Rational Tuning of Fluorobenzene Probes for Cysteine-Selective Protein Modification

Article abstract of DOI:10.1002/anie.201712589

Fluorobenzene probes for protein profiling through selective cysteine labeling have been developed by rational reactivity tuning. Tuning was achieved by selecting an electron-withdrawing para substituent in combination with variation of the number of fluo

Full text of DOI:10.1002/anie.201712589

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Accepted Article
Title: Rational tuning of fluorobenzene probes for cysteine-selective
protein modification
Authors: Ahmed M. Embaby, Sanne Schoffelen, Christian Kofoed,
Meldal Morten, and Frederik Diness
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201712589
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10.1002/anie.201712589
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Rational tuning of fluorobenzene probes for cysteine-selective
protein modification
Ahmed M. Embaby,[a] Sanne Schoffelen,[a] Christian Kofoed,[a] Morten Meldal[a],* and Frederik
Diness[a],*
Abstract: Fluorobenzene probes for protein profiling through
selective cysteine labeling have been developed by rational
reactivity tuning. Tuning was achieved by selecting an electron-
withdrawing para-substituent combined with variation of the
number of fluorine substituents. Optimized probes chemo-
selectively arylated cysteine residues in proteins under aqueous
conditions. Probes linked to azide, biotin or a fluorophore were
applicable to labeling of eGFP and albumin. Selective inhibition
of cysteine proteases was also demonstrated with the probes.
Additionally, probes were tuned for site selective labeling among
cysteine residues and for activity based protein profiling in cell
lysates.
general methodology for rational tuning of probes towards
selective modification of a given cysteine residue of interests is
needed. Based on experience with nucleophilic aromatic
substitution (SNAr) reactions,16 we hypothesized that a two
dimensional reactivity tuning would provide such a system. The
tuning relied, in one dimension, on the electron withdrawing
capacity of the paraꢀsubstituent on fluorobenzene and, in the
second dimension, on varying the number of fluorine
substituents. The reactivity of fluorobenzene derivatives towards
Nꢀtertꢀbutyloxycarbonyl (Boc) cysteine (1) was investigated as a
model system for exploring the concept (Scheme 1).
Scheme 1. Fluorobenzenes’ reactivity towards cysteine.
R
R
2a-q
0.8 eq.
Application of chemical tools for covalent modification of proteins
is a powerful approach for identification, quantification and
regulation of these biomolecules.1 This principle is utilized in
techniques such as ELISA, microarray screening, protein pullꢀ
down and activity based protein profiling (ABPP).2 It is also an
established therapeutic concept, exemplified by the marketed
pharmaceuticals aspirin, penicillin, omeprazole and neratinib.3
Cysteine is among the reactive target amino acids, and in spite
of its low occurrence in proteins (1.9%), it is often located in
functionally important sites.4 In addition to stabilizing proteins
through disulfide bonds, cysteines have diverse roles in
metabolic processes, e.g. in catalysis, allosteric regulation, and
metal binding.5 Numerous electrophiles designed to probe
cysteine residues have been described,6 including many applied
to cysteine proteases e.g. chloroꢀ, and acyloꢀmethylketones,7
epoxides,8 sulfonate esters,9 haloacetamides10 and Michael
acceptors11. In recent years, also Sꢀarylation has been explored
as a method for modifying cysteine.12 Notwithstanding these
efforts, cross reactivity with other nucleophilic amino acid side
chains, insufficient reactivity and lack of selectivity among
cysteine residues are still unsolved issues.9,13 It is noteworthy
