[001] Introduction

[002] Tryptophan (Trp) occupies a privileged place in biology owing to its versatile and unique chemistry (1,2). It is accounting for only 1% of amino acids observed in proteins compared to the expected 5% average amino acid frequency (2). As such, tryptophan is an attractive target for site-specific protein functionalization to enable diverse applications, including probes of protein function, activity-based proteomics for functional tryptophan identification and characterization, synthesis of covalent small-molecule inhibitors and activators, and antibodydrug conjugates (3-6). The distinct chemical reactivity of tryptophan arises from its indole moiety, which is hydrophobic and involved in a variety of bonding interactions (e.g., TI-TI, cation- 7i, and hydrogen bonds) (7,8). Compared to its low proteomic abundance, tryptophan is often enriched in functional sites in proteins, contributing to processes such as protein folding and signal transduction (1, 9-11). In this context, tryptophan residues are commonly thought of as buried within interior protein cores to hold/stabilize protein structures, but there is an increasing appreciation for the contributions of solvent-accessible surface tryptophan sites to protein function, including protein trafficking and protein-protein interactions (12,13). Indeed, post- translational modifications of surface-exposed tryptophans, such as oxidation and C- mannosylation, are critical nodes in signal transduction pathways. For example, irreversible oxidation of tryptophan by reactive oxygen species (ROS) can facilitate subsequent metalbinding and/or tryptophan dimerization processes resulting in loss of function (14-16). In contrast, tryptophan C-mannosylation, which occurs in a co-translational fashion in endoplasmic reticulum (ER) catalyzed by DPY19 enzymes, supports the formation of tryptophan-arginine ladders to enhance thermal stability and targeting to the secretory pathways (17,18).

[003] In contrast to the myriad of acid-base approaches for functionalizing cysteine and lysine residues, which are highly nucleophilic, methods for tryptophan modification remain underdeveloped. Despite advances in this area, including transition metal-catalyzed C-H activation (19-22) and photochemically- and electrochemically-induced radical processes (23- 26), reactions that enable site-selective tryptophan bioconjugation in proteins and proteomes and avoid harsh reaction conditions and/or side reactions are an unmet need (2,4). In particular, a major obstacle in developing an effective chemoselective tryptophan bioconjugation reaction under physiological conditions is its relatively weak nucleophilicity at neutral pH, precluding typical acid-base ligation processes. As such, we disclose a redox-based method, termed Tryptophan Chemical Ligation by Cyclization (Trp-CLiC), for selective and rapid tryptophan functionalization from peptides and proteins to proteomes (Fig. la-b, SI). [004] Summary of the Invention

[005] The invention provides methods and compositions for chemoselective cyclization conjugation to indole substrates, including oxidative cyclization reagents for chemoselective tryptophan bioconjugation, and methods of use.

[006] In an aspect the invention provides a redox-based strategy for bioconjugation of tryptophan, the rarest amino acid, using oxaziridine reagents that mimic oxidative cyclization reactions in indole -based alkaloid biosynthetic pathways to achieve highly selective and rapid tryptophan labeling.

[007] In embodiments the invention provides for appending payloads to solvent-accessible surface tryptophan residues on peptides and proteins, enabling functionalization of antibodies, detection of stress-induced protein unfolding, and profiling of hyperreactive tryptophan sites in whole proteomes.

[008] In an aspect, the invention provides a synthetic method for selective and rapid tryptophan bioconjugation in proteins and proteomes by functional mimicry of oxidative cyclization processes in indole-based alkaloid biosynthesis.

[009] In an aspect the invention provides a method of chemoselective conjugation comprising reacting an N-sulfonyl oxazirdine with an indole substrate in an oxidative cyclization reaction in an aqueous (preferably >90% or >95% water), biocompatible environment under conditions to form a resultant cycloadduct conjugation product.

[010] The biocompatible environment is non-denaturing and generally compatible with the preservation of protein structure and function; and in particular, as applied to the subject proteins of the reaction. The conditions are distinct from reactions in generally denaturing organic solvents, with simple indoles, where many different chemical products are formed.

[OH] In embodiments the N-sulfonyl oxaziridine is of formula I, the indole substrate is of formula II, and the cycloadduct is of the corresponding formula III: [012] wherein R1-R7 are independently selected from optionally substituted heteroatom and optionally substituted, optionally hetero-, optionally cyclic C1-C18 hydrocarbyl, and n is an integer 1-5, preferably 1-3 or 1-2.

[013] In embodiments:

[014] R1-R3 and R5-R7 are independently H, C1-C4 alkyl (Me, Et, Pr, Bu) or fully or partially fluorinated C1-C4 alkyl (e.g. CF ₃ ), sulfanyl or fluorosulfanyl (e.g. SF5), C1-C4 alkoxy/ether, ester or carboalkoxy (e.g. OMe, OOMe, or CXEMe), CN, NO2, or phenyl or substituted phenyl, with n substituents, preferably selected from C1-C4 alkyl (Me, Et, Pr, Bu), fully or partially fluorinated C1-C4 alkyl (e.g. CF3), sulfanyl or fluorosulfanyl (e.g. SF5), C1-C4 alkoxy/ether, ester or carboalkoxy (e.g. OMe, OOMe, or CO2Me), CN, NO2;

[015] R4 is an alpha carbon of an amino acid, preferably tryptophan, wherein the amine of the amino acid may be acetylated and the carboxyl maybe be O-methylated, and wherein the amino acid may be a residue of a protein;

[016] R4 is a beta carbon of tryptophan, wherein the tryptophan may be a residue of a protein (with a -CH2- group between the protein backbone and the indole);

[017] R2 is substituted or unsubstituted phenyl;

[018] R3 is H;

[019] R5 is H;

[020] R6 is H;

[021] R7 is H;

[022] R8 is H;

[023] R4 is a residue of a protein;

[024] the indole substrate is a tryptophan substrate, and the method provides a residue-specific bioconjugation strategy for tryptophan-based substrate functionalization;

[025] the indole substrate is a tryptophan substrate of a peptide, a polypeptide, or a protein [026] the indole substrate is a tryptophan substrate of a peptide, a polypeptide, or a protein and the method results in site- and residue-specific modification of the protein, with applications in synthesis and characterization of antibody-drug conjugates and related biologic therapeutics and imaging agents, chemoproteomics and inhibitor design, as well as modifications to study and improve upon protein function, including solubility, stability, and metabolism and pharmacokinetics ;

[027] the indole substrate is a tryptophan substrate of a peptide, a polypeptide, or a protein is an antibody, adeno-associated virus (AAV) capsid protein; the antibody is selected from a single-chain variable fragment antibody, a designed ankyrin repeat proteins (DARPin), and a single variable domain on a heavy chain (VHH) anitbody; and/or [028] the method is combined with stable isotope labeling with amino acid in cell culture (SILAC) or isotope coded affinity tag (ICAT); the method is combined with SILAC or ICAT for quantitative proteomics analysis of tryptophan function in vivo and in vitro by mass spectrometry, with application including but not limited to quantitative analysis of tryptophan reactivity, quantitative analysis of oxidative-sensitive tryptophan, quantitative analysis of C- mannosylated tryptophan and quantitative analysis of C-mannosyltransferase DPY 19 substrates. [029] In an aspect the invention provides a composition comprising an N-sulfonyl oxaziridine of formula I.

[030] In an aspect the invention provides a composition comprising a mixture of an N-sulfonyl oxaziridine of formula I, and an indole substrate of formula II.

[031] In embodiments:

[032] the indole substrate is a tryptophan substrate, preferably a tryptophan residue of a protein. [033] In an aspect, the invention provides a composition comprising a mixture of an N-sulfonyl oxaziridine of formula I, an indole substrate of formula II, and a cycloadduct of the corresponding formula III.

[034] In an aspect the invention provides a composition of a claim herein in an aqueous, biocompatible medium.

[035] In an aspect the invention provides a strategy for bioconjugation to tryptophan using N- sulfonyl oxaziridine -based reagents that mimic oxidative cyclization reactions on indole natural products to achieve highly selective, rapid, and robust tryptophan labeling. The invention provides broad utility of this method for tryptophan functionalization from proteins to proteomes, including synthesis of antibody-drug conjugates and identification of hyperreactive tryptophan sites in proteomes. Commercial applications include:

[036] 1. Therapeutic protein functionalization based on tryptophan bioconjugation such as therapeutic protein pegylation, antibody-drug conjugates, protein labeling for imaging and diagnosis, and alternative protein post translational modifications.

[037] 2. Therapeutic peptides functionalization based on tryptophan bioconjugation such as peptide pegylation, peptide-drug conjugates and other peptide post-translational modifications. [038] 3. Therapeutic intervention based on tryptophan bioconjugation for protein function activation and/or inhibition.

[039] 4. Biomolecule functionalization based on indole bioconjugation using N-sulfonyl oxaziridine compounds such as DNA, RNA, lipid and sugar bioconjugations.

[040] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited. [041] Brief Description of the Drawings

[042] Figs. la-g. Bioinspired strategy for chemoselective tryptophan bioconjugation based on indole oxidative cyclization chemistry, a) Biosynthesis of naturally occurring indole-based alkaloids through two-electron oxidation and subsequent cyclization, b) Bioinspired tryptophan bioconjugation, termed tryptophan chemical ligation by cyclization (Trp-CLiC), where N- sulfonyl oxaziridines promote a parallel two-electron oxidation and cyclization of the tryptophan indole side chain, c) Molecular orbital calculations revealed a higher HOMO energy of tryptophan relative to methionine, indicating that tryptophan is harder to oxidize, d) Molecular orbital calculations on disparate classes of oxaziridine oxidants revealed that the A-sulfonyl oxaziridine derivative has the lowest LUMO energy and is the most potent oxidant, e) Reaction scheme for the Trp-CLiC method. The cycloadduct and the hydroxyl tryptophan are the observed products, f) Evaluation of oxidative cyclization conjugation reactions for Trp-CLiC between Ac-Trp-OMe (50 mM) and various A-sulfonyl oxaziridine reagents (60 mM) for 10 min at room temperature by LC-MS. g) Chemical structures of optimized oxaziridines with alkyne or azide functional handles for addition of synthetic payloads for detection and/or enrichment.

[043] Figs. 2a-g. Trp-CLiC reagents for chemoselective tryptophan bioconjugation on peptides, proteins, and proteomes. a) Tryptophan modification of GLP-1 as a model peptide substrate (50 |tM) with various A-sulfonyl oxaziridine probes (250 pM). GLP-1 possesses one tryptophan, b) Peptide screening showed that the most effective A-sul I'onyl oxaziridine probe for peptide labeling is 0x-W18. c) Crystal structure of IL8 (PDB: 2il8) with one native tryptophan residue shown in stick form, d) Oxidative cyclization conjugation of the IL8 protein (50 pM) with 0x-W18 (300 pM). After denaturing with SDS to increase tryptophan solvent accessibility, IL8 could be labeled with a ca. 70% oxidative cyclization selectivity, e) In-gel fluorescence indicated that IL8 could be labeled by the Trp-CLiC reaction, and this reaction could be significantly inhibited by addition of free tryptophan as a scavenger, f) LC-MS/MS analysis further confirmed the site-specific bioconjugation on tryptophan residues, g) Proteome-wide labeling in HEK 293T cell lysates (1 mg/mL) with 0x-W18 (300 pM) showed that the Trp- CLiC method has high selectivity towards tryptophan over other competing amino acids.

