Chemoenzymatic synthesis of sialosides containing C7-modified sialic acids and their application in sialidase substrate specificity studies

Article abstract of DOI:10.1016/j.carres.2014.02.021

Modifications at the glycerol side chain of sialic acid in sialosides modulate their recognition by sialic acid-binding proteins and sialidases. However, limited work has been focused on the synthesis and functional studies of sialosides with C7-modified

Full text of DOI:10.1016/j.carres.2014.02.021

Carbohydrate Research 389 (2014) 100–111
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Chemoenzymatic synthesis of sialosides containing C7-modified
sialic acids and their application in sialidase substrate specificity
studies
à
⇑
Zahra Khedri , Yanhong Li, Saddam Muthana , Musleh M. Muthana, Ching-Wen Hsiao, Hai Yu, Xi Chen
Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Modifications at the glycerol side chain of sialic acid in sialosides modulate their recognition by sialic
acid-binding proteins and sialidases. However, limited work has been focused on the synthesis and
functional studies of sialosides with C7-modified sialic acids. Here we report chemical synthesis of
C4-modified ManNAc and mannose and their application as sialic acid precursors in a highly efficient
Received 27 December 2013
Accepted 20 February 2014
Available online 6 March 2014
one-pot three-enzyme system for chemoenzymatic synthesis of
a2–3- and a2–6-linked sialyl para-
Keywords:
nitrophenyl galactosides in which the C7-hydroxyl group in sialic acid (N-acetylneuraminic acid, Neu5Ac,
or 2-keto-3-deoxynonulosonic acid, Kdn) was systematically substituted by –F, –OMe, –H, and –N3
groups. Substrate specificity study of bacterial and human sialidases using the obtained sialoside library
containing C7-modified sialic acids showed that sialosides containing C7-deoxy Neu5Ac were selective
substrates for all bacterial sialidases tested but not for human NEU2. The information obtained from
sialidase substrate specificity can be used to guide the design of new inhibitors that are selective against
bacterial sialidases.
Chemoenzymatic synthesis
C7-modified sialic acid
Sialidase
Sialoside
Sialyltransferase
Substrate specificity
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
In order to facilitate the understanding of the significance of sia-
lic acid modifications in nature and to develop sialic acid-based
Sialic acids are common terminal monosaccharides on the
carbohydrate moieties of surface glycoconjugates on mammalian
cells. They are directly involved in numerous molecular recogni-
tion events involved in immune regulation, cell–cell interaction,
inflammation, bacterial, and viral infection. Three basic forms of
sialic acids are N-acetylneuraminic acid (Neu5Ac), N-glycolylneu-
raminic acid (Neu5Gc), and 2-keto-3-deoxynonulosonic acid
(Kdn) (Fig. 1). In nature, sialic acids can undergo additional
post-glycosylational modifications (modifications after the forma-
tion of glycosidic linkages)1 including mono- and multiple O-acet-
ylation at any hydroxyl groups, O-methylation and O-sulfation at
C-8, as well as O-lactylation and O-phosphorylation at C-9. So
far, more than 50 structurally distinct sialic acid forms have been
identified in nature.2–4 Although the functions of some sialic acid
modifications have been elucidated,2,3 the roles of the majority of
modified sialic acid residues remain unknown.
therapeutics, a highly efficient one-pot multienzyme chemoenzy-
matic sialylation system has been developed for the synthesis of
sialosides containing naturally occurring sialic acids and their
non-natural derivatives.5–7 In this system, N-acetylmannosamine
(ManNAc) and mannose derivatives are chemically or enzymati-
cally synthesized as sialic acid precursors. ManNAc, mannose,
and their derivatives are converted by a sialic acid aldolase
(NanA)-catalyzed reaction to form sialic acids and their derivatives,
which are activated by a CMP-sialic acid synthetase (CSS), and
transferred to proper acceptors by suitable sialyltransferases
(SiaTs) to form targeted sialosides with desired sialic acid forms,
sialyl linkages, and underlying glycans.5 The enzymatic reactions
containing three enzymes can be carried out in one-pot in a
buffered aqueous solution at room temperature or 37 °C without
the isolation of intermediates. It has been shown that all enzymes
used including recombinant bacterial NanAs, CSSs, and SiaTs
have quite good tolerance toward substrate modifications. Sialo-
sides with natural and non-natural modifications at C-5, C-9, and
⇑
Corresponding author. Tel.: +1 530 7546037; fax: +1 530 7528995.
C-8 of sialic acid have been successfully synthesized.6–12
and 2–6-Linked sialosides Sia 2-3/6GalbpNP containing C-5 and/
a2–3-
E-mail address: xiichen@ucdavis.edu (X. Chen).
Current address: Department of Pharmaceutical Chemistry, University of Kansas,
Lawrence, KS 66047, USA.
a
a
or C-9 modified sialic acids have been used to elucidate the sub-
à
Current address: Chemical Biology Laboratory, National Cancer Institute at Fred-
strate specificities of bacterial, human, and viral sialidases.9–11,13
erick, National Institutes of Health, Frederick, MD 21702, USA.
http://dx.doi.org/10.1016/j.carres.2014.02.021
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

Z. Khedri et al. / Carbohydrate Research 389 (2014) 100–111
101
HO
HO
HO
HO
OH
OH
CO2
OH
HO
OH
HO
OH
HO
HO
OH
HO
HO
-
-
CO2
O
O
-
O
CO2
NH
NH
HO
O
O
Neu5Ac
Neu5Gc
Kdn
Figure 1. Three basic forms of sialic acid.
HO
HO
HO
NHAc
O
HO
MeO
HO
NHAc
O
HO
N3
HO
NHAc
O
NHAc
O
F
HO
OH
OH
OH
OH
4-N3-ManNAc, 4a
4-F-ManNAc, 1a
4-O-Me-ManNAc, 2a 4-Deoxy-ManNAc, 3a
HO
HO
OH
O
OH
O
HO
HO
OH
O
OH
O
HO
MeO
HO
F
N3
HO
OH
OH
OH
HO
OH
4-N3-Man, 8a
4-F-Man, 5a
4-O-Me-Man, 6a
4-Deoxy-Man, 7a
Figure 2. Structures of C4-modified sialic acid precursors that are synthesized.
1) Tf2O, Pyridine, CH2Cl2
PhCH(OMe)2
HO
HO
N3
O
O
Ph
O
p-TsOH, DMF
2) NaN3, DMF
O
O
Ph
O
O
HO
OBn
HO
OBn
47% for two steps
87%
HO
OBn
OH
OH
9
10
11
1) p-TsOH, CH3OH
N3
N3
N3
O
BzO
BzO
NaH, BnBr, TBAI
90%
O
O
DAST, CH2Cl2
Ph
2) 2,6-Lutidine,BzCl
O
HO
F
O
35%
OBn
BnO
BnO
BnO
OBn
85%
OBn
12
13
14
1) H2, Pd/C, MeOH
2) NaOMe, MeOH
NHAc
HO
NHAc
BzO
AcSH, Py
O
O
F
F
67%
85%
BnO
HO
OBn
OH
15
1a
Scheme 1. Synthesis of 4-F-ManNAc 1a.
Selective sialidase inhibitors against human NEU2 or bacterial
sialidases have been designed and synthesized based on the struc-
tural features of the sialic acid modification obtained from sialidase
substrate specificity studies.14,15 Nevertheless, the method has not
been used for the synthesis of sialosides containing C7-modified
sialic acids.
Compared to sialosides containing C5- and C9-modified sialic
acids which are more commonly investigated, sialosides contain-
ing C7-modified sialic acids are underexplored. Neu5Ac analogs
containing C7-modification have been chemically synthesized16,17
and tested as substrates for CMP-sialic acid synthetase (CSS).17
C4-modified ManNAc derivatives have also been synthesized18,19
and used as substrates for sialic acid aldolase for the synthesis
of Neu5Ac analogs20–22 as well as Neu5Ac2en-derivatives as
transition state analog inhibitors19 or prodrugs23 against influenza
on cell surface glycoproteins, incubating UDP-N-acetylglucosamine
2-epimerase/ManNAc kinase (GNE) deficient HEK293 cells with
peracetylated 4-azido-ManNAc introduced 7-azido-Neu5Ac only
to the O-glycans of cell surface glycoproteins. Success in labeling
O-glycosylated proteins in live zebrafish embryos during develop-
ment was also achieved.25 Nevertheless, there are only a few
reports on the chemical synthesis of sialosides containing 7-deoxy
Neu5Ac26,27 and testing their binding to sialoside-binding pro-
teins.27 Enzymatic synthesis of sialosides containing C7-modified
sialic acid and their applications in investigating sialidase activities
have not been achieved.
Here we describe the highly efficient one-pot three-enzyme
chemoenzymatic synthesis of
a2–3- and a2–6-linked sialosides
containing C7-modified sialic acids with the C7-OH group of
Neu5Ac and Kdn being systematically replaced by –F, –OMe, –H,
and –N3 from the corresponding C4-modified ManNAc and man-
nose as sialic acid precursors. Good to excellent (53–98%) yields
have been obtained which illustrates again the high promiscuity
of bacterial sialoside biosynthetic enzymes in tolerating substrate
modifications. Sialidase substrate specificity studies using this set
of sialosides clearly show selective cleavage of sialosides contain-
ing 7-deoxy-Neu5Ac by bacterial enzymes but not by human cyto-
solic sialidase NEU2. Therefore, 7-deoxy-Neu5Ac2en is a potential
selective inhibitor against bacterial sialidases but not human
NEU2. We are in the process of synthesizing sialidase inhibitors
based on 7-deoxy-Neu5Ac and the results will be reported in due
course.
virus
(4-F-ManNAc or ManNAc4F) has been synthesized and used
as starting material for one-pot two-enzyme synthesis of
sialidases.
N-Acetyl-4-fluoro-4-deoxy-D-mannosamine
a
CMP-ManNAc4F.24 Recently, peracetylated N-acetyl-4-azido-4-
deoxymannosamine (peracetylated 4-azido-ManNAc) was used
successfully for metabolic engineering of mammalian cell surface
glycoproteins with 7-azido-Neu5Ac, indicating the tolerance of
4-azido-modified ManNAc by mammalian cellular enzymes
involved in sialic acid biosynthesis, activation, and transfer to gly-
coproteins.25 Unlike metabolic engineering of cultured mammalian
cells with peracetylated N-azidoacetylmannosamine (peracetylat-
ed ManNAz) which introduced Neu5Az to both N- and O-glycans

