Method for detecting an analyte of interest in a sample - Patent Application 20070123633

Abstract

The present invention relates to a method for determining at least one analyte of interest and its use.The present invention further relates to a kit, a conjugate, a method for synthesizing the conjugate, a monomer and their use for detecting an analyte of interest in a sample.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for determining at least one analyte of interest and its use.The present invention further relates to a kit, a conjugate, a method for synthesizing the conjugate, a monomer and their use for detecting an analyte of interest in a sample. [Background technology]

[0002] 2. Background of the Invention US Patent No. 9,511,150 reports sugar alcohols and cross-linking reagents, polymers, and therapeutic bioconjugates. US Patent Publication No. 2016 / 0250896 reports phosphonate and sulfonate and hydrophilic linkers and the use of such linkers for conjugation of drugs to cell-binding molecules. US Patent Publication No. 2010 / 0009902 reports conjugation with PEG (polyethylene glycol) of selected molecular weight. Vlahov IR et al., J. Org. Chem. 75 (2010) 3685-3691, reports a carbohydrate-based synthetic approach to control the toxicity profile of folate-drug conjugates. More specifically, this document discloses the incorporation of 1-amino-1-deoxy-d-glucitol-γ-glutamate subunits into a peptide backbone. The synthesis of Fmoc-3,4;5,6-di-O-isopropylidene-1-amino-1-deoxy-d-glycitol-γ-glutamate, suitable for Fmoc strategy solid-phase peptide synthesis (SPPS), was accomplished in four steps from δ-gluconolactone. Alternating glutamic acid and 3,4;5,6-di-O-isopropylidene-1-amino-1-deoxy-d-glycitol-γ-glutamate moieties were added onto a cysteine-supported resin, followed by addition of folate, deprotection, and cleavage to isolate the following new folate-spacer: Pte-γGlu-(Glu(1-amino-1-deoxy-d-glucitol)-Glu)2-Glu(1-amino-1-deoxy-d-glucitol)-Cys-OH.

[0003] A particular technical feature known in the art of polymer chemistry is polydispersity, which refers to the lack of uniformity in the amount of incorporated monomer and / or polymer chain length. A particular technical challenge is posed by the polydispersity often observed in linker-containing compounds and conjugates.

[0004] In particular, PEG-based linkers may have such shortcomings. Due to the technical features of the polymerization chemistry typically used, the resulting high molecular weight PEG molecules are characterized by substantial polydispersity. That is, a typical polymerization results in a mixture of molecules with different molecular weights. The use of such mixed molecular weight PEG molecules as linkers leads to the propagation of polydispersity between the resulting conjugates. As a result, any analysis of the conjugates is complicated, since the desired conjugates are defined as having a uniform molecular weight. However, such a uniform molecular weight is not achieved. Moreover, despite the hydrophilicity of the PEG moiety in the spacer, certain conjugates with PEG still lack sufficient solubility.

[0005] Polysaccharides also tend to be polydisperse and structurally variable due to the complexity and difficulty of sugar chemical synthesis of longer and more complex alcohols, requiring elaborate, low-yielding protecting group manipulations.

[0006] Therefore, there is an urgent need in the art to overcome the above problems.

[0007] For the present invention, certain substantially monodisperse linker molecules with polyols have been devised that can be used to advantageously crosslink functional molecules. The inventors have found that certain linker molecules with polyols not only provide better hydrophilicity than PEG-containing derivatives. In an exemplary situation, a conjugate containing such a linker that bridges an analyte-specific binding agent and a labeling compound results in an improved signal-to-noise ratio in an analyte detection assay. Furthermore, the linker and / or conjugate exhibits monodispersity, which preferably results from peptide synthesis and can be shown by HPLC chromatograms. The conjugates and / or monomers described herein are stable.

[0008] It is an object of the present invention to provide a method and its use for determining at least one analyte of interest.Furthermore, it is an object of the present invention to provide a kit, a conjugate, a method for synthesizing the conjugate, a monomer and their use for detecting an analyte of interest in a sample.

[0009] This object or these objects are solved by the subject matter of the independent claims. Further embodiments are subject to the dependent claims. Summary of the Invention

[0010] Summary of the Invention In the following, the present invention relates to the following aspects:

[0011] In a first aspect, the present invention provides a method for detecting an analyte of interest in a sample, comprising the steps of: a) providing said sample containing said analyte of interest; b) providing a conjugate comprising a linker, said linker covalently binding a labeling compound and an analyte-specific binding agent, said labeling compound being capable of generating a detectable signal, preferably a chemiluminescence-based signal, more preferably an electrochemiluminescence-based signal; c) binding said sample of step a) with said complex of step b); d) detecting the analyte of interest by using the detectable signal of the labeled compound; Including, The conjugate has formula I: [ka] is a compound of wherein A represents the label compound and B represents the analyte-specific binding agent, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2, 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3; and n is an integer of 1 to 20, preferably 1 to 15, or 2 to 20, preferably 2 to 15.

[0012] In a second aspect, the present invention relates to the use of the method according to the first aspect of the invention for detecting an analyte of interest in a sample.

[0013] In a third aspect, the present invention provides a kit for carrying out the detection of an analyte of interest in a sample, comprising, in separate containers: a) a solid phase capable of immobilizing said analyte; b) Formula I: [ka] A compound of the formula: wherein A represents the label compound and B represents the analyte-specific binding agent, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2 or 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3; n is an integer of 1 to 20 or 2 to 20, preferably 1 to 15, more preferably 2 to 15; The present invention relates to a kit comprising:

[0014] In a fourth aspect, the present invention relates to the use of a kit according to the third aspect of the invention for detecting an analyte of interest in a sample.

[0015] In a fifth aspect, the present invention provides a compound of formula I [ka] A complex of wherein A represents a labeling compound and B represents an analyte-specific binding agent, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2 or 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3; n is an integer of 1 to 20, preferably 1 to 15, or 2 to 20, preferably 2 to 15; Preferably, said compound relates to a complex suitable for detecting an analyte of interest in a sample.

[0016] In a sixth aspect, the present invention provides a method of synthesising a conjugate of the fifth aspect of the invention, comprising the steps of: a) providing a monomer or a derivative thereof, said monomer being an amino acid comprising an amino group, a carboxy group and at least two hydroxyl groups, said amino group or said carboxy group being protected by a first protecting group and said at least one hydroxyl group or each hydroxyl group being protected by a second protecting group; b) using said monomer in a process of solid phase peptide synthesis, cleaving said first and second protecting groups to obtain a compound of formula I [ka] wherein A represents a labeling compound and B represents an analyte-specific binding agent, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2 or 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3; forming a complex, wherein n is an integer from 1 to 20, preferably from 1 to 15, or from 2 to 20, preferably from 2 to 15; The present invention relates to a method comprising the steps of:

[0017] In an eighth aspect, the present invention provides a compound of formula II [ka] A monomer used for peptide-based synthesis comprising: In the formula, PG1, PG2, PG3, PG4, PG5, and PG6 are each independently a protecting group, and preferably, when PG1 is a protecting group, PG2=H, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2 or 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3, relating to the monomer. [Brief description of the drawings]

[0018] [Figure 1] Figure 1 shows the Elecsys ECL technology. [Diagram 2] FIG. 2 shows the conjugate stability of different compounds disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description of the Invention Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific embodiments and examples described herein, which may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0020] Several documents are cited herein. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. In the event of a discrepancy between a definition or teaching of such incorporated reference and a definition or teaching cited herein, the body of the present specification shall control.

[0021] Each element of the present invention will be described below. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. The specification should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application unless the context indicates otherwise.

[0022] definition It will be understood that the word "comprise", and variations such as "comprises" and "comprising", imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integers or steps or groups of integers or steps.

[0023] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0024] Percentages, concentrations, amounts, and other numerical data may be expressed or presented herein in a "range" format. It is understood that such range formats are used merely for convenience and brevity, and thus should be interpreted flexibly to include not only the numerical values ​​expressly recited as boundaries of the range, but also all of the individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were expressly recited. By way of illustration, a numerical range of "4%-20%" should be interpreted not only to include the explicitly recited value of 4%-20%, but also to include each individual value and subrange within the range indicated. Thus, this numerical range includes individual values ​​such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20%, and subranges such as 4-10%, 5-15%, 10-20%, etc. This same principle also applies to ranges reciting minimum or maximum values. Moreover, such interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0025] The term "about," when used in connection with a numerical value, is meant to encompass numerical values ​​in a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0026] As used herein, the term "detecting" an analyte of interest refers to the quantification or qualification of the analyte of interest, e.g., the presence or amount of the analyte of interest in a sample, using appropriate detection methods as described elsewhere herein.

[0027] The term "binding" the sample of step a) with the complex of step b) as used herein is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. The term may refer in particular, but not exclusively, to chemically binding the complex with the sample containing the analyte of interest. Preferably, binding or chemically binding means covalently or non-covalently binding the sample, preferably the analyte, with the complex.

[0028] In the context of this disclosure, the terms "analyte", "analyte molecule" or "analyte of interest" are used interchangeably to refer to a chemical species that is analyzed by a detectable label. A chemical species suitable for analysis by a detectable label, i.e., an analyte, can be any type of molecule present in a living organism, including, but not limited to, nucleic acids (e.g., DNA, mRNA, miRNA, rRNA, etc.), amino acids, peptides, proteins (e.g., cell surface receptors, cytosolic proteins, etc.), drug molecules, metabolites or hormones (e.g., testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (e.g., vitamin D), molecules characteristic of a particular modification of another molecule (e.g., sugar moieties or phosphoryl residues on proteins, methyl residues on genomic DNA), or substances internalized by an organism (e.g., therapeutic drugs, drugs of abuse, toxins, etc.) or metabolites of such substances. Such analytes may serve as biomarkers. In the context of the present invention, the term "biomarker" refers to a substance in a living system that is used as an indicator of the biological state of that system. An "analyte" can be any molecule that can be bound by an analyte-specific receptor. In an embodiment, the analyte is an antigen of an infectious agent. Examples of infectious agents are viral, bacterial and protozoan pathogens that infect humans. In an embodiment, the analyte is a viral antigen, in an embodiment, a hepatitis virus antigen or a human retrovirus antigen. In an embodiment, the analyte is a hepatitis C virus or a hepatitis B virus or an HIV antigen.

[0029] In general, the term "receptor" refers to any compound or composition that can recognize a specific spatial and polar configuration of a target molecule, i.e., an epitope site of an analyte. Thus, the term "analyte-specific receptor" referred to herein includes an analyte-specific reactant that can bind or complex with an analyte. This includes, but is not limited to, an antibody, particularly a monoclonal antibody or an antibody fragment. Such a receptor can act as a catcher of the analyte, for example, to immobilize the analyte. The epitope recognized by the antibody is bound, followed by the binding of a labeled antibody specific for another epitope of the analyte. Other receptors are known to those of skill in the art. The specific use of various receptors in receptor-based analyte assays will be understood by those of skill in the art with reference to this disclosure.

[0030] Analytes of interest or an analyte may be present in a sample, such as a biological sample or a clinical sample. The term "biological sample or clinical sample" is used interchangeably herein and refers to a part or piece of a tissue, organ or individual, usually smaller than such tissue, organ or individual, which is intended to represent the entire tissue, organ or individual. Upon analysis, the biological sample or clinical sample provides information about the state of the tissue, or the health or disease state of the organ or individual. Examples of biological or clinical samples include, but are not limited to, liquid samples such as blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva and lymphatic fluid, or solid biological or clinical samples such as dried blood spots and tissue extracts. Further examples of biological or clinical samples are cell cultures or tissue cultures.

