Tunable Heteroaromatic Sulfones Enhance in-Cell Cysteine Profiling

Article abstract of DOI:10.1021/jacs.9b08831

Heteroaromatic sulfones react with cysteine via nucleophilic aromatic substitution, providing a mechanistically selective and irreversible scaffold for cysteine conjugation. Here we evaluate a library of heteroaromatic sulfides with different oxidation st

Full text of DOI:10.1021/jacs.9b08831

Subscriber access provided by BIU Pharmacie | Faculté de Pharmacie, Université Paris V
Article
Tunable heteroaromatic sulfones enhance in-cell cysteine profiling
Hashim F Motiwala, Yu-Hsuan Kuo, Brittany L. Stinger, Bruce A. Palfey, and Brent R. Martin
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b08831 • Publication Date (Web): 27 Dec 2019
Downloaded from pubs.acs.org on December 28, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 21
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Tunable heteroaromatic sulfones enhance in-cell cysteine profiling
Hashim F. Motiwala†, Yu-Hsuan Kuo†,§, Brittany L. Stinger†, Bruce A. Palfey‡,§, Brent R. Martin†,§,||,
*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
†Department of Chemistry, §Program in Chemical Biology, University of Michigan, 930 N. University Ave, Ann Arbor, MI
48109. ‡Department of Biological Chemistry, University of Michigan Medical School, 5220E MSRB III 1150 W. Medical
Center Drive Ann Arbor, MI 48109, ||Department of Medicinal Chemistry, College of Pharmacy, University of Michigan,
428 Church St., Ann Arbor, MI 48109
ABSTRACT: Heteroaromatic sulfones react with cysteine via nucleophilic aromatic substitution, providing a mechanistically selec-
tive and irreversible scaffold for cysteine conjugation. Here we evaluate a library of heteroaromatic sulfides with different oxidation
states, heteroatom substitutions, and a series of electron donating and electron-withdrawing substituents. Select substitutions pro-
foundly influence reactivity and stability compared to conventional cysteine conjugation reagents, increasing the reaction rate by >3-
orders of magnitude. The findings establish a series of synthetically accessible electrophilic scaffolds tunable across multiple tunable
centers. New electrophiles and their corresponding alkyne-conjugates were profiled directly in cultured cells, achieving thiol satura-
tion in a few minutes at sub-millimolar concentrations. Direct addition of desthiobiotin-functionalized probes to cultured cells sim-
plified enrichment and elution to enable mass spectrometry discovery of >3000 reactive and/or accessible thiols labeled in their native
cellular environments in a fraction of the standard analysis time. Surprisingly, only 1/2 of annotated cysteines were identified by both
iodoacetamide-desthiobiotin and methylsulfonylbenzothiazole-desthiobiotin in replicate experiments, demonstrating complementary
detection by mass spectrometry analysis. These probes offer advantages over existing cysteine alkylation reagents, including accel-
erated reaction rates, improved stability, and robust ionization for mass spectrometry applications. Overall, heteroaromatic sulfones
provide modular tunability, shifted chromatographic elution times, and superior in-cell cysteine profiling for in-depth proteome-wide
analysis and covalent ligand discovery.
the methyl sulfonyl leaving group for thiopropionic acid12. De-
spite the potentially broad reactivity of these heteroaromatic
INTRODUCTION
First reported in 2012, methylsulfonylbenzothiazole (MSBT;
BT-1) reacts with cysteine by a nucleophilic aromatic substitu-
tion (SNAr) mechanism (Scheme 1 and Figure 1a).1 This class
of electrophiles was later expanded to include more efficient
oxadiazole (OD-1) and tetrazole (Tz-2) methyl sulfones (Fig-
ure 1a), which improve thiol blocking2 and form stable protein-
drug conjugates2a, 3. These heteroaromatic sulfone electrophiles
show advantages over maleimide for thiol blocking and anti-
body-drug conjugates since they demonstrate exquisite selec-
tivity for thiols (–SH) (i.e. no reaction with –SOH, –SNO, ly-
sine, etc.)2b and form exceptionally stable, irreversible adducts3.
In contrast to iodoacetamide and maleimide, the SNAr mecha-
nism prevents conjugation to sulfenic acids (R–SOH), provid-
ing a useful approach for thiolate selective cysteine conjuga-
tion, particularly for thiol blocking in cysteine oxidation analy-
sulfones, there are no systematic efforts to tune their reactivity,
explore different heterocycles, evaluate distinct oxidation
states, measure stability, quantify reaction rates, or expand their
utility for cysteine activity/reactivity-based profiling and other
proteomics applications.
Scheme 1
Current cysteine profiling methods use iodoacetamide-al-
kyne (IAM-alkyne) labeling in soluble cell or tissue homoge-
nates13. The TOP-ABPP (Tandem Orthogonal Proteolysis – Ac-
tivity-Based Protein Profiling)14 approach enables site-specific
analysis of cysteine accessibility and reactivity13b, redox modi-
fication status15, and/or covalent engagement16. Cells homoge-
nates are treated with differing concentrations of iodoacetam-
ide-alkyne to identify exceptionally reactive cysteines (enzyme
active site, redox sensitive sites) that resist dilution effects. Af-
ter click chemistry labeling with biotin affinity tags, a control
and an experimental sample of modified cysteine-peptides are
enriched and released (either chemically or proteolytically) for
analysis by mass spectrometry, resulting in comparative ratios
reflecting site-specific cysteine reactivity/occupancy. These
technically challenging conjugation and enrichment steps can
introduce significant variance. These sources of error are miti-
gated by introducing isotopic affinity tags, stable isotope label-
ing with amino acids in cell culture (SILAC), or other
sis2b, 4
.
The heteroaromatic pharmacophores are privileged structures
in medicinal chemistry, commonly found in many FDA-
approved drugs across diverse therapeutic applications.5 Hence,
beyond their utility as bioconjugation reagents3, 6 , similar scaf-
folds have also emerged in inhibitors for Glucagon-like Pep-
tide-1 Receptor (GLP-1R)7 and bacterial dihydrolipoamide S-
succinyltransferase8 as well as anti-microbial, anti-fungal, and
anti-cancer activities.9 In addition, 2-sulfonylpyrimidines have
been reported as covalent stabilizers of mutant p5310, as well as
thiol-reactive inducers of redox stress for treating tuberculo-
sis11. A recent picomolar potency Rho/MRTF/SRE pathway in-
hibitor reported an identical core scaffold to OD-1, substituting
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 21
quantitative labeling methods17. Once optimized, this approach
provides proteome-wide measurements of cysteine engagement
by covalent inhibitors or inhibitor fragments16, 18, greatly en-
hancing their preclinical development. Despite a wide number
of successes in vitro, the TOP-ABPP approach is constrained
by weak iodoacetamide-alkyne reactivity, which limits rapid in-
cell capture of accessible thiols.
incubated for 30 min at rt. BT-5, BT-6, BT-7, BT-9, BT-10, OD-1, and
TD-1 showed different degrees of insolubility. 5-TMRIA (tetramethyl-
rhodamine-5-iodoacetamide dihydroiodide) was added to each sample
and incubated for 30 min at rt (10% final DMSO content). The reaction
was quenched by the addition of 5X Laemmli sample buffer, heated at
~90 °C for 5 min, and separated by 12% SDS-PAGE. Gels were visu-
alized using the Cy3 channel on Amersham Typhoon Gel and Blot Im-
aging Systems. Gels were then stained with Coomassie to image pro-
tein loading using the Cy5 channel.
1
2
3
4
5
6
7
8
9
Here we report a systematic exploration of heteroaromatic
sulfone electrophiles, revealing general rules for tuning electro-
philicity, both through sulfur oxidation and electronic substitu-
tions that reproduce across different scaffolds. We report the
first application of heteroaromatic sulfones in bottom-up prote-
omics applications. Optimal scaffolds were selected for desthio-
biotin conjugation, allowing robust in-cell conjugation and site-
specific mass spectrometry analysis of accessible and/or reac-
tive cysteines in their native cellular environment. Overall,
these efforts complement iodoacetamide-based cysteine profil-
ing to measure an orthogonal subset of cysteine peptides. Such
chemical and physical complementarity establishes heteraro-
matic sulfones as a distinct class of synthetically accessible,
tunable covalent probes.
Live cell alkyne-probe labeling. HeLa cells were incubated with
either DMSO, IAM-alkyne, BT-alkyne, Tz-alkyne, and OD-alkyne in
cysteine-free DMEM media (without cystine, methionine, L-gluta-
mine, and sodium pyruvate) for 45 min at 37 °C. Cell lysate was pre-
pared and normalized to 1 mg/mL protein. Copper(I)-catalyzed alkyne-
azide cycloaddition (CuAAC) reaction was performed using 50 µg of
protein with a premixed solution at standard concentrations of TBTA
(tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) and copper (II)
sulfate, TAMRA-azide, and sodium ascorbate for 1 h in dark. The re-
action was quenched by the addition with 5X Laemmli sample buffer,
heated at ~85 °C for 5 min, and analyzed by 12% SDS-PAGE. Gels
were visualized using a Cy3 channel (TAMRA) and Cy5 (Coomassie)
on Amersham Typhoon Gel and Blot Imaging Systems.
