Multi-armed, monofunctional, and hydrolytically stable derivatives of poly (ethylene glycol) and related polymers for modification of surfaces and molecules

Abstract

Multi-armed, monofunctional, and hydrolytically stable polymers are described having the structure wherein Z is a moiety that can be activated for attachment to biologically active molecules such as proteins and wherein P and Q represent linkage fragments that join polymer arms poly a and poly b , respectively, to central carbon atom, C, by hydrolytically stable linkages in the absence of aromatic rings in the linkage fragments. R typically is hydrogen or methyl, but can be a linkage fragment that includes another polymer arm. A specific example is an mPEG disubstituted lysine having the structure where mPEG a and mPEG b have the structure CH 3 O—(CH 2 CH 2 O) n CH 2 CH 2 — wherein n may be the same or different for poly a - and poly b - and can be from 1 to about 1,150 to provide molecular weights of from about 100 to 100,000.

What is claimed is:  
     
         1 . A polymeric derivative represented by the structure  
       
         
           
           
               
               
           
         
       
       wherein poly a  and poly b  are nonpeptidic and substantially nonreactive water soluble polymeric arms that may be the same or different, wherein C is carbon, wherein P and Q comprise linkage fragments that may be the same or different and join polymeric arms poly a  and poly b , respectively, to C by hydrolytically stable linkages in the absence of aromatic rings in said linkage fragments, wherein R is a moiety selected from the group consisting of H, substantially nonreactive moieties, and linkage fragments having attached thereto by a hydrolytically stable linkage in the absence of aromatic rings one or more nonpeptidic and substantially nonreactive water soluble polymeric arms, and wherein Z comprises a moiety selected from the group consisting of moieties having a single site reactive toward nucleophilic moieties, sites that can be converted to sites reactive toward nucleophilic moieties, and the reaction product of a nucleophilic moiety and moieties having a single site reactive toward nucleophilic moieties.  
     
     
         2 . The polymeric derivative of  claim 1  wherein said hydrolytically stable linkages are selected from the group consisting of amide, amine, ether, carbamate, thiourea, urea, thiocarbamate, thiocarbonate, thioether, thioester, and dithiocarbamate linkages.  
     
     
         3 . The polymeric derivative of  claim 1  wherein said nucleophilic moieties are selected from the group consisting of amino, thiol, and hydroxyl moieties.  
     
     
         4 . The polymeric derivative of  claim 1  wherein said nucleophilic moiety is a biologically active molecule.  
     
     
         5 . The polymeric derivative of  claim 4  wherein said biologically active molecule is selected from the group consisting of polypeptides, polynucleotides, and lipids.  
     
     
         6 . The polymeric derivative of  claim 1  wherein said nucleophilic moiety is a solid surface or a particle.  
     
     
         7 . The polymeric derivative of  claim 6  wherein said solid particle is a liposome.  
     
     
         8 . The polymeric derivative of  claim 1  wherein Z is selected from the group consisting of carboxyl, hydroxyl, activated carboxyl, activated hydroxyl, and conjugates of activated carboxyl or hydroxyl sites and molecules having at least one reactive nucleophilic moiety.  
     
     
         9 . The polymeric derivative of  claim 1  wherein Z comprises a moiety selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters.  
     
     
         10 . The polymeric derivative of  claim 9  wherein said active ester is N-hydroxylsuccinimidyl ester and said active carbonates are selected from the group consisting of N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate.  
     
     
         11 . The polymeric derivative of  claim 1  wherein said nonpeptidic polymeric arms are selected from the group consisting of poly(alkylene oxides), poly(oxyethylated polyols), and poly(oxyethylated glucose).  
     
     
         12 . The polymeric derivative of  claim 1  wherein said nonpeptidic polymeric arms are selected from the group consisting of poly(ethylene glycol), poly(vinyl alcohol), poly(propylene glycol), poly(oxyethylated glycerol), poly(oxyethylated sorbitol), poly(oxyethylated glucose), poly(oxazoline), poly(acryloylmorpholine), and poly(vinylpyrrolidone).  
     
     
         13 . The polymeric derivative of  claim 1  wherein said nonpeptidic polymeric arms are linear mPEGs of molecular weight of from about 50 to 50,000.  
     
     
         14 . The polymeric derivative of  claim 1  wherein said linkage fragments P and Q comprise hydrolytically stable linkages in the absence of aromatic rings to one or more nonpeptidic and water soluble polymeric arms.  
     
