be 3rd level preference.
  • each independently is/selected from/preferred can not only mean that different categories can each independently be/selected from/preferred any option in the definition, but can also be added with "every time it appears”. When the same category appears in different locations or at different times, it is independently/selected from/any option in the preferred definition each time.
  • the two ts appearing in formula (2) are each independently 1- an integer of 6.
  • the "combination" of the listed items refers to any two or more combinations of the previously listed items; and the number of items is not limited.
  • the quantity can be zero, one or more than one.
  • the quantity of the same type of items is greater than 1, it can be the same or different specific forms that satisfy the item.
  • the alkaline reagent is selected from any one or a combination of any two or more of metal alkoxides, metal hydrides and metal hydroxides".
  • the combination of any two can be two specific metal alcohols.
  • the terminal substitution rate also known as terminal activity, refers to the ratio of PEG terminals containing active functional groups aldehyde groups.
  • a method for preparing polyethylene glycol acetal derivatives characterized in that its structure is as shown in formula (1) or formula (2):
  • n is an integer from 20 to 1000;
  • t is independently an integer from 1 to 6;
  • k is an integer from 1 to 8; when k is 1, R is selected from H, -CH 3 , -CH 2 CH 3 , -TBS, -Bn, -(CH 2 ) tp -COOH, -(CH 2 ) tp - Any one of COOtBu, -(CH 2 ) tp -N 3 , tp is an integer from 1 to 3; when k is an integer from 2 to 8, R is a multivalent branched structure, selected from divalent to octavalent;
  • the preparation method is as follows:
  • polyethylene glycol derivative I- 1 or I-3 containing the functional group React at 20-90°C for 2-24 hours to obtain the polyethylene glycol acetal derivative represented by formula (1) or formula (2);
  • the X 1 Each time the X 1 appears, it is independently any one of H, p-toluenesulfonyl, and methanesulfonyl; the X 2 each time it appears, it is independently any one of -OH, -SH, -NH 2 , - Any one of Cl, -Br, p-toluenesulfonyl, methanesulfonyl, trifluorosulfonyl, and trifluoroethylsulfonyl;
  • R is any one of the following situations:
  • Q: 1: k is 1, R is selected from H, -CH 3 , -CH 2 CH 3 , -TBS, -Bn, -(CH 2 ) tp -COOH, -(CH 2 ) tp -COOtBu, -(CH 2 ) Any one of tp NH 2 and -(CH 2 ) tp NHCbz;
  • the number average molecular weight corresponding to each polyethylene glycol chain is 900, 1000, 1500, 2000, 2500, 3000, 3350, 3400 , 3500, 4000, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 11000, 15000, Any one of 20,000, 25,000, or 30,000.
  • the alkaline reagent in the previous embodiment is selected from any one or a combination of any two or more of metal alkoxides, metal hydrides and metal hydroxides;
  • the metal alkoxide is selected from any one of sodium methoxide, sodium ethoxide and sodium tert-butoxide;
  • the metal hydride is potassium hydride or sodium hydride;
  • the metal hydroxide is sodium hydroxide or potassium hydroxide; more preferably Any of sodium hydroxide, potassium hydroxide, potassium hydride, and sodium hydride.
  • X 1 in formula (1) or formula (2) is H, p-toluenesulfonate group or methanesulfonyl ester group, and X 2 is -OH, -Cl or - Br.
  • X 2 is -Cl or -Br
  • the alkaline reagent is sodium hydroxide or potassium hydroxide.
  • X 1 is p-methanesulfonyl ester group and X 2 is -OH
  • the preparation process is as follows:
  • the organic solvent is not particularly limited, as long as both the raw material and the product can be dissolved, preferably from toluene, dichloromethane, chloroform, acetonitrile, dimethyl sulfoxide, tetrahydrofuran, N, Any one or a combination of any two or more of N'-dimethylformamide, N,N'-dimethylacetamide and 1,4-dioxane, more preferably toluene or tetrahydrofuran , more preferably anhydrous tetrahydrofuran.
  • the temperature of the alkaline reagent activation treatment in the aforementioned embodiment is preferably 20-60°C, more preferably 30-50°C, and most preferably 40°C; the activation treatment time is 6-24h, more preferably 8-12h; the molar ratio of the small molecule acetal derivative I-2 and the alkali reagent is 1:1-2:1; more preferably 1:1-1.5:1, more preferably 1 :1, 1.25:1 or 1.2:1.
  • the molar ratio of I-1 and the small molecule acetal derivative I-2 is preferably 1:10-1:160.
  • k is 1, the molar ratio of I-1 and I-2 The ratio is 1:10-1:30; the molar ratio of the polyethylene glycol derivative I-3 containing functional group X 1 and the small molecule acetal derivative I-2 is 1:20-1:100.
  • the reaction is carried out under an inert gas, and the inert gas is selected from any one of nitrogen, helium and argon and mixtures thereof.
  • the raw material I-1 or I-3 in the reaction is added to I-2 in a dropwise manner; the purification process involves precipitation or recrystallization; the precipitation reagent is
  • the ether reagent is selected from diethyl ether or methyl tert-butyl ether, and the recrystallization reagent is any one or a mixture of isopropyl alcohol and n-hexane; more preferably, the recrystallization reagent is a mixture of isopropyl alcohol and n-hexane, and The volume ratio of isopropyl alcohol and n-hexane is 2:1-5:1.
  • the structure of I-1 is preferably or Among them, the definition of R is consistent with that described in formula (1). They can all be obtained by mesylation or p-toluenesulfonylation of their corresponding alcoholates. Specific examples include, for example or Can be passed to It is obtained by mesylation or tosylation, can be passed to It is obtained by mesylation or p-toluenesulfonylation.
  • the structure of I-1 is preferably
  • the preferred structure of I-3 is same can be passed to It is obtained by mesylation or p-toluenesulfonylation.
  • the preferred structure of I-2 is Any of them.
  • a method for preparing polyethylene glycol aldehyde derivatives which is obtained by subjecting polyethylene glycol acetal derivatives to acid treatment.
  • the polyethylene glycol acetal derivatives are prepared by any of the aforementioned preparation methods. .
  • basically all polyethylene glycol acetal derivatives are converted into corresponding polyethylene glycol aldehyde derivatives, and there is no -CH ⁇ of the cyclic acetal group in the 1 HNMR of the polyethylene glycol aldehyde derivatives.
  • Protons chemical shift is about 4.80-5.00
  • contains characteristic peaks of aldehyde groups chemical shift is about 9.70-9.90).
  • the acid used in the preparation process of the polyglycol aldehyde derivative is selected from any one of hydrochloric acid, trifluoroacetic acid, formic acid and acetic acid and mixtures thereof.
  • the structure of the polyglycol aldehyde prepared according to the preparation method includes but is not limited to any one of the following structures:
  • the raw materials used in each preparation method can be purchased or synthesized by oneself.
  • a biologically relevant substance modified with a polyethylene glycol aldehyde derivative obtained according to the aforementioned preparation method characterized in that the biologically relevant substance is selected from the group consisting of peptides, polypeptides, proteins, polysaccharides, steroids, nucleotides, and oligonucleotides. Any of glycolic acid, polynucleotide, and fat; more preferably, the biologically related substance contains an active amino group, or contains at least one active amino group that can be coupled with the aldehyde group of the polyethylene glycol aldehyde derivative after modification.
  • the preparation methods of polyethylene glycol acetal derivatives and polyethylene glycol aldehyde derivatives will be further described below with reference to some specific examples. Specific examples are provided to further illustrate the present invention in detail, but do not limit the scope of the present invention.
  • the intermediates and final products prepared in the present invention can be purified by purification methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, etc.
  • purification methods including but not limited to nuclear magnetic resonance, electrophoresis, UV-visible spectrophotometer, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism spectrometry, etc. can be used.
  • 1 H-NMR for structural confirmation
  • GPC gel permeation chromatography
  • PDI polydispersity index
  • HPLC high performance liquid chromatography
  • the preparation process is as follows:
  • Step b In a dry and clean 500mL round-bottomed flask, add P1-1 obtained in step a, add 1N HCl solution, and stir for 8 hours in an ice-water bath. After the reaction is completed, adjust the pH of the solution to 6.4 ⁇ 0.2 with sodium bicarbonate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous magnesium sulfate, filter, and recrystallize from a mixed solution of anhydrous isopropyl alcohol and n-hexane to obtain mPEG-propionaldehyde-20k (P1-2, yield 98%).