Mass spectrometry analysis of labeled cysteines. Mouse brain sol-
uble proteome or probe-treated HeLa cell lysates (1 h treatment) in
DPBS (1X) was diluted with 50 mM HEPES (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid) buffer. The sample was heat denatured
at ~100 °C for 15 min followed by sonication of resulting hot cloudy
solution in an ultrasonic bath for ~1–2 min at ~40 °C. The denatured
sample was treated with 1 mM TCEP for 1 h at rt in dark followed by
incubation with respective electrophile for 30 min. Importantly, we
found TCEP reacts with IAM and most heteroaromatic sulfones, de-
creasing their actual concentrations by ~50% in 1 h. Next, the sample
was digested with trypsin (1:20 ratio), diluted with 50 mM TEAB (tri-
ethylammonium bicarbonate) buffer (pH 8.5), and incubated at 37 °C
for 18 h. The samples were desalted using an Oasis HLB SPE resin on
a Water Oasis µElution 96-well plate. The peptides were eluted with
acetonitrile, dried using a SpeedVac (45 °C, 5 mbar, 45 min), and stored
at –80 °C prior to analysis.
For live cell labeling, samples were extracted with chloroform/meth-
anol to remove salts and excess reagents and resuspended in 100 µL of
2 M urea in DPBS with 0.2% SDS and 88 µL of 50 mM TEAB by
sonication. The solution was then digested with ~1:30 trypsin at 37 °C
for 15 h, dried by SpeedVac, and resuspended in 300 µL of 1X DPBS.
For enrichment, the tryptic peptides were incubated with Pierce high
capacity streptavidin agarose resin (150 µL, 50% slurry in 1X DPBS)
for 4 h rotating at rt. After transfer to Bio-Rad micro Bio-Spin columns,
the samples were washed 5 times with 0.75 mL of DPBS and 9 times
with 0.75 mL of DI water. The peptides from each sample were eluted
from the resin by incubating the resin two times with 100 µL of the
elution buffer (1:1 ACN:water with 0.1% TFA, LCMS-grade) for ~5
min at rt and one time with 100 µL of the elution buffer for ~5 min at
60 °C. The total eluents (300 µL) containing the enriched peptides were
concentrated on a SpeedVac.
Peptides were reconstituted in water with 0.1% TFA and analyzed
by Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Scien-
tific). An Acclaim PepMap 100 C18 LC column (50 cm long x 75µm
diameter x 2 µm particle size) was used. LC solvents were: 0.1% FA in
H2O (Buffer A) and 0.1% FA in 80:20 ACN:H2O (Buffer B). Peptides
were eluted into the mass spectrometer at a flow rate of 300 nL/min
over a 120 minutes linear gradient (5-40% Buffer B from 2–95 min and
then 40-90% Buffer B from 95–115 min) at 35 °C. MS data was ac-
quired in a data-dependent mode. Full MS spectra were collected with
following parameters (Orbitrap resolution: 60,000; scan range: m/z
300-1800 (400–2000 for desthiobiotin probes); AGC target: 2.0e5;
maximum injection time: 50 ms). The MS2 spectra were collected with
following scan parameters (Orbitrap resolution: 15,000; AGC target:
1.0e5; maximum injection time: 22 ms; isolation window: m/z 1.6; CID
collision energy: 34%). Dynamic exclusion was set to 40 s with a mass
tolerance of 10 ppm. The MS data was analyzed with Thermo Proteome
Discoverer (V2.2.0.388) and searched against the mouse or human pro-
teome (Uniprot) and a common list of contaminants. The precursor
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL METHODS
Reaction rate determination. Reactions were performed in 20%
acetonitrile (ACN) in Dulbecco’s phosphate buffered saline (DPBS)
(2X) at pH 7.5 supplemented with 5 mM EDTA. Measurements were
performed using an Agilent Cary 100 UV-Visible spectrophotometer.
The compound (30 µM) was quickly mixed with N-acetyl-L-cysteine
methyl ester at five different concentrations, depending on the relative
absorbance. A UV-visible absorbance spectrum was acquired at the
most responsive wavelength for time-dependent rate measurements.
For more reactive electrophiles, a Hi-Tech Scientific KinetAsyst SF-61
DX2 stopped-flow spectrophotometer was used in single mixing mode
to monitor the rapid formation of cysteine-electrophile conjugate.
Pseudo first order rate constants were obtained by fitting the data using
“one-phase association/decay” in a GraphPad Prism. And second order
reaction rate was obtained by plotting the kobs at different concentra-
tions of cysteine.
Electrophile stability determination. HPLC analysis was per-
formed on Agilent 6520 Accurate-Mass Q-TOF LC/MS using a Waters
C18 column (4.6 x 75 mm; 3.5 µm particle size). LC solvents were:
0.1% formic acid (FA) and 10 mM ammonium formate (AF) in H2O
(Buffer A) and 0.1% FA and 10 mM AF in 90:10 ACN:H2O (Buffer
B). A standard gradient (5–95% buffer B over 11 min) at 0.40 mL/min
was used to analyze each electrophile (1 mM in PBS) at different time
points of incubation at room temperature (0, 4, and 24 h). Area under
the curve (either at 250 or 280 nm wavelength) was integrated at each
time point and analyzed using Agilent MassHunter software.
Cell and tissue lysate preparation. HeLa cells were maintained in
DMEM supplemented with 10% fetal bovine serum (FBS) and 1% Pen-
icillin–Streptomycin under 5% CO2 atmosphere at 37 °C. Cells were
harvested by scraping, washed, and sonicated at 4 °C. Mouse brains
(C57BL6) were dissected, frozen in liquid N2, and stored at –80 °C.
Tissues were later thawed on ice, dounce homogenized in DPBS (1X)
and centrifuged at 3,000 x g for 5 min at 4 °C to remove debris. The
supernatant was cleared by centrifugation (35,000 x g for 45 min at 4
°C) to yield the soluble proteome. Protein concentrations were meas-
ured using a DC assay (Bio-Rad).
Electrophile profiling in mouse brain soluble proteome. Mouse
brain soluble proteome in DPBS (1X) was adjusted with appropriate
amount of modified RIPA lysis buffer (pH 7.41, 50 mM Tris‧HCl, 150
mM NaCl, 2 mM EDTA, 0.10% SDS) to 1 mg/mL protein. The result-
ing lysate (50 µg of protein in 46 µL for each sample) was treated with
1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride) for 1 h at
room temperature (rt) in dark. TCEP was removed using Vivaspin® 500
µL ultrafiltration spin column and supplemented with 3.6% DMSO to
ensure compound solubility. Electrophiles (5mM) were added and
ACS Paragon Plus Environment

Page 3 of 21
Journal of the American Chemical Society
mass tolerance was set at 10 ppm, fragment mass tolerance was set to
efficiency of cysteine alkylation mirrored the measured reactiv-
ity trend with free cysteine (OD-1 > Tz-1 > BT-1 > BI-1). This
result conflicts with a separate report finding OD-1 as the least
efficient electrophile in cell lysates, which may reflect its mar-
ginal aqueous stability.2b Surprisingly, incubation with either 5
mM IAM or BT-1 was insufficient to block all thiols, while
NEM and OD-1 saturated all sites. 4-(5-(Methylsulfonyl)-1H-
tetrazol-1-yl)phenol (MSTP; Tz-1) displayed reactivity in be-
tween BT-1 and OD-1, while benzimidazole methyl sulfone
(BI-1) showed weak or no cysteine reactivity. Altogether, these
findings corroborate a direct relationship between the isolated
reaction rates and cysteine alkylation efficiency in complex pro-
teomes. Clearly modulating the heterocycle immensely influ-
ences the cysteine reactivity of the heteroaromatic sulfones.
0.1 Da, and maximum missed cleavage sites was set to 1. The false
discovery rate for peptides, proteins and sites identification was set to
1%. The minimum peptide length was set to five amino acids and pep-
tide re-quantification was enabled. The minimal number of peptides per
protein was set to one. Methionine oxidation, deamidation (N and Q),
N-acetylation on a protein terminus, and modification of cysteines or
other amino acids by a respective electrophile were searched as dy-
namic modifications.
