Visualizing metabolically labeled glycoconjugates of living cells by copper-free and fast huisgen cycloadditions

Article abstract of DOI:10.1002/anie.200705456

(Chemical Presented) Cu^I-free click: 4-Dibenzocyclooctynol reacts, in the absence of a Cu^I catalyst, exceptionally fast with azido-containing saccharides and amino acids to give stable triazoles. A biotin-modified derivative is ideal

Full text of DOI:10.1002/anie.200705456

Angewandte
Chemie
DOI: 10.1002/anie.200705456
Live-Cell Imaging
Visualizing Metabolically Labeled Glycoconjugates of Living Cells by
Copper-Free and Fast Huisgen Cycloadditions**
Xinghai Ning, Jun Guo, Margreet A. Wolfert, and Geert-Jan Boons*
Dedicated to Sir J. Fraser Stoddart on the occasion of his 65th birthday
Azides, which are extremely rare in biological systems, are
emerging as attractive chemical handles for bioconju-
gation.[1–5] In particular, the CuI-catalyzed 1,3-dipolar cyclo-
addition of azides with terminal alkynes to give stable
triazoles[6,7] has been employed for the tagging of a variety
of biomolecules,[8–12] activity-based protein profiling,[13] and
the chemical synthesis of microarrays and small-molecule
Figure 1. Reagents for labeling of azido-containing biomolecules.
libraries.[14]
An attractive approach for installing azides into biomol-
ecules is based on metabolic labeling, whereby an azide-
containing biosynthetic precursor is incorporated into bio-
molecules by using the cellsꢀ biosynthetic machinery.[15] This
approach has been employed for tagging proteins, glycans,
and lipids of living systems with a variety of reactive probes.
These probes can facilitate the mapping of saccharide-
selective glycoproteins and identify glycosylation sites.[16]
Alkyne probes have also been used for cell-surface imaging
of azide-modified biomolecules, and a particularly attractive
approach involves the generation of a fluorescent probe from
a nonfluorescent precursor by a [3+2]cycloaddition. [17]
The cellular toxicity of the CuI catalyst has precluded
applications wherein cells must remain viable,[18] and hence
there is a great need for the development of CuI-free [3+2]
cycloadditions.[19–21] In this respect, alkynes can be activated
by ring strain, and, for example, constraining an alkyne within
an eight-membered ring creates 18 kcalmolÀ1 of strain, much
of which is released in the transition state upon [3+2]
cyclcoaddition with an azide.[19,20] As a result, cyclooctynes
such as 1 react with azides at room temperature without the
need for a catalyst (Figure 1). The strain-promoted cyclo-
addition has been used to label biomolecules without
observable cytotoxicitiy.[20] The scope of the approach has,
however, been limited because of the slow rate of reaction.[22]
Appending electron-withdrawing groups to the octyne ring
can increase the rate of strain-promoted cycloadditions;
however, currently Staudinger ligation with phosphine 2
offers the most attractive reagent for cell-surface labeling
with azides.
It was envisaged that 4-dibenzocyclooctynols such as
compound 3 would be ideal for labeling living cells with
azides because the aromatic rings are expected to impose
additional ring strain and conjugate with the alkyne, thereby
increasing the reactivity of the alkyne in metal-free [2+3]
cycloadditions with azides. The compound should, however,
have excellent stability because the ortho hydrogen atoms of
the aromatic rings shield the alkyne from nucleophilic attack.
Furthermore, the hydroxy group of 3 provides a handle for the
incorporation of tags such as fluorescent probes and biotin.
Compound 3 could be prepared easily from known[23,24] 3-
hydroxy-1,2:5,6-dibenzocycloocta-1,5,7-triene (4) by protec-
tion of the hydroxy group as a TBS ether to give 5, which was
brominated to provide dibromide 6 in a yield of 60%
(Scheme 1). The TBS protecting group was lost during the
latter transformation, but the bromination was low yielding
when performed on alcohol 4. Dehydrobromination of 6 by
treatment with LDA in THF at 08C[25] gave the target
cyclooctyne 3 in a yield of 45%.
