Compositions and methods for chemoproteomic interaction site mapping

Abstract

Disclosed herein are compositions and methods of synthesizing chemoproteomics capture reagents.

Description

[0001] Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO COMPOSITIONS AND METHODS FOR CHEMOPROTEOMIC INTERACTION SITE MAPPING RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 654,562, filed May 31, 2024, the contents of which are incorporated herein by reference in their entirety. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under GM146246 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND Nearly all small molecule therapeutics exert their effects through binding to proteins. Therefore, deciphering the protein targets, precise sites, and modes of binding is a key aspect of most chemical probe and drug discovery campaigns. Complementary to structure-guided approaches, mass spectrometry-based chemoproteomics has emerged as enabling technology for target deconvolution. Numerous innovative methods including thermal protein profiling(TPP), limited proteolysis (LiP), stability of proteins from rates of oxidation (SPROX),covalent protein painting (CPP), and photoaffinity labeling (PAL), have yielded proteome-wide portraits of protein-protein, protein-oligonucleotide, protein-lipid, protein-metabolite,protein-cofactor, protein-small molecule, and even protein-drug interactions. However, suchinteractomic studies almost invariably stop short of capturing the precise sites and modes of interactions. A key reason for this gap is a lack of suitable photoaffinity-labeling (PAL) chemistries compatible with global mass spectrometry-based interaction site identification. PAL is a method used to chemically trap, identify, and study stable and transient protein interactions. For a molecule of interest (MOI)—typically a small molecule, but in some cases a larger biomolecule such as an nucleic acid, peptide, or protein—implementation of PAL requires incorporation of three new important functionalities: (1) A photo-activatable moiety (e.g. diazirine, aryl azide, or benzophenone), (2) an enrichment handle (e.g. an alkyne or biotin), and (3) a linker / spacer necessary for optimal positioning of the photo-activatable group (FIG.1A). To identify interacting proteins, a MOI bearing a photo-affinity group and enrichment handle is introduced to the sample of interest (e.g. cells, lysates, or purified Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO protein) and the mixture is irradiated with UV light, which triggers the rapid release of highly reactive intermediates, such as a radical, nitrene, or carbene. These high-energy species react quickly and irreversibly with proximal biomolecules. For proteins, labeling occurs via insertion into the peptide backbone or by modifying amino acid side chains. MOI-labeled proteins are then visualized by immunoblot or gel-based activity-based protein profiling(ABPP), and, after affinity purification, identified by mass spectrometry-basedchemoproteomics. PAL has been enthusiastically received and widely adopted by far reaching communities. However, its use in site-of-interaction studies has been hindered by complex and often convoluted fragmentation of crosslinked molecules during tandem MS (MS / MS)analysis (FIG. 1A), and the synthetic challenges associated with obtaining photoactivatableprobes, such as optimization of the linker length and photocrosslinker placement. In view of the foregoing, there is an ongoing, unmet need for the development of new photoactivatable probes for photoaffinity-labeling. SUMMARY OF THE INVENTION In certain aspects, the present disclosure provides compound having a structure represented by formula I, formula II, or a salt thereof: wherein: A is a binding partner for a target protein (e.g., asciminib or dasatinib) or is selected from aryl (e.g., phenyl), aralkyl, heteroaryl, and heterocyclyl; E is an enrichment handle (e.g., Lys(biotin)-OH); L1is selected from alkylenyl, -S-, -NH-, -CO-, -NH(CO)NH-, -O(CO)O-, -NH(CO)O-, -O(CO)NH-, -NRQ(CO)O-, -O(CO)NRQ-, -SO-, and -SO2-, or is absent; Y1, Y2, and Y3are each independently S or O; X1and X2are each independently CH or N; X3and X6are each independently O, S, or NR6; X4and X5are each independently alkylenyl; Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Xaa, Xab, and Xacare each natural or unnatural amino acids, each of which may be isotopically enriched with one or more instances of an isotope, e.g., 2H, 15N, 18O, or 13C;R1and R2are each independently alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl; R3and R4are each independently alkyl, aralkyl, or aryl; R5and R6are each independently H or alkyl; RQ, taken together with the intervening atoms, forms a cycloalkyl or a heterocyclyl; n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; n2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; n3 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; and n4 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some aspects, the present disclosure provides methods of synthesizing a chemoproteomic capture reagent comprising: contacting a solid support with an enrichment handle selected from an amino acid substituted with biotin, an antibody, a sugar, a ubiquitin tag, and a metal chelator, thereby creating a solid support-enrichment handle conjugate; contacting the solid support–enrichment handle conjugate with a solid–phase compatible cleavable linker, thereby creating a solid support–enrichment handle-solid-phase compatible cleavable linker conjugate; contacting the solid support–enrichment handle–solid–phase compatible cleavable linker conjugate with a click capture amino acid, thereby creating a solid support-enrichment handle–solid-phase compatible cleavable linker–click capture amino acid conjugate; contacting the solid support–enrichment handle–solid–phase compatible cleavable linker–click capture amino acid conjugate with a first isotopically labelled amino acid, thereby creating a solid support–enrichment handle-solid–phase compatible cleavable linker– click capture amino acid–first isotopically labelled amino acid conjugate; and contacting the solid support–enrichment handle–solid–phase compatible cleavable linker–click capture amino acid–first isotopically labelled amino acid conjugate with a second isotopically labelled amino acid, thereby creating the chemoproteomic capture reagent. In some aspects, the present disclosure provides methods of identifying a binding site comprising: Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO contacting a substrate with the compound of the present disclosure, or a salt thereof; thereby creating a substrate-diazirine-alkyne conjugate; contacting the substrate-diazirine-alkyne conjugate with a chemoproteomic capture reagent comprising an azide; thereby creating a substrate-chemoproteomic capture reagent conjugate; and determining the molecular weight of the substrate-chemoproteomic capture reagent conjugate; thereby identifying the binding site. BRIEF DESCRIPTION OF THE DRAWINGS FIGs.1A-1C show the strategic differences in photo-affinity labeling between conventional probes and SEE-CITE probes. FIG.1A shows that conventional photo-affinity probes yield MOI-dependent modification of proteins, which can fragment in unexpected manner. FIG.1B shows the design of Silyl Ether Enable Chemoproteomic Interaction and Target Engagement (SEE-CITE) probes. FIG.1C shows that SEE-CITE probes yield MOI- free modification of proteins, which makes MS analysis easier. FIGs.2A-2H depict the workflow towards establishing the SEE-CITE interaction site mapping platform using scout SEE-CITE probes. FIG.2A depicts a comparison of proteomes labeled by probes 1 versus 2a-d for cell-based compound treatment (20 μM, 1 h) in K562 cells, visualized by click conjugation to tetramethylrhodamine (TAMRA) azide and in-gel fluorescence. FIG.2B shows structural examples of the SEE-CITE probe-modified peptides clicked with C3- or C4-biotin azides. FIG.2C shows the optimization of the peptide- level enrichment sample preparation method with 2b (20 μM). Each bar graph shows the mean number of modified peptides across multiple biological replicates (n=2 or 3, per sample) for each condition. FIG.2D depicts a comparison of the number of the modified peptides between 2a (20 μM) and 2b (20 μM) under the optimized condition. Each data is represented as a mean of three biological replicates. Statistical significance was calculated with a two-tailed student’s t-test, **p < 0.01,***p < 0.001, and NS p > 0.05. FIG.2E shows the labeling preferences of 2a for each amino acid residue. The number of peptide-spectrum matches (PSMs) modified by 2a for each amino acid residue was extracted from each biological replicate data of 140 min gradient shown in FIG.2D. The mean of the triplicates is shown on the y-axis. FIG.2F shows a volcano plot of whole proteins identified in protein- level enrichment experiment by 2a (20 μM). The data was acquired by the label-free quant Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO (LFQ) method with triplicate samples on FragPipe and the raw data was analyzed by the label-free quant (LFQ) method on FragPipe and missing values were imputed by Perseus. The cutoff of the log2(FC) {FC: the protein intensities of the treated group / the control group} was set as >1.0, and the cutoff of the p-value was set as p < 0.05. Enriched proteins are highlighted in blue dots and unenriched proteins are highlighted in gray dots. FIG.2G shows that the Venn diagram exhibits overlap among the enriched proteins and the unenriched proteins identified in FIG.2F, and 2a-modified proteins without singletons extracted from each biological replicate data of 140 min gradient in FIG.2D. FIG.2H shows stratification by protein class for all identified proteins (left pie chart) and proteins enriched by 2a (right pie chart) in FIG.2F. FIGs.3A-3C depict the validation of BCR-ABL1 target engagement and labeling profile of dasatinib and asciminib probes. FIG.3A shows Western blots of dasatinib probes (4a-4c) in KCL-22. FIG.3B shows Western blots of asciminib probes (5a-5c) in KCL-22 cells. KCL-22 cells were incubated with dasatinib probes 4a-c for 2 h or with asciminib probes 5a-c for 4 h at the indicated concentration at 37 ℃ followed by 20-min UV irradiation on ice. The lysates of harvested cells were then resolved by SDS-PAGE and subjected to immunoblot analysis. FIG.3C shows labeling profiles of dasatinib and asciminib probes in either KCL-22 or K562 cell lines. Each cell line was incubated with the corresponding probes at the indicated concentration for 1 hour at 37 °C, then UV (365 nm) was irradiated on ice for 20 min. After harvesting and lysing the cells, CuAAC with TAMRA azide was conducted. Labeled proteins were visualized by TAMRA fluorescence scanning by the ChemiDoc imaging system after SDS-PAGE. FIGs.4A-4I show the interactome analysis of asciminib. FIG.4A shows that the volcano plot representing the protein abundance changes in UV± experiments with 5b (10μM) and asciminib (50 μM) in the KCL-22 cells. Data was acquired by the LFQ method withtriplicate samples on FragPipe. Missing values were imputed by Perseus. The cutoff of the log2(FC) was set as >1.0 for UV± experiments and set as >0.5 for competition experiments. The cutoff of the p-value was set as p < 0.05 for all experiments. Significantly enriched proteins are highlighted and kinases are annotated in black. FIG.4B shows that the volcano plot representing the protein abundance changes in competition experiments with 5b (10 μM) and asciminib (50 μM) in the KCL-22 cells. Data was acquired by the LFQ method with triplicate samples on FragPipe. Missing values were imputed by Perseus. The cutoff of the Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO log2(FC) was set as >1.0 for UV± experiments and set as >0.5 for competition experiments. The cutoff of the p-value was set as p < 0.05 for all experiments. Significantly enriched proteins are highlighted and kinases are annotated in black. FIG.4C depicts a heatmap of picked up off-target candidates for asciminib in K562 or KCL-22. FIG.4D depicts a heatmapof detection of BCR / ABL1 peptide by 4b (100 μM) or 5b (10 μM) in KCL-22 cells. FIG. 4Eshows a kinome tree annotation created by KinMap to highlight enriched kinases labeled by4b (100 μM) or 5b (10 μM) in KCL-22 cells. FIG. 4F shows that the volcano plotrepresenting the protein abundance changes in a probe vs probe experiment with 4b and 5b in K562 cells. K562 cells were treated with 10 μM of 4b or 5b for 1 h, then UV was irradiated. The data was acquired by the label-free quant (LFQ) method with triplicate samples and missing values were imputed by Perseus. The cutoff of the log2(FC) was set as >1.0 and the cutoff of the p-value was set as p < 0.05. Significantly enriched proteins by 4b are highlighted, proteins by 5b are highlighted and kinases are annotated in black. FIG.4G shows that the volcano plot representing the protein abundance changes in a UV± experiment with 5c in K562 cells. K562 was treated with 100 μM of 5c for 1 h, then UV was irradiated. The data was acquired by the label-free quant (LFQ) method with triplicate samples and missing values were imputed by Perseus. The cutoff of the log2(FC) was set as >1.0 and the cutoff of the p-value was set as p < 0.05. Significantly enriched proteins by 5c are highlighted and kinases are annotated in black. FIG.4H shows that the Venn diagram exhibits overlap between all the identified proteins by 5b in UV± experiments in K562 cells and those by 5c in UV± experiments in K562 cells (FIG.5F). FIG.4I shows that the Venn diagram exhibits overlap between significantly enriched proteins by 5b in UV± experiments in K562 and those by 5c in UV± experiments in K562 cells (FIG.5F). The y-axis stands for the average of unique counts of peptides or proteins modified by 5c across three replicates. FIGs.5A-5C show the SEE-CITE mapping of ABL binding sites. FIG.5A shows a gel-based AfBPP analysis comparing PAL-labeling of the kinase domain of recombinant ABL1 protein by dasatinib probes 4b / 4c and asciminib probes 5b / 5c.1 mg / mL HEK293T cell lysate spiked with or without 1 μM of the kinase domain of ABL1 recombinant protein was treated with DMSO or 10x competitors (parent kinase inhibitors) for 15 min prior to 10 μM treatment of photo-affinity probes at room temperature for 30 min. After UV irradiation for 20 min, CuAAC with TAMRA azide was conducted. Labeled proteins were visualized by TAMRA fluorescence scanning after separation of proteins by SDS-PAGE. FIG.5B shows Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO the photo-affinity labeling of recombinant ABL1 protein with dasatinib and asciminib probes at different concentrations.1 mg / mL HEK cell lysate spiked with or without 1 μM of the kinase domain of ABL1 recombinant protein was treated with 10 or 1 μM photo-affinity probes at room temperature for 30 min. After UV irradiation for 20 min, CuAAC with TAMRA azide was conducted. Labeled proteins were visualized by TAMRA fluorescence scanning after separation of proteins by SDS-PAGE. FIG.5C shows the workflow of quantitative AfBPP experiment with SEE-CITE probes 4c and 5c. In brief, 1 mg / mL HEK cell lysate spiked with 1 μM of the kinase domain of ABL1 recombinant protein was treated with 1 μM treatment of 4c or 5c at room temperature for 30 min. After UV irradiation for 20 min, CuAAC with light biotin azide (for 4c) or heavy biotin azide (for 5c) was conducted. After the click reaction, each mixture was combined into one and then peptide-level enrichment sample preparation was conducted. FIGs.6A-6L depict the SEE-CITE analysis of asciminib off-targets. FIG.6A shows a gel-based AfBPP analysis comparing 4c (100 μM) and 5c (100 μM) in K562 cells, 2a (20μM) and 5c (100 μM) in MOLT-4 cells. Each cell line was incubated with the correspondingprobes at the indicated concentration for 1 hour at 37 ℃, then UV (365 nm) was irradiated on ice for 20 min. After harvesting and lysing the cells, CuAAC with TAMRA azide was conducted. Labeled proteins were visualized by TAMRA fluorescence scanning by the ChemiDoc imaging system after SDS-PAGE. FIG.6B depicts scatter plots for probe- modified peptides whose log2(H / L) values were calculated in K562 cells (100 μM 4c vs 100 μM 5c). Dots stand for FECH peptides and black color means the average of log2(H / L) of 2 replicates was >0. FIG.6C shows mapping of peptides dominantly labeled by probe 5c in the crystal structure of FECH dimer (PDB: 2QD5). FIG.6D depicts scatter plots for probe- modified peptides whose log2(H / L) values were calculated in K562 cells (100 μM 4c vs 100 μM 5c). Dots stand for RTN4 peptides and black color means the average of log2(H / L) of 2 replicates was >0. FIG.6E depicts in-gel fluorescence analysis of 5c in NogoC (RTN4) WT / C1101S overexpressed HEK293T cells and western blotting (WB) of the same samples. FIG.6F depicts scatter plots for probe-modified peptides whose log2(H / L) values were calculated in STING overexpressed HEK293T cells (20 μM 2a vs 100 μM 5c). Dots stand for STING peptides and black color means the average of log2(H / L) of 2 replicates was >0 and dark grey color means <0. FIG.6G shows the mapping of peptides dominantly labeled by probe 5c in the crystal structure of STING dimer (PDB: 6NT5). FIG.6H shows scatter plots Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO for probe-modified peptides whose log2(H / L) values were calculated in K562 cells (100 μM 4c vs 100 μM 5c). Dots stand for COX5A peptides and black color means the average of log2(H / L) of 2 replicates was >0 and dark grey color means <0. FIG.6I shows scatter plots for probe-modified peptides whose log2(H / L) values were calculated in MOLT-4 cells (20 μM 2a vs 100 μM 5c). Dots stand for COX5A peptides and black color means the average of log2(H / L) of 2 replicates was >0 and dark grey color means <0. FIG.6J depicts the labeling site mapping of the crystal structure of human COX (PDB: 5Z62) by SEE-CITE probes and crystal structure of PSC in bovine heart COX (PDB: 1V54). T79 and Y80 residues in the human COX corresponded to G81 and Y82 residues in the bovine heart COX, respectively. FIG.6K shows the in situ labeling of COX5A-FLAG overexpressed in HEK293T cells using 5b via a concentration-dependent manner. FIG.6L shows competitive labeling of COX5A- FLAG overexpressed in HEK293T cells using 5b after pre-treatment of cells with asciminib at various concentrations. HEK293T cells overexpressing GFP-FLAG were also included in in-gel fluorescence experiments (K) and (L) as negative controls. FIGs.7A-7D show the stability of the prototype photo-affinity labeling probes via TLC. The stability of the prototype probes was analyzed under cellular experimental or elution from neutravidin condition. Each probe was put in an Eppendorf tube was mixed with the corresponding solvent (10 mM concentration, FBS- and antibiotics-free media) and subjected to the indicated temperature and time, with 1,000 rpm shaking for B and C. After that, the mixture was partitioned with ether and water, then the organic layer was monitored by TLC with KMnO4staining reagent. All probes were stable enough under general cellular labeling conditions. Probes except 2d were completely cleaved under the elution condition from neutravidin beads. FIG.7A shows the stability of the probes before treatment. FIG.7B shows the stability of the probes to RPMI at 37 ℃ for 1 hour. FIG.7C shows the stability of the probes to DMEM at 37 ℃ for 1 hour. FIG.7D shows the stability of the probes to 0.1% TFA / 80% CH3CN (aq.), room temperature, 10 minutes. FIGs.8A-8B show the X-ray crystal structure of kinase inhibitor-bound ABL kinase domain. FIG.8A depicts dasatinib (BMS-354825) Bound to Activated ABL Kinase Domain (PDB: 2GQG). FIG.8B depicts ABL1 kinase (T334I_D382N) in complex with asciminib and nilotinib (PDB: 5MO4). Arrows show the vectors for installing diazirine linkers. FIG.9 depicts an overview of diazirine-based photocrosslinking. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO FIGs.10A-10B depict the synthesis of the sCIP-DiLeu reagents. FIG.10A shows asymmetric dimethlleucine reagents with * atoms as positions of heavy isotope incorporation (15N,13C, or2H). FIG.10B shows the structure of 29-plex sCIP reagents. FIGs.11A-11G show that 29-plex sCIP-DiLeu reagents achieve accurate quantification. FIG.11A shows the synthesis of sCIP reagents. FIG.11B shows the masses of the 29-plex set of isobaric balancer and reporter combinations. FIG.11C shows the workflow for cysteine chemoproteomic sample preparation for reagent benchmarking. FIG. 11D shows a ratio spike in experiments demonstrating comparable ratios for all reagents. FIG.11E shows a ratio spike in experiments demonstrating comparable ratios for all reagents. FIG.11F shows ratios for spike in samples acquired with FAIMS-MS2 or SPS- MS3. FIG.11G shows a comparison of coefficients of variance for each acquisition method. FIG.12 depicts the workflow of the multiplexed sCIP-SEE-CITE chemoproteomics platform. Live cells are first treated with a reversible fragment for 0.5h followed by 0.5h probe treatment with SEE-CITE scout molecule 2a using serum- and antibiotic-free media at 37℃ and 5% CO2. Competitively treated samples then undergo UV irradiation at 365 nm for 20 minutes on ice. Samples are lysed, normalized and subjected to a copper catalyzed azide- alkyne cycloaddition reaction with in-house sCIP-DiLeu reagents for 1 hour at room temperature. Post-clicked samples are then combined into one single sample, which then underwent sample cleanup using chloroform-methanol precipitation, reduction with dithiothreitol (DTT), alkylation with iodoacetamide (IA) and tryptic digestion overnight. Digested peptides are next enriched with streptavidin for 2 hours at room temperature and eluted with 2% formic acid for 0.5 h at room temperature. Enriched peptides are then dried via speedvac, reconstituted in MS solvent with 5% acetonitrile and 1% formic acid) and analyzed by LC-MS / MS. FIGs.13A-13B depict the advances of sCIP-SEE-CITE proteomic with high- throughput screening of large libraries and improvement of coverage and sample preparation. FIG.13A shows the coverage comparison of SEE-CITE and sCIP-SEE-CITE done in MOLT4 cells with compounds 6 or 7 for SEE-CITE and compounds 1-6 for sCIP-SEE-CITE. FIG.13B shows the cell amounts required for SEE-CITE (60 million cells per condition) versus sCIP-SEE-CITE (2 million cells per condition). FIGs.14A-14C depicts fragment screening with the sCIP-SEE-CITE platform. FIG. 14A shows a focused panel of reversible fragments featuring various chemotypes. FIG.14B Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO shows high-value targets identified for fragment-screening experiment with compounds 1-5 (50 µM) and SEE-CITE scout probe 2a (10 µM). FIG.14C shows high-value targets identified for concentration-dependent sCIP-SEE-CITE with compounds 6 and 7 (1-50 µM) and SEE-CITE scout probe 2a (10 µM). FIG.15 shows structures of identified binding sites (magenta) for protein capture by sCIP-SEE-CITE. ECTL2, Alphafold ID AF-Q008S8; ADA, PDB ID: 3IAR; EPHX2, PDB ID: 1VJ5; HLA-A*03, PDB ID: 1AJK. FIG.16 depicts the structures of the sCIP-DiLeu reagents of this disclosure. Positions of isotopic labels are indicated by an asterisk (*). DETAILED DESCRIPTION OF THE INVENTION Most therapeutics are small molecules that function by interacting with specific protein targets. Consequently, delineating the protein targets, including the specific binding sites, for chemical probes and clinical candidates is essential to ensure potent on-target activity and minimize engagement of unfavorable off-targets. The pairing of mass spectrometry-based chemoproteomics with photoaffinity labeling (PAL) has emerged as a favored approach to generate proteome-wide target engagement maps for reversible compounds. However, a key limitation of most PAL-based proteomic platforms is the absence of strategies that report precise binding sites and enable direct head-to-head comparisons of relative target engagement at these sites by different lead compounds. This gap stems from a confluence of factors including the complex fragmentation patterns for crosslinked peptides, poor recovery of peptides crosslinked with large hydrophobic molecules, and modification masses that differ between molecules of interest (MOI). To address these challenges, here we establish the Silyl Ether Enables Chemoproteomic Interaction and Target Engagement (SEE-CITE) approach, which incorporates an unprecedented fully functionalized chemically cleavable cross-linking handle that enables precise site- of-labeling identification and head-to-head comparisons of relative binding site engagement by chemically diverse compounds. By benchmarking SEE-CITE using scout fragments and analogues of the FDA-approved kinase inhibitors dasatinib and asciminib, we identify known and novel binding sites, including for high impact targets, such as BCR- ABL1, STING, and COX5A. Nearly all small molecule therapeutics exert their effects through binding to proteins. Therefore, deciphering the protein targets, precise sites, and modes of binding is a key aspect of most chemical probe and drug discovery campaigns. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Complementary to structure-guided approaches, mass spectrometry-based chemoproteomics has emerged as enabling technology for target deconvolution. Numerous innovative methodsincluding thermal protein profiling (TPP), limited proteolysis (LiP), stability of proteins fromrates of oxidation (SPROX), covalent protein painting (CPP), and photoaffinity labeling(PAL), have yielded proteome-wide portraits of protein-protein, protein-oligonucleotide,protein-lipid, protein-metabolite, protein-cofactor, protein-small molecule, and even protein-drug interactions. However, such interactomic studies almost invariably stop short ofcapturing the precise sites and modes of interactions. A key reason for this gap is a lack of suitable photoaffinity-labeling (PAL) chemistries compatible with global mass spectrometry- based interaction site identification. PAL is a method used to chemically trap, identify, and study stable and transient protein interactions. For a molecule of interest (MOI)—typically a small molecule, but in some cases a larger biomolecule such as an nucleic acid, peptide, or protein—implementation of PAL requires incorporation of three new important functionalities: (1) A photo-activatable moiety (e.g., diazirine, aryl azide, or benzophenone), (2) an enrichment handle (e.g. an alkyne or biotin), and (3) a linker / spacer necessary for optimal positioning of the photo-activatable group (FIG.1A). To identify interacting proteins, a MOI bearing a photo-affinity group and enrichment handle is introduced to the sample of interest (e.g. cells, lysates, or purified protein) and the mixture is irradiated with UV light, which triggers the rapid release of highly reactive intermediates, such as a radical, nitrene, or carbene. These high-energy species react quickly and irreversibly with proximal biomolecules. For proteins, labeling occurs via insertion into the peptide backbone or by modifying amino acid side chains. MOI-labeled proteins are then visualized by immunoblot or gel-based activity-based protein profiling(ABPP), and, after affinity purification, identified by mass spectrometry-basedchemoproteomics. PAL has been enthusiastically received and widely adopted by far reaching communities. However, its use in site-of-interaction studies has been hindered by complex and often convoluted fragmentation of crosslinked molecules during tandem MS (MS / MS)analysis (FIG. 1A), and the synthetic challenges associated with obtaining photoactivatableprobes, such as optimization of the linker length and photocrosslinker placement. To overcome these limitations, focus has turned to synthesis of 'minimalized' alkyne‐ anddiazirine-containing alcohol 1 (Scheme 1), and to specialized methods aimed at improving the Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO MS / MS identification of crosslinked peptides, including custom searches of unassignedspectra and new open search algorithms, such as MSFragger. While useful for improvingcoverage of labeled peptides, such methods are frequently not scalable and additionally fail to address the bulkiness of most chemical crosslinkers, which preclude application to systems intolerant of large substitutions. Here, the development and application of Silyl Ether Enables Chemoproteomic Interaction and Target Engagement (SEE-CITE) for protein labeling is reported, which enables precise and high coverage interaction site mapping for elaborated and fragmentation prone drugs. In contrast with conventional diazirine-functionalized probes, the bulky drug- like molecule in SEE-CITE probes are easily released from the cross-linked peptide under low pH conditions (FIG.1C). After release, the SEE-CITE probes are distinguished by their minimalist silyl ether moiety, featuring 1 as a minimalist alcohol component. Implementation of this cleavage step enables unprecedented pair-wise quantitation of relative target engagement at specific binding sites. For the recently FDA-approved tyrosine kinase inhibitor(TKI) asciminib (ABL001), application of the SEE-CITE-method yielded precise interactionsites, including on-target (BCR-ABL1 myristoyl binding pocket) and de novo discovery of previously uncharacterized off- targets for this anti-chronic myelogenous leukemia (CML) agent. Diazirine-based photoaffinity labeling is widely adopted for mapping the protein interactome, including for deciphering the mode-of-action (MOA) of small molecules, and for mapping the naturally occurring interactome, spanning metabolite-, glycan-, lipid-, and protein-protein interactors. While identifying precisely where protein interactions occur is of high value for guiding downstream mechanistic studies, most PAL platforms fail to achieve high resolution interaction site mapping. Here, this gap is addressed by developing the SEE- CITE platform. SEE-CITE harnesses diazirine probes functionalized with a silyl ether-based acid cleavable linker, which allows for facile separation of the crosslinked molecule from the labeled peptide. The minimalist and compound agnostic molecular barcode that is left behind after cleavage both allows for improved binding site identification and quantitative comparison of the relative direct labeling for structurally diverse probes. To establish SEE-CITE, a synthetic route to access prototype “scout” SEE-CITE probes was first established. Using these probes, the proteomic labeling site identification was then optimized, including the sample preparation workflow, and LC-MS / MS acquisition. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO These studies revealed that the scout SEE-CITE probes showed a pronounced bias towards labeling acidic residues (FIG.2E), which is consistent with prior reports for such diazirine- based probes and points towards the presence of proximal acidic residues as favorable for efficient protein crosslinking. As this acidic residue labeling is generally appreciated to also occur via the diazo species, a broader radius of labeling is anticipated for alkyl diazirine compounds, in comparison to the aryl-fluorodiazirine counterparts which primarily reactthrough a carbene intermediate. Thus, the delineation of bona fide targets from more non-specific protein alkylation benefits from both probe-probe comparisons and competition studies. The SEE-CITE platform was then applied to compare the protein interactomes of two FDA approved TKIs, dasatinib and asciminib. Excitingly, it was found that the SEE-CITE platform accurately pinpoints the binding sites of these two compounds to the active and allosteric sites, respectively, of the clinically relevant target ABL kinase (FIG.5A). This performance is particularly noteworthy given that these sites were accurately identified despite some differences in the general relative reactivities of the asciminib and dasatinib probes. This high accuracy delineation of binding site engagement for a known target prompted us to then extend the SEE-CITE platform to characterize the asciminib interactome more broadly. These studies pinpoint intriguing binding sites for a number of high value targets, including STING, FECH, RTN4, and DHX9. The high concordance between theproteins captured by parent diazirine 4b and SEE-CITE probe 5c (FIG. 5B) provides furtherevidence that the comparatively bulky and lipophilic silyl ether functionality does not substantially alter a probe’s interactome. Intriguingly, for RNT4, this work demonstrates that mutation of a single highly reactive cysteine at the SEE-CITE identified binding site substantially reduced probe labeling. This finding points to the possibility that pKa-perturbed cysteines were readily modified by diazirine probes. The finding that asciminib engages directly with COX5A, which is a component of complex IV, hints at a possible mechanism for the previously reported decreased oxygen consumption rate (OCR) in response to asciminib As asciminib only received FDA approval two years ago, it is expected that these findings should add to the growing body of literature aimed at understanding the potential off-targets and therapeutic synergies and drug repurposing opportunities. These efforts to develop SEE-CITE highlight several important and still largely unfulfilled opportunities for interaction site mapping. One challenge that was encountered Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO while establishing SEE-CITE was the still generally modest coverage of labeled peptides afforded by the platform (~1,500 unique peptides (labeled sites?) / experiment). This finding aligns with the recently reported and conceptually related amine-reactive cleavable linkers, for which labeling site analysis was restricted to recombinant protein. The comparatively low coverage of BCR and ABL proteins identified using a vast excess (100 μM) of the non-SEE- CITE parent diazirine-based dasatinib probe, precluded SEE-CITE analysis of the endogenous fusion oncoprotein. This finding aligns with prior studies that sought to analyze BCR-ABL-dasatinib interactions, and provides compelling evidence to support the need for new protein interaction mapping technologies that afford enhanced labeling efficiency to better capture low abundance protein targets using clinically relevant probe concentrations. To demonstrate the utility of SEE-CITE, the technology has been paired with a newly established 29-plex isobaric chemoproteomic capture reagents (sCIP) to enable high throughput and multiplexed interaction site identification. The key advantages of this sCIP- SEE-CITE platform are reduced sample requirements, decreased sample prep time, multiplexing to allow for dose titrations and structure activity relationship (SAR) analysis within the same sample, and competition analysis to enable mapping of reversible compounds using generalized scout probes. By applying the sCIP-SEE-CITE platform binding sites are identified in high value targets, such as HLA, CCT6A, and ECT2L. In its first implementation, SEE-CITE sample preparation and data acquisition were conducted in a pairwise manner. Samples treated with different SEE-CITE probes that feature the silyl ether diazirine alkyne group followed by click conjugation to isotopically enriched (heavy versus light) biotin-azide enrichment handles. After sample pooling, tryptic digest and enrichment on streptavidin resin, the relative labeling was then quantified at the MS1 level based on the precursor ion intensities of light versus heavy peptides. Thus, this method, while fully functional, remained low throughput both for sample preparation and data acquisition. Here, the silane-based cleavable linkers for isotopically labeled proteomics (sCIP) method is combined with the SEE-CITE method to establish the sCIP-SEE-CITE platform that enhances sample throughput and enables multi-dimensional screening of many compounds in parallel. sCIP-SEE-CITE is then deployed to the screening of focused libraries of reversible fragments, which identifies a number of lead compounds for high value targets. Widespread utility for SEE-CITE in capturing novel drug binding sites is possible, including via multiplexed analysis, and, more broadly for interaction site mapping for larger Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO and highly gas-phase labile molecules, including lipids such as cholesterol, glycans, and even protein-protein interactions, using unnatural amino acids incorporated through genetic code expansion. In certain aspects, the present disclosure provides compounds having a structure represented by formula I, formula II, or a salt thereof: wherein: A is a binding partner for a target protein (e.g., asciminib or dasatinib) or is selected from aryl (e.g., phenyl), aralkyl, heteroaryl, and heterocyclyl; E is an enrichment handle (e.g., Lys(biotin)-OH); L1is selected from alkylenyl, -S-, -NH-, -CO-, -NH(CO)NH-, -O(CO)O-, -NH(CO)O-, -O(CO)NH-, -NRQ(CO)O-, -O(CO)NRQ-, -SO-, and -SO2-, or is absent; Y1, Y2, and Y3are each independently S or O; X1and X2are each independently CH or N; X3and X6are each independently O, S, or NR6; X4and X5are each independently alkylenyl; Xaa, Xab, and Xacare each natural or unnatural amino acids, each of which may be isotopically enriched with one or more instances of an isotope, e.g., 2H, 15N, 18O, or 13C;R1and R2are each independently alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl; R3and R4are each independently alkyl, aralkyl, or aryl; R5and R6are each independently H or alkyl; RQ, taken together with the intervening atoms, forms a cycloalkyl or a heterocyclyl; n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; n2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; n3 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; and n4 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the compound has a structure represented by formula I, formula II, or a salt thereof: Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO I II wherein: A is a binding partner for a target protein (e.g., asciminib or dasatinib) or is selected from aryl (e.g., phenyl), heteroaryl, and heterocyclyl; E is an enrichment handle (e.g., Lys(biotin)-OH); L1is selected from alkylenyl, -S-, -NH-, -CO-, -NH(CO)NH-, -O(CO)O-, -NH(CO)O-, -O(CO)NH-, -SO-, and -SO2-, or is absent; Y1, Y2, and Y3are each independently S or O; X1and X2are each independently CH or N; X3and X6are each independently O, S, or NR6; X4and X5are each independently alkylenyl; Xaa, Xab, and Xacare each natural or unnatural amino acids, each of which may be isotopically enriched with one or more instances of an isotope, e.g., 2H, 15N, 18O, or 13C;R1and R2are each independently alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl; R3and R4are each independently alkyl, aralkyl, or aryl; R5and R6are each independently H or alkyl; and n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; n2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; n3 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; and n4 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the compound has a structure represented by formula Ia, or a salt thereof: Ia. In certain preferred embodiments, A is a small molecule. Small molecules are low molecular weight organic compounds, generally with a molecular weight of less than about Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 900 daltons. One of skill in the art would understand which small molecules could be incorporated into the invention by considering several factors: 1. Molecular size and structure: The small molecule should be compatible with thesize constraints discussed herein, particularly in relation to the diazirine moiety and alkyne group for click chemistry reactions. 2. Binding affinity and specificity: The small molecule should demonstrateappropriate binding characteristics for the target protein of interest. 3. Photoreactivity compatibility: The chosen small molecule should not interferewith the photoreactive properties of the diazirine group. 4. Click chemistry compatibility: The small molecule should not contain functionalgroups that could compete with or inhibit the click chemistry reaction involving the alkyne group. 5. Stability and shelf-life: The small molecule should be stable under the conditionsrequired for synthesis, storage, and use of the invention. 6. Solubility and cell permeability: Depending on the intended application, the smallmolecule should possess appropriate physicochemical properties to function effectively in biological systems. By carefully considering these factors, one of skill in the art can select appropriate small molecules to incorporate into the invention, enhancing the molecules utility for chemoproteomic interaction site mapping and target engagement studies. Representative examples of small molecules include, but are not limited to asciminib, dasatinib, aspirin, imatinib, sofosbuvir, varenicline, abrocitinib, mitapivat, pacritinib, ganaxolone, futibatinib, daridorexant, deucravacitinib, sofosbuvir / velpatasvir, olutasidenib, sodium phenylbutyrate and taurursodiol, atorvastatin, metformin, oseltamivir, voriconazole, lenalidomide, clopidogrel, acetaminophen, ibuprofen, penicillin, duloxetine, sildenafil, desatinib, apixaban, rilpivirine, dexmethylphenidate, drospirenone, olanzapine, samidorphan, albuterol, simvastatin, felbamate, cyclopamine, actinomycin-D, clarithromycin, amoxicillin, vonoprazan, phorbol 12-myristate 13-acetate, retinoic acid, wrenchnolol, abacavir, lamivudine, efavirenz, tamoxifen, methotrexate, warfarin, rosuvastatin, fluoxetine, tramadol, carbamazepine, levetiracetam, omeprazole, pravastatin, lovastatin, odevixibat, oteseconazole, mavacamten, tapinarof, gadopiclenol, oomidenepag isopropyl, taurursodiol, amisulpride, risdiplam, vibegron, remimazolam, tucatinib, selpercatinib, nifurtimox, lonafarnib, Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO tirbanibulin, tazemetostat, oliceridine, opicapone, ozanimod, berotralstat, raltegravir, dolutegravir, bictegravir, tenofovir, emtricitabine, sofosbuvir / velpatasvir / voxilaprevir, daclatasvir, ledipasvir, elbasvir, grazoprevir, glecaprevir, pibrentasvir, boceprevir, telaprevir, simeprevir, paritaprevir, ombitasvir, dasabuvir, ribavirin, acyclovir, valacyclovir, ganciclovir, famciclovir, zidovudine, stavudine, didanosine, nevirapine, etravirine, rilpivirine, lopinavir, ritonavir, atazanavir, darunavir, indinavir, saquinavir, nelfinavir, fosamprenavir, tipranavir, entecavir, telbivudine, lamivudine, adefovir, cidofovir, foscarnet, maraviroc, enfuvirtide, dolutegravir, elvitegravir, abemaciclib, palbociclib, ribociclib, capecitabine, 5-fluorouracil, gemcitabine, cytarabine, decitabine, azacitidine, cladribine, fludarabine, mercaptopurine, thioguanine, pemetrexed, pralatrexate, temozolomide, dacarbazine, and procarbazine. In certain embodiments, the point of connection between A and L1is a bond. In certain embodiments, the point of connection between A and L1is alkyl. In certain embodiments, the point of connection between A and L1is an amine, such as a primary amine, a secondary amine, or a tertiary amine. In certain embodiments, the point of connection between A and L1is heterocyclyl. In some such embodiments, the point of connection between A and L1is a nitrogen-containing heterocyclyl, such as a piperidinyl, pyrrolidinyl, or a piperazinyl. In certain embodiments, A is aryl (e.g., phenyl). In some embodiments, A is heteroaryl. In certain embodiments, A is heterocyclyl. In certain preferred embodiments, A is asciminib. In other preferred embodiments, A is dasatinib. In some embodiments, A is aralkyl. In some such embodiments, A is benzyl. In some preferred embodiments, L1is alkylenyl, -S-, -NH(CO)NH-, -NH(CO)O-, - O(CO)NH-, -NRQ(CO)O-, or -O(CO)NRQ-; and wherein RQ, taken together with the intervening atoms, forms a heterocyclyl. In certain such embodiments, L1is alkylenyl, -S-, - NH(CO)NH-, -NH(CO)O-, or -O(CO)NH-. In certain embodiments, RQ, taken together with the intervening atoms, forms a piperazinyl. In some embodiments, RQ, taken together with the intervening atoms, forms an 8-azaspiro[4.5]decane. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO In some preferred embodiments, ; wherein represents the point of connection to the carbon atom connected to Si. In some preferred embodiments, ; wherein represents the point of connection to the carbon atom connected to Si. In certain preferred embodiments, L1is methylenyl, ethylenyl, propylenyl, butylenyl, pentylenyl, hexylenyl, heptylenyl, octylenyl, or nonylenyl. In some embodiments, L1is absent. In certain preferred embodiments, X1is N. In certain preferred embodiments, X2is N. In some embodiments, R1is selected from methyl, ethyl, isopropyl, and phenyl. In some embodiments, R2is selected from methyl, ethyl, isopropyl, and phenyl. In certain embodiments, n1 is 2. In certain embodiments, n1 is 3. In some embodiments, n2 is 2. In some embodiments, n3 is 2. In certain embodiments, the compound is selected from: Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO salt thereof. In certain embodiments, the compound has a structure represented by formula IIa, or a salt thereof: In some preferred embodiments, E is Lys(biotin)-OH. In certain embodiments, X3is S. In some embodiments, X4is alkyloxyalkyl. In some preferred embodiments, X4is substituted with alkyl, alkenyl, alkynyl, ester, amido, aryl, or heteroaryl. In certain especially preferred embodiments, X4is substituted with alkyl (e.g., methyl). In certain embodiments, X5is methylenyl, ethylenyl, propylenyl, butylenyl, pentylenyl, hexylenyl, heptylenyl, or octylenyl. In some preferred embodiments, X5is hexylenyl. In other preferred embodiments, X5is propylenyl. In certain embodiments, X5is substituted with alkylenyl, alkenylenyl, alkynylenyl, ester, amido, aryl, or heteroaryl. In certain embodiments, R5is H. In some embodiments, n4 is 2. In certain embodiments, X6is O. Alternatively, X6is NR6. In some such embodiments, R6is H. In certain preferred embodiments, Xaais 6-azido-lysine. In certain preferred embodiments, Xabis alanine. In other preferred embodiments, Xacis N,N-dimethyl-leucine. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO In certain embodiments, the compound has a structure represented by formula IIb or a salt thereof: IIb wherein: n5, n6, n7, and n8 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R7and R8are each independently alkyl, alkenyl, alkynyl, ester, amido, aryl, or heteroaryl. In certain embodiments, n5 is 2. In some embodiments, n6 is 3. In some embodiments, n7 is 1. In certain embodiments, n8 is 2. In some embodiments, n8 is 6. In certain preferred embodiments, R7is alkyl (e.g., methyl). In some preferred embodiments, R8is alkyl (e.g., methyl). In some preferred embodiments, R3is aryl (e.g., phenyl). In certain preferred embodiments, R4is aryl (e.g., phenyl). In some especially preferred embodiments, the compound is: ; or a salt thereof. In some aspects, the present disclosure provides methods of synthesizing a chemoproteomic capture reagent comprising: contacting a solid support with an enrichment handle selected from an amino acid substituted with biotin, an antibody, a sugar, a ubiquitin tag, and a metal chelator, thereby creating a solid support-enrichment handle conjugate; Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO contacting the solid support–enrichment handle conjugate with a solid–phase compatible cleavable linker, thereby creating a solid support–enrichment handle-solid-phase compatible cleavable linker conjugate; contacting the solid support–enrichment handle–solid–phase compatible cleavable linker conjugate with a click capture amino acid, thereby creating a solid support-enrichment handle–solid-phase compatible cleavable linker–click capture amino acid conjugate; contacting the solid support–enrichment handle–solid–phase compatible cleavable linker–click capture amino acid conjugate with a first isotopically labelled amino acid, thereby creating a solid support–enrichment handle-solid–phase compatible cleavable linker– click capture amino acid–first isotopically labelled amino acid conjugate; and contacting the solid support–enrichment handle–solid–phase compatible cleavable linker–click capture amino acid–first isotopically labelled amino acid conjugate with a second isotopically labelled amino acid, thereby creating the chemoproteomic capture reagent. In certain embodiments, the solid support is a resin. In some preferred embodiments, the resin is a chlorotrityl resin. In certain embodiments, the enrichment handle is an amino acid substituted with biotin (e.g., Lys(biotin)-OH). In some such embodiments, the amino acid is a naturally occurring amino acid (e.g., lysine or cysteine). In certain embodiments, the click capture amino acid is an azide containing amino acid (e.g., 6-azido-lysine). In some embodiments, the first isotopically labelled amino acid is a naturally occurring amino acid (e.g., valine or alanine). In some embodiments, the second isotopically labelled amino acid is an unnatural amino acid (e.g., N,N-dimethyl-leucine). In certain preferred embodiments, the first and second isotopically labelled amino acids are enriched with13C,15N, or2H. In some embodiments, the support-enrichment handle-solid-phase compatible cleavable linker-isotopically labelled amino acid conjugate is cleaved from the solid support using acid (e.g., hydrochloric acid). In some aspects, the present disclosure provides methods of identifying a binding site comprising: Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO contacting a substrate with the compound of the present disclosure, or a salt thereof; thereby creating a substrate-diazirine-alkyne conjugate; contacting the substrate-diazirine-alkyne conjugate with a chemoproteomic capture reagent comprising an azide; thereby creating a substrate-chemoproteomic capture reagent conjugate; and determining the molecular weight of the substrate-chemoproteomic capture reagent conjugate; thereby identifying the binding site. In certain embodiments, the substrate is a target protein (e.g., an enzyme). In some embodiments, the target protein is is purified from cell lysate. In certain preferred embodiments, the method further comprises incubating the substrate with the compound of the present disclosure. In certain embodiments, the chemoproteomic capture reagent is a compound of the present disclosure, or a salt thereof. In some embodiments, the methods further comprise irradiating the substrate- diazirine-alkyne conjugate; thereby covalently binding the diazirine-alkyne conjugate to the substrate. In some embodiments, the method further comprises cleaving a bond between Si and Y1. In some such embodiments, cleaving the bond between Si and Y1comprises contacting the conjugate with acid. In some embodiments, contacting the substrate-diazirine-alkyne conjugate with the chemoproteomic capture reagent of the present disclosure forms a triazole linking the chemoproteomic capture reagent to the substrate. In certain embodiments, the molecular weight of the substrate-chemoproteomic capture reagent conjugate is determined by tandem high performance liquid chromatography / mass spectrometry. In some embodiments, the methods further comprise digesting the substrate- chemoproteomic capture reagent conjugate; thereby creating a population of chemoproteomic capture reagent-labeled peptides and a population of unlabeled peptides. In some such embodiments, digesting the substrate-chemoproteomic capture reagent conjugate comprises contacting the chemoproteomic capture reagent-substrate conjugate with a digestion enzyme (e.g., trypsin). In some embodiments, the methods further comprise contacting the population of chemoproteomic capture reagent-labeled peptides and the population of unlabeled peptides with an enrichment agent selected from a metal ion, a lectin, an epitope tag, avidin, and Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO streptavidin; thereby creating an enriched population of chemoproteomic capture reagent- labeled peptides. In some such embodiments, the enrichment agent is streptavidin. In certain embodiments, the methods further comprise isolating the enriched population of chemoproteomic capture reagent-labeled peptides from the unlabeled peptides. In certain embodiments, the methods further comprise a step of eluting the enriched population of chemoproteomic capture reagent-labeled peptides from the enrichment agent, comprising contacting the enriched population of chemoproteomic capture reagent-labeled peptides with acid (e.g., formic acid). In some embodiments, the methods further comprise determining the molecular weight of the enriched population of chemoproteomic capture reagent-labeled peptides, thereby identifying the binding site. This disclosure also includes all suitable isotopic variations of a compound or chemoproteomic capture reagent of the disclosure. An isotopic variation of a compound or chemoproteomic capture reagent of the invention is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually or predominantly found in nature. Examples of isotopes that can be incorporated into a compound of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, such as2H (deuterium),3H (tritium),11C,13C,14C,15N,17O,18O,32P,33P,33S,34S,35S,36S,18F,36Cl,82Br,123I,124I,129I and131I, respectively. Accordingly, recitation of “hydrogen” or “H” should be understood to encompass1H (protium),2H (deuterium), and3H (tritium) unless otherwise specified. Certain isotopic variations of a compound of the invention, for example, those in which one or more radioactive isotopes such as3H or14C are incorporated, are useful in drug and / or substrate tissue distribution studies. Tritiated and carbon-14, i.e.,14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Such variants may also have advantageous optical properties arising, for example, from changes to vibrational modes due to the heavier isotope. Isotopic variations of a compound of the invention can generally be prepared by conventional procedures known by a person skilled in the art such as by the Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO illustrative methods or by the preparations described in the examples hereafter using appropriate isotopic variations of suitable reagents. In some embodiments, the compounds disclosed herein comprise stable isotopes of hydrogen, carbon, nitrogen, and oxygen in amounts greater than their natural abundance. For example, one or more hydrogen atoms may be enriched with2H in an amount greater than about 0.015% (e.g., 1.2-1.5%, 1.5-2%, 2-10%, or more than 10%). For example, one or more carbon atoms may be enriched with13C in an amount greater than about 1.1% (e.g., 1.2-1.5%, 1.5-2%, 2-10%, or more than 10%). One or more nitrogen atoms may be enriched with15N in an amount greater than about 0.4% (e.g., 0.5-1%, 1-2%, 2-10%, or greater than 10%). Likewise, one or more oxygen atoms may be enriched with16O in an amount greater than about 0.24% (e.g., 0.25-0.5%, 0.5-1%, 1-2%, 2-10%, or greater than 10%). Recitation of “hydrogen” or “H” should be understood to encompass1H (protium),2H (deuterium), and3H (tritium) unless otherwise specified. In some embodiments, the compounds disclosed herein have an isotopic purity of at least 50.0%, 60.0%, 70.0%, 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 97.0%, 98.0%, 99.0%, 99.5%, 99.7%, 99.8%, 99.9%, or 100%. In one embodiment, the compounds have an isotopic purity of at least 50.0%. In another embodiment, the compounds have an isotopic purity of at least 60.0%. In another embodiment, the compounds have an isotopic purity of at least 70.0%. In another embodiment, the compounds have an isotopic purity of at least 75.0%. In another embodiment, the compounds have an isotopic purity of at least 80.0%. In another embodiment, the compounds have an isotopic purity of at least 85.0%. In another embodiment, the compounds have an isotopic purity of at least 90.0%. In another embodiment, the compounds have an isotopic purity of at least 95.0%. In another embodiment, the compounds have an isotopic purity of at least 97.0%. In another embodiment, the compounds have an isotopic purity of at least 98.0%. In another embodiment, the compounds have an isotopic purity of at least 99.0%. In another embodiment, the compounds have an isotopic purity of at least 99.5%. In another embodiment, the compounds have an isotopic purity of at least 99.7%. In another embodiment, the compounds have an isotopic purity of at least 99.9%. Isotopic enrichment may be described as a percentage indicating the percent of isotopic atoms at a particular site on the molecule. The percentage can be referred to as the “isotopic purity” of the isotopically- labeled compound. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Definitions Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000). Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985). All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control. As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted. It is understood that substituents and substitution patterns on the compounds of the present invention can be selected by one of ordinary skilled person in the art to result chemically stable compounds which can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO groups may be on the same carbon or on different carbons, so long as a stable structure results. As used herein, the term “optionally substituted” refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent including,but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy,aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2- O-alkyl, -OP(O)(O-alkyl)2 or –CH2-OP(O)(O-alkyl)2. Preferably, “optionally substituted” refers to the replacement of one to four hydrogen radicals in a given structure with the substituents mentioned above. More preferably, one to three hydrogen radicals are replaced by the substituents as mentioned above. It is understood that the substituent can be further substituted. As used herein, the term “alkyl” refers to saturated aliphatic groups, including but not limited to C1-C10 straight-chain alkyl groups or C1-C10 branched-chain alkyl groups. Preferably, the “alkyl” group refers to C1-C6straight-chain alkyl groups or C1-C6branched- chain alkyl groups. Most preferably, the “alkyl” group refers to C1-C4straight-chain alkyl groups or C1-C4 branched-chain alkyl groups. Examples of “alkyl” include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1- octyl, 2-octyl, 3-octyl or 4-octyl and the like. The “alkyl” group may be optionally substituted. The term “acyl” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-. The term “acylamino” is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-. The term “acyloxy” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-. The term “alkoxy” refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like. The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl. The term “alkyl” refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1- 30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer. Moreover, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2- trifluoroethyl, etc. The term “Cx-y” or “Cx-Cy”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. C0alkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. A C1-6alkyl group, for example, contains from one to six carbon atoms in the chain. The term “alkylamino”, as used herein, refers to an amino group substituted with at least one alkyl group. The term “alkylthio”, as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-. The term “amido”, as used herein, refers to a group O R9N R10, wherein R9and R10each independently represent a hydrogen or hydrocarbyl group, or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure. The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by , wherein R9, R10, and R10’ each independently represent a hydrogen or a hydrocarbyl group, or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO The term “aminoalkyl”, as used herein, refers to an alkyl group substituted with an amino group. The term “aralkyl”, as used herein, refers to an alkyl group substituted with an aryl group. The term “aryl” as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7- membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like. The term “carbamate” is art-recognized and refers to a group , wherein R9and R10independently represent hydrogen or a hydrocarbyl group. The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group. The term “carbocycle” includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term “fused carbocycle” refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary “carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro- 1H-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one or more positions capable of bearing a hydrogen atom. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group. The term “carbonate” is art-recognized and refers to a group -OCO2-. The term “carboxy”, as used herein, refers to a group represented by the formula -CO2H. The term “cycloalkyl” includes substituted or unsubstituted non-aromatic single ring structures, preferably 4- to 8-membered rings, more preferably 4- to 6-membered rings. The term “cycloalkyl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is cycloalkyl and the substituent (e.g., R100) is attached to the cycloalkyl ring, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, denzodioxane, tetrahydroquinoline, and the like. The term “ester”, as used herein, refers to a group -C(O)OR9wherein R9represents a hydrocarbyl group. The term “ether”, as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O- heterocycle. Ethers include “alkoxyalkyl” groups, which may be represented by the general formula alkyl-O-alkyl. The terms “halo” and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo. The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to an alkyl group substituted with a hetaryl group. The terms “heteroaryl” and “hetaryl” include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heteroaryl” and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur. The term “heterocyclylalkyl”, as used herein, refers to an alkyl group substituted with a heterocycle group. The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heterocyclyl” and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like. The term “hydrocarbyl”, as used herein, refers to a group that is bonded through a carbon atom that does not have a =O or =S substituent, and typically has at least one carbon- hydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =O substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof. The term “hydroxyalkyl”, as used herein, refers to an alkyl group substituted with a hydroxy group. The term “lower” when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer atoms in the substituent, preferably six or fewer. A “lower alkyl”, for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent). The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”. Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7. The term “sulfate” is art-recognized and refers to the group –OSO3H, or a pharmaceutically acceptable salt thereof. The term “sulfonamido” is art-recognized and refers to the group represented by the general formulae , wherein R9and R10independently represents hydrogen or hydrocarbyl. The term “sulfoxide” is art-recognized and refers to the group–S(O)-. The term “sulfonate” is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof. The term “sulfone” is art-recognized and refers to the group –S(O)2-. The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. The term “thioalkyl”, as used herein, refers to an alkyl group substituted with a thiol group. The term “thioester”, as used herein, refers to a group -C(O)SR9or –SC(O)R9wherein R9represents a hydrocarbyl. The term “thioether”, as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur. The term “urea” is art-recognized and may be represented by the general formula ,wherein R9and R10independently represent hydrogen or a hydrocarbyl. Many of the compounds useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the compounds, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01 / 062726. Furthermore, certain compounds which contain alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers. In each instance, the disclosure includes both mixture and separate individual isomers. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO As used herein, the term “target protein” refers to a protein whose function is modulated by a biologically active (bioactive) small molecule, such as an agonist or antagonist of the protein. Preferably, the biological activity of a target protein has a desirable therapeutic effect, e.g., on the symptoms or etiology of a disease or condition. Non-limiting examples of a target protein include cyclooxygenases 1 & 2 (COX1 & 2), which are target proteins of aspirin (2-acetylsalicylic acid). As used herein, the term “enrichment handle” refers to a chemical moiety that, when used to label a target compound or biomolecule, enables the selective isolation and / or enrichment of the target compound or biomolecule from a population of unlabeled compounds or biomolecules. A non-limiting example of an enrichment handle is biotin, which enables enrichment through an interaction with, e.g., avidin or streptavidin. As used herein, the term “natural amino acid” refers to the 22 canonical proteinogenic amino acid, e.g., lysine, glycine, leucine, histidine, and the like. As used herein, the term “unnatural amino acid” refers to a non-proteinogenic amino acid that may or may not be chemically synthesized. Non-limiting examples of unnatural amino acids include 6-azidolysine and 2-naphthyl-alanine. As used herein, the term “binding partner” refers to a molecule to which another molecule can become specifically bound. A non-limiting example of a binding partner is glucose, which is the binding partner to hexokinase-1. EXAMPLES The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

