Atom transfer radical polymerization under biologically compatible conditions

Abstract

Methods for conducting controlled grafting-from radical polymerizations from biomolecules under conditions that are biologically compatible are described. The methods provide biomolecule-polymer conjugates with highly controlled structures and narrow polydispersities under aqueous reaction conditions and biological temperatures. Biomolecules, such as proteins and nucleotides can be conjugated to polymers with high levels of control.

1 . A process for forming a conjugate between a bioresponsive molecule and at least one polymer chain, the process comprising:
 polymerizing radically polymerizable monomers at a temperature of between about 4° C. and about 50° C. in the presence of an aqueous system comprising:
 a bioresponsive molecule having at least one site specific functional initiator comprising a radically transferable atom or group; 
 a transition metal that participates in a reversible reduction-oxidation cycle with at least one of the site specific functional initiator and a dormant polymer chain having a radically transferable atom or group, wherein the mole fraction of transition metal in a lower, activator oxidation state to transition metal in an higher, deactivator oxidation state is less than 20%; and 
 a ligand that forms a stable complex with the transition metal catalyst, 
 wherein the aqueous system comprises less than 30% by weight of organic solvent and monomer concentration and the total bioresponsive molecule concentration is less than about 3 mg/mL; and 
   forming a conjugate between the bioresponsive molecule and the at least one polymer chain, wherein the at least one polymer chain has a molecular weight distribution of less than 1.35.   
     
     
         2 . The process of  claim 1 , wherein the transition metal has a total concentration in the system of less than 1000 ppm. 
     
     
         3 . The process of  claim 1 , wherein the aqueous system comprises less than 20% by weight of organic solvent and monomer concentration. 
     
     
         4 . The process of  claim 1 , wherein the aqueous system further comprises a buffer. 
     
     
         5 . The process of  claim 4 , wherein the buffer is a phosphate buffer. 
     
     
         6 . The process of  claim 4 , wherein the buffer has a concentration of between 1 mM to 300 mM. 
     
     
         7 . The process of  claim 4 , wherein the buffer comprises the same counterion as the radically transferable atom or group. 
     
     
         8 . The process of  claim 1 , wherein the bioresponsive molecule is a molecule selected from the group consisting of a protein, an enzyme, a polypeptide, a peptide, a nucleic acid, a polynucleotide, a carbohydrate, a biologically active macromolecule, and combinations of any thereof. 
     
     
         9 . The process of  claim 1 , wherein the bioresponsive molecule is a protein. 
     
     
         10 . The process of  claim 9 , wherein the protein retains its structure, topology and activity in the conjugate. 
     
     
         11 . The process of  claim 1 , wherein the ligand is a strongly coordinating ligand. 
     
     
         12 . The process of  claim 1 , wherein a fraction of the transition metal is continuously reduced from the higher, deactivator oxidation state to the lower, activator oxidation state by controlled addition of a reducing agent or by controlled degradation of an added free radical initiator. 
     
     
         13 . The process of  claim 1 , wherein a fraction of the transition metal is continuously reduced from the higher, deactivator oxidation state to the lower, activator oxidation state by application of a potentiometric or galvanistic charge sufficient to maintain a targeted ratio of transition metal in the activator state to transition metal in the deactivator state. 
     
     
         14 . The process of  claim 12 , wherein the mole fraction of transition metal in the activator state to transition metal in the deactivator state is less than 10%. 
     
     
         15 . The process  claim 1 , wherein the concentration of the transition metal is from 10 ppm to 300 ppm. 
     
     
         16 . The process of  claim 1 , wherein the polymerizing radically polymerizable monomers comprises grafting from the at least one polymeric chain to the bioresponsive molecule. 
     
     
         17 . The process of  claim 1 , wherein the polymerizing radically polymerizable monomers is by a controlled radical polymerization process selected from a classic ATRP process, a reverse ATRP process, an AGET ATRP process, an ARGET ATRP process, an ICAR ATRP process, a RAFT polymerization process and an eATRP process. 
     
     
         18 . A conjugate between a bioresponsive molecule and one or more polymeric chains, wherein the one or more polymeric chains each have a molecular weight distribution of less than 1.20. 
     
     
         19 . The conjugate of  claim 18 , wherein the bioresponsive molecule is a molecule selected from the group consisting of a protein, an enzyme, a polypeptide, a peptide, a nucleic acid, a polynucleotide, a carbohydrate, a biologically active macromolecule, and combinations of any thereof. 
     
     
         20 . The conjugate of  claim 18 , wherein the bioresponsive molecule is a protein. 
     
     
         21 . The conjugate of  claim 18 , wherein the one or more polymeric chains have a structure resulting from a controlled radical polymerization process. 
     
     
         22 . The conjugate of  claim 18 , wherein the one or more polymeric chains are formed from radically polymerizable monomers. 
     
     
         23 . The conjugate of  claim 18 , wherein the one or more polymeric chains are linked to the bioresponsive molecule by a cleavable linkage. 
     
     
         24 . The conjugate of  claim 23 , wherein the cleavable linkage comprises a cleavable functionality selected from the group consisting of an ester functionality, a disulfide functionality, a phosphate functionality, and a thioester functionality. 
     
     
         25 . The process of  claim 13 , wherein the mole fraction of transition metal in the activator state to transition metal in the deactivator state is less than 10%. 
     
     
         26 . A conjugate, comprising:
 a bioresponsive molecule and one or more polymeric chains,
 wherein the one or more polymeric chains each have a molecular weight distribution of less than 1.20; 
 wherein the one or more polymeric chains have a structure resulting from a controlled radical polymerization process; and 
 wherein the bioresponsive molecule is a molecule selected from the group consisting of a protein, an enzyme, a polypeptide, a peptide, a nucleic acid, a polynucleotide, a carbohydrate, a biologically active macromolecule, and combinations of any thereof. 
   
     
     
         27 . The conjugate of  claim 26 , wherein the bioresponsive molecule is a protein. 
     
     
         28 . The conjugate of  claim 26 , wherein the one or more polymeric chains are formed from radically polymerizable monomers. 
     
     
         29 . The conjugate of  claim 26 , wherein the one or more polymeric chains are linked to the bioresponsive molecule by a cleavable linkage. 
     
     
         30 . The conjugate of  claim 29 , wherein the cleavable linkage comprises a cleavable functionality selected from the group consisting of an ester functionality, a disulfide functionality, a phosphate functionality, and a thioester functionality.