CN1660920A

CN1660920A - Polyglycolic acid or active ester with ω-amino acid connected at the end, its preparation method and application

Info

Publication number: CN1660920A
Application number: CN2005100237376A
Authority: CN
Inventors: 何明磊; 魏东芝
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2005-02-01
Filing date: 2005-02-01
Publication date: 2005-08-31

Abstract

An acid or active ester of polyethanediol, whose terminal end is linked with omega-amino acid has the structural formula: CH3-(OCh2CH2)n-CONH-(CH2)m-CO2-Q, 10-30 min of semi life in water and ideal reactive activity to amino.

Description

Terminal polyoxyethylene glycol acid or active ester and method for making and the application that connects omega-amino acid

Technical field

The present invention relates to polyoxyethylene glycol water-soluble polymers and its production and application, relate in particular to polyethylene active ester and its production and application.

Background technology

Use chemical process that the water-soluble polymers polyoxyethylene glycol is attached to molecule and in biotechnology, have great application value with the surface.Its most general form PEG is a kind of polymkeric substance of line style, and two ends are terminal polymkeric substance HO-CH with the hydroxyl ₂-CH ₂O-(CH ₂CH ₂O) n-CH ₂CH ₂OH.

Above-mentioned polymkeric substance α, the alpha, omega-dihydroxy polyoxyethylene glycol can be represented HO-PEG-OH by following skeleton symbol.Example as the biotechnology applications of PEG: the reactive derivative of some PEG is attached on the molecule of protein, enzyme, and PEG can be dissolved in organic solvent, and PEG is attached to and can makes the binding substances of PEG and enzyme be dissolved in organic solvent on the enzyme molecule; Compare with the protein of unmodified, PEG is connected to reduces immunogenicity on the protein, reduce kidney and get rid of speed, and may cause the binding substances huge increase of the half life in blood circulation; PEG is attached to the charged character that the surface can reduce the absorption of protein and cell and change the surface, and PEG is attached to and can makes its half life that very big increase is arranged on the liposome, and can increase its ability as the medium of medicament slow release.

PEG has only through overactivation and can be attached on molecule or the surface.Therefore, a series of activated derivatives is produced out: some have purposes widely, and some have special purpose, and some also are not proved to be useful or some problem has limited its purposes.

For example, the alpha, omega-dicarboxylic acid NHS ester of-PEG-is produced out, is used to haemoglobin molecule is coupled together, and NHS is the abbreviation of nitrogen N-Hydroxysuccinimide, and in the following description, the NHS ester of PEG acid is expressed as-COONHS.Have the easy hydrolysis of NHS ester of the PEG acid of ester bond, so the peg moiety on its binding substances comes off because of hydrolysis easily.

At present, many activatory carboxyl PEG that are used to modify have very poor reactive behavior: be not that to react too be exactly to react too slow soon.For example, the reactive behavior of the NHS ester of carboxymethylation PEG (CM-PEG) is too strong, so that be dissolved in hydrolysis at once in the water, this high reaction activity is the major defect of carboxymethylation PEG active ester.

Summary of the invention

The technical issues that need to address of the present invention are to disclose a kind of polyoxyethylene glycol acid or active ester and method for making and application that connects omega-amino acid, to overcome the defective that prior art exists, satisfy the needs of medical treatment and pharmacy field development.

The polyethylene active acid of terminal connection omega-amino acid of the present invention or the general structure of ester are as follows:

CH ₃-(OCH ₂CH ₂)n-CONH-(CH ₂)m-CO ₂-Q

N=113～680 wherein, m=2,3,5; Q represents in hydrogen, the tertiary butyl, N-O succinyl or the N-S succinyl, n=113～680;

When m=2, active ester is the active ester of ethylene glycol-Beta-alanine, and when m=3, active ester is the active ester of ethylene glycol-γ-An Jidingsuan, and when m=5, active ester is the active ester of ethylene glycol-epsilon-amino caproic acid;

When Q represents hydrogen, be acid;

Preferably:

