Compositions and systems for labeling based on intercellular and intracellular proximity.

Abstract

This invention provides a transition metal complex having a composition and electronic structure that allows for the generation of reactive labeling intermediates with a lifetime and diffusion radius advantageous for labeling based on the proximity of various biomolecular species, including proteins. [Solution] The transition metal complex of the following formula I is used. JPEG2026097836000012.jpg101168 (In the formula, M represents a transition metal, and A, D, E, G, Y, and Z are selected from C and N.)

Description

[Technical Field]

[0001] Related application data This application is a U.S. provisional patent application, number 62 / 982,366, filed on February 27, 2020, and a U.S. provisional patent application filed on September 10, 2020. The applicant claims priority under Article 8 of the Patent Cooperation Treaty to the U.S. Provisional Patent Application No. 63 / 076,658. These are such that each of them is incorporated herein by reference in its entirety.

[0002] Statement on the Rights of the Government This invention relates to the National Institutes of Health and the National Institute of General Medicine. This project was conducted with government support under grant No. 5R01GM103558-08 awarded by ical Sciences. The government has certain rights to that invention.

[0003] The present invention relates to a composition for proximity-based labeling, and a system for labeling. Regarding the method and, in particular, transitions for labeling based on intercellular and intracellular proximity Regarding metal catalysts. [Background technology]

[0004] Protein proximity labeling profiles protein interaction networks. It emerged as a powerful approach for this purpose. The accompanying protein or bystander protein The ability to label proteins by proximity labeling is related to the cellular environment of the target protein and This can be of great importance in further understanding its biological role. Current proximity labeling All methods involve transferring adjacent proteins to a small number of selected agents through diffusion or physical contact. This technology involves the use of an enzyme-generating reactive intermediate that labels a amino acid residue. Despite the transformative impact, phenoxymolga via peroxidase activation Ru (t 1 / 2 > 100μs) or biotin-AMP(t) mediated by biotin ligase 1 / 2 > 60s) etc. The inherent stability of these reactive intermediates can facilitate diffusion far from their source. As a result, these enzyme-producing reactive intermediates profile within a tight microenvironment. This presents challenges in labeling. Furthermore, the large enzyme size and the labeling requirements for specific amino acids are problematic. The dependence and the inability to control these labeling systems over time are limited to a specific spatial area. This presents further challenges to profiling within the region. Considering these limitations, proximity A new approach to sex-based labeling is needed. [Overview of the Initiative]

[0005] In one aspect, transition metal complexes are described herein, which are used in various biological systems, including proteins. This method produces reactive labeling intermediates with lifetimes and diffusion radii that are advantageous for labeling based on the proximity of molecular species. It has a composition and electronic structure to achieve the following. In some embodiments, the transition metal catalyst is below It 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 of these represents 1 to 4 optional additional ring substituents, and the 1 to 4 optional additional ring substituents Each of the substituents is independently selected from the group consisting of 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 , R 9 is alkyl; R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclo lene, and heteroarylene; L is a linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea ; R 2 is selected from the group consisting of 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, and R 8 and R 9 are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl , and N-succinimidyl ester, X - is a counterion and n is an integer from 0 to 20.

[0006] As further described herein, the polarity of a transition metal complex is R 3 ~R 7 Through the selection This allows it to adapt to a specific cellular environment. In some embodiments, for example, R 3 ~R 7 One or more of these are selected to exhibit hydrophilicity via charged and / or polar chemical moieties. In such embodiments, the transition metal complex is used to access the intercellular or extracellular aqueous environment. It can exhibit hydrophilicity suitable for placement. Alternatively, R 3 ~R 7 One or more of these are hydrophobic, lipophilic. Selected to exhibit polar or nonpolar properties. For example, in some embodiments, R 3 ~R 7 One or more of these may be alkyl, fluoro, or fluoroalkyl. Hydrophobic, The transition metal complexes described herein, which exhibit lipophilic or nonpolar properties, enter the intracellular environment. Suitable for arrangement or passage. Transition metal composites are based on the principles described herein. It can pass through the cell membrane to map the local intracellular environment accordingly. Therefore, such transition metal complexes are cell-permeable.

[0007] Furthermore, in some embodiments, the transition metal complex provides triplet energy exceeding 60 kcal / mol. It has a ghee state. In some embodiments, the metal center is selected from platinum group transition metals. It can be selected. For example, the metal center may be iridium. In some embodiments, formula I n is between 1 and 20.

[0008] Another aspect is protein-protein interactions on the cell membrane as well as intracellular proteins To selectively identify various features, including quality, nucleic acid and / or other biomolecular interactions. Compositions and methods for providing a micro-environment mapping platform that can be operated for a specific purpose. This is described herein. In some embodiments, the composition comprises a transition metal catalyst of formula I and The catalyst comprises a protein labeling agent and a transition metal catalyst which converts the protein labeling agent into a reactive intermediate. To activate. In some embodiments, the transition metal catalyst of formula I is used to activate a protein labeling agent. It can have an electronic structure that enables energy transfer and forms a reactive intermediate. The reactive intermediate reacts with proteins or other biomolecules within its diffusion radius. Alternatively, crosslinking occurs. If there are no proteins or other biomolecules within the above diffusion radius, a reactive intermediate is formed. The body is quenched by the surrounding environment. As further described herein, during the reaction The diffusion radius of the interstellar material can be adjusted to suit specific microenvironment mapping considerations. It can be limited to the nanometer scale. For example, in some embodiments, diffusion The radius can be less than 10 nm or less than 5 nm. Furthermore, in some embodiments, The reactive intermediate may have a half-life of less than 5 ns. In some embodiments, the protein label The identifying agent can be functionalized with markers, such as biotin or luminescent markers, to aid in analysis. Energy transfer from catalyst to protein labeling agent, including Dexter energy transfer, This can occur through various mechanisms further described herein.

[0009] Another aspect is that conjugates are intended for use in proximity-based labeling systems. It is described in detail. The conjugate is formed when a transition metal complex is coupled to a biomolecular binder. This includes the above, where the transition metal complex before coupling with the biomolecular binder is It is represented by formula I. As will be further detailed herein, biomolecular binders are Transition metal complexes for contact labeling and related analysis within desired intracellular or intercellular / cellular cells It can be used to position the biomolecular binders in the external environment. Labeling based on proximity of the intercellular / extracellular environment (including the cell membrane) and related micromapping The conjugate can be guided to the desired position for binding. Alternatively, biomolecular binding can be performed. The agent exhibits selective binding, including to various organelle environments as well as the environment near the nucleus, and to cells. Labeling based on proximity to the internal environment and related micromapping at desired locations It can lead to the formation of conjugates. Biomolecular binders include, for example, peptides and proteins. This may include sugars, small molecules, nucleic acids, or combinations thereof. Further details in this specification As described, transition metal complexes, including click chemistry, act as biomolecular binders. It may include reactive functional groups for plucking. In some embodiments, transition Metal complexes can be coupled to biomolecular binders in the absence of copper. (As described herein) The conjugate is for the cell proximity-based labeling system detailed above. It can be used in conjunction with protein labeling agents.

[0010] In a further aspect, a method of labeling based on proximity is described herein. The labeling method involves providing a transition metal catalyst of formula (I), and using this catalyst. This involves activating the protein labeling agent into a reactive intermediate. The reactive intermediate is a protein labeling agent. It couples or binds to proteins. In some embodiments, the protein labeling agent is used Selective placement or targeting of catalysts in specific environments for associated protein mapping. To achieve this, the transition metal catalyst is coupled to a biomolecular binder. The jugate and protein labeling agents have the composition described above and below in the detailed description. and / or may have the following characteristics.

[0011] These and other embodiments will be described further in the following detailed description. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the transition metal catalysts described herein according to several embodiments. [Figure 2] Figure 2 shows the transition metal catalysts and conjugates described herein according to several embodiments. [Figure 3] Figure 3 shows several embodiments of cell-permeable conjugates comprising a transition metal catalyst and a JQ1 biomolecular binder. [Figure 4] Figure 4 shows several embodiment synthesis schemes for producing the cell-permeable conjugate shown in Figure 3. [Figure 5] Figure 5A shows Western blots of intercellular labeling using the conjugates described herein, according to several embodiments. Figure 5B shows the results of densitometry analysis of the Western blots from Figure 5A. [Figure 6] Figure 6 shows the results of time-dependent labeling of BRD4 in HeLa cells. [Figure 7] Figure 7 shows a non-cell-permeable conjugate. [Figure 8] Figure 8 shows the BRD4 labeling results between the cell-permeable conjugate in Figure 3 and the non-cell-permeable conjugate in Figure 7. [Figure 9]Figure 9 shows the structure of the (-)-JQ1 conjugate and BRD4 labeling compared to the (+)-JQ1 conjugate in several embodiments. [Figure 10] Figures 10A–10C show volcano plots of significance versus fold enrichment for target bromodomain proteins using the conjugates described herein, according to several embodiments. [Figure 11] Figure 11 shows a synthesis route for the conjugate described herein, according to several embodiments. [Figure 12] Figure 12 provides volcano plots of significant versus fold enrichment for target tubulin protein in MCF-7 cells using the cell-permeable conjugate of Figure 11, according to several embodiments. [Figure 13] Figure 13 shows confocal microscopy images of intracellular labeling by the conjugate shown in Figure 3 at different time points, according to several embodiments. [Modes for carrying out the invention]

[0013] The embodiments described herein are described in the following detailed description as well as the examples and the above and This can be more easily understood by referring to the explanation below. However, The elements, apparatus, and methods described herein are presented in the detailed description and examples. The invention is not limited to any particular embodiment. These embodiments illustrate the principles of the present invention. It should be recognized that this is not the case. Modifications and adaptations will be readily apparent to those skilled in the art.

