Sulfonated [8,9] benzophenoxazine dyes and the use of their labelled conjugates

Claim 2

A labelling reagent comprising a sulfonated 3,7-diamino-[8,9]benzophenoxazine dye and a reactive linking group defined by the formula: including any associated counter ions, wherein: R 1 , R 2 , R 4 , R 6 , R 11 , R 12 , R 13 and R 14 , when taken alone, are separately hydrogen, sulfonate, carboxylate, phosphonate, phosphate, halogen, C 1 -C 6 alkyl, C 1 -C 6 aminoalkyl, C 5 -C 14 aryl, C 5 -C 14 aryl substituted with one or more of the same or different W groups, —OR A , —SR A , —NR A R B , —CN, —NO 2 , —C(O)R A or a reactive linking group; R 1 when taken together with R 2 is C 5 -C 14 aryleno or C 5 -C 14 aryleno substituted with one or more of the same or different W groups; R 3 , R 3′ , R 7 and R 7′ , when taken alone, are separately hydrogen, a reactive linking group, an aliphatic cationic chain, C 1 -C 6 alkyl or C 5 -C 14 aryl; R 3 when taken together with R 3′ is C 2 -C 8 alkyldiyl; R 7 when taken together with R 7′ is C 2 -C 8 alkyldiyl; R 11 and R 12 , when taken together, are C 5 -C 14 aryleno or C 5 -C 14 aryleno substituted with one or more of the same or different W groups; R 12 and R 13 , when taken together, are C 5 -C 14 aryleno or C 5 -C 14 aryleno substituted with one or more of the same or different W groups; R 13 and R 14 , when taken together, are C 5 -C 14 aryleno or C 5 -C 14 aryleno substituted with one or more of the same or different W groups; each W is independently hydrogen, sulfonate, carboxylate, phosphonate, phosphate, halogen, C 1 -C 6 alkyl, —OR A , —SR A , —NR A R B , —CN, —NO 2 or —C(O)R A ; and each R A and each R B is independently hydrogen or C 1 -C 6 alkyl; with the proviso that at least one of R 1 , R 2 , R 4 , R 6 , R 11 , R 12 , R 13 and R 14 is sulfonate, or R 12 and R 13 taken together are benzo containing at least one sulfonate attached to the benzo ring, or at least one of R 3 , R 3′ , R 7 and R 7′ is C 1 -C 6 alkylsulfonate or C 4 -C 10 arylsulfonate; and with the proviso that at least one of R 3 , R 3′ , R 7 , R 7′ , R 11 , R 12 , R 13 and R 14 is a reactive linking group.

Claim 3

The labelling reagent of claim 1 defined by the formula: wherein R 15 , R 16 , R 17 , and R 18 are separately hydrogen, sulfonate, carboxylate, phosphonate, phosphate, halogen, C 1 -C 6 alkyl, C 5 -C 14 aryl, C 5 -C 14 aryl substituted with one or more of the same or different W groups, —OR A , —SR A , NR A R B , —CN, —NO 2 , —C(O)R A or a reactive linking group.

Claim 4

The labelling reagent of claim 1 wherein R 2 and R 3 taken together with the C2-ring atom, C3 ring atom, and 3-nitrogen atom, form a 5- to 7-member ring whose ring atoms are selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; or R 3′ and R 4 taken together with the 3-nitrogen atom, C3-ring atom, and C4-ring atom, form a 5- to 7-member ring whose ring atoms are selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; or R 6 and R 7′ taken together with the C6-ring atom, C7-ring atom, and 7-nitrogen atom, form a 5- to 7-member ring whose ring atoms are selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; or R 7 and R 14 taken together with the 7-nitrogen atom, C7-ring atom, C8-ring atom, and C14-ring atom, form a 5- to 7-member ring whose ring atoms are selected from the group consisting of carbon, nitrogen, oxygen, and sulfur.

Claim 5

The labelling reagent of claim 3 wherein the 5- to 7-member ring includes a gem-disubstituted carbon atom.

