Monoazo dyes with cyclic amine as fluorescence quenchers

Abstract

The present disclosure provides reactive quencher dyes that can be used in the detection and/or quantification of desirable target molecules, such as proteins, nucleic acids and various cellular organelles. These dyes are essentially non-fluorescent but are efficient quenchers of various fluorescent dyes. Also, provided are methods of using the dyes, bio-probes incorporating dyes and methods of using the bio-probes. The quencher dyes described herein are modified to provide beneficial properties.

What is claimed is: 
     
         1 . A compound having the formula: 
       
         
           
           
               
               
           
         
         wherein at least one of R 1  or R 2  is a nitro group; 
         X is O, S, or NR 17 , wherein R 17  is H, an alkyl group that is saturated or unsaturated, linear or branched, unsubstituted, or further substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, thioamide, or Q, or an aryl group optionally substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, thioamide, or Q, 
         Q is a carboxyl group (CO 2   − ), a carbonate ester (COER 12 ), a sulfonate ester (SO 2 ER 12 ), a sulfoxide (SOR 12 ), a sulfone (SO 2 CR 12 R 13 R 14 ), a sulfonamide (SO 2 NR 12 R 13 ), a phosphate (PO 4   = ), a phosphate monoester (PO 3   − ER 12 ), a phosphate diester (PO 2 ER 12 ER 13 ), a phosphonate (PO 3   = ) a phosphonate monoester (PO 2   − ER 12 ) a phosphonate diester (POER 12 ER 13 ), a thiophosphate (PSO 3   = ), a thiophosphate monoester (PSO 2   − ER 12 ) a thiophosphate diester (PSOER 12 ER 13 ), a thiophosphonate (PSO 2   = ), a thiophosphonate monoester (PSO − ER 12 ) a thiophosphonate diester (PSER 12 ER 13 ), a phosphonamide (PONR 12 R 13 NR 15 R 16 ), its thioanalogue (PSNR 12 R 13 NR 15 R 16 ), a phosphoramide (PONR 12 R 13 NR 14 NR 15 R 16 ), its thioanalogue (PSNR 12 R 13 NR 14 NR 15 R 16 ), a phosphoramidite (PO 2 R 15 NR 12 R 13 ) or its thioanalogue (POSR 15 NR 12 R 13 ), wherein E is independently O or S; 
         R 1 , R 2 , R 6 , R 7 , R 8 , and R 9  are each independently H, F, Cl, Br, I, CN, nitro, azido, hydroxyl, amino, hydrazino, aryl, aroxyl, alkenyl, alkynyl, alkyl, alkoxy, alkylamino, dialkylamino, arylamino, diarylamino, alkylamino, alkylarylamino, alkanoylamino, alkylthio, alkylcarbonyl, aryl carbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkyloxycarbonyl, aroxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, alkyl(aryl)aminocarbonyl, arylcarboxamido, or Q, wherein the alkyl or alkoxy groups 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, and the aryl group is optionally substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, thioamide, or Q; 
         one or more of R 1  in combination with R 17 , R 6  in combination with R 7 , and R 8  in combination with R 9  form a saturated or unsaturated 5- to 10-member ring optionally substituted by one or more of a saturated or unsaturated, linear or branched, substituted or unsubstituted alky group, aryl, alkenyl, alkynyl, a saturated or unsaturated, branched or linear, substituted or unsubstituted alkoxy group, aroxyl, hydroxyl, F, Cl, Br, I, CN, nitro, alkylsulfonyl, arylsulfonyl, alkylsulfinyl, arylsulfinyl, carbonyl, thiocarbonyl, carboxylic acid, thiocarboxylic acid, carboxylic acid ester, thiocarboxylic acid ester, nitro, amino, amide, thioamide, azido, hydrazino, phosphonate or thiophosphonate, wherein the aryl group is optionally substituted with F, Cl, Br, I, CN, OH, alkyl, alkenyl, alkynyl, alkoxy, aryoxy, alkylthio, arylthio, nitro, azido, hydrazino, carboxyl, thiocarboxyl, carbonyl, thiocarbonyl, carboxylic acid ester, thiocarboxylic acid ester, unsubstituted or substituted amino, amide, thioamide, or Q; 
         R 10  and R 11  are each independently H, alkyl, aryl, alkylcarbonyl, aryl carbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkoxycarbonyl, aroxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, alkyl(aryl)aminocarbonyl, arylcarboxamido, or Q, wherein the alkyl group and the alkoxy group are each independently saturated or unsaturated, linear or branched, unsubstituted or further substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, thioamide, or Q, and the aryl group is unsubstituted or optionally substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, thioamide, or Q; 
         R 12 , R 13 , R 14 , R 15  and R 16  are each independently H, a halogen, an amino group, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkyl group, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryl group,
 or one or more of R 12  in combination with R 13 , R 14  in combination with R 16 , R 12  in combination with R 14 , R 12  in combination with R 15 , R 13  in combination with R 16 , and R 14  in combination with R 15  form a 5- to 10-member ring; 
 