[0014] [Definition] As used herein, either alone or in combination, the term "alkyl" is optional. This refers to a straight-chain or branched saturated hydrocarbon group that is substituted with one or more substituents. For example, A The lukyl group is C1-C 30 or C1-C 18 It is possible.

[0015] As used herein, the term "alkenil," either alone or in combination, means at least It also has one carbon-carbon double bond and is optionally substituted with one or more substituents, linear or fractional. This refers to branched hydrocarbon groups.

[0016] The term "alkynyl," as used alone or in combination herein, is at least It also has one carbon-carbon triple bond and is optionally substituted with one or more substituents, linear or modular. This refers to branched hydrocarbon groups.

[0017] In this specification, the term "aryl," used alone or in combination, may be any one of the following: This refers to aromatic monocyclic or polycyclic ring systems substituted with the ring substituents described above.

[0018] As used herein, either alone or in combination, the term "heteroaryl" refers to an aromatic compound. A monocyclic or polycyclic ring system in which one or more of the ring atoms are elements other than carbon (e.g., nitrogen). This refers to substances that are (such as boron, oxygen, and / or sulfur).

[0019] As used herein, either alone or in combination, the term "heterocyclic ring" refers to a monocyclic or polycyclic ring. A ring system in which one or more atoms of the ring system are elements other than carbon (e.g., boron, nitrogen, oxygen) This refers to a ring system that is (and / or sulfur or phosphorus, etc.), and the ring system is optionally one or more rings Substituting with substituents. Heterocyclic systems include rings with one or more unsaturated points, aromatic rings and / or may contain non-aromatic rings.

[0020] As used herein, either alone or in combination, the term "cycloalkyl" is optional. This refers to a non-aromatic monocyclic or polycyclic ring system that is substituted with one or more ring substituents.

[0021] The term "heterocycloalkyl" as used alone or in combination herein is , a non-aromatic monocyclic or polycyclic ring system in which one or more atoms of the ring system are elements other than carbon ( This refers to elements that are, for example, boron, nitrogen, oxygen, sulfur, or phosphorus (either individually or in combination). The ring system is optionally substituted with one or more ring substituents.

[0022] As used herein, either alone or in combination, the term "alkoxy" refers to RO- This refers to the part where R is an alkyl, alkenyl, or aryl as defined above. ru.

[0023] As used herein, either alone or in combination, the term "halo" refers to Family VIIA of the Periodic Table. This refers to the element (halogen). Depending on the chemical environment, halos are in a neutral or anionic state. It is possible.

[0024] Terms not specifically defined herein have their common meaning in the art. It is given.

[0025] [I. Transition Metal Complexes] In one aspect, transition metal complexes are described herein, which are used in various biological systems, including proteins. This method produces reactive labeling intermediates with lifetimes and diffusion radii that are advantageous for labeling based on the proximity of molecular species. It has a composition and electronic structure to achieve the following. In some embodiments, the transition metal catalyst is below It 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 of these represents 1 to 4 optional additional ring substituents, and the 1 to 4 optional additional ring substituents Each of the conversion groups can independently be alkyl, heteroalkyl, haloalkyl, or haloalkenyl. Halo, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 ,oh Yobi-R 9 Selected from the group consisting of OH, where R 8 Selected from the group consisting of hydrogen and alkyl Selected, R 9 is alkyl; R 1 These are directly bonded alkylenes, alkenylenes, and cycloacrylates. Cycloalkenylene, allylene, heteroalkylene, heteroalkenylene, heterocyclo Selected from the group consisting of len and heteroarrenes; L is an amide, ester, sulfonamide, sulfonate, carbamate, and urea. A connected part selected from the following groups; R 2 These include alkynes, amines, protective amines, azides, hydrazides, aryls, and heteroaryls. , cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydro Xy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O 2) R 9 , Selected from the group consisting of thiols, biotin, oxyamines, and haloalkyls, R 8 and R 9These are independently alkyl, haloalkyl, aryl, haloaryl, and N-succinate. Selected from the group consisting of midyl and N-succinimidyl esters, X - is a counterion n is an integer between 0 and 20.

[0026] Any substituent R 3 -R 7 If it does not exist, hydrogen will occupy the position on the aryl ring of formula I. This is understood. Furthermore, in some embodiments, the counterion (X - ) is tetraalkyl Borate, tetrafluoroborate, tetraphenylborate, PF6 - , and chloride They can be selected.

[0027] The polarity of transition metal complexes is R 3 -R 7 Adjusting to a specific cellular environment through selection This is possible. In some embodiments, for example, R 3 -R 7 One or more of these are charge and / or poles. The transition metal is selected to exhibit hydrophilicity via its chemical moiety. In such embodiments, the transition metal is selected to exhibit hydrophilicity via its chemical moiety. The complex can exhibit hydrophilicity suitable for placement in intercellular / extracellular environments. For example, in Figure 2... The transition metal complexes shown incorporate charged and polar chemical moieties for the aqueous intercellular environment. Yes, R 3 -R 7 One or more of these exhibit hydrophobic, lipophilic, or nonpolar properties. Selected. For example, in some embodiments, R 3 -R 7 One or more of these are alkyl, fluoro, Alternatively, it can be a fluoroalkyl. Figure 1 shows alkyl, fluoro, or fluoroalkyl. This shows a non-limiting embodiment of a transition metal complex containing an oroalkyl substituent. Hydrophobic, Transition metal complexes described herein, which exhibit lipophilic or nonpolar properties, enter the intracellular environment. Suitable for the arrangement. As shown in the examples herein, transition metal complexes are specified herein To map the local intracellular environment according to the principles described, through the cell membrane It can pass through. In some embodiments, for example, a cell-permeable transition metal complex of formula I It has a water solubility of less than 150 μM in 0.2% DMSO in pure water. In some embodiments, The transition metal complex of formula I has a water solubility of less than 100 μM. It exhibits hydrophobic, lipophilic, or nonpolar properties. The transition metal complex of formula I exhibiting properties is present in 0.2% DMSO in pure water at concentrations of 1 μM to 150 μM or 1 μM to 10 It may have 0 μM water solubility. Water solubility is determined by the retention time of the transition metal complex on a C18 column (HPLC). Therefore, it can be determined. The above-mentioned water solubility value is determined by the coupling of the transition metal complex with the biomolecular binder. This also applies to the conjugates described herein, including those that have been included.

[0028] The transition metal catalysts described herein enhance protein-protein interactions on cell membranes. Including a manipulative micro-environment mapping platform for selectively identifying various features It is used in compositions for providing a flume. In some embodiments, the composition is of formula I The solution comprises a transition metal catalyst and a protein labeling agent, wherein the transition metal catalyst reacts with the protein labeling agent. The intermediate is activated. In some embodiments, the transition metal catalyst of formula I is a reactive intermediate. It may have an electronic structure that allows for energy transfer to a protein labeling agent in order to form a body. The reactive intermediate reacts with or crosslinks with proteins or other biomolecules within its diffusion radius. If the protein or other biomolecule is not within the above diffusion radius, the reactive intermediate Quenched by the surrounding environment.

[0029] In some embodiments, energy transfer to the protein labeling agent is performed by a transition metal catalyst. This can originate from the excited state of the electronic structure. For example, the excited state of a catalyst may be a singlet excited state or a triplet excited state. It can be a multiplet excited state. The excited state of a catalyst is not limited to one, including energy absorption by the catalyst. It can be produced by the above mechanism. In some embodiments, the catalyst is a photocatalyst, and the excited state The state is induced by the absorption of one or more photons. In other embodiments, the catalyst is released into the surrounding environment. It can be placed into an excited state by interaction with one or more chemical species. Alternatively, a protein Energy transfer (including electron transfer) to the chlorine labeling agent originates from the ground state of the catalyst's electronic structure. It is possible.

[0030] Energy transfer (including electron transfer) to the protein labeling agent is a reaction of the protein labeling agent. A reactive intermediate is formed. The reactive intermediate contains proteins or within the diffusion radius of the reactive intermediate. It reacts with or crosslinks with other biomolecules. Furthermore, the reactive intermediate is quenched by the surrounding environment. The diffusion radius of the reactive intermediate is particularly Adjust to suit the considerations of a specific micro-environment mapping (proximity-based labeling). This can be done and can be limited to the nanometer scale. For example, in several embodiments Therefore, the diffusion radius of the reactive intermediate is less than 10 nm before quenching in the surrounding environment, and 5 It may be less than nm, less than 4 nm, less than 3 nm, or less than 2 nm. Therefore, it is a reactive intermediate. It reacts or crosslinks with proteins or other biomolecules within its diffusion radius, or If proteins or biomolecules are not present, the surrounding environment will quench the molecule. Then, through the coordinated efforts between the catalyst and the protein labeling agent, high resolution mapping of the local environment is achieved. It can be mixed. Furthermore, in some embodiments, the reactive intermediate is citric acid. Before ching, t less than 5 ns, less than 4 ns, or less than 2 ns 1 / 2 This may be demonstrated. In additional embodiments, By extending the half-life of the reactive intermediate, the diffusion radius can be expanded to 5-500 nm. can.

