Casein and casein analogue conjugates and their use for biological target staining procedures

Abstract

The present invention provides compounds and compositions suitable for target visualization, such as immunostaining procedures, that form covalent colorimetric staining and / or fluorescent staining. Certain disclosed compounds have Formula I, (P1) n-L-P2 wherein P1 is a first aromatic ring system comprising at least two fused rings wherein at least one of the fused rings comprises a structure as follows: P2 is a second aromatic ring system substituted with at least one-OH group, L is a linker, and n = 1-20. Particular examples of compounds satisfying both Formula I and Formula II include compounds according to the invention that can be used to form covalent conjugates at or around a biological target. Embodiments of a target staining regimen include providing at least one disclosed compound, and detecting a target in a sample using the compound. Kits for detecting biological targets are also disclosed. # imgabs0 #

Description

[0001] Related Applications

[0002] This application includes subject matter related to that disclosed in U.S. Provisional Application No. 63 / 477,653, filed on December 29, 2022, and U.S. Provisional Application No. 63 / 485,651, filed on February 17, 2023, which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to embodiments of compounds, compositions comprising such compounds, and methods of using such compounds and compositions to perform staining procedures to visualize biological targets, and more particularly to fused-ring tyramide conjugates, such as naphthol-tyramide conjugates, suitable for forming covalent colorimetric stains for in situ hybridization (ISH) and immunostaining procedures. Background Art

[0004] Methods for detecting targets, such as protein targets in biological samples, are commonly used for research purposes, and for analyzing pathological samples, such as formalin-fixed paraffin-embedded samples for diagnosis and tracking disease progression. Specific binding agents, such as antibodies, that specifically bind to the desired antigen can be produced. This allows the detection of small amounts of antigens within the sample. It is then necessary to visualize the antibody-antigen complex. This can be achieved in a variety of ways, such as by using a chromophore deposited at the target. By using permanent covalent labels, particularly labels that produce different colors, procedures for visualizing biological targets can be facilitated. For example, for IHC protocols involving hematoxylin, this counterstain may be difficult to distinguish from DAB (3,3'-diaminobenzidine) stains. In addition, other reagents (such as AEC (3-amino-9-ethylcarbazole)) are soluble in solvents commonly used to rinse samples, and the staining produced by the AEC IHC protocol is therefore washed away.

[0005] Therefore, there is a need for compounds and compositions suitable for staining procedures that form covalent colorimetric stains. These compounds and compositions should preferably provide a number of advantages, such as: (1) similar or clearer staining patterns and intensities than DAB or Fast Red; (2) permanent stains similar to those provided by DAB that are resistant to solvent washout and can be mounted in aqueous or organic mounting media; (3) covalent attachment to or around the target rather than simply producing a precipitate; (4) producing permanent stains of different colors, such as red, purple, blue, and green; and (5) allowing for fluorescence detection in addition to colorimetric detection. Summary of the invention

[0006] The present invention provides compounds and compositions suitable for target visualization, such as immunohistochemistry (IHC) staining procedures, which form covalent colorimetric stains. Certain disclosed embodiments involve the use of compounds that satisfy Formula I

[0007]

[0008] wherein L is a linker having from 1 to at least 50 atoms, more typically 2 to 20 atoms in the chain; 1 , P 2 and P 3 is an aromatic ring or an aromatic ring system; R is a chemical bond or independently an L group; and x and y are independently 0 to 5. 1 and P 3 The ring systems may be the same or different and each may contain at least two fused rings. 1 and / or P 3 At least one of the fused ring systems comprises the structure shown below: For certain embodiments, P 2 is a benzene ring substituted with at least one -OH group. The compounds suitable for the present invention may also satisfy the formula II

[0009] P 1 -LP 2

[0010] Formula II,

[0011] Among them, L, P 1 and P 2 As described in Formula I.

[0012] More specifically, P 1 and P 3 Typically hydrocarbon and / or heteroaryl ring systems. Typical hydrocarbon ring systems have 2 to 4 rings in a fused ring system. Suitable exemplary hydrocarbon ring systems include naphthalene, anthracenol, tetradecene alcohol, and phenanthranol. Heteroaryl fused ring systems are preferably electron-rich, examples include fused furan, fused thiophene, and fused pyrrole ring systems. 1 and / or P 3 may be substituted with at least one moiety selected to increase solubility in solvents commonly used in methods such as IHC, such as a water-soluble moiety selected to increase the water solubility of the compound. Exemplary solubilizing moieties include -SO 3 - 、-SO 3 H, -CO 2 - , -CO 2 H, -HPO 3 -2 , H 2PO 3 -1 , H 3 PO 3 , PEG, or their salt forms. L is selected from a covalent bond, C 1 -C 50 Aliphatic group, C 1 -C 50 Alkyl, C 1 -C 50 Alkylamide, C 1 -C 50 Ketone, C 1 -C 50 Diol or polyol. C 1 -C 50 The chain length is more usually C 1 -C 20 , or even more specifically C 3 -C 15 One exemplary linker family has the formula wherein n is 1 to 10, more typically 2-5, wherein a specific linker has L of

[0013] The compounds according to the present invention may also satisfy the formula III

[0014]

[0015] Regarding Formula III, R 1 and R 2 are independently selected from H and OH; R 3 -R 8 independently selected from H, -F, -Cl, -Br, -I, -OH, -OR 9 , where R 9 C 1 -C 6 Alkyl, -SO 3 - 、-SO 3 H, -CO 2 - , -CO 2 H, -HPO 3 -2 , H 2 PO 3 -1 , H 3 PO 3 , PEG, or their salt forms, wherein R 1 , R 2 , R 7 At least one of R 8 -R12 At least one of R is hydroxyl. 3 and R 4 together form at least one fused ring system; and / or R 4 and R 5 together form at least one fused ring system; and / or R 5 and R 6 together form at least one fused ring system; and n is 1 to 5.

[0016] Specific examples of compounds satisfying Formula I, II and / or III include

[0017]

[0018] The compounds according to the invention can be used to form covalent conjugates at or around a biological target. The biological target can be a cell, a tissue, a virus, a gene, an epitope, a target genetic sequence or a target protein sequence. Therefore, the invention also relates to a covalent conjugate comprising a biological sample containing a target and at least one compound according to formula I, formula II and / or formula III covalently bound to the target.

[0019] Also disclosed are embodiments of methods of using the disclosed compounds in target staining protocols. One embodiment of the method includes providing at least one compound according to Formula I, Formula II, and / or Formula III; and using the compound to detect a target in a sample. For example, the method may include contacting a biological sample so that a first compound is covalently bound to or substantially adjacent to the target. The first compound is then contacted with a diazo compound under conditions where the first compound is covalently linked to the diazo compound to form a conjugate, and the target is allowed to be detected. The first compound may exhibit a first optical signal when irradiated with light of an appropriate wavelength, and the conjugate may exhibit a second optical signal when irradiated with light of an appropriate wavelength, wherein the first optical signal and the second optical signal are different.

[0020] The method may include contacting the target with the first compound in the presence of a peroxidase to covalently couple the first compound to the target. Suitable peroxidases include, but are not limited to, horseradish peroxidase, microperoxidase, or ascorbate peroxidase (APEX). Exemplary diazo compounds include Fast Red, Fast Violet, and Fast Blue.

[0021] The method can be an IHC procedure. For example, the biological target can be recognized by a primary antibody. For these embodiments, the primary antibody can be coupled to a peroxidase. The method includes contacting the biological target with a primary antibody that binds to the target; and contacting the primary antibody with a secondary antibody directed against the primary antibody to form a primary-secondary antibody conjugate, wherein the secondary antibody is bound to a peroxidase. Exemplary primary antibodies include ESR1 antibodies, KRT15 antibodies, and / or CDH17 antibodies. For certain embodiments, the primary antibody is a mouse antibody, and the secondary antibody is poly-HRP-goat anti-mouse (GAM); or the primary antibody is a rabbit antibody, and the secondary antibody is poly-HRP-goat anti-rabbit (GAR). The target can be detected by colorimetry, such as by bright field microscopy, fluorescence, or both.

[0022] The present invention also provides a kit for detecting biological targets. An example of a kit according to the present invention includes a compound according to Formula I, Formula II and / or Formula III and a diazonium salt. A more specific example of a kit according to the present invention includes 3% bovine serum albumin; poly-HRP-goat anti-mouse (GAM), poly-HRP-goat anti-rabbit (GAR) and / or HRP-conjugated streptavidin; a compound according to Formula I, II and / or Formula III, such as a 5mM aqueous solution thereof; a buffer system for tyramide deposition, such as a 200mM citrate buffer at pH 4, and 7.5% stable hydrogen peroxide; a diazonium salt, such as Fast Red TR or Fast Blue BB; and a color development buffer, such as 100mM Tris at pH 8.5.

[0023] The above and other objects, features and advantages of the present invention will become more apparent from the following detailed description made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a flow chart of one embodiment of a method according to the present invention.

[0025] Figure 2 It is shown Figure 1 A flow chart of the steps of, but with reference to using a primary antibody that recognizes a target, using a secondary antibody conjugated to horseradish peroxidase (HRP) that recognizes the primary antibody, covalently depositing a tyramide compound according to the present invention, and then imaging using a diazonium salt that reacts with the tyramide compound.

[0026] Figure 3 is shown in horseradish peroxidase and H 2 O 2 Schematic representation of one embodiment of the method according to the invention for binding a tyramide compound according to the invention to a biological sample in the presence of .

[0027] Figure 4It is a bright field image of Peyer's patches (groups of lymphoid follicles in the small intestinal mucosa) stained with the tyramide compound according to the present invention.

[0028] Figure 5 is used with Figure 3 Comparative bright field images of Peyer's patches stained with DAB.

[0029] Figure 6 is a bright field image of intestinal villi stained with a tyramide compound according to the present invention.

[0030] Figure 7 is used with Figure 5 Comparative brightfield images of intestinal villi stained with DAB.

[0031] Figure 8 is a fluorescent image of intestinal villi stained with the tyramide compound according to the present invention.

[0032] Fig. 9 This is a DAPI counterstained image of intestinal villi.

[0033] Fig.10 is a bright-field image of intestinal villi counterstained with hematoxylin.

[0034] Fig.11 Provided are methods illustrating the use of formula I according to the present invention Staining images of the results obtained with compounds at concentrations of 0.5 mM and 1 mM and incubation times of 10 and 30 minutes.

[0035] Fig.12 Provided are staining images comparing the results obtained using the following substances: According to the first formula II [P 1 -LP 2 ] compound, i.e., in a 1 mM staining solution providing a 1 mM total compound concentration According to the second formula I of the present invention Compounds, i.e., in a 0.5 mM staining solution providing a 1 mM total compound concentration and the well-known diaminobenzidine (DAB).

[0036] Fig.13 Provided are histological staining images (20×) obtained using disclosed embodiments of the present invention showing that these embodiments are compatible for use with formalin-fixed paraffin-embedded (FFPE) tissue and organic mounting media.

[0037] Fig.14A histological staining image (20×) of mounted frozen tissue obtained using disclosed embodiments of the present invention is provided, showing that these embodiments are compatible for use with cryopreserved tissue.

[0038] Fig.15 Provided are histological staining images (20×) of FFPE tissue obtained using disclosed embodiments of the present invention, showing that these embodiments are compatible for use with aqueous mounting media.

[0039] Fig.16 Provided is a tissue staining image (20×) of FFPE breast tissue (ESRI) obtained using embodiments of the present disclosure showing fluorescence properties where the fluorescence is 3 times greater than the background intensity.

[0040] Fig.17 Provided are histologically stained images (20×) of FFPE tissue obtained using disclosed embodiments of the present invention, showing that these embodiments are compatible with hematoxylin.

[0041] Fig.18 Provided are histologically stained images (20×) of FFPE tissue obtained using disclosed embodiments of the present invention, showing that these embodiments are compatible with eosin.

[0042] Fig.19 Provided are histological staining images (20×) of FFPE tissue obtained using disclosed embodiments of the present invention, showing that these embodiments are compatible with methyl green.

[0043] Fig. 20 Provided are images (20×) of frozen tonsil tissue stained with Cytoseal 60 mounted using disclosed embodiments of the present invention showing double staining with DAB.

[0044] Fig.21 Provided are images (20×) of mouse intestinal tissue stained with Cytoseal 60 mounted using disclosed embodiments of the present invention showing double staining with DAB.

[0045] Fig. 22 Provided are images (20×) of mouse intestinal tissue stained with Cytoseal 60, obtained using disclosed embodiments of the present invention, showing dual staining capabilities.

[0046] Fig.23Provided are images (20×) of mouse intestinal tissue stained with Cytoseal 60 mounted using disclosed embodiments of the present invention, showing dual staining-fluorescence capabilities.

[0047] Fig.24 Provided are brain NeuN tissue staining images (20×) obtained using disclosed embodiments of the present invention showing blue immunohistochemistry (IHC) staining capabilities for aqueous mounting media.

[0048] Fig.25 Provided are brain NeuN tissue staining images (20×) obtained using disclosed embodiments of the present invention showing blue immunohistochemistry (IHC) staining capabilities against organic mounting media.

[0049] Fig.26 Provided are brain NeuN tissue staining images (20×) obtained using disclosed embodiments of the present invention showing red immunohistochemistry (IHC) staining capabilities for aqueous mounting.