  • the preparation process is as follows:
  • Step b In a dry and clean 1L round-bottomed flask, add P1-3 obtained in step a, add 1N HCl solution, and stir for 8 hours in an ice-water bath. After the reaction is completed, adjust the pH of the solution to 6.4 ⁇ 0.2 with sodium bicarbonate, and Extract twice with methyl chloride, combine the organic phases, dry over anhydrous magnesium sulfate, filter, and recrystallize from a mixed solution of anhydrous isopropanol and n-hexane to obtain mPEG-propionaldehyde-2k P1-4 (yield 98%).
  • the preparation process is as follows:
  • Step b In a dry and clean 500mL round-bottomed flask, add P2-1 obtained in step a, add 1N HCl solution, and stir for 8 hours in an ice-water bath. After the reaction is completed, adjust the pH of the solution to 6.4 ⁇ 0.2 with sodium bicarbonate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous magnesium sulfate, filter, and recrystallize from a mixed solution of anhydrous isopropyl alcohol and n-hexane to obtain mPEG-butyraldehyde-20k (P2-2, yield 97%).
  • the preparation process is as follows:
  • Step b In a dry and clean 2L round-bottomed flask, add P3-1 obtained in step a, add 1N HCl solution, and stir for 8 hours in an ice-water bath. After the reaction is completed, adjust the pH of the solution to 6.4 ⁇ 0.2 with sodium bicarbonate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous magnesium sulfate, filter, and recrystallize from a mixed solution of anhydrous isopropyl alcohol and n-hexane to obtain PEG-dibutyraldehyde-30k (P3-2, yield 97%).
  • the preparation process is as follows:
  • Step b In a dry and clean 2L round-bottomed flask, add P4-1 obtained in step a, add 1N HCl solution, and stir for 8 hours in an ice-water bath. After the reaction is completed, adjust the pH of the solution to 6.4 ⁇ 0.2 with sodium bicarbonate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous magnesium sulfate, filter, and recrystallize from a mixed solution of anhydrous isopropyl alcohol and n-hexane to obtain PEG-dipropionaldehyde-3.4k (P4-2, yield 97%).
  • the preparation process is as follows:
  • Step b In a dry and clean 2L round-bottomed flask, add P5-1 obtained in step a, add 1N HCl solution, and stir for 8 hours in an ice-water bath. After the reaction is completed, adjust the pH of the solution to 6.4 ⁇ 0.2 with sodium bicarbonate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous magnesium sulfate, filter, and recrystallize from a mixed solution of anhydrous isopropyl alcohol and n-hexane to obtain 4-arm-PEG-propionaldehyde-10k (P5-2, yield 97%).
  • the preparation process is as follows:
  • Step a Under nitrogen protection, add S1-1 (0.89g, 7.50mmol) and anhydrous THF (50mL) into a dry and clean 250mL round-bottomed flask, add NaOH (0.20g, 5.00mmol) under ice bath conditions, Stir until dissolved, stir and react at 40°C for 8 hours. After the reaction is completed, the reaction solution is lowered to room temperature, and TBSO-PEG-OMs-20k (5.00g, 0.25mmol) dissolved in anhydrous THF (50mL) is added dropwise to the aforementioned reaction solution. After the dropwise addition is completed, 40°C The reaction was stirred for 8 h.
  • Step b In a dry and clean 500mL round-bottomed flask, add P6-1 obtained in step a, dissolve it with tetrahydrofuran/acetic acid mixed solution, add TBAF under ice bath conditions, react at room temperature for 8 hours, extract, concentrate, and then add 1N HCl The solution was stirred and reacted in an ice-water bath for 8 hours.
  • the preparation process is as follows:
  • Step a Under nitrogen protection, add S1-1 (0.89g, 7.50mmol) and anhydrous THF (50mL) into a dry and clean 250mL round-bottomed flask, add NaOH (0.20g, 5.00mmol) under ice bath conditions, Stir until dissolved, stir and react at 40°C for 8 hours. After the reaction is completed, the reaction solution is lowered to room temperature, and N 3 -PEG-OMs-20k (5.00g, 0.25mmol) dissolved in anhydrous THF (50 mL) is added dropwise to the aforementioned reaction solution. After the dropwise addition is completed, 40 The reaction was stirred at °C for 8 hours.
  • Step b In a dry and clean 500mL round-bottomed flask, add P7-1 obtained in step a, then add 1N HCl solution, and stir for 8 hours in an ice-water bath. After the reaction is completed, adjust the pH of the solution to 6.4 ⁇ 0.2 with sodium bicarbonate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous magnesium sulfate, filter, and recrystallize from a mixed solution of anhydrous isopropyl alcohol and n-hexane to obtain N 3 -PEG-propionaldehyde-20k (P7-2, yield 95%).
  • the preparation process is as follows:
  • Anhydrous THF 50 mL
  • Step b In a dry and clean 2L round-bottomed flask, add P1-1 obtained in step a, add 1N HCl solution, and stir for 8 hours in an ice-water bath. After the reaction is completed, adjust the pH of the solution to 6.4 ⁇ 0.2 with sodium bicarbonate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous magnesium sulfate, filter, and recrystallize from a mixed solution of anhydrous isopropyl alcohol and n-hexane to obtain mPEG-propionaldehyde-20k (P1-2, yield 97%).
  • Example 8 The alkaline reagent in Example 8 was changed from NaH to NaOH, and other steps were the same as in Example 8. The final yield of P1-2 was 84%. After HPLC testing, the substitution rate of the aldehyde group at the P1-2 terminal was 38%.
  • the preparation process is as follows:
  • Example 8 The small molecule cyclic acetal raw material S5-1 in Example 8 was replaced with the small molecule linear acetal raw material S10-1. The other operating steps were the same as in Example 8. The final yield of P1-2 was about 83%. After HPLC testing, the substitution rate of the aldehyde group at the P1-2 terminal was 85%.
  • the preparation process is as follows:
  • Example 1.1 The small molecule cyclic acetal raw material S1-1 in Example 1.1 was replaced with the small molecule linear acetal raw material S11-1. The other operating steps were the same as in Example 1.1. The final yield of P1-2 was about 87%. After HPLC testing, the substitution rate of the aldehyde group at the P1-2 terminal was 87%.
  • Example 1.1 The raw material mPEG-OMs-20k in Example 1.1 is not added dropwise, but is added directly as a solid. The other operating steps are the same as those in Example 1.1, and the final yield of P1-2 is about 82%. After HPLC testing, the substitution rate of the aldehyde group at the P1-2 terminal was 91%.
  • the recrystallization step in step a in Example 1.1 was changed to use ether reagent (tert-butyl methyl ether) for beating and precipitation, that is, the crude product was dissolved in a small amount of DCM, and then added dropwise to the ether reagent (the volume of the ether reagent 20 times that of DCM), other operating steps were the same as in Example 1.1, and the final yield of P1-2 was about 87%. After HPLC testing, the substitution rate of the aldehyde group at the P1-2 terminal was 89%.
  • ether reagent tert-butyl methyl ether
  • Example 1.1 The recrystallization step in step a in Example 1.1 was changed to recrystallization with isopropyl alcohol.
  • the other operating steps were the same as in Example 1.1, and the final yield of P1-2 was about 89%.
  • the substitution rate of the aldehyde group at the P1-2 terminal was 93%.
  • Examples 1.1 and 8-14 are different preparation methods of mPEG-propionaldehyde-20k (P1-2), and Examples 9-14 are comparative methods. The comparison of each method is shown in Table 1 and Table 2 below:
  • Example 1.1 is used to dehydrogenate and activate small molecule acetals
  • the NaH in Example 8 and the NaOH in Example 9 are used to dehydrogenate and activate the hydrogen of mPEG-OH, and dehydrogenate mPEG-OH. It is better to use NaH than NaOH during hydrogen activation.
  • the substitution rate of the terminal aldehyde group of the product obtained by using NaH is 95%, while that of the product obtained by using NaOH is only 38%.
  • Example 1.1 As can be seen from Table 2, the addition method of raw materials and post-processing process have a significant impact on the yield and quality of the product.
  • Example 12 comparing Example 1.1 with Example 12, it can be seen that when polyethylene glycol raw material is added to an alkali metal-activated small molecule acetal derivative, the yield of adding dropwise is better than that of pouring directly. It is higher and the terminal functional group substitution rate of the product is also higher; it may be because the raw materials added by dripping can react with the activated other raw materials as soon as they are added, and the reaction is more complete and time-effective; secondly, the examples Comparing 1.1, 13 and 14, it can be seen that recrystallization during post-processing has a higher yield and better product quality than precipitation.