Western Blotting. SDS-PAGE gels were transferred to a polyvinyl-
idene difluoride (PVDF) membrane (75V, 2 h, ~4 °C) using a Mini
Trans-Blot Cell (Bio-Rad). The membrane was washed, blocked with
4% w/v of BSA in 1X TBST, and incubated with streptavidin-Cy 8
(1:3000) in 1X TBST for 1.5 h. Primary antibody incubation (1:1000
mouse alpha-tubulin for loading standard) was performed in 4% BSA
solution in 1X TBST for 2 h followed by further incubation with sec-
ondary antibody (1:4000 of Alexa Fluor 647 donkey anti-mouse) in 1X
TBST for 1 h on a rotator at rt in a dark. After washings, the membrane
was briefly dried in air and then imaged on Azure C600 Imager: 800
NIR channel and Cy5 channel were used for Streptavidin-Cy8 and
Alexa Fluor, respectively.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Heteroaromatic scaffold optimization. In order to assess
the potential to tune cysteine reactivity, we evaluated the struc-
ture-activity profile across heteroaromatic scaffolds. More than
20 different heteroaromatic sulfones were synthesized, assayed
by UV-absorbance kinetics, and profiled by IAM-TAMRA in-
gel competitive ABPP (Figures 2 and 3). This analysis re-
vealed a broad range of reactivities and provides general design
rules applicable across distinct classes of heteroaromatic sul-
fones.
RESULTS AND DISCUSSION
General heteroaromatic sulfones. Heteroaromatic sulfones
are validated reagents for bioconjugation, providing a platform
for generation of stable, serum-resistant antibody-drug conju-
gates3, 19. In addition, Tz-1 added to live cells prevents post-ly-
sis oxidation of S-sulfenylated peroxiredoxin2b, yet a brief (5
min) incubation with 10 mM Tz-1 fails to block the majority of
thiols in living cells2b. In order to more accurately quantify het-
eroaromatic sulfone reactivity, a UV absorbance assay was de-
veloped to monitor the rate of cysteine conjugation to different
electrophiles (Figure 1a). NEM yielded the fastest reaction rate
(1585 ± 109 M-1s-1), which proceeds >3-orders of magnitude
faster than IAM (0.902 ± 0.05 M-1s-1). Heteroaromatic sulfone
reaction rates were variable, where OD-1 was ~75x faster than
Tz-1, ~500x faster than IAM, and ~850x faster than BT-1.
Figure 2. Evaluation of cysteine reactivity across different het-
eroaromatic scaffolds. (a) Structures of heteroaromatic methyl sul-
fones evaluated. Measured reaction rates are shown in parentheses
below each electrophile. Standard deviations are derived from rep-
licates with multi-point curve-fitting. nd = not determined. (b)
Competitive IAM-TAMRA activity-based profiling of cysteine
electrophiles (5 mM) by in-gel fluorescence.
Figure 1. Competitive ABPP profile of cysteine electrophiles in
mouse brain soluble lysate. (a) Structures and rates of cysteine elec-
trophiles. Measured reaction rates are shown in parentheses below
each electrophile. Standard deviations are derived from replicates
with multi-point curve-fitting. nd = not determined. (b) In-gel flu-
orescence analysis of 5 mM heteroaromatic sulfone, IAM, or NEM
followed by incubation and detection with 20 µM IAM-TAMRA.
Remarkably, replacing the sulfur atom in benzothiazole to an
oxygen atom in benzoxazole (BO-1) increased reactivity by
>1600-fold, achieving rates 2x faster than OD-1 (Figure 2).
Conversely, substituting the oxadiazole with a thiadiazole (TD-
1) completely eliminated cysteine reactivity. Other five- and
six-membered heteroaromatic methyl sulfones, including
To validate the proteome reactivity of this class of electro-
philes, we evaluated reported heteroaromatic sulfones by gel-
based competitive activity-based protein profiling (ABPP) with
iodoacetamide-tetramethylrhodamine (IAM-TAMRA; 5-
TMRIA) in mouse brain homogenates (Figure 1b). The relative
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 21
phenyltriazole (Trz-1), quinoxaline (QX-1), quinazoline (QZ- rate and cysteine reactivity. Thus, not all scaffolds are as readily
1
2
3
4
5
6
7
8
1), pyrimidine (Pymd-1), and pyridine (Py-1), showed no sig-
nificant cysteine labeling. Despite a general loss of reactivity,
analogues with weak or no apparent in-gel competition may still
engage select cellular targets. While not generally reactive in
our assays, 2-sulfonylpyrimidines are reported to alkylate and
partially rescue mutant p53 activity.10 Clearly, subtle electron-
ics translate to major variations in heteroaromatic sulfone reac-
tivity, and thus imparting the capacity to derive selectivity
through rational reactivity tuning.
tunable with simple substitutions.
Benzothiazole scaffold optimization. Reported oxadiazole
(OD-1) and tetrazole (Tz-1) scaffolds differ from the benzothi-
azole and benzoxazole scaffolds, where a benzene ring is di-
rectly fused to the heterocycle2a. Since benzoxazole compound
BO-1 was highly reactive and unstable, we sought to explore if
benzothiazoles could be more effectively tuned to modulate re-
activity. A small series of benzothiazole derivatives were syn-
thesized covering a range of substituents on both the benzene
ring and sulfone (Figure 3a). Across the benzothiazole series,
subtle changes in electronics played a major role in tuning the
electrophilicity (Figure 3a-b). For example, the electron-donat-
ing methoxy-substituted analogue (BT-2) was ~20-fold slower
than unsubstituted BT-1. Electron-withdrawing substituents
such as carboxylic acid (BT-3), methyl ester (BT-4), and nitro
(BT-5) greatly accelerated the reaction rate by 5x, 300x, and
3200x, respectively. The reactivity of BT-5 mirrored NEM in
aqueous buffer, providing equivalent reactivity as the classic bi-
oconjugation reagent. A linear Hammett plot across substituted
benzothiazoles reported a positive slope (r = 4.464), validating
the electronic tunability of this reactive scaffold20 (Figure 3c).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We next examined if leaving group modifications could in-
fluence reactivity. To test this hypothesis, we synthesized an
electron-withdrawing trifluoromethylsulfonyl analogue (BT-6)
and a trifluoroethylsulfonyl analogue (BT-7). Compared to BT-
1, fluorine substitution increased the reaction rate of BT-6 by
~38-fold, while BT-7 was only 8-fold faster than its corre-
sponding non-fluorinated analogue (BT-3). Conversely, ethyl
(BT-8) and phenyl (BT-9) sulfonyl benzothiazoles were nearly
unreactive, corroborating reports that steric effects impact leav-
ing group ability. Presumably due to the high intrinsic reactivity
and instability, we were unable to synthesize a hybrid electro-
phile combining both the 6-methyl ester substituent on the ben-
zene ring with the trifluoromethylsulfonyl leaving group. This
again highlights a potential reactivity ceiling, where the intrin-
sic stability and hydrolysis requires a corresponding balance
with reactivity. Finally, the minimalist thiazole methyl sulfones
(T-1 and T-2) showed marginal or no cysteine reactivity, estab-
lishing the importance of a benzene-fused heterocycle.
Oxidation state profiling. Given the tunability of both the
benzothiazole scaffold and the sulfone leaving group, we next
examined the influence of different oxidation states of sulfur on
the reaction rate (Figure 4a-b). Oxidation of the 6-nitrobenzo-
thiazole thioether to the corresponding methyl sulfoxide (BT-
10) imparted robust cysteine reactivity, which was then further
enhanced by oxidation to the sulfone (BT-5). A similar trend
was observed with the phenyloxadiazole methyl sulfoxide (OD-
2) and benzoxazole methyl sulfoxide (BO-2), where the corre-
sponding thioethers were virtually non-reactive. Further oxida-
tion to the corresponding methyl sulfones OD-1 and BO-1 en-
hanced the reactivity by 10-15-fold. The 6-carboxylic acid ben-
zothiazole trifluoromethyl sulfoxide (BT-11) was more reactive
than the 2-methylsulfonyl benzothiazole-6-carboxylic acid BT-
3, but less reactive than the benzothiazole trifluoromethyl sul-
fone BT-6.
Figure 3. Substitutions on benzothiazoles modulate cysteine reac-
tivity. (a) Structures of benzothiazole analogues evaluated. Meas-
ured reaction rates are shown in parentheses below each electro-
phile. Standard deviations are derived from replicates with multi-
point curve-fitting. nd = not determined. (b) Competitive IAM-
TAMRA activity-based profiling of cysteine electrophiles (5 mM)
by in-gel fluorescence. (c) Hammett plot of benzothiazole deriva-
tives.
Next, we hypothesized that substitution of an electron-with-
drawing group on OD-1 or BO-1, such as –CO2H on the aryl-
ring, might enhance reactivity. Despite extensive efforts, nei-
ther products were stable and decomposed to corresponding hy-
drolysis product. Conversely, derivatives of phenyltetrazole
scaffold with either an electron-donating (–OH; Tz-1), neutral
(–H; Tz-2), or electron-withdrawing (–CO2H; Tz-3) group at
the para-position of the phenyl ring revealed little difference in
Further IAM-TAMRA competitive ABPP labeling at various
concentrations in mouse brain homogenates confirmed the
identification of several promising heteroaromatic sulfones and
sulfoxides with effective thiol blocking concentrations as low
as 0.10 mM (Figure S1). Overall, tuning the sulfur oxidation
state provides a simple strategy to dial electrophilicity across
ACS Paragon Plus Environment

Page 5 of 21
Journal of the American Chemical Society
general heterocyclic scaffolds. This finding is particularly rele- prevent mixed disulfide formation in proteomics applications.
