Two-color glycan labeling of live cells by a combination of diels-alder and click chemistry

Article abstract of DOI:10.1002/anie.201208991

One is not enough: Terminal alkenes are used as chemical reporters and ligation partners for 1,2,4,5-tetrazines in a Diels-Alder reaction with inverse electron demand (DARinv). Combination with strain-promoted azide-alkyne cycloaddition (SPAAC) allows the visualization of two different glycan structures in one experiment. Copyright

Full text of DOI:10.1002/anie.201208991

Angewandte
Chemie
DOI: 10.1002/anie.201208991
Chemical Glycobiology
Two-Color Glycan Labeling of Live Cells by a Combination of Diels–
Alder and Click Chemistry**
Andrea Niederwieser, Anne-Katrin Spꢀte, Long Duc Nguyen, Christian Jꢁngst, Werner Reutter,
and Valentin Wittmann*
Protein glycosylation is a complex form of posttranslational
modification and has been shown to be crucial for the
function of many proteins. Sialic acid is prominently posi-
tioned at the outer end of membrane glycoproteins. It plays
a critical role for the regulation of a myriad of cellular
functions and it forms a shield around the cell. Furthermore, it
constantly interacts with the environment of cells and
contributes to histocompatibility.[1] This makes studying
sialylation an interesting field of research, but monitoring
sialic acid in vivo is challenging. While proteins are routinely
labeled by genetic methods, such as expression as GFP fusion
proteins, comparable methods are not available for secondary
gene products, such as glycans of glycoconjugates. Metabolic
oligosaccharide engineering (MOE) is a successful new
strategy to visualize the localization of glycans in vitro and
in vivo.[2] In this approach, cells are cultivated in the presence
of non-natural monosaccharide derivatives that carry a chem-
ical reporter group and are nonetheless accepted by the
biosynthetic machinery of a cell. For instance, peracetylated
N-azidoacetylmannosamine (Ac4ManNAz) is taken up by the
cell, deacetylated by cellular esterases, and owing to the
promiscuity of the enzymes of sialic acid biosynthesis, is
converted into N-azidoacetyl neuraminic acid and incorpo-
rated into sialoglycoconjugates.[3] Once presented on the cell
surface, the azide-containing sialylated glycan can be visual-
ized through a bioorthogonal ligation reaction.[4] Besides
Ac4ManNAz, several monosaccharide derivatives of N-ace-
tylgalactosamine,[5] N-acetylglucosamine,[6] and l-fucose[7] are
suitable for MOE providing further insights into the role of
cellular structures and functions of glycans in the cell.
Currently, mainly Staudinger ligation[3] and azide–alkyne
[3+2] cycloaddition (copper-catalyzed[8] or strain-promoted,[9]
also known as the click reaction) are applied as ligation
reactions in MOE. However, both of them rely on the
reaction of azides and thus cannot be used for the concurrent
detection of two different metabolically incorporated carbo-
hydrates. A labeling strategy that can be carried out in the
presence of azides and alkynes would significantly expand the
scope of chemical labeling reactions in living cells and is thus
highly desirable.
Recently, it was shown that the Diels–Alder reaction with
inverse electron demand (DARinv) of 1,2,4,5-tetrazines[10]
with strained dienophiles, such as trans-cyclooctenes,[11] cyclo-
butenes,[12] norbornenes,[11d,f,13] cyclooctynes,[11d,f] and substi-
tuted cyclopropenes,[14] fulfills the requirements of a bioor-
thogonal ligation reaction and furthermore is orthogonal to
the azide–alkyne cycloaddition. However, these cyclic
alkenes or kinetically stable tetrazines[15] are expected to be
too large for being efficiently metabolized by the sialic acid
biosynthetic pathway, starting from the corresponding N-
acylmannosamine derivative. In search for smaller dieno-
philes suitable for MOE, we identified monosubstituted
(terminal) alkenes as a new class of chemical reporters. We
recently reported the successful application of the DARinv
between terminal alkenes and 1,2,4,5-tetrazines in the prep-
aration of carbohydrate microarrays.[13c] The fact that termi-
nal alkenes are hardly found in biological systems and are
completely absent in proteins makes them a promising
reporter group. Herein, we show that ManNAc derivatives
containing a terminal alkene in the acyl side chain are
metabolically incorporated into cell-surface sialic acids and
can subsequently be labeled by the DARinv (Figure 1).
