A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems

Article abstract of DOI:10.1021/ja044996f

Selective chemical reactions that are orthogonal to the diverse functionality of biological systems have become important tools in the field of chemical biology. Two notable examples are the Staudinger ligation of azides and phosphines and the Cu(I)-catal

Full text of DOI:10.1021/ja044996f

Published on Web 11/02/2004
A Strain-Promoted [3 + 2] Azide-Alkyne Cycloaddition for Covalent
Modification of Biomolecules in Living Systems
Nicholas J. Agard, Jennifer A. Prescher, and Carolyn R. Bertozzi*
Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, UniVersity of
California, and Materials Sciences DiVision, Lawrence Berkeley National Laboratory, Berkeley, California 94720
Received August 19, 2004; E-mail: crb@cchem.berkeley.edu
Selective chemical reactions that are orthogonal to the diverse
functionality of biological systems are now recognized as important
tools in chemical biology.1 As relative newcomers to the repertoire
of synthetic chemistry, these bioorthogonal reactions have inspired
new strategies for compound library synthesis,2 protein engineering,3
functional proteomics,4 and chemical remodeling of cell surfaces.5
The azide has secured a prominent role as a unique chemical handle
for bioconjugation. We have made extensive use of the azide as a
chemical reporter of glycosylation, employing the Staudinger
ligation with phosphines to tag azidosugars metabolically introduced
into cellular glycoconjugates.6 The Staudinger ligation can be
Figure 1. (A) Cu(I)-catalyzed Huisgen cycloaddition (“click” chemistry).
(B) Strain-promoted [3 + 2] cycloaddition of azides and cyclooctynes.
performed in living animals without physiological harm,7 suggesting
the potential for applications in noninvasive imaging and therapeutic
targeting. Still, the reaction is not without liabilities. The requisite
phosphines are susceptible to air oxidation, and their optimization
for improved water solubility and increased reaction rate has proven
to be synthetically challenging.
Scheme 1
The azide has an alternative mode of bioorthogonal reactivitys
the [3 + 2] cycloaddition with alkynes described by Huisgen.8 In
its classic form, this reaction has limited applicability in biological
systems due to the requirement of elevated temperatures (or
pressures) for reasonable reaction rates. Sharpless and co-workers
surmounted this obstacle with the development of a copper(I)-
catalyzed version, termed “click” chemistry, that proceeds readily
at physiological temperatures and in richly functionalized biological
environs (Figure 1A). This reaction has enabled the selective
modification of virus particles,3a nucleic acids,9 and proteins from
complex tissue lysates.4 Unfortunately, the mandatory copper
catalyst is toxic to both bacterial5b and mammalian cells,10 thus
precluding applications wherein the cells must remain viable.
Catalyst-free Huisgen cycloadditions of alkynes activated by
electron-withdrawing substituents have been reported to occur at
ambient temperatures.11 However, these compounds can undergo
Michael reaction with biological nucleophiles.
We considered an alternative means of activating alkynes for
catalyst-free [3 + 2] cycloaddition with azides: ring strain. In 1961,
Wittig and Krebs noted that the reaction between neat cyclooctyne,
the smallest of the stable cycloalkynes, and phenyl azide “proceeded
like an explosion to give a single product,” the triazole.12 The
massive bond angle deformation of the acetylene to 163°13 accounts
for nearly 18 kcal/mol of ring strain.14 This destabilization of the
ground state versus the transition state of the reaction provides a
dramatic rate acceleration compared to unstrained alkynes.15 Here
we report that the [3 + 2] cycloaddition of azides and cyclooctyne
derivatives (Figure 1B) occurs readily under physiological condi-
tions in the absence of auxiliary reagents. We employed the reaction
for the selective chemical modification of living cells without any
apparent toxicity.
essentially as described by Reese and Shaw.16 Briefly, compound
117 was treated with silver perchlorate to effect electrocyclic ring
opening to the trans-allylic cation, which was captured with methyl
4-(hydroxymethyl)-benzoate to afford bromo-trans-cyclooctene 2.
Base-mediated elimination of the vinyl bromide followed by
saponification yielded versatile intermediate 3, to which any
biological probe can be attached. Finally, compound 3 was coupled
to biotin analogue 418 bearing a PEG linker, providing target 5.
Cyclooctyne 3 was stable to mild acid (0.5 N HCl for 30 min),
base (0.8 M NaOMe for 30 min), and prolonged exposure to
biological nucleophiles such as thiols (120 mM 2-mercaptoethanol
for 12 h).
We performed model reactions with compound 3 and 2-azido-
ethanol, benzyl azide, or N-butyl R-azidoacetamide (see Supporting
Information for details). In all cases, the only products observed
were the two regioisomeric triazoles formed in approximately equal
amounts. We next applied the reaction for covalent labeling of
