Compositions and systems for inter- and intracellular proximity-based labeling

Abstract

In one aspect, transition metal complexes are described that have compositions and electronic structures to generate reactive labeled intermediates with lifetimes and diffusion radii that are favorable for proximity-based labeling of a variety of biomolecular species, including proteins, in intracellular and intercellular environments.

Description

[Technical Field]

[0001] Related application data This application claims priority under Patent Cooperation Treaty Article 8 to U.S. Provisional Patent Application No. 62 / 982,366, filed February 27, 2020, and U.S. Provisional Patent Application No. 63 / 076,658, filed September 10, 2020, each of which is incorporated herein by reference in its entirety.

[0002] Government Rights Statement This invention was made with government support under Grant No. 5R01GM103558-08 awarded by the National Institutes of Health and the National Institute of General Medical Sciences. The government has certain rights in the invention.

[0003] The present invention relates to compositions, systems, and methods for proximity-based labeling, and in particular to transition metal catalysts for inter- and intracellular proximity-based labeling. [Background technology]

[0004] Protein proximity labeling has emerged as a powerful approach for profiling protein interaction networks. The ability to label accompanying or bystander proteins via proximity labeling can have significant implications for further understanding the cellular environment and biological role of a protein of interest. Current proximity labeling methods all involve the use of enzyme-generated reactive intermediates that label neighboring proteins on a few selected amino acid residues through diffusion or physical contact. Despite the transformative impact of this technology, the generation of phenoxy radicals (t) via peroxidase activation remains a challenge. 1 / 2 > 100 μs) and biotin-AMP ligated via biotin ligase (t 1 / 2The inherent stability of these reactive intermediates (e.g., >60 s) can facilitate diffusion far from their source. As a result, these enzyme-generated reactive intermediates pose challenges for profiling within tight microenvironments. Furthermore, the large enzyme size, dependency on specific amino acids for labeling, and the inability to temporally control these labeling systems present further challenges for profiling within confined spatial regions. Given these limitations, new approaches to proximity-based labeling are needed. Summary of the Invention

[0005] In one aspect, transition metal complexes are described herein that have a composition and electronic structure to generate reactive labeled intermediates with lifetimes and diffusion radii that are favorable for proximity-based labeling of various biomolecular species, including proteins. In some embodiments, the transition metal catalyst is represented by Formula I: [ka] During the ceremony, M is a transition metal; A, D, E, G, Y, and Z are independently selected from C and N; R 3 ~R 7 each represent 1 to 4 optional ring substituents, each of the 1 to 4 optional ring substituents independently being alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 , and -R 9 OH, where R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; R 1 is a direct bond, alkylene, alkenylene, cycloalkenylene, Ruki Ren (cycloa lk ylene), selected from the group consisting of cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; L is a linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; R 2 is an alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O2 ) R 9 , thiol, biotin, oxyamine, and haloalkyl; R 8 and R 9 is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; and X - is a counterion, and n is an integer from 0 to 20.

[0006] As further described herein, the polarity of the transition metal complex is determined by the R 3 ~R 7 In some embodiments, the R 3 ~R 7 One or more of R are selected to exhibit hydrophilicity via charged and / or polar chemical moieties. In such embodiments, the transition metal complex may exhibit hydrophilicity suitable for placement in an intercellular or extracellular aqueous environment. Alternatively, R 3 ~R 7 One or more of R are selected to exhibit hydrophobic, lipophilic, or non-polar properties. For example, in some embodiments, R 3 ~R 7One or more of may be alkyl, fluoro, or fluoroalkyl. The transition metal complexes described herein that exhibit hydrophobic, lipophilic, or non-polar properties may be suitable for placement or passage into the intracellular environment. The transition metal complexes can pass through cell membranes for mapping the local intracellular environment according to the principles described herein. Thus, such transition metal complexes are cell-permeable.

[0007] Additionally, in some embodiments, the transition metal complex has a triplet energy state greater than 60 kcal / mol. The metal center, in some embodiments, can be selected from the platinum group of transition metals. For example, the metal center can be iridium. In some embodiments, n in Formula I is 1 to 20.

[0008] In another aspect, compositions and methods are described herein for providing a microenvironment mapping platform operable to selectively identify various features, including protein-protein interactions on cell membranes and protein, nucleic acid, and / or other biomolecular interactions within cells. In some embodiments, the composition includes a transition metal catalyst of Formula I and a protein labeling agent, where the transition metal catalyst activates the protein labeling agent to a reactive intermediate. In some embodiments, the transition metal catalyst of Formula I can have an electronic structure that allows energy transfer to the protein labeling agent to form the reactive intermediate. The reactive intermediate reacts or crosslinks with proteins or other biomolecules within the diffusion radius of the reactive intermediate. In the absence of proteins or other biomolecules within the diffusion radius, the reactive intermediate is quenched by the surrounding environment. As further described herein, the diffusion radius of the reactive intermediate can be tailored to specific microenvironment mapping considerations and can be limited to the nanometer scale. For example, in some embodiments, the diffusion radius can be less than 10 nm or less than 5 nm. Furthermore, in some embodiments, the reactive intermediate can have a half-life of less than 5 ns. In some embodiments, the protein labeling agent can be functionalized with a marker, such as biotin or a luminescent marker, to aid in analysis. Energy transfer from the catalyst to the protein labeling agent can occur via a variety of mechanisms, further described herein, including Dexter energy transfer.

[0009] In another aspect, conjugates for use in proximity-based labeling systems are described herein. The conjugates include those in which a transition metal complex is coupled to a biomolecule binding agent, where the transition metal complex prior to coupling to the biomolecule binding agent is represented by Formula I above. As further detailed herein, the biomolecule binding agent can be utilized to position the transition metal complex within a desired intracellular or intercellular / extracellular environment for proximity labeling and associated analysis. The biomolecule binding agent can exhibit selective binding to direct the conjugate to a desired location for proximity-based labeling and associated micromapping of the intercellular / extracellular environment (including the cell membrane). Alternatively, the biomolecule binding agent can exhibit selective binding to direct the conjugate to a desired location for proximity-based labeling and associated micromapping of the intracellular environment, including the perinuclear environment as well as various organelle environments. The biomolecule binding agent can include, for example, a peptide, a protein, a sugar, a small molecule, a nucleic acid, or a combination thereof. As further described herein, the transition metal complex can include a reactive functional group for coupling to a biomolecule binding agent, including click chemistry. In some embodiments, the transition metal complexes can couple to biomolecule binding agents in the absence of copper. The conjugates described herein can be used with protein labeling agents for the systems for cell proximity-based labeling detailed above.

[0010] In a further aspect, methods of proximity-based labeling are described herein. The methods of proximity-based labeling include providing a transition metal catalyst of formula (I) and using the catalyst to activate a protein labeling agent to a reactive intermediate. The reactive intermediate is coupled or bound to the protein. In some embodiments, the transition metal catalyst is coupled to a biomolecular binding agent to selectively position or target the catalyst to a specific environment for protein mapping with the protein labeling agent. The transition metal catalyst, conjugate, and protein labeling agent can have the compositions and / or properties described above and in the detailed description below.

[0011] These and other embodiments are further described in the detailed description below. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 illustrates a transition metal catalyst described herein, according to some embodiments. [Figure 2] FIG. 2 shows transition metal catalysts and conjugates described herein, according to some embodiments. [Figure 3] FIG. 3 shows a cell-permeable conjugate comprising a transition metal catalyst and a JQ1 biomolecule binder, according to some embodiments. [Figure 4] FIG. 4 shows a synthetic scheme for producing the cell-permeable conjugate of FIG. 3, according to some embodiments. [Figure 5] Figure 5A is a Western blot of intracellular labeling using a conjugate described herein, according to some embodiments, and Figure 5B shows the results of densitometry analysis of the Western blot in Figure 5A. [Figure 6] FIG. 6 shows the results of time-dependent labeling of BRD4 in HeLa cells. [Figure 7] FIG. 7 shows a non-cell-permeable conjugate. [Figure 8] FIG. 8 shows the BRD4 labeling results between the cell-permeable conjugate of FIG. 3 and the non-cell-permeable conjugate of FIG. [Figure 9] FIG. 9 shows the structure of a (−)-JQ1 conjugate and BRD4 labeling compared to a (+)-JQ1 conjugate, according to some embodiments. [Figure 10] 10A-10C show volcano plots of significance versus fold enrichment for a target bromodomain protein using conjugates described herein, according to some embodiments. [Figure 11]FIG. 11 shows a synthetic route for the conjugates described herein, according to some embodiments. [Figure 12] FIG. 12 provides a volcano plot of significance versus fold enrichment for the target tubulin protein in MCF-7 cells using the cell-permeable conjugate of FIG. 11, according to some embodiments. [Figure 13] FIG. 13 shows confocal microscopy images of intracellular labeling with the conjugate of FIG. 3 at different time points, according to some embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0013] The embodiments described herein can be more readily understood by reference to the following detailed description and examples and the above and following descriptions thereof. However, the elements, devices, and methods described herein are not limited to the specific embodiments presented in the detailed description and examples. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations will be readily apparent to those skilled in the art without departing from the spirit and scope of the present invention.

[0014] [Definition] The term "alkyl," as used herein, alone or in combination, refers to a straight-chain or branched saturated hydrocarbon group, optionally substituted with one or more substituents. For example, an alkyl group can be C1-C 30 or C1-C 18 It could be.

[0015] The term "alkenyl," as used herein, alone or in combination, refers to a straight-chain or branched-chain hydrocarbon group having at least one carbon-carbon double bond and optionally substituted with one or more substituents.

[0016] The term "alkynyl," as used herein, alone or in combination, refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon triple bond and optionally substituted with one or more substituents.

[0017] The term "aryl," as used herein, alone or in combination, refers to an aromatic monocyclic or polycyclic ring system optionally substituted with one or more ring substituents.

