Ethynylation of Cysteine Residues: From Peptides to Proteins in Vitro and in Living Cells

Article abstract of DOI:10.1002/anie.202002626

Current approaches to introduce terminal alkynes for bioorthogonal reactions into biomolecules still present limitations in terms of either reactivity, selectivity, or adduct stability. We present a method for the ethynylation of cysteine residues based on the use of ethynylbenziodoxolone (EBX) reagents. The acetylene group is directly introduced onto the thiol group of cysteine and can be used for copper-catalyzed alkyne-azide cycloaddition (CuAAC) without further processing. Labeling proceeded with reaction rates comparable to or higher than the most often used iodoacetamide on peptides or maleimide on the antibody trastuzumab, and high cysteine selectivity was observed. The reagents were also used in living cells for cysteine proteomic profiling and displayed improved coverage of the cysteinome compared to previously reported iodoacetamide or hypervalent iodine reagents. Fine-tuning of the EBX reagents allows optimization of their reactivity and physical properties.

Full text of DOI:10.1002/anie.202002626

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Ethynylation of Cysteines from Peptides to Proteins in Living
Cells
Authors: Romain Tessier, Raj Kumar Nandi, Brendan G. Dwyer, Daniel
Abegg, Charlotte Sornay, Javier Ceballos, Stephane Erb,
Sarah Cianferani, Alain Wagner, Guilhem Chaubet, Alexander
Adibekian, and Jerome Waser
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
Ethynylation of Cysteines from Peptides to Proteins in Living
Cells
Romain Tessier, Raj Kumar Nandi, Brendan G. Dwyer,[b]† Daniel Abegg, [b]† Charlotte Sornay[c]†,
[
a]
[a]†
[
a]
[d]
[d]
[c]
[c]
Javier Ceballos, Stéphane Erb, Sarah Cianferani, Alain Wagner, Guilhem Chaubet,* Alexander
[
b]
[a]
Adibekian,** and Jerome Waser***
Abstract: Efficient methods to introduce bioorthogonal groups, such
as terminal alkynes, into biomolecules are important tools for chemical
biology. State-of-the-art approaches are based on the introduction of
a linker between the targeted amino acid and the alkyne, and still
present limitations of either reactivity, selectivity or adduct stability.
Herein, we present an ethynylation method of cysteine residues
based on the use of ethynylbenziodoxolone (EBX) reagents. In
contrast to other approaches, the acetylene group is directly
introduced onto the thiol group of cysteine and can be used in one-
pot in a copper-catalyzed alkyne-azide cycloaddition (CuAAC) for
further functionalization. Labeling proceeded with reaction rates
comparable or higher than the most often used iodoacetamide on
peptides or maleimide on the antibody trastuzumab. Under optimized
conditions, high cysteine selectivity was observed. The reagents were
also used in living cells for cysteine proteomic profiling and displayed
an improved coverage of the cysteinome compared to previously
reported iodoacetamide or hypervalent iodine-reagent based probes.
Fine-tuning of the EBX reagents allowed optimization of their reactivity
and physical properties for the desired application.
Introduction
Selective chemical modifiers, combined to bioorthogonal reactive
handles, are crucial to study and alter biological systems.[ High
selectivity is key to their success. In addition, these reagents must
lead to high reaction rates in aqueous media under air, at neutral
pH and moderate temperature. Moreover, they should be non-
toxic and non-interfering with protein structures. Amongst them,
reagents that transfer terminal acetylenes to biomolecules are of
particular interest. Indeed, acetylenes are apolar and unable to
establish strong hydrogen bonds. They are therefore unlikely to
generate significant structural alterations once attached to
biomolecules. Ethylene is also the smallest suitable functional
group for bioorthogonal reactions and is frequently employed in
chemical biology,[ mostly through copper-catalyzed azide-alkyne
cycloadditions (CuAACs) (Scheme 1, path a).[ This well-known
1]
2]
3]
[
3+2] annulation has found numerous applications both in vitro
and in vivo.[ Other metal-assisted reactions also exploited the
unique reactivity of terminal alkynes to modify biomolecules. For
example, palladium-assisted Sonogashira cross-couplings were
applied to protein labeling in living systems (path b).[5]
4]
[
a]
Dr. R. Tessier, Prof. Dr. R. K. Nandi, Dr. J. Ceballos, Prof. Dr. J.
