Chromogenic peroxidase substrate

Abstract

The application describes chromogenic conjugates for color-based detection of targets. The conjugate includes a chromogenic moiety such as rhodamine, rhodol or fluorescein. The chromogenic moiety is linked to a peroxidase substrate. The chromogenic conjugate can be used for immunohistochemical analysis and in situ hybridization. The conjugate can be used to detect 1, 2, 3 or more targets in a sample by color.

Description

[0001] This application is a divisional application of the invention patent application with the application date of December 19, 2016, application number 201680074513.4 (international application number PCT / IB2016 / 001920), and name “Chromogenic peroxidase substrate”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of the filing date of and priority rights to U.S. Provisional Application No. 62 / 269,575, filed December 18, 2015. Technical Field

[0004] The present application relates to chromogenic conjugates, methods of chromogenic analysis such as immunohistochemistry (IHC) and chromogen in situ hybridization (CISH), and methods of preparing chromogenic conjugates. Background Art

[0005] When analyzing tissue samples on microscope slides, staining the tissue or certain parts of the tissue with colored dyes can help with the analysis. The ability to visualize or differentially identify microstructures is often enhanced by using histological stains. Hematoxylin and eosin (H&E) stains are the most commonly used stains in optical microscopy of histological samples. Hematoxylin is used to stain the nucleus blue, and eosin stains the cytoplasm and extracellular connective tissue matrix pink. In addition to H&E stains, other stains or dyes have been applied to provide more specific staining and provide a more detailed view of tissue morphology. Immunohistochemistry (IHC) stains have great specificity because they use peroxidase substrates or alkaline phosphatase (AP) substrates for IHC staining, providing a uniform staining pattern that appears to the observer as a uniform color with intracellular resolution of cell structures (e.g., cell membranes, cytoplasm, and nuclei).

[0006] Formalin-fixed paraffin-embedded (FFPE) tissue samples, metaphases or histological smears are usually analyzed by staining on slides, where specific biomarkers such as proteins or nucleic acids of interest can be stained with H&E and / or colored dyes, hereinafter referred to as "chromogens" or "chromogenic moieties". IHC staining is a common tool for evaluating tissue samples for the presence of specific biomarkers. IHC stains can accurately identify specific targets throughout the sample and allow quantification of these targets.

[0007] IHC staining uses chromogenic and / or fluorescent reporters that label targets in histological samples. This is done by linking the biomarker directly or indirectly to an enzyme (usually either horseradish peroxidase (HRP) or alkaline phosphatase (AP)), which then catalyzes the formation of an insoluble colored precipitate in the place of the biomarker (which expresses color) from a soluble, suitable enzyme substrate.

[0008] In a blot / capture assay, the biomarker is extracted from its original location into solution and then re-immobilized on a membrane, gel, or chip array, but the biomarker is also stained with a visible color (chromogen), usually through the action of the same HRP or AP enzyme.

[0009] Chromogens generally have much lower sensitivity than other detection techniques such as radiant energy, chemiluminescence or fluorescence, but have the advantage of permanent, clearly visible colors that can be observed visually, such as with bright field microscopy. Other limitations of enzyme-based chromogenic detection of targets in solid biological samples or targets immobilized on or into solid supports include: the number of chromogenic HRP and AP substrates available for target staining is very limited, which limits the use of these target visualization systems to detect multiple targets in a sample. Also, some chromogens such as the HRP substrate 3,3'-diaminobenzidine (DAB) are not characterized by well-defined spectral features, but rather an insoluble brown precipitate that absorbs light. In addition, when visualization of multiple targets is involved, it is usually necessary to use a combination of multiple enzyme-based visualization systems, such as HRP and AP. These limitations make the sample staining process complex, less robust and expensive, and also complicate automated detection of targets and image analysis of stained samples.

[0010] Rhodamine, rhodamine and fluorescein are strongly colored and fluorescent. Depending on the halogenation and / or substitution type, they have almost any color. They have been known for more than a century, and several are used as special dyes, which can dye tissue samples without any enzyme activity. For example, rhodamine 110 is used as a mitochondrial dye, which is tetrabromofluorescein, also known as eosin, widely used in hematoxylin / eosin (H and E) double dyes, wherein hematoxylin dyes the nucleus blue, and eosin dyes any protein basically pink or red. Therefore, although the derivatives of these compounds seem to be attractive as potential chromogens due to their unique and bright colors, nonspecific tissue staining, even in the absence of any enzyme activity, is an obstacle for them to be used as chromogens. Another obstacle is to provide derivatives with suitable solubility in aqueous environments.

[0011] Rhodamine and fluorescein compounds are also stable and require forcing conditions for further reaction. The solution is to introduce additional reactive groups, such as isothiocyanates, in fluorescein isothiocyanate ("FITC") and tetramethylrhodamine isothiocyanate ("TRITC") or carboxyfluorescein and sulforhodamine. However, increasing these reactive groups is not easy to do and a mixture of the two most inseparable isomers is obtained. FITC has become widely associated with reactive fluorescein and is a proven method for preparing fluorescein derivatives for antibodies and nucleic acid probes. However, such derivatives are expensive, some are prohibitively expensive.

[0012] In the past decade, progress has been made in the field of rhodamine and fluorescein 2'-ester derivatives. See, for example, Beija, Mariana et al., "Synthesis and applications of Rhodamine derivatives as fluorescent probes." Chem. Soc. Rev., 2009, 38, 2410-2433; Afonso, AM Carlos, et al., "An Expedient Synthesis of Cationic Rhodamine Fluorescent Probes Suitable for Conjugation to Amino Acids and Peptides." Synthesis, 2003, 17, 2647-2654; Xi Chen et al., "An efficient and versatile approach for the preparation of a rhodamine B ester bioprobe library." Dyes and Pigments 2012, 94, 296-303.

[0013] Both rhodamine and fluorescein have 2' carboxylic acids that can be derivatized to esters or amides under certain conditions. However, the 2' primary amides of rhodamine or fluorescein "fold" into colorless spirolactam or spirolactone tautomers, making these derivatives unsuitable for use as chromogens. Esters and amides of secondary amines do not undergo this tautomerization because they lack labile NH protons.

[0014] Methods for preparing amides of the secondary amine piperazine of rhodamine and fluorescein have been reported. See Nguyen T. et al., "Practical synthetic route to rhodamine dyes", Org. Lett. 2003, 18, 3245-48; Huang, Chusem, et al.; "Versatile Probes for the Selective Detection of Vicinal-Dithiol-Containing proteins: Design, Syntheses, and Applications in Living Cells". Chem. Eur. J. 2013, 19, 7739-7747.

[0015] Fluorescein isothiocyanate (FITC) is a derivative of fluorescein used in many applications utilizing fluorescence, such as flow cytometry. FITC consists of a fluorescein molecule functionalized at its 4' position with an isothiocyanate reactive group (-N=C=S) on a monocyclic phenyl group of the structure. The derivative is reactive toward nucleophiles on proteins, including amine and sulfhydryl groups.

[0016] Recently described the use of fluorescein as the detectable part of HRP substrate in the histochemical detection of target. WO2007 / 015168 of Lohse relates to the synthesis and use of the derivative of the linker of monomer or polymer linker molecule for biological and chemical applications, their synthesis and being conjugated to various detectable labels and other materials. The linker can be used for example with fluorescent label, nucleic acid or nucleic acid analog probe and solid phase system coupling, and is used to enhance the solubility of the molecule being conjugated. WO2009 / 036760, WO2010 / 094283, WO2010 / 094284, WO2011 / 047680 and WO2012 / 143010 relate to HRP substrate through the linker of WO2007 / 015168 being conjugated to fluorescein at its 4 ' position. The latter conjugate is colorless but fluorescent and can be used either for direct fluorescent detection of the target or for indirect histochemical detection of the target: the conjugate is enzymatically deposited at the target site labeled with HRP activity, and the deposited conjugate can then be detected either optically as a fluorescent dye or immunochemically as a hapten. Deposition of the conjugate by HRP requires the presence of a certain amount of DAB, ferulic acid or α-cyano-4-hydroxycinnamic acid (ACHCA) in the deposition medium. Summary of the invention

[0017] The present application provides chromogenic conjugate molecules capable of serving as substrates for enzymes having peroxidase activity and describes their use for detecting molecular targets in a sample.

[0018] This application involves the following technical solutions:

[0019] 1. A chromogenic conjugate comprising:

[0020] (a) a chromogenic moiety, and

[0021] (b) a peroxidase substrate portion,

[0022] wherein the chromogenic part and the peroxidase substrate part are linked together via a linker,

[0023] The conjugate is a compound of formula I:

[0024]

[0025] Where X is -OH, -OR X or -NR X R XX ,

[0026] Where Y is =O or =N + R Y R YY ;

[0027] Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R X , R XX , R Y , and R YY are independently selected from hydrogen and substituents having less than 40 atoms;

[0028] L is a linear linker comprising 5 to 29 consecutive linking atoms; and

[0029] PS is a peroxidase substrate moiety, wherein the peroxidase substrate moiety has the formula:

[0030]

[0031] in

[0032] R 21 Yes -H,

[0033] R 22 is -H, -OX, or -N(X) 2 ;

[0034] R 23 is -OH;

[0035] R 24 is -H, -OX, or -N(X) 2 ;

[0036] R 25 is -H, -OX, or -N(X) 2 ;

[0037] R 26 Yes-CON-(X) 2 , -CONH(X), or -COO(X);

[0038] wherein H is hydrogen; O is oxygen; N is nitrogen; and X is H, alkyl or aryl.

[0039] 2. The chromogenic conjugate according to item 1, wherein

[0040] R 1 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R 1 Can be used with R 2 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 The benzo, naphthyl or polycyclic arylene groups;

[0041] R 2 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R2 Can be used with R 1 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 benzo, naphtho or polycyclic arylene radicals, or, when X is -NR X R XX When R 2 Can be used with R X together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0042] R X When present, selected from hydrogen, optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R X Can be used with R 2 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0043] R XX When present, selected from optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R XX Can be used with R 3 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0044] R 3 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, when X is -NR X R XX When R 3 Can be used with R XX together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0045] R 4 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl of the group, or, when Y is -N+R Y R YY When R 4 Can be used with R yy together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group; R yy When present, selected from optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or R yy Can be used with R 4 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0046] R Y When present, selected from hydrogen, optionally substituted with one or more identical or different R 14The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R Y Can be used with R 5 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0047] R YY When present, selected from optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R XX Can be used with R 6 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0048] R 5 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R 5 Can be used with R 6 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 benzo, naphtho or polycyclic arylene radicals, or, when Y is -N+R Y R YY When R 5 Can be used with R y together to form optionally substituted with one or more identical or different R13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0049] R 6 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R 6 Can be used with R 5 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 The benzo, naphthyl or polycyclic arylene groups;

[0050] R 7 , R 8 and R 9 are each independently selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl of the group;

[0051] R 10 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, halogen, haloalkyl, -OR 12 , -SR 12 , -SOR 12 , -SO 2 R 12 , and nitrile;

[0052] R 11 Selected from -NR 15 R 15 , -OR 16 , -SR 16 , halogen, haloalkyl, -CN, -NC, -OCN, -SCN, -NO, -NO 2 , -N 3 , -S(O)R 16 , -S(O) 2 R 16 , -S(O) 2 OR 16 , -S(O)NR 15 R 15 , -S(O) 2 NR 15 R 15 , -OS(O)R 16 , -OS(O) 2 R 16 , -OS(O) 2 NR 15 R 15 , -OP(O) 2 R 16 , -OP(O) 3 R 16 R 16 , -P(O) 3 R 16 R 16 , -C(O)R 16 , -C(O)OR 16 , -C(O)NR 15 R 15 , -C(NH)NR 15 R 15 , -OC(O)R 16 , -OC(O)OR 16 , -OC(O)NR 15 R 15 and -OC(NH)NR 15 R 15 ;

[0053] R 12 is selected from (C1-C20)alkyl or heteroalkyl optionally substituted with a lipophilic substituent, (C5-C20)aryl or heteroaryl optionally substituted with a lipophilic substituent, and (C2-C26)arylalkyl or heteroarylalkyl optionally substituted with a lipophilic substituent;

[0054] R 13is selected from hydrogen, (C1-C8)alkyl or heteroalkyl, (C5-C20)aryl or heteroaryl, and (C6-C28)arylalkyl or heteroarylalkyl;

[0055] R 14 Selected from -NR 15 R 15 , = O, -OR 16 , = S, -SR 16 , =NR 16 , =NOR 16 , halogen, haloalkyl, -CN, -NC, -OCN, -SCN, -NO, -NO 2 , = N 2 , -N 3 , -S(O)R 16 , -S(O) 2 R 16 , -S(O) 2 OR 16 , -S(O)NR 15 R 15 , -S(O) 2 NR 15 R 15 , -OS(O)R 16 , -OS(O) 2 R 16 , -OS(O) 2 NR 15 R 15 , -OS(O) 2 OR 16 , -OS(O) 2 NR 15 R 15 , -C(O)R 16 , -C(O)OR 16 , -C(O)NR 15 R 15 , -C(NH)NR 15 R 15 , -OC(O)R 16 , -OC(O)OR 16 , -OC(O)NR 15 R 15 and -OC(NH)NR 15 R 15 ;

[0056] R 15 are independently hydrogen or R 16 , or, R 15Each together with the nitrogen atom to which it is bound forms a 5- to 8-membered saturated or unsaturated ring which may optionally include one or more identical or different additional heteroatoms and may optionally be substituted with one or more identical or different R 13 or R 16 Group;

[0057] R 16 Each independently is R 13 , or substituted with one or more identical or different R 13 or R 17 R 13 ;and

[0058] R 17 Each selected from -NR 13 R 13 , -OR 13 , = S, -SR 13 , =NR 13 , =NOR 13 , halogen, haloalkyl, -CN, -NC, -OCN, -SCN, -NO, -NO 2 , = N 2 , -N 3 , -S(O)R 13 , -S(O) 2 R 13 , -S(O) 2 OR 13 , -S(O)NR 13 R 13 , -S(O) 2 NR 13 R 13 , -OS(O)R 13 , -OS(O) 2 R 13 , -OS(O) 2 NR 13 R 13 , -OS(O) 2 OR 16 , -OS(O) 2 NR 13 R 13 , -C(O)R 13 , -C(O)OR 13 , -C(O)NR 13 R 13 , -C(NH)NR 15 R 13 , -OC(O)R 13 , -OC(O)OR 13 , -OC(O)NR 13 R 13and -OC(NH)NR 13 R 13 .

[0059] 3. The conjugate according to item 1, wherein the chromogenic moiety is selected from rhodamine and fluorescein, and salts thereof.

[0060] 4. The conjugate according to item 1, wherein the chromogenic moiety is selected from rhodamine, rhodamine 6G, tetramethylrhodamine, rhodamine B, rhodamine 101, rhodamine 110, fluorescein, and O-carboxymethylfluorescein.

[0061] 5. The conjugate according to any one of items 1 to 4, wherein the chromogenic moiety is a 2'-piperazineamide derivative.

[0062] 6. The chromogenic conjugate according to any one of items 1 to 5, wherein the chromogenic conjugate is selected from the molecules shown in Table 1 and salts of =N moieties.

[0063] 7. The conjugate according to any one of items 1 to 6, wherein R 23 is -OH and R 24 Yes -H.

[0064] 8. The conjugate according to any one of items 1 to 6, wherein R 21 or R 25 Any one of -OH, R 22 and R 24 Yes - H, R 23 It is -OH.

[0065] 9. The conjugate according to any one of items 1 to 6, wherein the peroxidase substrate is a residue of ferulic acid, cinnamic acid, caffeic acid, sinapic acid, 2,4-dihydroxycinnamic acid or 4-hydroxycinnamic acid (coumaric acid).

[0066] 10. The conjugate according to any one of items 1 to 9, wherein the linker is a compound comprising 1 or 2 repeating units of formula III:

[0067]

[0068] Where R 31 Selected from methyl, ethyl, propyl, OCH 2 , CH 2 OCH 2 , (CH 2 OCH 2 ) 2 , NHCH 2 NH(CH 2 ) 2 , CH 2 NHCH2 , cycloalkyl, alkyl-cycloalkyl, alkyl-cycloalkyl-alkyl, heterocyclyl (e.g., a nitrogen-containing ring having 4 to 8 atoms), alkyl-heterocyclyl, alkyl-heterocyclyl-alkyl, and wherein no more than 3 consecutive repetitions of ethoxy, R 32 and R 33 are independently selected from NH and O.

[0069] 11. The conjugate according to any one of items 1 to 10, wherein the linker is selected from a moiety of formula IIIa, IIIb, or IIIc:

[0070]

[0071]

[0072] 12. A composition comprising at least one chromogenic conjugate according to any one of items 1 to 11 and a solvent and optionally one or more of the following substances: (i) an organic modifier; (ii) an enzyme enhancer; (iii) an iron chelator; (iv) a detergent; (v) an antibacterial agent; (vi) an organic or inorganic salt; or (vii) an enzyme substrate.

[0073] 13. A kit for detecting a target having peroxidase activity or linked to a peroxidase in a sample, said kit comprising at least one chromogenic conjugate according to any one of items 1 to 11.

[0074] 14. A method for detecting a target in a sample by chromogenic detection, the method comprising:

[0075] incubating a sample presumably comprising a target in an aqueous solution, wherein the target comprises peroxidase activity, or the target is directly or indirectly linked to a peroxidase, wherein the aqueous solution comprises:

[0076] a) at least one chromogenic conjugate according to any one of items 1 to 11;

[0077] b) peroxide compounds;

[0078] A colored precipitate of the chromogenic conjugate in the sample is detected, thereby detecting the target in the sample.

[0079] 15. The method according to item 14, further comprising incubating the sample with a second chromogenic conjugate having Formula IX or Formula XIV:

[0080]

[0081] Where X is -OH, -OR X or -NR X RXX ,

[0082] Where Y is =O or =N + R Y R YY ;

[0083] Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R X , R XX , R Y , and R YY are independently selected from hydrogen and substituents having less than 40 atoms;

[0084] L is a linear linker comprising 5 to 29 consecutive linking atoms; and

[0085] PS is the peroxidase substrate moiety;

[0086]

[0087] Wherein X1 and X2 are selected from -OH, -OR X and-NR X R XX , X1 and X2 are preferably different,

[0088] Wherein Y1 and Y2 are selected from =O or =N + R Y R YY , Y1 and Y2 are preferably different,

[0089] Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R X , R XX , R Y , and R YY are independently selected from hydrogen and substituents having less than 40 atoms, or as defined elsewhere in this application; and

[0090] Where R 34 It is methyl, ethyl, propyl, OCH2 , CH 2 OCH 2 , (CH 2 OCH 2 ) 2 , NHCH 2 NH(CH 2 ) 2 , CH 2 NHCH 2 , cycloalkyl, alkyl-cycloalkyl, alkyl-cycloalkyl-alkyl, heterocyclyl (eg, a nitrogen-containing ring having 4 to 8 atoms), alkyl-heterocyclyl, or alkyl-heterocyclyl-alkyl.

[0091] 16. A method for performing chromogenic in situ hybridization, comprising:

[0092] contacting a nucleic acid target with a probe that hybridizes to the nucleic acid target under hybridization conditions, wherein the probe comprises (1) a nucleic acid sequence that is at least partially complementary to the nucleic acid target, and (2) a peroxidase, or a first member of a specific binding pair; wherein the target and the probe form a complex;

[0093] When the probe comprises (2), contacting the complex with a second member of the specific binding pair, wherein the second member of the specific binding pair is contacted, wherein the second member is directly or indirectly linked to a peroxidase and specifically binds to the first member;

[0094] Incubating the complex with at least one chromogenic conjugate according to any one of items 1 to 11; the incubation is performed for a time and temperature sufficient to form a colored precipitate at the target;

[0095] The colored precipitate is detected.

[0096] 17. A method for detecting two or more targets, comprising:

[0097] providing peroxidase activity at a first target in the sample;

[0098] contacting the sample with a first chromogenic conjugate;

[0099] forming a first colored precipitate at the first target;

[0100] removing the peroxidase activity from the first target;

[0101] removing unprecipitated first chromogenic conjugate from the sample;

[0102] providing peroxidase activity at a second target in the sample;

[0103] contacting the sample with a second chromogenic conjugate;

[0104] forming a second colored precipitate at the second target; and

[0105] detecting the first colored precipitate and the second colored precipitate, thereby detecting the first target and the second target in the sample,

[0106] At least one of the first chromogenic conjugate and the second chromogenic conjugate is the conjugate of any one of items 1 to 11, and wherein the first chromogenic conjugate molecule and the second chromogenic conjugate have one or more spectral characteristics that are different from each other.

[0107] 18. A method according to item 17, wherein the method further comprises: removing the peroxidase activity from the second target; removing the unprecipitated second chromogenic conjugate from the sample; providing peroxidase activity at a third target in the sample; contacting the sample with a third chromogenic conjugate, wherein the third chromogenic conjugate has one or more spectral characteristics different from the first chromogenic conjugate and the second chromogenic conjugate.

[0108] 19. A method for preparing a secondary amide of rhodamine or fluorescein, the method comprising:

[0109] The 2'-alkylcarboxymethyl derivative of rhodamine or fluorescein is reacted with an excess of a secondary amine at a suitable temperature in an anhydrous solvent to form a secondary amide.

[0110] 20. The method according to item 19, further comprising the step of preparing a 2'-alkylcarboxymethyl derivative by reacting rhodamine with a 2-haloacetyl ester. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] The teachings of the present invention can be best understood from the following detailed description when read with the accompanying drawings. Features are not necessarily drawn to scale. When practical, like reference numerals refer to like features.

[0112] Figure 1 are photomicrographs of tonsil tissue stained with Ki67. The left photo was stained with Compound 2 (described below); the right photo was stained with DAB.

[0113] Figure 2 The left photo was also stained with DAB; the right photo was stained with Compound 2.

[0114] Figure 3 The left photo was stained with DAB; the right photo was stained with Compound 2. DETAILED DESCRIPTION

[0115] the term

[0116] It should be understood that the terms used in this application are only for the purpose of describing specific embodiments and are not intended to be limiting. The defined terms are defined in addition to the technical and scientific meanings of the defined terms, as well as the meanings generally understood and accepted in the technical field taught by the present invention. For example, the definitions of commonly used terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

[0117] As used in the specification and the appended claims, the terms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise. Thus, for example, "a part" includes one device and a plurality of parts.

[0118] As used in the specification and appended claims and in addition to their common meanings, the terms "substantially" or "substantially" mean within a limit or degree acceptable to those skilled in the art. For example, "substantially deleted" means that the deletion is considered acceptable by those skilled in the art.

[0119] As used in the specification and the appended claims and in addition to their common meanings, the terms "about" and "approximately" mean within limits or amounts acceptable to one skilled in the art. The term "about" generally refers to a specified number ±15%. For example, "about 10" can indicate a range of 8.7 to 1.15. For example, "about the same" means that one skilled in the art would consider the items to be the same upon comparison.

