CN1629193A - Protein and polypeptide guanidine polyethyleneglycol reagent and its preparation process and use - Google Patents
Protein and polypeptide guanidine polyethyleneglycol reagent and its preparation process and use Download PDFInfo
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- CN1629193A CN1629193A CN 200410054144 CN200410054144A CN1629193A CN 1629193 A CN1629193 A CN 1629193A CN 200410054144 CN200410054144 CN 200410054144 CN 200410054144 A CN200410054144 A CN 200410054144A CN 1629193 A CN1629193 A CN 1629193A
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Abstract
The invention discloses a protein and polypeptide guanidine polyethyleneglycol reagent and its preparation process and use, wherein the preparation comprises, subjecting mPEG, potassium tert butoxide and bromized acetone for reaction in solvent at room temperature for 10-24 hours, obtaining mPEG acetone, subjecting the obtained mPEG acetone and acetic anhydride for reaction 6-10 hours at the presence of catalyst with boron trifluoride, then collecting mPEG substituted acetylacetone from the reaction yield.
Description
Technical field
The present invention relates to the Pegylation reagents and its production and use that a kind of new protein and polypeptide guanidine radicals or other contain the compound of guanidine radicals.
Background technology
Along with the fast development of biotechnology, the polypeptide of more and more biologically actives, protein, enzyme is used for clinical treatment by people.Yet these biomacromolecules exist some defectives when directly applying to human body.These defectives comprise: 1. get rid of fast, 2. biological degradation 3. causes immune response or the like.Therefore, protein particularly the chemically modified of medical protein remain the research field of a hot topic so far.Many natural and synthetic molecules are used as chemically modified.
Polyoxyethylene glycol, this synthetic molecule can reduce by the advantages such as antigenicity of modifier being widely used in protein, polypeptide, the modification of enzyme even some small-molecule drugs because its biocompatibility is nontoxic to human body.
Polyoxyethylene glycol (PEG) is a kind of water miscible polymkeric substance.When with protein (also having polypeptide, enzyme and other biological bioactive molecule) covalent attachment, polyoxyethylene glycol can remedy the deficiency of these molecules itself, enlarges its potential and uses.Compare with the contrast of unmodified, the pharmacological property that the PEG-protein conjugate is this have been improved has excited people to develop the enthusiasm of this binding substances as preparation.For example, the PEG-adenosine deaminase has obtained the approval of FDA; The cytokine that PEG modifies is made into, and the GM-CSF (macrophage colony stimulating factor) that PEG modifies has shown two kinds of unconnected biological properties.
The mono methoxy polyethylene glycol mPEG-OH that is used to modify is a kind of catenate polyether compound, forms the purpose that covalent attachment reaches modification by terminal hydroxyl is changed with proteinic amino acid side chain functional group when modifying protein.Owing to have a large amount of side chain amino in protein and the polypeptide, therefore the many terminal hydroxyls mPEG of people are changed into the chemical structure that is fit to modify amino up to now, such modification result often causes uncertain modified outcome, be difficult to reach medicinal purpose, this also is a reason of having only six kinds of successful modified medicaments so far.For this reason, people begin to make great efforts to develop the PEG reagent that can modify other side-chain radical, up to the present, have developed in the modifying protein reagent of thioether group in the PEG reagent of sulfydryl, the modifying protein.Yet, the present PEG reagent that does not also have arginine guanidine radicals in the modifying protein.
Summary of the invention
The technical issues that need to address of the present invention are Pegylation reagents that disclose a kind of protein and polypeptide guanidine radicals and its production and use, to satisfy the needs of relevant field development.
The contriver finds that through investigating arginic guanidine radicals can be with 1, and following reaction takes place under the condition of gentleness the 3-dicarbonyl compound, generates stable six-membered ring structure:
Therefore, if introduce 1 in mPEG-OH, the 3-dicarbonyl structure can obtain a kind of PEG reagent of novel guanidine radicals in can the modifying protein polypeptide.
The Pegylation reagents of protein of the present invention and polypeptide guanidine radicals is the single substitution product of mPEG to methyl ethyl diketone, and general structure is as follows:
Wherein, m=CH
3-,
n＝33～330；
The Pegylation reagents of protein of the present invention and polypeptide guanidine radicals can characterize by infared spectrum.
