Probe sialidase substrate specificity using chemoenzymatically synthesized sialosides containing C9-modified sialic acid

Article abstract of DOI:10.1039/c2cc17393j

A library of α2-3- and α2-6-linked sialyl galactosides containing C9-modified sialic acids was synthesized from C6-modified mannose derivatives using an efficient one-pot three-enzyme system. These sialosides were used in a high-throughput sialidase substrate specificity assay to elucidate the importance of C9-OH in sialidase recognition. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc17393j

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COMMUNICATION
Probe sialidase substrate specificity using chemoenzymatically
synthesized sialosides containing C9-modified sialic acidw
Zahra Khedri,z Musleh M. Muthana,z Yanhong Li,z Saddam M. Muthana,y Hai Yu,
Hongzhi Caoz and Xi Chen*
Received 27th November 2011, Accepted 7th February 2012
DOI: 10.1039/c2cc17393j
A library of a2–3- and a2–6-linked sialyl galactosides containing
C9-modified sialic acids was synthesized from C6-modified mannose
derivatives using an efficient one-pot three-enzyme system. These
sialosides were used in a high-throughput sialidase substrate
specificity assay to elucidate the importance of C9–OH in
sialidase recognition.
a hydrogen to form a C9-deoxy derivative can provide information
about the importance of C9–OH as both a hydrogen bond donor
and an acceptor. Substituting the C9–OH with a methoxy group
allows investigating the steric effect at C9 of sialic acid in protein
recognition. Finally, replacing the C9–OH with an azido group
introduces a chemical handle which can be directly used or reduced
to an amino group for coupling with diverse alkynyl, carbonyl or
acyl-containing molecules to expand the size and the application of
the resulting sialoside library.
Carbohydrate–protein interactions mediate a wide variety of
biological, pathological, and immunological processes.1,2
Many carbohydrates and glycoconjugates involved in the
protein recognition processes have terminal sialic acids. Sialic
acids form a family of more than 50 members of a-keto acids
with a 9-carbon backbone and three basic forms are the most
abundant, N-acetylneuraminic acid (Neu5Ac), non-human
N-glycolylneuraminic acid (Neu5Gc), and de-amino sialic acid
2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn).
These forms can be further acetylated, methylated, lactylated,
phosphorylated, or sulfated.3–5 The functions of sialic acids
are finely tuned by these naturally occurring modifications.5
Quite often, terminal sialic acid residues are modified at
carbon 9. To understand the importance of the C9–hydroxyl
group of sialic acids in protein recognition at the molecular
level, we planned to synthesize a library of a2–3- and a2–6-linked
sialyl para-nitrophenyl galactosides in which the C9–hydroxyl
group in the sialic acid was systematically substituted with F,
OMe, H, and N3, respectively. Substituting the C9–OH with a
fluorine allows the investigation of the importance of the hydrogen
bond donor effect of the hydroxyl group as fluorine can only
function as a hydrogen bond acceptor while the hydroxyl group
can behave both as a hydrogen bond acceptor and a hydrogen
bond donor despite their similar sizes.6 Replacing the C9–OH with
In order to achieve systematic synthesis of targeted a2–3-
and a2–6-linked sialosides containing C9-modified Neu5Ac,
Neu5Gc, and Kdn, a convenient and highly efficient one-pot
three-enzyme sialylation system established in our group7 was
used. This system contained a P. multocida sialic acid aldolase
(PmNanA),8 an N. meningitidis CMP-sialic acid synthetase
(NmCSS),9 and a bacterial sialyltransferase chosen from a
P. multocida multifunctional a2–3-sialyltransferase (PmST1)10
or a P. damselae a2–6-sialyltransferase (Pd2,6ST).11 It allowed
the synthesis of a2–3/6-linked sialosides containing diverse
sialic acid forms from simpler six-carbon sialic acid precursors
without purifying intermediates or using expensive or unavailable
sugar nucleotides. The six-carbon precursors (Scheme 1A) for
sialic acids and derivatives including N-acetylmannosamine
(ManNAc, 1a) derivatives 2a–5a, N-glycolylmannosamine
(ManNGc, 6a) derivatives 7a–10a, and mannose (Man, 11a)
derivatives 12a–15a were synthesized chemically, mostly for
the first time, using inexpensive glucose or mannose as a
starting material (see ESIw).
