Intracellular Linkage of a Photocatalyst for Protein Labeling Mediated by a Photoresponsive Probe

Abstract

Embodiments of the present disclosure relate to methods, compositions, and systems for photoactivated molecular labeling based on proximity. Molecules can be labeled by activation of a linked photocatalyst that can transfer energy to a nearby biomolecule labeling agent. Depending on the activation half-life and diffusion coefficient of the labeling agent, molecules within a specific proximity of the linked photocatalyst can be labeled, while molecules outside that proximity are not labeled.

Description

Technical Field

[0001] Incorporation by reference of all priority applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 366,463, filed on June 15, 2022, and U.S. Provisional Patent Application No. 63 / 384,767, filed on November 22, 2022, the contents of each of which are hereby incorporated by reference in their entirety.

Background Art

[0002] Field The present disclosure relates to compositions, systems, and methods for labeling biomolecules based on in vitro or in vivo proximity.

[0003] Description of Related Art Many disease conditions can be understood by elucidating localized biomolecular networks, or microenvironments. For this purpose, enzymatic proximity labeling platforms are widely applied to map the detailed spatial relationships of intracellular structures. These spatial relationships between these biomolecules are known to underlie fundamental biological processes. From the perspective of intracellular signaling, proteins may localize within defined landscapes, such as to the cell membrane, which can also be considered a microenvironment. Localized signaling within these microenvironments can play an important role in intercellular signaling communication. Therefore, the ability to accurately map these intracellular microenvironments can provide important insights into basic biology and potentially have a broad impact on human health and the development of new therapeutic strategies.

[0004] There are several platforms that facilitate specific labeling of proteins by leveraging spatial proximity. A review of existing methods for targeting small molecules to protein sequences has revealed that orthogonal approaches can complement known methods. These methods (including, but not limited to, APEX, Air-ID, BioID, SPPLAT, EMARS, and Turbo-ID) utilize a common design element in which a ligase is genetically fused to the protein of interest. This functional enzyme catalytically generates reactive species that target specific amino acid residues in adjacent systems by diffusion and / or physical contact. However, the reactive species produced by these techniques are known to have a relatively long half-life. Therefore, these reactive species may diffuse to distances comparable to or longer than the labeling, thereby labeling proteins outside the immediate vicinity of the ligase and causing non-specific results. Furthermore, the intermediate reactive species produced by these methods may exhibit selective (i.e., non-agnostic) binding to specific amino acid residues. From the perspective of intracellular signaling pathway mapping, since labeling depends on surface-exposed residues, the reactivity of selective binding to specific amino acid residues can lead to distorted results. Despite these limitations, these methods have revolutionized the ability to track the expression, localization, and structural changes of proteins and components in intracellular signaling pathways. Considering the essential value of understanding biological systems at the microenvironment level, there is a need for more accurate intracellular mapping methods.

[0005] In particular, the aforementioned approach is particularly vulnerable with respect to the identification of transiently and / or weakly interacting partners. Importantly, the identification of transiently and / or weakly interacting partners particularly involves problems in the field of targeted protein degradation (TPD). For example, all known molecular adhesives for TPD are brought about by an increase in the basic affinity that transiently and / or weakly interacts between the ligase and the protein of interest. Therefore, the pairing between the E3 ligase and the protein targeted for degradation is not well understood.

[0006] Accordingly, there is a need for methods for agnostically labeling intracellular proteins, peptides, chromatin, and nucleic acids with high levels of spatial resolution.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Summary of the Invention

[0008] The present disclosure provides systems, compositions, and methods related to the labeling of intracellular molecules proximal to a protein of interest.

[0009] In one aspect, the present disclosure is a method of labeling based on the proximity of intracellular molecules, comprising: introducing a binder complex into the cell intracellularly, the binder complex comprising a protein of interest coupled to a binder, the binder being capable of binding to a catalyst complex. A step of introducing a catalyst complex into a cell, thereby forming a biomolecular antenna containing the catalyst complex and a binder complex, A step of introducing a labeling agent containing a labeling portion and a reactive portion into the cell, wherein the reactive portion is configured to be activated to a reactive state by the catalyst complex, and A step of activating the catalyst complex, thereby activating the labeling agent by transfer of energy from the catalyst complex to the reactive portion, and binding the labeling agent to a biomolecule within the cell relates to a method comprising.

[0010] In some embodiments, the step of introducing the binder complex into the cell includes the step of introducing a nucleotide construct into the cell, the nucleotide construct encodes the binder complex, and the nucleotide construct can be expressed by the cell. In a further embodiment, the step of introducing the nucleotide construct into the cell includes the step of transfecting the cell.

[0011] In some embodiments, the biomolecule is an intracellular protein, peptide, chromatin, or nucleic acid.

[0012] In some embodiments, the method further includes the step of imaging a signal from the labeling portion.

[0013] In some embodiments, the catalyst complex includes a photocatalyst. In a further embodiment, the photocatalyst includes a transition metal complex. In still further embodiments, the step of activating the transition metal complex includes the step of photoactivating the transition metal complex. In still further embodiments, the step of photoactivating includes the step of illuminating the transition metal complex with light, and the light has a wavelength of about 380 nm to about 700 nm. In some embodiments, activating the catalyst complex causes Dexter energy transfer from the activated transition metal complex to the reactive portion to form a reactive intermediate.

[0014] In some embodiments, when the labeling agent binds to the biomolecule, the biomolecule is within a 10 nm radius of the biomolecular antenna.

[0015] In another aspect, the present disclosure is a system for labeling based on the proximity of intracellular molecules, comprising: a cell, a biomolecular antenna, comprising a photocatalyst complex comprising a photocatalyst and a ligand moiety, and a binder complex comprising a protein of interest coupled to a binder, wherein the binder is capable of binding to the ligand moiety of the photocatalyst complex, and a labeled agent comprising a labeling moiety and a reactive moiety, wherein the reactive moiety is configured to be activated to a reactive state by the photocatalyst complex. The biomolecular antenna and the labeled agent are each located intracellularly. In some embodiments, the binder comprises haloalkane dehalogenase. In further embodiments, the ligand moiety comprises an alkyl chloride. In still further embodiments, the alkyl chloride comprises six or more carbons. In some embodiments, the binder is coupled to the protein of interest at the N-terminus or C-terminus of the protein of interest.

[0016] In some embodiments, the ligand moiety comprises 4,4'-di-tert-butyl-2,2'-dipyridyl, 2,2'-bipyridine, diphenhydramine-2,2'-bipyridine, 4-4'-dimethoxy-2-2'-bipyridine, dinaphthalene-pyrene, phenanthroline, or diphenyl-phenanthroline.

[0017]

[0018]

[0019] ​​In some embodiments, the photocatalyst contains a transition metal. In further embodiments, the transition metal has a triplet energy state greater than 60 kcal / mol. In further embodiments, the transition metal is a platinum group metal. In still further embodiments, the transition metal is iridium or ruthenium. In further embodiments, the transition metal is in a 6-coordinate form. In further embodiments, the transition metal absorbs light having a wavelength of about 380 nm to about 700 nm. In further embodiments, the transition metal has a visible light absorption coefficient greater than 1000 M -1 cm -1 −1

[0020] In some embodiments, the photocatalyst is an organic catalyst that does not contain a transition metal. In further embodiments, the organic catalyst contains a thioxanthone group, a phenothiazine group, a flavin group, a phenoxazine group, a phthalazine group, a quinoxaline group, a quinazoline group, a benzophenothiazine group, a coumarin group, an acetophenone group, or a benzophenone group.

[0021] In some embodiments, the labeling agent is cell-permeable.

[0022] In some embodiments, the photocatalyst can activate the labeling agent to form a reactive intermediate. In further embodiments, the photocatalyst can activate the labeling agent to form a reactive intermediate via Dexter energy transfer. In further embodiments, the reactive intermediate has a diffusion radius of less than 10 nm before quenching. In further embodiments, the reactive intermediate has a half-life (t 1 / 2 ) of less than 2 ns.

[0023] In some embodiments, the binder is a protein, an E3 ligase, a polysaccharide, or a nucleic acid.

[0024] In some embodiments, the protein of interest is K-Ras, cMyc, Src, WRN, Slug, PARP1, Aβ, Tau, influenza hemagglutinin, or a viral nucleoprotein.

[0025] In another aspect, the present disclosure provides a biomolecular assembly comprising a dehalogenase and a photocatalyst coupled to a protein of interest.

[0026] In some embodiments, the photocatalyst is configured to activate a labeling agent comprising a labeling moiety and a reactive moiety. In further embodiments, the photocatalyst is configured to activate the labeling agent after absorbing light having a wavelength of from about 380 nm to about 700 nm.

[0027] In some embodiments, the photocatalyst comprises a transition metal. In further embodiments, the transition metal is a platinum group metal. In still further embodiments, the transition metal is iridium, tin, or ruthenium. In still further embodiments, the transition metal is of a six-coordinate type. In still further embodiments, the transition metal has a visible light absorption coefficient greater than 1000 M -1 cm -1 -1.

[0028] In some embodiments, the photocatalyst is an organic catalyst that does not contain a transition metal. In further embodiments, the organic catalyst comprises a thioxanthone group, a phenothiazine group, a flavin group, a phenoxazine group, a phthalazine group, a quinoxaline group, a quinazoline group, a benzophenothiazine group, a coumarin group, an acetophenone group, or a benzophenone group.

[0029] In some embodiments, the biomolecular assembly further comprises a photocatalyst complex, the photocatalyst complex comprising a photocatalyst and a ligand moiety. In further embodiments, the ligand moiety comprises an alkyl chloride. In still further embodiments, the alkyl chloride comprises six or more carbons.

[0030] In another aspect, the present disclosure is a method for detecting protein-protein interactions, comprising: intracellularly expressing a first protein in a cell, wherein the first protein is coupled to a binder capable of binding to a catalyst complex, and the catalyst complex is a photocatalyst; introducing the photocatalyst into the cell, thereby binding it to the first protein; A step of introducing a labeling agent containing a labeling moiety and a reactive moiety into a cell, activating a photocatalyst, thereby activating the labeling agent by energy transfer from the photocatalyst to the reactive moiety, binding the labeling agent to a second protein in the cell, and detecting the second protein by detecting the labeling moiety comprising wherein the photocatalyst is of formula (I):

[0031]

Chemical formula

[0032] (wherein, A1 is present 0 to 4 on the ring to which it is attached, and each A1 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1, each R1 is independently selected from H, a linear or branched alkyl group having 1 to 12 carbons, CHF2, and CF3, A2 is present 0 to 4 on the ring to which it is attached, and each A2 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1, A3 is present 0 to 4 on the ring to which it is attached, and each A3 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1, A4 is absent, a linear or branched alkyl group having 1 to 12 carbons, C 6~10 aryl, C 3~8 cycloalkyl, 4- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl, wherein the alkyl, aryl, cycloalkyl, heterocyclyl, and heteroaryl are optionally substituted with one or more C 1~6 alkyl, C 1~6 haloalkyl, halo, hydroxy, C 1~6 alkoxy, or amino, A5 is CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1~6Alkoxy, and

[0033]

Chemical formula

[0034] selected from A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a1(CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, each a1 is independently an integer from 0 to 10, and each a2 is independently an integer from 6 to 10. A9 is iridium. A10 is an anion selected from tetraalkylborate, tetrafluoroborate, tetraphenylborate, chloride, cyanide, hexafluorophosphate (PF6), bis(triphenylphosphine)iminium chloride, tetraphenylphosphonium chloride, and tetrabutylammonium. Each A11 and A12 is independently C or N coordinated to A9. Each A13 is independently CH or N) having the structure of. A method is provided.

[0035] In some embodiments, A3 may be present in 0 to 4 on the ring to which it is attached, and each A3 may be independently selected from CH3, CF3, F, Cl, and OR1. In some embodiments, A5 may be selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, and NHCOO. In some embodiments, A6 may be (PEG)a1(CH2)a2Cl, a1 may be an integer from 0 to 10, and a2 may be an integer from 6 to 10. In some embodiments, A13 may be CH. In some embodiments, the 5- to 6-membered heterocyclyl of A6 may be piperazine or pyrrolidine. In some embodiments, the photocatalyst has the structure:

[0036]

Chemical formula

[0037] has. In some embodiments, the photocatalyst has the structure:

[0038]

Chem.

[0039] has. In some embodiments, the photocatalyst has the structure:

[0040]

Chem.

[0041] has. In some embodiments, the photocatalyst has the structure:

[0042]

Chem.

[0043] has. In some embodiments, the photocatalyst has the structure:

[0044]

Chem.

[0045] has. In some embodiments, the photocatalyst has the structure:

[0046]

Chem.

[0047] has. In some embodiments, the photocatalyst has the structure:

[0048] [Chemical formula]

[0049] having. In some embodiments, the photocatalyst has the structure:

[0050] [Chemical formula]

[0051] having. In some embodiments, the photocatalyst has the structure:

[0052] [Chemical formula]

[0053] having. In some embodiments, the photocatalyst has the structure:

[0054] [Chemical formula]

[0055] having.

[0056] In another aspect, the present disclosure is a method for detecting protein-protein interactions, comprising: intracellularly expressing a first protein in a cell, wherein the first protein is coupled to a binder capable of binding to a catalytic complex; introducing a catalytic complex into the cell and thereby binding it to the first protein; introducing a labeling agent comprising a labeling moiety and a reactive moiety into the cell; activating the catalytic complex, thereby activating the labeling agent by transfer of energy from the catalytic complex to the reactive moiety and binding the labeling agent to a second protein in the cell; and A step of detecting a second protein by detecting a labeling part comprising wherein the labeling agent has the formula (III-a):

[0057]

Chemical formula

[0058] (wherein R 1' is selected from azide, methyldiazirine, trifluoromethyldiazirine, and phenyldiazirine, the Ar ring is selected from phenyl, pyridyl, pyrimidyl, pyrazinyl, pyridizinyl, naphthyl, and quinolinyl optionally substituted with one or more -OH, -OMe, -OEt, -OCF3, -OCF2H, -NHMe, -NMe2, -F, -Cl, -Br, -Me, or -Et, X is selected from O, NH, NR 2 , CH2NHCO, CONH, CONR 2 , SO2NH, and SO2NR 2 , R 2 is selected from H, OMe, Me, and Et, n is 0, 1, 2, 3, 4, 5, or 6, and Y is an amide bonded to biotin or a fluorescent dye bonded to an amide) A method having the structure is provided.

[0059] In some embodiments, the labeling agent has the structure:

[0060]

Chemical formula

[0061] having it.

[0062] In some embodiments of the methods disclosed herein, the first protein may be a ubiquitin ligase.

[0063] In another aspect, the present disclosure provides a method for detecting protein - protein interactions, comprising: intracellularly expressing a first protein, wherein the first protein is coupled to a binder capable of binding to a catalytic complex, and the first protein is a ubiquitin ligase; introducing the catalytic complex into the cell, thereby binding it to the first protein; introducing a labeling agent comprising a labeling moiety and a reactive moiety into the cell; activating the catalytic complex, thereby activating the labeling agent by transfer of energy from the catalytic complex to the reactive moiety and binding the labeling agent to a second protein in the cell; and detecting the second protein by detecting the labeling moiety. The present disclosure provides a method comprising the above steps.

[0064] In some embodiments of the methods disclosed herein, the catalytic complex is a photocatalyst. In some embodiments, the photocatalyst has the formula (I):

[0065]

Chemical formula

[0066] (wherein, A1 is present 0 - 4 times on the ring to which it is attached, and each A1 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1; each R1 is independently selected from H, a linear or branched alkyl group having 1 - 12 carbons, CHF2, and CF3; A2 is present 0 - 4 times on the ring to which it is attached, and each A2 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1; A3 is present 0 - 4 times on the ring to which it is attached, and each A3 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1; A4 is non-existent, a linear or branched alkyl group having 1 to 12 carbons, C 6~10 aryl, C 3~8 cycloalkyl, 4- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl, wherein said alkyl, aryl, cycloalkyl, heterocyclyl, and heteroaryl are each optionally substituted with one or more C 1~6 alkyl, C 1~6 haloalkyl, halo, hydroxy, C 1~6 alkoxy, or amino, A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1~6 alkoxy, and spiro C 1~6 alkoxy, A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a1(CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, where each a1 is independently an integer from 0 to 10 and each a2 is independently an integer from 6 to 10, A9 is a D9 metal selected from copper, vanadium, chromium, scandium, titanium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, and mercury, A10 is an anion selected from tetraalkylborate, tetrafluoroborate, tetraphenylborate, chloride, cyanide, hexafluorophosphate (PF6), bis(triphenylphosphine)iminium chloride, tetraphenylphosphonium chloride, and tetrabutylammonium, A11 and A12 are independently C or N coordinated to A9, each A13 is independently CH or N) having the structure of.

[0067] In some embodiments, there may be 0 to 4 A3 groups on the ring to which it is attached, and each A3 may be independently selected from CH3, CF3, F, Cl, and OR1. In some embodiments, A5 may be selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, and NHCOO. In some embodiments, A6 may be (PEG)a1(CH2)a2Cl, where a1 may be an integer from 0 to 10 and a2 may be an integer from 6 to 10. In some embodiments, A13 may be CH. In some embodiments, the 5- to 6-membered heterocyclyl of A6 may be piperazine or pyrrolidine. In some embodiments, the photocatalyst has the structure:

[0068] [Chemical Formula]

[0069] and has the structure: In some embodiments, the photocatalyst has the structure:

[0070] [Chemical Formula]

[0071] and has the structure: In some embodiments, the photocatalyst has the structure:

[0072] [Chemical Formula]

[0073] and has the structure: In some embodiments, the photocatalyst has the structure:

[0074] [Chemical Formula]

[0075] and has the structure: In some embodiments, the photocatalyst has the structure:

[0076]

Chemical formula

[0077] and has. In some embodiments, the photocatalyst has the structure:

[0078]

Chemical formula

[0079] and has. In some embodiments, the photocatalyst has the structure:

[0080]

Chemical formula

[0081] and has. In some embodiments, the photocatalyst has the structure:

[0082]

Chemical formula

[0083] and has. In some embodiments, the photocatalyst has the structure:

[0084]

Chemical formula

[0085] and has. In some embodiments, the photocatalyst has the structure:

[0086]

Chemical formula

[0087] has

[0088] In some embodiments of the methods disclosed herein, the labeling agent has the formula (III-a):

[0089]

Chemical formula

[0090] (wherein R 1' is selected from azido, methyldiazirine, trifluoromethyldiazirine, and phenyldiazirine, the Ar ring is selected from phenyl, pyridyl, pyrimidyl, pyrazinyl, pyridizinyl, naphthyl, and quinolinyl optionally substituted with one or more -OH, -OMe, -OEt, -OCF3, -OCF2H, -NHMe, -NMe2, -F, -Cl, -Br, -Me, or -Et, X is selected from O, NH, NR 2 , CH2NHCO, CONH, CONR 2 , SO2NH, and SO2NR 2 , R 2 is selected from H, OMe, Me, and Et, n is 0, 1, 2, 3, 4, 5, or 6, and Y is an amide bonded to biotin or a fluorescent dye bonded to an amide) has the structure of

[0091] In some embodiments, the labeling agent has the structure:

[0092]

Chemical formula

[0093] has

[0094] In some embodiments of the methods disclosed herein, the method is performed in the absence of an exogenous compound that promotes the interaction between the first protein and the second protein.

[0095] In some embodiments of the methods disclosed herein, the method is performed in the presence of a test compound, and the detection of the second protein or the level of the second protein indicates that the test compound promotes the interaction between the first protein and the second protein.

[0096] In some embodiments of the methods disclosed herein, during the activation of the catalytic complex, the cell is a living cell. In some embodiments of the methods disclosed herein, the binding agent comprises haloalkane dehalogenase. In some embodiments of the methods disclosed herein, activating the catalytic complex includes the step of illuminating the cell with light, and the light has a wavelength of about 380 nm to about 700 nm. In some embodiments of the methods disclosed herein, activating the catalytic complex causes Dexter energy transfer from the activated catalytic complex to the reactive moiety to form a reactive intermediate.