[*] X. Ning, Dr. J. Guo, Dr. M. A. Wolfert, Prof. Dr. G. J. Boons
Complex Carbohydrate Research Center
University of Georgia
315 Riverbend Road, Athens, GA 30602 (USA)
Fax: (+1)706-542-9161
E-mail: gjboons@ccrc.uga.edu
Homepage: http://cell.ccrc.uga.edu/~gjboons/boons/Home.htm
[**] This research was supported by the Research Resource Center for
Biomedical Complex Carbohydrates (P41-RR-5351). We thank Dr.
Heather Flanagan-Steet and Dr. Richard Steet for assistance with
confocal microscopy studies.
Scheme 1. Reagents and conditions. a) TBSCl, pyridine; b) Br2, CHCl3;
c) LDA, THF; d) 4-nitrophenyl chloroformate, pyridine, CH2Cl2;
e) DMF, Et3N. LDA=lithium diisopropylamide, TBS=tert-butyl-
dimethylsilyl.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Communications
Compound 3 has an excellent, long shelf life and after
(FITC) for 15 min at 48C. The efficiency of the two-step
cell-surface labeling was determined by measuring the
fluorescence intensity of the cell lysates. For comparison,
the cell-surface azido moieties were also labeled by Stau-
dinger ligation with biotin-modified phosphine 2 followed by
treatment with avidin–FITC. The labeling with 9 was almost
complete after an incubation time of 60 min (Figure 3a).
treatment did not react with nucleophiles such as thiols and
amines. However, upon exposure to azides a fast reaction
took place and gave the corresponding triazoles in high yield.
For example, triazoles 10–13 were obtained in quantitative
yields as mixtures of regioisomers by reaction of the
corresponding azido-containing sugar and amino acid deriv-
atives with 3 in methanol for 30 min (Figure 2). The progress
Figure 3. Cell-surface labeling with compounds 2 and 9. Jurkat cells
grown for three days in the absence or presence of Ac4ManNAz
(25 mm) were incubated a) with compounds 2 and 9 (30 mm) for 0–
180 min or b) with compounds 2 and 9 (0–100 mm) for 1 h at room
temperature. Next, the cells were incubated with avidin–FITC for
15 min at 48C, after which cell lysates were assessed for fluorescence
intensity. Samples are indicated as follows: blank cells incubated with
&
Figure 2. Metal-free cycloadditions of compound 3 with azido-contain-
ing amino acid and saccharides. Boc=tert-butoxycarbonyl, TDS=thex-
yldimethylsilyl.
of the reaction of 3 with benzyl azide in methanol and in a
mixture of water/acetonitrile (1:4 v/v) was monitored by
1H NMR spectroscopy by integration of the benzylic proton
signals, and second-order rate constants of 0.17 and 2.3mÀ1 sÀ1,
respectively, were determined. The rate constant of the
reaction with 3 in acetonitrile/water is approximately three
orders of magnitude greater than that with cyclooctyne 1.
Having established the superior reactivity of 3, we focused
our attention on the preparation of a derivative of 4-
dibenzocyclooctynol (9; Scheme 1), which is modified with
biotin. Such a reagent should make it possible to visualize
biomolecules after metabolically labeling cells with an azido-
containing biosynthetic precursor, followed by cycloaddition
with 9 and treatment with avidin modified with a fluorescence
probe. Alternatively, biotinylation of glycoconjugates with 9
should make it possible to isolate these derivatives for
glycocomics studies using avidin immobilized on a solid
support. Compound 9 could easily be prepared by a two-step
reaction involving treatment of 3 with 4-nitrophenyl chloro-
formate to give activated intermediate 7, followed by
immediate reaction with 8.
*
*
2 ( ) or 9 (&), and Ac4ManNAz cells incubated with 2 ( ) or 9 ( ).
Interestingly, under identical conditions phosphine 2[22] gave
significantly lower fluorescent intensities, indicating that cell-
surface labeling by Staudinger ligation is slower and less
efficient. In each case, the control cells exhibited very low
fluorescence intensities, demonstrating that background
labeling is negligible. It was found that the two-step labeling
approach with 9 had no effect on cell viability, as determined
by morphology and exclusion of trypan blue.
The concentration dependence of the cell-surface labeling
was studied by incubation of cells with various concentrations
of 2 and 9 followed by staining with avidin–FTIC (Figure 3b).
