Global affinity profiling of molecular interactions using competitive enrichment

Abstract

The present disclosure relates to a method of generating an affinity map, comprising: (a) contacting a sample, which comprises a set of proteins, with (i) a probe agent, which optionally comprises a protein-binding ligand Z, and (ii) a crosslinking agent, which upon activation is capable of crosslinking with a subset of protein(s) to which the protein-binding ligand is bound; and (b) activating the crosslinking agent, thereby forming a crosslinked sample comprising a subset of crosslinked proteins.

Description

[0001] Attorney Docket CUW-03825

[0002] GLOBAL AFFINITY PROFILING OF MOLECULAR INTERACTIONS USING COMPETITIVE ENRICHMENT

[0003] Related Applications

[0004] This application claims the benefit of U. S. Provisional Application No.

[0005] 63 / 720,063, filed November 13, 2024, the contents of which are fully incorporated by reference herein.

[0006] Government Support

[0007] This invention was made with government support under Grant Nos. GM147449 and GM 136640 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

[0008] Background

[0009] Methods for measuring the strength and specificity of protein-ligand interactions are of central importance in biology. Ligands can interact with many different proteins, with aberrant interactions causing disease and undesired therapeutic side effects. Extensive profiles of binding affinity across the proteome are therefore required to understand ligand functions and binding selectivity, especially in drug development. Affinity measurements are typically performed separately for individual protein-ligand pairs, either using purified proteins, cell-based reporter protein systems, or competitive binding with a selective radioactive ligand.1These methods can efficiently measure the binding affinities of many different molecules for a single protein target, making them indispensable in drug development. However, target-based assays are only available for a fraction of the proteome, preventing an accurate assessment of binding selectivity in complex biological systems (Fig. 1, top).

[0010] Current approaches for proteome-wide affinity measurement rely on competitive binding to bead surfaces or solid supports or shifts in protein stability as mass spectrometry (MS)-quantifiable readouts of protein binding site occupancy. Bead-based methods (e.g. Kinobeads)6 11can offer affinity profiles for select categories of proteins such as kinases, but are not easily used with membrane proteins or live cells. While more general in scope, methods based on ligand-altered protein stability (cellular thermal shift assay, CETSA;

[0011] FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0012] limited proteolysis, LiP; peptide-centric local stability assay, PELSA) capture the subset of interactions which materially alter protein melting point (Am) or susceptibility to proteolytic degradation.12 20Specialized methods are also available for measuring DNA / RNA-protein binding affinities21,22and profiling the kinetic reactivity of covalent molecules with nucleophilic amino acids,23but these are not applicable to druglike noncovalent ligands. There remains an unmet need for a general affinity profiling platform equally applicable to diverse categories of noncovalent ligands, protein targets, and biological samples.

[0013] Summary

[0014] Photoaffinity labeling offers a potentially advantageous proteomic readout for affinity profiling. Like a radioligand binding assay24, photocrosslinking works with membrane and non-membrane proteins, is compatible with live cells, and is not impacted by protein physical properties such as molecular weight or melting point.25,26Recently, photocatalytic labeling platforms (e.g. pMap, Photag, Lux -MS, MultiMap)27 31have been introduced that amplify crosslinking efficiency, leading to their widespread adoption. Despite their versatility, photolabeling methods have only been used to qualitatively nominate potential binding targets rather than quantitatively measure binding affinity.32,33The present invention uses labeling by photocatalytic probes to accurately measure the protein binding affinities (Ka) of unmodified, non-photocatalytic ligands, enabling robust affinity profiling. If protein labeling is proportional to binding site occupancy by a photocatalyst-conjugated probe, treatment with a competing ligand would afford a dosedependent reduction in labeling. The relationship between labeling signal and probe / ligand concentrations can therefore be modeled using standard competitive binding kinetics. Such a method would be akin to a global radioligand competition assay with a protein-level readout, enabling general Ka determination via MS measurement of the photocatalytically labeled proteome.

[0015] In certain aspects, the present disclosure provides a new platform, Affinity Map, which combines efficient labeling chemistry, high-throughput MS proteomics, and a tailored data processing pipeline to measure binding affinities between ligands and the proteome. To demonstrate the generality of this method, its compatibility with major

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[0017] types of therapeutic protein-binding ligands (small molecules, linear peptides, cyclic peptides, and antibodies), applicability to both membrane and non-membrane protein targets, and ability to measure binding affinities in cell lysate, organ extracts, and live cells (Fig. 1, bottom) is shown.

[0018] In certain aspects, the present disclosure provides a complex comprising Iridium (Ir) and a ligand of formula (I):

[0019] SO?

[0020] N A.

[0021] ( / y

[0022]

[0023] crA

[0024] (i),

[0025] wherein:

[0026] L is a linker; and

[0027] Z is a protein-binding ligand.

[0028] In certain aspects, the present disclosure provides a complex comprising Iridium (Ir) and a ligand of structure (IA):

[0029] ^. Sof

[0030] NA

[0031] ( / y

[0032]

[0033] OH

[0034] (IA).

[0035] In certain aspects, the present disclosure provides a method of generating an affinity map, comprising:

[0036] (a) contacting a sample, which comprises a set of proteins, with (i) a probe agent, which optionally comprises a protein-binding ligand Z, and (ii) a crosslinking agent, which upon activation is capable of crosslinking with a subset of protein(s) to which the protein-binding ligand is bound; and

[0037] (b) activating the crosslinking agent, thereby forming a crosslinked sample comprising a subset of crosslinked proteins.

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[0039] Brief Description of the Drawings

[0040] FIG. 1 shows a high fidelity platform for simultaneous protein-ligand interaction identification and affinity measurement. Top: Current methods for measuring proteinligand binding affinity use recombinant proteins, engineered cells, or provide incomplete coverage of interactions in the proteome. Bottom: Photocatalytic affinity labeling combined with a quantitative competitive ligand binding model enables robust, modalityagnostic Kd profiling in lysate, organ extracts, and live cells.

[0041] FIG. 2A-C show an experimental design and small molecule affinity profiling in lysate. A) EC50 values measured at each probe concentration are used to obtain the free ligand dappvalue. B) Neutral, hydrophilic photocatalyst 1 with low plasma protein binding (PPB). C) Affinity Map workflow in lysate.

[0042] FIG. 2D-H show an experimental design and small molecule affinity profiling in lysate. D) Comparison of dasatinib affinity profiles using various probes (0.01% FDR).

[0043] E) Volcano plot showing enriched proteins in a two-group comparison (log2(FC) = log2(l pM l-dasatinib)-log2(l pM 1-dasatinib + 10 pM dasatinib) color-coded by measured binding affinity. F) Correlation of dasatinib-target log2(FC) vs. Affinity Map-measured binding affinity. G) Correlation of kinase binding affinities measured by Affinity Map using both Cheng-Prusoff modeled (Kdllgand / Kdprobevalue significant, p < 0.1, or confidence interval < ±0.25) and not non-modeled values vs. Kinobeads. H) Dasatinib kinase targets identified by Affinity Map, Kinobeads, and CETSA.

[0044] FIG. 3A-D show affinity profiling of (+)-JQ-l, a selective bromodomaincontaining protein inhibitor. A) Validation of newly identified dasatinib binding targets RIPK1, PKN3, and CK1D and binding affinity via in vitro activity assays (PhosphoSens) at [ATP] = Km. B) Chemical structures of both l-(+)-JQl and unmodified (+)-JQl. C) Western blot showing a [(+)-JQl] -dependent reduction in BRD4 biotinylation by l-(+)-JQ1. D) Normalized streptavidin intensity (relative to total protein) vs. [(+)-JQl ].

[0045] FIG. 3E-H show affinity profiling of (+)-JQ-l, a selective bromodomaincontaining protein inhibitor. E) (+)-JQl affinity profile in K562 lysate reflects selective binding to BRD proteins. F) Comparison of protein binding affinities measured using Affinity Map vs. isothermal calorimetry. G) (+)-JQl Affinity profile in pooled human liver S9 fraction. H) Chemical structures of both l-(+)-Flumazenil and unmodified

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[0047] Flumazenil, the workflow for mouse brain membrane isolation, flumazenil affinity profile in mouse brain membrane isolates, and sigmoidal curve fits for the oc / yGABA receptor subunits showing labeling of the flumazenil binding interface (PDB: 6X3U). Affinity profiles shown at 0.01% FDR.

[0048] FIG. 4A-F show linear peptide affinity mapping in K562 lysate. A) Probe molecules were synthesized by reacting an iodoacetamide- 1 derivative with peptides containing an appended N- or C-terminal cysteine. Peptides selected for analysis were either endogenous (ncORF, proteolytic fragment) or derived from ordered and disordered regions of proteins. Smac pdb: 1FEW; S0S1 structure from AlphaFold. B) An IAP-binding peptide fragment from Smac was profiled using Affinity Map. C) The affinity of the primary interactor of the SOS 1 -derived peptide was also measured by Affinity Map.

[0049] D-F) Affinity profiles for endogenous peptides (derived from HIV-1 gpl60 signal peptide, SNHG6 IncRNAORF, and STAT upstream ORF). *ATE1.2: protein group comprising peptides matched to both ATE1 and the ATE1-2 sequence variant. Affinity profiles shown at 0.01% FDR.

[0050] FIG. 5A-B show an affinity Map for a cyclic RGD peptide and IgG on live cell surfaces. A) Left: cyclo-RGDfK affinity profiling in live U87MG glioma cells was performed in 246-well plates, allowing for simultaneous affinity measurement and selectivity assessment on the surfaces of living, adherent cells in complete media. Right: Affinity Map measures binding to integrin 03 (ITGB3), consistent with known cyclo-RGDfK selectivity for the aV03 heterodimer. B) Left: IgG affinity profiling on live THP-1 cell surfaces was performed with suspended cells using a single 96 cluster-tube plate. Right: the affinity profile shows IgG affinities for several Fc-receptors. All plots shown are at 0.01% FDR.

[0051] FIG. 6 shows (+)-JQl binding profile in liver S9 fraction. (+) - JQ1 Affinity Plot from human liver S9 fractions (BioIVT, X008011) demonstrates low false positive rates for this selective molecule in human organ lysate, and detection of potential metabolic enzymes that bind and may metabolically alter this small molecule (5% FDR cutoff).

[0052] FIG. 7 shows the development of an improved photocatalyst for Affinity Map.

[0053] FIG. 8 shows diazirine photosensitization yields and logP for synthesized photocatalysts.

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[0055] FIG. 9A-E showCatalyst 1 has higher aqueous solubility and less nonspecific photocatalytic labeling than 2. A) Structure of catalysts used for photophysical property comparison. Thermodynamic solubilities listed in PBS and DI H2O. Binding data of each catalyst to human plasma proteins demonstrating increased non-specific binding of 2 compared to 1. Both catalysts are stable after incubation at 37 °C for 6 hours. B) Thermodynamic solubility comparison in PBS and H2O. C) U87MG and THP1 cell viability following incubation with 1 or 2 and 15-minute irradiation. 1 is not toxic to cells. D) Diazirine sensitization yields over time. E) Comparison of protein biotinylation measured by streptavidin enrichment and bottom-up MS proteomics by 2 and 1 vs. nocatalyst control at timepoints where 1 has greater diazirine consumption than 2 shows increased nonspecific protein labeling by 2, which results from its greater nonspecific protein binding.

[0056] FIG. 10 shows UV-Vis Absorption and Emission Spectra for 1 and Diazirine Sensitization at Multiple Concentrations

[0057] FIG. 11A-D show a small molecule target- ID: comparison between 1 and 2. A) Structures of 1 and 2 conjugated to the SRC / ABL kinase inhibitor dasatnib. B) Volcano plot comparing protein labeling by 2-dasatinib (1 pM) vs 2-dasatinib (1 pM) + dasatinib (10 pM, off-compete control). Kinases highlighted blue. C) Volcano plot of 1-dasatinib (1 pM) vs 1-dasatinib (1 pM) + dasatinib (10 pM, off-compete control). Kinases highlighted blue D) Comparison of log2FC from B and C. Kinases and nucleotide-binding proteins are more enriched by 1 than 2 despite lower diazirine sensitization yield for 1 (FIG.8, 9) due to lower background signal caused by nonspecific protein labeling.

[0058] B and C performed according to methods 1-7 with a single off-compete concentration (10 pM).

[0059] FIG. 12 shows Antibody-targeted photocatalytic proximity labeling at CD 19 using 1 and 2. Both catalysts 1 and 2 are able to enrich the target (CD19) and a known interactor (CR2)1A,2Awhen conjugated to a goat anti-mouse secondary antibody targeting live cells stained with mouse anti-CD19 vs. isotype control.

[0060] FIG. 13A-C show Dose-responsive competitive photocatalytic labeling of recombinant BRD4 (AAs 49-460). A) Structures of l-(+)-JQl and (+)-JQl B) Representative western blot showing dose-dependent labeling of recombinant BRD4

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[0062] BD1+BD2 (AAs 49-460) domains and BSA C) Quantified densitometry values for BSA and BRD4 bands from the blot in B normalized against total protein intensity. Experiment performed in triplicate.

[0063] FIG. 14 shows a binding affinity profiling data processing workflow.

[0064] FIG. 15A-E show Dasatinib-Diazirine-Alkyne Photoaffinity Probe Labeling Optimization. A) Structures of Dasatinib-Diazirine-Alkyne photoaffinity probe. B) Diazirine probe sensitization time course at 375 nm. Experimental procedures described in methods described herein. C) Volcano plots for labeling optimization of dasatinib photoaffinity probe irradiation time and concentration with CSK highlighted as a target kinase. CSK was selected to benchmark labeling as it is the only kinase detected in all samples. The Affinity Map in the main text FIG. 2 and FIG. 15E used for comparison to photocatalytic probes was done with 5-minute irradiation at a 10-fold higher starting concentration as this result gave the best enrichment of CSK. Data was collected according to Method 13 with a two-arm comparison between probe and vehicle versus probe and dasatinib at the indicated concentrations. Each arm of each volcano plot was conducted in triplicate. D) Affinity Map performed using Dasatinib-Diazirine-Alkyne probe at concentration identical to photocatalytic probes with 20-minute irradiation when >95% of probe is consumed. Data was obtained following Method 13 with the concentrations shown below. There is significant measurement of any kinases. E) Affinity Map performed with the same probe and conditions optimized for photoaffinity probe. The same map is depicted in Fig.2D and used to compare catalytic and noncatalytic probes. Kinase targets highlighted in blue.

[0065] FIG. 16 shows Competitive photolabeling of BTK by 1, 2, and diazirine- alkyne conjugates of dasatinib in lysate measured via mass spectrometry. Top: Structures of 1, 2, and diazirine-dasatinib conjugates. Bottom: K562 cell lysate was treated with diazirine-biotin at 200 pM, 1, 2, or diazirine alkyne conjugates of dasatinib at 1 pM, 100 nM, 10 nM, or 1 nM, and unmodified dasatinib at concentrations ranging from 100 pM to 10 pM in half-log increments in duplicate (96 total samples, Method 1-2, FIG. 24). After irradiation, protein precipitation, resuspension, reduction / alkylation, IP, and tryptic digestion, peptides were analyzed via nano-UHPLC / IM / MS / MS in DIA mode using a nanoElute 2 / timsTOF Pro 2 MS system and the resulting data processed using DIANN

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[0067] 1.8 with an in-silico generated spectral library (Methods 3-7 and 11, FIG. 24). After imputation (MinProb, MSnbase), and normalization (vsn, MSnbase) within each photocatalyst-dasatinib concentration sample group, integrated intensity data was fit to a logistic model to measure a EC50 for the dose-responsive reduction in intensity induced by competitive binding of unmodified dasatinib at each photocatalyst concentration for each protein identified in the proteome assuming a Hill coefficient of 1 for all proteins. These EC50 values were then used to generate a Schild plot to model competitive binding between dasatinib and dasatinib-photocatalyst conjugates, with the slope affording the ratio of dasatinib-protein and conjugate-protein dappand the intercept affording the dappof unmodified dasatinib. 1 performs better than 2, which gives reduced signal at low concentrations and elevated background signal at high concentrations due to high nonspecific binding, while diazirine-alkyne conjugate of dasatinib yields only weak signal intensity that enables marginal detection of the BTK-dasatinib interaction.

[0068] FIG. 17A-C shows Performance of 1, 2, and diazirine-alkyne for binding affinity profiling of dasatinib in cell lysate. A) Proteins and kinases with affinities passing FDR obtained using 1, 2, and diazirine-alkyne conjugates of dasatinib in K562 lysate, with the percentage of kinases among identified interactions. For dasatinib-diazirine- alkyne, a relaxed imputation filter was used to enable detection of affinities. B) Venn diagram of all kinases identified by each dasatinib conjugate. C) Venn diagram of kinases with dappidentified using a relaxed imputation filter. Relaxed imputation filter refers to constructing competition models on imputed intensity data for a given protein at a given probe concentration if at least one measured value is available, rather than >25% as is typically required for Affinity Map profiles. 1 afforded the largest number of proteins with significant measured dappvalues, the highest percentage of kinases among measured proteins, and identified all other kinases identified by 2 and diazirine-alkyne. The reduced number of measured dappvalues by 2 reflects its higher nonspecific binding, as demonstrated by lower competitive fold-changes for target proteins (FIG. 11), while the small number of identified values by non-photocatalytic diazirine-alkyne reflects inefficient crosslinking.

[0069] FIG. 18A-E shows Comparison of 1 vs. 2 for protein Kdappmeasurement, benchmarking against literature Kdappvalues measured using Kinobeads, and comparing

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[0071] log(FC) vs. measured affinity. A) Venn diagram of protein targets with significant measured Kdappvalues identified in K562 cell lysate with either 1-dasatinib or 2-dasatinib.

[0072] B, C) Comparison of p dappvalues determined by 1-dasatinib and 2-dasatinib and published p dappvalues measured using the Kinobeads method. 1 enables the measurement of more kinases than is possible using 2, and reports slightly more accurate pVi'11values. Notably, the slope is near one, indicating that photocatalytic binding affinity profiling yields accurate Kdappvalues3A. D) Volcano plot showing enriched proteins in a two-group comparison (log2(FC) = log2(l pM l-dasatinib)-log2(l pM 1-dasatinib + 10 pM dasatinib)) color-coded by Affinity Map-measured binding affinity. E) Comparison of logio( aapp) values determined by probe 1 and fold change measured for enriched proteins in D.

[0073] FIG. 19A-C show the benchmarking Kdappestimation workflows for proteins with two significant EC50 measurements. Comparison of how different methods of estimating Kdappperform for proteins that have only two significant EC50 determinations, benchmarked against reported pKdappvalues measured using the Kinobeads method3A. As the error in a linear fit cannot be accurately determined using two data points, three methods were benchmarked for extracting estimated Kdappvalues in such cases using dasatinib binding affinity profiling data generated from K562 cell lysate using 1-dasatinib. A) Error-weighted mean of the two EC50 values from Equation 5 reported as Kdapp. B) Fitted Kdappfrom Equation 4 reported as Kdapp. TNK2 is excluded from comparison as pKdappreported is 19.08 due to erroneous results that can arise from two-point linear fitting. C) EC50 value at the minimum photocatalytic probe concentration reported as Kdapp. Based on these data, which showed similar performance for all methods, use the minimum value reporting method was chosen as the measurement made at the lowest concentration of the photocatalytic probe is the closest value to the y-intercept of the Schild plot, affording a more accurate estimate than would be produced by a weighted average for the Kdappof proteins that may have comparable affinities for both photocatalyst conjugates and free molecules3Awhile avoiding nonphysical results and statistical uncertainties inherent in two-point line fitting.

[0074] FIG. 20 shows the Kdappvalues from photocatalytic vs. Kinobeads profiling by number of EC50 values used for Affinity Map measurement. Relationship between the number of EC50 values used for pKdappdetermination by 1-dasatinib in K562 cell lysate

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[0076] and the average p dappdeviance between measured and literature p f<r'ppdata (Kinobeads) 3A

[0077] FIG. 21A-F show the benchmarking Affinity Map vs. literature Kinobeads, recombinant protein assay, and CETSA datasets. A) Venn diagram of protein kinases with significant Kdappmeasurements made via 1-dasatinib and protein kinases with significant Kdappmeasurements obtained in a published dataset generated using the Kinobeads method4A. B) Comparison of protein kinases in K562 lysate with significant measured Kdappvalues made via 1-dasatinib and protein kinases with measurable ATmvalues from a literature dataset generated using the CETSA method in lysate and live cells5A. C) Comparison of protein kinases in K562 lysate identified by Gao et. al. as interactors of a more promiscuous kinase ligand, staurosporine, using a limited proteolysis (LiP)-based method and reported protein kinases with measurable ATmvalues identified via CETSA5A,6A. D) Comparison of pKdappvalues determined using 1-dasatinib and reported pKdappvalues measured using the Kinobeads method. E) Comparison of pKdappvalues determined using Affinity Map and 1-dasatinib and reported pKdappvalues measured using recombinant protein assays7A.

[0078] F. Comparison of reported pKdappvalues measured using Kinobeads and reported pVi values measured by recombinant protein assays7A,8A. In comparison with previously reported data, binding affinity profiling via 1-dasatinib affords a distinct set of kinases and proteins associated with measurable Kdappvalues and performs well when compared against assays using activity -based measurement and recombinant proteins. No protein targets of dasatinib identified exclusively by CETSA are kinases. The accuracy of Affinity Map via 1-dasatinib benchmarked against in-vitro assays using activity-based measurements and recombinant proteins is similar to that obtained using Kinobeads.

[0079] FIG. 22A-D shows the validation of novel dasatinib-kinase interactions and affinities. A) RIPK1 PhosphoSense activity assay dose response curve at [ATP] = Km. B) PKN3 PhosphoSense activity assay dose response curve at [ATP] = Km. C) CK1D / CSNK1D PhosphoSense activity assay dose response curve at [ATP] = Km. D) Summary of PhosphoSense assay IC50, ATP Km, calculated Ktvalue (Cheng-Prusoff), and Kdappvalues measured using 1-dasatinib via photocatalytic binding affinity profiling for each kinase.

[0080] FIG.23 shows potential protease cleavage sites / PTM sites in each peptide screened

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[0082] by Affinity Map. Each peptide has affinity for a unique set of proteases, consistent with known protease substrate specificity. Potential cleavage sites are noted for unique proteases that have affinity for specific peptidesl0A l6A.

[0083] FIG.24 shows a sample preparation workflow.

[0084] FIG. 25 shows a small molecule and peptide photocatalytic binding affinity profiling plate setup

[0085] FIG. 26 shows a 96-Well plate irradiation apparatus. Top: Uniform 96-well plate irradiation setup and ferrioxalate-based actinometry principle. Bottom: a 96-well plate was irradiated and the resulting iron(II) generated by irradiation (actinometric reaction) was measured by iron(II)-phenanthroline complex absorbance at 510 nm. Absorbance values were uniform across the plate, showing a standard deviation that was 1.1% of the mean absorbance, demonstrating even plate irradiation.

[0086] FIG. 27 shows an IgG Affinity Profiling Plate Setup.

[0087] FIG. 28A-C shows a Dasatinib Affinity Map with extended pre-irradiation incubation affords similar Kdappvalues. A) Dasatinib affinity profile in K562 lysate where cell lysate was incubated for 1 hour with dasatinib then 1 hour with 1-dasatinib. B) Dasatinib affinity profile in K562 lysate where cell lysate was incubated for 20 minutes with dasatinib then 20 minutes with 1-dasatinib. C) Comparison of p 2iHppvalues determined by either long incubation shown in A or short incubation shown in B. The detected p )iappis similar at 1 hour and 20-minute incubation timepoints, although a reduction in signal intensity was observed with longer incubation time (potentially due to protein degradation). All reported affinity measurements represent apparent binding affinities (7Gapp), because as the extent of dasatinib-target equilibration is not assessed, very slow on or off rates for ligand association / dissociation are possible.

[0088] FIG. 29 shows photocatalytic labeling of recombinant BRD4 BD1+BD2. A) Gel 1, samples 1-9 replicate 1 stained for streptavidin (left) and total protein loading control (right). B) Gel 2, samples 1-9 replicate 2 stained for streptavidin (left) and total protein loading control (right). C) Gel 3, samples 1-9 replicate 3 stained for streptavidin (left) and total protein loading control (right).

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[0090] Detailed Description

[0091] The present disclosure may be embodied as a system, a method, and / or a computer program product. The computer program product may include a computer readable storage medium (or media) having computer readable program instructions thereon for causing a processor to carry out aspects of the present disclosure.

[0092] The computer readable storage medium can be a tangible device that can retain and store instructions for use by an instruction execution device. The computer readable storage medium may be, for example, but is not limited to, an electronic storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any suitable combination of the foregoing. A non-exhaustive list of more specific examples of the computer readable storage medium includes the following: a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a static random access memory (SRAM), a portable compact disc read-only memory (CD-ROM), a digital versatile disk (DVD), a memory stick, a floppy disk, a mechanically encoded device such as punch-cards or raised structures in a groove having instructions recorded thereon, and any suitable combination of the foregoing. A computer readable storage medium, as used herein, is not to be construed as being transitory signals per se, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through a waveguide or other transmission media (e.g., light pulses passing through a fiber-optic cable), or electrical signals transmitted through a wire.

[0093] Computer readable program instructions described herein can be downloaded to respective computing / processing devices from a computer readable storage medium or to an external computer or external storage device via a network, for example, the Internet, a local area network, a wide area network and / or a wireless network. The network may comprise copper transmission cables, optical transmission fibers, wireless transmission, routers, firewalls, switches, gateway computers and / or edge servers. A network adapter card or network interface in each computing / processing device receives computer readable program instructions from the network and forwards the computer readable

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[0095] program instructions for storage in a computer readable storage medium within the respective computing / processing device.

[0096] Computer readable program instructions for carrying out operations of the present disclosure may be assembler instructions, instruction-set-architecture (ISA) instructions, machine instructions, machine dependent instructions, microcode, firmware instructions, state-setting data, or either source code or object code written in any combination of one or more programming languages, including an object oriented programming language such as Smalltalk, C++ or the like, and conventional procedural programming languages, such as the “C” programming language or similar programming languages. The computer readable program instructions may execute entirely on the user’s computer, partly on the user’s computer, as a stand-alone software package, partly on the user’s computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user’s computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider). In some embodiments, electronic circuitry including, for example, programmable logic circuitry, field-programmable gate arrays (FPGA), or programmable logic arrays (PLA) may execute the computer readable program instructions by utilizing state information of the computer readable program instructions to personalize the electronic circuitry, in order to perform aspects of the present disclosure.

[0097] Aspects of the present disclosure are described herein with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the disclosure. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer readable program instructions.

[0098] These computer readable program instructions may be provided to a processor of a general purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create

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[0100] means for implementing the functions / acts specified in the flowchart and / or block diagram block or blocks. These computer readable program instructions may also be stored in a computer readable storage medium that can direct a computer, a programmable data processing apparatus, and / or other devices to function in a particular manner, such that the computer readable storage medium having instructions stored therein comprises an article of manufacture including instructions which implement aspects of the function / act specified in the flowchart and / or block diagram block or blocks.

[0101] The computer readable program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other device to cause a series of operational steps to be performed on the computer, other programmable apparatus or other device to produce a computer implemented process, such that the instructions which execute on the computer, other programmable apparatus, or other device implement the functions / acts specified in the flowchart and / or block diagram block or blocks.

[0102] The flowchart and block diagrams in the Figures illustrate the architecture, functionality, and operation of possible implementations of systems, methods, and computer program products according to various embodiments of the present disclosure. In this regard, each block in the flowchart or block diagrams may represent a module, segment, or portion of instructions, which comprises one or more executable instructions for implementing the specified logical function(s). In some alternative implementations, the functions noted in the block may occur out of the order noted in the figures. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality involved. It will also be noted that each block of the block diagrams and / or flowchart illustration, and combinations of blocks in the block diagrams and / or flowchart illustration, can be implemented by special purpose hardwarebased systems that perform the specified functions or acts or carry out combinations of special purpose hardware and computer instructions.

[0103] The descriptions of the various embodiments of the present disclosure have been presented for purposes of illustration, but are not intended to be exhaustive or limited to

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[0105] the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

[0106] In certain aspects, the present disclosure provides a complex comprising Iridium (Ir) and a ligand of formula (I):

[0107]

[0108] (I),

[0109] wherein:

[0110] L is a linker; and

[0111] Z is a protein-binding ligand.

