Intracellular ligation for photoresponsive, probe-mediated protein-labeled photocatalysts

Abstract

Embodiments of the present disclosure relate to methods, compositions, and systems for proximity-based photoactivated molecular marking. Molecules may be labeled by activation of a grafted photocatalyst capable of delivering energy to adjacent biomolecule labeling agents. Depending on the activation half-life and diffusion coefficient of the labeling agent, molecules within a particular vicinity of the grafted photocatalyst may be labeled, but molecules other than the vicinity will not be labeled.

Description

[0001] Incorporation by reference into any priority application

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 366,463, filed on June 15, 2022, and U.S. Provisional Application No. 63 / 384,767, filed on November 22, 2022, the contents of each of which are incorporated herein by reference in their entirety.

[0003] background

[0004] field

[0005] The present disclosure relates to compositions, systems and methods for proximity-based in vitro or in vivo biomolecule labeling.

[0006] Related technical description

[0007] Many disease pathologies can be understood by elucidating local biomolecular networks or microenvironments. To this end, enzymatic proximity labeling platforms are widely used to map extended spatial relationships in subcellular structures. It is known that these spatial relationships between biomolecules underlie fundamental biological processes. In the context of intracellular signaling, proteins can be localized in defined environments, such as cell membranes, which can also be considered microenvironments. Local signaling in these microenvironments may play an important role in intercellular signaling communication. Therefore, the ability to accurately map these intracellular microenvironments may yield important insights into basic biology, with potentially broad implications for human health and the development of novel therapeutic strategies.

[0008] There are some platforms that promote specific labeling of proteins by exploiting spatial proximity. A survey of existing methods for targeting small molecules to protein sequences shows that orthogonal technologies may complement known methods. These technologies (including but not limited to APEX, Air-ID, BioID, SPPLAT, EMARS and Turbo-ID) adopt common design elements, in which the tethered enzyme is genetically attached to the target protein. This functional enzyme catalyzes the production of active species through diffusion and / or physical contact, which targets specific amino acid residues in the adjacent system. However, it is well known that the active species produced from these technologies have a relatively long half-life. Therefore, these active species may diffuse to a distance comparable to or greater than the distance of the label, thereby labeling proteins outside the immediate vicinity of the tethered enzyme, resulting in non-specific results. In addition, the intermediate active species produced by these technologies may exhibit preferential (i.e., non-unknown) binding to specific amino acid residues. In the context of mapping cell signaling pathways, preferential binding reactivity to specific amino acid residues may lead to biased results because the labeling depends on the residues on the exposed surface. Even with these limitations, these techniques have revolutionized the ability to track the expression, localization, and conformational changes of proteins and components in cellular signaling pathways. Given the intrinsic value of understanding biological systems at the microenvironmental level, there remains a need for cellular mapping techniques with greater operational precision.

[0009] In particular, the aforementioned techniques have shown special weaknesses in identifying transient and / or weakly interacting partners. Importantly, in the field of targeted protein degradation (TPD), the identification of transient and / or weakly interacting partners is particularly problematic. For example, all known molecular glues of TPD come from the increase of transient and / or weakly interacting basic affinity between the ligase and the target protein. Therefore, the pairing of the protein of E3 ligase and targeted degradation is still poorly understood.

[0010] Therefore, there remains a need for methods to agnostically label intracellular proteins, peptides, chromatin, and nucleic acids with high levels of spatial resolution. Summary of the invention

[0011] The present disclosure provides systems, compositions, and methods directed to labeling intracellular molecules in proximity to a protein of interest.

[0012] In one aspect, the present disclosure relates to a method for proximity-based labeling of intracellular molecules, comprising:

[0013] introducing a binding agent complex into a cell of a cell, wherein the binding agent complex comprises a protein of interest coupled to a binding agent, wherein the binding agent is capable of binding to the catalyst complex;

[0014] introducing the catalyst complex into the cell, thereby forming a biomolecular antenna comprising the catalyst complex and the binding agent complex;

[0015] introducing into the cell a labeling agent comprising a labeling moiety and an active moiety, wherein the active moiety is configured to be activated to an active state by the catalyst complex; and

[0016] The catalyst complex is activated, thereby activating the labeling agent by transferring energy from the catalyst complex to the active moiety and causing the labeling agent to bind to a biomolecule within the cell.

[0017] In some embodiments, introducing the binding agent complex into the cell comprises introducing a nucleotide construct into the cell, wherein the nucleotide construct encodes the binding agent complex, and wherein the nucleotide construct can be expressed by the cell. In further embodiments, introducing the nucleotide construct into the cell comprises transfecting the cell.

[0018] In some embodiments, the biomolecule is an intracellular protein, peptide, chromatin, or nucleic acid.

[0019] In some embodiments, the method further comprises imaging the signal from the labeling moiety.

[0020] In some embodiments, the catalyst complex comprises a photocatalyst. In further embodiments, the photocatalyst comprises a transition metal complex. In yet further embodiments, activating the transition metal complex comprises photoactivating the transition metal complex. In yet further embodiments, photoactivation comprises irradiating light onto the transition metal complex, wherein the light has a wavelength of about 380 nm to about 700 nm. In some embodiments, activating the catalyst complex causes a Dexter energy transfer from the activated transition metal complex to the active moiety to form an active intermediate.

[0021] In some embodiments, when the labeling agent is bound to the biomolecule, the biomolecule is within a 10 nm radius of the biomolecule antenna.

[0022] In another aspect, the present disclosure provides a system for proximity-based labeling of intracellular molecules, comprising:

[0023] cell;

[0024] A biomolecular antenna comprising:

[0025] A photocatalyst composite comprising a photocatalyst and a ligand portion, and

[0026] a binding agent complex comprising a protein of interest coupled to a binding agent, wherein the binding agent is capable of binding to the ligand portion of the photocatalyst complex; and

[0027] a labeling agent comprising a labeling portion and an active portion, wherein the active portion is configured to be activated to an active state by the photocatalyst composite;

[0028] wherein the biomolecule antenna and the labeling agent are each located within the cell.

[0029] In some embodiments, the binding agent comprises a haloalkane dehalogenase. In further embodiments, the ligand moiety comprises an alkyl chloride. In yet further embodiments, the alkyl chloride comprises 6 or more carbons.

[0030] In some embodiments, the binding agent is coupled to the protein of interest at the N-terminus or the C-terminus of the protein of interest.

[0031] In some embodiments, the ligand moiety includes 4,4'-di-tert-butyl-2,2'-bipyridine, 2,2'-bipyridine, diphenhydramine-2,2'-bipyridine, 4-4'-dimethoxy-2-2'-bipyridine, dinaphthyl-pyrene, phenanthroline, and biphenyl-phenanthroline.

[0032] In some embodiments, the photocatalyst comprises a transition metal. In further embodiments, the transition metal has a triplet energy 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 hexacoordinated. 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 extinction coefficient greater than 1000 nm. -1 cm -1 .

[0033] In some embodiments, the photocatalyst is an organic catalyst that does not include a transition metal. In further embodiments, the organic catalyst includes 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, and a benzophenone group.

[0034] In some embodiments, the labeling agent is cell permeable.

[0035] In some embodiments, the photocatalyst is capable of activating the marking agent to form a reactive intermediate. In further embodiments, the photocatalyst is capable of activating the marking agent by Dexter energy transfer to form the reactive intermediate. 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 ).

[0036] In some embodiments, the binding agent is a protein, an E3 ligase, a polysaccharide, or a nucleic acid.

[0037] In some embodiments, the protein of interest is K-Ras, cMyc, Src, WRN, Slug, PARP1, Aβ, Tau, influenza hemagglutinin, or viral nucleoprotein.

[0038] In another aspect, the present disclosure provides a biomolecular assembly comprising a dehalogenase and a photocatalyst coupled to a protein of interest.

[0039] In some embodiments, the photocatalyst is configured to activate a marking agent comprising a labeling moiety and an active moiety. In further embodiments, the photocatalyst is configured to activate the marking agent upon absorption of light having a wavelength of about 380 nm to about 700 nm.

[0040] 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 further embodiments, the transition metal is hexacoordinated. In further embodiments, the transition metal has a molecular mass greater than 1000 M. -1 cm -1 Visible light extinction coefficient.

[0041] In some embodiments, the photocatalyst is an organic catalyst that does not include a transition metal. In further embodiments, the organic catalyst includes 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, and a benzophenone group.

[0042] In some embodiments, the biomolecule assembly further comprises a photocatalyst complex, wherein the photocatalyst complex comprises the photocatalyst and a ligand moiety. In further embodiments, the ligand moiety comprises an alkyl chloride. In yet further embodiments, the alkyl chloride comprises 6 or more carbons.

[0043] In another aspect, the present disclosure provides a method for detecting a protein-protein interaction, comprising:

[0044] expressing a first protein within a cell of the cell, the first protein coupled to a binding agent capable of binding to a catalyst complex, wherein the catalyst complex is a photocatalyst;

[0045] introducing the photocatalyst into the cell so that it binds to the first protein;

[0046] introducing a labeling agent comprising a labeling moiety and an active moiety into the cell;

[0047] activating the photocatalyst, thereby activating the labeling agent by transferring energy from the photocatalyst to the active portion and causing the labeling agent to bind to a second protein within the cell; and

[0048] detecting the second protein by detecting the labeling portion;

[0049] The photocatalyst has a structure of formula (I):

[0050]

[0051] in:

[0052] A1 is present 0 to 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;

[0053] Each R1 is independently selected from H, a straight or branched chain alkyl group having 1 to 12 carbons, CHF2, and CF3;

[0054] A2 is present 0 to 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;

[0055] A3 is present 0 to 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;

[0056] A4 is selected from none, a straight or branched alkyl group having 1 to 12 carbon atoms, 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, halogen, hydroxyl, C 1-6 Alkoxy or amino substitution;

[0057] A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1-6 Alkoxy, and

[0058] A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a l (CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, wherein each a1 is independently an integer from 0 to 10, and each a2 is independently an integer from 6 to 10;

[0059] A9 is iridium;

[0060] A10 is an anion selected from tetraalkylborate, tetrafluoroborate, tetraphenylborate, chloride, cyanide, hexafluorophosphate (PF6), bis(triphenylphosphine)imide chloride, tetraphenylphosphine chloride and tetrabutylammonium;

[0061] Each of A11 and A12 is independently C or N coordinated to A9; and

[0062] Each A13 is independently CH or N.

[0063] In some embodiments, A3 can exist 0 to 4 times on the ring to which it is connected, and each A3 can be independently selected from CH3, CF3, F, Cl and OR1. In some embodiments, A5 can be selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH and NHCOO. In some embodiments, A6 can be (PEG)a1(CH2)a2Cl, a1 can be an integer from 0 to 10, and a2 can be an integer from 6 to 10. In some embodiments, A13 can be CH. In some embodiments, the 5 to 6 membered heterocyclic group of A6 can be piperazine or pyrrolidine. In some embodiments, the photocatalyst has the following structure:

[0064]

[0065] In some embodiments, the photocatalyst has the following structure:

[0066]

[0067] In some embodiments, the photocatalyst has the following structure:

[0068]

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

[0070] In some embodiments, the photocatalyst has the following structure:

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

[0072] In some embodiments, the photocatalyst has the following structure:

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

[0074]

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

[0076]

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

[0078]

[0079] In another aspect, the present disclosure provides a method for detecting a protein-protein interaction, comprising:

[0080] expressing a first protein within a cell of the cell, the first protein being coupled to a binding agent capable of binding to the catalyst complex;

[0081] introducing the catalyst complex into the cell so that it binds to the first protein;

[0082] introducing a labeling agent comprising a labeling portion and an active portion into the cell;

[0083] activating the catalyst complex, thereby activating the labeling agent by transferring energy from the catalyst complex to the active moiety and causing the labeling agent to bind to a second protein within the cell; and

[0084] detecting the second protein by detecting the labeling portion;

[0085] The marking agent has a structure of formula (III-a):

[0086]

[0087] in:

[0088] R1 'Selected from azide, methyl diazirine, trifluoromethyl diazirine and phenyl diazirine;

[0089] Ring Ar is selected from phenyl, pyridyl, pyrimidinyl, pyrazinyl, pyridizynyl, naphthyl and quinolinyl, optionally substituted with one or more -OH, -OMe, -OEt, -OCF3, -OCF2H, -NHMe, -NMe2, -F, -Cl, -Br, -Me or -Et;

[0090] X is selected from O, NH, NR 2 、CH2NHCO、CONH、CONR 2 , SO2NH and SO2NR 2 ;

[0091] R 2 is selected from H, OMe, Me and Et;

[0092] n is 0, 1, 2, 3, 4, 5 or 6; and

[0093] Y is a biotin-linked amide or an amide-linked fluorescent dye.

[0094] In some embodiments, the labeling agent has the following structure:

[0095]

[0096] In some embodiments of the methods disclosed herein, the first protein can be a ubiquitin ligase.

[0097] In another aspect, the present disclosure provides a method for detecting a protein-protein interaction, comprising:

[0098] expressing a first protein within a cell of the cell, the first protein being coupled to a binding agent capable of binding to the catalyst complex, wherein the first protein is a ubiquitin ligase;

[0099] introducing the catalyst complex into the cell so that it binds to the first protein;

[0100] introducing a labeling agent comprising a labeling moiety and an active moiety into the cell;

[0101] activating the catalyst complex, thereby activating the labeling agent by transferring energy from the catalyst complex to the active moiety and causing the labeling agent to bind to a second protein within the cell; and

[0102] By detecting the labeling moiety, the second protein is detected.

[0103] In some embodiments of the methods disclosed herein, the catalyst complex is a photocatalyst. In some embodiments, the photocatalyst has a structure of formula (I):

[0104]

[0105] in:

[0106] A1 is present 0 to 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;

[0107] Each R1 is independently selected from H, a straight or branched chain alkyl group having 1 to 12 carbons, CHF2, and CF3;

[0108] A2 is present 0 to 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;

[0109] A3 is present 0 to 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;

[0110] A4 is selected from none, a straight or branched alkyl group having 1 to 12 carbon atoms, 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, halogen, hydroxyl, C 1-6 Alkoxy or amino substitution;

[0111] A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1-6 Alkoxy and Helix C 1-6 Alkoxy;

[0112] A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a l (CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, wherein each a1 is independently an integer from 0 to 10, and each a2 is independently an integer from 6 to 10;

[0113] A9 is a D9 metal selected from the group consisting of 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;

[0114] A10 is an anion selected from tetraalkylborate, tetrafluoroborate, tetraphenylborate, chloride, cyanide, hexafluorophosphate (PF6), bis(triphenylphosphine)imide chloride, tetraphenylphosphine chloride and tetrabutylammonium;

[0115] A11 and A12 are independently C or N coordinated to A9; and

[0116] Each A13 is independently CH or N.

[0117] In some embodiments, A3 can exist 0 to 4 times on the ring to which it is connected, and each A3 can be independently selected from CH3, CF3, F, Cl and OR1. In some embodiments, A5 can be selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH and NHCOO. In some embodiments, A6 can be (PEG)a1(CH2)a2Cl, a1 can be an integer from 0 to 10, and a2 can be an integer from 6 to 10. In some embodiments, A13 can be CH. In some embodiments, the 5 to 6 membered heterocyclic group of A6 can be piperazine or pyrrolidine. In some embodiments, the photocatalyst has the following structure:

[0118]

[0119] In some embodiments, the photocatalyst has the following structure:

[0120]

[0121] In some embodiments, the photocatalyst has the following structure:

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

[0123] In some embodiments, the photocatalyst has the structure:

[0124] In some embodiments, the photocatalyst has the following structure:

[0125] In some embodiments, the photocatalyst has the following structure:

[0126] In some embodiments, the photocatalyst has the following structure:

[0127] In some embodiments, the photocatalyst has the following structure:

[0128] In some embodiments, the photocatalyst has the following structure:

[0129]

[0130] In some embodiments of the methods disclosed herein, the labeling agent has the structure of formula (III-a):

[0131]

[0132] in:

[0133] R 1 ' is selected from azide, methyldiaziridine, trifluoromethyldiaziridine and phenyldiaziridine;

[0134] Ring Ar is selected from phenyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, naphthyl and quinolinyl, optionally substituted with one or more -OH, -OMe, -OEt, -OCF3, -OCF2H, -NHMe, -NMe2, -F, -Cl, -Br, -Me or -Et;

[0135] X is selected from O, NH, NR 2 、CH2NHCO、CONH、CONR 2 , SO2NH and SO2NR 2 ;

[0136] R 2 is selected from H, OMe, Me and Et;

[0137] n is 0, 1, 2, 3, 4, 5 or 6; and

[0138] Y is a biotin-linked amide or an amide-linked fluorescent dye.

[0139] In some embodiments, the labeling agent has the following structure:

[0140]

[0141] In some embodiments of the methods disclosed herein, the methods are performed in the absence of an exogenous compound that promotes interaction between the first and second proteins.

[0142] In some embodiments of the methods disclosed herein, the methods are performed in the presence of a test compound, wherein detecting the second protein or the level of the second protein indicates that the test compound promotes the interaction between the first and second proteins.

[0143] In some embodiments of the methods disclosed herein, during the activation of the catalyst complex, the cell is a living cell. In some embodiments of the methods disclosed herein, the binding agent comprises a haloalkane dehalogenase. In some embodiments of the methods disclosed herein, activating the catalyst complex comprises irradiating light onto the cell, wherein the light has a wavelength of about 380 nm to about 700 nm. In some embodiments of the methods disclosed herein, activating the catalyst complex causes a Dexter energy transfer from the activated catalyst complex to the active portion to form an active intermediate.

[0144] In another aspect, the present disclosure provides a cell comprising:

[0145] a first protein coupled to a haloalkane dehalogenase;

[0146] A photocatalyst having the structure of formula (I):

[0147]

[0148] in:

[0149] A1 is present 0 to 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;

[0150] Each R1 is independently selected from H, a straight or branched chain alkyl group having 1 to 12 carbons, CHF2, and CF3;

[0151] A2 is present 0 to 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;

[0152] A3 is present 0 to 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;

[0153] A4 is selected from none, a straight or branched alkyl group having 1 to 12 carbon atoms, 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, halogen, hydroxyl, C1-6 Alkoxy or amino substitution;

[0154] A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1-6 Alkoxy, C 1-6 Alkoxy, and

[0155] A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a l (CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, wherein each a1 is independently an integer from 0 to 10, and each a2 is independently an integer from 6 to 10;

[0156] A9 is a D9 metal selected from the group consisting of 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;

[0157] A11 and A12 are independently C or N coordinated to A9; and

[0158] Each A13 is independently CH or N; and

[0159] A labeling agent having the structure of formula (III-a):

[0160]

[0161] in:

[0162] R 1 ' is selected from azide, methyldiaziridine, trifluoromethyldiaziridine and phenyldiaziridine;

[0163] Ring Ar is selected from phenyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, naphthyl and quinolinyl, optionally substituted with one or more -OH, -OMe, -OEt, -OCF3, -OCF2H, -NHMe, -NMe2, -F, -Cl, -Br, -Me or -Et;

[0164] X is selected from O, NH, NR 2 、CH2NHCO、CONH、CONR 2 , SO2NH and SO2NR 2 ;

[0165] R 2 is selected from H, OMe, Me and Et;

[0166] n is 0, 1, 2, 3, 4, 5 or 6; and

[0167] Y is a biotin-linked amide or an amide-linked fluorescent dye.