[0031] Transition metal catalysts exhibiting the aforementioned electronic structural properties for energy transfer and reactive intermediate formation. - The combination of protein labeling agents, and the binding of related proteins or biomolecules It can be used for microenvironment mapping. Several transition metal complexes of formula I are available. In this embodiment, a long-lived triple excitation promotes energy transfer to the protein labeling agent. The initial state (T1) can be shown. For example, the T1 state is t 0.2~2μs 1 / 2 It may have. The transition metal complexes described in the document may be photocatalytic, and in some examples, electromagnetic spectrum It absorbs light in the visible region of Toll. Absorption of electromagnetic radiation excites the transition metal complex to the S1 state. Subsequently, quantitative intersystem crossing occurs to the T1 state. The medium then undergoes short-range Dexter energy transfer to the protein labeling agent, returning to the ground state S0. It can return. Energy transfer to the labeling agent is reactive with proteins or other biomolecules. The labeling agent is activated in response. The T1 state of the transition metal complex is 60 in some embodiments. It can exceed kcal / mol. The metal center can be selected from, for example, platinum group transition metals. In some embodiments, the metal center may be iridium.

[0032] Figures 1 and 2 show various transition metal complexes described herein. As shown in Figure 1. , R 2 It can be selected as a reactive functional group for coupling biomolecular binders. In some embodiments, for example, R 2 BCN, DBCO, TCO, tetrazine, alkynes, Includes one or more click chemistry components, including but not limited to azide. As shown in Figure 1, R 2 These click chemistrys are directly coupled to the linker (L). It can also be cut via heteroatoms, aryls, or carbonyls. It can also be used to create a pull-out effect.

[0033] Protein labeling agents undergo energy transfer from transition metal catalysts to form reactive intermediates. The reactive intermediate reacts with proteins or other biomolecules within the diffusion radius of the reactive intermediate. Alternatively, crosslinking is performed. The diffusion radius of the reactive intermediate was described above. Specific protein labeling agents The identity of the catalyst is the identity of the catalyst, the properties of the reactive intermediate that is formed, The selection should be made according to several considerations, including the lifetime and diffusion radius of the reactive intermediate. can.

[0034] For example, in an embodiment where the transition metal catalyst is a photocatalyst, the protein labeling agent is diazirine. It is possible. Triplet energy transfer from the excited state photocatalyst can convert diazirine into a triplet (T1) state. This can be promoted. Diaziline triplet releases free triplet carbene upon N2 elimination. , then, after going through a spin equilibrium on a picosecond timescale, it becomes a reactive singlet state (t 1 / 2<1 n s) it cross-links with neighboring proteins or is quenched in an aqueous environment. In one embodiment, the extinction coefficient of the transition metal complex is diazirine. It's three to five orders of magnitude larger than that.

[0035] Any diazirine that conforms to the technical principles discussed herein. For example, diazirine Sensitization occurs with free carboxylic acids, phenols, amines, alkynes, carbohydrates, and biotin groups. Including meaningful payloads for microscopy and proteomics applications It can be extended to various p- and m-substituted aryltrifluoromethyldiazilines. Diazirine can be functionalized with markers such as biotin. In this embodiment, the marker is desthiobiotin. The marker is a protein labeling agent. This can be useful in identifying labeled proteins. For example, this marker can be used in Western blots. It may be useful in the results of analysis using lots and / or other analytical techniques. Markers and In addition to biotin and desthiobiotin, alkynes, azides, FLAG tags, and fluorocarbons are also used. It may contain phoe and chloroalkane functional groups.

[0036] In some embodiments where the transition metal catalyst is a photocatalyst, the protein labeling agent is an azide. It is possible. Triplet energy transfer from the excited photocatalyst promotes nitrene formation from the azide. It can proceed. Reactive nitrenes can crosslink with neighboring proteins or citrate in an aqueous environment. It is activated to undergo energy transfer with a transition metal photocatalyst for nitrene formation. Any possible azide can be used. In some embodiments, the azide is an aryl azide. It is Do.

[0037] [II. Conjugate] On another level, the conjugate is intended for use in proximity-based labeling systems. This will be described in the specification. The conjugate is a transition metal coupled to a biomolecular binder. The transition metal complex containing the complex, before being coupled to the biomolecular binder, is of the formula I described above. As will be explained in more detail herein, a biomolecular binder is used to transfer transition gold The catalyst is used for proximity labeling and related analysis and mapping in the desired cellular environment. It can be arranged in the following configuration. In some embodiments, the desired cellular environment is intercellular. In this embodiment, the desired environment is intracellular. The biomolecular binder is labeled based on proximity. and conjugate at desired locations for micromapping of the associated intercellular environment To derive this, we can demonstrate selective bonding.

[0038] The conjugate transition metal complex has the structure and / or characteristics described in Section I above. It can contain any transition metal complex having properties. Furthermore, the biomolecular binder is protein It may include polyvalent display systems containing substances, polysaccharides, or nucleic acids. In this embodiment, the biomolecular binder is biotin, or specific to the target protein It is a small molecule ligand with specific binding affinity. For example, a biomolecular binder can be an antibody. In some embodiments, the biomolecular binder is a primary antibody bound to a desired antigen and a phase It is a secondary antibody for interaction. Furthermore, the biomolecular binder is a photocatalytic transition metal complex. It is obtained by coupling in a bonded manner.

[0039] A biomolecular binder can be attached to a transition metal catalyst. In some embodiments, The catalyst contains a reactive handle or functional group for coupling a biomolecular binder. In some embodiments, for example, the catalyst is BCN, DBCO, TCO, tetrazine, alkyne, This includes, but is not limited to, one or more click chemistry components including azide. This can be done. Figures 1 and 2 show reactive functional groups for coupling biomolecular binders. Various transition metal photocatalysts of formula (I) are shown. As shown in Figures 1 and 2, the reactive vessel Linkers of various lengths can be used between the active group and the coordination ligand. For example, The length of the linker, such as a dol or polyamide linker, includes the steric conditions of the target site. The selection can be made according to several considerations. Furthermore, in some embodiments, Transition metal complexes can be coupled to biomolecular binders in the absence of copper.

[0040] In some embodiments, the conjugate is suitable for labeling applications in the intercellular environment. It exhibits polarity. Alternatively, the conjugate can pass through the cell membrane for intracellular labeling applications. The conjugate can also be made cell-permeable to enable this. In some embodiments, For example, conjugates are described in Section I above for cell-permeable transition metal complexes. It can show the water solubility value.

[0041] [III. Systems for labeling based on intracellular proximity] In another respect, a system for proximity-based labeling is described herein. The system includes, for example, a protein labeling agent and a transition metal catalyst, where the transition metal catalyst It has an electronic structure that allows electron transfer to protein labeling agents and provides a reactive intermediate. The reactive intermediate then reacts with proteins or other proteins in the local or immediate cellular environment. It can be coupled with biomolecules. In some embodiments, the transition metal complex is This pertains to Formula I as described in the specification.

[0042] In some embodiments, electron transfer involves a singlet or triplet excited state. This is due to the excited state of the medium electronic structure. In some embodiments, for example, the excited state of the catalyst is It can be photo-induced. Alternatively, electron transfer may originate from the ground state of the catalytic electronic structure. .

[0043] As described herein, electron transfer to a protein labeling agent provides a reactive intermediate. The reactive intermediate diffuses in accordance with the embodiments of proximity labeling described herein. The radius can be indicated. The diffusion radius is the rapid quenching of reactive intermediates by the surrounding aqueous environment. It may be limited or restricted by the quenching process. For example, reactive intermediates may be quenched in an aqueous environment. The diffusion radius before entching may be less than 5 nm, less than 3 nm, or less than 2 nm. Therefore The reactive intermediate reacts with proteins or other biomolecules within its diffusion radius. If bridging occurs, or if no proteins or biomolecules are present, then in an aqueous environment... And it is quenched. In this way, through the coordinated efforts of the catalyst and the protein labeling agent, It is possible to map the local environment with high resolution. In addition, in some embodiments The reactive intermediate undergoes a quenching period of less than 2 ns. 1 / 2 This may be demonstrated in additional embodiments. This allows the diffusion radius to be extended to 5-500 nm by extending the half-life of the reactive intermediate. ru.

[0044] Any catalyst-tamp exhibiting the aforementioned electron structure properties for electron transfer and reactive intermediate formation Combinations of crystalline labeling agents can be used for microenvironmental mapping. In this embodiment, the catalyst-protein labeling agent combination is a transition metal catalyst of formula I and a diazirine labeling agent. Contains an agent. The transition metal catalyst of formula I has any of the structures described in Section I above. It may have and / or properties. In some embodiments, the protein labeling agent is Diagiri can be functionalized with markers such as biotin or luminescence markers to aid in analysis. Sensitization involves free carboxylic acids, phenols, amines, alkynes, and carbohydrates. Materials, and biotin groups, etc., for microscopy and proteomics applications. Various p- and m-substituted aryltrifluoromethyldiazilines with suitable payloads It can be extended. The extinction coefficient of transition metal catalysts is used for sensitization with blue LEDs. At the wavelength at which it emits more light (450 nm), it is five orders of magnitude larger than that of diazirine. This explains the absence of background non-catalytic reactions.

[0045] In some embodiments, multiple protein labeling agents may be used together with a transition metal catalyst. In such embodiments, the transition metal catalyst is one or all of the protein labeling agents. The electronic structure is shown to enable electron transfer to and provide a reactive intermediate. The body may exhibit different diffusion radii in some embodiments, and therefore And bind to different proteins or biomolecules at different locations. Such implementations This enhances the resolution of the intracellular proximity-based labeling system described herein. It is possible.

[0046] Furthermore, the transition metal complexes in the system intended herein are as described in Section II above. As mentioned above, providing a conjugate by coupling with a biomolecular binder is possible. Yes, it is possible. The inclusion of biomolecular binders is achieved by separating the transition metal catalyst along with one or more protein binders. It can be guided to the desired cellular environment for analysis and mapping. Several implementations In this configuration, the system described herein comprises multiple conjugates and a protein labeling agent. This can be used, and here each conjugate and associated protein labeling agent is different It is specific to the intracellular environment.