1
2
3
4
5
6
vant as heterocyclic thioethers and sulfoxides are present in sev-
eral FDA-approved drugs, including omeprazole, cefazolin, and
latamoxef.21 These drugs can potentially undergo oxidation dur-
ing metabolism or due to cellular redox stress, which could im-
part significant reactivity to a generally inert scaffold.
Typical bottom-up proteomics workflows reduce protein sam-
ples with TCEP or DTT, followed by addition of excess (>20
mM) freshly prepared IAM.23 Based on the accelerated rate con-
stants and robust stability of new analogues, we selected IAM,
NEM, and several heteroaromatic sulfone probes for in-depth,
unenriched mass spectrometry profiling.
Mouse brain soluble proteome was heat-denatured, reduced
with TCEP, and incubated with different probes for 30 minutes.
The mixture was then digested with trypsin and analyzed by
high resolution liquid chromatography mass spectrometry (LC-
MS). Even without any enrichment, a short 2 h gradient yielded
>3000 unique cysteine-modified peptides in duplicate analyses
(Tables 1 and S1). While the number of unique cysteine-mod-
ified peptides identified for IAM was similar to BT-4, both OD-
1 and Tz-1 yielded more labeled cysteine peptides. Conversely,
combined analysis of both ring-opened and ring-closed NEM
yielded significantly fewer cysteine-modified peptides than the
other electrophiles. Overall, even without enrichment, this anal-
ysis identified >3000 peptides for each electrophile in a 2 h LC-
MS experiment and ~6600 unique peptides pooled across dif-
ferent electrophiles. This finding suggests leveraging electro-
phile orthogonality can enhance cysteine coverage.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Heteroaromatic methyl sulfoxides are attenuated cysteine
electrophiles. (a) Structures of heteroaromatic methyl sulfoxide an-
alogues evaluated. Measured reaction rates are shown in parenthe-
ses below each electrophile. Standard deviations are derived from
replicates with multi-point curve-fitting. (b) Competitive IAM-
TAMRA activity-based profiling of cysteine electrophiles (5 mM)
by in-gel fluorescence.
Table 1. Unenriched one-dimensional LC-MS proteomic evalu-
ation of optimized heteroaromatic sulfones in mouse brain soluble
proteome. The number of unique peptides from replicate analysis
(N = 2) are listed. Peptides identified as deamidated, oxidized, or
with different charge states are counted as one unique peptide. Ly-
sine-modified peptides represent 1% or less of the total annotated
peptides, or equivalent to assigned peptide false-discovery rate.24
Heteroaromatic sulfone stability in physiological buffer.
In comparison with NEM-cysteine conjugates, heteroaromatic–
cysteine2a and heteroaromatic–selenocysteine6b conjugates
demonstrate superior stability with no observable hydrolysis or
reversibility. However, the stability of unconjugated heteroaro-
matic sulfones in aqueous buffers has not been reported. There-
fore, we sought to establish any correlation between the ob-
served reactivity across different heterocyclic classes of het-
eroaromatic sulfones with their stability. Nine different electro-
philes were profiled in phosphate-buffered saline (PBS) at pH
7.4 under ambient conditions for 4 and 24 h by HPLC (Figure
S2). Based on its material safety data sheet, iodoacetamide is
light-sensitive, unstable, and requires fresh preparation at the
time of use.22 In contrast, we found IAM to be quite stable in
PBS over 24 h. On the other hand, only ~50% of NEM was in-
tact after 4 h. In agreement with their enhanced reactivity, OD-
1 and BO-1 were the least stable heteroaromatic sulfones, un-
dergoing considerable hydrolysis in the aqueous solution over
24 h. Therefore, disparate reports of OD-1 reactivity are likely
explained by a decomposed aliquot2. Interestingly, solid BO-1
was completely decomposed after 6 months storage at –20 °C,
while solid OD-1 was stable under similar conditions for at least
a year. In addition, benzothiazole and tetrazole methyl sulfones
were remarkably stable in PBS. Even though its reaction rate is
comparable to NEM, BT-5 maintained exceptionally high sta-
bility. In fact, the aqueous solution of Tz-1 and BT-4 were
benchtop stable for more than a week (data not shown), demon-
strating robust properties for long-term storage. Altogether, the
heterocyclic ring scaffolds contribute to the inherent stability of
these compounds.
Unique Unique Cys-modified Lys-modified
Electrophile
proteins peptidespeptides
peptides
none
IAM
NEM
Tz-1
OD-1
BT-4
3171
3217
2990
3170
3148
3253
21116 0
0
21602 3354
20020 2620
22348 3709
21909 3939
21764 3213
79
201
121
302
90
In addition, the ratios of annotated cysteine to annotated ly-
sine labeled peptides for Tz-1, BT-4, and IAM were 30–42-
fold. Conversely, the ratio for maleimide and OD-1 only
reached 13, suggesting reduced selectivity. Importantly, gel-
based competitive analysis with NHS-TAMRA did not reveal
any significant competition (Figure S3), implying the anno-
tated sites are either highly abundant, exhibit high reactivity, or
reflect false positives assigned within the 1% false-discovery
rate.
Despite the dramatically disparate reaction rates, the number
of modified peptides identified are similar across different elec-
trophiles at these labeling concentrations. Interestingly, just 1.5
mM iodoacetamide (rather than >20 mM) was sufficient to al-
kylate >95% of cysteines across the proteome. Presumably, the
remaining <5% are unreactive or inaccessible even in the pres-
ence of highly reactive electrophiles. To our surprise, different
electrophiles reported a distinct, yet reproducible subset of
Mass spectrometry-based proteomics with heteroaro-
matic sulfones. IAM is the primary alkylation reagent used to
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 21
cysteine peptides (Figure S4). Different probes reported only As expected, addition of 100 µM probe to cells yielded the
1
2
3
4
5
6
7
8
~55% overlap in peptide coverage. Since 81±1% of cysteine-
peptides overlapped between runs within the same sample
group, the observed orthogonality is unlikely derived from sam-
ple variation. Instead, the orthogonality is more likely attributed
to shifts to less complex chromatographic windows (Figure 5a)
or differences in fragmentation, which can directly influence
peptide assignment. The elution profile for IAM overlaps more
closely with unmodified peptides, while Tz, OD, and BT elute
significantly later in the chromatographic gradient, potentially
shifting their analysis away from peptide-dense elution times.
This effect would increase the probability of data-dependent ion
selection and improve ion purity (reduce co-isolation) to en-
hance peptide assignment.
highest labeling, albeit with subtle differences in the profile of
labeled proteins. Unique labeling profiles were observed at
lower probe concentrations in mouse brain soluble proteome,
which may better represent differences across the most reactive
cysteines (Figure S5). Despite large differences in reactivity,
Tz-alkyne and BT-alkyne achieved similar intracellular label-
ing at equivalent concentrations. IAM also achieved relatively
robust labeling, even though it is much less reactive than other
probes. Presumably, the most reactive thiols are not greatly in-
fluenced by changes in probe electrophilicity. In addition, OD-
alkyne achieved the lowest labeling in live cells, presumably
due to its poor solubility and/or accelerated hydrolysis in aque-
ous media. Based on this analysis, solubility and cell permea-
bility play important roles in the efficiency of intracellular cys-
teine labeling.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
It is also possible that different electrophiles have less bene-
ficial electrospray ionization properties, which could impact de-
tection of conjugated peptides. By averaging the ion current
across commonly identified peptides, we derived a relative
electrospray efficiency for each probe. Maleimide showed the
weakest mean ion current, which likely contributes to the lower
conjugate-peptide annotations. Otherwise, the relative elec-
trospray ionization of heteroaromatic sulfone probes equaled or
out-performed IAM (Figure 5b), further validating their ex-
panded utility in proteomics applications.
Figure 5. Electrophile profiling for optimal LC-MS analysis. (a)
Right-shifted reversed-phase elution of heteroaromatic-labeled
cysteine peptides. (b) Ion current intensity profile of labeled cyste-
ine-peptides compared to matching unmodified peptides.
Labeling cysteines in live cells. Given their drug-like prop-
erties, we next evaluated heteroaromatic sulfone cell permea-
bility and cysteine alkylation in live cultured HeLa cells. Al-
kyne-conjugated probes were synthesized to perform live cell
cysteine alkylation, followed by cell lysis, click chemistry, and
in-gel detection. These tools provide a modular platform for
TOP-ABPP enrichment and site-specific analysis. IAM-
Alkyne, Tz-Alkyne, and BT-Alkyne retained similar reaction
rates as their parent electrophile, whereas the rate of OD-alkyne
was 3-fold lower (Figure 6a), likely influenced by the less elec-
tronically favorable ether. Since the carboxylic acid of OD- and
BO- probes were too unstable to purify, OD-alkyne employs a
propargyl ether linkage with reduced reactivity.
