Mebenthiazine haptens, immunogens and protein conjugates and methods of production and use thereof

Abstract

Compositions, kits, and systems containing tolythiazine haptens, immunogens, and / or protein conjugates are disclosed. Methods of producing and using the tolythiazine haptens, immunogens, and conjugates are also disclosed.

Description

[0001] Citations of relevant applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 589977, filed October 12, 2023, pursuant to 35 USC § 119(e). The entire contents of the aforementioned patent application are expressly incorporated herein by reference. Background Technology

[0003] Toluidine is a sedative, anesthetic, muscle relaxant, and analgesic drug used in horses, cattle, and other non-human mammals. It is an analogue of clonidine and an alpha-2 adrenergic receptor agonist. Toluidine has become a commonly used over-the-counter drug in the United States, colloquially known as "tranq," particularly prevalent in Puerto Rico. Toluidine mixed with fentanyl is known on the street as a "sleep-cut," "zombie drug," and "tranq dope." Toluidine is also frequently found in "speedballs," which are mixtures of various drugs, typically including cocaine, heroin, or morphine, with fentanyl. From November 2021 to August 2022, in a needle exchange program in Maryland, 80% of drug paraphernalia that tested positive for fentanyl also contained toluidine.

[0004] Chao et al. (New Journal of Chemistry (2021) 45:4658-4665), Wei et al. (CN116003616A), and Xu et al. (CN111454912A) described the development of monoclonal antibodies against toluidine. However, despite the exponential increase in cases of toluidine abuse, there are currently no commercially available diagnostic immunoassays for detecting toluidine in biological samples. Attached Figure Description

[0005] Figure 1 Chemical structures containing various non-limiting embodiments of the toluenethiazine hapten constructed according to this disclosure, wherein the hapten contains linkers at different positions.

[0006] Figure 2Chemical structures comprising various non-limiting embodiments of the toluenethiazide immunogen and protein conjugate constructed according to this disclosure. As shown, non-limiting examples of "proteins" that can be conjugated with the illustrated toluenethiazide include keyhole hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), glucose-6-phosphate dehydrogenase (G6PDH), any carrier or labeled peptide or protein, and any combination thereof.

[0007] Figure 3 Synthesis schemes containing certain non-limiting embodiments of the XLZ1 hapten and conjugate for immunogen synthesis according to the present disclosure. Figure 3 The markings (1)-(10) shown are equivalent to the following chemical formulas disclosed herein: (1) Formula XI; (2) Formula II; (3) Formula XII; (4) Formula XIII; (5) Formula XIV; (6) Formula XV; (7) Formula XVI; (8) Formula XVII; (9) Formula XVIII; and (10) Formula XIX.

[0008] Figure 4 Another approach to the synthesis comprising certain non-limiting embodiments of the XLZ1 immunogen and conjugate according to this disclosure. Non-limiting examples of “proteins” that may be conjugated thereto include KLH, BSA, OVA, G6PDH and / or any carrier and / or labeled protein. Figure 4 The markings (3), (6) and (10) shown are equivalent to the following chemical formulas disclosed herein: (3) formula XII; (6) formula XV; and (10) formula XIX.

[0009] Figure 5 Synthetic schemes containing specific (but not limiting) embodiments of the toluenethiazine conjugates XLZ1-C6-3K G6PDH and XLZ1-C11-3K G6PDH conjugates. The chemical formulas disclosed herein are as follows: upper left structure, formula XVII; upper middle structure, formula XXII; upper right structure, formula XXIII; lower left structure, formula XVII; lower middle structure, formula XXIX; and lower right structure, formula XXX.

[0010] Figure 6 Synthetic regimens comprising specific (but not limiting) embodiments of the toluenethiazide hapten XLZ2-O4-C2S2 hapten.

[0011] Figure 7Another scheme for synthesis containing certain non-limiting embodiments of the XLZ2-O4-C2SAc-immunogen and conjugate according to this disclosure. Non-limiting examples of "proteins" that can be conjugated thereto include KLH, BSA, OVA, G6PDH and / or any carrier and / or labeled protein. The chemical formulas disclosed on the right side of the figure are as follows: top structure, formula XXVI; middle structure, formula XXVII; bottom structure, formula XXVIII (where R4 is G6PDH).

[0012] Figure 8 Another embodiment containing certain non-limiting embodiments of the present disclosure for the synthesis of XLZ2-O4-C8-Br and XLZ2-O4-C6-Br haptens.

[0013] Figure 9 Another embodiment containing certain non-limiting embodiments of the present disclosure for the synthesis of XLZ2-O4-C6-3K G6PDH and XLZ2-O4-C8-3K G6PDH conjugates.

[0014] Figure 10 Further embodiments comprising certain non-limiting implementations of the synthesis of toluenethiazide hapten / immunogen / conjugate according to this disclosure. Non-limiting examples of “proteins” that can be conjugated thereto include KLH, BSA, OVA, G6PDH and / or any carrier and / or labeled protein.

[0015] Figure 11 Another embodiment comprising certain non-limiting embodiments of the toluenethiazide hapten / immunogen / conjugate synthesized according to this disclosure. Non-limiting examples of "proteins" that can be conjugated thereto include KLH, BSA, OVA, G6PDH and / or any carrier and / or labeled protein.

[0016] Figure 12 Another embodiment comprising certain non-limiting embodiments of the synthesis of toluenethiazide hapten / immunogen / conjugate according to this disclosure. Detailed Implementation

[0017] Before explaining in detail at least one embodiment of this disclosure through exemplary language and results, it should be understood that the application of this disclosure is not limited to the details of the construction and arrangement of the components set forth in the following description. This disclosure can have other embodiments, or can be practiced or performed in various ways. Therefore, the language used herein is intended to be given the broadest possible scope and meaning; and these embodiments are exemplary and not exhaustive. Furthermore, it should be understood that the wording and terminology used herein are for descriptive purposes only and should not be considered limiting.

[0018] Unless otherwise defined herein, scientific and technical terms relating to this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. The techniques and procedures described above are generally performed according to conventional methods well known in the art, as set forth in the various general and more specific references cited and discussed herein. The relevant nomenclature rules, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are terms well known and commonly used in the art. Standard techniques are used in chemical synthesis and chemical analysis.

[0019] All patents, published patent applications, and non-patent publications mentioned in this specification demonstrate the skill level of a person skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent publications cited in any part of this application are hereby expressly incorporated by reference in their entirety to the same extent that each individual patent or publication is specifically and individually indicated to be incorporated by reference.

[0020] According to this disclosure, all articles, compositions, kits, and / or methods disclosed herein can be prepared and performed without excessive experimentation. While articles, compositions, kits, and / or methods have been described according to specific embodiments, those skilled in the art will understand that various variations may be applied to the steps or sequence of steps described herein without departing from the concept, spirit, and scope of this disclosure. All such similar alternatives and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of this disclosure as defined by the appended claims.

[0021] When used in accordance with this disclosure, unless otherwise stated, the following terms shall be understood to have the following meanings: In the claims and / or description, when the term "a" or "an" is used in conjunction with the term "comprising," it may mean "a," but it is also consistent with the meanings of "one or more," "at least one," and "more than one." Therefore, unless the context clearly indicates otherwise, the terms "a" or "an" and "the" include multiple references. Thus, for example, a reference to "compound" can refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or more than a number of compounds. The term "a plurality" means "two or more."

[0022] The term "at least one" will be understood to include one and any quantity more than one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can be extended to 100 or 1000 or more, depending on the terms attached to it; furthermore, the quantity of 100 / 1000 should not be considered a limitation, as higher limitations may also produce satisfactory results. Furthermore, the term "at least one of X, Y, and Z" will be understood to include individual X, individual Y, and individual Z, as well as any combination of X, Y, and Z. The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is used only to distinguish two or more items and does not imply any order or importance of one item relative to another, nor does it imply any order of addition.

[0023] The term "or" is used in claims to indicate an inclusive "and / or" unless it is explicitly stated that it refers only to substitutes or that substitutes are mutually exclusive. For example, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0024] As used herein, any reference to “one embodiment,” “an embodiment,” “some embodiments,” “an example,” “for example,” or “an example” means that a particular element, feature, structure, or characteristic associated with that embodiment is included in at least one embodiment. For example, the phrases “in some embodiments” or “an example” appearing in various places in the specification do not necessarily refer to the same embodiment. Furthermore, all references to one or more embodiments or examples should be construed as non-limiting to the claims.

[0025] In this application, the term "about" is used to indicate that a value includes inherent error variations in the composition / apparatus / equipment, variations between the method used to determine the value, or variations between the subjects of study. For example, but not limited to, when using the term "about," a specified value may differ from a specified value by plus or minus 20%, 15%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, as such variations are suitable for performing the disclosed methods and will be understood by those skilled in the art.

[0026] The terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include)”), or “containing” (and any form of containing, such as “contains” and “contain)”) as used in this specification and claims are inclusive or open-ended and do not exclude additional, unreferenced elements or method steps.

[0027] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB if the order is important in the particular context. Continuing this example, combinations containing repetitions of one or more items or terms are explicitly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that there is generally no limit to the number of items or terms in any combination unless it is obvious from the context.

[0028] As used herein, the term "substantially" means that the subsequently described event or situation occurs completely, or that the subsequently described event or situation occurs to a great extent or degree. For example, when associated with a particular event or situation, the term "substantially" means that the subsequently described event or situation occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" may mean that two items are 100% adjacent to each other, or that two items are very close to each other but not 100% adjacent, or that a portion of one of the two items is not 100% adjacent to the other item but is very close to it.

[0029] As used herein, the phrases “connected to” and “coupled to” include both direct and indirect connections / couplings between two parts. Non-limiting examples of connections / couplings include one part covalently bonded to another part by a direct bond or by a spacer group; one part non-covalently bonded to another part directly or by a specific binding pair member bound to that part; incorporating one part into another part, such as by dissolving one part in another structure or by synthesis; and coating one part onto another part.

[0030] The terms “analog” and “derivative” are used interchangeably herein and refer to a substance whose structure contains the same basic carbon skeleton and carbon functional groups as a given compound, but may also contain one or more substituents. As used herein, the term “substitution” will be understood to mean the replacement of at least one substituent on a compound with residue R. In some non-limiting embodiments, R may include H, hydroxyl, mercapto, halogen selected from fluorine, chlorine, bromine or iodine, optionally substituted linear, branched or cyclic alkyl, wherein the optional substituent is selected from one or more: alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl and arylheterocycloalkyl, each of which is optionally substituted, wherein the optional substituent is selected from one or more: alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, arylheterocycloalkyl, phenyl, cyano, hydroxyl, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl)2, carboxyl and -C(O))-alkyl.

[0031] The term "hydrocarbon group" refers to an organic group consisting only of carbon and hydrogen. Hydrocarbon groups can be unsaturated, or they can contain one or more carbon-carbon double bonds or one or more carbon-carbon triple bonds, or a mixture thereof. The term "hydrocarbon group" includes alkyl, alkenyl, and alkynyl groups.

[0032] The phrase "bulky organic" refers to an organic group whose molecular size is large relative to its weight. Bulk organic groups impede the ability of specific binding members to bind to molecular regions containing bulk organic groups. "Bulk hydrocarbon" refers to hydrocarbon groups whose molecular size is large relative to their weight, such as the molecular size of branched alkyl or cyclic alkyl groups.

[0033] The phrase "non-macro-sized organic group" refers to an organic group that does not exhibit a large molecular size relative to its weight. Non-macro-sized organic groups do not significantly impede the antibody's ability to bind to molecular regions containing non-macro-sized organic groups. The phrase "non-macro-sized hydrocarbon group" refers to a hydrocarbon group that does not exhibit a large molecular size relative to its weight, such as those exhibited by straight-chain alkyl groups.

[0034] The term "alkyl" refers to an organic group consisting only of single-bonded carbon and hydrogen atoms in a straight-chain, branched, or cyclic configuration. The number of carbon atoms in the organic group is 1 to 50, or 1 to 40, or 1 to 30, or 1 to 25, or 1 to 20, or 1 to 15, or 1 to 10, or 1 to 5, or 2 to 50, or 2 to 40, or 2 to 30, or 2 to 25, or 2 to 20, or 2 to 15, or 2 to 10, or 2 to 5, or 5 to 50, or 5 to 40, or 5 to 30, or 5 to 25, or 5 to 20, or 5 to 15, or 5 to 10. The term "lower alkyl" refers to an organic group having 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7. Alkyl groups of 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, or 8 to 9, or 9 to 10.