[0120] A "moiety" is a portion of a molecule that retains chemical and / or physical and / or functional characteristics of the overall molecule that are relevant to the properties of a chromogenic conjugate; for example, a "peroxidase substrate moiety" is a portion of a molecule that is capable of serving as a substrate for an enzyme having peroxidase activity; a "peroxidase moiety" is a portion of a molecule that has intrinsic peroxidase activity, e.g., an enzyme.

[0121] "Conjugate" refers to two or more molecules (or two or more portions of two or more molecules) covalently linked into a larger construct.

[0122] The term "linked" in the context of this application means connected via a chemical bond.

[0123] A "target" is an object in a test sample to be detected by using the chromogenic conjugates and methods of the invention; targets of the invention include chemical and biological molecules and structures. Embodiments of targets of the invention are discussed in this application.

[0124] "Biomarker" refers to one or more biological objects, such as molecules, molecular complexes, structures, particles or organisms, associated with features that are characteristic for a particular cell type, tissue, cell structure, physiological condition, etc. Such biological objects are generally considered to be markers for that particular cell type, tissue, cell structure, physiological condition. Non-limiting examples of such biomarkers include, but are not limited to, specific nucleotide sequences, proteins or other biomolecules, such as carbohydrates or lipids, chromosomes or cell membrane structures, viruses, bacteria, microorganisms, etc. In some embodiments, the term target is used interchangeably with the term biomarker and refers to a molecule, molecular complex, structure or particle that is characteristic for a particular cell type, tissue, physiological condition, etc., wherein the total population of any of the latter biomarkers in a test sample is considered to be a target.

[0125] A "spectral signature" is a characteristic of electromagnetic radiation emitted or absorbed by a molecule or moiety that causes a transition from one energy state to another, such as from a higher energy state to a lower energy state. Only certain colors appear in the emission spectrum of a molecule or moiety because certain frequencies of light are emitted and certain frequencies of light are absorbed. A spectral signature can be summarized or referred to as the color of a molecule or moiety.

[0126] The term "rhodamine" may refer to the family of related xanthene-based dyes, which includes rhodamine 6G and rhodamine B; or the term "rhodamine" may refer to the following specific compounds:

[0127]

[0128] As the context indicates.

[0129] The term "fluorescein" may refer to the family of related xanthene-based dyes, which include fluorescein isothiocyanate, NHS-fluorescein, and O-carboxyfluorescein; or the term "fluorescein" may refer to the following specific compounds:

[0130]

[0131] For brevity, certain abbreviations are used: "Rho" refers to rhodamine; "TMRho" refers to tetramethylrhodamine; "Flu" refers to fluorescein; "Pip" refers to piperazine; "Cou" refers to coumarin; "Caf" refers to caffeic acid; "Fer" refers to ferulic acid; "Cin" refers to cinnamic acid; "Tyr" refers to tyrosine; "Et" refers to ethyl. Other abbreviations may also appear in this application.

[0132] "Spectrally narrow" refers to a chromogen whose absorbance maximum has a width of less than 50 nm at half peak height as measured by UV-VIS spectroscopy, measured at a concentration of 10 μM in +99% water at pH 6-8. "Magenta chromogen" is a spectrally narrow chromogen with a peak absorbance between 525 and 536 nm, measured at a concentration of 10 μM in +99% water at pH 6.0-8.0. "Greenish yellow chromogen" is a chromogen with a peak absorbance below 475 nm and less than 10% of the absorbance at 530 nm or above the peak absorbance relative to the peak absorbance, measured at a concentration of 10 μM in +99% water at pH 6.0-8.0. A "yellow chromogen" is a chromogen having a peak absorbance below 505 nm and less than 10% of the absorbance at or above the peak absorbance relative to the peak absorbance, measured at a concentration of 10 μM in +99% water at a pH of 6.0-8.0. A "cyan chromogen" is a chromogen having an absorbance maximum above 615 nm and less than 10% of the relative absorbance relative to the peak absorbance at any wavelength between 530 and 400 nm, measured at a concentration of 10 μM in +99% water at a pH of 6.0-8.0. An "orange chromogen" is a "spectrally narrow" chromogen having an absorbance maximum between 495 and 520 nm, measured at a concentration of 10 μM in +99% water at a pH of 6-8. A "dichroic chromogen" is a chromogen having at least two absorbance maxima separated by a local minimum at least 50 nm wide and a further global minimum between 390 and 700 nm, measured in water at a pH of 6 to 8. DETAILED DESCRIPTION OF THE INVENTION

[0134] In the following detailed description of the invention, for the purpose of explanation and not limitation, representative embodiments disclosing specific details are set forth to provide a thorough understanding of the teachings of the present invention. Descriptions or details of known systems, compounds, materials, methods of use, and methods of manufacture may be omitted to avoid obscuring the example embodiments. However, systems, parts, and methods within the purview of those skilled in the art may be used according to the representative embodiments.

[0135] The present application provides chromogenic peroxidase substrate conjugates (interchangeably referred to herein as "chromogenic conjugates", "conjugate molecules" or "reporter molecules"). The conjugate molecules of the present invention combine one or more advantages of DAB without one or more disadvantages.

[0136] Embodiments of the chromogenic conjugates of the invention include molecules comprising a peroxidase substrate portion linked to a chromogenic moiety that (a) are non-toxic, (b) precipitate from solution as a bright and spectrally narrow and intense stain via an HRP-mediated reaction; (c) once mixed, are stable in aqueous solution for more than 24 hours; and / or (d) are insoluble in organic solvents when precipitated.

[0137] In some embodiments, the chromogenic conjugates of the present application absorb and / or emit light in the range of about 450 nm to about 600 nm. In some embodiments, the chromogenic conjugate absorbs light at 536 nm, and the color of the dye can be defined as magenta. In some embodiments, the chromogenic conjugate is yellow and absorbs light at 450 nm. In some embodiments, the chromogenic conjugate is purple and absorbs light close to 600 nm. Embodiments of the chromogenic conjugates of the present invention can be used as substrates for peroxidases, such as HRP, which are spectrally narrow, not dichroic, and do not change their spectral characteristics when precipitated; the dye prepared by enzyme precipitation of the chromogenic conjugate has poor solubility in water or organic solutions, if any, and does not drift when exposed to the light source used to image the stained sample. These features make the chromogenic conjugates of the present invention particularly suitable for automated image analysis and multiplexing. In addition, the molecules of the chromogenic conjugates have a well-defined chemical structure and can be easily prepared by the methods described in the present application.

[0138] Chromogenic conjugate molecules

[0139] Some embodiments of the chromogenic conjugates of the present invention can be represented by the general formula (I):

[0140] (S)-L-(Z),

[0141] Where S is the peroxidase substrate moiety,

[0142] Z is the chromogenic part,

[0143] L is a linking group,

[0144] The chromogenic conjugate molecule has one, two, three, four, five or all of the following characteristics, and preferably all of the following characteristics: (1) it includes a substrate portion of a peroxidase, such as an HRP substrate; (2) it includes a chromogenic portion, which is a Rho or Flu 2'-ester or 2'-secondary amide derivative; (3) the enzyme substrate and the chromogenic portion are linked together via a water-soluble linker compound and are separated from each other by a distance of at least 5 consecutive interconnected atoms, (4) the linker compound (also referred to in this application as a "linker molecule", "linker" or "L") includes a chain of 5-29 interconnected atoms (corresponding to the abbreviations "L5-L29"); wherein, in some preferred embodiments, the linker compound includes two consecutive carbon atoms followed by an oxygen or nitrogen atom; (5) the conjugate is substantially soluble in aqueous solution; (6) the conjugate is substantially stable both as a chromogen in solution and as a precipitate.

[0145] Table 1 shows some non-limiting examples of chromogenic conjugates of the present invention:

[0146] Table 1

[0147]

[0148]

[0149]

[0150]

[0151]

[0152] Table 2 lists the masses and absorption of these conjugates:

[0153] Table 2

[0154]

[0155]

[0156] As shown in Table 2, chromogenic conjugates with distinct absorption maxima have been prepared. This enables staining of tissue samples with one or more distinct colors. For example, a method of analyzing a sample can include: labeling a first target with compound 14, such that the first target is detected or identified at a location in the sample with a magenta dye, and labeling a second target with compound 5, such that the second target is detected and identified as a violet dye.

[0157] Unexpectedly, a systematic trend was observed in the peak absorbance of the chromogenic conjugates with piperazine and ether. The piperazine amide absorbs light about 15 nm higher than the corresponding rhodamine and fluorescein, and the ester-containing conjugate absorbs light about 10 nm higher than the corresponding rhodamine and fluorescein. This enables fine tuning of the hue of the chromogenic conjugates.

[0158] Chromogenic part

[0159] The chromogenic conjugate may include a chromogenic moiety capable of providing a visible color. The chromogenic moiety may be a xanthene derivative, such as a fluorescein, rhodol or rhodamine residue.

[0160] In some embodiments, the chromogenic conjugate comprises (a) a chromogenic portion, and (b) a peroxidase substrate portion, wherein the chromogenic portion and the peroxidase portion are linked together via a linker, and wherein the linker comprises at least one linear chain having at least 5 consecutively linked atoms. For example, the chromogenic conjugate can be a compound of formula I:

[0161]

[0162] Where X is -OH, -OR X or -NR X R XX ,

[0163] Where Y is =O or =N + R Y R YY ;

[0164] Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R X , R XX , R Y , and R YY are independently selected from hydrogen and substituents having less than 40 atoms;

[0165] L is a linker; and

[0166] PS is the substrate moiety of peroxidase.

[0167] In some embodiments, certain combinations of residues X and Y may be preferred. For example, if X is -OH or -OR X , then preferably Y is =O. In another preferred combination, if X is -NR X RXX , then Y is = N + R Y R YY .

[0168] In some embodiments of the chromogenic conjugate of Formula I, R 1 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R 1 Can be used with R 2 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 The benzo, naphthano or polycyclic arylene groups are benzo, naphthano or polycyclic arylene groups.

[0169] In some embodiments of the chromogenic conjugate of Formula I, R 2 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R 2 Can be used with R 1 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 benzo, naphtho or polycyclic arylene radicals, or, when X is -NR X R XX When R 2 Can be used with R X together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group.

[0170] In some embodiments of the chromogenic conjugate of Formula I, R X When present, selected from hydrogen, optionally substituted with one or more identical or different R 14The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R X Can be used with R 2 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group.

[0171] In some embodiments of the chromogenic conjugate of Formula I, R XX When present, selected from optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R XX Can be used with R 3 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group.

[0172] In some embodiments of the chromogenic conjugate of Formula I, R 3 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, when X is -NR X R XX When R 3 Can be used with R XX together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group.

[0173] In some embodiments of the chromogenic conjugate of Formula I, R 4 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl of the group, or, when Y is -N + R Y R YY When R 4 Can be used with R YY together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group; R yy When present, selected from optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R yy Can be used with R 4 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group.

[0174] In some embodiments of the chromogenic conjugate of Formula I, R Y When present, selected from hydrogen, optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R Y Can be used with R 5 together to form optionally substituted with one or more identical or different R 13 or suitable R 14A 5-membered or 6-membered ring of a group.

[0175] In some embodiments of the chromogenic conjugate of Formula I, R YY When present, selected from optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R XX Can be used with R 6 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group.

[0176] In some embodiments of the chromogenic conjugate of Formula I, R 5 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R 5 Can be used with R 6 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 benzo, naphtho or polycyclic arylene radicals, or, when Y is -N + R Y R YY When R 5 Can be used with R y together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group.

[0177] In some embodiments of the chromogenic conjugate of Formula I, R 6 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R 6 Can be used with R 5 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 The benzo, naphthano or polycyclic arylene groups are benzo, naphthano or polycyclic arylene groups.

[0178] In some embodiments of the chromogenic conjugate of Formula I, R 7 , R 8 and R 9 are each independently selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 The present invention can be a (C6-C40) arylalkyl or heteroarylalkyl group.

[0179] In some embodiments of the chromogenic conjugate of Formula I, R 10 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, halogen, haloalkyl, -OR 12 , -SR 12 , -SOR 12 , -SO 2 R 12 , and nitrile.

[0180] In some embodiments, R 11 Selected from -NR 15 R 15 , -OR 16 , -SR 16 , halogen, haloalkyl, -CN, -NC, -OCN, -SCN, -NO, -NO2 , -N 3 , -S(O)R 16 , -S(O) 2 R 16 , -S(O) 2 OR 16 , -S(O)NR 15 R 15 , -S(O) 2 NR 15 R 15 , -OS(O)R 16 , -OS(O) 2 R 16 , -OS(O) 2 NR 15 R 15 , -OP(O) 2 R 16 , -OP(O) 3 R 16 R 16 , -P(O) 3 R 16 R 16 , -C(O)R 16 , -C(O)OR 16 , -C(O)NR 15 R 15 , -C(NH)NR 15 R 15 , -OC(O)R 16 , -OC(O)OR 16 , -OC(O)NR 15 R 15 and -OC(NH)NR 15 R 15 .

[0181] In some embodiments, R 12 is selected from (C1-C20)alkyl or heteroalkyl optionally substituted with a lipophilic substituent, (C5-C20)aryl or heteroaryl optionally substituted with a lipophilic substituent, and (C2-C26)arylalkyl or heteroarylalkyl optionally substituted with a lipophilic substituent.

[0182] In some embodiments, R 13 is selected from hydrogen, (C1-C8)alkyl or heteroalkyl, (C5-C20)aryl or heteroaryl and (C6-C28)arylalkyl or heteroarylalkyl.

[0183] In some embodiments, R 14 Selected from -NR 15 R 15 , = O, -OR 16, = S, -SR 16 , =NR 16 , =NOR 16 , halogen, haloalkyl, -CN, -NC, -OCN, -SCN, -NO, -NO 2 , = N 2 , -N 3 , -S(O)R 16 , -S(O) 2 R 16 , -S(O) 2 OR 16 , -S(O)NR 15 R 15 , -S(O) 2 NR 15 R 15 , -OS(O)R 16 , -OS(O) 2 R 16 , -OS(O) 2 NR 15 R 15 , -OS(O) 2 OR 16 , -OS(O) 2 NR 15 R 15 , -C(O)R 16 , -C(O)OR 16 , -C(O)NR 15 R 15 , -C(NH)NR 15 R 15 , -OC(O)R 16 , -OC(O)OR 16 , -OC(O)NR 15 R 15 and -OC(NH)NR 15 R 15 .

[0184] In some embodiments, R 15 are independently hydrogen or R 16 , or, R 15 Each together with the nitrogen atom to which they are bonded forms a 5- to 8-membered saturated or unsaturated ring which may optionally include one or more identical or different further heteroatoms and may optionally be substituted with one or more identical or different R 13 or R 16 Group.

[0185] In some embodiments, R 16 Each independently is R 13, or substituted with one or more identical or different R 13 or R 17 R 13 .

[0186] In some embodiments, R 17 Each selected from -NR 13 R 13 , -OR 13 , = S, -SR 13 , =NR 13 , =NOR 13 , halogen, haloalkyl, -CN, -NC, -OCN, -SCN, -NO, -NO 2 , = N 2 , -N 3 , -S(O)R 13 , -S(O) 2 R 13 , -S(O) 2 OR 13 , -S(O)NR 13 R 13 , -S(O) 2 NR 13 R 13 , -OS(O)R 13 , -OS(O) 2 R 13 , -OS(O) 2 NR 13 R 13 , -OS(O) 2 OR 16 , -OS(O) 2 NR 13 R 13 , -C(O)R 13 , -C(O)OR 13 , -C(O)NR 13 R 13 , -C(NH)NR 15 R 13 , -OC(O)R 13 , -OC(O)OR 13 , -OC(O)NR 13 R 13 and -OC(NH)NR 13 R 13 .

[0187] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R6 , R 7 , R 8 , R 9 , and R 10 are independently selected from -H, -halogen, -methyl, -ethyl, -propyl, -isopropyl, -vinyl, -SO 3 H,-PO 3 H, -NO 2 , -COOH, -NH 2 , -CN, -OH, -OMe and -OEt.

[0188] In some embodiments, the chromogenic moiety can be selected from the group consisting of: rhodamine, rhodamine 6G, tetramethylrhodamine, rhodamine B, rhodamine 101, rhodamine 110, fluorescein, and O-carboxymethylfluorescein and salts thereof. In some other embodiments, the chromogenic moiety can be selected from the group consisting of rhodamine 116, rhodamine 123, and rhodamine 19. In some embodiments, the chromogenic moiety is a rhodamine salt comprising an anion selected from the group consisting of: Cl - ,Br - , TFA - , and ClO 4 - .

[0189] A dichroic chromogen is a chromogen that changes hue or color with concentration. An example of a known dichroic chromogen is pumpkin seed oil. Pumpkin seed oil absorbs mainly blue light, has an absorbance minimum for green light and a smaller second absorbance peak in the far-red wavelength. Due to the short light path through the oil, the oil appears slightly green. As the light path increases, the color changes from brown to red. This change is valuable if one wants to measure the concentration of pumpkin seed oil by spectroscopy. At low concentrations, an accurate measurement can be based on the blue absorbance. In addition, since the absorbance increases with increasing concentration, the blue absorbance becomes inaccurate (because blue light is essentially absorbed), and it is possible to switch to the red absorbance (which is obviously inaccurate at low concentrations because red light is essentially not absorbed). Therefore, the technical effect of the dichroic chromogen is that the dichroic chromogen has an expanded dynamic range of human and instrument perception.

[0190] A dichroic effect can be obtained by mixing two or more of the present chromogens. Because most are spectrally very narrow and absorb in the visible spectrum, any mixture of two chromogens with absorbance maxima that are sufficiently separated will be dichroic.

[0191] Various examples of mixing dichroic chromogens to produce chromogens that change hue with concentration, referred to as dichroic orange and dichroic red, are described below.

[0192] In some embodiments, two or more chromogenic moieties are linked to each other, thereby forming a molecule or conjugate with two different absorbances. Suitable compounds include those having Formula XIV:

[0193]

[0194] Wherein X1 and X2 are selected from -OH, -OR X and-NR X R XX , X1 and X2 are preferably different,

[0195] Wherein Y1 and Y2 are selected from =O or =N + R Y R YY , Y1 and Y2 are preferably different,

[0196] Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R X , R XX , R Y , and R YY are independently selected from hydrogen and substituents having less than 40 atoms, or as defined elsewhere in this application; and

[0197] Where R 34 It is methyl, ethyl, propyl, OCH 2 , CH 2 OCH 2 , (CH 2 OCH 2 ) 2 , NHCH 2 NH(CH 2 ) 2 , CH 2 NHCH 2 , cycloalkyl, alkyl-cycloalkyl, alkyl-cycloalkyl-alkyl, heterocyclyl (e.g., a nitrogen-containing ring having 4 to 8 atoms), alkyl-heterocyclyl, or alkyl-heterocyclyl-alkyl, preferably piperazine, piperidine, pyrrolidine, imidazolidine, pyrazolidine, azetidine, or other 4- to 8-membered (or 5- to 7-membered) ring or heterocyclic group, which optionally has an amine, carboxyl or ester substituent.

[0198] Preferred compounds of formula XIV absorb light with at least two distinct absorption maxima, for example a first absorption maximum and a second absorption maximum, which are separated by at least 5 nm, or by at least 10 nm, or by at least 20 nm.

[0199] For example, a two-color Rhodamine 6G-fluorescein chromogen has been prepared according to Formula XIVb:

[0200]

[0201] This compound exhibits an orange-red color. It still has the Boc group, so peroxidase substrates such as coumaric acid can be added.

[0202] In some embodiments in which two or more chromogenic moieties are linked to each other in the conjugate, the conjugate has two different absorptions. For example, the present application provides a FRET conjugate of formula XIVa:

[0203]

[0204] Wherein X1 and X2 are selected from -OH, -OR X and-NR X R XX , and X1 and X2 are preferably different,

[0205] Wherein Y1 and Y2 are selected from =O or =N + R Y R YY , and Y1 and Y2 are preferably different,

[0206] Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R X , R XX , R Y , and R YY are independently selected from hydrogen or a substituent having less than 40 atoms, or as defined elsewhere herein; and

[0207] Where R 38 It is a 4- to 8-membered cycloalkyl group or a 4- to 8-membered heterocyclic group (eg, a nitrogen-containing ring having 4 to 8 atoms), preferably a piperidinyl group or a piperazinyl group.

[0208] Preferred compounds of formula XIVa are those wherein the emission of the first chromophore moiety (eg, a fluorescein derivative) and the absorbance of the second chromophore moiety (eg, a rhodamine derivative) overlap.

[0209] In some embodiments of Formula XIV and XIVa, the conjugate comprises a first chromogenic moiety and a second chromogenic moiety, the first chromogenic moiety is carboxy-fluorescein, and the second chromogenic moiety is selected from rhodamine 6G and rhodamine B.

[0210] In some embodiments of Formulas XIV and XIVa, R 1 To R 10 One or more (preferably R 10 ) is optionally connected to a linker (L), and the linker is optionally connected to a peroxidase substrate (PS). Some embodiments of Formulas XIV and XIVa fall within the scope of Formula I when they include a chromogenic moiety that is connected to a peroxidase substrate via a linker of less than 30 atoms; such embodiments are not outside the scope of Formula I simply because they include a second chromogenic moiety. By building both colors into the same molecule, the ratio between the two chromogenic moieties at a given site is fixed. In contrast, when there are two chromogen mixtures with different colors, one chromogen can preferably precipitate based on diffusion, target intensity, and other factors, which can produce variable colors.

[0211] As yet another aspect, there is provided a chromogenic conjugate according to Formula IX:

[0212]

[0213] Where X is -OH, -OR X or -NR X R XX ,

[0214] Where Y is =O or =N + R Y R YY ;

[0215] Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R X , R XX , R Y , and R YY are independently selected from hydrogen and substituents having less than 40 atoms;

[0216] L is a linker comprising a linear chain having 5 to 29 consecutive linking atoms; and

[0217] PS is a peroxidase substrate moiety, such as a peroxidase substrate as defined herein.

[0218] More particularly, there is provided a chromogenic conjugate according to formula IXa:

[0219]

[0220] wherein L is a linker comprising a linear chain having 5 to 29 consecutive linking atoms; and

[0221] PS is a peroxidase substrate moiety, such as a peroxidase substrate as defined herein.

[0222] A new chromogen was prepared which was designated as compound 35 and had the following formula IXa:

[0223]

[0224] Compound 35 is a cyan chromogen with an absorbance maximum at 640 nm in neutral water. Compound 35 can be combined with other chromogenic conjugates described herein to stain a target or sample with a different color.

