Triple Orthogonal Labeling of Glycans by Applying Photoclick Chemistry

Article abstract of DOI:10.1002/cbic.201800740

Bioorthogonal labeling of multiple biomolecules is of current interest in chemical biology. Metabolic glycoengineering (MGE) has been shown to be an appropriate approach to visualizing carbohydrates. Here, we report that the nitrile imine–alkene cycloaddi

Full text of DOI:10.1002/cbic.201800740

Accepted Article
Title: Triple Orthogonal Labeling of Glycans Applying Photoclick
Chemistry
Authors: Verena F. Schart, Jessica Hassenrück, Anne-Katrin Späte,
Jeremias E. G. A. Dold, Raphael Fahrner, and Valentin
Wittmann
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Link to VoR: http://dx.doi.org/10.1002/cbic.201800740
A Journal of
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10.1002/cbic.201800740
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Triple Orthogonal Labeling of Glycans Applying Photoclick Chemistry
Verena F. Schart,[a] Jessica Hassenrück,[a] Anne-Katrin Späte,[a] Jeremias E. G. A. Dold,[a]
Raphael Fahrner,[a] and Valentin Wittmann*[a]
Dedicated to Professor Chi-Huey Wong on the occasion of his 70th birthday
[a] Dr. V. F. Schart, J. Hassenrück, Dr. A.-K. Späte, J. E. G. A. Dold, R. Fahrner, 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
Supporting Information and the ORCID identification numbers for the authors of this
article can be found under https://xxxx.
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Abstract: Bioorthogonal labeling of multiple biomolecules is of current interest in chemical
biology. For the visualization of carbohydrates, metabolic glycoengineering (MGE) has been
shown to be an appropriate approach. Here, we report that the nitrile imine–alkene
cycloaddition (photoclick reaction) is a suitable ligation reaction in MGE. Using a
mannosamine derivative with an acrylamide reporter group, that is efficiently metabolized by
cells and that quickly reacts in the photoclick reaction, we achieved the labeling of sialic acids
on the surface of living cells. Screening of several alkenes unraveled that a previously
reported carbamate-linked methylcyclopropene reporter, that is well suited for the inverse-
electron-demand Diels-Alder (DAinv) reaction, has a surprisingly low reactivity in the
photoclick reaction. This enabled us to achieve for the first time a triple labeling of glycans by
the combination of DAinv, photoclick, and copper-free click chemistry.
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Bioorthogonal ligation reactions have become an important tool in the field of chemical
biology.[1] Especially the azide-alkyne cycloaddition[2] and the inverse-electron-demand
Diels-Alder (DAinv) reaction[3] have been intensively studied and employed for a wide range
of applications. During the last ten years, another ligation reaction, the nitrile imine–alkene
cycloaddition (photoclick reaction), gained attention in bioconjugation.[4] In this reaction, UV
irradiation of a tetrazole forms in situ a nitrile imine that rapidly reacts with an alkene
(Scheme 1A). Advantages of the photoclick reaction comprise a high reaction rate, the
formation of a fluorescent product, and the possibility to control the reaction in a spatial and
temporal manner by selective irradiation. So far, the reaction has been successfully applied to
label proteins,[4b-e,5] DNA,[6] and RNA,[7] while labeling of glycans, which represent another
class of essential biomolecules, has not yet been explored.
An important technique to image cellular carbohydrates is metabolic glycoengineering
(MGE).[8] In this approach, an unnatural monosaccharide derivatized with a chemical reporter
group is metabolized by cells or organisms and incorporated into glycoconjugates. The
unnatural reporter group can then be reacted in a bioorthogonal ligation reaction to visualize
or isolate the glycoconjugate. High labeling efficiency is achieved with carbohydrate
derivatives that both are well accepted by the enzymes involved in glycan biosynthesis and
that undergo a fast labeling reaction. Employing the DAinv reaction for labeling, however,
both criteria are often not met at the same time. For example, we could show that alkene-
modified ManNAc derivatives with shorter spacers between the alkene (acting as the
dienophile) and the electron-withdrawing carbonyl group are better accepted by the enzymes
but react slower in the DAinv reaction with tetrazines compared to derivatives with longer
chain lengths.[9] The lower DAinv reactivity of the electron-poor short-chain derivatives is in
line with a reaction control by a LUMOdiene–HOMOdienophile interaction. Electron-poor
alkenes, however, are the preferred substrates for the photoclick reaction[4d,10] and the small
acrylamide group reacts especially fast in this ligation reaction.[4d,5b] This prompted us to
investigate the photoclick reaction as an interesting expansion of the MGE toolbox.