[0018] The term "heteroaryl," as used herein, alone or in combination, refers to an aromatic monocyclic or polycyclic ring system in which one or more of the ring atoms is an element other than carbon, such as, for example, nitrogen, boron, oxygen, and / or sulfur.

[0019] The term "heterocycle," as used herein alone or in combination, refers to a monocyclic or polycyclic ring system in which one or more atoms of the ring system are elements other than carbon (e.g., boron, nitrogen, oxygen, and / or sulfur or phosphorus), and the ring system is optionally substituted with one or more ring substituents. Heterocyclic ring systems can include aromatic and / or non-aromatic rings, including rings with one or more points of unsaturation.

[0020] The term "cycloalkyl," as used herein, alone or in combination, refers to a non-aromatic mono- or multicyclic ring system, optionally substituted with one or more ring substituents.

[0021] The term "heterocycloalkyl," as used herein, alone or in combination, refers to a non-aromatic monocyclic or polycyclic ring system in which one or more atoms of the ring system is an element other than carbon (e.g., boron, nitrogen, oxygen, sulfur, or phosphorus, alone or in combination), and the ring system is optionally substituted by one or more ring substituents.

[0022] The term "alkoxy," as used herein, alone or in combination, refers to the moiety RO--, where R is alkyl, alkenyl, or aryl as defined above.

[0023] The term "halo," as used herein alone or in combination, refers to an element of Group VIIA of the periodic table (the halogens). Depending on the chemical environment, a halo can be in a neutral or anionic state.

[0024] Terms not specifically defined herein are given their ordinary meaning in the art.

[0025] [I. Transition Metal Complexes] In one aspect, transition metal complexes are described herein that have a composition and electronic structure to generate reactive labeled intermediates with lifetimes and diffusion radii that are favorable for proximity-based labeling of various biomolecular species, including proteins. In some embodiments, the transition metal catalyst is represented by Formula I: [ka] During the ceremony, M is a transition metal; A, D, E, G, Y, and Z are independently selected from C and N; R 3 ~R 7 each represent 1 to 4 optional ring substituents, each of the 1 to 4 optional ring substituents independently being alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 , and -R 9 OH, where R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; R 1 is a direct bond, alkylene, alkenylene, cycloalkenylene, Ruki Ren (cycloa lk ylene), cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; L is a linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; R 2 is an alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O2 ) R 9 , thiol, biotin, oxyamine, and haloalkyl; R 8 and R 9 is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; and X - is a counterion, and n is an integer from 0 to 20.

[0026] Optional Substituent R 3 -R 7 When X is not present, it is understood that a hydrogen occupies a position on the aryl ring of formula I. Additionally, in some embodiments, a counterion (X - ) is tetraalkylborate, tetrafluoroborate, tetraphenylborate, PF6 - and chloride.

[0027] The polarity of the transition metal complex is R 3 -R 7 In some embodiments, the R 3 -R 7 One or more of R are selected to exhibit hydrophilicity via charged and / or polar chemical moieties. In such embodiments, the transition metal complex may exhibit hydrophilicity suitable for placement in an intercellular / extracellular environment. For example, the transition metal complex shown in Figure 2 incorporates charged and polar chemical moieties for aqueous intercellular environments. Alternatively, R 3 -R 7One or more of R are selected to exhibit hydrophobic, lipophilic, or non-polar properties. For example, in some embodiments, R 3 -R 7 One or more of the groups can be alkyl, fluoro, or fluoroalkyl. Figure 1 shows a non-limiting embodiment of a transition metal complex comprising an alkyl, fluoro, or fluoroalkyl substituent. Transition metal complexes described herein that exhibit hydrophobic, lipophilic, or nonpolar properties may be suitable for placement in intracellular environments. As shown in the examples herein, transition metal complexes can cross cell membranes to map the local intracellular environment according to the principles described herein. In some embodiments, for example, a cell-permeable transition metal complex of Formula I has a water solubility of less than 150 μM in 0.2% DMSO in pure water. In some embodiments, a transition metal complex of Formula I has a water solubility of less than 100 μM. Transition metal complexes of Formula I that exhibit hydrophobic, lipophilic, or nonpolar properties may have a water solubility of 1 μM to 150 μM or 1 μM to 100 μM in 0.2% DMSO in pure water. Water solubility can be determined by the retention time of the transition metal complex on a C18 column (HPLC). The above water solubility values ​​also apply to the conjugates described herein, including those in which a transition metal complex is coupled to a biomolecule binding agent.

[0028] The transition metal catalysts described herein are utilized in compositions to provide operable microenvironmental mapping platforms for selectively identifying various features, including protein-protein interactions on cell membranes. In some embodiments, the compositions include a transition metal catalyst of Formula I and a protein labeling agent, where the transition metal catalyst activates the protein labeling agent to a reactive intermediate. In some embodiments, the transition metal catalyst of Formula I can have an electronic structure that allows energy transfer to the protein labeling agent to form a reactive intermediate. The reactive intermediate reacts or crosslinks with proteins or other biomolecules within its diffusion radius. If a protein or other biomolecules is not within the diffusion radius, the reactive intermediate is quenched by the surrounding environment.

[0029] In some embodiments, energy transfer to the protein labeling agent can be from an excited state of the transition metal catalyst's electronic structure. For example, the excited state of the catalyst can be a singlet excited state or a triplet excited state. The excited state of the catalyst can be generated by one or more mechanisms, including energy absorption by the catalyst. In some embodiments, the catalyst is a photocatalyst, and the excited state is induced by the absorption of one or more photons. In other embodiments, the catalyst can be placed in the excited state by interaction with one or more chemical species in the surrounding environment. Alternatively, energy transfer (including electron transfer) to the protein labeling agent can be from the ground state of the catalyst's electronic structure.

[0030] Energy transfer (including electron transfer) to the protein labeling agent forms a reactive intermediate of the protein labeling agent. The reactive intermediate reacts or crosslinks with proteins or other biomolecules within the diffusion radius of the reactive intermediate. In the absence of proteins or other biomolecules within the diffusion radius, the reactive intermediate is quenched by the surrounding environment. The diffusion radius of the reactive intermediate can be tailored to specific microenvironment mapping (proximity-based labeling) considerations and can be limited to the nanometer scale. For example, in some embodiments, the diffusion radius of the reactive intermediate can be less than 10 nm, less than 5 nm, less than 4 nm, less than 3 nm, or less than 2 nm before quenching in the surrounding environment. Thus, the reactive intermediate reacts or crosslinks with proteins or other biomolecules within the diffusion radius, or is quenched by the surrounding environment in the absence of proteins or biomolecules. In this way, high-resolution mapping of the local environment can be achieved through coordinated efforts between the catalyst and the protein labeling agent. Furthermore, in some embodiments, the reactive intermediate exhibits a t of less than 5 ns, less than 4 ns, or less than 2 ns before quenching. 1 / 2 In additional embodiments, the diffusion radius can be extended to between 5 and 500 nm by extending the half-life of the reactive intermediate.

[0031] Transition metal catalyst-protein labeling agent combinations exhibiting the aforementioned electronic structure characteristics for energy transfer and reactive intermediate generation, as well as the binding of related proteins or biomolecules, can be utilized for microenvironment mapping. In some embodiments, the transition metal complexes of Formula I can exhibit a long-lived triplet excited state (T1) that facilitates energy transfer to the protein labeling agent. For example, the T1 state can have a t of 0.2-2 μs. 1 / 2 The transition metal complexes described herein can be photocatalytic and, in some embodiments, absorb light in the visible region of the electromagnetic spectrum. Absorption of electromagnetic radiation can excite the transition metal complex to the S1 state, followed by quantitative intersystem crossing to the T1 state. The transition metal catalyst can then undergo short-range Dexter energy transfer to the protein labeling agent to return to the ground state, S0. Energy transfer to the labeling agent activates the labeling agent for reaction with proteins or other biomolecules. The T1 state of the transition metal complex can, in some embodiments, be greater than 60 kcal / mol. The metal center can be selected, for example, from the platinum group of transition metals. In some embodiments, the metal center can be iridium.

[0032] 1 and 2 show various transition metal complexes described herein. As shown in FIG. 2 can be selected as a reactive functional group for coupling a biomolecule binding agent. In some embodiments, for example, R 2 R contains one or more click chemistry moieties, including, but not limited to, BCN, DBCO, TCO, tetrazine, alkyne, and azide. As shown in Figure 1, R 2 These click chemistries can be coupled directly to the linker (L) or can be coupled via a heteroatom, aryl, or carbonyl.

[0033] The protein labeling agent receives energy transfer from the transition metal catalyst to form a reactive intermediate. The reactive intermediate reacts or crosslinks with proteins or other biomolecules within the diffusion radius of the reactive intermediate. The diffusion radius of the reactive intermediate is described above. The specific identity of the protein labeling agent can be selected according to several considerations, including the identity of the catalyst, the nature of the reactive intermediate formed, and the lifetime and diffusion radius of the reactive intermediate.

[0034] For example, in embodiments where the transition metal catalyst is a photocatalyst, the protein labeling agent can be diazirine. Triplet energy transfer from the excited state photocatalyst can promote the diazirine to the triplet (T1) state. The diazirine triplet undergoes N2 elimination to release a free triplet carbene, which undergoes spin equilibration on the picosecond timescale to a reactive singlet state (t 1 / 2 <1 ns), which crosslinks with neighboring proteins or is quenched in an aqueous environment. In some embodiments, the extinction coefficient of the transition metal complex is 3 to 5 orders of magnitude greater than that of the diazirine.

[0035] Any diazirine that conforms to the technical principles discussed herein can be used. For example, diazirine sensitization can be extended to various p- and m-substituted aryltrifluoromethyldiazirines with meaningful payloads for microscopy and proteomics applications, including free carboxylic acids, phenols, amines, alkynes, carbohydrates, and biotin groups. Diazirines can be functionalized with markers such as biotin. In some embodiments, the marker is desthiobiotin. The marker can aid in the identification of proteins labeled by the protein labeling agent. For example, this marker can be useful in Western blot and / or other analytical techniques. In addition to biotin and desthiobiotin, markers can include alkyne, azide, FLAG tag, fluorophore, and chloroalkane functional groups.