Figure 6. Live-cell cysteine labeling with alkyne-conjugated het-
eroaromatic sulfones. (a) Structures of alkyne-conjugated probes
evaluated. Measured reaction rates are shown in parentheses below
each electrophile. Standard deviations are derived from replicates
with multi-point curve-fitting. (b) Concentration-dependent activ-
ity-based profiling of cysteine-labeling in HeLa cells. Visualized
by in-gel fluorescence following click chemistry to TAMRA-azide.
Site of labeling analysis. Typical TOP-ABPP analysis sample
preparation is manually intensive, involving extensive click
chemistry steps, washes, and TEV cleavage prior to LC-MS
analysis14a. Peptides are then analyzed across at least 5 separate
fractions, totaling >10 h of instrument analysis time. Recent
studies report cysteine peptide coverage anywhere from 800 to
5000 labeled cysteine peptides13b, 25. Alternatively, IAM-
desthiobiotin enabled directed enrichment and detection of
~2700 cysteine-labeled peptides in cell lysates when analyzed
across a short, 60 min LC-MS experiment26. A recent compara-
tive analysis confirmed IAM-desthiobiotin achieves compara-
ble cysteine coverage while shortening the sample preparation
workflow by >1 day.27 Offline fractionation and analysis of 7-
In situ labeling was performed at three alkyne-probe concen-
trations (10, 30, and 100 µM) for 45 min in cysteine- and me-
thionine-free Dulbecco's Modified Eagle Medium (DMEM). In
this experiment, protein labeling occurs in direct competition
with glutathione, cysteine and other thiol metabolites. At these
lower labeling concentrations in HeLa cells, there was no no-
ticeable morphological changes or cell death. After lysis, cell
lysates were conjugated to TAMRA-azide for in-gel cysteine
profiling (Figure 6b).
ACS Paragon Plus Environment

Page 7 of 21
Journal of the American Chemical Society
fractions by mass spectrometry further increased cysteine cov- peptides were common across both BT- and IAM-desthiobiotin
1
2
3
4
5
6
7
8
erage >5-fold. These improvements dramatically expand prote-
ome-wide assessment of cysteine occupancy while reducing the
extended sample preparation time and technical error.28 Based
on these reports, click chemistry and orthogonal proteolysis are
dispensable for profiling functional cysteines in complex ly-
sates.
probes (Figure 8b). These findings further corroborate orthog-
onality between different probe scaffolds in proteomic analysis.
Altogether, the combined coverage pooling both BT- and IAM-
desthiobiotin probes annotated ~5000 cysteine peptides en-
gaged in live cells. In comparison, incubation for 1 h with 200
µM photo-caged IAM-alkyne probe in live HeLa cells detected
~1300 cysteines.29
Accordingly, bypassing click chemistry labeling and cleava-
ble linkers would be expected to streamline and enhance cyste-
ine analysis. In order to explore this direct labeling and elution
strategy, we synthesized BT-desthiobiotin-1 (BT-D1) (Figure
7a). BT-D1 was incubated with live HeLa cells in cystine- and
methionine-free DMEM. Analysis of the resulting lysate found
near-saturated cysteine labeling within 5 minutes (Figure 7b).
Nonetheless, initial mass spectrometry analysis in mouse brain
soluble proteome reported widespread methyl ester hydrolysis
in cells (Table S2). To avoid the additional complexity intro-
duced by ester hydrolysis, a second-generation desthiobiotin
analogue was synthesized, BT-desthiobiotin-2 (BT-D2) with a
mono-PEG linker with equivalent cysteine reactivity. Both BT-
D1 and BT-D2 showed reductions in cysteine reactivity com-
pared to BT-4, likely introduced by the excess steric bulk of the
desthiobiotin conjugate, yet still achieved 35-40-times greater
reactivity than IAM-desthiobiotin (IAM-D).
Although heteroaromatic sulfones have been shown to be ex-
clusively selective for cysteine over other nucleophilic amino
acids such as lysine, serine, histidine, and tyrosine in THF/PBS
(1:1) at room temperature2a, their selectivity in the proteome un-
der biological conditions have not been demonstrated. Here we
corroborate these findings in live cells. Labeling was highly se-
lective towards cysteine, with marginal identifications on other
conjugated amino acids below the peptide false-discovery rate
(Figure S6).24
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 8. Orthogonal cysteine labeling with BT-desthiobiotin in
live HeLa cells. (a) Equivalent reversed-phase elution of IAM-D,
BT-D1, and BT-D2 modifications of cysteine peptides. (b) Profile
of annotated cysteine-conjugated peptides by LC-MS from separate
(N=3) analyses of BT- and IAM-desthiobiotin labeled peptides. (c)
In-cell competition of BT- and IAM-desthiobiotin probes. Peptides
identified as deamidated, oxidized, or with different charge states
are counted as one unique peptide.
Figure 7. Rapid live cell cysteine labeling with desthiobiotin-
linked probes. (a) Structures of desthiobiotin probes evaluated.
Measured reaction rates are shown in parentheses below each elec-
trophile. Standard deviations are derived from replicates with
multi-point curve-fitting. (b) Streptavidin detection of time-de-
pendent activity-based profiling in live HeLa cells.
In order to further evaluate in-cell labeling rates, we mixed
both BT- and IAM-desthiobiotin probes simultaneously at three
different concentrations (10, 50, or 200 µM) and added to live
HeLa cells. Since, the reaction rate for BT-D2 is ~36x faster
than IAM-D, all three competition experiments yielded a
greater number of BT-D2 labeled cysteine peptides (Figure 8c,
Figure S7, and Table S2). Prior to cysteine saturation at 10 µM,
both BT- and IAM-desthiobiotin probes yield equivalent num-
bers of cysteine peptides. At 50 µM each probe, BT-D2 begins
to dominate the peptide identifications, presumably through di-
rect competition with reactive and/or accessible thiols. At the
highest concentration, BT-D2 annotated nearly 3000 unique la-
beled peptides, with an additional ~1300 peptides in common
HeLa cells were then incubated with either BT- or IAM-
desthiobiotin probes for 1 h to evaluate in-cell cysteine profiles.
Following heat denaturation, TCEP reduction, and alkylation
with BT-4 to block free thiols, the lysates were digested with
trypsin and enriched with streptavidin resin. The eluted peptides
were analyzed with a 2 h gradient by mass spectrometry. In to-
tal, this workflow yielded >3000 unique cysteine-labeled pep-
tides profiled within their native cellular environment (Figure
8 and Table S2). Despite similar chromatographic elution pro-
files (Figure 8a) and peptide yields, only ~50% of the annotated
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 21
across both probes. Importantly, combining both BT- and IAM- these probes offer a chemically tractable alternative for tunable
1
2
3
4
5
6
7
8
desthiobiotin probes to cells simultaneously achieves >5000 an-
notated cysteine peptides, labeled in live cells, using a simpli-
fied 2 h LC-MS analysis. Therefore, co-incubating cells with
more than one electrophile enhances cysteine-peptide coverage
in a complex peptide analysis.
cysteine conjugation and in-depth cellular profiling.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Importantly, even after increasing the probe concentrations,
hundreds of peptides remained exclusive for a single probe.
These differences do not appear to reflect steric differences,
since established active site cysteines, including GAPDH, cys-
tatin-B, thioredoxin, and phosphoglycerate dehydrogenase are
modified by both probes across each tested concentration.
There was also no clear difference in GO term identifiers be-
tween probes, suggesting little or no intrinsic chemical orthog-
onality (Figure S8). Interestingly, similar orthogonality was
observed with ethynyl benziodoxolone (EBX) reagents, which
report only fractional overlap (2/3) with iodoacetamide-based
probes, suggesting physiochemical differences directly influ-
ence peptide-conjugate analysis.30
Synthetic methods and characterization. Supporting figures S1-S8
including electrophile titrations, stability, lysine reactivity, click
chemistry gel analysis, and comparative proteomics analysis.
(PDF)
Combined mouse brain proteomics data, Table S1. (XLSX)
Combined site-specific desthiobiotin-probe proteomics data, Table
S2. (XLSX)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
AUTHOR INFORMATION
Corresponding Author
*brentrm@umich.edu
Conclusions. Maleimide remains the primary cysteine bio-
conjugation reagent, despite significant reversibility31, ring hy-
drolysis32, and non-specific reactivity33 These issues are absent
from heteroaromatic sulfones, which form stable adducts with
cysteine3. Despite their promise, the reactivity and biological
utility of heteroaromatic sulfones have not been thoroughly ex-
plored. Here we report a suite of analogues that define the struc-
tural-activity relationship for cysteine conjugation across sev-
eral privileged heteroaromatic scaffolds. These analogues re-
veal synthetically accessible reactive centers capable of precise
reactivity tuning. This includes modulating the sulfide oxida-
tion state, modification of the leaving group, heteroatom substi-
tutions, or addition of electron withdrawing or electron donat-
ing groups. Current efforts in cysteine-directed covalent drug
discovery predominantly accesses a subset of validated electro-
philes (i.e. acrylamides and chloroacetamides) with few rational
routes to modulate tunability16, 34. Incorporation of heteroaro-
matic sulfide electrophiles as covalent warheads offers the po-
tential to optimize reactivity to match the nucleophilicity of a
targeted cysteine. Altogether, these approaches present a simple
scaffold able to span a wide range of reactivities by implement-
ing only minimal chemical modifications.
