Supramolecular modification of proteins

Abstract

The modification of biomolecules, small molecules, and other agents of via conjugation of excipients, tags, or labels is of great importance. For example, the modification of therapeutic agents can confer improved stability, solubility, duration of action, or pharmacological properties. Supramolecular chemistry utilizes specific, directional, reversible, non-covalent molecular recognition motifs in order to achieve organization of molecules, and can be used to complex tags to agents of interest (e.g., insulin, glucagon, antibodies). The present invention provides useful supramolecular complexes wherein an agent of interest is specifically bound to a host via non-covalent interactions, and wherein the host is conjugated to a tag. The present invention also provides methods and compounds useful in preparing supramolecular complexes, and methods of treating diseases using the supramolecular complexes.

What is claimed is: 
     
         1 . An agent associated with a tag, wherein:
 the agent is specifically bound to a host via non-covalent interactions; and   the host is conjugated to the tag.   
     
     
         2 . The agent associated with a tag of  claim 1 , wherein the agent is selected from the group consisting of proteins, peptides, nucleic acids, polysaccharides, lipids, small molecules, diagnostic agents, and imaging agents. 
     
     
         3 . The agent of  claim 2 , wherein the agent is selected from the group consisting of proteins, peptides, nucleic acids, and small molecules. 
     
     
         4 . The agent of  claim 3 , wherein the agent is a protein. 
     
     
         5 . The agent of  claim 4 , wherein the protein is a therapeutic protein. 
     
     
         6 . The agent of  claim 5 , wherein the protein is insulin. 
     
     
         7 . The agent of  claim 4 , wherein the protein is glucagon. 
     
     
         8 . The agent of any of  claims 1 - 7 , wherein the host is a macrocycle. 
     
     
         9 . The agent of  claim 8 , wherein the macrocyclic host is selected from the group consisting of cucurbiturils, cyclodextrins, calixarenes, pillararenes, porphyrins, metallacrowns, crown ethers, cyclotriveratrylenes, cryptophanes, cyclophanes and carcerands. 
     
     
         10 . The agent of  claim 9 , wherein the macrocyclic host is an optionally substituted cucurbituril. 
     
     
         11 . The agent of  claim 10 , wherein the cucurbituril is an optionally substituted cucurbit[n]uril (CB[n]), wherein n is an integer between 5-10, inclusive. 
     
     
         12 . The agent of  claim 11 , wherein n is 7; and the cucurbituril is optionally substituted cucurbit[7]uril (CB[7]). 
     
     
         13 . The agent of  claim 1 , wherein the tag is selected from the group consisting of polymers, therapeutic agents, targeting agents, radionuclides, fluorophores, chromophores, phosphorescent agents, dyes, chemiluminescent agents, particles, colorimetric labels, magnetic labels, haptens, biomolecules, fatty acids, hydrocarbon chains, small molecules, excipients, and diagnostic agents. 
     
     
         14 . The agent of  claim 13 , wherein the host is conjugated to the tag. 
     
     
         15 . The agent of  claim 14 , wherein the tag is a polymer. 
     
     
         16 . The agent of  claim 15 , wherein the polymer is a polyether. 
     
     
         17 . The agent of  claim 16 , wherein the polyether is polyethylene glycol (PEG). 
     
     
         18 . The agent of any of  claims 1 - 17 , wherein the host is conjugated to the tag via a linker. 
     
     
         19 . The agent of  claim 18 , wherein the linker is of Formula (L-1): 
       
         
           
           
               
               
           
         
       
       wherein:
 each of L 1  and L 2  is independently a bond, optionally substituted alkylene, or optionally substituted heteroalkylene; and 
 A is a bond, optionally substituted heterocyclyl, or optionally substituted heteroaryl. 
 
     
     
         20 . The agent of  claim 19 , wherein A is optionally substituted heteroaryl. 
     
     
         21 . The agent of  claim 20 , wherein A is an optionally substituted triazole moiety. 
     
