Terminal Alkenes as Versatile Chemical Reporter Groups for Metabolic Oligosaccharide Engineering

Article abstract of DOI:10.1002/chem.201404716

The Diels-Alder reaction with inverse electron demand (DAinv reaction) of 1,2,4,5-tetrazines with electron rich or strained alkenes was proven to be a bioorthogonal ligation reaction that proceeds fast and with high yields. An important application of the DAinv reaction is metabolic oligosaccharide engineering (MOE) which allows the visualization of glycoconjugates in living cells. In this approach, a sugar derivative bearing a chemical reporter group is metabolically incorporated into cellular glycoconjugates and subsequently derivatized with a probe by means of a bioorthogonal ligation reaction. Here, we investigated a series of new mannosamine and glucosamine derivatives with carbamate-linked side chains of varying length terminated by alkene groups and their suitability for labeling cell-surface glycans. Kinetic investigations showed that the reactivity of the alkenes in DAinv reactions increases with growing chain length. When applied to MOE, one of the compounds, peracetylated N-butenyloxycarbonylmannosamine, was especially well suited for labeling cell-surface glycans. Obviously, the length of its side chain represents the optimal balance between incorporation efficiency and speed of the labeling reaction. Sialidase treatment of the cells before the bioorthogonal labeling reaction showed that this sugar derivative is attached to the glycans in form of the corresponding sialic acid derivative and not epimerized to another hexosamine derivative to a considerable extent. Shine bright: Metabolic oligosaccharide engineering with peracetylated N-butenyloxycarbonylmannosamine 1 (n=2) leads to cell-surface glycoconjugate staining with higher intensity compared with previously reported terminal alkene reporters. Analogues with shorter (n=1) or longer (n=3-4) side chains are much less efficient for labeling.

Full text of DOI:10.1002/chem.201404716

DOI: 10.1002/chem.201404716
Full Paper
&
Bioorthogonal Chemistry
Terminal Alkenes as Versatile Chemical Reporter Groups for
Metabolic Oligosaccharide Engineering
Anne-Katrin Spꢀte, Verena F. Schart, Sophie Schçllkopf, Andrea Niederwieser, and
Valentin Wittmann*[a]
Abstract: The Diels–Alder reaction with inverse electron
demand (DAinv reaction) of 1,2,4,5-tetrazines with electron
rich or strained alkenes was proven to be a bioorthogonal li-
gation reaction that proceeds fast and with high yields. An
important application of the DAinv reaction is metabolic oli-
gosaccharide engineering (MOE) which allows the visualiza-
tion of glycoconjugates in living cells. In this approach,
a sugar derivative bearing a chemical reporter group is met-
abolically incorporated into cellular glycoconjugates and
subsequently derivatized with a probe by means of a bioor-
thogonal ligation reaction. Here, we investigated a series of
new mannosamine and glucosamine derivatives with carba-
mate-linked side chains of varying length terminated by
alkene groups and their suitability for labeling cell-surface
glycans. Kinetic investigations showed that the reactivity of
the alkenes in DAinv reactions increases with growing chain
length. When applied to MOE, one of the compounds, pera-
cetylated N-butenyloxycarbonylmannosamine, was especially
well suited for labeling cell-surface glycans. Obviously, the
length of its side chain represents the optimal balance be-
tween incorporation efficiency and speed of the labeling re-
action. Sialidase treatment of the cells before the bioorthog-
onal labeling reaction showed that this sugar derivative is at-
tached to the glycans in form of the corresponding sialic
acid derivative and not epimerized to another hexosamine
derivative to a considerable extent.
Introduction
field of bioorthogonal ligation reactions has been greatly ad-
vanced by the development of the inverse-electron-demand
Diels–Alder reaction (DAinv reaction) of 1,2,4,5-tetrazines and
electron rich or strained alkenes.[6]
Visualization of biomolecules is an important prerequisite for
fundamental biological as well as medical research. A popular
approach is the direct installation of fluorescent tags onto bio-
molecules.[1] However, since fluorescence dyes can be rather
bulky and might influence the properties of the target mole-
cule, the chemical reporter strategy[2] has gained more and
more importance to monitor biomolecules. In this strategy,
a small functional group, the chemical reporter, is (often meta-
bolically) incorporated into the biomolecule and in a subse-
quent step derivatized with the fluorescence probe by means
of a bioorthogonal ligation reaction.[3] Ideally, the chemical re-
porter is small and does not alter the function and properties
of the biomolecule (e.g., protein, DNA or carbohydrate). Fre-
quently employed bioorthogonal ligation reactions include the
Staudinger ligation[4] and the azide–alkyne cycloaddition[5]
(often referred to as “click” reaction). Within the last years, the
The DAinv reaction does not require metal catalysts and pro-
ceeds fast and in high yields, especially under aqueous condi-
tions.[7] Furthermore, the reaction was shown to be bioorthog-
onal and can be orthogonal to the azide–alkyne cycloaddition
which enables dual-labeling strategies.[8] In the past, a number
of dienophiles have been employed for DAinv reactions with
1,2,4,5-tetrazines,[9] among them trans-cyclooctenes,[6a,8b,10] nor-
bornenes,[6b,8c,10d,11] cyclobutenes,[6c,12] and cyclooctynes.[10c,d,13]
Beside protein modification the reaction has been used in
a whole range of applications[6e] including DNA modifica-
tion[11b,14] and microarray preparation.[11d,15]
The DAinv reaction and related tetrazine ligations have also
been appreciated for labeling metabolically engineered oligo-
saccharides. Metabolic oligosaccharide engineering (MOE) is
a valuable technique to visualize cellular glycoconjugates,
which has been challenging for a long time. MOE relies on the
promiscuity of cellular enzymes that can accept and thus incor-
porate unnatural sugar derivatives into glycoconjugates.[16] If
derivatized with a chemical reporter group, the incorporated
sugar can be ligated to a probe.[17] Reporter groups suitable
for MOE do not only need to be metabolically stable but must
also not exceed a certain size limit in order to be accepted by
the enzymes. In the past, a plethora of investigations has been
carried out with azides and alkynes but recently, also tetrazine
ligations were used. Since the fast-reacting trans-cyclooctene-
[a] A.-K. Spꢀte,+ V. F. Schart,+ S. Schçllkopf, Dr. A. Niederwieser,
Prof. Dr. V. Wittmann
University of Konstanz
Department of Chemistry and
Konstanz Research School Chemical Biology (KoRS-CB)
78457 Konstanz (Germany)
Fax: (+49)7531-88-4573
E-mail: mail@valentin-wittmann.de
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201404716.
