CN101580585A - Histidine selective water soluble polymer derivative - Google Patents
Histidine selective water soluble polymer derivative Download PDFInfo
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- CN101580585A CN101580585A CNA2009100332523A CN200910033252A CN101580585A CN 101580585 A CN101580585 A CN 101580585A CN A2009100332523 A CNA2009100332523 A CN A2009100332523A CN 200910033252 A CN200910033252 A CN 200910033252A CN 101580585 A CN101580585 A CN 101580585A
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Abstract
The invention relates to a histidine selective derivative of water-soluble polymers such as polyethylene glycol and the like, a preparation method thereof, and a coupling product with other molecules. The other molecules particularly refer to biomolecules of proteins or polypeptides and the like which have medicinal values.
Description
Technical field
The invention provides histidine selective derivative such as this type of water soluble polymerizer of polyoxyethylene glycol, and their preparation method.The present invention be more particularly directed to (1) preparation and on an end, have suitable and protein or other biological group of molecules propylhomoserin link coupled histidine selective polymer derivative at least, (2) histidine selective polymer derivative itself, (3) method of the coupled product of preparation histidine selective polymer derivative and biomolecules, (4) coupled product of polymer and biomolecules itself, the using method of (5) this class conjugate.
Background technology
Along with protein chemistry and development of molecular biology, increasing protein drug is applied in prevention and treatment of diseases.Though single-minded, the determined curative effect of the action site of protein drug is found through a large amount of clinical application, natural or reorganization without the protein drug of any modification because renal clearance is higher, easily by enzyme liberating, so plasma half-life is shorter in the body.Because protein has certain immunogenicity in vivo, easily produce neutralizing antibody again, have a strong impact on the curative effect of pharmaceutical grade protein.These have restricted its clinical widespread use because the shortcoming that the protein self-characteristic brings has limited the performance of its route of administration and drug effect.
Appearing at of protein and superpolymer coupling technology solved the defective that pharmaceutical grade protein exists to a great extent.After various water soluble polymers and the protein coupling, by increasing proteinic molecular weight, reduce the albumen glomerular filtration rate(GFR, improve protein plasma half-life.And since superpolymer to protein surface cover epitope and restriction enzyme site bridging effect, can reach and improve the purpose that the pharmaceutical grade protein transformation period is short, have defective such as immunogenicity, expanded the clinical application of pharmaceutical grade protein greatly.Wherein to become research the most thorough for polyoxyethylene glycol, the protein modification superpolymer that is most widely used.
Polyethyleneglycol modified technique functions come from later stage nineteen seventies (Davis, S.Clin.Exp Immunol.46 (1981), 649-652).PEG is that the state with high degree of hydration exists in solution, and a part oxyethyl group can adsorb 16 water moleculess, and (Chem.Eng.Sci 61 (2006), 924-939) for C.J.Fee, J.M.Van Alstine.There are some researches show that in addition PEG has the ability of the water molecule layer that influences several loose contacts, this has just explained that PEG exists bigger hydration radius in solution.Bigger hydration radius can repel other protein moleculars, makes the PEG molecule show as reduced immunogenicity and low antigenicity.After PEG and protein coupling, these characteristics can be transferred in the coupling molecule.Therefore PEG is highly suitable for the modification of protein molecule.
PEG modifies mainly and for example is positioned at the epsilon-amino on the protein lysine side-chain with amino in the protein as decorating site at present.The preferred option that this modifying method is studied is often used also extensive.Adopt amino modifier to comprise polyoxyethylene glycol-aldehyde, mixed acid anhydride, N-hydroxy-succinamide ester, carbonylic imidazole alkane and chlorine cyanurate as decorating site.Though modify the activity that is proved to be the energy retaining protein in the PEG of amino modification technique, yet in some cases, using amino is inappropriate as decorating site.The passivation that for example has report to work as some specific position Methionin to produce because modify can make the protein active forfeiture (Suzuki, T, Biochimica et Biophysica Acta 788 (1984), 248-255).
