Violet laser excitable dyes and their method of use

Abstract

The present invention provides dye compounds optimally excited at about 400 nm and have a Stokes shift of at least about 80 nm. These dyes find use in detection of analyte in a sample and the preparation of dye-conjugates.

1 . A compound according to the formula: 
       
         
           
           
               
               
           
         
         wherein 
         X is independently carbon, —CR 12 , nitrogen, —NR 14 , sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR 12 —CR 13 , NR 14 —NR 15 , or S—S; 
         Y is independently carbon, —CR 12 , nitrogen, —NR 14 , sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR 12 —CR 13 , NR 14 —NR 15 , or S—S; 
         Z is independently carbon, —CR 12 , nitrogen, —NR 14 , sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR 12 —CR 13 , NR 14 —NR 15 , or S—S;
 wherein at least one of X, Y and Z is other than carbon or C—C; 
 R 12  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 R 13  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 R 14  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 R 15  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 
         R 1  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 1  comprises a reactive group, carrier molecule or solid support; 
         R 2  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 2  comprises a reactive group, carrier molecule or solid support; 
         R 3  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 3  comprises a reactive group, carrier molecule or solid support; 
         R 4  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 4  comprises a reactive group, carrier molecule or solid support; 
         R 5  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 5  comprises a reactive group, carrier molecule or solid support; 
         R 6  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 6  comprises a reactive group, carrier molecule or solid support; 
         R 7  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 7  comprises a reactive group, carrier molecule or solid support; 
         R 8  is hydrogen, alkyl, substituted alkyl, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 8  comprises a reactive group, carrier molecule or solid support; 
         R 9  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 9  comprises a reactive group, carrier molecule or solid support; 
         R 10  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 10  comprises a reactive group, carrier molecule or solid support; or 
         a member selected from
 R 1  in combination with R 2 ; 
 R 2  in combination with R 3 ; 
 R 3  in combination with R 4 ; 
 R 4  in combination with R 5 ; 
 R 6  in combination with R 7    
 R 7  in combination with R 8 ; 
 R 8  in combination with R 9 ; and 
 R 9  in combination with R 10 ; 
 together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl. 
 
       
     
     
         2 . The compound according to  claim 1 , wherein R 8  is alkyl substituted with —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO 2 R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O) 2 R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O) 2 R′, —S(O) 2 NR′R″, —NRSO 2 R′, —CN and —NO 2 ; wherein R′, R″, R′″ and R″″ each independently are selected from the group consisting of hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkyl, alkoxy, thioalkoxy and arylalkyl. 
     
     
         3 . The compound according to  claim 1 , wherein R 8  is —(CH 2 ) 3 SO 3   − . 
     
     
         4 . The compound according to  claim 1 , wherein X is —CR 12 , Y is nitrogen, and Z is oxygen. 
     
     
         5 . The compound according to  claim 4 , wherein R 12  is hydrogen. 
     
     
         6 . The compound according to  claim 1 , wherein R 4  is —SO 3   − . 
     
     
         7 . The compound according to  claim 1 , wherein R 3  is alkoxy. 
     
     
         8 . The compound according to  claim 1 , wherein at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  or R 9  comprises a sulfo group. 
     
     
         9 . The compound according to  claim 1 , wherein at least two of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  or R 9  comprises a sulfo group. 
     
     
         10 . The compound according to  claim 1 , wherein at least three of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  or R 9  comprises a sulfo group. 
     
     
         11 . The compound according to  claim 1  wherein at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  or R 9  comprises a reactive group, carrier molecule or solid support. 
     
     
         12 . The compound according to  claim 1 , wherein R 3  or R 8  comprises a reactive group, carrier molecule or solid support. 
     
     
         13 - 19 . (canceled) 
     
     
         20 . A method for detecting an analyte in a sample, wherein the method comprises
 a. combining a compound with a sample to form a combined sample, wherein the compound has the formula   
       
         
           
           
               
               
           
         
         wherein 
         X is independently carbon, —CR 12 , nitrogen, —NR 14 , sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR 12 —CR 13 , NR 14 —NR 15 , or S—S; 
         Y is independently carbon, —CR 12 , nitrogen, —NR 14 , sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR 12 —CR 13 , NR 14 —NR 15 , or S—S; 
         Z is independently carbon, —CR 12 , nitrogen, —NR 14 , sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR 12 —CR 13 , NR 14 —NR 15 , or S—S;
 wherein at least one of X, Y and Z is other than carbon or C—C; 
 R 12  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 R 13  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 R 14  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 R 15  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 
         R 1  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 1  comprises a reactive group, carrier molecule or solid support; 
         R 2  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 2  comprises a reactive group, carrier molecule or solid support; 
         R 3  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 3  comprises a reactive group, carrier molecule or solid support; 
         R 4  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 4  comprises a reactive group, carrier molecule or solid support; 
         R 5  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 5  comprises a reactive group, carrier molecule or solid support; 
         R 6  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 6  comprises a reactive group, carrier molecule or solid support; 
         R 7  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 7  comprises a reactive group, carrier molecule or solid support; 
         R 8  is hydrogen, alkyl, substituted alkyl, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 8  comprises a reactive group, carrier molecule or solid support; 
         R 9  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 9  comprises a reactive group, carrier molecule or solid support; 
         R 10  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 10  comprises a reactive group, carrier molecule or solid support; or 
         a member selected from
 R 1  in combination with R 2 ; 
 R 2  in combination with R 3 ; 
 R 3  in combination with R 4 ; 
 R 4  in combination with R 5 ; 
 R 6  in combination with R 7    
 R 7  in combination with R 8 ; 
 R 8  in combination with R 9 ; and 
 R 9  in combination with R 10 ; 
 together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl; 
 
         b. incubating the combined sample for a sufficient amount of time for the compound to associate with the analyte in the sample to form an incubated sample; 
         c. illuminating the incubated sample with an appropriate wavelength to form an illuminated sample; and 
         d. detecting the illuminated sample whereby the analyte in the sample is detected. 
       