Waser
Laboratory of Catalysis and Organic Synthesis
Ecole Polytechnique Fédérale de Lausanne
EPFL SB ISIC LCSO, BCH 4306, 1015 Lausanne, Switzerland
E-mail: jerome.waser@epfl.ch
[
[
[
b]
c]
d]
B. Dwyer, Dr. D. Abegg, Prof. Dr. A. Adibekian
Department of Chemistry
The Scripps Research Institute
130 Scripps Way, Jupiter, Florida 33458, United-States
Email: aadibeki@scripps.edu
Charlotte Sornay, Dr Alain Wagner, Dr Guilhem Chaubet
Bio-Functional Chemistry (UMR 7199), LabEx Medalis, University of
Strasbourg,
74 Route du Rhin, 67400 Illkirch-Graffenstaden, France
E-mail: chaubet@unistra.fr
Stéphane Erb, Dr Sarah Cianferani
Scheme 1. Well-established alkyne-linker approach for cysteine
functionalization.
Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO),
Université de Strasbourg, CNRS, IPHC UMR 7178, 67000
Strasbourg, France
†
These authors contributed equally.
Dr. R. Tessier
Labeling reagents that carry terminal alkynes and selectively
modify cysteines are especially useful, as the latter are a long-
established target in chemical biology. So far, iodoacetamide-
Present address: Department of Chemical Biology
Max Planck Institute of Molecular Physiology
Otto-Hahn-Strasse 11, 44227 Dortmund, Germany
Prof. Dr. R. K. Nandi
Present address: Department of Chemistry
Diamond Harbour Women's University
Sarisha, South 24 Parganas, West Bengal-743368, India
[
6]
alkynes[ and maleimide derivatives are the most common
reagents (Scheme 1, paths c and d). Nevertheless, there are still
limitations to these approaches, such as insufficient reactivity or
selectivity, as well as low adduct stability, resulting in suboptimal
7]
[8]
Supporting information for this article is given via a link at the end of
the document.
[1]
coverage of the cysteinome. Beyond these traditional reagents,
only few studies described the one-step attachment of terminal
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RESEARCH ARTICLE
acetylenes to cysteine residues, based on addition to alkynones
displayed an improved coverage of the cysteinome compared to
our previous work using JW-RF-010 (4).
[
9]
(
path e) or photocatalyzed arylation using diazonium salts (path
[10]
[11]
[12]
f).
Recently, efficient palladium-
and nickel-assisted
arylations were described (paths g and h), but their application
remains limited by solubility and biocompatibility issues.[13]
Most importantly, all these methods share a common feature: a
linker between the cysteine residue and the terminal alkyne is
mandatory. However, this linker may have a dramatic impact on
the structure, the stability and the localization of the labeled
molecule.[14] Furthermore, labeling loss can be observed with
poorly stable linkers, such as succinimides[15] and vinyl
ketones,[9a] in particular through hydrolysis and/or external
organosulfur attack. Installing the terminal alkyne directly on the
sulfur atom without any linker would constitute the minimal
disturbance possible, and may lead to a uniquely reactive alkyne.
However, there is currently no one-step method to introduce an
ethynyl group onto cysteine under physiological conditions.
Over the last years, our group has focused on the
functionalization of thiols with hypervalent iodine compounds.
These reagents are particularly attractive for chemical biology, as
they combine high reactivity and selectivity, low toxicity and
sufficient stability.[16] We first reported
a chemoselective
alkynylation of thiols such as dipeptide 1, using
triisopropylsilylethynylbenziodoxolone (TIPS-EBX (2) Scheme
2
A).[17] The resulting silylated alkyne could then be deprotected to
give free terminal alkyne 3, which could be functionalized via
CuAAC. However, this multi-step procedure performed in organic
solvents was not suitable for chemical biology applications. We
therefore designed a reagent (JW-RF-010 (4)) with an additional
azide handle, which was applied for in vitro and in vivo native
cysteine labeling (Scheme 2B).[18,19] JW-RF-010 (4) displayed
high chemoselectivity, outperforming iodoacetamide, the gold
standard in cysteine targeting. However, the labeling by JW-RF-
Scheme 2. Previous and current cysteine labeling studies using EBX reagents.
