Chemiluminescent acridinium compounds and analogues thereof as substrates of hydrolytic enzymes

A chemiluminescent substrate of a hydrolytic enzyme, said substrate having the structure

• wherein
• P is PO3B or a sugar moiety and B is a divalent cation or two monovalent cations selected from the group consisting of Na2, H2, K2, Ca and Mg;
• M is oxygen;
• R1 is selected from the group consisting of methyl, sulfoalkyl and carboxymethyl;
• R2a, R2b, R2c, R3a, R3b, R3c and R3d can be the same or different and are selected from the group consisting of hydrogen, methyl, methoxy, halide and cyano (—CN);
• A− is a counter ion for the electroneutrality of the quaternary nitrogen of the acridinium compounds, said A− not being present if said R1 substituent contains a strongly ionizable group that can form an anion and pair with the quaternary ammonium cationic moiety;
• and
• X is selected from the group consisting of O, N and S, such that,
• when X is O or S, Y is selected from the group consisting of phenyl, (2′-methyl)phenyl, (2′-methoxy)phenyl, (2′,6′-dimethyl)phenyl, (2′-methyl-6′-methoxy)phenyl, (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl, (2′,6′-dimethoxy-4′-benzyloxycarbonyl)phenyl, (2′-methyl-6′-methoxy-4′-benzyloxycarbonyl)phenyl, (2′,6′-dimethyl-4′-carboxyl)phenyl, (2′,6′-dimethoxy-4′-carboxyl)phenyl and (2′-methyl-6′-methoxy-4′-carboxyl)phenyl;
• and Z is omitted;
• and
• when X is N, Z is toluenesulfonyl and Y is carboxypropyl.

A chemiluminescent substrate of a hydrolytic enzyme, said substrate having the structure

• wherein
• P is PO3B or a sugar moiety and B is a divalent cation or two monovalent cations selected from the group consisting of Na2, H2, K2, Ca and Mg;
• M is oxygen;
• R1 is selected from the group consisting of methyl, sulfoalkyl and carboxymethyl;
• R2a, R2b, R2c, R3a, and R3d can be the same or different and are selected from the group consisting of hydrogen, methyl, methoxy, halide and cyano (—CN);
• A− is a counter ion for the electroneutrality of the quaternary nitrogen of the acridinium compounds, said A− not being present if said R1 substituent contains a strongly ionizable group that can form an anion and pair with the quaternary ammonium cationic moiety;
• and
• X is selected from the group consisting of O, N and S, such that,
• when X is O or S, Y is selected from the group consisting of phenyl, (2′-methyl)phenyl, (2′-methoxy)phenyl, (2′,6′-dimethyl)phenyl, (2′-methyl-6′-methoxy)phenyl, (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl, (2′,6′-dimethoxy-4′-benzyloxycarbonyl)phenyl, (2′-methyl-6′-methoxy-4-benzyloxycarbonyl)phenyl, (2′,6′-dimethyl-4′-carboxyl)phenyl, (2′,6′-dimethoxy-4′-carboxyl)phenyl and (2′-methyl-6′-methoxy-4′-carboxyl)phenyl;
• and Z is omitted;
• and
• when X is N, Z is toluenesulfonyl and Y is carboxypropyl.

A chemiluminescent substrate of a hydrolytic enzyme, said substrate having the structure

• wherein
• P is PO3B or a sugar moiety and B is a divalent cation or two monovalent cations selected from the group consisting of Na2, H2, K2, Ca and Mg;
• M is oxygen;
• R1 is selected from the group consisting of methyl, sulfoalkyl and carboxymethyl;
• R2a, R2b, R2c, R3c and R3d can be the same or different and are selected from the group consisting of hydrogen, methyl, methoxy, halide and cyano (—CN);
• A− is a counter ion for the electroneutrality of the quaternary nitrogen of the acridinium compounds, said A− not being present if said R1 substituent contains a strongly ionizable group that can form an anion and pair with the quaternary ammonium cationic moiety;
• and
• X is selected from the group consisting of O, N and S, such that,
• when X is O or S, Y is selected from the group consisting of phenyl, (2′-methyl)phenyl, (2′-methoxy)phenyl, (2′,6′-dimethyl)phenyl, (2′-methyl-6′-methoxy)phenyl, (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl, (2′,6′-dimethoxy-4′-benzyloxycarbonyl)phenyl, (2′-methyl-6′-methoxy-4′-benzyloxycarbonyl)phenyl, (2′,6′-dimethyl-4′-carboxyl)phenyl, (2′,6′-dimethoxy-4′-carboxyl)phenyl and (2′-methyl-6′-methoxy-4′-carboxyl)phenyl;
• and Z is omitted;
• and
• when X is N, Z is toluenesulfonyl and Y is carboxypropyl.