[0036] In some embodiments where the transition metal catalyst is a photocatalyst, the protein labeling agent can be an azide. Triplet energy transfer from the excited state photocatalyst can promote nitrene formation from the azide. The reactive nitrene can crosslink with neighboring proteins or be quenched in an aqueous environment. Any azide that can be activated to undergo energy transfer with the transition metal photocatalyst to form nitrenes can be utilized. In some embodiments, the azide is an aryl azide.

[0037] II. Conjugates In another aspect, described herein are conjugates for use in proximity-based labeling systems. The conjugates include a transition metal complex coupled to a biomolecule binding agent, where the transition metal complex is of Formula I, as described above, prior to coupling to the biomolecule binding agent. As described in further detail herein, the biomolecule binding agent can be used to position the transition metal catalyst in a desired cellular environment for proximity labeling and associated analysis and mapping. In some embodiments, the desired cellular environment is intracellular. In other embodiments, the desired environment is intracellular. The biomolecule binding agent can exhibit selective binding to direct the conjugate to a desired location for proximity-based labeling and associated micromapping of the intercellular environment.

[0038] The transition metal complex of the conjugate can include any transition metal complex having the structure and / or properties described in Section I above. Furthermore, the biomolecule binder can include a multivalent display system comprising a protein, polysaccharide, or nucleic acid. In some embodiments, the biomolecule binder is biotin or a small molecule ligand having specific binding affinity for a target protein. For example, the biomolecule binder can be an antibody. In some embodiments, the biomolecule binder is a secondary antibody for interacting with a primary antibody bound to a desired antigen. Furthermore, the biomolecule binder can be covalently coupled to the photocatalytic transition metal complex.

[0039] Biomolecule binders can be attached to the transition metal catalyst. In some embodiments, the catalyst includes a reactive handle or functional group for coupling a biomolecule binder. In some embodiments, for example, the catalyst can include one or more click chemistry moieties, including, but not limited to, BCN, DBCO, TCO, tetrazine, alkyne, and azide. Figures 1 and 2 show various transition metal photocatalysts of Formula (I) with reactive functional groups for coupling a biomolecule binder. As shown in Figures 1 and 2, linkers of various lengths can be used between the reactive functional group and the coordinating ligand. The length of the linker, such as an amide or polyamide linker, can be selected according to several considerations, including the steric requirements of the targeting site. Furthermore, in some embodiments, the transition metal complex can be coupled to the biomolecule binder in the absence of copper.

[0040] In some embodiments, the conjugates exhibit polarity suitable for labeling applications in the intracellular environment. Alternatively, the conjugates can be made cell-permeable so that they can cross cell membranes for intracellular labeling applications. In some embodiments, for example, the conjugates can exhibit water solubility values ​​as described in Section I above for cell-permeable transition metal complexes.

[0041] III. Systems for Intracellular Proximity-Based Labeling In another aspect, a system for proximity-based labeling is described herein. The system includes, for example, a protein labeling agent and a transition metal catalyst, wherein the transition metal catalyst has an electronic structure that allows electron transfer to the protein labeling agent to provide a reactive intermediate. The reactive intermediate can then be coupled to proteins or other biomolecules in the local or immediate cellular environment. In some embodiments, the transition metal complex is of Formula I described herein.

[0042] In some embodiments, electron transfer is due to an excited state of the catalyst electronic structure, including a singlet excited state or a triplet excited state. In some embodiments, for example, the excited state of the catalyst can be photoinduced. Alternatively, electron transfer can be from the ground state of the catalyst electronic structure.

[0043] As described herein, electron transfer to the protein labeling agent provides a reactive intermediate. The reactive intermediate can exhibit a diffusion radius consistent with the proximity labeling embodiments detailed herein. The diffusion radius can be limited or restricted by rapid quenching of the reactive intermediate by the surrounding aqueous environment. For example, the reactive intermediate can have a diffusion radius of less than 5 nm, less than 3 nm, or less than 2 nm before quenching in the aqueous environment. Thus, the reactive intermediate reacts or crosslinks with proteins or other biomolecules within the diffusion radius, or is quenched by the aqueous environment in the absence of proteins or biomolecules. In this way, high-resolution mapping of the local environment can be achieved through the coordinated efforts of the catalyst and the protein labeling agent. Additionally, in some embodiments, the reactive intermediate exhibits a t of less than 2 ns before quenching. 1 / 2 In additional embodiments, the diffusion radius can be extended to 5-500 nm through the extension of the half-life of the reactive intermediate.

[0044] Any catalyst-protein labeling agent combination that exhibits the aforementioned electronic structure characteristics for electron transfer and reactive intermediate formation can be utilized for microenvironment mapping. In some embodiments, the catalyst-protein labeling agent combination comprises a transition metal catalyst of Formula I and a diazirine labeling agent. The transition metal catalyst of Formula I can have any of the structures and / or properties described in Section I above. In some embodiments, the protein labeling agent can be functionalized with markers such as biotin or luminescent markers to aid in analysis. Diazirine sensitization can be extended to a variety of p- and m-substituted aryltrifluoromethyldiazirines with useful payloads for microscopy and proteomics applications, such as free carboxylic acids, phenols, amines, alkynes, carbohydrates, and biotin groups. The extinction coefficient of the transition metal catalyst can be five orders of magnitude greater than that of the diazirine at the wavelength (450 nm) emitted by the blue LED used for sensitization, accounting for the absence of background non-catalyzed reactions.

[0045] In some embodiments, multiple protein labeling agents can be used with a transition metal catalyst. In such embodiments, the transition metal catalyst exhibits an electronic structure that allows electron transfer to one or all of the protein labeling agents to provide reactive intermediates. In some embodiments, the reactive intermediates can exhibit different diffusion radii, thereby binding to different proteins or biomolecules at different locations. Such embodiments can enhance the resolution of the intracellular proximity-based labeling system described herein.

[0046] Additionally, the transition metal complexes in the systems contemplated herein can be coupled to biomolecular binding agents to provide conjugates, as described in Section II above. The inclusion of a biomolecular binding agent, along with one or more protein binding agents, can direct the transition metal catalyst to a desired cellular environment for analysis and mapping. In some embodiments, the systems described herein can utilize multiple conjugates and protein labeling agents, where each conjugate and associated protein labeling agent is specific for a different cellular environment.

[0047] IV. Intracellular Proximity-Based Labeling Methods In a further aspect, methods for cell proximity-based labeling are described herein. In some embodiments, the methods include providing a protein labeling agent and a conjugate comprising a transition metal catalyst coupled to a biomolecule binding agent. The protein labeling agent is activated by the transition metal catalyst to a reactive intermediate, which couples with proteins or other biomolecules in the cellular environment. The methods described herein can further include detecting or analyzing proteins that couple to the reactive intermediate, thereby providing a map of the local cellular environment.

[0048] Protein labeling agents and conjugates can have any of the structures, compositions, and / or properties described in any of Sections I-III above.

[0049] These and other embodiments are further illustrated in the examples that follow. [Example]

[0050] Example 1 - Transition Metal Catalyst [Step 1] 3-(4'-methyl-[2,2'-bipyridin]-4-yl)propanoic acid [ka] 3-(4'-Methyl-[2,2']bipyridinyl-4-yl)-propionic acid ethyl ester, 4,4'-dimethyl-2,2'-bipyridyl (2.5 g, 13.5 mmol) was dissolved in dry THF (20 mL) under a nitrogen atmosphere in a flame-dried flask. The solution was cooled to -78 °C, and a solution of LDA (14.8 mmol, 1.1 equiv.) was added. The reaction mixture was allowed to warm to room temperature over 1.5 h. This solution was cannulated into a solution of ethyl 2-bromoacetate (2.3 mL, 20 mmol) in dry THF (15 mL) at -78 °C under N2. The reaction mixture was allowed to slowly reach room temperature overnight and quenched by the addition of saturated sodium bicarbonate solution. After workup with ethyl acetate, the mixture was dried over Na2SO4 and concentrated under reduced pressure to give the crude product. The crude residue was purified by column chromatography (silica gel; DCM:MeOH:NH4OH 95:5:0.5) to give the desired product in 69% yield.

[0051] [Step 2] 5-(4'-methyl-[2,2]bipyridinyl-4-yl)-pent-4-enoic acid The bipyridinyl ethyl ester from Step 1 was taken up in 1:1 THF:water, followed by the addition of LiOH (2 equiv.). The reaction mixture was stirred at room temperature for 16 h (complete by TLC) and then quenched by the addition of NH4Cl (until pH 5-6). The mixture was extracted with EtOAc, dried over Na2SO4, and concentrated under reduced pressure to give the desired product as an off-white powder (63% yield).

[0052] tert-Butyl (2-(3-(4'-methyl-[2,2'-bipyridin]-4-yl)propanamido)ethyl)carbamate [ka] To a 20 mL vial charged with bipyx (228 mg, 1 mmol, 1 equiv.), PyBOP (612 mg, 1.2 mmol, 1.2 equiv.), and tert-butyl (2-aminoethyl)carbamate (192 mg, 1.2 mmol, 1.2 equiv.) was added DMF (2 mL), followed by diisopropylethylamine (347 μL, 0.15 mmol, 3 equiv.). The reaction was stirred for 16 h. The resulting mixture was quenched by the addition of water and EtOAc. The layers were separated, and the organics were washed with saturated NaHCO3, H2O, and brine. The organic layer was then dried over Na2SO4 and concentrated under reduced pressure to give a yellow oil, which was purified by flash column chromatography (silica gel, 0-15% MeOH / CHCl2) to give the desired compound as a yellow solid (380 mg, 99%).

