Water soluble multi-biotin-containing compounds

Abstract

Water-soluble discrete multi-biotin-containing compounds with at least three (3) biotin moieties are disclosed. The water-soluble biotin-containing compounds may additionally comprise one or more moieties that confer resistance to cleavage by biotinidase or that is cleavable in vitro or in vivo. The discrete multi-biotin-containing compounds may include a reactive moiety that provides a site for reaction with yet another moiety, such as a targeting, diagnostic or therapeutic functional moiety. Biotinylation reagents comprising water-soluble linker moieties are also disclosed and may additionally comprise a biotinidase protective group. Methods for amplifying the number of sites for binding biotin-binding proteins at a selected target using multi-biotin compounds also are disclosed.

1 . A composition of matter, which comprises a discrete biotin-containing compound of the general structural formula:  
         C-(L-P-B) n   wherein, 
 C is a multifunctional cross-linking moiety having discrete size and containing n substituents, which are reactive, or can be made reactive, with L;  
 L is a water-soluble linker moiety of discrete size containing 6 to 50 atoms in length and is one or more of alkyl, heteroalkyl, aryl, and heteroaryl linker moieties containing one or more of ethers, hydroxyls, amines, thioethers, thiols, esters, maleimides, iodoacetamides, hydroxyl amines, acyl hydrazines, carboxylic acids, polyphosphoric acids, phenols, sulfonic acids, iodoacetamides, aldehydes, nitrophenylazides, polylysines, ammoniums, amides, ketones, decaboranes, dodedaboranes, boranes, closo-carboranes, or nido-carboranes;  
 P is a biotinidase protective group that blocks biotinidase activity and is one or more of α-amino acids, N-methyl moieties, α-alkyl moieties, or aryl moieties;  
 B is selected from biotin moieties; and  
 n ranges from 3 to 64.  
   
     
     
         2 . The composition of matter of  claim 1 , wherein n ranges from 3 to 32.  
     
     
         3 . The composition of matter of  claim 1 , wherein said cross-linker, C, is one or more of tri-functional aromatic compounds, starburst dendrimers, cascade dendrimers, polylysine, polyglutamic, or polyaspartic acids.  
     
     
         4 . The composition of matter of  claim 1 , wherein said water-soluble linker, L, moieties are one or more of ethers, hydroxyls, amines, thioethers, or thiols.  
     
     
         5 . The composition of matter of  claim 1 , wherein said biotin moiety, B, is one or more of biotin, homobiotin, norbiotin, desthiobiotin, biotin sulfone, iminobiotin, alkylated biotins, or acetylated biotins.  
     
     
         6 . The composition of matter of  claim 1 , wherein 
 L is one or more of ethers, hydroxyls, amines, thioethers, or thiols; and    B is one or more of biotin, homobiotin, norbiotin, desthiobiotin, biotin sulfone, iminobiotin, alkylated biotins, or acetylated biotins.    
     
     
         7 - 10 . (canceled)  
     
     
         11 . A composition of matter, which comprises a discrete biotin-containing compound of the general structural formula:  
         C-(L-B) n ; wherein 
 C is a multifunctional cross-linking moiety having discrete size and containing n substituents, which are reactive, or can be made reactive, with L;  
 L is a water-soluble linker moiety of discrete size containing 6 to 50 atoms in length and is one or more of alkyl, heteroalkyl, aryl, and heteroaryl linker moieties containing ethers, hydroxyls, amines, thioethers, thiols, esters, maleimides, iodoacetamides, hydroxyl amines, acyl hydrazines, carboxylic acids, polyphosphoric acids, phenols, sulfonic acids, iodoacetamides, aldehydes, nitrophenylazides, polylysines, ammoniums, amides, ketones, decaboranes, dodedaboranes, boranes, closo- and nido-carboranes; 
 B is selected from biotin moieties; and  
 
 n ranges from 3 to 64.  
   
