Ion mobility modifying tags for mass spectrometry

Abstract

The disclosure relates generally to compounds and methods for selective gas phase enrichment of modified peptides for reactive protein profiling.

Description

ION MOBILITY MODIFYING TAGS FOR MASS SPECTROMETRYCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of U.S. provisional Patent Application No. 63 / 669,296, filed July 10, 2024, which is incorporated by reference herein in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under Contract Nos. GM147449, OD028556 and OD016320 awarded by National Institutes of Health (NIH). The government has certain rights in the invention.BACKGROUND

[0002] Measuring the reactive proteome is critical for understanding physiological biological processes and disease phenotypes. Several widely used methods, including reactive cysteine profiling, proximity labeling, and mapping post-translational modifications (PTMs), depend on bottom-up mass spectrometry (MS) based proteomics to identify and quantify reactions at specific amino acids in proteins. Recent advances in MS technology have made these measurements increasingly routine. However, due to the staggering size of chemical space contained in a typical proteomic sample (>500,000 unique tryptic peptides from >20,000 proteins), small sample sizes (-100 pg / 106cells), and the wide dynamic range of protein abundance (1-107copies per cell), direct detection, sequencing, and quantification of the small percentage of peptides containing a reacted amino acid among the abundant and complex background of unreacted peptides is challenging.

[0003] Affinity-based isolation of modified peptides is commonly used to enrich samples for MS analysis and has enabled deep reactivity-based profiling. Existing enrichment methods use selective binding by solid sorbents to isolate modified peptides from native ones in solution, with reversible desthiobiotin (Dtb) / streptavidin binding (Ka ~ 10-11) being a privileged platform.1However, the improved depth enabled by enrichment comes at a cost: extensive wash, elution, cleanup, and buffer exchange steps increase process complexity, statistical variance, and sample losses (Fig. 1A). Moreover, costly sorbent materials are required, nonspecific binding of abundant endogenous peptides causes high background, and large amounts of sample input are required to avoid losses of low abundance modifiedpeptides to plastic and glass surfaces during handling.2These problems become acute at scale, where thousands of samples are analyzed. Isobaric tandem- mass tags (TMT, iTRAQ) can be used to multiplex samples to avoid handling low-ng / mL samples of reacted peptide and improve protein coverage, but these significantly increase sample processing complexity and cost, and preclude straightforward assembly of large datasets.3

[0004] In contrast to liquid phase affinity-based peptide enrichment prior to MS analysis, gas phase enrichment strategies for separating ionized peptides offer many potential advantages, including simplification of sample processing, elimination of highly dilute analyte solutions that cause sample losses, and facile integration with existing mass spectrometer technology for simultaneous enrichment and analysis. Recently, differential ion mobility (IM) in low- pressure gas has emerged as a useful separation modality in MS bottom-up proteomics, leading to the wide deployment of tandem IM / MS instruments.SUMMARY

[0005] We reasoned that a modification capable of significantly altering the ion mobility of a peptide, but with minimal impact on its mass, would enable efficient enrichment in the gas phase. Due to the centrality of reactive proteome profiling and the ubiquity of imperfect enrichment methods using solid / liquid phase separation in modem chemical biology, a direct method for selectively measuring modified peptides without in solution enrichment would be highly impactful. While charged modifications have been previously used to alter chromatographic retention time,4,5ionization efficiency,4 1 1peptide fragmentation,4,7,8and intact protein charge,9approaches that modulate ion mobility for gas phase enrichment of modified peptides have not been previously reported.

[0006] The instant disclosure relates to compounds of formula (I), (II), or (III):whereinA is a reactive warhead;Li and L2 are independently linkers or absent;Ri in each instance is independently selected from alkyl, (^)n"R2 in each instance is independently an alkyl;R in each instance is independently selected from the group consisting of alkyl, halo, alkoxy, amino, thio, arenyl, and heteroarenyl; n is independently an integer from 0 to 5; n’ is an integer from 0 to 5; and n” is independently an integer from 0 to 3, or salt thereof.

[0007] The instant disclosure also relates to compounds of formula (IV):whereinX is independently selected from H, halo, substituted or unsubstituted alkyl, aryl, heteroaryl, , cycloalkyl, and heterocyclylLi is a linker;A is a reactive warhead; and each Ri and R2 are independently selected from H, substituted or unsubstituted alkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl or Ri and R2 together with the atoms to which they are attached form a five- or six-membered heterocyclyl.

[0008] This disclosure further provides a method of modulating ion mobility for gas phase enrichment of a modified peptide or selectively measuring a modified peptide comprising: covalently modifying a peptide to generate a modified peptide; and spatially separating the modified peptide from an unlabeled peptide using ion mobility spectrometry in the gas phase.

[0009] These and other features, aspects and advantages of the present disclosure will become better understood with reference to the following figures, associated descriptions, and claims.BRIEF DESCRIPTION OF THE FIGURES

[0010] Fig. 1A-B show that the gas phase enrichment overcomes the limitations of liquid phase enrichment in bottom-up proteomics. Fig. 1A is a comparison between in spectrometer gas phase enrichment vs. liquid phase streptavidin enrichment. Fig. IB shows timSHIFT reagent: design and applications.

[0011] Fig. 2A-D show that the cationic poly-imidazolium species efficiently shift peptides in IM / MS mass spectrometry with limited increase in collisional cross-section. Fig. 2A shows the experimental workflow and synthesized timSHIFT azides. Fig. 2B. shows (Left) m / z vs. 1 / ko plots showing unlabeled vs. labeled peptide- spectrum matches. Colormaps show their density via kernel density estimation. (Right) Average AmL. vs. l / ko, and Az (colormap) between labeled and unlabeled matched peptides. Fig. 2C shows (Left) m / z vs. 1 / ko plot showing unlabeled (A) and labeled (13, •) matched peptides. Average charge (arithmetic) in colormap. (Right) m / z and 1 / ko shift effect between labeled and unlabeled matched peptides for probe 13. Values are arithmetic averages between PSMs having the same sequence, dashed grey lines represent averages. Fig. 2D shows the synthesis of cysteine-targeting probe 14. Boc, tert-butyloxycarbonyl; DMF, V,V-di methyl formamide; Pd-C, palladium on carbon; RT, room temperature; TFA, trifluoro acetic acid; Ts, p-toluenesulfonyl.

[0012] Fig. 3A-B show that the charge is responsible for IM-m / z shift poly-charged imidazolium species and optimized fragmentation polygon enhances peptide detection. Fig. 3A shows m / z vs. 1 / ko plots for bis-cationic 14 (left), mono-cationic 15 (center), and neutral 16 (right). Colormaps encode unlabeled and labeled PTMs’ density via kernel density estimation and color-codes are the same as in Fig. 2. Fig. 3B shows number of average (n = 3) total and labeled peptides detected under standard and optimized DDA windows. Errorbars are reported at ±2c (95% confidence). Data was checked by two-tailed unpaired / -test assuming unequal variances: **** P < 0.0001; ** P < 0.01.

[0013] Fig. 4A-B show that the timSHIFT permits the low input detection and quantitation of labeled peptides and outperforms desthiobiotin / streptavidin enrichment. Fig. 4A shows number of average (n = 3, independent replicates) quantified peptides bearing the corresponding modification, 5 pl injection. Error bars are reported at ±G (68.2% confidence). *One outlier was removed after evaluation with two-sided Grubbs’ test (P < 0.05). Data was checked by two-tailed unpaired / -test assuming unequal variances. Fig. 4B shows heatmap showing quantitation (logio Intensity, average) of cysteine-bearing peptides with timSHIFT- IAA and DTB-IAA.

[0014] Fig. 5A-B show that the timSHIFT labeling enables cysteine profiling of small molecule covalent inhibitors from 2 million to 20,000 cells input. Fig. 5A is an experimental workflow for intact cell / cell lysate activity profiling. Fig. 5B (left) timSHIFT shows Pinl- C113 engagement by sulfopin upon Ramos cells treatment both in DDA (2 million cells) and DIA (20,000 cells), (right) timSHIFT maps the dose-dependent response to sulfopin treatment and known and novel potential off-targets are detected. Error bars are reported at ±G (68.2% confidence).

[0015] Fig. 6A-B show that the timSHIFT-based in spectrometer enrichment applied to 96- well plate covalent fragment screening and study of oxidative stress. Fig. 6A shows volcano plots for F6 (left) and F16 (center) for significantly targeted cysteines. Molecular properties of screened covalent fragments (top right). Eocalization of selected cysteines targeted by F6 and F16 (bottom right). Fig. 6B is a heatmap showing cysteines targeted by >5 fragments (left) and a selection of relevant significantly targeted cysteines (right). C. Quantified cysteines under different cell oxidative stress and comparison between global protein expression and cysteine off-competition / reactivity increase. For Fig. 6A, cEogP was calculated using Chemaxon consensus model at 0.1M electrolyte concentrations. Targeted cysteines are reported when -logFOl and FDR <5%. For Fig. 6C, targeted cysteines are marked when -logFC >1 (red) or -logFC <-l (blue) and FDR <5%.

[0016] Fig. 7A-H show the extent of m / z and 1 / ko shift effect with different probes, m / z vs. 1 / ko plots and analysis of matched PSMs (A, unlabeled; •, labeled) for alkyne modification(Fig. 7A) and probes 1-7 (Fig. 7B-H). PSMs were obtained in triplicate and the arithmetic averages were used. Colormaps are used consistently with Fig. 2.

[0017] Fig. 8A-E show the extent of m / z and 1 / ko shift effect with different probes, m / z vs. 1 / ko plots and analysis of matched PSMs (A, unlabeled; • , labeled) for probes 8-12. PSMs were obtained in triplicate and the arithmetic averages were used. Colormaps are used consistently with Fig. 2.

[0018] Fig. 9A-C show the effect covalent probes on the collisional cross-section of modified peptides. Fig. 9A shows the effect of covalent probes on the collisional crosssection of selected peptides shared across all covalent probes. Fig. 9B shows the average (matched peptides) variation in collisional cross-section between modified peptides and vehicle vs. their mass difference (left) and their average ion mobility difference (right). Fig. 9C shows peptides’ collisional cross-section vs. their ion mobility for probe 13. For all panels, values for peptides are obtained from the corresponding PSMs in triplicate, with arithmetic averaging.

[0019] Fig. 10A-C show a comparison between bis-cationic probe 14 and mono-cationic 15 and neutral 16. Fig. 10A shows m / z vs. 1 / ko plots and analysis of matched PSMs ( A , unlabeled; • , labeled) for probes 14-16. Fig. 10B shows m / z vs. 1 / ko plots and analysis of matched modified PSMs between 14 and 15 or 14 and 16 (A, 15 or 16; •, 14). PSMs were obtained in triplicate and the arithmetic averages were used. Colormaps are used consistently with Fig. 2. Fig. 10C shows the effect of chromatographic length and peptide detection using probe 14 using standard vs. optimized fragmentation windows.

[0020] Fig. 11 shows the effect of labeling concentration on peptide detection. Bar plot showing the average (n = 3) labeled, unlabeled, and total peptides with timSHIFT reagent 14. Error bars are reported at ±G. * One outlier was removed after Grubbs' test for a single outlier (P = 0.05). Data were checked by unpaired / -test assuming unequal variances: * P < 0.05, ** P < 0.01 **** P < 10’4.

[0021] Fig. 12A-C show the comparison between DTB-IAA pulldown and timSHIFT in spectrometer pulldown. Fig. 12A is the number of average (n = 3, independent replicates) quantified peptides bearing the corresponding modification, 0.5 pL injection. Error bars arereported at ±c (68.2% confidence). Data was checked by two-tailed unpaired / -test assuming unequal variances. Fig. 12B is a heatmap showing quantitation (logio Intensity, average) of cysteine-bearing peptides with timSHIFT-IAA (blue) and DTB-IAA (red). Fig. 12C is ART vs. / ko, and Az (colormap) plot between 14- and DTB-IAA-labeled PSMs (left). Average Az vs. / ko. and Az plot between 14- and DTB-IAA-labeled PSMs (right). Values are arithmetic averages between PSMs having the same sequence, dashed grey lines represent averages.

[0022] Fig. 13A shows that the timSHIFT detects leptomycin B covalent targeting of XPO1.

[0023] Fig. 13B shows a covalent fragments library. Compounds Fl-14, F30-31 were synthesized, the other compounds were purchased from commercial vendors.

[0024] Fig. 14 shows waterfall plots mapping the target of reactive cysteines by Fl-16 (vs. vehicle). Cysteines are considered targeted (red) when -logFC>l and FDR < 5%.

[0025] Fig. 15 shows waterfall plots mapping the target of reactive cysteines by F17-31 (vs. vehicle). Cysteines are considered targeted (red) when -logFC>l and FDR < 5%.

[0026] Fig. 16A-D are heatmaps showing selected reactive cysteines targeted by Fl-31 (vs. vehicle). Fig. 16A shows cysteines belonging to selected kinases. Fig. 16B is cysteines belonging to selected ubiquitin ligases. Fig. 16C is cysteines belonging to selected ion channels. Fig. 16D shows cysteines belonging to selected disulfide isomerases and glutathione transferases. Cysteines are considered targeted when -logFC>l and FDR < 5%. The colormap used is the same as in Fig. 6.

[0027] Fig. 17 shows a schematic of cysteine reactivity profiling using liquid phase enrichment. Analyte loss after step-intensive pulldown and sample handling. Larger input amounts are necessary to avoid these losses.

[0028] Fig. 18 shows In spectrometer gas phase enrichment using timShift. Innately charged (2+), cysteine reactive probe. Targeted fragmentation.

[0029] Fig. 19 shows a bar graph of timShift-IAA vs DTB-IAA. timShift enhances enrichment at low inputs. 20X IDs at 20 pg. timShift requires 25-fold less protein input

[0030] Fig. 20A-L shows a set of schematics of the timsReact concept. Under normal conditions, ions in the TIMS are held in the center via electric field during separation. With excessive amounts of peptide (>500 ng), inter-ion repulsion overwhelms the coaxial field. Gas phase deprotonation removes endogenous ions, enabling higher injection quantities. Fig. 20A shows a standard injection amount of unlabeled and labeled peptides, which would notsaturate the TIMS cartridge. Fig. 20B shows labeled peptides being efficiently separated from the unlabeled peptides in the TIMS cartridge. Fig. 20C shows a large injection amount of unlabeled and labeled peptides, which will saturate the TIMS cartridge. Fig. 20D shows inefficient separation of labeled and unlabeled peptides due to excessive material in the TIMS cartridge. Fig. 20E shows that given an excess amount of peptide injection, inter-ion repulsion will overwhelm the coaxial field. Fig. 20F shows a saturated TIMS cartridge with positively charged labeled and unlabeled peptides. Gaseous base, depicted as an outlined circle, can enter the TIMS cartridge. Fig. 20G shows gaseous base inside the TIMS cartridge. Fig. 20H shows deprotonation of charged peptides by gaseous base. Fig. 201 shows the exit of protonated gas. Fig. 20J shows depletion of uncharged unlabeled peptides. Fig. 20K shows that labeled peptides remain innately charged and can be injected at a higher amount into the TIMS cartridge. Fig. 20L shows that given a large injection of only labeled peptides, efficient separation of labeled peptides can occur.

[0031] Fig. 21 shows charge distribution of labeled and unlabeled peptides with no added base at a 200 ng injection. Cell lysate from Ramos cells were prepared and labeled with timShift reagent. 200 ng of the sample was injected, and an analysis was performed examining the aggregated intensity of either labeled or unlabeled peptides at different charge states. Importantly, due to the innate charge of the timShift reagent, labeled peptides are mostly at a higher charge state compared to unlabeled peptides (3+versus 2+respectively).

[0032] Fig. 22 shows charge distribution of labeled and unlabeled peptides with triethylamine. 500 ng of timShift reagent labeled sample was injected and triethylamine gas was introduced into the TIMS cartridge. Deprotonation was seen for both the labeled and unlabeled peptides as they now mostly exist as 2+and 1+peptides respectively.

[0033] Fig. 23 shows charge distribution of labeled and unlabeled peptides with trimethylamine. 200 ng of timSHIFT reagent labeled sample was injected and trimethylamine gas was introduced. Compared to the charge states seen in nitrogen, the standard condition, efficient deprotonation was seen for the labeled peptides, but less for the unlabeled peptides.

[0034] Fig. 24 shows a comparison between different accumulation time of labeled and unlabeled peptides with trimethylamine. Using the same conditions described in Fig. 24, ion accumulation time on the TIMS settings was adjusted. Efficient deprotonation was seen for labeled peptides and partial deprotonation was seen for unlabeled peptides. This trend holds at different accumulation times.

[0035] Fig. 25 shows peptide intensity at different accumulation time of labeled and unlabeled peptides with nitrogen. 200 ng of timSHIFT labeled sample was injected without any gaseous base, thus in a standard nitrogen environment. Accumulation time resulted in a decreased in intensity of peptides, but charge distribution was largely unchanged.

[0036] Fig. 26 shows the number of peptide spectrum matches at different accumulation time of labeled and unlabeled peptides with triethylamine. 200 ng of timSHIFT labeled sample was injected with gaseous triethylamine in the TIMS cartridge.

[0037] Fig. 27 shows the number of peptide spectrum matches at different accumulation time of labeled and unlabeled peptides with DIPEA. 200 ng of timSHIFT labeled sample was injected with gaseous DIPEA in the TIMS cartridge.

[0038] Fig. 28 shows the number of peptide spectrum matches at different accumulation time of labeled and unlabeled peptides with TMG. 200 ng of timSHIFT labeled sample was injected with gasesous TMG in the TIMS cartridge.

[0039] Fig. 29 shows the number of peptide spectrum matches at different collision energy of labeled and unlabeled peptides with triethylamine. 200 ng of timSHIFT labeled sample was injected with gaseous triethylamine. Increasing collision energy results in reduced identification of labeled peptides and 1+unlabeled peptides, but not 2+unlabeled peptides.

[0040] Fig. 30 shows a zoomed-in view of the number of peptide spectrum matches of unlabeled peptides. There are slight changes in the number of identified 2+unlabeled peptides at different collision energies.

[0041] Fig. 31 shows the number of peptide spectrum matches using soft ionization of labeled and unlabeled peptides in the presence of triethylamine. Overall the number of identified peptides decreased.

[0042] Fig. 32 shows a schematic of how global acetylation and esterification is accomplished. Esterification using MeOH was performed to limit the consumption of gaseous base by endogenous carboxylic acid groups in peptides, which can enhance the potency of gas phase deprotonation. Afterwards, gas phase deprotonation is used.

[0043] Fig. 33 shows a schematic of the proton affinity range of bases for deprotonating modified peptides.

[0044] Fig. 34 shows the aggregated intensity of labeled and unlabeled peptides after global acetylation in the presence of various bases. Notably, the charge distribution of labeled andunlabeled peptides was reduced by bases with lower proton affinities, demonstrating that global acetylation facilitates ion-molecule proton transfer.

[0045] Fig. 35 shows the aggregated intensity of labeled and unlabeled peptides after global acetylation and esterification in the presence of various bases. Charge distribution of labeled and unlabeled peptides were reduced by bases with lower proton affinities, while methylation protects labeled peptides from total deionization by bases with high proton affinity.

[0046] Fig. 36 shows the aggregated intensity of labeled and unlabeled peptides after labeling with a 3+variant of the timSHIFT reagent, in the presence of various bases. Importantly, the charge of labeled peptides remains high (3+or more) even in the presence of bases with higher proton affinities.

[0047] Fig. 37 shows the aggregated intensity of labeled and unlabeled peptides after labeling with a 3+ variant of the timSHIFT reagent and global acetylation and esterification in the presence of various bases. Importantly, for all the bases used, the majority of labeled peptides retain a 3+charge while unlabeled peptides are mostly 1+.

[0048] Fig. 38 shows the aggregated intensity of labeled and unlabeled peptides after labeling with a 3+variant of the timSHIFT reagent and global acetylation in the presence of various bases. Importantly, for all based used, the majority of labeled peptides retain a 3+charge. For unlabeled peptides, both 2+and 1+charged peptides are present.

[0049] Fig. 39 shows that peptides labeled via timShift funnel into a uniform 2+ charge distribution when gas phase ions are combined with mild gas phase bases, while endogenous peptides become uniformly 1+ charge. Residual charges in unmodified peptides may be due to retention of protons by terminal arginines. This increases intensity for labeled peptides and a more pronounced ion mobility shift effect.DETAILED DESCRIPTION

[0050] While the concepts of the present disclosure are illustrated and described in detail in the figures and descriptions herein, results in the figures and their description are to be considered as examples and not restrictive in character; it being understood that only the illustrative embodiments are shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

[0051] Unless defined otherwise, the scientific and technology nomenclatures have the same meaning as commonly understood by a person in the ordinary skill in the art pertaining to this disclosure.

[0052] The entire contents of each and every patent publication, non-patent publication, and reference text cited herein are hereby incorporated by reference, except that in the event of any inconsistent disclosure or definition from the present specification, the disclosure or definition herein shall be deemed to prevail.

[0053] In each of the foregoing and each of the following embodiments, it is to be understood that the formulae include and represent not only all pharmaceutically acceptable salts of the conjugate formulae. It is appreciated that certain functional groups, such as the hydroxy, amino, and like groups form complexes and / or coordination conjugates with water and / or various solvents, in the various physical forms of the compound of formula (I) or (II). It is understood that the formulae depicted throughout the disclosure are include and represent hydrates and / or solvates of compounds of formula (I) or (II). It is also to be understood that the non-hydrates and / or non-solvates of compounds of formula (I) or (II) are described by such formula, as well as the hydrates and / or solvates of the compounds of formula (I) or (II).Compounds of the disclosureIn certain aspects, the present disclosure provides a compound of formula (I), (II), or (III):whereinA is a reactive warhead;Li and L2 are independently linkers or absent;Ri in each instance is independently selected from alkyl, (R)n"R2 in each instance is independently an alkyl;R in each instance is independently selected from the group consisting of alkyl, halo, alkoxy, amino, thio, arenyl, and heteroarenyl. n is independently an integer from 0 to 5; n’ is an integer from 0 to 5; and n” is independently an integer from 0 to 3, or salt thereof.In certain embodiments, Li and L2 are independently selected from CH2,substituted by one or more substituents, wherein m is an integer from 0-5; n is an integer from 0-5; o is an integer from 1-3; and p is an integer from 1-3.In certain embodiments, n’ is 0.In certain embodiments, n’ is 1. In certain embodiments, Li is alkyl. In certain embodiments, Li is -(CH2)6-.In certain embodiments, Li is. In certainembodiments, L2 is alkyl. In certain embodiments, L2 is -CH2CH2-. In certain embodiments, L2 is absent. In certain embodiments, n” is 0.In certain embodiments, Ri is alkyl. In certain embodiments, Ri is methyl.In certain embodiments, n’ is 0. In some embodiments, n’ is 1.In certain embodiments, the compound iscertain embodiments, the compoundIn certain aspects, the present disclosure provides a compound of Formula (IV):whereinX is independently selected from H, halo, substituted or unsubstituted alkyl, aryl, heteroaryl, , cycloalkyl, and heterocyclylLi is a linker;A is a reactive warhead; and each Ri and R2 are independently selected from H, substituted or unsubstituted alkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl or Ri and R2 together with the atoms to which they are attached form a five- or six-membered heterocyclyl.In certain embodiments, at least one of R1and R2is charged (e.g., ammonium).one or more substituents, wherein m is an integer from 0-5; n is an integer from 0-5; o is an integer from 1-3; and p is an integer from 1-3.In certain embodiments, Li is. In certain embodiments, each X is halo (e.g., iodo).In certain embodiments, Ri is H.In certain embodiments, R2 is substituted alkyl. In certain embodiments, R2 is substituted ethyl. In certain embodiments, R2 is ammonium-substituted ethyl.In certain embodiments, Ri and R2 together with the atoms to which they are attached form a six-membered heterocyclyl. In certain embodiments, Ri and R2 together with the atoms to which they are attached formIn certain embodiments, the compound is selected fromIn certain embodiments, the reactive warhead comprises N3. In certain embodiments, the reactive warhead comprises -N(H)C(0)CH2l, -N(H)C(O)CH2C1, a phenol, an aniline, an azide an alkyne, or a tetrazine.In certain embodiments, the compound comprises one or more unnatural isotopes of a carbon, a hydrogen, a nitrogen, or an oxygen atom.

[0054] The disclosure relates compounds of Formula (I) or (II):whereinA is a reactive warhead;Li and L2 are independently linkers or absent,R is independently a substituent; n is independently an integer from 0 to 5; n’ is an integer from 0 to 5; and n” is independently an integer from 0 to 3; or salt thereof.Li can be -CH2. Li can bewhich can be substituted by one or more substituents. Li can hich can be substituted by one or more substituents. Li can ich can be substituted by one or more substituents. Li can which can be substituted by one or more substituents. Li can hich can be substituted by one or more substituents. Li canch can be substituted by one or more substituents., y .embodiments, Li can be -(CEhV- In some embodiments, Li can beL2 can be -CH2. L2 can bewhich can be substituted by one or more substituents. L2 can hich can be substituted by one or more substituents. L2 can hich can be substituted by one or more substituents. L2 can which can be substituted by one or more substituents. L2 can hich can be substituted by one or more substituents. L2 canich can be substituted by one or more substituents.embodiments, L2 can be -CH2CH2-. In some embodiments, L2 can be absent.

[0055] n can be 0. n can be 1. n can be 2. n can be 3. n can be 4. n can be 5.

[0056] n’ can be 0. n’ can be 1. n’ can be 2. n’ can be 3. n’ can be 4. n’ can be 5.

[0057] n” can be 0. n” can be 1. n” can be 2. n” can be 3.

[0058] The reactive warhead can comprise N3

[0059] The reactive warhead can comprise -N(H)C(O)CH2l. The reactive warhead can comprise -N(H)C(O)CH2C1. The reactive warhead can comprise an alkyne. The reactive warhead can comprise a tetrazine.

[0060] The reactive warhead can be selected from Fig. 17.

[0061] The compound of Formula (I) or (II) can be

[0062] The compound of Formula (I) or (II) can bewherein A is a reactive warhead as described herein.

[0063] The compound of Formula (I) or (II) canwherein A is a reactive warhead as described herein.

[0064] The compound of Formula (I) or (II) can bereactive warhead as described herein.

[0065] The disclosure further relates to compounds selected fromwherein A is a reactive warhead as described herein.

[0066] The disclosure further relates to a compound of formulawherein A is a reactive warhead as described herein.

[0067] The disclosure further relates to compounds selected fromwherein A is a reactive warhead as described herein.

[0068] Any suitable reactive warhead may be used in the compounds described herein.Many known reactive warhead moieties are listed below. Those of skill in the art will be able to select an appropriate reactive warhead without undue experimentation.

[0069] In certain preferred embodiments the reactive warhead is wherein Y = NH, CH2, O; X is Cl, Br, I, OTs, OMs, OTf, OAryl; A and B = H, alkyl, heteroalkyl, haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0070] In certain embodiments the reactive warhead is , whereinY = NH, CH2, or O; A, B, and C = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, or C(O)OR.

[0071] In certain embodiments the reactive warhead is , whereinY = NH, CH2, or 0; A, B, and R = independently H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.RX..-

[0072] In certain embodiments the reactive warhead is , wherein Y =NH, CH2, O; R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0073] In certain embodiments the reactive warhead is , wherein Y = NH,CH2, O; X = N O; R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0074] In certain embodiments the reactive warhead is

[0075] In certain embodiments the reactive warhead is

[0076] In certain embodiments the reactive warhead is, whereinY = NH, CH2, O; Rn= H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0077] In certain embodiments the reactive warhead is , wherein Y= NH, CH2, O; R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0078] In certain embodiments the reactive warhead is wherein Y = NH, CH2, O; R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0079] In certain embodiments the reactive warhead is, wherein Y =NH, CH2, O; A, B = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0080] In certain embodiments the reactive warhead is, wherein Y =NH, CH2, O; A, B = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0081] In certain embodiments the reactive warhead is , whereinR = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0082] In certain embodiments the reactive warhead is wherein R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0083] In certain embodiments the reactive warhead is , wherein R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0084] In certain embodiments the reactive warhead is, wherein M,N, O, P = C, N; X = Cl, Br, I, OTs, OMs, OTf; R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0085] In certain embodiments the reactive warhead is , wherein R =H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0086] In certain embodiments the reactive warhead is , wherein X = N, O.

[0087] In certain embodiments the reactive warhead is , wherein R= H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0088] In certain embodiments the reactive warhead is , wherein R =H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0089] In certain embodiments the reactive warhead is , wherein R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0090] In certain embodiments the reactive warhead is

[0091] In certain embodiments the reactive warhead is , wherein Y isNH, CH2, O.

[0092] In certain embodiments the reactive warhead is4*

[0093] In certain embodiments the reactive warhead is

[0094] In certain embodiments the reactive warhead is wherein R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0095] In certain embodiments the reactive warhead is , whereinR = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0096] In certain embodiments the reactive warhead is , wherein R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0097] In certain embodiments the reactive warhead is , wherein R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0098] In certain embodiments the reactive warhead is , wherein R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0099] In certain embodiments the reactive warhead is, wherein Y = NH,CH2, or O; R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0100] In certain embodiments the reactive warhead is , whereinR = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, or C(O)OR, Y is NH, CH2, O.

[0101] In certain embodiments the reactive warhead is wherein R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0102] In certain embodiments the reactive warhead is, wherein R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0103] In certain preferred embodiments the reactive warhead is, wherein R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0104] In certain preferred embodiments the reactive warhead is, wherein R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, SCh', C(O)OR.

[0105] In certain embodiments the reactive warhead is, wherein R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0106] In certain embodiments the reactive warhead is wherein X = N, CH; R = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, C(O)OR.

[0107] In certain embodiments the reactive warhead is wherein X = Cl, F, Br, NO2, CN.

[0108] In certain embodiments the reactive warhead is, wherein R =H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, or C(O)OR; Y = NH, CH2, O.

