Exploring the Potential of Norbornene-Modified Mannosamine Derivatives for Metabolic Glycoengineering

Article abstract of DOI:10.1002/cbic.201600197

Metabolic glycoengineering (MGE) allows the introduction of unnaturally modified carbohydrates into cellular glycans and their visualization through bioorthogonal ligation. Alkenes, for example, have been used as reporters that can react through inverse-electron-demand Diels–Alder cycloaddition with tetrazines. Earlier, norbornenes were shown to be suitable dienophiles; however, they had not previously been applied for MGE. We synthesized two norbornene-modified mannosamine derivatives that differ in the stereochemistry at the norbornene (exo/endo linkage). Kinetic investigations revealed that the exo derivative reacts more than twice as rapidly as the endo derivative. Through derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) we confirmed that both derivatives are accepted by cells and incorporated after conversion to a sialic acid. In further MGE experiments the incorporated sugars were ligated to a fluorophore and visualized through confocal fluorescence microscopy and flow cytometry.

Full text of DOI:10.1002/cbic.201600197

DOI: 10.1002/cbic.201600197
Full Papers
Exploring the Potential of Norbornene-Modified
Mannosamine Derivatives for Metabolic Glycoengineering
Anne-Katrin Späte, Jeremias E. G. A. Dold, Ellen Batroff, Verena F. Schart, Daniel E. Wieland,
[
a]
Oliver R. Baudendistel, and Valentin Wittmann*
Metabolic glycoengineering (MGE) allows the introduction of
unnaturally modified carbohydrates into cellular glycans and
their visualization through bioorthogonal ligation. Alkenes, for
example, have been used as reporters that can react through
inverse-electron-demand Diels–Alder cycloaddition with tetra-
zines. Earlier, norbornenes were shown to be suitable dieno-
philes; however, they had not previously been applied for
MGE. We synthesized two norbornene-modified mannosamine
derivatives that differ in the stereochemistry at the norbornene
(exo/endo linkage). Kinetic investigations revealed that the exo
derivative reacts more than twice as rapidly as the endo deriva-
tive. Through derivatization with 1,2-diamino-4,5-methylene-
dioxybenzene (DMB) we confirmed that both derivatives are
accepted by cells and incorporated after conversion to a sialic
acid. In further MGE experiments the incorporated sugars were
ligated to a fluorophore and visualized through confocal fluo-
rescence microscopy and flow cytometry.
Introduction
Inverse-electron-demand Diels–Alder (DAinv) cycloaddition
represents a well-established class of bioorthogonal ligation re-
actions. In particular, 1,2,4,5-tetrazines have been shown to
[1]
react well with a variety of alkenes. The initial product of this
2+4] cycloaddition reacts instantly with release of nitrogen in
a retro Diels–Alder reaction, thus making the reaction irreversi-
[
[2]
ble. This is just one of the advantages that make it a suitable
ligation reaction; in addition, the DAinv reaction can be per-
formed under very mild conditions, such as in aqueous media,
[
3]
at physiological pH, and without addition of a catalyst. As ex-
diene
dienophile
pected for a LUMO –HOMO
proceeds efficiently with electron-rich alkenes as dienophiles.
Alternatively, high reaction rates can also be achieved by using
-controlled reaction, it
[
4]
Scheme 1. A) Strained alkenes used in the DAinv reaction. B) Strained al-
kenes applied as dienophiles in MGE.
[
4–5]
[1c,6]
strained alkenes.
As examples, trans-cyclooctenes,
nor-
[
1d,6c,e,7]
[1b]
[8]
bornenes,
cyclopropenes
Scheme 1A).
Out of its wide scope of applications, our group is interested
cyclobutenes, acylazetines, and (methyl)-
[
9]
have been applied in DAinv reactions
hydrates that bear chemical reporter groups. Through the em-
ployment of a bioorthogonal ligation reaction, the reporter
group can then be attached to a probe for visualization or
purification. Popular bioorthogonal ligation reactions for MGE
(
in the use of the DAinv reaction for metabolic glycoengineer-
ing (MGE). MGE is a powerful technique that allows the visuali-
[
11]
are the Staudinger ligation and azide–alkyne cycloaddition
[10]
[12]
zation of carbohydrates in cells and even animals. Thus, it
contributes to the elucidation of the function of glycosylation.
This approach exploits the fact that the enzymes involved in
sugar metabolism are promiscuous with regard to unnaturally
modified carbohydrates and, thus, will also incorporate carbo-
(AAC).
Additionally, tetrazine-based ligation reactions—the
DAinv reaction, for example—have been developed and em-
ployed for MGE. To this end, sugars have been successfully
[
13]
[14]
modified with terminal alkenes and isonitriles, as well as
[
9b–g]
with cyclopropenes.
Because tetrazine ligations can be or-
thogonal to AAC, it is possible to label two different reporters
[
9c–e,13a,14b]
in the same experiment.
Scheme 1B depicts the cy-
[
a] A.-K. Späte, J. E. G. A. Dold, E. Batroff, V. F. Schart, D. E. Wieland,
O. R. Baudendistel, Prof. Dr. V. Wittmann
clopropene-modified mannosamine derivatives reported in the
past.
University of Konstanz, Department of Chemistry
and Konstanz Research School Chemical Biology (KoRS-CB)
So far, cyclopropenes were the only strained alkenes to have
been employed for MGE. Even though norbornenes show high
DAinv reactivity, they had not previously been applied for
MGE, probably because it was assumed that they are too bulky
7
8457 Konstanz (Germany)
E-mail: mail@valentin-wittmann.de
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cbic.201600197.
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Full Papers
[
9b,13a]
to be metabolically incorporated.
Nevertheless, we hy-
and 11 were treated with mannosamine hydrochloride (12),
and the sugars were peracetylated for facilitated purification as
well as increased cell permeability, yielding Ac ManNNorboc
pothesized that, even if the incorporation rate was very low,
[5]
their high reactivity in the DAinv reaction could make norbor-
nenes an interesting modification for mannosamine deriva-
4
exo
(4) and Ac ManNNorboc
(5). For kinetic studies and the al-
endo
4
[1d]
tives. In addition, norbornenes are stable, synthetically readi-
ly accessible, and commercially available. Here we present two
new norbornene-modified mannosamine derivatives that we
have kinetically evaluated and applied for MGE.
dolase reaction, both sugars were deacetylated by treatment
with EtNMe , resulting in ManNNorboc (13) and ManNNor-
2
exo
1
bocendo (14).