CH ₃O-(CH ₂CH ₂O)n-CH ₂CONH-CH ₂CH ₂COOH、

CH ₃O-(CH ₂CH ₂O)n-CH ₂CONH-CH ₂CH ₂CH ₂COOH、

CH ₃O-(CH ₂CH ₂O)n-CH ₂CONH-(CH ₂) ₅COOH、

CH ₃O-(CH ₂CH ₂O)n-CH ₂CONH-CH ₂CH ₂COONHS、

CH ₃O-(CH ₂CH ₂O) n-CH ₂CONH-CH ₂CH ₂CH ₂COONHS or

CH ₃O-(CH ₂CH ₂O)n-CH ₂CONH-(CH ₂) ₅COONHS。

The polyethylene active acid or the ester of connection omega-amino acid provided by the invention have suitable reactive behavior, and the half life of active ester in water be greatly about 10～30 minutes, and the ester bond that does not contain other connects.

The use of propionic acid polyethylene active ester and butyric acid polyethylene active ester is for following 4 points:

1. propionic acid polyethylene active ester has the desirable activity that connects amino acid whose amino in the aqueous solution, though butyric acid polyethylene active ester still can be used for the modification reaction of the aqueous solution be not as active as the counterpart of propionic acid;

2. crosslinking reaction can Be Controlled as side reaction;

3. the stability to hydrolysis of ehter bond connection (ethylene glycol skeleton) has caused the stability of binding substances;

4. the stability to hydrolysis of ehter bond connection (ethylene glycol skeleton) has guaranteed that intermediate obtains being easy to separate by ion-exchange chromatography in aqueous medium.

The preparation method of the polyethylene active acid of connection omega-amino acid of the present invention comprises the steps:

(1) preparation of CM-PEG (carboxymethylation polyoxyethylene glycol):

Mono methoxy polyethylene glycol (glycol) is dissolved in the toluene, adds potassium tert.-butoxide back flow reaction 1～2h, add ethyl bromoacetate back flow reaction 2～4h, room temperature reaction 18～24h then, concentrate the back and use ether sedimentation, throw out basic hydrolysis acidifying then adopts conventional method to collect product;

(2) preparation of omega-amino acid carbethoxy hydrochloride:

With hydrogen chloride gas feed contain omega-amino acid ethanol up to saturated, collect the omega-amino acid carbethoxy hydrochloride then;

(3) the terminal preparation that connects the polyethylene active acid of omega-amino acid:

The CM-PEG that gets preparation in the step 1 is dissolved in methylene dichloride, add dicyclohexylcarbodiimide, omega-amino acid carbethoxy hydrochloride then successively, with triethylamine the pH value of solution is adjusted to 8～9 then, reacted 8～12 hours, filter, filtrate adds ether, the collecting precipitation product, with water-soluble after the product drying, add sodium hydroxide solution and regulate pH to 10～12, reaction 1～3h is adjusted to 2～4 with oxalic acid with pH value of solution then, collect product, obtain the terminal polyethylene active acid that connects omega-amino acid.

(4) product of getting step (3) is dissolved in methylene dichloride, add the dicyclohexylcarbodiimide of 1.5～3 times of molar weights and the nitrogen N-Hydroxysuccinimide or the thiol succinimide of 1.5～3 times of molar weights, 10～30 ℃ of stirring reaction 20～28h, collect product, obtain the terminal polyethylene active ester that connects omega-amino acid;

Said omega-amino acid is selected from Beta-alanine, γ-An Jidingsuan or epsilon-amino caproic acid;

The preferred molecular weight of mono methoxy polyethylene glycol is 5000～30000.

Terminal polyethylene active acid or the ester that connects omega-amino acid of the present invention, in water, have 10-30 minute half life, reactive behavior with ideal and biologically active substance amino, can be used for being connected with biologically active substance, preparation has the biologically active substance that PEG modifies, and is used to prepare the medium of slow releasing pharmaceutical.

