Fluorescent dyes
Provided are various compounds comprising the formula Also provided are fluorescent dyes comprising the above compound. Additionally, a fluorescence energy transfer system is provided that comprises the above-described fluorescent dye and a second dye, wherein the second dye is capable of energy transfer with the fluorescent dye. Further provided is a kit for labeling a target molecule, where the kit comprises the above-described fluorescent dye with additional reagents useful for labeling the target molecule. Additionally provided is a target molecule labeled with the above-described fluorescent dye. Methods of labeling a target molecule with the above-described fluorescent dye are also provided.
1. A compound having the formula
wherein
R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 are independently H, F, Cl, Br, I, CN, nitro, azido, hydroxyl, amino, hydrazino, (substituted) aryl, (substituted) aryloxy, alkenyl, alkynyl, alkyl, alkoxy, alkylamino, dialkylamino, arylamino, diarylamino, alkyl(aryl)amino, alkanoylamino, alkylthio, alkylcarbonyl, aryl carbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkyloxycarbonyl, aroxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, alkyl(aryl)aminocarbonyl, arylcarboxamido, or Q, the alkyl or alkoxy portions of which are saturated or unsaturated, linear or branched, unsubstituted or further substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, thioamide, or Q;
Q is selected from the group consisting of a carboxyl group (CO 2 − ), a carbonate ester (COER 11 ), a sulfonate ester (SO 2 ER 11 ), a sulfoxide (SOR 11 ), a sulfone (SO 2 CR 11 R 12 R 13 ), a sulfonamide (SO 2 NR 11 R 12 ), a phosphate, a phosphate monoester (PO 3 − ER 11 ), a phosphate diester (PO 2 ER 11 ER 12 ), a phosphonate, a phosphonate monoester (PO 2 − ER 11 ), a phosphonate diester (POER 11 ER 12 ), a thiophosphate, a thiophosphate monoester (PSO 2 − ER 11 ), a thiophosphate diester (PSOER 11 ER 12 ), a thiophosphonate, a thiophosphonate monoester (PSO − ER 11 ), a thiophosphonate diester (PSER 11 ER 12 ), a phosphonamide (PONR 11 R 12 NR 14 R 15 ), a phosphonamide thioanalogue (PSNR 11 R 12 NR 14 R 15 ), a phosphoramide (PONR 11 R 12 NR 13 NR 14 R 15 ), a phosphoramide thioanalogue (PSNR 11 R 12 NR 13 NR 14 R 15 S), a phosphoramidite (PO 2 R 14 NR 11 R 12 ), and a phosphoramidite thioanalogue (POSR 14 NR 11 R 12 ), where E can independently be O or S, and where the aryl portions of any of the above are optionally substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, or thioamide;
R 1 in combination with R 2 , R 3 in combination with R 4 , R 5 in combination with R 6 , or R 9 in combination with R 10 can independently form a 5-10 member ring structure which is saturated or unsaturated, and which is optionally further substituted with an alkyl, an aryl, an alkenyl, an alkynyl, an alkoxy, an aryloxy, a hydroxyl, an F, Cl, Br, I, CN, a nitro, an alkylsulfonyl, an arylsulfonyl, an alkylsulfinyl, an arylsulfinyl, a (thio)carbonyl, a (thio)carboxylic acid, a (thio)carboxylic acid ester, a nitro, an amino, a (thio)amide, an azido, a hydrazino, or a (thio)phosphonate where each alkyl group or alkoxy group is independently saturated or unsaturated, linear or branched, or substituted or unsubstituted and each aryl group wherein is independently optionally substituted with F, Cl, Br, I, CN, OH, an alkyl, an alkenyl, an alkynyl, an alkoxy, an aryoxy, an alkylthio, an arylthio, a nitro, an azido, a hydrazino, a carboxyl, a thiocarboxyl, a carbonyl, a thiocarbonyl, a carboxylic acid ester, a thiocarboxylic acid ester, or an amino, amide, thioamide, or Q;
