Acridinium compound with fused heterocycle

Abstract

Chemiluminescent acridinium conjugates and compounds capable of forming conjugates are disclosed. These chemiluminescent acridinium conjugates can be used as chemiluminescent tracers in immunoassays for the quantification and identification of certain analytes.

Description

[0001] Public Domain

[0002] The present disclosure relates to a group of chemiluminescent acridinium compounds containing one or more heterocyclic groups, such as 2,3-cyclic alkylenedioxy groups, fused to an acridinium ring system. These substituents result in high quantum yields and significantly improve chemiluminescent stability. Disclosed herein are structural features of acridinium compounds required for high quantum yields and improved chemiluminescent stability.

[0003] Background

[0004] The chemiluminescence of acridinium systems has led to their use in immunoassays for analytes in samples.By conjugating the acridinium system to a ligand or its binding partner, the chemiluminescence can be correlated to the presence or concentration of the analyte.

[0005] An early generation of stable acridiniums was an acridinium ester (AE), which was referred to as DMAE (dimethyl acridinium ester), as shown in U.S. Pat. Nos. 4,918,192 and 5,110,932, which are hereby incorporated by reference in their entirety, and particularly with respect to the acridinium esters disclosed therein, such as DMAE. DMAE contains two methyl groups on the phenyl moiety, which are located on either side of the acridinium ester ring and stabilize the ester bond between them until, for example, chemiluminescence is induced. By conjugation with a reactive functional group (e.g., N-hydroxysuccinimide (NHS) ester) that can covalently bind to an analyte or its binding partner, the DMAE-NHS label can be used to form a conjugate that can be used in an immunoassay. DMAE-NHS has the following structure:

[0006]

[0007] wherein the acridinium ring system is numbered as indicated herein, and there is A - Counter ion.

[0008] Modification of these acridinium compounds has resulted in changes in the properties of the compounds. For example, U.S. Pat. No. 5,656,426 (which is hereby incorporated by reference in its entirety) includes a hydrophilic modification of DMAE (referred to as NSP-DMAE-NHS), wherein the N-methyl group in DMAE is replaced with an N-sulfopropyl (NSP) group to form a zwitterionic compound. Zwitterions contain separated positive and negative charges within the same molecule, wherein the net charge is zero. NSP-DMAE-NHS has the following structure:

[0009]

[0010] Similarly, U.S. Pat. Nos. 8,778,624 and 9,575,062 (which are hereby incorporated by reference in their entirety) include AE ​​labels containing zwitterionic groups at one or more positions of the molecule and other modifications to the acridinium ring system. The three labels of this AE group are called ZAE, ISOZAE and ISODIZAE, and their structures are:

[0011]

[0012]

[0013] U.S. Patent No. 6,664,043 (which is hereby incorporated by reference in its entirety) provides hydrophilic acridinium esters, such as HEGAE (1), in which a polyethylene glycol (PEG) moiety is introduced into the leaving group as a linker to a ligand or binding partner for detecting an analyte. The incorporation of PEG increases the hydrophilicity of the label. HEGAE (1) has the following structure:

[0014]

[0015] U.S. Patent No. 11,332,445 (which is hereby incorporated by reference in its entirety) includes AE containing a branched PEG structure covalently linked to an acridinium ring system. The structural features of this group of molecules are given below:

[0016]

[0017] U.S. Patent Nos. 7,309,615 and 7,785,904, which are hereby incorporated by reference in their entirety, each detail acridinium compounds containing hydrophilic alkoxy groups at C2 and / or C7 of the acridinium ring, wherein the two oxygen atoms modify the chemiluminescent output. The hydrophilic groups attached to the two oxygens also increase water solubility. Specifically, the most useful labels, HQYAE (2) and TSPAE (3), have essential parameters that exceed those of most other acridiniums in immunoassays. HQYAE contains two hydrophilic polyethylene glycol substituents. TSPAE contains two hydrophilic, negatively charged N-sulfopropyl groups at specific positions on the acridinium ring system. Thus, TSPAE is a water-soluble label that renders its conjugate hydrophilic and is able to lower the isoelectric point (pI) value of the labeled conjugate. TSPAE and HQYAE are acridinium labels that are increasingly used in commercial immunoassays that require high sensitivity. The structures of these compounds are:

[0018]

[0019] Although HQYAE and TSPAE have been increasingly used in assays requiring high sensitivity, they suffer from stability issues. These labels need to be formulated in a weakly acidic pH buffer so that they maintain sufficient chemiluminescent stability during long-term storage.

[0020] There is a continuing need for chemiluminescent compounds that can provide high sensitivity for analyte detection while being stable.

[0021] Summary

[0022] According to the aforementioned purposes and other purposes, the present disclosure includes acridiniums that can be used for chemiluminescent assays. As shown herein, heterocyclic groups (e.g., 5 to 10 membered heterocyclic groups) are fused to acridinium ring systems to affect the functionality of the compound, and in some cases, stability and / or reaction kinetics and / or light output are increased compared to compounds with hydrogen or hydrogen and / or electron-donating groups at fused positions (e.g., C2 and C3). Chemiluminescent acridinium compounds of the present disclosure typically contain 2,3- cyclic alkylenedioxy (or dioxolyl (dioxolo)) substituents, thereby producing high quantum yields and significantly increased chemiluminescent stability. These compounds are used for assays due to high quantum yields and increased chemiluminescent stability.

[0023] For example, a compound (eg, a detectable conjugate of an analyte or a binding partner of an analyte (eg, a ligand that binds to an analyte)) having the structure of Formula (I) is provided:

[0024] AL-Ψ (I)

[0025] wherein A is the analyte or a binding partner of the analyte,

[0026] L is absent (i.e., it is a bond) or is a linker, and

[0027] Ψ is a chemiluminescent acridinium, which contains the following structure:

[0028]

[0029] where "j" is 1, 2, 3, 4, 5 or 6;

[0030] R 1 Hydrogen, –R, –X b ,–R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L1 –Z or –R L –L 1 –R L –Z;

[0031] R 2 and R 3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or

[0032] R 2 and R 3 Together they form a 5-10 membered fused heterocyclyl group;

[0033] Z is a zwitterionic group which, at each occurrence, independently has the following structure:

[0034]

[0035] Wherein "q" and "l" are independently 0 or 1;

[0036] "r" is independently an integer from 0 to 10;

[0037] L 1 is independently –O–, –S–, –NH–, –N(R N )–、–(CH 2 ) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH 2 ) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH 2 ) 1-3 –、–(CH 2 ) 1-3 –C(O)–N(R N )–,–NH–S(O) 1-2 –、–N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–、–S(O) 1-2 –NH–, –(CH 2 ) 1-3–NH–S(O) 1-2 –、–(CH 2 ) 1-3 –N(R N )–S(O) 1-2 –、–(CH 2 ) 1-3 –S(O) 1-2 –N(R N )–、–(CH 2 ) 1-3 –S(O) 1-2 –NH–, –O–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –O–, –S–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –S–, –NH–(CH 2 ) 1-4 –、–N(R N )–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –N(R N )–、–(OCH 2 ) 1-10 –、–(CH 2 O) 1-10 –、–(OCH 2 CH 2 ) 1-10 –, or –(CH 2 CH 2 O) 1-10 –

[0038] R L C independently at each occurrence 1-20 divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents);

[0039] R is independently hydrogen or C 1-35 A hydrocarbon (e.g., an alkyl, alkenyl, alkynyl, or arylalkyl), optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms, having 1-20 substituents);

[0040] R' and R" are independently hydrogen or C1-10 alkyl;

[0041] X b is independently an anionic group at each occurrence; and

[0042] R N Each occurrence is independently selected from hydrogen or C 1-5 Alkyl (e.g. methyl, ethyl, propyl); or

[0043] or a salt thereof (e.g., a halide salt, such as a chloride salt). 3 is hydrogen. In various embodiments, R 2 is an alkoxy group, which is optionally substituted at one or more (eg, one, two, three) positions with one or more independently selected substituents (eg, -X, such as -S(=O) 1-2 –R*, –O–S(=O) 2 –R*, –S(=O) 2 –OR*, –O–SO 3 、–O–S(=O) 2 –OR*, –O–S(=O)–OR*, –O–S(=O)–R*, –S(=O)–OR*, or –S(=O)–R*, where R* is H or C 1-10 hydrocarbon) substitution.

[0044] Compounds for forming conjugates are also provided. For example, the compound (e.g., a compound for conjugation with an analyte or a binding partner of an analyte (e.g., a peptide, protein, or macromolecule including an antibody)) can have a structure of formula (IV):

[0045] RFG-L-Ψ (IV)

[0046] wherein RFG is a reactive functional group for conjugation with an analyte or a binding partner of the analyte,

[0047] L is absent (i.e., it is a bond) or is a linker, and

[0048] Ψ is a chemiluminescent acridinium, which contains the following structure:

[0049]

[0050] where "j" is 1, 2, 3, 4, 5 or 6;

[0051] R 1 is hydrogen, –R, –X, –R L –X b ,–L 1 –R, –L 1 –X b , –Z, –RL –Z, –L 1 –Z or –R L –L 1 –R L –Z;

[0052] R 2 and R 3 are independently selected from hydrogen, -R, an electron donating group or -Z, or

[0053] R 2 and R 3 Together they form a 5-10 membered fused heterocyclyl group;

[0054] Z is a zwitterionic group which, at each occurrence, independently has the following structure:

[0055]

[0056] Wherein "q" and "l" are independently 0 or 1;

[0057] "r" is independently an integer from 0 to 10;

[0058] L 1 is independently –O–, –S–, –NH–, –N(R N )–、–(CH 2 ) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH 2 ) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH 2 ) 1-3 –、–(CH 2 ) 1-3 –C(O)–N(R N )–,–NH–S(O) 1-2 –、–N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–、–S(O) 1-2 –NH–, –(CH2 ) 1-3 –NH–S(O) 1-2 –、–(CH 2 ) 1-3 –N(R N )–S(O) 1-2 –、–(CH 2 ) 1-3 –S(O) 1-2 –N(R N )–、–(CH 2 ) 1-3 –S(O) 1-2 –NH–, –O–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –O–, S–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –S–, –NH–(CH 2 ) 1-4 –、–N(R N )–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –N(R N )–、–(OCH 2 ) 1-10 –、–(CH 2 O) 1-10 –、–(OCH 2 CH 2 ) 1-10 –, or –(CH 2 CH 2 O) 1-10 –

[0059] R L C independently at each occurrence 1-20 divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents);

[0060] R is independently hydrogen or C 1-35 A hydrocarbon (e.g., an alkyl, alkenyl, alkynyl, or arylalkyl), optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms, having 1-20 substituents);

[0061] R' and R" are independently hydrogen or C 1-10 alkyl;

[0062] X b is independently an anionic group at each occurrence; and

[0063] R N is independently selected at each occurrence from hydrogen or C 1-5 Alkyl (e.g. methyl, ethyl, propyl); or

[0064] or a salt thereof. In a specific embodiment, R 1 For –R, –X b ,–R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z or –R L –L 1 –R L –Z.

[0065] In various embodiments, the compound used to form the conjugate is selected from:

[0066]

[0067]

[0068]

[0069]

[0070] Methods for forming conjugates are provided. In some embodiments, the method can include reacting a compound for forming a conjugate with an analyte or a binding partner of an analyte (eg, an antibody) to form a conjugate.

[0071] In another aspect of the invention, a reagent for the detection of an analyte is provided, the analyte comprising a detectable conjugate having a chemiluminescent acridinium having a heterocyclic ring fused to the acridinium ring structure. The detectable conjugate may comprise one or more (e.g., one, two) zwitterionic functional groups. The concentration of the reagent may be selected with respect to the sensitivity of the assay so that an assay requiring higher sensitivity may have a high concentration. For example, a sample may have less than 10 -3 M of detectable analyte. In some embodiments, the sample may have 10 -15 M to 10 -3In various embodiments, the reagent may have a concentration of less than 10 -3 M of the detectable conjugate. In some embodiments, the reagent may have a concentration of 10 -15 Up to 10 -3 The concentration of the detectable conjugate is M.

[0072] In another aspect of the present invention, there is provided an assay method for detecting or quantifying an analyte in a sample, comprising:

[0073] (a) providing a detectable conjugate having a structure of formula (I);

[0074] (b) providing a solid support on which are immobilized molecules capable of forming a binding complex with the analyte and capable of forming a binding complex with the detectable conjugate;

[0075] (c) mixing the compound, the solid support and the sample;

[0076] (d) separating the solid support from the mixture;

[0077] (e) triggering chemiluminescence of any acridinium label coordinated to the solid phase;

[0078] (f) measuring the amount of light emitted using a photometer; and

[0079] (g) detecting the presence of the analyte or calculating the concentration of the analyte by comparing the amount of light emitted to a standard dose response curve that relates the amount of light emitted to a known concentration of the analyte.

[0080] In some embodiments, the sample is serum.

[0081]

[0013] These and other aspects of the present invention will be better understood by reference to the following detailed description including the appended claims. Brief Description of the Drawings

[0083] FIG. 1 ( 1A-1E ) provides light emission spectra measured for several acridinium compounds described herein. Figure 1A is the optical emission spectrum of ADOAE A(5). Figure 1B are the optical emission spectra of ADOAE D (7) and ADOAE E (8). Figure 1C is the optical emission spectrum of ADOAE G(10). Figure 1D is the optical emission spectrum of ADOAE I(12). Figure 1E is the optical emission spectrum of ADOAE K(14).

[0084] Figure 2The light emission kinetics of acridinium esters HEGAE (1), HQYAE (2), and ADOAEs D to L (7-15) are provided. As can be seen, ADOAE F (9), ADOAE H (11), ADOAE J (13), and ADOAE L (15) each have faster chemiluminescent reaction kinetics than the other tested compounds.

[0085] FIG. 3 (3A-3D) provides comparative acridinium ( Figure 3A ) and exemplary ADOAEs ( Figures 3B - 3D ) structure.

[0086] Detailed Description

[0087] For convenience, certain terms used in this specification (including embodiments and appended claims) are summarized here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which this disclosure belongs.

[0088] Unless expressly defined otherwise, the following terms and phrases in this disclosure are intended to have the following meanings:

[0089] All percentages given herein refer to the weight percentage of the particular component relative to the entire composition (including carrier), unless otherwise indicated. It will be understood that the sum of all weight percentages of the individual components in the composition will not exceed 100%.

[0090] As used herein, the term "a" or "an" means one or more. As used herein, the term "consisting essentially of" is intended to limit the invention to the specified materials or steps, and those that do not materially affect the basic and novel characteristics of the claimed invention, as understood from a reading of this specification. The expression "comprising" includes "consisting essentially of" and "consisting of". When a compound comprises an indicated chemical moiety, the chemical moiety will be part of the compound and include any number of substituents at any position occupied by hydrogen in the indicated structure. For example, a compound comprising the indicated structure (e.g., structure of formula (I), A, L, Ψ) may be independently, for example, optionally substituted C 1 -C 35 The hydrocarbon is substituted one or more times.

[0091] Unless otherwise stated, the following definitions of various groups or substituents will apply. The specific and general values ​​for groups, substituents, and ranges listed below are for illustration only; they do not exclude other definitions of groups and substituents or other values ​​within the defined ranges. Unless otherwise stated, alkyl, alkenyl, alkynyl, alkoxy, etc. represent straight chain, branched, and cyclic groups and any combination thereof.

[0092] The term hydrocarbon may refer to a radical or group containing carbon and hydrogen atoms that may be bonded at a specified position (e.g., R, R', R", L 1 , L C , R L , R C , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 ). Examples of hydrocarbon radicals include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, aryl-alkyl, alkyl-aryl, and any combination thereof (e.g., alkyl-aryl-alkyl). As used herein, unless otherwise indicated, hydrocarbon can be a monovalent or polyvalent (e.g., divalent, trivalent) hydrocarbon radical. If there are no unsaturated bonds between carbon atoms, –(CH 2 ) n – free radicals (including methylene radicals, i.e. –CH 2 -) are considered alkyl radicals. Unless otherwise specified, all hydrocarbon radicals (including substituted and unsubstituted alkyl, alkenyl, alkynyl, aryl, aryl-alkyl, alkyl-aryl) can have 1-35 carbon atoms. In other embodiments, the hydrocarbon will have 1-20 or 1-12 or 1-8 or 1-6 or 1-3 carbon atoms, including, for example, embodiments with one, two, three, four, five, six, seven, eight, nine or ten carbon atoms. The hydrocarbon can have 2 to 70 atoms or 4 to 40 atoms or 4 to 20 atoms.

[0093] The substituted hydrocarbon may have one or more hydrocarbon radicals, substituted hydrocarbon radicals as substituents, or may contain one or more heteroatoms. Any hydrocarbon substituent (e.g., R, R', R", L 1 , L C , R L , R C , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8) may optionally include 1-20 (e.g., 1-10, 1-5) heteroatoms. Examples of substituted hydrocarbon radicals include, but are not limited to, heterocycles, such as heteroaryl. Unless otherwise indicated, hydrocarbons substituted with one or more heteroatoms will contain 1-20 heteroatoms. In other embodiments, hydrocarbons substituted with one or more heteroatoms will contain 1-12 or 1-8 or 1-6 or 1-4 or 1-3 or 1-2 heteroatoms. Examples of heteroatoms include, but are not limited to, oxygen, nitrogen, sulfur, phosphorus, halogen (e.g., F, Cl, Br, I), boron or silicon. In some embodiments, heteroatoms will be selected from oxygen, nitrogen, sulfur, phosphorus and halogen (e.g., F, Cl, Br, I). In certain embodiments, heteroatoms may be selected from O, N or S. In some embodiments, heteroatoms or groups may replace carbon. In some embodiments, heteroatoms or groups may replace hydrogen. In some embodiments, the substituted hydrocarbons may contain one or more heteroatoms in the backbone or chain of the molecule (e.g., inserted between two carbon atoms, such as in the form of "oxo"). In some embodiments, the substituted hydrocarbons may contain one or more heteroatoms pendant to the backbone or chain of the molecule (e.g., covalently bonded to a carbon atom in the chain or backbone, such as in the form of "oxo").

[0094] When the indicated group is substituted with the indicated substituents, the designated group may be substituted with one or more of any or all of the named substituents. For example, when a group (e.g., an alkyl or heteroaryl group) is substituted with an "unsubstituted C 1 -C 20 alkyl or unsubstituted 2 to 20-membered heteroalkyl”, the group may contain one or more unsubstituted C 1 -C 20 alkyl and / or one or more unsubstituted 2 to 20 membered heteroalkyl. In addition, when a portion is substituted by an R substituent, the group may be referred to as "R substituted". When a portion is R substituted, the portion is substituted by at least one R substituent, and each R substituent is optionally different. If the indicated group is used multiple times in a chemical genus (e.g., an R group), it will be understood that each group is independently selected at each occurrence.

