Photocatalyst label induced by bioluminescence

Abstract

Disclosed herein are systems, methods, and compositions for bioluminescence-induced catalysis of bioorthogonal labeling chemistry in a proximity-dependent manner. In particular, bioluminescent proteins or complexes, their fluorophore substrates, photocatalysts or photosensitizers, activatable labels, and systems thereof, as well as methods for catalytically activating activatable labels via bioluminescence-induced catalysis are provided herein.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 338,322, filed May 4, 2022, which is incorporated herein by reference.

[0002] Provided herein are systems, methods, and compositions for catalysis induced by bioluminescence of a proximity - dependent in - vivo orthogonal labeling chemistry that can be operated within a biological system. In particular, bioluminescent proteins or complexes, their fluorophore substrates, photocatalysts, activatable labels, and their systems, as well as methods for catalytically activating an activatable label via bioluminescence - induced catalysis are provided herein.

Background Art

[0003] Due to the need to study dynamic microenvironments, signaling pathways, and molecular processes in a physiologically relevant context, there has been a demand for new functional biological tools that enable such analysis non - destructively in live cells and complex models.

Summary of the Invention

[0004] Provided herein are systems, methods, and compositions for catalysis induced by bioluminescence of a proximity - dependent in - vivo orthogonal labeling chemistry. In particular, bioluminescent proteins or complexes, their fluorophore substrates, photocatalysts, activatable labels, and their systems, as well as methods for catalytically activating an activatable label via bioluminescence - induced catalysis are provided herein.

[0005] In some embodiments, provided herein is a photo-driven photocatalyst that utilizes bioluminescence as a light source and uses it to activate a bioorthogonal label of a target molecule (e.g., a biopolymer) at a functional moiety for subsequent visualization, enrichment, or manipulation. Components of such a bioluminescence-driven photocatalyst system include a bioluminescence light source (e.g., a luminophore and luciferase or a bioluminescence complex), a pair comprising a photosensitive catalyst (photocatalyst), and an activatable label (e.g., a molecule comprising an activatable moiety and a functional moiety). In some embodiments, when exposed to a light stimulus from the bioluminescence light source, the excited catalyst participates in the activation of an adjacent activatable label, and subsequently, a highly reactive intermediate is generated that can be utilized to undergo covalent cross-linking with a biopolymer in the surrounding environment. Activation of the catalyst by absorption of visible light provides temporal control of the catalytic reactivity. The use of bioluminescence to cause photocatalysis in a proximity-dependent manner (e.g., requiring localization of the bioluminescence light source and the photocatalyst) provides a gentle and minimally disruptive light source for causing catalytic labeling in intact cells, reduced phototoxicity, and efficient light delivery, as well as spatial and temporal (+ luminophore) control over catalyst activation, thereby overall enhancing the spatiotemporal resolution of downstream covalent labeling chemistry. In some embodiments, the bioluminescence light source and the photocatalyst are bioconjugated to induce proximity between the light source and the catalyst.

[0006] One aspect of the present technology is the use of bioluminescence, which is light generated by the interaction of a bioluminescent protein or peptide(s) and / or a complex of polypeptides with a luminophore, for activating a photocatalyst. Other aspects of the present technology include the activation of an activatable label by a bioluminescence-activated photocatalyst, and the use of one or more conjugates of the components of the system herein that drive intracellular covalent labeling chemistry using spatiotemporally arranged components (e.g., via protein fusions, capture agents / elements, linkers, etc.) to assemble the components of the bioluminescence-driven system / method, thereby increasing specificity and reducing toxicity and the like.

[0007] In an exemplary embodiment, exposing a bioluminescent protein to a suitable luminophore generates light that induces local photocatalytic generation of reactive intermediates with a restricted diffusion radius. Those local reactive intermediates can form covalent bonds with adjacent residues for subsequent covalent modification of target molecules (e.g., biopolymers) by functional moieties. Such bioorthogonal labeling chemistry can be utilized for a wide range of spatiotemporally controlled phenotypic, proteomic, and genomic analyses, including interactome and chromatin mapping, modulation of protein-protein interactions, and targeted visualization / enrichment / engineering of proteins and nucleic acids.

[0008] In some embodiments, appropriate proximity between the bioluminescent protein and the photocatalyst is achieved by linking the photocatalyst to the bioluminescent protein (either directly or indirectly). In certain embodiments, the bioluminescent protein is made as a fusion with a capturer (e.g., a capture protein), and the photocatalyst is conjugated to the capture element (e.g., via a linker). Binding of the capturer to the capture element brings the bioluminescent protein and the photocatalyst into proximity, enabling light generated by the bioluminescent protein and the luminophore to activate the photocatalyst.

[0009] In some embodiments, instead of using a bioluminescent protein, a multi-component bioluminescent complex can be used as a light source for the photocatalytic systems or methods described herein. The use of a bioluminescent complex that produces significantly improved light output upon complementation of two or more components (e.g., peptide(s) and / or polypeptide(s)) provides several advantages to some of the systems and methods herein. For example, by conjugating (e.g., fusing, linking, etc.) one or more components of the bioluminescent complex directly or indirectly to other components of the system (e.g., photocatalyst, target, etc.), it is ensured that those components are in proximity to the bioluminescent complex during light generation. By linking two components of the system to separate components of the bioluminescent complex, it is ensured that those components are in proximity during light generation by the complex. When the photocatalyst is linked to a first component of the bioluminescent complex (e.g., LgBiT or circularly permuted LgBiT (see, e.g., U.S. Patent Application No. 17 / 105,925; incorporated by reference in its entirety)), the first component has a high affinity for a second component of the bioluminescent complex (e.g., HiBiT), and the second component is genetically fused to the target of interest, proximity between the photocatalyst and the second component of the bioluminescent complex is required for initiation of photocatalysis, thereby providing a higher spatiotemporal control over activation of the photocatalyst and an approach that does not rely on a modality for targeting the photocatalytic system to the site of interest (i.e., complementation and addition of a fluorophore).

[0010] In some embodiments, the components of the bioluminescent protein or multi-component bioluminescent complex are inserted into an internal position within a capture agent. In some embodiments, the position within the capture agent is selected to enhance the efficiency of bioluminescence activation of the catalyst by greater proximity or a favorable conformation.

[0011] In some embodiments, the components of the bioluminescent protein or multi-component bioluminescent complex are circularly permuted.

[0012] A bioluminescent protein, or a component of a bioluminescent complex (or a fusion thereof with other components of the systems herein), can be expressed intracellularly or delivered into cells, so such systems provide the generation of light to initiate photocatalytic action intracellularly.

[0013] In some embodiments, a component of the systems herein (e.g., a bioluminescent protein, or a component of a bioluminescent complex) is fused to a protein / peptide that results in specific localization of the component intracellularly. For example, the localization protein / peptide localizes within a cellular compartment, binds to a specific protein, binds to DNA or RNA, binds to a specific nucleic acid sequence, etc. By localizing the component intracellularly or binding the component to a specific cellular component, subsequent photocatalytic labeling chemistry is similarly localized. In some embodiments, by localizing the system to a specific cellular target (e.g., a protein, nucleic acid sequence, etc.), the activated label can react with or act on the cellular target.

[0014] The systems and methods herein provide bioluminescence-induced catalysis of bioorthogonal labeling chemistry in a proximity-dependent manner, providing new functional biological tools for studying dynamic environments and molecular processes, including live cells, complex cell models, and model organisms, in a physiologically relevant context. The techniques herein use a non-invasive endogenous light source to activate a photosensitive catalyst, which can further participate in the local generation of reactive intermediates that can form covalent bonds with adjacent biopolymers for subsequent modification by functional moieties. These bioorthogonal labeling chemistries can be utilized for a wide range of spatiotemporally controlled phenotypic, proteomic, and genomic analyses. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

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[0016] Definitions While it is possible to use any methods and materials similar or equivalent to those described herein when practicing or testing the embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, prior to describing these materials and methods, it should be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or procedures described herein, as certain molecules, compositions, methodologies, or procedures may vary according to routine experimentation and optimization. It should also be understood that the terms used in the description are for the purpose of describing only a particular version or embodiment and are not intended to limit the scope of the embodiments described herein.

[0017] 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 invention belongs. In case of conflict, the present specification, including definitions, will control. Accordingly, the following definitions apply in the context of the embodiments described herein.

[0018] As used herein, and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" refers to one or more peptides and equivalents thereof known to those of ordinary skill in the art.

[0019] As used herein, the term "and / or" includes any and all combinations of the individual listed items, including any one of the individually listed items. For example, "A, B, and / or C" includes A, B, C, AB, AC, BC, and ABC, each of which should be considered separately described with the specification of "A, B, and / or C".

[0020] As used herein, the term "comprising" and its linguistic variations mean the presence of the recited features(s), element(s), method step(s), etc., but do not preclude the presence of additional features(s), element(s), method step(s), etc. In contrast, the term "consisting of" and its linguistic variations mean the presence of the recited features(s), element(s), method step(s), etc., and exclude any unrecited features(s), element(s), method step(s), etc., other than the impurities that are normally associated therewith. The phrase "consisting essentially of" means, in addition to meaning the recited features(s), element(s), method step(s), etc., any additional features(s), element(s), method step(s), etc., that do not substantially affect the basic properties of the composition, system or method. Many embodiments herein are described using the non-limiting expression "comprising". Such embodiments include a plurality of limiting "consisting of" and / or "consisting essentially of" embodiments, which embodiments may alternatively be claimed or described using such expressions.

[0021] As used herein, the term "substantially" means that the recited characteristics, parameters, and / or values need not be achieved exactly, but that deviations or variations, including for example, tolerances, measurement errors, limits of measurement accuracy, and other factors known to those of ordinary skill in the art, may occur to the extent that they do not prevent the effects intended to be provided by the characteristics. A characteristic or feature that is substantially absent (e.g., substantially non-emissive) may be within noise, below background, below the detection capabilities of the assay being used, or a minor portion (e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%, <0.000001%, <0.0000001%) of a significant characteristic (e.g., the luminescence intensity of a bioluminescent protein or bioluminescent complex).

[0022] As used herein, the term "luminescence" refers to the emission of light by a substance as a result of a chemical reaction ("chemiluminescence") or an enzymatic reaction ("bioluminescence").

[0023] As used herein, the term "bioluminescence" refers to the production and emission of light by a reaction catalyzed by or made possible by an enzyme, protein, protein complex, or other biomolecule (e.g., a bioluminescence complex). In typical embodiments, a substrate for a bioluminescence entity (e.g., a bioluminescent protein or bioluminescence complex) is converted by the bioluminescence entity into an unstable form; subsequently, the substrate emits light.

[0024] As used herein, the term "lumophore" refers to a chemical moiety or compound that can be placed in an excited electronic state (e.g., by a chemical reaction or enzymatic reaction) and can emit light upon returning to the electronic ground state.

[0025] As used herein, the term "imidazopyrazine lumophore" refers to a class of lumophores that includes "native coelenterazine" as well as its syntheses (e.g., derivatives or variants) and natural analogs, including, in addition to those disclosed in WO2003 / 040100, U.S. Application No. 12 / 056,073 (paragraph

[0086] ), U.S. Patent No. 8,669,103, U.S. Provisional Application No. 63 / 379,573 (the disclosures of which are incorporated herein by reference in their entireties), frimazine, frimazine analogs (e.g., fluorofrimazine), coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine ("coelenterazine-hh"), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methylcoelenterazine.

[0026] As used herein, the term "coelenterazine" refers to a naturally occurring ("native") imidazopyrazine having the following structure. [Chemical]

[0027] As used herein, the term "furimazine" refers to a selenotriazine derivative having the following structure. [Chemical]

[0028] As used herein, the term "fluorofurimazine" refers to a furimazine derivative having the following structure [Chemical] (U.S. Application No. 16 / 548,214, which is incorporated herein by reference in its entirety).

[0029] As used herein, the term "luciferin" refers to a compound having the following structure. [Chemical]

[0030] As used herein, the term "bioluminescence resonance energy transfer" ("BRET") refers to a distance-dependent interaction in which energy is transferred from a donor bioluminescent protein / complex and a substrate to an acceptor molecule without emitting photons. The efficiency of BRET depends on the inverse sixth power of the intermolecular distance and is useful over distances on the order of the dimensions of biopolymers (e.g., within 30 - 80 Å depending on the degree of spectral overlap).

[0031] As used herein, the term "Oplophorus luciferase" ("OgLuc") refers to a luminescent polypeptide having significant sequence identity, structural conservation, and / or functional activity of luciferase produced by and derived from the deep-sea shrimp Oplophorus gracilirostris. Specifically, the OgLuc polypeptide has significant sequence identity, structural conservation, and / or functional activity of the 19 kDa mature subunit of the Oplophorus luciferase protein complex such as SEQ ID NO: 1 (NANOLUC), contains 10 β-strands (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10), and refers to a luminescent polypeptide that produces luminescence using a substrate such as coelenterazine or a derivative or analog of coelenterazine.

[0032] As used herein, the term "complementary" refers to the property of two or more structural elements (e.g., peptides, polypeptides, nucleic acids, small molecules, etc.) that can hybridize to each other, dimerize, or otherwise form a complex. For example, "complementary peptides and polypeptides" can together form one complex. Complementary elements may require assistance (facilitation) to form a complex (e.g., derived from interacting elements), such as placing the elements in an appropriate conformation for complementarity, jointly determining the positions of the complementary elements, lowering the interaction energy for complementarity, and overcoming low affinity for each other.

[0033] As used herein, the term "complex" refers to an aggregate or agglomerate of molecules (e.g., peptides, polypeptides, etc.) that are in direct and / or indirect contact with each other. In one aspect, "contacting" or more specifically "direct contact" means that two or more molecules are sufficiently close such that non-covalent interactions with attractive forces such as van der Waals forces, hydrogen bonds, ionic and hydrophobic interactions govern the interaction of the molecules. In such an aspect, a complex of molecules (e.g., peptides and polypeptides) is formed under assay conditions such that the complex is thermodynamically favorable (e.g., as compared to the non-aggregated or non-complexed state of its constituent molecules). As used herein, the term "complex" refers to an aggregate of two or more molecules (e.g., peptides, polypeptides, or combinations thereof), unless otherwise specified.

[0034] As used herein, the term "capture protein" or "capture agent" means a protein or other molecular element that forms a stable covalent bond upon interaction with its substrate, ligand, or other molecule. A capture protein can be a receptor that forms a covalent bond upon binding to its ligand, or an enzyme that forms a covalent bond with its substrate. An example of a capture protein suitable for use in embodiments of the present invention is the HALOTAG protein described in U.S. Patent No. 7,425,436, which is incorporated herein by reference in its entirety.

[0035] As used herein, the terms "capture ligand", "capture moiety", or "capture element" refer to ligands, substrates, etc. that form a covalent bond with a capture protein upon interaction with the capture protein. Examples of capture ligands suitable for use in embodiments of the present invention include, for example, the HALOTAG ligand described in U.S. Patent No. 7,425,436 (which is hereby incorporated by reference in its entirety). Portions found to be useful as HALOTAG ligands include haloalkane (HA) groups (e.g., chloroalkane (CA) groups). In embodiments described herein that specify an HA or CA capture ligand, other suitable capture ligands can be substituted unless otherwise specified.

[0036] As used herein, the term "activatable label" refers to a bifunctional molecule that includes an activatable moiety linked to a functional moiety. The functional moiety is suitable for detection or capture or subsequent ligation and functions as a label (e.g., fluorophore, chromophore, strained alkyne, haloalkane, biotin, etc.). The activatable moiety can be converted from an activatable form to a reactive (activated) form by a catalyst. In some embodiments, the activatable moiety is a photoreactive group. Examples of activatable moieties include phenyltrifluoro-methyldiazirine, phenyl azide, and psoralen. The activated form of the activatable moiety can form a covalent bond with a target molecule (e.g., a biopolymer), thereby labeling them with the functional moiety.

[0037] As used herein, the term "cellular target" refers to a cellular (e.g., intracellular or surface-exposed) entity (e.g., molecule, cellular compartment, complex, etc.) that can be labeled by the systems herein. Cellular targets can be macromolecules such as proteins, polypeptides, nucleic acids (e.g., DNA or RNA), lipids, polysaccharides, or polypeptides (s) containing any of these, such as complexes. Cellular targets can be composed of multiple components, subunits, or polypeptides, for example, a cellular target can be a protein complex. Examples of cellular targets can include receptors or enzymes.

[0038] As used herein, the term "bioactive agent" generally means a physiologically or pharmacologically active substance, or a substance suitable for detection. In some embodiments, the bioactive agent is a potential therapeutic compound (e.g., small molecule, peptide, nucleic acid, etc.), or a drug-like molecule. The bioactive agents for use in the embodiments described herein are not limited by size or structure.

[0039] As used herein, the term "photoreactive group" refers to a moiety that forms a covalent bond with a molecule or functional group in its vicinity (e.g., within a certain distance range (e.g., <120 nm, <110 nm, <100 nm, <90 nm, <80 nm, <70 nm, <60 nm, <50 nm, <40 nm, <30 nm, <20 nm, <10 nm, <5 nm, <4 nm, <3 nm, <2 nm, etc.)) when exposed to light (e.g., a specific wavelength or range of wavelengths of light, etc.).

[0040] As used herein, the term "photocatalyst" refers to a molecule that, upon absorption of light of an appropriate wavelength, participates in the activation of adjacent activatable label(s) via either an energy transfer or an electron transfer event, thereby reducing the activation energy and / or increasing the rate of a chemical labeling reaction. In some embodiments, the excited photocatalyst can regenerate itself after each energy transfer or electron transfer event, thereby repeatedly participating in the activation of adjacent activatable labels. In some embodiments, a photocatalyst that, upon absorption of light of an appropriate wavelength, participates in an energy transfer event with oxygen to generate reactive species (e.g., protons, singlet oxygen, etc.) for subsequent chemical modification of adjacent biopolymers is referred to as a "photosensitizer". Some embodiments of the present disclosure described in conjunction with photocatalysts may include or be limited to photosensitizers.

[0041] As used herein, the term "small molecule" refers to an organic compound having a molecular weight (e.g., <2000 Daltons, <1000 Daltons, <500 Daltons) and dimensions on the order of 1 nm (e.g., length, width, diameter, etc.). Larger structures such as peptides, proteins, and nucleic acids are not small molecules, although the monomers (ribo- or deoxyribonucleotides, amino acids, etc.) that make them up are considered small molecules.

[0042] As used herein, the term "cell permeable" refers to a compound or moiety that can effectively cross an unmodified cell membrane.

[0043] The definitions of certain functional groups and chemical terms are described in more detail below. For the purposes of the present disclosure, chemical elements are those identified in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 thIdentified according to Ed. (reply), and specific functional groups are generally defined as described therein. In addition, for organic chemistry and general principles of specific functional moieties and reactivity, see Sorrell, Organic Chemistry, 2 nd edition, University Science Books, Sausalito, 2006; Smith, March’s Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7 th Edition, John Wiley & Sons, Inc., New York, 2013; Larock, Comprehensive Organic Transformations, 3 rd Edition, John Wiley & Sons, Inc., New York, 2018; Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987, the entire contents of each of which are hereby incorporated by reference.

[0044] As used herein, the term “physiological conditions” refers to any conditions compatible with living cells, e.g., mainly aqueous conditions such as temperature, pH, salinity, chemical composition, etc. that are compatible with living cells.

[0045] As used herein, the terms “conjugated” and “conjugation” refer to a covalent bond (e.g., after synthesis and / or during synthetic production) between two molecular entities. The conjugated entity can be a peptide or protein “fused” by a peptide bond, or can include other molecular entities (e.g., nucleic acids, small molecules, etc.) linked directly or by a suitable linker.

[0046] The term "binding moiety" refers to a domain that specifically binds to an antigen or epitope, independently of a different epitope or antigen-binding domain. Binding moieties include antibodies, antibody fragments, receptor domains that bind to target ligands, proteins that bind to immunoglobulins (e.g., Protein A, Protein G, Protein A / G, Protein L, Protein M), binding domains of proteins that bind to immunoglobulins (e.g., Protein A, Protein G, Protein A / G, Protein L, Protein M), oligonucleotide probes, peptide nucleic acids, DARPins, anticalins, nanobodies, aptamers, affimers, purified proteins (the analyte itself or a protein that binds to the analyte), analyte-binding domain(s) of a protein, and the like. Table A provides a list of exemplary binding moieties that can be used alone or in various combinations in the methods, systems, and assays (e.g., immunoassays) of this specification. [Table 1]

[0047] As used herein, the term "antibody" refers to the whole antibody molecule or a fragment thereof (e.g., fragments such as Fab, Fab’, and F(ab’)2, variable light chain, variable heavy chain, Fv, etc.). Antibodies can be polyclonal or monoclonal or recombinant antibodies, chimeric antibodies, humanized antibodies, human antibodies, and the like. As used herein, when an antibody or other entity "specifically recognizes" or "specifically binds" to an antigen or epitope, the antibody or other entity preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules and binds to the antigen or epitope with a substantially higher affinity than other entities that do not present the antigen or epitope. In this regard, "substantially higher affinity" means an affinity that is high enough to enable the detection of an antigen or epitope that is distinguishable from the entity using the desired assay or measuring device. Typically, it is at least 10 7 M -1 (e.g., >10 7 M -1 、>10 8M -1 、 >10 9 M -1 、 >10 10 M -1 、 >10 11 M -1 、 >10 12 M -1 、 >10 13 M -1 etc.) binding constant (K a ) means a binding affinity. In certain such embodiments, the antibody can bind to different antigens as long as the different antigens contain their specific epitopes. In certain cases, for example, homologous proteins from different species may contain the same epitope.

[0048] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody that includes at least a portion of the antigen-binding region or variable region. Antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv, scFv, Fd, variable light chain, variable heavy chain, diabody, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, for example, Hudson et al. (2003) Nat. Med. 9:129-134 (which is hereby incorporated by reference in its entirety). In certain embodiments, the antibody fragment is generated by enzymatic or chemical cleavage of an intact antibody (e.g., papain digestion and pepsin digestion of the antibody) by recombinant DNA techniques or chemical polypeptide synthesis. For example, a "Fab" fragment includes one light chain and the C H1 and variable region. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab’" fragment includes one light chain and the C H1 domain and C H2It includes one heavy chain that contains an additional constant region extending between the domains. Inter-chain disulfide bonds can be formed between the two heavy chains of the Fab’ fragment to form an “F(ab’)2” molecule. The “Fv” fragment contains variable regions from both the heavy and light chains but lacks the constant regions. The single-chain Fv (scFv) fragment contains the heavy and light chain variable regions connected by a flexible linker and forms a single polypeptide chain together with the antigen-binding region. Exemplary single-chain antibodies are discussed in detail in WO88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, which are incorporated herein by reference in their entireties. In certain cases, a single variable region (e.g., the heavy chain variable region or the light chain variable region) may have the ability to recognize and bind an antigen. Other antibody fragments will be understood by those skilled in the art.

[0049] As used herein, the terms “biomolecule” or “biological molecule” mean molecules and ions that are present in living organisms and are essential for biological processes (plural) such as cell division, morphogenesis, or development. Biomolecules include large macromolecules (or polyanions) such as proteins, carbohydrates, lipids, and nucleic acids, as well as small molecules such as primary metabolites, secondary metabolites, and natural products. A more general name for this class of substances is biotic substances. Biomolecules are usually endogenous, but can also be exogenous. For example, a drug can be a natural product, a semi-synthetic (biopharmaceutical), or completely synthetic.

[0050] As used herein, the term “alkyl” means from 1 to 30 carbon atoms, for example, from 1 to 16 carbon atoms (C1-C 16 alkyl), from 1 to 14 carbon atoms (C1-C 14 alkyl), from 1 to 12 carbon atoms (C1-C 12 alkyl), from 1 to 10 carbon atoms (C1-C 10 alkyl), from 1 to 8 carbon atoms (C1-C8 alkyl), from 1 to 6 carbon atoms (C1-C6 alkyl), from 1 to 4 carbon atoms (C1-C4 alkyl), from 6 to 20 carbon atoms (C6-C 20(alkyl), or a straight-chain or branched-chain saturated hydrocarbon chain containing 8 to 14 carbon atoms (C8-C 14 means an alkyl group. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0051] As used herein, the term "amino" means an -NH2 group.

[0052] As used herein, the term "halogen" or "halo" means F, Cl, Br, or I. As used herein, the term "haloalkyl" means an alkyl group as defined herein in which at least one hydrogen atom (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms) is replaced by a halogen. In some embodiments, each hydrogen atom of the alkyl group is replaced by a halogen. Representative examples of haloalkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, and 3,3,3-trifluoropropyl.

[0053] As used herein, the term "heteroalkyl" means an alkyl group as defined herein in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced by a heteroatom group such as -NR-, -O-, -S-, -S(O)-, -S(O)2-, and the like, where R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl, or heterocyclyl, each of which may optionally be substituted. By way of example, one, two, or three carbon atoms may each independently be replaced by the same or different heteroatom groups. Examples of heteroalkyl groups include, but are not limited to, -OCH3, -CH2OCH3, -SCH3, -CH2SCH3, -NRCH3, and -CH2NRCH3, where R is hydrogen, alkyl, aryl, arylalkyl, heteroalkyl, or heteroaryl, each of which may optionally be substituted. Heteroalkyl also includes groups in which the carbon atoms of the alkyl are oxidized (i.e., -C(O)-).

[0054] As used herein, the term "alkenyl" means a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond. The double bond(s) can be located at any position within the hydrocarbon chain. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

[0055] As used herein, the term "alkynyl" means a straight or branched hydrocarbon chain containing at least one carbon-carbon triple bond. The triple bond(s) can be located at any position within the hydrocarbon chain. Representative examples of alkynyl include, but are not limited to, ethynyl, propynyl, and butynyl.

[0056] As used herein, the term "alkylene" means a divalent alkyl radical (e.g., -CH2CH2-). As used herein, the term "alkenylene" means a divalent alkenyl radical (e.g., -CH = CH-). As used herein, the term "alkynylene" means a divalent alkynyl radical (e.g., -C≡C-).

[0057] As used herein, the term "alkoxy" means an alkyl group as defined herein attached to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0058] As used herein, the term "amino" means the -NH2 group.

[0059] As used herein, the term "aminoalkyl" means an alkyl group as defined herein in which at least one hydrogen atom is replaced by an amino group as defined herein. Representative examples of aminoalkyl include, but are not limited to, aminomethyl, 2-aminoethyl, 2-aminopropyl, 3-aminopropyl, and 4-aminobutyl.

[0060] As used herein, the term "cyano" means the -CN group.

[0061] As used herein, the term "cyanoalkyl" means an alkyl group as defined herein in which at least one hydrogen atom is replaced by a cyano group as defined herein. Representative examples of cyanoalkyl include, but are not limited to, cyanomethyl, 2-cyanoethyl, 2-cyanopropyl, 3-cyanopropyl, and 4-cyanobutyl.

DETAILED DESCRIPTION OF THE INVENTION

[0062] Provided herein are systems, methods, and compositions for bioluminescence-induced catalysis of bioorthogonal chemistry in a proximity-dependent manner. In particular, provided herein are bioluminescent proteins or complexes, their luminophore substrates, photocatalysts or photosensitizers, activatable labels, and systems thereof, as well as methods for catalytically activating an activatable label via bioluminescence-induced catalysis.

[0063] Due to the need to study dynamic microenvironments, signaling pathways, and molecular processes in a physiologically relevant context, there is a need for new functional biological tools that enable such analysis non-destructively in live cells and complex models. Bioluminescence-induced catalysis of bioorthogonal labeling chemistry in a proximity-dependent manner provides a solution to this need by utilizing a non-invasive endogenous light source to drive bioorthogonal chemical labeling reactions in biological systems, followed by proximity-dependent modification of biomacromolecules by functional moieties. The components of such a photocatalytic system include a bioluminescent light source (e.g., luciferase or a bioluminescent complex (e.g., NanoBiT)), and pairs of (1) a photosensitive catalyst (transition metal or organic dye catalyst) and (2) an activatable label. Addition of a luminophore substrate causes the bioluminescent entity (e.g., NanoBiT, NanoLuc, etc.) to generate light that induces local photocatalytic generation of a reactive intermediate with a restricted diffusion range, thereby enabling the formation of covalent bonds with adjacent residues for subsequent modification by a functional moiety of a target molecule (e.g., a biomacromolecule). These bioorthogonal labeling chemistries can be utilized for a wide range of spatio-temporally controlled phenotypic, proteomic, and genomic analyses, including interactome and chromatin mapping, regulation of protein-protein interactions, and targeted visualization / enrichment / engineering of proteins and nucleic acids.

[0064] Advantages of using bioluminescence rather than all-optical emission (e.g., LED or laser) as a light source include the use of an endogenous light source that is gentle and minimally disruptive, reduction of phototoxicity, efficient light delivery for catalysis in intact cells and complex models, local and conditional (+lumophore substrate) light delivery for higher spatiotemporal resolution for catalytic activation and downstream labeling chemistry, and the ability to link the light source to target molecules and / or other components of the system (e.g., photocatalyst).

[0065] In some embodiments, provided herein are systems that include one or more of a structurally complementary component of a bioluminescent protein or bioluminescent complex, a lumophore (wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the lumophore by interaction with the lumophore), a photocatalyst (wherein the photocatalyst is activated upon absorption of light of the first wavelength), and an activatable label (wherein the activatable label is converted to an activated label upon proximity to the activated photocatalyst).

[0066] In some embodiments, a component of a bioluminescent protein or bioluminescent complex is linked to a photocatalyst. In some embodiments, the linkage of the photocatalyst to the light source provides appropriate proximity for activating the photocatalyst.

[0067] Bioluminescent protein or complex The present disclosure includes materials and methods related to bioluminescent polypeptides, bioluminescent complexes, and their components. Specifically, light emitted from a bioluminescent protein or complex (or from a lumophore acted upon by a bioluminescent protein or complex) is used to activate a photocatalyst.

[0068] NanoLuc In some embodiments, the systems and methods herein include a bioluminescent protein. In some embodiments, the bioluminescent protein is a luciferase enzyme. Suitable luciferase enzymes include those selected from the group consisting of: Photinus pyralis or North American firefly luciferase, Luciola cruciata or Japanese firefly or Genji firefly luciferase, Luciola italic or Italian firefly luciferase, Luciola lateralis or Japanese firefly or Heike luciferase, N. nambi luciferase, Luciola mingrelica or Eastern European firefly luciferase, Photuris pennsylvanica or Pennsylvania firefly luciferase, Pyrophorus plagiophthalamus or Comet beetle luciferase, Phrixothrix hirtus or railroad worm luciferase, Renilla reniformis or wild-type Renilla luciferase, Renilla reniformis Rluc8 mutant Renilla luciferase, Renilla reniformis green fluorescent Renilla luciferase, Gaussia princeps wild-type Gaussia luciferase, Gaussia princeps Gaussia-Dura luciferase, Cypridina noctiluca or Cypridina luciferase, Cypridina hilgendorfii or Cypridina or Vargula luciferase, Metridia longa or Metridia luciferase, TurboLuc (Auld et al. Biochemistry 2018, 57, 31, 4700-4706: incorporated by reference in its entirety), NanoLuc (Suzuki et al.Nature Communications volume 7, article number: 13718 (2016); which is incorporated by reference in its entirety), and Oplophorus luciferase (e.g., Oplophorus gracilirostris (OgLuc luciferase), Oplophorus grimaldii, Oplophorus spinicauda, Oplophorus foliaceus, Oplophorus noraezeelandiae, Oplophorus typus, Oplophorus noraezelandiae, or Oplophorus spinous).

[0069] In some embodiments, the bioluminescent protein is the luciferase of Oplophorus gracilirostris, NanoLuc® luciferase (Promega Corporation, U.S. Patent No. 8,557,970, U.S. Patent No. 8,669,103; which are incorporated by reference in their entirety herein). PCT Application No. PCT / US2010 / 033449, U.S. Patent No. 8,557,970, PCT Application No. PCT / 2011 / 059018 and U.S. Patent No. 8,669,103 (each of which is incorporated by reference in its entirety herein for all purposes) describe compositions and methods comprising bioluminescent polypeptides. Such polypeptides find use in the embodiments herein and can be used in conjunction with the compositions, assays, devices, systems, and methods described herein. In some embodiments, the compositions, assays, devices, systems, and methods provided herein comprise a bioluminescent polypeptide having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range therebetween) sequence identity to SEQ ID NO: 1. In some embodiments, any of the aforementioned bioluminescent proteins are bound (e.g., fused, chemically conjugated, etc.) to one or more other components of the assays and systems described herein (e.g., fused to a HALOTAG protein).

[0070] In some embodiments, the bioluminescent protein is a circularly permuted version of a native or modified bioluminescent protein (see, e.g., U.S. Patent No. 10,774,364; which is incorporated by reference in its entirety).

