Selective Engineering of Linkage-Specific α2,6-N-Linked Sialoproteins Using Sydnone-Modified Sialic Acid Bioorthogonal Reporters

Article abstract of DOI:10.1002/anie.201814266

The metabolic oligosaccharide engineering (MOE) strategy using unnatural sialic acids has recently enabled the visualization of the sialome in living systems. However, MOE only reports on global sialylation and dissected information regarding subsets of s

Full text of DOI:10.1002/anie.201814266

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Selective Engineering of Linkage Specific α2,6-N-Linked
Sialoproteins using Sydnone-Modified Sialic Acid Bioorthogonal
Reporters
Authors: Zoeisha S. Chinoy, Clément Bodineau, Camille Favre, Kelley
W. Moremen, Raúl V. Durán, and Frédéric Friscourt
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201814266
Angew. Chem. 10.1002/ange.201814266
Link to VoR: http://dx.doi.org/10.1002/anie.201814266
http://dx.doi.org/10.1002/ange.201814266

10.1002/anie.201814266
Angewandte Chemie International Edition
a
Selective Engineering of Linkage Specific 2,6-N-Linked Sialoproteins using
Sydnone-Modified Sialic Acid Bioorthogonal Reporters
Zoeisha S. Chinoy1,2, Clément Bodineau1,3, Camille Favre1,2, Kelley W. Moremen4,5, Raúl V.
Durán1,3,6 and Frédéric Friscourt*1,2
1Institut Européen de Chimie et Biologie, Université de Bordeaux, 2 rue Robert Escarpit, 33607
Pessac, France.
2Institut de Neurosciences Cognitives et Intégratives d’Aquitaine, CNRS UMR5287, Bordeaux,
France
3Institut Bergonié, INSERM U1218, Bordeaux, France
4Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA USA
5Complex Carbohydrate Research Center, University of Georgia, Athens, GA USA
6Current address: Centro Andaluz de Biología Molecular y Medicina Regenerativa, Consejo Superior
de Investigaciones Científicas - Universidad de Sevilla - Universidad Pablo de Olavide, Avda.
Américo Vespucio 24, 41092 Seville, Spain
* Email: f.friscourt@iecb.u-bordeaux.fr
Keywords: chemical reporter; click chemistry; metabolic incorporation; sialic acids; sydnone
Abstract: The metabolic oligosaccharide engineering (MOE) strategy using unnatural sialic acids
has recently enabled the visualization of the sialome in living systems. However, MOE only reports
on global sialylation and dissected information regarding subsets of sialoside is missing. Here we
describe the synthesis and utilization of sialic acids modified with a sydnone reporter for the
metabolic labeling of sialoconjugates. We showed that the positioning of the reporter on the sugar
significantly altered its metabolic fate. Further in vitro enzymatic assays revealed that the 9-modified
neuraminic acid is preferentially accepted by the sialyltransferase ST6Gal-I over ST3Gal-IV, leading
to the favored incorporation of the reporter into linkage specific a2,6-N-linked sialoproteins. This
sydnone-sugar raises the possibility of investigating the roles of specific sialosides.
1
This article is protected by copyright. All rights reserved.