[0047] [IV. Labeling methods based on intracellular proximity] In a further aspect, a method of labeling based on cell proximity is described herein. In some embodiments, the method involves a protein labeling agent and a transition metal catalyst that binds biomolecules. The provision includes providing a conjugate containing a protein that has been coupled to a protein. The labeling agent is activated into a reactive intermediate by a transition metal catalyst, and this reactive intermediate enters the cellular environment. The method described herein involves coupling with proteins or other biomolecules. This further includes detecting or analyzing proteins that couple to reactive intermediates. This allows for the mapping of the local cellular environment.

[0048] Protein labeling agents and conjugates are described in one of the sections I to III above. It can have any structure, composition, and / or properties.

[0049] These and other embodiments are further illustrated in the following examples. [Examples]

[0050] Example 1 - Transition Metal Catalyst [Step 1] 3-(4'-methyl-[2,2'-bipyridine]-4-yl)propanoic acid [ka] 3-(4'-methyl-[2,2']bipyridinyl-4-yl)-propionate ethyl ester 4,4'-dimethyl 2,2'-bipyridyl (2.5 g, 13.5 mmol) was placed in a flame-dried flask under a nitrogen atmosphere. Dissolved in dry THF (20 mL) under gas. Cooled the solution to -78°C and LDA (14.8 mmol, 1.1 equivalents) was prepared. The solution was added. The reaction mixture was heated to room temperature for 1.5 hours. This solution was then cooled under N2 at -78°C. Cannula in ethyl 2-bromoacetate solution (2.3 ml, 20 mmol) in dried THF (15 ml) The reaction mixture was introduced using a refractory method. The reaction mixture was allowed to slowly reach room temperature overnight, and saturated sodium bicarbonate solution was dissolved in it. Quenched by adding the solution. After post-treatment with ethyl acetate, it was dried on Na2SO4 and reduced. The crude product was concentrated under pressure. The crude residue was subjected to column chromatography (silica gel; DCM: The product was purified using MeOH:NH4OH (95:5:0.5) and the target product was obtained in 69% yield.

[0051] [Step 2] 5-(4'-methyl-[2,2]bipyridinyl-4-yl)-pento-4-enoic acid After incorporating the bipyridinyl ethyl ester from Step 1 into 1:1 THF:water, LiO H (2 equivalents) was added. The reaction mixture was stirred at room temperature for 16 hours (completed by TLC), and then NH4Cl was added. Quenched by adding (until pH 5-6). Extracted the mixture with alkyl salts and Na The mixture was dried with 2SO4 and concentrated under reduced pressure to obtain the desired product as an off-white powder. rate 63%).

[0052] tert-butyl(2-(3-(4'-methyl-[2,2'-bipyridine]-4-yl)propanamide)ethyl) Bamate [ka] bipy x (228 mg, 1 mmol, 1 equivalent), PyBOP (612 mg, 1.2 mmol, 1.2 equivalents), and tert - 20 mL filled with butyl(2-aminoethyl) carbamate (192 mg, 1.2 mmol, 1.2 equivalents) In the vial, add DMF (2 mL), then diisopropylethylamine (347 μL, 0.15 mmol, 3) An equivalent amount was added. The reaction mixture was stirred for 16 hours. The resulting mixture was then mixed with water and SiO2. Quenched. Separated the layers and washed the organic matter with saturated NaHCO3, then H2O, and finally brine. Then the organic layer was dried over Na2SO4 and concentrated under reduced pressure to obtain a yellow oil, which was then flattened. Purified by silica gel column chromatography (silica gel, 0-15% MeOH / CH2Cl2). The target compound was obtained as a yellow solid (380 mg, 99%).

[0053] [Ir-Catalyst X] [ka] bipy py (161 mg, 0.42 mmol, 1.05 equivalents) and Ir[dF(CO2H-CF3)ppy]MeCN2 (351 mg, 0.4 Add DCM / EtOH (4 mL, 4:1) to a round-bottom flask filled with mmol (1 equivalent) and prepare the reaction mixture 30 The mixture was stirred at °C for 16 hours. The resulting solution was concentrated directly onto silica gel under reduced pressure. The crude product was then prepared. Purification by flash column chromatography (silica gel, 0-25% MeOH / DCM) is performed to obtain the desired result. Ir catalyst (200 mg, 42% yield) was obtained.

[0054] [DBCO Ir catalyst] [ka] CH2Cl2 (500 μL) mg filled with Ir-cat (catalyst) X (9.4 mg, 0.008 mmol, 1 equivalent) After cooling the 5 mL vial (wrapped in black tape to block out light) to 0°C, then apply trifluor. 100 μL of acetic acid was added. The reaction mixture was warmed to room temperature and stirred until complete (for TLC and HRMS). Therefore, monitoring is performed. The completed reaction is concentrated under reduced pressure, the solid is slurryed with MeOH, and then under reduced pressure. Concentrated (removed more than three times to remove excess acid).

[0055] Then, the Ir catalyst-trifluoroacetate is dissolved in DMF (500 μL), and then diisopropyl Ethylamine (10 μL) was added. DBCO-NHS (6 mg, 0.016 mmol, 2 equivalents) was added to this solution. In addition, the solution was stirred in the dark for 3 hours. After completion, the reaction mixture was flushed (by HRMS / TLC). The target compound was directly purified by column chromatography (C18, 5-95% MeCN / H2O). It was obtained as a yellow solid (10 mg, 91%).

[0056] Example 2 - Transition Metal Catalyst [Step 1] 3-(4'-methyl-[2,2'-bipyridine]-4-yl)propanoic acid and Ir[dF(CF3)ppy] Add MeCN / H2O (4:1) to a round-bottom flask packed with MeCN2PF6, and allow the reaction mixture to rise at 70°C for 16 hours. The mixture was stirred. The resulting solution was concentrated under reduced pressure to obtain a yellow solid. The crude product was flash-coated. The target acid-containing Ir catalyst was purified by Lam chromatography (silica gel, 0-10% MeOH / DCM). We obtained (yield 55%).

[0057] [Step 2] (Regarding differential activation catalysts): Pack the Ir catalyst, PyBOP, and amine. DMF was added to the 20 mL vial. After spraying the reaction mixture with N2 in the dark for 10 minutes, diiso Propylethylamine was added. This reaction was stirred in the dark under an N2 atmosphere for 16 hours. The mixture was quenched by adding water and toluene. The layers were separated and the organic matter was saturated with 5% citric acid. The mixture was washed with sodium NaHCO3 and brine. The organic layer was then dried over sodium Na2SO4 and concentrated under reduced pressure. The target compound was obtained.

[0058] Example 3 - Cell-permeable conjugate, (+)-JQ1-PEG3-Ir The cell-permeable conjugate containing a transition metal complex and the JQ1 biomolecular binder shown in Figure 3 is described below. The synthesis was carried out according to the protocol. The synthesis scheme for the transition metal complex and the JQ1 biomolecular binder is also described. As shown in Figure 4, a stirred solution of (+)-JQ1-CO2H (177 mg, 0.44 mmol) in anhydrous DMF (4.5 mL) was mixed with HAT. U (176 mg, 0.46 mmol) was added, followed by DIPEA (230 μL, 1.32 mmol). Stir under N2 conditions at room temperature for 10 minutes, then add t-Boc-N-amide-PEG3-amine (143 m) to anhydrous DMF (0.5 mL). A solution of (g, 0.49 mmol) was added dropwise. The resulting mixture was stirred overnight, diluted with SiO2, and saturated with N2. Quenched by adding an aqueous solution of aHCO3. Remove the aqueous phase and add an additional saturated aqueous solution of NaHCO3. The organic layer was washed with water and dried over Na2SO4. The solvent was removed under vacuum, and the crude material was laid in silica. Purified by microchromatography (gradient elution: 0-10% MeOH / CH2Cl2), (+)-JQ1-PEG3-NHB oc was obtained as a yellowish-brown 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). 13 C NMR (12 5 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. m / z HRMS found [M] + = 675.29120, [C 32 H 44 ClF 10 N6O 6S] + This requires 675.27226.

[0059] (+)-JQ1-PEG3-NHBoc (146 mg, 0.22 mmol) is stirred in CH2Cl2 (2 mL) at 0°C, and TFA (3 A volume of (mL) was added dropwise. The reaction mixture was warmed to room temperature overnight, and the solvent was removed under vacuum. Saturated NaHCO3 The crude mixture was made basic using an aqueous solution, extracted with CH2Cl2, and the solvent was removed under vacuum to obtain (+)-JQ. 1-PEG3-NH2 was obtained as a yellowish-brown solid (125 mg, 99%), and immediately without further purification. I used it.

[0060] (+)-JQ1-PEG3-NH2 (32 mg, 56 μmol) and Ir-CO2H (61 μmol) in anhydrous DMF (2 mL) under N2 conditions in the dark. A stirred solution of DIPEA (30 μL, 172 μmol) and PyBOP (45 mg, 86 μmol) was added. l) was added. The resulting mixture was stirred overnight, diluted with HCl, and saturated NaHCO3 aqueous solution was added. Quenched by [method]. Remove the aqueous phase, and in the organic layer, add an additional saturated NaHCO3 aqueous solution and 5% citrate solution. The solution was washed with brine and dried on Na2SO4. The solvent was removed in a vacuum, and the crude material was dried on silica. Lamb chromatography (gradient elution: 0-3% MeOH / CH2Cl2) and C8 reversed-phase preparative HPLC (gradient elution) Elution: Purified by 30-100% MeCN / H2O (0.1% formic acid), and (+)-JQ1-PEG3-Iridium ( iridium was obtained 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). 13 C NMR (125 MHz, CDCl3)δ: 171.9, 170.8, 16 7.0 (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), 7 0.7, 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 H z), -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 Cl F 10 IrN 10 [O5S] + requires 1507.33876 (100), 1508.34202 (70), 1505.33634 (60), 1506.3 3969 (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 similarly prepared from (-)-JQ1-CO2H.