As expected, cells displaying azido moieties showed a dose-
dependent increase in fluorescence intensity. Reliable fluo-
rescent labeling was achieved at a 3 mm concentration of 9;
however, optimal results were obtained at concentrations
ranging from 30 to 100 mm. No increase in labeling was
observed at concentrations higher than 100 mm owing to the
limited solubility of 9.
Next, Jurkat cells were cultured in the presence of 25 mm
N-azidoacetylmannosamine (Ac4ManNAz) for three days to
metabolically introduce N-azidoacetylsialic acid (SiaNAz)
moieties into glycoproteins.[26] As a negative control, Jurkat
cells were employed that were grown in the absence of
Ac4ManNAz. The cells were exposed to a 30 mm solution of
compound 9 for various time periods, and after washing, the
cells were stained with avidin–fluorescein isothiocyanate
Next, attention was focused on visualizing azido-contain-
ing glycoconjugates of living cells by confocal microscopy.
Thus, adherent Chinese hamster ovary (CHO) cells were
cultured in the presence of Ac4ManNAz (100 mm) for three
days. The resulting cell-surface azido moieties were treated
with 9 (30 mm) for 1 h and then with avidin–AlexaFluor488
for 15 min at 48C. As expected, staining was observed only at
2254
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Chemie
Keywords: azides · carbohydrates ·
.
click chemistry · glycoconjugates
[1]H. C. Kolb, K. B. Sharpless, Drug Discov-
ery Today 2003, 8, 1128 – 1137.
[2]S. Dedola, S. A. Nepogodiev, R. A. Field,
Org. Biomol. Chem. 2007, 5, 1006 – 1017.
[3]J. E. Moses, A. D. Moorhouse, Chem. Soc.
Rev. 2007, 36, 1249 – 1262.
[4]H. Nandivada, X. W. Jiang, J. Lahann, Adv.
Mater. 2007, 19, 2197 – 2208.
[5]P. Wu, V. V. Fokin, Aldrichimica Acta 2007,
40, 7 – 17.
[6]V. V. Rostovtsev, L. G. Green, V. V. Fokin,
K. B. Sharpless, Angew. Chem. 2002, 114,
2708 – 2711; Angew. Chem. Int. Ed. 2002,
41, 2596 – 2599.
[7]C. W. Tornoe, C. Christensen, M. Meldal, J.
Org. Chem. 2002, 67, 3057 – 3064.
[8]J. W. Chin, T. A. Cropp, J. C. Anderson, M.
Mukherji, Z. W. Zhang, P. G. Schultz, Sci-
ence 2003, 301, 964 – 967.
[9]Q. Wang, T. R. Chan, R. Hilgraf, V. V.
Fokin, K. B. Sharpless, M. G. Finn, J. Am.
Chem. Soc. 2003, 125, 3192 – 3193.
[10]Y. Kho, S. C. Kim, C. Jiang, D. Barma, S. W.
Kwon, J. K. Cheng, J. Jaunbergs, C. Wein-
baum, F. Tamanoi, J. Falck, Y. M. Zhao,
Proc. Natl. Acad. Sci. USA 2004, 101,
12479 – 12484.
Figure 4. Fluorescence images of cells labeled with compound 9 and avidin–AlexaFluor488.
CHO cells grown for three days in the absence (d–f) or presence (a–c) of Ac4ManNAz
(100 mm) were incubated with compound 9 (30 mm) for 1 h at 48C (a, d) or room
temperature (b, c, e, f). Next, cells were incubated with avidin–AlexaFluor488 for 15 min at
48C and, after washing, fixing, and staining for the nucleus with the far-red-fluorescent dye
TO-PRO, imaged (a, b, d, e) or after washing incubated for 1 h at 378C before fixing,
nucleus staining, and imaging (c, f). Merged indicates that the images of cells labeled with
AlexaFluor (488 nm) and TO-PRO-3 iodide (633 nm) are merged and shown in green and
red, respectively.
the cell surface (Figure 4), and importantly, the labeling
[11]J. Gierlich, G. A. Burley, P. M. E. Gramlich, D. M. Hammond, T.
Carell, Org. Lett. 2006, 8, 3639 – 3642.
[12]A. J. Link, M. K. S. Vink, N. J. Agard, J. A. Prescher, C. R.
