Acridone derivatives as labels for fluorescence detection of target materials

Abstract

Disclosed are new acridone dye derivatives having characteristic fluorescence lifetimes. Also disclosed are methods for labelling target biological materials employing the acridone dyes and use of the labelled materials in biological assays. The acridone derivatives have the following structure:in which Z 1 and Z 2 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; 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, quatemary ammonium, the group —E—F and the group —(CH 2 —) n Y; R 1 is selected from hydrogen, mono- or di-nitro-substituted benzyl, C 1 -C 20 alkyl, aralkyl, the group —E—F and the group —(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; and n is an integer from 1 to 6. The invention also relates to a set of different fluorescent acridone dye derivatives, each dye having a different fluorescence lifetime, the set of dyes being particularly useful for multiparameter analysis.

1 - 29 . (cancelled)  
     
     
         30 . In a process for labelling and lifetime detection of a target material, the improvement comprising labeling with a dye of the 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 a one ring, a two fused ring, or a three fused ring aromatic or heteroaromatic system, wherein each ring includes five or six atoms selected from carbon atoms and 0-2 atoms selected from oxygen, nitrogen and sulphur;  
 R 2 , R 3 , R 4  and R 5  are independently 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 group -E-F and the group —(CH 2 —) n Y;  
 R 1  is selected from hydrogen, mono- or di-nitro-substituted benzyl, C 1 -C 20  alkyl, aralkyl, the group -E-F and the group —(CH 2 —) n Y;  
 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;  
 Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl; and n is an integer from 1 to 6;  
 And further wherein at least one of groups R 2 , R 3 , R 4  and R 5  is a water solubilising group selected from sulphonate, sulphate, quaternary ammonium and the group —(CH 2 —) n Y; and/or R 1  is the group —(CH 2 —) n Y.  
 
     
     
         31 . The method of  claim 30 , wherein said dye is a fluorescent dye 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; 
 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, the group -E-F and the group —(CH 2 —) n Y; and    R 1  is selected from hydrogen, C 1 -C 20  alkyl, aralkyl, the group -E-F and the group —(CH 2 —) n Y.    
     
     
         32 . The method of  claim 31 , wherein said fluorescent dye has a fluorescence lifetime in the range from 2 to 30 nanoseconds.  
     
     
         33 . The method of  claim 30 , wherein said dye is a non-fluorescent or substantially non-fluorescent dye wherein at least one of groups R 1 , R 2 , R 3 , R 4  and R 5  includes at least one nitro group.  
     
     
         34 . The method of  claim 30 , wherein at least one of groups R 1 , R 2 , R 3 , R 4  and R 5  is the group -E-F.  
     
     
         35 . The method of  claim 30 , wherein said target bonding group F includes a reactive group for reacting with a functional group on a target material, or a finctional group for reacting with a reactive group on a target material.  
     
     
         36 . The method of  claim 35 , wherein said reactive group is selected from carboxyl, succinimidyl ester, sulpho-succinimidyl ester, isothiocyanate, maleimide, haloacetamide, acid halide, hydrazide, vinylsulphone, dichlorotriazine and phosphoramidite.  
     
     
         37 . The method of  claim 35 , wherein said functional group is selected from hydroxy, amino, sulphydryl, imidazole, carbonyl including aldehyde and ketone, phosphate and thiophosphate.  
     
     
         38 . The method of  claim 30 , 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, or phenylene substituted with sulphonate, p is 1-20, q is 0-10,r is 1-10and s is 1-5.    
     
     
         39 . A method for labelling a target biological material, the method comprising: 
 i) adding to a liquid containing said target biological material a 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 a one ring, a two fused ring, or a three fused ring aromatic or heteroaromatic system, wherein each ring includes five or six atoms selected from carbon atoms and 0-2 atoms selected from oxygen, nitrogen and sulphur;    at least one of the groups R 1 , R 2 , R 3 , R 4  and R 5  is the group -E-F where E and F are hereinbefore defined;    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;    when group R′ is not said group -E-F, it is selected from hydrogen, C 1 -C 20  alkyl, aralkyl and the group —(CH 2 —) n Y;    and firther wherein at least one of remaining groups R 2 , R 3 , R 4  and R 5  is a water solubilising group selected from sulphonate, sulphate, quaternary ammonium and the group —(CH 2 —) n Y; and/or R 1  is the group —(CH 2 —) n Y, where Y and n are hereinbefore defined; and    ii) incubating said dye with said target biological material under conditions suitable for labelling said target.    
     
     
         40 . The method of  claim 39 , wherein said target biological material is selected from the group consisting of antibody, lipid, protein, peptide, carbohydrate, nucleotides which contain or are derivatized to contain one or more of an arnino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups, and oxy or deoxy polynucleic acids which contain or are derivatized to contain one or more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups, microbial materials, drugs, hormones, cells, cell membranes and toxins.  
     