[0031] In the context of this disclosure, the term "antibody" refers to an intact immunoglobulin molecule, specifically IgM, IgD, IgE, IgA or IgG, as well as Fab fragments or V L -, V H- or parts of such immunoglobulin molecules such as CDR regions. Furthermore, the term relates to modified and / or altered antibodies, such as chimeric and humanized antibodies. The term also relates to modified or altered monoclonal or polyclonal antibodies, as well as recombinantly or synthetically produced / synthesized antibodies. The term also relates to intact antibodies and antibody fragments / portions thereof, such as isolated light and heavy chains, Fab, Fab / c, Fv, Fab', F(ab')2. The term "antibody" also includes antibody derivatives, bifunctional antibodies and antibody constructs, such as single chain Fv (scFv), bispecific scFv or antibody fusion proteins.

[0032] In chemistry, "solid phase synthesis" is a method in which molecules are covalently attached onto a solid support material and synthesized stepwise in a single reaction vessel using selective protecting group chemistry. As a specific embodiment, solid phase peptide synthesis is a common technique that includes separate steps for the synthesis of peptides. This approach allows for the removal of unreacted reagents by washing without loss of product. Typically, peptides are synthesized from the carbonyl group (C-terminus) to the amino group (N-terminus) of the amino acid chain. In peptide synthesis, an amino-protected amino acid is attached to a solid phase material, such as, but not limited to, polystyrene beads, thereby forming a covalent bond, most often an amide or ester bond, between the carbonyl group and the resin. The amino group is then deprotected and reacted with the carbonyl group of the next amino-protected amino acid. The solid phase now carries a dipeptide. The cycle is repeated to form the desired peptide chain. After all reactions are complete, the synthesized peptide is cleaved from the solid phase.

[0033] More specifically, the carboxyl moiety of each incoming amino acid is activated by one of several strategies and coupled with the α-amino group of the preceding amino acid. The α-amino group of the incoming residue is temporarily blocked to prevent peptide bond formation at this site. The residue is deblocked at the start of the next synthesis cycle. In addition, reactive side chains on the amino acids are modified with appropriate protecting groups. The peptide chain is extended by repeated synthesis cycles. An excess of reagents is used to drive the reaction as close to completion as possible.

[0034] The "blocking group" or "protecting group" or "protection group" used to block the α-amino group determines both the synthetic chemistry used and the nature of the side chain protecting groups. The two most commonly used α-amino protecting groups are Fmoc (9-fluorenyl-methoxy-carbonyl) and Boc (tert-butoxycarbonyl). Protection of reactive groups in the side chain is provided by protecting groups independent of the protecting group used for the α-amino group, including but not limited to carbamates, ethers, esters, amides, acetals, and enamines.

[0035] After complete assembly of the peptide, side chain protecting groups are removed, if desired, and the peptide is cleaved from the solid support using conditions that cause minimal damage to labile residues.

[0036] The products can then be analyzed to verify the sequence. The synthetic peptides are usually purified by gel chromatography or HPLC.

[0037] The binding of the label and / or target molecule to the peptide can be by different methods. As a non-limiting example, a component suitable for SPPS can be incorporated into the peptide, which comprises a reactive group, optionally protected, that can be used to form a bond with a further compound selected after the SPPS process. Alternatively, the selected compound can already be bound to the component when entering the SPPS process. Other alternatives are also possible.

[0038] The Fmoc protecting group is base labile. It is usually removed by dilute base such as piperidine. The side chain protecting groups are removed by treatment with trifluoroacetic acid (TFA), which also cleaves the bond that anchors the peptide to the support. The Boc protecting group is removed by weak acid (usually dilute TFA). Hydrofluoric acid (HF) can be used both to deprotect the amino acid side chains and to cleave the peptide from the resin support. Fmoc is a milder method than Boc, since the peptide chain is not subjected to acid at each cycle, and has become the predominant method used in commercial automated peptide synthesis.

[0039] The amino protecting groups mainly used in peptide synthesis are the 9-fluorenylmethyloxycarbonyl group (Fmoc) and the t-butoxycarbonyl (Boc). Some amino acids carry functional groups in the side chain that must be specifically protected from reaction with the incoming N-protected amino acid. In contrast to the Boc and Fmoc groups, which must be stable throughout the course of the peptide synthesis, they are also removed during the final deprotection of the peptide.

[0040] A "label compound" includes a moiety that is detectable or can be made detectable. Those skilled in the art know a label as a compound or composition that can provide a detectable signal in conjunction with physical activation (or excitation) or chemical reagents and can be modified so that a particular signal is decreased or increased.

[0041] Particular embodiments of the labeling compound capable of generating a detectable signal include labels detectable by several commercially available instruments that utilize chemiluminescence, preferably electrochemiluminescence (ECL), for analytical measurements. Species that can be induced to emit ECL (ECL-active species) have been used as ECL labels. Examples of ECL labels include i) organometallic compounds from Group VIII noble metals, including, for example, Ru-containing, Ir-containing and / or Os-containing organometallic compounds, such as the tris-bipyridyl-ruthenium (BPRu) moiety, and ii) luminol and related compounds. Species involved in ECL labeling in the ECL process are referred to herein as ECL coreactants. Commonly used coreactants include tertiary amines for ECL from RuBpy (see, for example, U.S. Pat. No. 5,846,485), oxalates and persulfates, and hydrogen peroxide for ECL from luminol (see, for example, U.S. Pat. No. 5,240,863). The light generated by the ECL label can be used as a reporter signal in diagnostic procedures (Bard et al., U.S. Pat. No. 5,238,808). For example, the ECL label can be covalently attached to a binding agent such as an antibody, a nucleic acid probe, a receptor or a ligand. The participation of the binding reagent in the binding interaction can be monitored by measuring the ECL emitted from the ECL label. Alternatively, the ECL signal from an ECL-active compound can indicate the chemical environment (see, for example, U.S. Pat. No. 5,641,623, which describes an ECL assay that monitors the formation or destruction of an ECL coreactant).For further background regarding ECL, ECL labels, ECL assays, and instruments for performing ECL assays, see U.S. Pat. Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910; 5,641,623; 5,643,713; 5,679,519; 5,705,402; 5,846,485; 5,866,434; 5,786,141; 5,731,147; and U.S. Pat. See U.S. Patent Nos. 6,066,448; 6,136,268; 5,776,672; 5,308,754; 5,240,863; 6,207,369 and 5,589,136; WO 99 / 63347, WO 00 / 03233, WO 99 / 58962, WO 99 / 32662, WO 99 / 14599, WO 98 / 12539, WO 97 / 36931 and WO 98 / 57154.

[0042] The term "chemiluminescence-based signal" refers to a signal that is generated by the emission of light (luminescence) as a result of a chemical reaction. This signal is detectable, for example, by several commercially available instruments that utilize chemiluminescence. The term "electrochemiluminescence-based signal" refers to a signal that consists of the emission of light (e.g., luminescence) as a result of an electrochemical reaction, in which an excited state of a species can be obtained at an electrode.

[0043] In the context of the present disclosure, the term "conjugate" refers to a product produced by the reaction of a linker, a labeling compound, and an analyte-specific binding agent, which reaction may result in the formation of covalent bonds between the labeling compound and the linker on one side, and between the linker and the analyte-specific binding agent on the other side.

[0044] The term "linker" can refer to a compound that functions as a spacer between the labeling compound and the analyte-specific binding agent and / or affects the physicochemical properties of the complex, such as hydrophilicity and solubility.

[0045] The term "analyte-specific binding agent" refers to a (macro)molecule (protein, peptide, nucleic acid, etc.) that is able to specifically bind to an analyte of interest, e.g. a monoclonal antibody.

[0046] The term "A represents a labeled compound and B represents an analyte-specific binding agent, or vice versa" means that A of formula I represents a labeled compound and B of formula I represents an analyte-specific binding agent. Alternatively, B of formula I represents a labeled compound and A of formula I represents an analyte-specific binding agent.

[0047] The term "binding the sample of step a) with the complex of step b)" refers to the reaction of the sample, which comprises or contains the analyte of interest, with the complex of step b). Preferably, binding refers to a non-covalent bond between the sample, preferably the analyte of interest, and the complex.

[0048] The term "peptide" refers to molecules formed using naturally occurring L-amino acids or their analogs, such as D-amino acids or N-alkylated amino acids. Preferred amino acids are selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val. Other building blocks with carboxylic acids and amino groups are also possible. Additionally, modifications such as fluorescent dyes or biotin are also possible.

[0049] "Solid-phase peptide synthesis (SPPS)" is a well-established method. Merrifield et al. first developed a convenient strategy for constructing peptides by subsequently coupling amino acid monomers using a solid-phase resin as a heterogeneous reaction medium (RB Merrifield, J. Am. Chem. Soc. 85 (1963) 2149-2154).

[0050] As a major advantage compared to solution synthesis of peptides, SPPS can be easily automated and impurities or by-products, reagents as well as unreacted starting materials can be washed away while the products or intermediates remain anchored to the solid phase.

[0051] Typically, the Merrifield method described above begins with the attachment of the first C-terminal amino acid to a so-called "linker" of a cross-linked polystyrene resin, which serves as a bridging element between the resin and the C-terminal amino acid of the peptide being synthesized, and which contains an acid-sensitive bond that is used for detachment of the peptide after synthesis.

[0052] As an example of a typical SPPS protocol, the N-terminus may be protected by a 9-fluorenylmethoxycarbonyl (Fmoc) group, which is stable in acid but removable by base. To ensure that only the N-terminal amino group incorporated into the peptide backbone can react with the carboxylic acid group of the subsequent amino acid after removal of the Fmoc group, any side chain functional groups are protected by base-stable groups. As already mentioned, the first step after immobilization of the first amino acid is the deprotection of the amino function by removal of the Fmoc group with 20% piperidine in N,N-dimethylformamide (DMF). The amino function is coupled with an activated carboxylic acid via the O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) ester of the next amino acid in the presence of base to form a new amide bond. This process is repeated until the desired peptide is assembled on the resin. As a final step, the complete peptide is cleaved from the resin using a solution containing trifluoroacetic acid (TFA). The released peptides in solution can be precipitated and washed before further purification.

[0053] This "classical" method of SPPS has been optimized in recent years using modified resins, linkers, protecting groups, coupling chemistries and cleavage procedures, but the principles remain the same.

[0054] The term "solid phase" as used herein refers to a wide variety of materials including solids, semi-solids, gels, films, membranes, meshes, felts, composites, particles, resins, papers, etc. that are typically used by those skilled in the art to isolate molecules. A solid phase can be, for example, a material in a chromatography column, or, as a specific embodiment, a functionalized resin in a column of a device for solid phase synthesis. A solid phase can be non-porous or porous. A solid phase can be non-magnetic or magnetic (including diamagnetic, paramagnetic, and superparamagnetic characteristics).

[0055] The surface of the solid phase, as described above, can be modified to provide linking sites, for example by bromoacetylation, silylation, addition of amino groups using nitric acid, and attachment of intermediate proteins, dendrimers and / or star polymers. This list is not meant to be limiting and any method known to one of skill in the art can be used.

[0056] The term "polyol unit" refers to a monomer (e.g., an amino acid) that contains one or more OH groups. Such monomers can be covalently linked together to form homopolymers or heteropolymers.