Kinetics
Results and Discussion
Firstly, we investigated the reactivity of the new derivatives in
the DAinv reaction. An excess of sugar 13 or 14 was treated
Design of norbornene derivatives
[
13a]
with the water-soluble tetrazine Tz-PEG-OH (15)
in acetate
Recently, the laboratories of Carell and Knall investigated the
buffer in a cuvette (Scheme 4). The reaction was monitored in
each case by following the decrease of the tetrazine absorb-
ance, which has a maximum at 522 nm. We determined
pseudo-first-order rate constants (kobs) for different sugar con-
[15]
reactivity of several norbornene derivatives, of which (bicy-
clo[2.2.1]hept-5-en-2-yl)methanol appeared the most promis-
ing for our application because it reacts rapidly and the hy-
droxymethyl group allows functionalization of the norbornene.
Because the stereochemistry of the norbornene has been
shown to influence its reactivity (the exo derivative reacts
centrations (Figure 1), and second-order rate constants (k )
2
[
9c,d]
were calculated from these as previously described.
In a
[
15a]
about three times more rapidly than the endo derivative
)
we investigated exo and endo derivatives separately. As mono-
saccharide we chose mannosamine. In cells, N-acetylmannosa-
mine is converted into N-acetylneuraminic acid, which is found
as a terminal structure of glycoproteins and is thus readily
accessible for metabolic labeling, especially on cell surfaces.
We therefore designed the two mannosamine derivatives
Ac ManNNorboc (4, Scheme 2) and Ac ManNNorboc (5).
endo
4
exo
4
The hydroxy group of the norbornene allows attachment of
Figure 1. Pseudo-first-order rate constants (kobs) for the reactions between
Tz-PEG-OH (15) and either ManNNorbocexo (13) or ManNNorbocendo (14) at
different sugar concentrations. From these data second-order rate constants
[9c,d]
were determined as previously reported.
[
15a]
manner similar to that seen in the results of Vrabel et al.
with the free alcohols 7 and 8, the exo derivative 13 [k =
Scheme 2. Mannosamine derivatives 4 and 5 with norbornene units at-
tached through carbamate linkages.
2
À1 À1
(4.6Æ0.5)m s ] reacts more than twice as rapidly as the
À1 À1
endo derivative 14 [k =(2.0Æ0.3)m s ]. In comparison with
2
sugars bearing terminal alkenes, and showing rate constants in
À1 À1 [13]
the norbornene to the sugar through a carbamate linkage. We
and others have previously been able to show that alkenes
and alkynes attached through carbamate linkages are accepted
the range from 0.02 to 0.07m s , the norbornenes display
approximately 100-fold increased reactivity. DAinv reactivity is
also improved in comparison with deacetylated Ac ManNCyoc
4
[9d–f,13,16]
À1 À1 [9d]
by the cells’ enzymatic machinery
and can even pro-
(1, Scheme 1, k =1.0m s ).
2
[9a]
vide improved reaction kinetics.
Metabolic glycoengineering
Synthesis
Having ensured that the norbornene derivatives react rapidly
in the DAinv reaction, we investigated their suitability for visu-
A commercially available mixture of exo- and endo-bicy-
clo[2.2.1]hept-5-ene-2-carbaldehyde (exo-6 and endo-6) was
separated by silica gel column chromatography, and the iso-
mers were reduced to exo-(bicyclo[2.2.1]hept-5-en-2-yl)metha-
1
The two norbornenes exo-6 and endo-6 were each employed as a racemic
mixture. Thus, upon coupling to the sugar, two diastereoisomers (each with
a- and b-configuration at the sugar) are obtained. For simplification, we
depict only one of these isomers throughout the schemes of this manuscript.
All obtained stereoisomers are shown in Figure S1 in the Supporting Informa-
tion.
[
17]
nol (7) and its endo isomer 8, respectively (Scheme 3). Both
[7f]
7
and 8 were then activated by treatment with disuccinimid-
yl carbonate (9). The corresponding activated carbonates 10
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Scheme 3. Synthesis of norbornene-functionalized mannosamine derivatives. a) column chromatography; b) NaBH
MeOH; e) 10 or 11; f) Ac O, pyr; g) EtNMe
4 3 3
, NaOH, MeOH; c) Et N, CH CN; d) NaOMe,
2
2
.
Scheme 4. Reaction of norbornene derivatives 13 or 14 with Tz-PEG-OH (15)
to determine rate constants.
alization of cell-surface glycans. To this end, HEK 293T cells
4
were grown for 48 h in presence either of Ac ManNNorbocexo
Scheme 5. Strategy for MGE experiments. Cells were fed with sugars 4 or 5
and then treated with Tz-biotin (16).
(
4) or of Ac ManNNorboc
(5). As negative control, cells were
4
endo
treated with DMSO only. Cells were then incubated with Tz-
[
13a]
biotin (16)
and subsequently treated with streptavidin-Alexa
Fluor-555 to allow their visualization (Scheme 5). The results
used flow cytometry. HEK 293T cells were grown with the
showed that labeling both with Ac ManNNorbocexo (4, Fig-
sugar derivatives Ac ManNNorboc (4) or Ac ManNNorboc
endo
4
4
exo
4
ure 2A) and with Ac ManNNorbocendo (5, Figure 2B) leads to
(5) or with DMSO only. For fluorescence labeling, cells were
treated with Tz-biotin (16, 100 mm, 3 h at 378C) followed by
streptavidin-Alexa Fluor-647. Because streptavidin-Alexa Fluor-
647 is not cell-permeable, flow cytometry measurements allow
results limited to fluorescence staining of cell-surface glycocon-
jugates to be obtained. The flow cytometry results confirm
that both sugar derivatives show fluorescence staining relative
to cells that were grown without modified sugar, with use of
the exo derivative 4 giving a significantly higher labeling effi-
4
membrane staining whereas only negligible background stain-
ing occurs in the negative control (Figure 2C). That implies
that both derivatives were incorporated into cell-surface glyco-
conjugates and could be visualized with the aid of the DAinv
reaction.
Microscopy experiments had already indicated brighter
membrane staining for the exo derivative than for the endo de-
rivative. For a quantitative analysis of staining intensities we
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Figure 2. HEK 293T cells were grown with A) 100 mm Ac
4
ManNNorbocexo (4),
B) 100 mm Ac ManNNorbocendo (5), or C) DMSO for 48 h. Subsequently, cells
4
were incubated with Tz-biotin (16, 100 mm, 3 h, 378C) and then streptavidin-
Alexa Fluor-555. Nuclei were stained with Hoechst 33342. Scale bar: 30 mm.
ciency than that of the endo derivative 5 (Figure 3). This might
be due to the higher DAinv reactivity of the exo derivative 4.