The preparation method of the Pegylation reagents of protein of the present invention and polypeptide guanidine radicals comprises the steps:
A route of the present invention's design, high productivity synthesizes a kind of novel protein and peptide guanidine radicals PEGization reagent easily, and step is as follows:
(1) with mPEG, potassium tert.-butoxide and bromo acetone room temperature reaction 10～24 hours in solvent, obtains mPEG acetone;
Said bromo acetone can adopt the commercially available prod, or adopts following method to be prepared:
The preparation of bromo acetone: quantitative bromine slowly splashed into contain in a small amount of vitriolic excess acetone, need high degree of agitation when splashing into.With alkali reaction solution is transferred to pH=6.5～7.5 after bromine drips off, distill away excessive propanone, obtain bromo acetone, its chemical structural formula is as follows:
BrCH
2COCH
3
The molecular weight of preferred mPEG is 2000～20000;
Said solvent is selected from a kind of in toluene, benzene, the dioxane, and the consumption of solvent is also non-key, as long as can dissolve mPEG;
The molar ratio of each raw material is:
MPEG: potassium tert.-butoxide=1: 1～2;
MPEG: bromo acetone=1: 2～5;
(2) with the mPEG acetone that obtained in solvent with acetic anhydride in the presence of the catalyzer boron trifluoride, the methyl ethyl diketone that end product mPEG replaces is collected in 50～80 ℃ of reactions 6～10 hours then from reaction product, overall yield is more than 90%.
Said solvent is selected from a kind of in dioxane, benzene, the toluene;
The molar ratio of each raw material is:
MPEG acetone: acetic anhydride=1: 2～5;
MPEG acetone: boron trifluoride=1: 1～5;
The methyl ethyl diketone that adopts the prepared mPEG of method of the present invention to replace can directly be used in the PEGization of protein, polypeptide, amino acid guanidine radicals, and can be used for comprising the PEGization of other compound of guanidine radicals.
By above-mentioned disclosed technical scheme as seen, the present invention has very significant advantage, and synthetic route is reasonable in design; 2. cheap 3. overall yields of agents useful for same are more than 90%.
Description of drawings
Fig. 1 is the infared spectrum of intermediate product mPEG-CH2-CO-CH3.
Fig. 2 is the infared spectrum of end product mPEG-CH2-CO-CH2-CO-CH3.
Fig. 3 is the electrophorogram (SDS-PAGE) of the methyl ethyl diketone modified interferon alpha-2b of use end product mPEG replacement
Fig. 4 is the electrophorogram (SDS-PAGE) of the methyl ethyl diketone modified interleukin-2 of use end product mPEG replacement.
Embodiment
Be the preparation method of the methyl ethyl diketone that replaces of the mPEG of starting raw material with mPEG2000, its preparation process is:
1) preparation of bromo acetone: 2 milliliters of bromines are slowly splashed into contain in 150 milliliters of acetone of a small amount of vitriolic, need high degree of agitation when splashing into.With saturated sodium bicarbonate solution reaction solution is transferred to pH7 after bromine drips off,, obtain a bromo acetone with distilling away excessive propanone behind the anhydrous sodium sulfate drying.
2) getting mPEG2000 (Fluka) 10 grams (5mmol) is dissolved in 150 milliliters of toluene, distill out 50 milliliters of toluene to remove moisture, cool the temperature to 60 ℃ of potassium tert.-butoxide reactions that add 7.5 mmoles add preparation in 1 after 1 hour bromo acetone 7.5 mmole room temperature reactions 10～24 hours.With the ether sedimentation of reaction solution with 10 times, will precipitate again with 100 milliliters of methylene dichloride dissolvings, add proper amount of active carbon and diatomite filtration, filtrate is added excessive ether (10 times) precipitation, obtain the mPEG-acetone of 8.5 gram white powder.Its infared spectrum is seen accompanying drawing 1, is the characteristic absorbance of this product at 1600～1800cm-1 in the accompanying drawing 1.
3) will go up the mPEG acetone that obtains of step 4 grams is dissolved in dry 50 milliliters of dioxane crossing, add 4 mmole acetic anhydrides, adding the appropriate amount of catalysts boron trifluoride, after 6～10 hours temperature of reaction system is reduced to room temperature in 50～80 degree reactions, obtain the methyl ethyl diketone that molecular weight is 2000 end product mPEG replacement with excessive ether sedimentation.Its infared spectrum is seen accompanying drawing 2, is the characteristic absorbance of this product at 1600～1800cm-1 in the accompanying drawing 2.
Embodiment 2
Be the preparation method of the methyl ethyl diketone that replaces of the mPEG of starting raw material with mPEG5000, its preparation process is:
1) preparation of bromo acetone: 2 milliliters of bromines are slowly splashed into contain in 150 milliliters of acetone of a small amount of vitriolic, need high degree of agitation when splashing into.With saturated sodium bicarbonate solution reaction solution is transferred to pH7 after bromine drips off,, obtain a bromo acetone with distilling away excessive propanone behind the anhydrous sodium sulfate drying.