102
Z. Khedri et al. / Carbohydrate Research 389 (2014) 100–111
1) MeI, Ag2O, CH2Cl2,
NHAc
BzO
NHAc
O
NHAc
O
HO
HO
MeO
HO
BzCl, Py
85%
4 A M.S.
O
HO
HO
2) KOH, MeOH
58.5%
OH
BzO
OBz
HO
OH
ManNAc
16
2a
1) 1,1-Thiocarbonyldiimidazole
1,2-C2H4Cl2
2) AIBN, Bu3SnH, Toluene
3) NaOMe, MeOH
81%
NHAc
HO
HO
O
OH
3a
Scheme 2. Synthesis of 4-O-methyl ManNAc 2a and 4-deoxy ManNAc 3a.
NaN3, H+ resin
DMF
O
O
1) p-TsCl, Py
Ph3P, DIAD
THF
O
OH
O
OH
O
HO
HO
HO
NHAc
O
2) DBU, EtOH
O
NHAc
OH
NHAc
NHAc
57%
74%
68%
OH
O
(two steps)
17
18
N3 19
OH
ManNAc
O
OAc
O
NHAc
NHAc
O
AcO
HO
N3
HO
Ac2O, Py
95%
Ac2O, TMSOTf
77%
NaOMe, MeOH
83%
O
NHAc
N3
OAc
AcO
N3
20
21
4a
Scheme 3. Synthesis of 4-N3-ManNAc 4a.
HO
MeO
HO
OH
O
OH
6a
1. MeI, Ag2O
CH2Cl2, 4A M.S.
2. NaOMe, MeOH
63%
OBz
1. DAST, CH2Cl2
2. KOH, MeOH
1. Tf2O, Py, CH2Cl2
HO
BzO
HO
HO
BzO
OBz
O
OH
O
OH
HO
OBz
BzCl, Py
70%
2. Bu4NNO2, CH3CN
60%
O
O
HO
F
55%
HO
BzO
HO
OH
OBz
OBz
OH
Man
22
23
5a
1. 1,1-Thiocarbonyldiimidazole
1,2-C2H4Cl2
2. AIBN, Bu3SnH, Toluene
3. NaOMe, MeOH
1. Tf2O, Py, CH2Cl2
88%
65% 2. Bu4NN3, Toluene
3. KOH, MeOH
HO
HO
HO
N3
HO
OH
OH
O
O
OH
OH
7a
8a
Scheme 4. Synthesis of C4-modified mannose derivatives 5a–8a.
HO
R2
OH OH
O
-
CO2
HO
HO
HO
HO
OH
O
PmST1
O
O
O
NO2
O
R1
O
NO2
OH
R1
Chemical
R1
HO
R2
HO
HO
HO
HO
OH
Siaα2-3GalβpNP
modification
O
O
R2
Pyruvate, CTP, Mg2+
HO
HO
R1
-
OH
OH
CO2
R1 = NHAc, OH
R1 = NHAc, OH
OH
PmNanA, NmCSS
O
R2= OH, F, OMe, H, N3
Pd2,6ST
ManNAc
or Mannose
O
HO
HO
NO2
O
OH
Siaα2-6GalβpNP
Scheme 5. One-pot three-enzyme synthesis of sialosides containing C7-modified sialic acids.

Z. Khedri et al. / Carbohydrate Research 389 (2014) 100–111
103
Table 1
One-pot three-enzyme synthesis of a2–3/6-linked sialosides containing C7-modified sialic acid
Sialic acid precursors
Sialoside products
a2–3-Linked sialosides
a2–6-Linked sialosides
O
OH OH
O
OH
HO
-
-
F
CO2
F
CO2
HO
HO
OH
HO
NH
O
O
O
NO2
O
O
O
NH
NH
F
HO
O
HO
HO
HO
HO
OH
O
O
NO2
O
O
O
HO
1a
1b, 98%
OH
1c, 92%
OH
NH
HO
OH
O
-
-
CO2
OMe
HO
OMe
HO
CO2
HO
HO
HO
OH
NH
O
HO
MeO
HO
O
O
O
O
O
O
O
NO2
NH
O
HO
HO
O
NO2
OH
O
HO
2a
2b, 95%
2c, 57%
-
OH
HO
O
-
CO2
OH
NH
OH
O
CO2
HO
OH
HO
HO
NH
O
O
O
NO2
O
NH
O
HO
HO
O
NO2
HO
HO
N3
OH
O
O
HO
3a
3b, 80%
3c, 88%
OH
HO
NH
OH
NH
O
OH
O
-
-
N3
CO2
HO
HO
CO2
HO
OH
HO
N3
HO
NH
O
O
O
O
O
NO2
O
O
HO
HO
HO
F
O
NO2
HO
O
O
OH
HO
4b, 91%
4a
4c, 98%
OH
OH
O
OH
HO
OH
HO
-
-
CO2
CO2
F
HO
O
OH
F
O
O
O
O
O
O
NO2
HO
O
HO
OH
OH
HO
O
HO
HO
HO
HO
O
NO2
5a
HO
5b, 98%
5c, 95%
OH
O
OH
HO
HO
HO
MeO
HO
OH
HO
-
OH
O
-
CO2
OMe
HO
OMe
HO
CO2
HO
HO
OH
O
O
O
O
NO2
O
O
HO
HO
O
O
NO2
NO2
6a
HO
6b, 89%
6c, 53%
OH
HO
HO
HO
HO
-
OH
HO
-
OH
OH
O
CO2
CO2
O
HO
HO
HO
HO
OH
O
O
O
NO2
OH
OH
O
HO
HO
HO
HO
7a
HO
7b, 90%
7c, 58%
OH
HO
OH
HO
HO
HO
-
OH
-
OH
N3
CO2
N3
CO2
OH
N3
O
O
O
O
O
O
O
NO2
HO
HO
O
HO
HO
O
NO2
8a
HO
8b, 98%
HO
8c, 89%
The synthesis of 4-F-ManNAc (1a) was achieved by following a
previously reported method.24 As shown in Scheme 1, benzyl
glycopyranoside 9 readily prepared from
-glucose28 was selec-
tively protected at the hydroxyl groups of both C4 and C6 with
benzylidene to form 10. The C2-OH of 10 was triflated and con-
verted to azide 11 with concomitant reversal of the stereochemis-
try by an SN2 reaction using sodium azide in DMF. Benzylation of
the C3-OH of compound 11 formed 12 followed by hydrolysis of
the benzylidene acetal under acidic conditions to yield 4,6-diol,
which was selectively protected by benzoate at C6-OH to form
2. Results and discussion
2.1. Chemical synthesis of sialic acid precursors
In order to synthesize desired 2–3- and 2–6-linked sialosides
containing C7-modified sialic acids with the C7-OH group of
Neu5Ac and Kdn being systematically replaced by –F, –OMe, –H,
and –N3, we designed and synthesized eight ManNAc and mannose
derivatives 1a–8a in which the C4-OH is replaced with –F, –OMe,
–H, and –N3, respectively (Fig. 2).
D
a
a

104
Z. Khedri et al. / Carbohydrate Research 389 (2014) 100–111
13. Reacting the C4-OH of 13 with DAST in dichloromethane
produced fluorinated hexose 14. Converting the C2-azido group
in 14 to NHAc group formed 15 which was deprotected by catalytic
hydrogenation and the Zemplén reaction to form the desired
4-F-ManNAc 1a.
provided tri-benzoate 16 with a free hydroxyl group at the C-4. It
was treated with iodomethane and silver oxide to produce the
methylation product. After removing the benzoyl protection groups
by treating with KOH in methanol, the desired 4-O-methyl ManNAc
2a was obtained in a reasonable yield. On the other hand, treatment
of benzoate 16 with 1,10-thiocarbonyldiimidazole, followed by
reaction with tri-n-butylstannane and azobisisobutyronitrile
The preparation of 4-O-methyl-ManNAc 2a and 4-deoxy-Man-
NAc 3a is shown in Scheme 2. Selectively benzoylation of ManNAc
A. PmST1
B. Vibrio cholerae sialidase
1.4
1.2
1
0.8
0.6
0.4
0.2
0
C. Salmonella typhimurium sialidase
D. Streptococcus pneumoniae sialidase
1.2
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
0.8
0.6
0.4
0.2
0
E. Clostridium perfringens sialidase
F. NEU2
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1.2
1
0.8
0.6
0.4
0.2
0
Figure 3. Sialidase substrate specificity studies using
Kdn 2–3/6GalbpNP are used as controls. Results using
shown in black bars.
a
a
2–3-(1b–8b) and
a
2–6-linked (1c–8c) sialyl GalbpNP containing C7-modified sialic acids. Neu5Ac
a2–3/6GalbpNP and
a
2–3-linked sialosides as substrates are shown in white bars and results using a
2–6-linked sialosides as substrates are