10.1002/anie.201814266
Angewandte Chemie International Edition
Sialic acids are negatively charged carbohydrates that are usually found at the non-reducing end
of complex glycans decorating cell-surface proteins and lipids.[1] They are therefore ideally placed
for interacting with the cell’s exogenous environment and consequently for mediating physiological
and pathological events including cell adhesion, host-pathogen interactions and cancer progression.[2]
In mammals, the vast array of existing sialoconjugates are biosynthesized in a non-templated manner
by 20 sialytransferases (STs) that catalyze the transfer of the same donor substrate (cytidine-5′-
monophospho-N-acyl-neuraminic acid (CMP-Neu5Ac)) to various specific acceptor
glycoconjugates.[3]
By exploiting the donor promiscuity of STs, the metabolic oligosaccharide engineering (MOE)
strategy has emerged as a fundamental technology for interrogating the sialome in living systems.[4]
In this two-step approach, a bioorthogonal chemical handle is first introduced into sialoconjugates by
feeding cells a sialic acid modified with a reporter, which can then be selectively ligated to a
complementary bioorthogonal probe for direct visualization or affinity capture. The inconspicuous
organic azide has elegantly been employed as a versatile chemical reporter for the tagging of cell-
surface sialoconjugates[5] that can be detected in live cells and living animals under strict metal-free
conditions either by Staudinger ligation,[6] or by rapid 1,3-dipolar cycloaddition with tailored
cyclooctynes (SPAAC).[7, 8] Terminal[9] and strained alkenes[10] have recently been utilized for tagging
sialoconjugates in living cells, however they often suffer from poor reactivity with the complementary
tetrazine probe, which has so far precluded their application in living animals.
Despite the many attractive features of employing azido-sugars for the MOE, azides can be
reduced into amines in biological systems, by the action of thiols.[11] Controversially, this may lead
to the accumulation of secondary byproducts with unknown metabolic fate and biological effects. To
overcome this limitation, we envisaged that sialic acids fitted with a sydnone moiety, an aromatic
mesoionic 1,3-dipole, would be valuable unnatural sugars for addition to the MOE toolbox. We and
others have recently shown that sydnones can react in a metal-free strain-promoted click fashion with
cyclooctyne reagents for the in vitro bioconjugation of purified proteins.[12-16]
Herein, we report on the preparation and utilization of sialic acids bearing a sydnone reporter as
novel metabolic precursors for the labeling of sialoconjugates in living cells (Figure 1). We observed
that while 5-modified neuraminic acid with sydnone (Neu5SydCl) was not metabolically
incorporated into sialosides, a sialic acid analog having the sydnone moiety at its 9-position
(Neu9NSydCl) was well tolerated by the biosynthetic machinery of the cell, leading to robust display
of the reporter on cell-surface sialoconjugates. Interestingly, further in vitro enzymatic studies
revealed that unlike CMP-Neu5Ac, CMP-Neu9NSydCl is not a universal donor substrate of all the
STs involved in the synthesis of N-glycans, ultimately leading to the favored incorporation of the
reporter into linkage specific a2,6-N-linked sialoproteins, a beneficial outcome, since MOE, while
2
This article is protected by copyright. All rights reserved.

10.1002/anie.201814266
Angewandte Chemie International Edition
useful for reporting on global sialylation, is usually unable to differentiate among the various types
of sialosides.[17-19]
probe
CPS
click
OH
-
HO
CO2
9
CPS
HO
AcHN
O
OH
5
OH
Neu5Ac
5
CPS
9
CPS
Cl
N
CPS
O
N
O
Biosynthetic Pathway
of Sialic Acid-conjugates
Gal
GlcNAc
Figure 1. Chemical remodeling of cell-surface sialoconjugates using sydnone-modified sialic acids.
Performing organic reactions in living systems remains a formidable chemical challenge. Both
the chemical reporter and the associated probe must not only exhibit stringent biostability but also
fast and exquisite reactivity with each other. Consequently, we first examined the stability of
chlorosydnones under simulated physiological conditions in the presence of glutathione (GSH). GSH
is a cellular thiol-containing tripeptide present in millimolar concentrations in the cytosol of
mammalian cells. We focus our attention on chlorosydnone since we have previously shown that
introduction of a chlorine atom on the sydnone ring significantly enhanced its reactivity.[15, 16] In this
context, we incubated an aqueous solution of N-chlorosydnone-benzoyl mannosamine 1
(ManNSydCl), and for reference N-azidoacetyl mannosamine, having an anomeric p-methoxyphenyl
group (ManNAz-PMP 2, Scheme S1 for synthetic details), supplemented with 100-fold GSH at 37
°C, and monitored their respective stability over time by HPLC (Figures S1-S2). Gratifyingly, both
a- and b-anomers of 1 were found highly stable, as no decomposition was observed for over 72 h.
Conversely, incubating the azide 2, quickly led to the appearance of a new HPLC peak with shorter
retention time, that was identified as the expected reduced product by NMR spectroscopy and mass
spectrometry, highlighting the greater biostability of the sydnone reporter.
Next, to identify the most reactive cyclooctynyl-probe with the chlorosydnone reporter, we
determined the second-order rate constants of cycloadditions of three representative cyclooctynes
commonly used in bioconjugation, MeO-DIBO,[20] MeO-DIBAC[21] and BCN[22], with 4-chloro-3-
1
phenylsydnone (3) by H-NMR spectroscopy in CD3OD at 25 °C (Figures S3-S6). While 3 reacted
slowly with MeO-DIBO (k = 0.06 M−1 s−1), its rate constant with MeO-DIBAC (k = 0.30 M−1 s−1)
was on par with the cycloaddition of DIBAC with azide. Interestingly, 3 reacted notably faster with
the less sterically hindered BCN (k = 0.57 M-1 s-1), at a suitable rate constant for the labeling of
complex glycans in living cells.
3
This article is protected by copyright. All rights reserved.

