Method of Preparing Propionic Acid-Terminated Polymers

Abstract

The invention provides methods for preparing polymers bearing a terminal propionic acid. The method involves first reacting a water soluble and non-peptidic polymer comprising at least one hydroxyl group with a tertiary alkyl acrylate in the presence of a catalyst to form a propionic acid ester of the polymer, wherein the polymer has a weight average molecular weight of at least about 10,000 Da; and then treating the propionic acid ester of the polymer with a strong acid to form a propionic acid of the polymer.

1 . A composition comprised of a conjugate formed from the reaction of an active ester of PEG and a biologically active molecule, wherein the active ester of PEG is prepared by the method comprising:
 i) reacting a PEG polymer with a tertiary alkyl acrylate in the presence of a catalyst comprising a quaternary ammonium salt to form a propionic acid ester of the PEG polymer, wherein the PEG polymer comprises at least one hydroxyl group and said PEG polymer is a monofunctional PEG, difunctional PEG, or branched PEG molecule comprising 1 to about 25 hydroxyl groups and having a weight average molecular weight of at least about 10,000 Da; and   ii) treating the propionic acid ester of the PEG polymer with a strong acid to form a PEG polymer functionalized with at least one propionic acid group;   iii) optionally, chromatographically purifying the PEG polymer functionalized with at least one propionic acid group;   iv) derivatizing the PEG polymer functionalized with at least one propionic acid group to form an active ester of PEG; and   v) optionally, chromatographically purifying the active ester of PEG.   
     
     
         2 . The composition of  claim 1 , wherein the active ester is selected from the group consisting of N-succinimidyl ester, o-, m-, or p-nitrophenyl ester, 1-benzotriazolyl ester, imidazolyl ester, and N-sulfosuccinimidyl ester. 
     
     
         3 . The composition of  claim 1 , wherein the active ester is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
       wherein m is from 3 to 3000, and EC is a residue of a moiety selected from the group consisting of fluorescein, biotin, acrylate, vinylsulfone, maleimide, tert-butyl carbonyl, and 9-fluorenylmethoxycarbonyl. 
     
     
         4 . The composition of  claim 3 , wherein the active ester comprises the structure 
       
         
           
           
               
               
           
         
       
       wherein m is 3 to 3000. 
     
     
         5 . The composition of  claim 1 , wherein the biologically active molecule is selected from the group consisting of an interferon, a TNFR, erythropoietin, human growth hormone, and variants and mimetics of any of the foregoing. 
     
     
         6 . The composition of  claim 1 , wherein the tertiary alkyl acrylate is α- or β-substituted. 
     
     
         7 . The composition of  claim 6 , wherein the substituting group is selected from the group consisting of halo, hydroxyl, thiol, alkylthio, acyl, acyloxy, nitro, cyano, azido, trihalomethyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, and substituted heteroaryl. 
     
     
         8 . The composition of  claim 7 , wherein the substituting group is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and benzyl. 
     
     
         9 . The composition of  claim 1 , wherein the tertiary alkyl group of the tertiary alkyl acrylate is tert-butyl, tert-amyl, α,α′-dimethylbenzyl, trityl, 1-adamantyl, or 2-methyl-2-adamantyl. 
     
     
         10 . The composition of  claim 1 , wherein the tertiary alkyl acrylate has the structure: 
       
         
           
           
               
               
           
         
         wherein: 
         R 1  and R 2  are each independently selected from the group consisting of hydrogen, halo, hydroxyl, thiol, alkylthio, acyl, acyloxy, nitro, cyano, azido, trihalomethyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, and substituted heteroaryl; and 
         R 3 -R 5  are each independently alkyl, substituted alkyl, aryl or substituted aryl. 
       
     
     
         11 . The composition of  claim 10 , wherein R 3 , R 4 , and R 5 , are each methyl, ethyl, or phenyl. 
     
     
         12 . The composition of  claim 10 , wherein R 1  and R 2  are hydrogen. 
     
     
         13 . The composition of  claim 10 , wherein R 1  is hydrogen and R 2  is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and benzyl, 
     
     
         14 . The composition of  claim 1 , wherein the strong acid is trifluoroacetic acid, trifluoromethancsulfonic acid, formic acid, hydrochloric acid, or p-toluenesulfonic acid. 
     
     
         15 . The composition of  claim 1 , wherein the catalyst comprising the quaternary ammonium salt is selected from the group consisting of tetramethyl ammonium halide, tetraethyl ammonium halide, tetrapropyl ammonium halide, tetrabutyl ammonium halide, tetramethyl ammonium hydroxide, tetraethyl ammonium hydroxide, tetrapropyl ammonium hydroxide, and tetrabutyl ammonium hydroxide. 
     
     
         16 . The composition of  claim 1 , wherein the catalyst comprising the quaternary ammonium salt has the structure: 
       
         
           
           
               
               
           
         
       
       wherein each R is independently alkyl or substituted alkyl and X is a counter ion. 
     
     
         17 . The composition of  claim 16 , wherein each R is C1-C8 alkyl and X is halo or hydroxide. 
     
     
         18 . The composition of  claim 1 , wherein said reacting step and said treating step are conducted in the presence of an organic solvent. 
     
     
         19 . The composition of  claim 18 , wherein the organic solvent is selected from the group consisting of dichloromethane, tetrahydrofuran, dimethylformamide, acetonitrile, toluene, xylene, phenyl acetonitrile, nitrobenzene, tetrachloroethylene, anisole, and chlorobenzene. 
     
     
         20 . The composition of  claim 1 , wherein the weight average molecular weight is from about 10,000 to about 100,000 Da. 
     
     
         21 . The composition of  claim 20 , wherein the weight average molecular weight is from about 20,000 to about 60,000 Da, 
     
     
         22 . The composition of  claim 1 , wherein the step of chromatographically purifying the PEG polymer functionalized with at least one propionic acid group is performed. 
     
     
         23 . The composition of  claim 1 , wherein the step of chromatographically purifying the active ester of PEG is performed. 
     
     
         24 . The composition of  claim 1 , wherein the step of chromatographically purifying the PEG polymer functionalized with at least one propionic acid group is performed and the step of chromatographically purifying the active ester of PEG is performed.