     
         15 . The polymeric derivative of  claim 1  wherein R comprises a linkage fragment attached by a hydrolytically stable linkage in the absence of aromatic rings to a nonpeptidic and substantially nonreactive water soluble polymeric arm.  
     
     
         16 . The polymeric derivative of  claim 15  wherein R is represented by the general structure —M-poly d , wherein poly d  is said polymeric arm and M is said linkage fragment.  
     
     
         17 . The polymeric derivative of  claim 1  wherein Z further comprises a linkage fragment attached by a hydrolytically stable linkage in the absence of aromatic rings to a nonpeptidic and substantially nonreactive water soluble polymeric arm.  
     
     
         18 . A polymeric derivative represented by the structure  
       
         
           
           
               
               
           
         
       
       wherein poly a  and poly b  may be the same or different and are selected from the group consisting of linear poly(ethylene glycol), poly(vinyl alcohol), poly(propylene glycol), poly(oxyethylated glycerol), poly(oxyethylated sorbitol), poly(oxyethylated glucose), poly(oxazoline), poly(acryloylmorpholine), and poly(vinylpyrrolidone); wherein C is carbon; wherein P and Q comprise linkage fragments that may be the same or different and join polymeric arms poly a  and poly b , respectively, to C by hydrolytically stable linkages selected from the group consisting of amide, amine, ether, carbamate, thiourea, urea, thiocarbamate, thiocarbonate, thioether, thioester, and dithiocarbamate linkages; wherein R is a moiety selected from the group consisting of H, substantially nonreactive moieties, and linkage fragments having attached thereto by a hydrolytically stable linkage in the absence of aromatic rings one or more nonpeptidic and substantially nonreactive water soluble polymeric arms; and wherein Z comprises a moiety selected from the group consisting of carboxyl, hydroxyl, trifluoroethylsulfonate, isocyanate, isothiocyanate, N-hydroxylsuccinimidyl ester, N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, trichlorophenylcarbonate, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters.  
     
     
         19 . A multi-armed monofunctional polymeric derivative that is the reaction product of at least one monofunctional nonpeptidic polymer derivative and a linker moiety having two or more active sites that form linkages with said monofunctional nonpeptidic polymer derivatives in the absence of aromatic moieties, wherein said linkages between said linker moiety and said monofunctional nonpeptidic polymer derivatives are hydrolytically stable.  
     
     
         20 . The multi-armed monofunctional polymeric derivative of  claim 19  wherein said linker moiety is selected from the group consisting of monohydroxy alcohols and monocarboxylic acids.  
     
     
         21 . The multi-armed monofunctional polymer derivative of  claim 19  wherein said active sites on said linker moiety are nucleophilic moieties.  
     
     
         22 . The multi-armed monofunctional polymer derivative of  claim 21  wherein said nucleophilic moieties are selected from the group consisting of amino, thiol, and hydroxyl moieties.  
     
     
         23 . The multi-armed monofunctional polymer derivative of  claim 19  wherein said active sites on said linker moiety are electrophilic moieties.  
     
     
         24 . The multi-armed monofunctional polymer derivative of  claim 23  wherein said electrophilic moieties are selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters.  
     
     
         25 . The multi-armed monofunctional polymeric derivative of  claim 24  wherein said active esters are N-hydroxylsuccinimidyl ester and said active carbonates are selected from the group consisting of N-hydroxylsuccinimidyl carbonates, p-nitrophenylcarbonates, and trichlorophenylcarbonates.  
     
     
         26 . The multi-armed monofunctional polymeric derivative of  claim 19  wherein said hydrolytically stable linkages in the absence of aromatic rings are selected from the group consisting of amide, amine, ether, carbamate, thiourea, urea, thiocarbamate, thiocarbonate, thioether, thioester, and dithiocarbamate linkages.  
     
     
         27 . The multi-armed monofunctional polymeric derivative of  claim 19  wherein said monofunctionality is selected from the group consisting of carboxyl, hydroxyl, activated carboxyl, activated hydroxyl, and conjugates of activated carboxyl or hydroxyl sites and molecules having at least one reactive nucleophilic moiety.  
     
     
         28 . The multi-armed monofunctional polymeric derivative of  claim 19  wherein said monofunctionality is selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters.  
     
     
         29 . The multi-armed monofunctional polymeric derivative of  claim 28  wherein said active ester is N-hydroxylsuccinimide and said active carbonates are selected from the group consisting of N-hydroxylsuccinimide carbonates, p-nitrophenylcarbonates, and trichlorophenylcarbonates.  
     