[0062] Example 4 - Intracellular Microenvironment Mapping [Intracellular labeling] 12 × 10¹⁶ c⁻ In HeLa cells on m plates, 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. 3 plates The culture medium was incubated at 7°C for 3 hours, then removed and replaced. Diazirine-PEG3-biotin was added. The plate was incubated for another 20 minutes at 37°C with (250 μM) solution. Then, the plate was... The cells were irradiated in a bioreactor at 450 nM for 15 minutes (without the lid). The culture medium was removed and the cells were cooled. The cells were washed twice with DPBS (4°C). The cells were resuspended in cold DPBS (4°C), scraped off, and separated into another 50 mL container. Transferred to an Alcon tube. Pelletized the cells (1000 g for 5 minutes at 4°C) and added PMSF (1 mM). cOmplete EDTA-free protease inhibitor (1x) (Roche) suspended in 1 mL of cold RIPA buffer. The solution was clouded. The lysed cells were incubated on ice for 5-10 minutes and then sonicated (35%, 30%). (5 x 5s with pauses). Next, centrifuge the solution in 15 x 1000g at 4°C for 15 minutes and collect the supernatant. The concentration of the cell lysate was measured using a BCA assay and adjusted to an isoconcentration of 1 mg / mL. Control samples were taken from each plex (15 μL) and stored at -20°C for later analysis.

[0063] [Streptavidin pulldown] Remove the magnetic streptavidin beads (NEB) (250 μL / plex), and administer RIPA (0. Washed twice with 5 mL (incubated on a rotisserie for 5 minutes). Place the beads on a magnetic rack. Pelletize, dilute with sample (1 mL), and incubate overnight on a rotisserie at 4°C. The beads were pelletized on a magnetic rack, the supernatant was removed, and the control samples were taken from each plex. A pull (15 μL) was stored at -20°C for later analysis. The beads were then refracted in RIPA (0.5 mL). Once, three times with 1% SDS (0.5 mL) in DPBS, three times with 1 M NaCl (0.5 mL) in DPBS, in DPBS The samples were washed three times with 10% EtOH and once with RIPA (0.5 mL). The samples were then washed with each washing solution for 5 minutes. After incubation for several minutes, the beads were pelletized. The beads were resuspended in RIPA buffer (300 μL). Then, it was transferred to a new 1.5 mL Lo-bind tube.

[0064] [Western blot analysis] After the final washing and transfer procedures for pulldown, the beads are pelletized on a magnetic rack. Then, the supernatant was removed. The beads were gently centrifuged and collected at the bottom of the tube, and a fresh solution was prepared. Elution buffer (30 mM biotin, 6 M urea, 2 M thiourea, 2% SDS in DPBS, pH=11.5) (24 μg The 4 x Laemlli buffer (6 μL) containing BME was gently added while mixing. Heat the mixture at 95°C for 15 minutes, pelletize it on a magnetic rack, and remove the supernatant while it is still hot. The beads were discarded. The sample was allowed to cool to room temperature and centrifuged. Subsequently, the sample (17 μL) was subjected to BioRa Load all appropriate controls onto a Criterion 4-20% trisglycine gel, and freshly... The gels were run in the prepared Tris electrophoresis buffer (160 V, 60 minutes). The gels were washed (3 x MiliQ water). ), transferred onto the NC membrane via iBlot2. The membrane was washed again (3 x MiliQ water). ), after blocking at room temperature for 1 hour with Li-COR TBS blocking buffer, Pierce Prot Anti-BRD4 (A-7, Santa Cruz) (1:500) and antihistamines in ein-free blocking (1:2000) Incubate overnight at 4°C with H3 (polychloronal Invitrogen PA5-16183) (1:2000). The membrane was washed with TBST three times (5 minutes per wash) and MiliQ water five times, and Li- Pierce Protein-Free with COR secondary antibodies (goat anti-mouse 800) and (goat anti-rabbit 700). The membrane was resuspended in Blocking Buffer and locked at room temperature for 1 hour (1:12,500). The images were washed with TBST (5 minutes per wash) and MiliQ water five times, and then imaged.

[0065] Figure 5A shows the results of Western blot analysis, and Figure 5B shows the transition metal complex and BRD4 protein. Figure 5B shows the results of Western blot densitometry, which quantifies the binding. As shown, the cell-permeable conjugate (+)-JQ1-PEG3-Ir of Example 3 herein is viable in living organisms. Compared to transition metal complexes lacking molecular binders, the BRD4-labeled complex showed an increase of more than 2.5 times.

[0066] Example 5 - Time-dependent labeling of BRD4 using (+)-JQ1-PEG3-Ir Follow the intracellular labeling protocol described in Example 4. The degree of biotinylation over time. The irradiation time was varied to demonstrate this (2 minutes, 5 minutes, 15 minutes). The control reaction using UV light was performed. This process is performed using a photobox, where 254 nm light is used to irradiate the plate at 4°C for 20 minutes. 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 of this specification was used for 2, 5, and 15 minutes. This enabled labeling of BRD4 over time. In contrast, it was not functionalized with the JQ1 biomolecular binder. The transition metal catalyst failed to produce BRD4 labeling.

[0067] Example 6 – Comparison of labeling between cell-permeable and non-cell-permeable conjugates The non-cell-permeable conjugate shown in Figure 7 was prepared as follows. In this example, the non-cell-permeable conjugate was prepared. The sex conjugate is denoted as JQ1-(Gen1)-Ir. (+)-JQ1-CO2H (100 mg, 0.25 mmol), A Zido-PEG3-amine (60 mg, 0.27 mmol), 1-propanephosphonic anhydride (300 μL, 0.5 mm (ol, 50% solution in ethyl acetate, 1.07 g / mL) and diisopropylethylamine (130 μL, 0. 75 mmol of the reaction mixture was mixed in 0.6 mL of dichloromethane and stirred at room temperature for 3.5 hours. The solution was partitioned into ethyl acetate (15 mL) and water (15 mL). The aqueous layer was extracted with additional ethyl acetate. After combining the layers, washing with brine, drying on magnesium sulfate, filtering, and then under reduced pressure... The substance was concentrated below. Then, the obtained substance was subjected to normal-phase column chromatography (IS) in hexane. Purified using a CO RediSep Gold 12-column filter with 0-100% (3:1 ethyl acetate:ethanol). The material fraction was concentrated to obtain JQ1-PEG3-azide as a colorless oil (68 mg, 45% yield). 1 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 ¹³C NMR (125 MHz) , CDCl3):170.6, 163.9, 155.7, 149.9, 136.8, 136.7, 132.2, 130.9, 130.8, 130.5, 1 29.9, 128.7, 70.7, 70.7, 70.7, 70.4, 70.0, 69.8, 54.4, 50.7, 39.4, 39.2, 14.4, 1 3.1, 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 [1st generation] (2 Mix 1 mg (0.02 mmol) and DIPEA (16 μL, 0.1 mmol) in acetonitrile (0.2 mL). A cloudy suspension was obtained. To this suspension, freshly prepared copper sulfate (1.4 mg, 0.005 mmol) and When a suspension of sodium ascorbate (3.3 mg, 0.02 mmol) in water (0.3 mL) is added, The solution instantly turned yellow. This reaction mixture was stirred at room temperature for 5 hours, at which point 1.5 mL of D was added. Dilute with MSO and perform preparative HPLC (50-100% MeCN / water, 0.05% TFA, 20 mL / min, LUNA 5 over 10 minutes) The product was purified using a micron C18(2) filter (100 angstroms, 250 x 21.2 mm). The product fraction was frozen. The product was dried. Preparative HPLC (under the same conditions) was repeated, and the product fraction was freeze-dried to obtain JQ-1-PEG3-Ir. It was obtained 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, 2 H), 7.66 (d, J = 10.4 Hz, 2H), 7.50 - 7.43 (m, 2H), 7.40 (dd, J = 8.7, 3.9 Hz, 2 H), 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). 13 C 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 860.202 9 (M+2H) / 2. LC retention time: 1.23 min using Acquity Single pole LCMS with 2 channels (0 and 25 V). The flow rate was 0.6 ml / min on a 2.1 x 50 mm BEH 1.7 μM particle size column, and the gradient was from 5 to 100% MeCN for 1.8 min and held for 0.2 min.

[0069] The cell-permeable conjugate of Example 3 is provided in this example for BRD4 labeling comparison and is referred to as JQ1-(Gen2)-Ir. It follows the intracellular labeling protocol described in Example 4. 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) were added to HeLa cells in 80% confluent 12×10 cm plates in phenol red-free DMEM (Gibco) (4 mL). ​​​​The plate was incubated at 37 °C for 3 hours, the medium was removed and replaced. Diazirine-PEG3- biotin was added (250 μM), and the plate was incubated at 37 °C for an additional 20 minutes. Then , the plate was irradiated in the bioreactor at 450 nM for 20 minutes (without the lid). Streptoa vidin enrichment and Western blot were performed as described above. The results of labeling are shown in Fig. 8 . As shown in the results, JQ1-(Gen1)-Ir lacked the ability to enter cells and perform BRD4 labeling . On the other hand, JQ1-(Gen2)-Ir entered the intracellular environment for BRD4 labeling.