[044] Figs. 3a-h. Rapid surface-exposed tryptophan bioconjugation enables engineered antibody labelling and unfolding stress detection, a) Crystal structure of egg white lysozyme (PDB: lazf) with six native tryptophan residues shown in stick form, b) Time-dependent A260/A280 absorption ratio spectra and in-gel fluorescence imaging showed that oxidative cyclization reactions on tryptophan promoted by Trp-CLiC are rapid, going to completion in less than two min. c) Calibration of A260/A280 absorption ratios revealed that only one tryptophan residue on lysozyme is modified under native folded conditions, d) LC-MS/MS analysis showed that extent of tryptophan labeling correlated with solvent-accessibility of the tryptophan sites. Tryptophan residue solvent accessibility of egg white lysozyme was analyzed by the Discovery Studio program, indicating varying degrees of surface exposure, e) Crystal structure of Her2-Fab (PDB:lfve) with seven native buried tryptophan residues shown as blue stick and the surface- exposed T74 and T198 labeled in red. f) Comparison of labeling of wildtype Her2-Fab and Her2-Fab constructs with two engineered tryptophan sites (T74W or T198W) distributed over the surface established that Trp-CLiC enables site-specific labeling of antibodies with surface tryptophan site, g) Schematic for detection of stress-induced protein unfolding by Trp-CLiC. In the unfolded state, internal tryptophan residues are more solvent exposed and can react with oxaziridine oxidative cyclization reagents. In the folded state, only surface, solvent-accessible tryptophan sites are accessible and less labeling will occur, h) Trp-CLiC labeling of lysozyme under varying concentrations of guanidinium chloride (GdmCl) as a denaturant to induce unfolding showed a dose-dependent increase in tryptophan modifications with more added denaturant, i) HEK 293T cells treated with vehicle control, or with the proteasome inhibitor MG- 132 or the ER stress inducer Tunicamycin, showed increased Trp-CLiC labeling under stress-induced unfolding conditions in whole proteomes. Accumulation of more unfolded proteins leads to a higher extent of tryptophan modifications.

[045] Figs. 4a-g. Trp-CLiC for identification of hyperreactive tryptophan sites in whole proteomes using activity-based protein profiling (ABPP). a) Workflow for reactive and functional tryptophan profiling in proteomes using Trp-CLiC for ABPP, including treatment of proteomes with 0x-W18 probe, followed by CuAAC -based installation of acid-cleavable biotin tag, then enrichment with streptavidin magnetic beads, and sequential on-bead trypsin digestions to afford probe-labelled peptides for LC-MS/MS analysis, b) Proposed CID-cleavage mechanisms of the cycloadduct product. The reporter ions 231.17 and 425.16 could be utilized to further confirm the tryptophan bioconjugation, c) 190 Reactive tryptophan sites were identified in two or three replicates, d) Drug Bank analysis of the identified 1 1 proteins with hyperreactive tryptophan residues, showing that 58% of them are currently classified as nondruggable. e) The Gene Ontology cellular compartment enrichment analysis indicated that many phase- separated compartments are enriched, e.g., nucleolus, nuclear body, nuclear speckle, RNP granule, chromosome, and stress granule. Essential proteins NPM1 for nucleolus and NONO for nuclear speckle were chosen for further functional cellular studies, f) GAPDH and TBA1B were chosen as representative examples for further in vitro validation of ABPP results. As more Ox- W18 is added, more GAPDH and TBA1B could be labeled as analyzed by Western Blot, g) Motif analysis showed that lysine, which is hydrophilic and can form cation-Ti interaction with indole ring to stabilize surface-exposed tryptophan, is highly enriched in the sites close to probed tryptophan residues.

[046] Figs. 5a-i. Trp-CLiC identifies functional tryptophan sites involved in mediating protein phase separation, a) Crystal structure of NPM1 C-terminal domain (PDB: 211h) with two native tryptophan residues on the surface shown in stick form, b) Western blot showed that NPM1 labeling is diminished after tryptophan to alanine mutations, establishing the high specificity for Trp-CLiC method for identifying the two hyperreactive tryptophan residues of this protein target, c-d) Fluorescence imaging showed that tryptophan to alanine mutations cause a loss of phase separation and marked increase in diffuse fluorescence across the nuclear matrix, indicating that mutations of these two key tryptophan sites in the C-terminal region of NPM1 are critical for its physiological nucleolus phase separation. Scale bar: 5 pm. e) Crystal structure of NONO (PDB: 3sde) with one native tryptophan residues on the surface shown in stick form, f) Western blot showed that NONO could not be probed after mutation of the hyperreactive W271 site to alanine, g-i) Fluorescence imaging comparing wildtype and W271A mutant cells confirmed that the W271 site is functional for NONO phase separation and paraspeckle formation. A significant decrease in punctate-positive cells in W271A mutant relative to wildtype is observed, while in W271A cells that do form phase separated compartments, their puncta size is abnormally large compared to wildtype congeners. Scale bar: 5 pm.

[047] Fig. 6. Scheme of oxidative cyclization reagents for chemoselective tryptophan bioconjugation. This metal-free mild Trp-CLiC method is highly Trp-selective and rapid, which can generate products with diverse payloads.

[048] Figs. 7a-b. Screening oxidative cyclization reagents for biocompatible tryptophan conjugation, a). Model of Trp-CLiC reaction between 50 pM Ac-Trp-OMe and 60 pM oxaziridine for 10 min at room temperature in the co-solvent (PBS/MeOH =1:1). The cycloadduct and the hydroxyl tryptophan are the proposed products, b). The reactions were monitored by detecting the relative quantification of peaks intensity with LC-MS. Taken the reaction of Ac-Trp-OMe with 0x-W2 for example, the absorption peaks at 0.33, 0.65, and 1.60 min belong to the hydroxyl tryptophan, Ac-Trp-OMe and cycloadduct, respectively. The conversion and ratio between cycloadduct and hydroxyl product can thus be calculated by the relative peak integration of LC-MS trace.

[049] Figs. 8a-b. Proposed mechanism of Trp-CLiC and reactivity of V-sulfonyl oxaziridine probe towards other residues, a). The proposed Trp-CLiC reaction mechanism between indole and A-sulfonyl oxaziridine. b). Residue reactivity of A-sulfonyl oxaziridine was also tested on other representative amino acids. Methionine and cysteine could be oxidized in the fashion of traceless labeling. Meanwhile, negligible reaction was detected with lysine or tyrosine.

[050] Fig. 9. Chemical structures, molecular orbital analysis and cycloadduct yields of N- sulfonyl oxaziridine library. Finally, a TV- sulfonyl oxaziridine library was designed, synthesized, and screened. The LUMO energies and cycloadduct yields were shown below the chemical structures.

[051] Figs. lOa-e. Characterization of the reaction spectroscopic and kinetics between oxaziridine and tryptophan, a). We tested whether the absorption feature would change significantly upon formation of oxidative cycloadduct and observed a marked blue shift of this band from 280 nm to 245 nm upon treatment with 0x-W18. b). The UV-Vis absorbance of Trp- CLiC reaction between Ac-Trp-OMe with 0x-W18. Different ratios of 0x-W18 was added to the Ac-Trp-OMe (100 pM) in PBS buffer at 25 °C, and then the absorbance was detected after 20 min for reaction completion. The spectroscopic changes showed the absorbance decrease at 280 nm and the intensity increase at 260 nm. c). A260/A280 indicated a linear correlation of the Trp:Ox ratio, which could be utilized to calculate Trp-CLiC reaction process, d). The Trp-CLiC reaction was monitored by UV-Vis with the absorbance at 280 nm upon adding 0x-W18 (50 pM) into Ac-Trp-OMe (500 pM) in PBS buffer at 25 °C. The results were applied to fit the singleexponential equation under pseudo-first order reaction kinetics model to get the observed rate constant k’ . e). After obtained the observed rate constants k’ under different concentrations of Ac-Trp-OMe, the second-order rate constant could be determined by the slope of the k’ against the Ac-Trp-OMe concentrations, which is similar to that of Click chemistry.

[052] Figs, lla-b. Screening oxidative cyclization reactions on Glucagon peptide, a).

Compared to GLP-1 peptide, Glucagon peptide has one tryptophan as well as one methionine, b). Glucagon (50 pM) was reacted with diverse oxaziridines in HjO (5% DMSO) for 10 min. The screening on Glucagon indicated the same result with that on GLP-1 peptide. 0x-W18 had the highest efficiency to form cycloadduct on tryptophan residue. All methionine amino acid could be oxidized without labeling.

[053] Figs. 12a-e. Rapid oxidative cyclization reaction informs solvent-accessible tryptophan on Lysozyme protein, a). In-gel fluorescence imaging showed that oxidative cyclization reaction on BSA tryptophans was rapid, which completed in less than two min. b). The denaturing condition triggered an increase in the labeled tryptophan sites by Trp-CLiC. Ingel fluorescence imaging under denaturing conditions with 0x-W16 labeling showed that both the protein fluorescence signal and molecular weight increase, consistent with more tryptophan sites modified upon protein unfolding. As expected, excess free tryptophan added as a negative control could inhibit tryptophan bioconjugation, c). LC-MS/MS analysis showed that W62, the most surface-exposed site, could be labeled by Trp-CLiC under native folded conditions, whereas the adjacent but more buried site W63 could only be targeted after protein denaturation, d). LC-MS/MS analysis showed that, under denaturing conditions, W62 and W63 would both be labeled, e). Number of unique oxidative cyclization bioconjugation carrying peptides on representative amino acids, namely Trp, Met, Lys, Cys and Tyr by labeling denatured Lysozyme (50 pM) with 1 mM 0x-W18 for 10 min. All the six tryptophan residues in Lysozyme protein were probed by 0x-W18, while no methionine, lysine, cysteine and tyrosine would be targeted. [054] Figs. 13a-b. Stability test of labeled antibody, a). Tryptophan and methionine residue solvent accessibility of Her2-Fab were analyzed by Discovery Studio program, b). The stability assay showed that oxidative cycloadduct of Her2-Fab could time-dependently decompose. The half-life time of the labeled cycloadduct was about 7 hours.

[055] Figs. 14a-b. Investigating the proteome reactivity of V-sulfonyl oxaziridine probe, a). Oxidative cyclization bioconjugation was Trp-selective on cell lysate. Cell lysate (1 mg/ml) was labeled with 0x-W18 (1 mM), and then clicked with azide-dye. Fluorescence imaging showed that Trp-CLiC also works for cell proteome. Excess free Ac-Trp-OMe could significantly inhibit the Trp-CLiC labeling, indicating the chemoselectivity of our method, b). Dose-dependent labeling of cell lysate indicated that 300 pM 0x-W18 was enough for the labeling of proteome (1 mg/ml).

[056] Figs. 15a-d. Bioinformatic analysis of probed tryptophan sites and proteins, a).

Venn diagram of proteins identified in three samples. 151 proteins were probed in more than two replicates, b). Gene Ontology biological pathway enrichment analysis of these 151 proteins, c). Heat map of residue enrichment during motif analysis indicated that charged amino acids lysine, arginine and aspartic acid are enriched in sites close to probed tryptophan residues, d) Analysis of numbers of charged residues near probed tryptophans (+4 and -4, eight residues in total). Compared to two controls, probed tryptophan sites have more neighboring charged residues, which could help stabilize the surface-exposed tryptophan. Control 1 : Not labeled tryptophans identified by LC-MS/MS. Control 2: Not labeled tryptophans in targeted proteins. [057] Figs. 16a-c. Trp-CLiC targets surface-exposed tryptophan residues, a-c). Trp-CLiC strategy revealed hyperreactive tryptophan sites, with W196 on GAPDH (PDB: 1U8F), W346 on TBA1B (PDB: 5IJ0), and W324 and W148 on LDHA enzyme (PDB: lilO) as representative examples. Solvent accessibilities of tryptophan residues in these three target proteins were analyzed by the Discovery Studio program, indicating that the most surface-exposed tryptophan sites were probed whereas other tryptophan residues were not labeled.

[058] Figs. 17a-d. Domain architectures and multiple sequence alignments of NPM1 and NONO proteins, a). NPM1 W288 and W290 are located at the C-terminus, which is the RRM domain for nucleic acid binding, b). NPM1 W288 and W290 highlighted in red are highly conserved cross diverse species, c). NONO W271 is located in the NOPS domain, d). NONO W271 highlighted in red is highly conserved cross diverse species.

[059] Description of Particular Embodiments of the Invention

[060] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or. The examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[061] The term "alkyl" refers to a hydrocarbon group selected from linear and branched saturated hydrocarbon groups of 1-18, or 1-12, or 1-6 carbon atoms. Examples of the alkyl group include methyl, ethyl, 1 -propyl or n-propyl ("n-Pr"), 2-propyl or isopropyl ("i-Pr"), 1-butyl or n-butyl ("n-Bu"), 2- methyl- 1-propyl or isobutyl ("i-Bu"), 1 -methylpropyl or s-butyl ("s-Bu"), and 1,1 -dimethylethyl or t-butyl ("t-Bu"). Other examples of the alkyl group include 1-pentyl, 2 -pentyl, 3 -pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3 -methyl- 1-butyl, 2-methyl- 1-butyl, 1- hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3- pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl and 3,3-dimethyl-2-butyl groups.

[062] Lower alkyl means 1-8, preferably 1-6, more preferably 1-4 carbon atoms; lower alkenyl or alkynyl means 2-8, 2-6 or 2-4 carbon atoms.

[063] The term "alkenyl" refers to a hydrocarbon group selected from linear and branched hydrocarbon groups comprising at least one C=C double bond and of 2-18, or 2-12, or 2-6 carbon atoms. Examples of the alkenyl group may be selected from ethenyl or vinyl, prop-1- enyl, prop-2-enyl, 2-methylprop-l-enyl, but-l-enyl, but-2-enyl, but-3-enyl, buta- 1,3-dienyl, 2- methylbuta-l,3-diene, hex-l-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, and hexa- 1,3-dienyl groups.

[064] The term "alkynyl" refers to a hydrocarbon group selected from linear and branched hydrocarbon group, comprising at least one C=C triple bond and of 2-18, or 2-12, or 2-6 carbon atoms. Examples of the alkynyl group include ethynyl, 1-propynyl, 2-propynyl (propargyl), 1- butynyl, 2-butynyl, and 3-butynyl groups.

[065] The term "cycloalkyl" refers to a hydrocarbon group selected from saturated and partially unsaturated cyclic hydrocarbon groups, comprising monocyclic and polycyclic (e.g., bicyclic and tricyclic) groups. For example, the cycloalkyl group may be of 3-12, or 3-8, or 3-6 carbon atoms. Even further for example, the cycloalkyl group may be a monocyclic group of 3-12, or 3- 8, or 3-6 carbon atoms. Examples of the monocyclic cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, 1 -cyclopent- 1-enyl, l-cyclopent-2-enyl, l-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-l-enyl, l-cyclohex-2-enyl, l-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl groups. Examples of the bicyclic cycloalkyl groups include those having 7-12 ring atoms arranged as a bicycle ring selected from [4,4], [4,5], [5,5], [5,6] and [6,6] ring systems, or as a bridged bicyclic ring selected from bicyclo [2.2. l]heptane, bicyclo[2.2.2]octane, and bicyclo[3.2.2]nonane. The ring may be saturated or have at least one double bond (i.e. partially unsaturated), but is not fully conjugated, and is not aromatic, as aromatic is defined herein.

[066] The term “aryl” herein refers to a group selected from: 5- and 6- membered carbocyclic aromatic rings, for example, phenyl; bicyclic ring systems such as 7-12 membered bicyclic ring systems wherein at least one ring is carbocyclic and aromatic, selected, for example, from naphthalene, indane, and 1,2,3,4-tetrahydroquinoline; and tricyclic ring systems such as 10-15 membered tricyclic ring systems wherein at least one ring is carbocyclic and aromatic, for example, fluorene.

[067] For example, the aryl group is selected from 5- and 6-membered carbocyclic aromatic rings fused to a 5- to 7-membered cycloalkyl or heterocyclic ring optionally comprising at least one heteroatom selected from N, 0, and S, provided that the point of attachment is at the carbocyclic aromatic ring when the carbocyclic aromatic ring is fused with a heterocyclic ring, and the point of attachment can be at the carbocyclic aromatic ring or at the cycloalkyl group when the carbocyclic aromatic ring is fused with a cycloalkyl group. Bivalent radicals formed from substituted benzene derivatives and having the free valences at ring atoms are named as substituted phenylene radicals. Bivalent radicals derived from univalent polycyclic hydrocarbon radicals whose names end in "-yl" by removal of one hydrogen atom from the carbon atom with the free valence are named by adding "-idene" to the name of the corresponding univalent radical, e.g., a naphthyl group with two points of attachment is termed naphthylidene. Aryl, however, does not encompass or overlap with heteroaryl, separately defined below. Hence, if one or more carbocyclic aromatic rings are fused with a heterocyclic aromatic ring, the resulting ring system is heteroaryl, not aryl, as defined herein.

[068] The term "halogen" or “halo” refers to F, Cl, Br or I.

[069] The term "heteroalkyl" refers to alkyl comprising at least one heteroatom.

[070] The term "heteroaryl" refers to a group selected from:

[071] 5- to 7-membered aromatic, monocyclic rings comprising 1, 2, 3 or 4 heteroatoms selected from N, O, and S, with the remaining ring atoms being carbon; [072] 8- to 12-membered bicyclic rings comprising 1, 2, 3 or 4 heteroatoms, selected from N, 0, and S, with the remaining ring atoms being carbon and wherein at least one ring is aromatic and at least one heteroatom is present in the aromatic ring; and

[073] 11- to 14-membered tricyclic rings comprising 1, 2, 3 or 4 heteroatoms, selected from N, 0, and S, with the remaining ring atoms being carbon and wherein at least one ring is aromatic and at least one heteroatom is present in an aromatic ring.

[074] For example, the heteroaryl group includes a 5- to 7-membered heterocyclic aromatic ring fused to a 5- to 7-membered cycloalkyl ring. For such fused, bicyclic heteroaryl ring systems wherein only one of the rings comprises at least one heteroatom, the point of attachment may be at the heteroaromatic ring or at the cycloalkyl ring.

[075] When the total number of S and O atoms in the heteroaryl group exceeds 1, those heteroatoms are not adjacent to one another. In some embodiments, the total number of S and O atoms in the heteroaryl group is not more than 2. In some embodiments, the total number of S and O atoms in the aromatic heterocycle is not more than 1.

[076] Examples of the heteroaryl group include, but are not limited to, (as numbered from the linkage position assigned priority 1) pyridyl (such as 2-pyridyl, 3-pyridyl, or 4-pyridyl) , cinnolinyl, pyrazinyl, 2,4-pyrimidinyl, 3,5-pyrimidinyl, 2,4-imidazolyl, imidazopyridinyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, thiadiazolyl, tetrazolyl, thienyl, triazinyl, benzothienyl, furyl, benzofuryl, benzoimidazolyl, indolyl, isoindolyl, indolinyl, phthalazinyl, pyrazinyl, pyridazinyl, pyrrolyl, triazolyl, quinolinyl, isoquinolinyl, pyrazolyl, pyrrolopyridinyl (such as lH-pyrrolo[2,3-b]pyridin-5-yl), pyrazolopyridinyl (such aslH- pyrazolo[3,4-b]pyridin-5-yl), benzoxazolyl (such as benzo[d]oxazol-6-yl), pteridinyl, purinyl, 1- oxa-2,3-diazolyl, l-oxa-2,4-diazolyl, l-oxa-2,5-diazolyl, l-oxa-3,4-diazolyl, l-thia-2,3-diazolyl, l-thia-2,4-diazolyl, l-thia-2,5-diazolyl, l-thia-3,4-diazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, furopyridinyl, benzothiazolyl (such as benzo[d]thiazol-6-yl), indazolyl (such as fH-indazol-5-yl) and 5,6,7,8-tetrahydroisoquinoline.

[077] The term "heterocyclic" or "heterocycle" or "heterocyclyl" refers to a ring selected from 4- to 12-membered monocyclic, bicyclic and tricyclic, saturated and partially unsaturated rings comprising at least one carbon atoms in addition to 1, 2, 3 or 4 heteroatoms, selected from oxygen, sulfur, and nitrogen. “Heterocycle” also refers to a 5- to 7-membered heterocyclic ring comprising at least one heteroatom selected from N, O, and S fused with 5-, 6-, and/or 7- membered cycloalkyl, carbocyclic aromatic or heteroaromatic ring, provided that the point of attachment is at the heterocyclic ring when the heterocyclic ring is fused with a carbocyclic aromatic or a heteroaromatic ring, and that the point of attachment can be at the cycloalkyl or heterocyclic ring when the heterocyclic ring is fused with cycloalkyl.

[078] “Heterocycle” also refers to an aliphatic spirocyclic ring comprising at least one heteroatom selected from N, 0, and S, provided that the point of attachment is at the heterocyclic ring. The rings may be saturated or have at least one double bond (i.e. partially unsaturated). The heterocycle may be substituted with oxo. The point of the attachment may be carbon or heteroatom in the heterocyclic ring. A heterocyle is not a heteroaryl as defined herein. [079] Examples of the heterocycle include, but not limited to, (as numbered from the linkage position assigned priority 1) 1-pyrrolidinyl, 2-pyrrolidinyl, 2,4-imidazolidinyl, 2,3-pyrazolidinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2,5-piperazinyl, pyranyl, 2- morpholinyl, 3-morpholinyl, oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 1,2- dithietanyl, 1,3-dithietanyl, dihydropyridinyl, tetrahydropyridinyl, thiomorpholinyl, thioxanyl, piperazinyl, homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl, thiepanyl, 1,4-oxathianyl, 1,4-dioxepanyl, 1,4-oxathiepanyl, 1,4-oxaazepanyl, 1,4-di thiepanyl, 1,4-thiazepanyl and 1,4- diazepane 1 ,4-dithianyl, 1,4-azathianyl, oxazepinyl, diazepinyl, thiazepinyl, dihydrothienyl, dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydro thienyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H- pyranyl, 1,4-dioxanyl, 1,3-dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl, pyrazolidinylimidazolinyl, pyrimidinonyl, 1,1-dioxo-thiomorpholinyl, 3- azabicyco[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl and azabicyclo[2.2.2]hexanyl.

Substituted heterocycle also includes ring systems substituted with one or more oxo moieties, such as piperidinyl N-oxide, morpholinyl-N-oxide, 1 -oxo- 1 -thiomorpholinyl and 1, 1-dioxo-l- thiomorpholinyl.

[080] Substituents, such as R1-R8, particularly Rl, R3, R5-R8, are selected from: halogen, -R', -OR ¹ , =0, =NR', =N-0R', -NR'R", -SR', -SiR'R"R"', -OC(O)R', -C(O)R', -CO ₂ R', -CONR’R", - 0C(0)NR'R", -NR"C(0)R', -NR'-C(0)NR"R'", -NR'-S0 ₂ NR’", -NR"C0 ₂ R’, -NH-C(NH ₂ )=NH, -NR'C(NH ₂ )=NH, -NH-C(NH ₂ )=NR’, -S(O)R', -SO ₂ R', -SO ₂ NR’R ", -NR"SO ₂ R, -CN and -NO ₂ , -N3, -CH(Ph) ₂ , perfluoro(Cl-C4)alkoxy and perfluoro(Cl-C4)alkyl, in a number ranging from zero to three, with those groups having zero, one or two substituents being particularly preferred. R, R', R" and R'" each independently refer to hydrogen, unsubstituted (Cl-C8)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with one to three halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(Cl-C4)alkyl groups. When R’ and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6- or 7- membered ring. Hence, -NR’R" includes 1-pyrrolidinyl and 4-morpholinyl, "alkyl” includes groups such as trihaloalkyl (e.g., -CF3 and -CH ₂ CF3), and when the aryl group is 1,2, 3, 4- tetrahydronaphthalene, it may be substituted with a substituted or unsubstituted (C3- C7)spirocycloalkyl group. The (C3-C7)spirocycloalkyl group may be substituted in the same manner as defined herein for "cycloalkyl".

[081] Preferred substituents are selected from: halogen, -R', -OR', =0, -NR'R", -SR', - SiR’R"R’", -OC(O)R’, -C(O)R’, -CO ₂ R’, -CONR’R", -0C(0)NR’R", -NR"C(O)R’, -NR"C0 ₂ R', - NR’-SO ₂ NR"R’", -S(O)R’, -SO ₂ R’, -SO ₂ NR’R", -NR"SO ₂ R, -CN and -NO ₂ , perfluoro(Cl- C4)alkoxy and perfluoro(Cl-C4)alkyl, where R' and R" are as defined above.

[082] The term "fused ring" herein refers to a polycyclic ring system, e.g., a bicyclic or tricyclic ring system, in whcih two rings share only two ring atoms and one bond in common. Examples of fused rings may comprise a fused bicyclic cycloalkyl ring such as those having from 7 to 12 ring atoms arranged as a bicyclic ring selected from [4,4], [4,5], [5,5], [5,6] and [6,6] ring systems as mentioned above; a fused bicylclic aryl ring such as 7 to 12 membered bicyclic aryl ring systems as mentioned above, a fused tricyclic aryl ring such as 10 to 15 membered tricyclic aryl ring systems mentioned above; a fused bicyclic heteroaryl ring such as 8- to 12-membered bicyclic heteroaryl rings as mentioned above, a fused tricyclic heteroaryl ring such as 11- to 14-membered tricyclic heteroaryl rings as mentioned above; and a fused bicyclic or tricyclic heterocyclyl ring as mentioned above.

[083] The compounds may contain an asymmetric center and may thus exist as enantiomers. Where the compounds possess two or more asymmetric centers, they may additionally exist as diastereomers. Enantiomers and diastereomers fall within the broader class of stereoisomers.

All such possible stereoisomers as substantially pure resolved enantiomers, racemic mixtures thereof, as well as mixtures of diastereomers are intended to be included. All stereoisomers of the compounds and/or pharmaceutically acceptable salts thereof are intended to be included. Unless specifically mentioned otherwise, reference to one isomer applies to any of the possible isomers. Whenever the isomeric composition is unspecified, all possible isomers are included. [084] The compounds of the invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds, such as deuterium, e.g. - CD ₃ , CD ₂ H or CDH ₂ in place of methyl. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( ³ H), iodine- 125 ( ¹²⁵ I) or carbon- 14 ( ¹⁴ C). All isotopic variations of the compounds of the invention, whether radioactive or not, are intended to be encompassed within the scope of the invention.

[085] Redox-Based Method for Tryptophan Functionalization Through Bioinspired Oxidative Cyclization.

[086] Inspired by oxidative cyclization reactions on L-tryptophan that generate alkaloid natural products with a versatile hexahydropyrrolo[2,3-b]indole scaffold (27), we reasoned that such a redox-based approach might open a new avenue for tryptophan modification over conventional acid-base chemistry. As a starting point for developing oxidative cyclization warheads for tryptophan bioconjugation, we observed that A-alkoxycarbonyl oxaziridines, which exerted rapid reactivity and high biocompatibility for oxidative methionine functionalization from proteins to proteomes, had negligible reactivity for tryptophan oxidation (4). Indeed, molecular orbital calculations revealed that tryptophan is more difficult to oxidize relative to methionine, as the highest occupied molecular orbital (HOMO) energy of tryptophan (-7.45 eV) is higher than that of methionine (-8.25 eV) (Fig. 1c). These results pointed to the need to develop more reactive oxaziridine reagents for redox-based modification of tryptophan. We then calculated lowest unoccupied molecular orbital (LUMO) energies of several diverse classes of oxaziridines and found that the V-sulfonyl oxaziridine motif can accept electrons the most readily (Fig. Id). [087] Initial reactivity studies with the V-sulfonyl oxaziridine Ox-Wl and N- acetyl -L- tryptophan methyl ester (Ac-Trp-OMe) were then performed in PBS/organic solvent mixture for 10 min at room temperature and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis (Figs 7a-b). Tryptophan reacted with a -50% conversion. Two major products were observed: the desired indole cycloadduct and hydroxyl tryptophan (Fig. le, 8a). The cycloadduct was generated with 28% yield, consistent with simple indoles that can react with such oxaziridines in organic solution (28). Moreover, the reaction of Ox-Wl with methionine could only generate the traceless methionine sulfoxide adduct (Fig. 8b). We then synthesized and screened a library of 20 different oxaziridines for Trp-CLiC chemistry; Ox-WlO, 0x-W17, and 0x-W19 emerged as the top lead candidates with improved -90% selectivity and high reactivity for the indole cycloadduct product, with a measured reaction rate of 24.9 + 1.3MV that is on par with click chemistry reactions (29-31 ) (Fig. If-g, 9-10a-e).

[088] Oxidative Tryptophan Bioconjugation on Peptides, Proteins, and Proteomes.

[089] With lead oxaziridine candidates (0x-W16 to 0x-W21) in hand that bear synthetic handles for subsequent addition of functional payloads via copper-mediated alkyne-azide cyclization (CuAAC) chemistry, we evaluated these Trp-CLiC reagents for tryptophan modification of peptides, proteins, and proteomes. GLP-1, which has a single tryptophan site, was chosen as an initial model peptide substrate (Fig. 2a). From reaction screens between GLP-1 (50 pM) and oxaziridine (300 pM), Ox-Wl 8 which has best solubility showed the highest cycloadduct yield (74%) with quantitative conversion (Fig. 2b). Similar high selectivity and efficiency for tryptophan labeling was observed on Glucagon as a second model peptide substrate, which has one tryptophan and one methionine site (Figs, l la-b). We also observed formation of methionine sulfoxide on Glucagon from oxygen-atom transfer oxidation as a traceless side product. We then went on to label the model protein IL8, which has one partially buried tryptophan site (Fig. 2c). To our delight, this tryptophan residue could be modified only after treatment with a denaturant to increase its solvent accessibility (Fig. 2d-e), and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis confirmed formation of the oxidative cycloadduct product (Fig. 2f). Finally, we tested the reactivity and selectivity of tryptophan labeling with 0x-W18 in whole proteomes using human embryonic kidney 293T (HEK 293T) cell lysates. Trypsin digestion with subsequent LC-MS/MS analysis established that 0x-W18 has high selectivity for tryptophan modification in proteomes (Fig. 2g) (32). Taken together, the data indicate that Trp-CLiC is an effective strategy for tryptophan bioconjugation across peptide, protein, and proteome levels, with A-sull'onyl oxaziridine derivatives emerging as privileged reagent scaffolds for promoting oxidative cyclization reactions on tryptophan to yield the corresponding indole cycloadduct products.

[090] Oxidative Tryptophan Bioconjugation is Rapid and Targets Solvent-accessible Surface Tryptophan Sites.

[091] After successfully demonstrating tryptophan labeling on peptides, proteins, and proteomes using Trp-CLiC, we sought to characterize features of this novel bioconjugation method in more depth using lysozyme as a model protein substrate that contains six distinctive tryptophan residues (Fig. 3a). Because of its indole ring, tryptophan exhibits a unique absorption signature centered around 280 nm, which is commonly applied to measure protein concentrations (33). Based on spectroscopic analyses (Figs. 12a-e), we used the ratio of absorption signatures at 260 and 280 nm (A260/A280 ratio) to track tryptophan bioconjugation on lysozyme. Time-dependent modification of lysozyme with 0x-W18 (1 mg/mL denatured protein, 1 mM Ox reagent) monitored by A260/A280 ratio changes showed completion of the reaction in less than two minutes (Fig. 3b). In-gel fluorescence imaging assays on modified lysozyme and bovine serum albumin (BSA) after 0x-W18 labeling, followed by subsequent click functionalization with fluorescent dyes, further confirmed the rapid reactivity of 0x-W18 towards tryptophan (Fig. 3b, 12a).

[092] We next evaluated how many and which tryptophan residues on lysozyme are prioritized for redox-based Trp-CLiC labeling under native folded conditions. The observed A260/A280 ratio was 0.80, which correlated to a single tryptophan residue being labeled (Fig. 3c). Moreover, in-gel fluorescence imaging after denaturing showed that both the protein fluorescence signal and molecular weight increase, consistent with a greater number of tryptophan residues modified upon protein unfolding (Fig. 3c, 12b). We then moved on to identify which tryptophan sites are targeted and tested the hypothesis that the propensity of oxidative tryptophan modification reactivity might correlate with solvent accessibility. To this end, we used the Discovery Studio platform to calculate the relative solvent accessibility of the six tryptophan residues on lysozyme (Fig. 3d). Trypsin digestion and LC-MS/MS analysis of Ox-W18-labeled lysozyme revealed that W62, the most surface-exposed tryptophan site, was also the most targeted site for native labeling from the labeled/unlabeled peptide count ratio (Fig. 3d, 12c). In contrast, the adjacent W63 site could only be modified with the 0x-W18 reagent after denaturing the protein to leverage its solvent accessibility (Figs. 12d-e). These data establish that the Trp-CLiC oxidative cyclization method shows preferential labeling of surface-exposed surface tryptophan residues.

[093] Oxidative Tryptophan Bioconjugation Enables Modification of Engineered Antibody Scaffolds and Detection of Stress-Induced Protein Unfolding.

[094] As the Trp-CLiC method enables rapid and specific tryptophan bioconjugation and preferentially targets surface-exposed tryptophan residues, we sought to exploit this selectivity for various protein labeling applications. As a first example to showcase the Trp-CLiC approach in this context, we introduced tryptophan residues into solvent- accessible locations for single- site-selective bioconjugation of antibody scaffolds. First, the solvent accessibilities of native tryptophan residues within Her2-Fab were analyzed, indicating that all endogenous tryptophan residues in this scaffold appear to be partially or totally buried (Fig. 3e, 13a). With this information in hand, we engineered two Her2-Fab constructs (Her2-Fab T74W and T198W), each possessing one de novo tryptophan residue distributed over the surface of the antibody. Indeed, the Trp-CLiC reagent showed a clear preference to target solvent-accessible engineered tryptophan residue with high selectivity and reactivity (>80%). In contrast, negligible bioconjugation was detected on wildtype Her2-Fab (Fig. 3f). The modest stability of such constructs precluded further evaluation in vivo (Fig. 13b), but the results provide a starting point for antibody functionalization via site-specific tryptophan modification.

[095] In another application, we used Trp-CLiC as a general method to probe stress-induced protein unfolding. Indeed, under various environmental stresses, folded proteins will expose more hydrophobic residues as they unfold, and accumulation and aggregation of unfolded proteins is regarded as a signal of loss of proteostasis (34-37). Owing to its sterically demanding and hydrophobic properties, tryptophan residues are more likely to be buried in protein cores. We reasoned that this oxidative cyclization method could enable monitoring of increases in solvent accessibility of buried tryptophan residues during stress -induced protein unfolding (Fig. 3g). To test this hypothesis, we labeled lysozyme as a model protein in the presence of varying concentrations of guanidinium chloride (GdmCl) as a denaturant. As anticipated, changes in A260/A280 ratios consistent with tryptophan labeling were observed in a dose-dependent manner with increasing GdmCl concentrations without protein sequence bias, deciphering quantitative unfolding transitions which fit the three-state model (Fig. 3h). We then used Trp- CLiC to monitor stress -induced protein unfolding in whole cell proteomes. HeLa cells were treated with the proteasome inhibitor MG- 132, the ER stress inducer Tunicamycin to trigger unfolded protein stress responses, or vehicle control, then lysed and labeled with A-sulfonyl oxaziridine linked to desthiobiotin. Indeed, more desthiobiotin staining signal was observed in environmentally stressed cells compared to control counterparts, consistent with higher levels of tryptophan labeling due to increased solvent exposure of internal tryptophan residues during protein unfolding (Fig. 3i).

[096] Oxidative Tryptophan Bioconjugation Enables Activity-Based Profiling of Functional Tryptophan Sites in Proteomes.

[097] We next moved on to apply Trp-CLiC at the whole proteome level, specifically to identify new functional tryptophan sites in cells using activity-based protein profiling (ABPP). HEK 293 T cell lysates were treated with the 0x-W18 probe in a standard ABPP workflow, followed by installation of acid-cleavable biotin enrichment tag using CuAAC click chemistry, capture with streptavidin-labeled magnetic beads, on-bead trypsin digestion, and LC-MS/MS analysis of probe-modified peptides (Fig. 4a, 14a-b). Interestingly, we observed that the oxidative indole cycloadduct product after acid-cleavage is collision-induced dissociation (CID)- cleavable, providing two reporter ions at m/z: 213.17 and 425.16 that could be used to selectively identify the targeted tryptophan sites with higher confidence (Fig. 4b).

[098] From the ABPP studies with the Trp-CLiC method, 190 unique hyperreactive tryptophan sites from 151 probe-modified proteins were identified in more than two replicates (Fig. 4c, SlOa). These Ox-W18-labeled tryptophan sites were found in many classes of proteins, including enzymes, chaperones, and nucleoproteins, as well as many structural proteins.

Meanwhile, we noticed that many targets are classified as non-druggable and highly enriched in diverse phase-separated compartments, such as the nucleolus, nuclear body, paraspeckle and stress granule (Fig. 4d-e, 15b). Moreover, global analysis of hyperreactive tryptophan sites identified by Trp-CLiC ABPP showed a preference for surface-exposed tryptophan residues. For example, the hyperreactive W196 site identified on GAPDH, W346 site found on TBA1B, and W324 and W148 on LDHA enzyme are the most surface-exposed tryptophan residues in these proteins, whereas other tryptophan residues were not probed (Fig. 16a-c). Interestingly, the W196 site on GADPH is a crucial residue, as the corresponding W196F mutant leads to increased free radical-induced aggregation of this protein target (34). GAPDH and TBA1B were chosen as representative examples to showcase the Trp-CLiC method for ABPP. Increased Ox- W18 addition resulted in more GAPDH and TBA1B labeling as determined by Western Blot analysis (Fig. 4f). We then sought to further investigate the character of probed solvent- accessible tryptophan sites by performing motif analysis. Interestingly, we observed that lysine is highly enriched in sites near probe-modified tryptophan residues. Lysine is hydrophilic and has the capacity to form cation-n interactions with the indole tryptophan ring, providing a potential pathway for stabilizing hydrophobic tryptophan residues being surface-exposed (Fig. 4g, 15c-d) (38, 39).

[099] As we also observed that the probe-modified proteins from Trp-CLiC ABPP are highly enriched in phase- separated subcellular compartments, we hypothesized that this method could be employed to identify not only proteins involved in phase separation but specific functional tryptophan sites on those protein targets, where privileged, surface-exposed tryptophan residues could mediate protein phase separation and mutations of such sites could trigger disease-relevant dysregulation of physiological compartmentalization. In this context, NPM1 is the scaffold protein of granular component subcompartment of the nucleolus, and mutations of the two highly conserved tryptophan residues (W288 and W290) located on the C-terminal RRM domain surface are the most common genetic lesions found in acute myeloid leukemia (Fig. 5 a, 17a-b) (40, 41 ). Indeed, Western blot analysis detected decreased or negligible fluorescence signal after either or both tryptophan residues were mutated to alanine, indicating that these two tryptophan sites are essential for NPM1 labeling by the Trp-CLiC method (Fig. 5b). We then proceeded to perform fluorescence imaging assays in cells to monitor functional changes in NPM1 distributions between wildtype and alanine mutants. Interestingly, mutation of these C- terminal tryptophan sites resulted in an alteration of NPM1 localization and phase separation, leading to a more diffuse fluorescence signal detected across nucleus matrix that identified the importance of these functional residues (Fig. 5c-d). As a second example to showcase this Trp- CLiC method for identification of functional tryptophan sites in mediating phase separation, NONO protein is the necessary factor for paraspeckle formation and the mutation of the conserved W271 is related to mental retardation (Fig. 5e, 17c-d). (42) W271 was also Trp-CLiC targeted (Fig. 5f) and mutation of this single residue also markedly influenced NONO-mediated phase separation and paraspeckle formation (Fig. 5g-i). In detail, we observed a significant decrease in punctate-positive cells in W271 A mutant relative to wildtype, but in W271 A cells that did form phase separated compartments, their puncta size was abnormally large compared to the wildtype congeners. As such, the Trp-CLiC method provides an effective strategy for discovering and characterizing functional tryptophan residues in proteomes and their potential roles in disease-relevant processes.

[0100] Trp-CLiC provides a unique and general redox-based method that enables chemoselective tryptophan bioconjugation to complement traditional acid-base strategies for more nucleophilic amino acids such as cysteine and lysine. Biosynthesis of indole-based alkaloids provides inspiration for the development of oxaziridine reagents that enable selective and rapid oxidative cyclization on indole side chains of tryptophan from proteins to proteomes. Despite its rarity in eukaryotic organisms, tryptophan plays crucial roles in stabilizing protein secondary structures, promoting enzymatic activity, and mediating protein-protein interactions. Moreover, post-translational modifications such as tryptophan oxidation and C-mannosylation also contribute to signal transduction and protein secretion. Indeed, in this report we highlighted the use of Trp-CLiC to identify and characterize new functional disease-related tryptophan sites that regulate protein-mediated phase separation processes. Owing to its rapid and specific tryptophan redox reactivity with a preference for solvent-accessible surface tryptophan sites, Trp-CLiC offers a broadly useful chemical platform for precise addition of payloads to proteins, identifying and characterizing functional tryptophans in whole proteomes, and a starting point for therapeutic strategies that engage tryptophan reactivity via activation and/or inhibition.

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Electrophiles. ChemRxiv https://chemrxiv.org/engage/chemrxiv/article- details/60c755f2bb8cla7d393dc505 (2021).

[0134] 33. H. Edelhoch, Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948-1954, (1967).

[0135] 34. C. Hetz et al, Targeting the unfolded protein response in disease. Nat. Rev. Drug Discovery 12, 703-719, (2013).

[0136] 35. M. Z. Chen et al, A thiol probe for measuring unfolded protein load and proteostasis in cells. Nat. Commun. 8, 474-483, (2017).

[0137] 36. E. J. Walker et al, Global analysis of methionine oxidation provides a census of folding stabilities for the human proteome. Proc. Natl. Acad. Sci. U. S. A. 116, 6081-6090, (2019).

[0138] 37. A. L. Samson et al, Oxidation of an Exposed Methionine Instigates the Aggregation of Glyceraldehyde-3 -phosphate Dehydrogenase. J. Biol. Chem. 289, 26922-26936, (2014).

[0139] 38. J. C. Ma, D. A. Dougherty, The cation-7i interaction. Chem. Rev. 97, 1303-1324, (1997).

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[0142] 41. D. L. J. Lafontaine et al, The nucleolus as a multiphase liquid condensate. Nat. Rev. Mol. Cell Biol. 22, 165-182, (2020). [0143] 42. D. M. Passon et al, Structure of the heterodimer of human NONO and paraspeckle protein component 1 and analysis of its role in subnuclear body formation. Proc. Natl. Acad. Sci. U. S. A. 109, 4846-4850, (2012).

[0144] 1. Synthesis of V-sulfonyl oxaziridine library

[0145] 1.1 Characterization of indole cycloadduct

[0146] To a stirring suspension of oxaziridine (160 mg, 0.5 mmol, 1.0 equiv.) in MeCN (3 mL) was added Ac-Trp-OMe (130 mg, 0.5 mmol, 1.0 equiv.) as a solution in MeOH (2 mL). Additional MeOH (1 mL) was used to quantitatively transfer the reagent. The mixture was stirred at room temperature for 16 h, then the solvent removed in vacuo. Purification by silica gel chromatography afforded the desired cycloadduct (37 mg, 13%) as a white solid. One diastereomer could be isolated (18 mg, 10:1 d.r.) and characterized below.

[0147] ^] H NMR (600 MHz, MeOD) 5 7.95 - 7.89 (m, 2H), 7.48 - 7.42 (m, 2H), 7.27 - 7.23 (m, 2H), 7.21 (m, 1H), 7.17 (m, 1H), 7.07 (d, J = 8.0 Hz, 2H), 6.77 (t, J = 7.5 Hz, 1H), 6.68 (d, J = 8.0 Hz, 1H), 5.92 (s, 1H), 5.48 (s, 1H), 4.95 (dd, J = 9.5, 3.1 Hz, 1H), 3.71 (s, 3H), 2.76 (dd, J = 15.1, 3.1 Hz, 1H), 2.36 - 2.27 (m, 4H), 1.97 (s, 3H).

[0148] ¹³ C NMR (151 MHz, MeOD) 8 173.6, 173.3, 151.4, 150.1, 145.3, 142.4, 138.9, 132.0, 131.7, 130.2, 128.6, 127.6, 125.2, 123.8, 120.2, 110.7, 92.1, 89.5, 80.8, 53.1, 38.4, 22.6, 21.4. [0149] HRMS (ESI): Calculated for C28H29N4O8S [M+H]+: 581.1701. Found 581.1727.

[0150] 1.2 Synthesis of /V-sulfonyl oxaziridine library

[0151] General procedure A: 's' ** 'R2

1 .2 equiv. 1 equiv. R ¹ K ₂ CO ₃ (sat. aq.) R ¹ rt, 1 h

[0152] To a solution of sulfonamide (10 mmol, 1.0 equiv.) and aldehyde (12 mmol, 1.2 equiv.) in dry THF (30 mL) was added TiLPrOL (4.1 mL, 14 mmol, 1.4 equiv.) dropwise at room temperature. The reaction was monitored by 1H NMR using small aliquots dissolved in CeD ₆ . After 16-48 h, the reaction mixture was diluted with CH2CI2 (50 mL) and water (50 mL), and extracted with additional CH2CI2 (3 x 50 mL). The combined organic phases were concentrated in vacuo until a total volume of ~ 40 mL to afford a solution of crude imine which was used directly in the next step. [0153] In a separate round-bottom flask, mCPBA (75%, 6.9 g, 30 mmol, 3.0 equiv.) was prestirred in 1:1 CFhCh/sat. aq. K2CO3 (60 mL) at room temperature for 10 minutes. The solution of crude imine from the previous step was added via addition funnel over 5 minutes, using additional CH2CI2 (10 mL) rinses for a quantitative transfer. The reaction was monitored by TLC with staining with phosphomolybdic acid (PMA); the oxaziridine product stains as a distinctively dark spot. After completion of the reaction (~1 h), the reaction was diluted with CH2CI2 (50 mL) and washed with sat. aq. NaHCCL (30 mL) and brine (30 mL). The combined organic layers were dried over Na ₂ SO4, filtered and concentrated in vacuo. Purification by column chromatography afforded the corresponding oxaziridine.

[0154]

[0155] General Procedure A was followed starting from methanesulfonamide (951 mg, 10 mmol, 1.0 equiv.) and benzaldehyde (1.2 mL, 12 mmol, 1.2 equiv.). Purification by column chromatography (0% to 30% EtOAC/hexane) afforded the corresponding oxaziridine (437 mg, 22%) as a white solid. Spectral data matched literature reports. ¹

[0156] ^X H NMR (300 MHz, CDC1 ₃ ) 5 7.55 - 7.38 (m, 5H), 5.46 (s, 1H), 3.23 (s, 3H).

[0157]

[0158] (£)-A-benzylidenebenzenesulfonamide (1.0 g, 4.1 mmol, 1.0 equiv.) and NEt ₃ BnCl (112 mg, 0.49 mmol, 0.12 equiv.) were taken up in CHCI3 (4 mL) and sat. aq. NaHCCL, (4 mL). mCPBA (75%, 1.125 g, 4.89 mmol, 1.2 equiv.) added dropwise as a solution in CH2CI2 (10 mL) over 30 minutes and the reaction was stirred at room temperature for 3 h. The organic layer was separated and washed with 10% Na2SO3, sat. aq. NaHCCL, brine, dried over Na2SO4, filtered and concentrated in vacuo. Purification by column chromatography (0% to 20% EtOAc/Hex) afforded the corresponding oxaziridine (749 mg, 70%) as a white solid. Spectral data matched

2 literature reports.

[0159] ^X H NMR (300 MHz, CDC1 ₃ ) 5 8.10 - 8.03 (m, 2H), 7.76 (d, J = 14 Hz, 1H), 7.64 (dd, J = 8.5, 7.1 Hz, 2H), 7.53 - 7.37 (m, 5H), 5.49 (s, 1H).

[0160]

[0161] General Procedure A was followed starting from 4-methoxyphenylsulfonamide (1.1 g, 5.9 mmol, 1.0 equiv.) and benzaldehyde (0.73 mL, 7.1 mmol, 1.2 equiv.). Purification by column chromatography (0% to 40% EtOAc/hexane) afforded the corresponding oxaziridine (1.1 g, 37%) as a white solid. Spectral data matched literature reports.

[0162] ^X H NMR (300 MHz, CDCI3) 8 8.03 - 7.93 (m, 2H), 7.53 - 7.34 (m, 5H), 7.13 - 7.04 (m, 2H), 5.43 (s, 1H), 3.92 (s, 3H).

[0163]

[0164] 0x-W4 was prepared according to the standard procedure. The crude material was initially purified on a silica gel Biotage column (25G) with a gradient of 2-20% EtOAc/hexanes, but remained partially impure by TLC analysis. A portion of this material was dissolved in CH2Q2, loaded onto and purified on a preparatory plate (silica gel base) using 10% EtOAc/hexanes as the mobile phase. A clear color oil which crystallized to form a white solid (294 mg., 1.1 mmol, 37% yield from 3 mmol of sulfonamide starting material) was obtained upon storage at -20 °C.

[0165] ^X H NMR (400 MHz, Chloroform-^ 6 7.63 - 7.37 (m, 5H), 5.28 (s, 1H), 3.58 - 3.43 (m,

4H), 1.79- 1.68 (m, 4H), 1.68 - 1.59 (m, 2H). (Contains ~5% of the aldehyde starting material as an impurity).

[0166] ¹³ C NMR (126 MHz, CDC1 ₃ ) 5 131.3, 131.3, 128.9, 128.3, 76.4, 48.0, 25.5, 23.7.

[0167] HRMS: Calculated for Ci2Hi ₆ N ₂ O ₃ SNa [M+Na] ⁺ = 291.0774. Measured [M+Na] ⁺ =

291.0749

[0168]

[0169] General Procedure A was followed starting from 4-methylbenzenesulfonamide (1.71 g,

10 mmol, 1.0 equiv.) and 4-nitrobenzaldehyde (1.81 g, 12 mmol, 1.2 equiv.). Purification by column chromatography (0% to 40% EtOAc/hexane) afforded the corresponding oxaziridine (324 mg, 11%) as a white solid. Spectral data matched literature reports. ⁴ [0170] ^X H NMR (300 MHz, CDC1 ₃ ) 8 8.31 - 8.22 (m, 2H), 7.93 (d, J = 8.3 Hz, 2H), 7.64 (d, J =

8.8 Hz, 2H), 7.45 (d, J = 8.1 Hz, 2H), 5.56 (s, 1H), 2.51 (s, 3H).

[0171]

[0172] General Procedure A was followed starting from 4-methylbenzenesulfonamide (1.71 g, 10 mmol, 1.0 equiv.) and 2-nitrobenzaldehyde (1.81 g, 12 mmol, 1.2 equiv.). Purification by column chromatography (20% to 100% EtOAc/hexane) afforded the corresponding oxaziridine (1.8 g, 55%) as a white solid. Spectral data matched literature reports. ⁵

[0173] ^X H NMR (300 MHz, CDC1 ₃ ) 8 8.23 (dd, J = 8.1, 1.4 Hz, 1H), 8.02 - 7.92 (m, 2H), 7.75

- 7.55 (m, 3H), 7.43 (m, 2H), 5.98 (s, 1H), 2.48 (s, 3H).

[0174]

[0175] A modified General Procedure A was followed starting from 4- methylbenzenesulfonamide (2.4 g, 14 mmol, 1.0 equiv.), dry acetone (6 mL), and Ti(zPrO)4 (10 mL, 34 mmol, 2.4 equiv.) in CH2Q2 (20 mL). Purification by column chromatography (10% EtOAc/hexane) afforded the corresponding oxaziridine (256 mg, 8%) as a colorless oil. Spectral data matched literature reports. ⁶

[0176] ^X H NMR (300 MHz, CDC1 ₃ ) 8 7.93 - 7.83 (m, 2H), 7.43 - 7.32 (m, 2H), 2.46 (s, 3H),

2.08 (s, 3H), 1.56 (s, 3H).

[0177]

[0178] General Procedure A was followed starting from 4-methylbenzenesulfonamide (1.71 g,

10 mmol, 1.0 equiv.) and 4-trifluoromethylbenzaldehyde (1.6 mL, 12 mmol, 1.2 equiv.).

Purification by column chromatography (0% to 20% EtOAc/hexane) afforded the corresponding

■7 oxaziridine (1.9 g, 56%) as a white solid. Spectral data matched literature reports.

[0179] ^X H NMR (300 MHz, CDC1 ₃ ) 8 7.98 - 7.88 (m, 2H), 7.67 (d, 7 = 8.2 Hz, 2H), 7.62 - 7.52

(m, 2H), 7.50 - 7.39 (m, 2H), 5.51 (s, 1H), 2.51 (s, 3H).

[0180]

[0181] General Procedure A was followed starting from piperidine- 1 -sulfonamide (1.6 g, 10 mmol, 1.0 equiv.) and 4-trifluoromethylbenzaldehyde (1.6 mL, 12 mmol, 1.2 equiv.).

Purification by column chromatography (0% to 20% EtOAc/hexane) afforded the corresponding oxaziridine (2.6 g, 77%) as a white solid.

[0182] ^X H NMR (300 MHz, CDC1 ₃ ) 5 7.68 (d, J = 8.3 Hz, 2H), 7.59 (d, J= 8.2 Hz, 2H), 5.33 (s, 1H), 3.51 (dd, 7 = 6.3, 4.5 Hz, 4H), 1.79 - 1.55 (m, 6H).

[0183] ¹³ C NMR (126 MHz, CDC1 ₃ ) 5 135.2, 133.3 (q, 7 = 32.5 Hz), 128.7, 125.9 (q, 7 = 3.8

Hz), 123.7 (q, J = 273 Hz), 75.3, 48.04, 25.5, 23.6.

[0184] ¹⁹ F NMR (376 MHz, CDC1 ₃ ) 8 -62.2.

[0185] HRMS (ESI): Calculated for C13H19F3N3O3S [M+NH ₄ ]+: 354.1093. Found 354.1071.

[0186]

[0187] General Procedure A was followed starting from 4-methoxybenzenesulfonamide (562 mg, 3 mmol, 1.0 equiv.) and 2,4-dinitrobenzaldehyde (706 mg, 3.6 mmol, 1.2 equiv.).

Purification by column chromatography (10% to 100% EtOAc/hexane), followed by trituration with 1:1 pentane/Et ₂ O afforded the corresponding oxaziridine (281 mg, 25%) as a white solid. [0188] ^X H NMR (300 MHz, CDC1 ₃ ) 8 9.06 (d, 7 = 2.3 Hz, 1H), 8.54 (dd, J = 8.6, 2.3 Hz, 1H), 8.07 - 7.96 (m, 2H), 7.85 (d, 7 = 8.6 Hz, 1H), 7.15 - 7.04 (m, 2H), 6.06 (s, 1H), 3.93 (s, 3H). [0189] ¹³ C NMR (126 MHz, CDC1 ₃ ) 8 165.5, 149.2, 148.4, 134.0, 132.6, 131.0, 128.7, 123.9, 120.6, 115.0, 74.8, 56.1.

[0190] HRMS (ESI): Calculated for Ci ₄ HnN ₃ O ₈ SNa [M+Na]+: 404.0159. Found 404.0140.

[0191]

[0192] Ox-Wl 1 was prepared according to the standard procedure. The crude material was initially purified on a silica gel Biotage column (25G) with a gradient of 10-30% EtOAc/hexanes, but remained partially impure by TLC analysis. A portion of this material was dissolved in CH2CI2, loaded onto and purified on a preparatory plate (silica gel base) using 30% EtOAc/hexanes as the mobile phase. A yellow oil which crystallized to form a yellow-white solid (176 mg., 0.49 mmol, 16% yield from 3 mmol of sulfonamide starting material) was obtained upon storage at -20 °C.

[0193] ^X H NMR (500 MHz, Chloroform-^/) 8 9.07 (d, J = 2.3 Hz, 1H), 8.57 (dd, J = 8.5, 2.3 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 6.05 (s, 1H), 3.68 - 3.35 (m, 4H), 1.78 - 1.69 (m, 4H), 1.68 - 1.61 (m, 2H).

[0194] ¹³ C NMR (126 MHz, CDC1 ₃ ) 8 149.1, 148.5, 134.5, 131.0, 128.6, 120.6, 74.1, 48.2, 25.5, 23.6.

[0195] HRMS Calculated for Ci2Hi ₄ N ₄ O ₇ SNa [M+Na] ⁺ = 381.0475. Measured |M+Na| ⁺ = 381.0486.

[0196]

[0197] Synthesized according to a reported literature procedure. ⁸ Spectral data matched literature reports.

[0198] ^X H NMR (400 MHz, CDC1 ₃ ) 8 7.92 - 7.85 (m, 1H), 7.75 (m, 2H), 7.67 - 7.47 (m, 6H).

[0199]

[0200] Adapted from a literature procedure. ⁹ To a solution of 2-methylpropane-2-sulfinamide (485 mg, 4 mmol, 1.0 equiv.) and 4-nitroacetophenone (793 mg, 4.8 mmol, 1.2 equiv.) in dry THF (13 mL) was added Ti(z'PrO)4 (2.4 mL, 8 mmol, 2 equiv.) dropwise at room temperature. The reaction was sealed and heated at 70°C for 48 h. Water (10 mL) was added and the mixture was filtered washing with CH2Q2. The filtrate was extracted with CH2Q2, then the combined organic layers was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. Purification by column chromatography afforded the desired imine (775 mg, 72%) as a dark red oil.

[0201] Dry mCPBA (-85%, 223 mg, 1.1 mmol, 1.1 equiv.) was taken up in CH2O2 (2.5 mL), followed by the addition of KOH (powdered, 196 mg, 3.5 mmol, 3.5 equiv.) to form a white suspension which was stirred at room temperature 5 minutes. In a separate vial, the imine from the previous step (268 mg, 1 mmol, 1 equiv.) was dissolved in CH2CI2 (5 mL), followed by the addition of dry mCPBA (-85%, 203 mg, 1 mmol, 1 equiv.). The reaction was stirred at room temperature 1 minute, and then added to the initial stirring basic white mCPBA suspension. [0202] The reaction was monitored by 1H NMR using small aliquots dissolved in CeD^,. After 16^-8 h, the reaction mixture was diluted with CH2O2 (50 mL) and water (50 mL), and extracted with additional CH CI2 (3 x 50 mL). The combined organic phases were concentrated in vacuo until a total volume of ~ 40 mL to afford a solution of crude imine which was used directly in the next step. After 5 minutes, the reaction was concentrated in vacuo to afford a light yellow oil. Trituration with 2:1 Et ₂ O/pentane afforded the desired oxaziridine (300 mg, >99%) as an off-white solid (2.5 : 1 d.r.).

[0203] ^X H NMR (300 MHz, CDCI3) Major diastereomer: 8 8.25 (m, 2H, major), 7.69 - 7.62 (m, 2H), 2.40 (s, 3H), 1.56 (s, 9H). Minor diastereomer: 8 8.25 (m, 2H), 7.74 (m, 2H), 7.69 - 7.62 (m, 2H), 1.93 (s, 3H), 1.49 (s, 9H).

[0204] ^X3 C NMR (126 MHz, CDC1 ₃ ) major diastereomer: 8 148.8, 143.9, 127.7, 123.9, 82.7, 62.5, 24.0, 17.5.

[0205] HRMS (ESI): Calculated for Ci2H ₁₆ N ₂ O ₅ SNa [M+Na]+: 323.0672. Found 323.0653.

[0206]

[0207] General Procedure A was followed starting from diisopropylamine- 1 -sulfonamide (110 mg, 0.76 mmol, 1.0 equiv.) and 2,4-dinitrobenzaldehyde (179 mg, 0.91 mmol, 1.2 equiv.).

Purification by column chromatography (0% to 30% EtOAc/hexane) afforded the corresponding oxaziridine (38 mg, 15%) as a white solid.

[0208] ^X H NMR (300 MHz, CDC1 ₃ ) 8 9.06 (d, J = 2.3 Hz, 1H), 8.55 (dd, J = 8.5, 2.3 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 6.07 (s, 1H), 3.94 (hept, J = 6.9 Hz, 2H), 1.38 (m, 12H).

[0209] ¹³ C NMR (151 MHz, CDCI3) 8 149.0, 148.7, 134.8, 131.2, 128.5, 120.5, 74.4, 50.6, 22.4,

21.9.

[0210] HRMS (ESI): Calculated for Ci ₃ Hi ₈ N ₄ O ₇ SNa [M+Na]+: 397.0788. Found 397.0763.