     
     
         12 . The composition of matter of  claim 11 , wherein the water soluble linker moiety, L, is one or more of 4,7,10-trioxa-1,13-tridecanediamine, 2,2′-(ethylenedioxy)diethylamine, or 12-N-methylamino-4,7,10-trioxadodecanoic acid.  
     
     
         13 . The composition of matter of  claim 11 , wherein said biotin, B, bears a carboxylate group and which composition of matter additionally comprises a chemically-bound moiety adjacent to the biotin carboxylate group which chemically-bound moiety blocks the activity of biotinidase.  
     
     
         14 . The composition of matter of  claim 11 , wherein one or more of a diagnostic moiety, a therapeutic moiety, or a radionuclide, is one or more of chemically-bound to or chelated with C.  
     
     
         15 . The composition of matter of  claim 11 , wherein: 
 C is one or more of at least tri-functional aromatic compounds, starburst dendrimers, cascade dendrimers, polylysine, polyglutamic, or polyaspartic acids; and    B is one or more of biotin, homobiotin, norbiotin, desthiobiotin, biotin sulfone, iminobiotin, alkylated biotins, or acetylated biotins.    
     
     
         16 - 21 . (canceled)  
     
     
         22 . A composition of matter, which comprises a discrete biotin-containing compound comprising at least three (3) biotin moieties each joined by a water soluble linker moiety to a discrete size dendrimer, wherein said water soluble linker moieties are 8 to 20 atoms in length.  
     
     
         23 . The composition of matter of  claim 22 , wherein the water-soluble linker moieties are one or more of polyether, polyhydroxyl, or polyamino compounds.  
     
     
         24 . The composition of matter of  claim 22 , wherein said biotin-containing compound has water solubility of greater than about 0.2 mg/mL at neutral pH and ambient temperature.  
     
     
         25 . The composition of matter of  claim 24 , wherein said biotin-containing compound has water solubility of greater than about 5 mg/mL at neutral pH and ambient temperature.  
     
     
         26 . A composition of matter, which comprises a discrete multi-biotin-containing compound of the structural formula:  
         T m -C-(L-B) n   wherein 
 C is one or more of multifunctional cross-linking moieties having discrete size and containing n+m substituents which are reactive, or can be made reactive, with L and T;  
 L is a water-soluble linker moiety of discrete size containing 6 to 50 atoms in length and is one or more of alkyl, heteroalkyl, aryl, and heteroaryl linker moieties containing one or more of ethers, hydroxyls, amines, thioethers, thiols, esters, maleimides, iodoacetamides, hydroxyl amines, acyl hydrazines, carboxylic acids, polyphosphoric acids, phenols, sulfonic acids, iodoacetamides, aldehydes, nitrophenylazides, polylysines, ammoniums, amides, ketones, decaboranes, dodedaboranes, boranes, closo-carboranes, or nido-carboranes;  
 B is selected from biotin moieties; and  
 T is one or more of therapeutic, diagnostic, or targeting moieties;  
 n+m ranges from 3 to 64; and  
 m ranges from 1 to 5.  
   
     
     
         27 . The composition of matter of  claim 26 , wherein: 
 C is selected from the group consisting of at least tri-functional aromatic compounds, starburst dendrimers, cascade dendrimers, polylysine, polyglutamic and polyaspartic acids; and    B is selected from biotin, homobiotin, norbiotin, desthiobiotin, biotin sulfone, iminobiotin, alkylated biotins, and acetylated biotins.    
     
     
         28 . A composition of matter, which comprises a discrete multi-biotin-containing compound of the structural formula:  
         T m -C-(L- P   y -B) n   wherein 
 C is one or more of multifunctional cross-linking moieties having discrete size and containing n+m substituents which are reactive, or can be made reactive, with L and T;  
 L is a water-soluble linker moiety of discrete size containing 6 to 50 atoms in length and is one or more of alkyl, heteroalkyl, aryl, and heteroaryl linker moieties containing one or more of ethers, hydroxyls, amines, thioethers, thiols, esters, maleimides, iodoacetamides, hydroxyl amines, acyl hydrazines, carboxylic acids, polyphosphoric acids, phenols, sulfonic acids, iodoacetamides, aldehydes, nitrophenylazides, polylysines, ammoniums, amides, ketones, decaboranes, dodedaboranes, boranes, closo-carboranes, or nido-carboranes;  
 y is 0 or 1;  
 P is a biotinidase protective group that blocks biotinidase activity and is one or more of α-amino acids, N-methyl moieties, α-alkyl moieties, or aryl moieties;  
 B is selected from biotin moieties;  
 T is one or more of therapeutic, diagnostic, or targeting moieties;  
 n+m ranges from 3 to 64; and  
 m ranges from 1 to 5.  
   