[0071] In some embodiments, the systems and methods herein include a bioluminescent complex (e.g., two or more components (e.g., peptides and / or polypeptides) that combine by structural complementarity to form a complex capable of activating a lumophore to emit light). In some embodiments, the lumophore emits significantly more light in the presence of the bioluminescent complex than in the presence of any one of the components alone. In some embodiments, the bioluminescent complex is formed from fragments of a luciferase enzyme (e.g., peptides and / or polypeptides). In some embodiments, the bioluminescent complex is a circularly permuted version of a native or modified bioluminescent component (e.g., formed from two circularly permuted fragments of luciferase) (see, e.g., U.S. Patent No. 10,774,364; which is incorporated by reference in its entirety).

[0072] PCT Applications PCT / US14 / 26354, PCT / US19 / 036844, and PCT / US20 / 62499, U.S. Patent No. 9,797,889, U.S. Patent Application No. 16 / 439,565, and U.S. Publication No. 2021 / 0262941 (each incorporated by reference in its entirety for all purposes herein) describe compositions and methods for constructing bioluminescent complexes, and such complexes, as well as their peptide and polypeptide components, find use in the embodiments herein and can be used in combination with the assays and methods described herein.

[0073] In some embodiments, the peptide and polypeptide components are provided for the construction of a bioluminescent complex that can produce luminescence in the presence of a suitable substrate (e.g., coelenterazine or a coelenterazine analog such as furimazine, fluorofurimazine, etc.). In some embodiments, the complementary polypeptide(s) and peptide(s) collectively span the length of the luciferase base sequence (or >75% of its length, >80% of its length, >85% of its length, >90% of its length, >95% of its length, or more) (or have at least 40% sequence identity to the luciferase base sequence (e.g., including >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, or more). In some embodiments, the "complementary" polypeptide(s) and peptide(s) are separate molecules each corresponding to a portion of the luciferase base sequence. Due to structural complementarity, they come together to form a bioluminescent complex. Suitable luciferase base sequences can include SEQ ID NO: 1 or 2, or the sequence of any of the full-length luciferases listed above. In some embodiments, the bioluminescent complex includes the NANOBIT or NANOTRIP system (Promega; Madison, WI). In some embodiments, the peptide and / or polypeptide components of the bioluminescent complex collectively have at least 60% sequence identity (e.g., >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >99%) with SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, the peptide and / or polypeptide components of the bioluminescent complex include HIBIT (SEQ ID NO: 3), SMBIT (SEQ ID NO: 4), LGBIT (SEQ ID NO: 5), LGTRIP (SEQ ID NO: 6), and / or SMTRIP9 (SEQ ID NO: 7).In some embodiments, the peptide and / or polypeptide components of the bioluminescent complex have at least 60% sequence identity (e.g., >60%, >65%, >70%, >75%, >80%, >85%, 90%, >95%, >99%) with HIBIT (SEQ ID NO: 3), SMBIT (SEQ ID NO: 4), LGBIT (SEQ ID NO: 5), LGTRIP (SEQ ID NO: 6), and / or SMTRIP9 (SEQ ID NO: 7). In some embodiments, the peptide and / or polypeptide components of the bioluminescent complex include circularly permuted variants of HIBIT (SEQ ID NO: 3), SMBIT (SEQ ID NO: 4), LGBIT (SEQ ID NO: 5, e.g., cp site 67 / 68 (e.g., with or without the substitutions E4D, Q42M, M106K, and / or T144D), LGTRIP (SEQ ID NO: 6, e.g., cp site 67 / 68, cp site 49 / 50, etc.), and / or SMTRIP9 (SEQ ID NO: 7).

[0074] In some embodiments, any of the foregoing components of the bioluminescent complex are linked (e.g., fused, chemically conjugated, ligated, etc.) to one or more other components of the assays and systems described herein (e.g., fused to a HALOTAG protein).

[0075] There are various features of the bioluminescent complexes found to have uses in embodiments herein that can provide advantages in certain applications. For example, a bioluminescent complex (e.g., a complex formed by complementation of HiBiT / LgBiT) generates light only by complementation of its component peptides / polypeptides. Thus, by conjugating (e.g., fusing, linking, etc.) one or more components of the bioluminescent complex directly or indirectly to other components of the system (e.g., a photocatalyst, an activatable label, a target, etc.), proximity of that component to the bioluminescent complex during light generation is ensured. To separate the components of the bioluminescent complex, by linking two other components of the system (e.g., a photocatalyst and a target binder), proximity of these components during light generation by the complex is ensured. In some embodiments, the use of the bioluminescent complex provides improved spatio-temporal resolution via conditional activation at a specific site because two components need to come together to form the complex.

[0076] In some embodiments, the components of a bioluminescent protein or multi-component bioluminescent complex are inserted into an internal position within a capture agent. In some embodiments, the position within the capture agent is selected to enhance the efficiency of bioluminescent activation of the catalyst by virtue of higher proximity or a favorable conformation.

[0077] In some embodiments, the components of a bioluminescent protein or multi-component bioluminescent complex are rearranged in a cyclic manner.

[0078] Lumophore substrate In some embodiments, the systems and methods herein include a lumophore substrate that emits light upon interaction with the bioluminescent proteins and / or complexes described herein. A lumophore suitable for use with the bioluminescent proteins or complexes used in the present system or method will be understood. For example, firefly luciferin having the following structure

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0079] The pairing of a suitable bioluminescent protein or complex with a lumophore is understood in the art. In certain embodiments, bioluminescent proteins that utilize imidazopyrazine lumophores such as coelenterazine, furimazine, or fluorofurimazine are provided in the systems or methods herein (U.S. Application No. 16 / 548,214, which is incorporated by reference in its entirety). In some embodiments, the system or method comprises (1) a polypeptide from Oplophorus (e.g., NANOLUC) or a component of a bioluminescent complex from Oplophorus (e.g., NANOBIT, NANOTRIP), and an imidazopyrazine lumophore (e.g., coelenterazine, furimazine, fluorofurimazine, etc.). In some embodiments, the systems and methods herein include, in addition to those disclosed in WO2003 / 040100, U.S. Application No. 12 / 056,073 (paragraph

[0086] ) and U.S. Patent No. 8,669,103 (the disclosures of which are incorporated herein by reference in their entireties), imidazopyrazine lumophores such as native coelenterazine, furimazine, fluorofurimazine, coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine (“coelenterazine-hh”), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methylcoelenterazine.

[0080] In some embodiments, the fluorophore emits light by interacting with a bioluminescent protein or complex. In some embodiments, the fluorophore emits light in the visible light spectrum (e.g., from about 400 to about 700 nm (e.g., 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, or ranges therebetween)). In some embodiments, the fluorophore emits light at wavelengths between 400 and 500 nm (e.g., 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, or ranges therebetween).

[0081] Photocatalyst In some embodiments, the systems and methods herein include a photocatalyst that can absorb light emitted from a fluorophore (by interaction with a bioluminescent protein or complex) and then activate an adjacent activatable label. Any compound or moiety that receives the light energy emitted from the bioluminescent protein or complex-activated fluorophore and can then participate in the activation of the activatable label can be utilized in the embodiments herein. In some embodiments, the excited photocatalyst participates in the activation of an adjacent activatable label by Förster resonance energy transfer, Dexter energy transfer, single electron transfer, or any other suitable energy or electron transfer mechanism.

[0082] In some embodiments, the photocatalyst is an iridium-based or ruthenium-based photocatalyst (Bevernaegie et al., “A Roadmap Towards Visible Light Mediated Electron Transfer Chemistry with Iridium(III) Complexes,” ChemPhotoChem 2021, 5, 217; Day et al., “Advances in Photocatalysis: A Microreview of Visible Light Mediated Ruthenium and Iridium Catalyzed Organic Transformations,” Org. Process Res. Dev. 2016, 20, 1156 - 1163, incorporated herein by reference in their entireties). In some embodiments, the photocatalyst has the structure of formula (I); [Chemical formula] [wherein, each series of dashed lines [Chemical formula] represents the presence or absence of a fused six-membered ring, M is a transition metal, m1, m2, m3, n1, n2, n3, p1, p2, and p3 are each independently 0, 1, or 2, R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 3a , R 3b , and R 3c are each independently selected from halo, alkyl, haloalkyl, amino, heteroalkyl, and group-linker-Q, where Q is a capture element, X 1a , X 1b , X 2a , X 2b , X 3a , and X 3bEach is independently selected from N and C, where X 1a and X 1b at least one of which is N, X 2a and X 2b at least one of which is N, X 3a and X 3b at least one of which is N, X 1c , X 1d , X 2c , X 2d , X 3c and X 3d are each independently selected from CH and N, Z is an anion, q is 0, 1, or 2].

[0083] In some embodiments, the photocatalyst comprises a transition metal selected from Ru and Ir.

[0084] In some embodiments, the photocatalyst is an iridium-based photocatalyst selected from:

Chemical formula

[0085] In some embodiments, the photocatalyst is a ruthenium-based photocatalyst selected from:

Chemical formula

[0086] In some embodiments, M is Ru. In some embodiments, M is Ir.

[0087] In some embodiments, m2, n2, and p2 are each 0, and each series of dashed lines represents the absence of a fused 6-membered ring, i.e., the compound has the following formula: [Chemical Formula]

[0088] In some embodiments, X 1a is N, X 1b is C, X 2a is N, X 2b is C, X 3a is C, X 3b is N. In some embodiments, X 1a is N, X 1b is C, X 2a is N, X 2b is C, X 3a is N, X 3b is N.

[0089] In some embodiments, X 1c , X 1d , X 2c , X 2d , X 3c , and X 3d are each CH. In some embodiments, X 1c , X 1d , X 2c , X 2d , X 3c , and X 3d are each N.

[0090] In some embodiments, R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 3a , R 3b , and R 3c are each independently selected from fluoro, methyl, tert-butyl, trifluoromethyl, and group-linker-Q. In some embodiments, R 1a , R 1b , R1c , R 2a , R 2b , R 2c , R 3a , R 3b , and R 3c One or less of them is a group-linker-Q.

[0091] In some embodiments, the compound comprises one group-linker-Q, wherein Q is a capture element. In some embodiments, the capture element is an "affinity molecule" and the corresponding capture agent is an "acceptor" (e.g., a small molecule, protein, antibody, etc.) that selectively interacts with the affinity molecule. Examples of such pairs include an antigen as the capture element and an antibody as the capture agent, a small molecule as the capture element, and a protein (e.g., streptavidin and biotin) that has a high affinity for the small molecule as the capture agent.

[0092] In some embodiments, Q is a substrate of a dehalogenase, such as haloalkane dehalogenase. Systems comprising a mutant hydrolase (e.g., mutant dehalogenase) that covalently binds to a substrate (e.g., haloalkyl substrate) are described, for example, in U.S. Patent Nos. 7,238,842, 7,425,436, 7,429,472, 7,867,726 (each of which is incorporated herein by reference in its entirety). For example, HALOTAG is a commercially available modified dehalogenase enzyme that forms a stable (e.g., covalent) bond (e.g., an ester bond) with a haloalkyl substrate, and this is used in the embodiments herein.

[0093] In some embodiments, Q has the formula -(CH2) n -Y, wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and Y is a halogen (i.e., F, Cl, Br, or I). In some embodiments, n is 4, 5, 6, 7, or 8, and Y is Cl. In some embodiments, n is 6 and Y is Cl, such that Q has the formula -(CH2)6-Cl.

[0094] The linker includes various combinations of such groups, including ester (-C(O)O-), amide (-C(O)NH-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), phenylene (e.g., 1,4-phenylene), linear or branched alkylene, and / or oligo- and poly-ethylene glycol (-(CH2CH2O) x -), and can provide a linker having a bond such as this. In some embodiments, the linker can include two or more atoms (e.g., 2 to 200 atoms, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 atoms, or any range therebetween (e.g., 2 to 20, 5 to 10, 15 to 35, 25 to 100, etc.)). In some embodiments, the linker includes a combination of an oligoethylene glycol bond and a carbamate bond. In some embodiments, the linker has the formula -O(CH2CH2O) z1 -C(O)NH-(CH2CH2O) z2 -C(O)NH-(CH2) z3 -(OCH2CH2) z4 O-, where z1, z2, z3, and z4 are each independently selected from 0, 1, 2, 3, 4, 5, and 6. For example, in some embodiments, the linker has a formula selected from the following.

Chemical formula

[0095] In some embodiments, q is 0, 1, or 2. One of ordinary skill in the art will recognize that the value of q depends on the selection of other variables and is selected to balance the total charge at the remainder of the molecule. For example, if the total charge of the metal-based portion of the molecule is +1, in some embodiments, q is 1 and A is a monovalent anion (e.g., halide or hexafluorophosphate). In some embodiments, if the total charge of the metal-based portion of the molecule is +2, in some embodiments, q is 2 and A is a monovalent anion (e.g., halide or hexafluorophosphate).

[0096] An exemplary photocatalyst linked to a HALOTAG substrate is shown in FIG. 5. Alternative positions for binding to the photocatalyst, other photocatalysts, different linkers and linker lengths, etc. are understood to be within the scope of this specification.

[0097] In some embodiments, the photocatalyst is an organic photoredox catalyst. In some embodiments, the organic photoredox catalyst is selected from quinone, pyrylium, acridinium, and xanthene.

[0098] In some embodiments, the photocatalyst is a quinone-based organic photoredox catalyst selected from the following.

Chemical formula

[0099] In some embodiments, the photocatalyst is a pyrylium-based organic photoredox catalyst selected from the following.

Chemical formula

[0100] In some embodiments, the photocatalyst is an acridinium-based organic photoredox catalyst selected from the following.

Chemical formula

[0101] In some embodiments, any suitable position in the above photocatalyst structure can be used as the binding site for linker-Q.

[0102] In some embodiments, the photocatalyst is a thiazine-based organic photoredox catalyst selected from the following, [Chemical formula] wherein R is the binding site for linker-Q. In some embodiments, R is amine, carboxyl, tert-butyl, tert-butyl-methoxy, ether, hydroxyl, PEG, etc.

[0103] In some embodiments, the photocatalyst (e.g., a quinone-based, pyrylium-based, acridinium-based, xanthene-based, or thiazine-based photoredox catalyst) is conjugated to linker-Q. In some embodiments, the linker (e.g., linker-Q) is bound to the photocatalyst at any appropriate position on the photocatalyst structure. In some embodiments, positions suitable for binding of the photocatalyst are understood in the art.

[0104] Activatable label In some embodiments, the systems and methods herein include activatable labels that, when acted upon by an excited photocatalyst, generate reactive intermediates that can form covalent bonds with adjacent biopolymers (e.g., attaching an activatable group to another entity, attaching a reactive moiety to another entity, etc.). Embodiments herein are not limited by the chemical mechanism of molecular activation. In some embodiments, the activatable label is a bifunctional molecule that includes an activatable moiety and a functional moiety (e.g., a detectable moiety, a capture ligand, a handle, etc.).

[0105] In some embodiments, the excited photocatalyst transfers energy or electrons to the activatable label. In some embodiments, the photocatalyst transfers energy to the activatable label by Förster resonance energy transfer, Dexter energy transfer, single electron transfer, singlet oxygen, or any other suitable energy transfer or electron transfer mechanism.

[0106] In some embodiments, the photocatalyst facilitates the extraction of hydrogen from the activatable label. The extraction of a hydrogen atom is a chemical reaction in which a hydrogen free radical is extracted from the substrate (activatable label) and taken up by the photocatalyst or photosensitizer. In such a reaction, the photoactivation of the photocatalyst or photosensitizer results in the loss of a hydrogen free radical, thereby activating the photocatalyst or photosensitizer to extract hydrogen from the activatable label and returning the photocatalyst or photosensitizer to an inactive state. By the extraction of hydrogen, the activatable label is converted to an activated label.

[0107] In some embodiments, the transfer of energy or electrons to the activatable label converts an inactive activatable moiety to a reactive activated moiety.

[0108] In some embodiments, converting an activatable label to an activated label involves catalyzing a redox reaction using an activatable label as a substrate of the reaction. In such embodiments, a photocatalyst or photosensitizer absorbs light and is raised to a redox-active or excited state. As a result, the photocatalyst or photosensitizer can catalyze a redox reaction to activate the activatable label.

[0109] In some embodiments, the activatable label includes a photoreactive group. In some embodiments, upon activation of the photoreactive group by an excited photocatalyst, the activated label covalently binds to a target molecule. In some embodiments, the target molecule is a protein or a nucleic acid.

[0110] In some embodiments, the photoreactive group includes:

Chemical formula

Chemical formula

[0111] In some embodiments, the activatable benzyl diazirine variant is selected from the following,

Chemical formula

[0112] In other embodiments, the photoreactive group is

Chemical formula

[0113] In other embodiments, the photoreactive group is

Chemical formula

Chemical formula

[0114] In some embodiments, the activatable label is a compound of formula (I) [Chemical formula] or a salt thereof, wherein A is a photoreactive group and is selected from the following, [Chemical formula] wherein each n is independently 1, 2, 3, or 4, each R is independently selected from hydrogen, halo, C1-C4 alkyl, C2-C4 alkenyl, hydroxy, mercapto, amino, cyano, C1-C4-alkoxy, halo-C1-C4-alkyl, hydroxy-C1-C4-alkyl, amino-C1-C4-alkyl, mercapto-C1-C4-alkyl, cyano-C1-C4-alkyl, -C(O)-C1-C4-alkyl, -C(O)OH, and -C(O)NH2, Q is CH or N, G is -N3, -CH=CH-N3, or [Chemical formula] and Z is -CR 7 =CR 8 -C(X)-, -C(X)-, and a bond, wherein R 7 and R 8 are each independently hydrogen or C1-C4 alkyl, and X is O or S, L is a linker, Y is a functional moiety.

[0115] The compound of formula (I) or the group G in the PRG herein is or comprises an azide or diazirine moiety attached to either a phenyl or naphthyl group. Thus, the compound, upon exposure to light, generates a reactive group that can react with a biomolecule to effect covalent modification of the biomolecule by the compound of formula (I). For example, an aryl azide can undergo photoinduced activation to form a reactive nitrene group, and an aryl diazirine can undergo photoinduced activation to form a reactive carbene species.

[0116] In some embodiments, A is selected from:

Chemical formula

Chemical formula

[0117] In some embodiments, A is a group of the following formula,

Chemical formula

[0118] In some embodiments, A is a group of the following formula,

Chemical formula

[0119] In some embodiments, A has a formula selected from the following.

Chemical formula

[0120] In some embodiments, Z is -C(X)-. In some embodiments, Z is -C(O)-. In some embodiments, Z is a bond.

[0121] In some embodiments, Z is selected from -C(O)- and -CR 7 =CH-C(O)-, wherein R 7 is hydrogen or methyl.

[0122] The linker may include one or more groups independently selected from methylene (-CH2-), ethylene (-CH=CH-), ethynylene (-C≡C-), ether (-O-), amine (-NR-) (wherein R is hydrogen or an alkyl group), thioether (-S-), carbonyl (-C(O)-), thiocarbonyl (-C(S)-), sulfonyl (-S(O)2-), arylene, heteroarylene and heterocyclylene moieties, or any combination thereof. For example, by combining the above moieties, additional groups that may be included in the linker can be formed. For example, a carbonyl group and an ether group together can result in an ester moiety (-C(O)O-), a carbonyl group and two ether groups together can result in a carbonate moiety (-OC(O)O-), a carbonyl group and an unsubstituted amine group together can result in an unsubstituted amide moiety (-C(O)NH-), a carbonyl group and two unsubstituted amine groups together can result in an unsubstituted urea moiety (-NHC(O)NH-), a carbonyl group together with an unsubstituted amine group and an ester group can provide an unsubstituted carbamate moiety (-OC(O)NH-), a carbonyl group together with a thioether and an unsubstituted amine group can result in an S-thiocarbamate moiety, a thiocarbonyl group together with an ether and an unsubstituted amine group can result in an O-thiocarbamate moiety, and a plurality of methylene groups together can form an alkylene chain, and so on. In some embodiments, the linker includes one or more methylene, ether, ester, amide, carbamate, carbonate, urea, thioether, thioester, thioamide, thiocarbamate, thiocarbonate, thiourea, arylene, heteroarylene, or heterocyclylene moieties, or any combination thereof. In some embodiments, the linker includes one or more -CH2-, -O-, -C(O)O-, -C(O)NH-, -NHC(O)O-, -OC(O)O-, -NHC(O)NH-, -S-, -C(O)S-, -C(S)NH-, -NHC(S)O-, -OC(S)O-, -NHC(S)NH-, arylene, heteroarylene or heterocyclylene moieties, or any combination thereof.

[0123] In some embodiments, the linker comprises one or more moieties selected from straight-chain or branched alkylene, -O-, -NH-, -C(O)O-, -C(O)NH-, -NHC(O)O-, -NHC(O)NH-, and phenylene groups. In some embodiments, the linker comprises one or more moieties selected from straight-chain or branched alkylene, -O-, and -NH- groups. In some embodiments, the linker comprises one or more ethylene glycol units (-CH2CH2O-).

[0124] In some embodiments, the linker has the following formula, -NHCH2CH2(OCH2CH2) n NH- wherein n is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8.

[0125] The group Y in the compound of formula (I) is a functional moiety such as a capture element, a detectable moiety, or a reactive moiety. In some embodiments, Y is a capture element, which is a moiety such as a ligand or a substrate that forms a covalent or non-covalent bond with a protein (the "capture protein") upon interaction with the protein. In some embodiments, the capture element is a HALOTAG ligand, which is described, for example, in U.S. Patent No. 7,425,436, which is incorporated herein by reference in its entirety. Moieties found to be useful as HALOTAG ligands include haloalkane (HA) groups (e.g., chloroalkane (CA) groups). For example, in some embodiments, Y is of the formula -(CH2) n-A, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and A is a halogen (e.g., chloro). In such embodiments, the corresponding capture protein is a HALOTAG protein, which is described, for example, in U.S. Patent No. 7,425,436. Another example of a capture element is biotin. For example, in some embodiments, Y has the following formula.

Chemical formula

[0126] In some embodiments, Y is a detectable moiety such as a fluorescent moiety. Suitable fluorescent functional groups include, but are not limited to: xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, oxazine 170, etc.), acridine derivatives (e.g., proflavine, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porphyrin, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), dipyrromethene boron (BODIPY dyes, Invitrogen), ALEXA FLUOR (Invitrogen), DYLIGHT FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dyes (CYANDYE, LLC), SETAU AND SQUARE DYES (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT DYES (APC, RPE, PerCP, Phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate), quantum dot nanocrystals, etc.

[0127] In some embodiments, Y includes a fluorescence generating functional group, which produces an enhanced fluorescence signal upon association with a target (e.g., upon binding of a protein to a moiety conjugated to the fluorescence generating functional group). Upon binding of the target, a significantly increased fluorescence (e.g., 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or more) is produced, reducing the background signal. Exemplary fluorescence generating dyes for use in the embodiments herein include fluorophores of the JANELIA FLUOR family such as: JANELIA FLUOR 549:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0128] In some embodiments, Y includes a reactive functional group, which can undergo further reaction with a corresponding reactive moiety on another molecule and affect covalent bonding. For example, in some embodiments, Y includes a group selected from azide, alkyne, alkene, or 1,2,4,5-tetrazinyl moieties, all of which generally undergo copper-catalyzed or copper-free "click" reactions (e.g., the reaction of azide with alkyne, the reaction of azide with difluorocyclooctyne, or the reaction of 1,2,4,5-tetrazinyl group with trans-cyclooctene moiety), known as "click handles".

[0129] In some embodiments, the photoreactive group includes: [Chemical formula] wherein one of R1 to R3 is a bond to the remainder of the activatable moiety, and the other two of R1 to R3 are independently selected from H, Cl, F, Br, I, CH3, OH, SH, NH2, CN, CF3, CCl3, CH=CH2, -CH2-CH3, -CH2-OH, -CH2NH2, CH2SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, CH2Cl3, O-CH3, C(O)CH3, C(O)OH, and C(O)NH2.

[0130] In some embodiments, the photoreactive group includes furocoumarin. In some embodiments, the furocoumarin is selected from: [Chemical formula] wherein one of R1 to R2 is a bond to the remainder of the activatable moiety, and the other of R1 to R2 is independently selected from H, Cl, F, Br, I, CH3, OH, SH, NH2, CN, CF3, CCl3, CH=CH2, -CH2-CH3, -CH2-OH, -CH2NH2, CH2SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, CH2Cl3, O-CH3, C(O)CH3, C(O)OH, and C(O)NH2.

[0131] In some embodiments, the activatable label includes a functional moiety that enables downstream enrichment, detection, or further manipulation of a target molecule (e.g., a biopolymer) labeled via click ligation without copper of the functional moiety, a capture element (e.g., biotin, chloroalkane linker, etc.), a cleavable capture element, a fluorescent molecule, or a click handle (e.g., TCO, DBCO). In some embodiments, the fluorescent molecule is fluorogenic and is selected from the following.

Chemical formula

[0132] Localization element (HALOTAG) In some embodiments, two or more components of the systems herein are conjugated (e.g., linked, fused, etc.) to a molecular element that facilitates localization of the components. In certain embodiments, a bioluminescent protein (or complex) and a photocatalyst are linked together via a molecular localization element that binds to the bioluminescent protein (or complex) and the photocatalyst and positions the bioluminescent protein (or complex) and the photocatalyst in a state close enough to each other such that light from a lumophore that interacts with the bioluminescent protein (or complex) activates the photocatalyst.

[0133] In some embodiments, a bioluminescent protein or bioluminescent complex is fused to a first molecular entity and the photocatalyst is conjugated to a second molecular entity, and the interaction of the first molecular entity and the second molecular entity brings the bioluminescent protein or bioluminescent complex close enough to the photocatalyst such that light emitted by a lumophore upon interaction with the bioluminescent protein or bioluminescent complex activates the photocatalyst. In some embodiments, the first molecular entity is a capture agent (capture protein) and the second molecular entity is a capture element.

[0134] In some embodiments, the bioluminescent protein or bioluminescent complex is fused to a modified dehalogenase capable of forming a covalent bond with its substrate, and the photocatalyst is conjugated to the dehalogenase substrate (see FIG. 6A). In some embodiments, the binding of the modified dehalogenase to the dehalogenase substrate brings the bioluminescent protein or bioluminescent complex into sufficient proximity to the photocatalyst such that the light emitted by the luminophore upon interaction with the bioluminescent protein or bioluminescent complex activates the photocatalyst. In some embodiments, in combination with its highly specific and rapid binding, minimizing the effect on the cell permeability of the haloalkane enables intracellular ligation of the haloalkane conjugate to the HALOTAG fused to the components of the system, thereby reducing the overall dependence of the components on cell permeability and enabling localization of the system to specific cell compartments (FIGS. 6B - C).

[0135] In some embodiments, a commercially available HALOTAG system (Promega Corp.; Madison, WI) is utilized to link or associate two or more components of the systems and methods described herein (e.g., a bioluminescent protein or bioluminescent complex and a photocatalyst). HALOTAG is a 297 - residue self - labeling polypeptide (33 kDa) derived from a bacterial hydrolase (dehalogenase) enzyme that has been modified to covalently bind to its ligand, the haloalkane moiety. The HALOTAG ligand can be linked to a solid surface (e.g., beads) or a functional group (e.g., a fluorophore), and the HALOTAG polypeptide can be fused to various target proteins, enabling covalent attachment of the target protein to the solid surface or functional group.

[0136] The HALOTAG polypeptide is a hydrolase with a genetically modified active site that specifically binds to a haloalkane ligand or chloroalkane linker, resulting in an increased ligand binding rate (see Pries et al., The Journal of Biological Chemistry, 270(18):10405-11, which is incorporated herein by reference in its entirety). The reaction that forms a bond between the protein tag and the chloroalkane linker is fast and essentially irreversible under physiological conditions (see Waugh DS (June 2005), Trends in Biotechnology, 23(6):316-20, which is incorporated herein by reference in its entirety). In the native hydrolase enzyme, nucleophilic attack by the chloroalkane reactive linker results in the substitution of the halogen with an amino acid residue, leading to the formation of an alkyl-enzyme covalent intermediate. This intermediate is then hydrolyzed by an amino acid residue within the wild-type hydrolase (see Chen et al. (February 2005), Current Opinion in Biotechnology, 16(1):35-40, which is incorporated herein by reference in its entirety). This would lead to the regeneration of the enzyme after the reaction. However, in HALOTAG, a modified haloalkane dehalogenase, the reaction intermediate cannot be hydrolyzed due to mutations in the enzyme and thus cannot proceed through a second reaction. As a result, the intermediate persists as a stable covalent adduct with no associated reverse reaction (see Marks et al. (August 2006), Nature Methods, 3(8):591-6, which is incorporated herein by reference in its entirety).

[0137] The HaloTag fusion protein can be expressed using standard recombinant protein expression techniques (see Adams et al. (May 2002) Journal of the American Chemical Society. 124(21):6063-76, which is incorporated by reference in its entirety). The HaloTag polypeptide is a relatively small protein, and its reaction is exogenous to mammalian cells, so there is no interference by endogenous mammalian metabolic reactions (see Naested et al. The Plant Journal. 18(5):571-6, which is incorporated by reference in its entirety). When this fusion protein is expressed, a wide range of potential experimental areas are possible, including enzyme assays, cell imaging, protein arrays, determination of intracellular localization, and many additional possibilities (see Janssen DB (April 2004). Current Opinion in Chemical Biology. 8(2):150-9, which is incorporated by reference in its entirety).

[0138] A variety of HaloTag ligands, functional groups, fusions, assays, modifications, uses, etc. are described in U.S. Patent No. 8,748,148, U.S. Patent No. 9,593,316, U.S. Patent No. 10,246,690, U.S. Patent No. 8,742,086, U.S. Patent No. 9,873,866, U.S. Patent No. 10,604,745, U.S. Patent Application No. 2009 / 0253131, U.S. Patent Application No. 2010 / 0273186, 20130337539, U.S. Patent Application No. 2012 / 0258470, U.S. Patent Application No. 2012 / 0252048, U.S. Patent Application No. 2011 / 0201024, U.S. 2014 / 0322794, each of which is incorporated by reference in its entirety.

[0139] In some embodiments, the capture protein herein is a circularly permuted modified dehalogenase (see, e.g., U.S. Provisional Application No. 63 / 338,364 and / or U.S. Application No. 18 / 311,977, which are incorporated by reference in their entirety). In some embodiments, the capture protein herein provides a circularly permuted, HALOTAG (cpHT) dehalogenase complex. In some embodiments, the capture protein comprises a cp variant of a polypeptide having at least 70% sequence identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) with SEQ ID NO: 8. In some embodiments, the capture protein comprises (i) a first segment having at least 70% sequence identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) with a first portion of SEQ ID NO: 8, and (ii) a second segment having at least 70% sequence identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) with a second portion of SEQ ID NO: 8. In some embodiments, the first fragment and the second fragment together comprise an amino acid sequence corresponding to at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, 100%) of the length of SEQ ID NO: 8. In some embodiments, the amino acid of the polypeptide corresponding to position 297 of SEQ ID NO: 8 is peptide-bonded to the amino acid of the polypeptide corresponding to position 1 of SEQ ID NO: 8. In some embodiments, the amino acid of the polypeptide corresponding to position 297 of SEQ ID NO: 8 is connected to the amino acid of the polypeptide corresponding to position 1 of SEQ ID NO: 8 by a linker peptide. In some embodiments, the linker peptide is 2 to 100 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or a range therebetween). In some embodiments, the linker peptide is a cleavable element (e.g., a protease-cleavable site (e.g., TEV protease),It includes chemically cleavable sites, photocleavable sites, etc. In some embodiments, the capture protein is a circularly permuted variant corresponding to SEQ ID NO: 8 (e.g., having at least 70% sequence identity thereto), but at positions corresponding to positions between positions 5 and 290 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,Those having a cp site at a position corresponding to the 248th, 249th, 250th, 251st, 252nd, 253rd, 254th, 255th, 256th, 257th, 258th, 259th, 260th, 261st, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272nd, 273rd, 274th, 275th, 276th, 277th, 278th, 279th, 280th, 281st, 282nd, 283rd, 284th, 285th, 286th, 287th, 288th, 289th, or 290th position). In some embodiments, the capture protein corresponds to SEQ ID NO: 8, but positions 5 to 13 of SEQ ID NO: 8 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or the range therebetween), positions 36 to 51 (e.g., 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 11, or the range therebetween), positions 63 to 72 (e.g., 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or the range therebetween), positions 84 to 92 (e.g., 84, 85, 86, 87, 88, 89, 90, 91, 92, or the range therebetween), positions 104 to 130 (e.g., 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, or the range therebetween), positions 142 to 148 (e.g., 142, 143, 144, 145, 146, 147, 148, and the range therebetween), positions 160 to 174 (e.g., 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, or the range therebetween), positions 186 to 189 (e.g., 186, 187, 188, 189, or the range therebetween), positions 201 to 203 (e.g., 201, 202, 203, or the range therebetween), positions 221 to 229 (e.g., 221, 222, 223, 224, 225, 226, 227, 228, 229, or the range therebetween), or positions 269 to 290 (e.g., 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,has a cp site at a position corresponding to a position between (or within a range between) them.

