Photoaffinity probes

Abstract

To provide compositions and methods for photoaffinity labeling of molecular targets.SOLUTION: Provided herein are compositions and methods for photoaffinity labeling of molecular targets, in particular, probes that specifically interact with cellular targets that are based on their affinity and are then covalently linked to a cellular target via a photoreactive group (PRG) on the probe.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 821,147, filed March 20, 2019, which is incorporated herein by reference in its entirety.

[0002] Provided herein are compositions and methods for photoaffinity labeling of molecular targets, particularly probes that specifically interact with intracellular targets based on their affinity and then covalently link to the intracellular targets via a photoreactive group (PRG) on the probe. [Background technology]

[0003] Isolation and enrichment of intracellular targets of bioactive compounds is essential for both target identification and target profiling. It is well recognized that target binding by biologically active molecules in a live cell environment is often different from target binding in a biochemical context. Thus, there is a need for enrichment tools that are compatible with target binding within a cellular environment. Furthermore, chemoproteomic techniques are traditionally biased towards abundant and soluble protein targets. Targets with low expression levels and / or low affinity, as well as membrane proteins, especially those with multiple transmembrane domains, remain difficult to identify with chemoproteomic approaches. Summary of the Invention

[0004] Provided herein are compositions and methods for photoaffinity labeling of molecular targets, particularly probes that specifically interact with intracellular targets based on their affinity and then covalently link to the intracellular targets via photoreactive groups on the probe (e.g., UV-activated photoreactive groups).

[0005] In some embodiments, provided herein are compositions comprising a PRG / HA probe that includes a bioactive agent, a photoreactive group (PRG), a linker, and a haloalkane (HA) covalently linked to a single compound of the following general structure: [ka] (wherein R is a covalent bond and A is (CH2) n , n=4-10, X is a halogen, -AX is a substrate for a dehalogenase, and Q is CH or N). In some embodiments, the bioactive agent is a small molecule or peptide. In some embodiments, R comprises -NH-, -C(O)NH-, -OC(O)NH-, N, C(O)N, OC(O)N, or -O-. In some embodiments, R is a covalent bond resulting from the reaction of two reactive moieties (R' and R"). In some embodiments, R is -(CH2) 0-2 NH(CH2) 0-2 -, -(CH2) 0-2 C(O)NH(CH2) 0-2 -, -(CH2) 0-2 OC(O)NH(CH2) 0-2 -, (CH2) 0-2 N(CH2) 0-2 -, -(CH2) 0-2 C(O)N(CH2) 0-2 -, -(CH2) 0-2 OC(O)N(CH2) 0-2 - or -(CH2) 0-2 O(CH2) 0-2 In some embodiments, PRG is a photoreactive group, such as an aryl azide, an alkyl aryl azide (e.g., methyl aryl azide), a substituted aryl azide, or a substituted alkyl aryl azide (e.g., substituted methyl aryl azide). In some embodiments, PRG is covalently linked to the remainder of the compound by a PRG linker moiety. In some embodiments, the PRG linker moiety is (CH2) 0-4In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is chemically cleavable, enzymatically cleavable, or photocleavable. In some embodiments, the cleavable linker comprises a cleavable moiety selected from the group consisting of disulfide, tert-butyl carbamate, silyl ether, diazobenzene, 1,2-diol, and -C(O)OCH2CH=CHCH2OC(O)-. In some embodiments, the cleavable linker comprises a cleavable moiety flanked on one or both sides by an alkylene or heteroalkylene chain. In some embodiments, the alkylene or heteroalkylene chain is selected from the group consisting of C 1-6 In some embodiments, -AX is -(CH)Cl. ...

[0006] In some embodiments, the general structure of the PRG / HA probe is: [ka] Q includes CH or N; R includes -NH-, -C(O)NH-, -OC(O)NH-, -O-, -(CH2) 0-2 NH(CH2) 0-2 -, -(CH2) 0-2 C(O)NH(CH2) 0-2 -, -(CH2) 0-2 OC(O)NH(CH2) 0-2 - or -(CH2) 0-2 O(CH2) 0-2 In another embodiment, the general structure of the PRG / HA probe is: [ka] R is N, C(O)N, OC(O)N, (CH2) 0-2 N(CH2) 0-2 -, -(CH2) 0-2 C(O)N(CH2) 0-2 - or -(CH2) 0-2OC(O)N(CH2) 0-2 -It is.

[0007] In some embodiments, PRG is an aryl azide, an alkyl aryl azide (e.g., methyl aryl azide), a substituted aryl azide, or a substituted alkyl aryl azide (e.g., substituted methyl aryl azide). In some embodiments, PRG comprises the following structure: [ka] wherein A is an aromatic hydrocarbon ring (e.g., a 6-membered ring), an aromatic heterocyclic ring (e.g., a 5- or 6-membered ring), an aromatic ring system, or a heteroaromatic ring system, the N group(s) are present in any suitable position on the ring of A, and the optional Y groups, when present on the ring of A, are independently selected from halogen (e.g., Cl, F, Br, I), CH, OH, SH, NH, CN, CF, CCl, -CH-CH, -CH-OH, -CHNH, CHSH, CHCl, CHBr, CHF, CHF, CHCN, CHCF, CHCl, and CN. In some embodiments, A is selected from furan, benzofuran, isobenzofuran, pyrrole, indole, isoindole, thiophene, benzothiophene, benzo[c]thiophene, imidazole, benzimidazole, purine, pyrazole, indazole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, benzene, naphthalene, pyridine, quinolone, isoquinoline, pyrazine, quinoxaline, pyrimidine, quinazoline, pyridazine, cinnoline, phthalazine, thalidomide, triazine (e.g., 1,2,3-triazine, 1,2,4-triazine, 1,3,5 triazine), and thiadiazole. In some embodiments, PRG is a substituted phenyl or benzyl azide. In some embodiments, PRG is a substituted phenyl or benzyl azide, such as: [ka] wherein Y is selected from halogen (e.g., Cl, F, Br, I), CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2-OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, CH2Cl3, and CN. In some embodiments, the ring of A is a fused ring system, such as: [ka] wherein X is O, NH, or S. In some embodiments, PRG is selected from the group consisting of: [ka] Other exemplary ring structures of PRG and A are shown in FIG.

[0008] In some embodiments, provided herein are compositions comprising a reactive PRG / HA reagent that includes a photoreactive group (PRG), a linker, and a haloalkane covalently linked to a single compound of the following general structure: [ka] (wherein R' is a first reactive moiety capable of forming a covalent bond when reacted with a second reactive moiety (R"), and A is (CH2) n , n=4-10, X is a halogen, -AX is a substrate for a dehalogenase, and Q is CH or N. In some embodiments, R' comprises -NH2, NH, -C(O)OH, OC(O)O-phenyl, C(O)O-phenyl, OH, C=O, aldehyde, difluorosulfinate, sulfonyl fluoride, sulfonyl chloride, or halogen. In some embodiments, R' comprises -(CH2) 0-2 NH2, -(CH2) 0-2 C(O)OH, (CH2) 0-2 OC(O)O-phenyl, (CH2) 0-2 C(O)O-phenyl, (CH2) 0-2 OH, (CH2) 0-2 C=O, (CH2) 0-2Cl, or (CH2) 0-2 Br. In some embodiments, PRG is a photoreactive group, such as an aryl azide, alkyl aryl azide (e.g., methyl aryl azide), substituted aryl azide, or substituted alkyl aryl azide (e.g., substituted methyl aryl azide) moiety. In some embodiments, PRG is covalently connected to the remainder of the compound by a PRG linker moiety. In some embodiments, the PRG linker moiety is (CH2) 0-4 In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is chemically cleavable, enzymatically cleavable, or photocleavable. In some embodiments, the cleavable linker comprises a cleavable moiety selected from the group consisting of disulfide, tert-butyl carbamate, silyl ether, diazobenzene, 1,2-diol, and -C(O)OCH2CH=CHCH2OC(O)-. In some embodiments, the cleavable linker comprises a cleavable moiety flanked on one or both sides by an alkylene or heteroalkylene chain. In some embodiments, the alkylene or heteroalkylene chain is selected from the group consisting of C 1-6 In some embodiments, -AX is -(CH)Cl. ...

[0009] In some embodiments, the PRG / HA reagent has the following general structure: [ka] wherein R' comprises -NH2, -C(O)OH, OC(O)O-phenyl, C(O)O-phenyl, OH, aldehyde, difluorosulfinate, sulfonyl fluoride, sulfonyl chloride, or halogen. In other embodiments, the PRG / HA reagent has the following general structure: [ka] In the formula, R' includes -NH- or CO.

[0010] In some embodiments, PRG is an aryl azide, alkylaryl azide (e.g., methylaryl azide), substituted aryl azide, or substituted alkylaryl azide (e.g., substituted methylaryl azide) moiety. In some embodiments, PRG comprises the following structure: [ka] wherein A is an aromatic hydrocarbon ring (e.g., a 6-membered ring), an aromatic heterocyclic ring (e.g., a 5- or 6-membered ring), an aromatic ring system, or a heteroaromatic ring system, the N group(s) are present in any suitable position on the ring of A, and the optional Y groups, when present on the ring of A, are independently selected from halogen (e.g., Cl, F, Br, I), CH, OH, SH, NH, CN, CF, CCl, -CH-CH, -CH-OH, -CHNH, CHSH, CHCl, CHBr, CHF, CHF, CHCN, CHCF, CHCl, and CN. In some embodiments, A is selected from furan, benzofuran, isobenzofuran, pyrrole, indole, isoindole, thiophene, benzothiophene, benzo[c]thiophene, imidazole, benzimidazole, purine, pyrazole, indazole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, benzene, naphthalene, pyridine, quinolone, isoquinoline, pyrazine, quinoxaline, pyrimidine, quinazoline, pyridazine, cinnoline, phthalazine, thalidomide, triazine (e.g., 1,2,3-triazine, 1,2,4-triazine, 1,3,5 triazine), and thiadiazole. In some embodiments, PRG is a substituted phenyl or benzyl azide. In some embodiments, PRG is a substituted phenyl or benzyl azide, such as: [ka] wherein Y is selected from halogen (e.g., Cl, F, Br, I), CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2-OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, CH2Cl3, and CN. In some embodiments, the ring of A is a fused ring system, such as: [ka] wherein X is O, NH, or S. In some embodiments, PRG is selected from the group consisting of: [ka] Other exemplary ring structures of PRG and A are shown in FIG.

[0011] In some embodiments, provided herein are systems, kits, and reaction mixtures that include (i) a PRG / HA reagent described herein, and (ii) a bioactive agent that displays a second reactive moiety, R". In some embodiments, the bioactive agent is a small molecule or peptide. In some embodiments, the bioactive agent includes a drug or a naturally occurring compound that has been modified to display R". In some embodiments, R" includes -NH2, -OC(O)O-nitrophenyl, -OH, -C=O, -aldehyde, -difluorosulfinate, -sulfonyl fluoride, sulfonyl chloride, or -halogen. In some embodiments, R" includes -(CH2) 0-2 NH2, -(CH2) 0-2 OC(O)O-Nitrophenyl, -(CH2) 0-2 OH, -(CH2) 0-2 C=O, -(CH2) 0-2 Cl, or -(CH2) 0-2 Contains Br.

[0012] In some embodiments, provided herein is a method of making a PRG / HA probe, comprising contacting (i) a PRG / HA reagent described herein with (ii) a second reactive moiety, R″, of a bioactive agent. In some embodiments, provided herein is a PRG / HA probe produced by a method described herein.

[0013] In some embodiments, provided therein is a composition comprising a PRG / fluorophore probe comprising a bioactive agent, a photoreactive group (PRG), and a fluorophore covalently linked to a single compound of the following general structure: [ka] where R is a covalent bond and Q is CH or N. In some embodiments, the bioactive agent is a small molecule. In some embodiments, R comprises -NH-, -C(O)NH-, -OC(O)NH-, N, C(O)N, OC(O)N, or -O-. In some embodiments, R is a covalent bond resulting from the reaction of two reactive moieties (R' and R"). In some embodiments, R is -(CH2) 0-2 NH(CH2) 0-2 -, -(CH2) 0-2 C(O)NH(CH2) 0-2 -, -(CH2) 0-2 OC(O)NH(CH2) 0-2 -, (CH2) 0-2 N(CH2) 0-2 -, -(CH2) 0-2 C(O)N(CH2) 0-2 -, -(CH2) 0-2 OC(O)N(CH2) 0-2 - or -(CH2) 0-2 O(CH2) 0-2In some embodiments, PRG is a photoreactive group, such as an aryl azide, alkylaryl azide (e.g., methylaryl azide), substituted aryl azide, or substituted alkylaryl azide (e.g., substituted methylaryl azide) moiety. In some embodiments, PRG is covalently connected to the remainder of the compound by a PRG linker moiety. In some embodiments, the PRG linker moiety is (CH2) 0-4 In some embodiments, the fluorophore is covalently linked to the remainder of the compound by a fluorophore linker moiety. In some embodiments, the fluorophore linker moiety comprises a cleavable linker. In some embodiments, the general structure of the PRG / fluorophore probe is: [ka] Q includes CH or N; R includes -NH-, -C(O)NH-, -OC(O)NH-, -O-, -(CH2) 0-2 NH(CH2) 0-2 -, -(CH2) 0-2 C(O)NH(CH2) 0-2 -, -(CH2) 0-2 OC(O)NH(CH2) 0-2 - or -(CH2) 0-2 O(CH2) 0-2 In another embodiment, the general structure of the PRG / fluorophore probe is: [ka] R is N, C(O)N, OC(O)N, (CH2) 0-2 N(CH2) 0-2 -, -(CH2) 0-2 C(O)N(CH2) 0-2 - or -(CH2) 0-2 OC(O)N(CH2) 0-2 -It is.

[0014] In some embodiments, PRG is an aryl azide, alkyl aryl azide (e.g., methyl aryl azide), substituted aryl azide, or substituted alkyl aryl azide (e.g., substituted methyl aryl azide) moiety. In some embodiments, PRG comprises the following structure: [ka] wherein A is an aromatic hydrocarbon ring (e.g., a 6-membered ring), an aromatic heterocyclic ring (e.g., a 5- or 6-membered ring), an aromatic ring system, or a heteroaromatic ring system, the N group(s) are present in any suitable position on the ring of A, and the optional Y groups, when present on the ring of A, are independently selected from halogen (e.g., Cl, F, Br, I), CH, OH, SH, NH, CN, CF, CCl, -CH-CH, -CH-OH, -CHNH, CHSH, CHCl, CHBr, CHF, CHF, CHCN, CHCF, CHCl, and CN. In some embodiments, A is selected from furan, benzofuran, isobenzofuran, pyrrole, indole, isoindole, thiophene, benzothiophene, benzo[c]thiophene, imidazole, benzimidazole, purine, pyrazole, indazole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, benzene, naphthalene, pyridine, quinolone, isoquinoline, pyrazine, quinoxaline, pyrimidine, quinazoline, pyridazine, cinnoline, phthalazine, thalidomide, triazine (e.g., 1,2,3-triazine, 1,2,4-triazine, 1,3,5 triazine), and thiadiazole. In some embodiments, PRG is a substituted phenyl or benzyl azide. In some embodiments, PRG is a substituted phenyl or benzyl azide, such as: [ka] wherein Y is selected from halogen (e.g., Cl, F, Br, I), CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2-OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, CH2Cl3, and CN. In some embodiments, the ring of A is a fused ring system, such as: [ka] wherein X is O, NH, or S. In some embodiments, PRG is selected from the group consisting of: [ka] Other exemplary ring structures of PRG and A are shown in FIG.

[0015] In some embodiments, provided herein are reactive photoreactive group (PRG) / fluorophore reagents that include a PRG and a fluorophore connected to a single compound of the following general structure: [ka] where R' is a first reactive moiety capable of forming a covalent bond when reacted with a second reactive moiety (R") and Q is CH or N. In some embodiments, R' comprises -NH, NH, -C(O)OH, OC(O)O-phenyl, C(O)O-phenyl, OH, C=O, aldehyde, difluorosulfinate, sulfonyl fluoride, sulfonyl chloride, or halogen. In some embodiments, R' is -(CH) 0-2 NH2, -(CH2) 0-2 C(O)OH, (CH2) 0-2 OC(O)O-phenyl, (CH2) 0-2 C(O)O-phenyl, (CH2) 0-2 OH, (CH2) 0-2 C=O, (CH2) 0-2 Cl, or (CH2) 0-2Br. In some embodiments, PRG is a photoreactive group, such as an aryl azide, alkyl aryl azide (e.g., methyl aryl azide), substituted aryl azide, or substituted alkyl aryl azide (e.g., substituted methyl aryl azide) moiety. In some embodiments, PRG is covalently connected to the remainder of the compound by a PRG linker moiety. In some embodiments, the PRG linker moiety is (CH2) 0-4 In some embodiments, the fluorophore is covalently linked to the remainder of the compound by a fluorophore linker moiety. In some embodiments, the fluorophore linker moiety comprises a cleavable linker.

[0016] In some embodiments, the PRG / fluorophore reagent has the following general structure: [ka] wherein R' comprises -NH2, -C(O)OH, OC(O)O-phenyl, C(O)O-phenyl, OH, aldehyde, difluorosulfinate, sulfonyl fluoride, sulfonyl chloride, or halogen. In other embodiments, the PRG / fluor reagent has the following general structure: [ka] In the formula, R' includes -NH- or CO.

[0017] In some embodiments, PRG is an aryl azide, alkyl aryl azide (e.g., methyl aryl azide), substituted aryl azide, or substituted alkyl aryl azide (e.g., substituted methyl aryl azide) moiety. In some embodiments, PRG comprises the following structure: [ka] wherein A is an aromatic hydrocarbon ring (e.g., a 6-membered ring), an aromatic heterocyclic ring (e.g., a 5- or 6-membered ring), an aromatic ring system, or a heteroaromatic ring system, the N group(s) are present in any suitable position on the ring of A, and the optional Y groups, when present on the ring of A, are independently selected from halogen (e.g., Cl, F, Br, I), CH, OH, SH, NH, CN, CF, CCl, -CH-CH, -CH-OH, -CHNH, CHSH, CHCl, CHBr, CHF, CHF, CHCN, CHCF, CHCl, and CN. In some embodiments, A is selected from furan, benzofuran, isobenzofuran, pyrrole, indole, isoindole, thiophene, benzothiophene, benzo[c]thiophene, imidazole, benzimidazole, purine, pyrazole, indazole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, benzene, naphthalene, pyridine, quinolone, isoquinoline, pyrazine, quinoxaline, pyrimidine, quinazoline, pyridazine, cinnoline, phthalazine, thalidomide, triazine (e.g., 1,2,3-triazine, 1,2,4-triazine, 1,3,5 triazine), and thiadiazole. In some embodiments, PRG is a substituted phenyl or benzyl azide. In some embodiments, PRG is a substituted phenyl or benzyl azide, such as: [ka] wherein Y is selected from halogen (e.g., Cl, F, Br, I), CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2-OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, CH2Cl3, and CN. In some embodiments, the ring of A is a fused ring system, such as: [ka] wherein X is O, NH, or S. In some embodiments, PRG is selected from the group consisting of: [ka] Other exemplary ring structures of PRG and A are shown in FIG.

[0018] In some embodiments, provided herein are systems, kits, and reaction mixtures that include (i) a reactive PRG / fluorophore reagent described herein, and (ii) a bioactive agent that displays a second reactive moiety, R". In some embodiments, the bioactive agent is a small molecule or peptide. In some embodiments, the bioactive agent includes a drug or a naturally occurring compound that has been modified to display R". In some embodiments, R" includes -NH2, -OC(O)O-nitrophenyl, -OH, -C=O, -aldehyde, -difluorosulfinate, -sulfonyl fluoride, sulfonyl chloride, or -halogen. In some embodiments, R" includes -(CH2) 0-2 NH2, -(CH2) 0-2 OC(O)O-Nitrophenyl, -(CH2) 0-2 OH, -(CH2) 0-2 C=O, -(CH2) 0-2 Cl, or -(CH2) 0-2 Contains Br.

[0019] In some embodiments, provided herein is a method of making a PRG / fluorophore probe comprising contacting (i) a reactive PRG / fluorophore reagent described herein with (ii) a bioactive agent that displays a second reactive moiety, R″. In some embodiments, provided herein is a PRG / fluorophore probe produced by the methods described herein.

[0020] In some embodiments, provided herein are methods of capturing a target molecule, the method comprising contacting a sample containing the target molecule with a PRG / HA probe described herein, where a bioactive agent of the PRG / HA probe can associate with the target molecule; allowing the PRG / HA probe to associate with the target molecule; and irradiating the sample with light of a wavelength that converts PRG to a reactive form of PRG, where the reactive form of PRG forms a covalent bond with the target molecule. In some embodiments, the method further comprises contacting the sample with a modified dehalogenase enzyme that forms a covalent bond with a haloalkane substrate when associated with the substrate; and covalently binding the modified dehalogenase enzyme to the haloalkane of the PRG / HA probe. In some embodiments, the modified dehalogenase enzyme is immobilized on a solid surface, and when the modified dehalogenase enzyme binds to the haloalkane of the PRG / HA probe, the target molecule is immobilized on the solid surface. In some embodiments, the method further comprises (i) removing the solid surface from the sample; and / or (ii) washing non-immobilized components of the sample from the solid surface. In some embodiments, the solid surface is a bead, particle, chip, tube, plate, or membrane. In some embodiments, the solid surface is a paramagnetic particle. In some embodiments, the method further comprises cleaving the linker of the PRG / HA probe to release the target molecule bound to the PRG and bioactive agent from the solid surface. In some embodiments, the method further comprises detecting and / or quantifying the target molecule. In some embodiments, the target molecule is detected and / or quantified by mass spectrometry. In some embodiments, the sample comprises a cell, and the target molecule is located within or on the surface of the cell. In some embodiments, contacting the sample comprising the target molecule with the PRG / HA probe further comprises placing the PRG / HA probe into the cell. In some embodiments, the method further comprises lysing the cell after PRG forms a covalent bond with the target molecule. In some embodiments, the bioactive agent of the PRG / HA probe can non-covalently bind to the target molecule when the bioactive agent is associated with the target molecule, hi some embodiments, the bioactive agent is a small molecule or a peptide.In some embodiments, the target molecule is a target protein. In some embodiments, the target protein is a cell surface protein, a transmembrane protein, a cytoplasmic protein, a nuclear protein, or a mitochondrial protein. In some embodiments, the light of a wavelength that converts PRG to a reactive form of PRG is ultraviolet (UV) light (e.g., 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, or a range therebetween (e.g., 10-400 nm, 320-380 nm, etc.).

[0021] In some embodiments, provided herein is a method of capturing an intracellular target protein, the method comprising contacting a cell containing the target protein with a PRG / HA probe described herein, where a bioactive agent of the PRG / HA probe can bind to the target protein; placing the PRG / HA probe in the cell; allowing the PRG / HA probe to bind to the target protein; irradiating the cell with UV light at a wavelength that converts PRG to a reactive form of PRG, where the reactive form of PRG forms a covalent bond with the target protein; lysing the cell to form a lysate; contacting the lysate with a modified dehalogenase enzyme immobilized on a solid surface that forms a covalent bond with a haloalkane substrate when associated therewith; covalently binding the modified dehalogenase enzyme to the haloalkane of the PRG / HA probe; and removing the solid surface from the lysate and / or washing non-immobilized components of the lysate from the solid surface. In some embodiments, the method further comprises cleaving a linker of the PRG / HA probe to release the target protein bound to PRG and the bioactive agent from the solid surface. In some embodiments, the method further comprises detecting and / or quantifying the target molecule. In some embodiments, the target protein is a cell surface protein, a transmembrane protein, a cytoplasmic protein, a nuclear protein, or a mitochondrial protein.

[0022] In some embodiments, provided herein is a method for measuring photocrosslinking efficiency of PRG linked to a bioactive agent, comprising contacting a sample containing a target molecule with a PRG / fluorophore probe described herein, where the target molecule is tethered to a bioluminescent reporter, where the emission spectrum of the bioluminescent reporter overlaps with the excitation spectrum of the fluorophore; binding a bioactive agent of the PRG / fluorophore probe to the target molecule; irradiating the sample with light of a wavelength that converts PRG to a reactive form of PRG, where the reactive form of PRG can form a covalent bond with the target molecule; contacting the sample with a substrate for the bioluminescent reporter; detecting emission within the emission spectrum of the bioluminescent reporter and the fluorophore, where the fluorophore is capable of reacting with the target molecule. detecting that the emission from the fluorophore is a result of bioluminescence resonance energy transfer (BRET) from the bioluminescent reporter to the fluorophore, and the amount of emission from the fluorophore correlates to the amount of PRG / fluorophore probe bound to the target molecule; further contacting the sample with an untagged bioactive agent to competitively displace the PRG / fluorophore probe that is not covalently attached to the target; contacting the sample with a substrate for the bioluminescent reporter to detect emission within the emission spectrum of the bioluminescent reporter and the fluorophore, and the emission from the fluorophore is a result of bioluminescence resonance energy transfer (BRET) from the bioluminescent reporter to the fluorophore, and the amount of emission from the fluorophore correlates to the amount of PRG / fluorophore probe covalently attached to the target molecule. In some embodiments, the method further comprises calculating the percent crosslinking by normalizing the amount of PRG / fluorophore probe covalently attached to the target molecule to the amount of PRG / fluorophore probe bound to the target molecule prior to the addition of the untagged bioactive agent. In some embodiments, the method further comprises detecting an emission within the emission spectrum of the bioluminescent reporter. In some embodiments, the bioluminescent reporter is a bioluminescent protein or a peptide or polypeptide that is a component of a bioluminescent complex. In some embodiments, the concentration of the untagged bioactive agent in the sample is greater than the concentration of the PRG / fluorophore probe in the sample.

[0023] In some embodiments, provided herein are methods for measuring photocrosslinking efficiency of PRG linked to a bioactive agent, the methods including contacting a sample containing a target molecule with a PRG / HA probe described herein, where the target molecule is tethered to a bioluminescent reporter; allowing a bioactive agent of the PRG / HA probe to bind to the target molecule; irradiating the sample with light of a wavelength that converts PRG to a reactive form of PRG, where the reactive form of PRG can form a covalent bond with the target molecule; contacting the sample with an excess of a BRET reagent comprising a bioactive agent covalently tethered to a fluorophore, where the emission spectrum of the bioluminescent reporter overlaps with the excitation spectrum of the fluorophore; contacting the sample with a substrate for the bioluminescent reporter and detecting emission within the emission spectrum of the bioluminescent reporter and the fluorophore, where the emission from the fluorophore is a result of bioluminescence resonance energy transfer (BRET) from the bioluminescent reporter to the fluorophore, and the amount of emission from the fluorophore correlates to the amount of BRET reagent bound to the target molecule. In some embodiments, the amount of BRET reagent bound to the target molecule correlates to the amount of target molecule not bound and / or covalently crosslinked by the PRG / HA probe. In some embodiments, the method further comprises: not contacting a sample containing the target molecule with a PRG / HA probe described herein, where the target molecule is tethered to a bioluminescent reporter; irradiating the sample and then contacting the sample with an excess of a BRET reagent comprising a bioactive agent covalently tethered to a fluorophore, where the emission spectrum of the bioluminescent reporter overlaps with the excitation spectrum of the fluorophore; contacting the sample with a substrate for the bioluminescent reporter and detecting emission within the emission spectrum of the bioluminescent reporter and the fluorophore, where the emission from the fluorophore is a result of bioluminescence resonance energy transfer (BRET) from the bioluminescent reporter to the fluorophore.In some embodiments, the method further comprises comparing (i) an emission spectrum collected from a sample treated with a BRET reagent (e.g., a bioactive agent and a fluorophore) and a PRG / HA probe and illuminated with light of a wavelength that converts PRG to its reactive form, with (ii) an emission spectrum collected from a sample treated with a BRET reagent (e.g., a bioactive agent and a fluorophore) but not treated with PRG / HA and illuminated with light of the same wavelength that converts PRG to its reactive form, whereby the reduction in BRET due to pretreatment with PRG / HA inversely correlates to the photocrosslinking efficiency of PRG (photocrosslinking of the PRG / HA probe to the target prevents the probe from being displaced by excess BRET reagent, thereby reducing the BRET signal). In some embodiments, the method further comprises contacting a sample comprising a target molecule with a PRG / HA probe described herein, where the target molecule is tethered to a bioluminescent reporter; not irradiating the sample, and then contacting the sample with an excess of a BRET reagent comprising a bioactive agent covalently tethered to a fluorophore, where the emission spectrum of the bioluminescent reporter overlaps with the excitation spectrum of the fluorophore; contacting the sample with a substrate for the bioluminescent reporter and detecting emission within the emission spectrum of the bioluminescent reporter and the fluorophore, where the emission from the fluorophore is the result of bioluminescent resonance energy transfer (BRET) from the bioluminescent reporter to the fluorophore. In some embodiments, the method further comprises (i) comparing the emission spectrum collected from the irradiated sample to (ii) the emission spectrum collected from a non-irradiated sample, where the reduction in BRET due to irradiation is inversely correlated to the photocrosslinking efficiency of PRG (photocrosslinking of the PRG / HA probe to the target prevents the probe from being displaced by the excess BRET reagent, thereby reducing the BRET signal).

[0024] In some embodiments, provided herein is a composition comprising a photoreactive group (PRG) linked to a bioactive agent, wherein the PRG is selected from: [ka]

[0025] In some embodiments, provided herein are methods for covalently attaching a bioactive agent to a target protein, the method comprising: contacting the target molecule with a bioactive agent linked to a photoreactive group (PRG), where the bioactive agent can associate with the target molecule; allowing the bioactive agent to associate with the target molecule; and irradiating with light of a wavelength that converts the PRG to a reactive form of PRG, where the reactive form of PRG forms a covalent bond with the target molecule. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 is a schematic diagram of a general photoaffinity cleavable chloroalkane probe. [Diagram 2] FIG. 1 is a schematic diagram of the effect of cell lysis on affinity-driven binding equilibrium with intracellular targets. [Diagram 3] Common PRGs and their reactive intermediates. [Figure 4] FIG. 1 is a schematic diagram of UV-induced covalent cross-linking of a photoreactive chemical probe with its associated intracellular target, thereby "freezing" the affinity-driven binding equilibrium and preserving it upon cell lysis. [Figure 5A] FIG. 1 is a schematic showing exemplary derivatizable bioactive agents from a parent bioactive agent. [Figure 5B] FIG. 1 is a schematic showing reaction of a derivatizable bioactive agent with a PRG / fluorophore reagent to yield a PRG / fluorophore probe. [Figure 6A] FIG. 1 is a schematic diagram of exemplary derivatizable bioactive agents displaying a variety of suitable reactive groups. [Figure 6B] FIG. 1 is a schematic diagram of exemplary PRG / chloroalkane reagents displaying a variety of complementary reactive groups. [Figure 7] FIG. 1 is a schematic showing the attachment of a generic derivatizable bioactive agent to a PRG / CA reagent to form a PRG / CA probe. [Figure 8] FIG. 1 is a schematic showing the attachment of a generic derivatizable bioactive agent to a PRG / HA or PRG / fluorophore reagent to form a PRG / HA or PRG / fluorophore probe. [Figure 9] FIG. 1 is a schematic showing the use of photoreactive chloroalkane chemical probes to establish compound selectivity and affinity profiling in living cells. [Figure 10] FIG. 1 is a schematic diagram of a typical fluorescent photoaffinity probe. [Figure 11] (A) General scheme of the BRET assay to quantify cross-linking efficiency. BRET occurs upon binding of the PRG / fluorophore probe to a target tethered to NANOLUC. UV irradiation induces covalent cross-linking of the probe to its target. Treatment with excess parent compound displaces the non-covalently bound PRG / fluorophore probe, decreasing BRET. (B) Residual BRET resulting from probes that could not be competitively dissociated by excess parent compound is normalized to the maximal BRET from an equivalent sample that was not treated with excess parent compound and used to derive cross-linking efficiency. The normalized residual BRET correlates to cross-linking efficiency. [Figure 12] 1 is the structure of the SAHA-dye tracer. [Figure 13] Experimental setup to test the effect of UV irradiation on the signal intensity of both NanoLuc and fluorescent dyes. [Figure 14] Effect of UV irradiation on the bioluminescence signal intensity of NanoLuc. [Figure 15] Effect of UV irradiation on the fluorescence intensity of NCT, BODIPY, and TMR fluorophores conjugated to SAHA at multiple probe concentrations and different irradiation times. [Figure 16] 1A-1C are exemplary parent drugs and their modifiable cores for chemical probe synthesis. SAHA and propranolol are building blocks for probe synthesis containing suitable protecting groups. [Figure 17]General structures of fluorescent photoaffinity labeling probes and their respective targets for photoaffinity labeling studies. [Figure 18] Comparison of the photocrosslinking efficiency of SAHA and dasatinib PRG / fluorophore probes, which contain commonly used photoreactive groups and a simple aryl azide. [Figure 19] Comparison of photocrosslinking efficiency of SAHA and dasatinib PRG / fluorophore probes containing aryl azides with different substituents. [Figure 20] Comparison of photocrosslinking efficiency of SAHA and dasatinib PRG / fluorophore probes containing various aryl diazides. [Figure 21] Comparison of the photocrosslinking efficiency of selected PRG / fluorophore probes. In all six model systems, the performance of the o-tolyl azide probe outperformed the diazirine probe. [Figure 22] Reverse BRET approach to estimate photocrosslinking efficiency. (A) General scheme of the reverse BRET assay to quantify crosslinking efficiency. Binding of the PRG / HA probe to a NanoLuc-tethered target does not result in BRET. UV irradiation induces covalent crosslinking of the PRG / HA probe to its target. Treatment with excess BRET reagent displaces the non-covalently bound PRG / HA probe, resulting in BRET. (B) The decrease in BRET resulting from a covalently bound PRG / HA probe that could not be competed with the BRET reagent is normalized to the maximum BRET from an equivalent sample not treated with PRG / HA. The normalized decrease in BRET is inversely related to the crosslinking efficiency. The chloroalkane portion of the probe is not required for this experiment. [Figure 23] Estimation of the photocrosslinking efficiency of selected photoaffinity probes by "direct" and "reverse" BRET methods. [Figure 24] (A) Exemplary structures of photoaffinity cleavable chloroalkane probes tested. (B) Structures of the fluorescent probes used in reverse BRET experiments. [Diagram 25] Effect of the chloroalkane tag on the phenotypic potency of the compounds. [Figure 26] FIG. 1 is a schematic diagram of an exemplary workflow for target enrichment of bioactive compounds utilizing photoaffinity chloroalkane probes. [Figure 27] Western analysis comparing the effect of PRG groups on the efficiency of SAHA target enrichment from K562 cells in the presence and absence of UV irradiation. [Figure 28] Western analysis comparing the effect of PRG groups on the efficiency of dasatinib target enrichment from K562 cells in the presence and absence of UV irradiation. [Figure 29A] Synthesis of a carboxylate-modified bioactive agent, SAHA-carboxylate. [Figure 29B] Synthesis of a carboxylate-modified bioactive agent, dasatinib carboxylate. [Figure 29C] Synthesis of a carboxylate-modified bioactive agent, propranolol carboxylate. [Diagram 30] 1 is an exemplary general scheme for the synthesis of bifunctional PRG building blocks. [Figure 31-1] 1 is an exemplary synthesis of a bifunctional PRG building block. [Figure 31-2] 1 is an exemplary synthesis of a bifunctional PRG building block. [Figure 31-3] 1 is an exemplary synthesis of a bifunctional PRG building block. [Diagram 32] 1 is an exemplary general scheme for synthesizing drug-PRG conjugates. [Diagram 33] 1 is an exemplary general scheme for synthesizing drug-PRG-NCT and drug-PRG-CA chemical probes. [Figure 34A] Illustrative ring structures of PRG and A. [Figure 34B] Illustrative ring structures of PRG and A. [Figure 35A] Molecular structure of SAHA (vorinostat). [Figure 35B]1 is a molecular structure of 4-phenylazide, 4-Me-3-phenylazide, and diazirine chloroalkane probes. [Figure 35C] LC-MS / MS analysis of target enrichment of SAHA from K-562 cells using 4-phenylazide, 4-Me-3-phenylazide, and diazirine chloroalkane probes. [Figure 36A] The molecular structure of dasatinib. [Figure 36B] 1 is a molecular structure of 4-phenylazide, 4-Me-3-phenylazide, and diazirine chloroalkane probes. [Figure 36C] LC-MS / MS analysis of dasatinib target enrichment from K-562 cells using 4-phenylazide, 4-Me-3-phenylazide, and diazirine chloroalkane probes. [Figure 37A] The molecular structure of propranolol. [Figure 37B] 1 is a molecular structure of the diazirine and 4-phenylazidochloroalkane probes. [Figure 37C] Bioluminescence blot analysis of propranolol target enrichment from genome-edited PC3 cells expressing HiBiT-tagged β2-AR using diazirine and 4-phenylazidochloroalkane probes. [Figure 37D] LC-MS / MS analysis of target enrichment of propranolol from PC3 cells using a 4-phenylazidochloroalkane probe. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] definition Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of each embodiment described in the present invention, some preferred methods, compositions, devices, and materials are described herein. However, prior to describing the materials and methods of the present invention, it should be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or protocols described herein, as they may vary according to routine experimentation and optimization. It should also be understood that the terminology used in these descriptions is intended to describe only the specific forms or embodiments, and is not intended to limit the scope of the embodiments described herein.