Multiple-color imaging using bioorthogonal ligation reactions that are orthogonal to each
other is of current interest in biomolecular chemistry.[1c,11] The combination of click and
DAinv chemistry, for example, has been used many times including in MGE to achieve dual
labeling.[12] Examples, in which a triple labeling with three orthogonal reactions has been
achieved, however, are rare[13] and have not yet been reported in MGE. Prescher and
coworkers investigated the combination of the photoclick and the DAinv reaction to achieve
labeling of two species.[12d] They could show that 3,3-disubstituted cyclopropenes react
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readily with nitrile imines but not with tetrazines. In contrast, 1,3-disubstituted derivatives
react readily with both. An example of an alkene substrate that readily undergoes a DAinv
reaction but does not react in the photoclick reaction has not yet been reported.
Here we show that the photoclick reaction is a suitable ligation reaction for MGE.
Acrylamide-functionalized mannosamine derivative 1 (Scheme 1B) is efficiently metabolized
by cells due to its structural similarity to natural ManNAc and it quickly reacts with nitrile
imines allowing the visualization of sialic acids on the surface of living cells. Furthermore, we
found that the previously reported[12e,14] Ac4ManNCyoc (2), that is known to react readily
with tetrazines, has a surprisingly low reactivity in the photoclick reaction. This enabled us to
achieve for the first time a triple labeling of glycans by the combination of DAinv, photoclick,
and click chemistry.
For the synthesis of 1 we started from N-Boc-protected peracetylated mannosamine[15] that
was N-deprotected and subsequently reacted with acryloyl chloride as shown in the
Supporting Information (SI). To confirm that 1 is accepted by the biosynthetic pathway, we
applied it for MGE and analyzed the resulting sialic acids after labeling with 1,2-diamino-4,5-
methylenedioxybenzene (DMB) (Scheme 2, path A). In this technique, the sialic acids are
released from metabolically engineered cells by acetic acid treatment followed by selective
labeling of the α-keto acids with DMB.[16] The DMB-labeled sialic acids were analyzed by
RP-HPLC/MS and correlated to separately synthesized reference compounds (Figure S1). We
found that 43% of all released sialic acids bear the acrylamide functionality (Figures S2 and
S3), which demonstrates the high acceptance of this small reporter group by the enzymes. In
comparison, mannosamine derivatives modified with a bulky norbornene were found to be
incorporated with an efficiency of only 1%.[17] These findings also show that the acrylamide
group is stable during metabolization and not attacked by thiols in a Michael type addition.
This stability in the cellular environment is in line with results of the Liu lab.[4d,18] Being
stable in a cellular environment and featuring a high incorporation efficiency, Ac4ManNAcryl
(1) is a promising new derivative for MGE.
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A
N
Ar1
Ar2
N
Ar1
Ar2
N
h
R
N
Ar1
N N
Ar2
N
N
R
tetrazole
nitrile imine
pyrazoline
(fluorescent)
B
O
AcO
HN
O
AcO
AcO
OAc
1: Ac4ManNAcryl
O
O
AcO
AcO
AcO
AcO
AcO
AcO
HN
O
HN
O
O
OAc
OAc
2: Ac4ManNCyoc
3: Ac4ManNNorb
O
O
N3
AcO
AcO
AcO
AcO
HN
O
O
HN
O
AcO
AcO
OAc
OAc
4: Ac4ManNAloc
5: Ac4ManNAz
Scheme 1. A) Photoclick reaction of tetrazoles. B) Ac4ManNAcryl (1) and additional
mannosamine derivatives 2–5 for MGE.
Scheme 2. MGE strategy using DMB labeling to determine the incorporation efficiency (path
A) and the photoclick reaction as ligation reaction for visualization (path B).
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To be able to apply the photoclick reaction in MGE, we first developed suitable reaction
conditions. From the range of tetrazoles[4c,19] that have been used for varying applications, we
chose to use tetrazoles 6 and 7[20] (Scheme 3) because they are synthetically well accessible
and were previously successfully used in a biological environment.[5b,21] Furthermore, their
reaction with acrylamide in preparative scale has been reported to yield pyrazolines 8 and 9
upon irradiation at 302 nm in high yields,[10b] which we could confirm. The fluorescent
pyrazolines have an absorbance maximum at 360 nm (Figure S4) and fluorescence maxima at
445 nm (8) and 485 nm (9), respectively (Figure S5).
Scheme 3. Photoclick reaction of tetrazoles 6 and 7 to yield pyrazolines 8 and 9.
