Novel Ferrocene Labels for Electrochemical Assay and their Use in Analytical Methods

Abstract

Compounds of general formula I are used as labels in an electrochemical assay: (I) in which: Fc and Fc′ are substituted or unsubstituted ferrocenyl moieties, X is a C1 to C6 alkylene chain which is optionally interrupted by —O— or —NH—; Y is a C1 to C6 alkylene chain which is optionally interrupted by —O— or —NH—; Z is a C1 to C12 alkylene chain which may optionally be substituted and/or may optionally be interrupted by —O—, —S—, cycloalkyl, —CO—, —CON R1—, —NR1CO— or —NR1— in which R1 represents hydrogen or C1 to C4 alkyl; and R is a linker group. Compounds I are used to make labelled substrates, as well as functionalised compounds for making the labelled substrates.

1 . A method of detecting an electrochemically labelled substrate in an electrochemical assay comprising measuring the electrochemical activity of the label, wherein the label is a compound of general formula I: 
       
         
           
           
               
               
           
         
         wherein: 
       
       Fc is a substituted or unsubstituted ferrocenyl moiety, 
       Fc′ is a substituted or unsubstituted ferrocenyl moiety, and may be the same as or different from Fc; 
       X is a C1 to C6 alkylene chain which is optionally interrupted by —O—, —S—, or —NR 5 —, wherein R 5  represents hydrogen or C1 to C6 alkyl; 
       Y is a C1 to C6 alkylene chain which is optionally interrupted by —O—, —S—, or —NR 5 —, wherein R 5  represents hydrogen or C1 to C6 alkyl; 
       Z is a C 1  to C 12  alkylene chain which may optionally be substituted and/or may optionally be interrupted by —O—, —S—, cycloalkyl, —CO—, —CON R 1 —, —NR 1 CO— or —NR 1 —, wherein R 1  represents hydrogen or C1 to C4 alkyl; and 
       R is a linker group. 
     
     
         2 . The method of Use according to  claim 1 , wherein:
 X represents C1 to C6-alkylene optionally interrupted by oxygen;   Y represents C1 to C6-alkylene optionally interrupted by oxygen, and   Z represents C1 to C8 alkylene optionally interrupted by oxygen.   
     
     
         3 . The method of  claim 1 , wherein:
 X is —(CH 2 ) x , wherein x is 1 or 2;   Y is —(CH 2 ) y , wherein y is 1 or 2; and   Z is (CH 2 ) z , wherein z is from 1 to 8.   
     
     
         4 . The method of  claim 1 , wherein Fc and Fc′ are the same and X and Y are the same. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the substrate is biological molecule selected from nucleotides, nucleosides, oligonucleotides, and polynucleotides. 
     
     
         7 . The method of  claim 1 , wherein the substrate is a biological molecule selected from amino acids, peptides, and proteins. 
     
     
         8 . A method for manufacturing a functionalized labeling compound comprising a label moiety, comprising reacting a compound of general formula I: 
       
         
           
           
               
               
           
         
         wherein: 
         Fc is a substituted or unsubstituted ferrocenyl moiety, 
         Fc′ is a substituted or unsubstituted ferrocenyl moiety, and may be the same as or different from Fc; 
         X is a C1 to C6 alkylene chain which is optionally interrupted by —O—, —S—, or —NR 5 —, wherein R 5  represents hydrogen or C1 to C6 alkyl; 
         Y is a C1 to C6 alkylene chain which is optionally interrupted by —O—, —S—, or —NR 5 —, wherein R 5  represents hydrogen or C1 to C6 alkyl; 
         Z is a C 1  to C 12  alkylene chain which may optionally be substituted and/or may optionally be interrupted by —O—, —S—, cycloalkyl, —CO—, —CON R 1 —, —NR 1 CO— or —NR 1 — wherein R 1  represents hydrogen or C1 to C4 alkyl; and 
         R is a linker group comprising an oxygen atom 
       
       with a functionalising compound to obtain a funtionalised labelling compound of general formula III:
   A-L-F  III
 
 
       wherein A represents 
       
         
           
           
               
               
           
         
         wherein Fc, Fc′, X, Y and Z are as defined above, 
       
       F represents a functionalising moiety; and 
       L represents a linker moiety. 
     
