Sulfur-Fluoride Exchange(SuFEx)
Literature Seminar
2019/11/28
M2 Katsuya Maruyama
1Contents- Introduction- Synthesis of S(VI)-F unit
- Applications of SuFEx

2

K. B. Sharpless
Nobel Laureate K. Barry Sharpless became W. M. Keck Professorof Chemistry at The Scripps Research Institute and The Skaggs
Institute of Chemical Biology in 1990. Previously a professor at
MIT and Stanford, he was educated at Dartmouth College (BA1963), Stanford (PhD 1968 with E. E. van Tamelen; postdoc 1969
with J. P. Collman), and Harvard (postdoc 1970, K. E. Bloch).

The Nobel Prize in Chemistry (2001):"for his work on chirally catalyzed oxidation reactions."
Asymmetric epoxidation

Asymmetric dihydroxylation

Asymmetric aminohydroxylation

3

Click Chemistry2000

2001 2002-2007 2008

2014

Robust connection
High functional group tolerance
Fast kinetics
Easy operation

Nat. Rev. Chem. 2018, 2, 202.

4

SuFEx Reaction
General scheme

Stable S-F bond (necessity of activation)
Metal free
diversity of linkage“Context dependent” Click reaction
5

Property of Sulfonyl Fluoride
Oxidation/reduction

HeatingBDE
SO2F2 90.5 kcal/mol
SO2Cl2 46 kcal/mol

Substitution at S(VI) center(no fluorination)

Nucleophilic attack

Activation with water
Sharpless, K. et al. Angew. Chem. Int. Ed. 2014, 53, 9430.

SuFEx Reaction
Activation of S(VI)-F- Hydrogen bond donor (acid, HX)）

HB of HF2-: 40 kcal/mol- Silylated nucleophile

Reactivity: Et3N < DBU < BEMP

7

SuFEx Reaction
Activation with Ca(NTf)2 (Lewis acid)

Effect of Ca salt

Proposed mode of activation

Selectivity of nucleophile

JOC 2017, 82, 2294.
Am Ende, C. W.; Ball, N. D. et al. Org. Lett. 2018, 20, 3943. 8

Synthesis of SuFEx unit

9SuFEx Unit

Chinthakindi, P. K.; Arvidsson, P. I. Eur. J. Org. Chem. 2018, 3648.

10

Sulfonylfluoride (RSO2F)
Oldest conditions: via RSO2Cl
ArylDestabilized and nucleophilic F-Aliphatic

Harsh conditions

Sharpless, K. et al. Angew. Chem. Int. Ed. 2014, 53, 9430. 11

Sulfonylfluoride (RSO2F)
Pd catalyzed SO2 insertion

sulfinate ammonium
Bagley, S. W.; Willis, M. C. et al. Chem. Sci. 2017, 8, 1233.
via:Via sulfonate saltBall, N. D. et al. J. Org. Chem. 2017, 82, 2294.

- transition metal free
- one-pot
Sun, B.; Qin, H. L. et al. RSC Adv. 2019, 9, 13863.

12

Sulfonylfluoride (RSO2F)
Electrochemical synthesis

Mechanism

Investigation of active species

Noël, T. et al. J. Am. Chem. Soc. 2019, 141, 11832.

SuFEx Hubs
ESF: ethene sulfonyl fluoride- Michael addition- Heck-Matsuda reaction: access to β-aryl ESF

Wu, P.; Sharpless, K. B. et al. Angew. Chem. Int. Ed. 2016, 55, 14155.

14

SuFEx Hubs
- Oxidative Heck reaction

Arvidsson, P. I. et al. Org. Lett., 2017, 19, 480.

Qin, H. L. et al. Adv. Synth. Catal., 2017, 359, 3237.

- Pd catalyzed coupling with Ar-I

Qin, H. L.; Shalpless, K. B. et al. Angew. Chem., Int. Ed., 2017, 56, 4849.

- C-H activation
Org. Chem. Front., 2018, 5, 1411.
Eur. J. Org. Chem., 2018, 4407.
Org. Lett., 2018, 20, 4699.
Organometallics, 2019, 38, 76.