Comparison of different sugar concentrations revealed that
cells grown with 250 mm of modified sugars displayed even
more intensive membrane staining than cells grown with
Figure 3. A) Fluorescence intensity distributions of HEK 293T cells grown
with 250 mm Ac ManNNorbocexo (4, green) or with 250 mm Ac ManNNor-
4 4
bocendo (5, blue) or without addition of unnatural sugar (gray) and labeled
with Tz-biotin (16, 100 mm, 3 h, 378C) followed by addition of streptavidin-
Alexa Fluor-647. B) Column diagram of fluorescence of HEK 293T cells grown
1
00 mm of unnatural sugar (Figure 3B).
To include cytoplasmic proteins in our investigation as well,
we performed a western blot analysis of metabolically labeled
cells. Cells grown in the presence of sugar derivatives 4 or 5 or
with DMSO as negative control were lysed, and the soluble
fractions were incubated with Tz-biotin (16, 100 mm, 2 h, room
temperature). These experiments show that the norbornene
derivatives are incorporated into glycoconjugates and can be
visualized with the aid of the DAinv reaction (Figure S2). Thus,
both norbornene reporters can also be applied to label soluble
glycoproteins.
4 4
with 100/250 mm Ac ManNNorbocexo (4) or with 100/250 mm Ac ManNNor-
bocendo (5) or without addition of unnatural sugar. Cells were then incubated
with Tz-biotin (16, 100 mm, 3 h, 378C) and then with streptavidin-Alexa
Fluor-647. Data were statistically analyzed by means of a t-test (*p<0.05,
**p<0.01, ***p<0.001).
Firstly, reference compounds were chemoenzymatically pre-
pared (Scheme 6A). To this end the norbornene-modified man-
nosamine derivatives 13 and 14 were enzymatically converted
[
19]
by treatment with sialic acid aldolase to afford the corre-
sponding sialic acids, which were purified by RP-HPLC to yield
Neu5Norbocexo (18) and Neu5Norbocendo (19) in 52 and 28%
yield, respectively. Treatment of 18 or 19 or of unmodified N-
acetylneuraminic acid (20) with fluorogenic DMB (17) gave flu-
orescent products 21–23. Formation of 21–23 was monitored
by RP-HPLC with use of a fluorescence detector (excitation
372 nm, emission 456 nm) and confirmed by use of an ESI-MS
detector (Figures S3–S5).
Determination of incorporation efficiencies
Next, we wanted to ensure that the norbornene-modified
ManNAc derivatives would be incorporated into sialic acid de-
rivatives. Furthermore, we wanted to investigate whether the
incorporation rates would differ for the exo and the endo deriv-
atives or if the different fluorescence intensities observed in
the flow cytometry experiment were solely due to different
reaction rates of the exo and the endo derivatives. For this pur-
pose we employed a method that allows quantification of
incorporated sialic acids. In this approach, sialic acids are re-
leased from glycoproteins by mild acid treatment and subse-
quently allowed to react with 1,2-diamino-4,5-methylenedioxy-
benzene (DMB, 17). DMB selectively reacts with a-keto acids
and can thus be applied to fluorescent labeling of sialic
To release sialic acids, metabolically engineered cells were
treated with 3m acetic acid for 1.5 h at 808C (Scheme 6B).
Subsequently, the cleaved sialic acids were fluorescently la-
beled by treatment with DMB (17), and the labeled sialic acids
21–23 could be identified by RP-HPLC analysis with the aid of
a fluorescence detector (Figures S6–S8) and correlated to the
reference compounds. This confirmed the incorporation of the
norbornene-modified mannosamines into sialic acid analogues.
[
18]
acids.
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Scheme 6. A) Chemoenzymatic preparation of reference compounds with sialic acid aldolase followed by DMB labeling reaction and analysis. B) Release of
cellular sialic acids with 3m acetic acid followed by DMB labeling and RP-HPLC analysis.
Incorporation efficiencies of the norbornene sugars were deter-
mined from the ratios of the fluorescence integrals of DMB-
Neu5AcNorboc (21 or 22) to that of the labeled natural sialic
acid DMB-Neu5Ac (23). This revealed comparable incorporation
rates of about 1% for both the exo and the endo derivative.
The more intense labeling of Ac ManNNorbocexo (4) after the
4
DAinv reaction relative to the endo derivative 5 is therefore
likely due to its faster DAinv reactivity.
Dual labeling
Previously, it had been shown that the DAinv reaction and
strain-promoted azide–alkyne cycloaddition (SPAAC) can be
[7d,9b–e,13a,14b,20]
used in the same experiment.
To establish that
the norbornene sugar Ac ManNNorbocexo (4) is suitable for a
4
dual labeling experiment, we employed 4 in combination with
[21]
peracetylated azidoacetylglucosamine (Ac GlcNAz). HEK 293T
4
cells were grown in the presence of Ac ManNNorbocexo (4) and
4
Ac GlcNAz for 48 h. Subsequently, the cells were incubated
4
with Tz-biotin (16) and then with a mixture of streptavidin-
Alexa Fluor-555 (to visualize norbornene-labeled sialic acids),
DIBO-Alexa Fluor-488 (to visualize metabolized GlcNAz by
SPAAC), and Hoechst 33342 (to visualize nuclei, Figure S9).
Under the fluorescence microscope, cells showed distinct
membrane staining both in the DAinv and in the SPAAC chan-
nel (Figure 4A). Control experiments, in which only one sugar
was applied but both ligation reactions were performed, only
showed staining in the channel corresponding to the sugar
Figure 4. HEK 293T cells were grown with A) 100 mm Ac
4
ManNNorbocexo (4)
GlcNAz,
and 50 mm Ac GlcNAz, B) 100 mm Ac ManNNorbocexo (4), C) 50 mm Ac
4
4
4
and D) without addition of unnatural sugar for 48 h. Cells were incubated
with Tz-biotin (16, 100 mm, 3 h, 378C) and then with a mixture of streptavi-
À1
din-Alexa Fluor-555 (6.6 mgmL ), DIBO-Alexa Fluor-488 (20 mm), and Hoechst
À1
3
3342 (10 mgmL ) for 30 min at 378C. Scale bar: 30 mm.
(
Figure 4B and C). Cells grown without modified sugar did not
Even though the norbornenes display high reaction rates,
show any membrane staining (Figure 4D). This experiment
confirmed that the DAinv reaction with a norbornene-modified
carbohydrate can be performed together with SPAAC of an
azide-modified carbohydrate in the same experiment.
a rather long labeling time of 3 h for the DAinv reaction was
required in order to obtain significant staining. This is most
likely due to the low incorporation efficiency. In comparison
with previously published terminal-alkene-modified sugars,
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À1
which required labeling times of 6 h, we consider the norbor-
nene sugars superior, due to their faster reactivity, although
the labeling efficiency does not reach the level achievable with
cyclopropene-modified sugars. Norbornene-modified sugars,
however, are synthetically better accessible than cyclopropene-
modified sugars and more stable.
