SuFEx-enabled, agnostic discovery of covalent inhibitors of human neutrophil elastase

Article abstract of DOI:10.1073/pnas.1909972116

Sulfur fluoride exchange (SuFEx) has emerged as the new generation of click chemistry. We report here a SuFEx-enabled, agnostic approach for the discovery and optimization of covalent inhibitors of human neutrophil elastase (hNE). Evaluation of our ever-growing collection of SuFExable compounds toward various biological assays unexpectedly revealed a selective and covalent hNE inhibitor: benzene-1,2-disulfonyl fluoride. Synthetic derivatization of the initial hit led to a more potent agent, 2-(fluorosulfonyl)phenyl fluorosulfate with IC₅₀ 0.24 μM and greater than 833-fold selectivity over the homologous neutrophil serine protease, cathepsin G. The optimized, yet simple benzenoid probe only modified active hNE and not its denatured form.

Full text of DOI:10.1073/pnas.1909972116

SuFEx-enabled, agnostic discovery of covalent
inhibitors of human neutrophil elastase
Qinheng Zhenga,1, Jordan L. Woehlb,1, Seiya Kitamurab, Diogo Santos-Martinsc, Christopher J. Smedleyd, Gencheng Lia,
Stefano Forlic, John E. Mosesd, Dennis W. Wolanb,2, and K. Barry Sharplessa,2
aDepartment of Chemistry, The Scripps Research Institute, La Jolla, CA 92037; bDepartment of Molecular Medicine, The Scripps Research Institute, La Jolla,
CA 92037; cDepartment of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037; and dLa Trobe Institute for
Molecular Science, La Trobe University, Bundoora, Melbourne, VIC 3086, Australia
Contributed by K. Barry Sharpless, July 23, 2019 (sent for review July 9, 2019; reviewed by Peng R. Chen and Lei Wang)
Sulfur fluoride exchange (SuFEx) has emerged as the new gener-
ation of click chemistry. We report here a SuFEx-enabled, agnostic
approach for the discovery and optimization of covalent inhibi-
tors of human neutrophil elastase (hNE). Evaluation of our ever-
growing collection of SuFExable compounds toward various bio-
logical assays unexpectedly revealed a selective and covalent hNE
inhibitor: benzene-1,2-disulfonyl fluoride. Synthetic derivatization
of the initial hit led to a more potent agent, 2-(fluorosulfonyl)phenyl
fluorosulfate with IC50 0.24 μM and greater than 833-fold selectivity
over the homologous neutrophil serine protease, cathepsin G. The
optimized, yet simple benzenoid probe only modified active hNE
and not its denatured form.
unique ensemble of factors between the naturally folded protein
and its correct partner probe’s latent S–F electrophilic site.
When the perfect S–F probe for a given protein encounters the
denatured form of the latter, there is no detectable reaction (24,
33). In fact, in our experience with many S–F capture probes and
various denatured proteins, even including entire denatured
proteomes, is that misfolded proteins simply do not react with
S–F electrophiles at all (24). Among the main factors present in the
natural proteins to facilitate extraction and transport of fluoride
will be special groups, e.g., arginine side chains juxtaposed to
provide just so, hydrogen bonding networks (33). The latter are
closely related to local electric fields, electrostatic effects which are
unique for all natural proteins (44–47). Thus, it is not hard to
imagine that the transit of “F−” away from a SuFExable probe in a
capture event could be a very subtle outcome of electric field pulses
for the specific reactant pair at hand. And yet, the enormous range
of reactivity of, e.g., PhSO2–F (stable in “refluxing aniline test,”
vide supra), does argue for something quite special going on with
these nearly inert S–F-based covalent probes.
click chemistry SuFEx agnostic covalent inhibitor elastase
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ulfur fluoride exchange (SuFEx)—the new-generation click
S
chemistry, since first introduced in 2014 (1), has quickly
found diverse applications across an array of fields (DOI:
10.1039/C8CS00960K), including chemical synthesis (2–12),
material science (13–19), chemical biology (20–32), and drug
discovery (33, 34). SuFEx creates robust intermolecular links be-
tween modules. The fidelity of SuFEx stems from the ability of
otherwise very stable high oxidation state sulfur fluorides (35–39)
to exchange the S–F bonds with incoming nucleophiles on sepa-
rate modules under SuFEx catalysis conditions. The resulting new
sulfur hub sites constitute permanent connections. These unique
SuFEx reactions are thought to be made possible by the strict
requirements needed to stabilize the departing fluoride ion in its
transit away from the strong original covalent bond to sulfur. The
facilitating agents are “H+” or “R3Si+.” The process especially
favors a 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU)-type amine
catalyst (13, 40, 41), and is also thought to involve bifluoride
counterion species (16, 17, 42).
In this light, the awakening of a SuFExable probe by the cap-
tured protein (24, 33) mirrors very closely our earlier femtomolar,
freeze-frame, yet reversible, inhibitors made inside acetylcholin-
esterase (AChE) as a “reaction vessel” (48–51), regarding the little
Significance
An agnostic screen of our collection of stable sulfur fluoride
exchange (SuFEx)able derivatives discovers a promising class of
covalent and highly selective, elastase inhibitors. Their dor-
mant behavior is a reflection of an exquisite sensitivity in
choice of a protein reaction partner, like a “sleeping beauty
finding the right prince.” Hence a special SuFEx catalysis envi-
ronment is required for the perfect matchmaking event: a
naturally folded protein with precise electrostatic and geo-
metric setups, such that one of its own nucleophilic side chain
ends up replacing the probe’s S–F link to become permanently
covalent. In contrast, the same protein, when denatured, is
incapable of being covalently modified by the same probe.
In the context of selective in vitro or in vivo covalent capture
of proteins by compounds containing a S-F bond that can un-
dergo SuFEx reaction (i.e., SuFExable compounds), examples
are rapidly accumulating (20–31). However, we are still far from
making strong a priori inferences about the various factors that
might relate to a given capture’s occurrence. That said, there is
one remarkable fact shared by all these SuFEx-based protein
captures, namely that the exchangeable S–F bonds in these
probes are among the most demure electrophiles known to
chemistry (1, 36–39). For example, no reaction occurred in the
neat mixture of refluxing benzenesulfonyl fluoride (4 mL, ∼33
mmol) and aniline (45 mL, ∼500 mmol) at 184 °C for 3 h (35).