15

SuFEx Hubs
Radical addition to ESF

via:

Eosin Y-Na2

Selected scopes

Liao, S. et al. Nat. Commun. 2019, 10, 1.

16SuFEx Hubs
BESF: 1-bromoethene-1-sulfonyl fluorideConstruction of heterocycles

Moses, J. E. et al. Chem. Commun., 2018, 54, 6020.

- Suzuki-Miyaura cross coupling: access to α-aryl ESF

Qin, H. L. et al. Eur. J. Org. Chem. 2019, 6101.

Synthesis of Fluorosulfate (ROSO2F)
Sulfuryl fluoride (SO2F2): gas Reaction with alcohol/phenol

fluorosulfate

Aryl fluorosulfate is useful in terms of stability.

stable for- months (neutral buffer)- 2 weeks (buffer pH 10):for simple molecules
Selected scopes:for complex molecules

Sharpless, K. et al. Angew. Chem. Int. Ed. 2014, 53, 9430. 18

Ex Situ Generation of SO2F2 Gas
Precursor of SO2F227 examples, 85-99%
De Borggraeve, W. M. et al. Org. Lett. 2017, 19, 5244.

19

SO2F2 Equivalents
Fluorosulfuryl imidazolium salt
fluorosulfate
sulfamoyl
fluoride- bench-stable solid- higher reactivity than SO2F2- commercially available from Aldrich- applicable to 1° amine
Sharpless, K. B.; Dong, J. et al. Angew. Chem. Int. Ed. 2018, 57, 2605.

selectivity:
amine, aniline > phenol

AISF: [(acetylamino)phenyl]imidodisulfuryl difluoride- no need of SO2F2 for preparation- bench-stable solid- higher reactivity than SO2F2- rapid kinetics

selectivity:phenol > aniline
Am Ende, C. W. et al. Org. Lett. 2018, 20, 812.

20

Sulfonimidoyl Fluoride (R-SONHF)
Thionyl tetrafluoride (SOF4) gas

multidimensional hub

1st addition- 1° amine

Selectivity of nucleophile

Selected scopes

orthogonal reactivity
SO2F2: phenol > amine
SOF4: amine > phenol
Sharpless, K. B. et al. Angew. Chem. Int. Ed. 2017, 56, 2903.

21

Sulfonimidoyl Fluoride (R-SONHF)
2nd addition- 2° amine

sulfonimidoyl fluoride- Amino acid: hydrolytic loss of second S-F- Aryl silyl ether

22

Sulfonimidoyl Fluoride (R-N=S(O)F2)
Comparison of reactivity

23

Sulfonimidoyl Fluoride (R-SONHF)
3rd addition

BEMP as potent base to activate inert S-F bond
- Aryl silyl ether- 2° amine- One-pot procedure24Application
Medicinal chemistry
Chemical biology
New synthetic methodology
Material science25

RSO2F in Biological Context

electrophilic probe to modify proteins

H+ mediated switch-on-> specificity to microenvironment- Activation of S-F in special environment- Proximity driven reaction- Ser, Tyr, Lys, Thr, His, Cys
Examples- Covalent drugs- Target identification and validation- Mapping of enzyme binding sites, substrates and protein–protein interactions.
- Late-stage functionalization (LSF) of bioactive molecules
26

Inertness of Sulfonyl Fluoride
Nucleophilic stability of electrophiles in chemical biology

Conditions: electrophiles (1.0 mmol), aniline
1.3 mL (neat), 184 °C
Sharpless, K. B. et al. Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 18808.

27

Application to Bioconjugation
Modification of BSA with SO2F2 gas

BSA~4 modifications on Lys

Modification of BSA with fluorosulfate

Averick, S. et al. Polymer 2016, 99, 7.