Funding Sources
Financial support was provided by the National Institutes of
Health DP2 GM114848 (B.R.M) and the University of Michigan.
Mass spectrometry instrumentation was supported by the National
Institute of Health S10 OD021619.
ACKNOWLEDGMENT
We thank John E. Crellin (Michigan) for initial chemical synthesis,
Sarah Haynes (Michigan) and Jaimeen Majmudar (Pfizer) for as-
sisting with proteomics data analysis, and Gabriela Gregorian
(Michigan) for mass spectrometry support.
REFERENCES
1.
Zhang, D.; Devarie-Baez, N. O.; Li, Q.; Lancaster, J. R., Jr.;
Xian, M., Methylsulfonyl benzothiazole (MSBT): a selective protein
thiol blocking reagent. Org. Lett. 2012, 14 (13), 3396-9.
2.
(a) Toda, N.; Asano, S.; Barbas, C. F., 3rd, Rapid, stable,
chemoselective labeling of thiols with Julia-Kocienski-like reagents: a
serum-stable alternative to maleimide-based protein conjugation. An-
gew. Chem., Int. Ed. 2013, 52 (48), 12592-6; (b) Chen, X.; Wu, H.;
Park, C. M.; Poole, T. H.; Keceli, G.; Devarie-Baez, N. O.; Tsang, A.
W.; Lowther, W. T.; Poole, L. B.; King, S. B.; Xian, M.; Furdui, C. M.,
Discovery of Heteroaromatic Sulfones As a New Class of Biologically
Compatible Thiol-Selective Reagents. ACS Chem. Biol. 2017, 12 (8),
2201-2208.
In addition, we demonstrate both iodoacetamide and methyl-
sulfonylbenzothiazole desthiobiotin probes achieve live cell
cysteine engagement and direct enrichment without the need for
further click chemistry conjugation. These reagents provide a
simplified method to directly profile cellular thiols in their na-
tive environment without lysis and exposure to atmospheric ox-
ygen or disruption of multi-protein complexes. By direct profil-
ing of native thiols in live cells, druggable sites can be assayed
for target engagement in cells using competitive ABPP meth-
ods. Such in-cell assays provide a critical link between drugga-
ble cysteine discovery in lysates and validation in live cells, par-
ticularly with emerging bifunctional covalent ligands for tar-
geted protein degradation.35
3.
Patterson, J. T.; Asano, S.; Li, X.; Rader, C.; Barbas, C. F.,
3rd, Improving the serum stability of site-specific antibody conjugates
with sulfone linkers. Bioconjugate Chem. 2014, 25 (8), 1402-7.
4.
Reisz, J. A.; Bechtold, E.; King, S. B.; Poole, L. B.; Furdui,
C. M., Thiol-blocking electrophiles interfere with labeling and detec-
tion of protein sulfenic acids. FEBS J. 2013, 280 (23), 6150-61.
5.
(a) Gill, R. K.; Rawal, R. K.; Bariwal, J., Recent Advances
in the Chemistry and Biology of Benzothiazoles. Arch. Pharm. 2015,
348 (3), 155-178; (b) Davinder, K.; Virender, K.; Rakesh Marwaha and
Gajendra, S., Oxadiazole-An Important Bioactive Scaffold for Drug
Discovery and Development Process Against HIV and Cancer- A Re-
view. Curr. Bioact. Compd. 2019, 15 (3), 271-279; (c) Sharma, P. C.;
Sinhmar, A.; Sharma, A.; Rajak, H.; Pathak, D. P., Medicinal signifi-
cance of benzothiazole scaffold: an insight view. J. Enzyme Inhib.
Med. Chem. 2013, 28 (2), 240-266; (d) Myznikov, L. V.; Hrabalek, A.;
Koldobskii, G. I., Drugs in the tetrazole series. (Review). Chem. Het-
erocycl. Compd. 2007, 43 (1), 1-9; (e) Ostrovskii, V. A.; Trifonov, R.
E.; Popova, E. A., Medicinal chemistry of tetrazoles. Russ. Chem. Bull.
Finally, optimized heteroaromatic sulfones provide acceler-
ated reaction kinetics to afford more precise temporal control of
cysteine alkylation in live cells. These tools promise to enable
direct evaluation of cysteine oxidation at much shorter time
scales with improved selectivity within their native subcellular
environment2b. Furthermore, the more non-polar probes provide
a privileged route to directly profile cysteines in more hydro-
phobic environments, such as sites of S-palmitoylation. Overall,
2012, 61 (4), 768-780; (f) Velık, J.; Baliharová, V.; Fink-Gremmels, J.;
Bull, S.; Lamka, J.; Skálová, L., Benzimidazole drugs and modulation
of biotransformation enzymes. Res. Vet. Sci. 2004, 76 (2), 95-108; (g)
́
ACS Paragon Plus Environment

Page 9 of 21
Journal of the American Chemical Society
Musmade, D. S.; Pattan, S. R.; Yalgatti, M. S., Oxadiazole a nucleus
with versatile biological behaviour. Int. J. Pharm. Chem. 2015, 5 (1),
17.
Yang, F.; Gao, J.; Che, J.; Jia, G.; Wang, C., A Dimethyl-
Labeling-Based Strategy for Site-Specifically Quantitative Chemical
Proteomics. Anal Chem 2018, 90 (15), 9576-9582.
18.
1
2
3
4
5
6
7
8
11-20.
6.
(a) Farrukh, A.; Paez, J. I.; Salierno, M.; del Campo, A., Bi-
(a) Bar-Peled, L.; Kemper, E. K.; Suciu, R. M.; Vinogra-
oconjugating Thiols to Poly(acrylamide) Gels for Cell Culture Using
Methylsulfonyl Co-monomers. Angew. Chem., Int. Ed. 2016, 55 (6),
2092-2096; (b) Pedzisa, L.; Li, X.; Rader, C.; Roush, W. R., Assess-
ment of reagents for selenocysteine conjugation and the stability of se-
lenocysteine adducts. Org. Biomol. Chem. 2016, 14 (22), 5141-5147.
dova, E. V.; Backus, K. M.; Horning, B. D.; Paul, T. A.; Ichu, T. A.;
Svensson, R. U.; Olucha, J.; Chang, M. W.; Kok, B. P.; Zhu, Z.; Ihle,
N. T.; Dix, M. M.; Jiang, P.; Hayward, M. M.; Saez, E.; Shaw, R. J.;
Cravatt, B. F., Chemical Proteomics Identifies Druggable Vulnerabili-
ties in a Genetically Defined Cancer. Cell 2017, 171 (3), 696-709 e23;
(b) Grossman, E. A.; Ward, C. C.; Spradlin, J. N.; Bateman, L. A.;
Huffman, T. R.; Miyamoto, D. K.; Kleinman, J. I.; Nomura, D. K., Co-
valent Ligand Discovery against Druggable Hotspots Targeted by Anti-
cancer Natural Products. Cell Chem. Biol. 2017, 24 (11), 1368-1376
e4.
7.
Bueno, A. B.; Showalter, A. D.; Wainscott, D. B.; Stutsman,
C.; Marin, A.; Ficorilli, J.; Cabrera, O.; Willard, F. S.; Sloop, K. W.,
Positive Allosteric Modulation of the Glucagon-like Peptide-1 Recep-
tor by Diverse Electrophiles. J. Biol. Chem. 2016, 291 (20), 10700-15.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8.
Chen, B.; Long, Q.; Zhao, Y.; Wu, Y.; Ge, S.; Wang, P.;
Yang, C.-G.; Chi, Y.; Song, B.; Yang, S., Sulfone-Based Probes Un-
raveled Dihydrolipoamide S-Succinyltransferase as an Unprecedented
Target in Phytopathogens. J. Agric. Food Chem. 2019, 67 (25), 6962-
6969.
19.
Chiotellis, A.; Sladojevich, F.; Mu, L.; Müller Herde, A.;
Valverde, I. E.; Tolmachev, V.; Schibli, R.; Ametamey, S. M.; Mindt,
T. L., Novel chemoselective 18F-radiolabeling of thiol-containing bio-
molecules under mild aqueous conditions. Chem. Commun. 2016, 52
(36), 6083-6086.
9.