[0028] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by those skilled in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, shall control. Therefore, in the context of the embodiments described herein, the following definitions shall apply.

[0029] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" refers to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.

[0030] As used herein, the term "and / or" includes any and all combinations of the listed items, including any of the individual listed items. For example, "A, B, and / or C" includes A, B, C, AB, AC, BC, and ABC, each of which should be considered as being individually set forth by the statement "A, B, and / or C."

[0031] As used herein, the term "comprise" and linguistic variations thereof indicate the presence of the recited feature(s), element(s), method step(s), etc., without excluding the presence of additional feature(s), element(s), method step(s), etc. In contrast, the term "consisting of" and linguistic variations thereof indicate the presence of the recited feature(s), element(s), method step(s), etc., and excludes any unrecited feature(s), element(s), method step(s), etc., except for impurities normally associated therewith. The phrase "consisting essentially of" indicates the recited feature(s), element(s), method step(s), etc., and any additional feature(s), element(s), method step(s), etc. that do not substantially affect the basic nature of the composition, system, or method. Many embodiments herein are described using the open term "comprise." Such embodiments encompass several closed "consisting of" and / or "consisting essentially of" embodiments, which may alternatively be claimed or described using such terms.

[0032] As used herein, the term "substantially" means that the described property, parameter, and / or value need not be exactly achieved, but that deviations or deviations, including, for example, tolerances, measurement errors, measurement accuracy limits, and other factors known in the art, may occur in amounts that do not interfere with the effect of the property intended to be provided. A substantially absent property or characteristic may be a property that is within the noise of the property of interest, below the background, below the detection capabilities of the assay used, or only insignificant (e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%, <0.000001%, <0.0000001%).

[0033] As used herein, the term "photoreactive group" refers to a functional moiety that forms a covalent bond with a molecule in its immediate vicinity when exposed to light (e.g., light of a particular wavelength or range of wavelengths, UV light, etc.).

[0034] As used herein, the term "sample" is used in its broadest sense. In one sense, the term is meant to include specimens or culture fluids obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass liquids, solids, tissues, and gases. Biological samples include blood products such as plasma, serum, etc. Samples may also refer to cell lysates or purified forms of enzymes, peptides, and / or polypeptides as described herein. Cell lysates may include cells lysed with a lysing agent, or lysates such as rabbit reticulocyte or wheat germ lysates. Samples may also include cell-free expression systems. Environmental samples include environmental materials such as surface materials, soil, water, crystals, and industrial samples. However, such examples should not be construed as limiting the types of samples applicable to the present invention.

[0035] As used herein, the term "physiological conditions" encompasses any conditions compatible with living cells, for example, primarily aqueous conditions of temperature, pH, salinity, chemical composition, and the like, that are compatible with living cells.

[0036] As used herein, the terms "conjugated" and "conjugation" refer to the covalent attachment (e.g., post-synthesis and / or during synthetic production) of two molecular entities. Chemical (e.g., "chemically" conjugated) or enzymatic attachment of a peptide or small molecule tag to a protein or small molecule is an example of conjugation. Conjugation of a peptide to a biomolecule occurs when an SE-peptide reacts with an amine on the biomolecule.

[0037] The term "binding moiety" refers to a domain that specifically binds to an antigen or epitope, independent of a different epitope or antigen-binding domain. A binding moiety can be an antibody, an antibody fragment, a receptor domain that binds to a target ligand, a protein that binds to an immunoglobulin (e.g., Protein A, Protein G, Protein A / G, Protein L, Protein M), a binding domain of a protein that binds to an immunoglobulin (e.g., Protein A, Protein G, Protein A / G, Protein L, Protein M), an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein (whether the analyte itself or a protein that binds to an analyte), an analyte-binding domain(s) of a protein, and the like. Table A provides a list of exemplary binding moieties that may be used singly or in various combinations in the methods, systems, and assays (e.g., immunoassays) herein.

[0038] [Table 1]

[0039] As used herein, the term "antibody" refers to an entire antibody molecule or a fragment thereof (e.g., fragments such as Fab, Fab', and F(ab')2, variable light chain, variable heavy chain, Fv, etc.). An antibody may be polyclonal or monoclonal or recombinant, chimeric, humanized, human, etc. As used herein, when an antibody or other entity "specifically recognizes" or "specifically binds" to an antigen or epitope, the antibody or other entity selectively recognizes the antigen in a complex mixture of proteins and / or macromolecules and binds to the antigen or epitope with a significantly higher affinity than other entities that do not display the antigen or epitope. In this context, "significantly higher affinity than" means an affinity high enough to allow detection of the antigen or epitope as distinguished from the entity using a desired assay or measurement device. Typically, it is at least 10 7 M -1(For example, >10 7 M -1 , >10 8 M -1 , >10 9 M -1 , >10 10 M -1 , >10 11 M -1 , >10 12 M -1 , >10 13 M -1 etc.) binding constant (K a ) means a binding affinity having a specific epitope. In certain such embodiments, an antibody can bind to different antigens as long as the different antigens contain that particular epitope. In certain cases, for example, homologous proteins from different species may contain the same epitope.

[0040] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody that contains at least a portion of the antigen binding region or variable region. Antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, variable light chain, variable heavy chain, diabody, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, for example, Hudson et al. (2003) Nat. Med. 9:129-134, which is incorporated herein by reference in its entirety. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage (e.g., papain and pepsin digestion of antibodies) of intact antibodies produced by recombinant DNA technology or chemical polypeptide synthesis. For example, a "Fab" fragment contains one light chain and one C heavy chain. H1 and variable regions. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. An "Fab'" fragment contains one light chain and one H1 Domain and C H2and one heavy chain with an additional constant region extending between the domains. An interchain disulfide bond may be formed between the two heavy chains of the Fab' fragment to form an "F(ab')2" molecule. An "Fv" fragment contains the variable regions from both the heavy and light chains, but does not contain the constant regions. A single-chain Fv (scFV) fragment contains the variable regions of the heavy and light chains, which are connected by a flexible linker to form a single polypeptide chain with an antigen-binding region. Exemplary single-chain antibodies are discussed in detail in WO88 / 01649, and U.S. Pat. Nos. 4,946,778 and 5,260,203, which are incorporated herein by reference in their entirety. In certain cases, a single variable region (e.g., heavy or light chain variable region) may have the ability to recognize and bind to an antigen. Other antibody fragments will be appreciated by those skilled in the art.

[0041] As used herein, the term "biomolecule" or "biological molecule" refers to molecules and ions that are present in living organisms and are essential for biological process(es), such as cell division, morphogenesis, or development. Biomolecules include large macromolecules (or polyanions), such as proteins, carbohydrates, lipids, and nucleic acids, as well as small molecules, such as primary metabolites, secondary metabolites, and natural products. A more common name for this class of material is biomaterials. Biomolecules are usually endogenous, but may also be exogenous. For example, pharmaceuticals may be natural or semi-synthetic (biopharmaceuticals), or completely synthetic.

[0042] As used herein, the term "bioluminescence" refers to the production and emission of light by a chemical reaction catalyzed or enabled by an enzyme, protein, protein complex, or other biological molecule (e.g., a bioluminescent complex). In a typical embodiment, a substrate of a bioluminescent entity (e.g., a bioluminescent protein or bioluminescent complex) is converted by the bioluminescent entity into an unstable form. The substrate subsequently emits light.

[0043] As used herein, the term "non-luminescent" refers to an entity (e.g., a peptide, polypeptide, complex, protein, etc.) that exhibits the property of not emitting a detectable amount of light in the visible spectrum (e.g., in the presence of a substrate). For example, an entity may be referred to as non-luminescent if it does not exhibit detectable luminescence in a given assay. As used herein, the term "non-luminescent" is synonymous with the term "substantially non-luminescent". In some embodiments, an entity is considered "non-luminescent" if any luminescence is small enough that it does not create a background that interferes with a particular assay.

[0044] As used herein, the term "Oplophorus luciferase" ("OgLuc") refers to a light-emitting polypeptide having significant sequence identity, structural conservation, and / or functional activity of the luciferase derived from and produced by the deep-sea shrimp Oplophorus gracilirostris. In particular, OgLuc polypeptide refers to a light-emitting polypeptide having significant sequence identity, structural conservation, and / or functional activity of the mature 19 kDa subunit (e.g., without the signal sequence) of the Oplophorus luciferase protein complex, such as SEQ ID NOs: 1 (WT OgLuc) and 5 (NANOLUC), which contains 10 β-strands (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10) and utilizes a substrate, such as coelenterazine or a derivative or analog of coelenterazine, to generate light.

[0045] As used herein, the term "complex" refers to an assembly or aggregate of molecules (e.g., peptides, polypeptides, etc.) that are in direct and / or indirect contact with each other. In one aspect, "contact" or more specifically "direct contact" means that two or more molecules are in sufficient proximity such that non-covalent attractive interactions, such as van der Waals forces, hydrogen bonds, ionic and hydrophobic interactions, govern the interaction of the molecules. In such an aspect, a complex of molecules (e.g., peptides and polypeptides) is formed under assay conditions that thermodynamically favor the complex (e.g., compared to the non-aggregated or non-aggregated states of its component molecules). As used herein, the term "complex" refers to an assembly of two or more molecules (e.g., peptides, polypeptides, or combinations thereof), unless otherwise specified.

[0046] As used herein, the term "capture protein" refers to a protein that forms a stable covalent bond with its substrate and / or ligand when it interacts with them. A capture protein can be a receptor that forms a covalent bond when it binds to its ligand, or an enzyme that forms a covalent bond with its substrate. An example of a capture protein suitable for use in embodiments of the present invention is the HALOTAG protein described in U.S. Patent No. 7,425,436, which is incorporated herein by reference in its entirety.

[0047] As used herein, the term "capture ligand" or "capture moiety" refers to a ligand, substrate, etc. that forms a covalent bond with a capture protein when it interacts with it. An example of a capture ligand suitable for use in embodiments of the present invention is the HALOTAG ligand, described, for example, in U.S. Pat. No. 7,425,436, which is incorporated herein by reference in its entirety. Moieties that find use as HALOTAG ligands include haloalkane (HA) groups (e.g., chloroalkane (CA) groups). In embodiments described herein that specify HA or CA capture ligands, other suitable capture ligands may be substituted unless otherwise specified.

[0048] As used herein, the term "intracellular target" refers to a protein, a polypeptide, a nucleic acid (e.g., DNA or RNA), a polysaccharide, or a complex that includes any of these with a polypeptide(s). An intracellular target may be composed of multiple components, subunits, or polypeptides, for example, an intracellular target is a protein complex. Examples of intracellular targets may include receptors or enzymes.

[0049] As used herein, the term "bioactive agent" generally refers to any physiologically or pharmacologically active substance or substance suitable for detection. In some embodiments, the bioactive agent is a potential therapeutic compound (e.g., a small molecule, peptide, nucleic acid, etc.) or a drug-like molecule. Bioactive agents for use in the embodiments described herein are not limited by size or structure.

[0050] As used herein, the term "small molecule" refers to an organic compound of low molecular weight (e.g., <2000 Daltons, <1000 Daltons, <500 Daltons) having dimensions (e.g., length, width, diameter, etc.) on the order of 1 nm. Larger structures such as peptides, proteins, and nucleic acids are not small molecules, although their constituent monomers (ribo- or deoxyribonucleotides, amino acids, etc.) are considered small molecules.

[0051] As used herein, the term "cell-permeable" refers to a compound or moiety that can efficiently cross a cell membrane that has not been synthetically permeabilized.

[0052] Definitions of certain functional groups and chemical terms are described in more detail below. For purposes of this disclosure, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 thEd., and specific functional groups are generally defined as described therein. In addition, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Sorrell, Organic Chemistry, 2000, Vol. 1, No. 1, pp. 111-115, 1997. nd edition,University Science Books,Sausalito,2006;Smith,March's Advanced Organic Chemistry:Reactions,Mechanism,and Structure,7 th Edition,John Wiley&Sons,Inc.,New York,2013;Larock,Comprehensive Organic Transformations,3 rd Edition,John Wiley&Sons,Inc.,New York,2018;Carruthers,Some Modern Methods of Organic Synthesis,3 rd Edition, Cambridge University Press, Cambridge, 1987, the entire contents of each of which are incorporated herein by reference.

[0053] The term "alkoxy" as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0054] The term "alkyl" as used herein refers to an alkyl group containing 1 to 30 carbon atoms, e.g., 1 to 16 carbon atoms (C1-C 16 Alkyl), 1 to 14 carbon atoms (C1-C 14 Alkyl), 1 to 12 carbon atoms (C1-C 12 Alkyl), 1 to 10 carbon atoms (C1-C 10"alkyl" refers to a straight or branched saturated hydrocarbon chain containing 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), or 1 to 4 carbon atoms (C1-C4 alkyl). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0055] The term "alkenyl," as used herein, refers to a straight or branched hydrocarbon chain containing 2 to 30 carbon atoms and containing at least one carbon-carbon double bond. For example, an alkenyl group can be an alkyl group having 2 to 16 carbon atoms (C2-C 16 Alkenyl, 2 to 14 carbon atoms (C2-C 14 Alkenyl, 2 to 12 carbon atoms (C2-C 12 Alkenyl, 2 to 10 carbon atoms (C2-C 10 alkenyl), 2 to 8 carbon atoms (C2-C8 alkenyl), 2 to 6 carbon atoms (C2-C6 alkenyl), or 2 to 4 carbon atoms (C2-C4 alkenyl). Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

[0056] The term "alkynyl," as used herein, refers to a straight or branched hydrocarbon chain containing 2 to 30 carbon atoms and containing at least one carbon-carbon triple bond. For example, an alkynyl group can be an alkyl group having 2 to 16 carbon atoms (C2-C3). 16 Alkynyl, 2 to 14 carbon atoms (C2-C 14 Alkynyl, 2 to 12 carbon atoms (C2-C 12Alkynyl, 2 to 10 carbon atoms (C2-C 10 alkynyl), 2 to 8 carbon atoms (C2-C8 alkynyl), 2 to 6 carbon atoms (C2-C6 alkynyl), or 2 to 4 carbon atoms (C2-C4 alkynyl). Representative examples of alkynyl include, but are not limited to, ethynyl, propynyl, and butynyl.

[0057] The term "alkylene" as used herein refers to an alkylene group having 1 to 30 carbon atoms, for example, 1 to 16 carbon atoms (C1-C 16 Alkylene, 1 to 14 carbon atoms (C1-C 14 Alkylene, 1 to 12 carbon atoms (C1-C 12 Alkylene, 1 to 10 carbon atoms (C1-C 10 It refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 8 carbon atoms (C1-C8 alkylene), 1 to 6 carbon atoms (C1-C6 alkylene), or 1 to 4 carbon atoms (C1-C4 alkylene). Representative examples of alkylene include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH(CH3)CH2-, and -CH2CH2CH2CH2CH2-.

[0058] The term "aryl" as used herein refers to a phenyl group, or a bicyclic or tricyclic aromatic fused ring system. A bicyclic fused ring system is exemplified by a phenyl group attached to a parent molecular moiety and fused to a phenyl group. A tricyclic fused ring system is exemplified by a phenyl group attached to a parent molecular moiety and fused to two other phenyl groups. Representative examples of bicyclic aryl include, but are not limited to, naphthyl. Representative examples of tricyclic aryl include, but are not limited to, anthracenyl.

[0059] The term "cycloalkyl" as used herein refers to a saturated carbocyclic ring system containing 3 to 10 carbon atoms and 0 heteroatoms. Cycloalkyls may be monocyclic, bicyclic, bridged, fused, or spirocyclic. Representative examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl.

[0060] The term "cycloalkenyl," as used herein, means a non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and preferably having from 5 to 10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, or cycloheptenyl.

[0061] The term “halogen” or “halo” as used herein means F, Cl, Br, or I.

[0062] The term "haloalkyl" as used herein, means an alkyl group, as defined herein, in which 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms are replaced by halogen.

[0063] The term "heteroalkyl," as used herein, means an alkyl group, as defined herein, in which one or more of the carbon atoms is replaced by a heteroatom selected from S, O, P, and N. Representative examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkyl amines, and alkyl sulfides.

[0064] The term "heteroalkylene," as used herein, means an alkylene group, as defined herein, wherein one or more of the carbon atoms is replaced by a heteroatom selected from S, O, P, and N. Representative examples of heteroalkylene include, but are not limited to, -O-CH-, -NH-CH-, -S-CH-, -O-CH-CH-, -S-CH-CH-, -NH-CH-CH-, -CH-O-CH-, -CH-S-CH-, and -CH-NH-CH-.

[0065] The term "heteroaryl" as used herein refers to an aromatic monocyclic ring or aromatic bicyclic ring system or aromatic tricyclic ring system. An aromatic monocyclic ring is a 5- or 6-membered ring containing at least one heteroatom independently selected from the group consisting of N, O, and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). A 5-membered aromatic monocyclic ring has two double bonds, and a 6-membered aromatic monocyclic ring has three double bonds. A bicyclic heteroaryl group is exemplified by a monocyclic heteroaryl ring fused to a monocyclic aryl group as defined herein or a monocyclic heteroaryl group as defined herein. A tricyclic heteroaryl group is exemplified by a monocyclic heteroaryl group fused to two rings independently selected from a monocyclic aryl group as defined herein or a monocyclic heteroaryl group as defined herein. Representative examples of monocyclic heteroaryls include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl, isothiazolyl, thienyl, furanyl, oxazolyl, isoxazolyl, 1,2,4-triazinyl, and 1,3,5-triazinyl. Representative examples of bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzodioxolyl, benzofuranyl, benzoxadiazolyl, benzopyrazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxadiazolyl, benzoxazolyl, chromenyl, imidazopyridine, imidazothiazolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolinyl, naphthyridinyl, purinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, quinoxalinyl, thiazolopyridinyl, thiazolopyrimidinyl, thienopyrrolyl, and thienothienyl. Representative examples of tricyclic heteroaryl include, but are not limited to, dibenzofuranyl, and dibenzothienyl.The monocyclic, bicyclic, and tricyclic heteroaryls are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring.

[0066] The term "heterocycle" or "heterocyclic" as used herein means a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. A monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. A 3- or 4-membered ring contains zero or one double bond and one heteroatom selected from the group consisting of O, N, and S. A 5-membered ring contains zero or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A 6-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A 7- and 8-membered ring contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles include azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, piperazinyl, and piperidinyl. , pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl.A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiro heterocyclic group, or a bridged monocyclic heterocyclic ring system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Tricyclic heterocycles are exemplified by bicyclic heterocycles fused to a phenyl group, or bicyclic heterocycles fused to a monocyclic cycloalkyl, or bicyclic heterocycles fused to a monocyclic cycloalkenyl, or bicyclic heterocycles fused to a monocyclic heterocycle, or bicyclic heterocycles in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles include octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.1. 3,7 ]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7 ]decane). The monocyclic, bicyclic, and tricyclic heterocycles are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained in the ring.

[0067] The term "hydroxy" as used herein, means a -OH group.

[0068] In some cases, the number of carbon atoms in a group (e.g., alkyl, alkoxy, or cycloalkyl) is indicated by the prefix "C x -C y -" where x is the smallest number of carbon atoms in the group and y is the largest number. Thus, for example, "C1-C 3- "Alkyl" refers to an alkyl group containing 1 to 3 carbon atoms.

[0069] The term "substituent" refers to a group substituted on an atom of a designated group.

[0070] When a group or moiety is substitutable, the term "substituted" indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments, 1, 2, or 3; in other embodiments, 1 or 2) hydrogens of the group designated with the phrase "substituted" can be replaced with one selected from the designated group described or suitable groups known to those of skill in the art (e.g., one or more of the groups described below). Substituents include, but are not limited to, halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0071] For the compounds described herein, groups and substituents thereof may be selected according to allowed atom valences and substituents such that selection and substitution give rise to stable compounds and do not spontaneously undergo transformations such as, for example, by rearrangement, cyclization, elimination, and the like.

[0072] Provided herein are compositions and methods for photoaffinity labeling of molecular targets, particularly probes that specifically interact with intracellular targets based on their affinity and then covalently link to the intracellular targets via a photoreactive group (PRG) on the probe.

[0073] Provided herein is a chemoproteomic platform for both target identification and compound selectivity profiling. The platform is based on the generation of photoreactive chemical probe(s) derived from biologically active compounds (bioactive agents). In some embodiments, the probes are generated by chemically tagging the bioactive agents with chloroalkanes that are (i) cell-permeable, (ii) photoreactive, and (iii) cleavable (FIG. 1). The probes are designed / synthesized using a modular assembly of components (e.g., bioactive agent, PRG, cleavable linker, and capture moiety). PRGs with cleavable chloroalkanes can be assembled (e.g., attached to bioactive agent(s)) into a single reagent to create a library of PRG / HA-labeled bioactive agents. This modular, general approach exploits the robust photoreactivity of PRG as described herein, allowing for efficient photocrosslinking to target(s) without the need to individually tailor the attachment of PRG to each bioactive compound. Furthermore, PRG / HA probes have minimal effect on the cell permeability of bioactive compounds, allowing efficient photocrosslinking of the probes to target protein(s) in a cellular environment. In addition, PRG / HA probes incorporate a capture moiety (e.g., cleavable chloroalkane), allowing straightforward enrichment of interacting targets through capture on HALOTAG resin without the additional click ligation of capture moieties required in other systems (see, e.g., ACS Chem.Biol., 2015, 10(10), pp 2316-2324; ACS Chem.Biol., 2016, 11(9), pp 2608-2617; US Patent Application Publication No. 2014 / 0322794; US Patent Application Publication No. 2016 / 0355523; each of which is incorporated herein by reference in its entirety). The cleavable moiety of the probes herein allows efficient release of enriched targets from solid support, for example, for identification by mass spectrometry.

[0074] Despite advances in chemoproteomics technology, identifying binding interactions established in living cells remains a challenge. A major methodological problem with chemoproteomics approaches is that cell lysis is required for enrichment of interacting intracellular targets. The cell lysis step disrupts the affinity-driven drug-target equilibrium established in living cells, so the equilibrium established after lysis may not be the equilibrium in intact cells (Figure 2). The platforms, systems, probes, reagents, and methods herein overcome this problem, which existing technologies often face, by forming a covalent bond between the probe and the target after the bioactive agent portion of the probe interacts non-covalently with the target, thereby preventing dissociation upon lysis. The covalent bond is formed using a photoreactive group (e.g., aryl azide, diazirine, benzophenone, etc.; see, e.g., Figure 3) that is chemically inert until activated by light (photoactivation). Upon photolysis, the photoreactive group (PRG) generates a highly reactive species(s) that can photocrosslink to a target molecule (e.g., form a covalent bond with an amino acid in a target protein). Provided herein is a platform that provides a general tool to label any bioactive agent with a photoreactive group, thereby enabling UV-induced covalent crosslinking of the bioactive agent with its interacting target, thereby "freezing" the equilibrium within the cell and maintaining it even upon lysis (Figure 4).

[0075] Existing photocrosslinking chemistries often suffer from low labeling efficiency and therefore rely on tailoring the attachment of PRGs to bioactive compounds to achieve efficient crosslinking with different protein targets. This is further complicated by the current state of the art that employs cell-permeable, photoreactive clickable probes that allow for bioorthogonal click ligation of capture tags (e.g., biotin) to the probe after cell lysis. While these clickable probes address the common clash between biotin conjugation and cell permeability of bioactive compounds, most click reactions are incompatible with aryl azides, a useful PRG. This is due to the inherent reactivity of the azide functionality towards common click partners (e.g., strained alkynes, trans-cyclooctene (TCO), etc.). Such cross-reactivity is not an issue for chloroalkane tags, which encompass an incorporated orthogonally reactive capture moiety that is fully compatible with aryl azides and obviates the need for a click reaction. For example, the chloroalkane tag, HALOTAG ligand (Promega; Madison, WI), exhibits minimal impact on small molecule cell permeability and binding interactions. This feature, combined with the unique compatibility of chloroalkanes with a variety of photoreactive groups and their high reactivity with HALOTAG proteins, makes haloalkane capture moieties particularly useful for use in modular platforms that employ photoreactive groups with enhanced crosslinking properties.

[0076] In some embodiments, provided herein is a chemoproteomic platform for intracellular target binding, selectivity profiling, and quantitative target specific analysis of compounds. In some embodiments, the platform relies on the chemical inertness of capture tags (e.g., haloalkane tags, chloroalkane tags, etc.), which allows the use of aryl azides (see, e.g., Table 1). In some embodiments, the platform is based on modularly assembled photoreactive chloroalkane probes (Figure 1) that incorporate existing and novel photoreactive groups. UV irradiation-induced crosslinking of the photoreactive chloroalkane probes to targets in live cells "freezes" the intracellular equilibrium, which is preserved even upon cell lysis. Further capture of interacting targets on capture protein (e.g., HALOTAG) resin allows for complete covalent enrichment of these targets. In some embodiments, the captured targets are released from the solid support by gentle chemical cleavage of the photoreactive chloroalkane tags, followed by LC-MS / MS analysis of the enriched targets (Figure 9).

[0077] One of the difficulties facing the field is the lack of effective assays to estimate photocrosslinking efficiency. The complexity and structural diversity of biological systems, combined with the complex reactivity of the photogenerated species, often results in conflicting literature data on crosslinking efficiency.

[0078] Currently, the state of the art in photocrosslinking chemistry applied to chemoproteomics is dominated by the use of diazirines (both alkyl- and aryl-diazirines) as PRGs. Benzophenones are also used, but are not commonly used in many systems because they are bulky and can exhibit low crosslinking efficiency. Aryl azides are often avoided in chemoproteomics due to their incompatibility with many click chemistry handles.

[0079] In order to systematically evaluate numerous existing and novel photoreactive groups (PRGs), during the development of the embodiments herein, a BRET-based assay was developed to estimate photocrosslinking efficiency. This assay format utilizes a fluorescent photoaffinity probe incorporating a PRG and a fluorescent reporter dye (Figure 10). The fluorescent photoaffinity probe of Figure 10 differs from the probe of Figure 1 in the reporter tag. That is, the chloroalkane capture tag in the probe of Figure 1 is switched to a fluorescent reporter dye in the probe of Figure 10. A schematic diagram of an assay using the probe of Figure 10 is shown in Figure 11. The assay depicted in Figure 11 relies on the measurement of BRET between a target tethered to a luciferase (e.g., NANOLUC) and a fluorescent photoaffinity probe, and has the advantage that the photocrosslinking efficiency is estimated independently of other environmental factors. When a fluorescent photoaffinity probe binds to its target protein fused to NANOLUC, a BRET ratio for a given probe concentration is obtained (e.g., emission from a fluorophore as a result of energy transfer from luciferase, which is expressed as a BRET ratio). This BRET correlates to the maximum probe occupancy at a given probe concentration. Further treatment with excess parent bioactive compound displaces bound fluorescent photoaffinity probes, resulting in a decrease in BRET, which correlates to the minimum probe occupancy. If the sample is UV-irradiated after binding of the fluorescent photoaffinity probe to crosslink the probe to its target, subsequent treatment with excess parent bioactive compound will only displace non-covalently bound probe. The residual BRET resulting from probes that could not be displaced (normalized to the maximum and minimum probe occupancy) can be used to derive the occupancy of covalently bound probes, which reflects the photocrosslinking efficiency. Simple fluorescence readings do not always provide reliable information on crosslinking efficiency, since the fluorescence of the dyes is very sensitive to their environment and the same dyes can result in quite different fluorescence readings when attached to different chemical entities. Fluorescence, unlike other methods (e.g., BRET), does not depend on close association of the probe with the target protein.

[0080] I. Probe In some embodiments, provided herein are probes that include a bioactive agent (e.g., a peptide, a small molecule, etc.), a PRG, and a functional moiety that allows for detection, capture, monitoring, etc. of the probe before and / or after covalent attachment (e.g., via reaction of a PRG) to a target molecule, protein, etc. Provided herein is a platform that facilitates the use of the capture and detection methods described herein with any bioactive agent of interest.

[0081] A.PRG / HA probe In some embodiments, provided herein are probes comprising a bioactive agent, a photoreactive group (PRG), and a capture ligand (e.g., a haloalkane); reagents and methods for assembling such probes (e.g., for labeling a bioactive agent (e.g., a reactively derivatized bioactive agent) with a PRG and a capture ligand (e.g., a haloalkane); methods for using such probes to capture a target molecule (e.g., an intracellular protein target) in a sample (e.g., a complex sample (e.g., a cell, a cell lysate, etc.)); and compositions for determining the crosslinking efficiency of the probes herein (e.g., PRG / fluorophore probes, fusions of an intracellular target with a bioluminescent reporter, etc.). In some embodiments, after the capture probe is covalently conjugated to a target (e.g., a target molecule, a target protein, etc.), a capture protein (e.g., or a capture fusion protein) binds to the capture ligand to allow for isolation, enrichment, etc. of the captured target. In some embodiments, provided herein is a system that allows for the attachment of PRG and capture ligands (e.g., haloalkanes) to any bioactive agent (e.g., small molecules, peptides, etc.) in a reactively derivatized form once the bioactive agent has been generated.

[0082] In some embodiments, provided herein are compositions comprising a PRG / HA probe, which comprises a bioactive agent, a photoreactive group (PRG), a linker, and a capture ligand (e.g., a haloalkane) covalently linked to a single compound of the following general structure: [ka] where R is a covalent bond and Q is CH or N.

[0083] In some embodiments, R is a covalent bond resulting from the reaction of two reactive moieties (R' and R"). In some embodiments, R comprises -NH-, -C(O)NH-, -OC(O)NH-, N, C(O)N, OC(O)N, or -O-. In some embodiments, R is -(CH2) 0-2 NH(CH2) 0-2 -, -(CH2) 0-2 C(O)NH(CH2) 0-2 -, -(CH2) 0-2 OC(O)NH(CH2) 0-2 -, (CH2) 0-2 N(CH2) 0-2 -, -(CH2) 0-2 C(O)N(CH2) 0-2 -, -(CH2) 0-2 OC(O)N(CH2) 0-2 - or -(CH2) 0-2 O(CH2) 0-2 - Including.

[0084] In some embodiments, the general structure of the PRG / HA probe is: [ka] Q includes CH or N; R includes -NH-, -C(O)NH-, -OC(O)NH-, -O-, -(CH2) 0-2 NH(CH2) 0-2 -, -(CH2) 0-2 C(O)NH(CH2) 0-2 -, -(CH2) 0-2 OC(O)NH(CH2) 0-2 - or -(CH2) 0-2 O(CH2) 0-2 In another embodiment, the general structure of the PRG / HA probe is: [ka] R is N, C(O)N, OC(O)N, (CH2) 0-2 N(CH2) 0-2 -, -(CH2) 0-2 C(O)N(CH2) 0-2 - or -(CH2) 0-2 OC(O)N(CH2) 0-2- It is.

[0085] In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is chemically cleavable, enzymatically cleavable, or photocleavable. In some embodiments, the cleavable linker comprises a cleavable moiety selected from the group consisting of disulfide, tert-butyl carbamate, silyl ether, diazobenzene, 1,2-diol, and -C(O)OCH2CH=CHCH2OC(O)-. In some embodiments, the cleavable linker comprises a cleavable moiety flanked on one or both sides by an alkylene or heteroalkylene chain. In some embodiments, the alkylene or heteroalkylene chain is selected from the group consisting of C 1-6 -alkylene, -O-, -(CH2)2O-, and -OC(O)NH- groups.