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and norbornene-based dienophiles are expected to be too
bulky for application in MOE, smaller probes, such as cyclopro-
penes[8d,f,18] and isonitriles[8g,19] have been developed. Recently,
we have shown that terminal alkenes are suitable reporter
groups for MOE, and ManNAc derivatives 1 and 2 (Figure 1)
with a pentenoyl and hexenoyl side chain, respectively, are ac-
cepted by the biosynthetic pathway of HEK 293T and HeLa S3
cells.[8e] Together with an azide-labeled second sugar we were
able to employ Ac4ManNPtl 1 in a dual-labeling strategy. Fur-
thermore, we could show that carbamate derivatives, such as
Ac4ManNPeoc 5 are also well tolerated by the enzymatic ma-
chinery[8e] which is in line with observations by others.[18b,20] Al-
though the rate of the DAinv reaction of pentenoyl and hexe-
noyl groups with a phenyl-(pyrimidin-2-yl)-tetrazine does not
reach the value of the reaction of cyclopropenes and isoni-
triles, it is still one order of magnitude higher than the typical
rate constant[21] of the Staudinger ligation. In addition, terminal
alkenes are expected to be more stable than strained cyclic al-
kenes and they are easier accessible. Thus, they might be pre-
ferred probes in cases where fast reaction rates are not re-
quired.
ability of a ManNAc derivative for glycan labeling after MOE
and the optimal length is not easily predictable.
Here, we present the series 3–6 of ManNAc derivatives fea-
turing carbamate-linked terminal alkenes with varying chain
length and their suitability for labeling cell-surface glycans. Bu-
tenyloxycarbonyl-derivatized mannosamine 4 turned out to
have the perfect balance between metabolic incorporation effi-
ciency and DAinv reactivity for labeling cell-surface sialic acids
and leads to significantly improved labeling efficiency com-
pared to previously reported ManNAc derivative 5.
Results and Discussion
Synthesis of Mannosamine Derivatives
The synthesis of mannosamine derivatives 3–6 is depicted in
Scheme 1. Mannosamine hydrochloride 7 was neutralized with
NaOMe solution and reacted with alkenyl succinimidyl carbo-
nates 8–11. The crude products were peracetylated to yield
mannosamine derivatives 3–6 in yields between 50 and 80%.
For subsequent kinetic studies in acetate buffer, the com-
pounds were de-O-acetylated using ethyldimethylamine in
methanol to give carbohydrates 12–15.
Figure 1. Mannosamine derivatives for MOE. Aloc = allyloxycarbonyl, Beoc
= butenyloxycarbonyl, Peoc = pentenyloxycarbonyl, Heoc = hexenyloxycar-
bonyl.
Scheme 1. Synthesis of mannosamine derivatives 3–6 and deacetylation to
12–15.
DAinv reactions are controlled by a LUMOdiene–HOMOdienophile
interaction in the transition state.[22] Efficient reactions require
a high HOMO energy of the dienophile which arises from elec-
tron-donating substituents at the double bond. Electron-with-
drawing substituents, on the other hand, lower the HOMO
energy and, thus, decrease the DAinv reaction rate with
a given diene. It can be expected that carbamates of type 3–6
show a higher DAinv reactivity with an increasing distance be-
tween the double bond and the carbamate group with its in-
ductive (ÀI) effect (i.e., with increasing chain length). On the
other hand, an important feature of a sugar derivative suitable
for use in MOE is its acceptance by the cells’ biosynthetic ma-
chinery, and it was shown that ManNAc derivatives with longer
acyl chains than the natural acetyl group are metabolized with
reduced efficiency.[23] Taken together, varying the length of the
acyl chain is expected to have two opposite effects on the suit-
Kinetic Studies
To determine second-order rate constants of different terminal
alkenes in DAinv reactions, we selected water-soluble phenyl-
(pyrimidin-2-yl)-tetrazine 16[8e] as reaction partner (Scheme 2)
because this type of tetrazine had been proven to be suited
for a number of applications[6c,10d,11b,d] including MOE[8e] due to
its reactivity and, at the same time, stability in aqueous media.
For the measurements we exploited the fact that tetrazine 16
has a characteristic absorption maximum at 522 nm. Thus, the
DAinv reaction could be followed by measuring the decrease
of absorbance of 16 over time. Second-order rate constants k2
were calculated as reported earlier[8e] (see Figure 2 for repre-
sentative data points and Table 1 for calculated rate con-
stants).
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Table 1. Second-order rate constants k2 of DAinv reaction of tetrazine 16
with terminal alkenes.
Entry
Alkene
k2 [mÀ1 sÀ1
]
1
2
3
4
0.002Æ0.0002
0.011Æ0.001
0.034Æ0.003
0.080Æ0.006
5
6
7
8
ManNAloc 12
ManNBeoc 13
ManNPeoc 14
ManNHeoc 15
0.0015Æ0.0001
0.014Æ0.003
0.017Æ0.002[8e]
0.074Æ0.013
Scheme 2. DAinv reaction between terminal alkenes (alkenols or mannosa-
mine derivatives 12–15) and tetrazine 16. Only one isomer of the product is
shown.
