Fluorescent mimetics of CMP-Neu5Ac are highly potent, cell-permeable polarization probes of eukaryotic and bacterial sialyltransferases and inhibit cellular sialylation

Article abstract of DOI:10.1002/anie.201400394

Oligosaccharides of the glycolipids and glycoproteins at the outer membranes of human cells carry terminal neuraminic acids, which are responsible for recognition events and adhesion of cells, bacteria, and virus particles. The synthesis of neuraminic acid containing glycosides is accomplished by intracellular sialyl transferases. Therefore, the chemical manipulation of cellular sialylation could be very important to interfere with cancer development, inflammations, and infections. The development and applications of the first nanomolar fluorescent inhibitors of sialyl transferases are described herein. The obtained carbohydrate-nucleotide mimetics were found to bind all four commercially available and tested eukaryotic and bacterial sialyl transferases in a fluorescence polarization assay. Moreover, it was observed that the anionic mimetics intruded rapidly and efficiently into cells in vesicles and translocated to cellular organelles surrounding the nucleus of CHO cells. The new compounds inhibit cellular sialylation in two cell lines and open new perspectives for investigations of cellular sialylation. You can't run, you can't hide: Sialyltransferases cover cancer cells with neuraminic acids, enabling them to escape from tissues and to metastasize. Cell-permeable chemical probes targeting this class of enzymes might help to study, understand, and inhibit such processes.

Full text of DOI:10.1002/anie.201400394

Angewandte
Chemie
DOI: 10.1002/anie.201400394
Biomimetics of Carbohydrate Nucleotides
Fluorescent Mimetics of CMP-Neu5Ac Are Highly Potent,
Cell-Permeable Polarization Probes of Eukaryotic and Bacterial
Sialyltransferases and Inhibit Cellular Sialylation**
Johannes J. Preidl, Vinayaga S. Gnanapragassam, Michael Lisurek, Jçrn Saupe,
Rꢀdiger Horstkorte, and Jçrg Rademann*
Abstract: Oligosaccharides of the glycolipids and glycopro-
teins at the outer membranes of human cells carry terminal
neuraminic acids, which are responsible for recognition events
and adhesion of cells, bacteria, and virus particles. The
synthesis of neuraminic acid containing glycosides is accom-
plished by intracellular sialyl transferases. Therefore, the
chemical manipulation of cellular sialylation could be very
important to interfere with cancer development, inflamma-
tions, and infections. The development and applications of the
first nanomolar fluorescent inhibitors of sialyl transferases are
described herein. The obtained carbohydrate-nucleotide mim-
etics were found to bind all four commercially available and
tested eukaryotic and bacterial sialyl transferases in a fluores-
cence polarization assay. Moreover, it was observed that the
anionic mimetics intruded rapidly and efficiently into cells in
vesicles and translocated to cellular organelles surrounding the
nucleus of CHO cells. The new compounds inhibit cellular
sialylation in two cell lines and open new perspectives for
investigations of cellular sialylation.
tissues.[3] Hypersialylation is, therefore, strongly indicative of
a bad prognosis of neoplasia and inhibition of this event could
be an alternative therapeutic strategy against cancer.[4]
Sialylation of glycoconjugates proceeds in the Golgi
apparatus by the action of at least 20 distinct sialyltransferases
found in both the human and the murine genome.[5] The
degree of sialylation of cells is strongly linked to the
expression level of sialyltransferases, which can thus be
considered to be potential targets for pharmacological
interference.[6] For a more detailed understanding of the
functions and the significance of protein sialylation, protein-
binding probes for this enzyme class are highly desirable. In
addition, specific and generic inhibitors of this enzyme class
would be valuable for the validation of sialyltransferases as
potential targets for the treatment of metastasizing neo-
plasms.[7]
The development of inhibitors for sialyltransferases is
a demanding task for several reasons. First, few of the
genetically encoded enzymes have been expressed and
isolated as stable, soluble, and bioactive proteins for homo-
geneous assays so far. Secondly, the elaboration of functional
assays has been difficult, as many of the enzymes require
specific oligosaccharide substrates, which must be prepared or
isolated. Thirdly, the monitoring of the enzymatic reaction
typically requires product analysis by chromatography ren-
dering the assay time-consuming, expensive, and low through-
put,[8] though the recent progress in the area of glycan arrays
might lead to an alternative to solution assays in the future.[9]
We concluded from these considerations that a generic
assay for sialyltransferases can be realized only if not the
enzyme-specific conversion of acceptor substrates but binding
at the recognition site of the generic donor substrate of these
membrane-bound enzymes, 5-N-acetyl-neuraminic acid cyti-
dine monophosphate (CMP-Neu5Ac, Figure 1), was detected.