[0035] Bulk alkyl groups include branched alkyl groups and cyclic alkyl groups. Bulk branched alkyl groups are branched at or near a carbon atom attached to another molecule. Examples of bulk branched alkyl groups include, but are not limited to, sec-butyl, tert-butyl, triethylmethyl, diethylmethyl, tripropylmethyl, and dipropylmethyl. Cycloalkyl groups are alkyl groups containing one or more rings. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and norbornyl. Examples of non-bulk alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl.

[0036] The term "alkenyl" refers to a hydrocarbon chain having the above-mentioned number of carbon atoms in a straight or branched configuration and having at least one carbon-carbon double bond. It can appear at any point along the hydrocarbon chain, and examples include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, and dimethylpentenyl.

[0037] The term "alkynyl" refers to a hydrocarbon group having a hydrocarbon chain with the number of carbon atoms mentioned above, containing at least one carbon-carbon triple bond, including but not limited to, for example, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl and 2-butynyl.

[0038] The term "lower hydroxyl group" refers to an organic group having the number of carbon atoms described above, with a straight-chain, branched, or cyclic configuration, including ether oxygen groups used to attach a hydrocarbon group to a parent compound.

[0039] The term "lower alkoxy" refers to an organic group having the number of carbon atoms described above, in a straight-chain, branched, or cyclic configuration, wherein the organic group includes ether oxygens used to attach alkyl groups to the parent compound.

[0040] As used herein, the term "aryl" refers to an organic group derived from an aromatic hydrocarbon by removing one atom and containing one or more aromatic rings (such as, for example, but not limited to, 1 to 5 aromatic rings, or 1 to 4 aromatic rings, or 1 to 3 aromatic rings, or 1 to 2 aromatic rings, or 2 to 4 aromatic rings, or 2 to 3 aromatic rings). Examples of aryl groups include, but are not limited to, phenyl (from benzene), naphthyl (from naphthalene), and anthracene (from anthracene). Aryl groups can be substituted or unsubstituted. "Substituted aryl" refers to an aryl group containing one or more substituents, such as, but not limited to, bulky hydrocarbon groups, non-bulky hydrocarbon groups, and functional groups (e.g., chlorine, bromine, iodine, fluorine, nitro, and sulfone).

[0041] As used herein, “arylalkyl” means an organic group having a lower hydrocarbon group to which an aryl group is attached. As used herein, “aralkyl group” means an organic group having a lower alkyl group to which an aryl group is attached, such as, but not limited to, benzyl, phenethyl, 3-phenylpropyl and 1-naphthylethyl.

[0042] As used herein, the term "sample" will be understood to include any type of biological sample that may be used under this disclosure. Examples of available fluid biological samples include, but are not limited to, whole blood or any part thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, peritoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder irrigation fluid, semen, feces, pleural fluid, nasopharyngeal fluid, combinations thereof, etc.

[0043] The terms “specific binding chaperone” or “analyte-specific binder” will be understood to refer to any molecule capable of specifically binding to a target analyte. For example, but not limited to, binders / chaperones can be antibodies, receptors, ligands, aptamers, molecularly imprinted polymers (i.e., inorganic matrices), any fragments thereof, any combination or derivative thereof, and any other molecule capable of specifically binding to a target analyte.

[0044] The term "antibody" is used in the broadest sense and specifically (but not limited to) includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fragments of any of the above, and conjugates of any of the above, provided they exhibit the desired analyte binding biological activity. Therefore, the term "antibody" or "antibody peptide" refers to a full-length immunoglobulin molecule (i.e., an intact antibody) or its antigen-binding fragment that competes with an intact antibody for binding to a specific antigen. Antigen-binding fragments can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, Fd, biantibodies, single-chain antibodies, single-domain antibodies (such as, but not limited to, NANOBODIES®), and other antibody fragments or conjugates thereof that retain at least a portion of the variable region of an intact antibody, antibody substitute protein, or peptide (i.e., engineered binding protein / peptide), as well as combinations or derivatives thereof. See, for example, Hudson et al. (Nature Med. (2003) 9:129-134). Antibodies can be any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0045] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to bind to antigens. The antigen-binding function of an antibody can be achieved by fragments of the complete antibody. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, Fd, biantibodies, single-chain antibodies, single-domain antibodies (such as, but not limited to, NANOBODIES®), isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of the complete antibody. These antibody fragments are obtained using conventional recombinant and / or enzymatic techniques and are screened for antigen binding in the same manner as the complete antibody.

[0046] As used in this article, "antibody heavy chain" refers to the larger of the two polypeptide chains that exist in the natural conformation of all antibody molecules.

[0047] As used in this article, "antibody light chain" refers to the smaller of the two polypeptide chains that exist in the native conformation of all antibody molecules. κ and λ light chains refer to the two main isotypes of antibody light chains.

[0048] The term “CDR” and its plural “CDRs” refer to the complementarity-determining region (CDR) of an antibody or antibody fragment, which determines the binding properties of the antibody or antibody fragment. In most cases, three CDRs are located in the light chain variable region (CDRL1, CDRL2, and CDRL3), and three CDRs are located in the heavy chain variable region (CDRH1, CDRH2, and CDRH3). CDRs contribute to the functional activity of the antibody molecule and are separated by amino acid sequences that contain scaffold or framework regions. Among the various CDRs, the CDR3 sequence, especially CDRH3, is the most diverse and therefore contributes the most to antibody specificity. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (1987), the entire contents of which are incorporated herein by reference); and (2) methods based on crystallographic studies of antigen-antibody complexes (Chothia et al., Nature, 342:877 (1989), the entire contents of which are incorporated herein by reference).

[0049] The term "epitope" includes any protein determinant capable of specifically binding to immunoglobulins or T-cell receptors. In some embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. Epitope determinants typically include chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups. In some embodiments, an epitope may have specific three-dimensional structural features (e.g., "conformational epitopes") and specific charge features.

[0050] If a particular antibody specifically binds to two epitopes, the epitope is defined as "identical" to the other epitope. In some embodiments, polypeptides with different primary amino acid sequences may contain the same epitope. In some embodiments, the same epitope may have a different primary amino acid sequence. If different antibodies compete for specific binding to the epitope, they are said to bind to the same epitope.

[0051] When an antibody preferentially recognizes an antigen in a complex mixture of proteins and / or macromolecules, it "specifically binds" to the antigen. In some embodiments, the antibody contains an antigen-binding site that specifically binds to a particular epitope. In some such embodiments, the antibody is capable of binding to different antigens, as long as the different antigens contain a specific epitope or a closely related epitope. For example, in some cases, homologous proteins from different species may contain the same epitope. In some embodiments, the antibody binds at a concentration of no more than 10... -6 M, 10 -7 M, 10 -8 M or 10 -9The dissociation constant of M specifically binds to the antigen. When an antibody specifically binds to a receptor or ligand (i.e., the antireceptor), it can substantially inhibit receptor-ligand adhesion. As used herein, when an excess of antibody reduces the amount of receptor bound to the ligand by at least about 20%, 40%, 60%, 80%, 85%, or 90% (as measured in an in vitro competitive binding assay), the antibody substantially inhibits receptor-ligand adhesion.

[0052] "Isolated" antibodies refer to antibodies that have been isolated and / or recovered from the environmental components in which they were produced. Contaminant components of the production environment are substances that can interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the antibody will be purified to a measurable state by at least three different methods: 1) the antibody weight percentage, as determined by the Lowry method, is greater than 50%, such as greater than 75%, or greater than 85%, or greater than 95%, or greater than 99%; 2) sufficient to obtain at least 10 N-terminal or internal amino acid sequence residues, such as at least 15 sequence residues, using a twist-cup sequencer; or 3) homogenized by SDS-PAGE under reducing or non-reducing conditions using Coomassie Brilliant Blue or an alternative silver staining. Isolated antibodies include recombinant intracellular in situ antibodies because at least one component of the environment in which the antibody was produced will be absent. However, typically, isolated antibodies will be prepared through at least one purification step. Furthermore, "isolated antibodies" are substantially free of other antibodies with different antigen specificities. However, isolated antibodies may have some cross-reactivity with other relevant antigens.

[0053] The term "antibody mutant" refers to an amino acid sequence variant of an antibody in which one or more amino acid residues have been modified. Such mutants must have less than 100% sequence identity or similarity with an amino acid sequence that has at least 75% sequence identity or similarity with the amino acid sequence of the variable domain of the antibody heavy or light chain, such as at least 80%, or at least 85%, or at least 90%, or at least 95%.

[0054] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies that specifically bind to the same epitope; that is, individual antibodies comprising this population are identical, except for a small number of naturally occurring mutations. Each monoclonal antibody targets a single determinant on an antigen, unlike conventional (polyclonal) antibody formulations which typically comprise different antibodies targeting different determinants (epitaxes). In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized in a single production method using hybridoma cultures, thus avoiding contamination by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring production by any particular method. For example, in one embodiment, the monoclonal antibody produced according to this disclosure can be prepared using a hybridoma method first described by Kohler and Milstein (Nature, 256:495 (1975)).

[0055] The monoclonal antibodies used according to this disclosure can be produced by any method known in the art, including but not limited to the result of deliberate immunization protocols; the result of an immune response leading to the spontaneous production of antibodies during a disease or cancer process; phage-derived antibodies; and so on. In addition to the hybridoma production methods described above, the monoclonal antibodies of this disclosure can also be produced by a variety of other methods, such as, but not limited to, recombinant DNA methods (see, for example, U.S. Patent 4,816,567); isolating antibody fragments from phage display libraries (see, for example, Clackson et al., Nature (1991) 352:624-628; and Marks et al., J. Mol. Biol. (1991) 222:581-597); and various other monoclonal antibody production techniques (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)). Furthermore, many monoclonal antibodies that can be used with the conjugates and methods disclosed herein or otherwise contemplated are commercially available and therefore need not be described further.

[0056] As used herein, "substantially pure" means that the target substance is the dominant substance present (i.e., it is more abundant, in molar terms, than any other single substance in the composition). Typically, a substantially pure composition will contain more than 50% of all macromolecules present in the composition, such as more than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%. In one embodiment, the target substance is purified to substantially homogeneity (contaminant substances cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecule.

[0057] An analyte is a molecule that can be recognized by an analyte-specific binding partner, such as (but not limited to) an antibody. An analyte contains at least one antigenic determinant or "epitope," which is the region where the analyte binds to its analyte-specific binding partner (i.e., an antibody).

[0058] Turning now to the concept of the present invention, certain non-limiting embodiments of this disclosure include toluenethiazide haptens, immunogens and protein conjugates, as well as compositions, kits and systems comprising them, and methods for producing and using them.

[0059] Some non-limiting embodiments of this disclosure relate to a composition comprising a compound having the structure of Formula I:

[0060] Formula I

[0061] The condition is that at least one of R1, R2, and R3 is not H. In the natural toluenethiazide molecule, each of R1, R2, and R3 is H.

[0062] In certain specific (but not limiting) embodiments, at least one of R1, R2, and R3 that is not H includes at least one of aliphatic, hydrocarbon, bulky organic, non-bulky organic, alkyl, alkenyl, alkynyl, phenyl, aryl, substituted aryl, carbocyclic, heterocyclic, hydrocarboxyloxy, lower alkoxy, aryl hydrocarbon, halide, amide, imide, nitrate, nitrite, carbonyl, ketone, ester, carboxylic acid, hydroxide, thiol, sulfide, sulfate, sulfonate, peptide, protein, enzyme, linker, immunogenic carrier, label, or combination thereof.

[0063] In certain specific (but non-limiting) embodiments, the compound comprises a structure of one of formulas II, III, IV, V, XI, XII, XIII, XIV, XV, XVII, XVIII, XIX, XXII, or XXIX: .

[0064] In other specific (but not limiting) embodiments, the compound has a structure of one of formulas VI, VII, VIII, IX, XX, XXI, XXIII, or XXX:

[0065] R4 is a peptide or protein. Non-limiting examples of peptides or proteins that can be used according to this disclosure include vectors and labeled proteins. In one particular (but non-limiting) embodiment, R4 is an immunogenic vector.