[0095] Unless otherwise specified, any compound disclosed herein having one or more chiral centers may be in the form of a racemic mixture with respect to each chiral center, or may be present as a pure or substantially pure (e.g., greater than 98% ee) R or S enantiomer with respect to each chiral center, or may be present as a mixture of R and S enantiomers with respect to each chiral center, wherein the mixture contains an enantiomeric excess of one or the other configuration, such as greater than 60% enantiomeric excess (of R or S) or greater than 70% or greater than 80% or greater than 90% or greater than 95% or greater than 98% or greater than 99% enantiomeric excess. In some embodiments, any chiral center may be in the "S" or "R" configuration.

[0096] It will be understood that the description of compounds herein is limited by the principles of chemical bonding. Thus, when a group may be substituted with one or more substituents, such substituents are selected to conform to the principles of chemical bonding (e.g., with respect to valence) and to yield compounds that are not intrinsically unstable. For example, any carbon atom will bond to two, three, or four other atoms, depending on the four valence electrons of the carbon.

[0097] Substituent (free radical) prefix names may be derived from the parent hydride by (i) replacing the "ane" in the parent hydride with the suffix "yl", "diyl", "triyl", "tetrayl"; or (ii) replacing the "e" in the parent hydride with the suffix "yl", "diyl", "triyl", "tetrayl" (here, when specifying the atom(s) with free valence, the number given is as low as consistent with any established numbering of the parent hydride). Also used throughout this article are recognized abbreviations (e.g., adamantyl, naphthyl, anthracenyl, phenanthrenyl, furanyl, pyridinyl, isoquinolinyl, quinolinyl, and piperidinyl) as well as popular names (e.g., vinyl, allyl, phenyl, and thienyl).

[0098] Typically, anionic groups (X a and / or X b ) can provide anionic charge to counterbalance any cationic charge covalently attached directly or indirectly and to form a zwitterion. In some embodiments, X a and X b is, independently at each occurrence, a carboxylate (–C(O)O - ), sulfonate Sulfate Phosphate (–OP(O)(OR P ) - ) or oxygen ions (–O - ), and R P is hydrogen or C optionally substituted by up to 10 heteroatoms 1-12 hydrocarbon.

[0099] An alkyl group generally refers to a saturated hydrocarbon chain which may be straight or branched and contains the specified number of carbon atoms. 1 -C 6 Alkyl means that the group may have 1 to 6 (inclusive) carbon atoms. Any atom may be optionally substituted, for example, by one or more substituents. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, and tert-butyl. Any alkyl group (e.g., R, R', R", L" or R" mentioned herein) may be substituted with any of the following: 1 , L C , R L , R C , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 ) can have 1-35 carbon atoms. In other embodiments, the alkyl group will have 1-20 or 1-12 or 1-8 or 1-6 or 1-3 carbon atoms, including, for example, embodiments having one, two, three, four, five, six, seven, eight, nine or ten carbon atoms. The alkyl group can be a lower alkyl group (e.g., C 1 -C 4 The alkyl group substituted by one or more heteroatoms (e.g., N, O, halogen) may include a heteroalkyl group, such as an amino group (e.g., alkylamino, dialkylamino), an alkoxy group, or a halogenated alkyl group).

[0100] Haloalkyl group is typically an alkyl group in which at least one hydrogen atom is substituted by halo. In some embodiments, more than one (for example 2,3,4,5,6,7,8,9,10,11,12,13 or 14) hydrogen atom is substituted by halo. In these embodiments, hydrogen atom can be substituted by identical halogen (for example fluorine) each, or hydrogen atom can be substituted by the combination (for example fluorine and chlorine) of different halogens. Haloalkyl can include an alkyl moiety (sometimes referred to as perhaloalkyl in this article, for example perfluoroalkyl, such as trifluoromethyl) in which all hydrogens are substituted by halogen. Haloalkyl group can be optionally substituted.

[0101] Typically, an alkoxy group has the formula -O(alkyl). An alkoxy group can be, for example, a methoxy group (-OCH 3), ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, pentyloxy, 2-pentyloxy, 3-pentyloxy or hexyloxy. Similarly, the term "thioalkoxy" refers to a group of formula -S(alkyl). Finally, the terms "haloalkoxy" and "halothioalkoxy" refer to -O(haloalkyl) and -S(haloalkyl), respectively. The term "thiol" refers to -SH. As used herein, the term "hydroxyl", used alone or in combination with other terms, refers to a group of formula -OH. Any alkoxy, thioalkoxy or haloalkoxy group (e.g., R, R', R", L 1 , L C , R L , R C , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 ) can have 1-35 carbon atoms. In other embodiments, the alkoxy, thioalkoxy or haloalkoxy groups will have 1-20 or 1-12 or 1-8 or 1-6 or 1-3 carbon atoms, including, for example, embodiments having one, two, three, four, five, six, seven, eight, nine or ten carbon atoms. The alkoxy group can be a lower alkoxy group (e.g., C 1 -C 4 alkoxy).

[0102] Arylalkyl group generally refers to the group that the alkyl hydrogen atom in the alkyl part is replaced by an aryl group.One of the carbon atoms of the alkyl part serves as the connection point of the arylalkyl group and another part.Any ring or chain atom can be optionally substituted, for example, replaced by one or more substituents.The limiting examples of arylalkyl include benzyl, 2-phenylethyl and 3-phenylpropyl groups.The arylalkyl group replaced by one or more heteroatoms (for example, N, O, halogen) can include heteroarylalkyl groups, for example amino groups (for example arylamino), aryloxy groups or halogenated arylalkyl groups.

[0103] The term "alkenyl" may refer to a straight or branched hydrocarbon chain containing a specified number of carbon atoms and having one or more carbon-carbon double bonds. Any atom may be optionally substituted, for example, by one or more substituents. Alkenyl groups may include, for example, vinyl, allyl, 1-butenyl, and 2-hexenyl. One of the double bond carbons may optionally be the point of attachment for an alkenyl substituent. Any alkenyl group (e.g., R, R', R", L" or R" may be present in any of the above-mentioned groups. 1 , L C , R L , R C , R1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 ) can have 1-35 carbon atoms. In other embodiments, the alkenyl group will have 1-20 or 1-12 or 1-8 or 1-6 or 1-3 carbon atoms, including, for example, embodiments with one, two, three, four, five, six, seven, eight, nine or ten carbon atoms. The alkenyl group substituted by one or more heteroatoms (e.g., N, O, halogen) can include heteroalkenyl groups, such as amino groups (e.g., alkenylamino, alkenylalkylamino), alkenyloxy groups or haloalkenyl groups).

[0104] The term alkynyl may refer to a straight or branched hydrocarbon chain containing the specified number of carbon atoms and having one or more carbon-carbon triple bonds. 1 , L C , R L , R C , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 ) can be optionally substituted, for example, substituted by one or more substituents. Alkynyl groups can include, for example, ethynyl, propargyl and 3-hexynyl. One of the triple bond carbons can be optionally the point of attachment of the alkynyl substituent. Alkynyl groups substituted by one or more heteroatoms (for example N, O, halogen) can include heteroalkynyl groups, for example amino groups (for example alkynylamino, alkenylalkylamino), alkynyloxy groups or haloalkynyl groups.

[0105] The term heterocyclyl generally refers to a fully saturated, partially saturated or aromatic monocyclic, bicyclic, tricyclic or other polycyclic ring system having one or more independently selected from O, N (it is understood that one or two additional groups (e.g. R N) to complete the nitrogen valence and / or form a salt) or a constituent heteroatom ring atom of S. The present disclosure is based in part on installing one or more fused heterocyclic groups onto an acridinium. A heteroatom or ring carbon can be the point of attachment of a heterocyclic substituent to another moiety. Any atom can be optionally substituted, for example, by one or more substituents (e.g., a heteroatom or group X). Heterocyclic groups can include, for example, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl. For example, the phrase "a heterocycle containing 5 to 6 ring atoms, wherein 1 to 2 ring atoms are independently selected from N, NH, N(C 1 -C 6 alkyl), NC(O)(C 1 -C 6 alkyl), O and S; and wherein the heterocycle is optionally substituted with 1 to 3 independently selected R" will include, but are not limited to, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl and pyrrolidinyl.

[0106] The term heterocycloalkenyl generally refers to partially unsaturated monocyclic, bicyclic, tricyclic or other polycyclic hydrocarbon groups, which have one or more (e.g., 1-4) heteroatom ring atoms, which are independently selected from O, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and / or form a salt) or S. Ring carbon (e.g., saturated or unsaturated) or heteroatom may be the point of attachment of the heterocycloalkenyl substituent. Any atom may be optionally substituted, for example, by one or more substituents. The heterocycloalkenyl group may include, for example, dihydropyridyl, tetrahydropyridyl, dihydropyranyl, 4,5-dihydrooxazolyl, 4,5-dihydro-1H-imidazolyl, 1,2,5,6-tetrahydro-pyrimidinyl and 5,6-dihydro-2H-[1,3]oxazinyl.

[0107] Cycloalkyl groups can be completely saturated monocyclic, bicyclic, tricyclic or other polycyclic hydrocarbon groups. Any atom can be optionally substituted, for example, substituted by one or more substituents. The ring carbon is used as the connection point of the cycloalkyl group and another part. The cycloalkyl moiety can include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl and norbornyl (bicyclo [2.2.1] heptyl). The cycloalkyl group replaced by one or more heteroatoms (such as N, O, halogen) can include heterocycloalkyl groups, such as oxirane, oxetane, azetidine, aziridine, furanyl, pyranyl, pyrrolidinyl, piperidinyl, thiirane, thietanyl, tetrahydrothienyl (tetrahydrothiphenyl), thiopyranyl or halogenated cycloalkyl.

[0108] Cycloalkenyl groups can be partially unsaturated monocyclic, bicyclic, tricyclic or other polycyclic hydrocarbon groups. Ring carbon (e.g., saturated or unsaturated) is the point of attachment of cycloalkenyl substituents. Any atom can be optionally substituted, for example, substituted by one or more substituents. Cycloalkenyl moieties can include, for example, cyclohexenyl, cyclohexadienyl or norbornenyl. Cycloalkenyl groups substituted by one or more heteroatoms (e.g., N, O, halogen) can include heterocycloalkenyl groups, for example, oxirane, oxetane, azetidinyl, aziridine, furanyl, pyranyl, pyrrolidinyl, piperidinyl, thiirane, thietanyl, tetrahydrothienyl, thiopyranyl or halogenated cycloalkenyl.

[0109] Aryl groups are typically aromatic monocyclic, bicyclic (2 fused rings) or tricyclic (3 fused rings) or polycyclic (>3 fused rings) hydrocarbon ring systems. One or more ring atoms may be optionally substituted, for example, by one or more substituents. Aryl moieties include, for example, phenyl and naphthyl. Cycloalkenyl groups substituted by one or more heteroatoms (e.g., N, O, halogen) may include heteroaryl groups or halogenated aryl groups.

[0110] Heteroaryl groups are typically aromatic monocyclic, bicyclic (2 fused rings), tricyclic (3 fused rings) or polycyclic (>3 fused rings) hydrocarbon groups having one or more heteroatom ring atoms in the ring(s) independently selected from O, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and / or to form a salt) or S. One or more ring atoms may be optionally substituted, for example, with one or more substituents. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl, β-carbolinyl, carbazolyl, coumarinyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furanyl, imidazolyl, imidizolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiaz ... oxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl and xanthenyl.

[0111] In general, when a definition for a particular variable includes both hydrogen and non-hydrogen (halogen, alkyl, aryl) possibilities, the term "substituent(s) other than hydrogen" refers collectively to the non-hydrogen possibilities for that particular variable unless otherwise specified.

[0112] In general, the limits (endpoints) of any range described herein are within the scope of the present invention and are understood to be disclosed embodiments. In addition, any half-integer values ​​within the range are also contemplated. For example, a range of 0 to 4 explicitly discloses 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, and any subset within the range (e.g., from 1 to 2.5).

[0113] The term "substituent" may refer to a group "substituted" on a hydrocarbon (e.g., an alkyl, haloalkyl, cycloalkyl, heterocyclyl, heterocyclylalkenyl, cycloalkenyl, aryl, or heteroaryl group) replacing one or more atoms therein at any atom of the group. In one aspect, a group (e.g., R, R', R", L 1 , L C , R L , R C , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 ) is independently any one atom or any combination of two or more of the atoms or groups of atoms specified for the substituent. In another aspect, the substituent itself may be substituted with any of the above substituents. In addition, as used herein, the phrase "optionally substituted" refers to unsubstituted (e.g., substituted with H) or substituted. It is understood that the substituents at a given atom are limited by valence. Common substituents include halogens (e.g., F), C 1-12 Straight or branched alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-12 Cycloalkyl, C 6-12 Aryl, C 3-12 Heteroaryl, C 3-12 Heterocyclic group, C 1-12 alkylsulfonyl, nitro, cyano, -COOR, -C(O)NRR', -OR, -SR, -NRR' and oxo, for example, mono- or di- or tri-substituted with a moiety (e.g., trifluoromethoxy, chloro, bromo, fluoro, methyl, methoxy, pyridyl, furyl, triazolyl, piperazinyl, pyrazolyl, imidazolyl, etc.), each of which optionally contains one or more heteroatoms, such as halogen, N, O, S and P. R and R' are independently hydrogen, C 1-12 Alkyl, C 1-12 Haloalkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-12 Cycloalkyl, C 4-24 Cycloalkylalkyl, C6-12 Aryl, C 7-24 Arylalkyl, C 3-12 Heterocyclic group, C 3-24 Heterocycloalkyl, C 3-12 Heteroaryl or C 4-24 Heteroarylalkyl. Unless otherwise specified, all groups described herein optionally contain one or more common substituents to the extent permitted by valence. In addition, as used herein, the phrase "optionally substituted" refers to unsubstituted (e.g., substituted with H) or substituted. As used herein, the term "substituted" refers to hydrogen and / or carbon atoms being removed and replaced by substituents (e.g., common substituents). When a substituent (free radical) prefix name (e.g., alkyl) is used without the modifier "optionally substituted" or "substituted", it is understood to mean that the specific substituent is unsubstituted. However, when "haloalkyl" is used without the modifier "optionally substituted" or "substituted", it is still understood to mean an alkyl group in which at least one hydrogen atom is replaced by halogen and any other relevant substituents as required.

[0114] When a portion of a compound of the present disclosure is described as an analyte or its binding partner, it will be understood that a covalent bond is formed with the analyte or its binding partner (e.g., using a reactive functional group that forms a covalent bond), such as by replacing a hydrogen on the unconjugated analyte or its binding partner with a covalent bond connected to the indicated portion. The covalent bond on the analyte or its binding partner can be formed, for example, at a group on the analyte, its binding partner, or an analyte derivative containing a group for forming a bond. The group can be, for example, an amine group, a thiol group, a carboxyl group, a maleimido group, or a carbohydrate group. For example, if the covalent bond is formed by a primary amine of the analyte or its binding partner, the compound can have the following structure:

[0115]

[0116] Where the unconjugated analyte or binding partner A has the structure A'-NH 2 In some embodiments, if the covalent bond is formed through the thiol group of the analyte or its binding partner, the compound may have the following structure:

[0117] A′-SL-Ψ

[0118] The unconjugated analyte or binding partner A has the structure A'-SH.

[0119] In some embodiments, any hydrocarbon or substituted hydrocarbon (eg, R, R', R", L 1 , L C , R L, R C , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 ) may be substituted with one or more (e.g., 1-6 or 1-4 or 1-3 or one or two or three) substituents X, wherein X is independently selected at each occurrence from one or more (e.g., 1-20) heteroatoms or one or more (e.g., 1-10) heteroatom-containing groups, or X is independently selected at each occurrence from -F, -Cl, -Br, -I, -OH, -OR*, -NH 2 , –NHR*, –N(R*) 2 , –N(R*) 3 + , –N(R*)–OH, –N(→O)(R*) 2 ,–O–N(R*) 2 , –N(R*)–O–R*, –N(R*)–N(R*) 2 , –C=N–R*, –N=C(R*) 2 , –C=N–N(R*) 2 、–C(=NR*)(–N(R*) 2 ), –C(H)(=N–OH), –SH, –SR*, –CN, –NC, –CHF 2 , –CCl 3 , –CF 2 Cl, –CFCl 2 , –C(=O)–R*, –CHO, –CO 2 H, –C(O)CH 3 , –CO 2 - , –CO 2 R*, –C(=O)–S–R*, –O–(C=O)–H, –O–(C=O)–R*, –S–C(=O)–R*, –(C=O)–NH 2 、–C(=O)–N(R*) 2 、–C(=O)–NHNH 2 , –O–C(=O)–NHNH 2 , –C(=S)–NH 2 、–(C=S)–N(R*) 2, –N(R*)–CHO, –N(R*)–C(=O)–R*, –C(=NR)–OR*, –O–C(=NR*)–R*, –SCN, –NCS, –NSO, –SSR*, –N(R*)–C(=O)–N(R*) 2 , –CH 3 , –CH 2 –CH 3 , –CH 2 –CH 2 –CH 3 , –C(H)(CH 2 ) 2 , –C(CH 3 ) 3 , –N(R*)–C(=S)–N(R*) 2 , –S(=O) 1-2 –R*, –O–S(=O) 2 –R*, –S(=O) 2 –OR*, –N(R*)–S(=O) 2 –R*, –S(=O) 2 –N(R*) 2 ,–O–SO 3 、–O–S(=O) 2 –OR*, –O–S(=O)–OR*, –O–S(=O)–R*, –S(=O)–OR*, –S(=O)–R*, –NO, –NO 2 ,–NO 3 ,–O–NO,–O–NO 2 ,–N 3 ,–N 2 –R*, –N(C 2 H 4 ), –Si(R*) 3 , –CF 3 ,–O–CF 3 , –O–CHF 2 , –O–CH 3 、–O–(CH 2 ) 1-6 CH 3 、–OC(H)(CH 2 ) 2 –OC(CH 3 ) 3 , –PR* 2 、–O–P(=O)(OR*) 2 OR – P(=O)(OR*) 2 ; wherein, independently at each occurrence, R* may be H or C 1-10 or C 1-8or C 1-6 or C 1-4 Hydrocarbons include, but are not limited to, alkyl, alkenyl, alkynyl, aryl (e.g., phenyl), alkyl-aryl (e.g., benzyl), aryl-alkyl (e.g., tolyl). In some embodiments, X may include C 1 -C 8 or C 1 -C 6 or C 2 -C 4 In some embodiments, X can be C 1 -C 8 or C 2 -C 6 or C 3 -C 5 Heterocyclic rings (e.g., heteroaryl radicals). The term "halo" or "halogen" refers to any radical of fluorine, chlorine, bromine, or iodine. In some embodiments, X is independently selected at each occurrence from -OH, -SH, -NH 2 , –N(R*) 2 , –C(O)OR*, –C(O)NR*R*, –C(O)NR*R*, –C(O)OH, –C(O)NH 2 In some embodiments, X is F. R and R* can be independently saturated or unsaturated alkyl (e.g., C 1 -C 8 In some embodiments, R and R* are independently selected from hydrogen, methyl, ethyl, propyl, or isopropyl. In some embodiments, R and R* are independently selected from hydrogen, methoxy, ethoxy, propoxy, or isopropoxy. In some embodiments, X is -CF 3 or –O–CF 3 .