10.1002/anie.201814266
Angewandte Chemie International Edition
To validate the physiological applicability of our system, we next turned our attention to
employing the chlorosydnone reporter for the labeling of sialosides in living cells. Accordingly, we
synthesized two unnatural neuraminic acids modified either at their 5- or 9-position with a
chlorosydnone. Neu5SydCl (4) was prepared enzymatically from ManNSydCl (1) in 88% yield using
recombinant Sialic acid Aldolase from E. coli, in the presence of sodium pyruvate (Scheme 1A). On
the other hand, incorporation of the chlorosydnone onto the 9-position of sialic acid was achieved by
chemical modification of 9-azido neuraminic acid 5 (Neu9Az) (Scheme 1B). Briefly, Neu9Az[23] was
reduced into the amine 6 by hydrogenation over Pd/C which was subsequently used for the
incorporation of the chlorosydnone using classical N-hydroxysuccinimide coupling protocol,
providing the desired Neu9NSydCl (7). Following a similar approach, we also prepared the azido-
analog Neu9NAz (8).
A.
O
OH
HO
HO
-PhSydCl
Cl
E. Coli SA-aldolase
NaPyruvate
CO2H
OH
HN
PhSydCl
OH
HO
HO
HO
O
O
MgCl2 (20 mM)
PB (0.1M) pH 7.7
N
H
ClSydPh
HO
N
N
O
O
O
1
4
88%
B.
O
O
OH
HO
N3
OH
HO
NH
HO
OH
CO2H
R
H2N
CO2H
CO2H
OH
H2, Pd/C
NHS
R
HO
AcHN
O
OH
O
OH
HO
O
DMF
H2O
AcHN
AcHN
HO
R
5
6
94%
PhSydCl
CH2-N3
7
8
61%
48%
Scheme 1. Preparation of 5- and 9-modified neuraminic acids. A. Enzymatic synthesis of Neu5SydCl (4); B.
Chemical synthesis of Neu9NSydCl (7) and Neu9NAz (8).
Next, U2OS cells were incubated for 48 h with 2 mM of neuraminic acids, Neu5Ac (negative
control), Neu9NAz (8) (positive control), and both sydnone modified compounds Neu5SydCl (4) and
Neu9NSydCl (7). Importantly, even at high concentration, none of the unnatural sugars exhibited any
particular cytotoxicity (Figures S9-S10). Labeled cells were then reacted with BCN-biotin conjugate
(50 µM) for 1 h and stained with Streptavidin-AlexaFluor568 for analysis by confocal fluorescence
microscopy (Figure 2A). Robust cell surface staining was observed when Neu9NSydCl (7) was
employed, demonstrating the successful metabolic labeling of chlorosydnone into sialosides of living
cells. To note, while employing BCN has previously been reported to be unspecific due to cross-
reactivity with endogenous thiols,[24] we did not observe any substantial unspecific labeling by flow
cytometry (Figure 2B and Figure S8), perhaps because our protocol only reports on cell-surface
modification. Flow cytometry analysis also revealed that the cell-surface incorporation of
Neu9NSydCl (7) was as efficient as the azido-analog 8 (Figure 2B). In marked contrast, performing
the MOE with Neu5SydCl (4) resulted in no cell membrane staining (Figure 2), indicating that
positioning of the chemical reporter on the sialic acid scaffold is of prime importance for a successful
4
This article is protected by copyright. All rights reserved.