In order to realize such a binding assay for sialyltransferases,
generic probes for this enzyme class were required.
S
ialic acids like 5-N-acetylneuraminic acid (Neu5Ac) are
attached to glycoconjugates such as glycoproteins and glyco-
lipids at the outer surface of eukaryotic cells and act as
multivalent ligands in cell-adhesion events. The regulation of
sialylation is of general importance for the maintenance of
healthy cells and organisms.[1] Hypersialylation of cells is
found in inflammation and enables immune cells to intrude
into infected tissue.[2] Moreover, strongly hypersialylated
cancer cells are capable of leaving their primary tissue
environment, migrating, and forming metastases in remote
[*] Dr. J. J. Preidl, Dr. J. Saupe, Prof. Dr. J. Rademann
Medicinal Chemistry, Freie Universitꢀt Berlin
Kçnigin-Luise-Strasse 2+4, 14195 Berlin (Germany)
E-mail: j.rademann@fu-berlin.de
Homepage: http://www.bcp.fu-berlin.de/ag-rademann
Dr. J. J. Preidl, Dr. M. Lisurek, Prof. Dr. J. Rademann
Department of Medicinal Chemistry
Leibniz Institut fꢁr Molekulare Pharmakologie (FMP)
Robert-Rçssle-Strasse 10, 13125 Berlin (Germany)
Already two decades ago, Reutter et al. established that
the enzymes in sialic acid metabolism including the sialyl-
transferases are promiscuous with respect to donor substrates
carrying various substitutions in the C5 position.[10] Besides
the native N-acetyl group, larger amide residues and azides
are accepted, a finding that was subsequently exploited for
the on-cell derivatization of 5-azidosialic acids.[11] Potent
inhibitors of sialyltransferases were identified by the group of
R. R. Schmidt that contained phosphate esters of a-hydroxy-
benzylphosphonic acid derivatives.[12] Combination of these
distinct findings prompted us to propose arylic amide 1 as
Dr. V. S. Gnanapragassam, Prof. Dr. R. Horstkorte
Institute for Physiological Chemistry
Martin-Luther University
Hollystrasse 1, 06114 Halle (Germany)
[**] Support from the DFG (FOR 806, SFB 765, TRR 67), and the Berlin
School of Integrative Oncology is gratefully acknowledged.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201400394.
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CMP-Neu5Ac mimetic. Accordingly, a set of potential
fluorescence probes 1 for sialyltransferases was designed
(Figure 1, Scheme 1) containing variations with respect to the
substitution at the phenyl ring of the inhibitor (meta or para),
the introduction of a glycine linker between fluorescein and
the inhibitor, and the configuration at the a-hydroxymethyl-
phosphonate (R or S).
The designed probe molecules were synthesized starting
from 3- and 4-nitrobenzyl bromides 2-m and 2-p. Alkylation
of triallyl phosphite with these electrophiles furnished the
phosphonates 3-m and 3-p, which were deprotonated with
sodium hexamethyldisilazane (NaHMDS). The obtained
intermediary phosphoryl-stabilized benzyl anions were oxi-
dized stereoselectively employing either (ꢀ)- or (+)-[(8,8-
dichlorocamphoryl)sulfonyl]oxaziridine 4 as the oxidant[15]
and provided the corresponding (R)- and (S)-a-hydroxy-
methylphosphonates (R)-5-m or -p and (S)-5-m or -p,
respectively. The enantiomeric excesses (ee) were determined
by the preparation and NMR analysis of the derived Mosherꢀs
esters and were found to be between 83–87% (Mosherꢀs
esters not shown; for details see the Supporting Information).