[0050] Fig. 27 Provided are brain NeuN tissue staining images (20×) obtained using disclosed embodiments of the present invention showing red immunohistochemistry (IHC) staining capabilities for organic mounting.

[0051] Fig.28 ESRI Red stained images (20×) and DAB staining of frozen breast tissue obtained using disclosed embodiments of the present invention were compared relative to a no primary antibody control.

[0052] Fig.29 ESRI blue stained images (20×) and DAB staining of frozen breast tissue obtained using disclosed embodiments of the present invention were compared relative to a no primary antibody control.

[0053] Fig.30 The KRT15 red staining images (20×) and DAB staining of frozen tonsil tissue obtained using the disclosed embodiments of the present invention were compared relative to a no primary antibody control.

[0054] Fig.31 The KRT15 blue staining images (20×) and DAB staining of frozen tonsil tissue obtained using the disclosed embodiments of the present invention were compared relative to a no primary antibody control.

[0055] Fig.32 CDH17 red staining images (20×) and DAB staining of frozen colon tissue obtained using disclosed embodiments of the present invention were compared relative to a no primary antibody control.

[0056] Fig.33 CDH17 blue staining images (20×) and DAB staining of frozen colon tissue obtained using disclosed embodiments of the present invention are compared relative to a no primary antibody control.

[0057] Fig.34 Provided are no antibody control red and blue staining images (20×) and DAB staining of frozen colon tissue CDH17 obtained using disclosed embodiments of the present invention.

[0058] Fig.35 FFPE breast ESR1 blue staining images (20×) and DAB staining obtained using disclosed embodiments of the present invention are compared relative to a no primary antibody control.

[0059] Fig.36 FFPE tonsil KRT15 red staining images (20×) and DAB staining obtained using disclosed embodiments of the present invention are compared relative to a no primary antibody control.

[0060] Fig.37 FFPE tonsil KRT15 blue staining images (20×) and DAB staining obtained using disclosed embodiments of the present invention are compared relative to a no primary antibody control.

[0061] Fig.38 FFPE colon CDH17 red staining images (20×) and DAB staining obtained using disclosed embodiments of the present invention are compared relative to a no primary antibody control.

[0062] Fig.39 FFPE colon CDH17 blue staining images (20×) and DAB staining obtained using disclosed embodiments of the present invention are compared relative to a no primary antibody control.

[0063] Fig.40 FFPE breast ESR1 red staining images (20×) (aqueous mounting medium, no dehydration / clearing) and DAB staining obtained using disclosed embodiments of the present invention are compared relative to a no primary antibody control.

[0064] Fig.41 FFPE breast ESR1 red staining images (20×) (aqueous mounting medium, no dehydration / clearing) and DAB staining obtained using disclosed embodiments of the present invention are compared relative to a no primary antibody control.

[0065] Fig.42FFPE breast KRT15 red staining images (20×) (aqueous mounting medium, no dehydration / clearing) and DAB staining obtained using disclosed embodiments of the present invention are compared relative to a no primary antibody control.

[0066] Fig.43 FFPE tonsil KRT15 blue staining images (20×) (aqueous mounting medium, no dehydration / clearing) and DAB staining obtained using disclosed embodiments of the present invention are compared relative to a no primary antibody control.

[0067] Fig.44 FFPE colon CDH17 red staining images (20×) (aqueous mounting medium, no dehydration / clearing) and DAB staining obtained using disclosed embodiments of the present invention are compared relative to a no primary antibody control.

[0068] Fig.45 FFPE colon CDH17 blue staining images (20×) (aqueous mounting medium, no dehydration / clearing) and DAB staining obtained using disclosed embodiments of the present invention are compared relative to a no primary antibody control.

[0069] Fig.46 FFPE tonsil KRT15 red stained images (20×) (hematoxylin counterstain) and DAB images obtained using disclosed embodiments of the present invention are compared.

[0070] Fig.47 FFPE tonsil KRT15 blue stain images (20×) (eosin counterstain) and DAB images obtained using disclosed embodiments of the present invention are compared.

[0071] Fig.48 FFPE tonsil KRT15 blue stain images (20×) (methyl green counterstain) and DAB images obtained using disclosed embodiments of the present invention are compared.

[0072] Fig.49 FFPE tonsil Ki67 red stain images (20×) (hematoxylin counterstain) and DAB images obtained using disclosed embodiments of the present invention are compared.

[0073] Fig.50 FFPE tonsil Ki67 blue stain images (20×) (eosin counterstain) and DAB images obtained using disclosed embodiments of the present invention are compared.

[0074] Fig.51 FFPE tonsil Ki67 red and blue staining images (20×) (methyl green counterstain) obtained using disclosed embodiments of the present invention are compared. DETAILED DESCRIPTION

[0075] I. Abbreviations and terms

[0076] A. Abbreviations

[0077] AP: Ascorbate peroxidase

[0078] HRP: Horseradish peroxidase

[0079] IHC: Immunohistochemistry

[0080] ISH: In situ hybridization

[0081] B. Terminology

[0082] The following explanations of terms are provided to better describe the present disclosure and guide those of ordinary skill in the art in the practice of the present disclosure.

[0083] As used herein, "comprising" means "including" and the singular forms "a or an" or "the" include plural references unless the context clearly dictates otherwise.

[0084] Unless the context clearly indicates otherwise, the term "or" refers to either a single element or a combination of two or more elements of the stated alternative elements.

[0085] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although methods and materials similar to or equivalent to the methods and materials described herein can be used to practice or test the present disclosure, suitable methods and materials are described below. Materials, methods and examples are illustrative only and are not intended to be limiting. Based on the specific embodiments and claims, other features of the present disclosure will be apparent to those of ordinary skill in the art.

[0086] Unless otherwise indicated, the disclosure of a numerical range should be understood to refer to every discrete point within that range, inclusive of the endpoints.

[0087] Unless otherwise indicated, all numbers expressing the amount of components, molecular weights, percentages, temperatures, times, etc. as used in this specification or claims are to be understood as being modified by the term "about". Therefore, unless otherwise implicitly or explicitly indicated, or unless the context is properly understood by one of ordinary skill in the art to have a more explicit interpretation, the numerical parameters shown are approximate values, which may depend on the desired properties sought and / or the limits of detection under standard test conditions / methods known to one of ordinary skill in the art. When an embodiment is directly and clearly distinguished from the prior art being discussed, the embodiment numbers are not approximate unless the word "about" is recited.

[0088] Although there are alternatives to the various components, parameters, operating conditions, etc. set forth herein, this does not mean that these alternatives must be equivalent and / or perform identically. Nor is it meant that the alternatives are listed in order of priority unless otherwise stated.

[0089] The compounds of the present disclosure also include all isotopes of atoms present in the compounds, which may include but are not limited to deuterium, tritium, 18 F. 14 C, etc.

[0090] Definitions of common terms in chemistry may be found in Richard J. Lewis, Sr. (Ed.), Hawley's Condensed Chemical Dictionary, John Wiley & Sons, 2016 (ISBN 978-1-118-13515-0).

[0091] Definitions of commonly used terms in molecular biology may be found in Benjamin Lewin, Genes VII, Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Publishing, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, John μ Wiley & Sons, 1995 (ISBN 0471186341); and other similar references.

[0092] Absorbance: The retention of radiation of a specific wavelength by a compound or substance incident upon it; a measure of the amount of light of a specific wavelength absorbed when the light passes through a compound or substance or through a solution of a compound or substance.

[0093] Absorption: The retention of radiation of a particular wavelength by a compound or substance incident upon it; a measure of the amount of light of a particular wavelength absorbed when the light passes through a compound or substance or through a solution of a compound or substance.

[0094] Acyl: An organic functional group having the general formula -C(O)R, where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, aryl or heteroaryl.

[0095] Adsorption: The physical adhesion or binding of ions and molecules to the surface of another molecule. The adsorbed ions or molecules are called adsorbates. Adsorption can be characterized as either chemisorption or physisorption, depending on the nature and strength of the bond between the adsorbate and the substrate surface.

[0096] Alcohol: An organic compound containing at least one hydroxyl (-OH) group. An alcohol can be a monohydric alcohol (containing one -OH group), a dihydric alcohol (containing two -OH groups; diols, such as ethylene glycol), a trihydric alcohol (containing three -OH; triols, such as glycerol) groups, or a polyhydric alcohol (containing three or more -OH groups; polyols). The organic portion of the alcohol can be aliphatic, cycloaliphatic (alicyclic), heteroaliphatic, cycloheteroaliphatic (heterocyclic), polycyclic, aryl, or heteroaryl, and can be substituted or unsubstituted.

[0097] Aliphatic: A compound or group thereof that is essentially based on hydrocarbons (e.g., for a hexane group, C 6 H 13 ), including alkanes, alkenes, alkynes, including their cyclic forms, and also including straight chain and branched arrangements, as well as all stereoisomers and positional isomers. Unless otherwise expressly stated, aliphatic groups contain 1 to 25 carbon atoms (C 1-25 ); for example, 1 to 15 (C 1-15 ), 1 to 10 (C 1-10 ), 1 to 6 (C 1-6 ) or 1 to 4 (C 1-4 ) carbon atoms. The term "lower aliphatic" refers to a group containing 1 to 10 (C 1-10) carbon atoms. Unless explicitly referred to as "unsubstituted aliphatic", aliphatic groups can be unsubstituted or substituted. Aliphatic groups can be substituted with one or more substituents (up to two substituents for each methylene carbon in the aliphatic chain, or up to one substituent for each carbon of a -C=C- double bond in the aliphatic chain, or up to one substituent for the carbon of a terminal methine). Exemplary substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amide, amino, aminoalkyl, aryl, arylalkyl, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxy, oxo, sulfonamide, sulfhydryl, thioalkoxy or other functional groups.

[0098] Alkoxy: A group (or substituent) having the structure -OR, where R is a substituted or unsubstituted alkyl group. 3 ) are exemplary alkoxy groups. In substituted alkoxy, R is an alkyl group substituted with a non-interfering substituent. "Thioalkoxy" refers to -SR, where R is a substituted or unsubstituted alkyl. "Haloalkoxy" refers to the group -OR, where R is a haloalkyl group.

[0099] Alkyl: A hydrocarbon group having a saturated carbon chain. The chain may be cyclic, branched or unbranched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl. The term lower alkyl refers to a chain containing 1-10 carbon atoms. The terms alkenyl and alkynyl refer to hydrocarbon groups having a carbon chain containing one or more double or triple bonds, respectively.

[0100] Alkylamino: The chemical functional group -N(H)R, where R is an alkyl group.

[0101] Alkyl ammonium: having the formula [N(H)(R') 3 ] + A cation wherein each R' is independently H or an alkyl group.

[0102] Amide: An organic compound characterized by a carbonyl group (C=O) attached to a nitrogen atom and having the general formula wherein R, R' and R" are the same or different and are generally selected from hydrogen, aliphatic and aromatic groups.

[0103]

[0104] Acylamino: A chemical functional group -C(O)N(R)(R'), where R and R' are independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, alkylsulfanyl, or other functional groups.

[0105] Amino: A chemical functional group -N(R)R', where R and R' are independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, alkylsulfanyl, or other functional groups. A "primary amino" group is -NH 2 "Monosubstituted amino" refers to the group -N(H)R substituted as above, including, for example, methylamino, (1-methylethyl)amino, phenylamino, etc. "Disubstituted amino" refers to the group -N(R)R' substituted as above, including, for example, dimethylamino, methylethylamino, di(1-methylethyl)amino, etc.

[0106] Aminoalkyl: Chemical functional group - RNH 2 , wherein R is an alkyl group.

[0107] Antibody: "Antibody" generally refers to immunoglobulins or immunoglobulin-like molecules [including, for example, but not limited to, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any chordate (such as a vertebrate), for example, a mammal (such as a human, goat, rabbit and mouse)] and fragments thereof, which specifically bind to a target molecule (or a group of highly similar target molecules) while substantially excluding binding to other molecules. "Antibodies" generally contain a polypeptide ligand having at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigenic epitope. Immunoglobulins are composed of heavy and light chains, each of which has a variable region, called a variable heavy (V H ) area and variable lightness (V L ) area. H Area and V L The regions together are responsible for binding to the antigen recognized by the immunoglobulin. Exemplary immunoglobulin fragments include, but are not limited to, proteolytic immunoglobulin fragments [such as F(ab') known in the art 2 fragments, Fab' fragments, Fab'-SH fragments and Fab fragments], recombinant immunoglobulin fragments (such as sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)' 2Fragments, single-chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv"). Other examples of antibodies include diabodies and triabodies (as known in the art) and camelid antibodies. "Antibodies" also include genetically engineered molecules such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Edition, WH Freeman & Co., New York, 1997.

[0108] Aralkyl / arylalkyl: an aryl group (such as a phenyl group) attached to an alkyl group, including but not limited to benzyl, ethylbenzene, propylbenzene, butylbenzene, pentylbenzene, etc. In contrast, the term "phenylalkyl" refers to a phenyl group attached to an alkyl group. Aralkyl groups, such as benzyl groups, may be unsubstituted or substituted with one, two or three substituents independently selected from alkyl, heteroalkyl, aliphatic, heteroaliphatic, thioalkoxy, haloalkyl (such as -CF 3 ), halogen, nitro, cyano, -OR (wherein R is hydrogen or alkyl), -N(R)R' (wherein R and R' are independently hydrogen or alkyl), -COOR (wherein R is hydrogen or alkyl) or -C(O)N(R')R" (wherein R' and R" are independently selected from hydrogen or alkyl). Non-limiting examples include o-, m- and / or p-chlorobenzyl, o-, m- and / or p-methoxybenzyl, and o-, m- and / or p-(trifluoromethyl)benzyl.

