含单一功能端基的两臂聚环氧乙烷及其衍生物的合成方法研究
摘要
聚环氧乙烷(PEO)是目前最常用的生物相容性聚合物之一,具有安全、无毒的优点。由于分子量较小的线形PEO作为药物载体存在体内循环周期短等诸多问题,因此,对于PEO修饰药物技术的研究目前主要集中在支化PEO的合成和修饰,高分子量PEO的合成和修饰,定向PEO修饰和可释放型PEO修饰方面。其中,支化PEO的研究受到了研究者极大的关注；与相同分子量的线形PEO相比,支化PEO具有流体力学体积较小、在体内的循环期更长的特点。目前,支化PEO的合成主要是通过将含功能端基的线形mPEO通过多官能团核的多次偶联反应,将含功能端基的线形mPEO通过多次偶联反应偶合到一个多官能团的核上。但是这种偶联方法存在反应周期长,产物需经过复杂的分离纯化过程等缺点。这就造成了含功能端基PEO价格昂贵,且不能大批量生产。
本文通过制备高纯度的双羟基小分子引发剂[(1'-乙氧基)-乙氧基]-1,2-丙二醇(EEPD),与二苯甲基钾(DPMK)一起作为共引发剂,利用开环聚合(ROP)的方法引发环氧乙烷进行聚合,合成了在链段中间含一个被保护羟基的两臂聚环氧乙烷。利用惰性的苄基基团对PEO端羟基进行封端,再通过水解将被保护的羟基还原得到在链段中间含单一活性羟基基团的两臂聚环氧乙烷(PEO2-OH)。
然后,用丙烯腈在NaOH的存在下对羟基进行修饰,得到了功能化聚环氧乙烷PEO2-CN;并分别通过一系列的转变得到了链段中间含单一羧基基团的PEO2-COOH,并继续进行修饰制备了可用于蛋白药物修饰的功能化两臂PEO2-SC。利用GPC、1HNMR对上述反应产物进行了详细的表征。
利用两臂PEO2-OH链段中间的活性羟基与2-溴代异丁酰溴反应得到可用于原子转移自由基聚合(ATRP)的大分子引发剂,利用该大分子引发剂引发苯乙烯聚合,制备得到两臂为亲水性PEO和一臂为亲油性聚苯乙烯(PS)的星型聚合物starPE02PS。利用GPC、1HNMR对产物进行了详细的表征。
Poly(ethylene oxide) (PEO) is one of the most widely used biocompatible polymer due to its safety and non-toxicity for human body. PEO with low molecular weight used as drug carriers still had some disadvantages such as short circulation half-life in vivo. Recently, the research on the PEGylation of drugs had focused on the synthesis and modification of branched PEO, synthesis and modification of high molecular weight PEO, site-specific and releasable PEGylation. Among these, much attention had paid to the study of branched PEO, which shows smaller hydrodynamic volume and longer circulation half-life in vivo comparing to linear PEO with the same molecular weight. At present, branched PEO was usually synthesized by coupling linear PEOs with functional end group to a multi-functional core molecule. However, this coupling approach showed some shortages as long reaction period and complicated purification and separation procedures, that is the cause for the expensive price of this kind of PEOs and difficult production on large scale.
In this thesis, a small molecular compound with two hydroxyl groups 3-[(1'-Ethoxyethyl)-ethoxyethyl]-1,2-propanediol (EEPD) was firstly synthesized with high purity. It was used as co-initiator with diphenylmethyl potassium (DPMK) to initiate the ring opening polymerization (ROP) of ethylene oxide (EO) to synthesize two arms PEO with a protected hydroxyl group at the conjunction point. After the end hydroxyl groups on PEO arms were end-capped by benzyl groups, the protected hydroxyl group was hydrolyzed to get PEO2-OH.
Afterwards, PEO2-OH was reacted with acrylonitrile in the presence of sodium hydroxide to get PEO2-CN, Then the cyano group was converted into carboxyl group and N-chlorosuccinimide for PEO2-COOH and PEO2-SC, and the latter can be used for protein modification. The functional products were characterized by GPC and 1H NMR in detail.
The active hydroxyl at the conjunction point of PEO2-OH was reacted with 2-bromoisobutyryl bromide to synthesize PEO2-Br, which can initiate atom transfer radical polymerization (ATRP) of styrene. Thus the star-shaped polymers (starPEO2PS) were obtained with two hydrophilic PEO arms and one hydrophobic PS arm. The three-arm amphiphilic starPEO2PS was characterized by GPC and 1H NMR in detail.