  • recrystallization reagent also affects the yield and quality of the product. Isopropyl alcohol and n-hexane are used. The yield of recrystallization of the mixture is higher and the substitution rate of the terminal functional groups of the product is also higher.

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Abstract

一种聚乙二醇缩醛及醛衍生物的制备方法，所述聚乙二醇缩醛衍生物如式（1）或式（2）所示，具体制备方法为在碱试剂的存在下，以小分子环状缩醛衍生物和聚乙二醇衍生物为原料，制备了一系列高收率、高纯度、高末端取代率的单一醛基官能化、多个醛基官能化的线性和非线性聚乙二醇醛衍生物。

Description

一种聚乙二醇醛衍生物的制备方法 技术领域
本发明涉及高分子合成化学领域，特别涉及一种聚乙二醇缩醛或醛衍生物的制备方法。
背景技术
聚乙二醇化(PEGylation)是药物或生物相关物质修饰的重要手段之一，经修饰后的药物分子将具备聚乙二醇的许多优良性质(如亲水性、柔性、抗凝血性等)。其中，端基为醛基的聚乙二醇醛修饰剂是蛋白质领域非常重要的修饰剂，具有有高选择性和高活性保持度的优点。醛基对蛋白质的N端具有一定的选择性，因为蛋白质N端氨基比侧链胺基的PKa要低，当在一定的pH下，侧链氨基质子化，失去亲核进攻醛基的能力时，N端氨基仍未质子化，还具有亲核进攻的能力；醛基还可以与蛋白质N端氨基形成schiff碱经还原后可得到稳定的亚胺连接，并保持该氨基的正电荷，对维持蛋白质的结构与活性方面有重要作用。
现有技术US6465694B1公开了聚乙二醇醛可以通过氧化聚乙二醇的末端羟基而获得，将氧气添加至PEG和催化剂的混合物中以将-CH2OH基团氧化为-CHO。然而，在大多数氧化条件下，PEG链会分解，PEG链的端基转化率并不高。聚乙二醇醛也可以通过缩醛法来制备，通过在PEG链末端引入线性缩醛基团随后水解而获得，但是这种方法所用的线性缩醛原料不稳定，会产生副产物，具体地，CN1763122A中公开了3-羟基丙醛二乙基缩醛与PEG甲磺酸盐的碱催化反应会产生大量不稳定的PEG乙烯基醚的副反应，且反应收率低于85％-90％，分子间也会偶联使得制备的聚乙二醇醛产品纯度不高，从而影响到PEG醛对蛋白等药物的修饰效率。CN102037056A公开了一种高纯度聚乙二醇醛的制备方法，但是因为反应步骤繁琐而不适合商业化。
因此，有必要改进现有的制备方法，开发出反应步骤少且能获得末端高取代率、高收率、高纯度聚乙二醇醛类衍生物的制备方法。
发明内容
为了实现上述目的，本发明提供了一种制备式(1)或式(2)所示的聚乙二醇缩醛类衍生物的方法，实施方案如下:
一种聚乙二醇缩醛衍生物的制备方法，其特征在于，其结构如式(1)或式(2)所示：
其中，n为20-1000的整数；
t每次出现时各自独立地为1-6的整数；
k为1-8的整数；k为1时，R选自H、-CH3、-CH2CH3、-TBS、-Bn、-(CH2)tp-COOH、-(CH2)tp-COOtBu、-(CH2)tp-N3中任一种，tp为1-3的整数；k为2-8的整数时，R为多价支化结构，选自二价到八价；
所述制备方法如下：
将小分子缩醛衍生物I-2溶于有机溶剂后用碱试剂进行活化处理，然后加入溶于有机溶剂的含有官能团X1的聚乙二醇衍生物I-1或者I-3，在20-90℃下反应2-24小时，得到式(1)或式(2)所示的聚乙二醇缩醛衍生物；
或者将含有官能团X1的聚乙二醇衍生物I-1或者I-3溶于有机溶剂后用碱试剂进行活化处理，然后加入溶于有机溶剂的小分子缩醛衍生物I-2，在20-90℃下反应2-24小时，得到式(1)或式(2)所示的聚乙二醇缩醛衍生物；
所述X1每次出现时各自独立地为H、对甲苯磺酰基、甲磺酰基中任一种；所述X2每次出现时各自独立地为-OH、-SH、-NH2、-Cl、-Br、对甲苯磺酰基、甲磺酰基、三氟磺酰基、三氟乙基磺酰基中任一种；
本发明还提供了一种聚乙二醇醛衍生物的制备方法，对式(1)或式(2)所示的缩醛类衍生物进行酸处理后即可得到，且式(1)或式(2)所示的缩醛类衍生物按照前述的方法进行制备。
本发明还提供了一种按照前述制备方法获得的聚乙二醇醛衍生物修饰的生物相关物质，其特征在于，所述生物相关物质选自肽、多肽、蛋白质、多糖、甾类、核苷酸、低聚核苷酸、多核苷酸、脂肪中任一种。
本发明的有益效果
本发明提供了一种聚乙二醇缩醛和对应的聚乙二醇醛类衍生物的制备方法，在碱试剂的存在下，以小分子环状缩醛衍生物和聚乙二醇为原料，制备了一系列单一醛基官能化、多个醛基官能化的线性和非线性聚乙二醇醛衍生物。本发明的制备方法反应步骤较少且不需要进行柱层析分离纯化操作，没有可检测量的聚乙二醇原料和其他副产物，具有稳定、高效、绿色经济、适合规模化生产的优点，所得的产品有较高的纯度和高末端官能团取代率，能为聚乙二醇化修饰领域提供更多高质量的聚乙二醇醛修饰剂。
附图说明
图1为本发明实施例1所制备的mPEG-丙醛-20k(P1-2)的核磁共振谱图(1HNMR)；
图2为本发明实施例2所制备的mPEG-丁醛-20k(P2-2)的核磁共振谱图(1HNMR)；
图3为本发明实施例3所制备的PEG-二丁醛-30k(P3-2)的核磁共振谱图(1HNMR)；
图4为本发明实施例4所制备的PEG-二丙醛-3.4k(P4-2)的核磁共振谱图(1HNMR)；
图5为本发明实施例5所制备的四臂PEG-丙醛-10k(P5-2)的核磁共振谱图(1HNMR)；
图6为本发明实施例6所制备的HO-PEG-丙醛-20k(P6-2)的核磁谱图
图7为本发明实施例7所制备的N3-PEG-丙醛-20k(P7-2)的核磁谱图
图8为本发明实施例1所制备的mPEG-丙醛-20k(P1-2)的GPC谱图；
图9为本发明实施例2所制备的mPEG-丁醛-20k(P2-2)的GPC谱图；
图10为本发明实施例3所制备的PEG-二丁醛-30k(P3-2)的GPC谱图；
图11为本发明实施例4所制备的PEG-二丙醛-3.4k(P4-2)的GPC谱图；
图12为本发明实施例5所制备的四臂PEG-丙醛-10k(P5-2)的GPC谱图；
图13为测试本发明实施例1所制备的mPEG-丙醛-20k(P1-2)的末端取代率的HPLC谱图；
图14为测试本发明实施例2所制备的mPEG-丁醛-20k(P2-2)的末端取代率的HPLC谱图；
图15为测试本发明实施例3所制备的PEG-二丁醛-30k(P3-2)的末端取代率的HPLC谱图；
图16为测试本发明实施例4所制备的PEG-二丙醛-3.4k(P4-2)的末端取代率的HPLC谱图；
图17为测试本发明实施例5所制备的四臂PEG-丙醛-10k(P5-2)的末端取代率的HPLC谱图。
具体实施方式
术语解释
本发明对于具体的实施方式作出了详细描述，然而，应理解的是，其仅以说明性方式而非限制性方式给出，在本发明的范围内的各种变化和修改对于所属领域的技术人员将是显而易见的。
在本发明中，除非另有描述，否则本文中使用的所有技术和科学术语具有与本领域普通技术人员通常所理解的相同的含义。本文引用的所有专利和其他出版物的公开内容通过引用的方式整体并入本文。在本文术语的任何描述阐释与通过引用并入本文的任何文件相冲突的情况下，以下述术语的描述与阐释为准。除非另外指明，否则各术语具有以下含义。