[0140] In some embodiments, the capture protein of the present specification is a split modified dehalogenase (see, e.g., U.S. Provisional Application No. 63 / 338,323 and / or U.S. Application No. 18 / 312,117, which are incorporated by reference in their entirety). In such embodiments, the two components of the split capture protein can interact (with or without facilitation) to form a capture complex. In some embodiments, the two components of the split modified dehalogenase are assembled into an active modified dehalogenase complex by structural complementarity. In some embodiments, the components of the split capture protein can be linked (e.g., fused) to different components of the system of the present specification (e.g., an activatable label, a photocatalyst, a bioluminescent protein, a component of a bioluminescent complex, etc.), and upon assembly (with or without facilitation), the capture complex can bind to a capture element. In some embodiments, the split capture protein is a split HALOTAG (spHT). In some embodiments, the first and second components of the split capture protein together include at least 70% sequence similarity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) to SEQ ID NO: 8. In some embodiments, the first and second components of the split capture protein together include at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) sequence identity to SEQ ID NO: 8. In some embodiments, the split capture protein includes (i) a first fragment that includes at least 70% sequence similarity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) to the first part of SEQ ID NO: 8, and (ii) a second fragment that includes at least 70% sequence similarity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) to the second part of SEQ ID NO: 8.In some embodiments, the first fragment comprises at least about 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) sequence identity with the first portion of SEQ ID NO: 8. In some embodiments, the second fragment comprises at least about 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 100%) sequence identity with the second portion of SEQ ID NO: 8. In some embodiments, the first and second fragments together comprise an amino acid sequence corresponding to at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, 100%) of the length of SEQ ID NO: 8. In some embodiments, the split capture protein comprises a split ("sp") site at a position corresponding to any position (e.g., positions 19-34) between positions 5 and 290 of SEQ ID NO: 8.In some embodiments, the split capture protein has an sp site at a position corresponding to a position between positions 5 to 13 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or a range therebetween), 36 to 51 (e.g., 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or a range therebetween), 63 to 72 (e.g., 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or a range therebetween), 84 to 92 (e.g., 84, 85, 86, 87, 88, 89, 90, 91, 92, or a range therebetween), 104 to 130 (e.g., 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, or a range therebetween), 142 to 148 (e.g., 142, 143, 144, 145, 146, 147, 148, and a range therebetween), 160 to 174 (e.g., 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, or a range therebetween), 186 to 189 (e.g., 186, 187, 188, 189, or a range therebetween), 201 to 203 (e.g., 201, 202, 203, or a range therebetween), 221 to 229 (e.g., 221, 222, 223, 224, 225, 226, 227, 228, 229, or a range therebetween), or 269 to 290 (e.g., 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, or 290, or a range therebetween) of SEQ ID NO: 8. In some embodiments, the assembled complex of the components of the split capture protein can form a covalent bond with a haloalkane substrate.In some embodiments, the capture protein herein is a modified dehalogenase having an insertion within a surface loop (e.g., a bioluminescent protein, a component of a bioluminescent complex, a circularly permuted bioluminescent protein, a circularly permuted component of a bioluminescent complex, an extended loop sequence, etc.) (see, e.g., U.S. Provisional Application No. 63 / 338,369 and / or U.S. Application No. 18 / 312,441, which are incorporated by reference in their entirety). In some embodiments, the components of the system herein (e.g., a bioluminescent protein, a component of a bioluminescent complex, a circularly permuted bioluminescent protein, a circularly permuted component of a bioluminescent complex) are inserted between an N-terminal segment having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with one of SEQ ID NOs: 6-9 and a C-terminal segment having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with SEQ ID NOs: 10-13 (e.g., SEQ ID NOs: 6 / 10, 7 / 11, 8 / 123, 9 / 13, or other combinations). In some embodiments, the components of the system herein (e.g., a bioluminescent protein, a component of a bioluminescent complex, a circularly permuted bioluminescent protein, a circularly permuted component of a bioluminescent complex) are inserted between an N-terminal segment having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with one of SEQ ID NOs: 14-20 and a C-terminal segment having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with SEQ ID NOs: 21-27 (e.g., SEQ ID NOs: 14 / 21, 15 / 22, 16 / 23, 17 / 24, 18 / 25, 19 / 26, 20 / 27, or other combinations).In some embodiments, the components of the systems herein (e.g., bioluminescent proteins, components of bioluminescent complexes, circularly permuted bioluminescent proteins, circularly permuted components of bioluminescent complexes) are inserted (e.g., SEQ ID NO: 81 / 86, 82 / 87, 83 / 88, 84 / 89, 85 / 90, or other combinations) between an N-terminal segment having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with one of SEQ ID NOs: 81-85 and a C-terminal segment having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity with SEQ ID NOs: 86-90.

[0141] In some embodiments, the first component of the systems herein (e.g., a bioluminescent protein or a component of a bioluminescent complex) is fused (e.g., expressed as a fusion) to a modified dehalogenase (e.g., HALOTAG or a variant thereof) or inserted within a surface loop of the modified dehalogenase, and the second component of the systems herein (e.g., a photocatalyst) is linked (e.g., directly or via a linker) to a dehalogenase substrate (e.g., a haloalkane). For example, the structure of the photocatalyst linked to the dehalogenase substrate is P-linker-AX, wherein P is the photocatalyst and A is (CH2) 2-12wherein X is a halogen, and the linker is a linker moiety capable of linking P to A-X. In some embodiments, the linker is a polyatomic straight or branched chain containing C, N, S, or O, or a group containing one or more rings, such as a saturated or unsaturated ring (e.g., one or more aryl rings, heteroaryl rings, or any combination thereof). In some embodiments, the linker comprises a combination of -O(CH2)2-, -(CH2)O-, -CH2-, -NHC(O)O-, -OC(O)NH-, NHC(O)-, and -C(O)NH-. In some embodiments, the linker is 5 to 50 (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or in the range therebetween) atoms in length. In some embodiments, the length of the linker for linking the photocatalyst allows for optimization of the geometric shape (e.g., for energy transfer). Exemplary linker-A-X groups are illustrated in FIG. 4. In some embodiments, the first component of the system herein (e.g., a component of a bioluminescent protein or bioluminescent complex) is inserted (e.g., expressed as an internal fusion) into a modified dehalogenase (e.g., HALOTAG or a variant thereof) to increase proximity or provide a geometric shape favorable for energy transfer to the attached catalyst (see, e.g., U.S. Provisional Application No. 63 / 338,369, U.S. Application No. 18 / 312,441, which are incorporated by reference in their entirety). In some embodiments, the position for insertion within the modified dehalogenase (e.g., HALOTAG or a variant thereof) is selected to provide an optimal proximity and geometric shape for the desired interaction between the components while maintaining the function or activity of the modified dehalogenase (e.g., HALOTAG or a variant thereof) and the inserted component.

[0142] The scope of the embodiments of this specification is not limited by the types of linkers available. Components and portions thereof may be directly linked (e.g., the linker consists of a single covalent bond), or may be linked via a suitable linker. The embodiments are not limited to any particular linker group. A variety of linker groups are contemplated, and suitable linkers include alkyl groups, methylene carbon chains, ethers, polyethers, alkylamide linkers, peptide linkers, modified peptide linkers, poly(ethylene glycol) (PEG) linkers, streptavidin-biotin or avidin-biotin linkers, polyamino acids (e.g., polylysine), functionalized PEG, polysaccharides, glycosaminoglycans, dendrimers (from WO93 / 06868 and Tomalia et al., Angew. Chem. Int. Ed. Engl. 29:138-175 (1990) (incorporated herein by reference in their entirety)), PEG-chelate polymers (W94 / 08629, WO94 / 09056, and WO96 / 26754, incorporated herein by reference in their entirety), oligonucleotide linkers, phospholipid derivatives, alkenyl chains, alkynyl chains, disulfides, or combinations thereof, but are not limited thereto. In some embodiments, the linker is cleavable (e.g., enzymatically (e.g., TEV protease site), chemically, photoinduced, etc.).

[0143] In some embodiments, a modified dehalogenase (e.g., HALOTAG) and a dehalogenase ligand (e.g., haloalkane) are used to link any two components of the systems and methods described herein (i.e., not limited to the linking of components of a bioluminescent protein or bioluminescent complex to a photocatalyst). In other embodiments, components of a bioluminescent protein or bioluminescent complex are linked to a photocatalyst (or other components described herein) by another mechanism.

[0144] In some embodiments, a first component of the systems or methods herein is linked (e.g., fused) to a capture agent (e.g., a capture protein), and a second component of the systems or methods is linked to a capture element. Binding of the capture element by the capture agent (e.g., a capture protein) results in co-localization of the first and second components. In some embodiments, the capture agent is a modified dehalogenase and the capture element is a haloalkane. However, other capture agent / element pairs that can be used in the embodiments herein include streptavidin / biotin, antibodies (or Ab fragments) and antigens, and the like.

[0145] In other embodiments, the components herein are connected by chemical modification / conjugation, e.g., native chemical ligation, Staudinger ligation, "traceless" Staudinger ligation, amide coupling, methods using activated esters, methods targeting lysine, tyrosine, and cysteine residues, imine bond formation (with and without orthoboric acid), boronic acid / diol interaction, disulfide bond formation, copper / copper-free azide, diazo, and tetrazine "click" chemical reactions, UV-promoted thiol conjugation, diazirine photo-labeling, Diels-Alder cycloaddition, metathesis reaction, Suzuki cross-coupling, 2-cyanobenzothiazole (CBT) coupling, 2-pyridinecarboxaldehyde (PCA) coupling, and the like.

[0146] Target Molecules and Localization Elements In some embodiments, the activated label binds to a target molecule (e.g., a cell target, protein, nucleic acid). In some embodiments, the components of the systems herein are configured to bind to, co-localize with, or otherwise associate with a target molecule.

[0147] In some embodiments, the bioluminescent protein or complex is conjugated to a target binder, which can bind to a target molecule (e.g., a protein, nucleic acid, or other biomolecule (e.g., lipid, sugar, etc.)). In some embodiments, the target binder is a protein or peptide directly or indirectly fused to a component of the bioluminescent protein or bioluminescent complex. In some embodiments, the target molecule is a nucleic acid, and the target binder can specifically or non-specifically bind to the nucleic acid. In some embodiments, the target binder is a wild-type or modified Cas protein (e.g., Cas9, dCas9, dCas12, dCas13, etc.), and the target molecule is a nucleic acid modified by CRISPR. In some embodiments, the system further comprises a guide RNA (gRNA). In some embodiments, the target molecule is a target peptide or protein, and the target binder can bind to the target peptide or protein. In some embodiments, the target binder is a small molecule or nucleic acid directly or indirectly linked to a component of the bioluminescent protein or bioluminescent complex.

[0148] In a series of embodiments, the bioluminescent protein, a component of the bioluminescent complex, the photocatalyst, or the activatable label is linked to a specific ligand, nucleic acid, or targeting protein (e.g., Cas9, dCas9, dCas12, dCas13, etc.). Exemplary targeting ligands include small molecules / drugs / signal transduction molecules that specifically bind to a target. In some embodiments, the photocatalyst is linked to such a small molecule / drug / signal transduction molecule, thereby enabling localization of the photocatalyst with the protein of interest fused to HiBiT or NanoLuc. Other exemplary targeting proteins / ligands include antibodies, antibody fragments, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, oligonucleotide probes, peptide nucleic acids, DARPins, anticalins, nanobodies, aptamers, affimers, purified proteins, and analyte-binding domain(s) of a protein. Linking the catalyst to a binding domain that recognizes the target protein enables localization of the catalyst with the protein of interest already fused to HiBiT or NanoLuc. In some embodiments, for example, a functional moiety having general affinity for nucleic acids, a trifunctional molecule containing a photoreactive moiety, a functional moiety and a recognition moiety that directly bind to the target protein, etc. are used to increase the proximity to the activatable label.

[0149] In this case, complementation of HiBiT fused to the protein of interest with LgBiT-HaloTag-catalyst localizes the photocatalytic system to the protein of interest. Similarly, LgBiT fused to the protein of interest localizes the protein of interest to the HiBiT-HaloTag-photocatalytic system.

[0150] system In some embodiments, two or more (e.g., 2, 3, 4, or more) of the components of the systems described herein are conjugated together. In some embodiments, one or more pairs of the components of the systems described herein are conjugated together. For example, the following pairs of components may be conjugated (e.g., linked by a linker, gene fused, etc.): a bioluminescent protein and a capture protein, a photocatalyst and a capture ligand, a bioluminescent protein and a photocatalyst, a component of a bioluminescent complex and a capture protein, a component of a bioluminescent complex and a photocatalyst, a bioluminescent protein and a target molecule, a component of a bioluminescent complex and a target molecule, a bioluminescent protein and a target binder (e.g., a protein, an antibody, an antibody fragment, an antibody binder, a nucleic acid, a small molecule ligand, etc.), a component of a bioluminescent complex and a target binder (e.g., a protein, an antibody, an antibody fragment, an antibody binder, a nucleic acid, a small molecule ligand, etc.), a component of a bioluminescent complex and a photocatalyst, a component of a bioluminescent complex and a capture ligand, an activatable label and a capture ligand, a capture protein and a target binder (e.g., a protein, an antibody, an antibody fragment, an antibody binder, a nucleic acid, a small molecule ligand, etc.), etc.

[0151] The components of the systems described herein can be delivered, combined, and / or manufactured in any suitable manner for a particular application. In embodiments where the system is present within a cell, the components may be expressed intracellularly, added exogenously, enabled to enter the cell (i.e., cell permeable components), or delivered to the cell. Delivery of the components to the cell can be performed in any suitable delivery vehicle such as liposomes, micelles, nanoparticles, viruses, etc. In some embodiments, the components are tagged (e.g., conjugated to a membrane translocation motif) to facilitate delivery into the cell. In some embodiments, components are included to promote cell uptake and / or subsequent endosomal escape. Such additional components can include modified polyethyleneimine polymers and modified poly(amidoamine) dendrimers for use in delivering biomolecules to cells (e.g., delivery of components that cannot passively penetrate cells but can be expressed intracellularly (e.g., LgBiT / photocatalyst direct conjugates, etc.)) (see U.S. Publication No. 2020 / 0399660, which is incorporated by reference in its entirety).

[0152] Exemplary combinations of components of the systems within the scope of this specification include the following.

[0153] · HiBiT fused to a protein of interest, fusion of LGBIT and HALOTAG, a photocatalyst-HALOTAG conjugated to a haloalkyl HALOTAG ligand binds to the haloalkyl ligand; the high affinity of HIBIT for LGBIT forms a bioluminescent complex, localizes the photocatalytic system to the protein of interest, and bioluminescence induces the photocatalyst.

[0154] ·A peptide containing HIBIT-TRIP9 fused to a protein of interest, the fusion of LGTRIP and HALOTAG, and a photocatalyst-HALOTAG conjugated to a haloalkyl HALOTAG ligand binds to the haloalkyl ligand; the high affinity of HIBIT-TRIP9 for LGTRIP forms a bioluminescent complex, localizes the photocatalyst system to the protein of interest, and bioluminescence induces the photocatalyst.

[0155] ·A fusion of HIBIT and an antisense oligonucleotide, LGBIT fused to HALOTAG, and a photocatalyst-HALOTAG conjugated to a haloalkyl HALOTAG ligand binds to the haloalkyl ligand; the high affinity of HIBIT for LGBIT forms a bioluminescent complex, and the hybridization of the antisense oligonucleotide to the target nucleic acid sequence localizes the photocatalyst system to the target DNA / RNA, and bioluminescence induces the photocatalyst.

[0156] ·SMBIT fused to an antibody against a target analyte, LGBIT / HALOTAG fused to a common immunoglobulin binding moiety, and a photocatalyst-HALOTAG conjugated to a haloalkyl HALOTAG ligand binds to the haloalkyl ligand; the binding of the antibody promotes complementation, localizes the photocatalyst system to the target analyte, and bioluminescence induces the photocatalyst.

[0157] ·A fusion of HIBIT and Trip9 to an antisense oligonucleotide targeting the same DNA / RNA of interest, LGTRIP fused to HALOTAG, and a photocatalyst-HALOTAG conjugated to a haloalkyl HALOTAG ligand binds to the haloalkyl ligand; the hybridization of the antisense oligonucleotide to the target nucleic acid sequence promotes complementation, localizes the photocatalyst system to the target DNA / RNA, and bioluminescence induces the photocatalyst.

[0158] dCas9 or dCas12g1 fused to both HaloTag and NanoLuc, and a photocatalyst-HALOTAG conjugated to a haloalkyl HALOTAG ligand binds to the haloalkyl ligand; The gRNA targets the photocatalytic system to the DNA / RNA of interest, and bioluminescence induces the photocatalyst.

[0159] Use In some embodiments, the systems and methods herein are used to perform various applications (e.g., intracellularly). In addition to other applications, various functional proteomics and genomic analyses are made possible by the advantages of the systems and methods herein. Exemplary applications of the systems and methods herein that focus on proteomics include proximity-based protein labeling and subsequent detection of the dynamic microenvironment, protein-protein interactions, and cell-cell interactions under relevant physiological conditions; labeling of HiBiT fusion proteins and their proximal proteins with fluorophores for downstream sorting / detection of HiBiT fusion-expressing cells; labeling of HiBiT fusion proteins with fluorophores to maintain the active site free to interact with other ligands for subsequent monitoring of protein dynamics and transport; protein labeling using click handles for subsequent attachment of diverse functional groups such as small molecule drugs, PROTACs, etc. for downstream manipulation of the protein of interest, and the like.

[0160] Exemplary genome-focused uses of the systems and methods of this specification include the following: - the use of a photocatalytic system comprising HaloTag-NanoLuc linked to components of the system to facilitate proximity labeling of DNA or RNA loci fused to a Cas protein (e.g., Cas9, dCas9, dCas12, dCas13) and targeted by the Cas protein with either biotin for enrichment and subsequent sequence analysis, a fluorophore for detection and visualization, or a click handle for subsequent attachment of various functional groups. Such systems can be used to identify Cas9 off-target effects as well as to map nucleic acid-protein interactions.

[0161] In some embodiments, provided herein is a method of activation that depends on the proximity of an activatable label within a cell, the method comprising contacting the cell with a fluorophore under conditions that allow the fluorophore to enter the cell, the cell comprising (a) a fusion of a bioluminescent protein and a capture protein, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the fluorophore upon interaction with the fluorophore, (b) a conjugate of (A) a capture ligand and (B) a photocatalyst, wherein the capture protein forms a covalent bond with the capture ligand upon interaction with the capture ligand and the photocatalyst is activated upon exposure to light of the first wavelength, and (c) an activatable label that is converted to an activation label when in proximity to the activated photocatalyst.

[0162] In some embodiments, provided herein is a method of inducing proximity-dependent bioorthogonal chemical ligation, the method comprising contacting a cell with a fluorophore under conditions such that the fluorophore enters the cell, the cell comprising: (a) a fusion of a bioluminescent protein and a capture protein, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the fluorophore upon interaction with the fluorophore; (b) a conjugate of (A) a capture ligand and (B) a photocatalyst, wherein the capture protein forms a covalent bond with the capture ligand upon interaction therewith and the photocatalyst is activated upon exposure to light of the first wavelength; (c) an activatable label that is converted to an activation label when in proximity to the activated photocatalyst; and (d) a target molecule, wherein the activation label forms a covalent bond with the target molecule when in proximity thereto.

[0163] In some embodiments, provided herein is a method of activation that is dependent on the proximity of an activatable label within a cell, the method comprising: (a) expressing within the cell a fusion of a bioluminescent protein and a capture protein; (b) contacting the cell with a fluorophore under conditions such that the fluorophore enters the cell, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the fluorophore upon interaction therewith; (c) contacting the cell with a conjugate of (i) a capture ligand and (ii) a photocatalyst under conditions such that the conjugate enters the cell, wherein the capture protein forms a covalent bond with the capture ligand upon interaction therewith and the photocatalyst is activated upon exposure to light of the first wavelength; and (d) contacting the cell with an activatable label, wherein the activatable label is converted to an activation label when in proximity to the activated photocatalyst.

[0164] In some embodiments, provided herein is a method of inducing bioorthogonal chemical ligation that depends on proximity to a target molecule, the method comprising: (a) expressing intracellularly a fusion of a bioluminescent protein and a capture protein; (b) contacting the cell with a fluorophore under conditions that allow the fluorophore to enter the cell, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the fluorophore by interaction with the fluorophore; (c) contacting the cell with a conjugate of (i) a capture ligand and (ii) a photocatalyst under conditions that allow the conjugate to enter the cell, wherein the capture protein forms a covalent bond with the capture ligand by interaction with the capture ligand and the photocatalyst is activated by exposure to light of the first wavelength; and (d) contacting the cell with an activatable label that is converted to an activated label when in proximity to the activated photocatalyst, and the activated label forms a covalent bond with the target molecule when in proximity to the target molecule.

[0165] In some embodiments, provided herein is a method of activation that depends on proximity of a photocatalyst within a cell, the method comprising contacting the cell with a fluorophore under conditions that allow the fluorophore to enter the cell, the cell comprising: (a) a fusion of a bioluminescent protein and a capture protein, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the fluorophore by interaction with the fluorophore; and (b) a conjugate of (A) a capture ligand and (B) a photocatalyst, wherein the capture protein forms a covalent bond with the capture ligand by interaction with the capture ligand and the photocatalyst is activated by exposure to light of the first wavelength.

[0166] In some embodiments, provided herein is a method of activation that depends on the proximity of a photocatalyst within a cell, the method comprising: (a) expressing within the cell a fusion of a bioluminescent protein and a capture protein; (b) contacting the cell with a fluorophore under conditions such that the fluorophore enters the cell, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the fluorophore upon interaction with the fluorophore; and (c) contacting the cell with a conjugate of (i) a capture ligand and (ii) a photocatalyst under conditions such that the conjugate enters the cell, wherein the capture protein forms a covalent bond with the capture ligand upon interaction therewith and the photocatalyst is activated upon exposure to light of the first wavelength.

[0167] One exemplary general embodiment of the systems and methods herein is illustrated in FIG. 7. In this embodiment, a first component of a bioluminescence complex (e.g., the LgBiT component of NanoBiT) is conjugated (e.g., fused) to a capture protein (e.g., HALOTAG). In some forms of this embodiment, the first component of the bioluminescence complex and the capture protein are expressed as a fusion within the cell. The photocatalyst is linked to a capture ligand (e.g., including a haloalkane). In some embodiments, the photocatalyst linked to the capture ligand is added extracellularly and can enter the cell (e.g., without permeabilizing the cell) and form a covalent bond with the capture protein. Formation of the active bioluminescence complex and emission of light are effected by exposing the first component of the bioluminescence complex to a second component of the bioluminescence complex (e.g., HiBiT) and a suitable fluorophore (e.g., furimazine, fluorofurimazine, etc.). Exposure of the photocatalyst to the light emitted from the bioluminescence complex activates the photocatalyst. The activated photocatalyst then participates in an energy transfer event with a label activatable in its vicinity to generate a reactive intermediate capable of forming a covalent bond with an adjacent protein.

[0168] In the embodiment shown in FIG. 7, the bioluminescent complex is utilized as a light source. In other embodiments such as illustrated in FIG. 8, the bioluminescent protein is utilized in place of the bioluminescent complex. The selection of the bioluminescent complex or protein is determined based on the particular application. Where applicable, the embodiments described for use with one bioluminescent entity herein can also find use with other bioluminescent entities herein, or other bioluminescent entities as understood in the art.

[0169] In some embodiments, the use of complementation to form a bioluminescent complex provides various advantages compared to other systems (e.g., those utilizing a laser or LED as a light source), or the systems of this specification that utilize a bioluminescent protein as a light source. For example, the use of the HiBiT / LgBiT complementation system (or other NanoBiT-based complementation systems) as the main light source, combined with a wide toolkit of pairs of photocatalyst / activatable labels, provides multiple advantages in live cells or other biological systems. HiBiT is a small, minimally perturbing tag suitable for tagging endogenous target proteins. LgBiT can be linked to a photocatalyst (e.g., via a HALOTAG or another capture system), which can, for example, induce proximity between the catalyst and the HiBiT-tagged protein of interest, induce proximity between the catalyst and the bioluminescence source (HiBiT / LgBiT), generate higher spatiotemporal resolution through conditional activation (+ furimazine) at a specific site (HiBiT / LgBiT complementation), provide a convenient approach for linking the catalyst to the HaloTag-LgBiT fusion in a specific compartment for biochemical or cellular expression of the fusion using chloroalkane chemistry, and provide a modality-independent approach for delivering the photocatalyst system to the site of interest (e.g., intracellularly or extracellularly). The use of a bioluminescent complex (or bioluminescent protein) provides for the local delivery of light of an appropriate wavelength (e.g., blue light) for the activation of a catalyst inside intact cells or other complex models. Other embodiments of this specification utilize the SmBiT / LgBiT complementation system, or another complementation system that requires external complementation (e.g., facilitation) to form a bioluminescent complex. Since SmBiT and LgBiT do not form an active bioluminescent complex without facilitation, the use of such components in the systems / methods of this specification can be used requiring additional localization events (e.g., the binding of an element conjugated (directly or indirectly) to SmBiT to an element conjugated (directly or indirectly) to LgBiT) to generate the light for activating the photocatalyst.

[0170] In other embodiments, the use of bioluminescent proteins provides various advantages compared to other systems (e.g., those utilizing a laser or LED as a light source), or the systems of the present disclosure that utilize a bioluminescent complex as a light source. For example, a bioluminescent protein (e.g., NANOLUC) provides a single entity light source that can be expressed intracellularly (e.g., alone or as a fusion with other components of the systems of the present disclosure). In certain embodiments of the present disclosure, the improved simplicity / efficiency of the single entity light source is preferred over embodiments that require complementation.

[0171] Figure 7 shows a system that enables spatiotemporal protein labeling induced by bioluminescence in intact cells (where spatial relationships are maintained). Such labeling enables subsequent enrichment and identification by mass spectrometry or other detection / quantitative analysis. Such embodiments can be used in mapping dynamic interactomes and protein complexes, mapping the interactome of membraneless intracellular compartments, mapping protein trafficking and secretion, etc.

[0172] Figure 8 shows a system for activatable label bioluminescence induction for covalent cross-linking with adjacent dsDNA. In such an embodiment, a CRISPR enzyme conjugate (i.e., Cas-NanoLuc-HaloTag-catalyst) is used in combination with an sgRNA to target a specific DNA locus for spatiotemporal DNA / protein labeling in intact cells (where spatial relationships are preserved). Similar systems having other protein-binding or nucleic acid-binding proteins conjugated to the components of this system are within the scope of this specification. In the CRISPR / Cas system, a guide RNA (gRNA) binds to a specific target sequence within the genomic DNA of a cell. The Cas9 enzyme binds to the complex of the gRNA and the target sequence. The Cas9 enzyme then cleaves the target DNA at the target position. When the DNA is cleaved, the cell's DNA repair mechanism repairs the cleavage, resulting in a change customized to the target sequence. In some embodiments, by linking a bioluminescent protein (e.g., NanoLuc) or a component of a bioluminescent complex to a component of the CRISPR system and linking a photocatalyst to the bioluminescent protein (e.g., NanoLuc) or a component of the bioluminescent complex (e.g., directly or via a HaloTag and a HaloTag ligand), the photocatalyst is activated near the target DNA, and the site of CRISPR modification can be labeled by the system of this specification. Although Cas9 is the most frequently used enzyme, other enzymes (e.g., Cpf1) can also be used in the CRISPR system (and the labeling system of this specification).

[0173] Figure 9 shows a system for the bioluminescence-induced generation of singlet oxygen for the covalent labeling of neighboring nucleic acids. The CRISPR enzyme conjugate (e.g., Cas-NanoLuc-HaloTag-catalyst) is used in combination with an sgRNA to target specific DNA / RNA loci for spatiotemporal labeling in intact cells (where spatial relationships are preserved). This exemplary system generates short-lived diffusing singlet oxygen, mainly for the proximity functionalization of guanine bases. The lack of an external light source and the locally conditional generation of singlet oxygen greatly reduces the problem of cytotoxicity.

[0174] Figures 10A, B, and C show a system for the bioluminescence-induced covalent labeling of proteins genetically fused to HiBiT using fluorogenic molecules. Such embodiments enable the conversion of bioluminescence to fluorescence for cell sorting applications. Means for bioluminescence-based cell sorting are not currently available. The systems herein enable the bioluminescence-induced activation of fluorogenic molecules. For example, a fluorogenic dye quenched with an azide can be used to couple the activation of the dye to a covalent protein label. Similar systems (e.g., using the same EMA dye) are useful for the labeling of nucleic acids. A similar approach utilizing a genetic fusion of Cas-NanoLuc-HaloTag linked to a catalyst in combination with an sgRNA can be used for the fluorescent labeling of the DNA / RNA locus of interest.

[0175] Figures 11A and 11B show a system for the bioluminescence-induced proximity incorporation of click handles (e.g., TCO, tetrazine, DBCO, N3) for the subsequent bioorthogonal ligation of fluorophores or other functional moieties. (A) Shows the two-step coupling covalent incorporation of a click handle (i.e., TCO) and subsequent labeling with a fluorophore. Such a method addresses the potential quenching of the catalyst by the fluorophore. A similar approach can also be used to label the nucleic acid of interest.

[0176] Other uses that utilize the systems, methods, and components of this specification are within the scope of this technology.

[0177] Experiment Example 1 During the development of the bioluminescence-induced photocatalyst label described in this specification, experiments were conducted to evaluate the influence of modifications (R1 - R3) on the physicochemical properties of the iridium catalyst and its subsequent ability to drive the photocatalyst label (Figure 12). The model catalysts and modification sites are shown in Figure 12A. The structures and syntheses of these catalysts are included in Example 12.

[0178] The influence that the structural modifications described in Figure 12B have on the physiochemical properties of the catalyst and its subsequent ability to undergo energy transfer events with diazirine residues was determined using the following analysis.

[0179] a. Excitation and emission profiles: The excitation and emission spectra of 200 μM of the catalyst in 100% DMSO were monitored using a SPARK multimode plate reader with the following settings: 380 nm excitation for the emission scan and 480 nm, 560 nm, or 600 nm emission for the excitation scan.

[0180] b. Emission energy (EmE) was calculated from λ using Planck's equation. Em Calculated from. E (J) = (h x c) / λ Where h (Planck's constant) = 6.625 x 10 -34 J x s, c (speed of light) = 3 x 10 8 m / s, and λ (m) is λ Em is The energy in joules was converted to cal / mol by converting 1 J = 0.239 Cal:

Number

[0181] c. The efficiency of energy transfer from the excited catalyst to the diazirine residue was derived from Stern-Volmer quenching relationship analysis by monitoring the ability of increasing concentrations of diazirine-biotin (0 - 2 mM) to quench the luminescence of 5 μM catalyst in (TBS + 0.01% BSA).

Number

Number

Number

[0182] The effect of the physiochemical properties of a catalyst on its ability to absorb blue light and undergo an energy transfer event with a diazirine residue for subsequent covalent cross-linking with an adjacent protein was further evaluated by the labeling efficiency of a model protein (Figures 12C and D). For this purpose, reactions containing 3 - 5 μM HaloTag-NanoLuc fusion protein, 100 μM diazirine-biotin, and various concentrations of iridium catalyst (0 - 10 μM) were constructed in wells of a UV-transparent 96-well plate in TBS (pH 7.5), and then irradiated for 0 - 15 minutes at either 455 nm or 365 nm using an Efficiency Aggregators bioreactor (80 W). To evaluate the labeling efficiency, the proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was stained with Licor total protein fluorescent stain, blocked with 5% BSA (Promega) in TBST for 1 hour at room temperature, and then incubated overnight at 4°C with streptavidin-HRP (Invitrogen) in TBST supplemented with 5% BSA. After three washes with TBST, the membrane was first scanned in the Cy5 channel to detect total protein, then treated with ECL substrate (Promega W1001) and scanned in the chemiluminescence channel to detect biotin-labeled protein. These analyses showed that the photoactivation of diazirine and subsequent protein labeling upon 365 nm irradiation were catalyst-independent, whereas the photoactivation upon 455 nm irradiation was catalyst-dependent. Furthermore, the labeling efficiency was dependent on the catalyst concentration (concentration-driven proximity between the catalyst and diazirine-biotin), as well as the emission energy of the catalyst (EmE) and its ability to drive the photoactivation of diazirine (EmE ≧ 51 kcal / mol). The labeling efficiency is likely to be further affected by other catalyst properties such as the extinction coefficient (i.e., Ir-8844 has the same EmE as Ir-8870 but provides a significantly higher labeling efficiency than Ir-8870).