010 (4) provided only modest coverage of the cysteinome,
comparable to iodoacetamide alkyne. We then investigated a
selective modification of any cysteines- and other thiol-bearing
compounds in aqueous media. Our studies resulted in an efficient,
chemoselective and clean labeling of cysteine-containing
peptides and proteins without cleavage of the hypervalent bond
Results and Discussion
Our previous work with alkyl-substituted EBX reagents resulted in
the exclusive formation of VBX products.[20] Nevertheless, our
(
(
Scheme 2C).[20] Although the synthesized vinylbenziodoxolones
VBXs) opened the way for novel bioorthogonal transformations,
DFT calculations showed that the energy of the transition states
leading to the alkyne product depended strongly on the structure
the presence of a linker was mandatory and no alkyne bond
remained in the product. From this point of view, the method was
not fundamentally different from other reported alternatives. The
fact that different products were obtained for hyperreactive or
surface-exposed cysteines also limited the generality of the
method. In particular, mixtures were obtained for cysteines with
intermediate reactivity and/or surface localization.
Herein, we present the development and application of TMS-
EBXs that selectively ethynylate cysteine residues, in a single
step under physiological conditions (Scheme 2D). These
reagents bear a labile trimethylsilyl group and can be fine-tuned
to adjust the rate of product formation and the physical properties
of the reagent in dependence of the desired application. The
hypervalent iodine compounds were applied from simple peptides
to complex proteins such as antibodies. One-pot CuAAC was
possible with the obtained thioalkynes. The reagents could also
be used for cysteine labeling in living cells. Proteomic profiling
[21]
of the substituent on the alkyne. On one hand, alkyl-EBXs led
to the formation of
a relatively stable vinylic carbanion
intermediate that can be easily protonated. On the other hand, a
low energy pathway involving a 1,2-silicon shift was available for
silyl-EBXs. Therefore, silyl-EBXs should give alkyne products,
even in water. However, TIPS-EBX (2) cannot be used, as it is not
soluble in aqueous media. To start our investigations, we selected
the highly abundant glutathione (5a), a naturally occurring
tripeptide that plays an essential role in primary metabolism as
disulfide bond reductant,[22] and TMS-EBX (6a) in an aqueous
buffer solution (Scheme 3). The use of the smaller trimethylsilyl
group on the alkyne gave sufficient solubility in water. To our
surprise, unsubstituted glutathione bound VBX 7a was obtained
instead of the expected silylated alkyne. This was due to a rapid
and complete desilylation of TMS-EBX (6a) to give EBX (8) in a
few minutes. EBX (8) is typically unstable, prepared in situ and
employed at low temperature.
[23]
However, 8 displayed a
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RESEARCH ARTICLE
remarkable stability in aqueous media. While no degradation was
observed after 5 minutes, 86% of reagent 8 still remained after 2
hours in 10 mM Tris buffer at pH 8.2. This unusual stability might
be explained by a coordinating effect from the buffer that
stabilizes the reagent. Such effect was previously observed in
organic solvent and allowed to crystallize an EBX-ACN
complex.[ In contrast to the previous substituted alkyl-VBX
products,[20] 7a underwent rearrangement into the ethynylated
glutathione 9a, giving a first access to the targeted product under
physiological conditions. Nevertheless, the rearrangement was
slow and incomplete after extended reaction times.
(entry 7). We then examined the impact of the pH on the reactivity.
At pH 7.2, the desired product 5a was obtained in 90% yield within
15 minutes (entry 8). After only 15 minutes at pH 6.4, our labeling
process produced 58% yield of the desired product 9a (entry 9).
An extended reaction time of 60 minutes increased the yield of 9a
to 78%.
24]
Table 1. Evaluation of reaction conditions for the ethynylation of glutathione (5a).