本发明中，数值区间的释义，既包括短横线标记的数值区间(如1-6)，也包括波浪线标记的数值区间如(1～6)。本发明中，在没有特别说明的情况下，以区间形式标记的整数区间均可表示该区间范围内所有整数构成的组，且该范围包括两个端点。如整数范围1-6表示1、2、3、4、5、6构成的组。本发明中的数值范围，包括但不限于整数、非整数、百分数、分数表示的数值范围，如无特别说明，均包括两个端点。
本发明中，数值涉及“约”、“左右”一般指±10％的数值范围。
本发明中，当连接基的端基与连接基含有的取代基易发生混淆时，采用来标记连接基中连接其它基团的位置，如在结构式中，采用的来标记二价连接基中连接其它基团的两个位置，前述两个结构式分别表示-CH(CH2CH2CH3)2-、-CH2CH2CH(CH3)2-CH2CH2-。
本发明中，“分子量”表征一个化合物分子的质量大小，“平均分子量”表征宏观物质中通式化合物组分的质量大小，且没有特别规定时，“平均分子量”一般指“数均分子量”Mn。对于数均分子量，既可以为多分散性嵌段或物质的分子量，也可以为单分散性嵌段或物质的分子量。没有特别写明时，“分子量”与“平均分子量”的计量单位为道尔顿，Da。还可以用“聚合度”表征聚乙二醇链的分子量大小，具体指一个化合物分子中重复单元(氧化乙烯基单元、EO单元)的数量。相应地，用“平均聚合度”、优选“数均聚合度”来表征重复单元数量的平均值、数均值。
本发明中，对于多分散性情况，化合物单个分子的分子量/聚合度、宏观物质中化合物组分的数均分子量/数均聚合度的“相等”或“相同”或“等于”或“约等于”(包括其他形式的等价表达)，在没有特别指定的情况下，并不限定在数值上严格相等，而是指数值相接近或近似相等，所述相接近或近似相等优选偏差不超过±10％，更优选偏 差不超过±5，通常以预设数值为基数。例如mPEG的分子量为5kDa，优选指通式中单个分子的分子量数值在4500～5500Da之间，对应的制备产物相应组分的平均分子量为5kDa，也即平均分子量的数值在4500～5500Da之间时的产物为目标产物。
本发明中，除非特别说明，任两个对象的“选自”/“优选”各自独立，当具有多级的选自/优选情况时，任两个对象的选自/优选可以为同级或不同级。例如，“LA、LB各自独立地选自A、B、C”，可以是LA、LB均为A，也可以是LA为A而LB为B1(B1为B的一种下级情形)。又例如，“LA优选为A(1级优选)，更优选为A1～A3(2级优选)，最优选为A11～A13(3级优选)，LB优选为B(1级优选)，更优选为B1～B3(2级优选)，最优选为B11～B13(3级优选)”，优选可以是A为A1～A3(2级优选)而B为B11～B13(3级优选)，也可以是A、B均为3级优选。
本发明中，“各自独立地为/选自/优选”不仅可以指不同类目可以各自独立地为/选自/优选定义里的任一选项，还可以再加上“每次出现时”表示同一类目在不同位置或不同时间出现时每次独立地为/选自/优选定义里的任一选项，例如，式(2)中出现的两个t每次出现时各自独立地为1-6的整数。
本发明中，当列举了至少两项时，所列举的项的“组合”指前述列举的项中任两种或任两种以上的组合；且对项的数量不做限定，任一项的数量可以为零个、一个或大于一个，同种项的数量大于1时，可以是满足该项的相同或不同的具体形式。例如，“碱试剂选自金属醇盐、金属氢化物和金属氢氧化物中任一种或任两种、任两种以上的组合”，任两种的组合，可以是两种具体的金属醇盐或两种具体的金属氢化物或者两种具体的金属氢氧化物；也可以是一种金属醇盐和一种金属氢化物、一种金属醇盐和一种金属氢氧化物、一种金属氢化物和一种金属氢氧化物。
本发明中，除非特别说明，否则术语“包括”、“包含”和“含有”以及类似的表述应在本说明书和权利要求书中以开放性和包含性的含义解释为“包括但不限于”或“非限制性地包括”。
本发明中，末端取代率，又称末端活性，是指PEG末端含有活性官能团醛基的比率，末端取代率越高，醛PEG化的程度就越高，更有利于其作为聚乙二醇化修饰剂。
发明详述
一种实施方案：
一种聚乙二醇缩醛衍生物的制备方法，其特征在于，其结构如式(1)或式(2)所示：
其中，n为20-1000的整数；
t每次出现时各自独立地为1-6的整数；
k为1-8的整数；k为1时，R选自H、-CH3、-CH2CH3、-TBS、-Bn、-(CH2)tp-COOH、-(CH2)tp-COOtBu、-(CH2)tp-N3中任一种，tp为1-3的整数；k为2-8的整数时，R为多价支化结构，选自二价到八价；
所述制备方法如下：
将小分子缩醛衍生物I-2溶于有机溶剂后用碱试剂进行活化处理，然后加入溶于有机溶剂的含有官能团X1的聚乙二醇衍生物I-1或者I-3，在20-90℃下反应2-24小时，得到式(1)或式(2)所示的聚乙二醇缩醛衍生物；
或者将含有官能团X1的聚乙二醇衍生物I-1或者I-3溶于有机溶剂后用碱试剂进行活化处理，然后加入溶于有机溶剂的小分子缩醛衍生物I-2，在20-90℃下反应2-24小时，得到式(1)或式(2)所示的聚乙二醇缩醛衍生物；
所述X1每次出现时各自独立地为H、对甲苯磺酰基、甲磺酰基中任一种；所述X2每次出现时各自独立地为-OH、-SH、-NH2、-Cl、-Br、对甲苯磺酰基、甲磺酰基、三氟磺酰基、三氟乙基磺酰基中任一种；
本发明的一种具体的实施方式，优选R为以下情形中任一种：
情:1：k为1，R选自H、-CH3、-CH2CH3、-TBS、-Bn、-(CH2)tp-COOH、-(CH2)tp-COOtBu、-(CH2)tpNH2、-(CH2)tpNHCbz中任一种；
情形2：k为2时，R为-CH2CH2-；
情形3：k为3时，R为 中任一种；
情形4：k为4时，R为或者
情形5：k为6时，R为或者
情形6：k为8时，R为或者
本发明的一种具体实施方案中，优选式(1)或式(2)中，每条聚乙二醇链对应的数均分子量为900、1000、1500、2000、2500、3000、3350、3400、3500、4000、5000、5500、6000、6500、7000、7500、8000、8500、9000、9500、10000、11000、15000、 20000、25000、30000中任一种。
本发明的一种具体实施方案中，优选前述实施方案中的碱试剂选自金属醇盐、金属氢化物和金属氢氧化物中任一种或任两种、任两种以上的组合；所述金属醇盐选自甲醇钠、乙醇钠和叔丁醇钠中任一种；所述金属氢化物为氢化钾或氢化钠；所述金属氢氧化物为氢氧化钠或氢氧化钾；更优选为氢氧化钠、氢氧化钾、氢化钾、氢化钠中任一种。
本发明的一种具体实施方案中，优选式(1)或式(2)中的X1为H、对甲苯磺酸酯基或甲磺酰酯基，X2为-OH、-Cl或-Br。本发明的一种具体实施方案中，进一步优选当X1为对甲苯磺酰基或甲磺酰基，X2为-OH，所述碱性试剂为氢氧化钠或氢氧化钾；当X1为H，X2为-Cl或-Br时，所述碱性试剂为氢氧化钠或氢氧化钾。本发明的一种具体实施方案中，进一步优选当X1为对甲磺酰酯基，X2为-OH时，所述制备过程如下：
当X1为H，X2为-Br时，所述制备过程如下：
本发明的一种具体实施方案中，所述有机溶剂没有特别限制，只要原料和产物都可以溶解即可，优选自甲苯、二氯甲烷、氯仿、乙腈、二甲基亚砜、四氢呋喃、N,N'-二甲基甲酰胺、N,N'-二甲基乙酰胺、1,4-二氧六环中任一种或任两种、任两种以上的组合，更优选为甲苯或四氢呋喃，更优选为无水四氢呋喃。