[0097] In another aspect, the present disclosure provides a first protein coupled to haloalkane dehalogenase, Formula (I):

[0098]

Chemical formula

[0099] (wherein, A1 is present in 0 to 4 on the ring to which it is attached, and each A1 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1, each R1 is independently selected from H, a linear or branched alkyl group having 1 to 12 carbons, CHF2, and CF3, There are 0 to 4 A2 groups on the ring to which it is attached, and each A2 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1. There are 0 to 4 A3 groups on the ring to which it is attached, and each A3 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1. A4 is absent, a linear or branched alkyl group having 1 to 12 carbons, C 6~10 aryl, C 3~8 cycloalkyl, 4- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl, and the alkyl, aryl, cycloalkyl, heterocyclyl, and heteroaryl are optionally substituted with one or more C 1~6 alkyl, C 1~6 haloalkyl, halo, hydroxy, C 1~6 alkoxy, or amino. A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1~6 alkoxy, C 1~6 alkoxy, and

[0100]

Chemical formula

[0101] selected from A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a1(CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, each a1 is independently an integer from 0 to 10, and each a2 is independently an integer from 6 to 10. A9 is a D9 metal selected from copper, vanadium, chromium, scandium, titanium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, and mercury. A11 and A12 are, independently, C or N coordinated to A9, each A13 is, independently, CH or N) a photocatalyst having the structure of, and Formula (III-a):

[0102]

Chemical formula

[0103] (wherein, R 1' is selected from azide, methyldiazirine, trifluoromethyldiazirine, and phenyldiazirine, the Ar ring is selected from phenyl, pyridyl, pyrimidyl, pyrazinyl, pyridizinyl, naphthyl, and quinolinyl optionally substituted with one or more -OH, -OMe, -OEt, -OCF3, -OCF2H, -NHMe, -NMe2, -F, -Cl, -Br, -Me, or -Et, X is O, NH, NR 2 , CH2NHCO, CONH, CONR 2 , SO2NH, and SO2NR 2 selected from, R 2 is selected from H, OMe, Me, and Et, n is 0, 1, 2, 3, 4, 5, or 6, Y is an amide bonded to biotin or a fluorescent dye bonded to an amide) a labeling agent having the structure of to provide a cell containing.

[0104] In some embodiments, there may be 0 to 4 A3 groups on the ring to which it is attached, and each A3 may be independently selected from CH3, CF3, F, Cl, and OR1. In some embodiments, A5 may be selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, and NHCOO. In some embodiments, A6 may be (PEG)a1(CH2)a2Cl, where a1 may be an integer from 0 to 10 and a2 may be an integer from 6 to 10. In some embodiments, A13 may be CH. In some embodiments, the 5- to 6-membered heterocyclyl of A6 may be piperazine or pyrrolidine. In some embodiments, the photocatalyst has the structure:

[0105]

Chemical formula

[0106] and has the structure: In some embodiments, the photocatalyst has the structure:

[0107]

Chemical formula

[0108] and has the structure: In some embodiments, the photocatalyst has the structure:

[0109]

Chemical formula

[0110] and has the structure: In some embodiments, the photocatalyst has the structure:

[0111]

Chemical formula

[0112] and has the structure: In some embodiments, the photocatalyst has the structure:

[0113] [Chemical formula]

[0114] and has. In some embodiments, the photocatalyst has the structure:

[0115] [Chemical formula]

[0116] and has. In some embodiments, the photocatalyst has the structure:

[0117] [Chemical formula]

[0118] and has. In some embodiments, the photocatalyst has the structure:

[0119] [Chemical formula]

[0120] and has. In some embodiments, the photocatalyst has the structure:

[0121] [Chemical formula]

[0122] and has. In some embodiments, the photocatalyst has the structure:

[0123] [Chemical formula]

[0124] has. In some embodiments, the labeling agent has the structure:

[0125] [Chemical formula]

[0126] has.

[0127] In another aspect, the present disclosure is Structure: P1 - P2 - Cat (wherein P1 is a ubiquitin ligase, P2 is a haloalkane dehalogenase, Cat is of formula (I):

[0128] [Chemical formula]

[0129] (wherein A1 is present 0 to 4 on the ring to which it is attached, and each A1 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1, each R1 is independently selected from H, a linear or branched alkyl group having 1 to 12 carbons, CHF2, and CF3, A2 is present 0 to 4 on the ring to which it is attached, and each A2 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1, A3 is present 0 to 4 on the ring to which it is attached, and each A3 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1, A4 is absent, a linear or branched alkyl group having 1 to 12 carbons, C 6~10 aryl, C 3~8 cycloalkyl, 4 - to 10 - membered heterocyclyl, and 5 - to 10 - membered heteroaryl, and the alkyl, aryl, cycloalkyl, heterocyclyl, and heteroaryl are one or more C1~6 alkyl, C 1~6 haloalkyl, halo, hydroxy, C 1~6 optionally substituted with alkoxy, or amino, A5 is CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1~6 alkoxy, C 1~6 alkoxy, and

[0130]

Chemical formula

[0131] selected from A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a1(CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, each a1 is independently an integer from 0 to 10, and each a2 is independently an integer from 6 to 10, A9 is a D9 metal selected from copper, vanadium, chromium, scandium, titanium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, and mercury, A11 and A12 are independently C or N coordinated to A9, each A13 is independently CH or N) is a photocatalyst having the structure of Provided is a protein complex comprising

[0132] In some embodiments, there may be 0 to 4 A3 groups on the ring to which it is attached, and each A3 may be independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1. In some embodiments, A5 may be selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, and NHCOO. In some embodiments, A6 may be (PEG)a1(CH2)a2Cl, where a1 may be an integer from 0 to 10 and a2 may be an integer from 6 to 10. In some embodiments, A13 may be CH. In some embodiments, the 5- to 6-membered heterocyclyl of A6 may be piperazine or pyrrolidine. In some embodiments, Cat has the structure:

[0133]

Chemical formula

[0134] and has the structure: In some embodiments, Cat has the structure:

[0135]

Chemical formula

[0136] and has the structure: In some embodiments, Cat has the structure:

[0137]

Chemical formula

[0138] and has the structure: In some embodiments, Cat has the structure:

[0139]

Chemical formula

[0140] and has the structure: In some embodiments, Cat has the structure:

[0141]

Chemical formula

[0142] and has. In some embodiments, Cat has the structure:

[0143]

Chemical formula

[0144] and has. In some embodiments, Cat has the structure:

[0145]

Chemical formula

[0146] and has. In some embodiments, Cat has the structure:

[0147]

Chemical formula

[0148] and has. In some embodiments, Cat has the structure:

[0149]

Chemical formula

[0150] and has. In some embodiments, Cat has the structure:

[0151]

Chemical formula

[0152] has

[0153] In another aspect, the present disclosure provides a cell comprising a protein complex according to the present disclosure, and Formula (III-a):

[0154]

Chemical formula

[0155] (wherein R 1' is selected from azide, methyldiazirine, trifluoromethyldiazirine, and phenyldiazirine, the Ar ring is selected from phenyl, pyridyl, pyrimidyl, pyrazinyl, pyridizinyl, naphthyl, and quinolinyl optionally substituted with one or more -OH, -OMe, -OEt, -OCF3, -OCF2H, -NHMe, -NMe2, -F, -Cl, -Br, -Me, or -Et, X is selected from O, NH, NR 2 , CH2NHCO, CONH, CONR 2 , SO2NH, and SO2NR 2 , R 2 is selected from H, OMe, Me, and Et, n is 0, 1, 2, 3, 4, 5, or 6, and Y is an amide linked to biotin or a fluorescent dye linked to an amide) and a labeling agent having the structure of to provide a cell.

[0156] In another aspect, the present disclosure provides a cell comprising a nucleotide sequence that expresses a fusion protein comprising a ubiquitin ligase and a haloalkane dehalogenase.

[0157] In another aspect, the present disclosure provides Formula (I):

[0158]

Chemical formula

[0159] (wherein A1 is present in 0 to 4 on the ring to which it is attached, and each A1 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1, each R1 is independently selected from H, a linear or branched alkyl group having 1 to 12 carbons, CHF2, and CF3, A2 is present in 0 to 4 on the ring to which it is attached, and each A2 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1, A3 is present in 0 to 4 on the ring to which it is attached, and each A3 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1, A4 is absent, a linear or branched alkyl group having 1 to 12 carbons, C 6~10 aryl, C 3~8 cycloalkyl, a 4- to 10-membered heterocyclyl, and a 5- to 10-membered heteroaryl, and the alkyl, aryl, cycloalkyl, heterocyclyl, and heteroaryl are optionally substituted with one or more C 1~6 alkyl, C 1~6 haloalkyl, halo, hydroxy, C 1~6 alkoxy, or amino, A5 is CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1~6 alkoxy, and

[0160]

Chemical formula

[0161] selected from A6 is, independently, (PEG)a1(CH2)a2Cl or (PEG)a1(CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, each a1 is, independently, an integer from 0 to 10, and each a2 is, independently, an integer from 6 to 10, A9 is a D9 metal selected from copper, vanadium, chromium, scandium, titanium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, and mercury, A11 and A12 are, independently, C or N coordinated to A9, each A13 is, independently, CH or N) a photocatalyst having the structure of

[0162]

Chemical formula

[0163] which is not, to provide a photocatalyst.

[0164] In some embodiments, there are 0 to 4 A3 on the ring to which it is attached, and each A3 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1. In some embodiments, A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, and NHCOO. In some embodiments, A6 is (PEG)a1(CH2)a2Cl, a1 is an integer from 0 to 10, and a2 is an integer from 6 to 10. In some embodiments, A13 is CH. In some embodiments, the 5- to 6-membered heterocyclyl of A6 is piperazine or pyrrolidine. In some embodiments, the photocatalyst has the structure:

[0165]

Chemical formula

[0166] has. In some embodiments, the photocatalyst has the structure:

[0167]

Chem.

[0168] has. In some embodiments, the photocatalyst has the structure:

[0169]

Chem.

[0170] has. In some embodiments, the photocatalyst has the structure:

[0171]

Chem.

[0172] has. In some embodiments, the photocatalyst has the structure:

[0173]

Chem.

[0174] has. In some embodiments, the photocatalyst has the structure:

[0175]

Chem.

[0176] has. In some embodiments, the photocatalyst has the structure:

[0177]

Chem.

[0178] has. In some embodiments, the photocatalyst has the structure:

[0179]

Chem.

[0180] has. In some embodiments, the photocatalyst has the structure:

[0181]

Chem.

[0182] has. In some embodiments, the photocatalyst has the structure:

[0183]

Chem.

[0184] has.

[0185] Throughout the drawings, reference numbers are reused to indicate correspondence between referenced elements. The following drawings and associated descriptions are provided to assist in understanding the implementation of the present disclosure and are not intended to limit the claims.

Brief Description of the Drawings

[0186]

Figure 1

Figure 2

Figure 3

Figure 4A

Figure 4B

Figure 4C

Figure 5A

Figure 5B

Figure 5C

Figure 5D

Figure 5E

Figure 5F

Figure 6

Figure 7A

Figure 7B

Figure 8A

Figure 8B

Figure 9

Figure 10

Figure 11A

Figure 11B

Figure 12A

Figure 12B

Figure 13A

Figure 13B

Figure 14A

Figure 14B

Figure 15A

Figure 15B

Figure 16A

Figure 16B

Figure 17A

Figure 17B

Figure 18A

Figure 18B

Figure 18C

Figure 18D

Figure 18E

Figure 18F

Figure 18G

Figure 18H

Figure 19A

Figure 19B

Figure 20A

Figure 20B

Figure 21A

Figure 21B

DETAILED DESCRIPTION OF THE INVENTION

[0187] The present disclosure will now be described with reference to the accompanying drawings, where like numerals refer to like elements throughout. The following description is merely for the purpose of facilitating understanding and is in no way intended to limit the disclosure, its application, or its use. It should be understood that the steps within a method may be performed in a different order without changing the principles of the present disclosure. Further, the embodiments disclosed herein may include several new features, none of which alone contributes to the desirable characteristics or is essential for implementing the systems, devices, and methods disclosed herein.

[0188] The systems, compositions, and methods disclosed herein relate to techniques for labeling intracellular molecules proximal to a target protein. Such labeling may enable protein labeling based on proximity in vitro and in vivo. The labeling may be achieved by a photo-responsive catalyst coupled to the target protein for activating a cell-permeable labeling agent via Dexter energy transfer to activate the reactive moiety of the labeling agent. Specifically, when activated, the photo-responsive catalyst activates the reactive moiety of the cell-permeable labeling agent. The reactive moiety may then bind to various biomolecules near the target protein. The label of the labeling agent may then be detected, for example, by imaging to provide information regarding the intracellular location of the target protein.

[0189] More specifically, methods and systems by which a self-labeling system, such as HaloTag, can be utilized to incorporate a photosensitive catalyst, such as iridium, into a biological system for microenvironment mapping are disclosed herein. According to the present disclosure, when used with a suitable cell-permeable labeling agent, the photosensitive catalyst can use the incident light energy, transfer that energy, and activate the cell-permeable labeling agent to label components of the biomolecular microenvironment.

[0190] Systems and Compounds for Labeling Based on Intracellular Proximity In one aspect, a system for labeling based on the proximity of intracellular molecules includes a cell; a photocatalyst complex further including a photocatalyst and a ligand moiety, and a binder complex including a protein of interest coupled to a binder, wherein the binder can bind to the ligand moiety of the photocatalyst complex, the binder complex including a biomolecular antenna; and a labeling agent including a label moiety and a reactive moiety, wherein the reactive moiety is configured to be activated to a reactive state by the photocatalyst complex, the biomolecular antenna and the labeling agent being located intracellularly, respectively.

[0191] FIG. 1 is a drawing showing the formation of a biomolecular antenna 106 of an example of a labeling system based on proximity within a cell. The biomolecular antenna 106 includes a catalyst complex 102 and a binder complex 104. Next, the catalyst complex 102 includes a photocatalyst 112 and a ligand moiety 114. The binder complex 104 includes a protein of interest 108 and a binder 110. The binder 110 may be capable of binding to the ligand moiety 114 of the catalyst complex 102, whereby the catalyst complex 102 and the binder complex 104 can form a biomolecular antenna 106.

[0192] The catalyst complex 102 can be any suitable catalyst complex 102 for activating the labeling agent 216 (described below) according to this embodiment. In some embodiments, the catalyst complex 102 may include a transition metal complex containing a transition metal. In these embodiments, the transition metal complex may include one ligand or a plurality of ligands coordinated to the metal center. In certain embodiments, the transition metal may be a platinum group metal. In certain embodiments, the transition metal may be iridium or ruthenium. In some embodiments, the transition metal may have a triplet energy state greater than 1 kcal / mol, greater than 5 kcal / mol, greater than 10 kcal / mol, greater than 20 kcal / mol, greater than 30 kcal / mol, greater than 40 kcal / mol, greater than 50 kcal / mol, greater than 60 kcal / mol, greater than 75 kcal / mol, greater than 100 kcal / mol, greater than 150 kcal / mol, greater than 200 kcal / mol, greater than 250 kcal / mol, greater than 500 kcal / mol, greater than 1000 kcal / mol, or any value or range enclosed within or by any of these ranges or values, although values outside of these ranges or values may be used.

[0193] In some embodiments, the transition metal (also referred to herein as the transition metal catalyst) may be capable of absorbing one or more wavelengths of visible light (i.e., light having wavelengths within the range of approximately 380 nm to approximately 700 nm, although values outside of this range may be used). Absorption of visible light can excite the transition metal complex to the S1 state, followed by quantitative intersystem crossing to the long-lived triplet excited state (T1). The T1 state has a half-life t within the range of 50 ns to 5 μs, within the range of 0.1 μs to 2.5 μs, within the range of 0.2 μs to 2 μs, or any value or range enclosed within or by any of these ranges or values. 1 / 2It may have these values, but values outside of these values or ranges may also be used. In some embodiments, the transition metal catalyst can return to the ground state S0 via short-distance Dexter energy transfer to the labeling agent. The energy transfer to the labeling agent can activate the labeling agent for reaction with a protein or other biomolecule. As a specific example, when the labeling agent is diazirine, the triplet energy transfer to the labeling agent can form a carbene intermediate for reacting with a protein or other biomolecular species.

[0194] The photocatalytic transition metal complex can have any composition and structure that is consistent with the principles and energy transfers described above. In some embodiments, the photocatalytic transition metal complex is of the 6-coordinate type. The ligand moiety 114 may include one or more ligands that can bind to the binder 110. The ligands of the ligand moiety 114 of the catalyst complex 102, such as a transition metal complex, can include one or more pyridine moieties in some embodiments. In some embodiments, one or more ligands include bipyridine or a derivative thereof. Table I provides examples of suitable ligands.

[0195]

Table 1

[0196] In some embodiments, the photocatalytic transition metal complex is [Ir(dF(CF3)ppy)2(dtbbpy)](PF6), ([Ir(dF(CF3)ppy)2(bpy)](PF6)), or a derivative thereof. In some embodiments, the iridium photocatalyst described in Trowbridge et al., Small molecule photocatalysis enables drug target identification via energy transfer, bioRxiv (2021) https: / / doi.org / 10.1101 / 2021.08.02.454797, which is incorporated herein by reference, may be used.

[0197] In some embodiments, the photocatalytic transition metal complex has the formula I:

[0198]

Chemical formula

[0199] (wherein, A1 may be 0 to 4 substituents on the ring to which it is attached, and each A1 may independently be CH3, CF3, F, Cl, N(CH3)2, or OR1, R1 may independently be H, a linear or branched alkyl group having 1 to 12 carbons, CHF2, or CF3, A2 may be 0 to 4 substituents on the ring to which it is attached, selected from CH3, CF3, F, Cl, N(CH3)2, or OR1, A3 may be 0 to 4 substituents on the ring to which it is attached, selected from CH3, CF3, F, Cl, N(CH3)2, or OR1, A4 may be absent, a linear or branched alkyl group having 1 to 12 carbons, C 6~10 aryl, C 3~8 cycloalkyl, a 4- to 10-membered heterocyclyl, and a 5- to 10-membered heteroaryl, wherein the alkyl, aryl, cycloalkyl, heterocyclyl, and heteroaryl are optionally substituted with one or more C 1~6 alkyl, C 1~6 haloalkyl, halo, hydroxy, C 1~6 alkoxy, or amino, A5 may be CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1~6 alkoxy, and

[0200]

Chemical formula

[0201] may be, A6 may independently be (PEG)a1(CH2)a2Cl or (PEG)a1(CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl. For example, a1 may be defined as the number of polyethylene glycol (PEG) subunits by an integer a1 from 0 to 10, and a2 may be defined as the number of methylene subunits by an integer a2 from 6 to 10. In some examples, the 5- to 6-membered heterocyclyl of A6 may be piperazine or pyrrolidine. A9 may be a D9 metal such as copper, vanadium, chromium, scandium, titanium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, and mercury. A10 may be an anion for the metal of A9. A non-exclusive list of such anions includes tetraalkylborate, tetrafluoroborate, tetraphenylborate, chloride, cyanide, hexafluorophosphate, bis(triphenylphosphine)iminium chloride, tetraphenylphosphonium chloride, or tetrabutylammonium. A11 and A12 may independently be a C or N coordinated to A9. Each A13 may independently be CH or N) and may have the structure of.