[0109] Aromatic or aryl group: An unsaturated cyclic hydrocarbon with alternating single and double bonds. Aromatic systems can have a single ring, such as benzene, a 6-carbon ring containing three double bonds, or aromatic compounds can have multiple rings, such as fused ring systems including naphthalene, anthracene, or phenanthrene.

[0110] Aryl: Unless otherwise specified, a monovalent aromatic carbocyclic group of 6 to 20 carbon atoms having a single ring (e.g., phenyl) or multiple fused rings in which at least one ring is aromatic (e.g., quinoline, indole, benzodioxole, etc.), provided that the point of attachment is through an atom of the aromatic portion of the aryl group and the aromatic portion at the point of attachment contains only carbons in the aromatic ring. If any aromatic ring portion contains heteroatoms, the group is a heteroaryl rather than an aryl group. Aryl groups are monocyclic, bicyclic, tricyclic, or tetracyclic.

[0111] Aralkyl: an alkyl group in which the carbon atom (usually a terminal or sp 3A non-cyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced by an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethane-1-yl, naphthylmethyl, 2-naphthylethane-1-yl, naphthylbenzyl, 2-naphthylphenylethane-1-yl, etc. When a specific alkyl moiety is intended, the nomenclature arylalkyl, arylalkenyl, and / or arylalkynyl may be used.

[0112] Biomolecule: may include any molecule found in a biological system, including but not limited to synthetic or naturally occurring proteins, glycoproteins, lipoproteins, amino acids, nucleosides, nucleotides, nucleic acids, oligonucleotides, DNA, RNA, carbohydrates, sugars, lipids, fatty acids, haptens, etc.

[0113] Carbonyl: A group of the formula -C(O)-. Carbonyl-containing groups include any substituent containing a carbon-oxygen double bond (C=O), including acyl groups, amides, carboxyl groups, esters, ureas, carbamates, carbonates, and ketones and aldehydes, such as substituents based on -COR or -RCHO, where R is an aliphatic, heteroaliphatic, alkyl, heteroalkyl, hydroxyl, or a secondary, tertiary, or quaternary amine.

[0114] Chemifluorescence: Fluorescence that occurs when a chemical reaction converts non-fluorescent reactants into products that fluoresce upon exposure to an excitation source.

[0115] Chemical adsorption: A type of adsorption characterized by Adsorbate Strong interactions with the substrate. Strong interactions include the formation of covalent and / or ionic bonds, which change the electronic structure of the adsorbate. Chemisorption can be reversed by high temperatures or chemical reactions that break the bonds between the adsorbate and the substrate.

[0116] Chromophore: An aromatic compound containing a chemical group that imparts color to the compound by causing a shift or appearance of an absorption band in the visible spectrum. Exemplary chromophores include, but are not limited to, RN=N- (eg, azo dyes).

[0117] Chromogen: A compound that can be converted to a chromophore by chemical or other means. Exemplary chromogens include, but are not limited to, naphthols, aryl diazonium salts, and 1,3-diketones.

[0118] Conditions sufficient for detection: Any environment that allows the desired activity, e.g., any environment that allows the probe to bind to the target and the interaction to be detected. For example, such conditions include appropriate temperature, buffer solutions, and detection means (such as microscopes and digital imaging equipment).

[0119] Conjugate: Two or more moieties coupled together, directly or indirectly. For example, a first moiety can be covalently or non-covalently (e.g., electrostatically) coupled to a second moiety. Indirect linkage is possible, for example, by using a "linker" (a molecule or group of atoms located between the two moieties).

[0120] Contact: A placement that allows association, particularly direct physical association, between two or more parts, e.g., in solid form and / or in liquid form (e.g., placing a biological sample, such as a biological sample attached to a slide, in contact with a composition, such as a solution containing a composition disclosed herein).

[0121] Conjugate, join, bond or connect: To couple a first unit to a second unit. This includes, but is not limited to, covalently bonding one molecule to another, non-covalently bonding (e.g., electrostatically bonding) one molecule to another (see, e.g., U.S. Pat. No. 6,921,496, which discloses methods for electrostatic conjugation), non-covalently bonding one molecule to another via hydrogen bonding, non-covalently bonding one molecule to another via van der Waals forces, and any and all combinations of such couplings.

[0122] Counterstaining: A method of post-processing a sample after it has been stained with a reagent to detect one or more targets so that their structure can be more easily visualized. For example, a counterstain is optionally used before the coverslip is applied to make IHC staining more obvious. The counterstain is a different color from the primary stain. Many counterstains are well known, such as hematoxylin, eosin, methyl green, methylene blue, Giemsa, Alcian blue, DAPI, and nuclear fast red. In some examples, more than one stain can be mixed together to produce a counterstain. This provides flexibility and the ability to select stains. For example, a first stain having a specific property but not having a different desired property can be selected for the mixture. A second stain that exhibits a missing desired property can be added to the mixture. For example, toluidine blue, DAPI, and pontamine sky blue can be mixed together to form a counterstain.

[0123] Conjugated: The term "conjugated" means to link together directly or indirectly. A first atom or molecule can be directly conjugated to a second atom or molecule or indirectly conjugated. A secondary antibody provides an example of indirect conjugation. A specific example of indirect conjugation is a rabbit anti-hapten primary antibody conjugated by a mouse anti-rabbit IgG antibody, which in turn is conjugated by a goat anti-mouse IgG antibody covalently linked to a detectable label.

[0124] Detection: Determine whether an agent (e.g., a signal or a specific antigen, protein, or nucleic acid) is present, for example, in a sample. In some examples, this may further include quantification and / or localization, such as localization within a cell or a specific cell compartment. "Detection" refers to any method for determining whether something is present, such as determining whether a target molecule is present in a biological sample. For example, "detection" may include the use of visual or mechanical equipment to determine whether a sample displays a specific feature. In some examples, detection refers to visually observing a probe bound to a target, or observing that the probe does not bind to the target. For example, for the methods described herein, optical microscopy and other microscopic means are commonly used to detect chromogenic precipitates.

[0125] Detectable marker: A molecule or material that can generate a detectable (such as visual, electronic or other) signal that indicates the presence and / or concentration of a target (such as a target molecule) in a sample (such as a tissue sample). When conjugated to a specific binding molecule, a detectable marker can be used to locate and / or quantify the target to which the specific binding molecule is directed. Therefore, the presence and / or concentration of the target in the sample can be detected by detecting the signal generated by the detectable marker. The detectable marker can be detected directly or indirectly, and several different detectable markers conjugated to different specific binding molecules can be used in combination to detect one or more targets. A variety of detectable markers that can be detected separately can be conjugated to different specific binding molecules that specifically bind to different targets, to provide a multiple determination that can provide detection of multiple targets in a sample. Detectable markers include, but are not limited to, colored, fluorescent, phosphorescent and luminescent molecules and materials, and more specifically relate to colored and / or fluorescent markers for the present disclosure.

[0126] Emission or emission signal: Light of a specific wavelength generated from a light source. In a specific example, an emission signal is emitted from a fluorophore after the fluorophore absorbs light of its excitation wavelength.

[0127] Excitation or excitation signal: Light of a specific wavelength necessary and / or sufficient to excite an electron to transition to a higher energy level. In a specific example, excitation is light of a specific wavelength necessary and / or sufficient to excite a fluorophore to a state where the fluorophore will emit light of a wavelength different from (such as longer than) the wavelength of light from the excitation signal.

[0128] Fixation: A method of preserving cells and tissue components in a state as close to the living body as possible and allowing them to undergo preparation processes without being altered. Fixation arrests the autolytic and bacterial decomposition processes that begin when cells die, and stabilizes cells and tissue components so that they can withstand subsequent tissue processing stages, such as for IHC.

[0129] Tissues can be fixed by perfusion or immersion with a fixative such as an aldehyde (such as formaldehyde, paraformaldehyde, glutaraldehyde, etc.). Other fixatives include oxidants (e.g., metal ions and complexes such as osmium tetroxide and chromic acid), protein denaturants (e.g., acetic acid, methanol and ethanol), fixatives of unknown mechanism (e.g., mercuric chloride, acetone and picric acid), combination reagents (e.g., Carnoy's fixative, methacarn, Bouin's solution, B5 fixative, Rossman's solution and Randel's solution), microwaves and miscellaneous (e.g., excluded volume fixation and steam fixation). Additives such as buffers, detergents, tannic acid, phenol, metal salts (e.g., zinc chloride, zinc sulfate and lithium salts) and lanthanum can also be included in the fixative. A commonly used fixative for preparing samples for IHC is formaldehyde, usually in the form of a formalin solution (a buffered solution of 4% formaldehyde, referred to as 10% buffered formalin).

[0130] Fluorescence: the emission of visible radiation by an atom or molecule from a higher electronic state to a lower electronic state, with the time interval between absorption and emission of energy being 10 -8 Up to 10 -3 seconds. Fluorescence occurs when atoms or molecules absorb energy from an excitation source (for example, ultraviolet light) and then emit that energy as visible radiation.

[0131] Fluorophore: A functional group or moiety of a molecule that causes the molecule to fluoresce when exposed to an excitation source. The term "fluorophore" is also used to refer to fluorescent compounds used as dyes to label proteins with fluorescent markers.

[0132] Fluorogen: A compound that can be converted to a fluorophore by chemical or other means. Exemplary fluorogens include, but are not limited to, cryptogloss forms of common fluorophores such as rhodamine and fluorescein.

[0133] Functional group: A specific group of atoms in a molecule that is responsible for the characteristic chemical reactions of the molecule. Exemplary functional groups include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, halo (fluorine, chlorine, bromine, iodine), epoxide, hydroxyl, carbonyl (ketone), aldehyde, carbonate, carboxylate, carboxyl, ether, ester, peroxy, peroxyhydroxyl, carboxamide, amino (primary, secondary, tertiary), ammonium, imide, azide, cyanate, isocyanate, thiocyanate, nitrate, nitrite, nitrile, nitroalkyl, nitroso, pyridyl, phosphate, sulfonyl, sulfide, thiol (sulfhydryl), disulfide.

[0134] Halogen: fluorine, bromine, chlorine or iodine.

[0135] Hapten: A molecule, usually a small molecule, that can specifically bind to an antibody but is generally not substantially immunogenic except when bound to a carrier molecule. Examples of haptens include, but are not limited to, fluorescein, biotin, nitroaryls (including, but not limited to, dinitrophenol (DNP)), digoxigenin, oxazole, pyrazole, thiazole, benzofuran, urea, thiourea, rotenone, coumarins, and cyclolignans.

[0136] Heteroaryl: An aromatic compound or radical having at least one heteroatom, ie, one or more carbon atoms in the ring have been replaced by an atom having at least one lone pair of electrons, typically nitrogen, oxygen, phosphorus, silicon or sulfur.

[0137] Hydrocarbon: An organic compound composed of the elements carbon and hydrogen. Hydrocarbons are commonly derived from petroleum, coal tar, and plant sources. Hydrocarbons include aliphatics (alkanes, alkenes, alkynes, and cyclic versions thereof, including straight chain and branched arrangements), aromatics (unsaturated cyclic hydrocarbons having alternating single and double bonds), and combinations thereof (e.g., arylalkyl compounds).

[0138] Immunohistochemistry (IHC): A method for determining the presence or distribution of an antigen in a sample by detecting the interaction of an antigen with a specific binding agent or moiety (such as an antibody). Under conditions that allow antibody-antigen binding, a sample containing an antigen (e.g., a target antigen) is incubated with an antibody. Antibody-antigen binding can be detected by a detectable label conjugated to the antibody (direct detection) or by a detectable label conjugated to a secondary antibody produced for the primary antibody (e.g., indirect detection). Detectable labels include, but are not limited to, radioisotopes, fluorescent dyes (such as fluorescein, fluorescein isothiocyanate, and rhodamine), enzymes, and chromogenic molecules.

[0139] Lower: refers to organic compounds having 10 or fewer carbon atoms in the chain, including all branching and stereochemical variations, specifically including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl.

[0140] Moiety: A moiety is a fragment of a molecule, or a portion of a conjugate.

[0141] Target molecule: A molecule whose presence, location and / or concentration is to be determined. Examples of target molecules include proteins and nucleic acid sequences present in a tissue sample.

[0142] Multiplex detection: Multiple targets in a sample are detected substantially simultaneously or sequentially using multiple different conjugates as desired. Multiplex detection may include identification and / or quantification of nucleic acids (typically DNA, RNA), peptides, proteins, alone and in any and all combinations. Multiplex detection may also include detection of two or more of genes, messengers, and proteins in a cell in the context of the cell's anatomy.

[0143] Polypeptide: A polymer in which the monomers are amino acid residues linked together by amide bonds. When the amino acid is an α-amino acid, either the L-optical isomer or the D-optical isomer can be used. As used herein, the terms "polypeptide" or "protein" are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term "polypeptide" is specifically intended to encompass naturally occurring proteins as well as recombinantly or synthetically produced proteins. The terms "residue" or "amino acid residue" include reference to an amino acid incorporated into a protein, polypeptide, or peptide.

[0144] Probe: A substance used to detect or identify another substance in a sample.

