Method for preparing functionalized polymers from polymer alcohols

Abstract

The present invention provides, among other things, methods for preparing functionalized and other polymers from polymer alcohols such as poly(ethylene glycol)s. In addition, polymer compositions, conjugates, polymeric reagents, are also provided.

1 .- 48 . (canceled) 
     
     
         49 . A method of forming a functionalized polymer, comprising:
 (a) providing a polymer comprising a formula HO-POLY-OH, wherein POLY is a water-soluble and non-peptidic polymer;   (b) optionally, converting HO-POLY-OH to a mixture comprising HO-POLY-OH, HO-POLY-Z and Z-POLY-Z, wherein Z is a leaving group, under conditions effective to form no more than about 45 percent of Z-POLY-Z;   (c) reacting HO-POLY-OH of step (a) or the mixture of step (b) with a functionalizing reagent comprising the structure X—L 0,1 —Y, wherein X is a group that reacts with a hydroxyl, optionally in anionic form, or with a carbon atom to which the hydroxyl or leaving group is attached, L 0,1  is an optional linker, and Y is an ionizable group, optionally as a protected ionizable group, to form a mixture comprising HO-POLY-OH, HO-POLY-L 0,1 —Y, and Y—L 0,1 -POLY-L 0,1 —Y, under conditions effective to form preferably no more than about 45 percent of Y—L 0,1 -POLY-L 0,1 —Y;   (d) optionally, alkylating the mixture from step (b) or step (c); and   (e) after deprotection of the functional group Y, if necessary, purifying the mixture from step (c) or step (d) by ion exchange chromatography to provide substantially pure polymer comprising a single —L 0,1 —Y group.   
     
     
         50 . The method of  claim 49 , wherein X is a halogen or a sulfonate ester. 
     
     
         51 . The method of  claim 49 , wherein said purifying step comprises:
 passing the mixture from step (c) or step (d) through a first ion exchange column to provide an eluate, wherein said passing the mixture step is carried out under conditions effective to adsorb substantially all polymer species comprising two —L 0,1 —Y groups onto the first column;   passing the eluate through a second ion exchange column under conditions effective to adsorb substantially all of polymer species having a single —L 0,1 —Y group onto said second column;   washing the second column with a solution having low ionic strength to remove only polymer species containing no —Y groups; and   passing a solution having high ionic strength through the second column to desorb polymer species having a single —L 0,1 —Y group.   
     
     
         52 . The method of  claim 51 , wherein the optional linker, L 0,1 , is present and is hydrolytically stable. 
     
     
         53 . The method of  claim 51 , wherein the optional linker, L 0,1 , is present and has a structure —(CR 1 R 2 ) m —, wherein R 1  and R 2  are each independently H or alkyl, and m ranges from 0-10. 
     
     
         54 . The method of  claim 51 , wherein Y is an amine or carboxylic acid. 
     
     
         55 . The method of  claim 51 , wherein said alkylating step comprises treating the mixture with an alkylating agent selected from the group consisting of dialkylsulfate, alkyl sulfonate, diazoalkane, alkyl halide, N,N′-dimethylformamide dialkyl acetal, 3-alkyl-1-p-tolyltriazene, trimethylanilinium hydroxide, trialkyloxonium fluoroborate, trimethylsulfonium hexafluorophosphonate and alkyl trichloroacetimidate. 
     
     
         56 . The method of  claim 51 , wherein said alkylating step is effective to convert hydroxyl groups in said mixture to —OR′, wherein R′ is selected from the group consisting of C 1 -C 20  alkyl, substituted C 1 -C 20  alkyl, C 1 -C 20  alkylene-aryl, and substituted C 1 -C 20  alkylene-aryl. 
     
     
         57 . The method of  claim 56 , wherein R′ is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, hexyl, and benzyl. 
     
     
         58 . The method of  claim 51 , wherein X is a nucleophile effective to displace —Z. 
     
     
         59 . The method of  claim 58 , wherein Z is a leaving group selected from the group consisting of halogen and sulfonate esters. 
     
     
         60 . The method of  claim 53 , wherein X is selected from the group consisting of leaving groups, substituted vinyl groups, and unsubstituted vinyl groups, m is 1-3, and Y is a protected carboxylic acid. 
     
     
         61 . The method of  claim 60 , wherein the protected carboxylic acid group is selected from the group consisting of nitrile, amide, alkyl ester and ortho ester. 
     