     
     
         29 . The composition of matter of  claim 28 , wherein m is 1; y is 1; and n ranges from 3 to 32.  
     
     
         30 . The composition of matter of  claim 28 , wherein n ranges from 3 to 32.  
     
     
         31 . The composition of matter of  claim 28 , wherein 
 C is one or more of tri-functional aromatic compounds, starburst dendrimers, cascade dendrimers, polylysine, polyglutamic, or polyaspartic acids;    B is one or more of biotin, homobiotin, norbiotin, desthiobiotin, biotin sulfone, iminobiotin, alkylated biotins, or acetylated biotins;    and n ranges from 3 to 64.    
     
     
         32 . The composition of matter of  claim 28 , wherein L contains one or more of an ionized moeity or an ionizable moiety.  
     
     
         33 . The composition of matter of  claim 32 , wherein the ionized or ionizable functionality of L is at least three atoms away from the point of conjugation of L with B.  
     
     
         34 . The composition of matter of  claim 33 , wherein said ionized group is one or more of a sulfonate ion or an ammonium ion.  
     
     
         35 . The composition of matter of  claim 32 , wherein L contains one or more of an anionic borane molecule or a carborane cage molecule.  
     
     
         36 . A biotinylation reagent useful in preparing multi-biotin-containing compounds, which comprises a discrete multi-biotin-containing compound of the structural formula:  
         X-R-C-(L-P-B) n   C is one or more of multifunctional cross-linking moieties having discrete size and containing n+m substituents which are reactive, or can be made reactive, with L and T;    L is a water-soluble linker moiety of discrete size containing 6 to 50 atoms in length and is one or more of alkyl, heteroalkyl, aryl, and heteroaryl linker moieties containing one or more of ethers, hydroxyls, amines, thioethers, thiols, esters, maleimides, iodoacetamides, hydroxyl amines, acyl hydrazines, carboxylic acids, polyphosphoric acids, phenols, sulfonic acids, iodoacetamides, aldehydes, nitrophenylazides, polylysines, ammoniums, amides, ketones, decaboranes, dodedaboranes, boranes, closo-carboranes, or nido-carboranes;    P is a biotinidase protective group that blocks biotinidase activity and is one or more of α-amino acids, N-methyl moieties, α-alkyl moieties, or aryl moieties;    B is selected from biotin moieties;    n ranges from 3 to 64;    X is one or more of a nucleophilic group or an electrophilic group; and    R is optional and serves as a spacer having the ability to react with X and C in forming the biotinylation reagent.    
     
     
         37 . The biotinylation reagent of  claim 36 , wherein X is one or more of amines, sulfhydryl groups, alkoxides, carboxylates, mixed anhydrides, isothiocyanates, isocyanates, ketones, aldehydes, α,β-unsaturated ene-one moieties, alkyl halides, or benzyl halides.  
     
     
         38 . The biotinylation reagent of  claim 36 , wherein X carries a chemical protective group, which is not P.  
     
     
         39 . The biotinylation reagent of  claim 38 , wherein said chemical protective group is one or more of t-butyloxycarbonyl (t-Boc), t-Bu esters, benzyloxycarbonyl (Z), phthalimidio, or fluorenylmethyloxycarbonyl (Fmoc).  
     
     
         40 . The biotinylation reagent of  claim 37 , wherein said carboxylates are one or more of N-succinimidyl esters, tetrafluorophenyl esters.