Covalent modification of biological macromolecules

Abstract

The present disclosure provides a method of covalently modifying a biological macromolecule, the method comprising subjecting a reaction mixture comprising: (a) a biological macromolecule comprising one or more thiol groups; and (b) a molecule comprising one or more olefin or alkyne moieties to a radical reaction under conditions sufficient to produce the covalently modified biological macromolecule. The present disclosure also provides a method of covalently modifying a biological macromolecule, the method comprising subjecting a reaction mixture comprising: (a) a molecule comprising one or more thiol groups; and (b) biological macromolecule comprising one or more olefin or alkyne moieties to a radical reaction under conditions sufficient to produce the covalently modified biological macromolecule. The present disclosure further provides a covalently modified biological macromolecule prepared by any of disclosed methods. The covalently modified biological macromolecules may be further crosslinked to form a scaffold.

1 . A method of covalently modifying a biological macromolecule, the method comprising subjecting a reaction mixture comprising:
 (a) a biological macromolecule comprising one or more thiol groups; and   (b) a molecule comprising one or more olefin or alkyne moieties   to a radical reaction under conditions sufficient to produce the covalently modified biological macromolecule.   
     
     
         2 . The method of  claim 1 , wherein the biological macromolecule is a protein. 
     
     
         3 . The method of  claim 1 , wherein the radical reaction is a photoinitiated radical reaction. 
     
     
         4 . The method of  claim 1 , wherein the radical reaction is performed under non-denaturing conditions. 
     
     
         5 . The method of  claim 1 , wherein the biological macromolecule comprises one or more lysine side chains, and wherein the one or more thiol groups are introduced into the biological macromolecule by converting the one or more lysine side chains into the one or more thiol groups. 
     
     
         6 . The method of  claim 1 , wherein the one or more thiol groups are introduced into the biological macromolecule by site directed mutagenesis. 
     
     
         7 . The method of  claim 1 , wherein the biological macromolecule comprises one or more disulfide bonds, and wherein the one or more thiol groups are introduced into the biological macromolecule by converting the one or more disulfide bonds into the one or more thiol groups. 
     
     
         8 . The method of  claim 1 , wherein the one or more olefin containing moieties further comprises one or more groups selected from the group consisting of poly(lactic acid) (PLA), polyglycolide (PGA), copolymer of PLA and PGA (PLGA), poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG), poly(ethylene oxide), poly(ethylene oxide)-co-poly(propylene oxide) block copolymer (poloxamers, meroxapols), poloxamine, polyanhydride, polyorthoester, poly(hydroxy acid), polydioxanone, polycarbonate, polyaminocarbonate, poly(vinyl pyrrolidone), poly(ethyl oxazoline), carboxymethyl cellulose, hydroxyalkylated cellulose, hydroxyethyl cellulose, and methylhydroxypropyl cellulose. 
     
     
         9 . The method of  claim 1 , wherein the one or more olefin containing moieties further comprises one or more groups selected from the group consisting of polypeptide, polysaccharide, carbohydrate, polysucrose, hyaluranic acid, dextran, dextran derivative, heparan sulfate, chondroitin sulfate, heparin, and alginate. 
     
     
         10 . The method of  claim 1 , wherein the one or more olefin moieties further comprises one or more groups selected from the group consisting of gelatin, collagen, albumin, ovalbumin, fibrinogen, fibrin, laminin, fibronectin, vitronectin, fluorescent protein, green fluorescent protein, fluorescent dye, non-fluorescent dye, fluorescence quencher, polypeptide tag, His-tag, FLAG-tag, antibody, antibody fragment, nucleic acid aptamer, oligonucleotide and polynucleotide, ribonucleoprotein, and viral capsids. 
     
     
         11 . The method of  claim 1 , wherein the biological macromolecule is fibrinogen. 
     
     
         12 . The method of  claim 1 , wherein the biological macromolecule is chymopapain. 
     
     
         13 . A method of covalently modifying a biological macromolecule, the method comprising subjecting a reaction mixture comprising:
 (a) a molecule comprising one or more thiol groups; and   (b) a biological macromolecule comprising one or more olefin or alkyne moieties   to a radical reaction under conditions sufficient to produce the covalently modified biological macromolecule.   
     
     
         14 . The method of  claim 13 , wherein the biological macromolecule is a protein. 
     
     
         15 . The method of  claim 13 , wherein the radical reaction is a photoinitiated radical reaction. 
     
     
         16 . The method of  claim 13 , wherein the radical reaction is performed under non-denaturing conditions. 
     
     
         17 . The method of  claim 13 , wherein the biological macromolecule is fibrinogen. 
     
     
         18 . The method of  claim 13 , wherein the biological macromolecule is chymopapain. 
     
     
         19 . The covalently modified biological macromolecule prepared according to the method of  claim 1 . 
     
     
         20 . The covalently modified biological macromolecule prepared according to the method of  claim 13 .