As shown in Scheme 1B, a2–3- and a2–6-linked sialosides
Siaa2–3GalbpNP (1b–15b) and Siaa2–6GalbpNP (1c–15c)
containing Neu5Ac, Neu5Gc, and Kdn and their C9-derivatives
were readily available using p-nitrophenyl b-D-galactopyranoside
(16, GalbpNP) as the sialyltransferase acceptor. Except for
sialosides containing natural sialic acid forms Neu5Ac (1b and 1c),
Neu5Gc (6b and 6c), and Kdn (11b and 11c) which were reported
previously,12 sialosides containing C9–F, –OMe, –H, or –N3
modified sialic acids were obtained for the first time.
Department of Chemistry, University of California,
One Shields Avenue, Davis, California 95616, USA.
E-mail: chen@chem.ucdavis.edu; Fax: +1 530-752-8995;
Tel: +1 530-754-6037
w Electronic supplementary information (ESI) available: Experimental
details of cloning, expression, and purification of BbUSP, chemical
and enzymatic synthesis, NMR and HRMS data. See DOI: 10.1039/
c2cc17393j
All a2–3/6-linked sialyl GalbpNP containing C-9 modified
Neu5Ac (2b–5b and 2c–5c) or Neu5Gc (7b–10b and 7c–10c)
were prepared in 81–98% yields, except for Neu5Gc9O-
Mea2–3GalbpNP 8b (40%) and Neu5Gc9OMea2–6GalbpNP
8c (30%). The low yields for the synthesis of 8b and 8c seemed
to be due to the limitation of sialic acid aldolase-catalyzed
z These authors contributed equally.
y Current address: National Cancer Institute, Frederick, Maryland,
20702, USA.
z Current address: National Glycoengineering Research Center,
Shandong University, Jinan, Shandong 250012, P. R. China.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 3357–3359 3357

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were obtained in good yields. Interestingly, the a2–3-linked
sialosides Kdn9Deoxya2–3GalbpNP 14b and Kdn9N3a2–
3GalbpNP 15b were obtained in excellent 94% and 91% yields,
respectively. However, their corresponding a2–6-linked sialosides
Kdn9Deoxya2–6GalbpNP 14c (70%) and Kdn9N3a2–6GalbpNP
15c (59%) were synthesized in lower yields. These results indicated
that CMP-Kdn9Deoxy and CMP-Kdn9N3 were relatively good
substrates for PmST1, but poorer substrates for Pd2,6ST.
The thirty sialyl galactosides synthesized including Siaa2–3/
6GalbpNP containing Neu5Ac, Neu5Gc, Kdn, or their C9-
derivatives (1b–15b and 1c–15c) were used in a 384-well plate-
based high-throughput colorimetric assay for the substrate
specificity studies of six sialidases,13 including four commercially
available bacterial sialidases from S. typhimurium (Prozyme),
C. perfringens (Sigma), V. cholerae (Sigma), and S. pneumoniae
(Prozyme), a multifunctional P. multocida sialyltransferase
(PmST1) which possesses sialidase activity,10,12 and a human
cytosolic sialidase NEU2.14 All six sialidases tested did not show
obvious hydrolysis activity towards Kdn or its C9-derivatives
(data not shown) under the conditions used (see ESIw). Therefore,
only the results using sialosides containing Neu5Ac, Neu5Gc, and
Scheme 1 (A) Structures of sialic acid precursors including N-acetyl-
mannosamine 1a, N-glycolylmannosamine 6a, mannose 11a, and their
derivatives; (B) synthesis of pNP-tagged a2–3/6-linked sialosides using a
one-pot three-enzyme system containing a P. multocida sialic acid
aldolase (PmNanA),8 an N. meningitidis CMP-sialic acid synthetase
(NmCSS),9 and a bacterial sialyltransferase chosen from a P. multocida
multifunctional a2–3-sialyltransferase (PmST1)10 or a P. damselae
a2–6-sialyltransferase (Pd2,6ST).11
reaction as shown by thin-layer chromatography (TLC) assays
for aldolase-catalyzed reaction using sodium pyruvate and
6-O-Me-ManNGc 8a for the formation of its sialic acid form
Neu5Gc9OMe. In contrast to the low yields of sialosides 8b and 8c
containing Neu5Gc9OMe, excellent yields were achieved for the
synthesis of sialosides Neu5Ac9OMea2–3GalbpNP 3b (94%) and
Neu5Ac9OMea2–6GalbpNP 3c (92%) containing Neu5Ac9OMe.