[0225] Connector

[0226] In the present application, the linking group (" L ") is a water-soluble molecular part comprising a chain having 5 to less than 30 adjacent atoms, for example 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 adjacent atoms. In some embodiments, the chain of atoms can be linear, in other embodiments, it can include one or more ring structures. In a preferred embodiment, the linking group molecule comprises an adjacent chain having 5 to 29 atoms, wherein every two connected carbon atoms are followed by heteroatoms such as oxygen atoms or nitrogen-atoms. In some embodiments, the linking group has no more than two continuous repeating ethoxy groups.

[0227] In some embodiments, the linker is a compound comprising 1 or 2 repeating units of the formula:

[0228]

[0229] Where R 31 Selected from methyl, ethyl, propyl, OCH 2 , CH 2 OCH2 , (CH 2 OCH 2 ) 2 , NHCH 2 NH(CH 2 ) 2 , CH 2 NHCH 2 , cycloalkyl, alkyl-cycloalkyl, alkyl-cycloalkyl-alkyl, heterocyclyl (e.g., a nitrogen-containing ring having 4 to 8 atoms), alkyl-heterocyclyl, alkyl-heterocyclyl-alkyl, and no more than 3 consecutive repetitions of ethoxy, R 32 and R 33 is independently selected from NH and O. More particularly, in some embodiments, the linker is selected from Formula IIIa, IIIb or IIIc:

[0230]

[0231] The linker can be based on a linker precursor such as compound 22, wherein the linker precursor is adapted to react with a peroxidase substrate moiety and a chromogenic moiety or with a reactive precursor of a peroxidase substrate moiety and a chromogenic moiety. For example, compound 22 can be used to prepare a conjugate comprising a linker of formula IIIa to IIIc by reacting compound 22 with a peroxidase substrate moiety precursor such as COMU (activated coumaric acid) and a chromogenic moiety precursor such as tetramethylrhodamine piperazinamide hydrobromide.

[0232]

[0233] Compound 22 can be prepared as described in Example 3 below or by other procedures. Other linkers and linker precursors can be prepared by the procedures set forth in WO 2007 / 015168 to Lohse or elsewhere.

[0234] The properties of the linking group can be modified to obtain the desired performance, for example, by changing the length or branching of the linking group. In addition, the linking group can be chemically modified to carry various substituents. The substituents can be further chemically protected and / or activated to allow the linking group to be further derivatized.

[0235] Peroxidase substrate moiety

[0236] The chromogenic conjugates of the present invention include a peroxidase substrate moiety. The term "peroxidase" relates to an enzyme having enzymatic activity that catalyzes a reaction of the form:

[0237] ROOR'+electron donor (2e - )+2H + →ROH+R'OH

[0238] For many peroxidases, the best substrate is hydrogen peroxide, but others are more active, such as organic hydroperoxides such as organic peroxides. The nature of the electron donor depends greatly on the structure of the enzyme, for example horseradish peroxidase (HRP) / EC 1.11.1.7) can use a variety of organic compounds as both electron donors and acceptors. HRP has an accessible active site, and many compounds can reach the reactive site.

[0239] The enzyme with peroxidase activity may be represented by a peroxidase molecule or a fragment of the enzyme comprising the enzymatic activity, said enzyme molecule being directly or indirectly linked to a molecule of a binding agent, said fragment accounting for, for example, 51% to 99.9%, or less than 51% of the entire size of the peroxidase molecule.

[0240] Peroxidases can be conjugated directly or indirectly to other molecules, such as reagents capable of binding to useful targets in a sample (e.g., a biological sample, such as a histological sample). The term "direct conjugation" means that the enzyme moiety is chemically linked (e.g., chemically conjugated) to another molecule; the term "indirect conjugation" means that the peroxidase is linked to the molecule via a linker molecule having one chemical bond to the binding agent and another chemical bond to the peroxidase. Methods for conjugating enzyme moieties are well known in the art.

[0241] In one embodiment, the peroxidase portion is a portion of HRP, such as a whole HRP molecule, a fragment that is capable of showing HRP enzymatic activity. It can also be a recombinant protein comprising a portion of HRP having enzymatic activity, etc. In another embodiment, the peroxidase can be soybean peroxidase (SP).

[0242] Non-limiting examples of reagents comprising an enzyme having peroxidase activity can be antibody molecules, such as primary or secondary antibody molecules or derivatives thereof, such as Fab, directly or indirectly conjugated to one or more moieties of HRP, and nucleic acid binding agents conjugated to HRP. Such binding agents can bind directly or indirectly to a target and thereby form complexes, each of which comprises a target and one or more molecules of a binding agent comprising an enzyme having peroxidase activity.

[0243] For each binding agent molecule, the number of HRP or other peroxidase moieties may range from 1 to 10 or more, such as 20-50 or more.

[0244] The location of a solid sample (e.g., a histological sample) or a solid support (e.g., a membrane or microscope slide) that includes peroxidase activity is sometimes referred to herein as a "target site." In one embodiment, the target site may include peroxidase activity, such as a portion of a peroxidase that is directly immobilized on or within a solid support. In another embodiment, the target site may include peroxidase activity that is directly immobilized on or within a solid support, i.e., a portion of a peroxidase that is linked to a reagent that is capable of binding directly or indirectly to a target that is immobilized on or within a support.

[0245] In some embodiments, the peroxidase moiety is part of a substrate for horseradish peroxidase (HRP) or soybean peroxidase (SP). In some embodiments, the peroxidase moiety (also labeled S or PS in some formulas) is part of a non-colorogenic or colorless HRP or SP substrate. In some embodiments, the portion of the peroxidase substrate has the following formula (Formula II):

[0246]

[0247] in

[0248] R 21 is -H, -OX, or N(X) 2 ;

[0249] R 22 is -H, -OX, or -N(X) 2 ;

[0250] R 23 is -OH;

[0251] R 24 is -H, -OX, or -N(X) 2 ;

[0252] R 25 is -H, -OX, or N(X) 2 ;

[0253] R 26 Yes-CON-(X) (2) , -CONH(X), or COO(X);

[0254] wherein H is hydrogen; O is oxygen; N is nitrogen; and X is H, alkyl or aryl. In some embodiments, S or PS is a residue of ferulic acid. In other embodiments, S is a residue of caffeic acid. In other embodiments, S or PS is a residue of sinapic acid. In a preferred embodiment, S is a residue of coumaric acid.

[0255] Compositions comprising chromogenic conjugates

[0256] As another aspect, compositions are provided that include any of the chromogenic conjugates described herein (referred to herein as "chromogenic compositions" or "chromogenic media"). Some compositions include one of the chromogenic conjugates described herein. Other compositions include two or more of the chromogenic conjugates described herein, for example, exactly two chromogenic conjugates (i.e., two types of chromogenic molecules, not exactly two molecules) or exactly three chromogenic conjugates, or exactly four chromogenic conjugates.

[0257] The composition may include one or more solvents, salts, detergents, and other components. In various embodiments, the chromogenic composition may further include one or more of the following: (i) organic modifiers; (ii) enzyme enhancers; (iii) iron chelators; (iv) detergents; (v) antimicrobial agents; (vi) organic or inorganic salts; (vii) enzyme substrates, such as peroxidase substrates. The list of additives added to the chromogenic composition is not limiting, and any compound that can enhance or weaken the performance of the chromogenic conjugate of the present invention as a peroxidase substrate or chromogenic molecule can be part of the composition depending on the embodiment of its use.

[0258] In some embodiments, the chromogenic composition or medium can comprise any liquid solvent, preferably an aqueous solvent (water), in which the chromogenic conjugate is initially soluble but can react to form an insoluble precipitate at the site of peroxidase activity. The liquid solvent can comprise a primary solvent such as water and an organic cosolvent such as NMP or 2-pyrrolidone. The liquid solvent can comprise a buffer having an appropriate buffering capacity, for example, phosphate buffered saline (PBS), Tris buffer, and / or imidazole buffer. The composition can be a buffered aqueous solution having a pH of 3 to 9, or about 3 to about 6, or about 4 to about 7, or about 5 to about 8.

[0259] In some embodiments, the color-forming composition or medium may include an organic or inorganic salt. The inorganic salt may be selected from sodium chloride, magnesium chloride, potassium chloride, calcium chloride, sodium phosphate, or ammonium sulfate, and combinations thereof. The organic salt may be selected from sodium acetate, ammonium acetate, or an imidazole salt such as imidazole hydrochloride, or others. The concentration of the salt may be about 10 -3 M to saturation, for example, about 20 mM to about 200 mM, or about 25 mM to about 100 mM. In some embodiments, the medium may contain salt at a concentration of about 10 mM, 20 mM, 50 mM, 75 mM or 100 mM.

[0260] In some embodiments, the color-forming composition may include a detergent such as polyethylene glycol-p-isobutylphenyl ether (NP-40), or a surfactant such as a surfactant selected from the following: a surfactant based on polyethylene glycol sorbitan monolaurate (Tween), or a block copolymer-based surfactant (pluronic, etc.) or others. The amount of the detergent may be from about 0.001% to about 5%, or on a v / v basis or on a w / v basis.

[0261] In some embodiments, the organic modifier may be present in the composition in an amount of about 1% to about 20% (v / v or w / v), however, in some embodiments, a higher concentration of organic modifier may be required. The organic modifier may be, for example, polyethylene glycol (PEG). Other examples include, but are not limited to, organic modifiers selected from the following: C1-C4 alcohols, N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), monoethylene glycol and diethylene glycol, cyclopentane, N,N-dimethylformamide (DMF) and combinations thereof. In some embodiments, polyethylene glycol (PEG), for example, PEG2000, or propylene glycol may be advantageously used. The amount of polyethylene glycol or other organic modifiers in the composition may be about 0.1% (v / v) to about 20% (v / v), for example, about 1% (v / v) to about 15% (v / v), for example, 5-10% (v / v).

[0262] The term "enzyme enhancer" refers to any compound that enhances the catalytic activity of peroxidase. Such enzyme enhancers may be selected from phenylboronic acid derivatives and divalent metal ions such as nickel or calcium. The amount of the enzyme enhancer may be about 10 -7 to about 10 -3 M.

[0263] Examples of iron chelators include ethylenediaminetetraacetic acid (EDTA) or ethylenediaminehydroxyphenylacetic acid type chelators (EDHPA). The concentration of the iron chelator may be about 10 -9 to about 10 -6 M.

[0264] The chromogenic composition can be provided as a stable solution. The term "stable" in the context of the present invention refers to the ability of the chromogenic composition to serve as a reaction medium for peroxidase-mediated precipitation of the conjugate and the ability of the chromogenic conjugate to keep the same spectral characteristics essentially unchanged over a significant period of time; for example, the composition can be prepared and kept at room temperature for at least 4 hours before use. The composition can also be prepared and preserved for a longer period of time, such as 12 hours to 12 months, or a longer period of time. To extend the shelf life of the chromogenic composition, it may be useful to store the composition at a temperature below 20°C, such as at 4-10°C, and / or add an antibacterial compound to the composition. The antibacterial compound can be any antibacterial compound commonly used for chromogenic compounds.

[0265] The concentration of the chromogenic conjugate in the composition may be about 10 -9 M to about 10 -2 M, which depends on the characteristics of the method of use. For example, about 10 -5 M to about 10 -3 M, for example about 10 -4 M to about 10 -3 M.

[0266] In some embodiments, the color-forming composition may also include a peroxide compound. For example, the medium may include hydrogen peroxide (H 2 O 2 ), for example, at a concentration of 0.0002% to 0.04%, or about 0.5 mM to about 1.5 mM. In some embodiments, when the color-forming composition includes a peroxide compound, the stability of the composition may be reduced to, for example, 1-2 weeks of storage without the altered characteristics discussed above.

[0267] In some embodiments, the compositions of the invention are substantially free of peroxidase substrates and peroxide compounds other than the peroxidase substrate portion of the chromogenic conjugates of the invention.

[0268] As mentioned above, in some embodiments, the compositions of the present invention may include a detergent. In some preferred embodiments, the detergent is a non-ionic, non-denaturing detergent. In some embodiments, the composition includes 0.001% to 10% detergent, or 0.01% to 1%, or 0.05% to 0.5%.

[0269] In some embodiments, the two-component formulation is provided as a kit or for a method. The first component comprises the chromogenic conjugate described herein in a buffer having a relatively low pH (e.g., pH 4 to 5, e.g., pH 4.8) to avoid hydrolysis over time. The second component is a buffered solution of hydrogen peroxide having a relatively high pH (e.g., pH 7-8, e.g., pH 7.4). The two components form a working solution having an intermediate pH (e.g., pH 6-7, e.g., pH 6.8) when mixed. Such a two-component formulation can be mixed by an instrument or manually, and has a shelf life of more than 2 years at cold room temperature. The formulation has a 3-week stability when mixed together at room temperature.

[0270] In some embodiments, the chromogenic composition comprises an organic co-solvent, for example 2-10% v / v of an organic co-solvent, which has been found to give significantly enhanced dyeing intensity. Suitable organic co-solvents are 2-pyrrolidone and NMP.

[0271] How to use

[0272] As another aspect, methods are provided for using the chromogenic conjugates described herein and compositions comprising those conjugates in various analyses, techniques and procedures in which samples, tissues, or portions thereof are stained or colored. For example, the chromogenic conjugates of the invention are used to detect molecular targets such as biological or chemical molecules, molecular structures, etc. in samples using a range of experimental protocols for detecting and visualizing these targets, e.g., immunohistochemistry (IHC), in situ hybridization (ISH), ELISA, Southern blotting, Northern blotting, and Western blotting. In general, the chromogenic conjugates of the invention can be used in any analysis in which DAB has been used as a stain for visualizing the target. Compared to the conjugates described in WO2009 / 036760, WO2010 / 094283, WO2010 / 094284, WO2011 / 047680 and WO2012 / 143010, the chromogenic conjugates of the present invention do not require the presence of a cross-linking agent such as DAB, ACHC or ferulic acid to mediate the precipitation of the chromogenic conjugates in the presence of peroxidase. The chromogenic conjugates can be used to detect molecular targets in solid or semi-solid samples or targets immobilized on or in solid supports, such as microscope slides, nitrocellulose membranes, microarray chips, gels, and other supports.

[0273] For example, the chromogenic conjugates of the invention can be used to stain or color formalin-fixed paraffin-embedded (FFPE) tissue samples, metaphases, or histological "smears". The chromogenic conjugates of the invention can be used in immunohistochemical analysis methods, where useful proteins are detected or identified by color. The chromogenic conjugates of the invention can be used in chromogenic in situ hybridization (CISH), where useful nucleic acids are detected or identified by color.

[0274] The method of the invention is performed by linking the target to a molecule or moiety with peroxidase activity, typically horseradish peroxidase (HRP) or a fragment thereof, and then catalyzing the formation of an insoluble colored precipitate at the location of the target from any of the soluble chromogenic conjugates described herein.

[0275] The conjugates of the present invention are superior to chromogens such as DAB for cells, tissues and other types of samples with brown hues. For example, melanoma is already brown in nature, as are many lung cancer samples caused by smoking or urban air pollution. For these samples, DAB is particularly unsuitable for staining samples from patients suffering from these two major types of cancer. Therefore, as another aspect, a method for staining a sample with a natural brown color is provided, which is performed by applying a conjugate of the present invention, particularly a red, yellow or blue chromogenic conjugate. In some embodiments, one, two or more of the chromogenic conjugates of the present invention are contacted with a sample comprising brown tissue (e.g., a sample comprising lung cancer cells, melanoma cells, melanocytes, mole tissue, tonsil tissue, or liver tissue). Preferably, the conjugate is compound 2. The conjugates of the present invention can also be advantageously used to detect multiple targets in a sample.

[0276] In the method of the present invention, one or more binding agents may be applied to the sample before the conjugate of the present invention is applied to the sample. The term "binding agent" refers to a molecule that can bind directly or indirectly to a target, wherein the term "directly" means that the binding agent has affinity with the target and can recognize the target and specifically bind to the target, wherein the term "indirectly" means that the binding agent does not have a specific affinity with the target but has affinity with a substance associated with the target and can specifically bind to the substance. A binding agent that can directly bind to a target is sometimes referred to as a "primary binding agent". A binding agent that can indirectly bind to a target is sometimes referred to as a "secondary binding agent". A primary binding agent is typically used to contact a sample. It includes any molecule that will specifically bind to a target that is presumed to be present in a sample. A secondary binding agent can be any molecule that binds to a primary binding agent. The detection system using the conjugate of the present invention to visualize the target may include other binding agents, such as tertiary or quaternary binding agents; the detection system may include several primary binding agents for various targets in the detection sample, such as two or more different molecules (e.g., two or more proteins, or a protein and a nucleic acid), or several primary binding agents for the same target in the detection sample, such as multiple molecular probes for detecting useful genes. The detection system may also include several secondary binding agents, which may be molecules of the same type, such as antibodies, or molecules of different types, such as antibodies and nucleic acids.

[0277] In cases where the target does not inherently include peroxidase activity, at least one of the binding agents used to detect the target in a sample in the visualization system of the present invention includes peroxidase activity so as to mark the location of the target in the sample with peroxidase activity.

[0278] The conjugate molecules of the present invention can be particularly advantageously used in multiplexing methods, in which more than one target needs to be stained or colored. As mentioned above, the conjugate molecules of the present invention have favorable optical characteristics, which allow clear differences between targets stained with different colors, all by observing the microscope field of the stained sample such as histological sample and analyzing the captured image of the stained sample. Another advantage is that the conjugate is a substrate of the same enzyme (i.e., peroxidase), such as HRP or SP. This significantly simplifies the process of multiple targets in the stained sample and improves the robustness of the staining process, because this same procedure and the same reagent can be used to detect the target by utilizing different conjugates. It can also make the overall detection process cheaper and particularly suitable for automated staining, imaging of stained samples and analysis of staining results.

[0279] The method of the present invention and the kit of parts can include any binding agent capable of monitoring a target in a solid sample or a target fixed on or in a solid carrier. For example, the binding agent can be an immunospecific binding pair such as an antibody and an antigen, or a non-immune specific binding pair such as a nucleic acid probe and a complementary sequence, or another type such as biotin and avidin. Various types of binding agents are common knowledge in the art, and descriptions of the binding agents can be seen in Q. Ashton Acton, ed., "Antigens—Advances in Research and Application: 2013 Edition", Scholarly Editions or Ralph Raply, ed, "The Nucleic Acid Protocols Handbook", Humana Press.

[0280] Typically, in the target detection methods of the present invention, samples containing the target are successively incubated in one or more incubation media. The term "incubation medium" in the context of the present invention refers to an aqueous medium containing a specific compound, wherein the sample is maintained during a certain period of time (referred to herein as "incubation time") to allow the desired reaction between the specific compound in solution and the sample to occur. The incubation medium can be a medium in which the target is naturally found by the binding agent ("binding agent medium"), or it can be one of the chromogenic compositions of the present invention (e.g., those described in the above section).

[0281] The time for maintaining and / or cultivating the sample in the cultivation medium, i.e., the cultivation time, can be from about 3 seconds to overnight, for example, about 10 seconds, 20 seconds, 30 seconds, 1 minute, 2 minutes, etc., for example, 3-10 minutes, 10-20 minutes, 20-40 minutes, 40-60 minutes, 1-2 hours or longer, for example, overnight. In one embodiment, the cultivation time of all steps in the detection process can have the same duration, i.e., each cultivation can last for 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, etc. The time can be selected based on the embodiment used. In another embodiment, the cultivation time between different steps can vary, for example, the cultivation of the sample in the medium comprising the binding agent can last for 1 minute to overnight, and the cultivation of the sample in the medium of the chromogenic conjugate and the peroxide compound can last for 1 minute to 15 minutes or longer.

[0282] The incubation can be performed under various temperature conditions depending on the type of target, binding agent, conjugate, etc. The detection process is mainly dependent on temperature, however, if desired, the temperature can be used to adjust the duration of the incubation time, for example, a lower temperature can be used to extend the incubation time, and vice versa, a higher temperature can be used to shorten the incubation time.

[0283] Basically, the binding agent medium is a buffered aqueous solution of one or more binding agents, with a pH of 4 to 9. In some embodiments, the first incubation medium may contain organic or inorganic salts. The inorganic salt may be selected from, for example, sodium chloride, magnesium chloride, potassium chloride, calcium chloride, sodium phosphate, or ammonium sulfate. The organic salt may be selected from, for example, sodium acetate, ammonium acetate or imidazole salts such as imidazole hydrochloride, etc.

[0284] The amount of salt in the binder medium may be about 10 -3 M to saturation, for example, about 20mM to about 200mM, or about 50mM to about 500mM. In some embodiments, the medium may include a salt in an amount of about 10mM to 500mM. In other embodiments, the medium may be free of salt.

[0285] As mentioned, typically, the pH of the binding agent medium can be from about 4 to about 9, for example, between pH 3.5 and pH 9.5, for example, between pH 5 and pH 7, between pH 5.5 and pH 6.5, or between pH 6.5 and 7.5, or between pH 7 and pH 8, or between pH 7.5 and pH 8.5, or pH 8 and pH 9. Any buffer having a suitable buffering capacity can be used, for example, phosphate buffered saline (PBS) and imidazole buffer. Other suitable buffers can be found in Good, NE., et al (1966) Hydrogen ion buffers for biological research. Biochem. 5 (2), 467-477. The pH of the medium may be necessary for binding of the binding agent to the target; it can be optimized depending on the properties of the binding agent and the target.

[0286] In some embodiments, the binder medium may include an organic modifier (the term "organic modifier" means any non-aqueous solvent), for example, N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), monoethylene glycol and diethylene glycol, sulfolane, N,N-dimethylformamide (DMF), polyethylene glycol (PEG), propylene glycol, etc. The amount of organic modifier may vary in the range of about 1% to about 20% (v / v or w / v), or in some embodiments, greater than 20%.

[0287] In some embodiments, the binder medium may include a detergent, for example, polyethylene glycol-p-isobutylphenyl ether (NP-40), or a surfactant such as a surfactant selected from the following: a surfactant based on monolauric acid polyethylene glycol sorbitan ester (Tween), or a surfactant based on a block copolymer (pluronic, etc.). The amount of the detergent may be from about 0.001% to about 5%, or based on v / v or based on w / v. In some embodiments, the binder medium may include a stabilizer for the binder, for example, bovine serum albumin or dextran. The amount of the stabilizer may be from 0.01% to 20% (w / v).

[0288] In some embodiments, the binding agent medium may include an ion chelator (e.g., ethylenediaminetetraacetic acid (EDTA) or ethylenediaminehydroxyphenylacetic acid type chelators (EDHPA), etc.). The amount of the chelator may range from about 10 -9 M to about 10 -6 M changes.

[0289] In some embodiments, the binding agent medium may include one or more blocking agents for saturating non-specific binding sites, i.e., sites of the solid support that do not contain the target. Some non-limiting examples of blocking agents suitable for different embodiments may be Denhard solution, bovine serum albumin, skim milk, etc.