Moreover, we demonstrate that double labeling of two
differently modified, metabolically incorporated monosac-
charides is possible by combining the DARinv with strain-
promoted azide–alkyne cycloaddition (SPAAC).
[*] Dipl.-Chem. A. Niederwieser, M. Sc. A.-K. Spꢀte, M. Sc. C. Jꢁngst,
Prof. Dr. V. Wittmann
University of Konstanz, Department of Chemistry and
Konstanz Research School Chemical Biology (KoRS-CB)
78457 Konstanz (Germany)
E-mail: mail@valentin-wittmann.de
Dr. L. D. Nguyen, Prof. Dr. W. Reutter
Institut fꢁr Biochemie und Molekularbiologie, CBF
Charitꢂ Universitꢀtsmedizin
As the reaction rate of the DARinv of acyclic olefins with
tetrazines is very sensitive to steric hindrance, double bonds
with more than one substituent react very slowly.[16] Terminal
alkenes, on the other hand, can react rapidly without any
further activation. This prompted us to design mannosamine
derivatives 2 and 4 (Figure 2) that were synthesized in three
steps from mannosamine hydrochloride (see the Supporting
Information). Based on previous work by Keppler et al.,[2c] we
expected both derivatives to be accepted by cells with N-
pentenoylmannosamine 2 (owing to the shorter acyl side
chain) being incorporated with higher efficiency. On the other
14195 Berlin-Dahlem (Germany)
[**] This work was supported by the Deutsche Forschungsgemeinschaft
(SFB 969), the University of Konstanz, the Konstanz Research
School Chemical Biology, the Wilhelm Sander-Stiftung (L.D.N.,
W.R.), and the Sonnenfeld-Stiftung, Berlin (L.D.N.). We thank Prof.
M. Wießler for helpful discussions regarding the Diels–Alder
chemistry and the Bioimaging Center of the University of Konstanz
and Prof. A. Zumbusch for providing the fluorescence microscopy
instrumentation.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201208991.
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Figure 1. Strategy for MOE using terminal alkenes as chemical reporter
groups.
Scheme 1. DARinv between pentenamide 7 and diaryltetrazine 8. Only
one tautomeric form of 9a/b is shown.
Figure 2. Mannosamine derivatives employed in MOE. Hxl=hexenoyl,
Peoc=pentenyloxycarbonyl, Ptl=pentenoyl.
hand, the hexenoyl side chain of mannosamine derivative 4
was expected to react faster in the DARinv owing to the
longer distance between the double bond and the electron-
withdrawing carbonyl group.
As a reaction partner for the alkenes, we selected
a diaryltetrazine because these compounds have been pre-
viously shown to be sufficiently stable against hydrolysis
which is an important prerequisite for bioorthogonal chemis-
try. In a model reaction, pentenamide 7 was reacted in DMSO
with tetrazine 8 to give dihydropyridazines 9a/b that each
exist in several tautomeric forms in virtually quantitative yield
(Scheme 1 and the Supporting Information). LC-MS analysis
revealed that the products are easily oxidized to pyridazines
10a/b. To complete this conversion, we treated 9a/b with
isopentyl nitrite and obtained pyridazines 10a/b in a yield of
78% after chromatographic purification.
Having shown that the coupling between a terminal
alkene and a tetrazine proceeds in excellent yield, we
investigated the kinetics of this ligation reaction. It is known
that the DARinv is much faster in water than in organic
solvents.[17] Thus, we attached a tri(ethylene glycol) linker to
obtain water-soluble tetrazine 11 (Figure 3). Employing 11,
quantitative kinetic measurements were carried out in
aqueous buffer with ManNPtl (1) and ManNHxl (3; see the
Supporting Information), and second-order rate constants
were determined to be 0.021 LmolÀ1 sÀ1 for the pentenoyl and
0.041 LmolÀ1 sÀ1 for the hexenoyl compound. Although these
numbers indicate a lower reaction rate compared to strained
cyclic alkenes, they are comparable to the rate constants of
Figure 3. Tetrazine and dibenzocyclooctyne derivatives.
the Staudinger ligation (k = 0.002 LmolÀ1 sÀ1 in CD3CN/
MeOH 95:5)[18] and SPAAC of a dibenzocyclooctyne (k =
0.0565 LmolÀ1 sÀ1 in MeOH),[19] which have both been
demonstrated to be extremely valuable bioorthogonal label-
ing reactions.[20]
To test the suitability of the new ManNAc derivatives for
MOE, different cell lines were cultured in the presence of
Ac4ManNPtl (2) and Ac4ManNHxl (4), respectively. Subse-
quently, the cells were labeled with tetrazine–biotin conjugate
12 followed by incubation with AlexaFluor-647-labeled
streptavidin (AF647-streptavidin). Figure 4a shows the
results obtained with HEK293T cells grown in the presence
of Ac4ManNPtl (2). Confocal fluorescence microscopy clearly
showed intense labeling of the cell membrane, as expected for
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Figure 5. Dual-labeling strategy for MOE employing alkene- and azide-
labeled monosaccharides followed by DARinv and SPAAC with two
different labels.