2) get mPEG5000 (Fluka) 10 grams (2mmol) and be dissolved in 150 milliliters of toluene, distill out 50 milliliters of toluene, cool the temperature to 60 to go out moisture.The potassium tert.-butoxide reaction that C adds 3 mmoles adds the bromo acetone 3 mmole room temperature reactions 10～24 hours of preparation in 1 after 1 hour.With the ether sedimentation of reaction solution with 10 times, will precipitate again with 100 milliliters of methylene dichloride dissolvings, add proper amount of active carbon and diatomite filtration, filtrate is added excessive ether (10 times) precipitation, obtain the mPEG-acetone of 8.9 gram white powder.Its infared spectrum is seen accompanying drawing 1, is the characteristic absorbance of this product at 1600～1800cm-1 in the accompanying drawing 1.Measuring productive rate through carbonyl is 99%.
3) will go up the mPEG acetone that obtains of step 5 grams is dissolved in dry 50 milliliters of dioxane crossing, add 2 mmole acetic anhydrides, add the appropriate amount of catalysts boron trifluoride again, after 6～10 hours temperature of reaction system is reduced to room temperature in 50～80 degree reactions, obtain the methyl ethyl diketone that molecular weight is 5000 end product mPEG replacement with excessive ether sedimentation.Its infared spectrum is seen accompanying drawing 2, is the characteristic absorbance of this product at 1600～1800cm-1 in the accompanying drawing 2.Measuring productive rate through carbonyl is 90%
The modification of the methyl ethyl diketone (guanidine radicals PEGization reagent) that mPEG replaces is used
The methyl ethyl diketone that Interferon, rabbit alpha-2b and new synthetic reagent mPEG replace reacts under the room temperature at pH8.0.The SDS-PAGE electrophoretogram of product (accompanying drawing 3) shows have one or more to modify band.In the accompanying drawing 3, M is a molecular weight standard, and 1～3 for modifying sample.As can be seen, under different modification conditions, the methyl ethyl diketone that guanidine radicals PEGization reagent mPEG replaces presents mono-modified to Interferon, rabbit alpha-2b and the band of modifying more.
Embodiment 4
The modification of the methyl ethyl diketone (guanidine radicals PEGization reagent) that mPEG replaces is used
The methyl ethyl diketone that interleukin-2 and new synthetic reagent mPEG replace reacts under the room temperature at pH7.5.The SDS-PAGE electrophoretogram of product is seen accompanying drawing 4, and Fig. 4 shows have one or more to modify band.Among the figure, M is a molecular weight standard, and 0 is the interleukin-2 contrast of unmodified, and 1～4 for modifying sample.As can be seen, under different modification conditions, the methyl ethyl diketone that guanidine radicals PEGization reagent mPEG replaces presents mono-modified to interleukin-2 and the band of modifying more.
Claims (8)
1. the Pegylation reagents of protein and polypeptide guanidine radicals is characterized in that, is the single substitution product of mPEG to methyl ethyl diketone, and general structure is as follows:
Wherein, m=CH
3-,
n＝33～330。
2. the method for the Pegylation reagents of preparation described white matter of claim 1 and polypeptide guanidine radicals is characterized in that, comprises the steps:
(1) with mPEG, potassium tert.-butoxide and bromo acetone room temperature reaction 10～24 hours in solvent, obtains mPEG acetone;
(2) with the mPEG acetone that obtained and acetic anhydride in the presence of the catalyzer boron trifluoride, the methyl ethyl diketone that end product mPEG replaces is collected in 50～80 ℃ of reactions 6～10 hours then from reaction product.
3. method according to claim 2 is characterized in that, the molecular weight of mPEG is 2000～20000.
4. method according to claim 2 is characterized in that, said solvent is selected from a kind of in toluene, benzene, the dioxane.
5. method according to claim 2 is characterized in that, the molar ratio of each raw material is in the step (1):
MPEG: potassium tert.-butoxide=1: 1～2; MPEG: bromo acetone=1: 2～5.
6. method according to claim 2 is characterized in that, the molar ratio of each raw material of step (2) is:
MPEG acetone: acetic anhydride=1: 2～5;
MPEG acetone: boron trifluoride=1: 1～5.
7. the application of the Pegylation reagents of white matter according to claim 1 and polypeptide guanidine radicals is characterized in that, is used to comprise the PEGization of the compound of guanidine radicals.
8. the application of the Pegylation reagents of white matter according to claim 1 and polypeptide guanidine radicals is characterized in that, directly is used in the PEGization of protein, polypeptide or amino acid guanidine radicals.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410054144 CN1629193A (en) | 2004-08-31 | 2004-08-31 | Protein and polypeptide guanidine polyethyleneglycol reagent and its preparation process and use |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410054144 CN1629193A (en) | 2004-08-31 | 2004-08-31 | Protein and polypeptide guanidine polyethyleneglycol reagent and its preparation process and use |
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|---|---|
| CN1629193A true CN1629193A (en) | 2005-06-22 |
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|---|---|---|---|
| CN 200410054144 Pending CN1629193A (en) | 2004-08-31 | 2004-08-31 | Protein and polypeptide guanidine polyethyleneglycol reagent and its preparation process and use |
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Open date: 20050622 |