[0120] Compounds (eg, detectable conjugates of analytes or binding partners of analytes) having the structure of Formula (I) are provided:

[0121] AL-Ψ (I)

[0122] wherein A is the analyte or a binding partner of the analyte,

[0123] L is absent (i.e., it is a bond) or is a linker, and

[0124] Ψ is a chemiluminescent acridinium, which contains the following structure:

[0125]

[0126] where "j" is 1, 2, 3, 4, 5 or 6;

[0127] R 1 is hydrogen, –R, –X b ,–R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z or –R L –L 1 –R L –Z;

[0128] R 2 and R 3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or

[0129] R 2 and R 3 Together they form a 5-10 membered fused heterocyclyl group;

[0130] Z is a zwitterionic group which, at each occurrence, independently has the following structure:

[0131]

[0132] Wherein "q" and "l" are independently 0 or 1;

[0133] "r" is independently an integer from 0 to 10;

[0134] L 1 is independently at each occurrence –O–, –S–, –NH–, –N(R N )–、–(CH 2 ) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH 2 ) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH 2 ) 1-3 –、–(CH 2 ) 1-3 –C(O)–N(R N)–,–NH–S(O) 1-2 –、–N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–、–S(O) 1-2 –NH–, –(CH 2 ) 1-3 –NH–S(O) 1-2 –、–(CH 2 ) 1-3 –N(R N )–S(O) 1-2 –、–(CH 2 ) 1-3 –S(O) 1-2 –N(R N )–、–(CH 2 ) 1-3 –S(O) 1-2 –NH–, –O–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –O–, –S–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –S–, –NH–(CH 2 ) 1-4 –、–N(R N )–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –N(R N )–、–(OCH 2 ) 1-10 –、–(CH 2 O) 1-10 –、–(OCH 2 CH 2 ) 1-10 –, or –(CH 2 CH 2 O) 1-10 –

[0135] R L Each occurrence is independently C 1-20a divalent hydrocarbon group (e.g., an alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., having 1-10 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl group; having 1-10 substituents, such as one or more groups -X);

[0136] R is independently hydrogen or C 1-35 a hydrocarbon (e.g., an alkyl, alkenyl, alkynyl, or arylalkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., having 1-20 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl group; having 1-20 substituents, such as one or more groups -X);

[0137] R' and R" are independently hydrogen or C 1-10 alkyl;

[0138] X b is independently an anionic group at each occurrence; and

[0139] R N is independently selected at each occurrence from hydrogen or C 1-5 Alkyl (e.g. methyl, ethyl, propyl); or

[0140] or a salt thereof (e.g., a halide salt, such as a chloride salt). 3 is hydrogen. In various embodiments, R 2 is an alkoxy group, which is optionally substituted at one or more (eg, one, two, three) positions with one or more independently selected substituents (eg, -X, such as -S(=O) 1-2 –R*, –O–S(=O) 2 –R*, –S(=O) 2 –OR*, –O–SO 3 、–O–S(=O) 2 –OR*, –O–S(=O)–OR*, –O–S(=O)–R*, –S(=O)–OR*, or –S(=O)–R*, where R* is H or C 1-10 In some embodiments, R 1 For –R, –X b ,–R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z or –RL –L 1 –R L –Z.

[0141] The compound may have the structure of formula (Ia):

[0142]

[0143] where "j" is 1, 2, 3, 4, 5 or 6;

[0144] R 1 Hydrogen, –R, –X b ,–R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z or –R L –L 1 –R L –Z;

[0145] R 2 and R 3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or

[0146] R 2 and R 3 Together they form a 5-10 membered fused heterocyclyl group;

[0147] R 4 is independently selected at each occurrence from hydrogen and -R (e.g., R', R N , lower alkyl such as C 1 -C 4 alkyl);

[0148] Ω is S, O or N;

[0149] Y is selected from –R, –L 1 –R, –R L –Z, –L 1 –R L –Z, or if Ω is O or S, then Y does not exist;

[0150] Y' is absent (i.e. it is a bond) or is selected from -L 1 –, –R L –, –R L –L 1 –, –L 1 –L 1 –, –L 1 –R L –, –L1 –R L –L 1 or –R L –L 1 –R L –; and Y' contains one or more connections to L C or Z L Keys;

[0151] Z is a zwitterionic group having the following structure:

[0152]

[0153] Wherein "q" and "l" are independently 0 or 1;

[0154] "r" is independently an integer from 0 to 10;

[0155] L 1 is independently –O–, –S–, –NH–, –N(R N )–、–(CH 2 ) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH 2 ) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH 2 ) 1-3 –、–(CH 2 ) 1-3 –C(O)–N(R N )–,–NH–S(O) 1-2 –、–N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–、–S(O) 1-2 –NH–, –(CH 2 ) 1-3 –NH–S(O) 1-2 –、–(CH 2 ) 1-3 –N(R N )–S(O)1-2 –、–(CH 2 ) 1-3 –S(O) 1-2 –N(R N )–、–(CH 2 ) 1-3 –S(O) 1-2 –NH–, –O–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –O–, –S–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –S–, –NH–(CH 2 ) 1-4 –、–N(R N )–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –N(R N )–、–(OCH 2 ) 1-10 –、–(CH 2 O) 1-10 –、–(OCH 2 CH 2 ) 1-10 – or – (CH 2 CH 2 O) 1-10 –

[0156] R L C independently at each occurrence 1-20 divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl, combinations thereof), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., having 1-10 heteroatoms such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl groups; having 1-10 substituents);

[0157] R is independently hydrogen or C 1-35 A hydrocarbon (e.g., an alkyl, alkenyl, alkynyl, arylalkyl, combinations thereof) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl group; having 1-10 substituents);

[0158] R' and R" are independently hydrogen or C 1-10 alkyl;

[0159] X b is independently an anionic group at each occurrence; and

[0160] R N is independently selected at each occurrence from hydrogen or C 1-5 Alkyl (eg, methyl, ethyl, propyl).

[0161] Z is a zwitterionic group which, at each occurrence, independently has the following structure:

[0162]

[0163] Wherein "q" and "l" are independently 0 or 1;

[0164] "r" is independently an integer from 0 to 10;

[0165] L 1 is independently –O–, –S–, –NH–, –N(R N )–、–(CH 2 ) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH 2 ) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH 2 ) 1-3 –、–(CH 2 ) 1-3 –C(O)–N(R N )–,–NH–S(O) 1-2 –、–N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–、–S(O) 1-2 –NH–, –(CH 2 ) 1-3 –NH–S(O) 1-2 –、–(CH 2 ) 1-3 –N(R N )–S(O)1-2 –、–(CH 2 ) 1-3 –S(O) 1-2 –N(R N )–、–(CH 2 ) 1-3 –S(O) 1-2 –NH–, –O–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –O–, –S–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –S–, –NH–(CH 2 ) 1-4 –、–N(R N )–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –N(R N )–、–(OCH 2 ) 1-10 –、–(CH 2 O) 1-10 –、–(OCH 2 CH 2 ) 1-10 – or – (CH 2 CH 2 O) 1-10 –

[0166] R L C independently at each occurrence 1-20 a divalent hydrocarbon radical (e.g., an alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., having 1-10 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl group; having 1-10 substituents);

[0167] R is independently hydrogen or C 1-35 A hydrocarbon (e.g., an alkyl, alkenyl, alkynyl, or arylalkyl) radical optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl group; having 1-20 substituents);

[0168] R' and R" are independently hydrogen or C 1-10 alkyl;

[0169] X b is independently an anionic group at each occurrence; and

[0170] R N is independently selected at each occurrence from hydrogen or C 1-5 Alkyl (e.g. methyl, ethyl, propyl); or

[0171] or a salt thereof (e.g., a halide salt such as a chloride salt). In some embodiments, Ω is S, O, or N. In various embodiments, R 1 For –R, –X b ,–R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z or –R L –L 1 –R L –Z. In some respects, R 1 For –R, –X b ,–R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z or –R L –L 1 –R L –Z, and Ω is S, O or N. For example, each R in formula (Ia) 4 Can be hydrogen. In some embodiments, "j" is 2 or 3. In various embodiments, the compound has the structure of formula (II):

[0172]

[0173] Where Ω is S, O or N;

[0174] Y is selected from –R, –L 1 –R, –R L –Z, –L 1 –R L –Z; or, in the case where Ω is O or S, then Y does not exist;

[0175] Y' is absent (i.e. it is a bond) or is selected from -L 1 –, –R L –, –R L –L 1 –, –L1 –L 1 –, –L 1 –R L –, –L 1 –R L –L 1 or –R L –L 1 –R L –; and Y' comprises one or more connected to A or L (e.g., to L C or Z L ) key;

[0176] R 1 is hydrogen, –R, –X, –R L –X, –L 1 –R, –L 1 –X, –Z, –R L –Z, –L 1 –Z or –R L –L 1 –R L –Z;

[0177] R 2 and R 3 are independently selected from hydrogen, -R, an electron donating group or -Z;

[0178] Z is a zwitterionic group having the following structure:

[0179]

[0180] Wherein "q" and "l" are independently 0 or 1;

[0181] "r" is independently an integer from 0 to 10;

[0182] L 1 is independently –O–, –S–, –NH–, –N(R N )–、–(CH 2 ) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH 2 ) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(RN )–(CH 2 ) 1-3 –、–(CH 2 ) 1-3 –C(O)–N(R N )–,–NH–S(O) 1-2 –、–N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–、–S(O) 1-2 –NH–, –(CH 2 ) 1-3 –NH–S(O) 1-2 –、–(CH 2 ) 1-3 –N(R N )–S(O) 1-2 –、–(CH 2 ) 1-3 –S(O) 1-2 –N(R N )–、–(CH 2 ) 1-3 –S(O) 1-2 –NH–, –O–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –O–, –S–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –S–, –NH–(CH 2 ) 1-4 –、–N(R N )–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –N(R N )–、–(OCH 2 ) 1-10 –、–(CH 2 O) 1-10 –、–(OCH 2 CH 2 ) 1-10 – or – (CH 2 CH 2 O) 1-10 –

[0183] R L C independently at each occurrence 1-20divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl; having 1-10 substituents);

[0184] R is independently hydrogen or C 1-35 A hydrocarbon (e.g., an alkyl, alkenyl, alkynyl, or arylalkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl; having 1-10 substituents);

[0185] R' and R" are independently hydrogen or C 1-10 alkyl;

[0186] X b is independently an anionic group at each occurrence; and

[0187] R N is independently selected at each occurrence from hydrogen or C 1-5 In some embodiments, R 1 For –R, –X b ,–R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z or –R L –L 1 –R L -Z. In certain aspects, Ψ has the structure of formula (IIa)

[0188]

[0189] wherein "h" is 1, 2, 3, 4, 5 or 6. In some embodiments, the compound has the structure of Formula (IIb):

[0190]

[0191] Where R 5 -R 8 are independently hydrogen or C 1-35 A hydrocarbon radical (e.g., an alkyl radical, an alkenyl radical, an alkynyl radical, an aryl radical, an alkoxy radical, an alkylthio radical, an amino radical; and wherein L 1with A (eg, L absent) or L (eg, with L optionally having one or more (eg, 1-20) substitution sites C or Z L ) covalently bound. 1 It may be, for example, -NH-C(O)-, -C(O)-NH-, -C(O)-O-, or -O-C(O)-. In a specific embodiment, R 5 and R 6 Each is a lower alkyl group (e.g., C 1 -C 4 alkyl, methyl) and R 7 and R 8 Each is hydrogen.

[0192] In some embodiments, the compound may have the structure of Formula (IIc):

[0193]

[0194] Where "Y" does not exist or is -L 1 –, –R L –, –L 1 –R L –or–R L –L 1 –, where Y” is identical to A (e.g. L is absent) or L (e.g. C or Z L ) are covalently linked.

[0195] In some embodiments, the compound may have the structure of Formula (IId):

[0196]

[0197] Where "Y" does not exist or is -L 1 –, –R L –, –L 1 –R L –or–R L –L 1 –, where Y” is identical to A (e.g. L is absent) or L (e.g. C or Z L ) is covalently linked. In some embodiments, R in formula (IId) is an optionally substituted aryl group (e.g., C 6 -C 12 aryl, phenyl), which may be substituted by, for example, aryl. For example, the compound may have the structure of formula (IIe):

[0198]

[0199] Where "Y" does not exist or is -L 1–, –R L –, –L 1 –R L –or–R L –L 1 –, where Y” is identical to A (e.g. L is absent) or L (e.g. C or Z L ) are covalently linked.

[0200] In some embodiments, L has the structure -L C –(Z L ) z –, where “z” is 0 or 1; L C For divalent C 1-35 alkyl, alkenyl, alkynyl, aryl or arylalkyl, optionally substituted with 1 to 20 heteroatoms; and

[0201] Z L is a zwitterionic linker group having the following structure:

[0202]

[0203] "m" is 0 (i.e. it is a bond) or 1;

[0204] "n" and "p" are independently, at each occurrence, an integer from 0 (i.e., it is a bond) to 10;

[0205] X a It is an anionic group;

[0206] R L C independently at each occurrence 1-20 A divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) substitution points (e.g., substituted with 1-10 heteroatoms, substituted with 1-10 substituents); and

[0207] R' is hydrogen or C 1-10 Alkyl. C Can have the following structure:

[0208] –(X 1 ) 0-1 –(R L ) 0-5 –(X 2 ) 0-1 –(R L ) 0-5 –(X 3 ) 0-1 –(R L ) 0-5 –(X 4 ) 0-1–(R L ) 0-5 –

[0209] Where X 1 Selected from =N–, –O–, –S– or –NR N –

[0210] X 2 –X 4 Independently selected from -O-, -S-, -NR N –, –C(O)–, –NR N –C(O)–, –C(O)–NR N –, –O–C(O)– or –C(O)–O–, –S–C(O)– or –C(O)–S–; and

[0211] R L is independently selected at each occurrence from –(CH 2 ) 1-5 –、–(CH 2 CH 2 O) 1-5 – or – (OCH 2 CH 2 ) 1-5 – or C 5 -C 6 Optionally substituted cycloalkylene (e.g., cyclopentylene, cyclohexylene). In various embodiments, L C Between A and Ψ (or between A and Z L In some embodiments, L comprises a linking moiety associated with thiol conjugation. For example, when the reactive functional group is maleimide, X1 (or X 2 -X 4 ) can be:

[0212]

[0213] in represents the point of attachment to any adjacent group. For example, the point of attachment marked with "*" can be a bond to a thiol group on the analyte or its binding partner. In some embodiments, X a and X b can be, for example, independently at each occurrence a carboxylate (—C(O)O - ), sulfonate Sulfate Phosphate (–OP(O)(OR P ) - ) or oxygen ions (–O - ), and R P is hydrogen or C1-12 A hydrocarbon, which optionally has one or more (e.g., 1-10, 1-5) substitution points (e.g., 1-10 heteroatoms, 1-10 substituents). 1 Can contain (or be) (e.g., sulfopropyl). In some embodiments, R 1 In some embodiments, R 1 –S(O) 2 –NH–Z or –(CH 2 ) 1-3 –S(O) 2 -NH-Z. In various embodiments, R 2 and R 3 Each occurrence is independently hydrogen, alkyl or alkoxy (e.g., lower alkoxy such as C 1 -C 4 In some embodiments, R 2 and R 3 In other embodiments, R 2 or R 3 One of them is hydrogen, and R 2 or R 3 The other is an alkoxy group (e.g., a lower alkoxy group, such as C 1 -C 4 In some embodiments, X a Sulfonate m is 1, R L is propyl, and n and p are each 3, such that Z L Has the following structure:

[0214]

[0215] In specific embodiments, the compound has the structure of Formula (IIIa) or (IIIb):

[0216]

[0217]

[0218] In specific embodiments, "j" is 2 or 3.

[0219] Compounds can be used to detect the presence of a substance (e.g., an analyte (e.g., a biomolecule)) in a sample. In some embodiments, the analyte is a thyroid hormone (e.g., thyroid stimulating hormone, and, for example, A is a binding partner thereof, such as an anti-thyroid stimulating hormone monoclonal antibody (AntiTSH-mAb)), androgen, steroid hormone (e.g., androstenedione, testosterone), troponin, thyroglobulin, anti-thyroid peroxidase antibody, triiodothyronine (T3) hormone, thyroxine (T4) hormone, thyroxine binding globulin (TBG), neurofilament light chain (e.g., serum neurofilament light chain), vitamin (e.g., vitamin D, such as 25-hydroxyvitamin D) or virus (e.g., hepatitis) antibody.

[0220] Compounds for forming conjugates are also provided. For example, the compound (e.g., a compound for conjugation with an analyte or a binding partner of an analyte (e.g., a peptide, protein, or macromolecule including an antibody)) can have a structure of formula (IV):

[0221] RFG-L-Ψ (Four)

[0222] wherein RFG is a reactive functional group for conjugation with an analyte or a binding partner of the analyte,

[0223] L is absent (i.e., it is a bond) or is a linker, and

[0224] Ψ is a chemiluminescent acridinium, which contains the following structure:

[0225]

[0226] where "j" is 1, 2, 3, 4, 5 or 6;

[0227] R 1 Hydrogen, –R, –X b ,–R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z or –R L –L 1 –R L –Z;

[0228] R 2 and R 3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or

[0229] R 2 and R 3 Together they form a 5-7 membered fused heterocyclyl group;

[0230] Z is a zwitterionic group which, at each occurrence, independently has the following structure:

[0231]

[0232] Wherein "q" and "l" are independently 0 or 1;

[0233] "r" is independently an integer from 0 to 10;

[0234] L 1 is independently –O–, –S–, –NH–, –N(R N )–、–(CH 2 ) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH 2 ) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH 2 ) 1-3 –、–(CH 2 ) 1-3 –C(O)–N(R N )–,–NH–S(O) 1-2 –、–N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–、–S(O) 1-2 –NH–, –(CH 2 ) 1-3 –NH–S(O) 1-2 –、–(CH 2 ) 1-3 –N(R N )–S(O) 1-2 –、–(CH 2 ) 1-3 –S(O) 1-2 –N(R N )–、–(CH 2 ) 1-3 –S(O) 1-2 –NH–, –O–(CH2 ) 1-4 –、–(CH 2 ) 1-4 –O–, S–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –S–, –NH–(CH 2 ) 1-4 –、–N(R N )–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –N(R N )–、–(OCH 2 ) 1-10 –、–(CH 2 O) 1-10 –、–(OCH 2 CH 2 ) 1-10 – or – (CH 2 CH 2 O) 1-10 –

[0235] R L C independently at each occurrence 1-20 a divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., having 1-10 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl; having 1-10 substituents, such as a hydrocarbon having 1-10 substituents selected from one or more groups -X);

[0236] R is independently hydrogen or C 1-35 A hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or arylalkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms, having 1-20 substituents);

[0237] R' and R" are independently hydrogen or C 1-10 alkyl;

[0238] X b is independently an anionic group at each occurrence; and

[0239] R N is independently selected at each occurrence from hydrogen or C 1-5 Alkyl (e.g., methyl, ethyl, propyl). For example, the reactive functional group can be selected from:

[0240] –NCS, –NCO, –SO 2 Cl, –N 3 ,–N 2 + Cl - ,

[0241] –Cl, –Br, –I, –NH 2 or –COOH.

