Acridone derivatives as labels for fluorescence detection of target materials

Abstract

Disclosed are methods for assay of an analyte employing acridone dyes having the structure: Z 1 and Z 2 represent atoms necessary to complete one ring, two fused ring, three fused ring aromatic or heteroaromatic systems, each ring having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; R 2 , R 3 , R 4 and R 5 are selected from hydrogen, halogen, amide, hydroxyl, cyano, nitro, mono- or di-nitro-substituted benzyl, amino, mono- or di-C 1 -C 4 alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C 1 -C 6 alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C 1 -C 20 alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the groups -E-F and —(CH 2 —) n Y; R 1 is selected from hydrogen, mono- or di-nitro-substituted benzyl, C 1 -C 20 alkyl, aralkyl, the groups -E-F and —(CH 2 —) n Y; where E is a spacer group, F is a target bonding group; Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl.

1 - 36 . (canceled) 
     
     
         37 . A method for the assay of an analyte in a sample, the method comprising:
 i) contacting the analyte with a specific binding partner for said analyte under conditions suitable to cause the binding of at least a portion of said analyte to said specific binding partner to form a complex and wherein one of said analyte and said specific binding partner is labelled with a fluorescent dye of formula:   
       
         
           
           
               
               
           
         
         
           wherein: 
           groups R 2  and R 3  are attached to the Z 1  ring structure and groups R 4  and R 5  are attached to the Z 2  ring structure; 
           Z 1  and Z 2  independently represent the atoms necessary to complete one ring, two fused ring, or three fused ring aromatic or heteroaromatic systems, each ring having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; 
           at least one of groups R 1 , R 2 , R 3 , R 4  and R 5  is the group -E-F where E is a spacer group having a chain from 1-60 atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a target bonding group; 
           when any of said groups R 2 , R 3 , R 4  and R 5  is not said group -E-F, said remaining groups R 2 , R 3 , R 4  and R 5  are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C 1 -C 4  alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C 1 -C 6  alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C 1 -C 20  alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium and the group —(CH 2 —) n Y; and, 
           when group R 1  is not said group -E-F, it is selected from hydrogen, C 1 -C 20  alkyl, aralkyl and the group —(CH 2 —) n Y; 
           Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl; and n is an integer from 1 to 6; 
         
         ii) measuring the fluorescence lifetime of the labelled complex; and 
         iii) correlating the fluorescence lifetime with the presence or the amount of said analyte in said sample. 
       
     
     
         38 . The method of  claim 37 , wherein said fluorescent dye has a fluorescence lifetime in the range from 2 to 30 nanoseconds. 
     
     
         39 . The method of  claim 37 , wherein Z 1  and Z 2  independently represent the atoms necessary to complete a phenyl or a naphthyl ring structure. 
     
     
         40 . The method of  claim 37 , wherein said target bonding group F comprises either:
 i) a reactive group selected from carboxyl, succinimidyl ester, sulpho-succinimidyl ester, isothiocyanate, maleimide, haloacetamide, acid halide, hydrazide, vinylsulphone, dichlorotriazine and phosphoramidite; or   ii) a functional group selected from hydroxy, amino, sulphydryl, imidazole, carbonyl including aldehyde and ketone, phosphate and thiophosphate.   
     
     
         41 . The method of  claim 37 , wherein said spacer group E is selected from:
 —(CHR′) p —   —{(CHR′) q —O—(CHR′) r } s —   —{(CHR′) q —NR′—(CHR′) r } s —   —{(CHR′) q —(CH═CH)—(CHR′) r } s —   —{(CHR′) q —Ar—(CHR′) r } s —   —{(CHR′) q —CO—NR′—(CHR′) r } s —   —{(CHR′) q —CO—Ar—NR′—(CHR′) r } s —   where R′ is hydrogen, C 1 -C 4  alkyl or aryl, which may be optionally substituted with sulphonate, Ar is phenylene, optionally substituted with sulphonate, p is 1-20, preferably 1-10, q is 0-10, r is 1-10 and s is 1-5.   
     
     
         42 . The method of  claim 37 , wherein at least one of groups R 1 , R 2 , R 3 , R 4  and R 5  comprises the group —(CH 2 —) n Y, where Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl and n is zero or an integer from 1 to 6. 
     
     
         43 . The method of  claim 37 , wherein said analyte-specific binding partner pairs are selected from the group consisting of antibodies/antigens, lectins/glycoproteins, biotin/streptavidin, hormone/receptor, enzyme/substrate or co-factor, DNA/DNA, DNA/RNA and DNA/binding protein.