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

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 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 R 11 , R 12 , R 13 and R 14 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 R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 19 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.

1 . A compound of formula (I): 
       
         
           
           
               
               
           
         
       
       wherein:
 groups R 1 , R 2 , R 11 , R 12 , R 13  and R 14  are selected independently from -L-M, -L-P, C 1 -C 6  alkyl and —(CH 2 ) k —SO 3 H, where k is an integer from 1 to 10; 
 groups R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  are selected independently from hydrogen, -L-M, -L-P, —SO 3 H, —SO 2 —CF 3  and —(CF 2 ) m —F, where m is 0 or an integer from 1 to 4; 
 or R 3  taken in combination with R 4  or R 5  taken in combination with R 6  and/or R 7  taken in combination with R 8  or R 9  taken in combination with R 10  form a fused aromatic six-membered ring containing carbon atoms and being optionally substituted one or more times by —SO 3 H, —SO 2 —CF 3  or —(CF 2 ) 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 R 15  are hydrogen or two or more of R 15  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 R 15  are hydrogen; and 
 n is an integer from 1 to 3; 
 provided that: 
 i) at least one of groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10  R 11 , R 12 , R 13  and R 14  is -L-M or -L-P; 
 ii) at least one of groups R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  comprises fluorine; and 
 iii) when any one of R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  is —SO 3 H at least one of groups R 11 , R 12 , R 13  and R 14  is selected from -L-M, -L-P and —(CH 2 ) k —SO 3 H. 
 
     
     
         2 . The compound of  claim 1 , wherein:
 the groups R 1  and R 2  are selected independently from -L-M, -L-P, C 1 -C 6  alkyl and —(CH 2 ) k —SO 3 H;   groups R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  are selected independently from hydrogen, —SO 3 H, —SO 2 —CF 3  and —(CF 2 ) m —F, where m is 0 or an integer from 1 to 4; or R 3  taken in combination with R 4  or R 5  taken in combination with R 6  and/or R 7  taken in combination with R 8  or R 9  taken in combination with R 10  form a fused aromatic six-membered ring containing carbon atoms and being optionally substituted one or more times by —SO 3 H, —SO 2 —CF 3  or —(CF 2 ) m —F, where m is hereinbefore defined;   groups R 11 , R 12 R 13  and R 14  are selected independently from C 1 -C 6  alkyl and —(CH 2 ) k —SO 3 H; and   groups R 15 , L, M and P, k and n are defined as in  claim 1 ;   provided that:   i) at least one of groups R 1  and R 2  is -L-M or -L-P;   ii) at least one of groups R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  comprises fluorine; and   iii) when any one of R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  is —SO 3 H at least one of groups R 11 , R 12 , R 13  and R 14  is —(CH 2 ) k —SO 3 H.   
     
     
         3 . The compound of  claim 1 , wherein:
 groups R 11 , R 12 , R 13  and R 14  are selected independently from -L-M, -L-P, C 1 -C 6  alkyl and —(CH 2 ) k —SO 3 H,   groups R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  are selected independently from hydrogen, —SO 3 H, —SO 2 —CF 3  and —(CF 2 ) m —F, where m is 0 or an integer from 1 to 4; or R 3  taken in combination with R 4  or R 5  taken in combination with R 6  and/or R 7  taken in combination with R 8  or R 9  taken in combination with R 10  form a fused aromatic six-membered ring containing carbon atoms and being optionally substituted one or more times by —SO 3 H, —SO 2 —CF 3  or —(CF 2 ) m —F, where m is hereinbefore defined; and   groups R 1  and R 2  are selected independently from C 1 -C 6  alkyl and —(CH 2 ) k —SO 3 H;   provided that:   i) at least one of groups R 11 , R 12 , R 13  and R 14  is -L-M or -L-P;   and ii) at least one of groups R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  comprises fluorine; and   iii) when any one of R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  is —SO 3 H at least one of groups R 11 , R 12 , R 13  and R 14  is —(CH 2 ) k —SO 3 H.   
     
     
         4 . The compound of  claim 1 , wherein at least one of groups R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  is fluorine. 
     