[0002] Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Synthesis of Exemplary Chemoproteomics Capture Reagents Scheme 1: Design and synthesis of prototype CEE-SITE probes 2a-2d (A) Design of the four prototype SEE-CITE probes from styrene. (B) aReagents andconditions: a. Karstedt’s catalyst, neat, 0 ℃ to rt; b.1, NEt3, CH2Cl2, 0 ℃, 41% (for 2a, 2 steps from styrene), 63% (for 2b, 2 steps from 3b) 87% (for 2c, 2 steps from styrene); c. B(C6F5)3, CH2Cl2, rt - 40 ℃, 86% (for 3b), 60% (for 3d); d. trichloroisocyanuric acid, CH2Cl2, 0 ℃ to rt; e.1, imidazole, CH2Cl2, 0 ℃, 57% (for 2d, 2 steps from 3d). The synthesis of prototype SEE-CITE probes was undertaken with two initial goals (1) to establish a robust synthetic strategy to access the desired scaffolds and (2) to assess how the bulkiness of the substituents on the silicon atom would affect protein labeling efficiency in photo cross-linking.2a-d were envisioned as the prototype SEE-CITE probeswith simple structures which have all requisite elements (Scheme 1A). Synthesis of 2a and2c commenced with hydrosilylation reaction of styrene with the corresponding chlorosilanes by Karstedt’s catalyst followed by silyl ether formation with minimalist diazirine tag 1 in one-pot. Silane intermediates 3b and 3d were obtained via B(C6F5)3-catalyzed hydrosilylation reaction and the subsequent chlorosilane formation was achieved by treatment of the silane intermediate with trichloroisocyanuric acid in CH2Cl2. Use of imidazole instead of NEt3as a base was crucial for obtaining 2d in good yield at the silyl ether formation step (Scheme 1B). Compounds 2a-d were observed to be stable to most conditions assessed, including aqueous buffer and cell culturing media; as expected, rapid cleavage of the silyl ether moiety was observed under acidic conditions that mimic our anticipated neutravidin elution conditions Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO (FIG.7 and Table 1). SDS-PAGE gel-based analysis of photocrosslinked lysates confirmed that all four compounds robustly label proteins in a UV-dependent manner (FIG.2A). Providing further evidence that the silyl ether moieties are comparatively inert under physiological conditions, markedly different gel-based labeling patterns were observed by 2a-d in K562 cells when compared to the previously reported minimalist tag 1, which is released upon silyl ether cleavage. Elevated protein labeling was observed for dimethyl probe 2a when compared to bulkier silane analogues, consistent with decreased steric hindrance favoring increased protein labeling. Looking beyond an enhanced reactivity of 2a, the protein labeling patterns for 2a-d were observed to be generally similar, suggesting a high degree of similarity in their protein targets. General Synthetic Methods All reactions were performed in oven dried glassware under an inert atmosphere of dry N2 unless stated otherwise. Silica gel P60 (SiliCycle) was used for column chromatography and SiliCycle 60 F254 silica gel (precoated sheets, 0.25 mm thick) was used for analytical thin layer chromatography. Plates were visualized by fluorescence quenching under UV light or by staining reagents. Other reagents were purchased from Sigma-Aldrich (St. Louis, MO), Alfa Aesar (Ward Hill, MA), EMD Millipore (Billerica, MA), Fisher Scientific (Hampton, NH), Oakwood Chemical (West Columbia, SC), Combi-blocks (San Diego, CA), Click Chemistry Tools (Scottsdale, AZ) and Cayman Chemical (Ann Arbor, MI) and used without further purification.1H NMR and13C NMR spectra for characterization of new compounds and monitoring reactions were collected in CDCl3, DMSO-d6, or CD3OD (Cambridge Isotope Laboratories, Cambridge, MA) on a Bruker AV300, AV400, AV500 and NEO600 MHz spectrometer in the Department of Chemistry & Biochemistry at University of California, Los Angeles. All chemical shifts are reported in the standard notation of parts per million using the peak of residual proton signals of the deuterated solvent as an internal reference. Coupling constant units are in Hertz (Hz). Splitting patterns are indicated as follows: br, broad; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; dt, doublet of triplets. Low-resolution mass spectrometry was performed on an Agilent Technologies InfinitiyLab LC / MSD single quadrupole LC / MS (ESI source). High- resolution mass spectroscopy was performed on Waters LCT Premier TOF LC / MS withACQUITY UPLC in the Department of Chemistry & Biochemistry at University ofCalifornia, Los Angeles or the University of California, Irvine Mass Spectrometry Facility. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Data were collected on a Waters LCT Premier by flow injection analysis (FIA) in methanol and data analyzed with MassLynx v.4.1 software. In ESI+ mode, analyte m / z ions (M+H)+ or (M+Na)+ were validated to less than ± 5 ppm relative to the nearest sodiated polyethylene glycol (CAS: 25322-68-3, av. Mwt 400)) or sodiated methoxypolyethyleneglycol (CAS: 990- 74-4, av. Mwt 350) calibrant peak lockmass. In ESI- mode, analyte m / z ions (M-H)- were validated relative to the nearest calibrant peak from the sodium formate cluster series (m / z 100-2000). Cell culture reagents including Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM) / high glucose media, Roswell Park Memorial Institute (RPMI) media, trypsin-EDTA and penicillin / streptomycin (Pen / Strep) were purchased from Fisher Scientific. All protein concentrations were determined using a Bio- Rad DC protein assay kit using reagents from Bio-Rad Life Science (Hercules, CA). The LC- MS / MS samples were analyzed by liquid chromatography tandem mass spectrometry using a Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer. Synthesis of prototype SEE-CITE probes Minimalist diazirine tag 1 was synthesized according to literature procedure. 3-(but-3-yn-1-yl)-3-(2-{[dimethyl(2-phenylethyl)silyl]oxy}ethyl)-3H-diazirine (2a) To a stirred mixture of styrene (62.4 μL, 543 μmol) and chlorodimethylsilane (67 uL, 615 μmol) was added platinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex solution (Karstedt’s catalyst, 0.05 M, 7.24 uL, 0.362 μmol) at 0 ℃ and the resulting mixture was stirred at room temperature under N2 atmosphere overnight. The reaction mixture was cooled to 0 °C and then 1 (50.0 mg, 362 µmol) in dry CH2Cl2(1 mL) and triethylamine (101 µL, 724 µmol) were added in this order and the resulting mixture was stirred for 30 minutes under N2 atmosphere. Water was added and the resulting mixture was extracted with CH2Cl2 and the combined organic layers were washed with brine, dried over anhydrous Na2SO4. After filtration and evaporation of the organic solvents, the crude mixture was purified by silica gel column chromatography (hexane / EtOAc = 98 / 2) to afford 2a (44.6 mg, 148 µmol, 41 %) as colorless oil. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Colorless oil;1H NMR (CDCl3) δ 7.31-7.25 (m, 2H), 7.23-7.14 (m, 3H), 3.45 (t, J = 6.4 Hz, 2H), 2.72-2.63 (2H, m), 2.08-1.99 (2H, m), 1.96 (t, J = 2.8 Hz, 1H) 1.67 (t, J = 8.0 Hz, 2H), 1.61 (t, J = 6.4 Hz, 2H), 1.00-0.95 (2H, m), 0.12 (s, 6H);13C NMR (CDCl3) δ 144.75, 128.32, 127.79, 125.59, 82.89, 68.99, 57.20, 35.91, 32.90, 29.19, 26.80, 18.18, 13.30, -2.27.; HRMS (ESI) Anal. calcd. for C17H24N2OSiNa m / z 323.1555 [M+Na]+, found 323.1570. 3-(but-3-yn-1-yl)-3-(2-{[methyl(phenyl)(2-phenylethyl)silyl]oxy}ethyl)-3H-diazirine (2c) To a stirred mixture of styrene (62.4 μL, 543 μmol) and chloromethylphenylsilane (92.4 uL, 615 μmol) was added platinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex solution (Karstedt’s catalyst, 0.05 M, 7.24 uL, 0.362 μmol) at 0 °C and the resulting mixture was stirred at room temperature under N2atmosphere overnight. The reaction mixture was cooled to 0 °C and then 1 (50.0 mg, 362 µmol) in dry CH2Cl2 (1 mL) and triethylamine (101 µL, 724 µmol) were added in this order and the resulting mixture was stirred for 30 minutes under N2atmosphere. Water was added and the resulting mixture was extracted with CH2Cl2 and the combined organic layers were washed with brine, dried over anhydrous Na2SO4. After filtration and evaporation of the organic solvents, the crude mixture was purified by silica gel column chromatography (hexane / EtOAc = 98 / 2) to 2c (114.6 mg, 316 µmol, 87 %) as colorless oil. Colorless oil;1H NMR (CDCl3) δ 7.62-7.56 (m, 2H), 7.43-7.37 (m, 3H), 7.30-7.24 (m, 2H), 7.22-7.14 (m, 3H), 3.51-3.45 (m, 2H), 2.72 (t, J = 8.4 Hz, 2H), 2.04-1.99 (m, 2H), 1.95 (t, J = 2.8 Hz, 1H), 1.66 (t, J = 7.6 Hz, 2H), 1.63-1.58 (m, 2H), 1.31-1.16 (m, 2H), 0.40 (s, 3H);13C NMR (CDCl3) δ 144.59, 136.30, 133.67, 129.82, 128.31, 127.96, 127.79, 125.62, 82.86, 69.00, 57.71, 35.84, 32.78, 29.04, 26.77, 17.03, 13.29, -4.00; HRMS (ESI) Anal. calcd. for C22H26N2OSiNa m / z 385.1712 [M+Na]+, found 385.1694. Diethyl(2-phenylethyl)silane (3b) Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO To a stirred solution of tris(perfluorophenyl)borane (123 mg, 240 µmol) in anhydrous CH2Cl2 (5 mL) was added diethylsilane (933 µL, 7.20 mmol), followed by addition of styrene (550 µL, 4.80 mmol). The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with hexane and the resulting mixture was filtered through a short pad of silica gel. After washing with hexane, the filtrate was concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography (hexane = 100%) to afford 3b (797.5 mg, 4.15 mmol, 86 %) as colorless oil. Colorless oil;1H NMR (CDCl3) δ 7.32-7.26 (m, 2H), 7.24-7.15 (m, 3H), 3.75-3.69 (m, 1H), 2.73-2.65 (m, 2H), 1.05-0.95 (m, 8H), 0.67-0.59 (m, 4H);13C NMR (CDCl3) δ 144.97, 128.29, 127.78, 125.57, 30.76, 12.76, 8.15, 2.72; HRMS (ESI) Anal. calcd. for C12H19Si m / z 191.1256 [M-H]-, found 191.1248. (2-phenylethyl)[di(propan-2-yl)]silane (3d) To a stirred solution of tris(pentafluorophenyl)boron (123 mg, 240 µmol) in anhydrous CH2Cl2(5 mL) was added diisopropylsilane (1.18 mL, 7.20 mmol), followed by addition of styrene (550 µL, 4.80 mmol) and then the reaction mixture was stirred at 40 °C for 18 hours. After dilution with hexane, the resulting mixture was filtered through a short pad of silica gel, washing with hexane, the filtrate was concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography (hexane = 100%) to afford 3d (631.9 mg, 2.867 mmol, 60 %) as colorless oil. Colorless oil;1H NMR (CDCl3) δ 7.32-7.26 (m, 2H), 7.24-7.15 (m, 3H), 3.53-3.49 (m, 1H), 2.75-2.67 (m, 2H), 1.13-0.95 (m, 16H);13C NMR (CDCl3) [observed peaks] δ; 145.23, 128.32, 127.72, 125.58, 31.47, 19.07, 18.74, 10.76, 10.55; HRMS (ESI) Anal. calcd. for C14H23Si m / z 219.1569 [M-H]-, found 219.1559. 3-(but-3-yn-1-yl)-3-(2-{[diethyl(2-phenylethyl)silyl]oxy}ethyl)-3H-diazirine (2b) A solution of 3b (83.5 mg, 434 µmol) in anhydrous CH2Cl2 (2 mL) was cooled to 0 °C, then trichloroisocyanuric acid (33.6 mg, 145 µmol) was carefully added and then warmed Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO to room temperature and stirred for 1.5 hours under the N2atmosphere. The insoluble materials were removed by filtration and washed with hexane, then the filtrate was concentrated in vacuo to afford the crude chlorosilane.1 (40.0 mg, 289 µmol) and this crude chlorosilane were dissolved in anhydrous CH2Cl2(3 mL) and cooled to 0 °C. Triethylamine (121 µL, 868 µmol) was added and the resulting mixture was stirred for 30 minutes under N2atmosphere. Water was added and the resulting biphasic mixture was extracted with CH2Cl2 and the combined organic layers were washed with brine and then dried over anhydrous Na2SO4. After filtration and evaporation, the residue was purified by silica gel column chromatography (hexane / EtOAc = 98 / 2) to afford 2b (59.6 mg, 181 µmol, 63 %) as colorless oil. Colorless oil;1H NMR (CDCl3) δ 7.31-7.26 (m, 2H), 7.24-7.14 (m, 3H), 3.49 (t, J = 6.4 Hz, 2H), 2.72-2.65 (m, 2H), 2.07-2.00 (m, 2H), 1.96 (t, J = 2.8 Hz, 1H), 1.68 (t, J = 8.0 Hz, 2H), 1.60 (t, J = 6.4 Hz, 2H), 1.02-0.95 (m, 8H), 0.64 (q, J = 8.0 Hz, 4H);13C NMR (CDCl3) δ 145.01, 128.33, 127.74, 125.59, 82.89, 68.97, 57.50, 36.07, 32.91, 29.19, 26.84, 14.83, 13.31, 6.73, 4.70; HRMS (ESI) Anal. calcd. for C19H28N2OSiNa m / z 351.1869 [M+Na]+, found 351.1883. 3-(but-3-yn-1-yl)-3-[2-({(2-phenylethyl)[di(propan-2-yl)]silyl}oxy)ethyl]-3H-diazirine (2d) A solution of 3d (128 mg, 579 µmol) in anhydrous CH2Cl2 (2 mL) was cooled to 0 °C, then trichloroisocyanuric acid (45.1 mg, 194 µmol) was carefully added and then warmed to room temperature and stirred for 1.5 hours under N2atmosphere. The insoluble materials were removed by filtration and washed with hexane, then the filtrate was concentrated in vacuo to afford the crude chlorosilane.1 (40.0 mg, 289 µmol) and imidazole (78.8 mg, 1.16 mmol) were dissolved in anhydrous CH2Cl2(1.5 mL) and cooled to 0 °C, then the crude chlorosilane in anhydrous CH2Cl2 (1.5 mL) was added and the resulting mixture was stirred for 30 minutes under N2 atmosphere. Water was added and the resulting biphasic mixture was extracted with CH2Cl2and the combined organic layers were washed with brine and then dried over anhydrous Na2SO4. After filtration and evaporation, the residue was purified by Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO silica gel column chromatography (hexane / EtOAc = 100 / 0 to 98 / 2) to afford 2d (58.3 mg, 163 µmol, 57 %) as colorless oil. Colorless oil; 1H NMR (CDCl3) δ 7.32-7.26 (m, 2H), 7.24-7.15 (m, 3H), 3.57 (t, J = 6.4 Hz, 2H), 2.74-2.68 (m, 2H), 2.04 (td, J = 7.6, 2.8 Hz, 2H), 1.96 (t, J = 2.8 Hz, 1H), 1.70 (t, J = 7.6 Hz, 2H), 1.61 (t, J = 6.4 Hz, 2H), 1.10-0.98 (m, 16H);13C NMR (CDCl3) δ 145.32, 128.38, 127.65, 125.60, 82.89, 68.97, 58.11, 36.21, 32.88, 29.53, 26.90, 17.64, 13.32, 12.91, 12.43; HRMS (ESI) Anal. calcd. for C21H32N2OSiNa m / z 379.2182 [M+Na]+, found 379.2169. 2-{[6-(4-{2-[3-(but-3-yn-1-yl)-3H-diazirin-3-yl]ethyl}piperazin-1-yl)-2-methylpyrimidin- 4-yl]amino}-N-(2-chloro-6-methylphenyl)-1,3-thiazole-5-carboxamide (4a) S1 (100.0 mg, 225.3 µmol), S2 (58.5 mg, 270.3 µmol) and DIPEA (78.5 µL, 450.5µmol) in anhydrous DMF (1 mL) were stirred at 80 °C under N2 for 22 hours. After coolingto room temperature, water was added and the precipitate was filtered and washed with CH2Cl2 and dried in vacuo to afford 4a (44.3 mg, 78.5 µmol, 35 %) as colorless solid. Colorless solid;1H NMR (DMSO-d6) δ 11.37 (brs, 1H), 9.86 (s, 1H), 8.21 (s, 1H), 7.39 (dd, J = 7.6, 2.0 Hz, 1H), 7.30-7.21 (m, 2H), 6.04 (s, 1H), 3.54-3.45 (m, 4H), 2.83 (t, J = 2.8 Hz, 1H), 2.43-2.34 (m, 7H), 2.23 (s, 3H), 2.15 (t, J = 7.2 Hz, 2H), 2.02 (td, J = 7.6, 2.8 Hz, 2H), 1.62-1.54 (m, 4H);13C NMR (DMSO-d6) δ 165.18, 162.55, 162.34, 159.91, 156.95, 140.83, 138.82, 133.52, 132.43, 129.03, 128.18, 127.01, 125.71, 83.29, 82.63, 71.75, 52.05, 43.56, 31.72, 29.41, 27.65, 25.57, 18.30, 12.69; HRMS (ESI) Anal. calcd. for C27H31ClN9OS m / z 564.2055 [M+H]+, found 564.2040. 2-[3-(but-3-yn-1-yl)-3H-diazirin-3-yl]ethyl 4-[6-({5-[(2-chloro-6- methylphenyl)carbamoyl]-1,3-thiazol-2-yl}amino)-2-methylpyrimidin-4-yl]piperazine-1- carboxylate (4b) Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO To a stirred solution of S3 (80 mg, 0.34 mmol) in anhydrous DMF (2 mL) was addedS1 (100 mg, 225 µmol) and DIPEA (118 µL, 676 µmol) and then the resulting mixture wasstirred at 60oC under N2 atmosphere for 19 hours. The reaction mixture was diluted with EtOAc and washed with water three times, brine, and then dried over anhydrous Na2SO4. After filtration and evaporation, the residue was purified by silica gel column chromatography (CH2Cl2 / MeOH = 95 / 5), then the second purification (EtOAc / MeOH = 98 / 2) was conducted to afford 4b (38.4 mg, 63.1 μmol, 28%) as colorless solid. Colorless solid;1H NMR (DMSO-d6) δ 11.50 (brs, 1H), 9.87 (s, 1H), 8.21 (s, 1H), 7.39 (dd, J = 8.0, 2.0 Hz, 1H), 7.30-7.21 (m, 2H), 6.07 (s, 1H), 3.92 (t, 6.0 Hz, 2H), 3.63-3.39 (m, 8H), 2.84 (t, J = 2.8 Hz, 1H), 2.41 (s, 3H), 2.23 (s, 3H), 2.01 (td, J = 7.2, 2.8 Hz, 2H), 1.74 (t, J = 6.0 Hz, 2H), 1.62 (t, J = 7.2 Hz, 2H);13C NMR (DMSO-d6) δ 165.22, 162.51, 162.28, 159.90, 157.00, 154.25, 140.82, 138.81, 133.50, 132.43, 129.02, 128.17, 127.00, 125.77, 83.13, 82.84, 71.81, 60.01, 43.17, 31.69, 31.44, 26.87, 25.57, 18.30, 12.61; HRMS (ESI) Anal. calcd. for C28H31ClN9O3S m / z 608.1954 [M+H]+, found 608.1984. 3-(2-{[(3-bromopropyl)(dimethyl)silyl]oxy}ethyl)-3-(but-3-yn-1-yl)-3H-diazirine (7) A mixture of 1 (100 mg, 724 μmol) and triethylamine (303 μL, 2.17 mmol) in anhydrous CH2Cl2(5 mL) was cooled to 0 °C then 6 (234 mg, 1.09 mmol) in anhydrous CH2Cl (2 mL) was added and the resulting mixture was stirred at the same temperature for 10 min. Water was added and the resulting mixture was extracted with CH2Cl2 and the combined organic layers were washed with brine and then dried over anhydrous Na2SO4. After filtration and evaporation, the residue was purified by (hexane / EtOAc = 98 / 2) to afford 7 (172.4 mg, 543.3 μmol, 75 %) as pale yellow oil. Pale yellow oil;1H NMR (CDCl3) δ 3.46-3.39 (m, 4H), 2.06-2.00 (m, 2H), 1.98 (t, J = 2.8 Hz, 1H), 1.95-1.85 (m, 2H), 1.66 (t, J = 7.6 Hz, 2H), 1.62 (t, J = 6.4 Hz, 2H), 0.75-0.68 (m, 2H), 0.12 (s, 6H);13C NMR (CDCl3) δ 82.84, 69.00, 57.21, 36.88, 35.83, 32.86, 27.09, 26.74, 15.41, 13.27, -2.32; HRMS (ESI) expected m / z was not observed. 2-{[6-(4-{3-[{2-[3-(but-3-yn-1-yl)-3H-diazirin-3- yl]ethoxy}(dimethyl)silyl]propyl}piperazin-1-yl)-2-methylpyrimidin-4-yl]amino}-N-(2- chloro-6-methylphenyl)-1,3-thiazole-5-carboxamide (4c) Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO To a stirred mixture of S1 (80.0 mg, 180 μmol) and 7 (85.8 mg, 270 μmol) in anhydrous DMF (1 mL) was added DIPEA (94.2 μL, 541 μmol) and sodium iodide (40.5 mg, 270 μmol) and then the resulting solution was stirred at 50 °C under N2 atmosphere for 2.5 hours. The reaction temperature was cooled to room temperature and then the mixture was diluted with EtOAc and washed with water three times, brine and dried over anhydrous Na2SO4. After filtration and evaporation, the residue was purified by silica gel column chromatography (CH2Cl2 / MeOH = 95 / 5) to afford 4c (57.5 mg, 84.5 μmol, 47 %) as colorless solid. Colorless solid;1H NMR (DMSO-d6) δ 11.45 (s, 1H), 9.86 (s, 1H), 8.20 (s, 1H), 7.39 (dd, J = 7.6, 2.0 Hz, 1H), 7.31-7.20 (m, 2H), 6.04 (s, 1H), 3.56-3.44 (m, 4H), 3.40 (t, J = 6.0 Hz, 2H), 2.81 (t, J = 2.8 Hz, 1H), 2.45-2.34 (m, 4H), 2.39 (s, 3H), 2.33-2.25 (m, 2H), 2.23 (s, 3H), 2.00 (td, J = 7.6, 2.8 Hz, 2H), 1.62-1.41 (m, 6H), 0.60-0.49 (m, 2H), 0.07 (s, 6H);13C NMR (DMSO-d6) [observed peaks] δ 165.15, 162.56, 162.36, 159.91, 156.93, 140.81, 138.81, 133.52, 132.43, 129.01, 128.15, 126.99, 125.69, 83.21, 82.60, 71.65, 60.98, 56.81, 52.30, 43.60, 35.06, 32.07, 27.10, 25.57, 20.02, 18.30, 13.27, 12.66, -2.19; HRMS (ESI) Anal. calcd. for C32H43ClN9O2SSi m / z 680.2713 [M+H]+, found 680.2745. (3R)-1-[5-({4-[chloro(difluoro)methoxy]phenyl}carbamoyl)-3-(1H-pyrazol-5-yl)pyridin- 2-yl]pyrrolidin-3-yl {2-[3-(but-3-yn-1-yl)-3H-diazirin-3-yl]ethyl}carbamate (5a) To a stirred solution of S4 (70.0 mg, 131 µmol) in anhydrous CH2Cl2 (2 mL) was added triethylamine (54.8 µL, 393 µmol) and 4-nitrophenyl chloroformate (39.6 mg, 197 µmol) at 0 °C and the resulting mixture was stirred at room temperature under N2atmosphere for 68 hours. Additional 4-nitrophenyl chloroformate (39.6 mg, 197 µmol) and triethylamine Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO (54.8 µL, 3.00 Eq, 393 µmol) were added at 0 °C and the resulting mixture was stirred at room temperature for 27 hours. The reaction mixture was diluted with CH2Cl2 and washed with brine, then dried over anhydrous Na2SO4. After filtration and evaporation, the residue was purified by silica gel column chromatography (hexane / EtOAc = 1 / 1, Rf = 0.35) to afford the carbonate intermediate (60.9 mg, 87.1 µmol, 67 %) as a colorless solid. This intermediatewas dissolved in anhydrous DMF (1 mL) and then S5 (14.1 mg, 103 µmol) and triethylamine(35.9 μL, 257 μmol) were added. After stirring at room temperature under N2 atmosphere for 3.5 hours, the reaction mixture was diluted with EtOAc and washed with saturated NaHCO3aq., water, 1N HCl aq., water, saturated NaHCO3 aq. and brine successively, then dried over anhydrous Na2SO4. After filtration and evaporation, the crude THP-protected carbamate was obtained. To a stirred solution of the crude carbamate in CH2Cl2(1 mL) was added TFA (1 mL) and the resulting mixture was stirred at room temperature for 3 hours. The reaction mixture was neutralized by pouring into saturated NaHCO3 aq. and extracted CH2Cl2. The combined organic layers were washed with saturated NaHCO3aq., brine, dried over anhydrous Na2SO4. After filtration and evaporation, the residue was purified by silica gel column chromatography (CH2Cl2 / MeOH = 92 / 8) to afford 5a (38.9 mg, 63.5 µmol, 74 % in 2 steps) as colorless solid. Colorless solid;1H NMR (CD3OD) δ 8.82-8.68 (m, 1H), 8.13-8.04 (m, 1H), 7.84-7.65 (m, 3H), 7.32-7.21 (m, 2H), 6.52-6.42 (m, 1H), 5.21-5.08 (m, 1H), 3.63-3.17 (m, 4H), 2.97 (t, J = 4.8 Hz, 2H), 2.28-2.21 (m, 1H), 2.14-1.90 (m, 4H), 1.65-1.44 (m, 4H);13C NMR (DMSO-d6, observed peaks) δ 164.05, 156.94, 155.45, 147.55, 144.78, 138.95, 138.62, 128.99, 125.00 (t, J = 285.3 Hz), 121.77, 121.40, 117.89, 113.72, 105.98, 83.11, 72.89, 71.74, 54.88, 46.83, 35.26, 32.16, 31.23, 30.50, 27.06, 12.64; HRMS (ESI) Anal. calcd. for C28H28ClF2N8O4m / z 613.1885 [M+H]+, found 613.1883. 3-[chloro(dimethyl)silyl]propyl 4-nitrophenyl carbonate (9) A mixture of 8 (1.50 g, 6.72 mmol), [Ir(cod)Cl]2 (451 μg, 0.672 μmol) and 1,5- cyclooctadiene (16.5 μL, 134 μmol) was stirred at 75 °C until the SM melted down. After that, chlorodimethylsilane (1.1 mL, 10.1 mmol) was slowly added to the mixture for a period of 15 min at 75 ℃. After the completion of the addition, the mixture was heated at 80 °C for Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 1 h. After cooling to room temperature, the mixture was concentrated under reduced pressure to give crude 9 (2.05 g, 6.46 mmol, 96 %) as light brown oil, which was analytically pure enough and used in the next step without purification. Light brown oil;1H NMR (CDCl3) δ 8.28-8.22 (m, 2H), 7.39-7.34 (m, 2H), 4.28 (t, J = 6.8 Hz), 1.93-1.82 (m, 2H), 0.95-0.86 (m, 2H), 0.44 (s, 6H);13C NMR (CDCl3) δ 155.48, 152.41, 145.29, 125.20, 121.71, 71.02, 22.30, 14.69, 1.44. 3-[{2-[3-(but-3-yn-1-yl)-3H-diazirin-3-yl]ethoxy}(dimethyl)silyl]propyl 4-nitrophenyl carbonate (10) To a stirred mixture of 1 (100 mg, 724 μmol) and triethylamine (303 μL, 2.17 mmol) in anhydrous CH2Cl2 (5 mL) at 0 ℃ was added 9 (345 mg, 1.09 mmol) in anhydrous CH2Cl2 (2 mL) dropwise and the resulting mixture was stirred at the same temperature for 10 minutes. Water was added and the resulting mixture was extracted with CH2Cl2 and the combined organic layers were washed with brine and then dried over anhydrous Na2SO4. After filtration and evaporation under reduced pressure, the residue was purified by silica gel column chromatography (hexane / EtOAc = 9 / 1) to afford 10 (245.8 mg, 81 %) as pale yellow oil. Pale yellow oil;1H NMR (CDCl3) δ 8.31-8.24 (m, 2H), 7.42-7.35 (m, 2H), 4.27 (t, J = 9.2 Hz, 2H), 3.45 (t, J = 8.4 Hz, 2H), 2.06-1.99 (m, 2H), 1.97 (t, J = 3.6 Hz, 1H), 1.88-1.74 (m, 2H), 1.70-1.58 (m, 4H), 0.71-0.61 (m, 2H), 0.14 (s, 6H);13C NMR (CDCl3) δ 155.58, 152.51, 145.33, 125.27, 121.78, 82.83, 71.66, 69.02, 57.21, 35.77, 32.85, 26.76, 22.43, 13.26, 11.92, -2.38; HRMS (ESI) Anal. calcd. for C19H25N3O6SiNa m / z 442.1410 [M+Na]+, found 442.1398. tert-butyl {(3R)-1-[3-bromo-5-({4-[chloro(difluoro)methoxy]phenyl}carbamoyl)pyridin- 2-yl]pyrrolidin-3-yl}carbamate (S7) A mixture of S6 (2.59 g, 6.29 mmol), (R)-(+)-3-[N-(tert- butyloxycarbonyl)amino]pyrrolidine (1.40 g, 7.54 mmol), DIPEA (3.28 mL, 18.86 mmol) in Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO anhydrous DMSO (12 mL) was stirred at 120 °C for 1 hour. After cooling to room temperature, the reaction mixture was diluted with EtOAc and washed with water, 1N HCl aq., sat. NaHCO3 aq., water and brine successively, then dried over anhydrous Na2SO4. After filtration and concentration under reduced pressure, S7 (3.46 g, 6.15 mmol, 98 %) was obtained as brown solid. Brown solid;1H NMR (DMSO-d6) δ 10.23 (s, 1H), 8.68 (d, J = 2.0 Hz, 1H), 8.34 (d, J = 2.0 Hz, 1H), 7.88-7.82 (m, 2H), 7.36-7.30 (m, 2H), 7.21 (d, J = 6.8 Hz, 1H), 4.09-3.98 (m, 1H), 3.92-3.78 (m, 2H), 3.76-3.67 (m, 1H), 3.58 (dd, J = 10.8, 4.8 Hz, 1H), 2.11-1.98 (m, 1H), 1.91-1.78 (m, 1H), 1.38 (s, 9H);13C NMR (DMSO-d6) δ 162.81, 156.01, 155.26, 146.55, 144.92, 141.57, 138.36, 124.98 (t, J = 285 Hz), 121.79, 121.42, 120.29, 101.53, 77.89, 55.36, 49.89, 48.23, 30.43, 28.21; HRMS (ESI) Anal. calcd. for C22H25BrClF2N4O4m / z 563.0690 [M+H]+, found 563.0692. tert-butyl [(3R)-1-{5-({4-[chloro(difluoro)methoxy]phenyl}carbamoyl)-3-[1-(tetrahydro- 2H-pyran-2-yl)-1H-pyrazol-5-yl]pyridin-2-yl}pyrrolidin-3-yl]carbamate (S8) A mixture of S7 (600 mg, 1.07 mmol), 1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (386 mg, 1.39 mmol), K3PO4 (680 mg, 3.20 mmol) in toluene (5 mL) was degassed by sonication for a while. After that, Pd(PPh3)4(61.7 mg, 53.4 μmol) was added and the resulting mixture was stirred at 110 °C under N2atmosphere for 16 hours. After cooling to room temperature, the reaction mixture was diluted with EtOAc and washed with water, brine, and dried over anhydrous Na2SO4. After filtration and evaporation, the residue was purified by silica gel column chromatography (CH2Cl2 / EtOAc = 3 / 1) twice to afford S8 (345.1 mg, 545.1 μmol, 51 %) as pale yellow amorphous solid. Pale yellow solid;1H NMR (CD3OD) δ 8.81 (d, J = 2.4 Hz, 1H), 8.11-7.90 (m, 1H), 7.81-7.72 (m, 2H), 7.65 (d, J = 2.4 Hz, 1H), 7.30-7.21 (m, 2H), 6.47-6.41 (m, 1H), 5.14-4.97 (m, 1H), 4.12-3.87 (m, 2H), 3.58-3.03 (m, 5H), 2.45-2.22 (m, 1H), 2.10-1.93 (m, 2H), 1.91- 1.71 (m, 2H), 1.70-1.55 (m, 2H), 1.55-1.48 (m, 1H), 1.47-1.35 (m, 9H);13C NMR (CD3OD, obscure spectrum, observed peaks) 166.57, 158.88, 157.93, 150.32, 147.58, 142.59, 142.53, 141.58, 140.66, 139.15, 126.77 (t, J = 285 Hz), 123.28, 123.01, 119.20, 109.89, 86.17, 86.08, Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 80.29, 69.22, 55.49, 51.32, 48.04, 31.70, 28.72, 26.01, 23.88, 23.82; HRMS (ESI) Anal. calcd. for C30H36ClF2N6O5 m / z 633.2398 [M+H]+, found 633.2416. 