Claim 6

The labelling reagent of claim 4 wherein the gem disubstituted carbon atom is substituted with two C 1 -C 6 alkyl groups which may be the same or different.

Claim 7

The labelling reagent of claim 5 wherein the C 1 -C 6 alkyl groups are methyl.

Claim 8

The labelling reagent of claim 1 wherein R 1 , R 2 , R 4 and R 6 are each hydrogen.

Claim 9

The labelling reagent of claim 1 wherein R 3 and R 3′ are each independently C 1 -C 3 alkyl.

Claim 10

The labelling reagent of claim 1 wherein R 1 and R 2 together are [1,2]benzeno, [1,2]naphthaleno or [2,3]naphthaleno.

Claim 11

The labelling reagent of claim 1 wherein R 11 , R 12 , R 13 and R 14 are each hydrogen.

Claim 12

The labelling reagent of claim 1 wherein R 11 and R 12 together are [1,2]benzeno.

Claim 13

The labelling reagent of claim 1 wherein R 12 and R 13 together are [1,2]benzeno.

Claim 14

The labelling reagent of claim 1 wherein R 13 and R 14 together are [1,2]benzeno.

Claim 15

The labelling reagent of claim 1 wherein the aliphatic cationic chain is —(CH 2 ) n —NR 2 , —(CH 2 ) n — + NR 3 , —(CH 2 ) n — + —NR 2 —(CH 2 ) n —NR 2 or —(CH 2 ) n — + NR 2 —(CH 2 ) n — + NR 3 , each n is independently an integer from 2 to 3, and each occurrence of R is independently selected from hydrogen and C 1 -C 6 alkyl.

Claim 16

The labelling reagent of claim 1 wherein alkylsulfonate is —(CH 2 ) n —SO 3 H, and n is an integer from 1 to 6.

Claim 17

The labelling reagent of claim 1 wherein arylsulfonate is: and n is 0 or 1.

Claim 18

The labelling reagent of claim 1 defined by one of the following formulas:

Claim 19

The labelling reagent of claim 1 defined by the formula: wherein R 3 and R 3′ are independently hydrogen or C 1 -C 6 alkyl.

Claim 20

The labelling reagent of claim 1 defined by the formula: DYE is the sulfonated 3,7-diamino-[8,9]benzophenoxazine dye; L is a linker; R 24 and R 25 taken separately are C 1 -C 12 alkyl, C 4 -C 10 aryl, or cycloalkyl containing up to 10 carbon atoms; or R 24 and R 25 taken together with the phosphoramidite nitrogen atom form a saturated nitrogen heterocycle; and R 26 is a phosphite ester protecting group.

Claim 21

The labelling reagent of claim 19 wherein R 26 is methyl, 2-cyanoethyl, or 2-(4-nitrophenyl)ethyl.

Claim 22

The labelling reagent of claim 19 wherein R 24 and R 25 are each isopropyl.

Claim 23

The labelling reagent of claim 19 wherein L is C 1 -C 12 alkyldiyl.

Claim 24

The labelling reagent of claim 19 wherein L is attached to the 3-nitrogen atom or the 7-nitrogen atom of the sulfonated 3,7-diamino-[8,9]benzophenoxazine structure.

Claim 25

The labelling reagent of claim 19 defined by the formula:

Claim 26

The labelling reagent of claim 1 wherein the reactive linking group is selected from amino, thiol, hydroxyl, carboxylic acid, N-hydroxysuccinimidyl ester, disulfide, a halide, an isothiocyanate, sulfonyl chloride, a silyl halide, 2,6-dichlorotriazinyl, pentafluorophenyl ester, a phosphoramidite, maleimide, haloacetyl, epoxide, an alkylhalide, an allyl halide, aldehyde, ketone, acylazide, anhydride, and iodoacetamide.