[0112] In certain embodiments:

[0113] L is a linker comprising formula (II):

[0114] -(CHR5)n1-X1-(CHR6)n2-O-(CHR7-CHR8-O)n3-(CHR9)n4-X2-(CHR10)n5- (II); wherein:

[0115] R5, R6, R7, R8, R9, and R10are each independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl;

[0116] X1and X2are each independently a bond (absent), -O-, -CH2-, -NH-, -C(O)-, -NHC(O)-, or -C(O)NH-;

[0117] n1, n2, n3, n4, and n5 are each independently an integer selected from 0 to 100. In certain embodiments, the complex has the structure of formula (III):

[0118] - 15 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0119] R1

[0120] %N

[0121] R2

[0122] fx r o

[0123] XZk 6

[0124] fXXX °'L'z

[0125] RV °

[0126]

[0127] R* (III),

[0128] wherein:

[0129] R1, R2, R3, and R4are each independently alkyl optionally substituted with one or more hydroxy, alkoxy, or halo.

[0130] In certain embodiments, the complex has the structural formula (IV):

[0131] OH

[0132] 7 OH LOxx?

[0133] r » r n

[0134] F^= / Nv. |

[0135] / / l\T >(r< N" 7\

[0136] FtAcZ °~L'Z

[0137] HO / N'"'0

[0138] 7

[0139]

[0140] OH

[0141] (IV).

[0142] In certain embodiments, L comprises a polyethylene glycol chain terminated with an amine group.

[0143] In certain embodiments, L comprises a polyethylene glycol chain comprising from about 1 to about 100 ethylene glycol units.

[0144] - 16 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0145] In certain embodiments, L is: -(CH2)n2-O-(CH2-CH2-O)n3-(CH2)n4-.

[0146] In certain embodiments, L is: -(CH2)2-O-(CH2-CH2-O)n3-(CH2)2-.

[0147] In certain embodiments, the complex has the structural formula (V-A), formula (V-B), or formula (V-C):

[0148]

[0149] - 17 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0150]

[0151] In certain embodiments, the complex has the structural formula (VI-A), formula (VI-B), or formula (VI-C):

[0152]

[0153] (VI-A);

[0154] - 18 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0155]

[0156] (VI-C).

[0157] In certain embodiments, R1, R2, R3, and R4are -N(CH2CH2OH)2.

[0158] In certain embodiments, the protein-binding ligand is an antibody, protein, peptide, or small molecule.

[0159] In certain embodiments, Z is a small molecule selected from:

[0160] - 19 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0161]

[0162] wherein * indicates a bond to L of the compound of Formula (I).

[0163] In certain aspects, the present disclosure provides a method of characterizing one or more interactions between a protein-binding ligand and one or more proteins in a biological sample, comprising contacting the biological sample with the complexes described herein.

[0164] In certain embodiments, the method further comprises contacting the biological sample with a free protein-binding ligand corresponding to Z; and a crosslinking agent which comprises a labeling moiety (e.g. a diazirine-biotin probe).

[0165] In certain preferred embodiments, the crosslinking agents contain at least a photocatalyst-activatable moiety (e.g., diazirine, azide, aniline, phenol), and optionally, a linker connecting the crosslinking agent to a mass spectrometry-detectable moiety (e.g., timShift) or affinity handle (e.g., biotin, FLAG, FITC, etc.), which is advantageous in generating an affinity map.

[0166] In certain embodiments, the method further comprises irradiating the biological sample, thereby generating an activated crosslinking agent which crosslinks with one or more proteins in the biological sample.

[0167] In certain embodiments, the irradiating comprises a light of 440 nm for at least 60 seconds.

[0168] In certain embodiments, the biological sample comprises one or more cells or the lysates of one or more cells.

[0169] In certain embodiments, the method further comprises resuspending the biological sample from the cell, performing a streptavidin enrichment of the resuspended sample, and characterizing the resuspended sample (e.g. using mass spectrometry).

[0170] In certain embodiments, the method is repeated for two or more concentrations of either the free protein-binding ligand or the complex of any one of claims 1-13.

[0171] - 20 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0172] In certain embodiments, the method is performed in parallel with at least 10, 100, or at least 1,000 biological samples.

[0173] In certain embodiments, the characterization comprises proteomic binding analysis of at least 10, 100, or 1,000 proteins.

[0174] In certain embodiments, the characterization comprises measuring a binding affinity constant or a kinetic rate constant of at least 10 proteins, lipids, nucleic acids, or carbohydrates.

[0175] In certain embodiments, the concentrations of the free protein-binding ligand and the complex of any one of claims 1-13 have a ratio of 1:1, 2:1, 3:1, 4:1. 5:1. 10:1. 100:1, 1,000:1, 1:1000, 1:2, 1:3, 1:4. 1:5, 1:10. 1:100, and / or 1:1,000.

[0176] In certain embodiments, the one or more interactions comprise a protein-protein interaction, protein-DNA interaction, protein-RNA interaction, or protein-small molecule interaction.

[0177] In certain embodiments, the mass spectra measurement is processed by an algorithm, wherein the algorithm characterization has a false negative rate of less than 5%.

[0178] In certain embodiments, the algorithm has a true positive rate of characterization of at least 95%.

[0179] In certain aspects, the present disclosure provides a complex comprising Iridium (Ir) and a ligand of structure (IA):

[0180]

[0181] OH

[0182] (IA).

[0183] In certain embodiments, the compound has the structure:

[0184] - 21 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0185]

[0186] wherein:

[0187] R1, R2, R3, and R4are each independently alkyl optionally substituted with one or more hydroxy, alkoxy, or halo.

[0188] In certain aspects, the present disclosure provides a method of generating an affinity map, comprising:

[0189] (a) contacting a sample, which comprises a set of proteins, with (i) a probe agent, which optionally comprises a protein-binding ligand Z, and (ii) a crosslinking agent, which upon activation is capable of crosslinking with a subset of protein(s) to which the protein-binding ligand is bound; and

[0190] (b) activating the crosslinking agent, thereby forming a crosslinked sample comprising a subset of crosslinked proteins.

[0191] In certain embodiments, a single molecule comprises the probe agent and the crosslinking agent.

[0192] In certain embodiments, a single molecule comprises a probe agent moiety and a crosslinking agent moiety.

[0193] A single molecule comprising a probe agent and a crosslinking agent is advantageous because it is easier to synthesize and / or comprises moieties which are more readily available.

[0194] In preferred embodiments, the crosslinking agents (e.g., warheads) comprise diazirines, azido, or benzophenone moieties.

[0195] - 22 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0196] The use of photoaffinity probes (e.g., JQ1 -diazirine) in place of the photocatalytic probe - diazirine mixture is an advantageous option for affinity mapping.

[0197] In certain embodiments, the method further comprises, in step (a), contacting the sample with a free protein-binding ligand (e.g. wherein the free protein-binding ligand is the same as Z, or wherein the free protein-binding ligand is different from Z).

[0198] In certain embodiments, in step (b), activating the crosslinking agent comprises irradiating the sample, thereby activating the crosslinking agent.

[0199] In certain embodiments, in step (b) the crosslinking agent forms a covalent bond with the protein(s).

[0200] In certain embodiments, the cross-linking agent is a carbene precursor, nitrene precursor, carbocation precursor, carbon-centered radical precursor, amidyl radical precursor, acyl halide precursor, singlet oxygen, RNA, or a electrophilic (Michael acceptor) containing agent.

[0201] In certain embodiments, the crosslinking agent comprises a diazo, diazirine, azido, phenol, or benzophenone moiety.

[0202] In certain embodiments, the crosslinking agent is activatable by a suitable photocatalyst. This may accomplished by pairing crosslinking agents that can be activated by energy transfer (e.g., diazirine, aryl azide, or benzophenone) with high triplet energy catalysts, like iridium complexes, or those that can be activated by electron transfer, such as tetrafluorophenyl, phenyl, and napthyl azides, phenols, anilines, etc., with oxidizing or reducing photocatalysts (e.g., ruthenium, osmium complexes, or flavins). Azides react through either energy transfer or single electron reduction, while anilines and phenols react through single electron oxidation.

[0203] In preferred embodiments, the crosslinking agent used provides a tightly resolved spatial resolution, ideally with a radius lower than 100 nm from the site of the photocatalyst. Diazirines and azides are the tightest radius modalities in the above list and are preferred for affinity mapping.

[0204] In certain embodiments, the crosslinking agent comprises a diazirine.

[0205] In certain embodiments, the crosslinking agent comprises an azido.

[0206] In certain embodiments, the crosslinking agent is tetraphenylazide.

[0207] In certain embodiments, the crosslinking agent is nitrophenol.

[0208] - 23 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0209] In certain embodiments, the crosslinking agent, upon activation, generates an activated crosslinking agent comprising a carbene.

[0210] In certain embodiments, the crosslinking agent, upon activation, generates an activated crosslinking agent comprising a carbocation.

[0211] In certain embodiments, the crosslinking agent, upon activation, generates an activated crosslinking agent comprising a nitrene.

[0212] In certain embodiments, the crosslinking agent comprises a labeling moiety.

[0213] In certain embodiments, the labeling moiety is biotin, an azide, an alkyne, desthiobiotin, fluoroisothiocyanate (FITC), an antibody, an antibody conjugate, or a biorthogonal enrichment handle.

[0214] In certain embodiments, the method further comprises (c) isolating the subset of crosslinked proteins (e.g., by performing an immunoprecipitation, such as a streptavidin immunoprecipitation, a gas phase ion separation, or a biorthogonal enrichment modality).

[0215] In certain embodiments, the method further comprises (d) analyzing the subset of crosslinked proteins.

[0216] In certain embodiments, the crosslinked sample is analyzed by mass spectrometry, nanopore analysis, or reverse translation of peptide.

[0217] In certain aspects, the present disclosure provides a method of analysis, comprising performing the methods described herein on a plurality of samples at a plurality of probe agent and free protein-binding ligand concentrations, and

[0218] further comprising generating the affinity map based on the analysis results, the probe agent concentrations, and the free protein-binding ligand concentrations (e.g., comprising applying the Cheng-Prusoff model).

[0219] In certain aspects, the present disclosure provides a method of analysis, comprising performing the methods described herein on a plurality of samples at a plurality of probe agent and free protein-binding ligand concentrations, and

[0220] further comprising generating the affinity map based on the analysis results, the probe agent concentrations, and the free protein-binding ligand concentrations.

[0221] In certain embodiments, the method further comprises systematically varying the concentration of the probe agent and the concentration of the free-binding ligand.

[0222] - 24 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0223] In certain embodiments, systematically varying the concentration of the probe agent and the concentration of the free-binding ligand includes varying both dimensions across a matrix of samples, such that within each concentration of probe agent a range of concentrations of free-binding ligand are used.

[0224] In certain embodiments, systematically varying the concentration of the probe agent and the concentration of the free-binding ligand comprises diluting in PBS.

[0225] In certain embodiments, generating the affinity map comprises applying the Cheng-Prusoff equation to the analysis results, the probe agent concentrations, and the free protein-binding ligand concentrations.

[0226] In certain embodiments, the probe agent comprises a photocatalyst coupled or linked to a protein-binding ligand.

[0227] In certain embodiments, the photocatalyst is a metal complex.

[0228] In certain embodiments, the photocatalyst is an organic photocatalyst.

[0229] In certain embodiments, the photocatalyst is a metal complex comprising Iridium (Ir).

[0230] In certain embodiments, the photocatalyst is a complex comprising Iridium (Ir) and a ligand of formula (I):

[0231]

[0232] (I),

[0233] wherein:

[0234] L is a linker; and

[0235] Z is a protein-binding ligand.

[0236] In certain embodiments:

[0237] L is a linker comprising formula (II):

[0238] -(CHR5)n1-X1-(CHR6)n2-O-(CHR7-CHR8-O)n3-(CHR9)n4-X2-(CHR10)n5- (II); wherein:

[0239] R5, R6, R7, R8, R9, and R10are each independently selected from hydrogen, alkyl,

[0240] - 25 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0241] aryl, heteroaryl, cycloalkyl, and heterocyclyl;

[0242] X1and X2are each independently a bond (absent), -O-, -CH2-, -NH-, -C(O)-, -NHC(O)-, or -C(O)NH-;

[0243] n1, n2, n3, n4, and n5 are each independently an integer selected from 0 to 100. In certain embodiments, the photocatalyst has the structure of formula (III):

[0244]

[0245] (III),

[0246] wherein:

[0247] R1, R2, R3, and R4are each independently alkyl optionally substituted with one or more hydroxy, alkoxy, or halo.

[0248] In certain embodiments, the photocatalyst has the structural formula (IV):

[0249]

[0250] - 26 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0251] (IV).

[0252] In certain embodiments, L comprises a polyethylene glycol chain terminated with an amine group.

[0253] In certain embodiments, L comprises a polyethylene glycol chain comprising from about 1 to about 100 ethylene glycol units.

[0254] In certain embodiments, L is: -(CH2)n2-O-(CH2-CH2-O)n3-(CH2)n4-.

[0255] In certain embodiments, L is: -(CH2)2-O-(CH2-CH2-O)n3-(CH2)2-.

[0256] In certain embodiments, the photocatalyst has the structural formula (V-A), formula (V-B), or formula (V-C):

[0257] R1

[0258] (V-A); R1

[0259]

[0260] R4 (V-B); or

[0261] - 27 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0262]

[0263] In certain embodiments, the photocatalyst has the structural formula (VI-A), formula (VI-B), or formula (VI-C):

[0264]

[0265] (VI-A);

[0266] - 28 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0267] OH

[0268] OH

[0269] (VI-B); or

[0270]

[0271] (VI-C).

[0272] In certain embodiments, R1, R2, R3, and R4are -N(CH2CH2OH)2.

[0273] In certain embodiments, the protein-binding ligand is an antibody, protein, peptide, or small molecule.

[0274] In certain embodiments, Z is a small molecule selected from:

[0275]

[0276] - 29 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0277] wherein * indicates a bond to L of the compound of Formula (I).

[0278] In certain embodiments, the sample is a biological sample.

[0279] In certain embodiments, the biological sample comprises one or more cells or the lysates of one or more cells.

[0280] In certain embodiments, the affinity map identifies the binding affinity of the protein-binding ligand at the plurality of proteins.

[0281] Definitions

[0282] 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.

[0283] 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).

[0284] 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).

[0285] 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.

[0286] - 30 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0287] The term "culturing" refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell. By "expanded" is meant any proliferation or division of cells.

[0288] The terms "decrease", "reduced", "reduction", or "inhibit" are all used herein to mean a decrease by a statistically significant amount. In some embodiments, "reduce," "reduction" or "decrease" or "inhibit" typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given ligand) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduction" or "inhibition" does not encompass a complete inhibition or reduction as compared to a reference level. " Complete inhibition" is a 100% inhibition as compared to a reference level.

[0289] The terms "increased", "increase" or "enhance" or "activate" are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "enhance" or "activate" means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, at least about a 20-fold increase, at least about a 50-fold increase, at least about a 100-fold increase, at least about a 1000-fold increase or more as compared to a reference level.

[0290] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0291] - 31 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0292] As used herein, the term “interaction”, when referring to an interaction between two molecules, refers to the physical contact (e.g., binding) of the molecules with one another. Generally, such an interaction results in an activity (which produces a biological effect) of one or both of said molecules. The activity may be a direct activity of one or both of the molecules, (e.g., signal transduction).

[0293] As used herein, an “isolated protein” refers to a protein that is substantially free of other proteins, cellular material, separation medium, and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide or fusion protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of a target polypeptide (e.g., immunoglobulin) or fragment thereof, in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of target protein or fragment thereof, having less than about 30% (by dry weight) of nontarget protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-target protein, still more preferably less than about 10% of nontarget protein, and most preferably less than about 5% non-target protein. When antibody, polypeptide, peptide or fusion protein or fragment thereof, e.g., a biologically active fragment thereof, is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0294] 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.

[0295] - 32 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0296] 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 groups may be on the same carbon or on different carbons, so long as a stable structure results.

[0297] 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-CI -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.

[0298] 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-C4 straight-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.

[0299] The term “acyl” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0300] 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-.

[0301] - 33 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0302] The term “acyloxy” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0303] 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.

[0304] The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

[0305] The term “alkyl” refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted 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., Ci-30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer.

[0306] 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.

[0307] 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. Co alkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. A C1-6 alkyl group, for example, contains from one to six carbon atoms in the chain.

[0308] The term “alkylamino”, as used herein, refers to an amino group substituted with at least one alkyl group.

[0309] 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-.

[0310] The term “amide”, as used herein, refers to a group

[0311] 0

[0312]

[0313] - 34 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0314] 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.

[0315] 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 R9R9

[0316] N or N-R10

[0317]

[0318] R10R10'

[0319] 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.

[0320] The term “aminoalkyl”, as used herein, refers to an alkyl group substituted with an amino group.

[0321] The term “aralkyl”, as used herein, refers to an alkyl group substituted with an aryl group.

[0322] The term “aryl” as used herein includes optionally substituted (i.e., 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.

[0323] The term “carbamate” is art-recognized and refers to a group

[0324] 0 0

[0325]

[0326] R9R9

[0327] 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.

[0328] 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,

[0329] - 35 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0330] 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-lH-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted (i.e., are optionally substituted) at any one or more positions capable of bearing a hydrogen atom.

[0331] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

[0332] The term “carbonate” is art-recognized and refers to a group -OCO2-.

[0333] The term “carboxy”, as used herein, refers to a group represented by the formula -CO2H.

[0334] The term “ester”, as used herein, refers to a group -C(O)OR9wherein R9represents a hydrocarbyl group.

[0335] 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.

[0336] The terms “halo” and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo.

[0337] The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to an alkyl group substituted with a hetaryl group.

[0338] - 36 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0339] The terms “heteroaryl” and “hetaryl” includes optionally substituted (i.e., 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 heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls.

[0340] Heteroaryl groups include, for example, pyrrole, furan, thiophene, pyridone, pyrmidone, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

[0341] The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0342] The term “heterocyclylalkyl”, as used herein, refers to an alkyl group substituted with a heterocycle group.

[0343] The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer to optionally substituted (i.e., substituted or unsubstituted) non-aromatic ring structures, preferably 3-to 10-membered rings, more preferably 3- to 7-membered rings, more prefereably 4- to 5-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.

[0344] 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

[0345] - 37 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0346] 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.

[0347] The term “hydroxyalkyl”, as used herein, refers to an alkyl group substituted with a hydroxy group.

[0348] 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 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).

[0349] 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 optionally substituted (i.e., are 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.

[0350] The term “sulfate” is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.

[0351] The term “sulfonamide” is art-recognized and refers to the group represented by the general formulae

[0352] O R10o, / '°

[0353] ■S-N or

[0354] -N °

[0355]

[0356] 6 R9R9

[0357] wherein R9and R10independently represents hydrogen or hydrocarbyl.

[0358] The term “sulfoxide” is art-recognized and refers to the group-S(O)-.

[0359] - 38 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0360] The term “sulfonate” is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.

[0361] The term “sulfone” is art-recognized and refers to the group -S(O)2-.

[0362] 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 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 sulfonamide, 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.

[0363] The term “thioalkyl”, as used herein, refers to an alkyl group substituted with a thiol group.

[0364] The term “thioester”, as used herein, refers to a group -C(O)SR9or -SC(O)R9wherein R9represents a hydrocarbyl.

[0365] The term “thioether”, as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur.

[0366] The term “urea” is art-recognized and may be represented by the general formula

[0367] - 39 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0368] o

[0369]

[0370] R9R9

[0371] wherein R9and R10independently represent hydrogen or a hydrocarbyl.

[0372] 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.

[0373] 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.

[0374] Some of the compounds may also exist in tautomeric forms. Such forms, although not explicitly indicated in the formulae described herein, are intended to be included within the scope of the present disclosure.

[0375] EXAMPLES

[0376] The disclosure 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 disclosure, and are not intended to limit the disclosure.

[0377] In photocatalytic proximity labeling, a photocatalyst near a protein of interest is used to generate reactive intermediates. These reactive species then covalently tag proteins in close proximity to the photocatalyst, enabling their identification by MS. High spatial resolution methods use carbenes generated by sensitized photolysis of diazirines for proximity labeling due to their tight labeling radius (< 4 nm) and promiscuous reactivity. By using a small molecule targeting modality for the photocatalyst, small molecule binding targets are selectively labeled, while using a larger (ca. 30 nm) primary / secondary antibody

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[0379] targeting modality enables protein-protein interaction discovery. However, currentgeneration photocatalysts for diazirine activation are either highly hydrophobic and waterinsoluble (2) or are anionic at physiological pH, are challenging to synthesize, and can present stability liabilities. These shortcomings have hindered widespread use of diazirine-based proximity labeling, preventing high throughput applications. Catalysts based on Ir(dF(CF3)ppy)2(dmbpy) such as 2 are highly stable and synthetically tractable, leading to their common use in small molecule target-ID applications. However, hydrophobic photocatalysts exhibit high background labeling caused by nonspecific protein binding, limiting the maximum achievable difference in MS signal observed in competitive binding assays using small molecule photocatalyst conjugates and reducing the signal-to-noise ratio. An ideal photocatalyst for high throughput applications, such as binding affinity profiling, requires high solubility, bench stability, and good photocatalytic activity.

[0380] Described herein is the synthesis and characterization of 1, a novel iridium photocatalyst for proximity labeling. The complex is charge-neutral, light and heat stable, hydrophilic, and able to activate diazirine probes for catalytic labeling. This new catalyst demonstrated high aqueous solubility (Figs.8, 9), activates diazirine probes and maintains diazirine-sensitization activity after 30 minutes of blue light irradiation (Figs. 8, 9), has reduced nonspecific protein binding and associated MS background signal (Fig.9, 11), can effectively label the binding targets of small molecules using a small molecule targeting modality (Fig. 11), and identifies protein-protein interactions using a primary / secondary antibody targeting modality (Fig. 12). Further, it was shown that these improved properties enable enhanced binding affinity profiling (Fig. 13, 16) in comparison with 2. Overall, 1 represents a next-generation photocatalyst which exhibits improved physical properties and robust diazirine-sensitization activity for carbene-based proximity labeling applications in lysates / extracts and on cell surfaces, and is especially well-suited to high throughput methods such as binding affinity profiling.

[0381] Example 1. Design concept and analysis pipeline

[0382] Accurate affinity profiling requires that the MS signal introduced by photocatalytic labeling is highly specific to direct ligand binding targets. The pMap platform was selected, in which highly reactive carbenes selectively biotinylate the direct binding targets of a -41 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0383] photocatalyst-probe conjugate, to provide a MS readout to be used for modeling. The relationship between EC50, probe concentration, and KA values for both the probe and ligand are given by the Cheng-Prusoff equation34:

[0384] ligand

[0385] EC50= [probe]

[0386]

[0387] Kd

[0388] Using this relationship, linear regression can be used to obtain 7ifdllgandvalues by measuring EC50 at a series of probe concentrations. Notably, modification of a ligand to incorporate a bulky reporter moiety such as a photocatalyst typically weakens its binding affinity. When the relative affinity of the probe is much weaker than the ligand, its effect on the EC50 value approaches zero, such that EC50 = k'<illg:'"‘l. Therefore, accurate KALGANDmeasurement for the unmodified ligand would be possible even if the binding affinity of the probe is low, so long as sufficient MS signal from photocatalytic labeling is observed for a given protein.

[0389] A custom data processing pipeline was designed to determine proteome-wide unmodified ligand affinities, which was named Affinity Surveyor (Fig. 2A, 14). After normalization and imputation, MS data for a specific protein is subjected to logistic curve fitting at each probe concentration. EC50 values from each curve are then used to fit the ligand binding affinity using the Cheng-Prusoff equation, and p-values for all logistic models are combined to give the aggregate significance of data supporting a f<illga"<lmeasurement. For proteins with significant (p < 0.1) values for 7ifdllgand / ^dprobe, corrected values of 'dllgandare reported. In cases where KA1 lg and / 7ifdprobeis indistinguishable from zero (confidence interval < ±0.25) average EC50 values are reported as KAlgand. For proteins with insufficient data to model / Cll8a"d / / C|irolic. the EC50 measured at the lowest probe concentration is reported as a best estimate of KAlgandbecause incorporation of a bulky photocatalyst often makes 'dprobemuch larger than KA1 lgand. This is repeated for all detected proteins in the MS dataset, providing a proteome-wide binding affinity profile.

[0390] Example 2. Catalyst development

[0391] An optimized bioorthogonal photocatalyst, 1, was developed for the Affinity Map workflow (Fig. 2B). It was first attempted to use previously reported photocatalyst 232,33to assess competitive binding of small molecules. However, Ir(dF(CF3)ppy)2(dmbpy) based - 42 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0392] catalysts are hydrophobic, leading to nonspecific protein binding, off-target labeling, and low water solubility. Reported catalysts27,35containing carboxylic acid moieties are more hydrophilic, but are challenging to synthesize and use on large scales. Photocatalyst 1 has high aqueous solubility, decreased nonspecific interactions with the proteome by mass spectrometry and plasma protein binding (PPB), no net charge, and robust bench stability (Fig. 7-11).

[0393] Example 3. Small molecule-protein affinity profiling.

[0394] Dasatinib was selected as a model ligand for initial experiments due to its large number of reported kinase binding affinities. To provide sufficient statistical power for useful affinity profiling, an experimental matrix was designed in which twelve ligand concentrations (vehicle, 100 pM-10 pM) were used at four photocatalytic probe (1-dasatinib) concentration (1 nM-1 pM in duplicate, 96 samples, Fig. 25), with a 60-minute incubation time. To facilitate large scale experiments and increase sample-to-sample consistency, a high throughput, 96-well plate format, two-day protocol for parallel photocatalytic labeling driven by 440 nm light, streptavidin enrichment, tryptic digestion, and preparation for MS analysis was developed (Fig. 2C, 24). MS data acquisition (timsTOF Pro 2) and protein quantification (DIA-NN, in silico library) generally required three days per affinity profile. After protein quantification, the data was subjected to the Affinity Surveyor pipeline to determine dasatinib targets. Notably, as binding may not have reached equilibrium for some targets and target concentrations are unknown, all measured KA values are reported as apparent affinities ( Tilipp) (Fig. 28).

[0395] Using a photocatalytic 1-dasatinib conjugate and the Affinity Map workflow in K562 lysate, )iappfor dasatinib against 28 kinases and 8 other proteins (many of which contain nucleotide binding sites) was unambiguously measured (Fig. 2D). Qualitative inspection of data collected for target proteins such as CSK and BTK revealed clear sigmoidal dose response curves at all 1-dasatinib probe concentrations (Fig. 16). The performance of 1-dasatinib, 2-dasatinib, and a dasatinib-diazirine-alkyne conjugate as reporter probes for affinity profiling was also compared. Using the more hydrophobic photocatalyst 2, fewer kinases with measurable affinity values were observed. Inspection of dose-response curves reveals lower on-target labeling efficiency at low catalyst concentrations and higher - 43 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0396] background signal at higher catalyst concentrations, reflecting labeling caused by nonspecific catalyst binding. In contrast, activation of a non-photocatalytic dasatinib-diazirine probe with 375 nm light led to far less efficient protein labeling than observed with 1-dasatinib, enabling measurement of only four kinase affinities. Previously reported photoaffinity and photocatalytic labeling methods compare protein crosslinking by a probe against a control, with the most differentially enriched proteins flagged as potential binding targets. Whether differential enrichment from protein labeling is correlated with affinity was evaluated. Treating K562 lysate with 1-dasatinib and either vehicle control or 10-fold excess dasatinib, it was found that the difference in enrichment was not well-correlated with affinity (Fig. 2E-F, 18). Indeed, many proteins with high affinity to dasatinib, including the well-characterized target BCR-ABL, have relatively low fold change due to variable and protein-specific MS intensity background signal. Proteins can have significant fold change but lack competitive binding curves, potentially representing false-positive hits caused by nonspecific binding, which are indistinguishable from real targets in standard two-group comparison but easily identified by logistic modeling in Affinity Map. dappvalues obtained using Affinity Map were close to literature Kinobead and in vitro affinities (Fig. 2G, 21),6’7’36including both values where the Kdllgand / Kdprobeis fully modeled and well constrained or estimated due to a lack of sufficient data for Cheng-Prusoff modeling. Notably, Kdappvalues were measured for more kinase targets than were identified in a recently reported CETSA experiment performed in K562 lysate, which identified five kinases with measurable ATmvalues despite MS detection of many kinases that were successfully profiled by Affinity Map (Fig. 2H, 21). One protein identified by CETSA, ADK,13lacked a known affinity for dasatinib. It was confirmed this interaction using Affinity Map, measuring an affinity of 1.6 pM. Three kinase targets were observed via Affinity Map (PKN3, CKN1D, and RIPK1) that were previously unknown. Therefore, an in vitro activity assay (PhosphoSens) was used to validate these interactions. Two kinases gave values very similar to those measured via Affinity Map (RIPK1, 3.0 pM vs. 3.6 pM; CSNK1D, 4.1 pM vs. 3.6 pM), while PKN3 was assayed within approximately one log of the Affinity Map value (Fig. 3 A, 22).