     
         22 . The agent of  claim 18 , wherein the linker is of Formula (L-2a) or (L-2b): 
       
         
           
           
               
               
           
         
       
       wherein:
 each of L 1  and L 2  is independently a bond, optionally substituted alkylene or optionally substituted heteroalkylene; and 
 Y is CR a  or N; 
 m is an integer from 0-10, inclusive; 
 each instance of R a  is independently hydrogen, halogen, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted acyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, —OR aa , —SR aa , —N(R aa ) 2 , —NO 2 , or —CN, or two R a  attached to adjacent atoms are joined to form an optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl ring; 
 each occurrence of R aa  is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, optionally substituted sulfonyl, an oxygen protecting group, or a nitrogen protecting group, or two R aa  are joined to form an optionally substituted heterocyclic or optionally substituted heteroaryl ring. 
 
     
     
         23 . The agent of  claim 22 , wherein the linker is of Formula (L-3a) or (L-3b): 
       
         
           
           
               
               
           
         
       
     
     
         24 . The agent of  claim 23 , wherein L 1  is optionally substituted alkylene. 
     
     
         25 . The agent of  claim 23 , wherein L 2  is optionally substituted heteroalkylene. 
     
     
         26 . The agent of any one of  claims 22 - 25 , wherein the linker is of one of the following formulae: 
       
         
           
           
               
               
           
         
       
     
     
         27 . The agent of any one of  claims 1 - 26 , wherein the non-covalent interaction is a guest-host interaction. 
     
     
         28 . The agent of any one of  claims 1 - 26 , wherein the agent is a protein, and the host binds an amino acid residue on the protein. 
     
     
         29 . The agent of  claim 28 , wherein the host binds an N-terminal amino acid on the protein. 
     
     
         30 . The agent of  claim 28  or  29 , wherein the host binds an amino acid selected from the group consisting of phenylalanine, tryptophan, and tyrosine. 
     
     
         31 . The agent of  claim 28 , wherein the amino acid is phenylalanine. 
     
     
         32 . The agent of any one of  claims 28 - 31 , wherein the protein is insulin; and the host binds to the B1 phenylalanine of insulin. 
     
     
         33 . A compound comprising an optionally substituted cucurbituril conjugated to a tag via a linker, or a salt thereof; wherein:
 the tag is selected from the group consisting of polymers, therapeutic agents, targeting agents, radionuclides, fluorophores, chromophores, phosphorescent agents, dyes, chemiluminescent agents, particles, colorimetric labels, magnetic labels, haptens, biomolecules, small molecules, fatty acids, hydrocarbon chains, excipients, and diagnostic agents.   
     
     
         34 . The compound of  claim 33 , wherein the compound is of Formula (CB-1):
   CB-L 1 -A-L 2 -TAG  (CB-1),
   
       or a salt thereof, wherein:
 CB is an optionally substituted cucurbituril; 
 TAG is a tag selected from the group consisting of polymers, therapeutic agents, targeting agents, radionuclides, fluorophores, chromophores, phosphorescent agents, dyes, chemiluminescent agents, particles, colorimetric labels, magnetic labels, haptens, biomolecules, small molecules, fatty acids, hydrocarbon chains, excipients, and diagnostic agents; 
 each of L 1  and L 2  is independently a bond, optionally substituted alkylene, or optionally substituted heteroalkylene; and 
 A is a bond, optionally substituted heterocyclyl, or optionally substituted heteroaryl. 
 
     
     
         35 . The compound of  claim 34 , wherein A is optionally substituted heteroaryl. 
     
     
         36 . The compound of  claim 35 , wherein A is an optionally substituted triazole moiety. 
     