Claim 27

A compound comprising a substrate covalently attached by a bond or a linker to a sulfonated 3,7-diamino-[8,9]benzophenoxazine dye wherein the substrate is selected from a polynucleotide, a nucleotide, a nucleoside, a polypeptide, a carbohydrate, a ligand, a particle, and a surface; and the sulfonated 3,7-diamino-[8,9]benzophenoxazine dye is defined by the formula: including any associated counter ions, wherein: R 1 , R 2 , R 4 , R 6 , R 11 , R 12 , R 13 and R 14 , when taken alone, are separately hydrogen, sulfonate, carboxylate, phosphonate, phosphate, halogen, C 1 -C 6 alkyl, C 1 -C 6 aminoalkyl, C 5 -C 14 aryl, C 5 -C 14 aryl substituted with one or more of the same or different W groups, —OR A , —SR A , —NR A R B , —CN, —NO 2 , —C(O)R A or a linker; R 1 when taken together with R 2 is C 5 -C 14 aryleno or C 5 -C 14 aryleno substituted with one or more of the same or different W groups; R 3 , R 3′ , R 7 and R 7′ , when taken alone, are separately hydrogen, a linker, an aliphatic cationic chain, C 1 -C 6 alkyl or C 5 -C 14 aryl; R 3 when taken together with R 3′ is C 2 -C 8 alkyldiyl; R 7 when taken together with R 7′ is C 2 -C 8 alkyldiyl; R 11 and R 12 , when taken together, are C 5 -C 14 aryleno or C 5 -C 14 aryleno substituted with one or more of the same or different W groups; R 12 and R 13 , when taken together, are C 5 -C 14 aryleno or C 5 -C 14 aryleno substituted with one or more of the same or different W groups; R 13 and R 14 , when taken together, are C 5 -C 14 aryleno or C 5 -C 14 aryleno substituted with one or more of the same or different W groups; each W is independently hydrogen, sulfonate, carboxylate, phosphonate, phosphate, halogen, C 1 -C 6 alkyl, —OR A , —SR A , NR A R B , —CN, —NO 2 or —C(O)R A ; and each R A and each R B is independently hydrogen or C 1 -C 6 alkyl; with the proviso that at least one of R 1 , R 2 , R 4 , R 6 , R 11 , R 12 , R 13 and R 14 is sulfonate, or R 12 and R 13 taken together are benzo containing at least one sulfonate attached to the benzo ring, or at least one of R 3 , R 3′ , R 7 and R 7′ is C 1 -C 6 alkylsulfonate or C 4 -C 10 arylsulfonate.

Claim 28

A compound comprising a substrate covalently attached by a bond or a linker to a sulfonated 3,7-diamino-[8,9]benzophenoxazine dye wherein the substrate is selected from a polynucleotide, a nucleotide, a nucleoside, a polypeptide, a carbohydrate, a ligand, a particle, and a surface; and the sulfonated 3,7-diamino-[8,9]benzophenoxazine dye is defined by the formula: including any associated counter ions, wherein: R 1 , R 2 , R 4 , R 6 , R 11 , R 12 , R 13 and R 14 , when taken alone, are separately hydrogen, sulfonate, carboxylate, phosphonate, phosphate, halogen, C 1 -C 6 alkyl, C 1 -C 6 aminoalkyl, C 5 -C 14 aryl, C 5 -C 14 aryl substituted with one or more of the same or different W groups, —OR A , —SR A , —NR A R B , —CN, —NO 2 , —C(O)R A or a linker; R 1 when taken together with R 2 is C 5 -C 14 aryleno or C 5 -C 14 aryleno substituted with one or more of the same or different W groups; R 3 , R 3′ , R 7 and R 7′ , when taken alone, are separately hydrogen, a linker, an aliphatic cationic chain, C 1 -C 6 alkyl or C 5 -C 14 aryl; R 3 when taken together with R 3′ is C 2 -C 8 alkyldiyl; R 7 when taken together with R 7′ is C 2 -C 8 alkyldiyl; R 11 and R 12 , when taken together, are C 5 -C 14 aryleno or C 5 -C 14 aryleno substituted with one or more of the same or different W groups; R 12 and R 13 , when taken together, are C 5 -C 14 aryleno or C 5 -C 14 aryleno substituted with one or more of the same or different W groups; R 13 and R 14 , when taken together, are C 5 -C 14 aryleno or C 5 -C 14 aryleno substituted with one or more of the same or different W groups; each W is independently hydrogen, sulfonate, carboxylate, phosphonate, phosphate, halogen, C 1 -C 6 alkyl, —OR A , —SR A , NR A R B , —CN, —NO 2 or —C(O)R A ; and each R A and each R B is independently hydrogen or C 1 -C 6 alkyl; with the proviso that at least one of R 1 , R 2 , R 4 , R 6 , R 11 , R 12 , R 13 and R 14 is sulfonate, or R 12 and R 13 taken together are benzo containing at least one sulfonate attached to the benzo ring, or at least one of R 3 , R 3′ , R 7 and R 7′ is C 1 -C 6 alkylsulfonate or C 4 -C 10 arylsulfonate.

