“Doubly Orthogonal” Labeling of Peptides and Proteins

Article abstract of DOI:10.1016/j.chempr.2019.06.022

Herein, we report a cysteine bioconjugation methodology for the introduction of hypervalent iodine compounds onto biomolecules. Ethynylbenziodoxolones (EBXs) engage thiols in small organic molecules and cysteine-containing peptides and proteins in a fast and selective addition onto the alkynyl triple bond, resulting in stable vinylbenziodoxolone hypervalent iodine conjugates. The conjugation occurs at room temperature in an open flask under physiological conditions. The use of an azide-bearing EBX reagent enables a “doubly orthogonal” functionalization of the bioconjugate via strain-release-driven cycloaddition and Suzuki-Miyaura cross-coupling of the vinyl hypervalent iodine bond. We successfully applied the methodology on relevant and complex biomolecules, such as histone proteins. Through single-molecule experiments, we illustrated the potential of this doubly reactive bioconjugate by introducing a triplet-state quencher close to a fluorophore, which extended its lifetime by suppressing photobleaching. This work is therefore expected to find broad applications for peptide and protein functionalization. Understanding the molecular basis of life is essential in the search for new medicines. Chemical biology develops molecular tools for studying biological processes, setting the basis for new diagnostics and therapeutics, and relies heavily on the ability to selectively modify biomolecules. Two approaches have been especially fruitful: (1) selective modification of natural biomolecules and (2) selective reaction between non-natural functionalities in the presence of biomolecules (the so-called orthogonal bioconjugation). In our work, we contribute to both by transferring highly reactive hypervalent iodine reagents to cysteine residues in proteins and peptides. The obtained bioconjugates retain the reactive hypervalent bonds, which can be selectively functionalized via a metal-mediated reaction. Combined with a traditional azide tag, our approach allows a doubly orthogonal functionalization of biomolecules and is hence expected to be highly useful in chemical biology. Chemical biology develops molecular tools for studying biological processes, setting the basis for new diagnostics and therapeutics, and relies heavily on the ability to modify selectively biomolecules. In our work, we introduce hypervalent iodine bonds into peptides and proteins, via functionalization of cysteine, by using unique cyclic reagents developed in our group. The hypervalent bond can then be selectively modified in the presence of both natural and synthetic functional groups, opening new opportunities for applications in chemical biology.

Full text of DOI:10.1016/j.chempr.2019.06.022

Article
‘‘Doubly Orthogonal’’ Labeling of Peptides
and Proteins
Romain Tessier, Javier Ceballos,
Nora Guidotti, Raphael
Simonet-Davin, Beat Fierz,
Jerome Waser
beat.fierz@epfl.ch (B.F.)
jerome.waser@epfl.ch (J.W.)
HIGHLIGHTS
Selective cysteine bioconjugation
under physiological conditions
Formation of hypervalent iodine-
bound peptides and proteins
Stable bioconjugates from simple
amino acids to histones
Doubly orthogonal
functionalization via azide-alkyne
cycloaddition and cross-coupling
Chemical biology develops molecular tools for studying biological processes,
setting the basis for new diagnostics and therapeutics, and relies heavily on the
ability to modify selectively biomolecules. In our work, we introduce hypervalent
iodine bonds into peptides and proteins, via functionalization of cysteine, by using
unique cyclic reagents developed in our group. The hypervalent bond can then be
selectively modified in the presence of both natural and synthetic functional
groups, opening new opportunities for applications in chemical biology.
Tessier et al., Chem 5, 1–21
August 8, 2019 ª 2019 Published by Elsevier
Inc.
https://doi.org/10.1016/j.chempr.2019.06.022

Please cite this article in press as: Tessier et al., ‘‘Doubly Orthogonal’’ Labeling of Peptides and Proteins, Chem (2019), https://doi.org/10.1016/
j.chempr.2019.06.022
Article
‘‘Doubly Orthogonal’’ Labeling
of Peptides and Proteins
Romain Tessier,1 Javier Ceballos,1,3 Nora Guidotti,2,3 Raphael Simonet-Davin,1 Beat Fierz,2,
*
and Jerome Waser1,4,
*
SUMMARY
The Bigger Picture
Understanding the molecular
basis of life is essential in the
search for new medicines.
Herein, we report a cysteine bioconjugation methodology for the introduction
of hypervalent iodine compounds onto biomolecules. Ethynylbenziodoxolones
(EBXs) engage thiols in small organic molecules and cysteine-containing pep-
tides and proteins in a fast and selective addition onto the alkynyl triple bond,
resulting in stable vinylbenziodoxolone hypervalent iodine conjugates. The
conjugation occurs at room temperature in an open flask under physiological
conditions. The use of an azide-bearing EBX reagent enables a ‘‘doubly orthog-
onal’’ functionalization of the bioconjugate via strain-release-driven cycloaddi-
tion and Suzuki-Miyaura cross-coupling of the vinyl hypervalent iodine bond.
We successfully applied the methodology on relevant and complex biomole-
cules, such as histone proteins. Through single-molecule experiments, we illus-
trated the potential of this doubly reactive bioconjugate by introducing a
triplet-state quencher close to a fluorophore, which extended its lifetime by
suppressing photobleaching. This work is therefore expected to find broad ap-
plications for peptide and protein functionalization.
Chemical biology develops
molecular tools for studying
biological processes, setting the
basis for new diagnostics and
therapeutics, and relies heavily on
the ability to selectively modify
biomolecules. Two approaches
have been especially fruitful: (1)
selective modification of natural
biomolecules and (2) selective
reaction between non-natural
functionalities in the presence of
biomolecules (the so-called
orthogonal bioconjugation).
INTRODUCTION
Efficient, selective, and flexible labeling techniques are essential for the study and
manipulation of proteins.1 Particularly in the last decade, the importance of site-se-
lective modification has been highlighted in the quest for antibody-drug conju-
gates2–6 and further emphasized by the development of stapled peptides for thera-
peutic interventions.7–11 Novel bioconjugation techniques are still in high demand,
but their development is complicated by the need for fast kinetics, high efficiency,
and excellent selectivity under mild conditions. Nevertheless, several labeling
methods exploiting both natural and unnatural amino acids have emerged.12–17
In our work, we contribute to both
by transferring highly reactive
hypervalent iodine reagents to
cysteine residues in proteins and
peptides. The obtained
bioconjugates retain the reactive
hypervalent bonds, which can be
selectively functionalized via a
metal-mediated reaction.
Because of its key role in the structure and catalytic activity of proteins, cysteine has
been one of the most studied natural amino acids in chemical biology. Its low abun-
dance in proteins and its intrinsic high nucleophilicity make it an ideal target for che-
moselective modification techniques often based on site-directed mutagenesis
(Scheme 1).18–21 Accordingly, significant efforts have been made on the develop-
ment of reagents enabling cysteine labeling with high selectivity and efficiency.