[0053] [Ir-Catalyst X] [ka] To a round-bottom flask charged with bipypy (161 mg, 0.42 mmol, 1.05 equiv.) and Ir[dF(COH-CF)ppy]MeCN (351 mg, 0.4 mmol, 1 equiv.), DCM / EtOH (4 mL, 4:1) was added, and the reaction mixture was stirred at 30 °C for 16 h. The resulting solution was concentrated under reduced pressure directly onto silica gel. The crude product was purified by flash column chromatography (silica gel, 0-25% MeOH / DCM) to give the desired Ir catalyst (200 mg, 42% yield).

[0054] [DBCO Ir catalyst] [ka] A 5 mL vial (wrapped in black tape to protect from light) charged with Ir-cat(catalyst)X (9.4 mg, 0.008 mmol, 1 equiv.) in 500 μL of CHCl was cooled to 0 °C, followed by the addition of trifluoroacetic acid (100 μL). The reaction mixture was warmed to room temperature and stirred until completion (monitored by TLC and HRMS). The completed reaction was concentrated under reduced pressure, and the solid was slurried in MeOH and concentrated under reduced pressure (three more times to remove excess acid).

[0055] The Ir catalyst-trifluoroacetate salt was then dissolved in DMF (500 μL), followed by the addition of diisopropylethylamine (10 μL). To this solution was added DBCO-NHS (6 mg, 0.016 mmol, 2 equiv.), and the solution was stirred in the dark for 3 h. Upon completion (by HRMS / TLC), the reaction mixture was directly purified by flash column chromatography (C18, 5-95% MeCN / HO) to afford the desired compound as a yellow solid (10 mg, 91%).

[0056] Example 2 - Transition Metal Catalyst [Step 1] To a round-bottom flask charged with 3-(4'-methyl-[2,2'-bipyridin]-4-yl)propanoic acid and Ir[dF(CF3)ppy]MeCN2PF6, MeCN / HO (4:1) was added, and the reaction mixture was stirred at 70 °C for 16 h. The resulting solution was concentrated under reduced pressure to give a yellow solid. The crude product was purified by flash column chromatography (silica gel, 0-10% MeOH / DCM) to give the desired acid-containing Ir catalyst (55% yield).

[0057] [Step 2] (For differential activation catalyst): DMF was added to a 20 mL vial charged with Ir catalyst, PyBOP, and amine. The reaction mixture was sparged with N2 in the dark for 10 minutes, and then diisopropylethylamine was added. The reaction was stirred in the dark under a N2 atmosphere for 16 hours. The resulting mixture was quenched by the addition of water and EtOAc. The layers were separated, and the organics were washed with 5% citric acid, saturated NaHCO3, and brine. The organic layer was then dried over Na2SO4 and concentrated under reduced pressure to give the desired compound.

[0058] Example 3 - Cell-permeable conjugate, (+)-JQ1-PEG3-Ir The cell-permeable conjugates containing a transition metal complex and the JQ1 biomolecule binder (Figure 3) were synthesized according to the following protocol. The synthetic scheme for the transition metal complex and the JQ1 biomolecule binder is also shown in Figure 4. To a stirred solution of (+)-JQ1-COH (177 mg, 0.44 mmol) in anhydrous DMF (4.5 mL) was added HATU (176 mg, 0.46 mmol), followed by DIPEA (230 μL, 1.32 mmol). The reaction was stirred at room temperature under N for 10 min, and a solution of t-Boc-N-amido-PEG-amine (143 mg, 0.49 mmol) in anhydrous DMF (0.5 mL) was added dropwise. The resulting mixture was stirred overnight, diluted with EtOAc, and quenched by the addition of saturated aqueous NaHCO. The aqueous phase was removed, and the organic layer was washed with additional saturated aqueous NaHCO, brine, and dried over NaSO. The solvent was removed in vacuo and the crude material was purified by silica column chromatography (gradient elution: 0-10% MeOH / CH2Cl2) to afford (+)-JQ1-PEG3-NHBoc as a tan solid (171 mg, 57%). 1 H NMR (500 MHz, CDCl3)δ: 7.39 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.7 Hz, 2H), 7.20 (br. s, 1H), 5.35 (br. s, 1 H), 4.65 (t, J= 7.1 Hz, 1H), 3.69 - 3.46 (m, 15H), 3.36 (dd, J = 15.0, 6.8 Hz, 1H), 3.30 (m, 2H), 2.65 (s, 3H), 2.39 (s, 3H), 1.66 (s, 3H), 1.41 (s, 9H). 13C NMR (125 MHz, CDCl3)δ: 170.7, 164.0, 156.3, 155.7, 150.0, 136.9, 136.7, 132.2, 131.0, 131.0, 130.6, 130.0, 128.8, 79.2, 70.6, 70.6, 70.4, 70.2, 70.0, 54.5, 40.4, 39.5, 39.0, 28.5, 14.5, 13.2, 11.9. + = 675.29120, [C 32 H 44 ClF 10 N6O6S] + requires 675.27226.

[0059] To a stirred solution of (+)-JQ1-PEG3-NHBoc (146 mg, 0.22 mmol) in CHCl (2 mL) at 0 °C was added TFA (3 mL) dropwise. The reaction mixture was allowed to warm to room temperature overnight, and the solvent was removed in vacuo. The crude mixture was made basic with saturated aqueous NaHCO, extracted with CHCl, and the solvent was removed in vacuo to give (+)-JQ1-PEG3-NH as a tan solid (125 mg, 99%), which was used immediately without further purification.

[0060] To a stirred solution of (+)-JQ1-PEG3-NH2 (32 mg, 56 μmol), Ir-CO2H (61 mg, 56 μmol), and PyBOP (45 mg, 86 μmol) in anhydrous DMF (2 mL) under N2 in the dark was added DIPEA (30 μL, 172 μmol). The resulting mixture was stirred overnight, diluted with EtOAc, and quenched by the addition of saturated aqueous NaHCO3. The aqueous phase was removed, and the organic layer was washed with additional saturated aqueous NaHCO3, 5% aqueous citric acid, brine, and dried over Na2SO4. The solvent was removed in vacuo and the crude material was purified by silica column chromatography (gradient elution: 0-3% MeOH / CHCl) and C8 reverse-phase preparative HPLC (gradient elution: 30-100% MeCN / HO (0.1% formic acid)) to afford (+)-JQ1-PEG-iridium as a yellow solid (25 mg, 27%).1 H NMR (500 MHz, CDCl3)δ: 9.24 - 8.92 (m, 2H), 8.58 - 8.27 (m, 2H), 8.24 (s, 1H), 8.04 (dd, J= 12.2, 8.9 Hz, 2H), 7.79 - 7.66 (m, 2H), 7.62 (s, 1H), 7.55 (s, 1H), 7.49 (t, J= 5.1 Hz, 1H), 7.41 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 8.2 Hz, 2H), 6.63 (dd, J = 12.2 8.8 Hz, 2H), 5.62 (dd, J = 8.0, 2.3 Hz, 2H), 4.80 (br. s, 2H), 4.66 (t, J= 6.9 Hz, 1H), 3.70 - 3.30 (m, 18H), 3.24 - 3.15 (m, 2H), 2.93 - 2.77 (m, 2H), 2.66 (s, 3H), 2.63 (s, 3H), 2.39 (s, 3H), 1.66 (s, 3H). 13C NMR (125 MHz, CDCl3)δ: 171.9, 170.8, 167.0 (dd, J = 258.2, 16.8 Hz), 163.9, 162.7 (dd, J= 262.6, 14.2 Hz), 157.6, 155.6 (d, J = 9.9 Hz), 155.1 (dd, J = 6.9, 28.6 Hz), 154.5, 149.6, 149.1, 145.2 (d, J = 3.2 Hz), 136.8 (d, J= 2.8 Hz), 136.6, 131.1, 130.9, 130.7, 130.0, 129.9, 129.6, 128.8, 127.9, 126.6, 126.4, 126.2, 123.7 (d, J = 22.5 Hz), 121.7 (dd, J = 273.3, 8.9 Hz), 114.2 (ddd, J = 17.1, 10.1, 2.6 Hz), 100.1 (dt, J= 27.0, 9.7 Hz), 70.7, 70.4, 70.3, 69.9, 69.8, 54.5, 39.6, 39.2, 38.9, 35.2, 32.1, 29.8, 29.5, 22.8, 21.8, 14.6, 14.3, 14.3, 13.2, 12.0. 19 F NMR (376 MHz, CDCl3)δ: -62.7 (d, J = 3.1 Hz), -62.7 (s), -72.1 (d, J = 714.3 Hz), -101.6 (dtt, J = 59.3, 12.5, 8.8 Hz), -105.9 - -106.1 (m). m / z HRMS found [M] + = 1507.34161 (100), 1508.33996 (84), 1505.33212 (63), 1506.3 3296 (52), 1509.33644 (72), 1510.33378 (47) [C 65 H 57 ClF 10 IrN 10 O5S] +requires 1507.33876 (100), 1508.34202 (70), 1505.33634 (60), 1506.33969 (42), 1509.33572 (32), 1509.34538 (24), 1510.33907 (23). HPLC (Vydac 218TP C18 HPLC, gradient: 0-90% MeCN / H2O (0.1% TFA) 10 min, 5 min 90% MeCN (0.1% TFA), 1 mL / min, 254 nm): τ r =12.5 min.

[0061] The enantiomer was prepared similarly from (-)-JQ1-CO2H.

