CST-II's recognition domain for acceptor substrates in α-(2→8)- sialylations

Article abstract of DOI:10.1016/j.carres.2011.05.009

CST-II is a bacterial sialyltransferase known for its ability to perform α-(2→8)-sialylations using GM3 related trisaccharide substrates. Previously, we probed the enzyme's substrate specificity and developed an efficient synthesis for α-(2→8)-oligosialos

Full text of DOI:10.1016/j.carres.2011.05.009

Carbohydrate Research 346 (2011) 1692–1704
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CST-II’s recognition domain for acceptor substrates in a-(2?8)-sialylations
Wenling Li a, , Ping Zhang a, Amir J. Zuccolo a, Ruxiang Blake Zheng b, Chang-Chun Ling a,
⇑
a Department of Chemistry, University of Calgary, Alberta Ingenuity Centre for Carbohydrate Science, Calgary, Canada AB T2N 1N4
b Department of Chemistry, University of Alberta, Alberta Ingenuity Centre for Carbohydrate Science, Edmonton, Canada AB T6G 2G2
a r t i c l e i n f o
a b s t r a c t
Article history:
CST-II is a bacterial sialyltransferase known for its ability to perform
a
-(2?8)-sialylations using GM3
Received 8 May 2011
Accepted 10 May 2011
Available online 14 May 2011
related trisaccharide substrates. Previously, we probed the enzyme’s substrate specificity and developed
an efficient synthesis for -(2?8)-oligosialosides, and we suggested that CST-II could have a very small
a
substrate recognition domain. Here we report our full studies on CST-II’s recognition feature for acceptor
substrates. The current study further demonstrates the versatility of CST-II in preparing complex oligo-
saccharides that contain a-(2?8)-oligosialyl moieties.
Keywords:
a-(2?8)-Sialylation
Ó 2011 Elsevier Ltd. All rights reserved.
Chemoenzymatic synthesis
CST-II
Glycosylations
Enzyme binding Site
1. Introduction
of the enzyme. To date, only one crystal structure of CST-II was
available in the literature:1 the enzyme was co-crystallized with
a 2-fluoro analog of CMP-NANA. However, no acceptor substrate
was present in the solved crystal structure; therefore, the molecu-
lar recognition features were poorly understood for acceptor sub-
strates. Chen’s group12 has carried out an extensive study on
probing the acceptor tolerability of CST-II, they employed a series
of trisaccharides that had the sialic acid residue attached to either
the O-30 or O-60 position of lactose, they found that the enzyme ac-
cepted all the trisaccharide substrates and produced a series of
disialylated glycans; they also reported that the enzyme tolerated
various substitutions at C-5 and C-9 of the sialic acid residue from
both the donor side and acceptor side. The fact that the enzyme
recognized substrates as large as GM3-related trisaccharides, might
suggest that CST-II had a large binding domain for acceptors. Par-
allel to Chen’s work,12 we carried out our own probing studies
and reported our preliminary results as a communication.13 We
suggested that CST-II most likely had a very small recognition do-
main for the acceptor substrate, which is about the size of a
monosialoside, and we confirmed this by using compound 1, 2,
CST-II is an interesting sialyltransferase expressed by
Campylobacter jejuni OH4384, capable of both -(2?3)- and
-(2?8)-sialylations.1 The enzyme has been shown to transfer a
a
a
N-acetylneuraminic acid (sialic acid) residue from cytidine-50-
monophospho-N-acetylneuraminic acid (CMP-NANA) to either
the O-3 position of a terminal galactose residue or to the O-8 posi-
tion of a terminal N-acetylneuraminic acid residue, leading to the
production of a wide variety of lipopolysaccharide (LPS) structures
in C. jejuni that resemble human gangliosides.2 An immune re-
sponse to LPS has shown to cause Guillain–Barré Syndrome which
is an autoimmune neuropathy in some patients.3 CST-II’s dual sub-
strate specificity is remarkable. One feature that was most notable
for CST-II was that the enzyme is capable of taking GM3-related tri-
saccharides which has a
sequence as a substrate for conducting
up to three units of sialic acid residues were reported to sequen-
tially add to the O-8 position of the terminal sialic acid residue of
the parent oligosaccharides starting from GM3 (Fig. 1) and a series
a
-NeupNAc-(2?3)-b-Galp-(1?4)-b-Glcp
a
-(2?8)-sialylation;4–6
of complex gangliosides containing different numbers of
oligosialyl fragments were isolated.4 Considering the enormous
difficulties encountered in chemical
-(2?8)-sialylations,7–11 we
think that CST-II can serve as an extremely valuable tool for syn-
thetic carbohydrate chemists. In order to exploit the full potential
of CST-II, we carried out a systematic study to probe the active site
a
-(2?8)-
and an a-(2?6)-linked thiodisaccharide 13 as a substrate
(Scheme 1). From monosialosides 1 and 2, we isolated a series of
short oligosialosides 3–5 and 10–12 by HPLC in 5–15% yields,
and from 13, the corresponding trisaccharide 14 was isolated in
48% yield. We also found that if the enzymatic syntheses were car-
ried out at a higher substrate concentration, we were able to
achieve sialyl transfers of more than three units for the first time,
to obtain a mixture containing oligomers 3–9 which can be iso-
lated in pure form in yields ranging from 1.7% to 9.6%.
a
⇑
Corresponding author.
E-mail address: ccling@ucalgary.ca (C.-C. Ling).
Based on these results, we consider that CST-II could be used
as an important tool to organic chemists to prepare various
Present address: School of Chemical and Biological Engineering, Lanzhou Jiaotong
University, Lanzhou, Gansu 730070, PR China.
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.05.009