that the SꢀH bond has low dissociation energy,14 and that the
acidity, and thereby the reactivity of cysteines in proteins greatly
depends on the local environment. At the surface of proteins,
cysteine thiols have a pKa of ~8.5; whereas the pKa may be as
low as 2.5 for a catalytic thiol in an active site.15 Hence, a
SH
O
Fn
Fn
S
F
OH
BocHN
DIPEA
DMF
16 h
PBS/ACN (1:2 or 1:1)
Tested at pH 7, 8, 9, 10 and 11
16 h
OH
BocHN
or
O
1
3a-f, 3h-j
Fluorobenzene derivatives (Hammett p-constants):
Tuning by electron withdrawing
capacity of the para substituent
No reactions
Reacts in buffer and DMF
Reacts only in DMF
(0.78)
Reacts
more than
once in DMF
(0.65)
(0.54)
CF3
(0.36)
(0.23)
Br
(0.00)
(-0.63)
N
O
N
NO2
O
S
F
O
H
NH2
Fn
Fn
F
Fn
Fn
Fn
Fn
Fn
F
F
F
F
F
Fn=F4
:
2g
-
2a
2b
2i
2c
2j
2d
2k
-
2e
2l
2f
2m
2q
Fn=2,6-F2
Fn=2-F
:
:
2h
-
-
2n
2o
2p
Both promoting conditions using diisopropylethyl amine (DIPEA)
in dimethylformamide (DMF) and less promoting conditions
using a buffered mixture of water and acetonitrile (ACN) were
tested. The reactivity of the pentafluorobenzene derivatives (2aꢀ
g) could be directly linked to the electron withdrawing capacity of
the last substituent. Substituents with a Hammett σpꢀconstant
above 0.5 lead to reaction in both buffer and DMF.17 A σpꢀ
constant between 0.5 and 0.2 (2dꢀe) provided reaction in DMF
but not in buffer. Pentafluorobenzene 2f (σpꢀconstant of H = 0.0)
reacted only slowly in DMF, while pentafluoroaniline 2g (σpꢀ
constant of ꢀNH2 = ꢀ0.63) was completely unreactive. Reducing
the number of fluorine substituents from five to three significantly
reduced the reactivity of all the derivatives. The sulfonylamideꢀ
and trifluoromethylꢀsubstituted trifluorobenzenes (2i and 2j) were
not reactive with 1 in buffer solution in contrast to their
corresponding pentafluorobenzene derivatives (2b and 2c). The
amidoylꢀsubstituted trifluorobenzene 2k did not react with 1 in
DMF while the amidoylꢀsubstituted pentafluorobenzene 2d was
[a]
Mr. Ahmed M. Embaby, Dr. Sanne Schoffelen, Mr. Christian Kofoed,
Prof. Dr. Morten Meldal* and Prof. Dr. Frederik Diness*
Center for Evolutionary Chemical Biology, Department of Chemistry
University of Copenhagen
Universitetsparken 5, DK2100 Copenhagen, Denmark
Eꢀmail: fdi@chem.ku.dk, meldal@nano.ku.dk.
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/anie.201712589
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reactive. Finally, the reactivity was reduced even further by
having only two fluorine substituents. Thus, the sulfonylamide or
trifluoromethylꢀsubstituted difluorobenzene (2n and 2o) were not
reactive at all even in DMF. All the compounds that initially
displayed reactivity in buffer at pH 11 were further tested for
reactivity at pH 7, 8, 9 and 10, and even though the reaction
The methodology could also be adopted for selective inhibition
of cysteine proteases over other classes of proteases,
demonstrated herein by an onꢀbead inhibition screening assay
towards the cysteine protease papain or the serine protease
subtilisin (Figure 2). FRET substrates for the two proteases were
synthesized on PEGA1900 polymer beads. The enzymes were
rates were reduced, conversion still occurred at these pH values. preꢀincubated with the potential inhibitor (100 equiv.) for 1 h at
All reactions, except for the one with 2a in DMF, afforded one
major adduct as confirmed by product isolation.
37 °C. These mixtures were then added to the substrate beads.
After 30 minutes, beads started to light up in wells with papain
alone or papain preꢀincubated with 2d, 2g or 2i (Figure 2 and
Figure S20) while the beads in the wells with papain preꢀ
incubated with more reactive compounds 2a or 2b remained
dark, even after 24 h. In a parallel experiment with subtilisin
under identical conditions, none of the compounds were able to
inhibit the protease activity.
