Chemiluminescent compounds

1 . A compound of general formula (I)

wherein:

either:

R 1 is a reactive group capable of reacting with an amine or thiol moiety;

L 1 is a hydrocarbon linker moiety comprising 2-12 carbon atoms, optionally substituted with hydroxy, halo, nitro or C 1 -C 4 alkoxy; and

R 2 is hydrogen, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, aryl, fused aryl, C 1 -C 4 alkoxy, C 1 -C 4 acyl, halide, hydroxy or nitro;

or, alternatively:

the combination R 1 -L 1 - comprises a C 1 -C 4 alkyl group optionally substituted with hydroxy, halo, nitro or C 1 -C 4 alkoxy; and

R 2 comprises a group R 4 -L 1 -, where R 4 is a reactive group capable of reacting with an amine or thiol moiety; and L 1 is as defined above;

L 2 is —C(═O)O—, —C(═O)—S— or —C(═O)N(SO 2 R 5 )—,

wherein, in each case, the —C(═O) is linked to the ring carbon atom, and

R 5 is C 1 -C 8 alkyl, aryl, C 1 -C 8 alkoxy or C 1 -C 8 acyl;

R 3 is a phenyl group substituted independently at the 2 and 6 positions with nitro, fluorine, chlorine, bromine or trifluoromethyl groups, wherein at least one of the said substituents is electron-withdrawing such that the pKa of the conjugate acid of the leaving group formed from R 3 and the —O, —S or —N(SO 2 R 5 ) of the L 2 group is ≦ about 9.5; and

X − is an anion formed as the result of the synthesis and processing of the molecule; wherein the compound may contain one or more additional R 2 moieties on either or both outer rings, provided that only one of said R 2 moieties may comprise an R 4 -L 1 - group.

2 . A compound as claimed in claim 1 wherein R1, or R4 is an active ester, a maleimide or an active halide.

3 . A compound as claimed in claim 2 wherein R 1 or R 4 is a succinimidyl ester, an imidate ester, chlorocarbonyl, bromocarbonyl, iodocarbonyl, chlorosulfonyl or fluorodinitrophenyl.

4 . A compound as claimed in claim 1 wherein, independently or in any combination:

R 1 is an active ester, an imidate, or an active halide;

L 1 comprises 3 to 10 carbon atoms;

R 2 is hydrogen or C1-C4 alkyl;

L 2 is —C (═O) O—; and

X − is a halide or halide-containing anion.

5 . A compound as claimed in claim 4 wherein, independently or in any combination:

R 1 is a succinimidyl ester, an imidate ester, chlorocarbonyl, bromocarbonyl, iodocarbonyl, chlorosulfonyl or fluorodinitrophenyl moiety;

L 1 comprises 3 to 10 methylene units;

R 2 is hydrogen; and

X − is iodide, fluorosulfonate, trifluoromethanesulfonate or trifluoroacetate.

6 . A compound as claimed in claim 1 wherein, independently or in any combination:

R 1 is a maleimide moiety;

L 1 comprises 3 to 10 methylene units;

R 2 is hydrogen; and

X − is iodide, fluorosulfonate, trifluoromethanesulfonate or trifluoroacetate.

7 . A compound as claimed in any one of claims 1 to 6 wherein R 3 is chosen such that the pKa of the conjugate acid of the L 2 -R 3 leaving group is ≦3.

8 . A compound according to claim 1 comprising:

9-(2,6-Bis(trifluoromethyl)phenoxycarbonyl)-10-(10-succinimidyloxycarbonyldecyl) acridinium trifluoromethanesulfonate;

9-(2,6-dibromophenoxycarbonyl)-10-(10-succinimidyloxycarbonyldecyl)acridinium trifluoromethanesulfonate;

9-(2,6-bis(trifluoromethyl)phenoxycarbonyl)-10-(3-succinimidyloxycarbonylpropyl) acridinium trifluoromethanesulfonate;

9-(2,6-dibromophenoxycarbonyl)-10-(3-succinimidyloxycarbonylpropyl)acridinium trifluoromethanesulfonate;

9-(2,6-dinitrophenoxycarbonyl)-10-(10-succinimidyloxycarbonyldecyl)acridinium trifluoromethanesulfonate; or

9-(2,6-dinitrophenoxycarbonyl)-10-(3-succinimidyloxycarbonylpropyl)acridinium trifluoromethanesulfonate.

9 . A process for the preparation of a compound as claimed in claim 1 , the process comprising treating a compound of general formula (II):

wherein R 2 , L 2 and R 3 are as defined in general formula (I) with a compound of general formula (III)

X-L 1 -R 1   (III)

wherein L 1 and R 1 are as defined in general formula (I) and X is a leaving group.

10 . A method of detecting or monitoring the association or dissociation of a first and a second binding partner in a medium, wherein the first binding partner is labelled with a compound as claimed in claim 1 and the second binding partner is labelled with a quencher or is itself a quencher, such that when the first and second binding partners are associated, the chemiluminescent signal of the compound as claimed in claim 1 is modulated; the method comprising adding hydrogen peroxide to the medium while maintaining the pH at about 6-10 and detecting the emission of chemiluminescent radiation.

11 . A method as claimed in claim 10 , wherein the first and second binding partners are molecular species which associate or dissociate via a covalent reaction.

12 . A method as claimed in claim 10 , wherein the first and second binding partners are a ligand and an anti-ligand.