     
         5 . The compound of  claim 1 , wherein two or more groups R 15  form a hydrocarbon ring system of formula: 
       
         
           
           
               
               
           
         
       
       wherein n is an integer from 1 to 3 and p is 0 or 1; and remaining groups R 15  are hydrogen. 
     
     
         6 . The compound of  claim 1 , wherein each group R 15  is hydrogen. 
     
     
         7 . The compound of  claim 1 , wherein L is a linking group having the formula:
   —(CHR′) p -Q-(CHR′) r —   
       where Q is selected from: —CHR′—, —NR′—, —O—, —S—, CR′═CR′—, —Ar—, —C(O)—NR′— and —C(O)—O—, wherein R′ is hydrogen or C 1 -C 4  alkyl; Ar is phenylene, optionally substituted with sulphonate; p is 0-5 and r is 1-5. 
     
     
         8 . The compound of  claim 7 , wherein Q is selected from: —CHR′— and —C(O)—NH—. 
     
     
         9 . The compound of  claim 7 , wherein Q is —CHR′— and R′ is hydrogen. 
     
     
         10 . The compound of  claim 1 , wherein -L-M and/or -L-P comprise a carboxypentyl group. 
     
     
         11 - 54 . (canceled) 
     
     
         55 . 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).   
     
     
         56 - 57 . (canceled) 
     
     
         58 . The compound of  claim 1 , wherein —(CH 2 ) k —SO 3 H is selected from —(CH 2 ) 3 —SO 3 H and —(CH 2 ) 4 —SO 3 H. 
     
     
         59 . The compound of  claim 1 , wherein at least two of groups R 3 , R 4 , R 5  and R 6  and/or groups R 7 , R 8 , R 9  and R 10  are F and any remaining groups R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  or R 10  are H. 
     
     
         60 . The compound of  claim 1 , wherein groups R 3 , R 4 , R 5  and R 6  and/or groups R 7 , R 8 , R 9  and R 10  are F. 
     
     
         61 . The compound of  claim 1 , wherein at least one of groups R 3 , R 4 , R 5  and R 6  and/or groups R 7 , R 8 , R 9  and R 10  are perfluoro C 1 -C 4  alkyl and any remaining groups R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  are selected from H or F. 
     
     
         62 . The compound of  claim 61 , wherein not more than two of the R 3 , R 4 , R 5  and R 6  positions and/or the R 7 , R 8 , R 9  and R 10  positions are substituted by perfluoro C 1 -C 4  alkyl. 
     
     
         63 . The compound of  claim 61 , wherein said perfluoro C 1 -C 4  alkyl is trifluoromethyl. 
     
     
         64 . The compound of  claim 1 , wherein at least one of groups R 3 , R 4 , R 5  and R 6  and/or groups R 7 , R 8 , R 9  and R 10  is —SO 2 —CF 3  and any remaining groups R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  are selected from H or F. 
     
     
         65 . The compound of  claim 1 , wherein said target bonding group M comprises a reactive group for reaction with a functional group on a target material, or a functional group for reaction with a reactive group on a target material. 
     
     
         66 . The compound of  claim 65 , wherein said reactive group is selected from the group consisting of succinimidyl ester, sulpho-succinimidyl ester, 4-sulfo-2,3,5,6-tetrafluorophenol (STP) ester, isothiocyanate, maleimide, haloacetamide, acid halide, hydrazide, vinylsulphone, dichlorotriazine and phosphoramidite. 
     
     
         67 . The compound of  claim 65 , wherein said functional group is selected from the group consisting of hydroxy, amino, sulphydryl, imidazole, carbonyl including aldehyde and ketone, carboxylic acid and thiophosphate. 
     
     
         68 . The compound of  claim 1 , wherein said target bonding group M comprises an affinity tag. 
     
     
         69 . The compound of  claim 1 , wherein P is selected from the group consisting of antibody, lipid, protein, peptide, carbohydrate, nucleotides which contain or are derivatized to contain one or more of an amino, sulphydryl, carbonyl, hydroxyl, carboxylic acid and thiophosphate groups, and oxy or deoxy polynucleic acids which contain or are derivatized to contain one or more of an amino, sulphydryl, carbonyl, hydroxyl, carboxylic acid and thiophosphate groups, microbial materials, drugs, hormones, cells, cell membranes and toxins.