[0086] In some embodiments, the linkers herein (e.g., linkers of the probes herein (e.g., connecting a capture ligand to a Q / R group) comprise any suitable moiety that provides a desired structure / function (e.g., length, flexibility / rigidity, solubility, cell permeability, etc.). The probes herein are not limited to any particular linker moiety. Indeed, a variety of linker moieties are contemplated, and suitable linkers include, but are not limited to, alkyl groups, methylene carbon chains, ethers, polyethers, alkylamide linkers, peptide linkers, modified peptide linkers, poly(ethylene glycol) (PEG) linkers, streptavidin-biotin or avidin-biotin linkers, polyamino acids (e.g., polylysine), functionalized PEGs, polysaccharides, glycosaminoglycans, dendritic polymers (see WO 93 / 06868, and Tomalia et al. in Angew. Chem. Int. Ed. Engl. 29:138-175 (1990)). al., incorporated herein by reference in their entireties), PEG-chelator polymers (WO 94 / 08629, WO 94 / 09056, and WO 96 / 26754, incorporated herein by reference in their entireties), oligonucleotide linkers, phospholipid derivatives, alkenyl chains, alkynyl chains, disulfides, or combinations thereof. In some embodiments, the linker is an alkyl, alkenyl, alkynyl, amide, phenyl, benzyl, carbamate, halo, fluoro, or combinations thereof. The aromatic hydrocarbon group may comprise or be optionally substituted with any combination of fluoro, chloro, bromo, iodo, hydroxyl, carbonyl, aldehyde, haloformyl, carbonate, carboxylate, carboxyl, ester, hydroperoxy, peroxy, ether, hemiacetal, hemiketal, acetal, ketal, orthoester, amide, amine, imine, imide, azide, azo, cyanate, nitrate, nitrite, nitrile, nitro, nitroso, pyridine, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinic acid, sulfonic acid, thiocyanate, thione, thial, phosphine, phosphonic acid, phosphate, and / or phosphodiester groups.Any suitable linker utilizing any suitable functional group is within the scope of embodiments of the present invention. In some embodiments, the linker comprises a cleavable moiety and one or more of the functional groups / moieties described above.

[0087] In some embodiments, the capture ligand is a haloalkane (also known as an "alkyl halide"). In some embodiments, the capture ligand is a chloroalkane. In some embodiments, the capture ligand is -AX. In some embodiments, X is Cl. In some embodiments, -AX is -(CH2)6Cl. When the capture ligand is a haloalkane, the capture protein is typically a dehalogenase enzyme that has been modified to form a covalent bond with its substrate (see, e.g., U.S. Pat. Nos. 7,425,436; 7,429,472; 7,867,726; 7,888,086; 7,935,803; RE42,931; 8,168,405; 8,202,700; and 8,257,939, which are incorporated herein by reference in their entireties). One such modified dehalogenase is the commercially available HALOTAG protein. In some embodiments, the capture protein comprises a polypeptide having at least 70% sequence identity (e.g., 75% identity, 80% identity, 85% identity, 90% identity, 95% identity, 98% identity, 99% identity) to SEQ ID NO: 15. In some embodiments, the capture ligand comprises an alkyl chain (e.g., (CH2) 4-24 The capture ligand (e.g., haloalkane) is a haloalkane that comprises a halogen (e.g., Cl, Br, F, I, etc.) covalently attached to the end of the alkyl chain. In some embodiments, the other end of the alkyl chain is attached to a linker or other portion of the probe (e.g., PRG, bioactive agent, central connecting group, etc.). In some embodiments, the capture ligand (e.g., haloalkane) is attached to other portions of the probe by a linker. The linker may comprise an alkyl chain or a substituted alkyl chain (e.g., C=O, NH, S, O, carbamate, ethylene, etc.).

[0088] In some embodiments, the bioactive agent is a small molecule or peptide. In some embodiments, the bioactive agent is any molecule that binds or potentially binds to a protein, intracellular target, molecule of interest, etc. In some embodiments, the bioactive agent is a small molecule. In some embodiments, the bioactive agent is a peptide. In some embodiments, the bioactive agent is a drug or putative drug. In some embodiments, a suitable bioactive agent can be derivatized for attachment to other components of the probes herein (e.g., PRG, HA). In some embodiments, a suitable bioactive agent includes a functional group for attachment to other components of the probes herein (e.g., PRG, HA). In some embodiments, the derivatized or functional group on the bioactive agent used for attachment to the probe does not interfere with the activity of the bioactive agent and / or binding of the bioactive agent to a protein, intracellular target, molecule of interest, etc. In some embodiments, the bioactive agent includes a reactive moiety or a reactive moiety is disposed on the bioactive agent to generate a bioactive agent that can be attached to a reactive probe reagent. In some embodiments, the bioactive agent comprises or is modified to comprise a reactive group selected from NH2, NH, -C(O)OH, OC(O)O-phenyl, C(O)O-phenyl, OH, C=O, aldehyde, difluorosulfinate, sulfonyl fluoride, sulfonyl chloride, and halogen. In some embodiments, such reactive groups are suitable for reaction with one or more reactive groups presented on the reactive probe reagent. In some embodiments, the active group presented on the reactive probe reagent is -(CH2) 0-2 NH2, -(CH2) 0-2 C(O)OH, (CH2) 0-2 OC(O)O-phenyl, (CH2) 0-2 C(O)O-phenyl, (CH2) 0-2 OH, (CH2) 0-2 C=O, (CH2) 0-2 Cl, and (CH2) 0-2 Br.

[0089] In some embodiments, the PRG is selected from any of the PRGs described herein and / or understood in the art. In some embodiments, the PRG is a photoreactive group, such as an aryl azide, alkylaryl azide (e.g., methylaryl azide), substituted aryl azide, or substituted alkylaryl azide (e.g., substituted methylaryl azide) moiety. In some embodiments, the PRG is an aryl azide, alkylaryl azide (e.g., methylaryl azide), substituted aryl azide, or substituted alkylaryl azide (e.g., substituted methylaryl azide) moiety. In some embodiments, the PRG comprises the following structure: [ka] wherein A is an aromatic hydrocarbon ring (e.g., a 6-membered ring), an aromatic heterocyclic ring (e.g., a 5- or 6-membered ring), an aromatic ring system, or a heteroaromatic ring system, the N group(s) are present in any suitable position on the ring of A, and the optional Y groups, when present on the ring of A, are independently selected from halogen (e.g., Cl, F, Br, I), CH, OH, SH, NH, CN, CF, CCl, -CH-CH, -CH-OH, -CHNH, CHSH, CHCl, CHBr, CHF, CHF, CHCN, CHCF, CHCl, and CN. In some embodiments, A is selected from furan, benzofuran, isobenzofuran, pyrrole, indole, isoindole, thiophene, benzothiophene, benzo[c]thiophene, imidazole, benzimidazole, purine, pyrazole, indazole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, benzene, naphthalene, pyridine, quinolone, isoquinoline, pyrazine, quinoxaline, pyrimidine, quinazoline, pyridazine, cinnoline, phthalazine, thalidomide, triazine (e.g., 1,2,3-triazine, 1,2,4-triazine, 1,3,5 triazine), and thiadiazole. In some embodiments, PRG is a substituted phenyl or benzyl azide. In some embodiments, PRG is a substituted phenyl or benzyl azide, such as: [ka] wherein Y is selected from halogen (e.g., Cl, F, Br, I), CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2-OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, CH2Cl3, CH(CH3)2, (CH2)2CH3, and CN. In some embodiments, the ring of A is a fused ring system, such as: [ka] where X is O, NH, or S. Exemplary PRGs are shown in Table 1.

[0090] [Table 2] Other exemplary ring structures of PRG and A are shown in FIG.

[0091] In some embodiments, other photoreactive groups find use as the PRG moiety of the probes herein. Suitable PRGs include phenylazides, phenyldiazirines, and benzophenones, where the reactive intermediates formed when irradiated with light of a particular wavelength are nitrenes, carbenes, and diradicals, respectively. In some embodiments, PRGs are stable (e.g., non-reactive) in visible light. In some embodiments, the activation energy of PRGs is greater than the absorption wavelength of the protein. Proteins absorb UV light at 280 and 200 nm, where the absorbance at 280 nm is due to the aromatic amino acids tryptophan, tyrosine, and phenylalanine, and peptide bonds are responsible for the absorbance at 200 nm. Excessive absorption of UV light by proteins leads to the formation of electronically excited states of the molecule, which can then undergo degradative chemical transformations. In some embodiments, the PRG or photoreactive group includes diazo groups, diazocarbonyls, enones, sulfur radicals, halogenated substrates, nitrobenzenes, diazonium salts, alkyl derivatives of azides and diazirines, and the like. Fleming provides a comprehensive review of the various chemical reagents used in photoaffinity labeling (see Fleming SA. Chemical reagents in photoaffinity labelling. Tetrahedron. 1995; 51: 12479-12520, incorporated herein by reference in its entirety). Sakurai et al. recently reported a study comparing the main photoreactive groups and assessing their reactivity (see Sakurai et al. Comparison of the reactivity of carbohydrate photoaffinity probes with different photoreactive groups. Chem. Bio. Chem. 2014; 15: 1399-1403, incorporated herein by reference in its entirety).

[0092] In some embodiments, PRG is covalently linked to the remainder of the compound by a PRG linker moiety. In some embodiments, the PRG linker moiety is (CH2) 0-4 It is.

[0093] B.PRG / Fluorescent probe In some embodiments, provided herein are probes comprising a bioactive agent, a photoreactive group (PRG), and a fluorophore; reagents and methods for assembling such probes (e.g., for labeling a bioactive agent (e.g., a reactively derivatized bioactive agent) with a PRG and a fluorophore); methods for using such probes to tag a target molecule (e.g., an intracellular protein target) in a sample (e.g., a complex sample (e.g., cells, cell lysates, etc.)); and compositions for determining the target cross-linking efficiency of the probes herein (e.g., PRG / fluorophore probes, fusions of intracellular targets with bioluminescent reporters, etc.). In some embodiments, provided herein are systems that allow for the attachment of a PRG and a fluorophore to any bioactive agent (e.g., a small molecule, peptide, etc.) in a reactively derivatized form as the bioactive agent is generated.

[0094] In some embodiments, provided therein is a composition comprising a PRG / fluorophore probe comprising a bioactive agent, a photoreactive group (PRG), and a fluorophore covalently linked to a single compound of the following general structure: [ka] where R is a covalent bond and Q is CH or N.

[0095] In some embodiments, R is a covalent bond resulting from the reaction of two reactive moieties (R' and R"). In some embodiments, R comprises -NH-, -C(O)NH-, -OC(O)NH-, N, C(O)N, OC(O)N, or -O-. In some embodiments, R is -(CH2) 0-2 NH(CH2) 0-2 -, -(CH2) 0-2C(O)NH(CH2) 0-2 -, -(CH2) 0-2 OC(O)NH(CH2) 0-2 -, (CH2) 0-2 N(CH2) 0-2 -, -(CH2) 0-2 C(O)N(CH2) 0-2 -, -(CH2) 0-2 OC(O)N(CH2) 0-2 - or -(CH2) 0-2 O(CH2) 0-2 - Including.

[0096] In some embodiments, the general structure of the PRG / fluorophore probe is: [ka] Q includes CH or N; R includes -NH-, -C(O)NH-, -OC(O)NH-, -O-, -(CH2) 0-2 NH(CH2) 0-2 -, -(CH2) 0-2 C(O)NH(CH2) 0-2 -, -(CH2) 0-2 OC(O)NH(CH2) 0-2 - or -(CH2) 0-2 O(CH2) 0-2 In another embodiment, the general structure of the PRG / fluorophore probe is: [ka] R is N, C(O)N, OC(O)N, (CH2) 0-2 N(CH2) 0-2 -, -(CH2) 0-2 C(O)N(CH2) 0-2 - or -(CH2) 0-2 OC(O)N(CH2) 0-2 -It is.

[0097] In some embodiments, the bioactive agent is a small molecule or peptide. In some embodiments, similar to the PRG / HA probes described above, a bioactive agent suitable for use in the PRG / fluorophore probe is any molecule that binds or potentially binds to a protein, intracellular target, molecule of interest, etc. In some embodiments, the bioactive agent is a small molecule. In some embodiments, the bioactive agent is a peptide. In some embodiments, the bioactive agent is a drug or putative drug. In some embodiments, a suitable bioactive agent can be derivatized for attachment to other components of the probes herein (e.g., PRG, fluorophore). In some embodiments, a suitable bioactive agent includes a functional group for attachment to other components of the probes herein (e.g., PRG, fluorophore). In some embodiments, the derivatized or functional group on the bioactive agent used for attachment to the probe does not interfere with the activity of the bioactive agent and / or binding of the bioactive agent to a protein, intracellular target, molecule of interest, etc. In some embodiments, the bioactive agent includes a reactive moiety or a reactive moiety is disposed on the bioactive agent to generate a bioactive agent that can be attached to a reactive probe reagent. In some embodiments, the bioactive agent comprises or is modified to comprise a reactive group selected from NH2, NH, -C(O)OH, OC(O)O-phenyl, C(O)O-phenyl, OH, C=O, aldehyde, difluorosulfinate, sulfonyl fluoride, sulfonyl chloride, and halogen. In some embodiments, such reactive groups are suitable for reaction with one or more reactive groups presented on the reactive probe reagent. In some embodiments, the active group presented on the reactive probe reagent is -(CH2) 0-2 NH2, -(CH2) 0-2 C(O)OH, (CH2) 0-2 OC(O)O-phenyl, (CH2) 0-2 C(O)O-phenyl, (CH2) 0-2 OH, (CH2) 0-2 C=O, (CH2) 0-2 Cl, and (CH2) 0-2 Br.

[0098] In some embodiments, the PRG is selected from any of the PRGs described herein (e.g., suitable aryl azides, alkylaryl azides (e.g., methylaryl azides), substituted aryl azides, or substituted alkylaryl azides (e.g., substituted methylaryl azides)) and / or PRGs understood in the art. Exemplary PRGs are shown in Table 1 (above). In some embodiments, other photoaffinity labels and photoreactive groups find use as the PRG portion of the probes herein (e.g., those described above and elsewhere herein). For example, suitable PRGs include phenyl azides, phenyl diazirines, and benzophenones, where the reactive intermediates formed when irradiated with certain wavelengths of light are nitrenes, carbenes, and diradicals, respectively. In some embodiments, the PRG or photoreactive group is stable (e.g., non-reactive) in visible light. In some embodiments, the activation energy of the PRG or photoreactive group is greater than the absorption wavelength of the protein. Proteins absorb UV light at 280 and 200 nm, where the absorbance at 280 nm is due to the aromatic amino acids tryptophan, tyrosine, and phenylalanine, and peptide bonds are responsible for the absorbance at 200 nm. Excessive absorption of UV light by proteins leads to the formation of electronically excited states of the molecule, which can then undergo degradative chemical transformations. In some embodiments, PRG or photoreactive groups include diazo groups, diazocarbonyls, enones, sulfur radicals, halogenated substrates, nitrobenzenes, diazonium salts, alkyl derivatives of azides and diazirines, and the like. Fleming provides a comprehensive review of the various chemical reagents used in photoaffinity labeling (see Fleming SA. Chemical reagents in photoaffinity labelling. Tetrahedron. 1995;51:12479-12520, incorporated herein by reference in its entirety).Sakurai et al. recently reported a study comparing the main photoreactive groups and assessing their reactivity (see Sakurai et al. Comparison of the reactivity of carbohydrate photoaffinity probes with different photoreactive groups. Chem. Bio. Chem. 2014;15:1399-1403, which is incorporated herein by reference in its entirety).

[0099] In some embodiments, PRG is covalently linked to the remainder of the compound by a PRG linker moiety. In some embodiments, the PRG linker moiety is (CH2) 0-4 It is.

[0100] In some embodiments, the fluorophore is selected from any suitable fluorophore understood in the art (eg, a small molecule fluorescent dye). Suitable fluorophores include xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, oxazine 170, etc.), acridine derivatives (e.g., proflavine, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porphine, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA Fluor (Invitrogen), DYLIGHT Fluor (Thermo Scientific, Pierce), ATTO and TRACY dyes (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES dyes (Dyomics), SULFO CY dyes (CYANDYE, LLC), SETAU and SQUARE dyes (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT dyes (APC, RPE, PerCP, phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, and BPE dyes (Phyco-Biotech), and the like. In some embodiments, the fluorophores herein are rhodamine analogs (e.g., carboxyrhodamine analogs), such as those described in U.S. Patent Application Serial No. 13 / 682,589, which is incorporated herein by reference in its entirety.In some embodiments, a suitable fluorophore is selected based on its physical properties, such as size, emission spectrum, excitation spectrum, solubility, cell permeability, and the like.

[0101] In some embodiments, the fluorophore is covalently linked to the remainder of the compound by a fluorophore linker moiety. In some embodiments, the fluorophore linker moiety is (CH2) 0-4 In some embodiments, the fluorophore linker moiety comprises a cleavable linker. In some embodiments, the cleavable linker is chemically cleavable, enzymatically cleavable, or photocleavable. In some embodiments, the cleavable linker comprises a cleavable moiety selected from the group consisting of disulfide, tert-butyl carbamate, silyl ether, diazobenzene, 1,2-diol, and -C(O)OCH2CH=CHCH2OC(O)-. In some embodiments, the cleavable linker comprises a cleavable moiety flanked on one or both sides by an alkylene or heteroalkylene chain. In some embodiments, the alkylene or heteroalkylene chain is selected from the group consisting of C 1-6 -alkylene, -O-, -(CH2)2O-, and -OC(O)NH- groups.

[0102] In some embodiments, the linkers herein (e.g., linkers of the probes herein (e.g., linking fluorophores to Q / R groups) comprise any suitable moiety that provides the desired structure / function (e.g., length, flexibility / rigidity, solubility, cell permeability, etc.). The probes herein are not limited to any particular linker moiety. Indeed, a variety of linker moieties are contemplated, and suitable linkers include, but are not limited to, alkyl groups, methylene carbon chains, ethers, polyethers, alkylamide linkers, peptide linkers, modified peptide linkers, poly(ethylene glycol) (PEG) linkers, streptavidin-biotin or avidin-biotin linkers, polyamino acids (e.g., polylysine), functionalized PEGs, polysaccharides, glycosaminoglycans, dendritic polymers (see WO 93 / 06868, and Tomalia et al. in Angew. Chem. Int. Ed. Engl. 29:138-175 (1990)). al., incorporated herein by reference in their entireties), PEG-chelator polymers (WO 94 / 08629, WO 94 / 09056, and WO 96 / 26754, incorporated herein by reference in their entireties), oligonucleotide linkers, phospholipid derivatives, alkenyl chains, alkynyl chains, disulfides, or combinations thereof. In some embodiments, the linker is an alkyl, alkenyl, alkynyl, amide, phenyl, benzyl, carbamate, halo, fluoro, chloro, bromo, iodo, hydroxyl, carbonyl, aldehyde, haloformyl, carbonate ester, or a combination thereof. Linkers that utilize any suitable functional group include any combination of aryl, carboxylate, carboxyl, ester, hydroperoxy, peroxy, ether, hemiacetal, hemiketal, acetal, ketal, orthoester, amide, amine, imine, imide, azide, azo, cyanate, nitrate, nitrite, nitrile, nitro, nitroso, pyridine, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinic acid, sulfonic acid, thiocyanate, thione, thial, phosphine, phosphonic acid, phosphate, and / or phosphodiester groups. Any suitable linker utilizing any suitable functional group is within the scope of embodiments of the present invention.In some embodiments, the linker comprises a cleavable moiety and one or more of the functional groups / moieties mentioned above.

[0103] II. Reagents A key aspect of the platform described herein is the preparation of bioactive agents that can covalently interact with other reagent(s) to form probes. In some embodiments, the bioactive agent comprises a reactive group suitable for reaction with other reagent(s). However, in other embodiments, reactive derivatives of bioactive agents are made using the methods and reagents described herein. The reactive derivatives present a first reactive group (e.g., R") that can react with a second reactive group (R') presented on the other reagent(s) (e.g., linked PRG and capture moiety, linked PRG and fluorophore, etc.) to form a covalent bond (e.g., R). A variety of pairings of reactive groups have been contemplated and tested for attachment to bioactive agents and formation of multimodal probes within the scope described herein. For example, FIG. 5A shows the addition of reactive amine groups to several known drugs to form reactive derivatives of the parent bioactive agent. FIG. 5B shows the reactive derivatives reacting with a PRG / fluorophore reagent presenting a complementary reactive group (e.g., COOH) to form a PRG / fluorophore probe with a bioactive agent.

[0104] Suitable reactive groups for attachment to a parent bioactive agent to yield a derivatizable bioactive agent include NH, NH, -C(O)OH, OC(O)O-phenyl, C(O)O-phenyl, OH, C=O, aldehyde, difluorosulfinate, sulfonyl fluoride, sulfonyl chloride, and halogen. Examples of generic compounds that have been modified to display a variety of possible reactive groups are shown in Figure 6A. For example, an NH2 reactive group on a bioactive agent reacts with a PRG / CA (or PRG / fluorophore reagent) that presents a carboxylate group to produce an amide coupling; a COOH reactive group on a bioactive agent reacts with a PRG / CA (or PRG / fluorophore reagent) that presents an amine to produce an amide coupling; an OC(O)O-phenyl reactive group on a bioactive agent reacts with a PRG / CA (or PRG / fluorophore reagent) that presents an amine to produce a carbamate coupling; an OH reactive group on a bioactive agent reacts via alkylation with a PRG / CA (or PRG / fluorophore reagent) that presents an alkylating agent; an aldehyde reactive group on a bioactive agent reacts via reductive amination with a PRG / CA (or PRG / fluorophore reagent) that presents an amine; a halogen reactive group on a bioactive agent reacts via alkylation with a PRG / CA (or PRG / fluorophore reagent) that presents an amine; and so forth. An example of attaching a typical drug-like bioactive agent to a PRG / HA reagent via a complementary reactivity pair is shown in Figure 7. Another example of attaching a bioactive agent to a PRG / HA or PRG / fluorophore reagent via a complementary reactivity pair is shown in Figure 8. In some embodiments, any of the reactive groups shown and / or described herein as being disposed on a bioactive agent may also find use as a reactive group on a PRG / HA or PRG / fluorophore reagent. Similarly, any of the reactive groups shown and / or described herein as being disposed on a PRG / HA or PRG / fluorophore reagent may also find use as a reactive group on a bioactive agent.

[0105] Any bioactive agent that exhibits one of the reactive groups described herein, that can be modified to exhibit one of the reactive groups described herein, that exhibits another suitable reactive group, and / or that can be modified to exhibit a suitable reactive group will find use in the embodiments herein. Examples of PRG / chloroalkane reagents suitable for reaction with complementary derivatizable bioactive agents are shown in Figure 6B. Each of the possible reactive groups on the PRG / CA reagent can react with a commentary reactive group on a bioactive agent (see, e.g., Figure 7).

[0106] A key aspect of the platforms described herein are reagents that contain PRG linked to either a capture ligand (e.g., HA, CA, etc.) or a fluorophore, and that display a reactive moiety that can covalently interact with a complementary reactive moiety on a bioactive agent to form a probe. In some embodiments, a PRG / HA or PRG / fluorophore reagent displaying a suitable reactive moiety reacts with a bioactive agent displaying a complementary reactive moiety to generate a PRG / HA or PRG / fluorophore probe. In some embodiments, the reactive derivatives of PRG / HA or PRG / fluorophore reagents present a first reactive group (e.g., R') that can react with a second reactive group (R") presented on a bioactive agent to form a covalent bond (e.g., R). A variety of reactive group pairings have been contemplated and tested for attachment to bioactive agents and formation of multimodal probes within the scope described herein. Examples of suitable reactive moieties for PRG / HA or PRG / fluorophore reagents are selected from NH, NH, -C(O)OH, OC(O)O-phenyl, C(O)O-phenyl, OH, C=O, aldehyde, difluorosulfinate, sulfonyl fluoride, sulfonyl chloride, and halogen. In other embodiments, suitable reactive moieties for PRG / HA or PRG / fluorophore reagents are -(CH) 0-2 NH2, -(CH2) 0-2 C(O)OH, (CH2) 0-2 OC(O)O-phenyl, (CH2) 0-2 C(O)O-phenyl, (CH2) 0-2 OH, (CH2)0-2 C=O, (CH2) 0-2 Cl, or (CH2) 0-2 Br. The complementary reactive moieties are selected to allow the PRG / HA or PRG / fluorophore reagent and the bioactive agent to react to form a probe.

[0107] In some embodiments, the bioactive agent is modified by the user to display a reactive moiety. In some embodiments, a PRG / HA or PRG / fluorophore reagent displaying a complementary reactive moiety is synthesized by the user or obtained commercially. In some embodiments, the user reacts the reactively modified bioactive agent with the PRG / HA or PRG / fluorophore reagent to obtain a PRG / HA or PRG / fluorophore probe. The assembled probe is then ready for use in the various assays and applications described herein.

[0108] A.PRG / HA Reagent In some embodiments, provided herein are reagents comprising a photoreactive group (PRG) and a capture ligand (e.g., a haloalkane) for reacting with a bioactive agent to assemble the PRG / HA probes described herein. In some embodiments, the PRG / HA reagent comprises a linker moiety.

[0109] In some embodiments, provided herein are compositions comprising reactive PRG / HA reagents that include a photoreactive group (PRG), a linker, and a capture ligand (e.g., a haloalkane) covalently linked to a single compound of the following general structure: [ka] where R' is a first reactive moiety capable of forming a covalent bond when reacted with a second reactive moiety (R"), -AX is a substrate for a dehalogenase, and Q is CH or N.

[0110] In some embodiments, R' comprises -NH, NH, -C(O)OH, OC(O)O-phenyl, C(O)O-phenyl, OH, C=O, aldehyde, difluorosulfinate, sulfonyl fluoride, sulfonyl chloride, or halogen. In some embodiments, R' comprises -(CH) 0-2 NH2, -(CH2) 0-2 C(O)OH, (CH2) 0-2 OC(O)O-phenyl, (CH2) 0-2 C(O)O-phenyl, (CH2) 0-2 OH, (CH2) 0-2 C=O, (CH2) 0-2 Cl, or (CH2) 0-2 Contains Br.

[0111] In some embodiments, the PRG / HA reagent has the following general structure: [ka] wherein R' comprises -NH2, -C(O)OH, OC(O)O-phenyl, C(O)O-phenyl, OH, aldehyde, difluorosulfinate, sulfonyl fluoride, sulfonyl chloride, or halogen. In other embodiments, the PRG / HA reagent has the following general structure: [ka] wherein R' comprises NH or C=O.

[0112] In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is chemically cleavable, enzymatically cleavable, or photocleavable. In some embodiments, the cleavable linker comprises a cleavable moiety selected from the group consisting of disulfide, tert-butyl carbamate, silyl ether, diazobenzene, 1,2-diol, and -C(O)OCH2CH=CHCH2OC(O)-. In some embodiments, the cleavable linker comprises a cleavable moiety flanked on one or both sides by an alkylene or heteroalkylene chain. In some embodiments, the alkylene or heteroalkylene chain is selected from the group consisting of C 1-6 -alkylene, -O-, -(CH2)2O-, and -OC(O)NH- groups.

[0113] In some embodiments, the linkers herein (e.g., linkers of the probes herein (e.g., connecting capture ligands to Q / R′ groups) comprise any suitable moiety that provides a desired structure / function (e.g., length, flexibility / rigidity, solubility, cell permeability, etc.). The probes herein are not limited to any particular linker moiety. Indeed, a variety of linker moieties are contemplated, and suitable linkers include, but are not limited to, alkyl groups, methylene carbon chains, ethers, polyethers, alkylamide linkers, peptide linkers, modified peptide linkers, poly(ethylene glycol) (PEG) linkers, streptavidin-biotin or avidin-biotin linkers, polyamino acids (e.g., polylysine), functionalized PEGs, polysaccharides, glycosaminoglycans, dendritic polymers (see WO 93 / 06868, and Tomalia et al. in Angew. Chem. Int. Ed. Engl. 29:138-175 (1990)). al., incorporated herein by reference in their entireties), PEG-chelator polymers (WO 94 / 08629, WO 94 / 09056, and WO 96 / 26754, incorporated herein by reference in their entireties), oligonucleotide linkers, phospholipid derivatives, alkenyl chains, alkynyl chains, disulfides, or combinations thereof. In some embodiments, the linker is an alkyl, alkenyl, alkynyl, amide, phenyl, benzyl, carbamate, halo, fluoro, chloro, bromo, iodo, hydroxyl, carbonyl, aldehyde, haloformyl, carbonate ester, or a combination thereof. Linkers that utilize any suitable functional group include any combination of aryl, carboxylate, carboxyl, ester, hydroperoxy, peroxy, ether, hemiacetal, hemiketal, acetal, ketal, orthoester, amide, amine, imine, imide, azide, azo, cyanate, nitrate, nitrite, nitrile, nitro, nitroso, pyridine, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinic acid, sulfonic acid, thiocyanate, thione, thial, phosphine, phosphonic acid, phosphate, and / or phosphodiester groups. Any suitable linker utilizing any suitable functional group is within the scope of embodiments of the present invention.In some embodiments, the linker comprises a cleavable moiety and one or more of the functional groups / moieties mentioned above.

[0114] In some embodiments, the PRG is selected from PRGs described herein and / or understood in the art (e.g., aryl azides, alkyl aryl azides (e.g., methyl aryl azides), substituted aryl azides, or substituted alkyl aryl azides (e.g., substituted methyl aryl azides). Exemplary PRGs are shown in Table 1 (above). In some embodiments, other photoreactive groups find use as the PRG portion of the probes herein (e.g., those described above and elsewhere herein). For example, suitable photoreactive groups include phenyl azides, phenyl diazirines, and benzophenones, where the reactive intermediates formed when irradiated with light of a particular wavelength are nitrenes, carbenes, and diradicals, respectively. In some embodiments, the PRG or photoreactive group is stable (e.g., non-reactive) in visible light. In some embodiments, the activation energy of the PRG or photoreactive group is greater than the absorption wavelength of the protein. Proteins absorb UV light at 280 and 200 nm, where the absorbance at 280 nm is due to the aromatic amino acids tryptophan, tyrosine, and phenylalanine, and peptide bonds are responsible for the absorbance at 200 nm. Excessive absorption of UV light by proteins leads to the formation of electronically excited states of the molecule, which can then undergo degradative chemical transformations. In some embodiments, PRG or photoaffinity groups include diazo groups, diazocarbonyls, enones, sulfur radicals, halogenated substrates, nitrobenzenes, diazonium salts, alkyl derivatives of azides and diazirines, and the like. Fleming provides a comprehensive review of the various chemical reagents used in photoaffinity labeling (see Fleming SA. Chemical reagents in photoaffinity labelling. Tetrahedron. 1995;51:12479-12520, incorporated herein by reference in its entirety).Sakurai et al. recently reported a study comparing the main photoreactive groups and assessing their reactivity (see Sakurai et al. Comparison of the reactivity of carbohydrate photoaffinity probes with different photoreactive groups. Chem. Bio. Chem. 2014;15:1399-1403, which is incorporated herein by reference in its entirety).

[0115] In some embodiments, PRG is covalently linked to the remainder of the compound by a PRG linker moiety. In some embodiments, the PRG linker moiety is (CH2) 0-4 It is.

[0116] In some embodiments, the capture ligand is a haloalkane (e.g., a chloroalkane). In some embodiments, the capture ligand is -AX, where A is (CH2). n , n=4-10, and X is a halogen. In some embodiments, X is Cl. In some embodiments, -AX is -(CH2)6Cl.

[0117] In some embodiments, provided herein are systems, kits, and reaction mixtures that include (i) a PRG / HA reagent described herein, and (ii) a bioactive agent that displays a second reactive moiety, R". In some embodiments, the bioactive agent is a small molecule or peptide. In some embodiments, the bioactive agent includes a drug or a naturally occurring compound that has been modified to display R". In some embodiments, R" includes -NH2, -OC(O)O-nitrophenyl, -OH, -C=O, -aldehyde, -difluorosulfinate, -sulfonyl fluoride, sulfonyl chloride, or -halogen. In some embodiments, R" includes -(CH2) 0-2 NH2, -(CH2) 0-2 OC(O)O-Nitrophenyl, -(CH2) 0-2 OH, -(CH2) 0-2 C=O, -(CH2)0-2 Cl, or -(CH2) 0-2 Contains Br.

[0118] In some embodiments, provided herein is a method of making a PRG / HA probe, comprising contacting (i) a PRG / HA reagent described herein with (ii) a second reactive moiety, R″, of a bioactive agent. In some embodiments, provided herein is a PRG / HA probe produced by a method described herein.

[0119] B.PRG / phosphor reagent In some embodiments, provided herein are reagents comprising a photoreactive group (PRG) and a fluorophore for reacting with a bioactive agent to assemble the PRG / fluorophore probes described herein. In some embodiments, the PRG / fluorophore reagent comprises a linker moiety.

[0120] In some embodiments, provided herein are reactive photoreactive group (PRG) / fluorophore reagents that include a PRG and a fluorophore connected to a single compound of the following general structure: [ka] (wherein R' is a first reactive moiety capable of forming a covalent bond when reacted with a second reactive moiety (R"), and Q is CH or N.

[0121] In some embodiments, R' comprises -NH, NH, -C(O)OH, OC(O)O-phenyl, C(O)O-phenyl, OH, C=O, aldehyde, difluorosulfinate, sulfonyl fluoride, sulfonyl chloride, or halogen. In some embodiments, R' comprises -(CH) 0-2 NH2, -(CH2) 0-2 C(O)OH, (CH2) 0-2 OC(O)O-phenyl, (CH2) 0-2 C(O)O-phenyl, (CH2) 0-2 OH, (CH2) 0-2 C=O, (CH2) 0-2Cl, or (CH2) 0-2 Contains Br.

[0122] In some embodiments, the PRG / fluorophore reagent has the following general structure: [ka] wherein R' comprises -NH2, -C(O)OH, OC(O)O-phenyl, C(O)O-phenyl, OH, aldehyde, difluorosulfinate, sulfonyl fluoride, sulfonyl chloride, or halogen. In other embodiments, the PRG / fluor reagent has the following general structure: [ka] In the formula, R' includes -NH- or CO.

[0123] In some embodiments, the PRG is selected from PRGs described herein and / or understood in the art (e.g., aryl azides, alkyl aryl azides (e.g., methyl aryl azides), substituted aryl azides, or substituted alkyl aryl azides (e.g., substituted methyl aryl azides). Exemplary PRGs are shown in Table 1 (above). In some embodiments, other photoreactive groups find use as the PRG portion of the probes herein (e.g., those described above and elsewhere herein). For example, suitable PRGs include phenyl azides, phenyl diazirines, and benzophenones, where the reactive intermediates formed when irradiated with light of a particular wavelength are nitrenes, carbenes, and diradicals, respectively. In some embodiments, the PRG or photoreactive group is stable (e.g., non-reactive) in visible light. In some embodiments, the activation energy of the PRG or photoreactive group is greater than the absorption wavelength of the protein. Proteins absorb UV light at 280 and 200 nm, where the absorbance at 280 nm is due to the aromatic amino acids tryptophan, tyrosine, and phenylalanine, and peptide bonds are responsible for the absorbance at 200 nm. Excessive absorption of UV light by proteins leads to the formation of electronically excited states of the molecule, which can then undergo degradative chemical transformations. In some embodiments, PRG or photoaffinity groups include diazo groups, diazocarbonyls, enones, sulfur radicals, halogenated substrates, nitrobenzenes, diazonium salts, alkyl derivatives of azides and diazirines, and the like. Fleming provides a comprehensive review of the various chemical reagents used in photoaffinity labeling (see Fleming SA. Chemical reagents in photoaffinity labelling. Tetrahedron. 1995;51:12479-12520, incorporated herein by reference in its entirety).Sakurai et al. recently reported a study comparing the main photoreactive groups and assessing their reactivity (see Sakurai et al. Comparison of the reactivity of carbohydrate photoaffinity probes with different photoreactive groups. Chem. Bio. Chem. 2014;15:1399-1403, which is incorporated herein by reference in its entirety).