Claim 29

The compound of claim 26 wherein the substrate is defined by the formula: wherein DYE is the sulfonated 3,7-diamino-[8,9]benzophenoxazine dye; L is a linker; B is a nucleobase; R 19 is H, monophosphate, diphosphate, triphosphate, or phosphate analog thereof; and R 20 and R 21 , when taken alone, are each independently H, HO, F, a phosphoramidite group, or a moiety which blocks polymerase-mediated polymerization, or when taken together, form 2′-3′-didehydroribose.

Claim 30

The compound of claim 27 wherein B is uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, 7-deazaguanosine, 7-deaza-8-azaguanine, or 7-deaza-8-azaadenine.

Claim 31

The compound of claim 27 wherein L is: and n is 0, 1, or 2.

Claim 32

The compound of claim 27 which is enzymatically incorporatable.

Claim 33

The compound of claim 27 which is a terminator.

Claim 34

The compound of claim 31 wherein R 19 is triphosphate, α-thiotriphosphate, or triphosphate ester analog; and R 20 and R 21 , when taken alone, are each independently H, F, or a moiety which blocks polymerase-mediated polymerization, or when taken together, form 2′-3′-didehydroribose.

Claim 35

The compound of claim 27 which is enzymatically extendable.

Claim 36

The compound of claim 26 wherein the substrate is defined by the formula: wherein the polynucleotide comprises two or more nucleotides; DYE is the sulfonated 3,7-diamino-[8,9]benzophenoxazine dye; L is a linker; B is a nucleobase; R 21 is H, OH, halide, azide, amine, C 1 -C 6 aminoalkyl, C 1 -C 6 alkyl, allyl, C 1 -C 6 alkoxy, —OCH 3 , or —OCH 2 CH═CH 2 ; and R 22 and R 23 are independently H, phosphate, internucleotide phosphodiester, or internucleotide analog.

Claim 37

The compound of claim 26 wherein the substrate is defined by the formula: wherein the compound comprises two or more nucleotides; DYE is the sulfonated 3,7-diamino-[8,9]benzophenoxazine dye; L is a linker; X is O, NH, or S; B is a nucleobase; R 21 is H, OH, halide, azide, amine, C 1 -C 6 aminoalkyl, C 1 -C 6 alkyl, allyl, C 1 -C 6 alkoxy, —OCH 3 , or —OCH 2 CH═CH 2 ; and R 22 is internucleotide phosphodiester or internucleotide analog.

Claim 38

The compound of claim 35 wherein L is C 1 -C 12 alkyldiyl.

Claim 39

The compound of claim 35 wherein L comprises —(CH 2 CH 2 O) n —, and n is 1 to 100.

Claim 40

The compound of claim 26 defined by the formula: P—L—-DYE, wherein P is a polypeptide; L is a linker; and DYE is the sulfonated 3,7-diamino-[8,9]benzophenoxazine dye.

Claim 41

The compound of claim 38 wherein L is an amide bond.

Claim 42

The compound of claim 38 wherein L is attached to P through a carboxyl terminus, an amino terminus, a lysine sidechain, an aspartic acid sidechain, or a glutamic acid sidechain.

Claim 43

The compound of claim 38 wherein P is streptavidin, a caspase-cleavage substrate, or an antibody.