Novel hydrazone-based and oxime-based fluorescent and chromophoric/pro-fluorescent and pro-chromophoric reagents and linkers

Abstract

Novel Hydrazone-based Fluorescent and Pro-Fluorescent Reagents and Linkers, including conjugationally extended hydrazine compositions, fluorescent hydrazone compositions, methods of the formation of hydrazones from the reaction of conjugationally extended hydrazines with conjugationally extended carbonyls, and methods of their use in assays systems are described. Use of these conjugationally compositions for direct colorimetric and fluorometric assays wherein a chromophore or the fluorophore is incorporated into the linker that is positioned between a reactive linking moiety and a biotin molecule.

1 . A fluorescent hydrazone compound of formula I,
   (R 1 R 2 )NN═C(R 1 R 2 )  I
   
       wherein:
 R 1  are independently a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; 
 R 2  are independently a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine and wherein said composition has an emission frequency equal to or greater than 400 nm. 
 
     
     
         2 . A fluorescent hydrazone compound according to  claim 1  wherein one of R, one of R 1  or one of R 2  further comprises a linker moiety selected from the group consisting of an amino reactive moiety, a thiol reactive moiety, an ester moiety and a modified carbohydrate monomer moiety. 
     
     
         3 . A fluorescent hydrazone compound according to  claim 2  wherein said linker further comprises a biomolecule. 
     
     
         4 . A fluorescent composition of the formula III, 
       
         
           
           
               
               
           
         
       
       wherein;
 R 1  (which is R 2 ) is a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; 
 R 2  (which is R 3 ) is a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; 
 R 3  (which is R 4 ) is H or OH; 
 R 4  (which is R 6 ) is H or a nucleic acid moiety; and 
 R 5  (which is R 7 ) is PO 3  or a nucleic acid moiety. 
 
     
     
         5 . A profluorescent hydrazine compound of formula IV,
   (R 1 R 2 )N(H) n (NH 2 ) m   IV
   
       wherein:
 R1 is a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; 
 R 2  is a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; 
 n is 0 when m is 2 and 
 n is 1 when m is 1. 
 
     
     
         6 . A profluorescent hydrazine compound according to  claim 5  wherein R 1  or R 2  further comprise a linkable moiety selected from the group consisting of an amino reactive moiety, a thiol reactive moiety, an ester moiety and a modified carbohydrate monomer moiety. 
     
     
         7 . A profluorescent hydrazine compound according to  claim 6  wherein said linker further comprises a biomolecule. 
     
     
         8 . A composition comprising a polymer having one or more profluorescent hydrazine compounds according to  claim 5  bound to said polymer by one or more linker moieties. 
     
     
         9 . A polymer according to  claim 8  wherein said polymer is poly-lysine, poly-ornithine or polyethyleneglycol. 
     
     
         10 . A profluorescent oxyamine compound of formula VI,
   (R 1 R 2 )ONH 2   VI
   
       wherein:
 R 1  is a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; and 
 R 2  is a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and tertiary and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, quaternary amine. 
 
     
     
         11 . A profluorescent oxyamine compound according to  claim 10  wherein R 1  or R 2  further comprise a linkable moiety selected from the group consisting of an amino reactive moiety, a thiol reactive moiety, an ester moiety and a modified carbohydrate monomer moiety. 
     
     
         12 . A profluorescent oxyamine compound according to  claim 10  wherein said linker further comprises a biomolecule. 
     
     
         13 . A method of preparing a fluorescent hydrazone according to  claim 1  by combining a hydrazine of formula IV,
   (R 1 R 2 )N(H) n (NH 2 ) m   IV
 
 
       with a carbonyl of formula V:
   O═C(R 1 R 2 )  V
 
 
       wherein:
 R 1  are independently a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; 
 R 2  are independently a hydrogen, a straight chain aliphatic moiety of 1-10 carbon atoms, a branched aliphatic moiety of 1-10 carbon atoms, a cyclic aliphatic moiety of 1-10 carbon atoms, a substituted or unsubstituted conjugationally extended moiety wherein the unsubstituted conjugationally extended moiety is an alkenyl, alkynyl, aromatic, polyaromatic, heteroaromatic or polyheteroaromatic moiety and wherein the substituted conjugationally extended moiety may be substituted with any combination of one or more of the groups hydroxy, alkoxy, alkene, alkyne, nitro, carboxy, sulfo, unsubstituted amine and substituted primary, secondary, tertiary and quaternary amine; 
 n is 0 when m is 2 and 
 n is 1 when m is 1 
 for a time and under conditions that allow hydrazone formation. 
 
     
     
         14 . A method according to  claim 13  wherein R 1  or R 2  of formula IV further comprises a linkable moiety selected from the group consisting of an amino reactive moiety, a thiol reactive moiety, an ester moiety and a modified carbohydrate monomer moiety. 
     
     
         15 . A method according to  claim 13  wherein R 1  or R 2  of formula V further comprises a linkable moiety selected from the group consisting of an amino reactive moiety, a thiol reactive moiety, an ester moiety and a modified carbohydrate monomer moiety. 
     
     
         16 .- 24 . (canceled)