[0183] Example 2 In this example, the effect of the proximity between the catalyst and the target protein on the efficiency of the LED-induced photocatalytic protein labeling is described (Figure 13). In this example, the proximity between the catalyst and the target protein is promoted by the covalent bond of an iridium catalyst conjugated to chloroalkane and HaloTag genetically fused to the target protein (e.g., NanoLuc) (Figure 13A). The structure of the modifiable iridium catalyst and its derivatives further conjugated to chloroalkane is shown in Figure 13B. The synthesis of these catalysts is included in Example 12.

[0184] The physiochemical properties of the iridium catalysts and their ability to undergo energy transfer events with diazirine-biotin were determined as described in Example 1 and are shown in Figure 13C. To determine the effect of proximity on the labeling efficiency (Figure 13D), a reaction containing either 100 μM diazirine-biotin and a HaloTag-NanoLuc fusion protein linked to 1 μM of the Ir-8810 catalyst, or 1 μM of the HaloTag-NanoLuc fusion protein + 1 μM of the Ir-8673 catalyst, was constructed in TBS (pH 7.5) in the wells of a UV-transparent 96-well plate and then irradiated at either 455 nm or 365 nm for 0 - 30 minutes using an Efficiency Aggregators bioreactor (80 W). To evaluate the labeling efficiency, the proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and analyzed as described in Example 1. This analysis showed that the labeling was dependent on irradiation and subsequent activation of diazirine. The activation of diazirine by irradiation at 455 nm was not only catalyst-dependent but also the labeling efficiency was highly dependent on the proximity between the catalyst and the target protein. These results indicate that upon absorption of blue light, the excited catalyst participates in an energy transfer event with the proximate diazirine-biotin residue, generating a highly short-lived reactive intermediate that can further undergo covalent cross-linking with adjacent proteins before being quenched by water. As a result, the labeling efficiency is directly correlated with proximity, leading to an increase in the labeling of adjacent proteins.

[0185] Example 3 This example explains the influence of the proximity between the catalyst and the bioluminescence light source on the efficiency of bioluminescence vs. LED-induced labeling (Figure 14). In this example, the proximity between the catalyst and the bioluminescence light source that also functions as the target protein (e.g., NanoLuc) is promoted by the covalent bond between the catalyst conjugated to chloroalkane and HaloTag genetically fused to NanoLuc. The structures of two modifiable catalysts and their derivatives further conjugated to chloroalkanes of different lengths are shown in Figure 14A. The synthesis of these catalysts is included in Example 12.

[0186] The physiochemical properties of the iridium catalysts and their ability to undergo energy transfer events with diazirine-biotin were determined as described in Example 1 and are shown in Figure 14B. Furthermore, the efficiency of energy transfer from NanoLuc to the catalyst was determined for catalysts with increasing concentrations (0 - 125 μM) by monitoring the ability to quench the bioluminescence emission of 0.6 nM NanoLuc after the addition of 20 μM NanoGlo® live cell substrate (Promega Corporation, catalog number N205) in TBS + 0.01% BSA, using modified Stern-Volmer quenching relationship analysis.

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[0187] These analyses revealed that chloroalkanes have a positive effect on the efficiency of proximity-independent energy transfer. Chloroalkanes increased the efficiency of energy transfer from NanoLuc to the catalyst (2 - 7-fold) and from the catalyst to diazirine (0 - 2-fold) for the Ir-8673 and Ir-8844 derivatives, respectively. This analysis also showed that Ir-8844 and its derivatives exhibit higher emission energy (EmE), indicating that they are more suitable for diazirine activation and subsequent photocatalytic labeling of neighboring proteins.

[0188] To evaluate the ability of the catalyst to drive the labeling of the photocatalytic protein upon excitation by either 455 nm irradiation or bioluminescence (30 μM N205, NanoGlo® Live Cell Substrate) (Figure 14C), a reaction containing 500 μM diazirine-biotin, 0.6 μM blocking protein, and HaloTag-NanoLuc conjugated to either 0.06 μM of the Ir-8810, Ir-8972, or Ir-8973 catalyst was constructed in TBS (pH 7.5) in wells of either a UV-transparent 96-well plate (LED irradiation) or a white 96-well plate (bioluminescence). The plate was either irradiated at 455 nm for 15 minutes using an Efficiency Aggregators biophotoreactor (0 - 4.8 W) or treated with 30 μM NanoGlo® Live Cell Substrate for 15 minutes, and control wells were left untreated. To evaluate the labeling efficiency, the proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and analyzed as described in Example 1. This analysis demonstrated photocatalytic protein labeling induced by either 455 nm irradiation or NanoLuc bioluminescence. HaloTag-NanoLuc conjugated to the Ir-8844 derivatives (i.e., Ir-8972 and Ir-8973) resulted in significantly higher bioluminescence-induced labeling than HaloTag-NanoLuc conjugated to the Ir-8673 derivative (i.e., Ir-8810). This is due to the higher emission energy (EmE) and higher energy transfer efficiency (k SVBL and kSV ) is due to. Furthermore, the Ir-8844 catalyst conjugated to a longer chloroalkane (i.e., Ir-8973) showed a lower k SVBL but showed a higher labeling efficiency, indicating that for this construct (HaloTag-NanoLuc), a longer chloroalkane can induce a higher proximity of NanoLuc to the catalyst.

[0189] Example 4 During the development of the bioluminescence-induced photocatalyst label described herein, experiments were conducted to evaluate the effects of the length and structure of chloroalkanes on catalyst properties, including energy transfer efficiency (from NanoLuc to the catalyst and from the catalyst to diazirine), the binding reaction rate to HaloTag, cell permeability, and the ability to drive the bioluminescence-induced photocatalyst protein label (Figures 15 and 16). The structures of the modifiable catalyst Ir-8844 and its derivatives conjugated to chloroalkanes of different lengths are shown in Figure 15A. The synthesis of these catalysts is included in Example 12.

[0190] The effects of chloroalkanes on the physiochemical properties of the iridium catalyst and their ability to undergo energy transfer events were determined as described in Examples 1 and 3 and are shown in Figure 15B. These analyses revealed that chloroalkanes increase the energy transfer efficiency from NanoLuc to the catalyst (2 - 7 times) in a manner inversely correlated with the length of the chloroalkane. Furthermore, chloroalkanes doubled the energy transfer efficiency from the catalyst to diazirine regardless of length. Since chloroalkanes did not affect the emission energy (EmE) of the catalyst, these results indicate that chloroalkanes increased the ability of the catalyst to absorb light in a manner inversely correlated with the length of the chloroalkane.

[0191] Chloroalkanes provide a means of inducing proximity between a catalyst and a bioluminescent light source through covalent attachment of a chloroalkane-catalyst conjugate to a HaloTag genetically fused to the light source. The effect of chloroalkane length and structure on the rate of the binding reaction to HaloTag (Figure 15C) was evaluated by treating lysates prepared from cells expressing the HaloTag fusion protein with chloroalkane-catalyst conjugates at a final concentration of 2 μM. At incubation times of 0 to 120 minutes, fractions of each reaction (each containing a different chloroalkane-catalyst conjugate) were removed and treated with HaloTag TMR-fluorescent ligand (Promega) at a final concentration of 5 μM. This allowed binding of the fluorescent ligand to any unbound HaloTag fusion protein. Fractions at each time point were separated by SDS-PAGE and scanned with a Typhoon fluorescence imager (GE healthcare). Bands were quantified using ImageQuant (GE healthcare), and the binding reaction rate was determined as the percentage of binding over time compared to time zero when no chloroalkane-catalyst conjugate was added. All chloroalkane-catalyst conjugates showed similar binding reaction rates to HaloTag regardless of chloroalkane length, indicating that the length of the chloroalkane had only a minor effect on the binding reaction rate.

[0192] The effects of the length and structure of chloroalkanes on the cell permeability of the catalyst conjugate were further evaluated by their binding reaction rates to HaloTag inside the cells (Figure 15D). For this purpose, cells expressing the HaloTag fusion protein were treated with a chloroalkane-catalyst conjugate at a final concentration of 2 μM for 0 to 180 minutes and then further treated with a HaloTag TMR-fluorescent ligand at a final concentration of 5 μM for an additional 15 minutes. This enabled the binding of the fluorescent ligand to any unbound HaloTag fusion protein. The cells were then collected, lysed with a surfactant lysis buffer, and each time point was analyzed as described above. This analysis revealed that shorter chloroalkanes minimize the impact on cell permeability and enable a fast binding reaction rate to HaloTag inside the cells.

[0193] The efficiency of bioluminescence-induced catalyst activation depends on the luminescence intensity of the luciferase energy donor, the spectral overlap between luciferase luminescence and catalyst excitation, the proximity between luciferase and the catalyst, and the ability of the complex to adopt a favorable conformation for efficient energy transfer between luciferase and the catalyst. First, the effect of the fusion orientation of NanoLuc-HaloTag on the efficiency of bioluminescence resonance energy transfer (BRET) to the bound HaloTag TMR-fluorescent ligand was tested. For this purpose, the fusions, either unlinked or linked to the HaloTag TMR-fluorescent ligand, were diluted to a final concentration of 6.6 nM in TBS + 0.01% BSA and then treated with 10-fold fluorofurinazine at a final concentration of 20 μM. After a 3-minute incubation, the raw luminescence (total RLU) or filtered luminescence of the donor (e.g., 450 nm / 8 nm BP) and acceptor (600 nm LP) luminescence, respectively, was measured with a GloMax® Discover plate reader (Promega). The BRET ratio was further calculated for each sample by dividing the acceptor luminescence value by its donor luminescence value. Equivalent brightness was obtained in both directions, but one of the NanoLuc-HaloTags showed higher BRET efficiency (Figure 16A), which is likely due to a higher proximity between the substrate binding site of NanoLuc and the fluorescent ligand bound to it.

[0194] The two conjugation directions were further linked to the chloroalkane-catalyzed conjugate and compared for their ability to drive bioluminescence-induced photocatalyst protein labeling (Figure 16B). Reactions containing 100 μM diazirine-biotin, 0.1 mg / mL biotinylated protein-depleted K562 cell lysate, and 60 nM of either Ir-9049, Ir-8972, Ir-8973, or Ir-9050 linked to HaloTag-NanoLuc or NanoLuc-HaloTag were assembled in wells of a white 96-well plate in TBS (pH 7.5). Bioluminescence was induced by treatment with 10-fold fluorofurinazine at a final concentration of 100 μM, while the control wells were left untreated. After 15 minutes of incubation, samples were collected, separated by SDS-PAGE, transferred to a nitrocellulose membrane, and analyzed as described in Example 1. Consistent with the two-fold higher BRET efficiency observed with NanoLuc-HaloTag, all four catalysts linked to NanoLuc-HaloTag resulted in higher bioluminescence-induced photocatalyst protein labeling than their counterparts linked to HaloTag-NanoLuc. Furthermore, among the four chloroalkane-catalyzed conjugates, the NanoLuc-HaloTag fusion preferred the shorter chloroalkane (i.e., Ir-9049), suggesting that this orientation is likely to enhance the proximity between the substrate-binding site and the conjugation catalyst of NanoLuc. On the other hand, the HaloTag-NanoLuc in the other orientation preferred the longer chloroalkane, which is likely to mitigate the reduction in proximity between the substrate-binding site and the conjugation catalyst of NanoLuc.

[0195] Example 5 This example describes a further optimization of means for inducing proximity between two of a bioluminescent energy donor, a chloroalkane-catalyst conjugate, and a bioluminescent photocatalyst complex comprising HaloTag (FIG. 17). To increase proximity, a chimeric construct was designed that includes circularly permuted NanoLuc (e.g., cpNLuc at residues 67 / 68) inserted into the surface loop of HaloTag (between residues 178 - 179) proximal to the ligand interaction site (i.e., HT 178 -cpNLuc- 179 )(FIG. 17A). First, NanoLuc-HaloTag and the chimeric HT 178 -cpNLuc- 179 were compared for the efficiency of bioluminescence resonance energy transfer (BRET) to a conjugated HaloTag TMR-fluorescent ligand. For this purpose, NanoLuc-HaloTag and non-conjugated or conjugated HT 178 -cpNLuc- 179 were diluted to a final concentration of 6.6 nM in TBS + 0.01% BSA and then treated with 10-fold fluorofurinazine at a final concentration of 20 μM. After a 3-minute incubation, the raw luminescence (total RLU) or filtered luminescence of the donor (e.g., 450 nm / 8 nm BP) and acceptor (600 nm LP) emissions, respectively, were measured with a GloMax® Discover plate reader (Promega). The BRET ratio was further calculated for each sample by dividing the acceptor emission value by its donor emission value. HT 178 -cpNLuc- 179 was a 10-fold dimer but showed 24-fold higher BRET efficiency (FIG. 17B), indicating that the chimeric construct was able to induce higher proximity between the substrate-binding site of NanoLuc and the conjugated fluorescent ligand, adopt a conformation favorable for energy transfer between the two, or both.

[0196] HT 178 -cpNLuc- 179was further conjugated to the chloroalkane-catalyst conjugate and compared to NanoLuc-HaloTag:Ir-9049 for its ability to drive bioluminescence-induced photocatalytic protein labeling (Figure 17C). Reactions containing 100 μM diazirine-biotin, 0.1 mg / mL biotinylated protein-depleted K562 cell lysate, and 60 nM conjugate were assembled in wells of a white 96-well plate in TBS (pH 7.5). Bioluminescence was induced by treatment with 10-fold fluorofurinazine at a final concentration of 100 μM, while control wells were left untreated. After 15 minutes of incubation, samples were collected, separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed as described in Example 1. Consistent with a 24-fold higher BRET efficiency, all three catalysts conjugated to HT 178 -cpNLuc- 179 resulted in higher photocatalytic protein labeling than NanoLuc-HaloTag:Ir-9049. Furthermore, among the three chloroalkane-catalyst conjugates, HT 178 -cpNLuc- 179 also preferred a shorter chloroalkane (i.e., Ir-9049). Taken together, these results indicate that HT 178 -cpNLuc- 179 :Ir-9049 is well-suited for catalyst activation via bioluminescence resonance energy transfer. The chimeric structure likely induces a higher proximity between the substrate-binding site of NanoLuc and the binding catalyst, or adopts a favorable conformation for energy transfer between the two, or both.

[0197] Example 6 This example summarizes the optimization of a bioluminescent photocatalyst system that increased the overall bioluminescence-induced photocatalyst protein label by 900-fold (Figure 18). These included optimization of the catalyst core and NanoLuc substrate, as well as the composition of the bioluminescent photocatalyst complex. Reactions containing 100 μM diazirine-biotin, K562 cell lysate depleted of 0.1 mg / mL biotinylated protein, and 60 nM conjugate were constructed in wells of a white 96-well plate in TBS (pH 7.5). Treatment with NanoLuc substrate (furimazine or fluorofurimazine) at the indicated final concentrations induced bioluminescence, while control wells were left untreated. After 20 minutes of incubation, samples were collected, separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed as described in Example 1 (Figure 18A). To avoid signal saturation, the accumulation of light emitted by the bioluminescence energy donor over the 20-minute incubation was monitored for each condition using a filtered luminescence setup (610 nm / LP) on a Varioskan plate reader (Figure 18B). Optimization of the catalyst core, the orientation of the NanoLuc:HaloTag fusion, and the length of the chloroalkane resulted in an optimized NanoLuc-HaloTag:Ir-9049 complex and a total 22.5-fold increase in protein labeling efficiency (Condition 5). Further, replacement of the NanoLuc substrate furimazine with fluorofurimazine and increasing the substrate concentration to 100 μM increased the total light output 5-fold, followed by a further 5-fold increase in protein labeling (Condition 8). Finally, replacement of the NanoLuc-HaloTag:Ir-9049 complex with chimeric HT 178 -cpNLuc- 179 :Ir-9049 decreased the total light output 2.8-fold but increased protein labeling a further 8-fold, most likely because the efficiency of energy transfer from NanoLuc to the catalyst was higher.

[0198] Example 7 This example further demonstrates the optimized HT 178 -cpNLuc- 179: Demonstrate the efficiency of the Ir-9049 bioluminescent photocatalyst complex. Compare the efficiency of photocatalyst labeling caused by increasing either the LED output or bioluminescence (Figure 19). 100 μM diazirine-biotin, K562 cell lysate depleted of 0.1 mg / mL biotinylated protein, and 60 nM NanoLuc-HaloTag:Ir-9049 or HT 178 -cpNLuc- 179 : Reactants containing Ir-9049 were constructed in either a) wells of a UV-transparent 96-well plate that had been further irradiated at 455 nm for 5 minutes at 0 - 60 W (Efficiency Aggregators biophotoreactor), or b) wells of a white 96-well plate that had been further treated with 100 μM fluorofurimazine at a final concentration for 5 minutes while leaving the control wells untreated, in TBS (pH 7.5).

[0199] To evaluate the labeling efficiency, samples were collected, separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed as described in Example 1. Bands were quantified using ImageJ, and a calibration curve of labeling intensity versus watts was generated using further LED output titration. This analysis revealed that the NanoLuc-HaloTag:Ir-9049 and HT 178 -cpNLuc- 179 :Ir-9049 complexes were able to drive bioluminescence-induced protein labeling with efficiencies corresponding to 12.1 W and 55.4 W, respectively. This result further shows the efficiency of catalyst activation by localized energy transfer for overall irradiation.

[0200] Example 8 This example demonstrates the ability to expand the use of the bioluminescent photocatalyst system to other photoreactive moieties such as aryl azides that are more compatible with a wider range of downstream applications beyond diazirine. The structures of the aryl azide biotin analogs are shown in Figures 20A and 21A, and their synthesis is included in Example 13.

[0201] First, two photoreactive moieties, phenyl-trifluoro-methyldiazirine and phenyl-azide, were compared with respect to their biophysical properties and ability to undergo energy transfer events with the excited Ir-9049 catalyst. Absorbance profiles of 4 mM phenyl-diazirine-biotin and 20-fold lower concentration of phenyl-azide-biotin (i.e., 200 μM) in 8% or 1% DMSO, respectively, were monitored with a SPARK multimode plate reader (Figure 20B). Further, the ability of the two photoreactive moieties to undergo energy transfer events with the excited Ir-9049 catalyst was determined as described in Example 1 (Figure 20C). In general, the blue-shifted absorbance of phenyl-azide was associated with significantly more efficient light absorption ability and higher triplet state energy (TSE), but was associated with a decreased ability to undergo energy transfer events with the excited Ir-9049 catalyst.

[0202] The photocatalyst complex (i.e., NanoLuc-HaloTag:Ir-9049 and HT 178 -cpNLuc- 179 :Ir-9049) excited by either LED or bioluminescence was further investigated for its ability to undergo energy transfer events with phenyl-azide-biotin for subsequent crosslinking with a neighboring protein. 100 μM phenyl-azide-biotin (9069), 0.1 mg / mL biotinylated protein-depleted K562 cell lysate, and 60 nM NanoLuc or NanoLuc-HaloTag:Ir-9049 or HT 178 -cpNLuc- 179:The reactants containing Ir-9049 were constructed in either a) wells of a UV-transparent 96-well plate that was further irradiated with 455 nm light at 0 - 1.6 W for 5 minutes (Efficiency Aggregators biophotoreactor), or b) wells of a white 96-well plate that was further treated with 100 μM fluoroflumazine for 20 minutes while leaving the control wells untreated, in TBS (pH 7.5). To evaluate the labeling efficiency, samples were collected, separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed as described in Example 1. Unlike diazirene, both LED and bioluminescence showed distinct light-independent and light-dependent backgrounds. Photoactivation of phenyl-azide by only 455 nm light is likely to be minimal, yet it generates reactive intermediates with significantly longer lifetimes compared to those generated upon photoactivation of diazirene. These longer-lived intermediates can diffuse further before being quenched by water, resulting in a generally larger labeling radius and increased light-dependent background. Catalytic activation by either LED or bioluminescence energy transfer resulted in specific labeling of proximity model proteins (i.e., NanoLuc-HaloTag or HT 178 -cpNLuc-1 79 ). Notably, in the case of bioluminescence, specific proximity labeling was only evident for the chimeric photocatalyst complex, enabling more efficient bioluminescence-induced catalytic activation.

[0203] During the development of the embodiments of this specification, experiments were conducted to explore structural modifications to phenyl-azide moieties that can reduce the light-dependent background and increase labeling specificity by decreasing the lifetime of the photo-generated intermediates. Modifications were also explored that enable more efficient bioluminescence-induced photocatalyst activation by causing a red-shifted absorbance and then decreasing the triplet state energy. The structures and physicochemical properties of a subset of aryl azide analogs are shown in FIGS. 21A and 21B-C, respectively. The absorbance profile (200 μM in 1-2% DMSO) and the ability to undergo energy transfer events with excited Ir-9049 were determined as described above. In general, the analogs exhibit various red-shifted absorbances (6-42 nm) compared to phenyl-azide-biotin 9069, which is associated with an increased ability to undergo energy transfer events (1.3-10.5-fold), indicating easier activation by blue light and photocatalytic energy transfer, respectively.

[0204] Aryl azide-biotin analogs were further evaluated for the efficiency and specificity of bioluminescence-induced photocatalyst protein labeling and the light-independent background (FIG. 21D). For this purpose, 100 μM of aryl azide-biotin analog, 0.1 mg / mL of biotinylated protein-depleted K562 cell lysate, and 60 nM of HT 178 -cpNLuc- 179Reactants containing Ir-9049 were constructed in wells of a white 96-well plate in TBS (pH 7.5), and this was further treated with fluorofurimazine at a final concentration of 100 μM for an additional 45 minutes, with control wells left untreated. Next, the samples were collected, separated by SDS-PAGE, transferred to a nitrocellulose membrane, and analyzed as described in Example 1. The longer 45-minute incubation increased the light-independent background, but this background was significantly reduced by four structural modifications including naphthene (9043), hydroxyl substitution at the meta position of the phenyl ring (9046), and methyl substitution at vinyl groups designed to inhibit Michael addition (9162 and 9422). Furthermore, several substitutions on the phenyl ring increased the specificity of the label to varying degrees, presumably by interfering with the generation rate and / or lifetime of the reactive intermediate and resulting in an overall smaller label radius.

[0205] Finally, three analogs 9422 < 9162 < 9043, which showed the lowest light-independent background and increased the label radius, were further evaluated for the efficiency and specificity of LED-induced bioluminescence labeling and the light-dependent background (Figure 22). 100 μM of an aryl azide-biotin analog, a K562 cell lysate depleted of 0.1 mg / mL of biotinylated protein, and 60 nM of NanoLuc or HT 178 -cpNLuc- 179:Reactants containing any of Ir-9049 were constructed in either a) wells of a UV-transparent 96-well plate that had been further irradiated at 455 nm with 0 - 1.6 W for 10 minutes (Efficiency Aggregators biophotoreactor), or b) wells of a white 96-well plate that had been further treated with 100 μM fluorofurimazine for 20 minutes while leaving the control wells untreated. High LED-dependent background was observed for all three analogs, which is likely due to their red-shifted absorbance and overall high susceptibility to activation by blue light. The vinyl-phenyl-azide analog 9162 with a methyl modification on the vinyl group showed the highest light-dependent background, which correlated with its 20 nm red-shifted absorbance and high light absorption ability. Additional CN substitution on the phenyl ring (e.g., 9422) had a negligible effect on the absorbance profile but significantly reduced the light-dependent background, which is likely due to reducing the lifetime of the reactive intermediate. Subsequently, despite the high light-dependent background, the proximity HT 178 -cpNLuc- 179 LED-induced photocatalytic labeling was detected for all three analogs. On the other hand, bioluminescence induced efficient catalyst-dependent labeling with very little light-dependent background, further demonstrating the advantage of a mild and endogenous localized bioluminescence source. Additionally, the lifetime of the photo-generated reactive intermediate resulted in a broad labeling specificity that would be useful for a variety of applications designed to label specific proteins compared to the surrounding environment.

[0206] Example 9 This example demonstrates the versatility provided by a bioluminescent photocatalyst complex that depends on the LgBiT / HiBiT complementation reporter. Such a configuration provides a modality-independent approach for the proximity between the photocatalyst complex and the protein of interest genetically fused to HiBiT, as well as for higher spatial control over photocatalytic reactivity. First, the properties of a bioluminescent complementation reporter containing either the VS-HiBiT peptide and HaloTag-LgBiT or LgBiT-HaloTag were compared (Figs. 23A–C). To measure complementation affinity, equal amounts of VS-HiBiT peptide serially diluted to final concentrations of 200–0 nM in TBS + 0.01% BSA and HaloTag-LgBiT or LgBiT-HaloTag diluted to a final concentration of 0.2 nM in TBS + 0.01% BSA were combined in the wells of a white 96-well plate and mixed for 30 min. After treatment with either 10-fold furimazine or fluorofurimazine at a final concentration of 20 μM for 3 min, bioluminescence was measured with a GloMax® Discover plate reader (Promega), and the binding affinity (Kd) was derived from the saturation binding curve of luminescence versus VS-HiBiT concentration (Fig. 23A). To measure luminance and BRET efficiency, HaloTag-LgBiT and LgBiT-HaloTag, either unlinked or linked to a HaloTag TMR-fluorescent ligand, were diluted to a final concentration of 12 nM in TBS + 0.01% BSA and combined with an equal amount of VS-HiBiT peptide diluted to a final concentration of 120 nM in TBS + 0.01% BSA, and mixed for 30 min to allow complementation. After treatment with either 10-fold furimazine or fluorofurimazine at a final concentration of 20 μM for 3 min, the raw luminescence (total RLU, Fig. 23B) or filtered luminescence of the donor (e.g., 450 nm / 8 nm BP) and acceptor (600 nm LP) emissions was measured with a GloMax® Discover plate reader (Promega). The BRET ratio was further calculated for each sample by dividing the acceptor emission value by its donor emission value (Fig. 23C).Both constructs had similar brightness and preference for furimazine as a substrate, but the VS-HiBiT / LgBiT-HaloTag construct showed three-fold higher complementation affinity and resulted in 1.5- to 2-fold higher BRET efficiency (Figure 23C). This is consistent with higher BRET in the fusion orientation of NanoLuc-HaloTag, further suggesting that this fusion orientation results in an increase in proximity between the luminescent substrate binding site and the binding fluorescent ligand.

[0207] The LgBiT-HaloTag was further conjugated to Ir-9049, and its ability to drive labeling of the photocatalytic protein by complementation with HiBiT genetically fused to the protein of interest was evaluated (Figure 23D). HEK293 cell lysates with an estimated expression of 2000 nM of FKBP-HiBiT were serially diluted into control HEK293 cell lysates to generate four-fold lysate solutions with expression levels of 960 nM, 480 nM, 240 nM, and 12 nM while maintaining a constant concentration of total protein. 20 μL of the four-fold serial dilution cell lysates were combined with 20 μL of a four-fold LgBiT-HaloTag:Ir-9049 (i.e., 240 nM) solution in wells of a UV-transparent or white 96-well plate and mixed for 15 minutes for complementation. After treatment with 10-fold diazirine-biotin at a final concentration of 100 μM, the UV-transparent plate was subjected to 455 nm irradiation at 0 - 3.2 W for 15 minutes (Efficiency Aggregators bioreactor), the white plate was further treated with either 100 μM of furimazine or fluorofurimazine for an additional 30 minutes, and the control wells were left untreated. Samples were then collected, separated by SDS-PAGE, transferred to a nitrocellulose membrane, and analyzed as described in Example 1. Light-dependent labeling of both LgBiT-HaloTag and FKBP-HiBiT was detected by irradiation or treatment with either furimazine or fluorofurimazine. The highest labeling was achieved using bioluminescence as the endogenous light source and fluorofurimazine (although not the preferred substrate for HiBiT / LgBiT) as the substrate. This indicates that substrate properties other than brightness, such as substrate and signal stability over time, can play a role in labeling efficiency. Furthermore, equivalent fluorofurimazine-induced labeling of both LgBiT-HaloTag and FKBP-HiBiT over a 0 - 4-fold molar excess of HiBiT indicates that efficient complementation drives proximity labeling of FKBP-HiBiT.

[0208] Example 10 This example demonstrates the ability to construct a bioluminescent photocatalyst complex intracellularly and use it to drive the labeling of neighboring proteins with cleavable biotin for subsequent enrichment on streptavidin beads (Figure 24). As shown in Figure 15 above, the combination of rapid and highly specific binding to HaloTag by its covalent attachment, along with the minimal impact of chloroalkane on the cell permeability of Ir-9049, enables the conjugation to HaloTag and subsequent construction of the bioluminescent photocatalyst complex in live cells. Furthermore, proximity labeling using diazirine-biotin cleavable by palladium (Figure 24A) reduces the intrinsic background enrichment due to endogenous biotinylated proteins.

[0209] HeLa cells were transfected with a DNA construct encoding NanoLuc-HaloTag, 2x10 5Plated in wells of a 6-well plate at cells / mL and incubated overnight at 37 °C with 5% CO₂. The next day, the plates were treated with the Ir-9049 catalyst at a final concentration of 2 μM for 60 minutes to construct a bioluminescent photocatalyst complex. To remove the excess unreacted Ir-9049 catalyst, the cells were washed twice with HBSS buffer for 15 minutes each. The final HBSS wash was replaced with Opti-MEM medium supplemented with 2% serum and 20 μM cleavable diazirine-biotin. After a 30-minute incubation, the plates were exposed to 455 nm LED irradiation (3.2 watts) for 15 minutes, treated with 20 μM fluorofurimazine for 45 minutes, or left untreated (light-free control). To remove the excess unreacted cleavable diazirine-biotin, the cells were washed twice with HBSS buffer for 15 minutes each. The final HBSS wash was replaced with 1 mL of mammalian lysis buffer (Promega) supplemented with 10-fold diluted 10× RQ1-DNase buffer (Promega), 50-fold diluted RQ1-DNase (Promega), and 100-fold diluted protease inhibitor cocktail (Promega). After a 30-minute incubation at room temperature with constant mixing, the cell lysates were collected, and the biotinylated proteins were captured on 75 μL of high-performance Magne™ streptavidin beads (Promega), while washing away non-specific interactions. The labeled proteins were then released by incubation with a palladium cleavage reagent (Promega) for 30 minutes, separated on SDS-PAGE, transferred to a PVDF membrane, and subjected to Western analysis using an antibody against HaloTag (Promega). Western blot (Figure 24B) revealed efficient photocatalyst labeling in a complex cellular environment by either LED or bioluminescence.

[0210] Example 11 This example demonstrates the versatility of a bioluminescent photocatalyst complex constructed intracellularly and combined with a two-step labeling approach (Figure 25). Proximity protein labeling by click handles provides the flexibility to introduce diverse functions via copper-free bioorthogonal ligation. Furthermore, this approach may minimize potential interference with the photoredox catalysis of functional moieties such as fluorophores.

[0211] HeLa cells were transfected with a DNA construct encoding NanoLuc-HaloTag diluted 10-fold in promoterless carrier DNA and plated in flasks at 2 × 10 5 cells / mL and incubated at 37 °C and 5% CO2 for 16–18 h. The next day, the cells were collated and 2 × 10 5Re-plated in 24-well plates at cells / mL and incubated overnight at 37 °C and 5% CO2. The next day, the plates were treated with either the Ir-9049 catalyst or chloroalkane-biotin (control) at a final concentration of 2 μM to construct the bioluminescent photocatalyst complex. Subsequently, the cells were washed twice with HBSS buffer for 15 minutes each to remove the excess unreacted Ir-9049 catalyst or chloroalkane-biotin. The final HBSS wash was replaced with Opti-MEM medium supplemented with 2% serum and 20 μM diazirine-TCO (trans-cyclooctene). After a 30-minute incubation, the plates were exposed to LED irradiation at 455 nm (1.6 watts) for 15 minutes or treated with 20 μM fluoroflumazine for 45 minutes. To remove the excess unreacted diazirine-TCO, the cells were washed twice with HBSS buffer for 15 minutes each. The final HBSS wash was replaced with Opti-MEM medium supplemented with 2% serum and 1 μM tetrazine-Janelia-549 fluorophore conjugate (Tocris) and incubated for 15 minutes to ligate the TCO-tetrazine. After the cells were finally washed twice, they were imaged with a BZ-X800 Analyzer (Keyence). Fluorescent images revealed specific LED- or bioluminescence-driven photocatalyst labeling with signals higher than the background.