Under the same conditions, electrophiles commonly studied as
covalent “warheads” in medicinal chemistry including epoxide,
acrylamide, vinyl sulfone, chloroacetamide, chloromethyl ketone,
β-lactam, maleimide, and fluorophosphate do not survive (SI
Appendix, Table S3).
Author contributions: D.W.W. and K.B.S. designed research; Q.Z., J.L.W., and S.K. per-
formed research; D.S.-M., and S.F. contributed analytic tools; C.J.S. and G.L. contributed
new reagents; Q.Z., J.L.W., S.K., D.S.-M., S.F., D.W.W., and K.B.S. analyzed data; and Q.Z.,
J.L.W., J.E.M., D.W.W., and K.B.S. wrote the paper.
Reviewers: P.R.C., Peking University; and L.W., University of California San Francisco
Medical Center.
The authors declare no conflict of interest.
Published under the PNAS license.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.wwpdb.org (PDB ID code 6e69).
1Q.Z. and J.L.W. contributed equally to this work.
2To whom correspondence may be addressed. Email: wolan@scripps.edu or sharples@
scripps.edu.
Only a correctly folded and functionally active protein can
serve as a “catalyst” (43) for a SuFEx capture event upon one of
its own nucleophilic amino acid side chains (Fig. 1A). These
enzymic SuFEx capture reactions are, we posit, the result of a
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1909972116/-/DCSupplemental.
Published online September 4, 2019.
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followed up by short and simple syntheses of analogs for structure–
activity relationship evaluation. This “compound-bank” mode of
the SuFEx-enabled platform, itself contributes to the ever-growing
library of SuFExable modules and candidates, and like “com-
pounding interest,” rewards the principle library with expanding
opportunities for discovering new kinds of phenotypic modula-
tors, including useful new functions for medicines in the long
term (Fig. 1B).
Over the past 5 y, we (1, 5, 8, 56) and others (12, 57–60) have
found a collection of efficient methods to synthesize SuFExable
compounds from either simple or complex organic molecules
using highly connective SuFEx core electrophiles (e.g., SO2F2,
O = SF4, CH2 = CHSO2F). Collective work within the Sharpless
laboratory continues to build a library of SuFExable small mod-
ules. There are >1,000 compounds in this collection. The indi-
vidual compounds are dissolved as 10 mM dimethyl sulfoxide
(DMSO) stock solutions and stored in −20 °C freezer, with most
being stable at −20 °C as evidenced by periodic inspection by
liquid chromatography-mass spectrometry (LC-MS) of randomly
sampled compounds over 3 y. Thanks to the great stability of the
SuFExable S–F links toward water and oxygen, the oldest library
deposits (as DMSO solutions) are still pure. To date, the library
and/or its sublibraries have been screened with collaborators at
Scripps Research against multiple targets. Here we report a case
study using the SuFEx-enabled approach to the discovery of se-
lective, covalent inhibitors of human neutrophil elastase (hNE).
hNE, a member of the serine protease superfamily, is aber-
rantly active in cystic fibrosis, chronic obstructive pulmonary
disease, and inflammatory bowel diseases (61–77). This protease
is therefore a key target for the development of antiinflammatory
agents to combat these diseases. Alkyl and aryl sulfonyl fluorides
have a long history as rather promiscuous covalent inhibitors of
serine proteases (78–87). In the late 1970s, the Powers group
Fig. 1. Overview. (A) The SuFExable probes (e.g., sulfonyl fluoride) only
capture a specific, naturally folded protein where a nucleophilic side chain
(Nu) and hydrogen bonding donors (HBDs, including amino acid side chains
and bound or free water) constitute such precise networks that meet the
local geometric and electrostatic requirements (dipole moment changes in
transition state) for the protein’s own SuFEx reaction. (B) Schematic of the
SuFExable library-enabled covalent lead compound discovery process.
understood, yet enormous rate acceleration achieved by the AChE
protein. We named these enzyme templating processes in situ
click chemistry, more recently also termed kinetic target-guided
synthesis (52–54).
Taking inspiration from the original click chemistry manifesto
(55) that molecular diversity can be achieved with ease through
the connection of small modules, using just a few good reactions,
in sequences of usually no more than 3 steps, we demonstrate
here 2 distinct but logically connected, sequential SuFEx-enabled
entities: 1) efficient construction of a pool of SuFExable com-
pounds via click chemistry principles, and 2) bioprospecting this
library for covalent capture agents for important protein targets.
The expectation, based on previous experience, is that evaluation
of a SuFEx library often turns up a few hits. The latter can then be
Fig. 2. Efficient SuFEx derivatization of abundant building blocks yields a
SuFExable library with 4 subset groups, categorized according to their S–F
links’ inherent reactivity. (A) Aryl sulfonyl fluorides (Subset I). (B) Aryl
fluorosulfates (Subset II). (C) Alkyl sulfonyl fluorides (Subset III). (D) (E)-Vinyl
sulfonyl fluorides (Subset IV). Ar, aryl, or heteroaryl; NEt3, triethylamine; R1,2
,
H, alkyl, aryl, or heteroaryl; R3, alkenyl, aryl, or heteroaryl; Pd(OAc)2, palla-
dium (II) acetate; AgTFA, silver (I) trifluoroacetate.
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of ∼3,000:1. Reasoning that a covalent inhibitor, which targets a
catalytic residue, should completely inactivate the enzyme at
high compound concentration (200 μM), a high threshold (95%)
was chosen for hit identification. Under this criterion, the screen
yielded 7 hits as probable covalent inhibitors (Fig. 3A, 6.7%
overall hit rate and 23% hit rate within subset I). All 7 com-
pounds belong to subset I, which suggests a rough cutoff based
on the S-link’s inherent reactivity. After validation by NMR, LC-
MS, and dose-dependent response, benzene-1,2-disulfonyl fluoride
(1) proved to be the leading candidate, inhibiting hNE with IC50
3.3 1.0 μM (Fig. 3 B and C).