[0242] The compound may have the structure of Formula (IVa):

[0243]

[0244] where "j" is 1, 2, 3, 4, 5 or 6;

[0245] R 1 Hydrogen, –R, –X b ,–R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z or –R L –L 1 –R L –Z;

[0246] R 2 and R 3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or

[0247] R 2 and R 3 together form a 5-10 membered fused heterocyclyl group;

[0248] R 4 is independently selected at each occurrence from hydrogen and -R (e.g., R', R N , lower alkyl such as C 1 -C 4 alkyl);

[0249] Ω is S, O or N;

[0250] Y is selected from –R, –L 1 –R, –R L –Z, –L1 –R L –Z, or in the case where Ω is O or S, then Y does not exist;

[0251] Y' is absent (i.e. it is a bond) or is selected from -L 1 –, –R L –, –R L –L 1 –, –L 1 –L 1 –, –L 1 –R L –, –L 1 –R L –L 1 or –R L –L 1 –R L –; and Y' contains one or more C or Z L The key to the connection;

[0252] Z is a zwitterionic group having the following structure:

[0253]

[0254] Wherein "q" and "l" are independently 0 or 1;

[0255] "r" is independently an integer from 0 to 10;

[0256] L 1 is independently –O–, –S–, –NH–, –N(R N )–、–(CH 2 ) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH 2 ) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH 2 ) 1-3 –、–(CH 2 ) 1-3 –C(O)–N(R N )–,–NH–S(O)1-2 –、–N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–、–S(O) 1-2 –NH–, –(CH 2 ) 1-3 –NH–S(O) 1-2 –、–(CH 2 ) 1-3 –N(R N )–S(O) 1-2 –、–(CH 2 ) 1-3 –S(O) 1-2 –N(R N )–、–(CH 2 ) 1-3 –S(O) 1-2 –NH–, –O–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –O–, –S–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –S–, –NH–(CH 2 ) 1-4 –、–N(R N )–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –N(R N )–、–(OCH 2 ) 1-10 –、–(CH 2 O) 1-10 –、–(OCH 2 CH 2 ) 1-10 – or – (CH 2 CH 2 O) 1-10 –

[0257] R L C independently at each occurrence 1-20 divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl, combinations thereof), optionally substituted with 1-10 heteroatoms and / or substituents;

[0258] R is independently hydrogen or C 1-35A hydrocarbon (e.g., alkyl, alkenyl, alkynyl, arylalkyl, combinations thereof) radical, optionally substituted with 1-20 heteroatoms and / or substituents;

[0259] R' and R" are independently hydrogen or C 1-10 alkyl;

[0260] X b is independently an anionic group at each occurrence; and

[0261] R N is independently selected at each occurrence from hydrogen or C 1-5 Alkyl (eg, methyl, ethyl, propyl).

[0262] Z is a zwitterionic group which, at each occurrence, independently has the following structure:

[0263]

[0264] Wherein "q" and "l" are independently 0 or 1;

[0265] "r" is independently an integer from 0 to 10;

[0266] L 1 is independently at each occurrence –O–, –S–, –NH–, –N(R N )–、–(CH 2 ) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH 2 ) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH 2 ) 1- 4–,–(CH 2 ) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH 2 ) 1-3 –、–(CH 2 ) 1-3 –C(O)–N(R N )–,–NH–S(O) 1-2 –、–N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–、–S(O) 1-2–NH–, –(CH 2 ) 1-3 –NH–S(O) 1-2 –、–(CH 2 ) 1-3 –N(R N )–S(O) 1-2 –、–(CH 2 ) 1-3 –S(O) 1-2 –N(R N )–、–(CH 2 ) 1-3 –S(O) 1-2 –NH–, –O–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –O–, –S–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –S–, –NH–(CH 2 ) 1-4 –、–N(R N )–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –N(R N )–、–(OCH 2 ) 1-10 –、–(CH 2 O) 1-10 –、–(OCH 2 CH 2 ) 1-10 – or – (CH 2 CH 2 O) 1-10 –

[0267] R L C independently at each occurrence 1-20 A divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents);

[0268] R is independently hydrogen or C 1-35 A hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or arylalkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms, having 1-20 substituents);

[0269] R' and R" are independently hydrogen or C 1-10 alkyl;

[0270] X b is independently an anionic group at each occurrence; and

[0271] R N is independently selected at each occurrence from hydrogen or C 1-5 Alkyl (e.g. methyl, ethyl, propyl); or

[0272] or a salt thereof (e.g., a halide salt such as a chloride salt). In some embodiments, Ω is S, O, or N. In various embodiments, R 1 For –R, –X b ,–R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z or –R L –L 1 –R L –Z. In some respects, R 1 For –R, –X b ,–R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z or –R L –L 1 –R L -Z, and Ω is S, O or N. For example, each R in formula (IVa) 4 In some embodiments, "j" is 2 or 3.

[0273] The compounds used to form the conjugates may have the relevant groups described herein (eg, Ω, Y, Y', Y", R, R', R", L 1 , L C , R L , R C , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 ,X,X a, X b ). For example, the compound can have a structure of Formula (V), (Va), (Vb), (Vc), (Vd), (Ve), (VIa), (VIb), (VIc), (VId), or (VIe):

[0274]

[0275]

[0276]

[0277]

[0278] In various embodiments, the compound is selected from:

[0279]

[0280]

[0281]

[0282]

[0283] In some embodiments, the compounds of the present disclosure may be zwitterionic and include one or more zwitterionic groups. For example, R attached to the positively charged nitrogen of the acridinium 1 The group may be optionally substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, Br, F) and may thus combine with the positively charged acridinium nitrogen atom to form a zwitterionic group. For example, a sulfopropyl or sulfobutyl group attached to the acridinium nitrogen may form a zwitterionic pair. 1 The group can also be neutral (e.g., methyl) or zwitterionic in nature (e.g., R 1 For –Z, –R L –Z, –L 8 –Z or –R L –L 8 –R M In some embodiments, R 1 Has the following structure:

[0284]

[0285] When the compound is charged (e.g. R 1 The compound may be in the form of a salt thereof and may optionally include a counter ion to balance the positively charged nitrogen of the acridinium core. The counter ion may be selected from CH 3 SO 4 - 、FSO3 - CF 3 SO4 - , C 4 F 9 SO 4 - , CH 3 C 6 H 4 SO 3 - , halogens (such as Cl - 、F - Br - ), CF 3 COO - , CH 3 COO - or NO 3 - In some embodiments, R 1 is methyl, ethyl, propyl or isopropyl. In some embodiments, the acridinium compound can be a zwitterion by covalently linking to an anion. For example, R 1 Can contain –R L –X and –X is a sulfonate group In some embodiments, R 1 For –R L –X and –X is a sulfonate group In some embodiments, R 1 For –R L –X or –L 8 -Z. In some embodiments, L 8 –S(O) 2 –NH– or –(CH 2 ) 1-3 –S(O) 2 –NH–. R 1 May contain sulfopropyl groups In a specific embodiment, R 1 It is sulfopropyl.

[0286] In some embodiments, the chemiluminescent acridinium Ψ is an acridinium ester. For example, Ψ can have the following structure:

[0287]

[0288] Where Ω is S, O or N;

[0289] Y is selected from –R, –L 1 –R, –R L –Z, –L 1 –R L–Z, or in the case where Ω is O or S, then Y does not exist;

[0290] Y' is absent (i.e. it is a bond) or is selected from –L 1 –, –R L –, –R L –L 1 –, –L 1 –L 1 –, –L 1 –R L –, –L 1 –R L –L 1 or –R L –L 1 –R L –; and Y' contains one or more C or Z L The key to the connection;

[0291] R 1 is hydrogen, –R, –X b ,–R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z or –R L –L 1 –R L –Z;

[0292] R 2 and R 3 are independently selected from hydrogen, -R, an electron donating group or -Z;

[0293] Z is a zwitterionic group having the following structure:

[0294]

[0295] Wherein "q" and "l" are independently 0 or 1;

[0296] "r" is independently an integer from 0 to 10;

[0297] L 1 is independently at each occurrence –O–, –S–, –NH–, –N(R N )–、–(CH 2 ) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH 2 ) 1-3–、–C(O)–、–O–C(O)–、–C(O)–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –C(O)–、–C(O)–O–、–C(O)–N(R N )–、–C(O)–NH–、–N(R N )–C(O)–、–NH–C(O)–、–C(O)–N(R N )–(CH 2 ) 1-3 –、–(CH 2 ) 1-3 –C(O)–N(R N )–、–NH–S(O) 1-2 –、–N(R N )–S(O) 1-2 –、–S(O) 1-2 –N(R N )–、–S(O) 1-2 –NH–、–(CH 2 ) 1-3 –NH–S(O) 1-2 –、–(CH 2 ) 1-3 –N(R N )–S(O) 1-2 –、–(CH 2 ) 1-3 –S(O) 1-2 –N(R N )–、–(CH 2 ) 1-3 –S(O) 1-2 –NH–、–O–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –O–、–S–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –S–、–NH–(CH 2 ) 1-4 –、–N(R N )–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –N(R N )–、–(OCH 2 ) 1-10 –、–(CH 2 O) 1-10–、–(OCH 2 CH 2 ) 1-10 – or – (CH 2 CH 2 O) 1-10 –

[0298] R L C independently at each occurrence 1-20 divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally substituted with 1-10 heteroatoms (e.g., heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl) and / or substituents;

[0299] R is independently hydrogen or C 1-35 A hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or arylalkyl) radical, which is optionally substituted with 1-20 heteroatoms (e.g., heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl) and / or substituents;

[0300] R' and R" are independently hydrogen or C 1-10 alkyl;

[0301] X b is independently an anionic group at each occurrence; and

[0302] R N is independently selected at each occurrence from hydrogen or C 1-5 In a specific embodiment, Ω is S, O or N and / or R 1 For –R, –X b ,–R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z or –R L –L 1 –R L In some embodiments, Ψ can be a chemiluminescent acridinium comprising the following structure:

[0303]

[0304] wherein "h" is 1, 2, 3, 4, 5, or 6. In some embodiments, Ψ is a chemiluminescent acridinium comprising the structure:

[0305]

[0306] Substituents on chemiluminescent acridinium esters can be modified to change the rate and yield of light emission, thereby reducing nonspecific binding, increasing stability, or increasing hydrophilicity. Typically, these modifications will substantially interfere with the binding of the analyte and its binding partner. Examples of substituent variability are disclosed by Natrajan et al. in U.S. Pat. No. 7,309,615, which is hereby incorporated by reference herein, describing high quantum yield acridinium compounds containing alkoxy groups (OR*) at C2 and / or C7, wherein R* is a group comprising a sulfopropyl moiety or an ethylene glycol moiety or a combination thereof. In some embodiments, R 2 and / or R 3 It may be an alkoxy group (e.g., OR and / or OR*). Natrajan et al., in International Publication No. WO2015 / 006174 (hereby incorporated by reference in its entirety), also describe hydrophilic, high quantum yield, chemiluminescent acridinium esters that also have certain electron donating functional groups at the C2 and / or C7 positions. 1 and / or R 2 These electron-donating groups at may have the following structures:

[0307]

[0308] Where R 9 -R 14 In each occurrence, independently select the methyl group or the group –(CH 2 CH 2 O) a CH 3 , wherein a is an integer from 1 to 5. In some embodiments, R 2 and R 3 is independently hydrogen, alkyl (e.g., methyl, ethyl, propyl, isopropyl), or alkoxy (e.g., methoxy, ethoxy, propoxy, or isopropoxy) at each occurrence. 2 and R 3 In other embodiments, R 2 or R 3 is hydrogen, and R 2 or R 3 The other is an alkoxy group or an electron donating group.

[0309] The detectable conjugate or the compound used to form the detectable conjugate may comprise a chemiluminescent acridinium sulfonamide. For example, Ψ in the conjugate or in the compound used to form the detectable conjugate may have the structure of formula (IIa), (IIb), (IIc), (IId) or (IIe):

[0310]

[0311] where “h” is 1, 2, 3, 4, 5 or 6;

[0312]

[0313] Where R 5 -R 8 are independently hydrogen or C 1-35 alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio or amino; and wherein L 1 with A (e.g. L does not exist) or L (e.g. with L C or Z L ) covalently bound.

[0314]

[0315]

[0316] Where Y" does not exist or is -L 1 –, –R L –, –L 1 –R L –or–R L –L 1 –, where Y” is equal to L (e.g., C or Z L ) is covalently linked. In some embodiments, R L is an optionally substituted five-membered or six-membered divalent aromatic hydrocarbon. L Can have the following structure:

[0317]

[0318] Where R 15 is independently hydrogen, halogen, or R at each occurrence. In some embodiments, R L Has the following structure:

[0319]

[0320] Where R 5 -R 8 Independently for C 1-35 In some embodiments, R 7 and R 8 Each is hydrogen, and R 5 and R 6Each is a methyl group. In some embodiments, Ψ comprises two pendant methyl groups on the phenolic ester to stabilize the bond, as disclosed in Law et al., Journal of Bioluminescence and Chemiluminescence 4:88-89 (1989), which is hereby incorporated by reference in its entirety. In some embodiments, Ψ has the following structure:

[0321]

[0322] In some embodiments, A, L and Ψ are covalently linked. The covalent bond portion between A and Ψ can be formed by a reactive functional group for forming a covalent bond with a peptide, protein or macromolecule, wherein the functional group includes an electrophilic group, a nucleophilic group or a photoreactive group. The reactive functional group can be an amine reactive group, a thiol reactive group, a carboxyl reactive group, a maleimide reactive group or a carbohydrate reactive group. In some embodiments, the reactive functional group can therefore react with a functional group (e.g., a primary amine) of an analyte or a binding partner. The reactive functional group can include (or be) isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, acetaldehyde, glyoxal, epoxide, ethylene oxide, carbonate, aryl halide, maleimide, imidoester, carbodiimide, anhydride, fluorophenyl ester or a combination thereof. In various embodiments, the reactive functional group is acylated or alkylated to label the analyte or its binding partner. For example, the bond may be formed by a reactive group selected from:

[0323] –NCS, –NCO, –SO 2 Cl, –N 3 ,–N 2 + Cl - ,

[0324] –Cl, –Br, –I, –NH 2 In some embodiments, the compound comprises a linker group having the structure -NH-C(O)- or -C(O)-NH-. In preferred embodiments, the compound or a portion thereof (e.g., L C , Ψ) comprises at least one -NH-C(O)- or -C(O)-NH- linker group.

[0325] The covalent linkage between A and Ψ (eg, L) or between RFG and Ψ (eg, L) may comprise (or be) a divalent C 1-20 alkyl, alkenyl, alkynyl, aryl or arylalkyl, which is optionally substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, F, Br). In some embodiments, L comprises a zwitterionic linker. L may have the structure -L C –(Z L ) z –, where z is 0 or 1. L C Can have the following structure

[0326] –(X 1 ) 0-1 –(R L ) 0-5 –(X 2 ) 0-1 –(R L ) 0-5 –(X 3 ) 0-1 –(R L ) 0-5 –(X 4 ) 0-1 –(R L ) 0-5 –

[0327] Where X 1 Selected from –O–, –S–, –NR N –, –C(O)–, –NR N –C(O)–, –C(O)–NR N –, –O–C(O)– or –C(O)–O–, –S–C(O)– or –C(O)–S–, =N–, –O–, or –S–;

[0328] X 2 –X 4 Independently selected from -O-, -S-, -NR N –, –C(O)–, –NR N –C(O)–, –C(O)–NR N –, –O–C(O)–, or –C(O)–O–, –S–C(O)–, or –C(O)–S–; and

[0329] R L independently selected at each occurrence from ––(CH 2 ) 1-5 –、–(CH 2 CH 2 O) 1-5 – or – (OCH 2 CH 2 )1-5 - or optionally substituted cycloalkylene (eg, C 5 -C 6 Cycloalkylene) which is replaced at one or more positions by, for example, an alkyl group (e.g., C 1 -C 5 Alkyl), alkoxy (e.g. C 1 -C 5 alkoxy) or halogen (eg, F); for example, provided that L C Between A and Ψ (or A and Z L The chain contains at least one atom (or at least two atoms) between .

[0330] In some embodiments, L and / or Ψ comprises -C(O)-NH-. In some embodiments, L C Has the following structure:

[0331]

[0332] The detectable label may comprise a dimethyl acridinium ester (DMAE) moiety and a zwitterionic linker, which comprises a zwitterionic linker or a polyethylene glycol-derived linker to improve the properties of the compound. When Ψ comprises a zwitterionic linker or a polyethylene glycol-derived linker or a dimethylphenyl ester, such properties may be improved, such as non-specific binding, hydrophilicity, or compound stability. In some embodiments, Z L Has the following structure:

[0333]

[0334] In several embodiments, R' is hydrogen or lower alkyl (eg, methyl, ethyl, propyl).

[0335] In some embodiments, the detectable conjugate can have the structure of Formula (III):

[0336]

[0337] wherein z is 0 (ie, it is a bond) or 1. In some embodiments, the compound used to form the conjugate may have the following structure:

[0338]

[0339] Exemplary compounds for forming conjugates are disclosed in Table 1. In some embodiments, the detectable conjugate is formed by reacting a compound (e.g., a compound of Formula (IV), (Va), (Vb), (Vc), (Vd), (Ve), (VIa), (VIb), a compound in Table 1) with an analyte, a binding partner thereof, or a derivatized version of the foregoing that is capable of reacting with a reactive functional group. As used to describe the compounds, these are typically acridinium containing 2,3-cyclic alkylenedioxy ("ADO"). The compound may be an acridinium ester ("AE"). The compound name may include "Z" which may refer to a zwitterionic linker, "CMO" which may refer to a carboxymethyl oxime linker, "CME" which may refer to a carboxymethyl ether linker, "CETE" which may refer to a carboxyethyl sulfide, "ZAE" which may refer to a zwitterionic acridinium ester (typically N-sulfopropyl dimethyl acridinium ester ("NSP-DMAE") in the illustrated embodiment), "ZD" which may refer to an acridinium core having an isopropoxy functional group attached thereto and a complete zwitterionic group (comprising N- + and X - Both)'s "ISODIZAE".