[0057] A "kit" is any article of manufacture (e.g., package or container) that comprises at least one reagent, such as a drug for treating a disorder, or a probe for specifically detecting a biomarker gene or protein of the invention. The kit is preferably advertised, distributed, or sold as a unit for carrying out the method of the invention. Typically, the kit may further comprise a carrier means compartmentalized to receive in tight confinement one or more container means, such as a vial, tube, or the like. In particular, each of the container means contains one of the separate elements used in the method of the first aspect. The kit may further comprise one or more other reagents, including, but not limited to, a reaction catalyst. The kit may further comprise one or more other containers containing additional materials, including, but not limited to, buffers, diluents, filters, needles, syringes, and a package insert with instructions for use. A label may be affixed to the container to indicate that the composition is to be used in a particular application, and may indicate directions for either in vivo or in vitro use. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disc), or directly on a computer or data processing device. Additionally, the kits may contain standard amounts of biomarkers, as described elsewhere herein, for calibration purposes.

[0058] In this detailed description, reference to "one embodiment," "embodiment," or "in an embodiment" means that the referenced feature is included in at least one embodiment of the technology in all its aspects according to the present disclosure. Moreover, individual references to "one embodiment," "an embodiment," or "embodiments" do not necessarily refer to the same embodiment. However, such embodiments are not mutually exclusive unless otherwise stated and unless readily apparent to one of ordinary skill in the art. Thus, the technology in all its aspects according to the present disclosure can encompass any various combinations and / or integrations of the embodiments described herein.

[0059] Embodiment In a first aspect, the present invention provides a method for detecting an analyte of interest in a sample, comprising the steps of: a) providing said sample containing said analyte of interest; b) providing a conjugate comprising a linker, said linker covalently binding a labeling compound and an analyte-specific binding agent, said labeling compound being capable of generating a detectable signal, preferably a chemiluminescence-based signal, more preferably an electrochemiluminescence-based signal; c) binding said sample of step a) with said complex of step b); d) detecting the analyte of interest by using the detectable signal of the labeled compound; Including, The conjugate has formula I: [ka] is a compound of wherein A represents a labeling compound and B represents an analyte-specific binding agent, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2, 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3; and n is an integer of 1 to 20, preferably 1 to 15, or 2 to 20, preferably 2 to 15.

[0060] The inventors have surprisingly found that the subject matter of the present invention, in particular the method according to the first aspect of the present invention, shows conjugates with very good control over structure and polydispersity, especially those containing peptide-based polyol linkers. The use of a single molecular weight and pure linker reduces the complexity of product purification, characterization and improves the reproducibility of production. In particular, solid-phase peptide chemistry can be utilized to obtain the conjugates of the present invention. Moreover, the conjugates are stable.

[0061] The method referred to according to the present invention includes a method consisting essentially of the steps mentioned above or a method comprising further steps. Moreover, the method of the present invention is preferably an ex vivo, more preferably an in vitro method. Moreover, the method of the present invention may comprise steps in addition to those explicitly mentioned above. For example, the further steps may relate to the detection of further analytes of interest and / or sample pre-treatment, enrichment steps or evaluation of the results obtained by the method. The method may be performed manually or may be supported by automation. Preferably, steps (a), (b), (c) and / or (d) may be fully or partially supported by automation, for example by suitable robots and sensory equipment.

[0062] According to step (a), a sample is provided.

[0063] According to step b), a conjugate is provided. The conjugate is a compound of formula I. The conjugate comprises a linker. The linker covalently connects the label compound and the analyte-specific binding agent. The label compound is capable of generating a detectable signal. Preferably, the detectable label is a chemiluminescence-based signal, more preferably an electrochemiluminescence-based signal.

[0064] According to step c), the sample of step a) is combined with the complex of step b).

[0065] According to step d), the analyte of interest is detected by using the detectable signal of the labelled compound.

[0066] In an embodiment of the first aspect of the present invention, X=O, a=1 and b=0.

[0067] In an embodiment of the first aspect of the present invention, X=O, a=1 and b=1.

[0068] In an embodiment of the first aspect of the present invention, X=O, a=1 and b=2.

[0069] In an embodiment of the first aspect of the present invention, X=O, a=1 and b=3.

[0070] In an embodiment of the first aspect of the present invention, X=O, a=2 and b=0.

[0071] In an embodiment of the first aspect of the present invention, X=O, a=2 and b=1.

[0072] In an embodiment of the first aspect of the present invention, X=O, a=2 and b=2.

[0073] In an embodiment of the first aspect of the present invention, X=O, a=2 and b=3.

[0074] In an embodiment of the first aspect of the present invention, X=O, a=3 and b=0.

[0075] In an embodiment of the first aspect of the present invention, X=O, a=3 and b=1.

[0076] In an embodiment of the first aspect of the present invention, X=O, a=3 and b=2.

[0077] In an embodiment of the first aspect of the present invention, X=O, a=3 and b=3.

[0078] In an embodiment of the first aspect of the present invention, X=O, a=4 and b=0.

[0079] In an embodiment of the first aspect of the present invention, X=O, a=4 and b=1.

[0080] In an embodiment of the first aspect of the present invention, X=O, a=4 and b=2.

[0081] In an embodiment of the first aspect of the present invention, X=O, a=4 and b=3.

[0082] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=1 and b=0.

[0083] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=1 and b=1.

[0084] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=1 and b=2.

[0085] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=1 and b=3.

[0086] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=2 and b=0.

[0087] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=2 and b=1.

[0088] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=2 and b=2.

[0089] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=2 and b=3.

[0090] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=3 and b=0.

[0091] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=3 and b=1.

[0092] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=3 and b=2.

[0093] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=3 and b=3.

[0094] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=4 and b=0.

[0095] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=4 and b=1.

[0096] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=4 and b=2.

[0097] In an embodiment of the first aspect of the present invention, X=1,2,3-triazole, a=4 and b=3.

[0098] In an embodiment of the first aspect of the present invention, X=amide, a=1 and b=0.

[0099] In an embodiment of the first aspect of the present invention, X=amide, a=1 and b=1.

[0100] In an embodiment of the first aspect of the present invention, X=amide, a=1 and b=2.

[0101] In an embodiment of the first aspect of the present invention, X=amide, a=1 and b=3.

[0102] In an embodiment of the first aspect of the present invention, X=amide, a=2 and b=0.

[0103] In an embodiment of the first aspect of the present invention, X=amide, a=2 and b=1.

[0104] In an embodiment of the first aspect of the present invention, X=amide, a=2 and b=2.

[0105] In an embodiment of the first aspect of the present invention, X=amide, a=2 and b=3.

[0106] In an embodiment of the first aspect of the present invention, X=amide, a=3 and b=0.

[0107] In an embodiment of the first aspect of the present invention, X=amide, a=3 and b=1.

[0108] In an embodiment of the first aspect of the present invention, X=amide, a=3 and b=2.

[0109] In an embodiment of the first aspect of the present invention, X=amide, a=3 and b=3.

[0110] In an embodiment of the first aspect of the present invention, X=amide, a=4 and b=0.

[0111] In an embodiment of the first aspect of the present invention, X=amide, a=4 and b=1.

[0112] In an embodiment of the first aspect of the present invention, X=amide, a=4 and b=2.

[0113] In an embodiment of the first aspect of the present invention, X=amide, a=4 and b=3.

[0114] In an embodiment of the first aspect of the present invention, a is 1.

[0115] In an embodiment of the first aspect of the present invention, a is 2.

[0116] In an embodiment of the first aspect of the present invention, a is 3.

[0117] In an embodiment of the first aspect of the present invention, a is 4.

[0118] In an embodiment of the first aspect of the present invention, n is an integer of 1-20, preferably 1-15.

[0119] In an embodiment of the first aspect of the present invention, n is an integer from 2 to 20, preferably from 2 to 15. In an embodiment of the first aspect of the present invention, n is an integer. n is selected from the range of 1 to 20. Preferably, n is 2 or more, for example, 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20.

[0120] In an embodiment of the first aspect of the invention, step b) comprises a peptide-based synthesis, preferably solid phase peptide synthesis (SPPS).

[0121] In an embodiment of the first aspect of the invention, the labelling compound is selected from the group consisting of an enzyme, a fluorescent dye, a luminescent dye, a metal chelate complex and a moiety containing a radioisotope.

[0122] In an embodiment of the first aspect of the invention, the labelling compound may be induced to emit light when electrochemically oxidised or reduced.

[0123] In an embodiment of the first aspect of the invention, the labeling compound is Ru 2+ or Ir 3+ The metal ion is

[0124] Preferably, the labeling compound is selected from the following group: Ru or Ir.

[0125] In an embodiment of the first aspect of the invention, the labeling compound is covalently attached to the linker by a first conjugation method, the first conjugation method being selected from the following group: click chemistry, amide, ester, imide, carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime, dihydropyridazine, thiol-maleimide, cycloaddition, tetrazine ligation, photoclick, Staudinger ligation, Diels-Alder, cross-coupling, Pictet-Spengler, quadricyclane.

[0126] In an embodiment of the first aspect of the invention the analyte-specific binding agent is selected from the group consisting of antibodies, analyte-specific fragments and / or derivatives of antibodies, aptamers, spiegelmers, DARPins, lectins, ankyrin repeat-containing proteins and Kunitz-type domain-containing proteins.

[0127] In an embodiment of the first aspect of the invention, the analyte-specific binding agent is covalently attached to the linker by a second conjugation method selected from the following group: click chemistry, amide, ester, imide, carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime, dihydropyridazine, thiol-maleimide, cycloaddition, tetrazine ligation, photoclick, Staudinger ligation, Diels-Alder, cross-coupling, Pictet-Spengler, quadricyclane.

[0128] Preferably, the analyte-specific binding agent is selected from the following groups: antibodies, Fabs.

[0129] In an embodiment of the first aspect of the invention, A of formula I represents a labeling compound and B of formula I represents an analyte-specific binding agent.

[0130] In an embodiment of the first aspect of the invention, B of formula I represents a labeling compound and A of formula I represents an analyte-specific binding agent.

[0131] In an embodiment of the first aspect of the invention, the analyte is immobilised on a solid phase before, during or after step (c).

[0132] In an embodiment of the first aspect of the invention the sample is selected from the group consisting of sputum, saliva, fluid, urine, whole blood, hemolyzed whole blood, serum and plasma.

[0133] In an embodiment of the first aspect of the invention, the complex of step (b) is provided in dissolved form and step (c) is carried out in a liquid aqueous buffer.

[0134] In an embodiment of the first aspect of the invention the liquid aqueous buffer is selected from phosphate, Tris buffer, citrate, cacodylate, barbital, glycine, HEPES, MES, PIPES, MOPS, bis-trismethane, ADA, bis-trispropane, ACES, MOPSO, BES, AMPB, TES, DIPSO, MOBS, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, gycinamide, Gly-Gly, HEPBS, bicine, TAPS and mixtures thereof.

[0135] In an embodiment of the first aspect of the present invention, the liquid aqueous buffer is selected from phosphate, tris(hydroxymethyl)aminomethane (TRIS, preferably pH 6.0-7.4), and mixtures thereof.

[0136] In an embodiment of the first aspect of the present invention, the complex is selected from the group consisting of at least one compound having at least one of the following formulas 4-I to 4-X: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0137] In embodiments, the labeled compound does not contain or is free of folic acid or its derivatives.

[0138] In some embodiments, the labeled compound does not contain or does not include a folate receptor binding ligand.

[0139] In embodiments, the analyte-specific binding agent does not contain or does not include cysteine.

[0140] In embodiments, the method does not include a drug delivery objective, which may mean that the method is not aimed at improving drug delivery or that the objective of the method is not related to drug delivery.

[0141] In embodiments, the method is a diagnostic method, preferably an in vitro diagnostic method.

[0142] In an embodiment, n>2.

[0143] In an embodiment, n>4.

[0144] In some embodiments, the labeled compound does not include a drug compound, such as desacetylvinblastine hydrazide or a derivative thereof.