Figure 2. Kinetics of reaction of alkenols (5 mm) with tetrazine 16 (5 mm) in
100 mm acetate buffer (pH 4.8) monitored by measuring the decrease of ab-
^
sorption at 522 nm of 16 over time. A=absorbance at 522 nm, =allyl alco-
~
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*
hol, =butenol, =pentenol, =hexenol.
In orienting experiments, we first determined the second-
order rate constants k2 of four alkenols (allyl alcohol, butenol,
pentenol, and hexenol) with tetrazine 16 (Table 1, entries 1–4).
As expected, the rate constant increases with growing distance
between the alkene and the electron-withdrawing oxygen cor-
responding to a higher HOMO energy of the dienophile. The
biggest increase of k2 by a factor of 5.5 is observed between
allyl alcohol and butenol. Further elongation of the carbon
chain to pentenol and hexenol leads to a stepwise increase of
k2 by factors of 3.1 and 2.4, respectively. The same trend is ob-
served for the series of mannosamine derivatives 12–15 (en-
tries 5–8). In this case k2 increases by an factor of 9.3 when
going from the Aloc (entry 5) to the Beoc derivative (entry 6).
Further chain elongation ends up in a rate constant of
0.074mÀ1 sÀ1 for ManNHeoc 15 (entry 8) comparable to the k2
Scheme 3. DAinv reaction of alkene 17 and tetrazine 18[11d] followed by oxi-
dation to yield pyridazines 20a/b.
sponding pyridazines 20a/b (Figure S1). To facilitate NMR char-
acterization, the oxidation was completed by the addition of
isopentyl nitrite to give the pyridazines 20a/b in a yield of
77%.
Detection of Cell-Surface Oligosaccharides
To examine the suitability of the alkene-labeled mannosamine
derivatives 3–6 for labeling cell-surface oligosaccharides, the
sugars were applied for MOE. HEK 293T cells were grown in
the presence of one of the monosaccharides 3–6. Subsequent-
ly, the cells were incubated with tetrazine-biotin (Tz-biotin)
21[8e] (Figure 3) in order to react incorporated alkenes. After la-
beling with streptavidin-AlexaFluor647, confocal fluorescence
laser-scanning microscopy showed cell membrane staining for
all mannosamine derivatives (Figure 4B–E) while no staining
was detected when only DMSO was added to the cells (Fig-
ure 4A). Interestingly, Ac4ManNAloc 3 showed a weak mem-
brane staining even though its reactivity in the DAinv reaction
is rather low (Figure 4B). For Ac4ManNBeoc 4 the most intense
value of hexenol of 0.08mÀ1 À1 (entry 4).
s
DAinv Reaction in Preparative Scale
To characterize the product and reaction yield of a DAinv reac-
tion with a carbamate-modified terminal alkene, we choose
model carbamate 17 and tetrazine 18[11d] as reaction partners
and performed a DAinv reaction in DMSO (Scheme 3). During
this reaction, two isomeric dihydropyridazines 19a/b, that exist
in several tautomeric forms, were formed. LC-MS analysis re-
vealed that these products were partially oxidized to the corre-
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labeling was obtained (Figure 4C), which was significantly
brighter than the one for both Ac4ManNPeoc 5 (Figure 4D)
and Ac4ManNHeoc 6 (Figure 4E) even though 5 and 6 react
faster in the DAinv reaction. A plausible explanation for this
observation is a more efficient incorporation of Beoc derivative
4 having a shorter side chain than derivatives 5 and 6 that
overcompensates its lower reactivity. Within the series of sugar
derivatives 3–6, clearly Ac4ManNBeoc 4 is best suited for label-
ing cell-surface sialic acids.
corporated mannosamine derivative 4 is indeed converted into
a sialic acid and attached to glycans rather than being incorpo-
rated as glucosamine or galactosamine derivate. Thus, HEK
293T cells that had been cultivated with 100 mm Ac4ManNBeoc
4 for 48 h were incubated with sialidase (10 mUmLÀ1, 1 h,
378C) prior to staining. Since no cell-membrane staining was
detected after sialidase treatment (Figure 5B), we conclude,
that epimerization of compound 4 to the corresponding glu-
cosamine or galactosamine derivatives had not taken place to
a considerable extent (Figure 5).
Since monosaccharides can be isomerized in cells by action
of several epimerases,[20,24] we wanted to confirm that the in-
Figure 3. Structure of Tz-biotin 21.
Figure 5. HEK 293T cells were grown with 100 mm Ac4ManNBeoc 4 (A, B) or
with DMSO (C) for 48 h. For removal of the sialic acids, cells were treated
with sialidase (10 mU mLÀ1, 1 h, 378C) (B). Staining was achieved by incuba-
tion with Tz-biotin 21 (1 mm, 6 h, 378C) followed by addition of streptavi-
din-AlexaFluor647. Nuclei were stained with Hoechst33342. Scale bar:
30 mm.
Synthesis of Alkene-Modified Glucosamine Derivatives
Previously, it has been shown[25] that N-acetylglucosamine de-
rivatives with a reporter group in the acyl chain, such as N-azi-
doacetylglucosamine, are suitable probes to label O-GlcNAcy-
lated intracellular proteins[26] employing MOE. Thus, to extend
possible applications of terminal alkenes, we prepared the
series 23–26 of carbamate-modified glucosamine derivatives
with varying chain length starting from glucosamine hydro-
chloride 22 (Scheme 4). We employed all four glucosamine de-
rivatives for MOE, however, we experienced cell toxicity for all
compounds at the concentrations necessary for detection of
cell-surface glycans.