本文通过制备高纯度的双羟基小分子引发剂[(1'-乙氧基)-乙氧基]-1,2-丙二醇(EEPD),与二苯甲基钾(DPMK)一起作为共引发剂,利用开环聚合(ROP)的方法引发环氧乙烷进行聚合,合成了在链段中间含一个被保护羟基的两臂聚环氧乙烷。利用惰性的苄基基团对PEO端羟基进行封端,再通过水解将被保护的羟基还原得到在链段中间含单一活性羟基基团的两臂聚环氧乙烷(PEO2-OH)。
然后,用丙烯腈在NaOH的存在下对羟基进行修饰,得到了功能化聚环氧乙烷PEO2-CN;并分别通过一系列的转变得到了链段中间含单一羧基基团的PEO2-COOH,并继续进行修饰制备了可用于蛋白药物修饰的功能化两臂PEO2-SC。利用GPC、1HNMR对上述反应产物进行了详细的表征。
利用两臂PEO2-OH链段中间的活性羟基与2-溴代异丁酰溴反应得到可用于原子转移自由基聚合(ATRP)的大分子引发剂,利用该大分子引发剂引发苯乙烯聚合,制备得到两臂为亲水性PEO和一臂为亲油性聚苯乙烯(PS)的星型聚合物starPE02PS。利用GPC、1HNMR对产物进行了详细的表征。
Poly(ethylene oxide) (PEO) is one of the most widely used biocompatible polymer due to its safety and non-toxicity for human body. PEO with low molecular weight used as drug carriers still had some disadvantages such as short circulation half-life in vivo. Recently, the research on the PEGylation of drugs had focused on the synthesis and modification of branched PEO, synthesis and modification of high molecular weight PEO, site-specific and releasable PEGylation. Among these, much attention had paid to the study of branched PEO, which shows smaller hydrodynamic volume and longer circulation half-life in vivo comparing to linear PEO with the same molecular weight. At present, branched PEO was usually synthesized by coupling linear PEOs with functional end group to a multi-functional core molecule. However, this coupling approach showed some shortages as long reaction period and complicated purification and separation procedures, that is the cause for the expensive price of this kind of PEOs and difficult production on large scale.
In this thesis, a small molecular compound with two hydroxyl groups 3-[(1'-Ethoxyethyl)-ethoxyethyl]-1,2-propanediol (EEPD) was firstly synthesized with high purity. It was used as co-initiator with diphenylmethyl potassium (DPMK) to initiate the ring opening polymerization (ROP) of ethylene oxide (EO) to synthesize two arms PEO with a protected hydroxyl group at the conjunction point. After the end hydroxyl groups on PEO arms were end-capped by benzyl groups, the protected hydroxyl group was hydrolyzed to get PEO2-OH.
Afterwards, PEO2-OH was reacted with acrylonitrile in the presence of sodium hydroxide to get PEO2-CN, Then the cyano group was converted into carboxyl group and N-chlorosuccinimide for PEO2-COOH and PEO2-SC, and the latter can be used for protein modification. The functional products were characterized by GPC and 1H NMR in detail.
The active hydroxyl at the conjunction point of PEO2-OH was reacted with 2-bromoisobutyryl bromide to synthesize PEO2-Br, which can initiate atom transfer radical polymerization (ATRP) of styrene. Thus the star-shaped polymers (starPEO2PS) were obtained with two hydrophilic PEO arms and one hydrophobic PS arm. The three-arm amphiphilic starPEO2PS was characterized by GPC and 1H NMR in detail.
引文
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[1]黄骏廉,黄兆华,张海涛,多臂树杈型功能化聚乙二醇制备方法及它在药物中的应用.2002：CN 1381512 A.
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[5]Dreborg, S.Akerblom E.B. Immunotherapy with Monomethoxypolyethylene Glycol Modified Allergens [J]. Critical Reviews in Therapeutic Drug Carrier Systems,1990, 6(4):315-365.
[6]Abuchowski, A., Van E.T., Palczuk N.C. Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol [J]. J. Biol. Chem.,1977,252(11):3578-3581.
[7]Harris, J.M., Martin N.E., Modi M. PEOylation-A novel process for modifying pharmacokinetics [J]. Clinical Pharmacokinetics,2001,40(7):539-551.
[8]Yamaoka, T., Tabata Y., Ikada Y. Distribution and Tissue Uptake of Poly(Ethylene Glycol) with Different Molecular-Weights after Intravenous Administration to Mice [J]. Journal of Pharmaceutical Sciences,1994,83(4):601-606.
[9]Davis, F.F. Enzyme polyethylene glycol adducts:modified enzymes with unique properties [J]. Enzyme Eng.,1978,4:169-173.
[10]Nucci, M.L., Shorr R., Abuchowski A. The Therapeutic Value of Poly(Ethylene Glycol)-Modified Proteins [J]. Advanced Drug Delivery Reviews,1991,6(2):133-151.