[0109] In certain embodiments the reactive warhead is , whereinR = H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, or C(O)OR; Y = NH, CH2, O.

[0110] In certain embodiments the reactive warhead is, wherein X = Cl,Br, I, OTs, OMs, OTf, OAryl.

[0111] In certain embodiments, the reactive warhead i

[0112] In certain embodiments the reactive warhead iwhereinR is H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, or C(O)OR, provided that R is not at the positions ortho to the hydroxyl group.

[0113] In certain embodiments the reactive warhead iwhereinR is H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo, NO2, amine, amide, C(O)OH, or C(O)OR provided that R is not at the positions ortho to the amino group.

[0114] In certain embodiments the reactive warhead iwherein R is H, alkyl, heteroalkyl haloalkyl, hydroxy, alkoxy, aryl, heteroaryl, alkenyl, alkynyl, halo,NO2, amine, amide, C(O)OH, or C(O)OR, provided that R is not at the positions ortho to the azide group.

[0115] In certain preferred embodiments, the reactive warhead

[0116] In the above definitions of A, — denotes the point of attachment to compounds of Formula (I), (II), (III), and (IV). In certain embodiments, n is an integer from 0 to 4.

[0117] In preferred embodiments, the reactive warhead is

[0118] In especially preferred embodiments, the reactive warhead i

[0119] In especially preferred embodiments, the reactive warhead is

[0120] In especially preferred embodiments, the reactive warhead is

[0121] In especially preferred embodiments, the reactive warhead i

[0122] Any disclosed compound can comprise one or more isotope of a carbon, a hydrogen, a nitrogen, or an oxygen atom.

[0123] All diastereomers and enantiomers of the compounds of the formulae (I)-(II) are contemplated herein.

[0124] Also contemplated herein are isotopomers, which are compounds where one or more atoms in the compound has been replaced with an isotope of that atom. Thus, for example, the disclosure relates to compounds wherein one or more hydrogen atoms is replaced with a deuterium or wherein a12C atom is replaced with a13C atom.

[0125] Compounds of the disclosure can be synthesized by any method well known in the art.Methods of Use

[0126] The disclosure relates to a gas phase in spectrometer enrichment which leverages the effect of modification-induced alteration of peptide ion mobility. This platform does not suffer from endogenous biotin background, entails fewer steps, and minimizes material losses.

[0127] The disclosure relates to a general approach, termed timSHIFT labeling, for reactive amino acid profiling which exploits differential ion mobility in the gas phase to enrich label-modified peptides for analysis in a tandem IM / MS mass spectrometer.

[0128] The disclosure also relates to an aerodynamic (small collisional cross-section), dicationic, cysteine-reactive reagent which dramatically increases the ion mobility of labeledpeptides, spatially separating them from unlabeled peptides and enabling their selective fragmentation and sequencing (Fig. IB).

[0129] In certain aspects, the present disclosure provides a method of modulating ion mobility for gas phase enrichment of a modified peptide comprising: covalently modifying a peptide to generate a modified peptide; and spatially separating the modified peptide from an unlabeled peptide using ion mobility spectrometry in the gas phase.

[0130] In certain aspects, the present disclosure provides a method of selectively measuring a modified peptide comprising: covalently modifying a peptide to generate a modified peptide; and spatially separating the modified peptide from an unlabeled peptide using ion mobility spectrometry in the gas phase.

[0131] In certain embodiments, covalently modifying a peptide comprises reacting the peptide with a cationic (e.g., polycationic) residue-reactive agent. In certain embodiments, the cationic residue reactive agent has a small collisional cross-section.

[0132] In certain embodiments, covalently modifying the peptide comprises reacting the peptide with a residue-reactive agent having the structure of a compound of any one of claims 1-17.

[0133] In certain embodiments, covalently modifying the peptide comprises covalently modifying the peptide to alter its 1 / ko and m / z physical properties, rendering it different from a 2+ and 1+ charged unmodified peptide.

[0134] In certain embodiments, the method further comprising selectively fragmenting the modified peptide to generate reporter ions.

[0135] In certain embodiments, the method further comprising sequencing the modified peptide in a tandem IM / MS mass spectrometer. In certain embodiments, sequencing the modified peptide in a tandem IM / MS mass spectrometer comprises data-dependent acquisition or data-independent acquisition.

[0136] In certain embodiments, the method further comprising reacting the modified and the unlabeled peptide with a gas-phase base. In certain embodiments, the gas-phase base has a proton affinity of at least 230 kcal / mol. In certain embodiments, the gas-phase base is selected from the group consisting of acetone, acetophenone, dimethylacetamide, 2-methylpyridine, collidine, DIPEA, and TMG. In certain preferred embodiments, the gasphase base is selected from the group consisting of collidine, DIPEA, and TMG.

[0137] In certain embodiments, covalently modifying the physical properties of a peptide comprises minimal perturbation of collisional cross-section and mass, but high charge difference to increase its ion mobility.

[0138] In certain embodiments, the residue-reactive reagent reacts with lysine residues via an amine reactive electrophile (e.g., an NHS ester or PFP ester).

[0139] In certain embodiments, the residue-reactive reagent reacts with cysteine residues via a cysteine reactive electrophile (e.g., a haloacetamide, alpha / beta unsaturated ketone, activated nitrile). In certain preferred embodiments, the residue-reactive reagent reacts with cysteine residues via an iodoacetamide.

[0140] In certain embodiments, the residue reactive agent is a dicationic, cysteinereactive reagent. In certain embodiments, the cysteine-reactive agent has a small collisional cross-section. In certain embodiments, the cysteine -reactive reagent is an innately cationic polyimidazolium shift reagent.

[0141] The disclosure also relates to a method of selectively measuring modified peptides comprising: covalently modifying peptides to alter their 1 / ko and m / z physical properties, rendering them different from those of 2+ and 1+ charged unmodified peptides, to generate modified peptides; spatially separating the modified peptides from unlabeled peptides using ion mobility spectrometry in the gas phase; selectively fragmenting the modified peptides exploiting the spatial separation; and sequencing the modified peptides in a tandem IM / MS mass spectrometer, either via data-dependent acquisition or data-independent acquisition.

[0142] In data-dependent acquisition, fragmentation m / z vs 1 / ko windows can be selected to exclude unlabeled peptides from fragmentation and analysis, while enhancing selection of labeled peptides for fragmentation (Fig. 3B).

[0143] In data-independent acquisition, m / z vs 1 / ko scan windows can be used for enhanced sequencing of spatially separated peptides.

[0144] In embodiments, covalently modifying a peptide’ physical properties can comprise minimal perturbation of collisional cross-section and mass, but high charge difference to increase its ion mobility.

[0145] In embodiments covalently modifying a peptide’ physical properties can comprise reacting the peptide with an aerodynamic (small collisional cross-section), (poly)cationic, residue-reactive reagent.

[0146] Residues can include the side-chain of cysteine, lysine, tyrosine, serine, threonine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, arginine. Residues can be modified by reacting intact proteins and subsequently subject them to tryptic digestion or on peptides.

[0147] Covalent modification of an amino-acid side chain of the peptide can occur via two-step click chemistry (for example using copper-catalyzed alkyne-azide cycloaddition, strain-release azide-alkyne cycloaddition, tetrazine- alkene reaction), whereby the amino-acid side chain is selectively ligated using a residue-reactive reagent, followed by click chemistry with the ion-mobility modifying reagent.

[0148] In embodiments, residue-reactive reagent can react with lysines via an NHS ester, PFP ester, or analogous amine reactive electrophile.

[0149] In embodiments, residue-reactive reagent can react with cysteines via a haloacetamide, alpha / beta unsaturated ketone, activated nitrile, or analogous cysteine reactive electrophile.

[0150] In embodiments, residue-reactive reagent can react with cysteines via an iodoacetamide.

[0151] In embodiments, covalently modifying a peptide’ physical properties can comprise reacting the peptide to an aerodynamic (small collisional cross-section), dicationic, cysteine-reactive reagent.

[0152] In embodiments, the cysteine-reactive reagent is an innately cationic polyimidazolium shift reagent.

[0153] In embodiments, the residue -reactive reagent is a compound of the disclosure.

[0154] The disclosure relates to a method of quantifying modifying modified peptides by relative intensity ratios at MS2 level of reporter ions generated by fragmentation of the modification, which exploits isobaric reagents wherein the modification containing contains different isotopes of the compounds of the disclosure to achieve multiplexed quantification.

[0155] Each sample containing peptides to be quantified (channel) is reacted with a reagent having the same total mass (isobaric), but different isotopic distribution within the molecule, and the samples are subsequently pooled.

[0156] MS2 level fragmentation causes loss of mass-balance components of the modification and generates unique reporter ions for quantification of each specific channel. Residues can include the side-chain of cysteine, lysine, tyrosine, serine, threonine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, arginine, and ligation methods can occur via two-step click chemistry (for example, copper-catalyzed alkyne-azide cycloaddition, strain-release azide-alkyne cycloaddition, tetrazine-alkene reaction) , whereby the amino-acid side chain is selectively ligated using a residue -reactive reagent, followed by click chemistry with the ion-mobility modifying reagent.

[0157] The method disclosed can be applied to the reactivity study of specific aminoacidic residues towards covalent molecules, termed activity-based protein profiling; to the study of protein-protein interactions via proximity labeling; or to the mapping of binding site of proteins upon treatment with molecules.

[0158] Hence, the disclosure relates to a method of quantifying modified peptides comprising: fragmenting the modified peptides to generate reporter ions; determining the relative intensity ratios of the reporter ions generated by fragmentation of the modification, wherein the modified peptide comprises different isotopes of a compound of the disclosure to achieve multiplexed quantification.

[0159] The disclosure also relates method of modulating ion mobility for gas phase enrichment of modified peptides comprising: covalently modifying a peptide’ physical properties to a previously unoccupied regime of 1 / ko and m / z space to generate modified peptides; spatially separating the modified peptides from unlabeled peptides; selective fragmentating the modified peptides; and sequencing the modified peptides in a tandem IM / MS mass spectrometer.Certain Definitions

[0160] As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an agent” includes mixtures of two or more such agents, reference to “the component” includes mixtures of two or more such components, and the like.

[0161] The term “about” when immediately preceding a numerical value means a range (e.g., plus or minus 10% of that value). For example, “about 50” can mean 45 to 55, “about 25,000” can mean 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example, in a list of numerical values such as “about 49, about 50, about 55, ...”, “about 50” means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 52.5. Furthermore, the phrases “less than about” a value or “greater than about” a value should be understood in view of the definition of the term “about” provided herein. Similarly, the term “about” when preceding a series of numerical values or a range of values (e.g., “about 10, 20, 30” or “about 10-30”) refers, respectively to all values in the series, or the endpoints of the range.

[0162] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, to A only (optionally including elements other than B); or to B only (optionally including elements other than A); or yet, to both A and B (optionally including other elements); etc.

[0163] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of anumber or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0164] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); or to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); or yet, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0165] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0166] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to.

[0167] Various compounds contained in compositions of the present disclosure may exist in particular geometric or stereoisomeric forms. In addition, polymers of the present disclosure may also be optically active. The present disclosure contemplates all such compounds, including cis- and trans-isomers, R- and S -enantiomers, diastereomers, (d)-isomers, (l)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the disclosure. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this disclosure.

[0168] If, for instance, a particular enantiomer of compound of the present disclosure is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

[0169] Structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds produced by the replacement of a hydrogen with deuterium or tritium, or of a carbon with a13C- or14C- enriched carbon are within the scope of this disclosure.

[0170] An aliphatic chain comprises the classes of alkyl, alkenyl and alkynyl defined below. A straight aliphatic chain is limited to unbranched carbon chain moieties. As used herein, the term “aliphatic group” refers to a straight chain, branched chain, or cyclic aliphatic hydrocarbon group and includes saturated and unsaturated aliphatic groups, such as an alkyl group, an alkenyl group, or an alkynyl group.

[0171] “Alkyl” refers to a fully saturated cyclic or acyclic, branched or unbranched carbon chain moiety having the number of carbon atoms specified, or up to 30 carbon atoms if no specification is made. For example, alkyl of 1 to 8 carbon atoms refers to moieties such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl, and those moieties which are positional isomers of these moieties. Alkyl of 10 to 30 carbon atoms includes decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, trocontanyl, pentacosenyl, and hexacosenyl. A straight chain or branched chain alkyl can have 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), or 20 or fewer. Alkyl groups may be substituted or unsubstituted.

[0172] As used herein, the term “alkylene” refers to an alkyl group having the specified number of carbons, for example from 2 to 12 carbon atoms, that contains two points of attachment to the rest of the compound on its longest carbon chain. Non-limiting examples of alkylene groups include methylene -(CH2)-, ethylene -(CH2CH2)-, n-propylene -(CH2CH2CH2)-,isopropylene -(CH2CH(CH3))-, and the like. Alkylene groups can be cyclic or acyclic, branched or unbranched carbon chain moiety, and may be optionally substituted with one or more substituents.

[0173] "Cycloalkyl" means mono- or bicyclic or bridged or spirocyclic, or polycyclic saturated carbocyclic rings, each having from 3 to 12 carbon atoms. In various aspects, cycloalkyls have from 3-10 carbon atoms in their ring structure, or 3-6 carbons in the ring structure.Cycloalkyl groups may be substituted or unsubstituted.

[0174] Unless the number of carbons is otherwise specified, “lower alkyl,” as used herein, means an alkyl group, as defined above, but having from one to ten carbons, or from one to six carbon atoms in its backbone structure such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. A substituent designated herein as alkyl can be a lower alkyl.

[0175] “Alkenyl” refers to any cyclic or acyclic, branched or unbranched unsaturated carbon chain moiety having the number of carbon atoms specified, or up to 26 carbon atoms if no limitation on the number of carbon atoms is specified; and having one or more double bonds in the moiety. Alkenyl of 6 to 26 carbon atoms is exemplified by hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosoenyl, docosenyl, tricosenyl, and tetracosenyl, in their various isomeric forms, where the unsaturated bond(s) can be located anywhere in the moiety and can have either the (Z) or the (E) configuration about the double bond(s).

[0176] “Alkynyl” refers to hydrocarbyl moieties of the scope of alkenyl but having one or more triple bonds in the moiety.

[0177] The term “alkylthio” refers to an alkyl group, as defined above, having a sulfur moiety attached thereto. The “alkylthio” moiety can be represented by one of -(S)-alkyl, -(S)- alkenyl, -(S)-alkynyl, and -(S)-(CH2)m-Ri, wherein m and Ri are defined below. Representative alkylthio groups include methylthio, ethylthio, and the like. The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group, as defined below, having an oxygen moiety attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like. An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, -0-(CH2)m-Rio, where m and Rio are described below.

[0178] The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the formulae:

[0179] wherein Rn and R12 each independently represent a hydrogen, an alkyl, an alkenyl, -(CH2)m-Rio, or Rn and R12 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; Rio represents an alkenyl, aryl, cycloalkyl, a cycloalkenyl, a heterocyclyl, or a polycyclyl; and m is zero or an integer in the range of 1 to 8. In some instances, only one of Rn or R12 can be a carbonyl, e.g., Rn, R12, and the nitrogen together do not form an imide. Rn and R12 each independently can represent a hydrogen, an alkyl, an alkenyl, or -(CH2)m-Rio. Thus, the term “alkylamine” as used herein means an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of Rn and R12 is an alkyl group. An amino group or an alkylamine is basic, meaning it has a conjugate acid with a pKa > 7.00, i.e., the protonated forms of these functional groups have pKas relative to water above about 7.00.

[0180] The term “quaternary ammonium” refers to a cationic group having the formula - NR3+or -NR2+- where each R group, independently, is hydrogen or a substituted or unsubstituted alkyl, aryl, or aralkyl. In certain preferred embodiments, all R are alkyl. As will be recognized by those of skill in the art, quaternary ammoniums that do not include protons will resist deprotonating agents.

[0181] The term “iminium” refers to a cationic group having the formula =NR2+or =NRi+- where each R group, independently, is hydrogen or a substituted or unsubstituted alkyl, aryl, or aralkyl. In certain preferred embodiments, all R are alkyl. As will be recognized by those of skill in the art, iminiums that do not include protons will resist deprotonating agents.

[0182] The term “amide”, as used herein, refers to a group

[0183] wherein each R13 independently represent a hydrogen or hydrocarbyl group, or two R13 are taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0184] The term “aryl” as used herein includes 3- to 12-membered substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon (i.e., carbocyclic aryl) or where one or more atoms are heteroatoms (i.e., heteroaryl). In various aspects, aryl groups include 5- to 12-membered rings, or 6- to 10-membered rings 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. Carbocyclic aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like. Heteroaryl groups include substituted or unsubstituted aromatic 3- to 12- membered ring structures, 5- to 12-membered rings, or 5- to 10-membered rings, whose ring structures include one to four heteroatoms. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl and heteroaryl can be monocyclic, bicyclic, or polycyclic. Each instance of an aryl group may be independently optionally substituted, i.e., unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl") with one or more substituents; e.g., for instance from 1 to 5 substituents, 1 to 4 substituents, 1 to 3 substituents, 1 to 2 substituents or just 1 substituent. The aromatic ring may be substituted at one or more ring positions with one or more substituents, such as halogen, azide, alkyl, aryl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or hetero aromatic moieties, fluoroalkyl (such as trifluromethyl), cyano, or the like. For example, the aryl group can be an unsubstituted C5-C12 aryl or the aryl group can be a substituted C5-C10 aryl.

[0185] The term “halo”, “halide”, or “halogen” as used herein means halogen and includes, for example, and without being limited thereto, fluoro, chloro, bromo, iodo and the like, in both radioactive and non-radioactive forms. Halo can be selected from the group consisting of fluoro, chloro and bromo.

[0186] The terms “heterocyclyl” or “heterocyclic group” refer to 3- to 12-membered ring structures, 5- to 12-membered rings, or 5- to 10-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can be monocyclic, bicyclic, spirocyclic, or polycyclic. Heterocycles can be saturated or unsaturated. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine,isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, pheno thiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, dimethylpiperazine, and the like. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aryl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, sulfamoyl, sulfinyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, and the like.

[0187] A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase “heterocyclyl group” includes fused ring species including those that include fused aromatic and non-aromatic groups. Representative heterocyclyl groups include, but are not limited to, piperidynyl, piperazinyl, morpholinyl, furanyl, pyrrolidinyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, thiophenyl, tetrahydrofuranyl, pyrrolyl, oxazolyl, imidazolyl, triazyolyl, tetrazolyl, benzoxazolinyl, and benzimidazolinyl groups. For example, heterocyclyl groups include, without limitation:(C6-C2o)aryl or an amine protecting group (e.g., a t-butyloxy carbonyl group) and wherein the heterocyclyl group can be substituted or unsubstituted. A nitrogen-containing heterocyclyl group is a heterocyclyl group containing a nitrogen atom as an atom in the ring. In some embodiments, the heterocyclyl is other than thiophene or substituted thiophene. In some embodiments, the heterocyclyl is other than furan or substituted furan.

[0188] The term “carbonyl” is art-recognized and includes such moieties as can be represented by the formula:

[0189] wherein X’ is a bond or represents an oxygen, a nitrogen, or a sulfur, and Ru represents a hydrogen, an alkyl, an alkenyl, -(CH2)m-Rio or a pharmaceutically acceptable salt, R15 represents a hydrogen, an alkyl, an alkenyl or -(CH2)m-Rio, where m and Rio are as defined above. Where X’ is an oxygen and Ru or R15 is not hydrogen, the formula represents an “ester.” WhereX’ is an oxygen, and Ru is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when R is a hydrogen, the formula represents a “carboxylic acid”. Where X’ is an oxygen, and R15 is a hydrogen, the formula represents a “formate.” In general, where the oxygen atom of the above formula is replaced by a sulfur, the formula represents a “thiocarbonyl” group. Where X’ is a sulfur and Ru or R15 is not hydrogen, the formula represents a “thioester” group. Where X’ is a sulfur and Ru is a hydrogen, the formula represents a “thiocarboxylic acid” group. Where X’ is a sulfur and R15 is a hydrogen, the formula represents a “thioformate” group.On the other hand, where X’ is a bond, and R is not hydrogen, the above formula represents a “ketone” group. Where X’ is a bond, and R is a hydrogen, the above formula represents an “aldehyde” group.

[0190] The term “amido” as used herein refers to a group having the formula C(O)NRR, wherein R is defined herein and can each independently be, e.g., hydrogen, alkyl, aryl or each R, together with the nitrogen atom to which they are attached, form a heterocyclyl group.

[0191] As used herein, the term “nitro” means -NO2; the term “halogen” designates - F, -Cl, -Br, or -I; the term “sulfhydryl” means -SH; the term “hydroxyl” means -OH; the term “sulfonyl” means -SO2-; the term “azido” means -N3; the term “cyano” means -CN; the term “isocyanato” means -NCO; the term “thiocyanato” means -SCN; the term “isothiocyanate” means -NCS; and the term “cyanato” means -OCN.

[0192] As used herein, the definition of each expression, e.g., alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

[0193] 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 disclosure, 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 alkoxy carbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxy, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aryl, or an aromatic or hetero aromatic moiety. The substituents on substituted alkyls can be selected from Cl -6 alkyl, C3-6 cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. The substituents on substituted alkyls can be selected from fluoro, carbonyl, cyano, or hydroxyl. It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. Unless specifically stated as “unsubstituted,” references to chemical moieties herein are understood to include substituted variants. For example, reference to an “aryl” group or moiety implicitly includes both substituted and unsubstituted variants.

[0194] The term “substituted” as used herein also refers to a group that is substituted with one or more groups including, but not limited to, the following groups: halogen (e.g., F, Cl, Br, and I), R, OR, ROH (e.g., CH2OH), OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, methylenedioxy, ethylenedioxy, (C3-C2o)heteroaryl, N(R)2, Si(R)3, SR, SOR, SO2R, SO2N(R)2, SO3R, P(O)(OR)2, OP(O)(OR)2, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, C(O)N(R)OH, OC(O)N(R)2, C(S)N(R)2, (CH2)O-2N(R)C(0)R, (CH2)O-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, or C(=NOR)R wherein R can be hydrogen, (Ci-C2o)alkyl, (C6-C2o)aryl, heterocyclyl or polyalkylene oxide groups, such as polyalkylene oxide groups of the formula-(CH2CH2O)f-R-OR, -(CH2CH2CH2O)g-R-OR, -(CH2CH2O)f(CH2CH2CH2O)g-R-OReach of which can, in turn, be substituted or unsubstituted and wherein f and g are each independently an integer from 1 to 50 (e.g., 1 to 10, 1 to 5, 1 to 3 or 2 to 5). Substituted also includes a group that is substituted with one or more groups including, but not limited to, the following groups: fluoro, chloro, bromo, iodo, amino, amido, alkyl, hydroxy, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylamino sulfonyl, sulfonic acid, carboxylic acid, dialkylamino and dialkylamido. Where there are two or more adjacent substituents, the substituents can be linked to form a carbocyclic or heterocyclic ring. Such adjacent groups can have a vicinal or germinal relationship, or they can be adjacent on a ring in, e.g., an ortho-arrangement. Each instance of substituted is understood to be independent. For example, a substituted aryl can be substituted with bromo and a substituted heterocycle on the same compound can be substituted with alkyl. It is envisaged that a substituted group can be substituted with one or more non-fluoro groups. As another example, a substituted group can be substituted with one or more non-cyano groups. As another example, a substituted group can be substituted with one or more groups other than haloalkyl. As yet another example, a substituted group can be substituted with one or more groups other than tert-butyl. As yet a further example, a substituted group can be substituted with one or more groups other than trifluoromethyl. As yet even further examples, a substituted group can be substituted with one or more groups other than nitro, other than methyl, other than methoxymethyl, other than dialkylaminosulfonyl, other than bromo, other than chloro, other than amido, other than halo, other than benzodioxepinyl, other than polycyclic heterocyclyl, other than polycyclic substituted aryl, other than methoxycarbonyl, other than alkoxycarbonyl, other than thiophenyl, or other than nitrophenyl, or groups meeting a combination of such descriptions. Further, substituted is also understood to include fluoro, cyano, haloalkyl, tert-butyl, trifluoromethyl, nitro, methyl, methoxymethyl, dialkylaminosulfonyl, bromo, chloro, amido, halo, benzodioxepinyl, polycyclic heterocyclyl, polycyclic substituted aryl, methoxycarbonyl, alkoxycarbonyl, thiophenyl, and nitrophenyl groups.

[0195] Structures containing a substituent bonded in between multiple carbon atoms denotes a bond to any of the carbons in the chain. For example,

[0196] For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.EXAMPLES

[0197] 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.

[0198] Development of the timSHIFT platform. In an ion mobility spectrometer, ions are separated according to their mobility in an inert gas. As modeled by the Mason-Schamp equation, the only determinants of ion mobility under isothermal conditions are the ion’s charge (z), reduced mass u), and collisional cross-section (a): K =z na

[0199] After electrospray ionization (ESI), tryptic peptide ions are predominantly in the 1+ and 2+ charge state because trypsin selectively cleaves at the carboxyl side of the most abundant basic amino acids, lysine (K) and arginine (R), leaving each peptide one K / R AA and the N-terminus. Since longer peptides have both higher mass and larger collisional crosssections, there is a tight and approximately linear correlation between 1 / ko and m / z.nIn a configuration used in the timsTOF family of mass spectrometers, which were used to collect all mass spectrometry data, trapped ion mobility spectroscopy (TIMS)12 13devices separate ions by accelerating them against a flow of gas in an electric field gradient,12operating at high ion mobility resolution and affording flexible control of ion mobility accumulation windows and fragmentation selection rules (PASEF).12

[0200] We hypothesized that covalently modifying a peptide with highly charged moiety could sufficiently increase its ion mobility to clearly separate it from unmodified, lower ion mobility peptides, effectively shifting its physical properties to a previously unoccupied regime of 1 / ko and m / z space. Because the increase of fl and a cancel the effect of high z, we identified minimal perturbation of collisional cross-section and mass, but high charge difference, as key design principles for maximizing the modification-induced shift effect.14To facilitate rapid testing, we used a two-step ligation process: A-431 cell lysates were reduced and alkylated with N-(hex-5-yn-l-yl)-2-iodoacetamide, followed by treatment with azide-functionalized shift reagent candidates under copper alkyne-azide cycloaddition (CuAAC). Remaining cysteines were alkylated with iodoacetamide, and standard protocols for SP3 precipitation, on-bead tryptic digest, and C18 desalting were used to generate tryptic peptides for IM / MS proteomic analysis in data-dependent acquisition mode,15allowing structure-activity relationship assessment of reagent impact on peptide 1 / ko and m / z values (Fig. 2A).

[0201] We synthesized azide-functionalized derivatives of tris(2-aminoethyl)amine (TREN, 1 and 3), poly(ethylpiperazine) (2, 4, and 6-8), and 1,4,7, 10-tetraazacyclododecane (cyclen, 5) based on the idea that multiple basic nitrogen atoms could undergo extensive protonation under chromatographic conditions. We compared the effect of covalent modification by candidate reagents on peptide 1 / ko vs. m / z properties (Fig. 2B, left) by comparing their modified and unmodified values (Fig. 7-8), and the average effect on m / z (average Am / z), charge (average Az), and ion mobility (average / sl / ko) (Fig. 2B, right). Alkyne modification had a negligible impact on the three properties (average Dsm / z: +0.6; average Az: 0.10; average Al / ko: -0.01 Vs cm'2). Attaching probes via CuAAC with increasing numbers of basic nitrogen atoms modestly increased charge,9but inconsistently with dominant polycationic charge state in the 0.1% formic acid buffer present at ionization (Fig. 2B, right). We believe that reversible protonation during ESI to be the main cause of this observation. Despite the significant shift (average Dsm / z: -124 + 25; average Az: 1.07 + 0.20; average M / ko'. -0.112 + 0.018 Vs cm'2for 1-8) (Fig. 7B-H, Fig. 8A), labeled peptides showed scattered 1 / ko vs. m / z values (e.g., Fig. 2B, probe 5) and were poorly separated from unmodified peptides due to large residual 2+ion populations and significant collisional cross- section increase, which in turn correlates with mass inflation (average Am 151 + 37 A for 1-8, Fig. 9).

[0202] Based on these data, we next synthesized a range of innately cationic polyimidazolium shift reagent candidates. These do not undergo protonation-deprotonation equilibria, enforcing a minimum modified peptide charge and enable greatly improved discrimination between modified and unmodified peptides in comparison with polyamine o O scaffolds, and do not suffer from increased collisional cross-section penalty (Am 140 ± 24 A for 9-13) compared to polyamines 1-8. Ethyl bis-(imidazolium) 9 and 2,2’-biimidazolium 10 scaffolds afforded a unimodal distribution of shifted peptide 1 / ko and m / z values highly distinct from those of unmodified peptides (Am / z: -201; Az: 1.38; Al / ko: -0.219 Vs cm'2for 9, t±m / z'. -209; Az: 1.46; kl / ko'. -0.223 Vs cm'2for 10, Fig. 8B-C). The addition of an extra neutral ethyl-imidazole unit (11) did not foster any significant difference (Am / z: -184; Az: 1.42; Al / ko: -0.225 Vs cm'2, Fig. 8D), further establishing that the addition of protonatable sites alone does not drive significant charge increases under electrospray ionization conditions.9ATris-cationic poly-imidazolium 12 showed slightly larger m / z shift (Am / z: -251; Az: 1.70; M / ko’. -0.221 Vs cm'2, Fig. 8E), but with a less tight distribution of m / z and 1 / ko values which overlapped with endogenous peptides. Having chosen 2,2'-biimidazolium as an easily accessible and efficient ion mobility-m / z shifting moiety, we installed a PEG3 linker for optimal aqueous solubility and reactivity (13) and obtained similar performance (Am / z: -189; Az: 1.46; Al / ko: -0.212 Vs cm'2) to that shown by 10. Matched modified and unmodified peptides (Fig. 2C) confirmed that the modification imparted a significant shift effect and that 97.8% of peptides experienced a reduction in both m / z and 1 / ko .