Among those, alkylating halogenoalkanes, such as iodoacetamide, and maleimide
derivatives are the most frequently employed. Although their reactivity has been
extensively studied, significant limitations persist. For example, a lack of chemose-
lectivity is observed in the presence of nucleophilic amino acids, such as lysine, his-
tidine, tyrosine, or tryptophan.12,14,17 In the case of the widely used maleimides, a
critical disadvantage is the well-known instability of their conjugates toward hydro-
lysis and external organosulfur attacks. This is mainly due to the reversibility of the
Combined with a traditional azide
tag, our approach allows a doubly
orthogonal functionalization of
biomolecules and is hence
expected to be highly useful in
chemical biology.
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Recent progress
Most often used
Halogeno-
Thiol-ene and
-yne click26-29
S
S
R
S
S
alkanes18-21
R
O
Metal-free
SH
arylation32-34
Maleimides18-21
Fn
NR
O
Metal-assisted
arylation35-43
Oxidative
S
elimination19
R
CHALLENGES: • Selectivity • In water • Efficiency • Bioconjugate Stability
Scheme 1. State of the Art and Challenges in Cysteine Functionalization
thiol-maleimide reaction, which leads to loss of label.22–25 Therefore, labeling
methods leading to more stable adducts, including thiol-ene and -yne reac-
tions,26–29 oxidative elimination of cysteine to dehydroalanine followed by Michael
addition,19,30,31 metal-free arylations,32–34 or metal-assisted functionalizations,35–43
have been developed. Nevertheless, many of these methods suffer from low or un-
controlled reactivity, low chemoselectivity, or the need for organic co-solvents. Bet-
ter performances can be found with the use of metal catalysts and reagents;35–43
however, solubility and biocompatibility remain predominant challenges of metal-
based methodologies.44 Finally, many of these methods are based on chemical
compounds requiring challenging preparations, careful storage, and/or stringent
use of inert atmosphere.
In this context, hypervalent iodine reagents are interesting because they combine
high reactivity with sufficient stability and low toxicity.45 Nevertheless, applications
for peptide and protein functionalization remain rare, probably as a result of concur-
ring oxidation side reactions.46–53 In 2018, Gaunt, Suero, and co-workers reported
an elegant methionine functionalization using a hypervalent iodine reagent.53 Our
group previously achieved a highly efficient and chemoselective alkynylation of
thiols by using hypervalent iodine-based ethynylbenziodoxolone54–60 (EBX) re-
agents (1) in organic solvents, which was also successful for amino acids and dipep-
tides (Scheme 2A1).61,62 In collaboration with the Adibekian group, we successfully
applied JW-RF-010 (1a), an azide-bearing EBX, in the labeling of native cysteines
in vitro and in vivo to afford alkynes as major products (Scheme 2A2).63 1a displayed
an exceptional stability with less than 3% decomposition after 14 days in D2O, as well
as an exceptional chemoselectivity, outperforming iodoacetamide, the gold stan-
dard in cysteine targeting. However, only hyper-reactive cysteines were efficiently
functionalized in aqueous media, and the method is therefore not general. Common
to all reported approaches for biomolecule functionalization with hypervalent iodine
reagents is using the inherent reactivity of the hypervalent bond to perform the bio-
conjugation step.
1Laboratory of Catalysis and Organic Synthesis,
Institute of Chemical Sciences and Engineering,
´
Ecole Polytechnique Fe´ de´ rale de Lausanne, EPFL
SB ISIC LCSO, 1015 Lausanne, Switzerland
2Laboratory of Biophysical Chemistry of
Macromolecules, Institute of Chemical Sciences
´
and Engineering, Ecole Polytechnique Fe´ de´ rale
de Lausanne, EPFL SB ISIC LCBM, 1015
Lausanne, Switzerland
3These authors contributed equally
4Lead Contact
In contrast, we present herein a highly efficient, fast, chemoselective, and clean la-
beling of cysteine-containing peptides and proteins to give stable adducts of EBX
and thiols without cleaving the hypervalent bond (Scheme 2B). This methodology
*Correspondence: beat.fierz@epfl.ch (B.F.),
jerome.waser@epfl.ch (J.W.)
https://doi.org/10.1016/j.chempr.2019.06.022
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A Previous Work: Hypervalent bond REACTS during functionalization
A1) Thioalkynylation in organic solvents61-62
O
I
R1
Mild and fast
Functional group tolerant
X Small molecules up to dipeptides
X Only in organic solvents
O
EBX (1)
R
S
Base, THF
R
SH
23°C, <5 min
R1
45-99%
A2) Intracellular application (limited to hyper-reactive cysteines)63
O
I
N3
O
JW-RF-010 (1a)
SH*
S
PBS pH 7.4
23 °C, 4 h
N3
CuAAC
Proteomic target profiling
(*): Hyper-reactive
cysteine
Physiological conditions
On proteins
X Only hyper-reactive cysteines
B This Work: Hypervalent bond CONSERVED during functionalization
O
I
R
R
O
S
EBX (1)
SH
I
"Open-flask"
r.t., 5 min
O
Efficient and robust
labeling
O
2 bioorthogonal handles!
R1
One-pot labeling &
cross-coupling
R
(HO)2B
S
L2Pd(OAc)2
"Open-flask"
37 °C, 30 min
Bioorthogonal cross-coupling
R1
Fast under physiological conditions
On small molecules, peptides and proteins
Reactive hypervalent bond conserved
Double orthogonality enabled
Scheme 2. Reaction of Cysteine with EBX Reagents
Previous work on thiol functionalization with reaction of the hypervalent bond (A) and approach
conserving the hypervalent iodine bond presented in this work, enabling doubly orthogonal
functionalization (B).
can be applied to all cysteine- and other thiol-bearing compounds, including modi-
fied ubiquitin and histone proteins. The peptide- and protein-bound vinyl benzio-
doxolone reagents are stable, yet their inherent reactivity59,64–66 opens the door
for bioconjugations orthogonal to natural functional groups existing in biomole-
cules. As a proof of concept, we report a palladium-catalyzed Suzuki-Miyaura
cross-coupling reaction67–71 selective at the vinyl hypervalent iodine bond
(Scheme 2B).
