Branched Discreet PEG Constructs

Abstract

Disclosed are general and “substantially pure” branched discrete polyethylene glycol constructs useful in attaching to a variety of biologically active groups, for example, preferential locators, as well as biologics like enzymes, for use in diagnostics, e.g. imaging, therapeutics, theranostics, and moieties specific for other applications. In its simplest intermediate state, a branched discrete polyethylene glycol construct is terminated at one end by a chemically reactive moiety, “A”, a group that is reactive with a biologic material that creates “A”, which is a biologically reactive group, connected through to a branched core (BC) which has attached at least two dPEG-containing chains, indicated by the solid line, , having terminal groups, which can be charged, non-reactive or reactable moieties and containing between about 2 and 64 dPEG residues.

1 . A substantially pure compound represented by: 
       
         
           
           
               
               
           
         
         where, 
         (a) A is a biologically active group or a chemically reactive or reactable moiety; 
         (b) the wavy line,  , is a linear discrete polyethylene glycol chain containing between about 4 and 48 discrete ethylene oxide residues optionally substituted with N, S, Si, Se, or P, aryl groups, or alkyl groups; and having chemically reactive or reactable end groups that are independently reactable; and optionally having branching side chains which can contain diagnostic or therapeutic groups and being cleavable; 
         (c) BC is a branching core containing a carbon core with 3 or 4 reactable sites, one of said carbon reactable sites being unique, for attachment of   and the other 2 or 3 carbon reactable sites being independently reactive or reactable moieties, for attachment of  , respectively; and 
         (d) 
       
       
         
           
           
               
               
           
         
          respectively, where the solid lines,  , are discrete polyethylene glycol containing chains that have between about 2 and 64 ethylene oxide residues and have a terminal moiety, wherein the terminal moiety can be neutral, charged, or independently reactable. 
       
     
     
         2 . The substantially pure compound of  claim 1 , wherein A comprises a preferential locator that binds with a biological moiety, said preferential locator being one or more of a cell surface and matrix antigen, transport protein, or receptor protein. 
     
     
         3 . The substantially pure compound of  claim 2 , wherein said preferential locator A is one or more of an antibody, antibody fragment, engineered antibody, engineered fragment, peptide substrate, peptomimetic substrate, cytokine, aptamer, siRNA, vitamin, or steroid. 
     
     
         4 . The substantially pure compound of  claim 1 , wherein A is a nanoparticle, microparticle, engineered molecular scaffold that contains multiple functionalities, or an engineered chemically reactable group. 
     
     
         5 . The substantially pure compound of  claim 1 , wherein A is a chemically reactable moiety being one or more of an alkyl or aryl functionalized 1° or 2° amine, hydroxyl, aldehyde, ketone, carboxylate, active carboxylate ester, thiol, maleimide, haloacetyl, vinyl sulfone, oxyamine, hydrazide, acylhydrazide, azide, alkyne, or alkene, where the chemically reactive moiety can be in the form of a chemically reactable moiety. 
     
     
         6 . The substantially pure compound of  claim 1 , wherein the solid lines in 
       
         
           
           
               
               
           
         
       
       as part of 
       
         
           
           
               
               
           
         
       
       respectively are terminated by a charged group that is either negative, positive or zwitterionic. 
     
     
         7 . The substantially pure compound of  claim 6 , wherein said charged group is a carboxylate group. 
     
     
         8 . The substantially pure compound of  claim 6 , wherein said charged group contains one or more nitrogen atoms. 
     
     
         9 . The compound of  claim 6 , wherein said zwitterionic moiety contains both a nitrogen atom and a carboxylate group. 
     
     
         10 . The substantially pure compound of  claim 1 , represented by: 
       
         
           
           
               
               
           
         
       
       wherein, each BC group may have, independently, 
       
         
           
           
               
               
           
         
       
       attached. 
     