For an efficient screening of reaction conditions and of the reactivity of different substrates,
we exploited the fluorogenic character of the photoclick reaction and developed a plate reader
assay in a 96-well format with a fluorescent readout. A solution containing tetrazole (1 mM)
and alkene (100 µM) in ethanol was irradiated at 302 nm with a UV lamp. The relative
amount of formed pyrazolines was then quantified by determination of the fluorescence
intensity. We first varied the irradiation time between 10 s and 5 min using Ac4ManNAcryl
(1) as reaction partner (Figure S6) and found that an irradiation of one minute is sufficient and
also compatible with biological experiments. Next, a suitable solvent needed to be determined
because pure ethanol is cytotoxic. A well-established solvent for cell experiments is
phosphate-buffered saline (PBS) that maintains the osmotic pressure of the cells. PBS,
however, has a high content of chloride ions which were reported to slow down the photoclick
reaction.[22] Thus, we investigated the efficiency of the photoclick reaction in water and PBS
and found both solvents to be similarly suited (Figure S7).
We next applied the plate reader assay to compare the reactivity of different carbohydrate
derivatives in the photoclick reaction. Beside Ac4ManNAcryl (1) we employed
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Ac4ManNCyoc (2) and Ac4ManNNorb (3) (see SI for its synthesis). Norbornenes[4e,23] and
several amide-linked methylcyclopropenes[5b,12d,24] are known to undergo photoclick reaction.
However, carbamate-linked methylcyclopropenes, such as 2, have not yet been investigated in
this context. We also included the terminal alkene Ac4ManNAloc (4)[9a] because allyl ethers
have been applied for the photoclick reaction although with varying efficiency.[4d,7,21]
We detected remarkable differences in the reactivities of the sugars 1–4 in the photoclick
reaction with tetrazole 6 (Figure 1). The highest fluorescence signal was detected for
Ac4ManNAcryl (1). The signal intensity observed with Ac4ManNNorb (3) is less than half of
it which is, however, still well above background. Surprisingly, Ac4ManNCyoc (2) resulted in
only very low intensity. Hardly any signal was detected for Ac4ManNAloc (4). Similar trends
were obtained with tetrazole 7 (Figure S8). The unexpected low fluorescence intensity
obtained with cyclopropene derivative 2 is remarkable because all previously tested
cyclopropenes reacted efficiently in the photoclick reaction.[5b,12d,24] This lack of reactivity of
Ac4ManNCyoc (2) in the photoclick reaction prompted us to investigate whether it could be
exploited to achieve a dual or even triple orthogonal labeling of carbohydrates.
rel. F
)
)
)
)
4
(
e
1
2
3
v
(
(
(
i
t
l
c
c
y
b
a
r
o
r
o
o
g
l
c
y
C
e
A
A
N
n
N
N
N
N
n
n
n
n
a
a
a
a
M
M
M
M
4
c
4
4
c
4
c
c
A
A
A
A
Figure 1. Results of the plate reader assay with different alkene-modified mannosamines.
Solutions containing 1 mM tetrazole 6 and 100 µM carbohydrate in ethanol were irradiated
for 1 min (302 nm). In the negative control, the carbohydrate was omitted. Fluorescence was
detected at 445 nm upon excitation at 360 nm. Two independent experiments with two
replicates each were measured.
Next, we established the application of the photoclick reaction in MGE. Even though the
photoclick reaction generates fluorescent pyrazolines, we decided to apply a two-step labeling
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10.1002/cbic.201800740
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procedure because it allows the use of dyes that are better suited for fluorescence microscopy
of living cells. Thus, we synthesized tetrazole-biotin derivatives 10 (Figure 2) and 11 (SI) that
– after photoclick reaction – can be labeled with streptavidin-AlexaFluor® 555 (streptavidin-
AF555). Both derivatives feature an amino-oligo-ethylene glycol (OEG)-linker to ensure
solubility. Their synthesis started from tetrazoles 6 and 7, respectively, as shown in the SI.
The UV spectra of both derivatives were similar to those of 6 and 7 (Figure S4).
Figure 2. Structure of tetrazole-biotin 10.
For the MGE experiments we used Ac4ManNAcryl (1) and Ac4ManNNorb (3) because of
their high photoclick reactivity and Ac4ManNCyoc (2) to test whether its low reactivity
according to the plate reader assay can be exploited for orthogonal labeling. HEK 293T cells
were grown with the respective monosaccharide, treated with tetrazole-biotin 10[25] and
irradiated for 1 min by UV light (302 nm) followed by incubation at room temperature for 15
min. Then, the glycoconjugates were visualized by addition of streptavidin-AF555 (Scheme 2,
path B) and imaged by confocal fluorescence microscopy (Figure 3). Ac4ManNAcryl (1)
resulted in a clear membrane staining while both Ac4ManNCyoc (2) and Ac4ManNNorb (3)
showed no significant fluorescence similar to that of the negative control. Since
Ac4ManNNorb (3) according to the plate reader assay (Figure 1) is reasonably reactive in the
photoclick reaction, the lack of membrane staining is likely due to a low incorporation
efficiency. Interestingly, although it has been recently reported, that the nitrile imines formed
during the photoclick reaction can react with a variety of nucleophiles present in a biological
system,[26] we observed only low background signal under the conditions used.