Entry
Variations from the above conditions
None
Yield
1
2
3
4
5
6
7
8
9
99% (99%)
87% (95%)
87% (90%)
74% (82%)
81% (90%)
60% (76%)
0% (0%)
TMS-EBX (6a)
JW-RT-02 (6c)
Scheme 3. Formation of EBX (8) and vinylbenziodoxolone 7a, followed by its
rearrangement into terminal thioalkyne 9a.
JW-RT-03 (6d)
JW-RT-04 (6e)
We hypothesized that an electronic fine-tuning of the TMS-EBX
aromatic core might have a critical impact on the rearrangement
rate and efficiency. We therefore prepared several hypervalent
iodine derivatives containing both electron withdrawing and
donating groups (Table 1). These EBX compounds were
prepared in two synthetic steps from commercially available
compounds, without column chromatography purification and
could be handled under air without any noticeable degradation
TES-EBX (10)
TIPS-EBX (2)
200 mM PB pH 7.2
200 mM PB pH 6.4
50 mM PB pH 8.2
200 mM Tris pH 8.2
200 mM HEPES pH 8.2
200 mM TAPS pH 8.2
Reaction at room temperature
200 µM reaction molarity
1.2 equiv. of 6b
90% (93%)
58% (78%)
95% (99%)
79% (94%)
42% (75%)
52% (88%)
81% (94%)
97% (97%)
93% (97%)
10
(
see Supporting Information). After optimization, a fast and
11
practicable procedure could be developed for the thioethynylation
based on the use of the more reactive nitro-substituted reagent
JW-RT-01 (6b) (Table 1, entry 1). A stock solution of 6b in DMSO
was added to a non-degassed 200 mM phosphate buffer (PB) at
pH 8.2 for desilylation. After 2 minutes, a solution of glutathione
12
13
14
(
5a) in the same buffer was transferred to the hypervalent iodine
15
reagent. The reaction was then shaken at 37 °C for 15 minutes.
The reaction afforded the ethynylated glutathione 9a in 99%
yield.[25] Compound 9a is stable at neutral pH, but slowly
hydrolyzes in acidic media. Under the optimized reaction
conditions, a broad range of TMS-EBX reagents could be used.
Nevertheless, lower yields were obtained with TMS-EBX (6a),
JW-RT-02 (6c), JW-RT-03 (6d) and JW-RT-04 (6e) (entries 2-5).
A complete desilylation of TES-EBX (10) was also observed,
giving the desired product 9a in a moderate yield after 15 minutes
16
Labeling conditions: 1.00 µmol glutathione (5a), JW-RT-01 (6b) (2.0 equiv.) in
0.5 mL of non-degassed 200 mM phosphate buffer pH 8.2 (2% v/v DMSO),
37 °C, 15 minutes. Calibrated HPLC yields based on absorbance at 214 nm
(Figure S1). See Supporting Information for details. The yields in parentheses
correspond to the yields after one hour of reaction. For complete robustness
studies, see Supporting Information Tables S1 and S2.
We also investigated the tolerance of our ethynylation process to
different buffers. Employing a 50 mM phosphate buffer did not
alter significantly the yield and rate of the reaction (entry 10).
Although the reaction rate significantly slowed down in Tris buffer,
(
entry 6). Finally, no conversion was observed in presence of
TIPS-EBX (2) because of its lack of solubility in aqueous media
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RESEARCH ARTICLE
an extended reaction time of 60 minutes afforded 94% yield of the
desired product (entry 11). Employing HEPES or TAPS buffers
instead of a phosphate buffer resulted in a slowdown of the
ethynylation process and a decrease of the yield (entries 12 and
Steric hindrance around the cysteine residue did not alter the
reaction efficiency (9e and 9f). We then introduced cysteine on
the C-terminal position. When the C-terminal carbonyl of the
peptide was an amide, the desired product 9g was obtained in
94% yield. With a carboxylic acid in contrast, a mixture of products
was obtained (See compound 9v in Supporting Information). A N-
terminal cysteine (9h) was well tolerated. Quantitative labeling
was observed with cysteines in close proximity of aspartic acid
(9i) and asparagine (9j). The presence of a methionine residue
(9k) did not show any detrimental effect on the labeling.