[0397] To test the false positive discovery rate of Affinity Map, a highly selective small molecule, (+)-JQl (Fig. 3B) was profiled, which is an inhibitor of bromodomain containing

[0398] - 44 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0399] proteins BRD2, BRD3, and BRD4. Using (+)-JQl and l-(+)-JQl, competitive labeling at the intact protein level in the presence of BSA as a nonspecific binding control was measured. A clear sigmoidal dose-response curve was observed by streptavidin western blot (Fig. 3C-D, 13), further validating our logistic model of photocatalytic protein biotinylation. In K562 cell lysate, Affinity Map accurately captures the selectivity of this compound (Fig. 3E), exclusively reporting measurable binding affinities for these three proteins. Measured )iappvalues are close to previously reported values obtained by isothermal calorimetry with purified BRD domains (Fig. 3F).37

[0400] The applicability of the Affinity Map workflow to protein extracts from human organs was next evaluated (Fig. 3G). Liver microsomes generated through mechanical organ disruption contain metabolic enzymes and are commonly used to evaluate the stability of small molecules. (+)-JQl targeted Affinity Map was performed using pooled human liver S9 fraction, a minimally processed homogenate containing microsomes and globular proteins. )iappvalues were measured for BRD3, BRD4, AKR1B10 (a cytosolic aldo-keto reductase), in addition to microsome constituents CYP2J2 (a membrane-bound P450 enzyme) and UGT1A3 (a transmembrane glucuronosyltransferase). (+)-JQl affinity to SLCO1B 1 was further measured, a liver-specific plasma membrane organic ion transporter. These data highlight the potential utility of Affinity Map for profiling diverse sectors of the proteome directly in patient samples.

[0401] To further demonstrate the utility of Affinity Map in organ extracts, the interactions of flumazenil, a benzodiazepine antagonist, in mouse brain membrane isolates were profiled, prepared according to radioligand binding assay protocols where endogenous GABA is washed out (Fig. 3H).38Flumazenil binds heteropentameric GABA receptors at the a and y subunit interface, known as the benzodiazepine site. Here, significant sigmoidal curve fits and accurate )iappvalues39were measured for two GABA receptor subunits, Gabrg2 ( 2 subunit) and Gabral (al subunit). A single sigmoid was measured for the a3 subunit but did not pass FDR likely due to its low MS intensity. Detection of other subunits (pi-3 and a2 / 4 / 6) that lack sigmoidal dose-response curves demonstrates subunit resolved affinity measurements directly at the a / y binding interface. Notably, the pentameric form of the GABA receptor binding flumazenil in whole mouse brain is composed primarily of al / 3 subunits, consistent with previous literature.40It is anticipated that Affinity Map in brain

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[0403] membrane isolates may be instrumental for profiling other neuroactive ligand-receptor interactions and their affinities.

[0404] Example 4. Pevtide-yrotein affinity profilins

[0405] Beyond drug-like small molecules, Affinity Map was next applied to profile the binding affinities of endogenous linear peptides and minimal binding motifs in proteins. Photocatalyst-peptide probes were prepared by reacting an iodoacetamide derivative of 1 and peptides bearing an appended N or C-terminal cysteine residue (Fig. 4A), and profiled affinities for each peptide in K562 cell lysate.

[0406] Affinity Map was first applied to a fragment of Smac, a mitochondrial protein that promotes cell death when present in the cytosol by binding inhibitor of apoptosis (IAP) proteins, leading to the release of active caspase enzymes. Using a 9 amino acid peptide (Fig.4B) as a minimal lAP-binding motif from Smac, Affinity Map identified IAP proteins XIAP and BIRC2 as the most significant interactions with affinities of 3.3 pM and 2.0 pM respectively. The measured binding affinities were comparable to reported data obtained in vitro with purified BIR domains and the same peptide (XIAP BIR3 / BIR2 domains: 0.43 pM / 6.0 pM).41As the workflow uses protein level enrichment, the obtained affinities represent an ensemble measurement of all peptide-BIR domain interactions within XIAP and BIRC2, each of which contain 3 unique BIR domains with different affinities for Smac.41Other IAPS, such as BIRC6, were not observed, which require multi-valent interactions with full-length Smac for binding.42Additionally, Affinity Map was performed for a 14 amino acid minimal binding motif from the protein SOS1 that binds the SH3 domain of GRB243,44GRB2 (Fig. 4C) was identified as a low affinity (5.6 pM) interactor of this peptide, consistent with a previous affinity measurement of 4 pM43

[0407] The binding affinities of several endogenous peptides (Fig. 4D-F) were also profiled: 1) a signal peptide fragment cleaved from the nascent HIV-1 gpl60 envelope protein during infection,452) a peptide translated from a noncanonical open reading frame (ncORF) from the SNHG6 long noncoding RNA (IncRNA),46and 3) a translated peptide from an upstream ORF (uORF) of the STAT1 gene.44

[0408] An affinity for a known interactor of the HIV-1 envelope protein was measured47and also measure the affinities of these three peptides to proteases that likely mediate - 46 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0409] their degradation. Unique proteases were identified for each peptide, consistent with their known cleavage sites (Fig. 23). As an example, the arginine rich peptide from the SNGH6 IncRNA ORF binds NRDC protease, which cleaves sites containing dibasic motifs.48This demonstrates the ability of Affinity Map to identify proteases relevant to the stability of endogenous peptides.

[0410] Example 5. Protein-protein and cyclic peptide-protein affinity measurement on live cell surfaces

[0411] It was hypothesized that Affinity Map would be compatible with live cells, enabling the measurement of ligand binding affinities for cell surface proteins in their native environment. Initially, it was sought to demonstrate affinity profiling on live cell surfaces using cyclic peptides, an emerging category of therapeutics and tool compounds.

[0412] The macrocyclic cRGDfK peptide is a high affinity integrin alpha-v beta-3 (aVps) antagonist that has been explored as a drug scaffold for glioma treatment.49In vitro assays for integrin-RGD peptide binding affinity and selectivity require purified integrins, and reported measurements using surface adhesion to live cells cannot discriminate between different integrin affinities.50,51A photocatalytic conjugate of cRGDfK was prepared by functionalizing the lysine residue with 1 via an amide linkage and treated live U87MG cells adhered to 6-well culture plates (16 plates, 96 wells) with 4 different probe concentrations and 12 concentrations of unmodified cRGDfK in complete media (Fig. 5A). Following irradiation, the cells were harvested and subjected to the Affinity Map workflow. Integrin P3 (ITGB3) had the most significant measurable binding affinity, with clear sigmoidal competition curves at multiple probe concentrations. These data represent the first measurement of cRGDfK selectivity and binding affinity (268 nM) in live cells. Beyond small molecules, carbene-based photocatalytic labeling methods have been previously used to identify the binding targets of proteins such as antibodies and the SARS-COV-2 spike protein on live cell surfaces.27,35In these experiments, labeling by photocatalyst-conjugated proteins is contrasted with a control lacking a photocatalyst targeting modality. It was reasoned that the Affinity Map platform could be generalized to profile the binding affinity of globular proteins to proteins on live cell surfaces. Currently, protein-protein affinities are measured using cell-based reporter protein systems or recombinant protein assays, and - 47 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0413] no published methods are available for proteome-wide protein-protein binding affinity measurement.

[0414] As a proof of concept, it was aimed to profile the binding affinities between human IgG and proteins on live THP-1 cell surfaces (Fig. 5B). 1-IgG was synthesized as a photocatalytic protein probe using purified IgG from human serum and an activated NHS ester prepared through a strain-promoted click ligation between 1-DBCO and NHS-PEG3-N3 and used unmodified IgG as a competitive non-photocatalytic ligand. Live cells were then incubated with a 96-sample matrix of 1-IgG / IgG concentrations (Fig. 27), irradiated the mixtures for 15 minutes, lysed the cells, and subjected the lysate to the standard Affinity Map sample preparation and data processing pipeline.

[0415] Binding affinities closely matching literature values were measured for the major IgG receptors expressed by THP-1 cells, including FCGR1 A (55 nM; lit. 15 nM), FCGRIIB (2.9 pM; lit. 8.3 pM) and a set of peptides matching to both FCGRIIA and FCGRIIC (432 nM; lit. 190 nM / 8.3 pM).52The IgG affinity of TRIM21, an intracellular Fc receptor (136 nM; lit. -200 nM),53was also measured and additional proteins that are not known to directly bind IgG were identified, such as the monocyte checkpoint inhibitor proteins LILRB2 and LILRB4 at lower significance. Notably, these two proteins have closely aligned affinity values with Fc receptors, suggesting that they may represent distal Fc receptor interactions. Together, these data validate the ability of Affinity Map to measure the affinities between globular proteins and cell surface receptors, which are important metrics in the development of therapeutic antibodies.

[0416] Example 6. Chemical synthesis and characterization

[0417] Reaction temperatures refer to heating / cooling media (heating block, oil bath, or cryogenic bath). After preparative HPLC purification, fractions containing the product were evaporated using a SPGenevac EZ-24.0. Final compounds were characterized by1H NMR,13C NMR,19F NMR (when applicable), and ESLMS. Retention times refer to analytical LC-MS chromatography, which was performed via HPLC-MS-ELSD using a 5%-95% solvent B gradient over 10 minutes, unless otherwise stated. Isolated molecules are reported with complete analytical characterization when not previously reported in literature. Commercially available reagents were purchased from Ambeed, Oakwood,

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[0419] Tokyo Chemical Industry, Sigma Aldrich, or Thermo Fisher Scientific.

[0420] Solvents:

[0421] Hexanes (VWR, ACS reagent), CH2Cl2 (VWR, ACS reagent), CHCI3 (Merck, > 98%), ethyl acetate (VWR, ACS reagent), methanol (Sigma- Aldrich, HPLC grade, > 99.8%), DMF (Oakwood, ACS reagent), DMSO (Alfa-Aesar, > 99.8%), MeCN (Sigma-Aldrich, HPLC grade, > 99.9%), acetone (VWR, ACS reagent), ethanol (KOPTEC, USP 200 proof) were used without additional purification. Anhydrous THF was purchased from Sigma-Aldrich (anhydrous, > 99.9%) and distilled over sodium / benzophenone, then stored in a Schlenk tube under argon in the presence of activated 4A molecular sieves.

[0422] NMR spectroscopy:

[0423] Experiments were performed at the CLC Nuclear Magnetic Resonance Core Facility at Weill Cornell Medicine using a Bruker Avance III HD 500 MHz equipped with a 5 mm CPTCI CryoProbe or an Agilent Varian INOVA 600 MHz with a 5 mm BB0600S3 probe. The following sequences were used:1H NMR: zg30;13C NMR: zgpg30;19F NMR: zg30. The following solvents were used: CDCI3 (Sigma- Aldrich, 99.8% d), H2O (Cambridge Isotope Laboratories, 99.9% d), DMSO-rfc (Cambridge Isotope Laboratories, 99.9% d + 0.05 v / v TMS), CD3OD (Cambridge Isotope Laboratories, 99.8% d). Spectra were analyzed using MestReNova 14.3 by applying standard baseline and phase correction methods. Chemical shifts (8) for ’H and13C NMR spectra are given in parts per million (ppm) relative to residual solvent peaks.19F NMR spectra were calibrated using an absolute referencing system, as suggested by IUPAC.1H and13C NMR multiplicities that can be analyzed as first order multiplets are reported using the following abbreviations (or combinations thereof): s = singlet, d = doublet, t = triplet, q = quartet, p = quintet, h =sextet; hept = heptet; m = multiplet, br = broad.

[0424] Flash column chromatography:

[0425] Purifications were performed using a Teledyne ISCO CombiFlash RF+Lumen system equipped with reusable cartridges loaded with Cleanert Silica (particle size: 40 -

[0426] - 49 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0427] 60 pm, pore size: 60 A). Column volumes (CV) are noted which scale according to appropriate cartridge size.

[0428] HPLC purification details:

[0429] General method development:

[0430] For all HPLC analysis and purification a Waters AutoPurification System with 2424-ELS Detector, 2998-Photodiode Array Detector and SQ Detector 2 was used. All gradients begin at 5% solvent B and hold this for 30 seconds during compound loading. The gradient then increases to the specified percentage solvent B over the specified time window. For isocratic methods, the percentage solvent B is reached in 90 seconds using a linearly increasing gradient and held for the specified duration of the run. Sample sandwiching refers to the use of a specified volume of a solvent before and after sample uptake by the syringe directly prior to injection in order to prevent hydrophobic compound precipitation during column loading.

[0431] Solvent A: FBCkFormic acid (999:1)

[0432] Solvent B: MeCN: Formic acid (999:1)

[0433] Preparative column: Xbridge BEH C18 OBD Prep Column, 130A, 5 pm, 19 mm x 150 mm, 186002979

[0434] Preparative flow rate: 20 mL / min

[0435] Semi-preparative column: Xbridge BEH C18 OBD Prep Column, 130A, 5 pm, 10 mm x 150 mm, 186008165

[0436] Semi-preparative flow rate: 10 mL / min

[0437] Analytical column: Xbridge C18 column, 130A, 5 inn, 4.6 x 159 mm, 186003116 Analytical flow rate: 1.4 mL / min

[0438] Cbz-CI _ / x. _ -O.. _, _

[0439] HO O NH2NaHCOsHoO NHCbz

[0440]

[0441] 1:1 THF / H2O - 50 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0442] Benzyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate (6). The reaction was performed according to a modified literature procedure29A. A round-bottom flask equipped with a PTFE-coated stir bar was charged with amino-PEG4- alcohol (2.8 mL, 15.49 mmol, 1.0 equiv.), sodium bicarbonate (1.952 g, 23.24 mmol, 1.5 equiv.), and H2O (31 mL). The mixture was stirred at room temperature until homogeneous, then cooled to 0 °C for 10 minutes. Separately, benzyl chloroformate (2.65 mL, 18.59 mmol, 1.5 equiv.) was dissolved in THF (31 mL) and this solution was added dropwise to the amine solution with vigorous stirring at 0 °C. The reaction was stirred at 0 °C for 1 hour, then allowed to warm to room temperature and stirred overnight. Solvent was removed and the crude mixture was redissolved in EtOAc: H2O (1:1, 150 mL). The aqueous layer was extracted 3x with EtOAc (75 mL), and the EtOAc extracts washed with brine, dried with Na2SC>4, and concentrated. The oil was diluted in DCM and purified by flash column chromatography on silica (hexanes: EtOAc = 0% to 100% over 10 CVs) to afford the title compound as a clear oil (2.709 g, 53%). Spectral data are consistent with existing literature 29A

[0443] N=N

[0444] DIPEA

[0445]

[0446] DMF

[0447] 4-((2-(3-(But-3-yn-l-yl)-3H-diazirin-3-yl)ethyl)amino)-4-oxobutanoic acid (Carboxyl-Dz-Alkyne)

[0448] A scintillation vial equipped with a PTFE-coated stir bar was charged with amino diazirine alkyne (0.021 g, 0.15 mmol, 1.0 equiv.) and succinic anhydride (0.017 g, 0.165 mmol, 1.1 equiv.). The mixture was dissolved in DMF (1 mL). After stirring for 5 minutes DIPEA (0.130 mL, 0.75 mmol, 5 equiv.) was added and the reaction was stirred overnight at room temperature in the dark. The reaction was purified by semipreparative HPLC using a 20% -60% solvent B over 10-minute method. The product eluted at 3.6 minutes and solvent was removed to afford the title compound. (12.2 mg, 34%).

[0449] - 51 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0450] 1H NMR (500 MHz, CDCI3) 56.23 (s, 1H), 3.09 (q, J = 6.6 Hz, 2H), 2.58 (dt, J = 89.5, 6.6 Hz, 4H), 2.05 - 1.97 (m, 3H), 1.72 - 1.61 (m, 4H).

[0451] 13C NMR (126 MHz, CDCI3) 8 172.54, 82.89, 77.41, 77.16, 76.91, 69.59, 34.62, 32.52, 32.19, 31.03, 26.93, 13.33.

[0452] LC-MS Rt= 4.68 min; m / z calcd. For C11H15N3O3 ([M+H]+) = 238.1, found 238.1

[0453]

[0454] Ir[dF(CF3)(OBN)ppy] Dimer (15)

[0455] An oven-baked scintillation vial equipped with a PTFE-coated stir bar was charged with

[0456] 14 (0.1 g, 0.256 mmol, 2.5 equiv.), 2,4,6-Trimethylpyridine (0.050 g, 0.410 mmol, 4 equiv.) and iridium(III) chloride hydrate (0.032 g, 0.103 mmol, 1.0 equiv.). To the mixture 2-ethoxyethanol (4.364 mL, 23.5 mM) was added and the vial was placed under an argon atmosphere by degassing the solvent. The vial was sealed and heated to 120 °C and stirred for 16 hours. The reaction was cooled to room temperature and carried forward without further purification.

[0457] Ir[dF(CF3)(OBN)ppy]bipy-NHCbz complex (1)

[0458] The scintillation vial from the previous reaction containing 15 in 2-ethoxyethanol was charged with 9 (0.690 g, 0.123 mmol, 1.2 equiv.), and silver hexafluorophosphate (0.129 g, 0.513 mmol, 5.0 equiv.). The mixture was heated to 60 °C and stirred for 16 hours. The reaction was cooled to room temperature, filtered over celite, washed with MeOH / acetone (1: 1, 70 mL) and the solvent was removed. The crude mixture was purified by flash column chromatography on silica (DCM: MeOH, 0% to 25% over 35 CVs) to afford the title compound as yellow solid (0.152 g, 69% over 2 steps).

[0459] - 52 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0460] 1H NMR (500 MHz, DMSO) 5 9.06 (s, 1H), 8.58 (s, 1H), 8.50 (br s, 1H), 8.35 (s, 2H), 8.03 (s, 1H), 7.86 (s, 1H), 7.69 (s, 1H), 7.62 (s, 1H), 7.55 (s, 1H), 7.39 - 7.27 (m, 6H), 7.23 (t, J = 5.8 Hz, 1H), 7.12 - 7.00 (m, 1H), 6.69 (s, 1H), 5.98 - 5.54 (m, 2H), 4.98 (s, 3H), 4.83 (t, J = 5.1 Hz, 2H), 4.50 (s, 2H), 3.80 (t, J = 4.4 Hz, 3H), 3.64 - 3.46 (m, 16H), 3.12 (q, 7 = 5.9 Hz, 4H).

[0461] 19F NMR (471 MHz, DMSO) 5 -59.69 (s, 6F), -102.68 (t, 7 = 97.4 Hz, 2F), -105.68 - -106.46 (m, 2F).

[0462] LC-MS Rt= 5.34 min; m / z calcd. For CeoHssFioIrNyOisS ([M+H]+) = 1532.3, found 1532.6

[0463]

[0464] 2-(2,4-Difluorophenyl)-N, N-bis(2-hydroxyethyl)-5-(trifluoromethyl)isonicotinamide (14, dF(CF3)(OBN)ppy).

[0465] A scintillation vial equipped with a PTFE-coated stir bar was charged with 13 (4.6 g, 11.7 mmol, 1.0 equiv.) and bis(hydroxyethyl)amine (5.6 mL, 58.5 mmol, 5.0 equiv.). The mixture was heated to 70 °C and stirred for 16 hours. The reaction was cooled to room temperature and partitioned between EtOAc and water, and the aqueous layer acidified to pH 7 with HC1 (1 M). The aqueous layer was extracted 3x with EtOAc (100 mL) and the EtOAc extracts were washed with brine, dried over Na2SO4, and concentrated to remove solvent. The residue was purified by flash column chromatography on silica (DCM: MeOH = 0% to 10% over 8 CVs) to afford the title compound as an off-white powder (4.04 g, 88%).

[0466] 1H NMR (500 MHz, CDCl3) 8 9.00 (s, 1H), 8.10 (td, 7 = 8.8, 6.5 Hz, 1H), 7.84 (s, 1H), 7.04 (td, 1H), 6.93 (ddd, 7 = 11.3, 8.6, 2.5 Hz, 1H), 4.19 - 3.94 (m, 2H), 3.90 - 3.75 (m, 2H), 3.66 (dt, 7= 10.6, 4.5 Hz, 2H), 3.31 - 3.16 (m, 2H), 2.71 (br s, 1H).

[0467] 13C NMR (126 MHz, CDCI3) 8 168.07, 165.23, 165.13, 163.21, 163.12, 162.09, 161.99, 160.07, 159.97, 156.21, 147.85, 147.81, 147.77, 147.74, 143.26, 143.24, 132.57, 132.54,

[0468] - 53 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0469] 132.49, 132.46, 126.51, 124.33, 122.16, 122.07, 121.89, 121.86, 121.80, 121.77, 121.03, 120.77, 120.51, 120.25, 112.55, 112.52, 112.38, 112.35, 104.95, 104.74, 104.54.

[0470] 19F NMR (471 MHz, CDCl3) 5 -59.77 (s, 3F), -106.08 - -106.28 (m, IF), -111.48 (q, J = 9.7 Hz, IF).

[0471] LC-MS Rt= 5.11 min; m / z calcd. For C17H15F5N2O3 C17H15F5N2O3([M+H]+) = 391.1, found 391.5

[0472] H2, Pd / C MeOH

[0473]

[0474] 1-NH2

[0475] A scintillation vial equipped with a PTFE-coated stir bar was charged with 1 (0.154 g, 0.1 mmol, 1.0 equiv.) and 10% Pd / C, type 487 (1 mg) and suspended in MeOH (1 mL). The mixture was placed under H2 atmosphere using a balloon and stirred at room temperature for 16 hours. The mixture was filtered over celite and the filter cake was washed with MeOH (50 mL). Solvent was removed to afford the title compound which was carried forward without additional purification.

[0476] AgPF68:1 Acetone / H20 16h, 60SC

[0477]

[0478] I-CO2H

[0479] - 54 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0480] A scintillation vial equipped with a PTFE-coated stir bar was charged with 15 (0.788 g, 0.39 mmol, 1.0 equiv.), 11 (0.482 g, 0.86 mmol, 2.2 equiv.), and silver hexafluorophosphate (0.297 g, 1.17 mmol, 3.0 equiv.). Acetone: H2O (8:1, 7.8 mL) was then added, the mixture heated to 60 °C, and stirred at this temperature for 16 hours. The reaction was cooled to room temperature, filtered, and the solvent was removed. The crude mixture was purified by flash column chromatography on silica (EtOAc: MeOH (+0.1% formic acid) = 0% to 35% over 7 CVs) to afford the title compound as yellow solid (0.62 g, >95%).

[0481] 'H NMR (500 MHz, DMSO) 8 9.05 (s, 1H), 8.58 (s, 1H), 8.34 (s, 2H), 8.11 - 7.48 (m, 7H), 7.29 (s, 1H), 7.05 (ddt, J= 12.1, 9.5, 2.5 Hz, 2H), 5.75 (dd, J= 104.6, 41.1 Hz, 2H), 5.16 - 4.62 (br m, 4H), 4.58 - 4.46 (m, 2H), 3.84 - 3.74 (m, 4H), 3.61 - 3.55 (m, 6H), 3.52 (dd, J = 5.9, 3.7 Hz, 2H), 3.49 - 3.42 (m, 8H), 3.14 (q, J = 5.9 Hz, 4H), 2.59 (t, J = 5.7 Hz, 2H), 2.37 - 2.21 (m, 6H).

[0482] 19F NMR (471 MHz, DMSO) 8 -59.69 (s, 6F), -102.16 - -103.22 (m, 2F), -105.47 - - 106.60 (m, 2F).

[0483] LC-MS Rt= 4.68 min; m / z calcd. For CseHseFwIrNvOieS ([M+H]+) = 1498.3, found 1498.8

[0484]

[0485] 1-DBCO

[0486] A scintillation vial equipped with a PTFE-coated stir bar was charged with I-NH2 (0.025 g, 0.018 mmol, 1.0 equiv.), dibenzocyclooctyne-A-hydroxysuccinimidyl ester (DBCO- NHS, 8.0 mg, 0.021 mmol, 1.2 equiv.), and dissolved in DMF (0.4 mL). The reaction was stirred at room temperature for 5 minutes and DIPEA (0.03 mL, 0.09 mmol, 5.0 equiv.) was added. After 16 hours the reaction mixture was diluted with MeOH / H2O ( 1: 1, 0.4 mL) and purified by preparative HPLC using a 45% solvent B 15-minute isocratic method. The

[0487] - 55 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0488] product eluted at 4.25 minutes and was concentrated to afford the title compound as a yellow solid (4.8 mg, 16%).

[0489] 1H NMR (598 MHz, DMSO) 89.00 (dd, J = 35.7, 20.5 Hz, 1H), 8.65 - 8.25 (m, 4H), 8.00 - 7.91 (m, 1H), 7.85 (d, J = 24.9 Hz, 3H), 7.71 (d, J = 16.4 Hz, 3H), 7.59 (d, J = 33.4 Hz, 4H), 7.46 (s, 1H), 7.31 (s, 3H), 6.90 (d, J = 10.9 Hz, 2H), 6.27 (br, s, 1H), 6.13 (s, 1H), 5.60 - 5.39 (m, 1H), 4.71 (br, s, 3H), 4.52 (s, 2H), 3.90 - 3.35 (m, 34H).

[0490] 19F NMR (563 MHz, DMSO) 8 -61.02 - -62.06 (m, 6F), -95.36 - -97.44 (m, 2F), -102.30 - -103.82 (m, 2F).

[0491] LC-MS Rt= 5.54 min; m / z calcd. For CviHesFioIrNsOisS ([M+H]+) = 1685.4, found 1685.6

[0492]

[0493] 1-IAA

[0494] A scintillation vial equipped with a PTFE-coated stir bar was charged with I-NH2 (0.22 g, 0.16 mmol, 1.0 equiv.) and dissolved in l,4-dioxane: H2O (3:1, 2.5 mL). The solution was stirred at room temperature for 5 minutes then cooled to 0 °C and NaOH (1 M solution, 0.25 mL, 0.1 M final concentration) was added. Separately, iodoacetic anhydride (0.12 g, 0.35 mmol, 2.2 equiv.) was dissolved in dichloroethane (0.2 mL). The iodoacetic anhydride solution was added dropwise to the I-NH2 solution at 0 °C with vigorous stirring in 2 additions of 200 pL. The reaction was stirred at 0 °C for 10 minutes in the dark then warmed to room temperature for 50 minutes. Solvent was removed and the residue was redissolved in DMSO: H2O (9:1, 1 mL). The mixture was purified by preparative HPLC using a 30% solvent B 10-minute isocratic method. The product eluted at 6.0 minutes and was concentrated to afford the title compound as a yellow solid (56.3 mg, 23%).

[0495] Note: The iodoacetamide containing product is light and temperature sensitive, and should be stored at < -20 °C in the dark.

[0496] - 56 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0497] 1H NMR (598 MHz, CD3OD_SPE) 59.06 - 8.91 (m, 1H), 8.60 (ddd, J = 18.2, 8.0, 5.2 Hz, 1H), 8.51 (tt, J = 11.0, 2.7 Hz, 1H), 8.45 - 8.34 (m, 1H), 8.26 - 8.06 (m, 2H), 8.05 - 7.64 (m, 5H), 7.30 (ddq, J = 10.8, 6.1, 2.7 Hz, 1H), 6.84 - 6.70 (m, 2H), 6.15 (dddd, J = 31.9, 24.7, 7.6, 2.7 Hz, 1H), 5.78 - 5.54 (m, 1H), 4.51 (td, J = 7.2, 3.3 Hz, 2H), 3.99 - 3.46 (m, 28H), 3.36 (ddd, J = 13.6, 6.0, 3.0 Hz, 2H).

[0498] 19F NMR (563 MHz, CD3OD_SPE) 8 -63.76 - -64.92 (m, 6F), -97.41 - -98.03 (m, 2F), - 103.19 - -104.31 (m, 2F).

[0499] LC-MS Rt= 4.77 min; m / z calcd. For C H JFIOII'NVO^S ([M+H]+) = 1566.2, found 1566.3

[0500]

[0501] 1-Dasatinib

[0502] A scintillation vial equipped with a PTFE-coated stir bar was charged with I-CO2H (4.25 mg, 0.003 mmol, 1.0 equiv.), deshydroxyethyl-dasatinib (382 uE of a 0.01 M solution in DMF, 1.69 mg, 0.0039 mmol, 1.1 equiv.), PyBOP (5.4 mg, 0.01 mmol, 3.0 equiv.), and DMF (0.2 mF). The solution was stirred at room temperature for 5 minutes, then DIPEA (4.83 uE, 0.028 mmol, 8 equiv.) was added and the reaction stirred for 16 hours. The reaction mixture was diluted with MeCN / H2O (1:1, 0.2 mL) and purified by semipreparative HPLC using a 5%-60% solvent B 10 minute method. The product eluted at 4.8 minutes and was concentrated to afford the title compound as a yellow solid (1.7 mg, 30%).

