EP4634197A1 - Compositions and methods for chemoproteomics - Google Patents

Abstract

Disclosed herein are compositions and methods for performing chemoproteomics.

Description

COMPOSITIONS AND METHODS FOR CHEMOPROTEOMICS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.: 63/432,249, filed December 13, 2022, and U.S. Provisional Application No.: 63/471,142, filed June 5, 2023, the entire contents of each of which are incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under D19AP00041 awarded by the Defense Advanced Research Projects Agency, and GM067555 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Cysteine chemoproteomics is an enabling technology for functional biology and drug discovery. Such studies have yielded proteome-wide discovery of ligandable, potentially "druggable" cysteines in human and pathogen proteomes. Accurate identification of the precise sites of chemical modification and quantification of the fractional occupancy of these labeling events remain central challenges for chemoproteomics. A standard chemoproteomics workflow mitigates these challenges by combining capture of labeled peptides using biotinylated enrichment handles with isotopic differentiation of sample treatment groups. Numerous options for both enrichment handles and isotopic labeling reagents are now available, and judicious reagent selection is an essential step for achieving high coverage and accurate quantitation.

Nearly all chemoproteomics enrichment handles are composed of two features. First, a biotin or desthiobiotin moiety for capture on streptavidin, avidin, or neutravidin resin. Second, a capture handle, which is typically either an azide or alkyne group to enable bioorthogonal conjugation by copper-catalyzed azide-alkyne cycloaddition (CuAAC or ‘click’ chemistry), or a reactive group such as iodoacetamide that directly labels reactive amino acid side chains (e.g., cysteine thiol).

Most chemoproteomic studies continue to rely on stable isotope incorporation, either through metabolic labeling or custom isotopically labeled capture reagents, and MSI based quantification. These methods are widely adopted in large part due to their relatively reasonable cost, the compatibility of the resulting datasets with freely available software packages for analysis, and increased data reproducibility afforded by the ability to combine ‘treated’ and ‘control’ samples early in the sample preparation workflow.

As an alternative to early combination of differentiated samples, the late-stage modification of enriched peptides with isobaric tags for relative and absolute quantification (iTRAQ), demethylated leucine (DiLeu) reagents, and tandem mass tag (TMT) reagents allows for sample multiplexing enabled by MS 2/MS3 -level quantification. By acting as barcodes, these isobaric tags enable high sample throughput in chemoproteomics workflows. This has proven valuable for studies that profile a large number of compounds and treatment conditions. Complicating matters, the peptide-level labeling of these samples is performed late-stage in the sample preparation workflow, which increases both the amount of labor required and the variability of the afforded data. As showcased by recent applications of TMT labeling to chemoproteomics, this variability can in part be avoided through inclusion of multiple replicate samples within a multiplexed sample. However, reliance on such replicates decreases the number of channels available for different treatments and thus the complexity of samples amenable to MS2-level analysis. The prohibitive cost of TMT reagents further limits the widespread adoption of these reagents.

Further, although isobaric reagents drastically decrease instrument acquisition time, they simultaneously increase sample preparation time. This long sample preparation time is due to the reliance on peptide-level N-terminal nucleophilic displacement of the N-hydroxysuccinimide (NHS) ester on the isobaric tags. Because TMT/iTRAQ tags require peptide-level labeling, the samples cannot be combined until the very end of sample preparation workflows after enrichment. This late-stage isobaric labeling counteracts the increased throughput of isobaric strategies by making the sample preparation comparatively more laborious than MSI quantitation strategies.

Collectively, these limitations to existing approaches highlight the important unmet need for new isobaric labeling reagents that allow for early-stage combination of samples, afford highly reproducible data, and are cost effective. Thus, there is an ongoing, unmet need for improved reagents and methods of performing chemoproteomics. SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides chemoproteomic capture reagents having a structure represented by formula I or a salt thereof: wherein

Xi, X2, and X3 are each independently selected from NR6, O, and S;

Ri, R2, R3, R4, R5 are each independently selected from hydrogen, alkyl, aralkyl, aryl, and heteroaryl; each R⁶ is independently selected from hydrogen, alkyl, aralkyl, and aryl;

Yi is an amino acid or a first sequence of amino acids;

Y2 is an amino acid or a second sequence of amino acids; and nl, n2, n3, and n4 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In another aspect, the present disclosure provides methods of identifying a binding site comprising: contacting a substrate with a click chemistry moiety, thereby creating a substrate-click chemistry moiety conjugate; contacting the substrate-alkyne conjugate with the chemoproteomic capture reagent disclosed herein, thereby creating a chemoproteomic capture reagent-substrate conjugate; digesting the chemoproteomic capture reagent-substrate conjugate, thereby creating a digested substrate-chemoproteomic capture reagent conjugate; contacting the digested substrate-chemoproteomic capture reagent conjugate with an enrichment agent; cleaving the digested substrate-chemoproteomic capture reagent conjugate, thereby creating a digested substrate-amino acid conjugate; and determining the molecular weight of the digested substrate-amino acid conjugate, thereby identifying the binding site. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows that solid phase peptide synthesis (SPPS) enables the synthesis of chemoproteomic capture reagents.

FIG. IB shows prior approaches to prepare DADPS containing chemoproteomic capture reagents.

FIG. 1C shows the disclosed herein utilizing a solid-phase compatible DADPS reagent for high yielding synthesis of isotopically labeled chemically cleavable chemoproteomics capture reagents that enable quantitative chemoproteomic identification of cysteines accessible to modification by cysteine-reactive small molecules.

FIG. 2 shows the solid phase workflow for the synthesis of DADPS chemoproteomic capture reagents using NBIV-044 and NBIV-053.

FIG. 3 shows a cysteine profiling workflow resulting identified PSMs, unique peptides, and proteins identified by samples prepared using IAA and DADPS azide capture reagents NBIV- 009, NBIV- Oil, NBIV-022, and NBIV-027, together with biotin-azide.

FIGs. 4A & 4B show the competitive ABPP workflow for identification of cysteines labeled by cysteines- reactive compounds using DADPS reagents NBIV-009 and NBIV-010, with structures shown in 'B.'

FIG. 4C shows the distribution of Log2 ratios for all unique peptides identified from competitive ABPP experiment following the workflow shown in 'A' using HEK293T lysates subjected to 500 pM KB02. Samples were prepared in triplicated and ratios >2 indicate peptides that harbor cysteines significantly modified by KB02.

FIG. 4D shows unique and overlapping cysteines (left) and KB02-labeled cysteines (Log2 ratio > 2; right) identified in samples prepared using DADPS reagents NBIV-009 and NBIV-010 compared with samples prepared using heavy and light biotin azide capture reagents.

FIG. 4E shows the median ratios and 95% confidence interval for samples prepared and analyzed using 1 : 1 and 4: 1 DADPS capture reagents.

FIG. 5 shows an exemplary procedure for the solid-phase synthesis of peptides.

FIG. 6 shows the sCIP chemoproteomic platform for profiling ligandable cysteines. Cysteines are capped with isotopically labeled iodoacetamide alkyne (IAA) pan-cysteine alkylation reagents to act as a balancer for MS2 quantification. Labeled cysteines are then click- conjugated to isotopically labeled sCIP capture reagents. Both MSI and MS2 level quantification are achieved after sample pooling and SP3-FAIMS sample preparation and analysis. MS2-level quantification is enabled by gas-phase reagent fragmentation of the triazole moiety releasing a dihydrooxazolium reporter ion, which is balanced by isotopically labeled balancer derived from the iodoacetamide alkyne reagents. Red asterisk indicate potential positions of heavy isotopes.

FIG. 7A shows the predicted Fragmentation patterns of reagents NBIV-070, NBIII-169, and NBIV-0222.

FIG. 7B shows the mass spectra for a cysteine-containing peptide labeled with reagent NBIV-070 showing the respective fragment ions.

FIG. 7C shows the frequency and relative intensity analysis for each fragment ion. Median ratios and 95% confidence interval for each fragment ion is shown on the right.

FIG. 8A shows a workflow showing where the balancer section (IAA) and the reporter section (sCIP) of the isobaric system originate from in a cysteine profiling workflow generating a novel oxazolium reporter ion.

FIG. 8B shows a heat map describing the distribution of heavy isotopes for a 6-plex set of isobaric tags.

FIG. 8C shows the analysis of peptide coverage (left) and fragment Ion intensity (right) for the sCIP-Zero reagent.

FIG. 8D shows the median ratios and 95% confidence interval for samples prepared by mixing all channels of the 6-plex reagent set 1 : 1 (left) and the 5-plex reagent set 1 :4: 10:4: 1 (right).

FIG. 9A shows the ratio compression workflow comparing a 2-plex set of sCIP and a 2- plex set of TMT reagents.

FIG. 9B shows the observed ratio compression for both sets of isobaric tags.

FIG. 9C shows the cost comparison for sCIP vs TMT reagents.

FIG. 10A shows workflow for cysteine profiling using a 5-plex sCIP platform. In this workflow two aliquots of cell lysate are treated with vehicle (DMSO) and three aliquots are treated with compound. The treated lysates are then treated with one of three isotopically differentiated lAAs (L-IAA, ¹³CH-IAA, or ¹³C2H-IAA) followed by click conjugation to an individual 5-plex sCIP reagent (300, 301C, 301N, 302C, or 302N). Pairs of IAA and sCIP reagents are selected so that their aggregate masses are all isobaric. After samples are combined, the pooled sample is then subjected to SP3 cleanup, trypsin digestion, streptavidin enrichment, and LC-MS/MS analysis revealing different ratios of reporter ions for each peptide. FIG. 10B shows the structures of exemplary compounds analyzed.

FIG. IOC shows a comparison of Ratios generated for KB02 -treated lysates analyzed using MSI sCIP reagents NBIV-09 and NBIV-09 compared to MS2 5-plex sCIP workflow.

FIGs. 10D & 10E show the concordance of ratios obtained from single vs triplicate channel analysis for KB02 across all cysteines identified in both sample sets 'D' and for selected cysteines with known ligandable cysteines 'E.'

FIG. 11 shows the Structures of NBIV-009 and NBIV-010, used for MS1-MS2 comparisons. Structures of Light IAA (1), 13C-H-IAA (NBIV-069) and 13C2-H-IAA (NBIV-083) used for MS2 Quant. Structures of 6-Plex sCIP Reagents used for MS2 Quant.

FIG. 12 shows the structures and masses of modified peptides and fragment ions determined for reagents in FIG. 7 by diagnostic ion mining.

FIG. 13 shows relative Ion intensity for fragment ions identified in FIG. 12.

FIG. 14A shows the synthesis of isotopically enriched IAA (IAA, 1-¹³C-IAA, and 1,2¹³C2- IAA).

FIG. 14B shows the synthesis of 6-plex sCIP reagent set.

FIG. 15A shows the general structure of the 6-plex sCIP reagents and iodoacetamide alkyne probes where red indicates potential position of heavy atom.

FIG. 15B shows the structure and mass of cysteine modification for each sCIP 6-plex reagent combination.

FIG. 15C shows the structure and mass for sCIP 6-plex reporter ions.

FIG. 16 shows the yield of different DADPS reagents.

FIG. 17A shows the synthesis of sCIP-Gly-NEh using solid-phase peptide synthesis.

FIG. 17B shows the synthesis of sCIP-TMT using in situ tandem mass tag conjugation, with the possible positions of heavy isotopes indicated with asterisks.

FIG. 17C shows the structures of sCIP-TMT¹⁰ reagents formed in situ and their intact modification mass. The probable positions of heavy isotopes are indicated with asterisks.

FIG. 18A shows a comparison of TMT and sCIP-TMT profiling workflows in which cellular lysates are treated first with IAA, then subjected to click chemistry with either biotin azide (top panel labeled “TMT”) or the pre-formed sCIP-TMT conjugate (lower panel labeled “sCIP- TMT”). The sCIP-TMT samples can then be subsequently combined after click, cleaned up with SP3, enriched as a single sample, and subjected to LC-MS/MS analysis. TMT samples must separately be cleaned up and enriched before TMT labeling and late-stage combination.

FIG. 18B shows an analysis of the time (hours) and tubes saved using the sCIP-TMT workflow as multiplex channels increase.

FIG. 18C shows the PSM, peptide, cysteine, and protein coverage of sCIP-TMT¹⁰ labeled samples analyzed using FAIMS-MS². N=3 biological replicates.

FIG. 18D shows the comparison of ratios for samples mixed in both 1:1 and 1:5: 10: 15 ratios analyzed using FAIMS-MS². N=3 biological replicates.

FIG. 18E shows the comparison of ratios for samples mixed in both 1: 1 and 1 :5:10:15 ratios analyzed using SPS-MS³. N=3 biological replicates.

FIG. 19A shows the synthesis of cleavable dimethyl leucine-based isobaric tags.

FIG. 19B shows the cysteine profiling workflow using sCIP-DiLeu with the possible positions of heavy isotopes indicated with asterisks.

FIG. 19C shows a plan for synthesizing a 29-plex set of isobaric sCIP-DiLeu reagents.

FIG. 20 shows the synthesis of sCIP-TMTpro using in situ tandem mass tag conjugation, with the possible positions of heavy isotopes indicated with asterisks.

FIG. 21A shows the structures of four prototype electrophilic fragments, for use in testing the compatibility of sCIP-TMT with screening applications. The compounds include two chloroacetamide-containing molecules, KB0217-19 and KB 10, which had previously showed a substantially distinct labeling pattern and more attenuated reactivity when compared to KB02. The compounds additionally include methylphenyl propiolate (MPP) and methyl cinnamate (MC), which each have distinct proteomic reactivity, with MPP functioning as a potent cysteine protease inhibitor whereas MPA shows negligible protease inhibitory activity.

FIG. 2 IB shows the comparison of the Log2 ratios for cysteines identified using MSI analysis (x-axis) versus sCIP-TMT (y-axis) with scout fragment KB02. N=3 biological replicates.

FIG. 21C shows the reactivity ratio for each compound shown in FIG. 21A, calculated as the number of liganded cysteines for each compound out of the total number of cysteines. N=3 biological replicates.

FIG. 2 ID shows the structure-activity relationship of the four compounds shown in FIG. 21A across a panel of cysteines. N=3 biological replicates. FIG. 22 shows the synthesis of symmetric and asymmetric N,N-dimethyl-L-leucine, with the possible positions of heavy isotopes indicated with asterisks.

FIG. 23 shows the structure of all N,N-dimethyl-L-leucine derivatives synthesized in this study. Grey atoms indicate position of heavy isotopes.

FIG. 24 shows the differential mixing of 29-plex sCIP-DiLeu in 1: 1 and 1 :5:10:15:20 ratios. Box plots display 5th percentile, first quartile (QI), median, third quartile (Q3), and 95th percentile values of the sample.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are silane-based Cleavable linkers for Isotopically-labeled Proteomics (sCIP) methods that combine the advantages of the solid-phase compatible DADPS cleavable linker with the discovery of custom isobaric reporter ions with improved multiplexing capacity and comparable ratio compression when compared to existing technologies.

In one aspect, the present disclosure provides chemoproteomic capture reagents having a structure represented by formula I or a salt thereof: wherein

Xi, X2, and X3 are each independently selected from NR6, O, and S;

Ri, R2, R3, R4, R5 are each independently selected from hydrogen, alkyl, aralkyl, aryl, and heteroaryl; each R⁶ is independently selected from hydrogen, alkyl, aralkyl, and aryl;

Yi is an amino acid or a first sequence of amino acids;

Y2 is an amino acid or a second sequence of amino acids; and nl, n2, n3, and n4 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In certain embodiments, R¹ is aryl (e.g., phenyl). In certain embodiments, R² is aryl (e.g., phenyl). In certain embodiments, R³ is hydrogen. In certain embodiments, R⁴ is alkyl (e.g, methyl). In certain embodiments, R⁵ is alkyl (e.g., methyl). In certain embodiments, X¹ is O. In certain embodiments, X² is S. In certain embodiments, X³ is O. In certain embodiments, nl is 2. In certain embodiments, nl is 6. In certain embodiments, n2 is 1. In certain embodiments, n3 is 3. In certain embodiments, n4 is 2.

In certain embodiments, Yi is a first sequence of amino acids. In certain embodiments, the first sequence of amino acids comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain preferred embodiments, the first sequence of amino acids comprises 2 amino acids. In certain preferred embodiments, the first sequence of amino acids comprises 3 amino acids. In certain embodiments, the first sequence of amino acids comprises naturally occurring amino acids.

In certain embodiments, the first sequence of amino acids comprises an amino acid having a side chain comprising an alkene, alkyne, diazo or azido. In certain embodiments, the first sequence of amino acids comprises an amino acid having a side chain comprising azido. In certain embodiments, the first sequence of amino acids comprises an amino acid at the C-terminus having a side chain comprising an alkene, alkyne, diazo or azido. In certain embodiments, the first sequence of amino acids comprises an amino acid at the C-terminus having a side chain comprising azido. In certain preferred embodiments, the first sequence of amino acids comprises 6-azido- lysine (LysN3) at the C-terminus.

In certain embodiments, N-terminus of the first sequence of amino acids is protected with a nitrogen protecting group (e.g., acetyl). In certain embodiments, the first sequence of amino acids comprises a P-amino acid (e.g., β-glycine) at the N-terminus. In certain preferred embodiments, the P-amino acid is substituted with a heterocyclic acyl (e.g., piperidinyl acyl, such as dimethylpiperidinylacyl). In certain embodiments, the first sequence of amino acids comprises leucine at the N-terminus. In certain embodiments, the nitrogen of the leucine is substituted with alkyl. In certain preferred embodiments, the nitrogen of the leucine is substituted with two methyl groups. In certain preferred embodiments, the first sequence of amino acids comprises a demethyl leucine (DiLeu) at the N-terminus. In certain preferred embodiments, the first sequence of amino acids comprises an isobutyl proline at the N-terminus. In certain preferred embodiments, the first . In certain embodiments, the first sequence of amino acids comprises proline at the N-terminus. In certain embodiments, the nitrogen of the proline is substituted with alkyl. In certain preferred embodiments, the nitrogen of the proline is substituted with butyl (e.g., isobutyl).

In certain embodiments, the first sequence of amino acids is isotopically enriched. In certain embodiments, the first sequence of amino acids is isotopically enriched with ¹³C, ¹⁵N, or ¹⁸O. In certain embodiments, the first sequence of amino acids is isotopically enriched with ²H, ¹³C, ¹⁵N, or ¹⁸O.

In certain embodiments, Y2 is an amino acid. In certain embodiments, Y2 is an amino acid having a side chain comprising biotin (e.g., avidin or streptavidin). In certain embodiments, Y2 is an amino acid having a side chain comprising alkyl. In certain embodiments, Y2 is an amino acid having a side chain comprising alkylamidoalkyl. In certain embodiments, the side chain of the amino acid of Y2 is substituted with heterocyclyl (e.g., biotinyl).

In certain embodiments, the chemoproteomic capture reagent is selected from: In certain preferred embodiments, one or more hydrogen atoms are enriched for ²H. In certain preferred embodiments, one or more carbon atoms are enriched for ¹³C. In certain preferred embodiments, one or more nitrogen atoms are enriched for ¹⁵N. In certain embodiments, one or more oxygen atoms are enriched for ¹⁸O.

This disclosure also includes all suitable isotopic variations of a compound of the disclosure. An isotopic variation of a compound of the invention is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually or predominantly found in nature. Examples of isotopes that can be incorporated into a compound of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, such as ²H (deuterium), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³²P, ³³P, ³³S, ³⁴S, ³⁵S, ³⁶S, ¹⁸F, ³⁶C1, ⁸²Br, ¹²³I, ¹²⁴I, ¹²⁹I and ¹³¹I, respectively. Accordingly, recitation of “hydrogen” or “H” should be understood to encompass ¹H (protium), ²H (deuterium), and ³H (tritium) unless otherwise specified. Certain isotopic variations of a compound of the invention, for example, those in which one or more radioactive isotopes such as ³H or ¹⁴C are incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated and carbon- 14, i.e., ¹⁴C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Such variants may also have advantageous optical properties arising, for example, from changes to vibrational modes due to the heavier isotope. Isotopic variations of a compound of the invention can generally be prepared by conventional procedures known by a person skilled in the art such as by the illustrative methods or by the preparations described in the examples hereafter using appropriate isotopic variations of suitable reagents.

In some embodiments, the compounds disclosed herein comprise stable isotopes of hydrogen, carbon, nitrogen, and oxygen in amounts greater than their natural abundance. For example, one or more hydrogen atoms may be enriched with ²H in an amount greater than about 0.015% (e.g., 1.2-1.5%, 1.5-2%, 2-10%, or more than 10%). For example, one or more carbon atoms may be enriched with ¹³C in an amount greater than about 1.1% (e.g., 1.2-1.5%, 1.5-2%, 2- 10%, or more than 10%). One or more nitrogen atoms may be enriched with ¹⁵N in an amount greater than about 0.4% (e.g., 0.5-1%, 1-2%, 2-10%, or greater than 10%). Likewise, one or more oxygen atoms may be enriched with ¹⁶O in an amount greater than about 0.24% (e.g., 0.25-0.5%, 0.5-1%, 1-2%, 2-10%, or greater than 10%). Recitation of “hydrogen” or“H” should be understood to encompass ¹H (protium), ²H (deuterium), and ³H (tritium) unless otherwise specified.

In some embodiments, the compounds disclosed herein have an isotopic purity of at least 50.0%, 60.0%, 70.0%, 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 97.0%, 98.0%, 99.0%, 99.5%, 99.7%, 99.8%, 99.9%, or 100%. In one embodiment, the compounds have an isotopic purity of at least 50.0%. In another embodiment, the compounds have an isotopic purity of at least 60.0%. In another embodiment, the compounds have an isotopic purity of at least 70.0%. In another embodiment, the compounds have an isotopic purity of at least 75.0%. In another embodiment, the compounds have an isotopic purity of at least 80.0%. In another embodiment, the compounds have an isotopic purity of at least 85.0%. In another embodiment, the compounds have an isotopic purity of at least 90.0%. In another embodiment, the compounds have an isotopic purity of at least 95.0%. In another embodiment, the compounds have an isotopic purity of at least 97.0%. In another embodiment, the compounds have an isotopic purity of at least 98.0%. In another embodiment, the compounds have an isotopic purity of at least 99.0%. In another embodiment, the compounds have an isotopic purity of at least 99.5%. In another embodiment, the compounds have an isotopic purity of at least 99.7%. In another embodiment, the compounds have an isotopic purity of at least 99.9%. Isotopic enrichment may be described as a percentage indicating the percent of isotopic atoms at a particular site on the molecule. The percentage can be referred to as the “isotopic purity” of the isotopically-labeled compound.

In another aspect, the present disclosure provides methods of identifying a binding site comprising: contacting a substrate with a click chemistry moiety, thereby creating a substrate-click chemistry moiety conjugate; contacting the substrate-alkyne conjugate with the chemoproteomic capture reagent disclosed herein, thereby creating a chemoproteomic capture reagent-substrate conjugate; digesting the chemoproteomic capture reagent-substrate conjugate, thereby creating a digested substrate-chemoproteomic capture reagent conjugate; contacting the digested substrate-chemoproteomic capture reagent conjugate with an enrichment agent; cleaving the digested substrate-chemoproteomic capture reagent conjugate, thereby creating a digested substrate-amino acid conjugate; and determining the molecular weight of the digested substrate-amino acid conjugate, thereby identifying the binding site.

In certain embodiments, the substrate is a protein. In certain preferred embodiments, the substrate is a protein comprising a sulfur containing amino acid (e.g., cysteine or homocysteine). In certain embodiments, the protein is formed from cell lysation.

In certain embodiments, the click chemistry moiety is an alkene, alkyne, diazo, or azide. In certain embodiments, the click chemistry moiety is alkyne.

In certain embodiments, contacting the substrate-alkyne conjugate with any of the chemoproteomic capture reagents described herein forms a triazole linking the chemoproteomic capture reagent to the substrate.

In certain embodiments, digesting the chemoproteomic capture reagent-substrate conjugate comprises contacting the chemoproteomic capture reagent-substrate conjugate with a digestion enzyme (e.g., trypsin).

In certain embodiments, the enrichment agent is a protein that binds biotin (e.g., avidin or streptavidin).

In certain embodiments, cleaving the digested substrate-chemoproteomic capture reagent conjugate comprises contacting the digested substrate-chemoproteomic capture reagent conjugate with acid (e.g., formic acid).

In certain embodiments, the click chemistry moiety is isotopically enriched. In certain embodiments, the click chemistry moiety is isotopically enriched with ¹³C, ¹⁵N, or ¹⁸O. In certain preferred embodiments, the click chemistry moiety is isotopically enriched with ²H, ¹³C, ¹⁵N, or ¹⁸O.

Definitions

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.

It is understood that substituents and substitution patterns on the compounds of the present invention can be selected by one of ordinary skilled person in the art to result chemically stable compounds which can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

As used herein, the term “optionally substituted” refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O-alkyl, - OP(O)(O-alkyl)2 or -CH2-OP(O)(O-alkyl)2. Preferably, “optionally substituted” refers to the replacement of one to four hydrogen radicals in a given structure with the substituents mentioned above. More preferably, one to three hydrogen radicals are replaced by the substituents as mentioned above. It is understood that the substituent can be further substituted.

As used herein, the term “alkyl” refers to saturated aliphatic groups, including but not limited to C1-C10 straight-chain alkyl groups or C1-C10 branched- chain alkyl groups. Preferably, the “alkyl” group refers to C₁-C₆ straight-chain alkyl groups or C₁-C₆ branched-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.

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

The term “acylamino” is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

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

The term “alkoxy” refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.

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

The term “alkyl” refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer.

Moreover, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.

The term “C_x-y” or “C_x-C_y”, 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. Coalkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. A C_1-6alkyl group, for example, contains from one to six carbon atoms in the chain.

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

The term “alkylthio”, as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

The term “amido”, as used herein, refers to a group wherein R⁹ and R¹⁰ each independently represent a hydrogen or hydrocarbyl group, or R⁹ and R¹⁰ taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by wherein R⁹, R¹⁰, and R¹⁰ each independently represent a hydrogen or a hydrocarbyl group, or R⁹ and R¹⁰ taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

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

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

The term “aryl” as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like. The term “carbamate” is art-recognized and refers to a group wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbyl group.

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

The term “carbocycle” includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term “fused carbocycle” refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary “carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5 -cyclooctadiene, 1, 2,3,4- tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0] octane, 4,5,6,7-tetrahydro-lH-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one or more positions capable of bearing a hydrogen atom.

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

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

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

The term “cycloalkyl” includes substituted or unsubstituted non-aromatic single ring structures, preferably 4- to 8-membered rings, more preferably 4- to 6-membered rings. The term “cycloalkyl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is cycloalkyl and the substituent (e.g., R¹⁰⁰) is attached to the cycloalkyl ring, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, denzodioxane, tetrahydroquinoline, and the like.

The term “ester”, as used herein, refers to a group -C(O)OR⁹ wherein R⁹ represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include “alkoxyalkyl” groups, which may be represented by the general formula alkyl-O-alkyl.

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

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

The terms “heteroaryl” and “hetaryl” include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heteroaryl” and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

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

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

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heterocyclyl” and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

The term “hydrocarbyl”, as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 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 =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

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

The term “lower” when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer atoms in the substituent, preferably six or fewer. A “lower alkyl”, for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “poly cycle”, and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”. Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the poly cycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.

The term “sulfate” is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof. The term “sulfonamide” is art-recognized and refers to the group represented by the general formulae wherein R⁹ and R¹⁰ independently represents hydrogen or hydrocarbyl.

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

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

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

The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate 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 alkoxy 1, 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.

The term “thioalkyl”, as used herein, refers to an alkyl group substituted with a thiol group. The term “thioester”, as used herein, refers to a group -C(O)SR⁹ or -SC(O)R⁹ wherein R⁹ represents a hydrocarbyl.

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

The term “urea” is art-recognized and may be represented by the general formula wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbyl.

Many of the compounds useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the compounds, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01/062726.

Furthermore, certain compounds which contain alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers. In each instance, the disclosure includes both mixture and separate individual isomers.

“PSM”, as used herein, refers to the total number of peptide spectral matches.

“LysN3”, as used herein, refers to 6-azido-lysine.

EXAMPLES

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1 : Synthesis of Exemplary Chemoproteomics Capture Reagents

Establishing a route to a prototype DADPS building block.

While the utility of silyl groups for capture and release of cargo on the solid phase was previously demonstrated, the feasibility of DADPS group incorporation into polypeptides obtained through SPPS remains unexplored. Therefore, the first step was to develop a synthetic route to enable high yielding incorporation of the DADPS group into a range of peptide-based reagents. It was initially envisioned that the prototype DADPS reagent containing a free carboxylic acid for solid phase coupling could easily be obtained by reacting β-hydroxyisovaleric acid with N-cbz-1- amino- 6-hexanol (Scheme SI). However, under all reaction conditions tested, none of the desired product was observed and instead observed homo coupling of the primary alcohol was found. It was speculated that the free acid was likely not compatible with the basic conditions required for DADPS formation. Therefore, the next step was to generate ester protected substrates to assess the DADPS formation in the absence of the free acid moiety.

A benzyl carbamate (Cbz) protected amino alcohol was used as part of the initial investigation of the DADPS formation reaction indicated that the addition of 4- dimethylaminopyridine (DMAP) afforded increased yields, which rendered Fmoc-protected reagents incompatible with the basic DADPS formation conditions (Scheme S2). A panel of six prototype ester-based DADPS reagents were readily obtained upon condensation with Cbz protected aminohexanol 1, in yields ranging from 43 to 85% (Scheme S3). All attempts at hydrolysis of the DADPS ester moieties afforded only undesired Cbz deprotection or cleavage of the silane (Table SI). Silane cleavage predominated for cleavage conditions that required strong Lewis acids, which was ascribed to coordination of the silyl diether oxygens facilitating cleavage.

As no desired product was observed for any of the ester substrates, it was next evaluated whether a DADPS reagent that featured an activated ester could be both obtained and subjected to the necessary protecting group manipulations to afford the desired Fmoc protected DADPS activated ester. This strategy would obviate the need for hydrolysis as one could directly couple the activated ester onto solid phase through amide bond formation. Following established conditions, five different activated ester analogues of beta- hydroxy isovaleric acid (Scheme S4) were obtained in near quantitative yields (78-99%) and subjected those to DADPS formation conditions. In all instances the desired product was not obtained in any appreciable yield. In reactions utilizing the N-hydroxysuccinimide (NHS) and 2,3,5,6-tetrafluorophenol (TFP) esters, by LC-MS observed displacement of the activated ester (Scheme S5) was observed, indicating in situ formation of the product followed by reaction of the ester with the Cbz-amino alcohol. Changing the base from DMAP to TEA did afford conversion to the desired product, as detected by MS (Scheme S6). However, the prior observation that DMAP was required for high yield formation of DADPS reagents combined with the general observed instability of the activated ester building blocks tempered enthusiasm for this route.

Scheme 1. Synthesis of solid-phase compatible DADPS-Fmoc reagents NBIV-044 and NBIV-053.

Synthesis and validation of solid phase compatibility of the FMOC-DADPS-acids NBIV-044 and NBIV-053

Given the generally mild and orthogonal conditions required for thiol-ene chemistry and the availability of the allyl ester model substrate 2, it was tested whether 2 could be coupled to 3- mercaptopropionic acid (MPA) under photoinitiated reaction conditions. Gratifyingly, product formation in 61% yield (Scheme S7A) was observed. However, all efforts towards selective deprotection of the Cbz group in the presence of the allyl ester were unproductive (Scheme S7B). The Cbz group was thus replaced with phthalimide protected amine 3, which afforded allyl ester DADPS-Fmoc reagent 4 in 92% yield. Subsequent protecting group manipulation afforded Fmoc protected DADPS reagent 5 in 70% yield over two steps.

Compound 5 was then subjected to a photoinitiated thiol-ene reaction with MPA to form a thioether linkage and free carboxylic acid on the reagent, obtaining the solid phase compatible DADPS reagent 6 in 59% yield (Scheme S8). While the high yield formation of this model substrate was encouraging, a decision was made to modify the strategy to eliminate the ester moiety, due to its potential hydrolytic instability in esterase-containing cell lysates. Additionally, there was a concern about the aqueous media solubility of the reagent bearing the long hexyl chain. Accordingly, the sequence of DADPS formation was repeated with an allyl ether and making two reagents NBIV- 044 and NBIV-053., which differed by alkyl chain length. Notably, the thiol-ene was performed neat, providing the final solid-phase compatible reagent in 39% yield for the reagent bearing an ethyl chain NBIV-044 and 31% yield for the hexyl chain NBIV-053 over 4 steps.

SPS and proteomic benchmarking of a panel of DADPS functionalized chemoproteomics capture reagents NBIV-009, NBIV-011, NBIV-022, and NBIV-027.

With a working strategy for DADPS enrichment reagent synthesis in hand, the next step was to synthesize a panel of reagents. Three variables were explored, the linker length, the source of azide, and type of amino acid used for isotopic labeling reagent synthesis. The first to be prioritized linker length as the inventors wanted to assess how changes to the reagent size and solubility would impact coverage. For azide source, β-azidohomoalanine was compared with azidolysine with the goal of again determining how reducing the reagent size would impact proteomic coverage. It was speculated that there could be differences in the fragmentation pattern of azidohomoalanine and azidolysine based reagents, which could impact coverage of chemoproteomics detected peptides. Lastly, given the ready availability of various isotopically labeled amino acids, most notably valine and alanine, it was sought to assess whether incorporation of isotopically labeled amino acids would enable MSI -based quantification of enriched peptides and whether amino acid selection would impact reagent performance.

With these objectives in mind, a panel of 4 reagents (NBIV-009, NBIV-011, NBIV- 022, and NBIV-027) were synthesized in high yield and purity, (FIG. 7) with the goal of systematically comparing each ofthe aforementioned variables. Using HEK293T cell lysates, cysteine-containing peptides were captured and identified, using a modified version of the SP3 workflow for analysis of the cysteinome (FIG. 8). First cysteines were capped with the highly reactive cysteine alkylating reagent iodoacetamide alkyne (IAA). The alkyne-labeled lysates were then subjected to click conditions with each of the azido-DADPS capture reagents followed by SP3 sample cleanup, tryptic digest, capture of labeled peptides with streptavidin, followed by release of DADPS labeled peptides under mild acidic conditions. LC-MS/MS analysis revealed similar performance for all reagents, as indicated by the comparable numbers of PSMs, peptides, and protein identifications (FIG. 8). Similar performance for the alanine and valine reagents NBIV-009 and NBIV-011 was observed, indicating that the synthesis of isotopically labeled DADPS reagents should proceed smoothly using either heavy valine or heavy alanine building blocks. The use of neutravidin for samples prepared with biotin-azide in place of streptavidin resin for samples prepared with the DADPS reagents Slight differences in coverage and peptides identified using biotin-azide vs DAPDS reagents can be rationalized by the use of neutravidin vs streptavidin resin for the respective workflows.

MSl-based quantification to discover ligandable cysteines using isotopically differentiated reagents NBIV-009 and NBIV-010.

To obtain an isotopically enriched DADPS capture reagent, the synthesis and application of heavy L-valine (¹³C₅ ¹⁵N)- containing reagent NBIV-010 was focused on, as the relatively large +6 Da mass difference is ideal for MSl-based quantification. Further motivating the isotopic reagent design, a +6-mass difference is used in isoTOP-ABPP and isoDTB reagents together with the previously reported heavy and light azido- biotin reagents, which was envisioned could facilitate head-to-head comparisons. Using the same synthetic strategy, heavy reagent NBIV-010 was obtained in 58% yield. LC-MS analysis revealed comparable intensities of light and heavy reagents, when assayed as a 1 : 1 mixture. These reagents in a were further validated in a competitive ABPP workflow. Using HEK293T cell lysates, samples were subjected to either vehicle, DMSO, or 500 pM KB02. Upon labeling with IAA and clicking the samples with either heavy or light D DPS probe (NBIV-010 or NBIV-009, respectively; FIG. 4B) or heavy or light biotin azide, the samples were subjected to SP3 cleanup. After enrichment with either streptavidin resin, for DADPS labeled peptides, or NeutrAvidin resin, for biotin azide labeled peptides, the peptides were cleaved or eluted off resin, respectively, and analyzed by LC-MS/MS. Across three biological replicates, the DADPS reagents identified 5075 unique cysteines, with 404 found in peptides with Log2 MSl extracted ion chromatograph area ratios>2 (FIG. 4C) — these elevated ratios indicate cysteines that are modified by KB02. Comparable coverage and ratios were observed for samples prepared using heavy/light biotin-azide reagents, and 75% of identified cysteines were shared across both reagent datasets (FIG. 4D, left panel). Demonstrating the utility of assaying multiple capture reagents, 488 cysteines were uniquely identified by the DADPS capture reagents. The DADPS and biotin azide datasets shared 163 cysteines with ratios>2, which represent a high confidence dataset of KB02- labeled sites (FIG. 4D, right panel). The performance of the DADPS capture reagents was further vetted using reagent dilution experiments to generate datasets with expected Log2 ratios near zero, for 1: 1 samples and near two for 4: 1 samples (FIG. 4E). Chemistry Methods

General Methods

All reactions were performed in dried glassware under an atmosphere of dry N2 unless otherwise stated. Silica gel P60 (SiliCycle) was used for column chromatography. Plates were visualized by fluorescence quenching under UV light or by staining with iodine, KMnO₄, or bromocresol green. Other reagents were purchased from Sigma-Aldrich (St. Louis, MO), Alfa Aesar (Ward Hill, MA), EMD Millipore (Billerica, MA), Fisher Scientific (Hampton, NH), Oakwood Chemical (West Columbia, SC), Combi-blocks (San Diego, CA) and Cayman Chemical (Ann Arbor, MI) and used without further purification. ¹H NMR and ¹³C NMR spectra for characterization of new compounds and monitoring reactions were collected in CDCl₃, CD₃OD, CD₆CO or DMSO-d₆ (Cambridge Isotope Laboratories, Cambridge, MA) on a Bruker AV 500 MHz spectrometer or Brucker AV 400 MHz in the Department of Chemistry & Biochemistry at The University of California, Los Angeles. All chemical shifts are reported in the standard notation of parts per million using the peak of residual proton signals of the deuterated solvent as an internal reference. Coupling constant units are in Hertz (Hz). Splitting patterns are indicated as follows: br, broad; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; dt, doublet of triplets. Low-resolution mass spectrometry was performed on an Agilent Technologies InfinitiyLab LC/MSD single quadrupole LC/MS (ESI source). High-resolution mass spectrometry was performed on a Waters LCT Premier with ACQUITY LC and autosampler (ESI source). Cell culture reagents including Dulbecco’s phosphate- buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM)/high glucose media, Roswell Park Memorial Institute (RPMI) media, trypsin-EDTA and penicillin/streptomycin (Pen/Strep) were purchased from Fisher Scientific. All protein concentrations were determined using a Bio-Rad DC protein assay kit using reagents from Bio-Rad Life Science (Hercules, CA). l-(Allyloxy)-2-Methylpropan-2-ol To a 100 mL round-bottom flask was added allyl alcohol (10.2 g, 12.0 mL, 3 Eq, 176 mmol) and cooled to 0°C. To this solution was slowly added sodium hydride (2.4 g, 60% Wt, 1.0 Eq, 58.8 mmol) and then let stir at 0°C for 20 min. Next, 2,2-dimethyloxirane (4.24 g, 5.22 mL, 1 Eq, 58.8 mmol) was added and solution refluxed at 52°C. Upon completion of reaction as determined by TLC (3 hours) the reaction mixture was diluted with salt. NH₄C1 and extracted with Et2O (3x 30mL). Combined organic layers were dried over sodium sulfate and volatiles removed under reduced pressure. The crude residue was purified by vacuum distillation yielding the desired alcohol as a clear liquid (5.18g, 67.7%). All analyses were consistent with previously reported data.^{2 1}H NMR (400 MHz, CDCl₃) δ 5.96 - 5.82 (m, 1H), 5.30 - 5.14 (m, 2H), 4.02 (ddt, J = 5.1, 3.2, 1.4 Hz, 2H), 3.26 (d, J = 3.1 Hz, 2H), 1.20 (d, J = 3.4 Hz, 6H).

Benzyl (6-Hydroxyhexyl)carbamate

To a 250mL round-botom flask was added 6-aminohexan-l-ol (2.985 g, 1 Eq, 24.71 mmol), sodium carbonate (5.761 g, 2.2 Eq, 54.36 mmol), Water (35 mL), and THF (35 mL). The flask was purged with argon, cooled to 0°C, and benzyl chloroformate (4.636 g, 3.880 mL, 1.1 Eq, 27.18 mmol) added dropwise over 5 min. Solution was then let warm to room temperature overnight. Upon completion, the reaction mixture was diluted with water and extracted with ethyl acetate (3x40mL). Combined organic layers dried over sodium sulfate and solvent removed under reduced pressure to yield the desired product as a white solid (4.5g, 72%). All analyses were consistent with previously reported data.^{3 1}H NMR (400 MHz, CDCl₃) δ 7.40 - 7.29 (m, 5H), 5.09 (s, 2H), 3.63 (q, J = 6.3 Hz, 2H), 3.20 (q, J = 6.7 Hz, 2H), 1.53 (ddt, J = 20.4, 13.8, 6.6 Hz, 4H), 1.43 - 1.29 (m, 4H).

2-( 6-Hydroxyhexyl)isoindoline-l, 3-dione

6-aminohexan-l-ol (2.00 g, 1 Eq, 17.1 mmol) and phthalic anhydride (2.53 g, 1 Eq, 17.1 mmol) dissolved in Toluene (50 mL) were refluxed with a dean-stark trap. After the reaction was judged complete by TLC (2 hours) the reaction mixture was cooled to room temperature and volatiles removed under reduced pressure. Crude material was then purified by silica column chromatography (1 :1 to 2: 1 ethyl acetate: hexanes) to yield the desired product as a white crystalline solid (3.9g, 92%). All analyses were consistent with previously reported data. 'HNMR (400 MHz, CDCl₃) 8 7.82 (dd, J = 5.4, 3.1 Hz, 2H), 7.69 (dd, J = 5.4, 3.0 Hz, 2H), 3.69 -3.64 (m, 2H), 3.61 (t, J = 6.5 Hz, 2H), 1.77 - 1.61 (m, 3H), 1.60 - 1.50 (m, 2H), 1.45 - 1.29 (m, 3H).

Benzyl (6-((((2-methyl-l-phenylpropan-2-yl)oxy)diphenylsilyl)oxy)hexyl)carbamate

To an oven dried 50mL round-bottom flask was added 2-methyl-l -phenylpropan-2-ol (150 mg, 154 pL, 1 Eq, 1.00 mmol) and Base (2.25 Eq, 2.25 mmol). The flask was capped with rubber septa and purged with argon followed by addition of anhydrous DCM (5.00 mL). The solution was then cooled to 0°C and diphenyldi chlorosilane (317 mg, 257 pL, 1.25 Eq, 1.25 mmol) was added dropwise. After complete addition the reaction was allowed to warm to specified temperature and stir for 5 hours. After 5 hours the solution was then cooled back to 0°C and benzyl (6- hydroxyhexyl)carbamate (264 mg, 1.05 Eq, 1.05 mmol) was added. The solution was then allowed to warm to room temperature and stir for 16h. After completion, the reaction mixture was diluted with sat. sodium bicarbonate and extracted with CH2Q2 (3x1 OmL). Combined organic layers were washed with brine (1x1 OmL) and dried over sodium sulfate. Crude product was purified by silica column chromatography (1 :9 to 1:3 ethyl acetate: hexanes) to yield the pure product as a clear oil. ¹H NMR (400 MHz, CDCl₃) 87.60 (dt, J= 8.0, 1.7 Hz, 4H), 7.42 - 7.30 (m,11H), 7.28 - 7.21 (m, 5H), 5.10 (s, 2H), 3.60 (t, J= 6.5 Hz, 2H), 3.16 (q, J= 6.8 Hz, 2H), 2.85 (s, 2H), 1.48 (dt, J= 18.1, 7.0 Hz, 4H), 1.37 - 1.28 (m, 2H), 1.25 (d, J= 1.4 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) 8 156.49, 138.60, 136.81, 135.30, 135.13, 131.02, 129.89, 128.65, 128.27, 128.22, 127.84, 127.70, 126.23, 75.85, 66.72, 62.83, 51.20, 41.20, 32.32, 30.06, 29.78, 26.59, 25.58. HRMS (ESI-MS) m/z-. Calculated [M+Na]⁺ = 604.2859 , Found [M+Na]⁺ = 604.2869

To an oven dried 250mL round-bottom flask was added DMAP (2.228 g, 2.25 Eq, 18.23 mmol), l-(allyloxy)-2-methylpropan-2-ol (1.055 g, 1 Eq, 8.104 mmol) and capped with septa. The system was purged with argon and CH2Q2 (30.0 mL) was added. The solution was cooled to 0°C and diphenyldichlorosilane (2.565 g, 2.085 mL, 1.25 Eq, 10.13 mmol) was added dropwise. The solution was then allowed to warm to room temperature. After the first step was determined to be complete by TLC (5 hours), the solution was cooled to 0°C. The flask was uncapped and 2-(6- hydroxyhexyl)isoindoline- 1,3-dione (2.104 g, 1.05 Eq, 8.509 mmol) was added. The flask was recapped and the reaction was warmed to room temperature and stirred overnight. Upon completion the reaction was diluted with sat. sodium bicarbonate and extracted with CH2CI2 (3x 40mL). Combined organic layers were washed with brine and dried over sodium sulfate. Crude product was purified by silica column chromatography (1:9 to 1:3 ethyl acetate: hexanes) to yield the pure product as a clear oil (2.9g, 64%). ¹H NMR (400 MHz, CDCl₃) 87.84 (dd, J = 5.4, 3.1 Hz, 2H), 7.70 (dd, J = 5.5, 3.0 Hz, 2H), 7.66 -7.61 (m, 3H), 7.40 - 7.27 (m, 7H), 5.85 (ddd, J = 22.7, 10.6, 5.4 Hz, 1H), 5.27 - 5.09 (m, 3H), 3.93 (dt, J = 5.4, 1.5 Hz, 2H), 3.72 (t, J = 6.5 Hz, 2H), 3.65 (t, J = 7.3 Hz, 2H), 3.29 (s, 2H), 1.65 (p, J = 7.5 Hz, 2H), 1.59 - 1.52 (m, 5H), 1.44 - 1.29 (m, 2H), 1.27 (s, 5H). ¹³C NMR (101 MHz, CDCl₃) 8 168.56, 135.37 (d), 135.07, 134.89 (d), 133.96, 132.33, 129.88, 127.69, 123.28, 116.47, 79.22, 75.40, 72.41, 62.94, 38.15, 32.38, 28.76, 27.48, 26.80, 25.59. HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 580.2495, Found [M+Na]⁺ = 580.2493

(9H-Fluoren-9-yl)methyl (6, 6-dimethyl-8,8-diphenyl-4, 7,9-trioxa-8-silapentadec-l-en-15- yl) carbamate

To an oven dried lOOmL round-botom flask was added 2-(6,6-dimethyl-8,8-diphenyl- 4,7,9-trioxa- 8-silapentadec-l-en-15-yl)isoindoline-l, 3-dione (2.890 g, 1 Eq, 5.181 mmol). The vial was capped and purged with nitrogen followed by addition of MeOH (25.91 mL) and then dropwise addition of hydrazine hydrate (1.038 g, 1.005 mL, 4 Eq, 20.73 mmol). The solution was left to stir at room temperature overnight. Upon completion, the reaction mixture was diluted with IM sodium carbonate and IM oxalic acid and extracted with ethyl acetate (3x 30mL). Then, the combined organic extracts were washed with brine and dried over sodium sulfate. Volatiles were removed under reduced pressure and material used in the next step without further purification. To an oven dried 100 mL round-bottom flask was added Fmoc-osu (2.097 g, 1.2 Eq, 6.218 mmol), capped, and purged with argon. The crude amine from the first step was dissolved in dry CH2CI2 (30 mL) and added to this flask. Reaction mixture was then cooled to 0°C and tri ethylamine (1.258 g, 1.73 mL, 2.4 Eq, 12.44 mmol) added. The solution was left to stir at room temperature for 16 hours. Upon completion, the reaction was diluted with water and extracted with CH₂C12(3x30mL). Organic layers combined and washed with brine then dried over sodium sulfate. The crude material was purified by silica column chromatography (1:9 to 1 :3 ethyl acetate: hexanes) to yield the desired product as a pale yellow oil (2.23g, 66%). ¹H NMR (400 MHz, CDCl₃) 8 7.77 (d, J - 7.5 Hz, 3H), 7.68 - 7.63 (m, 5H), 7.63 - 7.58 (m, 3H), 7.43 - 7.28 (m, 14H), 5.86 (ddt, J = 17.3, 10.6, 5.4 Hz, 1H), 5.27 - 5.11 (m, 2H), 4.71 (s, 1H), 4.41 (d, J = 6.9 Hz, 2H), 4.22 (t, J = 6.9 Hz, 1H), 3.94 (dt, J = 5.4, 1.5 Hz, 2H), 3.74 (t, J = 6.5 Hz, 2H), 3.31 (s, 2H), 3.16 (q, J = 6.9 Hz, 2H), 1.63 - 1.53 (m, 2H), 1.46 (q, J = 7.6 Hz, 2H), 1.37 (td, J = 13.4, 11.8, 6.1 Hz, 2H), 1.29 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) 8 156.52, 144.17, 141.46, 135.27, 135.09, 134.90, 129.92, 127.70, 127.15, 125.16, 120.10, 116.50, 79.21, 75.42, 72.42, 62.93, 47.46, 41.19, 32.38, 30.09, 27.50, 26.62, 25.64. HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 672.3121, Found [M+Na]⁺ = 672.3126. l-(9H-Fluoren-9-yl)-14, 14-dimethyl-3-oxo-12, 12-diphenyl-2,ll, 13,16-tetraoxa-20-thia-4-aza- 12- silatricosan-23-oic acid

To an oven-dried 25mL round-bottom flask was added 2,2-dimethoxy-2- phenylacetophenone (94.5 mg, 0.5 Eq, 369 pmol), 3-mercaptopropanoic acid (235 mg, 193 pL, 3 Eq, 2.21 mmol), and (9H-fluoren-9-yl)methyl (6,6-dimethyl-8,8-diphenyl-4,7,9-trioxa-8- silapentadec-l-en-15- yl)carbamate (500 mg, 1 Eq, 738 pmol) which were dissolved in dry THF (2.95 mL). The solution was then sparged with nitrogen for 10 minutes to remove oxygen. The reaction mixture was then allowed to stir at room temperature under UV irradiation (365nm) until judged complete by TLC (4 hours). Upon completion the reaction mixture was diluted with sat. sodium bicarbonate and extracted with ethyl acetate (5x15mL). The combined organic layers were washed with sat. ammonium chloride and brine then dried over sodium sulfate. The crude material was purified by silica column chromatography (1 :1 to 100% ethyl acetate: hexanes) to yield the desired product as a pale yellow oil (360mg, 62%). ¹H NMR (400 MHz, CDCl₃) 8 7.76 (d, J = 7.5 Hz, 2H), 7.64 (dt, J = 6.6, 1.6 Hz, 4H), 7.58 (d, J = 7.5 Hz, 2H), 7.42 - 7.28 (m, 10H), 4.79 (br, 1H), 4.48 -4.33 (m, 2H), 4.21 (t, J = 6.9 Hz, 1H), 3.73 (t, J = 6.5 Hz, 2H), 3.43 (t, J = 6.0 Hz, 2H), 3.28 (d, J = 2.6 Hz, 2H), 3.14 (dt, J = 17.4, 8.7 Hz, 2H), 2.74 (s, 2H), 2.59 (q, J = 8.3 Hz, 4H), 1.79 (t, J = 6.8 Hz, 2H), 1.64 - 1.51 (m, 2H), 1.51 - 1.32 (m, 2H), 1.27 (m, 10H). ¹³C NMR (101 MHz, CDCl₃) 8 141.45, 135.04, 134.89, 134.84, 129.90, 127.80, 127.69, 127.15, 125.14, 120.09, 79.80, 77.36, 75.39, 69.75, 69.74, 66.66, 62.94, 47.42, 41.18, 34.69, 32.38, 29.80, 28.96, 27.45, 26.83, 26.58, 25.63. HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 778.3210, Found [M+Na]⁺ = 778.3209 2-( 6, 6-Dimethyl-4, 4-diphenyl-3, 5, 8-trioxa-4-silaundec- 10-en- l-yl)isoindoline- 1 , 3-dione

To an oven dried 250mL round-bottom flask was added DMAP (2.534 g, 2.25 Eq, 20.74 mmol), l-(allyloxy)-2-methylpropan-2-ol (1.200 g mg, 1 Eq, 9.217 mmol) and capped with septa. The system was purged with argon and dry CH2Q2 (32 mL) was added. The solution was cooled to 0°C followed by dropwise addition of diphenyldichlorosilane (2.917 g, 2.37 mL, 1.25 Eq, 11.52 mmol). The solution was then allowed to warm to room temperature. After 5h the first addition was determined complete by TLC. The solution was cooled to 0°C and N-(2-Hydroxyethyl) phthalimide (1.85 g, 1.05 Eq, 9.68 mmol) was added. The solution was allowed to slowly warm to r.t. and stir overnight. Upon completion the reaction was diluted with sat. sodium bicarbonate and extracted with CH2Q2 (3x 20mL). Combined organic layers were washed with brine and dried over sodium sulfate. Crude product was purified by silica column chromatography (1 :9 to 1 :3 ethyl acetate: hexanes) to yield the pure product as a clear oil (3.25 g, 70%). ¹H NMR (400 MHz, CDCl₃) 8 7.83 - 7.76 (m, 2H), 7.73 - 7.66 (m, 2H), 7.62 - 7.50 (m, 5H), 7.35 - 7.28 (m, 2H), 7.25 - 7.18 (m, 3H), 5.83 (ddt, J = 17.3, 10.7, 5.5 Hz, 1H), 5.24 - 5.09 (m, 2H), 4.02 - 3.97 (m, 2H), 3.92 - 3.86 (m, 4H), 3.26 (s, 2H), 1.23 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) 8 168.32, 135.18, 135.00, 134.45, 133.89, 132.33, 129.97, 127.68, 123.28, 116.51, 79.04, 75.58, 72.35, 60.05, 40.06, 27.39. HRMS (ESI-MS) m/z: calculated [M+Na]⁺ = 524.1870, Found [M+Na]⁺ = 524.1873

(9H-Fluoren-9-yl)methyl(6,6-dimethyl-4,4-diphenyl-3,5,8-trioxa-4-silaundec-10-en-l- yl)carbamate

To an oven dried 250mL round-bottom flask was added 2-(6,6-dimethyl-4,4-diphenyl- 3,5,8-trioxa- 4-silaundec-10-en-l-yl)isoindoline-l, 3-dione (2.64 g, 1 Eq, 5.26 mmol). The vial was capped and purged with nitrogen followed by addition of MeOH (26.3mL) and then drop wise addition of hydrazine hydrate (1.05 g, 1.02 mL, 4 Eq, 21.1 mmol). Solution let stir at room temperature overnight. Upon completion, reaction mixture diluted with IM sodium carbonate and IM oxalic acid and extracted with ethyl acetate (3x 30mL). Then, combined organic extracts washed with brine and dried over sodium sulfate. Volatiles were removed under reduced pressure and material used in the next step without further purification. To an oven dried 250 mL roundbottom flask was added Fmoc-osu (2.13 g, 1.2 Eq, 6.32 mmol), capped, and purged with argon. The crude amine from the first step was dissolved in dry CH₂C1₂ (26 mL) and added to this flask. Reaction mixture was then cooled to 0°C and triethylamine (1.28 g, 1.76 mL, 2.4 Eq, 12.6mmol) added. The solution was allowed to stir at room temperature for 16 hours. Upon completion, the reaction was diluted with water and extracted with CH₂C1₂ (3x30mL). Organic layers combined and washed with brine then dried over sodium sulfate. The crude material was purified by silica column chromatography (1:9 to 1:3 ethyl acetate: hexanes) to yield the desired product as a pale yellow oil (1.89g, 61%). ¹H NMR (400 MHz, CDCl₃) 8 7.80 (d, J = 7.6 Hz, 2H), 7.69 (dt, J = 6.7, 1.5 Hz, 4H), 7.67 - 7.63 (m, 2H), 7.47 - 7.29 (m, 10H), 5.87 (ddt, J = 16.3, 10.7, 5.6 Hz, 1H), 5.56 (d, J = 5.9 Hz, 1H), 5.29 - 5.12 (m, 2H), 4.42 (d, J = 6.9 Hz, 2H), 4.25 (t, J = 6.9 Hz, 1H), 3.94 - 3.85 (m, 4H), 3.41 (q, J = 5.3 Hz, 2H), 3.30 (s, 2H), 1.32 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) 8 156.59, 144.17, 141.41, 135.02, 130.15, 127.81, 127.74, 127.12, 125.17, 120.04, 116.85, 78.87, 75.60, 72.29, 66.67, 62.43, 47.37, 43.27, 27.45. HRMS (ESI-MS) m/z: calculated [M+Na]⁺ = 616.2495, Found [M+Na]⁺= 616.2496.

General procedure for synthesis of β-hydroxy isovaleric esters

To an oven dried round-bottom flask was added DMAP (0.2 Eq.), CH₂C1₂ (0.5M), 13- hydroxy Isovaleric Acid (1 Eq.), and alcohol/phenol (1.05-1.2 Eq.). The reaction vessel was lightly purged with argon, cooled to 0°C and DCC (1.5 Eq.) was added portionwise over 3 minutes. The reaction mixture was then allowed to warm to room temperature and stir until judged complete by TLC (2- 16 hours). Crude material was purified by silica column chromatography (1:9 to 1:3 ethyl acetate:hexanes). Ethyl 3-hydroxy-3-methylbutanoate

An oven dried 250 mL round-bottom flask was capped with rubber septa and purged with argon. Then anhydrous THF (51mL) and a 2M solution of LDA in THF (21.2 mL, 0.83 Eq, 42.5 mmol) was added. This mixture was cooled to -78°C and ethyl acetate (5 mL, 1 Eq, 51.2 mmol) was added dropwise with strong stirring and solution left stirring at -78°C for 1 hour. After this, acetone (3.79mL, 1 Eq, 51.2 mmol) was added at -78°C and the solution left to warm to room temperature and stir for 15 min. The reaction mixture was then diluted with 2M HC1 (30mL) and the organic layer separated. Aqueous layer was then extracted with ethyl acetate (3x30mL) and combined organic layers were washed with sat. sodium bicarbonate and brine then dried over sodium sulfate. Crude material was purified by vacuum distillation to yield the desired product as a colorless liquid (4.23g, 56%). All analyses were consistent with previously reported data.^{5 1}H NMR (400 MHz, CDCl₃) 84.18 (q, J = 7.2 Hz, 2H), 2.48 (s, 2H), 1.33 - 1.24 (m, 9H).

Benzyl 3-hydroxy-3-methylbutanoate

Following the general procedure with benzyl alcohol (0.53mL, 1.2 Eq, 5.08mmol) the desired product was obtained as a colorless oil (870mg, 99%). All analyses were consistent with previously reported data.^{6 1}H NMR (400 MHz, CDCl₃) 87.44 - 7.30 (m, 5H), 5.16 (s, 2H), 3.47 (s, 1H), 2.55 (s, 2H), 1.28 (s, 6H).

Allyl 3-hydroxy-3-methylbutanoate

Following the general procedure with allyl alcohol (1.21mL, 1.05 Eq, 17.8mmol) the desired product was obtained as a colorless oil (1.77g, 66%). ¹H NMR (400 MHz, CDCl₃) 8 5.89 (ddt, J = 17.1, 10.4, 5.8 Hz, 1H), 5.34 - 5.19 (m, 2H), 4.59 (dt, J = 5.8, 1.4 Hz, 2H), 3.49 (s, 1H), 2.49 (s, 2H), 1.26 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 172.59, 131.88, 118.76, 69.07, 65.30, 46.42, 29.23.

Methylbut-2-en-l-yl 3-hydroxy-3-methylbutanoate

Following the general procedure with prenyl alcohol (1.22mL, 1.2 Eq, 12mmol) the desired product was obtained as a colorless oil (1.72g, 92%). ¹H NMR (400 MHz, CDCl₃) δ 5.27 (ddp, J = 8.7, 5.7, 1.4 Hz, 1H), 4.54 (dt, J = 7.2, 0.9 Hz, 2H), 3.58 (s, 1H), 2.41 (s, 2H), 1.72 - 1.62 (m, 6H), 1.20 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) 8 172.81, 139.47, 118.25, 68.95, 61.41, 46.44, 29.11, 25.69, 17.97.

Tert-butyl 3-hydroxy-3-methylbutanoate

To a flame dried 250mL round-bottom flask purged with argon was added THF (40 mL) and diisopropylamine (3.66 g, 5.10 mL, 0.97 Eq, 36.2 mmol). The solution was subsequently cooled to 0°C and n-butyllithium (2.36 g, 14.8 mL, 2.5 molar, 0.99 Eq, 36.9 mmol) was slowly added with strong stirring. This mixture was allowed to react for one hour at 0°C after which the solution was cooled to -78°C and tert-butyl acetate (4.33 g, 5.00 mL, 1 Eq, 37.3 mmol) was added dropwise. After another hour of stirring at -78°C, acetone (2.17 g, 2.74 mL, 1 Eq, 37.3 mmol) was added within 30 seconds and the mixture was allowed to stir for a further 10 minutes. The mixture was then warmed to 0°C, diluted with 25mL water and acidified using 2M HC1 and the organic layer separated. The aqueous layer was stripped of its solvents under reduced pressure and extracted with ethyl acetate (3x 30mL). The combined organic extracts were washed with sat. sodium bicarbonate, brine, and dried over sodium sulfate. Material concentrated under reduced pressure to yield the desired product as a colorless oil (5.16g, 79%). All analyses were consistent with previously reported data.^{5 1}H NMR (400 MHz, CDCl₃) δ 3.80 (s, 1H), 2.39 (s, 2H), 1.47 (s, 9H), 1.26 (s, 6H).

2,5-Dioxopyrrolidin-l-yl 3-hydroxy-3-methylbutanoate

Following the general procedure with N-hydroxysuccinimide (1.22mL, 1.2 Eq, 12mmol) and EDC (575mg, 1 Eq, 3mmol) the desired product was obtained as a colorless oil (618mg, 95%). Column chromatography conditions (1: 1 to 2: 1 ethyl acetate: hexanes). All analyses were consistent with previously reported data. ¹H NMR (400 MHz, CDCl₃) 8 2.85 (br, J = 2.8 Hz, 4H), 2.77 (s, J = 1.4 Hz, 2H), 1.41 - 1.37 (s, 6H).

Methoxybenzyl 3-hydroxy-3-methylbutanoate

Following the general procedure with p-methoxybenzyl alcohol (684mg, 1.2 Eq, 4.95mmol) the desired product was obtained as a colorless oil (856mg, 87%). ¹H NMR (400 MHz, CDCl₃) 87.24 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 5.03 (s, 2H), 3.72 (_s, 3H), 3.58 (s, 1H), 2.46 (s, 2H), 1.22 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) 8 172.39, 159.56, 129.99, 127.60, 113.81, 68.89, 65.99, 55.01, 46.47, 29.01.

2,3,5, 6-Tetrafluorophenyl 3-hydroxy-3-methylbutanoate

Following the general procedure with 2,3,5,6-tetrafluorophenol (1.69g, 1.2 Eq, 10.2mmol) the desired product was obtained as a colorless oil (2.0g, 89%). ¹H NMR (400 MHz, CDCl₃) 8 7.01 (tt, J = 9.9, 7.1 Hz, 1H), 2.87 (s, 2H), 1.41 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) 8 168.25, 103.56 (t), 69.55, 46.25, 29.17. ¹⁹F NMR (376 MHz, CDCl₃) 8 -137.70 - -139.89 (m), 152.04 -153.39 (m). 2, 6-Difluorophenyl 3-hydroxy-3-methylbutanoate

Following the general procedure with 2, 6- difluorophenol (661mg, 1.2 Eq, 5.1mmol) the desired product was obtained as a colorless oil (760mg, 78%). ¹H NMR (400 MHz, CDCl₃) δ 7.21 - 7.13 (m, 1H), 7.01 - 6.93 (m, 2H), 2.99 (s, 1H), 2.84 (s, 2H), 1.40 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) 8 169.05, 156.40, 153.91, 126.65, 112.16, 111.99, 69.28, 46.22, 29.10. ¹⁹F NMR (376 MHz, CDCl₃) 8 -125.90.

2, 4-Difluorophenyl 3-hydroxy-3-methylbutanoate

Following the general procedure with 2,4-difluorophenol (661mg, 1.2 Eq, 5.1mmol) the desired product was obtained as a colorless oil (950mg, 97%). ¹H NMR (400 MHz, CDCl₃) δ 7.10 (td, J = 8.7, 5.5 Hz, 1H), 6.97 - 6.84 (m, 2H), 3.08 (br, 1H), 2.79 (s, 2H), 1.39 (s, 6H). ¹³C NMR (101 MHz, CDCl₃)δ 170.08, 160.38 (dd, J = 247.7, 10.5 Hz), 154.07 (dd, J = 251.9, 12.5 Hz), 134.08 (dd, J = 13.1, 4.1 Hz), 124.30 (dd, J = 9.9, 2.0 Hz), 111.43 (dd, J = 23.1, 3.8 Hz), 105.24 (dd, J = 27.0, 22.4 Hz), 69.34, 46.38, 29.25. ¹⁹F NMR (376 MHz, CDCl₃) 8 -112.17, -123.09.

3-Hydroxy-3-methyl-l-(lH-pyrazol-l-yl)butan-l-one

Following the general procedure with IH-pyrazole (176mg, 1.2 Eq, 2.59mmol) the desired product was obtained as a colorless oil (357mg, 98%). ¹H NMR (400 MHz, CDCl₃) δ 8.29 (dd, J = 2.9, 0.7 Hz, 1H), 7.73 (dd, J = 1.5, 0.7 Hz, 1H), 6.47 (dd, J = 2.9, 1.5 Hz, 1H), 3.34 (s, 2H), 1.37 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) 8 171.26, 144.52, 128.50, 110.16, 69.84, 46.06, 29.60. General procedure for the synthesis of dialkoxydiphenylsilanes (DADPS)

To an oven dried round-bottom flask was added DMAP (2.25 Eq.), B-hydroxy isovaleric ester (1 Eq.) and capped with septa. The system was purged with argon and dry CH2Q2 (0.2M) was added. The solution was cooled to 0°C followed by addition of diphenyldichlorosilane (1.25 Eq.). The solution was then allowed to warm to room temperature. Upon completion of the first step as monitored by TLC (5-16 hours) the solution was cooled back to 0°C. The flask was briefly uncapped and the protected amino alcohol (1.05 Eq.) was added in one portion. The flask was recapped, briefly purged with argon, and let warm to room temperature. Upon complete conversion, the reaction was diluted with sat. sodium bicarbonate and extracted with CH2Q2 (3x3 OmL). Organic extracts were combined and washed with brine and dried over sodium sulfate. Crude material was purified by silica column chromatography (1 :9 to 1 :3 ethyl acetate: hexanes).

Ethyl 14, 14-dimethyl- 3-oxo- 1, 12, 12-triphenyl-2,ll, 13-trioxa-4-aza-12-silahexadecan-16- oate

Using general procedure with ethyl 3-hydroxy-3-methylbutanoate (315mg, 2.15mmol, 1 Eq.) and benzyl (6-hydroxyhexyl)carbamate (567mg, 2.26mmol, 1.05 Eq.) the desired product was obtained as a colorless oil (567mg, 46%). ¹H NMR (400 MHz, CDCl₃) 87.73 - 7.63 (m, 6H), 7.44 - 7.30 (m, 11H), 4.16 - 4.07 (m, 3H), 3.75 (t, J = 6.5 Hz, 1H), 3.17 (q, J = 6.7 Hz, 1H), 2.65 (s, 2H), 2.59 (s, 1H), 1.63 - 1.54 (m, 1H), 1.52 - 1.44 (m, 1H), 1.42 (s, 3H), 1.42 (s, 6H), 1.36 - 1.25 (m, 1H), 1.25 - 1.20 (m, 3H). ¹³C NMR (101 MHz, CDC13) 8 171.99, 136.04, 135.05, 134.61, 130.01, 129.97, 128.59, 127.75, 127.72, 74.37, 74.31, 62.93, 60.69, 60.34, 49.41, 49.00, 32.29, 30.67, 30.20, 25.56, 14.22. HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 600.2758, Found [M+Na]⁺ = 600.2758. Benzyl 14, 14-dimethyl-3-oxo-l, 12, 12-triphenyl-2,ll, 13-trioxa-4-aza-12-silahexadecan- 16-oate

Using general procedure with benzyl 3 -hydroxy-3 -methylbutanoate (450mg, 2.15mmol, 1 Eq.) and benzyl (6-hydroxyhexyl)carbamate (567mg, 2.26mmol, 1.05 Eq.) the desired product was obtained as a colorless oil (585mg, 43%). ¹H NMR (400 MHz, CDCl₃) 87.68 - 7.60 (m, 4H), 7.45 - 7.27 (m, 16H), 5.12 (br, J = 1.9 Hz, 3H), 3.73 (t, J = 6.5 Hz, 2H), 3.18 (q, J = 6.9 Hz, 2H), 2.66 (s, 2H), 1.63 - 1.53 (m, 2H), 1.53 - 1.46 (m, 5H), 1.44 (s, 6H), 1.40 - 1.33 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) 8 170.76, 136.77, 135.96, 135.02, 134.80, 134.75, 129.98, 128.55, 128.53, 128.35, 128.18, 128.11, 127.70, 74.29, 66.59, 66.27, 62.88, 49.26, 41.10, 32.24, 30.19,29.95, 26.49, 25.50. HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 662.2914, Found [M+Na]⁺ = 662.2950.

Allyl 14, 14-dimethyl- 3-oxo- 1 , 12, 12-triphenyl-2,ll, 13-trioxa-4-aza-12-silahexadecan- 16-oate

Using general procedure with allyl 3 -hydroxy-3 -methylbutanoate (200mg, 1.26mmol, 1 Eq.) and benzyl (6-hydroxyhexyl)carbamate (334mg, 1.33mmol, 1.05 Eq.) the desired product was obtained as a colorless oil (484mg, 65%). ¹H NMR (400 MHz, CDCl₃) 87.67 - 7.62 (m, 4H), 7.43 - 7.30 (m, 13H), 5.88 (ddt, J = 17.2, 10.4, 5.8 Hz, 1H), 5.33 - 5.17 (m, 2H), 5.10 (s, 2H), 4.74 (br, 1H), 4.56 (dt, J = 5.8, 1.4 Hz, 2H), 3.72 (t, J = 6.5 Hz, 2H), 3.17 (q, J = 6.7 Hz, 2H), 2.61 (s, 2H), 1.62 - 1.52 (m, 2H), 1.47 (p, J = 7.4 Hz, 2H), 1.41 (s, 6H), 1.39 - 1.24 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) 8 170.69, 135.08, 134.83, 132.36, 130.04, 128.64, 128.20, 127.75, 118.43, 74.32, 66.70, 65.20, 62.97, 49.31, 41.18, 32.32, 30.23, 30.04, 26.57, 25.59. HRMS (ESI-MS) m/z Calculated [M+Na]⁺ = 612.2758, Found [M+Na]⁺ = 612.2758.

Methylbut-2-en-l-yl 14, 14-dimethyl-3-oxo-l, 12, 12-triphenyl-2,ll, 13-trioxa-4-aza-12- silahexadecan- 16-oate Using general procedure with 3-methylbut-2-en-l-yl 3-hydroxy-3-methylbutanoate (400mg, 2.15mmol, 1 Eq.) and benzyl (6-hydroxyhexyl)carbamate (567mg, 2.26mmol, 1.05 Eq.) the desired product was obtained as a pale yellow oil (1.08g, 81 %).¹H NMR (400 MHz, CDCl₃) 8 7.67 - 7.62 (m, 4H), 7.42 - 7.30 (m, 11H), 5.30 (tdt, J = 5.7, 2.9, 1.4 Hz, 1H), 5.10 (s, 2H), 4.77 (s, 1H), 4.57 (d, J = 7.2 Hz, 2H), 3.72 (t, J = 6.4 Hz, 2H), 3.17 (q, J = 6.9 Hz, 2H), 2.58 (s, 2H), 2.08 (s, 1H), 1.72 (s, 3H), 1.67 (s, 3H), 1.56 (m, 2H), 1.46 (m, 2H), 1.40 (s, 6H), 1.38-1.28 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) 8 171.08, 138.80, 135.08, 134.87, 134.48, 129.99, 128.62, 128.24, 128.19, 127.72, 118.82, 74.34, 66.70, 62.92, 61.33, 49.40, 41.17, 32.31, 30.19, 29.22, 26.56, 25.83, 25.57, 18.08. HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 640.3070, Found [M+Na]⁺ = 640.3069

Tert-butyl 14, 14-dimethyl-3-oxo-l, 12, 12-triphenyl-2,ll, 13-trioxa-4-aza-12-silahexadecan- 16-oate

Using general procedure with tert-butyl 3 -hydroxy-3 -methylbutanoate (400mg, 2.3mmol, 1 Eq.) and benzyl (6-hydroxyhexyl)carbamate (606mg, 2.41mmol, 1.05 Eq.) the desired product was obtained as a pale yellow oil (741mg, 53%). ¹H NMR (400 MHz, CDCl₃) 8 7.74 - 7.68 (m, 4H), 7.45 - 7.30 (m, 11H), 5.12 (s, 2H), 4.96 (q, J = 10.6, 8.2 Hz, 1H), 3.79 (t, J = 6.5 Hz, 2H), 3.18 (q, J = 6.7 Hz, 2H), 2.54 (s, 2H), 1.69 - 1.56 (m, 4H), 1.56 - 1.34 (m, 19H). ¹³C NMR (101 MHz, CDCl₃) 8 170.29, 156.45, 134.99, 134.83, 134.42, 129.89, 128.47, 128.09, 128.02, 127.61, 80.16, 74.37, 66.50, 62.82, 50.59, 47.36, 41.03, 32.21, 30.09, 29.07, 28.10, 26.44, 25.47. HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 628.3070, Found [M+Na]⁺ = 628.3075

Methoxybenzyl 14, 14-dimethyl-3-oxo-l, 12, 12-triphenyl-2, 11, 13-trioxa-4-aza-12- silahexadecan-

16-oate

Using general procedure with tert-butyl 3-hydroxy-3-methylbutanoate (41 Img, 1 ,72mmol, 1 Eq.) and benzyl (6-hydroxyhexyl)carbamate (477mg, 1.9mmol, 1.1 Eq.) the desired product was obtained as a colorless oil (982mg, 85%). ¹H NMR (400 MHz, CDCl₃) 87.65 - 7.61 (m, 4H), 7.43 - 7.29 (m, 11H), 7.21 (d, J = 8.7 Hz, 2H), 6.82 (d, J = 8.7 Hz, 2H), 5.11 (s, 2H), 5.03 (s, 2H), 4.75 (s, 1H), 3.79 (s, 3H), 3.70 (t, J = 6.5 Hz, 2H), 3.16 (q, J = 6.8 Hz, 2H), 2.61 (s, 2H), 1.59 - 1.51 (m, 2H), 1.46 (p, J = 7.1 Hz, 2H), 1.40 (s, 6H), 1.31 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) 8 170.91, 159.64, 156.48, 136.81, 135.08, 134.86, 134.84, 130.23, 130.01, 128.62, 128.23, 128.18, 127.74, 113.97, 74.36, 66.67, 66.10, 62.93, 55.37, 49.37, 41.16, 32.30, 30.23, 30.02, 26.55, 25.56. HRMS (ESI-MS) m/z-. Calculated [M+Na]⁺ = 692.3019, Found [M+Na]⁺ = 692.3018.

Using general procedure with allyl 3-hydroxy-3-methylbutanoate (lOOmg, 0.63mmol, 1 Eq.) and 2-(6-hydroxyhexyl)isoindoline-l, 3-dione (164mg, 0.66mmol, 1.05 Eq.) the desired product was obtained as a colorless oil (342mg, 92%). ¹H NMR (400 MHz, CDCl₃) 8 7.84 (dd, J = 5.4, 3.1 Hz, 2H), 7.70 (dd, J = 5.5, 3.0 Hz, 2H), 7.65 - 7.60 (m, 4H), 7.41 - 7.30 (m, 6H), 5.87 (ddt, J = 17.3, 10.4, 5.8 Hz, 1H), 5.32 - 5.16 (m, 2H), 4.55 (dt, J = 5.9, 1.4 Hz, 2H), 3.71 (t, J = 6.4 Hz, 2H), 3.66 (t, J = 7.3 Hz, 2H), 2.59 (s, 2H), 1.65 (p, J= 7.5 Hz, 2H), 1.61 - 1.52 (m, 3H), 1.39 (s, 6H), 1.42-1.35 (m, 2H), 1.35 - 1.25 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) 8 170.70, 168.57, 135.09, 134.84, 133.97, 132.33, 130.02, 127.75, 123.29, 118.43, 74.32, 65.20, 63.00, 49.31, 38.15, 32.35, 30.23, 28.75, 26.78, 25.59. HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 608.2444, Found [M+Na]⁺ = 608.2442

Allyl l-(9H-fluoren-9-yl)-14, 14-dimethyl-3-oxo-12, 12-diphenyl-2,ll, 13-trioxa-4-aza-12- silahexadecan- 16-oate To an oven dried lOOmL round-botom flask was added allyl 3-((((6-(l,3-dioxoisoindolin- 2- yl)hexyl)oxy)diphenylsilyl)oxy)-3-methylbutanoate (1.525 g, 1 Eq, 2.60 mmol). The vial was capped and purged with nitrogen followed by addition of MeOH (26 mL) and then dropwise addition of hydrazine hydrate (521mg, 0.51 mL, 4 Eq, 10.41 mmol). Solution let stir at room temperature overnight. Upon completion, reaction mixture diluted with IM sodium carbonate and IM oxalic acid and extracted with ethyl acetate (3x 30mL). Then, combined organic extracts washed with brine and dried over sodium sulfate. Volatiles were removed under reduced pressure and material used in the next step without further purification. To an oven dried 100 mL roundbotom flask was added Fmoc-osu (1.054 g, 1.2 Eq, 3.12 mmol), capped, and purged with argon. The crude amine from the first step was dissolved in dry CH2CI2 (30 mL) and added to this flask. Reaction mixture was then cooled to 0°C and triethylamine (632 mg, 0.87 mL, 2.4 Eq, 6.248 mmol) added. Solution let stir at room temperature for 16 hours. Upon completion, the reaction was diluted with water and extracted with CH₂C1₂ (3x3 OmL). Organic layers combined and washed with brine then dried over sodium sulfate. The crude material was purified by silica column chromatography (1 :9 to 1 :3 ethyl acetate: hexanes) to yield the desired product as a colorless oil (1.22g, 69%). ¹H NMR (400 MHz, CDCl₃) 87.78 (dt, J = 7.5, 1.0 Hz, 2H), 7.71 - 7.65 (m, 4H), 7.62 (dt, J = 7.4, 0.9 Hz, 2H), 7.45 - 7.30 (m, 10H), 5.91 (ddt, J = 17.3, 10.4, 5.8 Hz, 1H), 5.36 - 5.19 (m, 2H), 4.83 (t, J = 6.0 Hz, 1H), 4.60 (dt, J = 5.8, 1.4 Hz, 2H), 4.44 (d, J = 6.9 Hz, 2H), 4.24 (t, J = 6.9 Hz, 1H), 3.77 (t, J = 6.5 Hz, 2H), 3.18 (q, J = 6.7 Hz, 2H), 2.65 (s, 2H), 1.65 - 1.56 (m, 2H), 1.55 - 1.47 (m, 2H), 1.45 (s, 6H), 1.43 - 1.28 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) 8 170.62, 156.48, 144.11, 141.39, 135.04, 134.78, 132.30, 130.01, 127.71, 127.08, 125.09, 120.02, 118.38, 74.27, 66.51, 65.14, 62.94, 49.25, 47.40, 41.10, 32.29, 30.20, 29.99, 26.52, 25.55. HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 700.3070, Found [M+Na]⁺ = 700.3074. l-(9H-Fluoren-9-yl)-14, 14-dimethyl-3,16-dioxo-12, 12-diphenyl-2, 11, 13, 17-tetraoxa-21-thia-4- aza-12-silatetracosan-24-oic acid

To a 25mL round-bottom flask was added 2,2-dimethoxy-2-phenylacetophenone (19.1 mg, 0.5 Eq, 74.5 pmol), 3-mercaptopropanoic acid (47.4 mg, 39 pL, 3 Eq, 447 pmol), and (9H-fluoren- 9- yl)methyl (6,6-dimethyl-8,8-diphenyl-4,7,9-trioxa-8-silapentadec-l-en-15-yl)carbamate (500 mg, 1 Eq, 738 pmol) which were dissolved in dry THF (0.6 mL). The solution was then sparged with nitrogen for 10 minutes to remove oxygen. The reaction mixture was then allowed to stir at room temperature under UV irradiation (365nm) until judged complete by TLC (4 hours). Upon completion the reaction mixture was diluted with sat. sodium bicarbonate and extracted with ethyl acetate (5x15mL). The combined organic layers were washed with sat. ammonium chloride and brine then dried over sodium sulfate. The crude material was purified by silica column chromatography (1 : 1 to 100% ethyl acetate: hexanes) to yield the desired product as a pale yellow oil (69mg, 59%). ¹H NMR (400 MHz, CDCl₃) δ 7.76 (dt, J = 7.6, 1.0 Hz, 2H), 7.66 - 7.56 (m, 6H), 7.45 - 7.28 (m, 10H), 4.80 (br, 1H), 4.47 - 4.39 (m, 2H), 4.21 (m, 1H), 4.14 (m, 2H), 3.73 (t, J= 6.3 Hz, 2H), 3.20 - 3.06 (m, 2H), 2.72 (t, J= 7.0 Hz, 2H), 2.57 (ddt, J= 21.6, 14.5, 6.7 Hz, 6H), 1.86 (p, J = 6.8 Hz, 2H), 1.63 - 1.53 (m, 2H), 1.51-1.25 (m, 12H). ¹³C NMR (101 MHz, CDCl₃) 8 176.19, 171.01, 156.69, 144.13, 141.46, 135.07, 134.82, 130.10, 127.80, 127.16, 125.15, 120.10, 74.33, 66.69, 62.98, 60.56, 49.37, 47.44, 41.18, 34.55, 32.35, 30.25, 28.74, 26.82, 26.56, 25.59, 21.18, 14.33. HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 806.3159, Found [M+Na]⁺ = 806.3155 l-(9H-Fluoren-9-yl)- 10, 10-dimethyl-3-oxo-8,8-diphenyl-2, 7,9, 12-tetraoxa-16-thia-4-aza-8- silanonadecan- 19-oic acid

To an oven dried pressure tube was added (9H-fluoren-9-yl)methyl (6,6-dimethyl-4,4- diphenyl- 3,5,8-trioxa-4-silaundec-10-en-l-yl)carbamate (494 mg, 1 Eq, 832 pmol), 3- mercaptopropionic acid (88.3 mg, 72.4 pL, 1 Eq, 832 pmol), and DMPA (4.26 mg, 0.02 Eq, 16.6 pmol). The vial was placed under a nitrogen atmosphere through vacuum purge cycles (3 cycles) and then the vial was capped. The vial was then irradiated using UV light (365nm, 4W compact lamp) with slow stirring and the whole setup was wrapped in aluminum foil. After 24 hours, full conversion was observed by NMR. The crude mixture was then dissolved in ethyl acetate and washed with sat. sodium bicarbonate (3x 5mL), sat. ammonium chloride (lx 5mL), and brine. The organic layer was then dried over sodium sulfate and concentrated under reduced pressure to yield the desired product as a pale-yellow wax (541mg, 93%). ¹H NMR (400 MHz, CDCl₃) δ 7.78 (d, J = 7.6 Hz, 2H), 7.64 (dd, J= 19.6, 4.5 Hz, 6H), 7.37 (ddt, J= 25.2, 14.3, 5.2 Hz, 1 OH), 4.41 (d, J= 6.9 Hz, 2H), 4.23 (t, J = 6.8 Hz, 1H), 3.90 - 3.69 (m, 2H), 3.46 - 3.16 (m, 6H), 2.73 (t, J = 7.2 Hz, 2H), 2.57 (dt, J= 22.3, 7.3 Hz, 4H), 1.77 (t, J= 6.9 Hz, 1H), 1.27 (m, 8H). ¹³C NMR (101 MHz, CDCl₃) 8 156.67, 144.05, 141.36, 134.95, 134.46, 130.13, 127.78, 127.72, 127.09, 125.09, 120.00, 79.54, 75.56, 69.71, 66.70, 62.32, 47.28, 43.21, 34.66, 29.55, 28.83, 27.35, 26.80. HRMS (ESI- MS) m/z:. Calculated [M+Na]⁺ = 722.2584 , Found [M+Na]⁺ = 722.2601

3-((3-((3-hydroxy-3-methylbutanoyl)oxy)propyl)thio)propanoic acid

To a one-dram scintillation vial equipped with stir bar was added allyl 3-hydroxy-3- methylbutanoate (lOOmg, 0.63 mmol, 1 Eq.), 3 -mercaptopropionic acid (201mg, 165pL, 1.90mmol, 3 Eq.), DMPA (81mg, 0.32mmol, 0.5 Eq.), and THF (2.4mL). The solution was sparged with argon for 10 minutes and then subjected to UV irradiation (365nm, 6W handheld lamp) for 4h. The crude material was purified by silica column chromatography (1: 1 ethyl acetate: hexanes) to yield the desired product as a white solid (102mg, 61%). ¹H NMR (400 MHz, CDCl₃) δ 6.25 (br, 1H), 4.20 (t, J= 6.3 Hz, 2H), 2.77 (t, J= 7.2 Hz, 2H), 2.62 (dt, J= 11.6, 7.1 Hz, 4H), 2.50 (s, 2H), 1.92 (p, J= 6.7 Hz, 2H), 1.28 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) 8 176.82, 172.94, 69.49, 63.26, 46.42, 34.73, 29.20, 28.64, 28.53, 26.83. HRMS (ESI-MS) m/z'. Calculated [M+Na]⁺ = 287.0929 , Found [M+Na]⁺ = 287.0952.

Symmetric N, N-dimethyl-L-leucine

To a 10 mL pressure tube equipped with a magnetic stir bar was added L-Leucine (75 mg, 1 Eq, 0.57 mmol), methanol (2.3mL, 0.25M), and formaldehyde (0.19 g, 0.17 mL, 37% Wt, 4 Eq, 2.3 mmol). Under strong stirring, pyridine-borane complex (0.11 g, 0.12 mL, 2 Eq, 1.1 mmol) was added dropwise. Upon complete addition the reaction mixture was allowed to stir at ambient temperature for 48-72 hours. After completion anhydrous sodium sulfate was added directly into the tube and the contents were filtered over cotton. The solids were washed with methanol and filtrate concentrated under reduced pressure to yield crude product as a white solid. Residue was purified by 2 rounds of trituration with diethyl ether to yield pure N,N-dimethyl-L-Leucine as a white solid (90mg, 99%). Spectral analyses aligned with previously reported. ¹H NMR (400 MHz, D2O) 8 3.60 - 3.53 (m, IH), 2.88 (s, 6H), 1.78 - 1.59 (m, 3H), 0.97 (dd, J = 6.1, 4.8 Hz, 6H).

Asymmetric N,N-dimethyl-L-leucine

N-Cbz-L-leucine: To a lOmL pressure tube was added L-leucine (200 mg, 1 Eq, 1.50 mmol) and Aq. NaOH (60.1 mg, 751 pL, 2 molar, 1 Eq, 1.50 mmol). Vial was cooled to 0°C and, with strong stirring, Cbz-Cl (307 mg, 257 pL, 1.2 Eq, 1.80 mmol) and NaOH (72.1 mg, 901 pL, 2 molar, 1.2 Eq, 1.80 mmol) was added from two separate syringes simultaneously. After complete addition, the solution was left to stir at 0°C for Ih followed by warming to ambient temperature and stirring for an additional hour. After completion the reaction mixture extracted lx with Et20, acidified with IM HC1, and then extracted 3x with Et20. The latter ether portions were combined and dried over sodium sulfate, filtered over cotton, and concentrated down to yield the pure product as a clear oil (338mg, 85%). Spectral analyses aligned with previously reported.

N-Cbz-N-methyl-L-leucine: N-Cbz-L-leucine (140 mg, 1 Eq, 528 pmol) was dissolved in MeCN (1.76 mL) and cooled to 0°C. Upon cooling, NaH (65.4 mg, 60% Wt, 3.1 Eq, 1.64 mmol) was added followed by dropwise addition of Mel (532 mg, 234 pL, 7.1 Eq, 3.75 mmol) to form a thick liquid. The reaction mixture was then allowed to warm to ambient temperature and stirred aggressively (840rpm) overnight. After completion, EtOAc (15 ml) and water (5 ml) were added and the solvent evaporated off. The residue was redissolved in ether and water and extracted 2x with ether. Organic layers were combined and washed 2x with sat. sodium bicarb. Aq layers combined and acidified to a pH of 2 with IM HC1 then extracted 3x with Et2O. Combined Et2O layers washed lx with water, dried over sodium sulfate, and concentrated down. Pure product obtained after flash column chromatography (30 to 50% EtOAc/Hex) as a clear oil (lOOmg, 67%). Spectral analyses aligned with previously reported.

N,N-dimethyl-L-leucine: General Procedure A:_To an oven dried pwave vial was added N-Cbz-N-methyl-L-leucine (110 mg, 1 Eq, 392 pmol), MeOH (1.31 mL), formaldehyde (63.7 mg, 58.4 pL, 37% Wt, 2 Eq, 785 pmol), and Pd/C (12.5 mg, 10% Wt, 0.03 Eq, 11.8 pmol). The flask was thoroughly purged with argon then hydrogen was bubbled through the solution for one minute. Left stir under a hydrogen atmosphere at ambient temperature overnight. After reaction completion as determined by LC-MS, anhydrous sodium sulfate was added and reaction mixture filtered over celite. The filtrate was concentrated down and the resulting white solid was triturated 2x with Et2O to furnish the final product as a white solid (54mg, 86%).

N,N-dimethyl-L-leucine: General Procedure B (for D incorporation on second methyl group): To an oven dried pwave vial was added N-Cbz-N-methyl-L-leucine (110 mg, 1 Eq, 392 pmol), MeOH (1.31 mL), and Pd/C (12.5 mg, 10% Wt, 0.03 Eq, 11.8 pmol). The flask was thoroughly purged with argon then hydrogen was bubbled through the solution for one minute. Left stir under a hydrogen atmosphere at ambient temperature overnight. After reaction completion as determined by LC-MS, the reaction mixture filtered over celite and concentrated down. The resulting N-methyl-L-leucine was dissolved in MeOH (1.31 mL) followed by addition of formaldehyde (63.7 mg, 58.4 pL, 37% Wt, 2 Eq, 785 pmol). With strong stirring pyr*BD3 (53.4 mg, 1.5 Eq, 556 pmol) was added and reaction mixture allowed to stir for 24h. After reaction completion as determined by LC-MS, anhydrous sodium sulfate was added and reaction mixture was filtered over cotton. The filtrate was concentrated down and the resulting white solid was triturated 2x with Et2O to furnish the final product as a white solid (40mg, 66%).

N,N-dimethyl-l-¹³C-L-Leucine (114): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with l-¹³C-L-Leucine. (79mg, >99% yield). ¹H NMR (300 MHz, D2O) 8 3.56 (dd, J= 9.8, 4.7 Hz, 1H), 2.88 (s, 6H), 1.79 - 1.61 (m, 3H), 0.97 (dd, J = 6.0, 3.6 Hz, 6H). ¹³C NMR (75 MHz, D₂O) 8 173.65, 36.76, 25.07, 22.69, 20.57.

N,N-dimethyl-^I5N-L-Leucine (115N): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with ¹⁵N-L-Leucine. (79mg, >99% yield). ¹H NMR (300 MHz, D2O) 83.61 - 3.53 (m, 1H), 2.89 (d, J= 0.8 Hz, 6H), 1.79 - 1.58 (m, 3H), 0.97 (dd, J= 6.0, 3.6 Hz, 6H). ¹³C NMR (75 MHz, D2O) 8 173.57, 70.27, 36.74, 25.08, 22.69, 20.56. N,N-dimethyl-2-¹³C-L-Leucine (115C): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with ¹⁵N-L-Leucine. (76mg, 96% yield). ¹H NMR (300 MHz, D2O) 83.85 - 3.28 (m, 1H), 2.89 (d, J= 3.2 Hz, 6H), 1.80 - 1.60 (m, 3H), 0.97 (dd, J= 6.0,

3.5 Hz, 6H). ¹³C NMR (75 MHz, D₂O) 8 173.91, 70.27, 36.75(d), 25.07, 22.67, 20.58.

N,N-¹³C2-dimethyl-L-Leucine (116C2): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with ¹³C-Formaldehyde (20% Wt). (72mg, 98% yield). ¹H NMR (300 MHz, D2O) 8 3.51 - 3.43 (m, 1H), 3.03 (d, J = 3.9 Hz, 3H), 2.55 (d, J = 3.9 Hz, 3H), 1.70 - 1.49 (m, 3H), 0.87 (dd, J= 6.0, 3.6 Hz, 6H). ¹³C NMR (75 MHz, D2O) 8 173.56, 70.26, 36.73, 25.06, 22.69, 20.55.

N,N-D2-dimethyl-L-Leucine (116D2): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with freshly prepared pyr*BD< (76mg, 95% yield). ¹H NMR (400 MHz, D2O) 8 3.55 (dt, J= 9.5, 2.4 Hz, 1H), 2.85 (p, J= 1.8 Hz, 4H), 1.77 - 1.57 (m, 3H), 0.95 (td, J = 5.8, 2.6 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 173.57, 70.19, 36.72, 25.04, 22.67, 20.53.

N,N-D2-dimethyl-2-¹³C-L-Leucine (117C/D2): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with freshly prepared pyr*BD3 (79mg, 99% yield). ¹H NMR (400 MHz, D2O) 8 3.75 - 3.33 (m, 1H), 2.85 (dq, J = 3.4, 1.3 Hz, 4H), 1.78 - 1.58 (m, 2H), 0.95 (dd, J = 6.1, 4.8 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 173.29, 70.19, 36.87, 36.53, 25.04, 22.69, 20.53.

N,N-¹³C2-dimethyl-^I5N-L-Leucine (117N/C2): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with ¹³C-Formaldehyde (20% Wt) and ¹⁵N- L-Leucine. (80mg, >99% yield). ¹H NMR (400 MHz, D2O) 8 3.78 - 3.33 (m, 1H), 2.88 (dt, J = 143.8, 3.6 Hz, 6H), 1.78 - 1.58 (m, 3H), 0.96 (dd, J= 6.0, 4.7 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 171.02, 70.22, 36.63 (d), 25.07, 22.71, 20.55.

N,N-¹³C2-dimethyl-2-¹³C-L-Leucine (117C3): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with ¹³C-Formaldehyde (20% Wt) and 2- ¹³C-L-Leucine. (79mg, 98% yield). ¹H NMR (400 MHz, D2O) 8 3.56 (dddd, J = 144.5, 10.3, 4.9,

2.5 Hz, 1H), 2.88 (ddd, J= 143.8, 4.0, 3.2 Hz, 6H), 1.79 - 1.60 (m, 2H), 0.97 (dd, J = 6.1, 4.8 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 171.01, 70.25, 41.18, 25.06, 22.66, 20.55.

N,N-¹³C2-dimethyl-2-¹³C,^I5N-L-Leucine (118N/C3): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with ¹³C-Formaldehyde (20% Wt) and 2- ¹³C,¹⁵N-L-Leucine. (75mg, 94% yield). ¹H NMR (400 MHz, D2O) 8 3.56 (ddd, J= 9.9, 4.6, 2.3 Hz, 1H), 2.88 (ddd, J = 143.7, 3.9, 0.8 Hz, 6H), 1.77 - 1.60 (m, 2H), 0.96 (dd, J = 6.1, 4.8 Hz, 5H). ¹³C NMR (101 MHz, D₂O) 8 173.54, 171.00, 81.72, 70.25, 41.05, 25.05, 22.68, 20.55.

N,N-D2-dimethyl-L-Leucine (118C2/D2): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with ¹³C-Formaldehyde (20% Wt) and freshly prepared pyr•BD₃. (80mg, >99% yield). ¹H NMR (400 MHz, D2O) 8 3.56 (ddd, J = 9.9, 4.5, 2.3 Hz, 1H), 2.86 (ddt, 7= 143.4, 3.7, 1.7 Hz, 4H), 1.78 - 1.59 (m, 3H), 1.01 - 0.92 (m, 6H). ¹³C NMR (101 MHz, D2O) 8 173.54, 81.71, 70.19, 41.73, 40.00, 36.71, 31.76, 31.54, 31.32, 25.05, 22.68,

20.55.

N,N-D2-dimethyl-2-¹³C,^I5N-L-Leucine (118N/C/D2): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with 2-¹³C,¹⁵N-L-Leucine and pyr•BD₃. (80mg, >99% yield). ¹H NMR (400 MHz, D2O) 8 3.78 - 3.36 (m, 1H), 2.87 (s, 4H), 1.77 - 1.60 (m, 3H), 0.97 (dd, 7= 6.1, 4.8 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 173.28, 70.21, 36.88, 36.54, 25.07, 22.67, 20.57.

N,N-D₄-dimethyl-L-Leucine (118D4): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with D2-Formaldehyde (20% Wt in D2O). (65mg, 87% yield). ¹H NMR (300 MHz, D₂O) 8 3.51 - 3.42 (m, 1H), 2.76 (t, 7= 1.8 Hz, 6H), 1.69 - 1.51 (m, 3H), 0.87 (dd, 7= 6.0, 3.6 Hz, 6H). ¹³C NMR (75 MHz, D2O) 8 173.58, 70.14, 36.73, 25.05, 22.68,

20.56.

N,N-D₄-dimethyl-¹⁵N-L-Leucine (119N/D4): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with ¹⁵N-L-Leucine and D2-Formaldehyde (20% Wt in D2O). (75mg, 93% yield). ¹H NMR (300 MHz, D2O) 8 3.61 - 3.53 (m, 1H), 2.86 (s, 2H), 1.80 - 1.61 (m, 3H), 0.97 (dd, 7= 6.0, 3.6 Hz, 6H). ¹³C NMR (75 MHz, D2O) 8 173.56, 70.10, 36.72, 25.05, 22.69, 20.57.

N,N-D2-dimethyl-L-Leucine (119N/C2/D2): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with ¹⁵N-L-Leucine, ¹³C-Formaldehyde (20% Wt), and freshly prepared pyr•BD₃. (73mg, 90% yield). ¹H NMR (400 MHz, D2O) 83.56 (ddt, 7= 9.9, 4.6, 2.3 Hz, 1H), 3.10 - 2.61 (m, 4H), 1.78 - 1.58 (m, 3H), 0.96 (dd, 7 = 6.1, 4.8 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 173.54, 70.16, 41.89, 39.88, 36.72, 31.77, 31.50, 31.28, 25.05, 22.68, 20.55. N,N-D4-dimethyl-¹³C-L-Leucine (119C/D4): Performed according to the general procedure for synthesis of symmetric dimethyl leucine with 2-¹³C-L-Leucine and D2-Formaldehyde (20% Wt in D2O). (79mg, >99% yield). ¹H NMR (400 MHz, D2O) 8 3.77 - 3.34 (m, 1H), 2.85 (dt, J= 3.5, 1.7 Hz, 2H), 1.78 - 1.58 (m, 3H), 0.96 (dd, J = 6.1, 4.7 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 173.53(d), 70.14, 36.68(d), 25.05, 22.66, 20.55.

N,N-Di-dimethyl-L-Leucine (115D): Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with Di-iodomethane. ¹H NMR (400 MHz, D2O) 83.61 - 3.54 (m, 1H), 2.89 (s, 5H), 1.79 - 1.60 (m, 3H), 0.97 (dd, J = 6.1, 4.8 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 173.54, 70.24, 36.73, 25.07, 22.69, 20.57.

N,N-¹³C ,D i-dimethyl-L-Leucine (116C/D): Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with Di -iodomethane and ¹³C-Formaldehyde (20% Wt in H2O). ¹H NMR (400 MHz, D2O) 8 3.58 (ddd, J = 10.0, 4.8, 2.3 Hz, 1H), 3.11 - 2.66 (m, 5H), 1.79 - 1.62 (m, 3H), 0.97 (dd, J = 6.1, 4.8 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 173.56, 70.24, 42.28, 40.05, 36.72, 25.07, 22.69, 20.57.

^I5N,N-¹³C-dimethyl-L-Leucine (116N/C): Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with ¹⁵N-L-Leucine and ¹³C-Formaldehyde (20% Wt in H2O). ¹H NMR (400 MHz, D2O) 8 3.61 - 3.54 (m, 1H), 2.89 (td, J= 73.0, 72.4, 9.0 Hz, 6H), 1.79 - 1.58 (m, 3H), 0.98 (dd, J = 6.1, 4.8 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 173.55, 70.27, 42.28, 40.04, 36.72, 25.08, 22.69, 20.56.

^I5N,N-¹³C-dimethyl-L-Leucine (116N/D): Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with ¹⁵N-L-Leucine and Di-iodomethane. ¹H NMR (400 MHz, D2O) 8 3.60 - 3.55 (m, 1H), 2.89 (d, J= 9.5 Hz, 5H), 1.79 - 1.60 (m, 3H), 0.98 (dd, J = 6.1, 4.8 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 70.24, 36.72, 25.08, 22.69, 20.57.

^I5N-¹³C-N-Di-dimethyl-L-Leucine (117N/C/D): Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with ¹⁵N-L-Leucine, Di -iodomethane, and ¹³C-Formaldehyde (20 Wt% in H2O). ¹H NMR (400 MHz, D2O) 8 3.56 (ddd, J= 9.9, 4.7, 2.2 Hz, 1H), 3.09 - 2.63 (m, 5H), 1.78 - 1.59 (m, 3H), 0.96 (dd, J= 6.1, 4.8 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 173.55, 70.20, 42.27, 40.05, 36.72, 25.07, 22.69, 20.56.

N-¹³C,Di-N-2-¹³C-dimethyl-L-leucine (117C2/D): Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with 2-¹³C-L-Leucine, Di -iodomethane, and ¹³C-Formaldehyde (20 Wt% in H2O). ¹H NMR (400 MHz, D2O) 8 3.66 - 3.20 (m, 1H), 2.97 - 2.53 (m, 5H), 1.65 - 1.48 (m, 3H), 0.84 (dd, J= 6.1, 4.8 Hz, 6H). ¹³C NMR (101 MHz, D₂O) 8 163.73, 70.25, 52.24, 42.28, 40.04, 25.06, 22.71, 20.56.

N,N-D3-dimethyl-L-leucine (117D3): Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with D₃-iodomethane. ¹H NMR (400 MHz, D2O) 83.60 - 3.51 (m, OH), 2.88 (d, J= 8.4 Hz, 1H), 1.79 - 1.59 (m, OH), 0.96 (dd, J= 6.1, 4.8 Hz, 1H). ¹³C NMR (101 MHz, D2O) 8 173.55, 70.18, 36.72, 25.06, 22.69, 20.57.

N,N-Ds-dimethyl-L-Leucine (119D5): Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with D₃-iodomethane and D2-formaldehyde (20% Wt in D2O). ¹H NMR (400 MHz, D2O) 8 3.62 - 3.54 (m, 1H), 2.86 (s, 1H), 1.81 - 1.60 (m, 3H), 0.97 (dd, J= 6.1, 4.8 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 173.56, 70.12, 36.72, 25.06, 22.68, 20.57.

N,N-¹³C2,D-dimethyl-¹³C,^I5N-L-Leucine (119N/C3/D): Performed according to the general procedure B for synthesis of asymmetric dimethyl leucine with 2-¹³C,¹⁵N-L-Leucine, ¹³C- iodomethane„¹³C-formaldehyde (20% Wt), and freshly prepared pyr»BD3. ¹H NMR (400 MHz, D2O) 8 3.78 - 3.37 (m, 1H), 2.88 (dq, J= 143.7, 3.7 Hz, 6H), 1.78 - 1.60 (m, 3H), 0.97 (dd, J = 6.1, 4.8 Hz, 6H). ¹³C NMR (101 MHZ, D2O) 8 173.80, 173.27, 171.00, 81.73, 70.23, 48.86, 42.08, 40.07, 36.88, 36.54, 25.07, 22.71, 20.58.

N,N-¹³C2,D-dimethyl-¹⁵N-L-Leucine (118N/C2/D): Performed according to the general procedure B for synthesis of asymmetric dimethyl leucine with ¹⁵N-L-Leucine, ¹³C-iodomethane, ¹³C-formaldehyde (20% Wt), and pyr«BD₃. ¹H NMR (400 MHz, D2O) 83.60 - 3.53 (m, 1H), 2.88 (dd, J = 143.8, 4.0 Hz, 5H), 1.79 - 1.60 (m,3H), 0.97 (dd, J = 6.1, 4.9 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 81.74, 70.24, 42.18, 39.99, 36.73, 25.07, 22.69, 20.56.

N,N-¹³C2,D-dimethyl-l,2-¹³C2-L-Leucine (118C3/D): Performed according to the general procedure A for synthesis of asymmetric dimethyl leucine with 1,2-¹³C2-L-Leucine, ¹³C- iodomethane, ¹³C-Formaldehyde (20% Wt) and freshly prepared pyr•BD₃. ¹H NMR (400 MHz, D2O) 8 3.80 - 3.37 (m, 1H), 2.88 (dq, J= 143.2, 3.5 Hz, 5H), 1.78 - 1.60 (m, 2H), 0.97 (dd, J = 6.1, 4.9 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 173.81, 173.28, 81.73, 70.48, 69.96, 48.86, 41.01, 25.06, 22.67, 20.56.

N,N-¹³C,Ds-dimethyl-L-leucine (118C/D3): Performed according to the general procedure B for synthesis of asymmetric dimethyl leucine with ¹³C-iodomethane, D2-formaldehyde (20% Wt), and pyr»BD₃. ¹H NMR (400 MHz, D2O) 8 3.56 (ddd, J= 9.9, 4.8, 2.2 Hz, 1H), 2.88 (d, J = 143.8 Hz, 3H), 1.81 - 1.62 (m, 3H), 0.96 (dd, J= 6.1, 4.8 Hz, 6H). ¹³C NMR (101 MHz, D₂O) 8 70.18, 42.19, 40.20, 36.72, 25.06, 22.68, 20.56.

N,N-¹³C2,D3-dimethyl-L-leucine (II9C2/D3): Performed according to the general procedure B for synthesis of asymmetric dimethyl leucine with ¹³C,D3-iodomethane, deformaldehyde (20% Wt). ¹H NMR (400 MHz, D2O) 8 3.44 (ddd, J = 9.9, 4.4, 2.1 Hz, 1H), 2.75 (ddd, J= 144.2, 7.9, 3.4 Hz, 3H), 1.66 - 1.48 (m, 3H), 0.84 (dd, J = 6.1, 4.8 Hz, 6H). ¹³C NMR (101 MHz, D2O) 8 173.54, 70.18, 42.22, 39.98, 25.06, 22.69, 20.57.

N,N-¹³C,D3-dimethyl-¹⁵N-L-leucine (119N/C/D3): Performed according to the general procedure B for synthesis of asymmetric dimethyl leucine with ¹⁵N-L-Leucine, ¹³C-iodomethane, D₂-formaldehyde (20% Wt), and pyr«BD₃. ¹H NMR (400 MHz, D2O) 8 3.56 (ddd, J = 10.0, 4.8, 2.2 Hz, OH), 3.09 - 2.68 (m, OH), 1.78 - 1.61 (m, OH), 0.96 (dd, J= 6.1, 4.8 Hz, 1H). ¹³C NMR (101 MHz, D2O) 8 173.56, 70.14, 41.68, 40.22, 36.71, 25.05, 22.69, 20.57.

Scheme SI. (NBI-135) DADPS formation with 6-hydroxycaproic acid.

Scheme S2. Initial screen of DADPS formation conditions.

Scheme S3. Preparation of DADPS ester analogues.

Scheme S4. Preparation of activated ester analogues.

Scheme S5a. Attempted synthesis of a DADPS substrate containing a tetrafluorophenol activated ester.

Scheme S6. Attempted synthesis of a DADPS substrate containing a tetrafluorophenol activated ester using triethylamine as the base.

Scheme S7A. Model thiol-ene synthesis.

Scheme S7B. Deprotection condition for DADPS reagent bearing an allyl ester and Cbz- protected amine.

Scheme S8. Synthetic scheme of ester containing solid-phase compatible reagent 6.

Biology Methods

Cell culture and preparation of cell lysates.

Cell culture reagents including Dulbecco’s phosphate- buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM)/high glucose media, Roswell Park Memorial Institute (RPMI) media, trypsin-EDTA and penicillin/streptomycin (Pen/Strep) were purchased from Fisher Scientific. Fetal Bovine Serum (FBS) were purchased from Avantor Seradigm (lot # 214B17). All cell lines were obtained from ATCC and were maintained at a low passage number (< 20 passages). HEK293T (ATCC: CRL-3216) cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotics (Penn/Strep, 100 U/mL). H661 (ATCC: HTB-183), HCT-15 (ATCC: CCL- 225), Jurkat (ATCC: TIB-152), MOLT-4 (ATCC: CRL-1582) andH2122 (ATCC: CRL5985) cells were cultured in RPMI-1640 supplemented with 10% FBS and 1% antibiotics (Penn/Strep, 100 U/mL). HEC-l-B (ATCC: HTB-113) cells were cultured in EMEM supplemented with 10% FBS and 1% antibiotics (Penn/Strep, 100 U/mL). Media was filtered (0.22 pm) prior to use. Cells were maintained in a humidified incubator at 37 °C with 5% CO2. Cell lines were tested for mycoplasma using the Mycoplasma Detection Kit (InvivoGen). Cells were harvested by centrifugation (4,500 g, 5 min, 4 °C), washed twice with cold DPBS, resuspended in DPBS, sonicated, and clarified by centrifuging (21,000 g, 10 min, 4 °C). The lysates were then transferred to a new microcentrifuge tube. Protein concentrations were determined using a Bio-Rad DC protein assay kit from Bio-Rad Life Science (Hercules, CA) and the lysate diluted to the working concentrations indicated below.

Proteomic sample preparation.

HEK293T proteome (100 pL of 2 mg/mL) was first labeled with IAA 1 or other reagents (2 pL of 100 mM stock solution in DMSO, final concentration = 2 mM) for Ih at ambient temperature. CuAAC was performed with biotin-azide 2 or other reagents (2 pL of 200 mM stock in DMSO, final concentration = 4 mM), TCEP (2 pL of fresh 50 mM stock in water, final concentration = 1 mM), TBTA (6 pL of 1.7 mM stock in DMSO/t-butanol 1:4, final concentration = 100 pM), CuSO4 (2 pL of 50 mM stock in water, final concentration = 1 mM), and 0.2% SDS for Ih at ambient temperature. After CuAAC labeling, each sample was treated with 0.5 pL benzonase (Fisher Scientific, 70- 664-3) for 30 min at 37 °C. For each 100 pL sample (1 mg/mL protein concentration), 20 pL Sera-Mag SpeedBeads Carboxyl Magnetic Beads, hydrophobic (GE Healthcare, 65152105050250) and 20 pL Sera-Mag SpeedBeads Carboxyl Magnetic Beads, hydrophilic (GE Healthcare, 45152105050250) were mixed and washed with water for three times. The bead slurries were then transferred to the CuAAC samples, incubated for 5 min at RT with shaking (1000 rpm). Absolute ethanol (400 pL) was added to each sample, and the samples were incubated for 5 min at RT with shaking (1000 rpm). Samples were then placed on a magnetic rack, washed three times with 80% ethanol in water (400 pL). After washing, beads were resuspended in 200 pL 2 M urea in 0.5% SDS/PBS. DTT (10 pL of 200 mM stock in water, final concentration = 10 mM) was added into each sample and the sample was incubated at 65 °C for 15 min. Then, iodoacetamide (10 pL of 400 mM stock in water, final concentration = 20 mM) was added and the solution was incubated for 30 min at 37 °C with shaking in the dark. Absolute ethanol (400 pL) was added to each sample, and the samples were incubated for a further 5 min at RT with shaking (1000 rpm). Beads were washed three times with 80% ethanol in water (400 pL). Next, beads were resuspended in 200 pL 2 M urea in PBS and 2 pL trypsin solution (Worthington Biochemical, LS003740, 1 mg/mL in 666 pL of 50 mM acetic acid and 334 pL of 100 mM CaC12) was added. Digest was overnight at 37 °C with shaking. After digestion, ~ 4 mL acetonitrile (> 95% of the final volume) was added to each sample and the mixtures were incubated for 10 min at RT with shaking (1000 rpm). The beads were then washed three times with 1 mL acetonitrile each with a magnetic rack. Peptides were eluted from SP3 beads with 100 pL of 2% DMSO in Molecular Biology Grade (MB) water for 30 min at 37 °C with shaking (1000 rpm). The elution was repeated again with 100 pL of 2% DMSO in MB water. Two eluents were combined.

Avidin enrichment of labeled peptides.

For each sample clicked with biotin azide, 50 pL of NeutrAvidin Agarose resin slurry (Pierce, 29200) was washed one time in 10 mL IAP buffer (50 mM MOPS pH 7.2, 10 mM sodium phosphate, and 50 mM NaCl buffer) and then resuspended in 500 pL IAP buffer. Peptide solutions eluted from SP3 beads were then transferred to the NeutrAvidin Agarose resin suspension, and the samples were rotated for 2h at RT. For each sample clicked with a DADPS cleavable azide, 50 pL of Streptavidin Agarose resin slurry (Pierce, 20353) was washed one time in 10 mL PBS and then resuspended in 500 pL PBS. Peptide solutions eluted from SP3 beads were then transferred to the Streptavidin Agarose resin suspension, and the samples were rotated for 2h at RT.

After incubation, the beads were pelleted by centrifugation (21,000 g, 1 min) and washed twice with 1 mL PBS each and then twice with 1 mL water each. NeutrAvidin-bound peptides were eluted with 60 pL of 80% acetonitrile in MB water with 0.1 % FA for 10 min at RT. The elution was repeated for 10 min at 72 °C. The elution was repeated once more for 10 min at RT. Streptavidin- bound peptides were eluted with 200pL of 2% formic acid in MB water for 30 min at RT. The elution was repeated once more with 80% acetonitrile in MB water for 2 min at RT. The combined eluants were dried (SpeedVac), then reconstituted with 5% acetonitrile and 1% FA in MB water and analyzed by LC-MS/MS. Liquid-chromatography tandem mass-spectrometry (LC-MS/MS) analysis.

The samples were analyzed by liquid chromatography tandem mass spectrometry using a mass spectrometer or coupled with a High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. Peptides were fractionated S21 online using a 18cm long, 100 pM inner diameter (ID) fused silica capillary packed in-house with bulk Cl 8 reversed phase resin (particle size, 1.9 pm; pore size, 100 A; Dr. Maisch GmbH). The 70-minute water-acetonitrile gradient was delivered using a Thermo Scientific 1200 system at different flow rates (Buffer A: water with 3% DMSO and 0.1% formic acid and Buffer B: 80% acetonitrile with 3% DMSO and 0.1% formic acid). The detailed gradient includes 0 - 5 min from 3 % to 10 % at 300 nL/min, 5 - 64 min from 10 % to 50 % at 220 nL/min, and 64 -70 min from 50 % to 95 % at 250 nL/min buffer B in buffer A. Data was collected with charge exclusion (1 , 8, >8). Data was acquired using a Data- Dependent Acquisition (DDA) method consisting of a full MSI scan (Resolution = 120,000) followed by sequential MS2 scans (Resolution = 15,000) to utilize the remainder of the 1 second cycle time. Precursor isolation window and normalized collision energy were set as described in the study. Table S7. Conditions of Liquid-chromatography (LC) Parameter Condition Column 100 pM ID fused silica capillary packed in-house with bulk Cl 8 reversed phase resin (particle size, 1.9 pm; pore size, 100 A; Dr. Maisch GmbH) Mobile phase Buffer A: water with 3% DMSO and 0.1% formic acid Buffer B: 80% acetonitrile with 3% DMSO and 0.1% formic acid Gradient and flow rate 0 - 5 min, 3 - 10% B, 300 nL/min 5 - 64 min, 10 - 50% B, 220 nL/min 64 - 70 min, 40 - 95% B, 250 nL/min Run time 70 minutes Injection volume 5 uL.

Protein and peptide identification.

Raw data collected by LC-MS/MS were searched with MSFragger (v3.4 and v3.5) and FragPipe (vl7.1 and 18.0). For closed search, the proteomic workflow and its collection of tools was set as default. Precursor and fragment mass tolerance was set as 20 ppm. Missed cleavages were allowed up to 1. Peptide length was set 7 - 50 and peptide mass range was set 500 - 5000. Cysteine residues were searched with differential modifications as described in the study. For labile search, mass offsets were set restricted to cysteines. Y ion masses and diagnostic fragment masses were set for different proteomic samples. PTM-Shepherd was enabled for localization. A sample workflow can be found attached. Calibrated and deisotoped spectrum files produced by FragPipe were retained and reused for this analysis. Data analysis and processing.

After MS search with MSFragger, raw files and identification files were imported to PDV for MS spectra annotation. Frequency distribution and intensity of the fragment ions and peptide remainder ions were calculated based on the output of PTM Shepherd as mean of all replicates (See supplementary data tables). Mean of the number of PSMs and peptides of all replicates were reported as bar plots. Additionally, venn diagrams were constructed to display the number of common cysteine peptides identified shared between various experiments.

Example 2: Exemplary Chemoproteomics Studies

Identification of dihydrooxazolium signature fragment ion produced from sCIP labeled peptides that is compatible with MS2 quantification

The objective was to determine whether the sCIP modified peptides would afford characteristic fragment ions with suitable intensity and specificity to function as diagnostic ions. Such ions would enable the goal of harnessing those ions for quantification at the MS2 level. Discovery searches identified a substantial number of characteristic fragment ions (~29- 40/reagent), including both unique species and species shared between reagents. Prioritizing those ions derived from modified precursor ion scans that exhibited both high intensity and frequent detection, it was predicted that the likely fragmentation pathways for each of the reagents (FIG. 5A & 12). Exemplifying this process, for Ml precursors modified with reagent NBIV-22 (FIG. 11), a m/z peak of 510.3762 was identified, which was ascribed to formation of the Fl ammonium ion through N26-C27 amide bond cleavage. Analogously, the M2 and M3 precursor ions, modified by NBIII-169 and NBIV-070, afforded m/z 464.3293 and m/z 440.2980, which also match with ammonium species F5 and F9, respectively. Ml, M2, and M3 precursor ions also afforded fragment ions with m/z 370.27, m/z 328.2231, and m/z 300.1918, respectively. These ions were ascribed to the cyclic dihydrooxazolium species F2, F6, and F10 generated from C-N bond cleavage of the triazole followed by cyclization with the proximal carbonyl oxygen, analogous to the formation of previously reported oxonium-biotin characteristic ion. Ions with m/z 286.2125, and m/z 258.1812 match with the structures of cyclic dihydrooxazolium species F7, and Fll, which formed through fragmentation of the N-terminal acetamide moiety. Interestingly, this analogous ion for the Ml precursor ion, F3, was not detected in the discovery search. Lastly, it was expected that m/z 229.1911 and m/z 187.1441 ions likely stem from C-N and C-0 bond scission of the F2, F6, and F10 ions affording piperidinium F4 (for Ml) and aziridinium F8 (for M2 and M3).

Given the prior observation that oxonium-biotin characteristic ions can exhibit high intensity but low specificity for modified precursor ions, which make such ions ill-suited to function as diagnostic or reporter ions. It was next asked whether any of the observed ions would exhibit both high intensity and high (near 100%) frequency of detection in modified peptide spectra, while simultaneously having low background identification in unmodified peptide spectra due to precursor coisolation. Manual inspection of individual MS/MS spectra using an integrated proteomics data viewer (PDV) revealed marked differences in the relative intensities of prioritized ions (FIG. 7B) Dataset-wide analysis of both the relative intensities and frequency of detection for all prioritized ions (FIG. 7C) similarly revealed pronounced ion-type and reagent- dependent differences across the panel of reagents analyzed. The ammonium ions (Fl, F5, F9) generated from amide bond cleavage exhibited generally high (70%) frequency of detection and low (<5%) intensity. Unexpectedly, the oxazolium ions (F2, F6, and F10) exhibited marked differences in both frequency and intensity across the reagent panel assayed. Fragmentation of precursor ions functionalized with β-azidoalanine reagents NBIV-070 and NBIII-169 (M2 and M3) afforded robust production of a putative dihydrooxazolium characteristic fragment ions (F6 and F10) together with daughter ions F7 and Fll. All four ions were observed in >95% of modified spectra with F6 and F10 further distinguished by their relatively high >55% median relative intensity in modified spectra and <15% median relative intensity in unmodified spectra. Gratifyingly, the choice of amino acid at the n-terminus of the probes (valine versus alanine) did not have a significant effect on intensity for these dihydrooxazolium ions.

These analyses were extended to a second set of “short” reagents synthesized with DADPS- Fmoc reagents NBIV-009, NBIV-011, and NBIV-027, which revealed generally similar trends for the corresponding characteristic fragment ions. While both the short NBIII-169 and long NBIV-027 linker reagents produced the oxonium ion, a modest but significant increase in the signal to noise ratio of the corresponding ion using the longer linker reagent (FIG. 13), together with no appreciable specificity decrease was observed. By contrast, precursor ions modified with the shorter reagents showed more intense ions, corresponding to the water loss from the oxonium species (F16, F20, and F25). It was sought to increase this specificity through the use of on-line high-field asymmetric waveform ion mobility spectrometry (FAIMS), which has been shown to decrease ratio compression of MS2 quantification in TMT samples. Gratifyingly, the high specificity for F6 and F10 was further enhanced in data acquired using a FAIMS device and contrasts markedly with the poor specificity observed for the piperidinium and aziridinium ions (F4, and F8). Unexpectedly, formation of F8 was observed to be sensitive to proximal amino acids, highly disfavored for valine- containing reagents in comparison to alanine- containing reagents. These favorable ion properties did not extend to ion F2, which demonstrated a median relative intensity <10%. Well-established disfavored energetics and kinetics of eight-membered ring formation likely rationalize this observed low intensity.

Collectively across all ions analyzed, the F6 and F10 ions were distinguished by the combination of near 100% frequency of detection, high intensity, and significant specificity for modified precursors, which was further improved through FAIMS data acquisition. To further guide the selection of optimal reagent combination, reagent cost was considered, as well as availability of isotopologues and the signal to noise for the m/z window for each prioritized ion. Alanine-derived reagents proved superior on both cost and isotopologue availability.

6-plex isobaric labeling enabled by sCIP.

Given the ready availability of isotopically differentiated iodoacetamide alkyne reagents, including benzyl-¹⁴ and isopropyl-¹⁵ based reagents, the production of a novel isobaric labeling strategy was envisioned to be enabled by combining stable isotope labeled iodoacetamide alkyne balancers with sCIP-generated reporter ions. Isotopologues (¹³C vs ¹⁵N) can be employed for isobaric tagging, thus enabling the use of heavy carbon, nitrogen, and oxygen isotopes in for multiplexed quantification of peptide abundance at the MS2 and MS3 level. To realize the vision of a custom isobaric cysteine chemoproteomic labeling method, a panel of three isotopically differentiated iodoacetamide alkyne probes was synthesized to act as the balancer portion of the system (IAA, 1-¹³C-IAA, and 1,2¹³C2-IAA; FIGs. 14 & 15A) The SPPS workflow was then employed to furnish six isotopically enriched-alanine-based sCIP reagents, which function as the reporter region of the system (FIGs. 14B & 15A). To begin validating the sCIP system the light reagent (sCIP-Zero) was subjected to the cysteine chemoproteomics workflow (FIGs. 8A & 8B). Discovery ion search revealed a panel of fragment ions generated from the isobaric reagent sets (FIG. 15). Analysis of peptide coverage and fragment ion intensity at varying collision energies (CEs) (FIG. 8C) revealed peak ion intensity and number of peptides identified at 30% MS² higher energy C trap dissociation (HCD) CE. These findings prompted us to conduct the experiments at 30% CE.

HEK293T cell lysates were subjected to IAA treatment with one of the three isotopically differentiated lAAs (IAA, 1-¹³C-IAA, and 1,2-¹³C2-IAA; FIG. 14) followed by click conjugation to equimolar concentration of the complementary sCIP tag. The samples were then combined equally and subjected to tryptic digest, enrichment, and LC-MS/MS analysis. Gratifyingly, the ratios of median reporter ion intensities for each channel were found to be centered near one as expected (FIG. 8D, left panel). Of note, during the course of benchmarking these reagents, significant ion coalescence was observed at lower resolving powers (RPs). This finding is not unexpected due to the small (2.5 mDa) mass difference separating the 3020 and 302C channels. Although these ions can be resolved and the resulting ratio skew overcome with a 240K RP (at 200 m/z), this high resolution results in decreased coverage. Therefore, for initial applications of the sCIP system, the 3020 channel was excluded, which allowed analysis at 60K RP and increased peptide coverage. To validate the system’s ability to identify peptides at different abundances, the aforementioned experiment was repeated with the 5-plex system, mixing each channel in a 1:4: 10:4: 1 ratio (FIG. 8D, right panel) and gratifyingly observed that the ratios calculated from median reporter ion intensities closely matching expected values corresponding to the channel stochiometry.

Ratio compression for the sCIP reagents was then benchmarked and compared with conventional TMT. As ratio compression is caused by coisolation of precursor ions and can effectively mask small fold-change differences between treatment groups, it was sought to determine whether the sCIP workflow would outperform TMT, given the aforementioned high specificity of the dihydrooxazolium ion (FIG. 7). To compare these two classes of reagents, isotopically differentiated (SILAC) 'light' and 'heavy' cell lysates were subjected to labeling with heavy or light IAA reagents (IAA, 1-¹³C-IAA, and 1,2-¹³C2-IAA; FIG. 14), followed by click conjugation to the indicated sCIP reagents following the workflow shown in FIG. 9A. After mixing of labeled lysate volumes at the indicated ratios, the samples were combined and analyzed by LC-MS/MS. Samples subjected to TMT labeling were analyzed analogously, with the exception that the TMT labeling step occurs much later in the workflow and therefore the samples were maintained separate until the final step. Comparison of these samples revealed both similar ratio compression (FIG. 9B) and coefficient of variance (FIG. 9C). Head-to-head cost comparison supports the relative cost-effectiveness of sCIP compared with TMT (FIG. 9D)

MS2-based quantification to discover ligandable cysteines using 5-plex sCIP isobaric tags

A key advantage of the sCIP approach compared with TMT is it allows for samples to be combined early in the sample preparation workflow, enabling multiple technical and biological replicates to be prepared with relative ease using small amounts of proteome (e.g. 40ug/channel used here). To establish whether the sCIP reagents would faithfully capture ligandable or potentially druggable cysteines, MS2 quantification was benchmarked with the 5-plex sCIP tag set against MSI quantification using heavy and light sCIP reagents NBIV-009 and NBIV-010 (FIG. 11). Three samples of HEK293T cell lysates were subjected to treatment with cysteine-reactive compound KB02 (500 pM) and two samples to DMSO treatment. After IAA treatment and click conjugation to the respective tag, the samples were combined and subjected to SP3 preparation and streptavidin enrichment (FIGs. 5A & 5B). LC-MS/MS analysis showed high concordance between the sCIP identified targets and those identified using traditional MSI quantification (FIG. 10C) Given the aforementioned desirability of expanding multiplexing capabilities through the use of a single channel per treatment condition, it was next sought to investigate whether the single channel analysis would faithfully recapitulate triplicate treatments. Gratifyingly, comparison of single vs multi-channel analysis for KB02 -treated cell lysates revealed high concordance between both approaches (FIGs. 10D & 10E). Collectively these findings indicate that the sCIP platform can enable the cost-effective, high throughput, and high-fidelity profiling of ligandable cysteines.

Discussion

Here, the advantages of the DADPS cleavable linker with the high yield and high throughput nature of solid phase synthesis was combined with a custom isobaric labeling strategy to develop the silane-based Cleavable linkers for Isotopically-labeled Proteomics (sCIP) method (FIG. 6). The sCIP method is enabled by the highly innovative fluorenylmethyl carbamate (Fmoc) functionalized building blocks (DADPS-Fmoc reagents), which function analogously to Fmoc- protected amino acids commonly utilized in SPPS. Using the DADPS-Fmoc reagents, a panel of biotin enrichment reagents was obtained. Application of these reagents to cysteine chemoproteomics revealed a high intensity and highly specific dihydrooxazolium characteristic ion unique to a subset of sCIP reagents. The discovery of this dihydrooxazolium characteristic fragment ion guided the design of a built-in isobaric labeling strategy in which the mass balancer and reporter are incorporated into cysteine-reactive iodoacetamide alkyne probe and DADPS biotin capture handle, respectively. Synthesis of a panel of six isotopically balanced reagent combinations enabled high throughput 6-plex multiplexing and MS2 quantification. The sCIP platform is distinguished by the early sample combination step while still maintaining comparable ratio compression to TMT reagents, which together afford enhanced throughput and high reproducibility cysteine ligandability studies for >10,000 residues in single shot chemoproteomics experiments.

Biology Methods

Cell culture and preparation of cell lysates.

Cell culture reagents including Dulbecco’s phosphate- buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM)/high glucose media, Roswell Park Memorial Institute (RPMI) media, trypsin-EDTA and penicillin/streptomycin (Pen/Strep) were purchased from Fisher Scientific. Fetal Bovine Serum (FBS) were purchased from Avantor Seradigm (lot # 214B17). All cell lines were obtained from ATCC and were maintained at a low passage number (< 20 passages). HEK293T (ATCC: CRL-3216) cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotics (Penn/Strep, 100 U/mL). H661 (ATCC: HTB-183), HCT-15 (ATCC: CCL-225), Jurkat (ATCC: TIB-152), MOLT-4 (ATCC: CRL-1582) andH2122 (ATCC: CRL5985) cells were cultured in RPMI- 1640 supplemented with 10% FBS and 1% antibiotics (Penn/Strep, 100 U/mL). HEC-l-B (ATCC: HTB-113) cells were cultured in EMEM supplemented with 10% FBS and 1% antibiotics (Penn/Strep, 100 U/mL). Media was filtered (0.22 pm) prior to use. Cells were maintained in a humidified incubator at 37 °C with 5% CO2. Cell lines were tested for mycoplasma using the Mycoplasma Detection Kit (InvivoGen). Cells were harvested by centrifugation (4,500 g, 5 min, 4 °C), washed twice with cold DPBS, resuspended in DPBS, sonicated, and clarified by centrifuging (21,000 g, 10 min, 4 °C). The lysates were then transferred to a new microcentrifuge tube. Protein concentrations were determined using a Bio-Rad DC protein assay kit from Bio-Rad Life Science (Hercules, CA) and the lysate diluted to the working concentrations indicated below.

Proteomic sample preparation for sCIP 5/6-Plex reagents.

HEK293T proteome (100 pL of 2 mg/mL) was first labeled with either L-IAA (X), ¹³C-H- IAA (NBIV-069), or ¹³C₂-H-IAA (NBIV-083) (2 pL of 100 mM stock solution in DMSO, final concentration = 2 mM) for Ih at ambient temperature. CuAAC was performed with the complementary sCIP reagent (2 pL of 200 mM stock in DMSO, final concentration = 4 mM), TCEP (2 pL of fresh 50 mM stock in water, final concentration = 1 mM), TBTA (6 pL of 1.7 mM stock in DMSO/t-butanol 1:4, final concentration = 100 pM), CuSO4 (2 pL of 50 mM stock in water, final concentration = 1 mM), and 0.2% SDS for Ih at ambient temperature. After CuAAC labeling, each sample was treated with 0.5 pL benzonase (Fisher Scientific, 70- 664-3) for 30 min at 37 °C. For each 100 pL sample (1 mg/mL protein concentration), 20 pL Sera-Mag SpeedBeads Carboxyl Magnetic Beads, hydrophobic (GE Healthcare, 65152105050250) and 20 pL Sera-Mag SpeedBeads Carboxyl Magnetic Beads, hydrophilic (GE Healthcare, 45152105050250) were mixed and washed with water for three times. The bead slurries were then transferred to the CuAAC samples, incubated for 5 min at RT with shaking (1000 rpm). Absolute ethanol (400 pL) was added to each sample, and the samples were incubated for 5 min at RT with shaking (1000 rpm). Samples were then placed on a magnetic rack, washed three times with 80% ethanol in water (400 pL). After washing, beads were resuspended in 200 pL 2 M urea in 0.5% SDS/PBS. DTT (10 pL of 200 mM stock in water, final concentration = 10 mM) was added into each sample and the sample was incubated at 65 °C for 15 min. Then, iodoacetamide (10 pL of 400 mM stock in water, final concentration = 20 mM) was added and the solution was incubated for 30 min at 37 °C with shaking in the dark. Absolute ethanol (400 pL) was added to each sample, and the samples were incubated for a further 5 min at RT with shaking (1000 rpm). Beads were washed three times with 80% ethanol in water (400 pL). Next, beads were resuspended in 200 pL 2 M urea in PBS and 2 pL trypsin solution (Worthington Biochemical, LS003740, 1 mg/mL in 666 pL of 50 mM acetic acid and 334 pL of 100 mM CaC12) was added. Digest was overnight at 37 °C with shaking. After digestion, ~ 4 mL acetonitrile (> 95% of the final volume) was added to each sample and the mixtures were incubated for 10 min at RT with shaking (1000 rpm). The beads were then washed three times with 1 mL acetonitrile each with a magnetic rack. Peptides were eluted from SP3 beads with 50 pL of 2% DMSO in Molecular Biology Grade (MB) water for 30 min at 37 °C with shaking (1000 rpm). The elution was repeated with 50 pL of 2% DMSO in MB water. Two eluants were combined.

Streptavidin enrichment of labeled peptides.

For each lOOpL sample of 2mg/mL cellular lysates, 50 pL of Streptavidin Agarose resin slurry (Pierce, 20353) was washed one time in 8 mL PBS and then resuspended in 500 pL PBS. Peptide solutions eluted from SP3 beads were then transferred to the Streptavidin Agarose resin suspension, and the samples were rotated for 2h at RT. After incubation, the beads were pelleted by centrifugation (15,000 g, 1 min) and washed twice with 1 mL PBS each and then twice with 1 mL water each. Bound peptides were eluted via acidic cleavage of the DADPS linkage using 200 pL of 2% formic acid in MB water for 30 min at RT. The elution was repeated once more with 80% acetonitrile in MB water for 2 min at RT. The combined eluants were dried (SpeedVac), then reconstituted with 5% acetonitrile and 1% FA in MB water and analyzed by LC-MS/MS.

Compound Labeling Experiments using 5-Plex sCIP Reagents

For experiments with 3 replicates in the same sample (replicates in 3 different channels) HEK293T cellular lysates were first treated with either DMSO (300 and 301N channel) or Compound (301C, 302N, and 302C channel) at a concentration of 500pM for 1 hour at ambient temperature. After which lysates were subjected to IAA treatment (ImM final concentration) with the respective IAA reagent (302 channels get L-IAA; 301 channels get 13C-H-IAA(NBIV-069), and 300 channel gets 13C2-H-IAA(NBIV-083)) for 1 hour at ambient temperature. CuAAC was performed with the complementary sCIP reagent (final concentration = 2 mM), TCEP (2 pL of fresh 50 mM stock in water, final concentration = 1 mM), TBTA (6 pL of 1.7 mM stock in DMSO/t- butanol 1:4, final concentration = 100 pM), CuSO4 (2 pL of 50 mM stock in water, final concentration = 1 mM), and 0.2% SDS for Ih at ambient temperature. Samples were then prepared according to the above procedure.

For experiments with single replicates of multiple compounds, HEK293T cellular lysates were first treated with either DMSO (300 and 301N channel), Compound 1 (301C), Compound 2 (302N) and Compound 3 (302C) at a concentration of 500pM for 1 hour at ambient temperature. Samples were then subjected to same treatment as described above.

Liquid-chromatography tandem mass-spectrometry (LC-MS/MS) analysis.

The samples were analyzed by liquid chromatography tandem mass spectrometry using a Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer or coupled with a High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. Peptides were fractionated S21 online using a 18cm long, 100 pM inner diameter (ID) fused silica capillary packed in-house with bulk C18 reversed phase resin (particle size, 1.9 pm; pore size, 100 A; Dr. Maisch GmbH). The 70-minute water-acetonitrile gradient was delivered using a Thermo Scientific™ EASY- nLC™ 1200 system at different flow rates (Buffer A: water with 3% DMSO and 0.1% formic acid and Buffer B: 80% acetonitrile with 3% DMSO and 0.1% formic acid). The detailed gradient includes 0 - 5 min from 3 % to 10 % at 300 nL/min, 5 - 64 min from 10 % to 50 % at 220 nL/min, and 64 - 70 min from 50 % to 95 % at 250 nL/min buffer B in buffer A (Table S7). Data was collected with charge exclusion (1, 8, >8). Data was acquired using a Data-Dependent Acquisition (DDA) method consisting of a full MSI scan (Resolution = 120,000) followed by sequential MS2 scans (Resolution = 15,000) to utilize the remainder of the 1 second cycle time. Precursor isolation window and normalized collision energy were set as described in the study. Table S7. Conditions of Liquid-chromatography (LC) Parameter Condition Column 100 pM ID fused silica capillary packed in-house with bulk Cl 8 reversed phase resin (particle size, 1.9 pm; pore size, 100 A; Dr. Maisch GmbH) Mobile phase Buffer A: water with 3% DMSO and 0.1% formic acid Buffer B: 80% acetonitrile with 3% DMSO and 0.1% formic acid Gradient and flow rate 0 - 5 min, 3 - 10% B, 300 nL/min 5 - 64 min, 10 - 50% B, 220 nL/min 64 - 70 min, 40 - 95% B, 250 nL/min Run time 70 minutes Injection volume 5 uL

Protein and peptide identification.

Raw data collected by LC-MS/MS were searched with MSFragger (v3.4 and v3.5) and FragPipe (vl7.1 and 18.0). For closed search, the proteomic workflow and its collection of tools was set as default. Precursor and fragment mass tolerance was set as 20 ppm. Missed cleavages were allowed up to 1. Peptide length was set 7 - 50 and peptide mass range was set 500 - 5000. Cysteine residues were searched with differential modifications as described in the study. For labile search, mass offsets were set restricted to cysteines. Y ion masses and diagnostic fragment masses were set as in FIGs. 11 & 12 for different proteomic samples. PTM-Shepherd was enabled for localization. A sample workflow can be found attached. Calibrated and deisotoped spectrum files produced by FragPipe were retained and reused for this analysis.

Data analysis and processing.

After MS search with MSFragger, raw files and identification files were imported to PDV for MS spectra annotation. Frequency distribution and intensity of the fragment ions and peptide remainder ions were calculated based on the output of PTM Shepherd as mean of all replicates (See supplementary data tables). Mean of the number of PSMs and peptides of all replicates were reported as bar plots. Additionally, venn diagrams were constructed to display the number of common cysteine peptides identified shared between various experiments.

Chemistry Methods

General Methods All reactions were performed in dried glassware under an atmosphere of dry N2 unless otherwise stated. Silica gel P60 (SiliCycle) was used for column chromatography. Plates were visualized by fluorescence quenching under UV light or by staining with iodine, KMnO4, or bromocresol green. Other reagents were purchased from Sigma-Aldrich (St. Louis, MO), Alfa Aesar (Ward Hill, MA), EMD Millipore (Billerica, MA), Fisher Scientific (Hampton, NH), Oakwood Chemical (West Columbia, SC), Combi-blocks (San Diego, CA) and Cayman Chemical (Ann Arbor, MI) and used without further purification. Additionally, all isotopically enriched reagents were purchase from Sigma-Aldrich (St. Louis, MO) or Cambridge Isotope Laboratories (Cambridge, MA) and used without further purification. ¹H NMR and ¹³C NMR spectra for characterization of new compounds and monitoring reactions were collected in CDCl₃, CD3OD, D2O or DMSO-d₆ (Cambridge Isotope Laboratories, Cambridge, MA) on a Bruker AV 500 MHz spectrometer or Bruker AV 400 MHz in the Department of Chemistry & Biochemistry at The University of California, Los Angeles. All chemical shifts are reported in the standard notation of parts per million using the peak of residual proton signals of the deuterated solvent as an internal reference. Coupling constant units are in Hertz (Hz). Splitting patterns are indicated as follows: br, broad; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; dt, doublet of triplets. Low-resolution mass spectrometry was performed on an Agilent Technologies InfinitiyLab LC/MSD single quadrupole LC/MS (ESI source). High-resolution mass spectrometry was performed on a Waters LCT Premier with ACQUITY LC and autosampler (ESI source). Cell culture reagents including Dulbecco’s phosphate- buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM)/high glucose media, Roswell Park Memorial Institute (RPMI) media, trypsin-EDTA and penicillin/streptomycin (Pen/Strep) were purchased from Fisher Scientific. All protein concentrations were determined using a Bio-Rad DC protein assay kit using reagents from Bio-Rad Life Science (Hercules, CA).

(((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine-18O2

To an oven dried pwave vial was added 180 Water (444 pL) and 1,4-Dioxane (667 pL) followed by acetyl chloride (17.6 mg, 15.9 pL, 0.56 Eq, 224 pmol). To this solution was added N- Fmoc-L-alanine (125 mg, 1 Eq, 0.400 mmol), system purged with nitrogen, and let stir at 100°C overnight. After 24h the solution was warmed to room temperature and solvent evaporated under reduced pressure. Crude material was purified by recrystallization in hexanes and ethyl acetate to yield the desired product as a white solid. >90% oxygen enrichment observed by low resolution LC-MS. LC-MS (ESI) m/z: Calculated [2M-H]- = 629.2412 , Found [2M-H]- = 629.2.

Isotopically labeled lodoacetamide alkyne (IAA) were all prepared according to the following procedure:

N-(hex-5-yn-l-yl)-2-iodoacetamide (IAA)

To an oven dried 25mL round-bottom flask was added 2-iodoacetic acid (100 mg, 1 Eq, 538 pmol), DMAP (13.1 mg, 0.2 Eq, 108 pmol), and anhydrous CH2CI2 (1.34 mL). The vial was purged with nitrogen followed by addition of hex-5-yn-l-amine (67.9 mg, 1.3 Eq, 699 pmol) and cooled to 0°C. DCC (166 mg, 1.5 Eq, 807 pmol) added at 0°C and the solution was allowed to slowly warm to room temperature. After completion of the reaction as determined by TLC (1 :1 EtOAc: Hexanes) solids were filtered off over cotton and filtrate was concentrated down. Crude was purified by flash column chromatography (1 :9 to 1 :1 EtOAc: Hexanes) to yield the product as an off white solid (84mg, 59%). All spectral data agrees with previously published literature.^{2 1}H NMR (400 MHz, Chloroform-d) δ 6.56 (br, 1H), 3.68 (s, 2H), 3.27 (q, J = 6.8 Hz, 2H), 2.21 (td, J= 6.8, 2.6 Hz, 2H), 1.95 (t, J= 2.7 Hz, 1H), 1.70 - 1.50 (m, 4H). l-13C-N-(hex-5-yn-l-yl)-2-iodoacetamide (1-13C-IAA)

Prepared according to the general procedure with l-13C-2-iodoacetic acid (56mg, lEq, 300pmol). Product isolated as off-white solid (43mg, 54%).¹H NMR (400 MHz, Chloroform-d) δ 6.09 (br, 1H), 3.70 (d, J= 4.2 Hz, 2H), 3.34 - 3.27 (m, 2H), 2.24 (td, J= 6.8, 2.7 Hz, 2H), 1.97 (t, J = 2.7 Hz, 1H), 1.72 - 1.52 (m, 4H). HRMS (ESI-MS) m/z: Calculated [M+H]⁺ = 267.0075 , Found [M+Na]⁺ = 267.0089.