Undesired side reactivity was observed in presence of an arginine
residue, resulting in the formation of the desired product 9l in 59%
yield. In the case of tyrosine, the desired product 9m was obtained
in 84% yield due to the formation of side-products.
13). Nevertheless, 75 and 88% yield were obtained after 60
minutes. Ethynylation of glutathione (5a) could be performed at
room temperature, resulting in a 81% yield of the product 9a after
15 minutes (entry 14). The reaction was still efficient when diluted
to 200 µM (entry 15). Finally, a reduced amount of JW-RT-01 (6b)
furnished the desired product 9a in 93% yield (entry 16).
Once the robustness of our ethynylation process has been
demonstrated, we investigated its scope first on tetrapeptides
(
Scheme 4A). Labeling of peptides containing valine (9b),
phenylalanine (9c) and proline (9d) was obtained in 91-93% yield.
Scheme 4. A. Scope of the ethynylation of tetrapeptides and B. Application to larger peptides. [a]Reactions were carried out on a 1.00 µmol scale. [b]Reactions were
carried out on a 0.50 µmol scale. Yields were determined by relative integration based on HPLC-MS. See Supporting Information for details.
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The reaction was also chemoselective in presence of serine (9n)
and tryptophan (9o). When a carboxylic acid function was
introduced instead of the amide in C-terminal position (9p), the
product was obtained in 95% yield. This result confirms that a
terminal carboxylic acid is an issue only in the case of C-terminal
cysteine. Finally, no side reactivity occurred with tetramers
containing histidine (9q) and lysine (9r). We then extended our
ethynylation process to more complex peptides (Scheme 4B). On
Human Serum Albumin Leu55-His63 fragment, our optimized
conditions afforded the desired product 9s in 97% yield. After
treatment with JW-RT-01 (6b), the peptidic gap junction blocker
GAP26 furnished 68% yield of the alkynylated product 9t. Finally,
a Trp554-Ala566 fragment of the hepatitis C virus envelope
glycoprotein E2 was successfully converted in its corresponding
thioalkyne product 9u in 94% yield.
sodium ascorbate (NaAsc) as reducing agent, the fluorescent
probe Cy7-azide (Cy7-N ) was coupled to ethynylated glutathione
3
(5a) and Human Serum Albumin Leu55-His63 fragment 5s to give
the corresponding products 13a and 13b (Scheme 6A).[26]
In nature, the vast majority of cysteines are engaged in disulfide
bridges. It is therefore crucial to develop protocols to target these
cysteine residues. The current most widespread procedure is an
in situ cleavage of the disulfide bond, followed by labeling of the
reduced cysteines. In our case, we envisioned that a one-pot
protocol allowing disulfide bond reduction and subsequent
cysteine ethynylation would be of high interest for the labeling of
disulfide bound cysteines. Our investigations started with oxidized
glutathione,
as
a
model
system,
and
tris(2-
carboxyethyl)phosphine (TCEP), as reducing reagent (see
Supporting Information). In presence of 1.5 equivalent of TCEP,
oxidized glutathione was completely reduced within 60 minutes.
Subsequent addition of 4.0 equivalents of JW-RT-01 (6b) (2.0
equivalents per reduced cysteine) afforded the desired product 9a
in 97% yield. Notably, the ethynylation process was also achieved
in presence of 5.0 and 10 equivalents of the reducing reagent and
without increasing the amount of 6b. Although the hypervalent
iodine reagent 6b was significantly degraded in the presence of
an excess of TCEP, these reactions respectively afforded the
ethynylated glutathione 9a in 66% and 39% yield. These
promising results on oxidized glutathione prompted us to apply
our one-pot procedure to the natural bioactive peptide oxytocin
Scheme 6. A. Copper-catalyzed azide-alkyne cycloadditions between terminal
thioalkynes and Cy7-N . Cy7 = Cyanine7 dye. B. Alkyne hydration in acidic
3
(
11). To our delight, in situ reduction and subsequent ethynylation
media. C. One-pot thiol alkynylation and subsequent Sonogashira cross-
coupling. TPPTS = Triphenylphosphine-3,3',3''-trisulfonic acid trisodium salt.
See Supporting Information for further details about these procedures.
afforded peptide 12 in 86% yield (Scheme 5).