6-[(3R)-3-aminopyrrolidin-1-yl]-N-{4-[chloro(difluoro)methoxy]phenyl}-5-(1H-pyrazol- 5-yl)pyridine-3-carboxamide hydrochloride (1:1) (S9) To a solution of S8 (561 mg, 886 μmol) in CH2Cl2(5 mL) was added 4N hydrochloric acid / 1,4-dioxane (5 mL) and the resulting mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, the residue was triturated with ether and the solid was collected by filtration and washed with ether then dried in vacuo to afford S9 (428 mg, 882.5 μmol, 99.6%) as a colorless solid. Colorless solid;1H NMR (CD3OD) δ 8.72 (d, J = 2.4 Hz, 1H), 8.55 (d, J = 2.4 Hz, 1H), 7.92 (d, J = 2.4 Hz, 1H), 7.85-7.80 (m, 2H), 7.33-7.27 (m, 2H), 6.73 (d, J = 2.4 Hz, 1H), 4.08-3.96 (m, 1H), 3.86-3.60 (m, 3H), 3.52-3.38 (m, 2H), 3.46 (dd, J = 11.6, 4.0 Hz, 1H), 2.56-2.39 (m, 1H), 2.32-2.17 (m, 1H);13C NMR (CD3OD) δ 162.92, 151.99, 147.94, 146.90, 145.85, 139.38, 138.56, 132.21, 126.72 (t, J = 285 Hz), 123.37, 123.11, 121.46, 120.92, 108.73, 55.24, 51.25, 49.86, 29.78; HRMS (ESI) Anal. calcd. for C20H20ClF2N6O2 m / z 449.1299 [M+H]+, found 449.1308. 2-[3-(but-3-yn-1-yl)-3H-diazirin-3-yl]ethyl{(3R)-1-[5-({4- [chloro(difluoro)methoxy]phenyl}carbamoyl)-3-(1H-pyrazol-5-yl)pyridin-2- yl]pyrrolidin-3-yl}carbamate (5b) A mixture of S9 (120 mg, 247 μmol) and S3 (68.9 mg, 297 μmol) in anhydrous DMF (2 mL) was added DBU (112 μL, 742 μmol) and the resulting mixture was stirred at room temperature for 28 hours. The reaction mixture was diluted with EtOAc and washed with water, 1N HCl aq., sat. NaHCO3 aq., brine, then dried over anhydrous Na2SO4. After filtration and evaporation, the residue was purified by silica gel column chromatography (CH2Cl2 / MeOH = 95 / 5) to afford 5b (32.9 mg, 53.7 μmol, 49 %) as a colorless solid. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Colorless solid;1H NMR (CD3OD) δ 8.70 (d, J = 2.4 Hz, 1H), 8.25-8.18 (m, 1H), 7.82-7.71 (m, 3H), 7.31-7.23 (m, 2H), 6.58-6.48 (m, 1H), 4.24-4.09 (m, 1H), 3.93 (t, J = 6.0 Hz, 2H), 3.66-3.16 (m, 4H), 2.26 (t, J = 2.8 Hz, 1H), 2.18-2.07 (m, 1H), 2.02 (td, J = 7.6, 2.8 Hz, 2H), 1.98-1.89 (m, 1H), 1.71 (t, J = 6.0 Hz, 2H), 1.63 (t, J = 7.6 Hz, 2H);13C NMR (DMSO-d6) [observed peaks] δ 164.01, 156.94, 155.56, 147.55, 144.79, 138.99, 138.64, 127.30, 125.02 (t, J = 285 Hz), 122.74, 121.78, 121.41, 117.80, 106.12, 83.15, 71.80, 58.69, 54.19, 50.07, 47.17, 31.82, 31.51, 30.26, 26.78, 12.65; HRMS (ESI) Anal. calcd. for C28H28ClF2N8O4m / z 613.1885 [M+H]+, found 613.1886. 3-[{2-[3-(but-3-yn-1-yl)-3H-diazirin-3-yl]ethoxy}(dimethyl)silyl]propyl {(3R)-1-[5-({4- [chloro(difluoro)methoxy]phenyl}carbamoyl)-3-(1H-pyrazol-5-yl)pyridin-2- yl]pyrrolidin-3-yl}carbamate (5c) To a stirred mixture of S9 (150 mg, 309 μmol), DMAP (37.8 mg, 309 μmol) and DIPEA (108 μL, 618 μmol) in anhydrous CH2Cl2 (2 mL) was added 10 (169 mg, 402 μmol) in anhydrous CH2Cl2(2 mL) then the resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with CH2Cl2 and washed with water, brine and dried over anhydrous Na2SO4. After filtration and evaporation, the residue was purified by silica gel column chromatography (CH2Cl2 / MeOH = 96 / 4) to afford 5c (68.9 mg, 94.5 μmol, 31 %) as a colorless solid. Colorless solid;1H NMR (CD3OD) δ 8.81-8.68 (m, 1H), 8.12-8.04 (m, 1H), 7.89-7.68 (m, 3H), 7.26 (d, J = 8.8 Hz, 2H), 6.54-6.43 (m, 1H), 4.17-4.06 (m, 1H), 4.06-3.91 (m, 2H), 3.59-3.05 (m, 6H), 2.24 (t, J= 2.8 Hz, 1H), 2.13-1.96 (m, 3H), 1.93-1.78 (m, 1H), 1.74-1.50 (m, 6H), 0.69-0.53 (m, 2H), 0.14 (s, 6H);13C NMR (DMSO-d6) δ 164.14, 157.13, 155.94, 149.00, 147.41, 144.75, 138.78, 138.67, 128.86, 125.00 (t, J = 285 Hz), 121.76, 121.39, 117.76, 113.72, 105.94, 83.21, 71.66, 66.05, 56.84, 54.35, 49.98, 47.20, 34.99, 31.99, 30.28, 27.07, 22.54, 12.63, 11.61, -2.31; HRMS (ESI) Anal. calcd. for C33H40ClF2N8O5Si m / z 729.2542 [M+H]+, found 729.2548. 1-benzyl 4-(3-((2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethoxy)dimethylsilyl)propyl) piperazine-1,4-dicarboxylate Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO To a stirred mixture of 3-((2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethoxy)dimethylsilyl)propyl (4-nitrophenyl) carbonate (25 mg, 60 μmol) and benzylpiperazine-1-carboxylate (20 mg, 17 μL, 89 μmol) in anhydrous 1,2-Dichloroethane (600 μL) was added DIPEA (12 mg, 16 μL, 89 μmol), then the resulting mixture was stirred at room temperature for 6 hours. The reaction mixture was then concentrated under reduced pressure and directly subjected to purification by preparatory-HPLC to afford 1-benzyl 4-(3-((2-(3- (but-3-yn-1-yl)-3H-diazirin-3-yl)ethoxy)dimethylsilyl)propyl) piperazine-1,4-dicarboxylate (5.8 mg, 12 μmol, 20%) as a brown oil. Brown oil;1H NMR (300 MHz, CDCl3) δ 7.39 – 7.32 (m, 5H), 5.15 (s, 2H), 4.06 (t, J = 7.0 Hz, 2H), 3.52 – 3.44 (m, 8H), 3.43 (t, J = 6.3 Hz, 2H), 2.06 – 1.98 (m, 2H), 1.96 (t, J = 2.7 Hz, 1H), 1.69 – 1.56 (m, 6H), 0.64 – 0.54 (m, 2H), 0.12 (s, 6H). To a stirred solution of spiro[indene-2,4'-piperidin]-1(3H)-one hydrochloride (31 mg, 130 μmol) in anhydrous 1,2-Dichloroethane (400 μL) was added DIPEA (40 mg, 54 μL, 310 μmol) and the resulting mixture was stirred at room temperature under an Argon atmosphere for 45 minutes. Then, a solution of 3-((2-(3-(but-3-yn-1-yl)-3H-diazirin-3- yl)ethoxy)dimethylsilyl)propyl (4-nitrophenyl) carbonate (37 mg, 88 μmol) in anhydrous 1,2- Dichloroethane (400 μL) was added and the resulting mixture was stirred at room temperature for 5.5 hours. The reaction mixture was then concentrated under reduced pressure and directly subjected to purification by preparatory HPLC followed by purification by silica gel column chromatography (hexane / EtOAc = 4 / 1) to afford 3-((2-(3-(but-3-yn-1- yl)-3H-diazirin-3-yl)ethoxy)dimethylsilyl)propyl 1-oxo-1,3-dihydrospiro[indene-2,4'- piperidine]-1'-carboxylate (11.2 mg, 26%) as a clear oil. clear oil;1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 7.7 Hz, 1H), 7.62 (td, J = 7.8, 1.2 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 4.17 (bs, 2H), 4.07 (t, J = 7.0 Hz, 2H), 3.45 (t, J = 6.4 Hz, 2H), 3.14 – 3.03 (m, 4H), 2.03 (td, J = 7.6, 2.7 Hz, 2H), 1.98 – Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 1.88 (m, 3H), 1.69 – 1.59 (m, 6H), 1.41 (d, J = 13.0 Hz, 2H), 0.66 – 0.57 (m, 2H), 0.13 (s, 6H). Table 1: Stability of prototype probes under elution condition from neutravidin beads Synthesis of DiLeu Reagents N,N-dimethyl-L-leucine To an 10mL pressure tube equipped with a magnetic stir bar was added L-Leucine (75mg, 1 Eq, 0.57mmol), methanol (2.3mL, 0.25M), and formaldehyde (0.19 g, 0.17 mL, 37% Wt, 4 Eq, 2.3 mmol). Under strong stirring, pyridine-borane complex (0.11 g, 0.12 mL, 2 Eq, 1.1 mmol) was added dropwise. Upon complete addition the reaction mixture was Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO allowed to stir at ambient temperature for 48-72 hours. After completion anhydrous sodium sulfate was added directly into the tube and the contents were filtered over cotton. The solids were washed with methanol and filtrate concentrated under reduced pressure to yield crude product as a white solid. Residue was purified by 2 rounds of trituration with diethyl ether to yield pure N,N-dimethyl-L-Leucine as a white solid (90mg, 99%). Spectral analyses aligned with previously reported.1H NMR (400 MHz, Deuterium Oxide) δ 3.60 – 3.53 (m, 1H), 2.88 (s, 6H), 1.78 – 1.59 (m, 3H), 0.97 (dd, J = 6.1, 4.8 Hz, 6H). General Procedure for Synthesis of Asymmetric N,N-dimethyl-L-leucine N-Cbz-L-Leucine To a 10mL pressure tube was added L-leucine (200 mg, 1 Eq, 1.50 mmol) and Aq. NaOH (60.1 mg, 751 μL, 2 molar, 1 Eq, 1.50 mmol). Vial was cooled to 0°C and, with strong stirring, Cbz-Cl (307 mg, 257 μL, 1.2 Eq, 1.80 mmol) and NaOH (72.1 mg, 901 μL, 2 molar, 1.2 Eq, 1.80 mmol) was added from two separate syringes simultaneously. After complete addition, the solution was left to stir at 0°C for 1h followed by warming to ambient temperature and stirring for an additional hour. After completion the reaction mixture extracted 1x w / Et2O, acidified with 1M HCl, and then extracted 3x w / Et2O. The latter ether portions were combined and dried over sodium sulfate, filtered over cotton, and concentrated down to yield the pure product as a clear oil (338mg, 85%).Spectral analyses aligned with previously reported. N-Cbz-N-methyl-L-Leucine Performed following a published procedure. N-Cbz-L-leucine (140 mg, 1 Eq, 528 μmol) was dissolved in MeCN (1.76 mL) and cooled to 0°C. Upon cooling, NaH (65.4 mg, 60% Wt, 3.1 Eq, 1.64 mmol) was added followed by dropwise addition of MeI (532 mg, 234 μL, 7.1 Eq, 3.75 mmol) to form a thick liquid. The reaction mixture was then allowed to warm to ambient temperature and stirred aggressively (840rpm) overnight. After completion, Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO EtOAc (15 ml) and water (5 ml) were added and the solvent evaporated off. The residue was redissolved in ether and water and extracted 2x w / ether. Organic layers were combined and washed 2x w / sat. sodium bicarb. Aq layers combined and acidified to a pH of 2 w / 1M HCl then extracted 3x w / Et2O. Combined Et2O layers washed 1x w / water, dried over sodium sulfate, and concentrated down. Pure product obtained after flash column chromatography (30 to 50% EtOAc / Hex) as a clear oil (100mg, 67%).Spectral analyses aligned with previously reported. N,N-dimethyl-L-leucine General Procedure A: To an oven dried µwave vial was added N-Cbz-N-methyl-L-leucine (110 mg, 1 Eq, 392 μmol), MeOH (1.31 mL), formaldehyde (63.7 mg, 58.4 μL, 37% Wt, 2 Eq, 785 μmol), and Pd / C (12.5 mg, 10% Wt, 0.03 Eq, 11.8 μmol). The flask was thoroughly purged with argon then hydrogen was bubbled through the solution for one minute. Left stir under a hydrogen atmosphere at ambient temperature overnight. After reaction completion as determined by LC-MS, anhydrous sodium sulfate was added and reaction mixture filtered over celite. The filtrate was concentrated down and the resulting white solid was triturated 2x w / Et2O to furnish the final product as a white solid (54mg, 86%). General procedure B (For D incorporation on second methyl group): To an oven dried µwave vial was added N-Cbz-N-methyl-L-leucine (110 mg, 1 Eq, 392 μmol), MeOH (1.31 mL), and Pd / C (12.5 mg, 10% Wt, 0.03 Eq, 11.8 μmol). The flask was thoroughly purged with argon then hydrogen was bubbled through the solution for one minute. Left stir under a hydrogen atmosphere at ambient temperature overnight. After reaction completion as determined by LC-MS, the reaction mixture filtered over celite and concentrated down. The resulting N-methyl-L-leucine was dissolved in MeOH (1.31 mL) followed by addition of formaldehyde (63.7 mg, 58.4 μL, 37% Wt, 2 Eq, 785 μmol). With strong stirring pyr•BD3 (53.4 mg, 1.5 Eq, 556 μmol) was added and reaction mixture allowed to stir for 24h. After reaction completion as determined by LC-MS, anhydrous sodium sulfate was added and reaction mixture was filtered over cotton. The filtrate was concentrated down and the resulting white solid was triturated 2x w / Et2O to furnish the final product as a white solid (40mg, 66%). N,N-dimethyl-1-13C-L-Leucine (114) Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Performed according to the general procedure for synthesis of symmetric dimethyl leucine with 1-13C-L-Leucine. (79mg, >99% yield). 1H NMR (300 MHz, Deuterium Oxide) δ 3.56 (dd, J = 9.8, 4.7 Hz, 1H), 2.88 (s, 6H), 1.79 – 1.61 (m, 3H), 0.97 (dd, J = 6.0, 3.6 Hz, 6H).13C NMR (75 MHz, D2O) δ 173.65, 36.76, 25.07, 22.69, 20.57. N,N-dimethyl-15N-L-Leucine (115N) Performed according to the general procedure for synthesis of symmetric dimethyl leucine with15N-L-Leucine. (79mg, >99% yield). 1H NMR (300 MHz, Deuterium Oxide) δ 3.61 – 3.53 (m, 1H), 2.89 (d, J = 0.8 Hz, 6H), 1.79 – 1.58 (m, 3H), 0.97 (dd, J = 6.0, 3.6 Hz, 6H).13C NMR (75 MHz, D2O) δ 173.57, 70.27, 36.74, 25.08, 22.69, 20.56. N,N-dimethyl-2-13C-L-Leucine (115C) Performed according to the general procedure for synthesis of symmetric dimethyl leucine with15N-L-Leucine. (76mg, 96% yield). 1H NMR (300 MHz, Deuterium Oxide) δ 3.85 – 3.28 (m, 1H), 2.89 (d, J = 3.2 Hz, 6H), 1.80 – 1.60 (m, 3H), 0.97 (dd, J = 6.0, 3.5 Hz, 6H).13C NMR (75 MHz, D2O) δ 173.91, 70.27, 36.75(d), 25.07, 22.67, 20.58. N,N-13C2-dimethyl-L-Leucine (116C2) Performed according to the general procedure for synthesis of symmetric dimethyl leucine with13C-Formaldehyde (20% Wt). (72mg, 98% yield). 1H NMR (300 MHz, D2O) δ 3.51 – 3.43 (m, 1H), 3.03 (d, J = 3.9 Hz, 3H), 2.55 (d, J = 3.9 Hz, 3H), 1.70 – 1.49 (m, 3H), 0.87 (dd, J = 6.0, 3.6 Hz, 6H).13C NMR (75 MHz, D2O) δ 173.56, 70.26, 36.73, 25.06, 22.69, 20.55. N,N-D2-dimethyl-L-Leucine (116D2) Performed according to the general procedure for synthesis of symmetric dimethyl leucine with freshly prepared pyr•BD3. (76mg, 95% yield). 1H NMR (400 MHz, Deuterium Oxide) δ 3.55 (dt, J = 9.5, 2.4 Hz, 1H), 2.85 (p, J = 1.8 Hz, 4H), 1.77 – 1.57 (m, 3H), 0.95 (td, J = 5.8, 2.6 Hz, 6H).13C NMR (101 MHz, D2O) δ 173.57, 70.19, 36.72, 25.04, 22.67, 20.53. N,N-D2-dimethyl-2-13C-L-Leucine (117C / D2) Performed according to the general procedure for synthesis of symmetric dimethyl leucine with freshly prepared pyr•BD3. (79mg, 99% yield). Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 1H NMR (400 MHz, Deuterium Oxide) δ 3.75 – 3.33 (m, 1H), 2.85 (dq, J = 3.4, 1.3 Hz, 4H), 1.78 – 1.58 (m, 2H), 0.95 (dd, J = 6.1, 4.8 Hz, 6H).13C NMR (101 MHz, D2O) δ 173.29, 70.19, 36.87, 36.53, 25.04, 22.69, 20.53. N,N-13C2-dimethyl-15N-L-Leucine (117N / C2) Performed according to the general procedure for synthesis of symmetric dimethyl leucine with13C-Formaldehyde (20% Wt) and15N-L-Leucine. (80mg, >99% yield). 1H NMR (400 MHz, Deuterium Oxide) δ 3.78 – 3.33 (m, 1H), 2.88 (dt, J = 143.8, 3.6 Hz, 6H), 1.78 – 1.58 (m, 3H), 0.96 (dd, J = 6.0, 4.7 Hz, 6H).13C NMR (101 MHz, D2O) δ 171.02, 70.22, 36.63 (d), 25.07, 22.71, 20.55. N,N-13C2-dimethyl-2-13C-L-Leucine (117C3) Performed according to the general procedure for synthesis of symmetric dimethyl leucine with13C-Formaldehyde (20% Wt) and 2-13C-L-Leucine. (79mg, 98% yield). 1H NMR (400 MHz, Deuterium Oxide) δ 3.56 (dddd, J = 144.5, 10.3, 4.9, 2.5 Hz, 1H), 2.88 (ddd, J = 143.8, 4.0, 3.2 Hz, 6H), 1.79 – 1.60 (m, 2H), 0.97 (dd, J = 6.1, 4.8 Hz, 6H).13C NMR (101 MHz, D2O) δ 171.01, 70.25, 41.18, 25.06, 22.66, 20.55. N,N-13C2-dimethyl-2-13C,15N-L-Leucine (118N / C3) Performed according to the general procedure for synthesis of symmetric dimethyl leucine with13C-Formaldehyde (20% Wt) and 2-13C,15N-L-Leucine. (75mg, 94% yield). 1H NMR (400 MHz, Deuterium Oxide) δ 3.56 (ddd, J = 9.9, 4.6, 2.3 Hz, 1H), 2.88 (ddd, J = 143.7, 3.9, 0.8 Hz, 6H), 1.77 – 1.60 (m, 2H), 0.96 (dd, J = 6.1, 4.8 Hz, 5H).13C NMR (101 MHz, D2O) δ 173.54, 171.00, 81.72, 70.25, 41.05, 25.05, 22.68, 20.55. N,N-D2-dimethyl-L-Leucine (118C2 / D2) Performed according to the general procedure for synthesis of symmetric dimethyl leucine with13C-Formaldehyde (20% Wt) and freshly prepared pyr•BD3. (80mg, >99% yield). 1H NMR (400 MHz, Deuterium Oxide) δ 3.56 (ddd, J = 9.9, 4.5, 2.3 Hz, 1H), 2.86 (ddt, J = 143.4, 3.7, 1.7 Hz, 4H), 1.78 – 1.59 (m, 3H), 1.01 – 0.92 (m, 6H).13C NMR (101 MHz, D2O) δ 173.54, 81.71, 70.19, 41.73, 40.00, 36.71, 31.76, 31.54, 31.32, 25.05, 22.68, 20.55. N,N-D2-dimethyl-2-13C,15N-L-Leucine (118N / C / D2) Performed according to the general procedure for synthesis of symmetric dimethyl leucine with 2-13C,15N-L-Leucine and pyr•BD3. (80mg, >99% yield). Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 1H NMR (400 MHz, Deuterium Oxide) δ 3.78 – 3.36 (m, 1H), 2.87 (s, 4H), 1.77 – 1.60 (m, 3H), 0.97 (dd, J = 6.1, 4.8 Hz, 6H).13C NMR (101 MHz, D2O) δ 173.28, 70.21, 36.88, 36.54, 25.07, 22.67, 20.57. N,N-D4-dimethyl-L-Leucine (118D4) Performed according to the general procedure for synthesis of symmetric dimethyl leucine with D2-Formaldehyde (20% Wt in D2O). (65mg, 87% yield). 1H NMR (300 MHz, D2O) δ 3.51 – 3.42 (m, 1H), 2.76 (t, J = 1.8 Hz, 6H), 1.69 – 1.51 (m, 3H), 0.87 (dd, J = 6.0, 3.6 Hz, 6H).13C NMR (75 MHz, D2O) δ 173.58, 70.14, 36.73, 25.05, 22.68, 20.56. N,N-D4-dimethyl-15N-L-Leucine (119N / D4) Performed according to the general procedure for synthesis of symmetric dimethyl leucine with15N-L-Leucine and D2-Formaldehyde (20% Wt in D2O). (75mg, 93% yield). 1H NMR (300 MHz, Deuterium Oxide) δ 3.61 – 3.53 (m, 1H), 2.86 (s, 2H), 1.80 – 1.61 (m, 3H), 0.97 (dd, J = 6.0, 3.6 Hz, 6H).13C NMR (75 MHz, D2O) δ 173.56, 70.10, 36.72, 25.05, 22.69, 20.57. N,N-D2-dimethyl-L-Leucine (119N / C2 / D2) Performed according to the general procedure for synthesis of symmetric dimethyl leucine with15N-L-Leucine,13C-Formaldehyde (20% Wt), and freshly prepared pyr•BD3. (73mg, 90% yield). 1H NMR (400 MHz, Deuterium Oxide) δ 3.56 (ddt, J = 9.9, 4.6, 2.3 Hz, 1H), 3.10 – 2.61 (m, 4H), 1.78 – 1.58 (m, 3H), 0.96 (dd, J = 6.1, 4.8 Hz, 6H).13C NMR (101 MHz, D2O) δ 173.54, 70.16, 41.89, 39.88, 36.72, 31.77, 31.50, 31.28, 25.05, 22.68, 20.55. N,N-D4-dimethyl-13C-L-Leucine (119C / D4) Performed according to the general procedure for synthesis of symmetric dimethyl leucine with 2-13C-L-Leucine and D2-Formaldehyde (20% Wt in D2O). (79mg, >99% yield). 1H NMR (400 MHz, Deuterium Oxide) δ 3.77 – 3.34 (m, 1H), 2.85 (dt, J = 3.5, 1.7 Hz, 2H), 1.78 – 1.58 (m, 3H), 0.96 (dd, J = 6.1, 4.7 Hz, 6H).13C NMR (101 MHz, D2O) δ 173.53(d), 70.14, 36.68(d), 25.05, 22.66, 20.55. General Procedure for Synthesis of Asymmetric N,N-dimethyl-L-leucine: N,N-D1-dimethyl-L-Leucine (115D) Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with D1-iodomethane. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 1H NMR (400 MHz, Deuterium Oxide) δ 3.61 – 3.54 (m, 1H), 2.89 (s, 5H), 1.79 – 1.60 (m, 3H), 0.97 (dd, J = 6.1, 4.8 Hz, 6H).13C NMR (101 MHz, D2O) δ 173.54, 70.24, 36.73, 25.07, 22.69, 20.57. N,N-13C,D1-dimethyl-L-Leucine (116C / D) Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with D1-iodomethane and13C-Formaldehyde (20% Wt in H2O). 1H NMR (400 MHz, Deuterium Oxide) δ 3.58 (ddd, J = 10.0, 4.8, 2.3 Hz, 1H), 3.11 – 2.66 (m, 5H), 1.79 – 1.62 (m, 3H), 0.97 (dd, J = 6.1, 4.8 Hz, 6H).13C NMR (101 MHz, D2O) δ 173.56, 70.24, 42.28, 40.05, 36.72, 25.07, 22.69, 20.57.15N,N-13C-dimethyl-L-Leucine (116N / C) Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with15N-L-Leucine and13C-Formaldehyde (20% Wt in H2O). 1H NMR (400 MHz, Deuterium Oxide) δ 3.61 – 3.54 (m, 1H), 2.89 (td, J = 73.0, 72.4, 9.0 Hz, 6H), 1.79 – 1.58 (m, 3H), 0.98 (dd, J = 6.1, 4.8 Hz, 6H).13C NMR (101 MHz, D2O) δ 173.55, 70.27, 42.28, 40.04, 36.72, 25.08, 22.69, 20.56.15N,N-13C-dimethyl-L-Leucine (116N / D) Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with15N-L-Leucine and D1-iodomethane. 1H NMR (400 MHz, Deuterium Oxide) δ 3.60 – 3.55 (m, 1H), 2.89 (d, J = 9.5 Hz, 5H), 1.79 – 1.60 (m, 3H), 0.98 (dd, J = 6.1, 4.8 Hz, 6H).13C NMR (101 MHz, D2O) δ 70.24, 36.72, 25.08, 22.69, 20.57.15N-13C-N-D1-dimethyl-L-Leucine (117N / C / D) Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with15N-L-Leucine, D1-iodomethane, and13C-Formaldehyde (20 Wt% in H2O). 1H NMR (400 MHz, Deuterium Oxide) δ 3.56 (ddd, J = 9.9, 4.7, 2.2 Hz, 1H), 3.09 – 2.63 (m, 5H), 1.78 – 1.59 (m, 3H), 0.96 (dd, J = 6.1, 4.8 Hz, 6H).13C NMR (101 MHz, D2O) δ 173.55, 70.20, 42.27, 40.05, 36.72, 25.07, 22.69, 20.56. N-13C,D1-N-2-13C-dimethyl-L-leucine (117C2 / D) Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with 2-13C-L-Leucine, D1-iodomethane, and13C-Formaldehyde (20 Wt% in H2O). Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 1H NMR (400 MHz, Deuterium Oxide) δ 3.66 – 3.20 (m, 1H), 2.97 – 2.53 (m, 5H), 1.65 – 1.48 (m, 3H), 0.84 (dd, J = 6.1, 4.8 Hz, 6H).13C NMR (101 MHz, D2O) δ 163.73, 70.25, 52.24, 42.28, 40.04, 25.06, 22.71, 20.56. N,N-D3-dimethyl-L-leucine (117D3) Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with D3-iodomethane. 1H NMR (400 MHz, Deuterium Oxide) δ 3.60 – 3.51 (m, 0H), 2.88 (d, J = 8.4 Hz, 1H), 1.79 – 1.59 (m, 0H), 0.96 (dd, J = 6.1, 4.8 Hz, 1H).13C NMR (101 MHz, D2O) δ 173.55, 70.18, 36.72, 25.06, 22.69, 20.57. N,N-D5-dimethyl-L-Leucine (119D5) Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with D3-iodomethane and D2-formaldehyde (20% Wt in D2O). 1H NMR (400 MHz, Deuterium Oxide) δ 3.62 – 3.54 (m, 1H), 2.86 (s, 1H), 1.81 – 1.60 (m, 3H), 0.97 (dd, J = 6.1, 4.8 Hz, 6H).13C NMR (101 MHz, D2O) δ 173.56, 70.12, 36.72, 25.06, 22.68, 20.57. N,N-13C2,D-dimethyl-13C,15N-L-Leucine (119N / C3 / D) Performed according to the general procedure B for synthesis of asymmetric dimethyl leucine with 2-13C,15N-L-Leucine,13C-iodomethane,,13C-formaldehyde (20% Wt), and freshly prepared pyr•BD3. 1H NMR (400 MHz, Deuterium Oxide) δ 3.78 – 3.37 (m, 1H), 2.88 (dq, J = 143.7, 3.7 Hz, 6H), 1.78 – 1.60 (m, 3H), 0.97 (dd, J = 6.1, 4.8 Hz, 6H).13C NMR (101 MHz, D2O) δ 173.80, 173.27, 171.00, 81.73, 70.23, 48.86, 42.08, 40.07, 36.88, 36.54, 25.07, 22.71, 20.58. N,N-13C2,D-dimethyl-15N-L-Leucine (118N / C2 / D) Performed according to the general procedure B for synthesis of asymmetric dimethyl leucine with15N-L-Leucine,13C-iodomethane,13C-formaldehyde (20% Wt), and pyr•BD3. 1H NMR (400 MHz, Deuterium Oxide) δ 3.60 – 3.53 (m, 1H), 2.88 (dd, J = 143.8, 4.0 Hz, 5H), 1.79 – 1.60 (m,3H), 0.97 (dd, J = 6.1, 4.9 Hz, 6H).13C NMR (101 MHz, D2O) δ 81.74, 70.24, 42.18, 39.99, 36.73, 25.07, 22.69, 20.56. N,N-13C2,D-dimethyl-1,2-13C2-L-Leucine (118C3 / D) Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with 1,2-13C2-L-Leucine,13C-iodomethane,13C-Formaldehyde (20% Wt) and freshly prepared pyr•BD3. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 1H NMR (400 MHz, Deuterium Oxide) δ 3.80 – 3.37 (m, 1H), 2.88 (dq, J = 143.2, 3.5 Hz, 5H), 1.78 – 1.60 (m, 2H), 0.97 (dd, J = 6.1, 4.9 Hz, 6H).13C NMR (101 MHz, D2O) δ 173.81, 173.28, 81.73, 70.48, 69.96, 48.86, 41.01, 25.06, 22.67, 20.56. N,N-13C,D3-dimethyl-L-leucine (118C / D3) Performed according to the general procedure B for synthesis of asymmetric dimethyl leucine with13C-iodomethane, D2-formaldehyde (20% Wt), and pyr•BD3. 1H NMR (400 MHz, Deuterium Oxide) δ 3.56 (ddd, J = 9.9, 4.8, 2.2 Hz, 1H), 2.88 (d, J = 143.8 Hz, 3H), 1.81 – 1.62 (m, 3H), 0.96 (dd, J = 6.1, 4.8 Hz, 6H).13C NMR (101 MHz, D2O) δ 70.18, 42.19, 40.20, 36.72, 25.06, 22.68, 20.56. N,N-13C2,D3-dimethyl-L-leucine (119C2 / D3) Performed according to the general procedure B for synthesis of asymmetric dimethyl leucine with13C,D3-iodomethane,13C-formaldehyde (20% Wt). 1H NMR (400 MHz, Deuterium Oxide) δ 3.44 (ddd, J = 9.9, 4.4, 2.1 Hz, 1H), 2.75 (ddd, J = 144.2, 7.9, 3.4 Hz, 3H), 1.66 – 1.48 (m, 3H), 0.84 (dd, J = 6.1, 4.8 Hz, 6H).13C NMR (101 MHz, D2O) δ 173.54, 70.18, 42.22, 39.98, 25.06, 22.69, 20.57. N,N-13C,D3-dimethyl-15N-L-leucine (119N / C / D3) Performed according to the general procedure B for synthesis of asymmetric dimethyl leucine with15N-L-Leucine,13C-iodomethane, D2-formaldehyde (20% Wt), and pyr•BD3. 