“Sleeping Beauty” Phenomenon: SuFEx-Enabled Discovery of ...

doi.org/10.26434/chemrxiv.7842020.v1

“Sleeping Beauty” Phenomenon: SuFEx-Enabled Discovery of SelectiveCovalent Inhibitors of Human Neutrophil ElastaseQinheng Zheng, Jordan L. Woehl, Seiya Kitamura, Diogo Santos-Martins, Christopher J. Smedley, GenchengLi, Stefano Forli, John E. Moses, Dennis W. Wolan, K. Barry Sharpless

Submitted date: 14/03/2019 • Posted date: 15/03/2019Licence: CC BY-NC-ND 4.0Citation information: Zheng, Qinheng; Woehl, Jordan L.; Kitamura, Seiya; Santos-Martins, Diogo; Smedley,Christopher J.; Li, Gencheng; et al. (2019): “Sleeping Beauty” Phenomenon: SuFEx-Enabled Discovery ofSelective Covalent Inhibitors of Human Neutrophil Elastase. ChemRxiv. Preprint.

Sulfur-Fluoride Exchange (SuFEx) has emerged as the new generation of click chemistry. We report here aSuFEx-enabled approach exploiting the “sleeping beauty” phenomenon of sulfur fluoride compounds in thecontext of the serendipitous discovery of selective covalent human neutrophil elastase (hNE) inhibitors.Evaluation of an ever-growing collection of SuFExable compounds toward various biological assaysunexpectedly yielded a selective and covalent hNE inhibitor, benzene-1,2-disulfonyl fluoride. Derivatization ofthe initial hit led to a better agent, 2- triflyl benzenesulfonyl fluoride, itself made through a SuFExtrifluoromethylation process, with IC50 = 1.1 μM and ~200-fold selectivity over the homologous neutrophilserine protease, cathepsin G. The optimized probe only modified active hNE and not its denatured form,setting another example of the “sleeping beauty” phenomenon of sulfur fluoride capturing agents for thediscovery of covalent medicines.

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https://chemrxiv.org/articles/_Sleeping_Beauty_Phenomenon_SuFEx-Enabled_Discovery_of_Selective_Covalent_Inhibitors_of_Human_Neutrophil_Elastase/7842020/1?file=14603165



“Sleeping beauty” phenomenon: SuFEx-enabled discovery of selective 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; bDepartment of Molecular Medicine; cDepartment of Integrative

Structural and Computational Biology, The Scripps Research Institute, La Jolla, California 92037,

United States; dLa Trobe Institute for Molecular Science, La Trobe University, Bundoora,

Melbourne, VIC 3086, Australia

1Q.Z. and J.L.W. contributed equally to this work.

2To whom correspondence may be addressed. Email: [email protected] or

[email protected].

Keywords:

click chemistry; SuFEx; sulfonyl fluoride; elastase; covalent inhibitor

Abstract:

Sulfur-Fluoride Exchange (SuFEx) has emerged as the new generation of click chemistry. We

report here a SuFEx-enabled approach exploiting the “sleeping beauty” phenomenon of sulfur

fluoride compounds in the context of the serendipitous discovery of selective covalent human

neutrophil elastase (hNE) inhibitors. Evaluation of an ever-growing collection of SuFExable

compounds toward various biological assays unexpectedly yielded a selective and covalent hNE

inhibitor, benzene-1,2-disulfonyl fluoride. Derivatization of the initial hit led to a better agent, 2-

triflyl benzenesulfonyl fluoride, itself made through a SuFEx trifluoromethylation process, with IC50

= 1.1 μM and ~200-fold selectivity over the homologous neutrophil serine protease, cathepsin G.

The optimized probe only modified active hNE and not its denatured form, setting another

example of the “sleeping beauty” phenomenon of sulfur fluoride capturing agents for the discovery

of covalent medicines.

Sulfur fluoride exchange (SuFEx)—the new generation click chemistry, since first introduced in

2014 (1), has quickly found diverse applications across an array of fields including chemical

synthesis (2-12), material science (13-19), chemical biology (20-31), and drug discovery (32, 33).

SuFEx creates robust intermolecular links between modules. The extreme fidelity stems from the

ability of otherwise, very stable higher oxidation state sulfur fluorides (34-38) to exchange S–F

with incoming nucleophiles under SuFEx catalysis conditions, forming stable and irreversible

linkages united through a sulfur center. These SuFEx reactions are made possible by the unique

requirements set to stabilize a departing fluoride ion in its transit away from a strong covalent

bond, with “H+” or “R3Si+” mediators, and especially favored by DBU-type amine catalysts (13, 39,

40), and also thought to involve bifluoride counter ion species (16, 17, 41).

In the context of selective in vitro or in vivo covalent capture of proteins by SuFExable*

compounds, examples are accumulating rapidly (20-31), but we are still reluctant to make strong

a priori inferences about the factors which determine a given capture’s occurrence. This said,

there is one remarkable fact shared by all SuFEx-based protein captures that the probes’ special

S–F links are among the most demure electrophiles known† (1, 34-38). Only a correctly folded

and functionally active protein can serve as a catalyst‡ for a SuFEx capture event upon one of its

own nucleophilic amino acid sidechains (Fig. 1A). These bio-compatible SuFEx reactions are

presumably mediated by environmental factors unique to the “live” system, thereby facilitating

displacement of the otherwise stable sulfur-fluoride by the partner protein. In contrast, the

denatured proteins are completely inert to the same S–F probes (24, 32). We name this special

relationship between functional living proteins and SuFExable probes the “sleeping beauty”

phenomenon§, which mirrors very closely our earlier protein enabled Huisgen azide-alkyne

cycloaddition (i.e. in situ click chemistry) (42, 43), in terms of the “live” enzyme requirement.

Taking inspiration from the original click chemistry manifesto (44); 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 three steps, we demonstrate here two distinct but logically

connected, sequential SuFEx-enabled entities: (1) efficient construction of a pool of SuFExable

compounds via click chemistry principles, and (2) bioprospecting this library for covalent capture

agents for important protein targets via “sleeping beauty” reactivity. The expectation, based on

previous experience, is that evaluation of a SuFEx library will often result in multiple lead

compounds. The latter can usually be followed up by easily deployed SAR-based (structure-

activity-relationship-based) lead studies. This “compound-bank” mode of the SuFEx-enabled

platform, itself contributes to the ever-growing library of SuFExable modules and candidates, and

like “compounding-interest”, rewards the principle library with expanding opportunities for

discovering new kinds of phenotypic modulators; including useful new functions for medicines in

the long term (Fig. 1B).

Over the past five years, we (1, 5, 8, 45) and others (12, 46-49) have developed a valuable

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 modules. There are >1000 compounds in this collection¶. 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. The individual compounds are dissolved as 10 mM DMSO stock

solutions and stored in –20 ºC freezer, with most being stable at –20 ºC as evidenced by periodic

inspection by LC-MS of randomly sampled compound over 2 years. To date, the library and/or its

sub-libraries have been screened with collaborators at Scripps Research and other institutes

against multiple targets. Here we report a case study using the SuFEx-enabled approach to the

discovery of selective, covalent inhibitors of human neutrophil elastase (hNE).

Human neutrophil elastase; a member of the serine protease superfamily, is aberrantly active

in cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and inflammatory bowel

diseases (IBD) (50-66). This protease is therefore a key target for the development of anti-

inflammatory agents to combat these diseases. Alkyl and aryl sulfonyl fluorides have a long

history as rather promiscuous covalent inhibitors of serine proteases (67-76). In the late 1970’s,

the Powers group demonstrated the intrinsic reactivity differences across several serine

proteases, including elastase, with 2-amido(peptido) benzenesulfonyl fluoride inhibitors (77, 78).

Their results encouraged 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 four subsets by the nature of S–F functional groups

(Fig. 1C). 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 representative molecule of each

subset under SuFEx catalysis are 28.5 (I), 1.0 (II, reference), 14.1 (III), and 4.1 (IV), respectively

(SI Appendix, Figure S6). Aryl sulfonyl fluorides (I), despite their own extreme resistance toward

nucleophiles*, top the SuFEx reactivity hierarchy among the four 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 substrate

MeOSuc-AAPV-AMC (50 μM). Increase in fluorescence was measured for 30 min at 30 sec

intervals. The assay was well behaved, as evidenced by a Z’ of 0.86, signal-to-background of

~3000:1, and a hit cutoff of >95% inhibition. Reasoning that a covalent inhibitor, which presumably

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. 2A, 6.7% overall hit rate

and 23% hit rate within subset I). All 7 compounds belong to subset I, which suggests a rough cut

off 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. 2B,C).

The covalent inhibition of hNE by 1 was examined by high-resolution MALDI-TOF mass

spectrometry (Fig. 3A). Incubation of 1 (exact mass 242) with hNE yielded a peak shift from the

protein mass by ~223 Da. Increased mass corresponds to: (1) a single molecule of 1 covalently

captured by hNE; (2) the loss of one hydrogen from the protease (possibly from the catalytic

serine); (3) the loss of one fluorine from 1.

We also subjected hNE to co-crystallization with 1 in order to corroborate the covalent binding.

The structure was determined using molecular replacement with PDB ID 5adw and the co-

complex was refined to 2.33 Å resolution (SI Appendix, Table S2). Importantly the naïve Fo-Fc

electron density maps contoured to 4σ clearly position 1, as a result of the strong diffraction of

sulfurs (Fig. 3B). 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 catalytic Ser195, as highlighted

by continuous electron density and a bond distance of ~1.6 Å (Fig. 3C,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, Figure S2).

Considering the mono-covalent attachment mode of 1, with the second –SO2F intact, it was

envisaged that one of the two sulfonyl fluoride groups could be substituted so as to perhaps

improve the capture rate, and/or selective binding. A set of benzenesulfonyl fluoride cores

carrying ortho-substituents (8–23) was therefore examined. Compounds 8–18 were synthesized

by the efficient aqueous potassium bifluoride exchange procedure from commercially available

sulfonyl chlorides but showed poorer reactivity/binding with hNE#. Of the two improved

compounds found in Table 1, 19, the mono-vinylogous derivative of 1, 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 potassium bifluoride catalyzed SuFEx

trifluoromethylation of aryl sulfonyl fluorides (79) enabled us to quickly convert 1 to the

perfluoroalkyl sulfones (22, 23). Compound 22 with the ortho-triflyl group emerged as the best

lead molecule to date with IC50 = 1.1 ± 0.1 μM.

Compound R IC50 (µM)||

1 SO2F 3.3 ± 1.0

8 F ~120

9 Cl 82 ± 16

10 Br 20 ± 10

11 I 9.7 ± 1.2

12 Me >200

13 OMe 73 ± 4

14 CN 13.3 ± 0.5

15 CF3 60 ± 8

16 NO2 20 ± 1

17 CO2Me 37 ± 2

18 Ph 27 ± 1

19

2.2 ± 0.7

20

84.4 ± 0.6

21

>200

22 SO2CF3 1.1 ± 0.1

23 SO2(CF2)2CF3 48 ± 2

SO2F

R

SO2F

S NO

OO

S NO

ON S F

O

O

Table 1. Efforts of optimization of lead compound 1 with alternative ortho-substitutions.

High resolution MALDI-TOF mass spectrometry study supports the covalent inhibition

mechanism of 22 to be sulfonylation of hNE (+ ~273 Da) possibly at its active site serine (Fig.

4A,B). In contrast, the incubation of compound 22 with inactive denatured hNE led to no

detectable covalent modification of the enzyme (Fig. 4C). The requirement for the naturally folded

enzyme represents another example of the unusual “sleeping beauty” phenomenon of SuFEx,

wherein the sulfur fluoride probes are inert in biological fluids/buffer if the capture protein or

proteins presented are denatured (24, 32).