[0340] Table 1

[0341]

[0342]

[0343]

[0344]

[0345]

[0346] In some embodiments, the detectable conjugate can have one or more of the following structures:

[0347]

[0348]

[0349] where z is independently 0 or 1 at each occurrence;

[0350] y is independently 0, 1, 2, 3, 4, or 5 at each occurrence; and

[0351] A' is the analyte or its binding partner conjugated via the primary amine of the unconjugated analyte or its binding partner A;

[0352] in

[0353] X2 –X 4 Independently selected from -O-, -S-, -NR N –, –C(O)–, –NR N –C(O)–, –C(O)–NR N –, –O–C(O)– or –C(O)–O–, –S–C(O)– or –C(O)–S–; and

[0354] R L is independently selected at each occurrence from –(CH 2 ) 1-5 –、–(CH 2 CH 2 O) 1-5 – or – (OCH 2 CH 2 ) 1-5 -, or optionally substituted C 5 -C 6 For example, the conjugate may have the formula:

[0355]

[0356]

[0357] In various embodiments, the compound has the following structure:

[0358]

[0359] where z is independently 0 or 1 at each occurrence;

[0360] y is independently 0, 1, 2, 3, 4, or 5 at each occurrence; and

[0361] A' is the analyte or its binding partner conjugated via the thiol group of the unconjugated analyte or its binding partner A;

[0362] Where X 2 –X 4 Independently selected from -O-, -S-, -NR N –, –C(O)–, –NR N –C(O)–, –C(O)–NR N –, –O–C(O)– or –C(O)–O–, –S–C(O)– or –C(O)–S–; and

[0363] R L independently selected at each occurrence from –(CH 2 ) 1-5 –、–(CH 2 CH 2 O)1-5 –, or –(OCH 2 CH 2 ) 1-5 -, or optionally substituted C 5 -C 8 Cycloalkylene (eg cyclohexane).

[0364] In various embodiments, a portion of the linker can reduce the hydrolysis rate of the reactive functional group compared to an otherwise identical compound having, for example, an alkyl bond proximal to the reactive functional group. For example, and particularly for embodiments comprising a maleimide reactive functional group, the linker can include a portion proximal to the reactive functional group, such as an optionally saturated cycloalkyl group. For example, the compound can have the following structure:

[0365]

[0366] The compounds of the present disclosure can be characterized by their stability. For example, when the compound or conjugate is stored in an aqueous solution (generally in the pH range of 6-9), if there is a minimum loss of chemiluminescent activity measured by the loss of relative light units ("RLU"), the compound can be considered to be stable. Compared with another compound, a compound with increased instability may have a greater loss of chemiluminescent activity. For example, a compound of the present disclosure (e.g., a compound with a structure of formula (I)-(VI)) can be characterized as having increased stability at 4 ° C (common reagent storage temperature) and / or 37 ° C (accelerated temperature) over 33 days at pH 6 and / or 7 and / or 8. Compared with an otherwise identical compound that does not have a fused heterocycle conjugated to an acridinium system, the compound may have increased stability. In some embodiments, the compound can be characterized as having a change in chemiluminescent activity of less than (or from 1% to) 40% (e.g., less than 30%, less than 20%, from 10% to 40%, from 10% to 30%, from 10% to 20%) after storage at 37°C and pH 7 and / or pH 8 for 33 days.

[0367] The compounds of the present disclosure can also be characterized using comparative chemiluminescence quantum yields. Quantum yield can be measured as the observable chemiluminescence amount of a compound of a defined mass. The increase in the quantum yield of acridinium esters is one of the several advantageous properties of acridinium compounds of the present disclosure, which exhibit higher quantum yields relative to other acridinium compounds. Chemiluminescence can be measured in relative light units (RLU) on a photometer. The quantum yield of acridinium can be measured as the chemiluminescence amount (RLU) per mole of acridinium. Even if the mass of the acridinium ester required for detection is low (e.g., low-dose analyte in immunoassay), the increase in the quantum yield of acridinium will increase the possibility of its detection. The increase in the quantum yield of acridinium esters can therefore increase the sensitivity of immunoassays using acridiniums with high quantum yields. The relative quantum yield can be calculated as the ratio of the disclosed compounds compared to HEGAE. A value of the relative quantum yield greater than 1 indicates an enhancement of the quantum yield relative to HEGAE. In some embodiments, the compound has a relative quantum yield greater than 1.0 (or up to 5) (e.g., 1-6, 1-5, greater than 1.5, 1.5-4, 1.7-3.8, greater than 2, greater than 3) relative to HEGAE. In some embodiments, the compound has a relative quantum yield greater than 1 (or up to 3) (e.g., 1.1-1.5) relative to HQYAE.

[0368] The compound may also be characterized by the wavelength of its chemiluminescent emission.For example, the compounds of the present disclosure may have a maximum emission wavelength (λmax) of 430 nm to 460 nm.

[0369] In some embodiments, the compounds can be characterized by light emission kinetics. The compounds of the present disclosure typically complete emission within 5 seconds after chemiluminescence is triggered. In some embodiments, the compounds of the present disclosure may have faster light emission kinetics compared to other acridinium compounds, for example, emitting 90% of its light within 2 seconds measured over 5 seconds.

[0370] Table 2 provides exemplary characterizations of compounds for several species measured (dashed double bonds indicate fusion to the acridinium ring)

[0371] Table 2

[0372]

[0373]

[0374] The compound can also be characterized by its light output or signal-to-noise ratio in a chemiluminescent assay. For example, the compound can be characterized by a thyroid stimulating hormone assay, which has a greater signal-to-noise ratio than acridinium conjugates HQYAE or TSPAE similar to those shown in U.S. Patent Nos. 7,309,615 and 7,785,904, each of which is hereby incorporated by reference in its entirety (and in particular with respect to the quantum yield of TSPAE and HQYAE). In some embodiments, the compound can be characterized as having a relative signal-to-noise ratio greater than 1 (e.g., 1 to 2) compared to HQYAE or TSPAE (e.g., in a TSH assay). Typically, the higher the signal-to-noise ratio of the same amount of analyte, the better the assay sensitivity.

[0375] The compound can be prepared by using standard synthesis methods (except those provided herein) from commercially available starting materials, compounds known in the literature, or intermediates that are easily prepared. Standard synthesis methods and procedures for preparing organic molecules and functional group conversions and operations can be easily obtained from relevant scientific literature or from standard textbooks in the field. It will be understood that when typical or preferred process conditions (e.g., reaction temperature, time, molar ratio of reactants, solvent, pressure) are given, other process conditions may also be used unless otherwise stated. Optimum reaction conditions may vary with the specific reactants or solvents used, but those skilled in the art can determine such conditions by conventional optimization procedures. Those skilled in the art of organic synthesis will recognize that in order to optimize the formation of compounds described herein, the nature and order of the synthetic steps presented may be changed.

[0376] Synthetic chemistry transformations (including protecting group methods) used to synthesize the compounds described herein are known in the art and include, for example, those described in RC Larock, Comprehensive Organic Transformations, Second Edition, Wiley-VCH Publishers (1999); PGM Wuts and TW Greene, Protective Groups in Organic Synthesis, Fourth Edition, John Wiley and Sons (2007); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof, each of which is hereby incorporated by reference in its entirety.

[0377] The methods described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g. 1 H or 13 C), infrared spectroscopy (FT-IR), spectrophotometry (e.g., UV-Visible), or mass spectrometry (MS), or by chromatography (e.g., high pressure liquid chromatography (HPLC) or thin layer chromatography (TLC)).

[0378] The preparation of compounds may involve the protection and deprotection of various chemical groups. Those skilled in the art can easily determine the need for protection and deprotection and the selection of appropriate protecting groups. The chemical properties of protecting groups can be found in, for example, Greene et al., Protective Groups in Organic Synthesis, 2nd edition, Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.

[0379] The reaction of the method described herein can be carried out in a suitable solvent, and the solvent can be easily selected by the technician in the field of organic synthesis. Suitable solvents can be substantially free from reacting with starting materials (reactants), intermediates or products at the temperature at which the reaction is carried out (i.e., the temperature range can be from the freezing point temperature of the solvent to the boiling temperature of the solvent). A given reaction can be carried out in a solvent or a mixture of more than one solvent. Depending on the specific reaction steps, a solvent suitable for a specific reaction step can be selected.

[0380] The separation of the racemic mixture of a compound can be carried out by any of numerous methods known in the art. For example, the absolute configuration of a stereoisomer can be determined by 1D and 2D NMR techniques (e.g., COSY, NOESY, HMBC, and HSQC). Specific embodiments of these NMR techniques can be found in Hauptmann, H et al., Bioconjugate Chem. 11 (2000): 239-252 or Bowler, J. Steroids 54 / 1 (1989): 71-99, each of which is hereby incorporated by reference in its entirety. Another exemplary method includes preparing Mosher esters or amide derivatives of the corresponding alcohol or amine, respectively. Then, by protons and / or 19F NMR spectrum determines the absolute configuration of ester or amide.An exemplary method includes using "chiral resolution acid" (it is an optically active salt-forming organic acid) to carry out fractional recrystallization.The resolving agent suitable for fractional recrystallization is, for example, an optically active acid, such as D-type and L-type tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or various optically active camphorsulfonic acids.The resolution of the racemic mixture can also be carried out by eluting on a column equipped with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine).A person skilled in the art can determine a suitable elution solvent composition.

[0381] Generally, zwitterionic acridinium esters ("ZAE") containing reactive functional groups for forming covalent bonds, as described in U.S. Pat. Nos. 6,664,043 to Natrajan et al., 7,309,615 to Natrajan et al., 9,575,062 to Natrajan et al., or 9,487,480 to Natrajan, each of which is hereby incorporated by reference in its entirety, and particularly with respect to the zwitterionic acridinium esters and their syntheses described therein, can be used to synthesize the compounds disclosed herein. For example, the zwitterionic acridinium ester starting material can comprise an N-sulfopropyl ("NSP") group in the zwitterionic portion and / or comprise a charged nitrogen atom attached to a charged acridinium core ("DIZAE") and / or comprise a sterically stabilized dimethyl acridinium ester ("DMAE") and / or comprise an isopropoxy functionalized acridinium core ("ISO") and / or comprise a zwitterion ("Z") between the acridinium ester and the reactive functional group and / or a hexaethylene glycol derivative ("HEG") and / or a glutaric acid derivative (e.g., -C(O)-(CH 2 ) 3 –C(O)–) ​​linker. The reactive functional group can be NH 2 Or N-hydroxysuccinimide ester ("NHS") or maleimide derivatization. For example, the compound (e.g., a compound for conjugation with an analyte or a binding partner of an analyte (e.g., a peptide, protein, or macromolecule including an antibody)) can have the structure of formula (IV):

[0382] RFG-L-Ψ (IV)

[0383] wherein RFG is a reactive functional group for conjugation with an analyte or a binding partner of the analyte,

[0384] L is absent (i.e., it is a bond) or is a linker, and

[0385] Ψ is a chemiluminescent acridinium, which contains the following structure:

[0386]

[0387] where "j" is 1, 2, 3, 4, 5 or 6;

[0388] R 1 is hydrogen, –R, –X, –R L –X b ,–L 1 –R, –L 1 –X b , –Z, –R L –Z, –L 1 –Z or –R L –L 1 –R L –Z;

[0389] R 2 and R 3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or

[0390] R 2 and R 3 Together they form a 5-7 membered fused heterocyclyl group;

[0391] Z is a zwitterionic group which, at each occurrence, independently has the following structure:

[0392]

[0393] Wherein "q" and "l" are independently 0 or 1;

[0394] "r" is independently an integer from 0 to 10;

[0395] L 1 is independently –O–, –S–, –NH–, –N(R N )–、–(CH 2 ) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH 2 ) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH 2 ) 1-3 –、–(CH 2 ) 1-3–C(O)–N(R N )–,–NH–S(O) 1-2 –、–N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–、–S(O) 1-2 –NH–, –(CH 2 ) 1-3 –NH–S(O) 1-2 –、–(CH 2 ) 1-3 –N(R N )–S(O) 1-2 –、–(CH 2 ) 1-3 –S(O) 1-2 –N(R N )–、–(CH 2 ) 1-3 –S(O) 1-2 –NH–, –O–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –O–, S–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –S–, –NH–(CH 2 ) 1-4 –、–N(R N )–(CH 2 ) 1-4 –、–(CH 2 ) 1-4 –N(R N )–、–(OCH 2 ) 1-10 –、–(CH 2 O) 1-10 –、–(OCH 2 CH 2 ) 1-10 – or – (CH 2 CH 2 O) 1-10 –

[0396] R L C independently at each occurrence 1-20divalent hydrocarbon radicals (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl; having 1-10 substituents);

[0397] R is independently hydrogen or C 1-35 A hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or arylalkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl; having 1-20 substituents);

[0398] R' and R" are independently hydrogen or C 1-10 alkyl;

[0399] X b is independently an anionic group at each occurrence; and

[0400] R N is independently selected at each occurrence from hydrogen or C 1-5 Alkyl (eg, methyl, ethyl, propyl).

[0401] Chemiluminescent conjugates or compounds for forming conjugates can also be synthesized by using acridinium sulfonamide reactants. For example, the acridinium sulfonamides disclosed in U.S. Pat. No. 5,543,524 to Mattingly et al. (hereby incorporated by reference in its entirety) are useful starting materials for preparing the chemiluminescent compounds disclosed herein.

[0402] Chemiluminescent conjugates are useful as labels in assays for the detection or quantification of certain analytes that are capable of competing for binding with a binding partner.

[0403] For example, the assay method can be a competitive immunoassay, which is generally related to the use of binding molecules (e.g., antibodies) to detect macromolecules (also referred to as macromolecular analytes). The antibody is fixed or attached to a solid phase, such as a particle, a bead, a film, a microtiter plate, or any other solid surface. The analyte generally measured in such assay methods is generally some clinically relevant substances, and can encompass a wide range of molecules from large macromolecules (e.g., proteins, nucleic acids, viruses and bacteria) to small molecules (e.g., valproate, vitamins, steroids, hormones, therapeutic drugs).

[0404] The compounds disclosed herein can be used for sandwich immunoassays, which generally involve the use of two binding molecules (e.g., antibodies) to detect macromolecules (also referred to as macromolecular analytes). An antibody is fixed or attached to a solid phase, such as a particle, a bead, a membrane, a microtiter plate, or any other solid surface. The compound can also be used for competitive assays. In an example of competitive heterogeneous assays, a carrier having an antibody (e.g., 3C3, 3H10, 4G8 bovine monoclonal antibody) bound thereto is contacted with a medium containing a sample suspected of containing the analyte and a chemiluminescent conjugate (or "labeled analog") as described herein. The analyte from the sample competes with the labeled analog for binding to the analyte antibody. After separating the carrier and the medium, the labeling activity of the carrier or medium is determined by conventional techniques, and it is related to the amount of the analyte in the sample. In a variation of the above competitive heterogeneous assays, the carrier comprises an analyte analog, which competes with the analyte of the sample for binding to an antibody reagent according to the principles described herein. The labeled analyte analog can be covalently linked to a chemiluminescent or fluorescent molecule commonly referred to as a marker or tracer.

[0405] When a solid phase with fixed antibodies or other adhesives is mixed with a sample containing an analyte and a labeled analyte, a binding complex is usually formed between the analyte or the labeled analyte. The adhesive can be, for example, an antibody, an antibody fragment, a nucleic acid, a peptide, a binding protein, or a synthetic binding polymer. In some embodiments, the adhesive can be a protein, such as an intrinsic factor combined with vitamin B12 or a folate binding protein combined with folic acid. Due to the involvement of the solid phase, this type of assay is generally referred to as a heterogeneous assay. The chemiluminescent signal associated with the binding complex can then be measured, and the presence or absence of the analyte in the sample can be inferred. Typically, before the signal is generated, the binding complex is separated from the remainder of the binding reaction component (such as an excessive labeled analyte). For example, if the binding complex is associated with magnetic beads, a magnet can be used to separate the binding complex associated with the beads from the bulk solution.

[0406] By using a series of "standards", i.e., analyte at known concentrations, a "dose response" curve for a known labeled analyte can be generated. Thus, a dose response curve relates a certain amount of measured signal to a specific concentration of the analyte. In a competitive assay, if chemiluminescence from a bound complex is measured, then as the concentration of the analyte increases, the amount of signal decreases. The concentration of the analyte in the unknown sample can then be calculated by comparing the signal generated by an unknown sample containing the macromolecular analyte to the dose response curve.

[0407] The method of attaching binding molecules (such as antibodies) to solid phases generally involves mixing necessary components to induce attachment. For example, antibodies can be covalently attached to particles containing amines on their surfaces by using cross-linking molecules (such as glutaraldehyde). Attachment can also be non-covalent, and can involve simply adsorbing binding molecules to the surface of solid phases (such as polystyrene beads and microtiter plates). The labeling of binding molecules (such as antibodies and other protein-binding proteins) is also well known in the prior art, and is generally referred to as conjugation reaction, and labeled antibodies are generally referred to as conjugates. Generally, the amine reactive part on the label reacts with the amine on the antibody to form an amide bond. Other bonds (such as thioethers, esters, carbamates, etc.) between antibodies and labels can also be used.

[0408] In another aspect of the present invention, a reagent for detecting an analyte may be provided, the reagent comprising a chemiluminescent acridinium compound bound to the analyte or a binding partner. The reagent may have a luminescence intensity of less than 10 -3 M of the detectable conjugate. In some embodiments, the reagent may have a concentration of less than 10 -3 M (e.g. 10 -15 M to 10 -3 In some embodiments, the compound is provided in a reagent that further comprises a buffer.

[0409] Typically, assays used to detect or quantify analytes in a sample include:

[0410] (a) providing a detectable conjugate;

[0411] (b) providing a solid support on which are immobilized molecules capable of forming a binding complex with the analyte and capable of forming a binding complex with the detectable conjugate;

[0412] (c) mixing the compound, the solid support and the sample;

[0413] (d) separating the solid support from the mixture;

[0414] (e) triggering chemiluminescence of any acridinium label coordinated to the solid phase;

[0415] (f) measuring the amount of light emitted using a photometer; and

[0416] (g) detecting the presence of the analyte or calculating the concentration of the analyte by comparing the amount of light emitted to a standard dose response curve that relates the amount of light emitted to the known concentration of the analyte.

[0417] In some embodiments, the sample is derived from a mammal (e.g., a human). In some embodiments, the sample comprises saliva and / or blood and / or serum. In some embodiments, the sample is saliva and / or blood and / or serum.

[0418] In some assay methods, the sample to be analyzed is pretreated to release the analyte from endogenous binding substances (e.g., plasma or serum proteins that bind to the analyte). The release of the analyte from the endogenous binding substances can be performed, for example, by adding a digestive agent or a releasing agent or a combination of a digestive agent and a releasing agent used in sequence. The digestive agent can break down the endogenous binding substances so that they can no longer bind to the analyte.