[0212] Example 12 In this example, the synthesis of the catalysts described herein will be explained. [Table 2] [Table 3]

[0213] Synthesis of Ir catalyst: [Chemical formula] {Ir[dFCF3ppy]2Cl}2 is commercially available from Strem: www.strem.com / catalog / v / 77-0468 / 31 / iridium_870987-64-7, and {Ir[dFCF3(CO2H)ppy]2Cl}2 was synthesized according to the procedures reported in the literature: Science 367, 1091-1097 (2020).

[0214] GP1: The bi-Ir-Cl complex (0.1 mmol, 1.0 equiv) was combined with AgOTf (53 mg, 0.2 mmol, 2.0 equiv) in CH3CN (5 mL). The mixture was stirred overnight at room temperature in the dark. Next, the resulting suspension was filtered through Celite and concentrated. The residue was redissolved in DCM / MeOH (1 / 1, 10 mL), filtered through Celite, and concentrated to obtain intermediate 3 or 4 as a yellow film, which was used without further purification.

[0215] To a solution of intermediate 3 or 4 (0.1 mmol, 1.0 equiv) in DCM / MeOH (1 / 1, 2 mL), bpy was added as the reactant (0.12 mmol, 1.2 equiv). Then, the reaction mixture was stirred at room temperature for 16 h. LC-MS indicated that intermediate 3 or 4 was completely converted. The solution was evaporated on Celite and purified by silica gel chromatography.

[0216] Ir-8673: 1 H NMR (400 MHz, DMSO-d6) δ 8.81 (d, J = 11.2 Hz, 2H), 8.34 (s, 2H), 7.97 (t, J = 5.9 Hz, 2H), 7.89 (d, J = 6.0 Hz, 1H), 7.81 (d, J = 5.9 Hz, 1H), 7.34 (s, 2H), 7.09 (t, J = 11.0 Hz, 2H), 5.86 (d, J = 8.2 Hz, 2H), 3.83 - 3.53 (m, 10H), 3.38 (t, J = 5.2 Hz, 2H), 3.13 (s, 3H), 1.59 (s, 6H), 1.56 (s, 6H). LRMS [M + H] +1215.1。

[0217] Ir-8844: 1 H NMR (400 MHz, Methylene chloride-d2) δ 8.48 (d, J = 8.7 Hz, 2H), 8.37 (d, J = 2.5 Hz, 2H), 8.07 (d, J = 8.9 Hz, 2H), 7.70 (d, J = 6.2 Hz, 4H), 7.12 - 6.98 (m, 2H), 6.73 - 6.57 (m, 2H), 5.73 (dd, J = 8.1, 2.2 Hz, 2H), 4.65 - 4.43 (m, 4H), 3.94 (d, J = 4.5 Hz, 4H), 3.79 - 3.42 (m, 16H). LRMS [M] + 1161.1。

[0218] Ir-8870: 1 H NMR (400 MHz, Methanol-d4) δ 8.77 (s, 2H), 8.59 (d, J = 9.0 Hz, 2H), 8.33 (d, J = 8.8 Hz, 2H), 8.03 (d, J = 5.7 Hz, 2H), 7.76 (s, 2H), 7.70 (d, J = 5.7 Hz, 2H), 6.89 - 6.70 (m, 2H), 5.80 (d, J = 8.3 Hz, 2H), 4.92 (s, 4H). LRMS [M] + 924.8。

[0219] Ir-8871: 1 H NMR (400 MHz, Methanol-d4) δ 9.32 (s, 2H), 8.60 (d, J = 8.9 Hz, 2H), 8.32 (dd, J = 15.8, 7.3 Hz, 4H), 8.19 (d, J = 5.7 Hz, 2H), 7.78 (s, 2H), 6.86 (t, J = 10.9 Hz, 2H), 5.77 (d, J = 8.2 Hz, 2H), 4.08 (s, 6H). LRMS [M] + 980.8。

[0220] Synthesis of bpy-1 [Chemical formula] Intermediate 8 was synthesized from commercially available starting material 7 according to the literature procedure: Science 367, 1091-1097 (2020).

[0221] Intermediate 9: To a THF solution (7 mL) of Intermediate 8 (200 mg, 0.7 mmol, 1.0 equivalent) was added NaH (60 wt%, 56 mg, 1.4 mmol, 2.0 equivalents). The mixture was stirred at room temperature for 30 minutes. To the suspension was added dropwise NaI (11 mg, 0.07 mmol, 0.1 equivalent) and 2-(2-(2-(2-chloroethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran (350 mg, 1.4 mmol, 2.0 equivalents) in DMF (3 mL) over 10 minutes. The mixture was then heated at 60 °C for 48 hours. The reaction was cooled and quenched by the addition of saturated aqueous NH4Cl solution (10 mL). The quenched reaction was then concentrated in vacuo to remove the organic solvent and extracted with EtOAc (20 × 3 mL). The combined organic layers were washed with H2O (50 mL), brine (50 mL), dried over Na2SO4, and concentrated to give the crude product, which was used in the next step without further purification.

[0222] bpy-1: Intermediate 9 (50 mg, 0.1 mmol, 1.0 equivalent) and TsOH·H2O (19 mg, 0.1 mmol, 1.0 equivalent) were dissolved in MeOH (4 mL). The solution was stirred at room temperature for 2 hours. LC-MS indicated complete conversion. The reaction was concentrated through celite and the desired product was isolated using silica gel chromatography. 11H NMR (400 MHz, chloroform-d) δ 8.64 (d, J = 5.1 Hz, 2H), 8.38 (d, J = 5.5 Hz, 2H), 7.41 (ddd, J = 19.4, 5.0, 2.2 Hz, 2H), 3.83 - 3.53 (m, 10H), 3.38 (t, J = 5.2 Hz, 2H), 3.13 (s, 3H), 1.59 (s, 6H), 1.56 (s, 6H). LRMS [M + H] + 419.5。

[0223] Synthesis of bpy-2

Chem.

[0224] Synthesis of Ir-8810

Chem.

[0225] Intermediate 14: To a solution of Intermediate 13 (12 mg, 21 μmol, 1.0 equiv) in ACN (2 mL) were added NEt3 (34 μL, 0.21 mmol, 10 equiv) and chloroalkane Intermediate 14 (25 mg, 25 μmol, 1.2 equiv). The reaction mixture was stirred overnight at room temperature and concentrated over Celite. The desired product was isolated using silica gel chromatography. 1 H NMR (400 MHz, chloroform-d) δ 8.68 (d, J = 5.2 Hz, 2H), 8.42 (s, 2H), 7.46 (dd, J = 26.0, 5.1 Hz, 2H), 5.32 (br s, 2H), 4.25 (d, J = 4.9 Hz, 4H), 3.83 - 3.29 (m, 30H), 3.17 (s, 3H), 1.80 (p, J = 6.9 Hz, 2H), 1.66 (s, 6H), 1.62 (s, 6H), 1.52 - 1.15 (m, 4H). LRMS [M + H] + 844.5.

[0226] Ir-8810: Intermediate 4 (10.3 mg, 10 μmol, 1.0 equivalent) and Intermediate 14 (11 mg, 13 μmol, 1.3 equivalents) were dissolved in DCM / MeOH (1 / 1, 2 mL). The solution was stirred overnight at room temperature. The desired product was isolated using preparative HPLC with 0.1% TFA in H2O and ACN as the mobile phase. LRMS [M+H] + 1640.1。

[0227] Synthesis of Ir-8972

Chemical Structure

[0228] To a solution of the crude product 15 (54 mg, 87 μmol, 1.0 equivalent) in ACN (2 mL) were added the chloroalkane amine reagent (15 mg, 92 μmol, 1.1 equivalents) and NEt3 (0.2 mL). The solution was stirred overnight at room temperature and concentrated through celite. The desired product was isolated using silica gel chromatography. 11H NMR (400 MHz, methanol-d4) δ 8.47 (d, J = 5.8 Hz, 2H), 7.88 (d, J = 2.6 Hz, 2H), 7.07 (dd, J = 5.6, 2.6 Hz, 2H), 4.35 (t, J = 4.4 Hz, 4H), 4.17 (d, J = 5.0 Hz, 2H), 3.93 (q, J = 3.3 Hz, 4H), 3.77 - 3.64 (m, 12H), 3.61 - 3.43 (m, 12H), 3.30 - 3.28 (m, 2H), 1.76 (t, J = 7.1 Hz, 2H), 1.66 - 1.30 (m, 6H). LRMS [M + H] + 702.3

[0229] Ir-8972: Intermediate 3 (10.6 mg, 11 μmol, 1.0 equiv) and Intermediate 16 (8 mg, 12 μmol, 1.1 equiv) were dissolved in DCM / MeOH (1 / 1, 2 mL). The solution was stirred overnight at room temperature. The desired product was isolated using preparative HPLC with 0.1% TFA in H2O and ACN as the mobile phase. 1 1H NMR (400 MHz, methanol-d4) δ 8.56 (d, J = 9.0 Hz, 2H), 8.42 - 8.24 (m, 4H), 7.82 (d, J = 6.3 Hz, 4H), 7.25 (d, J = 6.8 Hz, 2H), 6.79 (t, J = 10.9 Hz, 2H), 5.77 (d, J = 8.1 Hz, 2H), 4.45 (d, J = 4.7 Hz, 4H), 4.07 (s, 2H), 3.90 (d, J = 4.7 Hz, 4H), 3.77 - 3.40 (m, 24H), 3.30 - 3.23 (d, J = 6.1 Hz, 2H), 1.78 - 1.66 (m, 2H), 1.56 (t, J = 7.2 Hz, 2H), 1.43 - 1.35 (m, 4H). LRMS [M] + 1410.4

[0230] Synthesis of Ir-8973 [Chemistry] Intermediate 17: To a solution of crude product 15 (30 mg, 49 μmol, 1.0 equiv) in ACN (2 mL), a chloroalkane amine reagent (23 mg, 49 μmol, 1.0 equiv) and NEt3 (0.2 mL) were added. The solution was stirred overnight at room temperature and concentrated over celite. The desired product was isolated using silica gel chromatography. 1 H NMR (400 MHz, methanol-d4) δ 8.47 (d, J = 5.8 Hz, 2H), 7.88 (t, J = 2.7 Hz, 2H), 7.06 (d, J = 5.7 Hz, 2H), 4.34 (d, J = 4.8 Hz, 4H), 4.16 (d, J = 4.9 Hz, 4H), 3.93 (h, J = 2.9 Hz, 4H), 3.82 - 3.44 (m, 38H), 3.33 - 3.25 (m, 2H), 1.77 (t, J = 7.3 Hz, 2H), 1.60 (t, J = 7.1 Hz, 2H), 1.44 (dq, J = 23.7, 7.7 Hz, 4H). LRMS [M + H] + 921.4.

[0231] Ir-8973: Intermediate 3 (4.3 mg, 4.6 μmol, 1.0 equiv) and Intermediate 17 (4.2 mg, 4.6 μmol, 1.0 equiv) were dissolved in DCM / MeOH (1 / 1, 2 mL). The solution was stirred overnight at room temperature. The desired product was isolated by silica column using DCM / MeOH as eluent. 11H NMR (400 MHz, methanol-d4) δ 8.56 (d, J = 8.9 Hz, 2H), 8.42 - 8.25 (m, 4H), 7.82 (d, J = 4.9 Hz, 4H), 7.25 (d, J = 6.4 Hz, 2H), 6.97 - 6.68 (m, 4H), 5.85 - 5.68 (m, 2H), 4.49 - 4.41 (m, 4H), 4.15 - 4.04 (m, 4H), 3.94 - 3.85 (m, 4H), 3.73 - 3.41 (m, 38H), 3.30 - 3.24 (m, 2H), 1.81 - 1.68 (m, 2H), 1.63 - 1.50 (m, 2H), 1.50 - 1.26 (m, 4H). LRMS [M + H] + 1410.4。

[0232] Synthesis of Ir-9049

Chem.

[0233] To a DMF solution (5 mL) of bpy-8:21 (25 mg, 75 μmol, 1.0 equiv), bromoethanol (47 mg, 374 μmol, 5.0 equiv), NaI (1.2 mg, 7.5 μmol, 0.1 equiv), and K2CO3 (31 mg, 224 μmol, 3.0 equiv) were added. The mixture was stirred at 60 °C overnight. After cooling, the mixture was diluted with EtOAc (50 mL), filtered through celite, and the filtrate was concentrated under reduced pressure to give the crude product. The desired product was isolated by silica gel chromatography. 1 1H NMR (400 MHz, chloroform-d) δ 8.66 - 8.44 (m, 2H), 8.13 - 7.75 (m, 2H), 7.00 - 6.85 (m, 2H), 6.79 (brs, 1H), 4.37 - 4.20 (m, 4H), 4.04 - 3.58 (m, 12H), 3.34 (s, 3H). LRMS [M + H] + 379.4

[0234] To a THF solution (4 mL) of bpy-8-CA:bpy-8 (16 mg, 0.04 mmol, 1.0 equiv), pyridine (0.5 mL) and p-nitrophenyl chloroformate (10 mg, 0.05 mmol, 1.2 equiv) were added. The solution was stirred at room temperature overnight. The reaction mixture was diluted with DCM (10 mL), filtered through celite, and the filtrate was concentrated under reduced pressure to give the crude product, which was used in the next step without further purification.

[0235] To the solution of the crude product from the previous step in ACN (2 mL), a chloroalkane amine reactant (39 mg, 150 μmol, 3 equiv) and NEt3 (0.2 mL) were added. The solution was stirred overnight at room temperature and concentrated over celite. The desired product was isolated using silica gel chromatography. 1 H NMR (400 MHz, methanol-d4) δ 8.48 - 8.46 (m, 2H), 7.95 - 7.74 (m, 2H), 7.17 - 6.92 (m, 2H), 4.47 - 4.25 (m, 6H), 3.95 - 3.42 (m, 20H), 3.35 (s, 3H), 3.29 - 3.17 (m, 2H), 1.77 - 1.50 (m, 2H), 1.63 - 1.50 (m, 2H), 1.47 - 1.21 (m, 4H). LRMS [M + H] + 628.3.

[0236] Ir-9049: Intermediate 3 (14 mg, 15 μmol, 1.0 equiv) and bpy-8-CA (9.4 mg, 15 μmol, 1.0 equiv) were dissolved in DCM / MeOH (1 / 1, 2 mL). The solution was stirred overnight at room temperature. The desired product was isolated by silica column using DCM / MeOH as the eluent. 1 H NMR (400 MHz, methanol-d4) δ 8.58 (d, J = 8.9 Hz, 2H), 8.39 (s, 2H), 8.33 (d, J = 8.9 Hz, 2H), 7.84 (d, J = 6.9 Hz, 4H), 7.27 (s, 2H), 6.81 (t, J = 10.9 Hz, 2H), 5.79 (d, J = 8.0 Hz, 2H), 4.47 - 4.25 (m, 4H), 3.92 - 3.78 (m, 2H), 3.75 - 3.58 (m, 20H), 3.32 (s, 3H), 1.78 - 1.70 (m, 2H), 1.63 - 1.50 (m, 2H), 1.47 - 1.37 (m, 4H). LRMS [M + H] + 1336.7.

[0237] Synthesis of Ir-9050 [Chemical formula] To a solution of 21 (25 mg, 75 μmol, 1.0 equiv) in bpy-9:DMF (5 mL), 2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol (63 mg, 374 μmol, 5.0 equiv), NaI (1.2 mg, 7.5 μmol, 0.1 equiv) and K2CO3 (31 mg, 224 μmol, 3.0 equiv) were added. The mixture was stirred at 60 °C overnight. After cooling, the mixture was diluted with EtOAc (50 mL), filtered through celite and the filtrate was concentrated under reduced pressure to obtain the crude product. The desired product was isolated by silica gel chromatography. 1 H NMR (400 MHz, methanol-d4) δ 8.60 - 8.35 (m, 2H), 7.99 - 7.55 (m, 2H), 7.15 - 6.94 (m, 2H), 4.38 - 4.27 (m, 4H), 3.90 (d, J = 4.7 Hz, 4H), 3.76 - 3.47 (m, 16H), 3.30 (s, 3H). LRMS [M + H] + 467.5

[0238] To a solution of bpy-9 (16 mg, 0.35 mmol, 1.0 equiv) in THF (4 mL), pyridine (0.5 mL) and p-nitrophenyl chloroformate (8.3 mg, 0.04 mmol, 1.2 equiv) were added. The solution was stirred at room temperature overnight. The reaction mixture was diluted with DCM (10 mL), filtered through celite and the filtrate was concentrated under reduced pressure to obtain the crude product, which was used in the next step without further purification.

[0239] To a solution of the crude product from the previous step in ACN (2 mL), chloroalkane amine reagent (54 mg, 150 μmol, 3 equiv) and NEt3 (0.2 mL) were added. The solution was stirred at room temperature overnight and concentrated through celite. The desired product was isolated using silica gel chromatography. LRMS[M+H] + 1154.6

[0240] Ir-9050: Intermediate 3 (14 mg, 15 μmol, 1.0 equiv) and bpy-9-CA (9.4 mg, 15 μmol, 1.0 equiv) were dissolved in DCM / MeOH (1 / 1, 2 mL). The solution was stirred overnight at room temperature. The desired product was isolated by silica column using DCM / MeOH as the eluent. 1 H NMR (400 MHz, methanol-d4) δ 8.58 (d, J = 8.9 Hz, 2H), 8.39 (s, 2H), 8.33 (d, J = 8.9 Hz, 2H), 7.84 (d, J = 6.9 Hz, 4H), 7.27 (s, 2H), 6.81 (t, J = 10.9 H

[0241] Synthesis of Ru catalyst: Ru-8975: The desired product was isolated as the diacetate. 1 H NMR (400 MHz, methanol-d4) δ 8.90 (d, J = 8.5 Hz, 1H), 8.83 - 8.70 (m, 4H), 8.31 (d, J = 8.5 Hz, 1H), 8.25 - 8.03 (m, 5H), 7.94 (dd, J = 12.4, 5.6 Hz, 2H), 7.78 (s, 1H), 7.75 - 7.62 (m, 3H), 7.60 - 7.48 (m, 3H), 7.36 (d, J = 6.4 Hz, 2H), 7.03 (s, 1H), 3.81 (t, J = 6.0 Hz, 2H), 3.59 (t, J = 7.1 Hz, 2H), 2.07 (t, J = 6.6 Hz, 2H), 1.90 (s, 6H). LRMS [M] 2+ / 2 333.6.

[0242] Synthesis of phenanthroline-1

Chemical Structure

[0243] Phenanthroline intermediate 12 (120 mg, 0.33 mmol, 1.0 equiv) was dissolved in MeOH / 6N aqueous HCl solution (1 / 1, 6 mL). The solution was stirred at room temperature for 6 hours. LC-MS indicated complete conversion. The desired product, phenanthroline-1, was isolated by silica gel chromatography. 11H NMR (400 MHz, methanol-d4) δ 9.07 (d, J = 4.2 Hz, 1H), 8.68 (dd, J = 20.8, 6.3 Hz, 2H), 8.15 (d, J = 8.1 Hz, 1H), 7.74 (dd, J = 9.0, 4.1 Hz, 1H), 7.61 - 7.46 (m, 1H), 6.80 (s, 1H), 3.91 - 3.80 (m, 2H), 3.52 (t, J = 7.1 Hz, 2H), 2.14 - 1.99 (m, 2H). LRMS [M + H] + 254.3。

[0244] Synthesis of Ru-8974

Chem.

[0245] To a solution of the crude product 18 (26 mg, 62 μmol, 1.0 equiv) in ACN (2 mL) were added chloroalkane amine reagent (16 mg, 62 μmol, 1.0 equiv) and NEt3 (0.2 mL). The solution was stirred at room temperature overnight and concentrated through celite. The desired product was isolated using silica gel chromatography. 11H NMR (400 MHz, methanol-d4) δ 9.07 (d, J = 4.3 Hz, 1H), 8.71 (t, J = 7.6 Hz, 2H), 8.16 (d, J = 8.1 Hz, 1H), 7.75 (dd, J = 8.8, 4.1 Hz, 1H), 7.68 - 7.44 (m, 1H), 6.80 (s, 1H), 4.29 (t, J = 6.3 Hz, 2H), 3.60 - 3.25 (m, 14H), 2.25 - 2.15 (d, J = 6.6 Hz, 2H), 1.81 - 1.68 (m, 2H), 1.63 - 1.50 (m, 2H), 1.43 - 1.17 (m, 6H). LRMS [M + H] + 503.3。

[0246] Ru-8974: (bpy)2RuCl2 (7.0 mg, 14 μmol, 1.1 equiv) and intermediate 19 (6.6 mg, 13 μmol, 1.0 equiv) were dissolved in MeOH (2 mL). The solution was stirred at 60 °C overnight. The desired product was isolated by silica column using DCM / MeOH as the eluent. 1 1H NMR (400 MHz, methanol-d4) δ 8.92 (d, J = 8.6 Hz, 1H), 8.81 - 8.60 (m, 4H), 8.32 (d, J = 8.4 Hz, 1H), 8.12 (dt, J = 28.6, 9.3 Hz, 5H), 7.94 (dd, J = 12.0, 5.6 Hz, 2H), 7.82 - 7.62 (m, 4H), 7.55 (p, J = 6.6, 5.9 Hz, 3H), 7.36 (d, J = 6.6 Hz, 2H), 7.04 (s, 1H), 4.36 - 4.15 (m, 2H), 3.67 - 3.41 (m, 14H), 3.30 - 3.17 (m, 2H), 2.25 - 2.15 (m, 2H), 1.81 - 1.68 (m, 2H), 1.63 - 1.50 (m, 2H), 1.43 - 1.17 (m, 6H). LRMS [M] 2+ / 2 458.1。

[0247] Synthesis of Ru-9003 [Chemical formula] Ru-9003: To a solution of Ru-8975 (10 mg, 13 μmol, 1.0 equiv) in ACN (2 mL) were added pyridine (0.5 mL) and p-nitrophenyl chloroformate (7.7 mg, 39 μmol, 3.0 equiv). The solution was stirred at room temperature overnight. The reaction mixture was diluted with DCM (10 mL), filtered through celite, and the filtrate was concentrated under reduced pressure to obtain crude product intermediate 20, which was used in the next step without further purification.

[0248] The solution of crude intermediate 20 was redissolved in ACN (3 mL), and chloroalkane intermediate (32 mg, 39 μmol, 3.0 equiv) and NEt3 (0.2 mL) were added. The solution was stirred at room temperature overnight. The desired product was isolated by silica column using DCM / MeOH as the eluent. 1 H NMR (400 MHz, methanol-d4) δ 8.93 (d, J = 8.6 Hz, 1H), 8.81 - 8.66 (m, 6H), 8.33 (d, J = 8.3 Hz, 1H), 8.23 - 8.05 (m, 7H), 7.95 (dd, J = 12.0, 5.6 Hz, 3H), 7.79 (t, J = 7.0 Hz, 1H), 7.68 (dd, J = 15.9, 9.3 Hz, 4H), 7.63 - 7.51 (m, 5H), 7.38 (d, J = 7.0 Hz, 3H), 7.05 (s, 1H), 4.34 - 4.22 (m, 2H), 4.19 - 4.05 (m, 2H), 3.82 - 3.42 (m, 22H), 3.30 - 3.17 (m, 2H), 2.25 - 2.15 (m, 2H), 1.80 - 1.66 (m, 2H), 1.66 - 1.52 (m, 2H), 1.45 - 1.19 (m, 4H). LRMS [M] 2+ / 2 545.8. z, 2H), 5.79 (d, J = 8.0 Hz, 2H), 4.49 - 4.41 (m, 4H), 4.15 - 4.04 (m, 4H), 3.94 - 3.85 (m, 4H), 3.79 - 3.47 (m, 57H), 3,24 - 3.15 (m, 2H), 1.90 - 1.67 (m, 2H), 1.67 - 1.47 (m, 2H), 1.46 - 1.28 (m, 4H). LRMS [M + H] + 1863.3.

[0249] Synthesis of organic catalyst: Synthesis of PS-9167

Chem.

[0250] Step 2. DBF (400 mg, 0.75 mmol, 1 equiv.) was added to 2 mL of acetic anhydride. 0.75 mL of dehydrated pyridine was added to the mixture. The suspension was stirred at 65 °C for 3 h until the starting material disappeared. The mixture was concentrated, redissolved in EtOAc, and washed with a saturated aqueous ammonium chloride solution. The organic layer was dried over Na2SO4, filtered, and concentrated to obtain 3′,6′-diacetoxy-4′,5′-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-carboxylic acid (426 mg, 92%) without further purification.

[0251] Step 3. 3’,6’-Diacetoxy-4’,5’-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9’-xanthene]-5-carboxylic acid (85 mg, 0.14 mmol, 1 eq) was dissolved in 2 mL of DCM. DIPEA, T3P, and 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate 2,2,2-trifluoroacetate were added to the solution. The mixture was stirred overnight at room temperature. The solvent was removed by rotary evaporator and the crude product was suspended in 2 mL of acetic anhydride. Dehydrated pyridine (0.5 mL) was added to the mixture and the suspension was stirred at 80 °C for 3 h to give a pale yellow solution. The mixture was concentrated by rotary evaporator, redissolved in ethyl acetate and washed with saturated aqueous ammonium chloride solution. The organic layer was dried over Na2SO4, filtered and concentrated by rotary evaporator. The pale yellow solid was purified by preparative HPLC to give the desired product 4’,5’-dibromo-5-((26-chloro-13-oxo-3,6,9,12,17,20-hexaoxa-14-azapentacosyl)carbamoyl)-3-oxo-3H-spiro[isobenzofuran-1,9’-xanthene]-3’,6’-diyl diacetate (54 mg, 38%). LRMS: [M+H] + 817.56。

[0252] Example 13 Synthesis of Compounds In this example, the synthesis of the compounds described in this specification including those shown in Table 1 will be described.

Table 4-1

Table 4-2

[0253] Synthesis of Diazirine-biotin Analogs: Synthesis of Compound 8672

Chem.

[0254] Synthesis of Compound 9107

Chemical Structure

[0255] Synthesis of Compound 9177 [Chemical formula] (E)-4-((Tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl (2-(2-(2-(((4-nitrophenoxy)carbonyl)oxy)ethoxy)ethoxy)ethyl)carbamate was prepared by Steps 1-4 according to the procedures described in the published literature (ACS Chem. Biol. 2016, 11, 9, 2608-2617.).

[0256] Step 5. Triethylamine (466 μL, 3.34 mmol, 3 eq) was added to a solution of (E)-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl (2-(2-(2-(((4-nitrophenoxy)carbonyl)oxy)ethoxy)ethoxy)ethyl)carbamate (570 mg, 1.11 mmol, 1 eq) and N-(2-aminoethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide hydrochloride (395 mg, 1.22 mmol, 1.1 eq). The resulting solution was stirred at room temperature for 20 h, at which point TLC analysis indicated complete consumption of the starting material. The solvent was removed under vacuum and the residue was purified by silica gel chromatography using MeOH / DCM to afford the desired product (E)-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl (10,15-dioxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-11,14-diazanonadecyl)carbamate (680 mg, 93%).

[0257] Step 6. (E)-4-((Tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl (10,15-dioxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-11,14-diazanonadecyl)carbamate (900 mg, 1.36 mmol, 1 equiv) was dissolved in EtOH in a 50 mL RBF. To this solution was added PPTS (34.2 mg, 136 μmol). The resulting solution was heated at 50 °C for 1 h. The solvent was removed under vacuum and the residue was purified by silica gel chromatography using MeOH / DCM to give the product (E)-4-hydroxybut-2-en-1-yl (10,15-dioxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-11,14-diazanonadecyl)carbamate (654 mg, 83%).

[0258] Step 7. (E)-4-Hydroxybut-2-en-1-yl (10,15-dioxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-11,14-diazanonadecyl)carbamate (640 mg, 1.11 equiv, 1 equiv) was dissolved in DCM. p-Nitrophenyl chloroformate (269 mg, 1.33 mmol, 1.2 equiv) and pyridine (146 μL, 1.81 mmol, 1.5 equiv) were added. The resulting solution was stirred at room temperature for 20 h. The solvent was removed by rotary evaporation and the crude product was loaded directly onto a silica gel column and purified by flash chromatography to give (E)-4-(((4-nitrophenoxy)carbonyl)oxy)but-2-en-1-yl (10,15-dioxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-11,14-diazanonadecyl)carbamate (450 mg, 55%).

[0259] Step 8. To a solution of (E)-4-(((4-nitrophenoxy)carbonyl)oxy)but-2-en-1-yl (10,15-dioxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-11,14-diazanonadecyl)carbamate (27 mg, 36.5 μmol, 1 equiv) in 3 mL of DCM and 1 mL of DMF were added (4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)methanamine hydrochloride (9.5 mg, 37.8 μmol, 1.04 equiv) and triethylamine (15.3 μL, 109.3 μmol, 3 equiv). The mixture was stirred overnight at room temperature. The solvent was removed by rotary evaporation and the crude product was directly loaded onto a silica gel column and purified by flash chromatography to give (E)-4-(((4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzyl)carbamoyl)oxy)but-2-en-1-yl (10,15-dioxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-11,14-diazanonadecyl)carbamate (16 mg, 54%). LRMS: [M+H] + 817.56。

[0260] Synthesis of aryl azide precursor compounds Synthesis of aryl azide 1:

Chemical formula

[0261] Synthesis of aryl azide 2:

Chemical formula

[0262] Synthesis of aryl azide 3:

Chemical Structure

[0263] Intermediate 6: To a solution of Intermediate 5 (63 mg, 0.2 mmol, 1.0 equiv) in MeOH (4 mL), Cu(OAc)2 (3.8 mg, 0.02 mmol, 0.1 equiv) and NaN3 (14 mg, 0.2 mmol, 1.0 equiv) were added at once. The solution was heated at 60 °C for 3 hours. LC-MS indicated complete conversion. After diluting the reaction with EtOAc (50 mL), saturated aqueous NH4Cl solution (10 mL) was added to quench. The aqueous layer was extracted with EtOAc (10 × 3 mL). The combined organic layers were washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4, and concentrated under reduced pressure. The desired product was purified by silica gel chromatography. 11H NMR (400 MHz, chloroform-d) δ 8.00 - 7.94 (m, 2H), 7.36 - 7.29 (m, 2H), 6.75 (dd, J = 13.8, 2.0 Hz, 1H), 6.29 (d, J = 13.8 Hz, 1H), 4.37 (q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H). LRMS [M + H] + 218.09。

[0264] Aryl azide 3: To a solution of intermediate 6 (25 mg, 0.12 mmol, 1.0 equiv) in THF (4 mL) was added LiOH (14 mg) previously dissolved in H2O (2 mL). The reaction mixture was stirred at room temperature for 3 h. LC-MS indicated complete conversion. The reaction was concentrated under reduced pressure to remove volatiles and diluted with H2O (20 mL). The aqueous suspension was adjusted to pH 4 and extracted with EtOAc (20 × 3 mL). The combined organic layers were washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4, and concentrated under reduced pressure to give the crude product, which was used in the next step without further purification. LRMS [M-H] - 188.05。

[0265] Synthesis of aryl azide 4:

Chemical formula

[0266] Intermediate 9: Intermediate 8 (285 mg, 1.0 mmol, 1.0 eq), CuI (9.5 mg, 0.05 mmol, 0.05 eq), and Na-ascorbic acid (20 mg, 0.1 mmol, 0.1 eq) were charged into a vial purged with N2. DMSO (5 mL) and DMEDA (17 μL, 0.15 mmol, 0.15 eq) were added to the mixture. Next, the mixture was stirred at room temperature for 15 minutes, and then NaN3 (98 mg, 1.5 mmol, 1.5 eq) was added. The reaction was heated at 100 °C for 16 hours. Next, the reaction was stirred at room temperature for 16 hours and quenched by adding saturated aqueous NH4Cl solution (20 mL). The quenched reaction was extracted with EtOAc (50 × 3 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to obtain the crude product. The desired product was isolated using silica gel chromatography. 1 1H NMR (400 MHz, chloroform-d) δ 7.90 (d, J = 16.1 Hz, 1H), 7.48 (d, J = 8.3 Hz, 1H), 6.74 - 6.60 (m, 1H), 6.56 - 6.38 (m, 2H), 4.25 (q, J = 7.2 Hz, 2H), 3.88 (s, 3H), 1.33 (t, J = 7.2 Hz, 3H). LRMS [M + H] + 248.10。

[0267] Aryl azide 4: To a solution of intermediate 9 (170 mg, 0.69 mmol, 1.0 equiv) in THF (4 mL) was added LiOH (32 mg, 1.38 mmol, 2.0 equiv) previously dissolved in H2O (2 mL). The reaction mixture was stirred at room temperature for 3 h. LC-MS indicated complete conversion. The reaction was concentrated under reduced pressure to remove volatiles, diluted with H2O (20 mL). The aqueous suspension was adjusted to pH 4 and extracted with EtOAc (20 × 3 mL). The combined organic layers were washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4, concentrated under reduced pressure to give the crude product. The product was isolated using silica gel chromatography. 1 H NMR (400 MHz, methanol-d4) δ 7.89 (d, J = 16.2, 1H), 7.60 (d, J = 8.4 Hz, 1H), 6.87 - 6.60 (m, 2H), 6.47 (dd, J = 16.1 Hz, 1H), 3.91 (s, 3H). LRMS [M - H] - 218.06.