Intriguingly, testing the lead compounds (1, 19, 22) against a panel of serine proteases, we

found that two (1, 22) among the three effective hNE inhibitors did not inactivate the homologous

serine protease, human cathepsin G (hCG), which has 37% sequence identical to hNE and 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 and 22 identified in this study showed 58

and 182-fold specificity for hNE over hCG, respectively (Table 2). The selective inhibition of hNE

could be partly attributed to a proximity factor as suggested by molecular modeling using a

reactive docking protocol (SI Appendix, Figure S5).

Compound Structure hNE IC50 (µM)|| hCG IC50 (µM)|| S**

1

3.3 ± 1.0 190 ± 40 58

19

2.2 ± 0.7 6.0 ± 0.7 2.7

22

1.1 ± 0.1 >200 >182

SO2F

SO2F

SO2F

SO2F

SO2F

SO2CF3

PMSF

24 ± 1 69 ± 6 2.5

Table 2. Selectivity of the most potent compounds 1, 19, 22 against hNE and hCG.

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 reversible inhibitors in the active site, but the library

of sulfonyl fluorides used in the screen was chosen without regard to any enzyme•potential ligand

relationships. In other words, agnostic of structural considerations, the library yielded two more

examples of the “sleeping beauty” phenomenon, and in this instance its successful outcome is

distinguished by lack of known design elements around the pendant S–F electrophiles on the

candidate “ligand” scaffolds. This successful sulfur fluoride library is being used and augmented

regularly at Scripps Research, and it will hopefully contribute to future SuFEx-driven covalent drug

discovery endeavors.

SO2F

Fig. 1. Overview of SuFEx-enabled covalent drug discovery. (A) The “sleeping beauty”

phenomenon: sulfur fluoride probes only capture a naturally folded protein where a nucleophilic

sidechain (Nu) and activating sidechains (Act) are correctly positioned. (B) Cartoon schematic of

the SuFEx-enabled covalent drug discovery process. (C) Efficient SuFEx derivatization of

abundant building blocks yields a SuFExable library with four subset groups, categorized

according to their sulfur-fluoride based functionalities.

Fig. 2. Screen of the primary SuFEx library toward elastase inhibitory activity. (A) Initial screen

with 105 SuFExable compounds yielded 7 hits with >95% inhibition at 200 µM. (B) Dose-response

curves of hit compounds against hNE (AAPV-AMC fluorescence assay). Each compound was

assessed over a two-fold logarithmic dilution series. (C) Structures and IC50 values of compounds

1–7 and PMSF (phenylmethane sulfonyl fluoride). IC50 values were measured based on 10 min

incubation and are shown in mean ± SD (n ≥ 3).

Fig. 3. Compound 1 is a covalent inhibitor of hNE. (A) MALDI-TOF mass spectrometry evidence

of covalent complex 1:hNE formation. (B) Naïve Fo-Fc map contoured at 1.5s (green) and 4s

(magenta) clearly delineate 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 (grey

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.

Fig. 4. The “sleeping beauty” phenomenon of sulfonyl fluoride 22 demonstrated by the MALDI-

TOF mass spectra of (A) naturally folded hNE; (B) naturally folded hNE incubated with 22; (C)

denatured hNE (boiled) incubated with 22.

Footnotes:

*SuFExable are defined to describe those compounds containing a sulfur fluoride link that can

undergo SuFEx reaction.

†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 ºC for 3 h. 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 are not stable. (SI Appendix, Table S3).

‡As long as the accelerating AA sidechains (including but not limited to His57) and reactive moiety

(Ser195) are separately considered, and the former remain overall unchanged, the intramolecular

activation mode can be seen as “catalytic”, even if the TON equals 1. For a similar case, see:

Kassem S, et al. (2017) Stereodivergent synthesis with a programmable molecular machine.

Nature 549(7672):374-378.

§“Sleeping beauty” here defines the phenomenon of the remarkable acceleration of a SuFEx

reaction for otherwise very stable higher valent sulfur fluoride compounds, by the right, naturally

folded protein catalyst for its own covalent capture. The term has been used by Izsvák and co-

workers in molecular biology to describe the use of a specifically designed transposon system

(i.e. Tc1/mariner-type transposase and transposon) that can selectively insert genes into the

genomes of vertebrates. The genome of humans as well as of other mammals encodes for non-

functional transposases and the introduction of an active transposase from lower vertebrate fish

and amphibian species “awakens the system from an evolutionary sleep”. For the original paper

on sleeping beauty transposon, see: Ivics Z, Hackett PB, Plasterk RH, & Izsvak Z (1997)

Molecular reconstruction of Sleeping beauty, a Tc1-like transposon from fish, and its transposition

in human cells. Cell 91(4):501-510.

¶We are very grateful to the following co-workers for their contribution to the SuFExable compound

library: Hua Wang (manager), Peng Wu and his group, Scripps Research, Jiajia Dong and his

group, SIOC, Larisa Krasnova, Suhua Li, Qinheng Zheng, Bing Gao, Grant A. L. Bare, John E.

Moses and his Group, La Trobe University, Hua-Li Qin and his group, Wuhan University of

Technology, En-Xuan Zhang and his group, AsymChem Inc., Gerui Ren, Gencheng Li, Feng Zhou,

Feng Liu, Hamid R. Safaei.

#At the time of manuscript preparation, Murthy and co-workers reported a study on 2-

nitrobenzenesulfonyl fluoride (19) and derivatives as covalent capture agents for new antibiotics

development. Compound 17 showed only moderate inhibitory potency in our elastase activity

assay. For Murthy’s paper, see: Sadlowski C, et al. (2018) Nitro sulfonyl fluorides are a new

pharmacophore for the development of antibiotics. Mol Syst Des Eng 3(4):599-603.

||IC50 values were measured based on 10 min incubation and are shown in mean ± SD (n ≥ 3).

**S value denotes the selectivity, defined by the ratio of IC50 (hCG) over IC50 (hNE).

Acknowledgements:

The authors gratefully acknowledge financial support from NIH R01 GM117145 (K.B.S.), The

Scripps Research Institute (D.W.W.), NIH T32 AI7354-27 (J.L.W.), NIH R01 GM069832 (D.S.M.,

S.F.). We thank H. Rosen for access to instrumentation and the staff of the Stanford Synchrotron

Radiation Lightsource. We thank Y. Su of the Molecular Mass Spectrometry Facility of UCSD for

assistance on APCI-MS.

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download fileview on ChemRxivZheng et al Sleeping Beauty 2019 ChemRxiv.pdf (3.27 MiB)



S1

Supporting Information

“Sleeping beauty” phenomenon: SuFEx-enabled discovery of

selective 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; bDepartment of Molecular Medicine; cDepartment of Integrative

Structural and Computational Biology, The Scripps Research Institute, La Jolla, California 92037,

United States; dLa Trobe Institute for Molecular Science, La Trobe University, Bundoora,

Melbourne, VIC 3086, Australia

1Q.Z. and J.L.W. contributed equally to this work.

2To whom correspondence may be addressed. Email: [email protected] or

[email protected].

S2

Table of Contents

Contents Page

1. General 4

2. Compound synthesis and characterizations 5

2.1. General procedures 5

2.2. Naphthalene-1,3,6-trisulfonyl trifluoride (2) 8

2.3. Naphthalene-2,3-disulfonyl difluoride (3) 9

2.4. Methyl 3-(fluorosulfonyl)thiophene-2-carboxylate (4) 11

2.5. 2,6-dichlorobenzenesulfonyl fluoride (5) 12


2.7. 2-Iodobenzenesulfonyl fluoride (11) 14

2.8. [1,1'-biphenyl]-2-sulfonyl fluoride (18) 15

2.9. (E)-2-(2-(fluorosulfonyl)vinyl)benzenesulfonyl fluoride (19) 16

2.10. 2-(Morpholinosulfonyl)benzenesulfonyl fluoride (20) 17

2.11 4-((2-(Fluorosulfonyl)phenyl)sulfonyl)piperazine-1-sulfonyl fluoride (21) 19

2.12. 2-((Trifluoromethyl)sulfonyl)benzenesulfonyl fluoride (22) 21

2.13. 2-((Perfluoropropyl)sulfonyl)benzenesulfonyl fluoride (23) 22

3. Protease activity assays and library screen 23

3.1. Methods 23

3.2. Screen results 24

3.3. Validation of permanent binding 35

3.4. Inhibitory activity of hit molecules against hCG. 36

4. Mass Spectrometry 37

4.1. Methods 37

4.2. Results 37

5. X-ray crystal structure 38

5.1. Methods 38

5.2. Results 39

6. Reactive docking 40

6.1. Methods 40

6.2. Results and discussion 40

7. Reactivities of SuFExable functional groups 42

8. NMR spectra 43

8.1. Naphthalene-1,3,6-trisulfonyl trifluoride (2) 43

S3

8.2. Naphthalene-2,3-disulfonyl difluoride (3) 46

8.3. Methyl 3-(fluorosulfonyl)thiophene-2-carboxylate (4) 49



8.6. 2-Iodobenzenesulfonyl fluoride (11) 58

8.7. [1,1'-biphenyl]-2-sulfonyl fluoride (18) 61

8.8. (E)-2-(2-(fluorosulfonyl)vinyl)benzenesulfonyl fluoride (19) 64

8.9. 2-(Morpholinosulfonyl)benzenesulfonyl fluoride (20) 67

8.10. 4-((2-(Fluorosulfonyl)phenyl)sulfonyl)piperazine-1-sulfonyl fluoride (21) 70

8.11. 2-((Trifluoromethyl)sulfonyl)benzenesulfonyl fluoride (22) 73

8.12. 2-((Perfluoropropyl)sulfonyl)benzenesulfonyl fluoride (23) 76

9. References 80

S4

1. General Synthetic reagents, catalysts, and solvents were used as purchased without further

purification, unless otherwise indicated. The extent of reaction was monitored by thin-layer

chromatography (TLC), performed on 250 μm silica gel G plates with F254 indicator. The TLC

plates were visualized by ultraviolet light (254 nm) and treatment with potassium permanganate

stain followed by gentle heating. Flash chromatography was performed using 40−63 μm (230−400

mesh) silica gel.

Unless otherwise noted, 1H, 13C, and 19F NMR spectra were recorded on Bruker DRX-500,

Bruker DRX-600, Bruker AMX-400 instruments. Data for 1H NMR spectra is reported as follows:

chemical shift (ppm, referenced to residual solvent peak), coupling constant (Hz), and integration.

Data for 13C NMR is reported in terms of chemical shift, δ (ppm) relative to residual solvent peak

(CDCl3 singlet at 77.0 ppm, DMSO multiplet at 39.5 ppm). Data for 19F NMR is reported in terms

of chemical shift (ppm) relative to added internal standard (CFCl3 at 0.65 ppm) (1). Accurate mass

spectrometry (a.k.a. HRMS) spectra were recorded using electrospray ionization (ESI) or

atmosphere-pressure chemical ionization (APCI) with a time-of-flight (TOF) analyzer. For some

entries (compounds 5, 7, 11, and 18), soft ionization like ESI and APCI failed to give a molecular

ion signal. Hence, GC-MS with electron impact ionization (EI) and quadrupole analyzer was used

and gave strong signals at [M]+ for these compounds. Melting points were measured on a

Barnstead Electrothermal 9300 digital capillary melting point apparatus and are uncorrected.

S5

2. Compound synthesis and characterizations 2.1. General procedures

Unless otherwise noted, compounds in the SuFEx library were synthesized by the following

general procedures (I, II, III, IV). Synthesis and characterizations of compounds 1, 6, 8–10, and

12–17 have been reported in the literature (2-6).