Compound 2b was further studied for selectivity towards
cysteine over other amino acids in aqueous buffer. Peptide 4
containing the most nucleophilic protein functionalities was
mixed with an excess of 2b which gave only the
monosubstituted adduct 5a (Scheme 2). Reaction of 4 with
iodoacetamide led to blocking of cysteine (5b) which prevented
further reaction with probe 2b added afterwards. In comparison,
the less activated probe 2d did not react under the same
conditions.
Scheme 2. Chemoꢀselective arylation of cysteine in peptide.
Functionalized derivatives of 2b were synthesized by reacting
pentafluorophenylsulfonyl chloride with aminoꢀfunctionalized
PEG linkers containing a tag, such as an azide group for click
chemistry (CuAAC or SPAAC), the sulforhodamine
B
fluorophore or biotin affinity probe (6a, 7, or 8, Figure 1). The
generated tagꢀcontaining probes were reacted with enhanced
green fluorescent protein (eGFP) comprising two cysteines
(Cys49 and Cys71), not involved in disulfide bonds;18 or with
bovine serum albumin (BSA), which has 17 conserved disulfide
bonds and one free thiol (Cys34).19 All three types of reporterꢀ
linked probes were capable of labeling eGFP and BSA in a
concentrationꢀdependent manner and proved compatible with
the functional click, the fluorescence or the affinity tag used for
detection.
Figure 2. Onꢀbead papain and subtilisin inhibition assays of probe 2a, 2b and
2d. Schematic reaction and pictures of assay beads.
Three fluorobenzene derivatives with different reactivities (2b, 2i
and 2k) were selected for investigation of their ability to react
selectively with one among several cysteine residues in the
same protein. Tobacco Etch Virus (TEV) protease (Hisꢀtagged
variant) was chosen as a model protease as it contains a free
cysteine exposed on the surface (Cys129) and another in the
catalytic site (Cys170).20 Native TEV protease was incubated
with fluoroaryl compounds 2b, 2i or 2k (~50 equiv.) (Figure 3).
Figure 1. Labeling of eGFP and BSA with pentafluorophenylsulfonamide
probes.
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The concept was also applied in activityꢀbased protein profiling
(ABPP). Native or heatꢀdenatured TEV protease was treated
with an azideꢀfunctionalized probe (6aꢀd, 10 ꢁM, Figure 4) at
37 °C for 16 h and then visualized by a cyanine “click” dye.
Following gel electrophoresis, the scanning demonstrated the
ability of probe 6b and 6c to label TEV protease in an activityꢀ
dependent manner, as these only labeled the native protease.
The reduced reactivity of the denaturated protease may be
explained by loss of the local activating eviroment in the active
site. In contrast to 6b and 6c, probe 6a was able to label both
active and denatured TEV protease, while probe 6d did not label
any of the samples. This clearly demonstrates the concept of
rational reactivity tuning. Thus, the excessively reactive
flourobenzene probe 6a could be “tuned down” by either
replacing the sulfonamide with the less electronꢀwithdrawing
amide 6c or by removing two fluoroꢀsubstituents (6b). However,
combining both effects reduces the reactivity of the probe (6d) to
a level that prohibits labeling of even the active protease.
Figure 3. Screening of probes for site specific cysteine arylation in TEV
protease. Schematic reaction and MS spectra showing peaks corresponding
to cysteineꢀcontaining fragments of nonꢀmodified TEV protease and TEV
protease treated with probes 2b and 2i.
Figure 4. Activityꢀbased protein profiling of TEV. Structures of 6aꢀd and their
labeling pattern of native or heat denatured TEV protease with the probes.
To demonstrate the application of the fluorobenzenes to ABPP
in a cellular environment, bacterial cell lysates were treated with
probe 6a, 6b and 6d with or without pretreatment with
iodoacetamide. This revealed that the most reactive probe 6a,
labeled many protein bands with great intensity, whereas probe
6b appeared much more selective. Probe 6d, the least reactive,
was not able to label any protein in the cell lysate. The
pretreatment with iodoacetamide completely inhibited any
labeling with the probes, supporting that the probes target the
free cysteine residues. Moreover, probe 6b was clearly able to
label TEV protease within a spiked cell lysate (Figure 5).