[0070] Example 8 - Comparison of labeling by (+)-JQ1 and (-)-JQ1 conjugates (-)-JQ1 serves as a negative control because it has no affinity for BRD-protein. Follow the intracellular labeling protocol described in Example 4. In DM EM (Gibco) (4 mL) without phenol red, to HeLa cells in a 12×10 cm plate at 80% confluence, (+)-J Q1-PEG3-Ir (Gen-2) (5 μM) (4 plates, A); (-)-JQ1-PEG3-Ir (Gen-2) (5 μM) (4 plates , B); and DMSO (4 plates, C) were added. The plates were incubated at 37 °C for 3 hours , the medium was removed and replaced. Diazirine-PEG3-biotin was added (250 μM), and the plate was incubated at 37 °C for an additional 20 minutes. Then, the plate was placed in the bioreactor and irradiated at 450 nM for 20 minutes (without the lid). Streptavidin enrichment and Western blot were performed as described above. The results are shown in Fig. 9.

[0071] Example 9 - Selective labeling of BRD4 protein using (+)-JQ1 conjugate [Proteomics preparation and isobaric labeling] The procedure performed was intracellular labeling for Western blot analysis in Example 4 and They are the same. 80% confluence in DMEM (Gibco) (4 mL) without phenol red HeLa cells in a 12×10 cm plate were treated with JQ1-PEG3-Ir(5μM) (6 plates, A) and Ir-PEG3-NH Boc (called Free-Ir during analysis) (6 plates, B) was added. The plates were heated to 37°C. Incubate for 3 hours, then remove and replace the culture medium. Add diazirin-PEG3-biotin (2 (50 μM), the plate was incubated for another 20 minutes at 37°C. Then the plate was turned over. The cells were irradiated at 450 nM for 15 minutes in an Oreactor (without the lid). The culture medium was removed and the cells were chilled in cold DPBS (4 Washed twice with water (°C). Resuspended the cells in cold DPBS (4°C), scraped them off, and placed in another 15 mL falco solution. The cells were transferred to tubes (2 plates per tube; 6 tubes in total). The cells were then pelletized. (1000 g at 4°C for 5 minutes), PMSF (1 mM) and cOmplete EDTA-free protease inhibitor (1x) The lysed cells were suspended in 2 mL of cold RIPA buffer containing Roche. The lysed cells were left on ice for 5-10 minutes. The solution was incubated and sonicated (5 x 5 s with a 30 s pause at 35%). Next, the solution was 4 The cells were centrifuged at 15 x 1000 g for 15 minutes at °C, and the supernatant was collected. The concentration of the cell lysates was measured using BCA assay. The concentration was measured using method I and adjusted to 1.5 mg / mL. Magnetic streptavidin beads (NEB) were then removed. Dispense (350 μL / Plex), wash twice with RIPA (0.5 mL) (rotisserie for 5 minutes) (Incubate). Pellet the beads on a magnetic rack, dilute with the sample (1 mL), and leave at 4°C. Then incubate overnight on a rotisserie. Pellet the beads on a magnetic rack and then... Remove the clear solution and store the control samples (15 μL) from each plex at -20°C for later analysis. Then, the beads were treated once with RIPA (0.5 mL), three times with 1% SDS (0.5 mL) in DPBS, and finally with DPBS. Three times with 1 M NaCl (0.5 mL), three times with 10% EtOH in DPBS, and once with RIPA (0.5 mL). Washed. The samples were incubated with each washing solution for 5 minutes before being pelletized. Beads The mixture was resuspended in RIPA buffer (300 μL) and transferred to a new 1.5 mL Lo-bind tube.

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

[0073] The reaction mixture was incubated at room temperature for 2 hours. The sample was quenched with 8 μL of 5% hydroxylamine. The samples were heated and incubated for 15 minutes. All samples were transferred to new Protein LoBind tubes. The samples were pooled and quenched with TFA (16 μL, Optima). The samples were then subjected to proteomics. The samples were stored at -80°C until the end of the process. Before the run, the samples were desalted and fractionated.

[0074] [LC-MS / MS / MS-based proteomics analysis] Mass spectra were obtained using Orbitrap Fusion at the Princeton Proteomics Facility. The analysis was performed using MaxQuant. The TMT-labeled peptide was dried with SpeedVac and hydrated in 300 μl of water. Redissolve in 0.1% TFA and use Pierce® High pH Reversed-Phase Peptide Fractionation Ki The sample was divided into eight fractions using t (#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 SpeedVac and then rinsed in 20 μl of 5% acetonitrile / water (0 Resuspended in 0.1% formic acid (pH = 3)). 2 μl (~360 ng) was injected for each run using an Easy-nLC 1200 UPLC system. Orbitrap Fusion Lumos (Thermo Scientific, USA) and an in-line metal emitter were engaged with a 1.9 um C18-AQ resin (Dr. Maisch, Germany) packed 45 cm long and 75 um inner diameter nano-capillary column, and the sample was directly loaded onto it. The column temperature was set at 45 °C, and a two-hour gradient method with a flow rate of 300 nl / min was used. The mass spectrometer was operated in data-dependent mode by the simultaneous precursor selection (SPS)-MS3 method [Anal Chem. 2014, 86 (14), 7150-7158], followed by a MS1 scan at 120,000 resolution in the Orbitrap (positive mode, profile file data type, intensity threshold 5.0e3, mass range 375 - 1600 m / z), CID fragmentation in the ion trap with 35% collision energy for MS2, and HCD fragmentation in the Orbitrap with 55% collision energy for MS3 (50,000 resolution ). The MS3 scan range was set to 100 - 500, and the injection time was 120 ms. The dynamic exclusion list was activated to exclude previously sequenced peptides for 60 seconds, and a maximum cycle time of 2.5 seconds was used. Peptides for fragmentation were isolated using a quadrupole (0.7 m / z isolation window). The ion trap was operated in Rapid mode.

[0075] The MS / MS / MS data was searched against the 2018 Uniprot human protein database containing common contaminants (forward and reverse). The sample was set into three fractions, and the database The following search criteria were applied: Variable modifications included methionine oxidation and N-terminal acetylation. For ionization and deamidation (NQ), the immobilization modification is set to cysteine ​​carbamide methylation, and pept A maximum of 5 modifications were allowed per cytoplasm. The most common cause of cleavage failure was cleavage failure with specific trypsin digestion (trypsin / P). Two large samples were selected. Peptide samples were matched between runs. The maximum peptide mass was 6000 Da. The minimum labeling ratio count was set to 2, and the unique peptide and razor peptide were selected. Quantification was performed using both FTMS and ITMS. The FTMS MS / MS match tolerance was set to 0.05 Da, and ITMS MS The / MS match tolerance was set to 0.6 Da. All other settings were left at their default values. .

[0076] After that, the proteinGroups.txt file was imported into Persues [main: corrected Reporting intensity; remaining entries were left at default. Then, the row was changed to a value of "+". Filter by category column, "Identified only by site", "Reverse direction", And rows that match based on the criterion of "potential contaminants" are reduced matrix It was deleted via ks. The resulting matrix was then transformed with log2(x), and the column correlation was > It was confirmed to be 0.9. From the previous matrix, annotate the rows to the corresponding experiments. (3×A, 3×B). Then normalize the matrix (subtract columns) and the corresponding data The data was plotted as a scatter plot (volcano plot). The FDR was analyzed using a two-sample t-test (Benjamini-Hochb). Determined by erg). The results are shown in the volcano plots in Figures 10A-10C. Figure 10 As shown in A-10C, the (+)-JQ1 conjugate is, compared to the comparison conjugate species, Significant enrichment of labeled proteins from the mododomain family was achieved.

[0077] Example 10 - Cell-permeable conjugate, taxol-Ir A cell-permeable taxol-Ir conjugate having the structure described herein is shown in Figure 11. It is prepared according to the synthesis scheme described below.

[0078] Ir-CO2H (75 mg, 69 μmol) and PyBOP (55 mg, 10) in anhydrous DMF (1 mL) under N2 conditions in the dark. DIPEA (30 μL, 172 μmol) was added to a 5 μmol stirred solution. The resulting mixture was left at room temperature for 10 minutes. The mixture was stirred for 1 minute, and a solution of Taxol-NH2 (66 mg, 70 μmol) in anhydrous DMF (1 mL) was added dropwise. The reaction was stirred overnight, diluted with ammonium phosphate, and quenched by adding saturated NaHCO3 aqueous solution. The aqueous phase was removed, and the organic layer was washed with additional saturated NaHCO3 aqueous solution, 5% citric acid aqueous solution, and brine. The material was then dried on Na2SO4. The solvent was removed under vacuum, and the crude material was subjected to silica column chromatography. Phy (gradient elution: 0-3% MeOH / CH2Cl2) and C8 reverse-phase preparative HPLC (gradient elution: 30-100% MeOH / CH2Cl2) The solution was purified using CN / H2O (0.1% formic acid) to obtain taxol-iridium as a yellow solid. (47 mg, 33%) 1 H 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), 7.8 2 (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, 7 H), 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). 13 C 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 H z), 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, 3 1.2, 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 IrN6O16 ] + 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 - Intracellular Microenvironment Mapping [Intracellular labeling] In RPMI 1640 (Gibco) (4 mL) without phenol red, 80% confluen C. added Taxol-Ir to MCF-7 cells in 10 transparent 10 cm plates (Example 10) (20 μM) (5 plates, A) and Ir-dF(CF3)(dMebpy)PF6 [referred to as Free-Ir during analysis] ](2μM)(5 plates, B) was added. The plates were incubated at 37°C for 3 hours. The base was removed and replaced. N-(4-(3-(trifluoromethyl)-3H-diazilin-3-yl)benzyl Add hex-5-inamide (250 μM) and incubate the plate at 37°C for a further 20 minutes. Then, the plate was irradiated in a bioreactor at 450 nM for 20 minutes (without the lid). The plate was then irradiated in a Merck bioreactor at 450 nM for 15 minutes (without the lid). Next, remove the culture medium and gently wash the cells with cold DPBS (2 x 5 mL), scraping off the cells (5 Combine and pelletize (in mL of cold DPBS at 4°C, 1000 g for 5 minutes). Remove the supernatant. , 1 mL of cold solution containing PMSF (1 mM) and cOmplete EDTA-free protease inhibitor (Roche) The cells were suspended in a lysis buffer (10 mM HEPES, 150 mM NaCl, 1% SDS in 1.3 mM MgCl2). Lysized cells were incubated on ice and sonicated (35%, with a 30s pause) for 4x 5s). Next, the lysate was centrifuged at 15 x 1000 g at 4°C for 15 minutes, and the supernatant was collected. Cell lysis The concentration of the substance was measured by BCA analysis (typically 3 mg / mL).