(I) Synthesis of aryl sulfonyl fluorides

Procedure I (2): Aryl sulfonyl chloride (from commercial sources or synthesized by established

methods) dissolved in acetonitrile (0.5–1 M) was treated with saturated potassium bifluoride

aqueous solution (~5 M, 1.5–2.5 equiv). The emulsion was stirred vigorously for 1–4 h before

partitioned between ethyl acetate and water. The organic solution was collected, dried over

anhydrous sodium sulfate, concentrated and purified by column chromatography, if necessary, to

yield desired aryl sulfonyl fluoride (33 examples, 90–100% isolated yield).

(II) Synthesis of aryl fluorosulfates

Procedure IIA (2): (CAUTION: the reaction must be performed in a well vented fume hood.

Sulfuryl fluoride gas is toxic.) Phenols (from commercial sources), and triethylamine (1.5 equiv)

were dissolved in DCM. The flask sealed with a rubber septum was evacuated to gentle vacuum

evidenced by bubbling of solvent, and a balloon filled with sulfuryl fluoride gas was introduced to

the flask via a needle. The reaction was stirred vigorously for 2 h. Upon completion, solvent was

removed in vacuo. Residue was partitioned between ethyl acetate and water. The organic phase

Ar SO

OCl CH3CN/H2O

rt, 1–6 h

KFHF Ar SO

OF+

33 samples90–100% yield

OH

F S F

O O

RO S

FO

O

RNN S

O

OF

OTf 32 examples82–99% yield

NEt3DCMrt, 2 h

NEt3acetonitrile

rt, 1 h

IIA

IIB

S6

was washed with brine, dried over anhydrous sodium sulfate, then concentrated and purified by

flash column chromatography to give desired aryl fluorosulfate (32 examples, 82–99% isolated

yield).

Procedure IIB (7): Phenols (from commercial sources), and N-fluorosulfonyl N’-methyl-2-

methylimidazolium triflate (1.2 equiv) were dissolved in acetonitrile (0.1–1 M). Triethylamine (1.5

equiv) was added dropwise via a syringe. The reaction was stirred at room temperature for 1 h.

Upon completion, solvent was removed in vacuo. Residue was partitioned between ethyl acetate

and water. The organic phase was washed with brine, dried over anhydrous sodium sulfate, then

concentrated and purified by flash column chromatography to give desired aryl fluorosulfate (32

examples, 82–99% isolated yield).

(III) Synthesis of alkyl sulfonyl fluorides by Michael addition

Procedure IIIA (2, 8): To a solution of primary or secondary alkyl amine in DCM (0.5–1 M),

ESF (2.2 equiv) was added dropwise (exotherm). The mixture was stirred at autogenous

temperature for 6–12 h. Upon completion, volatiles were removed in vacuo. The residue was

purified by flash column chromatography to give desired sulfonyl fluoride adducts of primary or

secondary amines. (30 examples, 85–98% isolated yield).

Procedure IIIB (2, 8): Anilines and ESF (2.2 equiv) were dissolved (or suspended at starting

point) in glacial acetic acid (10 mmol per 1 mmol). The mixture was heated to 50 ºC (or 80 ºC if

necessary) for 12 h. Upon completion, volatiles were removed in vacuo. The residue was

purified by either recrystallization in ethanol or flash column chromatography to give desired

sulfonyl fluoride adducts of anilines (30 examples, 85–98% isolated yield).

(IV) Synthesis of vinyl sulfonyl fluorides

+ S F

O

OR N S

FO

ORR

NHR IIIA: DCM, rt, 2–6 h

IIIB: AcOH, rt, 12 h

30 examples85–98% yield

R I + S F

O

O R SFO

O

Pd(OAc)2AgTFAacetone

60 ºC, 6–12 h10 examples59–99% yield

S7

Procedure IV (9): An oven-dried vessal was charged with (hetero)aryl iodide, AgTFA (1.2

equiv), Pd(OAc)2 (2 mol%), acetone, and ethenesulfonyl fluoride (ESF, 2 equiv) were added.

The resulting mixture was refluxed at 60 ºC. Upon full conversion of (hetero)aryl iodide (6–12 h),

solvent was removed in vacuo. Crude was purified by flash column chromatography to give

desired product (10 examples, 59–99% yield).

S8

2.2. Naphthalene-1,3,6-trisulfonyl trifluoride (2)

A 100-mL round bottom flask equipped with a condenser was charged with naphthalene-

1,3,(6,7)-trisulfonic acid trisodium salt hydrate (4.34 g, < 10 mmol), and phosphorus pentachloride

(21 g, 100 mmol). Upon shaken of the flask, gas (hydrochloride with moist) evolution and

exotherm were observed. The mixture liquefied after heated at 110 ºC for about 0.5 h. The

reaction was stirred at 110 ºC overnight and cooled to room temperature. The yellow suspension

was poured with care onto crushed ice (200 g). The mixture was extracted with chloroform (150

mL x 3). The combined organic phase was washed by brine and dried over anhydrous sodium

sulfate before evaporated to a yellow solid crude (2-int-mix).

The crude sulfonyl chloride was mixed with potassium fluoride (11.6 g, 200 mmol) in a 500-mL

round bottom flask. Acetone (200 mL) was added and the resulting suspension was stirred

overnight. Volatiles were removed in vacuo, and the solid was partitioned with chloroform/water.

Organic phase was collected, concentrated and purified by column chromatography (SiO2, eluted

with hexanes to 20% ethyl acetate in hexanes). The titled compound was isolated as a white

crystalline (1.53 g, 41% yield over 2 steps, not corrected for the contamination of water in starting

material). 1H NMR (500 MHz, CDCl3) δ 9.17 (d, J = 1.3 Hz, 1H), 9.01 (d, J = 1.9 Hz, 1H), 8.99 (d, J = 1.8

Hz, 1H), 8.96 (dd, J = 9.1, 1.8 Hz, 1H), 8.50 (dd, J = 9.1, 2.0 Hz, 1H). 13C NMR (126 MHz, CDCl3) δ 139.6, 134.8 (d, JCF = 27.7 Hz), 133.5, 133.1, 132.9, 132.2, 132.0,

131.1 (d, JCF = 2.5 Hz), 130.1, 127.6. 19F NMR (377 MHz, CDCl3) δ 67.7, 66.8, 64.9.

TLC Rf = 0.65 (17% ethyl acetate in hexanes).

Melting point 124 – 126 ºC.

Mass Spectrometry APCI-TOF accurate mass calculated for C10H5F3O6S3 [M]– 373.9206, found

373.9213.

HPLC purity (280 nm) 96.8% (tR = 5.320 min).

SO3Na

SO3NaNaO3S •xH2O(6,7) PCl5

110 ºC

SO2Cl

SO2ClClO2S(6,7) KF

acetonert

SO2F

SO2FFO2S

22-int-mix

S9

2.3. Naphthalene-2,3-disulfonyl difluoride (3)

A 250-mL round bottom flask equipped with a stir bar was charged with 2,3-

dibromonaphthalene (1.1 g, 3.82 mmol), and sodium 2-propylthiolate (90% tech. grade, 1.66 g,

15.3 mmol). Hexamethylphosphoramide (HMPA, 20 mL, anhydrous, stored over 4 Å molecular

sieves) was added under N2. Upon heated to 100 ºC, the suspension was rapidly stirred into an

orange solution, and about 15 min later to another suspension. The suspension was stirred for 8

h and cooled to room temperature. Partition the mixture between ether (100 mL) and water (100

mL). Aqueous phase was further extracted by ether (50 mL x 2). Combine all organic phase, wash

with water (150 mL x 2) and brine (150 mL). Solvent was removed in vacuo giving crude thioether

(3-i). 1H NMR (500 MHz, Chloroform-d) δ 7.76 (s, 2H), 7.72 (dd, J = 6.2, 3.3 Hz, 2H), 7.42 (dd, J

= 6.2, 3.2 Hz, 2H), 3.59 (hept, J = 6.7 Hz, 2H), 1.39 (d, J = 6.7 Hz, 12H). 13C NMR (126 MHz,

CDCl3) δ 135.8, 132.3, 129.2, 127.0, 126.2, 37.4, 23.0.

The crude product (>95 % purity by 1H NMR) was dissolved in HMPA (20 mL). Under N2 stream,

freshly cut sodium strip (about 0.3 g, 10 mmol) was added with care over 15 min. A dark solution

was obtained, which was further stirred at 100 ºC overnight. The reaction gradually turned light-

yellow. Cool the mixture to room temperature, concentrated HCl was added dropwise (Be very

careful!) to adjust the pH lower than 3. The resulting suspension was partitioned between tert-

butyl methyl ether (100 mL) and water (100 mL). The organic phase was washed by water (100

mL x 2) and brine (100 mL) and concentrated to a yellow solid crude (3-ii, 0.83 g, 4.3 mmol, >100%

over 2 steps, HMPA contaminated). 1H NMR (400 MHz, Chloroform-d) δ 7.90 (s, 2H), 7.66 (dd, J

= 6.3, 3.2 Hz, 2H), 7.41 (dd, J = 6.3, 3.2 Hz, 2H), 3.88 (s, 2H).

The yellow solid was suspended on methanol (10 mL). Gentle heating at 50 ºC helped to

dissolve the thiolphenol. Cooled back to room temperature, into the solution was added hydrogen

peroxide (30%, 10 mL, ~ 40 equiv). This process was found exotherm, and precipitates formed

instantly. The yellow suspension was stirred at room temperature, turned into a thick porridge

after 3 h, and then a clear yellow solution overnight. After 24 h, the clean conversion to 3-iii was

Br

Br

3

NaSiPrHMPA100 ºC

S

S

Na (metal)HMPA100 ºC

SH

SH

H2O2H2O/MeOH

SO

O OH

SO

O

OH

NaOHH2O

SO

O ONa

SO

O

ONa

PCl5POCl3110 ºC

SO

O Cl

SO

O

Cl

KFHFCH3CN/H2O

rt

SO

O F

SO

O

F

3-i 3-ii 3-iii

3-iv 3-v

S10

determined by 1H NMR. 1H NMR (400 MHz, Methanol-d4) δ 8.68 (s, 1H), 8.01 (dd, J = 6.1, 3.2

Hz, 1H), 7.67 (dd, J = 6.2, 3.2 Hz, 1H).

Adjust the pH of the acid solution with 2 mol L-1 NaOH (3 mL) to over 8 (exotherm). Catalytic

amount of manganese dioxide (25 mg) was added to digest excess amount of hydrogen peroxide.

When gas evolution ceased, the solvent was removed in vacuo, and crude product being

azeotropically dried by toluene (25 mL x 3).

Naphthalene-2,3-disulfonic acid disodium salt, with MnO2 contaminated, was mixed with

phosphorus pentachloride (1.5 g, 7.2 mmol) in a 100-mL round bottom flask. Reaction occurred

instantly releasing heat and fume. The mixture was heated at 110 ºC for 6 h, before cooled to

room temperature and poured onto crushed ice (50 g). Sulfonyl chloride was extracted by

chloroform (100 mL), and washed sequentially by cold water (100 mL), brine (100 mL). The

sulfonyl chloride was not stable on silica gel, neither TLC nor column was applicable. 1H NMR

showed > 90% purity. 1H NMR (400 MHz, Chloroform-d) δ 8.98 (s, 1H), 8.21 (dd, J = 6.2, 3.3 Hz,

1H), 7.98 (dd, 1H).

Dissolved in acetone (20 mL, insoluble in acetonitrle), naphthalene-2,3-disulfonyl chloride (3-v) was treated by finely powdered potassium fluoride (464 mg, 8.00 mmol). The suspension was

stirred overnight, and then concentrated. Partition the crude between chloroform and water.