After incubation for 10 h with the most reactive probe 2b, a
mixture of adducts were detected, with the highest mass
corresponding to arylation of all free cysteine residues (Figure
S24). Gratifyingly, when TEV protease was incubated with the
less reactive probe 2i, only the monosubstituted adduct was
identified (Figure S25). Lastly, TEV protease was not modified
by the least reactive probe 2k under the same conditions. After
incubation for 24 h, the selective arylation of the cysteine in the
catalytic site by probe 2i was confirmed by highꢀresolution mass
spectrometry of all samples after tryptic digestion (Figure 3). In
the native TEV protease, the peptide fragment containing the
unmodified cysteine in the active site (Cys170) was found at
1219.59 Da and that of the fragment with a surface exposed
cysteine (Cys129) at 1267.68 Da. Only the signal at 1219.59 Da
disappeared upon treatment of TEV protease with 2i, and a new
mass peak at 1478.65 corresponding to the modified fragment
appeared. In this sample the fragment with the surface exposed
cysteine (Cys129) remained unchanged and a signal of the
probeꢀfunctionalized fragment with an expected mass peak at
1526.72 Da was not detected. In contrast, both the 1219.59 Da
and the 1267.68 Da signals completely disappeared upon
treatment of TEV protease with compound 2b. In this reaction
two new mass peaks corresponding to modified peptide
fragments appeared at 1514.62 Da and at 1562.73 Da. As a
control experiment, both fragments were found to be modified
after TEV treatment with iodoacetamide (Figure S27ꢀ29). The
difference between the selective probe 2i and the nonꢀselective
probe 2b is the number of fluorine substituents.
Figure 5. The selectivity of probes 6a, 6b and 6d towards proteins in cell
lysate. Labeling of TEV protease (spiked) and identification of chloramphenicol
acetyl transferase (CAT) by probe 6b. Reaction conditions: Preꢀincubation of
cell lysate/Tris buffer 1:1 (10 ꢁL), with or without iodoacetamide (0.5 mM), 37
°C, 2 h, then 6a, 6b or 6d in Tris buffer (10 ꢁL, 20 ꢁM), 37 °C, 16 h.
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In summary, it has been demonstrated that probes for site
selective cysteine labeling, enzyme inhibition and protein
profiling can be developed by rational and accurate tuning of the
electron deficiency of fluorobenzene derivatives. The reactivity
of the flourobenzene derivatives towards cysteine proved to
correlate with the Hammett σpꢀconstant of the paraꢀsubstituent
and with the number of additional fluorine substituents. Based
on this knowledge, probes for selective cysteine labeling over all
other amino acids were developed. The probes were
demonstrated to be applicable for labeling of proteins with free
cysteine residues such as BSA and eGFP. Chemoselective
inhibition of a cysteine protease over a serine protease was also
demonstrated. Developing less reactive probes allowed
discrimination among cysteine residues with different
physicochemical properties in the TEV protease, giving arylation
only at the cysteine residue in the catalytic site in an activityꢀ
dependent manner. These probes were used for activity based
protein profiling and for identifying proteins containing reactive
cysteine residues in cellular lysates.
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Acknowledgements
We are grateful to The University of Copenhagen for support
through the UCPH
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Keywords: Fluorobenzene • Arylation • Protein Modification •
Enzyme • Inhibitor
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Entry for the Table of Contents
COMMUNICATION
The reactivity of chemical probes
for selective cysteine Sꢀarylation in
proteins has been accurately tuned
by rational selection of substituents
in a twoꢀdimensional fashion. The
optimized probes are demonstrated
applicable to protein labelling of
GFP and albumin, selective
inhibition of cysteine proteases,
selective functionalization among
cysteine residues and for activity
based protein profiling in cell
lysates.
Ahmed M. Embaby, Sanne
Schoffelen, Christian Kofoed, Morten
Meldal* and Frederik Diness*
Page No. – Page No.
Rational tuning of fluorobenzene
probes for cysteine-selective
protein modification
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