The four nitro-substituted (R)- and (S)-a-hydroxymethyl-
phosphonates of structure 5 were reduced to the correspond-
ing four aromatic amines (R)-6-m or (R)-6-p and (S)-6-m or
(S)-6-p using tin(II) chloride as the reducing agent. The
anilines 6 were not stable when isolated or stored for a longer
time and thus after aqueous workup they were immediately
coupled with pivaloyl-protected, 6-carboxyfluorescein[16] 7 or
with the glycine spacer derivative 7-G. Six stable secondary
benzylic alcohols of the general structure 8 were isolated, (R)-
8-m and (S)-8-m, (R)-8-p and (S)-8-p, and (R)-8-G-m and (S)-
8-G-m, the latter two containing the glycine spacer. All six
alcohols 8 were coupled with the protected cytidine phos-
phoramidite 9,[17] oxidized with tert-butyl hydroperoxide, and
then deprotected with [Pd(PPh3)4] and K2CO3 in MeOH,
a procedure removing all allyl, acetyl, pivaloyl, and cyano-
ethyl protecting groups in one step.[18] The six obtained
fluorescein-labeled inhibitors (R)- and (S)-1-m, (R)- and (S)-
1-p, and (R)- and (S)-1-G-m were purified by HPLC and
transformed into their sodium salts employing a cation-
exchange resin. The final diastereomeric excesses (de) of the
six potential FP probes 1 were determined by integration of
the signals of the cytidine H-5 or H-1’ protons in the 1H NMR
spectra and were between 71 and 84%. This indicated that no
significant epimerization had occurred throughout the multi-
step protocol from 6 to 1.
Figure 1. Design of potential probe molecules for sialyltransferases:
The native substrate of sialyltransferases, CMP-Neu5Ac (top), can be
varied considerably at the 5-position of neuraminic acid. Docking
studies of the derived biomimetic (R)-1-m with the bacterial sialyltrans-
ferase PmST1 (PDB: 2IHZ)[13] and later (see the Supporting Informa-
tion) with the mammalian sialyltransferase pST3GalI (PDB: 2WNB[14]).
a potential, generic structure for sialyltransferase probes
(Figure 1): While 1 was calculated to retain the binding mode
of the substrate, the substrate tolerance in the C5 position of
CMP-Neu5Ac should translate into variable substituents in
the meta and para positions of the inhibitors, which possibly
would enable the integration even of larger groups such as
fluorophores, functionalities for pull-down experiments, and
photoactive cross-linking groups. We decided to focus on the
development of fluorescent sialyltransferase probes first as
these molecules could be directly employed in a binding assay
exploiting shifts in fluorescence polarization observed during
the competitive replacement of such molecules.
For evaluating the binding of the probes to the CMP-
Neu5Ac pocket, two mammalian sialyltransferases were
selected, rat rST3GalII,[19] for sialylation of galactose in
position 3, and human hST6GalI,[20] for sialylation of gal-
actose in position 6; both of these enzymes were commer-
cially available in soluble form and recombinantly expressed
in insect cells. In addition, the two prokaryotic sialyltransfer-
ases PmST1 from Pasteurella multocida[21] and Pd2,6ST(N)
from Photobacterium damsela[22] for sialylation of galactose in
position 3 and position 6, respectively, were employed. Poten-
tial enzyme probes 1 were dissolved (10 nm) in buffer with
serial dilutions of the four sialyltransferases. The dissociation
constant KD of the ligand–protein complex was determined by
Docking simulations of the designed molecules in the
crystal structure of sialyltransferase PmST1 (PDB: 2IHZ[13]
)
and later the nonhomologous, mammalian enzyme pST3GalI
(PDB: 2WNB[14]) encouraged us to challenge this hypothesis
(Figure 1 and Figure S2). Several combinations of a spacer
and fluorescein as the fluorophore fitted well into the protein
and did not interfere with the recognition of the presumed
2
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Scheme 1. Reaction conditions for the synthesis of probes 1: a) P(OAll)3, toluene, microwaves, 1058C, 15 h; b) 1. NaHMDS, THF, ꢀ95!ꢀ458C,
1–3 h, 2. (ꢀ)- or (+)-4, THF, ꢀ958C!ꢀ788C, 3.5 h; ee 84–87%; c) SnCl2, EtOH, 20–408C, 1–3 d; d) 7 or 7-G, EDC, DCM, 2–18 h; e) 1. 9, 1H-
tetrazole, DCM, 2.5–18 h, 2. tBuOOH, 1–21 h; f) 1. [Pd(PPh3)4], K2CO3, MeOH, 4–24 h, 2. HPLC, 3. Na+, de 71–84%).
recording the anisotropy changes in fluorescence polarization
as a function of protein concentration. All probes proved to
be ligands of the four STs investigated. Binding affinities to
the mammalian STs were very high—in the two-digit nano-
molar range—with surprisingly low affinity differences
between the different probes. Best binding affinities with
respect to the bacterial STs were also in the lower nanomolar
range with more variations between the probes.