[0268] Synthesis of aryl azide 5:

Chemical formula

[0269] Intermediate 12: The desired product was similarly synthesized according to the procedure for intermediate 9. 11H NMR (400 MHz, chloroform-d) δ 7.49 (d, J = 16.0 Hz, 1H), 7.23 - 7.16 (m, 2H), 7.05 - 6.92 (m, 1H), 6.63 (d, J = 6.0 Hz, 1H), 4.27 (q, J = 7.1 Hz, 2H), 3.92 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H). LRMS [M + H] + 248.10。

[0270] Aryl azide 5: Following the procedure for intermediate aryl azide 4, the desired product was similarly synthesized. 1 1H NMR (400 MHz, chloroform-d) δ 7.48 (d, J = 16.0 Hz, 1H), 7.25 - 7.16 (m, 2H), 7.10 - 6.95 (m, 1H), 6.63 (d, J = 6.0 Hz, 1H), 3.97 (s, 3H). LRMS [M - H] - 218.06。

[0271] Synthesis of aryl azide 6:

Chemical formula

[0272] Intermediate 15: To a solution of Intermediate 14 (73 mg, 0.22 mmol, 1.0 equiv) in MeOH (4 mL) were added Cu(OAc)2 (4.0 mg, 0.022 mmol, 0.1 equiv) and NaN3 (14 mg, 0.22 mmol, 1.0 equiv). The reaction mixture was stirred at 60 °C for 30 minutes. Complete conversion was indicated by LC-MS. The reaction mixture was diluted with EtOAc (50 mL), quenched by addition of saturated aqueous NH4Cl solution (10 mL). The aqueous layer was extracted with EtOAc (10 × 3 mL). The combined organic layers were washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4, and concentrated under reduced pressure. The desired product was purified by silica gel chromatography and isolated as a mixture with the protodeboronation byproduct. 1 1H NMR (400 MHz, chloroform-d) δ 7.50 (d, J = 15.9 Hz, 1H), 7.08 (d, J = 8.7 Hz, 2H), 6.35 (d, J = 15.9 Hz, 1H), 4.32 - 4.23 (q, J = 7.2 Hz, 2H), 1.34 (t, J = 7.2 Hz, 3H). LRMS [M + H] + 254.07。

[0273] Aryl azide 6: To a solution of intermediate 15 (30 mg, 0.12 mmol, 1.0 equiv) in THF (4 mL) was added LiOH (80 mg, 2.0 mmol, 17 equiv) previously dissolved in H2O (2 mL). The reaction mixture was stirred at room temperature for 3 h. LC-MS indicated complete conversion. The reaction was concentrated under reduced pressure to remove volatiles, diluted with H2O (20 mL). The aqueous suspension was adjusted to pH 4 and extracted with EtOAc (20 × 3 mL). The combined organic layers were washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4, concentrated under reduced pressure to give the crude product. The desired product was isolated using silica gel chromatography. 1 1H NMR (400 MHz, methanol-d4) δ 7.54 (d, J = 15.9 Hz, 1H), 7.34 (d, J = 9.2 Hz, 2H), 6.49 (d, J = 16.0 Hz, 1H). LRMS [M - H] - 224.03.

[0274] Synthesis of aryl azide 7:

Chemical formula

[0275] Intermediate 18: Following the procedure of Intermediate 15, the desired product was similarly synthesized. The desired product was purified by silica gel chromatography and isolated as a mixture with the protodeboronation by-product. LRMS [M+H] + 236.18

[0276] Aryl azide 7: Following the procedure of Aryl azide 6, the desired product was similarly synthesized. 1 H NMR (400 MHz, methanol-d4) δ 7.61 (d, J = 15.9 Hz, 1H), 7.53 - 7.41 (m, 2H), 7.23 (d, J = 8.4 Hz, 1H), 6.49 (d, J = 15.6 Hz, 1H). LRMS [M - H] - 206.04

[0277] Synthesis of aryl azide 8:

Chemical formula

[0278] Intermediate 21: To a DMF solution (3 mL) of Intermediate 20 (149 mg, 0.68 mmol, 1.0 equiv), NaN3 (66 mg, 1.0 mmol, 1.5 equiv) was added all at once. The mixture was heated at 70 °C overnight. The reaction was cooled, diluted with EtOAc (50 mL), and poured into crushed ice. After partitioning, the aqueous layer was extracted with EtOAc (20 x 3 mL). The combined organic layers were washed with H2O (50 mL) and brine (50 mL), dried over Na2SO4, and concentrated under reduced pressure. The desired product was isolated using silica gel chromatography. 1 1H NMR (400 MHz, chloroform-d) δ 7.79 - 7.70 (m, 2H), 7.58 (d, J = 16.0 Hz, 1H), 7.28 (d, J = 9.2 Hz, 1H), 6.42 (d, J = 16.0 Hz, 1H), 4.28 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H). LRMS [M + H] + 243.1.

[0279] Aryl Azide 8: To a THF solution (4 mL) of Intermediate 21 (25 mg, 0.10 mmol, 1.0 equiv), LiOH (40 mg, 1.0 mmol, 10 equiv) previously dissolved in H2O (2 mL) was added. The reaction mixture was stirred at room temperature for 3 h. LC-MS indicated complete conversion. The reaction was concentrated under reduced pressure to remove volatiles, diluted with H2O (20 mL). The aqueous suspension was adjusted to pH 4 and extracted with EtOAc (20 x 3 mL). The combined organic layers were washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4, concentrated under reduced pressure to give the crude product. The desired product was isolated using silica gel chromatography. 1 1H NMR (400 MHz, methanol-d4) δ 7.96 (d, J = 11.4 Hz, 2H), 7.63 (d, J = 16.0 Hz, 1H), 7.57 - 7.44 (m, 1H), 6.66 - 6.44 (m, 1H). LRMS [M - H] - 213.04.

[0280] Synthesis of Aryl Azide 9 [Chemical formula] Intermediate 23: To a solution of the phosphonate reagent (0.57 mL, 2.9 mmol, 1.0 equiv), a solution of LiHMDS (3.1 mL, 1.0 N, 3.1 mmol, 1.1 equiv) was added dropwise over 10 minutes. After the mixture was stirred at room temperature for 30 minutes, the aldehyde intermediate 22 was added all at once. Next, the reaction mixture was stirred at room temperature for 16 hours and quenched by the addition of saturated aqueous NH4Cl solution (20 mL). The quenched reaction mixture was extracted with EtOAc (30 × 3 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give the crude product. The desired product was isolated using silica gel chromatography. 1 H NMR (400 MHz, chloroform-d) δ 7.50 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 6.11 (t, J = 1.3 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 2.54 (t, J = 1.1 Hz, 3H), 1.31 (t, J = 7.1 Hz, 3H). LRMS [M + H] + 269.02

[0281] Intermediate 24: Intermediate 23 (110 mg, 0.41 mmol, 1.0 equiv), CuI (7.8 mg, 0.04 mmol, 0.1 equiv), and Na-ascorbic acid (8.0 mg, 0.04 mmol, 0.1 equiv) were charged into a vial purged with N2. DMSO (5 mL) and DMEDA (6.6 μL, 0.06 mmol, 0.15 equiv) were added to the mixture. Next, the mixture was stirred at room temperature for 15 minutes, and then NaN3 (53 mg, 0.82 mmol, 2.0 equiv) was added. The reaction mixture was stirred at 100 °C for 16 hours. Next, the reaction mixture was stirred at room temperature for 16 hours and quenched by the addition of saturated aqueous NH4Cl (20 mL). The quenched reaction mixture was extracted with EtOAc (50 × 3 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to obtain the crude product. The desired product was isolated using silica gel chromatography. 1 H NMR (400 MHz, chloroform-d) δ 7.62 - 7.45 (m, 2H), 7.13 - 6.95 (m, 2H), 6.24 - 6.04 (m, 1H), 4.24 (q, J = 7.1 Hz, 2H), 2.58 (d, J = 1.3 Hz, 3H), 1.34 (t, J = 7.1 Hz, 3H). LRMS [M + H] + 232.11。

[0282] Aryl azide 9: To a solution of Intermediate 24 (30 mg, 0.12 mmol, 1.0 equiv) in THF (4 mL) was added LiOH (80 mg, 2.0 mmol, 17 equiv) previously dissolved in H2O (2 mL). The reaction mixture was stirred at room temperature for 3 hours. LC-MS indicated complete conversion. The reaction mixture was concentrated under reduced pressure to remove volatile substances and diluted with H2O (20 mL). The aqueous suspension was adjusted to pH 4 and extracted with EtOAc (20 × 3 mL). The combined organic layers were washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4, and concentrated under reduced pressure to obtain the crude product. The desired product was isolated using silica gel chromatography. 11H NMR (400 MHz, acetonitrile-d3) δ 7.64 (d, J = 7.8 Hz, 2H), 7.12 (d, J = 7.8 Hz, 2H), 6.17 (s, 1H), 2.54 (s, 3H). [M - H] - 202.04。

[0283] Synthesis of A-1 (E)-3-(6-azidonaphthalen-2-yl)acrylic acid

Chem.

[0284] Step 2: To a 20 mL vial, tert-butyl (E)-3-(6-bromonaphthalen-2-yl)acrylate (200 mg, 0.600 mmol), Pd(dppf)Cl2 (22.0 mg, 0.030 mmol), B2pin2 (183 mg, 0.720 mmol), potassium acetate (118 mg, 1.20 mmol), and dioxane (4 mL) were added. The mixture was degassed with nitrogen for 1 minute. The mixture was stirred and heated at 100 °C for 1 hour. The mixture was cooled to room temperature. The mixture was diluted with EtOAc and filtered through celite. The solvent was evaporated to give tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-yl)acrylate, which was carried on to the next step without further purification. LRMS [M+H-C4H4] + 325。

[0285] Step 3: To a 20 mL vial, tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-yl)acrylate (225 mg, 0.592 mmol), NaN3 (57.8 mg, 0.889 mmol), Cu(OAc)2 (215 mg, 1.18 mmol), and MeOH (3 mL) were added. The mixture was stirred vigorously at 65 °C for 90 minutes. The mixture was diluted with EtOAc and washed with 10% ammonia. The organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography using 0 - 30% EtOAc in heptane as the eluent to give tert-butyl (E)-3-(6-azidonaphthalen-2-yl)acrylate. LRMS [M+H-C4H4] + 240。

[0286] Step 4: To a 20 mL vial was added tert-butyl (E)-3-(6-azidonaphthalen-2-yl)acrylate (100 mg, 0.339 mmol), DCM (2 mL), and formic acid (1 mL). The mixture was stirred for 14 h. A solid precipitated. The solid was collected by filtration and washed with DCM to afford (E)-3-(6-azidonaphthalen-2-yl)acrylic acid of A-1. LRMS [M-H] - 238.

[0287] Synthesis of A-2 (E)-3-(6-azidonaphthalen-2-yl)but-2-enoic acid

Chemical formula

[0288] Step 2: To a 100 mL flask, PCC (1.09 g, 5.07 mmol), Celite (2.5 g), and DCM (20 mL) were added. To the stirred mixture, 1-(6-bromonaphthalen-2-yl)ethan-1-ol (424 mg, 1.69 mmol) was added. The mixture was stirred at room temperature for 1 hour. The mixture was filtered through Celite and washed with DCM. The solvent of the filtrate was evaporated. The residue was purified by silica gel chromatography using 0 - 30% EtOAc in heptane as the eluent to afford 1-(6-bromonaphthalen-2-yl)ethan-1-one. LRMS [M+H] + 249。

[0289] Step 3: To a 20 mL vial, tert-butyl 2-(diethoxyphosphoryl)acetate (0.307 mL, 1.38 mmol) and THF (5 mL) were added. The mixture was stirred under nitrogen. To the mixture, 1 M LHMDS in THF (1.38 mL, 1.38 mmol) was added dropwise over 5 minutes. To this mixture, a THF solution of 1-(6-bromonaphthalen-2-yl)ethan-1-one (343 mg, 1.38 mmol) was added dropwise over 5 minutes. After 5 minutes, the vial was sealed and then stirred and heated at 70 °C for 3 hours. The mixture was adsorbed onto Celite and purified by silica gel chromatography using 0 - 20% EtOAc in heptane as the eluent to give tert-butyl (E)-3-(6-bromonaphthalen-2-yl)but-2-enoate. LRMS [M+H-C4H4] + 291。

[0290] Step 4: To a 20 mL vial, tert-butyl (E)-3-(6-bromonaphthalen-2-yl)but-2-enoate (350 mg, 1.01 mmol), Pd(dppf)Cl2 (73.8 mg, 0.101 mmol), B2pin2 (307 mg, 1.21 mmol), potassium acetate (198 mg, 2.02 mmol), and dioxane (5 mL) were added. The mixture was degassed with nitrogen for 1 minute. The mixture was stirred and heated at 120 °C for 2.5 hours. The mixture was cooled to room temperature. The mixture was diluted with EtOAc and filtered through celite. The solvent was evaporated to give tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-yl)but-2-enoate, which was carried on to the next step without further purification. LRMS [M+H-C4H4] + 339。

[0291] Step 5: To a 20 mL vial, tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-yl)but-2-enoate (145 mg, 0.368 mmol), NaN3 (35.9 mg, 0.552 mmol), Cu(OAc)2 (134 mg, 0.736 mmol), and MeOH (8 mL) were added. The mixture was stirred vigorously at 65 °C for 90 minutes. The mixture was diluted with EtOAc and washed with 10% ammonia. The organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography using 0 - 50% EtOAc in heptane as the eluent to give tert-butyl (E)-3-(6-azidonaphthalen-2-yl)but-2-enoate. LRMS [M+H-C4H4] + 254。

[0292] Step 6: To a 20 mL vial was added tert-butyl (E)-3-(6-azidonaphthalen-2-yl)but-2-enoate (30.0 mg, 0.0970 mmol), DCM (2 mL), and formic acid (1 mL). The mixture was stirred for 14 h. The solvent was evaporated to give (E)-3-(6-azidonaphthalen-2-yl)but-2-enoic acid of A-2. LRMS [M-H] - 252.

[0293] Synthesis of A-3 (E)-3-(7-azidoquinolin-3-yl)acrylic acid

Chemical Structure

[0294] Step 2: To a 20 mL vial was added 3-(dimethoxymethyl)quinoline (500 mg, 2.46 mmol), [Ir(COD)OMe]2 (81.5 mg, 0.123 mmol), B2pin2 (937 mg, 3.69 mmol), and 4,4’-di-tert-butylbipyridine (dtbpy, 66.0 mg, 0.246 mmol). The vial was purged with nitrogen. To this mixture was added anhydrous THF (5 mL). The mixture was sparged with nitrogen for 1 min. The mixture was stirred at room temperature for 14 h. The solvent was evaporated to give crude 3-(dimethoxymethyl)-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline, which was carried on to the next step without purification. LRMS [M+H] + 330.

[0295] Step 3: To a 20 mL vial, 3-(dimethoxymethyl)-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline (810 mg, 2.46 mmol), NaN3 (240 mg, 3.69 mmol), Cu(OAc)2 (894 mg, 4.62 mmol), and MeOH (8 mL) were added. The mixture was stirred vigorously at 55 °C for 90 minutes. The mixture was diluted with EtOAc and washed with 10% ammonia. The organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography using 0 - 40% EtOAc in heptane as the eluent to afford 7-azido-3-(dimethoxymethyl)quinoline. LRMS [M+H] + 245.

[0296] Step 4: To a 20 mL vial, 7-azido-3-(dimethoxymethyl)quinoline (26.9 mg, 0.110 mmol), TFA (1 mL), and water (0.1 mL) were added. The mixture was stirred for 10 minutes. The solvent was evaporated to afford 7-azidoquinoline-3-carbaldehyde. LRMS [M+H] + 199.

[0297] Step 5: To a 20 mL vial, tert-butyl 2-(diethoxyphosphoryl)acetate (0.0278 mL, 0.110 mmol), 7-azidoquinoline-3-carbaldehyde (21.8 mg, 0.110 mmol), and MeOH (1 mL) were added. To the stirred mixture, tetramethylguanidine (TMG, 0.055 mL, 0.441 mmol) was added dropwise. After 20 minutes, the solvent was evaporated and the residue was purified by silica gel chromatography using 0 - 50% EtOAc in heptane as the eluent to afford tert-butyl (E)-3-(7-azidoquinolin-3-yl)acrylate. LRMS [M+H] + 297.

[0298] Step 6: tert-Butyl (E)-3-(7-azidoquinolin-3-yl)acrylate (23.7 mg, 0.0800 mmol) and TFA (1 mL) were added to a 20 mL vial. The mixture was stirred for 15 minutes. The solvent was evaporated to afford (E)-3-(7-azidoquinolin-3-yl)acrylic acid of A-3. LRMS [M+H] + 241.

[0299] Synthesis of A-4 (E)-3-(6-azidoquinolin-3-yl)acrylic acid

Chemical Structure

[0300] Step 2: To a 20 mL vial, tert-butyl (E)-3-(6-bromoquinolin-3-yl)acrylate (64.0 mg, 0.191 mmol), Pd(dppf)Cl2 (7.0 mg, 0.0096 mmol), B2pin2 (58.4 mg, 0.230 mmol), potassium acetate (37.6 mg, 0.383 mmol), and dioxane (4 mL) were added. The mixture was degassed with nitrogen for 1 minute. The mixture was stirred and heated at 100 °C for 2 hours. The mixture was cooled to room temperature. The mixture was diluted with EtOAc and filtered through celite. The solvent was evaporated to give tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinolin-3-yl)acrylate, which was carried on to the next step without further purification. LRMS [M+H] + 382。

[0301] Step 3: To a 20 mL vial, tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinolin-3-yl)acrylate (73.0 mg, 0.191 mmol), NaN3 (18.7 mg, 0.287 mmol), Cu(OAc)2 (69.6 mg, 0.383 mmol), and MeOH (3 mL) were added. The mixture was stirred vigorously at 65 °C for 90 minutes. The mixture was diluted with EtOAc and washed with 10% ammonia. The organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography using 0 - 50% EtOAc in heptane as the eluent to give tert-butyl (E)-3-(6-azidoquinolin-3-yl)acrylate. LRMS [M+H] + 297。

[0302] Step 4: To a 20 mL vial, tert-butyl (E)-3-(6-azidoquinolin-3-yl)acrylate, and 4 M HCl in dioxane (2 mL) were added. The mixture was stirred and heated at 70 °C for 2 hours. The solvent was concentrated to give (E)-3-(6-azidoquinolin-3-yl)acrylic acid of A-4. LRMS [M+H] + 241。

[0303] Synthesis of A-5 (E)-3-(6-azidoquinolin-3-yl)but-2-enoic acid

Chemical formula

[0304] Step 2: To a 20 mL vial, 1-(6-bromoquinolin-3-yl)ethan-1-ol (260 mg, 1.03 mmol), PCC (668 g, 3.10 mmol), celite (1 g), and DCM (6 mL) were added. The mixture was stirred at room temperature for 2 hours. The mixture was filtered through celite and washed with DCM. The solvent of the filtrate was evaporated. The residue was purified by silica gel chromatography using 0 - 70% EtOAc in heptane as the eluent to obtain 1-(6-bromoquinolin-3-yl)ethan-1-one. LRMS [M+H] + 250.

[0305] Step 3: To a 20 mL vial was added tert-butyl 2-(diethoxyphosphoryl)acetate (0.185 mL, 0.829 mmol) and THF (6 mL). The mixture was stirred under nitrogen. To the mixture was added 1 M LHMDS in THF (1.13 mL, 1.13 mmol) dropwise over 5 minutes. To this mixture was added dropwise a THF solution of 1-(6-bromoquinolin-3-yl)ethan-1-one (188 mg, 0.753 mmol) over 5 minutes. The mixture was stirred for 20 minutes. The mixture was adsorbed onto Celite and purified by silica gel chromatography using 0 - 50% EtOAc in heptane as the eluent to afford tert-butyl (E)-3-(6-bromoquinolin-3-yl)but-2-enoate. LRMS [M+H] + 348。

[0306] Step 4: To a 20 mL vial was added tert-butyl (E)-3-(6-bromoquinolin-3-yl)but-2-enoate (55.0 mg, 0.158 mmol), Pd(dppf)Cl2 (5.8 mg, 0.0079 mmol), B2pin2 (48.1 mg, 0.190 mmol), potassium acetate (31.0 mg, 0.316 mmol), and dioxane (1 mL). The mixture was degassed with nitrogen for 1 minute. The mixture was stirred and heated at 100 °C for 2 hours. The mixture was cooled to room temperature. The mixture was diluted with EtOAc and filtered through Celite. The solvent was evaporated to afford tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinolin-3-yl)but-2-enoate, which was carried on to the next step without further purification. LRMS [M+H] + 396。

[0307] Step 5: To a 20 mL vial was added tert-butyl (E)-3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinolin-3-yl)but-2-enoate (62.4 mg, 0.158 mmol), NaN3 (15.4 mg, 0.237 mmol), Cu(OAc)2 (57.3 mg, 0.316 mmol), and MeOH (3 mL). The mixture was stirred vigorously at 65 °C for 90 minutes. The mixture was diluted with EtOAc and washed with 10% ammonia. The organic layer was dried over sodium sulfate, filtered, and the solvent was evaporated. The residue was purified by silica gel chromatography using 0–50% EtOAc in heptane as the eluent to afford tert-butyl (E)-3-(6-azidoquinolin-3-yl)but-2-enoate. LRMS [M+H] + 311。

[0308] Step 6: To a 20 mL vial was added tert-butyl (E)-3-(6-azidoquinolin-3-yl)but-2-enoate (43.5 mg, 0.140 mmol) and 4 M HCl in dioxane (2 mL). The mixture was stirred and heated at 70 °C for 2 hours. The solvent was evaporated to afford (E)-3-(6-azidoquinolin-3-yl)but-2-enoic acid of A-5. LRMS [M+H] + 255。

[0309] Synthesis of A-6 (E)-3-(6-azido-7-cyanonaphthalen-2-yl)acrylic acid

Chemical formula

[0310] Step 2: To a 100 mL flask were added 2-(7-bromo-3-fluoronaphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.18 g, 3.35 mmol), Cu(NO3)2·3H2O (1.62 g, 6.70 mmol), Zn(CN)2 (1.18 g, 10.1 mmol), CsF (509 mg, 3.35 mmol), MeOH (20 mL), and water (8 mL). The reaction mixture was stirred and heated at reflux for 1 hour. The mixture was cooled to room temperature. The mixture was diluted with EtOAc and washed with 1 M aqueous ammonia. The organic layer was dried over sodium sulfate, filtered, and the solvent of the filtrate was evaporated. The residue was purified by silica gel chromatography using 0 - 20% EtOAc in heptane to obtain 7-bromo-3-fluoronaphthalene-2-carbonitrile. 11H NMR (400 MHz, DMSO-d6) δ 8.71 (d, J = 6.6 Hz, 1H), 8.39 (d, J = 1.9 Hz, 1H), 8.11 (d, J = 10.6 Hz, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.91 (dd, J = 8.8, 2.0 Hz, 1H).

[0311] Step 3: In a 20 mL vial, 7-bromo-3-fluoro-2-naphthonitrile (199 mg, 0.797 mmol), tert-butyl acrylate (0.146 mL, 0.996 mmol), triethylamine (0.222 mL, 1.59 mmol), Pd(OAc)2 (1.8 mg, 0.0080 mmol), tri-(o-tolyl)phosphine (9.7 mg, 0.032 mmol), and toluene (8 mL) were added. The mixture was purged with nitrogen for 1 minute. The mixture was stirred and heated at 100 °C under nitrogen for 3 hours. The solvent was evaporated and the residue was purified by silica gel chromatography using 0 - 20% EtOAc in heptane to give tert-butyl (E)-3-(7-cyano-6-fluoronaphthalen-2-yl)acrylate. LRMS [M+H+MeCN] + 339.

[0312] Step 4: In a 20 mL vial, tert-butyl (E)-3-(7-cyano-6-fluoronaphthalen-2-yl)acrylate (64.7 mg, 0.218 mmol), NaN3 (15.5 mg, 0.239 mmol), and DMSO (1 mL) were added. The mixture was stirred and heated at 100 °C for 2 hours. The mixture was diluted with 1:2 EtOAc / Et2O (12 mL), filtered through celite, and washed through with Et2O. The filtrate was washed with water (3 × 10 mL). The organic layer was dried over sodium sulfate, filtered, and the solvent of the filtrate was evaporated. The mixture was purified by silica gel chromatography using 0 - 30% EtOAc in heptane as the eluent to give tert-butyl (E)-3-(6-azido-7-cyanonaphthalen-2-yl)acrylate. LRMS [M+H-H2O] + 293.

[0313] Step 5: tert-Butyl (E)-3-(6-azido-7-cyanonaphthalen-2-yl) acrylate (37.4 mg, 0.117 mmol) and TFA (1 mL) were added to a 20 mL vial. The mixture was stirred for 15 minutes. The solvent was evaporated to obtain (E)-3-(6-azido-7-cyanonaphthalen-2-yl) acrylic acid of A-6. LRMS [M-H] - 263.

[0314] Synthesis of A-7 (E)-3-(6-azido-7-cyanonaphthalen-2-yl) but-2-enoic acid [Chemical formula] Step 1: To a solution of 7-bromo-3-fluoro-2-naphthonitrile (from step 2 of A-6) (266 mg, 1.07 mmol) in dioxane (2 mL) were added tributyl(1-ethoxyvinyl)tin (0.396 mL, 1.17 mmol) and Pd(PPh3)2Cl2 (37.4 mg, 0.0533 mmol). The mixture was purged with nitrogen for 2 minutes. The mixture was stirred and heated at 130 °C for 30 minutes. The mixture was cooled to room temperature and the mixture was diluted with EtOAc and filtered through celite. The solvent of the filtrate was evaporated. The residue was purified by silica gel chromatography using 0-30% EtOAc in heptane as the eluent to obtain 7-(1-ethoxyvinyl)-3-fluoro-2-naphthonitrile. LRMS [M+H] + 242.

[0315] Step 2: To a 20 mL vial were added 7-(1-ethoxyvinyl)-3-fluoro-2-naphthonitrile (154 mg, 0.637 mmol) and a 10% v / v aqueous solution of TFA (2 mL), and the mixture was stirred for 10 minutes. The solvent was evaporated to obtain 7-acetyl-3-fluoro-2-naphthonitrile.

[0316] Step 3: To a 20 mL vial was added tert-butyl 2-(diethoxyphosphoryl)acetate (0.152 mL, 0.679 mmol) and THF (3 mL). The mixture was stirred under nitrogen. To the mixture was added 1 M LHMDS in THF (0.679 mL, 0.679 mmol) dropwise over 5 minutes. To this mixture was added a THF solution of 7-acetyl-3-fluoro-2-naphthonitrile (145 mg, 0.679 mmol) dropwise over 5 minutes. After 5 minutes, the vial was sealed and then stirred and heated at 70 °C for 14 hours. The mixture was adsorbed onto celite and purified by silica gel chromatography using 0 - 30% EtOAc in heptane as the eluent to afford tert-butyl (E)-3-(7-cyano-6-fluoronaphthalen-2-yl)but-2-enoate. LRMS [M+H+MeCN] + 353。

[0317] Step 4: To a 20 mL vial was added tert-butyl (E)-3-(7-cyano-6-fluoronaphthalen-2-yl)but-2-enoate (40.5 mg, 0.130 mmol), NaN3 (9.3 mg, 0.14 mmol), and DMSO (1 mL). The mixture was stirred and heated at 100 °C for 2 hours. The mixture was diluted with 1:2 EtOAc / Et2O (12 mL), filtered through celite, and washed through with Et2O. The filtrate was washed with water (3 × 10 mL). The organic layer was dried over sodium sulfate, filtered, and the solvent of the filtrate was evaporated. The mixture was purified by silica gel chromatography using 0 - 30% EtOAc in heptane as the eluent to afford tert-butyl (E)-3-(6-azido-7-cyanonaphthalen-2-yl)but-2-enoate. LRMS [M+H-H2O] + 307。

[0318] Step 5: tert-Butyl (E)-3-(6-azido-7-cyanonaphthalen-2-yl)but-2-enoate (13.5 mg, 0.0404 mmol) and formic acid (1 mL) were added to a 20 mL vial. The mixture was stirred and heated at 40 °C for 15 minutes. The solvent was evaporated to give (E)-3-(6-azido-7-cyanonaphthalen-2-yl)but-2-enoic acid of A-7. LRMS [M-H] - 277.

[0319] Synthesis of A-8:

Chemical formula

[0320] Step 2: Ethyl (E)-3-(4-bromo-3-methoxyphenyl)acrylate (285 mg, 1.00 mmol), CuI (9.5 mg, 0.050 mmol), and sodium ascorbate (20 mg, 0.10 mmol) were charged into a vial purged with N2. DMSO (5 mL) and DMEDA (17 μL, 0.15 mmol) were added to the mixture. Next, after stirring the mixture at room temperature for 15 minutes, NaN3 (98 mg, 1.5 mmol) was added. The reaction was stirred at 100 °C for 16 hours. Next, the reaction was stirred at room temperature for 16 hours and quenched by adding saturated aqueous NH4Cl solution (20 mL). The quenched reaction was extracted with EtOAc (50 × 3 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to obtain the crude product. Ethyl (E)-3-(4-azido-2-methoxyphenyl)acrylate was isolated using silica gel chromatography. LRMS [M+H] + 248.

[0321] Step 3: To a solution of ethyl (E)-3-(4-azido-2-methoxyphenyl)acrylate (170 mg, 0.690 mmol) in THF (4 mL) was added LiOH (32.0 mg, 1.38 mmol) previously dissolved in H2O (2 mL). The reaction mixture was stirred at room temperature for 3 hours. The reaction was concentrated under reduced pressure and diluted with H2O (20 mL). The pH of the aqueous suspension was adjusted to 4 and extracted with EtOAc (20 × 3 mL). The combined organic layers were washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4, and concentrated under reduced pressure to obtain the crude product. A-8 was isolated using silica gel chromatography. LRMS [M-H] - 218.

[0322] Synthesis of A-9 [Chemical formula] In the same manner as the synthesis of A-8, 4-bromo-3-methoxybenzaldehyde was converted to A-9 in 3 steps. LRMS [M-H] - 218.

[0323] Synthesis of A-10 [Chemistry] Similar to the synthesis of A-8, 1-(4-bromophenyl)ethan-1-one was converted to A-10 in three steps. LRMS [M-H] - 202.

[0324] Synthesis of A-11 [Chemistry] Step 1: A 1 M solution of LHMDS in THF (1.7 mL, 1.7 mmol) was added dropwise to a solution of ethyl 2-(diethoxyphosphoryl)acetate (0.32 mL, 1.7 mmol) over 10 minutes. The mixture was stirred at room temperature for 30 minutes, then 3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde (402 mg, 1.50 mmol) was added all at once. Next, the reaction was stirred at room temperature for 16 hours and quenched by the addition of saturated aqueous NH4Cl (20 mL). The quenched reaction was extracted with EtOAc (30 × 3 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give the crude product. Ethyl (E)-3-(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acrylate was isolated using silica gel chromatography. LRMS [M+H] + 339.

[0325] Step 2: To a solution of ethyl (E)-3-(3,5-difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acrylate (73 mg, 0.22 mmol) in MeOH (4 mL) were added Cu(OAc)2 (4.0 mg, 0.022 mmol) and NaN3 (14 mg, 0.22 mmol). The reaction mixture was heated at 60 °C for 30 minutes. The reaction mixture was diluted with EtOAc (50 mL), and then quenched by the addition of saturated aqueous NH4Cl solution (10 mL). The aqueous layer was extracted with EtOAc (10 × 3 mL). The combined organic layers were washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4, and concentrated under reduced pressure. The desired product was purified by silica gel chromatography to give ethyl (E)-3-(4-azido-3,5-difluorophenyl)acrylate. LRMS [M+H] + 254.

[0326] Step 3: To a solution of ethyl (E)-3-(4-azido-3,5-difluorophenyl)acrylate (30 mg, 0.12 mmol) in THF (4 mL) was added LiOH (80 mg, 2.0 mmol) previously dissolved in H2O (2 mL). The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure and diluted with H2O (20 mL). The pH of the aqueous suspension was adjusted to 4, and the mixture was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4, and concentrated under reduced pressure to give the crude product. A-11 was isolated using silica gel chromatography. LRMS [M-H] - 224.