R 11 , R 12 , R 13 , R 14 and R 15 are independently a hydrogen, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, or substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, or substituted or unsubstituted, an aryl group wherein said aryl group is unsubstituted or substituted; wherein R 11 in combination with R 12 , R 14 in combination with R 15 , R 11 in combination with R 13 , R 11 in combination with R 14 , R 12 in combination with R 15 , or R 13 in combination with R 14 can independently form a 5-10 member ring;
X is O, OR 16 , NR 17 R 18 or N + R 17 R 18 ; Y is O, OR 16 , NR 19 R 20 or N + R 19 R 20 , wherein R 16 , R 17 , R 18 , R 19 and R 20 are independently H, alkyl, alkenyl, alkynyl, or aryl; or R 17 in combination with R 18 , or R 19 in combination with R 20 can independently form a 5-10 member ring structure which is optionally further substituted with alkyl, alkenyl, alkynyl, aryl, alkoxy, F, Cl, Br, I, carboxylic acid or carboxylic acid ester, where the alkyl group is saturated or unsaturated, linear or branched, and is optionally further substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, nitro, azido, hydrazino, alkoxy, aryoxy, alkylthio, arylthio, thiocarboxyl, carbonyl, thiocarbonyl, thiocarboxylic acid ester, amino, amide, thioamide, or Q, and the aryl group wherein is optionally substituted by F, Cl, Br, I, CN, OH, alkoxy, aryoxy, alkylthio, arylthio, nitro, azido, hydrazino, carboxyl, thiocarboxyl, carbonyl, thiocarbonyl, carboxylic acid ester, thiocarboxylic acid ester, amino, amide, thioamide, or Q;
R 17 in combination with R 6 , R 18 in combination with R 7 , R 19 in combination with R 8 , and R 20 in combination with R 9 , can independently form a 5- to 10-member ring structure that is saturated or unsaturated and optionally further substituted with an alkyl, an aryl, an alkenyl, an alkynyl, an alkoxy, an aryloxy, a hydroxyl, F, Cl, Br, I, CN, a nitro, a carbonyl, a thiocarbonyl, a thiocarboxylic acid, a thiocarboxylic acid ester, a nitro, an amino, a (thio)amide, an azido, a hydrazino, or Q, wherein the alkyl group herein is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group wherein the alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted; and wherein the aryl group is optionally substituted with F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkoxy, aryoxy, alkylthio, arylthio, nitro, azido, hydrazino, carboxyl, thiocarboxyl, carbonyl, thiocarbonyl, carboxylic acid ester, thiocarboxylic acid ester, amino, amide, thioamide, or Q;
-A-B—Z is
wherein n is 1-10, and
wherein Z is a reactive group selected from the group consisting of an isocyanate, an isothiocyanate, a monochlorotriazine, a dichlorotriazine, a 4,6-dichloro-1,3,5-triazines, a mono- or di-halogen substituted pyridine, a mono- or di-halogen substituted diazine, a maleimide, a haloacetamide, an aziridine, a sulfonyl halide, a carboxylic acid, an acid halide, a phosphonyl halide, a phosphoramidite (PO 2 R 14 NR 11 R 12 ), a phosphoramidite thioanalogue (POSR 14 NR 11 R 12 ), a hydroxysuccinimide ester, a hydroxysulfosuccinimide ester, an imido ester, an azido, a nitrophenol ester, an azide, a 3-(2-pyridyl dithio)-propionamide, a glyoxal, an aldehyde, a thiol, an amine, a hydrazine, a hydroxyl, a terminal alkene, a terminal alkyne, a platinum coordinate group and an alkylating agent; and
the carbon length for said alkenyl, alkynyl, and alkyl groups is from 1-16.
2. The compound of claim 1 , wherein n is 1-4.
3. The compound of claim 1 , wherein -A-B—Z is
4. The compound of claim 1 , wherein -A-B—Z is
5. The compound of claim 1 , having the structure:
6. The compound of claim 5 , wherein R 6 and R 9 are both H or CH 3 .
7. The compound of claim 5 , wherein X and Y are
(a) OH and O, respectively;
(b) NHCH 2 CH 3 and NCH 2 CH 3 , respectively; or
(c) N(CH 3 ) 2 and N + (CH 3 ) 2 , respectively.