Another defective of amido modified dose is can comprise Methionin epsilon-amino, N-terminal alpha-amino group in order to the amino One's name is legion of modifying in the protein that possible decorating site also comprises Histidine.Numerous decorating sites causes formed polymer conjugates to be mixed by single Pegylation, two polyoxyethylene glycolization, three polyoxyethylene glycolization etc. usually.The separation of these mixtures is difficulty relatively, and batch between component ratio often there are differences, be unfavorable for scale operation preparation.
A kind of method of these problems of avoiding just is to use the site selectivity polymer active derivative as target spot with other functional groups beyond the amino.Existing this type of common modifier is the polymer derivant that acts on sulfydryl on the protein cysteine residues.Polyethyleneglycol derivative with sulfydryl selective reaction end group comprises maleimide, thiazolinyl sulfone, iodo-acid amide, mercaptan and disulphide, and wherein maleimide is the most frequently used.(Zalipsky，S.Bioconjug?Chem?6(1995)，150-165；Greenwald，R.B.Crit.Rev.Ther.Drug?Carrier?Syst.17(2000)，101-161；Herman，S.Macromol.Chem.Phys.195(1994)，203-209；CN100343305C；CN1723232A)。But there is obvious defects in the sulfydryl modification agent on using.Do not have free sulfhydryl group in the most protein, major part is that the form with disulfide linkage exists, and protein function is played an important role.With the proteinic space structure of coupling PEG meeting havoc behind the disulfide bond reduction, protein function is reduced, even completely lose.Also have by mutation method and in protein, introduce the example that protein is carried out pointed decoration in conjunction with the mercaptan site of PEG, but this method cost is higher, and the mercaptan site of introducing also may produce disulfide linkage with the mercaptan site of protein itself, destroys proteinic structure and function.
The factor that another restriction polyoxyethylene glycol maleimide modifier is used is that there is hydrolytic instability in it in storage He in the candidate molecules coupling process.Particularly, no matter the PEG maleimide all can observe the significantly hydrolysis of maleimide ring before coupling or after the coupling.This phenomenon will cause the unhomogeneity of coupled product, though biological activity may be similar, pharmacokinetic property may there are differences in the body, and this is disadvantageous for administration.And the difference between may existing batch, also have difficulties in the quality control.
According to above-described content, the applicant has proposed a kind of very attractive target spot, Histidine in the protein.In protein, the content of Histidine generally lacks than Methionin, and alternative site is reduced a lot than amido modified dose.The active possibility that descends of protein conjugate after the modification that brings owing to passivation is just low much like this.And Histidine is with respect to the halfcystine that forms disulfide linkage, and the influence of protein steric structure is descended greatly, avoided the destruction of coupling to protein structure and function.
Summary of the invention
First purpose of the present invention is to provide the Compound I with following general formula:
X-Q-W-A-NH
2(I)
X representation polymer wherein, Q represents independently linking group, and W represents electron-withdrawing group, and A represents aromatic group, and wherein Q-W also can represent electron-withdrawing group jointly.
Polymkeric substance X is multipolymer, polysaccharide, polyglutamic acid, polylysine, Palmiticacid, polysialic acids, palmitinic acid or the lauric acid of polyalkylene glycol, Polyvinylpyrolidone (PVP), polyacrylic ester, poly-oxazoline, polyvinyl alcohol, polypropylene glycol, polyacrylamide or PMAm, HPMA multipolymer, polyester, polyacetal, poe, polycarbonate, poly-iminocarbonic ester, polymeric amide, divinyl ether-maleic anhydride or phenylethylene-maleic anhydride, and their wire, divide dendritic or amino reactive derivative.Preferred polyoxyethylene glycol and his wire, divide dendritic or amino reactive derivative
When polymkeric substance was polyoxyethylene glycol, polyoxyethylene glycol comprised any linearity, branch or multi-arm shape.The polyoxyethylene glycol segment comprises-(CH
2CH
2O) n-, wherein n be generally about 3～about 4000, or about 3～about 3000 or more preferably from about 20～about 1000.