Next, an FP-based displacement assay was to be estab-
lished for each of the sialyltransferases (Figure 2). Thus, for
each ST a probe was selected which provided an optimal
combination of high affinity, large dynamic anisotropy
window, and constant fluorescence quantum yield. This was
(S)-1-G-m for the rat enzyme rST3GalII, (S)-1-p for the
human protein hST6GalI, (R)-1-p for Pd2,6ST(N), and (S)-1-
p for PmST1. The initial anisotropy was optimized for each
binding assay with respect to the sensitivity by adjusting the
protein concentration.[24]
The configuration of the a-hydroxymethylphosphonate
had only little influence on the binding properties of the
probes toward the mammalian STs while the R-configurated
probes bound much more strongly to the bacterial STs than
the S-configurated. The position of the aromatic substituent
had only little influence on the affinities of the probes to the
mammalian STs but the para-substituted probes were much
stronger binders of the bacterial STs than the meta-substi-
tuted. Both the configuration of the benzylic stereocenter and
the aromatic substitution influenced the anisotropic proper-
ties of the probes regarding the maximal anisotropy and the
quantum yield of fluorescence. The glycine linker seemed not
to be crucial for the binding of the probes to the STs;
however, it increased significantly the dynamic range of
anisotropy for rST3GalII. Differences in the dynamic range
between the probes might arise from flexibility of the
fluorophore tag, different rotational relaxation times of the
probe–protein complex, and the influence of the protein
environment on the fluorescence lifetime.[23] The specificity of
the ST probes was scrutinized for one selected example. (S)-1-
G-m was incubated with increasing concentrations of the
commercially available b-1,4-galactosyltransferase from cow
milk and fluorescence polarization was measured. Even at the
maximal protein concentration of 250 mm only partial reten-
tion of the FP was recorded indicating KD > 250 for the probe
and a selectivity for the two eukaryotic sialyltransferases of
more than 10000.
Subsequently, the bound probes were replaced compet-
itively by addition of cytidine diphosphate (CDP) and the
simplified, nonfluorescent CMP-Neu5Ac biomimetic 1-Ac
(Figure 1, R = Me). The experiments clearly confirmed the
reversible binding mode of all selected probes. The inhibition
constants Ki were calculated by the method of Nikolovska-
Coleska et al.[25] and indicated inhibition of the mammalian
STs by CDP in the mm range, whereas 1-Ac was a sub-mm
inhibitor (see Table 1). Remarkably, the inhibition potencies
of CDP are similar to or even better than those of the
nonfluorescent derivative 1-Ac with both bacterial STs and
were in the low nm range. The displacement assays proved to
be robust against 1% DMSO, an important feature for the
application of this assay in the screening of chemical libraries.
Finally, the selected sialyltransferase probe (S)-1-G-m was
investigated in living Chinese Hamster Ovary (CHO) cells.
Commonly, the outer cell membrane is considered to be
impermeable toward polyanionic molecules such as com-
pounds 1, a general problem for the cellular availability of
organic phosphates, phosphate mimetics, nucleic acids, etc.
Therefore, we expected the need for using transfection agents
in order to translocate the probe (S)-1-G-m into cells. To our
great surprise, however, treatment of CHO cells with the
fluorescent probe (S)-1-G-m led to the rapid and prolonged
intracellular staining of the cells (Figure 3). The intensity and
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Figure 2. Fluorescence polarization binding curves of the most potent probes (left) and displacement by cytidine diphosphate (CDP) (middle)
and 1-Ac (right) (see the Supporting Information for binding plots of all probes and DMSO test for 1-Ac).
Table 1: Binding affinities (KD values) and anisotropy changes (DA) of fluorescent NeuAc5-CMP mimetics 1 as well as displacement properties (Ki
values) and assay window (DA) of inhibitors cytidine diphosphate (CDP) and 1-Ac with respect to four eukaryotic and bacterial sialyltransferases.