[0202] In some embodiments, A1 may be F. In some embodiments, A1 may be Cl. In some embodiments, A1 may be CH3. In some embodiments, A1 may be CF3. In some embodiments, A1 may be N(CH3)2. In some embodiments, A1 may be OR1. In some embodiments, A1 may be present in one on the ring to which it is attached. In some embodiments, A1 may be present in two on the ring to which it is attached. In some embodiments, A1 may be present in two on the ring to which it is attached and may be F. In some embodiments, A1 may be present in one on the ring to which it is attached and may be F. In some embodiments, A1 may be present in two on the ring to which it is attached and may be Cl. In some embodiments, A1 may be present in one on the ring to which it is attached and may be Cl. In some embodiments, A1 may be present in two on the ring to which it is attached and may be CH3. In some embodiments, A1 may be present in one on the ring to which it is attached and may be CH3. In some embodiments, A1 may be present in two on the ring to which it is attached and may be CF3. In some embodiments, A1 may be present in one on the ring to which it is attached and may be CF3. In some embodiments, A1 may be present in two on the ring to which it is attached and may be N(CH3)2. In some embodiments, A1 may be present in one on the ring to which it is attached and may be N(CH3)2. In some embodiments, A1 may be present in two on the ring to which it is attached and may be OR1. In some embodiments, A1 may be present in one on the ring to which it is attached and may be OR1. In some embodiments, A2 may be F. In some embodiments, A2 may be Cl. In some embodiments, A2 may be CH3. In some embodiments, A2 may be CF3. In some embodiments, A2 may be N(CH3)2. In some embodiments, A2 may be OR1.In some embodiments, there may be one A2 on the ring to which it is attached. In some embodiments, there may be two A2s on the ring to which it is attached. In some embodiments, there may be two A2s on the ring to which it is attached and it may be F. In some embodiments, there may be one A2 on the ring to which it is attached and it may be F. In some embodiments, there may be two A2s on the ring to which it is attached and it may be Cl. In some embodiments, there may be one A2 on the ring to which it is attached and it may be Cl. In some embodiments, there may be two A2s on the ring to which it is attached and it may be CH3. In some embodiments, there may be one A2 on the ring to which it is attached and it may be CH3. In some embodiments, there may be two A2s on the ring to which it is attached and it may be CF3. In some embodiments, there may be one A2 on the ring to which it is attached and it may be CF3. In some embodiments, there may be two A2s on the ring to which it is attached and it may be N(CH3)2. In some embodiments, there may be one A2 on the ring to which it is attached and it may be N(CH3)2. In some embodiments, there may be two A2s on the ring to which it is attached and it may be OR1. In some embodiments, there may be one A2 on the ring to which it is attached and it may be OR1. In some embodiments, A11 may be the C coordinated to A9. In some embodiments, A12 may be the N coordinated to A9. In some embodiments, one or more of the ligands can include a hydrophilic moiety to enhance the compatibility of the transition metal complex with water or an aqueous environment, such as that found within a cell. Further, one or more of the ligands may include a moiety, functional group, and / or handle for coupling to a binder 110, such as a protein, polysaccharide, or nucleic acid.For example, a moiety containing an appropriately long alkyl chloride, such as an alkyl chloride having 6 or more carbons, can be bound to the target protein 108 by a binder 110 containing a genetically encoded halodearhalogenase (such as HaloTag) so as to be generated. In certain embodiments, the photocatalytic transition metal complex has the formula II-XI:.

[0203]

Chemical formula

[0204]

Chemical formula

[0205]

Chemical formula

[0206] has the structure of.

[0207] In some embodiments, the transition metal has a visible light absorption coefficient greater than about 10 M -1 cm -1 or greater than about 100 M -1 cm -1 or greater than about 250 M -1 cm -1 or greater than about 500 M -1 cm -1 or greater than about 1000 M -1 cm -1 or greater than about 2000 M -1 cm -1 or greater than about 5000 M -1 cm -1 or greater than about 10,000 M -1 cm -1 or may have a visible light absorption coefficient of any value or range within or surrounded by any of these ranges or values, although values outside of these values or ranges may also be used.

[0208] In some embodiments, the photocatalyst 112 of the catalyst complex 102 is an organic catalyst. In some embodiments, such an organic catalyst may not contain a photo-responsive transition metal. As a specific example of such embodiments, the organic catalyst may include a thioxanthone group, a phenothiazine group, a flavin group, a phenoxazine group, a phthalazine group, a quinoxaline group, a quinazoline group, a benzophenothiazine group, a coumarin group, an acetophenone group, or a benzophenone group. However, any suitable organic catalyst capable of activating the labeling agent 216 in accordance with the present disclosure may be selected.

[0209] The target protein 108 may be a suitable intracellular molecule. For example, the target protein 108 may be involved in signal transduction pathways, intracellular and / or cell architecture, localized biomolecular networks, and / or microenvironments. In some embodiments, the target protein 108 may be encoded by a nucleotide construct introduced into and thereby expressed in the cell. In other embodiments, the target protein 108 may be permeable to the cell. In still other embodiments, the target protein 108 may not be permeable to the cell, but may be introduced into the cell by some suitable method, such as precipitation with liposomes. In various embodiments, the target protein may be involved in a disease state. As specific examples, proteins involved in cancer include K-Ras, cMyc, Src, WRN, Slug, and PARP1. As specific examples, proteins involved in neurodegeneration include Aβ and Tau. As specific examples, proteins involved in viral infection may include influenza hemagglutinin and viral nuclear protein. In some embodiments, the target protein 108 is a ubiquitin ligase (e.g., an E3 ligase).

[0210] The binder 110 can be any suitable molecule having specific binding to the ligand moiety 114. In some embodiments, the binder 110 may include a haloalkane dehalogenase. In certain embodiments, the binder 110 may be HaloTag®. The HaloTag® (Promega) purification system was developed as a recombinant haloalkane dehalogenase protein that can be naturally produced in cells. The HaloTag system can be used in a number of self-labeling processes for chemical biology. As a specific example, the HaloTag system can be used for protein purification and for the incorporation of TAMRA-alkyl chloride dyes for monitoring intracellular signaling. Since the haloalkane dehalogenase is non-mammalian, the incorporation of the background of the photoreactive catalyst by the dehalogenase mechanism is theoretically low, substantially absent, or absent. Thus, the HaloTag system can function as a useful vector for incorporating a photoreactive catalyst into the natural cellular environment for the identification of the microenvironmental interactome of the protein of interest. Thus, the HaloTag system may be useful for monitoring transient interactions in the intracellular environment.

[0211] In some embodiments, a nucleotide construct encoding a fusion protein of the protein of interest 108 and the binder 110 (i.e., the binder complex 104) may be introduced into the cell. The cell may express the binder complex 104 encoded by the construct. In some embodiments, the construct may encode a binder complex 104 that is a fusion protein in which the binder 110 is bound to the C-terminus of the protein of interest 108. In some embodiments, the construct may encode a binder complex 104 that is a fusion protein in which the binder 110 is bound to the N-terminus of the protein of interest 108. In some embodiments, the construct may encode a binder complex 104 in which the binder 110 is bound to the protein of interest 108 at a site that does not prevent, substantially prevents, or minimally prevents a certain interaction of the binder 110 with the biomolecule 208 of the protein of interest 108.

[0212] Figure 2 is a diagram of a proximity-based labeling scheme that includes the biomolecular antenna 106 described in FIG. 1. Further included is a labeling agent 216 that includes a reactive moiety 204 and a label moiety 206. The light source 214 can generate photons 210 that activate the catalyst complex 102. Next, the catalyst complex 102 can activate the labeling agent 216 via Dexter energy transfer 212 that activates the reactive moiety 204. When in an activated state, the labeling agent 216 may be able to bind the label moiety 206 to a biomolecule 208 in the vicinity.

[0213] According to the present disclosure, the labeling agent 216 can be activated via Dexter energy transfer 212. Diazirine and azide-based probes have been widely applied in small molecule target ID, but require direct excitation by UV light, thereby precluding the possibility of target-localized activation. These types of reactive moieties are known to have the ability to receive triplet energy via Dexter energy transfer. By binding a photocatalyst (e.g., the photocatalyst 112 of the catalyst complex 102) to a biomolecule (e.g., the protein 108 of interest) produced by a cell, it becomes possible to act as an antenna that absorbs light energy, such as visible light. The photocatalyst 112 is activated by absorbing light. The activated photocatalyst 112 can then activate the labeling agent 216 in the immediate vicinity of the protein 108 of interest by Dexter energy transfer 212. Thereby, the activated labeling agent 216 can covalently bind itself to a molecular structure (including, but not limited to, proteins, chromatin, and nucleic acids) in the immediate vicinity of the binder complex 104 by the activated reactive moiety 204. The distance at which the activated labeling agent 216 can bind to the label moiety 206 depends on the diffusibility of the labeling agent 216 and the half-life t 1 / 2 of the activation of the reactive moiety 204. For example, the half-life t 1 / 2The longer the time, the longer the time that the labeling agent 216 can move before binding to the biomolecule 208. Similarly, the greater the diffusibility of the labeling agent 216, the longer the distance that the labeling agent 216 can move in the activated state before quenching or binding to the biomolecule 208.

[0214] In embodiments that include a reactive moiety 204 to which the labeling agent 216 is bound to the labeling moiety 206, the binding of the reactive moiety 204 to the biomolecule 208 also results in a labeling moiety 206 that has been transferred to the biomolecule 208 (including, but not limited to, proteins, chromatin, and nucleic acids). Placing a label on the biomolecule 208 enables the use of orthogonal techniques (including, but not limited to, proteomic analysis, flow cytometry, radiographic imaging, and electron microscopy) to identify the characteristics, identity, and spatiotemporal aspects of the microenvironment. In some embodiments, the reactive moiety 204 may be cell permeable regardless of the presence or absence of the bound labeling moiety 206. In some embodiments, the labeling agent 216 is cell permeable.

[0215] In some embodiments, the labeling agent 216 has the formula III:

[0216]

Chemical formula

[0217] (wherein, R 1 may be a substituent capable of receiving triplet energy from an activated photocatalyst. For example, R 1 may be azide, methyldiazirine, trifluoromethyldiazirine, or phenyldiazirine, R 1 may be independently disposed on the aromatic ring at the positions of a, b, or c, The aromatic ring may be phenyl, pyridyl, pyrimidyl, pyrazinyl, or pyridizinyl. The aromatic ring may be naphthyl or quinoline and may include various further bonds. The aromatic ring can be substituted with an electron-withdrawing group or an electron-donating group to weaken the reactivity of the labeled species. As specific examples, the substituents at the a, b, or c positions may be -OH, -OMe, -OEt, -OCF3, -OCF2H, -NHMe, -NMe2, -F, -Cl, -Br, -Me, -Et, X is O, NH, NR 2 , CH2NHCO, CONH, CONR 2 , SO2NH, SO2NR 2 and may be, Furthermore, R 2 may be H, OMe, Me, Et, n is an integer indicating the number of PEG units. For example, n may be 0, 1, 2, 3, 4, 5, or 6, but other values of n may also be used, Y may have a structure as shown (which may be an amide bonded to biotin or an amide resulting from the linkage with a fluorescent dye).

[0218] In some embodiments, the labeling agent 216 has the formula (III-a):

[0219]

Chemical formula

[0220] (wherein, R 1' is selected from azide, methyldiazirine, trifluoromethyldiazirine, and phenyldiazirine, The Ar ring is selected from phenyl, pyridyl, pyrimidyl, pyrazinyl, pyridizinyl, naphthyl, and quinolinyl optionally substituted with one or more -OH, -OMe, -OEt, -OCF3, -OCF2H, -NHMe, -NMe2, -F, -Cl, -Br, -Me, or -Et, X is selected from O, NH, NR 2 , CH2NHCO, CONH, CONR 2 , SO2NH, and SO2NR 2 and is selected from, R2 is selected from H, OMe, Me, and Et, n is 0, 1, 2, 3, 4, 5, or 6, and Y is an amide linked to biotin or a fluorescent dye linked to an amide) has the structure of.

[0221] The reactive moiety 204 can include any chemical species operable to interact with the photocatalyst 112 to form a reactive intermediate (also referred to herein as an activated labeling agent) for coupling to a biomolecule. As a specific example, in embodiments where the photocatalyst 112 includes a photocatalytic transition metal, the reactive moiety 204 may be an aryl azide or an alkyl azide. In such embodiments, triplet energy transfer from the excited state photocatalyst 112 can promote the azide to its triplet (T1) state. The azide triplet releases free triplet nitrene via the elimination of N2 and, via a spin equilibrium on the picosecond time scale, becomes a reactive singlet state (t 1 / 2 <1ns half-life), resulting in either cross-linking with a nearby biomolecule 208 or quenching by the aqueous environment.

[0222] In certain embodiments, the labeling agent 216 has the structure shown in any one of Formulas III - IX:

[0223]

Chemical formula

[0224] and may include the structure shown in one of them.

[0225] The sensitization of azides can be extended to various substituents on the attached ring having a beneficial payload (e.g., a label) for microscopy and proteomics applications. Such payloads or labels include free carboxylic acids, phenols, amines, alkynes, carbohydrates, and biotin groups.

[0226] The extinction coefficient of the photocatalyst 112 may be orders of magnitude greater than that of the azide at wavelengths emitted by, for example, blue light (e.g., light having a wavelength of about 450 nm) used for sensitization, which ensures that there is no, substantially no, or minimal non-catalytic reaction in the background of the reactive moiety 204. In other embodiments where the reactive moiety 204 can be activated to a singlet or triplet energy state in the absence of the photocatalyst, it may be desirable for the labeling rate in the presence of the protein photocatalyst to be faster than the background reaction.

[0227] In some embodiments, the reactive intermediate (also referred to herein as the activated labeling agent) is formed by the interaction of the reactive moiety 204 and the photocatalyst 112. The interaction may be an energy transfer, such as Dexter energy transfer. The reactive intermediate may have a t of less than 50 ns, less than 10 ns, less than 5 ns, less than 2 ns, or less than 1 ns, or any value or range enclosed within or by any of these ranges or values 1 / 2 but values outside of these values or ranges may also be used. In some embodiments, the reactive intermediate has a half-life and diffusion constant such that it diffuses less than 100 nm before quenching, less than 75 nm before quenching, less than 50 nm before quenching, less than 40 nm before quenching, less than 30 nm before quenching, less than 25 nm before quenching, less than 20 nm before quenching, less than 15 nm before quenching, less than 12.5 nm before quenching, less than 10 nm before quenching, less than 7.5 nm before quenching, less than 6 nm before quenching, less than 5 nm before quenching, less than 4 nm before quenching, less than 3 nm before quenching, less than 2 nm before quenching, or less than 1 nm before quenching, or any value or range enclosed within or by any of these ranges or values, but values outside of these values or ranges may also be used. The mechanistic principles described above may be applied to the proximity labeling of various types of biomolecules including, but not limited to, proteins, polysaccharides, and / or nucleic acids.

[0228] In some embodiments, the reactive moiety 204 can be functionalized with a labeling moiety 206. As discussed, labeling of the biomolecule 208 can facilitate the use of orthogonal techniques. For example, in some embodiments, the labeling moiety 206 may include a tag that can act as a ligand for a tagged protein. In some of these embodiments, the labeling moiety 206 may include biotin, which can bind to a tagged protein, such as avidin. As a specific example, such a tagged protein may be used in an immunoprecipitation assay to isolate the biomolecule 208 to which the labeling moiety 206 is attached. As another specific example, the tagged protein may include a fluorophore, and as a result, the v protein may be imaged. In some embodiments, the labeling moiety 206 itself may include a fluorophore, and as a result, it may be imaged without adding a tagged protein.

[0229] Method of Labeling Based on Proximity In one aspect, the present disclosure provides a method of labeling based on the proximity of intracellular molecules, the method comprising introducing a binder complex, the binder complex comprising a protein of interest coupled to a binder, the binder being capable of binding to a catalyst complex; introducing the catalyst complex into a cell; forming a biomolecular antenna comprising the catalyst complex and the binder complex; introducing a labeling agent into the cell, the labeling agent comprising a labeling moiety and a reactive moiety, the reactive moiety being coupled to the labeling moiety; activating the catalyst complex, thereby activating the labeling agent by transfer of energy from the catalyst complex to the reactive moiety; and binding the labeling agent to a biomolecule within the cell.

[0230] Figure 3 is a schematic diagram of a flowchart of a method according to the present disclosure. By block 302, a nucleotide construct may optionally be introduced into a cell. In some embodiments, the nucleotide construct may be introduced by transformation and / or transfection. The nucleotide may encode a binding agent complex 104 that includes a protein of interest 108 and a binding agent 110. The construct may be configured such that the portion encoding the binding agent complex 104 is expressible by the cell (e.g., the encoding portion may be present in an open reading frame).

[0231] Next, by block 304, the binding agent complex 104 may be introduced into the cell. In embodiments where the nucleotide construct has been introduced into the cell by block 302, the binding agent complex 104 may be introduced by cellular expression of the nucleotide construct. Such production of the binding agent complex 104 may occur intracellularly using the cell's own transcription and / or translation processes to produce the binding agent complex 104 based on the sequences found in the nucleotide construct. In other embodiments, the binding agent complex 104 may be introduced into the cell by another suitable method. By way of example, the binding agent complex 104 may be introduced into the cell by liposomes.

[0232] By block 316, the catalytic complex 102 is introduced into the cell according to this embodiment. As disclosed herein, such a catalytic complex 102 may be cell permeable. Alternatively, the catalytic complex 102 may be introduced into the cell by another suitable method. By way of non-limiting example, the catalytic complex 102 may be introduced by a liposome carrier.

[0233] By block 318, a labeling agent 216 may be introduced as in block 318. As disclosed herein, such a labeling agent 216 may be cell permeable, but may be introduced into the cell by any suitable method.

[0234] Block 308 may cause the catalyst complex 102 to form a binder complex 104 and a biomolecular antenna 106. For example, the ligand portion 114 of the catalyst complex 102 may bind to the binder 110 of the binder complex 104. In some embodiments, the alkyl chloride of the ligand portion 114 may bind to the HaloTag of the binder 110.

[0235] After the formation of the catalyst complex, block 310 may activate the catalyst complex. In embodiments where the catalyst complex 102 includes a photocatalyst 112, activation may include irradiation with light of a wavelength known to activate a particular photocatalyst 112. In some embodiments, activation can be performed in living cells.

[0236] Next, block 310 may activate the labeling agent 204 by activating the catalyst complex 102. In some embodiments, the photocatalyst 112 may activate the labeling agent 204 via Dexter energy transfer 212.

[0237] Once activated, block 312 may enable the labeling agent 216 to bind the labeling moiety 206 to a biomolecule 208 in proximity. A given activated labeling agent 216 may be quenched before it can bind to a proximate biomolecule 208.

[0238] After the labeling of the biomolecule, the label may optionally be detected by block 314. In some embodiments where the label is detectable, for example, by fluorescence microscopy, the label may be imaged. Such imaging may show where the biomolecule 208 is localized even if the interaction between the biomolecule 208 and the protein of interest 108 is relatively short and / or the biomolecule 208 and the protein of interest 108 are not co-localized during imaging. In some embodiments, the label may not be fluorescent but may be a target for a second label that can emit fluorescence. In some embodiments, the label may be a target for a subsequent immunoprecipitation assay. Such an immunoprecipitation assay may be a tagged protein immunoprecipitation or a tag-based pull-down assay. Once isolated, the labeled target protein may be subjected to other orthogonal techniques. For example, the isolated labeled target protein may be analyzed quantitatively or semi-quantitatively by Western blot, or the isolated material may be quantified and identified by proteomics analysis.

[0239] In some embodiments, the methods described above can be used to detect protein-protein interactions. For example, in some embodiments, the binder complex includes a first protein coupled to the binder, and the first protein is the protein of interest desired to examine the protein-protein interaction. In some embodiments, the first protein is a ubiquitin ligase and the binder is a haloalkane dehalogenase, and as a result, the binder complex is a fusion protein including the ubiquitin ligase and the haloalkane dehalogenase. Then, the catalytic complex can be introduced into the cell to bind to the haloalkane dehalogenase.

[0240] In some embodiments, upon activation of the catalyst complex and subsequent activation of the labeling agent, the labeling agent binds to a second protein that is in close proximity to the first protein. Detection of the second protein by detection of the labeled portion of the labeling agent implies a protein-protein interaction between the first protein and the second protein because the second protein is labeled only if it is in close proximity to the first protein.

[0241] In some embodiments, the method for detecting a protein-protein interaction may be used in the absence of any exogenous test compound to determine the identity of a second protein that interacts with a first protein. In some embodiments, this method may be used to identify weak protein-protein interactions, which may then function as therapeutic targets by screening for compounds that increase the protein-protein interaction. For example, in some embodiments, the first protein is a ubiquitin ligase, and identification of a weak protein-protein interaction with the ubiquitin ligase means that the interaction can be exploited by finding a compound (e.g., a PROTAC or a molecular glue) that promotes the interaction.