[0145] Sample: The term "sample" refers to any liquid, semisolid or solid substance (or material) in or on which a target may be present. In particular, a sample may be a biological sample or a sample obtained from a biological material. A biological sample is any solid or fluid sample obtained from, excreted or secreted by any living organism, including but not limited to unicellular organisms (such as bacteria, yeast, protozoa and ameba, etc.), multicellular organisms (such as plants or animals, including samples from healthy or apparently healthy human subjects or human patients affected by a condition or disease (such as cancer) to be diagnosed or studied). For example, a biological sample may be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous humor or vitreous humor, or any body secretion, exudate, exudate (e.g., fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (e.g., a normal joint or a joint affected by a disease). Biological samples can also be samples obtained from any organ or tissue (including biopsy or autopsy samples, such as tumor biopsies), or can include cells (whether primary cells or cultured cells) or culture medium conditioned by any cells, tissues or organs. In some examples, the biological sample is a nuclear extract. In some examples, the biological sample is bacterial cytoplasm. In other examples, the sample is a test sample. For example, a test sample is a cell, tissue or cell mass section prepared from a biological sample obtained from a subject. In one example, the subject is a subject who is at risk of or has suffered from a specific condition or disease.

[0146] Section: A section of a tissue sample refers to a single portion or piece of a tissue sample, such as a thin slice of tissue or a cell cut from a tissue sample. Multiple sections of a tissue sample can be taken and analyzed according to the present invention. The section can be as small as one or two cells or can be thousands of cells.

[0147] Specific binding moiety or specific binding agent: A member of a specific binding pair. A specific binding pair is a pair of molecules characterized in that they bind to each other to the substantial exclusion of binding to other molecules (e.g., a specific binding pair may have a binding constant that is at least 10 greater than that of either member of the binding pair to other molecules in a biological sample). 3 M -1 , Big 10 4 M -1 or Big 10 5 M -1 Specific examples of specific binding moieties include specific binding proteins (e.g., antibodies, lectins, avidin (such as streptavidin) and protein A), nucleic acid sequences, and protein-nucleic acids. Specific binding moieties may also include molecules (or portions thereof) that are specifically bound by such specific binding proteins.

[0148] Specific binding: This term refers to the binding of an agent that preferentially binds to a defined target (such as the binding of an antibody to a specific antigen or the binding of a nucleic acid probe to a specific nucleic acid sequence). In the case of antigens, "specific binding" refers to the preferential binding of an antibody or other ligand, in whole or in part, to a specific polypeptide. In the case of nucleic acid sequences, "specific binding" refers to the preferential binding of a nucleic acid probe, in whole or in part, to a specific nucleic acid sequence.

[0149] The specific binding agent or part essentially binds only to a determined target. It should be recognized that a lesser degree of non-specific interaction may occur between a molecule (such as a specific binding agent or part) and a non-target polypeptide or non-target nucleic acid sequence. Although selectively reactive antibodies bind antigens, they can bind antigens with low affinity. Antibody-antigen specific binding generally results in an increase in the amount of antibodies or other ligands bound to the target polypeptide (per unit time) greater than 2 times, such as greater than 5 times, greater than 10 times or greater than 100 times, compared to binding to non-target polypeptides. A variety of immunoassay formats are suitable for selecting antibodies that specifically immunoreact with a particular protein. For example, solid phase ELISA immunoassays are commonly used to select monoclonal antibodies that specifically immunoreact with proteins. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988).

[0150] Specific binding of a nucleic acid probe to a nucleic acid sequence typically results in an increase in the amount of nucleic acid probe bound to the target nucleic acid sequence by greater than 2 times, such as greater than 5 times, greater than 10 times, or greater than 100 times, compared to binding to non-target nucleic acids. A variety of ISH conditions are suitable for selecting nucleic acid probes that specifically bind to a particular nucleic acid sequence.

[0151] Staining: Any biological or chemical entity that, when applied to a targeted molecule in a biological sample, renders the molecule detectable under microscopic examination. Staining agents include, but are not limited to, detectable nucleic acid probes, antibodies, dyes, and other agents that in combination or by themselves produce a colored end product (by bright field or fluorescent detection methods). "Staining" or "microscopic visualization" can be performed by any means, including but not limited to immunostaining, which involves antibody detection of the desired antigen, followed by an enzymatic reaction that produces a colored reaction product, and may involve computerized image analysis.

[0152] Substitution: As a result of the reaction, an atom or atom group of another atom in a molecule is substituted. The term "substituent" generally refers to an atom or atom group that replaces one hydrogen atom or two hydrogen atoms (if the substituent is connected via a double bond) on a parent hydrocarbon chain or ring. The term "substituent" can also encompass an atom group with multiple connection points to a molecule, for example, a substituent replaces two or more hydrogen atoms on a parent hydrocarbon chain or ring. In such examples, unless otherwise specified, the substituent can be connected to the parent hydrocarbon chain or ring in any spatial orientation. Exemplary substituents include, for example, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amido, amino, aminoalkyl, aryl, arylalkyl, arylamino, carbonate, carboxyl, cyano, cycloalkyl, dialkylamino, halogen, halogenated aliphatic (for example, haloalkyl), halogenated alkoxy, heteroaliphatic, heteroaryl, heteroalicyclic, hydroxyl, oxo, sulfonamide, sulfhydryl, sulfhydryl and thioalkoxy groups.

[0153] Substituted: A base compound having one or more substituents coupled thereto, such as an aryl or aliphatic compound or a group thereof, each substituent typically replacing a hydrogen atom on the base compound. One of ordinary skill in the art will recognize that, unless otherwise expressly stated or otherwise indicated by the context, the compounds disclosed herein may be described with reference to a particular structure and a substituent coupled to such a structure, and such a structure and / or substituent may also be further substituted. By way of example only and not limitation, a substituted aryl compound may have an aliphatic group coupled to a closed ring of an aryl base (such as coupled to toluene). Also by way of example only and not limitation, a long chain hydrocarbon may have a hydroxyl group bonded thereto.

[0154] Tissue: A collection of interconnected cells that perform similar functions within an organism. Any collection of cells that can be mounted on a standard glass microscope slide includes, but is not limited to, organ sections, tumor sections, body fluids, smears, frozen sections, cytological preparations, and cell lines.

[0155] Tyramine: having the formula C 8 H 11 NO compound, also known as 4-(2-aminoethyl)phenol.

[0156] Tyramide: A tyramine derivative in which the amine functional group of the tyramine molecule forms an amide bond with a carbonyl-containing functional group.

[0157] II. Introduction

[0158] Some disclosed embodiments relate to being connected to tyrosinamide covalent linker substrates to provide colorless covalently labeled chromogens, which can be deposited at or around the target area in a cell or tissue sample by HRP enzymatic reaction. After deposition, then by adding different reagents (such as diazonium salts) to develop color, these reagents react with the chromogens deposited at or around the target area to form colored compounds at or around the target area, which can be detected by bright field, fluorescence microscopy or both. Since the labeling is covalently bound, it is more stable than the classical precipitation substrates used with aqueous mounting agents or organic mounting agents. Since colorants such as diazonium salts are coupled with chromogens after the covalent labeling of the target, a single chromogen substrate can be used to generate a variety of detectable colors during the IHC process. Disclosed covalent chromogenic substrates provide (1) better signal stability in different mounting media, and (2) tighter staining patterns.

[0159] III. Compounds

[0160] Certain embodiments of the present invention involve the use of compounds satisfying the following formula I

[0161]

[0162] About Formula I, P 1 , P 2 and P 3 is an aromatic ring or an aromatic ring system, such as a fused ring system. 1 and P 3 The ring systems may be the same or different, and each may comprise at least two fused rings, wherein at least one of the fused rings comprises the structure shown below:

[0163]

[0164] P 2 is a second aromatic ring system, typically substituted with at least one -OH group. L is a linker having 1 to at least 50 atoms, more typically 2 to 20 atoms in the chain. L may be substituted or unsubstituted. If the R group is not present, L may be, for example, replaced by P 3 group substituted, or may be RP 3 R is a chemical bond, or independently an L group. x and y are independently 0 to 5, wherein in certain disclosed embodiments, x and y are 0 or 1.

[0165] Certain disclosed embodiments of the present invention may satisfy Formula II

[0166] P 1 -LP 2

[0167] Formula II,

[0168] Where P 1 , P 2 and L are as described in Formula I.

[0169] The aromatic ring system according to the present invention can be a hydrocarbon ring system or a heteroaryl ring system. For certain embodiments, P 1 and P 3 is an aromatic hydrocarbon fused ring system, such as a fused ring system having 2 to at least 4 rings in the fused ring system. 1 and / or P 3 The P may be substituted with at least one solubility moiety selected to increase the solubility of the compound in solvents commonly used in staining protocols such as IHC, such as aqueous solvent systems. 1 and P 3 Fused ring systems include, but are not limited to:

[0170]

[0171]

[0172] Regarding these exemplary P 1 and P 3 A fused ring system, m is 1 to 5, more typically 1-3, wherein in certain disclosed embodiments, m=1. Representative "solubility enhancing moieties", particularly water solubility enhancing moieties, may be selected from -SO 3 - 、-SO 3 H, -CO 2 - , -CO 2 H, -HPO 3 -2 , H 2 PO 3 -1 , H 3 PO 3 , PEG, and their salt forms.

[0173] As indicated, P 1 and / or P 3 At least one hydroxyl (-OH) group may be included on the ring system. The at least one hydroxyl group is usually in the ortho or meta position relative to L. Thus, certain disclosed compounds have the following characteristics: 1and / or P 3 is naphthol, L is coupled to the 2- or 3-position, at least one hydroxyl group is at the 3- and / or 4-positions, and at least one moiety (such as -SO 3 - or its salt form) is coupled to at least one of positions 5-8; 1 and / or P 3 is anthracenol, L is coupled to the 2-position or the 3-position, at least one hydroxyl group is at the 3-position and / or the 4-position, and at least one moiety selected to increase the solubility of the compound is coupled to at least one of the 6-9-positions; 1 and / or P 3 is a tetraenol, L is coupled to the 2-position or the 3-position, at least one hydroxyl group is at the 3-position and / or the 4-position, and at least one moiety selected to increase the water solubility of the compound is coupled to at least one of the 7-10-positions; or P 1 and / or P 3 is phenanthranol, L is coupled to the 2- or 3-position, at least one hydroxyl group is at the 3- and / or 4-positions, and at least one moiety selected to increase the solubility of the compound is coupled to at least one of the 5-8-positions.

[0174] P 1 and / or P 3 It may be a heteroaryl fused ring system, typically an electron-rich heteroaryl fused ring system. Examples of suitable heteroaryl fused ring systems include, but are not limited to, fused furan, fused thiophene and fused pyrrole ring systems, such as

[0175] P 2 For certain disclosed embodiments, P 2 Including phenol, and P 2 Even more specific examples of include phenols in which the hydroxyl group is at the para position of L.

[0176] L is a covalent bond, or is selected from C 1 -C 50 Aliphatic group, C 1 -C 50 Alkyl, C 1 -C 50 Alkylamide, C 1 -C 50 Alkyl esters, C 1 -C 50 Ketone, C 2 -C 50 Polyols (including diols) C 2 -C 50 Polyalkyl ethers (such as polyalkylene oxides, including, for example, PEG linkers) and C 2 -C 50Aliphatic amine. Certain embodiments of C 1 -C 50 The joint length is more usually C 1 -C 20 For specific disclosed embodiments, L is an alkylamide such as wherein n is 1-10, more typically 1-5, such as 2. As used herein, a wavy line Indicates other parts of the compound, such as P 1 and P 2 A specific example of L is For certain disclosed embodiments, L is an ether or polyether represented by Wherein o represents the number of ethoxy units and is 1 to 10, more specifically 3 to 5. One of ordinary skill in the art will appreciate that other alkoxy units, such as propoxy and butoxy ethers, may also form suitable linkers. Such polyalkoxylated groups may be attached to the aromatic ring or ring system using any acceptable functional group as one of ordinary skill in the art will appreciate, wherein in certain disclosed embodiments, an amide, amine, or ester is used to couple the L group to the aromatic ring or ring system, such as the following linker The wavy line also indicates the 1 , P 2 and P 3 The point of attachment is an aromatic ring or aromatic ring system. Another embodiment relates to aliphatic amine linkers, such as

[0177] Certain disclosed embodiments of compounds useful in the present invention satisfying Formula I and / or II include: 1 Selected from:

[0178] m is 1 to 3; P 2 comprising phenol; at least one solubilizing moiety selected to increase the water solubility of the compound and selected from -SO 3 - 、-SO 3 H, -CO 2 - , -CO 2 H, -HPO 3 -2 , H 2 PO 3 -1 , H 3 PO 3 , PEG, and L is selected from a covalent bond, C 1 -C 20 Alkyl, C 1 -C 20 Alkylamide, C1 -C 10 Ketone or C 1 -C 10 A diol or a polyol.

[0179] The disclosed embodiments may also have the following formula III

[0180]

[0181] Regarding Formula III, R 1 and R 2 are independently selected from H and OH; R 3 -R 8 independently selected from H, -F, -Cl, -Br, -I, -OH and -OR 9 , where R 9 C 1 -C 6 Alkyl, -SO 3 - 、-SO 3 H, -CO 2 - , -CO 2 H, -HPO 3 -2 , H 2 PO 3 -1 , H 3 PO 3 , PEG, R 1 , R 2 , R 7 At least one of R 8 -R 12 At least one of R is hydroxyl. 3 and R 4 together form at least one fused ring system; and / or R 4 and R 5 together form at least one fused ring system; and / or R 5 and R 6 together form at least one fused ring system; and n is 1 to 5. For a specific compound, if R 1 is H, then R 2 is OH, and if R 1 is OH, then R 2 is H; and R 3 , R 4 , R 5 and R 6 independently H or -SO 3 - , where R 3 , R4 , R 5 or R 6 At least one of them is -SO 3 - or a salt form thereof.