Probe
rST3GalII
hST6GalI
Pd2,6ST(N)
PmST1
KD [nm][a]
DA [mA][a]
KD/Ki [nm][a]
DA [mA][a]
KD/Ki [nm][a]
DA [mA][a]
KD/Ki [nm][a]
DA [mA][a]
(R)-1-m
(S)-1-m
(R)-1-p
(S)-1-p
(R)-1-G-m
(S)-1-G-m
41.0ꢁ1.9
44.8ꢁ0.8
24.5ꢁ0.7
78.9ꢁ4.5
23.9ꢁ0.6
23.5ꢁ0.9
181.5ꢁ6.1
144.1ꢁ1.6
131.5ꢁ2.7
180.3ꢁ6.7
194.3ꢁ2.9
214.5ꢁ5.4
22.2ꢁ0.4
9.0ꢁ0.3
213.2ꢁ2.5
184.8ꢁ2.8
152.2ꢁ2.7
205.0ꢁ2.7
205.0ꢁ2.0
208.9ꢁ2.3
924.6ꢁ157.5
>1000
83.9ꢁ5.1
590.7ꢁ48.5
53.2ꢁ13.9
>1000
193.3ꢁ15.5
180.1ꢁ28.5
159.2ꢁ4.5
163.2ꢁ4.7
75.1ꢁ11.6
182.1ꢁ9.4
33.6ꢁ1.8
183.8ꢁ7.9
11.5ꢁ0.6
26.5ꢁ1.0
12.7ꢁ1.0
184.6ꢁ21.0
160.7ꢁ6.2
140.3ꢁ2.7
133.7ꢁ4.2
157.7ꢁ2.8
139.0ꢁ4.1
104.3ꢁ5.2
21.5ꢁ0.6
11.3ꢁ0.2
17.9ꢁ0.3
12.8ꢁ0.2
rST3GalII
hST6GalI
Pd2,6ST(N)
PmST1
Inhibitor
Ki [mm][a]
DA [mA][a]
Ki [mm][a]
DA [mA][a]
Ki [mm][a]
DA [mA][a]
Ki [mm][a]
DA [mA][a]
CDP
1-Ac 0% DMSO
1-Ac 1% DMSO
32.83ꢁ4.31
0.66ꢁ0.03
0.69ꢁ0.03
129.5ꢁ4.1
128.4ꢁ4.0
115.5ꢁ0.7
3.50ꢁ0.41
0.78ꢁ0.09
0.88ꢁ0.13
104.6ꢁ8.0
101.9ꢁ9.5
96.8ꢁ11.2
0.09ꢁ0.00
0.36ꢁ0.02
0.39ꢁ0.01
74.2ꢁ2.5
72.5ꢁ2.5
70.0ꢁ4.8
0.15ꢁ0.01
0.08ꢁ0.01
0.09ꢁ0.01
77.4ꢁ5.6
75.8ꢁ5.6
80.0ꢁ7.0
[a]ꢁStandard error; FP probes in boldface were used for displacement studies.
homogeneity of cellular uptake was investigated by flow
cytometry (Figure 3E,F). After 24 h incubation with (S)-1-G-
m all CHO cells showed strong fluorescence (dark gray),
whereas control cells treated with 6-carboxyfluorescein at the
very same concentration showed less than 5% fluorescence
(light gray).
partments might be part of the trans-Golgi network, which is
the reported site for sialyltransferases. Therefore, the func-
tional effect of the probe on sialylation in living cells was
investigated. Two cell lines, CHO and B35 cells, were
incubated with a single dose of probe (S)-1-G-m (50 mm,
24 h). Cells were harvested and lyzed, and equal amounts of
cell lysate were analyzed by SDS-PAGE. The gel was blotted
onto a nitrocellulose membrane and sialylated glycoproteins
were detected by incubation with biotinylated sialolectin
limax flavus agglutinin (LFA) followed by treatment with
streptavidine labeled with horseradish peroxidase (Figure 4).
In both cell lines a reduction in the chemiluminescence
intensity of the LFA staining after incubation with the probe
(S)-1-G-m was observed, indicating a reduced sialylation on
the separated glycoproteins.
In summary, we have established compounds of general
structure 1 as generic and potent fluorescent molecular
probes for sialyltransferases. The developed probes were
highly active toward the four tested recombinant STs, which
were the four commercially available enzymes at the time of
this study both of mammalian and of bacterial origin. The
binding of the probes to STs was determined by fluorescence
polarization experiments. Anisotropy was employed to estab-
lish a robust, competitive binding assay for each of the STs
used in this study. The selected probe (S)-1-G-m was highly
Staining of cells with (S)-1-G-m was observed in subcel-
lular compartments only, while the cytoplasm was not
affected at all, indicating a vesicular uptake mechanism.