[0503] 1H NMR (500 MHz, DMSO) 8 11.51 (s, 1H), 9.88 (s, 1H), 9.07 (s, 1H), 8.60 (s, 1H), 8.39 (d, J = 32.7 Hz, 2H), 8.22 (s, 1H), 8.03 (s, 1H), 7.87 (t, J = 5.7 Hz, 1H), 7.70 (s, 1H), 7.63 (s, 1H), 7.55 (s, 1H), 7.40 (dd, J = 7.7, 1.8 Hz, 1H), 7.33 - 7.20 (m, 3H), 7.07 (ddt, J = 12.1, 9.4, 2.5 Hz, 2H), 6.07 (s, 1H), 5.77 (d, J = 103.5, 42.3 Hz, 3H), 4.90 (d, J = 86.1 Hz, 3H), 4.63 - 4.39 (m, 2H), 3.95 - 3.77 (m, 3H), 3.66 - 3.48 (m, 22H), 3.37 (d, J = 6.0 Hz,

[0504] - 57 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0505] 5H), 3.17 (d, J = 5.8 Hz, 3H), 2.55 (t, J = 7.2 Hz, 2H), 2.42 (s, 3H), 2.38 - 2.29 (m, 2H), 2.24 (s, 3H).

[0506] 19F NMR (471 MHz, DMSO) 5 -59.68 (s, 6F), -102.70 (t, 2F), -106.09 (q, J = 69.6 Hz, 2F).

[0507] LC-MS Rt= 5.39 min; m / z calcd. For CTeHyeClFioTrNwOieSz ([M+H]+) = 1923.4, found 1923.7

[0508]

[0509] l-(+)-JQl

[0510] A scintillation vial equipped with a PTFE-coated stir bar was charged with I-NH2 (0.04 g, 0.029 mmol, 1.0 equiv.), (+)-JQl-COOH (0.012 g, 0.031 mmol, 1.1 equiv.), PyBOP (0.045 g, 0.086 mmol, 3.0 equiv.), and DMF (0.6 mL). The solution was stirred at room temperature for 5 minutes, then DIPEA (0.04 mL, 0.23 mmol, 8 equiv.) was added and the reaction stirred for 16 hours. The reaction mixture was diluted with H2O (0.2 mL) and purified by semi-preparative HPLC using a 35% solvent B 25-minute isocratic method.

[0511] The product eluted at 5.3 minutes and solvent was removed to afford the title compound as a yellow solid (3 mg, 6%).

[0512] 1H NMR (500 MHz, CD3OD_SPE) 88.97 (d, J = 5.8 Hz, 1H), 8.50 (s, 2H), 8.40 (dd, J = 5.8, 2.6 Hz, 1H), 8.18 (s, 1H), 7.86 (d, J = 97.1 Hz, 4H), 7.42 (ddd, J = 33.5, 8.6, 3.2 Hz, 4H), 7.30 (d, J = 17.5 Hz, 1H), 6.79 (q, J = 10.2 Hz, 2H), 5.92 (d, J = 101.5 Hz, 2H), 4.58 (s, 2H), 4.56 - 4.45 (m, 2H), 3.97 - 3.37 (m, 30H), 2.66 (s, 3H), 2.44 (s, 3H), 1.69 (s, 3H).

[0513] 19F NMR (563 MHz, CD3OD_SPE) 8 -61.75 (d, J = 11.6 Hz, 6F), -103.57 (d, J = 153.1 Hz, 2F), -107.26 (s, 2F).

[0514] - 58 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0515] LC-MS Rt= 5.46 min; m / z calcd. For C7iH67ClFioIrNiiOi4S2 ([M+H]+) = 1780.3, found 1780.7

[0516]

[0517] 1-cRGDFK

[0518] A scintillation vial equipped with a PTFE-coated stir bar was charged with I-CO2H (0.007 g, 0.005 mmol, 1.0 equiv.), PyBOP (0.002 g, 0.005 mmol, 1.0 equiv.), and in DMF (0.25 mL). The solution was stirred at room temperature for 5 minutes, then DIPEA (0.007 mL, 0.038 mmol, 8 equiv.) was added. After 35 minutes, cRGDFK (0.003 g, 0.005 mmol, 1.1 equiv.) dissolved in DMF (0.12 mL) was added dropwise and the reaction was stirred at room temperature for 16 hours. The reaction mixture was then diluted with H2O (0.2 mL) and purified by semi-preparative HPLC using a 22% solvent B 25 minute isocratic method. The product eluted at 18.4 minutes and solvent was removed to afford the title compound as a yellow solid (0.5 mg, 5%).

[0519] - 59 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0520] LC-MS Rt= 4.65 min; m / z calcd. For CssHgsFioIrNieChzS ([M+2H]2+) = 1043.2, found 1043.2

[0521]

[0522] 2-CO2H

[0523] A flame-dried schlenk flask equipped with a PTFE-coated stir bar was charged with 2-(2,4-difhrorophenyl)-5-(trifhroromethyl)pyridine (2 g, 7.72 mmol, 2.1 equiv.) and iridium(III) chloride hydrate (1.095 g, 3.677 mmol, 1.0 equiv.). The flask was placed under an argon atmosphere and 2-ethoxyethanol: H2O (2:1, 74 mL) was added. The mixture was heated to 130 °C and stirred for 16 hours. The reaction was cooled to room temperature, and quenched with cold water (400 mL) to afford a precipitate. The precipitate was filtered and washed with excess cold water, then dried to afford the title compound as a bright orange powder which was carried forward without further purification. (2.590 g, 47%).

[0524] Dimer Crack

[0525] A scintillation vial equipped with a PTFE-coated stir bar was charged with the dimeric intermediate (0.055 g, 0.037 mmol, 1.0 equiv.), bipy-Me-PEGa-CChH (0.038 g, 0.074 mmol, 2 equiv.), and silver hexafluorophosphate (0.028 g, 0.111 mmol, 3.0 equiv.).

[0526] Acetone (0.74 mL) was added, and the mixture was heated to 60 °C and stirred for 16 hours. The reaction was cooled to room temperature, filtered to remove insoluble salts, and the solvent was removed. The crude mixture was dissolved in DMSO (1.2 mL) and purified by preparative HPLC using a 35%-95% solvent B gradient over 15 minutes with 75 pL MeCN sample sandwiching during injection. Solvent was removed to afford the title compound as a yellow solid (25 mg, 28%).

[0527] 1H NMR (598 MHz, CDCl3) 88.65 (d, J = 20.0 Hz, 2H), 8.48 (ddd, J = 18.5, 8.7, 3.0 Hz, 2H), 8.07 - 8.01 (m, 2H), 7.77 (dd, J = 25.2, 5.6 Hz, 2H), 7.60 (s, 1H), 7.51 (d, J = 6.2 Hz, 2H), 7.35 (d, J = 5.6 Hz, 1H), 7.07 (br, s, J = 10.5 Hz, 1H), 6.93 (br, s, J = 7.3 Hz, 1H),

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[0529] 6.64 (ddt, J = 12.2, 9.3, 3.1 Hz, 2H), 5.63 (dd, J = 6.8, 4.0 Hz, 2H), 3.68 - 3.59 (m, 9H), 3.54 (dd, J = 9.3, 4.9 Hz, 4H), 3.40 (dt, J = 20.2, 5.7 Hz, 4H), 3.22 - 3.14 (m, 2H), 2.75 (td, J= 7.5, 4.5 Hz, 2H), 2.67 (s, 3H), 2.61 - 2.50 (m, 4H).

[0530] 13C NMR (150 MHz, CDCl3) 8 174.34, 173.90, 168.22, 168.06, 165.90, 164.24, 164.16, 162.44, 161.93, 155.48, 155.32, 154.97, 154.75, 154.64, 154.59, 150.00, 149.33, 145.07, 136.67, 129.94, 129.76, 127.22, 126.48, 125.94, 124.02, 123.88, 123.83, 123.69, 122.66, 122.59, 120.85, 120.78, 114.33, 114.28, 114.21, 114.16, 100.22, 100.11, 77.37, 77.16, 76.95, 70.66, 70.51, 70.31, 69.37, 39.84, 39.60, 35.54, 31.44, 30.89, 30.82, 28.30, 21.56, 21.41, 0.12.

[0531] 19F NMR (563 MHz, CDCl3) δ -62.77 (s, 3F), -62.84 (s, 3F), -71.85 (d, J = 713.4 Hz, 5F), -101.40 (dd, J = 68.5, 12.8 Hz, 2F), -105.84 (dd, J = 78.8, 12.5 Hz, 2F).

[0532] LC-MS Rt= 3.63 min (70%-95% B over 10 minutes); m / z calcd. For Cso UeFioIrNeO? ([M+H]+) = 1226.3, found 1226.8

[0533]

[0534] 2-Dasatinib

[0535] A scintillation vial equipped with a PTFE-coated stir bar was charged with 2-CO2H (12.6 mg, 0.010 mmol, 1.0 equiv.), des-hydroxyethyl-dasatinib 1.131 mL, 0.01 M solution in DMF, 5.01 mg, 0.011 mmol, 1.1 equiv.), PyBOP (16.1 mg, 0.031 mmol, 3.0 equiv.), and DMF (0.2 mL). The solution was stirred at room temperature for 5 minutes, then DIPEA (14.33 uL, 0.082 mmol, 8 equiv.) was added and the reaction stirred for 16 hours. The

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[0537] reaction mixture was diluted with MeCN / H2O (1:1, 0.5 mL) and purified by semipreparative HPLC using a 55% solvent B 10 minute isocratic method. The product eluted at 3.2 minutes, and solvent was removed to afford the title compound as a yellow solid (4.6 mg, 27%).

[0538] 'H NMR (598 MHz, DMSO) 8 11.53 (br, s, 1H), 9.89 (s, 1H), 8.81 (s, 2H), 8.49 - 8.39 (m, 4H), 8.22 (s, 1H), 7.99 (t, 7 = 4.5 Hz, 1H), 7.88 (q, 7 = 5.8 Hz, 1H), 7.82 (dd,7= 10.1, 5.7 Hz, 2H), 7.65 (s, 1H), 7.58 (ddd, 7 = 10.4, 5.8, 1.6 Hz, 2H), 7.52 (s, 1H), 7.40 (d, 7 = 7.7 Hz, 1H), 7.27 (dt, 7 = 15.4, 7.6 Hz, 2H), 7.06 (ddd, 7 = 12.2, 9.4, 2.4 Hz, 2H), 6.78 (br, s, 2H), 6.07 (s, 1H), 5.77 (ddd, 7 = 8.6, 6.6, 2.3 Hz, 2H), 3.63 - 3.40 (m, 15H), 3.16 (dd, 7 = 7.6, 4.0 Hz, 3H), 3.06 (t, 7 = 7.6 Hz, 2H), 2.57 (s, 4H), 2.56 - 2.53 (m, 1H), 2.41 (s, 3H), 2.34 (t, 7 = 7.0 Hz, 2H), 2.24 (s, 3H).

[0539] 19F NMR (563 MHz, DMSO) 8 -61.57 (d, 7 = 56.2 Hz, 6F), -70.15 (d, 7 = 711.2 Hz, 3F), -103.31 (dd, 7 = 22.9, 11.8 Hz, 2F), -106.78 (d, 7= 12.1 Hz, 2F).

[0540] LC-MS Rt= 6.15 min; m / z calcd. For CvoHeeClFioIrNisOyS ([M+2H]2+) = 826.2, found 826.4

[0541] Carboxyl-Dz-Alkyne PyBOP DIPEA DMF

[0542]

[0543] Dasastinib-dz-alkyne

[0544] A scintillation vial equipped with a PTFE-coated stir bar was charged with carboxyldiazirine- alkyne (0.012 g, 0.051 mmol, 1.0 equiv.), PyBOP (0.080 g, 0.154 mmol, 3.0 equiv.), and DMF (0.264 mL). The solution was stirred at room temperature for 5 minutes then DIPEA (0.072 mL, 0.411 mmol, 8 equiv.) was added. After 25 minutes deshydroxyethyl-dasatinib (0.025 g, 0.057 mmol, 1.1 equiv.) dissolved in DMF (0.25 mL) was added dropwise. The reaction was stirred at room temperature for 16 hours in the dark.

[0545] The reaction mixture was purified by semi-preparative HPLC using a 30%-95% solvent B over 10 minutes method. The product eluted at 4.5 minutes and solvent was removed to afford the title compound (19 mg, 56%).

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[0547] 1H NMR (500 MHz, DMSO) 5 11.51 (s, 1H), 9.88 (s, 1H), 8.22 (s, 1H), 7.85 (t, J = 5.6 Hz, 1H), 7.40 (dd, J = 7.7, 1.8 Hz, 1H), 7.30 - 7.22 (m, 2H), 6.06 (s, 1H), 3.62 - 3.52 (m, 8H), 2.91 (q, J = 7.0 Hz, 2H), 2.83 (t, J = 2.6 Hz, 1H), 2.58 (t, J = 7.1 Hz, 2H), 2.42 (s, 3H), 2.33 (t, J = 7.1 Hz, 2H), 2.24 (s, 3H), 1.99 (td, J = 7.4, 2.6 Hz, 2H).

[0548] 13C NMR (126 MHz, DMSO) 8 171.34, 170.11, 165.20, 162.52, 162.23, 159.90, 156.96, 140.82, 138.81, 133.51, 132.42, 129.03, 128.18, 127.00, 125.73, 83.17, 82.75, 71.77, 44.04, 43.43, 43.19, 40.58, 40.11, 40.02, 39.94, 39.85, 39.78, 39.69, 39.61, 39.52, 39.44, 39.35, 39.19, 39.02, 33.58, 32.07, 31.35, 30.37, 27.82, 27.22, 25.58, 18.30, 12.68.

[0549] LC-MS Rt= 5.75 min; m / z calcd. For C31H35CIN10O3S ([M+H]+) = 663.2, found 663.4

[0550] Purification of peptide ligands lacking N or C-terminal cysteines:

[0551] Peptides were purchased as crude mixtures from GenScript at 9 mg scales. The lyophilized powders were dissolved in 100 pL DMSO. 50 pL of this solution was purified using semipreparative HPLC using a 20-60% B over 10 minutes method, and solvent was removed to afford pure peptides for competitive labeling experiments. Purity was validated by HPLC-MS-ELSD using a 5%-95% solvent B over 6 minutes method.

[0552]

[0553] Peptide-photocatalyst conjugation:

[0554] Peptides containing terminal cysteines were purchased as crude mixtures from GenScript at 9 mg scales. The lyophilized powders were dissolved in 100 pL DMSO. 25 pL of this solution was transferred to a 1.5 mL centrifuge tube. To this tube was added DMSO (25 pL), H2O (45 pL) and TCEP (5 pL, 100 mM in H2O, final concentration 5 mM). The mixture was sonicated for 10 minutes at room temperature to dissolve solids and reduce disulfide bonds. 1-IA (1 mg, 50 pL of 12.5 mM solution in DMSO) was added

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[0556] followed by carbonate / bicarbonate buffer (100 mM, pH = 10.6, 50 pL). The reactions were then inverted in the dark at 37 °C for 16 hours. Conjugated peptides were isolated using an appropriate semipreparative HPLC-MS method. Products were concentrated in 1.5 mL centrifuge tubes under vacuum then redissolved in 100 p L DMSO for characterization and purity analysis via HPLC-MS-ELSD using a 5%-95% solvent B over 6 minutes method. Concentrations of the resulting stock solutions were determined by absorbance at 400 nm.

[0557] Table 1. Peptide sequence and preparation conditions.

[0558] Catalyst

[0559] Peptide Conjugate or HPLC Retention Molecular Mass(es)

[0560] Name

[0561] Sequence Unconjugate Gradient Time (min.) Weight Observed d

[0562] SI-Pep-1 / 20%-50% [M+H]+= AVPIAQKSE

[0563] Smac solvent B 942.9 (SEQ ID NO: Unconjugated 1.96 942.07

[0564] peptide over 10 [M+2H]2+= 1)

[0565] min. 472.2 AVPIAQKSE 20%-50% [M+2H]2+=

[0566] SI-Pep-2 /

[0567] C solvent B 2484.21 1242.4

[0568] 1-Smac Conjugate 3.20

[0569] (SEQ ID NO: over 10 [M+3H]3+= peptide

[0570] 2) min. 828.6 MRVKEKYQ HLWRWGW 35%-95% [M+4H]4+=

[0571] SI-Pep-3 /

[0572] RWGTMLLG solvent B 755.8

[0573] HIV-1 gp160 Unconjugated 5.42 3018.5 (SEQ ID NO: over 10 [M+5H]5+=

[0574] SPF

[0575] 3) min. 604.9 MRVKEKYQ HLWRWGW 20%-50%

[0576] SI-Pep-4 /

[0577] RWGTMLLG solvent B [M+6H]6+=

[0578] 1 -HIV-1 Conjugate 3.87 4559 C over 10 728.8 gp160 SPF

[0579] (SEQ ID NO: min.

[0580] 4)

[0581] MPVWWRRR

[0582] 5%-95% [M+6H]6+= RLRARSWAL SI-Pep-5 /

[0583] solvent B 3231.89 539.7 RGARKPLR SNHG6 Unconjugated 2.71

[0584] over 10 [M+7H]7+= (SEQ ID NO: IncRNA ORF

[0585] min. 462.7 5)

[0586] CMPVWWR 20%-60%

[0587] SI-Pep-6 /

[0588] RRRLRARSW solvent B 4774.03 [M+7H]7+=

[0589] 1- SNHG6 Conjugate 2.41

[0590] ALRGARKPL over 10 682.9

[0591] IncRNA ORF

[0592]

[0593] R min.

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[0595] (SEQ ID NO:

[0596] 6)

[0597] EVPVPPPVP

[0598] 20%-50% [M+2H]2+= PRRRP SI-Pep-7 / solvent B 797.4 (SEQ ID NO: Unconjugated 2.19 1592.89

[0599] SOS1 over 10 [M+3H]3+= 7)

[0600] min. 532.0

[0601] [M+3H]3+= EVPVPPPVP 5%-95% 1046.0 PRRRPC SI-Pep-8 / solvent B [M+4H]4+= Conjugate 3.14 3135.03

[0602] (SEQ ID NO: 1-SOS1 over 10 784.64 8) min. [M+5H]5+=

[0603] 628.0 MPSSRAV 20%-60% [M+H]+= (SEQ ID NO: SI-Pep-9 / solvent B 747.7 Unconjugated 2.01 746.88

[0604] 9) STAT1 uORF over 10 [M+2H]2+= min. 374.8 20%-60%

[0605] MPSSRAVC SI-Pep-10 /

[0606] solvent B [M+2H]2+= (SEQ ID NO: 1-STAT1 Conjugate 3.34 2289.02

[0607] over 10 1145.5 10) uORF

[0608]

[0609] min.

[0610] Dimethyl-BiPy AgPF6Acetone 16h, 609C

[0611]

[0612] Ir[dF(CF3)ppy]2(dMebipy)PF6(16)

[0613] A flame-dried schlenk flask equipped with a PTFE-coated stir bar was charged with 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine (2 g, 7.72 mmol, 2.1 equiv.) and iridium(III) chloride hydrate (1.095 g, 3.677 mmol, 1.0 equiv.). The flask was placed under an argon atmosphere and 2-ethoxy ethanol: H2O (2:1, 74 mL) was added. The mixture was heated to 130 °C and stirred for 16 hours. The reaction was cooled to room temperature, quenched with cold water (300 mL), affording a precipitate. The precipitate was filtered and washed with excess cold water, then dried to afford the title compound as a bright orange powder which was carried forward without further purification. (2.590 g, 47%).

[0614] A scintillation vial equipped with a PTFE-coated stir bar was charged with the dimeric intermediate (0.060 g, 0.040 mmol, 1.0 equiv.), 4,4’ -dimethyl-2, 2’ -bipyridine

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[0616] (0.016 g, 0.009 mmol, 2.2 equiv.), and silver hexafluorophosphate (0.012 g, 0.031 mmol, 3.0 equiv.). Acetone (0.8 mL) was added, the mixture was heated to 60 °C, and subsequently stirred for 16 hours. The reaction was cooled to room temperature, filtered to remove salts, and solvent was removed. The crude mixture was dissolved in DMSO (0.7 mL) and purified by preparative HPLC using a 50-95% solvent B gradient over 15 minutes with 150 pL MeCN sample sandwiching during injection. The product eluted at 5.65 minutes and was concentrated to afford the title compound as a yellow solid (70 mg, 97%).

[0617] Spectral data were consistent with existing literature31A.

[0618]

[0619] Ir[dF(CF3)-CO2Bn-ppy]2(dMebipy)PFe complex (SI-21)

[0620] Dimer Formation

[0621] A flame-dried schlenk flask equipped with a PTFE-coated stir bar was charged with (0.2 g, 0.509 mmol, 2.1 equiv.) and Iridium(III)chloride hydrate (0.072 g, 0.242 mmol, 1.0 equiv.). The flask was placed under an argon atmosphere and 2-ethoxy ethanol: H2O (2:1, 4.85 mL) was added. The mixture was stirred and heated to 130 °C for 16 h. The reaction was cooled to room temperature, quenched with cold water (200 mL) which caused the product to precipitate out. The precipitate was filtered and washed with excess cold water then dried to afford the title compound as a bright orange powder which was carried forward without further purification. (0.157 g, 44%).

[0622] Dimer Crack

[0623] A scintillation vial equipped with a PTFE-coated stir bar was charged with dimer (0.050 g, 0.025 mmol, 1.0 equiv.), 4,4’ -Dimethyl-2, 2’ -bipyridine (0.010 g, 0.054 mmol, 2.2 equiv.), and silver hexafluorophosphate (0.018 g, 0.074 mmol, 3.0 equiv.). Added acetone (1 mL) and the mixture was stirred and heated to 60 °C for 16 h. The reaction

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[0625] was cooled to room temperature, filtered to remove salts, and the solvent was removed. The crude mixture was dissolved in MeCN / H2O / DMSO (2:1:4, 1 mL) and purified by preparative HPLC through direct injection using a 50-95% solvent B over 10 min. method with a 150 pL MeCN sample sandwich to afford the title compound as a yellow solid (42.2 mg, 74%). 'H NMR (598 MHz, CDCl3) 88.57 (s, 2H), 8.30 - 8.21 (m, 2H), 7.61 (d, J = 2.5 Hz, 4H), 7.35 (dq, J = 27.7, 3.0 Hz, 10H), 7.10 (d, J = 5.6 Hz, 2H), 6.51 (ddd, J= 11.5, 8.7, 2.3 Hz, 2H), 5.80 - 5.75 (m, 2H), 5.37 (s, 4H), 2.59 (s, 6H).19F NMR (563 MHz, CDCl3) δ -62.54 (s, 6F), -72.67 (d, 7 = 711.8 Hz, 4.8F), -96.45 (t, J= 13.6 Hz, 2F), -99.54 (dt, J = 16.4, 8.4 Hz, 2F).

[0626]

[0627] G3-Ir-dMebpy complex (SI-22)

[0628] A scintillation vial equipped with a PTFE-coated stir bar was charged with SI-(0.022 g, 0.019 mmol, 1.0 equiv.) and palladium on carbon (10% mol, 0.2 mg) and dissolved in MeOH (1 mL). The solution was placed under H2 atmosphere and stirred at room temperature for 16 h. The mixture was filtered over celite and the filter cake was washed with MeOH (50 mL). Solvent was removed to afford the title compound without additional purification. 'H NMR (598 MHz, CD3OD_SPE) δ 8.64 (s, 2H), 8.40 (s, 2H), 7.89 (d, J = 5.7 Hz, 2H), 7.76 (s, 2H), 7.53 (d, J = 5.6 Hz, 2H), 6.72 (t, J = 9.9 Hz, 2H), 5.90 (d, J = 7.9 Hz, 2H), 2.62 (s, 6H).19F NMR (563 MHz, CD3OD_SPE) δ -64.34 (s, 6F), -74.74 (d, J = 707.7 Hz, 6F), -95.54 (s, 2F), -103.82 (q, J = 9.6 Hz, 2F).

[0629]

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[0631] 2-(2,4-difluorophenyl)- ^-diethyl-5-(trifluoromethyl)isonicotinamide (17)

[0632] A scintillation vial equipped with a PTFE-coated stir bar was charged with 13 (0.1 g, 0.264 mmol, 1.0 equiv.). The solid was suspended in THF (1.31 mL) and aqueous LiOH (2 M, 1.31 mL, 10.0 equiv.) was added. The mixture was stirred vigorously at room temperature. After 16 hours solvent was removed and the reaction mixture partitioned between H2O (30mL) and EtOAc (30 mL). The aqueous layer was acidified to pH 2 with HC1, then extracted 3x with EtOAc (30 mL). The EtOAc extracts were washed with brine, dried with Na2SO4, and concentrated to remove solvent. The product was used without further purification.

[0633] A scintillation vial equipped with a PTFE-coated stir bar was charged with the hydrolysis product (0.075 g, 0.248 mmol, 1.0 equiv.) and PyBOP (0.644 g, 1.238 mmol, 5 equiv.). DMF (2.475 mL) and DIPEA (0.431 mL, 2.475 mmol, 10 equiv.) were added, and the solution was stirred at room temperature for 10 minutes. Diethylamine (0.102 mL, 0.990 mmol, 4 equiv.) was added dropwise and the reaction was heated to 70 °C and stirred for 16 hours. The reaction was cooled to room temperature and the solvent was removed. The crude residue was partitioned between H2O (30mL) and DCM (30 mL), and the aqueous layer was extracted 3x with DCM (30 mL) and the DCM extracts were washed 3x H2O (100 mL) to remove excess DMF. The organic phase was then washed with brine, dried with Na2SC>4, and concentrated to remove solvent. The crude mixture was purified by flash column chromatography on silica (Hex: EtOAc) = 0% to 10% over 5 CVs followed by 10% to 30% over 10 CVs to afford the title compound as white solid (0.075 g, 85%).

[0634] 1H NMR (598 MHz, CDCl3) δ 8.98 (s, 1H), 8.11 (s, 1H), 7.74 (s, 1H), 7.04 (s, 1H), 6.93 (s, 1H), 3.88 (s, 1H), 3.27 (s, 1H), 1.25 (d, J= 14.3 Hz, 1H), 1.11 (s, 1H).

[0635] 13C NMR (150 MHz, CDCl3) δ 165.70, 165.13, 165.05, 163.44, 163.36, 162.00, 161.92, 160.31, 160.23, 156.22, 147.98, 147.95, 147.91, 147.88, 144.07, 132.65, 132.62, 132.58, 132.56, 126.10, 124.28, 122.46, 122.01, 121.99, 121.94, 121.91, 121.24, 121.17, 120.84, 120.62, 112.64, 112.61, 112.50, 112.47, 104.97, 104.80, 104.62, 42.96, 39.00, 13.55, 12.17.

[0636] 19F NMR (563 MHz, CDCl3) δ -59.76 (s, 3F), -106.31 (s, IF), -111.75 (s, IF).

[0637] LC-MS Rt= 7.14 min; m / z calcd. For C17H15F5N2O ([M+H]+) = 359.1, found 359.3

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[0639] lrCI3AgPF6H2O / Ethoxyethanol Dimethyl Bipyridine 16h, 130QC Acetone 16h, 60QC

[0640]

[0641] Ir[diethylamideppy]2(dMebipy)PF6 (18)

[0642] A flame-dried schlenk flask equipped with a PTFE-coated stir bar was charged with

[0643] 17 (0.063 g, 0.035 mmol, 2.1 equiv.) and iridium(III) chloride hydrate (0.025 g, 0.083 mmol, 1.0 equiv.). The flask was placed under an argon atmosphere and 2-ethoxyethanol: H2O (2: 1, 1.67 mL) was added. The mixture was heated to 130 °C and stirred for 16 hours. The reaction was cooled to room temperature, quenched with cold water (200 mL), generating a precipitate. The precipitate was filtered and washed with excess cold water then dried to afford the title compound as a bright orange powder which was used without further purification. (0.066 g, 85%).