=
The covalent inhibition of hNE by 1 was examined by high-
resolution matrix-assisted laser desorption/ionization-time of
flight (MALDI-TOF) mass spectrometry (Fig. 4A). Incubation
of 1 (exact mass 242 Da) with hNE yielded a peak shift from the
protein mass by 222 Da. Increased mass corresponds to: 1) a single
molecule of 1 covalently captured by hNE; 2) the loss of 1
hydrogen from the protease (possibly from the catalytic ser-
ine); and (3) the loss of 1 fluorine from 1.
We also subjected hNE to cocrystallization with 1 in order to
corroborate the covalent binding. The structure was determined
using molecular replacement with Protein Data Bank (PDB) ID
code 5adw and the cocomplex was refined to 2.33-Å resolution
(SI Appendix, Table S2). Importantly the naive Fo-Fc electron
density maps contoured to 4σ clearly position 1, as a result of the
strong diffraction of sulfurs (Fig. 4B). The aryl group of 1 is
nestled into a hydrophobic pocket consisting of residues Phe192
and Val216 and the compound is covalently bound to the cata-
lytic Ser195, as highlighted by continuous electron density and
Fig. 3. Screen of the primary SuFEx library toward elastase inhibitory ac-
tivity. (A) Initial screen with 105 SuFExable compounds yielded hits
7
with >95% inhibition at 200 μM. (B) Dose–response curves of hit compounds
(1–7, and phenylmethanesulfonyl fluoride [PMSF] as a reference inhibitor)
against hNE (AAPV-AMC fluorescence assay). Each compound was assessed
over a 2-fold logarithmic dilution series. (C) Structures and IC50 values of
compounds 1–7 and PMSF. IC50 values were measured based on 10-min in-
cubation and are shown in mean SD (n ≥ 3).
demonstrated the intrinsic reactivity differences across several
serine proteases, including elastase, with 2-amido(peptido)
benzenesulfonyl fluoride inhibitors (88, 89). Their results en-
couraged us to investigate our library of SuFExable compounds
as potential covalent inhibitors of hNE.
Results and Discussion
A set of 105 compounds (SI Appendix, Table S1), many of which
appear to be new compounds, were selected without bias, to
form a primary library for the screen against hNE. The selected
compounds can be categorized into 4 subsets by the nature of S–F
functional groups (Fig. 2). Each group tends to have its own
intrinsic zone of reactivity in acid–base environments. To give a
general impression, the relative rate measured on a representa-
tive molecule of each subset under SuFEx catalysis was found to
be 28.5 (I), 1.0 (II, reference), 14.1 (III), and 4.1 (IV), re-
spectively (SI Appendix, Fig. S6). Aryl sulfonyl fluorides (I), de-
spite their own extreme resistance toward nucleophiles, top the
SuFEx reactivity hierarchy among the 4 subsets, while aryl
fluorosulfates (II) lie at the bottom.
Elastase (5 nM) was incubated with each entry of the primary
SuFEx library (final compound concentration 200 μM) for 10
min at room temperature prior to the addition of peptide sub-
strate MeOSuc-AAPV-AMC (50 μM). Increase in fluorescence
was measured for 30 min at 30-s intervals. The assay was well
behaved, as evidenced by a Z′ of 0.86, and signal-to-background
Fig. 4. Compound 1 (exact mass 241.95) is a covalent inhibitor of hNE. (A)
MALDI-TOF mass spectrometry evidence of covalent complex hNE:1 forma-
tion. (B) Naive Fo-Fc map contoured at 1.5σ (green) and 4σ (magenta) clearly
delineates the binding orientation of 1 to Ser195 and the specific location of
the sulfur groups, respectively. Residue of 1 is shown as a stick model with
yellow carbon, red oxygen, light-blue fluorine, mustard sulfur. (C) Active site
residues that provide potential hydrogen bonds (black dashes), repulsive
interactions (brown dash), and hydrophobic residues that bind 1 (gray
elastase carbon, nitrogen blue). (D) Schematic of bond distances between 1
and elastase with potential hydrogen bonds (black dashes) and negative
repulsive interactions (brown dash) with bond distances in Å. The covalent
bond (red) between Ser195 and the sulfur of 1 were set at 1.57 Å during
structure refinement.
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Table 2. Selectivity of the lead compounds 1, 19, 22, and 24
against hNE and hCG
a bond distance of ∼1.6 Å (Fig. 4 C and D). The covalent inhibition
of hNE via sulfonylation by 1 appeared to be permanent—dialyzing
away small molecules after incubation did not recover enzyme
function (SI Appendix, Fig. S2).
Considering the monocovalent attachment mode of 1, with the
second –SO2F intact, it was envisaged that 1 of the 2 sulfonyl
fluoride groups could be substituted so as to perhaps improve the
capture rate, and/or selective binding (Table 1). A set of ben-
zenesulfonyl fluoride cores carrying ortho-substituents (8–24)
was therefore examined. Compounds 8–18 (90) were synthesized
by the efficient aqueous potassium bifluoride exchange pro-
cedure from commercially available sulfonyl chlorides but
showed poorer reactivity/binding with hNE. The monovinylogous
derivative 19 was a more active inhibitor with IC50 = 2.2
0.7 μM. The ortho-sulfamoyl benzenesulfonyl fluorides (20, 21)
led to considerably lower activity. A recently developed potas-
sium bifluoride catalyzed SuFEx perfluoroalkylation of aryl sul-
fonyl fluorides (91) enabled us to quickly convert 1 to the
monoperfluoroalkyl sulfones (22, 23). Compound 22 with the
ortho-triflyl group was identified as a better lead molecule with
IC50 = 1.1 0.1 μM. To our surprise, a simple benzenoid com-
pound 24, where the ortho-substituent is another SuFEx func-
tionality, aryl fluorosulfate, emerged as the best agent to date
with much improved potency, IC50 = 0.24 0.02 μM.
*IC50 values were measured based on 10-min incubation and are shown in
mean SD (n ≥ 3).
Table 1. Optimization of the original hit 1 by examining 2-
substituted benzenesulfonyl fluoride
†S value denotes the selectivity, defined by the ratio of IC50 (hCG) over IC50
(hNE).