Fluorescing vesicles translocated rapidly to the proximity of
the nucleus, the nucleus itself was not stained. The dye Bodipy
tr C5 ceramide[26] was used in order to define the subcellular
localization of (S)-1-G-m more precisely. Bodipy tr C5
ceramide was reported to specifically stain extranuclear
compartments of cells, especially the endoplasmic reticulum
(ER) and the Golgi apparatus. Co-staining of CHO cells with
(S)-1-G-m (green), the nuclear dye Hoechst 33342 (blue), and
the ER/Golgi tracker (red) confirmed that the Neu5Ac
biomimetic was translocated to compartments surrounding
the cell nucleus. The compartments directly attached to the
nucleus, the ER and the closer cis-Golgi network, which were
most intensively stained with the tracker, did not contain the
sialyltransferase probe, whereas compartments directly adja-
cent to the strongest red-stained organelles contained the
highest concentration of the fluorescein probe. These com-
4
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cell-permeable and located to compartments surrounding the
cell nucleus, possibly to the trans-Golgi network. (S)-1-G-m
was active in inhibiting protein sialylation in two cell lines.
These findings generate novel scientific opportunities and
research lines and pose additional questions. First of all the
described cell-permeable probes will be investigated further
in functional biological studies in order to scrutinize their
potential as inhibitors of membrane sialylation in living cells
and to study the effects of altered cellular decoration with
sialic acid residues. Secondly, it will be rewarding to identify
the putative membrane receptor responsible for the vesicular
uptake of the probes. Finally, the probes are currently used for
the screening of chemical libraries in search for small-
molecule inhibitors of sialyltransferases, which could be
tested in the future as an innovative therapeutic approach
against cancer cells.[27]
Received: January 14, 2014
Published online: && &&, &&&&
Keywords: fluorescence polarization · metastasis ·
.
neuraminic acid · protein-binding probes · sialyltransferases
[1] a) N. M. Varki, A. Varki, Lab. Invest. 2007, 87, 851 – 857; b) A.
Varki, Glycoconjugate J. 2009, 26, 231 – 245; c) P. R. Crocker,
J. C. Paulson, A. Varki, Nat. Rev. Immunol. 2007, 7, 255 – 266;
d) J. N. Arnold, M. R. Wormald, R. B. Sim, P. M. Rudd, R. A.
Dwek, Annu. Rev. Immunol. 2007, 25, 21 – 50.
[2] a) K. Ley, Trends Mol. Med. 2003, 9, 263 – 268; b) R. Schauer,
Glycoconjugate J. 2001, 17, 485 – 499.
[3] a) T. Meyer, I. R. Hart, Eur. J. Cancer 1998, 34, 214 – 221; b) R.
Kannagi, M. Izawa, T. Koike, K. Miyazaki, N. Kimura, Cancer
Sci. 2004, 95, 377 – 384.
[4] a) H. C. Carcinomas, F. Schneider, W. Kemmner, W. Haensch, G.
Franke, S. Gretschel, U. Karsten, P. M. Schlag, Cancer Res. 2001,
61, 4605 – 4611; b) M. J. Vierbuchen, W. Fruechtnicht, S. Brack-
rock, K. T. Krause, T. J. Zienkiewicz, Cancer 1995, 76, 727 – 735;
c) S. Ogata, I. Ho, A. Chen, H. C. Mucosa, D. Dubois, J.
Maklansky, A. Singhal, S. Hakomori, S. H. Itzkowitz, Cancer
Res. 1995, 55, 1869 – 1874; d) M. A. Liebert, M. Lise, C. Belluco,
S. P. Perera, R. Patel, P. Thomas, L. E. T. Al, Hybridoma 2000,
19, 281 – 286.
Figure 3. Cellular uptake of (S)-1-G-m by CHO cells: A) staining with
Hoechst 33342 for nuclei and overlay with phase-contrast image;
B) staining with BODIPY TR C5-ceramide (5 mm) for labeling of the
endoplasmatic reticulum and the Golgi; C) staining showing vesicular
allocation of the probe; D) co-staining with three dyes (for pictures of
control and treatment with carboxyfluorescein see the Supporting
Information). Flow cytometry analysis of (S)-1-G-m (50 mm) uptake by
CHO cells compared to carboxyfluorescein (FAM) and control after
24 h of incubation. E) Logarithmic plot indicates significant fluores-
cence in all cells incubated with the probe. F) Bar graph of absolute
values (mean ꢁSEM, n=2).