[0168] In some embodiments, A3 can exist 0 to 4 times on the ring to which it is connected, and each A3 can be independently selected from CH3, CF3, F, Cl and OR1. In some embodiments, A5 can be selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH and NHCOO. In some embodiments, A6 can be (PEG)a1(CH2)a2Cl, a1 can be an integer from 0 to 10, and a2 can be an integer from 6 to 10. In some embodiments, A13 can be CH. In some embodiments, the 5 to 6 membered heterocyclic group of A6 can be piperazine or pyrrolidine. In some embodiments, the photocatalyst has the following structure:

[0169]

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

[0171]

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

[0173]

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

[0175]

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

[0177] In some embodiments, the photocatalyst has the following structure:

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

[0179] In some embodiments, the photocatalyst has the following structure:

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

[0181] In some embodiments, the photocatalyst has the following structure:

[0182] In some embodiments, the labeling agent has the following structure:

[0183] In another aspect, the present disclosure provides a protein complex comprising the structure: P1-P2-Cat

[0184] in:

[0185] P1 is a ubiquitin ligase;

[0186] P2 is a haloalkane dehalogenase;

[0187] Cat is a photocatalyst having the structure of formula (I):

[0188]

[0189] in:

[0190] A1 is present 0 to 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;

[0191] Each R1 is independently selected from H, a straight or branched chain alkyl group having 1 to 12 carbons, CHF2, and CF3;

[0192] A2 is present 0 to 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;

[0193] A3 is present 0 to 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;

[0194] A4 is selected from none, a straight or branched alkyl group having 1 to 12 carbon atoms, 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, halogen, hydroxyl, C 1-6 Alkoxy or amino substitution;

[0195] A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1-6 Alkoxy, C1-6 Alkoxy, and

[0196] A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a l (CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, wherein each a1 is independently an integer from 0 to 10, and each a2 is independently an integer from 6 to 10;

[0197] A9 is a D9 metal selected from the group consisting of 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;

[0198] A11 and A12 are independently C or N coordinated to A9; and

[0199] Each A13 is independently CH or N.

[0200] In some embodiments, A3 can exist 0 to 4 times on the ring to which it is connected, and each A3 can be independently selected from CH3, CF3, F, Cl, N(CH3)2 and OR1. In some embodiments, A5 can be selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH and NHCOO. In some embodiments, A6 can be (PEG)a1(CH2)a2Cl, a1 can be an integer from 0 to 10, and a2 can be an integer from 6 to 10. In some embodiments, A13 can be CH. In some embodiments, the 5 to 6 membered heterocyclic group of A6 can be piperazine or pyrrolidine. In some embodiments, Cat has the following structure:

[0201]

[0202] In some embodiments, Cat has the following structure:

[0203]

[0204] In some embodiments, Cat has the following structure:

[0205]

[0206] In some embodiments, Cat has the following structure:

[0207] In some embodiments, Cat has the following structure:

[0208] In some embodiments, Cat has the following structure:

[0209] In some embodiments, Cat has the following structure:

[0210] In some embodiments, Cat has the following structure:

[0211] In some embodiments, Cat has the following structure:

[0212] In some embodiments, Cat has the following structure:

[0213] In another aspect, the present disclosure provides a cell comprising:

[0214] A protein complex according to the present disclosure; and

[0215] A labeling agent having the structure of formula (III-a):

[0216]

[0217] in:

[0218] R 1 ' is selected from azide, methyldiaziridine, trifluoromethyldiaziridine and phenyldiaziridine;

[0219] Ring Ar is selected from phenyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, naphthyl and quinolinyl, optionally substituted with one or more -OH, -OMe, -OEt, -OCF3, -OCF2H, -NHMe, -NMe2, -F, -Cl, -Br, -Me or -Et;

[0220] X is selected from O, NH, NR 2 、CH2NHCO、CONH、CONR 2 , SO2NH and SO2NR 2 ;

[0221] R 2 is selected from H, OMe, Me and Et;

[0222] n is 0, 1, 2, 3, 4, 5 or 6; and

[0223] Y is a biotin-linked amide or an amide-linked fluorescent dye.

[0224] 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.

[0225] In another aspect, the present disclosure provides a photocatalyst having a structure of formula (I):

[0226]

[0227] in:

[0228] A1 is present 0 to 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;

[0229] Each R1 is independently selected from H, a straight or branched chain alkyl group having 1 to 12 carbons, CHF2, and CF3;

[0230] A2 is present 0 to 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;

[0231] A3 is present 0 to 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;

[0232] A4 is selected from none, a straight or branched alkyl group having 1 to 12 carbon atoms, 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, halogen, hydroxyl, C 1-6 Alkoxy or amino substitution;

[0233] A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1-6 Alkoxy, and

[0234] A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a l (CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, wherein each a1 is independently an integer from 0 to 10, and each a2 is independently an integer from 6 to 10;

[0235] A9 is a D9 metal selected from the group consisting of 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;

[0236] A11 and A12 are independently C or N coordinated to A9; and

[0237] Each A13 is independently CH or N, and

[0238] wherein the photocatalyst is not

[0239]

[0240] In some embodiments, A3 exists 0 to 4 times on the ring to which it is connected, and each A3 can be 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 heterocyclic group in A6 is piperazine or pyrrolidine. In some embodiments, the photocatalyst has the following structure:

[0241]

[0242] In some embodiments, the photocatalyst has the following structure:

[0243]

[0244] In some embodiments, the photocatalyst has the following structure:

[0245]

[0246] In some embodiments, the photocatalyst has the following structure:

[0247] In some embodiments, the photocatalyst has the following structure:

[0248] In some embodiments, the photocatalyst has the following structure:

[0249] In some embodiments, the photocatalyst has the following structure:

[0250] In some embodiments, the photocatalyst has the following structure:

[0251] In some embodiments, the photocatalyst has the following structure:

[0252] In some embodiments, the photocatalyst has the following structure:

[0253] BRIEF DESCRIPTION OF THE DRAWINGS

[0254] Throughout the drawings, reference numerals are repeated to indicate correspondence between reference elements.The following drawings and associated descriptions are provided to illustrate embodiments of the present disclosure and do not limit the scope of the claims.

[0255] Figure 1 is a diagram showing the formation of a catalyst-antenna assembly according to the present disclosure.

[0256] Figure 2 is a diagram of photoactivation of a photocatalyst which subsequently activates a labeling agent via energy transfer, resulting in binding of the label to a nearby biomolecule.

[0257] Figure 3 is a flow chart illustrating a process for labeling a biomolecule within a cell of interest according to the present disclosure.

[0258] FIG. 4A to FIG. 4C is an image of a Western blot result of the procedure described in Example 5, involving immunoprecipitation of HALO-tagged proteins.

[0259] FIG. 5A to FIG. 5F is an image of a Western blot result of the procedure described in Example 6, involving immunoprecipitation of biotinylated proteins.

[0260] Figure 6 is an image of a Western blot result showing that the HaloTag-cereblon fusion protein is able to ubiquitinate ikaros.

[0261] FIG. 7A to FIG. 7B are images of Western blot results demonstrating proximity tagging of CK1 by HaloTag-cereblon fusion protein.

[0262] FIG. 8A to FIG. 8B are images of Western blot results demonstrating proximity tagging of SALL4 by HaloTag-cereblon fusion protein.

[0263] Fig. 9 is an image of a western blot result, which shows that KEAP1 is not tagged by the HaloTag-cereblon fusion protein.

[0264] Fig.10 is an image of a Western blot result, which shows that β-tubulin is not labeled by the HaloTag-cereblon fusion protein.

[0265] FIG. 11A to FIG. 11B is an image of a Western blot result, which shows that PARP1 is not tagged by the HaloTag-cereblon fusion protein.

[0266] FIG. 12A to FIG. 12B is an image of a Western blot showing that a known oncoprotein was tagged with the HaloTag-cereblon fusion protein.

[0267] FIG. 13A to FIG. 13B are images of Western blot results, which show that IKZF1 is tagged with HaloTag-cereblon fusion protein.

[0268] FIG. 14A to FIG. 14B are images of western blot results demonstrating that cRAF was labeled by the immunoprecipitation pull-down method and the HaloTag-based proximity labeling method.

[0269] FIG. 15A to FIG. 15B are images of western blot results demonstrating that RSK1 was labeled by the HaloTag-based proximity labeling method but not by the immunoprecipitation pull-down method.

[0270] FIG. 16A to FIG. 16B are images of western blot results demonstrating that SOS1 was labeled by the HaloTag-based proximity labeling method but not by the immunoprecipitation pull-down method.

[0271] FIG. 17A to FIG. 17B are images of western blot results, which demonstrate that KEAP1 is neither labeled by the immunoprecipitation pull-down method nor by the HaloTag-based proximity labeling method.

[0272] 18A to 18H are images of Western blot results, which indicate that intracellular labeling of K-Ras interactors by HaloTag-KRAS protein may be light irradiation time-dependent.

[0273] FIG. 19A to FIG. 19B are images of Western blot results showing the photocatalytic biotinylation of bovine serum albumin (BSA) by using a bisaziridine-biotin conjugate with different Ir photocatalysts.

[0274] FIG. 20A to FIG. 20B are images of Western blot results showing the photocatalytic biotinylation of bovine serum albumin (BSA) by using a bisaziridine-biotin conjugate with different Ir photocatalysts.

[0275] FIG. 21A to FIG. 21B are images of Western blot results showing the cell permeability of different Ir photocatalysts determined by Halotag chaser.

[0276] Detailed Description

[0277] The present disclosure will now be described in conjunction with the accompanying drawings, in which like numbers refer to like elements throughout. The following description is merely illustrative in nature and is in no way intended to limit the present disclosure, its application or use. It should be understood that the steps in the method can be performed in a different order without changing the principles of the present disclosure. In addition, the embodiments disclosed herein may include several new features, no single feature of which is solely responsible for the desired attributes of the present invention, or no single feature is essential to practicing the systems, devices and methods disclosed herein.

[0278] The systems, compositions and methods disclosed herein relate to the technology of intracellular molecule labeling adjacent to the target protein. Such labeling can allow for protein labeling based on proximity in vitro and in vivo. Labeling can be accomplished by a photoresponsive catalyst coupled to the target protein to activate a cell permeable labeling agent through Dexter energy transfer, which can then label adjacent biomolecules. Specifically, when activated, the photoresponsive catalyst will activate the active portion on the cell permeable labeling agent. The active portion can then be combined with various biomolecules near the target protein. The label on the labeling agent can then be detected, for example, by imaging, to provide information about the intracellular location of the target protein.

[0279] More specifically, disclosed herein are methods and systems by which light-responsive catalysts such as iridium can be incorporated into living systems for microenvironment mapping using self-labeling systems such as HaloTag. According to the present disclosure, when used in conjunction with appropriate cell-permeable labeling agents, the light-responsive catalysts can utilize incident photon energy and transfer the energy to activate the cell-permeable labeling agents to label components of the biomolecular microenvironment.

[0280] Systems and compounds for proximity-based labeling in cells

[0281] In one aspect, a proximity-based system for labeling intracellular molecules comprises: a cell; a biomolecule antenna, the biomolecule antenna comprising a photocatalyst complex and a binding agent complex, the photocatalyst complex further comprising a photocatalyst and a ligand portion, the binding agent comprising a target protein coupled to the binding agent, wherein the binding agent is capable of binding to the ligand portion of the photocatalyst complex; and a labeling agent, the labeling agent comprising a labeling portion and an active portion, wherein the active portion is configured to be activated to an active state by the photocatalyst complex; wherein the biomolecule antenna and the labeling agent are each located within the cell.

[0282] Figure 1 1 is a diagram showing the formation of a biomolecular antenna 106 in an example intracellular proximity-based labeling system. The biomolecular antenna 106 includes a catalyst complex 102 and a binder complex 104. The catalyst complex 102 further includes a photocatalyst 112 and a ligand portion 114. The binder complex 104 includes a target protein 108 and a binder 110. The binder 110 is capable of binding to the ligand portion 114 of the catalyst complex 102, so that the catalyst complex 102 and the binder complex 104 can form the biomolecular antenna 106.

[0283] The catalyst complex 102 can be any catalyst complex 102 suitable for activating a marking agent 216 (described below) according to the present embodiment. In some embodiments, the catalyst complex 102 includes a transition metal complex comprising a transition metal. In these embodiments, the transition metal complex can include one or more ligands coordinated to a metal center. In certain embodiments, the transition metal can be a platinum group metal. In certain embodiments, the transition metal can be iridium or ruthenium. In some embodiments, the triplet energy of the transition metal is 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 within or bounded by these ranges or values, although values ​​outside of these values ​​or ranges may be used in some cases.

[0284] In some embodiments, the transition metal (also referred to herein as a transition metal catalyst) is capable of absorbing one or more wavelengths of visible light (i.e., light having a wavelength of about 380 nm to about 700 nm, although wavelengths outside this range may be used in some cases). The absorption of visible light may excite the transition metal complex to the S1 state, followed by quantitative intersystem crossing to a long-lived triplet excited state (T1). The half-life of the T1 state is T 1 / 2 Can be 50ns to 5μs, 0.1μs to 2.5μs or 0.2μs to 2μs, or any numerical value or range within these ranges or values, or any numerical value or range defined by these ranges or values, although numerical values ​​outside these numerical values ​​or ranges can be used in some cases. In some embodiments, the transition metal catalyst can undergo a short-range Dexter energy transfer to the labeling agent and return to ground state S0. The energy transfer to the labeling agent can activate the labeling agent for reacting with proteins or other biomolecules. If the labeling agent is diaziridine, as an illustrative example, a carbene intermediate can be formed to the triplet energy transfer to the labeling agent for reacting with proteins or other biomolecule species.

[0285] The photocatalytic transition metal complex can have any composition and structure consistent with the above principles and energy transfer. In some embodiments, the photocatalytic transition metal complex is hexacoordinated. The ligand portion 114 may include one or more ligands capable of binding to the binding agent 110. In some embodiments, the ligand of the ligand portion 114 of the catalyst complex 102, such as a transition metal complex, may include one or more pyridine moieties. In some embodiments, one or more ligands include bipyridine or a derivative thereof. Table 1 provides examples of suitable ligands:

[0286] Table I. Examples of photocatalytic transition metal complex ligands

[0287] 4,4'-Di-tert-butyl-2,2'-bipyridine (dtbbpy) 2,2'-Bipyridine (bpy) Diphenhydramine-2,2'-bipyridyl (diPh-bpy) 4-4'-Dimethoxy-2-2'-bipyridine (diOMe-bpy) di-naphth-Py Phenanthroline (phen) Biphenyl-phenanthroline (di-Ph-Phen)

[0288] 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, an iridium photocatalyst can be used, as 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.

[0289] In some embodiments, the photocatalytic transition metal complex has the structure of Formula I:

[0290] I.

[0291] 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;

[0292] R1 can independently be H, a straight or branched alkyl group having 1 to 12 carbons, CHF2, CF3;

[0293] A2 can be 0 to 4 substituents on the ring to which it is attached, selected from CH3, CF3, F, Cl, N(CH3)2 or OR1;

[0294] A3 can be 0 to 4 substituents on the ring to which it is attached, selected from CH3, CF3, F, Cl, N(CH3)2 or OR1;

[0295] A4 can be none, a straight or branched chain alkyl group having 1 to 12 carbon atoms, 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, halogen, hydroxyl, C 1-6 Alkoxy or amino substituted

[0296] A5 can be CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1-6 Alkoxy, and

[0297] A6 may independently be (PEG)a1(CH2)a2Cl or (PEG)a1(CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, wherein, for example, a1 may be defined as the number of polyethylene glycol (PEG) subunits, wherein a1 is an integer from 0 to 10, and a2 may be defined as the number of methylene subunits, wherein a2 is an integer from 6 to 10, and in some embodiments, the 5- to 6-membered heterocyclyl in A6 may be piperazine or pyrrolidine;

[0298] 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;

[0299] A10 may be an anion of a metal in A9; a non-exclusive list of such anions includes tetraalkylborate, tetrafluoroborate, tetraphenylborate, chloride, cyanide, hexafluorophosphate, bis(triphenylphosphine)imide chloride, tetraphenylphosphine chloride, or tetrabutylammonium; and

[0300] A11 and A12 may independently be C or N coordinated to A9; and

[0301] Each A13 is independently CH or N.

[0302] 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 once on the ring to which it is attached. In some embodiments, A1 may be present twice on the ring to which it is attached. In some embodiments, A1 may be present twice on the ring to which it is attached and may be F. In some embodiments, A1 may be present once on the ring to which it is attached and may be F. In some embodiments, A1 may be present twice on the ring to which it is attached and may be Cl. In some embodiments, A1 may be present once on the ring to which it is attached and may be Cl. In some embodiments, A1 may be present twice on the ring to which it is attached and may be CH3. In some embodiments, A1 may be present once on the ring to which it is attached and may be CH3. In some embodiments, A1 may be present twice on the ring to which it is attached and may be CF3. In some embodiments, A1 may be present once on the ring to which it is connected, and may be CF3. In some embodiments, A1 may be present twice on the ring to which it is connected, and may be N(CH3)2. In some embodiments, A1 may be present once on the ring to which it is connected, and may be N(CH3)2. In some embodiments, A1 may be present twice on the ring to which it is connected, and may be OR1. In some embodiments, A1 may be present once on the ring to which it is connected, 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, A2 may be present once on the ring to which it is connected. In some embodiments, A2 may be present twice on the ring to which it is connected. In some embodiments, A2 may be present twice on the ring to which it is connected, and may be F. In some embodiments, A2 may be present once on the ring to which it is connected, and may be F. In some embodiments, A2 can exist 2 times on the ring to which it is connected, and can be Cl. In some embodiments, A2 can exist 1 time on the ring to which it is connected, and can be Cl. In some embodiments, A2 can exist 2 times on the ring to which it is connected, and can be CH3. In some embodiments, A2 can exist 1 time on the ring to which it is connected, and can be CH3. In some embodiments, A2 can exist 2 times on the ring to which it is connected, and can be CF3. In some embodiments, A2 can exist 1 time on the ring to which it is connected, and can be CF3.In some embodiments, A2 may be present twice on the ring to which it is attached and may be N(CH3)2. In some embodiments, A2 may be present once on the ring to which it is attached and may be N(CH3)2. In some embodiments, A2 may be present twice on the ring to which it is attached and may be OR1. In some embodiments, A2 may be present once on the ring to which it is attached and may be OR1. In some embodiments, A11 may be a C coordinated to A9. In some embodiments, A12 may be an N coordinated to A9. In some embodiments, in some embodiments, in some embodiments, one or more ligands may include a hydrophilic portion for enhancing the compatibility of the transition metal complex with water or water-based environments (e.g., those found within cells). In addition, one or more ligands may include a portion, functionality, and / or handle for coupling the binding agent 110, such as a protein, polysaccharide, or nucleic acid. For example, a moiety containing an alkyl chloride of appropriate length, such as a chlorinated alkane having 6 or more carbons, can be bound by a binder 110 comprising a halodehalogenase (e.g., HaloTag) that is genetically encoded and thus produced with the protein of interest 108. In certain embodiments, the photocatalytic transition metal complex has a structure of Formula II-XI:

[0303]

[0304]

[0305]

[0306] In some embodiments, the visible light extinction coefficient of the transition metal may be greater than about 10 M -1 cm -1 , greater than about 100M -1 cm -1 , greater than about 250M -1 cm -1 , greater than about 500M -1 cm -1 , greater than about 1000M -1 cm -1 , greater than about 2000M -1 cm -1 , greater than about 5000M -1 cm -1 , greater than about 10,000M -1 cm -1 , or any value or range within these ranges or values, or any value or range bounded by these ranges or values, although values ​​outside these values ​​or ranges may be used in some cases.