Z. Khedri et al. / Carbohydrate Research 389 (2014) 100–111
105
(AIBN) provided a deoxy intermediate. Subsequent removal of
2.3. Sialidase substrate specificity studies
benzoyl groups by the Zemplén reaction produced the target com-
pound 4-deoxy-ManNAc 3a.
The synthesized sialosides 1b–8b and 1c–8c were used together
with Neu5Ac 2–3/6GalbpNP and Kdn 2–3/6GalbpNP to test the
substrate specificity of a recombinant human cytosolic sialidase
NEU2 and several bacterial sialidases including the 2–3-sialidase
4-N3-ManNAc 4a was prepared via a modified 1,6-anhydro
sugar similar to that described in a report by von Itzstein et al.18
Briefly, as shown in Scheme 3, tosylation of ManNAc and subse-
quent reaction with DBU produced 1,6-anhydro compound 17.
Compound 17 was treated with triphenylphosphine and diiso-
propyl azodicarboxylate (DIAD) to yield 3,4-epoxide 18. Treating
18 with sodium azide, followed by acetylation using acetic anhy-
dride and acetolysis using Ac2O and TMSOTf, yielded the 4-azido
product 21. Subsequent removal of the acetyl groups using
NaOMe in methanol produced the target compound 4-N3-Man-
NAc 4a.
The preparation of mannose derivatives 5a–8a (Scheme 4)
started from mannose. Selectively benzoylation of mannose29
produced the key intermediate tetra-benzoate 22. The remaining
hydroxyl group of 22 was then transformed into a triflate, fol-
lowed by the treatment of Bu4NNO2 to produce 23. Fluorination
of the 23 with DAST in CH2Cl2 gave 4-F-mannose 5a after depro-
tection. For the synthesis of 4-O-methyl-mannose 6a, benzoate 22
was treated with iodomethane and silver oxide followed by
removal of the benzoyl groups to produce the desired 6a. 4-Dex-
oy-mannose 7a was obtained via activation of 22 with 1,10-thi-
ocarbonyldiimidazole followed by treatment with tributyltin
hydride and azobisisobutyronitrile, and then de-benzoylation. In
order to synthesize 4-N3-mannose 8a, compound 23 was sub-
jected to triflation and concomitant treatment with TBAN3, the
target compound 8a was obtained after the Zemplén
debenzoylation.
a
a
a
function of PmST1 and four commercially available bacterial sialid-
ases such as Vibrio cholerae sialidase, Salmonella typhimurium sial-
idase, Streptococcus pneumoniae sialidase, and Clostridium
perfringens sialidase. As shown in Figure 3, while sialosides con-
taining C7-deoxy Neu5Ac are generally better or similarly good
substrates compared to those containing non-modified Neu5Ac
for bacterial sialidases, substitution of the C7-OH of Neu5Ac in
sialosides by hydrogen diminishes the activity of human NEU2.
Previously we have shown that replacing one of the methyl
hydrogen atoms in the C5-N-acetyl group of Neu5Ac in sialosides
by a fluorine atom does not affect the activities of either bacterial
sialidases tested10 or human NEU2.14 We have also shown that
replacing the C9-OH group of Neu5Ac in sialosides by a fluorine
atom, a hydrogen atom (deoxy-Neu5Ac), or an azido group does
not significantly change the activities of all bacterial sialidases
tested although human NEU2 is quite sensitive to C9-modifications
on Neu5Ac.11 Here we show that both Streptococcus pneumoniae
sialidase (Fig. 3D) and Clostridium perfringens sialidase (Fig. 3E) as
well as human NEU2 (Fig. 3F) are quite sensitive to Neu5Ac
C7-fluorine substitution which diminishes their activities signifi-
cantly. Azido-substitution at C7-OH of Neu5Ac is also not well
tolerated by either human NEU2 or bacterial sialidases tested
except for the
sialidase which remains a reasonable activity (50%) compared to
non-modified Neu5Ac 2–6GalbpNP substrate (Fig. 3E). Neu5Ac
a2–6-sialidase activity of Clostridium perfringens
a
C7-OMe modification is not tolerated well by any of the enzymes
tested.
2.2. Enzymatic synthesis of C7-modified sialosides
A
highly efficient one-pot three-enzyme chemoenzymatic
sialylation approach5 was used to synthesize a library of
2–3-
and 2–6-linked sialyl para-nitrophenyl galactosides in which
3. Conclusion
a
a
In conclusion, we report here that the one-pot three-enzyme
chemoenzymatic sialylation system is highly efficient (yields vary
from 53% to 98%) in producing sialosides containing C7-modified
sialic acids from a library of C4-modified ManNAc and mannose
derivatives containing various functional groups (–F, –OMe, –H,
and –N3). We demonstrate again the substrate promiscuity of bac-
terial sialoside biosynthetic enzymes and their application in the
synthesis of diverse sialosides. Substrate specificity studies of five
bacterial sialidases and a human cytosolic sialidase NEU2 indicate
that in general, substituting C7-OH of sialic acids in sialosides by –
OMe or –N3 diminishes the activity of sialidases tested signifi-
cantly. Quite interestingly, sialosides containing C7-deoxy Neu5Ac
are good substrates for all bacterial sialidases tested but not for hu-
man NEU2. Sialidase inhibitors based on C7-deoxy Neu5Ac could
be selective inhibitors against bacterial sialidases without affecting
the activity of human NEU2 significantly. We are in the process of
testing this hypothesis.
the C7-hydroxyl group in the sialic acid was systematically
substituted with –F, –OMe, –H, and –N3 groups. The enzymatic
system (Scheme 5) contained p-nitrophenyl b-galactopyranoside
as the sialyltransferase acceptor, a C4-modified ManNAc or man-
nose chosen from 1a–8a as the sialic acid precursor, sodium
pyruvate, CTP, and three enzymes including Pasteurella multocida
sialic acid aldolase (PmNanA), Neisseria meningitidis CMP-sialic
acid synthetase (NmCSS), and Pasteurella multocida sialyltransfer-
ase 1(PmST1) or Photobacterium damselae
(Pd2,6ST) for the synthesis of 2–3-linked sialosides or
2–6-linked sialosides, respectively.
As shown in Table 1, the 2–3-linked sialosides containing
a2–6-sialyltransferase
a
a
a
C7-modified Neu5Ac (1b–4b) or C7-modified Kdn (5b–8b) were
obtained in excellent yields (80–98%). Except for Neu5Ac7O-
Me
C7-modified Neu5Ac (1c and 3c–4c) were obtained in excellent
yields (88–98%). Except for Kdn7OMe 2–6GalpNP, 6c (53%) and
Kdn7deoxy 2–6GalpNP, 7c (58%), other 2–6-linked sialosides
containing C7-modified Kdn including Kdn7F 2–6GalpNP, 5c
(95%) and Kdn7N3 2–6GalpNP, 8c (89%) were also obtained in
excellent yields. The higher yields obtained for 2–3-linked sialo-
sides containing Neu5Ac7OMe (95%) and Kdn7OMe (89%) com-
pared to that for 2–6-linked counterparts (57% and 53%,
a2–6GalbpNP 2c (57%), other a2–6-linked sialosides containing
a
a
a
a
4. Experimental section
4.1. General
a
a
a
All chemicals were obtained from commercial suppliers and
used without further purification unless otherwise noted. Anhy-
drous solvents were used to carry out chemical reactions under in-
ert argon or nitrogen environment. 1H NMR (600 MHz) and 13C
NMR (150 MHz) spectra were recorded on a Varian Inova-600
spectrometer. High-resolution electrospray ionization (ESI) mass
spectra were obtained at the Mass Spectrometry Facility in the
respectively) indicates that the 7-O-methylation on CMP-sialic acid
is better tolerated by PmST1 compared to Pd2,6ST. Similarly,
replacing C7-OH in the Kdn of CMP-Kdn is also better tolerated
by PmST1 compared to Pd2,6ST leading to a significant difference
in the yields obtained (90% for Kdn7deoxy
58% for Kdn7deoxy 2–6GalpNP 7c).
a2–3GalpNP 7b and
a

106
Z. Khedri et al. / Carbohydrate Research 389 (2014) 100–111
University of California, Davis. Silica gel 60 Å (200–425 mesh,
Fisher Chemical) was used for flash column chromatography.
Thin-layer chromatography (TLC) was performed on silica gel
plates 60 GF254 (Sorbent technologies) using p-anisaldehyde sugar
stain. Gel filtration chromatography was performed using a col-
umn (100 cm ꢀ 2.5 cm) packed with Bio-gel P-2 fine resins (Bio-
Rad, Hercules, CA).
Compound 12 (2.20 g, 4.65 mmol) was dissolved in methanol
(40 mL). p-Toluenesulfonic acid (0.30 g, 1.58 mmol) was added
and the reaction mixture was stirred at room temperature for
3 h. The reaction was monitored by TLC (Hexanes/EtOAc = 3:1, by
volume). Triethylamine (0.25 mL) was added and stirred for
10 min to neutralize the reaction mixture. The reaction mixture
was concentrated, co-evaporated with toluene, and dried under
vacuum overnight. The residue was dissolved in CH2Cl2 (30 mL)
under nitrogen atmosphere, placed in an ice-bath at 0 °C. 2,6-Luti-
dine (0.81 mL, 6.95 mmol) was added followed by benzoyl chloride
(0.64 mL, 5.55 mmol). The reaction mixture was stirred at 0 °C for
3 h, washed with water (2 ꢀ 30 mL), and then once with brine. The
organic solution was dried over anhydrous magnesium sulfate and
concentrated. The residue was dissolved in CH2Cl2 and purified by
silica gel column (Hexanes/EtOAc = 10:1 to 5:1, by volume) to pro-
duce compound 13 (1.94 g, 85%). 1H NMR (600 MHz, CDCl3) d 8.09–
7.32 (m, 15H), 4.92 (s, 1H, H-1), 4.77 (d, J = 11.4 Hz, 1H), 4.73 (d,
J = 12.0 Hz, 1H), 4.67 (d, J = 11.4 Hz, 1H), 4.65 (dd, J = 4.8 and
12.0 Hz, 1H), 4.56 (dd, J = 1.8 and 12.0 Hz, 1H), 4.52 (d,
J = 12.0 Hz, 1H), 4.02–3.91 (m, 4H); 13C NMR (150 MHz, CDCl3) d
167.06, 137.60, 136.77, 133.40, 128.90, 128.82, 128.64, 128.44,
128.43, 128.37, 128.30, 97.68, 79.54, 72.88, 71.21, 69.57, 66.89,
64.00, 60.77.
In a 50 mL centrifuge tube, DAST (3.0 mL, 24.5 mmol) was
added to anhydrous CH2Cl2 (10 mL). A solution containing com-
pound 13 (1.5 g, 3.06 mmol) in anhydrous CH2Cl2 was slowly
added at ꢁ78 °C, and the mixture was stirred at room temperature
for 3 d. The reaction was checked by TLC (Hexanes/EtOAc = 3:1, by
volume), cooled down to ꢁ20 °C, and terminated by adding meth-
anol (2 mL). The mixture was transferred into a flask, concentrated,
re-dissolved in CH2Cl2, and washed with water. The organic
solution was dried over anhydrous MgSO4, filtered, concentrated,
and purified by silica gel column (Hexanes/EtOAc = 12:1 to 6:1,
by volume) to produce compound 14 (0.53 g, 35% yield).
Compound 14 (0.49 g, 1.00 mmol) was dissolved in pyridine
(4 mL), and thioacetic acid (8 mL) was added. The reaction was al-
lowed to stir at rt for overnight and monitored by TLC (Hexanes/
EtOAc = 1:1, by volume). The reaction mixture was concentrated
and the residue was purified by flash chromatography (Hexanes/
EtOAc = 4:1 to 1:1, by volume) to produce compound 15 (0.43 g,
85% yield). 1H NMR (600 MHz, CDCl3) d 8.06–7.28 (m, 15H), 5.89
(d, J = 8.4 Hz, 1H, NH), 5.00 (s, 1H, H-1), 4.79–4.46 (m, 8H), 4.20–
4.15 (m, 2H).
To a solution of 15 (204 mg, 0.40 mmol) in MeOH (10 mL) was
added 10% Pd–C (100 mg). The resulting mixture was hydroge-
nated on a hydrogenation apparatus for 18 h under hydrogen
atmosphere. The resulting suspension was filtered and concen-
trated. The residue was dissolved in dry MeOH (10 mL) containing
a catalytic amount of NaOMe. The mixture was stirred at room
temperature overnight. The reaction was neutralized using Dowex
(H+) resin. The resulting suspension was filtrated and concentrated.
Silica gel column purification (EtOAc/MeOH = 9:1, by volume) pro-
duced the desired product 4-F-ManNAc 1a (60 mg, 67% yield). 1H
NMR (600 MHz, D2O) d 5.13 (s, 0.7H), 4.49 (d, J = 1.8 Hz, 0.3H),
3.71–3.91 (m, 3H), 3.66–3.45 (m, 3H), 3.39 (s, 1H) 3.38 (s, 2H);
13C NMR (150 MHz, D2O) d 94.1, 93.8, 74.7, 73.1, 71.6, 71.3, 70.9,
70.8, 70.3, 67.1, 66.8, 58.6, 58.5.
4.2. Chemical synthesis of ManNAc and Man derivatives as
sialoside precursors 1a–8a
4.2.1. 2-Acetamido-2,4-dideoxy-4-fluoro-D-mannopyranoside
(4-F-ManNAc, 1a)
Benzyl glucopyranoside 928 (7.2 g, 26.64 mmol) was dissolved
in 30 mL of anhydrous DMF. PhCH(OMe)2 (6.0 mL, 1.5 equiv)
and p-toluenesulfonic acid (100 mg) were added. The reaction
was carried out at 50 °C for 1 h using rotavap at 70 mbar. Reac-
tion was monitored by TLC (CH2Cl2/MeOH = 10:0.2 by volume).
The solution mixture was concentrated, co-evaporated with tolu-
ene, and the residue was dissolved in CH2Cl2. The sample was
loaded to a silica gel column and washed with hexanes, CH2Cl2,
and a mixture of CH2Cl2 and MeOH (100:1–20:1) to produce
1-benzyl-4,6-benzylidene-D-glucopyranoside 10 (8.27 g, 87% yield).
1H NMR (600 MHz, CDCl3) d 7.91–7.26 (m, 10H), 5.48 (s, 1H), 4.93 (d,
J = 4.2 Hz, 1H, H-1), 4.72 (d, J = 12.0 Hz, 1H), 4.53 (d, J = 11.4 Hz, 1H),
4.19 (dd, J = 5.4 and 11.2 Hz, 1H), 3.91 (t, J = 11.2 Hz, 1H), 3.82–3.35
(m, 4H); 13C NMR (150 MHz, CDCl3) d 137.37, 137.14, 128.78,
128.72, 128.51, 128.50, 128.36, 128.32, 126.60, 126.59, 102.06,
98.56, 81.22, 73.07, 71.65, 70.28, 69.10, 62.96.
Compound 10 (4.23 g, 11.8 mmol) was dissolved in anhydrous
CH2Cl2 (50 mL) and pyridine (20 mL) was added. The solution mix-
ture was placed in acetone-dry ice (ꢁ20 °C), and trifluoromethane-
sulfonic acid anhydride (Tf2O) (2.35 mL, 14.16 mmol) was added
drop-wisely. The mixture was stirred for 2 h at ꢁ20 °C. The reac-
tion was monitored by TLC (Hexanes/EtOAc = 3:1, by volume).
Upon completion, the reaction was quenched and washed with
brine three times. The organic solution was concentrated, re-dis-
solved in CH2Cl2, dried over MgSO4, and filtered. The filtrate was
concentrated, co-evaporated with toluene, and the residue was
dried under vacuum. Without purification, the residue was dis-
solved in anhydrous CH2Cl2 (30 mL) under nitrogen. Sodium azide
(7.68 g, 118 mmol) was added and the suspension was stirred at
50 °C for 24 h. the reaction was monitored by TLC and terminated
by adding water (50 mL). The solution mixture was extracted with
ethyl acetate, dried over anhydrous MgSO4, filtered, concentrated,
and purified by silica gel column to produce 2-azido-2-deoxy-
4,6-benzylidene-1,3-dibenzylmannpyranoside 11 (2.11 g, 47%
yield). 1H NMR (600 MHz, CDCl3) d 7.51–7.32 (m, 10H), 5.58 (s,
1H), 4.88 (d, J = 1.2 Hz, 1H, H-1), 4.72 (d, J = 11.4 Hz, 1H), 4.53 (d,
J = 12.0 Hz, 1H), 4.31–4.29 (m, 1H), 4.24 (dd, J = 3.6 and 9.0 Hz,
1H), 3.96 (d, J = 3.6 Hz, 1H), 3.93 (t, J = 9.0 Hz, 1H), 3.87–3.79 (m,
2H); 13C NMR (150 MHz, CDCl3) d 137.24, 136.72, 129.58, 128.85,
128.64, 128.47, 128.31, 126.50, 102.50, 98.46, 79.25, 69.80, 69.16,
68.86, 63.97, 63.87.
Compound 11 (2.0 g, 5.22 mmol) was dissolved in anhydrous
DMF (20 mL) under nitrogen and the reaction was placed in an
ice bath at 0 °C. Sodium anhydride (0.19 g, 7.83 mmol), tetrabutyl-
ammonium iodide (0.12 g, 0.33 mmol), and benzyl bromide
(1.25 mL, 10.5 mmol) were added. The solution mixture was stir-
red at 0 °C for 2 h then at room temperature for overnight. The
reaction was terminated by adding methanol (2 mL) followed by
water (50 mL) and the mixture was extracted with ethyl acetate.
The organic solution was dried over anhydrous MgSO4, filtered,
concentrated, and purified by silica gel column (Hexanes/
EtOAc = 10:1 to 5:1, by volume) to produce compound 12 (2.23 g,
90% yield).
4.2.2. 2-Acetamido-2-deoxy-4-O-methyl-D-mannopyranoside
(4-O-Me-ManNAc, 2a)
To a solution of pre-dried ManNAc (6.0 g, 27.1 mmol) in
anhydrous pyridine (120 mL) was added benzoyl chloride
(13.5 mL, 108.0 mmol) at ꢁ40 °C drop-wisely for 20 min. The reac-
tion solution was stirred at ꢁ40 °C for 2 h. The reaction was
stopped by adding MeOH and the reaction mixture was concen-
trated. The concentrated residue was dissolved in CH2Cl2 and