[0062] Example 4 - Subcellular microenvironment mapping [Intracellular labeling] JQ1-PEG3-Ir (Example 3) (5 μM) (4 plates, A); Ir-PEG3-NHBoc (5 μM) (4 plates, B); and DMSO (4 plates, C) were added to 12 × 10 cm plates of HeLa cells at 80% confluency in phenol red-free DMEM (Gibco) (4 mL). The plates were incubated at 37°C for 3 hours, and the medium was removed and replaced. Diazirine-PEG3-biotin (250 μM) was added, and the plates were incubated for an additional 20 minutes at 37°C. The plates were then irradiated in a bioreactor (without the lid) for 15 minutes at 450 nM. The medium was removed, and the cells were washed twice with cold DPBS (4°C). The cells were resuspended in cold DPBS (4°C), scraped, and transferred to another 50 mL Falcon tube. Cells were pelleted (1000 g for 5 min at 4 °C) and suspended in 1 mL of cold RIPA buffer containing PMSF (1 mM) and cOmplete EDTA-free protease inhibitor (1x) (Roche). Lysed cells were incubated on ice for 5–10 min and sonicated (35%, 5 x 5 s with a 30 s pause). The lysate was then centrifuged at 15 x 1000 g for 15 min at 4 °C, and the supernatant was collected. The concentration of the cell lysate was measured by BCA assay and adjusted to an equivalent concentration of 1 mg / mL. A control sample (15 µL) was removed from each plex and stored at -20 °C for later analysis.

[0063] [Streptavidin pull-down] Magnetic streptavidin beads (NEB) were removed (250 μL / plex) and washed twice with RIPA (0.5 mL) (incubated on a rotisserie for 5 minutes). The beads were pelleted on a magnetic rack, diluted with the sample (1 mL), and incubated on a rotisserie overnight at 4°C. The beads were pelleted on a magnetic rack, the supernatant removed, and a control sample (15 μL) from each plex was stored at -20°C for later analysis. The beads were then washed once with RIPA (0.5 mL), three times with 1% SDS in DPBS (0.5 mL), three times with 1 M NaCl in DPBS (0.5 mL), three times with 10% EtOH in DPBS, and once with RIPA (0.5 mL). The sample was incubated with each wash for 5 minutes before pelleting. The beads were resuspended in RIPA buffer (300 μL) and transferred to a new 1.5 mL Lobind tube.

[0064] [Western blot analysis] After the final wash and transfer steps for the pulldown, the beads were pelleted on a magnetic rack and the supernatant removed. The beads were gently centrifuged to collect at the bottom of the tube, and 24 μL of freshly prepared elution buffer (30 mM biotin, 6 M urea, 2 M thiourea, 2% SDS in DPBS, pH = 11.5) and 6 μL of 4x Laemli buffer supplemented with BME were added with gentle mixing. The beads were heated at 95°C for 15 minutes, pelleted on a magnetic rack, and the supernatant was removed while still hot and the beads discarded. The sample was allowed to cool to room temperature and centrifuged. Subsequently, 17 μL of the sample was loaded onto a BioRad Criterion 4-20% Tris-glycine gel, along with all appropriate controls, and run in freshly prepared Tris running buffer (160 V, 60 min). The gel was washed (3x MiliQ water) and transferred to an NC membrane via iBlot2. The membrane was washed again (3x MiliQ water) and blocked with Li-COR TBS blocking buffer for 1 hour at room temperature, then incubated overnight at 4°C with anti-BRD4 (A-7, Santa Cruz) (1:500) and anti-histone H3 (polyclonal Invitrogen PA5-16183) (1:2000) in Pierce Protein-Free Blocking (1:2000). The membrane was washed three times with TBST (5 minutes per wash) and five times with MiliQ water, resuspended in Pierce Protein-Free Blocking Buffer with Li-COR secondary antibodies (goat anti-mouse 800) and (goat anti-rabbit 700) (1:12,500) and rocked at room temperature for 1 hour. The membrane was washed three times with TBST (5 minutes per wash) and five times with MiliQ water, and then imaged.

[0065] Figure 5A shows the results of Western blot analysis, and Figure 5B shows the results of Western blot densitometry quantifying the binding of the transition metal complex to the BRD4 protein. As shown in Figure 5B, the cell-permeable conjugate (+)-JQ1-PEG3-Ir of Example 3 herein showed a greater than 2.5-fold increase in BRD4 labeling compared to the transition metal complex lacking the biomolecule binder.

[0066] Example 5 - Time-dependent labeling of BRD4 using (+)-JQ1-PEG3-Ir The intracellular labeling protocol described in Example 4 was followed. The irradiation time was varied (2, 5, and 15 minutes) to demonstrate the degree of biotinylation over time. A control reaction using UV light was performed using a UV photobox, in which the plate was irradiated with 254 nm light for 20 minutes at 4°C. Figure 6 shows the results of time-dependent labeling of BRD4 in HeLa cells. As shown in Figure 6, the cell-permeable conjugate synthesized in Example 3 herein enabled labeling of BRD4 at times of 2, 5, and 15 minutes. In contrast, a transition metal catalyst not functionalized with the JQ1 biomolecule binder failed to produce BRD4 labeling.

[0067] Example 6 - Comparison of labeling with cell-permeable and non-cell-permeable conjugates The cell-impermeable conjugate shown in Figure 7 was prepared as follows. In this example, the cell-impermeable conjugate is designated JQ1-(Gen1)-Ir. (+)-JQ1-CO2H (100 mg, 0.25 mmol), azido-PEG3-amine (60 mg, 0.27 mmol), 1-propanephosphonic anhydride (300 μL, 0.5 mmol, 50% solution in ethyl acetate, 1.07 g / mL), and diisopropylethylamine (130 μL, 0.75 mmol) were mixed in dichloromethane (0.6 mL) and stirred at room temperature for 3.5 hours. The reaction mixture was partitioned between ethyl acetate (15 mL) and water (15 mL). The aqueous layer was extracted with additional ethyl acetate, and the organic layers were combined, washed with brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting material was then purified by normal-phase column chromatography in hexane (ISCO RediSep Gold 12 column, 0-100% (3:1 ethyl acetate:ethanol)). The product fractions were concentrated to give JQ1-PEG3-azide as a colorless oil (68 mg, 45% yield). 1H NMR (500 MHz, CDCl3)δ: 7.44 (d, 2H, J = 8.3 Hz), 7.36 (d, 2H, J = 8.4 Hz), 6.90 (bs, 1H), 4.68 (t, 1H, J = 7.0 Hz), 3.75 - 3.69 (m, 8H), 3.63 (m 2H), 3.55 (m, 2H), 3.45 - 3.37 (m, 2H), 2.69 (s, 3H), 2.43 (s, 3H), 1.70 (s, 3H). 13 C NMR (125 MHz, CDCl3):170.6, 163.9, 155.7, 149.9, 136.8, 136.7, 132.2, 130.9, 130.8, 130.5, 129.9, 128.7, 70.7, 70.7, 70.7, 70.4, 70.0, 69.8, 54.4, 50.7, 39.4, 39.2, 14.4, 13.1, 11.8. m / z HRMS found [M] + = 601.2125, [C 27 H 33 ClN8O4S] + requires 601.2125.

[0068] JQ1-PEG3-azide (11 mg, 0.02 mmol) and Ir-alkyne [generation 1] (21 mg, 0.02 mmol) with DIPEA (16 μL, 0.1 mmol) were mixed in acetonitrile (0.2 mL) to give a hazy suspension. A freshly prepared suspension of copper sulfate (1.4 mg, 0.005 mmol) and sodium ascorbate (3.3 mg, 0.02 mmol) in water (0.3 mL) was added, instantly forming a yellow solution. The reaction mixture was stirred at room temperature for 5 h, at which point it was diluted with 1.5 mL of DMSO and purified by preparative HPLC (50–100% MeCN / water, 0.05% TFA over 10 min, 20 mL / min, LUNA 5 micron C18(2) 100 Å, 250 x 21.2 mm). The product fraction was lyophilized. Preparative HPLC (under the same conditions) was repeated, and the product fraction was lyophilized to give JQ-1-PEG3-Ir as a yellow solid (6 mg, 20% yield). 1 H NMR (500 MHz, MeOH-d4)δ: 9.07 (s, 1H), 8.92 (s, 1H), 8.70 (s, 2H), 8.14 - 8.08 (m, 2H), 8.06 (s, 1H), 7.86 - 7.80 (m, 2H), 7.66 (d, J = 10.4 Hz, 2H), 7.50 - 7.43 (m, 2H), 7.40 (dd, J = 8.7, 3.9 Hz, 2H), 6.92 - 6.79 (m, 2H), 5.94 - 5.85 (m, 2H), 4.69 - 4.61 (m, 1H), 4.57 (q, J = 4.5 Hz, 2H), 4.53 - 4.43 (m, 2H), 3.89 (t, J = 4.8 Hz, 2H), 3.68 - 3.56 (m, 10H), 3.50 - 3.39 (m, 3H), 3.28 (dd, J = 14.9, 5.2 Hz, 1H), 3.24 (d, J = 2.5 Hz, 3H), 2.69 (d, J = 3.2 Hz, 3H), 2.46 (s, 3H), 1.69 (dd, J = 17.2, 3.9 Hz, 15H). 13C NMR (125 MHz, MeOH-d4)δ: 171.32, 168.40, 166.33, 164.93, 164.59, 164.17, 162.20, 161.83, 161.67, 159.62, 159.51, 159.32, 156.29, 156.16, 155.51, 155.25, 151.03, 150.77, 149.66, 146.48, 144.21, 142.69, 136.67, 136.51, 132.09, 130.71, 130.57, 130.03, 128.41, 126.38, 126.13, 124.42, 123.22, 123.05, 122.80, 122.61, 122.54, 120.37, 113.94, 99.64, 99.42, 99.21, 77.45, 76.60, 70.12, 70.10, 69.94, 69.17, 68.99, 56.80, 53.63, 49.97, 49.92, 39.15, 37.18, 26.78, 26.74, 26.42, 26.38, 25.81, 13.00, 11.53, 10.17. 19 F NMR (471 MHz, MeOH-d4)δ: -61.73, -77.07, -103.74, -107.98. m / z calcd. for C 73 H 66 ClF 10 IrN 12 O 10 S (1719.3958 found 1719.3947 (M+H) and 860.2029 (M+2H) / 2. LC retention time: 1.23 min using an Acquity Single pole LCMS with two channels (0 and 25 V). Flow rate: 0.6 ml / min on a 2.1 x 50 mm BEH 1.7 μM particle size column, gradient: 5 to 100% MeCN in 1.8 min, hold for 0.2 min.