[0203] Charge-dependent gas phase separation of timSHIFT-modified peptides enhances their direct, streamlined measurement via enriched ion fragmentation. We observed that even under optimized conditions, the CuAAC reaction did not completely conjugate alkyne modified peptides to azide-functionalized probes 1-13 and complicated sample processing. To directly map reactive cysteine residues without stepwise click chemistry, we created an innately electrophilic iodoacetamide derivative (timSHIFT-IAA, 14) based on the 2,2’-biimidazolium scaffold in 13 (Fig. 2D). In addition, we synthesized analogous monocationic (15) and neutral (16) reagents to isolate the effect of reagent’s charge on ion mobility-m-z shift. Reduced cysteine residues in the human proteome (A-431 cells) were reacted with 14, 15, and 16 (Fig. 3A) and underwent IM / MS analysis. Bis-cationic 14 afforded a large shift (Am / z: -215; Az: 1.40; A 1 / ko: -0.215 Vs cm'2, ACT = 148 A2) with few identifiable 2+ions, while monocationic 15 (Am / z: -150; Az: 0.96; Al / ko: -0.107 Vs cm'2, ACCS = 126 A2) and neutral 16 (Am / z: -136; Az: 0.86; Al / ko -0.104 Vs cm’2, A = 116 A ) showed reduced ion mobility shift and were not separated from endogenous 2 peptide ions (Fig. 10A). Comparing matched modified peptides, 14 exhibited larger ion mobility shifts lower charge 15 and 16 (Fig. 10B), with 68% and 77% of shared PSMs at both lower m / z and 1 / ko, respectively. Because 14 reacts directly with peptides and no solution phase enrichment is required, it enables streamlined sample processing: proteins are labeled, desalted with SP3 precipitation, subjected to on-bead tryptic digestion, and the digestate directly analyzed via LC / IM / MS.

[0204] Peptides containing cysteines modified by optimized reagent 14 occupy a clearly defined / / / c and m / z property space containing few unmodified peptides. Normally, peptide ions containing PTMs with unknown fragmentation spectra are sequenced and quantified in data-dependent acquisition (DDA) mode. In TIMS / MS instruments ions selected in a total IM / MS spectrum are isolated by both 1 / ko (axial separation and selective release in the TIMS) and m / z (isolated via an in-line quadrupole magnet), fragmentated in a collision cell, and fragment ion masses measured via MS. TIMS / MS can give focused proteomic sequence coverage by exclusively fragmenting ions that fall inside a polygon in 1 / ko - m / z space, and this is commonly used to specifically fragment 2+peptides and avoid wasting fragmentation cycles on 1+nonpeptidic impurities. We designed a polygon (see overlay in Fig. 3B) enriched in peptides which reacted with 14 and depleted in unmodified peptides and evaluated its ability to enhance detection of peptides containing reactive cysteines (Fig. 3C). Using 14, significant (P < 10‘4) enrichment of modified peptides (from 1109 to 2750 IDs) was observed when comparing standard and optimized fragmentation selection windows, confirming that gas phase separation via differential ion mobility can be used for enhanced reactive proteome profiling. In contrast, monocationic (15) and neutral (16) reagents lacking large ion mobility shifts did not significantly enhance detection, highlighting the importance of charge in achieving gas phase enrichment.

[0205] We next optimized reaction and HPLC analysis conditions for profiling reactive cysteines in native cell lysate using timSHIFT-IAA, the optimized fragmentation polygon, and streamlined sample processing. We found that a 30-minute chromatographic gradient was sufficient to maximize identification of reactive cysteines, with gas phase enrichment observed under all conditions, and reacting the proteome with 1.0 mM 14 for one hour was sufficient to optimize reactive cysteine capture (Fig. 10C— 11). Overall, sampleprocessing from cell treatment to data acquisition takes one day, with a per sample MS analysis throughput of 40 samples per day.

[0206] Comparing gas phase enrichment with timSHIFT and liquid phase enrichment with desthiobiotin / streptavidin. We next compared gas phase enrichment with 14 with liquid phase enrichment with desthiobiotin / streptavidin16at different proteome input amounts and injection volumes (Fig. 4 and 12). Cell lysate from Ramos cells underwent reactive cysteine labeling with either 14 or DTB-IAA. Reductive alkylation was followed by dilution to achieve the desired protein input. SP3 precipitation using carboxylate- functionalized beads was followed by on-bead tryptic digestion.15At this stage, samples treated with 14 were subjected to proteomic analysis with the optimized fragmentation polygon, and a set of samples treated with DTB-IAA were analyzed using a standard polygon. One set of samples treated with DTB-IAA was enriched using streptavidin-coated magnetic beads and a wash / elution protocol developed by Gygi et al.,16followed by analysis using a standard polygon. Each sample was reconstituted to the same volume prior to injection.

[0207] While timSHIFT labeling and gas phase enrichment afforded high sensitivity in reactive cysteine quantitation down to 5 pg of reacted lysate (3000-2700 peptide IDs), DTB-IAA labeling and streptavidin enrichment had surprisingly low sensitivity in this range (<200 peptide IDs). Notably, fewer DTB-modified peptide IDs were observed after enrichment in all cases and ID count was highly sensitive to input protein amount, reflecting nonspecific losses during sample processing and handling. To achieve comparable profiling depth, the DTB / streptavidin protocol required 25x more protein input (500 pg vs. 20 pg), highlighting the improved sensitivity and reduced sample handling enabled by gas-phase timSHIFT enrichment.

[0208] Small molecule-cysteine reactivity profiling via gas phase enrichment.Based on the high sensitivity, sample processing throughput, and sample-to- sample quantitation consistency enabled by timSHIFT-IAA labeling, we tested whether it could be used as a drop-in replacement for desthiobiotin-IAA in activity-based profiling small of molecule reactivity with the cysteinome. We evaluated its performance in three application spaces: (1) accurately profiling the single-cysteine reactivity of a known, selective small molecule, (2) high throughput cysteine reactivity profiling of 31 electrophilic molecularfragments in a 96-well format, and (3) measuring the impact of oxidative stressors on cysteines.

[0209] We used sulfopin, an irreversible inhibitor of Pinl known to specifically react with a single cysteine site in the proteome developed by Gray et al.,11as a selective cysteinereactive small molecule to test the applicability of timSHIFT labeling / gas phase peptide enrichment in traditional activity based protein profiling workflows. Two million live Ramos cells were treated with sulfopin (1-100 pM) for 1 hour, lysed, treated with 14 for 1 hour, and then subjected to our streamlined processing and proteomics protocol (Fig. 5A).15We mapped 7,310 reactive cysteines, with Pinl-C113 as the sole cysteine significantly targeted by sulfopin at 10 pM (Fig. 5B). In accordance to Gygi et al.,16we could detect additional Pinl-C57 targeting (1 pM, undetectable peptide at 10-100 pM), and previously elusive16 17liganded catalytic MGMT-C145 was detected at 100 pM concentration. Inspired by the high sensitivity of timSHIFT labeling, we used selective sulfopin reactivity to explore the useful detection limits of gas phase enrichment. Using data independent analysis powered by a small spectral library containing -24,000 peptides modified by 14 and -74,000 unmodified, a longer chromatographic gradient, and lower flow rates, we were able to measure selective engagement of Pinl-C113 among -900 cysteine sites using just 20,000 cells in each replicate — a number expected to yield just -2 pg of initial protein input. This miniaturization makes timSHIFT amenable to the study of rare cell phenotypes, where a limited number of (sorted) cells is available. timSHIFT labeling could map the targeting of exportin 1 (XPO1) C528 by leptomycin B, a Streptomyces metabolite (1 pM, Fig. 13A).18

[0210] To evaluate the performance of timSHIFT labeling in high throughput applications, we profiled the reactivity of a panel of 31 electrophilic fragment molecules with the cysteinome. The molecules were featuring reactive chloroacetamide (Fl-15, F19, F21-22, F29-31), acrylamide (F16-17, F25-27), cyanoacrylamide (F23-24), a,P- unsaturated hydrazone (F18), a,P-unsaturated ketone (F20, F28), and a plethora of carbocyclic and heterocyclic (e.g., pyridine, piperazine, 1,3,4-triazole, benzothiazole) cores (Fig. 13B). We designed the library to cover an array of physico-chemical properties i.e., molecular weight, cLogP, hydrogen-bond donor / acceptor properties (Fig. 6a). All processing steps were performed in 96-well plate format: compound treatment, reduction / alkylation, SP3 desalting, tryptic digestion, offline C18 desalting, and 40 sample per day LC / IM / MS analysis, with 500 ng of tryptic peptides were used per analysis.

[0211] By combining data from all 96 samples, a total of 8,286 reactive cysteines (for a total of 9,843 peptides containing reactive cysteines) were quantified. We assessed their reactivity with the small fragment covalent library (Fig. 14-15). A broad array of cysteine engagement could be observed (Fig. 6B), but chloroacetamides were overall more reactive and less selective than Michael acceptors (e.g., Fig. 6a, compound F6 vs. F16). Catalytic residues in disulfide isomerases, glutathione transferases (i.e., PDIAs, TXNDs, GSTP1, GSTO1, Fig. 16d), and zinc fingers (i.e., ZFAN6, CHD4). RNA-binding proteins (i.e., BOP1, HNRPL, ABC3C, HNRL2, XPOT), DNA replication proteins (i.e., BD1L1), among others, were frequently targeted.

[0212] Redox sensitive cysteines at chloride ion channels (CLIC1, CLIC4) were particularly susceptible to chloroacetamides, while voltage-dependent anion channels (VDACs) could be targeted by other electrophiles (Fig. 16C). Fragments Fl-2, F5-11, among others, targeted Cl 38 of an ATPase catalytic subunit, which Cravatt et al. proved targetable via chloroacetamides (ATP6V1A),19Ligandable C18 in REEP5 (Cravatt et al.)20and C369 in D-3-phosphoglycerate dehydrogenase (SERA) were frequent targets of covalent fragments. DNA repair protein methylguanine methyltransferase (MGMT) could be liganded at C145 by numerous fragments, a nucleophilic cysteine that remediates alkyl lesions (Fl-2, F14, F19, F21, F31).21C80 at TOE1 — whose targeting was suggested by Li and Cravatt to effect its function via allosteric mechanism — was significantly targeted by F21.22Acrylamide F16 reacted with highly nucleophilic C396 at chromodomain helicase DNA binding protein 4 (CHD4), a key component of the nucleosome remodeling and deacetylase complex (NuRD). C73, a catalytic residue in papain-type bleomycin hydrolase (BLMH), was liganded by F15. Additionally, specific residues of ubiquitin ligases (e.g., catalytic C4341 in HUWE1) and kinases (e.g., BTK-C481, CDK2-C17723) could be targeted by a narrow set of fragments, thus offering promise for increase potency and selectivity (Fig. 16A-B).

[0213] Analogously to the covalent fragment library study, we applied timSHIFT to quantify the redox- sensitivity of the reactive cysteinome under H2O2-, menadione-, and KBrOa-induccd cellular stress (Fig. 6C). We successfully captured both differential off- competition and reactivity increase of reduced cysteines upon treatment with different stressors and concentrations and gauged them against global protein expression. H2O2 exerted no significant effect, likely due to facile detoxification by catalase, and KB1O3 showed limited effect on Ramos cells. Conversely, menadione treatment (20 pM) caused bothsignificant increase in quantified off-competed (red, e.g., MTCH2-C297, ROCK1-C714) or more reactive (blue, e.g., SYAC-C671, PPIG-C33, BUD31-C134) cysteines. Most proteins did not show significant differences in expression between vehicle and treatment, thus hinting that changes in residue reactivity cause differential timSHIFT labeling (Fig. 6C, right).Example 1: Methods

[0214] Cell culture. Ramos (RA 1, CRL-1596) and A-431 (CRL-1555) cells were purchased from ATCC. Ramos cells were cultured at 37 °C and 5% CO2 in RPMI 1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin. A-431 cells were cultured at 37 °C and 5% CO2 in DMEM complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Subculturing and medium renewal were performed according to ATCC’s product sheets. Cells were grown in 25 cm2, 75 cm2or 150 cm2canted neck, vented cap sterile cell culture flasks (for suspended cells) or 15-cm 10-cm culture dishes (for adherent cells).

[0215] Cell lysate preparation. Ramos cells were grown until cell density reached ~1.5- 106cells-ml’1, collected by centrifugation (800g, 5 min, 20 °C), and washed three times with DPBS (10 ml- 10'8cell'1). A-431 cells were grown until >80% confluence, washed once with DPBS (5 ml), detached by incubation with TrypLE™ Express Enzyme (5 ml, 5 min, 37 °C), collected by centrifugation (800g, 5 min, 20 °C), and washed three times with DPBS (10 ml- 10'6cell'1). Alternatively, if immediate use was not planned, cell pellets were stored at -80 °C and thawed before use. Cells were resuspended in DPBS at the desired lysis concentration, then lysed by sonication (QSonica Q500 with cup horn, 4 °C, 60% Amplitude, 4 sec on / off, 12 min total for Ramos cells, 10 min total for A-431 cells) and checked under microscope (1:1 lysate:trypan blue 0.4%) to ensure complete lysis. Lysate concentrations were assessed by bicinchoninic acid assay (BCA) and normalized to the desired value with cold DPBS. If not immediately used, cell lysates were stored at -80 °C and thawed before use.

[0216] In-plate reductive alkylation, SP3 precipitation, and washing. DTT in water (10 pL, 100 mM) was added and incubated at 60 °C for 30 minutes. lodoacetamide in water (10 pL, 200 mM) was added and incubated at in the dark at room temperature for 30 min. Samples were transferred to a KingFisher 96 deep-well plate (plate 1), SpeedBead magnetic carboxylate beads (12 ul, 1:1 E3:E7 washed twice with water) were added followed by MeCN (54 ul), and the plate was gently mixed by manual agitation. After 15 minutes,beads were washed using a KingFisher Flex 96 type 710 magnetic purification system (Ref. 5400500) with KingFisher 96 deep- well plates loaded with EtOH 80% (plate 2, 180 pL- well’x), EtOH 80% (plate 3, 180 pL-well’1) and MeCN (plate 4, 250 pL-well’1). The following steps were implemented: (a) plate 1, collect beads (premix), (b) plate 2, wash (release beads, 30 sec, medium; wash beads, 1 min, medium), wash (release beads, 30 sec, fast dual mix; wash beads, 1 min, fast dual mix), wash (release beads, 30 sec, medium; wash beads, 1 min, medium), (c) plate 3, wash (release beads, 30 sec, medium; wash beads, 1 min, medium), wash (release beads, 30 sec, fast dual mix; wash beads, 1 min, fast dual mix), wash (release beads, 30 sec, medium; wash beads, 1 min, medium), (d) plate 4, wash (release beads, 30 sec, medium; wash beads, 1 min, medium), wash (release beads, 30 sec, fast dual mix; wash beads, 1 min, fast dual mix), wash (release beads, 30 sec, medium; wash beads, 1 min, medium). Beads were released in a KingFisher 96 deep- well plate loaded with trypsin (0.2 ug-welF1) in NH4HCO3 (plate 5, 40 pL-well’1, 5 ng- pL’1in 50 mM NH4HCO3). The following steps were implemented: (a) plate 4, collect beads (premix), (b) plate 5, wash (release beads, 30 sec, medium; wash beads, 30 sec, medium), wash (release beads, 30 sec, fast dual mix; wash beads, 30 sec, fast dual mix), wash (release beads, 30 sec, medium; wash beads, 30 sec, medium).

[0217] In-plate trypsin digestion. Samples from plate 5 were carefully transferred to a full- skirted 96- well PCR plate, capped, and incubated at 37 °C for 16h while inverted using a rotisserie apparatus. The plate was quickly centrifuged (200 rpm, 20 °C, 1 min) to ensure all the content sits at the bottom. Formic acid (5 pL well’1, 10% v / v in water) was added, beads were removed using a DynaMag™ 96 Side Skirted Magnet (5 minutes incubation), and the samples transferred to a new full-skirted 96-well PCR plate.

[0218] In-plate desalting. An Oasis HLB 96-well plate was conditioned by washing once with MeCN+0.1% v / v formic acid (300 pL well’1, elution at 200g for one minute) and twice with H2O+0.1% v / v formic acid (300 pL well’1, elution at 200g for 1 min). The analyte samples (300 pL well'1in H2O+0.1% v / v formic acid) were loaded and eluted twice at 200g for 1 min. Absorbed peptides were washed twice H2O+0.1% v / v formic acid (300 pL well’1, elution at 200g for 1 min), then eluted once with 80% MeCN+0.1% v / v formic acid (100 pL well'1, elution at 200g for 1 min). The volatiles were removed by SpeedVac vacuum concentration, and the samples were reconstituted in with 2% MeCN+0.1% v / v formic acid.

[0219] Evaluation of different timSHIFT probes via CuCAAC (Fig. 2, 7-9). To A- 431 cells lysate (5 pL, 2.0 pg-pL'1, prepared according to the described procedure), NaHCCh in H2O (1 pL, 1 M) and TCEP HC1 in H2O (1 pL, 10 mM) were added and incubated at room temperature for 20 min. Alkyne A-(hex-5-yn-l-yl)-2-iodoacetamide in DMSO (2 pL, 10 mM, or 2 pL of DMSO for vehicle) was added and incubated at room temperature for Ih. The appropriate azide 1-13 in H2O (3 pL, 10 mM, 3 pL of H2O for vehicle), followed by a mixture of CuSO4-5H2O in H2O (2 pL, 10 mM, 2 pL of H2O for vehicle) and sodium ascorbate in H2O (2 pL, 100 mM, 2 pL of H2O for vehicle), and the reaction was incubated at room temperature for Ih. DTT in water (5 pL, 100 mM) was added and incubated at 60 °C for 30 minutes. lodoacetamide in water (5 pL, 200 mM) was added and incubated at in the dark at room temperature for 30 min. SpeedBead magnetic carboxylate beads (4 pL, 1:1 E3:E7 washed twice with water) were added followed by MeCN (30 pL) and incubated for 15 minutes. Using a magnetic rack, the solution was removed, and beads were washed twice with EtOH 80% (70 pL each time), once with MeCN (70 pL). Beads were resuspended in NH4HCO3 (23 pL, 50 mM) supplemented with trypsin (0.3 pg) and incubated at 37°C for 16 h while inverted using a rotisserie apparatus. Beads were removed using a DynaMag™ 96 Side Skirted Magnet (5 minutes incubation), and the samples were diluted to 300 pL with H2O+0.1% V / V formic acid. Samples underwent in-plate desalting according to the abovedescribed procedure, and the samples transferred to a new full-skirted 96-well PCR plate for TIMS-MS / MS analysis.

[0220] Evaluation of imidazole-based reagents bearing different charges (Fig. 3, 10). To A-431 cells lysate (13 pL, 3.08 pg-pL'1, final concentration: 2.0 pg-pL'1in 20 pL, prepared according to the described procedure), NaHCOa in H2O (1 pL, 1 M) and TCEP HC1 in H2O (1 pL, 40 mM) were added and incubated at room temperature for 20 min. The appropriate iodoacetamide-based probe 14-16 (5 pL, 10 mM in H2O) was added and incubated at room temperature for Ih. Each compound was tested in triplicate. DTT in water (10 pL, 100 mM) was added and incubated at 60 °C for 30 minutes. lodoacetamide in water (10 pL, 200 mM) was added and incubated at in the dark at room temperature for 30 min. SpeedBead magnetic carboxylate beads (12 pL, 1: 1 E3:E7 washed twice with water) were added followed by MeCN (54 pL) and incubated for 15 minutes. Using a magnetic rack, the solution was removed, and beads were washed twice with EtOH 80% (80 pL each time), once with MeCN (100 pL). Beads were resuspended in NH4HCO3 (40 pL, 50 mM) supplementedwith trypsin (0.2 pg) and incubated at 37 °C for 16h while inverted using a rotisserie apparatus. Formic acid 10% v / v in water (5 pL sample'1) was added, beads were removed using a DynaMag™ 96 Side Skirted Magnet (5 minutes incubation), and the samples transferred to a new full-skirted 96-well PCR plate for tims-MS / MS analysis.

[0221] Comparison between timSHIFT and DTB-IAA / streptavidin enrichment (Fig. 4, 12). The experiment involved three arms i.e., arm-1: timSHIFT 14, arm-2: DTB-IAA without enrichment, arm-3: DTB-IAA with streptavidin enrichment. Ramos cells were grown and lysed in DPBS according to the general procedure and the lysate concentration was normalized to 2.67 pg-pL'1. In three separate Eppendorf tubes, cell lysate (final concentration: 2.0 pg-pL'1) was treated with the appropriate amount of timSHIFT 14 in H2O (arm-1, stock concentration: 10 mM, final concentration: 2.5 mM) or DTB-IAA in DPBS:DMSO 4:1 (arm-2-3, stock concentration: 2 mM, final concentration: 0.5 mM) at room temperature in the dark for Ih. The appropriate amount of DTT in H2O (stock concentration: 100 mM, final concentration: 33.3 mM) was added to each Eppendorf tube and the reaction was incubated at 60 °C for 30 minutes. The appropriate amount of iodoacetamide in water (stock concentration: 200 mM, final concentration: 50 mM) was added and incubated at in the dark at room temperature for 30 min. At this point, for each arm, samples containing decreasing amounts of protein input in 40 pL (each in triplicate) were prepared (dilutions were performed with DPBS). The amounts of input are the following: 40 pg, 20 pg, 10 pg, 5 pg, 1 pg, 0.5 pg. For arm-3, larger amounts were also included, i.e., 500 pg in 500 pL and 200 pg in 200 pL. SpeedBead magnetic carboxylate beads (12 pL, 1:1 E3:E7 washed twice with water, except for 500 pg and 200 pg samples, where 150 pL and 60 pL were used, respectively) were added followed by MeCN (54 ul, except for 500 pg and 200 pg samples, where 650 pL and 260 pL were used, respectively), and the samples were mixed once by aspiration. After 15 minutes, samples were placed on a magnetic rack and the aqueous solution was removed by aspiration. Beads were washed twice with EtOH 80% (180 pL, except for 500 pg and 200 pg samples, where 1000 pL and 500 pL were used, respectively) and once with MeCN (250 pL, except for 500 pg and 200 pg samples, where 1000 pL and 500 pL were used, respectively). Beads were resuspended in a solution of trypsin (0.2 ug-well'1) in NH4HCO3 (40 pL, 5 ng- pL'1in 50 mM NH4HCO3, except for 500 pg and 200 pg samples, where 500 pL and 200 pL were used, respectively). Samples were incubated at 37°C for 16 h while inverted using a rotisserie apparatus. Sampleswere quickly centrifuged to ensure all the content sit at the bottom. For arm- 1 and arm-2, formic acid (5 pL well’1, 10% v / v in water) was added, and beads were removed using a DynaMag™ 96 Side Skirted Magnet (5 minutes incubation). Solvent was removed by SpiroVac evaporation, then the samples were reconstituted in MeCN 2% v / v + 0.1% formic acid (45 pL) for TIMS-MS / MS analysis. For arm-3, beads were removed using a DynaMag™ 96 Side Skirted Magnet (5 minutes incubation), the solution collected, and beads were washed once with HEPES 0.1 M (pH 7.4, 40 pL, except for 500 pg and 200 pg samples, where 500 pL and 200 pL were used, respectively). SeraMag streptavidin beads medium capacity (50 pL, except for 500 pg and 200 pg samples, where 625 pL and 250 pL were used, respectively) were added and samples were incubated at room temperature for 3 h with inversion using a rotisserie apparatus. Samples were quickly centrifuged to ensure all the content sit at the bottom, then beads were pelleted on a DynaMag™ 96 Side Skirted Magnet (5 minutes incubation). The solution was discarded, then beads were washed twice with HEPES 0.1 M + 0.05% v / v NP-40 (pH 7.4, 50 pL, except for 500 pg and 200 pg samples, where 500 pL and 200 pL were used, respectively), three times with HEPES 0.1 M (pH 7.4, 50 pL, except for 500 pg and 200 pg samples, where 500 pL and 200 pL were used, respectively), and three times with NH4HCO3 0.05 M (50 pL, except for 500 pg and 200 pg samples, where 500 pL and 200 pL were used, respectively). Beads were eluted by three consecutive incubations with inversion using a rotisserie apparatus: MeCN 80% + 0.1% formic acid (50 pL, except for 500 pg and 200 pg samples, where 500 pL and 200 pL were used, respectively) at room temperature for 20 min, MeCN 80% + 0.1% formic acid (50 pL, except for 500 pg and 200 pg samples, where 500 pL and 200 pL were used, respectively) at room temperature for 10 min, MeCN 80% + 0.1% formic acid (50 pL, except for 500 pg and 200 pg samples, where 500 pL and 200 pL were used, respectively) at 60°C for 10 min. Solvent was removed by SpiroVac evaporation, then the samples were reconstituted in MeCN 2% v / v + 0.1% formic acid (45 pL) for tims-MS / MS analysis.

[0222] In situ cell treatment (Fig. 5, 6c, and 13a). Ramos cells were grown until cell density reached ~1.5- 106cells-ml’1, collected by centrifugation (800g, 5 min, 20 °C), resuspended in RPMI 1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin to achieve a concentration of 3- 106cells-ml'1, and transferred to 24- well culture plates (1 mL / well). For each compound or condition, treatment occurred in triplicate. Cells were treated by adding 1-2 pL of concentrated stock (or vehicle, 1 pL forsulfopin and leptomycin B, 2 pL for H2O2, menadione, and KBrC ) and incubated at 37 °C and 5% CO2 for the appropriate amount of time (Ih for sulfopin and leptomycin B, 7h for the stressors). Cells were cooled to 0 °C, transferred to Eppendorf tubes, collected by centrifugation (800g, 5 min, 4 °C), and media was removed. Cells were washed three times by resuspension in cold DPBS (500 pL) followed by centrifugation (800g, 5 min, 4 °C). If immediate use was not planned, cell pellets were stored at -80 °C and thawed before use. Treated cells were lysed according to the outlined procedure and lysate concentration was normalized to 2.67 pg-pL'1. In a 96-well skirted PCR plate, cell lysates (15 pL, final concentration: 2.0 pg-pL'1) was treated with timSHIFT 14 in H2O (5 pL of 10 mM stock, final concentration: 2.5 mM) at room temperature in the dark for Ih. The treatment was followed by in-plate reductive alkylation, SP3 precipitation, washing, trypsin digestion, and desalting, as previously detailed. The samples transferred to a new full-skirted 96-well PCR plate for tims-MS / MS analysis.

[0223] In situ treatment - 20,000 cells (Fig. 5). Ramos cells were grown until cell density reached ~1.5- 106cells-ml’1, collected by centrifugation (800g, 5 min, 20 °C), resuspended in RPMI 1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin- streptomycin, spiked with either sulfopin (1 pL of 10 mM for 1 mL of media, final concentration 10 pM) or DMSO (for vehicle, 1 pL for 1 mL of media), to achieve a concentration of 2- 105cells-ml'1, transferred to a 96-well culture (100 pL / well), and incubated at 37 °C and 5% CO2 for Ih. For each compound or condition, treatment occurred in triplicate. Cells were cooled in ice, transferred to Eppendorf LoBind 500 pL tubes, collected by centrifugation (1500g, 4 min, 4 °C), and the supernatant was removed. Cells were washed once by resuspension in cold DPBS (100 pL) followed by centrifugation (1500g, 4 min, 4 °C). Cell pellets were stored at -80 °C and thawed before use. Cells were resuspended in 15 pL of DPBS and lysed according to the outlined procedure. In the same Eppendorf LoBind 500 pL tubes, cell lysates (15 pL) were treated with timSHIFT 14 in H2O (5 pL of 10 mM stock, final concentration: 2.5 mM) at room temperature in the dark for 2h. DTT in water (10 pL, 100 mM) was added to each LoBind tube and incubated at 60 °C for 30 minutes. lodoacetamide in water (10 pL, 200 mM) was added to each LoBind tube and incubated at in the dark at room temperature for 30 min. SpeedBead magnetic carboxylate beads (12 pL, 1:1 E3:E7 washed twice with water) were added to each LoBind tube. MeCN (100 ul) was consecutively added the samples were mixed once by aspiration. After 15minutes, samples were placed on a magnetic rack and the aqueous solution was removed by aspiration. Beads were washed twice with EtOH 80% (180 pL) and once with MeCN (250 pL). Beads were resuspended in a solution of trypsin (0.1 ug- sample'1) in NH4HCO3 (20 pL, 5 ng-pL'1in 50 mM NH4HCO3). Samples were incubated at 37°C overnight and inverted using a rotisserie apparatus. Samples were quickly centrifuged to ensure all the content sits at the bottom, then formic acid (2.5 pL, 10% v / v in water) was added, and beads were removed using a DynaMag™ 96 Side Skirted Magnet (5 minutes incubation). Samples were desalted using Pierce™ C18 Spin Tips: tips were conditioned once with MeCN 80%+0.1% v / v formic acid (20 pL, elution at 1000g for one minute), twice with H2<D+0.1% v / v formic acid (20 pL, elution at 1000g for one minute). The analyte samples loaded and eluted twice at 1000g for 1 min. Absorbed peptides were washed twice H2<D+0.1% v / v formic acid (20 pL, elution at 1000g for 1 min), then eluted twice with 80% MeCN+0.1% v / v formic acid (20 pL first elution, 30 pL second elution, elution at 1000g for 1 min). The volatiles were removed by SpeedVac vacuum concentration, and the samples were reconstituted in 20 pL with 2% MeCN+0.1% v / v formic acid with sonication (10 min), followed by incubation at 37°C for 15 minutes.

[0224] In vitro lysate treatment (Fig. 6a-b, and 14-16). Ramos cell lysates were normalized with DPBS (3.08 pg- pL'1, final concentration: 2.0 pg- pL'1in 20 pL) and the lysate (13 pL) was transferred to a full-skirted 96-well PCR plate. A stock of the appropriate compound in DMSO (or vehicle DMSO, 2 pL, 3.75 mM, final concentration: 500 pM) was added and the plate was incubated at room temperature for Ih. Each compound was tested in triplicate. timSHIFT 14 in water (5 pL of 10 mM stock, final concentration: 2.5 mM) at room temperature in the dark for Ih. The treatment was followed by in-plate reductive alkylation, SP3 precipitation, washing, trypsin digestion, and desalting, as previously detailed. The samples transferred to a new full-skirted 96-well PCR plate for tims-MS / MS analysis.