[0242] In some embodiments, the method includes introducing a test compound into a cell to determine whether the test compound promotes a protein-protein interaction. In some such embodiments, the level of the second protein labeled by the labeling agent is measured to determine the amount by which the protein-protein interaction is promoted by the test compound. In other embodiments, the test compound is similarly evaluated to determine whether it inhibits a protein-protein interaction.

[0243] Method for verifying protein association Just as the methods disclosed herein can be used to identify biomolecules proximal to a protein of interest tagged with a binder, the methods can be used to identify the reverse: whether a protein of interest is proximal to a biomolecule tagged with a binder. Such an approach may be useful for verifying whether a protein of interest is proximal to a biomolecule. Thus, for example, the methods can be used to verify whether a protein that causes a target disease associates with a ubiquitin ligase by forming a complex between a binder and the protein that causes the disease.

[0244] In one aspect, the present disclosure provides a method for verifying an association between a protein that causes a disease and a ubiquitin ligase. For example, other screening methods may suggest the presence of an association, and it may be desirable to verify the interaction before screening for substances that promote the interaction, such as PROTACs or molecular adhesives. The method includes introducing a first binder complex into a cell, wherein the first binder complex includes a target protein that causes a disease coupled to a first binder, and the first binder is capable of binding to a first catalytic complex; introducing the first catalytic complex into the cell; forming a first biomolecular antenna including the first catalytic complex and the binder complex; introducing a first labeling agent including a first labeling moiety and a first reactive moiety into the cell, wherein the first reactive moiety is coupled to the first labeling moiety; activating the first catalytic complex, thereby activating the first labeling agent by transfer of energy from the first catalytic complex to the first reactive moiety, and binding the first labeling agent to a ubiquitin ligase in the cell. In some embodiments, the method further includes confirming by immunoprecipitation that the ubiquitin ligase has been labeled by the labeling agent. In some embodiments, the labeled protein of the cell lysate is first recovered using functional beads that bind to the labeling agent (e.g., streptavidin-functional beads if the labeling agent includes a biotin moiety). The presence of the desired ubiquitin ligase can then be determined using Western blot with an appropriate antibody against the ligase.

[0245] Cells and proteins Some embodiments include a fusion protein comprising a protein of interest and a haloalkane dehalogenase. In some embodiments, the protein of interest is a ubiquitin ligase. Some embodiments also include a protein complex comprising a fusion protein coupled to a photocatalyst described herein (e.g., a photocatalyst according to formula (I)). Thus, some embodiments have the structure: P1-P2-Cat (wherein, P1 is a ubiquitin ligase, P2 is a haloalkane dehalogenase, Cat is of formula (I):

[0246]

Chemical formula

[0247] (wherein, There are 0 to 4 A1s on the ring to which it is attached, and each A1 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1, Each R1 is independently selected from H, a linear or branched alkyl group having 1 to 12 carbons, CHF2, and CF3, There are 0 to 4 A2s on the ring to which it is attached, and each A2 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1, There are 0 to 4 A3s on the ring to which it is attached, and each A3 is independently selected from CH3, CF3, F, Cl, N(CH3)2, and OR1, A4 is absent, a linear or branched alkyl group having 1 to 12 carbons, C 6~10 aryl, C 3~8 cycloalkyl, 4- to 10-membered heterocyclyl, and 5- to 10-membered heteroaryl, wherein the alkyl, aryl, cycloalkyl, heterocyclyl, and heteroaryl are one or more C 1~6Alkyl, C 1~6 Haloalkyl, halo, hydroxy, C 1~6 Optionally substituted with alkoxy or amino, A5 is CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1~6 Alkoxy, and

[0248]

Chemical formula

[0249] Selected from A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a1(CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, each a1 is independently an integer from 0 to 10, and each a2 is independently an integer from 6 to 10, A9 is a D9 metal selected from copper, vanadium, chromium, scandium, titanium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, and mercury, A11 and A12 are independently C or N coordinated to A9, Each A13 is independently CH or N) Is a photocatalyst having the structure of) Including a protein complex containing

[0250] In some embodiments, the protein complex is present intracellularly. In some embodiments, the protein complex is formed by intracellularly expressing a fusion protein and then exposing the fusion protein to the photocatalyst. Accordingly, some embodiments also provide a cell comprising a nucleotide sequence that expresses a fusion protein comprising ubiquitin ligase and haloalkane dehalogenase.

[0251] Some embodiments include cells comprising a first protein coupled to a haloalkane dehalogenase and a photocatalyst as described above (e.g., a photocatalyst according to formula (I)). In some embodiments, the cells also include a labeling agent as described above (e.g., a labeling agent according to formula (III) or (III-a)).

[0252] The term As used herein, "biomolecular antenna" refers to a molecular structure formed by the binding of a catalyst complex to a binder complex.

[0253] As used herein, "probe" or "reactive moiety" refers to a molecule that (1) can be coupled to a label, (2) can be activated by a photocatalyst via Dexter energy transfer, and (3) can bind to various molecules when in an activated state.

[0254] As used herein, "binder" refers to a molecule that (1) can bind to a protein of interest or can be expressed as part of a fusion protein with the protein of interest, and (2) can bind to the ligand portion of a catalyst complex.

[0255] "Room temperature" is abbreviated as "RT" herein.

[0256] As used herein, "PEG" refers to the structure:

[0257]

Chemical formula

[0258] refers to a moiety having.

[0259] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). The alkyl group may have 1 to 20 carbon atoms (whenever it appears in this specification, the numerical range, such as "1 to 20", refers to each integer within the given range. For example, "1 to 20 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, but this definition also includes the occurrence of the term "alkyl" where no numerical range is specified). The alkyl group may also be a medium-sized alkyl having 1 to 9 carbon atoms. The alkyl group can also be a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be represented as "C 1~4 alkyl" or a similar name. Merely by way of example, "C 1~4 alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are by no means limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, etc.

[0260] As used herein, "heterocyclyl" or "heterocyclic" means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring skeleton. Heterocyclyl may be connected in a fused, bridged, or spiro-linked fashion. Heterocyclyl may have any degree of saturation provided that at least one ring in the ring system is not aromatic. The heteroatom may be present in either the non-aromatic or aromatic rings in the ring system. The heterocyclyl group may have from 3 to 20 ring members (i.e., the number of atoms constituting the ring skeleton, including carbon atoms and heteroatoms), but this definition also includes occurrences of the term "heterocyclyl" where no numerical range is specified. The heterocyclyl group may also be a medium-sized heterocyclyl having from 3 to 10 ring members. The heterocyclyl group may also be a heterocyclyl having from 3 to 6 ring members. The heterocyclyl group may be represented by "3-6 membered heterocyclyl" or a similar name. In a preferred 6-membered monocyclic heterocyclyl, the heteroatom is selected from 1 to 3 of O, N, or S, and in a preferred 5-membered monocyclic heterocyclyl, the heteroatom is selected from 1 or 2 heteroatoms selected from O, N, or S.Examples of heterocyclic rings include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl, pyrrolidionyl, 4 - piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3 - dioxinyl, 1,3 - dioxanyl, 1,4 - dioxinyl, 1,4 - dioxanyl, 1,3 - oxathianyl, 1,4 - oxathiinyl, 1,4 - oxathianyl, 2H - 1,2 - oxazinyl, trioxanyl, hexahydro - 1,3,5 - triazinyl, 1,3 - dioxolyl, 1,3 - dioxolanyl, 1,3 - dithiolyl, 1,3 - dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3 - oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro - 1,4 - thiazinyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline.

[0261] As used herein, "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent atoms) containing one or more heteroatoms, i.e., elements other than carbon including, but not limited to, nitrogen, oxygen, and sulfur, in the ring backbone. When heteroaryl is a ring system, all rings of the system are aromatic. A heteroaryl group may have from 5 to 18 ring members (i.e., the number of atoms constituting the ring backbone, including carbon atoms and heteroatoms), but this definition also includes occurrences of the term "heteroaryl" where no numerical range is specified. In some embodiments, a heteroaryl group has from 5 to 10 ring members or from 5 to 7 ring members. A heteroaryl group may be represented by the name "5- to 7-membered heteroaryl", "5- to 10-membered heteroaryl", or a similar name. Examples of heteroaryl rings include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl.

[0262] As used herein, "aryl" refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent carbon atoms) containing only carbon in the ring backbone. When aryl is a ring system, all rings of the system are aromatic. An aryl group may have from 6 to 18 carbon atoms, but this definition also includes occurrences of the term "aryl" where no numerical range is specified. In some embodiments, an aryl group has from 6 to 10 carbon atoms. An aryl group may be represented by the name "C 6~10 aryl", "C6 or C 10 aryl", or a similar name. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.

[0263] As used herein, "cycloalkyl" means a fully saturated carbocyclic ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0264] 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. The use of the terms "including" and its other forms, such as "include", "includes", and "included", is not limiting. The use of the terms "having" and its other forms, such as "have", "has", and "had", is not limiting. The terms "comprising", "including", "having", etc. are synonymous and are used in an open-ended, inclusive manner and do not exclude additional elements, features, acts, operations, etc. That is, the above terms are construed as synonymous with the expression "having at least" or "including at least". For example, when used in the context of a method, the term "comprising" means that the method includes at least the recited steps, but may also include additional steps. When used in the context of an apparatus, the term "comprising" means that the apparatus includes at least the recited features or components, but may also include additional features or components. Also, the term "or" is used in its inclusive sense (and not in its exclusive sense), and as a result, when used, for example, to connect a list of elements, the term "or" means one, some, or all of the elements in the list. Further, the term "each", as used herein, in addition to having its ordinary meaning, can also mean any part of a series of elements to which the term "each" applies.

[0265] Terms used to the extent employed herein, such as the terms "about", "approximately", "generally", and "substantially" in this specification, still represent values, amounts, or characteristics close to the stated values, amounts, or characteristics that perform the desired function or achieve the desired result. For example, the terms "about", "approximately", "generally", and "substantially" may refer to amounts within less than 10%, less than 5%, less than 1%, less than 0.1%, and less than 0.01% of the stated amount.

[0266] As used herein, the term "and / or" has its broadest non-limiting meaning and discloses A alone, B alone, both A and B, or alternatively A or B, but not both A and B are required, or one of A or one of B is required. As used herein, the expression "at least one of" A, B, "and" C should be construed to mean A or B or C based on logical thinking using non-exclusive disjunction.

[0267] Conditional terms used herein, such as, among others, "can", "could", "might", "may", "for example", etc., unless specifically stated otherwise or understood in the context in which they are used, usually convey that a feature, element, and / or step is optional. Thus, such conditional terms are not usually intended to imply that a feature, element, and / or step is required in any way, or that the presence or absence or the performance of one or more implementations of these features, elements, and / or steps is determined by logic that would include whether there are other provisions or incentives. The terms "comprising", "including", "having", etc. are synonymous and are used inclusively in an open-ended manner and do not exclude further elements, features, acts, operations, etc. Also, the term "or" is used in its inclusive sense (not its exclusive sense), and as a result, when used, for example, to connect a list of elements, the term "or" means one, some, or all of the elements in the list.

[0268] None of the methods disclosed in this specification need to be performed in the recited order. The methods disclosed in this specification include certain actions to be taken by an implementer, and can include any third-party instructions, whether explicit or implicit, regarding those actions.

Examples

[0269] Further embodiments are disclosed in more detail in the examples below, which are in no way intended to limit the scope of the claims. The cell labeling of K-Ras interaction partners by the generation of halo.KRAS in the SW48 cell line was obtained from Horizon, as described below.

[0270] (Example 1) Preparation of the Labeling Partner With reference to Scheme 1, the preparation of the labeling partner will be described.

[0271]

Chemical Formula

[0272] Biotin-PEG3-NH2 (123 mg, 0.2944 mmol), HATU (280 mg, 0.7361 mmol), and DIPEA (0.2 mL, 1.1042 mmol) were added to a stirred solution of Compound 1 (60 mg, 0.368 mmol) in DMF (5 mL) at 0 °C. The reaction mixture was stirred at RT for 16 h. The progress of the reaction was monitored by thin-layer chromatography (TLC) and liquid chromatography-mass spectrometry (LC-MS). After completion of the reaction, the reaction mixture was quenched with cold water (10 mL) and extracted with ethyl acetate (2 × 10 mL). The combined organic layers were washed with saturated NaHCO3 (10 mL) and brine (10 mL). The organic layer was dried over sodium sulfate, then filtered and concentrated under reduced pressure to obtain the crude compound. The crude compound was purified by Prep-HPLC to give 4-azido-N-(13-oxo-17-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-12-azapentadecyl)benzamide (45 mg, yield 21%) as an off-white solid.

[0273] (Example 2) Preparation of Photocatalyst 1 With reference to Scheme 2, the preparation of the photocatalyst will be described.

[0274] [Chemical formula]

[0275] As Step 1, LDA (2M in THF, 32.56 mL, 65.13 mmol, 1.2 equiv) was added dropwise to a stirred solution of Compound 2 (10 g, 54.277 mmol, 1.0 equiv) in dry THF (300 mL), and the mixture was stirred at -78 °C for 1.5 h. In a separate round-bottom flask (RBF), methyl 2-bromoacetate (12.5 g, 81.415 mmol, 1.5 equiv) was dissolved in THF (100 mL) and cooled to -78 °C. Then, the methyl 2-bromoacetate solution was slowly added at -78 °C, and the mixture was stirred at RT for 16 h. The progress of the reaction was monitored by LC-MS and TLC (30% ethyl acetate in hexanes). After completion of the reaction, the reaction mixture was quenched with saturated NaHCO3 solution (150 mL) and extracted with ethyl acetate (2 × 300 mL). The organic layer was dried over Na2SO4 and evaporated under reduced pressure to give the crude compound. Subsequently, the crude compound was purified by silica gel (230 - 400 mesh) column chromatography eluting with 0 - 2% methanol in DCM to give Compound 3 (7 g, 50% yield) as a light brown solid.

[0276] As Step 2, a 2M NaOH (1.64 g, 40.966 mmol, 1.5 equiv) solution at 0 - 10 °C was added to a stirred solution of Compound 3 (7 g, 27.31 mmol, 1.0 equiv) in a mixture of methanol (70 mL) and THF (21 mL), and the mixture was stirred at RT for 2 h. The progress of the reaction was monitored by LC-MS and TLC (10% methanol in DCM). After completion of the reaction, the reaction mixture was concentrated under reduced pressure, diluted with water (40 mL), and then adjusted to a pH of 3 - 4 with 10% citric acid solution (30 mL). The solid was filtered and dried under vacuum to give Compound 4 (5 g, 75.7% yield) as a light brown solid.

[0277] In Step 3, trimethylamine (1.7 mL, 12.396 mmol, 1.5 equiv) and N,N'-disuccinimidyl carbonate were added to a stirred solution of compound 4 (2 g, 8.264 mmol, 1 equiv) in dichloromethane (20 mL) at RT, and the mixture was stirred at RT for 16 h. The progress of the reaction was monitored by LC-MS and TLC (in DCM, 5% methanol). After completion of the reaction, the reaction mixture was diluted with water (20 mL) and extracted with dichloromethane (2×20 mL). The organic layer was dried over Na2SO4 and evaporated under reduced pressure to give the crude compound. The crude compound was purified by silica gel column chromatography eluting with 0 - 5% methanol in DCM as the mobile phase to give compound 5 (1.2 g, 43% yield) as a light brown liquid.

[0278] As Step 4, in a clean dry RBF, aqueous IrCl3 (600 mg, 2.009 mmol, 1 equiv) and compound 6 (1.145 g, 4.421 mmol, 2.2 equiv) were added, evacuated, and filled with N2 three times. Then, a degassed solution of 2-ethoxyethanol (24 mL) and water (8.4 mL) was added, and the mixture was heated to 120 °C for 16 h. The progress of the reaction was monitored by LC-MS. After completion of the reaction, the reaction mixture was cooled to RT, the yellow precipitate was filtered off, washed with water (120 mL) and hexane (50 mL), and dried under vacuum to give compound 7 (605 mg, 33% yield) as a yellow solid.

[0279] As Step 5, AgPF6 (143 mg, 0.564 mmol, 2.1 equiv) was added to a stirred solution of compound 8 (400 mg, 0.268 mmol, 1 equiv) in acetonitrile (30 mL) at RT, and the mixture was stirred for 16 h. The progress of the reaction was monitored by LC-MS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give the crude compound. Then, the crude compound was purified by trituration with diethyl ether (20 mL) to give compound 9 (350 mg, 55.7% yield) as a pale yellow solid.

[0280] As step 6, a stirred solution of compound 5 (130 mg, 0.384 mmol, 1.2 equiv) in dichloromethane was degassed with argon for 5 minutes. Then, a solution of compound 9 in dichloromethane (18 mL) was added at RT and stirred for 16 hours. The progress of the reaction was monitored by LC-MS and TLC (10% methanol in DCM). After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain the crude compound. The crude compound was purified by trituration with diethyl ether (10 mL) to obtain compound Int-8 (320 mg, 83% yield) as a yellow solid. mL mL mg mmol mL mL% mg

[0281] As step 7, DIPEA (0.1 mL, 0.251 mmol) and compound Int-8 (100 mg, 0.083 mmol) were added to a stirred solution of 18-chloro-3,6,9,12-tetraoxaoctadecane-1-amine hydrochloride (compound Int-6) in DMF and stirred at RT for 16 hours. The progress of the reaction was monitored by LC-MS and TLC. After completion of the reaction, the reaction mixture was diluted with water (10 mL) and extracted with EtOAc (2 × 10 mL). The organic layer was dried over Na2SO4 and evaporated under reduced pressure to obtain the crude compound. The crude compound was purified by flash column chromatography (FCC) using 5% MeOH in DCM, and the required fractions were concentrated under reduced pressure to obtain photocatalyst 1 (50 mg, 49% yield) as a pale yellow solid. Photocatalyst 1 is an example of a photocatalyst according to the present disclosure.

[0282] With reference to Scheme 3, the preparation of compound Int-6 will be described.

[0283]

Chemical formula

[0284] As Reaction Step 8, sodium hydride (1.308 g, 32.724 mmol, 1.2 eq) was added to a stirred solution of Compound 11 (8 g, 27.27 mmol, 1 eq) in THF (56 mL) at 0 °C and stirred for 30 minutes. Then, Compound 12 (8.06 g, 32.724 mmol, 1.2 eq) was slowly added at 0 - 5 °C and stirred at RT for 16 hours. The progress of the reaction was monitored by LC-MS and TLC (70% ethyl acetate in petroleum ether). After completion of the reaction, the reaction mixture was quenched with saturated ammonium chloride solution (80 mL) and extracted with ethyl acetate (2 × 100 mL). The organic layer was washed with water (100 mL), dried over Na2SO4, evaporated under reduced pressure to obtain the crude compound, and purified by silica gel column chromatography eluting with 0 - 50% ethyl acetate in petroleum ether as the mobile phase to obtain Compound 13 (3.7 g, yield 33%) as a light brown liquid.

[0285] As Step 9, 4 M HCl in dioxane (19 mL) at 0 - 5 °C was added to a stirred solution of Compound 13 (3.7 g, 12.612 mmol) in dichloromethane (37 mL) and stirred at RT for 5 h. The progress of the reaction was monitored by LC-MS and TLC (70% ethyl acetate in petroleum ether). After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain Compound Int-6 (3 g, yield 96%) and used in Step 7 of Scheme 2 without further purification.

[0286] (Example 3) Preparation of Photocatalyst 1D With reference to Scheme 4, the preparation of the photocatalyst will be described.

[0287]

Chem.

[0288] As step 1, a stirred solution of compound 1 (130 mg, 0.384 mmol, 1.2 equiv) in dichloromethane (45 mL) was degassed with argon for 5 minutes. Subsequently, a solution of compound 2 in dichloromethane (18 mL) was added at RT and stirred for 16 hours. The progress of the reaction was monitored by LC-MS and TLC (10% methanol in DCM). After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain a crude compound, which was purified by trituration with diethyl ether (10 mL) to further obtain compound Int-8 (320 mg, yield 83%) as a yellow solid.

