CN102964587A - Preparation method and application of acid or active ester of polyethylene glycol with tail end connected with proline - Google Patents

Abstract

R₃可以是H－，也可以是－N－丁二酰亚胺基。The invention discloses a preparation method and application of polyethylene glycol acid or active ester whose end is connected with proline, and the invention is applied in the field of medicine. The polymer active ester has a hydrolysis half-life of 10.9 minutes in water, and has an ideal activity of reacting with amino groups of biologically active substances. Its structural formula is as follows: R ₁ -(OCH ₂ CH ₂ ) _n -OR ₂ where n=43~680; R ₁ can be CH ₃ - or R ₂ The structure of R ₂ is as follows:

R ₃ may be H- or -N-succinimide.

Description

Preparation method and application of acid or active ester of polyethylene glycol connected to proline at the end

technical field

The invention relates to a polyethylene glycol water-soluble polymer and a preparation method and application thereof, in particular to a polyethylene glycol active ester and a preparation method and application thereof.

Background technique

The use of chemical methods to bind the water-soluble polymer polyethylene glycol (PEG) to molecules and surfaces has significant applications in biotechnology. PEG has a wide range of applications in the field of biotechnology: some active derivatives of PEG are bound to proteins and enzyme molecules, that is, enzymes are modified. Since PEG can be dissolved in organic solvents, PEG combined with enzyme molecules can make PEG and enzyme molecules The conjugate is soluble in organic solvents; compared with unmodified protein, linking PEG to the protein can reduce the immunogenicity of the protein, reduce the rate of renal elimination, and thereby increase the half-life of the conjugate in blood circulation; The binding of PEG to the surface can reduce the adsorption of proteins and cells and change the charged properties of the surface. The binding of PEG to liposomes can greatly increase its half-life and increase its ability as a medium for drug sustained release.

PEG can only bind to molecules or surfaces after activation. Therefore, a series of activated derivatives were prepared. At present, the widely used modifier activation type is the activated ester of carboxylic acid. This type of modifier is modified by activating the ester to acylate the free amino group of the protein, thereby binding the PEG chain to the protein molecule.

Currently, many activated carboxy-PEGs used for modification have poor reactivity. For example, the NHS ester of carboxymethylated PEG (CM-PEG) is so reactive that it hydrolyzes immediately upon dissolution in water (hydrolysis half-life of 0.75 min at pH 8, 25°C), and this high reactivity is A serious drawback of methylated PEG active esters. Although the activated SC-PEG in the first generation of modifiers has better reactivity (half-life of 20.4 min at pH 8, 25°C), due to the unstable ester bond in the molecule, it is now less use. Another example is PEG (succinimidyl succinate, SS-PEG) activated by succinic anhydride succinimide ester. The preparation of SS-PEG is through the esterification reaction between mPEG and succinic anhydride to open the ring of succinic anhydride, thereby forming a carboxyl group at the chain end of PEG, and then activating the carboxyl group into succinimide ester. The reagent still has an ester bond after it is bound to the protein, and the ester bond is very easily hydrolyzed when the PEG polymer is bound to the protein due to its inherent instability. This hydrolysis will not only detach the PEG chain from the protein and lose the benefits of PEG modification, but also the succinic acid tail left on the protein can act as a hapten to cause the immunogenicity of the protein. Harris et al. introduced propionate units at the end of mPEG, which increased the hydrolysis half-life of this propionate PEG active ester to 16.5 minutes, but this method involved the introduction of a cyano group at one end of PEG, which required further hydrolysis with strong acid and strong base. Obtained products are very likely to destroy the PEG main chain.

Currently, many activated carboxyl PEGs used for modification have poor reactivity: they either react too quickly or react too slowly. For example, the NHS ester of carboxymethylated PEG (CM-PEG) is so reactive that it hydrolyzes immediately upon dissolution in water, and this high reactivity is a serious drawback of the active ester of carboxymethylated PEG.

Contents of the invention

The technical problem to be solved in the present invention is to disclose a preparation method and application of a polyethylene glycol acid or active ester connected to proline at the end, so as to overcome the defects in the prior art and meet the development needs of the fields of medicine and bioengineering need.

