Communications
DOI: 10.1002/anie.201003761
Bimolecular Imaging
Readily Accessible Bicyclononynes for Bioorthogonal Labeling and
Three-Dimensional Imaging of Living Cells**
Jan Dommerholt, Samuel Schmidt, Rinske Temming, Linda J. A. Hendriks, Floris P. J. T. Rutjes,
Jan C. M. van Hest, Dirk J. Lefeber, Peter Friedl, and Floris L. van Delft*
The advent of chemical biology tools for imaging and tracking
of biomolecules (proteins, lipids, glycans) in their native
environment is providing unique insights into cellular pro-
cesses that are not achievable with traditional biochemical or
[
1]
molecular biology tools. Bioorthogonal labeling of biomol-
ecules has proven particularly useful for the detection and
[
2]
[3]
study of glycans and lipids, based on a highly selective
reaction between an abiotic functional tag and a designed
chemical probe. With respect to the abiotic tag, azide has been
used extensively because of its straightforward chemical
[4]
introduction, small size, and relative inertness. The finding
that azides react rapidly and cleanly with terminal acetylenes
in the presence of copper(I), the quintessential “click”
reaction, has found tremendous application in life and
[
5]
Scheme 1. Reactions and structures of cyclooctyne compounds for
strain-promoted cycloaddition. a) Cycloaddition with azide (SPAAC) or
nitrone (SPANC). b) Structures of the most commonly employed
cyclooctynes.
material sciences. However, because up to 20 mol% of
copper(I) species is typically used, such click chemistry is not
suitable for labeling of living systems without compromising
[
6]
cell function. Apart from that, the presence of copper may
[
7]
[8]
induce oligonucleotide and polysaccharide degradation.
To avoid the use of toxic metals, several metal-free bioor-
with high reaction rates in a so-called strain-promoted
[9]
[12]
thogonal labeling reaction have been developed. In partic-
ular, phosphines have been used for covalent ligation to
alkyne–azide cycloaddition (SPAAC).
The toolbox of
metal-free bioorthogonal reactions was most recently further
[
10]
[13]
[14]
azides, a procedure known as Staudinger ligation. How-
ever, owing to the oxygen sensitivity of phosphines, recent
focus of chemical ligation is shifting towards strain-promoted
expanded by our research group and others, by demon-
strating that cyclooctynes undergo even more rapid strain-
promoted cycloaddition with nitrones (SPANC), a procedure
that was found suitable for dual, irreversible, and site-specific
[11]
cycloaddition reactions with cyclooctynes (Scheme 1a).
Most prominently, azides were find to react with cyclooctynes
[
13]
N-terminal modification of proteins.
The broad application of metal-free cycloaddition in life
sciences is, however, hampered by the limited commercial
availability and lengthy synthetic routes for preparation of the
most common cyclooctynes (Scheme 1b). For example, eight
synthetic steps are required to generate second-generation
[
+]
[
*] J. Dommerholt, R. Temming, L. J. A. Hendriks, F. P. J. T. Rutjes,
J. C. M. van Hest, F. L. van Delft
Radboud University Nijmegen
Institute for Molecules and Materials
Heijendaalseweg 135, 6525 AJ, Nijmegen (The Netherlands)
[15]
[16]
DIFO (1), nine steps for DIBAC (3), and seven steps for
[
17]
[18]
BARAC (4), while yields are usually low (10% for 2,
E-mail: f.vandelft@science.ru.nl
[
+]
16% for 4). Additional modifications, such as dibenzoannu-
lation (compounds 2–4), increase lipophilicity and may,
therefore lead to non-specific binding to proteins.
Here we report bicyclo[6.1.0]nonyne (BCN) as a novel
ring-strained alkyne for metal-free cycloaddition reactions
with azides and nitrones. Bicyclononyne derivatives, which
were obtained in a highly straightforward process through
S. Schmidt, P. Friedl
Radboud University Nijmegen
Nijmegen Center for Molecular Life Sciences
Department of Cell Biology, 6500 HB Nijmegen (The Netherlands)
[
17]
D. J. Lefeber
Radboud University Medical Center
Department of Laboratory Medicine
6
525 ED Nijmegen (The Netherlands)
cyclopropanation of 1,5-cyclooctadiene, are C symmetrical
+
s
[
] These authors contributed equally to this work.
and display excellent reaction kinetics in strain-promoted
cycloaddition reactions. Functionalized derivatives of BCN
were applied in the labeling of proteins and glycans, as well as
in the three-dimensional visualization of living melanoma
cells.
[
**] This research has been financially supported (in part) by the Council
for Chemical Sciences of The Netherlands Organization for
Scientific Research (NWO-CW, to F.L.D). Dr. G. J. Boons (Complex
Carbohydrate Research Center, Athens, GA) is kindly acknowledged
for fruitful discussions.
Based on the known reactivity enhancement of cyclo-
propane fusion, we speculated that analogues of bicyclo-
[19]
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2010, 49, 9422 –9425