250 mm), the flow was 9 mLmin , and the mobile phase was
a gradient of acetonitrile with 0.1% formic acid (solvent B) in water
with 0.1% formic acid (solvent A). Analytical RP-HPLC-MS was per-
formed with a Shimadzu system LCMS2020 (pumps LC-20 AD, au-
tosampler SIL-20AT HAS, column oven CTO-20AC, UV/Vis detector
SPD-20 A, fluorescence detector RF-20A, controller CBM-20, ESI de-
tector, LCMS Software Solution). The column was a Nucleodure C18
Gravity 3 mm from Macherey–Nagel (125”4 mm), the flow was
À1
0
.4 mLmin , and the mobile phase was a gradient of acetonitrile
Conclusion
with 0.1% formic acid (solvent B) in water with 0.1% formic acid
solvent A). UV/Vis absorption was measured with a Cary 50 instru-
ment from Varian and software Cary WinUV scanning kinetics.
(
In summary, we have synthesized two new mannosamine de-
rivatives bearing norbornene reporters for use in metabolic
glycoengineering. In each of the derivatives the norbornene
unit is attached to the sugar through a carbamate linkage. The
compounds differ in the stereochemistry at the norbornene
unit (exo or endo configuration). Kinetic studies showed that
exo-(Bicyclo[2.2.1]hept-5-en-2-yl)methanol (7) and endo-(bicy-
clo[2.2.1]hept-5-en-2-yl)methanol (8): These compounds have in
the meantime become commercially available (FCH Group Re-
agents for Synthesis and Sigma–Aldrich). We started from an exo/
endo mixture of bicyclo[2.2.1]hept-5-ene-2-carbaldehyde (exo/endo-
6; each isomer as a racemic mixture) and separated the diastereo-
both derivatives react rapidly in the DAinv reaction with a
À1 À1
1
,2,4,5-tetrazine, the exo derivative (k =4.6m s ) more than
2
[17]
À1 À1
mers by column chromatography (petrol ether/diethyl ether) to
twice as rapidly as the endo derivative (k =2.0m s ). Per-
forming DMB labeling experiments, we found the metabolic
incorporation efficiencies to be comparable for both isomers
2
obtain exo-6 and endo-6, each as a racemic mixture. The isomers
were reduced with NaBH by a procedure previously described by
4
[17]
Blanco et al.
but, due to its higher reactivity, Ac ManNNorbocexo (4) gives
4
exo-(Bicyclo[2.2.1]hept-5-en-2-yl)methyl succinimidyl carbonate (10)
was synthesized as described previously by Neumaier et al.
more distinct membrane staining in labeling experiments.
Compound 4 can also be combined with a strained azide-
modified sugar by copper-free click chemistry, allowing label-
ing of two different carbohydrates in the same experiment. In
conclusion, we have been able to expand the toolbox of
sugars available for MGE with a rapidly reacting derivative that
is easily synthetically accessible and, despite its bulkiness, is ac-
cepted by the cellular metabolism.
[
7f]
endo-(Bicyclo[2.2.1]hept-5-en-2-yl)methyl succinimidyl carbon-
ate (11): endo-(Bicyclo[2.2.1]hept-5-en-2-yl)methanol (8, 1.04 g,
8.3 mmol) was stirred in dry acetonitrile (30 mL) and triethylamine
(3.3 mL, 24.2 mmol) under nitrogen at RT. After 30 min disuccini-
midyl carbonate (3.5 g, 13.8 mmol) was added to the solution. The
resulting mixture was stirred overnight and then concentrated
under vacuum. The product was purified by FC (silica, CH Cl ) to
2
2
1
yield 11 as a white solid (1.21 g, 55%). H NMR (400 MHz, CDCl ):
3
d=6.27–6.13 (m, 1H; H-2), 6.04–5.90 (m, 1H; H-3), 4.13–4.06 (m,
Experimental Section
1
2
H; OCH CH), 3.97–3.89 (m, 1H; OCH CH), 2.99–2.91 (m, 1H; H-4),
2
2
General methods: All chemicals were purchased from Sigma–Al-
drich, Apollo, Fluka, Dextra, and Carbosynth and used without
further purification. Sialidase was purchased from Carbosynth
succinimide
.88–2.73 (m, 5H; CH CH2
, H-1), 2.51 (m, 1H; H-5), 1.89
2
(
7
ddd, J=11.9, 9.3, 3.8 Hz, 1H; H-6), 1.49 (dd, J=8.4, 2.2 Hz, 1H; H-
), 1.35–1.17 (m, 1H; H-7), 0.58 ppm (ddd, J=11.8, 4.5, 2.6 Hz, 1H;
(
MS110801004). Alexa Fluor-555-labeled streptavidin, DIBO-Alexa
13
H-6); C NMR (101 MHz, CDCl ): d=168.8 (C=O), 151.7 (C=O), 138.2
3
Fluor-488, and Hoechst 33342 were purchased from Invitrogen.
Ac GlcNAz was synthesized according to a published procedure.
4
(
C-2), 132.2 (C-3), 74.9 (C-8), 49.6 (C-7), 43.9 (C-4), 42.3(C-1), 37.8(C-
[22]
succinimide
5), 28.9 (C-6), 25.6 ppm (CH CH2
).
2
Technical solvents were distilled prior to use. All reactions were car-
ried out in dry solvents. Reactions were monitored by TLC on silica
gel 60 F254 (Merck) with detection under UV light (l=254 nm).