8. A compound selected from the group consisting of:
wherein
A is O, S or NR 21 , wherein R 21 is a hydrogen, an alkyl, an aryl, an alkenyl, an alkynyl, an alkylcarbonyl, an arylcarbonyl, an alkylaminocarbonyl, or an arylaminocarbonyl, the alkyl or aryl portions of which is optionally substituted by an alkyl, an aryl, an alkenyl, an alkynyl, F, Cl, Br, I, CN, OH, an alkoxy, an aryoxy, an alkylthio, an arylthio, a nitro, an azido, a hydrazino, a thiocarboxyl, a carbonyl, a thiocarbonyl, a thiocarboxylic acid ester, or an amino, amide, thioamide, or Q;
B is an alkyl, an alkenyl, an alkynyl, or an aryl linker, the alkyl or aryl portions of which is optionally substituted by an alkyl, an alkenyl, an alkynyl, an aryl, F, Cl, Br, I, CN, OH, an alkoxy, an aryoxy, an alkylthio, an arylthio, a nitro, an azido, a hydrazino, a carboxyl, a thiocarboxyl, a carbonyl, a thiocarbonyl, a carboxylic acid ester, a thiocarboxylic acid ester, or an amino, amide, thioamide, or Q, or
B in combination with A form an amide, a thioamide, a carboxylic acid ester, a carboxylic acid thioester, a thiocarboxylic acid ester, an imine, a hyrazone, or Q;
Q is selected from the group consisting of a carboxyl group (CO 2 − ), a carbonate ester (COER 11 ), a sulfonate ester (SO 2 ER 11 ), a sulfoxide (SOR 11 ), a sulfone (SO 2 CR 11 R 12 R 13 ), a sulfonamide (SO 2 NR 11 R 12 ), a phosphate, a phosphate monoester (PO 3 − ER 11 ), a phosphate diester (PO 2 ER 11 ER 12 ), a phosphonate, a phosphonate monoester (PO 2 − ER 11 ), a phosphonate diester (POER 11 ER 12 ), a thiophosphate, a thiophosphate monoester (PSO 2 − ER 11 ), a thiophosphate diester (PSOER 11 ER 12 ), a thiophosphonate, a thiophosphonate monoester (PSO − ER 11 ), a thiophosphonate diester (PSER 11 ER 12 ), a phosphonamide (PONR 11 R 12 NR 14 R 15 ), a phosphonamide thioanalogue (PSNR 11 R 12 NR 14 R 15 ), a phosphoramide (PONR 11 R 12 NR 13 NR 14 R 15 ), a phosphoramide thioanalogue (PSNR 11 R 12 NR 13 NR 14 R 15 S), a phosphoramidite (PO 2 R 14 NR 11 R 12 ), and a phosphoramidite thioanalogue (POSR 14 NR 11 R 12 ), where E can independently be O or S, and where the aryl portions of any of the above are optionally substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, or thioamide;
R 11 , R 12 , R 13 , R 14 and R 15 are independently a hydrogen, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, or substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, or substituted or unsubstituted, an aryl group wherein said aryl group is unsubstituted or substituted; wherein R 11 in combination with R 12 , R 14 in combination with R 15 , R 11 in combination with R 13 , R 11 in combination with R 14 , R 12 in combination with R 15 , or R 13 in combination with R 14 can independently form a 5-10 member ring;
Z is a reactive group selected from the group consisting of an isocyanate, an isothiocyanate, a monochlorotriazine, a dichlorotriazine, a 4,6-dichloro-1,3,5-triazines, a mono- or di-halogen substituted pyridine, a mono- or di-halogen substituted diazine, a maleimide, a haloacetamide, an aziridine, a sulfonyl halide, a carboxylic acid, an acid halide, a phosphonyl halide, a phosphoramidite (PO 2 R 14 NR 11 R 12 ), a phosphoramidite thioanalogue (POSR 14 NR 11 R 12 ), a hydroxysuccinimide ester, a hydroxysulfosuccinimide ester, an imido ester, an azido, a nitrophenol ester, an azide, a 3-(2-pyridyl dithio)-propionamide, a glyoxal, an aldehyde, a thiol, an amine, a hydrazine, a hydroxyl, a terminal alkene, a terminal alkyne, a platinum coordinate group and an alkylating agent; and
the carbon length for said alkenyl, alkynyl, and alkyl groups is from 1-16.