Figure 4. a) HEK293T cells were grown for two days with 100 mm
Ac4ManNPtl (2; left) or without 2 (right). Living cells were labeled with
1 mm tetrazine biotin 12 at 378C for 6 h followed by incubation with
AF647-streptavidin for 20 min at RT. b) HEK293T cells treated in the
same way as described under (a) but with Ac4ManNHxl (4) instead of
Ac4ManNPtl (2). c) HeLa S3 cells grown for 3 days with 100 mm
Ac4ManNPeoc (6; left) or without 6 (right), fixed with paraformalde-
hyde, and then treated as described under (a). Scale bar: 30 mm.
galactosamine (Ac4GalNAz, 14; Figure 5). Whereas N-acyl-
mannosamine derivatives, such as 2, are biosynthetically
converted into sialic acids that are found as terminal
structures of glycoproteins, Ac4GalNAz (14) has been
shown to be incorporated into mucin-type O-glycans.[5a] As
control experiments, only one sugar or no sugar was added to
the culture medium. After three days the cells were stained by
both DARinv (with tetrazine-biotin conjugate 12 followed by
incubation with AF647-streptavidin) and SPAAC (with
AlexaFluor 488-dibenzocyclooctyne 13, AF488-DIBO) and
investigated by confocal fluorescence microscopy.
this significantly sialylated cell line. Control experiments in
which cells were cultured in absence of 2 but otherwise
treated in the same way showed a negligible background
staining. This is an important result confirming that tetrazine–
biotin conjugate 12 does not react with other cellular
components, such as unsaturated cell-membrane lipids,
under these conditions. Corresponding results obtained with
HEK293T cells and Ac4ManNHxl (4) are shown in
Figure 4b. Interestingly, both sugars 2 and 4 lead
to about the same intensity of cell-membrane
staining. Similar results were obtained with HeLa
S3 cells (see the Supporting Information, Figur-
es S15 and S16).
When both sugars were present in the culture medium, we
detected a distinct labeling of the cell membrane in both
channels (Figure 6b), whereas omission of one of the sugars
So far, all mannosamine derivatives employed
for MOE were equipped with an acyl chain
connected to the 2-amino group.[21] To investigate
whether the biosynthetic machinery also accepts
other functional groups, we employed pentenyl
carbamate 6. A carbamate group has the advant-
age that it facilitates the synthetic access to
functionalized mannosamines derived from alco-
hols, such as pentenol. MOE experiments carried
out with HeLa S3 cells showed that Ac4ManNPeoc
(6) leads to similar staining intensity compared to
the experiments with sugars 2 and 4 (Figure 4c).
The second-order rate constant for DARinv of
ManNPeoc (5) with tetrazine 11 was determined
to be 0.017 LmolÀ1 sÀ1.
Figure 6. a) HeLa S3 cells were grown for three days. Living cells were labeled with
tetrazine–biotin 12 (1 mm) for 6 h at 378C followed by incubation with AF647–
streptavidin for 20 min at RT and with AF488-DIBO (4 mm) for 30 min at RT. b) Cells
were grown with 100 mm Ac4ManNPtl (2) and 25 mm Ac4GalNAz (14) and then
stained in the same way as described under (a). c) Cells were grown with 25 mm
Ac4GalNAz (14) and then stained in the same way as described under (a). d) Cells
were grown with 100 mm Ac4ManNPtl (2) and then stained in the same way as
We next turned our attention to the possibility
to employ DARinv in combination with SPAAC
for the concurrent detection of two different
metabolically incorporated sugars. HeLa S3 cells
were grown in presence of both alkene-labeled
Ac4ManNPtl (2) and peracetylated N-azidoacetyl- described under (a). Scale bar: 35 mm.
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Keywords: bioorthogonal reactions · carbohydrates ·
.
click chemistry · metabolic engineering · tetrazines
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