SuFEx with Protein in Vivo
Fluorosulfate-L-tyrosine (FSY)

Incorporation of FSY
Pyrrolysyl-tRNA synthetase (PylRS) mutated library(mutation at A302, L305, Y306, L309, I322, N346,C348, Y384, V401, W417)
selection

6 hits PylRS mutant specific for FSY(mutation 302I, 346T, 348I, 384L, 417K)-> FSYRS (fluorosulfate tyrosyl-tRNA synthetase)
Coexpression of target protein genecontaining TAG codon with tRNAPylCUA/FSYRS- FSY was successfully introduced in the protein of E. coli and HELA- No toxicity to cells during the expression of FSY incorporated proteins.
Wang, L. et al. J. Am. Chem. Soc. 2018, 140, 4995.

SuFEx with Protein in Vivo
Afb-Z protein
MBP-Z24FSY, Afb-7X were coexpressed in E. coli.

yield 59％ 53％ 35％

Westernblot

His tag
purification-> SDS-PAGE

LC-MS/MS
Croslinking between FSY and Lys/His/Thr30

SuFEx with Protein in Vivo
Calmodulin

intramolecular crosslinking

Trx1-PAPS reductase

crosslinking with native Tyr

31

SuFEx Cross-linking for Mass Spectrometry
Cross-linking mass spectrometry (CXMS)
Identification of protein interaction partners“plant-and-cast” strategy

identification of interacting
proteins
Wang, L. et al. Proc. Natl. Acad. Sci. U. S. A. 2018, 115, 11162.

32

SuFEx Cross-linking for Mass Spectrometry
Reaction with peptide

CXMS analysis of BSA

GST(in crystal structure)

18 crosslinkings

15 crosslinkings

13 crosslinkings

9 crosslinkings

Cα-Cα distance (in the crystal structure)> side chain length*2 + linker length ~ 20 Å
Cross-linking occurred at different structure＝ from crystal structure.

Non-specific cross-linking is observed with BS2G.

33

SuFEx Cross-linking for Mass Spectrometry
Analysis of Enzyme-Substrate Interaction

genetically encode chemical crosslinkers (GECX)
thioredoxin (Trx)

Additionaly identified Trx-interacting peptides

34

Tyr Bioconjugation

protein(Fs=SO2F)

Mechanism of SuFEx proposed by authors

SuFEx

Deprotection of aryl silyl ether

Use of stoichiometric amount of base avoids the silyl ether?
Lee, Y.; Kim, B. M. et al. Chem. Eur. J. 2018, 24, 10948.

35

Tyr Bioconjugation
Screening of baseno silyl activation

Residue selectivity

Recombinant human erythropoietin (rhEPO)(5 eq)

PEG
Mn=2372Δm/z=198036

Tyr-Reactive Probe
Sulfur-Triazole EXchange (SuTEx)

ProbesProteome analysis

Hsu, K. et al. Nat. Chem. Biol. doi:10.1038/s41589-019-0404-5

37

Tyr-Reactive Probe
Residue selectivity

Tuning of LG for tyrosine selectivity

38

Covalent Inhibitor by SuFEx
Inhibitor of intracellular lipid binding protein (iLBP)
Evaluation of proteome reactivity of probes

HeLa cell + blank/1/ 2 or S1 (40 uM, 37 °C, 16 h),
CuAAC with cell lysate to attach fluolophore,
SDS-PAGE

Sulfonyl fluoride: highly reactivive, resulting in non-specific reactivity
Sharpless, K. B.; Kelly, J. W. et al. J. Am. Chem. Soc. 2016, 138, 7353.

39

Covalent Inhibitor by SuFEx
Stable isotope labeling by amino acids in cell culture (SILAC)
12C, 14N

HeLa cells

13C, 15N

Probe1:
fatty acid binding protein (a family of iLBPs)
FABP 3, FABP 5,(CRABP2) selective labeling
Probe2:non-specific labeling40

Covalent Inhibitor by SuFEx
Conserved Arg~Arg~Tyr module in iLBPs

LC-MS/MS to determine reactive residue

apo CRABP2 (green), FABP5(magenta), FABP4 (cyan)

All reacted at Tyr inArg~Arg~Tyr module.

41

Covalent Inhibitor by SuFEx
Introduction of biphenyl substructure
Improved reactivity for CRABP2
No reactivity with FABP3, 4, 5
Mechanism analysis of selectivity for CRABP2- Buffer pH dependence

Tyr134 pKa ~ 7.6
effect of Arg111, Arg132?