W. Li et al. / Carbohydrate Research 346 (2011) 1692–1704
1693
OH
HO
OH
can be used to probe the requirement of aglycone sizes by CST-II.
Compound 17 is an analog of 13 and also the S-analog of the sub-
strate used by Chen’s reported work.12
The monosialosides 15 was prepared according to Scheme 2.
Starting from the previously known thioglycoside 18,13 we reacted
it with 11-azidoundecanol 1914 in a mixture of acetonitrile-dichlo-
romethane using N-iodosuccinimide (NIS)–triflic acid as the acti-
HO
HO
CO2H
O
CO2H
S
O
O
AcHN
HO
AcHN
HO
N3
10
HO
Me
15
16
OMe
HO
vating reagent; the desired
a-glycoside 20 was obtained in 64%
OH
HO
CO2H
O
yield, together with the undesired b-glycoside 21 in 12% yield.
After a full de-O-acetylation by Zémplen transesterification, and
a saponification of the intermediate methyl ester, acceptor 15
was obtained in 64% yield over two steps.
HO
O
OH
O
S
AcHN
HO
O
HO
HO
OH
To prepare the methyl thioglycoside 16, we used the peracety-
lated thioneuraminic acid 2215 as the starting material. First we
tried a one-pot procedure by converting 22 to an intermediate thi-
oether 23 using in situ deacetylation–methylation procedures
(Scheme 2). The methyl ester of 23 was directly saponified to ob-
tain the desired thioglycoside 16. The yield was only 35% overall
two steps. To improve the yield, we attempted a stepwise route:
thus compound 22 was first treated with diethylamine in DMF
under argon, to generate a thiolate which was reacted with methyl
HO
17
Figure 1. Other monosialosides used to probe the scope of a-(2?8)-sialylations by
CST-II.
OH
HO
CO2H
iodide; the fully protected methyl a-thiosialoside 24 was isolated
in 67% yield. After full deprotections as 20, compound 16 was ob-
tained in 82% yield.
O
O
AcHN
HO
HO
CO2H
OH
HO
HO
HO
CO2H
X
To obtain trisaccharide 17, we prepared the 40,60-lactosyl diol 25
(Scheme 3) according to the literature.16 The primary hydroxyl
group was first selectively activated using 1.2 equiv of triflic anhy-
dride as the reagent to afford the intermediate 26 which was re-
acted with thioacetate 22 as above, to yield the trisaccharide 27
in 26% yield over two steps. The poor yield was due to a partial
migration of acetate from O-3 to O-4 which occurred as a side reac-
tion. Nevertheless, the obtained trisaccharide 27 was fully depro-
tected under similar conditions as 20 to obtain the desired 17 in
80% yield.
O
O
AcHN
HO
O
HO
a
CO2H
AcHN
HO
HO
n
O
X
AcHN
HO
10
1 X=O
2 X=S
HO
3 X=O, n=0 8 X=O, n=5
10
4 X=O, n=1
5 X=O, n=2
9 X=O, n=6
10 X=S, n=0
6 X=O, n=3 11 X=S, n=1
7 X=O, n=4 12 X=S, n=2
OH
HO
CO2H
Both compounds 15 and 16 were subjected to CST-II for
a-
OH
HO
O
CO2H
(2?8)-sialylations (Scheme 4). Thus according to our preliminary
report, we conducted the reaction at a higher substrate concentra-
tion (ꢀ8.5 M); using 6.8 equiv of CMP-NANA, and 2.4 U of CST-II,
oligomers ranging from dimer 28 to hexamer 32 were obtained
in 2.2–12.0% yield after isolation. The incorporation of a long
hydrophobic chain was the key to allow for the isolation of all olig-
omers by reverse-phase HPLC chromatography. Similarly, using S-
O
AcHN
HO
HO
CO2H
O
HO
S
SR
a
AcHN
HO
O
O
S
SR
OH
AcHN
HO
HO
HO
O
OH
HO
HO
HO
14
HO
13
Scheme 1. Initially published substrates recognized by CST-II for
lations. Reagents: (a) CMP-NeuNAc/CST-ll/MnCI2/alkaline phosphotase.
a-(2?8)-sialy-
methyl
a-monosialoside 16 as a substrate, we also observed the
formation of several oligomers according to TLC. Due to the fact
that compound 16 and all formed oligomers had a small methyl
aglycone, which is not hydrophobic enough to allow the formed
products to retain on C18 silica gel matrix, we were not able to em-
ploy reverse-phase HPLC to isolate the formed oligomers. Using
size-exclusion chromatography to purify the reaction mixture
was also proven to be difficult as the formed products were consis-
tently contaminated with other by-products (see later). However,
using normal phase chromatography on silica gel, we were able
to isolate dimer 33 and trimer 34 in 17% and 10.1% yields, respec-
tively. Attempts to isolate higher oligomers were not successful.
In an analogous fashion, trisaccharide 17 was also subjected to
enzymatic sialylation as above (Scheme 5). Since we were only
interested in finding out if 17 would be a substrate of the enzyme,
no effort to synthesize higher oligomers was attempted. Thus using
only 1.7 equiv of CMP-NeuNAc, we found that CST-II smoothly con-
verted compound 17 to a tetrasaccharide 35 which was isolated in
pure form and in 39% yield by size exclusion chromatography on
Sephadex LH-20. TLC showed that a small amount of higher oligo-
mer possibly with three sialyl residues was also formed but not
further characterized.
a
-(2?8)-linked oligosialosides. The downside is that the isolated
yields for higher oligomers were low; however, considering that
each oligosialoside could be obtained just in one step, this method-
ology did exhibit significant advantages over conventional chemi-
cal synthesis, which required many steps of selective chemical
protections, deprotections and glycosylations. To date, the longest
oligomer ever synthesized by chemical synthesis was a tetramer.11
In this paper, we wish to report our full studies in probing the
binding domain of CST-II for acceptor substrates as well as to
demonstrate the versatility of CST-II to catalyze the synthesis of
other a-(2?8)-linked oligosialosides.
2. Results and discussions
To demonstrate the scope of the current methodology, we de-
signed three other monosialosides with different features at the
anomeric center (Fig. 1). Compound 15 is an O-glycoside related
to substrate 1 with azido functional group embedded in the
aglycone that we will need for our future conjugation studies.
Compound 16 is a thioglycoside with a very small aglycone, which
The ability to accept both O- and S-monosialosides as its sub-
strates is the most remarkable feature of CST-II. Considering the