The ethynylation and cycloaddition steps could be conducted in a
one-pot manner. Under mild acidic conditions (pH 3), thioalkyne
9a, generated in situ from 5a, was converted to the corresponding
thioester 14 (Scheme 6B). Thioesters are key reactive
intermediates in chemical biology, both in natural metabolism, for
example for the production of fatty acids,[
27a]
and in in vitro
Scheme 5. Labeling of sulfur bridge-containing oxytocin (11). Conditions: 1.5
[27b]
methods to synthesize proteins.
They can also be converted
Finally, the thioalkyne moiety was
engaged in a Sonogashira coupling (Scheme 6C).[
.
equiv. TCEP HCl, 200 mM PB pH 8.2, r.t., 60 min, then 4.0 equiv. JW-RT-01
back to free cysteines.[
27c]
(6b), 200 mM PB pH 8.2 (2% v/v DMSO), 37 °C, 15 min. Reaction was carried
28]
out on a 0.10 µmol scale. Yield was determined by relative integration based on
HPLC-MS. See Supporting Information for details.
The
formation of the desired alkyne 15 was confirmed by HPLC-MS,
together with a bisalkyne product resulting from a Glaser coupling
and other side products (see Supporting Information for details).
These promising results prompted us to evaluate the reactivity
and selectivity of JW-RT-01 (6b) on more complex substrates. We
selected trastuzumab (16) (~150 kDa), an FDA-approved
Once the potential of our ethynylation process has been
demonstrated, we investigated the reactivity of the terminal
thioalkyne, starting with the well-known [3+2] cycloaddition
[29]
between
azides
and
triple
bonds.
Using
tris(3-
antibody used to fight breast cancer. To our surprise, treatment
hydroxypropyltriazolylmethyl)amine (THPTA) as ligand and
of reduced trastuzumab (16) with reagent 6b led to a complex
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RESEARCH ARTICLE
mixture after the CuAAC step. This result suggested a partial
decomposition of the antibody or side reactions occurring during
the ethynylation or the cycloaddition steps. Control experiments
conducted on the non-reduced antibody showed residual
reactivity of JW-RT-01 (6b) even in absence of cysteine, leading
to degradation of trastuzumab (16) as shown by native mass
spectrometry (MS) (Figure S2). While arginine or tyrosine
residues were also found to react with EBX reagents to some
extent on tetrapeptides (Scheme 4), the reason and exact nature
of this enhanced side-reactivity on antibody 16 are not clear at
this stage. The formation of terminal alkynes is unlikely, as
highlighted by the antibody’s lack of fluorescence when CuAAC
Table 2. Evaluation of the reactivity of TMS-EBX reagent 6a for the
bioconjugation of the antibody trastuzumab (16)
Entry
Conditions A
Av.
Conv.
(%)
DoC
reaction with
a TAMRA-azide probe was attempted after
treatment with JW-RT-01 (6b). Varying buffer composition and pH,
or decreasing reaction time, temperature and number of
equivalents, both on either reduced or non-reduced trastuzumab
1
2
3
4
5
6
7
8
9
2 equiv., PB buffer, pH 8.2, 37 °C, 5 min.
8 equiv., PB buffer, pH 8.2, 37 °C, 5 min.
16 equiv., PB buffer, pH 8.2, 37 °C, 5 min.
8 equiv., PBS buffer, pH 8.2, 37 °C, 5 min.
8 equiv., PBS buffer, pH 6.5, 37 °C, 5 min.
8 equiv., PBS buffer, pH 7.5, 37 °C, 5 min.
8 equiv., PBS buffer, pH 8.5, 37 °C, 5 min.
8 equiv., PBS buffer, pH 7.5, 25 °C, 5 min.
8 equiv., PBS buffer, pH 7.5, 25 °C, 2 min.
8 equiv., PBS buffer, pH 7.5, 4 °C, 5 min.
0.8
3.7
4.4
4.0
0.9
3.4
4.4
1.8
1.2
0.1
52
94
97
96
50
93
97
74
60
13
(
16), did not allow to obtain better results, urging us to use the
less reactive EBX reagents TMS-EBX (6a) and JW-RT-02 (6c)
instead of JW-RT-01 (6b).