[0289] As step 2, DIPEA (0.3 mL, 0.159 mmol, 2 equiv) at RT was added to a solution of compound Int-3 (25 mg, 0.0796 mmol, 1 equiv) in DMF (1.5 mL) and stirred for 10 minutes. Subsequently, compound Int-8 (95 mg, 0.0796 mmol, 1 equiv) was added and stirred at RT for 16 hours. The progress of the reaction was monitored by LC-MS and TLC (10% methanol in DCM). After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain a crude compound, which was purified by silica gel (230 - 400 mesh) column chromatography eluting with 0 - 5% methanol in DCM as the mobile phase to obtain photocatalyst 1D (1.25 g, yield 63%) as a yellow solid. Photocatalyst 1D is an example of a photocatalyst according to the present disclosure.

[0290] With reference to Scheme 5, the preparation of compound Int-3 is described.

[0291]

Chemical formula

[0292] Sodium hydride (302 mg, 8.181 mmol, 1.2 equiv) at 0 °C was added to a stirred solution of compound 4 (2 g, 6.817 mmol, 1 equiv) in a mixture of DMF (10 mL) and THF (10 mL), and the mixture was stirred at RT for 30 min. Then, compound 5 (1.73 g, 8.18 mmol, 1.2 equiv) was slowly added at 0 - 5 °C, and the mixture was stirred at RT for 16 h. The progress of the reaction was monitored by LC-MS and TLC (70% ethyl acetate in petroleum ether). After completion of the reaction, the reaction mixture was quenched with saturated ammonium chloride solution (20 mL) and extracted with ethyl acetate (2 × 20 mL). The organic layer was washed with water (30 mL), dried over Na2SO4, evaporated under reduced pressure to obtain the crude compound, and purified by silica gel column chromatography eluting with 0 - 50% ethyl acetate in petroleum ether as the mobile phase to further obtain compound 6 (700 mg, yield 28%) as a pale yellow sticky liquid.

[0293] (Example 4) Preparation of Photocatalysts 2, 3, and 4 With reference to Scheme 6, the preparation of the photocatalysts is described.

[0294] [Chemical formula]

[0295] According to Scheme 6, DIPEA (0.1 mL, 0.251 mmol) and compound Int-8 (see Example 2) (100 mg, 0.083 mmol) were added to a stirred solution of 2-(2-((6-chlorohexyl)oxy)ethoxy)ethane-1-amine hydrochloride (20 mg, 0.083 mmol) in DMF (10 mL), and the mixture was stirred at RT for 16 h. The progress of the reaction was monitored by TLC and LC-MS. After completion of the reaction, the reaction mixture was diluted with water (10 mL) and extracted with EtOAc (2 × 10 mL). The organic layer was dried over Na2SO4 and evaporated under reduced pressure to obtain the crude compound. The crude compound was purified by FCC using 5% MeOH in DCM, and the required fractions were concentrated under reduced pressure to obtain photocatalyst 2 (50 mg, yield 51%) as a pale yellow solid. Photocatalyst 2 is an example of a photocatalyst according to the present disclosure.

[0296] Referring to Scheme 7, the preparation of the photocatalyst is described.

[0297]

Chem.

[0298] According to Scheme 7, DIPEA (0.1 mL, 0.251 mmol) and compound Int-8 (see Example 2) (100 mg, 0.083 mmol) were added to a stirred solution of 24-chloro-3,6,9,12,15,18-hexaoxatetracosan-1-amine hydrochloride (20 mg, 1.30 mmol) in DMF (10 mL), and the mixture was stirred at RT for 16 h. The progress of the reaction was monitored by TLC and LC-MS. After completion of the reaction, the reaction mixture was diluted with water (10 mL) and extracted with EtOAc (2 × 10 mL). The organic layer was dried over Na2SO4 and evaporated under reduced pressure to give the crude compound. The crude compound was purified by FCC using 4 - 5% MeOH in DCM, and the required fractions were concentrated under reduced pressure to give photocatalyst 3 (38 mg, 40% yield) as an off-white solid. Photocatalyst 3 is an example of the photocatalyst according to the present disclosure.

[0299] Referring to Scheme 8, the preparation of the photocatalyst is described.

[0300]

Chem.

[0301] According to Scheme 8, DIPEA (0.1 mL, 0.251 mmol) and compound Int-8 (see Example 2) (100 mg, 0.083 mmol) were added to a stirred solution of 30-chloro-3,6,9,12,15,18,21,24-octaoxatriacontan-1-amine hydrochloride (100 mg, 0.087 mmol) in DMF (10 mL), and the mixture was stirred at RT for 16 h. The progress of the reaction was monitored by TLC and LC-MS. After completion of the reaction, the reaction mixture was diluted with water (10 mL) and extracted with EtOAc (2 × 10 mL). The organic layer was dried over Na2SO4 and evaporated under reduced pressure to give the crude compound. The crude compound was purified by FCC using 5% MeOH in DCM, and the required fractions were concentrated under reduced pressure to give photocatalyst 4 (29 mg, yield 40%) as an off-white solid. Photocatalyst 4 is an example of a photocatalyst according to the present disclosure.

[0302] (Example 5) Preparation of Photocatalyst 5 With reference to Scheme 9, the preparation of the photocatalyst will be described.

[0303] [Chemical formula]

[0304] [Chemical formula]

[0305] Referring to Step 1 of Scheme 9, CBr4 (6.349 g, 19.146 mmol, 1.2 equiv) was added to a stirred solution of (2-bromopyridin-4-yl)methanol 1 (3.0 g, 15.95 mmol, 1.0 equiv) and triphenylphosphine (5.022 g, 19.14 mmol, 1.2 equiv) in THF (30 mL), and the mixture was stirred at RT for 16 h. After completion of the reaction, the solution was diluted with ice-cooled H2O, extracted with ethyl acetate (3 × 100 mL), the organic layer was washed with brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude compound. The crude compound was purified by column chromatography using PE and ethyl acetate as eluents. The required fractions were concentrated under reduced pressure to obtain 2-bromo-4-(bromomethyl)pyridine 2 (3.2 g, yield 79.93%) as a white solid.

[0306] As Step 2, to a stirred solution of 2-bromo-4-(bromomethyl)pyridine 2 (2 g, 7.971 mmol, 1.0 equiv) was added methyl 1-hydroxycyclopropane-1-carboxylate 3 (

[0307]

Chemical formula

[0308] 1.111 g, 9.565 mmol, 1.2 equiv) and cesium carbonate (7.791 g, 23.912 mmol, 3.0 equiv) at RT, and the mixture was stirred for 16 h. After completion of the starting materials, the reaction mixture was filtered and dried under reduced pressure to obtain the crude compound, which was purified by column chromatography using petroleum ether and ethyl acetate as eluents. The required fractions were concentrated under reduced pressure to obtain methyl 1-((2-bromopyridin-4-yl)methoxy)cyclopropane-1-carboxylate 3 (2.1 g, yield 92%) as a white solid.

[0309] As step 3, 4-methyl-2-(tributylstannyl)pyridine (0.160 g, 0.41 mmol, 1.2 equiv) in toluene (1.0 mL) was added to a stirred solution of methyl 1-((2-bromopyridin-4-yl)methoxy)cyclopropane-1-carboxylate (0.1 g, 0.34 mmol, 1.0 equiv). The mixture was degassed with N2 for 15 minutes. Pd(PPh3)4 (0.081 g, 0.07 mmol, 0.2 equiv) was added and the mixture was heated to 110 °C for 16 hours. The progress of the reaction was monitored by LC-MS. After the starting material was completely consumed, the reaction mixture was diluted with petroleum ether (150 mL), filtered, and dried under reduced pressure to obtain 6-bromo-2,3-dihydro-[1,4]dioxino[2,3-f]quinolin-10-ol 5 (3 g, 81% yield) as an off-white solid.

[0310] As step 4, an aqueous LiOH solution was added to a stirred solution of methyl 1-((4'-methyl-[2,2'-bipyridin]-4-yl)methoxy)cyclopropane-1-carboxylate (0.1 g, 0.349 mmol, 1.0 equiv) in MeOH and THF at RT. The mixture was stirred for 3 hours. The progress of the reaction was monitored by LC-MS. After the starting material was completely consumed, the reaction mixture was concentrated to obtain the crude compound, which was then dissolved in water, acidified with KHSO4 solution (pH ~2), and extracted with EtOAc (3 × 30 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure to obtain 1-((4'-methyl-[2,2'-bipyridin]-4-yl)methoxy)cyclopropane-1-carboxylic acid 6 (3 g, 81% yield) as an off-white solid.

[0311] As step 5, DCC (1.6 g, 7.19 mmol, 1.1 eq) and 2,3,4,5,6-pentafluorophenol (1.4 g, 7.09 mmol, 1.1 eq) were added to a stirred solution of 1-((4'-methyl-[2,2'-bipyridin]-4-yl)methoxy)cyclopropane-1-carboxylic acid 7 (2 g, 7.04 mmol, 1 eq) in 1,4-dioxane (20 mL) at RT and stirred for 5 h. The progress of the reaction was monitored by LC-MS and TLC (40% ethyl acetate in petroleum ether). After completion of the reaction, the reaction mixture was diluted with water (50 mL) and extracted with diethyl ether (3×50 mL). The organic layer was dried over Na2SO4 and evaporated under reduced pressure to give the crude compound, which was purified by silica gel column chromatography eluting with 10 - 15% ethyl acetate in petroleum ether as the mobile phase. The required fractions were concentrated under reduced pressure to give compound 7 (1.3 g, 70% yield) as an off-white solid.

[0312] As step 6, compound 8 (0.3 g, 0.44 mmol, 1 eq) was added to a solution of compound 7 (0.2 g, 0.44 mmol, 1 eq) in DCM (5 mL) at RT and stirred for 16 h. The progress of the reaction was monitored by LC-MS and TLC (40% ethyl acetate in petroleum ether). After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give the crude compound. The crude product was stirred in pentane (10 mL) and filtered to give compound 9 (1.25 g, 63% yield) as a yellow solid.

[0313] As Step 7, compound 10 (60 g, 0.194 mmol, 1.5 equiv) and DIPEA (0.06 mL, 0.388 mmol, 3 equiv) were added to a solution of compound 9 (0.15 g, 0.129 mmol, 1 equiv) in DMF (2 mL) at RT and stirred for 16 h. The progress of the reaction was monitored by LC-MS and TLC (in petroleum ether, 40% ethyl acetate). After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain the crude compound, which was purified by silica gel column chromatography (230 - 400 mesh) by eluting with 0 - 5% methanol in DCM as the mobile phase. The required fractions were concentrated under reduced pressure to obtain photocatalyst 5 (0.05 g, yield 30%) as a yellow solid. Photocatalyst 5 is an example of a photocatalyst according to the present disclosure.

[0314] (Example 6) Preparation of Photocatalyst 6 With reference to Scheme 10, the preparation of the photocatalyst will be described.

[0315] [Chemical formula]

[0316] According to Scheme 10, compound 9 (see Example 5) (0.124 g, 0.345 mmol, 2 equivalents) and DIPEA (0.04 mL, 0.521 mmol, 3 equivalents) were added to a solution of 2-(3-(4-(2-((6-chlorohexyl)oxy)ethyl)piperazin-1-yl)propoxy)ethan-1-amine (0.2 g, 0.179 mmol, 1 equivalent) in DMF (2 mL) at RT. The resulting reaction mixture was stirred at RT for 16 h. The progress of the reaction was monitored by LC-MS and TLC (10% MeOH in DCM). After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain the crude compound, which was purified by silica gel (NH silica gel mesh) column chromatography by eluting with 0 - 5% methanol in DCM as the mobile phase. The compound fractions were collected and concentrated under reduced pressure to obtain photocatalyst 6 (0.083 g, yield 47%). A 28 mg (90% LC-MS) lot of photocatalyst 6 was purified by Prep HPLC to obtain pure photocatalyst 6 (19.3 mg, 97% LC-MS). Photocatalyst 6 is an example of a photocatalyst according to the present disclosure.

[0317] (Example 7) Preparation of Photocatalyst 7 With reference to Scheme 11, the preparation of the photocatalyst will be described.

[0318] [Chemical formula]

[0319] [Chemical formula]

[0320] As step 1 of Scheme 11, a solution of compound 1 (6 g, 0.0351 mmol) in THF was added dropwise to a stirred solution of t-butyllithium (70 mL, 0.0701 mmol) in THF at -78 °C, and the reaction mixture was stirred for 30 minutes. A ZnCl2 solution (11.9 g, 0.088 mmol) in THF (40 mL) was added, and the reaction mixture was stirred at RT for 2 hours. A pre-prepared solution of compound 2 (8 g, 0.0315 mmol) and tetrakistriphenylphosphine in THF was added dropwise at RT. The resulting reaction mixture was refluxed for 36 hours. The reaction was monitored by TLC and LC-MS. The reaction mixture was cooled to RT, quenched with a saturated solution of EDTA, and adjusted to pH = 8 with NaHCO3. The aqueous layer was extracted with EtOAc. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by column chromatography to obtain compound 3 as an off-white solid (4.2 g, 50% yield).

[0321] As step 2, benzoyl peroxide (0.0021 mmol), and then NBS (0.021 mmol) were added to a stirred solution of compound 3 (1 g, 0.0042 mmol) in CCl4, and the resulting reaction mixture was refluxed for 16 hours. The reaction was monitored by TLC. The reaction mixture was cooled to RT, quenched with water, and extracted with DCM. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain crude compound 4. The obtained crude compound 4 was used in the next step without further purification.

[0322] As step 3, CaCO3 (0.00261 mmol) was added to a stirred solution of compound 4 (crude, 2.6 g, 0.0066 mmol) in DMSO, and the resulting reaction mixture was stirred at 150 °C for 16 hours. The reaction was monitored by TLC and LC-MS. The reaction mixture was cooled to RT, quenched with water, and extracted with EtOAc. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by column chromatography to obtain compound 5 as a white solid (200 mg, 38% yield).

[0323] As step 4, NaH (47.6 mg, 1.98 mmol) was added portionwise to a stirred solution of triethyl phosphonoacetate acetate (449.8 mg, 1.98 mmol) in THF at 0 °C, and the reaction mixture was stirred for 30 minutes. A solution of compound 5 (200 mg, 0.793 mmol) in THF was added, and the reaction mixture was stirred at RT for 3 hours. The reaction was monitored by TLC. The reaction mixture was quenched with a saturated solution of NH4Cl at 0 °C. The aqueous layer was extracted with EtOAc. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by column chromatography to give compound 6 as a white solid (192 mg, 74% yield).

[0324] As step 5, palladium on carbon (5% mmol) was added to a stirred solution of compound 6 (120 mg, 0.372 mmol) in methanol at RT. The resulting suspension was stirred under a hydrogen atmosphere for 4 hours. The reaction was monitored by TLC. The reaction mixture was filtered through a Celite bed, and the filtrate was concentrated under reduced pressure to give crude compound 7. The obtained crude compound 7 was used in the next step without further purification.

[0325] As step 6, aqueous LiOH (50 mg, 1.23 mmol) was added to a stirred solution of compound 7 (crude, 200 mg, 0.6172 mmol) in a mixture of methanol and THF (1:1) at RT, and the resulting reaction mixture was stirred for 4 hours. The reaction was monitored by TLC. The reaction mixture was quenched with water. The aqueous layer was acidified with citric acid and extracted with DCM. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by column chromatography to give compound 8 as an off-white solid (144 mg, 79% yield).

[0326] As step 7, DCC (38 mg, 0.185 mmol), followed by N-hydroxysuccinimide (23.3 mg, 0.2026 mmol) was added to a stirred solution of compound 8 (50 mg, 0.168 mmol) in THF at RT. The resulting reaction mixture was stirred for 16 h. The reaction was monitored by TLC and LC-MS. The reaction mixture was quenched with water. The aqueous layer was extracted with EtOAc. The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The crude reaction mixture was purified by reverse-phase column chromatography to give compound 9 as a white solid (20 mg, 30% yield).

[0327] As step 8, DIPEA (59.2 mg, 0.458 mmol), followed by NH2-PEG4-halolinker (also called compound Int-6 of Example 2) (45 mg, 0.127 mmol) was added to a stirred solution of compound 9 (50 mg, 0.127 mmol) in DMF at RT. The resulting reaction mixture was stirred for 16 h. The reaction was monitored by TLC and LC-MS. The reaction mixture was quenched with water. The aqueous layer was extracted with EtOAc. The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The crude reaction mixture was purified by reverse-phase column chromatography to give compound Int-10 as a light brown solid (23 mg, 30% yield).

[0328] As step 9, dry ACN (0.8 mL), followed by AgPF6 (34.7 mg, 0.1377 mmol) was added to a stirred solution of compound 14 (100 mg, 0.0672 mmol) in dry DCM (4 mL) in a glove box. The resulting reaction mixture was stirred in a sealed tube at 40 °C for 20 h. The reaction was monitored by LC-MS. The reaction mixture was cooled to RT and concentrated under reduced pressure. The crude reaction mixture was dissolved in acetone, filtered and the filtrate was concentrated under reduced pressure to give compound 15 as a yellow solid (90 mg, 72% yield).

[0329] As step 10, compound Int-10 (20 mg, 0.0346 mmol) was added to a stirred solution of compound 15 (27 mg, 0.0289 mmol) in a mixture of solvent DCM (0.8 mL) and EtOH (0.02 mL) (25:1). The resulting reaction mixture was stirred at RT for 20 h. The reaction was monitored by LC-MS. The reaction mixture was concentrated under reduced pressure. The crude reaction mixture was diluted with acetone, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude compound was triturated with pentane to give photocatalyst 7 as a yellow solid (25 mg, 52% yield). Photocatalyst 7 is an example of a photocatalyst according to the present disclosure.

[0330] (Example 8) Preparation of Photocatalyst 8 With reference to Scheme 12, the preparation of the photocatalyst is described.

[0331] [Chemical formula]

[0332] With reference to step 1 of Scheme 12, a stirred solution of 4-methyl-2-(tributylstannyl)pyridine 1 (2.0 g, 5.23 mmol) and 2-bromo-4-fluoropyridine (1.38 g, 7.85 mmol) in toluene (40 mL) was degassed with argon for 10 min. Then, TPP (137 mg, 0.52 mmol), LiCl (0.66 g, 15.7 mmol), and Pd(PPh3)4 (604 mg, 0.52 mmol) were added, and the reaction mixture was stirred at 110 °C for 16 h. The reaction was monitored by TLC and LC-MS. The reaction mixture was cooled to RT, quenched with water, and extracted with EtOAc. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by column chromatography to give compound 3 as an off-white semi-solid (0.5 g, 51% yield).

[0333] As step 2, dimethylamine (2 M in MeOH, 2.66 mL, 5.31 mmol), followed by triethylamine (1.12 mL, 7.9 mmol) were added to a stirred solution of compound 3 (0.5 g, 2.65 mmol) in DMSO (10 mL), and the resulting reaction mixture was heated at 100 °C for 16 h. The reaction was monitored by TLC. The reaction mixture was cooled to RT and concentrated under reduced pressure to give a crude compound. The obtained crude compound was purified by reverse-phase column chromatography to give compound 5 as a white solid (270 mg, 48% yield).

[0334] As step 3, LDA (2 M in THF, 1.26 mL, 2.53 mmol) was added to a stirred solution of compound 5 (270 mg, 1.26 mmol) in THF (6 mL) at -78 °C. The reaction mixture was stirred at -78 °C for 45 min. Methyl 2-bromoacetate (380 mg, 2.53 mmol) at -78 °C was added and the reaction mixture was stirred at RT for 16 h. The reaction was monitored by TLC and LC-MS. The reaction mixture was quenched with aqueous NH4Cl and extracted with EtOAc. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give a crude compound. The obtained crude compound was purified by reverse-phase column chromatography to give compound 7 as a light brown liquid (82 mg, 22% yield).