Z. Khedri et al. / Carbohydrate Research 389 (2014) 100–111
107
washed sequentially with HCl (100 mL, 2 M), saturated NaHCO3
(60 mL), and water (100 mL). The product was purified by silica
gel column (Hexanes/EtOAc = 6:4) to give 16 (12.3 g, 85%). 1H
J = 3.6 Hz, 0.5H), 4.60 (d, J = 8.4 Hz, 0.5H), 4.11–4.08 (m, 0.5H),
3.99–3.95 (m, 0.5H), 3.77–3.48 (m, 4H), 2.03 (s, 1.5H), 2.02 (s,
1.5H), 1.51–1.41 (m, 2H); 13C NMR (150 MHz, D2O) d 174.99,
174.83, 95.36, 91.58, 72.69, 69.01, 68.59, 65.26, 63.83, 58.41,
55.61, 34.83, 34.51, 22.28, 22.01.
NMR (600 MHz, CDCl3)
d 7.99 (d, J = 7.8 Hz, 4H), 7.96 (d,
J = 7.2 Hz, 2H), 7.55–7.51 (m, 3H), 7.38 (t, J = 7.2 Hz, 6 H), 6.18 (s,
1H), 6.11 (d, J = 9.0 Hz, 1H), 5.34 (dd, J = 8.4 and 4.2 Hz, 1H), 5.01
(dd, J = 9.0 and 3.0 Hz, 1H), 4.69–4.61 (m, 2H), 3.97 (d, J = 6.0 Hz,
2H), 2.05 (s, 3H); 13C NMR (150 MHz, CDCl3) d 170.73, 167.04,
166.88, 164.40, 134.01, 133.66, 133.54, 130.24, 130.14, 129.99,
129.79, 129.60, 128.99, 128.75, 128.69, 128.65, 91.89, 75.74,
74.68, 66.60, 63.75, 50.00, 23.44.
Compound 16 (0.30 g, 0.56 mmol) and Ag2O (1.30 g, 5.6 mmol)
were dissolved in anhydrous CH2Cl2 (15 mL) containing 4 Å molec-
ular sieves (0.6 g). CH3I (0.35 mL, 5.6 mmol) was added drop-wise-
ly at 0 °C and the suspension was stirred for 48 h at room
temperature. The mixture was filtered over Celite and concen-
trated. Silica gel column purification (Hexanes/EtOAc = 3:2, by vol-
ume) produced the benzoyl-protected methylated intermediate
(0.27 g, 90%). 1H NMR (600 MHz, CDCl3) d 8.06–7.97 (m, 6H),
7.59–7.55 (m, 3H), 7.46–7.39 (m, 6H), 6.24 (s, 1H), 5.99 (d,
J = 9.0 Hz, 1H), 5.43 (dd, J = 8.4 and 4.2 Hz, 1H), 5.09–5.07 (m,
1H), 4.66–4.65 (m, 2H), 4.06–4.04 (m, 1H), 3.70 (t, J = 8.4 Hz, 1H),
3.52 (s, 3H), 2.05 (s, 3H); 13C NMR (150 MHz, CDCl3) d 170.17,
166.09, 165.57, 164.28, 133.84, 133.33, 130.07, 129.70, 128.57,
91.27, 74.93, 74.13, 73.64, 63.34, 60.47, 49.28, 23.32.
The intermediate obtained above (0.27 g, 0.49 mmol) was dis-
solved in a solution of KOH in MeOH (10 mL, 0.1 M) and stirred
at room temperature for 1 h. The mixture was neutralized using
Dowex (H+) resin and the resulting suspension was filtrated and
concentrated. Silica gel column purification (EtOAc/MeOH/
H2O = 10:1:0.2, by volume) afforded product 2a (75 mg, 65%). 1H
NMR (600 MHz, D2O) d 5.08 (s, 0.7H), 4.98 (s, 0.3H), 4.30–4.29
(m, 1H), 4.11–4.09 (m, 1H), 3.82 (s, 3H), 3.54 (s, 3H), 3.42–3.35
(m, 2H), 2.04 (s, 3H); 13C NMR (150 MHz, D2O) d 174.91, 93.11,
77.02, 72.06, 71.18, 68.94, 60.34, 53.53, 22.08.
4.2.4. 2-Acetamido-4-azido-2,4-dideoxy-D-mannopyranoside (4-
N3-ManNAc, 4a)
To a stirring solution of pre-dried ManNAc (8.2 g, 37.07 mmol)
in anhydrous pyridine (50 mL) at 0 °C was added a solution of
p-toluenesulfonyl chloride (11.31 g, 59.31 mmol) in pyridine
(20 mL) and the mixture was stirred overnight at room tempera-
ture. The reaction was stopped by adding MeOH (10 mL) and the
mixture was concentrated. Purification of the residue by column
chromatography (EtOAc/MeOH = 9:1, by volume) provided the
tosylated product (9.60 g, 69%). The resulting 6-O-Ts-ManNAc
(9.60 g, 25.57 mmol) was dissolved in anhydrous ethanol
(100 mL), and 1,8-diazabicycloundec-7-ene (DBU) (7.6 mL,
51.14 mmol) was added drop-wisely. The reaction mixture was
stirred at room temperature overnight. The crude product was
dried under reduced pressure and silica gel flash column purifica-
tion (EtOAc/MeOH = 9:1, by volume) provided 1,6-anhydro com-
pound 17 (4.31 g, 83%).
To a solution of 17 (4.31 g, 21.21 mmol) in dry THF (50 mL) was
added PPh3 (6.67 g, 25.45 mmol). Diisopropylazodicarboxylate
(DIAD) (4.0 mL, 25.45 mmol) was added to the solution drop-wise-
ly at 0 °C. The reaction was stirred for 1 h at 0 °C followed by 1 h at
room temperature. The mixture was concentrated and silica gel
column purification (EtOAc) afforded compound 18 (2.90 g, 74%).
1H NMR (600 MHz, CDCl3) d 5.91 (s, 1H), 5.24 (d, J = 3.6 Hz, 1H),
4.84 (t, J = 4.8 Hz, 1H), 4.36–4.34 (m, 1H), 3.93 (d, J = 6.0 Hz, 1H),
3.73 (t, J = 4.8 Hz, 1H), 3.53 (dd, J = 6.6 and 4.8 Hz, 1H), 3.30 (dd,
J = 3.6 and 1.2 Hz, 1H), 2.05 (s, 3H); 13C NMR (150 MHz, CDCl3) d
170.09, 97.04, 71.82, 63.99, 55.88, 49.21, 47.10, 23.22.
Compound 18 (2.90 g, 15.66 mmol) was dissolved in anhydrous
DMF (25 mL). Dowex (H+) resin (3 g) and NaN3 (4.07 g,
62.64 mmol) were added to the solution. The suspension was
heated at 95 °C overnight. The reaction mixture was cooled down
to room temperature, filtered through a plug of Celite, and washed
with ethyl acetate and methanol. Silica gel column purification
(EtOAc/MeOH = 9.5:0.5, by volume) provided compound 19
(2.43 g, 68%). 1H NMR (600 MHz, CDCl3) d 5.34 (s, 1H), 4.56 (d,
J = 5.4 Hz, 1H), 4.26 (d, J = 7.8 Hz, 1H), 4.14 (t, J = 9.0 Hz, 1H), 3.97
(d, J = 3.4 Hz, 1H), 3.76 (dd, J = 7.2 and 6.0 Hz, 1H), 3.55 (s, 1H),
1.98 (s, 3H); 13C NMR (150 MHz, CDCl3) d 170.04, 101.00, 74.01,
68.11, 65.63, 63.10, 48.59, 23.20.
To a solution of compound 19 (2.43 g, 10.65 mmol) in pyridine
(20 mL) at 0 °C was added acetic anhydride (4 mL). The mixture
was stirred at room temperature for 3 h. MeOH (4 mL) was added
and the solvents were removed under reduced pressure. Product
20 (2.73 g, 95%) was obtained after silica gel column purification
(Hexanes/EtOAc = 3:7, by volume). A mixture of compound 20
(2.73 g, 10.10 mmol) in acetic anhydride (15 mL) and trimethylsilyl
trifluoromethanesulfonate (TMSOTf) (1.84 mL, 10.10 mmol) was
stirred under nitrogen at 0 °C for 2 h. The reaction was stopped
by the addition of a saturated solution of NaHCO3 (50 mL) over
30 min. The mixture was extracted with ethyl acetate and washed
with a saturated solution of NaHCO3 and brine. The concentrated
residue was subjected to silica gel column chromatography (Hex-
anes/EtOAc = 1:9, by volume) to yield 21 (2.8 g, 77%). Compound
21 (2.8 g, 7.52 mmol) was dissolved in dry MeOH (30 mL) contain-
ing a catalytic amount of NaOMe. The mixture was stirred at room
temperature for 2 h. The reaction was neutralized using Dowex
(H+) resin. The resulting suspension was filtrated and concentrated.
Silica gel column chromatography (EtOAc/MeOH = 9:1) of the
crude product afforded final product 4a (1.53 g, 83%). 1H NMR
4.2.3. 2-Acetamido-2,4-dideoxy-
ManNAc, 3a)
D-mannopyranoside (4-Deoxy-
Compound 16 (0.97 g, 1.81 mmol) and 1,1-thiocarbonyldiimi-
dazole (0.64 g, 3.62 mmol) were dissolved in 1,2-dichloroethane
(50 mL). The mixture was refluxed at 90 °C for 24 h. The solvent
was removed and the concentrated residue was co-evaporated
with toluene and dried under vacuum. To the dry compound was
added azobisisobutyronitrile (AIBN) (59 mg, 0.36 mmol), Bu3SnH
(2.92 mL, 10.86 mmol), and toluene (80 mL). The resulting mixture
was refluxed at 110 °C for 5 h. After removing the solvent under re-
duced pressure, column chromatography on silica gel (Hexanes/
EtOAc = 3:2, by volume) afforded deoxygenated intermediate
(0.84 g, 90%). 1H NMR (600 MHz, CDCl3) d 8.03–7.99 (m, 7H),
7.58–7.42 (m, 11H), 7.18–7.14 (m, 1H), 6.38 (d, J = 1.8 Hz, 0.3H),
6.13 (d, J = 1.8 Hz, 0.7H), 6.05 (d, J = 9.6 Hz, 0.7H), 6.01 (d,
J = 9.6 Hz, 0.3H), 5.74–5.71 (m, 0.3H), 5.43–5.40 (m, 0.7H), 4.94
(dd, J = 9.0 and 2.4 Hz, 0.7H), 4.83 (dd, J = 9.6 and 2.4 Hz, 0.3H),
4.54–4.43 (m, 3H), 4.26–4.22 (m, 1H), 2.30–2.27 (m, 1H), 2.15 (s,
2.3H), 2.10 (s, 0.7H), 1.93–1.85 (m, 1H); 13C NMR (150 MHz, CDCl3)
d 170.56, 170.26, 166.37, 165.88, 164.42, 164.28, 133.99, 133.52,
130.24, 130.18, 130.01, 129.86, 129.77, 129.26, 129.08, 128.93,
128.75, 128.68, 128.45, 93.92, 92.22, 71.17, 69.44, 68.30, 67.30,
65.87, 49.04, 48.04, 28.65, 28.44, 23.63, 23.50.
The intermediate obtained above (0.84 g, 1.62 mmol) was dis-
solved in dry MeOH (15 mL) containing a catalytic amount of
NaOMe. The mixture was stirred at room temperature overnight
and neutralized using Dowex (H+) resin. The resulting suspension
was filtrated and concentrated. Silica gel column purification
(EtOAc/MeOH/H2O = 8:1:0.3, by volume) produced the desired
compound 3a (0.3 g, 90%). 1H NMR (600 MHz, D2O) d 5.21 (d,