In addition, excellent yields were achieved for the synthesis of
sialosides containing C9–fluorine Neu5Ac or Neu5Gc residue
Neu5Ac9Fa2–3GalbpNP 2b (98%), Neu5Ac9Fa2–6GalbpNP 2c
(95%), Neu5Gc9Fa2–3GalbpNP 7b (98%), Neu5Gc9Fa2–
6GalbpNP 7c (95%), as well as C9-deoxy Neu5Ac or Neu5Gc
residue Neu5Ac9Deoxya2–3GalbpNP 4b (90%), Neu5Ac9-
Deoxya2–6GalbpNP 4c (97%), Neu5Gc9Deoxya2–3GalbpNP 9b
(90%), and Neu5Gc9Deoxya2–6GalbpNP 9c (84%). These
indicated that F and H substitutions on C-9 of Neu5Ac/
Neu5Gc make them similar or even better substrates for
enzymes used in the biosynthesis of sialosides.
Fig. 1 Substrate specificity studies of five bacterial sialidases and
human cytosolic sialidase NEU2. Reactions were carried out at 37 1C
for 30 min. The reaction with GalbpNP and an excess amount of
b-galactosidase was used as a control. X-axis labels: 1, 1b and 1c; 2, 2b
and 2c; 3, 3b and 3c; 4, 4b and 4c; 5, 5b and 5c; 6, 6b and 6c, 7, 7b and
7c; 8, 8b and 8c; 9, 9b and 9c; 10, 10b and 10c. White bars represent
results using Siaa2–3GalbpNP 1b–10b as the sialidase substrates;
black bars represent results using Siaa2–6GalbpNP 1c–10c as the
sialidase substrates. Sialic acid residues in Siaa2–3GalbpNP (1b–10b)
and Siaa2–6GalbpNP (1c–10c) are Neu5Ac (1b, 1c); Neu5Ac9F
(2b, 2c); Neu5Ac9OMe (3b, 3c); Neu5Ac9Deoxy (4b, 4c); Neu5Ac9N3
(5b, 5c); Neu5Gc (6b, 6c); Neu5Gc9F (7b, 7c); Neu5Gc9OMe (8b, 8c);
Neu5Gc9Deoxy (9b, 9c); Neu5Gc9N3 (10b, 10c).
For the synthesis of pNP-tagged sialosides containing
C9-derivatives of Kdn (12b–15b and 12c–15c), higher yields
were achieved for both Kdn9Fa2–3GalbpNP 12b (94%) and
Kdn9Fa2–6GalbpNP 12c (98%) compared to Kdna2–3GalbpNP
11b (75%) and Kdna2–6GalbpNP 11c (80%) indicating that the
C9–fluorine modification in sialic acids made them better enzyme
substrates. Sialosides with C9-methoxy Kdn such as Kdn9OMea2–
3GalbpNP 13b (78%) and Kdn9OMea2–6GalbpNP 13c (62%)
c
3358 Chem. Commun., 2012, 48, 3357–3359
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their derivatives (1b–10b and 1c–10c) as sialidase substrates are
shown in Fig. 1.
cleavage (Fig. 1E). As shown before,12,13 a2–3-linked Neu5Gc-
containing sialoside was a better substrate than the corres-
ponding Neu5Ac-containing counterpart for the a2–3-sialidase
activity of PmST1. Nevertheless, C9-modifications on Neu5Gc
in Siaa2–3GalbpNP decreased the a2–3-sialidase activity of
PmST1. In general, Siaa2–3GalbpNP containing Neu5Gc or
its C9-derivatives were not good substrates for S. pneumoniae
sialidase (Fig. 1D) although some of the structures including
Neu5Gca2–3GalbpNP (6b), Neu5Gc9OMea2–3GalbpNP (8b),
and Neu5Gc9N3a2–3GalbpNP (10b) were weak substrates for
S. typhimurium sialidase (Fig. 1C).
In consistence with our previous reports,12,13 C. perfringens
sialidase (Fig. 1A), V. cholerae sialidase (Fig. 1B), and human
sialidase NEU2 (Fig. 1F) were able to cleave both a2–3- and
a2–6-linked sialosides, while S. typhimurium sialidase
(Fig. 1C), S. pneumoniae sialidase (Fig. 1D), and PmST1
(Fig. 1E) could only cleave a2–3-linked sialosides efficiently.
