CN102964587A - Preparation method and application of acid or active ester of polyethylene glycol with tail end connected with proline - Google Patents
Preparation method and application of acid or active ester of polyethylene glycol with tail end connected with proline Download PDFInfo
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- CN102964587A CN102964587A CN2012104460857A CN201210446085A CN102964587A CN 102964587 A CN102964587 A CN 102964587A CN 2012104460857 A CN2012104460857 A CN 2012104460857A CN 201210446085 A CN201210446085 A CN 201210446085A CN 102964587 A CN102964587 A CN 102964587A
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Abstract
The invention discloses a preparation method and application of acid or active ester of polyethylene glycol with the tail end connected with proline, belonging to the field of medicine. The hydrolytic half-time period of the polymer active ester in water is 10.9 minutes and has the ideal activity of reacting with the amino of bioactive substances. The structural formula is R1-(OCH2CH2)n-O-R2, wherein n is equal to 43-680; the R1 can be CH3- or R2; the structure of the R2 is shown in the specification; and the R3 can be H- or -N-succinimide.
Description
Technical field
The present invention relates to polyoxyethylene glycol water-soluble polymers and its preparation method and application, relate in particular to Active Ester of Polyethyen Glycol and its preparation method and application.
Background technology
Use chemical process that water-soluble polymers polyoxyethylene glycol (Polyethylene Glycol, PEG) is attached to molecule and in biotechnology, have great application value with the surface.PEG has a wide range of applications at biological technical field: the reactive derivative of some PEG is attached on the molecule of protein, enzyme, namely enzyme is modified, because PEG can be dissolved in organic solvent, PEG is attached to the enzyme molecule can make the binding substances of PEG and enzyme be dissolved in organic solvent; Compare with the protein of unmodified, PEG is connected to immunogenicity, the reduction kidney that can reduce albumen on the protein gets rid of speed, and can therefrom impel the binding substances increase of the transformation period 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 transformation period that very big increase is arranged on the liposome, and can increase it as the ability of the medium of medicament slow release.
PEG only has through overactivation and can be attached on molecule or the surface.Therefore, a series of activated derivatives is produced out.Using at present wider modifier activation type is the Acibenzolar class of carboxylic acid.Such modifier is by Acibenzolar the free amino group of albumen to be carried out acidylate, realizes on the protein molecule modifying thereby the PEG link is incorporated into.
At present, the carboxyl PEG of many activation for modifying has relatively poor reactive behavior.The reactive behavior of NHS ester for example, carboxymethylation PEG(CM-PEG) is too strong, so that be dissolved at once hydrolysis in the water (hydrolysising half-life under pH8,25 ° of C is 0.75 min), this high reaction activity is the major defect of carboxymethylation PEG active ester.Although and the SC-PEG that activates in the first-generation modifier has reactive behavior preferably (transformation period under pH8,25 ° of C is 20.4 min), owing to contain unsettled ester bond at molecule, now less use.And for example PEG(succinimidyl succinate, the SS-PEG of Succinic anhydried succimide ester activation).The preparation of SS-PEG is by mPEG and Succinic anhydried generation esterification, makes the Succinic anhydried open loop, thereby causes a carboxyl at the end of the chain of PEG, then again activated carboxylic is become the succimide ester.This reagent with protein bound after also have an ester bond, and ester bond since its inherent instability when the PEG polymer is attached on the protein, be very easy to be hydrolyzed.This hydrolysis not only can make the PEG chain come off from albumen, and forfeiture PEG modifies the benefit of bringing, and the Succinic Acid afterbody of staying on the protein also can be used as the immunogenicity that haptens causes protein.The people such as Harris introduce propionic acid unit at the end of mPEG, so that the hydrolysising half-life of this propionic acid PEG active ester is increased to 16.5 minutes, introduce cyano group but the method has related at PEG one end, the hydrolysis that need to carry out again strong acid and highly basic just can obtain product, very likely destroys the PEG main chain.
At present, the carboxyl PEG of many activation for modifying has very poor reactive behavior: be not that to react be exactly to react too slow too soon.The reactive behavior of NHS ester for example, carboxymethylation PEG(CM-PEG) is too strong, so that be dissolved at once hydrolysis 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 acid of the terminal polyoxyethylene glycol that connects proline(Pro) or method for making and the application of active ester, and the defective that exists to overcome prior art satisfies the needs of field of medicaments and bioengineering field development.
The active acid of the polyoxyethylene glycol of terminal connection proline(Pro) of the present invention or the structural formula of ester are 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.
The active ester of the polyoxyethylene glycol of connection proline(Pro) provided by the invention has suitable reactive behavior, and the hydrolysising half-life of active ester in water is 10.9 minutes, and does not contain other ester bond connection.