     
         30 . The multi-armed monofunctional polymeric derivative of  claim 19  wherein said nonpeptidic polymeric derivative is selected from the group consisting of poly(alkylene oxides), poly(oxyethylated polyols), and poly(oxyethylated glucose).  
     
     
         31 . The multi-armed monofunctional polymeric derivative of  claim 19  wherein said nonpeptidic polymer derivative is selected from the group consisting of activated poly(ethylene glycol), poly(vinyl alcohol), poly(propylene glycol), poly(oxyethylated glycerol), poly(oxyethylated sorbitol), poly(oxyethylated glucose), poly(oxazoline), poly(acryloylmorpholine), and poly(vinylpyrrolidone).  
     
     
         32 . The multi-armed monofunctional polymeric derivative of  claim 19  wherein said nonpeptidic polymer derivative is a linear mPEG of molecular weight of from about 50 to 50,000 and the multi-armed monofunctional polymeric derivative has two arms of said linear mPEG.  
     
     
         33 . A material comprising a solid surface or particle having attached thereto compounds of the structure claimed in  claim 19 .  
     
     
         34 . The material of  claim 33  wherein said solid surface or particle is a liposome.  
     
     
         35 . A biologically active structure comprising a biologically active molecule having attached thereto one or more compounds of the structure claimed in  claim 19 .  
     
     
         36 . The biologically active structure of  claim 35  wherein said biologically active molecule is selected from the group consisting of polypeptides, polynucleotides, and lipids.  
     
     
         37 . The biologically active structure of  claim 36  wherein said polypeptide is selected from the group consisting of asparaginase, catalase, ribonuclease, subtilisine, trypsin, and uricase.  
     
     
         38 . A two-armed polymeric derivative having a structure selected from the group consisting of:  
       
         
           
           
               
               
           
         
       
       wherein poly a  and poly b  may be the same or different and comprise moieties selected from the group consisting of poly(ethylene glycol), poly(vinyl alcohol), poly(propylene glycol), poly(oxyethylated glycerol), poly(oxyethylated sorbitol), poly(oxyethylated glucose), poly(oxazoline), poly(acryloylmorpholine), and poly(vinylpyrrolidone) moieties; and wherein Z comprises a moiety selected from the group consisting of moieties having a single site reactive toward nucleophilic moieties, sites that can be converted to sites reactive toward nucleophilic moieties, and the reaction product of a nucleophilic moiety and moieties having a single site reactive toward nucleophilic moieties.  
     
     
         39 . The two-armed polymeric derivative of  claim 38  wherein said reactive site is selected from the group consisting of carboxyl, activated carboxyl, hydroxyl, activated hydroxyl, and conjugates of activated carboxyl or hydroxyl sites and molecules having at least one reactive nucleophilic moiety.  
     
     
         40 . The polymeric derivative of  claim 38  wherein Z comprises a moiety selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters.  
     
     
         41 . The polymeric derivative of  claim 40  wherein said active ester is N-hydroxylsuccinimidyl ester and said active carbonates are selected from the group consisting of N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate.  
     
     
         42 . A molecule having the structure  
       
         
           
           
               
               
           
         
       
       wherein mPEG a  and mPEG b  have the structure CH 3 —(CH 2 CH 2 O) n CH 2 CH 2 —, wherein n equals from 1 to about 1,150, and wherein n may be the same or different for mPEG a  and mPEG b .  
     
     
         43 . The molecule of  claim 42  wherein n equals from 1 to about 570.  
     
     
         44 . A method of synthesizing a multi-armed, water soluble, monofunctional polymeric molecule comprising reacting one or more nonpeptidic monofunctional polymers of the structure poly-W, wherein W is an active moiety providing the monofunctionality for the polymer, with a linker moiety having two or more active sites with which W is reactive, and forming hydrolytically stable linkages in the absence of aromatic rings between the monofunctional polymer and the linker moiety at the linker moiety active sites, the linker moiety having a reactive site for which said active moiety —W is not reactive to provide the monofunctionality for the multi-armed molecule.  
     
     
         45 . The method of  claim 44  wherein the method further comprises activating the reactive site after the multi-armed polymeric compound is formed with an electrophilic moiety.  
     
     
         46 . The method of  claim 45  wherein the electrophilic moiety is reactive with nucleophilic moieties selected from the group consisting of amino, thiol, and hydroxyl moieties.  
     