Additionally, acidic ethanolic p-anisaldehyde solution or basic
1,3,4,6-Tetra-O-acetyl-2-[(exo-bicyclo[2.2.1]hept-5-en-2-yl)meth-
oxycarbonylamino]-2-deoxymannopyranose (Ac ManNNorbocexo,
4
4): Mannosamine hydrochloride (12, 306 mg, 1.42 mmol) was sus-
pended in dry MeOH (5.0 mL), neutralized with NaOMe in MeOH
(3.2 mL, 1.42 mmol), and stirred for 1 h at RT under nitrogen. A so-
lution of 10 (392 mg, 1.48 mmol) in dry MeOH (5 mL) was added
and the reaction mixture was stirred overnight at RT until TLC indi-
cated complete reaction [ManNNorbocexo: R =0.39 (CH Cl /MeOH
KMnO solution followed by gentle heating were used for visualiza-
4
tion. Preparative flash column chromatography (FC) was performed
with an MPLC-Reveleris system from Grace. NMR spectra were re-
corded at room temperature with Avance III 400 and Avance III 600
instruments from Bruker. Chemical shifts are reported relative to
solvent signals (CDCl : d =7.26 ppm, d =77.16 ppm; CD OD: d =
f
2
2
5:1)]. After complete removal of the solvent, the residual syrup
was dissolved in dry pyridine (4.5 mL), treated with acetic anhy-
dride (1.5 mL), and stirred for two days at RT. The solvent was
evaporated, and the residue was diluted with CH Cl . After washing
3
H
C
3
H
4
.87 ppm, d =49.00 ppm; D O: d =4.73 ppm). Signals were as-
C 2 H
signed by first-order analysis; when feasible, assignments were
1
1
1
13
supported by two-dimensional H, H and H, C correlation spec-
troscopy (COSY, HMBC, and HSQC). Numbering of norbornene de-
rivatives is given in the Supporting Information. High-resolution
mass spectra (HRMS) were recorded with a micrOTOF II instrument
from Bruker Daltonics. Semipreparative RP-HPLC was conducted
with a LC-20A prominence system (high-pressure pumps LC-20 AT,
autosampler SIL-20A, column oven CTO-20AC, detector SPD-M20A
and ELSD-LT II, fraction collector FRC-10 A, controller CBM-20A and
LC Software Solution) from Shimadzu under the following condi-
tions. The column was a Eurosphere 100 C18 from Knauer (16”
2
2
with KHSO (10%), sat. NaHCO , and brine, the organic layer was
4
3
dried over MgSO and concentrated. The product was purified by
4
FC (silica, petroleum ether/ethyl acetate 1:1) to deliver 4 as a white
solid (426 mg, 60%, a/b mixture). TLC: R =0.56 (petroleum ether/
f
1
ethyl acetate 1:1); H NMR (400 MHz, CDCl ): a-anomer: d=6.20–
3
6.02 (m, 3H; H-1, H-2’, H-3’), 5.32 (dd, J=10.2, 4.3 Hz, 1H; H-3),
5.20 (t, J=10.0 Hz, 1H; H-4), 4.99 (d, J=9.4 Hz, 1H; NH), 4.39–4.30
(m, 1H; H-2), 4.26 (dd, J=12.3, 4.4 Hz, 1H; H-6), 4.20–4.11 (m, 1H;
H-8’), 4.10–3.92 (m, 3H; H-6, H-5, H-8’), 2.84 (brs, 1H; H-1’ or H-4’),
ChemBioChem 2016, 17, 1374 – 1383
www.chembiochem.org
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
2
2
1
.73 (brs, 1H; H-4’ or H-1’), 2.18 (s, 3H; OAc), 2.11 (s, 3H; OAc),
.06 (s, 3H; OAc), 2.02 (s, 3H; OAc), 1.73 (brs, 1H; H-5’), 1.43–
.11 ppm (m, 4H; H-7’, H-6’); b-anomer: d=6.18–6.00 (m, 2H; H-2’,
(4, 550 mg, 1.11 mmol) was dissolved in dry MeOH (14 mL) under
nitrogen. EtNMe (3 mL, 26 mmol) was added to the solution, and
2
the mixture was stirred at RT for four days. Additional EtNMe2
(1.5 mL, 13 mmol) was added, and the mixture was stirred at RT for
another four days. The solvent was evaporated, and the crude
product was purified by FC (silica, 0–10% MeOH in CH Cl in
H-3’), 5.85 (d, J=1.7 Hz, 1H; H-1), 5.14 (dd, J=9.7, 2.7 Hz, 1H; H-3),
5
9
3
.11–5.07 (m, 1H; H-4), 5.05 (d, J=3.9 Hz, 1H; NH), 4.48 (d, J=
.4 Hz, 1H; H-2), 4.26 (dd, J=12.2, 4.4 Hz, 1H; H-6), 4.21–3.93 (m,
H; H-6, H-8’a, H-8’b), 3.78 (ddd, J=9.5, 5.0, 2.5 Hz, 1H; H-5), 2.84
2
2
10 min followed by 10% MeOH in CH Cl for 10 min). Product 13
2
2
(
brs, 1H; H-1’ or H-4’), 2.73 (brs, 1H; H-4’ or H-1’), 2.19 (s, 3H;
was obtained as a white solid (105 mg, 29%).
OAc), 2.12 (s, 3H; OAc), 2.09 (s, 3H; OAc), 2.03 (s, 3H; OAc), 1.73
2
-[(endo-Bicyclo[2.2.1]hept-5-en-2-yl)methoxycarbonylamino]-2-
deoxymannopyranose (ManNNorbocendo, 14): Ac ManNNorboc
endo
(
1
brs, 1H; H-5’), 1.42–1.22 (m, 3H; H-7’ab, H-6’), 1.20–1.08 ppm (m,
1
3
H; H-6’); C NMR (101 MHz, CDCl ): a- and b-anomer: d=170.7
4
3
(5, 320 mg, 0.64 mmol) was dissolved in dry MeOH (28 mL) under
(
(
C=O), 170.2 (C=O), 169.8 (C=O), 168.3 (C=O), 156.3 (CON), 137.1
C-2’ or C-3’), 136.3 (C-3’ or C-2’), 92.0 (C-1a), 90.9 (C-1b), 73.6 (C-
nitrogen. EtNMe (6 mL, 52 mmol) was added to the solution, and
the mixture was stirred at RT for four days. Additional EtNMe2
2
5
b), 71.7 (C-3b), 70.3 (C-5a), 69.8 (C-8’), 69.3 (C-3a), 65.5 (C-4), 62.1
(
3 mL, 26 mmol) was added, and the mixture was stirred at RT for
(
C-6), 51.5 (C-2b), 51.3 (C-2a), 45.1 (C-7’), 43.7 (C-4’ or C-1’), 41.7 (C-
another four days. The solvent was evaporated, and the crude
product was purified by FC (silica, 0–10% MeOH in CH Cl in
1
2
;
’ or C-4’), 38.4 (C-5’), 29.6 (C-6’), 21.0 (OAc), 21.0 (OAc), 20.9 (OAc),
+
0.8 ppm (OAc); HRMS: m/z calcd for C H NO : 498.1970 [M+H]
2
2
2
3
31
11
2
0 min). Product 15 was obtained as a white solid (112 mg, 53%).
found: 498.1974.