13 . A method of detecting the presence or absence in a medium of an analyte comprising a quencher for a compound as claimed in claim 1 , the method comprising:

a) adding to the medium a specific binding partner for the analyte, said specific binding partner labelled with a compound as claimed in claim 1 such that if analyte is present a specific binding complex is formed;

b) adding hydrogen peroxide to the medium while maintaining the pH at about 6-10; and

c) detecting the emission of chemiluminescent radiation, wherein a modulated signal of the compound as claimed in claim 1 indicates the presence of the analyte.

14 . A method of detecting the presence or absence in a medium of an analyte which is not a quencher for a compound as claimed in claim 1 , the method comprising:

a) adding to the medium a specific binding partner for the analyte, said specific binding partner labelled with a compound as claimed in claim 1 or a quencher for a compound as claimed in claim 1 such that if analyte is present a specific binding complex is formed;

b) adding to the medium simultaneously or sequentially a secondary binding reagent labelled with either a quencher for the compound as claimed in claim 1 used in step (a) or a compound as claimed in claim 1 if a quencher is used in step (a); and

c) detecting the presence of specific binding complex by a chemiluminescent method including the steps of adding hydrogen peroxide to the medium while maintaining the pH at about 6-10 and detecting the emission of chemiluminescent radiation, wherein a modulated signal of the compound as claimed in claim 1 indicates the presence of the analyte.

15 . A method of detecting the presence or absence in a medium of an analyte which is not a quencher for a compound as claimed in claim 1 , the method comprising:

a) adding to the medium a specific binding partner for the analyte labelled with a compound as claimed in claim 1 or with a quencher for a compound as claimed in claim 1 such that if analyte is present, a specific binding complex is formed;

b) adding to the medium simultaneously or sequentially a second specific binding partner labelled with either a quencher or a compound as claimed in claim 1 as appropriate to the first labelled specific binding partner; and

c) detecting the presence of specific binding complex by a chemiluminescent method including the steps of adding hydrogen peroxide to the medium while maintaining the pH at about 6-10 and detecting the emission of chemiluminescent radiation, wherein a modulated signal of the compound as claimed in claim 1 indicates the presence of the analyte.

16 . A method for detecting the presence of inhibitors or promoters of a binding process, the method comprising mixing a first and a second specific binding partner labelled respectively with a compound as claimed in claim 1 and a quencher, adding hydrogen peroxide to the medium while maintaining the pH at about 6-10 and detecting the emission of chemiluminescent radiation, wherein modulation of the chemiluminescent signal of the compound as claimed in claim 1 indicates promotion of the binding process and the presence of an unmodulated signal indicates inhibition of the binding process.

17 . A method as claimed in claim 16 for monitoring the cleavage of an enzyme substrate or the hybridisation of complementary nucleic acids.

18 . A method as claimed in any one of claims 10 to 17 wherein the quencher is methyl red.

19 . A reagent comprising a ligand or anti-ligand labelled with a compound as claimed in claim 1 .

20 . A reagent as claimed in claim 19 wherein the ligand or anti-ligand is a nucleic acid, a peptide or a protein.

21 . A reagent as claimed in claim 20 wherein the ligand or anti-ligand is further labelled with a quencher.

22 . A reagent as claimed in claim 21 comprising a nucleic acid molecule comprising up to about 40 bases having ends which are mutually complementary and a central section containing a sequence of about 15 to 30 bases which is complementary to a target sequence and wherein the molecule is labelled at one end with a compound as claimed in claim 1 and at the other end with a quencher.

23 . A reagent as claimed in claim 19 which comprises a nucleic acid duplex each strand of which is labelled respectively with a compound as claimed in claim 1 and a quencher such that attenuation of the light emission exists when the duplex is substantially intact but not when the duplex is rendered substantially non-intact by chemical or physical means.

24 . A reagent as claimed in claim 21 comprising an enzyme substrate labelled at sites respectively either side of a cleavage site with a compound as claimed in claim 1 and a quencher.

25 . A reagent as claimed in any one of claims 1 to 24 wherein the quencher is methyl red.

26 . A kit comprising a reagent as claimed in any one of claims 19 to 24 , hydrogen peroxide solution and a buffer adapted to maintain the pH of the analyte containing medium at pH 6-10.

27 . A kit as claimed in claim 26 further comprising a second reagent labelled with a quencher.

28 . A kit as claimed in claim 27 wherein either: the first reagent is a specific binding partner for an analyte and the second reagent is a secondary binding partner; or the first reagent is a secondary binding partner and the second reagent is a specific binding partner for an analyte.

29 . A kit as claimed in claim 28 wherein the quencher is methyl red.

30 . A compound selected from the group consisting of:

9-(2,6-Bis(trifluoromethyl)phenoxycarbonyl)-10-(10-succinimidyloxycarbonyldecyl) acridinium trifluoromethanesulfonate;

9-(2,6-dibromoplienoxycarbonyl)-10-(10-succinimidyloxycarbonyldecyl)acridinium trifluoromethanesulfonate;

9-(2,6-bis(trifluoromethyl)phenoxycarbonyl)-10-(3-succinimidyloxycarbonylpropyl) acridinium trifluoromethanesulfonate;

9-(2,6-dibromophenoxycarbonyl)-10-(3-succinimidyloxycarbonylpropyl)acridinium trifluoromethanesulfonate;

9-(2,6-dinitrophenoxycarbonyl)-10-(10-succinimidyloxycarbonyldecyl)acridinium trifluoromethanesulfonate; and

9-(2,6-dinitrophenoxycarbonyl)-10-(3-succinimidyloxycarbonylpropyl)acridinium trifluoromethanesulfonate.