Under physiological conditions, this efficient, robust, and fast methodology allows
swift peptide and protein modifications without the need for incorporation of
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A
NH2
NH2
O
NH2
O
S
H
N
H
N
HO
OH
HO
OH
1a
N
H
N
H
pH = 8.2
buffer
O
I
O
O
O
O
O
SH
O
O
2
3a
I
O
N3
H
HO
N
OH
H
N
H
O
O
O
O
S
N3
4
O
JW-RF-010 (1a)
not observed
N3
4
In organic solvent
-elimination
B
a
and S-1,2-shift
R
R
I
S
S
O
I
O
O
N3
1a
RSH
N3
O
II
-addition
Vinylic carbanion
intermediate
I
protonation
In water
b
3a
Scheme 3. Reaction of EBX 1a with Glutathione (2)
Formation of vinylbenziodoxolone 3a (A) and speculative reaction mechanism (B).
unnatural amino acids. In addition, we combined this powerful methodology with
metal-free strain-promoted alkyne-azide cycloaddition (SPAAC),72–74 which allowed
functionalization of the adduct without cleaving the hypervalent bond, leading to
‘‘doubly orthogonal’’ modifications. We demonstrated this method’s potential by
introducing both a fluorophore and a triplet-state quencher (TSQ) on neuropeptide
substance P, enabling single-molecule fluorescence experiments with increased
sensitivity.
RESULTS AND DISCUSSION
Discovery and Optimization of the Hypervalent Iodine Transfer Reaction
Our study started with the functionalization of the thiol-containing glutathione
(GSH, 2) as a model substrate. 2 is a naturally occurring tripeptide that plays an
essential role in primary metabolism as a disulfide-bond reductant and is present
in high concentrations in cells. It was therefore surprising that alkynylation of 2
was not observed in cell lysate or living cells.63 At that time, this was attributed to
the lower acidity of 2 than of hyper-reactive cysteines. When 2 was treated with
equimolar amounts of 1a in a more basic buffer (10 mM Tris [pH 8.2]), vinylbenzio-
doxolone (VBX) 3a was obtained instead of the expected alkynyl sulfide 4
(Scheme 3A). Our previous computational work on the mechanism of the thio-alky-
nylation reaction demonstrated that concerted b addition of sulfide via transition
state I is favored for EBX reagents bearing an alkyl substituent (Scheme 3B).75
With fully deprotonated sulfides in organic solvents, the formed vinylic carbanion
intermediate II undergoes rapid a elimination and 1,2-sulfur shift to give alkynyl sul-
fides (Scheme 3B, route a). However, under the new conditions in water solution,
direct protonation of II is favored to give VBX 3a (Scheme 3B, route b).
4
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Indeed, we recently demonstrated that nitrogen and phenol nucleophiles can also
be added to EBX reagents to give VBX compounds, and computational studies
showed a very low barrier for protonation.76 Solvent polarity also plays a key role,
and in the case of thiol nucleophiles, increasing amounts of alkynylation were
observed when DMSO was added to the reaction mixture both with a protected
amino acid and with a tetrapeptide (see Figures S4 and S5). Mixtures of alkynes
and VBXs were observed when water was added to organic solvents. Although
our previous work on cell lysates and living cells were performed in aqueous buffer,
the targeted cysteines were in protein active sites, whose properties are very
different from those of the bulk solution. The lower dielectricity constant observed
inside proteins77 indicates that the local polarity is closer to that of organic solvents.
2D NMR spectroscopic studies established the Z configuration of the olefin, as well
as the b addition regioselectivity, in agreement with our previous density functional
theory calculations.75 The developed transformation corresponds to the occurrence
of a formal trans-addition of the thiol on the triple bond of the EBX reagent with per-
fect regio- and stereoselectivity, which is difficult to achieve for standard thiol-yne
reactions.26–28 Furthermore, the adduct is obtained without the need for further re-
agents, such as radical initiators, and without any byproduct formation.
To evaluate the potential of VBX for applications in chemical biology, it was first
important to study the stability of adduct 3a. No degradation of 3a was observed
up to 3 weeks at room temperature when it was kept as a solution in DMSO-d6.
Furthermore, 3a exhibited a surprising resistance to high temperatures with no
detectable degradation when being heated up to 100ꢀC in pure water. Concerning
pH stability, 3a remained intact in an acetic acid buffer (10 mM [pH 4.0]) over 3 days.
Only a slight degradation was observed in a basic CAPS buffer (10 mM [pH 11.0],
86% of remaining product after 3 days). Interestingly, no degradation, further addi-
tion, or exchange reaction was observed in the presence of 15 equiv of an external
thiol nucleophile (30 mM tiopronin, 63 higher than the standard glutathione con-
centration in a cell) after 8 days. Finally, whereas the presence of a further 10 equiv
of 1a resulted in 14% degradation after 4 days, 5 equiv of 1a did not lead to any
decomposition.
Once the high stability of VBX 3a had been confirmed, we started to evaluate the
robustness of our labeling method (for a complete list of screened conditions, see
Supplemental Experimental Procedures Section 6). The reaction is user friendly
and easy to set up. 2 was simply dissolved in non-degassed Tris buffer (10 mM
[pH 8.2]), and then 1a was added from a DMSO stock solution. Then, the reaction
was vortexed for a few seconds or homogenized with a simple pipette mixing and
left to stand on the bench for 20 min. Unshaken and at room temperature, the reac-
tion furnished a remarkable 81% yield of the desired product without the need for
oxygen exclusion (Table 1, entry 1). 5 min of incubation already afforded the product
in 55% yield (entry 2), and an extended labeling time of 60 min increased the yield to
87% (entry 3). The only side reactivity observed was oxidation of 2 into the dimeric
disulfide. Interestingly, the absence of DMSO did not yield any significant loss of ef-
ficiency (entry 4), which could prove essential in the labeling of organic solvent-sen-
sitive proteins. The reaction exhibited high tolerance toward buffer molarity (entry 5)
and types of buffer (entries 6 and 7). The pH was found to be a crucial parameter for
the reaction. At physiological pH (7.4), labeling was slowed down but still efficient
(74% after 1 h, entry 8). At pH 6.5, only 29% of 3a was obtained after 1 h (entry 9).