108
Z. Khedri et al. / Carbohydrate Research 389 (2014) 100–111
(600 MHz, D2O) d 5.13 (s, 0.6H), 4.97 (s, 0.4H), 4.45 (d, J = 6.4 Hz,
0.4H), 4.31 (d, J = 6.4 Hz, 0.6H), 4.18–4.12 (m, 1H), 3.89–3.78 (m,
3H), 3.59–3.48 (m, 2H), 2.06 (s, 1.5H), 2.04 (s, 1.5H); 13C NMR
(150 MHz, D2O) d 174.70, 174.48, 93.12, 92.87, 74.63, 71.56,
70.25, 70.00, 62.26, 60.73, 60.67, 53.94, 53.71, 52.65, 21.83, 21.79.
NMR (150 MHz, D2O) d 93.89, 88.80, 87.63, 71.20 (d, J = 9.06 Hz),
69.71 (d, J = 24.16 Hz), 68.55 (d, J = 17.97 Hz), 60.25.
4.2.6. 4-O-Methyl-D-mannopyranoside (4-O-Me-Man, 6a)
Compound 22 (0.40 g, 0.67 mmol) and Ag2O (1.55 g, 6.7 mmol)
were dissolved in anhydrous CH2Cl2 (15 mL) containing 4 Å molec-
ular sieves (0.7 g). CH3I (0.42 mL, 6.7 mmol) was added drop-wise-
ly at 0 °C and the suspension was stirred for 48 h at room
temperature. The mixture was filtered over Celite and concen-
trated. Silica gel column purification (Hexanes/EtOAc = 7:3, by vol-
ume) yielded the methylated product (0.37 g, 90%). 1H NMR
(600 MHz, CDCl3) d 8.15–7.34 (m, 20H), 6.59 (d, J = 1.8 Hz, 1H),
6.00 (t, J = 9.6 Hz, 1H), 5.86–5.85 (m, 1H), 4.70 (dd, J = 12.0 and
1.8 Hz, 1H), 4.43 (dd, J = 12.0, 4.2 Hz, 2H), 4.14 (dd, J = 9.6, 3.0 Hz,
1H), 3.47 (s, 3H); 13C NMR (150 MHz, CDCl3) d 166.36, 165.63,
165.54, 164.06, 134.22, 133.73, 133.66, 133.23, 130.28, 130.25,
130.21, 130.19, 130.09, 130.03, 130.01, 129.00, 128.77, 128.75,
128.73, 128.53, 92.02, 77.60, 71.40, 68.20, 67.90, 63.20, 58.50.
The product obtained above (0.37 g, 0.60 mmol) was dissolved
in dry MeOH (10 mL) containing a catalytic amount of NaOMe.
The mixture was stirred at room temperature for 6 h and neutral-
ized using Dowex (H+) resin. The resulting suspension was filtrated
and concentrated. Silica gel column purification (EtOAc/MeOH/
H2O = 10:1:0.5, by volume) afforded product 6a (82 mg, 70%). 1H
NMR (600 MHz, D2O) d 5.20 (d, J = 1.8 Hz and 0.6H), 5.14 (d,
J = 1.2 Hz, 0.4H), 4.17–3.66 (m, 5H), 3.53–3.40 (m, 4H); 13C NMR
(150 MHz, D2O) d 94.17, 79.87, 77.12, 72.53, 71.57, 71.06, 70.23,
66.48, 65.91, 61.08, 56.36.
4.2.5. 4-Deoxy-4-fluoro-D-mannopyranoside (4-F-Man, 5a)
To a solution of pre-dried mannose (6.0 g, 33.30 mmol) in anhy-
drous pyridine (120 mL) was added benzoyl chloride (16.7 mL,
133.2 mmol) at ꢁ40 °C drop-wisely for 20 min. The reaction solu-
tion was stirred at ꢁ40 °C for 2 h. The reaction was stopped by add-
ing MeOH and the reaction mixture was concentrated. The residue
was dissolved in CH2Cl2 and washed sequentially with HCl
(100 mL, 2 M), saturated NaHCO3 (60 mL), and water (100 mL).
The product was purified by a silica gel column (Hexanes/
EtOAc = 8:2) to give 22 (13.9 g, 70%). 1H NMR (600 MHz, CDCl3) d
8.15–8.14 (m, 4H), 7.96–7.91 (m, 4H), 7.65–7.63 (m, 1H), 7.55–
7.45 (m, 6H), 7.34 (t, J = 7.2 Hz, 2H), 7.28–7.25 (m, 3H), 6.55 (s,
1H), 5.80 (s, 2H), 5.01 (dd, J = 12.0 and 3.0 Hz, 1H), 4.52 (dd,
J = 12.0 and 1.8 Hz, 1H), 4.37 (t, J = 9.6 Hz, 1H), 4.24 (d, J = 9.6 Hz,
1H), 3.22 (s, 1H); 13C NMR (150 MHz, CDCl3) d 167.39, 166.64,
165.26, 164.23, 134.23, 133.77, 133.64, 133.60, 130.32, 130.16,
130.07, 130.05, 129.83, 129.45, 129.38, 128.99, 128.77, 128.62,
91.75, 73.94, 72.30, 69.68, 65.61, 63.10.
To a solution of compound 22 (0.6 g, 1.00 mmol) in CH2Cl2
(20 mL) and pyridine (0.8 mL, 10.00 mmol), trifluoromethanesulfo-
nic acid anhydride (Tf2O) (0.33 mL, 2 mmol) was added drop-wise-
ly at ꢁ20 °C. The reaction mixture was stirred at ꢁ20 °C for 1 h and
then 0 °C to room temperature for 1 h. The solvent was removed
and the residue was dissolved in CH2Cl2 and washed sequentially
with HCl (5%), NaHCO3, and water. The organic phase was concen-
trated by rotavap, co-evaporated with toluene, and dried under
vacuum for overnight. The product was dissolved in CH3CN
(20 mL) and Bu4NNO2 (0.57 g, 2.00 mmol) was added. The reaction
was stirred at room temperature for 1 h and at 55 °C for 4 h. The
solvent was removed. The residue was dissolved in CH2Cl2 and
washed with a saturated brine solution. The crude product was
dried and purified using silica gel flash column chromatography
(Hexanes/EtOAc = 4:1, by volume) to yield 23 (0.36 g, 60%). 1H
NMR (600 MHz, CDCl3) d 8.11–8.05 (m, 4H), 7.99–7.93 (m, 4H),
7.67–7.60 (m, 2H), 7.56–7.45 (m, 6H), 7.41–7.34 (m, 4H), 6.64 (d,
J = 6.0 Hz, 1H), 5.77–5.73 (m, 2H), 4.75–4.71 (m, 1H), 4.65–4.61
(m, 1H), 4.56 (t, J = 6.0 Hz, 1H), 4.45–4.41 (m, 1H); 13C NMR
4.2.7. 4-Deoxy-D-mannopyranoside (4-Deoxy-Man, 7a)
Compound 22 (0.25 g, 0.42 mmol) and 1,1-thiocarbonyldiimi-
dazole (0.15 g, 0.84 mmol) were dissolved in 1,2-dichloroethane
(30 mL). The mixture was refluxed at 90 °C for 24 h. The solvent
was removed. The concentrated residue was co-evaporated with
toluene and dried under vacuum. To the dry compound was added
azobisisobutyronitrile (AIBN) (10 mg, 0.08 mmol), Bu3SnH
(0.68 mL, 2.52 mmol), and toluene (60 mL). The resulting mixture
was refluxed at 110 °C for 5 h. After removing the solvent under re-
duced pressure, silica gel column purification (Hexanes/
EtOAc = 8:2, by volume) provided the deoxygenated product
(0.23 g, 98%). 1H NMR (600 MHz, CDCl3) d 8.15–8.08 (m, 6H),
7.92 (dd, J = 8.4 and 1.2 Hz, 2H), 7.66–7.35 (m, 12H), 6.63 (d,
J = 1.8 Hz, 1H), 5.88–5.85 (m, 1H), 5.70 (s, 1H), 4.54 (s, 3H), 2.46
(dd, J = 24.0, 12.0 Hz, 1H), 2.32–2.26 (m, 1H); 13C NMR (150 MHz,
CDCl3) d 166.36, 165.74, 165.38, 164.12, 133.99, 133.67, 133.47,
133.33, 130.15, 130.01, 129.84, 129.52, 128.83, 128.74, 128.55,
92.40, 68.93, 67.42, 67.35, 65.94, 28.34.
The compound obtained above (0.23 g, 0.39 mmol) was dis-
solved in dry MeOH (15 mL) containing a catalytic amount of
NaOMe. The mixture was stirred at room temperature overnight
and neutralized using Dowex (H+) resin. The resulting suspension
was filtrated and concentrated. Silica gel column purification
(EtOAc/MeOH/H2O = 8:1:0.3, by volume) afforded product 7a
(58 mg, 90%). 1H NMR (600 MHz, D2O) d 5.19 (s, 0.5H), 4.73 (s,
0.5H), 4.09–3.59 (m, 5H), 1.69–1.46 (m, 2H); 13C NMR (150 MHz,
D2O) d 94.77, 94.12, 72.79, 70.02, 69.02, 68.82, 68.16, 64.85,
64.31, 64.01, 29.20, 28.61.
(150 MHz, CDCl3)
d 166.22, 165.85, 165.44, 165.42, 165.15,
165.12, 164.15, 163.80, 134.02, 133.97, 133.95, 133.90, 133.65,
133.60, 133.59, 133.22, 133. 18, 130.17, 130.01, 129.97, 129.89,
129.84, 129.72, 129.71, 129.60, 129.25, 129.06, 128.92, 128.89,
128.85, 128.76, 128.65, 128.50, 128.46, 128.39, 128.38, 94.40,
92.00, 71.33, 69.50, 68.87, 68.20, 67.74, 67.65, 67.01, 62.99, 62.50.
To a solution of 23 (0.36 g, 0.60 mmol) in anhydrous CH2Cl2
(15 mL) in a Teflon flask, DAST (0.59 mL, 4.83 mmol) was slowly
added at ꢁ40 °C. The reaction was stirred for 3 days at room tem-
perature. After cooling down the reaction mixture to ꢁ20 °C, MeOH
(1 mL) was added and the solvent was removed under reduced
pressure. The residue was diluted with CH2Cl2, washed with water
3 times, dried over MgSO4, and concentrated. Silica gel flash col-
umn purification (Hexanes/EtOAc = 8:2, by volume) yielded a fluo-
rinated product (0.23 g, 65%). The fluorinated product (0.27 g,
0.49 mmol) was dissolved in a solution of KOH in MeOH (10 mL,
0.1 M) and stirred at room temperature for 1 h. The mixture was
neutralized using Dowex (H+) resin. The resulting suspension was
filtrated and concentrated. Silica gel column chromatography
(EtOAc/MeOH/H2O = 10:1:0.2) of the crude product afforded prod-
uct 5a (70 mg, 85%). 1H NMR (600 MHz, D2O) d 5.16 (s, 1H), 4.59 (t,
J = 9.6 Hz, 0.4H), 4.51 (t, J = 10.2 Hz, 0.6H), 4.12–3.58 (m, 5H); 13C
4.2.8. 4-Azido-4-deoxy-D-mannopyranoside (4-N3-Man, 8a)
To a solution of compound 23 (0.42 g, 0.70 mmol) in CH2Cl2
(15 mL) and pyridine (0.56 mL, 7.0 mmol), trifluoromethanesulfo-
nic acid anhydride (Tf2O) (0.23 mL, 1.4 mmol) was added drop-wi-
sely at ꢁ20 °C. The reaction mixture was stirred at ꢁ20 °C for 1 h
and at 0 °C to room temperature for 1 h. The solvent was removed.
The residue was dissolved in CH2Cl2 and washed sequentially with
HCl (5%), NaHCO3, and water. The organic phase was concentrated