Figure 4. HEK 293T cells were grown with DMSO (A) or with 100 mm
Ac4ManNAloc 3 (B), Ac4ManNBeoc 4 (C), Ac4ManNPeoc 5 (D), and
Ac4ManNHeoc 6 (E), respectively, for 48 h. For visualization, cells were incu-
bated with Tz-biotin 21 (1 mm, 6 h, 378C) followed by streptavidin-Alexa-
Fluor647 (20 min, 378C). Nuclei were stained with Hoechst33342. Scale bar:
30 mm.
Conclusion
In summary, we present a series of new mannosamine and glu-
cosamine derivatives with carbamate-linked side chains of
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General Procedure for the Synthesis of Sugar Carbamates
The hexosamine hydrochloride (1 equiv) was dissolved in MeOH,
and NaOMe (0.5m in MeOH, 1 equiv) was added under nitrogen.
The reaction mixture was stirred at room temperature for 2 h. A so-
lution of the alkenyl succinimidyl carbonate (1.2 equiv) in MeOH
was added and the reaction mixture was stirred for 16 h. The sol-
vent was evaporated and the residual oil was dissolved in pyridine
(10 equiv) and acetic anhydride (10 equiv), and the reaction mix-
ture was stirred for 16 h. Then, the solvent was removed and the
residual oil was dissolved in CH2Cl2 (100 mL) and washed with 10%
aq. KHSO4 (2ꢂ100 mL), sat. aq. NaHCO3 (2ꢂ100 mL), and brine
(100 mL). The organic layer was dried (MgSO4) and the solvent
evaporated. The crude product was purified by FC.
Scheme 4. Synthesis of glucosamine derivatives 23–26.
varying length terminated by alkene groups. Kinetic investiga-
tions showed that the reactivity of the alkenes in DAinv reac-
tions increases with growing chain length. When applied to
MOE, all mannosamine derivatives could be employed to label
cell-surface carbohydrates on living HEK 293T cells, though
with substantially differing intensity as detected by confocal
fluorescence microscopy. Whereas the compound with the
shortest side chain (Ac4ManNAloc 3) gave only a faint staining
of the plasma membrane, probably due to its low reactivity in
the DAinv labeling reaction, the compounds Ac4ManNHeoc 6
and Ac4ManNPeoc 5 with longer side chains resulted in some-
what more intense labeling. Ac4ManNBeoc 4, however, clearly
gave the brightest staining. Obviously, the length of its side
chain represents the optimal balance between incorporation
efficiency and speed of the labeling reaction. Sialidase treat-
ment of the cells before the bioorthogonal labeling reaction
showed that Ac4ManNBeoc 4 is attached to the glycans in
form of the corresponding sialic acid derivative and not epi-
merized to another hexosamine derivative to a considerable
extent.
1,3,4,6-Tetra-O-acetyl-2-((allyloxycarbonyl)amino)-2-deoxy-d-
mannopyranose (Ac4ManNAloc) (3): Mannosamine hydrochloride
7 (500 mg, 2.31 mmol) and allyl succinimidyl carbonate 8 (574 mg,
2.88 mmol) were reacted in MeOH (20 mL) according to the gener-
al procedure. The crude product was purified by FC (petroleum
ether/ethyl acetate 1:1). To remove remaining N-hydroxysuccini-
mide (NHS), the combined product fractions were evaporated, re-
dissolved in CH2Cl2 and extracted three times with 1n NaOH.
Ac4ManNAloc 3 was obtained as mixture of anomers (0.5 g, 50%,
a/b 2:1). Rf =0.46 (petroleum ether/ethyl acetate 1:1). a-Anomer:
1H NMR (400 MHz, CDCl3): d=6.09 (d, J=1.9 Hz, 1H, H-1), 5.93
(ddt, J=16.6, 11.5, 5.9 Hz, 1H, CHCH2), 5.39–5.10 (m, 4H, H-4, NH,
CHCH2), 5.09–4.98 (m, 1H, H-3), 4.58 (dt, J=5.7, 1.5 Hz, 2H, CH2),
4.35 (dd, J=9.8, 4.3 Hz, 1H, H-2), 4.30–4.20 (m, 1H, H-6a), 4.17–
3.96 (m, 2H, H-5, H-6b), 2.18 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.06 (s,
3H, CH3), 2.02 (s, 3H, CH3). b-Anomer: 1H NMR (400 MHz, CDCl3):
d=5.93 (ddt, J=16.6, 11.5, 5.9 Hz, 1H, CHCH2), 5.85 (d, J=1.8 Hz,
1H, H-1), 5.39–5.10 (m, 4H, H-4, NH, CHCH2), 5.09–4.98 (m, 1H, H-
3), 4.58 (dt, J=5.7, 1.5 Hz, 2H, CH2), 4.48 (dd, J=8.2, 3.4 Hz, 1H, H-
2), 4.30–4.20 (m, 1H, H-6a), 4.17–3.96 (m, 1H, H-6b), 3.78 (ddd, J=
9.8, 4.9, 2.6 Hz, 1H, H-5), 2.12 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.06 (s,
3H, CH3), 2.02 (s, 3H, CH3). a/b-Anomers: 13C NMR (101 MHz,
CDCl3): d=170.7, 170.7, 170.2, 170.2, 169.7, 168.2, 155.8 (COa,b),
132.7 (CHCH2b), 132.5 (CHCH2a), 118.5 (CHCH2a), 118.1 (CHCH2b),
92.0 (C-1a), 90.8 (C-1b), 73.5, 71.7, 70.3, 69.3, 66.3, 66.2, 65.4, 65.3
(C-3a,b, C-4a,b, C-5a,b, CH2a,b), 62.0 (C-6a), 61.9 (C-6b), 51.3 (C-
2a,b), 21.0, 21.0, 20.9, 20.9, 20.8, 20.8 (4x CH3a,b); HRMS: m/z calcd
for C18H25NO11: 454.13198 [M+Na]+, found: 454.13046.