[0644] A scintillation vial equipped with a PTFE-coated stir bar was charged with dimeric intermediate (0.033 g, 0.018 mmol, 1.0 equiv.), 4,4’ -dimethyl-2, 2’ -bipyridine (0.007 g, 0.053 mmol, 2.2 equiv.), and silver hexafluorophosphate (0.013 g, 0.053 mmol, 3.0 equiv.).

[0645] Acetone (0.7 mL) was added, and the mixture heated to 60 °C for 16 hours with stirring.

[0646] The reaction was cooled to room temperature, filtered to remove salts, and the solvent was removed. The crude mixture was dissolved in MeCN / H2O / DMSO (1:1:2, 1.5 mL) and purified by preparative HPLC using a 35-95% solvent B over 10 minute method with a 150 pL MeCN sample sandwich method during injection. The product eluted at 6.0 minutes and solvent was removed to afford the title compound as a yellow solid (25.1 mg, 65%).

[0647] 1H NMR (598 MHz, DMSO) δ 8.78 (dd, J = 29.4, 13.0 Hz, 2H), 8.32 (d, J = 4.8 Hz, 1H), 8.27 (d, J = 8.7 Hz, 1H), 7.80 - 7.56 (m, 6H), 6.95 (q, J = 11.8 Hz, 2H), 6.04 (d, J = 7.9 Hz, 1H), 5.52 (dd, J = 35.9, 8.2 Hz, 1H), 3.48 (dtdd, J = 38.2, 31.5, 13.9, 6.5 Hz, 5H), 2.98 (s, 3H), 2.62 - 2.57 (m, 6H), 1.28 - 1.14 (m, 6H), 1.02 (ddt, J = 46.3, 14.9, 7.1 Hz, 6H).

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[0649] 19F NMR (563 MHz, DMSO) 8 -61.44 (dd, J = 44.7, 30.5 Hz, 6F), -70.36 (d, J = 711.4 Hz, 2F), -95.47 (dt, J = 32.9, 12.9 Hz, IF), -96.49 (d, J = 8.4 Hz, IF), -102.95 (dd, J = 44.9, 11.0 Hz, IF), -103.21 (dd, J = 140.9, 10.4 Hz, IF).

[0650] LC-MS Rt= 5.80 min; m / z calcd. For C46H4oFioIrNe02 ([M+H]+) = 1092.3, found 1092.6

[0651] AgPF6

[0652] Dimethyl Bipyridine

[0653] Acetone

[0654] 16h, 60QC

[0655]

[0656] Ir[dF(CF3)(OBN)ppy]2(dMebipy)PFe complex (19)

[0657] A scintillation vial equipped with a PTFE-coated stir bar was charged with dimeric intermediate (15) (0.041 g, 0.020 mmol, 1.0 equiv.), 4,4’-dimethyl-2,2’-bipyridine (0.009 g, 0.050 mmol, 2.2 equiv.), and silver hexafluorophosphate (0.015 g, 0.061 mmol, 3.0 equiv.). Acetone (1 mL) was added, the mixture was heated to 60 °C, and stirred at this temperature for 16 hours. The reaction was cooled to room temperature, filtered to remove salts, and solvent was removed. The crude mixture was dissolved in DMSO (0.8 mL) and purified by preparative HPLC using a 20-95% solvent B over 10 minutes method with a 150 pL MeCN sample sandwiching method during injection. The product eluted at 4.4 minutes and was concentrated to afford the title compound as a yellow solid (37 mg, 79%).

[0658] 'H NMR (598 MHz, DMSO) 8 8.74 (d, J = 48.8 Hz, 2H), 8.51 - 8.27 (m, 2H), 7.87 (d, J = 35.0 Hz, 2H), 7.75 - 7.35 (m, 4H), 7.04 (t, J = 10.6 Hz, 2H), 5.84 (br, s, 2H), 4.88 (br, s, 2H), 4.72 (s, 2H), 3.50 (m, 16H), 2.59 (s, 6H).

[0659] 19F NMR (563 MHz, DMSO) 8 -59.67 (s, 6F), -102.54 (s, 2F), -106.00 (s, 2F).

[0660] LC-MS Rt= 3.07 min (45%-95% B over 10 minutes); m / z calcd. For C46H4oFioIrNe06 ([M+H]+) = 1156.2, found 1156.7

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[0662]

[0663] Ir[dF(CF3)ppy]2(sulfo-ether-bipy) (20)

[0664] A scintillation vial equipped with a PTFE-coated stir bar was charged with dimer (16) (0.008 g, 0.005 mmol, 1.0 equiv.), 9 (0.006 g, 0.011 mmol, 2.2 equiv.), and silver hexafluorophosphate (0.004 g, 0.016 mmol, 3.0 equiv.). Acetone (0.5 mL) was added, the mixture was heated to 60 °C, and stirred for 16 hours. The reaction was cooled to room temperature, filtered to remove salts, and the solvent was removed. The crude mixture was dissolved in DMSO (0.7 mL) and purified by semipreparative HPLC using a 5-95% solvent B over 10 minutes method with a 75 pL MeCN sample sandwich method during injection to afford the title compound as a yellow solid (6.3 mg, 46%).

[0665] 'H NMR (598 MHz, DMSO) 89.00 (s, 1H), 8.52 (s, 1H), 8.47 (d, J = 9.4 Hz, 2H), 8.42 (d, J = 9.5 Hz, 1H), 7.99 (d, J = 6.2 Hz, 1H), 7.84 (d, J = 5.7 Hz, 1H), 7.69 (s, 3H), 7.37 -7.25 (m, 6H), 7.06 - 6.96 (m, H), 5.75 (dd, J = 24.1, 8.1 Hz, 2H), 5.00 (s, 2H), 4.51 (d, J = 4.9 Hz, 2H), 3.81 (d, J = 4.9 Hz, 2H), 3.60 (d, J = 4.7 Hz, 2H), 3.53 (d, J = 4.5 Hz, 2H), 3.49 (s, 4H), 3.43 - 3.39 (m, 2H), 3.13 (d, J = 6.9 Hz, 2H).

[0666] 19F NMR (563 MHz, DMSO) 8 -61.15 - -61.93 (m, 6F), -103.43 (t, J = 9.5 Hz, 2F), -106.79 (t, J = 12.4 Hz, 2F).

[0667] LC-MS Rt= 7.16 min; m / z calcd. For C₅₀H₄₄F₁₀IrN₅O₈S ([M+H]+) = 1270.2, found 1270.2

[0668]

[0669] - 71 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0670] 2

[0671] A scintillation vial equipped with a PTFE-coated stir bar was charged with dimer (16) (0.062 g, 0.042 mmol, 1.0 equiv.), bipy-Me-PEG3-NHCbz (0.050 g, 0.092 mmol, 2.2 equiv.), and silver hexafluorophosphate (0.031 g, 0.125 mmol, 3.0 equiv.). Acetone (0.83 mL) was added, the mixture was heated to 60 °C, and stirred for 16 hours. The reaction was cooled to room temperature, filtered to remove salts, and the solvent was removed. The crude mixture was dissolved in DMSO (1.2 mL) and purified by preparative HPLC using a 35-95% solvent B over 15 minutes method with a 75 pL MeCN sample sandwich method during injection to afford the title compound as a yellow solid (32.5 mg, 28%).

[0672] 'H NMR (598 MHz, CDCl3) 88.71 - 8.40 (m, 4H), 8.03 (q, J = 10.4 Hz, 2H), 7.80 - 7.66 (m, 2H), 7.59 (s, 1H), 7.54 - 7.42 (m, 2H), 7.32 (d, J = 8.0 Hz, 5H), 6.93 (s, 1H), 6.70 -6.58 (m, 2H), 5.63 (d, J = 7.8 Hz, 1H), 5.54 (s, 1H), 5.07 (s, 3H), 3.66 - 3.53 (m, 10H), 3.49 (t, J = 5.7 Hz, 2H), 3.36 (dt, J = 10.1, 5.3 Hz, 2H), 3.17 (dq, J = 14.0, 7.5 Hz, 2H), 2.76 - 2.70 (m, 4H), 1.99 (s, 3H).

[0673] 19F NMR (563 MHz, CDCI3) 8 -62.80 (d, J = 39.3 Hz, 6F), -71.98 (d, 7= 712.8 Hz, 6F), -101.41 (dd, 7 = 53.7, 12.5 Hz, 2F), -105.86 (dd, 7 = 41.7, 12.6 Hz, 2F).

[0674] LC-MS Rt= 4.68 min; m / z calcd. For C₅₄H₄₅F₁₀IrN₄O₄ ([M+H]+) = 1260.3, found 1260.8

[0675] Common reagents and starting materials

[0676]

[0677] Diazirine-biotin (4)

[0678] The reaction was performed according to a modified literature procedure28A. A scintillation vial containing a PTFE-coated stir bar was charged with diazirine amine (139 mg, 0.551 mmol, 1.0 equiv.) and biotin-PECL-NHS ester (300 mg, 0.551 mmol, 1.0 equiv.).

[0679] - 72 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0680] The solids were dissolved in DMF (2.2 mL) and diisopropylethylamine (DIPEA, 192 pL, 1.102 mmol, 2.0 equiv.) was added. The reaction was stirred at room temperature for 16 hours. Solvent was removed and the residue was purified by flash column chromatography on silica (DCM: MeOH = 0% to 80% over 12 CVs) to afford the title compound as an opaque oil (0.330 g, 93%). Spectral data are consistent with existing literature28A.

[0681] Note: Trifluoromethyl diazirines can easily isomerize to diazo compounds under typical laboratory conditions (ambient lighting, room temperature, exposure to Lewis acids and transition metals). The diazo isomers are electrophilic, have shifted absorption spectra, and can cause non-specific background protein biotinylation in the presence or absence of catalyst. This, in turn, decreases observed fold change and can obfuscate results. Care should be taken to handle this compound in the dark and avoid extended storage at room temperature, as even trace quantities of diazo isomer can cause irreproducible results. The presence of diazo isomers in stock solutions must be routinely assayed via19F NMR (-CF3: -65.5 ppm for diazirine, -57.6 ppm for diazo).

[0682] Tx. JL Br -40 Eq CsF► f^H

[0683] Br Il HTDMSO FIl FT

[0684] 130 °C, 6 d

[0685]

[0686] 4,4’-Difluoro-2,2’-bipyridine (7).

[0687] A scintillation vial equipped with a PTFE-coated stir bar was charged with 4,4’- dibromo-2, 2’ -bipyridine (10 g, 32 mmol, 1.0 equiv.), cesium fluoride (19.44 g, 128 mmol, 4 equiv.), and DMSO (25.6 mL). The mixture was heated to 150 °C and stirred vigorously for 6 days. The reaction mixture was cooled to room temperature and partitioned between H2O (200 mL) and DCM (200 mL). The aqueous layer was extracted 3x with DCM (100 mL) and the DCM extracts were washed 5x H2O (75 mL) to remove all DMSO. The DCM extract was washed with brine, dried with Na2SO4, and concentrated. The residue was purified by flash column chromatography on silica (hexanes: EtO Ac = 0% to 10% over 7 CVs) to afford the title compound as a white solid (2.75 g, 45%). The spectral data were consistent with existing literature30A.

[0688] - 73 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0689]

[0690] l-(4’-Methyl-[2,2’-bipyridin]-4-yl)-3,17-dioxo-7,10,13-trioxa-4,16-diazaicosan-20-oic acid (c)

[0691] Amide coupling: A scintillation vial equipped with a PTFE-coated stir bar was charged with 3-(4’-methyl-[2,2’-bipyridin]-4-yl)propanoic acid (0.280 g, 1.157 mmol, 1.0 equiv.), 3-(2-(2-(3-benzyloxycarbonylaminopropoxy)ethoxy)ethoxy)propylamine (0.415 g, 1.378 mmol, 1.1 equiv.), benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (1.806 g, 3.471 mmol, 3.0 equiv.), and dissolved in DMF (5.79 mL). The solution was stirred for 5 minutes, then DIPEA was added (0.806 mL, 4.628 mmol, 4.0 equiv.). After 16 hours solvent was removed and the residue partitioned between H2O (100 mL) and DCM (100 mL). The aqueous layer was extracted 3x with DCM (50 mL) and the DCM extract was washed 5x H2O (50 mL) to remove all DMF. The DCM extract was washed with brine, dried with Na2SO4, and concentrated to remove solvent.

[0692] The residue was purified by flash column chromatography on silica (MeOH: DCM = 5% over 8 CVs). The oil was redissolved in DMF (2.0 mL) and purified by preparative HPLC using a 30%-50% solvent B over 10 minutes. The product eluted at 3.5 minutes and solvent was removed to afford the protected intermediate (276 mg, 43%).

[0693] 1H NMR (598 MHz, CD3OD_SPE) 88.53 (dd, J = 16.9, 5.1 Hz, 3H), 8.17 (dd, J = 29.2, 1.8 Hz, 3H), 7.38 - 7.33 (m, 8H), 7.32 (dd, J = 5.2, 1.5 Hz, 2H), 7.29 (d, J = 5.1 Hz, 2H), 5.09 (d, J = 14.1 Hz, 4H), 3.60 (d, J = 5.6 Hz, OH), 3.58 (s, 6H), 3.56 (dd, J = 5.9, 3.1 Hz, 3H), 3.53 - 3.49 (m, 6H), 3.45 (t, J = 5.4 Hz, 3H), 3.36 - 3.33 (m, 4H), 3.05 (t, J = 7.5 Hz, 3H), 2.62 (t, J = 7.5 Hz, 3H), 2.46 (s, 5H), 1.96 (s, 1H).

[0694] 13C NMR (150 MHz, CD3OD_SPE) 8 174.18, 173.15, 158.64, 156.73, 156.48, 153.26, 150.57, 150.03, 149.76, 138.27, 129.40, 128.91, 128.75, 126.17, 125.44, 123.54, 122.78, 71.46, 71.41, 71.11, 71.09, 71.06, 70.83, 70.45, 70.41, 67.31, 67.29, 49.43, 49.28, 49.14, 49.00, 48.86, 48.71, 48.57, 41.68, 40.37, 40.31, 37.02, 32.08, 22.54, 21.25.

[0695] LC-MS Rt= 4.95 min; m / z calcd. For C30H38N4O6([M+H]+) = 551.3, found 551.5

[0696] - 74 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0697] Cbz deprotection: A scintillation vial equipped with a PTFE-coated stir bar was charged with protected intermediate (0.073 g, 0.132 mmol, 1.0 equiv.) and 10% Pd / C, type 487 (1.4 mg) and suspended in MeOH (1.32 mL). The solution was placed under H2 using a balloon and stirred at room temperature for 16 hours. The mixture was filtered over celite and the filter cake was washed with MeOH (80 mL). Solvent was removed to afford the deprotected intermediate as a clear oil which was carried forward without additional purification (52.8 mg, 96%)

[0698] Succinylation: A scintillation vial equipped with a PTFE-coated stir bar was charged with the deprotected intermediate (0.052 g, 0.125 mmol, 1.0 equiv.) and succinic anhydride (0.015 g, 0.15 mmol, 1.2 equiv.), and dissolved in DMF (0.83 mL). The reaction was stirred at room temperature for 5 minutes, then DIPEA (0.109 mL, 0.625 mmol, 5.0 equiv.) was added and the reaction was stirred for 16 hours. The product was purified by preparative HPLC using a 5%-55% solvent B gradient over 10 minutes. The product eluted at 4.1 minutes and solvent was removed to afford the title compound as a clear oil (36.2 mg, 56%).

[0699] 'H NMR (598 MHz, CDCI3) 8 8.51 (dd, J = 21.6, 5.0 Hz, 2H), 8.16 (dd, J = 4.4, 1.7 Hz, 2H), 7.17 (ddd, J = 9.2, 5.1, 1.7 Hz, 2H), 6.93 (t, J = 5.7 Hz, 1H), 6.87 (t, J = 5.7 Hz, 1H), 3.61 - 3.50 (m, 10H), 3.47 (t, J = 5.1 Hz, 2H), 3.38 (dq, J = 7.5, 5.3 Hz, 4H), 3.01 (t, J = 7.6 Hz, 2H), 2.66 (t, J = 6.7 Hz, 2H), 2.58 (t, J = 7.7 Hz, 2H), 2.48 (t, J = 6.8 Hz, 2H), 2.43 (s, 3H).

[0700] 13C NMR (150 MHz, CDCI3) 8 175.29, 172.39, 172.15, 155.62, 155.44, 151.49, 149.43, 149.27, 148.32, 125.08, 124.61, 122.97, 121.64, 77.37, 77.16, 76.95, 70.57, 70.32, 70.14, 70.10, 69.99, 69.79, 39.49, 39.35, 39.28, 36.60, 31.30, 31.07, 29.99, 21.36.

[0701] LC-MS Rt= 3.99 min; m / z calcd. For C26H36N4O7 ([M+H]+) = 517.3, found 517.5

[0702] - 75 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825 Scheme 1. Synthesis of 1: Overview

[0703]

[0704] - 76 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0705] HCX. NHCbz NaH THF

[0706] rt, 16 h Cbz-CI

[0707]

[0708] H2O, 20 min Benzyl (2-(2-(2-(2-((4’-fluoro-[2,2’-bipyridin]-4-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (8).

[0709] A scintillation vial equipped with a PTFE-coated stir bar was charged with 6 (1.98 g, 6.0 mmol, 1.0 equiv.), 7 (2.93 g, 15.2 mmol, 2.5 equiv.), sodium hydride (60% w / w in mineral oil, 1.16 g, 30.4 mmol, 5 equiv.) and cooled to 0 °C. The vial was sparged with argon. Anhydrous THF (12 mL) was added and the mixture stirred at 0 °C for 15 minutes.

[0710] The mixture was warmed to room temperature and stirred for 16 hours. The reaction was then cooled to 0 °C and quenched with water (5 mL). Benzyl chloroformate (0.507 mL, 3.58 mmol, 0.59 equiv.) was added dropwise, then the reaction was stirred at room temperature for 30 minutes and solvent was removed. The crude mixture was then partitioned between H2O (100 mL) and DCM (100 mL). The aqueous layer was extracted 3x with DCM (100 mL) and the DCM extracts were washed with brine, dried with Na2SC>4, and concentrated to remove solvent. The resulting residue was purified by flash column chromatography on silica (hexanes: EtO Ac = 0% to 100% over 7 CVs) to afford the title compound as a clear oil (1.55 g, 51%).

[0711] 'H NMR (598 MHz, CDCI3) 58.65 - 8.53 (m, 1H), 8.45 (d, J = 5.7 Hz, 1H), 8.14 (d, J = 10.4 Hz, 1H), 7.96 (s, 1H), 7.34 - 7.27 (m, 5H), 7.02 (s, 1H), 6.85 (s, 1H), 5.40 (br s, 1H), 5.08 (s, 2H), 4.23 (s, 2H), 3.84 (s, 2H), 3.70 (s, 2H), 3.68 - 3.63 (m, 2H), 3.60 (d, J = 6.9 Hz, 4H), 3.54 (s, 2H), 3.37 (d, J = 5.5 Hz, 2H).

[0712] 13C NMR (150 MHz, CDCI3) 8 170.48, 168.74, 165.91, 159.40 (d, J = 7.2 Hz), 156.68 (d, 7 = 3.8 Hz), 156.48, 151.33 (d, 7 = 6.8 Hz), 150.36, 136.63, 128.50, 128.10 (d, 7= 7.5 Hz), 111.69, 111.59 (d, 7= 16.6 Hz), 109.00 (d, 7= 18.6 Hz), 106.96, 70.91, 70.60 (d, 7 = 9.3 Hz), 70.26, 70.01, 69.32, 67.50, 66.63, 40.88.

[0713] 19F NMR (563 MHz, CDCh) 8 -102.00 (d, 7 = 9.1 Hz).

[0714] LC-MS Rt= 5.16 min; m / z calcd. For C26H30FN3O6 ([M+H]+) = 500.2, found 500.5

[0715] - 77 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0716]

[0717] 4’-((3-Oxo-l-phenyl-2,7,10,13-tetraoxa-4-azapentadecan-15-yl)oxy)-[2,2’-bipyridine]-4-sulfonate (9).

[0718] A scintillation vial equipped with a PTFE-coated stir bar was charged with 8 (0.81 g, 1.6 mmol, 1.0 equiv.), sodium sulfite (0.61 g, 4.86 mmol, 3 equiv.), 15-crown-5 (1.98 mL, 9.7 mmol, 6 equiv.) and EtOH: H2O (2:1, 13 mL). The vial was sparged with argon for 15 min. The reaction was then heated to 90 °C and stirred for 7 days. The mixture was cooled to room temperature, solvent was removed, and the residue purified by flash column chromatography on silica (EtOAc: MeOH (0.1% formic acid) = 0% to 25% over 8 CVs) to afford the title compound as an opaque oil (0.79 g, 87%).

[0719] 1H NMR (598 MHz, DMSO) 88.64 (d, J = 4.9 Hz, 1H), 8.59 (s, 1H), 8.50 (d, J = 5.6 Hz, 1H), 7.93 (s, 1H), 7.57 (d, J = 3.1 Hz, 1H), 7.37 - 7.27 (m, 5H), 7.04 - 7.01 (m, 1H), 5.02 (s, 2H), 4.29 (t, 2H), 3.80 (t, J = 4.6 Hz, 2H), 3.64 - 3.60 (m, 2H), 3.54 - 3.49 (m, 6H), 3.43 (t, J = 6.0 Hz, 2H), 3.16 (t, J = 6.0 Hz, 2H).

[0720] 13C NMR (150 MHz, DMSO) 8 166.07, 157.51, 157.31, 155.79, 151.06, 149.62, 137.74, 128.75, 128.13, 128.05, 120.88, 117.78, 111.45, 107.07, 72.84, 70.52, 70.34, 70.29, 70.25, 70.10, 69.66, 69.17, 67.99, 65.73, 60.81.

[0721] LC-MS Rt= 4.69 min; m / z calcd. For C26H30N3O9S ([M+H]+) = 561.2, found 562.3.

[0722]

[0723] 4’-(2-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)ethoxy)-[2,2’-bipyridine]-4-sulfonate (10)

[0724] A scintillation vial equipped with a PTFE-coated stir bar was charged with 9 (0.63 g, 0.41 mmol, 1.0 equiv.) and 10% Pd / C, type 487 (4.5 mg) and MeOH (4 mL) added. The solution was placed under H2 atmosphere using a balloon and stirred at room temperature for 16 hours. The mixture was filtered over celite and the filter cake was washed with - 78 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0725] MeOH (150 mL). Solvent was removed and the title compound was carried forward without additional purification.

[0726]

[0727] 4’-((15-Carboxy-13-oxo-3,6,9-trioxa-12-azapentadecyl)oxy)-[2,2’-bipyridine]-4- sulfonate (11)

[0728] A scintillation vial equipped with a PTFE-coated stir bar was charged with 10 (0.19 g, 0.44 mmol, 1.0 equiv.), succinic anhydride (0.048 g, 0.49 mmol, 1.1 equiv.), and DMF (3 mL). The reaction was stirred at room temperature for 5 minutes. DIPEA (0.38 mL, 2.23 mmol, 5.0 equiv.) was added and the reaction was stirred for 1 hour. Solvent was then removed to afford the title compound without additional purification.

[0729] 'H NMR (500 MHz, CD3OD_SPE) 88.77 - 8.73 (m, 1H), 8.71 (d, J = 0.8 Hz, 1H), 8.47 (d, J = 5.7 Hz, 1H), 7.94 (d, J = 2.6 Hz, 1H), 7.79 (dd, J = 5.0, 1.6 Hz, 1H), 7.04 (dd, J = 5.8, 2.6 Hz, 1H), 4.38 - 4.31 (m, 2H), 3.95 - 3.88 (m, 2H), 3.73 (dt, J = 4.8, 3.0 Hz, 2H), 3.68 (d, J = 2.0 Hz, 2H), 3.66 - 3.56 (m, 6H), 3.51 (t, J = 5.5 Hz, 2H), 3.34 (t, J = 5.6 Hz, 2H).

[0730] 13C NMR (126 MHz, CD3OD_SPE) 8 177.09, 176.49, 174.71, 167.84, 158.31, 157.78, 155.73, 151.66, 150.81, 121.65, 119.12, 112.32, 108.88, 71.81, 71.61, 71.59, 71.53, 71.29, 70.56, 70.48, 69.92, 68.95, 57.65, 57.48, 57.30, 55.85, 52.15, 49.63, 49.45, 49.28, 49.11, 43.79, 40.43, 31.71, 30.74, 30.62, 30.24, 30.04, 17.43, 17.28, 17.13, 13.17.

[0731] LC-MS Rt= 3.73 min; m / z calcd. For C₂₂H₂₈N₃O₁₀S ([M+H]+) = 528.2, found 528.3

[0732] BnBr

[0733] K2CO3

[0734] DMF

[0735]

[0736] 16h, 30eC

[0737] Benzyl 2-chloro-5-(trifluoromethyl)isonicotinate (12).

[0738] A round-bottom flask equipped with a PTFE-coated stir bar was charged with 2-chloro-5-(trifluoromethyl)isonicotinic acid (7.0 g, 31.0 mmol, 1.0 equiv.), potassium

[0739] - 79 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0740] carbonate (5.1 g, 37.2 mmol, 1.2 equiv.), and DMF (51 mL). The solution was cooled to 0 °C for 15 minutes and benzyl bromide (3.7 mL, 31.0 mmol, 1.0 equiv.) was added dropwise. The reaction was stirred at 0 °C for 30 minutes then warmed to room temperature and stirred for 3 hours. The solvent was removed and the crude mixture partitioned between EtOAc and H2O. The aqueous layer was extracted 4x with EtOAc (125 mL). The EtOAc extracts were washed with brine, dried with Na2SO4, and concentrated to remove solvent. The residue was purified by flash column chromatography on silica (hexanes: EtOAc = 0% to 10% over 7 CVs) to afford the title compound as a white powder (8.5 g, 87%).

[0741] 1H NMR (500 MHz, CDCI3) 88.76 (s, 1H), 7.67 (s, 1H), 7.40 (dt, J = 22.2, 7.3 Hz, 5H), 5.39 (s, 2H).

[0742] 19F NMR (471 MHz, CDCI3) 8 -59.27.

[0743] LC-MS Rt= 7.51 min; m / z calcd. For Ci4H9ClF3NO2([M+H]+) = 316.0, found 316.1

[0744]

[0745] Benzyl 2-(2,4-difluorophenyl)-5-(trifluoromethyl)isonicotinate (13).

[0746] A flame-dried three-neck round-bottom flask equipped with a PTFE-coated stir bar was charged with 12 (6.04 g, 19.2 mmol, 1.0 equiv.), 2,4-difluorophenyl boronic acid (3.33 g, 21.1 mmol, 1.1 equiv.) potassium carbonate (7.95 g, 57.5 mmol, 3.0 equiv.), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex (0.42 g, 0.58 mmol, 0.03 equiv.). The flask was placed under argon atmosphere, toluene: H2O (2:1, 75 mL) was added, and the solvent further sparged for 10 minutes. The solution was heated to 85 °C and stirred for 16 hours. The reaction was cooled to room temperature and quenched with cold water (200 mL) and the crude mixture partitioned between EtOAc and water. The aqueous layer was extracted 3x with EtOAc (100 mL) and the EtOAc extracts were washed with brine, dried with Na2SO4, and concentrated to remove solvent. The

[0747] - 80 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0748] residue was purified by flash column chromatography on silica (hexanes: DCM = 0% to 25% over 12 CVs) to afford the title compound as a white powder (6.6 g, 88%).

[0749] 'H NMR (500 MHz, CDCl3) 89.05 (s, 1H), 8.11 (dd, J = 10.1, 5.1 Hz, 2H), 7.48 - 7.34 (m, 5H), 7.05 (td, J = 8.2, 2.4 Hz, 1H), 6.96 (ddd, J = 11.3, 8.6, 2.4 Hz, 1H), 5.42 (s, 2H).

[0750] 19F NMR (471 MHz, CDCI3) 8 -59.25 (s, 3F), -105.97 - -106.05 (m, IF), -111.21 (q, J = 9.8 Hz, IF).

[0751] LC-MS Rt= 7.98 min; m / z calcd. For C20H12F5NO2 ([M+H]+) = 394.1, found 394.2

[0752] Small molecule photocatalyst conjugates

[0753]

[0754] Flumazenil-Ester-PEG3-NHCbz

[0755] A scintillation vial equipped with a PTFE-coated stir bar was charged with Flumazenil acid (0.013 g, 0.046 mmol, 1.0 equiv.). The vial was placed under an argon atmosphere and thionyl chloride (2.356 mL, 323 mmol, 7,000 equiv.) was added and the mixture refluxed at 65 °C and stirred for 1 hour. The reaction was cooled to room temperature, solvent was removed under a stream of nitrogen, and the residue redissolved in DCM (1 mL). To the reaction added OH-PEG3-NHbz (0.049 g, 0.150 mmol, 3.25 equiv.) and DMAP (0.045 g, 0.369 mmol, 8.0 equiv.) dissolved in DCM (1 mL) and stirred at room temperature for 5 hours. Solvent was removed and the crude mixture was purified by flash column chromatography on silica (EtOAc: MeOH = 0% to 5% over 10 CVs) to afford the title compound as a cloudy oil (0.015 g, 57%).