Z. Khedri et al. / Carbohydrate Research 389 (2014) 100–111
109
by rotavap, co-evaporated with toluene, and dried under vacuum
4.3.3. 4-Nitrophenyl O-(5-acetamido-3,5-dideoxy-7-O-methyl-
glycero- -galacto-2-nonulopyranosylonic acid)-(2?3)-O-b-
galactopyranoside (Neu5Ac7OMe 2–3GalbpNP, 2b)
D-
D-
overnight. A mixture containing the crude product and Bu4NN3
(0.24 g, 1.05 mmol) in anhydrous toluene (15 mL) was heated at
70 °C for 1 h and at 100 °C for 1.5 h. The mixture was cooled down
to room temperature, concentrated, and purified by silica gel flash
column chromatography (EtOAc/Hexanes = 2:8, by volume) to af-
ford azido-modified product (0.33 g, 76%). 1H NMR (600 MHz,
CDCl3) d 8.15–8.13 (m, 4H), 8.01–7.97 (m, 4H), 7.67–7.53 (m,
6H), 7.47 (t, J = 7.8 Hz, 2H), 7.41–7.36 (m, 4H), 6.57 (s, 1H), 5.86–
5.83 (m, 2H), 4.77 (dd, J = 12.6 and 6.6 Hz, 1H), 4.68 (dd, J = 12.0
and 1.8 Hz, 1H), 4.43 (t, J = 10.2 Hz, 1H), 4.15 (dd, J = 10.2 and
2.4 Hz, 1H); 13C NMR (150 MHz, CDCl3) d 166.05, 165.44, 164.89,
163.76, 134.12, 133.71, 133.66, 133.31, 130.10, 129.83, 129.80,
128.95, 128.83, 128.65, 128.57, 128.55, 91.24, 71.52, 71.36, 68.34,
62.83, 57.19.
The compound obtained above (0.28 g, 0.45 mmol) was
dissolved in a solution of KOH in dry MeOH (0.1 M, 15 mL). The
mixture was stirred at room temperature for 2 h and neutralized
using Dowex (H+) resin. The resulting suspension was filtrated
and concentrated. Silica gel column purification (EtOAc/MeOH/
H2O = 10:1:0.2, by volume) afforded product 8a (78 mg, 85%). 1H
NMR (600 MHz, D2O) d 5.18 (s, 1H), 3.95–3.72 (m, 5H), 3.63 (t,
J = 10.2 Hz, 0.65H), 3.54 (t, J = 10.2 Hz, 0.35H); 13C NMR
(150 MHz, D2O) d 94.02, 74.43, 72.41, 70.62, 69.93, 69.44, 61.11,
59.03.
a
-D
a
Yield, 95%; white solid. 1H NMR (600 MHz, D2O) d 8.14 (d,
J = 9.6 Hz, 2H), 7.12 (d, J = 9.6 Hz, 2H), 5.15 (d, J = 7.8 Hz, 1H),
4.06 (dd, J = 9.6 and 3.6 Hz, 1H), 3.89 (d, J = 3.0 Hz, 1H), 3.79–3.45
(m, 10H), 3.30 (dd, J = 10.2 and 1.8 Hz, 1H), 3.25 (s, 3H), 2.61 (dd,
J = 12.0 and 4.8 Hz, 1H), 1.90 (s, 3H), 1.69 (t, J = 12.0 Hz, 1H); 13C
NMR (150 MHz, D2O) d 174.52, 173.91, 161.98, 142.75, 126.35,
116.72, 100.34, 99.93, 78.29, 75.88, 75.69, 72.95, 71.51, 69.43,
69.06, 67.58, 62.43, 60.94, 60.35, 51.78, 39.77, 22.52; HRMS (ESI)
calculated for C24H33N2Oꢁ16 (MꢁH) 605.1836, found 605.1867.
4.3.4. 4-Nitrophenyl O-(5-acetamido-3,5,7-trideoxy-
-galacto-2-nonulopyranosylonic acid)-(2?3)-
O-b- -galactopyranoside (Neu5Ac7Deoxy 2–3GalbpNP, 3b)
D-glycero-a-
D
D
a
Yield, 80%; white solid. 1H NMR (600 MHz, D2O) d 8.26 (dd,
J = 12.6 and 3.6 Hz, 2H), 7.25 (dd, J = 10.2 and 2.4 Hz, 2H), 5.30 (d,
J = 7.8 Hz, 1H), 4.23 (dd, J = 10.2 and 3.6 Hz, 1H), 4.05–3.86 (m,
4H), 3.75 (d, J = 6.0 Hz, 4H), 3.71 (d, J = 6.0 Hz, 2H), 3.47 (dd,
J = 12.0 and 6.6 Hz, 1H), 2.75 (dd, J = 12.6 and 4.2 Hz, 1H), 2.01 (s,
3H), 1.82 (t, J = 11.4 Hz, 1H), 1.68–1.59 (m, 2H); 13C NMR
(150 MHz, D2O) d 175.02, 174.61, 161.94, 142.73, 126.28, 116.62,
100.22, 99.84, 75.79, 75.63, 71.18, 68.95, 68.37, 68.26, 67.66,
65.44, 60.71, 55.80, 39.76, 34.72, 22.30; HRMS (ESI) calculated
for C23H31N2Oꢁ15 (MꢁH) 575.1730, found 575.1761.
4.3. Enzymatic synthesis of sialosides 1b–8b and 1c–8c
4.3.5. 4-Nitrophenyl O-(7-azido-5-acetamido-3,5,7-trideoxy-
glycero- -galacto-2-nonulopyranosylonic acid)-(2?3)-
O-b- -galactopyranoside (Neu5Ac7N3 2–3GalbpNP, 4b)
D-
4.3.1. General procedure for one-pot three-enzyme preparative-
a-D
scale synthesis of
a2–3- and
a2–6-linked sialosides
D
a
GalpNP (1.0 equiv, 10 mM, 0.1 mmol), a sialic acid precursor
(1.5 equiv, 15 mM, 0.15 mmol) chosen from 1a–8a, sodium pyru-
vate (10.0 equiv, 100 mM, 1 mmol), and CTP (2.0 equiv, 20 mM,
0.2 mmol) were dissolved in water in a 50 mL centrifuge tube con-
taining Tris–HCl buffer (100 mM, pH 8.5) and MgCl2 (20 mM). After
adding appropriate amounts of Pasteurella multocida sialic acid
aldolase30 (PmNanA, 0.7–1.1 mg), Neisseria meningitides CMP-sialic
acid synthetase31 (NmCSS, 1.0–1.3 mg), and a sialyltransferase
(PmST16, 0.1–0.2 mg or Pd2,6ST7, 0.3–0.5 mg), water was added
to bring the volume of the reaction mixture to 10 mL. The reaction
was incubated at 37 °C in an isotherm incubator with agitating at
120 rpm for 3–16 h using PmST1 or 16–48 h using Pd2,6ST. The
product formation was monitored by TLC using EtOAc/MeOH/
H2O/AcOH = 7:2:0.5:0.1 (by volume) as the developing solvent
and stained with p-anisaldehyde sugar stain solution. The reaction
was terminated by adding the same volume (10 mL) of ice-cold
EtOH followed by incubation at 4 °C for 30 min. The mixture was
centrifuged to remove precipitates. The supernatant was concen-
trated and passed through a Bio-gel P-2 gel filtration column with
water as the eluant to obtain the desired product. Silica gel column
purification (EtOAc/MeOH/H2O = 7:2:0.6, by volume) was used for
further purification.
Yield, 91%; white solid. 1H NMR (600 MHz, D2O) d 8.12 (dd,
J = 9.0, 1.8 Hz, 2H), 7.10 (dd, J = 9.0 and 1.8 Hz, 2H), 5.15 (d,
J = 7.8 Hz, 1H), 4.04 (dd, J = 10.2 and 3.6 Hz, 1H), 3.88 (d,
J = 3.6 Hz, 1H), 3.86–3.83 (m, 1H), 3.80–3.72 (m, 4H), 3.69 (dd,
J = 10.2 and 1.8 Hz, 1H), 3.60 (d, J = 6.6 Hz, 2H), 3.56 (s, 2H), 3.34
(dd, J = 9.0 and 1.8 Hz, 1H), 2.61 (dd, J = 12.6 and 4.2 Hz, 1H),
1.89 (s, 3H), 1.73 (t, J = 12.6 Hz, 1H); 13C NMR (150 MHz, D2O) d
174.42, 173.49, 161.66, 142.44, 126.03, 116.39, 100.39, 99.58,
75.55, 75.35, 71.98, 70.78, 68.71, 68.23, 67.57, 62.40, 60.74,
60.57, 52.36, 39.14, 22.09; HRMS (ESI) calculated for C23H30N5O1ꢁ5
(MꢁH) 616.1744, found 616.1780.
4.3.6. 4-Nitrophenyl O-(3,7-dideoxy-7-fluoro-D-glycero-a-D-
galacto-2-nonulopyranosylonic acid)-(2?3)-
O-b-D-galactopyranoside (Kdn7Fa2–3GalbpNP, 5b)
Yield, 98%; white solid. 1H NMR (600 MHz, D2O) d 8.25 (d,
J = 9.0 Hz, 2H), 7.24 (d, J = 9.0 Hz, 2H), 5.29 (d, J = 7.8 Hz, 1H),
4.20 (dd, J = 9.6 and 3.0 Hz, 1H), 4.12–4.08 (m, 1H), 4.04 (d,
J = 3.0 Hz, 1H), 3.93–3.84 (m, 3H), 3.76 (d, J = 6.6 Hz, 3H), 3.73–
3.62 (m, 4H), 3.58 (t, J = 9.0 Hz, 1H), 2.74 (dd, J = 12.6 and 4.8 Hz,
1H), 1.82 (t, J = 11.4 Hz, 1H); 13C NMR (150 MHz, D2O) d 173.60,
161.69, 142.46, 126.09, 116.44, 100.29, 99.71, 88.43 (d,
J = 179.69 Hz), 75.74, 75.39, 73.05 (d, J = 17.96 Hz), 69.85, 69.34,
69.09 (d, J = 27.18 Hz), 68.77, 67.46, 61.94, 60.59, 39.05; (ESI) cal-
culated for C21H27NO15Fꢁ (MꢁH) 552.1370, found 552.1391.
4.3.2. 4-Nitrophenyl O-(7-fluoro-5-acetamido-3,5,7-trideoxy-
glycero- -galacto-2-nonulopyranosylonic acid)-(2?3)-
O-b- -galactopyranoside (Neu5Ac7F 2–3GalbpNP, 1b)
D-
a-D
D
a
Yield, 98%; white solid. 1H NMR (600 MHz, D2O) d 8.26 (dd,
J = 7.8 and 2.4 Hz, 2H), 7.24 (dd, J = 7.2 and 2.4 Hz, 2H), 5.30 (d,
J = 7.8 Hz, 1H), 4.49 (dd, J = 47.4 and 9.0 Hz, 2H), 4.22 (dd, J = 9.6
and 3.0 Hz, 1H), 4.09–4.05 (m, 2H), 3.94–3.64 (m, 9H), 2.77 (dd,
J = 12.6 and 4.8 Hz, 1H), 2.03 (s, 3H), 1.86 (t, J = 12.6 Hz, 1H); 13C
NMR (150 MHz, D2O) d 174.98, 173.65, 161.93, 142.69, 126.31,
116.65, 100.50, 99.89, 88.75 (d, J = 179.08 Hz), 75.99, 75.65,
71.79, 69.27, 68.99, 68.32, 67.70, 62.16, 60.81, 51.38, 39.59,
22.39; HRMS (ESI) calculated for C23H30FN2Oꢁ15 (MꢁH) 593.1636,
found 593.1659.
4.3.7. 4-Nitrophenyl O-(3-deoxy-7-O-methyl-
galacto-2-nonulopyranosylonic acid)-(2?3)-
O-b-D-galactopyranoside (Kdn7OMea2–3GalbpNP, 6b)
D-glycero-a-D-
Yield, 89%; white solid. 1H NMR (600 MHz, D2O) d 8.26 (dd,
J = 7.2 and 2.4 Hz, 2H), 7.24 (dd, J = 7.2 and 1.8 Hz, 2H), 5.28 (d,
J = 7.8 Hz, 1H), 4.18 (dd, J = 9.6 and 3.0 Hz, 1H), 4.01 (d, J = 3.0 Hz,
1H), 3.92–3.84 (m, 4H), 3.75 (d, J = 6.0 Hz, 2H), 3.72 (s, 2H), 3.67
(dd, J = 12.0, 6.0 Hz, 1H), 3.63 (dd, J = 10.2 and 1.8 Hz, 1H), 3.60–
3.57 (m, 1H), 3.52 (s, 3H), 2.72 (dd, J = 12.0 and 4.8 Hz, 1H), 1.78
(t, J = 12.0 Hz, 1H); 13C NMR (150 MHz, D2O) d 173.75, 161.62,