本发明的一种具体实施方案中，优选前述实施方案中的碱性试剂活化处理的温度为20-60℃，更优选为30-50℃，最优选为40℃；所述活化处理的时间为6-24h，更优选为8-12h；小分子缩醛衍生物I-2和碱试剂的摩尔比为1:1-2:1；更优选为1:1-1.5:1，更优选为1:1、1.25:1或者1.2:1。
本发明的一种具体实施方案中，优选I-1和小分子缩醛衍生物I-2的摩尔比为1:10-1:160，k为1时，I-1和I-2的摩尔比为1:10-1:30；所述含有官能团X1的聚乙二醇衍生物I-3和小分子缩醛衍生物I-2的摩尔比为1:20-1:100。
本发明的一种具体实施方案中，优选所述反应是在惰性气体下进行，所述惰性气体选自氮气、氦气和氩气中任一种及其混合物。
本发明的一种具体实施方案中，所述反应中原料I-1或I-3是以滴加的方式加入到I-2中；纯化过程进行了沉淀或重结晶处理；所述沉淀试剂为醚类试剂，选自乙醚或甲基叔丁基醚，所述重结晶试剂为异丙醇、正己烷中任一种或混合物；更优选重结晶试剂为为异丙醇和正己烷的混合物，且异丙醇和正己烷的体积比为2:1-5:1。
本发明的一种具体实施方案中，优选I-1的结构为 或者其中，R的定义与式(1)中所述的一致。都可以由其对应的醇化物进行甲磺酰化或者对甲苯磺酰化而得到，具体举例，例如或者可以通过对进行甲磺酰化或者甲苯磺酰化而得到，都可以通过对进行甲磺酰化或者对甲苯磺酰化而得到。
本发明的一种具体实施方案中，优选I-1的结构为
本发明的一种具体实施方案中，优选I-3的结构为 同样的都可以通过对进行甲磺酰化或者对甲苯磺酰化而得到。
本发明的一种具体实施方案中，优选I-2的结构为 中任一种。
本发明的另一种实施方案：
一种聚乙二醇醛衍生物的制备方法，通过对聚乙二醇缩醛衍生物进行酸处理而得到，所述聚乙二醇缩醛衍生物由前述的任一种制备方法进行制备得到。本发明中，基本上全部的聚乙二醇缩醛衍生物都转化为相应的聚乙二醇醛衍生物，聚乙二醇醛衍生物的1HNMR不存在环状缩醛基的-CH<质子(化学位移约为4.80-5.00)，且含有醛基的特征峰(化学位移约为9.70-9.90)。
本发明的一种具体实施方案中，聚乙二醇醛衍生物的制备过程中用到的酸选自盐酸、三氟乙酸、甲酸和乙酸中任一种及其混合物。
本发明的一种具体实施方案中，按照所述制备方法进行制备得到的聚乙二醇醛的结构包括但不限于以下结构中任一种：


本发明中，各制备方法中用到的原料可以购买获得或者自行合成获得。
本发明的另一种实施方案：
一种按照前述制备方法获得的聚乙二醇醛衍生物修饰的生物相关物质，其特征在于，所述生物相关物质选自肽、多肽、蛋白质、多糖、甾类、核苷酸、低聚核苷酸、多核苷酸、脂肪中任一种；更优选生物相关物质含有活性氨基，或者经过改性后至少含有一个能与聚乙二醇醛衍生物的醛基偶联的活性氨基。
下面结合一些具体实施例对聚乙二醇缩醛衍生物、聚乙二醇醛衍生物的制备方法做进一步描述。具体实施例为进一步详细说明本发明，非限定本发明的保护范围。本发明中制备的中间体、终产物都可通过包括但不限于萃取、重结晶、吸附处理、沉淀、反沉淀、等的纯化方法加以纯化。对终产物的结构、分子量的表征确认，可采用包括但不限于核磁、电泳、紫外-可见分光光度计、FTIR、AFM、GPC、HPLC、MALDI-TOF、圆二色谱法等表征方法。本发明中，优选用1H-NMR进行结构确认，用凝胶渗透色谱(GPC,gel permeation chromatography)测量数均分子量(Mn)和多分散指数(PDI)，用高效液相色谱(HPLC)测试末端末端取代率。
实施例1：mPEG-丙醛的制备
实施例1.1：mPEG-丙醛-20k(P1-2)的制备
制备过程如下所示：
步骤a：氮气保护下，在干燥洁净的250mL圆底烧瓶中，加入S1-1(0.89g,7.50mmol)和无水THF(50mL)，冰浴条件下加入NaOH(0.25g,6.25mmol)，搅拌至溶解，40℃下搅拌反应8h。反应完成后，反应液降至室温，将溶解于无水THF(50mL)的mPEG-OMs-20k(5.00g,0.25mmol,Mn＝20kDa)滴加到前述的反应液中，滴加完毕后，40℃下搅拌反应8h。反应结束后，反应液降至室温，加入10％NaCl溶液，用乙酸乙酯洗三次，再加入二氯甲烷萃取两次，合并有机相，无水硫酸钠干燥，过滤，浓缩，用无水异丙醇/正己烷(v/v＝3/1)重结晶得到mPEG-丙缩醛-20k(P1-1,4.66g,收率92.7％)。1HNMR(CDCl3,400MHz)δ(ppm)：1.87-1.99(-CH2CH2CH<,2H),3.36-3.39(-OCH3,3H),3.40-3.80(-OCH2CH2O-,PEG-H；-OCH2CH2CH<,2H),3.82-3.98(>CHOCH2CH2O<,4H),4.93-4.99(>CHCH2-,1H)。
步骤b：在干燥洁净的500mL圆底烧瓶中，加入步骤a中所得的P1-1，加入1N HCl溶液，冰水浴下搅拌反应8h。反应完成后，用碳酸氢钠调节溶液pH为6.4±0.2，用二氯甲烷萃取两次，合并有机相，无水硫酸镁干燥，过滤，无水异丙醇和正己烷的混合溶液重结晶，得到mPEG-丙醛-20k(P1-2,收率98％)。核磁氢谱1HNMR(CDCl3)中，缩醛基的特征峰消失并出现了醛基的特征峰9.76-9.80(-CHO,1H)。HPLC测试P1-2末端醛基的取代率为99％，进行GPC测试，Mn＝20.1kDa，PDI＝1.03。
实施例1.2：mPEG-丙醛-2k(P1-4)的制备
制备过程如下所示：
步骤a：氮气保护下，在干燥洁净的500mL圆底烧瓶中，加入S1-1(23.63g,200.00mmol)和无水THF(200mL)，冰浴条件下加入NaOH(8.00g,200.00mmol)，搅拌至溶解，40℃下搅拌反应8h。反应完成后，反应液降至室温，将溶解于无水THF(200mL)的mPEG-OMs-20k(20.00g,10.00mmol,Mn＝2kDa)滴加到前述的反应液中，滴加完毕后，40℃下搅拌反应8h。反应结束后，反应液降至室温，加入10％NaCl溶液，用乙酸乙酯洗三次，再加入二氯甲烷萃取两次，合并有机相，无水硫酸钠干燥，过滤，浓缩，用无水异丙醇/正己烷(v/v＝3/1)重结晶得到mPEG-丙缩醛-2k(P1-3，19.53g,收率93.0％)。1HNMR(CDCl3,400MHz)δ(ppm):1.86-1.99(-CH2CH2CH<,2H),3.35-3.38(-OCH3,3H),3.40-3.80(-OCH2CH2O-,PEG-H；-OCH2CH2CH2CH<,2H),3.83-3.98(>CHOCH2CH2O<,4H),4.93-4.99(>CHCH2-,1H)。
步骤b：在干燥洁净的1L圆底烧瓶中，加入步骤a中所得的P1-3，加入1N HCl溶液，冰水浴下搅拌反应8h。反应完成后，用碳酸氢钠调节溶液pH为6.4±0.2，用二 氯甲烷萃取两次，合并有机相，无水硫酸镁干燥，过滤，无水异丙醇和正己烷的混合溶液重结晶，得到mPEG-丙醛-2k P1-4(收率98％)。核磁氢谱1HNMR(CDCl3,400MHz)中，缩醛基的特征峰消失并出现了醛基的特征峰9.76-9.80(-CHO,1H)。HPLC测试P1-4末端醛基的取代率为99％，进行GPC测试，Mn＝2.1kDa，PDI＝1.03。
实施例2：mPEG-丁醛-20k(P2-2)的制备
制备过程如下所示：