         at least one of R 7  in combination with R 10 , or R 9  in combination with R 11  form a saturated or unsaturated 5- to 10-member ring optionally substituted with alkyl, aryl, alkenyl, alkynyl, alkoxy, aroxyl, hydroxyl, F, Cl, Br, I, CN, nitro, alkylsulfonyl, arylsulfonyl, alkylsulfinyl, arylsulfinyl, carbonyl, thiocarbonyl, carboxylic acid, thiocarboxylic acid, carboxylic acid ester, thiocarboxylic acid ester, nitro, amino, amide, thioamide, azido, hydrazino, phosphonate, or thiophosphonate wherein the alky group and the alkoxy group are each independently saturated or unsaturated, linear or branched, substituted or unsubstituted, and wherein the aryl group is unsubstituted or substituted with F, Cl, Br, I, CN, OH, alkyl, alkenyl, alkynyl, alkoxy, aryoxy, alkylthio, arylthio, nitro, azido, hydrazino, carboxyl, thiocarboxyl, carbonyl, thiocarbonyl, carboxylic acid ester, thiocarboxylic acid ester, unsubstituted or substituted amino, amide, thioamide, or Q; and 
         R 12 , R 13 , R 14 , R 15  and R 16  are each independently H, a halogen, an amino group, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkyl group, a saturated or unsaturated, branched or linear, substituted or unsubstituted alkoxy group, or an unsubstituted or substituted aryl group; or one or more of R 12  in combination with R 13 , R 14  in combination with R 16 , R 12  in combination with R 14 , R 12  in combination with R 15 , R 13  in combination with R 16 , and R 14  in combination with R 15  form a 5- to 10-member ring, 
         wherein at least one of R 1 , R 2 , R 6 , R 7  R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16  and R 17  comprises one or more reactive groups Z independently selected from isocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine, 4,6-dichloro-1,3,5-triazines, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, haloacetamide, aziridine, sulfonyl halide, carboxylic acid, acid halide, phosphonyl halide, phosphoramidite (PO 2 R 15 NR 12 R 13 ) or its thioanalogue (POSR 15 NR 12 R 13 ), hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, azido, nitrophenol ester, azide, 3-(2-pyridyl dithio)-propionamide, glyoxal, aldehyde, thiol, amine, hydrazine, hydroxyl, terminal alkene, a terminal alkyne, a platinum coordinate group and an alkylating agent, 
         wherein at least one of 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 , R 14 , R 15 , R 16  and R 17  further comprises one or more electrophilic reactive groups selected from the group consisting of isocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine, 4,6-dichloro-1,3,5-triazines, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, haloacetamide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenol, azide, 3-(2-pyridyl dithio)-propionamide, glyoxal, and aldehyde. 
       
     
     
         2 . A conjugate obtained by a process of reacting the compound of  claim 1  with a target molecule, wherein said target molecule is a nucleoside, a nucleotide, an oligonucleotide, a polynucleotide, a peptide nucleic acid, a protein, a peptide, an enzyme, an antigen, an antibody, a hormone, a hormone receptor, a cellular receptor, a lymphokine, a cytokine, a hapten, a lectin, avidin, strepavidin, digoxygenin, a carbohydrate, an oligosaccharide, a polysaccharide, a lipid, or a glycolipid. 
     
     
         3 . The conjugate of  claim 2 , wherein the target molecule is a nucleoside, nucleotide, oligonucleotide or polynucleotide. 
     
     
         4 . The conjugate of  claim 2 , wherein the compound is linked to the target molecule by a linker arm. 
     
     
         5 . The compound of  claim 1 , wherein each alkyl or alkoxy group independently comprises from 1-18 carbon atoms. 
     
     
         6 . The compound of  claim 5 , wherein each alkyl or alkoxy group independently comprises from 1-6 carbon atoms. 
     
     
         7 . The compound of  claim 1 , wherein R 12 , R 13 , R 14 , R 15  and R 16  are each independently H, a halogen, an amino group, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkyl group, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryl group. 
     
     
         8 . The compound of  claim 7 , wherein each alkyl or alkoxy group independently comprises from 1-18 carbon atoms. 
     