Nevertheless, this low reactivity could be easily overcome with the use of 10 equiv
of 1a (entry 10). To our delight, the labeling remained efficient at either lower or
higher temperatures (entries 11 and 12). Although diluting the reaction mixture
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Table 1. Evaluation of Reaction Conditions for the Labeling of Glutathione (2)
NH2
O
H
N
HO
OH
N
H
1a (1.2 equiv)
O
O
O
2
S
O
3a
10 mM Tris pH 8.2
(2% v/v DMSO)
r.t., 20 min
I
O
N3
Entry
1
Variations from the Above Conditions
none
Yield
81%
2
5 min reaction
55%
3
1 h reaction
87%
4
no DMSO
78% (86%)
78% (97%)
77% (85%)
75% (91%)
54% (74%)
9% (29%)
57% (88%)
67% (86%)
78% (87%)
45% (76%)
76% (93%)
98%
5
100 mM Tris (pH 8.2)
10 mM HEPES (pH 8.2)
10 mM PBS (pH 8.2)
10 mM PBS (pH 7.4)
10 mM PBS (pH 6.5)
10 mM PBS (pH 6.5)
4ꢀC reaction temperature
37ꢀC reaction temperature
200 mM reaction molarity
200 mM reaction molarity
3 equiv of 1a
6
7
8
9
10a
11
12
13
14b
15c
Labeling conditions: 1.0 mmol glutathione (2) and 1.2 equiv of JW-RF-010 (1a) in 0.5 mL of non-degassed
Tris buffer (pH 8.2; 2% v/v DMSO) at room temperature for 20 min. Calibrated HPLC yield is based on
absorbance at 214 nm (see Figure S1). The yields in parentheses correspond to the yields after 1 h of re-
action. For complete robustness studies, see Supplemental Experimental Procedures Section 6.
a10 equiv of 1a was used.
b3 equiv of 1a was used.
cThe reaction was analyzed after 5 min.
from 2 mM to 200 mM increased the reaction time, it barely affected the labeling
efficiency (entry 13). Furthermore, increasing the stoichiometry of 1a to 3 equiv
allowed for efficient labeling in 20 min and almost quantitative transformation in
1 h (entry 14) (for the complete list of screened conditions, see Table S1). Under
these conditions, reactivity could be observed down to 2 mM reaction molarity
(see Figures S2 and S3). A competition experiment between 1a and iodoacetamide
at pH 8.2 resulted in a 2:3 ratio of labeled products, showing that the two com-
pounds reacted at similar rates. Finally, the use of 3 equiv of 1a afforded quantitative
functionalization of 2 within 5 min at 2 mM (entry 15). We selected these conditions
to study the scope of the reaction.
Scope of the Bioconjugation Reaction
We therefore prepared a library of hypervalent iodine reagents, resulting in com-
pounds bearing azide, alkyne, alcohol, and various other functional groups.62,78
Most of these EBX compounds can be prepared in one synthetic step from commer-
cial reagents and are stable toward air, light, and water. They can be stored on the
bench under ambient atmosphere for weeks or at 4ꢀC under nitrogen for years
6
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NH2
O
O
I
R
H
N
HO
OH
N
H
3.0 equiv
O
1
O
O
O
S
O
2
I
O
10 mM Tris pH 8.2
(2% v/v DMSO)
r.t., 5 min
R
3
=
O
N3
R
3a, 98% (95%)
3b, 86% (80%)
3c, 84% (76%)
3f, 96% (86%)
Cl
HO
Me
3d, 99% (85%)
3e, 93% (92%)
3h, 89% (49%)
Me
tBu
3g, 89% (82%)
3i, 43% (37%)
C14H29
TIPS
3k, No conversion
3l, No conversion
3j, 28%
Scheme 4. Scope of Different EBX Reagents for Glutathione (2) Labeling
Reactions were carried out on a 1.00 mmol scale at a 2 mM concentration (see Supplemental
Experimental Procedures Section 8). Yields were derived by comparison of the integration area of
the product in its respective HPLC trace with that of the standard curve at 214 nm for 3a (see
Figure S1). Yields in parentheses represent those of the labeling reactions without DMSO as an
organic co-solvent.
without exhibiting noticeable degradation. Our bioconjugation technique demon-
strated a broad tolerance toward a large variety of functional groups (Scheme 4).
Indeed, azide (3a), terminal alkyne (3b) and alkene (3c), halogen (3d), alcohol (3e),
and non-functionalized alkyl substituents (3f–3h) could be rapidly attached to 2 in
excellent yields. To our delight, these reactions were also highly efficient without
DMSO as an organic co-solvent. Because of their lipophilicity, solubility issues
were observed with tBu-EBX (1i), Ph-EBX (1j), C14H29-EBX (1k), and TIPS-EBX (1l), re-
sulting in low yields or no conversion for products 3i–3l.
The method could not be used with non-polar small organic molecules because of
their low solubility in water. However, in such cases the reagents could be accessed
in good yields under the conditions recently developed for N- and O-nucleophiles
(catalytic cesium carbonate in ethanol; Scheme 5).76 Phenyl and benzyl thiols (5
and 6) and protected cysteine (7) were added smoothly to reagent 1a to give 8a,
9a, and 10, respectively. X-ray analysis of reagents 8a, 9a, and 9b confirmed the hy-
pervalent structure, as well as the regio- and stereoselectivity of the thiol addition
(see Data and Code Availability). The addition to other EBX reagents bearing a
methyl group, a longer alkyl chain, or a chlorine functional group was also successful
(products 8b–8d and 9b–9d).
We then investigated the scope of thiol reagents with 1a under physiological condi-
tions (Scheme 6). Starting with small molecules, we were pleased to observe quan-
titative labeling with tiopronin (11), 6-thioguanine (12), and thio-b-glucose (13)
(Scheme 6A). We then extended the scope to the use of tetrapeptides containing
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O
I
R2
R1
R
3.0 equiv
O
S
O
1
I
O
R1
SH
10 mol% Cs2CO3
Ethanol, r.t., 60 min
5-7
8-10
AcHN
N3
CO2Me
S
Bn
Ph
S
S
O
O
O
I
O
I
O
I
O
N3
N3
8a, 56%
9a, 47%
10, 41%
Ph
Ph
Bn
Ph
S
S
S
S
S
O
O
O
O
O
I
O
O
Me
I
O
Cl
Cl
I
O
O
Me
5
5
3
3
8b, 64%
8c, 68%
8d, 79%
Bn
Bn
S
O
I
Me
I
O
I
Me
9b, 46%
9c, 56%
9d, 73%
Scheme 5. Scope of Different EBX Reagents with Non-polar Small Organic Molecules in Ethanol
Reactions were carried out on a 0.50–11 mmol scale. Isolated yields after recrystallization or column
chromatography are reported (see Supplemental Experimental Procedures Section 9a).
various natural amino acids (Scheme 6B). Cysteines in close proximity to phenylala-
nine (14a) or proline (14b) exhibited excellent reactivity. A more hindered environ-
ment around the cysteine residue with phenylalanine, valine, and isoleucine amino
acids (14c and 14d) did not show any detrimental effect on the labeling.
Whereas C-terminal cysteine was found to be more prone to oxidation, affording a
lower efficiency (14e), N-terminal cysteine reacted efficiently (14f). Aspartic acid
(14 g), asparagine (14h), and methionine (14i) were well tolerated. Only in the case
of arginine (14j) was a lower yield obtained as a result of the formation of side
products. High chemoselectivity was also observed in the presence of serine (14k),
tyrosine (14l), and tryptophan (14 m). No side reactions occurred with the most
nucleophilic amino acids histidine (14n) and lysine (14o). Finally, efficient labeling
was still observed without DMSO for peptides 14b, 14e, 14h, and 14l.