[0225] Evaluation of different timSHIFT concentrations (Fig. 11). Ramos cells were grown and lysed in DPBS according to the general procedure. In triplicate for each concentration studied, cell lysate (15 pL of 2.0 pg- pL'1) was treated with solutions of timSHIFT 14 in H2O at appropriate concentrations (5 pL) at room temperature in the dark for Ih. The treatment was followed by in-plate reductive alkylation, SP3 precipitation, washing, trypsin digestion, as previously detailed.

[0226] LC-TIMS-MS / MS DDA. Samples were separated on a Bruker nanoElute® 2 UHPLC in two-column mode with the following column setups: (a) Thermo PepMap™ Cl 8 Neo Trap (5 mm x 300 pm, 5 pm particle size, Part No.: 174500), Bruker PepSep Ten C18 (10 cm x 75 pm, 1.9 pm particle size, Part No.: 1893472) (standard sensitivity); (b) Waters nanoEase M / Z Symmetry C18 Trap (2 cm x 180 pm, 5 pm particle size, Part No.: 186008821), lonOptiks Aurora™ Elite CSI C18 (15 cm x 75 pm, 1.7 pm particle size, Part No.: AUR3-15075C18-CSI). The second column was installed in a Bruker Column Toaster (50°C). Gradients: (a) 2% MeCN in H2O + 0.1% formic acid to 35% MeCN in H2O + 0.1% formic acid in 20 min, 500 nL min'1; (b) 2% MeCN in H2O + 0.1% formic acid to 35% MeCN in H2O + 0.1% formic acid in 30 min, 500 nL min'1; (c) 2% MeCN in H2O + 0.1% formic acid to 35% MeCN in H2O + 0.1% formic acid in 5, 10, 20, 30, 60, or 90 min, 500 nL min'1; (d) 2% MeCN in H2O + 0.1% formic acid to 35% MeCN in H2O + 0.1% formic acid in 90 min, 500 nL min'1TIMS-MS / MS analysis was performed on a timsTOF Pro 2 mass spectrometer in data-dependent PASEF mode, m / z scan width: 100-1700, positive ion polarity, Captive Spray source (1400 V, 3 L min'1, 180 °C), collision energy: 10 eV, collision RF: 1500 Vpp, quadrupole energy: 5 eV. Three specific settings were used: (a) no window (10 PASEF MS / MS scans cycle'1; 1 / ko range: 0.50-1.48 Vs cm'2; ramp time: 100 ms; accumulation time: 100 ms; charge range: 0-8; m / z vs. 1 / ko window: none); (b) standard window (10 PASEF MS / MS scans cycle'1; 1 / ko range: 0.60-1.48 Vs cm'2; ramp time: 100 ms; accumulation time: 100 ms; charge range: 0-5; m / z vs. 1 / ko window: (150, 0.60), (800, 1.20), (1200, 1.50), (1705, 1.50), (1705, 0.60); (c) optimized window (4 PASEF MS / MS scans cycle'1; 1 / ko range: 0.65-1.00 Vs cm'2; ramp time: 150 ms; accumulation time: 150 ms; charge range: 0-8; m / z vs. 1 / ko window: (179.14, 0.64), (750.15, 1.01), (811.55, 1.00), (813.68, 0.81), (477.94, 0.64). Other parameters were set as suggested by the instrument manufacturer.TIMS-MS / MSExperiment Figures Col. / Grad.Settings'Gradient (c) was used for Fig. 9C.2Optimized window (c) was used for Fig. 3C and Fig. 9C.3Samples treated with DTB-IAA were analyzed with (b), samples treated with timSHIFT-IAA were analyzed with (c).

[0227] Peptide identification and quantification - DDA. Peptide identification and quantification was performed using the suite FragPipe 21.1 (MSFragger1Aand IonQuant2A)3Awith the Uniprot human (Homo sapiens) proteome database with decoy sequence appended. Precursor and fragment mass tolerance was set to ±20 ppm, digestion rule set as “Strict Trypsin” (K / R cuts), a maximum of two missed cleavages, peptide length range of 7-50, and a mass range of 500-5000. Modifications were considered as variable (maximum mods for peptide: 3, maximum number of combinations: 5,000) for methionine oxidation (M, 15.9949, 3), acetylation of N-terminus ([A, +42.0106, 1), and some were specifically defined for the individual experiment (site, mass delta, maximum number of occurrences): A-(hex-5-yn-l- yl)-2-iodoacetamide (C, +137.1820, 3); 1 (C, +422.3118, 3); 3 (C, +432.2962, 3); 3 (C, +366.2492, 3); 4 (C, +418.2805, 3); 5 (C, +392.2648, 3); 6 (C, +530.3805, 3); 7 (C, +573.4227, 3); 8 (C, +531.3645, 3); 9 (C, +438.2845, 3); 10 (C, +438.2845, 3); 11 (C, +518.3220, 3); 12 (C, +532.3370, 3); 13 (C, +514.3005, 3); 14 (C, +391.2209, 3); 15 (C,+297.1683, 3); 16 (C, +283.1532, 3). All the other research settings were kept at default, with enabled MSBooster,4Afalse discovery rates for PSMs and proteins were both controlled at 1%.5lonQuant was run with MaxLFQ and match-between-runs enabled (FDR < 1%) enabled, and all the other settings were kept at default.

[0228] Peptide-spectrum matches analysis. Peptide- spectrum matches output files from FragPipe were plotted to visualize PSM’s density via Gaussian kernel density estimation. To calculate each probe’s 1 / ko, m / z and shifting effect, peptide-spectrum matches output files (triplicates) were combined, PTM entries from the same peptide were arithmetically averaged and matching peptides were compared with the vehicle, which underwent the same data filtration. To estimate the collisional cross-section ( ) for individual peptides, we derived constant — ■ ■ - for our instrument and experimental conditionsfrom known calibration points (1, a 121 A2, 1 / ko: 0.544 Vs cm'2, m / z: 118.0863; 2, : 202 A2, 1 / ko: 0.9915 Vs cm'2, m / z: 622.0290; 1, a: 243 A2, 1 / ko: 1.1986 Vs cm'2, m / z: 922.0098; 4, a: 282 A2, 1 / ko 1.3934 Vs cm'2, m / z: 1221.9906) and entered the averaged values obtained with the abovementioned procedure in the Mason-Schamp equation. All average differences between probes and vehicles were obtained from matched peptides (peptides that were detected across all samples, to maintain consistency).

[0229] Generation of a timSHIFT 14 DIA library. Ramos cells were lysed according to the above-mentioned protocol, lysate concentrations were assessed by bicinchoninic acid assay (BCA) and normalized to 2 mg ml'1with cold DPBS. Four samples at different concentrations of 14 vs. IAA were prepared (1, 100% 14; 2, 75% 14; 3, 50% 14;4, 0% 14). Cell lysates (20 pL) were treated with 100 mM DTT in 200 mM NH4HCO3 (1 pL) and incubated at 60 °C for 10 min. The appropriate mixture of 14:IAA 10 mM (30 pL) and was added to the appropriate lysate, immediately followed by 0.1 M NaHCOa in PBS (9 pL), and the reaction was incubated for 2h at room temperature. Samples were diluted with H2O (56 pL), then SpeedBead magnetic carboxylate beads (12 pL, 1:1 E3:E7 washed twice with water) were added, followed by MeCN (128 ul), and the samples were mixed once by aspiration. After 15 minutes, samples were placed on a magnetic rack and the aqueous solution was removed by aspiration. Beads were washed twice with EtOH 80% (180 pL) and once with MeCN (250 pL). Beads were resuspended in a solution of trypsin (0.2 ug-well'1) in NH4HCO3 (40 pL, 5 ng- pL'1in 50 mM NH4HCO3). Samples were incubated at 37°C for 16 hwhile inverted using a rotisserie apparatus. Samples were quickly centrifuged to ensure all the content sits at the bottom, then formic acid (5 pL, 10% v / v in water) was added, and beads were removed using a DynaMag™ 96 Side Skirted Magnet (5 minutes incubation). Samples were desalted using Pierce™ C18 Spin Tips: tips were conditioned once with MeCN 80%+0.1% v / v formic acid (20 pF, elution at 1000g for one minute), twice with H2O+0.1% v / v formic acid (20 pL, elution at 1000g for one minute). The analyte samples loaded and eluted twice at 1000g for 1 min. Absorbed peptides were washed twice H2<D+0.1% v / v formic acid (20 pL, elution at 1000g for 1 min), then eluted twice with 80% MeCN+0.1% v / v formic acid (40 pL, elution at 1000g for 1 min). The volatiles were removed by SpeedVac vacuum concentration, and the samples were reconstituted in 40 pL with 2% MeCN+0.1% v / v formic acid.

[0230] Samples were separated on a Bruker nanoElute® 2 UHPLC in two-column mode using a Waters nanoEase M / Z Symmetry C18 Trap (2 cm x 180 pm, 5 pm particle size, Part No.: 186008821), lonOptiks Aurora™ Frontier C18 (60 cm x 75 pm, 1.7 pm particle size, Part No.: AUR3-60075C18). The second column was installed in a Bruker Column Toaster (50°C), and the gradient was 2% MeCN in H2O + 0.1% formic acid to 35% MeCN in H2O + 0.1% formic acid in 180 min, 150 nL min'1. TIMS-MS / MS analysis was performed on a timsTOF Pro 2 mass spectrometer in data-dependent PASEF mode, m / z scan width: 100-1700, positive ion polarity, Captive Spray source (1400 V, 3 L min'1, 180 °C), collision energy: 10 eV, collision RF: 1500 Vpp, quadrupole energy: 5 eV. Five different ranges and windows were used in order to maximize the depth of labeled peptides: (a) 20 PASEF MS / MS scans cycle'1; 1 / ko range: 0.60-1.48 Vs cm'2; ramp time: 150 ms; accumulation time: 2 ms; charge range: 0-8 (b) 20 PASEF MS / MS scans cycle'1; 1 / ko range: 0.60-1.48 Vs cm'2; ramp time: 150 ms; accumulation time: 2 ms; charge range: 0-8, m / z window locked to 200-450; (c) 20 PASEF MS / MS scans cycle'1; 1 / ko range: 0.60-1.48 Vs cm'2; ramp time: 150 ms; accumulation time: 2 ms; charge range: 0-8, m / z window locked to 400-650; (d) 20 PASEF MS / MS scans cycle'1; 1 / ko range: 0.60-1.48 Vs cm'2; ramp time: 150 ms; accumulation time: 2 ms; charge range: 0-8, m / z window locked to 600-850; (e) 20 PASEF MS / MS scans cycle'1; 1 / ko range: 0.65-1.00 Vs cm'2; ramp time: 150 ms; accumulation time: 2 ms; charge range: 0-8, m / z vs. 1 / ko window: (179.14, 0.64), (750.15, 1.01), (811.55, 1.00), (813.68, 0.81), (477.94, 0.64)). Other parameters were set as suggested by the instrument manufacturer.

[0231] Library generation was performed using the suite FragPipe 21.1 (MSFragger1Aand IonQuant2A)3Awith the Uniprot human (Homo sapiens) proteome database with decoy sequence appended and the workflow “DIA pecLib_Quant” . Precursor and fragment mass tolerance was set to ±20 ppm, digestion rule set as “Strict Trypsin” (K / R cuts), a maximum of two missed cleavages, peptide length range of 7-50, and a mass range of 500-5000. Modifications were considered as variable (maximum mods for peptide: 3, maximum number of combinations: 5,000) for methionine oxidation (M, 15.9949, 3), acetylation of N-terminus ([A, +42.0106, 1), and 14 (C, +391.2209, 3). All the other research settings were kept at default, and false discovery rates for peptides and proteins were both controlled at 1%.

[0232] Peptide identification and quantification - DIA. Peptide identification and quantification was performed using the suite DiaNN 1.8 with the create in-house library and library precursors were reannotated using the FASTA database. Digestion rule set as “Trypsin” (K / R cuts), a maximum of two missed cleavages, peptide length range of 7-30, charge ranging from 1-4, and a mass range of 300-1800. Modifications were considered as variable for methionine oxidation (M, 15.9949), acetylation of N-terminus ([A, +42.0106), and 14 (C, +391.2209). All the other research settings were kept at default and false discovery rates in output were controlled at 1%.5A

[0233] Cysteine activity profiling. Identified and quantified peptides containing modification 14 were converted to the corresponding cysteines by summing the intensity of peptides belonging to the same protein and containing the same modified cysteine (for instance as consequence of different tryptic cleavages). The experimental arms (vehicle vs. treated) were independently normalized via variance-stabilizing transformation^ using the package “MSnbase” and imputed via linear model imputation7 Ausing the R package “Umma” with Benjamini-Hochberg moderated T for P- Adjust. For DDA, data were filtered on a one- arm basis, with both arms having at least one non-missing value. For DDA analysis of leptomycin B (Fig. 13A), data were filtered on a one-arm basis, with at least three nonmissing value across the two arms. For DIA, data were filtered on a one-arm basis, with at least three non-missing value across the two arms.Example 2: Gas-Phase Base DeliverProtocol 1: A gastight capped 1 liter bottle was filled with 200 mL of volatile gas phase base. Gas was delivered to the bottle at 0.2 bar, through a rigid tube extending through the cap halfway into the bottle’s headspace. An outlet tube extending into the bottle was connected tothe vacuum vessel of a mass spectrometer system, delivering gas containing entrained basic vapor into the path of peptide ions. This method enables the use of low- volatility bases such as tetramethylguanidine.Protocol 2: A gastight capped 1 liter bottle was filled with 200 mL of volatile gas phase base. An outlet tube extending into the bottle was connected to the vacuum vessel of a mass spectrometer system via a needle valve, delivering gas containing basic vapor into the path of peptide ions. In this application, basic vapor is delivered to the vacuum vessel via its dynamic vacuum, without the use of an entraining carrier gas. This method is ideal for high volatility bases, such as acetone.Protocol 3: A gastight capped 1 liter bottle was filled with 200 mL of volatile gas phase base. Gas was delivered to the bottle at 0.2 bar, through a rigid tube extending through the cap halfway into the bottle’s headspace. An outlet tube extending into the bottle was connected to the electrospray source, delivering base to the active spray region. This method is ideal for use with mass spectrometer systems that cannot accommodate gas delivery inside the vacuum vessel.Example 3: Organic Synthesis and Characterization

[0234] General information concerning synthesis and characterization. Unless otherwise stated, reaction temperatures refer to the ones of the heating / cooling media (heating block, oil bath, or cryogenic bath). Chemicals were purchased from the vendors specified below and used without further purification, unless otherwise detailed. After preparative HPLC / HILIC purification, fractions containing the product were evaporated using SPGenevac EZ-24.0. Final compounds were characterized byNMR,13C NMR,19F NMR (when applicable), and ESLMS. Intermediates are reported with complete analytical characterization when not previously reported in literature and their purity was found sufficient to allow unambiguous assignment. Compound names were generated using ChemDraw 21.Analytical techniques

[0235] Thin layer chromatography (TLC) was performed using Sigma- Aldrich TLC plates (250 pm on glass, Z122726) and visualization was accomplished with UV light (254 nm) and / or staining with basic KMnC solution.

[0236] Analytical LC (C18) was performed on a Waters AutoPurification with 2424- ELS Detector, 2998-Photodiode Array Detector and SQ Detector 2, column: XBridge C18 5 pm (Part no. 186003116), flow rate: 1.4 ml min'1, eluant: EfoChMeCN + 0.1 % formic acid, 95:5 for 0.5 min, gradient to 5:95 for 5.5 min, 5:95 for 2.0 min, gradient to 95:5 for 0.5 min, 95:5 for 1.5 min.

[0237] Analytical HILIC was performed on a Waters AutoPurification with 2424- ELS Detector, 2998-Photodiode Array Detector and SQ Detector 2, column: XBridge BEH Amide 5 m (Part no. 186006595), flow rate: 1.4 ml min'1, eluant: EfoChMeCN + 0.1 % formic acid, 5:95 for 0.5 min, gradient to 95:5 for 5.5 min, 95:5 for 2.0 min, gradient to 5:95 for 0.5 min, 5:95 for 1.5 min.

[0238] NMR spectroscopy was performed at the CLC Nuclear Magnetic Resonance Core Facility at Weill Cornell Medicine using a Bruker Avance III HD 500 MHz equipped with a 5 mm CPTCI CryoProbe or an Agilent Varian INOVA 600 MHz with a 5 mm BB0600S3 probe. The following sequences were used:1H NMR: zg30;13C NMR: zgpg30;19F NMR: zg30; ^^H COSY: cosygpppqf (magnitude-mode ge-2D COSY using purge pulses before dl); ^-^C HSQC: hsqcedetgp (phase- sensitive ge-2D multiplicity-edited HSQC using echo-antiecho); ^-^C HMBC: hmbcgplpndqf (magnitude-mode ge-2D HMBC using low-pass J-filter). The following solvents were used: CDCh (Sigma-Aldrich, 99.8% dp H2O (Cambridge Isotope Laboratories, 99.9% dp DMSO-<% (Cambridge Isotope Laboratories, 99.9% d + 0.05 v / v TMS), CD3OD (Cambridge Isotope Laboratories, 99.8% d). Spectra were analyzed using MestReNova 14.3 by applying standard baseline and phase correction. Chemical shifts (5) for1H and13C NMR spectra are given in parts per million (ppm) relative to tetramethylsilane (TMS) using the residual solvent signals as references and the values tabulated by Gottlieb, Kotlyar, and Nudelman.8AFor13C NMR spectra recorded in D2O, absolute referencing was used.19F NMR spectra were calibrated using absolute referencing system, as suggested by IUPAC.1H and13C NMR-signals multiplicities that can be analyzed as first order multiplets are reported using the following abbreviations (or combinations thereof): s = singlet, d = doublet, t = triplet, q = quartet, p = quintet, h =sextet; hept = heptet; m = multiplet, br = broad.Purification techniques

[0239] Preparative HPLC (method 1) was performed on a Waters AutoPurification with 2424-ELS Detector, 2998-Photodiode Array Detector and SQ Detector 2, column: XBridge Prep C18 5 pm OBD (Part no. 186002979), flow rate: 20 ml min'1, eluant: EhC MeCN + 0.1 % formic acid, 95:5 for 0.5 min, gradient to 5:95 for 5.5 min, 5:95 for 2.0 min, gradient to 95:5 for 0.5 min, 95:5 for 1.5 min.

[0240] Preparative HILIC (method 2) was performed on a Waters AutoPurification with 2424-ELS Detector, 2998-Photodiode Array Detector and SQ Detector 2, column: XBridge® BEH Amide OBD™ 5 pm (Part no. 186006605), flow rate: 20 ml min'1, eluant: H2O:MeCN + 0.1 % formic acid, 5:95 for 0.5 min, gradient to 95:5 for 5.5 min, 95:5 for 2.0 min, gradient to 5:95 for 0.5 min, 5:95 for 1.5 min.

[0241] Semi-preparative HPLC (method 3) was performed on a Waters AutoPurification with 2424-ELS Detector, 2998-Photodiode Array Detector and SQ Detector 2, column: XBridge-BEH C18 5 m OBD (Part no. 186008166), flow rate: 10 ml min'1, eluant: PhC MeCN + 0.1 % formic acid, 95:5 for 0.5 min, gradient to 5:95 for 5.5 min, 5:95 for 2.0 min, gradient to 95:5 for 0.5 min, 95:5 for 1.5 min.

[0242] Semi-preparative HILIC (method 4) was performed on a Waters AutoPurification with 2424-ELS Detector, 2998-Photodiode Array Detector and SQ Detector 2, column: XBridge-BEH Amide 5 pm OBD (Part no. 186008261), flow rate: 10 ml min'1, eluant: H2<D:MeCN + 0.1 % formic acid, 3:97 for 0.5 min, gradient to 46:54 for 4.0 min, 97:3 to 3:97 for 5.0 min, 3:97 for 0.5 min.

[0243] Semi-preparative HILIC (method 5) was performed on a Waters AutoPurification with 2424-ELS Detector, 2998-Photodiode Array Detector and SQ Detector 2, column: XBridge-BEH Amide 5 pm OBD (Part no. 186008261), flow rate: 10 ml min'1, eluant: H2<D:MeCN + 0.1 % formic acid, 3:97 for 0.5 min, gradient to 97:3 for 6.5 min, 97:3 for 1.40 min, 3:97 for 1.5 min.

[0244] Semi-preparative HILIC (method 6) was performed on a Waters AutoPurification with 2424-ELS Detector, 2998-Photodiode Array Detector and SQ Detector 2, column: XBridge-BEH Amide 5 pm OBD (Part no. 186008261), flow rate: 10 ml min'1, eluant: H2<D:MeCN + 0.1 % formic acid, 5:95 for 0.5 min, gradient to 95:5 for 6.5 min, 95:5 for 1.40 min, 5:95 for 1.5 min.

[0245] Flash column chromatography was performed using a Teledyne ISCO CombiFlash RF+Lumen system equipped with reusable cartridges loaded with Cleanert Silica (particle size: 40 - 60 pm, pore size: 60 A).Example 4: Synthetic Procedures And Characterization

[0246] Azidoacetic acid (SI-1). The reaction was performed according to a modified procedure from Ballster and co-workers.9A

[0247] A round-bottom flask equipped with a PTFE-coated stirring bar was charged with ethyl bromoacetate (22.1 ml, 0.20 mol, 1.0 equiv.) and DMF (34 ml), then NaNa (14.3 g, 0.22 mol, 1.1 equiv.) was added in small portions and the reaction was stirred at room temperature. After 20 hours, the reaction was quenched with water (120 ml) and was extracted twice with Et O (100 ml each time). The combined organic extracts were washed three times with water (40 ml each time), were dried over MgSCL and the solvent was carefully removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 50 mbar). The crude ethyl azidoacetate (23.2 g, 0.18 mol, 1.0 equiv.) was dissolved in TFffiFhO (1:1, 120 ml), KOH (20.2 g, 0.36 mol, 2.0 equiv.) was added in small portion (warning: dissolution is exothermic, add slowly) and the reaction was stirred at room temperature for 22 hours. The reaction was carefully quenched with HC1 37% until pH 1, then the aqueous layer was extracted twice with EtOAc (100 ml each time). The combined organic layers were dried over MgSO4, and the solvent was carefully removed by rotary evaporation (40 °C at 150 mbar, then 40 °C at 5-10 mbar). The crude product (8.45 g, 83.6 mmol, light yellow oil) was judged pure enough to be used without additional purification. 'H NMR (500 MHz, CDCh) 8 9.57 - 9.11 (br, 1H), 3.97 (s, 2H). The experimental data are in agreement with the literature report.10ANote. Small organic azides are known to be explosive. While we did not observe any detonation, reactions must be performed in the strict absence of metals and products must be stored in the dark in flame-proof freezers.

[0248] / ( / / -Butyl 4-(2-bromoethyl)piperazine-l-carboxylate (SI-2). The title compound was synthesized according to a modified procedure from Yang and co- workers.11AA round-bottom flask equipped with a PTFE-coated stirring bar was charged with / er / -butyl piperazine- 1 -carboxylate (9.31 g, 50.0 mmol, 1.0 equiv.), A,A-diAopropylethylamine (8.91 ml, 50.0 mmol, 1.0 equiv.), 1,2-dibromoethane (50 ml) and the reaction was heated to 30 °C for 3 days. The volatiles were removed by rotary evaporation (40 °C at 5-10 mbar), then the residue was partitioned between CH2CI2 and H2O (200 ml each). The aqueous layer was extracted twice with CH2Q2 (50 ml each time), then the combined organic layers were dried over MgSCU, and the solvent was carefully removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar). The residue was purified by flash column chromatography on silica (hexanes:EtOAc = 25% to 35%) to afford the title compound (6.72 g, white solid). 'H NMR (500 MHz, CDCh) 5 3.76 - 1.94 (br m, 12H), 1.46 (s, 9H). LC-MS: Rt, 4.26 min; m / z, 293.2 ([M+H]+).The spectroscopic data are in agreement with the literature report.11

[0249] (Tosylazanediyl)bis(ethane-2,l-diyl) bis(4-methylbenzenesulfonate) (SI-3).The title compound was synthesized according to an adapted version of the procedure from Sutherland and co- workers.12AA round-bottom flask equipped with a PTFE-coated stirring bar was charged with p-toluenesufonyl chloride (57.2 g, 0.30 mol, 3.0 equiv.) and CH2CI2 (75 ml), then cooled to 0 °C. A solution of diethanolamine (10.5 g, 0.10 mol, 1.0 equiv.) in water (2.5 ml), aliquat 336 TG (4.04 g, 10.0 mmol, 10 mol%) in water (2.5 ml), and NaOH (12.0 g, 0.30 mol, 3.0 equiv.) in water (70 ml) were consecutively added under vigorous stirring, then the reaction was warmed to room temperature. After 2 hours, the reaction was poured into water (150 ml), the organic layer was separated and was washed three times with water (50 ml each time). The organic layer was dried over MgSO4, and the solvent was removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar). The crude product (61.9 g, waxy solid) was judged pure enough to be used without further purification. 'H NMR (500 MHz, CDCh) 5 7.76 (d, J = 8.3 Hz, 4H), 7.61 (d, J = 8.3 Hz, 2H), 7.36 (d, J= 8.3 Hz, 4H), 7.29 (d, J= 8.1 Hz, 2H), 4.11 (t, J = 5.9 Hz, 4H), 3.37 (t, J= 5.9 Hz, 4H), 2.46 (s, 6H), 2.43(s, 3H). LC-MS: Rt, 7.92 min; m / z, 396.4 ([M+H]+). The experimental data are in agreement with the literature report.12

[0250] / ( / / -Butyl (2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethyl)carbamate (14-2).Part of this synthesis is adapted from Spiegel and co-workers,13AHedberg and co-workers,14AWeissleder and co-workers,15Aand Hirozane and co- workers.16A

[0251] Tosylation. In a round-bottom flask equipped with a PTFE-coated stirring bar, tetraethylene glycol (14-1, 34.5 ml, 0.20 mol, 10.0 equiv.) and EhN (4.18 g, 30.0 mmol 1.5 equiv.) were dissolved in CH2CI2 (38 ml), then the solution was cooled to 0 °C. p- Toluensulfonyl chloride (3.18 g, 20.0 mmol, 1.0 equiv.) was added in small portions, then the reaction was warmed to room temperature. After 48 hours, the reaction was quenched with water (50 ml), the layers were separated and the organic layer was washed twice with water (50 ml each time), then the organic layer was dried over MgSCE, and the solvent was removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar). The residue 2- (2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (6.18 g, faint yellow thick oil) was judged pure enough to be used without additional purification. 'H NMR (500 MHz, CDCh) 5 7.80 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 4.16 (dd, J = 5.8, 3.9 Hz, 2H), 3.82 - 3.52 (m, 14H), 2.45 (s, 3H). LC-MS: Rt, 5.15 min; m / z, 349.2 ([M+H]+). The experimental data are in agreement with the literature report.13A

[0252] Nucleophilic displacement. In a round-bottom flask equipped with a PTFE- coated stirring bar, 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (6.18 g, 17.7 mmol, 1.0 equiv.) was dissolved in DMF (68 ml), then NaNa (1.38 g, 21.3 mmol, 1.2 equiv.) was added in one portion, then the reaction was heated at 60 °C. After 20 hours, the reaction was cooled to room temperature, then most of the solvent was removed by rotary evaporation (60 °C at 5-10 mbar). The residue was diluted with water (40 ml), extracted three times with EtOAc (40 ml each time), the combined organic layers were washed once with water (40 ml), then the organic layer was dried over MgSCE, and the solvent was removed by rotary evaporation (40 °C at 120 mbar, then 40 °C at 5-10 mbar). The residue 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-l-ol (1.63 g, light yellow oil) was judged pure enough to be used without additional purification. 'H NMR (500 MHz, CDCh) 83.77 - 3.71 (m, 2H), 3.71 - 3.66 (m, 10H), 3.66 - 3.59 (m, 2H), 3.39 (q, J= 4.6 Hz, 2H), 2.39 (t, J= 6.2 Hz, 1H). LC-MS: Rt, 4.22 min; m / z, 220.1 ([M+H]+) The experimental data are in agreement with the literature report.13A

[0253] Hydrogenation and Boc-protection. In a round-bottom flask equipped with a PTFE-coated stirring bar, 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-l-ol (1.63 g, 7.43 mmol, 1.0 equiv.), Pd-C 10% w / w (60 mg, 0.056 mmol, 0.76 mol%), and MeOH (20 ml) were charged, then the vessel was capped with a rubber septum, and H2 (1 atm) was bubbled through the slurry for 10 minutes. Afterwards, the reaction was stirred under 1 atm of hydrogen for 18 hours. The reaction was filtered over a short pad of Celite, rinsing thoroughly with additional methanol. The solvent was removed by rotary evaporation (40 °C at 150 mbar, then 40 °C at 5-10 mbar), affording a partially pure residue of 2-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)ethan-l-ol (1.06 g, light yellow oil), which was immediately dissolved in EtOH (15 ml). A solution of BOC2O (1.32 g, 6.03 mmol, 1.1 equiv. based on weight of the impure intermediate) in EtOH (2 ml) was added dropwise over 5 minutes and the reaction was stirred at room temperature. After 3 hours, the solvent was removed by rotary evaporation (40 °C at 100 mbar, then 40 °C at 5-10 mbar), then the residue was taken- up with CH2Q2 (75 ml), washed twice with 2M HC1 (20 ml each time), washed once with saturated NaHCOa (20 ml), ), then the organic layer was dried over MgSO4, and the solvent was removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar), affording mostly pure tert-butyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate (1.04 g, colorless oil). 'H NMR (500 MHz, CDCh) 5 3.79 - 3.72 (m, 4H), 3.72 - 3.61 (m, 8H), 3.56 (t, J= 5.0 Hz, 2H), 3.52 - 3.29 (m, 2H), 1.47 (s, 9H). LC-MS: Rt, 4.71 min; m / z, 294.2 ([M+H]+).The experimental data are in agreement with the literature report.15A

[0254] Appel bromination. In a round-bottom flask equipped with a PTFE-coated stirring bar, tert-butyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate (3.14 g, 10.7 mmol, 1.0 equiv.) and triphenylphosphine (3.37 g, 12.8 mmol, 1.2 equiv.) were dissolved in anhydrous THF (30 ml). A solution of CBr4 (4.26 g, 12.8 mmol, 1.2 equiv.) in CH2Q2 (7 ml) was added using a syringe pump (0.3 ml min'1) under stirring at room temperature, then upon completion of the addition the reaction was stirred at room temperature for 18 hours. The reaction volume was reduced to 20 ml by rotary evaporation (40 °C at 150 mbar), then hexanes (100 ml) were added to precipitate most triphenylphosphine oxide. The reaction was filtered over a short pad of Celite, rinsing withhexanes:CH2C12 (4:1, 30 ml), then the solvent was removed by rotary evaporation (40 °C at 250 mbar, then 40 °C at 5-10 mbar). The residue was carefully purified by flash column chromatography on silica (hexanes :EtO Ac = 5% to 40%), affording the title compound (2.30 g, colorless oil). 'H NMR (500 MHz, CDCh) 5 5.10 - 4.91 (br s, 1H), 3.82 (t, J= 6.4 Hz, 2H), 3.74 - 3.59 (m, 8H), 3.54 (t, J = 5.1 Hz, 2H), 3.48 (t, J = 6.3 Hz, 2H), 3.32 (q, J = 5.5 Hz, 2H), 1.45 (s, 9H). LC-MS: Rt, 5.92 min; m / z, 356.1 ([M+H]+). The experimental data are in agreement with the literature report.16ANote. Elution must be performed at low gradient rate to prevent co-elution of triphenylphosphine oxide impurities.Example 5: Synthetic procedures and characterization - probes

[0255] 2-Azido-\,\-bis(2-(dimethylamino)ethyl)acetamide (1). The title compound was synthesized according to the following multistep procedure.