Next, we tested the lead compounds (1, 19, 22, and 24) against
a panel of serine proteases; we found that three (1, 22, and 24)
among the four effective hNE inhibitors did not inactivate the
homologous serine protease, human cathepsin G (hCG), which
has 37% sequence identity with hNE and a highly similar crystal
structure [root-mean-square deviation (rmsd) = 0.82 Å, max
rmsd = 5.89 Å for 180 out of 218 Cα residues of hNE]. Unlike
PMSF (PhCH2SO2F) long known for ablating the hydrolytic
activity of almost all serine proteases, the compounds 1, 22, and
24 identified in this study showed 58 and >182, and >833-fold
specificity for hNE over hCG, respectively (Table 2). The se-
lective inhibition of hNE could be partly attributed to a proximity
factor as suggested by molecular modeling using a reactive
docking protocol (SI Appendix, Fig. S5)
High-resolution MALDI-TOF mass spectrometry study sup-
ports the covalent inhibition mechanism of the more potent and
selective agents 22 and 24 to be sulfonylation of hNE (maroon
peaks, +273 Da for 22, and +238 Da for 24). In both cases, we
observed the hNE dehydration product peak (M – 18, turquoise)
suggesting both agents to effect the covalent modification at the
same catalytic serine as 1 did (29, 32, 92–94). To further dem-
onstrate the stringent dependence of SuFEx reactions on pro-
tein’s tertiary structure, compounds 22 and 24 were incubated,
respectively, with inactive denatured hNE and no covalent
modification of the enzyme was found (Fig. 5).
To conclude, we have demonstrated a SuFEx library-enabled
approach to discover covalent deactivators of an enzyme’s
function, the protein at hand being human neutrophil elastase.
Its structure is known, including complexed with (ir)reversible
inhibitors in the active site, but the library of sulfonyl fluorides
used in the screen was chosen without regard to any enzy-
me:potential ligand relationships. In other words, agnostic of
structural considerations, our approach rapidly identified 2
SuFExable probes (22, 24) that found and captured their own
protein, hNE in this instance. This useful sulfur fluoride library
is being used and augmented regularly at Scripps Research,
*IC50 values were measured based on 10-min incubation and are shown in
mean SD (n ≥ 3).
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removed in vacuo. The residue was purified by flash column chromatography
to give the desired sulfonyl fluoride adducts of primary or secondary amines
(30 examples, 85–98% isolated yield).
General Procedure IV for the Preparation of Vinyl Sulfonyl Fluorides (Fig. 2D).
An oven-dried Schlenk tube was charged with (hetero)aryl iodide (Sigma-
Aldrich, or Combi-Blocks), AgTFA (Acros, 1.2 equiv), Pd(OAc)2 (Alfa Aesar,
2 mol %), acetone (anhydrous over 4-Å molecular sieves, Acros), and ESF
(homemade, 2 equiv) were added. The resulting mixture was refluxed at
60 °C. Upon full conversion of (hetero)aryl iodide (6–12 h), solvent was re-
moved in vacuo. The crude was purified by flash column chromatography to
give the desired product (10 examples, 59–99% yield).
Protease Activity Assays and SuFExable Library Screen. hNE activity was
measured in a total volume of 100 μL in a reaction buffer of phosphate-
buffered saline (PBS) (pH 7.4) and 0.05% (vol/vol) Nonidet P 40 Substitute
(Sigma). Final composition of each reaction was 5 nM hNE (Elastin Products
Corp.), 50 μM MeOSuc-AAPV-AMC substrate (Millipore), ∼2.5% DMSO
(Fisher), and various concentrations of fragments as inhibitors. Elastase was
incubated with various concentrations of inhibitors for 10 min at room
temperature before addition of MeOSuc-AAPV-AMC. Residual proteolytic
activity was measured at 25 °C using an Envision microplate reader for a
total of 30 min at 30-s intervals. Only data points reflecting linear substrate
conversion were used to determine relative protease activity. IC50 values
were obtained by fitting the data to a dose–response inhibition, log (in-
hibitor) vs. response–variable slope (4 parameters) using GraphPad Prism 8.
The physicochemical parameters were obtained from a reference, and the
correlation analysis was carried out using GraphPad Prism 8.
Fig. 5. High-resolution MALDI-TOF mass spectrometry demonstrated
sulfonyl fluoride capture agents for hNE as selective, covalent inhibitors. (A)
Compound 22. (B) Compound 24.
2
MALDI-TOF Mass Spectrometry of SuFExable Probes Bound to Neutrophil
Elastase. Elastase was resuspended in 50 mM sodium acetate (pH 4.5),
100 mM NaCl to 0.2 mg/mL final concentration. DMSO solution of each
compound (10 mM) was diluted 1:10 in the above buffer, then added in a
compound:hNE ratio = 3:1 and incubated at rt for 1 h prior to analysis by
MALDI-TOF mass spectrometry.
and it will hopefully contribute to future SuFEx-driven covalent
drug discovery endeavors.
Crystallization and X-Ray Data Collection. Inhibitor 1 was added in a 1.2 molar
excess to hNE [10 mg/mL in 10 mM Hepes (pH 6.5)], incubated for 1 h at
25 °C, and immediately used for crystallization. Crystals were grown by sit-
ting drop-vapor diffusion by mixing equal volumes (1.5 μL) of hNE:1 complex
and reservoir solution consisting of 0.3 M ammonium citrate (pH 5.0), 14% (wt/vol)
PEG 3350 at 25 °C. Data were collected on single, flash-cooled crystals at 100 K
in cryoprotectant consisting of 0.2 M ammonium citrate (pH 5.0), 20% (wt/vol)
PEG 3350, and 20% (vol/vol) glycerol, and were processed with HKL2000 in
orthorhombic space group P212121. The calculated Matthews’ coefficient
(VM = 2.77 Å3Da−1) suggested 4 monomers per asymmetric unit with a solvent
content of 56%. X-ray data were collected to 2.33-Å resolution on beamline
12.2 at the Stanford Synchrotron Radiation Lightsource.