Polyoxyethylene glycol can be end capped, for example use inertia group that one end of polyoxyethylene glycol is sealed, inertia group comprises that alkoxyl group, substituted alkoxy, alkenyloxy, substituted alkenyl oxygen base, alkynyloxy group, replacement alkynyloxy group, aryloxy, substituted aryloxy, preferred capping group are methoxyl group, oxyethyl group and benzyloxy.Capping group can also comprise phosphatide, comprises Yelkin TTS choline etc.
In the Compound I, each linking group Q represents Direct Bonding independently, alkylidene group or times aryl or the heteroaryl that replace, wherein any one can be by one or more Sauerstoffatoms, sulphur atom, R represents alkyl or aryl-NR group, ketone group ,-O-CO-base and/or-CO-O-base end-blocking or interruption.Suitable aryl comprises phenyl and naphthyl, and suitable heteroaryl comprises pyridine, pyrroles, pyrans, imidazoles, oxazole, pyridazine, pyrimidine and purine.Can be connected to polymkeric substance by the key of hydrolytically unstable or by non-unsettled key.
W represents ketone or aldehyde radical CO, ester group-O-CO-or sulfuryl-SO in the Compound I
2-, or the group that obtains by this group reduction.For example CH-OH group, ether CH-OR, ester group CH-O-C (O) R, amido CH-NH
2, CH-NHR or CH-NR
2, or acid amides CH-NHC (O) R or CH-N (C (O) R)
2
In the Compound I, A represents phenolic compound, comprises appointing aryl or the heteroaryl that replaces.Suitable aryl comprises phenyl and naphthyl, and suitable heteroaryl comprises pyridine, pyrroles, pyrans, imidazoles, oxazole, pyridazine, pyrimidine and purine.
This patent has further comprised the preparation method of Compound I, it is characterized in that being undertaken by following step: under amino Boc protection, W-A-NH-Boc and X-Q carry out condensation reaction under condensation reagents such as DCC, take off the Boc protection again and obtain Compound I.
Second purpose of the present invention is to provide the Compound I I with following general formula
X-Q-W-A-N＝N-Z (II)
In general formula I I, X, Q, W, A have the implication identical with general formula I.The Z representative derives from the group of biomolecules, and each connects by Histidine.Z can derive from any desirable biomolecules, for example protein.Protein can make polypeptide, antibody, antibody fragment, enzyme, cytokine, chemokine or acceptor.For example Z includes but are not limited to Somat, tethelin, thymosin, parathyroid hormone, erythropoietin, interleukin, Interferon, rabbit, Endostatin, G CFS, oxyphorase, cytokine etc.
The present invention also provides a kind of preparation method of Compound I I, it is characterized in that being undertaken by following step:
Compound I is dissolved in the dilute hydrochloric acid, adds the aqueous solution of Sodium Nitrite under the optimal temperature condition, makes the aqueous solution of diazonium salt.Add the biomolecules molecule under optimum conditions and carry out coupling.
Suitable condition is meant is enough to make histidine selective water soluble polymer derivative and biomolecules to carry out conditions such as link coupled time, temperature, pH value, solvent.Concrete condition depends on and the kind of biomolecules, desirable coupling type etc.
Typical coupling condition comprises pH usually in the 6-10 scope, and preferred pH value is 7-9.Temperature of reaction depends on the active requirement of biomolecules, is generally 0-75 ℃, preferred 2-45 ℃, and more preferably 2-8 ℃.Higher temperature may make the biomolecules inactivation.
Use slight excessive polymer derivant generally speaking, for example use the polymer derivant of 1-20 times of mol ratio, the polymer derivant of preferred 1-10 times of mol ratio more has the polymer derivant that selects 1-3 times of mol ratio.Excessive polymer derivant can be removed by ion exchange chromatography easily.The mol ratio of typical feed ratio polymer derivant and biomolecules is 1: 1,1.5: 1,2: 1,3: 1.Can use SDS-PAGE or HPLC detection reaction process.Reach stable in case work as unreacted polymer derivative or coupled product, then think to react and finish.Concrete reaction conditions and method should make that under certain coupling yield it is active that biomolecules keeps a part at least.