     
         62 . The method of  claim 49 , wherein X is selected from the group consisting of leaving groups, substituted vinyl groups, and unsubstituted vinyl groups, m is 1-3, and Y is a carbonitrile. 
     
     
         63 . The method of  claim 51 , wherein X is a halogen, m is 0, and Y is p-tolylsulfonyl, methylsulfonyl, trifluoromethylsulfonyl, or trifluoroethylsulfonyl. 
     
     
         64 . The method of  claim 53 , wherein the functionalizing reagent is selected from the group consisting of X′—(CR 1 R 2 ) m —C(O)—O—Rp, CH 2 ═CY′—C(O)—O—Rp, X′—(CR 1 R 2 ) m —Z, CH 2 ═CY′—(CR 1 R 2 ) m —Z, X′—(CR 1 R 2 ) m —C═N, and CH 2 ═CY′—C═N, wherein X′ is halogen or sulfonate ester, Z is an ortho ester, Y′ is H, halogen, alkyl, substituted alkyl, alkoxy, or substituted alkoxy, and Rp is alkyl or substituted alkyl. 
     
     
         65 . The method of  claim 49 , wherein said converting comprises reacting HO-POLY-OH with X′—SO 2 —R 3 , wherein R 3  is alkyl, substituted alkyl, aryl, or substituted aryl, and X′ is Br or Cl. 
     
     
         66 . The method of  claim 65 , wherein R 3  is p-tolyl, methyl, trifluoromethyl, or trifluoroethyl. 
     
     
         67 . The method of  claim 49 , further comprising, after said purifying step, transforming Y of the substantially pure polymer to a different reactive moiety. 
     
     
         68 . The method of  claim 49 , wherein said reactive moiety comprises a functional group selected from the group consisting of hydroxyl, active ester, active carbonate, ortho ester, acetal, aldehyde, aldehyde hydrate, ketone, ketone hydrate, oxime, alkenyl, acrylate, methacrylate, nitrile, primary or secondary amide, imide, acrylamide, active sulfone, amine, hydrazide, thiol, carboxylic acid, isocyanate, isothiocyanate, maleimide, succinimide, vinylsulfone, dithiopyridine, vinylpyridine, amidate, 2-substituted-1,3-oxazoline, 2-substituted 1,3-(4H)-dihydrooxazines, 2-substituted-1,3-thiazoline, 2-substituted 1,3-(4H)-dihydrothiazines, hydroxylamine, iodoacetamide, orthopyridyl disulfide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate. 
     
     
         69 . The method of  claim 49 , wherein Y is a protected functional group and said method further comprises deprotecting the functional group prior to said purifying. 
     
     
         70 . A method of separating a mixture of polymer species by ion exchange chromatography, comprising:
 (a) providing a mixture of water-soluble and non-peptidic polymers, said mixture comprising a neutral polymer absent an ionizable functional group, a monofunctional polymer comprising a single ionizable functional group, and a difunctional polymer comprising two ionizable functional groups;   (b) passing the mixture through a first ion exchange column to provide an eluate, wherein said passing the mixture step is carried out under conditions effective to adsorb substantially all of the difunctional polymer onto the first column;   (c) passing the eluate through a second ion exchange column under conditions effective to adsorb substantially all of the monofunctional polymer onto said second column;   (e) washing the second column with water or a low ionic solution to remove neutral polymer absent an ionizable functional group in a wash solution; and   (f) passing a solution having high ionic strength through the second column to desorb the monofunctional polymer.   
     
     
         71 . The method of  claim 70 , wherein said second ion exchange column is connected in series to one or more additional ion exchange columns, and said washing step further comprises washing said second and one or more additional ion exchange columns and said passing the eluate step further comprises eluting adsorbed monofunctional polymer from said second and one or more additional columns. 
     
     
         72 . The method of  claim 70 , further comprising passing a solution having sufficient ionic strength through the first column to desorb the difunctional polymer. 
     
     
         73 . The method of  claim 70 , wherein the first column is of a size sufficient to adsorb substantially all of the difunctional polymer and a fraction of the monofunctional polymer. 
     
     
         74 . The method of  claim 73 , wherein the fraction of monofunctional polymer adsorbed on the first column is less than about 70 percent. 
     
     
         75 . The method of  claim 71 , further comprising monitoring the wash solution from the second and one or more additional columns during said washing step, to determine that substantially all of the neutral polymer has been removed. 
     