     
         10 . The substantially pure compound of  claim 1 , represented by: 
       
         
           
           
               
               
           
         
         wherein, BC is a trifunctional core and may be the same as BC 1 , or BC 2 ; 
         where BC 1  and BC 2  are not the same; and BC 1  and BC 2  independently may have attached either 
       
       
         
           
           
               
               
           
         
          where each is different for attachment to BC 1  and BC 2 , respectively, but can be either 
       
       
         
           
           
               
               
           
         
       
     
     
         11 . The substantially pure compound of  claim 1 , wherein A is one or more of 
       
         
           
           
               
               
           
         
       
     
     
         12 . The substantially pure compound of  claim 1 , represented by: 
       
         
           
           
               
               
           
         
         wherein A 1 , A 2 , and A 3  independently, may be the same or different; and 
         any BC, independently, may be the same or different. 
       
     
     
         13 . The substantially pure compound of  claim 1 , wherein each wavy line,  , contains between about 2 and 24 ethylene oxide groups. 
     
     
         14 . The substantially pure compound of  claim 1 , wherein each solid line,  , independently, contains between about 0 and 64 ethylene oxide groups. 
     
     
         16 . The substantially pure compound of  claim 10 , wherein each wavy line,  , attached to AC contains, independently, between about 3 and 64 ethylene oxide groups. 
     
     
         17 . The substantially pure compound of  claim 11 , wherein each wavy line,  , attached to AC contains, independently, between about 3 and 64 ethylene oxide groups. 
     
     
         18 . A substantially pure compound represented by: 
       
         
           
           
               
               
           
         
         wherein, 
         (a) AC is an attachment core and G is a chemically reactable moiety or a moiety useful for one or more diagnostic or therapeutic applications, and where x=1 to 10; and A is a biologically active group or a chemically reactive or reactable moiety; optionally G is a branched chain. 
         (b) the wavy line,  , is a linear discrete polyethylene glycol chain containing between about 4 and 48 discrete ethylene oxide residues optionally substituted with N, S, Si, Se, P, aryl groups, or alkyl groups; and having chemically reactive or reactable end groups that are independently reactable; and optionally having branching side chains and being cleavable; 
         (c) BC is a branching core containing a carbon core with 3 or 4 reactable sites, one of said carbon reactable sites being unique and the other 2 or 3 carbon reactable sites being independently reactive or reactable moieties; and 
         (d) 
       
       
         
           
           
               
               
           
         
          are part of 
       
       
         
           
           
               
               
           
         
          respectively, where the solid lines,  , are discrete polyethylene glycol containing chains that have between about 2 and 64 ethylene oxide residues and have a terminal moiety, wherein the terminal moiety can be neutral, charged, or independently reactable. 
       
     
     
         19 . The substantially pure compound of  claim 18 , wherein G is a diagnostic group being one or more of radiolabels, chelated radiolabels, chromogenic and fluorescent dyes, biotin and its derivatives, and combinations thereof. 
     
     
         20 . The substantially pure compound of  claim 18 , wherein G is a therapeutic group being one or more of radionuclides, toxic drugs, cytotoxins, cytokines, enzymes, and combinations thereof. 
     
     
         21 . The substantially pure compound of  claim 18 , represented by: 
       
         
           
           
               
               
           
         
         where any AC, independently, may be the same or different; and any G, independently, may be the same or different; 
         wherein, the solid lines in 
       
       
         
           
           
               
               
           
         
          as part of 
       
       
         
           
           
               
               
           
         
          respectively are terminated by a charged group that is either negative, positive or zwitterionic. 
       
     
     
         22 . The substantially pure compound of  claim 18 , wherein each wavy line,  , attached to AC contains, independently, between about 3 and 64 ethylene oxide groups. 
     
     
         23 . A method of externally imaging an antibody, antibody fragment, or a peptide attached to an externally imagable radiolabel, which comprises attaching said imagable radiolabel to and said antibody, antibody fragment, or a peptide to the structure of  claim 10 , wherein RG is said radiolabel and A comprises said antibody, antibody fragment, or a peptide. 
     