     
         9 . A method for manufacturing a labelled substrate, comprising reacting a compound of general formula III:
   A-L-F  III
   wherein A, F and L are as defined in  claim 8 ;   
       with a substrate to form a labelled substrate. 
     
     
         10 . The method of  claim 9 , wherein the substrate is selected from amino acids, nucleotides, nucleosides, sugars, peptides, proteins, oligonucleotides, polynucleotides, carbohydrates, microparticles and nanoparticles. 
     
     
         11 . The method of  claim 9 , wherein the substrate is a biological substrate selected from nucleotides, nucleosides, oligonucleotides, and polynucleotides. 
     
     
         12 . The method of  claim 10 , wherein the substrate is a biological substrate selected from amino acids, peptides, and proteins. 
     
     
         13 . The functionalised labelling compound for use in the method of  claim 9 , wherein 
       A represents a labelling moiety of general formula Ia: 
       
         
           
           
               
               
           
         
         wherein: 
         Fc is a substituted or unsubstituted ferrocenyl moiety, 
         Fc′ is a substituted or unsubstituted ferrocenyl moiety, and may be the same as or different from Fc; 
         X is a C1 to C6 alkylene chain which is optionally interrupted by —O—, —S—, or —NR 5 —, in which R 5  represents hydrogen or C1 to C6 alkyl; 
         Y is a C1 to C6 alkylene chain which is optionally interrupted by —O—, —S—, or —NR 5 —, in which R 5  represents hydrogen or C1 to C6 alkyl; 
         Z is a C1 to C12 alkylene chain which may optionally be substituted and/or may optionally be interrupted by —O—, —S—, cycloalkyl, —CO—, —CON R 1 —, —NR 1 CO— or —NR 1 — in which R 1  represents hydrogen or C1 to C4 alkyl; 
       
       L represents a linker moiety; 
       and wherein F represents a functionalising moiety for reacting with a substrate for attachment of the labelling moiety to the substrate. 
     
     
         14 . The method of  claim 13 , wherein F represents a functionalising moiety for reacting with a substrate selected from amino acids, nucleotides, nucleosides, sugars, peptides, proteins, oligonucleotides, polynucleotides, carbohydrates, microparticles and nanoparticles. 
     
     
         15 . The method of  claims 13 , wherein the functionalising group F is selected from succinimidyl ester groups, phosphoramidite groups, maleimide groups, biotin and azide groups. 
     
     
         16 . The method of  claim 13 , wherein the functionalizing moiety is or is derivable from a phosphoramidite moiety. 
     
     
         17 . A method of detecting an electrochemically labelled substrate in an electrochemical assay comprising measuring the electrochemical activity of the label, wherein the electrochemically labelled substrate is a compound of general formula IIIa:
   A-L-F′—[S]  IIIa
   
       wherein A represents 
       
         
           
           
               
               
           
         
         wherein: 
         Fc is a substituted or unsubstituted ferrocenyl moiety, 
         Fc′ is a substituted or unsubstituted ferrocenyl moiety, and may be the same as or different from Fc; 
         X is a C1 to C6 alkylene chain which is optionally interrupted by —O—, —S—, or —NR 5 —, in which R 5  represents hydrogen or C1 to C6 alkyl; 
         Y is a C1 to C6 alkylene chain which is optionally interrupted by —O—, —S—, or —NR 5 —, in which R 5  represents hydrogen or C1 to C6 alkyl; 
         Z is a C 1  to C 12  alkylene chain which may optionally be substituted and/or may optionally be interrupted by —O—, —S—, cycloalkyl, —CO—, —CON R 1 —, —NR 1 CO— or —NR 1 — in which R 1  represents hydrogen or C1 to C4 alkyl; 
       
       L-F′ represents a linking moiety; and 
       [S] represents a substrate. 
     