[0307] In some embodiments, the photocatalyst 112 of the catalyst complex 102 is an organic catalyst. In some embodiments, such an organic catalyst may not include a light-responsive transition metal. As illustrative examples of these 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 marking agent 216 according to the present disclosure may be selected.

[0308] The target protein 108 may be a suitable intracellular molecule. For example, the target protein 108 may be involved in a signal transduction pathway, a subcellular and / or cellular structure, a local biomolecular network and / or a microenvironment. In some embodiments, the target protein 108 may be encoded by a nucleotide construct introduced into a cell and expressed by the cell. In other embodiments, the target protein 108 may penetrate into the cell. In another embodiment, the target protein 108 may not penetrate into the cell, but may be introduced into the cell by some suitable method, such as by deposition via liposomes. In various embodiments, the target protein may be involved in a disease state. As an illustrative example, proteins associated with cancer include K-Ras, cMyc, Src, WRN, Slug, and PARP1. As an illustrative example, proteins associated with neurodegenerative lesions include Aβ and Tau. As an illustrative example, proteins associated with viral infection include influenza hemagglutinin and viral nucleoprotein. In some embodiments, the target protein 108 is a ubiquitin ligase (e.g., an E3 ligase).

[0309] The binding agent 110 can be any suitable molecule that has a specific binding to the ligand portion 114. In some embodiments, the binding agent 110 can include a haloalkane dehalogenase. In certain embodiments, the binding agent 110 can be (Promega) purification system is developed as an engineered halogenated dehalogenase protein, which can be naturally produced in cells. The HaloTag system can be used for many self-labeling processes in chemical biology. As an illustrative example, the HaloTag system can be used for protein purification and the incorporation of TAMRA-alkyl chloride dyes for monitoring intracellular signal transduction. Because halogenated dehalogenases are non-mammalian, through the dehalogenase mechanism, theoretically the background incorporation of photoresponsive catalysts should be low, substantially absent or absent. Therefore, the HaloTag system can be used as a useful carrier to incorporate photoresponsive catalysts into the natural cell environment for identifying the microenvironmental interaction group of the target protein. The HaloTag system can therefore be used to monitor transient interactions in the intracellular environment.

[0310] In some embodiments, a nucleotide construct encoding a fusion of a protein of interest 108 and a binding agent 110 (i.e., a binding agent complex 104) can be introduced into a cell. The cell can express the binding agent complex 104 encoded by the construct. In some embodiments, the construct can encode a binding agent complex 104 (which is a fusion protein) in which the binding agent 110 is linked to the C-terminus of the protein of interest 108. In some embodiments, the construct can encode a binding agent complex 104 (which is a fusion protein) in which the binding agent 110 is linked to the N-terminus of the protein of interest 108. In some embodiments, the construct can encode a binding agent complex 104 in which the binding agent 110 is linked to the protein of interest 108 at a site on the binding agent 110 that does not disrupt, does not substantially disrupt, or minimally disrupts certain interactions of the protein of interest 108 with the biomolecule 208.

[0311] Figure 2 Yes Figure 1 1 is a diagram of a proximity-based labeling scheme for a biomolecule antenna 106. Additionally included is a labeling agent 216 that includes an active moiety 204 and a labeling moiety 206. A light source 214 can generate photons 210 that activate the catalyst complex 102. Thus, the catalyst complex 102 can activate the labeling agent 216 via Dexter energy transfer 212, activating the active moiety 204. When in an activated state, the labeling agent 216 is capable of binding the labeling moiety 206 to a nearby biomolecule 208.

[0312] According to the present disclosure, a labeling agent 216 can be activated by Dexter energy transfer 212. Although probes based on diazirine and azide have been widely used in small molecule target recognition, they need to be directly excited with ultraviolet light, thereby excluding the possibility of target localization activation. It is known that these types of active parts have the ability to receive triplet energy by Dexter energy transfer. Attaching a photocatalyst (e.g., a photocatalyst 112 of a catalyst complex 102) to a biomolecule produced by a cell (e.g., a target protein 108) allows it to act as an antenna, absorbing the photon energy of light, such as visible light. The photocatalyst 112 is activated by absorbing light. The activated photocatalyst 112 can then activate a labeling agent 216 adjacent to the target protein 108 by Dexter energy transfer 212. The activated labeling agent 216 can thus covalently connect itself to a molecular structure (including but not limited to proteins, chromatin and nucleic acids) adjacent to the binding agent complex 104 by the activated active part 204. The distance at which the activated labeling agent 216 can attach to the labeling moiety 206 depends on the diffusivity of the labeling agent 216 and the activation half-life t of the active moiety 204. 1 / 2For example, the activation half-life of the active portion 204 is t 1 / 2 The longer the marker 216 is, the longer it can travel before attaching to the biomolecule 208. Similarly, the more diffusive the marker 216 is, the greater the distance the marker 216 will be able to travel in an activated state before quenching or binding to the biomolecule 208.

[0313] In embodiments where the labeling agent 216 comprises an active moiety 204 attached to a labeling moiety 206, attachment of the active moiety 204 to a biomolecule 208 also results in transfer of the labeling moiety 206 to the biomolecule 208 (including but not limited to proteins, chromatin, and nucleic acids). Placing a label on a 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 nature, characteristics, and spatiotemporal aspects of a microenvironment. In some embodiments, the active moiety 204 is cell permeable, with or without an attached labeling moiety 206. In some embodiments, the labeling agent 216 is cell permeable.

[0314] In some embodiments, the labeling agent 216 has the structure shown in Formula III:

[0315]

[0316] Where R 1 is a substituent that can accept triplet energy from the activated photocatalyst. For example, R 1 It may be azide, methyldiaziridine, trifluoromethyldiaziridine or phenyldiaziridine;

[0317] R1 is independently located at position a, b or c on the aromatic ring;

[0318] The aromatic ring may be phenyl, pyridyl, pyrimidyl, pyrazinyl or pyridizynyl. The aromatic ring may be naphthyl or quinoline, and may include various additional linkages.

[0319] The aromatic ring may be substituted with electron-withdrawing or electron-donating groups to reduce the reactivity of the labeled substance. As illustrative examples, the substituents at positions a, b, or c may be -OH, -OMe, -OEt, -OCF3, -OCF2H, -NHMe, -NMe2, -F, -Cl, -Br, -Me, -Et;

[0320] X can be O, NH, NR 2 、CH2NHCO、CONH、CONR 2 、SO2NH、SO2NR 2 ;

[0321] Further, R 2 It can be H, OMe, Me, Et;

[0322] n is an integer representing the number of PEG units; for example, n can be 0, 1, 2, 3, 4, 5, or 6, although other values ​​of n may be used in some cases;

[0323] Y can be a biotin-linked amide, or an amide derivatized via linkage to a fluorescent dye.

[0324] In some embodiments, the labeling agent 216 has the structure of formula (III-a):

[0325]

[0326] in

[0327] R1' is selected from azide, methyl diazirine, trifluoromethyl diazirine and phenyl diazirine;

[0328] Ring Ar is selected from phenyl, pyridyl, pyrimidinyl, pyrazinyl, pyridizynyl, naphthyl and quinolinyl, optionally substituted with one or more -OH, -OMe, -OEt, -OCF3, -OCF2H, -NHMe, -NMe2, -F, -Cl, -Br, -Me or -Et;

[0329] X is selected from O, NH, NR 2 、CH2NHCO、CONH、CONR 2 , SO2NH and SO2NR 2 ;

[0330] R 2 is selected from H, OMe, Me and Et;

[0331] n is 0, 1, 2, 3, 4, 5 or 6; and

[0332] Y is a biotin-linked amide or an amide-linked fluorescent dye.

[0333] The active moiety 204 may include any chemical species operable to interact with the photocatalyst 112 to form an active intermediate (also referred to herein as an activated labeling agent) for coupling with a biomolecule. As an illustrative example, in an embodiment where the photocatalyst 112 includes a photocatalytic transition metal, the active moiety 204 may be an aryl azide or an alkyl azide. In such an embodiment, triplet energy transfer from the excited state photocatalyst 112 may promote the azide to reach its triplet state (T1). The azide triplet state undergoes N2 elimination to release a free triplet nitryl radical, which undergoes spin equilibrium in a picosecond time span to reach its active singlet state (with t 1 / 2 <1 ns half-life), resulting in cross-linking to nearby biomolecules 208 or being quenched by the aqueous environment.

[0334] In certain embodiments, the labeling agent 216 comprises a structure shown in any one of Formulas III-IX:

[0335]

[0336] Azide sensitization can be extended to various substitutions on the ring with attachment of valuable payloads (e.g., labels) for microscopy and proteomics applications. Such payloads or labels include free carboxylic acids, phenols, amines, alkynes, carbohydrates, and biotin groups.

[0337] The extinction coefficient of the photocatalyst 112 can be several orders of magnitude greater than the extinction coefficient of azide at wavelengths such as blue light (e.g., light having a wavelength of about 450 nm) emitted for sensitization, ensuring that there is no, substantially no, or minimal background non-catalytic reaction of the active moiety 204. In other embodiments where the active moiety 204 can be activated to singlet or triplet energy in the absence of a photocatalyst, it may be desirable that the labeling rate in the presence of a protein photocatalyst is faster than the labeling rate of the background reaction.

[0338] In some embodiments, an active intermediate (also referred to herein as an activated labeling agent) is formed by the interaction of the active moiety 204 and the photocatalyst 112. The interaction may be an energy transfer, such as a Dexter energy transfer. 1 / 2Less than 50ns, less than 10ns, less than 5ns, less than 2ns or less than 1ns, or any value or range within or bounded by these ranges or values, although values ​​outside these values ​​or ranges may be used in some cases. In some embodiments, the reactive intermediate has a half-life and diffusion constant such that the diffusion of the reactive intermediate is 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 within or bounded by these ranges or values, although values ​​outside these values ​​or ranges may be used in some cases. The above mechanistic principles can be applied to proximity labeling of various types of biomolecules, including but not limited to proteins, polysaccharides, and / or nucleic acids.

[0339] In some embodiments, the active portion 204 can be functionalized with a labeling portion 206. As discussed, the labeling of the biomolecule 208 can facilitate the use of orthogonal techniques. For example, in some embodiments, the labeling portion 206 can include a label that can act as a ligand for labeling proteins. In a subset of these embodiments, the labeling portion 206 can include biotin, which can bind to a labeled protein, such as avidin. As an illustrative example, such a labeled protein can be used in an immunoprecipitation assay to separate the biomolecule 208 to which the labeling portion 206 is attached. As another illustrative example, the labeled protein can include a fluorophore so that the labeled protein can be imaged. In some embodiments, the labeling portion 206 itself can include a fluorophore so that it can be imaged without adding a labeled protein.

[0340] Proximity-based labeling

[0341] In one aspect, the present disclosure provides a method for proximity-based labeling of intracellular molecules, comprising: introducing a binding agent complex, wherein the binding agent complex comprises a target protein coupled to a binding agent, wherein the binding agent is capable of binding to a catalyst complex; introducing the catalyst complex into the cell; forming a biomolecular antenna comprising the catalyst complex and the binding agent complex; introducing a labeling agent comprising a labeling portion and an active portion into the cell, wherein the active portion is coupled to the labeling portion; activating the catalyst complex, thereby activating the labeling agent by transferring energy from the catalyst complex to the active portion, and causing the labeling agent to bind to the biomolecules in the cell.

[0342] Figure 3 A flow chart of a method according to the present disclosure is shown. According to box 302, a nucleotide construct can be optionally introduced into a cell. In some embodiments, the nucleotide construct can be introduced by transformation and / or transfection. The nucleotides can encode a binding agent complex 104 comprising a protein of interest 108 and a binding agent 110. The construct can be configured so that the portion encoding the binding agent complex 104 is expressible by the cell (e.g., the encoding portion can be in an open reading frame).

[0343] Then, according to box 304, the binding agent complex 104 can be introduced into the cell. In embodiments where the nucleotide construct is introduced into the cell according to box 302, the binding agent complex 104 can be introduced by the cell expressing the nucleotide construct. This production of the binding agent complex 104 can be intracellular, using the cell's own transcription and / or translation processes to produce the binding agent complex 104 based on the sequence found in the nucleotide construct. In other embodiments, the binding agent complex 104 can be introduced into the cell by another suitable method. As an illustrative example, the binding agent complex 104 can be introduced into the cell by liposomes.

[0344] According to block 316, according to this embodiment, the catalyst complex 102 is introduced into the cell. As disclosed herein, such a catalyst complex 102 can be cell permeable. Alternatively, another suitable method of introducing the catalyst complex 102 into the cell. As a non-limiting example, the catalyst complex 102 can be introduced via a liposome carrier.

[0345] According to block 318, a labeling agent 216 may be introduced, as shown at block 318. As disclosed herein, such a labeling agent 216 may be cell permeable, although it may be introduced into the cell by any suitable method.

[0346] According to block 308, the catalyst complex 102 can form a biomolecular antenna 106 with the binding agent complex 104. For example, the ligand portion 114 of the catalyst complex 102 can bind to the binding agent 110 of the binding agent complex 104. In some embodiments, the alkyl chloride of the ligand portion 114 can bind to the HaloTag of the binding agent 110.

[0347] After the catalyst complex is formed, the catalyst complex may be activated according to block 310. In embodiments where the catalyst complex 102 comprises a photocatalyst 112, activation may include irradiation with light of a known wavelength to activate the particular photocatalyst 112. In some embodiments, the activation may be performed on living cells.

[0348] According to block 310, activation of the catalyst complex 102 can in turn activate the marking agent 204. In some embodiments, the photocatalyst 112 can activate the marking agent 204 via Dexter energy transfer 212.

[0349] According to block 312, when activated, the labeling agent 216 is capable of binding the labeling moiety 206 to a nearby biomolecule 208. It is possible that a given activated labeling agent 216 is quenched before it is able to bind a nearby biomolecule 208.

[0350] After the biomolecule is labeled, the label may be optionally detected according to box 314. In some embodiments where the label is detectable by, for example, a fluorescence microscope, the label may be imaged. Such imaging may indicate the location of the biomolecule 208, even if the interaction between the biomolecule 208 and the target protein 108 is relatively short, and / or the biomolecule 208 and the target protein 108 are not co-localized when imaged. In some embodiments, the label may not be fluorescent, but may be the target of a second label capable of fluorescence. In some embodiments, the label may be the target of a subsequent immunoprecipitation assay. Such an immunoprecipitation assay may be an immunoprecipitation of a labeled protein or a pull-down assay based on a label. Once separated, the labeled target protein may be subjected to other orthogonal analyses. For example, the separated labeled target protein may be quantitatively or semi-quantitatively analyzed by a protein imprint, or the separated substance may be quantitatively and identified by proteomics analysis.

[0351] In some embodiments, the above process can be used to detect protein-protein interactions. For example, in some embodiments, the binding agent complex comprises a first protein coupled to a binding agent, wherein the first protein is a target protein, and it is desirable to detect the protein-protein interaction of the target protein. In some embodiments, the first protein is a ubiquitin ligase, and the binding agent is a haloalkane dehalogenase, so that the binding agent complex is a fusion protein comprising a ubiquitin ligase and a haloalkane dehalogenase. The catalyst complex can then be introduced into the cell and combined with the haloalkane dehalogenase.

[0352] In some embodiments, upon activation of the catalyst complex and subsequently activation of the labeling agent, the labeling agent binds to the second protein in close proximity to the first protein. Since the second protein is labeled only when it is in close proximity to the first protein, based on this, detection of the second protein by detecting the labeled portion on the labeling agent indicates a protein-protein interaction between the first and second proteins.

[0353] In some embodiments, the method of detecting protein-protein interactions can 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 can be used to identify weak protein-protein interactions, which can then be used as therapeutic targets by screening for compounds that increase protein-protein interactions. For example, in some embodiments, the first protein is a ubiquitin ligase, and the use of a ubiquitin ligase to identify weak protein-protein interactions indicates that the interaction is an interaction that can be developed by discovering compounds that enhance the interaction (e.g., PROTACs or molecular glues).

[0354] In some embodiments, the method includes introducing the test compound into the cell to determine whether the test compound enhances protein-protein interaction. In some such embodiments, the level of the second protein of the marker labeling is measured to determine the amount of the protein-protein interaction enhanced by the test compound. In other embodiments, the test compound is evaluated by a similar method to determine whether it inhibits protein-protein interaction.

[0355] Methods for verifying protein association

[0356] Just as the methods disclosed herein can be used to identify biomolecules that enter the vicinity of a binding agent-labeled protein of interest, the methods can be used to identify the reverse situation: whether the protein of interest enters the vicinity of a binding agent-labeled biomolecule. This method can be used to verify whether the protein of interest enters the vicinity of a biomolecule. Thus, for example, the method can be used to verify whether a target disease-causing protein associates with a ubiquitin ligase by forming a complex between the binding agent and the pathogenic protein.

[0357] In one aspect, the present disclosure provides a method for verifying the connection between a pathogenic protein and a ubiquitin ligase. For example, other screening methods may have shown that there is a connection, and it is desirable to verify the interaction before screening for agents that enhance the interaction (e.g., PROTAC or molecular glue). The method includes introducing a first binding agent complex into a cell, wherein the first binding agent complex includes a pathogenic target protein coupled to a first binding agent, wherein the first binding agent is capable of binding to a first catalyst complex; introducing the first catalyst complex into the cell; forming a first biomolecule antenna comprising the first catalyst complex and the binding agent complex; introducing a first labeling agent comprising a first labeling portion and a first active portion into the cell, wherein the first active portion is coupled to the first labeling portion; activating the first catalyst complex, thereby activating the first labeling agent by transferring energy from the first catalyst complex to the first active portion, and binding the first labeling agent to the ubiquitin ligase in the cell. In some embodiments, the method further includes confirming that the ubiquitin ligase has been labeled by the labeling agent by immunoprecipitation. In some embodiments, the labeled protein from the cell lysate is first collected using functionalized beads bound to a labeling agent (e.g., streptavidin functionalized beads if the labeling agent comprises a biotin moiety). The presence of the desired ubiquitin ligase can then be determined using Western blotting and an appropriate antibody to the ligase.

[0358] Cells and proteins

[0359] Some embodiments include fusion proteins comprising a target protein and a haloalkane dehalogenase. In some embodiments, the target protein 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 include a protein complex comprising this structure:

[0360] P1-P2-Cat

[0361] in:

[0362] P1 is a ubiquitin ligase;

[0363] P2 is a haloalkane dehalogenase;

[0364] Cat is a photocatalyst having the structure of formula (I):

[0365]

[0366] in:

[0367] A1 is present 0 to 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;

[0368] Each R1 is independently selected from H, a straight or branched chain alkyl group having 1 to 12 carbons, CHF2, and CF3;

[0369] A2 is present 0 to 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;

[0370] A3 is present 0 to 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;

[0371] A4 is selected from none, a straight or branched alkyl group having 1 to 12 carbon atoms, 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, halogen, hydroxyl, C 1-6 Alkoxy or amino substitution;

[0372] A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1-6 Alkoxy, and

[0373] A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a l (CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, wherein each a1 is independently an integer from 0 to 10, and each a2 is independently an integer from 6 to 10;

[0374] A9 is a D9 metal selected from the group consisting of 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;

[0375] A11 and A12 are independently C or N coordinated to A9; and

[0376] Each A13 is independently CH or N.