[0145] In a second aspect, the present invention relates to the use of the method according to the first aspect of the invention for detecting an analyte of interest in a sample.

[0146] All embodiments described for the first aspect of the invention apply to the second aspect of the invention and vice versa.

[0147] In a third aspect, the present invention provides a kit for carrying out the detection of an analyte of interest in a sample, comprising, in separate containers: a) a solid phase capable of immobilizing said analyte; b) Formula I: [ka] is a compound of wherein A represents a labeling compound and B represents an analyte-specific binding agent, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2 or 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3; and n is an integer of 1 to 20, preferably 1 to 15, or 2 to 20, preferably 2 to 15. The present invention relates to a kit comprising:

[0148] All embodiments described for the first aspect of the invention and / or the second aspect of the invention apply to the third aspect of the invention and vice versa.

[0149] In an embodiment of the third aspect of the invention the complex is embodied in a dissolved form.

[0150] In an embodiment of the third aspect of the present invention, at least one container or a plurality of containers are made from glass or plastic.

[0151] In a fourth aspect, the present invention relates to the use of a kit according to the third aspect of the invention for detecting an analyte of interest in a sample.

[0152] All embodiments described for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention apply to the fourth aspect of the invention and vice versa.

[0153] In a fifth aspect, the present invention provides a compound of formula I [ka] wherein A represents a labeling compound and B represents an analyte-specific binding agent, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2 or 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3; n is an integer of 1 to 20, preferably 1 to 15, or 2 to 20, preferably 2 to 15; Preferably, said compound relates to a complex suitable for detecting an analyte of interest in a sample.

[0154] All embodiments described for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention and / or the fourth aspect of the invention apply to the fifth aspect of the invention and vice versa.

[0155] In an embodiment of the fifth aspect of the invention A or B is selected from the group consisting of peptides, polypeptides and proteins.

[0156] In an embodiment of the fifth aspect of the invention, A comprises an analyte-specific binding agent and B comprises a labelled compound, or B comprises an analyte-specific binding agent and A comprises a labelled compound.

[0157] In an embodiment of the fifth aspect of the invention the analyte-specific binding agent is selected from the group consisting of antibodies, analyte-specific fragments and / or derivatives of antibodies, aptamers, spiegelmers, DARPins, lectins, ankyrin repeat-containing proteins, and Kunitz-type domain-containing proteins and the labelling compound is selected from the group consisting of enzymes, fluorescent dyes, luminescent dyes, metal chelate complexes, and moieties containing radioisotopes.

[0158] In a sixth aspect, the present invention provides a method of synthesising a conjugate of the fifth aspect of the invention, comprising the steps of: a) providing a monomer or a derivative thereof, said monomer being an amino acid comprising an amino group, a carboxy group and at least one or at least two hydroxyl groups, said amino group or said carboxy group being protected by a first protecting group and said at least one hydroxyl group or each hydroxyl group being protected by a second protecting group; b) using said monomer in a process of solid phase peptide synthesis, cleaving said first and second protecting groups to obtain a compound of formula I [ka] wherein A represents a labeling compound and B represents an analyte-specific binding agent, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2 or 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3; forming a complex, wherein n is an integer from 1 to 20, preferably from 1 to 15, or from 2 to 20, preferably from 2 to 15; The present invention relates to a method comprising the steps of:

[0159] All embodiments described for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention and / or the fourth aspect of the invention and / or the fifth aspect of the invention apply to the sixth aspect of the invention and vice versa.

[0160] In an embodiment of the sixth aspect of the invention, the order of the method steps may vary depending on the metal complex (e.g. Ru or Ir) added. In the case of Ru, the peptide is synthesized, the first protecting group is cleaved, the Ru label is attached, the peptide is cleaved from the solid phase at the same time as the second protecting group is cleaved, and finally the analyte binding agent is attached. In the case of Ir, the peptide is synthesized, the first and second protecting groups are cleaved together with the cleavage of the peptide from the solid phase. Finally, the Ir label is attached, and then the analyte binding agent is attached.

[0161] In an embodiment of the sixth aspect of the present invention the first and / or second protecting group is selected from the group of ester, ether, silyl ether, acetal, 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), allyloxycarbonyl (Alloc), amide and tertbutyl ester.

[0162] In an embodiment of the sixth aspect of the present invention the first and / or second protecting group or further protecting groups are selected from the group consisting of Fmoc, tBu (tert-butyl), Boc (tert-butoxycarbonyl), ether, ester and acetal.

[0163] In an embodiment of the sixth aspect of the present invention, the monomer or derivative thereof is selected from the following formulae m-1 to m-3: [ka] In the formula, Fmoc means a 9-fluorenylmethoxycarbonyl protecting group.

[0164] In a seventh aspect, the present invention provides a compound of formula II [ka] A monomer used for peptide-based synthesis comprising: In the formula, PG1, PG2, PG3, PG4, PG5, and PG6 each independently represent a protecting group. Preferably, when PG1 is a protecting group, PG2 = H, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2 or 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3, relating to the monomer.

[0165] All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention and / or the fourth aspect of the invention and / or the fifth aspect of the invention and / or the sixth aspect of the invention apply to the seventh aspect of the invention and vice versa.

[0166] In an embodiment of the seventh aspect of the present invention, the protecting groups PG1, PG2, PG3, PG4, PG5, PG6 are independently selected from the group consisting of Fmoc, tBu, Boc, ether, ester and acetal.

[0167] In an embodiment of the seventh aspect of the present invention, at least two of the protecting groups selected from the group consisting of PG1, PG2, PG3, PG4, PG5, PG6 are linked to each other, for example by at least one covalent bond, preferably a single, double or triple covalent bond.

[0168] In an embodiment of the seventh aspect of the present invention, the monomer or derivative thereof is selected from the following formulae m-1 to m-3: [ka]

[0169] In further embodiments, the present invention relates to the following aspects:

[0170] 1. A method for detecting an analyte of interest in a sample, comprising: a) providing said sample containing said analyte of interest; b) providing a conjugate comprising a linker, said linker covalently binding a labeling compound and an analyte-specific binding agent, said labeling compound being capable of generating a detectable signal, preferably a chemiluminescence-based signal, more preferably an electrochemiluminescence-based signal; c) binding said sample of step a) with said complex of step b); d) detecting the analyte of interest by using the detectable signal of the labeled compound; Including, The conjugate has formula I: [ka] is a compound of wherein A represents the label compound and B represents the analyte-specific binding agent, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2, 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3; The method, wherein n is an integer of 1 to 20, preferably 1 to 15, or 2 to 20, preferably 2 to 15.

[0171] 2. The method of embodiment 1, wherein X=O, a=1 and b=0.

[0172] 3. The method according to any one of aspects 1-2, wherein X=O, a=1 and b=1.

[0173] 4. The method of any one of aspects 1 to 3, wherein X=O, a=1 and b=2.

[0174] 5. The method of any one of aspects 1-4, wherein X=O, a=1 and b=3.

[0175] 6. The method of any one of aspects 1 to 5, wherein X=O, a=2 and b=0.

[0176] 7. The method of any one of aspects 1-6, wherein X=O, a=2 and b=1.

[0177] 8. The method of any one of aspects 1-7, wherein X=O, a=2 and b=2.

[0178] 9. The method of any one of aspects 1-8, wherein X=O, a=2 and b=3.

[0179] 10. The method of any one of aspects 1-9, wherein X=O, a=3 and b=0.

[0180] 11. The method of any one of aspects 1 to 10, wherein X=O, a=3 and b=1.

[0181] 12. The method of any one of aspects 1 to 11, wherein X=O, a=3 and b=2.

[0182] 13. The method of any one of aspects 1 to 12, wherein X=O, a=3 and b=3.

[0183] 14. The method of any one of aspects 1 to 13, wherein X=O, a=4 and b=0.

[0184] 15. The method of any one of aspects 1 to 14, wherein X=O, a=4 and b=1.

[0185] 16. The method of any one of aspects 1 to 15, wherein X=O, a=4 and b=2.

[0186] 17. The method of any one of the preceding aspects, wherein X=O, a=4 and b=3.

[0187] 18. The method of any one of aspects 1 to 17, wherein X=1,2,3-triazole, a=1 and b=0.

[0188] 19. The method of any one of aspects 1 to 18, wherein X=1,2,3-triazole, a=1 and b=1.

[0189] 20. The method of any one of aspects 1 to 19, wherein X=1,2,3-triazole, a=1 and b=2.

[0190] 21. The method of any one of aspects 1 to 20, wherein X=1,2,3-triazole, a=1 and b=3.

[0191] 22. The method of any one of aspects 1 to 21, wherein X=1,2,3-triazole, a=2 and b=0.

[0192] 23. The method of any one of aspects 1 to 22, wherein X=1,2,3-triazole, a=2 and b=1.

[0193] 24. The method of any one of aspects 1 to 23, wherein X=1,2,3-triazole, a=2 and b=2.

[0194] 25. The method of any one of aspects 1 to 24, wherein X=1,2,3-triazole, a=2 and b=3.

[0195] 26. The method of any one of aspects 1 to 25, wherein X=1,2,3-triazole, a=3 and b=0.

[0196] 27. The method of any one of the preceding aspects, wherein X=1,2,3-triazole, a=3 and b=1.

[0197] 28. The method of any one of the preceding aspects, wherein X=1,2,3-triazole, a=3 and b=2.

[0198] 29. The method of any one of the preceding aspects, wherein X=1,2,3-triazole, a=3 and b=3.

[0199] 30. The method of any one of the preceding aspects, wherein X=1,2,3-triazole, a=4 and b=0.

[0200] 31. The method of any one of aspects 1 to 30, wherein X=1,2,3-triazole, a=4 and b=1.

[0201] 32. The method of any one of the preceding aspects, wherein X=1,2,3-triazole, a=4 and b=2.

[0202] 33. The method of any one of aspects 1 to 32, wherein X=1,2,3-triazole, a=4 and b=3.

[0203] 34. The method of any one of the preceding aspects, wherein X=amide, a=1 and b=0.

[0204] 35. The method of any one of the preceding aspects, wherein X=amide, a=1 and b=1.

[0205] 36. The method of any one of the preceding aspects, wherein X=amide, a=1 and b=2.

[0206] 37. The method of any one of the preceding aspects, wherein X=amide, a=1 and b=3.

[0207] 38. The method of any one of the preceding aspects, wherein X=amide, a=2 and b=0.

[0208] 39. The method of any one of the preceding aspects, wherein X=amide, a=2 and b=1.

[0209] 40. The method of any one of the preceding aspects, wherein X=amide, a=2 and b=2.

[0210] 41. The method of any one of the preceding aspects, wherein X=amide, a=2 and b=3.

[0211] 42. The method of any one of the preceding aspects, wherein X=amide, a=3 and b=0.

[0212] 43. The method of any one of the preceding aspects, wherein X=amide, a=3 and b=1.

[0213] 44. The method of any one of the preceding aspects, wherein X=amide, a=3 and b=2.

[0214] 45. The method of any one of the preceding aspects, wherein X=amide, a=3 and b=3.

[0215] 46. ​​The method of any one of the preceding aspects, wherein X=amide, a=4 and b=0.

[0216] 47. The method of any one of the preceding aspects, wherein X=amide, a=4 and b=1.

[0217] 48. The method of any one of the preceding aspects, wherein X=amide, a=4 and b=2.

[0218] 49. The method of any one of the preceding aspects, wherein X=amide, a=4 and b=3.

[0219] 50. The method of any one of aspects 1 to 49, wherein a is 1.

[0220] 51. The method of any one of aspects 1 to 50, wherein a is 2.

[0221] 52. The method of any one of the preceding aspects, wherein a is 3.