     
         37 . The compound of  claim 33 , wherein the compound is of Formula (CB-2a) or Formula (CB-2b): 
       
         
           
           
               
               
           
         
       
       wherein:
 CB is an optionally substituted cucurbituril; 
 TAG is a tag selected from the group consisting of polymers, therapeutic agents, targeting agents, radionuclides, fluorophores, chromophores, phosphorescent agents, dyes, chemiluminescent agents, particles, colorimetric labels, magnetic labels, haptens, biomolecules, small molecules, fatty acids, hydrocarbon chains, excipients, and diagnostic agents; 
 each of L 1  and L 2  is independently a bond, optionally substituted alkylene, or optionally substituted heteroalkylene; and 
 Y is CR a  or N; 
 each instance of R a  is independently hydrogen, halogen, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted acyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, —OR aa , —SR aa , —N(R aa ) 2 , —NO 2 , or —CN, or two R a  attached to adjacent atoms are joined to form an optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl ring; 
 each occurrence of R aa  is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, optionally substituted sulfonyl, an oxygen protecting group, or a nitrogen protecting group, or two R aa  are joined to form an optionally substituted heterocyclic or optionally substituted heteroaryl ring. 
 
     
     
         38 . The compound of  claim 37 , wherein the compound is of Formula (CB-3a) or (CB-3b): 
       
         
           
           
               
               
           
         
       
     
     
         39 . The compound of  claim 38 , wherein L 1  is optionally substituted alkylene. 
     
     
         40 . The compound of  claim 38 , wherein L 2  is optionally substituted heteroalkylene. 
     
     
         41 . The compound of any one of  claims 37 - 40 , wherein the compound is of one of the following formulae: 
       
         
           
           
               
               
           
         
       
     
     
         42 . The compound of any of  claims 33 - 41 , wherein the cucurbituril is an optionally substituted cucurbit[n]uril (CB[n]), wherein n is an integer between 5-10, inclusive. 
     
     
         43 . The compound of  claim 42 , wherein the cucurbituril is optionally substituted cucurbit[7]uril (CB[7]). 
     
     
         44 . The compound of any one of  claims 33 - 43 , wherein the tag is a polymer. 
     
     
         45 . The compound of  claim 44 , wherein the polymer is a polyether. 
     
     
         46 . The compound of  claim 45 , wherein the polyether is polyethylene glycol (PEG). 
     
     
         47 . The compound of any one of  claims 33 - 46 , wherein the compound is of Formula (PEG-T-CB[7]): 
       
         
           
           
               
               
           
         
       
     
     
         48 . A method of preparing the agent associated with a tag of any one of  claims 1 - 31 , the method comprising:
 contacting an agent with a host;   wherein the host is conjugated to the tag; and   wherein the tag is selected from the group consisting of polymers, therapeutic agents, targeting agents, radionuclides, fluorophores, chromophores, phosphorescent agents, dyes, chemiluminescent agents, particles, colorimetric labels, magnetic labels, haptens, biomolecules, small molecules, fatty acids, hydrocarbon chains, excipients, and diagnostic agents.   
     
     
         49 . The method of  claim 48 , wherein the host is a compound of any one of  claims 31 - 47 . 
     
     
         50 . The method of  claim 48  or  49 , wherein the step of contacting is performed in vitro. 
     
     
         51 . The method of  claim 48  or  49 , wherein the step of contacting is performed in vivo. 
     
     
         52 . A method of preparing a compound of any one of  claims 31 - 47 , the method comprising contacting a cucurbituril derivative comprising a first click chemistry handle with a tag comprising a second click chemistry handle. 
     
     
         53 . The method of  claim 52 , the method comprising contacting a cucurbituril derivative comprising an azide moiety with a tag comprising an alkyne moiety. 
     
     
         54 . The method of  claim 53 , the method comprising coupling a compound of Formula (Az-1): 
       
         
           
           
               
               
           
         
       
       or salt thereof, with a compound of Formula (Ak-1): 
       
         
           
           
               
               
           
         
       
       or salt thereof, to yield a compound of Formula (CB-2a) or (CB-2b): 
       
         
           
           
               
               
           