[11]Kozlowski, A.Harris J.M. Improvements in protein PEOylation:PEOylated interferons for treatment of hepatitis C [J]. Journal of Controlled Release,2001,72(1-3): 217-224.
[12]Monfardini, C., Schiavon 0., Caliceti P., Morpurgo M., Harris J.M., Veronese F.M. A Branched Monomethoxypoly(Ethylene Glycol) for Protein Modification [J]. Bioconjugate Chemistry,1995,6(1):62-69.
[13]Kozlowski, A., Charles S.A., Harris J.M. Development of PEOylated interferons for the treatment of chronic hepatitis C [J]. Biodrugs,2001,15(7):419-429.
[14]胡永祥,牛津梁,张文博,高林.聚乙二醇修饰药物技术的研究进展[J]. 中国生化药物杂志,2004,25(6)：369-372.
[15]Hao, Y, Chen J.H., Wang X.C., Zhu H.W., Rong Z.G. Effects of site-specific polyethylene glycol modification of recombinant human granulocyte colony-stimulating factor on its biologic activities [J]. Biodrugs,2006,20(6):357-362.
[16]S. Zalipsky, C. Gilon, A. Zilkha. Eur. Polym. J.1983,19,1177.
[17]M. Mutter, E. Bayer. Academic Press, New York 1979, pp:285.
[18]J. M. Harris, E. C. Struck, M. G. Case, M. S. Paley. J. Polym. Sci.:Polym. Chem. 1984,22,341.
[19]T. Suzaki, Y Murakami, Y. Takegami, J. Polym Sci:Polym. Lett. Ed.1979,17,241.
[20]J. M. Harris, E. C. Struck, M. G. Case, M. S. Paley. J. Polym. Sci.:Polym. Chem. 1984,22,341.
[21]R. J. De. Vos, J. E. Goethals, Macromol. Chem., Rapid Commun.1985,6,53.
[22]J. M. Harris, E. C. Struck, M. G. Case, M. S. Paley. J. Polym. Sci.:Polym. Chem. 1984,22,341.
[23]K. Nilsson, K. Mosbach Methods Enzymol.1984,104,56.
[24]Samuel Zalipsky. Bioconjugate Chem.1995,6,150.
[25]G. Johansson, R. Gysin, S. D. Flanagan. J. Biol. Chem.1981,256,9126.
[26]M. Leonard, E. Dellacherie. Makromol. Chem.1988,189,1809.
[27]V. N. R. Pillai, M. Mutter, E. Bayer, I. Gatfield. J. Org.Chem.1980,45,5364.
[28]S. Zalipsky, C. Gilon, A. Zilkha. J. Eur. Polym.1983,19,1177.
[29]J. M. Harris, N. H. Hundley, T. G. Shannon, E. C. Struck. J. Org. Chem.1982,47, 4789
[30]J. M. Harris, E. C. Struck, M. G. Case, M. S. Paley. J. Polym. Sci.:Polym. Chem. 1984,22,341.
[31]I. N. Topchieva, A. I. Kuzaev, V. P. Zubov. Eur. Polym. J.1988,24,899-904.
[32]Harris, J. M., Dust, J. M., McGill, A., Harris, P. A., Edgel, M. J., Herrati, R. S., Karr, L. J.,and Donnelly, D. L. (1991) New polyethylene glycol for biomedical applications. Water-Soluble Polymers (S. W. Shalaby, C. L. McCormic, and G. B. Butler, Eds.) American Chemical Society:ACS Symposium Series, pp 418-429, American Chemical Society:Washington, DC.
[33]A.Buckmann, M. Morr, G. Johansson. Makromol. Chem.1981,182,1379.
[34]G. Johansson, J. Chr-matogr.1986,368,309.
[35]S. Matsushima, N. Yoda, S. Yoshikawa. Makromol. Chem. Rapid Commun.1989,10, 63.
[36]G. Johansson, J. Chr-matogr.1986,368,309.
[37]K. Geckeler, E.Bayer. Polym Bull 1980,3,347.
[38]G. Johansson, R. Gysin, S. D. Flanagan. J. Biol Chem.1981,256,9126.
[39]K. Geckeler, E.Bayer. Polym Bull,1979,1,691.
[40]Jing Li, W. John Kao. Biomacromolecules 2003,4,1005.
[41]V. P. Shanbhag, G. Johansson. Biochem. Biophys. Res. Commun 1974,61,1141.
[42]W. Kern, S. Iwabuchi, V. Bohmer. Macromol. Chem.1979,180,2539.
[43]S.Zalipsky, Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates, Bioconjugate Chem,1995,6:150-165
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