Materials and Methods
General Procedure I for the Preparation of Aryl Sulfonyl Fluorides (Fig. 2A). Aryl
sulfonyl chloride (commercially available from Sigma-Aldrich or synthesized
according to known procedures) dissolved in acetonitrile (Fisher HPLC grade,
0.5–1 M) was treated with saturated potassium bifluoride aqueous solution
(Sigma-Aldrich, ∼5 M, 1.5–2.5 equiv). The emulsion was stirred vigorously for
1–4 h before being partitioned between ethyl acetate and water. The or-
ganic solution was collected, dried over anhydrous sodium sulfate (Na2SO4),
concentrated, and purified by column chromatography, if necessary, to yield
the desired aryl sulfonyl fluoride (33 examples, 90–100% isolated yield).
General Procedure II for the Preparation of Aryl Fluorosulfates (Fig. 2B). Phe-
nols (Sigma-Aldrich), and triethylamine (Alfa Aesar, 1.5 equiv) were dissolved
in dichloromethane (DCM) (Fisher). The flask sealed with a rubber septum was
evacuated, and sulfuryl fluoride gas (SynQuest Laboratories, Inc.) in a balloon
was introduced to the flask via a needle. The reaction was stirred vigorously
for 2 h. Upon completion, solvent was removed in vacuo. The residue was
partitioned between ethyl acetate and water. The organic phase was washed
with brine, dried over anhydrous Na2SO4, then concentrated and purified by
flash column chromatography to give the desired aryl fluorosulfate (32 ex-
amples, 82–99% isolated yield).
ACKNOWLEDGMENTS. We are very grateful to the following coworkers for
their contribution to the SuFExable compound library: Hua Wang (library
manager); Larisa Krasnova; Suhua Li; Bing Gao; Grant A. L. Bare; Gerui Ren;
Feng Zhou; Feng Liu; Hamid R. Safaei (Department of Chemistry); Peng Wu
and his group (Department of Molecular Medicine, Scripps Research); Jiajia
Dong and his group (Shanghai Institute of Organic Chemistry); the J.E.M.
group (La Trobe University); Hua-Li Qin and his group (Wuhan University of
Technology); En-Xuan Zhang and his group (AsymChem Inc.); Bo Qin; and
Mike Petrassi (Calibr, Scripps Research). The current work was financially
supported by the National Institutes of Health R01GM117145 (K.B.S.),
R01GM069832 (S.F.), T32AI7354-27 (J.L.W.), The Scripps Research Institute
(D.W.W.), and the ARC FT170100156 (J.E.M.). We thank Prof. Hugh Rosen
and Prof. Jin-Quan Yu (Scripps Research) for access to instrumentation,
Dr. Yongxuan Su of the Molecular Mass Spectrometry Facility (University of
California San Diego) for atmospheric-pressure chemical ionization MS,
Dr. John Cappiello (Scripps Research) for proofreading, and the staff of the
Stanford Synchrotron Radiation Lightsource.
General Procedure III for the Preparation of Alkyl Sulfonyl Fluorides (Fig. 2C).
To a solution of primary or secondary alkyl amine (Sigma-Aldrich, or Combi-
Blocks) in DCM or glacial acetic acid (Sigma-Aldrich, 0.5–1 M), ethenesulfonyl
fluoride (ESF) (homemade, 2.2 equiv) (56) was added dropwise. The mixture
was stirred at room temperature for 6–12 h. Upon completion, volatiles were
1. J. Dong, L. Krasnova, M. G. Finn, K. B. Sharpless, Sulfur(VI) fluoride exchange (SuFEx):
Another good reaction for click chemistry. Angew. Chem. Int. Ed. Engl. 53, 9430–9448
(2014).
2. P. S. Hanley, M. S. Ober, A. L. Krasovskiy, G. T. Whiteker, W. J. Kruper, Nickel- and
Palladium-catalyzed coupling of aryl fluorosulfonates with aryl boronic acids enabled
by sulfuryl fluoride. ACS Catal. 5, 5041–5046 (2015).
3. H. L. Qin, Q. Zheng, G. A. L. Bare, P. Wu, K. B. Sharpless, A Heck-Matsuda process
for the synthesis of β-arylethenesulfonyl fluorides: Selectively addressable bis-
electrophiles for SuFEx click chemistry. Angew. Chem. Int. Ed. Engl. 55, 14155–
14158 (2016).
4. A. S. Barrow, J. E. Moses, Synthesis of sulfonyl azides via Lewis base activation of
sulfonyl fluorides and trimethylsilyl azide. Synlett 27, 1840–1843 (2016).
5. S. Li, P. Wu, J. E. Moses, K. B. Sharpless, Multidimensional SuFEx click chemistry: Se-
quential sulfur(VI) fluoride exchange connections of diverse modules launched from
an SOF4 hub. Angew. Chem. Int. Ed. Engl. 56, 2903–2908 (2017).
6. S. D. Schimler et al., Nucleophilic deoxyfluorination of phenols via aryl fluorosulfonate
intermediates. J. Am. Chem. Soc. 139, 1452–1455 (2017).
7. R. Zelli, S. Tommasone, P. Dumy, A. Marra, A. Dondoni, A click ligation based on SuFEx
for the metal-free synthesis of sugar and iminosugar clusters. Eur. J. Org. Chem. 2016,
5102–5116 (2016).
18812
|
www.pnas.org/cgi/doi/10.1073/pnas.1909972116
Zheng et al.

8. G. F. Zha et al., Palladium-catalyzed fluorosulfonylvinylation of organic iodides.
Angew. Chem. Int. Ed. Engl. 56, 4849–4852 (2017).
44. A. Warshel, R. P. Bora, Perspective: Defining and quantifying the role of dynamics in
enzyme catalysis. J. Chem. Phys. 144, 180901 (2016).
9. G. Ren, Q. Zheng, H. Wang, Aryl fluorosulfate trapped Staudinger reduction. Org.
Lett. 19, 1582–1585 (2017).
10. C. J. Smedley et al., Sulfur-fluoride exchange (SuFEx)-mediated synthesis of sterically
hindered and electron-deficient secondary and tertiary amides via acyl fluoride in-
termediates. Chemistry 23, 9990–9995 (2017).