[0756] 1H NMR (500 MHz, CDCI3) 87.85 (s, 1H), 7.77 (dd, J = 8.7, 2.9 Hz, 1H), 7.41 (dd, J = 8.8, 4.5 Hz, 1H), 7.35 - 7.32 (m, 5H), 7.30 - 7.27 (m, 1H), 5.08 (s, 1H), 4.49 (d, J = 26.1 Hz, 2H), 3.81 (t, J = 4.8 Hz, 2H), 3.69 - 3.57 (m, 10H), 3.54 (t, 7= 5.1 Hz, 2H), 3.39 -3.35 (m, 2H), 3.23 (s, 3H).

[0757] 19F NMR (471 MHz, CDCI3) 8 -111.12.

[0758] - 81 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0759] LC-MS Rt= 3.89 min (40%-95% B over 10 minutes); m / z calcd. For C29H33FN4O8([M+H]+) = 585.61, found 585.54

[0760]

[0761] 1-Flumazenil

[0762] A scintillation vial equipped with a PTFE-coated stir bar was charged with Flumazenil-Ester-PEG3-NHCbz (0.015 g, 0.026 mmol, 1.0 equiv.) and 10% Pd / C, type 487 (0.279 mg) and suspended in MeOH (1 mL). The mixture was placed under H2 atmosphere and stirred at room temperature for 3 hours. The mixture was filtered over celite and the filter cake was washed with MeOH (4 mL). Solvent was removed to afford the title compound which was carried forward without additional purification.

[0763] A scintillation vial equipped with a PTFE-coated stir bar was charged with I-CO2H (0.035 g, 0.023 mmol, 1.0 equiv.), crude Flumazenil-Ester-PEG3-NH2 synthesized above (0.012 g, 0.026 mmol, 1.1 equiv.), PyBOP (0.036 g, 0.070 mmol, 3.0 equiv.), and DMF (0.468 mL). The solution was stirred at room temperature for 5 minutes, then DIPEA (0.032 mL, 0.187 mmol, 8 equiv.) was added and the reaction stirred at room temperature for 16 hours. The reaction mixture was diluted with MeOH (0.12 mL) and purified by semipreparative HPLC using a 20%-65% solvent B gradient over 10 minutes. The product eluted at 4.63 minutes and solvent was removed to afford the title compound as a yellow solid (8.5 mg, 19%).

[0764] 1H NMR (500 MHz, DMSO) 89.07 (s, 1H), 8.59 (s, 1H), 8.40 - 8.24 (m, 3H), 8.03 (s, 1H), 7.91 - 7.78 (m, 4H), 7.75 - 7.61 (m, 4H), 7.56 (s, 1H), 7.30 (s, 1H), 7.06 (ddd, J = 12.4, 9.5, 2.7 Hz, 2H), 6.06 - 5.55 (m, 2H), 4.90 (br,m, J = 93.5 Hz, 4H), 4.57 - 4.47 (m, 2H), 4.37 (s, 2H), 3.88 - 3.69 (m, 6H), 3.62 - 3.00 (m, 48H), 2.27 (s, 3H).

[0765] - 82 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0766] 19F NMR (471 MHz, DMSO) 8 -59.68 (s, 6F), -102.33 - -103.08 (m, 2F), -106.09 (q, J = 65.5 Hz, 2F), -113.37 (s, IF).

[0767] LC-MS Rt= 3.99 min (25%-95% B over 10 minutes); m / z calcd. For C77H81F11IrN11O17S ([M+H]+) = 1930.49, found 1930.22

[0768] Example 7. Materials and methods

[0769] Photocatalytic diazirine sensitization assay:

[0770] Diazirine-biotin (4, 100 pM), photocatalyst (10 pM), and internal standard (IS) (2-chloro-5-trifluoromethylpyridine, 100 pM) were dissolved in 350 pL H2O in a 1.5 mL capped centrifuge tube. The tube was degassed with argon and placed 17.8 cm above two 440 nm, 100 W COB LED light (Chanzon, 1DGL-JC-100W-440) placed side-by-side (Fig.

[0771] 26). The samples were then irradiated for the appropriate amount of time. After irradiation, D2O (100 pL) was added, and the concentration of diazirine measured by19F NMR spectroscopy relative to the IS, comparing against a negative control in which the photocatalyst is absent. Experiments were performed in triplicate and average values are reported.

[0772] LogP measurement:

[0773] H2O (150 pL) and 1 -octanol (150 pL) were combined in a 1.5 mL capped centrifuge tube to which appropriate catalyst (3 pL of a 100 pM stock, final concentration 1 pM, 1% DMSO) was added. The sample was vortexed for 7 seconds, let equilibrate for 2 min., then centrifuged at 21,000 x g for 10 min. Organic and aqueous layers were separated, solvent was evaporated, and solids redissolved in DMSO (100 pL). The photocatalyst concentration was measured by absorbance at 375 nm. Each measurement was performed in triplicate and averaged for the reported value.

[0774] Thermodynamic solubility measurement:

[0775] 1.125 mg of catalyst (as a 5 mM stock in DMSO) was added to a 1.5 mL capped centrifuge tube and solvent was evaporated. The powder was resuspended in H2O or PBS (100 pL), sonicated for 5 min., then inverted end over end for 24 hours at 37 °C. The sample was centrifuged (21,000 x g for 10 min.) and supernatant was transferred to a fresh tube. Solvent was evaporated and solid redissolved in DMSO (100 pL). The photocatalyst

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[0777] concentration was determined by absorbance at 375 nm. Each measurement was performed in triplicate and averaged for the reported value.

[0778] K562 Cellular Lysate Experimental Protocol

[0779] K562 cell lysate was prepared according to method 1. 200 L of 1.5 mg / mL lysate containing 200 p M Dz-Bt was added to 1.5 mL capped centrifuge tube. Added 2 pL of catalyst (100 pM, DMSO) or vehicle and incubated on ice for 30 min. Samples were degassed with argon according to method 3 and irradiated for appropriate amount of time using the apparatus in method 3. Samples were then processed according to methods 4-7. Each experiment was performed in triplicate.

[0780] Human Plasma Protein Binding

[0781] Assay was conducted by Pharmaron according to company protocol:

[0782] The working solution of test compound and control compound were prepared in DMSO at the concentration of 1 mM. A basic solution was prepared by dissolving 14.2 g / L Na2HPO4 and 8.77 g / L NaCl in deionized water and the solution could be stored at 4°C for up to 7 days. An acidic solution was prepared by dissolving 12.0 g / L NaH2PO4 and 8.77 g / L NaCl in deionized water and the solution could be stored at 4°C for up to 7 days. The basic solution was titrated with the acidic solution to pH 7.4 and store at 4°C for up to 7 days. pH was checked on the day of experiment and was adjusted if outside specification of 7.4 ± 0.1. Set the temperature of water bath to 37°C. Thaw the frozen Plasma (stored at -80°C) immediately in a 37°C water bath. Soak the dialysis membranes in ultrapure water for 60 minutes to separate strips, then in 20% ethanol for 20 minutes, finally in dialysis buffer for 20 minutes. Load the prepared membranes into the dialysis device and install the device again following manufacturers guidelines. Turn on air bath and allow to pre-heat to 37°C. If the dialysis membranes are not used immediately, they can be store at 4°C for no longer than 4 weeks. Add 597 pL of blank plasma solution into each vial of a new plastic plate or separate plastic tube by addition of 3 pL of the working solution of test compound, vortex at 1000 rpm for 2 minutes. The final percent volume of organic solvent is 0.5% and the final concentration for test compound is 5 pM.

[0783] Immediately transfer 50 pL of the spiked plasma solution suspension to a 96- well plate to act as T=0 control sample. The samples are treated the same as the samples after incubation. Place all remaining spiked plasma solution in the incubator for the duration of

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[0785] the study. At the same time, the remaining spiked plasma solution sample in the plastic plate or separate plastic tube is incubated for 6 hours at 37 °C with 5% CO2 in the CO2 incubator.

[0786] At T=6 hours, transfer 50 pL of the original spiked plasma solution suspension to the 96- well plate for analysis. Assemble the dialysis set up following the manufacturer’s instructions. Load cells with 120 pL of plasma sample and dialyzed against equal volume of dialysis buffer (PBS). The assay is performed in duplicate. Cover the unit with gas permeable lid and incubate for 6 hours at 37 °C at 100 rpm with 5% CO2 on an orbital shaker in the CO2 incubator. At the end of incubation, remove lid and pipette 50 pL of post-dialysis samples from both buffer and plasma solution chambers into separated 96-well plate for analysis, respectively. Add 50 pL of plasma solution to the buffer samples, and an equal volume of PBS to the collected plasma solution samples. Shake the plate at 1000 rpm for 2 minutes and add 400 pL of acetonitrile containing an appropriate internal standard (IS) to precipitate protein and release compound. Vortex at 1000 rpm for 10 minutes. Centrifuge for 30 minutes at 3,220 g. Then transfer 100 pL of the supernatant to new 96-well plates for analysis. Add 100 pL of distilled water to each sample and mix for analysis by LC-MS / MS. All calculations are carried out using Microsoft Excel.

[0787] Determine the concentrations of test compound and control compound in the buffer and plasma solution chambers. Calculate the percentages of test compound(s) and control compound bound as follows: % Unbound = (Area ratio buffer chamber / Area ratio plasma solution chamber) x 100; % Bound = 100 - % Unbound; % Remaining = Area ratio 6hr / Area ratio Ohr X 100.

[0788] Preparation of Photocatalyst-Antibody Conjugate

[0789] Photocatalyst 3: 3-DBCO (20 pL, 5 mM DMSO) (3A, 4A) and Azido-PEG3-NHS ester (BroadPharm, BP-21605) (1 pL, 100 mM 9:1 MeCN / fUO) were mixed in a vial and allowed to react for 10 minutes in the dark. Separately, goat anti-mouse polyclonal antibody (Jackson ImmunoResearch 115-005-205) (252 pL, 0.5 mg / mL, PBS) was combined with NaHCO? (25.2 pL, 1 mM). 3-NHS ester (11 pL) was added and allowed to react for 1 hour at room temperature. The reaction was then passed through a ZEBA 40 kDa 0.5 mL desalting column (Thermo Fisher Scientific, A57760) that had been equilibrated with PBS. The antibody-catalyst solution was analyzed by BCA for protein

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[0791] concentration (50:1 solution A: B; A: 10 mg / mL Bicinchonic acid, 20 mg / mL Na2CO?, 1.6 mg / mL sodium tartarate, 4 mg / mL NaOH, 9.5 mg / mL NaHCCL. B: 40 mg / mL CuSCL) compared to a serial dilution of BSA. Catalyst concentration was measured by fluorescence of the sample at 485 nm following excitation at 380 nm compared to a serial dilution of 3-NHS. 0.50 mg / mL antibody concentration with 7.4 catalysts / antibody. Preparation of photocatalyst-antibody conjugate:

[0792] Catalyst 1: 1-DBCO (20 pL, 5 mM DMSO) and azido-PEGs-NHS ester (BroadPharm, BP-21605) (1 pL, 100 mM DMSO) were mixed in a vial and allowed to react for 10 minutes. Separately, goat anti-mouse polyclonal antibody (Jackson ImmunoResearch 115-005-205) (180 pL, 0.5 mg / mL, PBS) was combined with NaHCO? (18 pL, 1 M). The solution of 1-NHS ester (19 pL) generated through strain-promoted cycloaddition was added and allowed to react with the antibody for 1 hour at room temperature. The reaction was then passed through a ZEBA 40 kDa 0.5 mL desalting column (Thermo Fisher Scientific, A57760) that had been equilibrated with PBS. The antibody-catalyst solution was analyzed by BCA (50:1 solution A: B; A: 10 mg / mL Bicinchonic acid, 20 mg / mL Na2CCh, 1.6 mg / mL sodium tartarate, 4 mg / mL NaOH, 9.5 mg / mL NaHCO?. B: 40 mg / mL CUSO4) vs. a serial dilution of BSA. Catalyst concentration was measured by fluorescence of the sample at 485 nm following excitation at 380 nm compared to a serial dilution of 1-NHS. The measured concentration was 0.52 mg / mL antibody, with 12 catalysts / antibody.

[0793] Catalyst 2: 2-DBCO (20 pL, 5 mM DMSO) and azido-PEG24-NHS ester (BroadPharm, BP-23542) (1 pL, 100 mM DMSO) were mixed in a vial and allowed to react for 10 minutes. Separately, goat anti-mouse polyclonal antibody (Jackson ImmunoResearch 115-005-205) (450 pL, 0.5 mg / mL, 1: 1 PBS / DMSO) was combined with NaHCO? (45 pL, 1 M). 2-NHS ester solution (5.625 pL) was added and allowed to react for 1 hour at room temperature. The reaction was then passed through a ZEBA 40 kDa 0.5 mL desalting column (Thermo Fisher Scientific, A57760) that had been equilibrated with 1:1 PBS / DMSO. The antibody-catalyst solution was analyzed by BCA for protein concentration (50:1 solution A: B; A: 10 mg / mL Bicinchonic acid, 20 mg / mL Na2CO?, 1.6 mg / mL sodium tartarate, 4 mg / mL NaOH, 9.5 mg / mL NaHCO?. B: 40 mg / mL CUSO4) compared to a serial dilution of BSA. Catalyst concentration was measured by fluorescence

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[0795] of the sample at 485 nm following excitation at 380 nm compared to a serial dilution of 2-NHS. The measured concentration was 0.14 mg / mL antibody, with 3.85 catalysts / antibody.

[0796] Live cell labeling:

[0797] Comparison of catalysts each to isotype control in triplicate, 6 samples per catalyst, 12 samples total. 108Ramos (RA 1, CRL-1596) cells were harvested, cooled to 4 °C, pelleted, and washed 2x (DPBS 1% FBS), and pelleted (800 x g, 5 min). The following steps were all performed at 4 °C. The cell pellet was resuspended in DPBS 1% FBS (10 mL) and evenly split into 0.5 mL aliquots in 1.5 mL capped centrifuge tubes. Cells were pelleted (800 x g, 5 min) and resuspended in DPBS 1% FBS (0.2 mL) containing 5 pg anti-CD19 primary (SJ25C1, IgGl) or isotype control (mouse IgG, Santa Cruz sc-2025) and continuously inverted end over end at 4 °C for 1 hour. Samples were then pelleted (800 x g, 5 min), washed with DPBS 1% FBS (0.6 mL, lx), and resuspended in DPBS 1% FBS (0.2 mL) containing 10 pg goat-anti-mouse secondary antibody conjugated to appropriate catalyst and continuously inverted end over end at 4 °C for 2 hours. Samples were then pelleted (800 x g, 5 min), washed with DPBS (0.6 mL, lx), and resuspended in DPBS (0.2 mL) containing 250 pM Dz-Bt. Samples were irradiated with 440 nm light using the apparatus in Method 3 for 15 minutes, then washed with DPBS (0.6 mL, 3x). Cells were resuspended in RIPA (Millipore, 20-188) containing 1% SDS (0.4 mL) and sonicated in a cup horn sonicator (Method 1) for 15 minutes (20 seconds on, 10 seconds off at 60% intensity). Samples were heated to 95 °C for 5 minutes, then centrifuged (15,000 x g, 10 minutes) to remove insoluble debris. 375 pL of supernatant was transferred to new 1.5 mL tubes. DTT (20 pL, 100 mM) was added to each sample, and the samples heated to 95 °C for 5 minutes. After allowing the sample to cool to room temperature, iodoacetamide was added (15 pL, 200 mM) and incubated in the dark. After 30 minutes, 150 pL streptavidin beads (Cytiva, 30152104010350) were added to each sample and the samples inverted end over end for 2 days at room temperature. Samples were then processed according to Methods 5-9 and Method 11 and protein abundances separately compared between the antiCD 19 and isotype treated samples for each catalyst.

[0798] Photocatalytic labeling of recombinant BRD4 BD1+BD2:

[0799] To a 1.5 mL capped centrifuge tube was added (His)e-BRD4 (BD1+BD2 domains, AAs 49-460, BPSBioscience catalog no. 31045) (2 pL, 31.76 pM), bovine serum albumin

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[0801] (5.08 pL, 13.79 pM), Dz-Bt (186.7 pL, 400 pM) and lx PBS (46.12 pL, pH 7.4). To a separate 1.5 mL capped centrifuge tube was added (+)-JQl (12 pL of 4x desired concentration, in PBS + 2% DMSO) followed by a solution of the proteins described above (24 pL). This was incubated on ice for 15 minutes. l-(+)-JQl (12 pL, 0.4 pM, PBS + 1% DMSO) was added, and the mixture incubated on ice for 15 minutes (final volume 48 pL, 0.75% DMSO). Samples were then degassed with argon and irradiated with 440 nm light for 20 minutes according to Method 3. The sample was diluted with 4x Laemlli buffer with 10% BME (12 pL) and heated to 95 °C for 10 minutes. The samples were cooled to room temperature and centrifuged. The samples were loaded onto a Novex 10-20% tris-glycine gel with protein ladder (Biorad Cat. 1610373) in freshly prepared Tris running buffer and subjected to electrophoresis (200 V, 45 min). The gel was washed (3x DI H2O) and transferred to nitrocellulose membrane using a Trans-Blot Turbo Transfer System (BioRad 1704150). The membrane was immersed in REVERT total protein stain (Li-Cor 926-11011) for 10 minutes. Stain was removed, the membrane washed (33.5:150:316.5, AcOH: MeOH: H2O, 3x 5 minutes), rinsed with water, and imaged using a Li-Cor Odyssey CLx scanner at 700 nm. The membrane was immersed in 30% MeOH in 100 mM aqueous NaOH for 10 minutes, washed with this solution (2x), rinsed with water (3x), and incubated with Intercept Blocking Buffer (Li-Cor, 927-60001) for 1 hour. The blocking buffer was decanted and the membrane was incubated with 10 mL IX TBST containing 1:10,000 dilution of IRDye 800 CW streptavidin (Li-Cor 926-32230) for 1 hour. The buffer was decanted and the membrane washed with IX TBST (3x 5 minutes) and water before imaging using a Li-Cor Odyssey CLx scanner at 800 nm. Pixel densitometry was performed in Image Studio V. 5.5.4. Streptavidin 800 nm channel signal was divided by total protein signal in the 700 nm channel to determine the protein abundance-normalized biotinylation signal for each protein band. Experiment performed in triplicate, whole gel images shown in Fig. 29).

[0802] K562 cell lysate was treated with diazirine-biotin at 200 pM, 1, 2, or diazirine alkyne conjugates of Dasatinib at 1 pM, 100 nM, 10 nM, or 1 nM, and unmodified Dasatinib at concentrations ranging from 100 pM to 10 pM in half-log increments in duplicate (96 total samples, method 1-2, FIG. 21). After irradiation, protein precipitation, resuspension, reduction / alkylation, IP, and tryptic digestion, peptides were analyzed via

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[0804] nano-UHPLC / IM / MS / MS in DIA mode using a nanoElute 2 / timsTOF Pro 2 MS system and the resulting data processed using DIANN 1.8 with an in-silico generated spectral library (methods 3-10). After imputation (MinProb) (75A, 16A), and normalization (vsn) within each photocatalyst-Dasatinib concentration sample group, integrated intensity data was fit to a logistic model to measure a EC50 for the dose-responsive reduction in intensity induced by competitive binding of unmodified Dasatinib at each photocatalyst concentration for each protein identified in the proteome assuming a Hill coefficient of 1 for all proteins. These EC50 values were then used to generate a Schild plot to model competitive binding between Dasatinib and Dasatinib-photocatalyst conjugates, with the slope affording the ratio of Dasatinib-protein and conjugate-protein apparent Kas and the intercept affording the KA (app.) of unmodified Dasatinib. 1 performs better than 2, which gives reduced signal at low concentrations and elevated background signal at high concentrations due to high nonspecific binding, while diazirine-alkyne conjugate of Dasatinib yields only very weak signal intensity that enables only marginal detection of the BTK-Dasatinib interaction.

[0805] AssayQuant Kinase Activity Assay:

[0806] Three kinases, PKN3, CK1D, and RIPK1 were assayed for inhibition by dasatinib using the PhosphoSens CSox-based kinetic assay. Each kinase was treated with either vehicle or dasatinib at 11 concentrations across a three-fold dilution series (final concentration of dasatinib: 100 pM, 33.3 pM,..., 1.69 nM) in technical duplicate. Individual reactions were set up as follows: 11.7 pL reaction mixture (HEPES, Brij-35, EGTA, MgCl2, DTT, ATP, & CSx Substrate), 0.3 pL 50X inhibitor in 100% DMSO, 3.0 uL enzyme dilution buffer (EDB - 20 mM HEPES, pH 7.5, 0.01% Brij-35, 5% Glycerol, ImM EGTA, 1 mM DTT, 1 mg / ml BSA) or a 5X kinase in EDB. Final reaction volume was 15 pL and final reaction concentrations were the following: 50 mM HEPES, pH 7.5, ATP Km, 1.0 mM DTT, 0.01% Brij-35, 0.5 mM EGTA, 1% glycerol (from EDB), 10 mM MgCh, 0.20 mg / mL BSA (from EDB), 15 pM AQT sensor substrate (AQT0150; AQT0509; AQT0709), IX kinase (PKN3: 5 nM; CK1D: 1 nM; and RIPKl: 15 nM), and 2% DMSO. Reactions were run at 30 °C for 120 minutes, measured in Perkin Elmer 384-well low volume white ProxiPlates (Cat. #6059480) after sealing using optically clear adhesive film (TopSealA-Plus plate seal, PerkinElmer, Cat. #6050185) in aBiotek Synergy

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[0808] Neo 2 microplate reader with excitation at 360 nm and emission at 485 nm. Rates were fit to the following four parameter logistic equation, and nonlinear regression was performed using the Solver algorithm in Excel9A.

[0809] Top — Bottom

[0810] Y = Bottom H - - — — —,u1—

[0811]

[0812] 1 + Slope

[0813] Ki values were calculated using the Cheng-Prusoff equation, assuming a reversible, substrate-competitive mode of inhibition:

[0814] ; C50

[0815]

[0816] AssayQuant Recombinant Kinase Constructs

[0817] RIPK1, recombinant human protein amino acids (1-327), N-terminal GST tag, SignalChem (Cat / Lot#: R07-11G / E4165-6). PNK3, full length amino acids (1-889), N-terminal GST tag, Carna Biosciences (Cat / Lot #: 01-146 / 18CBS-0337 B). CK1D (CSNK1D), catalytic domain amino acids (1-294), N-terminal GST tag, Carna Biosciences (Cat / Lot# 03-103 / 09CBS-1197 L).

[0818] Method 1. Pooled cell lysate preparation

[0819] For a single 96-sample experiment, 2.2 x 108K562 cells were collected by centrifugation (800 x g, 5 min, 20 °C), and washed twice with 40 mL of cold PBS. Cells were resuspended in 10.6 mL cold (4 °C) PBS in a 50 mL conical tube and lysed by sonication (QSonica Q500 with cup horn, 4 °C, 3 cycles of 60% amplitude, 4 seconds on / off for 64 seconds). Lysate was examined under a brightfield microscope (1:1 lysate:trypan blue 0.4%) to ensure complete lysis. The lysate was cleared by centrifugation (2,500 x g, 15 min, 4 °C). Concentrated diazirine-PEG3-biotin (Dz-Bt) dissolved in cold PBS (600 p. M, 7.04 mL) was added to the cleared lysate and diluted with cold PBS to 21.1 mL to give a final concentration of 200p. M Dz-Bt and a 2 mg / mL protein concentration, determined by bicinchoninic acid (BCA) assay. Lysate was kept fresh on ice and was not frozen.

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[0821] Method 2. In vitro lysate treatment with photocatalyst-conjugated small molecules / peptides and free small molecules / peptides.

[0822] 200 pL (400 pg protein) of pooled K562 cell lysate was aliquoted into 128-strip cluster tubes (96 tubes) in a rack and kept on ice. Preprepared 96- well plates (compound plates, Fig. 25) containing 3 pL of a DMSO solution containing appropriate concentrations of compounds (or vehicle DMSO) were used for 1) free small molecule addition and 2) probe addition. 1) All wells in the first compound plate (free small molecules) were diluted with 27 pL room temperature PBS and then 20 pL of each well was added to the correspond cluster tube containing 200 pL of lysate (120-fold dilution) and incubated on ice for 20 minutes. 2) All wells in the second compound plate (probe) were diluted with 34.5 pL room temperature PBS and 20 pL of each well was added to the correspond cluster tube containing 200 pL of lysate (150-fold dilution) and incubated on ice for 20 minutes (final volume 240 pL, final concentrations small molecule / probe tabulated below). The plate setup presented here, and the concentration range used for most molecules in this manuscript (0.1 nM-10 pM ligand, 1 nM - 1 pM probe) is designed to capture affinities in the 1 nM-10 pM range. Based on predicted ligand binding regime a shifted or extended concentration range could be used. For example, for ligands with weaker affinities a higher concentration regime can be used (100 nM-1 mM range for ligand, 1 pM-100 pM for probe). In these experiments, ligand concentration may be increased as high as solubility allows. For the IgG and dasatinib-diazirine-alkyne experiments (Fig. 27, Methods 13 and 18) concentrations were increased by an order of magnitude in this way. Samples were then argon degassed, irradiated with 440 nm light, and protein was precipitated as detailed in the methods described herein.

[0823] The plate setup presented below and the concentration range used for most molecules in this manuscript (0.1 nM-10 pM ligand, 1 nM - 1 pM probe) is designed to capture affinities in the 1 nM-10 pM range. Based on predicted ligand binding regime a shifted or extended concentration range could be used. For example, for ligands with weaker affinities a higher concentration regime can be used (100 nM-1 mM range for ligand, 1 pM-100 pM for probe). In these experiments, ligand concentration may be increased as high as solubility allows. For the IgG and Dasatinib-diazirine-alkyne experiments (Methods 13 and 18) concentrations were increased by an order of magnitude - 91 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0824] in this way.

[0825] Table 2. Probe and off-compete small molecule / peptide concentrations in a typical 96- well plate

[0826] Probe [ ] (pM) Off-compete [ ] (pM) (in duplicate) Number of samples 0, 10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01,

[0827] Row A / B: 1 24

[0828] 0.00316, 0.001, 0.000316, 0.0001

[0829] 0, 10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01,

[0830] Row C / D: 0.1 24

[0831] 0.00316, 0.001, 0.000316, 0.0001

[0832] 0, 10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01,

[0833] Row E / F: 0.01 24

[0834] 0.00316, 0.001, 0.000316, 0.0001

[0835] Row G / H: 0, 10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01,

[0836] 24

[0837] 0.001 0.00316, 0.001, 0.000316, 0.0001

[0838]

[0839] Method 3. Argon degassing, 440 nm light irradiation, and protein precipitation Samples were placed in a vessel that was sealed and purged with water-saturated argon (Fig. 26) for 15 minutes. Samples were then irradiated using the apparatus shown below. This setup was shown to achieve uniform irradiation of 96 cluster tubes using ferrioxalate-based actinometry (Fig. 26). Immediately following irradiation, 240 pF of -20 °C MeOH and 60 pL of CHCI3 were added to each cluster tube and mixed, precipitating denatured protein. Samples were centrifuged (2,500 x g, 10 min, 4 °C), affording a protein disk suspended between the MeOH and CHCI3 layers. Solvent was carefully aspirated without disrupting the protein disk, then 500 pL of -20 °C MeOH was added to each cluster tube and the protein disk disrupted via sonication (Qsonica Q500 with cup horn, 4 °C, 3 cycles of 60% Amplitude, 4 sec on / off). Samples were centrifuged (2,500 x g, 10 min, 4 °C), resulting in a protein pellet at the bottom of each cluster tube. The MeOH was aspirated, and protein pellets were either carried forward or stored at -80 °C.