[0290] Because a wide variety of targets, binding agents, and assay formats can be used, the composition of the binding agent medium can vary and can be tailored to a particular embodiment using knowledge in the art.

[0291] The methods of the invention also include one or more washing steps, either before or after incubation of the sample with a washing medium, e.g., between the step of incubating the sample with a binding agent and the step of staining the sample with one or more of the chromogenic conjugates of the invention. Typically, the washing medium will be a composition that is the same or similar to a composition that has been used to incubate or otherwise treat the sample in a step prior to the washing step, wherein the washing medium lacks the active ingredient, i.e., the specific reagent of the incubation step, e.g., a binding agent, a conjugate molecule, etc.

[0292] The method of the present invention may include one or more of the following steps: incubating the sample in a medium that will quench any undesirable peroxidase activity, such as endogenous peroxidase activity in the sample or residual peroxidase activity associated with the target site. Typically, incubating the sample in a peroxidase activity quenching medium will precede the step of incubating the sample with a binding agent, or when detecting several targets in the sample, it will occur after visualizing the first target with a first conjugate and before incubating the sample with a second binding agent involving a second target. The peroxidase activity quenching medium will typically contain a certain amount of a peroxide compound, such as hydrogen peroxide. The amount of the peroxide compound in the medium can be 1% to 10% (v / v or w / v).

[0293] The methods and uses of the present invention include various forms of assays in which the chromogenic conjugates of the present invention are used. Typically, the assays in which the conjugates can be used are any of those in which DAB can be used. Some non-limiting embodiments of such assay formats are described below.

[0294] The targets or biomarkers may be present in cells or tissues, and they may be detected using the methods described herein in any suitable assay format, such as in immunohistochemistry (IHC), or chromogenic in situ hybridization (CISH), or ELISA.

[0295] In some embodiments, the target can be a protein, such as a cell membrane receptor or a cytoplasmic protein, and in other embodiments, the target can be a nucleic acid, such as a cytoplasmic nucleic acid. Any derivative of the latter mentioned target, such as a fragment, precursor, mutant of the target protein or nucleic acid, etc., can also be a target in some embodiments.

[0296] Thus, in various embodiments, the target can be a biological or chemical target molecule, or a particle, or a molecular or cellular complex, or a molecular or cellular structure, or a virus, or a microorganism, or a fragment of said target molecule, particle, complex, structure, virus or microorganism. Targets included in chemical and environmental samples can be, in particular, pollutants, toxins, members of molecular libraries, industrial hazardous waste compounds, etc.

[0297] In some embodiments, biological sample can be a suspension of cells or tissue sections. The target molecule in the suspension or the structure of the cell can be detected using ELISA, IHC or CISH. When ELISA, IHC or ISH are used to detect the cells of the suspension, they are connected to a solid carrier, for example, an ELISA plate or a slide.

[0298] Preparation and processing steps for cells, tissues or other samples in IHC or CISH are well known in the art. For description of various steps, see, for example, "Immunohistochemical Staining Methods", Dako IHC Guidebook, 6th Ed (2013); van der Loos, "User Protocol: Practical Guide to Multiple Staining", Cambridge Research & Instrumentation, Inc. (2009); "Immunohistochemistry (IHC) an Application Guide", Abcam, (2013); "Handbook of Practical Immunohistochemistry", Lin & Prichard ed. (2015); Immunohistochemistry and In Situ Hybridization of Human Carcinomas", Hayated. (2005).

[0299] In CISH, a sample is obtained and exposed to a nucleic acid binding agent, which hybridizes with complementary base pairs of a target nucleic acid. The target nucleic acid in the sample is usually denatured to expose the binding site. In the assay of the present invention, the binding agent has been linked to or is linked to a peroxidase, and one or more chromogenic conjugates of the present invention are then contacted with the sample. The presence or amount of the target nucleic acid is detected by manual or automatic visual recognition of a chromogen.

[0300] In IHC, a sample is obtained and exposed to a binding agent, such as an antibody or fragment thereof, which specifically binds to a target molecule, such as a cell surface protein. In the assay of the present invention, the binding agent, such as a secondary antibody, is contacted with the sample after being linked to or conjugated to a peroxidase, one or more chromogenic conjugates of the present invention. The presence or amount of the target molecule is detected by manual or automated visual recognition of the chromogen.

[0301] In the assay of the present invention, light microscopy, commonly referred to as optical microscopy, uses visible light and a lens system to magnify the image of a small sample containing a chromogenic conjugate. Bright field microscopy is a simple optical microscopy illumination technique. The sample is illuminated with white light, and contrast in the sample is induced by the absorbance of some of the transmitted light in dense areas of the sample. The typical appearance of a bright field microscope image is a chromogenically colored sample on a bright background.

[0302] Automated staining and visualization devices can be used in various embodiments of the methods of the present invention, for example, for detecting multiple biomarkers. The detection of multiple markers requires balancing the signals of different chromogenic parts of the atoms. Automated staining devices are known in the art, and the methods are applicable to these devices.

[0303] In the automated analysis method, a computer-controlled automatic test instrument is used to evaluate the dyed sample, which uses calculations to derive quantitative measurements from the image. A high-performance charge coupled device (CCD) camera can be used to visualize the chromogenic dyed sample with one or more colored precipitates. Image acquisition can be used in conjunction with advanced wide-field microscopes and various algorithms for image repair. Color separation can be obtained using a three-CCD device (3CCD) and a dichroic beam splitter prism, which splits the image into red, green and blue components. The three CCDs are each arranged to correspond to a specific color.

[0304] The analytical method described in the present application may include some or all of the following steps: (a) collecting a first tissue or cell sample from an individual diagnosed with or suspected of having cancer (particularly lung cancer cells, melanoma cells or liver cancer cells); (b) administering a binding agent for the target, wherein the binding agent has peroxidase activity; (c) staining the first tissue or cell sample with one or more chromogenic conjugates of the present invention; (d) measuring the optical density of the stained tissue or cell sample from step (c), wherein the stained tissue or cell sample is illuminated with light having a wavelength absorbed by one or more chromogenic conjugates.

[0305] In some embodiments, the method of the present invention is used to detect a target in a sample, for example, an object having peroxidase activity, a biomarker, etc., wherein the target or sample is immobilized on a solid support, wherein the method comprises the following steps: (a) incubating the sample, presumably comprising a target having one or more binding agents comprising peroxidase activity, wherein the one or more binding agents are capable of directly or indirectly binding to the target, and forming a complex comprising the target and the one or more binding agents having peroxidase activity; (b) incubating the sample in a solution comprising one or more chromogenic conjugates of the present invention; (c) detecting the precipitated chromogenic conjugate, and thereby detecting the target.

[0306] Yet another aspect of the present application is a method for dyeing a sample by mixing the chromogenic conjugate of the present application with other chromogens. For example, rhodamine 6G-L12-Cou can be mixed with a fluorescein chromogen to produce a yellow / orange color. This combination is advantageous because no single chromogen produces a suitable orange color. In addition, rhodamine derivatives such as rhodamine 101 derivatives are expensive and chemically cumbersome. It is believed that chromogens that produce yellow are weaker for the human eye because yellow chromogens based on fluorescein have a sharp upper absorbance peak and absorb very little green light, even at high concentrations / intensities of dye. In other words, the yellow color does not change; they remain yellow. On the other hand, yellow dyes with a wider absorbance that extends weakly into the green absorbance range exhibit dichroism, meaning that they produce two colors. At low concentrations, these dyes appear yellow because they fully absorb blue light; at higher concentrations, the color migrates toward red as green light is absorbed. Thus, by mixing green-absorbing Rhodamine 6G with a yellow dye, the mixture can produce a designer dichroic dye in which the hue varies slightly with intensity to produce color contrast that expands the dynamic range.

[0307] Compound 2 alone produces a stain that changes from a bright magenta at low intensities toward full red at higher intensities. In a mixture comprising 0.6 mM compound 2 in 15 mM imidazole, pH 6.8, 4% NMP, 0.1% NP40-Nonidet (octylphenoxypolyethyleneoxyethanol), 0.01% benzalkonium chloride, 0.03% hydrogen peroxide, the mixture is characterized by making DAB comparable in intensity in the low range by iso-staining of low-expressing targets. In contrast, the mixture never over-stains, even the highest intensity targets. This can be seen when using various primary antibodies and various types of tissue samples.

[0308] As shown above, various dichroic dyes of the present application can be prepared by mixing two or more chromogenic conjugates of the present application. The dichroic effect depends on the extremely high absolute absorbance of the dominant wavelength that appears to be color-rich to the eye and the instrument used to observe the dichroic effect of the dyed sample. If the dominant chromogen is magenta, as compound 2, there must be substantially no perceived green light left for people or even instruments at some points of increased intensity. It has been unexpectedly found that 4 μm thick tissue sample sections can be strongly dyed with a single dichroic chromogen so that the color change will be observed by the human eye. Therefore, it can be inferred that the change from magenta to purple will be particularly acceptable because the hue change will be harmoniously and gradually perceived, and by using compounds 2 and 32 that do not absorb blue, the color will remain clear. Another hypothesis, that the reflection on the brown precipitate is produced by the chromogen that appears orange in the solution, is a dichroic bright orange, whose absorbance minimum is about 505nm, composed of a green-yellow chromogen plus a magenta chromogen. Thus, it will be appreciated that dichroic chromogens provide an expanded dynamic range; because one information channel dries up, (ie, all light of one color has been absorbed at a moderate intensity), and then the absorbance of light at other wavelengths begins at a higher intensity; thus, a color change occurs.

[0309] Two useful dichroic dyes were prepared: a dichroic orange dye that shifts to red at high intensity, and a dichroic red dye that shifts to blue-violet at high intensity. These compounds have an expanded dynamic range with more distinct thresholds for intensity and color change.

[0310] The dichroic orange dye was prepared as follows: a dichroic orange with good intensity with clear color was prepared in 50 mM imidazole, pH 6.8, 10% NMP, 0.1% NP40-Nonidet (octylphenoxypolyethyleneoxyethanol), 0.01% benzalkonium chloride, 0.03% hydrogen peroxide, 1 mM compound 9 and 0.2 mM compound 2. The color change is from a dull, slightly salmon orange at low intensity, through a bright, deep orange at medium intensity, approaching a very bright red at the highest intensity. This is valuable because no single chromogen can produce a good orange color when staining tissue, and it is an excellent contrast color relative to hematoxylin. The dichroic effect further assists in determining the intensity difference. The degree of color change is moderate, as the local shallow minimum is only 72 nm wide, but the key point is that this minimum is about 505 nm. The dichroic dye absorbs blue light well and absorbs green light very well, but is still comparable to human color receptors, decreasing right in the middle of the critical point. The bright and vivid color is therefore far more pleasing and clear to the eye than the single orange chromogen. The dichroic chromogen measured in water at pH 6.8 has a local shallow minimum between 459 and 531 nm, 72 nm wide, and an overall minimum essentially devoid of any absorbance above 575 nm.

[0311] The dichroic red dye was prepared as follows: in 50 mM imidazole, pH 6.8, 10% NMP, 0.1% NP40-Nonidet, 0.01% benzalkonium chloride, 0.03% hydrogen peroxide, 1 mM compound 2 and 0.2 mM compound 35 produced intense and radically changing colors. The color changes from weak magenta, through low intensity bright red. At medium intensity, the color migrates significantly toward reddish purple, and finally to the highest intensity dark blue-purple. This migration is very clear on the Her2 control cell line. Due to the high intensity, even some +0 cells are weakly stained, +1 cells appear bright red, +2 cells are clearly stained reddish purple, and +3 cells are dark blue-purple.

[0312] Another striking example is generated by staining tonsils with anti-CD21 or anti-CD20. CD21 is a prototypical low-expression marker, whereas CD20 marks one of the most abundant proteins.

[0313] In liver tissue stained with cytokeratin, dynamic color changes can be observed at close range between very low-expressing membranes, which are weak but clear magenta, and very high-expressing ductal structures, which have color changes from intense red to dark purple.

[0314] Areas of technical tissue defects as folds that produce increased absorbance demonstrate a final color change toward blue-black. When measured in water at pH 6.8, the composition has a local shallow minimum between 532 nm and 642 nm, 110 nm wide, and essentially no overall minimum in absorbance below 460 nm.

[0315] The dichroic effect demonstrated by these dichroic chromogens and dyes provides an expanded dynamic range. In addition, for those dichroic conjugates where the critical point of intensity is supported by a color change, those conjugates provide a unique method of staining a target. In some embodiments, a chromogenic HRP substrate conjugate is provided having at least one local minimum of at least 50 nm wide and an overall minimum between 390 and 700 nm, measured in water between pH 6 and 8.

[0316] Although the conjugate molecules of the present invention are described for their chromogenic properties, it is also contemplated that the conjugate molecules may be used as fluorescent molecules in assays and methods for detecting fluorescence.

[0317] As yet another aspect, a conjugate is disclosed that provides a fluorescent Resonance Energy Transfer (FRET). These FRET conjugates have two different chromophores that absorb and emit light at different wavelengths (maximum values), with the radius of interaction being smaller than the wavelength of the emitted light. The excited (first) chromophore emits energy absorbed by the receiving (second) chromophore, which then emits light at a different wavelength. For FRET conjugates, the distance and angle between the two chromophores affect the energy transfer, so it is desirable to fix the distance and angle, at least so that any rotation and flipping around the bond is much slower than in a photochemical reaction. In the FRET conjugates of the present invention, the emission spectrum of the first chromophore and the absorbance of the second chromophore should overlap, because increased overlap produces better energy transfer and better fluorescence.

[0318] A suitable FRET conjugate is prepared by reacting the piperazinamide of rhodamine 6G with carboxy-fluorescein (shown below as Formula XV):

[0319]

[0320] Another FRET conjugate is prepared by reacting rhodamine B with carboxy-fluorescein. Both FRET conjugates are fluorescent and can Resonance Energy Transfer. These FRET conjugates all absorb light at the maximum of the fluorescein moiety, but emit light at different emission maxima of the rhodamine 6G and rhodamine B moieties.

[0321] In some embodiments, the FRET conjugate according to Formula XIVa, wherein R 38is piperazine or another 5- to 6-membered ring.

[0322]

[0323] To enable FRET, the two chromogens must be close, but not touching. It is expected that if a flexible linker is used between these chromogens, there will be no energy transfer between them, because the flexible linker will allow them to contact; the piperazine ring provides sufficient distance and rigidity, and other 4- to 8-membered rings or heterocyclic groups are also expected to provide sufficient distance and rigidity. The effect is that the fluorescence of the carboxy-fluorescein part is transferred to the rhodamine part. Although these chromogenic parts maintain two excitation maxima (corresponding to their performance as independent fluorophores), a single emission maximum corresponding to the rhodamine part is also observed.

[0324] Rhodamine B-carboxyfluorescein conjugate can be excited at 500nm and emitted at 585nm. FRET conjugates of the present invention are extremely useful because they allow several fluorophores to be excited at the same wavelength, but can be detected at different wavelengths. It is expected that FRET conjugates of the present invention can be used for multiplexing flow cytometry and DNA sequencing. These are structurally similar back-to-back fluorescein-rhodamine conjugates, which are further connected to triphosphate nucleotides. Additional descriptions of dye terminators based on FRET are shown in US2005 / 0255475.

[0325] The disadvantage is that the quantum yield is limited. This is consistent with FRET theory: the distance and angle are the same for both compounds but the overlap between fluorescein and rhodamine B is less than that between fluorescein and rhodamine 6G. However, the conjugate is strongly fluorescent.

[0326] It is believed that rhodamine B-carboxyl-fluorescein conjugates may potentially be the long-sought fourth color for fluorescence microscopy, i.e., a fluorophore that emits blue. FITC (fluorescein) has been used to provide green emission or signals, and rhodamine or CyDyes have been used to provide red emission or signals, but it is difficult to obtain yellow fluorophores in practice, without causing the yellow signal to overflow unacceptably into green or red signals. This has limitations on the purposes of the yellow fluorescent signal. Typically, infrared emitting fluorophores are used as the fourth color, but their disadvantage is that they are invisible to the human eye. The FRET conjugates based on rhodamine B and carboxyl-fluorescein will also be visible using a group of excitation and emission filters of red fluorophores. By using a green excitation filter and a red emission filter, only the FRET fluorophore will be visible. Digital image processing can be used to subtract the FRET image from the red image to display the required red signal.

[0327] The linker attached to the peroxidase substrate can be attached to the FRET conjugate at either the acid or phenolic attachment point, as shown in Formula XV above. It is expected that it will be preferred to attach the linker to the acid position, provided that such a connection can be made (see Figure) without eliminating the FRET ability. In addition, as chromogens, these conjugates have highly desirable properties; for example, the 6G compound is orange, while the rhodamine B analog is true red. These findings highlight the potential value of rhodamine piperazinamides because their color FRET ability is unexpected and beneficial.

[0328] Complete set of medicine boxes

[0329] Kits comprising any of the chromogenic conjugates of the invention or compositions comprising the same are described herein for use in detecting a target in a sample. For example, the kit comprises one or more of the conjugates described below as A1 to A19 or K1 to K5 or in the Examples, or two, three, four or more of those conjugates, in a single composition or container or in separate compositions or containers.

[0330] Because the methods of the present invention are suitable for detecting a wide variety of targets in a variety of samples in a variety of assay formats, kits can include many different items, but all kits include a chromogenic conjugate, either in solid form (powder, lyophilized, etc.) or as a composition comprising a chromogenic conjugate molecule described herein or an incubation medium comprising a chromogenic conjugate molecule described herein. The following are some non-limiting exemplary embodiments of kits.

[0331] In one embodiment, the kit may include: (i) a chromogenic conjugate as described herein, either in solid form (powder, lyophilized, etc.) or in a liquid medium; and (ii) one or more binding agents capable of directly or indirectly binding to a target, wherein the binding agent may be any binding agent described herein.

[0332] In another embodiment, the kit may include: (i) a solution of a first chromogenic conjugate as described herein, wherein the first conjugate has a first spectral characteristic; (ii) a solution of a second chromogenic conjugate as described herein, wherein the second chromogenic conjugate has one or more spectral characteristics different from one or more spectral characteristics of the first chromogenic conjugate. Either the first conjugate or the second conjugate may be one of the conjugates listed below as A1 to A19 or K1 to K5 or in the Examples.

[0333] The kit of parts of the present invention may include several components (1, 2, 3, 4, 5, 6 or more) which are solutions of different chromogenic conjugates as defined above, wherein each of the different conjugates has a unique spectral signature that is different from the spectral signatures of the other conjugates included in the kit.

[0334] In another embodiment, any of the above kits may further include one or more binding agents capable of directly or indirectly binding to a target, wherein the binding agent may be any binding agent described herein.

[0335] In another embodiment any of the above kits may further include instructions for use of the chromogenic conjugate, staining interpretation and / or scoring guidelines.

[0336] In other embodiments, any of the above kits may further include one or more of the following: an aqueous composition comprising DAB, ACHC or another peroxidase substrate; a chromogenically detectably labeled binding agent capable of specifically binding to the conjugate; a procedure for staining, visualizing and / or quantifying the target; one or more reference materials, such as a sample comprising the stained target; additional stains, such as a histological stain or a substrate solution for another enzyme other than peroxidase; a blocking medium; an incubation medium, a wash medium, etc.

[0337] In another embodiment, the kit may include (i) any or all of the items of the above embodiments; (ii) tools for target visualization and / or image capture, or references for these tools; (iii) software for controlling the instrument; (iv) software for image analysis; and (iv) a locked image analysis algorithm.

[0338] The composition of the kit of parts of the present invention can be designed to be suitable for any of the above-mentioned applications of the chromogenic conjugates of the present invention.

[0339] Manufacturing method

[0340] As one aspect of the present application, a method for preparing the chromogenic conjugate of the present invention is provided. Such a method is demonstrated in the following examples.

[0341] As another aspect of the present application, an efficient method for preparing the piperazinamide of rhodamine is provided. The reaction between rhodamine and 2-haloacetyl ester gives a 2'-alkylcarboxymethyl derivative of formula IV:

[0342]

[0343] Where R 1 To R 10 , R x , R xx , Rz , X and Y have the definitions set out in this application.

[0344] These 2'-alkylcarboxymethyl derivatives react smoothly with excess piperazine at about 100°C in anhydrous solvents such as acetonitrile or N-methylpyrrolidone to give the corresponding piperazine amides of general formula V and Va, which are useful as intermediates for the synthesis of chromogenic conjugates, as well as chromogens:

[0345]

[0346] Where R 1 To R 10 , R x , R xx , R Y and R YY The excess piperazine was removed by evaporation under reduced pressure, followed by a precipitation step with diethyl ether, and the piperazine amide was isolated in high yield and purity, with no formation of rhodamine dimers being observed.

[0347] As another aspect of the present application, a method for preparing secondary amides of rhodamine, fluorescein and derivatives thereof is provided. The reaction between secondary amines and various esters of rhodamine gives secondary amides of rhodamine. For example, an ester of formula VII or formula VIIa (which is rhodamine B) can react with an amine to give an amide of rhodamine:

[0348]

[0349] Wherein R35 can be one of the following compounds 33R1-33R9:

[0350]

[0351] In Formula VIIa, R1 to R10 are preferably H. Various esters of Formula VIIa (Rhodamine B) are listed as 33R1 to 33R9, and their reactivity with secondary amines is tested and listed in order of increasing reactivity with those secondary amines, where R1 is the least reactive compound 33R1 and 33R9 is the most reactive of compounds 33R1-33R9. 33R1 and 33R2 react slightly or not with secondary amines. Reactivity can be enhanced by stabilizing the cationic leaving group. Benzyl ester 33R3, shows low but certain reactivity. Alkyl-carboxyl-methyl derivatives 33R4, 33R5, and 33R6 react strongly with secondary amines. However, 33R4 and 33R5 are more prone to side reactions (i.e., hydrolysis), and 33R6 reacts slightly slower. 4-Nitrobenzyl ester 33R7 reacts with secondary amines but is also more prone to hydrolysis, while ethyl-carboxyl-methyl ester 33R8 reacts appropriately. 33R8 is prepared from rhodamine B and 2-bromoethyl acetate. 33R8 is stable up to about 140°C overnight. Derivatives such as 33R9 are also stable and are clearly preferred or suitable precursors for the final chromogen of the present application. Compounds such as rhodamine B with esters 33R8 or 33R9 are stable. They are slightly reactive esters because they include a β-carbonyl group, but the β-carbonyl group is a suitable leaving group that allows secondary amines to react with these rhodamine B conjugates.

[0352] As another aspect of the present application, a method for preparing a secondary amide as rhodamine is provided. The reaction between the ester of rhodamine esters such as compounds 33R1-33R9 and various secondary amines gives the secondary amide of rhodamine. The various secondary amines can be one of the following compounds 34A to 34L, or compounds 34G to 34L, or compounds 34J to 34L, or compounds 34G to 34I:

[0353]

[0354] The secondary amine may be piperazine, piperidine, pyrrolidine, imidazolidine, pyrazolidine, azetidine, or other 4- to 8-membered (or 5- to 7-membered) ring or heterocyclic group, optionally with amine, carboxyl or ester substituents.

