Boronate-mediated delivery of molecules into cells

Abstract

Cargo molecules carrying one or more phenylboronate moieties useful for cellular uptake of the cargo molecules. Phenylboronate can be ligated, crosslinked or otherwise bonded to the cargo molecules. Cargo molecules include peptides and proteins. The phenylboronate groups are optionally conjugated to the cargo molecule via linking moieties that can be selectively cleaved, such cleavable linkers can allow the phenylboronate groups to be removed from the cargo molecule after the boronated cargo molecule is introduced into the cell. The invention includes certain phenylboronates which are boronation reagents, certain boronated oligopeptides and certain boronated peptides and proteins. The invention also includes kits for enhancing cellular uptake of cargo molecules by boronation with one or more phenylboronates or boronated oligopeptides.

We claim: 
     
         1 . A cargo molecule to which one or more phenylboronate moieties are covalently bound. 
     
     
         2 . The cargo molecule of  claim 1  which is a peptide, a protein or a nuclic acid. 
     
     
         3 . The cargo molecule of  claim 1  wherein the one or more phenylboronate moieties are selected from those having formulas: 
       
         
           
           
               
               
           
         
         or salts thereof, 
         where: 
         x is 1 or 2; 
         R 1  and R 5  are independently selected from hydrogen, an optionally substituted straight-chain or branched aliphatic group having 1-8 carbon atoms, a —CO 2 R 10  group, a —OCO—R 10  group, a —CON(R 12 ) 2  group, a —N(R 12 ) 2  group, a —OR 10  group, a —(CH 2 ) 2 —OR 10  group, a —(CH 2 ) 2 —N(R 12 ) 2  group, a halogen, a nitro group, or a cyano group; 
         R 2 , R 3 , R 4  and R 6  are independently selected from hydrogen, a straight-chain or branched aliphatic group having 1-8 carbon atoms, an alicyclic group, an aryl group, a heterocyclic group, a heteroaryl group, a —CO 2 R 10  group, a —OCO—R 10  group, a —CON(R 12 ) 2  group, a —N(R 12 ) 2  group, a —OR 10  group, a —(CH 2 ) m OH group, a halogen, a nitro group, a cyano group, -L-, or two adjacent R 2 -R 6 , together with the ring carbons to which they are attached, optionally form a 5-8-member alicyclic, heterocyclic, aryl or heteroaryl ring moiety, each of which groups or moieties is optionally substituted,
 wherein: 
 each R 10  is independently selected from hydrogen, a straight-chain or branched aliphatic group having 1-8 carbon atoms, an alicyclic group, an aryl group, a heterocyclic group, or a heteroaryl group, each of which groups is optionally substituted; 
 each R 12  is independently selected from hydrogen, a straight-chain or branched aliphatic group having 1-8 carbon atoms, an alicyclic group, an aryl group, a heterocyclic group, a heteroaryl group, or where two R 12  together with the nitrogen to which they are attached can form a 5-8 member heterocyclic or heteroaryl ring moiety, each of which groups or moieties is optionally substituted; 
 m is an integer from 1-8; and 
 -L- is a linker through which the phenylboronate is covalently bonded to the peptide or protein; 
 wherein at least one of R 1 -R 6  is -L- and 
 wherein optional substitution is substitution by one or more substituents selected from halogen; a nitro group; a cyano group; a C1-C6 alkyl group; a C1-C6 alkoxy group; a C2-C6 alkenyl group; a C2-C6 alkynyl group; a 3-7 member alicyclic ring, wherein one or two ring carbons are optionally replaced with —CO— and which may contain one or two double bonds; an aryl group having 6-14 carbon ring atoms; a phenyl group; a benzyl group; a 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein a ring carbon is optionally replaced with —CO— and which may contain one or two double bonds; or a heteroaryl group having 1-3 heteroatoms (N, O or S); a —CO 2 R 13  group; —OCO—R 13  group; —CON(R 14 ) 2  group; —N(R 14 ) 2  group; or —OR 13  group, where each R 13  or R 14  is independently hydrogen; an unsubstituted C1-C6 alkyl group; an unsubstituted aryl group having 6-14 carbon atoms; an unsubstituted phenyl group; an unsubstituted benzyl group; an unsubstituted 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein a ring carbon is optionally replaced with —CO— and which may contain one or two double bonds; or a heteroaryl group having 1-3 heteroatoms (N, O or S) and in addition two R 14  together with the nitrogen to which they are attached can form a heterocyclic or heteroaryl ring moiety, each of which groups or moieties is optionally substituted; each of which R 13  and R 14  groups is in turn optionally substituted with one or more unsubstituted C1-C3 alkyl groups, halogens, nitro groups, cyano groups, —CO 2 H 15  groups, —OCO—R 15  groups, —CON(R 16 ) 2  groups, —N(R 16 ) 2  groups, or —OR 15  groups, where each of R 15  and R 16  independently are hydrogen, an unsubstituted C1-C6 alkyl group; an unsubstituted aryl group having 6-14 carbon ring atoms; an unsubstituted phenyl group; an unsubstituted benzyl group, an unsubstituted 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein a ring carbon is optionally replaced with —CO— and which may contain one or two double bonds; or a heteroaryl group having 1-3 heteroatoms (N, O or S) and a total of 5-14 ring atoms; and in addition two R 16  together with the nitrogen to which they are attached can form an unsubstituted heterocyclic or heteroaryl ring moiety. 
 