Advantage of the present invention is to have synthesized a kind of polymer derivant of histidine selective.Compare with traditional amino selective derivatization thing, alternative site is reduced a lot.So just reduced the active possibility that descends of protein conjugate after the modification that brings owing to passivation.This compound only is connected in biomolecules by Histidine, makes its pointed decoration agent that becomes protein or polypeptide, thereby can significantly reduce the generation of isomer.
Description of drawings
Fig. 1 is mPEG-NH-CO-Ph-NH
2With N,O-Diacetylmuramidase reaction solution SDS-PAGE electrophoresis coomassie brilliant blue staining result.Wherein 1 is protein molecular weight standard, the 2nd, and N,O-Diacetylmuramidase contrast, the 3rd, mPEG-NH-CO-Ph-NH
2Contrast, the 4th, mPEG-NH-CO-Ph-NH
2With the N,O-Diacetylmuramidase reaction solution.
Fig. 2 is mPEG-NH-CO-Ph-NH
2With N,O-Diacetylmuramidase reaction solution SDS-PAGE electrophoresis iodine staining result.Iodine can dye at peg moiety.Wherein 1 is the N,O-Diacetylmuramidase contrast, the 2nd, and mPEG-NH-CO-Ph-NH
2Contrast, the 3rd, mPEG-NH-CO-Ph-NH
2With the N,O-Diacetylmuramidase reaction solution.
Embodiment
1.Boc para-amino benzoic acid is synthetic.
Para-amino benzoic acid 6.8g is added to dioxane and water is in the 50ml solution of (1: 1), and ice bath makes solution temperature less than 10 degree, slowly drips triethylamine 10g, after treating to dissolve fully, drip Boc acid anhydride 10.8g, keeping the body choosing is that temperature is less than 10 degree, stir 30min, back stirred overnight at room temperature.Underpressure distillation goes out dioxane, adds entry 20ml, and the 10ml extracted with diethyl ether once discards ether.The aqueous solution is regulated PH to 4 with 3M hydrochloric acid, 20ml ethyl acetate extraction 3 times, and dried over mgso, evaporated under reduced pressure promptly get the 9.6g solid.
2.mPEG-NH-CO-Ph-NH-Boc synthetic
Get mPEG-NH
2(Mw 20kDa) 0.5g, Boc para-amino benzoic acid 0.06g is dissolved in methylene dichloride, adds DCC0.05g, DMAP0.03g, stirring at room 24h.Suction filtration is removed solid, and decompression is steamed to small volume, splashes among the anhydrous diethyl ether 300ml, separates out solid 0.5g, and suction filtration promptly.
3.mPEG-NH-CO-Ph-NH
2Synthetic
The mPEG-NH-CO-Ph-NH-Boc 0.4g that gets preparation in 50ml eggplant type bottle, 5ml trifluoroacetic acid/dichloromethane (50/50) solution stirring 4h, solution decompression steams to small volume, splashes among the anhydrous diethyl ether 100ml, separates out solid 0.3g, suction filtration, drying under reduced pressure.
4.mPEG-NH-CO-Ph-NH
2With the N,O-Diacetylmuramidase coupling
Get mPEG-NH-CO-Ph-NH
280mg is dissolved in the 2ml 2mM dilute hydrochloric acid, at the 0-10 ℃ of aqueous solution that adds 6.8 μ l, 5% Sodium Nitrite down, makes the aqueous solution of diazonium salt, stirs 10min, adds N,O-Diacetylmuramidase 40mg and regulates pH value to 8.5, reaction 3h under 4 ℃.Reaction back sampling carrying out SDS-PAGE electrophoresis, electrophoresis finish the back Xylene Brilliant Cyanine G and dye at protein portion, the results are shown in Figure 1.Iodine dyes at peg moiety, the results are shown in Figure 2.
Claims (10)
1. the compound that has following general formula:
X-Q-W-A-NH2 (I)
X-Q-W-A-N＝N-Z (II)
X representation polymer wherein, Q represents independently linking group, and W represents electron-withdrawing group, and A represents aromatic group, and wherein Q-W also can represent electron-withdrawing group jointly.The Z representative derives from the group of biomolecules, and each connects by Histidine.