10.1002/anie.201814266
Angewandte Chemie International Edition
MOE. To ensure that this noted difference in cell metabolization was specific to the reporter and not
cell-type dependent, we also performed the MOE on human lung carcinoma (A549) cells.
Fluorescence microscopy only showed cell-surface staining with Neu9NSydCl (7) (Figure S7),
suggesting that the cellular discrimination between the 5- and 9-modified sialic acid with the
chlorosydnone is an intrinsic feature of sialoconjugates production.
Figure 2. Metabolic glycan labeling of living U2OS cells with modified neuraminic acids. A. Fluorescence
microscope images of U2OS cells cultured with Neu5Ac, Neu9NAz (8), Neu5SydCl (4) and Neu9NSydCl (7),
treated with BCN-biotin and stained with Streptavidin-AlexaFluor568 (Red) and DAPI (Blue), Scale bar: 10
µm. B. Flow cytometry analysis of U2OS cells cultured respectively with No Sugar, Neu5Ac, Neu9NAz (8),
Neu5SydCl (4) and Neu9NSydCl (7). All cells were treated with BCN-biotin and Streptavidin-AlexaFluor568.
Intrigued by the non-metabolization of Neu5SydCl (4) by cells, we decided to investigate which
biosynthetic enzyme may be responsible for blocking its incorporation into sialosides. To be
integrated into complex glycans, cytosolic sialic acids must first be activated in the nucleus into their
CMP-analogs by the action of CMP-sialic acid synthetase (CSS), followed by their transport to the
Golgi where they are utilized by sialyltransferases (STs).[25] To investigate the substrate tolerance of
CSS, we devised an in vitro enzymatic-1H-NMR coupled assay using bacterial CSS from N.
meningitidis (NmCSS). We selected NmCSS over its mammalian analog due to its commercial
availability. Favorably, murine CSS and NmCSS have been shown to share a similar overall active-
site architecture involved in substrate binding.[26] Accordingly, Neu5Ac and its unnatural analogs
Neu5SydCl (4), Neu9NSydCl (7) and Neu9NAz (8) were individually incubated at 37 °C with
1
NmCSS and CTP (Figure 3A), and the crude mixture was analyzed by H-NMR spectroscopy to
determine the formation of the respective CMP-sialic acids (Figure 3B and Figures S11-S14). The
chemical shifts of the protons present on the C3-carbon atom of sialic acid (dashed lines in Figure 3)
provided distinct peaks changes between 1.6 and 2.6 ppm upon CMP-activation, ideal to detect
product formation. After 5 h, while over 90% of Neu5Ac was converted into CMP-Neu5Ac,
modification at the 9-position with either an azido (Neu9NAz) or a chlorosydnone (Neu9NSydCl)
moiety only slightly interfered with the CMP-activation with a product conversion of 75% and 50%,
5
This article is protected by copyright. All rights reserved.

10.1002/anie.201814266
Angewandte Chemie International Edition
respectively. However, having the chlorosydnone on the 5-position of sialic acid seemed to prevent
NmCSS from generating the desired CMP-Neu5SydCl, as no product was detected, even upon
prolonged reaction time (up to 24 h, Figure S13). Collectively, these results suggest that Neu5SydCl
(4) is not recognized by CSS as an appropriate substrate and is therefore not activated into its CMP-
analog, which precludes its incorporation into cell-surface glycans.
Figure 3. Enzymatic-1H-NMR coupled assay for the detection of CMP-sialic acids formation. A. Enzymatic
formation of CMP-sialic acid using NmCSS. B. 1H-NMR spectra of the corresponding crude reaction mixtures
for CMP-Neu5Ac, -Neu9NAz, -Neu5SydCl and -Neu9NSydCl. Protons on the C3-carbon atom of the starting
sialic acid (dashed blue line) and the CMP-sialic acid product (dashed orange line).
Having established that the presence of the sydnone reporter on sialic acid could have dramatic
impact on enzymes substrate recognition, we sought to examine the promiscuity of STs involved in
the biosynthesis of N-linked sialoproteins towards CMP-Neu9NSydCl (9). We chose to investigate
N-glycan structures because they are the most prevalent modifications found in vertebrate
glycoproteins.[27] Sialylation of N-glycoproteins is mainly controlled by ST6Gal-I and ST3Gal-IV
that adds a sialic acid residue to N-acetyllactosamine (LacNAc) termini of N-glycans in an a2,6- and
a2,3-linkage, respectively.
To determine the tolerance of human ST6Gal-I and ST3Gal-IV in terms of accepting CMP-sialic
acid analogs, we measured the kinetic parameters of both enzymes for the transfer of CMP-Neu5Ac
6
This article is protected by copyright. All rights reserved.