[0124] In some embodiments, PRG is covalently linked to the remainder of the compound by a PRG linker moiety. In some embodiments, the PRG linker moiety is (CH2) 0-4 It is.

[0125] In some embodiments, the fluorophore is selected from any suitable fluorophore understood in the art (eg, a small molecule fluorescent dye). Suitable fluorophores include xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, oxazine 170, etc.), acridine derivatives (e.g., proflavine, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porphine, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA Fluor (Invitrogen), DYLIGHT Fluor (Thermo Scientific, Pierce), ATTO and TRACY dyes (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES dyes (Dyomics), SULFO CY dyes (CYANDYE, LLC), SETAU and SQUARE dyes (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT dyes (APC, RPE, PerCP, phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, and BPE dyes (Phyco-Biotech), and the like. In some embodiments, the fluorophores herein are rhodamine analogs (e.g., carboxyrhodamine analogs), such as those described in U.S. Patent Application Serial No. 13 / 682,589, which is incorporated herein by reference in its entirety.In some embodiments, a suitable fluorophore is selected based on its physical properties, such as size, emission spectrum, excitation spectrum, solubility, cell permeability, and the like.

[0126] In some embodiments, the fluorophore is covalently linked to the remainder of the compound by a fluorophore linker moiety. In some embodiments, the fluorophore linker moiety is (CH2) 0-4 In some embodiments, the fluorophore linker moiety comprises a cleavable linker. In some embodiments, the cleavable linker is chemically cleavable, enzymatically cleavable, or photocleavable. In some embodiments, the cleavable linker comprises a cleavable moiety selected from the group consisting of disulfide, tert-butyl carbamate, silyl ether, diazobenzene, 1,2-diol, and -C(O)OCH2CH=CHCH2OC(O)-. In some embodiments, the cleavable linker comprises a cleavable moiety flanked on one or both sides by an alkylene or heteroalkylene chain. In some embodiments, the alkylene or heteroalkylene chain is selected from the group consisting of C 1-6 -alkylene, -O-, -(CH2)2O-, and -OC(O)NH- groups.

[0127] In some embodiments, the linkers herein (e.g., linkers of the probes herein (e.g., connecting capture ligands to Q / R′ groups) comprise any suitable moiety that provides a desired structure / function (e.g., length, flexibility / rigidity, solubility, cell permeability, etc.). The probes herein are not limited to any particular linker moiety. Indeed, a variety of linker moieties are contemplated, and suitable linkers include, but are not limited to, alkyl groups, methylene carbon chains, ethers, polyethers, alkylamide linkers, peptide linkers, modified peptide linkers, poly(ethylene glycol) (PEG) linkers, streptavidin-biotin or avidin-biotin linkers, polyamino acids (e.g., polylysine), functionalized PEGs, polysaccharides, glycosaminoglycans, dendritic polymers (see WO 93 / 06868, and Tomalia et al. in Angew. Chem. Int. Ed. Engl. 29:138-175 (1990)). al., incorporated herein by reference in their entireties), PEG-chelator polymers (WO 94 / 08629, WO 94 / 09056, and WO 96 / 26754, incorporated herein by reference in their entireties), oligonucleotide linkers, phospholipid derivatives, alkenyl chains, alkynyl chains, disulfides, or combinations thereof. In some embodiments, the linker is an alkyl, alkenyl, alkynyl, amide, phenyl, benzyl, carbamate, halo, fluoro, chloro, bromo, iodo, hydroxyl, carbonyl, aldehyde, haloformyl, carbonate ester, or a combination thereof. Linkers that utilize any suitable functional group include any combination of aryl, carboxylate, carboxyl, ester, hydroperoxy, peroxy, ether, hemiacetal, hemiketal, acetal, ketal, orthoester, amide, amine, imine, imide, azide, azo, cyanate, nitrate, nitrite, nitrile, nitro, nitroso, pyridine, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinic acid, sulfonic acid, thiocyanate, thione, thial, phosphine, phosphonic acid, phosphate, and / or phosphodiester groups. Any suitable linker utilizing any suitable functional group is within the scope of embodiments of the present invention.In some embodiments, the linker comprises a cleavable moiety and one or more of the functional groups / moieties mentioned above.

[0128] In some embodiments, provided herein are systems, kits, and reaction mixtures that include (i) a reactive PRG / fluorophore reagent described herein, and (ii) a bioactive agent that displays a second reactive moiety, R". In some embodiments, the bioactive agent is a small molecule or peptide. In some embodiments, the bioactive agent includes a drug or a naturally occurring compound that has been modified to display R". In some embodiments, R" includes -NH2, -OC(O)O-nitrophenyl, -OH, -C=O, -aldehyde, -difluorosulfinate, -sulfonyl fluoride, sulfonyl chloride, or a halogen. In some embodiments, R" includes -(CH2) 0-2 NH2, -(CH2) 0-2 OC(O)O-Nitrophenyl, -(CH2) 0-2 OH, -(CH2) 0-2 C=O, -(CH2) 0-2 Cl, or -(CH2) 0-2 Contains Br.

[0129] In some embodiments, provided herein is a method of making a PRG / fluorophore probe comprising contacting (i) a reactive PRG / fluorophore reagent described herein with (ii) a bioactive agent that displays a second reactive moiety, R″. In some embodiments, provided herein is a PRG / fluorophore probe produced by the methods described herein.

[0130] III. Acquisition method In some embodiments, the PRG / HA probes described herein find use in capturing target molecules bound to the bioactive agent of the probe. PRG / HA probes are particularly useful for capturing intracellular targets because upon irradiation, the probe becomes covalently bound to the target, such that the target associated with the probe (e.g., captured) is no longer affected by cell lysis or other treatments. In some embodiments, after capture (e.g., immobilization, enrichment, isolation, purification, etc.), it can be released through cleavage of a cleavable linker on the probe, allowing the target to be manipulated, analyzed, or used for downstream applications (e.g., assays).

[0131] In some embodiments, provided herein is a method of capturing a target molecule, comprising contacting a sample containing a target molecule (e.g., a target protein, an intracellular target, etc.) with a PRG / capture ligand probe (e.g., a PRG / HA probe) described herein, where a bioactive agent can associate with the target molecule; allowing the bioactive agent to associate with the target molecule; and irradiating the sample with light of a wavelength that converts PRG to a reactive form of PRG, where the reactive form of PRG forms a covalent bond with the target molecule. In some embodiments, the sample contains cells, and the target molecule is on the surface of the cells or within the cells.

[0132] In some embodiments, the method further comprises lysing cells of the sample to form a lysate comprising the target molecules covalently attached to the probes.

[0133] In some embodiments, the method further includes contacting the sample with a capture protein (e.g., a modified dehalogenase enzyme) that binds to the capture ligand (e.g., forms a covalent bond with the haloalkane substrate when associated therewith); and covalently binding the capture protein (e.g., the modified dehalogenase enzyme) to the capture ligand (e.g., a haloalkane) of the probe.

[0134] In some embodiments, the capture protein (e.g., modified dehalogenase enzyme) is immobilized on a solid surface and the target molecule is immobilized on the solid surface when the capture protein (e.g., modified dehalogenase enzyme) binds to the capture ligand (e.g., haloalkane). In some embodiments, the method further comprises (i) removing the solid surface from the sample; and / or (ii) washing non-immobilized components of the sample from the solid surface. In some embodiments, the solid surface is a bead, particle, chip, tube, plate, or membrane. In some embodiments, the solid surface is a paramagnetic particle. Suitable solid surfaces include, but are not limited to, glass and modified or functionalized glass, plastics (e.g., acrylic resins, polystyrene and copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon, etc.), polyvinylpyrrolidine (PVP), copolymers of vinyl and acrylamide, polystyrene, polystyrene cross-linked with divinylbenzene, etc. (see Merrifield Biochemistry 1964,3,1385-1390, which is incorporated herein by reference in its entirety), polysaccharides, cellulose or derivatives thereof, nylon or nitrocellulose, resins, silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glass, plastics, fiber optic bundles, and various other polymers. In some embodiments, the paramagnetic beads comprise a paramagnetic resin, such as those described in Example 9 of WO2014093671, which is incorporated herein by reference in its entirety.

[0135] In some embodiments, the method further comprises cleaving the linker of the probe to release the target molecule bound to the PRG and bioactive agent (e.g., from the capture protein, solid surface, etc.). In some embodiments, the method further comprises detecting and / or quantifying the target molecule. In some embodiments, the target molecule is detected and / or quantified by mass spectrometry.

[0136] In some embodiments, the sample comprises a cell, and the target molecule is located within or on the surface of the cell. In some embodiments, contacting the sample comprising the target molecule with the probe further comprises placing the probe into the cell. In some embodiments, the method further comprises lysing the cell after the PRG forms a covalent bond with the target molecule. In some embodiments, the bioactive agent can non-covalently bind to the target molecule when the bioactive agent is associated with the target molecule. In some embodiments, the target protein is a cell surface protein, a transmembrane protein, a cytoplasmic protein, a nuclear protein, or a mitochondrial protein.

[0137] In some embodiments, provided herein are methods of capturing intracellular target proteins, the methods including contacting a cell containing the target protein with a probe (e.g., a PRG / capture ligand probe (e.g., a PRG / HA probe) described herein), where a bioactive agent can bind to the target protein; allowing the probe to enter the cell; allowing the bioactive agent to bind to the target protein; irradiating the cell with UV light at a wavelength that converts PRG to a reactive form of PRG, where the reactive form of PRG forms a covalent bond with the target protein; lysing the cells to form a lysate; contacting the lysate with a capture protein (e.g., a modified dehalogenase enzyme) immobilized on a solid surface that forms a covalent bond when associated with a capture ligand (e.g., a haloalkane substrate); covalently binding the capture protein (e.g., a modified dehalogenase enzyme) to the capture ligand (e.g., a haloalkane) of the probe; and removing the solid surface from the lysate and / or washing non-immobilized components of the lysate from the solid surface. In some embodiments, the method further comprises cleaving the linker of the probe to release the target protein bound to the PRG and bioactive agent from the solid surface. In some embodiments, the method further comprises detecting and / or quantifying the target molecule. In some embodiments, the target protein is a cell surface protein, a transmembrane protein, a cytoplasmic protein, a nuclear protein, or a mitochondrial protein.

[0138] In some embodiments, provided herein is a method for measuring photocrosslinking efficiency of PRG linked to a bioactive agent, comprising contacting a sample containing a target molecule with a PRG / fluorophore probe described herein, where the target molecule is tethered to a bioluminescent reporter, and the emission spectrum of the bioluminescent reporter overlaps with the excitation spectrum of the fluorophore; binding the bioactive agent to the target molecule; irradiating the sample with light (e.g., UV light) of a wavelength that converts PRG to a reactive form of PRG, where the reactive form of PRG can form a covalent bond with the target molecule; contacting the sample with a substrate for the bioluminescent reporter and detecting emission within the emission spectrum of the bioluminescent reporter and the fluorophore, where emission from the fluorophore is detected. The method includes detecting, where the light is the result of bioluminescence resonance energy transfer (BRET) from the bioluminescence reporter to the fluorophore, and the amount of emission from the fluorophore correlates to the amount of PRG / fluorophore probe bound to the target molecule; further contacting the sample with an untagged bioactive agent to competitively displace the PRG / fluorophore probe that is not covalently attached to the target; contacting the sample with a substrate for the bioluminescence reporter to detect emission within the emission spectrum of the bioluminescence reporter and the fluorophore, where the emission from the fluorophore is the result of bioluminescence resonance energy transfer (BRET) from the bioluminescence reporter to the fluorophore, and the amount of emission from the fluorophore correlates to the amount of PRG / fluorophore probe covalently attached to the target molecule. In some embodiments, the method further includes calculating the percent crosslinking by normalizing the amount of PRG / fluorophore probe covalently attached to the target molecule to the amount of PRG / fluorophore probe bound to the target molecule before adding the untagged bioactive agent. In some embodiments, the method further includes detecting emission within the emission spectrum of the bioluminescence reporter. In some embodiments, the bioluminescent reporter is a bioluminescent protein or a peptide or polypeptide that is a component of a bioluminescent complex. In some embodiments, the concentration of the untagged bioactive agent in the sample is greater than the concentration of the PRG / fluorophore probe in the sample.

[0139] In some embodiments, provided herein are methods for measuring photocrosslinking efficiency of PRG linked to a bioactive agent, the methods including contacting a sample containing a target molecule with a PRG / HA probe described herein, where the target molecule is tethered to a bioluminescent reporter; binding the bioactive agent to the target molecule; irradiating the sample with light of a wavelength that converts PRG to a reactive form of PRG, where the reactive form of PRG can form a covalent bond with the target molecule; contacting the sample with an excess of a BRET reagent containing a bioactive agent covalently tethered to a fluorophore, where the emission spectrum of the bioluminescent reporter overlaps with the excitation spectrum of the fluorophore; contacting the sample with a substrate for the bioluminescent reporter and detecting emission within the emission spectrum of the bioluminescent reporter and the fluorophore, where the emission from the fluorophore is a result of bioluminescence resonance energy transfer (BRET) from the bioluminescent reporter to the fluorophore, and the amount of emission from the fluorophore correlates to the amount of BRET reagent bound to the target molecule. In some embodiments, the method further comprises: not contacting a sample comprising a target molecule with a PRG / HA probe described herein, where the target molecule is tethered to a bioluminescent reporter; irradiating the sample and then contacting the sample with an excess of a BRET reagent comprising a bioactive agent covalently tethered to a fluorophore, where the emission spectrum of the bioluminescent reporter overlaps with the excitation spectrum of the fluorophore; contacting the sample with a substrate for the bioluminescent reporter and detecting emission within the emission spectrum of the bioluminescent reporter and the fluorophore, where the emission from the fluorophore is a result of bioluminescence resonance energy transfer (BRET) from the bioluminescent reporter to the fluorophore.In some embodiments, the method further comprises comparing (i) an emission spectrum collected from a sample treated with a PRG / HA probe and illuminated with light of a wavelength that converts PRG to its reactive form, with (ii) an emission spectrum collected from a sample not treated with a PRG / HA probe and illuminated with light of the same wavelength that converts PRG to its reactive form, whereby reduction in BRET by pretreatment with a PRG / HA probe inversely correlates to the photocrosslinking efficiency of PRG (photocrosslinking of the PRG / HA probe to the target prevents displacement of the probe by excess BRET reagent, thereby reducing the BRET signal).

[0140] IV. PRG / Bioactive Agent Conjugates In some embodiments, provided herein are PRGs and / or PRG / bioactive agent conjugates for conjugation to a bioactive agent that provide distinct and / or favorable properties (e.g., covalent binding efficiency, reactivity, solubility, size, cell permeability, etc.) compared to available PRGs. In some embodiments, provided herein are compositions comprising a photoreactive group (PRG) linked to a bioactive agent, where the PRG is selected from: [ka]

[0141] In some embodiments, provided herein is a method of covalently attaching a bioactive agent to a target protein, the method comprising: contacting the target molecule with a bioactive agent linked to one of the photoaffinity labels (PRGs) described above, where the bioactive agent is capable of associating with the target molecule; allowing the bioactive agent to associate with the target molecule; and irradiating with light of a wavelength that converts the PRG into a reactive form of PRG, where the reactive form of PRG forms a covalent bond with the target molecule.

[0142] V. Bioluminescence Reporters The present disclosure includes bioluminescent polypeptides, bioluminescent complexes and their components, as well as materials and methods related to bioluminescence resonance energy transfer (BRET). For example, embodiments herein include BRET displacement assays, in which BRET is measured between PRG / fluorophore probes and fusions of bioactive agent targets and bioluminescent reporters (e.g., bioluminescent proteins, bioluminescent complexes, etc.). Some embodiments herein include a BRET-based approach to systematically estimate the photocrosslinking efficiency of multiple PRGs. Experiments conducted during the development of embodiments herein have demonstrated that the estimation of photocrosslinking efficiency by this approach correlates well with the enrichment efficiency of target proteins from living cells in several model systems. These methods have demonstrated that aryl azides generally provide higher photocrosslinking efficiency than other commonly used PRGs. Furthermore, certain substituted aryl azides have consistently provided higher crosslinking efficiency than the commonly used diazirines and benzophenones.

[0143] Any embodiment utilizing conjugates, assays, etc. with the bioluminescent proteins or probes described herein is within the scope of the present invention. The following paragraphs describe bioluminescent proteins (e.g., NANOLUC, etc.), bioluminescent conjugates (e.g., NANOBIT, NANOTRIP, etc.), and resonance energy transfer thereto (e.g., NANOBRET) that may find use in embodiments herein.

[0144] In some embodiments, provided herein are assays, methods, compositions, reagents, systems, etc., incorporating bioluminescent polypeptides and / or bioluminescent complexes (of peptide(s) and / or polypeptide components) based (e.g., structurally, functionally, etc.) on the luciferase of Oplophorus gracilirostris, NanoLuc® luciferase (Promega Corporation; U.S. Pat. No. 8,557,970; U.S. Pat. No. 8,669,103; incorporated herein by reference in their entireties), the NanoBiT (U.S. Pat. No. 9,797,889; incorporated herein by reference in their entireties) or the NanoTrip (U.S. Provisional Patent Application No. 62 / 684,014) complementation system, and / or the NanoBRET (U.S. Pat. No. 10,067,149; U.S. Pat. No. 10,024,862; incorporated herein by reference in their entireties) system. As described below, in some embodiments, the compositions, assays, reagents, methods, and systems herein incorporate commercially available NANOLUC-based technologies (e.g., NANOLUC luciferase, NANOBRET, NANOBIT, NANOTRIP, NANOGLO, etc.), while in other embodiments, various combinations, variations, or derivatives of commercially available NANOLUC-based technologies are used.

[0145] International Application No. PCT / US2010 / 033449, U.S. Patent No. 8,557,970, PCT International Application No. PCT / 2011 / 059018, and U.S. Patent No. 8,669,103 (each of which is incorporated by reference herein in their entirety and for all purposes) describe compositions and methods that include bioluminescent polypeptides. Such polypeptides find use in embodiments herein and can be used in conjunction with the compositions, assays, reagents, systems, and methods described herein.

[0146] In some embodiments, the compositions, assays, devices, systems, and methods provided herein comprise SEQ ID NO:5 or a bioluminescent polypeptide having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any range therebetween) sequence identity to SEQ ID NO:5. In some embodiments, provided herein is a fusion of (i) a bioluminescent polypeptide having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any range therebetween) sequence identity to SEQ ID NO:5 and (ii) a target protein (e.g., an intracellular target).

[0147] In some embodiments, the bioluminescent polypeptide is linked (e.g., fused, chemically linked, etc.) to a molecule of interest, a protein of interest, or another component of the assays and systems described herein. In some embodiments, the bioluminescent polypeptide described herein, or any of its fusions or conjugates, is immobilized to a solid surface or device.

[0148] PCT Application No. PCT / US14 / 26354 and U.S. Patent No. 9,797,889 (each of which is incorporated herein by reference in their entirety and for all purposes) describe compositions and methods for assembling bioluminescent complexes, and such complexes, as well as peptide and polypeptide components thereof, find use in embodiments herein and can be used in conjunction with the assays and methods described herein. Some embodiments herein use NANOBIT-based technology.

[0149] In some embodiments, provided herein are polypeptide components of bioluminescent complexes, which have at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity to SEQ ID NO:9, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, provided herein are peptide components of bioluminescent complexes, wherein the peptide has at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity to SEQ ID NO:10, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:8. In some embodiments, provided herein are peptide components of bioluminescent complexes, which have at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity to SEQ ID NO:11, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:8. In some embodiments, the peptide or polypeptide components of the bioluminescent complexes are linked (e.g., fused, chemically linked, etc.) to target molecules, intracellular targets, or other components of the compositions, assays, reagents, methods, and systems described herein. In some embodiments, a peptide or polypeptide component of a bioluminescent complex, or a fusion or conjugate thereof, is immobilized on a solid surface or portion of a device.

[0150] U.S. Provisional Patent Application No. 62 / 684,014; U.S. Patent Application No. 16 / 439,565; and U.S. Provisional Patent Application No. 62 / 941,255 (incorporated herein by reference in their entirety for all purposes) describe compositions, systems, and methods for assembling bioluminescent complexes, e.g., from three or more peptide / polypeptide components. Such complexes, as well as their peptide and polypeptide components (e.g., NanoTrip-based peptides, polypeptides, and complexes), find use in embodiments herein and can be used in conjunction with the assays and methods described herein.

[0151] In some embodiments, provided herein are polypeptide components of bioluminescent complexes, which have at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity to SEQ ID NO:12, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:6, and SEQ ID NO:9. In some embodiments, provided herein are peptide components of bioluminescent complexes, wherein the peptide has at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity to SEQ ID NO:11, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:8. In some embodiments, provided herein are peptide components of bioluminescent complexes, wherein the peptide has at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity to SEQ ID NO:13, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7. In some embodiments, provided herein are peptide components of bioluminescent complexes, wherein the peptide has at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity to SEQ ID NO:14, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:8.

[0152] In some embodiments, one or more of the NanoTrip-based peptides or polypeptides are linked (e.g., fused, chemically linked, etc.) to a target molecule, intracellular target, or other component of the compositions, methods, reagents, assays, and systems described herein. In some embodiments, one or more of the above-mentioned NanoTrip-based peptides or polypeptides are immobilized to a solid surface or device.

[0153] PCT Application No. PCT / US13 / 74765, U.S. Patent No. 10,067,149, and U.S. Patent No. 10,024,862 (incorporated herein by reference in their entirety for all purposes) describe bioluminescence resonance energy transfer (BRET) compositions, systems, and methods (e.g., incorporating NANOLUC-based technology), and such compositions, systems, and methods, and their bioluminescent polypeptide and fluorophore-conjugated components, find use in embodiments herein and can be used in conjunction with the compositions, systems, reagents, assays, and methods described herein. In some embodiments, these NANOBRET technologies find use with the compositions, systems, and methods described herein in detecting and analyzing intracellular binding of bioactive agents to intracellular targets.

[0154] U.S. Patent No. 10,168,323 and U.S. Patent No. 9,551,705 (incorporated herein by reference in their entirety for all purposes) describe compositions and methods for capturing and identifying intracellular targets of bioactive agents, and in particular, these patents describe bioactive agents tethered to capture ligands, intracellular targets (optionally tagged with reporters), capture proteins (optionally present as capture dimers), surfaces (e.g., displaying capture ligands, capture proteins, or capture dimers), and methods of using same to capture and identify intracellular targets of bioactive agents. In some embodiments, these techniques find use with the compositions, systems, and methods described herein in detecting and analyzing intracellular binding of bioactive agents to intracellular targets.

[0155] In some embodiments, any of the NANOLUC-based, NANOBIT-based, and / or NanoTrip-based peptides, polypeptides, complexes, fusions, and conjugates may find use in BRET-based applications with the compositions, assays, methods, reagents, and systems described herein. BRET applications of the technology described herein utilize one or more energy acceptors. As used herein, the term "energy acceptor" refers to any small molecule (e.g., chromophore), macromolecule (e.g., autofluorescent protein, phycobiliprotein, nanoparticle, surface, etc.), or molecular complex that produces a readily detectable signal in response to energy absorption (e.g., resonance energy transfer). In certain embodiments, the energy acceptor is a fluorophore or other detectable chromophore.Suitable fluorophores include xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, oxazine 170, etc.), acridine derivatives (e.g., proflavine, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porphine, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA Fluor (Invitrogen), DYLIGHT Fluor (Thermo Scientific, Pierce), ATTO and TRACY dyes (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES dyes (Dyomics), SULFO CY dyes (CYANDYE, LLC), SETAU and SQUARE dyes (SETA BioMedicals), QUASAR and CAL Fluor dyes (Biosearch Technologies), SURELIGHT dyes (APC, RPE, PerCP, phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, and BPE dyes (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate), quantum dot nanocrystals, and the like. In some embodiments, the fluorophore is a rhodamine analog (e.g., a carboxyrhodamine analog), such as those described in U.S. Patent Application Serial No. 13 / 682,589, which is incorporated herein by reference in its entirety. In some embodiments, the fluorophore is linked to the bioactive agent and the PRG in the PRG-fluorophore probes described herein. EXAMPLES

[0156] Example 1 During the development of the BRET approach described herein to estimate crosslinking efficiency, experiments were performed to determine the effect of UV irradiation on the bioluminescence intensity of the energy donor NANOLUC and the fluorescence intensity of three fluorophores that can be used as energy acceptors (Figures 12-15). The model system for these experiments (Figure 13) was the interaction between HDAC6 fused to NANOLUC and SAHA (vorinostat), where SAHA binds to HDAC6 and was conjugated with one of three fluorophores NCT, TAMRA, and BODIPY576 (Figure 12). In these experiments, HEK293T cells transiently expressing HAD6-NANOLUC fusions were treated with serial dilutions of SAHA-fluorophore conjugates for 2 hours and then UV-irradiated for 0-30 minutes, followed by treatment with NANOGLO detection reagent. As shown in Figure 14, the NANOLUC signal was unaffected by UV irradiation for up to 30 minutes, thereby indicating that NANOLUC can function as a robust bioluminescence donor under UV-irradiated conditions. On the other hand, the measurement of fluorescence intensity showed that the fluorescence intensity of SAHA-BODIPY576 and SAHA-TMR decreased with UV irradiation in a manner dependent on the UV irradiation time (FIG. 15). In contrast, the fluorescence intensity of SAHA-NCT was not significantly affected by irradiation for up to 30 minutes. Therefore, NANOLUC and NCT were selected as the BRET donor and acceptor pair for the assay to estimate the photocrosslinking efficiency. It should be noted that other luciferase and fluorophore combinations are within the scope of the contemplated scope and within the scope of the embodiments herein.

[0157] Example 2 This example (Figures 16-21) demonstrates the ability of the BRET approach shown in Figure 11 to estimate the crosslinking efficiency of various photoreactive groups. Three model compounds were selected for these experiments: SAHA, dasatinib, and propranolol (Figures 16 and 17). Upon introduction of a carboxylic acid functional group at the highlighted position (Figure 16), an amide coupling reaction can be used to attach functional elements (e.g., dyes, chloroalkanes, PRG, etc.) via the probe reagent without significantly affecting the binding properties of the bioactive agent to its target.

[0158] The synthesis of NCT / PRG chemical probes of dasatinib, SAHA, and propranolol was accomplished as shown in Figures 32, 33, and in Example 7. The structures of the NCT photoaffinity probes used in the BRET assay and their respective targets (fused to NANOLUC) are shown in Figure 17.

[0159] Experiments conducted during the development of embodiments herein investigated the cross-linking efficiency of photoaffinity probes to their respective protein targets fused to NANOLUC. Briefly, as shown in FIG. 11, triplicates of cells expressing protein targets fused to NANOLUC or lysates therefrom were plated in wells of a 96-well plate and treated with (a) 0.3 or 1 μM NCT / PRG probe (control for maximum probe occupancy at a given concentration), (b) 0.3 or 1 μM NCT / PRG probe (experimental sample), and (c) 0.3 or 1 μM NCT / PRG probe and 40 μM untagged bioactive compound (control for minimal probe occupancy) for 90 minutes protected from light, followed by 15 minutes of UV irradiation. After irradiation, the experimental sample was treated with 40 μM untagged bioactive compound for 60 minutes to competitively displace non-covalently bound NCT / PRG probe, while the control sample was left untreated. BRET was then measured upon treatment with NANOGLO detection reagent. Residual BRET values ​​in experimental samples resulting from probes that were not competitively displaced were normalized to maximum and minimum probe occupancy controls. These normalized residual BRET values, which reflect the occupancy of covalently bound probes, were used to derive crosslinking efficiencies. An identical 96-well plate treated in a similar manner but not UV-irradiated was used to derive background values, which were then subtracted from the calculated crosslinking efficiencies. The data in Figures 18-21 are presented as background-corrected crosslinking efficiencies.

[0160] First, SAHA and dasatinib probes containing commonly used PRGs (benzophenone and diazirine, both aryl and alkyl) and monosubstituted aryl azides were tested for crosslinking efficiency. Figure 18 illustrates the photocrosslinking efficiency of these probes to their targets (HDAC1 and HDAC10 for SAHA probes, CSK and MAPK14 for dasatinib probes), as determined by the BRET assay described herein. In each case, both benzophenone and trifluoromethylaryl diazirines showed the lowest levels of photocrosslinking to their targets. Alkyl diazirines and aryl azides showed significantly improved crosslinking efficiency across targets tested compared to both benzophenone and aryl diazirine. 4-phenyl azide provided consistent crosslinking efficiency across all targets.

[0161] The position of the azide group relative to the attachment point on the phenyl ring influenced the photocrosslinking efficiency of the probe (Figure 18). To further explore the effect of aryl ring substitution on photocrosslinking efficiency, eight additional aryl azides were tested in the BRET assay. The results in Figure 19 demonstrate that for both model compounds, two ortho-methyl substituted aryl azides consistently provided improved crosslinking efficiency, surpassing the performance of simple 4-phenyl azide. Other phenyl or benzyl azides, including the known PRG-4-azido-2,3,5,6-tetrafluorobenzyl, provided poor or inconsistent crosslinking efficiency.

[0162] Next, the effect of the second azide group on the cross-linking efficiency of PRG was investigated (Figure 20). Among the simple aryl diazides, an ortho relationship between the azide groups resulted in a lower cross-linking efficiency than the simple 4-phenyl azide. On the other hand, a meta relationship between the two azide functional groups showed improved cross-linking efficiency compared to 4-phenyl azide. Surprisingly, the introduction of an o-methyl substituent on the o-aryl diazide moiety resulted in poor cross-linking efficiency. In contrast, the introduction of an o-fluoro substituent next to the o-aryl diazide resulted in superior performance.

[0163] Figure 21 summarizes the cross-linking efficiencies for the common PRG and the superior aryl azide-containing probes in six model systems. The results clearly show that aryl azides significantly enhance the photocross-linking efficiency compared to diazirine. Furthermore, ortho-tolyl azide outperformed other PRG groups in all six model systems. Furthermore, similar photocross-linking efficiencies were determined for two different concentrations of NCT / PRG probes, suggesting that these efficiencies are an inherent property of PRG rather than being concentration-driven.

[0164] Example 3 In this example, we further confirm the cross-linking efficiency estimated by the BRET approach. It is generally known that the fluorescent signal from the dye can be sensitive to the environment, and therefore the fluorescent readings from the dye may lead to misleading results for various fluorescent probes. To address this, we developed a "reverse BRET" approach, described in FIG. 22, to complement the "direct BRET" method for estimating the cross-linking efficiency of PRG. Unlike direct BRET, reverse BRET relies on the fluorescent readings from a single fluorescent tracer (probe) that does not undergo any chemical changes during UV irradiation (e.g., a bioactive agent is linked to a fluorophore, but does not have PRG), thus eliminating any possible effect of UV irradiation on fluorescence. Briefly, as shown in FIG. 22, triplicate wells of cells or lysates therefrom transiently expressing the target fused to NANOLUC were plated in wells of a 96-well plate and protected from light and treated with (a) nothing (control for maximum occupancy of the fluorescent tracer), (b) 0.3 or 1 μM HA / PRG probe (experimental samples), and (c) 40 μM untagged bioactive compound (control for minimal occupancy of the fluorescent tracer) for 90 min, followed by 15 min of UV irradiation. After irradiation, all samples were treated with 3 μM fluorescent tracer for 60 min, which competitively displaced the experimental samples non-covalently bound to the HA / PRG probe. BRET was then measured upon treatment with NANOGLO detection reagent. To calculate cross-linking efficiency, the normalized BRET values ​​of the experimental samples relative to the control were subtracted from the theoretical maximum cross-linking efficiency of 100%. An identical 96-well plate treated in a similar manner but without UV irradiation was used to derive a background value which was subtracted from the calculated cross-linking efficiency. The data in Figure 23 are presented as background corrected cross-linking efficiency.

[0165] In the reverse BRET approach, the fluorophore of the fluorophore / PRG probe was replaced with a cleavable chloroalkane (the presence of chloroalkane should not be required in this experiment, the bioactive agent / PRG conjugate should be sufficient). The chemical structure of the probe is shown in FIG. 24. FIG. 23 shows a comparison of the photocrosslinking efficiencies estimated by both the reverse BRET and direct BRET methods. The results show that both methods give very similar results, thus corroborating the data provided by each BRET method. Furthermore, similar photocrosslinking efficiencies were determined for two different concentrations of NCT / PRG or HA / PRG probes, suggesting that these efficiencies are not concentration driven but are an intrinsic property of PRG.

[0166] Example 4 This example shows that PRG / cleavable chloroalkanes generally have little effect on the phenotypic potency of three model compounds (Figure 25). SAHA is a broad histone deacetylase (HDAC) inhibitor. K562 cells (2x10 5 cells / mL) were treated with serial dilutions of SAHA or derivatives bearing a cleavable chloroalkane tag (+ / -PRG) for 2 h and then tested for HDAC activity in cells using the non-lytic HDAC-Glo™ I / II Assay (Promega). Results revealed high cellular potency against HDAC class I / IIb, with IC50 values ​​of 0.1 μM and 0.3-1 μM (depending on the PRG group) for SAHA and chloroalkane derivatives, respectively. Dasatinib is a broad-spectrum kinase inhibitor that targets the BCR-ABL oncogenic pathway, thereby reducing downstream STAT5 activation. K-562 cells (2 × 10 5cells / mL) were treated with serial dilutions of dasatinib or derivatives bearing a cleavable chloroalkane tag (+ / -PRG) for 24 h and then tested for expression of the STAT5 reporter. Results showed that expression of the STAT5 reporter was inhibited, with IC50 values ​​of 0.4 nM and 2.4–13 nM (depending on the PRG group) for dasatinib and dasatinib derivatives, respectively. Propranolol is a beta-blocker that targets the β-adrenergic receptor. PC3 cells (2 × 10 ) stimulated with 400 nM isoproterenol, transiently expressing the CRE reporter, were cultured at 100 °C for 24 h. 5 Cells (100 / mL) were treated with serial dilutions of propranolol or derivatives bearing a cleavable chloroalkane tag (+ / -PRG) for 6 h and then tested for expression of the CRE reporter. Results showed that expression of the CRE reporter was inhibited, with IC50 values ​​of 96 nM and 0.5-3.7 μM (depending on the PRG group) for propranolol and the chloroalkane derivatives, respectively. Taken together, these results demonstrate that the effect of the photoreactive chloroalkane tag on the cellular potency and membrane permeability of the compounds is generally small.