步骤a：氮气保护下，在干燥洁净的250mL圆底烧瓶中，加入S2-1(0.99g,7.50mmol)和无水THF(100mL)，冰浴条件下加入NaOH(0.20g,5.00mmol)，搅拌至溶解，40℃下搅拌反应8h。反应完成后，反应液降至室温，将溶解于无水THF(50mL)的mPEG-OMs-20k(5.00g,0.25mmol,Mn＝20kDa)滴加到前述的反应液中，滴加完毕后，55℃下搅拌反应8h。反应结束后，反应液降至室温，加入10％NaCl溶液，用乙酸乙酯洗三次，再加入二氯甲烷萃取两次，合并有机相，无水硫酸钠干燥，过滤，浓缩，用无水异丙醇/正己烷(v/v＝3/1)重结晶得到mPEG-丁缩醛-20k(P2-1，4.68g,收率93.1％)。1HNMR(CDCl3,400MHz)δ(ppm):1.73-1.82(-CH2CH2CH2CH<,4H),3.32-3.37(-OCH3,3H),3.38-3.80(-OCH2CH2O-,PEG-H；-OCH2CH2CH2CH<,2H),3.84-4.09(>CHOCH2CH2O<,4H),4.82-4.90(>CHCH2-,1H)。
步骤b：在干燥洁净的500mL圆底烧瓶中，加入步骤a中所得的P2-1，加入1N HCl溶液，冰水浴下搅拌反应8h。反应完成后，用碳酸氢钠调节溶液pH为6.4±0.2，用二氯甲烷萃取两次，合并有机相，无水硫酸镁干燥，过滤，无水异丙醇和正己烷的混合溶液重结晶，得到mPEG-丁醛-20k(P2-2，收率97％)。核磁氢谱1HNMR(CDCl3,400MHz,400MHz)中，缩醛基的特征峰消失并出现了醛基的特征峰9.75-9.78(-CHO,1H)。HPLC测试P2-2末端醛基的取代率为99％，进行GPC测试，Mn＝20.1kDa，PDI＝1.03。
实施例3：PEG-30k-二丁醛(P3-2)的制备
制备过程如下所示：
步骤a：在干燥洁净的1L圆底烧瓶中，加入S2-1(4.36g,33.00mmol)和无水THF(50mL)，冰浴条件下加入NaOH(4.26g,106.00mmol)，搅拌至溶解，40℃下搅拌反应8h。反应完成后，反应液降至室温，将溶解于无水THF(100mL)的 MsO-PEG-OMs-30k(9.90g,0.33mmol,Mn＝30kDa)滴加到前述的反应液中，滴加完毕后，55℃下搅拌反应8h。反应结束后，反应液降至室温，加入10％NaCl溶液，用乙酸乙酯洗三次，再加入二氯甲烷萃取两次，合并有机相，无水硫酸钠干燥，过滤，浓缩，用无水异丙醇/正己烷(v/v＝3/1)重结晶得到PEG-二丁缩醛-30k(P3-1,9.31g,收率93.8％)。1HNMR(d6-DMSO,400MHz)δ(ppm):1.50-1.65(-OCH2CH2CH2CH<,8H),3.35-3.65(-OCH2CH2O-,PEG-H；-OCH2CH2OCH2-,4H),3.70-3.90(>CHOCH2CH2O<,8H),4.75-4.85(>CHCH2-,2H)。
步骤b：在干燥洁净的2L圆底烧瓶中，加入步骤a中所得的P3-1，加入1N HCl溶液，冰水浴下搅拌反应8h。反应完成后，用碳酸氢钠调节溶液pH为6.4±0.2，用二氯甲烷萃取两次，合并有机相，无水硫酸镁干燥，过滤，无水异丙醇和正己烷的混合溶液重结晶，得到PEG-二丁醛-30k(P3-2,收率97％)。核磁氢谱1HNMR(d6-DMSO,400MHz)中，缩醛基的特征峰消失并出现了醛基的特征峰9.79-9.83(-CHO,2H)。HPLC测试P3-2末端醛基的取代率为99％，进行GPC测试，Mn＝30.2，PDI＝1.04。
实施例4：PEG-二丙醛-3.4k(P4-2)的制备
制备过程如下所示：
步骤a：氮气保护下，在干燥洁净的1L圆底烧瓶中，加入S1-1(93.56g,792.00mmol)和无水THF(300mL)，冰浴条件下加入NaOH(21.12g,528.00mmol)，搅拌至溶解，40℃下搅拌反应8h。反应完成后，反应液降至室温，将溶解于无水THF(300mL)的MsO-PEG-OMs-3.4k(29.92g,8.80mmol,Mn＝3.4kDa)滴加到前述的反应液中，滴加完毕后，40℃下搅拌反应8h。反应结束后，反应液降至室温，加入10％NaCl溶液，用乙酸乙酯洗三次，再加入二氯甲烷萃取两次，合并有机相，无水硫酸钠干燥，过滤，浓缩，用无水异丙醇/正己烷(v/v＝3/1)重结晶得到PEG-二丙缩醛-3.4k(P4-1,28.14g,收率92.2％)。1HNMR(CDCl3,400MHz)δ(ppm):1.86-1.99(-CH2CH2CH<,4H),3.38-3.80(-OCH2CH2O-,PEG-H；-OCH2CH2CH<,4H),3.83-3.98(>CHOCH2CH2O<,8H),4.93-4.99(>CHCH2-,2H)。
步骤b：在干燥洁净的2L圆底烧瓶中，加入步骤a中所得的P4-1，加入1N HCl溶液，冰水浴下搅拌反应8h。反应完成后，用碳酸氢钠调节溶液pH为6.4±0.2，用二氯甲烷萃取两次，合并有机相，无水硫酸镁干燥，过滤，无水异丙醇和正己烷的混合溶液重结晶，得到PEG-二丙醛-3.4k(P4-2，收率97％)。核磁氢谱1HNMR(CDCl3,400MHz)中，缩醛基的特征峰消失并出现了醛基的特征峰9.74-9.85(-CHO,2H)。HPLC测试P4-2末端醛基的取代率为99％，进行GPC测试，Mn＝3.6kDa，PDI＝1.03。
实施例5:四臂聚乙二醇丙醛的制备
实施例5.1：四臂聚乙二醇丙醛4-arm-PEG-pALD-10k(P5-2)的制备
制备过程如下所示：
步骤a：氮气保护下，在干燥洁净的2L圆底烧瓶中，加入4-arm-PEG-OH(25.00g,2.50mmol，Mn＝10kDa)，甲苯中共沸除水，将NaH(16.00g,400.00mmol)溶解于无水THF(1L)中，冰浴条件下加到前述的4-arm-PEG-OH溶液中，温度升至85℃回流反应8h。反应完成后，反应液降至室温，然后加入NaI(1.50g,10.00mmol)和S5-1(72.41g,400.00mmol)，温度升至85℃回流反应8h。反应结束后，将反应置于冰浴下，缓慢滴加氯化铵水溶液淬灭反应，浓缩去除THF，加入10％NaCl溶液，用乙酸乙酯洗三次，再加入二氯甲烷萃取两次，合并有机相，无水硫酸钠干燥，过滤，浓缩，用无水异丙醇/正己烷(v/v＝3/1)重结晶得到4-arm-PEG-丙缩醛-10k(P5-1,23.59g,收率90.7％)。1HNMR(CDCl3,400MHz)δ(ppm):1.85-1.97(-CH2CH2CH<,8H),3.40-3.80(-OCH2CH2O-,PEG-H；-OCH2CH2CH<,8H；-CCH2-,8H),3.83-3.98(>CHOCH2CH2O<,16H),4.91-4.97(>CHCH2-,4H)。
步骤b：在干燥洁净的2L圆底烧瓶中，加入步骤a中所得的P5-1，加入1N HCl溶液，冰水浴下搅拌反应8h。反应完成后，用碳酸氢钠调节溶液pH为6.4±0.2，用二氯甲烷萃取两次，合并有机相，无水硫酸镁干燥，过滤，无水异丙醇和正己烷的混合溶液重结晶，得到4-arm-PEG-丙醛-10k(P5-2,收率97％)。核磁氢谱1HNMR(CDCl3,400MHz)中，缩醛基的特征峰消失并出现了醛基的特征峰9.76-9.82(-CHO,4H)。HPLC测试P5-2末端醛基的取代率为97％，进行GPC测试，Mn＝10.4kDa，PDI＝1.04。
实施例5.2：四臂聚乙二醇丙醛4-arm-PEG-pALD-20k的制备
按照实施例5.1中的操作步骤，将原料4-arm-PEG-OH-10k换成4-arm-PEG-OH-20k，最终得到4-arm-PEG-丙缩醛-20k(P5-3)和4-arm-PEG-丙醛-20k(P5-4)。结构同样通过1HNMR、GPC和HPLC验证。