1,2-13C2-N-(hex-5-yn-l-yl)-2-iodoacetamide (1,2-13C2-IAA) Prepared according to the general procedure with l,2-13C2-2-iodoacetic acid (50mg, lEq, 270pmol). Product isolated as off-white solid (46mg, 64%).¹H NMR (400 MHz, Chloroform-d) 8 6.07 (s, 1H), 3.70 (dd, J = 151.5, 4.2 Hz, 2H), 3.31 (ddt, J = 10.3, 7.0, 3.4 Hz, 2H), 2.24 (td, J = 6.8, 2.7 Hz, 2H), 1.97 (t, J = 2.7 Hz, 1H), 1.70 - 1.54 (m, 4H).HRMS (ESI-MS) m/z: Calculated [M+H]⁺ = 268.0109 , Found [M+Na]⁺ = 268.0111.

General Procedure for solid-phase synthesis of peptides

Peptides were manually synthesized according to the following general procedure.

Loading of the 2-chlorotrityl chloride resin

2-chlorotrityl chloride resin (100-200 mesh, 0.1-0.9mmol/g) was added to a solid-phase vessel and swelled in dry CH2CI2 for 1 hr. The CH2CI2 was vacuum filtered off and first fmoc protected amino acid (2 Eq) was dissolved in dry CH2CI2 and diisopropylethylamine (DIPEA) (3Eq.) and loaded onto resin. This was left to incubate for Ihr, after which the solution was vacuum filtered off and resin washed thoroughly with CH2CI2, DMF, and MeOH. In the case of N-Fmoc- Biotin-Lys, the loading step was performed in DMF.

Amino Acid Coupling

Coupling of standard amino acids was carried out through treatment of the deprotected resin with 3 equivalents of Fmoc-protected amino acids, 3 equivalents of A,A,A',A'-Tetramethyl- O -( 1H-benzotriazol- l -yl)uronium hexafluorophosphate (HBTU), and 6 equivalents of DIPEA in DMF for 30 min. Each coupling was performed twice unless the amino acid used was valuable in which case the coupling was left longer. In between coupling and deprotection steps the resin was washed thoroughly with CH2CI2, then DMF, MeOH, and CH2CI2. Coupling of compounds to resin was monitored using the Kaiser test. Coupling completed with valuable amino acid bulding blocks, i.e. isotopically enriched amino acids, were carried out using 1.1 equivalents HBTU, 1.1 equivalents Fmoc-protected amino acid, and 2.2 equivalents of DIPEA. These couplings were repeated once with l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3- oxide hexafluorophosphate (HATU).

Fmoc Deprotection

Removal of A-terminal Fmoc protecting groups was carried out by treating the resin with 50% 4-methylpiperidine in DMF (3 x 1 min). After deprotection the resin was washed thoroughly with CH2CI2, then DMF and CH2CI2. Complete deprotection was monitored using the Kaiser test. Acetate Capping

Capping of the N-termini was carried out by treating the deprotected resin bearing the full peptide with acetic anhydride (5 Eq.) and DIPEA (5 Eq.) in CH2CI2 (2 x 20min). After deprotection the resin was washed thoroughly with CH2CI2, then DMF and CH2CI2. Complete capping was monitored using the Kaiser test.

Resin Cleavage

Fully assembled peptides were then thoroughly washed with CH2CI2 and the dried resin was incubated with 20% hexafluoroisopropanol (HFIP) for 10 minutes. The resulting solution was collected into a round-bottom flask and the process was repeated two times. The collected peptide solution was concentrated down to 2-3 mL and precipitated into cold diethyl ether. The ether was decanted off and the peptide dissolved in water. The desired peptides were obtained as fluffy whitepale yellow solids after lyophilization. LC-MS analysis revealed only minor impurities and tags were used without further purification.

NBIV-010: HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 1155.5411 , Found [M+Na]⁺ = 1155.2639.

NBIV-011: HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 1121.4960 , Found [M+Na]⁺ = 1121.2201.

NBIV-022: HRMS (ESI-MS) m/z: Calculated [M+H]⁺ = 1183.6079 , Found [M+H]⁺ = 1183.6276. NBIV-027: HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 1107.4798 , Found [M+Na]⁺ = 1107.4849.

NBIV-070: HRMS (ESI-MS) m/z: Calculated [M+H]⁺ = 1113.5291 , Found [M+H]⁺ = 1113.2329. NBIV-071: HRMS (ESI-MS) m/z: Calculated [M+H]⁺ = 1114.5325 , Found [M+H]⁺ = 1114.2303. NBIV-087: HRMS (ESI-MS) m/z: Calculated [M+H]⁺ = 1114.5262 , Found [M+H]⁺ = 1114.2172. NBIV-088: HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 1137.5115 , Found [M+Na]⁺ = 1137.1945.

NBIV-089: HRMS (ESI-MS) m/z: Calculated [M+H]⁺ = 1115.5334 , Found [M+H]⁺ = 1115.2336. NBIV-090: HRMS (ESI-MS) m/z: Calculated [M+Na]⁺ = 1137.5178 , Found [M+Na]⁺ = 1137.2007. Example 3: Synthesis of Additional Exemplary Chemoproteomics Capture Reagents and Their Use in Exemplary Chemoproteomics Studies

It was postulated that incorporating an uncapped glycine at the N-terminus of the sCIP reagent (sCIP-Gly-NH2) makes for a substantially nucleophilic amine with minimal steric hindrance (FIG. 17A). sCIP-Gly-NH2 can then be conjugated to an amine-reactive isobaric tag, such as TMT, to make a sCIP-TMT conjugate that can be used directly for click conjugation to alkyne labeled proteins (FIG. 17B). Using this approach, this probe can be seamlessly incorporated into a 10-plex TMT workflow to demonstrate the ability to quantify different ratios of each reporter. sCIP-TMT conjugates with each of the 10 tags (126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, and 131; shown in FIG. 17B and FIG. 17C) were subjected to a copper catalyzed azide alkyne cycloaddition (CuAAC or click) with IAA labeled HEK293T cell lysates. Using these click-compatible isobaric tags samples can be combined early in the sample preparation workflow. This becomes important with higher levels of multiplexing as demonstrated by the comparative sCIP-TMT and TMT workflows in FIG. 18A. Furthermore, the number of eppendorf and falcon tubes required for each workflow as well as the time (excluding digestion) indicates that the sCIP-TMT workflow can save up to 86 tubes and 7.3 hours of preparation time (FIG. 18B) This discrepancy can be attributed to the late-stage sample combination and the various extra cleanup steps needed for TMT labeling.

TMT reporters were next quantified in both low and high abundance to compare against the expected characteristics of cysteine-reactive electrophile profiling experiments. To do this HEK293T lysates labeled with one of each of the sCIP-TMT¹⁰ reagents were combined in equimolar and varying ratios. These samples could then be analyzed using the TMT workflows in the already established and freely available FragPipe with MSFragger software. Doing this analysis revealed good PSM, peptide, cysteine, and protein coverage for all samples (FIG. 18C) comparable to the established streamlined cysteine activity-based protein profiling (SLC-ABPP) method. Furthermore, the observed ratios centered around 1 for all 10 reporters mixed in equal ratios, and the expected ratios were observed for those mixed in 1 : 5: 10: 15 ratios (FIG. 18D, FIG. 18E, and Table S2) Table S2. Mixing ratios of sCIP-TMT¹⁰ reagents for FIG. 18

The compatibility of the workflow with other commonly employed analysis methods such as MS³ quantitation with synchronous precursor selection (SPS) was then verified. Subjecting the same samples as above to SPS-MS3 data acquisition resulted in similar results to those acquired using MS2-FAIMS albeit with tighter ratio spread (FIG. 18D, FIG. 18E and Table S2) and decreased number of identifications as expected.

As cysteine chemoproteomics is widely utilized in pinpointing ligandable or potentially druggable cysteine residues, the compatibility of sCIP-TMT with screening applications was assessed. Four prototype electrophilic fragments were selected (FIG. 21A), including two chlor oacetamide-containing molecules, the widely utilized KB02 and KB 10, which had previously shown a substantially distinct labeling pattern and more attenuated reactivity when compared to KB02. Additionally, methylphenyl propiolate (MPP) and methyl cinnamate (MC) were selected, as previous work had revealed distinct proteomic reactivity for each molecule, with MPP functioning as a potent cysteine protease inhibitor whereas MPA showed negligible protease inhibitory activity. HEK293T cell lysates were subjected to either vehicle (DMSO) or each compound (500 pM) in duplicate. Compound treatments were performed in cell lysates to avoid the recently reported pervasive protein aggregation observed in cell-based analysis using comparatively high doses of electrophilic compounds. After treatment the lysates were subjected to the sCIP-TMT workflow (as shown in FIG. 18A). In total, 10733 peptides, 8515 cysteines, and 3787 proteins were identified. The vast majority (>96%) of enriched peptides harbored the sCIP modification, consistent with efficient capture of labeled peptides. 790 high confidence cysteines were detected with log2 ratios >1 for at least one compound, consistent with covalent modification at these sites.

It was next explored whether specific targets and SAR reported by the sCIP-TMT dataset could add to the burgeoning set of available cysteine chemoproteomic datasets. Three aspects of the dataset were of particular interest: (1) corroborating prior reports of cysteine ligandability; (2) de novo identification of ligandable cysteines; and (3) assessing the proteome-wide reactivity of different cysteine-reactive electrophiles. Comparison to the previous dataset generated using KB02 and MS 1 -based quantification revealed substantial overlap between the targets identified by both approaches together with high concordance (r² = 0.63) in the measured ratios, with some unavoidable ratio compression observed for the sCIP-TMT dataset (FIG. 21B), which was acquired using FAIMS-MS2. Exemplifying established labeling sites, it was observed that GSTO1 Cys32 was labeled to near completion by both KB02 and KB 10, consistent with the high ligandability of this cysteine, as reported by a number of previous studies (FIG. 21C). Additional targets that proved highly consistent with prior reports include Creatine Kinase Cys283²³, which is an established target of KB02 and related analogues, and PIN1 Cysl 13, for which several highly potent inhibitors have been reported. Taken together these findings provide compelling evidence that sCIP-TMT faithfully captures cysteine ligandability sites.

With the goal of demonstrating the utility of the sCIP reagents paired with a wide range of established isobaric tags, the work was expanded to tags that would allow for higher degrees of multiplexing than what is commercially available. The previously described dimethyl leucine (DiLeu) reporter was a perfect choice with an already established 4, 8, 12, and 21-plex systems. Furthermore, the building blocks for these tags are all amino acids making them amenable to solidphase peptide synthesis and thus could be easily incorporated into the established synthetic workflow. Since balancing this reporter with an amino acid, such as alanine, has also been reported, this balancer/reporter combination was used for the set of fully functionalized sCIP- DiLeu isobaric tags built using SPPS (FIG. 19A). These reagents are able to be incorporated into the established sCIP cysteine profiling workflow (FIG. 19B), mapping out an unprecedented 29- plex system with mass differences as low as 2.92 mDa (FIG. 19C).

To this end 29 unique isotopologues of N,N-dimethyl-L-leucine (DiLeu) were synthesized using a modified route to previously published procedures (FIG. 22) in good overall yields (30- 99%). These isotopologues of DiLeu (FIG. 23) were used along with L-alanine as a balancer to synthesize a 29-plex set of sCIP-DiLeu clickable isobaric tags using SPPS (FIG. 19A). As before these reagents were validated in a differential mixing experiment where HEK293T cellular lysates were labeled first with IAA (500pM) and then click to one of the 29 reagents. The labeled lysates were then mixed in 1: 1 and 1:5: 10: 15:20 ratios and analyzed using LC-MS/MS. Analysis of this data showed good concordance between the expected and observed ratios (FIG. 24).

The utility of sCIP reagents was further demonstrated with the synthesis of sCIP-TMTpro, which contains an isobutyl proline, adding another potential reagent to the repertoire of mass tags (FIG. 20)

Preparation of sCIP-TMT capture reagents for CuAAC.

Each TMT channel (29mM in MeCN) was mixed in an equimolar ratio with sCIP-Gly- NH2 (29mM in DMSO) and let react for Ih at ambient temperature. After Ih, 0.5 equivalents of hydroxylamine (lOmM in DMSO) was added and let react for 15 minutes after which the sCIP- TMT conjugate was ready to be used for CuAAC.

Generation and preparation of cell lysate samples and mass spectrometry methods

Additional relevant methods relating to the isolation and analysis of the HEK293T proteome are found in Example 1.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

CLAIMS We claim:

1. A chemoproteomic capture reagent having a structure represented by formula I or a salt thereof: wherein

Xi, X2, and X3 are each independently selected from NR6, O, and S;

Ri, R2, R3, R4, R5 are each independently selected from hydrogen, alkyl, aralkyl, aryl, and heteroaryl; each R⁶ is independently selected from hydrogen, alkyl, aralkyl, and aryl;

Yi is an amino acid or a first sequence of amino acids;

Y2 is an amino acid or a second sequence of amino acids; and nl, n2, n3, and n4 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

2. The chemoproteomic capture reagent of claim 1, wherein R¹ is aryl (e.g., phenyl).

3. The chemoproteomic capture reagent of claim 1 or 2, wherein R² is aryl (e.g., phenyl).

4. The chemoproteomic capture reagent of any one of claims 1-3, wherein R³ is hydrogen.

5. The chemoproteomic capture reagent of any one of claims 1-4, wherein R⁴ is alkyl (e.g., methyl).

6. The chemoproteomic capture reagent of any one of claims 1-5, wherein R⁵ is alkyl (e.g., methyl).

7. The chemoproteomic capture reagent of any one of claims 1-6, wherein X¹ is O.

8. The chemoproteomic capture reagent of any one of claims 1-7, wherein X² is S.

9. The chemoproteomic capture reagent of any one of claims 1-8, wherein X³ is O.

10. The chemoproteomic capture reagent of any one of claims 1-9, wherein nl is 2.

11. The chemoproteomic capture reagent of any one of claims 1-9, wherein nl is 6.

12. The chemoproteomic capture reagent of any one of claims 1-11, wherein n2 is 1.

13. The chemoproteomic capture reagent of any one of claims 1-12, wherein n3 is 3.

14. The chemoproteomic capture reagent of any one of claims 1-13, wherein n4 is 2.

15. The chemoproteomic capture reagent of any one of claims 1-14, wherein Yi is a first sequence of amino acids.

16. The chemoproteomic capture reagent of any one of claims 1-15, wherein the first sequence of amino acids comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

17. The chemoproteomic capture reagent of claim 15, wherein the first sequence of amino acids comprises 2 amino acids.

18. The chemoproteomic capture reagent of claim 15, wherein the first sequence of amino acids comprises 3 amino acids.

19. The chemoproteomic capture reagent of any one of claims 1-18, wherein the first sequence of amino acids comprises naturally occurring amino acids.

20. The chemoproteomic capture reagent of any one of claims 1-19, wherein the first sequence of amino acids comprises an amino acid having a side chain comprising an alkene, alkyne, diazo or azido.

21. The chemoproteomic capture reagent of any one of claims 1-19, wherein the first sequence of amino acids comprises an amino acid having a side chain comprising azido.

22. The chemoproteomic capture reagent of any one of claims 1-21, wherein the first sequence of amino acids comprises an amino acid at the C-terminus having a side chain comprising an alkene, alkyne, diazo or azido.

23. The chemoproteomic capture reagent of any one of claims 1-21, wherein the first sequence of amino acids comprises an amino acid at the C-terminus having a side chain comprising azido.

24. The chemoproteomic capture reagent of any one of claims 1-21, wherein the first sequence of amino acids comprises 6-azido-lysine (LysN3) at the C-terminus.

25. The chemoproteomic capture reagent of any one of claims 1-24, wherein the N-terminus of the first sequence of amino acids is protected with a nitrogen protecting group (e.g., acetyl).

26. The chemoproteomic capture reagent of any one of claims 1-24, wherein the first sequence of amino acids comprises a P-amino acid (e.g., β-glycine) at the N-terminus.

27. The chemoproteomic capture reagent of claim 26, wherein the P-amino acid is substituted with heterocyclic acyl (e.g., piperidinyl acyl, such as dimethylpiperidinylacyl).

28. The chemoproteomic capture reagent of any one of claims 1-24, wherein the first sequence of amino acids comprises leucine at the N-terminus.

29. The chemoproteomic capture reagent of claim 28, wherein the nitrogen of the leucine is substituted with alkyl.

30. The chemoproteomic capture reagent of claim 29, wherein the nitrogen of the leucine is substituted with two methyl groups.

31. The chemoproteomic capture reagent of any one of claims 1 -24, wherein the first sequence of amino acids comprises

32. The chemoproteomic capture reagent of any one of claims 1-24, wherein the first sequence of amino acids comprises proline at the N-terminus.

33. The chemoproteomic capture reagent of claim 32, wherein the nitrogen of the proline is substituted with alkyl.

34. The chemoproteomic capture reagent of claim 33, wherein the nitrogen of the proline is substituted with butyl (e.g., isobutyl).

35. The chemoproteomic capture reagent of any one of claims 1-34, wherein the first sequence of amino acids is isotopically enriched.

36. The chemoproteomic capture reagent of any one of claims 1-35, wherein the first sequence of amino acids is isotopically enriched with ¹³C, ¹⁵N, or ¹⁸O.

37. The chemoproteomic capture reagent of any one of claims 1-36, wherein the first sequence of amino acids is isotopically enriched with ²H, ¹³C, ¹⁵N, or ¹⁸O.

38. The chemoproteomic capture reagent of any one of claims 1-37, wherein Y2 is an amino acid.

39. The chemoproteomic capture reagent of any one of claims 1-38, wherein Y2 is an amino acid having a side chain comprising biotin (e.g., avidin or streptavidin).

40. The chemoproteomic capture reagent of any one of claims 1-39, wherein Y2 is an amino acid having a side chain comprising alkyl.

41. The chemoproteomic capture reagent of any one of claims 1-40, wherein Y2 is an amino acid having a side chain comprising alkylamidoalkyl.

42. The chemoproteomic capture reagent of claim 40 or 41 , wherein the side chain of the amino acid of Y2 is substituted with heterocyclyl (e.g., biotinyl).

The chemoproteomic capture reagent of any one of claims 1-42, wherein the chemoproteomic capture reagent is selected from:

44. The chemoproteomic capture reagent of any one of claims 1-43, wherein one or more hydrogen atoms are enriched for ²H.

45. The chemoproteomic capture reagent of any one of claims 1-44, wherein one or more carbon atoms are enriched for ¹³C.

46. The chemoproteomic capture reagent of any one of claims 1-45, wherein one or more nitrogen atoms are enriched for ¹⁵N.

47. The chemoproteomic capture reagent of any one of claims 1-46, wherein one or more oxygen atoms are enriched for ¹⁸O.

48. A method of identifying a binding site comprising: contacting a substrate with a click chemistry moiety, thereby creating a substrate-click chemistry moiety conjugate; contacting the substrate-alkyne conjugate with the chemoproteomic capture reagent of any one of claims 1-40, thereby creating a chemoproteomic capture reagent-substrate conjugate; digesting the chemoproteomic capture reagent-substrate conjugate, thereby creating a digested substrate-chemoproteomic capture reagent conjugate; contacting the digested substrate-chemoproteomic capture reagent conjugate with an enrichment agent; cleaving the digested substrate-chemoproteomic capture reagent conjugate, thereby creating a digested substrate-amino acid conjugate; and determining the molecular weight of the digested substrate-amino acid conjugate, thereby identifying the binding site.

49. The method of claim 48, wherein the substrate is a protein.

50. The method of claim 48, wherein the substrate is a protein comprising a sulfur containing amino acid (e.g., cysteine or homocysteine).

51. The method of any one of claims 48-50, wherein the protein is formed from cell lysation.

52. The method of any one of claims 48-51, wherein the click chemistry moiety is an alkene, alkyne, diazo, or azide.

53. The method of any one of claims 48-52, wherein the click chemistry moiety is alkyne.

54. The method of claim 53, wherein contacting the substrate-alkyne conjugate with the chemoproteomic capture reagent of any one of claims 1-47 forms a triazole linking the chemoproteomic capture reagent to the substrate.

55. The method of any one of claims 48-54, wherein digesting the chemoproteomic capture reagent-substrate conjugate comprises contacting the chemoproteomic capture reagent-substrate conjugate with a digestion enzyme (e.g., trypsin).

56. The method of any one of claims 48-55, wherein the enrichment agent is a protein that binds biotin (e.g., avidin or streptavidin).

57. The method of any one of claims 48-56, wherein cleaving the digested substrate- chemoproteomic capture reagent conjugate comprises contacting the digested substrate- chemoproteomic capture reagent conjugate with acid (e.g., formic acid).

58. The method of any one of claims 48-57, wherein the click chemistry moiety is isotopically enriched.

59. The method of any one of claims 48-58, wherein the click chemistry moiety is isotopically enriched with ¹³C, ¹⁵N, or ¹⁸O.

60. The method of any one of claims 48-59, wherein the click chemistry moiety is isotopically enriched with ²H, ¹³C, ¹⁵N, or ¹⁸O.