Bertozzi, D. A. Tirrell, Proc. Natl. Acad. Sci. USA 2006, 103,
10180 – 10185.
[13]A. E. Speers, G. C. Adam, B. F. Cravatt, J. Am. Chem. Soc. 2003,
125, 4686 – 4687.
[14]X. L. Sun, C. L. Stabler, C. S. Cazalis, E. L. Chaikof, Bioconju-
gate Chem. 2006, 17, 52 – 57.
[15]J. A. Prescher, C. R. Bertozzi, Nat. Chem. Biol. 2005, 1, 13 – 21.
[16]S. R. Hanson, T. L. Hsu, E. Weerapana, K. Kishikawa, G. M.
Simon, B. F. Cravatt, C. H. Wong, J. Am. Chem. Soc. 2007, 129,
7266 – 7267.
[17]K. Sivakumar, F. Xie, B. M. Cash, S. Long, H. N. Barnhill, Q.
Wang, Org. Lett. 2004, 6, 4603 – 4606.
[18]A. J. Link, D. A. Tirrell, J. Am. Chem. Soc. 2003, 125, 11164 –
11165.
[19]R. B. Turner, P. Goebel, B. J. Mallon, A. D. Jarrett, J. Am. Chem.
Soc. 1973, 95, 790 – 792.
[20]N. J. Agard, J. A. Prescher, C. R. Bertozzi, J. Am. Chem. Soc.
2004, 126, 15046 – 15047.
[21]S. S. van Berkel, A. T. J. Dirks, M. F. Debets, F. L. van Delft,
J. J. L. M. Cornelissen, R. J. M. Nolte, F. P. J. T. Rutjes, Chem-
BioChem 2007, 8, 1504 – 1508.
[22]N. J. Agard, J. M. Baskin, J. A. Prescher, A. Lo, C. R. Bertozzi,
ACS Chem. Biol. 2006, 1, 644 – 648.
[23]M. E. Jung, A. B. Mossman, M. A. Lyster, J. Org. Chem. 1978,
43, 3698 – 3701.
[24]M. E. Jung, S. J. Miller, J. Am. Chem. Soc. 1981, 103, 1984 – 1992.
[25]G. Seitz, L. Pohl, R. Pohlke, Angew. Chem. 1969, 81, 427 – 428;
Angew. Chem. Int. Ed. Engl. 1969, 8, 447 – 448.
[26]S. J. Luchansky, C. R. Bertozzi, ChemBioChem 2004, 5, 1706 –
1709.
procedure was equally efficient when performed at either
ambient temperature or 48C. Furthermore, blank cells
exhibited very low fluorescence staining, confirming that
background labeling is negligible.
Cell-surface glycoconjugates are constantly recycled by
endocytosis, and to monitor this process, metabolically
labeled cells were reacted with 9 and avidin–AlexaFluor488
according to the standard protocol and incubated at 378C for
1 h before examination by confocal microscopy. We observed
that a significant quantity of labeled glycoproteins had been
internalized into vesicular compartments.
At the completion of these studies, Bertozzi and co-
workers reported a difluorinated cyclooctyne (DIFO) that
reacts with azides at almost the same reaction rate as
compound 3.[27] DIFO linked to AlexaFluor was employed
to investigate the dynamics of glycan trafficking. It was found
that after incubation for 1 h, labeled glycans colocalized with
markers for endosomes and Golgi.
4-Dibenzocyclooctynols such as 3 and 9 have several
advantageous features for researchers such as ease of
chemical synthesis and the possibility to further enhance the
rate of cycloaddition by functionalization of the aromatic
moieties. Modifying the aromatic rings may also offer an
exciting opportunity to obtain reagents that become fluores-
cent upon [3+2]cycloaddition with azido-containing com-
pounds, which will make it possible to monitor in real time the
trafficking of glycoproteins and other biomolecules in living
cells.
[27]J. M. Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V.
Chang, I. A. Miller, A. Lo, J. A. Codelli, C. R. Bertozzi, Proc.
Natl. Acad. Sci. USA 2007, 104, 16793 – 16797.
Received: November 29, 2007
Published online: February 14, 2008
Angew. Chem. Int. Ed. 2008, 47, 2253 –2255
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
2255