[0167] Example 5 This example demonstrates the ability of aryl azide-cleavable chloroalkane probes to enrich for endogenous targets of SAHA. Figure 26 shows a schematic of the enrichment workflow. Briefly, K562 cells were plated at 2 x 10 in 100 mm dishes. 6Cells / mL were plated and treated with PRG-cleavable chloroalkane SAHA probe at a final concentration of 1 μM in the presence or absence of 20 μM SAHA. After 2.5 hours of equilibrium binding, cells were irradiated for 15 minutes, whereas controls were not. The medium was then removed, cells were washed with PBS, lysed, and centrifuged at 3000×g for 1 minute. The cleared lysate was added to 30 μL of precipitated paramagnetic HaloTag® beads and incubated overnight with constant mixing. After binding, unbound moieties were removed, HaloTag® paramagnetic beads were washed five times, and captured targets were released from the beads by palladium-catalyzed cleavage (ACS Chem.Biol., 2016, 11, 2608, incorporated herein by reference in its entirety). The released targets were subjected to Western blot analysis with anti-HDAC1 antibody (ABCAM), anti-HDAC2 antibody (ABCAM), anti-HDAC6 antibody (Cell signaling), anti-HDAC3 antibody (Cell signaling); anti-HDAC8 antibody (ABCAM); and anti-HDAC10 antibody (ABCAM) (Figure 27). The results in Figure 27 demonstrate the requirement of UV irradiation for efficient enrichment and the ability of the aryl azide probe to enhance enrichment of all known SAHA targets, including low affinity targets (HDAC8) and low abundance targets (HDAC3, HDAC6, and HDAC10). 3-Fluoro-4,5-phenyldiazide exhibits relatively high enrichment of most targets even without irradiation, suggesting an additional mechanism for stabilizing the probe-target interaction.

[0168] Example 6 This example demonstrates the ability of aryl azide-cleavable chloroalkane probes to enrich for endogenous targets of dasatinib. Figure 26 shows a schematic of the enrichment workflow. Briefly, K562 cells were plated at 2 x 10 in 100 mm dishes. 6Cells / mL were plated and treated with PRG-cleavable chloroalkanatinib probe at a final concentration of 1 μM in the presence or absence of 20 μM dasatinib. After 2.5 h of equilibrium binding, cells were irradiated for 15 min, whereas controls were not. The medium was then removed, and cells were then washed with PBS, lysed, and centrifuged at 3000×g for 1 min. The cleared lysate was added to 30 μL of precipitated paramagnetic HaloTag® beads and incubated overnight with constant mixing. After binding, unbound moieties were removed, HaloTag® paramagnetic beads were washed five times, and captured targets were released from the beads by palladium-catalyzed cleavage. Released targets were subjected to Western blot analysis with anti-MAPK14 (ABCAM), anti-PKMYT1 (Cell signaling), and anti-LYN (Cell signaling) antibodies (FIG. 28). The results in Figure 28 demonstrate the requirement of UV irradiation for efficient enrichment and the ability of the aryl azide probe to enhance enrichment of known dasatinib targets. 3-Fluoro-4,5-phenyldiazide exhibits relatively high enrichment of most targets even in the absence of irradiation, suggesting an additional mechanism for stabilizing the probe-target interaction.

[0169] Example 7 Synthesis and characterization of probes and reagents Synthesis of Carboxy-SAHA: [ka] To a solution of methyl 4-aminobenzoate (190 mg, 1.26 mmol) in DMF (75 mL) was added HATU (597 mg, 1.57 mmol) and N-methylmorpholine (0.9 mL, 8.8 mmol), followed by SL_0964 (524 mg, 1.26 mmol). The resulting solution was stirred at 22 °C for 24 h, at which point LCMS analysis indicated complete consumption of starting material. The solvent was removed under reduced pressure and the crude residue was purified by silica gel chromatography (0 → 10% MeOH / DCM, silica was neutralized with 0.5% trimethylamine in DCM during equilibration) to give 350 mg (49% yield) of the anilide SL_0995 as a white solid. 1 H-NMR (400 MHz, DMSO-d6) δ 10.20 (s, 1H), 10.17 (s, 1H), 7.90 (apparent d, J = 8.7 Hz, 2H), 7.73 (apparent d, J = 8.7 Hz, 2H), 7.47 - 7.08 (m, 15H), 3.81 (s, 3H), 2.29 (t, J = 7.4 Hz, 2H), 1.77 (t, J = 7.3 Hz, 2H), 1.49 (p, J = 7.4 Hz, 2H), 1.28 - 1.07 (m, 4H), 1.03 - 0.93 (m, 2H);MS (SI) C 35 H 35 N2O5[MH]- Calculated value 563.26, measured value 563.40.

[0170] [ka] To a suspension of SL_0995 (350 mg, 620 μmol) in MeOH (25 mL) and HO (5 mL) was added KOH (70 mg, 1.2 mmol). The resulting suspension was gently heated until a clear solution formed and left stirring at 40° C. for 24 h, at which point LCMS analysis indicated complete consumption of starting material. The reaction mixture was passed through a pad of DOVEX 50WX4 resin. The resin was washed with an additional 50 mL of MeOH. The solutions were combined and concentrated to give 330 mg (97% yield) of acid SL_1005 as a white solid. 1H-NMR (400 MHz, DMSO-d6) δ 12.68 (s, 1H), 10.16 (s, 2H), 7.87 (d, J = 8.6 Hz, 2H), 7.70 (d, J = 8.7 Hz, 2H), 7.32 (d, J = 4.2 Hz, 15H), 2.28 (t, J = 7.5 Hz, 1H), 1.77 (m, 2H), 1.49 (m, J = 7.6 Hz, 2H), 1.15 (m, 4H), 0.99 (m, 2H);MS (SI) C 34 H 33 N2O5[MH]- Calculated value 549.24, measured value 549.07.

[0171] Synthesis of Carboxy-Dasatinib: [ka] To a solution of trifluoroacetylpiperazine (2.00 g, 11.0 mmol) in dry MeCN (60 mL) was added K2CO3 (3.04 g, 22.0 mmol), followed by tert-butyl 2-bromoacetate (1.62 mL, 11.0 mmol). The resulting solution was heated to 60 °C under Ar for 22 h, at which point LCMS analysis indicated complete consumption of starting material. The reaction mixture was filtered through a pad of Celite, and the pad was washed with additional MeCN (100 mL) and combined with the filtrate. The solvent was removed under reduced pressure, and the crude residue was purified by silica gel chromatography (0 → 100% EtOAc / heptane) to give 2.34 g (72% yield) of the ester SL-1243 as a white solid. 1 H-NMR (400 MHz, DMSO-d6) δ 3.59 - 3.52 (m, 4H), 3.19 (s, 2H), 2.70 - 2.56 (m, 4H), 1.41 (s, 9H);MS (SI) C 12 H 20 F3N2O3[M+H]+ calculated 297.14, found 297.07.

[0172] [ka] To a solution of SL-1243 (2.34 g, 7.90 mmol) in MeOH (40 mL) and water (4 mL) was added K2CO3 (2.18 g, 15.8 mmol). The resulting suspension was stirred at 50 °C for 2 h, at which point HPLC analysis indicated complete consumption of starting material. MeOH was removed under reduced pressure and the residue was partitioned between water (50 mL) and EtOAc (100 mL). The aqueous layer was further washed with EtOAc (100 mL) and the organics were combined, dried over MgSO4 and the solvent removed under reduced pressure to give 605 mg (38% yield) of a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 3.05 (s, 2H), 2.76 - 2.68 (m, 4H), 2.45 - 2.41 (m, 4H), 1.40 (s, 9H).

[0173] [ka] A 50 mL round bottom flask equipped with a stir bar and condenser was charged with 2-((6-chloro-2-methylpyrimidin-4-yl)amino)-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide (380 mg, 1.90 mmol), SL-1249 (471 mg, 7.59 mmol), isopropyl alcohol (3 mL), ethylene glycol (3 mL), and EtN(iPr)2 (1 mL). The resulting suspension was heated to 110 °C for 20 h, at which point HPLC analysis indicated complete consumption of starting material. The precipitate was filtered off, and both the filtrate and precipitate were purified by preparative RP HPLC (5→95% MeCN / H2O buffered with 0.5% TFA) to give 375 mg (35% yield) of the ester SL-1272 as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.62 (s, 1H), 9.89 (s, 1H), 8.23 ​​(s, 1H), 7.40 (dd, J = 7.4, 2.0 Hz, 1H), 7.35 - 7.09 (m, 2H), 6.12 (s, 1H), 4.42 - 3.49 (m, 4H), 3.27 - 2.98 (m, 4H), 2.44 (s, 3H), 2.24 (s, 3H), 1.48 (s, 9H).

[0174] [ka] To a suspension of SL-1272 in DCM (10 mL) was added TiPS (0.15 mL) followed by TFA (5 mL) and the resulting solution was stirred for 15 min and an additional 5 mL of TFA was added. The resulting solution was stirred at 22° C. for 3 h, at which point HPLC analysis indicated complete consumption of the starting material. The volatiles were removed in vacuo and the residue was filtered and dried under vacuum to give 180 mg (96% yield) of SL-1338 as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 11.63 (s, 1H), 9.90 (s, 1H), 8.24 (s, 1H), 7.64 - 7.36 (m, 1H), 7.36 - 7.13 (m, 2H), 6.13 (s, 1H), 4.11 (s, 4H), 2.44 (br. s, 4H), 2.44 (s, 3H). 2.24 (s, 3H);MS (SI) C 22 H 24 ClN7O3S [M+H]+ calculated 501.14, found 502.12.

[0175] Synthesis of Carboxy-(S)-Propranolol: [ka] To a solution of (S)-2-((naphthalen-1-yloxy)methyl)oxirane (308 mg, 1.54 mmol, prepared according to J. Org. Chem. 1989, 54, 1295-1304) and β-alanine t-butyl ester hydrochloride (699 mg, 3.85 mmol) in 9:1 DMF-water (10 mL) was added NaHCO3 (323 mg, 3.85 mmol). The stirred suspension was heated to 85 °C for 21 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was filtered and the volatiles removed under reduced pressure. The crude residue was purified by silica gel chromatography (0→20% MeOH / DCM) to give 181 mg (34% yield) of the ester SL-1457 as a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 8.23 ​​(ddd, J = 7.4, 2.3, 0.9 Hz, 1H), 7.89 - 7.77 (m, 1H), 7.50 (dtd, J = 8.1, 6.8, 5.2 Hz, 2H), 7.45 (dt, J = 8.5, 0.9 Hz, 1H), 7.40 (dd, J = 8.3, 7.4 Hz, 1H), 6.93 (dd, J = 7.5, 1.2 Hz, 1H), 5.08 (d, J = 4.8 Hz, 1H), 4.12 (dd, J = 9.2, 4.4 Hz, 1H), 4.06 - 3.97 (m, 1H), 2.80 - 2.72 (m, 3H), 2.72 - 2.58 (m, 1H), 2.34 (t, J = 6.7 Hz, 2H), 1.83 (s, 1H), 1.37 (s, 9H);HRMS (SI) C 20 H 28 NO4[M+H]+ calculated 346.2013, found 346.2013.

[0176] [ka] To a solution of SL-1457 (430 mg, 1.24 mmol) in DCM (20 mL) was added TiPS (0.26 mL) followed by TFA (6 mL). The resulting solution was heated at 30° C. for 4 h, at which point HPLC analysis indicated complete consumption of starting material. The volatiles were removed under reduced pressure and the crude residue was dissolved in 10 mL of MeOH and the volatiles were removed in vacuo. The crude residue was dried under high vacuum and used in the next step without further purification.

[0177] [ka] To a solution of SL-1514 (540 mg, 1.34 mmol) in dioxane (25 mL) was added trimethylamine (1.31 mL, 9.37 mmol), followed by Boc2O (877 mg, 4.02 mmol). The resulting solution was stirred at 22 °C for 21 h, at which point HPLC analysis indicated complete consumption of starting material. The volatiles were removed under reduced pressure and the crude residue was dissolved in 10 mL of MeOH and the volatiles were removed in vacuo. The crude residue was purified by silica gel chromatography (0 → 20% MeOH / DCM) to afford 397 mg (76% yield) of the acid SL-1517 as a clear glassy residue. 1 H NMR (400 MHz, DMSO-d6, reported for mixture of rotamers) δ 1 H NMR (400 MHz, DMSO-d6) δ 12.18 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H), 7.98 - 7.74 (m, 1H), 7.59 - 7.49 (m, 2H), 7.47 (d, J = 8.2 Hz, 1H), 7.41 (t, J = 7.9 Hz, 1H), 6.94 (m, 1H), 5.31 (m, 1H), 4.14 (m, 1H), 4.05 (m, 2H), 3.65 - 3.58 (m, 1H), 3.48 (m, 2H), 3.30 - 3.20 (m, 1H), 1.39 (s, 4.5H), 1.35 (s, 4.5H); MS (SI) C 21 H 26NO6[MH]- Calculated value 388.18, measured value 388.09.

[0178] Synthesis of photoreactive bifunctional building blocks: [ka] To a solution of N-Boc-ethylenediamine (571 mg, 3.57 mmol) in MeCN (25 mL) was added Na2CO3 (378 mg, 3.57 mmol) followed by 4-(bromomethyl)benzophenone (327 mg, 1.19 mmol). The resulting solution was stirred at 22 °C for 22 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was filtered and volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 → 10% MeOH / DCM) to give 350 mg (83% yield) of amine SL-1203. 1 H NMR (400 MHz, DMSO-d6) δ 7.82 - 7.60 (m, 5H), 7.60 - 7.53 (m, 2H), 7.53 - 7.37 (m, 2H), 6.91 - 6.64 (m, 1H), 3.79 (s, 2H), 3.04 (q, J = 6.2 Hz, 2H), 2.55 (t, J = 6.5 Hz, 2H), 1.37 (s, 9H);MS (SI) 21 H 27 N2O3[M+H]+ calculated 355.20, found 354.62.

[0179] [ka] To a solution of N-Boc-ethylenediamine (250 mg, 1.56 mmol) in MeCN (30 mL) was added K2CO3 (215 mg, 1.56 mmol) followed by 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl bromide (145 mg, 520 μmol). The resulting solution was stirred at 22 °C for 24 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was filtered and the volatiles removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 → 20% MeOH / DCM) to give 115 mg (62% yield) of the amine SL-1201. MS (SI) C 16 H 22 F3N4O2[M+H]+ calculated 359.17, found 359.47.

[0180] [ka] To a solution of N-Boc-ethylenediamine (101 mg, 629 μmol) in MeCN (10 mL) was added K2CO3 (130 mg, 944 μmol), and KI (10.4 mg, 629 μmol), followed by SL-1259 (80.0 mg, 315 μmol, prepared according to J. Am. Chem. Soc. 2014, 136, 10777-10782). The resulting solution was stirred at 80 °C for 19 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was filtered and volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 → 100% MeOH / DCM) to give 66 mg (87% yield) of the amine SL-1358. 1 H NMR (400 MHz, methanol-d4) δ 3.20 (t, J = 6.2 Hz, 2H), 2.74 (t, J = 5.9 Hz, 2H), 2.61 (t, J = 7.4 Hz, 2H), 1.57 (dd, J = 8.3, 7.2 Hz, 2H), 1.44 (s, 9H), 1.03 (s, 3H). MS (SI) C 11 H 23N4O2[M+H]+ calculated 243.18, found 243.05.

[0181] [ka] To a solution of N-Boc-ethylenediamine (962 mg, 6.00 mmol) in MeCN (150 mL) was added K2CO3 (1.11 g, 8.00 mmol) followed by 4-bromobenzyl bromide (1.00 g, 4.00 mmol). The resulting solution was stirred at 22 °C for 23 h, at which point HPLC analysis indicated complete consumption of the starting material. The reaction mixture was filtered and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 → 20% MeOH / DCM, column equilibrated with 0.5% TEA / DCM) to give 1.03 g (78% yield) of the amine SL-0997 as a clear oil. 1 H NMR (400 MHz, DMSO-d6) δ 7.65 - 7.41 (m, 2H), 7.41 - 7.11 (m, 2H), 6.74 (t, J = 5.8 Hz, 1H), 3.63 (s, 2H), 3.00 (q, J = 6.3 Hz, 2H), 2.48 (m, 2H, overlap with DMSO-d5), 2.16 (s, 1H), 1.36 (s, 9H);MS (SI) C 21 H 27 N2O3[M+H]+ calculated 329.09, found 329.13.

[0182] A 50 mL round bottom flask equipped with a stir bar and a condenser with a rubber septum was charged with SL-0997 (510 mg, 1.55 mmol), sodium ascorbate (15.3 mg, 77.5 μmol), NaN3 (201 mg, 3.10 mmol), and CuI (59 mg, 310 μmol). The flask was evacuated and backfilled with Ar (3 times) and placed under Ar atmosphere. Through the septum, degassed EtOH (14 mL) and degassed water (6 mL) were added, followed by 1,2-dimethylethylenediamine (21 mg, 233 μmol). The resulting suspension was heated to 90° C. for 45 min, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was adsorbed onto Celite and dried under high vacuum. Purification by silica gel chromatography (0→100% MeOH / DCM) afforded 353 mg (78% yield) of the amine SL-1002 as a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 7.44 - 7.28 (m, 2H), 7.13 - 6.98 (m, 2H), 6.77 (t, J = 5.8 Hz, 1H), 3.66 (s, 2H), 3.01 (q, J = 6.3 Hz, 2H), 2.48 (m, 2H, overlap with DMSO-d5), 1.37 (s, 9H);MS (SI) C 21 H 27 N2O3[M+H]+ calculated 292.18, found 292.19.

[0183] [ka] To a solution of N-Boc-ethylenediamine (606 mg, 3.78 mmol) in DMF (60 mL) was added K2CO3 (697 mg, 5.04 mmol) followed by 3-bromobenzyl bromide (630 g, 2.52 mmol). The resulting solution was stirred at 22 °C for 25 h, at which point HPLC analysis indicated complete consumption of the starting material. The reaction mixture was filtered and the volatiles removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 → 20% MeOH / DCM) to afford 670 mg (71% yield) of the amine SL-1088 as a clear oil. 1 H NMR (400 MHz, DMSO-d6) δ 7.53 (t, J = 1.9 Hz, 1H), 7.41 (dt, J = 7.6, 1.7 Hz, 1H), 7.36 - 7.18 (m, 2H), 6.73 (t, J = 5.7 Hz, 1H), 3.66 (s, 2H), 3.01 (q, J = 6.3 Hz, 2H), 2.49 (m, 2H, overlap with DMSO-d5), 2.27 (s, 1H), 1.37 (s, 9H);MS (SI) C 21 H 27 N2O3[M+H]+ calculated 329.09, found 329.18.

[0184] A 50 mL round bottom flask equipped with a stir bar and a condenser with a rubber septum was charged with SL-1088 (600 mg, 1.82 mmol), sodium ascorbate (18.1 mg, 91.1 μmol), NaN3 (237 mg, 3.64 mmol), and CuI (69 mg, 360 μmol). The flask was evacuated and backfilled with Ar (three times) and placed under an Ar atmosphere. Through the septum, degassed EtOH (10 mL) and degassed water (6 mL) were added, followed by a degassed solution of 1,2-dimethylethylenediamine (24 mg, 273 μmol) in EtOH (4 mL). The resulting suspension was heated to 90 °C for 70 min, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was poured into 10% aqueous Na2CO3 (50 mL) and extracted three times with 75 mL of DCM. The organics were combined, dried over MgSO4, filtered, and the solvent removed in vacuo. Purification by silica gel chromatography (0→10% MeOH / DCM) afforded 388 mg (73% yield) of the amine SL-1092 as a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 7.34 (t, J = 7.8 Hz, 1H), 7.13 (d, J = 7.6 Hz, 1H), 7.09 (t, J = 2.0 Hz, 1H), 6.96 (dd, J = 8.1, 2.3 Hz, 1H), 6.73 (t, J = 5.8 Hz, 1H), 3.67 (s, 2H), 3.01 (q, J = 6.3 Hz, 2H), 1.36 (s, 9H);MS (SI) C 21 H 27 N2O3[M+H]+ calculated 292.18, found 292.24.

[0185] [ka] To a solution of N-Boc-ethylenediamine (491 mg, 3.07 mmol) in DCM (100 mL) and 2-azidobenzaldehyde (410 mg, 2.79 mmol, prepared according to Journal of the American Chemical Society, 2014, 136, 15138) was added NaBH(OAc)3 (1.48 g, 6.97 mmol). The resulting solution was stirred at 22 °C for 5 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was quenched with 1 M aqueous Na2CO3 (100 mL) and the resulting biphasic mixture was stirred for 1 h. The organic layer was separated and the aqueous layer was extracted with additional DCM (100 mL). The organics were combined, dried over MgSO4, filtered, and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0→100% MeOH / DCM) to afford 553 mg (68% yield) of the amine SL-1053. 1 H NMR (400 MHz, DMSO-d6) δ 7.39 (dd, J = 7.5, 1.6 Hz, 1H), 7.34 (td, J = 7.6, 1.7 Hz, 1H), 7.25 (dd, J = 7.9, 1.2 Hz, 1H), 7.15 (td, J = 7.4, MS (SI) C 14 H 22 N5O2[M+H]+ calculated 292.18, found 291.80.

[0186] [ka] A 25 mL round bottom flask equipped with a stir bar and a condenser with a rubber septum was charged with 3-bromo-4-methylbenzaldehyde (795 mg, 3.99 mmol), sodium ascorbate (39.6 mg, 0.20 mmol), NaN3 (519 mg, 7.99 mmol), and CuI (152 mg, 799 μmol). The flask was evacuated and backfilled with Ar (three times) and placed under an Ar atmosphere. Through the septum, degassed EtOH (7 mL) and degassed water (3 mL) were added, followed by 1,2-dimethylethylenediamine (23 mg, 600 μmol). The resulting suspension was heated to 90 °C for 3 h, at which point HPLC monitoring indicated the reaction had stopped. The reaction mixture was poured into 10% aqueous Na2CO3 (100 mL) and extracted three times with 75 mL of DCM. The organics were combined, dried over MgSO4, filtered, and the solvent removed in vacuo. Purification by silica gel chromatography (0→10% EtOAc / heptane) afforded 57 mg (7% yield) of aldehyde SL-1173. 1 H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 7.74 (d, J = 1.4 Hz, 1H), 7.64 (dd, J = 7.7, 1.5 Hz, 1H), 7.48 (d, J = 7.7 Hz, 1H), 2.25 (s, 3H); 13 C NMR (100 MHz, DMSO-d6) δ 192.2, 139.0, 136.0, 135.6, 131.8, 126.0, 118.6, 17.4;MS (SI) C 21 H 27 N2O3[M+H]+ calculated 292.18, found 292.24.

[0187] To a solution of N-Boc-ethylenediamine (47 mg, 290 μmol) and SL-1173 (57 mg, 270 μmol) in DCM (5 mL) was added NaBH(OAc)3 (140 mg, 660 μmol). The resulting solution was stirred at 22 °C for 20 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was quenched with 1 M aqueous Na2CO3 (15 mL) and the resulting biphasic mixture was stirred for 1 h. The organic layer was separated and the aqueous layer was extracted with additional DCM (2 x 100 mL). The organics were combined, dried over MgSO4, filtered, and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 -> 10% MeOH / DCM) to give 68 mg (84% yield) of the amine SL-1175. 1 H NMR (400 MHz, DMSO-d6) δ 7.20 (d, J = 1.6 Hz, 1H), 7.16 (d, J = 7.7 Hz, 1H), 7.02 (dd, J = 7.7, 1.6 Hz, 1H), 6.72 (t, J = 5.8 Hz, 1H), 3.66 (s, 2H), 3.01 (q, J = 6.3 Hz, 2H), 2.13 (s, 3H), 1.37 (s, 9H); 13 HRMS (SI) C 15 H 24 N5O6[M+H]+ calculated 306.1925, found 306.1922.

[0188] [ka] To a solution of 3-methyl-4-nitrobenzaldehyde (1.00 g, 6.06 mmol) in DMF (25 mL) was added NaN3 (1.18 g, 18.2 mmol). The resulting suspension was heated at 60° C. for 19 h. The reaction mixture was cooled to room temperature, filtered, and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0→20% EtOAc / heptane) to give 223 mg (23% yield) of azide SL-1064. 1 H NMR (400 MHz, DMSO-d6) δ 9.92 (s, 1H), 7.75 (dd, J = 8.2, 2.0 Hz, 1H), 7.69 (dd, J = 1.8, 0.9 Hz, 1H), 7.24 (s, 1H), 2.27 (s, 3H).

[0189] To a solution of N-Boc-ethylenediamine (244 mg, 1.52 mmol) and SL-1064 (223 mg, 1.38 mmol) in DCM (50 mL) was added NaBH(OAc)3 (733 mg, 3.46 mmol). The resulting solution was stirred at 22 °C for 2 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was quenched with an aqueous solution of K2CO3 (10%, 50 mL) and the resulting biphasic mixture was stirred for 1 h. The organic layer was separated and the aqueous layer was extracted with additional DCM (2 x 75 mL). The organics were combined, dried over MgSO4, filtered, and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 -> 100% MeOH / DCM) to give 263 mg (62% yield) of the amine SL-1066. 1 H NMR (400 MHz, DMSO-d6) δ 7.22 (dd, J = 8.1, 2.0 Hz, 1H), 7.18 (d, J = 1.9 Hz, 1H), 7.15 (d, J = 8.1 Hz, 1H), 6.86 - 6.56 (m, 1H), 3.61 (s, MS (SI) C 15 H 24N5O6[M+H]+ calculated 306.19, found 306.18.

[0190] [ka] A 25 mL round bottom flask equipped with a stir bar and a condenser with a rubber septum was charged with 2-bromo-3-methylbenzaldehyde (672 mg, 3.38 mmol), sodium ascorbate (33.4 mg, 169 μmol), NaN3 (439 mg, 6.75 mmol), and CuI (129 mg, 675 μmol). The flask was evacuated and backfilled with Ar (three times) and placed under an Ar atmosphere. Through the septum, degassed EtOH (10 mL) and degassed water (4 mL) were added, followed by 1,2-dimethylethylenediamine (45 mg, 500 μmol). The resulting suspension was heated to 90 °C for 60 min, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was poured into 10% aqueous Na2CO3 (100 mL) and extracted three times with 75 mL of DCM. The organics were combined, dried over MgSO4, filtered, and the solvent removed in vacuo. Purification by silica gel chromatography (0→30% EtOAc / heptane) afforded 256 mg (49% yield) of aldehyde SL-1593. 1 H NMR (400 MHz, DMSO-d6) δ 10.38 (d, J = 0.5 Hz, 1H), 7.76 (dd, J = 7.8, 1.7 Hz, 1H), 7.47 (ddq, J = 7.5, 1.5, 0.7 Hz, 1H), 7.29 (d, J = 7.9 Hz, 1H), 2.50 (s, 3H); 13 C NMR (100 MHz, CDCl3) δ 190.0, 140.1, 137.1, 133.5, 129.2, 129.0, 125.8, 17.8.

[0191] To a solution of N-Boc-ethylenediamine (120 mg, 751 μmol) and SL-1593 (110 mg, 680 μmol) in DCM (5 mL) was added NaBH(OAc)3 (362 mg, 1.71 mmol). The resulting solution was stirred at 22 °C for 18 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was quenched with an aqueous solution of K2CO3 (10%, 15 mL) and the resulting biphasic mixture was stirred for 1 h. The organic layer was separated and the aqueous layer was extracted with additional DCM (2 x 100 mL). The organics were combined, dried over MgSO4, filtered and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 -> 30% MeOH / DCM) to give 188 mg (90% yield) of the amine SL-1594 as a clear oil. 1 H NMR (400 MHz, DMSO-d6) δ 7.23 (dd, J = 7.4, 1.9 Hz, 1H), 7.18 - 7.00 (m, 2H), 6.72 (t, J = 5.6 Hz, 1H), 3.72 (s, 2H), 3.02 (q, J = 6.2 Hz, 2H), 2.54 (m, 2H, overlap with DMSO-d5), 2.33 (s, 3H), 1.99 (broad s, 1H), 1.36 (s, 9H);HRMS (SI) C 15 H 24 N5O6[M+H]+ calculated 306.1925, found 306.1932.

[0192] [ka] To a solution of 3,4-difluorobenzaldehyde (1.47 g, 10.4 mmol) in DMF (10 mL) was added NaN3 (2.02 g, 31.0 mmol). The resulting suspension was heated at 60° C. for 19 h. The reaction mixture was cooled to room temperature, filtered through Celite, and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0→20% EtOAc / heptane) to afford 1.16 mg (61% yield) of the azide SL-1281 as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.93 (d, J = 1.9 Hz, 1H), 7.92 - 7.67 (m, 2H), 7.55 (t, J = 8.2 Hz, 1H); 13 C NMR (100 MHz, DMSO-d6) δ 190.97 (d, J = 2.0 Hz), 153.67 (d, J = 250.0 Hz), 133.90 (d, J = 5.5 Hz), 133.68 (d, J = 11.0 Hz), 127.12 (d, J = 3.4 Hz), 122.12 (d, J = 1 Hz), 116.51 (d, J = 19.1 Hz).

[0193] To a solution of N-Boc-ethylenediamine (380 mg, 2.37 mmol) and SL-1281 (347 mg, 2.15 mmol) in DCM (5 mL) was added NaBH(OAc)3 (1.14 g, 5.38 mmol). The resulting suspension was stirred at 22 °C for 4 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was quenched with an aqueous solution of K2CO3 (10%, 50 mL) and the resulting biphasic mixture was stirred for 1 h. The organic layer was separated and the aqueous layer was extracted with additional DCM (3 x 50 mL). The organics were combined, dried over MgSO4, filtered and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 -> 20% MeOH / DCM) to give 481 mg (72% yield) of the amine SL-1289. 1 H NMR (400 MHz, DMSO-d6) δ 7.27 (dd, J = 12.4, 1.7 Hz, 1H), 7.22 (t, J = 8.2 Hz, 1H), 7.17 (dd, J = 8.2, 1.7 Hz, 1H), 6.73 (t, J = 6.0 Hz, MS (SI)C 14 H 21FN5O2[M+H]+ calculated 310.18, found 310.18.

[0194] [ka] To a solution of 4-fluoro-3-(trifluoromethyl)benzaldehyde (1.73 g, 9.01 mmol) in DMF (40 mL) was added NaN3 (1.76 g, 27.0 mmol). The resulting suspension was heated at 60° C. for 19 h. The reaction mixture was cooled to room temperature, filtered through Celite, and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0→30% EtOAc / heptane) to give 1.56 g (81% yield) of the azide SL-1115 as a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 9.99 (d, J = 0.5 Hz, 1H), 8.16 (dt, J = 2.0, 0.6 Hz, 1H), 8.10 (ddd, J = 8.3, 1.9, 0.6 Hz, 1H), 7.45 (dq, J = 8.3, 0.6 Hz, 1H).

[0195] To a solution of N-Boc-ethylenediamine (803 mg, 5.01 mmol) and SL-1115 (980 mg, 4.56 mmol) in DCM (200 mL) was added NaBH(OAc)3 (2.41 g, 11.4 mmol). The resulting suspension was stirred at 22 °C for 4 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was quenched with an aqueous solution of K2CO3 (10%, 200 mL) and the resulting biphasic mixture was stirred for 1 h. The organic layer was separated and the aqueous layer was extracted with additional DCM (2 x 100 mL). The organics were combined, dried over MgSO4, filtered, and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 -> 100% [3:1 EtOAc:EtOH] / DCM) to give 1.31 g (80% yield) of the amine SL-1116. 1H NMR (400 MHz, DMSO-d6) δ 7.69 - 7.65 (m, 2H), 7.51 (d, J = 8.2 Hz, 1H), 6.74 (t, J = 5.7 Hz, 1H), 3.72 (s, 2H), 3.02 (q, J = 6.3 Hz, 2H), 2.49 (m, 1H, overlap with DMSO-d5), 1.37 (s, 9H);MS (SI) C 15 H 21 F3N5O2[M+H]+ calculated 360.16, found 360.10.

[0196] [ka] A 25 mL round bottom flask equipped with a stir bar and a condenser with a rubber septum was charged with 4-bromo-3,5-dimethylbenzaldehyde (650 mg, 3.05 mmol), sodium ascorbate (30.0 mg, 153 μmol), NaN3 (397 mg, 6.10 mmol), and CuI (116 mg, 610 μmol). The flask was evacuated and backfilled with Ar (3 times) and placed under Ar atmosphere. Through the septum, degassed EtOH (10 mL) and degassed water (4 mL) were added, followed by 1,2-dimethylethylenediamine (40 mg, 458 μmol). The resulting suspension was heated to 90 °C for 3 h (the reaction was stopped before complete consumption of starting material). The reaction mixture was filtered through Celite and the solvent was removed under vacuum. Purification by silica gel chromatography (0→10% EtOAc / heptane) afforded 157 mg (29% or 45% yield depending on recovered starting material) of aldehyde SL-1212 as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 7.67 - 7.39 (m, 2H), 2.44 (d, J = 0.7 Hz, 6H); 13 C NMR (100 MHz, CDCl3) δ 191.3, 142.6, 133.4, 132.7, 130.4, 18.3.

[0197] To a solution of N-Boc-ethylenediamine (158 mg, 986 μmol) and SL-1212 (157 mg, 896 μmol) in DCM (15 mL) was added NaBH(OAc)3 (475 mg, 2.24 mmol). The resulting suspension was stirred at 22 °C for 17 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was quenched with an aqueous solution of K2CO3 (10%, 15 mL) and the resulting biphasic mixture was stirred for 1 h. The organic layer was separated and the aqueous layer was extracted with additional DCM (3 x 50 mL). The organics were combined, dried over MgSO4, filtered and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 -> 30% MeOH / DCM) to give 212 mg (74% yield) of the amine SL-1214. 1 H NMR (400 MHz, DMSO-d6) δ 7.02 (s, 2H), 6.72 (t, J = 5.8 Hz, 1H), 3.57 (s, 2H), 3.00 (q, J = 6.3 Hz, 2H), 2.47 (m, 2H, overlap with DMSO-d5), 2.29 (s, 6H), 2.11 (br. s, 1H);MS (SI) C 16 H 26 N5O2[M+H]+ calculated 320.17, found 320.17.

[0198] [ka] To a solution of 3,4,5-trifluorobenzaldehyde (1.00 g, 6.25 mmol) in DMF (10 mL) was added NaN3 (1.22 g, 18.7 mmol). The resulting suspension was heated at 60° C. for 20 h. The reaction mixture was cooled to room temperature, filtered through Celite, and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0→15% EtOAc / heptane) to give 364 mg (32% yield) of azide SL-1274 (as a white solid) and 170 mg (13% yield) of SL-1274-diazide, as well as 350 mg of a mixed fraction of SL-1274 and SL-1274-diazide.