实施例6：羟基-PEG-丙醛-20k(P6-2)的制备
制备过程如下所示：
步骤a：氮气保护下，在干燥洁净的250mL圆底烧瓶中，加入S1-1(0.89g,7.50mmol)和无水THF(50mL)，冰浴条件下加入NaOH(0.20g,5.00mmol)，搅拌至溶解，40℃下搅拌反应8h。反应完成后，反应液降至室温，将溶解于无水THF(50mL)的TBSO-PEG-OMs-20k(5.00g,0.25mmol)滴加到前述的反应液中，滴加完毕后，40℃下搅拌反应8h。反应结束后，反应液降至室温，加入10％NaCl溶液，用乙酸乙酯洗三次，再加入二氯甲烷萃取两次，合并有机相，无水硫酸钠干燥，过滤，浓缩，用无水异丙醇/正己烷(v/v＝3/1)重结晶得到TBSO-PEG-丙缩醛-20k(P6-1,4.65g,收率93.0％)。 1HNMR(CDCl3,400MHz)δ(ppm):0.21(-Si(CH3)2-,6H),0.98(-SiC(CH3)3,9H),1.75-1.84(-CH2CH2CH<,2H),3.35-3.72(-OCH2CH2O-,PEG-H；-OCH2CH2CH2CH<,2H),3.71-3.90(>CHOCH2CH2O<,4H),4.80-4.90(>CHCH2-,1H)。
步骤b：在干燥洁净的500mL圆底烧瓶中，加入步骤a中所得的P6-1，用四氢呋喃/乙酸混合溶液溶解，冰浴条件下加入TBAF，室温反应8h，萃取，浓缩、然后加入1N HCl溶液，冰水浴下搅拌反应8h。反应完成后，用碳酸氢钠调节溶液pH为6.4±0.2，用二氯甲烷萃取两次，合并有机相，无水硫酸镁干燥，过滤，无水异丙醇和正己烷的混合溶液重结晶，得到羟基-PEG-丙醛-20k(P6-2,收率95％)。核磁氢谱1HNMR(CDCl3,400MHz)中，缩醛基和TBS的特征峰都消失并出现了醛基的特征峰9.73-9.80(-CHO,1H)。HPLC测试P6-2末端醛基的取代率为97％，进行GPC测试，Mn＝20.1kDa，PDI＝1.04。
实施例7：N3-PEG-丙醛-20k(P7-2)的制备
制备过程如下所示：
步骤a：氮气保护下，在干燥洁净的250mL圆底烧瓶中，加入S1-1(0.89g,7.50mmol)和无水THF(50mL)，冰浴条件下加入NaOH(0.20g,5.00mmol)，搅拌至溶解，40℃下搅拌反应8h。反应完成后，反应液降至室温，将溶解于无水THF(50mL)的N3-PEG-OMs-20k(5.00g,0.25mmol)滴加到前述的反应液中，滴加完毕后，40℃下搅拌反应8h。反应结束后，反应液降至室温，加入10％NaCl溶液，用乙酸乙酯洗三次，再加入二氯甲烷萃取两次，合并有机相，无水硫酸钠干燥，过滤，浓缩，用无水异丙醇/正己烷(v/v＝3/1)重结晶得到N3-PEG-丙缩醛-20k(P7-1,4.65g,收率93.0％)。1HNMR(CDCl3,400MHz)δ(ppm):1.75-1.84(-CH2CH2CH<,2H),3.35-3.72(-OCH2CH2O-,PEG-H；-OCH2CH2CH2CH<,2H；-OCH2CH2N3,4H),3.71-3.90(>CHOCH2CH2O<,4H),4.80-4.90(>CHCH2-,1H)。
步骤b：在干燥洁净的500mL圆底烧瓶中，加入步骤a中所得的P7-1，然后加入1N HCl溶液，冰水浴下搅拌反应8h。反应完成后，用碳酸氢钠调节溶液pH为6.4±0.2，用二氯甲烷萃取两次，合并有机相，无水硫酸镁干燥，过滤，无水异丙醇和正己烷的混合溶液重结晶，得到N3-PEG-丙醛-20k(P7-2，收率95％)。核磁氢谱1HNMR(CDCl3,400MHz)中，缩醛基的特征峰都消失并出现了醛基的特征峰9.73-9.80(-CHO,1H)。HPLC测试P7-2末端醛基的取代率为99％，进行GPC测试，Mn＝20.1kDa，PDI＝1.03。
实施例8：mPEG-丙醛-20k(P1-2)的制备方法2
制备过程如下所示：
步骤a：氮气保护下，在干燥洁净的2L圆底烧瓶中，加入mPEG-OH(5.00g,0.25mmol，Mn＝20kDa)，甲苯中共沸除水，将NaH(0.20g,5.00mmol)溶解于无水THF(50mL)中，冰浴条件下加到前述的mPEG-OH溶液中，温度升至85℃回流反应8h。反应完成后，反应液降至室温，然后加入S5-1(1.36g,7.50mmol)，温度升至85℃回流反应8h。反应结束后，将反应置于冰浴下，缓慢滴加氯化铵水溶液淬灭反应，浓缩去除THF，加入10％NaCl溶液，用乙酸乙酯洗三次，再加入二氯甲烷萃取两次，合并有机相，无水硫酸钠干燥，过滤，浓缩，用无水异丙醇/正己烷(v/v＝3/1)重结晶得到mPEG-丙缩醛-20k(P1-1,2.30g,收率88.5％)。结构同样通过核磁进行表征。
步骤b：在干燥洁净的2L圆底烧瓶中，加入步骤a中所得的P1-1，加入1N HCl溶液，冰水浴下搅拌反应8h。反应完成后，用碳酸氢钠调节溶液pH为6.4±0.2，用二氯甲烷萃取两次，合并有机相，无水硫酸镁干燥，过滤，无水异丙醇和正己烷的混合溶液重结晶，得到mPEG-丙醛-20k(P1-2,收率97％)。核磁氢谱1HNMR(CDCl3,400MHz)中，缩醛基的特征峰消失并出现了醛基的特征峰9.76-9.82(-CHO,1H)。经HPLC测试，P1-2末端醛基的取代率为95％。
实施例9：mPEG-丙醛-20k(P1-2)的制备方法3
将实施例8中的碱性试剂由NaH换为NaOH，其他步骤与实施例8中的相同，得到P1-2的最终收率为84％。经HPLC测试，P1-2末端醛基的取代率为38％。
实施例10：mPEG-丙醛-20k(P1-2)的制备方法4
制备过程如下所示：
将实施例8中的小分子环状缩醛原料S5-1换为小分子线状缩醛原料S10-1，其他操作步骤与实施例8中的相同，得到P1-2的最终收率约为83％。经HPLC测试，P1-2末端醛基的取代率为85％。
实施例11：mPEG-丙醛-20k(P1-2)的制备方法5
制备过程如下所示：
将实施例1.1中的小分子环状缩醛原料S1-1换为小分子线状缩醛原料S11-1，其他操作步骤与实施例1.1中的相同，得到P1-2的最终收率约为87％。经HPLC测试，P1-2末端醛基的取代率为87％。
实施例12：mPEG-丙醛-20k(P1-2)的制备方法6
将实施例1.1中的原料mPEG-OMs-20k不以滴加的方式加入，而是固体直接加入，其 他操作步骤与实施例1.1中的相同，得到P1-2的最终收率约为82％。经HPLC测试，P1-2末端醛基的取代率为91％。
实施例13：mPEG-丙醛-20k(P1-2)的制备方法7
将实施例1.1中步骤a中的重结晶步骤改为用醚类试剂(叔丁基甲基醚)进行打浆沉淀，即将粗产物溶解于少量的DCM中，再滴加到醚试剂中(醚试剂的体积为DCM的20倍)，其他操作步骤与实施例1.1中的相同，得到P1-2的最终收率约为87％。经HPLC测试，P1-2末端醛基的取代率为89％。
实施例14：mPEG-丙醛-20k(P1-2)的制备方法8
将实施例1.1中步骤a中的重结晶步骤改为用异丙醇进行重结晶，其他操作步骤与实施例1.1中的相同，得到P1-2的最终收率约为89％。经HPLC测试，P1-2末端醛基的取代率为93％。
上述实施例1.1和实施例8-14为mPEG-丙醛-20k(P1-2)的不同制备方法，实施例9-14为对比方法，各方法的对比如下表1和表2所示：
表1
表2