[0211]

[0212] General Procedure A was followed starting from 4-methoxy-2,6- dimethylbenzenesulfonamide (646 mg, 3 mmol, 1.0 equiv.) and 2,4-dinitrobenzaldehyde (706 mg, 3.6 mmol, 1.2 equiv.). Purification by column chromatography (10% to 50% EtOAc/hexane) afforded the corresponding oxaziridine (218 mg, 18%) as a white solid. ^X H NMR (300 MHz, CDC1 ₃ ) 5 9.07 (d, 7 = 2.2 Hz, 1H), 8.55 (dd, J = 8.5, 2.2 Hz, 1H), 7.86 (d, 7 = 8.6 Hz, 1H), 6.72 (s, 2H), 6.25 (s, 1H), 3.86 (s, 3H), 2.75 (s, 6H). ¹³ C NMR (126 MHz, CDC1 ₃ ) 8 163.5, 149.1, 148.5, 145.3, 134.4, 131.1, 128.6, 123.2, 120.6, 116.7, 73.5, 55.6, 23.9. HRMS (ESI):

Calculated for CieH^OsS [M+NH ₄ ]+: 427.0918. Found 427.0916.

[0213]

[0214] General Procedure A was followed starting from 4-ethynylbenzenesulfonamide (91 mg, 0.5 mmol, 1.0 equiv.) and 4-trifluoromethylbenzaldehyde (0.082 mL, 0.6 mmol, 1.2 equiv.). Purification by column chromatography (0% to 20% EtOAc/hexane) afforded the corresponding oxaziridine (72 mg, 41%) as a beige solid. ^X H NMR (300 MHz, CDCI3) 8 8.01 (d, 7 = 8.4 Hz, 2H), 7.78 - 7.64 (m, 4H), 7.58 (d, 7 = 8.2 Hz, 2H), 5.56 (s, 1H), 3.36 (s, 1H). ¹³ C NMR (151 MHz, CDCI3) 8 134.4, 133.6 (q, 7 = 32.8 Hz), 133.1, 129.7, 129.5, 128.9 (2C), 125.9 (q, 7 = 3.7 Hz), 123.6 (q, 7 = 273 Hz) 82.4, 81.7, 75.4. ¹⁹ F NMR (376 MHz, CDC1 ₃ ) 8 -62.2.

[0215] HRMS (ESI): Calculated for C16H14F3N2O3S [M+NH ₄ ]+: 371.0671. Found 371.0659.

[0216]

[0217] General Procedure A was followed starting from 4-ethynylbenzenesulfonamide (91 mg, 0.5 mmol, 1.0 equiv.) and 2-nitrobenzaldehyde (91 mg, 0.6 mmol, 1.2 equiv.). Purification by column chromatography (10% to 50% EtOAc/hexane) afforded the corresponding oxaziridine (48 mg, 29%) as a white solid. H NMR (300 MHz, CDC1 ₃ ) 8 8.25 (d, 7 = 8.0 Hz, 1H), 8.05 (d, 7 = 8.5 Hz, 2H), 7.74 - 7.69 (m, 3H), 7.65 (m, 1H), 7.60 (m, 1H), 6.06 (s, 1H), 3.35 (s, 1H). [0218] ¹³ C NMR (126 MHz, CDC1 ₃ ) 8 148.0, 134.8, 133.5, 133.0, 131.6, 130.0, 129.6, 129.0, 127.7, 125.3, 82.4, 81.8, 75.4. HRMS (ESI): Calculated for Ci ₅ Hi ₀ N2O ₅ SNa [M+Na]+: 353.0202. Found 353.0181.

[0219]

[0220] General Procedure A was followed starting from the corresponding sulfonamide (74 mg, 0.5 mmol, 1.0 equiv.) and 2-nitrobenzaldehyde (91 mg, 0.6 mmol, 1.2 equiv.). Purification by column chromatography (10% to 30% EtOAc/hexane) afforded the corresponding oxaziridine (101 mg, 68%) as a white solid. ^X H NMR (500 MHz, CDCI3) 5 8.29 - 8.23 (m, 1H), 7.75 (td, J = 7.6, 1.3 Hz, 1H), 7.70 - 7.62 (m, 2H), 6.02 (s, 1H), 4.25 - 4.17 (m, 2H), 3.19 (s, 3H), 2.39 (t, J = 2.5 Hz, 1H). ¹³ C NMR (126 MHz, CDC1 ₃ ) 5 148.2, 134.7, 131.4, 129.1, 128.1, 125.2, 76.7, 75.2, 74.6, 40.9, 35.6. HRMS (ESI): Calculated for C11H15N4O5S [M+NH ₄ ]+: 315.0757. Found 315.0763.

[0221]

[0222] General Procedure A was followed starting from the corresponding sulfonamide (74 mg, 0.5 mmol, 1.0 equiv.) and 2,4-dinitrobenzaldehyde (118 mg, 0.6 mmol, 1.2 equiv.).

Purification by column chromatography (5% to 30% EtOAc/hexane) afforded the corresponding oxaziridine (75 mg, 44%) as a white solid.

[0223] ^X H NMR (500 MHz, CDC1 ₃ ) 5 9.08 (d, J = 2.2 Hz, 1H), 8.57 (dd, J = 8.5, 2.3 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 6.09 (s, 1H), 4.23 (t, J = 2.4 Hz, 2H), 3.19 (s, 3H), 2.41 (t, J = 2.5 Hz, 1H). ^X3 C NMR (151 MHz, CDC1 ₃ ) 5 149.2, 148.5, 134.1, 131.0, 128.7, 120.6, 76.4, 74.8, 74.3, 40.9, 35.6. HRMS (ESI): Calculated for CiiHi ₀ N ₄ O ₇ SNa [M+Na]+: 365.0162. Found 365.0137.

[0224]

[0225] General Procedure A was followed starting from the corresponding sulfonamide (103 mg, 0.50 mmol, 1.0 equiv.) and 2-nitrobenzaldehyde (91 mg, 0.60 mmol, 1.2 equiv.).

Purification by column chromatography (20% to 80% EtOAc/hexane, then 70% to 100% DCM/hexane) afforded the corresponding oxaziridine (110 mg, 62%) as a colorless oil.

[0226] ^X H NMR (500 MHz, Chloroform-d) 5 8.25 (dd, J = 8.0, 1.3 Hz, 1H), 7.76 (td, J = 7.5, 1.3 Hz, 1H), 7.70 - 7.60 (m, 2H), 6.00 (s, 1H), 4.01 - 3.60 (m, 3H), 3.55 - 3.43 (m, 2H), 2.16 - 1.92 (m, 2H), 1.87 - 1.72 (m, 2H).

[0227] ^X3 C NMR (126 MHz, CDC1 ₃ ) 8 148.1, 134.7, 131.4, 129.0, 128.1, 125.2, 75.4, 56.2, 44.4, 44.4, 30.2, 30.2. HRMS Calculated for Ci2Hi ₄ N ₆ O ₅ SNa [M+Na]+ = 377.0639. Found [M+Na]+ = 377.0611.

[0228] [0229] General Procedure A was followed starting from the corresponding sulfonamide (44 mg, 0.16 mmol, 1.0 equiv.) and 2,4-dinitrobenzaldehyde (37 mg, 0.19 mmol, 1.2 equiv.). Purification by column chromatography (20% to 80% EtOAc/hexane) afforded the corresponding oxaziridine (27 mg, 36%) as a white solid.

[0230] ^X H NMR (500 MHz, CDC1 ₃ ) 8 9.05 (d, J = 2.2 Hz, 1H), 8.53 (dd, J = 8.5, 2.2 Hz, 1H), 8.04 - 7.97 (m, 2H), 7.84 (d, J = 8.5 Hz, 1H), 7.15 - 7.09 (m, 2H), 6.04 (s, 1H), 4.28 - 4.23 (m, 2H), 3.94 - 3.88 (m, 2H), 3.75 (m, 2H), 3.41 (m, 2H). ¹³ C NMR (126 MHz, CDC1 ₃ ) 6 164.7, 149.2, 148.4, 134.0, 132.6, 131.0, 128.7, 124.2, 120.6, 115.5, 74.4, 70.6, 69.5, 68.2, 50.8.

HRMS (ESI): Calculated for C17H20N7O9S [M+NH ₄ ]+: 498.1037. Found 498.1020.

[0232] To a solution of tert-butyl (piperazin- l-ylsulfonyl)carbamate (275 mg, 1.04 mmol, 1.0 equiv.) in DMF (10 mL) was added desthiobiotin (223 mg, 1.04 mmol, 1.0 equiv.), EDC’HCl (307 mg, 1.6 mmol, 1.5 equiv.), HOBT’lLO (245 mg, 1.6 mmol, 1.5 equiv.), and DIPEA (0.37 mL, 2.1 mmol, 2.0 equiv.). After 72 h stirring at room temperature, the crude reaction mixture was directly subjected to column chromatography (6% to 10% MeOH/CFECE) to afford the desired product A (110 mg, 23%) as a white solid containing a small HOBT impurity (-10%). [0233] Compound A (100 mg, 0.22 mmol, 1.0 equiv.) was dissolved in CH2Q2 (3.6 mL) and TFA (0.9 mL). After stirring at room temperature for 1.5 h, the solvent was removed in vacuo. Purification by column chromatography (10% MeOH/CILCL ) afforded the corresponding sulfonamide B (76 mg, 98%) as a white solid. [0234] To a solution of sulfonamide B (52 mg, 0.14 mmol, 1.0 equiv.) and 2-nitrobenzaldehyde (25 mg, 0.17 mmol, 1.2 equiv.) in dry THF (0.7 mL) and CH2CI2 (0.7 mL) was added Ti(zPrO)4 (59 pL, 0.20 mmol, 1.4 equiv.) dropwise at room temperature. The mixture was heated to 40 °C for 12 h. The solvent was removed in vacuo to afford crude imine which was used directly in the next step.

[0235] In a 25 mL rbf, mCPBA (75%, 50 mg, 0.38 mmol, 5.4 equiv.) was prestirred in 1:1 CH ₂ C12/sat. aq. K2CO3 (2 mL) at room temperature for 10 minutes. The solution of crude imine from the previous step (in 1 mL CH2Q2) was added drop wise, using additional CH2O2 (1 mL) rinses for a quantitative transfer. After 2.5 h, the reaction was diluted with CH2Q2 (50 mL) and sat. aq. NaHCCL (15 mL), and extracted with CH2Q2 (2 x 30 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated in vacuo.

Purification by column chromatography (10% MeOH/CPLCk) afforded the oxaziridine linked to Desthiobiotin (38 mg, 53%) as a white solid.

[0236] ^X H NMR (600 MHz, CDC1 ₃ ) 6 8.25 (dd, J = 8.2, 1.2 Hz, 1H), 7.75 (td, J = 7.6, 1.3 Hz, 1H), 7.66 (td, J = 7.8, 1.5 Hz, 1H), 7.62 (dd, J = 7.7, 1.5 Hz, 1H), 6.01 (s, 1H), 5.30 (s, 1H), 4.87 (s, 1H), 3.82 (m, 1H), 3.78 - 3.70 (m, 2H), 3.67 (m, 1H), 3.64 - 3.49 (m, 6H), 2.34 (t, J = 7.5 Hz, 2H), 1.64 (p, J = 7.3 Hz, 2H), 1.54 - 1.22 (m, 6H), 1.10 (d, J = 6.4 Hz, 3H). ¹³ C NMR (126 MHz, CDCI3) 5 171.7, 163.7, 148.0, 134.7, 131.5, 128.9, 127.9, 125.2, 75.5, 56.1, 51.5, 47.1, 47.0, 45.3, 41.2, 33.0, 29.6, 29.3, 26.3, 24.9, 15.8. HRMS (ESI): Calculated for C2iH ₃ iN ₆ O ₇ S [M+H]+: 511.1970. Found 511.1956.

[0237] 2. Selectivity and reactivity of Ac-Trp-OMe with V-sulfonyl oxaziridines

[0238] General procedure

[0239] A stock solution of Ac-Trp-OMe (100 mM) in MeOH was mixed with oxaziridine (100 mM) in MeOH (v/v ratio = 1 : 1.2), followed by the immediate addition of PBS (1 volume equiv. to make 1: 1 PBS/MeOH). For some oxaziridines with low solubility, the reaction was performed in PBS/MeOH/MeCN (1:1:1) co-solvent. The reaction was stirred for 10 min at room temperature, and then analyzed by LC-MS. It is important the initial stock solutions of Ac-Trp- OMe and oxaziridine are fully homogeneous prior to mixing.