[0377] In some embodiments, the protein complex is present in a cell. In some embodiments, the protein complex is formed by expressing a fusion protein in a cell and then exposing the fusion protein to a photocatalyst. Therefore, some embodiments also provide a cell comprising a nucleotide sequence expressing a fusion protein comprising a ubiquitin ligase and a haloalkane dehalogenase.

[0378] 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 cell further comprises a labeling agent as described above (e.g., a labeling agent according to formula (III) or (III-a)).

[0379] the term

[0380] As used herein, "biomolecular antenna" refers to a molecular structure formed by the association of a catalyst complex and a binding agent complex.

[0381] As used herein, "probe" or "active moiety" refers to a molecule that is capable of (1) coupling to a label, (2) being activated by a photocatalyst via Dexter energy transfer, and (3) binding to various molecules in the activated state.

[0382] As used herein, "binding agent" refers to a molecule that (1) can be linked to a protein of interest or expressed as part of a fusion protein with a protein of interest, and (2) can bind to the ligand portion of a catalyst complex.

[0383] "Room temperature" is abbreviated herein as "RT".

[0384] As used herein, "PEG" refers to a moiety having the structure:

[0385] As used herein, "alkyl" refers to a fully saturated straight or branched chain hydrocarbon (i.e., without double or triple bonds). An alkyl group can have 1 to 20 carbon atoms (whenever it appears in this article, a numerical range such as "1 to 20" refers to every integer in the given range; for example, "1 to 20 carbon atoms" means that the alkyl group can be composed of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although this definition also covers those terms "alkyl" that do not specify a numerical range). The alkyl group can also be a medium-sized alkyl group having 1 to 9 carbon atoms. The alkyl group can also be a low-sized alkyl group having 1 to 4 carbon atoms. The alkyl group can be named "C 1-4 Alkyl" or similar designation. For example only, "C 1-4"Alkyl" means that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Common alkyl groups include, but are by no means limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like.

[0386] As used herein, "heterocyclyl" or "heterocycle" refers to a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. The heterocyclyl group can be connected together in a fused, bridged or spirally connected manner. The heterocyclyl group can have any degree of saturation, as long as at least one ring in the ring system is not aromatic. The heteroatom can be present in the non-aromatic or aromatic ring in the ring system. The heterocyclyl group can have 3 to 20 ring members (i.e., the number of atoms constituting the ring backbone, including carbon atoms and heteroatoms), although this definition also covers those terms "heterocyclyl" that do not specify a numerical range. The heterocyclyl group can also be a medium-sized heterocyclyl with 3 to 10 ring atoms. The heterocyclyl group can also be a heterocyclyl with 3 to 6 ring atoms. The heterocyclyl group can be named "3 to 6-membered heterocyclyl" or similar names. In preferred six-membered monocyclic heterocyclyls, the heteroatoms are selected from one to three of O, N or S, and in preferred five-membered monocyclic heterocyclyls, the heteroatoms are selected from one or two heteroatoms of O, N or S. Examples of heterocyclyl rings include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxiranyl, thiepanyl, piperidinyl, piperazinyl, dioxypiperazinyl, pyrrolidinyl, pyrrolidonyl, prrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,4-dioxanyl, 1,3-dioxathiinyl, 1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxane, hexahydro-1,3,5-triazine, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl, 1,3-dithiolyl 1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinone, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiomorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline.

[0387] 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, each ring in the system is aromatic. The heteroaryl group can have 5 to 18 ring atoms (i.e., the number of atoms constituting the ring backbone, including carbon atoms and heteroatoms), although this definition also encompasses those terms "heteroaryl" in which no numerical range is specified. In some embodiments, the heteroaryl group has 5 to 10 ring atoms or 5 to 7 ring atoms. The heteroaryl group can be designated as "5 to 7-membered heteroaryl", "5 to 10-membered heteroaryl" or similar names. Examples of heteroaryl rings include, but are not limited to, furanyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolyl, isoquinolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothiophenyl.

[0388] As used herein, "aryl" refers to an aromatic ring or ring system containing only carbon in the ring backbone (i.e., two or more fused rings that share two adjacent carbon atoms). When aryl is a ring system, each ring in the system is aromatic. Aryl groups can have 6 to 18 carbon atoms, although this definition also encompasses those where no numerical range is specified for the term "aryl". In some embodiments, aryl groups have 6 to 10 carbon atoms. Aryl groups can be designated as "C 6-10 Aryl", "C6 or C 10 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.

[0389] As used herein, "cycloalkyl" refers to a fully saturated carbocyclic ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0390] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. The use of the term "including" and other forms, such as "include", "includes" and "included", is not limiting. The use of the term "having" and other forms, such as "have", "has" and "had", is not limiting. The terms "comprising", "including", "having", etc. are synonymous and are used inclusively in an open manner and do not exclude other elements, features, behaviors, operations, etc. That is, the above terms are interpreted synonymously with the phrase "having at least" or "including at least". For example, when used in the context of a process, the term "comprising" means that the process includes at least the steps listed, but may 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. Furthermore, the term "or" is used in its inclusive sense (and not in its exclusive sense), so that when used, for example, to link 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 its ordinary meaning, may refer to any subset of a group of elements to which the term "each" applies.

[0391] Language of degree as used herein, such as the terms "approximately," "about," "generally," and "substantially" as used herein, means a value, amount, or feature that is close to a stated value, amount, or feature and still performs a desired function or achieves a desired result. For example, the terms "approximately," "about," "generally," and "substantially" may refer to an amount within 10%, within 5%, within 1%, within 0.1%, and within 0.01% of a stated amount.

[0392] As used herein, the term "and / or" has its broadest least restrictive meaning, which means that the disclosure includes A alone, B alone, A and B together, or either A or B, but does not require both A and B or does not require one of A or one of B. As used herein, the phrase at least one of A, B, and C should be construed to mean a logical A or B or C, using a non-exclusive logical or statement.

[0393] Conditional language used herein, for example, including, “can,” “could,” “might,” “may,” “eg,” and the like, unless specifically stated otherwise or otherwise understood in the context of use, is generally intended to convey that certain features, elements, and / or steps are optional. Thus, such conditional language generally does not mean that features, elements, and / or steps are required in any way, or that one or more implementations necessarily include logic for determining whether to include such features, elements, and / or steps or whether to always perform such features, elements, and / or steps, regardless of whether there are other inputs or prompts. The terms “comprising,” “including,” “having,” and the like are synonymous and are used inclusively in an open manner and do not exclude other elements, features, behaviors, operations, and the like. In addition, the term “or” is used in its inclusive sense (and not in its exclusive sense), so when used, for example, to connect a list of elements, the term “or” refers to one, some, or all of the elements in the list.

[0394] Any method disclosed herein does not require execution in the order listed.The methods disclosed herein include certain actions taken by a practitioner; however, they may also include any third-party instructions for these actions, whether explicit or implicit. Example

[0395] Additional embodiments are disclosed in further detail in the following examples, which do not in any way limit the scope of the claims.The following describes cellular labeling of K-Ras interacting partners by production of halo.KRAS in the SW48 cell line obtained from Horizon.

[0396] Example 1. Preparation of labeling partner

[0397] The preparation of the labeling partner is described in Scheme 1:

[0398] Solution 1

[0399]

[0400] At 0 ℃, by biotin-PEG3-NH2(123mg, 0.2944mmol), HATU(280mg, 0.7361mmol) and DIPEA(0.2mL, 1.1042mmol) are added in the DMF(5mL) stirring solution of compound 1(60mg, 0.368mmol).Reaction mixture is stirred at room temperature for 16 hours.The progress of reaction is monitored by thin layer chromatography (TLC) and liquid chromatography-mass spectrometry (LC-MS).After the reaction is completed, reaction mixture is quenched with cold water (10mL), and extracted with ethyl acetate (2x 10mL).The organic layer merged is washed with saturated NaHCO3(10mL) and saline solution (10ml).Organic layer is dried over sodium sulfate, then filtered and concentrated under reduced pressure, to obtain 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-azaheptadecanyl)benzamide (45 mg, 21% yield) as an off-white solid.

[0401] Example 2. Preparation of Photocatalyst 1

[0402] The description of the preparation of the photocatalyst refers to the description of Scheme 2:

[0403] Solution 2

[0404]

[0405]

[0406] For step 1, LDA (2M in THF, 32.56mL, 65.13mmol, 1.2 equivalents) is added dropwise to a stirred solution of compound 2 (10g, 54.277mmol, 1.0 equivalents) in anhydrous THF (300mL), and stirred for 1.5h at -78 ° C. In another round-bottomed flask (RBF), 2-bromoacetic acid methyl esters (12.5g, 81.415mmol, 1.5 equivalents) are dissolved in THF (100mL) and cooled to -78 ° C. Then 2-bromoacetic acid methyl ester solution is slowly added at -78 ° C, and stirred for 16h at room temperature. The progress of the reaction is monitored by LC-MS&TLC (30% ethyl acetate in hexane). After the reaction is completed, saturated NaHCO solution (150mL) is used to quench the reaction mixture, and extracted with ethyl acetate (2x 300mL). Organic layer is dried with Na2SO4, and evaporated under reduced pressure to obtain crude compound. The crude compound was then purified by silica gel (230 to 400 mesh) column chromatography eluting with 0 to 2% methanol in DCM to afford compound 3 (7 g, 50% yield) as a light brown solid.

[0407] For step 2, 0 to 10 ℃ of 2M NaOH (1.64g, 40.966mmol, 1.5 equivalents) solution was added to a stirred solution of compound 3 (7g, 27.31mmol, 1.0 equivalents) in a mixture of methanol (70mL) and THF (21mL), and stirred at room temperature for 2 hours. The progress of the reaction was monitored by LC-MS and TLC (10% methanol in DCM). After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and diluted with water (40mL), then 10% citric acid solution (30mL) was used to adjust pH to 3-4. Filter the solid, dry under vacuum, and obtain compound 4 (5g, yield 75.7%) of a light brown solid.

[0408] For step 3, at room temperature trimethylamine (1.7mL, 12.396mmol, 1.5 equivalents) and N, N'-disuccinimidyl carbonate are added to a stirred solution of compound 4 (2g, 8.264mmol, 1 equivalent) in dichloromethane (20mL), and stirred for 16 hours at RT. The progress of the reaction is monitored by LC-MS and TLC (5% methanol in DCM). After the reaction is complete, the reaction mixture is diluted with water (20mL), and extracted with dichloromethane (2x 20mL). Organic layer is dried with Na2SO4, and evaporated under reduced pressure to obtain a crude compound. By being purified by silica gel column chromatography crude compound, using 0 to 5% methanol in DCM as mobile phase elution, a light brown liquid compound 5 (1.2g, yield 43%) is obtained.

[0409] For step 4, in clean and dry RBF, add IrCl aqueous solution (600mg, 2.009mmol, 1 equivalent) and compound 6 (1.145g, 4.421mmol, 2.2 equivalents), and evacuate and use N ventilate three times.Then add the degassed solution of 2-ethoxyethanol (24mL) and water (8.4mL), and be heated to 120 ℃ 16 hours. Monitor the progress of reaction by LC-MS. After reaction is completed, reaction mixture is cooled to RT, and filter yellow precipitate, wash with water (120mL) and hexane (50mL), and dry under vacuum, obtain compound 7 (605mg, productive rate 33%) of yellow solid.

[0410] For step 5, AgPF6 (143mg, 0.564mmol, 2.1 equivalents) was added to a stirred solution of compound 8 (400mg, 0.268mmol, 1 equivalent) in acetonitrile (30mL) at RT and stirred for 16 hours. The progress of the reaction was monitored by LC-MS. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to obtain a crude compound. Subsequently, the crude compound was purified by slurrying with diethyl ether (20mL) to obtain a light yellow solid compound 9 (350mg, 55.7% yield).

[0411] For step 6, the stirring solution of compound 5 (130mg, 0.384mmol, 1.2 equivalents) in dichloromethane is degassed with argon for 5 minutes.Then a solution of compound 9 in dichloromethane (18mL) is added and stirred for 16 hours at RT. The progress of the reaction is monitored by LC-MS and TLC (10% methanol in DCM). After the reaction is completed, the reaction mixture is concentrated under reduced pressure to obtain a crude compound. By slurrying the crude compound with diethyl ether (10mL), a yellow solid compound Int-8 (320mg, 83% yield) is obtained.

[0412] For 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-tetrahydrooctadecane-1-amine hydrochloride (compound Int-6) in DMF and stirred for 16 hours at RT. The progress of the reaction was monitored by LC-MS and TLC. After the reaction was complete, the reaction mixture was diluted with water (10 mL) and extracted with EtOAc (2x10 mL). The organic layer was dried with Na2SO4 and evaporated under reduced pressure to obtain a crude compound. The crude compound was purified by flash column chromatography (FCC) using 5% MeOH in DCM, and the desired fraction was concentrated under reduced pressure to obtain a light yellow solid photocatalyst 1 (50 mg, 49% yield). Photocatalyst 1 is an exemplary photocatalyst according to the present disclosure.

[0413] The description of the preparation of compound Int-6 refers to the description of Scheme 3:

[0414] Solution 3

[0415]

[0416] For reactions step 8, sodium hydride (1.308g, 32.724mmol, 1.2 equivalents) is added to a stirred solution of compound 11 (8g, 27.27mmol, 1 equivalent) in THF (56mL) at 0°C, and stirred for 30 minutes. Then, slowly add compound 12 (8.06g, 32.724mmol, 1.2 equivalents) at 0 to 5°C, and stir at room temperature for 16 hours. Monitor reaction progress by LC-MS and TLC (petroleum ether of 70% ethyl acetate). After the reaction is complete, quench the reaction mixture with saturated ammonium chloride solution (80mL), and extract with ethyl acetate (2x 100mL). Wash the organic layer with water (100mL), use Na2SO4 dry, and evaporate under reduced pressure to obtain crude compound, which is purified by silica gel column chromatography, eluted with the petroleum ether of 0-50% ethyl acetate as mobile phase, and obtain compound 13 (3.7g, yield 33%) of light brown liquid.

[0417] For step 9, 4M HCl in dioxane (19 mL) was added to a stirred solution of compound 13 (3.7 g, 12.612 mmol) in dichloromethane (37 mL) at 0 to 5 ° C and stirred for 5 h at RT. The reaction progress was monitored by LC-MS and TLC (70% ethyl acetate in petroleum ether). After the reaction was completed, the reaction mixture was concentrated under reduced pressure to give compound Int-6 (3 g, yield 96%), which was used for step 7 of scheme 2 without further purification.

[0418] Example 3. Preparation of Photocatalyst 1D

[0419] The description of the preparation of the photocatalyst refers to the description of Scheme 4:

[0420] Solution 4

[0421]

[0422] For step 1, the stirring solution of compound 1 (130mg, 0.384mmol, 1.2 equivalents) in dichloromethane (45mL) is degassed with argon for 5 minutes. Then, a solution of compound 2 in dichloromethane (18mL) is added and stirred for 16 hours at RT. The progress of the reaction is monitored by LC-MS and TLC (10% methanol in DCM). After the reaction terminates, the reaction mixture is concentrated under reduced pressure to obtain a crude compound, which is purified with diethyl ether (10mL) to further obtain compound Int-8 (320mg, 83% yield) as a yellow solid.

[0423] For step 2, DIPEA (0.3 mL, 0.159 mmol, 2 eq) was added to a solution of compound Int-3 (25 mg, 0.0796 mmol, 1 eq) in DMF (1.5 mL) at RT and stirred for 10 minutes. Compound Int-8 (95 mg, 0.0796 mmol, 1 eq) was then added and stirred for 16 hours at RT. The progress of the reaction was monitored by LC-MS and TLC (10% methanol in DCM). After the reaction was completed, the reaction mixture was concentrated under reduced pressure to give a crude compound, which was purified by silica gel (230 to 400 mesh) column chromatography, eluting with 0 to 5% methanol in DCM as the mobile phase to give a photocatalyst 1D (1.25 g, 63% yield) as a yellow solid. Photocatalyst 1D is an exemplary photocatalyst according to the present disclosure.

[0424] The description of the preparation of compound Int-3 refers to the description of Scheme 5:

[0425] Solution 5

[0426]

[0427] Sodium hydride (302mg, 8.181mmol, 1.2 equivalents) is added to a stirred solution of compound 4 (2g, 6.817mmol, 1 equivalent) in a mixture of DMF (10mL) and THF (10mL) at 0°C, and stirred for 30 minutes at RT. Then slowly add compound 5 (1.73g, 8.18mmol, 1.2 equivalents) at 0 to 5°C, and stir for 16 hours at RT. Monitor the progress of the reaction by LC-MS and TLC (petroleum ether of 70% ethyl acetate). After the reaction is completed, the reaction mixture is quenched with saturated ammonium chloride solution (20mL), and extracted with ethyl acetate (2x 20mL). The organic layer is washed with water (30mL), dried with Na2SO4, and evaporated under reduced pressure to obtain a crude compound, which is purified by silica gel column chromatography, eluted with petroleum ether of 0 to 50% ethyl acetate as mobile phase, and further obtains compound 6 (700mg, yield 28%) of light yellow viscous liquid shape.

[0428] Example 4. Preparation of Photocatalysts 2, 3 and 4

[0429] The description of the preparation of the photocatalyst refers to the description of Scheme 6:

[0430] Solution 6

[0431]

[0432] According to Scheme 6, DIPEA (0.1 mL, 0.251 mmol) and compound Int-8 (reference 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 stirred at RT for 16 hours. The reaction progress 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 x 10 mL). The organic layer was dried over Na2SO4 and evaporated under reduced pressure to give a crude compound. The crude compound was purified by FCC using 5% MeOH in DCM, and the desired fraction was concentrated under reduced pressure to give photocatalyst 2 (50 mg, 51% yield) as a light yellow solid. Photocatalyst 2 is an exemplary photocatalyst according to the present disclosure.

[0433] The description of the preparation of the photocatalyst refers to the description of Scheme 7:

[0434] Solution 7

[0435]

[0436] According to Scheme 7, DIPEA (0.1 mL, 0.251 mmol) and compound Int-8 (reference example 2) (100 mg, 0.083 mmol) were added to a stirred solution of 24-chloro-3,6,9,12,15,18-hexaoxa tetracosane-1-amine hydrochloride (20 mg, 1.30 mmol) in DMF (10 mL) and stirred for 16 hours at RT. The reaction progress was monitored by TLC and LC-MS. After the reaction was complete, the reaction mixture was diluted with water (10 mL) and extracted with EtOAc (2 x 10 mL). The organic layer was dried with Na2SO4 and evaporated under reduced pressure to obtain a crude compound. The crude compound was purified by FCC using 4 to 5% MeOH in DCM, and the desired fraction was concentrated under reduced pressure to obtain a photocatalyst 3 (38 mg, 40% yield) as an off-white solid. Photocatalyst 3 is an exemplary photocatalyst according to the present disclosure.

[0437] The description of the preparation of the photocatalyst refers to the description of Scheme 8:

[0438] Solution 8

[0439]

[0440] According to Scheme 8, DIPEA (0.1 mL, 0.251 mmol) and compound Int-8 (reference example 2) (100 mg, 0.083 mmol) were added to a stirred solution of 30-chloro-3,6,9,12,15,18,21,24-octaoxatriacontane-1-amine hydrochloride (100 mg, 0.087 mmol) in DMF (10 mL) and stirred for 16 hours at RT. The reaction progress was monitored by TLC and LC-MS. After the reaction was complete, the reaction mixture was diluted with water (10 mL) and extracted with EtOAc (2 x 10 mL). The organic layer was dried with Na2SO4 and evaporated under reduced pressure to obtain a crude compound. The crude compound was purified by FCC using 5% MeOH in DCM, and the desired fraction was concentrated under reduced pressure to obtain a photocatalyst 4 (29 mg, 40% yield) in the form of an off-white solid. Photocatalyst 4 is an exemplary photocatalyst according to the present disclosure.