The preparation method of the active ester of the polyoxyethylene glycol of connection proline(Pro) of the present invention comprises the steps:
(1) preparation CM-PEG(carboxymethylation polyoxyethylene glycol):
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, use ether sedimentation after concentrated, then acidifying of throw out basic hydrolysis adopts conventional method to collect product;
(2) preparation of ethyl prolinate hydrochloride:
Hydrogen chloride gas passed into the ethanol that contains proline(Pro) until saturated, then collect the ethyl prolinate hydrochloride;
(3) the terminal preparation that connects the polyoxyethylene glycol acid derivative of proline(Pro):
The CM-PEG that gets preparation in the step (1) is dissolved in methylene dichloride, then adds 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, 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 polyoxyethylene glycol acid 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).
Said proline(Pro) is D type, L-type or racemic modification;
The preferred molecular weight of polyoxyethylene glycol is 2000 ~ 30000 Da.
The terminal active ester that connects the polyoxyethylene glycol of proline(Pro) of the present invention, in water, have 10.9 minutes hydrolysising half-life, have desirable and reactive behavior biologically active substance amino, can be used for being connected with biologically active substance, the biologically active substance that preparation PEG modifies is for the preparation of the medium of slow releasing pharmaceutical etc.
The invention still further relates to a kind of said terminal polyethylene active acid of proline(Pro) or binding substances of ester and biologically active substance of connecting, general structure is as follows:
Wherein: n be 1 or 2, NH-Pro partly represent 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 end of aforesaid method preparation to connect the Active Ester of Polyethyen Glycol of proline(Pro), have good reactive behavior, can be used for being connected with the molecule of protein or enzyme, and for the preparation of the medium of slow releasing pharmaceutical.
Embodiment
Embodiment 1
Be the active ester synthesis of proline(Pro) that the mono methoxy PEG of 5000 Da is connected with amido linkage with molecular weight
Step 1:CM-PEG(carboxymethylation polyoxyethylene glycol) preparation:
Be the mono methoxy polyethylene glycol 15 g(3 mmol of 5000 Da with molecular weight) be dissolved in the toluene, distill out a part of toluene with azeotropic water removing, then the potassium tert.-butoxide back flow reaction 1.5 hours that adds 4 mmol, the ethyl bromoacetate back flow reaction 3 hours that then slowly adds 4 mmol, then room temperature reaction is 21 hours, remove by filter the rear pressure reducing and steaming solvent of precipitation, residue adds a small amount of methylene dichloride dissolving, be settled out product with dry ether, this product is dissolved in the deionized water, add 0.1 mol/L sodium hydroxide solution until the pH value stabilization of solution at pH10, then use the pH to 3 of the hydrochloric acid conditioning solution of 0.1 mol/L, the chloroform extraction solution of usefulness equivalent three times merges organic phase and uses anhydrous sodium sulfate drying, obtain the CM-PEG that molecular weight is 5000 Da with the ether sedimentation drying after concentrated, molecular formula is as follows: CH
3O-(CH
2CH
2O)
n-CH
2COOH.
Step 2: the preparation of ethyl prolinate hydrochloride:
20 g proline(Pro) are joined in the 300 mL dehydrated alcohols, under agitation pass into dry hydrogen chloride gas until saturated, then add dehydrated alcohol and carry out underpressure distillation, unnecessary hydrogenchloride is removed, then solution is concentrated into suitable volume, obtains the ethyl prolinate hydrochloride with dry anhydrous ether sedimentation.
Step 3: the synthetic mPEG-proline(Pro) conjugate that connects with amido linkage:
Getting 7.5 g(1.5 mmol) CM-PEG of preparation in the step (1) is dissolved in the methylene dichloride of 40 mL dryings, then the dicyclohexylcarbodiimide that adds successively 2 mmol, the ethyl prolinate hydrochloride of 2 mmol, then with triethylamine the pH value of solution is adjusted to 8, stirring reaction 12 hours, after removing by filter precipitation, add the anhydrous diethyl ether precipitated product, to be dissolved in deionized water after the product drying, add 0.1 mol/L sodium hydroxide solution and regulate pH to 11, reacted 2 hours, then with oxalic acid pH value of solution is adjusted to 3, with chloroform extraction solution three times, the combining extraction liquid anhydrous sodium sulfate drying obtains the mPEG-proline(Pro) conjugate with the amido linkage connection that molecular weight is 5000 Da with the anhydrous diethyl ether precipitation after solution is concentrated again.
Step 4:
The molecular weight of getting step 3 preparation is the mPEG-proline(Pro) of 5000 Da, be dissolved in the dry refining anhydrous methylene chloride, add the dicyclohexylcarbodiimide (DCC) of 1.2 times of molar weights and the N-maloyl imines of 1.2 times of molar weights, stirring at room reaction 24 hours, stopped reaction filtering precipitated dicyclohexylurea (DCU), solution obtains the N-maloyl imines ester that molecular weight of product is the mPEG-proline(Pro) of 5000 Da with the anhydrous diethyl ether precipitation.