     
         76 . The method of  claim 75 , wherein said monitoring step comprises analyzing the wash solution by a method selected from the group consisting of ion exchange chromatography, size exclusion chromatography, and high performance liquid chromatography, thin layer chromatography, and infrared analysis. 
     
     
         77 . The method of  claim 75 , wherein said polymer species are PEG and said monitoring step comprises treating a sample of said wash solution with 1% polyacrylic acid (Mn 250,000) in 1 N HCl to determine the presence of polymer. 
     
     
         78 . The method of  claim 70 , wherein said washing step is carried out with distilled water. 
     
     
         79 . The method of  claim 70 , wherein the polymer mixture in step (a) is provided in an aqueous solution having low ionic strength to allow adsorption of functionalized polymer on the ion-exchange gel. 
     
     
         80 . The method of  claim 70 , wherein the neutral polymer absent an ionizable functional group has the structure, HO-POLY-OH or R′O-POLY-OR′; the monofunctional polymer has the structure, HO-POLY-L 0,1 —Y or R′O-POLY-L 0,1 —Y; and the difunctional polymer has the structure, Y—L 0,1 -POLY-L 0,1 —Y, wherein L is an optional linker, Y is a functional group and R— is selected from the group consisting of C 1 -C 20  alkyl, substituted C 1 -C 20  alkyl, C 1 -C 20  alkylene-aryl, and substituted C 1 -C 20  alkylene-aryl. 
     
     
         81 . The method of  claim 64 , wherein the mixture in step (a) comprises CH 3 O-POLY-OCH 3 , in combination with H 3 CO-POLY-L 0,1 —Y, and Y—L 0,1 -POLY-L 0,1 —Y, wherein L is an optional linker and Y is a functional group. 
     
     
         82 . The method of  claim 81 , wherein the mixture comprises no more than about 40% by weight of HO-POLY-OH and Y—L 0,1 -POLY-L 0,1 —Y in combination. 
     
     
         83 . The method of  claim 82 , wherein the mixture comprises no more than about 10% by weight of HO-POLY-OH and Y—L 0,1 -POLY-L 0,1 —Y in combination. 
     
     
         84 .- 86 . (canceled) 
     
     
         87 . An ion exchange chromatography apparatus, comprising:
 a supply of solution of a water-soluble and non-peptidic polymer mixture comprising a neutral polymer absent ionizable functional groups, a monofunctional polymer comprising a single ionizable functional group, and a difunctional polymer comprising two ionizable functional groups;   a first ion exchange column comprising a first inlet, a first outlet, and a first ion exchange media, said first inlet being in fluid communication with said supply;   a second ion exchange column comprising a second inlet, a second outlet, and a second ion exchange media, said second inlet being in fluid communication with said first outlet; and   at least one product recovery vessel in fluid communication with said second outlet, adapted to receive eluent exiting from said second ion exchange column.   
     
     
         88 . The apparatus of  claim 87 , wherein said first ion exchange column is of a size sufficient to adsorb substantially all of the difunctional polymer. 
     
     
         89 . The apparatus of  claim 87 , further comprising a product recovery vessel in fluid communication with said first outlet, adapted to receive eluent exiting from said first ion exchange column. 
     
     
         90 . The apparatus of  claim 87 , further comprising a salt solution supply in fluid communication with at least one of said first and second inlets. 
     
     
         91 . The apparatus of  claim 87 , further comprising a neutral solution supply in fluid communication with at least one of said first and second inlets. 
     
     
         92 . The apparatus of  claim 87 , further comprising one or more additional ion exchange columns connected in series, and in fluid communication with said second outlet. 
     
     
         93 . A compound of the following structure: 
       
         
           
           
               
               
           
         
       
       wherein (n) is from 2 to 3,000, R 4  is H or an organic radical, and (n′) is 3 to 10. 
     
     
         94 . The compound of  claim 93 , wherein (n) is from 10 to 2,000. 
     
     
         95 . The compound of  claim 93 , wherein R 4  is alkyl. 
     
     
         96 . The compound of  claim 95 , wherein alkyl is methyl or benzyl. 
     
     
         97 . The compound of  claim 93 , wherein (n′) is 3. 
     
     
         98 .- 102 . (canceled) 
     
     
         103 . A composition comprising monomethoxy end-capped polyethylene glycol) (“mPEG-OH”), wherein the composition has a polyethylene glycol) diol content of less than 0.3 wt. %. 
     
     
         104 . The composition of  claim 103 , wherein the composition has a polydispersity less than about 1.025. 
     
     
         105 .- 112 . (canceled)