These promising results on tetramers prompted us to apply our labeling technique
to more complex biomolecules. After treatment with 1a, human serum albumin
Leu55-His63 fragment (15) was converted to the corresponding hypervalent iodine
bioconjugate 16 in 88% yield (Scheme 6C). We then decided to employ our method
to modify cysteines in a protein context. As a model system, we chose the protein
ubiquitin, which carries an N-terminal hexahistidine tag followed by a single cysteine
residue (His6-Cys ubiquitin, 17). To our delight, the treatment of 17 with only 2 equiv
of 1a under native conditions afforded a quantitative transformation into VBX 18 in
8
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A a
OH
R
S
H
N
O
O
H2N
CO2H
N
3.0 equiv 1a
O
R
I
O
Me
HO
HO
SH
N3
N
H
SH
10 mM Tris pH 8.2 (2% v/v DMSO)
r.t., 5 min
N
N
OH
SH
12, 100%
11, 95%
13, 100%
SH
B a
SH
SH
SH
SH
O
O
O
O
H2N
H2N
Cys
Ala Cys Phe Ala
Ala
Pro Ala
H2N
H2N
Ile Cys
Cys
Phe
Ala
Ala
Val
Val
NH2
NH2
NH2
NH2
14a, 97%
14b, 97% (98%)
Me
Me
14c, 95%
Me
Me
14d, 99%
Me
O
NH2
Me
Me
SH
Me
SH
SH
O
O
O
O
H2N
Cys
Me
H2N
Ala
O
Cys Ile
Glu
Ala Gln Leu
H2N
Cys Asp
H2N
Ala
Ala
O
Ala Cys Asn Ala
O
NH2
NH2
NH2
NH2
SH
14f, 92%
OH
14g, 96%
14h, 98% (96%)
14e, 79% (69%)
Me
OH
NH2
SH
SH
SH
SH
O
O
O
O
H2N
Cys
Ala
Ala Cys
Ala
H2N
Ala Cys
Tyr Ala
Ala
Met
H2N
H2N
Ala Cys Arg Ala
NH
Ser
NH2
NH2
NH2
NH2
OH
14i, 97%
SH
14j, 84%
14k, 96%
SH
14l, 95% (95%)
H2N
N
H
S
SH
Me
OH
O
O
O
Ala
His
Ala
Cys
Ala
H2N
Lys
H2N
Cys Trp
H2N
Cys
Ala
Ala
Ala
NH2
NH2
NH2
NH2
N
14m, 99%
14n, 92%
14o, 99%
NH
NH
C b,c
N3
O
I
SH
O
I
N3
O
S
H2N
O
Leu Gln
Gln
0.91 equiv
Cys
Phe
1a
H2N
Leu Gln
Gln
Glu
Cys
Phe
Pro
15
Glu
10 mM Tris pH 8.2 (2% v/v DMSO)
r.t., 60 min
HO
O
16, 88%
Pro
Asp
His
HO
O
His Asp
D c,d
O
I
N3
SH
N3
O
I
O
O
2.0 equiv
1a
S
10 mM Tris pH 8.2 (2% v/v DMSO)
r.t., 60 min
His6-Cys-Ub (17)
18, 99%
Scheme 6. Reaction with Peptides and Proteins
Scope of the functionalization of small molecules (A) and tetrapeptides (B) and applications on human serum albumin Leu55-His63 sequence (15) (C) and
His6-Cys-ubiquitin (17) (D).
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Scheme 6. Continued
aReactions were carried out on a 1.00 mmol scale at a 2 mM concentration. Yields were determined by relative integration based on HPLC-MS (see
Supplemental Experimental Procedures Section 9b). Yields in parentheses represent the labeling reactions without DMSO.
bReaction was carried out on a 1.30 mmol scale at 3 mM concentration. EBX (1a) was used substoichiometrically for ease of chromatogram analysis.
cYields were determined by relative integration based on HPLC traces at 214 nm (see Supplemental Experimental Procedures Section 9b).
dReaction was carried out on a 50.0 nmol scale at a 0.5 mM concentration.
less than 1 h (Scheme 6D). To the best of our knowledge, this constitutes the first
example of a protein-bound hypervalent iodine reagent. This efficient conjugation
was also compatible with a protein-denaturing buffer (see Supplemental Experi-
mental Procedures Section 9e). Of note, side reactions due to arginine were not
observed in ubiquitin, which contains five arginine residues. We thus conclude
that such side reactions, as observed for 14j, occur only when arginines are immedi-
ately next to the cysteine. Importantly, no reaction was observed with native
(cysteine-free) ubiquitin (see Supplemental Experimental Procedures Section 9f),
further demonstrating the cysteine specificity of the reaction in a protein context.
Moreover, the correct modification site in ubiquitin was further confirmed by top-
down mass spectrometry (see Supplemental Experimental Procedures Section 9e).
It should be noted that because the reaction kinetics are dependent on the reagent
concentrations, for larger proteins an increased concentration of EBX reagent might
be required.
In nature, cysteine residues are known to form disulfide bonds, particularly in
secreted, extracellular proteins. When these disulfides are targeted with cysteine la-
beling reagents, the standard protocol is an in situ disulfide cleavage with reducing
reagents, such as tris(2-carboxyethyl)phosphine (TCEP). Therefore, a one-pot proto-
col allowing disulfide bond cleavage followed by reaction with EBX reagents would
be highly useful for the labeling of disulfide-bound cysteines. As a proof of concept,
we treated oxidized 2 with 1.5 equiv of TCEP followed by 6 equiv (3 equiv per reduced
cysteine) of 1a. To our delight, the product 3a was obtained with 92% yield (see Sup-
plemental Experimental Procedures Section 10a). Furthermore, even in the presence
of 5 and 10 equiv of the reducing agent and no increase in the amount of 1a, the prod-
uct was still obtained in good yields (82% and 60%, respectively). Although a direct
reaction between TCEP (reducing) and the EBX reagent (oxidizing) did take place,
it was slower than the labeling reaction. Importantly, the hypervalent iodine conju-
gate 3a, which still possesses reactive iodine and azide groups, did not show any
degradation after 24 h in the presence of TCEP. With these exciting results in
hand, we applied our one-pot reduction-labeling technique to larger peptides con-
taining disulfide bridges. We were pleased to see excellent yields on challenging nat-
ural bioactive peptides, such as oxytocin (19) and somatostatin (20) (products 21 and
22; Scheme 7) (see Supplemental Experimental Procedures Sections 10b and 10c).