     
         18 . The method of  claim 17 , wherein the substrate is selected from biological molecules, microparticles and nanoparticles. 
     
     
         19 . The method of  claim 17 , wherein the substrate is a biological molecule selected from amino acids, nucleotides, nucleosides, sugars, peptides, proteins, oligonucleotides, polynucleotides, and carbohydrates. 
     
     
         20 . The method of  claim 17 , wherein the substrate is a biological molecule selected from nucleotides, nucleosides, oligonucleotides, and polynucleotides. 
     
     
         21 . The method of  claim 20 , wherein the substrate is or comprises an oligonucleotide. 
     
     
         22 . The method of  claim 19 , wherein the substrate is a biological molecule selected from amino acids, sugars, peptides, and proteins. 
     
     
         23 . An assay kit for determining the presence of an assay target, wherein the assay kit comprises a labelled substrate according to  claim 17 . 
     
     
         24 . A compound according to general formula I 
       
         
           
           
               
               
           
         
         wherein: 
         Fc is a substituted ferrocenyl moiety, 
         Fc′ is a substituted ferrocenyl moiety, and may be the same as or different from Fc; 
         X is a C1 to C6 alkylene chain which is optionally interrupted by —O—, —S—, or —NR 5 —, wherein R 5  represents hydrogen or C1 to C6 alkyl; 
         Y is a C1 to C6 alkylene chain which is optionally interrupted by —O—, —S—, or —NR 5 —, wherein R 5  represents hydrogen or C1 to C6 alkyl; 
         Z is a C1 to C12 alkylene chain which may optionally be substituted and/or may optionally be interrupted by —O—, —S—, cycloalkyl, —CO—, —CON R 1 —, —NR 1 CO— or —NR 1 — wherein R 1  represents hydrogen or C1 to C4 alkyl; and 
         R is a linker group. 
       
     
     
         25 . A compound according to  claim 24 , wherein each ferrocenyl moiety is substituted by at least one substituent selected from halo, C1 to C4-alkyl, haloalkyl, aryl, C1 to C4 alkenyl, and cyano. 
     
     
         26 . The compound of  claim 24 ,
 wherein:   Fc and Fc′ are each an unsubstituted ferrocenyl moiety.   
     
     
         27 . A compound according to  claim 24 , wherein:
 X represents —(CH 2 ) x — in which x is from 1 to 6; and   Y represents —(CH 2 ) y — in which y is from 1 to 6.   
     
     
         28 . A compound according to  claim 24 , wherein Z represents C6 to C8 alkylene optionally interrupted by oxygen. 
     
     
         29 . A compound according to  claim 24 , wherein the linker group R comprises a group capable of reacting with a compatible group of a functionalising moiety or of a substrate to attach the compound to said functionalising moiety or said substrate. 
     
     
         30 . A compound selected from 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         31 . N,N-diferrocenylmethyl-6-aminohexanol or an analog thereof, wherein both ferrocenyl groups are substituted by one or more substituents. 
     
     
         32 . A labeled substrate comprising a labeling moiety derived from N,N-diferrocenylmethyl-6-aminohexanol or from an analog thereof, wherein both ferrocenyl groups are substituted by one or more substituents. 
     
     
         33 . A method of detecting a nucleic acid comprising contacting the nucleic acid with a complementary nucleic acid probe under conditions to allow hybridization between the probe and amplicon, followed by the step of selectively degrading either hybridized or unhybridized probe, wherein said probe is labelled with compound according to  claim 24 , and wherein the method provides the step of measuring the electrochemical activity of the compound labelling the probe of wherein said electrochemical activity is dependent either quantitatively or qualitatively on the extent of degradation of the probe. 
     
     
         34 . A method of detecting an amino acid, peptide or protein labelled with a compound according to  claim 24 , comprising the step of measuring the electrochemical activity of the compound.