     
     
         21 . The method according to claim  17 , wherein the illumination and/or detecting step comprises a flow cytometer. 
     
     
         22 - 23 . (canceled) 
     
     
         24 . A method for evaluating the viability of cells in a sample, comprising
 a. combining a compound with a sample to form a combined sample, wherein the compound comprises an amine reactive group and has the formula   
       
         
           
           
               
               
           
         
         
           wherein 
           X is independently carbon, —CR 12 , nitrogen, —NR 14 , sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR 12 —CR 13 , NR 14 —NR 15 , or S—S; 
         
         Y is independently carbon, —CR 12 , nitrogen, —NR 14 , sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR12-C13, NR14-NR15, or S—S; 
         Z is independently carbon, —CR 12 , nitrogen, —NR 14 , sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR 12 —CR 13 , NR 14 —NR 15 , or S—S;
 wherein at least one of X, Y and Z is other than carbon or C—C; 
 R 12  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 R 13  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 R 14  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 R 15  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 
         R 1  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 1  comprises a reactive group, carrier molecule or solid support; 
         R 2  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 2  comprises a reactive group, carrier molecule or solid support; 
         R 3  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 3  comprises a reactive group, carrier molecule or solid support; 
         R 4  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 4  comprises a reactive group, carrier molecule or solid support; 
         R 5  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 5  comprises a reactive group, carrier molecule or solid support; 
         R 6  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 6  comprises a reactive group, carrier molecule or solid support; 
         R 7  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 7  comprises a reactive group, carrier molecule or solid support; 
         R 8  is hydrogen, alkyl, substituted alkyl, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 8  comprises a reactive group, carrier molecule or solid support; 
         R 9  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 9  comprises a reactive group, carrier molecule or solid support; 
         R 10  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 10  comprises a reactive group, carrier molecule or solid support; or 
         a member selected from
 R 1  in combination with R 2 ; 
 R 2  in combination with R 3 ; 
 R 3  in combination with R 4 ; 
 R 4  in combination with R 5 ; 
 R 6  in combination with R 7    
 R 7  in combination with R 8 ; 
 R 8  in combination with R 9 ; and 
 R 9  in combination with R 10 ; 
 
         together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl; 
         b. incubating the combined sample for a sufficient amount of time for the compound to form a covalent bond with amine groups on a cell membrane of live cells and to form covalent bonds with amine groups inside dead cells in the sample to form an incubated sample; 
         c. illuminating the incubated sample with an appropriate wavelength to form an illuminated sample; and 
         d. detecting the illuminated sample whereby the viability of cells in a sample is evaluated. 
       
     
     
         25 . A kit for detecting an analyte in a sample, wherein the kit comprises:
 a. a compound having the formula   
       
         
           
           
               
               
           
         
         wherein 
         X is independently carbon, —CR 12 , nitrogen, —NR 14 , sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR 12 —CR 13 , NR 14 —NR 15 , or S—S; 
         Y is independently carbon, —CR 12 , nitrogen, —NR 14 , sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR 12 —CR 13 , NR 14 —NR 15 , or S—S; 
         Z is independently carbon, —CR 12 , nitrogen, —NR 14 , sulfur, oxygen, selenium, phosphorous, silicon, arsenic, amido, C—C, CR 12 —CR 13 , NR 14 —NR 15 , or S—S;
 wherein at least one of X, Y and Z is other than carbon or C—C; 
 R 12  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 R 13  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 R 14  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 R 15  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, or thioether; 
 
         R 1  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 1  comprises a reactive group, carrier molecule or solid support; 
         R 2  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 2  comprises a reactive group, carrier molecule or solid support; 
         R 3  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 3  comprises a reactive group, carrier molecule or solid support; 
         R 4  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 4  comprises a reactive group, carrier molecule or solid support; 
         R 5  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 5  comprises a reactive group, carrier molecule or solid support; 
         R 6  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 6  comprises a reactive group, carrier molecule or solid support; 
         R 7  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 7  comprises a reactive group, carrier molecule or solid support; 
         R 8  is hydrogen, alkyl, substituted alkyl, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 8  comprises a reactive group, carrier molecule or solid support; 
         R 9  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 9  comprises a reactive group, carrier molecule or solid support; 
         R 10  is hydrogen, alkyl, substituted alkyl, alkoxy, hydroxy, sulfo, halogen, amino, substituted amino, aldehyde, carboxylic acid, ester, azido, nitro, nitroso, cyano, thioether, 5-, 6- or 7-aromatic or heteroaromatic ring, substituted 5-, 6- or 7-aromatic or heteroaromatic ring or R 10  comprises a reactive group, carrier molecule or solid support; or 
         a member selected from
 R 1  in combination with R 2 ; 
 R 2  in combination with R 3 ; 
 R 3  in combination with R 4 ; 
 R 4  in combination with R 5 ; 
 R 6  in combination with R 7    
 R 7  in combination with R 8 ; 
 R 8  in combination with R 9 ; and 
 R 9  in combination with R 10 ; 
 
         together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl; and 
         b. instructions for detecting the analyte. 
       
     
     
         26 . (canceled)