[0182] An example of a compound satisfying Formula I above (positively charged counterions such as Na are not shown) + The metal counter ion) is

[0183]

[0184] (R)-7,7′-(((6-((4-hydroxyphenethyl)amino)-6-oxohexane-1,5-diyl)bis(azadiyl))bis(carbonyl))bis(6-hydroxynaphthalene-2-sulfonate).

[0185] Examples of compounds satisfying Formula I-III (again without counterions shown) are

[0186]

[0187] 6-Hydroxy-7-((4-hydroxyphenethyl)carbamoyl)naphthalene-2-sulfonate

[0188] Compounds exemplifying the P1, P2, L and R features of the general formula discussed above are provided in Table 1 below.

[0189] Table 1

[0190]

[0191]

[0192]

[0193] Compounds according to formula I-III can be used to mark targets in biological samples. Therefore, the application also relates to covalent conjugates, which include a biological sample containing a target and at least one compound according to formula I, II and / or III covalently bound to the target, as shown in formula IV below.

[0194]

[0195] A specific example of a tyramide-based conjugate satisfying Formula IV is provided as Formula V below.

[0196]

[0197] Compounds according to Formula I-III can be reacted with chromogens and / or fluorogens to provide conjugates that can be visualized, such as by using bright field and / or fluorescence microscopy. Therefore, the present application also includes compounds satisfying Formula I, II and / or III coupled to chromogens and / or fluorogens, as indicated by the following Formula VI,

[0198]

[0199] A more specific example of a tyramide-based conjugate is provided as Formula VII, wherein "ring" A refers to an aromatic system as defined above, and "water-soluble moiety" is also defined above.

[0200]

[0201] Azo dyes provide specific examples of chromophores and / or fluorophores. And / or in this context indicate that certain azo dyes can produce a specific color for viewing by, for example, bright field microscopy, and can also be fluorescent agents suitable for viewing using fluorescence microscopy. Again with specific reference to tyramide compounds as examples, such tyramide-based compounds can have the following general formula VIII

[0202]

[0203] Examples of specific azo dye conjugates are discussed in more detail below with reference to embodiments of methods of using such compounds.

[0204] Conjugates comprising at least one compound according to the invention coupled to a biological target and further coupled to a chromogen and / or fluorogen (such as an azo dye) are also within the scope of the invention. Such compounds are exemplified by the following formula IX

[0205]

[0206] The tyramide-based compound coupled to the biological target and the azo dye chromophore / fluorophore may have the following formula X

[0207]

[0208] IV. Synthesis of compounds satisfying formula I, II and III

[0209] Compounds satisfying Formula I and II can be prepared by any suitable synthetic scheme. One example of a method for preparing compounds according to Formula I and II is provided below as Scheme I. Additional details regarding the synthesis of compounds according to Formula I and II are provided in the following Examples.

[0210]

[0211] Scheme I

[0212] Commercially available carboxyhydroxynaphthalene (2) is sulfonated by treatment with sulfuric acid at elevated temperature, such as 120° C. A salt form of 4, such as the sodium salt, can be precipitated from solution as a substantially pure product using NaCl.

[0213] Compound 4 can then be coupled to amide 6 with tyramide using the reagent hexafluorophosphate azabenzotriazole tetramethyluronium (HATU) that can be used to form amide bonds. Compound 4 is mixed with anhydrous DMF, triethylamine and HATU under an inert atmosphere. Tyramine is added to the reaction mixture, which is sealed and stirred at room temperature overnight. Volatiles are removed, and the residue is dissolved in water and filtered. Saline is added to the filtrate, and the product (6) is precipitated as a thin light yellow solid.

[0214] V. How to use

[0215] A. Background Technology

[0216] Also disclosed are embodiments of methods for using disclosed compounds to visualize biological targets in staining schemes (such as immunostaining schemes). Biological targets can be cells, tissues, viruses, genes, epitopes, target genetic sequences, or target protein sequences. The present invention can be implemented using specific binding moieties (such as proteins, including antibodies), and can be multiple determinations of multiple targets in samples using multiple binding agents (such as 2-50 different binding agents (such as antibodies), wherein each binding agent recognizes specific antigens), to allow for simultaneous detection of multiple targets. The method can be performed on histologically intact tissues (e.g., formalin-fixed paraffin-embedded tissues (FFPET)) to measure proteins and other molecules on intact tissues preserved. The complete target tissue can be the entire histological section of the target, or alternatively can be a specific type of cell separated from a histological section by technology (such as laser capture microdissection).

[0217] The present invention can be used to detect proteins in human, animal and / or plant tissues by epitope binding using antibodies or antibody-like molecules. These detectable proteins include, for example, prognostic and / or predictive factors in cancer, proteins involved in general biological pathways and / or structural proteins. Certain embodiments of the present invention allow screening of samples (such as patient samples) for disease markers (such as tumor-associated markers). In specific embodiments, the binding agent comprises at least one primary antibody and may include a secondary enzyme-linked antibody, particularly a peroxidase, such as HRP.

[0218] 1. Processing of tissue samples

[0219] Biological samples can be fixed with fixatives such as formalin (formaldehyde) and glutaraldehyde, but other fixation techniques such as alcohol immersion can also be used. Samples can also be embedded in paraffin, and in certain embodiments, samples are formalin-fixed paraffin-embedded. The preparation of tissue blocks for IHC and immunostaining procedures is well known to those of ordinary skill in the art. Any intact organ or tissue can be cut into small pieces and incubated in various fixatives until the tissue is "fixed".

[0220] 2. Sample deparaffinization

[0221] Deparaffinization is the substantial or complete removal of paraffin from a paraffin embedded sample. Many deparaffinization techniques are known, but deparaffinization generally involves washing the sample with an organic solvent (such as benzene, toluene, ethylbenzene, xylene and mixtures thereof) to dissolve the paraffin.

[0222] 3. Rehydration

[0223] After deparaffinization, the sample can be rehydrated, usually using an aqueous, lower alcohol solution of reduced concentration. Ethanol is the preferred lower alcohol for rehydration. The sample is mixed with the alcohol solution and centrifuged.

[0224] 4. Preprocessing

[0225] The sample can be pretreated to prepare the target for detection. Pretreatment techniques include, for example, heat-induced epitope retrieval or proteolytic enzyme-mediated treatment. Citrate buffer, Tris and EDTA base can be used in heat-induced protocols. Proteolytic enzymes include pepsin, proteinase K and trypsin.

[0226] 5. Antibody conjugates

[0227] Certain embodiments of the present invention relate to the use of antibodies that bind to proteins, polypeptides and peptides. The antibody can be a monoclonal antibody. The antibody can be conjugated to a reporter molecule (such as an enzyme). Any antibody with sufficient selectivity, specificity or affinity can be used, as can be evaluated using conventional immunological screening methods known to those of ordinary skill in the art. Antibody conjugates include antibody conjugates connected to an enzyme tag that produces a colored product when contacted with a chromogenic substrate. Any suitable enzyme can be used, such as urease, alkaline phosphatase and glucose oxidase, but the preferred enzyme for the present invention is a peroxidase, such as horseradish peroxidase (HRP), which has a molecular weight of 40kD and is isolated from the root of the horseradish plant. HRP has an iron-containing heme group (heme) as its active site, which forms a complex with hydrogen peroxide. The non-covalent binding of HRP to antibodies is known to those of ordinary skill in the art, as described in detail by Sternberger.

[0228] Antibodies can be used for immunodetection of wild-type and / or mutant ligand proteins, polypeptides and / or peptides. Immunodetection methods include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays, fluorescent immunoassays, chemiluminescent assays, bioluminescent assays and Western blots. Various useful immunodetection methods are described in the scientific literature, including: Doolittle MH and Ben-Zeev O, 1999; Gulbis B and Galand P, 1993; De Jager R et al., 1993; and Nakamura et al., 1987, each of which is incorporated herein by reference.

[0229] Immunobinding generally involves obtaining a sample comprising a protein, polypeptide and / or peptide target. The sample is contacted with a primary anti-target antibody under conditions effective to form an immune complex, which generally involves adding the antibody composition to the sample and incubating the mixture for a sufficient time to allow the antibody to form an immune complex with the antigen present in the sample. Thereafter, the sample-antibody composition (such as a tissue section or a Western blot) is washed to remove any non-specifically bound antibody species to allow detection of only those antibodies involved in the primary immune complex.

[0230] B. Embodiments of the Invention

[0231] One embodiment of the method includes providing at least one compound according to Formula I and / or Formula II; and using the compound to detect a target in a sample. The method may include, for example, contacting a biological sample containing a target with a first compound according to Formula I and / or Formula II under conditions selected to covalently bind the first compound to the target or substantially adjacent to the target. The first compound is then contacted with a diazo compound under conditions where the first compound is covalently linked to the diazo compound to form a conjugate. The conjugate thus formed is then detected using a suitable technique (such as bright field or fluorescence microscopy). The first compound may exhibit a first optical signal when irradiated with light of an appropriate wavelength, and the conjugate may exhibit a second optical signal when irradiated with light of an appropriate wavelength, wherein the first optical signal and the second optical signal are different.

[0232] Figures 1 to 3 Various aspects of the disclosed method are shown. Figure 1In a first step 10, a biological sample having a target of interest is contacted with a 1° antibody that binds to the antigenic portion of the target. Exemplary primary antibodies include Ki67 antibodies. The 1° antibody-biological target is then contacted with a 2° antibody directed against the primary antibody in step 12. By way of example only, the 1° antibody can be a mouse antibody and the 2° antibody can be a poly-HRP-goat anti-mouse (GAM) antibody. As another example, the 1° antibody can be a rabbit antibody and the secondary antibody can be a poly-HRP-goat anti-rabbit (GAR). The 2° antibody is conjugated to an enzyme, particularly a peroxidase. Suitable peroxidases include, but are not limited to, horseradish peroxidase, microperoxidase, and ascorbate peroxidase (APEX).

[0233] In step 14, the biological sample bound to the 1° antibody-2° antibody construct can optionally be washed with a buffer solution (such as buffered Tween 20). 2 O 2 ) is incubated with a compound of formula I and / or formula II according to the present invention in the presence of a molecule of the present invention. This process deposits the compound of formula I and / or formula II on the desired biological target or in close proximity to the desired biological target.

[0234] The deposition process Figure 3 In the case of HRP and H 2 O 2 Compound 30 was incubated with biological samples in the presence of HRP and H 2 O 2 The reaction of the exemplary tyramide compound with the target is catalyzed to form a tyramide compound-biological sample conjugate 32 .

[0235] Continue to refer Figure 1 In step 16, the compound-biological sample conjugate is then incubated with a visualization element (such as a colorimetric and / or fluorescent agent, such as a diazonium salt chromogen). In step 18, the sample with the visualization element covalently bound thereto is then optionally washed and then imaged, such as by using bright field or fluorescence microscopy.

[0236] Similar immunostaining procedures were performed by Figure 2Schematically shown. A biological target 20 is bound to a 1° antibody 22 to form a biological target-1° antibody conjugate. The biological target-1° antibody conjugate is incubated with a 2° antibody 24 conjugated to a peroxidase (such as HRP26 shown) to form a 1° antibody-2° antibody-HRP conjugate. The 1° antibody-2° HRP conjugate is then incubated with a compound 30 according to the present invention to covalently attach the compound to the target 20. One of ordinary skill in the art will appreciate that multiple such compounds 30 are covalently attached to the target. This is followed by reaction with a diazonium salt to form a compound 32 covalently attached to the target 20. The process can be repeated as needed, using different 1° antibodies and visualization elements to detect and distinguish multiple different targets in a single sample.

[0237] Figures 1 to 3 The illustrated embodiment involves contacting the target with the first compound in the presence of a peroxidase to covalently couple the first compound to the target. Suitable peroxidases include, but are not limited to, horseradish peroxidase or ascorbate peroxidase (APEX).

[0238] Figures 1 to 3 The illustrated embodiments also involve coupling the compounds according to the invention with a visualizing agent such as a diazonium salt, which typically has a structure such as RN 2 X - , wherein R is an organic group, more generally an organic group having at least one aromatic ring. It will be appreciated by those of ordinary skill in the art that any diazonium salt can be used according to the present invention. This allows the user to select the color that the user wants to be associated with a specific target, such as red, purple, blue or green. Exemplary diazo compounds include Fast Red TR, Fast Violet LB and Fast Blue. Conjugates containing such azo dyes after reacting with exemplary compounds according to the present invention are as follows.

[0239]

[0240]

[0241] The green color is most ideally produced using anthracene derivatives as indicated below, where R 1 and R 2 For example

[0242]

[0243] These conjugates can be detected colorimetrically, such as by using bright field microscopy. The conjugates may also fluoresce and thus may also be detected using fluorescence microscopy.

[0244] The compounds according to the invention can also be used in Western blotting protocols. A complete Western blotting procedure can be performed and the blot visualized with a standard secondary antibody-HRP conjugate using a covalent chromogenic substrate. An embodiment of a Western blotting protocol is described in more detail in Example 5 below.