[0335] As step 4, aqueous LiOH (13.4 mg, 0.56 mmol) was added to a stirred solution of compound 7 (80 mg, 0.28 mmol) in methanol (0.4 mL) and THF (0.4 mL) at RT. The resulting reaction mixture was stirred for 16 h. The reaction was monitored by TLC. Volatiles were evaporated under reduced pressure to give a crude residue. The crude residue was purified by reverse-phase column chromatography to give compound 8 as a light brown liquid (52 mg, 68% yield).

[0336] As step 5, DCC (76 mg, 0.36 mmol), followed by N-hydroxysuccinimide (42 mg, 0.36 mmol) was added to a stirred solution of compound 8 (50 mg, 0.18 mmol) in THF:DMF (2:1, 1.5 mL) at RT. The resulting reaction mixture was stirred at RT for 16 h. The reaction was monitored by TLC and LC-MS. The reaction mixture was quenched with water and extracted with EtOAc. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to afford compound 9 as a white semi-solid (62 mg, 91% yield).

[0337] As step 6, DIPEA (80 μL, 0.48 mmol), followed by NH2-PEG4-halolinker (50 mg, 0.16 mmol) was added to a stirred solution of compound 9 (60 mg, 0.16 mmol) in DMF (1 mL) at RT. The resulting reaction mixture was stirred for 16 h. The reaction was monitored by TLC and LC-MS. The reaction mixture was purified by reverse-phase column chromatography to afford compound Int-11 as a light brown mucus (21 mg, 23% yield).

[0338] As step 7, compound 12 (33 mg, 0.035 mmol) was added to a stirred solution of compound Int-11 (20 mg, 0.035 mmol) in DCM (0.8 mL) and EtOH (20 μL) at RT. The resulting reaction mixture was stirred at RT for 16 h. The reaction was monitored by LC-MS. The reaction mixture was concentrated under reduced pressure. The crude reaction mixture was diluted with acetone, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude compound was purified by reverse-phase column chromatography to afford photocatalyst 8 as a yellow solid (7.7 mg, 17% yield). Photocatalyst 8 is an example of a photocatalyst according to the present disclosure.

[0339] (Example 9) Preparation of Photocatalyst 9 With reference to Scheme 13, the description of the preparation of the photocatalyst is provided.

[0340] [Chemical formula]

[0341] Referring to Step 1 of Scheme 13, DIPEA (0.047 mL, 0.36 mmol) was then added to a stirred solution of Compound 1 (50 mg, 0.12 mmol) in DMF at RT, followed by NH2-PEG4-halolinker (47 mg, 0.13 mmol). The resulting reaction mixture was stirred at RT for 16 h. The reaction was monitored by TLC and LC-MS. The reaction mixture was purified by reverse-phase column chromatography to give Compound 3 as a light brown mucus (30 mg, 43% yield).

[0342] As Step 2, Compound 4 (32.61 mg, 0.034 mmol) was added to a stirred solution of Compound 3 (20 mg, 0.034 mmol) in a mixture of DCM (0.8 mL) and EtOH (0.02 mL) (25:1). The resulting reaction mixture was stirred at RT for 16 h. The reaction was monitored by LC-MS. The reaction mixture was concentrated under reduced pressure to give a crude compound. The crude compound was diluted with acetone and filtered, and the filtrate was concentrated under reduced pressure, triturated with MTBE, and then lyophilized to give photocatalyst 9 as a yellow solid (24 mg, 54% yield). Photocatalyst 9 is an example of a photocatalyst according to the present disclosure.

[0343] (Example 10) Preparation of Photocatalyst 10 With reference to Scheme 14, the description of the preparation of the photocatalyst is described.

[0344] [Chemical formula]

[0345] Referring to Step 1 of Scheme 14, K2CO3 (1.8 g, 12.00 mmol, 3 eq) was added to a stirred solution of (2,4-difluorophenyl)boronic acid (1.0 g, 4.40 mmol, 1.0 eq) and 2-bromo-5-(trifluoromethyl)pyrimidine (1.3 g, 8.80 mmol, 2 eq) in 1,4-dioxane (10 mL). The mixture was purged with argon gas for 10 minutes. Pd(PPh3)4 was added and the mixture was heated at 80 °C for 16 hours. After completion of the reaction, the reaction mixture was filtered through a Celite bed and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude compound was purified by column chromatography using petroleum ether and ethyl acetate as eluents. The required fractions were concentrated under reduced pressure to obtain 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyrimidine (Compound 3, 0.8 g, 70%) as a white solid.

[0346] As Step 2, IrCl3.H20 (600 mg, 2.009 mmol, 1 eq) and Compound 8 (1.145 g, 4.421 mmol, 2.2 eq) were added to an RBF, evacuated and filled with N2 three times. Then, a degassed solution of 2-ethoxyethanol (24 mL) and water (8.4 mL) was added and heated to 120 °C for 16 hours. The progress of the reaction was monitored by LC-MS. After completion of the reaction, the reaction mixture was cooled to RT. The yellow precipitate was filtered, washed with water (120 mL) and hexane (50 mL), and dried under vacuum to obtain Compound 4 (605 mg, yield 33%) as a yellow solid.

[0347] As Step 3, AgPF6 (143 mg, 0.564 mmol, 2.1 eq) was added to a stirred solution of Compound 4 (400 mg, 0.268 mmol, 1 eq) in acetonitrile (30 mL) at RT and stirred for 16 hours. The progress of the reaction was monitored by LC-MS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and the crude compound was purified by trituration with diethyl ether (20 mL) to obtain Compound 5 (350 mg, 55.7%) as a pale yellow solid.

[0348] As step 4, compound 6 (0.11 g, 0.269 mmol, 1 equivalent) was added to a solution of compound 5 (0.17 g, 0.269 mmol, 1 equivalent) in DCM (5 mL) at RT, and the mixture was stirred for 16 h. The progress of the reaction was monitored by LC-MS and TLC (in petroleum ether, 70% ethyl acetate). After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a crude compound. The crude compound was stirred in pentane (10 mL) and filtered to give compound 7 (0.2 g, 83% yield) as a yellow solid.

[0349] As step 5, compound 8 (NH2-PEG4-halolinker, 0.05 g, 0.356 mmol, 2 equivalents) and DIPEA (0.1 mL, 0.496 mmol, 3 equivalents) were added to a solution of compound 7 (0.2 g, 0.175 mmol, 1 equivalent) in DMF (2 mL) at RT. The mixture was stirred for 16 h. The progress of the reaction was monitored by LC-MS and TLC (in DCM, 10% MeOH). After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a crude compound. The crude compound was purified by silica gel (NH silica gel mesh) column chromatography eluting with 0 - 5% methanol in DCM as the mobile phase. The required fractions were concentrated under reduced pressure to give photocatalyst 10 (0.035 g, 20% yield) as a brown sticky compound. The compound was purified by pre-LC. Photocatalyst 10 is an example of a photocatalyst according to the present disclosure.

[0350] (Example 11) Photoresponsive labeling in living cells First, reagents were prepared. An Ir-Cl photocatalyst master stock solution was prepared at 10 mM in 100% DMSO. The specific Ir-Cl photocatalyst used in this example was the photocatalyst prepared according to Example 2. To prepare a working solution of the Ir-Cl catalyst, a portion of the Ir-Cl catalyst master stock solution was diluted in 1×DPBS, resulting in a final Ir-Cl catalyst concentration of 10 μM and a DMSO concentration of 0.1%. A labeled agent master stock having a concentration of 25 mM in 100% DMSO was also prepared. The specific labeled agent used was the one prepared according to Example 1. To prepare a working solution of the labeled agent, a portion of the labeled agent master stock solution was diluted in 1×DPBS, resulting in a final concentration of the labeled agent of 250 μM and a DMSO concentration of 1%.

[0351] A human KRAS (G12D HaloTag® / +) cell line (HD-103-021, Horizon Discovery) with a protein reporter was cultured until 80 - 90% confluent. The cells were harvested and pelleted by centrifugation at 1000 g for 5 minutes at 4°C (Eppendorf Centrifuge 5427 R). The supernatant was removed, and the cell pellet was resuspended and mixed in ice-cold 1×DPBS (Gibco®). Then, the cells were pelleted again by centrifugation at 1000 g for 5 minutes at 4°C. The supernatant was removed, and fresh ice-cold 1×DPBS was added to the pellet and mixed.

[0352] For each set of experimental samples, approximately 7×10 6 cells were used. The Ir-Cl catalyst working solution was added to the cell pellet, and the pellet was gently resuspended and mixed. The cells were incubated with the Ir-Cl catalyst for 30 minutes at 4°C while continuously rotating end-to-end (15 rpm, TARSONS - ROTOSPIN). After incubation, the cells were pelleted by centrifugation at 1000 g for 5 minutes at 4°C. The supernatant was removed, and 1×DPBS was added to the pellet. The pellet was resuspended and mixed, and then centrifuged at 1000 g for 5 minutes at 4°C.

[0353] The supernatant was removed, and the pellet was resuspended in the working labeling agent in 1× DPBS (final DMSO concentration of 1%). The pellet was gently disrupted and mixed, and immediately irradiated with light at a wavelength of 450 nm for 10 minutes at 100% output using an M2 photoreactor (Penn PhD Photoreactor M2 from Sigma Aldrich). After irradiation, the cells were pelleted by centrifugation at 1000 g for 5 minutes at 4°C.

[0354] The supernatant was removed, and the pellet was resuspended in 1× cell lysis buffer (Cell Signaling Technology) supplemented with protease - phosphatase inhibitor (Halt™ Protease and Phosphatase Inhibitor Cocktail (100X)) and gently mixed. Then, this mixture was incubated on ice for 15 minutes.

[0355] After incubation, the cell lysate was sonicated using a probe - sonicator (SONICS vibra - cell Fisher Scientific) for 3 cycles of 15 s on, 10 s off, at 30% amplitude. The cell lysate was pelleted by centrifugation at 18,000 g for 15 minutes at 4°C. The supernatant was collected and then used for immunoprecipitation (IP) by coating with specific antibodies (HALO - tagged protein - HALO - tag IP) and biotinylated proteins (IP based on streptavidin magnetic beads) according to Example 12 and Example 13, respectively.

[0356] (Example 12) Immunoprecipitation of HALO - tagged protein (K - Ras interacting partner protein) 50 μl of Dynabead™ magnetic bead slurry (Dynabeads™ Protein G from Thermo Fisher Scientific) was measured. The beads were collected by standing on a magnetic stand and washed 3 times with wash buffer (PBST). Then, the beads were collected by standing on a magnetic stand and the supernatant was removed.

[0357] To coat the beads with the antibody, anti-HaloTag® monoclonal antibody from Promega was added to PBST at a final concentration of 1:50. The beads were incubated for 1 hour at RT with continuous end-to-end rotation. Then, the beads were collected by standing them on a magnetic rack and washed three times with the wash buffer.

[0358] After this, the beads were incubated for 1 hour at RT with continuous end-to-end rotation in a pre-clearing solution (5% BSA, 2.5 μM carbonic anhydrase). The beads were washed three times with the wash buffer to remove the antibody that had bound in excess.

[0359] The cell lysate obtained according to Example 11 was added to and mixed with the beads coated with the anti-HaloTag antibody. The lysate and the beads were incubated for 3 hours at RT with continuous end-to-end rotation. After incubation, the tubes were placed on a magnetic rack and the beads were collected. The supernatant was removed. Then, the beads were washed three times with the wash buffer.

[0360] For elution, 50 μl of SDS-Laemmli buffer (containing 1 M DTT at 1:10) was added to and mixed with the beads. This mixture was held at 95 °C for 10 minutes. Then, the tubes were placed on a magnetic rack and the supernatant, which is the final pull-down eluate sample, was collected in each tube.

[0361] Next, the pull-down eluate sample was subjected to Western blotting. 10 μl of the pull-down eluate sample was loaded into each lane of a 4–15% Tris-Glycine gel (Bio-Rad). Gel electrophoresis was performed at 80 V for the first 10 minutes and then at 100 V until the front of the dye reached the end of the gel.

[0362] The protein was transferred from the gel to a nitrocellulose membrane at 4°C with a constant current of 300 mA for 90 minutes using a Bio-Rad Wet Transfer apparatus. The membrane was washed once with TBST and blocked with 5% milk in TBST for 60 minutes at RT. After blocking, the membrane was washed with TBST and a primary antibody specific to one of the potential K-Ras interaction partner proteins was added (1:1000) and incubated overnight at 4°C in a shaker. The next day, the membrane was washed 3 times with TBST for 10 minutes each at RT with incubation for each wash.

[0363] An HRP-tagged secondary antibody specific to the primary antibody was added and incubated for 120 minutes at RT with constant shaking. After incubation, the membrane was washed with TBST (3 times for 10 minutes). Thereafter, the membrane was developed using a chemiluminescence reagent (Clarity(™) Western ECl Substrate) with a Bio-Rad ChemiDoc imaging system.

[0364] Figure 4A is an image of the results of a Western blot according to the above procedure using an anti-PI3k primary antibody. Each column of the gel represents different conditions. For example, the presence of a labeling agent (WH8), the presence of a photocatalyst (IR), and irradiation were varied in each column. Further, the cell line used (006 or 021) was varied in one of the columns. The condition containing the labeling agent, photocatalyst, and irradiation for cell line 021 resulted in a distinct band for PI3 kinase P110α. Without being bound by a particular theory, it is contemplated that the observed band of a larger molecular weight suggests the formation of an aggregate containing PI3 kinase P110α.

[0365] Figure 4B is an image of the results of a Western blot according to the above procedure using an SPRED1 antibody. Similar to Figure 4A, the various combinations represent combinations of labeling agent, photocatalyst, irradiation, and cell line. The condition including the labeling agent, photocatalyst, irradiation, and cell line 021 resulted in the formation of distinct bands. Without being bound by a particular theory, it is conceivable that the observed bands of larger molecular weight suggest the formation of aggregates containing SPRED1.

[0366] Figure 4C is an image of the results of a Western blot according to the above procedure using an anti-cRAF antibody. Similar to Figure 4A, the various combinations represent combinations of labeling agent, photocatalyst, irradiation, and cell line. The condition including the labeling agent, photocatalyst, irradiation, and cell line 021 resulted in the formation of distinct bands.

[0367] (Example 13) Immunoprecipitation of Biotinylated Protein (Biotinylated K-Ras Interaction Partner Protein) Weighed 50 μl of Pierce Streptavidin magnetic bead slurry, stood the beads on a magnetic stand to collect them, and washed them three times with wash buffer (PBST). The beads were incubated for 1 hour at RT with continuous end-to-end rotation in a pre-clearing solution (5% BSA, 2.5 μM carbonic anhydrase). Then, the beads were stood on a magnetic rack to collect them and washed three times with wash buffer.

[0368] The cell lysate obtained according to Example 11 was added to streptavidin beads and mixed. This mixture was incubated for 3 hours at RT with continuous end-to-end rotation. After incubation, the tube was placed on a magnetic rack to collect the beads. The supernatant was removed. Then, the beads were washed three times with wash buffer.

[0369] For elution, 50 μl of SDS-Laemmli buffer (containing 1 M DTT at 1:10) was added to the beads and mixed. This mixture was held at 95 °C for 10 minutes. Subsequently, the tubes were placed on a magnetic rack and the supernatants were collected into their respective tubes. These were the final pull-down eluate samples.

[0370] Subsequently, the pull-down eluate samples were subjected to Western blotting. 10 μl of the pull-down eluate was loaded into each lane of a 4–15% Tris-Glycine gel (Bio-Rad). The gel was run at 80 V for the first 10 minutes and then at 100 V until the front of the dye reached the end of the gel.

[0371] Proteins were transferred from the gel to a nitrocellulose membrane at 4 °C and a constant current of 300 mA for 90 minutes using a Bio-Rad Wet Transfer apparatus. The membrane was washed once with TBST and blocked with 5% milk in TBST for 60 minutes at RT. After blocking, the membrane was washed with TBST, the necessary antibody was added (1:1000), and it was held on a shaker at 4 °C overnight. The next day, the membrane was washed three times with TBST, with a 10-minute incubation for each wash at RT.

[0372] Each HRP-tagged secondary antibody was added and held at RT with constant shaking for 120 minutes. After the 120-minute incubation, the membrane was washed with TBST (10-minute washes, 3 times). After this, the membrane was developed using a chemiluminescence reagent (Clarity(™) Western ECl Substrate) on a Bio-Rad ChemiDoc imaging system.

[0373] Figure 5A is an image of the results of a Western blot following the above procedure using an anti-biotin primary antibody. Each lane of the gel represents different conditions with respect to irradiation. The left lane represents cells that received 10 minutes of irradiation, while for the right lane, the cells did not receive irradiation. In the irradiated cells, there is a prominent band starting from the ~55 kDa mark and going upwards. The non-irradiated cells show only faint bands suggesting some level of non-specific binding.

[0374] Figure 5B is an image of the results of a Western blot according to the above procedure using an anti-RSK1 antibody. Similar to Figure 5A, the two columns of the gel represent irradiated cells or non-irradiated cells. A distinct band of the predicted molecular weight is present in the irradiated cells, but no visible band is present in the non-irradiated cells.

[0375] Figure 5C is an image of the results of a Western blot according to the above procedure using an anti-cRAF antibody. Similar to Figure 5A, the two columns of the gel represent irradiated cells or non-irradiated cells. A distinct band of the predicted molecular weight is present in the irradiated cells, but no visible band is present in the non-irradiated cells.

[0376] Figure 5D is an image of the results of a Western blot according to the above procedure using an anti-cRAF antibody. Similar to Figure 5A, the two columns of the gel represent irradiated cells or non-irradiated cells. A distinct band of the predicted molecular weight is present in the irradiated cells, but no visible band is present in the non-irradiated cells.

[0377] Figure 5E is an image of the results of a Western blot using cell lysates and an anti-GAPDH antibody. Similar to Figure 5A, the two columns of the gel represent irradiated cells or non-irradiated cells. Distinct bands of the predicted molecular weight are present in both the irradiated and non-irradiated cells. This confirms that the blots shown in Figures 5A - 5D had similar cell numbers under irradiated and non-irradiated conditions.

[0378] Figure 5F is an image of the results of a Western blot according to the above procedure using an anti-SOS1 antibody. In this gel, three types of cells were run: KRAS(G12DHaloTag® / +)-protein reporter (HD-103021), KRAS HaloTag-protein reporter control (HD-103-037), and SW48 Parental (HD-PAR-006). In the photocatalyst (IR-Cl), labeling partner (WH8), and irradiated KRAS(G12DHaloTag® / +)-protein reporter cells, a distinct SOS1 band indicated by the arrow is observed. In the two control conditions (HD-103-037 and HD-PAR-006), no distinct band is observed. Notably, other methods for detecting protein associations have not previously been able to identify SOS1 as a protein that associates with KRas.

[0379] (Example 14) Fusion of HaloTag to cereblon does not prevent ubiquitination by cereblon of ikaros Using Jurkat cells, a stable cell line was generated in which endogenous cereblon (CRBN) was knocked out and a fusion of HaloTag protein and CRBN was expressed (referred to herein as the "HALO.CRBN Jurkat cell line"). CRBN is known to ubiquitinate ikaros (IKZF1) in the presence of lenalidomide and / or pomalidomide. Figure 6 is an image of the results of a Western blot according to the above procedure using lysates of the HALO.CRBN Jurkat cell line, where the cells were exposed to various concentrations of pomalidomide or lenalidomide. The gel was stained with a mouse anti-IKZF1 antibody. The dilution was 1:1000 and the exposure was approximately 10 seconds. The columns for 10 μM pomalidomide and 10 μM lenalidomide contain significantly less IKZF1 than the control or 1 μM pomalidomide columns, suggesting that the HaloTag / cereblon fusion protein can ubiquitinate IKZF1 in the presence of lenalidomide and / or pomalidomide.

[0380] (Example 15) Detection of Cereblon Interaction Using the HALO.CRBN Jurkat cell line of Example 14, cell lysates were prepared for a series of gels shown in FIGS. 7A - 13B to examine the interaction between CRBN and other proteins.