1H NMR (400 MHz, Deuterium Oxide) δ 3.56 (ddd, J = 10.0, 4.8, 2.2 Hz, 0H), 3.09 – 2.68 (m, 0H), 1.78 – 1.61 (m, 0H), 0.96 (dd, J = 6.1, 4.8 Hz, 1H).13C NMR (101 MHz, D2O) δ 173.56, 70.14, 41.68, 40.22, 36.71, 25.05, 22.69, 20.57. Chemoproteomic Interaction Site Mapping As achieving high coverage interaction site mapping remains a challenging proposition for established proteomic workflows, the feasibility of peptide capture was next tested with the prototype SEE-CITE reagents. Using the established single-pot solid-phase enhanced sample preparation (SP3) workflow, in situ labeling using 2b in K562 cells was performed, and lysate was subjected to click conjugation biotinylation with the previously reported C3-biotin azide capture handle, followed by SP3 cleanup, tryptic digestion, enrichment on neutravidin resin, and simultaneous acid-mediated peptide elution andcleavage of the silyl ether moiety (FIG. 2B). Gratifyingly, LC-MS / MS analysis detectedcrosslinked peptides modified with the expected +436.2256 mass shift, matching the click conjugated and acid-cleaved SEE-CITE adduct M1. However, the number of modified Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO peptides identified by FragPipe search, 379 unique peptides, was unexpectedly low when compared to our cysteine enriched chemoproteomic samples prepared with SP3 cleanup (typically >10,000 unique cysteine-containing peptides). Initially, it was speculated that incomplete reagent cleavage under the comparatively mild elution conditions might contribute to this low coverage. However, open search analysis with FragPipe supported complete cleavage of the silyl ether, with no peptides detected with modifications matching the parent compound mass. Some of the coverage differences between cysteine chemoproteomics and SEE-CITE labeling can be ascribed to the comparatively low diazirine crosslinking efficiency (i.e. rapid quenching by water or rearrangement). However, it was hypothesized that additional parameters could be tailored to enhance the coverage. Therefore, factors that could impact the coverage were next investigated, including those that had been previously found to contribute to chemoproteomic coverage for other samples (1) the nature of the biotin capture reagent, (2) the amount of input lysate, and (3) the choice of biotin decontamination method, and factors specific to the SEE-CITE system including (4) relative labeling of different amino acid residues, (5) and the MS acquisition parameters. To test whether scaleup of protein input could improve peptide coverage, the lysate volume was increased to achieve an eight-fold (400 μg to 3,200 μg) increase in protein input(FIG. 2B). The initial scaleup efforts, using SP3 cleanup, failed to afford a significantincrease in coverage. As it has been found that SP3 cleanup performs poorly at increased protein loading, it was next investigated whether chloroform methanol (CHCl3 / MeOH) sample cleanup would yield more favorable coverage results upon scaleup. Somewhat unexpectedly, given that it was previously found, for cysteine chemoproteomics, modestly decreased coverage for samples prepared with CHCl3 / MeOH when compared to SP3, for SEE-CITE labeled samples, a robust increase in modified peptides identified from 3,200 μg protein input (FIG.2C) was observed. An additional increase in detectable modified peptides was observed for samples labeled with C4-biotin—these results are consistent with the prior findings of improved gas phase fragmentation properties and increased coverage for the C4- biotin reagent. It was next asked whether further improvements to the sample preparation workflow, liquid chromatography, and acquisition parameters could further enhance the coverage. Using repeat injections of the same sample, the MS2 resolving power was varied from15K to 60K, Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO which revealed a modest increase in coverage for acquisition at 30K resolving power. Motivated by recent reports of container dependent differences in coverage, the coverage obtained for samples prepared using microtubes from two different vendors was compared, and it was found that a 1.8-fold increase in peptide coverage could be achieved through container selection. Building upon these coverage improvements, the coverage of the methyl 2a and ethyl 2b probes for datasets (FIG.2D) was compared. It was found that the methyl 2a scout probe affords a ~1.5-fold increase in coverage, which is consistent with the increasedlabeling observed for the gel-based analysis (FIG. 2A). A slight but not significant 1.2-foldfurther increase in coverage was achieved by lengthening the LC gradient from 70 to 140 min acquired using 70 min and 140 min liquid chromatography gradients (FIG.2D). In situ photoaffinity labeling 5.0×106K562 or KCL-22 cells grown under the aforementioned conditions was resuspended in 1 mL of RPMI without FBS and penicillin-streptomycin in a 6 cm plate or a 6-well plate and the probe solution in DMSO was added to achieve the desired concentration (DMSO never exceeded 1%). After 1 hour incubation at 37 °C / 5% CO2, the plate was put on ice and then UV (350 nm) was irradiated for 20 minutes without a lid. For competitive experiments, competitor or DMSO was added and incubated at 37 °C for 30 minutes prior to 30-minute probe treatment. The cells were harvested to a 1.5 mL Eppendorf tube and rinsed with 0.5 mL cold PBS. After centrifuge at 1,000 x g, 4 °C for 3 minutes, the supernatant was aspirated. The cell pellet was gently resuspended in 1 mL cold-PBS and centrifuged again at 1,000 x g, 4 ℃ for 3 minutes. After discarding the supernatant, 0.3% CHAPS in PBS was added to the cell pellet and pipetted up and down at least 10 times, then incubated at 4 °C for 30 minutes. The mixture was spun down at 15,000 x g at 4 °C for 15 minutes. The supernatant was taken to a new 1.5 mL Eppendorf tube and the protein concentration of the lysate was determined by BioRad DC protein assay. The lysate was diluted to 1 mg / mL with PBS and 25 uL was mixed with 3 μL of CuAAC cocktail (freshly prepared from 1.5 uL 1.75 mM of TBTA intBuOH / DMSO = 4 / 1, 0.5 uL 50 mM of CuSO4 in MB water, 0.5 μL 50 mM of TCEP in water, 0.5 uL 1.25 mM of TAMRA-azide in DMSO. The reagents were mixed in this order). After incubation at room temperature in the dark for 1 hour, 12 μL 4x loading dye was added and the mixture was boiled around 100 °C for 5 minutes. Proteins were separated by SDS−PAGE (4-12% gradient Bis-Tris gel, MOPS running buffer) at 160 V and then visualized by in-gel fluorescence scanning on a ChemiDoc Imaging System. After that, the Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO gel was stained by coomassie and incubated at room temperature for 1 hour or in the cold room overnight. After that, it was imaged by coomassie staining setting on a ChemiDoc Imaging System. Preparation of NeutrAvidin enrichment samples The probe-labeled K562 proteome was prepared as described in 4. For SP3 purification, 200 μL 2.0 mg / mL proteome was treated with 24 μL CuAAC cocktail (freshly prepared from 12 μL of 5 mM TBTA intBuOH / DMSO = 4 / 1, 4 uL of 50 mM CuSO4 in MB water, 4 uL of 50 mM TCEP in water, 4 uL of 1.25 mM the corresponding biotin-azide in DMSO (final biotin concentration was 2-fold of the probe concentration, but at least 100 μM or more). The reagents were mixed in this order). After CuAAC labeling, each sample was treated with 20 μL 10% SDS (final concentration of SDS ≒ 1%) and then 0.5 μL benzonase (Fisher Scientific, 70-664-3) for 30 minutes at 37 °C. For each 200 μL sample (2 mg / mL protein concentration), 20 μL Sera-Mag Speed- Beads Carboxyl Magnetic Beads, hydrophobic (GE Healthcare, 65152105050250) and 20 μL Sera-Mag Speed- Beads Carboxyl Magnetic Beads, hydrophilic (GE Healthcare, 45152105050250) were mixed and washed with water for three times. The bead slurries were then transferred to the CuAAC samples and incubated for 10 min at room temperature with shaking (1000 rpm). Absolute ethanol (500 μL) was added to each sample, and the samples were incubated for 5 min at room temperature with shaking (1,000 rpm). Samples were then placed on a magnetic rack, aspirated the supernatant and washed three times with 80% ethanol in water (400 μL). After washing, beads were resuspended in 200 μL 2 M urea in 0.5% SDS / PBS. DTT (10 μL of 200 mM stock in water, final concentration was 10 mM) was added into each sample and the sample was incubated at 65 °C for 15 min. Then, iodoacetamide (10 μL of 400 mM stock in water, final concentration was 20 mM) was added and the solution was incubated for 30 min at 37 °C with shaking. After that, absolute ethanol (400 μL) was added to each sample, and the samples were incubated for 5 min at room temperature with shaking (1,000 rpm). Beads were then again washed three times with 80% ethanol in water (400 μL). Next, beads were resuspended in 200 μL of 2 M urea in PBS and 3 μL 1 mg / mL trypsin solution was added. Digest was overnight at 37 °C with shaking (200 rpm, 14-16 h). After digestion, 3.8 mL acetonitrile was added to each sample and the mixtures were incubated for 10 min at room temperature with shaking (1,000 rpm). The beads were then washed with 1 mL acetonitrile three times with a magnetic rack. Peptides were eluted from SP3 beads with 100 μL of 2% Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO DMSO in water for 30 min at 37 °C with shaking (1,000 rpm). The elution was repeated again with 100 μL of 2% DMSO in water. For each sample, 50 μL of NeutrAvidin Agarose resin slurry (Pierce, 29200) washed three times in IAP buffer (50 mM MOPS pH 7.2, 10 mM sodium phosphate, and 50 mM NaCl buffer) and then resuspended in 500 uL IAP buffer and added to the peptide solutions eluted from SP3 beads, and the samples were rotated for 2 hours at room temperature. After incubation, the beads were pelleted by centrifugation (1,800 g, 2 minutes) and washed (3 × 1 mL PBS, 3 × 1 mL water). Bound peptides were eluted twice with 80 μL of 80% acetonitrile in water containing 0.1% TFA. The first 10 min incubation at room temperature and the second one at 72 °C. The beads were washed again with 40 uL solvent. The combined eluents (200 uL) were dried (SpeedVac), then reconstituted with 5% acetonitrile and 1% formic acid in MB water and analyzed by LC-MS / MS. For CHCl3 / MeOH purification, CuAAC was performed the same as in SP3 purification (CuAAC cocktail was added to 2.0 mg / mL proteome in proportion to the volume of lysate. For example, 96 uL CuAAC cocktail for 800 uL of 2 mg / mL lysate). After incubation at room temperature for 1 hour, 3x volume MeOH, 1x volume CHCl3, and 3x volume of water were added to the CuAAC mixture in this order and then centrifuged at 4 °C for 10 minutes (4,200 x g). The top layer was aspirated off without disturbing the white protein disc and 3x volume MeOH was added. After centrifugation at 4oC for 10 minutes (4,200 x g), the supernatant was removed to give protein pellets. The pellet was washed with gentle sonication in 1x volume cold MeOH and centrifuged at 4,200 x g for 10 minutes to pellet protein and remove supernatant (two times). The protein pellets were resuspended in 6M urea (A half volume of the lysate, for example, 400 μL 6M urea for 800 uL of the lysate. Similarly, 600 μL 6M urea for 1,200 uL of the lysate, 800 μL 6M urea for 1,600 μL of the lysate) and 200 mM DTT in MB water was added so that the final [DTT] was 10 mM (20 μL for 800 μL / 30 μL for 1,200 μL / 40 μL for 1,600 μL of lysate). After incubation at 65 °C for 15 minutes, 400 mM of iodoacetamide (IA) in MB water was added so that the final [IA] was 20 mM (20 μL for 800 μL / 30 μL for 1,200 μL / 40 μL for 1,600 μL of lysate) and incubated at 37 °C for 30 minutes. PBS was added to adjust [urea] ca.2M (760μL for 800 μL of lysate / 1,140 μL for 1,200 μL of lysate / 1,520 μL for 1,600 μL of lysate) then 8 μL of 5 mg / mL trypsin was added to the mixture and digested overnight at 37 °C with shaking (200 rpm, 14-16 h). After digestion, 10% SDS was added (for example, 270 uL for 1,600 μL lysate) and heated at 60°C Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO for 5 minutes to solubilize the insoluble substances. After that, PBS was added to adjust to ca. 0.2% SDS (urea concentration ~0.36 M). For each sample, 50 μL of NeutrAvidin Agarose resin slurry (Pierce, 29200) was washed three times in 10 mL IAP buffer (50 mM MOPS pH 7.2, 10 mM sodium phosphate, and 50 mM NaCl buffer) and then resuspended in 500 μL IAP buffer and added to the peptide solutions above, and the samples were rotated for 2 hours at room temperature. After incubation, the beads were pelletized by centrifugation at 1,800 x g for 2 minutes and washed with 1 mL PBS (two times) then 1 mL water (three times). Bound peptides were eluted two times with 80 μL of 80% acetonitrile in MB water containing 0.1% TFA. The first 10 minutes incubation was at room temperature and the second one was at 72 °C, then rinsed with 40 μL of eluate. The combined eluents were dried (SpeedVac), then reconstituted with 5% acetonitrile and 1% FA in MB water and analyzed by LC-MS / MS. Preparation of streptavidin enrichment samples The probe-labeled proteome was prepared as described in 4.200 μL 1.0 mg / mL labeled proteome was treated with 24 μL CuAAC cocktail (freshly prepared from 12 μL of 1.75 mM TBTA intBuOH / DMSO = 4 / 1, 4 μL of 50 mM CuSO4 in MB water, 4 μL of 50 mM TCEP in water, 4 μL of 5 mM the corresponding biotin-azide in DMSO (final biotin concentration was 2-fold of the probe concentration, but at least 100 μM or more). The reagents were mixed in this order). After CuAAC labeling, each sample was treated with 20 μL 10% SDS (final concentration of SDS ca.1%). For each 200 μL sample (1.0 mg / mL protein concentration), 10 μL Sera-Mag Speed- Beads Carboxyl Magnetic Beads, hydrophobic (GE Healthcare, 65152105050250) and 10 μL Sera-Mag Speed- Beads Carboxyl Magnetic Beads, hydrophilic (GE Healthcare, 45152105050250) were mixed and washed with water for three times. The bead slurries were then transferred to the CuAAC samples and incubated for 10 minutes at room temperature with shaking (1,000 rpm). Absolute ethanol (400 μL) was added to each sample, and the samples were incubated for 10 min at room temperature with shaking (1,000 rpm). Samples were then placed on a magnetic rack, aspirated the supernatant and washed three times with 80% ethanol in water (400 μL). After washing, beads were resuspended in 50 uL of 0.2% SDS in PBS and incubated for 30 min at room temperature with shaking (1,000 rpm). Samples were then placed on a magnetic rack and the supernatant was transferred to a new 1.5 mL Eppendorf tube (two times, 100 uL in total). For each sample, 50 μL of Streptavidin Agarose resin slurry (Pierce, 20353) washed Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO in PBS buffer and then resuspended in 500 uL PBS buffer and added to the protein solutions eluted from SP3 beads, and the samples were rotated for 2 hours at room temperature. After incubation, the beads were pelleted by centrifugation at 1,800 x g for 2 minutes and washed with 1 mL PBS (two times) then 1 mL water (two times). After washing, beads were resuspended in 200 μL 6 M urea in PBS. DTT (10 μL of 200 mM stock in water, final concentration was 10 mM) was added into each sample and the sample was incubated at 65 °C for 15 min. Then, iodoacetamide (10 μL of 400 mM stock in water, final concentration was 20 mM) was added and the solution was incubated for 30 min at 37 °C. After that, 400 uL PBS was added to dilute urea concentration to ~2M, then the beads were spun down at 1,800 x g for 2 minutes, and aspirated the supernatant. The beads were resuspended in 200 μL 2 M urea in PBS and 3 μL 1 mg / mL trypsin solution was added and incubated overnight at 37 °C with shaking (200 rpm, 14-16 h). After digestion, 10 μL of 10% trifluoroacetic acid (TFA) was added to each ~200 μL samples to achieve the final concentration of 0.5%. Samples were then cleaned up with Pierce C18 spin tips (Thermo Fisher, Cat. No.87784) according to the manufacturer’s protocol. The samples were dried by SpeedVac, reconstituted with a mass spectrometry buffer (5% acetonitrile and 1% formic acid in molecular biology- grade water) and analyzed by LC-MS / MS. Delineation of protein targets and labeling sites Satisfied with the improved coverage afforded by the methodological optimization, attention then turned to stratifying the protein targets and labeling sites captured by the SEE-CITE probe 2a. Using the FragPipe workflow for PTMProphet localization site analysis, theamino acid residues identified as labeled in 2a-modified peptides were analyzed. Consistent with prior reports, a marked enrichment for diazirine labeling at acidic residues (FIG.2E) was observed. Nucleophilic cysteine, histidine and tyrosine residues were also frequently modified, whereas basic arginine and lysine residues were rarely detected as labeled by 2a. Covalent modification of lysine and arginine residues would cause a missed cleavage event and generate comparatively long and possibly non-proteotypic peptides. Therefore, the overlap between 2a-enriched proteins and 2a-labeled peptides M2 was assessed. It was found that 1,304 proteins were significantly enriched by 2a in a UV-dependent manner, as identified by label free quantification (LFQ) analysis of K562 cells treated with 2a (log2(FC)>1 and p < 0.05, FC = fold change of the intensity of the UV-treated group / the control group, FIG.2F). Gratifyingly, 2a binding sites were identified for 32% of enriched Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO proteins (417 total proteins). Unexpectedly, 515-total labeled peptides were identified matching proteins that were either not identified (158 total) or not enriched in the LFQ datasets (357 total) (FIG.2G). The majority of these sites were ascribed to low occupancy labeling sites, likely stemming from comparatively modest affinity of 2a for the engaged proteins, due to the small size and lack of hydrogen bonding moieties in this scout probe (FIG.2G). Comparing all identified proteins to enriched proteins, protein class analysis revealed a slight increase in enzymes and a marked decrease in transcription factors (TFs) enriched by scout probe 2a, consistent with the general paucity of small molecule binding pockets in TFs relative to enzymes (FIG.2H). Liquid-chromatography tandem mass-spectrometry (LC-MS / MS) analysis Table 2: Chromatography settings for LC-MS / MS acquisition The samples were analyzed by liquid chromatography tandem mass spectrometry using a Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer. Peptides were fractionated online using a 18 cm long, 100 μM inner diameter (ID) fused silica capillary packed in-house with bulk C18 reversed phase resin (particle size, 1.9 μm; pore size, 100 Å; Dr. Maisch GmbH). The 70-minute or 140-min water acetonitrile gradient was delivered using a Thermo Scientific™ EASY-nLC™ 1200 system at different flow rates (Buffer A: water with 3% DMSO and 0.1% formic acid and Buffer B: 80% acetonitrile with 3% DMSO Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO and 0.1% formic acid). The detailed gradient includes 0 - 5 min from 3% to 10% at 300 nL / min, 5 – 64 min from 10% to 50% at 220 nL / min, and 64 – 70 min from 50% to 95% at 250 nL / min buffer B in buffer A for 70 min gradient, or 0 - 6 min from 3 % to 20 % at 300 nL / min, 6 - 130 min from 20 % to 38 % at 220 nL / min, and 130 – 140 min from 38% to 95% at 250 nL / min buffer B in buffer A for 140 min gradient. Data was collected with charge exclusion (1, 8,>8). Data was acquired using a Data-Dependent Acquisition (DDA) method comprising a full MS1 scan (Resolution = 120,000) followed by sequential MS2 scans (Resolution = 15,000, 30,000, 60,000) to utilize the remainder of the 3 second cycle time. Time between master scans was set 1s and 3s for compound labeling datasets, validation datasets, and fractionation datasets. HCD collision energy of MS2 fragmentation was 30%. Data compilation and statistics Raw data collected by LC-MS / MS were searched with MSFragger and FragPipe (version 20). The proteomic workflow and its collection of tools was set as default. Precursor and fragment mass tolerance was set as 20 ppm. Missed cleavages were allowed up to 1. Peptide length was set 7 - 50 and peptide mass range was set 500 - 5000. For SEE-CITE isoTOP-ABPP experiments (peptide-level analyses), MS1 labeling quant was enabled with Light set as *+436.2256 and Heavy set as *442.2633. MS1 intensity ratio of heavy and light labeled peptides were reported. For protein-level analyses quantified using label-free quantification, LFQ-MBR workflow was run with turning MSBooster and Percolater. Identified proteins were filtered by Perseus 2.0.11 to retain only proteins identified in at least 2 replicates (for experiments with 3 replicates / condition) or 3 replicates (for experiments with 6 replicates / condition) of at least 1 experimental condition per experiment. Missing values were then imputed by Perseus based on the normal distribution. For experiments utilizing MS1 quantitation (SEE-CITE based isoTOP-ABPP), custom python scripts were implemented to compile labeled peptide and protein datasets. Unique proteins and unique peptides were quantified for each dataset. Unique proteins were established based on UniProt protein IDs. Unique peptides were found based on sequences containing modified residues. Unique modified peptides were classified by an identifier consisting of a UniProt protein ID, the modified residue, and the corresponding amino acid number (ProteinID_*#); residue numbers were found by aligning the peptide sequence to the corresponding UniProt protein sequence. When there are multiple modified residues in one peptide, all the modified residue numbers will be reported as ProteinID_*#_*#. Statistical values including the exact n, Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO statistical test, and significance are reported in the brief description of the drawings. Statistical significance was defined as p-value < 0.05 and unless indicated otherwise determined by an unpaired Student’s t-test. Statistical analysis was performed using GraphPad Prism (v10) for Mac (GraphPad Software). Statistical values including the exact n, statistical test, and significance are reported in the brief description of the drawings. Statistical significance was defined as p value < 0.05 and unless indicated otherwise determined by an unpaired Student’s t-test. Synthesis of dasatinib and asciminib photoaffinity-labeling probes Encouraged by the high proteomic coverage afforded by the prototype probes (2a-d),the next steps were focused on expanding the SEE-CITE methodology to more structurally complex MOIs. Dasatinib and asciminib were selected as ideal drugs for proof-of-concept SEE-CITE studies. Both are FDA approved drugs that treat CML by inhibiting the BCR- ABL1 oncogenic kinase expressed from the Philadelphia chromosome (Ph). The modes of action of these two highly efficacious drugs are distinct. Dasatinib is an ATP competitive type I kinase inhibitor. In contrast, asciminib is an allosteric inhibitor with a novel Specifically Targeting the Abl Myristoyl Pocket (STAMP) mode of inhibition. The off- targets of dasatinib have been widely characterized, including by chemoproteomics, and it has been shown to have off-target activity across a number of other Src family kinases and several serine / threonine kinases. As asciminib only recently received FDA approval in 2021, its off-targets remain less well characterized. Intriguingly, kinobead analysis with the relatedGNF-5 compound failed to capture any kinase off-targets including BCR-ABL1, whichillustrates the challenges with capturing bonafide asciminib targets using existing technology. To guide the synthesis of dasatinib and asciminib SEE-CITE probes and to further enable our off-target analysis efforts, PAL probes that lacked the silyl ether cleavable linkage were also synthesized. Available co-crystal structures of dasatinib and asciminib bound to activated Abl kinase domain (PDB: 2GQG and 5MO4, respectively) were leveraged to identify solvent accessible derivatization sites for each drug. For dasatinib, both alkyl- and carbamate-linked probes 4a and 4b were synthesized, which were both obtained by functionalizing the solvent accessible piperazine nitrogen of the dasatinib core (FIG.8A-8B, Scheme 2 and Scheme 3). SEE-CITE dasatinib probe 4c was similarly obtained by alkylation of the piperazine core with alkyl bromide silyl ether diazirine handle 7. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO For asciminib, it was chosen to derivatize the solvent accessible hydroxy pyrrolidine moiety. Similar to the synthetic route of the dasatinib probe, it was first sought to obtain ether-linked probes by alkylating the hydroxy pyrrolidine with diazirine handles (FIG.8A- 8B, Scheme 2 and Scheme 4). In contrast to the synthesis of the dasatinib probes, for asciminib, the alkylation strategy failed to afford appreciable amounts of the desired probes. Therefore, the diazirine handle was instead installed via carbamate linkage, which proceeded smoothly to furnish asciminib probe 5a. Synthesis of the corresponding SEE-CITE probe was, however, hampered by difficulties in synthesis of corresponding amine-containing silylether diazirine intermediate (Scheme 4). Therefore, the strategy was revised, and reversecarbamate probe 5b and SEE-CITE intermediate 10 were generated. Asciminib SEE-CITEprobe 5c was then obtained using the reverse carbamate linkage S9 to 10 (Scheme 4).