[0066] According to this disclosure, any immunogenic vector known in the art or otherwise contemplated herein may be used. Non-limiting examples include keyhole hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), egg ovalbumin, bovine gamma globulin (BGG), thyroglobulin, fibrinogen, polysaccharides, glucose-6-phosphate dehydrogenase (G6PDH), carrier proteins, marker proteins, and combinations thereof.

[0067] Some non-limiting embodiments of this disclosure relate to a composition comprising a compound having the structure of formula X:

[0068] One of R5 and R6 is present and includes at least one of aliphatic, hydrocarbon, bulky organic group, non-bulky organic group, alkyl, alkenyl, alkynyl, phenyl, aryl, substituted aryl, carbocyclic, heterocyclic, hydrocarbon carbonyloxy, lower alkoxy, aryl hydrocarbon, halide, amide, imide, nitrate, nitrite, carbonyl, ketone, ester, carboxylic acid, hydroxide, thiol, sulfide, sulfate, sulfonate, peptide, protein, enzyme, linker, immunogenic carrier, label, or combination thereof.

[0069] In certain specific (but non-limiting) embodiments, the compound has a structure of one of the formulas XXIV, XXV, XXVI, XXVII, XXVIII, XXXI, XXXII, XXXIII, XXXIV, XXXV, or XXXVI:

[0070] R4 is a peptide or protein.

[0071] In certain specific (but not limiting) implementations, R5 or R6 contains an immunogenic vector, such as (but not limited to) any immunogenic vector disclosed herein or otherwise contemplated.

[0072] The compositions disclosed herein may include one or more additional ingredients, such as (but not limited to) at least one excipient.

[0073] Certain non-limiting embodiments of this disclosure relate to methods for preparing any compositions disclosed herein or otherwise contemplated. In certain specific (but non-limiting) embodiments, the method includes, as Figure 3-9 One or more steps are shown. For example (but not limited to), the method may include the step of attaching a tert-butyloxycarbonyl (Boc) protecting group to the amine of a toluenethiazide molecule to attach various R groups directly to the positions shown, rather than to the amine group.

[0074] Some non-limiting embodiments of this disclosure relate to methods for preparing antibodies. In this method, an antibody-producing animal is immunized with an immunogen comprising any composition disclosed herein or otherwise contemplated, and antibodies that specifically bind to the immunogen are isolated from the animal.

[0075] The immunization and isolation steps can be performed by any method known in the art or otherwise contemplated herein. In certain specific (but not limiting) embodiments, the antibody isolation step further includes the steps of: isolating antibody-producing spleen cells from an antibody-producing animal; selecting at least one antibody-producing spleen cell that secretes a monoclonal antibody that specifically binds to an immunogen; and preparing a hybrid cell line by fusing the selected antibody-producing spleen cell from the antibody-producing animal with a suitable fusion partner, thereby the hybrid cell line secreting a monoclonal antibody.

[0076] Some non-limiting embodiments of this disclosure relate to at least one antibody produced by methods disclosed herein or otherwise contemplated.

[0077] Some non-limiting embodiments of this disclosure relate to a coupling composition comprising any composition disclosed herein or otherwise contemplated, and having an enzyme coupled to the composition. According to this disclosure, any enzyme known in the art or otherwise contemplated herein for use in diagnostic assay systems may be utilized. Non-limiting examples of available enzymes include glucose-6-phosphate dehydrogenase (G6PDH), alkaline phosphatase, β-galactosidase, and horseradish peroxidase.

[0078] Some non-limiting embodiments of this disclosure relate to a kit containing at least one of the hapten, immunogen, conjugate, and / or antibody disclosed herein or otherwise contemplated. One particular (but non-limiting) embodiment includes a kit containing at least one antibody and at least one conjugate.

[0079] Some non-limiting embodiments of this disclosure relate to a method for detecting toluenethiazide in a sample. In this method, a sample suspected of containing toluenethiazide is bound to any antibody disclosed herein or otherwise contemplated, and the presence of toluenethiazide in the sample is detected based on the antibody's specific binding to it.

[0080] Some non-limiting embodiments of this disclosure relate to a method for detecting toluenethiazide in a sample. In this method, a sample suspected of containing toluenethiazide is mixed with one or more components of any kit disclosed herein or otherwise contemplated, and the presence of the enzyme reaction product of the conjugate is detected. When a reduction in the enzyme reaction product of the conjugate is observed compared to that detected in the absence of the sample, the presence of toluenethiazide in the sample is determined.

[0081] U.S. Patent No. 7,022,492 discloses various additional R groups, conjugates, systems, kits, and methods for use in combination with the compositions disclosed herein and / or for the detection of toluenethiazides (the entire contents of which are expressly incorporated herein by reference).

[0082] Example

[0083] Examples are provided below. However, it should be understood that this disclosure is not limited to its application to the specific experiments, results, and laboratory procedures disclosed below. Rather, these examples are provided merely as one of various implementations and are exemplary, not exhaustive.

[0084] Example 1: General Synthesis Procedure

[0085] Materials and Equipment: Compounds were purified on a Shimadzu HPLC system (Riverwood, MD) equipped with a silica bond-C18 reversed-phase column and a Biotage LC (Charlotte, NC). Chemical reactions were monitored by TLC (thin-layer chromatography) using silica gel plates from Analtech, Inc. (Newark, DE) and an ESI-MS Waters HPLC (Milford, MA). Silica gel plates were visualized using UV short-wavelength (254 nm). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), Fluka (Waltham, MA), ThermoScientific (Waltham, MA), and VWR (Radnor, PA) and used as is. 1 ¹H NMR was recorded on a Bruker UltraShield™ 600 MHz spectrometer (Bruker, Billerica, MA). Chemical shifts are reported in parts per million (ppm, δ) and correlated with tetramethylsilane or deuterated solvent as an internal reference. The NMR abbreviations used are: s (singleton), brs (broad singleton), d (doublet), t (triplet), q (quartet), dd (double doublet), qui (quintet), J (coupling constant), Hz (Hertz). ESI-MS spectra were recorded on a Water UPLC instrument (Milford, MA) at Siemens Healthineers Research & Development (Newark, DE). UV: Cary 60 for OD. 280 And NanoDrop 2000.

[0086] The following abbreviations have the following meanings: ACN-acetonitrile AcOH-acetic acid Boc2O-di-tert-butyl dicarbonate BrAcSu-bromoacetic acid N-hydroxysuccinimide ester BSA (Bovine Serum Albumin) CA-cellulose acetate filter calcd - calculated value CDCN- d 3 - Deuterated acetonitrile with 3 deuterium atoms (for NMR spectroscopy) CDCl3-deuterated chloroform (used in nuclear magnetic resonance spectroscopy) CV-cylinder volume DCM-dichloromethane DI water - deionized water DIPEA-N,N-Diisopropylethylamine DMF-N,N-dimethylformamide DTT-Dithiothreitol EDC or EDC HCl - N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride eq. - molar equivalent EDTA-ethylenediaminetetraacetic acid EtOAc - Ethyl acetate g-gram G6PDH - Glucose-6-phosphate native enzyme G6PDNa2- disodium glucose-6-phosphate (substrate) GMBS - N-γ-maleimide butyroyl-oxosuccinimide ester h-hours 1 H-proton (used in NMR) HPLC-High Performance Liquid Chromatography KLH-Keypore Hemocyanin LC-Liquid Chromatography min MeOH-methanol MeOD- d 4 - Deuterated methanol with 4 deuterium atoms (for NMR spectroscopy) mg μg-micrograms μL MHz - Megahertz mmol, mM (millimoles) MWCO - Molecular Weight Cutoff mL-ml NaOAc-sodium acetate β-NADH-nicotinamide adenine dinucleotide NEt3-Triethylamine G6PDH - Natural glucose-6-phosphate dehydrogenase NMR - Nuclear Magnetic Resonance OVA - ovalbumin isolated from egg white (Sigma) PB-phosphate buffer RPM - Revolutions per minute rt - room temperature 3K G6PDH-Recombinant G6PDH Enzyme SuOH-N-hydroxysuccinimide TCEP HCl - Tris(2-carboxyethyl)phosphine hydrochloride TFA-trifluoroacetic acid TRIS-Tris(hydroxymethyl)aminomethane UV - ultraviolet rays v / v - volume ratio XLZ1-Toluenethiazide Series I Conjugates / Immunogen XLZ2-Toluenethiazide Series II Conjugates / Immunogen

[0087] Example 2: Experimental synthesis of XLZ 1 hapten from immunogen and natural G6PDH conjugate ( Figure 3 )

[0088] Preparation of XLZ1-C4-OtBu(1) In a vial equipped with a magnetic stir bar and cap, toluenethiazide (95 mg, 0.43 mmol) was dissolved in DMF (500 µL); then, K₂CO₃ (120 mg, 0.87 mmol, 2 equivalents) and tert-butyl 4-iodobutyrate (0.65 mmol, 1.5 equivalents, 142 µL) were added. The resulting reaction mixture was heated at 50–70 °C for 6 days, and 90% of the toluenethiazide was reacted. The reaction was continued until completion (an additional 3 days). The product was purified by Biotage LC using DCM / MeOH 70 / 30 v / v. The fractions containing the product were combined and concentrated under vacuum to give 123 mg (0.34 mmol) of oily product in 80% yield. [C] 20 H 31 N2O2S + The calculated MS value is 363.21, and the measured value is 363.33.

[0089] 1 H NMR (600 MHz, CDCl3) 7.12 (t, J = 7.86 Hz, 1H), 7.05 (d, J = 7.60 Hz, 2H), 3.66 - 3.64 (m, 2H), 3.60 - 3.57 (m, 2H), 2.89 - 2.87 (m, 2H), 2.23 (s, 6H), 2.20 - 2.19 (m, 2H), 1.89 - 1.76 (m, 4H), 1.43 (m, 2H), 1.37 (s, 9H).

[0090] Preparation of XLZ1-C4-OH(2): In a vial equipped with a magnetic stir bar, XLZ1-C4-OtBu (80 mg, 0.22 mmol) was dissolved in DCM (200 µL), followed by the addition of TFA (200 µL). The resulting reaction mixture was stirred at room temperature (rt.) for 5 days. The solvent was evaporated on a rotary evaporator to obtain an oil, which was further dried on an oil pump. [C] 16 H23 N2O2S + The calculated MS value is 307.15, and the measured value is 307.30.

[0091] Preparation of XLZ1-C4-OSu(3): In a vial equipped with a magnetic stir bar and cap, XLZ1-C4-OH(2) (7.8 mg, 0.025 mmol) was dissolved in DMF (156 µL), and then EDC was added. HCl (6 mg, 0.031 mmol) and SuOH (6 mg, 0.052 mmol). The resulting reaction mixture was stirred at room temperature for two days and monitored by UHPLC MS. [C] 20 H 26 N3O4S + The calculated MS value was 404.16, and the measured value was 404.32. Once OSu activation was complete, DMF (0.156 mL) was added to prepare a 25 mg / mL XLZ1-C4OSu(3)-DMF solution. This solution was used for immunogen synthesis.

[0092] Preparation of XLZ1-C5-OtBu(4) In a vial equipped with a magnetic stir bar and cap, toluenethiazide (130 mg, 0.59 mmol) was dissolved in DMF (500 µL), followed by the addition of 160 mg (1.18 mmol, 2 equivalents) of K₂CO₃, then 209 mg (1.5 equivalents), 160 µL of tert-butyl 5-bromopentanoate and KI (approximately 50 mg). The resulting reaction mixture was heated at 100–120 °C for 6 days, and 90% of the toluenethiazide was reacted. The product was purified by reverse-phase chromatography and lyophilized overnight to give 180 mg (0.48 mmol) of product (4), in a yield of 81%.

[0093] Mobile phase: Solvent A: Water + 0.1% AcOH; Solvent B: ACN + 0.1% AcOH; LC purification program: 0.00-1.00 min isocratic elution with solvent B 10%, 1.00-24.05 min gradient elution with solvent B to 80%, 24.05-28.00 min isocratic elution with solvent B 80%, 28.00-32.00 min isocratic elution with solvent B to 10%, 32.05 min stop collection. The fraction collected within 12-15 minutes contains the desired product.