- Mutation experiment42

Covalent Inhibitor by SuFExcrystal structure
4-CRABP2

retinoic acid (RA)-CRABP2(PDB: 2FR3)
large hydrophobic binding
pocket to accommodate
Phenyl ring/ trimethyl
cyclohexene ring(selectivity of 4 to CRABP2)

43

Covalent Inhibitor by SuFEx
Covalent inhibitors of human neutrophil elastase (hNE).

PNAS 2019 116 18808

Sharpless, K. B. et al. Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 18808. 44

Covalent Inhibitor by SuFEx
Categolized SuFExable library
105 compounds

relative ratewith ArOTBS
28.5

11st screen
Incubation with enzyme for 10 min
Addition of substrate peptide(MeOSuc-AAPV-AMC)
Measure increase in fluorescence

14.1

4.1

45

Covalent Inhibitor by SuFEx
Hit compounds

2nd screen

Crystal structure hNE+146

Covalent Inhibitor by SuFEx
Selectivity between serine proteases

Effect of denaturation
MALDI-TOF-MS

47

SummarySuFEx-Advocated by Sharpless in 2014.
Stable S(VI)-F bondContext dependent activation

New synthetic methodology
Proteome analysis
Analysis of PPI
Medicinal chemistry
Polymer synthesis

Challenges
Mild/sharp activation of S-F bondTuning of leaving group instead of FElucidation of mechanism48

Appendix

49

Unstable Sulfonyl Fluoride
PMSF: serine protease inhibitor
short half-life under aqueous conditions
pH 7: 110 min
pH 7.5: 55 min
pH 8: 35 min
unstable under basic conditions(forms sulfene via E1cB elimination)

Aliphatic sulfonyl fluoride encounter the risk of hydrolysis
under basic conditions.

50

Stability under Electrolytic Conditions

Electrochemical instability of 1,2,3-triazole is demonstrated with CV.
51

Radical Addition to ESF
Entire mechanism

Liao, S. et al. Nat. Commun. 2019, 10, 1.

52

Sulfonylfluoride (RSO2F)
Cyclic alkenylsulfonyl fluoride53

RSO2F as Synthetic Reagent
PyFluor: deoxyfluorination

Coupling reagent

JACS 2015, 137, 9571

54

RSO2F as Synthetic Reagent
ArOSO2F as cross-coupling partner
toxitic

SO2F2, AISF, imidazolium salt
can be alternative.

✔ high reactivity-OSO2F ~ -OTf, -Br > -OTs, -OMs

by Hanley-Br ≥ -OTf > -OSO2F > -Cl

by Sharpless

Additive free Suzuki-Miyaura coupling

55

RSO2F as Synthetic Reagent
1,1-Dihydrofluoroalkylation of Amines- no reaction with –I, -OTs, -OMs

Synthesis of sulfonyl azide

Synlett 2016, 27, 1840

56

RSO2F as Synthetic Reagent
Diazotizing reagent: primary amine/sulfonamide to azide/sulfonylazide> 40 examples
orthogonal to most of function(including 2°/3° amines)

diazotransfer is reversible

Sharpless, K. B.; Dong, J. et al. Nature 2019, 574, 86.

57

RSO2F as Synthetic Reagent

Sharpless, K. B.; Dong, J. et al. Nature 2019, 574, 86.

58

Synthesis of Sulfotyrosine
Tyr O-sulfation: seen in PTMs

ACIE 2016, 55, 1835

59

Iminosulfur Oxidifluoride under Aqueous Conditions
Derivatization under aqueous conditions

ACIE 2019, 58, 8

80–98%

88-97%
iminosulfur oxidifluoride

stable at pH 2-14, 37 °C, 96 h

95-99% for aryl R1
19% for aliphatic R1

stable at pH 6-8, 37 °C, 48 h,hydrolyzed in other range of pH60

Iminosulfur Oxidifluoride under Aqueous Conditions
DNA modification

Protein modification~30%61