[0222] 53. The method of any one of the preceding aspects, wherein a is 4.

[0223] 54. The method of any one of aspects 1-53, wherein the labeling compound is selected from the group consisting of an enzyme, a fluorescent dye, a luminescent dye, a metal chelate complex, and a moiety containing a radioisotope.

[0224] 55. The method of any one of aspects 1 to 54, wherein the labeling compound can be induced to emit light when electrochemically oxidized or reduced.

[0225] 56. The labeled compound is Ru 2+ or Ir 3+ 56. The method of any one of the preceding embodiments, comprising a metal ion which is

[0226] 57. The method of any one of aspects 1 to 56, wherein the labeling compound is covalently attached to the linker by a first conjugation method, and the first conjugation method is selected from the following group: click chemistry, amide, ester, imide, carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime, dihydropyridazine, thiol-maleimide, cycloaddition, photoclick, Staudinger ligation, Diels-Alder, tetrazine ligation, cross-coupling, Pictet-Spengler, quadricyclane.

[0227] 58. The method of any one of aspects 1 to 57, wherein the analyte-specific binding agent is selected from the group consisting of antibodies, analyte-specific fragments and / or derivatives of antibodies, aptamers, spiegelmers, DARPins, lectins, ankyrin repeat-containing proteins, and Kunitz-type domain-containing proteins.

[0228] 59. The method of any one of aspects 1 to 58, wherein the analyte-specific binding agent is covalently attached to the linker by a second conjugation method, the second conjugation method being selected from the following group: click chemistry, amide, ester, imide, carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime, dihydropyridazine, thiol-maleimide, cycloaddition, tetrazine ligation, photoclick, Staudinger ligation, Diels-Alder, cross-coupling, Pictet-Spengler, quadricyclane.

[0229] 60. The method of any one of the preceding aspects, wherein A of formula I represents a labeling compound and B of formula I represents an analyte-specific binding agent.

[0230] 61. The method of any one of the preceding aspects, wherein B of formula I represents a labeling compound and A of formula I represents an analyte-specific binding agent.

[0231] 62. The method of any one of aspects 1-61, wherein the analyte is immobilized on a solid phase before, during or after step (c).

[0232] 63. The method according to any one of aspects 1 to 62, wherein the sample is selected from the group consisting of sputum, saliva, fluid, urine, whole blood, hemolyzed whole blood, serum and plasma.

[0233] 64. The method of any one of aspects 1-63, wherein the complex of step (b) is provided in dissolved form and step (c) is carried out in a liquid aqueous buffer.

[0234] 65. The method of any one of aspects 1-64, wherein the liquid aqueous buffer is selected from phosphate, Tris buffer, citrate, cacodylate, barbital, glycine, HEPES, MES, PIPES, MOPS, Bis-Trismethane, ADA, Bis-Trispropane, ACES, MOPSO, BES, AMPB, TES, DIPSO, MOBS, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, gycinamide, Gly-Gly, HEPBS, bicine, TAPS, and mixtures thereof.

[0235] 66. The complex is represented by the following formulas 4-I to 4-X: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] The method of any one of the preceding aspects, wherein the compound is selected from the group consisting of at least one compound having at least one of the following formulas:

[0236] 67. The method according to any one of aspects 1 to 66, wherein step b) comprises peptide-based synthesis, preferably solid-phase peptide synthesis (SPPS).

[0237] 68. Use of a method according to any one of aspects 1 to 67 for detecting an analyte of interest in a sample.

[0238] 69. A kit for carrying out the detection of an analyte of interest in a sample, comprising, in separate containers: a) a solid phase capable of immobilizing said analyte; b) Formula I: [ka] wherein A represents a labeling compound and B represents an analyte-specific binding agent, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2 or 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3; and n is an integer of 1 to 20, preferably 1 to 15, or 2 to 20, preferably 2 to 15. Including the kit.

[0239] 70. The kit according to embodiment 69, wherein the complex is embodied in a dissolved form.

[0240] 71. Use of a kit according to any one of claims 69 to 70 for detecting an analyte of interest in a sample.

[0241] 72. Formula I: [ka] wherein A represents a labeling compound and B represents an analyte-specific binding agent, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2 or 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3; n is an integer of 1 to 20, preferably 1 to 15, or 2 to 20, preferably 2 to 15; Preferably, the compound is suitable for detecting an analyte of interest in a sample.

[0242] 73. A conjugate according to aspect 72, wherein A or B is selected from the group consisting of peptides, polypeptides and proteins.

[0243] 74. A complex according to any one of aspects 72-73, wherein A comprises an analyte-specific binding agent and B comprises a labeled compound, or B comprises an analyte-specific binding agent and A comprises a labeled compound.

[0244] 75. The conjugate of any one of aspects 72-74, wherein the analyte-specific binding agent is selected from the group consisting of antibodies, analyte-specific fragments and / or derivatives of antibodies, aptamers, spiegelmers, DARPins, lectins, ankyrin repeat-containing proteins, and Kunitz-type domain-containing proteins, and the labeling compound is selected from the group consisting of enzymes, fluorescent dyes, luminescent dyes, metal chelate complexes, and moieties containing radioisotopes.

[0245] 76. A method for synthesizing a conjugate according to any one of aspects 72 to 75, comprising: a) providing a monomer or a derivative thereof, said monomer being an amino acid comprising an amino group, a carboxy group and at least one hydroxyl group or at least two hydroxyl groups, said amino group or said carboxy group being protected by a first protecting group and said at least one hydroxyl group or each hydroxyl group being protected by a second protecting group; b) using said monomer in a process of solid phase peptide synthesis, cleaving said first and second protecting groups to obtain a compound of formula I [ka] wherein A represents a labeling compound and B represents an analyte-specific binding agent, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2 or 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3; forming a complex, wherein n is an integer from 1 to 20, preferably from 1 to 15, or from 2 to 20, preferably from 2 to 15; A method comprising:

[0246] 77. The method of embodiment 76, wherein the first and / or second protecting group is selected from the group consisting of Fmoc, tBu, Boc, ethers, esters and acetals.

[0247] 78. The monomer or a derivative thereof is selected from the following formulae m-1 to m-3: [ka] 78. The method of any one of aspects 76 to 77, wherein Fmoc means a 9-fluorenylmethoxycarbonyl protecting group.

[0248] 79. The following formula II [ka] A monomer used for peptide-based synthesis comprising: In the formula, PG1, PG2, PG3, PG4, PG5, and PG6 each independently represent a protecting group. Preferably, when PG1 is a protecting group, PG2 = H, or vice versa; X is selected from the group consisting of O, S, CH2, SO, SO2 or 1,2,3-triazole and amide; a is 1 to 5, preferably 1 to 4, more preferably 1 to 2; b is 0 to 3;

[0249] 80. The monomer according to embodiment 79, wherein the protecting groups PG1, PG2, PG3, PG4, PG5, PG6 are independently selected from the group consisting of Fmoc, tBu, Boc, ether, ester and acetal.

[0250] 81. The monomer according to any one of aspects 79 to 80, wherein at least two of the protecting groups selected from the group consisting of PG1, PG2, PG3, PG4, PG5, PG6 are linked to each other, for example by at least one covalent bond.

[0251] 82. The monomer or derivative thereof is represented by the following formulae m-1 to m-3: [ka] 82. The monomer according to any one of embodiments 79 to 81, selected from: EXAMPLES

[0252] The following examples are offered to illustrate, but not to limit, the invention claimed herein.

[0253] Example 1: Synthesis of "clicked" side chain derivatives

[0254] [ka] Scheme 1. Synthesis of compound 4. i) MsCl, DMAP (catalyst), anhydrous pyridine, 1.5 h; ii) NaN3, DMF, 6 h; iii) Fmoc-L-propargylglycine, CuSO4, sodium L-ascorbate, DMF / H2O, 3 h.

[0255] Synthesis of compound 2 2,3:4,5-Di-O-isopropylidene-D-arabitol (267 mg, 1.19 mmol) was dissolved in anhydrous pyridine (3 mL) and cooled in an ice bath. 4-(Dimethylamino)pyridine (catalyst) and methanesulfonyl chloride (110 μl, 1.43 mmol) were added. The reaction mixture was stirred at 0° C. for 1.5 h. Pyridine was evaporated in vacuum, the residue was dissolved in CHCl and washed with HO and saturated NaCl solution. The organic phase was dried over anhydrous NaSO, the solvent was evaporated in vacuum and the oily residue was purified by flash column chromatography (SiO, n-hexane:EtOAc 7:3) to give 318 mg of mesylate 2 (86%); 1 H NMR (400 MHz, chloroform-d) δ ppm 1.32 (s, 3H) 1.36-1.43 (m, 9H) 3.01-3.12 (s, 3H) 3.66-3.75 (m, 1H) 3.91-3.97 (m, 1H) 4.00-4.07 (m, 1H) 4.11-4.20 (m, 2H) 4.26-4.33 (m, 1H) 4.47-4.58 (m, 1H).

[0256] Synthesis of compound 3 Mesylate 2 (1.29 g, 4.16 mmol) was dissolved in DMF (20 mL), NaN3 (324 mg, 4.99 mmol) was added and the reaction mixture was stirred at 85 °C for 6 h. The solvent was evaporated in vacuum and the residue was dissolved in EtOAc and washed with H2O and NaCl saturated solution. The organic phase was dried over anhydrous Na2SO4, the solvent was evaporated in vacuum and the residue was purified by flash column chromatography (SiO2, n-hexane: EtOAc 9:1) to give 788 mg of azide 3 (74%); 1 H NMR (400 MHz, chloroform-d) δ ppm 1.32 (s, 3H) 1.38 (m, 6H) 1.44 (s, 3H) 3.31 (m, 1H) 3.65 (m, 1H) 3.71-3.78 (m, 1H) 3.91-3.98 (m, 1H) 3.98-4.04 (m, 1H) 4.06-4.17 (m, 2H).

[0257] Synthesis of compound 4 Azide 3 (1.63 g, 6.34 mmol) and Fmoc-L-propargylglycine (2.13 g, 6.34 mmol) were dissolved in 25 mL of DMF and 0.5 mL of HO. CuSO4 (253 mg, 1.58 mmol) and sodium L-ascorbate (3.13 g, 18.8 mmol) were added and the reaction mixture was stirred at room temperature for 3 h. HO was added and the mixture was extracted three times with Et2O. The organic phase was dried over anhydrous Na2SO4, the solvent was evaporated in vacuum and the residue was purified by chromatography (RP-C18AQ, 9:1 to 1:9 HO:ACN gradient elution) to give 2.14 g of Fmoc-protected amino acid 4 (57%); 1 H NMR (400MHz, chloroform-d) δppm 1.25-1.52(m,12H)3.16-3.38(m,1H)3.38-3.50(m,1H)3.50-3.61(m,1H )3.91-3.98(m,1H)4.00-4.08(m,1H)4.10-4.17(m,1H)4.19-4.28(m,2H) 4.31-4.46(m,3H)4.48-4.68(m,1H)4.69-4.95(m,1H)5.78-6.15(m,1H) 7.27-7.34(m,2H)7.35-7.44(m,2H)7.53-7.63(m,3H)7.70-7.87(m,2H).

[0258] Example 2: Synthesis of Asp / Glu derivatives [ka]

[0259] Scheme 2. Synthesis of Asp / Glu derivatives. i) H2, Pd / C (catalyst), methanol, 8 h; ii) Fmoc-Asp-OAll or Glu-Asp-OAll, HBTU, DIPEA, anhydrous DMF, 14 h; iii) Pd(PPh3)4, morpholine, anhydrous THF, 30 min.