[0094] [C 20 H 33 N2O2S + The calculated MS value is 377.23, and the measured value is 377.38. 1H NMR (600 MHz, CDCl3) 7.14 - 7.11 (m, 1H), 7.05 (d, J = 7.61 Hz, 2H), 3.66 - 3.63 (m, 2H), 3.56 - 3.53 (m, 2H), 2.89 -2.87 (m, 2H), 2.23 (s, 6H), 2.18 (t, J = 6.96 Hz, 2H), 1.79 - 1.75 (m, 2H), 1.53 - 1.50 (m, 2H), 1.45 - 1.44 (m, 2H), 1.39 (s, 9H).

[0095] Preparation of XLZ1-C5-OH(5): In a vial equipped with a magnetic stir bar and cap, XLZ1-C5-OtBu (180 mg, 0.48 mmol) was dissolved in DCM (300 µL), followed by the addition of TFA (300 µL). The resulting reaction mixture was stirred at room temperature for 7 days. After LC-MS analysis, the solvent was evaporated to dryness to give 152 mg XLZ1-C5-OH (5) (99% yield). [C] 16 H 25 N2O2S + The calculated MS value is 321.16, and the measured value is 321.26. 1 H NMR (600 MHz, CDCl3) 10. 28 (s, 1H), 7.28 (t, J = 7.71 Hz, 1H), 7.16 (d, J = 7.57 Hz, 2H), 3.78-3.76 (m, 2H), 3.71 (t, J = 5.35Hz, 2H), 3.04 (t, J = 6.02 Hz, 2H), 2.37 (t, J = 6.25 Hz, 2H), 2.20 (s, 6H), 2.17 - 2.16 (m, 2H), 1.62 - 1.61 (m, 4H).

[0096] Preparation of XLZ1-C5-OSu(6): In a vial equipped with a magnetic stir bar and cap, dissolve XLZ1-C5-OH (12 mg, 0.037 mmol) in DMF (0.240 mL), then add EDC. HCl (9 mg, 0.047 mmol) and SuOH (9 mg, 0.078 mmol). The resulting reaction mixture was stirred at room temperature for two days, and the reaction was monitored by LC MS. The reaction was complete after 8 h. [C] 21 H 28 N3O4S+ The calculated MS value was 418.18, and the measured value was 418.35. Once OSu activation was complete, DMF (0.240 mL) was added to prepare a 25 mg / mL XLZ1-C5-OSu(6)-DMF solution. This solution was used to prepare the immunogen and G6PDH conjugate.

[0097] Preparation of XLZ1-C3-NHBoc(7): In a vial equipped with a magnetic stir bar and cap, toluenethiazide (140 mg, 0.64 mmol) was dissolved in DMF (750 µL), followed by the addition of K₂CO₃ (175 mg, 1.27 mmol, 2 equivalents), 3-(amino)propyl bromide (182 mg, 0.77 mmol, 1.2 equivalents), and KI (100 mg). The resulting reaction mixture was heated to 120 °C for 7 days. The solvent was removed using a rotary evaporator, and the crude product was suspended in EtOAc (20 mL) and washed with deionized water (2 x 10 mL). The organic layer was collected, concentrated, dissolved in acetonitrile (2 mL), and purified using a Shimadzu LC-patch with a C18 column.

[0098] Mobile phase: Solvent A: water + 0.1% AcOH, Solvent B: acetonitrile + 0.1% AcOH, detection at 250 nm; flow rate: 20 mL / min; LC purification program: 0-1 min isocratic solvent B 20%, 1-20 min gradient solvent B 60%, 20-24 min isocratic solution B 60%, 24-26 min gradient solution B 20%, 26-28 min isocratic liquid B 20%, controller stops at 28.01 min.

[0099] The fraction containing the product was collected, concentrated, and freeze-dried to obtain 160 mg of product (7) (main peak 120 mg, peak tail 40 mg). [C 20 H 32 N3O2S + The calculated MS value is 378.22, and the measured value is 378.38.

[0100] 1 H NMR (600 MHz, CDCl3): 8.14 (brs, 2H), 7.22 (t, J = 7.62 Hz, 1H), 7.10 (d, J =7.58 Hz, 2H), 3.77 (t, J = 7.71 Hz, 2H), 3.73 (t, J = 5.26 Hz, 2H), 3.13 (brs, 2H), 2.95 (t, J= 5.90 Hz, 2H), 2.21 (s, 6H), 2.06 (s, 3H), 1.98 (s, 2H), 1.76 (t, J = 6.77 Hz, 2H), 1.43 (s, 9H).

[0101] Synthesis of XLZ1-C3-NH2(8) In a vial equipped with a magnetic stir bar and cap, XLZ1-C3NHBoc(7) (40 mg) and DCM / TFA 1 / 1 v / v 300 µL were added. The resulting reaction mixture was stirred for 3 days. The solvent was removed on a rotary evaporator and then lyophilized to give 30 mg of a colorless, oily XLZ1-C3NH2 TFA salt (8). [C 15 H 24 N3S + The calculated MS value is 278.44, and the measured value is 278.25.

[0102] Preparation of XLZ1-C8-OH(9) In a vial equipped with a magnetic stir bar and cap, XLZ1-C3NH2 TFA salt (8) (30 mg, 0.076 mmol) was dissolved in THF (0.2 mL), DIPEA (20 µL) was added, followed by succinic anhydride (8 mg, 0.08 mmol). The resulting reaction mixture was stirred overnight at room temperature, concentrated, dissolved in ACN, and purified by reversed-phase HPLC with a C18 column. The fraction containing product XLZ1-C8-OH (9) was collected, concentrated, and lyophilized (33.5 mg). [C 19 H 28 N3O3S + The calculated MS value is 378.19, and the measured value is 378.30.

[0103] Mobile phase: Solvent A: water + 0.05% TFA, Solvent B: acetonitrile + 0.05% TFA; Detection at 250 nm; Flow rate: 7 mL / min; LC purification program: 0-2 min isocratic solvent B 20%, 2-14 min gradient solvent B 70%, 14-16 min isocratic solution B 70%, 16-17 min gradient solvent A 20%, 17-21 min isocratic liquid B 20%.

[0104] 1 H NMR (600 MHz, CDCl3): 9.94 (s, 1H), 8.51 (s, 2H), 7.66 (s, 1H), 7.29 (t, J = 7.65Hz, 1H), 7.16 (d, J = 7.60 Hz, 2H), 3.78 (t, J= 7.46 Hz, 2H), 3.73 (brs, 2H), 3.26 (s, 2H), 3.05 (t, J = 5.55 Hz, 2H), 2.66 (brs, 2H), 2.55 (brs, 2H), 2.19 (m, 8H), 1.75 (m, 2H). After lyophilization, 22.5 mg of white powdery product was recovered.

[0105] Preparation of XLZ1-C8-OSu(10) In a glass vial equipped with a magnetic stir bar and cap, XLZ1-C8-OH (9) (11 mg) was dissolved in DMF (200 µL), followed by the addition of EDC (9 mg) and SuOH (9 mg). The resulting reaction mixture was stirred for 3 days, followed by the addition of additional EDC (9 mg) and SuOH (6 mg). The reaction mixture was stirred by UPLC-MS until complete, yielding product (10). [C 23 H 31 N4O5S + The calculated MS value was 475.58, and the measured value was 475.40. Then, DMF (240 µL) was added to prepare a 25 mg / mL hapten-DMF solution.

[0106] Example 3: Bioconjugation

[0107] Bioconjugation buffer

[0108] Buffer 1: 50 mM phosphate buffer, pH 8.00; Buffer 2: 100 mM phosphate buffer, pH 8.00; Buffer 3: 55 mM TRIS buffer, pH 8.00; Buffer 4: 50 mM phosphate buffer, pH 7.00; Buffer 5: 55 mM TRIS buffer, pH 7.00; Buffer 6: 50 mM phosphate buffer, 1 mM EDTA, pH 7.25; Buffer 7: 50 mM phosphate buffer, 1 mM EDTA, pH 7.25, 0.025 mM DTT; Buffer 8: 50 mM phosphate buffer, pH 7.5.

[0109] Preparation of XLZ1-C4-OVA conjugate (11a):In a plastic tube, OVA (10 mg) was suspended in buffer 1 (2 mL), and then XLZ1-C4-OSu(3) (1.9 mg, 75 µL, equivalent to a 20 x mol excess at MW 42700 Da) was added dropwise. The resulting reaction mixture was shaken at room temperature for 16 h, and then purified on a G25M column (CV = 60 mL) pre-equilibrated with buffer 4. 7 mL of the conjugate solution was collected and concentrated to 4.0 mL on an Amicon stirred ultrafiltration cup MW CO 10 kDa, to a concentration of 0.72 mg / mL.

[0110] Bioconjugation method of XLZ1-C4 immunogen ( Figure 4 )

[0111] Preparation of XLZ1-C4-BSA immunogen (11b): In a glass vial equipped with a magnetic stir bar and cap, BSA (20 mg) was suspended in buffer 1 (3 mL), followed by the addition of XLZ1-C4-OSu (3) (2.34 mg, 94 µL, equivalent to a 20 x mol excess at MW 69000 Da). The resulting reaction mixture was stirred at room temperature for 16 h and then purified on a G25M column (CV = 60 mL) pre-equilibrated with buffer 4. 15 mL of the conjugate solution was collected and concentrated to 5.0 mL (concentration = 3.82 mg / mL) on an Amicon stirred ultrafiltration cup MW CO 10 kDa.

[0112] Preparation of XLZ1-C4-KLH immunogen (11c): In a glass vial equipped with a magnetic stir bar and cap, KLH (20 mg) was suspended in Buffer 1 (3 mL), followed by the addition of XLZ1-C4-OSu (3) (2.56 mg, 102 µL, equivalent to a 200 x mol excess at an average MW 635000 Da). The resulting reaction mixture was stirred at room temperature for 16 h and then purified on a G25M column (CV = 60 mL) pre-equilibrated with Buffer 4. A 15 mL volume of the conjugate solution was collected and concentrated to 3.8 mL (concentration = 5.0 mg / mL) using an Amicon stirred ultrafiltration cup with a MW CO2 10 kDa filter.

[0113] Bioconjugation method of XLZ1-C5 immunogen ( Figure 4 )

[0114] Preparation of XLZ1-C5-OVA conjugate (12a):In a glass vial equipped with a magnetic stir bar and cap, OVA (20 mg) was suspended in buffer 2 (3 mL), followed by the dropwise addition of XLZ1-C5-OSu (2.71 mg, 108 µL, equivalent to a 20 x mol excess at MW42700 Da). The resulting reaction mixture was stirred at room temperature for 16 h, and then purified on a G25M column (CV = 60 mL) pre-equilibrated with buffer 4. A 15 mL volume of the conjugate solution was collected and concentrated to 7.0 mL (concentration = 2.97 mg / mL) using an Amicon stirred ultrafiltration cup MW CO2 10 kDa.

[0115] Preparation of XLZ1-C5-BSA immunogen (12b): In a glass vial equipped with a magnetic stir bar and cap, BSA (20 mg) was suspended in buffer 2 (3 mL), and then XLZ1-C5-OSu (3.72 mg, 148 µL, equivalent to a 40 x mol excess at MW 69000 Da) was added dropwise. The resulting reaction mixture was shaken at room temperature for 16 h, and then purified on a G25M column (CV = 55 mL) pre-equilibrated with buffer 4. 15 mL of the conjugate solution was collected and concentrated to 5.2 mL (concentration = 4.0 mg / mL) on an Amicon stirred ultrafiltration cup MW CO 10 kDa.

[0116] Preparation of XLZ1-C5-KLH immunogen (12c): In a glass vial equipped with a magnetic stir bar and cap, KLH (20 mg) was suspended in Buffer 2 (3 mL), and then XLZ1-C5-OSu (2.02 mg, 81 µL, equivalent to a 200 x mol excess of an average MW 635000 Da) was added dropwise. The resulting reaction mixture was shaken at room temperature for 16 h, and then purified on a G25M column (CV = 55 mL) pre-equilibrated with Buffer 4. 18 mL of the conjugate solution was collected and then concentrated to 4.0 mL (concentration = 5.0 mg / mL) on an Amicon stirred ultrafiltration vessel.