Organic phase was collected, concentrated, and purified by column chromatography (SiO2, 10%

to 33% ethyl acetate in hexanes) to give the titled compound as a yellow solid (208 mg, 0.711

mmol, 19% over 6 steps). 1H NMR (600 MHz, CDCl3) δ 9.00 – 8.96 (m, 2H), 8.20 (dt, J = 6.4, 3.2 Hz, 2H), 8.02 – 7.97 (m,

2H). 13C NMR (151 MHz, CDCl3) δ 137.2, 133.7, 132.9, 130.2, 126.8 (d, JCF = 30.2 Hz). 19F NMR (377 MHz, CDCl3) δ 72.8.

TLC Rf = 0.52 (33% ethyl acetate in hexanes, UV).

Melting point 156 – 158 ºC (ether).

Mass spectrometry APCI-TOF accurate mass calculated for C10H6F2O4S2NH4 [M + NH4]+

310.0014, found 310.0013.


S11

2.4. Methyl 3-(fluorosulfonyl)thiophene-2-carboxylate (4)

The title compound was synthesized by General Procedure I. Saturated potassium bifluoride

solution effected the conversion of methyl 3-(chlorosulfonyl)thiophene-2-carboxylate (Combi-

Blocks, 1.20 g, 5.00 mmol) to methyl 3-(fluorosulfonyl)thiophene-2-carboxylate (4, 1.06 g, 4.73

mmol, 95%) as a brown solid. 1H NMR (600 MHz, CDCl3) δ 7.65 (dd, J = 5.3, 0.6 Hz, 1H), 7.61 (d, J = 5.3 Hz, 1H), 3.99 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 159.1, 137.3, 134.6 (d, JCF = 30.2 Hz), 130.9 (d, JCF = 1.5 Hz), 130.6

(d, JCF = 1.5 Hz), 53.6. 19F NMR (377 MHz, CDCl3) δ 63.0.


Melting point 78 – 80 ºC (decompoused).

Mass spectrometry ESI-TOF accurate mass calculated for C6H5FO4S2Na [M + Na]+ 246.9505,

found 246.9505.

HPLC purity (254 nm) 100% (tR = 4.307 min).

SO

O Me

SO

O Cl

SO

O Me

SO

O F

KFHFacetonitrile/H2O

rt2 h

4

S12

2.5. 2,6-dichlorobenzenesulfonyl fluoride (5)


solution effected the conversion of 2,6-dichlorobenzenesulfonyl chloride (Combi-Blocks, 1.23 g,

5.00 mmol) to 2,6-dichlorobenzenesulfonyl fluoride (5, 1.11 g, 4.85 mmol, 97%) as a yellow

crystalline. 1H NMR (600 MHz, CDCl3) δ 7.63 – 7.44 (m, 3H). 13C NMR (151 MHz, CDCl3) δ 136.2, 135.0, 131.6, 131.0 (d, JCF = 24.2 Hz). 19F NMR (377 MHz, CDCl3) δ 68.2.


Melting point 70 – 72 ºC (hexane/ethyl acetate).

Mass spectrometry EI-Q mass calculated for C6H3Cl2FO2S [M]+ 227.92, found 227.9 (100), 132.9

(80), 229.9 (78), 109.0 (61), 74.0 (53), 160.9 (38), 144.9 (43).


Cl

Cl

SO

OFCl

Cl

SO

OClKFHF

acetonitrile/H2Ort

2 h5

S13



solution effected the conversion of 3,4-dichlorobenzenesulfonyl chloride (Combi-Blocks, 1.23 g,

5.00 mmol) to 3,4-dichlorobenzenesulfonyl fluoride (7, 1.03 g, 4.50 mmol, 90%) as a colorless

liquid. 1H NMR (600 MHz, CDCl3) δ 8.10 (d, J = 2.2 Hz, 1H), 7.85 (dd, J = 8.5, 2.2 Hz, 1H), 7.73 (dd, J

= 8.5, 1.0 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 140.7, 134.2, 132.0 (d, JCF = 27.2 Hz), 131.3, 129.8, 126.9. 19F NMR (377 MHz, CDCl3) δ 66.89, 0.65.


Mass spectrometry EI-MS calculated for C6H3Cl2FO2S [M]+ 227.92, found 227.9.


Cl

SO

O F

7

ClCl

SO

O Cl

Cl

KFHFCH3CN/H2O

rt2 h

S14

2.7. 2-Iodobenzenesulfonyl fluoride (11)

Neutralizing 2-iodobenzene sulfonic acid hydrate with sodium bicarbonate gave 2-

iodobenzene sulfonic acid sodium salt (11-i) as a brown crystalline. A 100-mL round bottom flask

equipped with a condenser and a tail gas absorbing water tank was charged with 2-iodobenzene

sulfonic acid sodium salt (5.00 g, 16.3 mmol) and phosphorus pentachloride (5.10 g, 24.5 mmol).

The solid mixture liquefied partially after shaken with exotherm. Phosphoryl chloride (2 mL) was

added to help the stirring of the syrup-like viscous mixture. The reaction was heated at 110 ºC for

4 h before cooled to room temperature. The dark red mixture was poured onto ice (150 g) and

extracted with ether (100 mL x 3). The organic phase was washed with cold water and dried over

anhydrous sodium sulfate. TLC indicated the purity of the crude product (11-ii) about 90%. The

ether solution was concentrated and remaining thick oil was re-dissolved into acetonitrile (10 mL).

Saturated potassium bifluoride solution (~ 5 mol L-1, 6.5 mL) was added, and the resulting biphasic

mixture being stirred into an emulsion for 3 h. Partition the mixture with water (100 mL) and ethyl

acetate (100 mL). The organic phase was concentrated and purified by column chromatography

(SiO2, hexanes to 17% ethyl acetate in hexanes) to give an off-white crystalline (2.73 g, 9.54

mmol, 59% over 3 steps). 1H NMR (500 MHz, CDCl3) δ 8.18 (dd, J = 7.9, 1.1 Hz, 1H), 8.15 (dd, J = 8.0, 1.5 Hz, 1H), 7.59

(tt, J = 7.9, 1.3 Hz, 1H), 7.38 (td, J = 7.7, 1.6 Hz, 1H). 13C NMR (126 MHz, CDCl3) δ 143.3, 137.8 (d, JCF = 28.7 Hz), 135.8, 132.1 (d, JCF = 1.3 Hz),

128.8, 92.3. 19F NMR (377 MHz, CDCl3) δ 56.9.


Melting point 61 – 62 ºC (MeOH).

Mass spectrometry EI-MS calculated for C6H4FIO2S [M]+ 285.9, found 285.6.


IS

O

O FIS

O

O OH NaOHH2O

IS

O

O ONa PCl5POCl3110 ºC

IS

O

O Cl KFHFCH3CN/H2O

rt1111-i 11-ii

S15

2.8. [1,1'-biphenyl]-2-sulfonyl fluoride (18)


solution effected the conversion of [1,1'-biphenyl]-2-sulfonyl chloride (Alfa-Aesar, 1.25 g, 5.00

mmol) to [1,1'-biphenyl]-2-sulfonyl fluoride (18, 1.10 g, 4.66 mmol, 93%) as a white crystalline. 1H NMR (600 MHz, CDCl3) δ 8.19 (d, J = 8.0 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.60 (t, J = 7.8 Hz,

1H), 7.51 – 7.43 (m, 5H), 7.41 – 7.35 (m, 3H). 13C NMR (151 MHz, CDCl3) δ 143.2, 138.0, 134.9, 133.2 (d, JCF = 1.5 Hz), 132.5 (d, JCF = 22.7

Hz), 130.1 (d, JCF = 1.5 Hz), 129.1 (d, JCF = 1.5 Hz), 128.7, 128.2 (d, JCF = 10.6 Hz). 19F NMR (377 MHz, CDCl3) δ 67.6.

TLC Rf = 0.60 (17% ethyl acetate in hexanes).

Melting point 76 – 77 ºC (CH3CN/H2O)

Mass spectrometry EI-MS calculated for C12H9FO2S [M]+ 236.0, found 235.8.


SO

O Cl SO

O FKFHF

CH3CN/H2Ort

18

S16

2.9. (E)-2-(2-(fluorosulfonyl)vinyl)benzenesulfonyl fluoride (19)

The title compound was synthesized by General Procedure IV. Palladium acetate/silver

trifluoroacetate effected the conversion of 11 (286 g, 1.00 mmol) and ESF (220 mg, 2.00 mmol)

to (E)-2-(2-(fluorosulfonyl)vinyl) benzenesulfonyl fluoride (19, 173.8 mg, 0.648 mmol, 65%) as a

white crystalline. 1H NMR (600 MHz, CDCl3) δ 8.50 (d, J = 15.3 Hz, 1H), 8.22 (d, J = 7.9 Hz, 1H), 7.88 (t, J = 7.6

Hz, 1H), 7.78 (dd, J = 12.8, 7.7 Hz, 3H), 6.98 – 6.91 (m, 1H). 13C NMR (151 MHz, CDCl3) δ 143.2, 136.1, 132.8 (d, JCF = 25.7 Hz), 132.3, 131.6, 131.3, 129.7,

124.8 (d, JCF = 30.2 Hz), 117.4 (d, JCF = 336.73 Hz). 19F NMR (377 MHz, CDCl3) δ 67.1, 61.6.


Melting point 91 – 92 ºC (hexanes/ethyl acetate).

Mass spectrometry APCI-TOF accurate mass calculated for C8H6F2O4S2 [M]– 267.9681, found

267.9678.


SO

O F

SO

O FIS

O

O F+ S F

O

O

Pd(OAc)2AgOC(O)CF3

acetone60 ºC

19

S17

2.10. 2-(Morpholinosulfonyl)benzenesulfonyl fluoride (20)

A 4-mL vial equipped with an egg-shape stir bar was charged with 2-nitrobenzenesulfonyl

chloride (222 mg, 1.00 mmol), morpholine (95.8 mg, 1.10 mmol), and dichloromethane (1.0 mL).

Triethylamine (152 mg, 1.50 mmol) was added dropwise to give a yellow suspension. The mixture

was stirred at room temperature for 4 h, before partitioned between hydrochloric acid (1 mol L-1,

50 mL) and ethyl acetate (50 mL). The organic phase was collected and concentrated to give

virtually pure sulfonamide (20-i, 270 mg, 0.99 mmol).

Into a reaction tube charged with the crude sulfonamide (270 mg), palladium on carbon (27

mg, 10% wt.), tetrahydrofuran (2 mL) and ethanol (2 mL) were added. Hydrogen gas was

introduced by a balloon, and bubbled for 10 min. The suspension was stirred under hydrogen for

another 6 h, before diluted by methanol (50 mL). The methanolic solution was passed through a

pad of celite and concentrated to give a yellow oil (20-ii). A 20-mL scintillation vial was charged with the crude aniline. Concentrated hydrochloric acid

(0.4 mL) was added, and the suspension was cooled to 0 ºC. At the same temperature, sodium

nitrite solution (40%, 0.4 mL) was added dropwise via a syringe. The diazotization process took

30 min to complete at 0 ºC. In another 20-mL scintillation vial, copper(I) chloride (30 mg, 0.31

mmol) was dissolved in hydrochloric acid (1 mL), and to which, sodium bisulfite solution (40%, 1

mL) was added dropwise to make a yellow suspension. Add the diazonium solution to the SO2

solution at 0 ºC via a glass pipette dropwise. The resulting mixture was allowed to be warmed to

5 – 10 ºC and stirred for a further 30 min, before extracted by dichloromethane (20 mL).

Concentration of the dichloromethane solution gave crude sulfonyl chloride (20-iii, ~ 80% purity).