[0069] The cell-permeable conjugate from Example 3 is provided for BRD4 labeling comparison in this example and is referred to as JQ1-(Gen2)-Ir. The intracellular labeling protocol described in Example 4 was followed. HeLa cells were plated at 80% confluency in phenol red-free DMEM (Gibco) (4 mL) in 12 × 10 cm plates. JQ1-PEG3-Ir (Gen-2) (5 μM) (4 plates, A); JQ1-PEG3-Ir (Gen-1) (5 μM) (4 plates, B); and DMSO (4 plates, C). The plates were incubated at 37°C for 3 hours, and the medium was removed and replaced. Diazirine-PEG3-biotin was added (250 μM), and the plates were incubated for an additional 20 minutes at 37°C. The plates were then irradiated in the bioreactor (without the lid) for 20 minutes at 450 nM. Streptavidin enrichment and Western blotting were performed as described above. The labeling results are shown in Figure 8. As shown in the results, JQ1-(Gen1)-Ir lacked the ability to enter cells and carry out BRD4 labeling, whereas JQ1-(Gen2)-Ir entered the intracellular environment for BRD4 labeling.

[0070] Example 8 - Comparison of labeling with (+)-JQ1 and (-)-JQ1 conjugates (-)-JQ1 has no affinity for BRD-protein and serves as a negative control. The intracellular labeling protocol described in Example 4 was followed. HeLa cells were plated at 80% confluency in phenol red-free DMEM (Gibco) (4 mL) in 12 × 10 cm plates. (+)-JQ1-PEG3-Ir (Gen-2) (5 μM) (four plates, A); (-)-JQ1-PEG3-Ir (Gen-2) (5 μM) (four plates, B); and DMSO (four plates, C). The plates were incubated at 37°C for 3 hours, and the medium was removed and replaced. Diazirine-PEG3-biotin was added (250 μM), and the plates were incubated for an additional 20 minutes at 37°C. The plates were then irradiated in the bioreactor (without the lid) for 20 minutes at 450 nM. Streptavidin enrichment and Western blotting were performed as described above. The results are shown in Figure 9.

[0071] Example 9 - Selective labeling of BRD4 protein using (+)-JQ1 conjugate [Proteomics preparation and isobaric labeling] The procedure used was the same as that for intracellular labeling for Western blot analysis in Example 4. JQ1-PEG3-Ir (5 μM) (6 plates, A) and Ir-PEG3-NHBoc (referred to as Free-Ir in the analysis) (6 plates, B) were added to 80% confluent 12 × 10 cm plates of HeLa cells in phenol red-free DMEM (Gibco) (4 mL). The plates were incubated at 37 °C for 3 hours, and the medium was removed and replaced. Diazirine-PEG3-biotin (250 μM) was added, and the plates were incubated for an additional 20 minutes at 37 °C. The plates were then irradiated in a bioreactor (without a lid) for 15 minutes at 450 nM. The medium was removed, and the cells were washed twice with cold DPBS (4 °C). The cells were resuspended in cold DPBS (4 °C), scraped, and transferred to separate 15 mL Falcon tubes (2 plates per tube; a total of 6 tubes). Cells were pelleted (1000 g for 5 min at 4°C) and suspended in 2 mL of cold RIPA buffer containing PMSF (1 mM) and cOmplete EDTA-free protease inhibitor (1x) (Roche). Lysed cells were incubated on ice for 5–10 min and sonicated (35%, 5 x 5 s with a 30 s pause). The lysate was then centrifuged at 15 x 1000 g for 15 min at 4°C, and the supernatant was collected. The concentration of the cell lysate was determined by BCA assay and adjusted to 1.5 mg / mL. Magnetic streptavidin beads (NEB) were removed (350 μL / plex) and washed twice with RIPA (0.5 mL) (incubated on a rotisserie for 5 min). The beads were pelleted on a magnetic rack, diluted with the sample (1 mL), and incubated on a rotisserie overnight at 4°C. The beads were pelleted on a magnetic rack, the supernatant removed, and a control sample (15 μL) from each plex was stored at -20°C for later analysis. The beads were then washed once with 0.5 mL of RIPA, three times with 0.5 mL of 1% SDS in DPBS, three times with 0.5 mL of 1 M NaCl in DPBS, three times with 10% EtOH in DPBS, and once with 0.5 mL of RIPA. The samples were incubated with each wash for 5 minutes before pelleting.The beads were resuspended in RIPA buffer (300 μL) and transferred to a new 1.5 mL Lo-bind tube.

[0072] The supernatant was removed, and the beads were washed three times with 0.5 mL of DPBS and three times with 0.5 mL of 100 mM NH4HCO3. The beads were resuspended in 500 μL of 6 M urea in DPBS, and 25 μL of 200 mM DTT in 25 mM NH4HCO3 was added. The beads were incubated at 55°C for 30 minutes. 30 μL of 500 mM IAA in 25 mM NH4HCO3 was then added and incubated at room temperature in the dark for 30 minutes. The supernatant was removed, and the beads were washed three times with 0.5 mL of DPBS and three times with 0.5 mL of 50 mM TEAB. The beads were resuspended in 0.5 mL of 50 mM TEAB, transferred to a new Protein LoBind tube, pelleted, and the supernatant was removed. The beads were resuspended in 40 μL TEAB (50 mM), 1.2 μL trypsin (1 mg / mL in 50 mM acetic acid) was added, and the beads were incubated overnight on a rotisserie at 37°C. After 16 h, an additional 0.8 μL of trypsin was added, and the beads were incubated for an additional hour on a rotisserie at 37°C. Meanwhile, TMT10 plex labeling reagent (0.8 mg) (Thermo) was equilibrated to room temperature, diluted with 41 μL of anhydrous acetonitrile (Optima grade; vortex for 5 min), and centrifuged. The beads were then pelleted, and the supernatant was transferred to the corresponding TMT-labeling reagent. A1: 127N B1: 128C C1: 130N A2: 127C B2: 129N C2: 130C A3: 128N B3: 129C C3: 131

[0073] Reactions were incubated for 2 hours at room temperature. Samples were quenched with 8 μL of 5% hydroxylamine and incubated for 15 minutes. All samples were pooled into new Protein LoBind tubes and quenched with TFA (16 μL, Optima). Samples were stored at -80°C until proteomics were performed. Samples were desalted and fractionated prior to running.

[0074] [LC-MS / MS / MS-based proteomics analysis] Mass spectra were acquired using an Orbitrap Fusion at the Princeton Proteomics Facility and analyzed using MaxQuant. TMT-labeled peptides were dried in a SpeedVac, redissolved in 300 μl of 0.1% TFA in water, and fractionated into eight fractions using the Pierce™ High pH Reversed-Phase Peptide Fractionation Kit (#84868). Fractions 1, 4, and 7 were combined as Sample 1. Fractions 2 and 6 were combined as Sample 2. Fractions 3, 5, and 8 were combined as Sample 3. The three combined samples were completely dried in a SpeedVac and resuspended in 20 μl of 5% acetonitrile / water (0.1% formic acid, pH 3). 2 μl (~360 ng) was injected per run using an Easy-nLC 1200 UPLC system. Samples were loaded directly onto a 45 cm long, 75 μm i.d. nanocapillary column packed with 1.9 μm C18-AQ resin (Dr. Maisch, Germany) coupled to an Orbitrap Fusion Lumos (Thermo Scientific, USA) with an in-line metal emitter. The column temperature was set at 45 °C, and a two-hour gradient method was used with a flow rate of 300 nL / min. The mass spectrometer was operated in data-dependent mode using synchronous precursor selection (SPS)-MS3 [Anal Chem. 2014, 86 (14), 7150-7158]. MS1 scans (positive mode, profile data type, intensity threshold 5.0e3, mass range 375–1600 m / z) at 120,000 m / z resolution in the Orbitrap were followed by CID fragmentation in the ion trap at 35% collision energy for MS2 and HCD fragmentation in the Orbitrap at 55% collision energy for MS3 (50,000 m / z resolution). The MS3 scan range was set to 100-500 with an injection time of 120 ms. A dynamic exclusion list was activated to exclude previously sequenced peptides for 60 s, and a maximum cycle time of 2.5 s was used. The quadrupole was used to isolate peptides for fragmentation (0.7 m / z isolation window). The ion trap was operated in Rapid mode.

[0075] MS / MS / MS data were searched (forward and reverse) against the 2018 Uniprot human protein database, including common contaminants. Samples were divided into three fractions, and database search criteria were applied as follows: variable modifications were methionine oxidation and N-terminal acetylation and deamidation (NQ), and fixed modifications were cysteine ​​carbamidomethylation, with a maximum of five modifications per peptide. A maximum of two missed cleavages was allowed for specific trypsin digestion (trypsin / P). Peptide samples were matched between runs. The maximum peptide mass was 6000 Da. The minimum label ratio count was set to 2, and quantification was performed using both unique and razor peptides. The FTMS MS / MS match tolerance was set to 0.05 Da, and the ITMS MS / MS match tolerance was set to 0.6 Da. All other settings were left at default.

[0076] The proteinGroups.txt file was then imported into Persues [Main: Corrected Reported Intensity; remaining entries were left as default]. Rows were then filtered by the categorical column with "+" values, and matching rows based on the criteria of "Identified by Site Only," "Reverse Orientation," and "Potential Contaminant" were removed via a reduced matrix. The resulting matrix was then log2(x) transformed, and column correlations were confirmed to be >0.9. From the previous matrix, rows were annotated to corresponding experiments (3xA, 3xB). The matrix was then normalized (column subtraction), and the corresponding data were plotted as a scatter plot (volcano plot). FDR was determined by a two-sample t-test (Benjamini-Hochberg). The results are shown in the volcano plots in Figures 10A-10C. As shown in Figures 10A-10C, the (+)-JQ1 conjugate significantly enriched labeled proteins of the bromodomain family compared to the control conjugate species.