[0003] Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Scheme 2: Design of dasatinib and asciminib photo-affinity probes and silyl ether linkers Reagents and conditions: a.1, NEt3, CH2Cl2, 0 ℃, 75%; b. chlorodimethylsilane, 1,5- cyclooctadiene, [Ir(cod)Cl]2 (0.01 mol%), 75-80 ℃, 96%; c.1, NEt3, CH2Cl2, 0 ℃, 81%. Scheme 3: Synthetic route to dasatinib probes Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Scheme 4: Synthetic route to asciminib probes

[0004] Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Inhibition of BCR-ABL1 with compounds of this disclosure Table 3: Unique kinases enriched by 4b (100 µM) in KCL-22 cells Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Table 4: Unique kinases enriched by 5b (10 µM) in KCL-22 cells Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Table 5: Shared kinases enriched by 4b (100 µM) and 5b (10 µM) in KCL-22 cells With photoaffinity probes 4a-c and 5a-c in hand, it was next sought to validate that all probes retained BCR-ABL1 inhibitory activity and cell membrane permeability. Immunoblot analysis of compound-treated KCL-22 cells, a Ph+ CML cell line established from a patient with blast phase disease, revealed robust inhibition of BCR-ABL1 activity (FIG.3A, FIG. 3B). Treatment with dasatinib probes 4a-c completely abolished both BCR-ABL1 autophosphorylation, STAT5 phosphorylation at Y694, and phosphorylation of CRKL substrate at Y207 (FIG.3A). As these three phosphosites are established indicators of BCR-ABL1 kinase activity, these findings are consistent with maintained BCR-ABL1 inhibitoryactivity and cell permeability for all three dasatinib analogues. Similar analysis for the asciminib probes 5a-c again revealed total blockade of phosphorylation of both BCR-ABL1 and STAT5 (FIG.3B). Consistent with prior reports for asciminib, these analogues, with the exception of the the slight decrease in signal observed for 5c, afforded no appreciable decrease in CRKL phosphorylation. These findings are consistent with prior reports for asciminib activity. To further assess the behavior of these probes, including their relative photoaffinity labeling capacity, gel-based affinity-based protein profiling (AfBPP) was utilized. Cell-based Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO labeling of KCL-22 cells followed by UV-based photo crosslinking, click conjugation, and in-gel fluorescence analysis revealed striking differences in the relative labeling intensitiesfor the dasatinib probes (FIG. 3C). Probe 4b, which features the carbamate linkage showedmarkedly increased labeling when compared with alkyl analog 4a or 4c. Notably SEE-CITE probe 5c showed similar banding patterns to 5a and 5b, albeit at an increased compound dose required to achieve comparable labeling. Taken together these findings highlight the potential impact that small changes in PAL probe structure can have on relative proteome-wide labeling. Identification of novel asciminib binding targets Guided by photoaffinity-based mode of action studies, the PAL and SEE-CITE probes were then deployed to capture the asciminib binding proteome and compare the identified targets to those engaged by dasatinib. Two complementary datasets were generated to delineate high- and low-occupancy target engagement. First, using LFQ quantification, 703 proteins were identified in KCL-22 cells and 941 proteins were identified in K562 cells that showed UV-dependent labeling by asciminib probe 5b; 373 proteins shared across both cells lines and 330 and 568 proteins unique to KCL-22 cells and K562 cells, respectively (FIG. 4A-B). Both KCL-22 and K562 cells were then subjected to competition-based analysis in which asciminib-induced blockade of 5b labeling was assessed using LFQ. In total, 282 competing proteins were identified in KCL-22 cells and 313 proteins in K562 cells, including 96 shared between both cell lines (FIG.4A-B). Several proteins with important functional activities stood out as both enriched and competed. The mitochondrial ferrochelatase (FECH), which catalyzes the final step in heme biosynthesis, was strongly enriched and competed by asciminib in KCL-22 cells, with a high competition ratio of 2.50 (FIG.4A-B). The ATP-dependent RNA helicase A (DHX9) was also observed to engage asciminib, albeit with a more modest competition ratio of 1.11 and no detection in UV / no-UV experiment in KCL-22 cells (FIG.4A-B). Notably, the therapeutic activity of DHX9 inhibitors has been reported for microsatellite instability-high colorectal cancers. STING (TMEM173), an important regulator of innate immunity which elicits anti-tumor immune responses and a target of a number of chemical probe and drug development campaigns, was also enriched and competed only in K562 cells (FIG.2F). Reticulon-4 (RTN4) was also observed as a target of asciminib, due to the presence of a hyperreactive cysteine residue in this key regulator of endoplasmic reticulum structure (FIG. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 4A-B). Strikingly absent from the proteins significantly enriched by 5b was the bona fide target of asciminib BCR-ABL1. Endogenous BCR-ABL1 has been captured by prior photoaffinity labeling studies, including those using diazirine, tetrazine, and iridium catalyst-based probes. However, the BCR-ABL peptide coverage reported by these studies was low, hinting at more generalized limitations to achieving high coverage of endogenous BCR-ABL1. Therefore, a multi- pronged approach was pursued to improve the detection of BCR-ABL1 by AfBPP- chemoproteomics, with the goal of mapping the binding sites using the dasatinib and asciminib SEE-CITE probes. By lengthening the LC gradient from 70 mins to 140 mins, it was expected that overall coverage would improve, including detection of BCR-ABL1 byasciminib probe 5b. Despite these modifications, BCR-ABL was still not enrichedsignificantly (FIG.4C and Table 2). As asciminib is a less potent inhibitor of BCR-ABL when compared to dasatinib, it was hypothesized that BCR-ABL may be detected using the dasatinib probe 4b. Gratifyingly, using the 140 minute gradient, robust enrichment and competition of ABL1 and, to a lesser extent, BCR proteins (FIG.4C and Table 2) was observed. However, despite the high probe concentration utilized, only 2 peptides from ABL1 were identified. With the hypothesis that coelution of peptides derived from abundant, low affinity targets, might be decreasing coverage of ABL1, protein capture was also tested at a more modest 1 μM 4b compound dose. However, neither BCR nor ABL1 was identified at this lower probe concentration. Armed with the dasatinib and asciminib datasets, the enriched targets of each probe were then compared, with the goal of identifying proteins bound preferentially by asciminib. Consistent with dasatinib’s activity as an active-site directed type I kinase inhibitor, 44 of 67 total kinases enriched by one or both probes were uniquely captured by dasatinib (FIG.4Dand Tables 3-5). Despite the asciminib’s more limited kinase engagement, 40 out of those 67kinases were highly enriched by 5b. As the stochastic nature of data-dependent acquisition (DDA) can result in sample-to-sample variability, a probe-versus-probe dataset was then generated in which the relative enrichment of each probe was directly compared. In total, 67 proteins were preferentially enriched (log2(FC) > 1.0, p < 0.05) by asciminib 5b and 164 bydasatinib 4b (log2(FC) < -1.0, p < 0.05) (FIG. 4E). Several of the aforementioned proteinsthat were both enriched and competed by asciminib, including FECH and RTN4, showed comparable enrichment by dasatinib probe 4b, suggestive of promiscuous binding. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Intriguingly, this data hints at preferential labeling of STING by dasatinib, albeit below the threshold of significance. In contrast, DHX9 was observed to be preferentially enriched by asciminib (log2(FC) = 1.34, p = 0.0021). In contrast with the somewhat modest DHX9 enrichment ratio, COX5A, a subunit of complex IV in the mitochondrial electron transport chain, stood out as a highly asciminib-specific target (FIG.4E, log2(FC)=6.71, p=0.0000343). COX5A was additionally observed to show UV-dependent enrichment by 5b, and this enrichment was blocked by pretreatment with excess asciminib in KCL-22 and K562 cells (FIG.4A-B). These findings are particularly intriguing given the recent reports of asciminib-induced decreased mitochondrial respiration. To vet the SEE-CITE methodology for target hunting, the overlap of proteins captured by SEE-CITE probe 5c to those by the non-SEE-CITE probe 5b in K562 cells (FIG.4F) were assessed. Of the 2095 whole identified proteins by 5c, 1906 had an overlap with2593 of those by 5b, and 459 out of 839 significantly enriched proteins by 5c overlapped with those by 5b (FIG.4G, H). Enrichment with Neutravidin using 5c identified an average of 522 peptides and 312 proteins in the three replicates, a much lower coverage than that of 2a (FIG.2D, G), but this is in accordance with the fact that asciminib is a more elaborate probe oriented toward a specific target (i.e. BCR-ABL1) compared to the non-specific binding nature of the scout probe 2a (FIG.4I). High-accuracy quantification of relative binding site engagement The low PAL coverage of endogenous BCR-ABL1-derived peptides prompted reconsideration of the strategy for benchmarking SEE-CITE’s capacity to accurately identify binding sites. To improve coverage and to facilitate quantification of the relative binding of dasatinib and asciminib to the active and allosteric (STAMP) binding sites, recombinant ABL1 kinase domain was next subjected to SEE-CITE analysis. Gratifyingly, gel-basedanalysis revealed that ABL1 was labeled robustly by both 4b and 5b probes; this labeling wasalso fully competed by pre-treatment with excess parent compound (FIG.5A). These findings align with PAL-labeling at the same binding site occupied by the parent inhibitor. Initial gel-based analysis with SEE-CITE probes 4c and 5c revealed more attenuated labeling of a band at the same molecular weight as ABL1, which was not appreciably competed by excess of parent compound (FIG.5A). While the gel-based performance difference between parent and SEE-CITE PAL probes was initially unexpected, it was hypothesized that the SEE-CITE probes were labeling Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO both ABL1 and a lower affinity probe-interacting protein with the same apparent molecular weight, with the latter protein being responsible for the lack of competition. To test this hypothesis, the PAL probe concentrations were decreased to 1 μM and the AfBPP-gel basedanalysis was repeated (FIG. 5B). The signal for the putative co-migrating protein band wassubstantially reduced at this lower probe concentration, and, gratifyingly, at this lower probe concentration, gel-based competitive labeling consistent with 4c and 5c labeling ABL1 was observed. With protein labeling conditions established, LC-MS / MS quantification of the binding sites labeled by dasatinib 4c and asciminib 5c probes was undertaken. Following the workflow shown in FIG.5C, recombinant ABL1 protein was subjected to UV crosslinking and click conjugation with light- and heavy-biotin azide reagents, for 4c and 5c, respectively. Uniquely enabled by the compound-agnostic universal variable peptide modificationgenerated by the silane-based cleavable linker in the SEE-CITE reagents, the relative labelingof individual ABL1 peptides by each compound was then quantified based on the MS1precursor ion intensity ratios (Heavy / Light). Of the 43 total modified peptides that were assigned to quantification, three labeled amino acids (E258 / G372 / D381) were identified, located proximal to the ATP binding site. Consistent with dasatinib’s active site-directed mode of inhibition, all three residues exhibited extremely negative MS1 ratios, indicative of preferential crosslinking by 4b. Similarly, three residues (P480 / E481 / E526) localized to the myristoyl pocket engaged by asciminib were identified that all exhibited highly positive MS1 ratios, indicative of preferential labeling by 5c at this allosteric site. Thus, the highly precise compound-specific delineation of these known compound binding sites provides a compelling case for the use of SEE-CITE in proteome-wide binding site analysis. Off-target binding site analysis SEE-CITE’s robust delineation of the ABL kinase dasatinib and asciminib binding sites, motivated the extension of the platform to de novo off-target binding site discovery. Given the comparatively recent FDA approval of asciminib and the absence of extensive prior proteomic off-target analysis, attention was focused on mapping asciminib binding sites proteome-wide. Three parallel lines of SEE-CITE off-target inquiry were pursued: (1) analysis of endogenous proteins labeled in cells, (2) heterologous overexpression of targets identified by our AfBPP analysis and mutational analysis to confirm labeling sites binding Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO sites, and (3) in vitro gel-based ABPP analysis recombinant protein to corroborate cell-based labeling. For cell-based labeling of endogenous proteins, K562 cells were subjected to either 4c or 5c followed by conjugation to heavy or light biotin azide and SEE-CITE analysis. Guided by weaker intensity of SEE-CITE probes in gel experiments and the dose-dependent identification of ABL using the dasatinib probe 4b (FIG.4C), a high compound dose of 100 μM was selected to maximize coverage of binding sites. In total 1603 peptides were identified, and 651 proteins across 2 replicate experiments. Consistent with the analysis of diazirine probes 4c and 5c in KCL-22 cells (FIG.3C), it was observed that asciminib SEE- CITE probe 5c showed elevated proteome labeling, both by gel-based ABPP (FIG.6A) and by LC-MS / MS analysis (FIG.6B). As the calculated logP values for 4c and 5c are 5.29 and 6.38, respectively, it is expected that the observed differences in reactivity may be attributed to a combination of several factors, for example intramolecular quenching of diazirine and differences in cellular permeability. Highlighting the added value of the peptide labeling studies, FECH was not significantly enriched by either 4b or 5b in the protein-based probe vs probe streptavidin enrichment experiment (FIG.4E). FECH did however show site-specific differential labeling by 4b and 5c and the SEE-CITE analysis. This difference is rationalized as likely stemming from the presence of multiple probe binding sites, which are uniquely identified by the SEE- CITE approach (FIG.6B, FIG.6C). It was found that 5c dominantly modified peptides proximal to the HEME binding site such as H263, which plays the role of proton acceptor. Nearly identical amino acid labeling patterns were identified for both probes, which is suggestive of generally similarly binding poses. Also aligning with the AfBPP analysis (FIG. 4B, FIG.4C), it was found that RTN4, also widely referred to as Neurite outgrowth inhibitor (NOGO), was preferentially captured by asciminib SEE-CITE probe 5c. The strongest preference for 5c-labeling of RTN4 was at A1075, followed by dominant labeling at several amino acid residues including C1101 (FIG.6D, FIG.6E). Notably, this cysteine has recently been implicated in endoplasmic reticulum (ER) morphology and cancer. Proteomic and gel- based analysis of 5c-treated HEK293T cells heterologously overexpressing epitope tagged RTN4 was then conducted. The SEE-CITE analysis revealed robust labeling of RTN4 at C1101, consistent with the behavior of the endogenous protein (FIG.6F). It was found that a single point mutation (Cys1101Ser) affords diminished labeling by 5c, consistent with this Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO cysteine functioning as a primary probe labeling site. Whether or not a defined small molecule binding site is present in RTN4 is still unclear, as the protein remains structurally unresolved in the PDB and is highly disordered in the AlphaFold model (FIG.8; ID: AF- Q9NQC3-F1). Notably, Cys1101 exhibits hyperreactivity towards the cysteine-reactive probe iodoacetamide alkyne (IAA), which is suggestive that cysteine reactive may be a central driver of RTN4 labeling by the asciminib probes. Binding sites were identified for only a fraction of the proteins enriched in the aforementioned AfBPP analysis. Notable proteins that were not identified by the SEE-CITE probes included high value targets STING and DHX9. Therefore, the next focus was on extending the SEE-CITE platform to these targets. Given the differences in reactivities of the 4c and 5c probes, the labeling of scout probe 2a to 5c was instead compared. To favor DHX9 binding site discovery, MOLT4 cells were selected, which are distinguished by high DHX9 expression as reported by Protein Atlas. SEE-CITE analysis revealed that DHX9 was labeled robustly by both scout probe 2a and asciminib probe 5c at three sites. The measured negative log2(H / L) values for all identified peptides are consistent with preferential binding of the scout compound when compared to asciminib probe 5c. In contrast, and aligning with the above comparison of 4c and 5c, FECH showed positive log2(H / L) values, consistent with asciminib preferential labeling (FIG.6B). Taken together these analyses illustrate the value in elevated protein expression for ensuring binding site identification via SEE-CITE. As STING was not identified in the SEE-CITE K562 or MOLT4 cells datasets, a heterologous overexpression system was employed, with the goal of boosting STING expression and coverage. SEE-CITE analysis of HEK293T cells heterologously overexpressing STING-HA revealed that 5c exhibited slightly increased labeling when compared to the scout probe at two key cysteine residues, C91 (Log2(H / L) = 0.34, 0.43) and C148 (Log2(H / L) = 0.48), respectively (FIG.6G, FIG.6H). C91 residue is known as the palmitoylation site, and C148 also plays a key role in inducing the STING polymerization for its full activation through the exposure of two cysteine residues (C148) in the linker domain to form a disulfide bond bridging the two dimers. While immunoblot-based analysis confirmed robust STING expression, no significant band was detected for STING in an in-gel fluorescence analysis. Taken together, these findings align with recent reports of promiscuous STING small molecule binding and labeling by diazirine-containing compounds. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO One target that stood out in both the protein-directed AfBPP (FIG.4) and SEE-CITE datasets (FIG.6) in K562 and MOLT-4 cells was COX5A. SEE-CITE probe 4c wasobserved to modify COX5A at T79 / Y80, which are located proximal to an establishedphosphatidylsulfocholine (PSC) binding site detected in previously reported bovine cytochrome C co-crystal structure (PDB: 5Z62 for human and 1V54 for bovine, respectively) (FIG.6I-J). In-gel fluorescence analysis with 5b in HEK293T cells transiently overexpressing human COX5A-FLAG resulted in robust COX5A labeling in a probe- concentration-dependent manner (FIG.6K-L). Elaborately, 5b was found to label COX5A efficiently at 1 μM, the lowest concentration tested, and achieved saturated labeling at 10 μM. Pre-treating transfected cells with asciminib at equimolar or 1.5-fold increase inconcentration blocked the subsequent binding of 5b probe to COX5A, thus leading to anearly complete abolishment of COX5A engagement by 5b as anticipated (FIG.6L). Supporting the LC-MS / MS results, these findings validated the direct COX5A engagement of asciminib and may be linked to a previously reported impairment in mitochondrial respiratory by asciminib. Exemplary molecular biology procedures Cloning of plasmids pDONR221 containing COX5A sequence (DNASU plasmid #HsCD00042518) or pRK5 containing GFP sequence was subcloned using GateWay cloning into C-terminal FLAG destinator vector. For GateWay cloning, the donor vector (300 ng) was combined with the destination vector (150 ng) in a microcentrifuge tube followed by the addition of TE buffer (8 μL). GateWayTMLR ClonaseTMII Enzyme Mix (Invitrogen, 11791020) (2 mL) was added to the mixture to give a total mixture volume of 10 uL. The mixture was vortexed briefly and incubated overnight at room temperature. Proteinase K solution (1 μL) was added to terminate the reaction followed by a brief vortex and 10-minute incubation at 37oC. Transformation was immediately carried out by transferring the quenched reaction mixture (5 μL) into competent TOP10 cells. After 30-minute incubation on ice, the cells were heat shocked at 42oC for 35 seconds and left on ice for another 5-mins post heat-shock. SOC buffer (250 μL) was added before growing more cells in LB media at 37oC with 100 mg / mL ampicillin for 1 hour. Cells containing plasmid of interest were plated onto agar plates with 100 mg / mL ampicillin for selection. Isolated colonies were selected for purifying plasmid DNAs, and successful cloning was confirmed by sequencing. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO In-gel fluorescence analysis of COX5A using 5b HEK293T cells were plated into a 6-well plate 24-hour prior to transfection and were transiently transfected at 70-80% confluency. Briefly, COX5A-FLAG or GFP-FLAG plasmid (1.5 μg) and PEI MAX-Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000) (Polysciences, Inc., 24765-1, 7.5 μL) were each diluted with reduced-serum medium OptiMEMTM(75 μL). After 5-minute equilibration at room temperature, the two diluted mixtures were mixed gently and incubated for another 20 minutes at room temperature. The transfection cocktail was then added dropwise to the cells and incubated for 24 hours. If any, transfection reagents were scaled according to the volume of mediate in plates of various sizes. 24-hour post-transfection, transiently transfected cells were treated with DMSO or 5b at 1, 10 and 25 μM for 30 minutes at 37oC. For competitively treated samples, cells were pre- treated with DMSO or asciminib at 10 and 25 μM followed by the treatment with 5b probe at 10 μM for another 30 minutes at 37oC. Treated cells were then UV irradiated at 350 nm for 20-minutes and harvested in cold 1x PBS (pH 7.4) by centrifugation at 1,000 g for 5 minutes. Cell lysis was done with 8M urea prepared in 1x PBS followed by 3 freeze-thaw cycles using liquid nitrogen. Cell pellets were spun down at 3,000 g for 15 minutes. Protein concentrations were determined using a BioRad DC protein assay kit from BioRad Life Science (Hercules, CA). All samples were normalized to the same 1 mg / mL concentration prior to later steps. Normalized samples were subjected to biorthogonal copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction with TAMRA-azide (Click Chemistry Tools, AZ109- 5) for 1 hour at room temperature in the dark. Click reaction was performed by adding 2.4 μL of a click master mix prepared from 1.74 mM TBTA, 1.25 mM TAMRA-azide, 50 mM CuSO4and 50 mM TCEP into each 1 mg / mL normalized sample (20 μL). The click reaction was terminated by adding 4x Laemmli sample loading buffer (BioRad, 6.7 μL) with 10% b- mercaptoethanol and subjected to 5-minute heat denaturation at 95oC. Denatured proteins were resolved by SDS-PAGE using 4-12% CriterionTMXT Bis-Tris Protein Gel (BioRad) and MOPS SDS running buffer (Invitrogen) at 200V. Fluorescence was visualized with a BioRad ChemiDoc Imaging System. The gel was transferred to the nitrocellulose membrane (BioRad) using a semi-dry tranfer system (BioRad Transblot) with a low molecular weight setting. The membrane was blocked with 5% (w / v) milk in 1x Tris-buffered saline (TBS) for 1 hour at room temperature. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO The membrane was incubated with the primary antibody, anti-FLAG antibody (Cell Signaling Technology, 14793), at a ratio of 1:3000 in 5% (w / v) milk in 1x TBS overnight at 4oC, washed with 1x TBS for 10 minutes three times. The membrane was then incubated with the secondary antibody, IRDye® 800CW Goat anti-Rabbit Secondary Antibody (Li-Cor Biotechnology, 102673-330), at a ratio of 1:5000 in 5% (w / v) milk in 1x TBST (TBS with 0.1% Tween20) room temperature for 1 hour, washed with 1x TBS for 10 minutes three times. The membrane was imaged using a BioRad ChemiDoc Imaging System. General procedure of Western blot In a 6-well plate, KCL-22 grown in 5 mL of RPMI media supplemented with FBS and penicillin-streptomycin were incubated with DMSO solution of each probe (DMSO never exceeded 1%) at the indicated concentration for 2 hours (dasatinib probes) or 4 hours (asciminib probes). After that, the media was transferred to a 15 mL Eppendorf tube and rinsed with 5 mL cold PBS. After centrifuge at 1,000 x g, 4 °C for 3 minutes, the supernatant was aspirated and the cell pellet was gently resuspended in 1 mL cold-PBS and transferred to a 1.5 mL Eppendorf tube. The falcon tube was rinsed with 0.5 mL of cold PBS and it was combined to the same Eppendorf tube. After centrifuging at 1,000 x g, 4 °C for 3 minutes, the supernatant was aspirated and the cell pellet was lysed with 0.3% CHAPS supplemented with protease and phosphatase inhibitors in PBS and pipetted up and down at least 20 times, then incubated at 4 °C for more than 30 minutes. The mixture was spun down at 15,000 x g at 4 °C for 15 minutes, then the supernatant was transferred to a new 1.5 mL of Eppendorf tube and the protein concentration of the lysate was determined by BioRad DC protein assay. The lysate containing 80 ug of proteins was mixed with 4x loading dye so that the loading dye was adjusted to 1x concentration. After boiling each sample at 100 °C for 5 minutes, proteins were separated by SDS−PAGE (Bio-Rad TGX Stain-Free gel, SDS running buffer) at 140V. After transferring proteins to a membrane, each protein was detected using the following antibodies: c-Abl (Cell Signaling #2862), phospho-c-Abl (Y245) (Cell Signaling #2861), STAT5 (D2O6Y) (Cell Signaling #94205), phospho-STAT5 (Y694) (ABclonal #AP0758), CRKL (ABclonal #A11735), phospho-CRKL (Y207) (ABclonal #AP0824) and β-Actin (8H10D10) (Cell Signaling #3700). Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO Pairing sCIP-DiLeu with SEE-CITE For the sCIP platform, the key innovation was the synthesis of a 29-plex set of isobaric reagents that feature biotin and an alkyne handle for enrichment, a dialkoxydiphenylsilane (DADPS) cleavable linker for release of labeled peptides, and isobaric dimethylleucine and alanine reporter and balancer groups (FIG.10A-B). Using these reagents, high accuracy and quantitative proteomics were demonstrated using a cysteine chemoproteomic readout (FIG.11A-11G). These results confirmed the robust performance of the 29-plex sCIP reagents. With sCIP reagents in hand, the next step was to establish the sCIP-SEE-CITE platform. Using the prototype scout reagent 2a, the sCIP-SEE-CITE chemoproteomics screening platform was next optimized with 2b and a small panel of 12 sCIP-DiLeu reagents. sCIP-SEE-CITE chemoproteomic mapping of fragment-protein interactions was carried out in a competitive format (FIG.9). Elaborately, cells were sequentially treated with fragments featuring unique chemotypes and the SEE-CITE probe 2b. Competitively treated samples then underwent UV irradiation followed by conjugation with sCIP-DiLeu reagents via a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Post-clicked samples from different conditions were combined and proceeded with sample cleanup, tryptic digestion, enrichment and acid-based peptide elution prior to LC- MS / MS analysis (FIG.12). Gratifyingly, this analysis yielded 826 total peptides from 434 unique proteins, identified to be off-competed by one or more compounds (FIG.13A). While generating these datasets, a key observation was that the amount of cellular material could be reduced substantially per treatment condition, as a result of the multiplexing. Exemplifying this improvement, in the first generation SEE-CITE platform required ~60-million cells or 3,200 µg of cellular material for optimal coverage of labeled peptides. This large sample amount is incompatible with some rarer cell types, such as primary patient derived samples. Thus, with the sCIP-SEE-CITE platform, because of the sample multiplexing only ~2 million cells are required for each treatment condition, which represents a substantial improvement over the prior method (FIG.13B). The robust coverage achieved by the first screening experiment was modestly improved compared to 700 modified peptides or 426 corresponding modified proteins identified from the SEE-CITE platform with the same experimental condition (FIG.13A). This comparability of the two platforms suggested that the conjugation of the sCIP reagent Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO onto SEE-CITE labeled proteins resulted in little to no difference in desired improved coverage. Next, using a 12-plex set sCIP-DiLeu reagents, MOLT4, a T-lymphoblast cell line, was subjected to competition-based screen using a focused fragment library featuring compounds with a privileged benzimidazole scaffold (compounds 1-3), known for a wide range of biological activities, a pair of stereo-methyl-piperazine chemotype (compounds 4-5) and fused-ring molecules (compounds 6-7) (FIG.14A). Duplicates of six competitively treated samples using compounds 1-5 and controls were clicked to 12 isobaric sCIP-DiLeu reagents via CuAAC and further subjected to later sCIP-SEE-CITE sample-preparation steps detailed above. As anticipated, compared to conventional chemoproteomics or advanced SEE-CITE proteomics platform, which could quantify labeled peptides pooled from only two different conditions, sCIP-SEE-CITE with isobaric tags demonstrates advances in quantitation of multiple screening conditions in a single mass spectrometry experiment via MS / MS fragmentation, confirming its feasibility and compatibility with screening reversible fragment molecules. Thus, multiplexed quantitative sCIP-SEE-CITE proteomics advances the convenient assessment of protein labeling profiles of various chemotypes in one single experiment, as well as an early-stage combination of samples after a click reaction. These advantages of sCIP-SEE-CITE not only substantially decrease sample preparation as stated above but also data acquisition time, allowing high throughput screening of large compound libraries and accelerating ligand- target identification process, which has been traditionally known as time-consuming. Taking advantage of multiplexing advances of sCIP-SEE-CITE, we demonstrated that sCIP-SEE-CITE could be applied to further evaluate fragment hits via concentration- dependent chemoproteomics (FIG.14C). Samples treated with the same fragment hits in a concentration-dependent manner could be combined and analyzed by LC-MS / MS as one mass-spec sample to assess their labeling potency for high-value ligandable targets. The structure activity relationship of a focused set of compounds was next assessed. Pleasingly, a number of targets were identified for which no inhibitors are currently available. Exemplifying these types of proteins, a striking SAR was observed for Epithelial cell transforming sequence 2 oncogene-like (ECT2L), which is related to the transforming factor ECT2 and is highly expressed in a subset of neuroendocrine cancers. Only the closely related fragments 4 and 5 competed labeling of ECT2L (FIG.14B). Further supporting the capacity of the platform to accurately delineate likely targets, good concordance is observed more Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO broadly for the targets identified for compounds 4 and 5, including ADA, which is an essential regulator of immune cell activation, HPCAL1, a regulator of neuronal signaling, and CCT6A, a component of the T complex that functions as a major cellular chaperone. Inspection of the binding sites (FIG.15) for these four proteins revealed a large putative pocket in ECT2L. Similarly, for ADA, labeling was observed at a small allosteric pocket that is co-crystallize with a nitrate anion. Clearly illustrating the added value of this approach, for EPHX2, the labeled site was observed to be immediately proximal to a co- crystallized inhibitor. Another class of proteins that showed strong SAR are the human leukocyte antigen (HLA), which are key regulators of the adaptive immune system. We identified molecules that engage both HLA-A and HLA-C (FIG.14B) proximal to the peptide binding site (FIG. 15) and close to known binder abacivir. As abacivir binding leads to enhanced antigenicity, it is expected that these compounds may function similarly for other HLA alleles, beyond HLA-B*57. In summary, sCIP-SEE-CITE pairs the custom sCIP isobaric reagents with SEE-CITE site of labeling analysis to enable straightforward and high coverage screening of multiple reversible compounds proteome-wide. The key features that distinguish sCIP-SEE-CITE from prior chemoproteomic PAL platforms are the direct head-to-head quantitative measures of sites of labeling and the early-stage combination, which together make the platform uniquely suited to small molecule screening. These features decrease sample preparation and data acquisition time, to facilitate the high throughput screening of large compound libraries and to accelerate ligand-target identification and SAR analysis. INCORPORATION BY REFERENCE All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. EQUIVALENTS While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO We claim:

1. A compound having a structure represented by formula I, formula II, or a salt thereof:I II wherein: A is a binding partner for a target protein (e.g., asciminib or dasatinib) or is selected from aryl (e.g., phenyl), aralkyl, heteroaryl, and heterocyclyl; E is an enrichment handle (e.g., Lys(biotin)-OH); L1is selected from alkylenyl, -S-, -NH-, -CO-, -NH(CO)NH-, -O(CO)O-, -NH(CO)O-, -O(CO)NH-, -NRQ(CO)O-, -O(CO)NRQ-, -SO-, and -SO2-, or is absent; Y1, Y2, and Y3are each independently S or O; X1and X2are each independently CH or N; X3and X6are each independently O, S, or NR6; X4and X5are each independently alkylenyl; Xaa, Xab, and Xacare each natural or unnatural amino acids, each of which may be isotopically enriched with one or more instances of an isotope, e.g., 2H, 15N, 18O, or 13C;R1and R2are each independently alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl; R3and R4are each independently alkyl, aralkyl, or aryl; R5and R6are each independently H or alkyl; RQ, taken together with the intervening atoms, forms a cycloalkyl or a heterocyclyl; n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; n2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; n3 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; and n4 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 2. The compound of claim 1, wherein the compound has a structure represented by formula I, formula II, or a salt thereof:wherein: A is a binding partner for a target protein (e.g., asciminib or dasatinib) or is selected from aryl (e.g., phenyl), heteroaryl, and heterocyclyl; E is an enrichment handle (e.g., Lys(biotin)-OH); L1is selected from alkylenyl, -S-, -NH-, -CO-, -NH(CO)NH-, -O(CO)O-, -NH(CO)O-, -O(CO)NH-, -SO-, and -SO2-, or is absent; Y1, Y2, and Y3are each independently S or O; X1and X2are each independently CH or N; X3and X6are each independently O, S, or NR6; X4and X5are each independently alkylenyl; Xaa, Xab, and Xacare each natural or unnatural amino acids, each of which may be isotopically enriched with one or more instances of an isotope, e.g., 2H, 15N, 18O, or 13C;R1and R2are each independently alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl; R3and R4are each independently alkyl, aralkyl, or aryl; R5and R6are each independently H or alkyl; and n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; n2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; n3 is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; and n4 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