Experimental Section
General Methods
Chemicals were purchased from Aldrich, Acros Organics, Fluka, and
Dextra and used without further purification. AlexaFluor 647-la-
beled streptavidin and Hoechst33342 were purchased from Invitro-
gen. Technical solvents were distilled prior to use. All reactions
were carried out in dry solvents. Thin-layer chromatography (TLC)
was performed on silica gel 60 F254 coated aluminum sheets
(Merck) with detection by UV light (l=254 nm). Additionally, the
sheets were stained by dipping in acidic ethanolic p-anisaldehyde
solution or basic KMnO4 solution followed by gentle heating. Prep-
arative flash column chromatography (FC) was performed with an
MPLC-Reveleris system from Grace. Nuclear magnetic resonance
(NMR) spectra were recorded at room temperature on Avance III
400 and Avance III 600 instruments from Bruker. Chemical shifts
are reported relative to solvent signals (CDCl3: dH =7.26 ppm, dC =
77.16 ppm). Signals were assigned by first-order analysis and,
when feasible, assignments were supported by two-dimensional
1,3,4,6-Tetra-O-acetyl-2-((but-3-en-1-yl-oxycarbonyl)amino)-2-
deoxy-d-mannopyranose (Ac4ManNBeoc) (4): Mannosamine hy-
drochloride 7 (840 mg, 3.9 mmol) and but-3-en-1-yl succinimidyl
carbonate 9 (1 g, 4.7 mmol) were reacted in MeOH (30 mL) accord-
ing to the general procedure. The crude product was purified by
FC (petroleum ether/ethyl acetate 1:2). To remove remaining NHS,
the combined product fractions were evaporated, redissolved in
CH2Cl2 and extracted three times with 2n NaOH. Ac4ManNBeoc 4
was obtained as mixture of anomers (1.4 g, 80%, a/b 2:1). Rf =0.37
1
(petroleum ether/ethyl acetate 1:1). a-Anomer: H NMR (400 MHz,
CDCl3): d=6.09 (d, J=1.5 Hz, 1H, H-1), 5.83–5.72 (m, 1H, CHCH2),
5.31 (dd, J=10.2, 4.3 Hz, 1H, H-3), 5.23–5.05 (m, 3H, H-4, CHCH2),
5.02 (d, J=10.1 Hz, 1H, NH), 4.34 (dd, J=9.6, 3.0 Hz, 1H, H-2),
4.30–4.20 (m, 1H, H-6a), 4.20–4.06 (m, 3H, H-6b, OCH2), 4.04–3.97
(m, 1H, H-5), 2.46–2.31 (m, 2H, CH2), 2.18 (s, 3H, CH3), 2.10 (s, 3H,
CH3), 2.06 (s, 3H, CH3), 2.03 (s, 3H, CH3). b-Anomer: 1H NMR
(400 MHz, CDCl3): d=5.84 (d, J=1.9 Hz, 1H, H-1), 5.83–5.72 (m, 1H,
CHCH2), 5.24–5.05 (m, 4H, H-3, H-4, CHCH2), 5.03 (d, J=9.7 Hz, 1H,
NH), 4.47 (d, J=9.0 Hz, 1H, H-2), 4.30–4.20 (m, 1H, H-6a), 4.20–4.06
(m, 3H, H-6b, OCH2), 3.78 (ddd, J=9.6, 4.9, 2.5 Hz, 1H, H-5), 2.46–
2.31 (m, 2H, CH2), 2.12 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.06 (s, 3H,
CH3), 2.04 (s, 3H, CH3). a/b-Anomers: 13C NMR (101 MHz, CDCl3):
1
1H,1H and H,13C correlation spectroscopy (COSY, HMBC and HSQC).
High-resolution mass spectrometry (HRMS) was carried out on a mi-
crOTOF II instrument from Bruker Daltonics. UV/Vis Absorption was
measured using a Carry 50 instrument from Varian and software
scanning kinetics. Microscopy was performed using a point laser
scanning confocal microscope (Zeiss LSM 510 Meta) equipped with
Meta detector for spectral imaging.
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d=170.6, 169.9, 168.4, 168.1, 156.6 (COa,b), 134.0 (CHCH2), 117.3
(CHCH2), 91.9 (C-1a), 90.7 (C-1b), 73.4 (C-5b), 71.6 (C-3b), 70.2 (C-
5a), 69.1 (C-3a), 65.3, 65.2 (C-4a,b), 64.5 (OCH2), 61.9, 61.8 (C-6a,b),
51.3 (C-2a,b), 33.4 (OCH2CH2), 20.9 (CH3), 20.8 (CH3), 20.8 (CH3), 20.7
(CH3), 20.7 (CH3), 20.6 (CH3); HRMS: m/z calcd for C19H27NO11:
468.14763 [M + Na]+, found: 468.14524.
Sialidase Experiments
HEK 293T cells were treated in the same way as for fluorescence
microscopy. Prior to incubation with Tz-biotin 21, sialidase
(0.5 UmLÀ1 in Opti-MEM, Roche) was added for 1 h at 378C. Cells
were washed two times with PBS and labeled as described previ-
ously.