Application for Fluorescent Protein Labeling
Next, we investigated the potential of the obtained bioconjugates for further func-
tionalization. At first, we examined whether standard methods for azide modification
could be used in the presence of the reactive hypervalent iodine center. In order to
avoid the use of metal catalysts, we chose the well-known copper-free SPAAC.72–74
In principle, two strategies could be followed: (1) labeling of the peptide or protein
with 1a followed by cycloaddition with a functionalized alkyne or (2) prior reaction of
1a with the alkyne followed by reaction with the peptide or protein. The former ap-
peared easier given that the reactivity of the hypervalent iodine of the VBX products
is lower, whereas the latter is attractive for applications because it would allow prep-
aration of multi-functionalized labeling reagents in situ starting from 1a and
commercially available alkynes. As a model system, we again chose 2 together
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Scheme 7. Labeling of Sulfur-Bridge-Containing Peptides
Conditions: 1.5 equiv of TCEP$HCl and 100 mM Tris (pH 8.2) at room temperature for 60 min; then
6.0 equiv of 1a and 100 mM Tris (pH 8.2; 2% v/v DMSO) at room temperature for 5 or 10 min. Yields
were determined by relative integration based on HPLC traces at 214 nm (see Supplemental
Experimental Procedures Sections 10b and 10c).
aReaction was carried out on a 0.50 mmol scale.
bReaction was carried out on a 0.10 mmol scale.
with broadly used dibenzocylooctynes 2379 bearing a free acid (23a), a biotin (23b),
or cyanine dyes (23c and 23d) (Scheme 8A). Gratifyingly, both the cysteine addition
followed by click reaction (a) and the click reaction followed by cysteine addition (b)
approaches were successful with all cyclooctynes to give products 24a–24d. This
demonstrated that azide-cyclooctyne cycloaddition is fully orthogonal to hyperva-
lent iodine reactivity. Both approaches of labeling also performed well on 15 and
17 to give 25 and 26 in quantitative yield (Scheme 8B).
To showcase the compatibility of our methodology with proteins more complex than
ubiquitin and to determine whether the resulting conjugates are well tolerated, we
generated fluorescently labeled nucleosomes. Nucleosomes consist of a central pro-
tein octamer formed from histones H2A, H2B, H3, and H4 and ꢁ150 bp of DNA that is
wrapped around the protein complex.80 For labeling, we mutated glutamate 63 in H4
to cysteine (E63C) and expressed and purified the protein. We then assembled his-
tone octamer complexes by using the remaining other human histone proteins
(H2A, H2B, and H3C110A; see Supplemental Experimental Procedures Section
3d). The resulting protein complex 27 contained two cysteine residues, which effi-
ciently reacted with 1a within 60 min under aqueous conditions to yield 28 (Figure 1A).
We then successfully labeled 28 with 23d Cy5-DBCO via azide-cyclooctyne cycload-
dition to afford product 29 (Figure 1A). SDS-PAGE analysis of product 29 revealed the
high specificity of the labeling procedure given that only histone H4 exhibited a fluo-
rescence signal (Figure 1B). The selectivity of the reaction was also corroborated by
high-performance liquid chromatography (HPLC) and top-down tandem mass spec-
trometry (see Supplemental Experimental Procedures Section 9g).
To demonstrate that the protein functionality was not affected by the modification,
we continued to reconstitute full nucleosomes, which will only form if the histone oc-
tamer structure is not disrupted. We thus combined 28 with a 170-bp-long segment
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Scheme 8. Combination of Cysteine Labeling with Click Cycloaddition
(A) Labeling of glutathione (2) via cysteine addition followed by click reaction (a) and click reaction followed by cysteine addition (b) approaches.
(B) Application to the labeling of 15 and 17. All reactions were monitored by HPLC, and no side products were observed except <20% oxidation to
disulfide during the thiol addition step. Reaction conditions were modified for 15 and 17 (See Supplemental Experimental Procedures Sections 11c and
11e–11g). HIR, hypervalent iodine reagent.
of the 601-nucleosome positioning sequence81 and reconstituted nucleosomes via
dialysis from high (2M NaCl) to low (10 mM KCl) salt (Figure 1C). To our delight, nu-
cleosomes were readily formed, yielding both unmodified or EBX-modified (280) nu-
cleosomes with equal efficiency, as judged by native PAGE (Figure 1D). Finally,
nucleosome assembly also proceeded smoothly with Cy5-labeled octamers to yield
fluorescent nucleosomes 290 (Figures 1E and 1F). Together, these experiments
demonstrate that EBX modification at exposed cysteines is compatible with protein
function and a viable strategy for highly efficient protein labeling.
Development of the ‘‘Doubly Orthogonal’’ Functionalization
After having demonstrated that selective reaction of the azide was possible, we
investigated functionalization of the hypervalent bond. Encouraged by recent prog-
ress in the use of palladium catalysis for the functionalization of peptides and
proteins,67–71,82,83 we opted for a Suzuki-Miyaura cross-coupling. Although avoiding
transition metals in biomolecule functionalization is preferred, it should be noted
that the palladium content can easily be reduced down to 1.0 ppm through scav-
enging and size-exclusion chromatography.70 Furthermore, palladium catalysis
has even been used in living cells, in which it shows low toxicity.69,82 After an exten-
sive study on various palladium catalytic systems (see Tables S2–S8) with boronic
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Figure 1. Labeling of Histones and Formation of Nucleosomes
(A) Functionalization of reconstituted histone octamers with EBX reagent 1a. Reactions were carried out on a 2.4 nmol scale at a 40 mM concentration of
histone octamers. Yields were determined by relative integration based on HPLC traces at 214 nm (see Supplemental Experimental Procedures
Section 9h).
(B) Analysis of histone octamers by SDS-PAGE followed by staining with Coomassie blue or imaging of Cy5 fluorescence.
(C) Reconstitution of nucleosomes 280 from EBX-labeled histone octamers (28) and the 170 bp 601 DNA sequence.
(D) Analysis of EBX-labeled nucleosomes 280 and unmodified nucleosomes 270 by native PAGE followed by GelRed staining.
(E) Reconstitution of nucleosomes 290 from Cy5-labeled histone octamers (29) and the 170 bp 601 DNA sequence.