110
Z. Khedri et al. / Carbohydrate Research 389 (2014) 100–111
142.46, 126.02, 116.37, 99.95, 99.56, 77.78, 75.50, 75.38, 74.01,
71.17, 70.42, 69.94, 68.72, 67.ꢁ21, 62.13, 60.60, 60.21, 39.13; HRMS
(ESI) calculated for C22H30NO16 (MꢁH) 564.1570, found 564.1594.
142.42, 125.97, 116.48, 100.27, 99.58, 73.99, 72.27, 70.65, 70.24,
68.37, 68.08, 67.84, 65.13, 55.75, 40.46, 34.33, 21.99; HRMS (ESI)
calculated for C23H31N2Oꢁ15 (MꢁH) 575.1730, found 575.1759.
4.3.8. 4-Nitrophenyl O-(3,7-dideoxy-
nonulopyranosylonic acid)-(2?3)-O-b-
(Kdn7Deoxy 2–3GalbpNP, 7b)
D
-glycero-
a-D-galacto-2-
4.3.13. 4-Nitrophenyl O-(7-azido-5-acetamido-3,5,7-trideoxy-
glycero- -galacto-2-nonulopyranosylonic acid)-(2?6)-
O-b- -galactopyranoside (Neu5Ac7N3 2–6GalbpNP, 4c)
D-
D-galactopyranoside
a
-D
a
D
a
Yield, 90%; white solid. 1H NMR (600 MHz, D2O) d 8.26 (dd,
J = 8.4, 1.2 Hz, 2H), 7.25 (dd, J = 8.4 and 1.2 Hz, 2H), 5.29 (d,
J = 7.8 Hz, 1H), 4.20 (dd, J = 7.2 and 2.4 Hz, 1H), 4.03 (s, 2H),
3.92–3.52 (m, 8H), 3.23 (t, J = 10.2 Hz, 1H), 2.71 (dd, J = 12.6 and
4.8 Hz, 1H), 1.92 (t, J = 12.0 Hz, 1H), 1.78 (t, J = 12.6 Hz, 1H), 1.66
(t, J = 11.4 Hz, 1H); 13C NMR (150 MHz, D2O) d 174.14, 161.92,
142.73, 126.28, 116.61, 100.21, 99.88, 75.66 (2C), 74.45, 72.26,
69.24, 68.99, 68.51, 67.61, 65.48, 60.77, 39.58, 34.71; HRMS (ESI)
calculated for C21H28NOꢁ15 (MꢁH) 534.1464, found 534.1476.
Yield, 98%; white solid. 1H NMR (600 MHz, D2O) d 8.29 (d,
J = 9.0 Hz, 2H), 7.26 (d, J = 9.6 Hz, 2H), 5.18 (d, J = 7.8 Hz, 1H),
4.02 (d, J = 3.0 Hz, 1H), 3.99 (dd, J = 7.2 and 4.2 Hz, 1H), 3.96–3.92
(m, 1H), 3.89–3.83 (m, 4H), 3.77 (dd, J = 9.6 and 3.6 Hz, 1H), 3.72
(s, 1H), 3.68–3.60 (m, 3H), 3.50 (dd, J = 9.0 and 1.8 Hz, 1H), 2.73
(dd, J = 12.0 and 4.2 Hz, 1H), 2.02 (s, 3H), 1.68 (t, J = 12.0 Hz, 1H);
13C NMR (150 MHz, D2O)
d 174.33, 173.18, 161.78, 142.43,
126.05, 116.44, 100.56, 99.78, 73.93, 72.27, 71.70, 70.97, 70.23,
68.30, 68.16, 63.09, 62.38, 61.03, 52.50, 40.04, 22.08; HRMS (ESI)
calculated for C23H30N5Oꢁ15 (MꢁH) 616.1744, found 616.1775.
4.3.9. 4-Nitrophenyl O-(7-azido-3,7-dideoxy-
galacto-2-nonulopyranosylonic acid)-(2?3)-
O-b- -galactopyranoside (Kdn7N3 2–3GalbpNP, 8b)
D-glycero-a-D-
4.3.14. 4-Nitrophenyl O-(3,9-dideoxy-7-fluoro-D-glycero-a-D-
D
a
galacto-2-nonulopyranosylonic acid)-(2?6)-
O-b-D-galactopyranoside (Kdn7Fa2–6GalbpNP, 5c)
Yield, 98%; white solid. 1H NMR (600 MHz, D2O) d 8.27 (dd,
J = 9.6 and 2.4 Hz, 2H), 7.25 (dd, J = 9.0 and 1.8 Hz, 2H), 5.29 (d,
J = 7.8 Hz, 1H), 4.17 (dd, J = 9.6 and 3.0 Hz, 1H), 4.05–4.02 (m,
2H), 3.93–3.87 (m, 3H), 3.76–3.73 (m, 4H), 3.67 (dd, J = 9.6 and
2.4 Hz, 1H), 3.65–3.59 (m, 2H), 2.72 (dd, J = 12.6 and 4.8 Hz, 1H),
1.87 (t, J = 12.6 Hz, 1H); 13C NMR (150 MHz, D2O) d 173.85,
161.76, 142.50, 126.17, 116.54, 100.73, 99.73, 75.52, 75.39, 73.77,
70.74, 70.59, 70.09, 68.79, 67.83, 62.56, 60.67, 59.80, 38.66; HRMS
(ESI) calculated for C21H27N4Oꢁ15 (MꢁH) 575.1478, found 575.1505.
Yield, 95%; white solid. 1H NMR (600 MHz, D2O) d 8.26 (dd,
J = 7.2 and 1.8 Hz, 2H), 7.25 (dd, J = 7.2 and 2.4 Hz, 2H), 5.18 (d,
J = 7.8 Hz, 1H), 4.06–3.58 (m, 12H), 3.48 (t, J = 10.2 Hz, 1H), 2.71
(dd, J = 12.0 and 4.8 Hz, 1H), 1.63 (t, J = 12.6 Hz, 1H); 13C NMR
(150 MHz, D2O) d 173.10, 161.78, 142.50, 126.09, 116.49, 100.77,
99.82, 88.50 (d, J = 179.2 Hz), 73.96, 72.87 (d, J = 17.9 Hz), 72.32,
70.29, 69.84, 69.49 (d, J = 4.9 Hz), 69.11 (d, J = 27.6 Hz), 68.39,
63.36, 61.90, 40.01; HRMS (ESI) calculated for C21H27NO15Fꢁ
(MꢁH) 552.1370, found 552.1386.
4.3.10. 4-Nitrophenyl O-(7-fluoro-5-acetamido-3,5,7-trideoxy-
glycero- -galacto-2-nonulopyranosylonic acid)-(2?6)-
O-b- -galactopyranoside (Neu5Ac7F 2–6GalbpNP, 1c)
D-
a-D
4.3.15. 4-Nitrophenyl O-(3-deoxy-7-O-methyl-D-glycero-a-D-
D
a
Yield, 92%; white solid. 1H NMR (600 MHz, D2O) d 8.15 (dd, J = 6.6
and 1.8 Hz, 2H), 7.14 (dd, J = 6.6 and 3.0 Hz, 2H), 5.06 (d, J = 7.8 Hz,
1H), 4.34 (dd, J = 47.4 and 8.4 Hz, 1H), 3.92–3.48 (m, 12H), 2.62
(dd, J = 12.6 and 4.8 Hz, 1H), 1.89 (s, 3H), 1.54 (t, J = 12.0 Hz, 1H);
13C NMR (150 MHz, D2O) d 174.88, 173.28, 162.06, 142.76, 126.35,
116.75, 101.02, 100.09, 88.36 (d, J = 180.89 Hz), 74.19, 72.57,
71.73 (d, J = 16.31 Hz), 70.55, 69.22 (d, J = 28.69 Hz), 68.63, 68.39,
63.57, 62.13, 51.49, 40.57, 22.42; HRMS (ESI) calculated for C23H30-
FN2Oꢁ15 (MꢁH) 593.1636, found 593.1665.
galacto-2-nonulopyranosylonic acid)-(2?6)-
O-b-D-galactopyranoside (Kdn7OMea2–6GalbpNP, 6c)
Yield, 53%; white solid. 1H NMR (600 MHz, D2O) d 8.28 (dd,
J = 7.2 and 2.4 Hz, 2H), 7.25 (dd, J = 7.2 and 2.4 Hz, 2H), 5.17 (d,
J = 7.2 and 2.4 Hz, 1H), 3.99–3.98 (m, 1H), 3.89 (t, J = 10.2 Hz, 1H),
3.86–3.82 (m, 2H), 3.77 (dd, J = 10.2 and 4.8 Hz, 1H), 3.71 (s, 3H),
3.68–3.53 (m, 5H), 3.44 (s, 2H), 3.40 (t, J = 10.2 Hz, 1H), 2.69 (dd,
J = 12.6 and 4.8 Hz, 1H), 1.59 (t, J = 12.6 Hz, 1H); 13C NMR
(150 MHz, D2O) d 173.48, 161.77, 142.48, 126.06, 116.32, 100.21,
99.75, 77.77, 73.94, 73.67, 72.25, 71.33, 70.35, 70.20, 70.08,
68.32, 62.95, 61.34, 60.20, 39.77, HRMS (ESI) calculated for C22H30-
NOꢁ16 (MꢁH) 564.1570, found 564.1585.
4.3.11. 4-Nitrophenyl O-(5-acetamido-3,5-dideoxy-7-O-methyl-
D
-glycero-
a-
D-galacto-2-nonulopyranosylonic acid)-(2?6)-
O-b- -galactopyranoside (Neu5Ac7OMe
D
a2–6GalbpNP, 2c)
Yield, 57%; white solid. 1H NMR (600 MHz, D2O) d 8.29 (d,
J = 7.2 Hz, 2H), 7.26 (d, J = 7.2 Hz, 2H), 5.18 (d, J = 7.2 Hz, 1H),
4.01 (s, 2H), 3.93–3.58 (m, 10H), 3.39 (d, J = 7.8 Hz, 1H), 3.33 (s,
3H), 2.71 (dd, J = 11.0 and 1.8 Hz, 1H), 2.02 (s, 3H), 1.66 (t,
J = 12.0 Hz, 1H); 13C NMR (150 MHz, D2O) d 174.48, 173.37,
162.06, 142.77, 126.35, 116.61, 100.41, 100.04, 78.26, 74.19,
72.53, 72.25, 71.58, 70.47, 69.17, 68.58, 63.23, 62.67 (2C), 51.88,
4.3.16. 4-Nitrophenyl O-(3,7-dideoxy-
nonulopyranosylonic acid)-(2?6)-O-b-
(Kdn7Deoxy 2–6GalbpNP, 7c)
D
-glycero-
a-D-galacto-2-
D-galactopyranoside
a
Yield, 58%; white solid. 1H NMR (600 MHz, D2O) d 8.29 (d,
J = 9.6 Hz, 2H), 7.27 (d, J = 9.0 Hz, 2H), 5.18 (d, J = 7.8 Hz, 1H),
4.01 (d, J = 3.6 Hz, 1H), 3.99–3.97 (m, 2H), 3.88 (t, J = 10.8 Hz,
1H), 3.84 (t, J = 10.2, 1H), 3.77 (dd, J = 10.2 and 3.6 Hz, 1H), 3.70–
3.65 (m, 1H), 3.60–3.52 (m, 2H), 3.46 (dd, J = 11.4 and 6.6 Hz,
1H), 3.31 (t, J = 18.0 Hz, 1H), 3.16 (d, J = 18.0 Hz, 1H), 3.11 (t,
J = 9.6 Hz, 1H), 2.69 (dd, J = 12.6 and 4.8 Hz, 1H), 1.87–1.83 (m,
1H), 1.61 (t, 1H), 1.45–1.42 (m, 1H); 13C NMR (150 MHz, D2O) d
173.53, 161.73, 142.42, 125.99, 116.47, 100.34, 99.65, 74.54,
73.95, 72.24, 71.65, 70.24, 69.56, 68.35, 68.21, 65.21, 63.17,
40.12, 22.43; HRMS (ESI) calculated for
605.1836, found 605.1869.
C
24H33N2Oꢁ16 (MꢁH)
4.3.12. 4-Nitrophenyl O-(5-acetamido-3,5,7-trideoxy-
-galacto-2-nonulopyranosylonic acid)-(2?6)-
O-b- -galactopyranoside (Neu5Ac7Deoxy 2–6GalbpNP, 3c)
D-glycero-
a-D
D
a
Yield, 88%; white solid. 1H NMR (600 MHz, D2O) d 8.29 (d,
J = 9.0 Hz, 2H), 7.27 (d, J = 9.0 Hz, 2H), 5.18 (d, J = 7.8 Hz, 1H),
4.02 (d, J = 3.6 Hz, 1H), 3.99–3.89 (m, 2H), 3.84 (t, J = 7.8 Hz, 1H),
3.78 (dd, J = 9.6 and 3.0 Hz, 1H), 3.71–3.68 (m, 3H), 3.60–3.56 (m,
1H), 3.52 (dd, J = 12.0 and 3.0 Hz, 1H), 3.47 (t, J = 9.6 Hz, 1H), 3.40
(dd, J = 11.4 and 7.2 Hz, 1H), 2.73 (dd, J = 12.6 and 4.8 Hz, 1H),
1.98 (s, 3H), 1.64 (t, J = 12.0 Hz, 1H), 1.59–1.54 (m, 1H), 1.36–1.30
(m, 1H); 13C NMR (150 MHz, D2O) d 174.59, 173.48, 161.72,
40.11, 34.40; HRMS (ESI) calculated for
534.1464, found 534.1471.
C
21H28NOꢁ15 (MꢁH)
4.3.17. 4-Nitrophenyl O-(7-azido-3,7-dideoxy-
galacto-2-nonulopyranosylonic acid)-(2?6)-
D-glycero-a-D-
O-b-D-galactopyranoside (Kdn7N3a2–6GalbpNP, 8c)
Yield, 89%; white solid. 1H NMR (600 MHz, D2O) d 8.28 (d,
J = 9.0 Hz, 2H), 7.25 (d, J = 9.0 Hz, 2H), 5.18 (d, J = 7.2 Hz, 1H),