[0117] Preparation of XLZ1-C5-Natural G6PDH Couplings

[0118] G6PDH enzyme (24 mg, approximately 2 mL) was centrifuged at 9.0 g at 4°C for 30 minutes on a benchtop centrifuge. The supernatant was discarded, and the white filter cake at the bottom was dissolved in Buffer 3 (3 mL). The resulting clear solution was then transferred to a dialysis bag and the buffer was exchanged with Buffer 3 (3 x 500 mL) in a refrigerated room. After dialysis, 3 mL of enzyme solution was recovered; the concentration was measured at 40x dilution and found to be 7.5 mg / mL. The concentration was then adjusted to 5 mg / mL by adding Buffer 3 (1.5 mL); the concentration was measured again at 40x dilution and found to be 5.1 mg / mL.

[0119] Cool the enzyme solution on an ice bath, then add G6PNa2 (30 mg). Slowly mix the resulting reaction mixture until the salt dissolves, then add β-NADH (40 mg). Mix the solution until all solids dissolve.

[0120] G6PDH enzyme (0.98 mL, 5 mg) was placed in four test tubes, and XLZ1-C5-OSu hapten (6) (25 mg / mL in DMF) was added to each tube in excess at 5 x, 10 x, 15 x, and 20 x molar amounts. The resulting reaction mixture was shaken at room temperature (18.2 °C) for 90 min, and then quenched with an excess of 30 x molar L-lysine (1.0 M) relative to the hapten. The conjugates were then purified using buffer 5 on a G50-M column (CV = 60 mL). 15 mL of each conjugate was collected and concentrated to approximately 3.0 mL; the concentration of each conjugate was 1.1 to 1.5 mg / mL.

[0121] Table 1

[0122] Preparation of XLZ1-C8-OVA conjugate (14a) In a glass vial equipped with a magnetic stir bar and cap, OVA (10 mg) was suspended in Buffer 2 (2 mL), followed by the dropwise addition of XLZ-C8-OSu (10) (2.23 mg, 110 µL, equivalent to a 20 x mol excess). The resulting reaction mixture was stirred at room temperature for 16 h, and then purified on a G25M column (CV = 60 mL) pre-equilibrated with Buffer 4. 15 mL of the conjugate solution was collected and concentrated to 7.0 mL (concentration = 2.5 mg / mL) using an Amicon stirred ultrafiltration cup MW CO2 10 kDa.

[0123] Preparation of XLZ1-C8-BSA immunogen (14b):In a glass vial equipped with a magnetic stir bar and cap, BSA (20 mg) was dissolved in Buffer 2 (3 mL) to prepare a 7 mg / mL solution. The resulting mixture was cooled to 4 °C on an ice bath, and then hapten-DMF solution (160 µL, 4.01 mg, 25 x mol excess) was added dropwise. The resulting reaction mixture was stirred at room temperature for 16 h, and then purified on a G25M-dextran gel column (CV = 55 mL) using Buffer 4. A 14 mL volume of the conjugate solution was collected and concentrated to 5.0 mL (concentration = 3.71 mg / mL) using an Amicon stirred ultrafiltration cup (MW CO 10000 Da).

[0124] Preparation of XLZ1-C8-KLH immunogen (14c): In a glass vial equipped with a magnetic stir bar and cap, KLH (20 mg) was dissolved in Buffer 2 (3 mL) to prepare a 7 mg / mL solution. The resulting mixture was cooled to 4°C on an ice bath, and then hapten-DMF solution (160 µL, 4.01 mg) was added dropwise. The resulting reaction mixture was stirred at room temperature for 16 h, and then purified on a G25M-dextran gel column (CV = 70 mL) using Buffer 4. A 14 mL volume of the conjugate solution was collected and concentrated to 5.0 mL (concentration = 4.0 mg / mL) using an Amicon stirred ultrafiltration cup (MW CO 10.000 Da).

[0125] Preparation of XLZ1-C8-natural G6PDH conjugate (15a-d): G6PDH enzyme (3.8 mL, 40 mg) was centrifuged at 9.0 g for 30 minutes at 4°C on a benchtop centrifuge; the supernatant was discarded, and the white filter cake at the bottom was dissolved in Buffer 3 (5 mL). The resulting clear solution was then transferred to a dialysis bag and the buffer was exchanged with Buffer 3 (3 x 500 mL) in a refrigerated room. After dialysis, 5 mL of enzyme solution was recovered; the concentration was measured at a 40x dilution and found to be 7.71 mg / mL. The concentration was then adjusted to 5 mg / mL by adding Buffer 3 (1.5 mL).

[0126] Cool the enzyme solution on an ice bath, then add G6PNa2 (50 mg) and gently mix the resulting reaction mixture until all substrate is dissolved; then add β-NADH (50 mg). Mix the solution until all solids are dissolved.

[0127] G6PDH enzyme solution (5 mg) was placed in four test tubes, and then XLZ1-C8-OSu hapten (10) (20 mg / mL hapten in DMF solution) was added to each tube in excess at 5 x, 10 x, 15 x, and 20 x molar amounts. The resulting reaction mixture was shaken at room temperature (18.2 °C) for 90 min, and then quenched with an excess of 30 x molar L-lysine (1.0 M). The conjugates were then purified using buffer 5 on a G25-M column (CV = 60 mL). Approximately 13 mL of each conjugate was collected (Table 2).

[0128] Table 2

[0129] Example 4: Synthesis of XLZ1-C3SAc 3K G6PDH conjugate ( Figure 5 )

[0130] Preparation of XLZ1-C3-AcBr hapten (16) In a vial equipped with a magnetic stir bar and cap, XLZ-C3-NH2(8) (40 mg, 0.102 mmol) was dissolved in degassed DMF (200 µL), followed by the addition of NEt3 (30 µL, 2 equivalents). The resulting mixture was cooled on an ice bath, and then BrAcSu solution (30 mg, 1.2 equivalents, dissolved in 200 µL THF) was added dropwise over 2 minutes. The resulting reaction mixture was warmed to room temperature and stirred for 1 h. LC-MS confirmed the formation of the product. The calculated m / z values ​​for C17H25BrN3OS+ were 400.09 and 398.09, and the measured values ​​were 400.09 and 398.12 (as two major Br isotopes). The crude reaction mixture was injected into a Shimadzu preparative HPLC system packed with a Zorbax C18 column. The fraction containing the product was collected, concentrated, and freeze-dried to give 47 mg of brown oily product 16 (0.091 mmol of TFA salt, yield 90%).

[0131] Solvent A: Water + 0.1% TFA, Solvent B: ACN + 0.1% TFA, UV detection 254 nm.

[0132] LC purification procedure: 0-2 minutes isocratic solvent B 10%, 2-28 minutes gradient solvent B 70%, 28-30 minutes isocratic solution B 70%, 30-33 minutes gradient solvent A 10%, stop collection at 34 minutes.

[0133] Preparation of XLZ1-SC6-3K-G6PDH conjugate (17) Figure 5 ): Place 3K G6PDH enzyme (50 mg, approximately 5 mL) into a dialysis bag and exchange the buffer with buffer 6 (2 x 550 mL) in a refrigerated room. Then adjust the concentration to 5 mg / mL by adding buffer 6.

[0134] Reduction of 3K-G6PDH enzyme: 3K-G6P1H enzyme (5 mg / mL, 1.1 mL) was loaded into a plastic tube, and then DTT (12 µL, 0.5 M solution) was added. The resulting reaction mixture was covered with argon gas and then shaken in a refrigerator for 16 h. The next day, the buffer was exchanged with buffer 7 on an Akta Starter fitted with G25 2 x 5 mL HiTrap columns in a refrigerator. Fractions containing reduced enzyme were collected (1.90 mg / mL, 2 mL, 3.80 mg).

[0135] In a separate vial, XLZ1-C3-AcBr hapten 16 (3 mg) was dissolved in degassed DMF (300 µL), and then 0.89 µL of this solution (equivalent to a 50 x hapten molar excess) was added dropwise to reduced 3K G6PDH enzyme on an ice bath. The resulting reaction mixture was covered with argon and stirred in a refrigerator for 16 h.

[0136] The reaction mixture was then purified on a G25M column (CV = 55 mL) using buffer 4. A 15 mL fraction of the conjugate was collected and concentrated to 2.1 mL (1.5 mg / mL) using an Amicon stirred ultrafiltration cup MW CO 10000.

[0137] Preparation of XLZ1-C11-MAL hapten (18) In a vial equipped with a magnetic stir bar and cap, XLZ-C3-NH2 (102 mg, 0.26 mmol, MW 391.45, a TFA salt) was dissolved in DMF (400 µL), followed by the addition of NEt3 (70 µL). GMBS (73 mg, 0.26 mmol, MW 280) was dissolved in DMF (200 µL) and added dropwise. The resulting reaction mixture was stirred at room temperature and, after 20 minutes, analyzed by uPLC. The reaction mixture was then diluted with 1.1 mL of ACN / water (1 / 1 v / v) and injected into a preparative LC.

[0138] Solvent A: Water + 0.1% AcOH, Solvent B: Acetonitrile + 0.1% AcOH.

[0139] LC purification procedure: 0-1 min isocratic solvent A 10%, 1-20 min gradient solvent B 80%, 20-24 min isocratic solution B 80%, 24-28 min gradient solution B 10%, stop collection at 28.01.

[0140] The product was eluted to a sharp peak at 9 minutes, fractions 7 to 9 were collected, concentrated and lyophilized to give 70.2 mg of colorless powder (0.159 mmol, 61% yield).

[0141] The product is packaged in 5 vials: vial 1 - 15.8 mg, vial 2 - 15.3 mg, vial 3 - 15.5 mg, vial 4 - 15.1 mg, and vial 5 - 8.5 mg.

[0142] [C 23 H 30 N4O3S +1 The calculated MS value was 443.21, and the measured value was 443.68 (retention time 2.473 minutes).

[0143] 1 H NMR (600 MHz, CDCl3) -d ): 7.78 (s, 1H), 7.28 - 7.26 (m, 1H), 7.14 (d, J = 7.59Hz, 2H), 6.66 (s, 2H), 3.92- 3.89 (m, 2H), 3.81 - 3.79 (m, 2H), 3.55 (t, J = 6.79 Hz, 2H), 3.27 (q, J = 5.76 Hz, 2H), 3.05 - 3.03 (m, 2H), 2.23 - 2.16 (9m, 6H), 2.15 - 2.14 (m, 2H), 2.06 (s, 1H), 1.75 -1.73 (m, 2H).

[0144] Preparation of XLZ1-C11-3K G6PDH conjugate (19): The 3K G6PDH enzyme was reduced according to the procedure described in conjugate 17. 3K G6PDH enzyme (1.1 mL, 5 mg / mL) was placed in a plastic tube, and then DTT (11 µL, 0.5 M) was added. The resulting reaction mixture was covered with argon gas and shaken in a refrigerator for 16 h.

[0145] The next day, the reduced enzyme was exchanged with buffer 7 on a G25-M dextran gel column (CV = 28 mL). A 7 mL volume of the reduced enzyme was collected and concentrated to approximately 1.5 mL using an Amicon stirred ultrafiltration vessel (30000 MW CO2). A DMF (10 mg / mL) solution of XLZ-C11-MAL was prepared in a separate vial. The enzyme was cooled on ice and then 46 µL of the hapten solution was added dropwise. The resulting reaction mixture was covered with argon and stirred overnight (16 h) in a refrigerator (2–8 °C) with stirring, and then purified on a G25-M dextran gel column (CV = 40 mL) using buffer 4. 16 mL volumes of each conjugate were collected and concentrated to 2.80 mL (1.31 mg / mL) using an Amicon stirred ultrafiltration vessel (MW CO2 30000 cutoff).

[0146] Example 5: Synthesis of XLZ2-O4-C2S2 hapten ( Figure 6 )

[0147] Preparation of HO-4-XLZ2-Boc(20): In a vial equipped with a magnetic stir bar and cap, 137 mg of 4-hydroxytoluidine (0.58 mmol) was suspended in 0.5 mL of DMF, followed by the addition of NEt3 (cat, 20 µL) and Boc2O / THF (290 µL, 1 equivalent of 2THF solution). The resulting reaction mixture was stirred for 4 hours, then diluted with EtOAc (30 mL) and washed with deionized water (2 x 20 mL). The organic layer was evaporated to dryness to give 196 mg of product. [C] 17 H 24 N2O3S +1 The calculated MS value was 337.16, the measured value was 280 (as the major fragment, Boc cleavage), and 337.12. The product was then purified by Shimadzu HPLC.