从上述表1和表2可以看出不同的制备方法，收率和末端官能团取代率存在差异，且末端取代率的差异更大，本领域技术人员知晓末端取代率对高分子产品的质量更为重要，末端取代率高的对应的产品纯度更高。
从表1可以看出，聚乙二醇醛衍生物的制备过程中，小分子缩醛试剂和碱试剂的类型对产品的收率和质量有着重大影响。首先，实施例1.1、8、9和实施例10、11相比可知，与线性缩醛小分子试剂相比，采用环状缩醛小分子试剂的收率更高、末端取代率也更高，这是因为环状缩醛基比线性缩醛基稳定，制备过程的副反应更少；其次，实施例1.1、8、9的对比可知，不同的小分子缩醛试剂对应不同的较优金属碱试剂，实施例1.1中的NaOH是对小分子缩醛进行脱氢活化，实施例8中的NaH和实施例9中的NaOH是对mPEG-OH的氢进行脱氢活化，对mPEG-OH进行脱氢活化时用NaH比用NaOH好，用NaH得到的产品的末端醛基取代率为95％，而用NaOH的只有38％。
从表2可以看出，原料的加入方式和后处理过程对产品的收率和质量有着重大影响。首先，实施例1.1和实施例12相比可知，对将聚乙二醇原料加入到碱金属活化了的小分子缩醛衍生物中时，滴加加入的方式比直接倒入的方式的收率更高且产品的末端官能团取代率也更高；可能是因为滴加方式加入的原料，一加入就能马上和活化了的另一原料反应，反应更充分且更具有时效性；其次，实施例1.1、13和14对比可知，后处理时用重结晶比用沉淀法收率更高且产品质量更佳，且重结晶试剂的选择对产品的收率和质量也有影响，采用异丙醇和正己烷的混合物进行重结晶的收率更高且产品的末端官能团取代率也更高。
综上可知，与对比例实施例9-14相比，本申请的制备方法(实施例1.1)得到的产品收率更高、纯度更高、质量更佳(更高的末端醛基取代率)。

Claims (16)

  1. 一种聚乙二醇缩醛衍生物的制备方法，其特征在于，其结构如式(1)或式(2)所示：
    其中，n为20-1000的整数；
    t每次出现时各自独立地为1-6的整数；
    k为1-8的整数；k为1时，R选自H、-CH3、-CH2CH3、-TBS、-Bn、-(CH2)tp-COOH、-(CH2)tp-COOtBu、-(CH2)tp-N3中任一种，tp为1-3的整数；k为2-8的整数时，R为多价支化结构，选自二价到八价；
    所述制备方法如下：
    将小分子缩醛衍生物I-2溶于有机溶剂后用碱试剂进行活化处理，然后加入溶于有机溶剂的含有官能团X1的聚乙二醇衍生物I-1或者I-3，在20-90℃下反应2-24小时，得到式(1)或式(2)所示的聚乙二醇缩醛衍生物；
    或者将含有官能团X1的聚乙二醇衍生物I-1或者I-3溶于有机溶剂后用碱试剂进行活化处理，然后加入溶于有机溶剂的小分子缩醛衍生物I-2，在20-90℃下反应2-24小时，得到式(1)或式(2)所示的聚乙二醇缩醛衍生物；
    所述X1每次出现时各自独立地为H、对甲苯磺酰基、甲磺酰基中任一种；所述X2每次出现时各自独立地为-OH、-SH、-NH2、-Cl、-Br、对甲苯磺酰基、甲磺酰基、三氟磺酰基、三氟乙基磺酰基中任一种；
    或者
  2. 根据权利要求1所述的聚乙二醇缩醛衍生物的制备方法，其特征在于，R选自H、-CH3、-CH2CH3、-Bn、-(CH2)tp-COOH、-(CH2)tp-COOtBu、-(CH2)tpNH2、-(CH2)tpNHCbz、-CH2CH2-、 中任一种。
  3. 根据权利要求1所述的聚乙二醇缩醛衍生物的制备方法，其特征在于，每条聚乙二醇链对应的数均分子量为900、1000、1500、2000、2500、3000、3350、3400、3500、4000、5000、5500、6000、6500、7000、7500、8000、8500、9000、9500、10000、11000、15000、20000、25000、30000中任一种。
  4. 根据权利要求1所述的聚乙二醇缩醛衍生物的制备方法，其特征在于，所述碱试剂选自金属醇盐、金属氢化物和金属氢氧化物中任一种或任两种、任两种以上的组合；所述金属醇盐选自甲醇钠、乙醇钠和叔丁醇钠中任一种；所述金属氢化物为氢化钾或氢化钠；所述金属氢氧化物为氢氧化钠或氢氧化钾；更优选为氢氧化钠、氢氧化钾、氢化钾、氢化钠中任一种。
  5. 根据权利要求1所述的聚乙二醇缩醛衍生物的制备方法，其特征在于，X1为H、对甲苯磺酸酯基或甲磺酰酯基，X2为-OH、-Cl或-Br。
  6. 根据权利要求5所述的聚乙二醇缩醛衍生物的制备方法，其特征在于，当X1为对甲苯磺酰基或甲磺酰基，X2为-OH时，所述碱性试剂为氢氧化钠或氢氧化钾；当X1为H，X2为-Cl或-Br时，所述碱性试剂为氢氧化钠或氢氧化钾。
  7. 根据权利要求6所述的聚乙二醇缩醛衍生物的制备方法，其特征在于，
    当X1为对甲磺酰酯基，X2为-OH时，所述制备过程如下：
    或者
    当X1为H，X2为-Br时，所述制备过程如下：
    或者
  8. 根据权利要求1所述的聚乙二醇缩醛衍生物的制备方法，其特征在于，所述有机溶剂选自甲苯、二氯甲烷、氯仿、乙腈、二甲基亚砜、四氢呋喃、N,N'-二甲基甲酰胺、N,N'-二甲基乙酰胺、1,4-二氧六环中任一种或任两种、任两种以上的组合，更优选为甲苯或四氢呋喃，更优选为无水四氢呋喃。
  9. 根据权利要求1所述的聚乙二醇缩醛衍生物的制备方法，其特征在于，所述活化处理的温度为20-60℃，更优选为30-50℃，最优选为40℃；所述活化处理的时间为6-24h，更优选为8-12h；小分子缩醛衍生物I-2和碱试剂的摩尔比为1:1-2:1；更优选为1:1-1.5:1，更优选为1:1、1.25:1或者1.2:1。
  10. 根据权利要求1所述的聚乙二醇缩醛衍生物的制备方法，其特征在于，所述含有官能团X1的聚乙二醇衍生物I-1和小分子缩醛衍生物I-2的摩尔比为1:10-1:160，k为1时，I-1和I-2的摩尔比为1:10-1:30；所述含有官能团X1的聚乙二醇衍生物I-3和小分子缩醛衍生物I-2的摩尔比为1:20-1:60。
  11. 根据权利要求1所述的聚乙二醇缩醛衍生物的制备方法，其特征在于，所述反应是在惰性气体下进行，所述惰性气体选自氮气、氦气和氩气中任一种及其混合物。
  12. 根据权利要求1所述的聚乙二醇缩醛衍生物的制备方法，其特征在于，所述反应中原料I-1或I-3是以滴加的方式加入到I-2中；纯化过程进行沉淀或重结晶处理；更优选进行重结晶处理；所述沉淀试剂为醚类试剂，选自乙醚或甲基叔丁基醚，所述重结晶试剂为异丙醇、正己烷中任一种或混合物；更优选重结晶试剂为为异丙醇和正己烷的混合物，且异丙醇和正己烷的体积比为2:1-5:1。
  13. 一种聚乙二醇醛衍生物的制备方法，其特征在于，通过对聚乙二醇缩醛衍生物进行酸处理而得到，所述聚乙二醇缩醛衍生物由权利要求1-12中任一项所述的制备方法进行制备。
  14. 根据权利要求12所述的聚乙二醇醛衍生物的制备方法，其特征在于，所述酸选自盐酸、三氟乙酸、甲酸和乙酸中任一种及其混合物。
  15. 根据权利要求12所述的聚乙二醇醛衍生物的制备方法，其特征在于，所述聚乙二醇醛衍生物的结构如下：


  16. 一种权利要求14-15中任一项所述的聚乙二醇醛衍生物修饰的生物相关物质，其特征在于，所述生物相关物质选自肽、多肽、蛋白质、多糖、甾类、核苷酸、低聚核苷酸、多核苷酸、脂肪中任一种；更优选生物相关物质含有活性氨基，或者经过改性后至少含有一个能与聚乙二醇醛衍生物的醛基偶联的活性氨基。
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