       
     
     
         4 . A boronation reagent having the formula: 
       
         
           
           
               
               
           
         
         or salts thereof, 
         where: 
         x is 1 or 2; 
         R 1  is selected from hydrogen, an optionally substituted straight-chain or branched aliphatic group having 1-8 carbon atoms, a —CO 2 R 10  group, a —OCO—R 10  group, a —CON(R 12 ) 2  group, a —N(R 12 ) 2  group, a —OR 10  group, a —(CH 2 ) 2 —OR 10  group, a —(CH 2 ) 2 —N(R 12 ) 2  group, a halogen, a nitro group, or a cyano group; 
         R 2 , R 3 , and R 4  are independently selected from hydrogen, a straight-chain or branched aliphatic group having 1-8 carbon atoms, an alicyclic group, an aryl group, a heterocyclic group, a heteroaryl group, a —CO 2 R 10  group, a —OCO—R 10  group, a —CON(R 12 ) 2  group, a —N(R 12 ) 2  group, a —OR 10  group, a —(CH 2 ) m OH group, a halogen, a nitro group, a cyano group, or two adjacent R 2 -R 6 , together with the ring carbons to which they are attached, optionally form a 5-8-member alicyclic, heterocyclic, aryl or heteroaryl ring moiety, each of which groups or moieties is optionally substituted,
 wherein: 
 each R 10  is independently selected from hydrogen, a straight-chain or branched aliphatic group having 1-8 carbon atoms, an alicyclic group, an aryl group, a heterocyclic group, or a heteroaryl group, each of which groups is optionally substituted; 
 each R 12  is independently selected from hydrogen, a straight-chain or branched aliphatic group having 1-8 carbon atoms, an alicyclic group, an aryl group, a heterocyclic group, a heteroaryl group, or where two R 12  together with the nitrogen to which they are attached can form a 5-8 member heterocyclic or heteroaryl ring moiety, each of which groups or moieties is optionally substituted; and 
 m is an integer from 1-8; 
 
         R 7  and R 8  are selected from from hydrogen or a optionally substituted C1-C3 alkyl group; 
         R 30  and R 31  are H, alkyl or R 30  and R 31  together with the intervening C═C moiety form a 6-8 member carbocyclic or heterocyclic ring wherein one of the ring carbons can be replaced with —CO—; 
         X—CO— is an activated ester; 
         —O—Y is —O—CO—R 24 , where R 24  is an optionally substituted alkyl or aryl group, a —O—PO 3 H 2  or a corresponding phosphate ester thereof or a —O—SO 2 —R 25  group, where R 25  is an optionally substituted alkyl or aryl group; and 
         wherein optional substitution is substitution by one or more substituents selected from halogen; a nitro group; a cyano group; a C1-C6 alkyl group; a C1-C6 alkoxy group; a C2-C6 alkenyl group; a C2-C6 alkynyl group; a 3-7 member alicyclic ring, wherein one or two ring carbons are optionally replaced with —CO— and which may contain one or two double bonds; an aryl group having 6-14 carbon ring atoms; a phenyl group; a benzyl group; a 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein a ring carbon is optionally replaced with —CO— and which may contain one or two double bonds; or a heteroaryl group having 1-3 heteroatoms (N, O or S); a —CO 2 H 13  group; —OCO—R 13  group; —CON(R 14 ) 2  group; —N(R 14 ) 2  group; or —OR 13  group, where each R 13  or R 14  is independently hydrogen; an unsubstituted C1-C6 alkyl group; an unsubstituted aryl group having 6-14 carbon atoms; an unsubstituted phenyl group; an unsubstituted benzyl group; an unsubstituted 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein a ring carbon is optionally replaced with —CO— and which may contain one or two double bonds; or a heteroaryl group having 1-3 heteroatoms (N, O or S) and in addition two R 14  together with the nitrogen to which they are attached can form a heterocyclic or heteroaryl ring moiety, each of which groups or moieties is optionally substituted; each of which R 13  and R 14  groups is in turn optionally substituted with one or more unsubstituted C1-C3 alkyl groups, halogens, nitro groups, cyano groups, —CO 2 H 15  groups, —OCO—R 15  groups, —CON(R 16 ) 2  groups, —N(R 16 ) 2  groups, or —OR 15  groups, where each of R 15  and R 16  independently are hydrogen, an unsubstituted C1-C6 alkyl group; an unsubstituted aryl group having 6-14 carbon ring atoms; an unsubstituted phenyl group; an unsubstituted benzyl group, an unsubstituted 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein a ring carbon is optionally replaced with —CO— and which may contain one or two double bonds; or a heteroaryl group having 1-3 heteroatoms (N, O or S) and a total of 5-14 ring atoms; and in addition two R 16  together with the nitrogen to which they are attached can form an unsubstituted heterocyclic or heteroaryl ring moiety; 
         with the proviso that in formula IIA one of R 2  or R 3  is —B(OH) 2  or R 2  and R 3  together are: 
       