[0256] Boc-protection. A round-bottom flask equipped with a PTFE-coated stirring bar was charged with tris(2-aminoethyl)amine (749 pl, 5.0 mmol, 1.0 equiv.) and CH2Q2 (17 ml). BOC2O (1.15 ml, 5.0 mmol, 1.0 equiv.) in CH2CI2 (5 ml) was added dropwise over 90 minutes, then the reaction was stirred for 18 hours. The reaction was quenched with water (0.5 ml), then the volatiles were removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar). The residue was taken-up with MeCN:H2O (1:1, 4 ml) and purified by HPLC (method 1, only part of the solution was purified), affording intermediate tert-butyl (2- (bis(2-aminoethyl)amino)ethyl)carbamate (SI-4) as formate salt (69 mg, colorless gum). 'H NMR (500 MHz, D2O) 5 3.19 (t, J= 6.6 Hz, 2H), 3.12 (t, J= 6.3 Hz, 4H), 2.87 (t, J = 6.3 Hz, 4H), 2.69 (t, J = 6.6 Hz, 2H), 1.44 (s, 9H). The experimental data are in agreement with the literature report.17A

[0257] Reductive amination and amidation. In a scintillation vial equipped with a PTFE-coated stirring bar, SI-4 (68.7 mg, 0.287 mmol, 1.0 equiv.) was dissolved in MeCN (7 ml), then formaldehyde 37% in water (207 pl, 2.8 mmol, 10.0 equiv.) was added in one portion and the reaction was stirred at room temperature. After 40 minutes, NaCNBHa (52.5mg, 0.83 mmol, 3.0 equiv.) was added in one portion and the reaction was stirred at room temperature for 2 hours. The reaction was quenched with NaOH 2M (2 ml), then the volatiles were removed by spiral evaporation (40 °C). The crude residue was taken-up with MeCN:H2O (1:1, 1 ml) and purified by HPLC (method 1), affording intermediate SI-5 (14.8 mg, colorless gum). Intermediate SI-5 was dissolved in HCI 2M (1 ml) and stirred for 20 hours. Water was removed by spiral evaporation (40 °C) and further dried under vacuum (W2mbar) to afford deprotected intermediate SI-6 (10.0 mg, colorless gum). In a scintillation vial equipped with a PTFE-coated stirring bar, intermediate SI-6 (10.0 mg, 0.049 mmol, 1.0 equiv.), azidoacetic acid SI-1 (7.3 pl, 0.097 mmol, 2.0 equiv.), and N,N- dizsopropylethylamine (59.1 pl, 0.339 mmol, 7.0 equiv.) were dissolved in DMF (1 ml), then PyBOP (75.6 mg, 0.145 mmol, 3.0 equiv.) was added in one portion under vigorous stirring. The reaction was stirred for 18 hours, then the solvent was removed using spiral evaporation (40 °C). The crude residue was taken-up with MeCN:H2O (1:1, 1 ml) and purified by HPLC (method 1), followed by HILIC (method 5) affording the title compound as formate salt (0.8 mg, colorless film). 'H NMR (500 MHz, D2O) 5 4.05 (s, 2H), 3.37 (t, J = 6.4 Hz, 2H), 3.22 (t, J= 6.8 Hz, 4H), 2.95 (t, J= 6.8 Hz, 4H), 2.87 (s, 18H), 2.75 (t, J= 6.5 Hz, 2H).13C{XH}NMR (126 MHz, D20) 5 171.0, 170.8 (formate), 54.8, 51.9, 51.7, 48.3, 43.2, 36.7. LC-MS(HILIC): Rt, 3.00 min; m / z, 286.4 ([M+H]+).

[0258] 2-Azido-l-(4-(2-(4-methylpiperazin-l-yl)ethyl)piperazin-l-yl)ethan-l-one(2). The title compound was synthesized according to the following multistep procedure. Part of the synthetic procedure was adapted from Yang and co- workers.18A

[0259] Nucleophilic displacement and Boc-deprotection. A round-bottom flask equipped with a PTFE-coated stirring bar was charged with SI-2 (2.63 g, 8.97 mmol, 2.0 equiv.), A-Me-piperazine (1.99 ml, 17.9 mmol, 2.0 equiv.), KI (3.13 g, 18.8 mmol, 2.1 equiv.), and MeOH (90 ml). The reaction was heated at 40 °C for 21 hours, then the volatiles were removed by rotary evaporation (40 °C at 120 mbar, then 40 °C at 5-10 mbar). The residue was partially redissolved with CH2Cl2:MeOH 9:1 (100 ml), then the insoluble solids were removed by filtration. The solvent fraction was concentrated by rotary evaporation (40°C at 700 mbar, then 40 °C at 5-10 mbar), then the residue was purified by flash column chromatography on silica (CH2Cl2:MeOH = 10% to 20%) to afford the intermediate tertbutyl 4-(2-(4-methylpiperazin-l-yl)ethyl)piperazine-l -carboxylate (SI-7) (3.68 g, yellow waxy solid), which contained significant impurities and was used without further purification. The intermediate was resuspended in HC1 2M (40 ml) and stirred at room temperature for 24 hours, then the solvent was removed by rotary evaporation (50 °C at 5-10 mbar) and further dried under vacuum (10‘2mbar). Product SI-8 (4.28 g, white solid) was used for the following transformations without additional purification.1H NMR analysis is hampered by the presence of extremely broad signals in both D2O and DMSO-<%.

[0260] Amidation. In a scintillation vial equipped with a PTFE-coated stirring bar, intermediate SI-8 (70.0 mg, 0.195 mmol, 1.0 equiv.), azidoacetic acid SI-1 (29.3 pl, 0.391 mmol, 2.0 equiv.), and N, A-dizsopropylethylamine (238 pl, 1.37 mmol, 7.0 equiv.) were dissolved in DMF (3 ml), then PyBOP (305 mg, 0.586 mmol, 3.0 equiv.) was added in one portion under vigorous stirring. The reaction was stirred for 18 hours, then the solvent was removed using spiral evaporation (40 °C). The crude residue was taken-up with MeCN:H2O (1:1, 1 ml) and purified by HPLC (method 1), followed by HILIC (method 4). (6.5 mg, colorless film). 'H NMR (500 MHz, D2O) 5 4.27 (s, 2H), 3.99 - 3.83 (br, 2H), 3.79 (t, J =5.4 Hz, 2H), 3.59 - 3.32 (m, 8H), 3.26 - 3.05 (br, 4H), 3.01 - 2.84 (m, 5H), 2.74 - 2.39 (br, 2H).13C{XH} NMR (126 MHz, D2O) 5 168.5, 53.1, 52.8, 51.30, 51.29, 50.5, 50.4, 49.4, 42.7, 41.6, 38.9. LC-MS (HILIC): Rt, 3.60 min; m / z, 296.6 ([M+H]+).

[0261] 2-Azido-2V-(2-(bis(2-aminoethyl)amino)ethyl)acetamide (3). In a scintillation vial equipped with a PTFE-coated stirring bar, tris(2-aminoethyl)amine (89.9 pl, 0.60 mmol, 3.0 equiv.), azidoacetic acid SI-1 (15.0 pl, 0.20 mmol, 1.0 equiv.), and N,N- dizsopropylethylamine (209 pl, 1.20 mmol, 6.0 equiv.) were dissolved in DMF (2 ml), then PyBOP (312 mg, 0.60 mmol, 3.0 equiv.) was added in one portion under vigorous stirring. The reaction was stirred for 18 hours, then the solvent was removed using spiral evaporation (40 °C). The crude residue was taken-up with MeCN:H2O (1:1, 1 ml) and purified by HPLC (method 1), followed by HILIC (method 4). The title compound was obtained as formate salt (3.2 mg, colorless film). 'H NMR (500 MHz, D2O) 5 8.46 (br s, 1H), 4.04 (s, 2H), 3.36 (t, J= 6.6 Hz, 2H), 3.10 (t, J= 6.3 Hz, 4H), 2.86 (t, J= 6.3 Hz, 4H), 2.73 (t, J= 6.6 Hz, 1H).13C{XH} NMR (126 MHz, D2O) 5 170.9, 170.6 (formate), 51.9, 51.6, 50.5, 36.9, 36.6. LC- MS (HILIC): Rt, 5.31 min; m / z, 230.3 ([M+H]+).

[0262] 2-Azido-l-(4-(2-(piperazin-l-yl)ethyl)piperazin-l-yl)ethan-l-one (4). The title compound was synthesized according to the following multistep procedure. Part of the synthesis was adapted from patent W02007042815.

[0263] Nucleophilic displacement and Boc-deprotection. A round-bottom flask equipped with a PTFE-coated stirring bar was charged with tert-butyl piperazine- 1- carboxylate (3.73 g, 20.0 mmol, 2.0 equiv.), MeCN (143 ml), 1,2-dibromoethane (866 pl, 10.0 mmol, 1.0 equiv.), and NaHCOa (4.20 g, 50.0 mmol, 5.0 equiv.). The vessel was equipped with a reflux condenser and the reaction was heated at reflux (90 °C) overnight. The reaction was cooled to room temperature, then the volatiles were removed by rotary evaporation (40 °C at 150 mbar, then 40 °C at 5-10 mbar). The residue was taken-up with EtOAc (80 ml), washed once with water (80 ml) and brine (80 ml). The organic layer was dried over MgSCE, then the solvent was removed by rotary evaporation (40 °C at 150 mbar, then 40 °C at 5-10 mbar). Intermediate di-tert-butyl 4,4'-(ethane-l,2-diyl)bis(piperazine-l- carboxylate) (SI-9) (3.33 g, white solid) was transferred to a round-bottom flask equipped with a PTFE-coated stirring bar and dissolved in a MeOH:HCl 2M in water (2:1, 120 ml) mixture. HCI 37 % (5 ml) was added, and the reaction was stirred at room temperature for 2 hours, then the reaction was heated at 70 °C for 18 hours. The reaction was cooled to room temperature, then the solvent was removed by rotary evaporation (50 °C at 5-10 mbar) and further dried under vacuum (10‘2mbar). Intermediate SI-10 (3.05 g, white solid) was used for the following transformations without additional purification. 'H NMR (500 MHz, D2O) 5 3.61 - 3.53 (m, 10H), 3.53 - 3.47 (m, 10H). ^CfH} NMR (126 MHz, D2O) 5 51.1, 49.0, 41.3.

[0264] Amidation. In a scintillation vial equipped with a PTFE-coated stirring bar, intermediate SI-10 (137.7 mg, 0.40 mmol, 2.0 equiv.), SI-1 (15.0 pl, 0.20 mmol, 1.0 equiv.),and A,A-diz‘5opropylethylamine (418 pl, 2.40 mmol, 12.0 equiv.) were dissolved in DMF (6 ml), then PyBOP (312 mg, 0.60 mmol, 3.0 equiv.) was added in one portion under vigorous stirring. The reaction was stirred for 18 hours, then the solvent was removed using spiral evaporation (40 °C). The crude residue was taken-up with MeCN:H2<D (1:1, 1 ml) and purified by HPLC (method 1), followed by HILIC (method 4). (3.5 mg, colorless film). 'H NMR (500 MHz, D2O) 5 4.27 (s, 2H), 4.00 - 3.85 (br, 2H), 3.80 (t, J= 5.3 Hz, 2H), 3.48 - 3.41 (br, 4H), 3.39 (t, J= 6.3 Hz, 2H), 3.31 (t, J= 5.2 Hz, 4H), 2.91 (t, J = 6.4 Hz, 2H), 2.87 - 2.79 (br, 4H). ^CfH] NMR (126 MHz, D2O) 5 168.5, 52.7, 51.3, 50.9, 50.4, 49.0, 43.1, 41.5, 38.8. LC-MS (HILIC): Rt, 4.55 min; m / z, 282.3 ([M+H]+).

[0265] 2-Azido-l-(l,4,7,10-tetraazacyclododecan-l-yl)ethan-l-one (5). The title compound was synthesized according to the following multistep procedure. Parts of this synthesis are adapted from the work of Sherry and co-workers.19

[0266] Boc-protection. A round-bottom flask equipped with a PTFE-coated stirring bar was charged with cyclen (1.00 g, 5.80 mmol, 1.0 equiv.), CHCh (28 ml) and EhN (2.45 ml, 17.6 mmol, 3.03 equiv.). A solution of BocaO (3.84 g, 15.6 mmol, 3.03 equiv.) in CHCh (22 ml) was added dropwise over 60 minutes, then the reaction was stirred for 13 hours at room temperature. The reaction was quenched with water (20 ml), then the layers were separated, and the organic layer was washed twice with water (40 ml each time). The organic layer was dried over MgSCU, and the solvent was removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar). The crude intermediate di-tert-butyl 4-(3,3- dimethylbutanoyl)- 1 ,4,7, 10-tetraazacyclododecane- 1 ,7-dicarboxylate (SI-11), containing trace amounts of EtsN, was used without further purification (2.87 g, white foam). 'H NMR (500 MHz, CDCh) 5 3.80 - 2.70 (br m, 16H), 1.47 (s, 18H), 1.45 (s, 9H). The experimental data are in agreement with the literature report.19A

[0267] Amidation. In a scintillation vial equipped with a PTFE-coated stirring bar, SI-1 (60.6 mg, 0.60 mmol, 1.0 equiv.) was dissolved in CH2CI2 (4 ml) with one drop of DMF, then oxalyl chloride (66.9 pl, 0.78 mmol, 1.3 equiv.) was added dropwise. The reaction was stirred for 2 hours, then the volatiles were removed by spiral evaporation (40°C). The residue was redissolved in CH2Q2 (4 ml), then a solution of intermediate SI-11 (284 mg, 0.60 mmol, 1.0 equiv.) and DIPEA (418 pl, 2.4 mmol, 4.0 equiv.) in in CH2CI2 (4 ml) was added dropwise. The reaction was stirred at room temperature for 60 minutes, then the volatiles were removed by spiral evaporation (40 °C). The residue was partitioned between in CH2Q2 (20 ml) and water (20 ml). The organic layer was washed twice with water (10 ml each time), then the organic layer was dried over MgSCU, and the solvent was removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar mbar). The crude residue was taken-up with MeCNithO (1:1, 3 ml) and purified by HPLC (method 1), affording partially purified di-tert-butyl 4-(2-azidoacetyl)-10-(3,3-dimethylbutanoyl)-l,4,7,10- tetraazacyclododecane-l,7-dicarboxylate (SI-12) (60.3 mg, colorless gum). 'H NMR (500 MHz, CDCh) 5 3.86 (s, 2H), 3.72 - 3.09 (br m, 16H), 1.58 - 1.35 (m, 27H).

[0268] Boc-deprotection. Intermediate SI-12 was dissolved in H2<D:MeCN (5:2, 7 ml) and HC1 37% (2 ml) was added, then the reaction was stirred at room temperature for 5 hours, then the volatiles were removed by spiral evaporation (40 °C). The crude residue was taken-up with MeCN:H2<D (1:1, 2 ml) and purified by HILIC (method 6), affording the title compound (24.8 mg, colorless oil). 'H NMR (500 MHz, D2O) 5 4.28 (s, 2H), 3.70 - 3.61 (m, 4H), 3.38 - 3.33 (m, 4H), 3.29 - 3.20 (m, 6H), 3.19 - 3.13 (m, 2H). ^CfH} NMR (126 MHz, D2O) 5 172.2, 51.3, 46.8, 46.2, 45.8, 45.6, 44.9, 44.1, 43.7, 43.3, 43.2. LC-MS (HILIC): Rt, 4.43 min; m / z, 256.3 ([M+H]+), 173.3 ([M+H-azidoacetyl]+).

[0269] 2-Azido-l-(4-(2-(4-(2-(piperazin-l-yl)ethyl)piperazin-l-yl)ethyl)piperazin- l-yl)ethan-l-one (6). The title compound was synthesized according to the following multistep procedure.

[0270] Nucleophilic displacement and Boc-deprotection. A round-bottom flask equipped with a PTFE-coated stirring bar was charged SI-2 (2.66 g, 9.07 mmol, 2.0 equiv.), piperazine (391 mg, 4.54 mmol, 1.0 equiv.), TBAI (7.04 g, 19.1 mmol, 2.1 equiv.), and MeOH (45 ml). The reaction was stirred at 40 °C for 7 hours, then the volatiles were removed by rotary evaporation (40 °C at 150 mbar, then 40 °C at 5-10 mbar). The residue was partitioned between in CH2Q2 (20 ml) and aqueous NaOH 2M (20 ml), the layers wereseparated, and the aqueous layer was extracted twice with CH2Q2 (20 ml each time). The combined organic layers were dried over MgSCL. and the solvent was removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar). The residue was purified by flash column chromatography on silica (CH2Ch:MeOH = 0% to 5% to 20%), affording intermediate di- tert-butyl 4,4'-(piperazine- 1 ,4-diylbis(ethane-2, 1 -diyl))bis(piperazine- 1 - carboxylate) (SI-13) (1.85 g, white solid), which contained residual TBAI. The crude residue was dissolved in H2O:MeOH (4:3, 35 ml) and HC1 37% (4 ml) was added. The reaction was heated at 80 °C under stirring for 18 hours, then the solvent was removed by rotary evaporation (40 °C at 5-10 mbar) and under high vacuum (10‘2mbar), affording intermediate l,4-bis(2-(piperazin-l-yl)ethyl)piperazine hexahydrochloride (SI-14) (2.04 g, yellow solid), containing residual traces of TBAI. LC-MS (HILIC): Rt, 5.26 min; m / z, 311.3 ([M+H]+)

[0271] Amidation. In a scintillation vial equipped with a PTFE-coated stirring bar, crude SI-14 (211.7 mg, 0.4 mmol, 2.0 equiv.), azidoacetic acid SI-1 (15.0 pl, 0.20 mmol, 1.0 equiv.), and A,A-diAopropylethylamine (1.05 ml, 6.0 mmol, 15.0 equiv.) were dissolved in DMFithO (4:1, 5 ml), then PyBOP (156 mg, 0.40 mmol, 2.0 equiv.) was added in one portion under vigorous stirring and the reaction was stirred at 50 °C for 5 minutes. The reaction was cooled to room temperature and stirred for 18 hours, then the solvent was removed using spiral evaporation (40 °C). The crude residue was taken-up with MeCNithO (1:1, 1 ml) and purified by HPLC (method 1), followed by HILIC (method 6). The title compound was obtained as formate salt (27.8 mg, colorless film). 'H NMR (500 MHz, D2O) 5 4.28 (s, 2H), 4.02 - 3.73 (m, 3H, exchangeable protons), 3.65 - 3.13 (m, 24H).I3C{'H} NMR (126 MHz, D2O) 5 168.6, 165.8, 51.69, 51.66, 51.6, 51.0, 50.8, 50.5, 50.1, 49.0, 41.6, 38.9. LC-MS (HILIC): Rt, 3.56 min; m / z, 394.4 ([M+H]+).

[0272] 2-Azido-l-(4-(2-(bis(2-(piperazin-l-yl)ethyl)amino)ethyl)piperazin-l- yl)ethan-l-one (7). The title compound was synthesized according to the following multistep procedure. Part of the synthetic sequence was adapted from the work of Bencini and coworkers.2^

[0273] Cyclization. A round-bottom flask equipped with a PTFE-coated stirring bar was charged SI-3 (17.0 g, 30.0 mmol, 3.0 equiv.), TREN (1.50 ml, 10.0 mmol, 1.0 equiv.), K2CO3 (13.8 g, 0.1 mol, 10.0 equiv.), and MeCN (50 ml). The reaction was heated at reflux for 18 hours, then cooled to room temperature and quenched with water (100 ml). The aqueous layer was extracted three times with CH2CI2 (70 ml each time), then the combined organic layers were washed once with brine (50 ml), then the layers were dried over MgSCU, and the solvent was removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar). The residue was dissolved in EtOAc (100 ml), washed once with water (100 ml), then the organic layer was dried over MgSCU, and the solvent was removed by rotary evaporation (40 °C at 150 mbar, then 40 °C at 5-10 mbar). The residue was recrystallized from boiling ethanol, affording intermediate tris(2-(4-tosylpiperazin-l-yl)ethyl)amine (SI-15) (2.68 g, white solid). 'H NMR (500 MHz, CDCh) 5 7.61 (d, J= 8.1 Hz, 6H), 7.32 (d, J = 8.0 Hz, 6H), 3.05 - 2.88 (m, 11H), 2.59 - 2.28 (m, 34H).13CfH} NMR (126 MHz, CDCh) 5 143.9, 132.3, 129.8, 128.0, 56.2, 52.8, 52.5, 46.1, 21.7.

[0274] Tosylamide deprotection. In a round-bottom flask equipped with a PTFE- coated stirring bar and a reflux condenser, intermediate SI-15 (2.19 g, 2.68 mmol, 1.0 equiv.) was suspended in HBr 48% in water (25 ml), then the reaction was vigorously stirred at 100 °C for 4 hours. The volatiles were removed by rotary evaporation (60 °C at 5-10 mbar), then the residue was triturated with Et2O:MeOH (4:1, 30 ml), collected by filtration, and washed twice with Et2<D. Intermediate tris(2-(piperazin-l-yl)ethyl)amine heptahydrobromide (SI-16) (2.53 g, white solid) was used without additional purification. 'H NMR (500 MHz, D2O) 5 3.81 - 3.62 (m, 24H), 3.53 (t, J= 7.3 Hz, 6H), 3.18 (t, J= 7.3 Hz, 6H).13CfH} NMR (126 MHz, D2O) 5 53.4, 49.2, 47.3, 40.9. LC-MS (HILIC): Rt, 5.29 min; m / z, 354.4 ([M+H]+).

[0275] Amidation. In a scintillation vial equipped with a PTFE-coated stirring bar, SI-16 (368 mg, 0.4 mmol, 2.0 equiv.), azidoacetic acid SI-1 (15.0 pl, 0.20 mmol, 1.0 equiv.), and A,A-diAopropylethylamine (697 pl, 4.0 mmol, 20.0 equiv.) were dissolved in DMF (4 ml), then PyBOP (156 mg, 0.30 mmol, 1.5 equiv.) was added in one portion under vigorous stirring and the reaction was stirred at room temperature and stirred for 1 hour, then the solvent was removed using spiral evaporation (40 °C). The crude residue was taken-up with MeCN:H2<D (1:1, 1 ml) + 0.1% TFA and purified by HPLC (method 1), followed by HILIC (method 5). The title compound was obtained as formate (26.0 mg, colorless film) and TFA salt (21.4 mg, colorless film). Formate: 'H NMR (500 MHz, D2O) 5 4.30 (s, 2H), 3.91 - 3.73(m, 10H), 3.73 - 3.63 (m, 10H), 3.55 (t, J= 7.3 Hz, 6H), 3.48 (dd, J = 8.1, 6.1 Hz, 4H), 3.15 (dd, J= 9.7, 4.8 Hz, 6H).13C{XH} NMR (126 MHz, D2O) 5 168.6, 165.7, 53.5, 53.1, 51.7, 50.5, 49.1, 47.04, 46.95, 41.6, 40.6, 38.9. Trifluoroacetate: 'H NMR (500 MHz, D2O) 5 4.30 (s, 2H), 3.83 (s, 4H), 3.66 (s, 16H), 3.58 - 3.37 (m, 10H), 3.15 (q, J= 7.3 Hz, 6H). ^CfH} NMR (126 MHz, D2O) 5 168.6, 162.9 (q, J= 35.4 Hz), 116.3 (q, J= 291.7 Hz), 53.4, 53.0, 51.7, 50.5, 49.0, 47.2, 47.0, 41.6, 40.8, 38.9.19F NMR (471 MHz, D2O) 5 -75.5. LC-MS(HILIC): Rt, 4.26 min (formate), 4.67 min (trifluoroacetate); m / z, 437.5 ([M+H]+).1 K CO 3 i M CN fl

[0276] 2-Azido-l-(4-(2-(4-(2-morpholinoethyl)piperazin-l-yl)ethyl)piperazin-l- yl)ethan-l-one (8). The title compound was synthesized according to the following multistep procedure.

[0277] Nucleophilic displacement, Boc-deprotection, nucleophilic displacement, Boc-deprotection. A round-bottom flask equipped with a PTFE-coated stirring bar and a reflux condenser was charged with chloroethylmorpholine HCI (844 mg, 4.54 mmol, 1.0 equiv.), Boc-piperazine (845 mg, 4.54 mmol, 1.0 equiv.), K2CO3 (1.88 g, 13.6 mmol, 3.0 equiv.), and MeCN (10 ml), then the reaction was heated at reflux. After 2 hours, methanol (5 ml) was added, and the reaction was stirred for 3 additional days at reflux. The reaction was cooled to room temperature and the volatiles were removed by rotary evaporation (40 °C at 120 mbar, then 40 °C at 5-10 mbar). The residue was taken-up with methanol (30 ml) and filtered to remove insoluble K2CO3 and salts, then the solvent was removed by rotary evaporation (40 °C at 120 mbar, then 40 °C at 5-10 mbar). The white solid residue was dissolved in H2O:MeOH (10:3, 60 ml), then HCI 37% (4 ml) was added and the reaction was stirred overnight at room temperature. Additional HCI 37% (3 ml) was added, and the temperature was increased to 60 °C for 2 hours. Upon cooling to room temperature, the volatiles were removed by rotary evaporation (50 °C at 5-10 mbar) and then by high vacuum (10‘2mbar). An a round-bottom flask equipped with a PTFE-coated stirring bar, the crude residue (2.82 g, assumed 9.15 mmol, 1.0 equiv.) was dissolved in H2O (15 ml), then NaOH (1.10 g, 27.4 mmol, 3.0 equiv.) was added and the solution was stirred for 20 minutes, then the volatiles were removed by rotary evaporation (50 °C at 5-10 mbar). SI-2 (2.68 g, 9.14mmol, 1.0 equiv.), TBAI (3.37 g, 9.14, 1.0 equiv.), and MeOH (46 ml) were added, and the reaction was heated at 50 °C for 18 hours. The volatiles were removed by rotary evaporation (40 °C at 120 mbar, then 40 °C at 5-10 mbar), then the residue was partitioned between CH2Q2 and water (50 ml each). The aqueous layer was extracted twice with CH2Q2 (50 ml each time), then the combined organic layers were dried over MgSCU, and the solvent was removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar). The residue was partially purified by flash column chromatography on silica (CH2Cl2:MeOH = 10% to 20% with 1% NH4OH), affording impure intermediate tert-butyl 4-(2-(4-(2- morpholinoethyl)piperazin-l-yl)ethyl)piperazine-l -carboxylate (SI-17) (1.68 g, white solid). In a round-bottom flask equipped with a PTFE-coated stirring bar and a reflux condenser, the intermediate SI-17 was dissolved in MeOH:H2<D (1:1, 30 ml), then HC1 37% (4 ml) was added, and the reaction was heated at 70 °C for 3 hours. The reaction was cooled to room temperature, then the volatiles were removed by rotary evaporation (40 °C at 100 mbar, then 50 °C at 5-10 mbar), and residual water was stripped by adding toluene and removing the volatiles by rotary evaporation (azeotropic removal, 50 °C at 5-10 mbar). The impure intermediate 4-(2-(4-(2-(piperazin- l-yl)ethyl)piperazin- l-yl)ethyl)morpholine pentahydrochloride (SI-18) (1.92 g, off-white solid) was directly submitted to the next step. LC-MS (HILIC): Rt, 5.15 min; m / z, 312.3 ([M+H]+)

[0278] Amidation. In a scintillation vial equipped with a PTFE-coated stirring bar, impure SI-18 (98.8 mg, 0.2 mmol, 1.0 equiv.), azidoacetic acid SI-1 (15.0 pl, 0.20 mmol, 1.0 equiv.), and A,A-diAopropylethylamine (418 pl, 2.4 mmol, 12.0 equiv.) were dissolved in DMFiFhO (5:1, 2.4 ml), then PyBOP (156 mg, 0.30 mmol, 1.5 equiv.) was added in one portion under vigorous stirring and the reaction was stirred at room temperature and stirred for 18 hours, then the solvent was removed using spiral evaporation (40 °C). The crude residue was taken-up with MeChkFhO (1:1, 1 ml) and purified by HPLC (method 1), followed by HILIC (method 5). The title compound was obtained as formate (18.5 mg, white foam). 'H NMR (500 MHz, D2O) 5 4.25 (s, 2H), 3.97 (t, J = 4.7 Hz, 4H), 3.79 (t, J= 5.0 Hz, 2H), 3.68 (t, J = 5.3 Hz, 2H), 3.41 (t, J = 6.7 Hz, 2H), 3.35 (t, J= 4.8 Hz, 4H), 3.29 - 2.95 (br m, 18H).13C{XH} NMR (126 MHz, D2O) 5 168.5, 168.1, 63.9, 52.2, 52.1, 51.74, 51.66, 51.5, 51.1, 50.5, 50.4, 50.2, 42.6, 40.0. LC-MS (HILIC): Rt, 3.50 min; m / z, 395.3 ([M+H]+).