     
         24 . The method of  claim 23 , wherein said antibody has been generated against a tumor associated glycoprotein. 
     
     
         25 . The method of  claim 24 , wherein said antibody comprises B72.3, CC49, V59, or 3E8. 
     
     
         26 . The method of  claim 25 , wherein said antibody comprises CC49, domain deleted CC49, CC49 fragments, including Fab′, CC49 diabodies, CC49 scFv, and single chains derived therefrom. 
     
     
         27 . The method of  claim 23 , wherein said antibody comprises 3E8, domain deleted 3E8, 3E8 fragments, 3E8 diabodies, and single chains derived therefrom. 
     
     
         28 . A substantially pure compound represented by: 
       
         
           
           
               
               
           
         
         where, 
         (a) A is a biologically active group or a chemically reactive or reactable moiety; 
         (b) the first wavy line,  , is a linear discrete polyethylene glycol chain containing between about 4 and 48 discrete ethylene oxide residues optionally substituted with N, S, Si, Se, P, aryl groups, or alkyl groups; and having chemically reactive or reactable end groups that are independently reactable; and optionally having branching side chains and being cleavable; 
         (c) when x=1, the compound is part of a second  ; 
         (d) when x=2 or 3, the aryl core is part of a unique BC, where O   A is a solid line,  , with a terminal group; and 
         (e) R is any group known in the art that can be added as an additional attachment point for another A; 
         wherein, the solid line,  , is a discrete polyethylene glycol-containing chain that has between about 2 and 64 ethylene oxide residues and have a terminal moiety, wherein the terminal moiety can be neutral, charged, or independently reactable; and BC is a branching core containing a carbon core with 3 or 4 reactable sites, one of said carbon reactable sites being unique, for attachment of   and the other 2 or 3 carbon reactable sites being independently reactive or reactable moieties, for attachment of  , respectively. 
       
     
     
         29 . The substantially pure compound of  claim 28 , wherein said aryl core is one or more of tyrosine, methyl or benzyl ester of tyrosine, 2,3-hydroxybenzoic acid, 2,4-hydroxybenzoic acid, 2,3-aminophenol, 4-aminophenol, 3,5-dihyroxybenzoic acid, 2,4-dihyroxybenzoic acid, or 2,4,6-trihydroxybenzoic acid. 
     
     
         30 . A substantially pure compound, being one or more of:
 A-dPEG x -Tris(A) 3 , A-dPEG x -Tris(-dPEG x -m) 3 , x same or different; x=2-64;   A-dPEG x -Tris(-dPEG x -A) 3 , x same or different; x=2-64;   A-dPEG x -Tris(-dPEG x -Tris(m-dPEG x ) 3 ) 3 , x same or different; x=2-64;   A-dPEG x -Tris(-dPEG x -TFP ester) 3 , x same or different; x=2-64;   A-dPEG x -Tris(-dPEG x -Tris(-A/CO 2 H) 3 ) 3 , x same or different; x=2-64;   A-dPEG x -Tris(-dPEG x -Tris(-dPEG x -Tris(-dPEG x -m) 3 ) 3 ) 3 , x same or different; x=2-64;   TBE-dPEG x -Lys(-dPEG x -A) 2 , TBE-dPEG x -Lys(-dPEG x -MAL) 2 ;   TBE-dPEG x -Lys(-dPEG x -Lys(-dPEG x -A) 2 ) 2 ;   A-dPEGx-Tyr(-dPEGx-A)-OH/TFP, x same or different; x=2-64;   A-dPEG x -Tyr(-dPEG x -Tris(-dPEG x -A)-dPEG x -Tris(-dPEG x -m/A) 3 , x same or different, x=2-64; or   A-dPEG 4 -(AC)-dPEG 4 -(AC)-dPEG 4 -(AC)-dPEG 4 -(AC)-OH.   
     