The invention still further relates to a kind of said terminal polyethylene active acid of omega-amino acid or modifier of ester and biologically active substance of connecting, general structure is as follows:

CH ₃-(OCH ₂CH ₂)n-CONH-(CH ₂)m-CONH-Pro

Wherein: NH-Pro partly represents the amino sites of biologically active substance.

Biologically active substance is selected from protein, enzyme, polypeptide, medicine, dyestuff, nucleosides, ester class or liposome.

Adopt the polyethylene active ester of the end connection omega-amino acid of method for preparing, has good reaction activity, being dissolved in the water not can very fast hydrolysis, thereby can be used for being connected with the molecule of protein or enzyme, be used to prepare the medium of slow releasing pharmaceutical, can satisfy the needs of pharmacy field development.

Description of drawings

Fig. 1 is to use the electrophorogram (SDS-PAGE) of the active ester Interferon, rabbit alpha-2b of PEG-γ-An Jidingsuan.

Embodiment 1

Molecular weight is the mPEG-CH of 5kD ₂CONH-CH ₂CH ₂COONHS's is synthetic

The preparation of step 1:CM-PEG (carboxymethylation polyoxyethylene glycol):

With molecular weight is that mono methoxy polyethylene glycol (glycol) 10g (2 mmole) of 5kD is dissolved in the toluene, distill out a part of toluene with azeotropic water removing, the potassium tert.-butoxide back flow reaction 1.5h that adds 3 mmoles then, the ethyl bromoacetate back flow reaction 3h that then slowly adds 3 mmoles, room temperature reaction 21h then, remove by filter post precipitation pressure reducing and steaming solvent, residue adds a small amount of methylene dichloride dissolving, be settled out product with dry ether, this product is dissolved in the deionized water, the pH value stabilization of the sodium hydroxide solution until solution of adding 0.1mol/L is at pH10, use the pH to 3 of the hydrochloric acid conditioning solution of 0.1mol/L then, the chloroform extraction solution of usefulness equivalent three times merges organic phase and uses anhydrous sodium sulfate drying, concentrate the back and obtain the CM-PEG 9.5g that molecular weight is 5kD with the ether sedimentation drying, molecular formula is as follows: CH3O-(CH ₂CH ₂O) n-CH ₂COOH

Step 2:

The preparation of Beta-alanine carbethoxy hydrochloride:

20 gram Beta-alanines are joined in the 300mL dehydrated alcohol, under agitation feed the exsiccant hydrogen chloride gas up to saturated, add dehydrated alcohol then and carry out underpressure distillation, unnecessary hydrogenchloride is removed, then with solution concentration to suitable volume, obtain the Beta-alanine carbethoxy hydrochloride with dry anhydrous ether sedimentation.

Step 3: synthetic mPEG-CH ₂CONH-CH ₂CH ₂COOH:

The CM-PEG that gets preparation in 5g (1 mmole) step 1 is dissolved in the 20mL exsiccant methylene dichloride, the dicyclohexylcarbodiimide that adds 2 mmoles then successively, the Beta-alanine carbethoxy hydrochloride of 2 mmoles, with triethylamine the pH value of solution is adjusted to 8 then, stirring reaction 12h, remove by filter post precipitation, add the anhydrous diethyl ether precipitated product, to be dissolved in deionized water after the product drying, the sodium hydroxide solution that adds 0.1mol/L is regulated pH 11, reaction 2h is adjusted to 3 with oxalic acid with pH value of solution then, uses chloroform extraction solution 3 times, the combining extraction liquid anhydrous sodium sulfate drying, obtaining molecular weight with anhydrous diethyl ether precipitation again after the solution concentration is 5000 mPEG-CH ₂CONH-CH ₂CH ₂COOH, i.e. the PEG propanoic derivatives that is connected with PEG with amido linkage.