The structural formula of the active acid or ester of polyethylene glycol whose end is connected to proline of the present invention is as follows:

R ₁ -(OCH ₂ CH ₂ ) _n -O-R ₂

where n=43~680;

R ₁ can be CH ₃ - or R ₂

The structure of _R2 is as follows:

R ₃ may be H- or -N-succinimide.

The active ester of polyethylene glycol linked to proline provided by the invention has suitable reactivity, the hydrolysis half-life of the active ester in water is 10.9 minutes, and does not contain other ester linkages.

The preparation method of the active ester of polyethylene glycol linked to proline of the present invention comprises the steps:

(1) Preparation of CM-PEG (carboxymethylated polyethylene glycol):

Dissolve polyethylene 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 precipitate with ether, the precipitate Alkaline hydrolysis and then acidification, using conventional methods to collect the product;

(2) Preparation of proline ethyl ester hydrochloride:

Pass hydrogen chloride gas into ethanol containing proline until saturated, then collect proline ethyl ester hydrochloride;

(3) Preparation of polyethylene glycol acid derivatives with proline connected at the end:

Take the CM-PEG prepared in step (1) and dissolve it in dichloromethane, then add dicyclohexylcarbodiimide and proline ethyl ester hydrochloride in sequence, and then adjust the pH value of the solution to 8 with triethylamine ~9, react for 8~12 hours, filter, add diethyl 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 10~12, react for 1~3 hours, and then dilute the solution with oxalic acid The pH was adjusted to 2-4, and the product was collected to obtain a polyethylene glycol acid derivative whose end was linked to proline.

(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 N-hydroxysuccinimide, 10 to 3 times the molar amount The reaction was stirred at 30°C for 20-28 hours, and the product was collected to obtain a polyethylene glycol active ester whose end was linked to proline.

Said proline is D-form, L-form or racemate;

The preferred molecular weight of polyethylene glycol is 2000～30000 Da.

The active ester of polyethylene glycol whose end is connected to proline has a hydrolysis half-life of 10.9 minutes in water, has ideal reactivity with the amino group of biologically active substances, and can be used to link with biologically active substances to prepare PEG modification Biologically active substances, media for the preparation of sustained-release drugs, etc.

The present invention also relates to a combination of said polyethylene glycol active acid or ester connected to proline at the end and a biologically active substance, the general structural formula of which is as follows:

Wherein: n is 1 or 2, and the NH-Pro part represents the amino site of the biologically active substance.

Biologically active substances are selected from proteins, enzymes, polypeptides, drugs, dyes, nucleosides, esters or liposomes.

The proline-linked polyethylene glycol active ester prepared by the above method has good reactivity and can be used for linking with protein or enzyme molecules and for preparing a medium for slow-release medicine.

Detailed ways

Example 1

Synthesis of proline active ester linked with monomethoxy PEG with a molecular weight of 5000 Da via amide bond

Step 1: Preparation of CM-PEG (Carboxymethylated Polyethylene Glycol):

Dissolve 15 g (3 mmol) of monomethoxypolyethylene glycol with a molecular weight of 5000 Da in toluene, distill off part of the toluene to remove water azeotropically, then add 4 mmol of potassium tert-butoxide to reflux for 1.5 hours, and then Slowly add 4 mmol of ethyl bromoacetate to reflux reaction for 3 hours, then react at room temperature for 21 hours, remove the precipitate by filtration, evaporate the solvent under reduced pressure, dissolve the residue with a small amount of dichloromethane, precipitate the product with dry ether, dissolve the product In deionized water, add 0.1 mol/L sodium hydroxide solution until the pH value of the solution is stable at pH 10, then adjust the pH of the solution to 3 with 0.1 mol/L hydrochloric acid, extract the solution three times with an equal amount of chloroform, and combine the organic The phases were combined and dried with anhydrous sodium sulfate, concentrated and then precipitated and dried with ether to obtain CM-PEG with a molecular weight of 5000 Da, the molecular formula is as follows: CH ₃ O-(CH ₂ CH ₂ O) _n -CH ₂ COOH.