     
         9 . The compound of  claim 8 , wherein each alkyl or alkoxy group independently comprises from 1-6 carbon atoms. 
     
     
         10 . The compound of  claim 1 , wherein
 X is O, S, or NR 17 , wherein R 17  is H, an alkyl group that is saturated or unsaturated, linear or branched, unsubstituted, or further substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, thioamide, or an aryl group optionally substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, or thioamide,   R 1 , R 2 , R 6 , R 7 , R 8 , and R 9  are each independently H, F, Cl, Br, I, CN, nitro, azido, hydroxyl, amino, hydrazino, aryl, aroxyl, alkenyl, alkynyl, alkyl, alkoxy, alkylamino, dialkylamino, arylamino, diarylamino, alkylamino, alkylarylamino, alkanoylamino, alkylthio, alkylcarbonyl, aryl carbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkyloxycarbonyl, aroxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, alkyl(aryl)aminocarbonyl, or arylcarboxamido, wherein the alkyl or alkoxy groups are saturated or unsaturated, linear or branched, unsubstituted or further substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, or thioamide, and the aryl group is optionally substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, or thioamide;   one or more of R 1  in combination with R 17 , R 6  in combination with R 7 , and R 8  in combination with R 9  form a saturated or unsaturated 5- to 10-member ring optionally substituted by one or more of a saturated or unsaturated, linear or branched, substituted or unsubstituted alky group, aryl, alkenyl, alkynyl, a saturated or unsaturated, branched or linear, substituted or unsubstituted alkoxy group, aroxyl, hydroxyl, F, Cl, Br, I, CN, nitro, alkylsulfonyl, arylsulfonyl, alkylsulfinyl, arylsulfinyl, carbonyl, thiocarbonyl, carboxylic acid, thiocarboxylic acid, carboxylic acid ester, thiocarboxylic acid ester, nitro, amino, amide, thioamide, azido, hydrazino, phosphonate or thiophosphonate, wherein the aryl group is optionally substituted with F, Cl, Br, I, CN, OH, alkyl, alkenyl, alkynyl, alkoxy, aryoxy, alkylthio, arylthio, nitro, azido, hydrazino, carboxyl, thiocarboxyl, carbonyl, thiocarbonyl, carboxylic acid ester, thiocarboxylic acid ester, unsubstituted or substituted amino, amide, or thioamide;   R 10  and R 11  are each independently H, alkyl, aryl, alkylcarbonyl, aryl carbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkoxycarbonyl, aroxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, alkyl(aryl)aminocarbonyl, or arylcarboxamido, wherein the alkyl group and the alkoxy group are each independently saturated or unsaturated, linear or branched, unsubstituted or further substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, or thioamide, and the aryl group is unsubstituted or optionally substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, or thioamide; and   at least one of R 7  in combination with R 10 , or R 9  in combination with R 11  form a saturated or unsaturated 5- to 10-member ring optionally substituted with alkyl, aryl, alkenyl, alkynyl, alkoxy, aroxyl, hydroxyl, F, Cl, Br, I, CN, nitro, alkylsulfonyl, arylsulfonyl, alkylsulfinyl, arylsulfinyl, carbonyl, thiocarbonyl, carboxylic acid, thiocarboxylic acid, carboxylic acid ester, thiocarboxylic acid ester, nitro, amino, amide, thioamide, azido, hydrazino, phosphonate, or thiophosphonate wherein the alky group and the alkoxy group are each independently saturated or unsaturated, linear or branched, substituted or unsubstituted, and wherein the aryl group is unsubstituted or substituted with F, Cl, Br, I, CN, OH, alkyl, alkenyl, alkynyl, alkoxy, aryoxy, alkylthio, arylthio, nitro, azido, hydrazino, carboxyl, thiocarboxyl, carbonyl, thiocarbonyl, carboxylic acid ester, thiocarboxylic acid ester, unsubstituted or substituted amino, amide, or thioamide.   
     
     
         11 . The compound of  claim 10 , wherein each alkyl or alkoxy group independently comprises from 1-18 carbon atoms. 
     
     
         12 . The compound of  claim 11 , wherein each alkyl or alkoxy group independently comprises from 1-6 carbon atoms. 
     
     
         13 . The compound of  claim 10 , wherein R 12 , R 13 , R 14 , R 15  and R 16  are each independently H, a halogen, an amino group, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkyl group, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryl group. 
     
     
         14 . The compound of  claim 13 , wherein each alkyl or alkoxy group independently comprises from 1-18 carbon atoms. 
     
     
         15 . The compound of  claim 14 , wherein each alkyl or alkoxy group independently comprises from 1-6 carbon atoms.