[0441] Example 5. Preparation of Photocatalyst 5

[0442] The description of the preparation of the photocatalyst refers to the description of Scheme 9:

[0443] Solution 9

[0444]

[0445]

[0446] With reference to scheme 9, step 1, CBr4 (6.349 g, 19.146 mmol, 1.2 equivalents) is added to a stirred solution of (2-bromopyridin-4-yl)methanol 1 (3.0 g, 15.95 mmol, 1.0 equivalents) and triphenylphosphine (5.022 g, 19.14 mmol, 1.2 equivalents) in THF (30 mL), and stirred for 16 hours at RT. After the reaction is complete, the solution is diluted with ice-cold H2O, and extracted with ethyl acetate (3 x 100 mL), the organic layer is washed with brine (50 mL), and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude compound. Using PE and ethyl acetate as eluents, the crude compound is purified by column chromatography. The desired fraction is concentrated under reduced pressure to obtain 2-bromo-4-(bromomethyl)pyridine 2 (3.2 g, 79.93% yield) as a white solid.

[0447] For step 2, to a stirred solution of 2-bromo-4-(bromomethyl)pyridine 2 (2 g, 7.971 mmol, 1.0 equiv) was added 1-hydroxycyclopropane-1-carboxylic acid methyl ester 3 ( To the 4-thiazolinyl group-2-yl)-2-bromopyridine-4-yl)-methoxy-cyclopropane-1-methyl-formiate 3 (2.1 g, 92% yield) was added to the 4-thiazolinyl group-2-yl)-2-bromopyridine-4-yl)-methoxy-cyclopropane-1-methyl-formiate 3 (2.1 g, 92% yield) in ...

[0448] For step 3, a solution of 4-methyl-2-(tributyltinyl)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 reaction progress was monitored by LC-MS. After the raw material was completely consumed, the reaction mixture was diluted with petroleum ether (150 mL), filtered, and dried under reduced pressure to give 6-bromo-2,3-dihydro-[1,4]dioxa[2,3-f]75uinoline-10-ol 5 (3 g, 81% yield) as an off-white solid.

[0449] For step 4, aqueous LiOH was added to a stirred solution of methyl 1-((4'-methyl-[2,2'-bipyridyl]-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 reaction progress was monitored by LC-MS. After complete consumption of the starting material, the reaction mixture was concentrated to give the crude compound, which was then dissolved in water, acidified with KHSO4 solution (pH ~ 2), and extracted with EtOAc (3 x 30 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure to give 1-((4'-methyl-[2,2'-bipyridyl]-4-yl)methoxy)cyclopropane-1-carboxylic acid 6 (3 g, 81% yield) as an off-white solid.

[0450] For step 5, at RT, 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'-bipyridyl]-4-yl)methoxy)cyclopropane-1-carboxylic acid 7 (2 g, 7.04 mmol, 1 eq) in 1,4-dioxane (20 mL) and stirred for 5 hours. 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 x 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 to 15% ethyl acetate in petroleum ether as mobile phase and the desired fractions were concentrated under reduced pressure to give compound 7 (1.3 g, 70% yield) as off-white solid.

[0451] For step 6, at RT, compound 8 (0.3g, 0.44mmol, 1 equivalent) is added to a solution of compound 7 (0.2g, 0.44mmol, 1 equivalent) in DCM (5mL), and stirred for 16 hours. The progress of the reaction is monitored by LC-MS and TLC (petroleum ether of 40% ethyl acetate). After the reaction is completed, the reaction mixture is concentrated under reduced pressure to obtain a crude compound. The crude product is stirred and filtered in pentane (10mL) to obtain compound 9 (1.25g, yield 63%) as a yellow solid.

[0452] For step 7, compound 10 (60g, 0.194mmol, 1.5 equivalents) and DIPEA (0.06mL, 0.388mmol, 3 equivalents) were added to a solution of compound 9 (0.15g, 0.129mmol, 1 equivalent) in DMF (2mL) at RT and stirred for 16 hours. The progress of the reaction was monitored by LC-MS and TLC (40% ethyl acetate in petroleum ether). After the reaction was completed, the reaction mixture was concentrated under reduced pressure to obtain a crude compound, which was purified by silica gel column chromatography (230 to 400 mesh) using 0 to 5% methanol in DCM as a mobile phase. The desired fraction was concentrated under reduced pressure to obtain a photocatalyst 5 (0.05g, 30% yield) in the form of a yellow solid. Photocatalyst 5 is an exemplary photocatalyst according to the present disclosure.

[0453] Example 6. Preparation of Photocatalyst 6

[0454] The description of the preparation of the photocatalyst refers to the description of Scheme 10:

[0455] Solution 10

[0456]

[0457] According to Scheme 10, compound 9 (reference 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)piperazine-1-yl)propoxy)ethane-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 hours. The progress of the reaction was monitored by LC-MS and TLC (10% MeOH in DCM). After the reaction was completed, the reaction mixture was concentrated under reduced pressure to give a crude compound, which was purified by silica gel (NH silica gel mesh) column chromatography, eluting with 0 to 5% methanol in DCM as the mobile phase. The compound fractions were collected and concentrated under reduced pressure to give photocatalyst 6 (0.083 g, 47% yield). A batch of 28 mg (90% LC-MS) of Photocatalyst 6 was purified by preparative HPLC to give pure Photocatalyst 6 (19.3 mg, 97% LC-MS). Photocatalyst 6 is an exemplary photocatalyst according to the present disclosure.

[0458] Example 7. Preparation of Photocatalyst 7

[0459] The description of the preparation of the photocatalyst refers to the description of Scheme 11:

[0460] Solution 11

[0461]

[0462]

[0463] For 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 tert-butyl lithium (70 mL, 0.0701 mmol) in THF at -78 ° C, and the reaction mixture was stirred for 30 minutes. A solution of ZnCl2 (11.9 g, 0.088 mmol) in THF (40 mL) was added, and the reaction mixture was stirred at RT for 2 hours. A solution of pre-prepared compound 2 (8 g, 0.0315 mmol) and tetrakis triphenylphosphine in THF was added dropwise at room temperature. 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 and quenched with a saturated EDTA solution, and pH=8 was adjusted with NaHCO3. The aqueous layer was extracted with EtOAc. The organic layer was dried with Na2SO4, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by column chromatography to obtain compound 3 (4.2 g, 50% yield) as an off-white solid.

[0464] For step 2, benzoyl peroxide (0.0021mmol) and then NBS (0.021mmol) are joined to compound 3 (1g, 0.0042mmol) in CCl4 stirring solution, and the resulting reaction mixture is refluxed for 16 hours. Monitor the reaction by TLC. The reaction mixture is cooled to RT, quenched with water, extracted with DCM. By organic layer Na2SO4 drying, filter and concentrate under reduced pressure, obtain crude compound 4. The crude compound 4 obtained is used for next step without further purification.

[0465] For step 3, CaCO (0.00261mmol) was added to a stirred solution of compound 4 (crude product 2.6g, 0.0066mmol) 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 with Na2SO4, filtered and concentrated under reduced pressure. The crude reaction mixture was purified by column chromatography to obtain compound 5 (200mg, 38% yield) as a white solid.

[0466] For step 4, at 0 ℃, NaH (47.6mg 1.98mmol) was added to a stirred solution of triethyl phosphoacetate (449.8mg, 1.98mmol) in THF in batches, and the reaction mixture was stirred for 30 minutes. A solution of compound 5 (200mg, 0.793mmol) in THF was added, and the reaction mixture was stirred for 3 hours at RT. The reaction was monitored by TLC. The reaction mixture was quenched with NH4Cl saturated solution at 0 ℃. The water layer was extracted with EtOAc. The organic layer was dried with Na2SO4, filtered and concentrated under reduced pressure. The crude reaction mixture was purified by column chromatography to obtain compound 6 (192mg, 74% yield) as a white solid.

[0467] For step 5, palladium 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 for 4 hours under a hydrogen atmosphere. The reaction was monitored by TLC. The reaction mixture was filtered through a diatomaceous earth bed, and the filtrate was concentrated under reduced pressure to give a crude compound 7. The obtained crude compound 7 was used in the next step without further purification.

[0468] For step 6, at RT, LiOH aqueous solution (50mg, 1.23mmol) was added to a stirred solution of compound 7 (crude product 200mg, 0.6172mmol) in a mixture of methanol and THF (1: 1), 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 with Na2SO4, filtered and concentrated under reduced pressure. The crude reaction mixture was purified by column chromatography to obtain compound 8 (144mg, 79% yield) as an off-white solid.

[0469] For step 7, DCC (38 mg, 0.185 mmol) and then N-hydroxysuccinimide (23.3 mg, 0.2026 mmol) were added to a stirred solution of compound 8 (50 mg, 0.168 mmol) in THF at room temperature. The resulting reaction mixture was stirred for 16 hours. 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 with Na2SO4, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by reverse phase column chromatography to obtain compound 9 (20 mg, 30% yield) as a white solid.

[0470] For step 8, DIPEA (59.2 mg, 0.458 mmol) was added to a stirred solution of compound 9 (50 mg, 0.127 mmol) in DMF followed by NH2-PEG4-halogen linker (referred to as compound Int-6 in Example 2) (45 mg, 0.127 mmol) at RT. The resulting reaction mixture was stirred for 16 hours. 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 with Na2SO4, filtered and concentrated under reduced pressure. The crude reaction mixture was purified by reverse phase column chromatography to give compound Int-10 (23 mg, 30% yield) as a light brown solid.

[0471] For step 9, dry ACN (0.8mL) was added to a stirred solution of compound 14 (100mg, 0.0672mmol) in dry DCM (4mL) in a glove box followed by AgPF6 (34.7mg, 0.1377mmol). The resulting reaction mixture was stirred at 40°C for 20 hours in a sealed tube. 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 (90mg, 72% yield) as a yellow solid.

[0472] For 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 (25: 1) of solvent DCM (0.8 mL) and ethanol (0.02 mL). The resulting reaction mixture was stirred at RT for 20 hours. 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 slurried with pentane to obtain photocatalyst 7 (25 mg, yield 52%) as a yellow solid. Photocatalyst 7 is an exemplary photocatalyst according to the present disclosure.

[0473] Example 8. Preparation of Photocatalyst 8

[0474] The description of the preparation of the photocatalyst refers to the description of Scheme 12:

[0475] Solution 12

[0476]

[0477]

[0478] Reference scheme 12, step 1, 4-methyl-2-(tributyltin)pyridine 1 (2.0g, 5.23mmol) and 2-bromo-4-fluoropyridine (1.38g, 7.85mmol) in toluene (40mL) were stirred with argon for 10 minutes. Then TPP (137mg, 0.52mmol), LiCl (0.66g, 15.7mmol) and Pd (PPh3) 4 (604mg, 0.52mmol) were added, and the reaction mixture was stirred at 110°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 with Na2SO4, filtered, and concentrated under reduced pressure. The crude reaction mixture was purified by column chromatography to obtain compound 3 (0.5g, 51% yield) in an off-white semi-solid state.

[0479] For step 2, dimethylamine (2M methanol, 2.66mL, 5.31mmol), then triethylamine (1.12mL, 7.9mmol) was added to a stirred solution of compound 3 (0.5g, 2.65mmol) in DMSO (10mL), and the resulting reaction mixture was heated at 100°C for 16 hours. The reaction was monitored by TLC. The reaction mixture was cooled to RT and concentrated under reduced pressure to obtain a crude compound. The crude compound obtained by reverse phase column chromatography was purified to obtain compound 5 (270mg, 48% yield) as a white solid.

[0480] For step 3, at -78 ° C, LDA (2M 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). The reaction mixture was stirred at -78 ° C for 45 minutes. 2-bromoacetic acid methyl ester (380 mg, 2.53 mmol) at -78 ° C was added, and the reaction mixture was stirred at RT for 16 hours. The reaction was monitored by TLC and LC-MS. The reaction mixture was quenched with NH4Cl aqueous solution, and extracted with EtOAc. The organic layer was dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude compound. The crude compound obtained was purified by reverse phase column chromatography to obtain compound 7 (82 mg, yield 22%) as a light brown liquid.

[0481] For step 4, LiOH aqueous solution (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 hours. The reaction was monitored by TLC. The volatiles were evaporated under reduced pressure to obtain a crude residue. The crude residue was purified by reverse phase column chromatography to obtain compound 8 (52 mg, 68% yield) as a light brown liquid.

[0482] For step 5, DCC (76 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, followed by N-hydroxysuccinimide (42 mg, 0.36 mmol). The resulting reaction mixture was stirred for 16 hours at RT. 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 give compound 9 (62 mg, 91% yield) as a white semisolid.

[0483] For step 6, DIPEA (80 μL, 0.48mmol) was added to a stirred solution of compound 9 (60mg, 0.16mmol) in DMF (1mL) at RT, followed by NH2-PEG4-halogen linker (50mg, 0.16mmol). The resulting reaction mixture was stirred for 16 hours. The reaction was monitored by TLC and LC-MS. The reaction mixture was purified by reverse phase column chromatography to obtain compound Int-11 (21mg, 23% yield) as a light brown viscous liquid.

[0484] For 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 for 16 hours at RT. 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 obtain photocatalyst 8 (7.7 mg, yield 17%) as a yellow solid. Photocatalyst 8 is an exemplary photocatalyst according to the present disclosure.

[0485] Example 9. Preparation of Photocatalyst 9

[0486] The description of the preparation of the photocatalyst refers to the description of Scheme 13:

[0487] Solution 13

[0488]

[0489]

[0490] With reference to scheme 13, step 1, DIPEA (0.047 mL, 0.36 mmol) was added to a stirred solution of compound 1 (50 mg, 0.12 mmol) in DMF at RT, followed by NH2-PEG4-halogen linker (47 mg, 0.13 mmol). The resulting reaction mixture was stirred for 16 hours at RT. The reaction was monitored by TLC and LC-MS. The reaction mixture was purified by reverse phase column chromatography to give compound 3 (30 mg, 43% yield) as a light brown viscous liquid.

[0491] For step 2, compound 4 (32.61 mg, 0.034 mmol) was added to a stirred solution of compound 3 (20 mg, 0.034 mol) in a mixture (25: 1) of DCM (0.8 mL) and ethanol (0.02 mL). The resulting reaction mixture was stirred for 16 hours at RT. The reaction was monitored by LC-MS. The reaction mixture was concentrated under reduced pressure to obtain a crude compound. The crude compound was diluted with acetone and filtered, and the filtrate was concentrated under reduced pressure and pulped with MTBE, and then freeze-dried to obtain a yellow solid photocatalyst 9 (24 mg, yield 54%). Photocatalyst 9 is an exemplary photocatalyst according to the present disclosure.

[0492] Example 10. Preparation of photocatalyst 10

[0493] The description of the preparation of the photocatalyst refers to the description of Scheme 14:

[0494] Solution 14

[0495]

[0496] Referring to Scheme 14, step 1, K2CO3 (1.8 g, 12.00 mmol, 3 equiv) was added to a stirred solution of (2,4-difluorophenyl)boric acid (1.0 g, 4.40 mmol, 1.0 equiv) and 2-bromo-5-(trifluoromethyl)pyrimidine (1.3 g, 8.80 mmol, 2 equiv) in 1,4-dioxane (10 mL). The mixture was purged with argon for 10 minutes. Pd(PPh3)4 was added, and the mixture was heated to 80°C for 16 hours. After the reaction was complete, the reaction mixture was filtered through a diatomaceous earth bed, and the filtrate was concentrated under reduced pressure to give a crude product. The crude compound was purified by column chromatography using petroleum ether and ethyl acetate as eluents. The desired fraction was concentrated under reduced pressure to give 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyrimidine (compound 3, 0.8 g, 70%) as a white solid.

[0497] For step 2, IrCl3 . H2O (600mg, 2.009mmol, 1 equivalent) and compound 8 (1.145g, 4.421mmol, 2.2 equivalents) are added in RBF, and evacuate and use N2 to ventilate three times. Then add the degassed solution of 2-ethoxyethanol (24mL) and water (8.4mL), and be heated to 120 ℃ for 16 hours. Monitor the progress of reaction by LC-MS. After the reaction is complete, the reaction mixture is cooled to RT. Filter the yellow precipitate, wash with water (120mL) and hexane (50mL), and vacuum dry to obtain compound 4 (605mg, 33% yield) as a yellow solid.

[0498] For step 3, AgPF6 (143 mg, 0.564 mmol, 2.1 equivalents) was added to a stirred solution of compound 4 (400 mg, 0.268 mmol, 1 equivalent) in acetonitrile (30 mL) at RT and stirred for 16 hours. The progress of the reaction was monitored by LC-MS. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the crude compound was purified by slurrying with diethyl ether (20 mL) to obtain compound 5 (350 mg, 55.7%) as a light yellow solid.

[0499] For step 4, at RT, compound 6 (0.11g, 0.269mmol, 1 equivalent) is added to a solution of compound 5 (0.17g, 0.269mmol, 1 equivalent) in DCM (5mL), and stirred for 16 hours. The progress of the reaction is monitored by LC-MS and TLC (petroleum ether of 70% ethyl acetate). After the reaction is completed, the reaction mixture is concentrated under reduced pressure to obtain a crude compound. The crude compound is stirred and filtered in pentane (10mL) to obtain compound 7 (0.2g, yield 83%) as a yellow solid.

[0500] For step 5, compound 8 (NH2-PEG4-halogen linker, 0.05 g, 0.356 mmol, 2 eq.) and DIPEA (0.1 mL, 0.496 mmol, 3 eq.) were added to a solution of compound 7 (0.2 g, 0.175 mmol, 1 eq.) in DMF (2 mL) at room temperature. The mixture was stirred for 16 hours. The progress of the reaction was monitored by LC-MS and TLC (10% MeOH in DCM). After the reaction was completed, the reaction mixture was concentrated under reduced pressure to obtain a crude compound. The crude compound was purified by silica gel (NH Silicagel net) column chromatography, eluting with 0 to 5% methanol in DCM as the mobile phase. The desired fraction was concentrated under reduced pressure to obtain a photocatalyst 10 (0.035 g, 20% yield) of a brown colloidal compound. The compound was purified by preparative-LC. Photocatalyst 10 is an exemplary photocatalyst according to the present disclosure.

[0501] Example 11. Photoresponsive labeling in living cells

[0502] First, prepare the reagents. Prepare a 10mM Ir-Cl photocatalyst master stock solution with 100% DMSO. The specific Ir-Cl photocatalyst used in this example is the photocatalyst prepared according to Example 2. In order to produce a working solution of the Ir-Cl catalyst, a portion of the master stock solution of the Ir-Cl catalyst was diluted in 1x DPBS so that the final concentration of the Ir-Cl catalyst was 10μM and the DMSO concentration was 0.1%. A marker master stock solution was also prepared, and its concentration in 100% DMSO was 25mM. The specific marker used was the marker prepared according to Example 1. In order to produce a working solution of the marker, a portion of the marker master stock solution was diluted in 1x DPBS so that the final concentration of the marker was 250μM and the DMSO concentration was 1%.