[0260] Synthesis of compound 5 Azide 3 (2.19 g, 8.5 mmol) was dissolved in methanol (30 mL) and then palladium(0) on activated carbon was added (catalyst). After evacuating the air and saturating the atmosphere with H2, the reaction mixture was vigorously stirred under a constant supply of H2 for 8 h. The catalyst was removed by filtration through a pad of Celite and the filtrate was dried under reduced pressure to give 1.95 g of amine 5, which was used directly in the next step without further purification (98%). 1 H NMR (400 MHz, chloroform-d) δ ppm 1.27 (s, 3H) 1.30 (s, 3H) 1.31-1.35 (m, 6H) 2.77 (dd, J = 13.4, 6.4 Hz, 1H) 2.97 (dd, J = 13.4, 3.6 Hz, 1H) 3.55 (t, J = 8.0 Hz, 1H) 3.82-3.92 (m, 2H) 3.93-4.01 (m, 1H) 4.02-4.11 (m, 1H).

[0261] Synthesis of compound 6 In a three-neck round-bottom flask under argon atmosphere, Fmoc-Asp-OAll (2.28 g, 5.76 mmol) was dissolved in anhydrous DMF (7 mL), followed by the addition of (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 2.4 g, 6.34 mmol) and diisopropylethylamine (DIPEA, 1.54 mL, 8.64 mmol). After stirring at room temperature for 10 min, amine 2 (1.47 g, 6.34 mmol), pre-dissolved Dry anhydrous DMF (3 mL) was added and the mixture was stirred at room temperature for 15 h. The reaction mixture was poured into H2O and the aqueous phase was extracted three times with EtOAc. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was then purified twice by flash column chromatography (SiO2, n-hexane / EtOAc gradient elution from 3:2 to 2:3) and (SiO2, n-hexane / EtOAc gradient elution from 3:2 to 1:4) to give 2.88 g of product (82%); 1H NMR (400MHz, chloroform-d) δ ppm 1.28-1.40 (m, 9H) 1.42 (s, 3H) 2.74 (dd, J = 15.9, 4.4Hz, 1H) 2.98 (dd, J = 15.8, 4.4Hz, 1H) 3.48-3.57 (m, 3H) 3.89-4.03 (m, 3H) 4.07-4.18 (m, 1H) 4.18-4.34 (m, 2H) 4.37-4.52 (m, 1H) 4.52-4.77 (m, 3H) 5.23 (dd, J = 10.4, 1.1Hz, 1H) 5.32 (br d,J=17.2Hz,1H)5.83-5.97(m,1H)6.03-6.30(m,2H)7.27-7.34(m,2H)7.34-7.44(m,2H)7.52-7.66(m,2H)7.75(d,J=7.5Hz,2H).

[0262] Synthesis of compound 7 The allyloxycarbonyl-protected Glu derivative 7 was prepared using the same procedure as for compound 6, starting from Fmoc-Glu-OAll (3.19 g, 7.81 mmol), HBTU (3.26 g, 8.6 mmol), DIPEA (2 mL, 11.7 mmol) and amine 5 (1.987 g, 8.6 mmol). After work-up, purification was carried out by flash column chromatography (SiO2, 1:1 to 1:4 n-hexane / EtOAc gradient elution) to give 4.75 g of the desired product (97%); 1 H NMR(400MHz,chloroform-d)δppm 1.30-1.38(m,9H)1.41(s,3H)1.95-2.35(m,4H)3.49-3.59(m,3H)3.91-4.05(m,3H)4.13(dd ,J=8.5,6.1Hz,1H)4.18-4.25(m,1H)4.31-4.46(m,3H)4.64(d,J=5.6Hz,2H)5.25(dd,J=10.4 ,1.1Hz,1H)5.32(d,J=17.2Hz,1H)5.74(d,J=7.4Hz,1H)5.83-5.96(m,1H)6.18-6.28(m,1H) 7.31(tt,J=7.4,1.1Hz,2H)7.39(t,J=7.5Hz,2H)7.59(t,J=6.3Hz,2H)7.76(d,J=7.1Hz,2H).

[0263] Synthesis of compound 8 In a three-necked round-bottom flask under argon atmosphere, compound 6 (2.46 g, 4.04 mmol) and tetrakis(triphenylphosphine)palladium(0) (462 mg, 0.40 mmol) were dissolved in anhydrous THF (60 mL), followed by the addition of morpholine (0.524 mL, 6.06 mmol). The mixture was stirred for 30 min, after which 20 mL of H2O and 20 mL of saturated NaHCO3 solution were added. The formed precipitate was removed by filtration, and the filtrate was acidified to pH ∼1-2 using 2N HCl. The mixture was then extracted three times with EtOAc, and the organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified twice by flash column chromatography (RP-C18AQ, 3:2 to 2:3 HO / ACN gradient elution) and (RP-C18AQ, 3:2 to 3:7 HO / ACN gradient elution) to give 1.60 g of product (70%). 1 H NMR(400MHz,chloroform-d)δppm 1.29-1.51(m,12H)2.75-2.87(m,1H)2.94-3.08(m,1H)3.48-3.65(m,3H)3.92-4.06(m,2H)4.07-4.17(m,2H)4.18-4.25(m,1H)4.28-4. 43(m,2H)4.49-4.66(m,1H)6.19-6.37(m,1H)6.67-6.84(m,1H)7.25-7.34(m,2H)7.33-7.45(m,2H)7.52-7.67(m,2H)7.70-7.81(m,2H).

[0264] Synthesis of compound 9 Starting from protected compound 7 (4.62 g, 7.42 mmol), tetrakis(triphenylphosphine)palladium(0) (429 mg, 0.37 mmol) and morpholine (0.770 mL, 8.90 mmol), the deprotected Glu derivative 9 was prepared using the same procedure as for compound 8. After workup, the crude product was purified by flash column chromatography (RP-C18AQ, 3:2 to 2:3 H2O / ACN gradient elution) to give 3.64 g of product (84%); 1H NMR (400MHz, chloroform-d) δ ppm 1.31-1.41 (m, 9H) 1.42-1.48 (m, 3H) 2.06-2.16 (m, 1H) 2.17-2.30 (m, 1H) 2.38-2.49 (m, 1H) 2.50-2.63 (m, 1H) 3.50-3.65 (m, 3H) 3.94-4.08 (m, 3H) 4.13-4.19 (m, 1H) 4.20-4.26 (m, 1H) 4.30-4.48 (m, 3H) 6.04 (br d,J=6.8Hz,1H)6.42-6.57(m,1H)7.32(td,J=7.4,0.8Hz,2H)7.41(t,J=7.5Hz,2H)7.60(t,J=6.3Hz,2H)7.77(d,J=7.5Hz,2H).

[0265] Example 3: Synthesis of Ser derivatives Example 3 shows the synthesis of Ser derivatives (Scheme 3). [ka]

[0266] Scheme 3. Synthesis of Ser derivative 14. i) NaH, allyl bromide, anhydrous THF, 14 h; ii) 4N HCl in dioxane, 2 h; iii) FmocOSu, Na2CO3, H2O / ACN 1:1, 3 h; iv) AD-mix β, H2O / tert-BuOH 1:1, 5 days; v) 2,2-dimethoxypropane, pTsOH, anhydrous EtOAc, 3 days.

[0267] Synthesis of compound 11 Under Ar atmosphere, NaH (60% dispersion in mineral oil, 1.46 g, 36.5 mmol) was added in small portions to anhydrous THF (20 mL) pre-cooled to 0 °C. After stirring for 5 min, Boc-L-serine (10, 2.5 g, 12.2 mmol) was added slowly over 30 min. After the addition was complete, the mixture was stirred for an additional 30 min, then allyl bromide (1.84 g, 15.25 mmol) was added. The reaction mixture was stirred overnight (14 h) and slowly heated to room temperature. The reaction was quenched by adding 5 mL of H2O and the solvent was evaporated under reduced pressure. The residue was dissolved in H2O and cooled in an ice bath, after which the solution was acidified to pH approx. 1-2 with 2 N HCl. The aqueous phase was extracted 5 times with EtOAc and the organic layer was dried over anhydrous Na2SO4 and evaporated in vacuum. After dissolving the residue in CH2Cl2 (10 mL), 5 mL of 4N HCl in 1,4-dioxane was added and the mixture was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure and the residue was treated with Et2O to give a white precipitate, which was separated by filtration and dried in vacuum. 1.92 g of the product was obtained as the hydrochloride salt (87%); 1 H NMR (400MHz, heavy water) δppm 3.66-3.78(m,1H)3.79-4.04(m,3H)4.04-4.32(m,1H)4.08-4.20(m,1H)5.06-5.32(m,2H)5.64-5.87(m,1H).

[0268] Synthesis of compound 12 The allyl Ser derivative 11 (1.79 g, 9.92 mmol) was dissolved in a 1:1 mixture of H2O / ACN (30 mL), followed by the addition of Na2CO3 (2.10 g, 19.8 mmol) and Fmoc-N-hydroxysuccinimide ester (FmocOSu, 3.68 g, 10.9 mmol). The reaction mixture was stirred at room temperature for 3 h, and then the organic solvent was evaporated under reduced pressure. The pH was adjusted to 1-2 with 6N HCl, and the aqueous layer was extracted five times with EtOAc. The organic phase was then dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (RP-C18AQ, 3:2 to 2:3 H2O / ACN gradient elution). The fractions containing the product were pooled and the organic solvent was evaporated under reduced pressure. The aqueous phase was then extracted with EtOAc, and the organic layer was dried in vacuum to give 3.63 g of Fmoc-protected derivative 12 (96%); 1 H NMR(400MHz,chloroform-d)δppm 3.60-3.79(m,1H)3.87-4.16(m,3H)4.24(br t,J=7.1Hz,1H)4.32-4.61(m,3H)5.21(d,J=10.3Hz,1H)5.27(d,J=17.2Hz,1H)5.63-5.77(m, 1H)5.77-5.93(m,1H)7.27-7.34(m,2H)7.35-7.43(m,2H)7.51-7.65(m,2H)7.68-7.80(m,2H).

[0269] Synthesis of compound 13 Fmoc-Ser derivative 12 (3.63 g, 9.90 mmol), asymmetric dihydroxylation mix β (AD mix β, 17.86 g) and Na2CO3 (1.05 g, 9.90 mmol) were suspended in a 1:1 mixture of H2O / tert-BuOH (60 mL) and the reaction mixture was stirred at 4 °C for 5 days. The OsO4 present in AD mix β was neutralized by adding 4 g of Na2S2O3 and stirring at room temperature for 10 min. After dilution with 100 mL of H2O, 30 mL of EtOAc was added and the two phases formed were separated. The pH of the aqueous phase was adjusted to 1-2 units using 6 N HCl and the aqueous layer was extracted four times with EtOAc. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. Finally, the residue was purified by flash column chromatography (RP-C18AQ, 3:1 to 1:1 H2O / ACN gradient elution) to give 2.24 g of vicinal diol 13 (56%); 1 H NMR (400MHz, acetone) δppm 3.31(s,2H)3.47-3.63(m,4H)3.71-3.86(m,2H)3.91-4.03(m,1H)4.22-4.30(m,1H)4.30-4.39(m,2H)4.40-4 .52(m,1H)6.85(d,J=8.4Hz,1H)7.29-7.36(m,2H)7.37-7.44(m,2H)7.64-7.75(m,2H)7.86(d,J=7.5Hz,2H).