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

[0081] Add 32 μL of click cocktail to 1 mL of cell lysate in a 1.5 mL Lo-bind tube. The resulting solution was vortexed and incubated on a rotisserie at room temperature for 1 hour, and 5 Quenched by adding μL of 250 mM Na4EDTA. The mixture was cooled to 0°C and 15 mL of tubular saturation solution was added. Transfer to a bowl and dilute with 4.2 mL of ice-cold acetone. Allow the sample to precipitate overnight at -20°C (3 hours). However, satisfactory results were found to be obtainable), 4.5 × 1000 g, centrifuged at 4°C for 20 minutes, The supernatant was removed. The pellet was sonicated (20% for 2 s) in ice-cold methanol (1 mL) until completely dissolved. The mixture was suspended in 4.5 × 1000 g and incubated at -20°C for 30 minutes. After that time, the mixture was divided into 4.5 × 1000 g and 4 The pellets were centrifuged at ℃ for 20 minutes, and the supernatant was removed. This procedure was repeated. The pellets were left at room temperature for 20 minutes. The sample was air-dried, redissolved in 300 μL of 1% SDS (at room temperature for 1 hour), and heated at 95°C for 5 minutes. The material was cooled and diluted with 900 μL of RIPA buffer. 250 μL of streptavidin magnetic beads ( Thermo Fisher, cat. 88817) to Protein LoBind microcentrifuge tube (Eppendorf, cat. 022431081) In addition to the above, the surface was washed twice with 1 mL of RIPA buffer (Thermo Fisher, cat. 89900). Add approximately 1.0 mg of cell lysate to treptavidin magnetic beads and incubate at room temperature for 3 hours. The beads were pelletized using a magnetic rack, and the supernatant of the dissolved material was removed. The beads are mixed with 1 mL of 1% SDS, 1 mL of 1 M NaCl, and 1 mL of 10% EtOH (all of which are 1x DPB). Each was washed three times with (prepared during S), and incubated for 5 minutes between washes. Final wash Purification was performed with 1 mL of RIPA buffer. Then, the beads were treated with 3 solutions containing 20 mM DTT and 25 mM biotin. The sample was resuspended in 0 μL of 4x Laemmli sample buffer (Boston BioProducts, cat. BP-110R). The beads were heated at 95°C for 10 minutes, then placed on a magnetic rack. The supernatant was then used to store fresh protein. The sample was transferred to a LoBind microcentrifuge tube and stored at -80°C. Quantitative proteomics sample preparation and The analysis was conducted by IQ Proteomics (Cambridge, Massachusetts).

[0082] In LC-MS analysis at IQ Proteomics, EASY nanoLC-1000 (or nanoLC-1200) (Thermo Fisher) Orbitrap Fusion Lumos coupled to a liquid chromatography system Mass spectra were obtained. Sepax GP-C18 resin (1.8 μm, 150 Å, Sepax) was self-filled. A 75 μm capillary column was packed with approximately 2 μg of peptide to a final length of 35 cm. 0.1% Peptides were separated using a linear gradient of acetonitrile from 8% to 28% in acid for 110 minutes. The mass spectrometer was operated in data-dependent mode. The scan sequence was FTMS1 Spectrometer. Tor (Resolution = 120,000; Mass range 350-1400 m / z; Maximum injection time 50 ms; AGC target 1·1) 0 6 Dynamic exclusion was initiated after 60 seconds with a + / - 10 ppm window. The ten strongest precursor ions are extracted via collision-induced dissociation (CID) in an ion trap using MS2. The selected model for analysis had a normalized collision energy (NCE) of 35, a maximum injection time of 100 ms, and a 0.7 D a isolation window; AGC target 1.5·10 4 ). After obtaining MS2, during Orbitrap In the analysis, synchronous precursor selection (SPS) MS3 was performed to determine high-energy collision-induced dissociation (HCD). Eight MS2 product ions were selected (NCE=55; resolution=50,000; maximum injection time=86 ms) AGC Target 1.4·10 5 The isolation window is 1.2 Da for +2 m / z, +3 (1.0 Da for m / z, 0.8 Da for +4 to +6 m / z). All mass spectra are ReA Converted to mzXML using a modified version of dW.exe. Using the SEQUEST algorithm, Concatenated 2018 human Uniprot protein data including contaminants (forward + reverse sequences) MS / MS spectra were searched against the base. The database search criteria were as follows: Ru: Complete triptych with two cutting errors; precursor mass tolerance 50 ppm and flag Menthion ion tolerance 1 Da; methionine oxidation (15.9949 Da) was used as the differential modification. Static modification was Carboxamide methylation of cysteine ​​(57.0214) and the N-terminus of lysine and peptides The TMT at the end was (229.1629). Peptide spectral agreement was confirmed using linear discriminant analysis. The samples were filtered and adjusted to a peptide false discovery rate (FDR) of 1%.

[0083] All bioinformatics analyses of LC-MS / MS data were performed in the R statistical computing environment. Using peptide-level abundance data, the peptides corresponding to proteins in the experiment The number was identified. To reduce the possibility that outliers could affect downstream proximal calls, a single PEP Proteins with cytoplasmic quantification were removed. Subsequently, peptide-level abundance data were obtained. Each sample was separately normalized to the total abundance. Then, these sums were... The abundance data is then recalculated by multiplying the average and normalized abundance values ​​of each protein by this average. It was kaled. Next, by taking the median of all peptides corresponding to the protein... Then, the peptide-level data was merged with the protein-level data. Next, the protein Filtering by quality, known contaminants identified from database searches, and known Proteins that are antibody contaminants (for example, those with the gene symbol IGK, IGK, or IGH) (Those present and containing immunoglobulins in the Uniprot description) were removed. Then, the data was processed. Although PRNPs were filtered out, they were consistently detected in almost all experiments. This is a known false positive protein. The protein abundance was converted to log2 and linearized using Limma. Shape modeling analysis was performed. Limma employs an empirical Bayesian approach, and per group This program enables a realistic distribution of biological variance with small sample sizes. Furthermore, by using the complete dataset, the observed sample variance is converted to a pooled estimate. It shrinks in that direction. By borrowing the dispersion information between proteins in this way, the true dispersion is more This improved the ability to make accurate estimates and to detect actual differences between groups. Regarding the substance, the abundance data was generated using the lmFit function in a linear model with the experimental group as the input variable. The model was fitted. The log2FC value was estimated, and the p-value was calculated to determine significance. Next, Benj The rate of false detection (FDR) method by Amini and Hochberg was used to correct p-values ​​for multiple comparisons. The volcano plot was generated in R using the ggplot2 library. log2FC and p from Limma The estimated values ​​were subsetted to those that reached the specified log2FC cutoff. Quality is determined by whether it is above or below the log2x (fold) cutoff threshold, and statistically... The data was color-coded based on whether the result was statistically significant (FDR-corrected p-value < 0.05).

[0084] Figure 12 shows MCF-7 using the cell-permeable conjugate from Example 10 for labeling. Significance vs. enrichment ratio for targeted tubulin proteins in cells We will provide lots.

[0085] Example 12 - Confocal Microscope HeLa in a 35 mm glass-bottom microscope dish containing DMEM (phenol red-free) Cells were seeded and treated with (+)-JQ1-PEG3-Ir (Example 3) (5 μM), Ir-PEG3-NHBoc [referred to as Free-Ir]. The plates were treated with ](5μM) and DMSO. The plates were incubated at 37°C for 3 hours, and the culture medium was then removed. Removed and replaced. Diazirin-PEG3-biotin (250 μM) was added, and the plate was left for another 20 minutes. The plates were incubated at 37°C. Afterward, the plates were subjected to different concentrations in a bioreactor at 450 nM. The cells were irradiated for an extended period (without a lid). The culture medium was removed and the cells were washed with PBS. Next, the cells were finely irradiated. The cells were fixed in 400 μL of 4% paraformaldehyde in PBS at 37°C for 20 minutes. The cells were washed three times with PBS. The cells were purified and permeabilized with 0.1% Triton X-100 in 400 μL of PBS for 20 minutes at RT. The cells were then washed with PBS. The cells were purified and blocked with 2% BSA in 400 μL of PBS for 20 minutes using RT. The cells were washed three times with PBS and then rinsed in 400 μL. Streptoavidin-Alexa Fluor 488 diluted 1:500 in PBS and Hoechst diluted 1:10,000 They were incubated together. Confocal microscopy observation was performed using a Nikon A1 / HD 25 microscope (Nikon Instruments). The procedure was performed at 40x magnification using nts, Inc. (Melville, New York). See Figure 13 for the image. These are representative cross-sectional images taken during each session.