[0148] Solvent A: Water + 0.1% AcOH, Solvent B: Acetonitrile + 0.1% AcOH

[0149] LC purification method: 0-1 min isocratic solvent B 10%, 1-20 min gradient solvent B 80%, 20-24 min isocratic solution B 80%, 24-28 min gradient solvent B 10%. The product was eluted with a broad peak for 14-18 min, the fraction containing the product was collected, concentrated and lyophilized to give 108 mg of white crystalline compound 20.

[0150] 1 H NMR (600 MHz, CDCl3) -d ): 6.47 (s, 2H), 4.7 (width s, 1H), 3.79 (t, J= 7.2 Hz, 2H), 2.75 (t, J = 6.0 Hz, 2H), 2.07 - 2.03 (m, 8H), 1.48 (s, 9H).

[0151] Preparation of BocXLZ2-O4-C2S2(21): In a vial equipped with a magnetic stir bar and cap, HO-4-XLZ2-Bc (20) (85 mg, 0.253 mmol) was dissolved in DMF (0.5 mL), followed by the addition of K2CO3 (80 mg, 0.58 mmol), and then dropwise the addition of a BrAcS2 linker solution (44 mg, 0.11 mmol in 0.2 mL of DMF). The resulting reaction mixture was stirred at room temperature for 2 days and then further heated to 60 °C for 3 days. The crude mixture was extracted with EtOAc (20 mg) and washed with deionized water (2 x 20 mL). The organic layer was concentrated on a rotary evaporator and purified by LC column chromatography over 15 min with hexane / ethyl acetate 1:0 to 1 / 1 v / v to remove nonpolar byproducts. The column was further washed with EtOAc / MeOH 9 / 1 v / v (20 mL). A yellow oily compound (21) (50 mg, 0.055 mmol, 44% yield) was given. [C 42 H 60 N6O8S4Na + The calculated mass value is 927.33, and the measured mass value is 927.51.

[0152] 1 H NMR (600 MHz, CDCl3) 7.07 (t, J = 5.97 Hz, 2H), 6.62 (s, 4H), 4.46 (s, 4H), 3.87- 3.83 (m, 4H), 3.66 (q, J = 6.37 Hz, 4H), 2.85 (t, J = 6.44 Hz, 4H), 2.81 (t, J = 6.68Hz, 4H), 2.13 (s, 12H), 2.12 – 2.10 (m, 4H), 1.55 (s, 18H).

[0153] Preparation of XLZ2-O4-C2S2(22): Compound (21) was then deprotected in DCM (0.5 mL) and TFA (0.1 mL) for 4 hours and purified by reversed-phase HPLC with a C18 column using solvent A: water + 0.1% AcOH and solvent B + 0.1% AcOH, monitored at 254 and 260 nm, at a flow rate of 7.00 mL / mL.

[0154] HPLC purification procedure: 0-2 min isocratic solvent B 10%, 2-15 min gradient solvent B 70%, 15-17 min isocratic solution B 70%, 17-19 min gradient solvent B 10%, 19-22 min gradient solvent B 10%. Elute the product over 8-10 min.

[0155] After purification, 37.5 mg (0.053 mmol, 96% yield) of a light yellow powder, XLZ2-O4-C2-S2 hapten (13), was isolated. [C 32 H 44 N6O4S4H + The calculated mass was 705.99, the measured mass was 705.40, and the [MNa+] mass was 727.39, with a major fragmentation mass of 353.44 [M+2 / 2]. (2.987 and 2.98 minutes)

[0156] 1 H NMR (600 MHz, CD3CN-) d 3 7.34 (t, J = 5.71 Hz, 2H), 6.74 (s, 4H), 4.42 (s, 4H), 3.51 (q, J = 6.52 Hz, 4H), 3.46 - 3.44 (m, 5H), 3.04 - 3.02 (m, 4H), 2.84 (t, J = 6.67Hz, 4H), 2.19 - 2.17 (m, 12H), 2.08 - 2.06 (m, 4H), 1.86 (s, 2H).

[0157] AcONa buffer preparation - 25 mM is prepared using 75 µL acetic acid in 50 mL deionized water, and then NaOH is added to adjust the pH to 4.25-4.30.

[0158] Hapten activation (23): In a glass vial equipped with a magnetic stir bar and cap, XLZ2-O4-C2-S2(22) (9 mg, 0.0127 mmol) was dissolved in EtOH (200 µL), followed by the addition of AcONa buffer (200 µL), and then 4 mg of TCEP. HCl. The resulting reaction mixture was covered with Ar and stirred for 1 h. The formation of the reduction product was confirmed by UPLC-MS. [C 16 H 24 N3O2S2 +The calculated MS value for compound 14 was 354.13, the measured value was 354.15, and the retention time was 2.33 minutes (the retention time of the starting disulfide was 2.53 minutes). Compound 14 was freshly prepared before use in bioconjugation.

[0159] Example 6: Bioconjugation method of XLZ2-O4-C2SAc-immunogen and G6PDH conjugate ( Figure 7 )

[0160] BrAcSu (8 mg, 0.033 mmol) was dissolved in DMF (400 µL) to prepare a 20 mg / mL solution.

[0161] XLZ2 Immunogen Synthesis - Protein Activation: BrAcOVA protein activation (24a) In a vial equipped with a magnetic stir bar and cap, suspend OVA (15 mg) in Buffer 2 (2 mL), then place the protein solution in the refrigerator and add BrAcSu (1.65 mg 74 µL, 20x molar excess). Stir the resulting reaction mixture for 90 minutes, then dialyze against Buffer 8 (2 x 250 mL) in the refrigerator. The next day, transfer the protein to a vial equipped with a stir bar and cap.

[0162] Preparation of XLZ2-O4-C2SAc-OVA (25a): Activated hapten 23 (2.47 mg, 110 µL, 20 x excess) was added dropwise to the protein solution. The resulting reaction mixture was covered with argon gas, stirred on an ice bath, heated to room temperature, and stirred for 16–20 h. The conjugate was then purified with buffer 4 on a dextran gel G25-M column; 15 mL of the conjugate fraction was collected and concentrated to approximately 6.5 mL (2.42 mg / mL) using an Amicon stirred ultrafiltration vessel. The conjugate was then filtered out through a CA filter (0.20 µm).

[0163] BrAcBSA protein activation (24b): In a vial equipped with a magnetic stir bar and cap, BSA (20 mg) was suspended in Buffer 2 (3 mL). The protein solution was then placed in the refrigerator, and BrAcSu (2.81 mg 125 µL, 40x mol excess) was added dropwise. The resulting reaction mixture was stirred in the refrigerator for 90 minutes, and then dialyzed against Buffer 8 (2 x 250 mL) in the refrigerator. The next day, the protein was transferred to a vial equipped with a stir bar and cap.

[0164] Preparation of XLZ2-O4-C2SAc-BSA(25b):Activated hapten 23 (3.66 mg, 163 µL, 40 x excess) was added dropwise to the protein solution. The resulting reaction mixture was covered with argon gas, stirred on an ice bath, heated to room temperature, and stirred for 16–20 h. The conjugate was then purified with buffer 4 on a dextran gel G25-M column, and 15 mL of the conjugate was collected and concentrated to approximately 8.0 mL (2.60 mg / mL) using an Amicon stirred ultrafiltration vessel. The conjugate was then filtered out through a CA filter (0.20 µm).

[0165] BrAcKLH protein activation (24c): In a vial equipped with a magnetic stir bar and cap, KLH (20 mg) was suspended in Buffer 2 (3 mL), and the protein solution was placed in the refrigerator. BrAcSu (2.23 mg 100 µL) was then added dropwise. The resulting reaction mixture was stirred for 90 minutes and then dialyzed against Buffer 8 (2 x 250 mL) in the refrigerator. The next day, the protein was transferred to a vial equipped with a stir bar and cap.

[0166] Preparation of XLZ2-O4-C2SAc-KLH(25c): Activated hapten 23 (100 µL) was added dropwise to the protein solution. The resulting reaction mixture was covered with argon gas, stirred on an ice bath, heated to room temperature, and stirred for 16–20 h. The conjugate was then purified with buffer 4 on a dextran gel G25-M column, and a fraction of 15 mL of immunogen solution was collected and concentrated to approximately 5.5 mL (3.60 mg / mL) using an Amicon stirred ultrafiltration vessel. The conjugate was then filtered out through a CA filter (0.20 µm).

[0167] Preparation of XLZ2-O4-C2SAc-G6PDH conjugate (27a-e): The G6PDH enzyme suspension (3 mL, 30 mg) was centrifuged at 40 °C to 9 g for 40 minutes. The supernatant was discarded; the resulting white precipitate was dissolved in buffer 1 (3.5 mL), and then dialyzed against buffer 1 (2 x 250 mL); 3.5 mL of enzyme at a concentration of 8.33 mg / mL was recovered.

[0168] Cool the enzyme on an ice bath, then add G6PDNa (30 mg) and gently mix the mixture until all solids dissolve; then add β-NADH (30 mg) and gently mix the mixture until all solids dissolve.

[0169] Transfer the reaction mixture to a refrigerated container. Add BrAcSu (1.62 mg, 80 µL, 30 x mol excess, 6 mg dissolved in 300 µL DMF). Stir the resulting reaction mixture in the refrigerated container for 16 h, then transfer it to a dialysis bag and exchange the buffer with Buffer 1 (2 x 250) in the refrigerated container. The next day, purify the activated BrAcG6PDH enzyme using a G-25 M column (CV = 55 mL); collect 15 mL of BrAcG6PDH enzyme (26) and concentrate it to 3.3 mL (concentration = 7.67 mg / mL) on an Amicon stirred ultrafiltration cup. Then adjust the concentration to 5 mg / mL using Buffer 1 (1.6 mL).

[0170] For the second batch hapten activation of natural G6PDH conjugates: In a vial equipped with a magnetic stir bar and cap, XLZ2-O4-C2-S2(23) (2.0 mg) was dissolved in EtOH (150 µL), followed by the addition of 25 mM NaOAc (100 µL) at pH 4.3, and then TCEP. HCl (0.81 mg, 28 µL, from 5.8 mg in 200 µL NaOAc buffer). The resulting reaction mixture was covered with argon and stirred at room temperature for 1 h, but uPLC only completed 75% of the thiol reduction; then an additional volume of 28 µL TCEP was added. The reaction mixture was covered with argon and then placed in a refrigerator and stirred overnight. The reaction was complete the next day (6.5 mg / mL activated hapten 24 solution).

[0171] The BrAcG6PDH activating enzyme was then placed in five vials (4 to 5 mg of BrAcG6PDH enzyme per vial) (Table 3); the enzyme solution was then cooled on an ice bath. An activated hapten (14) at a molar ratio of 5 to 25 x to the enzyme was then added dropwise to each vial. The resulting reaction mixture was covered with argon gas and placed in a refrigerator with stirring for 16 h. The next day, the resulting conjugate was purified using buffer 4 on a G25-M column (CV = 55 mL); the amount in column 6 was recovered (Table 3).

[0172] Table 3

[0173] Example 7

[0174] Synthesis of XLZ2-Boc-O4-C8Br (29) Figure 8 ):In a vial equipped with a magnetic stir bar and cap, N,N′-1,2-ethylenedimethylbis[2-bromoacetamide] linker (28) (80 mg, 0.266 mmol, 1.5 equivalents) was dissolved in DMF (0.4 mL), followed by the addition of K2CO3 (50 mg, 0.361 mmol). The resulting mixture was stirred for 5 minutes, followed by the dropwise addition of a DMF solution of HO-4-XLZ2-Bc (60 mg, 0.178 mmol, 0.6 mL DMF) over 4 h. The resulting reaction mixture was stirred at 40 °C for 4 days. The reaction mixture yielded a mixture of approximately 50% of the single reaction product (the desired product) and approximately 20% of the second reaction byproducts. The volatiles were removed on a rotary evaporator. The resulting residue was dissolved in EtOAc (20 mL) and washed with a saturated solution of sodium dihydrogen phosphate (20 mL). The organic layer was concentrated on a rotary evaporator to give a crude oil, which was further purified by reverse-phase HPLC using a C18 column. [C 23 H 33 BrN4O5S +1 The calculated MS values ​​for ]+ are 557.14 and 559.14, while the measured values ​​are 557.38 and 559.39.