N-[(exo-Bicyclo[2.2.1]hept-5-en-2-yl)methoxycarbonyl]neuramin-
ic acid (Neu5Norbocexo 18): ManNNorbocexo (13, 29 mg,
.09 mmol) and sodium pyruvate (154 mg, 1.44 mmol) were dis-
solved in phosphate buffer (0.89 mL, 100 mm, pH 7.1). Sialic acid al-
dolase (2 U) was added, and the mixture was incubated at RT and
1
,3,4,6-Tetra-O-acetyl-2-[(endo-bicyclo[2.2.1]hept-5-en-2-yl)meth-
,
oxycarbonylamino]-2-deoxymannopyranose (Ac ManNNor-
bocendo, 5): Mannosamine hydrochloride (12, 375 mg, 1.73 mmol)
was suspended in dry MeOH (6.0 mL), neutralized with NaOMe
4
0
(
3.9 mL, 1.73 mmol), and stirred for 1 h at RT under nitrogen. A so-
3
00 rpm for 17 days. The mixture was lyophilized, and the product
lution of 11 (480 mg, 1.8 mmol) in dry MeOH (5.0 mL) was added
and the reaction mixture was stirred overnight at RT until TLC indi-
cated complete reaction [ManNNorbocendo: R =0.34 (CH Cl /MeOH
was purified by RP-HPLC (18–26% B in 20 min, t =12.5 min) to
R
yield 18 as a white solid (19 mg, 52%) and in the form of an
f
2
2
anomeric mixture (a/b 1:20). TLC: R =0.28 (EtOAc/MeOH/EtOH/
f
5
:1)]. After complete removal of the solvent the residual syrup was
dissolved in dry pyridine (5.5 mL), treated with acetic anhydride
1.8 mL), and stirred overnight at RT. The solvent was evaporated,
and the remainder was dissolved in CH Cl . After washing with
1
H O/AcOH 2:1:1:1:0.1); H NMR (400 MHz, D O): b-anomer: d=
2
2
6
.25–5.94 (m, 2H; H-2’, H-3’), 4.22–4.10 (m, 1H; H-8’a), 4.09–3.96
(
(
m, 3H; H-8’b, H-4, H-6), 3.89–3.82 (m, 1H; H-9a), 3.75 (ddd, J=9.2,
2
2
6
1
1
5
.5, 2.7 Hz, 1H; H-8), 3.70–3.59 (m, 3H; H-5, H-7, H-9b), 2.85 (brs,
H; H-1’), 2.72 (d, J=11.3 Hz, 1H; H-4’), 2.28 (dd, J=13.0, 4.9 Hz,
H; H-3 ), 1.86 (app.t, J=12.3 Hz, 1H; H-3 ), 1.80–1.66 (m, 1H; H-
KHSO (10%), sat. NaHCO , and brine, the organic layer was dried
over MgSO4 and concentrated. The product was purified by FC
4
3
eq
ax
(
(
silica, petroleum ether/ethyl acetate 1:1) to yield 5 as a white solid
1
3
’), 1.38–1.27 (m, 2H; H-7’), 1.27–1.16 ppm (m, 2H; H-6’); C NMR
542 mg, 63%). TLC: R =0.45 (petroleum ether/ethyl acetate 1:1);
f
(
151 MHz, D O) b-anomer: d=174.4 (C-1), 158.8 (C-9’), 137.3 (C-2’),
1
2
H NMR (600 MHz, CDCl ): a-anomer: d=6.10–6.05 (m, 1H; H-2’),
3
1
6
3
36.5 (C-3’), 95.6 (C-2), 70.6 (C-6), 70.3 (C-8), 69.6 (C-8’), 68.3 (C-7),
7.0 (C-4), 63.2 (C-9), 53.4 (C-5), 44.4 (C-7’), 43.3 (C-4’), 41.3 (C-1’),
6
4
.03–5.98 (m, 1H; H-1), 5.92–5.83 (m, 1H; H-3’), 5.22 (dd, J=10.1,
.1 Hz, 1H; H-3), 5.15–5.08 (m, 1H; H-4), 4.88 (d, J=9.4 Hz, 1H;
9.0 (C-3), 37.9 (C-5’), 28.6 ppm (C-6’); HRMS: m/z calcd for
NH), 4.25 (dd, J=8.5, 4.6 Hz, 1H; H-2), 4.18 (dd, J=12.0, 5.1 Hz,
+
C H NO : 418.1708 [M+H] ; found: 418.1696.
18
27
10
1
2
2
2
H; H-6), 4.02 (dd, J=12.5, 2.6 Hz, 1H; H-6), 3.94 (ddd, J=10.1, 4.6,
.5 Hz, 1H; H-5), 3.82–3.72 (m, 1H; H-8’), 3.63–3.53 (m, 1H; H-8’),
.81 (brs, 1H; H-4’), 2.74 (brs, 1H; H-1’), 2.38–2.27 (m, 1H; H-5’),
.15–1.88 (m, 12H; OAc), 1.79–1.72 (m, 1H; H-6’), 1.42–1.32 (m, 1H;
N-[(endo-Bicyclo[2.2.1]hept-5-en-2-yl)methoxycarbonyl]neura-
minic acid (Neu5Norbocendo, 19): ManNNorbocendo (14, 20.5 mg,
0
.06 mmol) and sodium pyruvate (109 mg, 0.93 mmol) were dis-
H-7’), 1.21–1.13 (m, 1H; H-7’), 0.53–0.38 ppm (m, 1H; H-6’); b-
anomer: d=6.10–6.05 (m, 1H; H-2’), 5.92–5.83 (m, 1H; H-3’), 5.76
solved in phosphate buffer (0.63 mL, 100 mm, pH 7.1). Sialic acid al-
dolase (2 U) was added, and the mixture was incubated at RT and
(
(
(
2
2
(
1
1
app.t, J=1.6 Hz, 1H; H-1), 5.06 (dt, J=9.6, 4.8 Hz, 1H; H-4), 4.97
d, J=9.7 Hz, 1H; NH), 4.94 (dd, J=10.9, 3.8 Hz, 1H; H-3), 4.41–4.35
m, 1H; H-2), 4.18 (dd, J=12.0, 5.1 Hz, 1H; H-6), 3.98 (dd, J=12.4,
.5 Hz, 1H; H-6), 3.82–3.72 (m, 1H; H-8’), 3.70 (ddd, J=9.3, 5.2,
.6 Hz, 1H; H-5), 3.63–3.53 (m, 1H; H-8’), 2.81 (brs, 1H; H-4’), 2.74
3
00 rpm for 26 days. The mixture was lyophilized, and the product
was purified by RP-HPLC (18–26% B in 20 min, t =12.5 min) to
yield 19 as a white solid (7 mg, 28%) and in the form of an anome-
ric mixture (a/b 1:20). TLC: R =0.28 (EtOAc/MeOH/EtOH/H O/AcOH
2
5
4
3
R
f
2
1
:1:1:1:0.1); H NMR (600 MHz, D O): b-anomer: d=6.23 (dd, J=
2
brs, J=3.3 Hz, 1H; H-1’), 2.38–2.27 (m, 1H; H-5’), 2.15–1.88 (m,
.8, 3.0 Hz, 1H; H-2’), 6.04–5.96 (m, 1H; H-3’), 4.08–3.98 (m, 2H; H-
, H-6), 3.90–3.80 (m, 2H; H-8’a, H-9a), 3.80–3.73 (m, 1H; H-8’),
.71–3.55 (m, 4H; H-9b, H-8’b, H-7, H-5), 2.89 (brs, 1H; H-4’), 2.83
2H; OAc), 1.79–1.72 (m, 1H; H-6’), 1.42–1.32 (m, 1H; H-7’), 1.21–