         
       
       or salt thereof, wherein:
 CB is an optionally substituted cucurbituril; 
 TAG is a tag selected from the group consisting of polymers, therapeutic agents, targeting agents, radionuclides, fluorophores, dyes, chemiluminescent agents, particles, colorimetric labels, magnetic labels, haptens, biomolecules, small molecules, fatty acids, hydrocarbon chains, excipients, and diagnostic agents; 
 each of L 1  and L 2  is independently a bond, optionally substituted alkylene or heteroalkylene; 
 m is an integer from 0-10, inclusive; 
 each instance of R a  is independently hydrogen, halogen, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted acyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, —OR aa , —SR aa , —N(R aa ) 2 , —NO 2 , or —CN, or two R a  attached to adjacent atoms are joined to form an optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl ring; and 
 each occurrence of R aa  is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, optionally substituted sulfonyl, an oxygen protecting group, or a nitrogen protecting group, or two R aa  are joined to form an optionally substituted heterocyclic or optionally substituted heteroaryl ring. 
 
     
     
         55 . The method of  claim 54 , comprising contacting a compound of Formula (Az-1), or salt thereof, with a compound of one of the following formulae: 
       
         
           
           
               
               
           
         
       
       or a salt thereof. 
     
     
         56 . The method of any one of  claim 54  or  55 , wherein the compound of Formula (Az-1) is of Formula (Az-2): 
       
         
           
           
               
               
           
         
       
     
     
         57 . The method of  claim 56 , wherein CB is optionally substituted cucurbit[7]uril (CB[7]). 
     
     
         58 . The method of any one of  claims 52 - 57 , wherein the tag comprising a click chemistry handle is of Formula (PEG-DBCO): 
       
         
           
           
               
               
           
         
       
     
     
         59 . The method of any one of  claims 52 - 58 , wherein the step of coupling is performed in the presence of copper. 
     
     
         60 . The method of any one of  claims 52 - 59 , wherein the step of coupling is a copper-free reaction. 
     
     
         61 . A pharmaceutical composition comprising an agent of any one of  claims 1 - 32 , or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. 
     
     
         62 . A method of treating a disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent of any one of  claims 1 - 32 , or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of  claim 61 . 
     
     
         63 . The method of  claim 62 , wherein the disease is a proliferative disease, genetic disorder, a disease associated with angiogenesis, an inflammatory disease, cardiovascular disease, hepatic disease, spleen disease, pulmonary disease, painful condition, hematological disease, neurological disease, psychiatric disorder, autoimmune disease, infectious disease, metabolic disease, or endocrine disease. 
     
     
         64 . The method of  claim 62 , wherein the disease is a diabetic condition. 
     
     
         65 . The method of  claim 64 , wherein the disease is Type I diabetes. 
     
     
         66 . A kit comprising an agent of any one of  claims 1 - 32 , or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of  claim 61 , and instructions for administering the compound or composition to a subject. 
     
     
         67 . A kit comprising a compound comprising a cucurbituril and a click chemistry handle, or salt thereof; instructions for preparing a compound of any one of  claims 33 - 47 ; and optionally a second compound comprising a tag and a click chemistry handle, or salt thereof. 
     
     
         68 . The agent of  claim 4 , wherein the agent is an antibody. 
     
     
         69 . The agent of  claim 68 , wherein the agent is a monoclonal antibody 
     
     
         70 . The agent of  claim 68 , wherein the agent is an antibody directed against human CD20. 
     
     
         71 . The method of  claim 63 , wherein the disease is a proliferative disease. 
     
     
         72 . The method of  claim 71 , wherein the proliferative disease is cancer. 
     
     
         73 . The method of  claim 72 , wherein the cancer is a hematological cancer. 
     
     
         74 . The method of  claim 72 , wherein the cancer is a lymphoma, leukemia, or multiple myeloma. 
     
     
         75 . The method of  claim 72 , wherein the cancer is leukemia. 
     
     
         76 . An agent according to any one of  claims 1 - 32 , or a pharmaceutical composition of  claim 61 , for use in treating a disease or condition in a subject in need thereof. 
     
     
         77 . Use of an agent according to any one of  claims 1 - 32 , or a pharmaceutical composition of  claim 61 , for the preparation of a medicament. 
     
     
         78 . Use of an agent according to any one of  claims 1 - 32 , or a pharmaceutical composition of  claim 61 , for use in treating a disease or condition in a subject in need thereof.