Step 2: Preparation of proline ethyl ester hydrochloride:

Add 20 g of proline to 300 mL of absolute ethanol, pass in dry hydrogen chloride gas under stirring until saturated, then add absolute ethanol for vacuum distillation to remove excess hydrogen chloride, and then concentrate the solution to a suitable volume, and precipitated with dry anhydrous ether to obtain proline ethyl ester hydrochloride.

Step 3: Synthesis of mPEG-proline conjugate linked by amide linkage:

Take 7.5 g (1.5 mmol) of CM-PEG prepared in step (1) and dissolve in 40 mL of dry dichloromethane, then add 2 mmol of dicyclohexylcarbodiimide and 2 mmol of ethyl proline Hydrochloride, then adjust the pH value of the solution to 8 with triethylamine, stir for 12 hours, filter to remove the precipitate, add anhydrous ether to precipitate the product, dry the product and dissolve it in deionized water, add 0.1 mol/L Sodium hydroxide solution adjusted the pH to 11, reacted for 2 hours, then adjusted the pH of the solution to 3 with oxalic acid, extracted the solution three times with chloroform, combined the extracts and dried them with anhydrous sodium sulfate, concentrated the solution and then precipitated with anhydrous ether to obtain the molecular weight 5000 Da amide-linked mPEG-proline conjugate.

Step 4:

Take the mPEG-proline with a molecular weight of 5000 Da prepared in step 3, dissolve it in dry and refined anhydrous dichloromethane, add 1.2 times the molar amount of dicyclohexylcarbodiimide (DCC) and 1.2 times the molar amount of N-hydroxysuccinimide, stirred at room temperature for 24 hours, stopped the reaction and filtered off the precipitated dicyclohexyl urea (DCU), and the solution was precipitated with anhydrous ether to obtain the N-hydroxyl group of mPEG-proline with a molecular weight of 5000 Da Succinimide ester.

Example 2

Synthesis of proline active ester linked with PEG diacid with molecular weight of 20000 Da by amide bond

Step 1: Preparation of CM-PEG diacid:

Dissolve 20 g (1 mmol) of monomethoxypolyethylene glycol with a molecular weight of 20,000 Da in an appropriate amount of toluene, distill a part of the toluene to remove water azeotropically, and then add 3 mmol of potassium tert-butoxide to reflux for 2 hours , and then slowly add 3 mmol of ethyl bromoacetate to reflux reaction for 4 hours, then react at room temperature for 18 hours, filter and remove the precipitate, evaporate the solvent under reduced pressure, add dichloromethane to the residue to dissolve, and precipitate the product with dry ether. Dissolve in deionized water, gradually add 0.1 mol/L sodium hydroxide solution until the pH value of the solution is stable at pH 10, then adjust the pH of the solution to 3 with 0.1 mol/L hydrochloric acid, extract the solution three times with an equal amount of chloroform, The organic phases were combined and dried with anhydrous sodium sulfate, concentrated and then precipitated and dried with ether to obtain a CM-PEG diacid with a molecular weight of 20000 Da. The chemical formula is as follows: HOOCCH ₂ O-(CH ₂ CH ₂ O) _n -CH ₂ COOH.

Step 2: Preparation of proline ethyl ester hydrochloride:

Add 20 g of proline to 300 mL of absolute ethanol, pass in dry hydrogen chloride gas under stirring until saturated, then add absolute ethanol for vacuum distillation to remove excess hydrogen chloride, and then concentrate the solution to a suitable volume, and precipitated with dry anhydrous ether to obtain proline ethyl ester hydrochloride.

Step 3: Synthesis of PEG-diproline conjugates linked by amide bonds:

Take 10 g (0.5 mmol) of the CM-PEG diacid prepared in step (1) and dissolve it in 40 mL of dry dichloromethane, then add 2 mmol of dicyclohexylcarbodiimide (DCC), 2 mmol of Proline ethyl ester hydrochloride, then adjust the pH value of the solution to 8 with triethylamine, stir the reaction overnight, filter to remove the precipitate, add anhydrous ether to precipitate the product, dry the product and dissolve it in deionized water, add Adjust the pH to 11 with 0.1 mol/L sodium hydroxide solution, then adjust the pH of the solution to 3 with oxalic acid, extract the solution three times with chloroform, dry the combined extracts with anhydrous sodium sulfate, concentrate the solution and then precipitate with anhydrous ether to obtain the molecular weight PEG-diproline of 20000 Da.