1,3,4,6-Tetra-O-acetyl-2-((hex-5-en-1-yl-oxycarbonyl)amino)-2-
deoxy-d-mannopyranose (Ac4ManNHeoc) (6): Mannosamine hy-
drochloride 7 (740 mg, 3.45 mmol) and hex-5-en-1-yl succinimidyl
carbonate 11 (1 g, 4.14 mmol) were reacted in MeOH (30 mL) ac-
cording to the general procedure. The crude product was purified
by FC (petroleum ether/ethyl acetate 1:1). To remove remaining
NHS, the combined product fractions were evaporated, redissolved
in CH2Cl2 and extracted three times with 2 n NaOH. Ac4ManNHeoc
6 was obtained as mixture of anomers (1.06 g, 64%, a/b 4:3). Rf =
0.84 (petroleum ether/ethyl acetate 1:1). a-Anomer: 1H NMR
(400 MHz, CDCl3): d=6.08 (s, 1H, H-1), 5.81–5.70 (m, 1H, CHCH2),
5.30 (dd, J=10.6, 3.9 Hz, 1H, H-3), 5.22–5.11 (m, 1H, H-4), 5.10–4.94
(m, 3H, NH, CHCH2), 4.33 (dd, J=9.8, 4.2 Hz, 1H, H-2), 4.28–4.22
(m, 1H, H-6a), 4.13–3.98 (m, 4H, H-6b, H-5, OCH2), 2.17 (s, 3H, CH3),
2.11 (s, 3H, CH3), 2.10–2.06 (m, 2H, CH2CHCH2), 2.05 (s, 6H, 2x CH3),
1.70–1.58 (m, 2H, OCH2CH2CH2), 1.46 (q, J=7.1, 6.5 Hz, 2H,
Kinetic Measurements
For deacetylation of 3–6, the respective mannosamine derivative
(0.5 g) was dissolved in dry MeOH (28 mL), and EtNMe2 (6 mL) was
added. The reaction mixture was stirred for 8 d at RT during which
additional EtNMe2 (3 mL) was added two times (day 2 and 4). The
solvent was evaporated and the brown residue was purified by FC
(CH2Cl2/MeOH 10:1) yielding free sugars 12--15. For kinetic meas-
urements, stock solutions (10 mm in 100 mm acetate buffer,
pH 4.8) of alkenols, mannosamine derivatives 12–15, and tetrazine
16 were prepared. The reaction partners were mixed in a quartz
cuvette immediately before the measurement. To monitor the reac-
tion over time, the absorption at lmax =522 nm was measured and
the tetrazine concentration was calculated using Beer–Lambert
law. The second-order rate constant was determined by plotting
the inverse tetrazine concentration versus time followed by linear
regression analysis as reported earlier.[8e]
1
OCH2CH2CH2). b-Anomer: H NMR (400 MHz, CDCl3): d=5.84 (s, 1H,
H-1), 5.81–5.70 (m, 1H, CHCH2), 5.22–5.11 (m, 1H, H-4), 5.10–4.94
(m, 4H, NH, H-3, CHCH2), 4.46 (dd, J=9.3, 3.5 Hz, 1H, H-2), 4.28–
4.22 (m, 1H, H-6a), 4.13–3.98 (m, 3H, H-6b, OCH2), 3.77 (ddd, J=
9.6, 5.0, 2.5 Hz, 1H, H-5), 2.10–2.06 (m, 8H, 2x CH3, CH2CHCH2), 2.02
(s, 3H, CH3), 2.01 (s, 3H, CH3), 1.70–1.58 (m, 2H, OCH2CH2CH2), 1.46
(q, J=7.1, 6.5 Hz, 2H, OCH2CH2CH2). a/b-Anomers: 13C NMR
(101 MHz, CDCl3): d=170.7, 170.2, 169.7, 168.2, 156.9 (COa,b),
138.4 (CHCH2), 115.0 (CHCH2), 92.0, 90.8 (C-1a,b), 73.5, 71.7, 70.3,
Acknowledgements
This work was supported by the Deutsche Forschungsgemein-
schaft (SFB 969 and SPP 1623), the University of Konstanz, and
69.3 (C-3a,b, C-5a,b), 65.7, 65.5, 62.1, 62.0 (C-4a,b, OCH2a,b), 51.4, the Konstanz Research School Chemical Biology. We thank the
51.2 (C-2a,b), 33.43, 33.40 (CH2CHCH2a,b), 28.51, 28.46
(OCH2CH2CH2a,b), 25.20, 25.18 (OCH2CH2CH2a,b), 21.0, 20.94, 20.87,
20.85, 20.79, 20.76 (4x CH3a,b); HRMS: m/z calcd for C21H31NO11:
496.17893 [M+Na]+, found: 496.17665.
Bioimaging Center of the University of Konstanz for providing
the fluorescence microscopy instrumentation.
Keywords: bioorthogonal chemistry
·
carbohydrates
·
·
cycloaddition
tetrazines
· metabolic oligosaccharide engineering
Cell Growth Conditions
HEK 293T cells were grown in Dulbecco’s modified essential
medium (DMEM) supplemented with 5% fetal bovine serum (FBS),
100 units mLÀ1 penicillin and 100 mgmLÀ1 streptomycin. All cells
were incubated with 5% carbon dioxide in a water-saturated incu-
bator at 378C. Wells were coated with 0.01% poly-l-lysine (Sigma)
and 25 mgmLÀ1 fibronectin (Sigma) in phosphate-buffered saline
(PBS) for 1 h at 378C and rinsed with PBS prior to cell seeding.
[1] G. T. Hermanson, Bioconjugate Techniques, 3edrd edAcademic Press,
New York, 2013.
[2] J. A. Prescher, C. R. Bertozzi, Nat. Chem. Biol. 2005, 1, 13–21.
[3] E. M. Sletten, C. R. Bertozzi, Angew. Chem. Int. Ed. 2009, 48, 6974–6998;
Angew. Chem. 2009, 121, 7108–7133.
[4] E. Saxon, C. R. Bertozzi, Science 2000, 287, 2007–2010.
[5] a) V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem.
Int. Ed. 2002, 41, 2596–2599; Angew. Chem. 2002, 114, 2708–2711;
b) C. W. Tornøe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057–
3064; c) N. J. Agard, J. A. Prescher, C. R. Bertozzi, J. Am. Chem. Soc. 2004,
126, 15046–15047; d) X. Ning, J. Guo, M. A. Wolfert, G.-J. Boons, Angew.