[0245] VI. Kit

[0246] The present invention also provides a kit for detecting biological targets. An example of a kit according to the present invention includes a compound according to formula I and / or II and a diazonium salt. A more specific example of a kit according to the present invention includes a blocking buffer, such as 3% bovine serum albumin; poly-HRP-goat anti-mouse (GAM), poly-HRP-goat anti-rabbit (GAR) and / or HRP-conjugated streptavidin; a compound according to formula I, II and / or formula III, such as a 5mM aqueous solution; a 2 buffer system for tyramide deposition, such as a 200mM citrate buffer at pH 4, and 7.5% stable hydrogen peroxide; a diazonium salt, such as Fast Red TR or Fast Blue BB; and a color development buffer, such as 100mM Tris at pH 8.5.

[0247] VII. Examples

[0248] The following examples are provided to illustrate certain specific features of the disclosed embodiments of the invention. It will be appreciated by those of ordinary skill in the art that the scope of the invention is not limited to these specific features.

[0249] Example 1 - Synthesis of Chromogen Substrate

[0250] This example concerns the synthesis of a chromogen substrate, as indicated below.

[0251]

[0252] 3-Hydroxynaphthalene-2-carboxylic acid (1.0056 g, 5.3438 mmol) was added to a round bottom flask at 0°C, followed by anhydrous tetrahydrofuran (10 mL), followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.70 mL, 7.8 mmol) and 1-hydroxybenzotriazole (0.8351 g, 6.057 mmol) and 0.3 mL of triethylamine via a syringe. The reaction mixture was stirred at 0°C for 1 hour and then warmed to room temperature. Tyramine (5.3438 mmol) was added in one portion and the reaction mixture was stirred overnight under an inert atmosphere. Volatiles were removed and the product was purified using flash chromatography and an elution system containing ethyl acetate and hexanes. Pure fractions were combined to give 3-hydroxy-N-(4-hydroxyphenethyl)-2-naphthamide (400 mg, 25% of theory) as a light yellow crystalline solid. 1H NMR: (400MHz, DMSO-d6): δ12.00, (br s,1H),9.21,(s,1H),9.07,(t,1H),8.48,(s,1H),7.84,(d,1H),7.73,(d,1H),7.50,(t, 1H),7.35,(t,1H),7.26(s,1H),7.07(d,2H),6.68,(d,2H),3.52(dt,2H),2.79,(t,1H).

[0253] Example 2—Synthesis of Chromogen Substrate

[0254] This example concerns the synthesis of a second ethoxylated chromogen substrate designed to increase the solubility of the substrate in solvents commonly used in IHC protocols.

[0255]

[0256] To a vial was added 2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]acetic acid (152.5 mg, 0.4963 mmol), followed by anhydrous N,N-dimethylformamide (4 mL), followed by N,N-diisopropylethylamine (0.18 mL, 1.0 mmol), followed by HATU (387 mg, 0.997444 mmol). The reaction mixture was stirred for 15 minutes. 4-(2-aminoethyl)phenol (72.1 mg, 0.526 mmol) was then added to the reaction mixture in one portion. The reaction mixture was stirred at room temperature overnight under an inert atmosphere. The volatiles were removed and the residue was purified by flash chromatography and H 2 The reaction mixture was purified by O / MeCN. The pure fractions were combined to give tert-butyl (1-(4-hydroxyphenyl)-4-oxo-6,9,12-trioxa-3-azatetradec-14-yl)carbamate (191.2 mg, 90% of theory) as a yellow oil. 1 H NMR: (400MHz, DMSO-d6): δ7.05, (d,2H), 6.72, (d,2H), 3.97, (s,2H)3.7-3.0, (m,16H)1.38, (s,9H).

[0257]

[0258] In a vial containing 100 mg of tert-butyl (1-(4-hydroxyphenyl)-4-oxo-6,9,12-trioxa-3-azatetradecane-14-yl)carbamate was added 5 mL of dichloromethane followed by 1 mL of trifluoroacetic acid. The mixture was stirred for 2 hours until consumption of the starting material was confirmed by thin layer chromatography (TLC). Volatiles were removed and anhydrous N,N-dimethylformamide (1 mL) was added to the reaction mixture.

[0259] In a separate vial, 3-hydroxynaphthalene-2-carboxylic acid (60 mg, 0.319 mmol), anhydrous N,N-dimethylformamide (5 mL), N,N-diisopropylethylamine (0.1 mL, 0.6 mmol) and finally HATU (241 mg, 0.621 mmol) were mixed. The reaction mixture was then stirred for 15 minutes. A solution of 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-N-(4-hydroxyphenethyl)acetamide in DMF was added and the reaction mixture was stirred at room temperature overnight under an inert atmosphere. The volatiles were removed and the residue was purified by flash chromatography using H 2 The reaction mixture was purified by O / MeCN. The pure fractions were combined to give 3-hydroxy-N-(1-(4-hydroxyphenyl)-4-oxo-6,9,12-trioxa-3-azatetradec-14-yl)-2-naphthamide (80.1 mg, 53% of theory) as a sticky light yellow solid. 1 H NMR: (400MHz, CD3OD): δ8.69,(s,1H),8.07,(d,1H),7.97,(d,1H),7.69,(t,1H),7.59,(t,1H),7 .00,(d,2H),6.70,(d,2H),4.31,(t,2H),3.89-3.75(m,4H),3.70-3.25,(m,12H),2.68,(t,2H).

[0260] Example 3—Synthesis of Chromogen Substrate

[0261] This example concerns the synthesis of a third sulfonated chromogen substrate designed to increase the solubility of the substrate in solvents commonly used in IHC protocols.

[0262]

[0263] Add 42 mL of 85% H 2 SO 4. The flask was heated to 120° C. with stirring, then 17.4 g of 3-hydroxy-2-naphthoic acid was added, and the resulting reaction was stirred for 30 minutes. The reaction mixture was then stopped from heating and allowed to cool to room temperature. The reaction mixture was diluted with 275 mL of deionized water and heated to 60° C., then 16 g of NaCl was added. The solution was stirred for 10 minutes, then the heating was stopped and allowed to cool to room temperature with stirring. The reaction mixture was stirred at room temperature for 1 hour. The product, sodium 7-carboxy-6-hydroxynaphthalene-2-sulfonate, precipitated as a fine white solid and was collected by vacuum filtration and dried overnight under high vacuum. Sodium 7-carboxy-6-hydroxynaphthalene-2-sulfonate (26.3 g, 97% of theoretical value) was isolated as a free-flowing white solid. 1 H NMR: (400MHz, DMSO-d6): δ11.01, (br s, 1H), 8.56 (s, 1H), 8.16 (s, 1H), 7.74 (m, 2H), 7.33 (s, 1H).

[0264]

[0265] 26 grams of 7-carboxy-6-hydroxynaphthalene-2-sodium sulfonate were added to a round bottom flask under an inert atmosphere, followed by 100 mL of anhydrous DMF and 27 mL of triethylamine. After 10 minutes, HATU (35 g) was added and the reaction mixture was stirred for another 10 minutes. 12.61 grams of tyramine were added to the reaction mixture, which was sealed and stirred overnight at room temperature. Volatiles were removed and the residue was dissolved in 300 mL of water and filtered. 60 mL of brine was added to the filtrate and the resulting solution was allowed to stand for 30 minutes. The product precipitated as a fine light yellow solid, which was collected by vacuum filtration and dried via high vacuum. Sodium 6-hydroxy-7-((4-hydroxyphenylethyl)carbamoyl)naphthalene-2-sulfonate (13 g, 35% of theoretical value) was isolated as a free-flowing light yellow solid. 1 H NMR: (400MHz, DMSO-d6): δ12.16(br s,1H),9.22,(s,1H),9.08(t,1H),8.51,(s,1H),8.07,(s,1H),7.69(m, 2H),7.26(s,1H),7.77(d,2H),6.70(d,2H),3.53(dt,2H),2.80(t,2H).

[0266] Example 4—Tissue Processing and Brightfield Imaging

[0267] Fixed tissues were processed via deparaffinization and heat-induced epitope retrieval (HIER). Tissues were then permeabilized with buffered 0.1% triton for 30 minutes and blocked with appropriate normal serum based on the type of primary antibody for 1 hour at room temperature. Primary antibodies were diluted to the desired concentration and added to the tissues. Tissues were then incubated in a humidified chamber at room temperature for the desired time. Slides were then washed in buffered 0.05% Tween 20. Secondary antibody conjugates (SuperBoost TM Poly HRP (with appropriate species to match the primary antibody) was diluted to the desired concentration and added to the tissue, which was incubated in a humidified chamber at ambient temperature for 1 hour. Slides were washed in buffered 0.05% Tween 20. Sodium 6-hydroxy-7-((4-hydroxyphenethyl)carbamoyl)naphthalene-2-sulfonate was added in 3.75% Tween 20. 2 O 2 The slides were diluted in citrate buffer and incubated at ambient temperature for the desired time in a humidified chamber. The slides were then washed in buffered 0.05% Tween 20. The color was then developed for a period of time by selecting the appropriate reagent (red, blue, purple or DAB control) to obtain the desired intensity. When the desired staining intensity was reached, the color development was stopped using SHAM. The samples were co-stained with Gill's hematoxylin and / or DAPI as needed, and the slides were imaged using bright field microscopy. Figures 4 to 10 Images of samples stained according to this example are provided.

[0268] Example 5—Western Blot

[0269] This example describes the use of a covalent chromogenic substrate for protein visualization in a western blot application. A complete western blot procedure can be performed and the blot visualized with a standard secondary antibody-HRP conjugate using a covalent chromogenic substrate.

[0270] The target protein extract was diluted to the appropriate desired concentration with Laemmli buffer (20% glycerol, 2.1% SDS, 0.125M Tris at pH 6.8, 0.73M 2-mercaptoethanol, bromophenol blue), and the solution was then heated in a 95-100°C water bath for 5 minutes, cooled to room temperature and loaded onto an SDS-PAGE gel (using acrylamide percentage appropriate for the target protein plus stacking gel (8% acrylamide containing bromophenol blue)). Prestained protein molecular weight standards (10-170 kDa) may also be added as molecular weight references. Electrophoresis was performed at 200 V until the dye front ran out of the gel, which was usually about 1. The protein was then transferred from the gel to a PVDF membrane at 6°C and 100 V for 1 hour. The PVDF membrane was washed twice with TTBS (5 minutes each time), and then the membrane was blocked with a TTBS solution of milk powder and then incubated with the primary antibody selected for the target protein target. The PVDF was then washed again and incubated with the secondary antibody-HRP conjugate. By 2 O 2 The covalent reaction of the substrate is carried out by incubating the covalent chromogenic substrate (compound 5) in HRP buffer, followed by washing with TTBS and then incubating the diazonium salt of the desired color. The reaction with the diazonium salt is rapid.

[0271] Compared to the procedure of using traditional colorimetric probes to visualize protein targets using NBT / BCIP, proteins can be detected with a direct method that does not require any additional equipment or time. In addition to the colorimetric signal, some of the diazonium salts are also fluorescent. This method can be easily expanded for multiplex detection and is widely applicable to protein analysis.

[0272] Example 6

[0273] This example evaluates the use of formula I according to the present invention The results obtained for the compounds, i.e.

[0274]

[0275] This compound was evaluated to determine the effect of chromogen concentration and incubation time on the staining results. Fig.11 It is shown that the chromogens according to the invention provide very suitable staining results for typical staining protocols. Higher chromogen concentrations provide stronger staining results, as shown by comparing the results produced using 0.5 mM and 1 mM concentrations. Fig.11 However, as can be seen by comparing the images produced using a 10 minute incubation time with a 30 minute incubation time, increased incubation times can also lead to overdevelopment and less intense staining results.

[0276] Example 7

[0277] This example compares the results obtained by staining Ki67 tonsil tissue using the following positions: (1) the first IIP according to the present invention 1 -LP 2 Compound, i.e., in a 1 mM staining solution providing a 1 mM total compound concentration According to the second formula I of the present invention Compounds, i.e., in a 0.5 mM staining solution providing a 1 mM total compound concentration and the well-known diaminobenzidine (DAB), Although all three tests produced suitable staining images (see Fig.12 ), but the second compound appears to provide a stronger Fast Red stain than the first compound and can be used in a wider range of staining protocols than DAB. Figure 13 to Figure 51 As shown, the second compound was also demonstrated to be a viable and robust histochemical stain detectable and multiplexed in blue and red via brightfield and fluorescence imaging, compatible with a variety of tissue types, counterstains, and mounting media.

[0278] Example 8

[0279] This example relates to the synthesis of di-tert-butyl (6-((4-hydroxyphenethyl)amino)-6-oxohexane-1,5-diyl)(S)-dicarbamate according to the following scheme.