1694
W. Li et al. / Carbohydrate Research 346 (2011) 1692–1704
Cl
OAc
S
AcO
O
N3
10
CO2Me
AcHN
OAc
OAc
O
AcO
AcO
CO2Me
O
AcO
OAc
O
18
a
O
+
N3
10
CO2Me
AcHN
AcO
AcHN
AcO
+
OAc
OAc
(12%)
N3
10
(64%)
21
20
HO
b
19
OH
HO
CO 2H
O
O
AcHN
HO
N3
10
HO
(64%)
15
OH
HO
CO2Me
S
O
Ac HN
HO
OH
Me
d
OAc
c
OH
23
AcO
CO2Me
HO
CO2H
S
(35%)
O
O
SAc
AcHN
AcO
AcHN
HO
e
OAc
AcO
CO2Me
OAc
HO
Me
b
22
O
16
S
AcHN
AcO
(82%)
OAc
Me
(67%)
24
Scheme 2. Preparations of acceptors 15 and 16. Reagents and conditions: (a) HO(CH2)11N3/NIS/TfOH/CH3CN–CH2Cl2 (2:1), ꢁ50 °C to rt; (b) NaOMe/MeOH, then H2O; (c) (1)
KOBu-t/MeOH, ꢁ40 °C, then (2) Mel/DMF, rt; (d) NaOH/MeOH; (e) MeI/Et2NH/DMF.
OAc
AcO
CO2Me
OMe
OAc
AcO
O
SAc
AcHN
AcO
OAc
CO2Me
AcO
O
OAc
b
AcO
O
22
S
O
AcHN
AcO
O
OAc
HO
OR
O
HO
AcO
OAc
OAc
AcO
AcO
O
OMe
(26% from25)
O
27
OAc
OAc
c
25 R=H
26 R=CF3SO2
a
OMe
HO
OH
HO
CO2H
O
HO
O
OH
O
S
AcHN
HO
O
HO
HO
OH
HO
(80%)
17
Scheme 3. Preparation of acceptors 17. Reagents and conditions: (a) Tf2O/CH2CI2-Py; (b) Et2NH/DMF, 0 °C to rt; (c) NaOMe/MeOH, then H2O.