[0381] HALO.CRBN Jurkat cells were cultured in suspension until 80 - 90% confluent. The cells were either (1) treated with 10 μM pomalidomide for 16 hours, (2) treated with 10 μM lenalidomide for 16 hours, or (3) left untreated.

[0382] The cells were harvested and pelleted by centrifugation at 1000 g for 5 minutes at 4°C (Eppendorf Centrifuge 5427 R). The supernatant was removed, and the cell pellet was resuspended and mixed in ice - cold 1×DPBS (Gibco™). Then, the cells were pelleted again by centrifugation at 1000 g for 5 minutes at 4°C. The supernatant was removed, and fresh ice - cold 1×DPBS was added to the pellet and mixed. For each set of experimental samples, approximately 7×10 6 cells were used.

[0383] An Ir - Cl photocatalyst master stock solution was prepared at 10 mM in 100% DMSO. The specific Ir - Cl photocatalyst used in this example was the photocatalyst prepared according to Example 2. To prepare a working solution of the Ir - Cl catalyst, a portion of the Ir - Cl catalyst master stock solution was diluted in 1×DPBS, resulting in a final Ir - Cl catalyst concentration of 10 μM and a DMSO concentration of 0.1%. A labeled agent master stock with a concentration of 25 mM in 100% DMSO was also prepared. The specific labeled agent used was prepared according to Example 1. To prepare a working solution of the labeled agent, a portion of the labeled agent master stock solution was diluted in 1×DPBS, resulting in a final labeled agent concentration of 250 μM and a DMSO concentration of 1%.

[0384] The Ir-Cl catalyst working solution was added to the cell pellet, and the pellet was loosened and mixed. The cells were incubated with the Ir-Cl catalyst for 30 minutes at 4°C with continuous end-to-end rotation (15 rpm, TARSONS - ROTOSPIN). After incubation, the cells were pelleted by centrifugation at 1000 g for 5 minutes at 4°C. The supernatant was removed, and 1× DPBS was added to the pellet. The pellet was resuspended and mixed, and then centrifuged at 1000 g for 5 minutes at 4°C. The supernatant was removed, and the pellet was resuspended in the working labeling agent (final 1% DMSO concentration) in 1× DPBS. The pellet was loosened, mixed, and immediately irradiated with light of wavelength 450 nm at 100% output for 10 minutes using an M2 photoreactor (Penn PhD Photoreactor M2 from Sigma Aldrich). After irradiation, the cells were pelleted by centrifugation at 1000 g for 5 minutes at 4°C.

[0385] The supernatant was removed, and the pellet was resuspended in 1× cell lysis buffer (Cell Signaling Technology) supplemented with protease - phosphatase inhibitor (Halt(™) Protease and Phosphatase Inhibitor Cocktail (100X)) and gently mixed. Then, this mixture was incubated on ice for 15 minutes.

[0386] After incubation, the cell lysate was sonicated using a probe - sonicator (SONICS vibra - cell Fisher Scientific) with 15 s on, 10 s off, 30% amplitude, for 3 cycles. The cell lysate was centrifuged at 18,000 g at 4°C for 15 minutes. The supernatant was collected and then used for protein content assessment (Pierce(™) BCA Protein Assay Kit). Then, the supernatant containing approximately 200 μg of protein was used for each IP.

[0387] For IP, 50 μl of streptavidin magnetic bead slurry (Pierce Streptavidin Magnetic Beads, ThermoFisher Scientific) was measured. The beads were collected by standing them on a magnetic stand and washed three times with wash buffer (PBST). Then, the beads were collected by standing them on a magnetic stand and the supernatant was removed.

[0388] Thereafter, the beads were incubated with the pre-clearing solution (5% BSA, 2.5 μM carbonic anhydrase) for 1 hour at RT with continuous end-to-end rotation. The beads were washed three times with wash buffer.

[0389] The cell lysate obtained from irradiated HALO.CRBN Jurkat cells as described above was added to streptavidin beads and mixed. The lysate and beads were incubated for 3 hours at RT with continuous end-to-end rotation. After incubation, the tubes were placed on a magnetic rack and the beads were collected. The supernatant was removed. Thereafter, the beads were washed three times with wash buffer.

[0390] For elution, 50 μl of SDS-Laemmli buffer (containing 1 M DTT at 1:10) was added to the beads and mixed. This mixture was held at 95 °C for 10 minutes. Then, the tubes were placed on a magnetic rack and the supernatant, which was the final pull-down eluate sample, was collected in each tube.

[0391] Subsequently, the pull-down eluate sample was subjected to Western blot. 10 μl of the pull-down eluate sample was loaded into each lane of a 4–15% Tris-Glycine gel (Bio-Rad). Gel electrophoresis was performed at 80 V for the first 10 minutes and then at 100 V until the front of the dye reached the end of the gel.

[0392] The protein was transferred from the gel to a nitrocellulose membrane at 4°C with a constant current of 300 mA for 90 minutes using a Bio-Rad Wet Transfer apparatus. The membrane was washed once with TBST and blocked with 5% milk in TBST for 60 minutes at RT. After blocking, the membrane was washed with TBST and a primary antibody specific to one of the potential CRBN interaction partner proteins was added (1:1000) and incubated overnight at 4°C in a shaker in the room. The next day, the membrane was washed 3 times with TBST, incubated for 10 minutes each time with shaking at RT.

[0393] Each HRP-tagged secondary antibody specific to the primary antibody was added and incubated for 120 minutes with constant shaking at RT. After incubation, the membrane was washed with TBST (3 times for 10 minutes each). Then, the membrane was developed using a chemiluminescence reagent (Clarity(™) Western ECl Substrate) with a Bio-Rad ChemiDoc imaging system. These images are shown in Figures 7A - 12B.

[0394] Figures 7A and 7B are images of gels performed using lysates and stained with an anti-CK1 polyclonal rabbit antibody. The gels shown in Figures 7A and 7B were stained at a 1:1000 dilution. The exposure of the gel in Figure 7A was approximately 20 seconds, while the exposure of the gel in Figure 7B was approximately 60 seconds. Observable CK1 bands are present in columns 7 and 8 corresponding to the immunoprecipitation lysates of cells using a photocatalyst, a labeling agent, and irradiation (light), suggesting that CK1 was proximity-labeled by the labeling agent, thereby indicating a protein - protein interaction between CRBN and CK1.

[0395] Figures 8A and 8B are images of gels that were performed using immunoprecipitated proteins and stained with an anti-SALL4 monoclonal rabbit antibody. The gels shown in Figures 8A and 8B were stained with a 1:500 antibody dilution. The exposure of the gel in Figure 8A was approximately 10 seconds, while the exposure of the gel in Figure 8B was approximately 30 seconds. Observable SALL4 bands are present in columns 7 and 8 corresponding to the immunoprecipitation lysates of cells using a photocatalyst, a labeling agent, and irradiation (light), suggesting that SALL4 was proximity-labeled by the labeling agent, thereby indicating a protein-protein interaction between CRBN and SALL4.

[0396] Figure 9 is an image of the results of a Western blot that was performed using lysates and stained with an anti-KEAP1 antibody at a 1:1000 dilution. The exposure was approximately 30 seconds. Observable KEAP1 bands are present in columns 13 and 1 corresponding to the cell lysates using a photocatalyst, a labeling agent, and irradiation (light) cell lysates, but no observable KEAP1 bands were present in the columns (5-8) of the immunoprecipitation pull-down method. This suggests that the KEAP1 protein was not proximity-labeled, and thus, there was no protein-protein interaction between CRBN and KEAP1 in the absence of the molecular adhesive.

[0397] Figure 10 is an image of the results of a Western blot that was performed using lysates and stained with an anti-β-tubulin rabbit antibody at a 1:1000 dilution. The exposure was approximately 5 seconds. Observable β-tubulin bands are present in columns 13 and 1 corresponding to the cell lysates using a photocatalyst, a labeling agent, and irradiation (light) cell lysates, but no observable β-tubulin bands were present in the columns (5-8) of the immunoprecipitation pull-down method. This suggests that the β-tubulin protein was not proximity-labeled, and thus, there was no protein-protein interaction between CRBN and β-tubulin in the absence of the molecular adhesive.

[0398] Figures 11A and 11B are images of gels that were performed using lysates and stained with an anti-PARP1 polyclonal rabbit antibody. The gels shown in Figures 11A and 11B were stained with a 1:1000 antibody dilution. The exposure of the gel in Figure 11A was approximately 20 seconds, while the exposure of the gel in Figure 11B was approximately 120 seconds. Observable PARP1 bands are present in columns 13 and 1 corresponding to cell lysates using a photocatalyst, a labeling agent, and irradiated (light) cell lysates, but no observable PARP1 bands were present in the columns (5-8) of the immunoprecipitation pull-down method. This suggests that the PARP1 protein was not proximity-labeled, and thus there was no protein-protein interaction between CRBN and PARP1 in the absence of the molecular glue.

[0399] Figures 12A and 12B are images of gels that were performed using lysates and stained with an antibody specific for a known oncogenic protein. The gels shown in Figures 12A and 12B were stained with a 1:1000 antibody dilution. The exposure of the gel in Figure 12A was approximately 45 seconds, while the exposure of the gel in Figure 12B was approximately 60 seconds. Observable bands corresponding to the oncogenic protein are present in columns 7 and 8 corresponding to cell lysates using a photocatalyst, a labeling agent, and irradiation (light), suggesting that the oncogenic protein was proximity-labeled by the labeling agent, thereby indicating a protein-protein interaction between CRBN and the oncogenic protein.

[0400] Figures 13A and 13B are images of gels that were performed using lysates and stained with an anti-IKZF1 monoclonal mouse antibody. The gels shown in Figures 13A and 13B were stained with a 1:100 antibody dilution. The exposure of the gel in Figure 13A was approximately 10 seconds, while the exposure of the gel in Figure 13B was approximately 30 seconds. Observable bands of IKZF1 are present in columns 7 and 8 corresponding to the immunoprecipitated lysates of cells using a photocatalyst, a labeling agent, and irradiation (light), suggesting that IKZF1 was proximity-labeled by the labeling agent, thereby indicating a protein-protein interaction between CRBN and IKZF1.

[0401] (Example 16) The tagging approach based on proximity was able to detect proteins associated with K-Ras that were not detected by alternative approaches. The light-responsive proximity-based tagging approach disclosed herein was compared with immunoprecipitation pull-down, an existing approach for detecting protein-protein interactions. Two cell lines were used. The first was a stable cell line expressing a HaloTag / K-Ras G12D fusion protein (Horizon Discovery, catalog number HD-103-021). The second was a stable cell line expressing K-Ras G12D and HaloTag separately (Horizon Discovery, catalog number HD-103-0037).

[0402] Cells were cultured to 80 - 90% confluence. Cells were washed with 1×PBS and then 0.25% Trypsin-EDTA was added. The cells were then incubated at 5% CO2, 37 °C for approximately 2 minutes. Once the cells were detached, the trypsin was neutralized with complete medium. The cells were pelleted by centrifugation at 1000 g for 5 minutes at 4 °C (Eppendorf Centrifuge 5427 R). The supernatant was removed and the cell pellet was resuspended and mixed in ice-cold 1×DPBS (Gibco™). The cells were then pelleted again by centrifugation at 1000 g for 5 minutes at 4 °C. The supernatant was removed and fresh ice-cold 1×DPBS was added to the pellet and mixed.

[0403] For each set of experimental samples, 7×10 6Individual cells were used. A 10 μM Ir-Cl catalyst (Compound 1, see Scheme 1) was added to the cell pellet, and the pellet was loosened and mixed. The cells were incubated with the Ir-Cl catalyst for 30 minutes at 4 °C while continuously rotating end-to-end at 15 rpm (TARSONS - ROTOSPIN). After incubation, the cells were pelleted by centrifugation at 1000 g for 5 minutes at 4 °C. The supernatant was removed, and 1× DPBS was added to the pellet. The pellet was resuspended and mixed, and then centrifuged at 1000 g for 5 minutes at 4 °C. The cell pellet was resuspended in 900 μl of 1× PBS and transferred to a 6-well plate. A labeling agent according to Example 1 was added to the cell suspension at a final concentration of 250 μM in 1× DPBS (final 1% DMSO concentration).

[0404] The 6-well plate containing the cells was placed in a CLIGHT photoreactor. Irradiation was usually carried out for 5 minutes. For SOS1, irradiation was carried out for 10 minutes.

[0405] After irradiation, the cell suspension was transferred to a microfuge tube and pelleted by centrifugation at 1000 g for 5 minutes at 4 °C. The supernatant was removed, and the pellet was resuspended in 1× cell lysis buffer (Cell Signaling Technology) supplemented with protease - phosphatase inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktail (100X), sodium fluoride, PMSF) and gently mixed. This was incubated on ice for 15 minutes.

[0406] After incubation, the lysate was sonicated using a probe - sonicator (SONICS vibra - cell Fisher Scientific) for 15 s on, 10 s off, 30% amplitude, 3 cycles. The cell lysate was centrifuged at 18,000 g at 4 °C for 15 minutes. The supernatant containing the cell lysate was collected in a tube.

[0407] The protein content of the lysates was evaluated (Pierce™ BCA Protein Assay Kit). The cell lysates were then used for immunoprecipitation (IP) by coating them with specific antibodies (HALO-tag IP) and biotinylated proteins (IP based on streptavidin magnetic beads).

[0408] HALO-tag IP 50 μl of Dynabead™ magnetic bead slurry (Dynabeads™ Protein G Thermo Fisher Scientific) was measured. The beads were placed on a magnetic stand for collection and washed three times with wash buffer (PBST). After washing, the beads were placed on a magnetic stand for collection and the supernatant was removed.

[0409] To coat the beads with the antibody, the anti-HaloTag® monoclonal antibody from Promega (source: mouse) was added to PBST at a final concentration of 1:50. The beads were incubated for 1 hour at RT with continuous end-to-end rotation with the antibody. The beads were then placed on a magnetic rack for collection and washed three times with wash buffer. After this, the beads were incubated for 1 hour at RT with continuous end-to-end rotation with the pre-clearing solution (5% BSA, 2.5 μM carbonic anhydrase). The beads were washed three times with wash buffer to remove the excess bound antibody.

[0410] 200 μg of each irradiated cell lysate obtained as described above was added to and mixed with the beads coated with the HALO antibody. The mixture of the lysate and the beads was incubated for 3 hours at RT with continuous end-to-end rotation.

[0411] After incubation, the tubes were placed on a magnetic rack and the beads were collected. The supernatant was removed. The beads were washed three times with wash buffer. 50 μl of SDS-Laemmli buffer (containing 1 M DTT at 1:10) was added to the beads and mixed. This mixture was heated to 95 °C for 10 minutes. Then, the tubes were placed on a magnetic rack to isolate the beads, and the supernatant was collected into each tube. The collected supernatant was then used for Western blot.

[0412] Streptavidin bead IP Using 50 μl of Pierce Streptavidin magnetic bead slurry, the beads were collected by standing them on a magnetic stand and washed three times with wash buffer (PBST). The beads were incubated for 1 hour at RT with rotation end-to-end continuously in a pre-clearing solution (5% BSA, 2.5 μM carbonic anhydrase). The beads were collected by standing them on a magnetic rack and washed three times with wash buffer. The cell lysate obtained from irradiated cells was added to the streptavidin beads and mixed. The mixture of the lysate and the beads was incubated for 3 hours at RT with rotation end-to-end continuously.

[0413] After incubation, the tubes were placed on a magnetic rack and the beads were collected. The supernatant was removed. The beads were washed three times with wash buffer. 50 μl of SDS-Laemmli buffer (containing 1 M DTT at 1:10) was added to the beads and mixed, and then heated to 95 °C for 10 minutes. The tubes were placed on a magnetic rack and the supernatant was collected into each tube. The collected supernatant was then used for Western blot.

[0414] Western blot The eluate samples from the HALO-tag IP and streptavidin bead IP were subjected to Western blot. 10 μl of the pull-down eluate sample was loaded into each lane of a 4–15% Tris-Glycine gel (Bio-Rad). The gel was run at 80 V for the first 10 minutes and then at 100 V until the front of the dye reached the end of the gel. The proteins in the gel were transferred to a nitrocellulose membrane at 4 °C and a constant current of 300 mA for 90 minutes using a Bio-Rad Wet Transfer apparatus.

[0415] The membrane was washed once with TBST and blocked with 5% milk in TBST for 60 minutes at RT. After blocking, the membrane was washed with TBST and a primary antibody specific for one of the potential KRAS interaction partner proteins was added at 1:1000 and incubated overnight at 4 °C on a shaker.

[0416] The next day, the membrane was washed at RT with TBST (3 times for 10 minutes). Each HRP-tagged secondary antibody specific for the primary antibody was added and incubated for 120 minutes at RT with constant shaking. The membrane was then washed with TBST (3 times for 10 minutes). The membrane was developed using a chemiluminescence reagent (Immobilon® Forte Western HRP Substrate) with a Bio-Rad ChemiDoc imaging system to generate Figures 14A–17B.

[0417] Figures 14A and 14B show the gels stained with the anti-c-RAF antibody.

[0418] In Figure 14A, the rabbit anti-c-RAF antibody was diluted 1:750. The exposure was approximately 60 seconds. The lysate was subjected to the Halo-Tag IP discussed above. The gel shows a weak cRAF band in lane 5 corresponding to immunoprecipitation with the Halo antibody, while lanes 6 and 7 of the total lysate show distinct cRAF bands.

[0419] In Figure 14B, the gel was stained with rabbit anti-c-RAF antibody diluted 1:1000. The exposure was about 30 seconds. The lysate was subjected to IP based on streptavidin beads discussed above. The gel shows a distinct cRAF band in column 5 corresponding to immunoprecipitation using photocatalyst, irradiation, and labeling agent. Columns 6 and 7 of the total lysate also show distinct cRAF bands. This demonstrates that the approach disclosed herein was more effective than immunoprecipitation pull-down method in identifying protein-protein interactions.

[0420] Figures 15A and 15B show gels stained with anti-RSK1 antibody.

[0421] In Figure 15A, rabbit anti-RSK1 antibody was diluted 1:1000. The exposure was about 90 seconds. The lysate was subjected to Halo-Tag IP discussed above. The gel does not show observable bands in column 5 corresponding to immunoprecipitation with Halo antibody, while columns 6 and 7 of the total lysate show distinct RSK1 bands. No observable bands were seen even with longer exposure of about 180 seconds.

[0422] In Figure 15B, the gel was stained with rabbit anti-RSK1 antibody diluted 1:1000. The exposure was about 30 seconds. The lysate was subjected to IP based on streptavidin beads discussed above. The gel shows a distinct RSK1 band in column 5 corresponding to immunoprecipitation using photocatalyst, irradiation, and labeling agent. Columns 6 and 7 of the total lysate also show distinct RSK1 bands. The results shown in Figures 15A and 15B indicate that the approach disclosed herein was able to identify RSK1 near K-Ras, which could not be achieved with immunoprecipitation pull-down approach.

[0423] Figures 16A and 16B show gels stained with anti-SOS1 antibody.

[0424] In Figure 16A, the rabbit anti-SOS1 antibody was diluted 1:1000. The exposure was approximately 45 seconds. The lysate was subjected to Halo-Tag IP discussed above. The gel did not show observable bands in column 5 corresponding to immunoprecipitation by Halo antibody, while columns 6 and 7 of the total lysate showed distinct SOS1 bands.

[0425] In Figure 16B, the gel was stained with a 1:1000 diluted rabbit anti-SOS1 antibody. The exposure was approximately 60 seconds. The lysate was subjected to IP based on streptavidin beads discussed above. The gel shows distinct SOS1 bands in column 5 corresponding to immunoprecipitation using photocatalyst, irradiation, and warhead. Columns 6 and 7 of the total lysate also show distinct RSK1 bands. The results shown in Figures 16A and 16B indicate that the approach disclosed herein was able to identify SOS1 near K-Ras, which could not be achieved with the immunoprecipitation pull-down approach.

[0426] Figures 17A and 17B show gels stained with anti-KEAP1 antibody.