[5] A. Harduin-Lepers, V. Vallejo-Ruiz, M.-A. Krzewinski-Recchi,
B. Samyn-Petit, S. Julien, P. Delannoy, Biochimie 2001, 83, 727 –
737.
[6] a) M. M. Fuster, J. D. Esko, Nat. Rev. Cancer 2005, 5, 526 – 542;
b) F. DallꢀOlio, M. Chiricolo, Glycoconjugate J. 2001, 18, 841 –
850; c) A. Cazet, S. Julien, M. Bobowski, M.-A. Krzewinski-
Recchi, A. Harduin-Lepers, S. Groux-Degroote, P. Delannoy,
Carbohydr. Res. 2010, 345, 1377 – 1383.
[7] Several steroid derivatives have been reported as inhibitors of
2,3-ST with promiscuous effects: a) C.-Y. Wu, C.-C. Hsu, S.-T.
Chen, Y.-C. Tsai, Biochem. Biophys. Res. Commun. 2001, 284,
466 – 473; b) W.-W. Chang, C.-Y. Yu, T.-W. Lin, P.-H. Wang, Y.-C.
Tsai, Biochem. Biophys. Res. Commun. 2006, 341, 614 – 619;
c) C.-H. Chiang, C.-H. Wang, H.-C. Chang, S. V. More, W.-S. Li,
W.-C. Hung, J. Cell. Physiol. 2010, 792 – 799.
[8] For a recent report on screening for sialyltransferase inhibitors
based on substrate turnover see: C. D. Rillahan, S. J. Brown,
A. C. Register, H. Rosen, J. C. Paulson, Angew. Chem. 2011, 123,
12742 – 12745; Angew. Chem. Int. Ed. 2011, 50, 12534 – 12537.
[9] a) A. Beloqui, J. Calvo, S. Serna, S. Yan, I. B. Wilson, M. Martin-
Lomas, N. C. Reichardt, Angew. Chem. 2013, 125, 7625 – 7629;
Figure 4. Lectin blot. CHO or B35 cells were grown in the absence (ꢀ)
or presence (+) of 1-G-m (50 mm) for 24 h. Cells were harvested and
homogenized in RIPA buffer. Equal amounts (50 mg) of cell lysate in
each lane were subjected to SDS-PAGE. Proteins were blotted and
sialylated proteins were detected using biotinylated limax flavus
agglutinin (LFA) (1:5000) and streptavidin labeled with horseradish
peroxidase (1:20000).
Angew. Chem. Int. Ed. 2014, 53, 1 – 7
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

.
Angewandte
Communications
Angew. Chem. Int. Ed. 2013, 52, 7477 – 7481; b) L. Ban, N. Pettit,
L. Li, A. D. Stuparu, L. Cai, W. Chen, W. Guan, W. Han, P. G.
Wang, M. Mrksich, Nat. Chem. Biol. 2012, 8, 769 – 773.
[18] D. R. Vutukuri, P. Bharathi, Z. Yu, K. Rajasekaran, M.-H. Tran,
S. Thayumanavan, J. Org. Chem. 2003, 68, 1146 – 1149.
[19] Y. Lee, Y. Kim, K.-Y. Lee, K.-S. Kim, B. Kim, H.-N. Kim, C.-H.
Kim, S.-O. Do, Arch. Biochem. Biophys. 1998, 360, 41 – 46.
[20] M. A. Williams, H. Kitagawa, A. K. Datta, J. C. Paulson, J. C.
Jamieson, Glycoconjugate J. 1995, 12, 755 – 761.
[21] H. Yu, H. Chokhawala, R. Karpel, H. Yu, B. Wu, J. Zhang, Y.
Zhang, Q. Jia, X. Chen, J. Am. Chem. Soc. 2005, 127, 17618 –
17619.
[22] M. Sun, Y. Li, H. A. Chokhawala, R. Henning, X. Chen,
Biotechnol. Lett. 2008, 30, 671 – 676.
[23] A. Song, J. Zhang, M. Zhang, T. Shen, J. Tang, Colloids Surf. A
2000, 167, 253 – 262.