[0280]

[0281] To N 2 ,N 6 To a solution of -bis(tert-butyloxycarbonyl)-L-lysine (1 g, 2.9 mmol) in anhydrous DMF (30 mL) was added triethylamine (0.5 mL, 3.6 mmol, 1.2 eq), followed by HATU (1.323 g, 3.5 mmol, 1.2 eq) and tyramine (477 mg, 3.5 mmol, 1.2 eq), and the solution was stirred for 18 hours. The volatiles were removed by rotary evaporation, and the residue was dissolved in EtOAc (100 mL). The solution was washed with 0.5 M HCl (2×50 mL), deionized water (1×50 mL), saturated NaHCO 3 The organic layer was washed with Na 2 SO 4 Dry, filter, and remove volatiles by rotary evaporation. Flash chromatography (mobile phase MeOH / CHCl 3) to give di-tert-butyl (6-((4-hydroxyphenethyl)amino)-6-oxohexane-1,5-diyl)(S)-dicarbamate (951 mg, 70% of theory) as a brown oil. 1 H NMR,DMSO-d6:7.01(d,2H),6.82(d,2H),6.15(br s,1H),5.22(br s,1H),4.75(br s,1H),4.00(br s,1H),3.60(br s,1H),3.40(br s,1H),3.01(m,2H),2.71(m,2H),1.62(br s,2H),1.51-1.25(m,20H),1.12(br s,2H).

[0282] Example 9

[0283] This example relates to the synthesis of (S)-6-((4-hydroxyphenethyl)amino)-6-oxohexane-1,5-diammonium 2,2,2-trifluoroacetate according to the following scheme.

[0284]

[0285] To a solution of di-tert-butyl (6-((4-hydroxyphenethyl)amino)-6-oxohexane-1,5-diyl)(S)-dicarbamate (950 mg, 2.0 mmol) in dichloromethane (20 mL) was added trifluoroacetic acid (5 mL, 65.4 mmol). The solution was stirred for 3 hours before the volatiles were removed via rotary evaporation. Drying under high vacuum for 18 hours gave (S)-6-((4-hydroxyphenethyl)amino)-6-oxohexane-1,5-diammonium 2,2,2-trifluoroacetate as a sticky brown solid (quantitative conversion).

[0286] Example 10

[0287] This example relates to the synthesis of triethylammonium (S)-7,7'-(((6-((4-hydroxyphenethyl)amino)-6-oxohexane-1,5-diyl)bis(imino))bis(carbonyl))bis(6-hydroxynaphthalene-2-sulfonate according to the following scheme.

[0288]

[0289] To a solution of sodium 7-carboxy-6-hydroxynaphthalene-2-sulfonate (1.161 g, 4.0 mmol, 2 eq) in anhydrous DMF (30 mL) was added HATU (1.597 g, 4.2 mmol, 2.1 eq) and triethylamine (0.8 mL, 5.8 mmol, 2.9 eq). After stirring for 1 hour, a 200 mM solution of (S)-6-((4-hydroxyphenethyl)amino)-6-oxohexane-1,5-diammonium 2,2,2-trifluoroacetate (987 mg, 2 mmol) in anhydrous DMF was added, followed by an equal portion of TEA (0.8 mL, 5.8 mmol, 2.9 eq). The solution was stirred for 18 hours, and then the volatiles were removed via rotary evaporation. Purification by reverse phase flash chromatography (mobile phase MeOH / water) gave (S)-7,7′-(((6-((4-hydroxyphenethyl)amino)-6-oxohexane-1,5-diyl)bis(imino)bis(carbonyl))bis(6-hydroxynaphthalene-2-sulfonate) (1.065 g, 55% of theory) as a light yellow solid. 1 H NMR, DMSO-d6:9.13(s,1H),9.03(t,1H),8.64(s,1H),8.52(s,1H),8.18(t,1H),8.07(d,2H),7.75-7.6(m,4H),7.26(d ,2H),6.97(d,2H),6.64(d,2H),4.53(q,1H),3.15-3.4(m,4H),2.6(t,2H),1.9-1.7(m,2H),1.63(m,2H),1.41(m,2H).

[0290] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the embodiments shown are only preferred examples of the invention and should not be considered to limit the scope of the invention. In general, in the following clauses and claims, the terms used should not be interpreted as limiting the clauses and claims to the specific embodiments disclosed in the specification and claims, but should be interpreted as including all possible embodiments and the full range of equivalents authorized by such clauses and claims. Therefore, the clauses and claims are not limited by the present invention. The inventor therefore claims that the present invention fully belongs to the scope and spirit of the claims. In addition, the embodiments may be according to the following numbered clauses.

[0291] 1. A compound, the compound satisfies formula I

[0292]

[0293] in:

[0294] L is a linker having 1 to at least 50 atoms;

[0295] P 1 , P 2 and P 3 is an aromatic ring or an aromatic ring system;

[0296] R is a chemical bond or independently an L group; and

[0297] x and y are independently 0 to 5.

[0298] 2. The compound according to clause 1, wherein P 1 and P 3 The ring systems are the same or different and each may comprise at least two fused rings, wherein at least one of the fused rings comprises the structure shown below:

[0299]

[0300] 3. A compound according to clause 1 or 2, wherein P 2 is a phenyl ring substituted with at least one -OH group.

[0301] 4. A method according to any one of clauses 1 to 3, wherein:

[0302] L is a linker having 2 to 20 atoms in the chain;

[0303] R is a chemical bond or independently an L group; and

[0304] x and y are independently 0 or 1.

[0305] 5. The compound according to clause 1, which has a structure selected from the following:

[0306]

[0307] 6. The compound according to item 1, which satisfies formula II

[0308] P 1 -LP 2 ,

[0309] Formula II

[0310] in:

[0311] P 1 is a first aromatic ring system comprising at least two fused rings, wherein at least one of the fused rings comprises a structure as shown below:

[0312]

[0313] P 2 is a second aromatic ring system substituted with at least one -OH group; and

[0314] L is a connector.

[0315] 7. The compound according to clause 6, wherein P 1 There are 2 to 4 rings in the fused ring system.

[0316] 8. A compound according to any one of clauses 1 to 7, wherein P 1 and / or P 3 is substituted with at least one water soluble moiety selected to increase the water solubility of the compound.

[0317] 9. A compound according to any one of clauses 1 to 8, wherein P 1 Selected from:

[0318] Wherein m is 1 to 5.

[0319] 10. The compound according to clause 8 or 9, wherein the water-soluble part is selected from SO 3 - 、-SO 3 H, -CO 2 - , -CO 2 H, -HPO 3 -2 , H 2 PO 3 -1 , H 3 PO 3 , PEG,

[0320] 11. A compound according to any one of clauses 1 to 10, wherein P 1 The hydroxyl group on L is at the ortho or meta position.

[0321] 12. The compound according to clause 9, wherein:

[0322] P 1 is naphthol, L is coupled to the 2- or 3-position, at least one hydroxyl group is at the 3- and / or 4-positions, and the at least one moiety selected to increase the water solubility of the compound is coupled to at least one of the 5-8-positions;

[0323] P 1 is anthraquinone, L is coupled to the 2-position or the 3-position, at least one hydroxyl group is at the 3-position and / or the 4-position, and the at least one moiety selected to increase the water solubility of the compound is coupled to at least one of the 6-9-positions;

[0324] P 1is chrysene alcohol, L is coupled to the 2-position or the 3-position, at least one hydroxyl group is at the 3-position and / or the 4-position, and the at least one moiety selected to increase the water solubility of the compound is coupled to at least one of the 7-10-positions; or

[0325] P 1 is phenanthranol, L is coupled to the 2- or 3-position, at least one hydroxyl group is at the 3- and / or 4-positions, and the at least one moiety selected to increase the water solubility of the compound is coupled to at least one of the 5-8-positions.

[0326] 13. The compound according to clause 1, wherein P 1 and / or P 3 It is a heteroaryl fused ring system.

[0327] 14. The compound according to clause 13, wherein the heteroaryl fused ring system is electron rich.

[0328] 15. The compound according to clause 13 or 14, wherein the heteroaryl fused ring system is selected from fused furan, fused thiophene and fused pyrrole ring systems.

[0329] 16. A compound according to any one of clauses 13-15, wherein P 1 and / or P 3 Selected from

[0330] 17. A compound according to any one of clauses 13-16, wherein P 1 and / or P 3 is substituted with at least one moiety selected to increase the water solubility of the compound.

[0331] 18. A compound according to any one of clauses 1 to 17, wherein P 2 It is an aromatic hydrocarbon.

[0332] 19. A compound according to any one of clauses 1 to 18, wherein P 2 Including phenol.

[0333] 20. The compound according to clause 19, wherein the phenol has a hydroxyl group at the para position of L.

[0334] 21. A compound according to any one of clauses 1-20, wherein L is selected from C 1 -C 50 Aliphatic group, C 1 -C 50 Alkyl, C 1 -C 50 Alkylamide, C 1 -C 50 Alkyl esters, C1 -C 50 Ketone, C 2 -C 50 Polyols, C 2 -C 50 Polyalkyl ether and C 2 -C 50 Aliphatic amines.

[0335] 22. A method according to any one of clauses 1 to 21, wherein:

[0336] P 1 Selected from

[0337] Where m is 1 to 3; P 2 including phenol;

[0338] The at least one moiety selected to increase the water solubility of the compound is selected from -SO 3 - 、-SO 3 H, -CO 2 - , -CO 2 H, -HPO 3 -2 , H 2 PO 3 -1 , H 3 PO 3 , PEG, and

[0339] L is selected from covalent bonds, C 1 -C 10 Alkyl, C 1 -C 10 Alkylamide, C 1 -C 10 Ketone or C 1 -C 10 Diol.

[0340] 23. The compound according to clause 22, wherein:

[0341] P 1 For naphthol;

[0342] m is 1; and

[0343] The at least one moiety selected to increase water solubility is -SO 3 - .

[0344] 24. The compound according to clauses 21-23, wherein L is And n is 1 to 5.

[0345] 25. The compound according to any one of clauses 21-23, wherein m is 1 and n is 2.

[0346] 26. The compound according to clause 1, wherein:

[0347] P 1 For naphthol;

[0348] P 2 for phenol;

[0349] The at least one moiety selected to increase water solubility is -SO 3 - ;and

[0350] L is

[0351] 27. A compound having the structure

[0352]

[0353] in:

[0354] R 1 and R 2 are independently selected from H and OH;

[0355] R 3 -R 8 are independently selected from H, -F, -Cl, -Br, -I, -OH,

[0356] -OR 9 , where R 9 C 1 -C 6 Alkyl, -SO 3 - 、-SO 3 H, -CO 2 - , -CO 2 H, -HPO 3 -2 , H 2 PO 3 -1 , H 3 PO 3 , PEG, Where R 1 , R 2 , R 7 At least one of R 8 -R 12 At least one of is a hydroxyl group; or

[0357] R3 and R 4 together form at least one fused ring system; and / or

[0358] R 4 and R 5 together form at least one fused ring system; and / or

[0359] R 5 Together with R, form at least one fused ring system; and / or

[0360] n is 1 to 30.

[0361] 28. The compound according to clause 27, wherein if R 1 is H, then R 2 is OH, and if R 1 is OH, then R 2 For H.

[0362] 29. The compound according to clause 27 or 28, wherein R 3 , R 4 , R 5 and R 6 independently H or -SO 3 - .

[0363] 30. The compound according to clause 28, wherein:

[0364] R 1 is -OH; and

[0365] R 5 Yes SO 3 - or SO 3 H.

[0366] 31. A compound according to any one of clauses 27-28, wherein R 10 It is -OH.

[0367] 32. The compound according to clause 27, which has the structure

[0368]

[0369] 33. A compound or a salt thereof, wherein the compound or the salt thereof has the structure

[0370]

[0371] 34. A compound or a salt thereof, wherein the compound or the salt thereof has the structure

[0372]

[0373] 35. A covalent conjugate comprising:

[0374] a biological sample comprising a target; and

[0375] At least one compound according to any one of clauses 1-34 covalently bound to the target.

[0376] 36. The conjugate according to clause 35, which has the formula

[0377]

[0378] 37. The conjugate according to clause 35, which has the formula

[0379]

[0380]

[0381] 38. The conjugate according to clause 35, which has the formula

[0382]

[0383] 39. The conjugate according to clause 38, which has the formula

[0384]

[0385] 40. A method comprising:

[0386] at least one providing a compound according to any one of clauses 1-34; and

[0387] The compound is used to detect the target in a sample.

[0388] 41. The method according to clause 40, wherein the method is an immunostaining method.

[0389] 42. A method comprising:

[0390] contacting a biological sample comprising a target with a first compound according to any one of clauses 1 to 34 under conditions selected such that the first compound is covalently bound to or substantially adjacent to the target;

[0391] contacting the first compound with a diazo compound under conditions such that the first compound is covalently linked to the diazo compound to form a conjugate; and

[0392] The target is detected.

[0393] 43. The method of clause 42, wherein the first compound exhibits a first optical signal when illuminated with light of an appropriate wavelength.

[0394] 44. A method according to any of clauses 42-43, wherein the conjugate exhibits a second optical signal when illuminated with light of an appropriate wavelength, wherein the first optical signal and the second optical signal are different.

[0395] 45. A method according to any one of clauses 42 to 44, wherein the biological target is a cell, a tissue, a virus, a gene, an epitope, a target genetic sequence or a target protein sequence.

[0396] 46. ​​The method according to any one of clauses 42 to 45, comprising contacting the target with the first compound in the presence of a peroxidase to covalently couple the first compound to the target.

[0397] 47. The method according to clause 46, wherein the peroxidase is horseradish peroxidase or ascorbate peroxidase (APEX).

[0398] 48. The method according to clause 46, wherein the peroxidase is a heme-containing peptide.

[0399] 49. The method according to clause 46, wherein the peroxidase is a microperoxidase.

[0400] 50. A process according to any one of clauses 42 to 49 wherein the diazo compound is selected from Fast Red, Fast Violet and Fast Blue.