Chem. Int. Ed. 2008, 47, 2253–2255; Angew. Chem. 2008, 120, 2285–
2287.
[6] a) M. L. Blackman, M. Royzen, J. M. Fox, J. Am. Chem. Soc. 2008, 130,
13518–13519; b) N. K. Devaraj, R. Weissleder, S. A. Hilderbrand, Biocon-
jugate Chem. 2008, 19, 2297–2299; c) K. Braun, M. Wiessler, V. Ehemann,
R. Pipkorn, H. Spring, J. Debus, B. Didinger, M. Koch, G. Muller, W. Wal-
Fluorescence Microscopy
HEK 293T cells (22000 cells per well) were seeded in 4-well ibiTreat
m-Slides (ibidi) and allowed to attach for 12 h. Cells were then incu-
bated with 100 mm of the corresponding mannosamine derivative
(3–6) for 48 h. The sugars were prepared as stock solutions in
DMSO (100 mm) and diluted into media. DMSO only was added as
negative control. Cells were washed two times with PBS and then
treated with Tz-biotin 21 (6 h, 1 mm) at 378C. After two washes
with PBS, cells were incubated with AlexaFluor 647-labeled strepta-
vidin (6.6 mgmLÀ1) and Hoechst 33342 (10 mgmLÀ1) for 20 min at
378C in the dark. Cells were washed twice with PBS, and DMEM
was added for microscopy. A Zeiss microscope LSM 510 Meta
equipped with a 40ꢂ1.3 NA Plan-Neofluar oil DIC immersion ob-
jective was employed for imaging. Analysis of the obtained data
was performed using Image J software version 1.45S.
ˇ
ˇ
˙
deck, Drug Des. Dev. Ther. 2008, 2, 289–301; d) J. Seckute, N. K. Devaraj,
Curr. Opin. Chem. Biol. 2013, 17, 761–767; e) A.-C. Knall, C. Slugovc,
Chem. Soc. Rev. 2013, 42, 5131–5142.
[7] J. W. Wijnen, S. Zavarise, J. B. F. N. Engberts, J. Org. Chem. 1996, 61,
2001–2005.
[8] a) M. R. Karver, R. Weissleder, S. A. Hilderbrand, Angew. Chem. Int. Ed.
2012, 51, 920–922; Angew. Chem. 2012, 124, 944–946; b) J. Schoch, M.
Staudt, A. Samanta, M. Wiessler, A. Jꢀschke, Bioconjugate Chem. 2012,
23, 1382–1386; c) L. I. Willems, N. Li, B. I. Florea, M. Ruben, G. A. van der
&
&
Chem. Eur. J. 2014, 20, 1 – 8
www.chemeurj.org
6
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Paper
Marel, H. S. Overkleeft, Angew. Chem. Int. Ed. 2012, 51, 4431–4434;
Angew. Chem. 2012, 124, 4507–4510; d) D. M. Patterson, L. A. Nazarova,
B. Xie, D. N. Kamber, J. A. Prescher, J. Am. Chem. Soc. 2012, 134, 18638–
18643; e) A. Niederwieser, A.-K. Spꢀte, L. D. Nguyen, C. Jꢃngst, W. Reut-
ter, V. Wittmann, Angew. Chem. Int. Ed. 2013, 52, 4265–4268; Angew.
[14] a) H. Bußkamp, E. Batroff, A. Niederwieser, O. S. Abdel-Rahman, R.
Winter, V. Wittmann, A. Marx, Chem. Commun. 2014, 50, 10827–10829;
b) U. Rieder, N. W. Luedtke, Angew. Chem. Int. Ed. 2014, 53, 9168–9172;
Angew. Chem. 2014, 126, 9322–9326.
[15] C.-J. Zhang, C. Y. J. Tan, J. Ge, Z. Na, G. Y. J. Chen, M. Uttamchandani, H.
Sun, S. Q. Yao, Angew. Chem. Int. Ed. 2013, 52, 14060–14064; Angew.
Chem. 2013, 125, 14310–14314.
ˇ
ˇ
˙
Chem. 2013, 125, 4359–4363; f) C. M. Cole, J. Yang, J. Seckute, N. K. De-
varaj, ChemBioChem 2013, 14, 205–208; g) Y. A. Wainman, A. A. Neves,
S. Stairs, H. Stçckmann, H. Ireland-Zecchini, K. M. Brindle, F. J. Leeper,
Org. Biomol. Chem. 2013, 11, 7297–7300.
[16] O. T. Keppler, R. Horstkorte, M. Pawlita, C. Schmidt, W. Reutter, Glycobiol-
ogy 2001, 11, 11R–18R.
[9] J. Sauer, D. K. Heldmann, J. Hetzenegger, J. Krauthan, H. Sichert, J.
Schuster, Eur. J. Org. Chem. 1998, 2885–2896.
[17] D. H. Dube, C. R. Bertozzi, Curr. Opin. Chem. Biol. 2003, 7, 616–625.
[18] a) A.-K. Spꢀte, H. Bußkamp, A. Niederwieser, V. F. Schart, A. Marx, V. Witt-
mann, Bioconjugate Chem. 2014, 25, 147–154; b) D. M. Patterson, K. A.
Jones, J. A. Prescher, Mol. BioSyst. 2014, 10, 1693–1697.
[19] a) A. A. Neves, H. Stçckmann, Y. A. Wainman, J. C. H. Kuo, S. Fawcett, F. J.
Leeper, K. M. Brindle, Bioconjugate Chem. 2013, 24, 934–941; b) S.
Stairs, A. A. Neves, H. Stçckmann, Y. A. Wainman, H. Ireland-Zecchini,
K. M. Brindle, F. J. Leeper, ChemBioChem 2013, 14, 1063–1067.
[20] L. A. Bateman, B. W. Zaro, K. N. Chuh, M. R. Pratt, Chem. Commun. 2013,
49, 4328–4330.