[0175] LC purification procedure: 0–1 min isocratic solvent B 10%, 1–20 min gradient solvent B 80%, 20–24 min isocratic solution B 80%, 24–28 min gradient solution B 10%. The product was eluted over 16–19 min, the 20–24 min fraction was collected, concentrated and lyophilized to give 50 mg of white powder (0.089 mmol, 50% yield).

[0176] 1 H NMR (600 MHz, MeOD) -d 4 : 6.70 (s, 2H), 4.85 (s, 8H water peak), 4.44 (s, 2H), 3.84-3.82 (s, 5H), 3.44-3.37 (m, 4H), 3.31 (s, 5H), 2.12 (s, 6H), 1.54 (s, 9H).

[0177] Synthesis of XZ2-O4-C8Br hapten (30)In a glass vial equipped with a stir bar, XLZ2Boc-O4-C8Br (29) (50 mg, 0.089 mol) was suspended in EtOAc (300 µL). In another vial, acetyl bromide (80 µL, 132 mg, 1.09 mmol) was added dropwise to EtOAc / MeOH (1 / 1 v / v 300 µL) on an ice bath. The acetyl bromide was allowed to react with methanol for 10 min. Then, a Boc-protected hapten-EtOAc solution (50 mg) was added dropwise. The resulting reaction mixture was stirred for 4 h. The reaction mixture was concentrated on a rotary evaporator to give 40 mg (0.088 mmol, quantitative yield) of a pale yellow oily XLZ2-O4-C8Br hapten (30). [C 18 H 25 BrN4O3S +1 The calculated MS values ​​for ]+ are 457.09 and 459.09, while the measured values ​​are 457.29 and 459.29.

[0178] Synthesis of XLZ2Boc-O4-C2NHBoc(31) HO-4XZL2-Boc (50 mg, 0.149 mmol, dissolved in 0.5 mL DMF) was placed in a vial, followed by K2CO3 (70 mg, 0.506 mmol), then 2-(Boc-amino)ethyl bromide linker (66.7 mg, 0.29 mmol) and KI (approximately 20 mg). The resulting reaction mixture was capped with Ar, heated to 50 °C for 3 days. Volatile substances were removed under vacuum to obtain a brown oily substance, which was dissolved in EtOAc (20 mL) and washed with deionized water (2 x 20 mL). The organic layer was then concentrated and suspended in ACN (2 mL) and purified by preparative LC to give 60 mg XLZ2Boc-O4-C2NHBoc (0.125 mmol), in 84% yield.

[0179] Solvent A: Water + 0.1% AcOH, Solvent B: Acetonitrile + 0.1% AcOH

[0180] Purification by LC: 0-1 min isocratic solvent B 15%, 1-20 min gradient solvent B 70%, 20-26 min isocratic solution B 70%. The product was eluted in 70% solvent B (23-25 ​​min). The fraction containing the product was collected, concentrated, and lyophilized overnight to give 60 mg of a white powder (0.125 mmol, 84% yield).

[0181] [C 24 H 37 N3OS +1 The calculated MS value for ]+ is 480.25, and the measured value is 480.48.

[0182] 1 H NMR (600 MHz, CDCl3) -d ): 6.58 (s, 2H), 3.98 - 3.96 (m, 2H), 3.85 (t, J = 6.84 Hz, 2H), 3.50 - 3.49 (m, 2H), 2.80 (t, J = 6.57 Hz, 2H), 2.12 (m, 8H), 1.54 (s, 9H), 1.44 (s, 9H).

[0183] Synthesis of XLZ2-O4-C2NH2(32) XLZ2BocO4-C2NHBoc 31 (60 mg, 0.125 mmol) was dissolved in DCM / TFA (0.3 mL, 1 / 1 v / v) and stirred for 16 h. Boc deprotection was confirmed by uHPLC. Volatiles were removed on a rotary evaporator to give 55 mg of XLZ2-O-C2NH2 in the form of TFA salt (0.108 mmol, 86% yield). This intermediate could be used for the next step without further purification. [C] 14 H 22 N3OS +1 The calculated MS value for ]+ is 280.15, and the measured value is 280.29 (retention time 0.510 minutes).

[0184] Synthesis of XLZ2-O4-C6Br hapten (33) : XLZ2-O-C2NH2 TFA (55 mg, 0.108 mmol) was dissolved in DMF (0.8 mL), and then NEt3 (45 µL, 3 equivalents) was added. The pH of the reaction solution was approximately 8 as measured by pH paper. The resulting reaction mixture was cooled on an ice bath, and then BrAcSu (30 mg, 0.127 mmol, 250 µL of 120 mg / mL BrAcSu-DMF solution) was added dropwise over 5 minutes, followed by the addition of additional NEt3 (20 µL). The reaction mixture was allowed to warm to room temperature; after 10 minutes, product formation was confirmed by uPLC. [C] 16 H 22 BrN3O2S +1 MS calculated value of 401.34+ ACN (41), measured value of acetonitrile adduct 442.27, retention time (2.37 min).

[0185] The product was purified by preparative LC. The product was eluted with a broad peak, and the fraction containing the product was collected, concentrated, and lyophilized overnight to give 10 mg of product (0.025 mmol), with a yield of 20%.

[0186] Solvent A: Water + 0.1% TFA, Solvent B: Acetonitrile

[0187] Purification LC program: 0-2 min isocratic solvent B 10%, 2-20 min gradient solvent B 60%, 20-23 min isocratic solution B 60%, 23-27 min gradient solvent A 10%, 27-28 min isocratic liquid B 10%. Elute the product with a broad peak for 7 to 14 min.

[0188] 1 H NMR (600 MHz CD3CN) -d 3 ): 6.82 (s, 2H), 4.59 (t, s, 2H), 4.07 (t, J =5.42 Hz (2H), 3.84 - 3.82 (m, 4H), 3.58 - 3.56 (m, 2H), 3.33 - 3.31 (m, 2H), 2.57 (s, 3H), 2.21 (s, 6H). Separated into acetate.

[0189] Example 8: Preparation of XLZ2-O4-C8-3K G6PDH (34) and XLZ2-O4-C6-3K G6PDH (35) (Figure) 9）： 3K G6PDH enzyme (1 mL, 10.8 mg / mL) was placed in a dialysis bag and the buffer was exchanged with buffer 6 (2 x 250 mL, 4 h interval) in a refrigerated room. The concentration was then adjusted to 5 mg / mL using buffer 6. The final volume of the enzyme solution was 2 mL.

[0190] Enzyme reduction: In a plastic tube, place 2 mL of 3K G6PDH enzyme solution, followed by 20 µL of DTT (0.5 M solution). Cover the resulting reaction mixture with Ar and agitate in the refrigerator for 16 h. The next day, degas Buffer 7 in the refrigerator for 30 min, then dialyze the enzyme with Buffer 7 (250 mL) in the refrigerator, followed by exchanging the buffer with Buffer 7 using a Sephadex G25M column (CV = 28 mL). After column chromatography, recover 7 mL of enzyme and concentrate it to 2.6 mL (c = 5.13 mg / mL) on an Amicon stirred ultrafiltration flask (MW CO30000 Da). The enzyme solution is then placed in two vials (4 mg enzyme each), covered with Ar, and cooled on an ice bath.

[0191] The hapten was then dissolved in DMF to prepare a 10 mg / mL solution. For this purpose, the XLZ2-O4-C6-Br hapten (1.22 mg) was dissolved in DMF (0.122 mL), and the XLZ2-O4-C8-Br hemiprotein (1.40 mg) was dissolved in DMF (0.140 mL).

[0192] Each hapten-DMF solution was then added dropwise at a 40 x mol excess to the corresponding reducing enzyme (4 mg each) (Column 2 of Table 4); the resulting reaction mixture was covered with Ar and stirred overnight in a refrigerator. The conjugates were then purified using buffer 4 through a Sephadex G25M column (CV 40 mL), filtered through a Corning CA 0.20 µm filter, and concentrated to the volume shown in column 3 of Table 4.

[0193] Table 4

[0194] Example 9

[0195] Figure 10-11 Further embodiments comprising certain non-limiting implementations of the synthesis of toluenethiazide hapten / immunogen / conjugate according to this disclosure. Non-limiting examples of “proteins” that can be conjugated thereto include KLH, BSA, OVA, G6PDH, and any carrier and / or labeled protein.

[0196] Preparation of XLZ3-CH2-NH2(39) Figure 12 ): Synthesis of XLZ3-CH2-NH2 (39): 1,1-Dimethylethyl N-[3-amino-2-(hydroxymethyl)propyl]carbamate (37) (125 mg, 0.61 mmol) was dissolved in THF (620 µL). The solution was cooled on an ice bath, and then 2,6-dimethylphenyl isothiocyanate (38) (100 µL, 101 mg, 0.98 equivalent 0.6 mmol) was added dropwise over 5 minutes. The resulting reaction mixture was warmed to room temperature and stirred for 3 h. Volatiles were removed on a rotary evaporator, and then deionized water (200 µL) was added, followed by HCl (10%, 600 µL). The resulting reaction mixture was refluxed for 2 h. The formation of XLZ3-CH2NH2 (39) was confirmed by uPLC. [C 13 H 20 N3S + The calculated m / z value was 250.14, and the measured value was 250.13. NaOH (5 N) was then added dropwise until the pH was approximately 10, followed by extraction of intermediate 39 with ethyl acetate (2 x 20 mL). The solvent was removed on a rotary evaporator to give 160 mg (0.64 mmol) of crude 39. Compound 39 was too polar to be purified by normal-phase or reverse-phase methods.

[0197] Compound 39 (160 mg, 0.64 mmol) was suspended in DMF (500 µL), followed by the addition of Boc2O (640 µL, 2 equivalents, 1.28 mmol, 2 M THF solution) and NET3 (93 µL, 0.66 mmol). The resulting reaction mixture was stirred for 4 h to form Boc-XLZ3-CH2-NHBoc(40). [C 23 H 36 N3O4S + The calculated m / z value of [Na] was 472.24, and the measured value was 472.31. Intermediate 40 was then purified by LC using ethyl acetate / hexane to give 201 mg (0.45 mmol) of colorless oily Boc-XLZ3-CH2-NHBoc, with a yield of 74%.

[0198] 1H NMR (600 MHz, CDCl3-d) 6.95 (d, J = 7.01 Hz, 2H), 6.86 (t, J = 7.48 Hz, 1H), 4.96 (s, 1H), 3.74 - 3.66 (m, 2H), 3.16 - 3.06 (m, 2H), 2.76 - 2.72 (m, 1H), 2.56 - 2.52 (m, 1H), 2.44 - 2.42 (m, 1H), 2.08 (s, 3H), 2.05 (s, 3H), 1.48 (s, 9H), 1.45 (s, 9H).

[0199] Boc-XLZ3-CH2-NHBoc(40) (201 mg, 0.45 mmol) was then suspended in 1 mL of DCM / TFA 1 / 1 v / v and stirred overnight. The volatiles were removed under vacuum to give a pale yellow oily 39 TFA (120 mg, 0.33 mmol) (as a TFA salt).

[0200] Synthesis of XLZ3-C7-S2 hapten (42) formula XXXV Figure 12 ): Compound 39 TFA (35 mg, 0.096 mmol) was dissolved in DMF (500 µL), NEt3 (41 µL, 3 equivalents) was added, followed by 4-butyrolactone (41) (15 µL, 1.5 equivalents). The resulting reaction mixture was heated at 60 °C for 3 days. Volatiles were removed under vacuum, followed by the addition of EtOH (200 µL), and then a catalytic amount of I2. The resulting reaction mixture was heated to 40 °C for 3 h, and then purified by reversed-phase LC to give 13 mg of white crystals XLZ3-C7-S2 (42) in a yield of 43%. [C 34 H 49 N6O2S 4+The calculated value of m / z is 701.28, and the measured value is [m+2 / 2] = 351.49.