3. The compound of claim 1 or 2, wherein the compound has a structure represented by formula Ia, or a salt thereof:Ia.Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 4. The compound of any one of claims 1-3, wherein A is a small molecule.

5. The compound of any one of claims 1-3, wherein A is aryl (e.g., phenyl).

6. The compound of any one of claims 1-3, wherein A is heteroaryl.

7. The compound of any one of claims 1-3, wherein A is heterocyclyl.

8. The compound of any one of claims 1-3, wherein A is asciminib.

9. The compound of any one of claims 1-3, wherein A is dasatinib.

10. The compound of any one of claims 1-3, wherein A is aralkyl.

11. The compound of claim 10, wherein A is benzyl.

12. The compound of any one of claims 1-11, wherein L1is alkylenyl, -S-, -NH(CO)NH-, -NH(CO)O-, -O(CO)NH-, -NRQ(CO)O-, or -O(CO)NRQ-; and wherein RQ, taken together with the intervening atoms, forms a heterocyclyl.

13. The compound of claim 12, wherein L1is alkylenyl, -S-, -NH(CO)NH-, -NH(CO)O-, or -O(CO)NH-.

14. The compound of claim 12, wherein RQ, taken together with the intervening atoms, forms a piperazinyl.

15. The compound of claim 12, wherein RQ, taken together with the intervening atoms, forms an 8-azaspiro[4.5]decane.

16. The compound of claim 12 or 14, wherein; wherein represents the point of connection to the carbon atom connected to Si.Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 17. The compound of claim 12 or 15, wherein; wherein represents the point of connection to the carbon atom connected to Si.

18. The compound of claim 12 or 13, wherein L1is methylenyl, ethylenyl, propylenyl, butylenyl, pentylenyl, hexylenyl, heptylenyl, octylenyl, or nonylenyl.

19. The compound of any one of claims 1-11, wherein L1is absent.

20. The compound of any one of claims 1-19, wherein X1is N.

21. The compound of any one of claims 1-20, wherein X2is N.

22. The compound of any one of claims 1-21, wherein R1is selected from methyl, ethyl, isopropyl, and phenyl.

23. The compound of any one of claims 1-22, wherein R2is selected from methyl, ethyl, isopropyl, and phenyl.

24. The compound of any one of claims 1-23, wherein n1 is 2.

25. The compound of any one of claims 1-23, wherein n1 is 3.

26. The compound of any one of claims 1-25, wherein n2 is 2.

27. The compound of any one of claims 1-26, wherein n3 is 2.Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 28. The compound of claim 1, wherein the compound is selected from:

29. The compound of claim 2, wherein the compound is selected from:Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO30. The compound of claim 1 or 2, wherein the compound has a structure represented by formula IIa, or a salt thereof:

31. The compound of claim 30, wherein E is Lys(biotin)-OH.

32. The compound of claim 30 or 31, wherein X3is S.

33. The compound of any one of claims 30-32, wherein X4is alkyloxyalkyl.

34. The compound of any one of claims 30-33, wherein X4is substituted with alkyl, alkenyl, alkynyl, ester, amido, aryl, or heteroaryl.

35. The compound of any one of claims 30-33, wherein X4is substituted with alkyl (e.g., methyl).Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 36. The compound of any one of claims 30-35, wherein X5is methylenyl, ethylenyl, propylenyl, butylenyl, pentylenyl, hexylenyl, heptylenyl, or octylenyl.

37. The compound of claim 36, wherein X5is hexylenyl.

38. The compound of claim 36, wherein X5is propylenyl.

39. The compound of any one of claims 30-38, wherein X5is substituted with alkylenyl, alkenylenyl, alkynylenyl, ester, amido, aryl, or heteroaryl.

40. The compound of any one of claims 30-39, wherein R5is H.

41. The compound of any one of claims 30-40, wherein n4 is 2.

42. The compound of any one of claims 30-41, wherein X6is O.

43. The compound of any one of claims 30-41, wherein X6is NR6.

44. The compound of claim 43, wherein R6is H.

45. The compound of any one of claims 30-44, wherein Xaais 6-azido-lysine.

46. The compound of any one of claims 30-45, wherein Xabis alanine.

47. The compound of any one of claims 30-46, wherein Xacis N,N-dimethyl-leucine.Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 48. The compound of any one of claims 30-47, wherein the compound has a structure represented by formula IIb or a salt thereof:IIb wherein: n5, n6, n7, and n8 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R7and R8are each independently alkyl, alkenyl, alkynyl, ester, amido, aryl, or heteroaryl.

49. The compound of claim 48, wherein n5 is 2.

50. The compound of claim 48 or 49, wherein n6 is 3.

51. The compound of any one of claims 48-50, wherein n7 is 1.

52. The compound of any one of claims 48-51, wherein n8 is 2.

53. The compound of any one of claims 48-51, wherein n8 is 6.

54. The compound of any one of claims 48-53, wherein R7is alkyl (e.g., methyl).

55. The compound of any one of claims 48-54, wherein R8is alkyl (e.g., methyl).

56. The compound of any one of claims 48-55, wherein R3is aryl (e.g., phenyl).

57. The compound of any one of claims 48-56, wherein R4is aryl (e.g., phenyl).Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 58. The compound of claim 1 or 2, wherein the compound is:a salt thereof.

59. A method of synthesizing a chemoproteomic capture reagent comprising: contacting a solid support with an enrichment handle selected from an amino acid substituted with biotin, an antibody, a sugar, a ubiquitin tag, and a metal chelator, thereby creating a solid support-enrichment handle conjugate; contacting the solid support–enrichment handle conjugate with a solid–phase compatible cleavable linker, thereby creating a solid support–enrichment handle-solid-phase compatible cleavable linker conjugate; contacting the solid support–enrichment handle–solid–phase compatible cleavable linker conjugate with a click capture amino acid, thereby creating a solid support-enrichment handle–solid-phase compatible cleavable linker–click capture amino acid conjugate; contacting the solid support–enrichment handle–solid–phase compatible cleavable linker– click capture amino acid conjugate with a first isotopically labelled amino acid, thereby creating a solid support–enrichment handle-solid–phase compatible cleavable linker–click capture amino acid–first isotopically labelled amino acid conjugate; and contacting the solid support–enrichment handle–solid–phase compatible cleavable linker– click capture amino acid–first isotopically labelled amino acid conjugate with a second isotopically labelled amino acid, thereby creating the chemoproteomic capture reagent.

60. The method of claim 59, wherein the solid support is a resin.

61. The method of claim 60, wherein the resin is a chlorotrityl resin.Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 62. The method of any of claims 59-61, wherein the enrichment handle is an amino acid substituted with biotin (e.g., Lys(biotin)-OH).

63. The method of claim 62, wherein the amino acid is a naturally occurring amino acid (e.g., lysine or cysteine).

64. The method of any one of claims 59-63, wherein the click capture amino acid is an azide containing amino acid (e.g., 6-azido-lysine).

65. The method of any one of claims 59-64, wherein the first isotopically labelled amino acid is a naturally occurring amino acid (e.g., valine or alanine).

66. The method of any one of claims 59-65, wherein the second isotopically labelled amino acid is an unnatural amino acid (e.g., N,N-dimethyl-leucine).

67. The method of claim 65 or 66, wherein the first and second isotopically labelled amino acids are enriched with13C,15N, or2H.

68. The method of any one of claims 59-67, wherein the support-enrichment handle-solid- phase compatible cleavable linker-isotopically labelled amino acid conjugate is cleaved from the solid support using acid (e.g., hydrochloric acid).

69. A method of identifying a binding site comprising: contacting a substrate with the compound of any one of claims 1-29, or a salt thereof; thereby creating a substrate-diazirine-alkyne conjugate; contacting the substrate-diazirine-alkyne conjugate with a chemoproteomic capture reagent comprising an azide; thereby creating a substrate-chemoproteomic capture reagent conjugate; and determining the molecular weight of the substrate-chemoproteomic capture reagent conjugate; thereby identifying the binding site.

70. The method of claim 69, wherein the substrate is a target protein (e.g., an enzyme).Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 71. The method of claim 70, wherein the target protein is purified from cell lysate.

72. The method of any one of claims 69-71, wherein the method further comprises incubating the substrate with the compound of any one of claims 1-29.

73. The method of any one of claims 69-72, wherein the chemoproteomic capture reagent is the compound of any one of claims 1 or 2 and 30-58, or a salt thereof.

74. The method of any one of claims 69-73, wherein the method further comprises irradiating the substrate-diazirine-alkyne conjugate; thereby covalently binding the diazirine- alkyne conjugate to the substrate.

75. The method of claim 74, wherein the method further comprises cleaving a bond between Si and Y1.

76. The method of claim 75, wherein cleaving the bond between Si and Y1comprises contacting the conjugate with acid.

77. The method of any one of claims 69-76, wherein contacting the substrate-diazirine- alkyne conjugate with the chemoproteomic capture reagent of any one of claims 1 and 19-47 forms a triazole linking the chemoproteomic capture reagent to the substrate.

78. The method of any one of claims 69-77, wherein the molecular weight of the substrate-chemoproteomic capture reagent conjugate is determined by tandem high performance liquid chromatography / mass spectrometry.

79. The method of any one of claims 69-78, wherein the method further comprises digesting the substrate-chemoproteomic capture reagent conjugate; thereby creating a population of chemoproteomic capture reagent-labeled peptides and a population of unlabeled peptides.Attorney Docket No.: UCH-40225 UCLA Ref. No.: [UCLA 2024-194-2] WO 80. The method of claim 79, wherein digesting the substrate-chemoproteomic capture reagent conjugate comprises contacting the chemoproteomic capture reagent-substrate conjugate with a digestion enzyme (e.g., trypsin).

81. The method of claim 79 or 80, wherein the method further comprises contacting the population of chemoproteomic capture reagent-labeled peptides and the population of unlabeled peptides with an enrichment agent selected from a metal ion, a lectin, an epitope tag, avidin, and streptavidin; thereby creating an enriched population of chemoproteomic capture reagent-labeled peptides.

82. The method of claim 81, wherein the enrichment agent is streptavidin.

83. The method of claim 81 or 82, wherein the method further comprises isolating the enriched population of chemoproteomic capture reagent-labeled peptides from the unlabeled peptides.

84. The method of claim 82 or 83, wherein the method further comprises a step of eluting the enriched population of chemoproteomic capture reagent-labeled peptides from the enrichment agent, comprising contacting the enriched population of chemoproteomic capture reagent-labeled peptides with acid (e.g., formic acid).

85. The method of any one of claims 81-84, wherein the method further comprises determining the molecular weight of the enriched population of chemoproteomic capture reagent-labeled peptides, thereby identifying the binding site.

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