11. B. Gao, S. Li, P. Wu, J. E. Moses, K. B. Sharpless, SuFEx chemistry of thionyl tetraflu-
oride (SOF4) with organolithium nucleophiles: Synthesis of sulfonimidoyl fluorides,
sulfoximines, sulfonimidamides, and sulfonimidates. Angew. Chem. Int. Ed. Engl. 57,
1939–1943 (2018).
45. I. T. Suydam, C. D. Snow, V. S. Pande, S. G. Boxer, Electric fields at the active site of an
enzyme: Direct comparison of experiment with theory. Science 313, 200–204 (2006).
46. S. D. Fried, S. G. Boxer, Electric fields and enzyme catalysis. Annu. Rev. Biochem. 86,
387–415 (2017).
47. S. D. Fried, S. Bagchi, S. G. Boxer, Extreme electric fields power catalysis in the active
site of ketosteroid isomerase. Science 346, 1510–1514 (2014).
48. W. G. Lewis et al., Click chemistry in situ: Acetylcholinesterase as a reaction vessel for
the selective assembly of a femtomolar inhibitor from an array of building blocks.
Angew. Chem. Int. Ed. Engl. 41, 1053–1057 (2002).
12. T. Guo et al., A new portal to SuFEx click chemistry: A stable fluorosulfuryl imidazolium
salt emerging as an “F-SO2+” donor of unprecedented reactivity, selectivity, and scope.
Angew. Chem. Int. Ed. Engl. 57, 2605–2610 (2018).
13. J. Dong, K. B. Sharpless, L. Kwisnek, J. S. Oakdale, V. V. Fokin, SuFEx-based synthesis of
polysulfates. Angew. Chem. Int. Ed. Engl. 53, 9466–9470 (2014).
14. J. Yatvin, K. Brooks, J. Locklin, SuFEx on the surface: A flexible platform for post-
polymerization modification of polymer brushes. Angew. Chem. Int. Ed. Engl. 54,
13370–13373 (2015).
15. J. S. Oakdale, L. Kwisnek, V. V. Fokin, Selective and orthogonal post-polymerization
modification using sulfur(VI) fluoride exchange (SuFEx) and copper-catalyzed azide-
alkyne cycloaddition (CuAAC) reactions. Macromolecules 49, 4473–4479 (2016).
16. B. Gao et al., Bifluoride-catalysed sulfur(VI) fluoride exchange reaction for the syn-
thesis of polysulfates and polysulfonates. Nat. Chem. 9, 1083–1088 (2017).
17. H. Wang et al., SuFEx-based polysulfonate formation from ethenesulfonyl fluoride-
amine adducts. Angew. Chem. Int. Ed. Engl. 56, 11203–11208 (2017).
18. D. Gahtory et al., Quantitative and orthogonal formation and reactivity of SuFEx
platforms. Chemistry 24, 10550–10556 (2018).
19. K. Brooks et al., SuFEx postpolymerization modification kinetics and reactivity in
polymer brushes. Macromolecules 51, 297–305 (2018).
20. N. P. Grimster et al., Aromatic sulfonyl fluorides covalently kinetically stabilize
transthyretin to prevent amyloidogenesis while affording a fluorescent conjugate. J.
Am. Chem. Soc. 135, 5656–5668 (2013).
49. Y. Bourne et al., Freeze-frame inhibitor captures acetylcholinesterase in a unique
conformation. Proc. Natl. Acad. Sci. U.S.A. 101, 1449–1454 (2004).
50. H. D. Agnew et al., Iterative in situ click chemistry creates antibody-like protein-
capture agents. Angew. Chem. Int. Ed. Engl. 48, 4944–4948 (2009).
51. H. D. Agnew et al., Protein-catalyzed capture agents. Chem. Rev. 10.1021/acs.
chemrev.8b00660. (2019).
52. E. Oueis, C. Sabot, P.-Y. Renard, New insights into the kinetic target-guided synthesis
of protein ligands. Chem. Commun. (Camb.) 51, 12158–12169 (2015).
53. D. Bosc, J. Jakhlal, B. Deprez, R. Deprez-Poulain, Kinetic target-guided synthesis in
drug discovery and chemical biology: A comprehensive facts and figures survey. Fu-
ture Med. Chem. 8, 381–404 (2016).
54. M. Jaegle et al., Protein-templated fragment ligations—From molecular recognition
to drug discovery. Angew. Chem. Int. Ed. Engl. 56, 7358–7378 (2017).
55. H. C. Kolb, M. G. Finn, K. B. Sharpless, Click chemistry: Diverse chemical function from
a few good reactions. Angew. Chem. Int. Ed. Engl. 40, 2004–2021 (2001).
56. Q. Zheng, J. Dong, K. B. Sharpless, Ethenesulfonyl fluoride (ESF): An on-water pro-
cedure for the kilogram-scale preparation. J. Org. Chem. 81, 11360–11362 (2016).
57. C. Veryser, J. Demaerel, V. Bieliu Nas, P. Gilles, W. M. De Borggraeve, Ex situ gener-
ation of sulfuryl fluoride for the synthesis of aryl fluorosulfates. Org. Lett. 19, 5244–
5247 (2017).
58. H. Zhou et al., Introduction of a crystalline, shelf-stable reagent for the synthesis of
sulfur(VI) fluorides. Org. Lett. 20, 812–815 (2018).
59. C. J. Smedley et al., 1-Bromoethene-1-sulfonyl fluoride (BESF) is another good con-
nective hub for SuFEx click chemistry. Chem. Commun. (Camb.) 54, 6020–6023 (2018).
60. J. Leng, H. L. Qin, 1-Bromoethene-1-sulfonyl fluoride (1-Br-ESF), a new SuFEx clickable
reagent, and its application for regioselective construction of 5-sulfonylfluoro iso-
xazoles. Chem. Commun. (Camb.) 54, 4477–4480 (2018).
21. A. Baranczak et al., A fluorogenic aryl fluorosulfate for intraorganellar transthyretin
imaging in living cells and in Caenorhabditis elegans. J. Am. Chem. Soc. 137, 7404–
7414 (2015).
