Angewandte
Chemie
DOI: 10.1002/anie.201403121
Protein Modifications
Allenamides as Orthogonal Handles for Selective Modification of
Cysteine in Peptides and Proteins**
Ata Abbas, Bengang Xing,* and Teck-Peng Loh*
Abstract: In this study, a remarkably simple and direct strategy
has been successfully developed to selectively label target
cysteine residues in fully unprotected peptides and proteins.
The strategy is based on the reaction between allenamides and
the cysteine thiol, and proceeds swiftly in aqueous medium
with excellent selectivity and quantitative conversion, thus
forming a stable and irreversible conjugate. The combined
simplicity and mildness of the process project allenamide as
robust and versatile handles to target cysteines and has
potential use in biological systems. Additionally, fluorescent-
labeling studies demonstrated that the installation of a C-
terminal allenamide moiety onto various molecules of interest
may supply a new methodology towards the site-specific
labeling of cysteine-containing proteins. Such a new labeling
strategy may thus open a window for its application in the field
of life sciences.
where most notable works involve cross-metathesis of allyl
sulphides using the ruthenium catalyst reported by the group
[3]
of Davis, and thiol–allene coupling using a gold catalyst
[4]
reported by the group of Che. In recent years, some notable
efforts to successfully target cysteine residues can be summed
[3]
up as cross-metathesis of allyl sulfides, and conjugation of
[
5]
thiol groups with allyl selenosulfate salts, electron-deficient
[
6]
[7]
[8]
alkynes, or bromomaleimides, dithiomaleimide deriva-
tives, and PEGylation of native disulfide bond using a double
[
9]
cysteine alkylating reagent. However, most strategies suffer
from one or more drawbacks and the search for a versatile
methodology remains elusive. While the cross-reactivity of
classical reagents, including a-halo acyl analogues and
maleimides, with other amino acids such as histidine and
lysine residues potentially affects the selectivity, their rever-
sibility/irreversibility is a matter of interest in newer findings.
For example, bromomaleimides, dithiomaleimides, and elec-
tron-deficient alkynes form a bond with cysteine thiols, and
can be reversed with excess glutathione, DTT, and other
reactants. This reversibility, though extremely useful, may
pose a limitation when in vivo application is required, because
of the presence of albumin in blood serum and glutathione in
the intracellular environment. Therefore, an urgent need
exists to find promising orthogonal handles and related
labeling strategies which can selectively and irreversibly bind
with cystein. The groups of Ovaa and Mootz have recently
demonstrated, simultaneously and independently, that termi-
nal alkynes (in modified ubiquitin) can be used to inhibit
cysteine proteases irreversibly through in situ thiol–alkyne
S
elective chemical modification of protein structure is of
significant importance to directly visualize protein dynamics,
clearly understand their interaction mechanisms, and pre-
cisely mediate protein–protein interactions under single-cell
and molecule resolution. Among the various remarkable
labeling strategies, the sulfhydryl group in peptides and
proteins has remained an attractive target for site-selective
[
1]
modification owing mainly to its higher nucleophilicity and
[
2]
relatively lower natural abundance. Briefly, the direct
modification of cysteine can be mainly represented by two
typical chemical pathways, that is, the nucleophilic substitu-
tion of a leaving group through the thiol of cysteine, as is the
case for a-halocarbonyl derivatives (e.g. 2-iodoacetic acid and
related variants), and the Michael addition of a thiol group to
Michael acceptors like a,b-unsaturated systems. Another less
common strategy is the metal-catalyzed cystein modification
[10]
coupling,
which is quite impressive and surprising as
alkynes have largely been considered inert under biological
conditions. Activation of alkynes by a positively charged
protein pocket (oxoanion hole) has been proposed to be
[
11]
responsible for this unexpected reactivity.
With the long-term goal to understand and regulate
protein functions through their chemical modification, our
group has previously reported modification of the N-terminus
of peptides and proteins using Mukaiyama aldol condensa-
[
*] Prof. T. P. Loh
Department of Chemistry
University of Science and Technology of China
Hefei, Anhui 230026 (China)
[
12]
tions, and herein we, for the first time, present an efficient
way to utilize C-substituted terminal allenamide moieties as
an “orthogonal handle” to selectively and irreversibly modify
cysteine residues in fully unprotected peptides and proteins
which have other reactive amino-acid side chains. The
reaction is characterized by quantitative yields, very high
selectivity, mild reaction conditions, and high reaction rates.
In a typical reaction, simple pipette mixing of a cysteine-
containing peptide or protein with allenamide in aqueous
buffer of pH 8.0 at room temperature gives the conjugation
product with quantitative conversion within 10–20 minutes.
LCMS was used to confirm quantitative conversion while
A. Abbas, Prof. B. Xing, Prof. T. P. Loh
Division of Chemistry and Biological Chemistry
School of Physical and Mathematical Sciences
Nanyang Technological University
21 Nanyang Link, Singapore-637371 (Singapore)
E-mail: teckpeng@ntu.edu.sg
[
**] Nanyang Technological University and the Singapore Ministry of
Education Academic Research Fund. This research was supported
financially by Nanyang Technological University (New Initiative
Funding) and the National Environment Agency (NEA-ETRP Project
Ref. No. 1002 111).
Angew. Chem. Int. Ed. 2014, 53, 1 – 5
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
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