10.1002/anie.201814266
Angewandte Chemie International Edition
and its unnatural analogs CMP-Neu9NSydCl (9) and CMP-Neu9Az (10) to LacNAc-butyl (11), as a
model acceptor (Table 1 and Figures S15-S16). ST6Gal-I seemed to be promiscuous in regards to
utilizing CMP-sialic acid analogs with a slight preference for sydnone 9, exhibiting kcat/KM values of
similar order. In contrast, the activity of ST3Gal-IV was clearly more impacted by the presence of
the reporters. ST3Gal-IV displayed a 10-fold decrease in kcat/KM values for sydnone 9 (kcat/KM = 1.7
± 1.4 mM-1 min-1, the lack of recognition of 9 by ST3Gal-IV prevented Vmax to be reached (Figure
S16) and therefore the KM(app) and kcat(app) values should be considered estimates) compared to azide
10 and the natural CMP-Neu5Ac substrate, suggesting that in a physiological context, while CMP-
Neu9Az (10) would similarly be employed by both enzymes, CMP-Neu9NSydCl (9) would be
preferentially utilized by ST6Gal-I over ST3Gal-IV.
Table 1. Kinetic constants of the sialylation of LacNAc-butyl (11) by human ST6Gal-I or ST3Gal-
IV in the presence of CMP-Neu5Ac, -Neu9Az (10) or -Neu9NSydCl (9)a
ST6Gal-I
kcat(app)
ST3Gal-IV
kcat(app)
KM(app)
[µM]
KM(app)
[µM]
kcat/KM
kcat/KM
Donor
[min-1]
[mM-1.min-1]
[min-1]
[mM-1.min-1]
CMP-Neu5Ac
CMP-Neu9Az (10)
CMP-Neu9SNSydCl (9)
11 ± 0.4
18 ± 1.7
10 ± 0.7
92 ± 25
47 ± 19
116 ± 33
1.4 ± 0.1
3.4 ± 0.3
5.2 ± 3.7
4.7 ± 1.5
14.3 ± 5.1
1.7 ± 1.4
119 ± 16
385 ± 88
86 ± 21
300 ± 66
237 ± 59
3057 ± 2720
a
Data obtained using fixed enzyme and LacNAc-butyl concentrations while varying the
concentrations of the CMP-sialic acid donors.
To further validate the preferential incorporation of Neu9NSydCl (7) into a2,6-N-sialoproteins in
a more complex system, we incubated asialofetuin, a model glycoprotein displaying LacNAc
terminated N-glycans, with human ST6Gal-I or ST3Gal-IV in the presence of either CMP-Neu5Ac
or its unnatural analogs chlorosydnone 9 and azide 10 overnight at 37 °C in cacodylate buffer (50
mM, pH 7.5). The labeled proteins were then treated with BCN-FITC conjugate at 37 °C for 3 h to
reveal the presence of the reporters by in gel-fluorescence imaging (Figure 4). A robust fluorescent
band, corresponding to enzymatically sialylated asialofetuin, was clearly detected when both azido-
and sydnone-modified CMP-sialic acids were employed in the presence of ST6Gal-I, confirming the
promiscuity of this enzyme. In sharp contrast, when ST3Gal-IV was utilized, fluorescent labeling was
only observed in the presence of azide 10, highlighting the selective incorporation of Neu9NSydCl
(7) into a2,6-N-sialoproteins in a more complex biochemical context.
7
This article is protected by copyright. All rights reserved.