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Figure 3. Confocal fluorescence microscopy images of labeled cells using the photoclick
reaction. HEK 293T cells were incubated with 100 µM Ac4ManNAcryl (1), Ac4ManNCyoc
(2) or Ac4ManNNorb (3) for 2 days. As solvent control, DMSO was added. For visualization
cells were reacted with tetrazole-biotin 10 (1 min, 302 nm; 15 min, rt) followed by
streptavidin-AF555. Nuclei were stained with Hoechst 33342. Scale bar 30 µm.
Since Ac4ManNCyoc (2) does not lead to a membrane staining after photoclick reaction and
Ac4ManNAcryl (1) does not react in the DAinv reaction (Figure S9), we were able to use the
two carbohydrates in an orthogonal dual labeling strategy (Figure S10). Furthermore, we
could show that the photoclick reaction is also orthogonal to the strain-promoted azide-alkyne
cycloaddition by combining Ac4ManNAcryl (1) and Ac4ManNAz (5) in an MGE experiment
(Figure S11). These results set the stage for the exploration of a triple orthogonal labeling of
glycans combining all three derivatives 1, 2, and 5 (Figure 4A). Thus, we grew HEK 293T
cells with 80 µM Ac4ManNAcryl (1), 100 µM Ac4ManNCyoc (2), and 10 µM Ac4ManNAz
(5). The different concentrations were chosen to obtain comparable cell surface staining
intensities in all three channels. For triple labeling, we first performed the DAinv reaction
using a tetrazine-Cy5 conjugate and the click reaction using a dibenzocyclooctyne-
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AlexaFluor® 488 conjugate (DIBO-AF488) (for formulas see SI page S3) simultaneously,
followed by the photoclick reaction. As shown in Figure 4B, first row, a clear membrane
staining was detected in all three channels. Omission of one of the sugars resulted in missing
membrane staining in the respective channel (rows 2–4) confirming the absence of cross
reactivity. When pure DMSO was added (negative control), membrane staining could not be
detected in any channel. With these experiments, a triple labeling was achieved for the first
time in MGE.
In summary, we have demonstrated that the photoclick reaction can be used to visualize cell-
surface carbohydrates of metabolically engineered living cells. Ac4ManNAcryl (1), which is
modified with the smallest chemical reporter group used in MGE so far, turned out to be
metabolically stable enough to be incorporated into sialoglycans with high efficiency and
subsequently be labeled with a tetrazole derivative. Labeling by the photoclick reaction is not
only a useful expansion of the toolbox of labeling reactions that can be used to visualize
carbohydrates. Due to the possibility to achieve spatiotemporal control by selective
irradiation, the reaction opens new avenues in studying carbohydrate-dependent processes in a
space- and time-resolved manner. The surprising observation that Ac4ManNCyoc (2) has only
a low reactivity in the photoclick reaction enabled us to achieve a triple labeling in MGE for
the first time. Since the simultaneous visualization of multiple biomolecules is of general
interest in chemical biology, we expect the triple-labeling approach to have many potential
applications.
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Figure 4. A) Triple labeling strategy combining DAinv, click, and photoclick chemistry. B)
Confocal fluorescence microscopy of triply labeled cells. HEK 293T cells were incubated
with Ac4ManNAz (5, 10 µM), Ac4ManNCyoc (2, 100 µM) and Ac4ManNAcryl (1, 80 µM)
for 48 h. As controls, two sugars or DMSO only was added. For labeling, cells were reacted
with DIBO-AF488 (20 µM, 1 h, 37 °C) and tetrazine-Cy5 (40 µM, 1 h, 37 °C) followed by
addition of tetrazole-biotin 10 (1 min, 302 nm; 15 min, rt) and streptavidin-AF555. Nuclei
were stained with Hoechst 33342. Scale bar 30 µm.
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Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (SPP 1623 and SFB 969,
project B05), the University of Konstanz and the Konstanz Research School Chemical
Biology (stipend for RF). We thank Felix Englmaier for synthetic support and the Bioimaging
Center of the University of Konstanz for providing the fluorescence microscopy
instrumentation.
Keywords: bioorthogonal reactions • carbohydrates • click chemistry • metabolic engineering
• tetrazines
_______________
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Entry for the table of contents
All good things come in threes: The nitrile imine–alkene cycloaddition (photoclick reaction)
was shown to be a suitable ligation reaction for metabolic glycoengineering. In combination
with the inverse-electron-demand Diels-Alder reaction and the strain-promoted azide-alkyne
cycloaddition, a triple labeling of glycans was achieved for the first time.
This article is protected by copyright. All rights reserved.