| CPC C07H 21/00 (2013.01) [C07H 19/04 (2013.01); C09B 11/02 (2013.01); C09B 11/06 (2013.01); C09B 11/24 (2013.01); C12Q 1/707 (2013.01)] | 20 Claims |
|
1. A method for fluorescently labeling a target molecule, comprising the steps of: providing a fluorescent dye compound having the formula:
 wherein
R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 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;
wherein Q is selected from the group consisting of a carboxyl group (CO2−), a carbonate ester (COER11), a sulfonate ester (SO2ER11), a sulfoxide (SOR11), a sulfone (SO2CR11R12R13), a sulfonamide (SO2NR11R12), a phosphate (PO4=), a phosphate monoester (PO3−ER11), a phosphate diester (PO2ER11ER12), a phosphonate (PO3=), a phosphonate monoester (PO2−ER11) a phosphonate diester (POER11ER12), a thiophosphate (PSO3=), a thiophosphate monoester (PSO2−ER11), a thiophosphate diester (PSOER11ER12), a thiophosphonate (PSO2=), a thiophosphonate monoester (PSO−ER11), a thiophosphonate diester (PSER11ER12), a phosphonamide (PONR11R12NR14R15), a phosphonamide thioanalogue (PSNR11R12NR14R15), a phosphoramide (PONR11R12NR13NR14R15), a phosphoramide thioanalogue (PSNR11R12NR13NR14R15S), a phosphoramidite (PO2R14NR11R12), and a phosphoramidite thioanalogue (POSR14NR11R12),
wherein E can independently be O or S, and
wherein the aryl portions of any of the above are optionally substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, or thioamide;
wherein R1 in combination with R2, R3 in combination with R4, R5 in combination with R6, or R9 in combination with R10 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, 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;
R11, R12, R13, R14 and R15 are independently 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 R11 in combination with R12, R14 in combination with R15, R11 in combination with R13, R11 in combination with R14, R12 in combination with R15, or R13 in combination with R14 can independently form a 5-10 member ring;
X is O, OR16, NR17R18 or N+R17R18; Y is O, OR16, NR19R20 or N+R19R20, wherein R16, R17, R18, R19 and R20 are independently H, alkyl, alkenyl, alkynyl, or aryl; or R17 in combination with R18, or R19 in combination with R20 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;
wherein R17 in combination with R6, R18 in combination with R7, R19 in combination with R8, and R20 in combination with R9, 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 is O, S or NR21, wherein R21 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; and
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 (PO2R14NR11R12), a phosphoramidite thioanalogue (POSR14NR11R12), 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,
wherein the carbon lengths for said alkenyl, alkynyl, and alkyl groups are in the range of 1 to 16;
providing a target molecule to be labeled, wherein the target molecule is a peptide or a protein, and;
contacting the fluorescent dye compound with the target molecule such that reactive group Z reacts with the target molecule to form a covalent bond between reactive group Z and the target molecule.
|