N

O

SO

O

SO

O

F

20

N

O

SO

O

SO

O

Cl

KFHFCH3CN/H2O

rt

NaNO2, HClthen CuCl, NaHSO3

H2O0 ºC

NO2

S ClO

O

NH

O

NEt3, DCMrt NO2

SNO

O

OH2, Pd/C

THF/EtOHrt

NH2

SNO

O

O

20-i 20-ii

20-iii

S18

The crude sulfonyl chloride was dissolved into acetonitrile (2 mL) and treated with saturated

potassium bifluoride solution (1 mL). The biphasic mixture was stirred at room temperature for 2

h and partitioned between ethyl acetate (50 mL) and water (50 mL). The organic phase was

concentrated and purified by chromatography to give the titled compound as a yellow solid (142

mg, 0.459 mmol, 46% over 4 steps). 1H NMR (600 MHz, CDCl3) δ 8.35 (dd, J = 7.9, 1.4 Hz, 1H), 8.24 (dd, J = 7.9, 1.4 Hz, 1H), 7.91

(td, J = 7.7, 1.4 Hz, 1H), 7.84 (tt, J = 7.7, 1.3 Hz, 1H), 3.75 – 3.71 (m, 4H), 3.34 – 3.29 (m, 4H). 13C NMR (151 MHz, CDCl3) δ 138.5, 135.4, 133.5, 133.2, 132.9 (d, J = 1.5 Hz), 132.1 (d, J = 27.2

Hz), 66.46, 46.14. 19F NMR (377 MHz, CDCl3) δ 64.0.



Mass spectrometry Accurate mass (ESI-TOF) calculated for C10H13FNO5S2 [M + H]+ 310.0214,

found 310.0212.


S19

2.11. 4-((2-(Fluorosulfonyl)phenyl)sulfonyl)piperazine-1-sulfonyl fluoride (21)

A 4-mL vial equipped with an egg-shape stir bar was charged with 2-nitrobenzenesulfonyl

chloride (222 mg, 1.00 mmol), N-Boc-piperazine (205 mg, 1.10 mmol), and dichloromethane (1.0

mL). Triethylamine (152 mg, 1.50 mmol) was added dropwise to give a yellow suspension. The

mixture was stirred at room temperature for 4 h, before partitioned between hydrochloric acid (0.1

mol L-1, 50 mL) and ethyl acetate (50 mL). The organic phase was collected and concentrated to

give virtually pure sulfonamide (21-i, 364 mg, 0.98 mmol).

Treating the crude sulfonamide with hydrochloric acid in 1,4-dioxane (4 mol L-1, 10 mL)

overnight cleanly cleaved the Boc group. The resulting hydrochloride salt was obtained in >98%

purity by evaporation, and then mixed with 2,3-dimethyl-1-fluorosulfonylimidozolium triflate (492

mg, 1.50 mmol) in a 20-mL scintillation vial. Acetonitrile (5 mL) and triethylamine (303 mg, 3.00

mmol) were added, and the solution being stirred for 3 h at room temperature before quenched

by water. The sulfamoyl fluoride (21-ii) was isolated by column chromatography as a white

crystalline (328 mg, 0.921 mmol, 92% over 2 steps). The structure of this compound was

determined by NMR and LRMS.

Into a reaction tube charged with the crude sulfamoyl fluoride (328 mg, 0.921 mmol), palladium

on carbon (27 mg, 10% wt.), tetrahydrofuran (2 mL) and ethanol (2 mL) were added. Hydrogen

gas was introduced by a balloon, and bubbled for 10 min. The suspension was stirred under

hydrogen for another 6 h, before diluted by methanol (50 mL). The methanolic solution was

passed through a pad of Celite and concentrated to give a yellow solid (21-iii). A 20-mL scintillation vial was charged with the crude aniline (21-iii). Concentrated hydrochloric

acid (0.4 mL) was added, and the suspension was cooled to 0 ºC. At the same temperature,

sodium nitrite solution (40%, 0.4 mL) was added dropwise via a syringe. The diazotization process

took 30 min to complete at 0 ºC. In another 20-mL scintillation vial, copper(I) chloride (30 mg, 0.31

NN

SO

OS

O

O F

SO

O

F21

KFHFCH3CN/H2O

rt

NaNO2, HClthen CuCl, NaHSO3

H2O0 ºC

NO2

S ClO

O

NH

BocN

NEt3, DCMrt NO2

SNO

O

NBocH2, Pd/C

THF/EtOHrt

NH2

SNO

O

N

HCl (dioxane)then NEt3

N N SO2F

OTf

NO2

SNO

O

N SFO

O

NN

SO

OS

O

O F

SO

O

Cl

SFO

O

21-i 21-ii

21-iii 21-iv

S20

mmol) was dissolved in hydrochloric acid (1 mL), and to which, sodium bisulfite solution (40%, 1

mL) was added dropwise to make a yellow suspension. Add the diazonium solution to the SO2

solution at 0 ºC via a glass pipette dropwise. The resulting mixture was allowed to be warmed to

5 – 10 ºC and stirred for a further 30 min, before extracted by dichloromethane (20 mL).

Concentration of the dichloromethane solution gave crude sulfonyl chloride (21-iv, ~ 80% purity).

The crude sulfonyl chloride (21-iv) was dissolved into acetonitrile (2 mL) and treated with

saturated potassium bifluoride solution (1 mL). The biphasic mixture was stirred at room

temperature for 2 h and partitioned between ethyl acetate (50 mL) and water (50 mL). The organic

phase was concentrated and purified by chromatography to give the titled compound as an off-

white solid (210 mg, 0.538 mmol, 58% over 3 steps). 1H NMR (600 MHz, Chloroform-d) δ 8.36 (dd, J = 7.9, 1.4 Hz, 1H), 8.31 (dd, J = 7.8, 1.4 Hz, 1H),

7.94 (td, J = 7.7, 1.4 Hz, 1H), 7.90 – 7.86 (m, 1H), 3.54 (dd, J = 7.2, 3.1 Hz, 4H), 3.51 (dd, J = 6.6,

3.6 Hz, 4H). 13C NMR (151 MHz, CDCl3) δ 138.4, 135.7, 134.0, 133.4, 133.2 (d, J = 1.5 Hz), 131.8 (d, J = 25.7

Hz), 46.8, 45.0. 19F NMR (377 MHz, CDCl3) δ 64.1, 41.4.



Mass spectrometry APCI-TOF accurate mass calculated for C10H12F2N2O6S3NH4 [M + NH4]+

408.0164, found 408.0160.


S21

2.12. 2-((Trifluoromethyl)sulfonyl)benzenesulfonyl fluoride (22)

The reaction condition was adapted from Smedley et al (10). A 10-mL vial sealed with a

septum was charged with benzene-1,2-disulfonyl fluoride (242 mg, 1.00 mmol), finely powdered

potassium bifluoride (1.6 mg, 0.02 mg). Under N2 atmosphere, anhydrous DMSO (4.0 mL) and

trifluoromethyl trimethylsilane (200 mg, 1.4 mmol) were added via syringe. The resulting mixture

was stirred at room temperature for 2 h (distribution of starting material, mono-trifluoromethylated

product, di-trifluoromethylated product was estimated by analytical HPLC as 41/52/7), before

directly loaded onto a prep-HPLC. The titled compound was isolated as a white crystalline (104

mg, 0.304 mmol, 30%). 1H NMR (600 MHz, Chloroform-d) δ 8.53 – 8.47 (m, 1H), 8.47 – 8.42 (m, 1H), 8.16 – 8.02 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 137.0, 136.6, 136.1, 134.9 (d, JCF = 28.7 Hz), 133.8 (d, JCF = 1.5

Hz), 132.2 (d, JCF = 1.5 Hz), 118.6 (qd, JCF3 = 336.7 Hz, JCF = 359.4 Hz). 19F NMR (376 MHz, CDCl3) δ 67.1 (s, 1F), -73.5 (s, 3F).

TLC Rf = 0.45 (33% ethyl acetate in hexanes, UV)

Melting point 91 – 93 ºC (MeOH).

Mass spectrometry APCI-TOF accurate mass calculated for C7H4F4O4S2NH4 [M + NH4]+

309.9825, found 309.9821.


SO

O

SO

O F

FF

F

22

SO

O

SO

O F

F

+ SiF

FF

Me

MeMe KFHF

DMSOrt

S22

2.13. 2-((Perfluoropropyl)sulfonyl)benzenesulfonyl fluoride (23)

The reaction condition was adapted from Smedley et al (10). A 10-mL vial sealed with a septum

was charged with benzene-1,2-disulfonyl fluoride (242 mg, 1.00 mmol), finely powdered

potassium bifluoride (1.6 mg, 0.02 mg). Under N2 atmosphere, anhydrous DMSO (2.0 mL) and

perfluoropropyl trimethylsilane (242 mg, 1.0 mmol) were added via syringe. The resulting mixture

was stirred at room temperature for 2 h (distribution of starting material, mono-perfluoropropylated

product, di-perfluoropropylated product was estimated by analytical HPLC as 50/45/5). The titled

compound was isolated by prep-TLC (SiO2, 33% ethyl acetate in hexanes) as a white crystalline

(43 mg, 0.110 mmol, 11%). 1H NMR (600 MHz, CDCl3) δ 8.53 – 8.48 (m, 1H), 8.46 – 8.42 (m, 1H), 8.12 – 8.05 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 136.5, 136.2, 135.5, 134.46 (d, JCF = 28.7 Hz), 133.2 (JCF = 1.5 Hz),

131.9, 120 – 106 (m, perfluoropropyl). 19F NMR (377 MHz, CDCl3) δ 67.1 (s, 1F), -80.8 (t, J = 9.3 Hz, 3F), -107.38 (qt, J = 8.8, 4.2 Hz,

2F), -124.78 – -124.81 (m, 2F).


Melting point 78 – 79 ºC (hexanes/ethyl acetate).

Mass spectrometry APCI-TOF accurate mass calculated for C9H3F8O4S2 [M]– 390.9350, found

390.9339.


SO

O

SO

O F

FF

F FFFF

SO

O

SO

O F

FFF

F FFFF

Me3SiKFHFDMSOrt

+

23

S23

3. Protease activity assays and library screen 3.1. Methods

Activity of hNE was measured in a total volume of 100 µL in a reaction buffer of PBS (pH

7.4) and 0.05% (v/v) NonidetTM P 40 Substitute (Sigma). Final composition of each reaction was

5 nM hNE (Elastin Products Corp.), 50 µM AAPV-aminomethylcoumarin (AMC) substrate

(Millipore), ~2.5% dimethyl sulfoxide (Fisher), and various concentrations of compounds as

inhibitors. hNE was incubated with inhibitors for 10 min at room temperature before addition of

AAPV-AMC (11, 12). Residual proteolytic activity was measured kinetically at 25 °C using an

Envision microplate reader for a total of 30 min at 30 sec 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 (inhibitor) vs. response – variable

slope (four parameters) using GraphPad Prism. Human Cathepsin G (hCG) activity was

measured in a comparable manner to hNE, except that the final concentration of protease in

each reaction was 15 nM and 50 µM of the substrate, AAPF-AMC (Millipore) (11, 12).

S24

3.2. Screen results

Table S1. Randomly picked SuFExable molecules and corresponding hNE inhibitory activity.