[0503] Human KRAS with reporter protein ( / +) cell line (HD-103-021, Horizon Discovery) was grown to 80% to 90% confluence. The cells were collected and pelleted by centrifugation at 1000 g for 5 minutes at 4°C (Eppendorf centrifuge 5427R). The supernatant was removed and the cell pellet was placed in ice-cold 1X DPBS (Gibco TM ) and mixed. Subsequently, the cells were pelleted again by centrifugation at 1000 g for 5 minutes at 4°C. The supernatant was removed and new ice-cold 1X DPBS was added to the pellet and mixed.

[0504] For each experimental sample group, approximately 7 × 10 6 cells. Add the Ir-Cl catalyst working solution to the cell pellet, then break up the pellet and mix. Incubate the cells with the Ir-Cl catalyst at 4°C for 30 minutes under continuous end-to-end rotation (15 rpm, TARSONS-ROTOSPIN). After incubation, centrifuge at 1000g for 5 minutes at 4°C to pellet the cells. Remove the supernatant and add 1X DPBS to the pellet. Resuspend the pellet and mix, then centrifuge at 1000g for 5 minutes at 4°C.

[0505] The supernatant was removed and the pellet was resuspended in the labeling agent in 1X DPBS (DMSO final concentration 1%). The pellet was broken up, mixed, and immediately irradiated with light at a wavelength of 450 nm for 10 minutes at 100% power 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.

[0506] The supernatant was removed and the pellet was added to a 5% flask supplemented with protease-phosphatase inhibitors (Halt TM The cells were resuspended in 1X cell lysis buffer (Cell Signaling Technology) containing protease and phosphatase inhibitor cocktail (100X) and mixed briefly. The mixture was then incubated on ice for 15 minutes.

[0507] After incubation, the cell lysate was sonicated using a probe sonicator (SONICS vibra cell Fisher Scientific) with an amplitude of 30%, 15 seconds on, 10 seconds off, for three cycles. The cell lysate was centrifuged at 18000 g for 15 minutes at 4°C. The supernatant was collected and subsequently used for immunoprecipitation (IP) by coating with specific antibodies (HALO-labeled protein-HALO-tag IP) and biotinylated proteins (based on streptavidin magnetic beads IP) according to Examples 12 and 13, respectively.

[0508] Example 12. Immunoprecipitation of HALO-tagged protein (K-Ras interacting partner protein)

[0509] Aliquot 50μl Dynabead TM Dynabeads TM Protein G Thermo Fisher Scientific). The beads were collected on a magnetic stand and washed three times with wash buffer (PBST). The beads were then collected on a magnetic stand and the supernatant was removed.

[0510] To coat the beads with antibodies, antibodies from Promega were used. The monoclonal antibodies were added to a final concentration of 1:50 in PBST. The beads were incubated with the antibodies for 1 hour at RT with continuous end-to-end rotation. The beads were then collected on a magnetic stand and washed three times with wash buffer.

[0511] Thereafter, the beads were incubated with pre-clearing solution (5% BSA, 2.5 μM carbonic anhydrase) for 1 hour at RT with continuous end-over-end rotation. The beads were washed three times with wash buffer to remove excess bound antibody.

[0512] The cell lysate obtained according to Example 11 was added to the anti-HaloTag antibody coated beads and mixed. The lysate and beads were incubated at RT for 3 hours with continuous end-to-end rotation. After incubation, the test tube was placed on a magnetic rack and the beads were collected. The supernatant was removed. The beads were then washed three times with wash buffer.

[0513] For elution, 50 μl of SDS-Laemmli buffer (containing 1:10 1M DTT) was added to the beads and mixed. The mixture was kept at 95°C for 10 minutes. The tubes were then placed on a magnetic stand and the supernatant representing the final pull-down eluate sample was collected in separate tubes.

[0514] The pull-down eluate samples were then subjected to Western blotting. 10 μl of the pull-down eluate samples were loaded into each lane of a 4% to 15% Tris-Glycine gel (Bio-Rad). The gel electrophoresis was run at 80 V for the first 10 minutes and then at 100 V until the dye front reached the end of the gel.

[0515] The proteins were transferred from the gel to the nitrocellulose membrane using a Bio-Rad wet transfer apparatus at 4°C with a constant current of 300 mA for 90 minutes. The membrane was rinsed 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 (1:1000) specific for a potential K-Ras interaction partner protein was added and kept overnight in a 4°C space on a shaker. The next day, the membrane was washed 3 times with TBST, with each wash incubated at room temperature for 10 minutes.

[0516] HRP-labeled secondary antibody specific for the primary antibody was added and incubated for 120 min at RT with constant shaking. After incubation, the membrane was washed with TBST (10 min, 3 times). Thereafter, chemiluminescent reagent (Clarity TM Western ECl Substrate) developed film.

[0517] Figure 4A is an image of a protein blotting result using an anti-PI3k primary antibody according to the above procedure. Each column of the gel represents a different condition. For example, the presence of a labeling agent (WH8), the presence of a photocatalyst (IR), and the irradiation are varied for each column. In addition, the cell line (006 or 021) used is varied for each column. The conditions including the labeling agent, the photocatalyst, and the irradiation of the cell line 021 result in a distinct band of PI3 kinase P110α. Without being bound by a particular theory, it is generally believed that when a high molecular weight band is observed, it indicates the formation of aggregates including PI3 kinase P110α.

[0518] Figure 4B is an image of a Western blot using an anti-SPRED1 antibody according to the above procedure. Figure 4A Likewise, the various combinations represent different combinations of labeling agents, photocatalysts, irradiation, and cell lines. The conditions including labeling agents, photocatalysts, irradiation, and cell line 021 resulted in the formation of distinct bands. Without being bound by a particular theory, it is believed that when high molecular weight bands are observed, this indicates the formation of aggregates including SPRED1.

[0519] Figure 4C is an image of a Western blot result using an anti-cRAF antibody according to the above procedure. Figure 4A Likewise, the various combinations represent different combinations of labeling agents, photocatalysts, irradiation, and cell lines. The conditions including labeling agents, photocatalysts, irradiation, and cell line 021 resulted in the formation of distinct bands.

[0520] Example 13. Immunoprecipitation of biotinylated protein (biotinylated K-Ras interaction partner protein)

[0521] 50 μl of Pierce Streptavidin magnetic bead slurry was aliquoted and beads were collected on a magnetic stand and washed three times with wash buffer (PBST). The beads were incubated with pre-clearing solution (5% BSA, 2.5 μM carbonic anhydrase) for 1 hour at RT with continuous end-to-end rotation. The beads were then collected on a magnetic stand and washed three times with wash buffer.

[0522] The cell lysate obtained according to Example 11 was added to the streptavidin beads and mixed. The mixture was incubated for 3 hours at RT with continuous end-to-end rotation. After incubation, the tube was placed on a magnetic rack and the beads were collected. The supernatant was removed. The beads were then washed three times with wash buffer.

[0523] For elution, add 50 μl of SDS-Laemmli buffer (containing 1:10 1M DTT) to the beads and mix. Keep this mixture at 95°C for 10 minutes. Then place the tubes on a magnetic stand and collect the supernatants in their own tubes. These are the final pull-down eluate samples.

[0524] The pull-down eluate samples were then subjected to Western blotting. 10 μl of the pull-down eluate samples were loaded into each lane of a 4% to 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 dye front reached the end of the gel.

[0525] Proteins were transferred from the gel to a nitrocellulose membrane using a Bio-Rad wet transfer apparatus at a constant current of 300 mA at 4°C for 90 minutes. The membrane was rinsed once with TBST and blocked with 5% milk in TBST for 60 minutes at RT. After blocking, the membrane was washed with TBST and the desired antibodies (1:1000) were added and kept overnight at 4°C on a shaker. The next day, the membrane was washed 3 times with TBST, with each wash incubated at room temperature for 10 minutes.

[0526] The corresponding HRP-labeled secondary antibody was added and kept at RT with continuous shaking for 120 minutes. After incubation for 120 minutes, the membrane was washed with TBST (3 times for 10 minutes each). Thereafter, chemiluminescence reagent (Clarity TM Western ECl Substrate) developed film.

[0527] Figure 5Ais an image of a Western blot using an anti-biotin primary antibody according to the above procedure. Each column of the gel represents a different irradiation condition: the left column represents cells that underwent 10 minutes of irradiation, while for the right column, the cells did not undergo irradiation. For the irradiated cells, there are distinct bands starting from the approximately 55 kDa marker and upwards. Non-irradiated cells show only faint bands, indicating some nonspecific level of binding.

[0528] Figure 5B is an image of a Western blot result using an anti-RSK1 antibody according to the above procedure. Figure 5A Again, the two columns in the gel represent irradiated or non-irradiated cells. The irradiated cells have a distinct band of the expected molecular weight, but the non-irradiated cells have no visible band.

[0529] Figure 5C is an image of a Western blot result using an anti-cRAF antibody according to the above procedure. Figure 5A Again, the two columns in the gel represent irradiated or non-irradiated cells. The irradiated cells have a distinct band of the expected molecular weight, but the non-irradiated cells have no visible band.

[0530] Figure 5D is an image of a Western blot result using an anti-cRAF antibody according to the above procedure. Figure 5A Again, the two columns in the gel represent irradiated or non-irradiated cells. The irradiated cells have a distinct band of the expected molecular weight, but the non-irradiated cells have no visible band.

[0531] Figure 5E is an image of a Western blot using cell lysate and anti-GAPDH antibody. Figure 5A Again, the two columns in the gel represent irradiated or non-irradiated cells. Both irradiated and non-irradiated cells have distinct bands of the expected molecular weight. This confirms that FIG. 5A to FIG. 5D The blots shown have similar numbers of cells in irradiated and non-irradiated conditions.

[0532] Fig. 5F is an image of a Western blot result using an anti-SOS1 antibody according to the above procedure. For this gel, three types of cells were electrophoresed: KRAS ( / +) reporter protein (HD-103021), KRAS HaloTag reporter protein control (HD-103-037), and SW48 parental (HD-PAR-006). / +) reporter protein cells, a distinct SOS1 band was observed, as indicated by the arrow. For the two control conditions (HD-103-037 and HD-PAR-006), no distinct band was observed. Notably, other methods for detecting protein associations have previously failed to identify SOS1 as a protein associated with KRas.

[0533] Example 14. Fusion of HaloTag to cereblon does not prevent ubiquitination of ikaros by cereblon.

[0534] A stable cell line was created using Jurkat cells in which native cereblon (CRBN) was knocked out and a fusion of HaloTag protein and CRBN was expressed (referred to herein as "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 a western blot using lysates of the HALO.CRBN Jurkat cell line that had been exposed to varying concentrations of pomalidomide or lenalidomide. The gel was stained using a mouse anti-IKZF1 antibody. The dilution was 1:1000 and the exposure time was approximately 10 seconds. The 10 μM pomalidomide and 10 μM lenalidomide lanes contained significantly less IKZF1 than the control or 1 μM pomalidomide lanes, indicating that the HaloTag / cereblon fusion protein can ubiquitinate IKZF1 in the presence of lenalidomide and / or pomalidomide.

[0535] Example 15. Detection of cereblon interactions

[0536] The HALO.CRBN Jurkat cell line of Example 14 was used to produce 7A to 13B Shown are a series of gels of cell lysates used to examine the interactions between CRBN and other proteins.

[0537] HALO.CRBN Jurkat cells were grown in suspension to 80% to 90% confluence. 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.

[0538] The cells were collected and pelleted by centrifugation at 1000 g for 5 minutes at 4°C (Eppendorf centrifuge 5427R). The supernatant was removed and the cell pellet was placed in ice-cold 1X DPBS (Gibco TM ) and mix. Subsequently, the cells were pelleted again by centrifugation at 1000 g for 5 min at 4°C. The supernatant was removed and fresh ice-cold 1X DPBS was added to the pellet and mixed. For each experimental sample group, approximately 7 × 10 6 cells.

[0539] A 10 mM Ir-Cl photocatalyst master stock solution was prepared in 100% DMSO. The specific Ir-Cl photocatalyst used in this example was the photocatalyst prepared according to Example 2. To produce a working solution of the Ir-Cl catalyst, a portion of the Ir-Cl catalyst master stock solution was diluted in 1x DPBS so that the final concentration of the Ir-Cl catalyst was 10 μM and the DMSO concentration was 0.1%. A marker master stock solution was also prepared, which had a concentration of 25 mM in 100% DMSO. The specific marker used was the marker prepared according to Example 1. To produce a working solution of the marker, a portion of the marker master stock solution was diluted in 1x DPBS so that the final concentration of the marker was 250 μM and the DMSO concentration was 1%.

[0540] The Ir-Cl catalyst working solution was added to the cell pellet, and the pellet was broken up and mixed. The cells were incubated with the Ir-Cl catalyst at 4°C with continuous end-to-end rotation (15 rpm, TARSONS-ROTOPIN) for 30 minutes. After incubation, the cells were pelleted by centrifugation at 1000g for 5 minutes at 4°C. The supernatant was removed and 1X DPBS was added to the pellet. The pellet was resuspended and mixed, and then centrifuged at 1000g for 5 minutes at 4°C. The supernatant was removed and the pellet was resuspended in the labeling agent in 1X DPBS (DMSO final concentration 1%). The pellet was broken up, mixed, and immediately irradiated with light of a wavelength of 450nm for 10 minutes at 100% power using an M2 photoreactor (Penn PhDPhotoreactor M2 from Sigma-Aldrich). After irradiation, the cells were pelleted by centrifugation at 1000g for 5 minutes at 4°C.

[0541] The supernatant was removed and the pellet was resuspended in medium supplemented with protease-phosphatase inhibitors (Halt TM The cells were added to 1X Cell Lysis Buffer (Cell Signaling Technology) containing protease and phosphatase inhibitor cocktail (100X) and mixed briefly. The mixture was then incubated on ice for 15 minutes.

[0542] After incubation, the cell lysate was sonicated using a probe sonicator (SONICS vibra cell Fisher Scientific) at 30% amplitude, 15 seconds on, 10 seconds off, for three cycles. The cell lysate was centrifuged at 18000 g for 15 minutes at 4°C. The supernatant was collected and subsequently used for protein content estimation (Pierce TM BCA protein assay kit). The supernatant containing approximately 200 μg of protein was then used for each IP.

[0543] For IP, aliquot 50 μl of streptavidin magnetic bead slurry (Pierce streptavidin magnetic beads, Thermo Fisher Scientific). Collect the beads on a magnetic stand and wash three times with wash buffer (PBST). Then collect the beads on a magnetic stand and remove the supernatant.

[0544] Thereafter, the beads were incubated with pre-clearing solution (5% BSA, 2.5 μM carbonic anhydrase) for 1 hour at room temperature with continuous end-over-end rotation. The beads were washed three times with wash buffer.

[0545] Cell lysates obtained from irradiated HALO.CRBN Jurkat cells as described above were added to the streptavidin beads and mixed. The lysates and beads were incubated at RT for 3 hours with continuous end-to-end rotation. After incubation, the test tubes were placed on a magnetic rack and the beads were collected. The supernatant was removed. The beads were then washed three times with wash buffer.

[0546] For elution, 50 μl of SDS-Laemmli buffer (containing 1:10 1M DTT) was added to the beads and mixed. The mixture was kept at 95°C for 10 minutes. The tubes were then placed on a magnetic stand and the supernatant representing the final pull-down eluate sample was collected in separate tubes.

[0547] The pull-down eluate samples were then subjected to Western blotting. 10 μl of the pull-down eluate samples were loaded into each lane of a 4% to 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 dye front reached the end of the gel.

[0548] The proteins were transferred from the gel to the nitrocellulose membrane using a Bio-Rad wet transfer apparatus at a constant current of 300 mA at 4°C for 90 minutes. The membrane was rinsed 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 a potential CRBN interaction partner protein (1:1000) was added and kept overnight in a 4°C space on a shaker. The next day, the membrane was washed 3 times with TBST, with each wash incubated at room temperature for 10 minutes.

[0549] HRP-labeled secondary antibody specific for the primary antibody was added and incubated for 120 min at RT with constant shaking. After incubation, the membrane was washed with TBST (10 min each, 3 times). Thereafter, chemiluminescent reagent (Clarity TM These images are shown in 7A to 12B .

[0550] Fig. 7A and Figure 7B are images of a gel run using lysates and stained with anti-CK1 polyclonal rabbit antibody. Fig. 7A and Figure 7B The gels shown were stained at a 1:1000 dilution. Fig. 7A The exposure of the gel was about 20 seconds, and Figure 7B The exposure of the gel was about 60 seconds. There are observable CK1 bands in lanes 7 and 8, corresponding to the lysates from the immunoprecipitated cells using the photocatalyst, labeling agent, and irradiation (light), indicating that CK1 is proximal labeled by the labeling agent, thereby indicating a protein-protein interaction between CRBN and CK1.

[0551] Fig. 8A and Figure 8B are images of a run using immunoprecipitated proteins and stained with anti-SALL4 polyclonal rabbit antibody. Fig. 8A and Figure 8B The gels shown were stained with an antibody dilution of 1:500. Fig. 8A The exposure of the gel was approximately 10 sec, while Figure 8B The exposure of the gel was about 30 seconds. There were observable SALL4 bands in lanes 7 and 8, corresponding to the lysates from the immunoprecipitated cells using the photocatalyst, the labeling agent, and irradiation (light), indicating that SALL4 was proximal labeled by the labeling agent, thereby indicating a protein-protein interaction between CRBN and SALL4.

[0552] Fig. 9 is an image of a western blot using lysate and staining with an anti-KEAP1 antibody at a 1:1000 dilution. The exposure time was approximately 30 seconds. There are observable KEAP1 bands in lanes 13 and 1, corresponding to lysates from cells using a photocatalyst, labeling agent, and irradiated (light) cell lysate, although there are no observable KEAP1 bands in the immunoprecipitation pull-down lanes (5-8). This indicates that the KEAP1 protein is not proximally labeled and therefore there is no protein-protein interaction between CRBN and KEAP1 in the absence of a molecular glue.

[0553] Fig.10is an image of a western blot using lysate and staining with an anti-β-tubulin rabbit antibody at a dilution of 1:1000. The exposure time was approximately 5 seconds. There are observable β-tubulin bands in lanes 13 and 1, corresponding to lysates from cells using a photocatalyst, a labeling agent, and irradiation (light) of the cell lysate, although there are no observable β-tubulin bands in the immunoprecipitation pull-down lanes (5-8). This indicates that β-tubulin is not proximal labeled, and therefore there is no protein-protein interaction between CRBN and β-tubulin in the absence of a molecular glue.

[0554] Fig.11A and Fig. 11B is an image of a gel run using lysate and stained with anti-PARP1 polyclonal rabbit antibody. Fig.11A and Fig. 11B The gels shown were stained with an antibody dilution of 1:1000. Fig.11A The gel exposure is about 20 seconds, and Fig. 11B The gel exposure was approximately 120 seconds. There were observable PARP1 bands in lanes 13 and 1, corresponding to the lysates from cells treated with photocatalyst, labeling agent, and irradiated (light), although there were no observable PARP1 bands in the immunoprecipitation pull-down lanes (5-8). This suggests that the PARP1 protein was not proximity labeled and therefore there was no protein-protein interaction between CRBN and PARP1 in the absence of the molecular glue.

[0555] Fig. 12A and Fig. 12B Figure 2 is an image of a gel run using lysates and stained with antibodies specific for known oncoproteins. Fig. 12A and Fig. 12B The gels shown were stained with an antibody dilution of 1:1000. Fig. 12A The gel exposure time is about 45 seconds, while Fig. 12B The gel exposure time was about 60 seconds. There were observable bands corresponding to oncogenic proteins in lanes 7 and 8, corresponding to lysates from cells treated with photocatalyst, labeling agent, and irradiation (light), indicating proximity labeling of the oncogenic proteins by the labeling agent, thereby indicating a protein-protein interaction between CRBN and the oncogenic proteins.