We synthesized biotinylated cyclooctyne 5 as shown in Scheme
1. Construction of the substituted cyclooctyne core was achieved
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C O M M U N I C A T I O N S
Figure 2. Labeling of azide-modified GlyCAM-Ig with alkyne probes.
Purified GlyCAM-Ig was treated with buffer (-), 250 µM 5, or 250 µM 6
alone or in the presence (cat) of CuSO4, TCEP, and a triazolyl ligand,
overnight at room temperature. Reaction mixtures were quenched with
2-azidoethanol and analyzed by Western blot probing with HRP-R-biotin
(upper panel). The blot was then stripped and reprobed with HRP-R-Ig
(lower panel).
biomolecules. The recombinant glycoprotein GlyCAM-Ig19 was
expressed in CHO cells in the presence of peracetylated N-
azidoacetylmannosamine (Ac4ManNAz), leading to metabolic
incorporation of the corresponding N-azidoacetyl sialic acid (Sia-
NAz) into its glycans.20 Control samples of GlyCAM-Ig were
expressed in the absence of azido sugar. The purified GlyCAM-Ig
samples were incubated with 250 µM 5 overnight, the unreacted
cyclooctyne was quenched with excess 2-azidoethanol, and the
samples were analyzed by Western blot probing with HRP-R-biotin
(Figure 2). Robust biotinylation was observed for GlyCAM-Ig
modified with SiaNAz. Native GlyCAM-Ig lacking azides showed
no background labeling, underscoring the exquisite selectivity of
the strain-promoted cycloaddition.
As a point of comparison, we performed similar reactions with
biotin-modified terminal alkyne 6 (Scheme 1). In the absence of
reagents for copper catalysis, no glycoprotein labeling was observed
(Figure 2). As expected on the basis of reports from Sharpless,
Finn, and Cravatt,3a,4 addition of CuSO4, TCEP, and a triazolyl
ligand resulted in facile labeling of the azide-modified glycoprotein.
The blot was stripped and reprobed with HRP-R-IgG to confirm
equal protein loading. Interestingly, we consistently observed
diminished R-IgG immunoreactivity for azide-modified GlyCAM-
Ig labeled with 6 in the presence of copper. It is possible that the
combination of triazole products and copper damages the epitope
recognized by HRP-R-IgG.
Figure 3. Cell-surface labeling with compound 5. Jurkat cells were
incubated in the presence (+Az) or absence (-Az) of 25 µM Ac4ManNAz
for 3 d. (A) The cells were reacted with various concentrations of 5 for 1
h at room temperature and treated with FITC-avidin; mean fluorescence
intensity (MFI) was determined by flow cytometry. (B) Cells were incubated
with 250 µM 5 at room temperature and analyzed as in A. (C) Cells were
incubated with 100 µM probe for 1 h at room temperature and analyzed as
in A. Error bars represent the standard deviation from three replicates. AU
) arbitrary fluorescence units.
fellowship. This work was supported by grants to C.R.B. from the
National Institutes of Health (GM58867) and the U.S. Department
of Energy under Contract No. DE-AC03-76SF00098.
Supporting Information Available: Synthetic procedures and
spectral data, and protocols for kinetic analyses and biological
experiments. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
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14, 603-609.
Finally, we investigated the utility of the strain-promoted reaction
for live cell labeling. Jurkat cells were incubated with 25 µM Ac4-
ManNAz for 3 d to introduce SiaNAz residues into their cell-surface
glycoproteins.5a,21 The cells were reacted with various concentrations
of 5 for 1.5 h at room temperature and then stained with FITC-
avidin and analyzed by flow cytometry. As shown in Figure 3A,
cells displaying azides showed a dose-dependent increase in
fluorescence upon treatment with the cyclooctyne probe. No
detectable labeling of cells lacking azides was observed. The cell-
surface reaction was also dependent on duration of incubation with
5 (Figure 3B) and the density of cell-surface azides (Supporting
Information). No negative effects on cell viability were observed.
Curious as to how the strain-promoted cycloaddition compares to
the Staudinger ligation, we reacted azide-labeled cells with either
compound 5 or a previously reported phosphine-biotin probe.6b
As shown in Figure 3C, the cell-surface reactions were comparable,
with the Staudinger ligation proceeding about twice as efficiently.
In summary, the strain-promoted [3 + 2] azide-alkyne cycload-
dition can be used for selective modification of biomolecules and
living cells without apparent physiological harm. An important next
step will be its extension to living animals. In the future, ring strain
might be more thoroughly explored as a means to promote otherwise
reticent reactions with potential bioorthogonality.
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Acknowledgment. We thank Scott Laughlin for providing
GlyCAMIg, Tanya Leavy for model azides, and Patrick Holder for
the triazolyl ligand. J.A.P. was supported by a HHMI predoctoral
(21) Saxon, E.; Luchansky, S. J.; Hang, H. C.; Yu, C.; Lee, S. C.; Bertozzi, C.
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