     
         47 . The method of  claim 44  wherein the active moiety W is an electrophilic moiety selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters.  
     
     
         48 . The method of  claim 47  wherein the active ester is N-hydroxylsuccinimidyl ester and the active carbonates are selected from the group consisting of N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate.  
     
     
         49 . The method of  claim 44  wherein the active moiety W is a nucleophilic moiety selected from the group consisting of amino, thiol, and hydroxyl moieties.  
     
     
         50 . The method of  claim 44  wherein the active sites on the linker moiety are nucleophilic moieties selected from the group consisting of amino, thiol, and hydroxyl moieties.  
     
     
         51 . The method of  claim 44  wherein the active sites on the linker moiety are electrophilic moieties selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters.  
     
     
         52 . The method of  claim 51  wherein the active ester is N-hydroxylsuccinimidyl ester and the active carbonates are selected from the group consisting of N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate.  
     
     
         53 . The method of  claim 44  wherein the hydrolytically stable linkages are selected from the group consisting of amide, amine, ether, carbamate, thiourea, urea, thiocarbamate, thiocarbonate, thioether, thioester, and dithiocarbamate linkages.  
     
     
         54 . A method for preparing a polymeric derivative represented by the structure  
       
         
           
           
               
               
           
         
       
       comprising the steps of: 
 a) reacting nonpeptidic, water soluble, monofunctional polymers of the structure poly a -W and poly b -W with a linker moiety having at least two active sites for which W is selective, a reactive site Z for which W is not selective, and a moiety R which is substantially nonreactive, wherein W is an active electrophilic moiety selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters, and may be the same or different on poly a  and poly b , wherein poly a  and poly b  are polymer moieties selected from the group consisting of poly(ethylene glycol), poly(vinyl alcohol), poly(propylene glycol), poly(oxyethylated glycerol), poly(oxyethylated sorbitol), poly(oxyethylated glucose), poly(oxazoline), poly(acryloylmorpholine), and poly(vinylpyrrolidone) and may be the same or different, and wherein the active sites of the linker moiety are nucleophilic sites selected from the group consisting of amino, thiol, and hydroxyl; and  
 b) forming hydrolytically stable linkages P and Q, which may be the same or different, in the absence of aromatic rings between the polymer and the linker moiety that are selected from the group consisting of amide, amine, ether, carbamate, thiourea, urea, thiocarbamate, thiocarbonate, thioether, thioester, and dithiocarbamate linkages.  
 
     
     
         55 . The method of  claim 54  wherein the linker moiety is substituted with polymer at each active site in one step.  
     
     
         56 . The method of  claim 55  wherein the linker moiety is substituted with polymer at each active site in more than one step.  
     
     
         57 . The multi-armed polymeric derivative of  claim 54  wherein said linker moiety is selected from the group consisting of monohydroxy alcohols and monocarboxilic acids having two or more active moieties selected from the group consisting of thiol, amino, and hydroxyl moieties.  
     
     
         58 . The multi-armed polymeric derivative of  claim 1  wherein Z is selected from the group consisting of carboxyl, hydroxyl, activated carboxyl, activated hydroxyl, and conjugates of precursor activated carboxyl or hydroxyl sites and molecules having sites for which said precursor activated sites are active.  
     
     
         59 . A method for forming monofunctional monomethoxy-poly(ethylene glycol) disubstituted lysene comprising the following step:  
       
         
           
           
               
               
           
         
       
     
     
         60 . The method of  claim 59  wherein the reaction takes place in water at a pH of about 8.0.  
     
     
         61 . The method of  claim 59  further comprising the steps of  
       
         
           
           
               
               
           
         
       
     
     
         62 . The method of  claim 61  wherein steps a) and b) take place in methylene chloride.  
     
     
         63 . The method of  claim 59  further comprising the steps of activating the carboxyl moiety and reacting the activated carboxyl moiety with an active moiety to join the disubstituted lysine to the active moiety.  
     
     
         64 . A method for forming a monofunctional monomethoxy-poly(ethylene glycol) disubstituted compound comprising the following steps:  
       
         
           
           
               
               
           
         
       
     
     
         65 . The method of  claim 64  further comprising the steps of activating the carboxyl moiety and reacting the activated carboxyl moiety with an active moiety to join the disubstituted lysine to the active moiety.  
     
     
         66 . The method of  claim 64  wherein step a) takes place in aqueous buffer.  
     
     
         67 . The method of  claim 64  wherein step b) takes place in methylene chloride.