[0355] Unexpectedly and unexpectedly, piperazine (compound 34B) reacts with these esters (which are only slightly reactive), and N,N'-dimethylethylenediamine (compound 34A) also reacts. Further experiments demonstrated that compounds 34C-L also unexpectedly react with these esters. Compounds 34A-L are listed from most reactive to least reactive according to their experimental (or expected) reactivity with the esters, with compound 34A being the most reactive and compound 34L expected to be the least reactive. Compound 34A, i.e., N,N'-dimethylethylenediamine, is an alternative to piperazine and absorbs at 531 nm, which is the same absorbance as the esters. Secondary amines 34B, 34C, 34D, 34E, and 34F will not undergo further reaction when reacted with rhodamine, which is used to prepare the conjugate with the peroxidase substrate attached thereto. It is observed that the reactivity of the 7-ring from the 5-ring of 34C to the 34E drops sharply, and the absorbance of the rhodamine 6G derivative of 34C is between β-carbonyl ester (531nm) and other secondary amides (535nm) (533nm). Therefore, it is expected that 34L will react in a similar manner. 34G, 34H and 34I also reflect the efforts of the carboxylic acid derivatives of the secondary amide of rhodamine, and 34G reacts acceptably. It is expected that azetidine compounds such as 34J, 34K and 34L will continue to have a positive trend in reactivity. In particular, it is expected that compounds such as 34K or 34L including azetidine-3 carboxyl groups react to form useful synthetic precursors, which can thus include a novel family of desired rhodamine carboxylic acid derivatives. 34G, 34H and 34I also respond to the demand for carboxylic acid derivatives of the secondary amide of rhodamine that is fully reacted.

[0356] Thus, it has been unexpectedly discovered that the reactivity between similar secondary amines and β-carbonyl esters of rhodamine can vary by several orders of magnitude. In some cases, steric hindrance appears to affect reactivity. In addition, this may affect the stability of these secondary amides, which appear to be unusually unstable to aqueous bases, so acid-labile tert-butyl protecting groups were used on compounds 34G and 34H. This instability / reactivity also extends to β-carbonyl esters that are easily hydrolyzed.

[0357] As another aspect of the present application, a method for preparing a secondary amide of rhodamine is disclosed. The reaction between a β-carbonyl ester of rhodamine and a secondary amine gives a secondary amide of rhodamine of general formula VIII:

[0358]

[0359]

[0360] wherein R1 to R10 are as defined herein, but are preferably H, and are preferably substituted with (alkyl) carboxylic acids or esters. 36or R 37 In some embodiments, R 36 and R 37 Piperazine, piperidine, pyrrolidine, imidazolidine, pyrazolidine, azetidine or other 4-8 yuan (or 5-7 yuan) ring or heterocyclic group can be formed, which optionally has amine, carboxyl or ester substituents. These secondary amides can be prepared on any scale and purified by crystallization. Less reactive derivatives that do not include further functional groups can be used as chromogens; because they combine color and fluorescence with stable and crystalline chemical structures, these are suitable for use as dyes / inks / pigments / lasers / LEDs and in compositions and uses.

[0361] Yet another aspect is a method of staining a tissue sample for the absence of a target. It has been recognized that if certain portions of tissue are stained with the chromogens of the present application or with DAB, that portion of tissue may not be stained with a different color unless the first stain is very weak and the second stain is very strong. It should be understood that double stains of co-located targets with mixed colors can be prepared.

[0362] However, in such cases of co-localized targets, the first chromogen reduces the number of antigens available for recognition by the second antibody and also reduces the number of first primary antibodies (not recognized by HRP visualization in the first step) that may be visualized in a different color in the second step.

[0363] This effect can be performed to stain for an absent target, such as a marker. This was observed in the staining of colon cancer using two chromogenic conjugates of the present invention. Colon tissue is generally positive for cytokeratin 18. Colon tissue was stained using the following method: cytokeratin 18 was stained with a chromogenic conjugate having a first color, and then stained with a chromogenic conjugate having a second color to stain all cytokeratins in the colon tissue. For example, the first and second chromogenic conjugates can be precipitated in the same binder, and they can contact the colon tissue simultaneously or in succession. Alternatively, this can be accomplished by having two different binders, one of which is specific for cytokeratin 18 and the other is pan-specific for cytokeratin (both normal colon and colon cancer are positive for cytokeratin), wherein the first of the two chromogenic conjugates is precipitated in the first binder and the second of the two chromogenic conjugates is precipitated in the second binder. After staining colon cancer tissue with the first and second chromogenic conjugates having different colors, it was observed that in many cancer tissues, small areas were recognized by the second chromogenic conjugate, that is, they were stained with the second color. If the cancerous tissue is stained with only the first color, these areas may easily be overlooked, and the sample may be mistakenly considered to be negative for cytokeratin 18, even though the absence of the stain may have been caused by many factors, such as bubbles in the staining process, necrotic tissue, poor fixation, or other reasons. Therefore, the technical effect of the second stain is that it positively proves the absence of cytokeratin 18, for example indicating that the cells have mutated and lost their ability to produce cytokeratin 18. It is well known that colon cancer undergoes such mutations. Therefore, this staining method allows the use of the first and second chromogenic conjugates to identify cancerous tissue with such mutations.

[0364] Yet another aspect of the present application is compound 22 (Formula XIa) and other compounds of Formula XI:

[0365]

[0366] Where R 18 is a halogen; R 19 is a nitrogen atom protecting group (eg, a tert-butyloxycarbonyl (BOC) group; p is 0 to 4; q is 0 to 4.

[0367] The reaction between compound 22 and rhodamine provides rhodamine derivatives of general formula VI:

[0368]

[0369] Similar rhodamine derivatives can be prepared from other compounds according to formula XI.

[0370] The reactions proceed to completion in anhydrous solvents at about 100°C with virtually no formation of any by-products. In a single step, these reactions provide rhodamine derivatives with enhanced water solubility, with extended linkers and appropriately protected primary amino groups, which are then deprotected with TFA and can react with HRP substrates to give efficient chromogenic HRP substrates in only three steps.

[0371] More than 20 different chromogens were prepared from compound 22. Compound 22 reacts with the relatively low-reactivity natural carboxylic acid group of each rhodamine and fluorescein tested. It also reacts with the phenolic oxygen on fluorescein. It readily and selectively reacts with the piperazinamide of fluorescein and rhodamine. As described in Formula X, compound 22 reacts with the phenolic group next to the two sulfonic acid groups in Patent Blue V.

[0372] Similarly, compound 22 or other compounds of Formula IX can be reacted with fluorescein to obtain fluorescein derivatives.

[0373] Representative Implementation Methods

[0374] A1. A chromogenic conjugate comprising (a) a chromogenic portion and (b) a peroxidase substrate portion, wherein the chromogenic portion and the peroxidase portion are linked together via a linker,

[0375] Wherein the conjugate is a compound of formula I:

[0376]

[0377] Where X is -OH, -OR X or -NR X R XX ,

[0378] Where Y is =O or =N + R Y R YY ;

[0379] Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R X , R XX , R Y , and R YY are independently selected from hydrogen and substituents having less than 40 atoms;

[0380] L is a linker that is a compound comprising a chain of at least 5 consecutive linked atoms (e.g., 5 to 29 consecutive atoms); and

[0381] PS is a peroxidase substrate moiety. For example, in one embodiment, X is -OH, -OR X , Y is =O. In another embodiment, X is -NR X R XX , Y is = N + R Y R YY .

[0382] A2. The chromogenic conjugate of embodiment A1, wherein

[0383] R 1 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R 1 Can be used with R 2 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 The benzo, naphthyl or polycyclic arylene groups;

[0384] R 2 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R 2 Can be used with R 1 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 benzo, naphtho or polycyclic arylene radicals, or, when X is -NR X R XX When R 2 Can be used with RX together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0385] R X When present, selected from hydrogen, optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R X Can be used with R 2 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0386] R XX When present, selected from optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R XX Can be used with R 3 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0387] R 3 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, when X is -NR X R XX When R3 Can be used with R XX together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0388] R 4 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl of the group, or, when Y is -N+R Y R YY When R 4 Can be used with R yy together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group; R yy When present, selected from optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or R yy Can be used with R 4 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0389] R Y When present, selected from hydrogen, optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R Y Can be used with R5 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0390] R YY When present, selected from optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R XX Can be used with R 6 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0391] R 5 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R 5 Can be used with R 6 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 benzo, naphtho or polycyclic arylene radicals, or, when Y is -N+R Y R YY When R 5 Can be used with R y together to form optionally substituted with one or more identical or different R 13 or suitable R 14 A 5-membered or 6-membered ring of a group;

[0392] R 6 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R 6 Can be used with R 5 together to form optionally substituted with one or more identical or different R 13 or suitable R 14 The benzo, naphthyl or polycyclic arylene groups;

[0393] R 7 , R 8 and R 9 are each independently selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl of the group;

[0394] R 10 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or suitable R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or suitable R 14 (C6-C40) arylalkyl or heteroarylalkyl, halogen, haloalkyl, -OR 12 , -SR 12 , -SOR 12 , -SO 2 R 12 , and nitrile;

[0395] R 11 Selected from -NR 15 R 15 , -OR 16 , -SR 16 , halogen, haloalkyl, -CN, -NC, -OCN, -SCN, -NO, -NO 2 , -N 3 , -S(O)R 16, -S(O) 2 R 16 , -S(O) 2 OR 16 , -S(O)NR 15 R 15 , -S(O) 2 NR 15 R 15 , -OS(O)R 16 , -OS(O) 2 R 16 , -OS(O) 2 NR 15 R 15 , -OP(O) 2 R 16 , -OP(O) 3 R 16 R 16 , -P(O) 3 R 16 R 16 , -C(O)R 16 , -C(O)OR 16 , -C(O)NR 15 R 15 , -C(NH)NR 15 R 15 , -OC(O)R 16 , -OC(O)OR 16 , -OC(O)NR 15 R 15 and -OC(NH)NR 15 R 15 ;

[0396] R 12 is selected from (C1-C20)alkyl or heteroalkyl optionally substituted with a lipophilic substituent, (C5-C20)aryl or heteroaryl optionally substituted with a lipophilic substituent, and (C2-C26)arylalkyl or heteroarylalkyl optionally substituted with a lipophilic substituent;

[0397] R 13 is selected from hydrogen, (C1-C8)alkyl or heteroalkyl, (C5-C20)aryl or heteroaryl, and (C6-C28)arylalkyl or heteroarylalkyl;

[0398] R 14 Selected from -NR 15 R 15 , = O, -OR 16 , = S, -SR 16 , =NR 16 , =NOR 16, halogen, haloalkyl, -CN, -NC, -OCN, -SCN, -NO, -NO 2 , = N 2 , -N 3 , -S(O)R 16 , -S(O) 2 R 16 , -S(O) 2 OR 16 , -S(O)NR 15 R 15 , -S(O) 2 NR 15 R 15 , -OS(O)R 16 , -OS(O) 2 R 16 , -OS(O) 2 NR 15 R 15 , -OS(O) 2 OR 16 , -OS(O) 2 NR 15 R 15 , -C(O)R 16 , -C(O)OR 16 , -C(O)NR 15 R 15 , -C(NH)NR 15 R 15 , -OC(O)R 16 , -OC(O)OR 16 , -OC(O)NR 15 R 15 and -OC(NH)NR 15 R 15 ;

[0399] R 15 are independently hydrogen or R 16 , or, R 15 Each together with the nitrogen atom to which it is bound forms a 5- to 8-membered saturated or unsaturated ring which may optionally include one or more identical or different additional heteroatoms and may optionally be substituted with one or more identical or different R 13 or R 16 Group;

[0400] R 16 Each independently is R 13 , or substituted with one or more identical or different R 13 or R 17 R 13 ;and

[0401] R17 Each selected from -NR 13 R 13 , -OR 13 , = S, -SR 13 , =NR 13 , =NOR 13 , halogen, haloalkyl, -CN, -NC, -OCN, -SCN, -NO, -NO 2 , = N 2 , -N 3 , -S(O)R 13 , -S(O) 2 R 13 , -S(O) 2 OR 13 , -S(O)NR 13 R 13 , -S(O) 2 NR 13 R 13 , -OS(O)R 13 , -OS(O) 2 R 13 , -OS(O) 2 NR 13 R 13 , -OS(O) 2 OR 16 , -OS(O) 2 NR 13 R 13 , -C(O)R 13 , -C(O)OR 13 , -C(O)NR 13 R 13 , -C(NH)NR 15 R 13 , -OC(O)R 13 , -OC(O)OR 13 , -OC(O)NR 13 R 13 and -OC(NH)NR 13 R 13 .

[0402] A3. The conjugate of any one of embodiments A1 and A2, wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 10are independently selected from -H, -halogen, -methyl, -ethyl, -propyl, -isopropyl, -vinyl, -SO 3 H,-PO 3 H, -NO 2 , -COOH, -NH 2 , -CN, -OH, -OMe and -OEt.

[0403] A4. The conjugate of any one of embodiments A1 to A3, wherein the chromogenic moiety is selected from rhodamine and fluorescein, and salts thereof.

[0404] A5. The conjugate of any one of embodiments A1-A4, wherein one or more of the chromogenic moieties is selected from rhodamine, rhodamine 6G, tetramethylrhodamine, rhodamine B, rhodamine 101, rhodamine 110, fluorescein, and O-carboxymethylfluorescein.

[0405] A6. The conjugate of any one of embodiments A1 to A5, wherein the moiety is selected from rhodamine 116, rhodamine 123, and rhodamine 19.

[0406] A7. The conjugate of any one of embodiments A1 to A6, wherein the moiety is a rhodamine salt and comprises an anion selected from the group consisting of: Cl - ,Br - , TFA - , and ClO 4 - .

[0407] A8. The conjugate of any one of embodiments A1 to A7, wherein the chromogenic moiety is a 2'-piperazineamide derivative.

[0408] A9. The conjugate of any one of embodiments A1 to A8, wherein R 10 is selected from the group consisting of alkyl, heteroalkyl, alkoxy, halogen, haloalkyl, amino, alkylthio, cyano, isocyano, cyanooxy, mercaptocyano, nitro, and sulfinyl.

[0409] A10. The chromogenic conjugate of any one of embodiments A1 to A9, wherein the chromogenic conjugate is selected from the molecules shown in Table 1 and salts of =N groups.

[0410] A11. The conjugate according to any one of embodiments A1 to A10, wherein the peroxidase moiety has the following formula:

[0411]

[0412] R 21 Yes -H,

[0413] R 22 is -H, -OX, or -N(X) 2 ;

[0414] R 23 is -OH;

[0415] R 24 is -H, -OX, or -N(X) 2 ;

[0416] R 25 is -H, -OX, or N(X) 2 ;

[0417] R 26 is -CON-(X)2, -CONH(X), or COO(X);

[0418] wherein H is hydrogen; O is oxygen; N is nitrogen; and X is H, an alkyl or aryl group or a bond to L.

[0419] A12. The conjugate of any one of embodiments A1 to A11, wherein R 23 Yes -OH, R 24 Yes -H.

[0420] A13. The conjugate of any one of embodiments A1 to A12, wherein R 21 or R 25 Any of -OH, R 22 and R 24 Yes - H, R 23 It is -OH.

[0421] A14. The conjugate of any one of embodiments A1 to A13, wherein the peroxidase substrate is a residue of ferulic acid, cinnamic acid, caffeic acid, sinapic acid, 2,4-dihydroxycinnamic acid or 4-hydroxycinnamic acid (coumaric acid).

[0422] A15. The conjugate of any one of embodiments A1 to A14, wherein the linker is a compound comprising 1 or 2 repeating units of formula III:

[0423]

[0424] Where R 31 Selected from methyl, ethyl, propyl, OCH 2 , CH 2 OCH 2 , (CH 2 OCH 2 ) 2 , NHCH 2 NH(CH 2 ) 2 , CH 2 NHCH 2, cycloalkyl, alkyl-cycloalkyl, alkyl-cycloalkyl-alkyl, heterocyclyl (e.g., a nitrogen-containing ring having 4 to 8 atoms), alkyl-heterocyclyl, alkyl-heterocyclyl-alkyl, and no more than 3 consecutive repetitions of ethoxy, R 32 and R 33 are independently selected from NH and O.

[0425] A16. The conjugate of any one of embodiments A1-14, wherein the linker is selected from:

[0426]

[0427]

[0428] A17. Chromogenic conjugate according to formula IX:

[0429]

[0430] Where X is -OH, -OR X or -NR X R XX ,

[0431] Where Y is =O or =N + R Y R YY ;

[0432] Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R X , R XX , R Y , and R YY are independently selected from hydrogen and substituents having less than 40 atoms;

[0433] L is a linear linker comprising 5 to 29 consecutive linking atoms; and

[0434] PS is the substrate moiety of peroxidase.

[0435] A18. The conjugate of embodiment A17, wherein the conjugate has the structure of Formula IXa:

[0436]

[0437] A19. The conjugate of embodiment A17, wherein the conjugate has the structure of Formula IXb:

[0438]

[0439] B1. A kit for detecting a target having peroxidase activity or linked to a peroxidase in a sample, the kit comprising at least one solution of any of the chromogenic conjugates according to the preceding embodiments or any of embodiments K1 to K5 or the conjugates of the examples. The term "having peroxidase activity" means that the target either has intrinsic peroxidase activity or has a peroxidase activity chemically linked to (i.e., via a chemical bond) the target, for example, a target without intrinsic peroxidase activity is processed and chemically coupled to the peroxidase activity; the term "linked" in the context of the present invention means that the peroxidase activity is indirectly linked to the target, for example, via a target binding agent comprising / having peroxidase activity, such as an antibody / nucleic acid probe-HRP conjugate.

[0440] B2. A kit according to embodiment B1, wherein the kit comprises two or more aqueous solutions, wherein each of the solutions comprises a chromogenic conjugate according to any one of embodiments A1-A19 or K1 to K5, wherein the chromogenic conjugates of each of the two or more solutions differ from each other in one or more of their spectral characteristics.

[0441] B3. The kit according to embodiment B1, wherein the solution comprises at least two chromogenic conjugates of embodiments A1-A19 or K1 to K5.

[0442] C1. A method for detecting a target in a sample by a chromogenic detection method, comprising:

[0443] (i) incubating a sample presumably containing a target in an aqueous solution, wherein the target comprises peroxidase activity, or the target is directly or indirectly linked to a peroxidase, for a time and temperature sufficient to form a colored precipitate of a chromogenic conjugate, wherein the aqueous solution comprises:

[0444] a) at least one (or at least two, or at least three, or at least four, and / or not more than four, three or two) chromogenic conjugate according to any one of embodiments A1 to A19 or K1 to K5,

[0445] b) peroxide compounds,

[0446] and

[0447] (ii) detecting the colored precipitate of the chromogenic conjugate in the sample, thereby detecting the target in the sample.

[0448] C2. The method of embodiment C1, wherein the colored precipitate is detected using an optical microscope.

[0449] C3. The method of any of embodiments C1-C2, wherein the colored precipitate is detected by visual observation by a human observer.

[0450] C4. The method of any one of embodiments C1-C3, wherein the colored precipitate is detected by an automatic imaging instrument.

[0451] C5. The method of any one of embodiments C1 to C4, wherein the aqueous solution comprises 0.001% to 0.005% hydrogen peroxide and 0.1 mM to 10 mM of the at least one chromogenic conjugate.

[0452] C6. The method of any one of embodiments C1 to C5, wherein the amount of at least one chromogenic conjugate is from about 0.01 mM to about 10 mM, such as from about 0.05 mM to about 5 mM, such as from about 0.2 to about 2 mM, such as from about 0.4 to about 1 mM.

[0453] C7. The method of any one of embodiments C1-C6, wherein the peroxidase activity is associated with horseradish peroxidase.

[0454] C8. The method of any one of embodiments C1-C7, wherein the horseradish peroxidase is directly or indirectly linked to the target.

[0455] C9. The method of any one of embodiments C1-C8, wherein the target is a polypeptide, a nucleic acid, a carbohydrate, a lipid or a derivative thereof, a molecular complex, a particle, a eukaryotic cell or a prokaryotic cell or a microorganism.

[0456] C10. The method of any one of embodiments C1-C9, wherein the sample is a biological sample, an environmental sample, or a chemical sample.

[0457] C11. The method according to embodiment C10, wherein the sample is fixed on a solid support.

[0458] C12. The method of any one of embodiments C1-C11, further comprising directly or indirectly binding the target to an antibody linked to a peroxidase.

[0459] C13. The method of any one of embodiments C1-C12, comprising one or more additional steps, such as a washing step; a step of quenching unwanted peroxidase activity; a step of incubating the sample with one or more additional binding agents, such as a binding agent capable of binding to another target in the sample, or a binding agent capable of binding to the chromogen of the chromogenic conjugate.

[0460] C14. The method of any one of embodiments C1-C13, wherein the sample comprises at least two different targets, or at least two different subpopulations of the same target, wherein the at least two targets or at least two different subpopulations of the same target comprise peroxidase activity or are directly or indirectly linked to a peroxidase, and the method comprises

[0461] (i) incubating the sample in an aqueous solution for a time and at a temperature sufficient to form a colored precipitate of a first chromogenic conjugate, wherein the aqueous solution comprises:

[0462] a1) a first chromogenic conjugate according to any one of embodiments A1 to A19 or K1 to K5;

[0463] b1) peroxide compounds;

[0464] (ii) incubating the sample (i) in an aqueous solution for a time and at a temperature sufficient to form a colored precipitate of a second chromogenic conjugate, wherein the aqueous solution comprises:

[0465] a2) a second chromogenic conjugate of any one of embodiments A1 to A19 or K1 to K5;

[0466] b2) peroxide compounds;

[0467] wherein the first chromophore conjugate has a first chromophore characteristic, the second chromophore conjugate has a second chromophore characteristic, and wherein the first and second chromophore characteristics have one or more different spectral characteristics;

[0468] (iii) optionally, incubating the sample with a further aqueous solution of a chromogenic conjugate to stain a third, fourth, etc. target in the sample, wherein the further aqueous solution comprises a chromogenic conjugate having a chromogenic characteristic that allows the first, second, third, fourth, etc. targets to be distinguishably stained from one another by color.

[0469] (iv) detecting the first, second, third, fourth, etc. targets by detecting a colored precipitate of the corresponding chromogenic conjugate in the sample.

[0470] C15. The method of any of embodiments C1-C14, wherein the peroxidase activity is linked to the target via a target-specific binding agent comprising a peroxidase, such as one or more HRP moieties.