[10] a) N. K. Devaraj, S. Hilderbrand, R. Upadhyay, R. Mazitschek, R. Weissled-
er, Angew. Chem. Int. Ed. 2010, 49, 2869–2872; Angew. Chem. 2010, 122,
2931–2934; b) R. Rossin, P. Renart Verkerk, S. M. van den Bosch, R. C. M.
Vulders, I. Verel, J. Lub, M. S. Robillard, Angew. Chem. Int. Ed. 2010, 49,
3375–3378; Angew. Chem. 2010, 122, 3447–3450; c) K. Lang, L. Davis,
S. Wallace, M. Mahesh, D. J. Cox, M. L. Blackman, J. M. Fox, J. W. Chin, J.
Am. Chem. Soc. 2012, 134, 10317–10320; d) T. Plass, S. Milles, C. Koehler,
´
J. Szymanski, R. Mueller, M. Wießler, C. Schultz, E. A. Lemke, Angew.
Chem. Int. Ed. 2012, 51, 4166–4170; Angew. Chem. 2012, 124, 4242–
4246; e) M. T. Taylor, M. L. Blackman, O. Dmitrenko, J. M. Fox, J. Am.
Chem. Soc. 2011, 133, 9646–9649.
[21] E. M. Sletten, C. R. Bertozzi, Acc. Chem. Res. 2011, 44, 666–676.
[22] R. Sustmann, Pure Appl. Chem. 1974, 40, 569–593.
[23] O. T. Keppler, P. Stehling, M. Herrmann, H. Kayser, D. Grunow, W. Reutter,
M. Pawlita, J. Biol. Chem. 1995, 270, 1308–1314.
[24] a) J. Du, M. A. Meledeo, Z. Wang, H. S. Khanna, V. D. P. Paruchuri, K. J.
Yarema, Glycobiology 2009, 19, 1382–1401; b) M. Boyce, I. S. Carrico,
A. S. Ganguli, S.-H. Yu, M. J. Hangauer, S. C. Hubbard, J. J. Kohler, C. R.
Bertozzi, Proc. Natl. Acad. Sci. USA 2011, 108, 3141–3146; c) B. W. Zaro,
Y.-Y. Yang, H. C. Hang, M. R. Pratt, Proc. Natl. Acad. Sci. USA 2011, 108,
8146–8151.
[11] a) K. Lang, L. Davis, J. Torres-Kolbus, C. Chou, A. Deiters, J. W. Chin, Nat.
Chem. 2012, 4, 298–304; b) J. Schoch, M. Wiessler, A. Jꢀschke, J. Am.
Chem. Soc. 2010, 132, 8846–8847; c) E. Kaya, M. Vrabel, C. Deiml, S. Prill,
V. S. Fluxa, T. Carell, Angew. Chem. Int. Ed. 2012, 51, 4466–4469; Angew.
Chem. 2012, 124, 4542–4545; d) H. S. G. Beckmann, A. Niederwieser, M.
Wiessler, V. Wittmann, Chem. Eur. J. 2012, 18, 6548–6554.
[12] a) W. Waldeck, M. Wiessler, V. Ehemann, R. Pipkorn, H. Spring, J. Debus,
B. Didinger, G. Mueller, J. Langowski, K. Braun, Int. J. Med. Sci. 2008, 5,
273–284; b) R. Pipkorn, W. Waldeck, B. Didinger, M. Koch, G. Mueller, M.
Wiessler, K. Braun, J. Pept. Sci. 2009, 15, 235–241; c) S. B. Engelsma, L. I.
Willems, C. E. van Paaschen, S. I. van Kasteren, G. A. van der Marel, H. S.
Overkleeft, D. V. Filippov, Org. Lett. 2014, 16, 2744–2747.
[25] D. J. Vocadlo, H. C. Hang, E.-J. Kim, J. A. Hanover, C. R. Bertozzi, Proc.
Natl. Acad. Sci. USA 2003, 100, 9116–9121.
[26] G. W. Hart, M. P. Housley, C. Slawson, Nature 2007, 446, 1017–1022.
[13] W. Chen, D. Wang, C. Dai, D. Hamelberg, B. Wang, Chem. Commun.
2012, 48, 1736–1738.
Received: August 3, 2014
Published online on && &&, 0000
Chem. Eur. J. 2014, 20, 1 – 8
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These are not the final page numbers! ÞÞ

Full Paper
FULL PAPER
&
Bioorthogonal Chemistry
A.-K. Spꢀte, V. F. Schart, S. Schçllkopf,
A. Niederwieser, V. Wittmann*
&& – &&
Terminal Alkenes as Versatile
Chemical Reporter Groups for
Metabolic Oligosaccharide
Engineering
Shine bright: Metabolic oligosaccharide
engineering with peracetylated N-bute-
nyloxycarbonylmannosamine 1 (n=2)
leads to cell-surface glycoconjugate
staining with higher intensity compared
with previously reported terminal
alkene reporters. Analogues with short-
er (n=1) or longer (n=3–4) side chains
are much less efficient for labeling.
Chemically modified…
mannosamine derivatives with side
chains of different length
terminated by alkenes were applied
for metabolic oligosaccharide
engineering (MOE). During MOE the
sugars are transformed by cells into
the corresponding sialic acids and
incorporated into cell-surface
glycoconjugates (see picture). As
terminal alkenes can function as
dienophiles during inverse-electron-
demand Diels–Alder reactions, the
sugars can be fluorescently labeled
by ligation to a tetrazine conjugate.
The compound with
a butenyloxycarbonyl side chain
leads to the optimal balance
between incorporation efficiency
and speed of the ligation reaction
resulting in the most intense
fluorescence signal. For more
details, see the Full Paper by V.
Wittmann et al. on p. && ff.
&
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Chem. Eur. J. 2014, 20, 1 – 8
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8
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!