Among three sialidases that could cleave both a2–3- and
a2–6-linked sialosides including C. perfringens sialidase
(Fig. 1A), V. cholerae sialidase (Fig. 1B), and human NEU2
(Fig. 1F), V. cholerae sialidase (Fig. 1B) had the highest
substrate promiscuity. It could use all 20 compounds
(1b–10b and 1c–10c) as substrates and only the 9-O-methyl
modification on either Neu5Ac or Neu5Gc decreased its
activity significantly. 9-F and 9-deoxy modification on
Neu5Gc decreased its activity moderately and 9-N3 modification
on Neu5Gc or 9-F, 9-deoxy, and 9-N3 modifications on Neu5Ac
only affected its activity weakly. In comparison, although
C9-modifications on Neu5Ac were well tolerated by C. perfringens
sialidase (Fig. 1A), sialosides containing Neu5Gc or its
C9-derivatives were not good substrates for this enzyme. Quite
interestingly, although human do not synthesize Neu5Gc-
containing structures but present substantial amount of Neu5Ac
on oligosaccharides and glycoconjugates, human cytosolic sialidase
NEU2 (Fig. 1F) cleaved Neu5Gc- and Neu5Gc9N3-containing
sialosides more efficiently than Neu5Ac-containing sialosides.
Sialosides containing C9-modified Neu5Ac derivatives and
9-F, 9-methoxy, and 9-deoxy derivatives of Neu5Gc were
not suitable substrates for human NEU2. This indicated that
while NEU2 was able to tolerate some modifications at C5 as
shown previously,14 it was more restricted to C9-modifications. It
is unclear why the sialosides with C9-azido Neu5Gc (10b and 10c)
were tolerable substrates for NEU2 but other modifications
on Neu5Gc protected it from being cleaved by NEU2. Future
studies are needed to test whether C9-azido modification on
the Neu5Gc in sialosides changes the conformation of NEU2.
All three sialidases that were specific for a2–3-linked sialosides
including S. typhimurium sialidase (Fig. 1C), S. pneumoniae
sialidase (Fig. 1D), and PmST1 (Fig. 1E) had good tolerance
towards C9-modifications on Neu5Ac in sialoside substrates. No
significant changes were seen for S. typhimurium sialidase
(Fig. 1C) and S. pneumoniae sialidase (Fig. 1D) when a2–3-linked
sialosides containing Neu5Ac or its C9-derivatives were used
as the substrates. Quite interestingly, 9-F, 9-deoxy, and 9-N3
modifications on Neu5Ac in Siaa2–3GalbpNP (2b, 4b and 5b,
respectively) provided better substrates than non-modified
Neu5Aca2–3GalbpNP (1b) for the a2–3-sialidase activity
of PmST1 with about 2-fold activity increase in sialic acid
In conclusion, a library of thirty pNP-tagged sialosides
containing Neu5Ac, Neu5Gc, and Kdn derivatives, in which
the C9–OH on the sialic acid moiety was systematically
substituted with F, OMe, H, and N3, was chemoenzymatically
synthesized using an efficient one-pot three-enzyme approach.
These synthetic sialosides were applied in a 384-well plate-
based colorimetric high-throughput screening platform as
important probes for elucidating the importance of sialic
acid C9-modifications and identifying substrate specificity of
various sialidases.
This work was supported by NIH grant R01HD065122,
NSF grant CHE-1012511, and Beckman Young Investigator
Award. X. Chen is a Camille Dreyfus Teacher-Scholar and a
UC-Davis Chancellor’s Fellow.
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c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 3357–3359 3359