[0419] Conditions for measuring a portion of a sample according to principles described herein can include measuring in an aqueous buffered medium of medium pH, which generally provides the best assay sensitivity. The aqueous medium can be water only, or can include 0.1 to 40% cosolvent by volume. The pH of the medium can be in the range of 4 to 11, or 5 to 10, or 6.5 to 9.5, or 7 to 8. Typically, the pH of the solution will be a compromise between the best binding of any particular binding pair of binding members, the best pH of other reagents (such as members of a signal generating system) of the assay, etc. Various buffers can be used to achieve the desired pH and maintain pH during the assay. Illustrative buffers include, for example, borate, phosphate, carbonate, TRIS, barbiturate, PIPES, HEPES, MES, ACES, MOPS, and BICINE.

[0420] Various auxiliary materials can be used in the assay method. For example, in addition to the buffer, the medium can include stabilizers for the medium and for the reagents used. In some embodiments, the medium can include proteins (e.g., albumin), organic solvents (e.g., formamide), quaternary ammonium salts, polyanions (e.g., dextran sulfate), binding enhancers (e.g., polyalkylene glycols), polysaccharides (e.g., dextran, trehalose), and combinations thereof.

[0421] Triggering the chemiluminescence of the analog can be performed by adding a chemiluminescent triggering reagent. The chemiluminescent triggering reagent can be acidic or alkaline. A variety of chemiluminescent triggering reagents can be added in sequence. For example, an acidic solution (e.g., an acidic solution containing hydrogen peroxide) can be added first, followed by an alkaline solution (e.g., an alkali metal hydroxide containing a surfactant). In some embodiments, the chemiluminescent triggering reagent comprises hydrogen peroxide, a salt of hydrogen peroxide, nitric acid, a nitrate, sodium hydroxide, an ammonium salt, a surfactant, or a combination thereof. Example

[0422] The following examples illustrate the synthesis of a representative number of compounds, the characterization of parameters relevant in assay development, and the use of these compounds in the measurement of samples in heterogeneous competitive assays. Therefore, the examples are intended to illustrate but not limit the present disclosure. Additional compounds not specifically exemplified can be synthesized using conventional methods in combination with the methods described herein.

[0423] Example 1: Quantum Yield

[0424] The comparative chemiluminescence quantum yield of the new structure of acridinium ester has been measured. The increase of the quantum yield from acridinium ester is one of several favorable properties of acridinium esters (relative to those showing lower quantum yield) showing higher quantum yield. Chemiluminescence is measured in relative light units (RLU) on a photometer. The quantum yield of acridinium ester is measured as the chemiluminescence amount (RLU) per mole of acridinium ester. Therefore, quantum yield is the observable chemiluminescence amount of acridinium ester of a defined mass. The increase of the quantum yield of acridinium ester increases its detection possibility, particularly in the case of gradually decreasing low-quality acridinium esters, such as and under low-dose analytes in immunoassays. The increase of the quantum yield of acridinium ester can therefore increase the sensitivity of immunoassays using high quantum yield acridinium esters. The relative quantum yield is calculated as a ratio with the quantum yield of HEGAE as the denominator. A value of the relative quantum yield greater than 1 indicates an increase in the quantum yield relative to HEGAE.

[0425] The relative quantum yields of the novel ADO acridinium esters of the present invention and HEGAE and HQYAE for comparison are listed in Table 3. With the exception of ADOAE A (4), the measured quantum yields of the novel ADOAEs are significantly higher (1.7 to 3.8 times) than that of HEGAE.

[0426] Table 3

[0427] Compound Quantum Yield Relative to HEGAE HEGAE(1) 1.0 HQYAE(2) 2.8 ADOAEA(4) 0.4 ADOAEC(6) 1.9 ADOAED(7) 2.5 ADOAEE(8) 3.5 ADOAEF(9) 3.8 ADOAEG(10) 1.7 ADOAEH(11) 2.7

[0428] Compounds ADOAE A (4) and ADOAE C (6) are very similar in structure. The only difference is that the former contains a five-membered ADO ring, while the latter has a six-membered ADO ring at its corresponding 2- and 3-positions. However, the quantum yield of ADOAE A (4) is only 1 / 5 of that of ADOAE C (6). This confirms the finding that the ring size is related to the performance of the acridinium ester and does not eliminate chemiluminescence. Without wishing to be bound by theory, increasing the ring size of the 2,3-ring substituent (e.g., six-membered, seven-membered) will increase the feasibility of the compound in immunoassays.

[0429]

[0430] Example 2: Emission Wavelength

[0431] The light emission spectra of the new compounds were measured using a PR-740FSSS spectral camera, which is capable of measuring light emission intensity in the wavelength range of 380–780 nm. The emission wavelengths of HEGAE and HQYAE were measured for comparison. It was found that the maximum emission wavelength (λmax) of the new acridinium ester was 440–450 nm, which is longer than the wavelength of HEGAE (425 nm) and shorter than the wavelength of HQYAE (475 nm).

[0432] All acridinium compounds were diluted with DMF and prepared as 1 mg / mL stock solutions. A 20 μL aliquot was placed in a glass tube and further diluted with 250 μL of DMF. Next, 300 μL of flash reagent 1 was added to the sample and the glass tube was placed in front of a PR-740FSSS spectrophotometer. The emission spectra of all compounds were recorded within a 5 second time window after the addition of 300 μL of flash reagent 2. The emission spectra were recorded at Figure 1A Table 4 provides the measured λmax for several acridinium esters of the present disclosure.

[0433] Table 4

[0434]

[0435]

[0436] Example 3: Emission Kinetics

[0437] The chemiluminescent emission kinetics of the new acridinium esters were measured using an AutoLumat Plus LB953 photometer (LB953) from Berthold Technologies. HEGAE and HQYAE were included in the measurements for comparison. ADOAEs and TSPAE aliquots were initially diluted with DMF and prepared at a concentration of 1 mg / ml. The concentration was diluted 100-fold to 10 -2 The solution was subjected to a rapid buffer from 10 -2 times up to 10 -8 10 μL of diluted sample was used to measure the chemiluminescence kinetics on the AutoLumat Plus LB953 for a total of 5 seconds with 50 data points, where the chemiluminescence value was within 10 5 Up to 10 6 A range of RLU / 10 μL is acceptable for good linearity.

[0438] The results of the measurements are Figure 2As can be seen, all tested acridinium esters completed light emission within 5 seconds. The new compounds ADOAEF (9), ADOAE H (11), ADOAE J (13) and ADOAE L (15) emitted ∼90% of their light within 2 seconds, which is significantly faster than HEGAE and HQYAE. The fast light emission kinetics are suitable for short-cycle light detection, which is desirable for high-throughput instruments.

[0439] Example 4: Thyroid Stimulating Hormone (TSH) Assay

[0440] AntiTSH-mAb conjugates of the new acridinium esters 7-11 and 13-15 were prepared as well as TSPAE (3) for comparison. TSPAE (3) is one of the best high quantum yield AEs for the assay, having a light output similar to that of HQYAE of U.S. Patent Nos. 7,309,615 and 7,785,904, which are hereby incorporated by reference in their entireties (and particularly with respect to light output of HQYAE in immunoassay measurements). The concentrations of the conjugates were measured using Micro BCA TM Protein Assay Determination Acridinium ester incorporation onto antiTSH mAb was measured by MALDI-TOF mass spectrometry.

[0441] The functionality of the conjugates in an immunoassay was evaluated on an ADVIA Centaur XPT using the Siemens-Healthineers Centaur TSH3 UL assay, where the Lite reagent was replaced by the experimental antiTSH-mAb conjugate, while all other test reagents remained the same. The relative light units of each test compound were measured over 10 different standards - each with a known concentration of TSH.

[0442] Briefly, anti-TSH conjugates of several TSH-conjugated AEs were diluted to 0.3 mg / mL in TSH3-UL Lite reagent buffer (SAP procedure 42196). Commercially available TSH3-UL reagent (REF 06491072 lot 332) was used for the study. Anti-FITC solid phase and FITC auxiliary reagents from lot 332 were recovered and paired with each TSH-AE Lite reagent. The reagents were then measured on an ADVIA Centaur XPT (equipment ID: B1072). The system automatically performed the following operations:

[0443] • Dispense 100 μL of sample (standard) into the cuvette.

[0444] • Dispense 50 μL of Auxiliary Reagent and 50 μL of Lite Reagent and incubate at 37°C for 2.75 minutes.

[0445] • Dispense 200 μL of solid phase and incubate at 37°C for 5.5 minutes.

[0446] Separate, aspirate and wash the cuvette with Wash 1.

[0447] • Dispense 300 μL each of the acidic reagent (Flash Reagent 1) and the alkaline reagent (Flash Reagent 2) to initiate the chemiluminescent reaction.

[0448] • Use the in-house TSH3-UL master curve standard lot 19031 as samples and calculate the average RLUs.

[0449] As shown in Tables 5 and 6, the conjugates tested were shown to be functional in the assay.

[0450] Table 5

[0451]

[0452] Table 6

[0453]

[0454]

[0455] The signal-to-noise ratio of each compound under each test condition is also calculated based on the RLU values ​​from Table 5 and Table 6. Tables 7 and 8 provide the signal-to-noise ratios of the measured conjugates. Most conjugates have low background signals under zero dose TSH standards. Compared with TSPAE (3), compounds 7, 9, 11 and 15 have equal or higher signals at the high dose end (e.g., S10). The overall signal-to-noise ratio of compounds 9, 13 and 15 relative to zero dose is higher than that of TSPAE. Higher signal-to-noise ratios are generally associated with better assay sensitivity provided by the disclosed compounds.

[0456] Table 7

[0457]

[0458] Table 8

[0459]

[0460]

[0461] Example 5: Chemiluminescence Stability

[0462] Excellent chemiluminescent stability (negligible instability) is one of several favorable properties of acridinium esters used as labels in immunoassays, which ensures that assay-derived clinical data will not change and become invalid during the life of the test kit. For example, the main mechanism of chemiluminescent instability of acridinium esters in aqueous solution is hydrolysis of the phenolic ester by hydroxide anions and other nucleophiles. High quantum yield acridinium esters (such as HQYAE and TSPAE) contain two hydrophilic alkoxy groups at the 2 and 7 positions and have been observed to be less stable than unsubstituted acridinium esters, which may be due to an additional mechanism of chemiluminescent instability.

[0463] The comparative chemiluminescent stability of the novel ADO acridinium esters conjugated to anti-hTSH monoclonal antibodies was measured by activation of the acridinium ester benzoic acid group with N-hydroxysuccinimide. The rate of chemiluminescent instability of the acridinium esters was measured by the loss of chemiluminescence over a set period of time under conditions close to the storage and handling conditions expected for the assay test kit.

[0464] In addition, heating the acridinium ester alone at temperatures exceeding the recommended storage conditions was intended to give an estimate of the long-term instability of the product. In addition to the stability evaluation of the freshly prepared acridinium ester conjugates, a control conjugate from TSPAE was tested, which represents a commercially available high-quantum acridinium ester. The incubation buffers were formulated to narrow the interpretation of the results to only the loss of chemiluminescence caused by temperature and pH. Therefore, these buffers do not include complex biological components, nor do they include excessive surfactants and other potential stabilizers that could complicate the interpretation of the results.

[0465] The comparative chemiluminescent instability of acridinium esters was measured at two temperature ranges (4°C (standard refrigeration) and 37°C (accelerated heating)) and at three pH values ​​of 6.0, 7.0 and 8.0 in each of these two temperature ranges. The buffer in which the comparative chemiluminescent stability of acridinium ester-antibody conjugates was measured consisted of 0.10 M sodium phosphate (pH buffer), 0.15 M sodium chloride (ionic strength agent), 7.7 mM sodium azide (antimicrobial preservative) and 0.1% (w / v) bovine serum albumin (protein conjugate stabilizer). Three volumes of this buffer were then adjusted to the three specified pHs, respectively. Chemiluminescence was measured in relative light units (RLU) on a photometer. Residual chemiluminescence measurements of each acridinium ester-antibody conjugate were performed using an ADVIA Centaur XPT, where the conjugate was initially diluted to approximately 5×10 6The target chemiluminescent concentration of RLU / 25 μL was used as the appropriate dilution. This chemiluminescent level provides a sufficiently high starting value to allow a measurable decline and is completely within the linear region of the Centaur photometer. Chemiluminescence was measured regularly on the Centaur using five replicates of 25 μL for each time point over a period of about one month. 0.30 mL of flash reagent 1 was added sequentially, and then 0.30 mL of flash reagent 2 was added after 60 seconds to trigger the chemiluminescent reaction in the cuvette. The chemiluminescent acquisition time was a nominal 3.500 seconds. The dark count time was 2.000 seconds. Chemiluminescence is reported as net chemiluminescence, i.e., total chemiluminescence minus the adjusted dark count. The residual chemiluminescence percentage associated with the initial chemiluminescence was calculated and tabulated by the average of the five replicates collected at each time point.

[0466] Two sets of experiments were performed. Tables 9 and 10 show the stability of several representative ADO acridinium esters under three pH conditions (pH 6, 7, and 8) at 4°C (common reagent storage temperature, Table 9) and 37°C (accelerated temperature, Table 10), the first set of experiments over 33 days and the second set of experiments over 35 days. Under nominal 4°C storage, all ADOAEs exhibited better chemiluminescent stability than TSPAE. At 37°C, where the instability of acridinium esters is accelerated by elevated temperature, all new ADOAEs exhibited significantly better chemiluminescent stability than TSPAE. This was particularly evident at pH 7 and pH 8; most immunoassays are performed in this pH range. For example, at day 33 at 37°C, the chemiluminescent activity of TSPAE dropped to less than 1% of the original chemiluminescence at pH 7 and pH 8, while the chemiluminescent activity of the new ADOAEs was still maintained at values ​​as high as 73% and 85%, respectively.

[0467] Table 9

[0468]

[0469]

[0470] Table 10

[0471]

[0472]

[0473] Example 6: Synthesis of ADOAE A(4) and ADOAE B(5)

[0474]

[0475] The synthesis of compounds ADOAE A (4) and ADOAE B (5) was started from the known starting material 5-methoxyisatin (4B). N-arylation of 5-methoxyisatin was carried out on a 2 g scale with 12 (2.1 g, 11 mmol) at 150°C in DMF using NaH (11 mmol) as base and CuI (22 mmol) as coupling agent over 8 h. LC / MS analysis showed that 70% of the N-arylisatin underwent further rearrangement to acridinium 9-carboxylic acid (4C). At this stage, DMF was removed from the reaction mixture at 60°C using high vacuum, 10% KOH solution was added, and reflux was continued at 120°C for 2 h. LCMS analysis confirmed that the reaction intermediate had been completely converted to 4C. The mixture was filtered through a sintered funnel, and the resulting filtrate was cooled to room temperature and acidified to pH = 2 with concentrated HCl. The orange precipitate was separated, filtered and dried under vacuum at 5 °C to obtain acridinium 9-carboxylic acid (4C) in 85% yield over two steps. Esterification of acridinium 9-carboxylic acid (4C) (1.32 g, 4.44 mmol) with phenol derivative 4D (400 mg, 2.22 mmol) was completed by toluenesulfonyl chloride in pyridine at 35 °C over 8 hours to give acridinium 9-carboxylate 4E in 70% yield. N-sulfopropanation of 4E (50 mg, 0.11 mmol) was carried out in a microwave reactor at 155 °C by ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate [BMIM][PF6] and 10 equivalents of 1,3-propanesultone in 2,6-di-tert-butylpyridine as base. 60% of the reaction was complete within 6 hours, at which stage 2N HCl was added to the reaction mixture and stirring was continued at 120 °C for 2 hours to afford ADOAE A (4) with an overall yield of 14%. Next, the active NHS-ester syntheses were prepared for protein conjugation. ADOAE A (9 mg, 0.015 mmol) was treated with TSTU and N,N-diisopropylethylamine in DMF at room temperature for 30 minutes to afford the final ADOAE B (5) as a yellow solid.

[0476] Example 7: Synthesis of ADOAE C(6) and ADOAE D(7)

[0477]

[0478] The synthesis of ADOAE C (6) and ADOAE D (7) was initiated from the starting materials 5-methoxyisatin (4B) and the bromo derivative 6A. The coupling between 4B (2 g, 11 mmol) and the bromo derivative 6A (2.1 g, 11 mmol) was completed in 8 hours in DMF at 150°C using NaH (11 mmol) as base and CuI (22 mmol) as coupling agent. LC / MS analysis confirmed that 50% of the N-arylisatin product rearranged to acridinium-9-carboxylic acid 6B. At this stage, DMF was removed from the reaction mixture using high vacuum at 60°C, 10% KOH solution was added, and reflux was continued at 120°C for 2 hours. Complete conversion of the N-arylisatin intermediate to acridinium-9-carboxylic acid was confirmed by LCMS analysis. The mixture was filtered through a sintered funnel, and the resulting filtrate was cooled to room temperature and further acidified to pH = 2 with concentrated HCl. After 30 min at 5 °C, the orange-yellow precipitate was filtered off and dried under vacuum to afford acridinium 9-carboxylic acid 6B as an orange powder in 86% yield over two steps. The acid 6B (1.44 g, 4.44 mmol) was esterified with the phenol derivative 4D (400 mg, 2.22 mmol) by p-toluenesulfonyl chloride in pyridine over 8 h at room temperature to afford the acridinium ester 6C in 70% yield. N-sulfopropylation of 6C (50 mg, 0.11 mmol) was carried out in a microwave reactor at 155 °C by the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate [BMIM][PF6] and 10 molar equivalents of 1,3-propane sultone in 2,6-di-tert-butylpyridine as base. 80% of the reaction was complete within 6 h, at which stage 2N HCl was added to the reaction mixture and stirring was continued at 105 °C for 2 h to afford ADOAE C (6) in 50% overall yield. Next, the active NHS-ester synthesis was prepared. Compound 6 (10 mg, 0.017 mmol) was treated with TSTU (0.051 mmol) and N,N-diisopropylethylamine (0.034 mmol) in DMF for 30 min to afford ADOAE D (7) in 52% yield after preparative HPLC purification.