[0086] Various embodiments of the present invention have been described to achieve various objectives of the present invention. It should be recognized that the form is merely illustrative of the principle of the present invention. Many modifications and adaptations thereof can be easily made by those skilled in the art without departing from their purpose and scope. This will become clear.

Claims

1. The following transition metal complex is 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 of these represents 1 to 4 optional additional ring substituents, and the 1 to 4 optional additional ring substituents Each of the conversion groups can independently be alkyl, heteroalkyl, haloalkyl, or haloalkenyl. Halo, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 ,oh Yobi-R 9 Selected from the group consisting of OH, where R 8 Selected from the group consisting of hydrogen and alkyl Selected, R 9 is alkyl; R 1 These include direct bonding, alkylene, alkenylene, and cycloacrylate. Cycloalkenylene, allylene, heteroalkylene, heteroalkenylene, heterocyclo Selected from the group consisting of len and heteroarrenes; L is an amide, ester, sulfonamide, sulfonate, carbamate, and urea. It is a connected part selected from the following groups; R 2 is alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl , cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydro Xy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O 2) R 9 , Selected from the group consisting of thiols, biotin, oxyamines, and haloalkyls, R 8 and R 9 These are independently alkyl, haloalkyl, aryl, haloaryl, and N-succinate. Selected from the group consisting of midyl and N-succinimidyl esters, X - is a counterion n is an integer from 0 to 20. Transition metal complex.

2. The transition metal complex according to claim 1, wherein M is a platinum group metal.

3. The transition metal complex according to claim 2, wherein M is iridium.

4. The transition metal complex according to claim 1, having an absorption spectrum in the visible region of its electromagnetic spectrum. 。

5. R 2 The present invention is described in claim 1, wherein the part is selected to include a portion for coupling biomolecules. A transition metal complex.

6. R 2 The transition metal complex according to claim 5, wherein the click chemistry portion is the click chemistry portion.

7. The click chemistry portion is BCN, DBCO, TCO, tetrazine, alkyne, and A transition metal complex according to claim 6, selected from the group consisting of zides.

8. The transition metal complex according to claim 7, wherein the biomolecule is an antibody.

9. The transition metal complex according to claim 1, wherein the transition metal complex is cell-permeable.

10. The transition metal complex according to claim 1, wherein its water solubility in 0.2% DMSO in pure water is 1 μM to 150 μM. body.

11. A conjugate comprising a transition metal complex coupled to a biomolecular binder, The transition metal complex before coupling with the biomolecular binder is represented by the following formula I. can be, 【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 of these represents 1 to 4 optional additional ring substituents, and the 1 to 4 optional additional ring substituents Each of the conversion groups can independently be alkyl, heteroalkyl, haloalkyl, or haloalkenyl. Halo, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 ,oh Yobi-R 9 Selected from the group consisting of OH, where R 8 Selected from the group consisting of hydrogen and alkyl Selected, R 9 is alkyl; R 1 These include direct bonding, alkylene, alkenylene, and cycloacrylate. Cycloalkenylene, allylene, heteroalkylene, heteroalkenylene, heterocyclo Selected from the group consisting of len and heteroarrenes; L is an amide, ester, sulfonamide, sulfonate, carbamate, and urea. It is a connected part selected from the following groups; R 2 These include alkynes, amines, protective amines, azides, hydrazides, aryls, and heteroaryls. , cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydro Xy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O 2) R 9 , Selected from the group consisting of thiols, biotin, oxyamines, and haloalkyls, R 8 and R 9 These are independently alkyl, haloalkyl, aryl, haloaryl, and N-succinate. Selected from the group consisting of midyl and N-succinimidyl esters, X - is a counterion n is an integer from 0 to 20. Conjugate.

12. The transition metal complex and the biomolecular binder are coupled via click chemistry. The conjugate according to claim 11.

13. The conjugate according to claim 11, wherein M is a platinum group metal.

14. Claim 11, wherein the transition metal complex has an absorption spectrum in the visible region of the electromagnetic spectrum. The conjugate described above.

15. The conjugate according to claim 11, wherein the conjugate is cell-permeable.

16. The conjugate according to claim 11, wherein the water solubility in 0.2% DMSO in pure water is 1 μM to 150 μm. gate.

17. Protein labeling agent, Transition metal catalysts and A system for proximity labeling, including, The transition metal catalyst enables electron transfer to the protein labeling agent, thereby transforming the reactive intermediate. The transition metal catalyst has the electronic structure provided, and is represented by the following formula I: 【Transformation 3】 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 of these represents 1 to 4 optional additional ring substituents, and the 1 to 4 optional additional ring substituents Each of the conversion groups can independently be alkyl, heteroalkyl, haloalkyl, or haloalkenyl. Halo, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 ,oh Yobi-R 9 Selected from the group consisting of OH, where R 8 Selected from the group consisting of hydrogen and alkyl Selected, R 9 is alkyl; R 1 These include direct bonding, alkylene, alkenylene, and cycloacrylate. Cycloalkenylene, allylene, heteroalkylene, heteroalkenylene, heterocyclo Selected from the group consisting of len and heteroarrenes; L is an amide, ester, sulfonamide, sulfonate, carbamate, and urea. It is a connected part selected from the following groups; R 2 These include alkynes, amines, protective amines, azides, hydrazides, aryls, and heteroaryls. , cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydro Xy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O 2) R 9 , Selected from the group consisting of thiols, biotin, oxyamines, and haloalkyls, R 8 and R 9 These are independently alkyl, haloalkyl, aryl, haloaryl, and N-succinate. Selected from the group consisting of midyl and N-succinimidyl esters, X - is a counterion n is an integer from 0 to 20. system.

18. The system according to claim 17, wherein the electron transfer is due to the excited state of the electronic structure of the catalyst. Hmm.

19. The cis according to claim 18, wherein the electron transfer is due to the triplet state of the electronic structure of the catalyst. Tem.

20. The system according to claim 17, wherein the diffusion radius of the reactive intermediate is 1 to 500 nm.

21. The system according to claim 20, wherein the diffusion radius is 1 to 10 nm.

22. The transition metal catalyst is coupled to a biomolecular binder as described in claim 17. Stem.

23. The claim states that the biomolecule binder includes peptides, proteins, sugars, small molecules, or nucleic acids. The system described in item 22.

24. The transition metal complex and the biomolecular binder are coupled via click chemistry. The system according to claim 22.

25. The system according to claim 17, wherein the protein labeling agent is diazirine.

26. The system according to claim 25, wherein the diazirine contains a molecular marker.

27. The system according to claim 25, wherein the reactive intermediate is a carbene.

28. The system according to claim 22, wherein the transition metal catalyst is cell-permeable.

29. A method for proximity labeling, A conduit containing a protein labeling agent and a transition metal catalyst coupled to a biomolecular binder. To provide a gate; Activating the protein labeling agent into a reactive intermediate using the transition metal catalyst; call The reactive intermediate is coupled to a protein in the cellular environment. This includes, where the transition metal complex is represented by the following formula I, 【Chemistry 4】 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 of these represents 1 to 4 optional additional ring substituents, and the 1 to 4 optional additional ring substituents Each of the conversion groups can independently be alkyl, heteroalkyl, haloalkyl, or haloalkenyl. Halo-3, hydroxyl, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 ,oh Yobi-R 9 Selected from the group consisting of OH, where R 8 Selected from the group consisting of hydrogen and alkyl Selected, R 9 is alkyl; R 1 These include direct bonding, alkylene, alkenylene, and cycloacrylate. Cycloalkenylene, allylene, heteroalkylene, heteroalkenylene, heterocyclo Selected from the group consisting of len and heteroarrenes; L is an amide, ester, sulfonamide, sulfonate, carbamate, and urea. It is a connected part selected from the following groups; R 2 These include alkynes, amines, protective amines, azides, hydrazides, aryls, and heteroaryls. , cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydro Xy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O 2) R 9 , Selected from the group consisting of thiols, biotin, oxyamines, and haloalkyls, R 8 and R 9 These are independently alkyl, haloalkyl, aryl, haloaryl, and N-succinate. Selected from the group consisting of midyl and N-succinimidyl esters, X - is a counterion n is an integer from 0 to 20. method.

30. Activating the protein labeling agent is performed by converting the transition metal catalyst to the protein labeling agent. The method according to claim 29, comprising electron transfer to the agent.

31. The method according to claim 30, wherein the electron transfer is due to the excited state of the electronic structure of the catalyst.

32. The method according to claim 31, wherein the excited state is a triplet state.

33. The energy state of the triplet state is at least 60 kcal / mol, as described in claim 32. The method.

34. The method according to claim 29, wherein the protein labeling agent is diazirine.

35. The method according to claim 34, wherein the diazirine is functionalized with a marker.

36. The method according to claim 29, wherein the diffusion radius of the reactive intermediate is 1 to 10 nm.

37. The reactive intermediate is quenched outside the diffusion radius, and biomolecules outside the diffusion radius The method according to claim 36, wherein coupling with is prevented.

38. The method according to claim 29, wherein the biomolecular binder comprises a protein, a sugar, or a nucleic acid. Law.

39. The biomolecular binder positions the transition metal complex within or adjacent to the cell nucleus. The method according to claim 29.

40. Further detection or analysis of the protein coupled to the reactive intermediate The method according to claim 28, including the method described in claim 28.

41. The excited state is generated by the absorption of light by the transition metal catalyst, as described in claim 31. Method of description.

42. The transition metal complex and the biomolecular binder are coupled via click chemistry. The method according to claim 29.

43. The method according to claim 29, wherein the cellular environment is an intracellular environment.

44. The method according to claim 29, wherein the cellular environment is an intercellular environment.

45. The biomolecular binder is coupled to the transition metal catalyst in the absence of copper, claim. Method 29.

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