[0427] In Figure 17A, the rabbit anti-KEAP1 antibody was diluted 1:1000. The exposure was approximately 60 seconds. The lysate was subjected to Halo-Tag IP discussed above. The gel did not show observable bands in column 5 corresponding to immunoprecipitation by Halo antibody, while columns 6 and 7 of the total lysate showed distinct SOS1 bands.

[0428] In Figure 17B, the gel was stained with a 1:1000 diluted rabbit anti-KEAP1 antibody. The exposure was approximately 60 seconds. The lysate was subjected to IP based on streptavidin beads discussed above. The gel did not show observable bands in column 5 corresponding to immunoprecipitation using photocatalyst, irradiation, and labeling agent. Columns 6 and 7 of the total lysate showed distinct KEAP1 bands. These results suggest that there is no protein-protein interaction between KRAS G12D and KEAP1.

[0429] (Example 17) Proximity labeling of KRAS interactors may depend on irradiation time Figures 18A - 18D are images of Western blots using both lysates and streptavidin pull - down proteins stained with an anti - RSK1 polyclonal rabbit antibody. The antibody dilution was 1:1000. The gel exposures for Figures 6A and 6C were about 10 seconds, while the gel exposures for Figures 6B and 6D were about 60 seconds. There are observable bands of RSK1 in lanes 1 of Figures 6B and 6D corresponding to immunoprecipitation lysates of cells using a photocatalyst, a labeling agent, and irradiation (light). The presence of these bands suggests that RSK1 was proximity - labeled by the labeling agent. The observed bands of RSK1 are darker in the 5 - minute irradiation sample (Figure 6D) than in the 2.5 - minute irradiation sample (Figure 6B). Without being bound by a particular theory, it is considered that these results indicate that the proximity labeling of KRAS interactors may depend on the irradiation time.

[0430] Figures 18E - 18H are images of Western blots using both lysates and streptavidin pull - down proteins stained with an anti - SOS1 polyclonal rabbit antibody. The antibody dilution was 1:1000. The gel exposures for Figures 6E and 6G were about 10 seconds, while the gel exposures for Figures 6F and 6H were about 30 seconds. There are observable bands of SOS1 in lanes 1 of Figures 6F and 6H corresponding to immunoprecipitation lysates of cells using a photocatalyst, a labeling agent, and irradiation (light). The presence of these bands suggests that SOS1 was proximity - labeled by the labeling agent. The observed bands of SOS1 are darker in the 5 - minute irradiation (Figure 6H) than in the 2.5 - minute irradiation sample (Figure 6F). Without being bound by a particular theory, it is considered that these results indicate that the proximity labeling of KRAS interactors may depend on the irradiation time.

[0431] (Example 18) System for testing the Ir - photocatalytic activity Catalyst - dependent biotinylation of bovine serum albumin The photocatalyst was combined with the diazirine-labeled compound 8 or 9 and BSA in DPBS to obtain a reaction mixture with a total solution volume of 200 μL. The BSA concentration was 10 μM, the diazirine-labeled compound was fixed at 100 μM, and the final catalyst concentrations were set at 5 μM and 10 μM, respectively. These samples were then either placed in the dark or irradiated with visible light (450 nm) at 100% intensity for 5 minutes in an M2 photoreactor. Next, 60 μL of the sample was taken out, combined with 20 μL of 4× reducing SDS-Laemmli buffer (containing 1 M DTT at 1:10), mixed, and heated at 95 °C for 5 minutes. Then, 15 μL of each sample was analyzed by fluorescence Western blot.

[0432] Fluorescence Western blot The samples were loaded onto a 4–20% Tris-Glycine gel (Bio-Rad). The gel was run at 120 V for 80 minutes or until the front of the dye reached the end of the gel. The proteins were transferred from the gel to a nitrocellulose membrane using an iBlot 2 semi-dry Transfer device. The membrane was used to detect total protein for normalization according to the manufacturer's manual (Revert™ 700 Total Protein Stain, LI-COR). Subsequently, the membrane was washed once with TBST and blocked with Intercept® Blocking Buffer for 1 hour at RT with gentle shaking. After blocking, the membrane was washed with TBST, the necessary antibody was added (1:1000), and it was held in a shaker at 4 °C overnight. The next day, the membrane was washed 3 times with TBST for 10 minutes of incubation each time at RT. IRDye® 800CW secondary antibody or IRDye® 680RD secondary antibody was added and held at RT with constant shaking for 1 hour. After 1 hour of incubation, the membrane was washed with TBST (10-minute washes, 3 times). Thereafter, the membrane was scanned with an Odyssey Imager (LI-COR Odyssey M Imaging System).

[0433] Figures 19A-19B show the results of Western blots of the biotinylation of BSA by various photocatalysts in combination with the labeling reagent WH8. Figure 19A shows the total protein staining results for all treatment conditions. The same amount of BSA was used in each treatment. Figure 19B shows the results of a fluorescent Western blot using a rabbit anti-biotin antibody. There do not appear to be bands in the samples that were not irradiated (lanes 6 and 8-11). There appear to be bands for biotinylated BSA in both the 5 μM and 10 μM photocatalyst treatments in the samples that were irradiated for 5 minutes.

[0434] Figures 20A-20B show the results of Western blots of the biotinylation of BSA by various photocatalysts in combination with the labeling reagent WH9. Figure 20A shows the total protein staining results for all treatment conditions. The same amount of BSA was used in each treatment. Figure 20B shows the results of a fluorescent Western blot using a rabbit anti-biotin antibody. There do not appear to be bands in the samples that were not irradiated (lanes 6 and 8-11). There appear to be bands for biotinylated BSA in both the 5 μM and 10 μM photocatalyst treatments in the samples that were irradiated for 5 minutes.

[0435] (Example 19) Cell permeability of Ir-photocatalyst In the same manner as the procedure described in Example 11, intracellular photo-responsive labeling was performed as described. An Ir-photocatalyst master stock solution was prepared at 10 mM or 5 mM in 100% DMSO. KRAS (G12DHaloTag® / +) cells (HD-103-021, Horizon Discovery) were cultured to 90% confluence in a 6-well cell culture dish using phenol-free RPMI1640 medium (Gibco®). Photocatalyst IR1 (photocatalyst 1), IR23 (photocatalyst 3), IR24 (photocatalyst 4), IR42 (photocatalyst 5), or DMSO was added to the appropriate wells at a final photocatalyst concentration of 5 or 10 μM. The cells were incubated at 37 °C for 1 or 2 hours. Then, the medium was aspirated, and the cells were gently washed twice with 2 mL of fresh RPMI1640 medium with a 10-minute incubation at 37 °C for each wash. TAMRA-Cl fluorescent dye (Promega, G8251) was diluted with OptiMEM to a 5 μM working solution. 0.8 mL of the working dye solution was added to the cells and incubated at 37 °C for 15 minutes. The cells were gently washed twice with 2 mL of cold DPBS, aspirated, and then 200 μL of RIPA supplemented with a protease-phosphatase inhibitor (Halt® Protease and Phosphatase Inhibitor Cocktail (100X)) was added and gently mixed. The cell lysate was collected in a 1.5 mL tube. The cell lysate was sonicated using a probe-sonicator (SONICS vibra-cell Fisher Scientific) for 15 s on, 10 s off, 30% amplitude, for 3 cycles. The cell lysate was centrifuged at 18,000 g at 4 °C for 20 minutes. The supernatant was collected and adjusted to 1 mg / ml in concentration by BCA assay (Pierce® BCA Protein Assay Kit, Thermofisher). The cell lysate was processed and analyzed by fluorescence Western blot using an anti-TAMRA antibody and an anti-β-actin antibody (for normalization). Without being bound by a particular theory, the TAMRA signal may be inversely proportional to the Ir binding to intracellularly expressed Halotag-KRAS (G12D), and thus may be inversely proportional to the Ir cell permeability.

[0436] Figures 21A and 21B show the results of a Halo-tracking assay fluorescence Western blot using anti-TAMRA antibody and anti-β-actin antibody. The mouse anti-TAMRA antibody was diluted 1:1000. The rabbit anti-β-actin antibody was diluted 1:5000 depending on the loading control. Photocatalysts IR1, IR23, IR24, and IR42 were used.

[0437] In Figure 21A, the samples were treated with the catalyst for 1 hour or 2 hours. In the 1-hour samples, compared to the DMSO control, IR1-5μM and IR1-10μM showed a slightly weaker binding to HaloTag, while the other three photocatalysts (IR23, IR24, and IR42) appeared to bind completely to HaloTag. Without being bound by a particular theory, these results may indicate that IR24, IR42, and IR23 have higher cell permeability than IR1.

[0438] In Figure 21B, at photocatalyst concentrations of 0.5μM and 1μM, they did not seem to bind completely to HaloTag. 5μM of IR42 showed complete binding. 5μM of IR1 showed most of the binding. Without being bound by a particular theory, these results seem to indicate that IR42 has higher cell permeability than IR1.

Claims

1. A method for labeling intracellular molecules based on their proximity, A step of expressing a first protein intracellularly within a cell, wherein the first protein is coupled to a binder that can bind to a catalytic complex, and the catalytic complex is a photocatalyst. The process involves introducing a photocatalyst into cells and thereby causing it to bind to a first protein. A step of introducing a labeling agent containing a labeling portion and a reactive portion into cells. A process of activating a photocatalyst, thereby activating a labeling agent through energy transfer from the photocatalyst to the reactive part, and binding the labeling agent to a second protein in the cell, and A process to detect a second protein by detecting the labeled portion. Includes, The photocatalyst is given by formula (I): 【Chemistry 1】 (In the formula, A1 is present in 0 to 4 units on the ring to which it is bonded, and each A1 is CH 3 , CF 3 F, Cl, N(CH 3 ) 2 , and OR 1 Selected independently from, Each R 1 H, a linear or branched alkyl group having 1 to 12 carbon atoms, CHF 2 , and CF 3 Selected independently from, There are 0 to 4 A2s on the ring to which it is attached, and each A2 is CH 3 , CF 3 , F, Cl, N(CH 3 ) 2 , and OR 1 is independently selected from, A3 is present in 0 to 4 units on the ring to which it is bonded, and each A3 is CH 3 , CF 3 F, Cl, N(CH 3 ) 2 , and OR 1 Selected independently from, A4 is absent, a linear or branched alkyl group having 1 to 12 carbon atoms, C 6～10 Ariel, C 3～8 Selected from cycloalkyls, 4-10 membered heterocyclines, and 5-10 membered heteroaryls, wherein the alkyl, aryl, cycloalkyl, heterocycline, and heteroaryl are one or more C 1～6 Alkyl, C 1～6 Haloalkyl, halo, hydroxy, C 1～6 It is optionally substituted with alkoxy or amino acids. A5 is CONH, NHCO, SONH, SO 2 NH, NHSO, NHSO 2 , NH, OCONH, NHCOO, C 1～6 alkoxy, and 【Chemistry 2】 Selected from, A6 is independently (PEG)a 1 (CH 2 )a 2 Cl or (PEG)a 1 (CH 2 )a 2 (5-6 member heterocycline) (PEG)a 1 (CH 2 )a 2 Cl and each a 1 These are independent integers from 0 to 10, and each a 2 These are, independently, integers between 6 and 10. A9 is iridium, A10 is tetraalkylborate, tetrafluoroborate, tetraphenylborate, chloride, cyanide, hexafluorophosphate (PF 6 ), an anion selected from bis(triphenylphosphine)iminium chloride, tetraphenylphosphonium chloride, and tetrabutylammonium, Each of A11 and A12 is independently a C or N coordinated to A9. Each A13 is independently either CH or N. A method having the structure of

2. The structure in which the photocatalyst is selected from one of the following: 【Chemistry 3-1】 【Chemistry 3-2】 【Chemistry 3-3】 The method according to claim 1, comprising:

3. There are 0 to 4 A3 atoms on the ring to which it is bonded, and each A3 atom is CH 3 , CF 3 , F, Cl, and OR 1 Selected independently of, A5 is selected from CONH, NHCO, SONH, SO 2 NH, NHSO, NHSO 2, NH, OCONH, and NHCOO, or A6 is (PEG)a 1 (CH 2 )a 2 Cl, where a1 is an integer from 0 to 10, and a2 is an integer from 6 to 10, or A13 is CH, or The 5-6 member heterocycline of A6 is piperazine or pyrrolidine. The method according to claim 1.

4. The labeling agent is given by formula (III-a): 【Chemistry 4】 (In the formula, R 1' It is selected from azide, methyldiaziline, trifluoromethyldiaziline, and phenyldiaziline. The Ar ring is one or more -OH, -OMe, -OEt, -OCF 3 , -OCF 2 H, -NHMe, -NMe 2 Selected from phenyl, pyridyl, pyrimidyl, pyrazinyl, pyrididine, naphthyl, and quinolinyl, which are optionally substituted with -F, -Cl, -Br, -Me, or -Et. X is O, NH, NR 2 CH 2 NHCO, CONH, CONR 2 , SO 2 NH and SO 2 NR 2 Selected from, R 2 It is selected from H, OMe, Me, and Et. n is 0, 1, 2, 3, 4, 5, or 6. Y is a biotin-bound amide or an amide-bound fluorescent dye. The method according to claim 1, having the structure described above.

5. The labeling agent has a structure: 【Transformation 5】 The method according to claim 4, having the following characteristics.

6. The method is carried out in the absence of an exogenous compound that promotes the interaction between the first protein and the second protein, or The method according to any one of claims 1 to 5, wherein the method is carried out in the presence of a test compound, and the detection of a second protein or the level of a second protein indicates that the test compound promotes the interaction between the first protein and the second protein.

7. The method according to any one of claims 1 to 5, wherein the cells are living cells during the activation of the catalytic complex.

8. The method according to any one of claims 1 to 5, wherein the binder comprises a haloalkane dehalogenase.

9. The method according to any one of claims 1 to 5, wherein activating the catalyst complex includes the step of illuminating the cells with light, the light having a wavelength of about 380 nm to about 700 nm.

10. The first protein coupled to haloalkane dehalogenase, Equation (I): 【Transformation 6】 (In the formula, A1 is present in 0 to 4 units on the ring to which it is bonded, and each A1 is CH 3 , CF 3 F, Cl, N(CH 3 ) 2 , and OR 1 Selected independently from, Each R 1 H, a linear or branched alkyl group having 1 to 12 carbon atoms, CHF 2 , and CF 3 Selected independently from, A2 exists in 0 to 4 units on the ring to which it is bonded, and each A2 is a CH 3 , CF 3 F, Cl, N(CH 3 ) 2 , and OR 1 Selected independently from, A3 is present in 0 to 4 units on the ring to which it is bonded, and each A3 is CH 3 , CF 3 F, Cl, N(CH 3 ) 2 , and OR 1 Selected independently from, A4 is absent, a linear or branched alkyl group having 1 to 12 carbon atoms, C 6～10 Ariel, C 3～8 Selected from cycloalkyls, 4-10 membered heterocyclines, and 5-10 membered heteroaryls, wherein the alkyl, aryl, cycloalkyl, heterocycline, and heteroaryl are one or more C 1～6 Alkyl, C 1～6 Haloalkyl, halo, hydroxy, C 1～6 It is optionally substituted with alkoxy or amino acids. A5 is CONH, NHCO, SONH, SO 2 NH, NHSO, NHSO 2 , NH, OCONH, NHCOO, C 1～6 alkoxy, and 【Transformation 7】 Selected from, A6 is independently (PEG)a 1 (CH 2 )a 2 Cl or (PEG)a 1 (CH 2 )a 2 (5-6 member heterocycline) (PEG)a 1 (CH 2 )a 2 Cl and each a 1 These are independent integers from 0 to 10, and each a 2 These are, independently, integers between 6 and 10. A9 is a D9 metal selected from copper, vanadium, chromium, scandium, titanium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, and mercury. A11 and A12 are independently C or N coordinated to A9. Each A13 is independently either CH or N. A photocatalyst having the structure, and Equation (III-a): 【Transformation 8】 (In the formula, R 1' is selected from azide, methyldiazirine, trifluoromethyldiazirine, and phenyldiazirine, The Ar ring is one or more -OH, -OMe, -OEt, -OCF 3 , -OCF 2 H, -NHMe, -NMe 2 Selected from phenyl, pyridyl, pyrimidyl, pyrazinyl, pyrididine, naphthyl, and quinolinyl, which are optionally substituted with -F, -Cl, -Br, -Me, or -Et. X is O, NH, NR 2 , CH 2 NHCO, CONH, CONR 2 , SO 2 NH, and SO 2 NR 2 and is selected from R 2 It is selected from H, OMe, Me, and Et. n is 0, 1, 2, 3, 4, 5, or 6. Y is a biotin-bound amide or an amide-bound fluorescent dye. Labeling agent having the structure Cells that include this.

11. The structure in which the photocatalyst is selected from one of the following: 【Chemistry 9-1】 【Chemistry 9-2】 【Chemistry 9-3】 A cell according to claim 10, having the characteristics of the cell.

12. There are 0 to 4 A3 atoms on the ring to which it is bonded, and each A3 atom is CH 3 , CF 3 , F, Cl, and OR 1 A cell according to claim 10, independently selected from the above.

13. A5 is CONH, NHCO, SONH, SO 2 NH, NHSO, NHSO 2 The cells according to claim 10, selected from NH, OCONH, and NHCOO.

14. A6 is (PEG)a 1 (CH 2 )a 2 Cl and a 1 a is an integer between 0 and 10, and 2 is an integer between 6 and 10, or The 5-6 member heterocycline of A6 is piperazine or pyrrolidine. The cell according to claim 10.

15. The cell according to claim 10, wherein A13 is CH.

16. Labeling agent structure: 【Chemistry 10】 A cell according to any one of claims 10 to 15, having the characteristics of the cell.

17. structure: P1-P2-Cat (In the formula, P1 is a ubiquitin ligase, P2 is a haloalkane dehalogenase, Cat is given by equation (I): 【Chemistry 11】 (In the formula, A1 is present in 0 to 4 units on the ring to which it is bonded, and each A1 is CH 3 , CF 3 F, Cl, N(CH 3 ) 2 , and OR 1 Selected independently from, Each R 1 H, a linear or branched alkyl group having 1 to 12 carbon atoms, CHF 2 , and CF 3 Selected independently from, A2 exists in 0 to 4 units on the ring to which it is bonded, and each A2 is a CH 3 , CF 3 F, Cl, N(CH 3 ) 2 , and OR 1 Selected independently from, A3 is present in 0 to 4 units on the ring to which it is bonded, and each A3 is CH 3 , CF 3 F, Cl, N(CH 3 ) 2 , and OR 1 Selected independently from, A4 is absent, a linear or branched alkyl group having 1 to 12 carbon atoms, C 6～10 Ariel, C 3～8 Selected from cycloalkyls, 4-10 membered heterocyclines, and 5-10 membered heteroaryls, wherein the alkyl, aryl, cycloalkyl, heterocycline, and heteroaryl are one or more C 1～6 Alkyl, C 1～6 Haloalkyl, halo, hydroxy, C 1～6 It is optionally substituted with alkoxy or amino acids. A5 is CONH, NHCO, SONH, SO 2 NH, NHSO, NHSO 2 , NH, OCONH, NHCOO, C 1～6 alkoxy, and 【Chemistry 12】 Selected from, A6 is independently (PEG)a 1 (CH 2 )a 2 Cl or (PEG)a 1 (CH 2 )a 2 (5-6 member heterocycline) (PEG)a 1 (CH 2 )a 2 Cl and each a 1 These are independent integers from 0 to 10, and each a 2 These are, independently, integers between 6 and 10. A9 is a D9 metal selected from copper, vanadium, chromium, scandium, titanium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, and mercury. A11 and A12 are independently C or N coordinated to A9. Each A13 is independently either CH or N. (A photocatalyst having the structure) A protein complex containing [the specified ingredient].

18. The structure in which Cat is selected from one of the following: 【Chemistry 13-1】 【Chemistry 13-2】 The protein complex according to claim 17, having the following characteristics.

19. The structure in which Cat is selected from one of the following: 【Chemistry 14】 The protein complex according to claim 17, having the following characteristics.

20. A structure selected from one of the following: 【Chemistry 15-1】 【Chemistry 15-2】 【Chemistry 15-3】 Photocatalysts.

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