[0199] Price SL-1274: 1 H NMR (400 MHz, DMSO-d6) δ 9.93 (d, J = 2.0 Hz, 1H), 7.72 (t, J = 1.4 Hz, 1H), 7.64 (dd, J = 10.7, 1.7 Hz, 1H); 13 C NMR (100 MHz, DMSO-d6) δ 190.60 (d, J = 2.2 Hz), 155.90 (J = 247.0 Hz), 134.03 (d, J = 4.0 Hz), 133.16 (d, J = 7.0 Hz), 124.30 (d, J = 14.0 Hz), 117.58 (d, J = 2.8 Hz), 112.29 (d, J = 20.7 Hz); 19 F NMR (376 MHz, DMSO-d6) δ-122.2 (ddd, J = 10.8, 1.8, 1.3 Hz).

[0200] Ranger SL-1274: 1 H NMR (400 MHz, DMSO-d6) δ 9.89 (t, J = 1.9 Hz, 1H), 7.79–7.71 (m, 2H); 13 C NMR (100 MHz, DMSO-d6) δ 190.14 (s), 155.07 (dd, J = 250.6, 4.5 Hz), 132.60 (t, J = 7.5 Hz), 123.03 (s), 115.14 - 105.17 (m); 19 F NMR (376 MHz, DMSO-d6) δ-121.15-121.23 (m).

[0201] SL-1280: To a solution of N-Boc-ethylenediamine (191 mg, 1.19 mmol) and azide SL-1274 (175 mg, 1.09 mmol) in DCM (25 mL) was added NaBH(OAc)3 (575 mg, 2.71 mmol). The resulting suspension was stirred at 22 °C for 2 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was quenched with an aqueous solution of K2CO3 (10%, 25 mL) and the resulting biphasic mixture was stirred for 1 h. The organic layer was separated and the aqueous layer was extracted with additional DCM (3 x 25 mL). The organics were combined, dried over MgSO4, filtered and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 -> 20% MeOH / DCM) to give 41 mg (12% yield) of amine SL-1280. 1 H NMR (400 MHz, DMSO-d6) δ 7.27 - 6.95 (m, 2H), 6.74 (t, J = 5.7 Hz, 1H), 3.65 (s, 2H), 2.99 (q, J = 6.3 Hz, 2H), 2.47 (m, 2H, overlap with DMSO-d5), 1.36 (s, 9H);MS (SI) C 14 H 20 F2N5O2[M+H]+ calculated 328.16, found 328.17.

[0202] SL-1279: To a solution of N-Boc-ethylenediamine (191 mg, 1.19 mmol) and diazide SL-1274 (175 mg, 1.09 mmol) in DCM (25 mL) was added NaBH(OAc)3 (575 mg, 2.71 mmol). The resulting suspension was stirred at 22 °C for 2 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was quenched with an aqueous solution of K2CO3 (10%, 25 mL) and the resulting biphasic mixture was stirred for 1 h. The organic layer was separated and the aqueous layer was extracted with additional DCM (3 x 25 mL). The organics were combined, dried over MgSO4, filtered and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 -> 20% MeOH / DCM) to give 41 mg (12% yield) of amine SL-1279.1 H NMR (400 MHz, DMSO-d6) δ 7.30 - 6.92 (m, 2H), 6.73 (t, J = 5.5 Hz, 1H), 3.66 (s, 2H), 3.00 (q, J = 6.3 Hz, 2H), 2.47 (m, 2H, overlap with DMSO-d5), 1.36 (s, 9H);MS (SI) C 14 H 20 FN8O2[M+H]+ calculated 351.17, found 351.18.

[0203] [ka] To an ice-cold solution of N-Boc-ethylenediamine (828 mg, 5.17 mmol) in MeCN (200 mL) was added K2CO3 (953 mg, 6.90 mmol) followed by 1-(bromomethyl)-2,3,4,5,6-pentafluorobenzene (900 mg, 3.45 mmol). The resulting solution was allowed to warm to 22 °C and stirred for 25 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was filtered and the volatiles removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 → 10% MeOH / DCM) to give 1.22 g (quantitative) of the amine SL-1266 as a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 6.69 (t, J = 5.7 Hz, 1H), 3.79 (s, 2H), 2.98 (q, J = 6.2 Hz, 2H), 2.47 (m, 2H, overlap with DMSO-d5), 2.17 (s, 1H), 1.36 (s, 9H); 19 F NMR (376 MHz, DMSO-d6) δ -143.92 (dd, J = 24.3, 8.3 Hz), -156.98 (t, J = 22.1 Hz), -161.43 - -164.35 (m).

[0204] To a solution of SL-1266 (660 mg, 1.94 mmol) in DMF (8 mL) was added NaN3 (378 mg, 5.82 mmol). The resulting suspension was heated at 80 °C for 71 h. The reaction mixture was cooled to room temperature, filtered through Celite, and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 -> 10% MeOH / DCM) to give 298 mg (42% yield) of the azide SL-1271 as a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 6.72 (s, 1H), 3.79 (s, 2H), 2.99 (q, J = 6.2 Hz, 2H), 1.37 (s, 9H); 19 F NMR (376 MHz, DMSO-d6) δ -144.70 (s), -152.86 (dd, J = 22.9, 9.8 Hz);HRMS (SI) C 14 H 18 F4N5O2[M+H]+ calculated 364.1391, found 364.1386.

[0205] [ka] To a solution of 2,4-dinitrobenzaldehyde (240 mg, 1.22 mmol) in DMF (25 mL) was added NaN3 (398 mg, 6.12 mmol). The resulting suspension was heated at 22 °C for 17 h. The reaction mixture was poured into brine (25 mL) and extracted with DCM (3 x 50 mL). The organics were combined, dried over MgSO4, filtered, and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 -> 10% EtOAc / heptane) to give 164 mg (71% yield) of the azide SL-1069 as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.24 (d, J = 0.8 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 6.91 (ddd, J = 8.4, 2.0, 0.8 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H).

[0206] To a solution of N-Boc-ethylenediamine (154 mg, 959 μmol) and SL-1069 (164 mg, 872 μmol) in DCM (50 mL) was added NaBH(OAc)3 (462 mg, 2.18 mmol). The resulting suspension was stirred at 22 °C for 2 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was quenched with an aqueous solution of K2CO3 (10%, 50 mL) and the resulting biphasic mixture was stirred for 1 h. The organic layer was separated and the aqueous layer was extracted with additional DCM (2 x 75 mL). The organics were combined, dried over MgSO4, filtered and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 -> 10% MeOH / DCM) to give 206 mg (71% yield) of the amine SL-1070. 1 H NMR (400 MHz, DMSO-d6) δ 7.42 (d, J = 8.0 Hz, 1H), 7.05 - 6.87 (m, 2H), 6.73 (t, J = 5.8 Hz, 1H), 3.57 (s, 2H), 3.00 (q, J = 6.3 Hz, 2H), 2.48 (m, 2H, overlap with DMSO-d5), 1.37 (s, 9H);MS (SI) C 14 H 21 N8O6[M+H]+ calculated 333.18, found 333.18.

[0207] [ka] To a solution of N-Boc-ethylenediamine (390 mg, 2.44 mmol) in DMF (45 mL) was added K2CO3 (449 mg, 3.25 mmol) followed by 1,3-dibromo-5-(bromomethyl)benzene (534 mg, 1.62 mmol). The resulting solution was stirred at 22 °C for 20 h, at which point HPLC analysis indicated complete consumption of the starting material. The reaction mixture was filtered and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 → 10% MeOH / DCM) to give 568 mg (86% yield) of amine SL-1040 as a clear oil. 1H NMR (400 MHz, DMSO-d6) δ 7.67 (t, J = 1.8 Hz, 1H), 7.55 (d, J = 1.8 Hz, 2H), 6.74 (t, J = 5.5 Hz, 1H), 3.67 (s, 2H), 3.01 (q, J = 6.3 Hz, 2H), 2.47 (m, 2H, overlap with DMSO-d5), 2.30 (s, 1H), 1.37 (s, 9H);MS (SI) C 14 H 21 Br2N2O2[M+H]+ calculated 407.00, found 406.92.

[0208] A 25 mL round bottom flask equipped with a stir bar and a condenser with a rubber septum was charged with SL-1040 (260 mg, 637 μmol), sodium ascorbate (12.6 mg, 63.7 μmol), NaN3 (166 mg, 2.55 mmol), and CuI (49 mg, 255 μmol). The flask was evacuated and backfilled with Ar (three times) and placed under an Ar atmosphere. Through the septum, degassed EtOH (6 mL) and degassed water (3 mL) were added, followed by a degassed solution of 1,2-dimethylethylenediamine (16.9 mg, 191 μmol) in EtOH (1.5 mL). The resulting suspension was heated to 90 °C for 2 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was poured into 10% aqueous Na2CO3 (20 mL) and extracted three times with 50 mL of DCM. The organics were combined, dried over MgSO4, filtered, and the solvent removed in vacuo. Purification by silica gel chromatography (0→40% MeOH / DCM) afforded 134 mg (63% yield) of diazide SL-1083 as a clear oil. 1 H NMR (400 MHz, DMSO-d6) δ 6.93 (d, J = 2.1 Hz, 2H), 6.80 - 6.69 (m, 1H), 6.66 (t, J = 2.1 Hz, 1H), 3.67 (s, 2H), 3.00 (q, J = 6.3 Hz, 2H), 2.47 (m, 2H, overlap with DMSO-d5), 1.36 (s, 9H);MS (SI) C14 H 21 N8O2[M+H]+ calculated 333.18, found 333.18.

[0209] [ka] To a solution of 3-fluoro-4-nitrobenzaldehyde (400 mg, 2.37 mmol) in DMF (5 mL) was added NaN3 (461 mg, 7.10 mmol). The resulting suspension was heated at 35° C. for 22 h. The reaction mixture was poured into brine (5 mL) and extracted with DCM (3×10 mL). The organics were combined, dried over MgSO4, filtered, and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0→20% EtOAc / heptane) to give 115 mg (29% yield) of diazide SL-1062 as a white solid. 1 H NMR (400 MHz, CDCl3) δ 9.93 (s, 1H), 7.66 (dd, J = 8.1, 1.7 Hz, 1H), 7.64 (d, J = 1.6 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H, overlapped with CDCl3); 19 F NMR (376 MHz, CDCl3) δ-no signal.

[0210] To a solution of N-Boc-ethylenediamine (108 mg, 672 μmol) and SL-1062 (115 mg, 611 μmol) in DCM (50 mL) was added NaBH(OAc)3 (324 mg, 1.53 mmol). The resulting suspension was stirred at 2° C. for 3 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was quenched with an aqueous solution of K2CO3 (10%, 50 mL) and the resulting biphasic mixture was stirred for 1 h. The organic layer was separated and the aqueous layer was extracted with additional DCM (2×50 mL). The organics were combined, dried over MgSO4, filtered, and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0→10% MeOH / DCM) to give 134 mg (66% yield) of the amine SL-1063. 1H NMR (400 MHz, DMSO-d6) δ 7.24 (d, J = 1.7 Hz, 1H), 7.21 (d, J = 8.1 Hz, 1H), 7.17 (dd, J = 8.2, 1.7 Hz, 1H), 6.72 (t, J = 5.7 Hz, 1H), 3.66 (s, 2H), 3.00 (q, J = 6.3 Hz, 2H), 2.47 (m, 2H, overlap with DMSO-d6), 2.25 (s, 1H), 1.36 (s, 9H);MS (SI) C 14 H 21 N8O6[M+H]+ Calculated 333.18, Found 333.18

[0211] [ka] To a stirred solution of methyl 3,5-dibromo-4-methylbenzoate (643 mg, 2.09 mmol) in THF (15 mL) under argon was added LiBH4 solution (4 M in THF, 6.26 mmol). The resulting solution was allowed to react at 22 °C for 18 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was quenched with water (10 mL) and the resulting biphasic mixture was stirred for 3 h and extracted with EtOAc (3 x 75 mL). The organics were combined, dried over MgSO4, filtered, and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 -> 30% EtOAc / heptane) to afford 539 mg (92% yield) of the alcohol SL-1598 as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.52 (s, 2H), 4.63 (d, J = 0.9 Hz, 2H), 2.56 (s, 3H); MS (SI) C8H8Br2OLi [M+Li]+ Calculated value 284.91, Actual value 284.36.

[0212] To a stirred solution of SL-1598 (447 mg, 1.60 mmol) in DCM (25 mL) was added DMP (Dess-Martin periodinane) (711 mg, 1.68 mmol). The resulting solution was stirred at 22° C. for 3 h, at which point HPLC analysis indicated complete consumption of the starting material. The reaction mixture was loaded onto a silica gel column and purified by silica gel chromatography (0→10% EtOAc / heptane) to give 391 mg (88% yield) of the aldehyde as a white solid of SL-1601. 1 H NMR (400 MHz, CDCl3) δ 9.87 (s, 1H), 8.00 (s, 2H), 2.65 (s, 3H); 13 C NMR (100 MHz, CDCl3) δ 189.0, 144.4, 136.1, 132.6, 126.1, 24.3.

[0213] A 25 mL round bottom flask equipped with a stir bar and a condenser with a rubber septum was charged with SL-1601 (179 mg, 644 μmol), sodium ascorbate (12.8 mg, 64.4 μmol), NaN3 (168 mg, 2.58 mmol), and CuI (24.5 mg, 129 μmol). The flask was evacuated and backfilled with Ar (3 times) and placed under Ar atmosphere. Degassed water (1.2 L) was added through the septum, followed by a degassed solution of 1,2-dimethylethylenediamine (40 mg, 458 μmol) in EtOH (3 mL). The resulting suspension was heated at 90 °C for 7 h. The reaction mixture was adsorbed onto Celite and purified by silica gel chromatography (0 → 20% EtOAc / heptane) to give 37 mg (28% yield) of the diazide SL-1400 as a white solid. 1 H NMR (400 MHz, CDCl3) δ 9.95 (s, 1H), 7.42 (s, 2H), 2.15 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ 190.1, 141.4, 135.6, 128.1, 114.6, 12.1;HRMS (SI) C8H7N6O [M+H]+ calcd 203.0676, found 203.0673.

[0214] To a solution of N-Boc-ethylenediamine (55.8 mg, 348 μmol) and SL-1400 (64 mg, 317 μmol) in DCM (10 mL) was added NaBH(OAc)3 (168 mg, 791 μmol). The resulting suspension was stirred at 22 °C for 20 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was quenched with an aqueous solution of K2CO3 (10%, 10 mL) and the resulting biphasic mixture was stirred for 1 h. The organic layer was separated and the aqueous layer was extracted with additional DCM (2 x 25 mL). The organics were combined, dried over MgSO4, filtered and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 -> 10% MeOH / DCM) to give 96 mg (88% yield) of the amine SL-1605. 1 H NMR (400 MHz, DMSO-d6) δ 7.05 (s, 2H), 6.82 - 6.58 (m, 1H), 3.69 (s, 2H), 3.01 (q, J = 6.3 Hz, 2H), 2.5 (overlapped with 2H DMSO-d5), 1.99 (s, 3H), 1.36 (s, 9H);MS (SI) C 15 H 23 N8O2[M+H]+ calculated 347.19, found 347.21.

[0215] [ka] To a stirred solution of methyl 3,4-dibromo-5-methylbenzoate (264 mg, 857 μmol) in THF (8 mL) under argon was added LiBH4 solution (643 μL, 4 M in THF, 2.57 mmol). The resulting solution was allowed to react at 22 °C for 17 h. The reaction mixture was quenched with water (2 mL) and the resulting biphasic mixture was stirred for 1 h and extracted with EtOAc (3 × 15 mL). The organics were combined, dried over MgSO4, filtered, and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 → 30% EtOAc / heptane) to give 192 mg (80% yield) of the alcohol SL-1596 as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.49 (d, J = 2.0 Hz, 1H), 7.19 - 7.16 (m, 1H), 4.62 (s, 2H), 2.48 (s, 3H); 13 C NMR (100 MHz, CDCl3) δ 141.0, 140.8, 129.4, 127.7, 125.9, 125.6, 63.9, 25.0;MS (SI) C8H7Br2[M-OH]+ calcd 260.8909, found 260.8913.

[0216] To a stirred solution of SL-1596 (122 mg, 436 μmol) in DCM (7 mL) was added DMP (Dess-Martin periodinane) (194 mg, 456 μmol). The resulting solution was stirred at 22° C. for 1.5 h, at which point HPLC analysis indicated complete consumption of the starting material. The reaction mixture was loaded onto a silica gel column and purified by silica gel chromatography (0→10% EtOAc / heptane) to afford 99 mg (82% yield) of the aldehyde SL-1601 as a white solid. 1 H NMR (400 MHz, CDCl3) δ 9.91 (s, 1H), 7.95 (dd, J = 1.9, 0.7 Hz, 1H), 7.66 (dd, J = 1.9, 0.8 Hz, 1H), 2.57 (t, J = 0.7 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 190.1, 141.9, 135.7, 134.3, 132.0, 129.4, 126.7, 25.1.

[0217] A 25 mL round bottom flask equipped with a stir bar and a condenser with a rubber septum was charged with SL-1602 (141 mg, 507 μmol), sodium ascorbate (10.5 mg, 50.7 μmol), NaN3 (132 mg, 2.03 mmol), and CuI (19.3 mg, 102 μmol). The flask was evacuated and backfilled with Ar (3 times) and placed under Ar atmosphere. Degassed water (1.2 L) was added through the septum, followed by a degassed solution of 1,2-dimethylethylenediamine (13.4 mg, 152 μmol) in EtOH (3 mL). The resulting suspension was heated to 90 °C for 3 h. The reaction mixture was adsorbed onto Celite and purified by silica gel chromatography (0 → 10% EtOAc / heptane) to give 10 mg (10% yield) of the diazide SL-1404 as a white solid. 1 H NMR (400 MHz, CDCl3) δ 9.88 (s, 1H), 7.52 (d, J = 1.8 Hz, 1H), 7.43 (d, J = 1.8 Hz, 1H), 2.30 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 190.1, 134.7, 134.2, 133.2, 133.1, 129.1, 116.8, 18.1.

[0218] To a solution of N-Boc-ethylenediamine (5.1 mg, 32 μmol) and SL-1404 (64 mg, 29 μmol) in DCM (5 mL) was added NaBH(OAc)3 (16 mg, 73 μmol). The resulting suspension was stirred at 22 °C for 7 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was quenched with an aqueous solution of K2CO3 (10%, 10 mL) and the resulting biphasic mixture was stirred for 1 h. The organic layer was separated and the aqueous layer was extracted with additional DCM (2 x 10 mL). The organics were combined, dried over MgSO4, filtered and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0 -> 10% MeOH / DCM) to give 3.5 mg (35% yield) of the amine SL-1417. 1 H NMR (400 MHz, MeOD) δ 7.12 (d, J = 1.8 Hz, 1H), 6.98 (d, J = 1.8 Hz, 1H), 3.71 (s, 2H), 3.19 (t, J = 6.3 Hz, 2H), 2.66 (t, J = 6.3 Hz, 2H), 2.22 (s, 3H), 1.43 (s, 9H); 13 C NMR (100MHz, MeOD) δ 158.6, 138.9, 135.0, 133.9, 128.7, 128.5, 118.1, 80.1, 53.2, 40.7, 30.8, 28.7, 18.0. HRMS (SI) C 15 H 23 N8O2[M+H]+ calculated 347.1938, found 347.1942.

[0219] Synthesis of fluorescent SAHA-PRG-NCT conjugate: [ka] [ka]

[0220] Synthetic scheme for the synthesis of SAHA-PRG-NCT probe Probes: SL-1231, SL-1011, SL-1001, SL-1090, SL-1187, SL-1087, SL-1136, SL-1233, SL-1089, SL-1080 (SAHA-PRG-NCT probe numbers: 1, 4, 5, 6, 7, 8, 11, 12, 16, 18) were synthesized according to the general scheme SAHA-I.

[0221] Probes: SL-1237, SL-1270, SL-1056, SL-1306, SL-1295, SL-1294, SL-1277, SL-1104, SL-1414, SL-1421 (SAHA-PRG-NCT probe numbers: 2, 3, 9, 10, 13, 14, 15, 17, 19, 20) were synthesized according to the general scheme SAHA-II.

[0222] [ka]

[0223] [ka]

[0224] General Scheme: Exemplary Synthesis Procedures for SAHA-I Synthesis of probe SL-1231 (SAHA-PRG-NCT probe no. 1): [ka] To a solution of SL-1203 (8.0 mg, 22 μmol) in DCM (4 mL) was added TiPS (50 μL) followed by TFA (1 mL). The resulting solution was stirred at 22° C. for 1 h, at which point HPLC analysis indicated complete consumption of the starting material. The volatiles were removed under reduced pressure and the crude residue was dissolved in 5 mL of MeOH and the volatiles were removed in vacuo. The crude residue was dried under high vacuum and used in the next step without further purification. MS (SI) C 16 H 18 N2ONa [M+H]+ calculated 277.13, found 277.03.

[0225] [ka] To a solution of SL-1223 (7.8 mg, 21 μmol) and DIPEA (13 μL, 70 μmol) in DMF (6 mL) was added NCT-TFP (5 mg, 7 μmol, prepared according to ACS Chem. Biol., 2016, 11, 2608-2617). The resulting solution was stirred at 22 °C for 1 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to give 4.5 mg (70% yield) of the amine SL-1224 as a deep blue film. HPLC: 97% purity at 254 nm; MS (SI) C 51 H 51 N4O5[M+]+ calculated 799.38, found 799.45.

[0226] [ka] To a solution of SL_1005 (3 mg, 5 μmol), HATU (2.3 mg, 6.2 μmol), and N-methylmorpholine (6 μL, 50 μmol) in DMF (5 mL) was added SL-1224 (4.5 mg, 4.9 μmol). The resulting solution was stirred at 22 °C for 18 h, at which point LCMS analysis indicated complete consumption of starting material. The reaction mixture was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to give 4.0 mg (61% yield) of the conjugate SL-1228 as a deep blue film. HPLC: 99% purity at 254 nm; MS (SI) C 85 H 83 N6O9[M+]+ calculated 1331.62, found 1331.51.

[0227] [ka] SL-1228 (4.0 mg, 3 μmol) was treated with 4 mL of the cleavage cocktail DCM:TFA:TiPS (95:5:0.1). The resulting solution was stirred at 22 °C for 30 min, at which point HPLC analysis indicated complete consumption of starting material. Volatiles were removed under reduced pressure and the crude residue was dissolved in 5 mL of MeOH and purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to give 2.2 mg (67% yield) of the conjugate SL-1231 as a deep blue film. HPLC: 99% purity at 254 nm; HRMS (SI) C 66 H 69 N6O9[M+]+ calculated 1089.5121, found 1089.5119.

[0228] General Scheme: Exemplary Synthesis Procedures for SAHA-II Synthesis of probe SL-1237 (SAHA-PRG-NCT probe no. 2): [ka] To a solution of SL_1005 (20 mg, 37 μmol), HATU (16 mg, 42 μmol), and N-methylmorpholine (26 μL, 0.24 mmol) in DMF (6 mL) was added SL-1201 (12 mg, 34 μmol). The resulting solution was stirred at 22 °C for 2 h, at which point LCMS analysis indicated complete consumption of the starting material. The reaction mixture was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to give 14 mg (47% yield) of the conjugate SL-1234 as a clear film. HPLC: 99% purity at 254 nm; MS (SI) C 50 H 54 F3N6O6[M+]+ calculated 891.41, found 891.46.

[0229] [ka] To a solution of SL-1234 (14 mg, 16 μmol) in DCM (9 mL) was added TiPS (100 μL) followed by TFA (1 mL). The resulting solution was stirred at 22° C. for 2 h, at which point HPLC analysis indicated complete consumption of starting material. The volatiles were removed under reduced pressure and the crude residue was dissolved in 5 mL of MeOH and the volatiles were removed in vacuo. The crude residue was dried under high vacuum and used in the next step without further purification. MS (SI) C 26 H 32 F3N6O4[M+H]+ calculated 549.24, found 549.17.

[0230] [ka] To a solution of SL-1235 (9 mg, 14 μmol) and NMM (8 μL, 70 μmol) in DMF (7 mL) was added NCT-TFP (5 mg, 7 μmol, prepared according to ACS Chem. Biol., 2016, 11, 2608-2617). The resulting solution was stirred at 22 °C for 1 h, at which point HPLC analysis indicated complete consumption of the starting material. The reaction mixture was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to give 4.8 mg (62% yield) of the conjugate SL-1237 as a deep blue film. HPLC: 99% purity at 254 nm; HRMS (SI) C 61 H 64 F3N8O8[M+]+ calculated 1093.4794, found 1093.4778.

[0231] Characterization data for the SAHA-PRG-NCT probe: [ka] HPLC: 99% purity at 254 nm; 1H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.37 (d, J = 8.2 Hz, 1H), 8.20 - 8.13 (m, 1H), 8.12-8.10 (m, 1H), 7.74 - 7.64 (m, 1H), 7.56 (m, 1H), 7.39 (m, 1H), 7.21 (m, 2H), 6.67 (m, 2H), 3.79 - 3.50 (m, 12H), 3.07 (s, 3H), 2.79 (m, 4H), 2.35 (m, 2H), 2.08 (m, 5H), 1.99 - 1.76 (m, 7H), 1.75 - 1.57 (m, 5H), 1.49 - 1.25 (m, 7H), 1.09 (s, 1H), 1.01 (t, J = 7.3 Hz, 1H), 0.76 (s, 1H);HRMS (SI) C 56 H 65 N8O8[M+]+ calculated 977.4920, found 977.4910.

[0232] [ka] HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.37 - 6.60 (multiplet 13H), 4.80 (m, 1H), 4.59 (m, 1H), 3.79 - 3.51 (multiplet, 12H), 3.11 - 3.00 (m, 4H), 2.88 - 2.68 (m, 4H), 2.44 - 2.21 (m, 3H), 2.17 - 2.02 (m, 5H), 2.00 - 1.88 (m, 4H), 1.84 - 1.73 (m, 4H), 1.72 - 1.58 (m, 4H), 1.47 - 1.33 (m, 4H);HRMS (SI) C 59 H 64 N9O8[M+]+ calculated 1026.4872, found 1026.4898.

[0233] [ka] HPLC: 99% purity at 254nm; HRMS (SI) C 59 H 64 N9O8[M+]+ calculated 1026.4872, found 1026.4879.

[0234] [ka] HPLC: 99% purity at 254nm; HRMS (SI) C 59 H 64 N9O8[M+]+ calculated 1026.4872, found 1026.4874.

[0235] [ka] HPLC: 96% purity at 254nm; MS (SI) C 60 H 66 N9O8[M+]+ calculated 1040.50, found 1040.36.

[0236] [ka] HPLC: 99% purity at 254nm; HRMS (SI) C 60 H 66 N9O8[M+]+ calculated 1040.5029, found 1040.5048.

[0237] [ka] HPLC: 99% purity at 254nm; HRMS (SI) C 60 H 66 N9O8[M+]+ calculated 1040.5029, found 1040.5027.

[0238] [ka] HPLC: 99% purity at 254 nm;1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.43 - 6.61 (multiplet 12H), 4.77 (m, 1H), 4.58 (m, 1H), 3.79 - 3.49 (multiplet, 12H), 3.11 - 3.00 (m, 4H), 2.87 - 2.68 (m, 4H), 2.40 - 2.21 (m, 3H), 2.17 - 2.02 (m, 5H), 2.00 - 1.88 (m, 4H), 1.84 - 1.73 (m, 4H), 1.72 - 1.58 (m, 4H), 1.47 - 1.33 (m, 4H);MS (SI) C 59 H 63 FN9O8[M+]+ calculated 1044.48, found 1044.89.

[0239] [ka] HPLC: 99% purity at 254nm; HRMS (SI) C 60 H 63 F3N9O8[M+]+ calculated 1094.4746, found 1094.4740.

[0240] [ka] HPLC: 99% purity at 254nm; HRMS (SI) C 61 H 68 N9O8[M+]+ calculated 1054.5185, found 1054.5186.

[0241] [ka] HPLC: 96% purity at 254 nm; 1H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.44 - 6.62 (multiplet 11H), 4.75 (m, 1H), 4.59 (m, 1H), 3.82 - 3.52 (multiplet, 12H), 3.12 - 2.99 (m, 4H), 2.91 - 2.65 (m, 4H), 2.44 - 2.23 (m, 3H), 2.17 - 2.02 (m, 5H), 2.00 - 1.88 (m, 4H), 1.84 - 1.73 (m, 4H), 1.72 - 1.54 (m, 4H), 1.47 - 1.30 (m, 4H);HRMS (SI) C 59 H 62 F2N9O8[M+]+ calculated 1062.4684, found 1062.4715.

[0242] [ka] HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.42 - 6.61 (multiplet 11H), 4.77 (m, 1H), 4.57 (m, 1H), 3.82 - 3.52 (multiplet, 12H), 3.10 - 2.99 (m, 4H), 2.88 - 2.65 (m, 4H), 2.42 - 2.23 (m, 3H), 2.14 - 2.02 (m, 5H), 2.00 - 1.88 (m, 4H), 1.84 - 1.73 (m, 4H), 1.72 - 1.54 (m, 4H), 1.47 - 1.30 (m, 4H);HRMS (SI) C 59 H 62 FN 12 O8[M+]+ calculated 1085.4792, found 1085.4817.

[0243] [ka] HPLC: 96% purity at 254 nm; 1H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.40 - 6.57 (multiplet 9H), benzyl CH2 is under HOD peak, 3.82 - 3.51 (multiplet, 12H), 3.12 - 2.97 (m, 4H), 2.91 - 2.65 (m, 4H), 2.44 - 2.23 (m, 3H), 2.17 - 2.02 (m, 5H), 2.00 - 1.88 (m, 4H), 1.84 - 1.73 (m, 4H), 1.72 - 1.54 (m, 4H), 1.47 - 1.30 (m, 4H);HRMS (SI) C 59 H 60 F4N9O8[M+]+ calculated 1098.4495, found 1098.4505.

[0244] [ka] HPLC: 99% purity at 254nm; HRMS (SI) C 59 H 63 N 12 O8[M+]+ calculated 1067.4886, found 1067.4889.

[0245] [ka] HPLC: 99% purity at 254nm; HRMS (SI) C 59 H 63 N 12 O8[M+]+ calculated 1067.4886, found 1067.4878.

[0246] [ka] HPLC: 97% purity at 254nm; HRMS (SI) C 59 H 63 N 12 O8[M+]+ calculated 1067.4886, found 1067.4899.

[0247] [ka] HPLC: 99% purity at 254 nm; 1H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.37 (d, J = 8.2 Hz, 1H), 8.11 (m, 1H), 7.74 (m, 1H), 7.49 (m, 2H), 7.23 (d, J = 8.3 Hz, 2H), 7.02 (s, 1H), 6.67 (m, 3H), 4.62 (s, 1H), 3.83 - 3.62 (m, 6H), 3.56 (m, 6H), 3.03 (m, 4H), 2.82 (m, 4H), 2.42 - 2.24 (m, 3H), 2.14 - 2.05 (m, 5H), 2.03 - 1.96 (m, 3H), 1.96 - 1.88 (m, 4H), 1.85 - 1.74 (m, 4H), 1.75 - 1.57 (m, 4H), 1.46 - 1.33 (m, 4H);HRMS (SI) C 60 H 65 N 12 O8[M+]+ calculated 1081.5043, found 1081.5041.

[0248] [ka] HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.36 - 6.64 (multiplet 11H), 4.79 (m, 1H), 4.56 (m, 1H), 3.82 - 3.50 (multiplet, 12H), 3.10 - 2.97 (m, 4H), 2.88 - 2.65 (m, 4H), 2.42 - 2.26 (m, 2H), 2.26 - 2.14 (m, 3H), 2.14 - 2.02 (m, 6H), 2.00 - 1.88 (m, 4H), 1.84 - 1.73 (m, 4H), 1.72 - 1.54 (m, 4H), 1.47 - 1.30 (m, 4H);HRMS (SI) C60 H 65 N 12 O8[M+]+ calculated 1081.5043, found 1081.5030.

[0249] Synthesis of fluorescent dasatinib-PRG-NCT conjugates: [ka]

[0250] [ka]

[0251] Exemplary synthesis procedure for Dasatinib-PRG-NCT probe Synthesis of probe SL-1269 (dasatinib-PRG-NCT probe no. 3): [ka] To a solution of SL-1338 (9.0 mg, 18 μmol), HATU (8.5 mg, 22 μmol), and DIPEA (16 μL, 0.09 mmol) in DMF (7 mL) was added SL-1358 (4.3 mg, 18 μmol). The resulting solution was stirred at 22° C. for 19 h, at which point LCMS analysis indicated complete consumption of starting material. The reaction mixture was purified by preparative RP HPLC (5→95% MeCN / H2O buffered with 0.5% TFA) to give 11 mg (85% yield) of conjugate SL-1264 as a clear film. HPLC: 99% purity at 254 nm; 1H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.18 (s, 1H), 7.36 (dd, J = 7.3, 2.2 Hz, 1H), 7.31 - 7.14 (m, 2H), 6.17 (s, 1H), 4.35 (s, 1.4H), 4.27 (s, 0.6 H), 4.21 - 3.63 (m, 3H), 3.60 - 3.40 (m, 5H), 3.29 - 3.20 (m, 3H), 2.52 (s, 3H), 2.33 (s, 3H), 1.73 - 1.65 (m, 0.6 H), 1.60 (t, J = 7.4 Hz, 1.4 H), 1.47 - 1.40 (m, 9H), 1.07 (m, 3H);HRMS (SI) C 33 H 45 ClN 11 O4S [M+H]+ calculated 726.3060, found 726.3082.

[0252] [ka] To a solution of SL-1264 (11 mg, 15 μmol) in DCM (8 mL) was added TiPS (100 μL) followed by TFA (2 mL). The resulting solution was stirred at 22° C. for 1.5 h, at which point HPLC analysis indicated complete consumption of starting material. The volatiles were removed under reduced pressure and the crude residue was dissolved in 5 mL of MeOH and the volatiles were removed in vacuo. The crude residue was dried under high vacuum and used in the next step without further purification. HRMS (SI) C 28 H 37 ClN 11 O2S [M+H]+ calculated 626.2535, found 626.2531.

[0253] [ka] To a solution of SL-1268 (11 mg, 15 μmol) and DIPEA (6.0 μL, 34 μmol) in DMF (6 mL) was added NCT-TFP (3.5 mg, 5 μmol, prepared according to ACS Chem. Biol., 2016, 11, 2608-2617). The resulting solution was stirred at 22 °C for 1 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to give 1.3 mg (23% yield) of the conjugate SL-1269 as a deep blue film. HPLC: 97% purity at 254 nm; 1H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.42 - 8.36 (m, 1H), 8.25 - 8.07 (m, 2H), 7.90 - 7.72 (m, 1H), 7.44 - 7.31 (m, 1H), 7.29 - 7.16 (m, 2H), 6.66 (s, 1H), 6.64 (s, 1H), 6.10 (s, 0.6 H), 6.00 (s, 0.4 H), 4.48 (s, 1H), 4.26 (s, 1H), 4.16 - 3.80 (br. s. 2H) 3.80 - 3.62 (m, 5H), 3.62 - 3.51 (m, 5H), 3.51 - 3.42 (m, 4H), 3.33 (m, 2H, overlap with CD2HOD), 3.27 (m, 2H, overlap with CD2HOD), 3.10 - 2.98 (m, 4H), 2.86 - 2.62 (m, 4H), 2.51 - 2.43 (m, 3H), 2.33 (s, 3H), 2.05 (m, 4H), 1.93 (m, 4H), 1.88 - 1.75 (m, 4H), 1.75 - 1.65 (m, 1H), 1.61 (m, 1H), 1.06 - 1.03 (m, 3H);HRMS (SI) C 63 H 69 ClN 13 O6S [M+]+ calculated 1170.4898, found 1170.4881.