[0556] Fig.13A and Fig. 13B are images of a run using lysates and stained with anti-IKZF1 monoclonal mouse antibody. Fig.13A and Fig. 13B The gels shown were stained with an antibody dilution of 1:100. Fig.13A The gel exposure is about 10 seconds, while Fig. 13BThe gel was exposed for approximately 30 seconds. There are observable IKZF1 bands in lanes 7 and 8, corresponding to the immunoprecipitated lysates from cells with the photocatalyst, labeling agent, and irradiation (light), indicating proximity labeling of IKZF1 by the labeling agent, thereby indicating a protein-protein interaction between CRBN and IKZF1.

[0557] Example 16. Proximity-based labeling method can detect K-Ras-related proteins that are not detected by alternative methods. White matter.

[0558] For detecting protein interactions, the light-responsive, proximity-based labeling method disclosed herein was compared with existing immunoprecipitation pull-down methods. Two cell lines were used. The first was a stable cell line expressing the 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).

[0559] Grow cells to 80-90% confluence. Rinse cells with 1X PBS and then add 0.25% trypsin-EDTA. Then incubate cells at 5% CO2, 37°C for about 2 minutes. Once cells are detached, neutralize trypsin with complete medium. Pellet cells by centrifugation at 1000g for 5 minutes at 4°C (Eppendorf centrifuge 5427R). Remove supernatant and resuspend cell pellet in ice-cold 1X DPBS (Gibco TM ) and mixed. Thereafter, the cells were pelleted again by centrifugation at 1000 g for 5 minutes at 4°C. The supernatant was removed, and new ice-cold 1X DPBS was added to the pellet and mixed.

[0560] For each experimental sample group, take 7x10 6 cells. 10 μM Ir-Cl catalyst (Compound 1, see Scheme 1) is added to the cell pellet, and the pellet is dispersed and mixed. The cells are incubated with the Ir-Cl catalyst at 4°C with continuous end-to-end rotation (TARSONS-ROTOPIN) at 15 rpm for 30 minutes. After incubation, the cells are precipitated by centrifugation at 1000g for 5 minutes at 4°C. The supernatant is removed and 1X DPBS is added to the pellet. The pellet is resuspended and mixed, and then centrifuged at 1000g for 5 minutes at 4°C. The cell pellet is resuspended in 900 μl 1X PBS and transferred to a 6-well plate. The labeling agent according to Example 1 is added to the cell suspension at a final concentration of 250 μM in 1X DPBS (DMSO final concentration 1%).

[0561] The 6-well plate with cells was placed in the CLIGHT photoreactor. Typically, irradiation was performed for 5 minutes. For SOS1, 10 minutes of irradiation was performed.

[0562] After irradiation, the cell suspension was transferred to a microcentrifuge tube and pelleted by centrifugation at 1000 g for 5 min at 4°C. The supernatant was removed and the pellet was resuspended in 4% paraformaldehyde supplemented with protease-phosphatase inhibitors (Halt TM The cells were added to 1X Cell Lysis Buffer (Cell Signaling Technology) containing protease and phosphatase inhibitor cocktail (100X), Fluorite, PMSF) and mixed briefly. They were incubated on ice for 15 min.

[0563] After incubation, the lysate was sonicated using a probe sonicator (SONICS vibra cell Fisher Scientific) at 30% amplitude, 15 seconds on, 10 seconds off, for three cycles. The cell lysate was centrifuged at 18000 g for 15 minutes at 4°C. The supernatant containing the cell lysate was collected in a test tube.

[0564] Evaluation of protein content of lysates (Pierce TM BCA protein assay kit). The cell lysate was then used for immunoprecipitation (IP) by coating with specific antibodies (HALO-tag IP) and biotinylated proteins (streptavidin magnetic beads-based IP).

[0565] HALO-tapIP

[0566] Aliquot 50μl Dynabead TM Dynabeads TM Protein G Thermo Fisher Scientific). The beads were collected on a magnetic stand and washed three times with wash buffer (PBST). After washing, the beads were collected on a magnetic stand and the supernatant was removed.

[0567] To coat the beads with antibodies, antibodies from Promega (origin: mouse) were added. Monoclonal antibodies were added to PBST at a final concentration of 1:50. The beads were incubated with the antibody at RT for 1 hour with continuous end-to-end rotation. The beads were then collected on a magnetic rack and washed three times with wash buffer. Thereafter, the beads were incubated with a pre-clearing solution (5% BSA, 2.5 μM carbonic anhydrase) at RT for 1 hour with continuous end-to-end rotation. The beads were washed three times with wash buffer to remove excess bound antibodies.

[0568] 200 μg of each irradiated cell lysate obtained as described above was added to the HALO antibody coated beads and mixed. The lysate and bead mixture was incubated at RT for 3 hours with continuous end-over-end rotation.

[0569] 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 1:10 1M DTT) was added to the beads and mixed. The mixture was heated to 95°C for 10 minutes. The tubes were then placed on a magnetic rack to separate the beads and the supernatant was collected in separate tubes. The collected supernatant was then used for Western blotting.

[0570] Streptavidin beads IP

[0571] Take 50 μ l Pierce streptavidin magnetic beads slurry, collect beads on magnetic stand, and wash three times with washing buffer (PBST). At room temperature, with continuous end-to-end rotation, beads and pre-clearing solution (5% BSA, 2.5 μ M carbonic anhydrase) are hatched for 1 hour. Beads are collected on magnetic stand, and washed three times with washing buffer. The cell lysate obtained from irradiated cells is added to the streptavidin beads and mixed. Lysate and bead mixture are hatched for 3 hours with continuous end-to-end rotation at RT.

[0572] 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 (with 1:10 1M DTT) was added to the beads and mixed, then heated to 95°C for 10 minutes. The tubes were placed on a magnetic rack and the supernatant was collected from each tube. The collected supernatant was then used for Western blotting.

[0573] Western blotting

[0574] Western blotting was performed on the eluate samples from HALO-tag IP and streptavidin bead IP. 10 μl of the pull-down eluate sample was loaded into a 4%-15% Tris-Glycine gel (BIO-RAD) in each lane. The gel was first run at 80V for 10 minutes, then at 100V until the dye front reached the end of the gel. The proteins in the gel were transferred to a nitrocellulose membrane using a Bio-Rad wet transfer apparatus at 4°C, at a constant current of 300 mA for 90 minutes.

[0575] The membrane was rinsed once with TBST and blocked with 5% milk in TBST for 60 min at RT. After blocking, the membrane was washed with TBST and primary antibody specific for a potential KRAS interacting partner protein was added at 1:1000 and kept at 4°C overnight on a shaker.

[0576] The next day, the membrane was washed with TBST (10 minutes, 3x) at RT. The respective HRP-labeled secondary antibodies specific to the primary antibody were added and kept for 120 minutes at RT with continuous shaking. Afterwards, the membrane was washed with TBST (10 minutes, 3 times). The membrane was imaged in the BIO-RAD ChemiDoc imaging system using a chemiluminescent reagent ( Forte Western HRP Substrate) to generate FIG. 14A to FIG. 17B .

[0577] Fig.14A and Fig. 14B Shown is a gel stained with anti-cRAF antibody.

[0578] for Fig.14A , rabbit anti-cRAF antibody was diluted to 1:750. Exposure was approximately 60 seconds. The lysate was subjected to Halo Tag IP as discussed above. The gel showed a weak cRAF band in lane 5, corresponding to immunoprecipitation with the Halo antibody, while total lysate lanes 6 and 7 showed distinct cRAF bands.

[0579] for Fig. 14B , the gel was stained with a rabbit anti-cRAF antibody diluted 1:1000. The exposure was approximately 30 seconds. The lysates were subjected to the streptavidin bead-based IP discussed above. The gel showed a distinct cRAF band in lane 5, corresponding to the immunoprecipitation using a photocatalyst, irradiation, and labeling agent. The total lysate lanes 6 and 7 also showed a distinct cRAF band. This suggests that the method described herein is more efficient than the immunoprecipitation pull-down in identifying protein-protein interactions.

[0580] Fig.15A and Fig. 15B Shown is a gel stained with anti-RSK1 antibody.

[0581] for Fig.15A , rabbit anti-RSK1 antibody was diluted to 1:1000. The exposure was about 90 seconds. The lysate was subjected to the Halo-Tag IP discussed above. The gel showed no observable bands, corresponding to immunoprecipitation using the Halo antibody, while total lysate lanes 6 and 7 showed clear RSK1 bands. A longer exposure of about 180 seconds also produced no observable bands.

[0582] for Fig. 15B, the gel was stained with a rabbit anti-RSK1 antibody diluted 1:1000. The exposure was approximately 30 seconds. The lysate was subjected to streptavidin bead-based IP as discussed above. The gel shows a distinct RSK1 band in lane 5, corresponding to immunoprecipitation using a photocatalyst, irradiation, and a labeling agent. Total lysate lanes 6 and 7 also show distinct RSK1 bands. Fig.15A and Fig. 15B The results shown in show that even though the immunoprecipitation pull-down method cannot identify RSK1 adjacent to K-Ras, the method disclosed herein can.

[0583] Fig.16A and Fig. 16B Shown is a gel stained with anti-SOS1 antibody.

[0584] for Fig.16A , rabbit anti-SOS1 antibody was diluted to 1:1000. Exposure was approximately 45 seconds. The lysate was subjected to Halo Tag IP as discussed above. The gel showed no observable band in lane 5, corresponding to immunoprecipitation using Halo antibody, while total lysate lanes 6 and 7 showed clear SOS1 bands.

[0585] for Fig. 16B , the gel was stained with a rabbit anti-SOS1 antibody diluted 1:1000. The exposure was about 60 seconds. The lysate was subjected to the streptavidin bead-based IP discussed above. The gel shows a distinct SOS1 band in lane 5, corresponding to the immunoprecipitation using photocatalyst, irradiation, and warhead. The total lysate lanes 6 and 7 also show a distinct RSK1 band. Fig.16A and Fig. 16B The results shown in show that even though the immunoprecipitation pull-down method cannot identify SOS1 adjacent to K-Ras, the method disclosed herein can.

[0586] Fig.17A and Fig. 17B Shown is a gel stained with anti-KEAP1 antibody.

[0587] for Fig.17A , rabbit anti-KEAP1 antibody was diluted to 1:1000. Exposure was approximately 60 seconds. The lysate was subjected to the Halo-Tag IP discussed above. The gel showed no observable band in lane 5, corresponding to immunoprecipitation using the Halo antibody, while total lysate lanes 6 and 7 showed a clear KEAP1 band.

[0588] for Fig. 17B, the gel was stained with a rabbit anti-KEAP1 antibody diluted 1:1000. The exposure time was approximately 60 seconds. The lysates were subjected to the streptavidin bead-based IP discussed above. The gel showed no observable band in lane 5, corresponding to the immunoprecipitation using a photocatalyst, irradiation, and labeling agent. The total lysate lanes 6 and 7 showed a distinct KEAP1 band. These results indicate that there is no protein-protein interaction between KRAS G12D and KEAP1.

[0589] Example 17. Proximity labeling of KRAS interactors may depend on irradiation time.

[0590] 18A to 18D is an image of a western blot using lysate and streptavidin pull-down protein, which was stained with anti-RSK1 polyclonal rabbit antibody. The antibody dilution was 1:1000. Figure 6 A and Figure 6 The gel exposure for C is about 10 seconds, while Figure 6 B and Figure 6 The gel exposure for D was approximately 60 seconds. Figure 6 B and Figure 6 There are observable RSK1 bands in lane 1 of D, corresponding to the lysate from the immunoprecipitated cells treated with the photocatalyst, labeling agent, and irradiation (light). The presence of these bands indicates that RSK1 is proximally labeled by the labeling agent. The 5-minute irradiation sample ( Figure 6 D) showed a larger RSK1 band than the 2.5 min irradiation sample ( Figure 6 The RSK1 band observed in B) is thicker. Without being bound by a particular theory, it is believed that these results suggest that proximity labeling of KRAS interactors may depend on irradiation time.

[0591] FIG. 18E to FIG. 18H is a western blot image of lysate and streptavidin pull-down protein stained with anti-SOS1 polyclonal rabbit antibody. The antibody dilution was 1:1000. Figure 6 The gel exposure for E and 6G was approximately 10 seconds, while Figure 6 The gel exposure for F and 6H was approximately 30 seconds. Figure 6 F and Figure 6 There are observable SOS1 bands in lane 1 of H, corresponding to the lysate from the immunoprecipitation of cells treated with the photocatalyst, labeling agent, and irradiation (light). The presence of these bands indicates that SOS1 is proximally labeled with the labeling agent. The 5-minute irradiation ( Figure 6 H) The observed SOS1 band is larger than that of the sample irradiated for 2.5 minutes ( Figure 6 F) The observed RSK1 band is thicker. Without being bound by a particular theory, these results suggest that proximity labeling of KRAS interactors may depend on irradiation time.

[0592] Example 18. System for detecting Ir photocatalyst activity

[0593] Catalyst-dependent biotinylation of bovine serum albumin

[0594] The photocatalyst was combined with 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; the final catalyst concentrations were set to 5 μM and 10 μM, respectively. These samples were then placed in the dark or irradiated with visible light (450 nm) at 100% intensity for 5 minutes in an M2 photoreactor. 60 μL of the sample was then removed, combined with 20 μL of 4x reducing SDS-Laemmli buffer (with 1:10 1M DTT), mixed and heated at 95°C for 5 minutes. 15 μL of each sample was then analyzed by fluorescent protein blotting.

[0595] Fluorescent Western Blot

[0596] The samples were loaded into 4% to 20% Tris-Glycine gels (Bio-Rad). The gels were run at 120 V for 80 minutes or until the dye front reached the end of the gel. Proteins were transferred from the gel to nitrocellulose membranes using an iBlot 2 semi-dry transfer apparatus. The membranes were used to detect total protein for standardization, which was performed according to the manufacturer's manual (Revert TM 700 total protein staining, LI-COR). Thereafter, the membrane was rinsed once with TBST and Block with blocking buffer for 1 hour at RT with gentle shaking. After blocking, wash the membrane with TBST and add the desired antibody (1:1000) and keep overnight on a shaker at 4°C. The next day, wash the membrane 3 times with TBST at RT with a 10 minute incubation per wash. Add 800CW secondary antibody or 680RD secondary antibody and kept at RT for 1 hour with continuous shaking. After incubation for 1 hour, the membrane was washed with TBST (washed for 10 minutes, 3 times). Thereafter, the membrane was scanned on Odyssey Imager (LI-COR Odyssey M imaging system).

[0597] FIG. 19A to FIG. 19B are the results of western blotting showing the biotinylation of BSA by different photocatalysts combined with the labeling reagent WH8. Fig.19A The total protein staining results for all treatment conditions were shown in Table 1. The same amount of BSA was used in each treatment. Fig.19BThe results are fluorescent western blots using rabbit anti-biotin antibodies. The samples that were not irradiated have no bands (lanes 6 and 8 to 11). For the samples that were irradiated for 5 minutes, biotinylated BSA bands were present in both 5 μM and 10 μM catalyst treatments.

[0598] FIG. 20A to FIG. 20B are Western blot results showing biotinylation of BSA by different photocatalysts combined with the labeling agent WH9. Fig. 20A Total protein staining results for all treatment conditions are shown. The same amount of BSA was used in each treatment. Fig. 20B Fluorescent Western blot results using rabbit anti-biotin antibody are shown. Samples that did not receive irradiation have no bands (lanes 6 and 8 to 11). For samples that were irradiated for 5 minutes, biotinylated BSA bands were present at both 5 μM and 10 μM catalyst treatments.

[0599] Example 19. Ir Photocatalyst Cell Permeability

[0600] Light-responsive labeling was performed in cells according to a procedure similar to that described in Example 11. 10 mM or 5 mM Ir photocatalyst stock solutions were prepared in 100% DMSO. Phenol-free RPMI1640 medium (Gibco TM ) in a 6-well cell culture dish. / +) cells (HD-103-021, Horizon Discovery) were cultured to 90% confluence. Photocatalysts IR1 (photocatalyst 1), IR23 (photocatalyst 3), IR24 (photocatalyst 4), IR42 (photocatalyst 5), or DMSO were added to appropriate wells with a final photocatalyst concentration of 5 μM or 10 μM. The cells were incubated at 37°C for 1 hour or 2 hours. The culture medium was then aspirated and the cells were gently washed twice with 2 mL of fresh RPMI 1640, with each wash incubated at 37°C for 10 minutes. TAMRA-Cl fluorescent dye (Promega, G8251) was diluted to a 5 μM working solution with OptiMEM. 0.8 mL of the working dye solution was added to the cells and allowed to incubate at 37°C for 15 minutes. The cells were gently washed twice with 2 mL of cold DPBS, aspirated, and then 200 μL of DPBS supplemented with protease-phosphatase inhibitors (Halt TMThe cell lysate was collected in a 1.5 mL tube using RIPA with protease and phosphatase inhibitor cocktail (100X) and mixed briefly. The cell lysate was sonicated using a probe sonicator (SONICSvibra cell Fisher Scientific) at 30% amplitude, 15 seconds on, 10 seconds off, for three cycles. The cell lysate was centrifuged at 18,000 g for 20 minutes at 4°C. The supernatant was collected and analyzed by BCA assay (Pierce TM BCA protein assay kit, Thermofisher) was adjusted to a concentration of 1 mg / ml. Cell lysates were processed and analyzed by fluorescent western blot with anti-TAMRA and anti-β-actin (for normalization) antibodies. Without being bound by a particular theory, the TAMRA signal may be inversely proportional to Ir binding to Halotag-KRAS (G12D) expressed intracellularly and may therefore be inversely proportional to Ir cell permeability.

[0601] Fig.21A and Fig.21B The results of fluorescent western blots were measured using Halo-chaser with anti-TAMRA and anti-β-actin antibodies. Mouse anti-TAMRA antibody was diluted to 1:1000. Rabbit anti-β-actin antibody was diluted to 1:5000, corresponding to the internal control. Photocatalysts IR1, IR23, IR24, and IR42 were used.

[0602] for Fig.21A , samples were treated with catalysts for either 1 hour or 2 hours. For the 1 hour samples, IR1-5μM and IR1-10μM showed slightly less binding to the Halo tag compared to the DMSO control, while the other three photocatalysts (IR23, IR24, and IR42) appeared to bind completely to the Halo tag. Without being bound by a particular theory, these results may indicate that IR24, IR42, and IR23 are more cell permeable than IR1.

[0603] for Fig.21B , 0.5 μM and 1 μM photocatalyst concentrations did not appear to fully bind the Halotag. 5 μM of IR42 showed complete binding. 5 μM of IR1 showed majority binding. Without being bound by a particular theory, these results suggest that IR42 has a higher cell permeability than IR1.

Claims

1. A method for labeling intracellular molecules based on proximity, characterized in that: include: introducing a binding agent complex into a cell of a cell, wherein the binding agent complex comprises a protein of interest coupled to a binding agent, wherein the binding agent is capable of binding to the catalyst complex; introducing the catalyst complex into the cell, thereby forming a biomolecular antenna comprising the catalyst complex and the binding agent complex; introducing into the cell a labeling agent comprising a labeling moiety and an active moiety, wherein the active moiety is configured to be activated by the catalyst complex to an active state; as well as The catalyst complex is activated, thereby activating the labeling agent by transferring energy from the catalyst complex to the active moiety and causing the labeling agent to bind to a biomolecule within the cell.