13
.13 (m, 1H; H-7’), 0.53–0.38 ppm (m, 1H; H-6’); C NMR (151 MHz,
CDCl ) a- and b-anomers: d=170.59 (C=O), 170.08 (C=O), 169.65
(
3
(d, J=4.0 Hz, 1H; H-1’), 2.51–2.36 (m, 1H; H-5’), 2.28 (dd, J=13.0,
C=O), 168.15 (C=O) 156.78 (NÀC=O), 156.08 (NÀC=O), 137.71 (C-
4
1
6
9
.9 Hz, 1H; H-3 ), 1.91–1.78 (m, 2H; H-6’a, H-3 ), 1.44–1.36 (m,
e
q
a
x
2
3
5
2
2
’), 132.24 (C-3’), 91.91 (C-1a), 90.76 (C-1b), 73.38 (C-5a), 71.57 (C-
b), 70.21 (C-5b), 69.04 (C-3a), 65.22 (C-8’), 62.03 (C-6), 51.30 (C-2b),
1.08 (C-2a), 49.42 (C-7’), 43.86 (C-4’), 42.23 (C-1’), 38.08 (C-5’),
8.92 (C-6’), 20.90 (C(O)CH ), 20.84 (C(O)CH ), 20.76 (C(O)CH ),
H; H-7’), 1.28 (d, J=8.3 Hz, 1H; H-7’), 0.59–0.47 ppm (m, 1H; H-
13
’b); C NMR (151 MHz, D O): b-anomer: d=174.4 (C-1), 158.8 (C-
2
’), 137.9 (C-2’), 132.2 (C-3’), 95.6 (C-2), 70.6 (C-6), 70.3 (C-8), 69.0
3
3
3
(C-8’), 68.3 (C-7), 67.0 (C-4), 63.2 (C-9), 53.4 (C-5), 48.8 (C-7’), 43.5
(C-4’), 41.9 (C-1’), 39.0 (C-3), 37.5 (C-5’), 28.0 ppm (C-6’); HRMS: m/z
calcd for C H NO : 418.1708 [M+H] ; found: 418.1684.
0.66 ppm (C(O)CH ); HRMS: m/z calcd for C H NO : 498.1970
3
23 31
11
+
[M+H] ; found: 498.1976.
+
18
27
10
2
-[(exo-Bicyclo[2.2.1]hept-5-en-2-yl)methoxycarbonylamino]-2-
Kinetic measurements: For kinetic studies, stock solutions of Tz-
[13a]
deoxymannopyranose (ManNNorbocexo, 13): Ac ManNNorbocexo
PEG-OH (15),
ManNNorbocexo (13), and ManNNorbocendo (14)
4
ChemBioChem 2016, 17, 1374 – 1383
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1380
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
were prepared in acetate buffer (100 mm, pH 4.7, 208C) and mixed
in a quartz cuvette to afford final concentrations of 1 mm Tz-PEG-
OH (15) and 10, 13.3, or 16.6 mm of sugar. The reactions were
monitored through the decreasing absorption of the tetrazine at
vent control, DMSO was added instead of the sugar. The cells were
cultured for 48 h with or without the added sugar. Cells were tryp-
sinated, resuspended in PBS (5 mL), and pelleted by centrifugation
(5 min, 400g). The supernatant was discarded, and the pellet was
resuspended in PBS (1 mL) and pelleted by centrifugation (5 min,
400g). The cells were lysed in lysis buffer (250 mL) containing Triton
X-100 (0.5%), DNase (30 mgmL ), RNase (30 mgmL ), b-glycero-
phosphate (20 mm), sodium fluoride (20 mm), sodium orthovana-
date (0.3 mm), complete X protease inhibitor (Roche; 1”), NaCl
(300 mm), Tris·HCl (pH 7.4, 25 mm), EDTA (5 mm), and 2-acetamido-
2-deoxy-d-glucopyranosylidenamino N-phenylcarbamate [PUGNAc
(O-GlcNAc-b-N-acetylglucosaminidase inhibitor to maintain O-
GlcNAcylation during lysis), Sigma–Aldrich, 100 mm], and incuba-
tion at 48C was carried out for 30 min. The lysate was cleared by
centrifugation (22000g, 30 min, RT). Tz-biotin (16) was added to
the lysate to afford a final concentration of 100 mm. The samples
were incubated for 2 h at RT, SDS-sample buffer (3”55.5 mL) was
added, and the sample was heated at 908C for 15 min. Proteins
were separated by SDS-polyacrylamide gel electrophoresis with
5
22 nm. Pseudo-first-order rate constants were determined for
every concentration of sugar by plotting ln(A /A ) over time. For
o
t
À1
À1
the determination of A , a 1 mm solution of only Tz-PEG-OH (15) in
o
acetate buffer was used. A is the absorption of the reaction mix-
t
ture at time point t. Analysis by linear regression provided pseudo-
first-order rate constants. Second-order rate constants were deter-
mined by plotting the pseudo-first-order rate constants against the
corresponding sugar concentration, followed by linear regression.
All measurements were carried out at least in triplicate. Sufficient
stability of Tz-PEG-OH (15) had previously been verified in our lab
by measuring the absorption at 522 nm of a solution of Tz-PEG-OH
[13a]
(15) in acetate buffer.
Cell growth conditions: HEK 293T (human embryonic kidney) cells
were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) con-
taining fetal bovine serum (FBS, 10%), and penicillin and strepto-
1
0% polyacrylamide gels and transferred to nitrocellulose mem-
À1
mycin (each 100 UmL ). Cells were incubated under carbon diox-
branes (BioRad). Transfer efficiency was analyzed with Ponceau S
staining. The membranes were blocked in milk (5% in PBS-T) for
ide (5%) in a water-saturated incubator at 378C. The cells were di-
luted every three days by washing with PBS buffer and detaching
with trypsin and EDTA.