In presence of non-reduced trastuzumab, TMS-EBX (6a) showed
still some side reactivity, but to a lower extent than JW-RT-01 (6b).
Notably, under the previously optimized conditions (phosphate
buffer pH 8.2, 37 °C), the reduced antibody afforded clean native
mass spectra, allowing us to determine average degrees of
conjugation (DoC) and conversion rates (Table 2, see Supporting
Information Table S3 for detailed results). We observed that
native MS profiles were identical after 5 or 15 minutes of
incubation with 2 equivalents of 6a. Increasing the number of
equivalents of TMS-EBX (6a) from 2 to 8 and 16 resulted in
increased DoC – from 0.8 to 3.7 and 4.4 – and conversion values
10
Conditions B: TAMRA-N
4 h.
3 4
, CuSO , THPTA, NaAsc, PBS (1X, pH 7.4), 25 °C,
–
from 52% to 94% and 97% (Table 2, entries 1-3), highlighting a
2
reactivity comparable to that of classical maleimides.[30] Switching
from PB to PBS buffer had a negligible impact on the reactivity of
6
a (entry 4). In contrast, pH and temperature had a profound
We then evaluated the proteome-wide labeling of cysteines by the
TMS-EBX reagents 6a-e. HeLa cell lysates were treated for one
hour at 37 °C with 6a-e before CuAAC-mediated installation of a
TAMRA dye and in-gel SDS-PAGE fluorescence scanning.
Coomassie Brilliant Blue (CBB) staining was used as a loading
control (Figure 1A). All five of the TMS-EBX reagents labeled
HeLa lysates at 10 µM with JW-RT-02 (6c) showing the strongest
labeling followed by TMS-EBX (6a) and JW-RT-03 (6d) (Figure
1B). To confirm that the compounds reacted with cysteines in a
chemoselective manner, lysates were first treated with the
cysteine blocking reagent iodoacetamide (IAA). To our delight,
IAA pre-treatment abolished labeling of JW-RT-02 (6c),
demonstrating that TMS-EBX reagents label proteomic cysteines
impact on the bioconjugation outcome (entries 5-8). Poor
reactivity was found at pH 6.5, resulting in low average DoC and
conversion values compared to pH ≥ 7.5, with the highest
reactivity being found at pH 8.5, in line with the pKa of cysteine
thiols (~8.0). Logically, reactivity decreased upon diminishing the
reaction medium temperature (entry 8). While clean native mass
spectra were obtained for all these experiments, side reactivity
was still observed under these conditions on the non-reduced
antibody. Therefore, we cannot definitely rule out side reactions
on reduced trastuzumab (16), even though it could be outpaced
by cysteine modification. In order to find out conditions at which
6a would not react with the non-reduced antibody, different
incubation times and temperatures were screened again. With 8
equivalents of 6a in PBS buffer at pH 7.5, intact trastuzumab (16)
could still be obtained after 2 minutes at 25 °C, and up to 5
in a highly chemoselective manner (Figure 1C). Having
demonstrated the utility of TMS-EBX in vitro, we evaluated the in
situ reactivity of the three best reagents in living HeLa cells. Cells
were treated with the reagents at 3 or 10 µM concentration for 1.5
hours, without any noticeable toxicity – greater than 93 percent
remaining viability was observed by imaging and by quantification
with the WST1 absorbance assay (Figure S3). The cells were
then collected, lysed, subjected to CuAAC-mediated installation
of the TAMRA dye, and the probe-labeled proteins were
visualized by fluorescence in-gel scanning. Interestingly, the
probe JW-RT-03 (6d) exhibited the strongest labeling in situ, likely
due to the stronger hydrophobicity of this probe enhancing the
minutes at
4 °C. Employing reduced trastuzumab, these
conditions yielded an average DoC value of 1.2 with a conversion
up to ~60% in just 2 minutes at 25 °C (entry 9). At 4 °C, only very
low conversion was observed (Entry 10). Applied to the 4-F
derivative JW-RF-02 (6c), similar results were obtained, in
coherence with its reactivity comparable to the one of the
unsubstituted parent compound 6a (see Supporting Information
Table S4 for detailed results).