[0840] Method 4. Protein pellet redissolving, reduction, alkylation, and streptavidin bead enrichment

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[0842] Cluster tubes containing protein pellets were resuspended in 80 uL 600 mM Tris-HC1 Buffer with 4% SDS (pH = 8). Samples were boiled at 95 °C for 15 minutes, sonicated in a bath sonicator at room temperature for 10 minutes, boiled again at 95°C for 5 minutes to dissolve the protein pellet, and then 140 uL of MQ H2O was added to each sample. Dithiothreitol (DTT) in water (50 pL, 54 mM stock, 10 mM final concentration) was added and the mixture incubated at 95 °C for 10 minutes. After the mixture was allowed to cool to room temperature, iodoacetamide in water (50 pL, 128 mM stock, 20 mM final concentration) was added and incubated in the dark at room temperature for 30 minutes. Samples were centrifuged (2,500 x g, 10 min) to ensure any insoluble material was removed, transferred to a 0.5 mL / well 96-well plate, and 50 pL of Sera-Mag™ Medium Capacity Streptavidin beads (Cytiva, 30152104010350) were added to each sample. The plate was sealed with a silicone 96-well plate sealing mat (VWR, 76311-636) and incubated overnight with continuous inversion.

[0843] Method 5. Automated streptavidin bead washing

[0844] Samples were transferred to a KingFisher 96 deep-well plate (Thermo Fisher Scientific 95040450B) (plate 1). Beads were washed using a Qiagen BioSprint 96 workstation (Qiagen 9000852) with KingFisher 96 deep-well plates loaded with four wash buffers: 1) 3 x PBS with 1% SDS washes (plate 2, 3, and 4, 250 pL / well), 2) 3 x PBS with 1 M NaCl (plate 5, 6, and 7, 250 pL / well), 3) 3 x PBS with 10% EtOH (plate 8, 9, 10, 250 pL / well), and 4) 3 x 50 mM ammonium bicarbonate in MQ H2O (plate, 11, 12, 13, 250 pL / well). The following steps were implemented for each washing buffer (4 rounds with plate 1-4; 4-7; 7-10; and 10-13): (a) first plate (1, 4, 7, or 10) collect beads (premix), (b) plate 2, 5, 8, or 11, wash (release beads, 30 sec, medium; wash beads, 1 min, medium), wash (release beads, 30 sec, fast dual mix; wash beads, 1 min, fast dual mix), wash (release beads, 30 sec, medium; wash beads, 1 min, medium), (c) plate 3, 6, 9, or 12 wash (release beads, 30 sec, medium; wash beads, 1 min, medium), wash (release beads, 30 sec, fast dual mix; wash beads, 1 min, fast dual mix), wash (release beads, 30 sec, medium; wash beads, 1 min, medium), (d) plate 4, 7, 10, or 13, wash (release beads, 30 sec, medium; wash beads, 1 min, medium), wash (release beads, 30 sec, fast dual mix; wash beads, 1 min, fast dual mix), wash (release beads, 30 sec, medium; wash beads, 1 min, medium) (Fig. 24). After the final wash, beads from plate 13 were released in a KingFisher 96 deep- well plate loaded

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[0846] with trypsin (0.2 pg / well, Pierce, Thermo part #90058) in 50 rnM ammonium bicarbonate (plate 14, 60 pE / well, 3.3 ng / pE final trypsin concentration in 50 mM ammonium bicarbonate). The following steps were implemented: (a) plate 4, collect beads (premix), (b) plate 5, wash (release beads, 30 sec, medium; wash beads, 30 sec, medium), wash (release beads, 30 sec, fast dual mix; wash beads, 30 sec, fast dual mix), wash (release beads, 30 sec, medium; wash beads, 30 sec, medium).

[0847] Method 6. Trypsin digestion

[0848] Beads suspended in trypsin-containing 50 mM ammonium bicarbonate (0.2 pg of trypsin per sample) were transferred to a full-skirted 96-well PCR plate, capped, and incubated at 37 °C overnight with continuous inversion. The plate was centrifuged (200 x g, 20 °C, 1 min) to collect liquid / beads at the bottom of each well, then beads pelleted using a magnetic rack (DynaMag™ 96 Side Skirted Magnet) for 5 minutes. Formic acid (5 pl / well, 5% v / v in water) was added to a fresh full-skirted 96-well PCR plate and the supernatant from the trypsin digest was transferred to this plate.

[0849] Method 7. Proteomic data acquisition (standard sensitivity)

[0850] All proteomic samples were analyzed using a Bruker nanoElute 2 / timsTOF Pro 2 nano-UHPLC-IM / MS / MS system using a two-column nano-UHPLC separation method and data independent analysis (DIA) MS method. Acidified and digested samples (5 pL) were loaded on a trap column (Thermo-Fisher, Cat. No. 174500, PepMap Neo C18, 5 pm particle size, 300 pm ID, 5 mm length) using 12 volume equivalents of H2O / 0.1% FA. Flow through the trap was then reversed, and peptides eluted through a separation column (Bruker, Cat. No. 1893472, Bruker 10 C18, 1.9 pm particle size, 75 pm ID, 10 cm length) using a 2-35% gradient (H2O / 0.1% FA - MeCN / 0.1% FA) at a 500 nE / min flow rate. A 20 pm ID CaptiveSpray emitter was used at 1400 V capillary voltage. MS analysis was performed using the instrument default short gradient DIA method in HyStar v. 6.2 without alteration.

[0851] Method 8. In-plate desalting

[0852] An Oasis HLB 96-well plate (Waters) was conditioned by washing once with MeCN+0.1% formic acid (300 pE / well, elution at 200 x g for one minute) and twice with H2O+0.1% formic acid (300 pE / well, elution at 200 x g for 1 min). The tryptic peptide solutions from on-bead digestion were loaded and eluted twice at 200 x g for 1 min.

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[0854] Adsorbed tryptic peptides were washed twice with H2O+0.1% v / v formic acid (300 pL / well, elution at 200 x g for 1 min), then eluted once with 80% MeCN+0.1% v / v formic acid (100 pL / well, elution at 200 x g for 1 min). Volatiles were removed by vacuum concentration, and the samples reconstituted in 40 pL 2% MeCN+0.1% formic acid. Method 9. Proteomic data acquisition (high sensitivity).

[0855] Samples (IgG Affinity Map experiment only) were analyzed using a Bruker nanoElute 2 / timsTOF Pro 2 nano-UHPLC-IM / MS / MS system using a two-column nano-UHPLC separation method and data independent analysis (DIA) MS method. Oasis HLB plate desalted samples (5 pL) were loaded on a trap column (Waters nanoEase M / Z Symmetry C18 Trap, 2 cm x 180 pm, 5 pm particle size, Part No.: 186008821) using 12 volume equivalents of H2O / 0.1% FA. Flow through the trap was then reversed, and peptides eluted through a separation column with an integrated emitter tip (lonOptiks Aurora Ultimate CSI Cl 8 (25 cm x 75 pm, 1.7 pm particle size, Part No.: AUR3-25075C18-CSI) using a 60 minute 2-35% gradient (H2O / 0.1% FA - MeCN / 0.1% FA) at 150 nE / min flow rate with 1400 V capillary voltage. MS analysis was performed using the default short gradient DIA method in HyStar v. 6.2 without alteration.

[0856] Method 10. Proteomic data acquisition (high sensitivity, 90 minute gradient).

[0857] Samples (cycloRGDfK Affinity Map experiment only) were analyzed using a Bruker nanoElute 2 / timsTOF Pro 2 LC / IM / MS2 system using a two-column nano-UHPLC separation method and data independent analysis (DIA) MS method. Oasis HLB plate desalted samples (5 pL) were loaded on a trap column (Waters nanoEase M / Z Symmetry C18 Trap, 2 cm x 180 pm, 5 pm particle size, Part No.: 186008821) using 12 volume equivalents of H2O / 0.1% FA. Flow through the trap was then reversed, and peptides eluted through a separation column with an integrated emitter tip (lonOptiks AuroraTM Ultimate CSI C18 (25 cm x 75 pm, 1.7 pm particle size, Part No.: AUR3-25075C18-CSI) using a 90 minute 2-35% gradient (H2O / 0.1% FA - MeCN / 0.1% FA) at 150 nL / min flow rate with 1400 V capillary voltage. MS analysis was performed using the default short gradient DIA method in HyStar v. 6.2 without alteration.

[0858] Method 11. Proteomics data independent analysis using DIA-NN.

[0859] Data was analyzed using DIA-NN 1.8, 1.9, or 2.0.54A Human FASTA sequence database from UniProt (accession date: 12-05-2022) was used. A trypsin-digested in silico spectral

[0860] - 95 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0861] library generated in DIA-NN was used for analysis. DIA-NN settings: MSI accuracy and mass accuracy tolerance: ±12.5 ppm; missed cleavages: 1; maximum number of variable modifications: 1; peptide length: 7-30; precursor charge range: 1-4; precursor m / z range: 300-1800; fragment ion m / z range: 200-1800; modifications: C carbamidomethylation, N-term M excision, Ox(M), Ac(N-term); Precursor FDR (%) = 1.0; match between runs enabled, neural network classifier: double-pass mode; protein inference: genes; quantification strategy: robust LC (high precision); cross-run normalization: off; librarygeneration: smart profiling.

[0862] Method 12. Cell culture (K562 and THP-1 cells)

[0863] K-562 (CCL-243) and THP-1 (TIB-202) cells were purchased from ATCC. K-562 and THP-1 cells were cultured at 37 °C and 5% CO2 in RPMI 1640 complete medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillin-streptomycin (100 U / mL final concentration, Gibco). Subculturing and medium renewal were performed according to ATCC’s product sheets for each cell line. Unless otherwise noted, cells were grown in 25 cm2, 75 cm2or 150 cm2canted neck, vented cap sterile cell culture flasks. Method 13. Dasatinib-diazirine-alkyne labeling

[0864] K562 cell lysate was prepared according to Method 1. Lysate (0.2 mL, 2 mg / mL) was transferred to cluster tubes and treated with compounds as described in Method 2 (using dasatinib-dz-alkyne instead of 1-dasatinib at the concentrations shown) and irradiated using the same apparatus as Fig.26 with 100-watt 375 nm LEDs for 5 minutes.

[0865] 34 pL of 7x protease inhibitor cocktail (Roche, 11836170001 ) was then added, and biotin-PEG4-azide (2.74 pL, 10 mM in DMSO), CuSO4(1.37 pL, 50 mM in H2O), THPTA (6.85 pL of 10 mM in H2O), and sodium ascorbate (13.7 pL, 50 mM in H2O, freshly made) added, in that order. Note that order of addition is important, adding the first three reagents together as a cocktail and sodium ascorbate last. Samples were inverted end over end for 1 hour at room temperature. Then 280 pL of -20 °C MeOH and 70 pL of CHCL were added to each cluster tube and mixed, precipitating denatured protein. Samples were centrifuged (2,500 x g, 10 min, 4 °C), affording a protein disk suspended between the MeOH and CHCI3 layers. Solvent was carefully aspirated without disrupting the protein disk, then 500 pL of -20 °C MeOH was added to each cluster tube and the protein disk disrupted via sonication (Qsonica Q500 with cup horn, 4 °C, 3 cycles of 60% Amplitude, 4 sec on / off).

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[0867] Samples were centrifuged (2,500 x g, 10 min, 4 °C), resulting in a protein pellet at the bottom of each cluster tube. The MeOH was aspirated, and protein pellets were stored at - 80 °C overnight then processed and analyzed using Methods 4-7 and 11.

[0868] Table 3. Probe and off-compete concentrations for the dasatinib-dz-alkyne Affinity Map experiment

[0869] Probe [ ] (pM) Off-compete [ ] (pM) (in duplicate) Number of samples 0, 100, 31.6, 10, 3.16, 1, 0.316, 0.1,

[0870] Row A / B: 10 24

[0871] 0.0316, 0.01, 0.00316, 0.001

[0872] 0, 100, 31.6, 10, 3.16, 1, 0.316, 0.1,

[0873] Row C / D: 1 24

[0874] 0.0316, 0.01, 0.00316, 0.001

[0875] 0, 10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01,

[0876] Row E / F: 0.1 24

[0877] 0.00316, 0.001, 0.000316, 0.0001

[0878] 0, 10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01,

[0879] Row G / H: 0.01 24

[0880] 0.00316, 0.001, 0.000316, 0.0001

[0881]

[0882] Method 14. Preparation of liver S9 fraction for (+)-JQl Affinity Map.

[0883] Frozen, mixed gender pooled liver S9 fractions were purchased from BioIVT (X008011). The purchased samples contained 40 mg of protein total, which was diluted to ~2 mg / mL with PBS containing 200 pM diazrine-PEG3 -biotin (Dz-Bt) and was aliquoted across 96 cluster tubes (200 pL homogenate, 400 pg protein / cluster tube). For compound treatment, the in vitro lysate treatment method described herein (method 2) was followed (plate layout in Fig. 25). The samples were then processed using methods 3-7, and 11.

[0884] Method 15. Cell culture (U87MG cells)

[0885] U87MG cells (HTB-14) were purchased from ATCC. Cells were cultured at 37 °C and 5% CO2 in DMEM 1640 complete medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillin- streptomycin (100 U / mL final concentration, Gibco). Subculturing and medium renewal were performed according to ATCC’s product sheets

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[0887] for each cell line. Unless otherwise noted, cells were grown in 10 cm or 15 cm diameter cell culture treated plates.

[0888] Method 16. Live U87MG cell treatment with 1-cRGDfK and free cRGDfk and labeling with 440 nm light irradiation.

[0889] 15 cm plates with U87MG cells were washed once with DPBS (10 mL), detached by incubation with TrypLE™ Express Enzyme (Gibco, 5 mL, 5 min, 37 °C), and collected by centrifugation (800 x g, 5 min, 20 °C). Cells were resuspended in DMEM 1640 complete medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillin- streptomycin (100 U / mL final concentration, Gibco) and passed through cell strainers to help dissociate clumping cells. 4 x 6-well treated cell culture plates were seeded with 5 x 105 U87MG cells and allowed to grow until confluent (~3 days). The media was aspirated and 400 L of phenol red-free media containing 200 p. M diazrine- PEG3-biotin (Dz-Bt) was pipetted over the adherent cells in each well. The appropriate concentration of unconjugated cRGDfK was added (40 pL) or a vehicle control to each well and cells incubated on ice for 20 minutes. Cells were then treated with the appropriate concentration of 1-cRGDfK (40 p. L) and incubated on ice for 20 minutes (final volume 240 p. L, 160 p. M Dz-Bt, final concentrations free cRGDFk / probe tabulated in SI Method 16 and shown below). Each 6-well plate was then irradiated for 15 minutes at 4 °C. Following irradiation, cells were released from the plate with 1 mL of cold PBS, transferred to a 1.5 mL tube, and pelleted by centrifugation (800 x g, 5 min, 4 °C). The supernatant was aspirated, and cell pellets were frozen at -80 °C. This experiment was repeated for each concentration of 1-cRGDfK (1 pM, 100 nM, 10 nM, or 1 nM), thus four separate groups of four plates were seeded then treated on separate days (4 concentrations x 4 plates x 6 wells = 96 wells total).

[0890] Table 4. Probe and off-compete concentrations for cRGDfK affinity profiling

[0891] Probe [ ] (p. M) Off-compete [ ] (pM) (in duplicate) Number of samples Plate 1 (day 1): 0, 100, 31.6, 10, 3.16, 1, 0.316, 0.1,

[0892] 24

[0893] 1 0.0316, 0.01, 0.00316, 0.001

[0894]

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[0896] Plate 2 (day 2): 0, 100, 31.6, 10, 3.16, 1, 0.316, 0.1,

[0897] 24

[0898] 0.1 0.0316, 0.01, 0.00316, 0.001

[0899] Plate 3 (day 3): 0, 100, 31.6, 10, 3.16, 1, 0.316, 0.1,

[0900] 24

[0901] 0.01 0.0316, 0.01, 0.00316, 0.001

[0902] Plate 4 (day 4): 0, 100, 31.6, 10, 3.16, 1, 0.316, 0.1,

[0903] 24

[0904] 0.001 0.0316, 0.01, 0.00316, 0.001

[0905]

[0906] Method 17. U87MG cell lysis, reduction, alkylation, and streptavidin enrichment Cell pellets were thawed for 1 hour at 4 °C before resuspension in RIPA lysis buffer containing 1% SDS and protease inhibitor cocktail (200 pL). Cells were incubated in RIPA buffer for 30-minutes at room temperature followed by sonication (QSonica Q500 with cup horn, 4 °C, 4 cycles of 60% amplitude, 4 seconds on / off for 64 seconds). Immediately following lysis, insoluble material was pelleted (800 x g, 5 min, 4 °C) and the supernatant was transferred to cluster tubes. Dithiothreitol (DTT) in water (50 pL, 50 mM stock, 10 mM final concentration) was added and the mixture incubated at 95 °C for 10 minutes. After the mixture was allowed to cool to room temperature, iodoacetamide in water (50 pL, 120 mM stock, 20 mM final concentration) was added and incubated at in the dark at room temperature for 30 minutes. Samples were centrifuged once more (2,500 x g, 10 min), to ensure any insoluble material was removed, transferred to a 0.5 mL / well 96-well plate, and 50 pL of Sera-Mag™ Medium Capacity Streptavidin beads (Cytiva, 30152104010350) were added to each sample. The plate was sealed with a silicone 96-well plate sealing mat (VWR, 76311-636) and incubated overnight with continuous inversion. Sample were then subject to automated streptavidin bead washes described herein (e.g., processed using methods 5, 6, 8, 10, and 11).

[0907] Method 18. Conjugation of 1 to human serum-derived IgG

[0908] 1-DBCO (120 pL, 5 mM in DMSO) and azido-PEGs-NHS ester (BroadPharm, BP- 21605, 6 pL, 100 mM in DMSO) were mixed and allowed to react for 10 minutes. Purified IgG (Sigma Aldrich, 14506, 300 pL, 20 mg / mL in PBS) was mixed with NaHCO? (30 pL, IM in H2O) and the click reaction mixture (120 uL, 4.76 mM in DMSO) and allowed to react for 2 hours in the dark. The resulting iridium labeled IgG was desalted twice (to - 99 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0909] ensure no free catalyst remained) using Thermo Scientific™ Zeba™ Spin Desalting Columns, 40K MWCO (catalog no. PIA57759) that had been equilibrated with PBS. The desalted mixture was analyzed by BCA for protein concentration (50:1 solution A: B; A: 10 mg / mL bicinchonic acid, 20 mg / mL NaaCCb, 1.6 mg / mL sodium tartarate, 4 mg / mL NaOH, 9.5 mg / mL NaHCCL. B: 40 mg / mL CuSCL) compared to a BSA standard. Photocatalyst concentration was measured by fluorescence of the sample at 485 nm following excitation at 380 nm compared to a standard of 1. An extent of labeling of 4 1 / IgG was obtained.

[0910] Method 19. Treatment of live THP-1 cells with 1-IgG and free IgG, labeling with 440 nm light irradiation

[0911] 2.1 x 108THP-1 cells were collected by centrifugation (800 x g, 5 min, 20 °C), and washed twice with 40 mL of cold PBS. Cells were resuspended in concentrated diazrine- PEGs-biotin (Dz-Bt) dissolved in cold PBS (570 p. M, 2.2 mL) and aliquoted into 128-strip cluster tubes (96 tubes) in a rack and kept on ice (21 p. L / aliquot, approximately 400 p.g of protein after lysis). The appropriate concentration of free IgG was added (29 p. L) or a vehicle PBS control to each cluster tube and incubated on ice for 20 minutes. Cells were then treated with the appropriate concentration of IgG-Ir (10 pL / aliquot) and incubated on ice for 20 minutes (final volume 60 p, L, 200 p. M Dz-Bt, final concentrations free IgG / probe tabulated in Fig. 27 and below). Samples were then irradiated for 15 minutes at 4 °C. Following irradiation, 200 p. L of cold PBS was added to each cluster tube and cells were pelleted by centrifugation (800 x g, 5 min, 4 °C). The supernatant was aspirated, and cells were pelleted and washed with another 200 p. L of cold PBS. Cell pellets were frozen at -80 °C prior to lysis.

[0912] Table 5. Probe and off-compete concentrations for the IgG Affinity Map experiment

[0913] Probe [ ] (|iM) Number of samples Off-compete [ ] (p. M) (in duplicate)

[0914] 0, 100, 31.6, 10, 3.16, 1, 0.316, 0.1,

[0915] Row A / B: 10 24

[0916] 0.0316, 0.01, 0.00316, 0.001

[0917]

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[0919] 0, 100, 31.6, 10, 3.16, 1, 0.316, 0.1,

[0920] Row C / D: 1 24

[0921] 0.0316, 0.01, 0.00316, 0.001

[0922] 0, 100, 31.6, 10, 3.16, 1, 0.316, 0.1,

[0923] Row E / F: 0.1 24

[0924] 0.0316, 0.01, 0.00316, 0.001

[0925] 0, 100, 31.6, 10, 3.16, 1, 0.316, 0.1,

[0926] Row G / H: 0.01 24

[0927] 0.0316, 0.01, 0.00316, 0.001

[0928]

[0929] Method 20. THP-1 cell lysis

[0930] Cell pellets were thawed for 1 hour at 4 °C before resuspension in RIPA lysis buffer containing 1% SDS and protease inhibitor cocktail (100, L). Cells were incubated in RIPA buffer for 30-minutes at room temperature followed by sonication (QSonica Q500 with cup horn, 4 °C, 4 cycles of 60% amplitude, 4 seconds on / off for 64 seconds). Immediately following lysis, 140. L of additional H2O was added to each cluster tube (final volume 240. L) and MeOH / CHCh precipitation was carried out according to method 3. The samples were then processed using methods 4, 5, 6, 8, 9, and 11.

[0931] Method 21. Ferrioxalate actinometry for assessment of plate-based 440 nm irradiation

[0932] Based on a modified literature procedure17A, 240. L of actinometric solution containing potassium ferrioxalate (200 mM H2SO4, 20 mM K₃Fe(C₂O₄)₃ in H₂O) was aliquoted into 128-strip cluster tubes (96 tubes) in a rack and irradiated with 440 nm light using the plate irradiation apparatus (Fig. 26) for 30 seconds. During the irradiation, 187 uL of spectrophotometric analysis solution (300 mM AcONa, 130 mM H2SO4, and 277 pM phenanthroline) was added to a 96-well plate. Following irradiation, the actinometric solution was diluted with 760 p. L H2O and 25 p. L of this diluted solution transferred to the plate containing the spectrophotometric solution. After the solutions were mixed, the plate was incubated in the dark at room temperature for 1 hour to equilibrate. Absorbance of each individual well was read at 510 nm to detect the light induced formation of iron(II) phenanthroline complexes and assess the evenness of plate irradiation.

[0933] C. THP-1

[0934] - 101 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0935] 3 x IO5THP-1 cells were collected by centrifugation (800 x g, 5 min, 20 °C), and washed twice with 40 mL of cold PBS. Cells were resuspended in cold PBS (0.75 mL) and aliquoted into 8-strip cluster tubes (12 tubes) in a rack and kept on ice (50 pL / aliquot, approximately 20,000 cells) for 20 minutes. Cells were then treated with free catalyst (1 and 2) or a vehicle control (10 pL / aliquot, 6 uM) and incubated on ice for 20 minutes (final volume 60 pL, final concentration of catalyst 1 pM). Samples were then irradiated with 440 nm light for 15 minutes at 4 °C. Following irradiation, cells were stained with trypan blue (1:1 with 0.4%, VWR) and cell viability was calculated with a Countess 3 (Invitrogen).

[0936] U87MG

[0937] 2 x 6-well treated cell culture plates were seeded with 5 x 105U87MG cells and allowed to grow until confluent (~3 days). The media was aspirated and 400 pL of phenol red-free media was pipetted over the adherent cells in each well. DMSO (40 pL) was added to each well and cells were incubated on ice for 20 minutes. Cells were then treated with the free catalysts (1 and 2) or a vehicle control (40 pL, 120 pM) and incubated on ice for 20 minutes (final volume 480 pL, 10 pM catalyst). Each 6-well plate was then irradiated for 15 minutes at 4 °C. Following irradiation, cells were released from the plate, stained with trypan blue (1:1 with 0.4%, VWR), and cell viability was calculated with a Countess 3 (Invitrogen).

[0938] E. K562 cell lysate was prepared according to method 1. 200 pL of 1.5 mg / mL lysate containing 200 pM Dz-Bt was added to 1.5 mL capped centrifuge tube. Added 2 pL of catalyst (100 pM, DMSO) or vehicle and incubated on ice for 30 min. Samples were degassed with argon according to method 3 and irradiated for appropriate amount of time using the apparatus in method 3. Samples were then processed according to methods 4-7. Each experiment was performed in triplicate. Human Plasma Protein Binding: Assay was conducted by Pharmaron.

[0939] Photocatalytic diazirine sensitization assay with different concentrations:

[0940] - 102 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0941] A stock solution of diazirine-biotin (4, final concentration 100 |iM), 2-chloro-5-trifluoromethylpyridine IS (final concentration 100 pM), and water was aliquoted into 15 1.5 mL centrifuge tubes. The catalyst (1) was then added to the tubes at the appropriate concentration (final volume 350 pL), vortexed for 7 seconds to mix, and spun down at 200 x g for 10 seconds. The tubes were degassed with argon, placed 17.8 cm above two 440 nm 100 W COB LED lights (Fig.26), and irradiated for 15 minutes. 100 pL of deuterium oxide was added to the tubes, and samples were transferred to NMR tubes and analyzed by19F NMR spectroscopy. Quantitation was done relative to IS and comparing against a negative control in which the photocatalyst is absent.

[0942] Method 22. Affinity profiling dasatinib with longer incubation time

[0943] K562 cell lysate was prepared according to Method 1. Lysate (0.2 mL, 4 mg / mL) was transferred to cluster tubes and treated with compounds as described in Method 2, but incubation periods were extended to 1 hour instead of 20 minutes for each compound treatment. Sample were irradiated, prepped, and subjected to mass spectrometry according to Methods 3-7, and analyzed using Method 11.

[0944] Method 23. Pooled membrane preparation from whole mouse brains (Flumazenil Affinity Map)

[0945] Based on a modified literature procedure18A, 3 male CD-I (ICR) mouse brains (BioIVT, MSEOOBRAIN-0102904) were thawed on ice for 25 minutes then resuspended in 10 volumes ice cold lysis buffer (50 mM Tris-HCl pH 7.4, containing protease inhibitor cocktail - Roche, 11836170001, approximately 5 mL per brain). The organs were homogenized (Bio-Gen PRO200 Homogenizer) on ice using at medium intensity (6 pulses x 10 seconds). As an initial clearing step, the homogenate was centrifuged (1,000 x g for 10 min at 4 °C) to obtain supernatant. The supernatant was transferred to ultracentrifuge tubes and centrifuged (40,000 x g for 20 minutes at 4 °C; Beckman SW 32 Ti swinging bucket rotor). The supernatant was decanted and discarded and replaced with fresh ice-cold lysis buffer. The pellet was resuspended by homogenization (5 pulses x 10 seconds) and the centrifugation-homogenization was repeated 2 additional times for a total of 3 ultracentrifugations to ensure complete homogenization and wash out of endogenous

[0946] - 103 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0947] ligands (specifically GABA). The final pellet was resuspended in a minimal amount of ice- cold protease-inhibitor-free lysis buffer and protein concentration was determined by bicinchoninic acid (BCA) assay. The freshly prepared membrane suspension was never frozen and used as prepared. Concentrated diazirine-PEG7 -biotin (Dz-Bt) dissolved in cold Tris-HCl was added for a final concentration of 200 pM and a 1 mg / mL protein concentration. The membrane suspension was then aliquoted across 96 cluster tubes (200 pL, 200 pg per sample). Compound treatment and sample processing was then performed according to methods 2-6, 8, 24, and 11 (plate layout in Fig. 25).

[0948] Method 24. Proteomic data acquisition (medium sensitivity).

[0949] Samples (flumazenil Affinity Map experiment only) were analyzed using a Bruker nanoElute 2 / timsTOF Pro 2 nano-UHPLC-IM / MS / MS system using a two- column nano-UHPLC separation method and data independent analysis (DIA) MS method. Oasis HLB plate desalted samples (5 pL) were loaded on a trap column (Waters nanoEase M / Z Symmetry C18 Trap, 2 cm x 180 pm, 5 pm particle size, Part No.:

[0950] 186008821) using 12 volume equivalents of H2O / 0.1% FA. Flow through the trap was then reversed, and peptides eluted through a separation column with an integrated emitter tip (lonOptiks Aurora Ultimate CSI C18 (15 cmx 75 pm, 1.7 pm particle size, Part No.: AUR3-15075C18-CSI) using a 40 minute 2-35% gradient (H2O / 0.1% FA - MeCN / 0.1% FA) at 150 nE / min flow rate with 1400 V capillary voltage. MS analysis was performed using the default short gradient DIA method in HyStar v. 6.2 without alteration.