This product ¹H NMR (solvent: methyl-sulphoxide): 2.43ppm (t, 4H ,-CH2-COOH); 3.21ppm (s ,-OCH ₃), 3.50ppm (s ,-OCH ₂, CH ₂O-), and 7.18ppm (t, 1H, CONH), IR spectrum: 1629cm ^-1

(CONH)，1740cm ^-1(COOH)

Step 4:

The molecular weight of getting step 3 preparation is PEG-acyl γ-L-Ala 5g of 5kD, be dissolved in the dry purified anhydrous methylene chloride, add the dicyclohexylcarbodiimide (DCC) of 2 times of molar weights and the nitrogen N-Hydroxysuccinimide of 2 times of molar weights, stirring at room reaction 24h, stopped reaction filters out precipitation (DCU) dicyclohexylurea (DCU), and solution obtains the nitrogen hydroxysuccinimide eater that molecular weight of product is PEG-acyl γ-L-Ala of 5kD with the anhydrous diethyl ether precipitation.Molecular formula is as follows:

CH ₃O-(CH ₂CH ₂O)n-CH ₂CONH-CH ₂CH ₂COONHS

IR spectrum: 1629cm ^-1(CONH), 1729cm ^-1(COONHS)

Embodiment 2

Synthetic molecular weight is the mPEG-CH of 12kD ₂CONH-CH ₂CH ₂CH ₂COOH

Step 1:

The preparation of CM-PEG (carboxymethylation polyoxyethylene glycol):

With molecular weight is that the mono methoxy polyethylene glycol 12g (1 mmole) of 12kD is dissolved in an amount of toluene, distill out a part of toluene with azeotropic water removing, the potassium tert.-butoxide back flow reaction 2h that adds 2 mmoles then, the ethyl bromoacetate back flow reaction 4h that then slowly adds 2 mmoles, room temperature reaction 18h then, remove by filter post precipitation pressure reducing and steaming solvent, residue adds the methylene dichloride dissolving, be settled out product with dry ether, this product is dissolved in the deionized water, the pH value stabilization of sodium hydroxide solution until solution that adds 0.1mol/L gradually is at pH10, use the pH to 3 of the hydrochloric acid conditioning solution of 0.1mol/L then, the chloroform extraction solution of usefulness equivalent three times merges organic phase and uses anhydrous sodium sulfate drying, concentrate the back and obtain the CM-PEG that molecular weight is 12kD with ether sedimentation, molecular formula is as follows: CH3O-(CH ₂CH ₂O) n-CH ₂COOH

Step 2:

The preparation of γ-An Jidingsuan hydrochloride: the 20g γ-An Jidingsuan is joined in the 300mL dehydrated alcohol, under agitation feed the exsiccant hydrogen chloride gas up to saturated, add dehydrated alcohol then and carry out underpressure distillation, unnecessary hydrogenchloride is removed, then with solution concentration to suitable volume, obtain the γ-An Jidingsuan carbethoxy hydrochloride with dry anhydrous ether sedimentation.

Step 3:

Synthetic mPEG-CH ₂CONH-CH ₂CH ₂COOH:

The CM-PEG that gets preparation in 5g (1 mmole) step 1 is dissolved in the 20mL exsiccant methylene dichloride, the dicyclohexylcarbodiimide (DCC) that adds 2 mmoles then successively, the γ-An Jidingsuan hydrochloride of 2 mmoles, with triethylamine the pH value of solution is adjusted to 8 then, stirring reaction spends the night, remove by filter post precipitation, add the anhydrous diethyl ether precipitated product, to be dissolved in deionized water after the product drying, the sodium hydroxide solution that adds 0.1mol/L is regulated pH 11, with oxalic acid pH value of solution is adjusted to 3 then, with chloroform extraction solution 3 times, the combining extraction liquid anhydrous sodium sulfate drying obtains the mPEG-CH that molecular weight is 12kD ²CONH-CH ₂CH ₂CH ₂COOH, i.e. the PEG butyric acid that is connected with PEG with amido linkage, IR spectrum: 1629cm ^-1(CONH), 1740cm ^-1(COOH)

Step 4:

The molecular weight of getting step 3 preparation is the PEG-acyl γ-An Jidingsuan 6g (0.5 mmole) of 12kD, be dissolved in the dry purified anhydrous methylene chloride, add the dicyclohexylcarbodiimide (DCC) of 2 times of molar weights and the nitrogen N-Hydroxysuccinimide of 2 times of molar weights, stirring at room reaction 24h, stopped reaction filters out precipitation (DCU) dicyclohexylurea (DCU), and solution obtains the nitrogen hydroxysuccinimide eater that molecular weight of product is PEG-acyl-γ-An Jidingsuan of 12kD with the anhydrous diethyl ether precipitation.Molecular formula is as follows:

CH ₃O-(CH ₂CH ₂O)n-CH ₂CONH-CH ₂CH ₂CH ₂COONHS

IR spectrum: 1629cm ^-1(CONH), 1729cm ^-1(COONHS)

Embodiment 3

Synthetic molecular weight is the mPEG-CH of 20kD ₂CONH-(CH ₂) ₅COOH

Step 1:

The preparation of CM-PEG (carboxymethylation polyoxyethylene glycol):

With molecular weight is that the mono methoxy polyethylene glycol 20g (1 mmole) of 20kD is dissolved in an amount of toluene, distill out a part of toluene with azeotropic water removing, the potassium tert.-butoxide back flow reaction 2h that adds 3 mmoles then, the ethyl bromoacetate back flow reaction 4h that then slowly adds 1.5 mmoles, room temperature reaction 24h then, remove by filter post precipitation pressure reducing and steaming solvent, residue adds a small amount of methylene dichloride dissolving, be settled out product with dry ether, this product is dissolved in the deionized water, the pH value stabilization of sodium hydroxide solution until solution that adds 0.1mol/L gradually is at pH11, use the pH to 3 of the hydrochloric acid conditioning solution of 0.1mol/L then, the chloroform extraction solution of usefulness equivalent three times merges organic phase and uses anhydrous sodium sulfate drying, concentrate the back and obtain the CM-PEG that molecular weight is 20kD with ether sedimentation, molecular formula is as follows: CH ₃O-(CH ₂CH ₂O) n-CH ₂COOH

Step 2:

The preparation of epsilon-amino caproic acid hydrochloride:

Epsilon-amino caproic acid is partly joined in the 300mL dehydrated alcohol, under agitation feed the exsiccant hydrogen chloride gas up to saturated, add dehydrated alcohol then and carry out underpressure distillation, unnecessary hydrogenchloride is removed, then with solution concentration to suitable volume, obtain the epsilon-amino caproic acid carbethoxy hydrochloride with dry anhydrous ether sedimentation.

Step 3:

Synthetic mPEG-CH ₂CONH-CH ₂CH ₂COOH:

The CM-PEG that gets preparation in 10g (0.5 mmole) step 1 is dissolved in the 20mL exsiccant methylene dichloride, the dicyclohexylcarbodiimide (DCC) that adds 1 mmole then successively, the epsilon-amino caproic acid hydrochloride of 1 mmole, with triethylamine the pH value of solution is adjusted to 8 then, stirring reaction spends the night, remove by filter post precipitation, add the anhydrous diethyl ether precipitated product, to be dissolved in deionized water after the product drying, the sodium hydroxide solution that adds 0.1mol/L is regulated pH 10, with oxalic acid pH value of solution is adjusted to 3 then, with chloroform extraction solution 3 times, combining extraction liquid anhydrous sodium sulfate drying, obtain molecular weight and be 5000 mPEG-CH ₂CONH-(CH2) ₅COOH, i.e. the PEG caproic acid that is connected with PEG with amido linkage.IR spectrum: 1629cm ^-1(CONH), 1740cm ^-1(COOH)

Step 4

The molecular weight of getting step 3 preparation is the PEG-acyl gamma-amino caproic acid 10g of 20kD, be dissolved in the dry purified anhydrous methylene chloride, add the dicyclohexylcarbodiimide (DCC) of 2 times of molar weights and the nitrogen N-Hydroxysuccinimide of 2 times of molar weights, stirring at room reaction 24h, stopped reaction filters out precipitation (DCU) dicyclohexylurea (DCU), and solution obtains the nitrogen hydroxysuccinimide eater that molecular weight of product is the PEG-acyl gamma-amino caproic acid of 20kD with the anhydrous diethyl ether precipitation.Molecular formula is as follows:

CH ₃O-(CH ₂CH ₂O)n-CH ₂CONH-(CH ₂) ₅COONHS

IR spectrum: 1629cm ^-1(CONH), 1729cm ^-1(COONHS)

Embodiment 4

The hydrolysis rate of PEG active ester

In order to determine the correlated response activity of different PEG-NHS active ester, monitor that by UV Spectroscopy the formation speed of formation HO-NHS under 290nm is measured.Under typical experiment condition, the PEG-NHS ester of 7～10mg is dissolved in the 0.1mol/L phosphoric acid buffer of pH8 of 3.0mL, absorption value track record under the 290nm, Log value with the absorption value of time 0min～t min was mapped to the time, the collinear slope that forms is exactly the first order rate constant of hydrolysis reaction, this rate constant is exactly the half life of hydrolysis divided by 0.693, and all rate determinations all carry out twice, the mapping of averaging.Table 1 is depicted as the hydrolysis half life of a series of different PEG-NHS, comprises the half life of new synthetic and more known compounds.

Table 1 hydrolysis half life pH8,25 ℃

PEG-NHS ester half life

PEG-O-(CH2)3COONHS 23.3min

PEG-O-(CH2)2COONHS 16.5min

PEG-CONH-(CH2)2-COONHS 9.1min

PEG-CONH-(CH2)3-COONHS 17.4min

PEG-CONH-(CH2)5-COONHS 29.8min

Embodiment 5

The nitrogen hydroxysuccinimide eater of PEG-acyl-γ-An Jidingsuan of 12kD is connected with proteinic

Interferon, rabbit INF-α-2b is mixed with the solution of 1mg/mL with the phosphoric acid buffer of pH6.0, get the test tube of 8 2mL, every pipe adds the interferon solution that 1mL prepares, and the molecular weight that add different amounts are that the nitrogen hydroxysuccinimide eater of the PEG-γ-An Jidingsuan of 12kD makes that the mol ratio of Interferon, rabbit and Acibenzolar was respectively 1: 11: 0.5 in 1～8 pipe, 1: 3,1: 5,1: 7,1: 10,1: 20,1: 0.Concussion reaction 0.5h, with the glycine termination reaction of 2M, every pipe takes a morsel and does the SDS electrophoresis, and electrophoresis result is seen accompanying drawing 1.Among the figure:

The Interferon, rabbit of 9-----unmodified (molecular weight 19000)

The Interferon, rabbit (molecular weight 31000) that 10----is mono-modified

The two Interferon, rabbit of modifying (molecular weight 43000) of 11----

The two Interferon, rabbit of modifying (molecular weight 43000) of 12-----

To be respectively the nitrogen hydroxysuccinimide eater mol ratio of Interferon, rabbit and PEG-γ-An Jidingsuan be 1: 0.5 to swimming lane 1～7 among the figure, and 1: 1,1: 3,1: 5,1: 7,1: 10,1: 20 modification sample, swimming lane 8 were Interferon, rabbit in the same old way.As can be seen, along with the increase of active ester, the modified protein quality increases, and begins to occur two modified outcomes when mol ratio reaches 1: 7, and when mol ratio further increases (1: 10,1: 20), begins to occur the bands of modifying more.

The reaction solution of each test tube is passed through the S-200 gel separation, and mono-modified the carry sample that obtains measured the variation of the interferon biological activity before and after modifying through the vlSv-wbh cell system.Mono-modified activity is 6.39 * 10 ⁷(IU/mg), the ratio work of unmodified protein is 1.73 * 10 ⁸(IU/mg), therefore, the activity of mono-modified Interferon Alpha-2b is left 36.9%.