[0471] C16. The method of any of embodiments C1-C15, wherein the target is a protein or a nucleic acid.

[0472] C17. The method of any one of embodiments C1-C16, wherein the sample is stained manually, automatically or semi-automatically.

[0473] C18. The method of any of embodiments C1-C17, wherein staining is assessed by image analysis.

[0474] C19. The method of any of embodiments C1-C18, wherein the target is a nucleic acid and the sample is incubated to produce chromogenic in situ hybridization.

[0475] C20. The method of any one of embodiments C1-C19, wherein the method comprises detecting one or more targets by a process comprising the steps of:

[0476] providing peroxidase activity at a first target in the sample;

[0477] contacting the sample with a first chromogenic conjugate having a first color;

[0478] forming a first colored precipitate at the first target;

[0479] removing the peroxidase activity from the first target;

[0480] removing unprecipitated first chromogenic conjugate from the sample;

[0481] providing peroxidase activity at a second target in the sample;

[0482] contacting the sample with a second chromogenic conjugate having a second color;

[0483] forming a second colored precipitate at the second target; and

[0484] The first colored precipitate and the second colored precipitate are detected, thereby detecting the first target and the second target in the sample.

[0485] D1. A method for performing chromogenic in situ hybridization, the method comprising:

[0486] contacting a nucleic acid target with a probe that hybridizes to the nucleic acid target under hybridization conditions, wherein the probe comprises (1) a nucleic acid sequence that is at least partially complementary to the nucleic acid target, and (2) a peroxidase, or a first member of a specific binding pair; wherein the target and the probe form a complex;

[0487] When the probe comprises (2), contacting the complex with a second member of the specific binding pair, wherein the second member is directly or indirectly linked to a peroxidase and specifically binds to the first member;

[0488] Incubating the complex with at least one chromogenic conjugate of any one of embodiments A1 to A19 or K1 to K5; the incubation is performed for a time and temperature sufficient to form a colored precipitate at the target; and

[0489] The colored precipitate is detected.

[0490] D2. The method of embodiment D1, wherein the first member of the specific binding pair is an antigen or a hapten and the second member is an antibody or an antibody fragment.

[0491] D3. The method of any one of embodiments D1 or D2, wherein the target nucleic acid is present in a nucleic acid duplex, and further comprising a step of denaturing the nucleic acid duplex.

[0492] D4. The method of any one of embodiments D1-D3, further comprising a step of denaturing the PNA probes using one or more FITC-labeled PNA probes.

[0493] D5. The method of any one of embodiments D1-D4, further comprising counterstaining with a non-specific stain such as hematoxylin.

[0494] D6. The method of any one of embodiments D1 to D5, further comprising one or more of the following steps: deparaffinization; washing with a buffer before the contacting step; stringent washing after hybridization; blocking endogenous peroxidase; washing and dehydration after the incubation step.

[0495] D7. A method according to embodiment D3, wherein the probe is a nucleic acid analog fragment comprising a peptide-nucleic acid (PNA) backbone.

[0496] E1. A method for precipitating a chromogenic conjugate of any one of embodiments A1 to A19 or K1 to K5 in a target site comprising peroxidase activity, the method comprising incubating the target site in a solution comprising the chromogenic conjugate and a peroxide compound, and thereby precipitating the conjugate molecule at the target site.

[0497] E2. The method according to embodiment E1, wherein the peroxidase activity is associated with horseradish peroxidase (HRP) present at the target site.

[0498] E3 The method of any of embodiments E1 or E2, wherein the target site comprises a target and a peroxidase directly or indirectly linked to the target.

[0499] E4. The method of any of embodiments E1 or E3, wherein the target site comprises a biomarker.

[0500] E5. The method of any of embodiments E1 or E4, comprising precipitating at least two chromogenic conjugates according to any of embodiments A1 to A19 or K1 to K5 at the target site.

[0501] F1. A method for detecting one or more targets in a sample, the method comprising:

[0502] providing peroxidase activity at a first target in the sample;

[0503] contacting the sample with a first chromogenic conjugate;

[0504] forming a first colored precipitate at the first target;

[0505] removing the peroxidase activity from the first target;

[0506] removing unprecipitated first chromogenic conjugate from the sample;

[0507] providing peroxidase activity at a second target in the sample;

[0508] contacting the sample with a second chromogenic conjugate;

[0509] forming a second colored precipitate at the second target; and

[0510] detecting the first colored precipitate and the second colored precipitate, thereby detecting the first target and the second target in the sample,

[0511] wherein at least one of the first chromogenic conjugate or the second chromogenic conjugate is selected from any chromogenic conjugate of any one of embodiments A1-A16, and wherein the first chromogenic conjugate and the second chromogenic conjugate have one or more spectral characteristics that are different from each other.

[0512] F2. The method of embodiment F1, wherein the method further comprises: removing the peroxidase activity from the second target; removing the unprecipitated second chromogenic conjugate from the sample; providing peroxidase activity at a third target in the sample; contacting the sample with a third chromogenic conjugate, wherein the third chromogenic conjugate has one or more spectral characteristics that are different from each of the first chromogenic conjugate and the second chromogenic conjugate.

[0513] F3. The method of any one of embodiments F1 to F2, wherein the first chromogenic conjugate and the second chromogenic conjugate are each a conjugate of any one of embodiments A1 to A19 or K1 to K5.

[0514] F4. The method of any one of embodiments F1 to F3, further comprising the elements described in any one of embodiments C1-C13 or C15-C19.

[0515] G1. A chromogenic medium comprising: a chromogenic conjugate of any one of embodiments A1 to A19 or K1 to K5; a salt comprising an organic cation, anion or both; a non-ionic, non-denaturing detergent; wherein the pH of the medium is about 3 to about 9.

[0516] G2. The medium of embodiment G1, wherein the organic salt is selected from: salts of substituted and unsubstituted imidazoles, salts of substituted and unsubstituted pyridines, salts of substituted and unsubstituted pyrimidines, salts of substituted and unsubstituted pyrazines, salts of substituted and unsubstituted pyridazines, and salts of tertiary and quaternary amines.

[0517] G3. The medium of embodiment G1 or G2, wherein the organic salt is an imidazolium salt.

[0518] G4. The medium of embodiments G1, G2 or G3, wherein the detergent is 4-nonylphenyl-polyethylene glycol.

[0519] G5. The medium of any one of embodiments G1-G4, wherein the pH is from about 3 to about 6, or from about 4 to about 7, or from about 5 to about 8.

[0520] G6. The medium of any one of embodiments G1-G5, wherein the medium comprises 1 mM to 100 mM imidazole, or 10 mM to 75 mM, or 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM imidazole.

[0521] G7. The medium of any one of embodiments G1-G6, wherein the medium comprises N-methylpyrrolidone (NMP) or pyrrolidone.

[0522] G8. The medium of any one of embodiments G1 to G7, wherein the medium comprises 0.1 to 2 mM of the chromogenic conjugate, 10 to 50 mM of imidazole, 1% to 10% of NMP, and 0.01% to 1% of octylphenoxypolyethyleneoxyethanol.

[0523] H1. A method for preparing a secondary amide of rhodamine (e.g., piperazinamide), the method comprising:

[0524] The 2'-alkylcarboxymethyl derivative according to formula IV is reacted with an excess (preferably a moderate excess) of a secondary amine such as piperazine at a suitable temperature (e.g., about 90° C. to about 110° C.) in an anhydrous solvent (e.g., acetonitrile or NMP) to form a secondary amide of formula Va:

[0525]

[0526] Where R 1 To R 10 , R X , R XX , R Y and R YYHaving the definitions set out in this application. Excess piperazine was removed by evaporation under reduced pressure, followed by a precipitation step with diethyl ether, and piperazine amide could be isolated in high yield and purity, and no formation of rhodamine dimers was observed.

[0527] H2. The method of embodiment H1, wherein the 2'-alkylcarboxymethyl derivative and the secondary amine are reacted in acetonitrile or N-methylpyrrolidone at a temperature of about 100°C.

[0528] H3. The method according to embodiment H1 or H2, further comprising the steps of removing excess secondary amine by evaporation under reduced pressure and precipitating the secondary amide with diethyl ether. The secondary amide can be isolated in high yield and purity.

[0529] H4. The method of any one of embodiments H1 to H3, wherein the secondary amide is substantially free of rhodamine dimers.

[0530] H5. The method of any one of embodiments H1 to H5, further comprising preparing a 2'-alkylcarboxymethyl derivative of formula IV by reacting rhodamine and a 2-haloacetyl ester.

[0531] H6. The method of any one of embodiments H1 to H5, wherein the secondary amine is piperazine, piperidine, pyrrolidine, imidazolidine, pyrazolidine, azetidine, or other 4- to 8-membered ring or heterocyclic group, which optionally has an amine, carboxyl or ester substituent.

[0532] H7. The method of any one of embodiments H1 to H5, wherein the alkyl group of the 2'-alkylcarboxymethyl derivative is selected from compounds 33R1 to 33R9, or compounds 33R4 to 33R9, or compounds 33R7 to 33R9.

[0533] H8. The method of any one of embodiments H1 to H5, wherein the secondary amine is selected from compounds 34A to 34L, or compounds 34G to 34L, or compounds 34J to 34L, or compounds 34G to 34I.

[0534] I1. A method for staining a sample with a dichroic stain, the method comprising:

[0535] providing peroxidase activity at a first target in the sample;

[0536] contacting the sample with a first chromogenic conjugate at a first concentration (e.g., 0.1 mM to 10 mM, or 1 mM), wherein the first chromogenic conjugate is selected from the chromogenic conjugate of any one of embodiments A1-A19 or K1-K5;

[0537] forming a precipitate of a first chromogenic conjugate at the first target;

[0538] Check the color of the precipitate;

[0539] The target in the sample is measured by the detected colors, wherein a first detected color indicates a lower amount of target and a second detected color indicates a higher amount of target.

[0540] I2. The method of embodiment I1, wherein the first chromogenic conjugate is Compound 2.

[0541] I3. The method of any one of embodiments I1 to I2, further comprising contacting the sample with a second chromogenic conjugate at a second concentration (e.g., 0.03 mM to 10 mM, or 0.3 mM or 1 mM), wherein the first chromogenic conjugate and the second chromogenic conjugate have one or more spectral characteristics that are different from each other.

[0542] I4. The method of any one of embodiments I2 to I3, wherein the second chromogenic conjugate is selected from any chromogenic conjugate of any one of embodiments A1 to A16.

[0543] I5. The method of any one of embodiments I3 to I4, wherein the first chromogenic conjugate is compound 9 and the second chromogenic conjugate is compound 10.

[0544] I6. A method according to embodiment I5, further comprising contacting the sample with a third chromogenic conjugate at a third concentration (e.g., 0.03 mM to 10 mM, or 0.3 mM or 1 mM), wherein the third chromogenic conjugate can be compound 35.

[0545] I7. The method of any one of embodiments I3 to I4, wherein the first chromogenic conjugate is compound 35 and the second chromogenic conjugate is compound 10.

[0546] I8. The method of any one of embodiments I3 to I4, wherein the first chromogenic conjugate is compound 5 and the second chromogenic conjugate is compound 35.

[0547] J1. Compounds of formula XI:

[0548]

[0549] Where R 18 is a halogen; R 19 is a nitrogen atom protecting group (e.g., a tert-butyloxycarbonyl (BOC) group; p is 0 to 4; q is 0 to 4.

[0550] J2. Compounds of embodiment J1, wherein R 18 is bromine; R 19 is a BOC group; p is 1 to 3; and q is 1 to 3.

[0551] J3. The compound of embodiment J2, which has the structure of Formula XIa:

[0552]

[0553] J4. A method for preparing a chromogen, comprising: reacting rhodamine (e.g., rhodamine 6G or rhodamine B), fluorescein (e.g., fluorescein isothiocyanate, NHS-fluorescein, or O-carboxyfluorescein), or a 2'ester or amide derivative of rhodamine or fluorescein with a compound of any one of embodiments J1 to J3 to obtain an intermediate compound of formula XII:

[0554]

[0555] Where R 19 is a nitrogen atom protecting group (e.g., a tert-butyloxycarbonyl (BOC) group; R 20 is O or NH; p is 0 to 4; q is 0 to 4.

[0556] J5. An intermediate for the synthesis of a chromogenic compound, wherein the intermediate is a compound according to formula XII:

[0557]

[0558] Where R 19 is a nitrogen atom protecting group (e.g., a tert-butyloxycarbonyl (BOC) group; R 20 is O or NH; p is 0 to 4; q is 0 to 4.

[0559] J6. An intermediate for the synthesis of a chromogenic compound, wherein the intermediate is a compound according to formula V or Va:

[0560]

[0561] Where R 1 To R 10 , R X , R XX , R Y and R YY The definitions set out in this application are as follows.

[0562] J7. An intermediate for the synthesis of a chromogenic compound, wherein the intermediate is a compound according to formula XIII:

[0563]

[0564] Where R 19 is a nitrogen atom protecting group (e.g., a tert-butyloxycarbonyl (BOC) group; R 20 is O or NH; p is 0 to 4; q is 0 to 4.

[0565] J8. The intermediate of embodiment J7, wherein the intermediate is a compound according to formula XIIIa:

[0566]

[0567] Where R 19 is a nitrogen atom protecting group (e.g., a tert-butyloxycarbonyl (BOC) group; R 20 is O or NH; p is 0 to 4; q is 0 to 4.

[0568] K1. A conjugate having two chromogenic moieties according to formula XIV:

[0569]

[0570] Wherein X1 and X2 are selected from -OH, -OR X and-NR X R XX , X1 and X2 are preferably different,

[0571] Wherein Y1 and Y2 are selected from =O or =N + R Y R YY , Y1 and Y2 are preferably different,

[0572] Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R X , R XX , R Y , and R YY are independently selected from hydrogen and substituents having less than 40 atoms, or as defined elsewhere in this application; and

[0573] Where R 34 Selected from methyl, ethyl, propyl, OCH 2 , CH 2 OCH 2 , (CH 2 OCH 2 ) 2 , NHCH 2 NH(CH 2 ) 2 , CH 2 NHCH 2, cycloalkyl, alkyl-cycloalkyl, alkyl-cycloalkyl-alkyl, heterocyclyl (eg, a nitrogen-containing ring having 4 to 8 atoms), alkyl-heterocyclyl, or alkyl-heterocyclyl-alkyl, preferably piperidinyl or piperazinyl.

[0574] K2. FRET conjugate of formula XIVa:

[0575]

[0576] Wherein X1 and X2 are selected from -OH, -OR X and-NR X R XX , X1 and X2 are preferably different,

[0577] Wherein Y1 and Y2 are selected from =O or =N + R Y R YY , Y1 and Y2 are preferably different,

[0578] Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R X , R XX , R Y , and R YY are independently selected from hydrogen and substituents having less than 40 atoms, or as defined elsewhere in this application; and

[0579] Where R 38 It is a 4- to 8-membered cycloalkyl group or a 4- to 8-membered heterocyclic group (eg, a nitrogen-containing ring having 4 to 8 atoms), preferably a piperidinyl group or a piperazinyl group.

[0580] Preferred compounds of Formula XIVa are those in which the emission of the first chromophore moiety (eg, a fluorescein derivative) and the absorption of the second chromophore moiety (eg, a rhodamine derivative) overlap.

[0581] K3. The conjugate according to embodiment K1 or K2, wherein the conjugate comprises a first chromogenic moiety and a second chromogenic moiety, the first chromogenic moiety is carboxy-fluorescein, and the second chromogenic moiety is selected from rhodamine 6G and rhodamine B.

[0582] K4. A conjugate according to any one of embodiments K1 to K3, wherein X1 is selected from -OH and -OR X , X2 is -NR X R XX, Y1 is = O, Y2 is = N + R Y R YY .

[0583] K5. The conjugate according to any one of embodiments K1 to K4, wherein one or more of R1 to R10 (preferably R10) is optionally linked to a linker (L), and the linker is optionally linked to a peroxidase substrate (PS).

[0584] K6. A method for detecting a target in a sample by fluorescence detection, the method comprising:

[0585] (i) incubating a sample presumably comprising a target in an aqueous solution, wherein the target comprises peroxidase activity or the target is directly or indirectly linked to a peroxidase, wherein the aqueous solution comprises:

[0586] The FRET conjugate according to any one of embodiments K2 to K5, wherein the FRET conjugate comprises a first chromogenic moiety and a second chromogenic moiety, the first chromogenic moiety (e.g., a fluorescein derivative) having an absorption spectrum, the second chromogenic moiety (e.g., a rhodamine derivative) having an emission spectrum, and the light absorption spectrum and the light emission spectrum overlap,

[0587] (ii) exciting the precipitate of the FRET conjugate;

[0588] (iii) Detecting fluorescence from the emission spectrum in the sample, thereby detecting the target in the sample.

[0589] K7. A method according to embodiment K6, wherein the sample is incubated for a time and temperature sufficient to form a precipitate of the FRET conjugate.

[0590] K8. The method according to embodiment K6, further comprising incubating the sample with at least two, or at least three, other fluorescent molecules. The other fluorescent molecules have a different color or absorption maximum than the FRET conjugate.

[0591] L1. A method for detecting a target in a sample comprising brown tissue by chromogenic detection, the method comprising:

[0592] (i) incubating a sample presumably comprising a target in aqueous solution for a time and temperature sufficient to form a colored precipitate of a chromogenic conjugate, wherein the target comprises peroxidase activity or the target is directly or indirectly linked to a peroxidase,

[0593] The aqueous solution comprises:

[0594] a) the chromogenic conjugate of any one of embodiments A1 to A17, or K1 to K5,

[0595] b) peroxide compounds;

[0596] wherein the cancer cells are stained (slecand),

[0597] (ii) detecting the colored precipitate of the chromogenic conjugate in the sample, thereby detecting the target in the sample.

[0598] L2. A method according to embodiment L1, wherein the brown tissue is selected from tonsil, liver, melanoma, cell, lung cancer, or other.

[0599] Example

[0600] Examples 1 to 26 are non-limiting illustrations of the synthetic methods of conjugates and intermediates used to prepare some selected chromogenic conjugates, and their practical use in detecting molecular targets in IHC and ISH assay formats.

[0601] Example 1

[0602] Tert-butyl N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate (Compound 20) was prepared according to WO2007 / 015168.

[0603]

[0604] Example 2

[0605] "Boc-L15" (Compound 21) was prepared according to US20100055761.

[0606]

[0607] Example 3

[0608] Tert-butyl N-[2-[2-[2-[(2-bromoacetyl)amino]ethoxy]ethoxy]ethyl]carbamate (Compound 22) was prepared as follows: 214 mmol of 2-bromoacetic anhydride in about 700 mL of dichloromethane (DCM) was prepared as follows: 214 mmol of dicyclohexylcarbodiimide (DCC) and 428 mmol of bromoacetic acid were reacted in 600 mL of DCM at 4° C. for 20 h, and dicyclohexylurea (DCU) was filtered off with the DCM washings.

[0609] To the ice-cooled 214mmol 2-bromoacetic anhydride prepared above in about 700mL DCM, 321mmol lutidine was added, and then 214mmol compound 20 dissolved in 106mL DCM was added dropwise over 30 minutes. After another 10 minutes, the ice-cold reaction mixture was extracted with 400mL, then extracted with 50mL 1M citrate (pH 4.5), and finally extracted with 50mL water. DCM was evaporated below 40°C, and two more portions of 200mL DCM were evaporated to obtain 84g of oil, to which 170mL ether was added to further precipitate DCU, which was filtered off. A dense microcrystalline off-white precipitate was produced overnight at -18°C, which was filtered off, washed with ether and dried in vacuo. The yield was 60g, 76% of 22. The single pure product was analyzed by TLC as 0.5 in 5% methanol in ethyl acetate, with weak UV activity and strong ninhydrin reaction. Mass spectrometry provided data consistent with the bromine isotope pattern. Purity was >99% as determined by HPLC @ 210 nm.

[0610]

[0611] Example 4

[0612] Rhodamine 6G hydrochloride (Compound 23) was prepared as follows: 50 g of Rhodamine 6G ethyl ester hydrochloride (Sigma-Aldrich catalog # R4127) was hydrolyzed by refluxing in 320 mL of water, 30 mL of 10 M sodium hydroxide, and 350 mL of ethanol for 45 minutes. The reaction mixture was cooled and acidified with 75 mL of 4 M HCl to produce an intense red precipitate, which was filtered off, washed thoroughly with water, and dried in vacuo over sodium hydroxide pellets at 90° C. The yield was 46 g of Rhodamine 6G hydrochloride, 99.3% purity, as measured by HPLC@260.

[0613]

[0614] The name "Rhodamine 6G" is the common name and commercial name for the ethyl ester. The free acid is commercially available from Sigma Aldrich as the perchlorate salt under the name "Rhodamine 19". To avoid confusion with common names, abbreviations, and / or commercial names that may vary with substitution patterns, the parent structure will be referred to throughout this application as "Rhodamine 6G", regardless of the common name used for the specific derivative.

[0615] Example 5

[0616] Tetramethylrhodamine was prepared by sulfuric acid catalyzed condensation of phthalic anhydride and 3-(dimethylamino)-phenol similar to prior art methods. After extractive work-up, it was further purified by chromatography with 25% methanol in DCM to give 17 g of 98% HPLC pure product @ 260 nm. Rhodamine B base #234141, Rhodamine 101 inner salt #83694, and Rhodamine 110 chloride #83695 were obtained from Sigma-Aldrich.

[0617] Example 6

[0618] This example describes an exemplary method for the synthesis of ethyl-carboxymethylrhodamine B bromide (Compound 24).

[0619]

[0620] 4.42g (10mmol) of Rhodamine B base (Sigma Aldrich) was added to a 250mL flask containing 9mL acetonitrile and 3.5mL N,N-diisopropylethylamine (DIPEA) (20mmol). The solution was stirred at 90°C until all dissolved, and then 1.67mL of ethyl bromoacetate (15mmol) was added. After further reaction at 90°C for 1 hour, the reaction mixture was cooled to room temperature and 100mL of ether was added dropwise to produce a thin dark powder precipitate. It was filtered off, washed with 2x20mL of ether, and dried overnight in a vacuum. This obtained 6.93g (98%) of dark purple powder with 99% HPLC purity @ 260nm.

[0621] Example 7

[0622] This example describes an exemplary method for the synthesis of Rhodamine B piperazinamide bromide (Compound 25).

[0623]

[0624] 10 g of compound 24 (16 mmol) and 10 g of piperazine were dissolved in 40 mL of acetonitrile and reacted at 90° C. with stirring under nitrogen for 1 hr. 1 / 2 hours. Evaporate the acetonitrile and excess piperazine on a rotary evaporator at 70-80°C. Add two more 25 mL portions of acetonitrile and evaporate the acetonitrile again to drive off the remaining piperazine. Finally, dissolve the product in 25 mL acetonitrile and add 100 mL ether dropwise with stirring to produce a fine dark powder. Filter it off and dry overnight in a dryer with an oil pump to give a purple-gold solid. The yield is 7.64 g (82%) with a purity of 98% as measured by HPLC @ 260 nm.