BBS Code* (cmpd # in text) Subset Structure % inhibition†

(IC50)‡

BBS-63 III

20%

BBS-64 III

12%

BBS-65 III

22%

BBS-66 III

17%

BBS-67 III

39%

BBS-68 III

19%

BBS-70 III

12%

BBS-244 III

11%

BBS-245 III

7%

BBS-246 III

37%

BBS-247 III

28%

FO2SN

SO2F

S

FO2SN

SO2F

FO2SN

SO2F

FO2SN

SO2F

FO2SN

SO2F

FO2SN

SO2F

O

FO2SN

SO2F

NH

FO2SN N

SO2F

SO2F SO2F

NSO2F

NSO2FFO2S

N

NSO2FFO2S

SO2FFO2S

S25

BBS-248 III

25%

BBS-249 III

31%

BBS-250 III

10%

BBS-254 III

0%

BBS-255 III

36%

BBS-256 III

36%

BBS-257 III

9%

BBS-258 III

16%

BBS-268 III

12%

BBS-297 III

5%

BBS-298 III

10%

BBS-299 III

41%

BBS-300 III

3%

BBS-310 III

40%

N

SO2F

SO2FN

SO2F

FO2S

NSO2F

N CO2H

SO2F

NH

F

SO2F

NH

F3C

SO2F

NH

O2N

SO2F

NH

NC

SO2F

NH

F5S

SO2F

NH

Br

SO2F

NN

SO2FFO2S

SO2F

SO2F

N

NSO2F

FO2S

N

SO2F

SO2F

FO2S

SO2F

O2N

SO2F

N

O

S26

BBS-311 III

24%

BBS-418 III

26%

BBS-421 III

1%

BBS-422 III

1%

BBS-424 III

6%

BBS-430 III

44%

BBS-432 III

14%

BBS-420 III

25%

BBS-216 III

0%

BBS-219 IV

24%

BBS-222 IV

0%

BBS-224 IV

10%

BBS-225 IV

21%

BBS-226 IV

15%

O2N

SO2F

N

NHBoc

N

N

FO2S

SO2F

Boc

N

O

SO2F

N

NSO2F

Boc

N

O

N

N

Cl

SO2F

SO2F

FO2S CN

MeO2C

SO2F

FO2S CN

NC

NSO2FFO2S

OMe

MeO

Cl

SO2F

SO2F

Br

SO2F

SO2F

F3CO

SO2F

HO2C

SO2F

F3C

S27

BBS-235 IV

9%

BBS-228 IV

40%

BBS-232 IV

18%

BBS-417 IV

12%

BBS-241 I

0%

BBS-295 I

(>200)

BBS-305 (1) I

(3.3 ± 1.0)

BBS-306 I

(20 ± 10)

BBS-416 I

(>150)

BBS-433 (10) I

(20 ± 10)

BBS-434 I

(>150)

BBS-114 II

0%

BBS-116 II

0%

BBS-240 II

0%

BBS-263 II

34%

BBS-266 II

12%

BBS-272 II

0%

BBS-274 II

0%

SO2F

OSO2F

O2N

SO2FF3C

SO2F

F5S

SO2F

SO2F

SO2F

SO2F

SO2F

Br

SO2F

O2N

SO2F

Br

SO2F

I

OSO2F

N

OSO2F

OSO2F

CO2H

OSO2FFO2SO

H2N

OSO2F

OSO2F

OCH3

OSO2F

OCH3

SO O

S28

BBS-275 II

3%

BBS-282 II

10%

BBS-290 II

10%

BBS-291 II

13%

BBS-292 II

26%

BBS-296 II

16%

BBS-312 II

12%

BBS-319 II

2%

BBS-320 II

3%

BBS-321 II

5%

BBS-413 II

8%

BBS-414 II

15%

BBS-415 II

11%

OSO2F

CO2H

FO2SO

NH2

OSO2FFO2SO

OSO2FFO2SO

P OSO2F

FO2SO

H

OH

HH

FO2SO

H

HH

O

FO2SO

HN

FO2SO

N

FO2SO

OH

FO2SO

HN

O

FO2SO

SNH2

O

O

FO2SO

NNH

NN

S29

BBS-419 II

11%

BBS-423 II

11%

BBS-425 II

10%

BBS-426 II

15%

BBS-427 II

3%

BBS-428 II

7%

BBS-429 II

17%

BBS-301 II

(9.5 ± 3.5)

BBS-1001 I

(~200)

BBS-740 I

(10 ± 4)

BBS-743 I

(>200)

BBS-1002 II

(>400)

BBS-1003 (8) I

(~120)

BBS-1004 (9) I

(82 ± 16)

FO2SO

NH

SO

O O

PPhO

Ph

FO2SO

OH

O

FO2SOOH

O

FO2SO

NH

NH

O N

O

F3C CF3

OSO2FFO2SO

FO2SO

N3

FO2SO

MeO

N

N

FO2S

O

O

S OO

F

S OO

F

SO

O

F

S OO

F

NSO

O F

F

SO

O F

Cl

SO

O F

S30

BBS-1005 (12) I

(>200)

BBS-1006 (13) I

(73 ± 4)

BBS-1007 (14) I

(13.3 ± 0.5)

BBS-1008 (15) I

(60 ± 8)

BBS-1009 (16) I

(20 ± 1)

BBS-1010 (17) I

(37 ± 2)

BBS-1011 (2) I

(17.5 ± 1.1)

BBS-1012 (18) I

(27 ± 2)

BBS-1013 (22) I

(1.1 ± 0.1)

BBS-1014 (11) I

(9.7 ± 1.2)

BBS-1015 (19) I

(2.2 ± 0.7)

BBS-1016 I

(7.7 ± 0.7)

CH3SO

O F

O

SO

O F

H3C

SO

O F

N

SO

O F

FF

F

SO

O FN+

O-O

SO

O F

OO

CH3

S OO

F

SO

O

FS

O

OF

SO

O F

SO

O

SO

O F

FF

F

I

SO

O F

SO

O F

SO

O F

S

O

O

F S

O

O

F

S31

BBS-1017 I

(51 ± 7)

BBS-1018 I

(>200)

BBS-1019 II

(>200)

BBS-1020 II

(>200)

BBS-1021 I

(~200)

BBS-1022 I

(~200)

BBS-1023 I

(>200)

BBS-1024 N/A

(~200)

BBS-1025 N/A

(~200)

BBS-1026 N/A

(~200)

BBS-1027 (23) I

(48 ± 2)

BBS-1028 I

(36 ± 3)

S

O

O

F S

O

O

N

O

O

S OO

F

SO O

F

S

FF

FF

F

OS

O

O

F

S

FF

FF

FO

SO

O F

OS

O

O

F

SO

OF

SO

O

FSO

O

F

S OO

F

SO

OF

O

OSO

O

O

OSO

O

O

OSO

O

SO

O

SO

O F

FF

F FF

FF

S OO

F

SO

O

FS

O

OF

S32

BBS-1029 II

(~200)

BBS-1030 (3) I

(6.8 ± 1.1)

BBS-1031 I

(>200)

BBS-1032 I

(>200)

BBS-1033 N/A

(>200)

BBS-1034 N/A

(59 ± 15)

BBS-1035 II

(>200)

BBS-1036 N/A

(>200)

BBS-1037 I

(>200)

BBS-1038 I

(67 ± 19)

BBS-1039 (4) I

(17 ± 2)

BBS-1040 (5) I

(5.4 ± 0.5)

OS

O

O

FF

F

O

SO O

F

SO

O F

SO

O

F

SF

FF

F

F

SO

O

F

SF

FF

F

F

S OO

F

O

OS O

O

O

OSO

O

OO

CH3

O O

OSO

O

F

OS

O O

SOO

F

Br

SO

O F

F

F SO

O F

SO

O Me

SO

O F

Cl

Cl

SO

OF

S33

BBS-1041 I

(105 ± 17)

BBS-1042 I

(>200)

BBS-1043 (6) I

(5.9 ± 1.1)

BBS-406 I

(89 ± 4)

BBS-1044 (20) I

(84 ± 1)

BBS-1045 II

(>200)

BBS-1048 I

(60 ± 10)

BBS-1049 (7) I

(49 ± 3)

BBS-1050 (21) I

(>200)

PMSF (ref.) N/A

(24 ± 1)

*BBS-numbering is assigned for every SuFExable compound for internal (within TSRI) reference. †Per cent inhibition of hNE activity was measured based on a compound concentration of 200 μM. ‡IC50 values were measured based on 10 min incubation and are shown in mean ± SD (n ≥ 3).

F

F

F

SO

O F

S Cl

Cl

S

O

O

F

N+O

O-

N+

O

O-S O

O

F

OH

SO

OF

SO

O

F

SOO

F

N

O

SO

O

SO

O

F

SF

FF

F

FO

SO

O

F

Cl

Cl

SO

O F

Cl

SO

O F

Cl

N

N

SO

O

SO

O F

SO

O

F

SO

O

F

S34

Fig S1. Scatterplot of in-house sulfonyl fluoride and fluorosulfate compounds screened against human neutrophil elastase. Residual hNE activity toward a fluorescent-peptide

substrate (AAPV-AMC) after incubation of 5 nM hNE with 200 µM compound from a 10mM DMSO

stock. Percent residual activity normalized against a positive control of hNE plus substrate and

negative control of substrate alone.

1 11 21 31 41 51 61 71 81 91 101

111

121

131

0

25

50

75

100

125

Arbitrary Cmpd #

Nor

mal

ized

hN

E A

ctiv

ity (%

)

candidates of covalent inhibitors

S35

3.3. Validation of permanent binding

To test for covalency and the possibility of hydrolytic activity returning to hNE over time, hNE

was resuspended in the above buffer to 0.1 mg/ml. Compounds 1 and 22 were added to hNE in

a 50:1 ratio and incubated for 30 min at RT. Complexes were dialyzed using 0.5 mL Slide-A-Lyzer

MINI dialysis units (Thermo) in the above buffer for 24 hrs. At time points, t = 0, 1, 2, and 24 hr(s),

an aliquot was removed, hNE concentration determined, two-fold dilution series of hNE in PBS

(7.4), AAPV-AMC substrate added, and residual proteolytic activity measured kinetically at 25 °C

using an Envision microplate reader for a total of 30 mins at 30 sec intervals. Dilution series curves

made using GraphPad Prism and hNE activity at 5 nM determined through Michaelis-Menton

kinetics for each time point (Fig S2).

Fig S2. Covalency test, dialysis of hNE complexed with compunds 1 and 22 over time. hNE

was resuspended to 0.1 mg/mL and complexed with compounds 1 and 22 in a 50:1

(compound:hNE) ratio for 30 mins at RT. Complexes were dialyzed, with hNE control, in

resuspension buffer for 24 hr. Aliquots taken at time points shown below and residual hNE activity

was tested at 5 nM protein concentration. No change in hNE hydrolytic activity is observed over

time. Data presented here are taken from a representative trial of at least three independent

experiments. Legend is inset.

S36

3.4. Inhibitory activity of hit molecules against hCG.

Fig S3. Dose-response curves of top hNE inhibitors against human cathepsin G. Each

compound was assessed over a two-fold logarithmic dilution series. Data presented here are

taken from a representative trial of at least three independent experiments. Legend is inset.

S37

4. Mass Spectrometry 4.1. Methods

Enzyme hNE was resuspended in 50mM 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.

4.2. Results

Fig S4. MALDI-TOF mass spectrometry analysis of hNE with or without compounds, 1 and 22. hNE was incubated alone (top left) and in a 1:3 ratio with compounds 1 (top right) and 22

(bottom) for 1 hr at RT and analyzed by high accuracy MALDI-TOF mass spectrometry. Peaks

correspond to the mass of hNE apo and ± compound in Da.