[0327] Synthesis of A-12

Chemical formula

[0328] Synthesis of A-13 [Chemistry] Step 1: A 1 M LHMDS solution in THF (3.1 mL, 3.1 mmol) was added dropwise to a solution of ethyl 2-(diethoxyphosphoryl)acetate (0.57 mL, 2.9 mmol) over 10 minutes. After the mixture was stirred at room temperature for 30 minutes, 2-fluoro-5-formylbenzonitrile (425 mg, 2.85 mmol) was added all at once. Next, the reaction was stirred at room temperature for 16 hours and quenched by the addition of saturated aqueous NH4Cl (20 mL). The quenched reaction was extracted with EtOAc (30 × 3 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give the crude product. Ethyl (E)-3-(3-cyano-4-fluorophenyl)acrylate was isolated using silica gel chromatography. LRMS [M+H] + 220.

[0329] Step 2: NaN3 (66 mg, 1.0 mmol) was added all at once to a solution of ethyl (E)-3-(3-cyano-4-fluorophenyl)acrylate (149 mg, 0.68 mmol) in DMF (3 mL). The mixture was heated at 70 °C overnight. The reaction was cooled, diluted with EtOAc (50 mL) and poured into crushed ice. After partitioning in a separatory funnel, the aqueous layer was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with H2O (50 mL) and brine (50 mL), dried over Na2SO4 and concentrated under reduced pressure. Ethyl (E)-3-(4-azido-3-cyanophenyl)acrylate was isolated using silica gel chromatography. LRMS [M+H] + 243.

[0330] Step 3: To a solution of ethyl (E)-3-(4-azido-3-cyanophenyl)acrylate (25 mg, 0.10 mmol) in THF (4 mL) was added LiOH (40 mg, 1.0 mmol) previously dissolved in H2O (2 mL). The reaction mixture was stirred at room temperature for 3 h. The reaction was concentrated under reduced pressure and diluted with H2O (20 mL). The pH of the aqueous suspension was adjusted to 4 and extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4, concentrated under reduced pressure to give the crude product. A-13 was isolated using silica gel chromatography. LRMS [M-H] - 213.

[0331] Synthesis of A-14

Chemical formula

[0332] Synthesis of biotin compounds Synthesis of 9069

Chemical formula

[0333] Synthesis of 9042 The desired product was synthesized in the same manner as the procedure for 9069 using aryl azide 1. 11H NMR (400 MHz, chloroform-d) δ 8.30 - 8.04 (m, 2H), 6.78 (d, J = 8.4 Hz, 1H), 6.24 (br s, 1H), 5.64 (br s, 1H), 4.63 - 4.49 (m, 1H), 4.44 - 4.26 (m, 1H), 3.96 (s, 3H), 3.75 - 3.39 (m, 20H), 3.13 (d, J = 21.2 Hz, 2H), 2.96 - 2.87 (m, 1H), 2.74 (d, J = 12.9 Hz, 1H), 2.23 (t, J = 7.1 Hz, 2H), 1.57 - 1.37 (m, 6H). LRMS [M + H] + 638.31。

[0334] Synthesis of 9043 The desired product was similarly synthesized using aryl azide 2 according to the procedure of 9069. 1 1H NMR (400 MHz, methanol-d4) δ 8.40 (s, 1H), 8.03 (d, J = 8.8 Hz, 1H), 7.96 - 7.86 (m, 2H), 7.62 (s, 1H), 7.30 (d, J = 8.9 Hz, 1H), 4.56 - 4.40 (m, 1H), 4.32 - 4.20 (m, 1H), 3.77 - 3.66 (m, 20H), 3.22 - 3.07 (m, 1H),2.92 (dd, J = 13.1, 4.8 Hz, 1H), 2.75 - 2.60 (m, 2H), 2.20 (t, J = 7.4 Hz, 2H), 1.80 - 1.45 (m, 6H). LRMS [M + H] + 658.32。

[0335] Synthesis of 9046 The desired product was similarly synthesized using commercially available 4-azido-2-hydroxybenzoic acid according to the procedure of 9069. 11H NMR (400 MHz, methanol-d4) δ 7.80 (dd, J = 8.3, 2.1 Hz, 1H), 6.63 - 6.53 (m, 2H), 4.56 - 4.40 (m, 1H), 4.32 - 4.20 (m, 1H), 3.70 - 3.47 (m, 20H), 3.22 - 3.07 (m, 1H), 2.91 (dd, J = 12.8, 4.8 Hz, 1H), 2.69 (d, J = 12.7 Hz, 1H), 2.20 (t, J = 7.3 Hz, 3H), 1.80 - 1.50 (m, 4H), 1.46 - 1.38 (m, 2H). LRMS [M + H] + 624.72。

[0336] Synthesis of 9044 The desired product was similarly synthesized using commercially available (E)-3-(4-azidophenyl)acrylic acid according to the procedure of 9069. 1 1H NMR (400 MHz, methanol-d4) δ 7.61 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 15.7 Hz, 1H), 7.12 (d, J = 8.1 Hz, 2H), 6.62 (d, J = 15.5 Hz, 1H), 4.56 - 4.40 (m, 1H), 4.32 - 4.20 (m, 1H), 3.74 - 3.46(m, 20H), 3.22 - 3.07 (m, 1H), 2.93 (dd, J = 12.7, 5.0 Hz, 1H), 2.71 (d, J = 12.7 Hz, 1H), 2.22 (t, J = 7.4 Hz, 2H), 1.88 - 1.57 (m, 4H), 1.53 - 1.38 (m, 2H). LRMS [M + H] + 634.28。

[0337] Synthesis of 9047 The desired product was similarly synthesized using aryl azide 3 according to the procedure of 9069. 11H NMR (400 MHz, chloroform-d) δ 7.80 (d, J = 7.9 Hz, 2H), 7.35 (br s, 1H), 7.33 (d, J = 7.9 Hz, 2H), 6.73 (d, J = 14.1 Hz, 1H), 6.27 (d, J = 13.8 Hz, 1H), 5.97 (s, 1H), 5.01 (s, 1H), 4.63 - 4.49 (m, 1H), 4.44 - 4.26 (m, 1H), 3.71 - 3.38 (m, 20H), 3.16 - 3.09 (m, 1H), 2.91 (dd, J = 13.3, 4.9 Hz, 1H), 2.71 (d, J = 12.9 Hz, 1H), 2.20 (t, J = 7.1 Hz, 2H), 1.80 - 1.45 (m, 6H). LRMS [M + H] + 634.28。

[0338] Synthesis of 9157 The desired product was similarly synthesized using aryl azide 5 according to the procedure of 9069. 1 1H NMR (400 MHz, methanol-d4) δ 7.50 (d, J = 15.7 Hz, 1H), 7.27 - 7.11 (m, 2H), 7.05 - 6.92 (m, 1H), 6.63 (d, J = 15.8 Hz, 1H), 4.56 - 4.40 (m, 1H), 4.32 - 4.20 (m, 1H), 3.94 (s, 3H), 3.74 - 3.46(m, 20H), 3.22 - 3.07 (m, 1H), 3.02 - 2.86 (m, 1H), 2.71 (d, J = 12.8 Hz, 1H), 2.22 (t, J = 7.7 Hz, 2H), 1.88 - 1.57 (m, 4H), 1.53 - 1.38 (m, 2H). LRMS [M + H] + 664.28。

[0339] Synthesis of 9158 The desired product was similarly synthesized using aryl azide 4 according to the procedure of 9069. 11H NMR (400 MHz, methanol-d4) δ 7.76 (d, J = 15.9 Hz, 1H), 7.56 (d, J = 8.2 Hz, 1H), 6.80 - 6.58 (m, 3H), 4.52 - 4.40 (m, 1H), 4.32 - 4.22 (m, 1H), 3.90 (s, 3H), 3.74 - 3.46 (m, 20H), 3.22 - 3.09 (m, 1H), 2.91 (dd, J = 12.8, 5.0 Hz, 1H), 2.69 (d, J = 12.7 Hz, 1H), 2.20 (t, J = 7.4 Hz, 2H), 1.88 - 1.57 (m, 4H), 1.53 - 1.38 (m, 2H). LRMS [M + H] + 664.30。

[0340] Synthesis of 9159 The desired product was similarly synthesized using aryl azide 8 according to the procedure of 9069. 1 1H NMR (400 MHz, methanol-d4) δ 8.00 - 7.81 (m, 2H), 7.57 - 7.44 (m, 2H), 6.77 - 6.63 (m, 1H), 4.52 - 4.40 (m, 1H), 4.32 - 4.22 (m, 1H), 3.74 - 3.46 (m, 20H), 3.24 - 3.16 (m, 1H), 2.94 (dd, J = 12.8, 5.0 Hz, 1H), 2.72 (d, J = 12.8 Hz, 1H), 2.22 (t, J = 7.5 Hz, 2H), 1.88 - 1.57 (m, 4H), 1.53 - 1.38 (m, 2H). LRMS [M + H] + 659.28。

[0341] Synthesis of 9160 The desired product was similarly synthesized using aryl azide 7 according to the procedure of 9069. 11H NMR (400 MHz, methanol-d4) δ 7.52 - 7.33 (m, 3H), 7.19 (t, J = 8.5 Hz, 1H), 6.61 (d, J = 15.9 Hz, 1H), 4.52 - 4.40 (m, 1H), 4.32 - 4.22 (m, 1H), 3.74 - 3.56 (m, 16H), 3.53 - 3.43 (m, 4H), 3.22 - 3.07 (m, 1H), 2.91 (d, J = 12.7 Hz, 1H), 2.69 (d, J = 13.0 Hz, 1H), 2.20 (t, J = 7.5 Hz, 2H), 1.88 - 1.57 (m, 4H), 1.53 - 1.38 (m, 2H). LRMS [M + H] + 652.31。

[0342] Synthesis of 9161 The desired product was similarly synthesized using aryl azide 6 according to the procedure of 9069. 1 1H NMR (400 MHz, methanol-d4) δ 7.41 (d, J = 15.7 Hz, 1H), 7.28 (d, J = 9.3 Hz, 2H), 6.62 (d, J = 15.7 Hz, 1H), 4.55 - 4.40 (m, 1H), 4.32 - 4.22 (m, 1H), 3.76 - 3.57 (m, 16H), 3.53 - 3.43 (m, 4H), 3.24 - 3.12 (m, 1H), 2.91 (dd, J = 12.9, 4.9 Hz, 1H), 2.69 (d, J = 12.8 Hz, 1H), 2.20 (t, J = 7.4 Hz, 2H), 1.88 - 1.57 (m, 4H), 1.53 - 1.38 (m, 2H). LRMS [M + H] + 670.28。

[0343] Synthesis of 9162 The desired product was similarly synthesized using aryl azide 9 according to the procedure of 9069. 11H NMR (400 MHz, methanol-d4) δ 7.57 (dd, J = 8.5 Hz, 2H), 7.17 - 7.01 (d, J = 8.5, 2H), 6.25 (s, 1H), 4.59 - 4.41 (m, 1H), 4.32 - 4.22 (m, 1H), 3.76 - 3.43 (m, 20H), 3.24 - 3.12 (m, 1H), 2.92 (d, J = 4.5 Hz, 1H), 2.71 (d, J = 12.7 Hz, 1H), 2.51 (s, 3H), 2.22 (t, J = 7.3 Hz, 2H), 1.88 - 1.57 (m, 4H), 1.53 - 1.38 (m, 2H). LRMS [M + H] + 648.32。

[0344] Synthesis of 9422

Chem.

[0345] Step 2: To a solution of ethyl (E)-3-(3-cyano-4-fluorophenyl)but-2-enoate (265 mg, 1.14 mmol, 1.0 equiv) in DMF (5 mL) was added NaN3 (111 mg, 1.70 mmol, 1.5 equiv) all at once. The mixture was heated at 70 °C overnight. The reaction was cooled, diluted with EtOAc (50 mL) and poured into crushed ice. After partitioning, the aqueous layer was extracted with EtOAc (30 x 3 mL). The combined organic layers were washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4 and concentrated in vacuo. The crude product was concentrated and purified through a silica gel column using DCM / MeOH to afford the desired product, ethyl (E)-3-(4-azido-3-cyanophenyl)but-2-enoate (121 mg, 42%).

[0346] Step 3: To a solution of ethyl (E)-3-(4-azido-3-cyanophenyl)but-2-enoate (50 mg, 0.20 mmol, 1.0 equiv) in MeOH (2 mL) was added LiOH (9.3 mg, 0.39 mmol, 2 equiv) pre-dissolved in H2O (2 mL). The reaction mixture was stirred at room temperature for 3 h. LC-MS indicated complete conversion. The reaction was concentrated in vacuo to remove volatiles, diluted with H2O (20 mL). The aqueous suspension was adjusted to pH 4 and extracted with EtOAc (20 x 3 mL). The combined organic layers were washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4 and concentrated in vacuo to afford the crude product. The desired product was isolated using silica gel chromatography.

[0347] Step 4: The product from Step 3 (50 mg, 0.22 mmol, 1 equivalent) was dissolved in ACN. To this solution, biotin-PEG4-amine (111 mg, 0.24 mmol, 1.1 equivalents), TEA (92 μL, 0.66 mmol, 3 equivalents), and T3P (261 μL, 0.44 mmol, 2 equivalents) were added, and the reaction mixture was stirred at room temperature overnight. The crude product was concentrated and purified by passing through a silica gel column using DCM / MeOH to obtain the desired product, 4-azido-N-(2-(2-(2-(2N-((E)-18-(4-azido-3-cyanophenyl)-16-oxo-3,6,9,12-tetraoxa-15-azanonadeca-17-en-1-yl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (112 mg, 76%). LRMS: [M+H] + 673.34。

[0348] Synthesis of Compound 9575

Chemical formula

[0349] Synthesis of Further Biotin Compounds Compounds 9582, 9595, 9917, 9559, 9615, and 9616 were synthesized from the corresponding aryl azide intermediates (or commercially available aryl azides) and biotin-PEG4-amine in the same manner as the synthesis of 9575. The mass spectrometry data are shown in Table 1 above.

[0350] Synthesis of Activatable Labels Containing Click Handles or Caged Fluorophores Synthesis of 9140 [Chemical Structure Diagram] Step 1. tert-Butyl 3-oxo-1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)-4,7,10,13-tetraoxa-2-azapentadecane-16-oate (200.0 mg, 0.72 mmol, 1 equiv) was dissolved in 5 mL of THF at 0 °C. By maintaining the reaction mixture in an ice bath, CDI (174.8 mg, 1.08 mmol, 1.5 equiv) was added to the solution. The reaction mixture was stirred for 1 h. Then, (4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)methanamine (309.2 mg, 1.44 mmol, 2.0 equiv) and TEA (300.9 μL, 2.16 mmol, 3 equiv) were added to the reaction mixture, and the reaction was continued for an additional 12 h. The crude product was concentrated and purified by silica gel chromatography using heptane / EtOAc as the eluent to afford the desired product tert-butyl 3-oxo-1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)-4,7,10,13-tetraoxa-2-azapentadecane-16-oate (305.0 mg, 82%).

[0351] Step 2. tert-Butyl 3-oxo-1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)-4,7,10,13-tetraoxa-2-azapentadecane-16-oate (220 mg) was dissolved in 2 mL of DCM. To this solution, 1 mL of TFA was added and the reaction mixture was stirred at room temperature for 1 h until LC-MS indicated no remaining starting material. The desired product, 3-oxo-1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)-4,7,10,13-tetraoxa-2-azapentadecane-16-oic acid (159 mg) was obtained without further purification.

[0352] Step 3.3-oxo-1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)-4,7,10,13-tetraoxa-2-azahexadecane-16-oic acid (60.0 mg, 0.13 mmol, 1 equiv) was dissolved in 5 mL of THF. To this solution, HATU (73.8 mg, 0.19 mmol, 1.5 equiv), DIPEA (67.6 μL, 0.39 mmol, 3 equiv), and (E)-cyclooct-4-en-1-yl(3-aminopropyl)carbamate hydrochloride (34.0 mg, 0.13 mmol, 1 equiv) were added, and the reaction was stirred at room temperature overnight. Silica gel purification was performed using DCM / MeOH to give the final product, (E)-1-(cyclooct-4-en-1-yloxy)-1,7-dioxo-10,13,16-trioxa-2,6-diazaoctadecane-18-yl(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzyl)carbamate (9140) (67 mg, 77%). LRMS: [M+H] + 672.89。

[0353] Synthesis of 9086

Chemical formula

[0354] Synthesis of 9421

Chemical formula

[0355] Synthesis of 9476

Chemical formula

[0356] Example 14 In this example, the synthesis of additional activatable labels and catalysts described herein is explained.

[0357] Synthesis of Compound 9578

Chemical formula

[0358] Step 2: To a 100 mL flask were added biotin-NHS ester (2.90 g, 8.49 mmol), DMF (8 mL), and DIPEA (2.97 mL, 17.0 mmol). The mixture was stirred for 18 hours. The solvent was evaporated and the residue was purified by silica gel chromatography using 0 - 10% MeOH in DCM to obtain tert-butylmethyl (2-(N-methyl-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)penta-amido)ethyl)carbamate. LRMS [M+H] + 415.

[0359] Step 3: To a 100 mL flask, tert-butyl methyl (2-(N-methyl-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)penta-amide)ethyl)carbamate (3.50 g, 8.44 mmol) and TFA (5 mL) were added. The mixture was stirred for 30 minutes. The solvent was evaporated and the residue was co-evaporated with toluene three times to give N-methyl-N-(2-(methylamino)ethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentaamide. LRMS [M+H] + 315。

[0360] Step 4: To a 20 mL vial, 4-(((tert-butoxycarbonyl)amino)methyl)benzoic acid (400 mg, 1.59 mmol), N-methyl-N-(2-(methylamino)ethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentaamide (600 mg, 1.91 mmol), DIPEA (1.11 mL), and DCM (4 mL) were added. To the mixture, T3P (50% in MeCN, 1.46 mL, 2.39 mmol) was added. The mixture was stirred for 1 hour. The mixture was purified by silica gel chromatography using 0-10% MeOH in DCM as the eluent to give a product containing a substantial amount of DIPEA-TFA salt. The material was vigorously triturated twice with Et2O. Each time, the ether was removed by decantation, leaving an oily solid behind. The solid was then dried under vacuum to give tert-butyl (4-(methyl(2-(N-methyl-5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentaamide)ethyl)carbamoyl)-benzyl)carbamate. LRMS [M+H] + 548。

[0361] Step 5: To a 20 mL vial, tert-butyl (4-(methyl(2-(N-methyl-5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentaamido)ethyl)carbamoyl)-benzyl)carbamate (488 mg, 0.891 mmol) and TFA (3 mL) were added. The mixture was stirred for 15 minutes. The solvent was evaporated and the residue was co-evaporated twice with toluene to give 4-(aminomethyl)-N-methyl-N-(2-(N-methyl-5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentaamido)ethyl)benzamide. LRMS [M+H] + 448.

[0362] Step 6: To a 100 mL flask, 4-(aminomethyl)-N-methyl-N-(2-(N-methyl-5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentaamido)ethyl)benzamide (398 mg, 0.889 mmol), (E)-but-2-ene-1,4-diylbis(4-nitrophenyl) bis(carbonate) (from step 1, 744 mg, 1.78 mmol), and DMF (10 mL) were added. The slurry was stirred. To the mixture, DIPEA (1.24 mL, 7.11 mmol) was added. After 30 minutes, the mixture was concentrated. The residue was diluted in DCM and filtered. The filtrate was loaded onto a silica gel column and purified using 0 - 15% MeOH in DCM as the eluent. The product residue was triturated three times with Et2O and the supernatant was poured off from the oily solid formed each time. The remaining solid was dried under vacuum to give (E)-4-(((4-nitrophenoxy)carbonyl)oxy)but-2-en-1-yl (4-(methyl(2-(N-methyl-5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentaamido)ethyl)carbamoyl)benzyl)carbamate. LRMS [M+H] + 727.

[0363] Step 7: To a 20 mL vial, add (4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)methanamine hydrochloride (40.0 mg, 0.159 mmol), (E)-4-(((4-nitrophenoxy)carbonyl)oxy)but-2-en-1-yl (4-(methyl(2-(N-methyl-5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentaamide)ethyl)carbamoyl)benzyl)carbamate (116 mg, 0.159 mmol), DMF (4 mL), and DIPEA (0.278 mL, 1.59 mmol). Stir the mixture for 2 hours. Purify the mixture by RP HPLC (MeCN / water with 0.1% TFA) to obtain compound 9578. LRMS [M+H] + 803。 1 H NMR (400 MHz, DMSO-d6) δ 7.92 - 7.75 (m, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.34 - 7.23 (m, 6H), 6.43 (s, 2H), 5.83 (d, J = 3.7 Hz, 2H), 4.50 (t, J = 3.4 Hz, 4H), 4.30 (t, J = 6.3 Hz, 2H), 4.22 (d, J = 6.1 Hz, 4H), 4.17 - 4.08 (m, 2H), 3.58 (d, J = 12.6 Hz, 3H), 3.37 (s, 2H), 3.10 (td, J = 7.4, 4.8 Hz, 1H), 3.01 (s, 1H), 2.95 (d, J = 16.8 Hz, 1H), 2.88 (s, 2H), 2.86 - 2.80 (m, 1H), 2.68 (s, 1H), 2.58 (d, J = 12.4 Hz, 1H), 2.34 (d, J = 7.2 Hz, 1H), 2.23 (q, J = 15.7, 11.6 Hz, 2H), 1.66 - 1.29 (m, 6H).

[0364] Synthesis of Compound 9643

Chemical Structure

[0365] Step 2: To a 20 mL vial, tert-butyl (E)-(2-(3-(6-azidonaphthalen-2-yl)-N-methylacrylamide)ethyl)-(methyl)carbamate (60.0 mg, 0.146 mmol) and formic acid (2 mL) were added and the mixture was stirred for 2 hours. The solvent was evaporated to obtain (E)-3-(6-azidonaphthalen-2-yl)-N-methyl-N-(2-(methylamino)ethyl)acrylamide. LRMS [M+H] + 310.

[0366] Step 3: To a 20 mL vial, (E)-3-(6-azidonaphthalen-2-yl)-N-methyl-N-(2-(methylamino)ethyl)acrylamide (45.3 mg, 0.146 mmol), (E)-4-(((4-nitrophenoxy)carbonyl)oxy)but-2-en-1-yl (4-(methyl(2-(N-methyl-5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide)ethyl)carbamoyl)benzyl)carbamate (from step 6 of 9578, 106 mg, 0.146 mmol), DMF (4 mL), and DIPEA (0.256 mL, 1.46 mmol) were added. The mixture was stirred for 2 hours. The mixture was purified by RP HPLC (MeCN / water with 0.1% TFA) to obtain compound 9643. LRMS [M+H] +897 1 H NMR (400 MHz, DMSO-d6) δ 8.23 (t, J = 5.9 Hz, 1H), 8.08 (s, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.94 (d, J = 8.7 Hz, 1H), 7.81 (q, J = 6.2, 5.3 Hz, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.70 (d, J = 2.3 Hz, 1H), 7.57 (d, J = 15.7 Hz, 1H), 7.39 - 7.25 (m, 6H), 6.72 (d, J = 15.8 Hz, 1H), 6.39 (d, J = 27.5 Hz, 2H), 5.83 (d, J = 3.0 Hz, 2H), 4.59 - 4.40 (m, 4H), 4.30 (t, J = 6.6 Hz, 1H), 4.22 (d, J = 5.9 Hz, 2H), 4.12 (q, J = 7.7, 6.5 Hz, 1H), 3.38 (br s, 12H), 3.26 (q, J = 6.3 Hz, 2H), 3.12 (q, J = 6.9, 6.4 Hz, 2H), 3.00 (s, 1H), 2.95 (d, J = 15.9 Hz, 1H), 2.87 (s, 1H), 2.82 (dd, J = 11.7, 4.5 Hz, 1H), 2.58 (d, J = 12.3 Hz, 1H), 2.21 (d, J = 20.6 Hz, 2H), 1.67 - 1.28 (m, 6H).

[0367] Synthesis of Compound 9679

Chem.

[0368] Step 2: tert-Butyl (15-((6-aminoacridin-3-yl)amino)-15-oxo-3,6,9,12-tetraoxapentadecyl)carbamate (139.9 mg, 0.25 mmol) was added to a 20 mL vial, and 3 mL of AcOH was added and dissolved in 4.5 mL of water. NaNO2 (29.5 mg, 0.43 mmol) was dissolved in 1 mL of water and added to the reaction at 0 °C. After 5 minutes, NaN3 (32.7 mg, 0.50 mmol) was dissolved in 1 mL of water and added dropwise to the reaction at 0 °C. Bubbles were observed. When the bubbles disappeared, the temperature was raised to room temperature. After 30 minutes, the pH of the aqueous layer was adjusted to 7 with NaHCO3. The reaction was quenched by adding brine and extracted three times with 30 mL of EtOAc. The organic layer was collected, and the crude product was purified by RP HPLC (MeCN / water with 0.1% TFA) to obtain tert-butyl (15-((6-azidoacridin-3-yl)amino)-15-oxo-3,6,9,12-tetraoxapentadecyl)carbamate. LRMS [M+H] + 583。

[0369] Step 3: tert-Butyl (15-((6-azidoacridin-3-yl)amino)-15-oxo-3,6,9,12-tetraoxapentadecyl)carbamate (93.0 mg, 0.16 mmol) was added to a 20 mL vial, and 0.6 mL of TFA was added and dissolved in 2.4 mL of DCM at 0 °C. After 30 minutes, the reaction mixture was diluted with DMF and purified by RP HPLC (MeCN / water with 0.1% TFA) to obtain 1-amino-N-(6-azidoacridin-3-yl)-3,6,9,12-tetraoxapentadecane-15-amide. LRMS [M+H] + 483。

[0370] Step 4: 1-Amino-N-(6-azidoacridin-3-yl)-3,6,9,12-tetraoxapentadecane-15-amide (86.7 mg, 0.18 mmol) was dissolved in 3 mL of anhydrous DMF in a 20 mL vial. DIPEA (94 μL, 0.54 mmol) was added to the reaction at room temperature. Biotin-NHS (92.0 mg, 0.27 mmol) was added to the reaction. After 10 minutes, the reaction mixture was diluted with DMF and purified by RP HPLC (MeCN / water with 0.1% TFA) to obtain Compound 9679. LRMS [M+H] + 709。 11H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 9.56 (s, 1H), 8.84 (s, 1H), 8.42 (d, J = 9.0 Hz, 1H), 8.34 (d, J = 9.1 Hz, 1H), 7.81 (t, J = 5.7 Hz, 1H), 7.73 (dd, J = 9.1, 1.9 Hz, 1H), 7.71 (d, J = 2.1 Hz, 1H), 7.57 (dd, J = 9.0, 2.2 Hz, 1H), 6.42 (s, 1H), 6.37 (s, 1H), 4.30 (dd, J = 7.8, 4.9 Hz, 1H), 4.12 (dd, J = 7.8, 4.4 Hz, 1H), 3.79 (t, J = 6.1 Hz, 2H), 3.57 - 3.46 (m, 16 H), 3.35 (t, J = 5.9 Hz, 2H), 3.08 (m, 1H), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.75 (t, J = 6.1 Hz, 2H), 2.05 (t, J = 7.4 Hz, 2H), 1.66 - 1.54 (m, 1H), 1.53 - 1.39 (m, 2H), 1.36 - 1.20 (m, 2H).

[0371] Synthesis of Compound 9680

Chem.

[0372] Synthesis of PS-9850

Chem.

[0373] Example 15 This example demonstrates the ability to further expand a toolkit of photoreactive groups that are compatible with bioluminescent photocatalytic systems to vinyl-naphthyl-azide. The structures of vinyl-naphthyl-azide-biotin and vinyl-quinoline-azide-biotin are shown in Figure 26A, and their synthesis is included in Example 13. The absorbance profiles of 200 μM of the activatable label in 2% DMSO were monitored with a SPARK multimode plate reader (Figures 26B - C). Generally, substitution onto naphthyl resulted in a range of absorbances that were red-shifted compared to the two absorbance peaks (λmax = 255 and 305 nm) of the parent naphthyl-azide 9043, suggesting a higher ability to absorb light and subsequently easier activation by bioluminescence-induced photocatalytic energy transfer. Naphthyl-azide analogs were further evaluated for their ability to receive bioluminescence-induced photocatalytic protein labels, as well as their light-dependent, light-independent, and catalyst-independent backgrounds (Figure 26D). For this purpose, 100 μM of naphthyl-azide biotin analogs, 0.1 mg / mL of biotinylated protein-depleted K562 lysate, and 60 nM of HT 178 -cpNLuc- 179 : The reaction containing the Ir-9049 conjugate was constructed in TBS (pH 7.5) in the wells of a white 96-well plate. The control reaction contained either no HT 178 -cpNLuc- 179 : Ir-9049 conjugate (light-independent and catalyst-independent background) or HT 178 -cpNLuc- 179:The Ir-9049 conjugate was replaced with NanoLuc (catalyst-free, light-dependent background). Treatment with 100 μM fluorofurimazine for 20 minutes induced bioluminescence. To evaluate the labeling efficiency, samples were collected, separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed as described in Example 1. Western analysis revealed that, except for 9615, the red-shifted absorbance associated with vinyl substitution resulted in an increase in labeling efficiency without a significant increase in light-dependent and light-independent backgrounds compared to the parent naphthyl-azide 9043. Furthermore, methyl substitution of the vinyl group designed to inhibit Michael addition decreased the background and increased the labeling specificity, particularly for 9615. This decrease in background was generally associated with a blue-shifted absorbance compared to equivalent vinyl-naphthyl-azide analogs.

[0374] The absorbance profiles of the quinoline-azide analogs and their ability to undergo bioluminescence-induced protein labeling, as well as the light-dependent and light-independent backgrounds, were strongly affected by the position of the azide. Positions associated with 15 nm and 25 nm red-shifted absorbance (i.e., 9595) showed very high backgrounds, while positions that resulted in a single blue-shifted absorbance peak (i.e., 9917 and 9599) showed weak protein labeling.

[0375] Example 16 During the development of the bioluminescence-induced photocatalyst label described in this specification, an experiment was conducted to evaluate the ability of the ruthenium catalyst to drive the bioluminescence-induced photocatalyst protein label (Figs. 27-28). The structure of the ruthenium catalyst Ru-8974 conjugated to chloroalkane is shown in Fig. 27A, and its synthesis is included in Example 12. The physiochemical properties of the iridium catalyst and the ruthenium catalyst, as well as their ability to undergo activation by bioluminescence energy transfer, were determined as described in Examples 1 and 3 and are shown in Fig. 27B. These analyses revealed that the red-shifted excitation of ruthenium and the greater overlap with NanoLuc luminescence result in a significantly higher energy transfer efficiency from NanoLuc to the catalyst. At the same time, its red-shifted luminescence results in a lower emission energy (EmE) and a lower ability to undergo subsequent energy transfer events with activatable labels compared to the iridium catalyst.

[0376] The ability of the ruthenium catalyst to drive the bioluminescence-induced photocatalyst protein label was further evaluated with respect to a subset of photoreactive groups (Figs. 27D and 28B). The structures of the photoreactive groups included in this screening are shown in Figs. 27C and 28A, and their synthesis is included in Example 13. For this purpose, 100 μM of the activatable label, 0.1 mg / mL of biotinylated protein-depleted K562 lysate, and 60 nM of HT 178 -cpNLuc- 179 :Ru-8974 conjugate-containing reactants were constructed in TBS (pH 7.5) in the wells of a white 96-well plate. Control reactants were either HT 178 -cpNLuc- 179 :Ru-8974 conjugate-free (light-independent and catalyst-independent background) or HT 178 -cpNLuc- 179: The Ru-8974 conjugate was replaced with NanoLuc (catalyst-free, light-dependent background). Treatment with 100 μM fluorofurimazine for 20 minutes induced bioluminescence. To evaluate the labeling efficiency, samples were collected, separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed as described in Example 1. Western analysis revealed that ruthenium could drive bioluminescence-induced photocatalytic protein labeling with several activatable labels, regardless of a wide range of efficiencies and light-dependent and light-independent backgrounds. Some substitutions increased the specificity of the label to varying degrees, presumably by interfering with the rate of formation and / or lifetime of the reactive intermediate, resulting in an overall smaller labeling radius.