[0951] Table 6. 1 -Flumazenil probe and flumazenil off-compete concentrations in a 96-well plate

[0952] Probe [ ] (pM) Off-compete [ ] (pM) (in duplicate) Number of samples 0, 10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01,

[0953] Row A / B: 1 24

[0954] 0.00316, 0.001, 0.000316, 0.0001

[0955] 0, 10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01,

[0956] Row C / D: 0.1 24

[0957] 0.00316, 0.001, 0.000316, 0.0001

[0958]

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[0960] 0, 10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01,

[0961] Row E / F: 0.01 24

[0962] 0.00316, 0.001, 0.000316, 0.0001

[0963] Row G / H: 0, 10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01,

[0964] 24

[0965] 0.001 0.00316, 0.001, 0.000316, 0.0001

[0966]

[0967] Computational Methods - Affinity Surveyor

[0968] Peptide identification and quantification - DIA

[0969] Peptide identification and quantification were performed using DIANN 1.8 or 1.9 19A,20A using a library generated in-silico from the human proteome (Uniprot, 12 / 05 / 2022). Digestion was set as “Trypsin / P” (K / R cuts), a maximum of one missed cleavage was allowed, peptide length set to 7-30, charge set to 1-4, precursor mass range set to 300-1800, and using a fragment ion m / z range of 200-1800. Modifications were considered as variable for methionine oxidation, acetylation of N-terminus, N-terminal methionine excision, and cysteine carbamidomethylation. The precursor false discovery rate was set to 1%, mass accuracy and MSI accuracy to 12.5 ppm, and scan window set to 0 with likely inferences removed. Isotopologues and match between runs were enabled. Searches were conducted in double-pass mode using robust, high precision liquid chromatography quantification strategy, protein inference set to genes, library generation set to smart profiling, speed and RAM usage set to optimal results, and with cross-run normalization disabled.

[0970] Preprocessing

[0971] Preprocessing of DIANN output files (report.pg_matrix.tsv, report.pr_matrix.tsv) was done in R (4.4.1) using the MSnbase package separately for each set of samples treated with a given concentration of photocatalyst conjugate. Proteomics data was subjected to variance stabilization normalization (‘vsn’) and left-censored missing value imputation (‘MinProb’) using default settings2IA-22A, reducing noise introduced by sample injection and enabling improved fitting performance for low abundance proteins, and the number of peptides used to quantify each protein determined from the peptide-level report and then added as metadata. Once complete, individually processed MSnSet datasets were combined into a single table.

[0972] - 105 - FOLE YHO AGUS 13146434.108 Attorney Docket CUW-03825

[0973] Affinity Surveyor data preprocessing.

[0974] Preprocessing of DIANN output files (report.pg_matrix.tsv, report.pr_matrix.tsv) was done in R (4.4.1) using the MSnbase package separately for each set of samples treated with a given concentration of photocatalyst conjugate. Proteomics data was subjected to variance stabilization normalization (‘vsn’) and left-censored missing value imputation (‘MinProb’) using default settings,55,56and the number of peptides used to quantify each protein determined from the peptide-level report and then added as metadata. Once complete, individually processed MSnSet datasets were combined into a single table.

[0975] Affinity Surveyor.

[0976] Our approach for apparent (non-equilibrium) binding affinity (A)iapp) elucidation is analogous to a proteome-wide radioligand competitive binding assay, in which streptavidin IP / MS intensity is used as an indirect readout of competitive protein binding site occupancy by a photocatalyst conjugate probe and a competing ligand molecule with distinct Af<iHppvalues (7<fdllgandand Af<iprohc). As photocatalytic proximity labeling measured by mass spectrometry is more complex than a simple radioactivity readout corresponding to receptor occupancy, two assumptions are made to constrain the kinetic model: 1) the MS intensity difference of proteins obtained from streptavidin / biotin IP induced by photocatalyst off-compete by a non-photocatalytic ligand is directly proportional to the occupancy of a single binding site by the photocatalyst conjugate, and 2) protein labeling does not materially alter affinity for the probe or ligand molecules. With these constraints in place, a rearranged Cheng-Prusoff equation can be used to independently solve for the 'dappvalues for each molecule58using protein intensities derived from photocatalytic labeling experiments where the concentrations of both the probe and ligand molecules are systematically varied. All reported affinity profiling experiments use four concentrations of the photocatalyst conjugate and 12 concentrations of competitive non-photocatalytic molecule in duplicate, for a total of 96 samples.

[0977] Affinities were measured in Python (3.12.2). The EC50 of competitive binding of a ligand molecule to each protein in the proteome at each photocatalytic probe concentration

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[0979] for which < 25% of values are imputed is modeled using Equation 1, assuming a Hill coefficient of 1:

[0980] > (1)

[0981]

[0982] i + where [L] = competitive ligand molecule concentration, y = integrated MS intensity for a protein, and a (maximum intensity minus minimum intensity), c (minimum intensity), and the EC50 (concentration of ligand molecule displacing 50% of the quantity of probe molecule present in the absence of competition) are all freely optimized parameters. Nonlinear fitting for Equation 1 was performed using Imfit.59

[0983] During optimization, the initial value for c is set to the global minimum intensity detected from all proteins and samples and the initial a value is set to the maximum of the signal intensity value in samples used for curve fitting. The significance (p-value) of the fit is determined by performing a one-way F-test with the SciPy ‘stats’ module, comparing Equation 1 to the reduced model y = a, representing a null hypothesis in which there is no relationship between ligand molecule concentration and signal intensity. Each competitive model is fit 30 times with different initial values set for the EC50 parameter drawn from a random uniform distribution between -5 and 3 (two order of magnitude beyond the range of concentrations used), retaining the result with the most significant fit. The impact of removing the data point corresponding to treatment with vehicle in place of the unmodified ligand was then tested by repeating the nonlinear fitting process and evaluating whether inclusion of vehicle is required for the fit to remain significant. Fits whose significance depends on the inclusion of the vehicle are removed.

[0984] The relationship between KA and EC50 for pseudo-first order binding kinetics, i.e. at low concentrations of protein ([Protein] « [Ligand]), is described by the Cheng-Prusoff equation34according to Equation 2:

[0985] ligand > >

[0986]

[0987]

[0988] [probe]1probe

[0989]

[0990] where [probe] = probe concentration, A)illgand= dissociation constant of ligand molecule, 'dprobe> dissociation constant of photocatalytic probe, and EC50 = EC50 of competitive binding by the ligand molecule at each probe molecule concentration. Equation 2 can be expressed as Equation 3, a Schild plot:

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[0992] jr ligand

[0993] ECS0= ■ [probe] (3)

[0994]

[0995] Ad

[0996] As EC50 errors are measured in logarithmic space, the Schild plot is modeled in logarithmic space as well (Equation 4) using curve_fit in the scipy. optimize package60:

[0997] ( ligand \

[0998] Vg,and+ ^dprobe• [probe] J (4)

[0999]

[1000] The significance of the modeled 'dllgand / A iprohcvalue is measured with a one-way F-test using the SciPy ‘stats’ module, comparing Equation 4 to a reduced model logio ECso) = a, representing the null hypothesis that there is no relationship between the value of EC50 and the concentration of the probe photocatalyst conjugate. The 'dappof the free molecule (often a drug, metabolite, peptide, or protein of interest) will be different from an observed EC50 by an amount proportional to the ratio of the probe and ligand affinities multiplied by the concentration of its photocatalyst conjugate. As the affinity or concentration of a probe photocatalyst conjugate becomes weaker, the / C1'83"'1value of the unconjugated molecule approaches the measured EC50 value.

[1001] For each protein observed, EC50 determination was performed at each photocatalytic probe concentration and use significant (p < 0.001) values of the ECso to measure 'dprobeand A'<il lgandvia the Cheng-Prusoff equation. Individual proteins have widely varying affinity for the photocatalyst conjugated probe and unmodified ligand molecule; the vast majority do not display any significant EC50 values at any value of [probe], reflecting that they do not bind either the probe or ligand molecules. Other proteins which do bind both the probe and ligand molecules within the range of concentrations used show significant EC50 values. These are then subjected to Cheng-Prusoff analysis to calculate ATiprohcand Kd1 lgand. A tiered approach is used to estimate binding affinities for proteins based on the number of significant EC50 values observed for each protein:

[1002] 1. Three or more significant EC50 values at different probe concentrations, measurable K1 lgand / k'<i|irolic: A iprohcand K,\lgandare directly measured by fitting the data to Equation 4. If the significance of the modeled slope ( f<illga"<l / iprohc) is significant (p < 0.1), both values are reported along with the error from fitting. If the slope is not significant (p > 0.1) and data is tightly distributed (confidence interval < ±0.25), the relative affinity for the probe ligand vs. the ligand molecule is too low to accurately measure. At this limit

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[1004] (^dligand / '(|iirolic« 0), each EC50 ~ '<ill8a"(l; k'<ill8a"(lis therefore reported as the weighted average of the EC50 values in these cases. The weighted average of EC50 values is determined by Equation 5, where / is the weighted average, N is the total number of EC50 used for fitting, i indicates the EC50 values and cr (standard error) of EC50 from fitting in Equation 1 at each value of [probe]. All such proteins are flagged as having modeled A)illgand / ^dprobevalues.

[1005] =

[1006]

[1007] '' Sf I / O,)22. Three or more significant EC50 values at different probe concentrations, insufficient data to measure k'<ill8a"‘l / '<i|irolic: Where the slope is not significant (p > 0.1) and data is not tightly distributed (confidence interval > ±0.25), '(illga"‘l / '<i|iroliccannot be empirically determined. The weighted average EC50 value (calculated using Equation 5) is therefore reported, reflecting the null hypothesis A)illgand / '<i|irolic~ 0).

[1008] 3. Two significant EC50 values at different probe concentrations: Curve fitting with two points cannot afford an accurate estimation of error in the resulting fit. The value measured at the lowest photocatalytic probe concentration is therefore reported as a best estimate of 'dllgandas it is the closest empirical value to the y-intercept in the Schild plot.

[1009] 4. One significant EC50 value at one probe concentration: '(illga"‘lis reported as the EC50.

[1010] 5. Zero significant EC50 values: The most significant EC50 value is reported as 7Cillgand.

[1011] For each protein, the total significance of the data used for A)illgandestimation is reported as the Fisher combined p-values of all fitted EC50 values observed, and this aggregated p-value is then subjected to Benmamini-Hochburg FDR estimation to account for multiple hypothesis testing and represents the significance of all evidence used to support a reported k'<ill8a"‘lvalue. For most proteins, k'<ill8a"‘l / 'dprobeis small because attaching the photocatalyst to a ligand significantly reduces its affinity ( '(illga"‘l« 'dprobe), consistent with the impact of attaching other types of photoaffinity labels; in such cases, weighted average EC50 values give very good estimates of )iappbecause the influence of photocatalytic probe concentration on any observed EC50 value is small. Since proteins that have measurable EC50 values only at high probe concentration (scenarios 2, 3, and 4 above)

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[1013] likely have small 'dllgand / k'<i|irolicratios, and most protein-ligand systems have small 'dllgand / 'dproberatios, single EC50 values (scenario 4) can be used as acceptable best estimates of k'(illg:"l‘l. In scenarios with exactly two significant EC50 values, using the EC50 value obtained at the lower value of photocatalyst concentration provides an improved estimate as it is measured closer to the value of 'dllgandin the Schild plot.

[1014] It was found that the assumption of small values for f<i1 l a"'1 / k'<i|irolicis valid in data generated using photocatalytic affinity profiling for dasatinib, which shows good correlation with reported affinities for kinases with 'dllgandvalues measured using three or more, two, or one significant EC50 value (Fig. 20, Figure 2G). Indeed, correlation between Xdllgandvaiuesmeasured by photocatalytic affinity profiling and literature values is good and has a slope near one, reflecting the validity of both the broader assumptions made to constrain the kinetic model used for analysis and the tiered data processing pipeline approach described herein.

[1015] Dasatinib-diazirine-alkyne labeling.

[1016] K562 cell lysate was prepared according to Method 1. Lysate (0.2 mL, 2 mg / mL) was transferred to cluster tubes and treated with compounds (two experiments detailed in SI Method 13 and Fig. 15) and irradiated using the same apparatus as Fig. 26 with 100-watt 375 nm LEDs for 5 minutes. 34 pL of 7x protease inhibitor cocktail (Roche, 11836170001) was then added, and biotin-PEG4-azide (2.74 pL, 10 mM in DMSO), CuSO4 (1.37 pL, 50 mM in H2O), THPTA (6.85 pL of 10 mM in H2O), and sodium ascorbate (13.7 pL, 50 mM in H2O, freshly made) added, in that order. Note that order of addition is important, adding the first three reagents together as a cocktail and sodium ascorbate last. Samples were inverted end over end for 1 hour at room temperature. Then 280 pL of -20 °C MeOH and 70 pL of CHC13 were added to each cluster tube and mixed, precipitating denatured protein. Samples were centrifuged (2,500 x g, 10 min, 4 °C), affording a protein disk suspended between the MeOH and CHC13 layers. Solvent was carefully aspirated without disrupting the protein disk, then 500 pL of -20 °C MeOH was added to each cluster tube and the protein disk disrupted via sonication (Qsonica Q500 with cup horn, 4 °C, 3 cycles of 60% Amplitude, 4 sec on / off). Samples were centrifuged (2,500 x g, 10 min, 4 °C), resulting in a protein pellet at the bottom of each

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[1018] cluster tube. The MeOH was aspirated, and protein pellets were stored at -80 °C overnight then processed by the protein pellet redissolving, reduction, alkylation, and streptavidin bead enrichment method described herein.

[1019] Sensitization of Das-Dz-alk with UV in Fig 15B:

[1020] 200 pL Dasatinib-dz-alkyne and 4,4’-dimethyl-2,2’-bipyridine IS (as a 50 pM stock in PBS) were aliquoted into 8-strip cluster tubes (15 tubes). The samples were irradiated 17.8 cm above two 375 nm 100 W COB LED lights for the appropriate amount of time (Fig.26). The samples were then analyzed by HPLC, and peaks were quantified by absorbance at 325 nm and standardized based off IS. Each measurement was performed in triplicate, comparing against a negative control with no irradiation.

[1021] Table 8. Probe and off-compete concentrations for the dasatinib-dz-alkyne Affinity Map experiment shown in Fig. 15D

[1022] Probe [ ] (pM) Off-compete [ ] (pM) (in duplicate) Number of samples 0, 10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01,

[1023] Row A / B: 1 24

[1024] 0.00316, 0.001, 0.000316, 0.0001

[1025] 0, 10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01,

[1026] Row C / D: 0.1 24

[1027] 0.00316, 0.001, 0.000316, 0.0001

[1028] 0, 10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01,

[1029] Row E / F: 0.01 24

[1030] 0.00316, 0.001, 0.000316, 0.0001

[1031] Row G / H: 0, 10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01, 24

[1032] 0.001 0.00316, 0.001, 0.000316, 0.0001

[1033]

[1034] Volcano plot construction: pMap target-ID processing for two-sample group comparison

[1035] Processing of DIANN output files (report.pg_matrix.tsv, report.pr_matrix.tsv) was done in R (4.4.1) using the MSnbase package separately for each set of samples treated with given concentration of photocatalyst conjugate and off-compete molecule or vehicle.

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[1037] Proteomics data was subjected to variance stabilization normalization (‘vsn’) in independent experimental groups21 A-22A, imputation using MinProb in MSnbase, and the number of peptides used for each protein inference determined and added as metadata. Once complete, individually processed MSnSet datasets were combined into a single table. Differential expression analysis was performed using linear models (‘limma’)27 Awith Benmamini-Hochburg FDR adjustment to identify significantly enriched proteins. A volcano plot comparing the log2 fold change and -logio(p-value) was generated, with significantly enriched proteins colored by affinity measured using Affinity Map.

[1038] Discussion

[1039] Affinity Map enables improved measurement of polypharmacology at critical decision points in drug development, identifying off-target interactions which contribute to toxicity and undesired side effects. Many such interactions cannot be detected with existing affinity profiling methods due to incompatibility between specific protein physical properties and assay requirements. Hits from qualitative target-ID experiments can either lack activity- based assays or can be challenging to isolate, preventing direct affinity measurement using in vitro approaches. Affinity Map bypasses these limitations by offering a completely target- agnostic binding affinity measurement platform.

[1040] Notably, Affinity Map requires the synthesis of a photocatalytic probe for competition experiments with the corresponding unmodified ligand of interest. Incorporation of the photocatalyst can disrupt target binding, requiring assessment of structure- activity relationships in probe design, and some molecules cannot be easily modified; these represent key limitations of all label-based methods in chemical biology. Affinity Map may be viewed as a complementary approach to label-free methods such as CETSA, which do not require probe molecules as competitive ligands but can fail to identify target proteins if they lack significant ligand-induced stability shifts.

[1041] In developing Affinity Map, the first scalable photocatalytic proximity labeling workflow (>1,200 samples were used to generate data in Figs. 2-5) is also introduced, enabling high throughput applications in target identification for drug-like molecules.

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[1043] Incorporation by Reference

[1044] 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.

[1045] Equivalents

[1046] 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. 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.

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[1148] - 118 - FOLE YHO AGUS 13146434.108

Claims

Attorney Docket CUW-03825CLAIMSWe claim:

1. A complex comprising Iridium (Ir) and a ligand of formula (I):(I),wherein:L is a linker; andZ is a protein-binding ligand.

2. The complex of claim 1, wherein:L is a linker comprising formula (II):-(CHR5)n1-X1-(CHR6)n2-O-(CHR7-CHR8-O)n3-(CHR9)n4-X2-(CHR10)n5- (II); wherein:R5, R6, R7, R8, R9, and R10are each independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl;X1and X2are each independently a bond (absent), -O-, -CH2-, -NH-, -C(O)-, -NHC(O)-, or -C(O)NH-;n1, n2, n3, n4, and n5 are each independently an integer selected from 0 to 100.

3. The complex of claim 1 or 2, having the structure of formula (III):- 119 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-03825(III),wherein:R1, R2, R3, and R4are each independently alkyl optionally substituted with one or more hydroxy, alkoxy, or halo.

4. The complex of any one of claims 1-3, having structural formula (IV):(IV).

5. The complex of any one of claims 1-4, wherein L comprises a polyethylene glycol chain terminated with an amine group.- 120 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-038256. The complex of any one of claims 1-5, wherein L comprises a polyethylene glycol chain comprising from about 1 to about 100 ethylene glycol units.

7. The complex of any one of claims 2-4, wherein L is:-(CH2)n2-O-(CH2-CH2-O)n3-(CH2)n4-.

8. The complex of any one of claims 2-4, wherein L is:-(CH2)2-O-(CH2-CH2-O)n3-(CH2)2-.

9. The complex of any one of claims 1-8, having structural formula (V-A), formula (V-B), or formula (V-C):R1R4(V-A); R1R4(V-B); or- 121 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-03825(V-C).

10. The complex of any one of claims 1-9, having structural formula (VI-A), formula (VI-B), or formula (VI-C):(VI-A);- 122 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-03825(VI-C).

11. The complex of claim 3 or 9, wherein R1, R2, R3, and R4are -N(CH2CH2OH)2.

12. The complex of any one of claims 1-11, wherein the protein-binding ligand is an antibody, protein, peptide, or small molecule.

13. The complex of any one of claims 1-12, wherein Z is a small molecule selected from:- 123 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-03825wherein * indicates a bond to L of the compound of Formula (I).

14. A method of characterizing one or more interactions between a protein-binding ligand and one or more proteins in a biological sample, comprising contacting the biological sample with a complex of any one of claims 1-13.

15. The method of claim 14, further comprising contacting the biological sample with a free protein-binding ligand corresponding to Z; and a crosslinking agent which comprises a labeling moiety (e.g. a diazirine-biotin probe).

16. The method of claim 15, further comprising irradiating the biological sample, thereby generating an activated crosslinking agent which crosslinks with one or more proteins in the biological sample.

17. The method of any one of claim 16, wherein the irradiating comprises a light of 440 nm for at least 60 seconds.

18. The method of any one of claims 14-17, wherein the biological sample comprises one or more cells or the lysates of one or more cells.

19. The method of claim 18, further comprising resuspending the biological sample from the cell, performing a streptavidin enrichment of the resuspended sample, and characterizing the resuspended sample (e.g. using mass spectrometry).- 124 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-0382520. The method of any one of claims 14-19, wherein the method is repeated for two or more concentrations of either the free protein-binding ligand or the complex of any one of claims 1-13.

21. The method of any one of claims 14-20, wherein the method is performed in parallel with at least 10, 100, or at least 1,000 biological samples.

22. The method of any one of claims 14-21, wherein the characterization comprises proteomic binding analysis of at least 10, 100, or 1,000 proteins.

23. The method of any one of claims 14-22, wherein the characterization comprises measuring a binding affinity constant or a kinetic rate constant of at least 10 proteins, lipids, nucleic acids, or carbohydrates.

24. The method of any one of claims 14-23, wherein the concentrations of the free protein-binding ligand and the complex of any one of claims 1-13 have a ratio of 1:1, 2:1, 3:1, 4:

1. 5:

1. 10:

1. 100:1, 1,000:1, 1:1000, 1:2, 1:3, 1:

4. 1:5, 1:

10. 1:100, and / or 1:1,000.

25. The method of any one of claims 14-24, wherein the one or more interactions comprise a protein-protein interaction, protein-DNA interaction, protein-RNA interaction, or protein-small molecule interaction.

26. The method of any one of claims 14-25, wherein the mass spectra measurement is processed by an algorithm, wherein the algorithm characterization has a false negative rate of less than 5%.

27. The method of claim 26, wherein the algorithm has a true positive rate of characterization of at least 95%.

28. A complex comprising Iridium (Ir) and a ligand of structure (IA):- 125 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-03825OH(IA).

29. The complex of claim 28 having the structure:wherein:R1, R2, R3, and R4are each independently alkyl optionally substituted with one or more hydroxy, alkoxy, or halo.

30. A method of generating an affinity map, comprising:(a) contacting a sample, which comprises a set of proteins, with (i) a probe agent, which optionally comprises a protein-binding ligand Z, and (ii) a crosslinking agent, which upon activation is capable of crosslinking with a subset of protein(s) to which the protein-binding ligand is bound; and(b) activating the crosslinking agent, thereby forming a crosslinked sample comprising a subset of crosslinked proteins.- 126 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-0382531. The method of claim 30, wherein a single molecule comprises the probe agent and the crosslinking agent.

32. The method of claim 30, wherein a single molecule comprises a probe agent moiety and a crosslinking agent moiety.

33. The method of claim 30, further comprising, in step (a), contacting the sample with a free protein-binding ligand (e.g. wherein the free protein-binding ligand is the same as Z, or wherein the free protein-binding ligand is different from Z).

34. The method of any one of claims 30-33, wherein, in step (b), activating the crosslinking agent comprises irradiating the sample, thereby activating the crosslinking agent.

35. The method of any one of claims 30-34, wherein, in step (b) the crosslinking agent forms a covalent bond with the protein(s).

36. The method of any one of claims 30-35, wherein the cross-linking agent is a carbene precursor, nitrene precursor, carbocation precursor, carbon-centered radical precursor, amidyl radical precursor, acyl halide precursor, singlet oxygen, RNA, or a electrophilic (Michael acceptor) containing agent.

37. The method of any one of claims 30-36, wherein the crosslinking agent comprises a diazo, diazirine, azido, phenol, or benzophenone moiety.

38. The method of claim 37, wherein the crosslinking agent comprises a diazirine.

39. The method of claim 37, wherein the crosslinking agent comprises a azido.

40. The method of claim 37, wherein the crosslinking agent is tetraphenylazide.- 127 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-0382541. The method of claim 37, wherein the crosslinking agent is nitrophenol.

42. The method of any one of claims 30-41, wherein the crosslinking agent, upon activation, generates an activated crosslinking agent comprising a carbene.

43. The method of any one of claims 30-41, wherein the crosslinking agent, upon activation, generates an activated crosslinking agent comprising a carbocation.

44. The method of any one of claims 30-41, wherein the crosslinking agent, upon activation, generates an activated crosslinking agent comprising a nitrene.

45. The method of any one of claims 30-44, wherein the crosslinking agent comprises a labeling moiety.

46. The method of claim 45 wherein the labeling moiety is biotin, an azide, an alkyne, desthiobiotin, fluoroisothiocyanate (FITC), an antibody, an antibody conjugate, or a biorthogonal enrichment handle.

47. The method of any one of claims 30-46, further comprising (c) isolating the subset of crosslinked proteins (e.g., by performing an immunoprecipitation, such as a streptavidin immunoprecipitation, a gas phase ion separation, or a biorthogonal enrichment modality).

48. The method of any one of claims 30-47, further comprising (d) analyzing the subset of crosslinked proteins.

49. The method of any one of claims 30-48, wherein the crosslinked sample is analyzed by mass spectrometry, nanopore analysis, or reverse translation of peptide.- 128 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-0382550. A method of analysis, comprising performing the method of any one of claims SO-49 on a plurality of samples at a plurality of probe agent and free protein-binding ligand concentrations, andfurther comprising generating the affinity map based on the analysis results, the probe agent concentrations, and the free protein-binding ligand concentrations.

51. The method of claim 50, further comprising systematically varying the concentration of the probe agent and the concentration of the free-binding ligand.

52. The method of claim 51, wherein systematically varying the concentration of the probe agent and the concentration of the free-binding ligand comprises diluting in PBS.

53. The method of any one of claims 50-52, wherein generating the affinity map comprises applying the Cheng-Prusoff equation to the analysis results, the probe agent concentrations, and the free protein-binding ligand concentrations.

54. The method of any one of claims 30-53, wherein the probe agent comprises a photocatalyst coupled or linked to a protein-binding ligand.

55. The method of claim 54, wherein the photocatalyst is a metal complex.

56. The method of claim 54, wherein the photocatalyst is an organic photocatalyst.

57. The method of claim 54 or 55, wherein the photocatalyst is a metal complex comprising Iridium (Ir).

58. The method of claim 57, wherein the photocatalyst is a complex comprising Iridium (Ir) and a ligand of formula (I):- 129 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-03825(I),wherein:L is a linker; andZ is a protein-binding ligand.

59. The method of claim 58, wherein:L is a linker comprising formula (II):-(CHR5)n1-X1-(CHR6)n2-O-(CHR7-CHR8-O)n3-(CHR9)n4-X2-(CHR10)n5- (II); wherein:R5, R6, R7, R8, R9, and R10are each independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl;X1and X2are each independently a bond (absent), -O-, -CH2-, -NH-, -C(O)-, -NHC(O)-, or -C(O)NH-;n1, n2, n3, n4, and n5 are each independently an integer selected from 0 to 100.

60. The method of claim 58 or 59, having the structure of formula (III):R4(III),- 130 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-03825wherein:R1, R2, R3, and R4are each independently alkyl optionally substituted with one or more hydroxy, alkoxy, or halo.

61. The method of any one of claims 58-60, having structural formula (IV):OHOH(IV).

62. The method of any one of claims 58-61, wherein L comprises a polyethylene glycol chain terminated with an amine group.

63. The method of any one of claims 58-62, wherein L comprises a polyethylene glycol chain comprising from about 1 to about 100 ethylene glycol units.

64. The method of any one of claims 58-62, wherein L is:-(CH2)n2-O-(CH2-CH2-O)n3-(CH2)n4-.

65. The method of any one of claims 58-64, wherein L is:-(CH2)2-O-(CH2-CH2-O)n3-(CH2)2-.- 131 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-0382566. The method of any one of claims 58-65, having structural formula (V-A), formula (V-B), or formula (V-C):- 132 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-03825R1(V-C).

67. The method of any one of claims 58-66, having structural formula (VI-A), formula (VI-B), or formula (VI-C):(VI-A);- 133 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-03825(VI-C).

68. The method of claim 60 or 66, wherein R1, R2, R3, and R4are -N(CH2CH2OH)2.

69. The method of any one of claims 58-68, wherein the protein-binding ligand is an antibody, protein, peptide, or small molecule.

70. The method of any one of claims 58-69, wherein Z is a small molecule selected from:- 134 - FOLE YHO AGUS 13146434.108Attorney Docket CUW-03825wherein * indicates a bond to L of the compound of Formula (I).

71. The method of any one of claims 58-70, wherein the sample is a biological sample.

72. The method of claim 71, wherein the biological sample comprises one or more cells or the lysates of one or more cells.

73. The method of any one of claims 30-72, wherein the affinity map identifies the binding affinity of the protein-binding ligand at the plurality of proteins.- 135 - FOLE YHO AGUS 13146434.108

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