Z. Khedri et al. / Carbohydrate Research 389 (2014) 100–111
111
4.01 (d, J = 3.0 Hz, 1H), 3.99–3.93 (m, 2H), 3.91–3.77 (m, 5H), 3.74–
3.66 (m, 3H), 3.62–3.57 (m, 1H), 3.49 (t, J = 9.0 Hz, 1H), 2.71 (dd,
J = 12.6 and 4.2 Hz, 1H), 1.66 (t, J = 12.0 Hz, 1H); 13C NMR
(150 MHz, D2O) d 173.32, 161.75, 142.41, 126.01, 116.42, 100.56,
99.72, 73.93, 73.33, 72.24, 70.86, 70.72, 70.21, 69.84, 68.29,
63.08, 62.41, 60.14, 39.68; HRMS (ESI) calculated for C21H27N4O1ꢁ5
(MꢁH) 575.1478, found 575.1498.
Teacher-Scholar and a UC-Davis Chancellor’s Fellow. The authors
thank the organizers of the EuroCarb17 especially Professor Timor
Baasov.
Supplementary data
Supplementary data (1H and 13C spectra of sialosides 1b–8b and
1c–8c) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.carres.2014.02.021.
4.4. Sialidase substrate specificity assays
4.4.1. Materials
References
Clostridium perfringens sialidase (type VI), Vibrio cholerae
sialidase (type III), and b-galactosidase from Aspergillus oryzae were
purchased from Sigma. Salmonella typhimurium sialidase and Strep-
tococcus pneumoniae sialidase were bought from Prozyme. All
these enzymes were used without further purification. PmST16
and NEU214 were expressed in Escherichia coli and purified as
described previously. Sodium pyruvate (Fisher Biotech), N-acetyl-
mannosamine (ManNAc, Sigma), mannose (Acros Organics), para-
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4.4.2. Assays
All sialidase assays were carried out at 37 °C in duplicate in
384-well plates in a final volume of 20
lL containing a sialoside
substrate (0.3 mM) and the b-galactosidase (12
lg, 126 mU). The
amount of the b-galactosidase required to completely hydrolyze
GalbpNP (0.3 mM) within the time frame of the assay was prede-
termined. The assay conditions for various sialidases were as
follows: Salmonella typhimurium sialidase (6 mU) in sodium
acetate buffer (100 mM, pH 5.5) containing NaCl (100 mM);
Clostridium perfringens sialidase (4 mU) in MES buffer (100 mM,
pH 5.0); Vibrio cholerae sialidase (2.8 mU) in sodium acetate buffer
(100 mM, pH 5.5) containing NaCl (150 mM) and CaCl2 (10 mM);
Streptococcus pneumoniae sialidase (0.8 mU) in sodium acetate buf-
fer (100 mM, pH 6.0); PmST1 (25
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NanH2 (10 ng) in sodium acetate buffer (100 mM, pH 5.0). The
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l
l
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Acknowledgements
This work was supported by National Institutes of Health Grant
R01HD065122, National Science Foundation Grant CHE-1012511,
and Beckman Young Investigator Award. X.C. is a Camille Dreyfus