(a) Lad, N. P.; Manohar, Y.; Mascarenhas, M.; Pandit, Y. B.;
Kulkarni, M. R.; Sharma, R.; Salkar, K.; Suthar, A.; Pandit, S. S., Me-
thylsulfonyl benzothiazoles (MSBT) derivatives: Search for new po-
tential antimicrobial and anticancer agents. Bioorganic & Medicinal
Chemistry Letters 2017, 27 (5), 1319-1324; (b) Xu, W.; Yang, S.; Bha-
dury, P.; He, J.; He, M.; Gao, L.; Hu, D.; Song, B., Synthesis and bio-
activity of novel sulfone derivatives containing 2,4-dichlorophenyl
substituted 1,3,4-oxadiazole/thiadiazole moiety as chitinase inhibitors.
Pestic. Biochem. Physiol. 2011, 101 (1), 6-15; (c) Su, S.; Zhou, X.;
Zhou, Y.; Liao, G.; Shi, L.; Yang, X.; Zhang, X.; Jin, L., Synthesis and
Biological Evaluation of Novel Sulfone Derivatives Containing 1,3,4-
Oxadiazole Moiety. World J. Org. Chem. 2014, 2 (1), 18-27.
20.
Hansch, C.; Leo, A.; Taft, R. W., A survey of Hammett sub-
stituent constants and resonance and field parameters. Chem. Rev.
1991, 91 (2), 165-195.
21.
K.; Skånberg, I.; Weidolf, L., Stereoselective Metabolism of Omepra-
zole by Human Cytochrome P450 Enzymes. Drug Metab. Dispos.
2000, 28 (8), 966; (b) Lipsky, J. J., Mechanism of the inhibition of the
gamma-carboxylation of glutamic acid by N-methylthiotetrazole-con-
taining antibiotics. Proc. Natl. Acad. Sci. U. S. A. 1984, 81 (9), 2893.
(a) Äbelö, A.; Andersson, T. B.; Antonsson, M.; Naudot, A.
22.
Aitken, A.; Learmonth, M., Carboxymethylation of Cysteine
Using Iodoacetamide/Iodoacetic Acid. In The Protein Protocols Hand-
book, Walker, J. M., Ed. Humana Press: Totowa, NJ, 1996; pp 339-
340.
10.
Bauer, M. R.; Joerger, A. C.; Fersht, A. R., 2-Sulfonylpy-
rimidines: Mild alkylating agents with anticancer activity toward p53-
compromised cells. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (36),
E5271-80.
23.
Suttapitugsakul, S.; Xiao, H.; Smeekens, J.; Wu, R., Evalu-
ation and optimization of reduction and alkylation methods to maxim-
ize peptide identification with MS-based proteomics. Mol. BioSyst.
2017, 13 (12), 2574-2582.
11.
Coulson, G. B.; Johnson, B. K.; Zheng, H.; Colvin, C. J.;
Fillinger, R. J.; Haiderer, E. R.; Hammer, N. D.; Abramovitch, R. B.,
Targeting Mycobacterium tuberculosis Sensitivity to Thiol Stress at
Acidic pH Kills the Bacterium and Potentiates Antibiotics. Cell Chem.
Biol. 2017, 24 (8), 993-1004 e4.
24.
Chalkley, R. J., When target-decoy false discovery rate esti-
mations are inaccurate and how to spot instances. J Proteome Res 2013,
12 (2), 1062-4.
25.
M.; Zaro, B. W.; Bradshaw, J. M.; Brameld, K. A.; Cravatt, B. F., The
Proteome-Wide Potential for Reversible Covalency at Cysteine. An-
gew. Chem., Int. Ed. 2019, 58 (33), 11385-11389.
12.
Kahl, D. J.; Hutchings, K. M.; Lisabeth, E. M.; Haak, A. J.;
Senkane, K.; Vinogradova, E. V.; Suciu, R. M.; Crowley, V.
Leipprandt, J. R.; Dexheimer, T.; Khanna, D.; Tsou, P. S.; Campbell,
P. L.; Fox, D. A.; Wen, B.; Sun, D.; Bailie, M.; Neubig, R. R.; Larsen,
S. D., 5-Aryl-1,3,4-oxadiazol-2-ylthioalkanoic Acids: A Highly Potent
New Class of Inhibitors of Rho/Myocardin-Related Transcription Fac-
tor (MRTF)/Serum Response Factor (SRF)-Mediated Gene Transcrip-
tion as Potential Antifibrotic Agents for Scleroderma. J .Med. Chem.
2019, 62 (9), 4350-4369.
26.
Patricelli, M. P.; Janes, M. R.; Li, L. S.; Hansen, R.; Peters,
U.; Kessler, L. V.; Chen, Y.; Kucharski, J. M.; Feng, J.; Ely, T.; Chen,
J. H.; Firdaus, S. J.; Babbar, A.; Ren, P.; Liu, Y., Selective Inhibition
of Oncogenic KRAS Output with Small Molecules Targeting the Inac-
tive State. Cancer Discovery 2016, 6 (3), 316-29.
13.
(a) Qian, Y.; Weerapana, E., A Quantitative Mass-Spec-
trometry Platform to Monitor Changes in Cysteine Reactivity. Methods
Mol. Biol. 2017, 1491, 11-22; (b) Weerapana, E.; Wang, C.; Simon, G.
M.; Richter, F.; Khare, S.; Dillon, M. B.; Bachovchin, D. A.; Mowen,
K.; Baker, D.; Cravatt, B. F., Quantitative reactivity profiling predicts
functional cysteines in proteomes. Nature 2010, 468 (7325), 790-5.
27.
Zanon, P. R. A.; Lewald, L.; Hacker, S. M., Isotopically La-
beled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of
the Bacterial Cysteinome. Angew. Chem. Int. Ed. Engl. 2019.
28.
Bianco, G.; Yamashita, Y.; Crowley, V. M.; Remillard, D.; Lum, K.
M.; Simon, G. M.; Kemper, E. K.; Lazear, M. R.; Yin, S.; Blewett, M.
M.; Dix, M. M.; Nguyen, N.; Shokhirev, M. N.; Chin, E.; Lairson, L.;
Forli, S.; Teijaro, J. R.; Cravatt, B. F., An activity-guided map of elec-
trophile-cysteine interactions in primary human immune cells. bioRxiv
2019, 808113.
Vinogradova, E. V.; Lazar, D. C.; Suciu, R. M.; Wang, Y.;
14.
(a) Weerapana, E.; Speers, A. E.; Cravatt, B. F., Tandem or-
thogonal proteolysis-activity-based protein profiling (TOP-ABPP)--a
general method for mapping sites of probe modification in proteomes.
Nat. Protoc. 2007, 2 (6), 1414-25; (b) Speers, A. E.; Cravatt, B. F., A
tandem orthogonal proteolysis strategy for high-content chemical pro-
teomics. J. Am. Chem. Soc. 2005, 127 (28), 10018-9.
29.
Abo, M.; Bak, D. W.; Weerapana, E., Optimization of Caged
15.
Zhou, Y.; Wynia-Smith, S. L.; Couvertier, S. M.; Kalous, K.
Electrophiles for Improved Monitoring of Cysteine Reactivity in Liv-
ing Cells. ChemBioChem 2017, 18 (1), 81-84.
30.
Waser, J.; Adibekian, A., Proteome-Wide Profiling of Targets of Cys-
teine reactive Small Molecules by Using Ethynyl Benziodoxolone Re-
agents. Angew. Chem. Int. Ed. Engl. 2015, 54 (37), 10852-7.
S.; Marletta, M. A.; Smith, B. C.; Weerapana, E., Chemoproteomic
Strategy to Quantitatively Monitor Transnitrosation Uncovers Func-
tionally Relevant S-Nitrosation Sites on Cathepsin D and HADH2. Cell
Chem. Biol. 2016, 23 (6), 727-37.
Abegg, D.; Frei, R.; Cerato, L.; Prasad Hari, D.; Wang, C.;
16.
Backus, K. M.; Correia, B. E.; Lum, K. M.; Forli, S.; Horn-
ing, B. D.; Gonzalez-Paez, G. E.; Chatterjee, S.; Lanning, B. R.; Tei-
jaro, J. R.; Olson, A. J.; Wolan, D. W.; Cravatt, B. F., Proteome-wide
covalent ligand discovery in native biological systems. Nature 2016,
534 (7608), 570-4.
31.
Baldwin, A. D.; Kiick, K. L., Tunable degradation of malei-
mide-thiol adducts in reducing environments. Bioconjugate Chem.
2011, 22 (10), 1946-53.
32.
Matsui, S.; Aida, H., Hydrolysis of some N-alkylmalei-
mides. J. Chem. Soc., Perkin Trans. 2 1978, (12), 1277-1280.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 21
33.
(a) Kuo, Y. H.; Konopko, A. M.; Borotto, N. B.; Majmudar,
H.; von Delft, F.; London, N., Rapid Covalent-Probe Discovery by
J. D.; Haynes, S. E.; Martin, B. R., Profiling Protein S-Sulfination with
Maleimide-Linked Probes. ChemBioChem 2017, 18 (20), 2028-2032;
(b) Zhang, Y.; Zhou, X.; Xie, Y.; Greenberg, M. M.; Xi, Z.; Zhou, C.,
Thiol Specific and Tracelessly Removable Bioconjugation via Michael
Addition to 5-Methylene Pyrrolones. J. Am. Chem. Soc. 2017, 139
(17), 6146-6151; (c) Ravasco, J. M. J. M.; Faustino, H.; Trindade, A.;
Gois, P. M. P., Bioconjugation with Maleimides: A Useful Tool for
Chemical Biology. Chem.: Eur. J. 2019, 25 (1), 43-59.