[0279] 3-(6- Azidohexyl)- l-(2-(3-methyl-lH-imidazol-3-ium- l-yl)ethyl)-LH- imidazol-3-ium dibromide (9). The multistep synthesis proceeded via two separate fragments, that were coupled as last step.

[0280] Fragment 1 (SI-20). In a Shlenk tube equipped with a PTFE-coated stirring bar and an argon inlet, 177-imidazole (1.36 g, 20.0 mmol, 1.0 equiv.) was dissolved in anhydrous THF (15 ml), then the solution was cooled to 0 °C. NaH 60% w / w in mineral oil (960 mg, 24.0 mmol, 1.2 equiv.) was added in small portions, and the reaction was stirred at the same temperature for 15 minutes. 1,6-Dibromohexane (9.23 ml, 60.0 mmol, 3.0 equiv.) was added in one portion, then the reaction was warmed to room temperature and stirred for 5 hours. The reaction was carefully quenched with water (10 ml), then the aqueous layer was extracted three times with CH2Q2 (50 ml each time), then the combined organic layers were dried over MgSCU, and the solvent was removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar). The residue was partially purified by flash column chromatography on silica (hexanes 100%, then CFhChiMeOH 5%) to afford impure l-(6- bromohexyl)-177-imidazole (SI-19) (2.27g, colorless thick oil). In a round-bottom flask equipped with a PTFE-coated stirring bar, the residue was dissolved in acetone:DMF (4:1, 25 ml), then NaNa (958 mg, 14.7 mmol, 1.5 equiv.) was added, and the reaction was heated at 40 °C under stirring for 26 hours. Upon cooling to room temperature, acetone was removed by rotary evaporation (40 °C at 200 mbar, then 40 °C at 5-10 mbar), then the oily residue was partitioned between CH2Q2 and water (70 ml each). The aqueous layer was extracted twice with CH2Q2 (50 ml each time), then the combined organic layers were washed three times with water (70 ml each time), were dried over MgSCU, and the solvent was removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar). The intermediate l-(6- azidohcxyl)- 1 / 7-imidazolc (SI- 20), containing residual traces of DMF, was obtained (611 mg, faint yellow oil). 'H NMR (500 MHz, CDCh) 5 7.46 (s, 1H), 7.06 (t, J = 1.3 Hz, 1H), 6.90 (t, J = 1.3 Hz, 1H), 3.94 (t, J = 7.1 Hz, 2H), 3.26 (t, J = 6.8 Hz, 2H), 1.80 (p, J= 7.2 Hz, 2H),1.63 - 1.56 (m, 2H), 1.46 - 1.37 (m, 2H), 1.37 - 1.28 (m, 2H). LC-MS: Rt, 4.20 min; m / z, 194.2 ([M+H]+).

[0281] Fragment 2 (SI-21). In a round-bottom flask equipped with a PTFE-coated stirring bar, 177-imidazole (6.81 g, 0.1 mol, 1.0 equiv.) and powdered NaOH (6.00 g, 0.15 mol, 1.5 equiv.), and DMSO (50 ml) were charged and heated at 50 °C. After 10 minutes, methyl iodide (6.54 ml, 0.105 mol, 1.05 equiv.) was added dropwise over 10 minutes, then the reaction was stirred at 50 °C for 18 hours. The reaction was cooled to room temperature, diluted with water (200 ml) and extracted three times with CH2Q2 (100 ml each time). The combined organic layers were washed three times with water (50 ml each time), then dried over MgSCU, and the solvent was removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 20 mbar). The intermediate / V- Me- imidazole (2.25 g, colorless liquid) was pure enough to be used without further purification. 'H NMR (500 MHz, CDCh) 8 7.43 (s, 1H), 7.05 (t, J = 1.2 Hz, 1H), 6.88 (t, J = 1.3 Hz, 1H), 3.69 (s, 3H).21AIn a scintillation vial equipped with a PTFE-coated stirring bar, intermediate 1-Me-imidazole (2.10 g, 25.6 mmol, 1.0 equiv.) was dissolved in acetone: 1,2-dibromoethane (1.32:1, 12.2 ml), then heated at 90 °C for 20 hours. Upon cooling to room temperature, a white solid precipitated and was collected by filtration, then washed twice with acetone (10 ml each time). Intermediate l-(2-bromoethyl)-3-methyl- 177-imidazol-3-ium bromide (5.44 g, white solid), containing l,l'-(ethane-l,2-diyl)bis(3- methyl-177-imidazol-3-ium) dibromide (2.5:1 product:byproduct ratio, judged by ’ H NMR) was used for the final coupling step without additional purification. LC-MS: Rt, 3.58 min; m / z, 188.9 (85%, [M]+).

[0282] Final coupling. In a scintillation vial equipped with a PTFE-coated stirring bar, intermediate l -(6-azidohcxyl)- 1 / 7-imidazolc SI-20 (135 mg, 0.70 mmol, 1.0 equiv.) and l-(2-bromoethyl)-3-methyl-177-imidazol-3-ium bromide SI-21 (312 mg, 0.70 mmol based on 2.5:1 product:byproduct ratio, 1.0 equiv.) and MeCN:DMF (1:1, 6 ml) were stirred at 80 °C for 19 hours. Upon cooling to room temperature, the volatiles were removed by spiral evaporation (40 °C) and under high vacuum (10‘2mbar). The crude residue was taken-up with MeCN:H2<D (1:1, 4 ml) and purified by HPLC (method 1), affording the title compound (219 mg, white foam). 'H NMR (500 MHz, D2O) 8 8.82 (t, J= 1.7 Hz, 1H), 8.81 (t, J= 1.8 Hz, 1H), 7.61 (t, J= 1.8 Hz, 1H), 7.52 (apt dt, J = 3.7, 1.9 Hz, 2H), 7.42 (t, J = 1.9 Hz, 1H), 4.79 - 4.75 (overlapped with DHO resonance, m, 4H), 4.22 (t, J= 7.1 Hz, 2H), 3.91 (s, 3H), 3.33 (t, J= 6.8 Hz, 2H), 1.87 (p, J= 7.2 Hz, 2H), 1.60 (p, J= 7.0 Hz, 2H), 1.49 - 1.36 (m,2H), 1.36 - 1.21 (m, 2H).13C{XH} NMR (126 MHz, D20) 5 136.6, 135.9, 124.5, 123.5, 122.3, 122.2, 51.0, 49.8, 48.8, 48.7, 36.0, 29.0, 27.7, 25.2, 24.8. LC-MS: Rt, 3.64 min; m / z, 302.2 ([M-H]+), 177.2 ([M-chain]2+).

[0283] l-(6-Azidohexyl)-l',3,3'-trimethyl-177,l'77-[2,2'-biimidazole]-3,3'-diium diiodide (10). The title compound was synthesized according to the following multistep procedure. Part of the synthesis was adapted from Heller and co- workers.22A

[0284] Cyclization. In a round-bottom flask equipped with a PTFE-coated stirring bar, glyoxal 40% in water (9.18 ml, 0.20 mol, 1.0 equiv.) was cooled to 0 °C, then ammonium hydroxide (43 ml) was added dropwise. Upon completion of the addition, the vessel was equipped with a reflux condenser and the reaction was heated at 50 °C for 24 hours. The reaction was cooled to room temperature, and the tan precipitate that formed was collected by filtration, rinsed once with water (15 ml) and once with acetone (15 ml), then dried under high vacuum (10‘2mbar), affording analytically pure 2,2’ -biimidazole (430 mg, tan solid). 'H NMR (500 MHz, DMSO-d6) 8 12.64 (br s, 2H), 7.13 (s, 2H), 6.99 (s, 2H). The experimental data are in agreement with the literature report.23A

[0285] Nucleophilic displacement. In a scintillation vial equipped with a PTFE- coated stirring bar, 2,2’ -biimidazole (112 mg, 0.83 mmol, 1.0 equiv.) was suspended in DMF (7 ml), then powdered NaOH (40.1 mg, 1.00 mmol, 1.2 equiv.) was added in one portion, and the reaction was stirred at room temperature. After 20 minutes, 1,6-dibromohexane (154 pl, 1.00 mmol, 1.2 equiv.) was added in one portion, and the reaction was stirred at room temperature. After 7 hours, the reaction was carefully quenched with water (10 drops), the volatiles were removed by spiral evaporation (40 °C). The crude residue was taken-up with MeCN:H2O (1:1, 1 ml) and purified by HPLC (method 1), affording 1 -(6 -bromohexyl) - 177, 1'77-2, 2'-biimidazole (SI- 22) as formate salt (11 mg, colorless oil).1!! NMR (500 MHz, CDCh) 8 7.27 - 7.26 (overlaps with CHCh, m, 1H), 7.24 (s, 2H), 7.05 (d, 7= 1.9 Hz, 1H), 5.03 - 4.79 (br, 2H), 4.75 (t, 7 = 7.4 Hz, 2H), 3.37 (t, 7 = 6.6 Hz, 2H), 1.90 (p, 7= 6.9 Hz, 2H), 1.84 (p, 7 = 6.9 Hz, 2H), 1.57 - 1.45 (m, 2H), 1.45 - 1.31 (m, 2H). The experimental data are in agreement with the literature reports.24A

[0286] Nucleophilic displacement and exhaustive methylation. In a scintillation vial equipped with a PTFE-coated stirring bar, l-(6-bromohexyl)- 177, 1'77-2, 2'-biimidazole SI- 22 (11 mg, 0.037 mmol, 1.0 equiv.) was dissolved in DMF (1 ml), then NaNa (7.2 mg, 0.11 mmol, 3.0 equiv.) was added and the reaction was stirred at room temperature. After 1 hour, the volatiles were removed by spiral evaporation (40 °C), the residue was dissolved in MeCN:DMF (5:2, 1.4 ml), then K2CO3 (11.8 mg, 0.085 mmol, 2.3 mmol) and methyl iodide (140 pl, 2.26 mmol, 61 equiv.) were added and the reaction was heated at 80 °C. After 18 hours, the volatiles were removed by spiral evaporation (40 °C), the residue was dissolved in MeCNithO (1:1, 1 ml) and purified by HPFC (method 1), affording the title compound (1.8 mg, brown gum). 'H NMR (500 MHz, D2O) 5 8.17 (d, J= 2.1 Hz, 1H), 8.11 (d, J= 2.1 Hz, 1H), 8.09 (s, 2H), 4.20 (t, J = 7.4 Hz, 2H), 3.95 (s, 6H), 3.93 (s, 3H), 3.30 (t, J = 6.8 Hz, 2H), 1.88 (p, J= 7.5 Hz, 2H), 1.65 - 1.49 (m, 2H), 1.43 - 1.34 (m, 2H), 1.32 - 1.21 (m, 2H). ^CfH] NMR (126 MHz, D2O) 5 128.8, 128.4, 126.8, 124.8, 124.2, 51.0, 50.3, 36.6, 36.5, 29.0, 27.7, 25.4, 25.0. LC-MS: Rt, 4.86 min; m / z, 302.2 ([M-H]+), 177.1 ([M-chain]2+).

[0287] 1 -(2-( 177- 1111 idazol- 1 -y 1 )ethyl-y3-( 2-( 1 -( 6-az idoli exy 1 )- 177-i ni idazol-3- i 11 ni -3- yl)ethyl)-177-imidazol-3-ium dibromide (11). The title compound was synthesized according to the following multistep procedure. Part of the synthesis was adapted from Barnard and co- workers.25A

[0288] Nucleophilic displacement, nucleophilic displacement. In a Schlenk tube equipped with a PTFE-coated stirring bar and an argon inlet, 177-imidazole (1.36 g, 20.0 mmol, 1.0 equiv.) was dissolved in anhydrous THF (15 ml), then the solution was cooled to 0 °C. NaH 60% w / w in mineral oil (960 mg, 24.0 mmol, 1.2 equiv.) was added in small portions, and the reaction was stirred at the same temperature for 15 minutes. 1,6- Dibromohexane (9.23 ml, 60.0 mmol, 3.0 equiv.) was added in one portion, then the reaction was warmed to room temperature and stirred for 5 hours. The reaction was carefullyquenched with water (10 ml), then the aqueous layer was extracted three times with CH2Q2 (50 ml each time), then the combined organic layers were dried over MgSC , and the solvent was removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar). The residue was partially purified by flash column chromatography on silica (hexanes 100%, then CthChiMeOH 5%) to afford impure l-(6-bromohexyl)-177-imidazole (SI-19) (4.45 g, colorless thick oil). In a round-bottom flask equipped with a PTFE-coated stirring bar, the residue was dissolved in acetone :DMF:H2O (40:3:5, 48 ml), then NaNa (1.88 g, 28.9 mmol, 1.5 equiv.) was added, and the reaction was heated at 40 °C under stirring for 16 hours. Upon cooling to room temperature, acetone was removed by rotary evaporation (40 °C at 200 mbar, then 40 °C at 5-10 mbar), then the oily residue was partitioned between CH2Q2 and water (70 ml each). The aqueous layer was extracted twice with CH2Q2 (50 ml each time), then the combined organic layers were washed three times with water (70 ml each time), were dried over MgSCU, and the solvent was removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar). The intermediate l -(6-azidohcxyl)- 1 / 7-imidazolc (SI-20), containing residual traces of DMF, was obtained (2.07 g, faint yellow oil). 'H NMR (500 MHz, CDCh) 5 7.46 (s, 1H), 7.06 (t, J = 1.3 Hz, 1H), 6.90 (t, J = 1.3 Hz, 1H), 3.94 (t, J = 7.1 Hz, 2H), 3.26 (t, J= 6.8 Hz, 2H), 1.80 (p, J= 7.2 Hz, 2H), 1.63 - 1.56 (m, 2H), 1.46 - 1.37 (m, 2H), 1.37 - 1.28 (m, 2H). LC-MS: Rt, 4.20 min; m / z, 194.2 ([M+H]+).

[0289] Nucleophilic displacement. In a round-bottom flask equipped with a PTFE- coated stirring bar, 177-imidazole (13.6 g, 0.20 mol, 2.0 equiv.), finely powdered KOH (13.5 g, 0.24 mol, 2.4 equiv.), and TBAB (1.94 g, 6.01 mmol, 6 mol%) were charged and stirred at 40 °C. 1,2-Dibromoethane (8.66 ml, 0.10 mol, 1.0 equiv.) was added in one portion and the reaction was stirred for 24 h. Additional KOH (6.7 g, 0.12 mol, 1.2 equiv.) and 1,2- dibromoethane (8.66 ml, 0.10 mol, 1.0 equiv.) were added and the reaction was stirred for 24 h. Upon cooling to room temperature, the reaction was extracted four times with CHCh (40 ml each time), then the solvent was removed by rotary evaporation (40 °C at 200 mbar, then 40 °C at 5-10 mbar). The residue was resuspended in EtOAc (100 ml), then hexanes (20 ml) was added to induce precipitation. The off-white solid was collected by filtration and washed with a minimal amount of hexanes :EtO Ac 5:1, affording SI-23 (4.26 g, off-white solid). 'H NMR (500 MHz, CDCh) 5 7.28 (t, J= 1.2 Hz, 2H), 7.07 (d, J = 1.2 Hz, 2H), 6.67 (t, J = 1.4 Hz, 2H), 4.27 (s, 4H). LC-MS: Rt, 1.36 min; m / z, 163.2 ([M+H]+). The experimental data are in agreement with the literature report.25A

[0290] Nucleophilic displacement, nucleophilic displacement. In a scintillation vial equipped with a PTFE-coated stirring bar, SI-20 (2.07 g, 10.7 mmol, 1.0 equiv.) was dissolved in MeCN (50 ml), then 1,2-dibromoethane (4.64 ml, 53.6 mmol, 5.0 equiv.) was added and the reaction was stirred at 60 °C for 16 h. The reaction was cooled to room temperature, then the volatiles were removed by rotary evaporation (40 °C at 120 mbar, then 40 °C at 5-10 mbar). The gummy residue was washed with boiling hexanes (three times, 50 ml each time) to remove residual 1,2-dibromohexane and impurities. The crude intermediate was dried under high vacuum (10‘2mbar), then resuspended in MeCN (50 ml), SI- 23 (1.91 g, 11.8 mmol, 1.1 equiv.) and the reaction was heated at 80 °C for 16 h. The reaction was cooled to room temperature, the solvent was removed by rotary evaporation (40 °C at 120 mbar, then 40 °C at 5-10 mbar). The crude product was purified by HPLC (method 1), affording the title compound (253 mg, colorless gum) with some residual formate. 'H NMR (598 MHz, D2O) 5 8.98 (s, 1H), 8.89 (s, 1H), 8.73 (s, 1H), 8.40 (s, 1H, formate), 7.61 (s, 1H), 7.56 (s, 1H), 7.55 (s, 1H), 7.53 (s, 1H), 7.50 - 7.47 (m, 2H), 4.86 - 4.78 (m, 8H, overlaps with H2O), 4.23 (t, J= 7.3 Hz, 2H), 3.33 (t, J= 6.8 Hz, 2H), 1.88 (p, J = 7.0 Hz, 2H), 1.60 (p, J = 7.0 Hz, 2H), 1.41 (p, J = 7.6 Hz, 2H), 1.32 (p, 7 = 7.7 Hz, 2H).13C{XH} NMR (150 MHz, D2O) 5 169.5 (formate), 137.0, 136.1, 135.6, 123.5, 123.4, 123.3, 122.4, 121.7, 121.3, 51.1, 49.9, 49.2, 49.0, 48.6, 48.2, 29.1, 27.8, 25.3, 24.9. LC-MS: Rt, 2.30 min; m / z, 382.2 ([M- H]+).

[0291] l-(6-Azidohexyl)-3-(2-( l-(2-(3-methyl- lH-imidazol-3-ium-l-yl)ethyl)- 1H- imidazol-3-ium-3-yl)ethyl)- lH-imidazol-3-ium trihalide (12). In a scintillation vial equipped with a PTFE-coated stirring bar, 11 (90 mg, 0.166 mmol, 1.0 equiv.) was dissolved in MeCN:DMF 5:1 (2.4 ml), then Mel (412 pl, 6.63 mmol, 40 equiv.) was added and the reaction was stirred at room temperature for 200 hours. The solvent was removed by spiralevaporation (30 °C), then the crude product was purified by HPLC (method 1), affording the title compound (46.7 mg, white solid) with some residual formate. 'H NMR (598 MHz, D2O) 5 9.12 (s, 1H), 8.98 (s, 1H), 8.90 (s, 1H), 8.34 (s, formate), 7.65 (s, 1H), 7.64 (s, 1H), 7.60 (s, 1H), 7.57 (s, 1H), 7.56 (s, 1H), 7.52 (s, 1H), 4.91 - 4.82 (m, 8H), 4.27 (t, J = 7.2 Hz, 2H), 3.95 (s, 3H), 3.36 (t, J = 6.8 Hz, 2H), 1.93 (p, J= 7.3 Hz, 2H), 1.64 (p, J = 7.0 Hz, 2H), 1.45 (p, J= 7.2 Hz, 2H), 1.36 (p, J= 7.4 Hz, 2H).13C NMR (150 MHz, D2O) 5 167.4 (formate), 137.1, 136.9, 136.1, 124.8, 123.7, 123.6, 123.5, 122.5, 122.4, 51.1, 50.1, 49.3, 49.2, 48.7, 48.5, 36.5, 29.1, 27.9, 25.4, 25.1. LC-MS: Rt, 3.30 min; m / z, 396.3 ([M-2H]+), 198.8 ([M- H]2+).

[0292] l-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl)-l',3,3'-trimethyl- 1H, VH-[2,2'-biimidazole]-3,3'-diium bis-iodide (13). The title compound was synthesized according to the following multistep procedure. Parts of this synthesis were adapted from the report of Spiegel and co-workers,13AGreenberg and co-workers,26Aand Oisaki, Kanai and co- workers.27A

[0293] Tosylation. In a round-bottom flask equipped with a PTFE-coated stirring bar, tetraethylene glycol (34.5 ml, 0.20 mol, 10.0 equiv.) and EhN (4.18 g, 30.0 mmol 1.5 equiv.) were dissolved in CH2CI2 (38 ml), then the solution was cooled to 0 °C. Tosyl chloride (3.18 g, 20.0 mmol, 1.0 equiv.) was added in small portions, then the reaction was warmed to room temperature. After 48 hours, the reaction was quenched with water (50 ml), the layers were separated and the organic layer was washed twice with water (50 ml each time), then the organic layer was dried over MgSCU, and the solvent was removed by rotary evaporation (40 °C at 700 mbar, then 40 °C at 5-10 mbar). The residue 2-(2-(2-(2- hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (SI-24) (6.02 g, colorless oil) was judged pure enough to be used without additional purification. 'H NMR (500 MHz,CDCh) 5 7.80 (d, J= 8.0 Hz, 2H), 7.34 (d, J= 8.0 Hz, 2H), 4.16 (dd, J = 5.8, 3.9 Hz, 2H), 3.82 - 3.52 (m, 14H), 2.45 (s, 3H). LC-MS: Rt, 5.26 min; m / z, 349.2 ([M+H]+). The experimental data are in agreement with the literature report.13A

[0294] Nucleophilic displacement. In a round-bottom flask equipped with a PTFE- coated stirring bar, 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate SI-24 (6.00 g, 17.2 mmol, 1.0 equiv.) was dissolved in MeCN (43 ml), then NaNa (1.68 g, 25.8 mmol, 1.5 equiv.) was added in one portion, then the reaction was heated at 80 °C. After 20 hours, additional NaNa (560 mg, 8.61 mmol, 0.5 equiv.) was added and the reaction was heated at 80 °C for additional 20 hours. The reaction was cooled to room temperature, then the solvent was removed by rotary evaporation (40 °C at 100 mbar). The residue partitioned between CH2Q2 (100 ml) and water (40 ml), then the aqueous layer was extracted once with CH2Q2 (40 ml). The combined organic layers were washed once with brine (40 ml), then dried over MgSCU, and the solvent was removed by rotary evaporation (40 °C at 100 mbar, then 40 °C at 5-10 mbar). The residue (SI-25) (3.44 g, faint yellow oil) was judged pure enough to be used without additional purification. 'H NMR (500 MHz, CDCh) 8 3.75 - 3.72 (m, 2H), 3.71 - 3.65 (m, 10H), 3.65 - 3.60 (m, 2H), 3.40 (t, J= 5.0 Hz, 2H). LC-MS: Rt, 4.20 min; m / z, 220.1 ([M+H]+) The experimental data are in agreement with the literature1 report.

[0295] Appel bromination. In a round-bottom flask equipped with a PTFE-coated stirring bar, intermediate SI-25 (3.44 g, 15.7 mmol, 1.0 equiv.) and CBr4 (6.24 g, 18.8 mmol, 1.2 equiv.) were dissolved in CH2Q2 (78 ml) and cooled to 0 °C, then PPI13 (4.94 g, 18.8 mmol, 1.2 equiv.) was added in small portions. Upon completion of the addition, the reaction was warmed to room temperature and stirred for 6 hours. The reaction volume was reduced to 20 ml by rotary evaporation (40 °C at 600 mbar), then hexanes (100 ml) were added to precipitate most triphenylphosphine oxide, which was removed by decantation. The solvent was removed by rotary evaporation (40 °C at 250 mbar, then 40 °C at 5-10 mbar). The residue was purified by flash column chromatography on silica (hexanes / EtOAc = 20% to 35%), affording SI-26 (3.84 g, faint yellow oil). 'H NMR (500 MHz, CDCh) 8 3.82 (t, J = 6.4 Hz, 2H), 3.72 - 3.63 (m, 10H), 3.48 (t, J = 6.4 Hz, 2H), 3.39 (t, J = 5.1 Hz, 2H). LC-MS: Rt, 5.76 min; m / z, 254.0 (100%, [M+H-N2]+). The experimental data are in agreement with the literature report.27A

[0296] Nucleophilic displacement. In a scintillation vial equipped with a PTFE- coated stirring bar, 2,2'-biimidazole (625 mg, 4.66 mmol, 1.0 equiv.) and finely powdered NaOH (280 mg, 6.99 mmol, 1.5 equiv.) were charged, followed by DMF (46 ml). The reaction was stirred at room temperature for 30 minutes, then bromide SI-26 (1.45 g, 5.13 mmol, 1.1 equiv.) in DMF (4 ml) was added and the reaction was stirred at room temperature for 3 days. The solvent volume was reduced to 9-10 ml by rotary evaporation (50 °C), then the residual solution was purified by HPFC (method 1), affording the intermediate SI- 27 (434 mg, dark red gum). 'H NMR (500 MHz, CDCh) 5 7.38 (s, 1H, NH), 7.31 (s, 1H), 7.18 (s, 1H), 5.00 (t, J = 5.0 Hz, 2H), 4.01 (t, J = 5.0 Hz, 2H), 3.81 - 3.69 (m, 10H), 3.48 (t, J= 5.0 Hz, 2H). One aromatic proton is obscured by residual CHCI3. ^CfH} NMR (126 MHz, CDCh) 5 168.4, 138.0, 137.9, 125.4, 123.2, 70.83, 70.76, 70.71, 70.65, 70.5, 70.2, 50. 8, 48.0. One aromatic carbon is missing due to overlap. LC-MS: Rt, 4.06 min; m / z, 336.2 ([M+HD-

[0297] Exhaustive methylation. In a scintillation vial equipped with a PTFE-coated stirring bar, l-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)- 177, 1'77-2, 2'-biimidazole (SI- 27) (434 mg, 1.29 mmol, 1.0 equiv.) was dissolved in MeCN:DMF (1:1, 14 ml), then K2CO3 (411 mg, 2.98 mmol, 2.3 mmol) and methyl iodide (5 ml, 2.97 mmol, 62 equiv.) were added and the reaction was heated at 80 °C. After 22 hours, the volatiles were removed by spiral evaporation (40 °C), the residue was dissolved in MeCN:H2O (1:1, 1 ml) and purified by HPFC (method 1), affording the title compound (312 mg, orange waxy gum). The compound contains traces of DMF that cannot be removed under prolonged exposure to vacuum. 'H NMR (500 MHz, D2O) 5 8.26 (s, 1H), 8.17 (s, 1H), 8.11 (s, 2H), 4.46 (t, 7 = 4.7 Hz, 2H), 3.99 (s, 9H), 3.93 (t, 7 = 4.7 Hz, 2H), 3.77 - 3.70 (m, 4H), 3.70 - 3.63 (m, 6H), 3.53 (t, 7 = 4.8 Hz, 2H).13C{XH} NMR (126 MHz, D2O) 5 128.9, 128.3, 127.0, 125.1, 125.0, 70.1, 69.64, 69.62, 69.4, 69.3, 67.9, 50.7, 50.3, 37.1, 37.0, 36.9. One aromatic carbon is missing due to overlap. LC-MS: 7?f, 2.30 min; m / z, 189.6 ([M]2+), 378.3 ([M-H]+).

[0298] l-(l-Iodo-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-l',3,3'-trimethyl- 1H, l'H-[ 2,2' -biimidazole ]-3,3 '-diium bistrifluoroacetate (14).

[0299] Nucleophilic displacement. In a scintillation vial equipped with a PTFE- coated stirring bar, 2,2'-biimidazole (323 mg, 2.41 mmol, 1.0 equiv.) and finely powdered NaOH (116 mg, 2.89 mmol, 1.2 equiv.) were charged, followed by DMF (24 ml). The reaction was stirred at room temperature for 30 minutes, then bromide 14-2 (1.03 g, 2.89 mmol, 1.2 equiv.) in DMF (1 ml) was added and the reaction was stirred at room temperature for 16 hours. The solvent volume was reduced to 5 ml by spiral evaporation, then the residual solution was purified by HPLC (method 1), affording intermediate SI- 28 (313.3 mg, black gum). 'H NMR (500 MHz, CDCh) 5 7.18 - 7.12 (m, 3H), 7.06 (s, 1H), 4.87 (t, J= 5.0 Hz, 2H), 3.88 (t, J = 5.0 Hz, 2H), 3.69 - 3.22 (m, 12H), 1.44 (s, 9H).

[0300] Exhaustive methylation. In a scintillation vial equipped with a PTFE-coated stirring bar, SI-28 (313 mg, 0.764 mmol, 1.0 equiv.) was dissolved in MeCN:DMF (1:1, 10 ml) with K2CO3 (243 mg, 1.76 mmol, 2.3 equiv.), then Mel (3.0 ml, 48.2 mmol, 63.0 equiv.) was added and the reaction was stirred at 80 °C for 20 hours. Upon cooling to room temperature, the insoluble solids were removed by filtration over Celite®, then the reaction volume was reduced to 2 ml by spiral evaporation. The residual solution was purified by HPLC (method 1), affording SI-29 (335.6 mg, brown gum). 'H NMR (500 MHz, D2O) 5 8.22 (d, J= 2.1 Hz, 1H), 8.13 (d, J= 2.0 Hz, 1H), 8.08 (s, 2H), 4.42 (d, J = 4.7 Hz, 1H), 3.95 (s, 9H), 3.89 (t, J = 4.7 Hz, 2H), 3.68 - 3.56 (m, 10H), 3.27 (t, J= 5.3 Hz, 2H), 1.43 (s, 9H).