     
         41 . A method for the assay of an analyte in a sample which method comprises: 
 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 a one ring, a two fused ring, or a three fused ring aromatic or heteroaromatic system, wherein each ring includes five or six atoms selected from carbon atoms and 0-2 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;    and further wherein at least one of remaining groups R 2 , R 3 , R 4  and R 5  is a water solubilising group selected from sulphonate, sulphate, quaternary ammonium and the group —(CH 2 —) n Y; and/or R 1  is the group —(CH 2 —) n Y, where Y and n are hereinbefore defined;    ii) measuring the emitted fluorescence of the labelled complex; and    iii) correlating the emitted fluorescence with the presence or the amount of said analyte in said sample.    
     
     
         42 . The method of  claim 41 , wherein said analyte-specific binding partner is selected from the group consisting antibodies/antigens, lectins/glycoproteins, biotin/streptavidin, honnone/receptor, enzyme/substrate or co-factor, DNA/DNA, DNA/RNA and DNA/binding protein.  
     
     
         43 . A set of two or more different fluorescent dyes, each dye of said set of dyes having the 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 a one ring, a two fused ring, or a three fused ring aromatic or heteroaromatic system, wherein each ring includes five or six atoms selected from carbon atoms and 0-2 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;  
 and further wherein at least one of remaining groups R 2 , R 3 , R 4  and R 5  is a water solubilising group selected from sulphonate, sulphate, quaternary ammonium and the group —(CH 2 —) n Y; and/or R 1  is the group —(CH 2 —) n Y, where Y and n are hereinbefore defined; and  
 wherein each dye of said set has a distinguishably different fluorescence lifetime compared with the lifetimes of the remaining dyes of the set.  
 
     
     
         44 . The set of  claim 43  including four different dyes, each dye of the set having a different fluorescence lifetime.  
     
     
         45 . The set of  claim 43 , wherein each of the fluorescent dyes in the set has a fluorescence lifetime in the range from 2 to 30 nanoseconds.  
     
     
         46 . The set of  claim 43 , wherein the difference in the lifetimes of the fluorescent emission of at least two dyes is at least 15% of the value of the shorter lifetime dye.  
     
     
         47 . The method of  claim 33 , wherein the fluorescence of said dye is enhanced by reducing at least one nitro group to —NHOH or —NH 2    
     
     
         48 . The method of  claim 33 , for detecting nitroreductase enzyme activity in a composition further comprising: 
 i) mixing under conditions to promote nitroreductase activity said composition with the dye; and    ii) measuring an increase in fluorescence wherein said increase is a measure of the amount of nitroreductase activity.    
     
     
         49 . The method of  claim 33 , further comprising: 
 i) binding one component of a specific binding pair to a surface;    ii) adding a second component of the specific binding pair under conditions to promote binding between the components, said second component being labelled with a nitroreductase enzyme;    iii) adding the dye; and    iv) detecting binding of the second component to the first component by measuring an increase in fluorescence as a measure of bound nitroreductase activity.    
     
     
         50 . The method of  claim 49 , wherein said specific binding pair is selected from the group consisting of antibodies/antigens, lectins/glycoproteins, biotin/streptavidin, hornone/receptor, enzyme/substrate, DNA/DNA, DNA/RNA and DNA/binding protein.  
     
     
         51 . The method of  claim 33 , further comprising: 
 i) contacting a host cell which has been transfected with a nucleic acid molecule comprising expression control sequences operably linked to a sequence encoding a nitroreductase, with the dye; and    ii) measuring an increase in fluorescence as a measure of nitroreductase gene expression.    
     
     
         52 . The method of  claim 51 , wherein said method is conducted in the presence of a test agent whose effect on gene expression is to be determined.  
     
     
         53 . A dye of the 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, wherein each ring includes five or six atoms selected from carbon atoms and 0-2 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; and, when any of said groups R 1 , 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, nitro, 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, mono- or di-nitro-substituted benzyl, 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;  
 and further wherein at least one of remaining groups R 2 , R 3 , R 4  and R 5  is a water solubilising group selected from sulphonate, sulphate, quaternary ammonium and the group —(CH 2 —) n Y; and/or R 1  is the group —(CH 2 —) n Y, where Y and n are hereinbefore defined.  
 
     
     
         54 . The dye of  claim 53 , wherein said target bonding group F comprises: 
 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.    
     
     
         55 . The dye of  claim 53 , wherein said spacer group E is selected from: 
 —(CHR′) p —   —{(CHR′) q —O—(CHR′) r } s —   —{(CHR′) q —NR′—(CHR′)} 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, q is 0-10, r is 1-10 and s is 1-5.