Electrophile-Fragment Screening. J. Am. Chem. Soc. 2019, 141 (22),
8951-8968.
1
2
3
4
5
6
7
8
35.
(a) Spradlin, J. N.; Hu, X.; Ward, C. C.; Brittain, S. M.;
Jones, M. D.; Ou, L.; To, M.; Proudfoot, A.; Ornelas, E.; Woldegiorgis,
M.; Olzmann, J. A.; Bussiere, D. E.; Thomas, J. R.; Tallarico, J. A.;
McKenna, J. M.; Schirle, M.; Maimone, T. J.; Nomura, D. K., Harness-
ing the anti-cancer natural product nimbolide for targeted protein deg-
radation. Nat. Chem. Biol. 2019, 15 (7), 747-755; (b) Zhang, X.; Crow-
ley, V. M.; Wucherpfennig, T. G.; Dix, M. M.; Cravatt, B. F., Electro-
philic PROTACs that degrade nuclear proteins by engaging DCAF16.
Nat. Chem. Biol. 2019, 15 (7), 737-746.
34.
Resnick, E.; Bradley, A.; Gan, J.; Douangamath, A.; Krojer,
T.; Sethi, R.; Geurink, P. P.; Aimon, A.; Amitai, G.; Bellini, D.; Ben-
nett, J.; Fairhead, M.; Fedorov, O.; Gabizon, R.; Gan, J.; Guo, J.; Plot-
nikov, A.; Reznik, N.; Ruda, G. F.; Diaz-Saez, L.; Straub, V. M.; Szom-
mer, T.; Velupillai, S.; Zaidman, D.; Zhang, Y.; Coker, A. R.; Dowson,
C. G.; Barr, H. M.; Wang, C.; Huber, K. V. M.; Brennan, P. E.; Ovaa,
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 11 of 21
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 12 of 21
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
Me
NH
H
R
N
R
SH
Me
S
+
Het
MeSO2H
+
S
Het
O
HN
O
O
O
O
O
S
N
S
Me
HN
O
150
M-1s-1
kobs
30
direct
enrichment
10
LC-MS/MS
>3000
IAM
O
1
NEM
O
N
S
N
S
S
O
S
O
Cys-peptides (2 h)
Me
O2
N
Me
ACS Paragon Plus Environment

Page 13 of 21
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
R
buffer
MeSO2H
+
Het
R SH
Me
S
+
Het
S
pH 7.4
O
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 14 of 21
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
b
20 µM IAM-TAMRA
O
O
N
S
S
O
I
NH2
Me
IAM
BT-1 (MSBT)
(0.52 ± 0.02 M -1s-1
250
150
(0.90 ± 0.05 M-1s-1
)
)
100
75
N
N
N
HO
N
O
O
N
O
S
O
50
37
Et
Me
NEM
Tz-1 (MSTP)
(6.03 ± 0.55 M -1s-1
)
(1585 ± 109 M-1s-1
)
O
N
N
N
O
25
20
S
O
S
O
O
N
H
Me
Me
OD-1
(451 ± 46 M-1s-1
BI-1
(nd)
Coomassie
)
ACS Paragon Plus Environment

Page 15 of 21
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
b
20 µM IAM-TAMRA
N
N
N
HO
N
O
S
Me
O
250
150
Tz-1
(6.03 ± 0.55 M-1s-1
)
100
75
N
N
N
N
O
S
O
Tz-2
Me
50
37
(12.1 ± 0.67 M-1s-1
)
N
N
HO2C
N
N
25
O
S
O
Me
Tz-3
(11.2 ± 0.46 M-1s-1
Coomassie
)
N
N
N
N
N N
O
N
O
S
Me
O
S
O
S
O
S
Me
O
S
N
O
O
O
O
Me
H
Me
OD-1
(451 ± 46 M-1s-1
TD-1
(nd)
BO-1
Trz-1
(nd)
)
(845 ± 14 M-1s-1
)
OMe
N
N
N
O
O
S
N
O
O
N
S
N
S
O
CF2H
O
MeO
N
S
Me
O
Me
O
Me
QX-1
(nd)
QZ-1
(nd)
Pymd-1
(nd)
Py-1
(nd)
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 16 of 21
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
b
O
20 µM IAM-TAMRA
N
S
O
S
Me
BT-1
(0.52 ± 0.02 M-1s-1
)
250
150
O
N
S
S
O
100
75
MeO
Me
BT-2
(0.027 ± 0.005 M-1s-1
)
50
O
S
N
S
O
37
HO2C
Me
BT-3
(2.39 ± 0.10 M-1s-1
)
25
O
N
S
O
S
MeO2C
Me
Coomassie
BT-4
(156 ± 5 M-1s-1
)
O
S
O
S
O
S
N
S
N
N
O
O
O
S
S
O2N
Me
CF3
HO2C
CH2CF3
BT-5
BT-6
BT-7
(1651 ± 45 M-1s-1
)
(19.5 ± 0.10 M-1s-1
)
(17.4 ± 0.15 M-1s-1
)
O
O
N
O
N
O
N
N
S
S
O
S
O
S
O
S
O
S
S
S
Me
Et
Ph
Me
HO2C
BT-8
BT-9
(nd)
T-1
(nd)
T-2
(0.072 ± 0.014 M -1s-1
)
(0.018 ± 0.002 M -1s-1
)
c
4.0
R2 = 0.9639
3.0
–NO2
–CONHR
2.0
1.0
–CO2 Me
-
–CO2
ρ = 4.4641
0.0
-1.0
-2.0
–H
O
N
S
S
O
R
Me
–OMe
-0.3 -0.2 -0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
σ
ACS Paragon Plus Environment

Page 17 of 21
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
b
20 µM IAM-TAMRA
O
S
Me
N
S
O2N
(865 ± 17 M-1s-1
N
)
250
150
O
S
100
75
S
CF3
)
HO2C
BT-11
(10.0 ± 0.50 M-1s-1
50
37
N
N
O
S
O
Me
OD-2
(31.2 ± 1.6 M-1s-1
)
25
O
S
N
O
Me
BO-2
(78.2 ± 3.6 M-1s-1
)
Coomassie
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 18 of 21
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
b
Peptide elution profile
unmodified
IAM
NEM
Tz-2
OD-1
BT-4
Relative ionization profile
1.00
0.75
0.50
0.25
0.00
4
3
2
1
0
IAM NEM Tz-1 OD-1 BT-4
0
40
80
120
retention time (min)
ACS Paragon Plus Environment

Page 19 of 21
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
N
N
O
O
N
N
I
N
H
O
S
Me
O
IAM-Alkyne
Tz-alkyne
(6.68 ± 0.21 M -1s-1
)
(0.63 ± 0.04 M -1s-1
)
O
S
N
S
N
N
O
S
H
N
Me
O
Me
O
O
O
O
BT-alkyne
OD-alkyne
(163 ± 12 M-1s-1
)
(102 ± 11 M-1s-1
)
CuAAC (TAMRA-azide)
Tz-alkyne BT-alkyne OD-alkyne
b
IAM-alkyne
(µM)
250
150
100
75
50
37
25
20
Coomassie
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 20 of 21
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
b
Me
H
N
BT-D1
(100 µM)
NH
O
O
HN
OMe
HN
O
S
N
S
O
Me
O
min
250
O
BT-deshtiobiotin-1 (BT-D1)
(47.5 ± 2.7 M -1s-1
)
150
100
75
Me
NH
H
N
O
HN
O
O
O
S
N
S
Me
50
37
HN
O
O
BT-desthiobiotin-2 (BT-D2)
(42.1 ± 1.1 M -1s-1
)
Me
NH
H
N
O
HN
O
O
O
HN
I
25
O
O
IAM-desthiobiotin (IAM-D)
(1.16 ± 0.17 M -1s-1
)
ACS Paragon Plus Environment

Page 21 of 21
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
b
Peptide elution profile
Desthiobiotin-adduct
IAM-D
1.00
0.75
0.50
0.25
0.00
IAM-D
BT-D2
Both
BT-D2
unmodified
200 µM IAM-D (N=3)
200 µM BT-D2 (N=3)
1529
1667
1775
4971 peptides
0
40
80
120
retention time (min)
c
10 µM IAM-D
+ 10 µM BT-D2
(N=3)
50 µM IAM-D
+ 50 µM BT-D2
(N=3)
200 µM IAM-D
+ 200 µM BT-D2
(N=3)
236
707
301
444
393
1353
2991
1074
292
1037 peptides
1703 peptides
5051 peptides
ACS Paragon Plus Environment