[0077] Example 10 - Cell-permeable conjugate, taxol-Ir Cell-permeable taxol-Ir conjugates having the structures described herein were made according to the synthetic scheme in Figure 11 and are described below.

[0078] To a stirred solution of Ir-COH (75 mg, 69 μmol) and PyBOP (55 mg, 105 μmol) in anhydrous DMF (1 mL) under N in the dark was added DIPEA (30 μL, 172 μmol). The resulting mixture was stirred at room temperature for 10 minutes, and a solution of Taxol-NH (66 mg, 70 μmol) in anhydrous DMF (1 mL) was added dropwise. The reaction was stirred overnight, diluted with EtOAc, and quenched by the addition of saturated aqueous NaHCO. The aqueous phase was removed, and the organic layer was washed with additional saturated aqueous NaHCO, 5% aqueous citric acid, brine, and dried over NaSO. The solvent was removed in vacuo and the crude material was purified by silica column chromatography (gradient elution: 0–3% MeOH / CH2Cl2) and C8 reverse-phase preparative HPLC (gradient elution: 30–100% MeCN / HO (0.1% formic acid)) to afford taxol-iridium as a yellow solid (47 mg, 33%). 1H NMR (500 MHz, CDCl3)δ: 8.77 (d, J = 7.3 Hz, 1H), 8.75 (s, 1H), 8.77 - 8.65 (m, 1H), 8.48 (t, J = 10.5 Hz, 2H), 8.14 - 7.99 (m, 4H), 7.92 - 7.77 (m, 2H), 7.82 (d, J = 7.3 Hz, 2H), 7.74 (t, J = 7.3 Hz, 2H), 7.66 - 7.28 (m, 13H), 7.04 - 6.94 (m, 1H), 6.64 (t, J = 9.4 Hz, 2H), 6.16 (s, 1H), 6.10 (t, J = 8.4 Hz, 1H), 5.79 - 5.68 (m, 1H), 5.67 - 5.57 (m, 3H). 5.55 - 5.45 (m, 1H), 5.29 (s, 1H), 4.90 (d, J = 9.6 Hz, 1H), 4.84 (d, J = 3.6 Hz, 1H), 4.27 (d, J= 8.9 Hz, 1H), 4.15 (d, J = 7.9 Hz, 1H), 3.87 (d, J = 7.9 Hz, 1H), 3.16 (app. s, 4H), 2.95 - 2.58 (m, 7H), 2.58 - 2.50 (m, 1H), 2.35 (app. s, 3H), 2.26 - 2.09 (m, 5H), 1.86 - 1.63 (m, 7H), 1.25 (app. s, 3H), 1.16 (s, 3H), 1.13 (s, 3H). 13C NMR (125 MHz, CDCl3)δ: 202.03, 172.7, 172.5, 171.5 (d, J = 3.2 Hz), 170.5, 169.6, 169.5, 168.2 - 168.0 (m), 167.3, 167.0, 165.0 (dd, J = 262.5, 13.0 Hz), 262.7 (dd, J = 263.7, 13.0 Hz), 157.7, 153.4 - 155.2 (m), 155.1 - 155.0 (m), 154.8 - 154.6 (m), 149.7, 149.3, 145.1 - 144.8 (m), 140.8 (d, J = 2.6 Hz), 138.7 (d, J = 2.0 Hz), 136.8 - 136.6 (m), 134.1 (d, J = 2.1 Hz), 133.9, 132.8, 131.8, 130.3, 130.1 (d, J = 6.1 Hz), 129.8, 129.3, 128.9, 128.8, 128.7, 128.1, 127.4, 126.4, 126.2, 123.9 (t, J = 21.3 Hz), 122.7 (d, J = 9.1 Hz), 120.6 (d, J = 9.1 Hz), 114.2 (dd, J = 16.5, 6.7 Hz), 100.1 (td, J = 27.0, 9.8 Hz), 84.1, 81.0, 78.6, 76.5, 75.4, 74.5, 73.5, 71.6, 71.5, 71.5, 56.2, 55.9, 55.8, 53.6, 47.1, 43.3, 38.8, 35.5, 35.4, 35.3, 33.4, 31.2, 29.8, 26.5, 26.4, 23.8, 23.8, 22.7, 21.6, 21.0, 20.9, 14.6, 11.0. 19 F NMR (376 MHz, CDCl3)δ: -62.7 (d, J = 5.6 Hz), -62.8 (d, J = 5.0 Hz), -71.0, -72.9, -101.3 - -101.5 (m), -105.7 - -105.9 (m). m / z HRMS found [M] += 1871. 51783 (100), 1872.51899 (89), 1869.51134 (55), 1870.51373 (55), 1873.51932 (52), 1874.52130 (22), [C 89 H 80 F 10 IrNO 16 ] + requires 1871.50949 (100), 1872.51284 (96), 1869.50715 (60), 1870.51051 (57), 1873.51620 (46), 1874.51955 (14). HPLC(Vydac 218TP C18 HPLC, gradient: 0-90% MeCN / H2O (0.1% TFA) 10 min, 5 min 90% MeCN (0.1% TFA), 1 mL / min, 254 nm):t r =13.3 minutes.

[0079] Example 11 - Subcellular microenvironment mapping [Intracellular labeling] MCF-7 cells in ten clear 10 cm plates at 80% confluency in phenol red-free RPMI 1640 (Gibco) (4 mL) were treated with Taxol-Ir (Example 10) (20 μM) (5 plates, A) and Ir-dF(CF3)(dMebpy)PF6 (referred to as Free-Ir in the analysis) (2 μM) (5 plates, B). The plates were incubated at 37°C for 3 hours, and the medium was removed and replaced. N-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzyl)hex-5-ynamide was added (250 μM), and the plates were incubated at 37°C for an additional 20 minutes. The plates were then irradiated in a bioreactor (without a lid) at 450 nM for 20 minutes. The plates were then irradiated in a Merck bioreactor (without a lid) at 450 nM for 15 minutes. The medium was then removed, and the cells were gently washed with cold DPBS (2 x 5 mL). The cells were scraped (into 5 mL of cold DPBS), combined, and pelleted (4 °C, 1000 g for 5 min). The supernatant was removed, and the cells were suspended in 1 mL of cold lysis buffer (10 mM HEPES, 150 mM NaCl, 1.3 mM MgCl2, 1% SDS) containing PMSF (1 mM) and cOmplete EDTA-free protease inhibitors (Roche). The lysed cells were incubated on ice and sonicated (35%, 4 x 5 s with a 30 s pause). The lysate was then centrifuged at 15 x 1000 g for 15 min at 4 °C, and the supernatant was collected. The concentration of the cell lysate was determined by BCA analysis (typically 3 mg / mL).

[0080] [CuAAC reaction] Click cocktail for 3-plex: In a 0.5 mL Lobind tube, 6.2 μL of 500 mM CuSO4 was added to 62 μL of 100 mM THPTA and vortexed. Next, 15.5 μL of 5 mM biotin-PEG 7-azide (Broadpharm) was added, followed by 15.5 μL of freshly prepared 1 M sodium ascorbate (Important: Add reagents in this order).

[0081] To the cell lysate (1 mL) in a 1.5 mL Lo-bind tube, 32 μL of click cocktail was added. The resulting solution was vortexed and incubated on a rotisserie at room temperature for 1 hour, then quenched by adding 5 μL of 250 mM Na4EDTA. The mixture was cooled to 0°C, transferred to a 15 mL tube, and diluted with 4.2 mL of ice-cold acetone. The sample was precipitated overnight at -20°C (3 hours was found to provide satisfactory results), centrifuged at 4.5 × 1000 g for 20 minutes at 4°C, and the supernatant was removed. The pellet was thoroughly suspended in ice-cold methanol (1 mL) by sonication (20% for 2 seconds) and incubated at -20°C for 30 minutes. After that time, the mixture was centrifuged at 4.5 × 1000 g for 20 minutes at 4°C, and the supernatant was removed. This procedure was repeated. The pellet was air-dried at room temperature for 20 minutes, redissolved in 300 μL 1% SDS (1 hour at room temperature), and heated at 95°C for 5 minutes. The sample was cooled and diluted with 900 μL RIPA buffer. 250 μL of streptavidin magnetic beads (Thermo Fisher, cat. 88817) were added to a Protein LoBind microcentrifuge tube (Eppendorf, cat. 022431081) and washed twice with 1 mL of RIPA buffer (Thermo Fisher, cat. 89900). Approximately 1.0 mg of cell lysate was added to the unwashed streptavidin magnetic beads and incubated at room temperature for 3 hours. The beads were pelleted using a magnetic rack, and the lysate supernatant was removed. The beads were then washed three times with 1 mL of 1% SDS, 1 mL of 1 M NaCl, and 1 mL of 10% EtOH (all prepared in 1x DPBS), with a 5-minute incubation between washes. A final wash was performed with 1 mL of RIPA buffer. The beads were then resuspended in 30 μL of 4x Laemmli sample buffer (Boston BioProducts, cat. BP-110R) containing 20 mM DTT and 25 mM biotin. The beads were heated at 95°C for 10 minutes and then placed on a magnetic rack. The supernatant was transferred to a new Protein LoBind microcentrifuge tube and stored at -80°C.Quantitative proteomics sample preparation and analysis was performed by IQ Proteomics (Cambridge, MA).