10.1002/anie.201814266
Angewandte Chemie International Edition
Figure 4. In-gel visualization of asialofetuin (2.4 mg/mL) incubated with human ST6Gal-I (19.5 µg/mL) or
ST3Gal-IV (19.5 µg/mL) in the presence of CMP-Neu5Ac (1.2 mg/mL) or its unnatural analogs CMP-Neu9Az
(10) (1.2 mg/mL) or CMP-Neu9NSydCl (9) (1.7 mg/mL), or no reagent (−). The presence of the chemical
reporter was detected by fluorescence imaging (incubation with BCN-FITC (top row; λexc: trans UV)) and by
Coomassie Blue stain to reveal total protein content (bottom row).
In summary, we have demonstrated that chlorosydnones are valuable chemical reporters for the
MOE of living cells. In particular, they not only proved to react positively fast with strained
cyclooctynes but also showed high stability in the presence of thiols. In addition, we showed that
unlike azide, the chlorosydnone reporter is not an innocent bystander and its presence on sialic acid
was not unnoticed by the biosynthetic pathway of sialoconjugates. For instance, while employing
Neu9NSydCl led to robust cell-surface labeling, Neu5SydCl was not metabolized by cells and we
identified CMP-sialic acid synthetase as the enzymatic roadblock. Furthermore, CMP-Neu9NSydCl
was shown to be preferentially accepted by the sialyltransferase ST6Gal-I over ST3Gal-IV, leading
to the favored incorporation of the sydnone reporter into linkage specific a2,6-N-linked sialoproteins.
Neu9NSydCl raises therefore the possibility to selectively track and capture a subset of sialosides, a
desirable outcome, since MOE with existing azido-sugars can only report on global sialylation.
Further studies are currently being performed in our laboratory to explore the impact of
chlorosydnone on a larger panel of human sialyltransferases.
Acknowledgments
Financial support from CNRS (ATIP-Avenir program to F.F.) is gratefully acknowledged. This study
has also received financial support from the French State in the frame of the "Investments for the
Future" Programme IdEx Bordeaux (ANR-10-IDEX-03-02 to F.F.) and from the Laboratory of
Excellence TRAIL (ANR-10-LABX-57 to F.F.), EraNet Neuron TRAINS, and US National Institutes
of Health grants (P41GM103390, P01GM107012, and R01GM130915 to K.W.M.).
8
This article is protected by copyright. All rights reserved.