[0301] Boc-deprotection. In a scintillation vial equipped with a PTFE-coated stirring bar, the alkylated intermediate (313 mg, 0.442 mmol, 1.0 equiv.) was dissolved in MeOH (10 ml), then trifluoroacetic acid (1.02 ml, 13.3 mmol, 30.0 equiv.) was added, the reaction was stirred at room temperature for 3 days, then the temperature was increased to 50 °C. After 22 hours, the solvent was removed by spiral evaporation, the residue was dissolved in MeCN:H2<D (1:1, 2 ml) and purified by HPLC (method 1), affording the corresponding deprotected intermediate SI-30 (91.7 mg, brown gum). 'H NMR (500 MHz, D2O) 5 8.21 (d, J = 2.1 Hz, 1H), 8.13 (d, J = 2.1 Hz, 1H), 8.07 (s, 2H), 4.42 (t, J = 4.7 Hz, 2H), 3.94 (s, 9H), 3.89 (t, J= 4.7 Hz, 2H), 3.76 (t, J= 5.2 Hz, 2H), 3.71 - 3.66 (m, 4H), 3.65 - 3.61 (m, 4H), 3.21 (t, J = 5.1 Hz, 2H). Note. Deprotection with HC1 should be avoided since chloride ions can generate chloroacetamide impurities during the next step.

[0302] lodoacetamidation. In a scintillation vial equipped with a PTFE-coated stirring bar, free amine intermediate (35.0 mg, 50.5 pmol, 1.0 equiv.) was dissolved in MeCN:H2O (1:1, 2 ml), then NaOH IM in H2O (222 pl) was added and the solution was stirred at 0 °C. lodoacetic anhydride (89.3 mg, 0.252 mmol, 5.0 equiv.) was added in one portion and the reaction was warmed up to room temperature. After 16 hours, the reaction was purified by HPLC (method 2), affording the title product (22.8 mg, yellow gum). 'H NMR (598 MHz, D2O) 5 8.24 (s, 1H), 8.16 (s, 1H), 8.10 (s, 2H), 4.45 (s, 2H), 3.97 (s, 9H), 3.92 (q, J= 4.5 Hz, 2H), 3.78 (s, 2H), 3.71 - 3.61 (m, 10H), 3.41 (q, J= 4.5 Hz, 2H). ^CfH] NMR (150 MHz, D2O) 5 172.1, 166.1, 128.8, 128.2, 126.9, 125.0, 124.9, 70.0, 69.6, 69.5, 69.4, 68.4, 67.8, 50.5, 39.6, 36.8, 36.7, -2.0. [One aliphatic resonance overlaps]19F NMR (563 MHz, D2O) 5 -75.5. LC-MS (HILIC): Rt, 4.86 min; m / z, 520.2 ([M-H]+), 260.6 ([M]2+). Note. We observed that 2.0 equiv. of iodoacetic anhydride are sufficient to drive the acylation to completion.

[0303] l-( l-Iodo-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-3-methyl-lH- imidazol-3-ium formate (15).

[0304] Nucleophilic displacement. In a scintillation vial equipped with a PTFE- coated stirring bar, 1-Me-imidazole (17.3 pl, 0.217 mmol, 1.5 equiv.), bromide 14-2 (51.6 mg, 0.145 mmol, 1.0 equiv.), and K2COa (40.0 mg, 0.290 mmol, 2.0 equiv.) were suspended in DMF:MeCN (1:1, 1 ml) and heated at 80 °C. After 2 hours, additional 1-Me-imidazole (17.3 pl, 0.217 mmol, 1.5 equiv.) was added and the reaction was heated at 80 °C for additional 20 hours. The reaction was cooled at room temperature and directly purified by preparative HPLC (method 1), affording SI-31 (23.7 mg, colorless gum) as mixed bromide / formate salt. 'H NMR (500 MHz, CDCh) 8 9.85 (s, 1H), 8.49 (formate, s, 2H), 7.56 (overlaps with water, s, 1H), 7.28 (s, 1H), 5.26 - 5.00 (br s, 1H), 4.48 (t, J= 4.4 Hz, 2H), 3.98 (s, 3H), 3.84 (t, J = 4.6 Hz, 2H), 3.65 - 3.61 (m, 2H), 3.61 - 3.56 (m, 6H), 3.50 (t, J = 5.5 Hz, 2H), 3.26 (q, J = 5.9 Hz, 2H), 1.40 (s, 9H).13CfH} NMR (126 MHz, CDCh) 8 167.2(formate), 156.2, 138.2, 123.5, 122.7, 79.4, 70.4, 70.31, 70.30, 70.2, 69.1, 49.7, 40.4, 36.4, 28.5. LC-MS: Rt, 4.29 min; m / z, 358.3 ([M]+).

[0305] Boc-deprotection. In a scintillation vial equipped with a PTFE-coated stirring bar, SI-31 (23.7 mg, 0.055 mmol based on bromide, 1.0 equiv.) was dissolved in TFA (1 ml) and the reaction was heated at 65 °C under stirring. After 15 hours, the reaction was cooled to room temperature and the volatiles were removed by spiral evaporation (40 °C). The residue SI-32 was used without additional purification. 'H NMR (500 MHz, D2O) 5 8.61 (s, 1H), 7.39 (s, 1H), 7.31 (s, 1H), 4.26 (t, J= 4.9 Hz, 2H), 3.81 - 3.71 (m, 5H), 3.63 (t, J = 5.1 Hz, 2H), 3.59 - 3.51 (m, 8H), 3.08 (t, J= 4.9 Hz, 2H).13CfH} NMR (126 MHz, D2O) 5 162.4 (trifluoroacetate, q, J = 36.2 Hz), 136.2, 123.4, 122.5, 116.1 (trifluoroacetate, q, J= 290.9 Hz), 69.50, 69.47, 69.39, 69.38, 68.3, 66.3, 48.9, 39.0, 35.6. LC-MS: Rt, 1.69 min; m / z, 258.2 ([M]+), 215.2 ([M-aminoethyl]+). Note. Deprotection with HC1 should be avoided since chloride ions can generate chloroacetamide impurities during the next step.

[0306] lodoacetamidation. In a scintillation vial equipped with a PTFE-coated stirring bar, intermediate SI-32 (0.055 mmol based on bromide, 1.0 equiv.) was dissolved in MeCN:H2O (1:1, 2 ml), then NaOH IM (222 pl) was added and the reaction was cooled to 0 °C. After 10 minutes, iodoacetic anhydride (38.9 mg, 0.11 mmol, 2.0 equiv.) was added in one portion, and the reaction was warmed at room temperature under darkness. After 24 hours, the reaction volume was reduced to 1 ml by spiral vacuum (room temperature), and the reaction was purified by preparative HPLC (method 1) to afford the title compound (30.9 mg, colorless gum) as formate salt. 'H NMR (500 MHz, D2O) 5 8.75 (s, 1H), 8.30 (s, 1H), 7.53 (t, J= 1.9 Hz, 1H), 7.45 (t, J= 1.9 Hz, 1H), 4.40 (t, J= 4.9 Hz, 2H), 3.91 (d, J = 7.8 Hz, 5H), 3.75 (s, 2H), 3.72 - 3.65 (m, 8H), 3.62 (t, J= 5.4 Hz, 2H), 3.39 (t, J= 5.4 Hz, 2H).13CfH} NMR (126 MHz, D2O) 5 172.1, 167.3, 136.4, 123.5, 122.7, 69.7, 69.6, 69.50, 68.47, 68.4, 4

[0307] N-(2-(2-(2-(2-( IH-imidazol- l-yl)ethoxy)ethoxy)ethoxy)ethyl)-2- iodoacetamide formate adduct (16).

[0308] Nucleophilic displacement. In a scintillation vial equipped with a PTFE- coated stirring bar, 1 - / / -imidazole (9.8 mg, 0.144 mmol, 1.0 equiv.) and powdered NaOH (6.9 mg, 0.172 mmol, 1.2 equiv.) were suspended in DMF (500 pl) and stirred at room temperature for 30 minutes. Afterwards, bromide 14-2 (56.4 mg, 0.158 mmol, 1.1 equiv.) was added in one portion and the reaction was stirred at room temperature for 3 hours. The reaction was directly purified by preparative HPLC (method 1), affording the corresponding alkylated intermediate SI-33 (32.0 mg, colorless oil) with some formate adduct. 'H NMR (500 MHz, CDC13) 5 7.71 (S, 1H), 7.04 (s, 1H), 6.99 (s, 1H), 5.29 - 5.09 (br, 1H), 4.11 (t, J = 5.1 Hz, 2H), 3.73 (t, J = 5.1 Hz, 2H), 3.60 - 3.55 (m, 8H), 3.51 (t, J = 5.3 Hz, 2H), 3.33 - 3.23 (m, 2H), 1.41 (s, 9H). ^CfH] NMR (126 MHz, CDCh) 5 156.1, 137.4, 127.9, 119.7, 79.2, 70.7, 70.63, 70.57, 70.4, 70.33, 70.31, 47.4, 40.4, 28.5. LC-MS: Rt, 4.29 min; m / z, 344.4 ([M+H]+).

[0309] Boc-deprotection. In a scintillation vial equipped with a PTFE-coated stirring bar, alkylated intermediate SI-33 (32.0 mg, 0.093 mmol based on freebase, 1.0 equiv.) was dissolved in TFA (1 ml) and the reaction was heated at 65 °C under stirring. After 17 hours, the reaction was cooled to room temperature and the volatiles were removed by spiral evaporation (40 °C). The residue SI-3 4 was used without additional purification. 'H NMR (500 MHz, D2O) 5 8.68 (s, 1H), 7.47 (s, 1H), 7.39 (s, 1H), 4.36 (t, J = 4.9 Hz, 2H), 3.84 (t, J = 4.9 Hz, 2H), 3.67 (t, J= 5.0 Hz, 2H), 3.64 - 3.58 (m, 8H), 3.12 (t, J= 5.0 Hz, 2H).13C{XH} NMR (126 MHz, D2O) 5 162.6 (trifluoroacetate, q, J = 36.2 Hz), 134.8, 122.1, 119.5, 116.2 (trifluoroacetate, q, J = 291.1 Hz), 69.53, 69.52, 69.44, 69.41, 68.4, 66.3, 48.8, 39.1. LC-MS: Rt, 1.69 min; m / z, 244.2 ([M+H]+), 201.1 ([M-aminoethyl+H]+). Note. Deprotection with HC1 should be avoided since chloride ions can generate chloroacetamide impurities during the next step.

[0310] lodoacetamidation. In a scintillation vial equipped with a PTFE-coated stirring bar, deprotected intermediate SI-34 (0.093 mmol based on freebase, 1.0 equiv.) was dissolved in MeCN:H2O (1:1, 2 ml), then NaOH IM (222 pl) was added and the reaction was cooled to 0 °C. After 10 minutes, iodoacetic anhydride (65.8 mg, 0.19 mmol, 2.0 equiv.) was added in one portion, and the reaction was warmed at room temperature under darkness. After 24 hours, the reaction volume was reduced to 1 ml by spiral vacuum (room temperature), and the reaction was purified by preparative HPEC (method 1) to afford the title compound (30.9 mg, colorless gum) as formate adduct. 'H NMR (500 MHz, D2O) 58.77 (s, 1H), 8.33 (s, 1H), 7.56 (t, J= 1.9 Hz, 1H), 7.48 (t, J = 1.9 Hz, 1H), 4.44 (t, J= 4.9 Hz, 2H), 3.93 (t, J= 4.9 Hz, 2H), 3.75 (s, 2H), 3.73 - 3.63 (m, 8H), 3.61 (t, J = 5.3 Hz, 2H), 3.38 (t, J= 5.3 Hz, 2H). ^CfH} NMR (126 MHz, D2O) 5 172.1, 168.3, 135.0, 122.2, 119.63, 69.68, 69.65, 69.54, 69.51, 68.5, 68.4, 49.0, 39.6, -2.3. LC-MS: Rt, 4.20 min; m / z, 425.2 ([M+H]+).

[0311] 2V-(2-(2-Iodoacetamido)ethyl)-6-((4 / ?,5S)-5-methyl-2-oxoimidazolidin-4- yl)hexanamide (DTB-IAA).

[0312] Desthiobiotin. The procedure was adapted from Ball and co-workers.28In a round-bottom flask equipped with a PTFE-coated stirring bar, biotin (2.68 g, 10.4 mmol, 1.0 equiv.) and Na2COa (1.12 g, 10.6 mmol, 1.02 equiv.) were suspended in water (40 ml) and heated to 75 °C until complete dissolution, then Raney nickel (22 g) was added and the reaction was stirred at 75 °C overnight, then the temperature was increased to 100 °C for 8 hours, then to 110 °C for an additional night. The reaction was cooled to room temperature, filtered over Celite®, and the filter cake was thoroughly washed with water. Water was removed by rotary evaporation (50 °C at 5-10 mbar), then the residue was redissolved in water (15 ml) and HC1 37% (2 ml) was added, causing precipitation of a white solid. Crude desthiobiotin was recrystallized from boiling water (1.02 g, white solid). 'H NMR (500 MHz, DMSO-d6) 5 11.98 (s, 1H), 6.31 (s, 1H), 6.11 (s, 1H), 3.60 (p, 7 = 6.5 Hz, 1H), 3.47 (td, J= 7.6, 4.1 Hz, 1H), 2.20 (td, J = 7.5, 3.5 Hz, 2H), 1.48 (p, J = 7.3 Hz, 2H), 1.39 - 1.15 (m, 6H), 0.95 (d, J = 6.4 Hz, 3H). LC-MS: Rt, 4.26 min; m / z, 215.0 ([M+H]+). The experimental data are in accordance with the literature report.28ANote. Any residual biotin is inseparable from desthiobiotin. Therefore, the reaction must be carried to completion to ensure high purity.

[0313] Amidation. In a scintillation vial equipped with a PTFE-coated stirring bar, desthiobiotin (127 mg, 0.59 mmol, 1.0 equiv.), DIPEA (258 pl, 1.48 mmol, 2.5 equiv.) and Boc-ethylenediamine (114 mg, 0.71 mmol, 1.2 equiv.) were dissolved in DMF (2 ml), then PyBOP (401 mg, 0.77 mmol, 1.3 equiv.) was added in one portion and the reaction wasstirred at room temperature for 48 h. The solvent was removed by spiral vacuum (40 °C). The residue was redissolved in MeCN (1 ml) and purified by HPLC (method 1), affording tertbutyl (2-(6-((47?,5S)-5-methyl-2-oxoimidazolidin-4-yl)hexanamido)ethyl)carbamate SI-35 (170.5 mg, white solid). 'H NMR (500 MHz, DMSO-d6) 8 7.76 (t, J = 5.6 Hz, 1H), 6.76 (t, J = 5.7 Hz, 1H), 6.29 (s, 1H), 6.11 (s, 1H), 3.60 (p, J= 6.8 Hz, 1H), 3.50 - 3.45 (m, 1H), 3.04 (q, J= 6.3 Hz, 2H), 2.95 (q, J= 6.3 Hz, 2H), 2.03 (t, J= 7.4 Hz, 2H), 1.47 (p, J = 7.1 Hz, 2H), 1.37 (s, 9H), 1.34 - 1.13 (m, 6H), 0.95 (d, J = 6.4 Hz, 3H). LC-MS: Rt, 4.71 min; m / z, 357.7 ([M+H]+), 257.7 ([M-Boc+H]+).

[0314] Boc-deprotection. In a scintillation vial equipped with a PTFE-coated stirring bar, tert-butyl (2-(6-((4 / ?,5.S')-5-mcthyl-2-oxoimidazolidin-4-yl)hcxanamido)cthyl)carbamatc SI-35 (170 mg, 0.48 mmol, 1.0 equiv.) was dissolved in CH2CI2 (3 ml), then TFA (1 ml) was added in one portion and the reaction was stirred at room temperature for 90 minutes. The solvent was removed by spiral vacuum (40 °C). The residue was redissolved in DMF (1 ml) and purified by HPLC (method 1) to afford A-(2-aminoethyl)-6-((4R,5S)-5-methyl-2- oxoimidazolidin-4-yl)hexanamide trifluoroacetate SI-36 (140.6 mg, colorless gum). 'H NMR (598 MHz, DMSO-d6) 8 7.98 - 7.88 (m, 1H), 7.69 (br s, 3H), 6.30 (s, 1H), 6.13 (s, 1H), 3.61 (p, J= 6.5 Hz, 2H), 3.25 (q, J= 6.8 Hz, 2H), 2.83 (q, J= 6.3 Hz, 2H), 2.09 (t, J = 7.4 Hz, 2H), 1.50 (h, J= 7.4 Hz, 2H), 1.38 - 1.12 (m, 6H), 0.98 - 0.90 (m, 3H). LC-MS: Rt, 3.52 min; m / z, 257.5 ([M+H]+).

[0315] lodoacetamidation. In a scintillation vial equipped with a PTFE-coated stirring bar, A-(2-aminoethyl)-6-((47?,5S)-5-methyl-2-oxoimidazolidin-4-yl)hexanamide trifluoroacetate SI-36 (35 mg, 94.5 pmol, 1.0 equiv.) was dissolved in MeCN:H2<D (1:1, 2 ml), then NaOH IM in H2O (220 pl) was added and the solution was cooled to 0 °C. lodoacetic anhydride (167 mg, 0.47 mmol, 5.0 equiv.) was added and the reaction was warmed to room temperature and stirred for 16 h. The reaction was purified by HILIC (method 5), affording the title compound (23.4 mg, white solid). 'H NMR (500 MHz, DMSO-d6) 8 8.26 (s, 1H), 7.78 (s, 1H), 6.29 (s, 1H), 6.11 (s, 1H), 3.65 - 3.57 (m, 3H), 3.48 (t, J= 7.3 Hz, 1H), 3.08 (s, 4H), 2.04 (t, J = 7.5 Hz, 2H), 1.48 (p, J = 7.5 Hz, 2H), 1.41 - 1.11 (m, 6H), 1.00 - 0.91 (m, 3H). LC-MS: Rt, 3.71 min; m / z, 412.1 ([M+H]+). The experimental data agree a commercial vendor report (MedChemExpress, cat. No. HY-150230).

[0316] Synthetic procedures and characterization - covalent fragments

[0317] General procedure. In a scintillation vial equipped with a PTFE-coated stirring bar, the appropriate amine (1.0 equiv.) and pyridine (1.5 equiv.) were dissolved in CH2Q2 (0.2 M) or CH2Q2 / DMF (0.2 M, if stated), then chloroacetyl chloride (1.5 equiv.) was added dropwise and the reaction was stirred at room temperature for 30 minutes. The volatiles were subsequently removed by spiral evaporation (30 °C), the residue was redissolved in DMF (500 uL) and purified by preparative HPLC (method 1).

[0318] 2-Chloro-l-(4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-l-yl)ethan-l-one(Fl). Synthesized from l-(5-(trifluoromethyl)pyridin-2-yl)piperazine ([CAS 132834-58-3], Aldrich, 95%, 61 mg, 0.26 mmol), obtained as a white solid (1.6 mg). 'H NMR (500 MHz, CDCh) 5 8.42 (s, 1H), 7.68 (d, J = 8.7 Hz, 1H), 6.67 (d, J = 8.9 Hz, 1H), 4.12 (s, 2H), 3.84 - 3.73 (m, 4H), 3.68 - 3.63 (m, 4H).19F NMR (471 MHz, CDCh) 5 -61.3. 'H NMR (500 MHz, DMSO-d6) 8 8.43 (d, J= 2.7 Hz, 1H), 7.82 (dd, J= 9.0, 2.6 Hz, 1H), 6.98 (d, J= 9.1 Hz, 1H), 4.44 (s, 2H), 3.72 - 3.66 (m, 4H), 3.63 - 3.54 (m, 4H).19F NMR (471 MHz, DMSO-d6) 8 -59.4.13C{XH} NMR (126 MHz, DMSO-d6) 5 164.9, 160.0, 145.2 (q, J= 4.5 Hz), 134.6 (q, J = 3.2 Hz), 124.8 (q, J = 270.1 Hz), 113.6 (q, J= 32.3 Hz), 106.5, 44.6, 44.0, 43.7, 42.0, 41.2. LC-MS: Rt, 5.87 min; m / z, 308.2 ([M+H]+).

[0319] N-(2-(lH-Pyrazol-l-yl)pyridin-3-yl)-2-chloroacetamide (F2). Synthesized from 2-(177-pyrazol-l-yl)pyridin-3-amine ([CAS 172784-50-8], Enamine, 56.9 mg, 0.36 mmol), obtained as a white solid (37.1 mg), white solid. 'H NMR (500 MHz, CDCh) 8 12.72 (s, 1H), 9.04 (dd, J = 8.1, 1.6 Hz, 1H), 8.64 (d, J = 2.7 Hz, 1H), 8.19 (dd, J = 4.7, 1.6 Hz, 1H), 7.80 (d, J= 1.9 Hz, 1H), 6.51 (t, J = 2.3 Hz, 1H), 4.22 (s, 2H).13C NMR (126MHz, CDCh) 5 166.1, 142.8, 141.1, 138.9, 130.6, 129.4, 125.8, 122.2, 107.2, 43.4. LC-MS: Rt, 6.18 min; m / z, 237.1 ([M+H]+).

[0320] 2-Chloro-2V-(4-phenyloxazol-2-yl)acetamide (F3). Synthesized from 4- phenyloxazol-2-amine ([CAS 33119-65-2], Enamine, 95%, 33.2 mg, 0.21 mmol), obtained as a white solid (24.5 mg). 'H NMR (500 MHz, CDCh) 5 9.48 - 8.52 (br, 1H), 7.75 (s, 1H), 7.68 (d, J= 7.6 Hz, 2H), 7.42 (t, J= 7.5 Hz, 2H), 7.34 (t, J= 7.3 Hz, 1H), 4.70 - 3.86 (br s, 2H).13CfH} NMR (126 MHz, CDC13:CD3OD 6:1) 5 168.6 (br), 156.7 (br), 144.0 (br), 134.1 (br), 132.7, 132.3, 129.3, 46.5. [Due to limited solubility in organic solvents, the13C NMR spectrum was recorded in CDC13:CD3OD, which causes some signal broadening. One aromatic signal is missing due to broadening] LC-MS: Rt, 5.51 min; m / z, 237.0 ([M+H]+).

[0321] 2-Chloro-l-(3-(trifluoromethyl)-5,6-dihydro-[l,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)ethan-l-one (F4). Synthesized from 3-(trifluoromethyl)-5,6,7,8-tetrahydro- [l,2,4]triazolo[4,3-a]pyrazine ([CAS 486460-21-3], ChemCruz, 38.2 mg, 0.20 mmol), obtained as a colorless gum (37.5 mg). 'H NMR (500 MHz, CDC13) 5 5.06 (s, 2H), 4.40 - 4.27 (m, 1H), 4.26 - 4.15 (m, 3H), 4.15 - 4.09 (m, 1H), 4.08 - 3.97 (m, 1H). LC-MS: Rt, 4.66 min; m / z, 269.0 ([M+H]+). The experimental data are in accordance with patent EP2270009, 2011.

[0322] 2-Chloro-l-(4-(4-chlorophenyl)piperazin-l-yl)ethan-l-one (F5).Synthesized from l-(4-chlorophenyl)piperazine ([CAS 38212-33-8], ChemCruz, 45.0 mg, 0.23 mmol), obtained as a white solid (46.1 mg). 'H NMR (500 MHz, CDC13) 5 7.26 - 7.12 (m, 2H), 6.93 - 6.74 (m, 2H), 4.11 (s, 2H), 3.79 (t, J= 5.2 Hz, 2H), 3.68 (t, J = 5.0 Hz, 2H),3.21 (t, J= 5.1 Hz, 2H), 3.15 (t, J= 5.6 Hz, 2H).13C{XH} NMR (126 MHz, CDCh) 5 165.3, 149.5, 129.3, 125.9, 118.1, 49.8, 49.4, 46.3, 42.1, 40.9. LC-MS: Rt, 6.20 min; m / z, 273.1 ([M+HD-

[0323] N-(7-Bromo-[l,2,4]triazolo[l,5-a]pyridin-2-yl)-2-chloroacetamide (F6).Synthesized from 7-bromo-[l,2,4]triazolo[l,5-a]pyridin-2-amine ([CAS 947248-68-2], Ark Pharm Inc., 47.5 mg, 0.22 mmol). Reaction performed in CH2Ch:DMF (4:1, 0.2 M).Obtained as a white solid (45.9 mg). 'H NMR (500 MHz, CDCh) 5 8.99 (s, 1H), 8.71 (s, 1H), 7.63 (d, J= 9.3 Hz, 1H), 7.55 (d, J= 9.4 Hz, 1H), 4.30 (br s, 2H).13CfH} NMR (126 MHz, CDCh:CD3OD 6:1) 5 157.9, 148.5, 134.3, 129.0, 115.60, 115.57, 107.8, 42.6. [Due to limited solubility in organic solvents, the13C NMR spectrum was recorded in CDCh:CD3OD] LC-MS: Rt, 4.59 min; m / z, 289.0 ([M+H]+).

[0324] 2-Chloro-2V-(4-(pyridin-4-yl)thiazol-2-yl)acetamide (F7). Synthesized from 4-(pyridin-4-yl)thiazol-2-amine ([CAS 30235-28-0], Chem-Impex, 45.2 mg, 0.26 mmol). Reaction performed in CH2Ch:DMF (4:1, 0.2 M). Obtained as a yellow gum (2.4 mg). 'H NMR (500 MHz, CDCh) 5 8.67 (d, J = 4.7 Hz, 2H), 7.74 (d, J = 5.2 Hz, 2H), 7.45 (s, 1H), 4.33 (s, 2H). The compound undergoes decomposition over time in the available NMR solvents, therefore no clean13C NMR spectrum could be obtained. LC-MS: Rt, 4.00 min; m / z, 254.1 ([M+H]+)

[0325] l-(8-Oxa-3-azabicyclo[3.2.1]octan-3-yl)-2-chloroethan-l-one (F8).Synthesized from 8-oxa-3-azabicyclo[3.2.1]octane hydrochloride ([CAS 54745-74-3], J&W Pharmalab, 96%, 50.2 mg, 0.34 mmol), obtained as colorless thick oil (51.6 mg). 'H NMR (500 MHz, CDCh) 8 4.59 (d, J= 6.9 Hz, 1H), 4.14 (d, J= 6.1 Hz, 1H), 4.09 - 3.96 (m, 2H), 3.74 (dd, J= 14.4, 11.0 Hz, 2H), 3.71 - 3.60 (m, 2H), 2.16 - 1.98 (m, 3H), 1.97 - 1.86 (m, 1H).13C{XH} NMR (126 MHz, CDCh) 8 162.3, 72.8, 72.2, 57.3, 53.6, 41.3, 27.8, 26.1. LC- MS: Rt, 4.18 min; m / z, 190.1 ([M+H]+).

[0326] 2V-(Benzo[rf]thiazol-6-yl)-2-chloroacetamide (F9). Synthesized from benzo[7]thiazol-6-amine ([CAS 533-30-2], Alfa-Aesar, 98+%, 53.3 mg, 0.35 mmol).Reaction performed in CEhChiDMF (4:1, 0.2 M). Obtained as white solid (34.7 mg). 'H NMR (500 MHz, CDCh) 5 8.96 (s, 1H), 8.55 (d, J= 2.2 Hz, 1H), 8.42 (br s, 1H), 8.10 (d, J = 8.7 Hz, 1H), 7.43 (dd, J= 8.7, 2.2 Hz, 1H), 4.25 (s, 2H).13C{XH} NMR (126 MHz, CDCh) 5 164.1, 154.1, 150.7, 135.0, 134.6, 123.9, 119.3, 113.1, 43.0. LC-MS: Rt, 4.85 min; m / z, 227.0 ([M+H]+).

[0327] 2-Chloro-N-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)acetamide (F10).Synthesized from 7-amino-4-(trifluoromethyl)-277-chromen-2-one ([CAS 53518-15-3], Oakwood, 48.7 mg, 0.21 mmol). Reaction performed in CH2Ch:DMF (4:1, 0.2 M). Obtained as off-white solid (30.8 mg). 'H NMR (500 MHz, CDCh) 8 8.46 (s, 1H), 7.89 (d, J = 2.2 Hz, 1H), 7.71 (dd, J = 8.8, 1.9 Hz, 1H), 7.46 (dd, J = 8.8, 2.2 Hz, 1H), 6.74 (s, 1H), 4.24 (s, 2H).13C{XH} NMR (126 MHz, CDCh:CD3OD 6:1) 8 165.8, 159.6, 155.1, 142.3, 141.4 (q, J = 33.1 Hz), 125.9 (d, 7= 3.0 Hz), 121.4 (q, J = 275.4 Hz), 116.6, 113.9 (q, 7 = 5.8 Hz), 109.8, 107.8, 42.9.19F NMR (471 MHz, CDCh:CD3OD 6:1) 8 -61.0. [Due to limited solubility inorganic solvents,13C NMR and19F NMR spectra were recorded in CDC13:CD3OD] LC-MS: Rt, 6.03 min; m / z, 306.1 ([M+H]+).

[0328] 2-Chloro-\-(6-chloropyridazin-4-yl (acetamide (Fll). Synthesized from 6- chloropyridazin-4-amine ([CAS 29049-45-4], Enamine, 95%, 43.6 mg, 0.34 mmol). Reaction performed in CEhChiDMF (4:1, 0.2 M). Obtained as light orange solid (8.2 mg). 'H NMR (500 MHz, CDCh) 5 9.14 (d, J = 2.3 Hz, 1H), 8.77 (s, 1H), 8.24 (d, J = 2.3 Hz, 1H), 4.27 (s, 2H).13C{XH} NMR (126 MHz, CDC13:CD3OD 6:1) 5 167.2, 157.1, 142.6, 139.6, 115.8, 42.6. [Due to limited solubility in organic solvents, the13C NMR spectrum was recorded in CDC13:CD3OD] LC-MS: Rt, 4.57 min; m / z, 206.0 ([M+H]+).