[0254] Characterization data of the Dasatinib-PRG-NCT probe: [ka] HPLC: 97% purity at 254nm; HRMS (SI) C 73 H 73 ClN 11 O7S [M+]+ calculated 1282.5098, found 1282.5079.

[0255] [ka] HPLC: 95% purity at 254nm; HRMS (SI) C 68 H 68 ClF3N 13 O6S [M+]+ calculated 1286.4771, found 1286.4779.

[0256] [ka] HPLC: 99% purity at 254nm; HRMS (SI) C 68 H 68 ClN 14 O6S [M+]+ calculated 1219.4850, found 1219.4853.

[0257] [ka] HPLC: 98% purity at 254nm; HRMS (SI) C 68 H 68 ClN 14 O6S [M+]+ calculated 1219.4850, found 1219.4848.

[0258] [ka] HPLC: 97% purity at 254nm; HRMS (SI) C 68 H 68 ClN 14O6S [M+]+ calculated 1219.4850, found 1219.4862.

[0259] [ka] HPLC: 98% purity at 254nm; HRMS (SI) C 67 H 70 ClN 14 O6S [M+]+ calculated 1233.5007, found 1233.4992.

[0260] [ka] HPLC: 95% purity at 254nm; MS (SI) C 67 H 70 ClN 14 O6S [M+]+ calculated 1233.50, found 1234.2.

[0261] [ka] HPLC: 99% purity at 254nm; HRMS (SI) C 67 H 70 ClN 14 O6S [M+]+ calculated 1233.5007, found 1233.4998.

[0262] [ka] HPLC: 99% purity at 254 nm; 11H NMR (400 MHz, methanol-d4, reported for a mixture of rotamers) δ 8.38 (m, 1H), 8.18 (s, 1H), 8.11 (m, 1H), 7.82 (d, J = 1.8 Hz, 0.4 H), 7.76 (d, J = 1.8 Hz, 0.5 H), 7.37 (m, 1H), 7.33 - 7.21 (m, 2H), 7.21 - 7.07 (m, 3H), 6.69 (s, 1H), 6.64 (s, 1H), 6.11 (s, 0.4 H), 5.99 (s, 0.3 H), 4.68 (s, 1H), 4.60 (s, 1H), 4.57 (s, 1H), 4.27 (s, 1H), 4.19 - 3.77 (m, 3H), 3.78 - 3.62 (m, 5H), 3.62 - 3.42 (m, 8H), 3.08 - 2.98 (m, 4H), 2.84 - 2.71 (m, 4H), 2.47 (m 3H), 2.33 (s, 3H), 2.11 - 2.03 (m, 4H), 1.97 - 1.90 (m, 4H), 1.86 - 1.76 (m, 4H); MS (SI) C 66 H 67 ClFN 14 O6S [M+]+ calculated 1237.48, found 1238.07.

[0263]

Chem.

[0264]

Chem.

[0265] [ka] HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.38 (m, 1H), 8.18 (s, 1H), 8.11 (m, 1H), 7.80 (m, 1H), 7.37 (m, 1H), 7.31 - 7.18 (m, 2H), 7.03 (m, 2H), 6.66 (m, 2H), 6.05 (m, 1H), 4.67 (s, 1H), 4.58 (s, 2H), 4.26 (s, 1H), 4.16 - 3.80 (m, 3H), 3.69 (m, 6H), 3.64 - 3.41 (m, 10H), 3.11 - 2.98 (m, 4H), 2.79 (m, 4H), 2.47 (m, 3H), 2.33 (s, 3H), 2.06 (m, 4H), 1.93 (m, 4H), 1.81 (m, 4H);HRMS (SI) C 66 H 66 ClF2N 14 O6S [M+]+ calculated 1255.4662, found 1255.4682.

[0266] [ka] HPLC: 99% purity at 254 nm; 11H NMR (400 MHz, methanol-d4, reported for a mixture of rotamers) δ 8.27 (m, 1H), 8.08 (s, 1H), 7.98 (m, 1H), 7.73 - 7.65 (m, 1H), 7.26 (m, 1H), 7.22 - 7.03 (m, 2H), 7.03 - 6.75 (m, 2H), 6.58 (m, 2H), 6.05 - 5.93 (m, 1H), 4.57 (s, 1H), 4.47 (m, 2H), 4.21 (s, 1H), 3.88 (s, 3H), 3.70 - 3.53 (m, 5H), 3.53 - 3.28 (m, 9H), 3.02 - 2.91 (m, 4H), 2.75 - 2.57 (m, 4H), 2.37 (m, 3H), 2.23 (s, 3H), 2.05 - 1.89 (m, 4H), 1.84 (m, 4H), 1.72 (m, 4H); HRMS (SI) C 66 H 66 ClFN 17 O6S [M+]+ calculated 1278.4770, found 1278.4789.

[0267]

Chemical Structure

[0268] [ka] HPLC: 95% purity at 254nm; HRMS (SI) C 66 H 67 ClN 15 O6S [M+]+ calculated 1260.4864, found 1260.4837.

[0269] [ka] HPLC: 97% purity at 254nm; MS (SI) C 66 H 67 ClN 15 O6S [M+]+ calculated 1260.49 found 1260.66.

[0270] [ka] HPLC: Purity 96% at 254 nm; HRMS (SI) C 66 H 67 ClN 15 O6S [M+]+ Calculated value 1260.4864, Measured value 1260.4861.

[0271]

Chem.

[0272]

Chem.

[0273] Synthesis of fluorescent propranolol-PRG-NCT conjugate: [ka]

[0274] [ka]

[0275] Exemplary synthesis procedure for the propranolol-PRG-NCT probe Synthesis of probe SL-1475 (Propranolol-PRG-NCT probe no. 3): [ka] To a solution of SL-1517 (14 mg, 35 μmol), HATU (17 mg, 44 μmol), and DIPEA (31 μL, 170 μmol) in DMF (5 mL) was added SL-1358 (8.5 mg, 35 μmol). The resulting solution was stirred at 22° C. for 19 h, at which point LCMS analysis indicated complete consumption of starting material. The reaction mixture was purified by preparative RP HPLC (5→95% MeCN / H2O buffered with 0.5% TFA) to give 20 mg (93% yield) of conjugate SL-1469 as a clear film. HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.33 (s, 1H), 7.85 - 7.72 (m, 1H), 7.55 - 7.41 (m, 3H), 7.40 - 7.30 (m, 1H), 6.90 (m, 1H), 4.31 (s, 1H), 4.23 - 4.04 (m, 2H), 3.71 (m, 1H), 3.68 - 3.58 (m, 2H), 3.51 - 3.33 (m, 4H), 3.17 (m, 2H), 2.71 (m, 2H), 1.59 (m, 1H), 1.53 - 1.20 (m, 20H), 1.03 (s, 1H), 0.97 (s, 2H);HRMS (SI) C 32 H 48 N5O7[M+H]+ calculated 614.3548, found 614.3546.

[0276] [ka] To a solution of SL-1469 (22 mg, 36 μmol) in DCM (8 mL) was added TiPS (100 μL) followed by TFA (2 mL). The resulting solution was stirred at 22° C. for 1 h, at which point HPLC analysis indicated complete consumption of the starting material. The volatiles were removed under reduced pressure and the crude residue was dissolved in 5 mL of MeOH and the volatiles were removed in vacuo. The crude residue was dried under high vacuum and used in the next step without further purification. MS (SI) C 22 H32 N5O3[M+H]+ calculated 414.25, found 414.27.

[0277] [ka] To a solution of SL-1471 (6.0 mg, 14 μmol) and DIPEA (9.0 μL, 51 μmol) in DMF (6 mL) was added NCT-TFP (5.2 mg, 7.3 μmol, prepared according to ACS Chem. Biol., 2016, 11, 2608-2617). The resulting solution was stirred at 22 °C for 2 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to give 3.9 mg (56% yield) of conjugate SL-1475 as a deep blue film. HPLC: 97% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.35 (m, 1H), 8.20 (m, 1H), 8.13 (m, 1H), 7.92 - 7.67 (m, 2H), 7.55 - 7.18 (m, 4H), 6.87 (dd, J = 7.7, 1.0 Hz, 0.5H), 6.78 (dd, J = 7.7, 1.0 Hz, 0.5H), 6.63 (d, J = 2.1 Hz, 1H), 6.59 (s, 1H), 6.54 (s, 1H), 4.38 (m, 1H), 4.26 - 3.98 (m, 2H), 3.71 (m, 4H), 3.68 - 3.58 (m, 3H), 3.55 (m, 4H), 3.52 - 3.33 (m, 7H), 3.11 - 3.03 (m, 1H), 2.97 (m, 4H), 2.93 - 2.85 (m, 1H), 2.79 (m, 4H), HRMS (SI) C 57 H64 N7O7[M+]+ calculated 958.4862, found 958.4869.

[0278] Characterization data for the Propranolol-PRG-NCT probe: [ka] HPLC: 98% purity at 254nm; HRMS (SI) C 67 H 68 N5O8[M+]+ calculated 1070.5062, found 1070.5071.

[0279] [ka] HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.34 (m, 1H), 8.21 (m, 1H), 8.09 (m, 1H), 7.87 - 7.66 (m, 2H), 7.54 - 7.12 (m, 8H), 6.98 - 6.73 (m, 1H), 6.68 - 6.47 (m, 2H), 4.75 (s, 1H), 4.69 (s, 1H), 4.43 - 4.29 (m, 1H), 4.29 - 3.99 (m, 2H), 3.70 (m, 4H), 3.67 - 3.57 (m, 3H), 3.54 (m, 4H), 3.51 - 3.34 (m, 4H), 3.27 - 3.07 (m, 2H), 3.07 - 2.90 (m, 4H), 2.86 (m, 1H), 2.82 - 2.64 (m, 3H), 2.06 (m, 4H), 1.94 (m, 4H), 1.82 (m, 4H);HRMS (SI) C 57 H 64 N7O7[M+]+ calculated 1074.4736 found 1074.4744.

[0280] [ka] HPLC: Purity 99% at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for a mixture of rotamers) δ 8.35 (m, 1H), 8.23 (m, 1H), 8.10 (m, 1H), 7.88 - 7.66 (m, 2H), 7.51 - 7.44 (m, 1H), 7.44 - 7.36 (m, 2H), 7.36 - 7.23 (m, 3H), 7.12 - 6.94 (m, 2H), 6.94 - 6.77 (m, 1H), 6.67 (d, J = 1.5 Hz, 1H), 6.62 (s, 0.5H), 6.54 (s, 0.5H), 4.68 (s, 1H), 4.66 (s, 1H), 4.40 (m, 1H), 4.19 (m, 1H), 4.11 (m, 1H), 3.72 (m, 4H), 3.69 - 3.59 (m, 4H), 3.56 (m, 4H), 3.53 - 3.38 (m, 4H), 3.30 - 3.11 (m, 2H), 3.01 (m, 4H), 2.94 (m, 1H), 2.80 (m, 4H), 2.08 (m, 4H), 1.96 (m, 4H), 1.85 (m, 4H); HRMS (SI) C 57 H 64 Calculated for C

[0281]

Chemical Structure

[0282]

Chemical Structure

[0283] [ka] HPLC: 99% purity at 254 nm; 1H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.33 (m, 1H), 8.21 (m, 1H), 8.06 (m, 1H), 7.92 - 7.65 (m, 2H), 7.52 - 7.23 (m, 4H), 7.16 (m, 1H), 7.11 - 7.00 (m, 1H), 7.00 - 6.93 (m, 1H), 6.83 (m, 1H), 6.65 (m, 1H), 6.60 (s, 0.5H), 6.53 (s, 0.5H), 4.72 - 4.56 (m, 2H), 4.40 (m, 1H), 4.18 (m, 1H), 4.10 (m, 1H), 3.81 - 3.37 (m, 17H), 3.24 - 3.16 (m, 1H), 3.08 - 2.88 (m, 5H), 2.77 (m, 4H), 2.13 (s, 3H), 2.07 (m, 4H), 1.93 (m, 4H), 1.82 (m, 4H);HRMS (SI) C 57 H 64 N7O7[M+]+ Calculated 1021.4971 Found 1021.4993.

[0284] [ka] HPLC: 99% purity at 254 nm; 1H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 1H NMR (400 MHz, methanol-d4) δ 8.33 (m, 1H), 8.27 - 8.15 (m, 1H), 8.06 (m, 1H), 7.84 - 7.66 (m, 2H), 7.52 - 7.23 (m, 4H), 7.23 - 7.00 (m, 3H), 6.82 (m, 1H), 6.65 (m, 1H), 6.60 (s, 0.5 H), 6.51 (s, 0.5 H), 4.69 - 4.59 (m, 2H), 4.38 (m, 1H), 4.18 (m, 1H), 4.09 (m, 1H), 3.70 (m, 4H), 3.65 - 3.35 (m, 11H), 3.20 (m, 1H), 2.95 (m, 5H), 2.86 - 2.57 (m, 4H), 2.13 (m, 3H), 2.11 - 1.99 (m, 4H), 1.99 - 1.87 (m, 4H), 1.82 (s, 4H);HRMS (SI) C 57 H 64 N7O7[M+]+ Calculated 1021.4971 Found 1021.4972.

[0285] [ka] HPLC: 99% purity at 254 nm; 11H NMR (400 MHz, methanol-d4, reported for a mixture of rotamers) δ 8.32 (m, 1H), 8.21 (m, 1H), 8.11 (m, 1H), 7.87 - 7.67 (m, 2H), 7.54 - 7.41 (m, 1H), 7.38 (m, 2H), 7.33 - 7.24 (m, 1H), 7.24 - 7.01 (m, 3H), 6.83 (m, 1H), 6.65 (s, 1H), 6.61 (s, 0.5H), 6.55 (s, 0.5H), 4.71 (m, 2H), 4.39 (m, 1H), 4.18 (m, 1H), 4.10 (m, 1H), 3.78 - 3.65 (m, 5H), 3.65 - 3.43 (m, 11H), 3.40 (m, 4H), 3.07 - 2.84 (m, 5H), 2.79 (m, 4H), 2.40 (m, 3H), 2.06 (m, 4H), 1.93 (m, 4H), 1.82 (m, 4H); HRMS (SI) C 57 H 64 N7O7[M+]+ calculated 1021.4971, found 1021.4976.

[0286]

Chem.

[0287]

Chemical Structure

[0288]

Chemical Structure

[0289] [Chemical Structure] HPLC: Purity 99% at 254 nm; 11H NMR (400 MHz, methanol-d4, reported for a mixture of rotamers) δ 8.33 (m, 1H), 8.20 (m, 1H), 8.03 (m, 1H), 7.86 - 7.63 (m, 2H), 7.54 - 7.19 (m, 4H), 7.00 (m, 2H), 6.93 - 6.70 (m, 1H), 6.65 (m, 1H), 6.62 (s, 0.5H), 6.49 (s, 0.5H), 4.67 - 4.48 (m, 2H), 4.38 (m, 1H), 4.24 - 3.97 (m, 2H), 3.71 (m, 4H), 3.68 - 3.59 (m, 3H), 3.58 - 3.51 (m, 5H), 3.51 - 3.38 (m, 3H), 3.29 - 3.16 (m, 2H), 3.05 - 2.92 (m, 4H), 2.92 - 2.84 (m, 1H), 2.78 (m, 4H), 2.15 - 2.00 (m, 4H), 1.94 (m, 4H), 1.83 (m, 4H); HRMS (SI) C 60 H 61 F2N8O7[M+]+ Calcd 1043.4626, Found 1043.4641.

[0290] [Chemical formula] HPLC: Purity 99% at 254 nm; 11H NMR (400 MHz, reported for a mixture of rotamers in methanol-d4) δ 8.33 (m, 1H), 8.26 - 8.13 (m, 1H), 8.01 (m, 1H), 7.85 - 7.62 (m, 2H), 7.51 - 7.19 (m, 4H), 7.01 - 6.70 (m, 3H), 6.70 - 6.44 (m, 2H), 4.70 - 4.50 (m, 2H), 4.38 (m, 1H), 4.25 - 4.01 (m, 2H), 3.83 - 3.58 (m, 8H), 3.58 - 3.37 (m, 8H), 3.25 - 3.15 (m, 1H), 2.98 (m, 4H), 2.92 (m, 1H), 2.88 - 2.66 (m, 4H), 2.08 (m, 4H), 1.94 (s, 4H), 1.83 (m, 4H); HRMS (SI) C 60 H 61 FN 11 O7[M+]+ calculated 1066.7434, found 1066.4728.

[0291]

Chemical Structure

[0292] [ka] HPLC: 99% purity at 254 nm; 11H NMR (400 MHz, methanol-d4, reported for a mixture of rotamers) δ 8.35 (m, 1H), 8.27 - 8.16 (m, 1H), 8.09 (m, 1H), 7.91 - 7.64 (m, 2H), 7.47 - 7.25 (m, 5H), 6.93 - 6.84 (m, 1H), 6.84 - 6.75 (m, 2H), 6.66 (d, J = 1.6 Hz, 1H), 6.61 (s, 0.5H), 6.54 (s, 0.5H), 4.64 - 4.46 (m, 2H), 4.39 (m, 1H), 4.28 - 4.01 (m, 2H), 3.83 - 3.67 (m, 4H), 3.67 - 3.58 (m, 3H), 3.58 - 3.51 (m, 5H), 3.51 - 3.37 (m, 4H), 3.19 - 3.15 (m, 1H), 3.07 - 2.87 (m, 5H), 2.78 (m, 4H), 2.14 - 1.99 (m, 4H), 1.93 (m, 4H), 1.82 (m, 4H); HRMS (SI) C 60 H 62 N 11 O7 [M+]+ calculated 1048.4828, found 1048.4824.

[0293]

Chem.

[0294]

Chem.

[0295]

Chemical Structure

[0296]

Chem.

[0297] Synthesis of cleavable chloroalkane photoreactive probes: [ka]

[0298] General scheme for cleavable chloroalkane photoreactive probes [ka]

[0299] Exemplary Synthesis of Cleavable Chloroalkane Photoreactive Probes: [ka] To a solution of SL-1235 (2.5 mg, 3.7 μmol) and NMM (3 μL, 27 μmol) in DMF (6 mL) was added CA-pNO2Ph-carbamate (3.2 mg, 3.8 μmol, prepared according to ACS Chem. Biol., 2016, 11, 2608-2617). The resulting solution was stirred at 22 °C for 20 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to give 2.3 mg (48% yield) of conjugate SL-1363 as a clear film. HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 7.64 (m, 2H), 7.50 (s, 1H), 7.39 (m, 2H), 7.27 (m, 3H), 5.84 (m, 2H), 4.66 (m, 1H), 4.52 (m, 4H), 4.29 - 4.01 (m, 4H), 3.81 - 3.45 (m, 28H), 3.42 (s, 2H), 3.27 (m, 7H), 2.38 (m, 2H), 2.09 (t, J = 7.4 Hz, 2H), 1.86 - 1.53 (m, 8H), 1.53 - 0.97 (m, 8H);HRMS (SI)C 56 H 84 ClF3NO 18 [M+H]+ calculated 1262.5569, found 1262.5573.

[0300] Characterization data for the SAHA-PRG-chloroalkane probe: [ka] HPLC: 99% purity at 254 nm; 1H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 7.67 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 5.85 (m, 2H), 4.70 - 4.41 (m, 4H), 4.37 - 3.92 (m, 4H), 3.79 - 3.35 (m, 31H), 3.30 - 3.22 (m, 6H), 3.15 (m, 1H), 2.39 (t, J = 7.4 Hz, 2H), 2.11 (t, J = 7.3 Hz, 2H), 1.87 - 1.51 (m, 10H), 1.51 - 1.33 (m, 8H);HRMS (SI) C 51 H 85 ClNO 18 [M+H]+ calculated 1146.5696, found 1146.5699.

[0301] [ka] HPLC: 99% purity at 254 nm; 1H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 7.65 (m, 2H), 7.41 (m, 3H), 7.19 (m, 1H), 7.08 (m, 2H), 5.84 (m, 2H), 4.78 (m, 1H), 4.52 (m, 5H), 4.26 - 3.94 (m, 4H), 3.80 - 3.46 (m, 28H), 3.40 (m, 2H), 3.27 (m, 6H), 2.38 (m, 2H), 2.09 (m, 2H), 1.90 - 1.52 (m, 8H), 1.52 - 1.18 (m, 8H);HRMS (SI) C 54 H 84 ClN 10 O 18 [M+H]+ calculated 1195.57, found 1195.41.

[0302] [ka] HPLC: 99% purity at 254 nm; 1H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 7.67 (m, 2H), 7.40 (m, 2H), 7.31 - 7.01 (m, 2H), 7.01 - 6.74 (m, 1H), 5.83 (m, 2H), 4.79 (s, 1H), 4.67 - 4.42 (m, 5H), 4.31 - 4.03 (m, 4H), 3.79 - 3.33 (m, 30H), 3.27 (m, 7H), 2.38 (m, 2H), 2.18 (m, 3H), 2.09 (m, 2H), 1.84 - 1.51 (m, 8H), 1.51 - 1.18 (m, 8H);HRMS (SI) C 55 H 86 ClN 10 O 18 [M+H]+ calculated 1209.5805, found 1209.5814.

[0303] [ka] HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 7.66 (m, 2H), 7.40 (m, 2H), 7.34 - 6.81 (m, 3H), 5.84 (m, 2H), 4.74 (m, 1H), 4.54 (m, 5H), 4.16 (m, 4H), 3.79 - 3.44 (m, 28H), 3.40 (s, 2H), 3.27 (m, 5H), 3.20 (s, 1H), 2.38 (m, 2H), 2.34 - 1.98 (m, 5H), 1.89 - 1.54 (m, 8H), 1.51 - 1.34 (m, 8H);HRMS (SI) C 55 H 85 ClN 10 NaO 18 [M+Na]+ calculated 1231.5624, found 1231.5594.

[0304] [ka] HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 7.69 (m, 2H), 7.56 - 7.24 (m, 2H), 7.23 - 6.54 (m, 2H), 5.86 (m, 2H), 4.75 (m, 1H), 4.56 (m, 5H), 4.17 (m, 4H), 3.70 (m, 4H), 3.67 - 3.56 (m, 15H), 3.52 (m, 11H), 3.31 - 3.13 (m, 6H), 2.40 (m, 2H), 2.11 (m, 2H), 1.86 - 1.56 (m, 8H), 1.56 - 1.34 (m, 8H);HRMS (SI) C 54 H 82 ClFN 13 O 18 [M+H]+ calculated 1254.5552, found 1254.5568.

[0305] Characterization data for the Dasatinib-PRG-chloroalkane probe: [ka] HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.18 (m, 1H), 7.46 - 7.29 (m, 4H), 7.29 - 7.19 (m, 3H), 6.14 (m, 1H), 5.83 (m, 2H), 4.69 (m, 2H), 4.61 - 4.26 (m, 6H), 4.17 (m, 4H), 3.86 - 3.41 (m, 29H), 3.41 - 3.34 (m, 3H), 3.26 (m, 5H), 2.52 (m, 3H), 2.33 (s, 3H), 1.76 (m, 2H), 1.58 (m, 2H), 1.52 - 1.35 (m, 4H); HRMS (SI) C 54 H 82 ClFN13 O 18 [M+H]+ calculated value 1455.5510, measured value 1455.5547.

[0306]

Chem.

[0307]

Chem.

[0308]

Chem.

[0309] [ka] HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.18 (s, 1H), 7.36 (m, 1H), 7.30 - 7.18 (m, 2H), 7.01 (m, 1H), 7.00 - 6.73 (m, 1H), 6.17 (m, 1H), 5.83 (s, 2H), 4.63 - 4.27 (m, 7H), 4.27 - 4.05 (m, 5H), 3.78 - 3.43 (m, 32H), 3.43 - 3.35 (m, 3H), 3.27 (m, 6H), 2.52 (m, 3H), 2.33 (s, 3H), 1.87 - 1.67 (m, 2H), 1.59 (m, 2H), 1.52 - 1.33 (m, 4H);HRMS (SI) C 61 H 85 Cl2FN 18 O 16 S [M+H]+ calculated 1447.5545, found 1447.5547.

[0310] Characterization data for the Propranolol-PRG-Chloroalkane probe: [ka] HPLC: 99% purity at 254 nm; 1H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.35 - 8.19 (m, 1H), 7.83 (m, 1H), 7.58 - 7.42 (m, 3H), 7.42 - 7.31 (m, 3H), 7.27 (m, 1H), 7.20 (m, 1H), 6.95 (m, 1H), 5.82 (s, 2H), 4.68 (m, 2H), 4.60 - 4.33 (m, 5H), 4.28 (m, 1H), 4.24 - 4.06 (m, 5H), 3.85 - 3.37 (m, 33H), 3.37 - 3.31 (m, 15H), 3.28 - 3.18 (m, 5H), 3.04 (m, 1H), 2.84 (m, 1H), 1.85 - 1.67 (m, 2H), 1.59 (m, 2H), 1.52 - 1.26 (m, 4H);MS (SI) C 57 H 82 ClF3NO 17 [M+H]+ calculated 1243.55, found 1243.78.

[0311] [ka] HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.39 - 8.17 (m, 1H), 7.89 - 7.69 (m, 1H), 7.58 - 7.43 (m, 3H), 7.43 - 7.24 (m, 1H), 6.95 (m, 1H), 5.83 (s, 2H), 4.52 (s, 4H), 4.44 (m, 1H), 4.28 (m, 1H), 4.19 (m, 5H), 3.77 - 3.34 (m, 39H), 3.28 - 3.16 (m, 5H), 2.90 (m, 2H), 1.93 - 1.69 (m, 2H), 1.68 - 1.33 (m, 9H), 1.06 (s, 1H), 1.02 (s, 2H);MS (SI) C 52 H 84 ClNO17 [M+H]+ calculated 1127.56, found 1227.86.

[0312] [ka] HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.43 - 8.15 (m, 1H), 7.91 - 7.71 (m, 1H), 7.58 - 7.43 (m, 3H), 7.43 - 7.35 (m, 1H), 7.35 - 7.20 (m, 2H), 7.12 - 7.04 (m, 1H), 7.04 - 6.98 (m, 1H), 6.95 (m, 1H), 5.82 (s, 2H), 4.62 (m, 2H), 4.57 - 4.38 (m, 6H), 4.28 (m, 1H), 4.25 - 4.08 (m, 5H), 3.78 - 3.36 (m, 37H), 3.26 (m, 6H), 3.09 - 2.96 (m, 1H), 2.96 - 2.83 (m, 1H), 1.82 - 1.66 (m, 2H), 1.58 (m, 2H), 1.52 - 1.33 (m, 5H);HRMS (SI) C 55 H 83 ClNO 17 [M+H]+ calculated 1176.5590, found 1176.5583.

[0313] [ka] HPLC: 99% purity at 254 nm; 1H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 8.34 - 8.14 (m, 1H), 7.88 - 7.73 (m, 1H), 7.56 - 7.33 (m, 4H), 7.27 - 6.82 (m, 4H), 5.82 (m, 2H), 4.63 (m, 2H), 4.58 - 4.32 (m, 5H), 4.32 - 4.04 (m, 6H), 3.73 - 3.36 (m, 32H), 3.29 - 3.19 (m, 6H), 3.10 - 2.95 (m, 1H), 2.89 (m, 1H), 2.16 (m, 3H), 1.84 - 1.67 (m, 2H), 1.58 (m, 2H), 1.50 - 1.32 (m, 4H);HRMS (SI) C 56 H 85 ClNO 17 [M+H]+ calculated 1190.5746, found 1190.5721.

[0314] [ka] HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4, reported for mixture of rotamers) δ 1H NMR (400 MHz, Methanol-d4) δ 8.37 - 8.10 (m, 1H), 7.90 - 7.74 (m, 1H), 7.57 - 7.43 (m, 3H), 7.40 (t, J = 7.9 Hz, 1H), 7.21 - 7.04 (m, 3H), 6.95 (ddd, J = 7.7, 3.0, 1.1 Hz, 1H), 5.82 (s, 2H), 4.59 (d, J = 7.5 Hz, 2H), 4.55 - 4.35 (m, 5H), 4.29 (ddd, J = 10.6, 5.9, 4.8 Hz, 1H), 4.25 - 4.07 (m, 5H), 3.74 - 3.36 (m, 33H), 3.35 (s, 1H), 3.27 - 3.20 (m, 4H), 3.09 - 2.97 (m, 1H), 2.97 - 2.81 (m, 1H), 2.17 (d, J = 9.4 Hz, 3H), 1.83 - 1.68 (m, 2H), 1.58 (p, J = 6.7 Hz, 2H), 1.52 - 1.32 (m, 4H);HRMS (SI) C 56 H 85 ClN9O 17 [M+H]+ calculated value 1190.5746, measured value 1190.5721.

[0315]

change

[0316] Synthesis of SAHA dye tracer: [ka] SAHA-NCT was synthesized according to the protocol published in ACS Chemical Biology, 2015, 10, 2316-2324.

[0317] [ka] SAHA-TMR was synthesized according to the protocol published in PCT International Application No. 2014093677 (June 19, 2014).

[0318] [ka] To a solution of SL_0825 (6.0 mg, 11 μmol, prepared according to ACS Chemical Biology, 2015, 10, 2316-2324) and NMM (8 μL, 60 μmol) in DMF (4 mL) was added BODIPY576 SE (4.8 mg, 11 μmol). The resulting solution was allowed to react at 22 °C for 2 h, at which point HPLC analysis indicated complete consumption of starting material. The solvent was removed under vacuum and the crude residue (SL_0830) was used in the next step without further purification. MS(SI)C 5o H 50 BF2N6O4[M + H]+ calculated 847.40, found 847.47.

[0319] Crude SL_0830 (9.5 mg, 11 μmol) was dissolved in DCM (5 mL) and the stirred solution was cooled to 0° C. TiPS (3 drops) was added followed by TFA (1 drop). The resulting solution was left stirring at 0° C. for 3 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was purified by preparative RP HPLC (5→95% MeCN / H2O buffered with 0.5% TFA) to give 1.9 mg (28% yield) of SAHA-BODIPY 576 as a purple film. HPLC: 95% purity at 254 nm; 1 H NMR (300 MHz, methanol-d4) δ 10.60 (s, 1H), 7.39 (dd, J = 8.5, 2.9 Hz, 2H), 7.15 - 7.06 (m, 6H), 6.91 (t, J = 4.9 Hz, 1H), 6.79 (dd, J = 7.1, 4.0 Hz, 1H), 6.25 (m, 1H), 6.22 - 6.03 (m, 1H), 4.23 (d, J = 7.6 Hz, 2H), 2.58 (td, J = 7.7, 4.2 Hz, 2H), 2.36 - 2.09 (m, 3H), 1.97 (m, 1H), 1.58 (m, 4H), 1.28 (m, 4H); MS (SI) C 31 H 36 BF2N6O4[M+H]+ calculated 605.29, found 605.50.

[0320] Synthesis of Propranolol Dye Tracer: [ka] A solution of (S)-2-((naphthalen-1-yloxy)methyl)oxirane (330 mg, 1.65 mmol, prepared according to J. Org. Chem. 1989, 54, 1295-1304) and tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (1.02 g, 4.12 mmol) in 9:1 DMF-water (10 mL) was heated to 85° C. for 21 h, at which point HPLC analysis indicated complete consumption of starting material. Volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (0→20% MeOH / DCM) to afford 535 mg (72% yield) of the amine SL-1325 as a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 8.39 - 8.16 (m, 1H), 8.04 - 7.75 (m, 1H), 7.64 - 7.48 (m, 2H), 7.46 (d, J = 8.3 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 6.95 (dd, J = 7.6, 1.1 Hz, 1H), 6.76 (t, J = 5.7 Hz, 1H), 5.13 (d, J = 4.5 Hz, 1H), 4.24 - 3.89 (m, 3H), 3.55 - 3.42 (m, 6H), 3.36 (t, J = 6.1 Hz, 2H), 3.05 (q, J = 6.0 Hz, 2H), 2.88 - 2.67 (m, 4H), 1.36 (s, 9H);MS (SI) C 24 H 37 N2O6[M+H]+ calculated 449.27, found 449.64.

[0321] To a solution of SL-1325 (313 mg, 698 μmol) in DCM (16 mL) was added TiPS (200 μL) followed by TFA (4 mL). The resulting solution was stirred at 22° C. for 2 h, at which point HPLC analysis indicated complete consumption of starting material. The volatiles were removed under reduced pressure and the crude residue was dissolved in 20 mL of MeOH and the volatiles were removed in vacuo. The crude residue was dried under high vacuum and used in the next step without further purification. 1 H NMR (400 MHz, DMSO-d6) δ 8.64 (d, J = 27.2 Hz, 2H), 8.26 (dd, J = 7.9, 1.8 Hz, 1H), 8.04 - 7.87 (m, 1H), 7.79 (s, 3H), 7.64 - 7.50 (m, 3H), 7.50 - 7.29 (m, 1H), 7.06 - 6.87 (m, 1H), 4.35 (dtd, J = 9.9, 5.1, 2.9 Hz, 1H), 4.16 (dp, J = 8.6, 4.8 Hz, 2H), 3.73 (ddd, J = 6.2, 4.3, 2.2Hz, 2H), 3.68 - 3.52 (m, 6H), 3.35 (m, 1H), 3.22 (m, 3H), 2.97 (h, J = 5.7 Hz, 2H);MS (SI) C 19 H 29 N2O4[M+H]+ calculated 349.21, found 349.28.

[0322] To a solution of 6-carboxy-NCT (10 mg, 18 μmol, prepared according to Org. Lett. 2016, 18, 5316-5319), DIPEA (22 μL, 120 μmol), and TSTU (5.5 mg, 18 μmol) in DMF (5 mL) was added SL-1327 (10 mg, 18 μmol). The resulting solution was stirred at 22 °C for 2 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to give 7 mg (44% yield) of conjugate SL-1328 as a deep blue film. HPLC: 95% purity at 254 nm;1 H NMR (400 MHz, methanol-d4) δ 8.35 (d, J = 8.2 Hz, 1H), 8.24 - 8.02 (m, 2H), 7.87 (d, J = 1.8 Hz, 1H), 7.74 (d, J = 8.1 Hz, 1H), 7.49 - 7.36 (m, 2H), 7.36 - 7.14 (m, 2H), 6.78 (d, J = 7.6 Hz, 1H), 6.58 (d, J = 2.6 Hz, 2H), 4.41 (dtd, J = 10.2, 5.4, 3.0 Hz, 1H), 4.04 (qd, J = 9.7, 5.4 Hz, 2H), 3.88 - 3.75 (m, 2H), 3.75 - 3.59 (m, 12H), 3.59 - 3.41 (m, 5H), 3.26 (dd, J = 12.7, 10.0 Hz, 3H), 2.94 (dddd, J = 25.0, 16.2, 7.2, 4.0 Hz, HRMS (SI) C 57 H 64 N7O7[M+]+ calculated 893.4484, found 893.4485.