[0270] Synthesis of compound 14 The diol derivative 13 (2.19 g, 5.46 mmol) was suspended in anhydrous EtOAc (30 mL), followed by the addition of 2,2-dimethoxypropane (27 mL, 218.3 mmol). Finally, p-toluenesulfonic acid (105 mg, 0.55 mmol) was added, and the reaction mixture was stirred at room temperature for 3 days. After the addition of 30 mL of 5% NaHCO3 solution, the organic solvent was evaporated under reduced pressure, and the aqueous phase was directly injected for chromatographic separation (RP-C18AQ, 4:1 to 1:1 H2O / ACN gradient elution). The fractions containing the product were pooled, and the organic solvent was removed under reduced pressure. After the remaining solution was cooled in an ice bath, the pH was adjusted to 1-2 units using 1N HCl, and the resulting mixture was extracted three times with EtOAc. The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuum to give 1.96 g of the acetal-protected product 14 (81%); 1 H NMR(400MHz,chloroform-d)δppm 1.29-1.50(m,6H)3.43-3.60(m,2H)3.65-3.83(m,2H)3.94-4.07(m,2H)4.1 7-4.30(m,2H)4.31-4.48(m,2H)4.50-4.62(m,1H)5.79-5.91(m,1H)6.04(br s,1H)7.26-7.34(m,2H)7.35-7.44(m,2H)7.54(br s,1H)7.75(d,J=7.5Hz,2H).

[0271] Example 4: Synthesis of hexahydroxyGlu derivatives Example 4 shows the synthesis of hexahydroxyGlu derivatives (Scheme 4). [ka]

[0272] Scheme 4. Synthesis of Glu derivative 19. i) PPh3, DIAD, phthalimide, anhydrous THF, 15 h; ii) 1M N2H2 in ethanol, ethanol, 3 h; iii) Fmoc-Glu-OAll, HBTU, DIPEA, anhydrous DMF, 14 h; iv) Pd(PPh3)4, morpholine, anhydrous THF, 30 min.

[0273] Synthesis of compound 16 Triisopropylideneheptol (15, 1.08 g, 3.25 mmol) was dissolved in anhydrous THF (12 mL) and the solution was cooled to -15 °C before adding triphenylphosphine (1.11 g, 4.22 mmol) and phthalimide (621 mg, 4.22 mmol). Finally, diisopropyl azodicarboxylate (DIAD, 0.831 mL, 4.22 mmol) was added dropwise over 5 min, and after the addition was complete, the mixture was slowly heated to room temperature and stirred for 15 h. After evaporating the solvent under reduced pressure, the residue was treated with 2N NaOH and the resulting mixture was extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure and the crude product was purified by flash column chromatography (SiO2, n-hexane / EtOAc gradient elution from 4:1 to 2:3) to give 1.31 g of product 16 (87%); 1 H NMR (400 MHz, chloroform-d) δ ppm 1.25 (s, 3H) 1.31 (s, 3H) 1.36 (s, 3H) 1.39 (s, 3H) 1.43 (s, 3H) 1.51 (s, 3H) 3.68 (dd, J = 14.2, 2.9 Hz, 1H) 3.94-4.14 (m, 5H) 4.18 (dd, J = 14.1, 10.2 Hz, 1H) 4.33 (dd, J = 6.5, 2.0 Hz, 1H) 4.54-4.61 (m, 1H) 7.63-7.71 (m, 1H) 7.76-7.84 (m, 1H).

[0274] Synthesis of compound 17 The phthalimide derivative 16 (1.76 g, 3.82 mmol) was dissolved in ethanol (30 mL) followed by the addition of 1 M hydrazine in ethanol (7.64 mL, 7, 64 mmol). The reaction mixture was then heated to reflux temperature and stirred for 3 h. After cooling to room temperature, a large amount of white precipitate was removed by filtration and the filtrate was concentrated under reduced pressure. The resulting residue was suspended in 1 M KOH solution (30 mL) and the aqueous phase was extracted five times with EtOAc. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. Finally, the crude product was purified by flash column chromatography (SiO2, gradient elution from EtOAc / methanol 98:2+1%Et3N to EtOAc / methanol 9:1+1%Et3N) to give 1.08 g of amine 17 (86%). 1 H NMR(400MHz,chloroform-d)δppm1.26-1.34(m,9H)1.35-1.40(m,6H)1.42(br s,2H)1.47(s,3H)2.87(dd,J=13.3,3.9Hz,1H)3.03(dd,J=13.2,8.1Hz,1H)3.82(dd,J=7.7,1.5Hz,1H )3.86-3.95(m,2H)3.95-4.02(m,1H)4.06-4.12(m,1H)4.13-4.20(m,1H)4.25(dd,J=6.8,1.6Hz,1H).

[0275] Synthesis of compound 18 In a three-necked round-bottom flask under argon atmosphere, Fmoc-Glu-OAll (2.12 g, 5.19 mmol) was dissolved in anhydrous DMF (5 mL), followed by the addition of HBTU (2.16 g, 5.71 mmol) and DIPEA (1.36 mL, 7.78 mmol). After stirring at room temperature for 10 min, amine 17 (1.89 g, 5.71 mmol), predissolved in anhydrous DMF (5 mL) was added, and the mixture was stirred at room temperature for 15 h. The reaction mixture was poured into H2O, and the aqueous phase was extracted three times with EtOAc. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was then purified twice by flash column chromatography (SiO2, 1:1 to 1:4 n-hexane / EtOAc gradient elution). Fractions containing impurities were pooled and purified again by flash column chromatography (SiO2, 3:2 to 1:4 n-hexane / EtOAc gradient elution). 3.22 g of product (86%) was obtained; 1 H NMR (400MHz, chloroform-d) δ ppm 1.27-1.36 (m, 9H) 1.36-1.41 (m, 6H) 1.43-1.51 (m, 3H) 2.15-2.30 (m, 3H) 3.24-3.36 (m, 1H) 3.81-4.02 (m, 5H) 4.05-4.13 (m, 2H) 4.16-4.30 (m, 3H) 4.32-4.44 (m,3H)4.62(d,J=5.5Hz,2H)5.22(d,J=10.4Hz,1H)5.31(d,J=17.3Hz,1H)5.77-5.96(m,2H )6.19-6.32(m,1H)7.26-7.33(m,2H)7.34-7.42(m,2H)7.51-7.64(m,2H)7.70-7.78(m,2H).

[0276] Synthesis of compound 19 In a three-necked round-bottom flask under argon atmosphere, allyloxycarbonyl protected derivative 18 (3.16 g, 4.37 mmol) and tetrakis(triphenylphosphine)palladium(0) (252 mg, 0.22 mmol) were dissolved in anhydrous THF (30 mL), morpholine (0.454 mL, 5.24 mmol) was added, and the mixture was then stirred at room temperature for 20 min. After addition of 5% NaHCO3 solution (20 mL), the formed precipitate was removed by filtration and the filtrate was acidified to pH approx. 1-2 with 2N HCl. The mixture was then extracted three times with EtOAc, and the organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (RP-C18AQ, 3:2 to 2:3 H2O / ACN gradient elution). The fractions containing the product were pooled and the organic solvent was evaporated under reduced pressure. Finally, the remaining aqueous layer was extracted with EtOAc three times, and the organic layer was dried over anhydrous Na2SO4 and concentrated to dryness to give 2.56 g of the deprotected product 19 (86%); 1 H NMR(400MHz,acetonitrile-d3)δppm1.27-1.32(m,6H)1.32-1.40(m,9H)1.45(s,3H)1.85-1.94(m,1H)2.01-2.20(m,2H)2.25-2.38(m,2H)3.24-3.36 (m,1H)3.60-3.72(m,1H)3.81-3.93(m,2H)3.92-4.01(m,1H)4.03-4.13 (m,2H)4.11-4.20(m,1H)4.21-4.40(m,5H)6.37(d,J=7.7Hz,1H)6.72(br s,1H)7.29-7.40(m,2H)7.39-7.49(m,2H)7.69(m,J=7.2,3.90Hz,2H)7.85(d,J=7.5Hz,2H).

[0277] Example 5: Peptide synthesis Peptides were synthesized by fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis on a multiple peptide synthesizer, e.g. from Multisyntech. For this, 4.0 equivalents of each amino acid derivative were used. The amino acid derivatives were dissolved in N-methylpyrrolidone containing 1 equivalent of 1-hydroxy-7-azabenzotriazole. Peptides were synthesized on Tentagel R resin. The coupling reaction was carried out for 5 min in dimethylformamide as reaction medium with 4 equivalents of HATU and 8 equivalents of N,N-diisopropylethylamine (DIPEA) relative to the resin loading. The Fmoc group was cleaved after each synthesis step for 8 min using 25% piperidine in dimethylformamide. In the case of labels exhibiting an alkyne functionality, the alkyne group was introduced by coupling of Fmoc-propargylglycine. In the case of labels containing maleimide-functionalized lysine, the lysine was introduced by coupling of Fmoc-ivDdelysine. After synthesis, the resin was treated with 2% hydrazine in DMF for 2 × 30 min to release the ivDde-protected lysine, after which 6-maleimidohexanoic acid N-hydroxysuccinimide ester (10 equiv.) and DIPEA (10 equiv.) were added to the resin and incubated for 1 h, followed by washing 3 times with DMF.

[0278] Release of the peptide from the synthesis resin and cleavage of the acid-labile protecting groups were achieved with a cocktail containing trifluoroacetic acid, triisopropylsilane and water (38:1:1) at room temperature for 3 h. The peptide was then precipitated by mixing the reaction solution with chilled diisopropyl ether. The precipitate was filtered, rinsed again with cold diisopropyl ether, dissolved in a small amount of aqueous acetic acid and lyophilized. The resulting crude material was purified by preparative RP-HPLC using an acetonitrile / water gradient containing 0.1% trifluoroacetic acid. The identity of the purified material was confirmed by ion spray mass spectrometry.

[0279] Release of the peptide from the synthesis resin and cleavage of the acid-labile protecting groups were achieved with a cocktail containing trifluoroacetic acid, triisopropylsilane and water (38:1:1) at room temperature for 3 h. The peptide was then precipitated by mixing the reaction solution with chilled diisopropyl ether. The precipitate was filtered, rinsed again with cold diisopropyl ether, dissolved in a small amount of aqueous acetic acid and lyophilized. The resulting crude material was purified by preparative RP-HPLC using an acetonitrile / water gradient containing 0.1% trifluoroacetic acid. The identity of the purified material was confirmed by ion spray mass spectrometry.

[0280] Example 6: Metal Complex Linker Assembly General protocol for Ir / Ru linker conjugation: [ka] In a 25 mL flask, polyol linker (9.5 μmol, 1 equiv.) was dissolved in 9 mL of anhydrous DMF: DIPEA (19 μmol, 2 equiv.) and metal complex NHS ester (9.5 μmol, 1 equiv.) (dissolved in 1 mL of DMF) were added. The reaction was allowed to react overnight at room temperature. After that, the solvent was evaporated in vacuum and the resulting red solid was dissolved in 2 mL of DMSO, and the product was purified by HPLC-prep to obtain a red solid. X can be the same as defined above. X can be selected from the group consisting of O, S, CH2, SO, SO2 or 1,2,3-triazole and amide.

[0281] HPLC preparative method for Ru complexes: Column C18 column, flow rate 10mL / min: 0 min: 98%H2O 0.05%TFA, 2%CH3CN 0.05%TFA; 0~10 minutes: 98%H2O 0.05%TFA, 2%CH3CN 0.05%TFA; 10~60 minutes: 50%H2O 0.05%TFA; 50%CH3CN 0.05%TFA; 60~90 minutes: 0%H2O 0.05%TFA;100%CH3CN 0.05%TFA; HPLC preparative method for Ir complexes: Column C18 column, flow rate 10mL / min: 0 min: 98% H2O; 2% CH3CN; 0–10 min: 98% H2O; 2% CH3CN; 10–60 min: 50% H2O; 50% CH3CN; 60–90 min: 0% H2O; 100% CH3CN;

[0282] Compound 20: BPRu-[FA41]5-Pra-amide [ka] 16.0 mg of product, 73% yield, red oil, TFA salt HPLC-MS: (m / z) [M] 2+ / 2Calculated value 1037.40, actual value 1037.77.