[10] a) H. Kayser, R. Zeitler, C. Kannicht, D. Grunow, R. Nuck, W.
Reutter, J. Biol. Chem. 1992, 267, 16934 – 16938; b) O. T.
Keppler, R. Horstkorte, M. Pawlita, C. Schmidt, W. Reutter,
Glycobiology 2001, 11, 11R – 18R; c) C. Oetke, R. Brossmer,
L. R. Mantey, S. Hinderlich, R. Isecke, W. Reutter, O. T.
Keppler, M. Pawlita, J. Biol. Chem. 2002, 277, 6688 – 6695.
[11] E. Saxon, C. R. Bertozzi, Science 2000, 287, 2007 – 2010.
[12] a) B. Mꢁller, C. Schaub, R. R. Schmidt, Angew. Chem. 1998, 110,
3021 – 3024; Angew. Chem. Int. Ed. 1998, 37, 2893 – 2897;
b) K. H. Jung, R. Schwçrer, R. R. Schmidt, Trends Glycosci.
Glycotechnol. 2003, 15, 275 – 289.
[24] X. Huang, A. Aulabaugh, Methods Mol. Biol. 2009, 565, 127 –
143.
[25] Z. Nikolovska-Coleska, R. Wang, X. Fang, H. Pan, Y. Tomita, P.
Li, P. P. Roller, K. Krajewski, N. G. Saito, J. A. Stuckey, et al.,
Anal. Biochem. 2004, 332, 261 – 273.
[13] L. Ni, H. A. Chokhawala, H. Cao, R. Henning, L. Ng, S. Huang,
H. Yu, X. Chen, A. J. Fisher, Biochemistry 2007, 46, 6288 – 6298.
´
[14] F. V. Rao, J. R. Rich, B. Rakic, S. Buddai, M. F. Schwartz, K.
Johnson, C. Bowe, W. W. Wakarchuk, S. DeFrees, S. G. Withers,
N. C. J. Strynadka, Nat. Struct. Mol. Biol. 2009, 16, 1186 – 1188.
[15] D. Pogatchnik, D. Wiemer, Tetrahedron Lett. 1997, 38, 3495 –
3498.
[16] F. M. Rossi, J. P. Y. Kao, Bioconjugate Chem. 1997, 8, 495 – 497.
[17] a) D. M. Whitfield, S. P. Douglas, T.-H. Tang, I. G. Csizmadia,
H. Y. S. Pang, F. L. Moolten, J. J. Krepinsky, Can. J. Chem. 1994,
72, 2225 – 2238; b) Y. Kajihara, T. Ebata, K. Koseki, H. Kodama,
H. Matsushita, H. Hironobu, J. Org. Chem. 1995, 60, 5732 – 5735.
[26] R. E. Pagano, O. C. Martin, H. C. Kang, R. P. Haugland, J. Cell
Biol. 1991, 113, 1267 – 1279.
[27] For a recent development and application of metabolic inhibitor
of cellular sialylation see: a) C. D. Rillahan, A. Antonopoulos,
C. T. Lefort, R. Sonon, P. Azadi, K. Ley, A. Dell, S. M. Haslam,
J. C. Paulson, Nat. Chem. Biol. 2012, 8, 661 – 668; b) C. Bꢁll, T. J.
Boltje, M. Wassink, A. M. A. de Graaf, F. L. van Delft, M. H.
den Brok, G. J. Adema, Mol. Cancer Ther. 2013, 12, 1935 – 1946.
6
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ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 7
These are not the final page numbers!

Angewandte
Chemie
Communications
Biomimetics of Carbohydrate Nucleo-
tides
J. J. Preidl, V. S. Gnanapragassam,
M. Lisurek, J. Saupe, R. Horstkorte,
J. Rademann*
&&&& — &&&&
Fluorescent Mimetics of CMP-Neu5Ac
Are Highly Potent, Cell-Permeable
Polarization Probes of Eukaryotic and
Bacterial Sialyltransferases and Inhibit
Cellular Sialylation
You can’t run, you can’t hide: Sialyl-
transferases cover cancer cells with neu-
raminic acids, enabling them to escape
from tissues and to metastasize. Cell-
permeable chemical probes targeting this
class of enzymes might help to study,
understand, and inhibit such processes.
Angew. Chem. Int. Ed. 2014, 53, 1 – 7
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!