[0479] Example 8: Synthesis of ADOAE E(8)

[0480]

[0481] The synthesis of ADOAE E (8) was initiated by the intermediate acridinium 9-ester (6C). 10 equivalents of BBr were used at 0 °C. 3 (1M, CH 2 Cl 2) was subjected to methyl ether cleavage of 6C (400 mg, 0.084 mmol) within 5 h to afford the hydroxy derivative 8A in 75.5% yield. Next, dialkylation was performed with 1,3-propane sultone in an ionic liquid at elevated temperature. Compound 8A (50 mg, 0.11 mmol) was reacted with 20 equivalents of 1,3-propane sultone, 10 equivalents of 2,6-di-tert-butylpyridine and K in 1-butyl-3-methylimidazolium hexafluorophosphate [BMIM][PF6]. 2 CO 3 (2 equivalents) were reacted at 160 ° C for 6 hours in a microwave reactor. 80% of the reaction was completed within 6 hours. At this stage, the crude reaction mixture was hydrolyzed with 2N HCl at 120 ° C for 2 h. The resulting acid compound was directly purified on preparative HPLC. The lyophilized HPLC fractions produced pure acid 8B (17 mg, yield 23%). Finally, NHS-ester synthesis was performed by using 3 equivalents of TSTU and 2 equivalents of N, N-diisopropylethylamine in DMF. After preparative HPLC purification of the crude reaction mixture, 8 mg of ADOAE E (8) was obtained.

[0482] Example 9: Synthesis of ADOAE F(9)

[0483]

[0484] The acid derivative 6 (10 mg, 0.015 mmol) was coupled directly to the HEG-amine spacer in one step by in situ activation of the acid with TSTU acid within 30 minutes, and the reaction mixture was then added to diamino-HEG in DMF. After 2 hours, the reaction mixture was purified on preparative HPLC and the preparative HPLC fractions were lyophilized for 48 hours to give the amine compound 9A in 79% yield. The resulting amine derivative (10 mg, 0.11 mmol) was converted to NHS ester at room temperature by using 3 equivalents of DSG in DMF and phosphate buffer at pH = 7.2. The reaction mixture was directly purified on HPLC and the preparative fractions were lyophilized to give 6.2 mg of ADOAE F (9) as a yellow compound.

[0485] Example 10: Synthesis of ADOAE G(10) and ADOAE H(11)

[0486]

[0487] The synthesis of symmetrical dioxane-AEs started with the known commercially available (5,6)-ethylenedioxy-isatin 10A. Isatin (1 g, 4.47 mmol) and bromo derivative 6A (1.83 g, 8.94 mmol) were coupled together in the presence of CuI, NaH in anhydrous DMF at 155 °C for 12 hours, but at this point the reaction proceeded further and rearranged to acridinium-9-carboxylic acid. The crude product was purified by using acid / base extraction to produce the desired pure acid 10B in 30% yield. This was followed by an esterification reaction where the acid derivative 10B (200 mg, 0.59 mmol) was reacted with the phenol derivative 4D (106 mg, 0.59 mmol) by using the solvent CHCl. 2 Cl 2 Reaction with toluenesulfonyl chloride in a mixture of pyridine (9:1, 10 mL) gave 10C in 70% yield. The synthesis of the NSP-AE-acid synthetase involved two reactions in a one-pot synthesis. First step: N-alkylation of acridinium-9-carboxylate 10C (80 mg, 0.16 mmol) with 1,3-propanesultone in a microwave reactor, the reaction was monitored on LCMS and the N-alkylation was complete within 8 h. Second step: Methyl ester hydrolysis was performed with 2N HCl at 120 °C within 2 h and the acid compound was purified using preparative HPLC to give 19 mg of 10D in 20% yield. Finally, the HPLC-purified material 10D (6 mg, 0.001 mmol) was treated with 3 equivalents of TSTU and 2 equivalents of N,N-diisopropylethylamine in DMF in the NHS-ester synthesis. After 30 min, the reaction was purified on preparative HPLC and 8 mg of ADOAE G (10) was obtained.

[0488] The synthesis of ADOAE H (11) started with the acid derivative 10D. The acid derivative (10 mg, 0.016 mmol) was coupled with HEG diamine (13.4 mg, 0.048 mmol) by acid activation with TSTU, followed by amide formation with HEG-diamine. The reaction was complete in 30 minutes. The crude product was directly purified on preparative HPLC to give 2 mg of the terminal amine 11A. The synthesis of the final NHS ester was performed by treating 11A (2 mg, 0.0028 mmol) with DSG in DMF and pH = 7.5 phosphate buffer at room temperature for 30 minutes. The crude product was purified on preparative HPLC to give 2 mg of ADOAE H (11) in 64% yield.

[0489] Example 11: Synthesis of ADOAE I(12) and ADOAE J(13)

[0490]

[0491] The synthesis of ADOAE I (12) and ADOAE J (13) started with commercially available 5-methoxyisatin. N-arylation of 5-methoxyisatin 4B (2 g, 13.6 mmol) with bromine 12A (1 g, 5.64 mmol) was carried out using CuI, NaH in anhydrous DMF at 150°C for 12 hours, but at this time the maximum reaction (90%) proceeded further and rearranged to acridinium-9-carboxylic acid. At this stage, DMF was removed from the reaction mixture under reduced pressure at 60°C, and the crude mixture was refluxed with 10% KOH (10 mL) for 30 minutes. The crude acid product was acidified with concentrated hydrochloric acid to give 1.2 g of acridinium 9-carboxylic acid 12B in an overall yield of 67%. Next, 12B (0.5 g, 1.53 mmol) was reacted with the phenol derivative 4D (221 mg, 1.23 mmol) using CH 2 Cl 2 Esterification was carried out with toluenesulfonyl chloride in a solvent mixture of 2-(4-(2-pyridine)-6-ol (9:1) at 35 °C overnight to afford acridinium 9-phenylcarboxylate 12C in 67% yield. The synthesis of NSP-AE-acid 12D involved two reactions in a one-pot synthetic scheme. Step 1: Acridinium 9-carboxylate 12C (100 mg, 0.205 mmol) was N-alkylated with 10 equivalents of 1,3-propanesultone in a microwave reactor. The reaction was monitored on LCMS and the N-alkylation was completed within 8 h. Step 2: Methyl ester hydrolysis was carried out with 2N HCl (10 mL) at 105 °C within 2 h in a microwave reactor and the acid compound was purified by using preparative HPLC to afford acridinium NSP-DMAE-acid 12D in 17% yield. Next, the HPLC purified material 12D (10 mg, 0.017 mmol) was treated with 3 equivalents of TSTU and 2 equivalents of N,N-diisopropylethylamine in DMF. After 30 minutes, the reaction was purified on preparative HPLC and 8 mg of ADOAE I (12) was obtained.

[0492] HEG-amine synthesis was carried out in two steps from acid 12D. Step 1: Acid 12D (8 mg, 0.015 mmol) was treated with TSTU (7.7 mg, 0.026 mmol) and 2 equivalents of DIPEA in DMF and the reaction was complete in 30 minutes by LCMS mass spectrometry. At this stage, the NHS-ester intermediate mixture was transferred to a stirred mixture of diamino-PEG6 and 4 equivalents of DIPEA in DMF at room temperature. After 2 hours, the mixture was purified on HPLC to produce 3 mg of HEG-amine 13A in 26% yield. The resulting HEG-amine derivative 13A (3 mg, 0.0035 mmol) was reacted with DSG (3.4 mg) in DMF and 5 equivalents of pH=7.5 buffer as base at room temperature. After stirring at room temperature for 30 minutes, the final product was purified on HPLC to give 2.8 mg of ADOAE J (13) in 76% yield.

[0493] Example 12: Synthesis of ADOAE K(14) and ADOAE L(15)

[0494]

[0495] The synthesis of ADOAE K (14) started with known commercially available isatin (14A) and 6-bromo-1,4-benzodioxane 6A. Isatin (2 g, 13.6 mmol) was N-arylated with bromine 6A (4.36 g, 20.4 mmol) using CuI, NaH in anhydrous DMF at 150 °C over 12 h, but the maximum reaction proceeded further and completely rearranged to acridinium-9-carboxylic acid. At this stage, DMF was removed from the reaction mixture under reduced pressure at 60 °C and the crude acid product was acidified with concentrated hydrochloric acid to give 2 g of acridinium-9-carboxylic acid 14B in 52% overall yield. Next, 14B (0.5 g, 1.77 mmol) was reacted with the phenol derivative 4D (256 mg, 1.54 mmol) at 35 °C using CH 2 Cl 2Esterification was carried out with toluenesulfonyl chloride in pyridine (9:1) solvent overnight to afford acridinium 9-methyl formate 14C in 76% yield. The synthesis of the NSP-AE-acid synthetase involved two reactions in a one-pot process. Step 1: N-alkylation of acridinium 9-carboxylate 14C (120 mg, 0.127 mmol) with 10 equivalents of 1,3-propane sultone in a microwave reactor. The reaction was monitored on LCMS and the N-alkylation was complete within 8 h. Step 2: Methyl ester hydrolysis was carried out with 2N HCl (10 mL) at 110 °C in a microwave reactor for 2 h. The resulting crude product was filtered through a sinter funnel and the filtrate was purified on preparative HPLC to afford acridinium NSP-AE-acid 14D in 56% yield. The HEG-amine synthesis was carried out in two steps from acid 14D (30 mg, 0.054 mmol). Step 1: 20 mg of the acid derivative was treated with TSTU (24 mg, 0.082 mmol) in DMF and 2 equivalents of DIPEA base, the reaction was complete within 30 min and it was confirmed by LCMS. At this stage, 50% of the reaction mixture was directly purified on preparative HPLC and 6 mg of ADOAE K (14) was isolated. Step 2: The remaining 50% of the reaction mixture (step 1) was reacted with diamino-PEG6 in DMF and 4 equivalents of DIPEA. The mixture was purified on HPLC and lyophilized after which 5 mg of pure material of HEG-amine (15A) was obtained in preparative fraction. The final NHS ester synthesis was carried out with DSG in DMF and 5 equivalents of pH=7.5 buffer as base and the final product was purified on HPLC to give 3 mg of ADOAE L (15).

[0496] Example 13: Synthesis of ADOAE M(16), ADOAE N(17), ADOAE P(19) and ADOAE Q(20)

[0497]

[0498]

[0499] General Synthesis: The synthesis of ADOAE M (16), ADOAE N (17), ADOAE P (19), and ADOAE Q (20) was carried out in two steps from AE-acid intermediates 14D, 10D, ADOAE C (6), and 12D.

[0500] Step 1: 3 mg of AE-acid of each compound was activated with 2 equivalents of TSTU and 2 eq of DIPEA in DMF, respectively, and the reaction was complete within 30 minutes by LCMS mass spectrometry. At this stage, the NHS-activated mixture (AE-NHS ester) was transferred dropwise to a stirred mixture of 1.5 equivalents of HEG-diamine and DIPEA (2 eq) in DMF at 0°C. The reaction temperature was slowly raised to room temperature over 30 minutes. After 2 hours at room temperature, LCMS indicated complete conversion of the AE-NHS ester to the AE-HEG-amine product. Step 2: 2 equivalents of Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) were added to the reaction mixture, warmed to 40°C, and the reaction was complete within 1 hour by LCMS mass spectrometry. The resulting crude mixture was directly purified on preparative HPLC. Lyophilized HPLC fractions yielded pure ADOAE M (16): 3 mg (53%), ADOAE N (17): 2 mg (37.2%), ADOAE P (19): 1.5 mg (28.3%) and ADOAE Q (20): 2 mg (37%).

[0501] Example 14. Synthesis of ADOAE O(18)

[0502]

[0503] The synthesis of ADOAE O (18) was carried out in one step from acid 8B. Acid 8B (3.5 mg, 0.015 mmol) was treated with TSTU (2.3 mg, 0.076 mmol) and DIPEA in DMF and the reaction was complete within 30 minutes by LCMS mass spectrometry. At this stage, aminoethylmaleimide (3.81 mg, 0.015 mmol) was added to the reaction mixture at room temperature. After 2 hours, the mixture was purified on HPLC to produce 2.5 mg of ADOAE O (18) with a yield of 60.6%.

[0504] Example 15. Synthesis of ADOAE R(21)

[0505]

[0506] The synthesis of ADOAE R (21) started from the acid derivative 8B. The acid derivative (5 mg, 0.009 mmol) was coupled with HEG diamine (3.64 mg, 0.013 mmol) by acid activation with TSTU, followed by amide formation with HEG-diamine. The reaction was completed in 30 minutes. The crude product was directly purified on preparative HPLC and 3 mg of terminal amine 21A was obtained in 43% yield. The synthesis of the final acridinium ester-maleimidocyclohexanecarboxylate (AE-MCC) was carried out by reacting 21A (3 mg, 0.0028 mmol) with 1.5 equivalents of SULFO-SMCC in DMF / pH == 7.5 phosphate buffer at room temperature for 30 minutes. The crude product was directly purified on preparative HPLC to produce 2 mg of ADOAE R (21) in 54% yield.

[0507] Example 16: Preparation of Acridinium Ester - antiTSH Antibody Conjugate

[0508] The following is a typical procedure for producing the conjugates described herein. A solution of AntiTSH-mAb (2 mg) in 1 mL of 0.1 M phosphate buffer (pH = 8) was treated with 10 equivalents of acridinium esters (TSPAE (2), ADOAE D (7), ADOAE E (8), ADOAE F (9), ADOAE G (10), ADOAE H (11), ADOAE J (13), ADOAE K (14) and ADOAE L (15)) added as a solution in DMSO (0.033 mL of a 4 mmol / L DMSO solution) ( Figures 3A - 3C The reaction was stirred at room temperature in the dark at 2 to 5 ° C for 16 hours. The labeled reaction was transferred to an Amicon Ultra-4 30kDa molecular weight cutoff filter and diluted with 3mL of deionized water. The filter was centrifuged at 5000×G for 10 minutes to reduce the volume to about 0.2mL. This process was repeated four more times. The final conjugate in about 0.2mL was added to a total of 1mL with deionized water to obtain a 2mg / mL solution. The AE-antiTSH mAb protein concentration was determined by micro BCA assay. The acridinium ester incorporated on the antiTSH mAb was measured by MALDI-TOF mass spectrometry.

[0509] All references cited herein (including patent applications and publications) are incorporated herein by reference and for all purposes to the same extent as if each individual publication or patent or patent application was expressly and individually indicated as being incorporated by reference in its entirety for all purposes. As will be apparent to those skilled in the art, many modifications and variations may be made to the present invention without departing from the spirit and scope of the present invention. The specific embodiments described herein are provided by way of example only, and the present invention is limited only by the terms of the appended claims and the full scope of equivalents to which such claims are entitled.

Claims

1. A compound having the structure of Formula (I) (eg, a detectable conjugate of an analyte or a binding partner of an analyte): AL-Ψ (I) wherein A is the analyte or a binding partner of the analyte, L is absent (ie it is a bond) or optionally contains a group L C or Z L of the connector, and Ψ is a chemiluminescent acridinium, which contains the following structure: where "j" is 1, 2, 3, 4, 5, or 6; R1 is hydrogen, –R, –X, –R L –X b , –L1–R, –L1–X b , –Z, –R L –Z, –L1–Z or –R L –L1–R L –Z; R2 and R3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or R2 and R3 together form a 5-7 membered fused heterocyclic group; Z is a zwitterionic group which, at each occurrence, independently has the following structure: Wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10 (e.g., 1 to 10); L1 is independently –O–, –S–, –NH–, –N(R N )–、–(CH2) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–,–NH–S(O) 1-2 –、–N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–、–S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –(CH2) 1-3 –S(O) 1-2 –N(R N )–、–(CH2) 1-3 –S(O) 1-2 –NH–, –O–(CH2) 1-4 –, –(CH2) 1-4 –O–, –S–(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –、–N(R N )–(CH2) 1-4 –, –(CH2) 1-4 –N(R N )–、–(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 – or – (CH2CH2O) 1-10 – R L C independently at each occurrence 1-20 a divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) sites of substitution (e.g., having 1-10 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl; having 1-10 substituents, such as the group —X); R is independently hydrogen or C 1-35 a hydrocarbon (e.g., an alkyl, alkenyl, alkynyl, or arylalkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., having 1-20 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl group; having 1-20 substituents, such as a group -X); R' and R" are independently hydrogen or C 1-10 alkyl; X b is independently an anionic group at each occurrence; R N is independently selected at each occurrence from hydrogen or C 1-5 Alkyl (e.g., methyl, ethyl, propyl); L C For divalent C 1-35 A hydrocarbon, optionally having one or more (e.g., 1-10, 1-5) substitution sites, one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl; having 1-20 substituents); and Z L is a zwitterionic linker group having the following structure: "m" is 0 (i.e. it is a bond) or 1; "n" and "p" are independently at each occurrence an integer from 0 (i.e., it is a bond) to 10; X a is independently an anionic group at each occurrence; R L C independently at each occurrence 1-20 A divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., substituted with 1-10 heteroatoms, substituted with 1-10 substituents); and R' is hydrogen or C 1-10 Alkyl; or Salts thereof (eg halide salts such as chloride salts).

2. The compound according to claim 1, wherein R1 is -R, -X, -R L –X b , –L1–R, –L1–X b , –Z, –R L –Z, –L1–Z, or –R L –L1–R L –Z.

3. A compound according to claim 1 or claim 2, wherein R3 is hydrogen.

4. A compound according to any one of claims 1 to 3, wherein R2 is an alkoxy group, which is optionally substituted at one or more (e.g., one, two, three) positions with one or more independently selected substituents (e.g., -X, such as -S(=O)1-2-R*, -O-S(=O)2-R*, -S(=O)2-OR*, -O-SO3, -O-S(=O)2-OR*, -O-S(=O)-OR*, -O-S(=O)-R*, -S(=O)-OR* or -S(=O)-R*, wherein R* is H or C 1-10 hydrocarbon) substitution.

5. The compound according to claim 1, wherein Ψ comprises the following structure: where "h" is 1, 2, 3, 4, 5, or 6.

6. The compound according to any one of claims 1 to 5, wherein Ψ comprises the following structure:

7. A compound according to any one of claims 1 to 6, wherein Ψ has a structure of formula (II): Where Ω is S, O or N; Y is selected from –R, –L1–R, –R L –Z, –L1–R L –Z, or in the case where Ω is O or S, then Y does not exist; Y' is absent (i.e. it is a bond), or is selected from -L1-, -R L –, –R L –L1–, –L1–L1–, –L1–R L –, –L1–R L –L1 or –R L –L1–R L –; and Y′ contains one or more residues that are identical to A (e.g., when L is absent) or L (e.g., C or Z L ) connection key; R1 is hydrogen, –R, –X, –R L –X, –L1–R, –L1–X, –Z, –R L –Z, –L1–Z or –R L –L1–R L –Z; R2 and R3 are independently selected from hydrogen, -R, an electron donating group or -Z; Z is a zwitterionic group having the following structure: Wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L1 is independently –O–, –S–, –NH–, –N(R N )–、–(CH2) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–,–NH–S(O) 1-2 –、–N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–、–S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –(CH2) 1-3 –S(O) 1-2 –N(R N )–、–(CH2) 1-3 –S(O) 1-2 –NH–, –O–(CH2) 1-4 –, –(CH2) 1-4 –O–, –S–(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –、–N(R N )–(CH2) 1-4 –, –(CH2) 1-4 –N(R N )–、–(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 – or – (CH2CH2O) 1-10 – R L C independently at each occurrence 1-20 divalent hydrocarbons (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally substituted with 1-10 heteroatoms; R is independently hydrogen or C 1-35 a hydrocarbon (eg, alkyl, alkenyl, alkynyl, or arylalkyl) radical optionally substituted with 1 to 20 heteroatoms; R' and R" are independently hydrogen or C 1-10 alkyl; X b is independently an anionic group at each occurrence; and R N is independently selected at each occurrence from hydrogen or C 1-5 Alkyl (eg, methyl, ethyl, propyl).