2. the described compound of claim 1, wherein polymkeric substance X is a polyalkylene glycol, Polyvinylpyrolidone (PVP), polyacrylic ester, poly-oxazoline, polyvinyl alcohol, polypropylene glycol, polyacrylamide or PMAm, the HPMA multipolymer, polyester, polyacetal, poe, polycarbonate, poly-iminocarbonic ester, polymeric amide, the multipolymer of divinyl ether-maleic anhydride or phenylethylene-maleic anhydride, polysaccharide, polyglutamic acid, polylysine, Palmiticacid, polysialic acids, palmitinic acid or lauric acid, and their wire, divide dendritic or amino reactive derivative.
3. the described compound of claim 2, polymkeric substance wherein is a polyoxyethylene glycol.
4. each described compound among the claim 1-4, wherein each linking group Q represents Direct Bonding independently, alkylidene group or times aryl or the heteroaryl that replace, wherein any one can be by one or more Sauerstoffatoms, sulphur atom, R represents alkyl or aryl-NR group, ketone group ,-O-CO-base and/or-CO-O-base end-blocking or interruption.
5. each described compound among the claim 1-5, wherein W represents ketone or aldehyde radical CO, ester group-O-CO-or sulfuryl-SO
2-, or the group that obtains by this group reduction.
6. each described compd A is represented phenolic compound among the claim 1-6, comprises appointing aryl or the heteroaryl that replaces.
7. the conjugate structure X-Q-W-A-N=N-Z after the modifier of claim 1-6 is modified, the Z representative comes from the group of biomolecules, comprises Somat, tethelin, thymosin, parathyroid hormone, erythropoietin, interleukin, Interferon, rabbit, Endostatin, G CFS, oxyphorase, cytokine etc.
8. the described compound of claim 7, wherein said or each albumen connects by Histidine.
9. the method for preparing each described compound among the claim 1-8, comprising under amido protecting, the condensation reaction of W-A-NH-protection and X-Q, deprotection obtains I again, forms the method for II under the diazotization condition.
10. pharmaceutical composition, comprise the compound of each described physiological tolerance among the claim 1-9 and pharmaceutically acceptable carrier and compound as the purposes of medicine.
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| CNA2009100332523A CN101580585A (en) | 2009-06-16 | 2009-06-16 | Histidine selective water soluble polymer derivative |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105001413A (en) * | 2015-08-06 | 2015-10-28 | 青岛大学 | Preparation method for polyethylene glycol diazonium polymer and application thereof |
| WO2019158037A1 (en) * | 2018-02-13 | 2019-08-22 | 上海时莱生物技术有限公司 | Amphiphilic block copolymer, preparation method thereof and nanomicelle drug-loading system |
| CN114716662A (en) * | 2021-01-06 | 2022-07-08 | 北京键凯科技股份有限公司 | Acrylic acid and derivative functionalized water-soluble polymer thereof and application thereof |
-
2009
- 2009-06-16 CN CNA2009100332523A patent/CN101580585A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105001413A (en) * | 2015-08-06 | 2015-10-28 | 青岛大学 | Preparation method for polyethylene glycol diazonium polymer and application thereof |
| WO2019158037A1 (en) * | 2018-02-13 | 2019-08-22 | 上海时莱生物技术有限公司 | Amphiphilic block copolymer, preparation method thereof and nanomicelle drug-loading system |
| EP3753966A4 (en) * | 2018-02-13 | 2021-03-31 | Selection Bioscience LLC | Amphiphilic block copolymer, preparation method thereof and nanomicelle drug-loading system |
| US11225551B2 (en) | 2018-02-13 | 2022-01-18 | Selection Bioscience Llc | Amphiphilic block copolymer, preparation method thereof and nanomicelle drug-loading system |
| CN114716662A (en) * | 2021-01-06 | 2022-07-08 | 北京键凯科技股份有限公司 | Acrylic acid and derivative functionalized water-soluble polymer thereof and application thereof |
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Application publication date: 20091118 |