(F) Analysis of the Cy5-labeled nucleosome 290 assembly by native PAGE followed by staining with GelRed or imaging of Cy5 fluorescence. NCP,
nucleosome core particle. The gel migration is indicated in relation to a DNA ladder.
acid 30a, air- and moisture-stable bis-lithium-2-(dimethylamino)-4,6-dihydroxylate-
pyrimidine palladium diacetate complex 31a82 was found to be the most suitable
catalyst-ligand system for our model reaction with VBX 3a. Cross-coupling product
32a was obtained in 70% yield (Table 2, entry 1). No degradation of the azide group
was observed in the presence of the palladium catalyst. The reaction was run under
air in a non-degassed phosphate buffer at 37ꢀC for 30 min. Although DMSO helped
to solubilize the boronic acid in aqueous media, it was non-essential for the reaction
(entry 2). The reaction gave the desired product 32a in satisfactory yields from pH 7.4
to 9.0 (entries 3 and 4). Using HEPES buffer instead of phosphate buffer resulted in a
slightly less efficient coupling (entry 5). Tris buffer significantly slowed down the re-
action rate, but a longer reaction time resulted in a satisfactory 51% yield (entry 6). A
more concentrated buffer could also be used (entry 7). Cooling down to room
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Table 2. Evaluation of the Reaction Conditions for the Cross-Coupling between 3a and 30a
NMe2
Pd(OAc)2
N
N
NH2
O
S
LiO
OLi
H
N
2
31a
HO
O
OMe
N
H
CO2H
1.0 equiv
3a
O
OMe
50 mM PB pH 8.2
(2% v/v DMSO)
37 °C, 30 min
(HO)2B
30a
10.0 equiv
32a
N3
Entry
Variations from the Optimized Conditions
none
Yield
1
2
3
4
5
6
7
8
9
70%
51%
50%
59%
51%
no DMSO
50 mM PB (pH 7.4)
50 mM PB (pH 9.0)
50 mM HEPES (pH 8.2)
50 mM Tris (pH 8.2)
100 mM PB (pH 8.2)
room temperature
200 mM reaction molarity
19% (51%a)
58%
53%
63%
Reactions were carried out on a 1.00 mmol scale. Calibrated HPLC yield is based on absorbance at 214 nm
(see Figure S6). For a complete list of examined catalysts, ligands, and conditions, see Tables S2–S9.
aThe reaction was analyzed after 2 h.
temperature still gave 32a in 53% yield (entry 8). When the reaction was diluted from
2 mM to 200 mM, product 32a could be obtained in 63% yield.
Once the robustness of the cross-coupling had been demonstrated, we examined
the scope of boronic acids (Scheme 9). Different electron-rich phenyl boronic acid
substrates, such as (3,5-dimethoxyphenyl)boronic acid (30b), (4-hydroxyphenyl)
boronic acid (30c), or (4-methylphenyl)boronic acid (30d), could be coupled
with 3a. Electron-deficient substrates, such as (4-methoxycarbonylphenyl)boronic
acid (30e) or (4-cyanophenyl)boronic acid (30f), as well as fluorinated arenes
such as para-fluorobenzene (30 g) or 3,5-bis(trifluoromethyl)benzene (30h) were
also successful. Cross-coupling of furyl (30i) and hexenyl (30j) boronic acids
was possible as well. In contrast, heterocyclic boronic acids (30k–30n) could not
be used.
We then applied our Suzuki-Miyaura cross-coupling to the more complex case of
modified ubiquitin 17 (Scheme 10). To our delight, the cross-coupling worked in a
one-pot labeling-cross-coupling approach to give 33. Finally, both reactive handles
were successfully employed in a one-pot labeling-SPAAC-Suzuki-Miyaura process,
on
a proof-of-principle scale, to afford doubly functionalized Cys-labeled
ubiquitin 34.
Application of ‘‘Doubly Orthogonal’’ Functionalization to Stabilize
Fluorescent Dyes
We then exploited our reagents for fluorescent labeling of receptors on living cells.
To this end, we synthesized substance P (35), a neuropeptide and a high-affinity
ligand of the neurokinin 1 (NK1) receptor,84 carrying an additional N-terminal
14
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NMe2
N
N
Pd(OAc)2
HO
NH2
O
S
LiO
OLi
H
N
2
31a
N
H
CO2H
1.0 equiv
(HO)2B
R
3a
O
O
50 mM PB pH 8.2
(2% v/v DMSO)
37 °C, 30 min
30b-n
10.0 equiv
32b-n
R
N3
Successful substrates:
OMe
R =
OH
Me
CO2Me
CN
OMe
30b
30c
30d
30e
30f
CF3
F
O
CF3
30g
30j
30h
30i
Unsuccessful substrates:
N
N
S
O
30k
30l
30m
30n
Scheme 9. Scope of Different Boronic Acid Reagents (30b–30d) for Cross-Coupling with 3a
Reactions were carried out on a 1.00 mmol scale. For all successful substrates, 32 was the major
product observed by HPLC (see Supplemental Experimental Procedures Section 15).
cysteine residue (Figure 2A) (see Supplemental Experimental Procedures Section
18a). We functionalized 35 with EBX reagent 1a and then labeling it with Cy5-
DBCO (23d), producing the labeled peptide 36 (Figure 2B). We then tested the func-
tionality of this construct by using a previously generated human embryonic kidney
(HEK) cell line expressing green fluorescent protein (GFP)-tagged NK1 receptor at
the cell surface85,86 (Figure 2C). Substrate 36 readily labeled the cells and exhibited
a distinct colocalization with the GFP-tagged NK1 receptor (Figure 2D), demon-
strating the stability of our bioconjugate for experiments on live cells.
Fluorescent dyes often suffer from poor photophysics and photochemistry, resulting
in dye bleaching. We envisioned that our dual-modification scheme might enable
the attachment of photoprotection compounds that positively affect the photophys-
ical and photochemical properties of nearby fluorophores.
We thus decided to exploit the doubly orthogonal functionalization scheme to in-
crease the photostability of cyanine dye Cy5, which should increase dye brightness,
decrease bleaching kinetics, and allow for longer tracks in single-particle tracking
applications. To this end, we decided to place the TSQ 6-hydroxy-2,5,7,8-tetrame-
thylchroman-2-carboxylic acid (Trolox) in close proximity to the Cy5 fluorophore.87
Such positioned TSQs reduce blinking rates, photobleaching rates, and dark-state
lifetimes.88 We therefore synthesized a boronic-acid adduct of Trolox (37). We
then functionalized 35 with 1a and coupled 37 via Suzuki-Miyaura cross-coupling
(Figure 2E). The obtained product 38 was further labeled with DBCO-Cy5 as
described above. Finally, a biotin moiety was attached to Lys5 for subsequent
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Scheme 10. One-Pot Labeling-Suzuki-Miyaura and Labeling-SPAAC-Suzuki-Miyaura
Functionalization of His6-Cys Ubiquitin (17)
n.d., yield not determined. Formation of 34 was confirmed by HPLC-MS.
surface immobilization using reagent 39 to peptides 36 and 38, yielding the final
peptides 40 (without Trolox) and 41 (with Trolox).