8. The compound according to claim 7, wherein Ω is S, O or N, and R1 is -R, -X, -R L –X, –L1–R, –L1–X, –Z, –R L –Z, –L1–Z, or –R L –L1–R L –Z.

9. A compound according to any one of claims 6 to 8, wherein Ψ has the structure of formula (IIa) where "h" is 1, 2, 3, 4, 5, or 6.

10. The compound according to any one of claims 1 to 9, wherein Ψ has the structure of formula (IIb): Wherein R5-R8 are independently hydrogen or C 1-35 alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio or amino; and wherein L1 is identical to A (eg, L is absent) or L (eg, identical to L C or Z L ) covalently bound. The compound according to claim 10 , wherein L1 is —NH—C(O)—, —C(O)—NH—, —C(O)—O—, or —O—C(O)—.

12. The detectable conjugate of claim 10 or 11, wherein R5 and R6 are each lower alkyl (eg, C1-C4 alkyl, methyl) and R7 and R8 are each hydrogen.

13. A compound according to any one of claims 1 to 9, wherein Ψ has the structure of formula (IIc): Where Y” does not exist, or is –L1–, –R L –, –L1–R L –or–R L –L1–, where Y” is with A (e.g. L is absent) or L (e.g. with L C or Z L ) are covalently linked.

14. A compound according to any one of claims 1 to 9, wherein Ψ has the structure of formula (IId): Where Y” does not exist, or is –L1–, –R L –, –L1–R L –or–R L –L1–, where Y” is with A (e.g. L is absent) or L (e.g. with L C or Z L ) are covalently linked.

15. A compound according to any one of claims 1 to 9 and 14, wherein Ψ has the structure of formula (IIe): Where Y” does not exist, or is –L1–, –R L –, –L1–R L –or–R L –L1–, where Y” is with A (e.g. L is absent) or L (e.g. with L C or Z L ) are covalently linked.

16. The compound according to any one of claims 1 to 15, wherein L has the structure -L C –(Z L ) z –, where "z" is 0 or 1; L C For divalent C 1-35 an alkyl, alkenyl, alkynyl, aryl or arylalkyl radical, optionally substituted with 1 to 20 heteroatoms; and Z L is a zwitterionic linker group having the following structure: "m" is 0 (i.e. it is a bond) or 1; "n" and "p" are independently, at each occurrence, an integer from 0 (i.e., it is a bond) to 10; X a It is an anionic group; R L C independently at each occurrence 1-20 A divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) substitution points (e.g., substituted with 1-10 heteroatoms, substituted with 1-10 substituents); and R' is hydrogen or C 1-10 alkyl.

17. The compound according to claim 16, wherein L C Has the following structure: –(X1) 0-1 –(R L ) 0-5 –(X2) 0-1 –(R L ) 0-5 –(X3) 0-1 –(R L ) 0-5 –(X4) 0-1 –(R L ) 0-5 – Wherein X1 is selected from =N-, -O-, -S-, or -NR N –, –C(O)–, –NR N –C(O)–, –C(O)–NR N –, –O–C(O)–, or –C(O)–O–, –S–C(O)–, or –C(O)–S–, or X1 is a group in represents the point of attachment to any adjacent group; X2-X4 are independently selected from -O-, -S-, -NR N –, –C(O)–, –NR N –C(O)–, –C(O)–NR N –, –O–C(O)–, or –C(O)–O–, –S–C(O)–, or –C(O)–S–; and R L is independently selected at each occurrence from –(CH2) 1-5 –, –(CH2CH2O) 1-5 – or – (OCH2CH2) 1-5 –.

18. A compound according to any one of claims 1 to 17, wherein X a and X b is, independently at each occurrence, a carboxylate (–C(O)O - ), sulfonate Sulfate Phosphate (–OP(O)(OR P )O–) or oxygen ions (–O - ), and R P is hydrogen or optionally has one or more (e.g., 1-10, 1-5) substitution sites (e.g., has 1-10 heteroatoms, has 1-10 substituents) C 1-12 hydrocarbon.

19. The compound according to any one of claims 1 to 18, wherein R1 comprises -R L –SO3 - .

20. The compound according to any one of claims 1-18, wherein R1 comprises (or is) a sulfopropyl group.

21. The compound according to any one of claims 1 to 18, wherein R1 is -S(O)2-NH-Z or -(CH2) 1-3 –S(O)2–NH–Z.

22. A compound according to any one of claims 1-21, wherein R2 and R3 are independently hydrogen, alkyl or alkoxy (e.g., lower alkoxy, such as C1-C4 alkoxy, methoxy, ethoxy, propoxy, isopropoxy) at each occurrence.

23. A compound according to any one of claims 1-21, wherein R2 and R3 are each hydrogen.

24. according to the compound described in any one of claims 1-21, wherein one of R2 or R3 is hydrogen, and the other of R2 or R3 is alkoxy (for example lower alkoxy, such as C1-C4 alkoxy, methoxy, ethoxy, propoxy, isopropoxy).

25. A compound according to any one of claims 16-24, wherein X a Sulfonate m is 1, R L is propyl, and n and p are each 3, such that Z L Has the following structure:

26. A compound according to any one of claims 1 to 25, wherein the compound has a structure of formula (IIIa) or (IIIb): Where "z" is 0 or 1.

27. according to the compound described in any one of claims 1-26, wherein analyte is thyroid hormone (for example thyrotropin, and for example A is its binding partner, for example anti-thyroid stimulating hormone monoclonal antibody (AntiTSH-mAb)), troponin, steroid hormone (for example androstenedione, testosterone), thyroglobulin, anti-thyroid peroxidase antibody, triiodothyronine (T3) hormone, thyroxine (T4) hormone, thyroxine binding globulin (TBG), neurofilament light chain (for example serum neurofilament light chain), vitamin (for example vitamin D, such as 25-hydroxyvitamin D) or virus (for example hepatitis) antibody.

28. The compound of any one of claims 1-12 and 16-27, wherein the compound is formed by reacting an analyte or its binding partner with:

29. A reagent composition for the detection of an analyte, comprising a compound according to any one of claims 1 to 28 in a pH buffered medium.

30. An assay for the detection or quantification of an analyte in a sample, comprising: (a) providing a detectable conjugate according to any one of claims 1 to 28; (b) providing a solid support on which a molecule capable of forming a binding complex with the analyte and / or capable of forming a binding complex with the detectable conjugate is immobilized; (c) mixing the compound, the solid support and the sample; (d) separating the solid support from the mixture; (e) triggering chemiluminescence of any acridinium label coordinated to the solid phase; (f) measuring the amount of light emitted using a photometer; and (g) detecting the presence of the analyte or calculating the concentration of the analyte by comparing the amount of light emitted to a standard dose response curve that relates the amount of light emitted to a known concentration of the analyte.

31. A compound having the structure of formula (IV) (eg, for conjugation with an analyte or a binding partner for an analyte): RFG—L-Ψ (IV) wherein RFG is a reactive functional group for conjugation with an analyte or a binding partner of the analyte, L is absent (ie it is a bond) or optionally contains a group Z L or L C of the connector, and Ψ is a chemiluminescent acridinium, which contains the following structure: where "j" is 1, 2, 3, 4, 5, or 6; R1 is hydrogen, –R, –X b , –R L –X b , –L1–R, –L1–X b , –Z, –R L –Z, –L1–Z or –R L –L1–R L –Z; R2 and R3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or R2 and R3 together form a 5-7 membered fused heterocyclyl group; Z is a zwitterionic group which, at each occurrence, independently has the following structure: Wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L1 is independently –O–, –S–, –NH–, –N(R N )–、–(CH2) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–,–NH–S(O) 1-2 –、–N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–、–S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –(CH2) 1-3 –S(O) 1-2 –N(R N )–、–(CH2) 1-3 –S(O) 1-2 –NH–, –O–(CH2) 1-4 –, –(CH2) 1-4 –O–, –S–(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –、–N(R N )–(CH2) 1-4 –, –(CH2) 1-4 –N(R N )–、–(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 – or – (CH2CH2O) 1-10 – R L C independently at each occurrence 1-20 divalent hydrocarbons (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl, having 1-10 substituents); R is independently hydrogen or C 1-35 A hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or arylalkyl) radical optionally having one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., having 1-20 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl, having 1-20 substituents); R' and R" are independently hydrogen or C 1-10 alkyl; X b is independently an anionic group at each occurrence; R N is independently selected at each occurrence from hydrogen or C 1-5 Alkyl (e.g., methyl, ethyl, propyl); L C For divalent C 1-35 A hydrocarbon (e.g., an alkyl, alkenyl, alkynyl, aryl, or arylalkyl) having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl, having 1-10 substituents); and Z L is a zwitterionic linker group having the following structure: "m" is 0 (i.e. it is a bond) or 1; "n" and "p" are independently, at each occurrence, an integer from 0 (i.e., it is a bond) to 10; X a It is an anionic group; R L C independently at each occurrence 1-20 A divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl, having 1-10 substituents); and R' is hydrogen or C 1-10 Alkyl; or Salts thereof (eg halide salts such as chloride salts).

32. The compound according to claim 29, wherein R1 is -R, -X, -R L –X b , –L1–R, –L1–X b , –Z, –R L –Z, –L1–Z, or –R L –L1–R L –Z.

33. A compound according to claim 31 or 32, wherein R3 is hydrogen.

34. according to the compound described in any one of claims 31-35, wherein R2 is alkoxy, it is optionally substituted by one or more independently selected substituents (for example -X, such as -S (=O)) at one or more (for example one, two, three) positions. 1-2 –R*, –O–S(=O)2–R*, –S(=O)2–OR*, –O–SO3, –O–S(=O)2–OR*, –O–S(=O)–OR*, –O–S(=O)–R*, –S(=O)–OR* or –S(=O)–R*, where R* is H or C 1-10 hydrocarbon) substitution.

35. The compound of claim 31, wherein Ψ comprises the structure: Where "h" is 1, 2, 3, 4.

36. A compound according to any one of claims 31-35, wherein Ψ comprises the structure:

37. A compound according to any one of claims 31-36, wherein Ψ has a structure of formula (V) Where Ω is S, O or N; Y is selected from –R, –L1–R, –R L –Z, –L1–R L –Z, or in the case where Ω is O or S, then Y does not exist; Y' is absent (i.e. it is a bond), or is selected from -L1-, -R L –, –R L –L1–, –L1–L1–, –L1–R L –, –L1–R L –L1 or –R L –L1–R L –; and Y′ contains one or more residues that are identical to A (e.g., when L is absent) or L (e.g., L C or Z L ) connection key; R1 is hydrogen, –R, –X, –R L –X, –L1–R, –L1–X, –Z, –R L –Z, –L1–Z or –R L –L1–R L –Z; R2 and R3 are independently selected from hydrogen, -R, an electron donating group or -Z; Z is a zwitterionic group having the following structure: Wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L1 is independently –O–, –S–, –NH–, –N(R N )–、–(CH2) 1-10 –, –S(=O) 1-2 –, –C=C–, –C=C–(CH2) 1-3 –, –C(O)–, –O–C(O)–, –C(O)–(CH2) 1-4 –, –(CH2) 1-4 –C(O)–, –C(O)–O–, –C(O)–N(R N )–, –C(O)–NH–, –N(R N )–C(O)–, –NH–C(O)–, –C(O)–N(R N )–(CH2) 1-3 –, –(CH2) 1-3 –C(O)–N(R N )–,–NH–S(O) 1-2 –、–N(R N )–S(O) 1-2 –, –S(O) 1-2 –N(R N )–、–S(O) 1-2 –NH–, –(CH2) 1-3 –NH–S(O) 1-2 –, –(CH2) 1-3 –N(R N )–S(O) 1-2 –, –(CH2) 1-3 –S(O) 1-2 –N(R N )–、–(CH2) 1-3 –S(O) 1-2 –NH–, –O–(CH2) 1-4 –, –(CH2) 1-4 –O–, –S–(CH2) 1-4 –, –(CH2) 1-4 –S–, –NH–(CH2) 1-4 –、–N(R N )–(CH2) 1-4 –, –(CH2) 1-4 –N(R N )–、–(OCH2) 1-10 –, –(CH2O) 1-10 –, –(OCH2CH2) 1-10 – or – (CH2CH2O) 1-10 – R L C independently at each occurrence 1-20 divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl, having 1-10 substituents); R is independently hydrogen or C 1-35 a hydrocarbon (e.g., an alkyl, alkenyl, alkynyl, or arylalkyl) radical, optionally having one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-20 or 1-20 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl, having 1-20 or 1-10 substituents); R' and R" are independently hydrogen or C 1-10 alkyl; X b is independently an anionic group at each occurrence; and R N is independently selected at each occurrence from hydrogen or C 1-5 Alkyl (eg, methyl, ethyl, propyl).

38. The compound of claim 37, wherein Ω is S, O or N, and R1 is -R, -X, -R L –X, –L1–R, –L1–X, –Z, –R L –Z, –L1–Z, or –R L –L1–R L –Z.

39. A compound according to any one of claims 35-38, wherein Ψ has a structure of formula (Va) where "h" is 1, 2, 3, 4, 5 or 6; 40. A compound according to any one of claims 31-39, wherein Ψ has a structure of formula (Vb): Wherein R5-R8 are independently hydrogen or C 1-35 hydrocarbon (eg, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio or amino); and wherein L1 is identical to A (eg, L is absent) or L (eg, identical to L C or Z L) Covalently bonded.

41. The compound of claim 40, wherein L1 is -NH-C(O)-, -C(O)-NH-, -C(O)-O-, or -O-C(O)-.

42. A compound according to claim 40 or 41, wherein R5 and R6 are each lower alkyl (e.g., C1-C4 alkyl, methyl) and R7 and R8 are each hydrogen.

43. A compound according to any one of claims 31-39, wherein Ψ has a structure of formula (Vc): Where Y” does not exist, or is –L1–, –R L –, –L1–R L –or–R L –L1–, where Y” is with A (e.g. L is absent) or L (e.g. with L C or Z L ) are covalently linked.

44. A compound according to any one of claims 31-39, wherein Ψ has a structure of formula (Vd): Where Y” does not exist, or is –L1–, –R L –, –L1–R L –or–R L –L1–, where Y” is with A (e.g. L is absent) or L (e.g. with L C or Z L ) are covalently linked.

45. A compound according to any one of claims 31-39 and 44, wherein Ψ has a structure of formula (Ve): Where Y” does not exist, or is –L1–, –R L –, –L1–R L –or–R L –L1–, where Y” is with A (e.g. L is absent) or L (e.g. with L C or Z L ) are covalently linked.

46. A compound according to any one of claims 31-45, wherein L has the structure -L C –(Z L ) z –, where "z" is 0 or 1; L C For divalent C 1-35 an alkyl, alkenyl, alkynyl, aryl or arylalkyl radical optionally substituted with 1 to 20 heteroatoms; and Z L is a zwitterionic linker group having the following structure: "m" is 0 (i.e. it is a bond) or 1; "n" and "p" are independently, at each occurrence, an integer from 0 (i.e., it is a bond) to 10; X a It is an anionic group; R L C independently at each occurrence 1-20 A divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) substitution points (e.g., substituted with 1-10 heteroatoms, substituted with 1-10 substituents); and R' is hydrogen or C 1-10 alkyl.

47. The compound according to claim 46, wherein L C Has the following structure: –(X1) 0-1 –(R L ) 0-5 –(X2) 0-1 –(R L ) 0-5 –(X3) 0-1 –(R L ) 0-5 –(X4) 0-1 –(R L ) 0-5 – Wherein X1 is selected from =N-, -O-, -S-, or -NR N –, –C(O)–, –NR N –C(O)–, –C(O)–NR N –, –O–C(O)–, or –C(O)–O–, –S–C(O)–, or –C(O)–S–; X2-X4 are independently selected from -O-, -S-, -NR N –, –C(O)–, –NR N –C(O)–, –C(O)–NR N –, –O–C(O)–, or –C(O)–O–, –S–C(O)–, or –C(O)–S–; and R L is independently selected at each occurrence from –(CH2) 1-5 –, –(CH2CH2O) 1-5 –, –(OCH2CH2) 1-5 -, or an optionally substituted cycloalkylene (e.g., C5 cycloalkylene, C6 cycloalkylene).

48. A compound according to any one of claims 31-47, wherein X a and X b is, independently at each occurrence, a carboxylate (–C(O)O - ), sulfonate (–SO3 - ), sulfate (–OSO3 - ), phosphate (–OP(O)(OR P ) - ) or oxygen ions (–O - ), and R P is hydrogen or C 1-12 A hydrocarbon optionally having one or more (eg, 1-10, 1-5) substitution sites (eg, having 1-10 heteroatoms, having 1-10 substituents).

49. A compound according to any one of claims 31-48, wherein R1 comprises -R L –SO3 - .

50. A compound according to any one of claims 31-49, wherein R1 comprises (or is) a sulfopropyl group.

51. A compound according to any one of claims 31-50, wherein R1 is -S(O)2-NH-Z or -(CH2) 1-3 –S(O)2–NH–Z.

52. according to the compound described in any one in claim 31-51, wherein R2 and R3 are independently hydrogen, alkyl or alkoxy (for example lower alkoxy, such as C1-C4 alkoxy, methoxyl group, ethoxyl group, propoxyl group, isopropoxy) at each occurrence.

53. A compound according to any one of claims 31-51, wherein R2 and R3 are each hydrogen.

54. according to the compound described in any one of claims 31-51, wherein R2 or R3 one is hydrogen, and R2 or R3 another one is alkoxy (for example low alkoxy such as C1-C4 alkoxy, methoxyl group, ethoxyl group, propoxyl group, isopropoxy).

55. A compound according to any one of claims 47-54, wherein X a Sulfonate (-SO3 - ), m is 1, R L is propyl, and n and p are each 3, such that Z L Has the following structure:

56. A compound according to any one of claims 31-42 and 47-55, having a structure of formula (VIa) or (VIb):

57. A compound according to any one of claims 31-56, wherein the reactive functional group is selected from: –NCS, –NCO, -SO2Cl, –N3, –N2 + Cl - , –Cl, –Br, –I, –NH2 or –COOH.

58. A compound according to any one of claims 31-42 and 46-57, wherein the compound is:

59. A method comprising reacting a compound according to any one of claims 31 to 58 with an analyte or a binding partner for the analyte (eg an antibody).

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