S38

5. X-ray crystal structure 5.1. Methods

Crystallization and x-ray data collection. Crystallization of hNE in complex with our

covalent capture agent was set up similarly to Hansen et al. and their structure of a

dihydropyrimidone inhibitor (13). In short, inhibitor 1 was added in a 1.2 molar excess to human

neutrophil elastase (Elastin Products Co.) [10 mg/ml in 10 mM HEPES (pH 6.5)], incubated for 1

hr at 25°C and immediately used for crystallization. Crystals were grown by sitting 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% (w/v) PEG 3350 at 25°C. Data was collected on single,

flash-cooled crystals at 100 K in cryoprotectant consisting of 0.2 M ammonium citrate (pH 5.0),

20% (w/v) PEG3350, and 20% (v/v) glycerol, and were processed with HKL2000 in orthorhombic

space group P212121. The calculated Matthews’ coefficient (VM = 2.77 Å3Da-1) suggested four

monomers per asymmetric unit with a solvent content of 56%. X-ray data was collected to 2.33 Å

resolution on beamline 12.2 at the Stanford Synchrotron Radiation Lightsource (SSRL) (Menlo

Park, CA). Data collection and processing statistics are summarized in Table S2. Structure solution and refinement. The hNE:1 structure was determined by molecular

replacement (MR) with Phaser (14) using the previously published apo structure (PDB ID: 5abw)

as the initial search model. The structure was manually built with Coot (15) and iteratively refined

using Phenix (16) with cycles of conventional positional refinement with isotropic B-factor

refinement. Non-crystallographic symmetry (NCS) constraints were applied for the initial rounds

of refinement. The electron density maps clearly identified that 1 was covalently attached to

Ser195 within the active site (Fig. 3B-D). Water molecules were automatically positioned by

Phenix using a 2.5σ cutoff in Fo-Fc maps and manually inspected. The final Rcryst and Rfree are

19.3% and 24.1%, respectively (Table S2). The model was analyzed and validated with

PROCHECK (17), WHATCHECK (18), and Molprobity (19) on the JCSG webserver. Analysis of

backbone dihedral angles with the program PROCHECK indicated that all residues are located in

the most favorable and additionally allowed regions in the Ramachandran plot. Coordinates and

structure factors have been deposited in the PDB with accession entry 6e69. Structure refinement

and statistics are shown in Table S2. X-ray structure data deposition. The atomic coordinates and structure factors have been

deposited in the Protein Data Bank, www.wwpdb.org (PDB ID: 6e69).

S39

5.2. Results

Table S2. Data collection and refinement statistics.

Structure 6E69 Space group P 212121

Cell dimensions a, b, c; Å 69.5, 124.6, 126.7 α, β, γ; ⁰ 90, 90, 90

Data Processing Resolution, Å (outer shell) 50.0-2.33 (2.37-2.33)

Completeness, % 96.8 (72.5) Unique reflections 46,266 (1,697)

Redundancy 2.7 (2.6) Rmeas (%)* 6.7 (44.9) Rmerge (%)† 4.7 (31.7) Rp.i.m. (%)‡ 7.1 (31.2)

Average I / Average s (I) 13.0 (3.2) CC1/2 87.4 (100)

Refinement Resolution, Å (outer shell) 50.0-2.33 (2.38-2.33) No. reflections (test set)§ 46,245 (2,386)

Rcryst (%)¶ 19.3 (21.3) Rfree (%) 24.1 (28.5)

Protein atoms / Waters 6,711 / 267 CV coordinate error (Å)# 0.28

Rmsd bonds (Å) / angles ⁰ 0.006 / 0.9 B-values protein/waters/ligands (Å2) 39 / 34 / 53

Ramachandran Statistics (%) Preferred 97.1 Allowed 2.9 Outliers 0

*Rmeas = /ΣhklΣi Ii(hkl), where Ii(hkl) are the observed intensities, <I(hkl)> are the average intensities and

N is the multiplicity of reflection hkl. †Rmerge = ΣhklΣi|Ii(hkl) -<I(hkl)>|/ ΣhklΣiIi(hkl) where Ii(hkl) is the ith

measurement of reflection h and < I(hkl)> is the average measurement value. ‡Rp.i.m. (precision-

indicating Rmerge) = Σhkl[1/( N hkl – 1) ]1/2Σi|Ii(hkl) - <I(hkl)>|/ΣhklΣiIi(hkl). §Reflections with I > 0 were used

for refinement (15). ¶Rcryst = Σh||Fobs|-|Fcalc||/Σ|Fobs|, where Fobs and Fcalc are the calculated and

observed structure factor amplitudes, respectively. Rfree is Rcryst with 5.0% test set structure factors. #Cross-validated (CV) Luzzati coordinate errors.

S40

6. Reactive docking 6.1. Methods

The structures of hNE (PDB ID: 6e69) and hCG (PDB ID: 1t32) (20) were superposed and

prepared according to the standard AutoDock protocol (21). Hydrogens, protonation states, and

side chain conformations of Gln, Asn, and His residues were calculated with Reduce (22). Ligand

3D coordinates were generated from SMILES strings using custom Python scripts based on

OpenBabel (23). The reactive docking protocol was applied, as described previously (24, 25). The

reactive potential was defined for the sulfonyl-fluoride sulfur and the serine Og, with e value of

0.241. For each ligand, 10 independent ga_runs were performed with the default GA settings.

Results were clustered, and the lowest energy result was selected.

6.2. Results and discussion

Fig S5. Reactive docking results. Residues defining binding sites are shown as sticks, and

volumes represented as semi-transparent surfaces. Ligands are shown as sticks. (a) Compound

1 (yellow) docked in hNE (white; PDB ID 6e69); pseudo-covalent bond between 1 and Ser195 is

shown as dashed lines (green). (b). Compound 1 (yellow) docked in hCG (green; PDB ID 1t32);

S41

(c) Compound 19 (yellow) docked in hCG (green; PDB ID 1t32), and the experimental coordinates

of beta-ketophosphonate 1 (JNJ-10311795; purple), a potent non-covalent hCG inhibitor.

Superposition of hCG (PDB ID: 1t32) and our hNE:1 co-complex (PDB ID: 6e69) crystal

structures provides insight as to how 1 is selective for hNE over hCG. The active sites are

conserved in shape, topography, and sequence. However, key differences in residues flanking

the catalytic site are likely responsible for selectivity of 1 for hNE, including substitutions of Phe192

to Lys, Val216 to Gly, and that hCG lacks the disulfide bond formed by hNE Cys191 and Cys220

(Fig S5a). We applied a reactive docking protocol to identify the determinants of selectivity by 1

and the promiscuity of 19. Modeling the near-attack conformations (NAC), which precede

formation of irreversible adducts, suggests that the Val216 side chain of hNE is important for

binding the free compound and stabilizing the proper reactive geometry. The side chain of Val216

engages the aromatic ring of 1 and 19 and orients the sulfonyl fluoride at an optimal distance for

nucleophilic attack by Ser195. The substitutions of hNE’s Val216 for a Gly in hCG and Ala213 for

a Val, expand the active site cavity and promote repulsion with the compound’s aromatic ring,

respectively (Fig S5b). Our docking results suggest the combined substitutions in hCG result in

a shift of the aromatic ring binding interaction away from Ser195 and a significant increase in

distance for nucleophilic attack by the active site serine (from 2.2 ± 0.2 Å in hNE to 3.4 ± 0.1 Å in

hCG). Importantly, compounds with the reactive warhead attached to the phenyl group via one or

more carbons (i.e., 19, and PMSF) have optimal distance between the sulfonyl fluoride and

aromatic ring to react with the serine and interact with the larger hydrophobic pocket of hCG (1.8

± 0.1 Å for 19), respectively, as demonstrated by the orientation of the naphthyl group of JNJ-

10311795 in the x-ray co-complex structure with hCG (Fig S5c) (20).

S42

7. Reactivities of SuFExable functional groups

Fig S6. SuFEx reactions of various sulfur fluoride species with aryl silyl ether. a) Relative

rates of various sulfur fluoride species were measured by competition with phenyl fluorosulfate.

By limiting the amount of 4-phenyl tert-butyldimethylsilyl ether (5%), all reactions were carried to

about 2.5% conversion (according to the amount of total “S–F” groups) in the presence of DBU

catalyst. The ratio of respective product (krel) was determined by 19F NMR. Relative rates of

compounds i–iv and v (PMSF) in SuFEx reactions offer a general impression of the reactivity of

library subsets I–IV and PMSF in the main text; b) reactions for determining relative rates.

S43

8. NMR spectra 8.1. Naphthalene-1,3,6-trisulfonyl trifluoride (2)

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5

f1(ppm)

-200000

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

zqh-1684-500.1.fid—50mMcyclosporine

7.26

8.49

8.49

8.51

8.51

8.95

8.96

8.97

8.97

8.99

8.99

9.01

9.01

9.17

9.18

8.48.58.68.78.88.99.09.19.29.3f1(ppm)

8.49

8.49

8.51

8.51

8.95

8.96

8.97

8.97

8.99

8.99

9.01

9.01

9.17

9.18

SO2F

SO2FFO2S

2

S44

-100102030405060708090100110120130140150160170180190200210220230

f1(ppm)

-200000

-100000

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

1800000

zqh-1684-500.2.fid—

76.91

77.16

77.41

127.62

130.08

131.12

131.14

132.01

132.25

132.91

133.13

133.52

134.73

134.95

139.55

126128130132134136138140142f1(ppm)

127.62

130.08

131.12

131.14

132.01

132.25

132.91

133.13

133.52

134.73

134.95

139.55

SO2F

SO2FFO2S

2

S45

-15-10-505101520253035404550556065707580f1(ppm)

-50

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

zqh-1684-40-F.33.fid—

0.65

64.85

66.79

67.65

64.565.065.566.066.567.067.568.0f1(ppm)

0

100

200

300

400

500

600

64.85

66.79

67.65 SO2F

SO2FFO2S

2

S46

8.2. Naphthalene-2,3-disulfonyl difluoride (3)

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5

f1(ppm)

0

50

100

150

200

250

300

350

400

450

zqh-1782-600.1.fid—H-1Routine,CP-DCH,AVIIIHD-600

2.0

72.

05

2.0

0

7.26

7.9

87.

99

8.0

08

.00

8.0

18

.01

8.1

98

.19

8.2

08

.20

8.2

18

.21

8.9

68

.97

8.9

88

.98

8.9

9

3

SO

O F

SO

O

F

S47

0102030405060708090100110120130140150160170180190200210220

f1(ppm)

-100

0

100

200

300

400

500

600

700

800

900

zqh-1782-600.2.fid—

76.95

77.16

77.37

126.66

126.86

130.16

132.87

133.72

137.17

3

SO

O F

SO

O

F

S48

-10-50510152025303540455055606570758085f1(ppm)

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

zqh-1782-400-F.1.fid—F-19,CDCl3,DPX-400QNPProbe.CF3ClasRefat0ppm.

7.26

72.84

3

SO

O F

SO

O

F

S49

8.3. Methyl 3-(fluorosulfonyl)thiophene-2-carboxylate (4)

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5

f1(ppm)

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500

1600

1700

1800


3.3

5

1.0

21.

00

3.9

9

7.26

7.6

17.

62

7.6

47.

64

7.6

57.

65

SO

O Me

SO

O F

4

S50

0102030405060708090100110120130140150160170180190200210220

f1(ppm)

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

zqh-1792-600.2.fid—

53.59

76.95

77.16

77.37

130.56

130.57

130.89

130.90

134.55

134.75

137.32

159.08

SO

O Me

SO

O F

4

S51

5101520253035404550556065707580859095100f1(ppm)

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

zqh-1792-F.1.fid—F-19,CDCl3,DPX-400QNPProbe.CF3ClasRefat0ppm.