10.1002/anie.201814266
Angewandte Chemie International Edition
References
[1]
A. Varki, R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart,
M. E. Etzler, Essentials of Glycobiology, 2nd ed., Cold Spring Harbor Laboratory Press, 2009.
A. Varki, Nature 2007, 446, 1023-1029.
A. Harduin-Lepers, V. Vallejo-Ruiz, M. A. Krzewinski-Recchi, B. Samyn-Petit, S. Julien, P.
Delannoy, Biochimie 2001, 83, 727-737.
[2]
[3]
[4]
[5]
[6]
[7]
P. R. Wratil, R. Horstkorte, W. Reutter, Angew. Chem. Int. Ed. 2016, 55, 9482-9512.
E. Saxon, C. R. Bertozzi, Science 2000, 287, 2007-2010.
J. A. Prescher, D. H. Dube, C. R. Bertozzi, Nature 2004, 430, 873-877.
P. V. Chang, J. A. Prescher, E. M. Sletten, J. M. Baskin, I. A. Miller, N. J. Agard, A. Lo, C.
R. Bertozzi, Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 1821-1826.
F. Friscourt, G. J. Boons, in Click Chemistry in Glycoscience: New Developments and
Strategies, 2013, pp. 211-233.
[8]
[9]
A. Niederwieser, A. K. Spate, L. D. Nguyen, C. Jungst, W. Reutter, V. Wittmann, Angew.
Chem. Int. Ed. 2013, 52, 4265-4268.
[10] D. M. Patterson, L. A. Nazarova, B. Xie, D. N. Kamber, J. A. Prescher, J. Am. Chem. Soc.
2012, 134, 18638-18643.
[11] A. L. Handlon, N. J. Oppenheimer, Pharm. Res. 1988, 5, 297-299.
[12] S. Wallace, J. W. Chin, Chem. Sci. 2014, 5, 1742-1744.
[13] M. K. Narayanam, Y. Liang, K. N. Houk, J. M. Murphy, Chem. Sci. 2016, 7, 1257-1261.
[14] S. Bernard, D. Audisio, M. Riomet, S. Bregant, A. Sallustrau, L. Plougastel, E. Decuypere, S.
Gabillet, R. A. Kumar, J. Elyian, M. N. Trinh, O. Koniev, A. Wagner, S. Kolodych, F. Taran,
Angew. Chem. Int. Ed. 2017, 56, 15612-15616.
[15] C. Favre, L. de Cremoux, J. Badaut, F. Friscourt, J. Org. Chem. 2018, 83, 2058-2066.
[16] C. Favre, F. Friscourt, Org. Lett. 2018, 20, 4213-4217.
[17] H. Moller, V. Bohrsch, J. Bentrop, J. Bender, S. Hinderlich, C. P. R. Hackenberger, Angew.
Chem. Int. Ed. 2012, 51, 5986-5990.
[18] N. E. Mbua, X. R. Li, H. R. Flanagan-Steet, L. Meng, K. Aoki, K. W. Moremen, M. A.
Wolfert, R. Steet, G. J. Boons, Angew. Chem. Int. Ed. 2013, 52, 13012-13015.
[19] N. Nischan, J. J. Kohler, Glycobiology 2016, 26, 789-796.
[20] F. Friscourt, P. A. Ledin, N. E. Mbua, H. R. Flanagan-Steet, M. A. Wolfert, R. Steet, G. J.
Boons, J. Am. Chem. Soc. 2012, 134, 5381-5389.
[21] M. F. Debets, J. S. Prins, D. Merkx, S. S. van Berkel, F. L. van Delft, J. C. M. van Hest, F.
Rutjes, Org. Biomol. Chem. 2014, 12, 5031-5037.
[22] J. Dommerholt, S. Schmidt, R. Temming, L. J. A. Hendriks, F. Rutjes, J. C. M. van Hest, D.
J. Lefeber, P. Friedl, F. L. van Delft, Angew. Chem. Int. Ed. 2010, 49, 9422-9425.
[23] S. Han, B. E. Collins, P. Bengtson, J. C. Paulson, Nat. Chem. Biol. 2005, 1, 93-97.
[24] R. van Geel, G. J. M. Pruijn, F. L. van Delft, W. C. Boelens, Bioconjugate Chem. 2012, 23,
392-398.
[25] J. Du, M. A. Meledeo, Z. Wang, H. S. Khanna, V. D. P. Paruchuri, K. J. Yarema, Glycobiology
2009, 19, 1382-1401.
[26] S. Krapp, A. K. Munster-Kuhnel, J. T. Kaiser, R. Huber, J. Tiralongo, R. Gerardy-Schahn, U.
Jacob, J. Mol. Biol. 2003, 334, 625-637.
[27] K. W. Moremen, M. Tiemeyer, A. V. Nairn, Nat. Rev. Mol. Cell Biol. 2012, 13, 448-462.
9
This article is protected by copyright. All rights reserved.

10.1002/anie.201814266
Angewandte Chemie International Edition
TOC
In search of selectivity: Incorporation of a clickable sydnone reporter onto sialic acid had a profound
impact on its metabolic fate. While the 5-modified sugar was not utilized by cells, the 9-modified
analog was strongly displayed on cell-surface sialosides. Further studies revealed that the 9-modified
sialic acid is preferentially accepted by the sialyltransferase ST6Gal-I over ST3Gal-IV, leading to the
incorporation of the reporter into linkage specific a2,6-N-linked sialoproteins.
10
This article is protected by copyright. All rights reserved.