[0625] The piperazinamides of rhodamine 6G and tetramethylrhodamine were prepared according to compound 25 via the corresponding ethyl-carboxymethyl esters, which were then reacted with piperazine.

[0626] Example 8

[0627] This example describes an exemplary method for synthesizing tetramethylrhodamine-Pip-L12-Cou (Compound 7). 100 mg of tetramethylrhodamine piperazinamide hydrobromide (see Example 7) and 100 mg of compound 22 were suspended in 1 mL of NMP and 50 microliters of DIPEA. After suspending at 80° C. for 4 hours, the reaction mixture was precipitated with ether to obtain the intermediate tetramethylrhodamine-Pip-L12-Boc. It was dissolved in 1.5 mL of TFA and kept for 30 minutes to remove the Boc-group, precipitated with ether, and mixed with COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethylamino-morpholino-carbonyl) 2-nitropropene ... The mixture was reacted with coumaric acid activated with hexafluorophosphate for 10 minutes and then purified by HPLC. The yield was 73 mg (50%).

[0628] Example 9

[0629] This example describes an exemplary method collectively known as Rhodamine 6G-Et-Pip-L12-Cou (Compound 14). 100 mg of Rhodamine 6G was reacted with 3 equivalents of N-hydroxy-succinimide, 3 equivalents of diisopropylcarbodiimide, and 3 equivalents of N-Boc-N'-[2-hydroxyethyl]-piperazine in NMP at 80°C overnight. This gave the intermediate Rhodamine 6G-Et-Pip-Boc:

[0630]

[0631] The crude intermediate was isolated by precipitation with ether, then deprotected with TFA, precipitated with ether, alkylated with compound 22, Boc deprotected, and finally reacted with COMU activated coumaric acid and purified by HPLC. The yield was 7 mg of compound 14, about 5% for all steps combined.

[0632] Example 10

[0633] This example describes an exemplary method collectively known as Rhodamine 6G-L12-Boc (Compound 26).

[0634]

[0635] 2.07 g of rhodamine 6G inner salt and 5.0 mmol of compound 23 were dissolved in 15 mL of anhydrous NMP and 1.7 mL of DIPEA (2 equivalents) at 100 ° C. Once all dissolved, 2.76 g of compound 22 (1.5 equivalents) were added and the reaction mixture was stirred at 100 ° C for 3 hours. The reaction was cooled to room temperature and 200 mL of ethyl acetate was added dropwise under vigorous stirring, and precipitation began after about half of the ethyl acetate had been added. The mixture was gently stirred at 4 ° C overnight to obtain a bright red precipitate, which was filtered off, washed with a small amount of cold ethyl acetate, and dried overnight in a vacuum. This produced 3.4 g of compound 26 with a yield of 99% and a purity of 97%, as measured by HPLC @ 260 nm.

[0636] Embodiment 11

[0637] This example describes an exemplary method collectively known as Rhodamine 6G-L12-TFA salt (Compound 27).

[0638]

[0639] 2 g of compound 26 (rhodamine 6G-L12-Boc) was dissolved in 12 mL of TFA at room temperature. After 30 minutes, 100 mL of ethyl acetate was first added, followed by 100 mL of ether, with ice cooling. This produced a deep red, extremely hygroscopic precipitate, which was filtered off, washed with a small amount of ether, and immediately dried in a vacuum. The yield was 1.05 g (50%) with a purity of 97%, measured by HPLC @ 260 nm.

[0640] Example 12

[0641] This embodiment describes an exemplary method for synthesizing rhodamine 6G-L12-Cou (compound 2). 100mg of compound 27 was dissolved in 1mL of NMP. 41mg of coumaric acid and 100mg of COMU (0.95 equivalent) were dissolved in 0.5mL of NMP and activated for 30 seconds by 43 microliters of DIPEA (1 equivalent). The activated mixture was added to the solution of compound 27, and then another 100 microliters of DIPEA were added immediately. The reaction was completed in 10 minutes, and the product was precipitated with 15mL of ether. It was partially dissolved in 50mL of 2% TFA and 25% acetonitrile in water, and purified by RP-HPLC. Each fraction containing the product was separated by mass spectrometry, and the pure fraction was collected and freeze-dried. The yield was 40mg (38%). The mutagenesis of compound 2 has been tested, and the results show that it does not have significant mutagenesis. Compound 2 was tested on rats in the LD50 experiment, and it is expected to have low toxicity, especially compared with DAB.

[0642] Example 13

[0643] Compound 1 (Rhodamine 110-L12-Cou); Compound 3 (TetraRhodamine-L12-Cou); Compound 4 (Rhodamine B-L12-Cou) and Compound 5 (Rhodamine 101-L12-Cou) were prepared in the same manner as Compound 2 (see Example 12) by alkylating the starting Rhodamine compound with Compound 22 in NMP and excess DIPEA at 100°C for 3-4h, followed by precipitation with ether. The intermediate was Boc-deprotected in neat TFA for 30-60 minutes and precipitated as a TFA salt by addition of ether. It was reacted with COMU (activated coumaric acid) and then purified by HPLC to give the final chromogenic conjugate.

[0644] Embodiment 14

[0645] In this example, other 4-hydroxy-coumaric acid derivatives of rhodamine 6G (compounds 16-19) were prepared by COMU-mediated coupling with the TFA salt compound 27 in a manner similar to the method for preparing compound 2 (Example 12). Compound 16 (rhodamine 6G-L12-Caf) was prepared from compound 27 and COMU-activated caffeic acid; compound 17 (rhodamine 6G-L12-2,4-OH-Cin) was prepared from compound 27 and COMU-activated 2,4-dihydroxycinnamic acid; compound 18 (rhodamine 6G-L12-Fer) was prepared from compound 27 and COMU-activated ferulic acid; and compound 19 (rhodamine 6G-L12-Sin) was prepared from compound 27 and COMU-activated cinnamic acid. In all preparations, COMU activation of the cinnamic acid derivatives was performed in NMP with a slight excess of free cinnamic acid and exactly one equivalent of DIPEA. Compound 27 was dissolved in NMP, activated cinnamic acid was added, and then excess DIPEA was added, just as was done in the preparation of compound 2. This minimized side reactions at the unprotected hydroxyl groups.

[0646] Embodiment 15

[0647] This example describes an exemplary method for synthesizing chromogenic conjugates with shortened and extended linkers. Compound 15 (Rhodamine 101-L27-Cou) was carried out with compound 21 (Boc-L15-OH) by adding a coupling step. To 70 mg of about 0.1 mmol of compound 27 suspended in 0.5 mL of NMP was added 0.13 mmol of compound 21 (Boc-L15-OH) activated with 0.95 equivalents of COMU and 1 equivalent of DIPEA. After 10 minutes, the intermediate Rhodamine 6G-L27-Boc was precipitated with 15 mL of ether; it was then dissolved in pure TFA and kept for 30 minutes to remove the Boc group, followed by ether precipitation, and finally reacted with COMU-activated coumaric acid. Compound 13 was prepared in a similar manner to compound 2 by alkylation of rhodamine 6G with N-(2-bromoacetyl)-N'-Boc-ethylenediamine, followed by TFA-mediated Boc deprotection and coupling with COMU-activated coumaric acid.

[0648]

[0649] Example 16

[0650] This example describes an exemplary method for synthesizing fluorescein-based chromogenic conjugates. 2,7-dichlorofluorescein-based compounds 9, 10, and 11 were prepared by alkylating the parent fluorescein with compound 22 in a manner similar to the preparation of rhodamine (see Examples 12-13).

[0651] Embodiment 17

[0652] This example describes an exemplary method for the synthesis of compound 28 (Flu-L12-Boc). 1.66 g of fluorescein and 1.85 g of compound 22 (1 equivalent) were dissolved in 3 mL of NMP and 1.3 mL of DIPEA (1.5 equivalents) and stirred at 100° C. overnight. The reaction mixture was taken up in 50 mL of DCM and washed with 5 mL of saturated NaHCO 3 The product of 2-(4-(4-(4-(4-(4-(4-phenyl)-2-yl)-2-nitropropene)) was added to 4-(4-(4-(4-phenyl)-2-nitropropene)-1-nitropropene-2-yl)-4 ...

[0653]

[0654] Embodiment 18

[0655] This example describes an exemplary method for synthesizing compound 10 (Flu-L12-Cou). 62 mg of compound 28 (Flu-L12-Boc) was dissolved in 0.5 mL of TFA at room temperature. After 30 minutes, the deprotected intermediate Flu-L12 was separated by precipitation with 8 mL of ether. It was dissolved in 0.3 mL of NMP and reacted with 2 equivalents of COMU-activated coumaric acid in 0.4 mL of NMP. After 10 minutes, the crude product was precipitated with ether and purified by HPLC. The yield was 40 mg (60%).

[0656] Compound 11 (2,7-dichloro-Flu-L12-Cou) was prepared in the same manner as compound 10 starting from 2,7-dichlorofluorescein and compound 22. Isolation and characterization of the correct isomer was performed by mass spectrometry / UV, Boc-deprotection was performed, and it was coupled with coumaric acid.

[0657] Embodiment 19

[0658] This example describes an exemplary method for the synthesis of compound 9 ((O-carboxymethyl)-Flu-L12-Cou). 310 mg of compound 28 was reacted with 111 microliters of tert-butyl bromoacetate (1.5 equivalents) at 100° C. in 1 mL of NMP and 170 microliters of DIPEA for 4 hours. The reaction mixture was directly applied to a small silica gel column and the intermediate compound was eluted with 10% MeOH in DCM. The yield was 220 mg (60%).

[0659]

[0660] 73 mg of compound 32 was dissolved in 0.5 mL of TFA. After 1 hour, mass spectrometry showed that the Boc group and tert-butyl ester had been cleanly removed. The intermediate was precipitated with ether and reacted with COMU-activated coumaric acid and purified by HPLC to give 29 mg of compound 9 (40%).

[0661] Embodiment 20

[0662] This example describes an exemplary method for synthesizing compound 12 (Flu-Pip-L12-Cou). Fluorescein piperazinamide was prepared according to reference 3. 400 mg of fluorescein piperazinamide and 370 mg of compound 22 (1 equivalent) were dissolved in 2 mL of NMP and 340 microliters of DIPEA and reacted at 60° C. for 1 hour. Column chromatography allowed the separation of 240 mg of Flu-Pip-L12-Boc (35%). 69 mg of this intermediate was Boc-deprotected with TFA, reacted with COMU-activated coumaric acid, and then HPLC purified to give 40 mg (55%) of compound 12 (Flu-Pip-L12-Cou).

[0663] Embodiment 21

[0664] This example describes IHC assays performed using many of the chromogenic conjugates described herein. IHC assays were performed using Dako reagents and instrumentation and according to the manufacturer's instructions. Compounds 1-19 were initially purified by HPLC to +98% purity and then assayed in these IHC assays.

[0665] As a pretreatment, multiple sample slides containing FFPE human tissues (including 10 different types of normal and cancer tissues) and FFPE slides containing Her2 control cell lines expressing 5 different levels of Her2 protein were deparaffinized in xylene and alcohol, and then the targets were retrieved in the Dako PT-Link module according to the manufacturer's instructions and stained on the DakoAutostainer Link.

[0666] Target retrieval was also tested in a microwave oven (10 min boiling in HEPES pH 8, target retrieval) in combination with staining on an Autostainer Plus. Autostainer staining was performed by applying the reagents while the slide was placed horizontally at room temperature.

[0667] Alternatively, all pretreatment and staining are performed automatically on the onboard Dako Omnis. Omnis staining is performed as follows: the reagents are applied in the capillary gap between the slide and the lid, and the lid is shaken to mix the reagents. This is performed at 32°C.

[0668] The samples were stained using the following protocol. Dako RTU primary antibodies (FLEX-IR series) were used according to manufacturer's instructions. For HER2 staining, monoclonal rabbit anti-HER2 was used at 1 mg / L. To visualize the primary antibodies, DakoEnvision+Dual link HRP visualization (K4061) was used, which includes both goat-anti-mouse-HRP and goat-anti-rabbit-HRP conjugates. DAB was used as a reference to gradually develop staining, and compounds 1 to 19 were tested as DAB alternatives on these samples.

[0669] An exemplary staining protocol includes: (1) Block endogenous peroxidase with Dako (S2023) peroxide blocker, 5 minutes. (2) Primary RTU antibody, 20 minutes. (3) Envision+ Dual Link HRP, 20 minutes. (4) DAB (Dako K3468), or one of the chromogenic conjugates of the invention, 5 minutes. (5) Counterstaining with hematoxylin (Dako S3301) for 5 minutes. Between each step, washes were performed with wash buffer (Dako S3006). Slides were dehydrated and coverslipped on a Sakura TissueTech Film coverslipper.

[0670] It was observed that the chromogenic substrate buffer commonly used for DAB gave unsatisfactory results with the chromogenic conjugate of the present invention. Better results were obtained with a buffer composition comprising 50 mM Imidazole: HCl pH 6.8 with 0.1% NP40-Non-idet as detergent. Extensive titration with hydrogen peroxide showed a wide concentration range with no visible differences between 0.002%, 0.003% and 0.004%.

[0671] Several different derivatives of rhodamine 6G were prepared and tested. Comparison between compound 2 and compounds 16-19 and five different 4-hydroxycinnamic acid derivatives showed that compound 16 with caffeic acid and compound 19 with sinapinic acid performed poorly. Compound 18 with ferulic acid performed acceptable, and compound 17 with 2,4-dihydroxycinnamic acid gave strong staining, but also some background. Compound 2 with 4-hydroxycinnamic acid was the best performing of the five chromogenic conjugates, giving both strong and crisp staining with no background. The other chromogenic moieties were then prepared using 4-hydroxycinnamic acid as the peroxidase substrate moiety.

[0672] At 0.5 mM, Compound 2 completely matched DAB in intensity and crispness across multiple primary antibody stainings, both as cell membrane, cytoplasm and nuclear markers, with essentially the same format as DAB with the same high expression / low expression balance. In stating that DAB matches in intensity, the qualification is that this refers to staining and detection of low and moderately expressed targets. This is what primarily determines clinical utility.

[0673] In many cases very highly expressed targets appear almost black with DAB, whereas the chromogenic conjugates of the present invention are spectrally narrow, yet allow light with wavelengths that they do not absorb to pass through. For this reason, several trained observers, including practicing pathologists, found that Compound 2 actually performed better and more dynamically in the high expression range, whereas DAB tended to overstain and "blot out" morphological details. This was found in Figure 1-3 is reflected in the micrographs. Figure 1 are photomicrographs of tonsil tissue stained with Ki67. The left photo was stained with Compound 2 (described below); the right photo was stained with DAB. Figure 2 The left photo is also stained with DAB; the right photo is stained with Compound 2. Figure 3 The left photo was stained with DAB; the right photo was stained with Compound 2.

[0674] The comparison of compound 2 and other rhodamine 6G derivatives with different linkers between rhodamine 6G and coumaric acid shows great similarity in spectral properties. Only compound 6 with piperazinamide stands out by absorbing 536nm light compared to others, which is about 5nm higher than the absorption wavelength of compounds 2, 13, 14 and 15 containing esters, and its maximum absorbance is all within 531-533nm. Compounds 6, 14 and 15 have significantly increased solubility in substrate buffer relative to compound 2, while compound 13 has a shortened linker with reduced solubility. In all cases, NMP is added to the buffer to increase chromogen solubility. See the results in the table below, which is a functional relationship between the solubility of 2, 13 and 15 and the percentage of NMP as an organic cosolvent, 0% represents pure aqueous buffer 50mM imidazole: HCl, pH 6.8, 3% and 10% represent the buffer of the percentage.

[0675] Table 3

[0676] Compound Connector 0％ 3％ 10% NMP 13 L6 0.5mM 0.9mM 1.8mM 2 L12 1.0mM 1.2mM 3.6mM 15 L27 1.7mM 1.9mM 6-7mM

[0677] Embodiment 22

[0678] This example describes the different colors provided by different embodiments of the chromogenic conjugates of the present invention. When tested at 0.4 to 1 mM, the following chromogens worked effectively: Compound 2 (0.5 mM), Compound 13 (1 mM) and Compound 14 (1 mM) produced red staining that was comparable to DAB in intensity and crispness. Compound 6 produced a slightly purple-red staining that was comparable to DAB in intensity and crispness. Compound 4 (0.4 mM) produced a purple staining that was comparable to DAB in intensity and crispness. Compound 3 (1 mM) produced a slightly reddish-purple staining that was more comparable to DAB in intensity and crispness. Compound 8 (1 mM) produced a slightly bluish-purple staining that was more comparable to DAB in intensity and crispness. Compound 15 (1 mM) produced a dark blue staining that was superior to the rival DAB in intensity and crispness. Compound 9 (1 mM) produced a slightly green-yellow staining that was most comparable to DAB in intensity.

[0679] Embodiment 23

[0680] This example demonstrates a method for immunohistochemical staining of three different targets using the conjugates of the invention. An exemplary triple staining method uses a combination of DAB and compounds 2 and 8, performed on a Dako Autostainer at room temperature. The steps of the method include:

[0681] 1. Block endogenous peroxidase with peroxide blocking reagent (Dako S2023) for 5 minutes.

[0682] 2. Anti-Ki67 RTU antibody (Dako IR626), 20 minutes

[0683] 3. Envision+dual Link HRP (Dako K4061), 20 minutes

[0684] 4.DAB (Dako K3468) 5 minutes.

[0685] 5. HRP was quenched with peroxide blocking reagent (Dako S2023) and 5 mg / mL α-cyanocinnamic acid was added for 5 minutes.

[0686] 6. Anti-CD20cy RTU antibody (Dako IR604), 20 minutes

[0687] 7. Envision+dual Link HRP (Dako K4061), 20 minutes

[0688] 8. 0.5 mM compound 2 in 50 mM imidazole:HCl pH 6.8, 0.003% hydrogen peroxide and 2.5% NMP, 5 min.

[0689] 9. HRP was quenched with peroxide blocking reagent (Dako S2023) and 5 mg / mL α-cyanocinnamic acid was added for 5 minutes.

[0690] 10. Anti-Her2 (Dako vial ST301 from HercepTest kit SK001) for 20 minutes.

[0691] 11. Envision+dual Link HRP (Dako K4061), 20 minutes

[0692] 12. 0.5 mM compound 8 in 50 mM imidazole:HCl pH 6.8, 0.003% hydrogen peroxide and 2.5% NMP, 5 min.

[0693] 13. Counterstaining with hematoxylin (Dako S3301)

[0694] This staining procedure produces: brown ki67 nuclear staining (DAB), with some cell staining in all tissues; purple CD20 cell membrane staining (compound 8), mainly in tonsils; and Her2 cell membrane staining (compound 2) combined with blue nuclei (hematoxylin) in breast cancer and colon. Control experiments with single and double staining demonstrated that triple staining correctly produced the same pattern as observed with single staining. It was found that the addition of α-cyanocinnamic acid to the peroxidase blocking agent greatly enhanced its HRP quenching efficiency; that is, there was no significant color spillover at all, DAB was not contaminated in any way by the subsequent purple and red staining, and the purple staining was not contaminated in any way by the subsequent red staining. All four colors, including blue hematoxylin, contrasted clearly with each other.

[0695] The chromogenic conjugates of the present application contrast well with brown DAB, except for the two blue conjugates based on Rhodamine 101 (Compound 5 and Compound 15), but they also contrast well with hematoxylin. Combined with the possibility of very efficient quenching of HRP activity and their narrow absorption spectrum, this makes the chromogenic conjugates of the present invention disclosed in the present application exceptionally suitable for multiplex staining and analysis.

[0696] Embodiment 24

[0697] This example describes the use of different embodiments of the chromogenic conjugates of the invention in chromogen in situ hybridization (CISH).These chromogenic conjugates and the fact that they are substrates for HRP rather than alkaline phosphatase appear to be particularly useful for CISH applications.

[0698] Slides containing multiple human tissue samples were subjected to the following rapid CISH protocol:

[0699] 1. Deparaffinization in xylene and then in ethanol

[0700] 2. Boil in the microwave for 10 minutes.

[0701] 3. Wash with buffer, water, and then dehydrate in ethanol.

[0702] 4. Denature IQFISH HER2 (Dako GM333, including FITC-labeled PNA centrosome 17 probe) at 66°C for 10 minutes

[0703] 5. Hybridize at 45°C for 15 minutes.

[0704] 6. Stringent wash at 63°C for 10 minutes.

[0705] 7. Wash buffer at room temperature.

[0706] 8. Block endogenous peroxidase with Dako (S2023) peroxide blocking reagent for 5 minutes.

[0707] 9. a. 20 nM anti-FITC-HRP or b. 20 nM anti-FITC-alkaline phosphatase for 20 min.

[0708] 10. a. 1 mM 2 in substrate buffer pH 6.8, 0.003% hydrogen peroxide, for 5 minutes; or b. Liquid Permanent Red (Dako K0640), for 10 minutes.

[0709] 11. Counterstain with hematoxylin (Dako S3301) for 5 min.

[0710] 12. Wash with water and dehydrate in anhydrous ethanol for 1 minute.

[0711] The slides incubated with anti-FITC-HRP in step 9 were stained with compound 2 in step 10, and the slides incubated with anti-FITC-alkaline phosphatase were stained with Liquid Permant Red. The stained slides are shown in Figure 1 This protocol was not according to the manufacturer's instructions, as the incubation time was reduced and no proteolytic pretreatment was used.

[0712] Results: Slides stained with anti-FITC-AP and Liquid Permanent Red showed small but clear and distinct punctate signals in 6-hour formalin-fixed tonsil tissue, with very small puncta in some areas of 24-hour fixed tonsil tissue. No consistent signals were detected in other types of tissue.

[0713] Slides stained with anti-FITC-HRP and Compound 2 produced large and distinct dots in both tonsil tissues, and the expected presence of 1-2 unique dots in all other tissues, including normal liver, pancreas, kidney, colon, and cerebellum, as well as in breast cancer, melanoma, and benign tumors. In only one tissue (malignant colon cancer tissue) were the dots very small.

[0714] While an equal number of nuclei were detected with Ki67, and liver membranes were equally weakly stained with DAB and Compound 2, small highly expressing structures in the liver, especially CDX-2 colonic structures, were overstained with DAB, often covering the entire cell, while nuclei were clearly highlighted when stained with Compound 2.

[0715] This example illustrates the highly useful properties of the combination of the chromogenic conjugates of the present invention with an appropriate HRP conjugate in analytical methods performed by CISH. Since HRP is a significantly smaller enzyme (40 kDa compared to 140 kDa for alkaline phosphatase), it penetrates well into, even deep into, the cell nucleus, and performs very well with the intense chromogens disclosed herein even with very short hybridization and incubation steps.