[0401] 51. The method according to clause 42, wherein the biological target is recognized by a primary antibody and the primary antibody is conjugated to a peroxidase.

[0402] 52. The method according to clause 42, comprising:

[0403] contacting the biological target with a primary antibody that binds to the target; and

[0404] The primary antibody is contacted with a secondary antibody directed against the primary antibody to form a primary-secondary antibody conjugate, wherein the secondary antibody is conjugated to peroxidase.

[0405] 53. The method according to any one of clauses 51 or 52, wherein the primary antibody is a Ki67 antibody, an ESR1 antibody, a KRT15 antibody and / or a CDH17 antibody.

[0406] 54. A method according to clause 52, wherein:

[0407] The primary antibody is a mouse antibody, and the secondary antibody is poly-HRP-goat anti-mouse (GAM); or

[0408] The primary antibody was a rabbit antibody, and the secondary antibody was poly-HRP-goat anti-rabbit (GAR).

[0409] 55. A method comprising:

[0410] contacting a biological sample having a target of interest with a primary antibody that binds to the target;

[0411] contacting the primary antibody with a secondary antibody comprising a peroxidase compound to form a primary antibody-secondary antibody conjugate;

[0412] contacting the sample with a compound satisfying Formula I under conditions suitable for covalently bonding the compound to the sample

[0413]

[0414] wherein L is a linker having 1 to at least 50 atoms, P 1 , P 2 and P 3 is an aromatic ring or aromatic ring system, R is a chemical bond or independently an L group, and x and y are independently 0 to 5;

[0415] contacting the sample with a diazonium salt; and

[0416] The target is detected.

[0417] 56. A method according to clause 55, wherein the target is detected by colorimetry, fluorescence, or both.

[0418] 57. A method according to clause 55 or 56, wherein the diazonium salt is selected from Fast Red, Fast Violet or Fast Blue.

[0419] 58. A method according to clause 55, comprising co-locating a plurality of different color combinations at or around a target.

[0420] 59. The method of clause 55, comprising detecting two or more targets in the sample.

[0421] 60. The method of clause 55, wherein the compound is fluorescent, and the method further comprises detecting the target using fluorescence.

[0422] 61. The method according to clause 55, wherein the compound has the formula P 1 -LP 2and is a naphthol derivative, and the diazonium salt is a fast blue to produce a green color suitable for detecting the target.

[0423] 62. The method according to clause 55, wherein the peroxidase is horseradish peroxidase or ascorbate peroxidase.

[0424] 63. A kit, comprising:

[0425] The compound according to clause 1; and

[0426] Diazonium salt.

[0427] 64. A kit comprising:

[0428] Blocking buffer;

[0429] poly-HRP-goat anti-mouse (GAM), poly-HRP-goat anti-rabbit (GAR), and / or HRP-conjugated streptavidin;

[0430] The compound according to clause 1;

[0431] Hydrogen peroxide;

[0432] Reaction buffer;

[0433] Diazonium salts; and

[0434] Reaction termination reagent.

[0435] 65. A kit according to clause 64, wherein the diazonium salt is Fast Red, Fast Violet and / or Fast Blue.

Claims

1. A compound, the compound satisfies formula I in: L is a linker having 1 to at least 50 atoms; P 1 , P 2 and P 3 is an aromatic ring or an aromatic ring system; R is a chemical bond or independently an L group; and x and y are independently 0 to 5.

2. The compound according to claim 1, wherein P 1 and P 3 The ring systems are the same or different and each may comprise at least two fused rings, wherein at least one of the fused rings comprises the structure shown below:

3. The compound according to claim 1 or 2, wherein P 2 is a phenyl ring substituted with at least one -OH group.

4. The compound according to claim 1, wherein: L is a linker having 2 to 20 atoms in the chain; R is a chemical bond or independently an L group; and x and y are independently 0 or 1.

5. The compound according to claim 1, which has a structure selected from the following:

6. The compound according to claim 1, which satisfies Formula II P 1 -L-P 2 , Formula II in: P 1 is a first aromatic ring system comprising at least two fused rings, wherein at least one of the fused rings comprises a structure as shown below: P 2 is a second aromatic ring system substituted with at least one -OH group; and L is a connector.

7. The compound according to claim 6, wherein P 1 There are 2 to 4 rings in the fused ring system.

8. The compound according to claim 1, wherein P 1 and / or P 3 is substituted with at least one water soluble moiety selected to increase the water solubility of the compound.

9. The compound according to claim 1, wherein P 1 Selected from: Wherein m is 1 to 5.

10. The compound according to claim 8 or 9, wherein the water-soluble part is selected from SO3 - , -SO3H、-CO2 - 、-CO2H、-HPO3 -2 、H2PO3 -1 、H3PO3、PEG、 11. The compound according to claim 1, wherein P 1 The hydroxyl group on L is at the ortho or meta position.

12. The compound according to claim 9, wherein: P 1 is naphthol, L is coupled to the 2- or 3-position, at least one hydroxyl group is at the 3- and / or 4-positions, and the at least one moiety selected to increase the water solubility of the compound is coupled to at least one of the 5-8-positions; P 1 is anthraquinone, L is coupled to the 2-position or the 3-position, at least one hydroxyl group is at the 3-position and / or the 4-position, and the at least one moiety selected to increase the water solubility of the compound is coupled to at least one of the 6-9-positions; P 1 is chrysene alcohol, L is coupled to the 2-position or the 3-position, at least one hydroxyl group is at the 3-position and / or the 4-position, and the at least one moiety selected to increase the water solubility of the compound is coupled to at least one of the 7-10-positions; or P 1 is phenanthranol, L is coupled to the 2- or 3-position, at least one hydroxyl group is at the 3- and / or 4-positions, and the at least one moiety selected to increase the water solubility of the compound is coupled to at least one of the 5-8-positions.

13. The compound according to claim 1, wherein P 1 and / or P 3 It is a heteroaryl fused ring system.

14. The compound of claim 13, wherein the heteroaryl fused ring system is electron rich.

15. The compound according to claim 13 or 14, wherein the heteroaryl fused ring system is selected from fused furan, fused thiophene and fused pyrrole ring systems.

16. The compound according to claim 13, wherein P 1 and / or P 3 Selected from 17. The compound according to claim 13, wherein P 1 and / or P 3 is substituted with at least one moiety selected to increase the water solubility of the compound.

18. The compound according to claim 13, wherein P 2 It is an aromatic hydrocarbon.

19. The compound according to claim 1, wherein P 2 Including phenol.

20. The compound of claim 19, wherein the phenol has a hydroxyl group at the para position of L.

21. The compound according to claim 1, wherein L is selected from C1-C 50 Aliphatic, C1-C 50 Alkyl, C1-C 50 Alkylamide, C1-C 50 Alkyl esters, C1-C 50 Ketone, C2-C 50 Polyols, C2-C 50 Polyalkyl ether and C2-C 50 Aliphatic amines.

22. The compound according to claim 1, wherein: P 1 Selected from Wherein m is 1 to 3; P 2 including phenol; The at least one moiety selected to increase the water solubility of the compound is selected from -SO3 - 、-SO3H、-CO2 - 、-CO2H、-HPO3 -2 、H2PO3 -1 、H3PO3、PEG、 and L is selected from a covalent bond, a C1-C 10 Alkyl, C1-C 10 Alkylamide, C1-C 10 Ketone or C1-C 10 Diol.

23. The compound according to claim 22, wherein: P 1 For naphthol; m is 1; and The at least one moiety selected to increase water solubility is -SO3 - .

24. The compound according to claim 21, wherein L is And n is 1 to 5.

25. The compound of claim 21, wherein m is 1 and n is 2.

26. The compound according to claim 1, wherein: P 1 For naphthol; P 2 for phenol; The at least one moiety selected to increase water solubility is -SO3 - ;and L is 27. A compound having the structure in: R 1 and R 2 are independently selected from H and OH; R 3 -R 8 are independently selected from H, -F, -Cl, -Br, -I, -OH, -OR 9 , where R 9 C1-C6 alkyl, -SO3 - 、-SO3H、-CO2 - 、-CO2H、-HPO3 -2 、H2PO3 -1 、H3PO3、PEG、 Where R 1 , R 2 , R 7 At least one of R 8 -R 12 At least one of is a hydroxyl group; or R3 and R4 together form at least one fused ring system; and / or R4 and R5 together form at least one fused ring system; and / or R5 and R together form at least one fused ring system; and / or n is 1 to 30.

28. The compound of claim 27, wherein if R1 is H, then R2 is OH, and if R1 is OH, then R2 is H.

29. A compound according to claim 27 or claim 28, wherein R3, R4, R5 and R6 are independently H or -SO3 - .

30. The compound according to claim 28, wherein: R1 is -OH; and R5 is SO3 - or SO3H.

31. The compound according to claim 27, wherein R 10 It is -OH.

32. The compound according to claim 27, wherein the compound has the structure 33. A compound or a salt thereof, wherein the compound or the salt thereof has the structure 34. A compound or a salt thereof, wherein the compound or the salt thereof has the structure 35. A covalent conjugate, comprising: a biological sample comprising a target; and At least one compound according to claim 1 covalently bound to the target.

36. The conjugate of claim 35, wherein the conjugate has the formula 37. The conjugate of claim 35, wherein the conjugate has the formula 38. The conjugate of claim 35, wherein the conjugate has the formula 39. The conjugate of claim 38, wherein the conjugate has the formula 40. A method comprising: Providing at least one compound according to claim 1; as well as The compound is used to detect the target in a sample.

41. The method of claim 40, wherein the method is immunostaining.

42. A method comprising: contacting a biological sample comprising a target with a first compound according to claim 1 under conditions selected so that the first compound is covalently bound to or substantially adjacent to the target; contacting the first compound with a diazo compound under conditions such that the first compound is covalently linked to the diazo compound to form a conjugate; as well as The target is detected.

43. The method of claim 42, wherein the first compound exhibits a first optical signal when illuminated with light of an appropriate wavelength.

44. The method of claim 42, wherein the conjugate exhibits a second optical signal when illuminated with light of an appropriate wavelength, wherein the first optical signal and the second optical signal are different.

45. The method of claim 42, wherein the biological target is a cell, a tissue, a virus, a gene, an epitope, a target genetic sequence, or a target protein sequence.

46. ​​The method of claim 42, comprising contacting the target with the first compound in the presence of a peroxidase to covalently couple the first compound to the target.

47. The method of claim 46, wherein the peroxidase is horseradish peroxidase or ascorbate peroxidase (APEX).

48. The method of claim 46, wherein the peroxidase is a heme-containing peptide.

49. The method of claim 46, wherein the peroxidase is a microperoxidase.

50. The method of claim 42, wherein the diazo compound is selected from the group consisting of Fast Red, Fast Violet, and Fast Blue.

51. The method of claim 42, wherein the biological target is recognized by a primary antibody, and the primary antibody is conjugated to a peroxidase.

52. The method of claim 42, comprising: contacting the biological target with a primary antibody that binds to the target; as well as The primary antibody is contacted with a secondary antibody directed against the primary antibody to form a primary-secondary antibody conjugate, wherein the secondary antibody is conjugated to peroxidase.

53. The method according to any one of claims 51 or 52, wherein the primary antibody is Ki67 antibody, ESR1 antibody, KRT15 antibody and / or CDH17 antibody.

54. The method of claim 52, wherein: The primary antibody is a mouse antibody, and the secondary antibody is poly-HRP-goat anti-mouse (GAM); or The primary antibody was a rabbit antibody, and the secondary antibody was poly-HRP-goat anti-rabbit (GAR).

55. A method comprising: contacting a biological sample having a target of interest with a primary antibody that binds to the target; contacting the primary antibody with a secondary antibody comprising a peroxidase compound to form a primary antibody-secondary antibody conjugate; contacting the sample with a compound satisfying Formula I under conditions suitable for covalently bonding the compound to the sample wherein L is a linker having 1 to at least 50 atoms, P 1 , P 2 and P 3 is an aromatic ring or aromatic ring system, R is a chemical bond or independently an L group, and x and y are independently 0 to 5; contacting the sample with a diazonium salt; and The target is detected.

56. The method of claim 55, wherein the target is detected colorimetrically, fluorescently, or both.

57. The method of claim 55 or 56, wherein the diazonium salt is selected from Fast Red, Fast Violet or Fast Blue.

58. The method of claim 55, comprising co-locating a plurality of different color combinations at or around the target.

59. The method of claim 55, comprising detecting two or more targets in the sample.

60. The method of claim 55, wherein the compound is fluorescent and the method further comprises detecting the target using fluorescence.

61. The method of claim 55, wherein the compound has the formula P 1 -LP 2 and is a naphthol derivative, and the diazonium salt is a fast blue to produce a green color suitable for detecting the target.

62. The method of claim 55, wherein the peroxidase is horseradish peroxidase or ascorbate peroxidase.

63. A kit, comprising: The compound according to claim 1; and Diazonium salt.

64. A kit comprising: Blocking buffer; poly-HRP-goat anti-mouse (GAM), poly-HRP-goat anti-rabbit (GAR), and / or HRP-conjugated streptavidin; The compound according to claim 1; Hydrogen peroxide; Reaction buffer; Diazonium salts; and Reaction termination reagent.

65. A kit according to claim 64, wherein the diazonium salt is Fast Red, Fast Violet and / or Fast Blue.

Images