1
(
4
h at RT, followed by incubation with anti-biotin antibody
Abnova, Anti-Biotin mAb clone SB58c, 1:2000 dilution in milk) at
8C overnight or anti-a-tubulin antibody (AA4.3, hypridoma super-
Sugar stock solutions: The sugars were prepared as stock solu-
tions (100 mm) in DMSO and stored at À208C. They were freshly
diluted into media on the day of the experiment.
natant in 1% FCS, 1:200 dilution in milk) for 1 h at RT. The mem-
branes were washed (3”, 10–15 min, PBS-T), incubated with secon-
dary horseradish-peroxidase-conjugated anti-mouse antibody [Dia-
nova, goat anti-mouse igG (H+L)-HRP, 1:50000 dilution in milk,
MGE and fluorescence microscopy: In an approach similar to that
[
9d,13b]
used in previously described experiments,
(
HEK 293T cells
1
h, RT] and washed again (3”, 10–15 min, PBS-T). Blots were
À2
17500 cells cm ) were seeded in 4-well ibiTreat m-Slides (ibidi)
developed by use of an ECL detection system (clarity Western ECL
substrate, BioRad) and visualized with a CCD camera (Raytest-
Ph+ coated with poly-l-lysine (0.0025%, 1 h at 378C or o/n at
8C). After 12 h, cells were incubated for 48 h with labeled man-
nosamine [Ac ManNNorbocexo (4) or Ac ManNNorboc (5),
00 mm]. DMSO only was added as solvent control. Cells were
4
1
000, Fujifilm).
4
4
endo
1
Flow cytometry analysis: HEK 293T cells were seeded in 12-well
plates (170000 cells/well) coated with poly-l-lysine (0.0025%, 1 h,
[13a]
washed twice with PBS and then treated with Tz-biotin (16,
1
00 mm) for 1–3 h at 378C. After two washes with PBS, cells were
3
78C). After 12 h cells were incubated for 48 h with labeled man-
À1
incubated with streptavidin-Alexa Fluor-555 (6.6 mgmL ) and
Hoechst 33342 (10 mgmL ) for 20 min at 378C in the dark. Cells
nosamine [Ac ManNNorboc (4), Ac ManNNorboc (5), 100 or
4
exo
4
endo
À1
2
50 mm] A corresponding amount of DMSO was added as solvent
were washed three times with PBS, and DMEM was added for
microscopy. Fluorescence microscopy was performed with a Zeiss
LSM 780 instrument equipped with a 40”1.4 NA Plan-Apochromat
oil immersion objective and a GaAsP-detector array for spectral
imaging. Analysis of the obtained data was performed by use of
Image J software version 1.45 S.2.
control. Cells were washed with PBS (2”) and then treated with Tz-
[13a]
biotin (16,
cells were
6.6 mgmL ) for 30 min at 378C in the dark. Cells were washed
with PBS, then blocked with FACS buffer [PBS+ FCS (10%)] for
min and again washed three times with PBS. Then cells were re-
100 mm) for 3 h at 378C. After two washes with PBS,
incubated with streptavidin-Alexa Fluor-647
À1
(
5
À2
leased with trypsin-EDTA (100 mL/well) and resuspended in FACS
buffer (400 mL/well). 50000 cells were counted per measurement.
For flow cytometry analysis BD FACSCalibur was used, and the ob-
tained data were evaluated with FlowJo Software version 8.8.7 and
processed with Graphpad prism (the averaging called median was
used). Experiments were performed in triplicate. Data were statisti-
cally analyzed by means of a t-test (*p<0.05, **p<0.01, ***p<
Dual labeling experiments: HEK 293T cells (17500 cells cm
)
were seeded in 4-well ibiTreat m-Slides (ibidi) Ph+ coated with
poly-l-lysine (0.0025%, 1 h at 378C or o/n at 48C) and allowed to
attach for 16 h. Cells were then incubated with Ac ManNNorbocexo
4
(
4, 100 mm) and Ac GlcNAz (50 mm) for 48 h. Either no sugar or only
4
one sugar was added as negative control. Cells were washed twice
with PBS and then treated with Tz-biotin (16, 100 mm) for 3 h. Un-
bound Tz-biotin (16) was washed away (2” with PBS) and cells
were incubated with a mixture of DIBO-Alexa Fluor-488 (20 mm),
streptavidin-Alexa Fluor-555 (6.6 mgmL ), and Hoechst 33342
10 mgmL ) for 30 min at 378C in the dark. Cells were washed
0
.001).
À1
Preparation of reference compounds for DMB labeling experi-
ments: The sialic acid derivative [Neu5Norbocexo (18), Neu5Nor-
bocendo (19), or Neu5Ac (20), 0.1 mg] was dissolved in DMB solution
À1
(
three times with PBS, and DMEM was added for microscopy. Mi-
croscopy was performed as described above.
[
DMB·2HCl (5.3 mm), Na S O (16 mm), TFA (40 mm) in MilliQ water,
2 2 4
2
65 mL]. The mixture was incubated for 2.5 h at 568C in a thermo-
Western blot analysis: Western blot analysis was performed in
mixer (300 rpm) in the dark. Subsequently, the mixture was cooled
on ice for 10 min and neutralized with NaOH (0.5m, 21 mL). For an-
alytical RP-HPLC-MS measurements, the reaction mixture was dilut-
ed with MilliQ water (1:2), filtered, and injected (10 mL). For fluores-
cence detection (l =372 nm, l =456 nm) a higher dilution was
[9f]
a modified version of the previously described protocol. HEK
93T cells were seeded (1200000 cells/10 cm dish), and after 16 h
the media was exchanged with media containing labeled sugar
Ac ManNNorboc (4) or Ac ManNNorboc (5), 100 mm]. As a sol-
2
[
4
exo
4
endo
ex
em
ChemBioChem 2016, 17, 1374 – 1383
www.chembiochem.org
1381
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
necessary (1:400) and only 3 mL were injected. A gradient of 10–
0.6% B in 55 min was used. Chromatograms are shown in Fig-
ures S3–S5.
[5] F. Thalhammer, U. Wallfahrer, J. Sauer, Tetrahedron Lett. 1990, 31, 6851–
6854.
3
[
6] a) N. K. Devaraj, S. Hilderbrand, R. Upadhyay, R. Mazitschek, R. Weissled-
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25 31
3
10
+
[
M+H ]; found: 534.2067.
Derivatization of Neu5Norbocendo (19): Product DMB-Neu5Norbo-
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[
M+H ]; found: 534.2060.
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08C for 90 min in a thermomixer (300 rpm). The solution was di-
luted with MilliQ H O (500 mL) and neutralized through addition of
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This work was supported by the Deutsche Forschungsgemein-
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