9. A compound selected from the group consisting of:
wherein
A is O, S or NR 21 , wherein R 21 is a hydrogen, an alkyl, an aryl, an alkenyl, an alkynyl, an alkylcarbonyl, an arylcarbonyl, an alkylaminocarbonyl, or an arylaminocarbonyl, the alkyl or aryl portions of which is optionally substituted by an alkyl, an aryl, an alkenyl, an alkynyl, F, Cl, Br, I, CN, OH, an alkoxy, an aryoxy, an alkylthio, an arylthio, a nitro, an azido, a hydrazino, a thiocarboxyl, a carbonyl, a thiocarbonyl, a thiocarboxylic acid ester, or an amino, amide, thioamide, or Q,
B is an alkyl, an alkenyl, an alkynyl, or an aryl linker, the alkyl or aryl portions of which is optionally substituted by an alkyl, an alkenyl, an alkynyl, an aryl, F, Cl, Br, I, CN, OH, an alkoxy, an aryoxy, an alkylthio, an arylthio, a nitro, an azido, a hydrazino, a carboxyl, a thiocarboxyl, a carbonyl, a thiocarbonyl, a carboxylic acid ester, a thiocarboxylic acid ester, or an amino, amide, thioamide, or Q, or
B in combination with A form an amide, a thioamide, a carboxylic acid ester, a carboxylic acid thioester, a thiocarboxylic acid ester, an imine, a hyrazone, or Q,
Q is selected from the group consisting of a carboxyl group (CO 2 − ), a carbonate ester (COER 11 ), a sulfonate ester (SO 2 ER 11 ), a sulfoxide (SOR 11 ), a sulfone (SO 2 CR 11 R 12 R 13 ), a sulfonamide (SO 2 NR 11 R 12 ), a phosphate, a phosphate monoester (PO 3 − ER 11 ), a phosphate diester (PO 2 ER 11 ER 12 ), a phosphonate, a phosphonate monoester (PO 2 − ER 11 ), a phosphonate diester (POER 11 ER 12 ), a thiophosphate, a thiophosphate monoester (PSO 2 − ER 11 ), a thiophosphate diester (PSOER 11 ER 12 ), a thiophosphonate, a thiophosphonate monoester (PSO − ER 11 ), a thiophosphonate diester (PSER 11 ER 12 ), a phosphonamide (PONR 11 R 12 NR 14 R 15 ), a phosphonamide thioanalogue (PSNR 11 R 12 NR 14 R 15 ), a phosphoramide (PONR 11 R 12 NR 13 NR 14 R 15 ), a phosphoramide thioanalogue (PSNR 11 R 12 NR 13 NR 14 R 15 S), a phosphoramidite (PO 2 R 14 NR 11 R 12 ), and a phosphoramidite thioanalogue (POSR 14 NR 11 R 12 ), where E can independently be O or S, and where the aryl portions of any of the above are optionally substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, or thioamide,
R 11 , R 12 , R 13 , R 14 and R 15 are independently a hydrogen, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, or substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, or substituted or unsubstituted, an aryl group wherein said aryl group is unsubstituted or substituted; wherein R 11 in combination with R 12 , R 14 in combination with R 15 , R 11 in combination with R 13 , R 11 in combination with R 14 , R 12 in combination with R 15 , or R 13 in combination with R 14 can independently form a 5-10 member ring,
Z is a reactive group selected from the group consisting of an isocyanate, an isothiocyanate, a monochlorotriazine, a dichlorotriazine, a 4,6-dichloro-1,3,5-triazines, a mono- or di-halogen substituted pyridine, a mono- or di-halogen substituted diazine, a maleimide, a haloacetamide, an aziridine, a sulfonyl halide, a carboxylic acid, an acid halide, a phosphonyl halide, a phosphoramidite (PO 2 R 14 NR 11 R 12 ), a phosphoramidite thioanalogue (POSR 14 NR 11 R 12 ), a hydroxysuccinimide ester, a hydroxysulfosuccinimide ester, an imido ester, an azido, a nitrophenol ester, an azide, a 3-(2-pyridyl dithio)-propionamide, a glyoxal, an aldehyde, a thiol, an amine, a hydrazine, a hydroxyl, a terminal alkene, a terminal alkyne, a platinum coordinate group and an alkylating agent, and
the carbon length for said alkenyl, alkynyl, and alkyl groups is from 1-16;
wherein E − is an anion.
10. The compound of claim 1 , selected from the group consisting of:
wherein E − is an anion.
11. The compound of claim 1 , wherein the carbon length for said alkenyl, alkynyl, and alkyl groups is from 1-10.