22. A. Narayanan, L. H. Jones, Sulfonyl fluorides as privileged warheads in chemical bi-
ology. Chem. Sci. (Camb.) 6, 2650–2659 (2015).
61. P. Birrer et al., Protease-antiprotease imbalance in the lungs of children with cystic
fibrosis. Am. J. Respir. Crit. Care Med. 150, 207–213 (1994).
23. E. C. Hett et al., Rational targeting of active-site tyrosine residues using sulfonyl
fluoride probes. ACS Chem. Biol. 10, 1094–1098 (2015).
62. A. M. Cantin, D. Hartl, M. W. Konstan, J. F. Chmiel, Inflammation in cystic fibrosis lung
disease: Pathogenesis and therapy. J. Cyst. Fibros. 14, 419–430 (2015).
63. S. Gehrig et al., Lack of neutrophil elastase reduces inflammation, mucus hyperse-
cretion, and emphysema, but not mucus obstruction, in mice with cystic fibrosis-like
lung disease. Am. J. Respir. Crit. Care Med. 189, 1082–1092 (2014).
64. R. L. Gibson, J. L. Burns, B. W. Ramsey, Pathophysiology and management of pul-
monary infections in cystic fibrosis. Am. J. Respir. Crit. Care Med. 168, 918–951 (2003).
65. N. Mayer-Hamblett et al., Association between pulmonary function and sputum
biomarkers in cystic fibrosis. Am. J. Respir. Crit. Care Med. 175, 822–828 (2007).
66. H. Nakamura, K. Yoshimura, N. G. McElvaney, R. G. Crystal, Neutrophil elastase in
respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-
8 gene expression in a human bronchial epithelial cell line. J. Clin. Invest. 89, 1478–
1484 (1992).
67. D. P. Nichols, J. F. Chmiel, Inflammation and its genesis in cystic fibrosis. Pediatr.
Pulmonol. 50 (suppl. 40), S39–S56 (2015).
68. S. D. Sagel, J. F. Chmiel, M. W. Konstan, Sputum biomarkers of inflammation in cystic
fibrosis lung disease. Proc. Am. Thorac. Soc. 4, 406–417 (2007).
69. S. D. Sagel, B. D. Wagner, M. M. Anthony, P. Emmett, E. T. Zemanick, Sputum bio-
markers of inflammation and lung function decline in children with cystic fibrosis.
Am. J. Respir. Crit. Care Med. 186, 857–865 (2012).
70. M. S. Twigg et al., The role of serine proteases and antiproteases in the cystic fibrosis
lung. Mediators Inflamm. 2015, 293053 (2015).
24. W. Chen et al., Arylfluorosulfates inactivate intracellular lipid binding protein(s)
through chemoselective SuFEx reaction with a binding site Tyr residue. J. Am. Chem.
Soc. 138, 7353–7364 (2016).
25. W. Chen et al., Synthesis of sulfotyrosine-containing peptides by incorporating fluo-
rosulfated tyrosine using an Fmoc-based solid-phase strategy. Angew. Chem. Int. Ed.
Engl. 55, 1835–1838 (2016).
26. C. Hoppmann, L. Wang, Proximity-enabled bioreactivity to generate covalent peptide
inhibitors of p53-Mdm4. Chem. Commun. (Camb.) 52, 5140–5143 (2016).
27. O. Fadeyi et al., Chemoselective preparation of clickable aryl sulfonyl fluoride
monomers: A toolbox of highly functionalized intermediates for chemical biology
probe synthesis. ChemBioChem 17, 1925–1930 (2016).
28. S. H. Li et al., Direct introduction of R-SO2F moieties into proteins and protein-
polymer conjugation using SuFEx chemistry. Polymer 99, 7–12 (2016).
29. O. O. Fadeyi et al., Covalent enzyme inhibition through fluorosulfate modification of
a noncatalytic serine residue. ACS Chem. Biol. 12, 2015–2020 (2017).
30. N. Wang et al., Genetically encoding fluorosulfate-L-tyrosine to react with lysine,
histidine, and tyrosine via SuFEx in proteins in vivo. J. Am. Chem. Soc. 140, 4995–4999
(2018).
31. B. Yang et al., Proximity-enhanced SuFEx chemical cross-linker for specific and multitargeting
cross-linking mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 115, 11162–11167 (2018).
32. B. Yang et al., Genetically introducing biochemically reactive amino acids dehy-
droalanine and dehydrobutyrine in proteins. J. Am. Chem. Soc. 141, 7698–7703 (2019).
33. D. E. Mortenson et al., “Inverse drug discovery” strategy to identify proteins that are
targeted by latent electrophiles as exemplified by aryl fluorosulfates. J. Am. Chem.
Soc. 140, 200–210 (2018).
34. Z. Liu et al., SuFEx click chemistry enabled late-stage drug functionalization. J. Am.
Chem. Soc. 140, 2919–2925 (2018).
35. C. Suter, “Derivatives of aromatic sulfonic acids. 1. Sulfonyl halides, esters, and an-
hydrides” in The Organic Chemistry of Sulfur: Tetracovalent Sulfur Compounds (Wi-
ley, New York, 1944), pp. 452–458.
71. C. J. Wagner, C. Schultz, M. A. Mall, Neutrophil elastase and matrix metalloproteinase
12 in cystic fibrosis lung disease. Mol. Cell Pediatr. 3, 25 (2016).
72. P. J. Barnes, Cytokines as mediators of chronic asthma. Am. J. Respir. Crit. Care Med.
150, S42–S49 (1994).
73. P. J. Barnes, Mediators of chronic obstructive pulmonary disease. Pharmacol. Rev. 56,
515–548 (2004).
74. K. C. Pandey, S. De, P. K. Mishra, Role of proteases in chronic obstructive pulmonary
disease. Front. Pharmacol. 8, 512 (2017).
75. Y. Qiu et al., Biopsy neutrophilia, neutrophil chemokine and receptor gene expression
in severe exacerbations of chronic obstructive pulmonary disease. Am. J. Respir. Crit.
Care Med. 168, 968–975 (2003).