A chemiluminescent substrate of a hydrolytic enzyme, said substrate having the structure

• wherein
• P is PO3B or a sugar moiety and B is a divalent cation or two monovalent cations selected from the group consisting of Na2, H2, K2, Ca and Mg;
• M is oxygen;
• R1 is selected from the group consisting of methyl, sulfoalkyl and carboxymethyl;
• R2a, R2b, R2c, R3a and R3b can be the same or different and are selected from the group consisting of hydrogen, methyl, methoxy, halide and cyano (—CN);
• A− is a counter ion for the electroneutrality of the quaternary nitrogen of the acridinium compounds, said A− not being present if said R1 substituent contains a strongly ionizable group that can form an anion and pair with the quaternary ammonium cationic moiety;
• and
• X is selected from the group consisting of O, N and S, such that,
• when X is O or S, Y is selected from the group consisting of phenyl, (2′-methyl)phenyl, (2′-methoxy)phenyl, (2′,6′-dimethyl)phenyl, (2′-methyl-6′-methoxy)phenyl, (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl, (2′,6′-dimethoxy-4′-benzyloxycarbonyl)phenyl, (2′-methyl-6′-methoxy-4′-benzyloxycarbonyl)phenyl, (2′,6′-dimethyl-4′-carboxyl)phenyl, (2′,6′-dimethoxy-4′-carboxyl)phenyl and (2′-methyl-6′-methoxy-4′-carboxyl)phenyl;
• and Z is omitted;
• and
• when X is N, Z is toluenesulfonyl and Y is carboxypropyl.

A chemiluminescent substrate of a hydrolytic enzyme, said substrate having the structure

• wherein
• P is PO3B or a sugar moiety and B is a divalent cation or two monovalent cations selected from the group consisting of Na2, H2, K2, Ca and Mg;
• M is oxygen;
• R1 is selected from the group consisting of methyl, sulfoalkyl and carboxymethyl;
• R2a, R2b, R2c, R3a, R3b, R3c and R3d can be the same or different and are selected from the group consisting of hydrogen, methyl, methoxy, halide and cyano (—CN);
• A− is a counter ion for the electroneutrality of the quaternary nitrogen of the acridinium compounds, said A− not being present if said R1 substituent contains a strongly ionizable group that can form an anion and pair with the quaternary ammonium cationic moiety;
• and
• X is selected from the group consisting of O, N and S, such that,
• when X is O or S, Y is selected from the group consisting of phenyl, (2′-methyl)phenyl, (2′-methoxy)phenyl, (2′,6′-dimethyl)phenyl, (2′-methyl-6′-methoxy)phenyl, (2′,6′-dimethyl-4′-benzyloxycarbonyl)phenyl, (2′,6′-dimethoxy-4′-benzyloxycarbonyl)phenyl, (2′-methyl-6′-methoxy-4′-benzyloxycarbonyl)phenyl, (2′,6′-dimethyl-4′-carboxyl)phenyl, (2′,6′-dimethoxy-4′-carboxyl)phenyl and (2′-methyl-6′-methoxy-4′-carboxyl)phenyl;
• and Z is omitted;
• and
• when X is N, Z is toluenesulfonyl and Y is carboxypropyl.

A chemiluminescent substrate of a hydrolytic enzyme, said substrate having the structure

• wherein
• P is PO3B or a sugar moiety and B is a divalent cation or two monovalent cations selected from the group consisting of Na2, H2, K2, Ca and Mg;
• M is oxygen;
• R1 is selected from the group consisting of methyl, sulfoalkyl and carboxymethyl;
• R2a, R2b, R2c, R3a, R3b, R3c and R3d can be the same or different and are selected from the group consisting of hydrogen, methyl, methoxy, halide and cyano (—CN);
• R11 is selected from the group consisting of hydrogen, —R, substituted or unsubstituted aryl, halides, nitro, sulfonate, sulfate, phosphonate, —CO2H, —C(O)OR, cyano (—CN), —SCN, —OR, —SR, —SSR, —C(O)R, —C(O)NHR, ethylene glycol and polyethyelene glycol, where R is an alkyl group having 1 to 6 carbon atoms;
• A− is a counter ion for the electroneutrality of the quaternary nitrogen of the acridinium compounds, said A− not being present if said R2 substituent contains a strongly ionizable group that can form an anion and pair with the quaternary ammonium cationic moiety;
• and
• X1 and X2 are the same or different and are selected from the group consisting of O, N and S, such that,
• when at least one of X1 and X2 is O or S, the corresponding Z1 or Z2 is omitted;
• and
• when at least one of X1 and X2 is N, the corresponding Z1 or Z2 is hydrogen, alkyl, aryl or toluenesulfonyl.