[0377] Example 17 This example demonstrates the ability to assemble a bioluminescent photocatalytic complex intracellularly for subsequent protein labeling and enrichment (Figure 29). The structures of activatable labels containing either phenyl-trifluoro-methyldiazirine or vinyl-naphthyl-azide linked to biotin cleavable by palladium are shown in Figure 29A, and their synthesis is included in Example 13. Briefly, HeLa cells were transfected with a DNA construct encoding HT 178 -cpNLuc- 179 and 2x10 5Plated in wells of a 6-well plate at cells / mL and incubated overnight at 37 °C and 5% CO2. The next day, the plates were treated with the Ir-9049 or Ru-8974 catalyst at a final concentration of 3 μM for 90 minutes to construct the bioluminescent photocatalyst complex. To remove the excess unreacted catalyst, the cells were washed twice in HBSS buffer for 15 minutes each. The last HBSS wash was replaced with Opti-MEM medium supplemented with 2% serum and 20 μM of the cleavable activatable label. After a 30-minute incubation, the plates were treated with 20 μM fluorofurimazine for up to 60 minutes, and the control cells were left untreated. To remove the excess unreacted activatable catalyst, the cells were washed twice in HBSS buffer for 15 minutes each. The last HBSS wash was replaced with 1 mL of mammalian lysis buffer (Promega) supplemented with 10-fold diluted 10×RQ1-DNase buffer (Promega), 50-fold diluted RQ1-DNase (Promega), and 100-fold diluted protease inhibitor cocktail (Promega). After incubation at room temperature for 30 minutes in a constant mixing state, the cell lysates were collected, and the biotinylated proteins were captured on 75 μL of high-performance Magne (registered trademark) streptavidin beads (Promega), while the non-specific interactions were washed away. The labeled proteins were then released by incubation with a palladium cleavage reagent (Promega) for 30 minutes, separated on SDS-PAGE, transferred to a PVDF membrane, and subjected to Western analysis using an antibody against HaloTag (Promega). Western blot (Figure 29B) revealed significantly more efficient chimeric iridium-driven photocatalyst labeling and enrichment by either diazirine or vinyl-naphthyl azide.

[0378] Example 18 In this example, we will describe the further optimization of the complementation-based bioluminescent photocatalyst complex and its use as a means to target the photocatalytic system to an endogenous HiBiT-tagged target (Figs. 30 and 31). To increase the efficiency of bioluminescent energy transfer to the conjugated catalyst, a circularly permuted LgBiT mutant (E4D, Q42M, M106K, T144D (i.e., cpmLgBiT circularly permuted at residues 67 / 68)) from LgTrip, which is inserted into the surface loop of HaloTag (between residues 178 - 179) and is close to the ligand interaction site, is incorporated into a chimeric structure (i.e., HT 178 -cpmLgBiT- 179 )(Fig. 30A). First, the chimeric was compared to a simple LgBiT-HaloTag fusion for its brightness and the efficiency of bioluminescence resonance energy transfer (BRET) to the conjugated HaloTag TMR-fluorescent ligand. For this purpose, the LgBiT-HaloTag fusion and the chimeric were diluted to a final concentration of 13 nM in TBS + 0.01% BSA and complemented with an equal amount of 130 nM VS-HiBiT peptide for 60 minutes. After complementation, the reaction was either further incubated with 10× HaloTag-TMR ligand at a final concentration of 300 nM or left untreated. After treatment with 10x fluorofurinazine at a final concentration of 20 μM, the raw luminescence (total RLU) or filtered luminescence of the donor (e.g., 450 nm / 8 nm BP) and acceptor (600 nm LP) was measured with a GloMax® Discover plate reader (Promega). The BRET ratio was further calculated for each sample by dividing the acceptor luminescence value by the donor luminescence value. HT 178 -cpmLgBiT- 179 was a 100-fold dimer but showed a 10-fold higher BRET efficiency (Fig. 30B), indicating that the chimeric structure was able to induce a higher proximity between the luminescent substrate binding site and the conjugated fluorescent ligand, or adopt a conformation favorable for energy transfer between the two, or both.

[0379] To demonstrate the specificity driven by a system that links the BRET activation of a conjugated ligand (e.g., chloroalkane conjugated to a fluorophore, chloroalkane conjugated to a photosensitive catalyst, etc.) and the complementation of a bioluminescent energy donor, cells expressing endogenous HiBiT-tagged EGFR localized to the cell membrane were transfected with HT 178 -cpmLgBiT- 179 DNA encoding the chimera, plated in 35 mm glass-bottom dishes (MatTek Corporation), and incubated in a tissue culture incubator for 24 hours. The next day, the cells were either left unlabeled or labeled with HaloTag JF-549-fluorescent ligand at a final concentration of 1 μM for 1 hour. After two washes for 15 minutes each, the cells were treated with fluorofurinazine at a final concentration of 20 μM and imaged with an Olympus LV200 bioluminescence microscope (Olympus). Appropriate fields of view were identified based on imaging of the total luminescence signal of the donor. To image BRET events, luminescence images of the donor and acceptor were sequentially acquired using a 460 / 80 bandpass filter and a 590 nm longpass filter, respectively. Image analysis revealed that the total luminescence and BRET signals were localized to the cell membrane and that there was efficient energy transfer to the fluorescent acceptor.

[0380] The ability of a complementation-based bioluminescent photocatalyst system to drive the labeling of endogenous HiBiT-tagged GAPDH was further evaluated (Figure 31). The structure of an activatable label containing either phenyl-trifluoro-methyl diazirine or vinyl-naphthyl-azide linked to palladium-cleavable biotin is shown in Figure 31A. Briefly, HeLa cells expressing endogenous HiBiT-tagged GAPDH were transfected with HT 178 -cpmLgBiT- 179 DNA constructs encoding, and 2x10 5Plated in wells of a 6-well plate at cells / mL and incubated overnight at 37 °C and 5% CO2. The next day, the plates were treated with the Ir-9049 or Ru-8974 catalyst at a final concentration of 3 μM for 90 minutes to construct a bioluminescent photocatalyst complex. Bioluminescence induction labeling and concentration were carried out as described in Example 17 above. The concentrated protein was separated on SDS-PAGE, transferred to a PVDF membrane, and subjected to Western analysis using an antibody against either HaloTag (Promega) or HiBiT (Promega). Western blot (Figure 31B) revealed significantly more efficient iridium-driven photocatalyst labeling and concentration of both chimeric and endogenous HiBiT-GAPDH by either diazirine or vinyl-naphthyl azide.

[0381] Example 19 During the development of the bioluminescence-induced photocatalyst labeling described herein, experiments were conducted to evaluate the ability to drive intracellular labeling of adjacent proteins (Figure 32). In this evaluation, constructs encoding the gene fusion of EGFR-HT 178 -cpNLuc- 179 localized to the cell membrane were utilized (Figure 32A). Total luminescence emitted over time from cells expressing either the chimeric alone or the EGFR-HT 178 -cpNLuc- 179 fusion indicated that the fusion was significantly dimeric (Figure 32B).

[0382] Cells expressing the chimeric were further evaluated for their ability to drive fluorophimine-dependent labeling of adjacent proteins. For this purpose, HeLa cells were transfected with a DNA construct encoding the EGFR-HT 178 -cpNLuc- 179 fusion, and 1×10 6Cells were plated in 10 cm dishes at a cell / dish density and incubated overnight at 37 °C with 5% CO₂. The next day, the plates were treated with the Ir-9049 catalyst at a final concentration of 3 μM for 90 minutes to construct the bioluminescent photocatalyst complex. After two washes to remove excess unreacted catalyst, the cells were treated with 20 μM cleavable diazirine-biotin for 30 minutes. Bioluminescence was initiated by treatment with 20 μM fluorofurimazine, and the control cells were left untreated. After a 60-minute incubation, the cells were washed to remove excess unreacted cleavable diazirine-biotin and then collected in 10 mM MOPS buffer (pH 7.4) supplemented with a 100-fold diluted protease inhibitor cocktail (Promega). After sonication, 1% DDM at a final concentration and 150 mM NaCl at a final concentration were added to the lysate, and the mixture was incubated for 60 minutes with constant mixing and then centrifuged briefly to remove cell debris. A small portion of the lysate from replicates that were either treated or untreated with fluorofurimazine was subjected to Western analysis using antibodies against HaloTag (Promega) or EGFR (Cell Signaling) to verify equivalent processing between replicates. This analysis also revealed significantly lower expression of the overexpressed EGFR-HT 178 -cpNLuc- 179 fusion compared to endogenous EGFR (Figure 32C). The labeled proteins were further concentrated by overnight capture on 120 μL of high-performance Magne® streptavidin beads (Promega). The next day, nonspecific interactors were washed out, and the labeled proteins were released by incubation with a palladium cleavage reagent (Promega) for 45 minutes. Western analysis of the eluted proteins using antibodies against HaloTag (Promega) and EGFR (Cell Signaling) revealed fluorofurimazine-dependent enrichment of the chimeras despite relatively low expression and faint luminescence (Figure 32D).

[0383] To detect the enrichment of adjacent proteins, the lysate was further subjected to mass spectrometry. For this purpose, the lysate was incubated with silica HMBC beads (Promega) in LCMS-grade acetonitrile at a final concentration of 80% for 30 minutes. After washing three times in 80% ethanol, the captured proteins were subjected to 30 minutes of reduction, followed by 30 minutes of alkylation, and then on-bead digestion with trypsin-LysC was carried out overnight. The tryptic peptides were removed, quenched with 5% formic acid, desalted, and subjected to mass spectrometry. This analysis revealed not only a significant fluorophirimazine-dependent enrichment of EGFR but also a significant enrichment of other membrane-localized proteins and proteins associated with degradation, suggesting that the EGFR-chimeras are continuously phosphorylated and degraded (Figure 32E).

[0384] Example 20 During the development of the bioluminescence-induced photocatalyst label described herein, experiments were conducted to evaluate the ability of the activatable label to undergo covalent cross-linking with adjacent nucleic acids (Figures 33 - 34). The structures of the photoreactive groups evaluated are shown in Figures 33C and 34A, and their synthesis is described in Example 13.

[0385] Briefly, experimental and control reactants containing (1) 0.1 μg / μL DNA or RNA marker (Promega) and 50 μM activatable label, or (2) 0.1 μg / μL DNA or RNA marker (Promega), 50 μM activatable label, and 100 μM catalyst (Ir-9049 or Ru-8974) were constructed in wells of a UV-transparent 96-well plate in TE buffer at pH 7.4. The plate was either kept in the dark (control) or irradiated at 455 nm (2% LED; ~1.6 W) for 20 minutes using an Efficiency Aggregators bioreactor. To assess the covalent cross-linking efficiency, after removing the reactants from the non-cross-linked activatable label using a Zeba column (ThermoFisher), spots were made on a nitrocellulose membrane using a slot blot apparatus. The membrane was blocked with 5% BSA (Promega) in TBST for 1 hour at room temperature and then incubated overnight at 4 °C with an anti-biotin antibody (Invitrogen) in TBST. The next day, the membrane was washed three times with TBST and then incubated for 1 hour with a secondary anti-goat HRP antibody (Jackson laboratories). After three washes with TBST, the membrane was treated with an ECL substrate (Promega) and scanned in the chemiluminescence channel to detect biotin-labeled DNA or RNA. The band amounts quantified using Image J software were normalized as follows: Ir-9049-driven DNA or RNA labeling efficiency was derived from normalization against a label using 9069 as the activatable label, and Ru-8974-driven DNA or RNA labeling efficiency was derived from normalization against a label using 9616 as the activatable label. These analyses revealed overall more efficient iridium-driven activation and cross-linking to DNA (Figure 33D) and RNA (Figure 34B). Furthermore, some vinyl-naphthyl-azide and vinyl-quinoline-azide showed relatively high labeling efficiencies.

[0386] Example 21 Since it has been shown that the activatable label cross-links to both proteins and nucleic acids, further experiments were perfor...

Claims

1. A system comprising: (a) a bioluminescent protein; (b) a fluorophore, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the fluorophore by interaction with the fluorophore; (c) a photocatalyst, wherein the photocatalyst is activated by exposure to light of the first wavelength; and (d) an activatable label, wherein the activatable label is converted to an activated label when in proximity to the activated photocatalyst. The system of claim 1, comprising:

2. A system comprising: (a) a bioluminescent protein; (b) a fluorophore, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the fluorophore by interaction with the fluorophore; and (c) a photocatalyst, wherein the photocatalyst is activated by exposure to light of the first wavelength. The system of claim 2, comprising:

3. The system according to claim 1 or 2, wherein the bioluminescent protein is luciferase.

4. The system according to claim 3, wherein the luciferase is an Oplophorous-derived polypeptide having at least 70% sequence identity with SEQ ID NO:

1.

5. The system according to claim 4, wherein the luciferase has 100% sequence identity with SEQ ID NO:

1.

6. The system according to claim 3, wherein the luciferase is a variant of the Oplophorous-derived polypeptide with a cyclic rearrangement.

7. The system according to claim 6, wherein the luciferase comprises a first segment having at least 70% sequence identity with the first portion of SEQ ID NO: 1 and a second segment having at least 70% sequence identity with the second portion of SEQ ID NO:

1.

8. The system according to claim 7, wherein the first segment has 100% sequence identity with the first portion of SEQ ID NO: 1 and the second segment has 100% sequence identity with the second portion of SEQ ID NO:

1.

9. A system comprising: (a) a structural complementing component of a bioluminescence complex; (b) a fluorophore, wherein the bioluminescence complex catalyzes the emission of light of a first wavelength from the fluorophore by interaction with the fluorophore; (c) a photocatalyst, wherein the photocatalyst is activated by exposure to light of the first wavelength; and (d) An activatable label that is converted into an activated label when the activatable label is in proximity to the activated photocatalyst. The system comprising the above.

10. A system comprising: (a) A structural complementing component of a bioluminescence complex; (b) A luminophore, wherein the bioluminescence complex catalyzes the emission of light of a first wavelength from the luminophore by interaction with the luminophore; and (c) A photocatalyst that is activated upon exposure to light of the first wavelength. The system comprising the above.

11. The system according to claim 10, wherein the structural complementing components comprise two or more peptides and / or polypeptides that can bind to each other to form an active bioluminescence complex.

12. The system according to claim 11, wherein the structural complementing components collectively comprise at least 70% sequence identity with SEQ ID NO:

2.

13. The system according to claim 12, wherein the structural complementing components comprise a peptide having at least 70% sequence identity with SEQ ID NO: 3 or 4, and a polypeptide having at least 70% sequence identity with SEQ ID NO:

5.

14. The system according to claim 13, wherein the structural complementing components comprise a peptide having 100% sequence identity with SEQ ID NO: 3 or 4, and a polypeptide having 100% sequence identity with SEQ ID NO:

5.

15. The system according to claim 12, wherein the structural complementing components comprise a peptide having at least 70% sequence identity with SEQ ID NO: 3 or 4, a polypeptide having at least 70% sequence identity with SEQ ID NO: 6, and a peptide having at least 70% sequence identity with SEQ ID NO:

7.

16. The system according to claim 15, wherein the structural complementing components comprise a peptide having 100% sequence identity with SEQ ID NO: 3 or 4, a polypeptide having 100% sequence identity with SEQ ID NO: 6, and a peptide having 100% sequence identity with SEQ ID NO:

7.

17. The system according to any one of claims 13 to 16, wherein the polypeptide component is cyclically rearranged.

18. The system according to any one of claims 1 to 17, wherein the luminophore is a luciferin or coelenterazine molecule.

19. The system according to claim 18, wherein the coelenterazine molecule is furimazine.

20. The system according to claim 19, wherein the selenotriazine molecule is frimazine or fluorofrimazine.

21. The system according to any one of claims 1 to 20, wherein the photocatalyst is an iridium-based or ruthenium-based photocatalyst.

22. The photocatalyst has the structure of formula (I) 【Chemical 1】 wherein each series of dashed lines 【Chemical 2】 represents the presence or absence of a fused six-membered ring, M is a transition metal, m1, m2, m3, n1, n2, n3, p1, p2, and p3 are each independently 0, 1, or 2, R 1a 、 R 1b 、 R 1c 、 R 2a 、 R 2b 、 R 2c 、 R 3a 、 R 3b 、 and R 3c are each independently selected from halo, alkyl, haloalkyl, amino, heteroalkyl, and group-linker-Q, where Q is a capture element, X 1a , X 1b , X 2a , X 2b , X 3a , and X 3b are each independently selected from N and C, where at least one of X 1a and X 1b is N, and at least one of X 2a and X 2b is N, and at least one of X 3a and X 3b is N, X 1c 、 X 1d 、 X 2c 、 X 2d 、 X 3c 、 and X 3d is independently selected from CH and N, respectively, Z is an anion, q is 0, 1, or 2, the system according to claim 21.

23. The system according to claim 22, wherein the transition metal is selected from Ru and Ir.

24. The photocatalyst is 【Chemical 3】 an iridium-based photocatalyst selected from, or a derivative thereof, and the compound is functionalized with at least one group - linker - Q, wherein Q is a capture element, the system according to claim 23.

25. The photocatalyst is 【Chemical Formula 4】 a ruthenium-based photocatalyst selected from, or a derivative thereof, and the compound is functionalized with at least one group - linker - Q, wherein Q is a capture element, the system according to claim 23.

26. The photocatalyst has the formula 【Chemical Formula 5】 the system according to claim 22.

27. R 1a 、R 1b 、R 1c 、R 2a 、R 2b 、R 2c 、R 3a 、R 3b 、and R 3c The system according to claim 22, wherein one of R

28. The system according to claim 27, wherein Q is a capture element.

29. The system according to claim 28, wherein Q is a haloalkane.

30. The system according to claim 27, wherein the linker comprises an ester (-C(O)O-), an amide (-C(O)NH-), a carbamate (-NHC(O)O-), a urea (-NHC(O)NH-), a phenylene (e.g., 1,4-phenylene), a linear or branched alkylene, an oligo- or poly-ethylene glycol (-(CH 2 CH 2 O) x -), or a combination thereof.

31. The linker is -O(CH 2 CH 2 O) z1 -C(O)NH-(CH 2 CH 2 O) z2 -C(O)NH-(CH 2 z3 -(OCH 2 CH 2 z4 O-, wherein z1, z2, z3, and z4 are each independently selected from 0, 1, 2, 3, 4, 5, and 6, the system according to claim 30.​​

32. The linker is [Chemical Formula 6] selected from, the system according to claim 31.

33. The system according to any one of claims 1 to 32, wherein the photocatalyst is an organic photoredox catalyst.

34. The system according to claim 33, wherein the organic photoredox catalyst is selected from quinone, pyrylium, acridinium, and xanthene.

35. The organic photoredox catalyst is ​ a quinone selected from, the system according to claim 34.

36. The organic photoredox catalyst is 【Chemical 8】 a pyrylium selected from, the system according to claim 34.

37. The organic photoredox catalyst is 【Chemical Formula 9】 an acridinium selected from, the system according to claim 34.

38. The organic photoredox catalyst is 【Chemical 10】 a xanthene selected from, the system according to claim 34.

39. The system according to claim 34, wherein the organic photoredox catalyst is a thiazine-based organic photoredox catalyst.

40. The system according to any one of claims 1 to 39, wherein the light of the first wavelength is within the visible light spectrum.

41. The system according to claim 40, wherein the light of the first wavelength is 400 to 500 nm.

42. The system according to claim 1 or 9, wherein the photocatalyst promotes (i) energy transfer to the activatable label, (ii) extraction of hydrogen from the activatable label, or (iii) catalysis of a photoredox reaction.

43. The system according to claim 1 or 9, wherein the photocatalyst transfers energy to the activatable label by Förster resonance energy transfer, Dexter energy transfer, single electron transfer, or singlet oxygen.

44. The system according to claim 1 or 9, wherein the conversion of the activatable label to the activated label includes converting an inactive moiety on the activatable label to a reactive moiety on the activated label.

45. The system according to claim 1 or 9, wherein the activatable label includes a fluorogenic dye, a scavenger, a cleavable scavenger, and a click handle.

46. The system according to claim 1 or 9, wherein the activatable label includes a photoreactive group and a functional group.

47. The system according to claim 46, wherein the activated label covalently binds to a target molecule upon activation of the photoreactive group by the photocatalyst or photosensitizer.

48. The system according to claim 47, further comprising a target molecule with which the activated label can react.

49. The system according to claim 48, wherein the target molecule is a protein, carbohydrate, lipid, or nucleic acid.

50. The system according to claim 46, wherein the photoreactive group includes benzoyl azide, benzyldiazirine, or an activatable variant thereof.

51. The activatable benzoyl azide variant is 【Chemical 11】 selected from wherein R is selected from H, Cl, F, Br, I, CH 3 , OH, SH, NH 2 , CN, CF 3 , CCl 3 , CH═CH 2 , -CH 2 -CH 3 , -CH 2 -OH, -CH 2 NH 2 , CH 2 SH, CH 2 Cl, CH 2 Br, CH 2 F, CHF 2 , CH 2 CN, CH 2 CF 3 , CH 2 Cl 3 , O-CH3, C(O)CH 3 , C(O)OH, and C(O)NH 2 and is selected from wherein X is O or S, the system according to claim 50.

52. The activatable benzyldiazirine variant is 【Chemical 12】 selected from wherein R is H, Cl, F, Br, I, CH 3 , OH, SH, NH 2 , CN, CF 3 , CCl 3 , CH=CH 2 , -CH 2 -CH 3 , -CH 2 -OH, -CH 2 NH 2 , CH 2 SH, CH 2 Cl, CH 2 Br, CH 2 F, CHF 2 , CH 2 CN, CH 2 CF 3 , CH 2 Cl 3 , O-CH3, C(O)CH 3 , C(O)OH, and C(O)NH 2 The system according to claim 50, selected from

53. The photoreactive group is 【Chemical 13】 (2-Aryl-5-carboxytetrazole (ACT)), or an ACT derivative, wherein R is H, Cl, F, Br, I, CH 3 , OH, SH, NH 2 , CN, CF 3 , CCl 3 , CH=CH 2 , -CH 2 -CH 3 , -CH 2 -OH, -CH 2 NH 2 , CH 2 SH, CH 2 Cl, CH 2 Br, CH 2 F, CHF 2 , CH 2 CN, CH 2 CF 3 , CH 2 Cl 3 , O-CH3, C(O)CH 3 , C(O)OH, and C(O)NH 2 The system according to claim 46, selected from

54. The photoreactive group is 【Chemical 14】 comprising, wherein one of R1 to R3 is a bond to the remainder of the activatable moiety, and the other two of R1 to R3 are independently H, Cl, F, Br, I, CH 3 , OH, SH, NH 2 , CN, CF 3 , CCl 3 , CH=CH 2 , -CH 2 -CH 3 , -CH 2 -OH, -CH 2 NH 2 , CH 2 SH, CH 2 Cl, CH 2 Br, CH 2 F, CHF 2 , CH 2 CN, CH 2 CF 3 , CH 2 Cl 3 , O-CH3, C(O)CH 3 , C(O)OH, and C(O)NH 2 selected from, the system according to claim 46.

55. The system according to claim 46, wherein the photoreactive group includes furocoumarin.

56. the furocoumarin is 【Chemical Formula 15】 selected from, wherein one of R1 to R2 is a bond to the remainder of the activatable moiety, and the other of R1 to R2 is independently H, Cl, F, Br, I, CH 3 , OH, SH, NH 2 , CN, CF 3 , CCl 3 , CH═CH 2 , -CH 2 -CH 3 , -CH 2 -OH, -CH 2 NH 2 , CH 2 SH, CH 2 Cl, CH 2 Br, CH 2 F, CHF 2 , CH 2 CN, CH 2 CF 3 , CH 2 Cl 3 , O-CH3, C(O)CH 3 , C(O)OH, and C(O)NH 2 selected from, the system according to claim 55.

57. the photoreactive group is 【Chemical 16】 including, in the formula, each n is independently 1, 2, 3, or 4, Each R is independently hydrogen, halo, C 1 -C 4 -alkyl, C 2 -C 4 -alkenyl, hydroxy, mercapto, amino, cyano, C 1 -C 4 -alkoxy, halo-C 1 -C 4 -alkyl, hydroxy-C 1 -C 4 -alkyl, amino-C 1 -C 4 -alkyl, mercapto-C 1 -C 4 -alkyl, cyano-C 1 -C 4 -alkyl, -C(O)-C 1 -C 4 -alkyl, -C(O)OH, and -C(O)NH 2 selected from, Q is CH or N, G is -N 3 , -CH=CH-N 3 or 【Chemical 17】 The system according to claim 46.

58. the photoreactive group includes Z-A, in the formula, A is 【Chemical Formula 18】 being wherein each n is independently 1, 2, 3, or 4, and each R is independently hydrogen, halo, C 1 -C 4 alkyl, C 2 -C 4 alkenyl, hydroxy, mercapto, amino, cyano, C 1 -C 4 -alkoxy, halo-C 1 -C 4 -alkyl, hydroxy-C 1 -C 4 -alkyl, amino-C 1 -C 4 -alkyl, mercapto-C 1 -C 4 -alkyl, cyano-C 1 -C 4 -alkyl, -C(O)-C 1 -C 4 -alkyl, -C(O)OH, and -C(O)NH 2 selected from, Q is CH or N, and G is -N 3 , -CH=CH-N 3 , or 【Chemical Formula 19】 being Z is -CR 7 =CR 8 -C(X)-, -C(X)-, and a bond, where R 7 and R 8 are each independently hydrogen or C 1 to C 4 alkyl, and X is O or S, the system of claim 46.

59. The system according to claim 46, wherein the functional group is selected from a scavenger, a cleavable scavenger, a fluorescent molecule, or a click handle.

60. the functional group is 【Chemical 20】 a fluorogenic fluorescent molecule selected from

61. The components of the bioluminescent protein or the bioluminescent complex are fused to a first molecular entity, the photocatalyst or photosensitizer is linked to a second molecular entity, and the interaction between the first molecular entity and the second molecular entity causes the bioluminescent protein or bioluminescent complex to be sufficiently close to the photocatalyst or photosensitizer such that the light emitted by the lumophore due to the interaction with the bioluminescent protein or bioluminescent complex activates the photocatalyst or photosensitizer. The system according to any one of claims 1 to 60.

62. The system according to claim 61, wherein the first molecular entity is a scavenger and the second molecular entity is a capture element.

63. The system according to claim 62, wherein the components of the bioluminescent protein or bioluminescent complex are fused to the N-terminus, C-terminus, or internal site within the scavenger.

64. The components of the bioluminescent protein or the bioluminescent complex are fused to a modified dehalogenase capable of forming a covalent bond with its substrate or inserted into the modified dehalogenase, and the photocatalyst or photosensitizer is linked to the dehalogenase substrate. The system according to claim 61.

65. The binding of the modified dehalogenase to the dehalogenase substrate sufficiently proximate the photocatalyst or photosensitizer such that the light emitted by the lumophore due to the interaction with the bioluminescent protein or bioluminescent complex activates the photocatalyst or photosensitizer. The system according to claim 64.

66. The system according to claim 64, wherein the modified dehalogenase includes at least 70% sequence identity with SEQ ID NO:

8.

67. The system according to claim 66, wherein the modified dehalogenase comprises 100% sequence identity with SEQ ID NO:

8.

68. The system according to claim 64, wherein the dehalogenase substrate is a haloalkane.

69. The structure of the photocatalyst linked to the dehalogenase substrate is P - linker - AX, where P is the photocatalyst, A is (CH 2 ), 2-12 and X is a halogen, and the linker is a linker moiety capable of linking P to A - X. The system according to claim 64.

70. The system according to claim 69, wherein the linker is a polyatomic straight-chain or branched-chain containing C, N, S, or O, or a group containing one or more rings, such as a saturated or unsaturated ring, such as one or more aryl rings, heteroaryl rings, or any combination thereof.

71. wherein the linker is -O(CH 2 ), 2 --(CH2)O--, --CH 2 --, --NHC(O)O--, --OC(O)NH--, NHC(O)--, and --C(O)NH--, the system according to claim 70, comprising a combination of

72. The system according to claim 70 or 71, wherein the linker has a length of 5 to 50 atoms.

73. The system according to claim 61, wherein the bioluminescent protein or complex is conjugated to a target binder, and the target binder is capable of binding to the target molecule.

74. The system according to claim 73, wherein the target binder is a protein or peptide directly or indirectly fused to a component of the bioluminescent protein or the bioluminescent complex.

75. The system according to claim 73, wherein the target molecule is a nucleic acid target binder capable of specifically or non-specifically binding to a nucleic acid.

76. The system according to claim 75, wherein the target binder is a Cas protein and the target molecule is a nucleic acid modified by CRISPR.

77. The system according to claim 76, further comprising a guide RNA (gRNA).

78. The system according to claim 74, wherein the target molecule is a target peptide or protein, and the target binder is capable of binding to the target peptide or protein.

79. The system according to claim 73, wherein the target binder is a small molecule or nucleic acid directly or indirectly linked to a component of the bioluminescent protein or the bioluminescent complex.

80. A cell comprising the system according to any one of claims 1 to 79.

81. A method of activation dependent on the proximity of molecules within a cell, comprising contacting the cell with a fluorophore under conditions that allow the fluorophore to enter the cell, wherein the cell (a)A fusion of a bioluminescent protein and a capture protein, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the fluorophore by interaction with the fluorophore, said fusion; (b)A conjugate of (A) a capture ligand and (B) a photocatalyst or photosensitizer, wherein the capture protein forms a covalent bond with the capture ligand by interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of the first wavelength, said conjugate, and (c)An activatable label, wherein the activatable label is converted to an activated label when in proximity to the activated photocatalyst or photosensitizer, said activatable label, comprising said method.

82. A method of inducing proximity-dependent bioorthogonal chemical ligation, comprising contacting a cell with a fluorophore under conditions such that the fluorophore enters the cell, wherein the cell (a)A fusion of a bioluminescent protein and a capture protein, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the fluorophore by interaction with the fluorophore, said fusion; (b)A conjugate of (A) a capture ligand and (B) a photocatalyst or photosensitizer, wherein the capture protein forms a covalent bond with the capture ligand by interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of the first wavelength, said conjugate, (c)An activatable label, wherein the activatable label is converted to an activated label when in proximity to the activated photocatalyst or photosensitizer, said activatable label, and (d)A target molecule, wherein the activated label reacts with the target molecule when in proximity to the target molecule, said target molecule, comprising said method.

83. The method according to claim 82, wherein the activated label reacts with the target molecule by forming a bond with the activated label.

84. A method of activation dependent on proximity of an activatable label within a cell, comprising (a)expressing a fusion of a bioluminescent protein and a capture protein within the cell, (b) contacting the cell with a fluorophore under conditions such that the fluorophore enters the cell, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the fluorophore by interaction with the fluorophore, said contacting; (c) contacting the cell with a conjugate of (i) a capture ligand and (ii) a photocatalyst or photosensitizer under conditions such that the conjugate enters the cell, wherein the capture protein forms a covalent bond with the capture ligand by interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of the first wavelength, said contacting, and (d) contacting the cell with an activatable label, wherein the activatable label is converted to an activated label when in proximity to the activated photocatalyst or photosensitizer, said contacting, comprising the method. [

85. ] A method for inducing a bioorthogonal chemical reaction that depends on proximity to a target molecule, (a) expressing a fusion of a bioluminescent protein and a capture protein intracellularly, (b) contacting the cell with a fluorophore under conditions such that the fluorophore enters the cell, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the fluorophore by interaction with the fluorophore, said contacting, (c) contacting the cell with a conjugate of (i) a capture ligand and (ii) a photocatalyst or photosensitizer under conditions such that the conjugate enters the cell, wherein the capture protein forms a covalent bond with the capture ligand by interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of the first wavelength, said contacting, (d) contacting the cell with an activatable label, wherein the activatable label is converted to an activated label when in proximity to the activated photocatalyst or photosensitizer, said contacting, comprising wherein the activated label reacts with the target molecule when in proximity to the target molecule, the method. [

86. ] The method according to claim 85, wherein the activated label reacts with the target molecule by forming a bond with the activated label. [

87. ] A method of activation dependent on the proximity of a photocatalyst or photosensitizer within a cell, comprising contacting the cell with a lumophore under conditions such that the lumophore enters the cell, wherein the cell is (a) a fusion of a bioluminescent protein and a capture protein, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the lumophore by interaction with the lumophore, said fusion (b) a conjugate of (A) a capture ligand and (B) a photocatalyst or photosensitizer, wherein the capture protein forms a covalent bond with the capture ligand by interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of the first wavelength, said conjugate The method comprising.

88. A method of activation dependent on the proximity of a photocatalyst or photosensitizer within a cell, comprising: (a) expressing a fusion of a bioluminescent protein and a capture protein within the cell; (b) contacting the cell with a lumophore under conditions such that the lumophore enters the cell, wherein the bioluminescent protein catalyzes the emission of light of a first wavelength from the lumophore by interaction with the lumophore, said contacting, and (c) contacting the cell with a conjugate of (i) a capture ligand and (ii) a photocatalyst or photosensitizer under conditions such that the conjugate enters the cell, wherein the capture protein forms a covalent bond with the capture ligand by interaction with the capture ligand, and the photocatalyst or photosensitizer is activated by exposure to light of the first wavelength, said contacting The method comprising.

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