Embodiment 2
Be the active ester synthesis of proline(Pro) that the PEG diacid of 20000 Da is connected with amido linkage with molecular weight
The preparation of step 1:CM-PEG diacid:
Be the mono methoxy polyethylene glycol 20 g(1 mmol of 20000 Da with molecular weight) be dissolved in an amount of toluene, distill out a part of toluene with azeotropic water removing, then the potassium tert.-butoxide back flow reaction 2 hours that adds 3 mmol, the ethyl bromoacetate back flow reaction 4 hours that then slowly adds 3 mmol, then room temperature reaction is 18 hours, remove by filter the rear pressure reducing and steaming solvent of precipitation, residue adds the methylene dichloride dissolving, be settled out product with dry ether, this product is dissolved in the deionized water, add gradually 0.1 mol/L sodium hydroxide solution until the pH value stabilization of solution at pH10, then use the pH to 3 of the hydrochloric acid conditioning solution of 0.1 mol/L, with the chloroform extraction solution of equivalent three times, merge organic phase and use anhydrous sodium sulfate drying, obtain the CM-PEG diacid that molecular weight is 20000 Da with the ether sedimentation drying after concentrated, chemical formula is as follows: HOOCCH
2O-(CH
2CH
2O)
n-CH
2COOH.
Step 2: the preparation of ethyl prolinate hydrochloride:
20 g proline(Pro) are joined in the 300 mL dehydrated alcohols, under agitation pass into dry hydrogen chloride gas until saturated, then add dehydrated alcohol and carry out underpressure distillation, unnecessary hydrogenchloride is removed, then solution is concentrated into suitable volume, obtains the ethyl prolinate hydrochloride with dry anhydrous ether sedimentation.
Step 3: the synthetic PEG-two proline(Pro) conjugates that connect with amido linkage:
Get 10 g(0.5 mmol) the CM-PEG diacid of preparation in the step (1) is dissolved in the methylene dichloride of 40 mL dryings, then the dicyclohexylcarbodiimide (DCC) that adds successively 2 mmol, the ethyl prolinate hydrochloride of 2 mmol, then with triethylamine the pH value of solution is adjusted to 8, stirring reaction spends the night, after removing by filter precipitation, add the anhydrous diethyl ether precipitated product, to be dissolved in deionized water after the product drying, add 0.1 mol/L sodium hydroxide solution and regulate pH to 11, then with oxalic acid pH value of solution is adjusted to 3, with chloroform extraction solution three times, the combining extraction liquid anhydrous sodium sulfate drying obtains PEG-two proline(Pro) that molecular weight is 20000 Da with the anhydrous diethyl ether precipitation after solution is concentrated again.
Step 4:
The molecular weight of getting step (3) preparation is PEG-two proline(Pro) of 20000 Da, be dissolved in the dry refining anhydrous methylene chloride, add the dicyclohexylcarbodiimide (DCC) of 3 times of molar weights and the N-maloyl imines of 3 times of molar weights, stirring at room reaction 24 hours, stopped reaction filtering precipitated dicyclohexylurea (DCU), solution obtains the N-maloyl imines ester that molecular weight of product is PEG-two proline(Pro) of 20000 Da with the anhydrous diethyl ether precipitation.
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.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5965566A (en) * | 1993-10-20 | 1999-10-12 | Enzon, Inc. | High molecular weight polymer-based prodrugs |
| CN1660920A (en) * | 2005-02-01 | 2005-08-31 | 华东理工大学 | Polyethyleneglycol acid or active ester connected to omega amino acid at their ends, preparation method and application |
| CN101870769A (en) * | 2010-06-13 | 2010-10-27 | 河北科技大学 | PEG (Polyethylene Glycol), mPEG (Methoxy Polyethylene Glycol) chemical modifier and method thereof for preparing water-soluble resveratrol prodrug |
-
2012
- 2012-11-09 CN CN2012104460857A patent/CN102964587A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5965566A (en) * | 1993-10-20 | 1999-10-12 | Enzon, Inc. | High molecular weight polymer-based prodrugs |
| CN1660920A (en) * | 2005-02-01 | 2005-08-31 | 华东理工大学 | Polyethyleneglycol acid or active ester connected to omega amino acid at their ends, preparation method and application |
| CN101870769A (en) * | 2010-06-13 | 2010-10-27 | 河北科技大学 | PEG (Polyethylene Glycol), mPEG (Methoxy Polyethylene Glycol) chemical modifier and method thereof for preparing water-soluble resveratrol prodrug |
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Application publication date: 20130313 |