[0283] Compound 21:BPRu-[FA41] 10 -Pra-amide [ka] 4.8 mg of product, 35% yield, red oil, TFA salt HPLC-MS: (m / z) [M] 2+ / 2 calculated value 1692.69, found value 1694.03; (m / z) [M+H] 3+ / 3 Calculated value 1128.79, measured value 1129.05

[0284] Compound 22: Ir-[FA41]5-Pra-amide [ka] 12.1 mg of product, 37% yield, red oil, DIPEA salt HPLC-MS: (m / z) [M+2H] 2+ / 2Calculated value 1400.91, measured value 1401.87

[0285] Compound 23:Ir-[FA41]10 -Pra-amide [ka] 10.4 mg of product, 56% yield, red oil, DIPEA salt HPLC-MS: (m / z) [M+3H] 3+ / 3 Calculated value 1371.14, measured value 1371.74

[0286] Compound 24: BPRu-[FA40]5-Pra-amide [ka] 5.0 mg of product, 24% yield, red oil, TFA salt HPLC-MS: (m / z) [M] 2+ / 2Calculated value 1002.36, actual value 1002.55.

[0287] Compound 25: BPRu-[FA40] 10 -Pra-amide [ka] 0.9 mg product, 7% yield, red oil, TFA salt HPLC-MS: (m / z) [M] 2+ / 2 calculated value 1622.61, found value 1623.44; (m / z) [M+H] 3+ / 3 Calculated value 1082.07, measured value 1082.75

[0288] Compound 26: Ir-[FA40]5-Pra-amide [ka] 5.5 mg product, 17% yield, red oil, DIPEA salt HPLC-MS: (m / z) [M+2H] 2+ / 2Calculated value 1365.87, measured value 1366.82;

[0289] Compound 27: Ir-[FA40] 10 -Pra-amide [ka] 4.4 mg of product, 24% yield, red oil, DIPEA salt HPLC-MS: (m / z) [M+3H] 3+ / 3 Calculated value 1324.41, measured value 1125.08

[0290] Compound 28:BPRu-[S773]5-K(MH)-NH2 [ka] 3.0 mg product, 3% yield HPLC-MS: (m / z) [M+2H]2+ / 2 calculated 1176.7, found 1176.7;

[0291] Compound 29:sRu-[FA30]5-Pra-NH2 [ka] 6.9 mg product, 7% yield HPLC-MS: (m / z) [M+2H]2+ / 2 calculated 974.0, found 973.8;

[0292] Example 7: Non-site-specific conjugation of labels Elecsys high sensitivity troponin-T (HS Tn-T) clone 5D8 was used for conjugation and ELC (electrochemiluminescence) performance evaluation of newly synthesized Ru (ruthenium) or Ir (iridium) complexes containing various polyol linkers. For labels with alkyne functionality, labeling was performed by click reaction. To generate clickable antibodies, azide functionality was introduced to IgG by conjugating azide-PEG4-NHS at a stoichiometry of 1:5 or 1:10 (IgG:label), and MABs were prepared using the same method. <tn-t>Chim-5D8-IgG-PEG4-N3 (1:5 or 1:10) was obtained. The azido antibody was then conjugated with alkynyl-polyol-Ru / Ir labels via copper-catalyzed click chemistry by using tetrakis(acetonitrile)copper(I) tetrafluoroborate as the copper I source and THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) as the water-soluble copper ligand. Click reactions were carried out in 50 mM KPP, 150 mM KCl, pH 7.4, containing 5% acetonitrile.

[0293] For labels with maleimide functionality, thiol functionality was introduced onto IgG by conjugation with N-succinimidyl-S-acetylthiopropionate (SATP) at a stoichiometry of 1:5 or 1:10 (IgG:SATP), followed by MAB <tn-t>Chim-5D8-IgG-SATP (1:5 or 1:10) was obtained. The acetyl protection from sulfur was removed by hydroxylamine treatment to obtain the final sulfhydryl-containing antibody. This SH antibody was then conjugated with maleimide-polyol-Ru / Ir label in 50 mM KPP, 150 mM KCl, pH 7.4, containing 5% DMSO.

[0294] Conjugates of Troponin-T (HS Tn-T) clone 5D8 with compound 30 and compound 31 were also obtained following the same procedure and used as a comparison for Elecsys performance.

[0295] Compound 30: BPRu-MEA [ka]

[0296] Compound 31: BPRu-DADOO-PEG24-MH [ka]

[0297] Example 8: Elecsys Performance All assay variants were run on a Cobas E170 Module using the Troponin T hs assay protocol with a blank control (Diluent Multiassay, Id. 11732277122, Roche Diagnostics GmbH, Mannheim, Germany), Cal1 and Cal2 from the Troponin T hs CalSet (Id. 05092752190, Roche Diagnostics GmbH, Mannheim, Germany) using the Troponin T hs assay specifications.

[0298] These conjugates were then used in the Troponin T hs Elecsys assay variant (Id.05092744190, Roche Diagnostics GmbH, Mannheim, Germany) replacing the original R2 reagent at a concentration of 2.5 μg / ml. ECL measurements were performed after incubation of the conjugates for 1 week at 4° C. in TnT R2 buffer.

[0299] The incorporation rates of PEG and polyol-based linkers are similar (see average labeling rates). New polyol linkers such as Ru-[FA41]10-Pra-NH2 (compound 20) and Ir-[FA41]10-Pra-NH2 (compound 23) show comparable signal-to-noise ratios in the low (Cal1 / MA) and high (Cal2 / MA) ranges of TnT analytes when compared to reference compounds 30 and 31. MA: serum blank; Cal1: 18 ng / L; Cal2: 4200 ng / L. [Table 1]

[0300] Example 9: Conjugate stability FIG. 2 shows the conjugate stability of different compounds disclosed herein. After the initial measurement, the stability of the conjugates was evaluated by measuring the ECL signal after incubation of the samples for 1 and 3 weeks at 35° C. Signal recovery was calculated by comparing the obtained signal with that of the sample incubated for 1 week at 4° C. The new labels (Ir-[FA41]10-Pra-NH2) (compound 23) and (Ru-[FA41]10-Pra-NH2) (compound 21) showed significantly improved stability over the last generation polyol-based label (BPRu-(MF74)5-K(MH)amide). In addition, a comparison of the linkers conjugated via maleimide chemistry with those conjugated by click chemistry shows the improved stability of the latter over time. [Table 2]

[0301] This patent application claims priority to European Patent Application No. 22157541.8, the contents of which are incorporated herein by reference.

Claims

1. 1. A method for detecting an analyte of interest in a sample, comprising: a) providing said sample containing said analyte of interest; b) providing a conjugate comprising a linker, said linker covalently binding a label compound and an analyte-specific binding agent, said label compound capable of generating a detectable signal, preferably a chemiluminescence-based signal, more preferably an electrochemiluminescence-based signal; c) allowing the sample of step a) to bind to the complex of step b); d) detecting the analyte of interest by using the detectable signal of the labeled compound; Including, The conjugate has formula I: 【Chemistry 1】 wherein A represents the labeled compound and B represents the analyte-specific binding agent, or vice versa; X is O, S, or CH ２ , SO, SO ２ , 1,2,3-triazoles and amides; a is 1 to 5, preferably 1 to 4, more preferably 1 or 2; b is 0 to 3; The method wherein n is an integer from 2 to 20.

2. The conjugate has the following formulae 4-I to 4-X: 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 2. The method of claim 1, wherein the compound is selected from the group consisting of at least one compound having at least one of the following properties:

3. 10. The method of claim 1, wherein the labeling compound is selected from the group consisting of an enzyme, a fluorescent dye, a luminescent dye, a metal chelate complex, and a moiety containing a radioisotope.

4. The labeling compound is Ru ２＋ or Ir ３＋ 10. The method of claim 1, comprising a metal ion wherein

5. 2. The method of claim 1, wherein the analyte-specific binding agent is selected from the group consisting of antibodies, analyte-specific fragments and / or derivatives of antibodies, aptamers, spiegelmers, DARPins, lectins, ankyrin repeat-containing proteins, and Kunitz-type domain-containing proteins.

6. 10. The method of claim 1, wherein the analyte is immobilized to a solid phase before, during, or after step (c).

7. The method according to any one of claims 1 to 6, wherein step b) comprises a peptide-based synthesis, preferably solid phase peptide synthesis (SPPS).

8. Use of the method of any one of claims 1 to 6 for detecting said analyte of interest in said sample.

9. 1. A kit for carrying out the detection of an analyte of interest in a sample, comprising, in separate containers: a) a solid phase capable of immobilizing said analyte; b) Formula I: 【Chemistry 12】 wherein A represents the labeled compound and B represents the analyte-specific binding agent, or vice versa; X is O, S, or CH ２ , SO, SO ２ or selected from the group consisting of 1,2,3-triazoles and amides; a is 1 to 5, preferably 1 to 4, more preferably 1 or 2; b is 0 to 3; n is an integer of 1 to 20, preferably 1 to 15, or 2 to 20, preferably 2 to 15; Includes a kit.

10. 10. Use of the kit of claim 9 for detecting the analyte of interest in the sample.

11. Formula I, 【Chemistry 13】 wherein A represents a labeled compound and B represents an analyte-specific binding agent, or vice versa; X is O, S, or CH ２ , SO, SO ２ or selected from the group consisting of 1,2,3-triazoles and amides; a is 1 to 5, preferably 1 to 4, more preferably 1 or 2; b is 0 to 3; n is an integer from 1 to 20, preferably from 1 to 15, or from 2 to 20, preferably from 2 to 15; Preferably, the compound is suitable for detecting an analyte of interest in a sample.

12. A method for synthesizing the conjugate of claim 11, comprising: a) providing a monomer or a derivative thereof, said monomer being an amino acid comprising an amino group, a carboxy group and at least two hydroxyl groups, said amino group or said carboxy group being protected by a first protecting group and said at least one hydroxyl group or each hydroxyl group being protected by a second protecting group; b) using said monomer in a process of solid phase peptide synthesis, cleaving said first protecting group and said second protecting group, to obtain a compound of formula I 【Chemistry 14】 wherein A represents the labeled compound and B represents the analyte-specific binding agent, or vice versa; X is O, S, or CH ２ , SO, SO ２ or selected from the group consisting of 1,2,3-triazoles and amides; a is 1 to 5, preferably 1 to 4, more preferably 1 or 2; b is 0 to 3; forming a complex, wherein n is an integer from 1 to 20, preferably from 1 to 15, or from 2 to 20, preferably from 2 to 15; A method comprising:

13. Formula II below 【Chemistry 15】 A monomer used for peptide-based synthesis comprising: In the formula, PG1, PG2, PG3, PG4, PG5, and PG6 each independently represent a protecting group. Preferably, when PG1 is a protecting group, PG2 = H, or vice versa; X is O, S, or CH ２ , SO, SO ２ or selected from the group consisting of 1,2,3-triazoles and amides; a is 1 to 5, preferably 1 to 4, more preferably 1 or 2; b is 0 to 3;

14. 14. The monomer of claim 13, wherein at least two of the protecting groups selected from the group consisting of PG1, PG2, PG3, PG4, PG5, and PG6 are linked to each other, e.g., by at least one covalent bond.

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