[0201] Solvent A: Water + 0.1% AcOH, Solvent B: ACN + 0.1% AcOH, UV detector at 237 nm.

[0202] LC purification program: 0-2 minutes isocratic solvent B 10%; 2-20 minutes gradient solvent B 70%; 20-24 minutes gradient solvent A 70%; 24-28 minutes isocratic solvent B 10%.

[0203] 1H NMR (600 MHz, CD3CN-d3) 8.40 (s, 1H), 7.11 - 7.68 (m, 6H), 6.80 (s, 2H), 3.46 (dd, J = 13.35, 2.9 Hz, 2H), 3.26 (t, J = 6.41 Hz, 4H), 3.18 (dd, J = 13.3, 8.4 Hz, 2H), 3.05-3.02 (m, 2H), 2.88 (dd, J = 12.41, 8.82 Hz, 2H), 2.74 (t, J = 7.3 Hz, 4H), 2.28 -2.26 (m, 6H), 2.19-2.18 (m, 12H), 1.95 -1.93 (m, 6H).

[0204] Bioconjugation of the XLZ3-C7-S2 hapten (42, formula XXXV) has been performed according to a similar procedure previously ( Figure 7 ) proceed, and obtain as follows Figure 12 The XXXVI coupling shown is an example.

[0205] Non-limiting exemplary implementation

[0206] Exemplary Embodiment 1. A composition comprising a compound having the structure of Formula I:

[0207] The condition is that at least one of R1, R2, and R3 is not H.

[0208] Exemplary Embodiment 2. The composition according to Exemplary Embodiment 1, wherein at least one of R1, R2 and R3 that is not H comprises at least one of aliphatic group, hydrocarbon group, bulky organic group, non-bulky organic group, alkyl, alkenyl, alkynyl, phenyl, aryl, substituted aryl, carbocyclic, heterocyclic, hydrocarbon carbonyloxy, lower alkoxy, aryl hydrocarbon, halide, amide, imide, nitrate, nitrite, carbonyl, ketone, ester, carboxylic acid, hydroxide, thiol, sulfide, sulfate, sulfonate, peptide, protein, enzyme, linker, immunogenic carrier, label, or combination thereof.

[0209] Exemplary Embodiment 3. The composition according to Exemplary Embodiment 1 or 2, wherein the compound comprises a structure of one of Formula II, III, IV, V, XI, XII, XIII, XIV, XV, XVII, XVIII, XIX, XXII, or XXIX: .

[0210] Exemplary Embodiment 4. A composition according to any one of Exemplary Embodiments 1-3, wherein the compound has a structure of one of Formulas VI, VII, VIII, IX, XX, XXI, XXIII or XXX:

[0211] R4 is a peptide or protein.

[0212] Exemplary Embodiment 5. The composition according to Exemplary Embodiment 4, wherein R4 is an immunogenic vector.

[0213] Exemplary Embodiment 6. The composition according to Exemplary Embodiment 5, wherein the immunogenic carrier is selected from the group consisting of: keyhole cyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), egg ovalbumin, bovine gamma globulin (BGG), thyroglobulin, fibrinogen, polysaccharide, glucose-6-phosphate dehydrogenase (G6PDH), carrier protein, marker protein, and combinations thereof.

[0214] Exemplary Implementation 7. A composition comprising a compound having the structure of Formula X:

[0215] One of R5 and R6 is present and includes at least one of the following: aliphatic group, hydrocarbon group, bulky organic group, non-bulky organic group, alkyl, alkenyl, alkynyl, phenyl, aryl, substituted aryl, carbocyclic, heterocyclic, hydrocarbon carbonyloxy, lower alkoxy, aryl hydrocarbon, halide, amide, imide, nitrate, nitrite, carbonyl, ketone, ester, carboxylic acid, hydroxide, thiol, sulfide, sulfate, sulfonate, peptide, protein, enzyme, linker, immunogenic carrier, label, or combination thereof.

[0216] Exemplary Embodiment 8. The composition according to Exemplary Embodiment 7, wherein the compound has a structure of one of the formulas XXIV, XXV, XXVI, XXVII, XXVIII, XXXI, XXXII, XXXIII, XXXIV, XXXV, or XXXVI:

[0217] R4 is a peptide or protein.

[0218] Exemplary Embodiment 9. The composition according to Exemplary Embodiment 7 or 8, wherein R5 or R6 comprises an immunogenic vector.

[0219] Exemplary Embodiment 10. The composition according to Exemplary Embodiment 9, wherein the immunogenic carrier is selected from the group consisting of: keyhole cyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), egg ovalbumin, bovine gamma globulin (BGG), thyroglobulin, fibrinogen, polysaccharide, glucose-6-phosphate dehydrogenase (G6PDH), carrier protein, labeling protein, and combinations thereof.

[0220] Exemplary Embodiment 11. The composition according to any one of Exemplary Embodiments 1-10 further comprises at least one excipient.

[0221] Exemplary Embodiment 12. A method for preparing an antibody, comprising the steps of: immunizing an animal that produces antibodies with an immunogen comprising a composition according to any one of Exemplary Embodiments 1-11; and isolating the antibody from the animal, wherein the antibody specifically binds to the immunogen.

[0222] Exemplary Implementation 13. The method according to Exemplary Implementation 12, wherein the step of isolating antibodies further comprises the following steps: isolating antibody-producing spleen cells from an antibody-producing animal; selecting at least one antibody-producing spleen cell that secretes a monoclonal antibody that specifically binds to an immunogen; and preparing a hybrid cell line by fusing the selected antibody-producing spleen cell from the antibody-producing animal with a suitable fusion partner, wherein the hybrid cell line secretes a monoclonal antibody.

[0223] Exemplary Embodiment 14. A coupling composition comprising: a composition according to any one of Exemplary Embodiments 1-11; and an enzyme coupled to the composition.

[0224] Exemplary Embodiment 15. The coupling composition according to Exemplary Embodiment 14, wherein the enzyme is selected from the group consisting of: glucose-6-phosphate dehydrogenase (G6PDH), alkaline phosphatase, β-galactosidase and horseradish peroxidase.

[0225] Exemplary Embodiment 16. The coupling composition according to Exemplary Embodiment 15, wherein the enzyme is G6PDH.

[0226] Exemplary Implementation 17. An antibody produced by the method according to Exemplary Implementation 12 or 13.

[0227] Exemplary Implementation 18. A kit comprising the antibody according to Exemplary Implementation 17.

[0228] Exemplary Embodiment 19. The kit according to Exemplary Embodiment 18 further comprises the coupling composition according to any one of Exemplary Embodiments 14-16.

[0229] Exemplary Implementation 20. A method for detecting toluenethiazide in a sample, the method comprising the steps of: binding the sample to an antibody according to Exemplary Implementation 17; and detecting the presence of toluenethiazide in the sample based on the specific binding of the antibody to it.

[0230] Exemplary Implementation 21. A method for detecting toluenethiazide in a sample, the method comprising the steps of: combining the sample with a kit according to Exemplary Implementation 19; detecting the presence of an enzymatic reaction product of the conjugate; and determining that toluenethiazide is present in the sample when a reduction in the enzymatic reaction product of the conjugate is observed compared to the enzymatic reaction product detected when the sample is absent.

[0231] Therefore, according to this disclosure, compositions, kits, apparatuses, and systems that fully satisfy the objectives and advantages described above, as well as methods for producing and using them, are provided. Although this disclosure has been described in conjunction with the specific figures, experiments, results, and language described above, it will be apparent to those skilled in the art that many alternatives, modifications, and variations will be readily apparent. Therefore, this disclosure is intended to cover all such alternatives, modifications, and variations that fall within the spirit and broad scope of this disclosure.

Claims

1. A composition comprising a compound having the structure of Formula I: Formula I The condition is that at least one of R1, R2, and R3 is not H.

2. The composition according to claim 1, wherein, At least one of R1, R2, and R3 that is not H includes at least one of aliphatic groups, hydrocarbon groups, bulky organic groups, non-bulky organic groups, alkyl, alkenyl, alkynyl, phenyl, aryl, substituted aryl, carbocyclic, heterocyclic, hydrocarbon carbonyloxy, lower alkoxy, aryl hydrocarbon, halide, amide, imide, nitrate, nitrite, carbonyl, ketone, ester, carboxylic acid, hydroxide, thiol, sulfide, sulfate, sulfonate, peptide, protein, enzyme, linker, immunogenic carrier, label, or combination thereof.

3. The composition according to claim 1, wherein, The compound comprises a structure of one of formulas II, III, IV, V, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XXII, or XXIX: 。 4. The composition according to claim 1, wherein, The compound has a structure of one of formulas VI, VII, VIII, IX, XX, XXI, XXIII or XXX: R4 is a peptide or protein.

5. The composition according to claim 4, wherein, R4 is an immunogenic vector.

6. The composition according to claim 5, wherein, The immunogenic vector is selected from the group consisting of: keyhole hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), egg ovalbumin, bovine gamma globulin (BGG), thyroglobulin, fibrinogen, polysaccharide, glucose-6-phosphate dehydrogenase (G6PDH), carrier protein, marker protein, and combinations thereof.

7. A composition comprising a compound having the structure of formula X: One of R5 and R6 is present and includes at least one of the following: aliphatic group, hydrocarbon group, bulky organic group, non-bulky organic group, alkyl, alkenyl, alkynyl, phenyl, aryl, substituted aryl, carbocyclic, heterocyclic, hydrocarbon carbonyloxy, lower alkoxy, aryl hydrocarbon, halide, amide, imide, nitrate, nitrite, carbonyl, ketone, ester, carboxylic acid, hydroxide, thiol, sulfide, sulfate, sulfonate, peptide, protein, enzyme, linker, immunogenic carrier, label, or combination thereof.

8. The composition according to claim 7, wherein, The compound has a structure of one of the formulas XXIV, XXV, XXVI, XXVII, XXVIII, XXXI, XXXII, XXXIII, XXXIV, XXXV, or XXXVI: R4 is a peptide or protein.

9. The composition according to claim 7, wherein, R5 or R6 contains an immunogenic vector.

10. The composition according to claim 9, wherein, The immunogenic vector is selected from the group consisting of: keyhole hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), egg ovalbumin, bovine gamma globulin (BGG), thyroglobulin, fibrinogen, polysaccharide, glucose-6-phosphate dehydrogenase (G6PDH), carrier protein, marker protein, and combinations thereof.

11. The composition according to any one of claims 1-10, further comprising at least one excipient.

12. A method for preparing antibodies, comprising the following steps: Animals that produce antibodies are immunized with an immunogen comprising any one of the compositions according to claims 1-11; Antibodies are isolated from the animal, wherein the antibodies specifically bind to the immunogen.

13. The method according to claim 12, wherein, The process of isolating antibodies further includes the following steps: Isolate antibody-producing spleen cells from the antibody-producing animals; Select at least one spleen cell that produces antibodies by secreting at least one monoclonal antibody that specifically binds to the immunogen. A hybrid cell line is prepared by fusing selected antibody-producing spleen cells from the antibody-producing animal with a suitable fusion partner, thereby the hybrid cell line secreting the monoclonal antibody.

14. A coupling composition comprising: The composition according to any one of claims 1-11; and An enzyme coupled to the composition.

15. The coupling composition according to claim 14, wherein, The enzyme is selected from the group consisting of: glucose-6-phosphate dehydrogenase (G6PDH), alkaline phosphatase, β-galactosidase, and horseradish peroxidase.

16. The coupling composition according to claim 15, wherein, The enzyme is G6PDH.

17. An antibody produced by the method according to claim 12 or 13.

18. A kit comprising the antibody according to claim 17.

19. The kit according to claim 18, further comprising the coupling composition according to any one of claims 14-16.

20. A method for detecting toluenethiazide in a sample, the method comprising the following steps: Bind the sample to the antibody according to claim 17; as well as The presence of toluenethiazide in the sample is detected based on the specific binding of the antibody to it.

21. A method for detecting toluenethiazide in a sample, the method comprising the following steps: Combine the sample with the kit according to claim 19; Detect the presence of the enzyme-catalyzed reaction product of the conjugate; as well as When a decrease in the enzymatic reaction product of the conjugate is observed compared to the enzymatic reaction product detected when the sample is absent, the presence of toluenethiazide in the sample is determined.

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