     
         31 . The substantially pure compound of  claim 30 , being one or more of:
 H 2 NTris(TBE) 3 ;   PhthN-dPEG x -Tris(CO 2 H);   PhthN-dPEG x -Tris(TFP/active ester) 3 , x=8, 12, 24;   NH 2 -dPEG x -Tris(m-dPEG 11  and m-dPEG 24 ) 3 , x=8, 12, 24;   PhthN-dPEG x -Tris(-dPEG y N 3 ) 3 ; x=8, 12, 24; y=7, 11, 23, 35;   MAL-dPEG 12 -Tris(-dPEG 24 -CO 2 H) 3 ;   MAL-dPEG 12 -Tris(m-dPEG 24 ) 3 ;   H 2 N-dPEG 12 -Tris(-dPEG 12 -Tris(-dPEG 11 -m) 3 ) 3 ;   MAL-dPEG 12 -Tris(-dPEG 12 -Tris(m-dPEG 11 ) 3 ) 3 ;   H 2 N-dPEG 24 -Tris(-dPEG 12 -Tris(-dPEG 24 -m) 3 ) 3 ;   MAL-dPEG 24 -Tris(-dPEG 12 -Tris(m-dPEG 24 ) 3 ) 3 ;   MAL-dPEG 24 -Tris(-dPEG 24 -Tris(m-dPEG 24 ) 3 ) 3 ;   PhthN-dPEG 12 -Tris(-dPEG 12 -TFP ester) 3 ;   PhthN-dPEG 24 -Tris(-dPEG 12 -TFP ester) 3 ;   PhthN-dPEG 12 -Tris(-dPEG 12 -Tris(-CO 2 H) 3 ) 3 ;   PhthN-dPEG 12 -Tris(-dPEG 12 -Tris(-dPEG 12 -Tris(-dPEG 11 -m) 3 ) 3 ) 3 ) 3 ;   PhthN-dPEG 24 -Tris(-dPEG 12 -Tris(-dPEG 12 -Tris(-dPEG 11 -m) 3 ) 3 ) 3 ) 3 ;   (A 1 ) 2 : (MAL-dPEG x ) 2 -Lys-dPEG 4 -amido-dPEG 12 -Tris(-dPEG 24 -m) 3 , x=0, 2, 12, 24;   (A 1 ) 2 : (Ex-4-S-MAL-dPEG 2 )-Lys-dPEG 4 -amido-dPEG 12 -Tris(-dPEG 24 ) 3 ;   (A1) 4 : ((MAL-dPEG x ) 2 -Lys-dPEG 4 -)Lys-dPEG 4 -amido-dPEG 12 -Tris(-dPEG 24 -m) 3 ; x=0, 2, 12, 24;   PhthN-dPEGx-Tyr(-dPEGx-TBE)-OH/TFP; x=4, 8, 12, 24;   PhthN-dPEG x -Tyr(-dPEG x -Tris(-dPEG x -A)-dPEG x -Tris(-dPEG x -m/A) 3 ;   PhthN-dPEG 12 -Tyr(-dPEG 4 -Tris(-dPEG 24 -CO 2 H)-dPEG 12 -Tris(-dPEG 24 -m) 3 ;   H 2 N-dPEG 12 -Tyr(-dPEG 12 -Gu(Boc) 2 )-OBn   HO-dPEG 12 -Gu(Boc) 2      PhthN-dPEG 12 -Tyr(-dPEG x -TBE)-dPEG x -Tyr(-dPEG x -NH-boc)-OH; x=0, 4, 812, 24.   Fmoc-NH-dPEG 4 -(Lys)-dPEG 4 -(Lys)-dPEG 4 -(Lys)-dPEG 4 -(Lys)-OH; or   Fmoc-NH-dPEG 4 -(Lys)-dPEG 4 -(Lys)-dPEG 4 -(Lys)-OH.   DOTA/DOTA-TBE-Tyr(-dPEG x -A)-OH/TBE; x=4, 8, 12, 24, 36.