We then surface immobilized 40 and 41 (see Supplemental Experimental Procedures
Section 19a) and observed the stability of individual Cy5 molecules by single-mole-
cule total internal reflection (TIRF) microscopy (Figure 2F). Individual fluorescent
molecules were imaged over time under conditions of oxygen exclusion. After
50 s of imaging, most Cy5 dyes were bleached in the absence of coupled Trolox
in 40, whereas for the Trolox-containing molecule 41, a large portion of Cy5 was still
fluorescent (Figure 2G). When measuring bleaching time constants for both condi-
tions (Figures 2H and 2I), we detected a 3-fold increase in the dye’s bleaching
time constant as a result of the proximity to the Trolox moiety (from 17.9 G 8.8 s
for 40 to 55.9 G 18.6 s for 41). Together, these results demonstrate that a doubly
orthogonal functionalization strategy can be exploited in a modular fashion to label
proteins with fluorophores stabilized by a covalently coupled TSQ.
Conclusion
In summary, we have reported a general cysteine labeling protocol for the installa-
tion of a hypervalent iodine structure on both peptides and proteins. The obtained
bioconjugate contains two reactive groups, an azide and a hypervalent iodine, which
are orthogonal in reactivity to each other and to existing natural functional groups in
peptides and proteins.
The cysteine labeling protocol proceeds with high efficiency, chemoselectivity, and
functional-group tolerance under native conditions. The addition of thiols onto
EBX reagents proceeds with perfect Z stereoselectivity on simple thiols and small
peptides containing cysteine, but further studies will be needed to establish the
stereoselectivity of the reaction on larger peptides and proteins. In contrast
to previous methods based on the use of hypervalent iodine reagents, the reactive
carbon-iodine bond does not react and is transferred intact to the biomolecule. A
16
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Figure 2. Application of the ‘‘Doubly Orthogonal’’ Functionalization to Stabilize Fluorescent Dyes
(A) Substance P (35) carrying N-acetylation and C-amidation with additional N-terminal Gly and Cys residues.
(B) Labeling and SPAAC of 35 (see Supplemental Experimental Procedures Section 11d).
(C) Scheme of the NK1-R-GFP-fusion-expressing cells and the cell-labeling experiment.
(D) Imaging of cells expressing GFP-NK1-receptor, which was labeled with 35-Cy5 (SP-Cy5, 36). Scale bar, 50 mm.
(E) Labeling-SPAAC-Suzuki-Miyaura functionalization with Trolox (TX) of 35 followed by biotin (bt) modification (see Supplemental Experimental
Procedures Section 19b).
(F) Scheme of the single-molecule TIRF experiment.
(G) Single-molecule images of immobilized 40 and 41 at the indicated time points. Scale bar, 5 mm.
(H) Averaged fluorescence photobleaching kinetics for 40 and 41.
(I) Fluorescence bleaching time constants from six experiments for 40 and five experiments for 41. Error bars indicate the standard deviation (n = 5 or 6,
p = 0.007, Student’s t test).
wide range of peptidic-hypervalent iodine conjugates and a protein-bound hyperva-
lent iodine compound were efficiently generated. Importantly, the obtained conju-
gates are compatible with protein structure and function, as demonstrated by the
assembly of nucleosome particles.
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Additionally, the methodology could be extended to the functionalization of cyste-
ines in disulfide bridges after a simple reductive pretreatment. The obtained bio-
conjugates allow ‘‘doubly orthogonal’’ functionalization: an azide group can be
selectively functionalized by cycloaddition with cyclooctynes without affecting the
hypervalent bond. Alternatively, the hypervalent iodine structure could be success-
fully engaged in an aqueous palladium-catalyzed cross-coupling with boronic acids
without losing the azido group.
This provides a means for interesting and highly useful dual functionalization. Here,
we demonstrated the usefulness of the approach by improving the photophysics of
cyanine dyes by the attachment of a TSQ by using the dual-reactive handle. We thus
provide a modular method for stabilizing organic dyes in biomolecules.
Altogether, our approach allows fast and selective peptide and protein modifica-
tion. We are convinced that our work has just started to unravel the potential of hy-
pervalent iodine reagents in biomolecule labeling, and the stage is now set for
developing
a day-to-day use in chemical biology and potential medicinal
applications.
DATA AND CODE AVAILABILITY
Crystal structures for compounds 8a, 9a, and 9b are available as Data S1, S2, and S3,
respectively, and also at the Cambridge Crystallographic Data Centre under acces-
sion numbers CCDC: 1912427 (8a), CCDC: 1912430 (9a), and CCDC: 1862137 (9b).
Single-molecule data are available at https://zenodo.org/ (https://doi.org/10.5281/
zenodo.3246280).
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.chempr.
2019.06.022.
ACKNOWLEDGMENTS
We thank the European Research Council (Starting Grant iTools4MC 334840 and
Consolidator Grant SeleCHEM 771170), the Swiss National Science Foundation
´
(National Centre of Competence in Research, ‘‘chemical biology’’), and Ecole Poly-
technique Fe´ de´ rale de Lausanne (EPFL) for financial support. We thank Dr. Carolin
Lechner for contributing to peptide chemistry at the early stages of the project,
Dr. Andreas L. Bachmann for providing the His6-Cys ubiquitin construct, Dr. Ruud
Hovius for providing the NK1-receptor-expressing cell lines and histone octamers,
Maxime Mivelaz for providing nucleosome DNA and helping with TIRF experiments,
Karthik Maddi for providing histone mutants, and Kristina Makasheva for helping
with TIRF experiments. We also thank Dr. Daniel Ortiz from the EPFL Institute of
Chemical Sciences and Engineering for helping with the low-concentration labeling
experiments. We thank Laure Menin and Natalia Gasilova for top-down mass spec-
trometry analysis.
AUTHOR CONTRIBUTIONS
R.T. discovered and designed the reaction; synthesized the reagents and substrates;
performed the optimization studies, the scope of the reaction on peptides, and the
modifications studies on peptides; and contributed to the redaction of the manu-
script. J.C. synthesized the reagents and substrates, performed the modification
studies on peptides, and contributed to the redaction of the manuscript. N.G. de-
signed and performed the reactions on larger peptides; modified ubiquitin,
18
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j.chempr.2019.06.022
nucleosomes, and substance P; performed the cell experiments and single-molecule
photobleaching studies; and contributed to the redaction of the manuscript. R.S.D.
made several experiments important for the discovery and optimization of the reac-
tion on glutathione. B.F. devised and supervised the experiments on larger pep-
tides, ubiquitin, nucleosomes, cells, and single-molecule studies and participated
in the redaction of the manuscript. J.W. managed the overall project, designed
the reactions, planned the experiments on small molecules and peptides, and
participated in the redaction of the manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: January 28, 2019
Revised: May 21, 2019
Accepted: June 22, 2019
Published: July 2, 2019
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