Step 4:

Take the PEG-diproline with a molecular weight of 20000 Da prepared in step (3), dissolve it in dry and refined anhydrous dichloromethane, add 3 times the molar amount of dicyclohexylcarbodiimide (DCC) and 3 times Molar amount of N-hydroxysuccinimide, stirred at room temperature for 24 hours, stopped the reaction and filtered off the precipitated dicyclohexyl urea (DCU), and the solution was precipitated with anhydrous ether to obtain PEG-diproline with a molecular weight of 20000 Da of N-hydroxysuccinimide esters.

Claims (8)

1. terminal acid or active ester that connects the polyoxyethylene glycol of proline(Pro) is characterized in that structure is as follows:

R ₁－(OCH ₂CH ₂) _n－O－R ₂

N=43 ~ 680 wherein;

R ₁Can be CH ₃-, also can be R ₂

R ₂Structure as follows:

R ₃Can be H-, also can be-N-succimide base.

2. end according to claim 1 connects active acid or the ester of the polyoxyethylene glycol of proline(Pro), it is characterized in that said acid or ester are:

NHS represents N-maloyl imines herein.

3. the active acid of the polyoxyethylene glycol of terminal connection proline(Pro) according to claim 1 and 2 or the preparation method of ester comprise the steps:

(1) polyoxyethylene glycol is dissolved in the toluene, added the potassium tert.-butoxide back flow reaction 1 ~ 2 hour, added the ethyl bromoacetate back flow reaction 2 ~ 4 hours, then room temperature reaction is 18 ~ 24 hours, uses ether sedimentation after concentrating, and then acidifying of throw out basic hydrolysis obtains CM-PEG;

(2) hydrogen chloride gas is passed into the ethanol that contains proline(Pro) until saturated, then collect the ethyl prolinate hydrochloride;

(3) get the CM-PEG for preparing in the step (1) and be dissolved in methylene dichloride, then add successively dicyclohexylcarbodiimide, ethyl prolinate hydrochloride, then with triethylamine the pH value of solution is adjusted to 8 ~ 9, 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, reacted 1 ~ 3 hour, then with oxalic acid pH value of solution is adjusted to 2 ~ 4, collect product, obtain the terminal polyethyleneglycol derivative that connects proline(Pro);

(4) product of getting step (3) is dissolved in methylene dichloride, add the dicyclohexylcarbodiimide of 1.5 ~ 3 times of molar weights and the N-maloyl imines of 1.5 ~ 3 times of molar weights, 10 ~ 30 ℃ of stirring reactions 20 ~ 28 hours are collected product, obtain the terminal Active Ester of Polyethyen Glycol that connects proline(Pro).

4. method according to claim 3 is characterized in that, proline(Pro) can be D type, L-type or racemic modification.

5. according to claim 3 or 4 described methods, it is characterized in that described polyoxyethylene glycol can be mono methoxy polyethylene glycol, also can be two hydroxyl polyoxyethylene glycol, the molecular weight of described polyoxyethylene glycol is 2000 ~ 30000 Da.

6. terminal connect the polyethylene active acid of proline(Pro) or the application of ester according to claim 1 and 2 is characterized in that, be used for being connected with protein or enzyme, preparation has protein or the enzyme that PEG modifies.

7. a claim 1 or the 2 described terminal polyethylene active acid of proline(Pro) or the binding substancess of ester and biologically active substance of connecting is characterized in that general structure is as follows:

Wherein: n be 1 or 2, NH-Pro partly represent the amino sites of biologically active substance.

8. modifier according to claim 7 is characterized in that biologically active substance is selected from protein, enzyme, polypeptide, medicine, dyestuff, nucleosides, ester class or liposome.