[0240] For LC-MS analysis, solvent A was water and solvent B was methanol. The linear gradient was 40-100% B in 6.5 min and 100% B for 1.5 min. [0241] 3. Molecular orbital calculations

[0242] Molecular orbital calculations for model oxaziridines were computed using the Spartan ’20 program from Wavefunction, Inc. Molecules were built and their geometries minimized using the Spartan 20’ GUI and submitted to an ‘Equilibrium Geometry’ calculation at ‘Ground’ state in ‘Water’ with ‘Density Functional wB97X-D’ method and a ‘6-31G*’ basis set. The LUMO values in eV obtained from these calculations are plotted in Figure 1c and S3.

[0243] 4. Characterization of the spectroscopic and kinetics of the Trp-CLiC reactions

[0244] The absorbance of unlabeled Ac-Trp-OMe and purified cycloadduct in PBS buffer were detected by UV-Vis equipment. To gain the A260/A280 ratio against the Trp/Ox ratio, different concentrations of 0x-W18 from 0 to 100 pM was added to the Ac-Trp-OMe (100 pM) in PBS buffer at 25 °C, and then the absorbance was detected after 20 min for reaction completion.

[0245] For kinetics study of the Trp-CLiC reaction, the absorbance at 280 nm was time- dependently monitored by UV-Vis upon adding 0x-W18 (50 pM) into different concentrations of Ac-Trp-OMe (500 pM, 550 pM and 600 pM) in PBS buffer at 25 °C. Then the second-order reaction rate constant could be obtained.

[0246] 5. Oxaziridine screening on peptides

[0247] Peptides were dissolved in 5% DMSO solution. Different V-sull'onyl oxaziridines (100 mM stock in DMF) were added to 50 pl peptide solution (50 pM) and the final concentration was 250 pM. The reactions were performed for 10 min at room temperature and then quenched by addition of excess Ac-Met-OMe solution. The labeled peptides were subjected to Q-TOF analysis with a Proswift RP-4H (monolithic phenyl, 1.0 mm x 50 mm, Dionex) column. The Solvent A was MilliQ water with 0.1% (v/v) formic acid, while the Solvent B was ACN with 0.1% (v/v) formic acid. The LC system was set at the 0.30 mL/min with a gradient of 5-100% solvent B in solvent A over 8 min. MassHunter Quantitative Analysis Version software was used for ion chromatogram extraction and Maximum Entropy deconvolution.

[0248] 6. Protein labelling with A -sulfonyl oxaziridine

[0249] Proteins were dissolved in Tris (50 mM, pH 8.0) buffer. 0x-W18 (100 mM stock in DMF) was added to 30 pL protein solution (50 pM) and the final concentration was 300 pM. The reactions were performed for 10 min at room temperature and then quenched by addition of excess Ac-Met-OMe solution. For protein labelling under denaturing condition, the protein solution was treated with 0.5% SDS and then boiled for 5 min at 95 °C before 0x-W18 addition. For competition assay, free Ac-Trp-OMe (2 mM) was added beforehand. For protein labelling under different concentrations of GdmCl, high-concentration Lysozyme was diluted into GdmCl buffers and final protein concentration was set as 50 pM. Lysozyme protein would be stored in GdmCl buffers for three hours to form stable conformations before labelling with 0x-W18. After tryptophan labelling with 0x-W18, the proteins were desalted with Zeba™ Spin Desalting Columns (7K MWCO, 0.5 mL) before subsequent Q-TOF analysis and UV-Vis analysis or further click reaction. In-gel fluorescence imaging was also applied to test the tryptophan labelling efficiency. For the copper-mediated alkyne-azide cyclization reaction of fluorescence dye, 100 pM CuSO ₄ , 100 pM TBTA, 100 pM azide-dye and 2 mM sodium ascorbate was added to Ox-W18-labeled proteins. This reaction was performed at room temperature for 1 h.

[0250] 7. Chemoselective tryptophan profiling

[0251] The desired proteome sample was diluted to a 2.5 mg/mL solution. Then 0x-W18 probe was added to each sample, typically 1 mM as the final concentration. After incubation at room temperature for half one hour, this reaction was quenched by excess Ac-Met-OMe. For click reaction, 40 pl 100 mM sodium ascorbate, 8 pl 50 mM DADPS azide-biotin in DMSO and 200 ul Cu-TBTA mixture [ 200 ul TBTA ligand solution (0.9 mg/mL in DMSO:/-butanol=l:4) + 20 ul 100 mM CuSO ₄ ] were added to 1.8 mL sample solution and then incubated for one hour. The proteome was precipitated by MeOH/CHCk (4:1.5). After washed with pre-chilled cold methanol for three times, the pellet was re-dissolved in 1 mL 0.2% SDS/PBS (w/v). Biotin labeled proteome was enriched with pre- washed high-capacity agarose beads on a rotator at 4 °C overnight. Then on-beads digestion was performed. 800 pL fresh 6M Urea/PBS was added to the beads (total volume is about 1 ml), and then the proteome reduction (5 mM DTT) and IA alkylation (10 mM I A A) were done in order. Then the buffer was changed to IM Urea/PBS (with 0.1% SDS, 1 mM CaCF). After addition of 8 pL of 0.5 pg/pl Trypsin solution, the mixture was Incubated on a rotator at 37 °C for 16- 18h. Then the beads were washed very carefully followed by acid-cleavage in 5% formic acid in water to afford the probed tryptophan- containing peptides. Liquid was removed using SpeedVac and lyophilizer. Before mass spectrometric analysis, peptides were reconstituted in 200 pL 0.1% FA solution.

[0252] 8. In-solution digestion of IL8, lysozyme, and proteome samples

[0253] The precipitated protein or proteome were re-dissolved in the dissolving buffer (PBS, 6 M urea, pH 7.5). 100 mM DTT was added to a final concentration of 5 mM, and then reacted for 30 min at 37 °C. 1 M IAA was added to a final concentration of 15 mM, and then reacted for 30 min at room temperature and in the dark. After reduction and alkylation, Trypsin was added at a 50:1 proteimprotease ratio (w/w), then mixed and incubated for 18 hours at 37 °C. The insolution digestion was terminated by adding trifluoroacetic acid to a final concentration of 0.5- 1%. Particulate material was removed by centrifuging at 16,000 g for 10 min. The collected peptides could be examined by LC-MS/MS.

[0254] 9. LC-MS/MS analysis [0255] Trypsin digested peptides were analyzed on a Thermo Scientific Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer coupled with an Aligent 1260 Infinite LC system. The mobile phases were A: 0.1% formic acid in 95% H ₂ O-20% ACN; B: 0.1% formic acid in 80% ACN-20% H ₂ O. The MS/MS analysis was performed under the positive-ion mode with a full-scan m/z range from 400 to 1,800 and a mass resolution of 70,000. Peptides were eluted using a 90 min gradient (0-5 min 100% A; 60 min 50% A; 70 min 100% A; 70-90 min 100% A) with a flow of 0.1 mL/min. MS/MS fragmentation was performed in a data-dependent mode, of which TOP 15 most intense ions are selected for MS2 analysis with a resolution of 17,500 using the collision mode of CID. Other important parameters: isolation window, 1.6 mlz units; default charge, +2, charge exclusion, unassigned, 8, >8 ; normalized collision energy, 27%; maximum IT, 50 ms; dynamic exclusion, 60.0 s.

[0256] 10. Peptide identification in proteomics experiments

[0257] For protein ID and modification identification, all MS/MS raw files were analyzed using pFind platform. Alkylation of cysteine (+57.0215 Da) was set as the fixed modification, and oxidation of methionine and tryptophan (+15.9949 Da) was assigned as the variable modifications. Besides, for MS/MS analysis of IL8 or Lysozyme, tryptophan oxidative cycloadduct was also seen as the modification (+ 297.0419 for tryptophan cycloadduct and - 281.0470 Da for neutral loss); for ABPP analysis, product after acid-cleavage was regarded as the final modification (+ 440.1478 for tryptophan cycloadduct and - 424.1529 Da for neutral loss). This software was set up to search peptides with precursor mass accuracy at + 20 ppm, fragment ion mass accuracy at + 20 ppm, and the results were filtered by applying a 1% FDR.

[0258] 11. Preparation and characterization of Her2-Fab antibodies

[0259] All tryptophan mutants were made using QuikChange to introduce single codon mutations onto Fab. Fabs were expressed and purified by an optimized autoinduction protocol previously described. ¹⁰ In brief, C43 (DE3) Pro+ E. coli containing expression plasmids were grown in TB autoinduction media at 37°C for 6 h, then cooled to 30°C for 16 to 18 h. The bacterial cells were spun down and lysed with B-PER detergent, followed by incubation at 60°C for 20 minutes. Following a hard spin at 30,000g for 20 minutes, the Fabs in the lysed supernatant were purified by Protein A affinity chromatography. Fab purity and integrity was assessed by SDS-PAGE and intact protein mass spectrometry using a Xevo G2-XS Mass Spectrometer (Waters) equipped with a LockSpray (ESI) source and Acquity Protein BEH C4 column (2.1 mm inner diameter, 50 mm length, 300 A pore size, 1.7 pm particle size) connected to an Acquity I-class liquid chromatography system (Waters). Deconvolution of mass spectra was performed using the maximum entropy (MaxEnt) algorithm in MassLynx 4.1 (Waters).

[0260] 12. Her2-Fab labeling with 0x-W18 and stability studies [0261] The Fab antibodies were incubated at 50 [1M with 3 molar equivalents of the 0x-W18 (150 pM). After labeling for 2 hours, the Fabs were buffer exchanged into PBS using a 0.5-mL Zeba 7-kDa desalting column (Thermo Fisher Scientific). Conjugation was monitored by intact protein mass spectrometry using a Xevo G2-XS Mass Spectrometer (Waters). For the stability studies, the Fabs were labeled as stated above and placed in an incubator at 37 °C. 5 mL of the sample were taken at the indicated timepoints, flash frozen, and stored at -20°C. All time points were measured by intact protein mass spectrometry.

[0262] 13. Unfolding stress detection of diverse inhibitors in living cells

[0263] HeLa cells were treated with MG- 132 (5 pg/ml) or Tunicamycin (5 pM) for 8 hours. Then cells were harvested and lysed in PBS buffer (1% Triton, with protease inhibitors). The proteome concentration was adjusted to 1 mg/ml and A-sulfonyl oxaziridine with desthiobiotin (250 pM) was added for proteome labeling. Reactions were quenched by excess Ac-Met-OMe solution. The labeling results were detected by Western blot analysis against biotin.

[0264] 14. Immunofluorescence imaging

[0265] HEK 293T cells in 8-well chamber slides were cultured in DMEM contained with 10% FBS. NPM1 and NONO WT and mutant plasmids were transfected at the point of 50% confluency by Lipofectamine 2000 Transfection Reagent. After 6 h, the cells were changed to fresh DMEM (10% FBS) medium. Then cells were Hoechst stained after 24 h expression for 20 min. The immunofluorescence imaging processes were performed using a Zeiss LSM700 laser scanning confocal microscope with a 63x oil-immersion objective lens using Zen 2009 software (Carl Zeiss). Image analysis and quantification was performed using Fiji.

[0266] 15. NMR spectra. See priority application.

[0267] Supplemental References

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[0271] 4. X. Gu, Y. Zhang, Z.-J. Xu, C.-M. Che, Chem. Commun. 50, 7870-7873, (2014).

[0272] 5. L. Lykke, C. Rodriguez-Escrich, K. A. Jorgensen, J. Am. Chem. Soc. 133, 14932- 14935, (2011).

[0273] 6. W. B. Jennings, S. P. Watson, D. R. Boyd, J. Chem. Soc., Chem. Commun. 14, 931- 932, (1988).

[0274] 7. R. W. F. Kerr et al, Tetrahedron Asymmetry 28, 125-134. 2017

[0275] 8. B. Song et al, Org. Lett. 17, 190-193, (2015).

[0276] 9. J. L. G. Ruano, J. Aleman, C. Fjardo, A. Parra, Org. Lett. 7, 5493-5496, (2005). [0277] 10. S. K. Elledge et al. Proc. Natl. Acad. Sci. U. S. A. 117, 5733-5740, (2020).