       
         
           
           
               
               
           
         
       
     
     
         5 . A boronation reagent of formula: 
       
         
           
           
               
               
           
         
         or salts thereof 
         x is 1 or 2; 
         R 1  is selected from hydrogen, an optionally substituted straight-chain or branched aliphatic group having 1-8 carbon atoms, a —CO 2 R 10  group, a —OCO—R 10  group, a —CON(R 12 ) 2  group, a —N(R 12 ) 2  group, a —OR 10  group, a —(CH 2 ) 2 —OR 10  group, a —(CH 2 ) 2 —N(R 12 ) 2  group, a halogen, a nitro group, or a cyano group; 
         R 2 , R 3 , and R 4  are independently selected from hydrogen, a straight-chain or branched aliphatic group having 1-8 carbon atoms, an alicyclic group, an aryl group, a heterocyclic group, a heteroaryl group, a —CO 2 R 10  group, a —OCO—R 10  group, a —CON(R 12 ) 2  group, a —N(R 12 ) 2  group, a —OR 10  group, a —(CH 2 ) m OH group, a halogen, a nitro group, a cyano group, or two adjacent R 2 -R 6 , together with the ring carbons to which they are attached, optionally form a 5-8-member alicyclic, heterocyclic, aryl or heteroaryl ring moiety, each of which groups or moieties is optionally substituted,
 wherein: 
 each R 10  is independently selected from hydrogen, a straight-chain or branched aliphatic group having 1-8 carbon atoms, an alicyclic group, an aryl group, a heterocyclic group, or a heteroaryl group, each of which groups is optionally substituted; 
 each R 12  is independently selected from hydrogen, a straight-chain or branched aliphatic group having 1-8 carbon atoms, an alicyclic group, an aryl group, a heterocyclic group, a heteroaryl group, or where two R 12  together with the nitrogen to which they are attached can form a 5-8 member heterocyclic or heteroaryl ring moiety, each of which groups or moieties is optionally substituted; and 
 m is an integer from 1-8; 
 
         R 7  and R 8  are selected from from hydrogen or a optionally substituted C1-C3 alkyl group; 
         X—CO— is an activated ester; 
         —O—Y is —O—CO—R 24 , where R 24  is an optionally substituted alkyl or aryl group, a —O—PO 3 H 2  or a corresponding phosphate ester thereof or a —O—SO 2 —R 25  group, where R 25  is an optionally substituted alkyl or aryl group; and 
         wherein optional substitution is substitution by one or more substituents selected from halogen; a nitro group; a cyano group; a C1-C6 alkyl group; a C1-C6 alkoxy group; a C2-C6 alkenyl group; a C2-C6 alkynyl group; a 3-7 member alicyclic ring, wherein one or two ring carbons are optionally replaced with —CO— and which may contain one or two double bonds; an aryl group having 6-14 carbon ring atoms; a phenyl group; a benzyl group; a 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein a ring carbon is optionally replaced with —CO— and which may contain one or two double bonds; or a heteroaryl group having 1-3 heteroatoms (N, O or S); a —CO 2 H 13  group; —OCO—R 13  group; —CON(R 14 ) 2  group; —N(R 14 ) 2  group; or —OR 13  group, where each R 13  or R 14  is independently hydrogen; an unsubstituted C1-C6 alkyl group; an unsubstituted aryl group having 6-14 carbon atoms; an unsubstituted phenyl group; an unsubstituted benzyl group; an unsubstituted 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein a ring carbon is optionally replaced with —CO— and which may contain one or two double bonds; or a heteroaryl group having 1-3 heteroatoms (N, O or S) and in addition two R 14  together with the nitrogen to which they are attached can form a heterocyclic or heteroaryl ring moiety, each of which groups or moieties is optionally substituted; each of which R 13  and R 14  groups is in turn optionally substituted with one or more unsubstituted C1-C3 alkyl groups, halogens, nitro groups, cyano groups, —CO 2 H 15  groups, —OCO—R 15  groups, —CON(R 16 ) 2  groups, —N(R 16 ) 2  groups, or —OR 15  groups, where each of R 15  and R 16  independently are hydrogen, an unsubstituted C1-C6 alkyl group; an unsubstituted aryl group having 6-14 carbon ring atoms; an unsubstituted phenyl group; an unsubstituted benzyl group, an unsubstituted 5- or 6-member ring heterocyclic group having 1-3 heteroatoms and wherein a ring carbon is optionally replaced with —CO— and which may contain one or two double bonds; or a heteroaryl group having 1-3 heteroatoms (N, O or S) and a total of 5-14 ring atoms; and in addition two R 16  together with the nitrogen to which they are attached can form an unsubstituted heterocyclic or heteroaryl ring moiety;
 with the proviso that in formula IVA one of R 2  or R 3  is —B(OH) 2  or R 2  and R 3  together are: