ULTRA BRIGHT DIMERIC OR POLYMERIC DYES WITH SPACING LINKER GROUPS

1 . A method, comprising:

admixing a compound, or a stereoisomer, salt or tautomer thereof, with an antibody, the compound having the following structure (I):

wherein:

M is, at each occurrence, independently a fluorescent dye;

L′ is at each occurrence, independently either: i) an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker; or ii) a linker comprising a functional group capable of formation by reaction of two complementary reactive groups;

L 2 and L 3 are, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker;

L 4 is, at each occurrence, independently a polyethylene oxide linker;

R 1 is, at each occurrence, independently H, alkyl or alkoxy;

R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkylether, heteroalkyl, —OP(═R a )(R b )R c , Q, or a protected form thereof, or L′;

R 4 is, at each occurrence, independently OH, SH, O—, S—, OR a or SR a ;

R 5 is, at each occurrence, independently oxo, thioxo or absent;

R a is O or S;

R b is OH, SH, O—, S—, OR a or SR a ;

R c is OH, SH, O − , S − , OR a , OL′, SR a , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether;

R d is a counter ion;

Q is, at each occurrence, independently a moiety comprising a reactive group, or protected form thereof, capable of forming a covalent bond with the antibody;

L′ is, at each occurrence, independently a linker comprising a covalent bond to Q, a linker comprising a covalent bond to the antibody;

m is, at each occurrence, independently an integer of one or greater; and

n is an integer of one or greater,

provided that at least one occurrence of either R 2 and R 3 is —OP(═R a )(R b )R c , Q, or a protected form thereof, or L′, wherein R c is OL′; and

forming a conjugate of the first compound to the antibody.

2 . The method of claim 1 , wherein Q comprises a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino or maleimide functional group.

3 . The method of claim 1 , wherein Q has one of the following structures:

wherein each X is independently a halogen.

4 . The method of claim 1 , wherein Q has the following structure:

wherein R is an optionally substituted alkyl group.

5 . The method of claim 1 , wherein Q is a disulfide moiety having the following structure:

wherein n′ is an integer from 1 to 10.

6 . The method of claim 1 , wherein Q has the following structure:

7 . The method of claim 6 , wherein a sulfur atom on the antibody reacts with Q to form the following linking structure:

8 . A method for preparing a conjugate of a compound, comprising:

admixing a compound or a stereoisomer, salt or tautomer thereof, with an antibody, wherein the compound is conjugated to a sulfur atom of the antibody, and wherein the first compound has the following structure (I):

wherein:

M is, at each occurrence, independently a fluorescent dye;

L′ is at each occurrence, independently either: i) an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker; or ii) a linker comprising a functional group capable of formation by reaction of two complementary reactive groups;

L 2 and L 3 are, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker;

L 4 is, at each occurrence, independently a polyethylene oxide linker;

R 1 is, at each occurrence, independently H, alkyl or alkoxy;

R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkylether, heteroalkyl, —OP(═R a )(R b )R c , Q, or a protected form thereof, or L′;

R 4 is, at each occurrence, independently OH, SH, O—, S—, OR d or SR d ;

R 5 is, at each occurrence, independently oxo, thioxo or absent;

R a is O or S;

R b is OH, SH, O, S—, OR d or SR d ;

R c is OH, SH, O, S—, OR d , OL′, SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether;

R a is a counter ion;

Q is, at each occurrence, independently a moiety comprising a reactive group, or protected form thereof, capable of forming a covalent bond with the antibody;

L′ is, at each occurrence, independently a linker comprising a covalent bond to Q, a linker comprising a covalent bond to the antibody;

m is, at each occurrence, independently an integer of one or greater, and

n is an integer of one or greater,

provided that at least one occurrence of either R 2 and R 3 is —OP(═R a )(Rs)R c , Q, or a protected form thereof, or L′, wherein R c is OL′.

9 . The method of claim 8 , wherein L′ has one of the following structures:

10 . The method of claim 8 , wherein M is, at each occurrence, independently pyrene, perylene, perylene monoimide or 6-FAM or derivative thereof.

11 . The method of claim 8 , wherein M, at each occurrence, independently has one of the following structures:

12 . The method of claim 8 , wherein Q comprises a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino or maleimide functional group.

13 . The method of claim 8 , wherein R 2 or R 3 has one of the following structures:

14 . The method of claim 8 , wherein the compound has one of the following structures:

wherein:

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each A is independently the antibody;

each m″ is independently 4 or 10; and

F, F, F″ and dT have the following structures, respectively:

15 . A method of conjugating a polymeric dye compound to an analyte or targeting moiety, comprising:

admixing the polymeric dye compound, or a stereoisomer, salt or tautomer thereof, with the analyte or targeting moiety having specificity for the analyte, wherein the dye compound is conjugated to a sulfur atom of the analyte or targeting moiety, and wherein the polymeric dye compound has the following structure of (I):

wherein:

M is, at each occurrence, independently a fluorescent dye;

L′ is at each occurrence, independently either: i) an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker; or ii) a linker comprising a functional group capable of formation by reaction of two complementary reactive groups;

L 2 and L 3 are, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker;

L 4 is, at each occurrence, independently a polyethylene oxide linker;

R 1 is, at each occurrence, independently H, alkyl or alkoxy;

R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkylether, heteroalkyl, —OP(═R a )(R b )R c , Q, or a protected form thereof, or L′;

R 4 is, at each occurrence, independently OH, SH, O—, S—, OR d or SR d ;

R 5 is, at each occurrence, independently oxo, thioxo or absent;

R a is O or S;

R b is OH, SH, O—, S—, OR d or SR d ;

R c is OH, SH, O—, S—, OR d , OL′, SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether;

R d is a counter ion;

Q is, at each occurrence, independently a moiety comprising a reactive group, or protected form thereof, capable of forming a covalent bond with the analyte or targeting moiety;

L′ is, at each occurrence, independently a linker comprising a covalent bond to Q, a linker comprising a covalent bond to the analyte or targeting moiety;

m is, at each occurrence, independently an integer of one or greater; and

n is an integer of one or greater,

provided that at least one occurrence of either R 2 and R 3 is —OP(═R a )(R b )R c , Q, or a protected form thereof, or L′, wherein R c is OL′.

16 . The method of claim 15 , wherein the analyte is a biomolecule.

17 . The method of claim 16 , wherein the biomolecule is an antibody, antigen, nucleic acid sequence, amino acid or a polymer thereof, enzyme, protein, receptor, receptor ligand, glycoprotein, aptamer, prion, or cell surface receptor antagonist.

18 . The method of claim 15 , wherein the targeting moiety is an antibody or cell surface receptor antagonist.

19 . The method of claim 15 , wherein one of R 2 or R 3 is OH or —OP(═R a )(R b )R c , and the other of R 2 or R 3 is a linker comprising a covalent bond to the analyte or targeting moiety.

20 . The method of claim 15 , wherein the following linking structure:

is formed from Q reacting with the sulfur atom of the analyte or targeting moiety.