[0323] [ka] To a solution of SL-1327 (4.1 mg, 7.1 μmol) and DIPEA (9 μL, 50 μmol) in DMF (4 mL) was added a solution of BODIPY576 SE (3.0 mg, 7.0 μmol) in DMF (4 mL). The resulting solution was allowed to react at 22° C. for 3 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was purified by preparative RP HPLC (5→95% MeCN / H2O buffered with 0.5% TFA) to give 4 mg (74% yield) of the conjugate SL-1329 as a deep blue film. HPLC: 96% purity at 254 nm; 1H NMR (400 MHz, メタノール-d4) δ 8.25 (dd, J = 8.2, 1.5 Hz, 1H), 7.81 (dd, J = 7.8, 1.5 Hz, 1H), 7.58 - 7.41 (m, 3H), 7.37 (t, J = 7.9 Hz, 1H), 7.24 - 7.16 (m, 3H), 7.14 (d, J = 4.5 Hz, 1H), 6.98 (d, J = 4.6 Hz, 1H), 6.88 (dd, J = 5.7, 4.2 Hz, 2H), 6.41 - 6.21 (m, 2H), 4.40 (dtd, J = 10.1, 5.2, 3.1 Hz, 1H), 4.17 (dd, J = 9.9, 4.9 Hz, 1H), 4.11 (dd, J = 9.9, 5.4 Hz, 1H), 3.75 (t, J = 5.0 Hz, 2H), 3.69 - 3.57 (m, 4H), 3.53 (t, J = 5.5 Hz, 2H), 3.44 - 3.34 (m, 3H), 3.29 - 3.18 (m, 4H), 2.64 (t, J = 7.7 Hz, 2H);HRMS (SI) C 35 H 41 BF2N5O5[M+H]+ calculated value 660.3163, measured value 660.3158.

[0324] Synthesis of ダサチニブpigment トレーサーの:

change

[0325] Synthesis of cleavable chloroalkane conjugates: [ka] To a solution of SL_0825 (10 mg, 19 μmol, prepared according to ACS Chemical Biology, 2015, 10, 2316-2324) and NMM (8 μL, 60 μmol) in DMF (5 mL) was added p-nitrophenyl carbonate-CA-T4E (16 mg, 19 μmol, prepared according to ACS Chemical Biology 2016, 11, 2608-2617). The resulting solution was stirred at 22 °C for 20 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to give 16 mg (69% yield) of conjugate SL-1790 as a clear film. HPLC: 97% purity at 254 nm; MS (SI) C 64 H 90 ClNO 17 [M+H]+ calculated 1249.61, found 1249.54.

[0326] To a solution of SL-1790 (16 mg, 13 μmol) in DCM (9 mL) was added TiPS (100 μL) followed by TFA (1 mL). The resulting solution was stirred at 22° C. for 30 min, at which point HPLC analysis indicated complete consumption of starting material. Volatiles were removed under reduced pressure and the crude residue was dissolved in 5 mL of MeOH and volatiles were removed under vacuum. The crude residue was purified by preparative RP HPLC (5→95% MeCN / H2O buffered with 0.5% TFA) to give 12 mg (90% yield) of conjugate SL-1799 as a white solid. HPLC: 99% purity at 254 nm; 1 H NMR (400 MHz, methanol-d4) δ 9.74 (s, 1H), 7.50 (dd, J = 8.4, 2.1 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 5.84 (s, 2H), 4.52 (s, 4H), 4.24 (s, 2H), 4.20 (t, J = 4.7 Hz, 2H), 4.16 (t, J = 4.8 Hz, 2H), 3.68 (q, J = 5.7 Hz, 4H), 3.62 (d, J = 2.0 Hz, 4H), 3.61 - 3.55 (m, 10H), 3.52 (td, J = 6.2, 5.5, 1.8 Hz, 6H), 3.48 (s, 2H), 3.29 - 3.23 (m, 6H), 2.36 (t, J = 7.4 Hz, 2H), 2.10 (s, 1H), 1.84 - 1.73 (m, 2H), 1.69 (s, 2H), 1.59 (m, 4H), 1.52 - 1.44 (m, 2H), 1.40 (d, J = 5.7 Hz, 6H).MS (SI) C 45 H 76 ClNO 17 [M+H]+ calculated 1007.50, found 1007.44.

[0327] [ka] To a solution of SL-1006 (10 mg, 16 μmol, prepared according to Cell Chemical Biology 2018, 25, 206-214) and DIPEA (8 μL, 60 μmol) in DMF (8 mL) was added p-nitrophenyl carbonate-CA-T4E (16 mg, 19 μmol, prepared according to ACS Chemical Biology 2016, 11, 2608-2617). The resulting solution was stirred at 22 °C for 20 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to give 15 mg (78% yield) of the conjugate SL-1118 as a clear film. HPLC: 99% purity at 254 nm; MS (SI) C 54 H 84 Cl2N 11 O 16 S [M+H]+ calculated 1244.52, found 1244.66.

[0328] [ka] A solution of (S)-2-((naphthalen-1-yloxy)methyl)oxirane (328 mg, 1.64 mmol, prepared according to J. Org. Chem. 1989, 54, 1295-1304) and tert-butyl (2-aminoethyl)carbamate (656 mg, 4.10 mmol) in 9:1 DMF-water (10 mL) was heated to 85° C. for 24 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was filtered and volatiles removed under reduced pressure. The crude residue was purified by silica gel chromatography (0→20% MeOH / DCM) to afford 450 mg (76% yield) of amine SL-1326 as a yellow oil. 1H NMR (400 MHz, DMSO-d6) δ 8.24 (dd, J = 7.7, 1.9 Hz, 1H), 7.95 - 7.78 (m, 1H), 7.58 - 7.45 (m, 3H), 7.40 (t, J = 7.9 Hz, 1H), 6.95 (dd, J = 7.6, 1.1 Hz, 1H), 6.75 (t, J = 5.7 Hz, 1H), 5.09 (d, J = 4.6 Hz, 1H), 4.12 (dd, J = 9.2, 4.3 Hz, 1H), 4.09 - 3.96 (m, 2H), 3.02 (q, J = 6.2 Hz, 2H), 2.79 (dd, J = MS (SI) C 20 H 29 N2O4[M+H]+ calculated 361.21, found 361.76.

[0329] To a solution of SL-1326 (16 mg, 44 μmol) in DCM (4 mL) was added TiPS (50 μL) followed by TFA (1 mL). The resulting solution was stirred at 22° C. for 2 h, at which point HPLC analysis indicated complete consumption of the starting material. The volatiles were removed under reduced pressure and the crude residue was dissolved in 5 mL of MeOH and the volatiles were removed in vacuo. The crude residue was dried under high vacuum and used in the next step without further purification. 1H NMR (400 MHz, methanol-d4) δ 8.38 - 8.23 ​​(m, 1H), 7.92 - 7.75 (m, 1H), 7.60 - 7.44 (m, 3H), 7.40 (t, J = 7.9 Hz, 1H), 6.95 (dd, J = 7.6, 1.0 Hz, 1H), 4.49 (dtd, J = 10.1, 5.2, 3.0 Hz, 1H), 4.27 (dd, J = 9.9, 4.9 Hz, 1H), 4.21 (dd, J = 9.9, 5.5 Hz, 1H), 3.62 - 3.47 (m, 3H), 3.47 - 3.36 (m, 3H);MS (SI) C 15 H 21 N2O2[M+H]+ calculated 261.16, found 261.11.

[0330] To a solution of SL-1802 (15 mg, 40 μmol) and DIPEA (23 μL, 130 μmol) in DMF (8 mL) was added p-nitrophenyl carbonate-CA-T4E (11 mg, 13 μmol, prepared according to ACS Chemical Biology 2016, 11, 2608-2617). The resulting solution was stirred at 22 °C for 2 h, at which point HPLC analysis indicated complete consumption of starting material. The reaction mixture was purified by preparative RP HPLC (5 → 95% MeCN / H2O buffered with 0.5% TFA) to give 9.3 mg (74% yield) of conjugate SL-1803 as a clear film. HPLC: 99% purity at 254 nm; 1H NMR (400 MHz, methanol-d4) δ 8.38 - 8.15 (m, 1H), 7.94 - 7.69 (m, 1H), 7.61 - 7.44 (m, 3H), 7.39 (t, J = 7.9 Hz, 1H), 6.94 (dd, J = 7.6, 1.0 Hz, 1H), 5.83 (s, 2H), 4.52 (d, J = 2.9 Hz, 4H), 4.43 (dt, J = 8.8, 4.1 Hz, 1H), 4.26 (dd, J = 9.9, 4.9 Hz, 1H), 4.23 - 4.05 (m, 5H), 3.67 (q, J = 5.0 Hz, 4H), 3.64 - 3.54 (m, 14H), 3.54 - 3.40 (m, 12H), 3.28 (m, 8H), 1.88 - 1.68 (m, 2H), 1.59 (p, J = 6.8 Hz, 2H), 1.52 - 1.32 (m, 4H);HRMS (SI)C 45 H 73 ClNO 16 [M+H]+ calculated 974.4735, found 974.4726.

[0331] Example 8 Enrichment experiments conducted during the development of the embodiments described herein demonstrated enhanced enrichment of endogenous targets of SAHA with 4-phenylazide and 4-Me-3-4-phenylazide-cleavable chloroalkane probes (SL-1373 and SL-1388, respectively). Figure 26 shows a schematic of the enrichment workflow. K562 cells were cultured at 2 x 10 in 100 mm dishes. 6Cells were plated at 10 ... This analysis demonstrates that cleavable chloroalkane probes bearing PRG 4-phenylazide or 4-Me-3-phenylazide enhance enrichment of relevant SAHA targets over diazirine. 4-Phenylazide and 4-Me-3-phenylazide-cleavable chloroalkane probes allowed enrichment of all predicted HDAC class I / IIB, which are primary targets of SAHA, as well as other proteins associated with them that are part of megadalton HDAC complexes. In addition, 4-Me-3-phenylazide and especially 4-phenylazide allowed enrichment of SAHA off-targets, many of which bind divalent metal ions. SAHA binds to HDACs by chelating to HDAC-bound zinc ions and can interact in a manner similar to other metal ion-binding proteins.

[0332] Example 9 Enrichment experiments conducted during the development of embodiments described herein demonstrated enhanced enrichment of endogenous targets of dasatinib with 4-phenylazide-cleavable probes (SL-1377). Figure 26 shows a schematic of the enrichment workflow. K562 cells were plated at 2 x 10 in 100 mm dishes.6 Cells were plated at 10 ... Putative targets identified by mass spectrometry are defined as those enriched at least 4-fold over the control (e.g., +20 μM dasatinib) (Figure 36). This analysis demonstrates enhanced enrichment of dasatinib targets, especially off-targets, with a cleavable chloroalkane probe bearing PRG 4-phenylazide (SL-1377). 4-Phenylazide, 4-Me-3-phenylazide, and diazirine-cleavable chloroalkane probes (SL-1377, SL-1387, and SL-1355, respectively) allowed enrichment of predicted primary targets, including the Src family kinase BCR / ABL1, and other kinases. Here, the 4-phenylazide probe (SL-1377) also allowed enhanced enrichment of off-targets, many of which were purine-binding proteins. Dasatinib is a type I kinase inhibitor that occupies the kinase ATP-binding pocket and can interact in a similar manner with other purine-binding proteins.

[0333] Example 10 Enrichment experiments conducted during the development of embodiments described herein demonstrated enhanced enrichment of endogenous targets of propranolol with a 4-phenylazide-cleavable probe (SL-1484). Figure 26 shows a schematic of the enrichment workflow. Genome-edited PC3 cells expressing HiBiT-tagged β2-AR were plated at 2×10 in a 100 mm dish. 6 Cells were plated at 1000 μL / mL and treated in triplicate with the PRG-cleavable chloroalkane propranolol probe at a final concentration of 1 μM in the absence or presence of competitor 40 μM propranolol, which served as a control for enrichment specificity. After 2 h of equilibrium binding, cells were irradiated for 15 min. The medium was then removed, cells were washed with PBS, lysed, and centrifuged at 3000×g for 1 min. The cleared lysate was added to 30 μL of precipitated paramagnetic HaloTag-coated beads and incubated overnight with constant mixing. After binding, the unbound fraction was removed, the HaloTag-coated paramagnetic beads were washed five times, and the captured targets were released from the beads by palladium-catalyzed cleavage. The released targets were subjected to bioluminescence blot analysis ( FIG. 37C ) and LC-MS / MS analysis ( FIG. 37D ). Bioluminescence blot analysis relying on HiBiT / LgBiT complementation demonstrates the superiority of the 4-phenylazide-cleavable chloroalkane probe (SL-1484) over the diazirine-cleavable chloroalkane probe (SL-1473) in enriching the β2-AR (GPCR), the primary target of propranolol. LC-MS / MS analysis further demonstrates the ability of the 4-phenylazide-cleavable chloroalkane probe (SL-1484) to enrich and identify not only the β2-AR but also other putative off-targets, most of which are membrane proteins.

[0334] References The following references, some of which are cited above, are incorporated herein by reference in their entireties.

[0335] 1. ACS Chem.Biol.,2015,10(10),pp 2316-2324 2. ACS Chem.Biol.,2016,11(9),pp 2608-2617 3. US20140322794 4. US20160355523 5. Photo-induced covalent cross-linking for the analysis of biomolecular interactions.Chem.Soc.Rev 2013,42,3289 6. Target identification of natural products and bioactive compounds using affinity-based probes. Pan,S.;Zhang,H.;Wang,C.;Yao,S.C.;Yao,S.Q.Nat Prod Rep 2016,33,612-620. 7. Photoaffinity labeling in target- and binding-site identification. Smith,E.;Collins,I.Future Med Chem 2015,7,159-183. 8. Development and Leading-Edge Application of Innovative Photoaffinity Labeling. Hatanaka,Y.Chem Pharm Bull(Tokyo)2015,63,1-12. 9. Chemical Reagents in Photoaffinity Labeling. A.Fleming,S.Tetrahedron 1995,51,12479-12520. 10. Recent developments and applications of clickable photoprobes in medicinal chemistry and chemical biology. Lapinsky,D.J.;Johnson,D.S.Future Med Chem 2015,7,2143-2171. 11. Chemical proteomics approaches for identifying the cellular targets of natural products. Wright,MH;Sieber,SANat Prod Rep 2016,33,681-708

[0336] array WT OgLuc (SEQ ID NO: 1) MTLADF WT OgLuc Lg (SEQ ID NO: 2) MFTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSGENGLKADIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIYDERLINPD WT OgLuc β9 (SEQ ID NO:3) GSLLFRVTIN WT OgLuc β10 (SEQ ID NO: 4) GVTGWRLCENILA NanoLuc (SEQ ID NO:5) MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA NanoLuc Lg (SEQ ID NO: 6) MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPD NanoLuc β9 (SEQ ID NO: 7) GSLLFRVTINV NanoLuc β10 (SEQ ID NO:8) GVTGWRLCERILA LgBiT (SEQ ID NO: 9) MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN SmBiT (SEQ ID NO: 10) VTGYRLFEEIL HiBiT (SEQ ID NO: 11) VSGWRLFKKIS LgTrip(3546) (SEQ ID NO: 12) MKHHHHHHVFTLDDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIMRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPD SmTrip9 (SEQ ID NO: 13) GSMLFRVTINS β9 / β10 dipeptide (SEQ ID NO: 14) GSMLFRVTINSVSGWRLFKKIS HALOTAG protein (SEQ ID NO: 15) MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARET FQAFRTTDVGRKLIIDQNVFIEGTLMGVVRPLTEEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISG

Claims

1. A composition comprising a PRG / HA probe, which comprises a bioactive agent, a photoreactive group (PRG), a linker, and a haloalkane covalently linked to a single compound of the following general structure: 【Chemistry 1】 (wherein R is a covalent bond and A is (CH 2 ) n , n=4-10, X is a halogen, -AX is a substrate for a dehalogenase, and Q is CH or N.

2. The composition of claim 1 , wherein the bioactive agent is a small molecule.

3. 2. The composition of claim 1, wherein R comprises -NH-, -C(O)NH-, -OC(O)NH-, N-, C(O)N, OC(O)N, or -O-.

4. R is -(CH 2 ) 0-2 NH (CH 2 ) 0-2 -, -(CH 2 ) 0-2 C(O)NH(CH 2 ) 0-2 -, -(CH 2 ) 0-2 OC(O)NH(CH 2 ) 0-2 -, (CH 2 ) 0-2 N (CH 2 ) 0-2 , (CH 2 ) 0-2 C(O)N(CH 2 ) 0-2 , (CH 2 ) 0-2 OC(O)N(CH 2 ) 0-2 , or -(CH 2 ) 0-2 O (CH 2 ) 0-2 The composition of claim 3 comprising:

5. 2. The composition of claim 1, wherein the PRG is selected from the group consisting of: 【Chemistry 2】

6. The composition of claim 5 , wherein the PRG is covalently connected to the remainder of the compound by a PRG linker moiety.

7. The PRG linker portion is (CH 2 ) 0-4 The composition of claim 6, wherein

8. The composition of claim 1 , wherein the linker is a cleavable linker.

9. The composition of claim 8 , wherein the cleavable linker is chemically cleavable, enzymatically cleavable, or photocleavable.

10. The cleavable linker can be a disulfide, a tert-butyl carbamate, a silyl ether, a diazobenzene, a 1,2-diol, or a -C(O)OCH 2 CH=CHCH 2 10. The composition of claim 9, comprising a cleavable moiety selected from the group consisting of OC(O)-.

11. The composition of claim 10 , wherein the cleavable linker comprises the cleavable moiety flanked on one or both sides by an alkylene or heteroalkylene chain.

12. The alkylene or heteroalkylene chain is 1-6 -Alkylene, -O-, -(CH 2 ) 2 The composition of claim 11 comprising any suitable combination of -O-, and -OC(O)NH- groups.

13. The composition of claim 1 , wherein X is Cl.

14. -A-X is -(CH 2 ) 6 The composition of claim 13, wherein said compound is Cl.

15. A composition comprising a reactive PRG / HA reagent comprising a photoreactive group (PRG), a linker, and a haloalkane covalently linked to a single compound of the following general structure: 【Chemistry 3】 where R' is a first reactive moiety capable of forming a covalent bond when reacted with a second reactive moiety (R"), and A is (CH 2 ) n , n=4-10, X is a halogen, -AX is a substrate for a dehalogenase, and Q is CH or N.

16. R' is -NH 2 , NH, -C(O)OH, OC(O)O-phenyl, C(O)O-phenyl, OH, C=O, aldehyde, difluorosulfinate, sulfonyl fluoride, sulfonyl chloride, or halogen.

17. R' is -(CH 2 ) 0-2 N.H. 2 , -(CH 2 ) 0-2 C(O)OH, (CH 2 ) 0-2 OC(O)O-phenyl, (CH 2 ) 0-2 C(O)O-phenyl, (CH 2 ) 0-2 OH, (CH 2 ) 0-2 C=O, (CH 2 ) 0-2 Cl, or (CH 2 ) 0-2 17. The composition of claim 16 comprising Br.

18. 16. The composition of claim 15, wherein the PRG is selected from the group consisting of: 【Chemistry 4】

19. The composition of claim 18 , wherein the PRG is covalently connected to the remainder of the compound by a PRG linker moiety.

20. The PRG linker portion is (CH 2 ) 0-4 20. The composition of claim 19, wherein

21. The composition of claim 15 , wherein the linker is a cleavable linker.

22. 22. The composition of claim 21, wherein the cleavable linker is chemically cleavable, enzymatically cleavable, or photocleavable.

23. The cleavable linker can be a disulfide, a tert-butyl carbamate, a silyl ether, a diazobenzene, a 1,2-diol, or a -C(O)OCH 2 CH=CHCH 2 23. The composition of claim 22, comprising a cleavable moiety selected from the group consisting of OC(O)-.

24. 24. The composition of claim 23, wherein the cleavable linker comprises the cleavable moiety flanked on one or both sides by an alkylene or heteroalkylene chain.

25. The alkylene or heteroalkylene chain is 1-6 -Alkyl, -O-, -(CH 2 ) 2 25. The composition of claim 24 comprising any suitable combination of -O-, and -OC(O)NH- groups.

26. 16. The composition of claim 15, wherein X is Cl.

27. -A-X is -(CH 2 ) 6 27. The composition of claim 26, wherein said compound is Cl.

28. A system, kit or reaction mixture comprising (i) a PRG / HA reagent according to one of claims 15 to 27, and (ii) a bioactive agent presenting a second reactive moiety R″.

29. 30. The system, kit, or reaction mixture of claim 28, wherein the bioactive agent is a small molecule.

30. 30. The system, kit, or reaction mixture of claim 29, wherein the bioactive agent comprises a drug or a naturally occurring compound modified to display R".

31. R″ is —NH 2 30. The system, kit, or reaction mixture of claim 29, comprising: -OC(O)O-nitrophenyl, -OH, -C=O, -aldehyde, -difluorosulfinate, -sulfonyl fluoride, sulfonyl chloride, or -halogen.

32. R" is -(CH 2 ) 0-2 N.H. 2 , -(CH 2 ) 0-2 OC(O)O-nitrophenyl, -(CH 2 ) 0-2 OH, -(CH 2 ) 0-2 C=O, -(CH 2 ) 0-2 Cl, or -(CH 2 ) 0-2 32. The system, kit, or reaction mixture of claim 31 , comprising Br.

33. A method of making a PRG / HA probe, comprising contacting (i) a PRG / HA reagent according to one of claims 15 to 24 with (ii) a second reactive moiety R″ of a bioactive agent.

34. 34. A PRG / HA probe produced by the method of claim 33.

35. A composition comprising a PRG / fluorophore probe comprising a bioactive agent, a photoreactive group (PRG), and a fluorophore covalently linked to a single compound of the following general structure: 【Chemistry 5】 where R is a covalent bond and Q is CH or N.

36. The composition of claim 35 , wherein the bioactive agent is a small molecule.

37. 36. The composition of claim 35, wherein R comprises NH-, -C(O)NH-, -OC(O)NH-, N, C(O)N, OC(O)N, or -O-.

38. R is -(CH 2 ) 0-2 NH (CH 2 ) 0-2 -, -(CH 2 ) 0-2 C(O)NH(CH 2 ) 0-2 -, -(CH 2 ) 0-2 OC(O)NH(CH 2 ) 0-2 -, (CH 2 ) 0-2 N (CH 2 ) 0-2 -, -(CH 2 ) 0-2 C(O)N(CH 2 ) 0-2 -, -(CH 2 ) 0-2 OC(O)N(CH 2 ) 0-2 - or - (CH 2 ) 0-2 O (CH 2 ) 0-2 The composition of claim 37, comprising:

39. 36. The composition of claim 35, wherein the PRG is selected from the group consisting of: 【Chemistry 6】

40. 40. The composition of claim 39, wherein the PRG is covalently connected to the remainder of the compound by a PRG linker moiety.

41. The PRG linker portion is (CH 2 ) 0-4 41. The composition of claim 40, wherein

42. 36. The composition of claim 35, wherein the fluorophore is covalently linked to the remainder of the compound by a fluorophore linker moiety.

43. 43. The composition of claim 42, wherein the fluorophore linker moiety comprises a cleavable linker.

44. Reactive PRG / fluorophore reagents, comprising a photoreactive group (PRG) and a fluorophore attached to a single compound of the following general structure: 【Chemistry 7】 where R' is a first reactive moiety capable of forming a covalent bond when reacted with a second reactive moiety (R"), and Q is CH or N.

45. R' is -NH 2 , NH, -C(O)OH, OC(O)O-phenyl, C(O)O-phenyl, OH, C=O, aldehyde, difluorosulfinate, sulfonyl fluoride, sulfonyl chloride, or halogen.

46. R' is -(CH 2 ) 0-2 N.H. 2 , -(CH 2 ) 0-2 C(O)OH, (CH 2 ) 0-2 OC(O)O-phenyl, (CH 2 ) 0-2 C(O)O-phenyl, (CH 2 ) 0-2 OH, (CH 2 ) 0-2 C=O, (CH 2 ) 0-2 Cl, or (CH 2 ) 0-2 46. ​​The composition of claim 45 comprising Br.

47. 45. The composition of claim 44, wherein the PRG is selected from the group consisting of: 【Chemistry 8】

48. 48. The composition of claim 47, wherein the PRG is covalently connected to the remainder of the compound by a PRG linker moiety.

49. The PRG linker portion is (CH 2 ) 0-4 49. The composition of claim 48, wherein

50. 45. The composition of claim 44, wherein the fluorophore is covalently linked to the remainder of the compound by a fluorophore linker moiety.

51. 51. The composition of claim 50, wherein the fluorophore linker moiety comprises a cleavable linker.

52. 51. A system, kit or reaction mixture comprising (i) a reactive PRG / fluorophore reagent according to one of claims 44 to 50, and (ii) a bioactive agent presenting said second reactive moiety R″.

53. 53. The system, kit, or reaction mixture of claim 52, wherein the bioactive agent is a small molecule.

54. 54. The system, kit, or reaction mixture of claim 53, wherein the bioactive agent comprises a drug or a naturally occurring compound modified to display R".

55. R″ is —NH 2 52. The system, kit, or reaction mixture of claim 51, comprising: -OC(O)O-nitrophenyl, -OH, -C=O, -aldehyde, -difluorosulfinate, -sulfonyl fluoride, sulfonyl chloride, or -halogen.

56. R" is -(CH 2 ) 0-2 N.H. 2 , -(CH 2 ) 0-2 OC(O)O-nitrophenyl, -(CH 2 ) 0-2 OH, -(CH 2 ) 0-2 C=O, -(CH 2 ) 0-2 Cl, or -(CH 2 ) 0-2 56. The system, kit, or reaction mixture of claim 55, comprising Br.

57. 51. A method for making a PRG / fluorophore probe comprising contacting (i) a reactive PRG / fluorophore reagent according to one of claims 43 to 50 with (ii) a bioactive agent presenting said second reactive moiety R″.

58. 58. A PRG / fluorophore probe produced by the method of claim 57.

59. 1. A method for capturing a target molecule, comprising: contacting a sample containing said target molecule with a PRG / HA probe according to one of claims 1 to 14, wherein said bioactive agent is capable of associating with said target molecule; Associating the bioactive agent with the targeting molecule; and The method includes irradiating the sample with light of a wavelength that converts the PRG into a reactive form of the PRG, wherein the reactive form of the PRG forms a covalent bond with the target molecule.

60. contacting the sample with a modified dehalogenase enzyme that forms a covalent bond with a haloalkane substrate when associated therewith; and 60. The method of claim 59, further comprising covalently binding the modified dehalogenase enzyme to a haloalkane of the PRG / HA probe.

61. 61. The method of claim 60, wherein the modified dehalogenase enzyme is immobilized on a solid surface and the target molecule is immobilized on the solid surface when the modified dehalogenase enzyme binds to the haloalkane of the PRG / HA probe.

62. (i) removing the solid surface from the sample; and / or 62. The method of claim 61, further comprising (ii) washing non-immobilized components of the sample from the solid surface.

63. 62. The method of claim 61 , wherein the solid surface is a bead, particle, chip, tube, plate, or membrane.

64. 63. The method of claim 62, wherein the solid surface is a paramagnetic particle.

65. The method of any one of claims 61 to 64, further comprising cleaving the linker of the PRG / HA probe to release the target molecule bound to the PRG and bioactive agent from the solid surface.

66. The method according to one of claims 59 to 65, further comprising detection and / or quantification of the target molecule.

67. 67. The method of claim 66, wherein the target molecule is detected and / or quantified by mass spectrometry.

68. The method according to one of claims 59 to 67, wherein the sample comprises cells and the target molecule is located within or on the surface of the cells.

69. 69. The method of claim 68, wherein contacting the sample containing the target molecule with the PRG / HA probe further comprises introducing the PRG / HA probe into the cells.

70. 69. The method of claim 68, further comprising lysing the cell after the PRG forms a covalent bond with the target molecule.

71. 60. The method of claim 59, wherein the bioactive agent is capable of non-covalently binding to the target molecule when the bioactive agent is associated with the target molecule.

72. 60. The method of claim 59, wherein the bioactive agent is a small molecule.

73. 60. The method of claim 59, wherein the target molecule is a target protein.

74. 71. The method of claim 70, wherein the target protein is a cell surface protein, a transmembrane protein, a cytoplasmic protein, a nuclear protein, or a mitochondrial protein.

75. 60. The method of claim 59, wherein the wavelength of light that converts the PRG to a reactive form of the PRG is ultraviolet (UV) light.

76. A method for capturing an intracellular target protein, comprising the steps of: contacting a cell containing said target protein with the PRG / HA probe according to one of claims 1 to 14, wherein said bioactive agent is capable of binding to said target protein; introducing said PRG / HA probe into said cells; binding the bioactive agent to the target protein; irradiating the cells with UV light at a wavelength that converts the PRG into a reactive form of the PRG, wherein the reactive form of the PRG forms a covalent bond with the target protein; lysing the cells to form a lysate; contacting the lysate with a modified dehalogenase enzyme immobilized on a solid surface that forms a covalent bond with a haloalkane substrate when associated therewith; covalently attaching the modified dehalogenase enzyme to a haloalkane of the PRG / HA probe; and removing said solid surface from said lysate and / or washing non-immobilized components of said lysate from said solid surface.

77. 74. The method of claim 73, further comprising cleaving the linker of the PRG / HA probe to release the target protein bound to the PRG and bioactive agent from the solid surface.

78. 75. The method of claim 74, further comprising detecting and / or quantifying the target molecule.

79. 74. The method of claim 73, wherein the target protein is a cell surface protein, a transmembrane protein, a cytoplasmic protein, a nuclear protein, or a mitochondrial protein.

80. 1. A method for measuring photocrosslinking efficiency of PRG linked to a bioactive agent, comprising: contacting a sample containing a target molecule with the PRG / fluorophore probe according to one of claims 35 to 43, wherein the target molecule is tethered to a bioluminescent reporter, the emission spectrum of the bioluminescent reporter overlapping with the excitation spectrum of the fluorophore; binding said bioactive agent to said targeting molecule; irradiating the sample with light of a wavelength that converts the PRG into a reactive form, the reactive form of the PRG capable of forming a covalent bond with the target molecule; contacting said sample with a substrate for a bioluminescent reporter; detecting emission within the emission spectrum of said bioluminescent reporter and said fluorophore, where the emission from said fluorophore is the result of bioluminescence resonance energy transfer (BRET) from said bioluminescent reporter to said fluorophore, and where the amount of emission from said fluorophore (and / or the BRET ratio) correlates to the amount of PRG / fluorophore probe bound to a target molecule; contacting the sample with an untagged bioactive agent; and monitoring emission within the emission spectra of the bioluminescent reporter and the fluorophore, wherein emission from the fluorophore is the result of bioluminescence resonance energy transfer (BRET) from the bioluminescent reporter to the fluorophore, and the amount of emission from the fluorophore (and / or the BRET ratio) correlates to the amount of PRG / fluorophore probe covalently attached to the target molecule.

81. 78. The method of claim 77, comprising calculating the percentage of crosslinking by normalizing the amount of PRG / fluorophore probe covalently attached to the target molecule to the maximum amount of PRG / fluorophore probe bound to the target molecule.

82. 78. The method of claim 77, further comprising detecting an emission within the emission spectrum of the bioluminescent reporter.

83. 78. The method of claim 77, wherein the bioluminescent reporter is a bioluminescent protein or a peptide or polypeptide that is a component of a bioluminescent complex.

84. 78. The method of claim 77, wherein a concentration of said untagged bioactive agent in said sample is greater than a concentration of PRG / fluorophore probe in said sample.

85. 1. A method for measuring photocrosslinking efficiency of PRG linked to a bioactive agent, comprising: contacting a sample containing a target molecule with a PRG / HA probe according to one of claims 1 to 14, wherein said target molecule is tethered to a bioluminescent reporter; binding said bioactive agent to said targeting molecule; irradiating the sample with light of a wavelength that converts the PRG into a reactive form, the reactive form of the PRG capable of forming a covalent bond with the target molecule; contacting the sample with a BRET reagent comprising the bioactive agent covalently tethered to a fluorophore, wherein an emission spectrum of the bioluminescent reporter overlaps with an excitation spectrum of the fluorophore; contacting said sample with a substrate for a bioluminescent reporter; detecting emission within the emission spectrum of the bioluminescent reporter and the fluorophore, wherein the emission from the fluorophore is the result of bioluminescence resonance energy transfer (BRET) from the bioluminescent reporter to the fluorophore.

86. not contacting a sample containing a target molecule with a PRG / HA probe, said target molecule being tethered to a bioluminescent reporter; irradiating the sample with light of a wavelength that converts the PRG into a reactive form, the reactive form of the PRG capable of forming a covalent bond with the target molecule; contacting the sample with a BRET reagent comprising the bioactive agent covalently tethered to a fluorophore, wherein an emission spectrum of the bioluminescent reporter overlaps with an excitation spectrum of the fluorophore; contacting said sample with a substrate for a bioluminescent reporter; 78. The method of claim 77, further comprising detecting an emission within the emission spectrum of the bioluminescent reporter and the fluorophore, wherein the emission from the fluorophore is the result of bioluminescence resonance energy transfer (BRET) from the bioluminescent reporter to the fluorophore.

87. 79. The method of claim 78, further comprising (i) comparing an emission spectrum collected from a sample treated with a PRG / HA probe and illuminated with light of a wavelength that converts the PRG to a reactive form of the PRG with an emission spectrum collected from a sample not treated with the PRG / HA and illuminated with light of the same wavelength that converts the PRG to a reactive form of the PRG, wherein the reduction in BRET due to pretreatment with the PRG / HA is inversely correlated to the efficiency of photocrosslinking efficiency of the PRG.

88. 1. A composition comprising a photoaffinity label (PRG) linked to a bioactive agent, wherein the PRG is selected from: 【Chemistry 9】

89. 1. A method for covalently attaching a bioactive agent to a target protein, comprising: contacting the target molecule with the composition of claim 80, wherein the bioactive agent is capable of associating with the target molecule; Associating the bioactive agent with the targeting molecule; and The method includes irradiating the PRG with light of a wavelength that converts the PRG into a reactive form, wherein the reactive form of the PRG forms a covalent bond with the target molecule.

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