[0716] Embodiment 25

[0717] A new chromogen was prepared to further prepare a dichroic chromogen. The new chromogen was called compound 35 and has the following formula IX:

[0718]

[0719] Compound 35 is prepared by patent blue V sodium salt and compound 22 of the present application. Make patent blue V sodium salt (116mg, 0.2mmol) and compound 22 (154mg, 0.4mmol) together in 1mL NMP and 170 microlitres of DIPEA (1mmol) at 100 ℃ of reactions 16 hours. The intermediate product is precipitated with 7mL ether, and it is dissolved in 1mL TFA. After 1 hour, make intermediate once more as thin blue dust precipitation with 7mL ether, wash several times with ether. It is dried and dissolved in 3mL NMP, add 600 microlitres of DIPEA simultaneously, make it react with 600 microlitres of 0.5mM coumaric anhydride.

[0720] After 10 minutes of reaction, the mixture was precipitated with 25 mL of ether and purified by preparative HPLC as a TFA salt. The yield for the three steps (assuming a molar extraction coefficient of 80.000 @ 640 nm) was 46 mg, 26% s. C 44 H 54 N 4 O 12 S 2(cationic salt), calculated value 896.0565, found value 895.91. The maximum absorbance of compound 35 in neutral water is 640 nm.

[0721] The following synthetic scheme illustrates the preparation of compound 35 from an intermediate of formula X and intermediate compound 22:

[0722]

[0723] Embodiment 26

[0724] This embodiment demonstrates that compound 35 can be mixed with other chromogens, which include but are not limited to other chromogenic conjugates disclosed in the present application. These mixtures of yellow and cyan chromogens produce shades between yellow, blue or pure balanced green. The mixture of yellow and magenta allows the preparation of intermediate colors between orange and red. The mixture of magenta and cyan produces shades between increasing blue-violet, blue and blue-cyan. Therefore, compound 35 and other cyan chromogens can be mixed with chromogens of other colors to produce pure colors (rainbow colors) and all colors of the purple system between red and blue.

[0725] This example also describes mixing dichroic chromogens to prepare several examples of chromogens (referred to as Example Dichroic Orange and Dichroic Red) that change hue with concentration as described below. In such an example, the chromogen buffer consists of: 50 mM imidazole, pH 6.8, 10% NMP, 0.1% NP40-Nonidet, 0.01% benzalkonium chloride, 0.03% hydrogen peroxide. The example also includes Example Yellow, Example Green, Example Blue, which are also mixtures that produce the desired colors.

[0726] Example Yellow: 1 mM Compound 9 mixed with 0.3 mM Compound 10 produces a clear bright yellow stain at low intensities and a deep sunflower yellow stain at high intensities that contrasts very well with blue hematoxylin. The brown hue of Compound 10 alone at high concentrations is avoided, as is the unpleasant greenish yellow of Compound 9 alone. Because these chromogens are so spectrally narrow, the combination of the two chromogens better matches the absorbance of a wide range of retinal color receptors. In other words, the combination of the chromogens mixed together in this ratio produces a stain and a precipitate that has an almost constant absorbance from 430 to 500 nm and undergoes a sharp drop at higher wavelengths.

[0727] Example Green: 1 mM Compound 9 mixed with 0.3 mM Compound 10 + 0.3 mM Compound 35 (cyan) produces a beautiful bright green that looks a bit fuzzy but very bright at low intensities. At higher intensities, the saturation increases, resulting in a very strong and full green. The combined yellow chromogen peaks at 487nm and the cyan peaks at 642nm. At low intensities, some blue and red light are also transmitted, but in a balanced manner. As the intensity increases and the saturation of yellow and cyan decreases, the perceived saturation of green increases less, but proportionally more green light is transmitted.

[0728] Example Blue: 1 mM Compound 5 mixed with 1 mM Compound 35 (cyan) produced a bright but very intense sky blue stain. Again, as with Example Yellow, the effect is that by mixing two chromogens that are spectrally narrowly spaced, a most preferred match is produced with the cones of the human eye that absorb red and green light, leaving an almost preferred blue light. The contrast with the blue hematoxylin is not the best, but the hematoxylin-stained nuclei suddenly look somewhat reddish in contrast to this blue. This is because hematoxylin also has a small element of red transmission, which suddenly becomes apparent when contrasted with this pure blue.

[0729] Example Cyan: Perfect blue and green colors can be made with a mixture including compound 35, and the new cyan chromogen shows its great utility. But like green-yellow alone, cyan alone is slightly weakly perceived at low intensities and unpleasant to the human eye at high intensities. Even 1-2 mM compound 32 alone in chromogen buffer does not produce convincing results. This is understandable, because both chromogens absorb only near our color vision, leaving most of the light free to transmit at low intensities.

[0730] It is expressly contemplated that any chromogenic conjugate in the present application can be used in any of the methods of use, compositions, and kits of parts in the present application.

[0731] In this application, numerical ranges include the numbers defining the range. In this application, wherever the wording "comprising" is found, it is contemplated that the wording "consisting essentially of" or "consisting of" may be used in appropriate locations. It should be appreciated that chemical structures and formulae may be extended and expanded for illustrative purposes.

[0732] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0733] Any publications cited are only for their disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publications. Furthermore, the publication dates provided may be different from the actual publication dates that can be independently verified.

[0734] In view of the present application, it should be noted that the methods and apparatus are performed in accordance with the teachings of the present invention. In addition, various components, materials, structures and parameters are included only as examples and embodiments, without any limiting meaning. In view of the present application disclosure, the teachings of the present invention can be performed in other applications, and other components, materials, structures and instruments for performing these applications can be determined, while remaining within the scope of the appended claims.

Claims

1. A chromogenic conjugate comprising: (a) a chromogenic moiety, and (b) a peroxidase substrate moiety PS, The chromogenic conjugate is a compound of formula I: Where X is –OH, -OR X or –NR X R XX , Wherein Y is =O, =NR Y or =N + R Y R YY ; R 1 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl of the group; R 2 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, when X is -NR X R XX When R 2 Can be used with R X together to form optionally substituted with one or more identical or different R 13 or R 14 A 5-membered or 6-membered ring of a group; R X is selected from hydrogen, optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R X Can be used with R 2 together to form optionally substituted with one or more identical or different R 13 or R 14 A 5-membered or 6-membered ring of a group; R XX Selected from optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R XX Can be used with R 3 together to form optionally substituted with one or more identical or different R 13 or R 14 A 5-membered or 6-membered ring of a group; R 3 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, when X is -NR X R XX When R 3 Can be used with R XX together to form optionally substituted with one or more identical or different R 13 or R 14 A 5-membered or 6-membered ring of a group; R 4 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl of the group; R YY Selected from optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl of the group, or when Y is =N + R Y R YY When R YY Can be used with R 4 together to form optionally substituted with one or more identical or different R 13 or R 14 A 5-membered or 6-membered ring of a group; R Y is selected from hydrogen, optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R Y Can be used with R 5 together to form optionally substituted with one or more identical or different R 13 or R 14 A 5-membered or 6-membered ring of a group; R 5 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, when Y is =N + R Y R YY When R 5 Can be used with R Y together to form optionally substituted with one or more identical or different R 13 or R 14 A 5-membered or 6-membered ring of a group; R 6 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl of the group; R 7 , R 8 and R 9 are each independently selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl of the group; R 10 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl, halogen, -OR 12 , -SR 12 , -SOR 12 , -SO 2 R 12 , and nitrile; R 11 selected from -NR 15 R 15 ,-OR 16 ,-SR 16 ,halogen, -CN, -SCN, -NO 2 ,-N 3 ,-S(O)R 16 ,-S(O) 2 R 16 ,-S(O) 2 OR 16 ,-S(O)NR 15 R 15 ,-S(O) 2 NR 15 R 15 ,-OS(O)R 16 ,-OS(O) 2 R 16 ,-OS(O) 2 NR 15 R 15 ,-OP(O) 2 R 16 ,-OP(O) 3 R 16 R 16 ,-P(O) 3 R 16 R 16 ,-C(O)R 16 ,-C(O)OR 16 ,-C(O)NR 15 R 15 ,-C(NH)NR 15 R 15 ,-OC(O)R 16 ,-OC(O)OR 16 ,-OC(O)NR 15 R 15 and -OC(NH)NR 15 R 15 ; R 12 is selected from (C1-C20)alkyl or heteroalkyl, (C5-C20)aryl or heteroaryl and (C2-C26)arylalkyl or heteroarylalkyl; R 13 is selected from hydrogen, (C1-C8)alkyl or heteroalkyl, (C5-C20)aryl or heteroaryl, and (C6-C28)arylalkyl or heteroarylalkyl; R 14 Selected from -NR 15 R 15 , = O, -OR 16 , = S, -SR 16 , =NR 16 , =NOR 16 ,halogen, -CN, -SCN, -NO 2 , -N 3 , -S(O)R 16 , -S(O) 2 R 16 , -S(O) 2 OR 16 , -S(O)NR 15 R 15 , -S(O) 2 NR 15 R 15 , -OS(O)R 16 , -OS(O) 2 R 16 , -OS(O) 2 NR 15 R 15 , -OS(O) 2 OR 16 , -OS(O) 2 NR 15 R 15 , -C(O)R 16 , -C(O)OR 16 , -C(O)NR 15 R 15 , -C(NH)NR 15 R 15 , -OC(O)R 16 , -OC(O)OR 16 , -OC(O)NR 15 R 15 and -OC(NH)NR 15 R 15 ; R 15 are independently hydrogen or R 16 , or, R 15 Each together with the nitrogen atom to which it is bound forms a 5- to 8-membered saturated or unsaturated ring which may optionally include one or more identical or different additional heteroatoms and may optionally be substituted with one or more identical or different R 13 or R 16 Group; R 16 Each independently is R 13 , or substituted with one or more identical or different R 13 or R 17 R 13 ;and R 17 each independently selected from -NR 13 R 13 ,-OR 13 ,=S,-SR 13 ,=NR 13 ,=NOR 13 ,halogen, -CN, -SCN, -NO 2 ,-N 3 ,-S(O)R 13 ,-S(O) 2 R 13 ,-S(O) 2 OR 13 ,-S(O)NR 13 R 13 ,-S(O) 2 NR 13 R 13 ,-OS(O)R 13 ,-OS(O) 2 R 13 ,-OS(O) 2 NR 13 R 13 ,-OS(O) 2 OR 16 ,-OS(O) 2 NR 13 R 13 ,-C(O)R 13 ,-C(O)OR 13 ,-C(O)NR 13 R 13 ,-C(NH)NR 15 R 13 ,-OC(O)R 13 ,-OC(O)OR 13 ,-OC(O)NR 13 R 13 and -OC(NH)NR 13 R 13 , wherein the chromogenic portion and the peroxidase substrate portion are connected together via a linker L, wherein the linker L comprises a straight chain of 5 to 29 consecutive connected atoms, wherein the linker L is a water-soluble molecular portion, wherein the linker is linked to the chromogenic moiety via an ester or secondary amide linkage to the 2' carboxylic acid of the chromogenic moiety, The peroxidase substrate part PS is the following formula: in R 21 Yes -H, R 22 is -H, -OX, or -N(X) 2 ; R 23 is -OH; R 24 is -H, -OX, or -N(X) 2 ; R 25 is -H, -OX, or -N(X) 2 ; R 26 is -CONH-, or -COO-; Wherein H is hydrogen; O is oxygen; N is nitrogen; X is H.

2. The chromogenic conjugate according to claim 1, wherein the linker is a compound comprising 1 or 2 repeating units of formula III: Where R 31 OCH 2 , R 32 and R 33 are independently selected from NH, CH 2 NH and O.

3. The chromogenic conjugate of claim 1, wherein the linker is selected from Formula IIIa, IIIb, or IIIc: 。 4. A chromogenic conjugate comprising: (a) a chromogenic moiety, and (b) a peroxidase substrate moiety PS, wherein the chromogenic moiety is selected from the group consisting of rhodamine, rhodamine 6G, tetramethylrhodamine, rhodamine B, rhodamine 110, rhodamine 101, fluorescein, and O-carboxymethylfluorescein; wherein the chromogenic part and the peroxidase substrate part are linked together via a linker L, wherein the linker L comprises a straight chain of 5 to 29 consecutively linked atoms, wherein the linker L is a water-soluble molecular part, wherein the linker is linked to the chromogenic moiety via an ester or secondary amide linkage to the 2' carboxylic acid of the chromogenic moiety, and the peroxidase substrate moiety PS is of the formula: in R 21 Yes -H, R 22 is -H, -OX, or -N(X) 2 ; R 23 is -OH; R 24 is -H, -OX, or -N(X) 2 ; R 25 is -H, -OX, or -N(X) 2 ; R 26 is -CONH-, or -COO-; Wherein H is hydrogen; O is oxygen; N is nitrogen; X is H.

5. A composition comprising at least one chromogenic conjugate according to any one of claims 1 to 4 and a solvent and optionally one or more of the following substances: (i) an organic modifier; (ii) an enzyme enhancer; (iii) an iron chelator; (iv) a detergent; (v) an antibacterial agent; (vi) an organic or inorganic salt; or (vii) an enzyme substrate, wherein the organic modifier is selected from polyethylene glycol, C1-C4 alcohol, N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), sulfolane, N,N-dimethylformamide (DMF) and combinations thereof; and The enzyme enhancer is selected from phenylboronic acid derivatives and divalent metal ions. The composition according to claim 5 , wherein the organic modifier is monoethylene glycol or diethylene glycol.

7. A kit for detecting a target having peroxidase activity or linked to a peroxidase in a sample, the kit comprising at least one chromogenic conjugate according to any one of claims 1 to 4.

8. Use of the chromogenic conjugate according to any one of claims 1 to 4 in the preparation of a kit for detecting a target in a sample by chromogenic detection, wherein the detection include: Incubating a sample presumably comprising a target in an aqueous solution, wherein the target comprises peroxidase activity or is directly or indirectly linked to a peroxidase, for a time and temperature sufficient to form a colored precipitate of a chromogenic conjugate, wherein the aqueous solution comprises: a) at least one chromogenic conjugate according to any one of claims 1 to 4; b) peroxide compounds; A colored precipitate of the chromogenic conjugate in the sample is detected, thereby detecting the target in the sample.

9. The use according to claim 8, further comprising incubating the sample with a second chromogenic conjugate having Formula IX or Formula XIV: Where X is -OH, -OR X or -NR X R XX , Where Y is =O or =N + R Y R YY ; R 1 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl of the group; R 2 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, when X is -NR X R XX When R 2 Can be used with R X together to form optionally substituted with one or more identical or different R 13 or R 14 A 5-membered or 6-membered ring of a group; R X is selected from hydrogen, optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R X Can be used with R 2 together to form optionally substituted with one or more identical or different R 13 or R 14 A 5-membered or 6-membered ring of a group; R XX Selected from optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R XX Can be used with R 3 together to form optionally substituted with one or more identical or different R 13 or R 14 A 5-membered or 6-membered ring of a group; R 3 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, when X is -NR X R XX When R 3 Can be used with R XX together to form optionally substituted with one or more identical or different R 13 or R 14 A 5-membered or 6-membered ring of a group; R 4 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl; R YY Selected from optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl of the group, or when Y is =N + R Y R YY When R YY Can be used with R 4 together to form optionally substituted with one or more identical or different R 13 or R 14 A 5-membered or 6-membered ring of a group; R Y is selected from hydrogen, optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, R Y Can be used with R 5 together to form optionally substituted with one or more identical or different R 13 or R 14 A 5-membered or 6-membered ring of a group; R 5 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl, or, when Y is =N + R Y R YY When R 5 Can be used with R Y together to form optionally substituted with one or more identical or different R 13 or R 14 A 5-membered or 6-membered ring of a group; R 6 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl of the group; R 7 , R 8 and R 9 are each independently selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl of the group; R 10 Selected from hydrogen, R 11 , optionally substituted with one or more identical or different R 14 The (C1-C20)alkyl or heteroalkyl group is optionally substituted with one or more identical or different R 13 or R 14 (C5-C20) aryl or heteroaryl, and optionally substituted with one or more identical or different R 13 or R 14 (C6-C40) arylalkyl or heteroarylalkyl, halogen, -OR 12 , -SR 12 , -SOR 12 , -SO 2 R 12 , and nitrile; R 11 selected from -NR 15 R 15 ,-OR 16 ,-SR 16 ,halogen, -CN, -SCN, -NO 2 ,-N 3 ,-S(O)R 16 ,-S(O) 2 R 16 ,-S(O) 2 OR 16 ,-S(O)NR 15 R 15 ,-S(O) 2 NR 15 R 15 ,-OS(O)R 16 ,-OS(O) 2 R 16 ,-OS(O) 2 NR 15 R 15 ,-OP(O) 2 R 16 ,-OP(O) 3 R 16 R 16 ,-P(O) 3 R 16 R 16 ,-C(O)R 16 ,-C(O)OR 16 ,-C(O)NR 15 R 15 ,-C(NH)NR 15 R 15 ,-OC(O)R 16 ,-OC(O)OR 16 ,-OC(O)NR 15 R 15 and -OC(NH)NR 15 R 15 ; R 12 is selected from (C1-C20)alkyl or heteroalkyl, (C5-C20)aryl or heteroaryl and (C2-C26)arylalkyl or heteroarylalkyl; R 13 is selected from hydrogen, (C1-C8)alkyl or heteroalkyl, (C5-C20)aryl or heteroaryl, and (C6-C28)arylalkyl or heteroarylalkyl; R 14 Selected from -NR 15 R 15 , = O, -OR 16 , = S, -SR 16 , =NR 16 , =NOR 16 ,halogen, -CN, -SCN, -NO 2 , -N 3 , -S(O)R 16 , -S(O) 2 R 16 , -S(O) 2 OR 16 , -S(O)NR 15 R 15 , -S(O) 2 NR 15 R 15 , -OS(O)R 16 , -OS(O) 2 R 16 , -OS(O) 2 NR 15 R 15 , -OS(O) 2 OR 16 , -OS(O) 2 NR 15 R 15 , -C(O)R 16 , -C(O)OR 16 , -C(O)NR 15 R 15 , -C(NH)NR 15 R 15 , -OC(O)R 16 , -OC(O)OR 16 , -OC(O)NR 15 R 15 and -OC(NH)NR 15 R 15 ; R 15 are independently hydrogen or R 16 , or, R 15 Each together with the nitrogen atom to which it is bound forms a 5- to 8-membered saturated or unsaturated ring which may optionally include one or more identical or different additional heteroatoms and may optionally be substituted with one or more identical or different R 13 or R 16 Group; R 16 Each independently is R 13 , or substituted with one or more identical or different R 13 or R 17 R 13 ;and R 17 each independently selected from -NR 13 R 13 ,-OR 13 ,=S,-SR 13 ,=NR 13 ,=NOR 13 ,halogen, -CN, -SCN, -NO 2 ,-N 3 ,-S(O)R 13 ,-S(O) 2 R 13 ,-S(O) 2 OR 13 ,-S(O)NR 13 R 13 ,-S(O) 2 NR 13 R 13 ,-OS(O)R 13 ,-OS(O) 2 R 13 ,-OS(O) 2 NR 13 R 13 ,-OS(O) 2 OR 16 ,-OS(O) 2 NR 13 R 13 ,-C(O)R 13 ,-C(O)OR 13 ,-C(O)NR 13 R 13 ,-C(NH)NR 15 R 13 ,-OC(O)R 13 ,-OC(O)OR 13 ,-OC(O)NR 13 R 13 and -OC(NH)NR 13 R 13 , L is a linear linker comprising 5 to 29 consecutive linking atoms; and PS is a peroxidase substrate moiety, and the peroxidase substrate moiety PS is the following formula: in R 21 Yes -H, R 22 is -H, -OX, or -N(X) 2 ; R 23 is -OH; R 24 is -H, -OX, or -N(X) 2 ; R 25 is -H, -OX, or -N(X) 2 ; R 26 is -CONH-, or -COO-; Wherein H is hydrogen; O is oxygen; N is nitrogen; X is H; Wherein X1 and X2 are selected from -OH, -OR X and-NR X R XX , Wherein Y1 and Y2 are selected from =O or =N + R Y R YY , Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R X , R XX , R Y , and R YY are independently selected from hydrogen and substituents having less than 40 atoms; and Where R 34 OCH 2 , NHCH 2 , or NH(CH 2 ) 2 .

10. The use according to claim 9, wherein Y1 and Y2 are different.

11. The use according to claim 9, wherein X1 and X2 are different.

12. Use of the chromogenic conjugate according to any one of claims 1 to 4 in the preparation of a kit for performing chromogenic in situ hybridization, wherein the chromogenic in situ hybridization include: contacting a nucleic acid target with a probe that hybridizes to the nucleic acid target under hybridization conditions, wherein the probe comprises (1) a nucleic acid sequence that is at least partially complementary to the nucleic acid target, and (2) a peroxidase and optionally a first member of a specific binding pair; wherein the nucleic acid target and the probe form a complex; When the probe comprises a first member of the specific binding pair, contacting the complex with a second member of the specific binding pair, wherein the second member of the specific binding pair is directly or indirectly linked to a peroxidase and specifically binds to the first member; Incubating the complex with at least one chromogenic conjugate according to any one of claims 1 to 4; the incubation is performed for a time and temperature sufficient to form a colored precipitate at the nucleic acid target; The colored precipitate is detected.

13. Use of the chromogenic conjugate according to any one of claims 1 to 4 in the preparation of a kit for detecting two or more targets, wherein include: Binding a peroxidase molecule or an enzyme fragment containing peroxidase activity to a first target via a binding agent to provide peroxidase activity at the first target in the sample; contacting the sample with a first chromogenic conjugate; forming a first colored precipitate at the first target; removing the peroxidase activity from the first target; removing unprecipitated first chromogenic conjugate from the sample; Binding the peroxidase molecule or the enzyme fragment containing peroxidase activity to a second target via a binding agent to provide peroxidase activity at the second target in the sample; contacting the sample with a second chromogenic conjugate; forming a second colored precipitate at the second target; and detecting the first colored precipitate and the second colored precipitate, thereby detecting the first target and the second target in the sample, Wherein the first chromogenic conjugate and the second chromogenic conjugate are chromogenic conjugates according to any one of claims 1 to 4, and wherein the first chromogenic conjugate and the second chromogenic conjugate have one or more spectral characteristics that are different from each other.

14. The use according to claim 13, further comprising: include: removing the peroxidase activity from the second target; removing unprecipitated second chromogenic conjugate from the sample; The peroxidase molecule or the enzyme fragment containing peroxidase activity is bound to a third target by a binding agent to provide peroxidase activity at the third target in the sample; the sample is contacted with a third chromogenic conjugate, wherein the third chromogenic conjugate has one or more spectral characteristics different from the first chromogenic conjugate and the second chromogenic conjugate.

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