[0082] For LC-MS analysis at IQ Proteomics, mass spectra were acquired on an Orbitrap Fusion Lumos coupled to an EASY nanoLC-1000 (or nanoLC-1200) (Thermo Fisher) liquid chromatography system. Approximately 2 μg of peptides were loaded onto a 75 μm capillary column packed with Sepax GP-C18 resin (1.8 μm, 150 Å, Sepax) to a final length of 35 cm. Peptides were separated using a 110-minute linear gradient of 8% to 28% acetonitrile in 0.1% formic acid. The mass spectrometer was operated in data-dependent mode. The scan sequence consisted of FTMS1 spectra (resolution = 120,000; mass range 350-1400 m / z; maximum injection time 50 ms; AGC target 1·10). 6 Dynamic exclusion was initiated for 60 seconds with a + / - 10 ppm window. The 10 most intense precursor ions were selected for MS2 analysis via collision-induced dissociation (CID) in the ion trap (normalized collision energy (NCE) = 35; maximum injection time = 100 ms; 0.7 Da isolation window; AGC target 1.5·10 4 After MS2 acquisition, synchronous precursor selection (SPS) MS3 was performed to select eight MS2 product ions for high-energy collision-induced dissociation (HCD) in the Orbitrap (NCE = 55; resolution = 50,000; maximum injection time = 86 ms; AGC target 1.4 10 5The isolation window was 1.2 Da for +2 m / z, 1.0 Da for +3 m / z, and 0.8 Da for +4 to +6 m / z. All mass spectra were converted to mzXML using a modified version of ReAdW.exe. MS / MS spectra were searched against the concatenated 2018 human Uniprot protein database, including common contaminants (forward and reverse sequences), using the SEQUEST algorithm. Database search criteria were: full tryptic with two missed cleavages; precursor mass tolerance of 50 ppm and fragment ion tolerance of 1 Da; oxidation of methionine (15.9949 Da) was used as a differential modification. Static modifications were carboxyamidomethylation of cysteine ​​(57.0214) and TMT (229.1629) on lysine and the N-terminus of the peptide. Peptide-spectrum matches were filtered using linear discriminant analysis, adjusted to a 1% peptide false discovery rate (FDR).

[0083] All bioinformatics analyses of the LC-MS / MS data were performed in the R statistical computing environment. Peptide-level abundance data were used to identify the number of peptides corresponding to proteins in an experiment. Proteins with single-peptide quantification were removed to reduce the possibility of outliers affecting downstream proximal calls. Peptide-level abundance data were then normalized to the sum total abundance for each sample separately. These sums were then averaged, and each normalized protein abundance value was multiplied by this average to rescale the abundance data. The peptide-level data were then merged with the protein-level data by taking the median of all peptides corresponding to a protein. Proteins were then filtered to remove known contaminants identified from database searches and proteins that are known antibody contaminants (e.g., those with IGK, IGK, or IGH in their gene symbol and immunoglobulin in their Uniprot descriptor). Data were then filtered to remove PRNPs, which are known false positives consistently detected in nearly all experiments. Protein abundances were log2 transformed and linear modeling analysis was performed using Limma. Limma employs an empirical Bayesian approach, allowing for realistic distribution of biological variance with small sample sizes per group. The program further utilizes the complete dataset to shrink the observed sample variance toward a pooled estimate. Borrowing variance information between proteins in this way allows for a more accurate estimation of true variance, improving our ability to detect actual differences between groups. For each protein, abundance data were fitted to a linear model with experimental group as an input variable using the lmFit function. log2FC values ​​were estimated, and p-values ​​were calculated to determine significance. The false discovery rate (FDR) method by Benjamini and Hochberg was then used to correct p-values ​​for multiple comparisons. Volcano plots were generated in R using the ggplot2 library. The log2FC and p-value estimates from Limma were subset to those that reached a specified log2FC cutoff.Proteins were colored based on whether they were above or below the log2 fold cutoff threshold and whether they were statistically significant (FDR corrected p-value <0.05).

[0084] FIG. 12 provides a volcano plot of significance versus fold enrichment for targeting tubulin protein in MCF-7 cells using the cell-permeable conjugate of Example 10 for labeling.

[0085] Example 12 - Confocal Microscopy HeLa cells were seeded in 35 mm glass-bottom microscope dishes containing DMEM (phenol red-free) and treated with (+)-JQ1-PEG3-Ir (Example 3) (5 μM), Ir-PEG3-NHBoc [referred to as Free-Ir] (5 μM), and DMSO. The plates were incubated at 37°C for 3 hours, and the medium was removed and replaced. Diazirine-PEG3-biotin was added (250 μM), and the plates were incubated for an additional 20 minutes at 37°C. The plates were then irradiated in a bioreactor (without a lid) at 450 nM for different times. The medium was removed, and the cells were washed with PBS. Next, the cells were fixed with 400 μL of 4% paraformaldehyde in PBS at 37°C for 20 minutes. The cells were washed three times with PBS and permeabilized with 400 μL of 0.1% Triton X-100 in PBS at RT for 20 minutes. Cells were washed with PBS and blocked with 400 μL of 2% BSA in PBS for 20 minutes at room temperature. Cells were washed three times with PBS and incubated with 1:500 dilution of streptavidin-Alexa Fluor 488 and 1:10,000 dilution of Hoechst in 400 μL of PBS. Confocal microscopy was performed at 40x magnification using a Nikon A1 / HD 25 microscope (Nikon Instruments, Inc., Melville, NY). The images in Figure 13 are representative of multiple cross-sectional images taken during each session.

[0086] <Additional Notes> (Appendix 1) A transition metal complex of formula I:

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Claims

1. a protein labeling agent; a transition metal catalyst, The transition metal catalyst has an electronic structure that allows electron transfer to the protein labeling agent to provide a reactive intermediate for binding to and labeling the protein, the transition metal catalyst being represented by Formula I: 【Chemistry 1】 During the ceremony, M is a transition metal; A, D, E, G, Y, and Z are independently selected from C and N; R 3 ~R 7 each represent 1 to 4 optional ring substituents, each of said 1 to 4 optional ring substituents independently being alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 , and -R 9 OH, where R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; L is a linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; R 2 is an alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O 2 )R 9 , thiol, biotin, oxyamine, and haloalkyl; R 8 and R 9 is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; and X - is a counterion, and n is an integer from 0 to 20. system.

2. The system of claim 1 , wherein the electron transfer is due to an excited state in the electronic structure of the catalyst.

3. The system of claim 2 , wherein the electron transfer is due to a triplet state in the electronic structure of the catalyst.

4. The system of claim 1, wherein the diffusion radius of the reactive intermediate is 1 to 500 nm.

5. The system of claim 4, wherein the diffusion radius is 1 to 10 nm.

6. The system of claim 1 , wherein the transition metal catalyst is coupled to a biomolecule binding agent.

7. The system of claim 6 , wherein the biomolecule binding agent comprises a peptide, a protein, a sugar, a small molecule, or a nucleic acid.

8. R 2 is selected from the group consisting of cycloalkylnyl, cycloalkenyl, heteroaryl, alkyne, azide, thiol, maleimide, oxyamine, and hydrazide, and the transition metal catalyst and biomolecule binder are coupled via click chemistry.

9. The system of claim 1 , wherein the protein labeling agent is diazirine.

10. The system of claim 9 , wherein the diazirine comprises a molecular marker.

11. The system of claim 9 , wherein the reactive intermediate is a carbene.

12. The system of claim 6, wherein the transition metal catalyst is cell-permeable and has a water solubility of 1 μM to 150 μM in 0.2% DMSO in pure water.

13. 1. A method of proximity labeling, comprising: providing a conjugate comprising a protein labeling agent and a transition metal catalyst coupled to a biomolecule binding agent; activating the protein labeling agent to a reactive intermediate with the transition metal catalyst; and coupling the reactive intermediate to a protein in a cellular environment to label the protein, wherein the transition metal catalyst is represented by Formula I: 【Chemistry 2】 During the ceremony, M is a transition metal; A, D, E, G, Y, and Z are independently selected from C and N; R 3 ~R 7 each represent 1 to 4 optional ring substituents, each of said 1 to 4 optional ring substituents independently being alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 , and -R 9 OH, where R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; L is a linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; R 2 is an alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O 2 )R 9 , thiol, biotin, oxyamine, and haloalkyl; R 8 and R 9 is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; and X - is a counterion, and n is an integer from 0 to 20. method.

14. 14. The method of claim 13, wherein activating the protein labeling agent comprises electron transfer from the transition metal catalyst to the protein labeling agent.

15. 15. The method of claim 14, wherein the electron transfer is due to an excited state in the electronic structure of the catalyst.

16. 16. The method of claim 15, wherein the excited state is a triplet state.

17. 17. The method of claim 16, wherein the triplet state energy state is at least 60 kcal / mol.

18. 14. The method of claim 13, wherein the protein labeling agent is diazirine.

19. The method of claim 18 , wherein the diazirine is functionalized with a marker.

20. 14. The method of claim 13, wherein the diffusion radius of the reactive intermediate is 1 to 10 nm.

21. 21. The method of claim 20, wherein the reactive intermediate is quenched outside the diffusion radius, preventing binding to biomolecules outside the diffusion radius.

22. The method of claim 13 , wherein the biomolecule binding agent comprises a protein, a sugar, or a nucleic acid.

23. 14. The method of claim 13, wherein the biomolecule binding agent positions the transition metal catalyst in or adjacent to the cell nucleus.

24. 14. The method of claim 13, further comprising detecting or analyzing the protein coupled to the reactive intermediate.

25. 16. The method of claim 15, wherein the excited state is generated by absorption of light by the transition metal catalyst.

26. 14. The method of claim 13, wherein the transition metal catalyst and biomolecule binder are coupled via click chemistry.

27. The method of claim 13, wherein the cellular environment is an intracellular environment.

28. The method of claim 13, wherein the cellular environment is an intercellular environment.

29. The method of claim 13, wherein the biomolecule binding agent is coupled to the transition metal catalyst in the absence of copper.

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