2. The method according to claim 1, characterized in that Introducing the binding agent complex into the cell comprises introducing a nucleotide construct into the cell, wherein the nucleotide construct encodes the binding agent complex, and wherein the nucleotide construct is expressed by the cell.

3. The method according to claim 2, characterized in that Introducing a nucleotide construct into a cell involves transfecting the cell.

4. The method according to claim 1, characterized in that The biomolecules are intracellular proteins, peptides, chromatin or nucleic acids.

5. The method according to claim 1, characterized in that Further comprising imaging the signal from the labeling moiety.

6. The method according to claim 1, characterized in that The catalyst composite includes a photocatalyst.

7. The method according to claim 6, characterized in that The photocatalyst includes a transition metal complex.

8. The method according to claim 7, characterized in that Activating the transition metal complex includes photoactivating the transition metal complex.

9. The method according to claim 8, characterized in that The photoactivation includes irradiating light onto the transition metal complex, wherein the light has a wavelength of about 380 nm to about 700 nm.

10. The method according to claim 8, characterized in that Activating the catalyst complex results in a Dexter energy transfer from the activated transition metal complex to the active moiety to form an active intermediate.

11. The method according to claim 1, characterized in that When the labeling agent binds the labeling moiety to the biomolecule, the biomolecule is within a 10 nm radius of the biomolecule antenna.

12. A proximity-based system for labeling intracellular molecules, characterized in that: include: cell; A biomolecular antenna comprising: A photocatalyst composite comprising a photocatalyst and a ligand portion, and a binding agent complex comprising a protein of interest coupled to a binding agent, wherein the binding agent is capable of binding to the ligand portion of the photocatalyst complex; as well as a labeling agent comprising a labeling portion and an active portion, wherein the active portion is configured to be activated to an active state by the photocatalyst composite; wherein the biomolecule antenna and the labeling agent are each located within the cell.

13. The system of claim 12, wherein: The binding agent includes a haloalkane dehalogenase.

14. The system of claim 13, wherein: The ligand moiety includes an alkyl chloride.

15. The system of claim 14, wherein: The alkyl chloride contains 6 or more carbons.

16. The system of claim 12, wherein: The binding agent is coupled to the target protein at the N-terminus or C-terminus of the target protein.

17. The system of claim 12, wherein: The ligand moieties include 4,4'-di-tert-butyl-2,2'-bipyridine, 2,2'-bipyridine, diphenhydramine-2,2'-bipyridine, 4-4'-dimethoxy-2-2'-bipyridine, dinaphthyl-pyrene, phenanthroline and biphenyl-phenanthroline.

18. The system of claim 12, wherein: The photocatalyst includes a transition metal.

19. The system of claim 18, wherein: The transition metal has a triplet energy greater than 60 kcal / mol.

20. The system of claim 18, wherein: The transition metal is a platinum group metal.

21. The system of claim 20, wherein: The transition metal is iridium or ruthenium.

22. The system of claim 21, wherein: The transition metal is hexacoordinated.

23. The system of claim 18, wherein: The transition metal absorbs light having a wavelength of about 380 nm to about 700 nm.

24. The system of claim 18, wherein: The transition metal has a value greater than 1000 M -1 cm -1 Visible light extinction coefficient.

25. The system of claim 12, wherein: The photocatalyst is an organic catalyst that does not include a transition metal.

26. The system of claim 25, wherein: The organic catalyst includes 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, and a benzophenone group.

27. The system of claim 12, wherein: The labeling agent is cell permeable.

28. The system of claim 12, wherein: The photocatalyst is capable of activating the marking agent to form a reactive intermediate.

29. The system of claim 28, wherein: The photocatalyst is capable of activating the labeling agent via Dexter energy transfer to form the active intermediate.

30. The system of claim 28, wherein: The reactive intermediate has a diffusion radius of less than 10 nm before quenching.

31. The system of claim 28, wherein: The active intermediate has a half-life (t 1 / 2 ).

32. The system of claim 12, wherein: The binding agent is a protein, an E3 ligase, a polysaccharide or a nucleic acid.

33. The system of claim 12, wherein: The target protein is K-Ras, cMyc, Src, WRN, Slug, PARP1, Aβ, Tau, influenza hemagglutinin or viral nucleoprotein.

34. A biomolecule component, characterized in that It contains a dehalogenase and a photocatalyst coupled to a target protein.

35. The biomolecule assembly of claim 34, wherein: The photocatalyst is configured to activate a marking agent comprising a marking portion and an active portion.

36. The biomolecule assembly of claim 35, wherein The photocatalyst is configured to activate the marking agent upon absorbing light having a wavelength of about 380 nm to about 700 nm.

37. The biomolecule assembly of claim 35, wherein: The photocatalyst includes a transition metal.

38. The biomolecule assembly of claim 37, wherein The transition metal is a platinum group metal.

39. The biomolecule assembly of claim 38, wherein The transition metal is iridium, tin or ruthenium.

40. The biomolecule assembly of claim 37, wherein The transition metal is hexacoordinated.

41. The biomolecule assembly of claim 37, wherein The transition metal has a value greater than 1000 M - 1 cm -1 Visible light extinction coefficient.

42. The biomolecule assembly of claim 34, wherein: The photocatalyst is an organic catalyst that does not include a transition metal.

43. The biomolecule assembly of claim 42, wherein: The organic catalyst includes 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.

44. The biomolecule assembly of claim 34, wherein Further included is a photocatalyst composite, wherein the photocatalyst composite comprises the photocatalyst and a ligand moiety.

45. The biomolecule assembly of claim 44, wherein The ligand moiety includes an alkyl chloride.

46. ​​The biomolecule assembly of claim 45, wherein The alkyl chloride contains 6 or more carbons.

47. A method for detecting protein-protein interactions, characterized in that include: expressing a first protein within a cell of the cell, the first protein coupled to a binding agent capable of binding to a catalyst complex, wherein the catalyst complex is a photocatalyst; introducing the photocatalyst into the cell so that it binds to the first protein; introducing a labeling agent comprising a labeling portion and an active portion into the cell; activating the photocatalyst, thereby activating the labeling agent by transferring energy from the photocatalyst to the active portion and causing the labeling agent to bind to a second protein within the cell; as well as detecting the second protein by detecting the labeling portion; The photocatalyst has a structure of formula (I): in: A1 is present 0 to 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 straight or branched chain alkyl group having 1 to 12 carbons, CHF2, and CF3; A2 is present 0 to 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 to 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 selected from none, a straight or branched alkyl group having 1 to 12 carbon atoms, 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, halogen, hydroxyl, C 1-6 Alkoxy or amino substitution; A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1-6 Alkoxy, and A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a l (CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, wherein 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)imide chloride, tetraphenylphosphine chloride and tetrabutylammonium; Each of A11 and A12 is independently C or N coordinated to A9; and Each A13 is independently CH or N.

48. The method of claim 47, wherein: The photocatalyst has the following structure:

49. The method of claim 47, wherein: The photocatalyst has the following structure:

50. The method of claim 47, wherein: The photocatalyst has the following structure:

51. The method of claim 47, wherein: The photocatalyst has the following structure:

52. The method of claim 47, wherein: The photocatalyst has the following structure:

53. The method of claim 47, wherein: The photocatalyst has the following structure:

54. The method of claim 47, wherein: The photocatalyst has the following structure:

55. The method of claim 47, wherein: The photocatalyst has the following structure:

56. The method of claim 47, wherein: The photocatalyst has the following structure:

57. The method of claim 47, wherein: The photocatalyst has the following structure:

58. The method of claim 47, wherein: A3 is present 0 to 4 times on the ring to which it is attached, and each A3 is independently selected from CH3, CF3, F, Cl and OR1.

59. The method of claim 47, wherein: A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH and NHCOO.

60. The method of claim 47, wherein: A6 is (PEG)a1(CH2)a2Cl, a1 is an integer from 0 to 10, and a2 is an integer from 6 to 10.

61. The method of claim 47, wherein: A13 is CH.

62. The method of claim 47, wherein: The 5- to 6-membered heterocyclic group of A6 is piperazine or pyrrolidine.

63. A method for detecting protein-protein interactions, characterized in that include: expressing a first protein within a cell of the cell, the first protein being coupled to a binding agent capable of binding to the catalyst complex; introducing the catalyst complex into the cell so that it binds to the first protein; introducing a labeling agent comprising a labeling portion and an active portion into the cell; activating the catalyst complex, thereby activating the labeling agent by transferring energy from the catalyst complex to the active moiety and causing the labeling agent to bind to a second protein within the cell; as well as detecting the second protein by detecting the labeling portion; The marking agent has a structure of formula (III-a): in: R 1 ' is selected from azide, methyldiaziridine, trifluoromethyldiaziridine and phenyldiaziridine; Ring Ar is selected from phenyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, 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 a biotin-linked amide or an amide-linked fluorescent dye.

64. The method of claim 63, wherein: The marking agent has the following structure:

65. The method of any one of claims 47 to 64, wherein: The first protein is a ubiquitin ligase.

66. A method for detecting protein-protein interactions, characterized in that include: expressing a first protein within a cell of the cell, the first protein being coupled to a binding agent capable of binding to the catalyst complex, wherein the first protein is a ubiquitin ligase; introducing the catalyst complex into the cell so that it binds to the first protein; introducing a labeling agent comprising a labeling portion and an active portion into the cell; activating the catalyst complex, thereby activating the labeling agent by transferring energy from the catalyst complex to the active moiety and causing the labeling agent to bind to a second protein within the cell; as well as The second protein is detected by detecting the label moiety.

67. The method according to any one of claims 63 to 66, characterized in that The catalyst complex is a photocatalyst.

68. The method of claim 67, wherein: The photocatalyst has a structure of formula (I): in: A1 is present 0 to 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 straight or branched chain alkyl group having 1 to 12 carbons, CHF2, and CF3; A2 is present 0 to 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 to 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 selected from none, a straight or branched alkyl group having 1 to 12 carbon atoms, 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, halogen, hydroxyl, C 1-6 Alkoxy or amino substitution; A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1-6 Alkoxy, and A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a l (CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, wherein 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 the group consisting of 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)imide chloride, tetraphenylphosphine chloride and tetrabutylammonium; A11 and A12 are independently C or N coordinated to A9; and Each A13 is independently CH or N.

69. The method of claim 68, wherein: The photocatalyst has the following structure:

70. The method of claim 68, wherein: The photocatalyst has the following structure:

71. The method of claim 68, wherein: The photocatalyst has the following structure:

72. The method of claim 68, wherein: The photocatalyst has the following structure:

73. The method of claim 68, wherein: The photocatalyst has the following structure:

74. The method of claim 68, wherein: The photocatalyst has the following structure:

75. The method of claim 68, wherein: The photocatalyst has the following structure:

76. The method of claim 68, wherein: The photocatalyst has the following structure:

77. The method of claim 68, wherein: The photocatalyst has the following structure:

78. The method of claim 68, wherein: The photocatalyst has the following structure:

79. The method of claim 68, wherein: A3 is present 0 to 4 times on the ring to which it is attached, and each A3 is independently selected from CH3, CF3, F, Cl and OR1.

80. The method of claim 68, wherein: A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH and NHCOO.

81. The method of claim 68, wherein: A6 is (PEG)a1(CH2)a2Cl, wherein a1 is an integer from 0 to 10, and a2 is an integer from 6 to 10.

82. The method of claim 68, wherein: A13 is CH.

83. The method of claim 68, wherein: The 5- to 6-membered heterocyclic group in A6 is piperazine or pyrrolidine.

84. The method of any one of claims 47, 63 and 66, wherein: The marking agent has a structure of formula (III-a): in: R 1 ' is selected from azide, methyldiaziridine, trifluoromethyldiaziridine and phenyldiaziridine; Ring Ar is selected from phenyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, 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 a biotin-linked amide or an amide-linked fluorescent dye.

85. The method of claim 84, wherein: The marking agent has the following structure:

86. The method of any one of claims 47 to 85, wherein: The method is performed in the absence of exogenous compounds that promote interaction between the first and second proteins.

87. The method of any one of claims 47 to 85, wherein: The method is performed in the presence of a test compound, wherein detecting the second protein or the level of the second protein indicates that the test compound promotes the interaction between the first and second proteins.

88. The method of any one of claims 47 to 87, wherein: During activation of the catalyst complex, the cells are living cells.

89. The method according to any one of claims 47 to 88, wherein: The binding agent includes a haloalkane dehalogenase.

90. The method according to any one of claims 47 to 89, wherein: Activating the catalyst complex comprises shining light onto the cell, wherein the light has a wavelength of about 380 nm to about 700 nm.

91. The method of any one of claims 47 to 90, wherein: Activating the catalyst complex causes a Dexter energy transfer from the activated catalyst complex to the active moiety to form an active intermediate.

92. A cell, characterized in that Include: a first protein coupled to a haloalkane dehalogenase; A photocatalyst having the structure of formula (I): in: A1 is present 0 to 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 straight or branched chain alkyl group having 1 to 12 carbons, CHF2, and CF3; A2 is present 0 to 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 to 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 selected from none, a straight or branched alkyl group having 1 to 12 carbon atoms, 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, halogen, hydroxyl, C 1-6 Alkoxy or amino substitution; A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1-6 Alkoxy, and A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a l (CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, wherein 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 the group consisting of 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; and A11 and A12 are independently C or N coordinated to A9; Each A13 is independently CH or N; and A labeling agent having the structure of formula (III-a): in: R 1 ' is selected from azide, methyldiaziridine, trifluoromethyldiaziridine and phenyldiaziridine; Ring Ar is selected from phenyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazine, 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 a biotin-linked amide or an amide-linked fluorescent dye.

93. The cell of claim 92, wherein The photocatalyst has the following structure:

94. The cell of claim 92, wherein The photocatalyst has the following structure:

95. The cell of claim 92, wherein The photocatalyst has the following structure:

96. The cell of claim 92, wherein The photocatalyst has the following structure:

97. The cell of claim 92, wherein The photocatalyst has the following structure:

98. The cell of claim 92, wherein The photocatalyst has the following structure:

99. The cell of claim 92, wherein The photocatalyst has the following structure:

100. The cell of claim 92, wherein The photocatalyst has the following structure:

101. The cell of claim 92, wherein The photocatalyst has the following structure:

102. The cell of claim 92, wherein The photocatalyst has the following structure:

103. The cell of claim 92, wherein A3 is present 0 to 4 times on the ring to which it is attached, and each A3 is independently selected from CH3, CF3, F, Cl and OR1.

104. The cell of claim 92, wherein A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH and NHCOO.

105. The cell of claim 92, wherein A6 is (PEG)a1(CH2)a2Cl, wherein a1 is an integer from 0 to 10, and a2 is an integer from 6 to 10.

106. The cell of claim 92, wherein A13 is CH.

107. The cell of claim 92, wherein The 5- to 6-membered heterocyclic group in A6 is piperazine or pyrrolidine.

108. The cell of any one of claims 92-107, wherein The marking agent has the following structure:

109. A protein complex, characterized in that Contains this structure: P1-P2-Cat in: P1 is a ubiquitin ligase; P2 is a haloalkane dehalogenase; Cat is a photocatalyst having the structure of formula (I): in: A1 is present 0 to 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 straight or branched chain alkyl group having 1 to 12 carbons, CHF2, and CF3; A2 is present 0 to 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 to 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 selected from none, a straight or branched alkyl group having 1 to 12 carbon atoms, 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, halogen, hydroxyl, C 1-6 Alkoxy or amino substitution; A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1-6 Alkoxy, and A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a l (CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, wherein 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 the group consisting of 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; and Each A13 is independently CH or N.

110. The protein complex of claim 109, wherein Cat has the following structure:

111. The protein complex of claim 109, wherein Cat has the following structure:

112. The protein complex of claim 109, wherein Cat has the following structure:

113. The protein complex of claim 109, wherein Cat has the following structure:

114. The protein complex of claim 109, wherein Cat has the following structure:

115. The protein complex of claim 109, wherein Cat has the following structure:

116. The protein complex of claim 109, wherein Cat has the following structure:

117. The protein complex of claim 109, wherein Cat has the following structure:

118. The protein complex of claim 109, wherein Cat has the following structure:

119. The protein complex of claim 109, wherein Cat has the following structure:

120. The protein complex of claim 109, wherein A3 is present 0 to 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.

121. The protein complex of claim 109, wherein A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH and NHCOO.

122. The protein complex of claim 109, wherein A6 is (PEG)a1(CH2)a2Cl, wherein a1 is an integer from 0 to 10, and a2 is an integer from 6 to 10.

123. The protein complex of claim 109, wherein A13 is CH.

124. The protein complex of claim 109, wherein The 5- to 6-membered heterocyclic group in A6 is piperazine or pyrrolidine.

125. A cell, characterized in that Include: The protein complex of any one of claims 109-119; and A labeling agent having the structure of formula (III-a): in: R 1 ' is selected from azide, methyldiaziridine, trifluoromethyldiaziridine and phenyldiaziridine; Ring Ar is selected from phenyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, 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 a biotin-linked amide or an amide-linked fluorescent dye.

126. A cell, characterized in that The invention comprises a nucleotide sequence for expressing a fusion protein comprising a ubiquitin ligase and a haloalkane dehalogenase.

127. A photocatalyst having a structure of formula (I): in: A1 is present 0 to 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 straight or branched chain alkyl group having 1 to 12 carbons, CHF2, and CF3; A2 is present 0 to 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 to 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 selected from none, a straight or branched alkyl group having 1 to 12 carbon atoms, 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, halogen, hydroxyl, C 1-6 Alkoxy or amino substitution; A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH, NHCOO, C 1-6 Alkoxy, and A6 is independently (PEG)a1(CH2)a2Cl or (PEG)a l (CH2)a2(5- to 6-membered heterocyclyl)(PEG)a1(CH2)a2Cl, wherein 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 the group consisting of 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; and Each A13 is independently CH or N, wherein the photocatalyst is not 128. The photocatalyst according to claim 127, characterized in that A3 is present 0 to 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.

129. The photocatalyst according to claim 127, characterized in that A5 is selected from CONH, NHCO, SONH, SO2NH, NHSO, NHSO2, NH, OCONH and NHCOO.

130. The photocatalyst according to claim 127, wherein A6 is (PEG)a1(CH2)a2Cl, wherein a1 is an integer from 0 to 10, and a2 is an integer from 6 to 10.

131. The photocatalyst according to claim 127, wherein A13 is CH.

132. The photocatalyst according to claim 127, wherein The 5- to 6-membered heterocyclic group in A6 is piperazine or pyrrolidine.

133. The photocatalyst according to claim 127, characterized in that The photocatalyst has the following structure:

134. The photocatalyst according to claim 127, wherein The photocatalyst has the following structure:

135. The photocatalyst according to claim 127, wherein The photocatalyst has the following structure:

136. The photocatalyst according to claim 127, characterized in that The photocatalyst has the following structure:

137. The photocatalyst according to claim 127, wherein The photocatalyst has the following structure:

138. The photocatalyst according to claim 127, wherein The photocatalyst has the following structure:

139. The photocatalyst according to claim 127, wherein The photocatalyst has the following structure:

140. The photocatalyst according to claim 127, wherein The photocatalyst has the following structure:

141. The photocatalyst according to claim 127, wherein The photocatalyst has the following structure:

142. The photocatalyst according to claim 127, wherein The photocatalyst has the following structure:

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