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cellular permeability (Figure 1D). This result further demonstrated
the importance of having a library of fine-tuned EBX reagents.
Figure 2. Proteomic chemoselectivity and reactivity of JW-RT-03 (6d) using LC-
Figure 1. Gel-based proteomic evaluation of TMS-EBXs reactivity. A. HeLa cell
lysate in PBS (pH 7.4) was treated with the TMS-EBX reagents before CuAAC
installation of the TAMRA dye and in-gel SDS-PAGE fluorescence scanning. B.
Fluorescence image of HeLa lysate treated with 10 µM TMS-EBX reagents. C.
IAA (30 mM) competed the labeling of HeLa lysate by JW-RT-02(6c). D. JW-
RT-03 (6d) exhibited the strongest in situ labeling using live HeLa cells after 1.5
h of treatment.
MS/MS analysis. A. MS-based capture-and-release strategy using a
photocleavable biotin linker to identify peptides modified by reagent 6d. B.
Chemoselectivity of 6d towards nucleophilic amino acids in HeLa lysate (n = 6).
Error bars are S.E.M.
In comparison, we previously only found 2257 and 2184 peptides
respectively enriched by 10 µM JW-RF-010 (4) and IAA alkyne
from HeLa. Therefore, JW-RT-03 (6d) exhibits a greater coverage
of reactive cysteines in the proteome than either of these probes.
JW-RT-03 (6d) also exhibited good chemoselectivity towards
cysteines (90.2%) relative to other nucleophilic amino acids (Asp,
Glu, His, Lys, Ser, Thr, Tyr, Arg), as determined by searching for
the corresponding mass adducts on all of these residues using
the Sequest HT algorithm (Figure 2B).[31] Although arginine and
tyrosine reacted with JW-RT-01 (6b) to some extent on
tetrapeptides, minimal reactivity towards arginine (0.3%) and
tyrosine (1.49%) was observed with JW-RT-03 (6d) throughout
the proteome. Taken together, these results demonstrate that
JW-RT-03 (6d) has greater coverage of reactive proteomic
cysteines than JW-RF-010 or IAA alkyne, the current gold
standard in the cysteinomics field, while maintaining high
selectivity towards cysteines relative to other nucleophilic amino
Having identified the most reactive probe in situ using gel-based
proteomic techniques, we evaluated the chemoselectivity and
cysteinome coverage of 6d using mass spectrometry (MS)-based
analysis. Using our established catch-and-release enrichment
method,[19] HeLa lysates were treated with 10 µM of JW-RT-03
(
6d) for one hour, followed by CuAAC-mediated conjugation to
photocleavable biotin linker 17, enrichment using streptavidin
beads, tryptic digestion, release of probe-bound peptides, with UV
irradiation, and LC-MS/MS analysis (Figure 2A). In total, 4325
cysteine-containing peptides were found to be labeled by JW-RT-
03 (6d).
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acids. Therefore, the TMS-EBX reagents offer tremendous
potential as broad profile cysteine-reactive probes in
chemoproteomics.
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J. W. thanks ERC (European Research Council, Starting Grant
iTools4MC, number 334840 and Consolidator Grant SeleCHEM,
number 771170) and EPFL for financial support. C. S thanks
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Keywords: bioconjugation • cysteine ethynylation • hypervalent
iodine • antibody functionalization • cysteine proteomic
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RESEARCH ARTICLE
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RESEARCH ARTICLE
10.1002/anie.202002626
Romain Tessier, Raj Kumar Nandi,
Brendan G. Dwyer, Daniel Abegg,
Charlotte Sornay, Javier Ceballos,
Stéphane Erb, Sarah Cianferani, Alain
Wagner, Guilhem Chaubet,*Alexander
Adibekian,** and Jerome Waser***
It always reaches its target! A direct ethynylation method for cysteines using
hypervalent iodine reagents is reported. The reaction proceeds on peptides and
antibodies, and can also be used for cysteine proteomic profiling in the living cell,
displaying an improved coverage of the cysteinome compared to previously
reported probes.
Page No. – Page No.
Ethynylation of Cysteines from
Peptides to Proteins in Living Cells
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