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Publication Number
US-12673938-B2
Publication Date
2026-07-07
Expiration Date
Abstract
Disclosed herein are detectable moieties and detectable conjugates comprising one or more detectable moieties. In some embodiments, the disclosed detectable moieties have a narrow wavelength and are suitable for multiplexing. Also disclosed are methods of labeling one or more targets within a biological specimen using any of the detectable conjugates and/or detectable moieties described herein.
Core Innovation
The invention relates to detectable conjugates for in situ detection and signal-amplification chemistries such as tyramide signal amplification, catalyzed reporter deposition, and quinone methide signal amplification. The compounds of Formula (I) link a tissue reactive moiety to a detectable moiety through a linker group Q and an index m, where Z is a tyramide, a tyramide derivative, a quinone methide precursor, or a functional group capable of participating in a click chemistry reaction.
In the compounds of Formula (I), Q is a branched or unbranched, linear or cyclic, substituted or unsubstituted group having between 2 and 40 carbon atoms and optionally one or more heteroatoms selected from O, N, or S, and m is 0, 1, or 2. The detectable moiety W is defined by Formula (VIIA) with R substituents constrained to specific alkyl and related group definitions, and at least one relevant substituent terminates in a moiety capable of participating in a click chemistry reaction.
The disclosure further provides alternative conjugate formats such as Formula (VIII) where X is a tissue reactive moiety and W is a detectable moiety, and variants where the click-reactive counterpart is on Y in Formula (XII) with Y being a click chemistry member and W being a detectable moiety. The document also describes immunohistochemical use, kits containing multiple conjugates, biological samples with stained targets, biomarker detection by in situ hybridization and immunohistochemistry, and multiplex brightfield immunohistochemistry using chromogens based on tyramide and quinone methide precursors.
Claims Coverage
The consolidated claim coverage centers on one independent claim for a compound of Formula (I) with Z, Q, m, and W constrained by tissue-reactive and click-chemistry-capable features. The inventive features include the Z selection, the constrained Q group, the W framework of Formula (VIIA), and dependent refinements to R^m, R^q, R^r, and quinone methide precursor options.
Formula (I) compound with tissue-reactive or click-chemistry-capable Z
A compound of Formula (I) in which Z is a tissue reactive moiety selected from a tyramide, a tyramide derivative, or a quinone methide precursor, or a functional group capable of participating in a click chemistry reaction, including dibenzocyclooctyne, trans-cyclooctene, an alkyne, an alkene, an azide, a tetrazine, a maleimide, a N-hydroxysuccinimide, a thiol, a 1,3-nitrone, an aldehyde, a ketone, a hydrazine, a hydroxylamine, or an amino group.
Constrained Q group with 2-40 carbons and optional heteroatoms
Q is a branched or unbranched, linear or cyclic, substituted or unsubstituted group having between 2 and 40 carbon atoms and optionally one or more heteroatoms selected from O, N, or S, and m is 0, 1, or 2.
W defined by Formula (VIIA) with terminal click-capable moieties
W has Formula (VIIA), where R^x is H or a branched or unbranched C1-C4 alkyl group optionally substituted with halogen atoms; R^m is H or a branched or unbranched C1-C4 alkyl group optionally substituted with halogen atoms and/or one or more —S(O)(O)(OH) groups, or a branched or unbranched C1-C20 alkyl group optionally including one or more heteroatoms selected from O or N and optionally including one or more carbonyl groups, provided that the C1-C20 alkyl group terminates in a moiety capable of participating in a click chemistry reaction; and R^q and R^r are each independently H, a branched or unbranched C1-C4 alkyl group optionally substituted with halogen atoms, or a group R^s that terminates in a moiety capable of participating in a click chemistry reaction.
Substituent relationship constraints for Rq, Rr, and Rs
At least one of R^q or R^r comprises R^s, R^q and R^r are both not R^s, and R^s is a saturated or unsaturated C1-C20 alkyl group comprising at least one amide group and optionally substituted with one or more heteroatoms.
Quinone methide precursor structure options
The quinone methide precursor is specified to correspond to either Formula (IXA) or Formula (IXE), with substituent groups R1-R7 specified and w ranging from 1 to 12.
The claim coverage is directed to Formula (I) compounds that combine a tissue-reactive moiety or click-chemistry-capable functional group with a defined Q group and a Formula (VIIA) W component, together with dependent limitations on R^m, R^q, R^r, and quinone methide precursor structure options.
Stated Advantages
Minimal visual cross-talk and faithful pattern replication in comparison to gold-standard DAB staining on serial sections.
Orthogonal reactivity for Cu(I)-free strain-promoted azide-alkyne cycloaddition (SPAAC) and TCO-tetrazine ligation.
Specified optical absorbance performance with narrow first-peak full width at half maximum (FWHM).
Documented Applications
Detecting cancers and infectious agents, including gene rearrangements, tumor suppressor gene deletions, multiple solid-tumor deletions, and virus or microorganism-derived nucleic acids.
In situ hybridization and immunohistochemistry.
Multiplex brightfield immunohistochemistry.
Immunohistochemical use of the conjugates to produce biological samples with stained targets.
Kits containing multiple conjugates for immunohistochemical use.
Staining one or more targets.
A biological sample is stained via an immunohistochemical assay using at least one of the conjugates.
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