[0329] Methyl 5-bromo-2-(2-chloroacetamido)nicotinate (F12). Synthesized from methyl 2-amino-5-bromonicotinate ([CAS 52727-57-8], Aldrich, 96%, 40.0 mg, 0.17 mmol).Obtained as yellow solid (24.4 mg). 'H NMR (500 MHz, CDC13) 5 11.48 (s, 1H), 8.68 (d, J = 2.4 Hz, 1H), 8.47 (d, J= 2.5 Hz, 1H), 4.36 (s, 2H), 4.00 (s, 3H).13CfH} NMR (126 MHz, CDCh) 5 165.9, 164.8, 153.9, 150.3, 142.4, 114.3, 113.2, 53.5, 44.0. LC-MS: Rt, 5.68 min; m / z, 307.0 ([M+H]+).

[0330] 2-Chloro-N-(5-(furan-2-yl)-l,3,4-oxadiazol-2-yl)acetamide (F13).Synthesized from 5-(furan-2-yl)-l,3,4-oxadiazol-2-amine ([CAS 7659-06-5], Chem-Impex, 99.2%, 56.6 mg, 0.37 mmol). Reaction performed in CH2Ch:DMF (4:1, 0.2 M). Obtained aswhite solid (43.2 mg). 'H NMR (500 MHz, DMSO-d6) 5 12.27 (br s, 1H), 8.04 (d, J = 1.8 Hz, 1H), 7.25 (d, J = 3.6 Hz, 1H), 6.78 (dd, J = 3.6, 1.8 Hz, 1H), 4.44 (s, 2H).13C{XH} NMR (126 MHz, DMSO-d6) 8 164.4, 156.3, 153.7, 146.7, 138.3, 113.8, 112.6, 43.0. LC-MS: Rt, 4.47 min; m / z, 228.0 ([M+H]+).

[0331] ( / ?)-2-Chloro-l-(3-phenoxypiperidin-l-yl)ethan-l-one (F14). Synthesized from (R)-3-phenoxypiperidine ([CAS 1885089-47-3], Ambeed, 85.6 mg, 0.40 mmol). Obtained as colorless thick gum (92.8 mg). 'H NMR (500 MHz, CDCh) 5 7.32 - 7.27 (m, 2H), 7.02 - 6.88 (m, 3H), 4.45 (tt, J = 6.1, 3.2 Hz), 4.34 (dd, J= 13.2, 3.7 Hz), 4.27 (tt, J =7.8, 3.6 Hz), 4.11 (ap q, J = 12.2 Hz, 2H), 4.00 (ap dd, J = 45.2, 12.2 Hz, 2H), 3.81 - 3.74 (m), 3.72 - 3.61 (m), 3.47 (ddd, J= 12.6, 8.0, 3.5 Hz), 3.32 (ddd, J= 13.5, 9.8, 3.3 Hz), 3.20 (dd, J= 13.0, 8.1 Hz), 2.18 - 2.10 (m), 2.05 - 1.85 (m), 1.85 - 1.74 (m), 1.72 - 1.61 (m), 1.60 - 1.50 (m). ^CfH} NMR (126 MHz, CDCh) 5 165.9, 165.6, 157.3, 156.8, 130.0,129.8, 121.8, 121.6, 116.2, 116.0, 71.3, 70.8, 50.3, 46.8, 46.5, 43.1, 41.3, 41.2, 30.1, 29.1, 23.5, 21.4. [Due to rotamers and non-equivalent axial-equatorial positions, more signals are observed. Integrals are reported when unambiguously meaningful] LC-MS: Rt, 6.08 min; m / z, 254.1 ([M+H]+).

[0332] 2-Chloro- \ -(6-methoxybenzo|d|thiazol-2-yl (acetamide (F30). Synthesized from 6-methoxybenzo[d]thiazol-2-amine ([CAS 1747-60-0], Aldrich, 98%, 48.8 mg, 0.27 mmol). Reaction performed in CH2Cl2:DMF (4:1, 0.2 M). Obtained as white solid (44.3 mg). 'H NMR (500 MHz, DMSO-d6) 8 12.58 (s, 1H), 7.66 (d, J= 8.8 Hz, 1H), 7.59 (s, 1H), 7.05 (d, J= 8.8 Hz, 1H), 4.44 (s, 2H), 3.81 (s, 3H).13CfH} NMR (126 MHz, DMSO-d6) 8 165.6,156.3, 155.4, 142.6, 132.8, 121.4, 115.1, 104.8, 55.6, 42.5. LC-MS: Rt, 5.63 min; m / z, 257.0 ([M+H]+).

[0333] 2V,A-Dibenzyl-2-chloroacetamide (F31). Synthesized from dibenzylamine([CAS 103-49-1], Aldrich, 97%, 59.1 mg, 0.30 mmol). Obtained as colorless thick gum (59.1 mg). 'H NMR (500 MHz, CDCh) 5 7.43 - 7.28 (m, 6H), 7.22 (d, J = 7.3 Hz, 2H), 7.17 (d, J = 7.4 Hz, 2H), 4.62 (s, 2H), 4.52 (s, 2H), 4.15 (s, 2H).13CfH} NMR (126 MHz, CDCh) 5 167.4, 136.6, 135.8, 129.3, 128.9, 128.4, 128.1, 127.8, 126.7, 50.4, 48.8, 41.5. LC-MS: Rt,6.75 min; m / z, 274.1 ([M+H]+).

[0334] 2,4,6-Triiodobenzene-l,3,5-tricarbonyl trichloride.

[0335] Part of the synthesis was adapted from Bai and co-workers (Iriorg. Chem. 2020, 59, 8081-8098).

[0336] In a round-bottom flask equipped with a PTFE-coated stirring bar, 1,3,5- triiodomesitylene (synthesized according to J. Organomet. Chem. 1998, 569, 195-202, 10.95 g, 22.0 mmol, 1.0 equiv.) was dissolved in pyridine:H2O (2.4: 1, 186 ml) and heated at 90 °C, then KMnCU (65.7 g, 0.24 mol, 11.0 equiv.) was added in four portions every 50 minutes. After 24 hours at 90 °C, the reaction was hot filtered over Celite, washed with 5% aqueous KOH (200 ml), and pyridine was azeotropically removed by rotary evaporation (50 °C at 5- 10 mbar). The liquid residue was acidified up to pH 1 with HC1 37%, cooled to 0 °C and the resulting precipitate was collected by filtration, washed once with ice-cold water, and dried under high vacuum (10‘2mbar), affording 2,4,6-triiodobenzene-l,3,5-tricarboxylic acid (5.18 g, white solid). HPLC-MS: Rt, 1.49 min; m / z, 542.7 (100%, [M-C02]’), 1174.5 (50%, [2M- H]-).

[0337] In a scintillation vial equipped with a PTFE-coated stirring bar, 2,4,6- triiodobenzene-l,3,5-tricarboxylic acid (252 mg, 0.43 mmol, 1.0 equiv.) was suspended in thionyl chloride (3 ml) with catalytic DMF (10 pl) and heated at 65 °C for 6 hours. The volatiles were removed by spiral evaporation (40 °C) and under high vacuum (10‘2mbar), affording the title compound (276 mg, off-white solid) which was used without additional purification.

[0338] 2,2'-((5-((3-Azidopropyl)carbamoyl)-2,4,6- triiodoisophthaloyl)bis(azanediyl))bis(N,N,N-trimethylethan-l-aminium) bisformate.

[0339] In a scintillation vial equipped with a PTFE-coated stirring bar, 2,4,6- triiodobenzene-l,3,5-tricarbonyl trichloride (104 mg, 0.16 mmol, 1.0 equiv.) was dissolved in DMF (2 ml), then DIPEA (70 pl, 0.40 mmol, 2.5 equiv.) and 3 -azidopropan- 1 -amine (15.7 pl, 0.16 mmol, 1.0 equiv.) was added in one portion and the reaction was stirred at room temperature. After 30 minutes, cholamine chloride hydrochloride (112.7 mg, 0.64 mmol, 4.0 equiv.) and DIPEA (140 pl, 0.80 mmol, 5.0 equiv.) were added and the reaction was stirred at room temperature. After 24 hours, water (100 pl) was added, then the volatiles were removed by spiral evaporation (40 °C), the residue was dissolved in MeCN:H2O (1 : 1, 1 ml) and purified by HPLC (method 1) and HILIC (method 5), affording the title compound (4.8 mg, colorless film). ’H NMR (500 MHz, D2O) 5 8.40 (formate, s, 2H), 4.02 - 3.88 (m, 4H), 3.71 (t, J= 7.1 Hz, 4H), 3.55 (t, J= 6.7 Hz, 2H), 3.51 (t, J= 6.6 Hz, 2H), 3.27 (s, 18H), 1.97 (p, J = 6.6 Hz, 2H).13CfH} NMR (126 MHz, D2O) 5 172.5, 172.4, 172.0, 171.9, 171.0, 148.6, 147.83, 147.80, 90.8, 90.3, 63.2, 53.5, 48.7, 37.2, 34.1, 27.2. Additional splitting was observed due to hindered rotation around amide bonds. HILIC-MS: Rt, 3.34 min; no molecular ion detected.

[0340] 4,4'-(5-((3-Azidopropyl)carbamoyl)-2,4,6-triiodoisophthaloyl)bis(l,l- dimethylpiperazin-l-ium) bisformate.

[0341] In a scintillation vial equipped with a PTFE-coated stirring bar, N-Boc- piperazine (1.86 g, 10.0 mmol, 1.0 equiv.), K2CO3 (1.38 g, lO.Ommol, 1.0 equiv.) were suspended in acetone (15 ml), then Mel (6.23 ml, 0.10 mol, 10.0 equiv.) was added dropwise over 5 minutes, then the reaction was heated to 60 °C. After 3 hours the reaction was warmed to room temperature and the volatiles were removed by spiral evaporation (40 °C). The residual solid was extracted three times with CHCI3 (30 ml each time), then the combined organic extracts were evaporated to dryness via rotary evaporation (40 °C at 250 mbar, then 40 °C at 5-10 mbar). The impure waxy residue containing 4-(tert-butoxycarbonyl)-l,l- dimethylpiperazin-l-ium iodide (3.40 g, supposedly 9.94 mmol, 1.0 equiv.) was suspended in water (10 ml), then HC1 37% (5 ml) was added, and the reaction was stirred at room temperature. After 24 hours, the volatiles were removed via rotary evaporation (50 °C at 5-10 mbar), affording a yellow gummy solid. The residue was triturated with boiling acetone:water (15: 1, 32 ml), then cooled down and the white solid predominantly containing 1,1- dimethylpiperazin-l-ium iodide hydrochloride (2.13 g) was collected by filtration.1H NMR (500 MHz, D2O) 5 3.87 - 3.82 (m, 4H), 3.81 - 3.75 (m, 4H), 3.39 (s, 6H). HILIC-MS: Rt, 4.25 min; m / z, 115.1 (100%, [M]+). The experimental data agrees with the literature report (J. Mol. Struct. 2015, 1094, 210-236).

[0342] In a scintillation vial equipped with a PTFE-coated stirring bar, 2,4,6- triiodobenzene-l,3,5-tricarbonyl trichloride (104 mg, 0.16 mmol, 1.0 equiv.) was dissolved in DMF (2 ml), then DIPEA (70 pl, 0.40 mmol, 2.5 equiv.) and 3 -azidopropan- 1 -amine (15.7 pl, 0.16 mmol, 1.0 equiv.) was added in one portion and the reaction was stirred at room temperature. After 30 minutes, 1,1-dimethylpiperazin-l-ium iodide hydrochloride (179 mg, 0.64 mmol, 4.0 equiv.) and DIPEA (140 pl, 0.80 mmol, 5.0 equiv.) were added and the reaction was stirred at room temperature. After 24 hours, water (100 pl) was added, then the volatiles were removed by spiral evaporation (40 °C), the residue was dissolved inMeCNifhO (1:1, 1 ml) and purified by HPLC (method 1) and HILIC (method 5), affording the title compound (2.0 mg, colorless film).!H NMR (500 MHz, D2O) 5 8.40 (s, 2H), 4.41 - 3.16 (br m, 27H), 3.00 (s, 3H), 1.97 (p, J = 6.7 Hz, 2H). Signal broadening due to rotamers does not allow the acquisition of a13C NMR spectrum. HILIC-MS: Rt, 3.29 min; no molecular ion detected.A suitable bisimidazole is alkylated with a heterobifunctional bromo-ester optionally including a PEG linker. The alkylated bisimidazole is then permethylated with excess methyl iodide, then the ester converted to an acid either using lithium hydroxide or trifluoroacetic acid. Following deesterification, the product (a bisimidazolium connected to a carboxylic acid via a flexible linker) is then converted to a lysine-reactive electrophile through functionalization (DCC / EDCI, base, n-hydroxysuccinimide or sulfo-n-hydroxysuccinimide, hexafluorobenzene, acetyl chloride, or thionyl chloride), affording bisimidazolium conjugates bearing NHS, sulfo-NHS, pentafluorophenyl ester, acid anhydride, or acid chloride functionality.

[0343]

[0344]

[0345]

[0346]

[0347] REFERENCES1. Dundas, C. M., Demonte, D. & Park, S. Streptavidin-biotin technology: improvements and innovations in chemical and biological applications. Appl. Microbiol. Biotechnol. 97, 9343-9353 (2013).2. Niinae, T., Ishihama, Y. & Imami, K. Biotinylation-based proximity labelling proteomics: basics, applications and technical considerations. J. Biochem. (Tokyo) 170, 569- 576 (2021).3. Rozanova, S. et al. Quantitative Mass Spectrometry-Based Proteomics: An Overview, in Quantitative Methods in Proteomics (eds. Marcus, K., Eisenacher, M. & Sitek, B.) 85-116 (Springer US, New York, NY, 2021). doi:10.1007 / 978-l-0716-1024-4_8.4. Zhang, J., Al-Eryani, R. & Ball, H. L. Mass Spectrometry Analysis of 2- Nitrophenylhydrazine Carboxy Derivatized Peptides. J. Am. Soc. Mass Spectrom. 22, (2011).5. Mirzaei, H. & Regnier, F. Enhancing Electrospray Ionization Efficiency of Peptides by Derivatization. Anal. Chem. 78, 4175-4183 (2006).6. Zabet-Moghaddam, M., Shaikh, A. L. & Niwayama, S. Peptide peak intensities enhanced by cysteine modifiers and MALDI TOF MS. J. Mass Spectrom. 47, 1546-1553 (2012).7. Vasicek, L. & Brodbelt, J. S. Enhanced Electron Transfer Dissociation through Fixed Charge Derivatization of Cysteines. Anal. Chem. 81, 7876-7884 (2009).8. Ko, B. J. & Brodbelt, J. S. Enhanced Electron Transfer Dissociation of Peptides Modified at C-terminus with Fixed Charges. J. Am. Soc. Mass Spectrom. 23, 1991-2000 (2012).9. Frey, B. L. et al. Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation. J. Am. Soc. Mass Spectrom. 24, 1710-1721 (2013).10. Xu, Y., Zhang, L., Lu, H. & Yang, P. Mass Spectrometry Analysis of Phosphopeptides after Peptide Carboxy Group Derivatization. Anal. Chem. 80, 8324-8328 (2008).11. Qiao, X. et al. Piperazines for peptide carboxyl group derivatization: effect of derivatization reagents and properties of peptides on signal enhancement in matrix-assisted laser desorption / ionization mass spectrometry. Rapid Commun. Mass Spectrom. 25, 639-646 (2011).12. Meier, F., Park, M. A. & Mann, M. Trapped Ion Mobility Spectrometry and Parallel Accumulation-Serial Fragmentation in Proteomics. Mol. Cell. Proteomics 20, 100138 (2021).13. Dodds, J. N. & Baker, E. S. Ion Mobility Spectrometry: Fundamental Concepts, Instrumentation, Applications, and the Road Ahead. J. Am. Soc. Mass Spectrom. 30, 2185— 2195 (2019).14. Huang, T., Armbruster, M. R., Coulton, J. B. & Edwards, J. L. Chemical Tagging in Mass Spectrometry for Systems Biology. Anal. Chem. 91, 109-125 (2019).15. Hughes, C. S. et al. Ultrasensitive proteome analysis using paramagnetic bead technology. Mol. Syst. Biol. 10, 757 (2014).16. Yang, K. et al. Accelerating multiplexed profiling of protein-ligand interactions: High-throughput plate-based reactive cysteine profiling with minimal input. Cell Chem. Biol. 31, 565-576 (2023).17. Dubiella, C. et al. Sulfopin is a covalent inhibitor of Pinl that blocks Myc-driven tumors in vivo. Nat. Chem. Biol. 17, 954-963 (2021).18. Kudo, N. et al. Leptomycin B inactivates CRMl / exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc. Natl. Acad. Sci. 96, 9112-9117 (1999).19. Chen, Y.-C. et al. Covalent Modulators of the Vacuolar ATPase. J. Am. Chem. Soc. 139, 639-642 (2017).20. Senkane, K. et al. The Proteome-Wide Potential for Reversible Covalency at Cysteine. Angew. Chem. Int. Ed. 58, 11385-11389 (2019).21. Pegg, A. E. Multifaceted Roles of Alkyltransferase and Related Proteins in DNA Repair, DNA Damage, Resistance to Chemotherapy, and Research Tools. Chem. Res. Toxicol. 24, 618-639 (2011).22. Li, H. et al. Assigning functionality to cysteines by base editing of cancer dependency genes. Nat. Chem. Biol. 19, 1320-1330 (2023).23. Craven, G. B. et al. High-Throughput Kinetic Analysis for Target-Directed Covalent Ligand Discovery. Angew. Chem. Int. Ed. 57, 5257-5261 (2018).1A. Kong, A. T., Leprevost, F. V., Avtonomov, D. M., Mellacheruvu, D. & Nesvizhskii, A. I. MSFragger: ultrafast and comprehensive peptide identification in mass spectrometrybased proteomics. Nat. Methods 14, 513-520 (2017).2A. Yu, F., Haynes, S. E. & Nesvizhskii, A. I. lonQuant Enables Accurate and Sensitive Label-Free Quantification With FDR-Controlled Match-Between-Runs. Mol. Cell.Proteomics 20, 100077 (2021).3 A. Yu, F. et al. Fast Quantitative Analysis of timsTOF PASEF Data with MSFragger and lonQuant. Mol. Cell. Proteomics 19, 1575-1585 (2020).4A. Yang, K. L. et al. MSBooster: improving peptide identification rates using deep learning-based features. Nat. Commun. 14, 4539 (2023).5A. da Veiga Leprevost, F. et al. Philosopher: a versatile toolkit for shotgun proteomics data analysis. Nat. Methods 17, 869-870 (2020).6A. Huber, W., von Heydebreck, A., Siiltmann, H., Poustka, A. & Vingron, M. Variance stabilization applied to microarray data calibration and to the quantification of differential expression. Bioinformatics 18, S96-S104 (2002).7A. Smyth, G. K. limma: Linear Models for Microarray Data, in Bioinformatics and Computational Biology Solutions Using R and Bioconductor (eds. Gentleman, R., Carey, V. J., Huber, W., Irizarry, R. A. & Dudoit, S.) 397-420 (Springer, New York, NY, 2005). doi: 10.1007 / 0-387-29362-0_23.8A. Gottlieb, H. E., Kotlyar, V. & Nudelman, A. NMR Chemical Shifts of Common Laboratory Solvents as Trace Impurities. J. Org. Chem. 62, 7512-7515 (1997).9A. Hernandez-Alonso, D., Zankowski, S., Adriaenssens, L. & Ballester, P. Water-soluble aryl-extended calix[4]pyrroles with unperturbed aromatic cavities: synthesis and binding studies. Org. Biomol. Chem. 13, 1022-1029 (2015).10A. Lo, K. K.-W. et al. Cyclometalated iridium(III) polypyridine dibenzocyclooctyne complexes as the first phosphorescent bioorthogonal probes. Chem. Commun. 49, 4271-4273 (2013).11 A. Lai, W. et al. Design, Synthesis, and Bioevaluation of a Novel Hybrid Molecular Pyrrolobenzodiazepine-Anthracenecarboxyimide as a Payload for Antibody-Drug Conjugate. J. Med. Chem. 65, 11679-11702 (2022).12A. Wilson, J. M. et al. Synthesis, characterisation and anti-protozoal activity of carbamate-derived poly azamacrocycles. Org. Biomol. Chem. 5, 3651-3656 (2007).13 A. Chirkin, E. et al. Neutralization of Pathogenic Fungi with Small-Molecule Immuno therapeutic s. Angew. Chem. Int. Ed. 56, 13036-13040 (2017).14A. Heller, K. et al. Covalent Protein Labeling by Enzymatic Phosphocholination. Angew. Chem. Int. Ed. 54, 10327-10330 (2015).15A. Yang, K. S., Budin, G., Tassa, C., Kister, O. & Weissleder, R. Bioorthogonal Approach to Identify Unsuspected Drug Targets in Live Cells. Angew. Chem. Int. Ed. 52, 10593-10597 (2013).16A. Hirozane, Y. et al. Structure-based rational design of staurosporine-based fluorescent probe with broad-ranging kinase affinity for kinase panel application. Bioorg. Med. Chem. Lett. 29, 126641 (2019).17A. Pham, T. A., Xu, J. & Raymond, K. N. A Macrocyclic Chelator with Unprecedented Th4+ Affinity. J. Am. Chem. Soc. 136, 9106-9115 (2014).18A. Liang, Y.-K. et al. Natural product-based design, synthesis and biological evaluation of anthra[2,l-d]thiazole-6, 11-dione derivatives from rhein as novel antitumour agents. Eur. J. Med. Chem. 84, 505-515 (2014).19A. Woods, M. et al. Synthesis, Relaxometric and Photophysical Properties of a New pH- Responsive MRI Contrast Agent: The Effect of Other Ligating Groups on Dissociation of a p-Nitrophenolic Pendant Arm. J. Am. Chem. Soc. 126, 9248-9256 (2004).20A. Bencini, A. et al. A large cavity cryptand for recognition of dianionic substrates in aqueous solution. Tetrahedron Lett. 38, 5327-5330 (1997).21A. Ye, H. et al. A Safe and Facile Route to Imidazole- 1- sulfonyl Azide as a Diazotransfer Reagent. Org. Lett. 15, 18-21 (2013).22A. Mao, F., Mano, N. & Heller, A. Long Tethers Binding Redox Centers to Polymer Backbones Enhance Electron Transport in Enzyme “Wiring” Hydrogels. J. Am. Chem. Soc. 125, 4951-4957 (2003).23A. Leval, A. et al. Hydrogen production from formic acid catalyzed by a phosphine free manganese complex: investigation and mechanistic insights. Green Chem. 22, 913-920 (2020).24A. Matsumoto, S., Watanabe, M. & Akazome, M. Incrementing Stokes Shifts through the Formation of 2,2'-Biimidazoldiium Salts. Org. Lett. 20, 3613-3617 (2018).25A. Li, Z. et al. Synthesis, conformational analysis and antibacterial activity of Au(I)- Ag(I) and Au(I)-Hg(II) heterobimetallic N-heterocyclic carbene complexes. Dalton Trans. 49, 12820-12834 (2020).26A. Jacinto, M. P., Fried, S. D. & Greenberg, M. M. Intracellular Formation of a DNA Damage-Induced, Histone Post-Translational Modification Following Bleomycin Treatment. J. Am. Chem. Soc. 144, 7600-7605 (2022).27A. Maruyama, K. et al. Protein Modification at Tyrosine with Iminoxyl Radicals. J. Am. Chem. Soc. 143, 19844-19855 (2021).28A. Mangubat-Medina, A. E., Martin, S. C., Hanaya, K. & Ball, Z. T. A Vinylogous Photocleavage Strategy Allows Direct Photocaging of Backbone Amide Structure. J. Am. Chem. Soc. 140, 8401-8404 (2018).INCORPORATION BY REFERENCE

[0348] All of the U.S. patents and U.S. patent application publications cited herein are hereby incorporated by reference.EQUIVALENTS

[0349] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the various embodiments of the disclosure described herein. Such equivalents are encompassed by the following claims.

Claims

We claim:

1. A compound of formula (I), (II), or (III):whereinA is a reactive warhead;Li and L2 are independently linkers or absent;R2 in each instance is independently an alkyl;R in each instance is independently selected from the group consisting of alkyl, halo, alkoxy, amino, thio, aryl, and heteroaryl; each n is independently an integer from 0 to 5; n’ is an integer from 0 to 5; and n” is independently an integer from 0 to 3; or salt thereof.

2. The compound of claim 1, wherein Li and L2 are independently selected from CH2,, each of which can be substituted by one or more substituents, wherein m is an integer from 0-5; n is an integer from 0-5; o is an integer from 1-3; and p is an integer from 1-3.

3. The compound of claim 1 or 2 wherein n’ is 0.

4. The compound of claim 1 or 2 wherein n’ is 1.

5. The compound of any of claims 1-4, wherein Li is -(CH2)6-6. The compound of any of claims 1-4, wherein Li7. The compound of any of claims 1-6, wherein L2 is -CH2CH2-.

8. The compound of any of claims 1-6, wherein L2 is absent.

9. The compound of any of claims 1-8, wherein n” is 0.

10. The compound of claim 1, wherein the compound is11. The compound of claim 1, wherein the compound is12. The compound of claim 1, wherein the compound isalt thereof.

13. The compound of claim 1, wherein the compound is14. A compound of formula (IV):wherein each X is independently selected from H, halo, substituted or unsubstituted alkyl, aryl, heteroaryl, cycloalkyl, and heterocyclylLi is a linker;A is a reactive warhead; and each Ri and R2 are independently selected from H, substituted or unsubstituted alkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl or Ri and R2 together with the atoms to which they are attached form a five- or six-membered heterocyclyl; or a salt thereof.

15. The compound of claim 14, provided that at least one of R1and R2is charged (e.g., a group comprising a quaternary ammonium or iminium).more substituents, wherein m is an integer from 0-5; n is an integer from 0-5; o is an integer from 1-3; and p is an integer from 1-3.

17. The compound of any one of claims 14-16, wherein Li is18. The compound of any one of claims 14-17, wherein each X is halo (e.g., iodo).

19. The compound of any one of claims 14-18, wherein Ri is H.

20. The compound of claim 19, wherein R2 is substituted alkyl.

21. The compound of claim 19 or 20, wherein R2 is substituted ethyl.

22. The compound of any one of claims 19-21, wherein R2 is ammonium-substituted ethyl.

23. The compound of any one of claims 14-18, wherein Ri and R2 together with the atoms to which they are attached form a six-membered heterocyclyl.

24. The compound of claim 23, wherein Ri and R2 together with the atoms to which they are attached form25. The compound of claim 14, wherein the compound is selected fromor a salt thereof.

26. The compound of any of claims 1-25, wherein the reactive warhead comprises N3.

27. The compound of any of claims 1-25, wherein the reactive warhead comprises -N(H)C(0)CH2l, -N(H)C(O)CH2C1, a phenol, an aniline, an azide, an alkyne, or a tetrazine.

28. The compound of any of claims 1-27, wherein the compound comprises one or more unnatural isotopes of a carbon, a hydrogen, a nitrogen, or an oxygen atom.

29. A method of modulating ion mobility for gas phase enrichment of a modified peptide or selectively measuring a modified peptide comprising:covalently modifying a peptide to generate a modified peptide; and spatially separating the modified peptide from an unlabeled peptide using ion mobility spectrometry in the gas phase.

30. The method of claim 29, wherein covalently modifying a peptide comprises reacting the peptide with a cationic (e.g., polycationic) residue-reactive agent.

31. The method of claim 30, wherein the cationic residue reactive agent has a small collisional cross-section.

32. The method of claim 30 or 31, wherein covalently modifying the peptide comprises reacting the peptide with a residue-reactive agent having the structure of a compound of any one of claims 1-17.

33. The method of any one of claims 29-32, wherein covalently modifying the peptide comprises covalently modifying the peptide to alter its 1 / ko and m / z physical properties, rendering it different from a 2+ and 1+ charged unmodified peptide.

34. The method of any one of claims 29-33, the method further comprising selectively fragmenting the modified peptide to generate reporter ions.

35. The method of any one of claims 29-34, the method further comprising sequencing the modified peptide in a tandem IM / MS mass spectrometer.

36. The method of claim 35, wherein sequencing the modified peptide in a tandem IM / MS mass spectrometer comprises data-dependent acquisition or data-independent acquisition.

37. The method of any one of claims 29-36, the method further comprising reacting the modified and the unlabeled peptide with a gas-phase base.

38. The method of claim 37, wherein the gas-phase base has a proton affinity of at least 230 kcal / mol.

39. The method of claim 37 or 38, wherein the gas-phase base is selected from the group consisting of collidine, DIPEA, and TMG.

40. The method of claim 23, wherein covalently modifying the physical properties of a peptide comprises minimal perturbation of collisional cross-section and mass, but high charge difference to increase its ion mobility.

41. The method of claim 19, wherein the residue-reactive reagent reacts with lysine residues via an amine reactive electrophile (e.g., an NHS ester or PFP ester).

42. The method of claim 30, wherein the residue-reactive reagent reacts with cysteine residues via a cysteine reactive electrophile (e.g., a haloacetamide, alpha / beta unsaturated ketone, activated nitrile).

43. The method of claim 42, wherein the residue-reactive reagent reacts with cysteine residues via an iodoacetamide.

44. The method of claim 19, wherein the residue reactive agent is a dicationic, cysteinereactive reagent.

45. The method of claim 44, wherein the cysteine-reactive agent has a small collisional cross-section.

46. The method of claim 44 or 45, wherein the cysteine-reactive reagent is an innately cationic polyimidazolium shift reagent.

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