76. A. Belaaouaj et al., Mice lacking neutrophil elastase reveal impaired host defense
against gram negative bacterial sepsis. Nat. Med. 4, 615–618 (1998).
77. J. P. Motta et al., Food-grade bacteria expressing elafin protect against inflammation
and restore colon homeostasis. Sci. Transl. Med. 4, 158ra144 (2012).
78. D. K. Myers, A. Kemp, Jr, Inhibition of esterases by the fluorides of organic acids.
Nature 173, 33–34 (1954).
36. W. Steinkopf, Aromatic sulphuric fluoride. J. Prakt. Chem. 117, 1–82 (1927).
37. W. Steinkopf, On aromatic sulpho-fluoride. J. Prakt. Chem. 128, 63–88 (1930).
38. W. Davies, J. H. Dick, Aliphatic sulphonyl flurorides. J. Chem. Soc. 1932, 483–486 (1932).
39. W. Davies, J. H. Dick, Benzenesulphonyl fluoride derivatives. J. Chem. Soc. 1932, 2042–
2046 (1932).
40. V. Gembus, F. Marsais, V. Levacher, An efficient organocatalyzed interconversion of
silyl ethers to tosylates using DBU and p-toluenesulfonyl fluoride. Synlett 2008, 1463–
1466 (2008).
41. E. J. Choi, D. Jung, J. S. Kim, Y. Lee, B. M. Kim, Chemoselective tyrosine bioconjugation
through sulfate click reaction. Chemistry 24, 10948–10952 (2018).
42. T. Hmissa et al., Autocatalytic synthesis of bifluoride ionic liquids by SuFEx click
chemistry. Angew. Chem. Int. Ed. Engl. 57, 16005–16009 (2018).
79. D. E. Fahrney, A. M. Gold, Sulfonyl fluorides as inhibitors of esterases. 1. Rates of
reaction with acetylcholinesterase, α-chymotrypsin, and trypsin. J. Am. Chem. Soc. 85,
997–1000 (1963).
43. J. L. Bourassa, E. P. Ives, A. L. Marqueling, R. Shimanovich, J. T. Groves, Myoglobin
catalyzes its own nitration. J. Am. Chem. Soc. 123, 5142–5143 (2001).
80. A. M. Gold, D. Fahrney, Sulfonyl fluorides as inhibitors of esterases. 2. Formation and
reactions of phenylmethanesulfonyl α-chymotrypsin. Biochemistry 3, 783–791 (1964).
Zheng et al.
PNAS
|
September 17, 2019
|
vol. 116
|
no. 38
|
18813

81. A. M. Gold, Sulfonyl fluorides as inhibitors of esterases. 3. Identification of serine as
site of sulfonylation in phenylmethanesulfonyl α-chymotrypsin. Biochemistry 4, 897–
901 (1965).
82. B. R. Baker, J. A. Hurlbut, Irreversible enzyme inhibitors. CXIV. Proteolytic enzymes. IV.
Additional active-site-directed irreversible inhibitors of α-chymotrypsin derived from
phenoxyacetamides bearing a terminal sulfonyl fluoride. J. Med. Chem. 11, 241–245
(1968).
83. B. R. Baker, J. A. Hurlbut, Irreversible enzyme inhibitors. 113. Proteolytic enzymes. 3. Active-
site-directed irreversible inhibitors of α-chymotrypsin derived from phenoxyacetamides
with an N-fluorosulfonylphenyl substituent. J. Med. Chem. 11, 233–241 (1968).
84. B. R. Baker, E. H. Erickson, Irreversible enzyme inhibitors. CXV. Proteolytic enzymes. V.
Active-site-directed irreversible inhibitors of trypsin derived from p-(phenoxyalkoxy)
benzamidines with a terminal sulfonyl fluoride. J. Med. Chem. 11, 245–249 (1968).
85. B. R. Baker, Specific irreversible enzyme inhibitors. Annu. Rev. Pharmacol. 10, 35–50
(1970).
86. R. Laura, D. J. Robison, D. H. Bing, (p-Amidinophenyl)methanesulfonyl fluoride, an
irreversible inhibitor of serine proteases. Biochemistry 19, 4859–4864 (1980).
87. D. A. Shannon et al., Sulfonyl fluoride analogues as activity-based probes for serine
proteases. ChemBioChem 13, 2327–2330 (2012).
88. M. O. Lively, J. C. Powers, Specificity and reactivity of human granulocyte elastase and
cathepsin G, porcine pancreatic elastase, bovine chymotrypsin and trypsin toward
inhibition with sulfonyl fluorides. Biochim. Biophys. Acta 525, 171–179 (1978).
89. T. Yoshimura, L. N. Barker, J. C. Powers, Specificity and reactivity of human leukocyte
elastase, porcine pancreatic elastase, human granulocyte cathepsin G, and bovine
pancreatic chymotrypsin with arylsulfonyl fluorides. Discovery of a new series of potent
and specific irreversible elastase inhibitors. J. Biol. Chem. 257, 5077–5084 (1982).
90. C. Sadlowski et al., Nitro sulfonyl fluorides are a new pharmacophore for the de-
velopment of antibiotics. Mol. Syst. Des. Eng. 3, 599–603 (2018).
91. C. J. Smedley et al., Bifluoride ion mediated SuFEx trifluoromethylation of sulfonyl
fluorides and iminosulfur oxydifluorides. Angew. Chem. Int. Ed. Engl. 58, 4552–4556
(2019).
92. I. Photaki, Transformation of serine to cysteine–β-elimination reactions in serine de-
rivatives. J. Am. Chem. Soc. 85, 1123–1126 (1963).
93. D. H. Strumeyer, W. N. White, D. E. Koshland, Jr, Role of serine in chymotrypsin ac-
tion–Conversion of active serine to dehydroalanine. Proc. Natl. Acad. Sci. U.S.A. 50,
931–935 (1963).
94. H. Weiner, W. N. White, D. G. Hoare, D. E. Koshland , Jr, The formation of anhydrochymotrypsin
by removing the elements of water from the serine at the active site. J. Am. Chem.
Soc. 88, 3851–3859 (1966).
18814
|
www.pnas.org/cgi/doi/10.1073/pnas.1909972116
Zheng et al.