0.65

62.97

SO

O Me

SO

O F

4

S52


-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5

f1(ppm)

-20

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340


1.0

0

7.26

7.50

7.52

7.53

7.56

7.57Cl

Cl

SO

OF

5

S53

0102030405060708090100110120130140150160170180190200210220

f1(ppm)

-20

0

20

40

60

80

100

120

140

160

180

200

220

240

260

zqh-1793-600.2.fid—C-13Routine1D,CPDCHCryoProbe,AVIII-600

76.95

77.16

77.37

130.95

131.11

131.60

134.97

136.15

Cl

Cl

SO

OF

5

S54

-50510152025303540455055606570758085f1(ppm)

0

100

200

300

400

500

600

700

800

900zqh-1793-F.1.fid—F-19,CDCl3,DPX-400QNPProbe.CF3ClasRefat0ppm.

0.65

68.19

Cl

Cl

SO

OF

5

S55


-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5

f1(ppm)

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

3400

3600


1.0

31.

02

1.0

0

7.26

7.72

7.72

7.74

7.74

7.8

47.

84

7.8

57.

86

8.1

08

.10

Cl

SO

O F

7

Cl

S56

0102030405060708090100110120130140150160170180190200210220

f1(ppm)

-20

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360

zqh-1824-600.2.fid—

76.35

76.56

76.77

126.87

129.84

131.34

131.87

132.05

134.25

140.69

Cl

SO

O F

7

Cl

S57

-50510152025303540455055606570758085f1(ppm)

0

500

1000

1500

2000

2500

3000

zqh-1824-F.1.fid—F-19,CDCl3,DPX-400QNPProbe.CF3ClasRefat0ppm.

0.65

66.89

Cl

SO

O F

7

Cl

S58

8.6. 2-Iodobenzenesulfonyl fluoride (11)

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5

f1(ppm)

-100000

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

1800000

1900000

2000000

2100000

zqh-1690-500.1.fid—50mMcyclosporine

1.00

1.00

1.00

0.96

7.26

7.36

7.37

7.38

7.38

7.39

7.40

7.57

7.58

7.58

7.59

7.59

7.59

7.60

7.61

7.61

8.14

8.14

8.15

8.16

8.17

8.17

8.19

8.19

ISO

O F

11

S59

-100102030405060708090100110120130140150160170180190200210220230

f1(ppm)

-200000

-100000

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

1800000zqh-1690-500.2.fid—AVNEO500,C-13Routine,5-25-2017

76.91

77.16

77.41

92.28

128.77

132.07

132.08

135.82

137.66

137.85

143.30

ISO

O F

11

S60

-15-10-5051015202530354045505560657075

f1(ppm)

-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

16000

17000

18000

19000zqh-1690-400.3.fid—

0.65

56.88I

SO

O F

11

S61

8.7. [1,1'-biphenyl]-2-sulfonyl fluoride (18)

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5

f1(ppm)

0

50

100

150

200

250

300

350

400

450

500


2.0

04

.09

1.0

71.

03

1.0

0

7.26

7.3

87.

38

7.3

97.

45

7.4

57.

46

7.4

67.

47

7.4

97.

597.

60

7.6

27.

757.

767.

788

.18

8.1

9

7.27.37.47.57.67.77.87.98.08.18.28.3f1(ppm)

2.0

0

4.0

9

1.0

7

1.0

3

1.0

0

7.26

7.3

87.

38

7.3

97.

45

7.4

57.

46

7.4

67.

47

7.4

97.

597.

60

7.6

2

7.75

7.76

7.78

8.1

88

.19S

O

O F

18

S62

0102030405060708090100110120130140150160170180190200210220

f1(ppm)

-50

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700


76.95

77.16

77.37

128.13

128.20

128.70

129.06

129.07

130.12

130.13

132.40

132.55

133.16

133.17

134.89

138.03

143.23

SO

O F

18

S63

-15-10-5051015202530354045505560657075

f1(ppm)

-50

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

zqh-1685-400-F.33.fid—F-19,CDCl3,DPX-400QNPProbe.CF3ClasRefat0ppm.

0.65

67.62

19FNMR(377MHz,CDCl3)δ67.62,0.65.

SO

O F

18

S64

8.8. (E)-2-(2-(fluorosulfonyl)vinyl)benzenesulfonyl fluoride (19)

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5

f1(ppm)

-20

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300


1.0

4

1.9

61.

01

0.9

4

1.0

0

6.9

36

.93

6.9

56

.96

7.26

7.76

7.78

7.79

7.8

07.

87

7.8

87.

90

8.2

28

.23

8.4

98

.51

SO

O F

SO

O F

19

S65

0102030405060708090100110120130140150160170180190200210220

f1(ppm)

-50

0

50

100

150

200

250

300

350

400

450

500

550

600

zqh-1691-600.2.fid—

76.95

77.16

77.37

116.33

124.70

124.90

129.73

131.30

131.62

132.25

132.76

132.93

136.14

143.23

115120125130135140145f1(ppm)

116.33

124.70

124.90

129.73

131.30

131.62

132.25

132.76

132.93

136.14

143.23

SO

O F

SO

O F

19

S66

-15-10-5051015202530354045505560657075

f1(ppm)

-1000

-500

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

zqh-1691-400-F.3.fid—AV400BBOFProbe,F-19inCDCl3,CFCl3=0ppm.

0.65

61.56

67.07

SO

O F

SO

O F

19

S67

8.9. 2-(Morpholinosulfonyl)benzenesulfonyl fluoride (20)

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5

f1(ppm)

-20

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280


3.8

0

3.8

0

1.0

11.

02

1.0

01.

00

3.3

13

.32

3.3

23

.32

3.3

23

.33

3.7

33

.73

3.7

4

7.26

7.8

47.

84

7.8

47.

85

7.9

07.

90

7.9

17.

91

7.9

27.

93

8.2

38

.23

8.2

48

.24

8.3

48

.35

8.3

68

.36

N

O

SO

O

SO

O

F

20

S68

0102030405060708090100110120130140150160170180190200210220

f1(ppm)

0

50

100

150

200

250

300

350

400

450


46.14

66.46

76.95

77.16

77.37

132.02

132.20

132.89

132.90

133.17

133.46

135.44

138.47

N

O

SO

O

SO

O

F

20

S69

-15-10-505101520253035404550556065707580

f1(ppm)

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500zqh-1806-400-f.1.fid—F-19,CDCl3,DPX-400QNPProbe.CF3ClasRefat0ppm.

0.65

64.00

N

O

SO

O

SO

O

F

20

S70

8.10. 4-((2-(Fluorosulfonyl)phenyl)sulfonyl)piperazine-1-sulfonyl fluoride (21)

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5

f1(ppm)

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400zqh-1828-600.1.fid—H-1Routine,CP-DCH,AVIIIHD-600

3.9

03

.85

1.10

1.12

1.0

21.

00

3.5

03

.50

3.5

13

.51

3.5

43

.54

3.5

53

.55

7.26

7.8

77.

87

7.8

87.

88

7.8

87.

89

7.8

97.

89

7.9

37.

93

7.9

47.

94

7.9

57.

96

8.3

08

.30

8.3

18

.32

8.3

68

.36

8.3

78

.37

NN

SO

OSO

O F

SO

O

F21

S71

0102030405060708090100110120130140150160170180190200210220

f1(ppm)

0

50

100

150

200

250


44.95

46.83

76.95

77.16

77.37

131.70

131.87

133.17

133.18

133.36

133.97

135.70

138.40

NN

SO

OSO

O F

SO

O

F21

S72

-15-10-5051015202530354045505560657075

f1(ppm)

-5

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85zqh-1828-400-f.1.fid—F-19,CDCl3,DPX-400QNPProbe.CF3ClasRefat0ppm.

0.65

41.41

64.11

NN

SO

OSO

O F

SO

O

F21

S73

8.11. 2-((Trifluoromethyl)sulfonyl)benzenesulfonyl fluoride (22)

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5

f1(ppm)

-50

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

zqh-1688-p2-600.1.fid—H-1Routine,CP-DCH,AVIIIHD-600

2.0

0

1.0

01.

01

7.26

8.0

78

.08

8.0

88

.09

8.1

08

.11

8.4

58

.45

8.4

68

.46

8.4

78

.48

8.4

98

.49

8.5

08

.50

8.5

08

.51

7.98.08.18.28.38.48.58.6f1(ppm)

2.0

0

1.0

0

1.0

1

8.0

78

.07

8.0

88

.08

8.0

98

.10

8.1

18

.11

8.4

48

.45

8.4

58

.46

8.4

68

.47

8.4

88

.49

8.4

98

.50

8.5

08

.50

8.5

1

SO

O

SO

O F

FF

F

22

S74

0102030405060708090100110120130140150160170180190200210220

f1(ppm)

-50

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

zqh-1688-p2-600.2.fid—

76.95

77.16

77.37

116.33

116.59

118.56

118.77

120.71

120.94

123.12

132.16

132.17

133.83

133.84

134.78

134.97

136.10

136.63

136.96

115120125130135f1(ppm)

115.70

115.96

117.92

118.13

120.08

120.31

122.48

131.52

131.54

133.20

133.21

134.15

134.34

135.99

136.33

114115116117118119120121122123124f1(ppm)

116.33

116.59

118.56

118.77

120.71

120.94

123.12

CF3

SO

O

SO

O F

FF

F

22

S75

-110-100-90-80-70-60-50-40-30-20-10010203040506070

f1(ppm)

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

zqh-1688-p2-400.1.fid—F-19,CDCl3,DPX-400QNPProbe.CF3ClasRefat0ppm.

2.81

1.00

-73.48

0.65

67.11

SO

O

SO

O F

FF

F

22

S76

8.12. 2-((Perfluoropropyl)sulfonyl)benzenesulfonyl fluoride (23)

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5

f1(ppm)

-20

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280


1.9

3

0.9

91.

00

7.26

8.0

88

.09

8.0

98

.10

8.1

08

.11

8.4

38

.43

8.4

48

.45

8.4

98

.50

8.5

08

.51

8.5

18

.52

SO

O

SO

O F

FF

F FFFF

23

S77

0102030405060708090100110120130140150160170180190200210220

f1(ppm)

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300zqh-1749-600.2.fid—C-13Routine1D,CPDCHCryoProbe,AVIII-600

76.34

76.55

76.76

106.33

106.59

106.80

107.86

107.91

108.12

108.17

108.33

108.38

108.59

108.64

109.70

109.91

110.17

111.81

112.04

112.28

113.78

113.84

114.07

114.30

115.47

115.69

115.87

115.91

116.10

116.33

117.39

117.61

117.83

117.95

119.53

119.76

131.88

133.20

133.21

134.37

134.56

135.48

136.22

136.45

104106108110112114116118120f1(ppm)

106.59

108.12

108.17

108.38

108.59

110.17

111.81

112.04

113.84

114.07

114.30

115.47

115.69

115.87

115.91

116.10

116.33

117.39

117.61

117.83

117.95

119.53

SO

O

SO

O F

FF

F FFFF

23

S78

-15-10-5051015202530354045505560657075

f1(ppm)

0

50

100

150

200

250

300

350

400

zqh-1749-400.1.fid—F-19,CDCl3,DPX-400QNPProbe.CF3ClasRefat0ppm.

0.65

67.08

SO

O

SO

O F

FF

F FFFF

23

S79

-140-135-130-125-120-115-110-105-100-95-90-85-80-75-70-65-60-55-50-45f1(ppm)

0

50

100

150

200

250

300

350

400

450

zqh-1749-400.4.fid—F-19,CDCl3,DPX-400QNPProbe.CF3ClasRefat0ppm.

-124.80

-124.79

-107.43

-107.42

-107.41

-107.40

-107.39

-107.37

-107.36

-107.35

-107.34

-80.84

-80.81

-80.79

-107.55-107.45-107.35-107.25f1(ppm)

-107.43

-107.42

-107.41

-107.40

-107.39

-107.37

-107.36

-107.35

-107.34

-80.95-80.85-80.75-80.65f1(ppm)

-80.84

-80.81

-80.79

SO

O

SO

O F

FF

F FFFF

23

S80

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