Study On The Branched PEG Derivatives For Site-specific Modification Of Peptide Drugs

PEG modification(PEGylation) now plays an important role in the field of biological medicine,and it is currently considered one of the most successful techniques in enhancing the potentials of peptides and proteins as therapeutic agents.It is proved by the research that the branched PEG has the advantage of a lower inactivation of the proteins during conjugation,and the conjugate's activity is preserved to a larger extent as compared to linear PEG.Furthermore,and more important,it is more effective also in protecting proteins from proteolysis and hydrolysis that giving a more prolonged residence in body,in preventing the activity loss of the conjugate,and in reducing immunogenicity.Therefore,the research of the beanched PEG became the trend of the PEGylation.In the thesis,there are two methods,divergent and convergent,are used to research the synthesis of the branched PEG respectively.Divergent is a general approach in which branch points are constructed in a stepwise fashion from a central core.That is to say multifunctional compounds were used as initiators or transfer agents to initiate ethylene oxide(EO) polymerization.Two initiate systems were adopted:alkali-polyols and Lewis acid-polyots.Through the control of reaction conditions,branched PEG with the controlled molecule weight was obtained.The reaction mechanisms and product structures were also researched from the process of some elementary reaction steps of polymerization such as chain initiation, chain propagation,chain transfer and chain termination.Branched PEG was obtained when the alkali-polyols initiate system used.Further more,polymerization products with different active end groups could be synthesized when the different termination ways were adopted.But the molecule weight distribution of the product was wideLewis acid andβ-cyclodextrin(β-CD) has been adopted as initiate system to synthesize PEG at the first time in this thesis,and branched PEG with high molecule weight was obtained.The results also broke the rule that using cationic polymerization can't get PEG with high molecule weight.Three kinds of Lewis acid,ZnCl₂,TiCl₄ and ZrCl₄,were observed.The polymerization products with different structures were synthesized by the mole ratio change of Lewis acid toβ-CD.In addition,through the mole ratio change ofβ-CD to EO,the branched PEGs with different molecule weight were obtained.The more important fact is many different polymerization products with all kinds of active end groups could be got easily by the use of different termination ways in theory.This can enhance the potential application of the branched PEG that synthesized by divergent.Moreover,the polymerization mechanism was studied and the cationic coordination polymerization mechanism was presented by the analysis of lots of experiment phenomenon and results.Convergent is another general approach in which branch segments are constructed separately and then joined to a multifunctional core.The branched structure was obtained by several linear mPEG react with one multifunctional compound.The glutamic acid(Glu) and the tris(hydroxymethyl)aminomethane(Tris) were used as core, and di-branched and tri-branched molecules are synthesized respectively in this thesis. The reaction condition and mechanisms of the branched PEG derivative synthesized by convergent were researched.Glu is used as core,Two linear monomethoxypoly(ethy lene glycol)(mPEG) chains linked together through the two carboxylic group of the Glu with the 4-Dimethylaminopyridine(DMAP)/Dicyclohexylcarbodiimide(DCC) as the catalytic system.When Tris is used as core,The first step is protecting the amino group of Tris by t-butyloxycarbonyl anhydride.The amino group was reserved that can be used as functional radical to modifying the protein later,mPEG was activated by changing hydroxide terminal to acid chloride and carboxylation of mPEG was fulfilled before.A novel tri-branched macromolecule was synthesized from the reaction between Boc-Tris and mPEG-COCI.Carboxyl-monomethoxypoly(ethylene glycol) was prepared with the initial materials of mPEG and acrylonitrile in water.This is a novel method putting forward at the first time in this thesis.A general space arm molecule,6-maleimide caproic acid(6-MICA),is synthesized and introduced in the branched PEG that synthesized by convergent to reduce the steric hindrance.The scheme of the synthesis of 6-MICA is divided into two steps.Firstly,the intermediate,6-maleic amic caproic acid(6-MACA),is synthesized by open ring reaction between maleic anhydride and 6-aminocaproic acid.Secondly,6-MICA is synthesized through the close ring reaction of the intermediates.The reaction of first step is carry out in the two-phase solution.The reaction conditions are optimized by orthogonal design.The yield can over 90%.The conditions and the catalyst of the reaction of the second step is selected,and the optimized scheme is screened out.The catalyst is TEA.The solvent is toluene.The reaction is carried out under reflux condition.6-MICA was used to improve the di-branched PEG.The conjugated reaction between 6-MICA and the di-branched PEG is catalyzed by DCC/HOBt.The peptide drug,Tαl,was modified by the improved di-branched PEG,and the reaction goes fast. The metabolic activity of the conjugated peptides was tested.The result showed that the conjugated drugs have nice pharmacokinetic properties.The conjugated peptides reduced the shake of the concentration in serum and prolonged the half life term compared with the native peptides.