US 2010/0249385 A1Published application

Water-soluble fluoro-substituted cyanine dyes as reactive fluorescence labelling reagents

AssigneeGe Healthcare UK Limited

InventorsMICHAEL EDWARD COOPER, NICHOLAS JOHN GARDNER, PETER GORDON LAUGHTON

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Abstract

Disclosed are cyanine dyes that are useful for labelling and detecting biological and other materials. The dyes are of formula (I): in which at least one of groups R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 R11, R12, R13 and R14 is -L-M or -L-P, where L is a linking group, M is a target bonding group and P is a conjugated component, and at least one of groups R3, R4, R5, R6, R7, R8, R9 and R19 comprises fluorine. The use of cyanine dyes substituted by fluorine and having additional substitution with three or more sulphonic acid groups for labelling biological target molecules results in a labelled product in which there is reduced dye-dye aggregation and improved photostability, compared with cyanine dyes having no such substitutions. The dyes of the present invention are particularly useful in assays involving fluorescence detection where continual or repeated excitation is a requirement, for example in kinetic studies, or in microarray analyses where microarray slides may need to be reanalysed over a period of days.

Claims25

Dependent claims

A compound of formula (I):

wherein:
groups R1, R2, R11, R12, R13 and R14 are selected independently from -L-M, -L-P, C1-C6 alkyl and —(CH2)k—SO3H, where k is an integer from 1 to 10;
groups R3, R4, R5, R6, R7, R8, R9 and R10 are selected independently from hydrogen, -L-M, -L-P, —SO3H, —SO2—CF3 and —(CF2)m—F, where m is 0 or an integer from 1 to 4;
or R3 taken in combination with R4 or R5 taken in combination with R6 and/or R7 taken in combination with R8 or R9 taken in combination with R10 form a fused aromatic six-membered ring containing carbon atoms and being optionally substituted one or more times by —SO3H, —SO2—CF3 or —(CF2)m—F, where m is hereinbefore defined;
L is a linking group having a chain from 1-20 linked atoms selected from the group consisting of carbon, nitrogen, oxygen and sulphur atoms;
M is a target bonding group;
P is a conjugated component;
groups R15 are hydrogen or two or more of R15 groups are combined to form a one-ring or two-fused hydrocarbon ring system each ring having five or six carbon atoms, and
remaining groups R15 are hydrogen;
and
n is an integer from 1 to 3;
provided that:
i) at least one of groups R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 R11, R12, R13 and R14 is -L-M or -L-P;
ii) at least one of groups R3, R4, R5, R6, R7, R8, R9 and R10 comprises fluorine;
and
iii) when any one of R3, R4, R5, R6, R7, R8, R9 and R10 is —SO3H at least one of groups R11, R12, R13 and R14 is selected from -L-M, -L-P and —(CH2)k—SO3H.

13 more elements

A compound selected from:

i) 2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium (Cy5F) (Compound 1);
ii) 2-{(1E,3E,5E,7E)-7-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]hepta-1,3-5-trienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium (Cy7F) (Compound 2);
iii) 3-(5-Carboxypentyl)-3-methyl-5-sulfo-1-(4-sulfobutyl)-2-{1E,3E,5E}-5-[4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3H-indolium (Compound 3);
iv) 1-(5-Carboxypentyl)-3-methyl-5-sulfo-3-(4-sulfobutyl)-2-{(1E,3E,5E)-5-[4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3H-indolium (Compound 4);
v) 2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,6-difluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium (Compound 5);
vi) 2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium (Compound 6);
vii) 1-(5-Carboxypentyl)-2-{(1E,3E,5E)-5-[1-(5-carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-3H-indolium (Compound 7);
viii) 2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-1,3-bis(4-sulfobutyl)-5-[(trifluoromethyl)sulfonyl]-3H-indolium (Compound 8);
ix) 2-{(1E,3E,5E)-5-[1-(4-Sulfobutyl)-4,6-bis(trifluoromethyl)-3-methyl-3-(5-carboxypentyl))-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,6-difluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium (Compound 9);
x) 2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-1,3-bis(4-sulfobutyl)-6-(trifluoromethyl)-3H-indolium (Compound 10);
xi) 2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7,8,9-hexafluoro-1,1-dimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium (Compound 11);
xii) 2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-6-fluoro-3-methyl-1,3-bis(4-sulfobutyl)-4-(trifluoromethyl)-3H-indolium (Compound 12);
xiii) 2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,6-difluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium (Compound 13);
xiv) 2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-5-sulfo-1,3-bis(4-sulfobutyl)-3H-indolium (Compound 14);
xv) 2-{(1E,3E,5E)-5-[3-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-1-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis-(4-sulfobutyl)-3H-indolium (Compound 15);
xvi) 2-{(1E,3E,5E)-5-[3-(5-Carboxypentyl)-6-fluoro-3-methyl-1-(4-sulfobutyl)-4-(trifluoromethyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium (Compound 16);
xvii) 2-{(1E,3E,5E)-5-[3-(5-Carboxypentyl)-4-trifluoromethyl-6-fluoro-3-methyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-(5-carboxypentyl)-4-trifluoromethyl-6-fluoro-3-methyl-3H-indolium (Compound 17);
xviii) 2-{(1E,3E,5E,7E)-7-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]hepta-1,3,5-trienyl}-3-methyl-1,3-bis(4-sulfobutyl)-4,6-bis(trifluoromethyl)-3H-indolium (Compound 18);
xix) 2-{(1E,3E,5E,7E)-7-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]hepta-1,3,5-trienyl}-3-methyl-1,3-bis(4-sulfobutyl)-5-trifluoromethyl-3H-indolium (Compound 19);
xx) 2-{(1E,3E)-3-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]prop-1-enyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium (Compound 20);
and
xxi) 1-(5-Carboxypentyl)-2-{(1E,3E)-3-[1-(5-carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]prop-1-enyl}-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-3H-indolium (Compound 21).

19 more elements

Dependencies are inferred from the claim text.