Claims

1. a kind of polyglycolic acid or the active ester that terminal links ω-amino acid, it is characterized in that, general structural formula is as follows:

CH ₃ -(OCH ₂ CH ₂ )n-CONH-(CH ₂ )m-CO ₂ -Q

Where n=113-680, m=2, 3, 5; Q represents one of hydrogen, tert-butyl, N-O succinyl or N-S succinyl, n=113-680.

2. the active acid of polyethylene glycol or the ester of terminal link ω-amino acid according to claim 1, it is characterized in that, said acid or ester is:

_CH3O- ( _CH2CH2O ) _n-CH2CONH _- _CH2CH2COOH _,

_CH3O- ( _CH2CH2O ) _n _- _CH2CONH - _{CH2CH2CH2COOH} _,

CH ₃ O-(CH ₂ CH ₂ O)n-CH ₂ CONH-(CH ₂ ) ₅ COOH,

CH ₃ O-(CH ₂ CH ₂ O)n-CH ₂ CONH-CH ₂ CH ₂ COONHS,

CH ₃ O-(CH ₂ CH ₂ O)n-CH ₂ CONH-CH ₂ CH ₂ CH ₂ COONHS or

CH ₃ O—(CH ₂ CH ₂ O)n—CH ₂ CONH—(CH ₂ ) ₅ COONHS.

NHS here stands for nitrogen hydroxysuccinimide.

3. according to claim 1 and 2, the preparation method of the active acid of polyethylene glycol or the ester that the end connects omega-amino acid comprises the steps:

(1) Dissolve monomethoxypolyethylene glycol in toluene, add potassium tert-butoxide to reflux for 1-2 hours, add ethyl bromoacetate to reflux for 2-4 hours, then react at room temperature for 18-24 hours, concentrate and diethyl ether Precipitation, alkali hydrolysis and acidification of the precipitate to obtain CM-PEG;

(2) Hydrogen chloride gas is passed into the ethanol containing ω-amino acid until saturated, and then the ω-amino acid ethyl ester hydrochloride is collected;

(3) Dissolve the CM-PEG prepared in step 1 in dichloromethane, then add dicyclohexylcarbodiimide, ω-amino acid ethyl ester hydrochloride successively, and then adjust the pH value of the solution to 8 to 9, react for 8 to 12 hours, filter, add ether to the filtrate, collect the precipitated product, dry the product and dissolve it in water, add sodium hydroxide solution to adjust the pH to 10 to 12, react for 1 to 3 hours, and then dilute the solution with oxalic acid The pH is adjusted to 2-4, and the product is collected to obtain polyethylene glycol active acid whose terminal is connected with ω-amino acid.

(4) Dissolve the product of step (3) in dichloromethane, add 1.5 to 3 times the molar amount of dicyclohexylcarbodiimide and 1.5 to 3 times the molar amount of nitrogen hydroxysuccinimide or thiohydroxysuccinimide Imine, stirred and reacted at 10-30°C for 20-28 hours, and the product was collected to obtain polyethylene glycol active ester with ω-amino acid connected at the end.

4. The method according to claim 3, wherein said ω-amino acid is selected from β-alanine, γ-aminobutyric acid or ε-aminocaproic acid.

5. The method according to claim 3 or 4, characterized in that the molecular weight of monomethoxypolyethylene glycol is 5000-30000.

6. The application of polyethylene glycol active acid or ester with ω-amino acid connected at the end according to claim 1 or 2, characterized in that it is used to link with protein or enzyme to prepare protein or enzyme with PEG modification.

7. A modification of polyethylene glycol active acid or ester and biologically active substance whose end is connected to an omega-amino acid according to claim 1 or 2, wherein the general structural formula is as follows:

CH ₃ -(OCH ₂ CH ₂ )n-CONH-(CH ₂ )m-CONH-Pro

Wherein: the NH-Pro part represents the amino site of the bioactive substance.

8. The modification according to claim 7, characterized in that the biologically active substance is selected from proteins, enzymes, polypeptides, drugs, dyes, nucleosides, esters or liposomes.