W. Li et al. / Carbohydrate Research 346 (2011) 1692–1704
1695
OH
HO
CO2H
O
O
AcHN
HO
HO
CO 2H
HO
O
O
AcHN
HO
OH
HO
HO
CO2H
O
HO
CO2H
O
HO
a
O
O
AcHN
AcHN
HO
n
HO
N3
10
HO
N3
10
28 n=0
29 n=1
30 n=2
31 n=3
32 n=4
(12.0%)
(7.7%)
(5.3%)
(4.5%)
(2.2%)
(64%)
15
OH
HO
HO
CO2H
O
O
AcHN
HO
OH
HO
HO
CO2H
HO
CO2H
S
a
O
O
O
AcHN
HO
HO
AcHN
HO
CO2H
HO
Me
O
S
AcHN
16
n
HO
HO
Me
(17.0%)
(10.1%)
33 n=0
34 n=1
Scheme 4.
a-(2?8)-Sialylations of substrates 15 and 16 with CMP-NeuNAc and CST-II. Reagents: (a) CMP-NeuNAc/CST-II/MnCl2/alkaline phosphotase.
had no aglycone attached to the reducing end neuraminic acid res-
idue. Thus it appeared that the enzyme had recognized natural sia-
lic acid (36) which had no aglycone, and used it as a substrate to
OMe
HO
OH
HO
HO
CO2H
O
HO
O
OH
conduct a-(2?8)-sialylations. The source of the sialic acid present
O
S
AcHN
HO
in the reaction media could be traced back to our CMP-NeuNAc,
which was obtained by reacting cytidine 50-triphosphate (CTP)
with 5-N-acetyl-neuraminic acid 36 as published procedure,4,5
and used as crude; it was sometimes contaminated with a small
amount of unconverted 36. However, even when we employed a
batch of CMP-NeuNAc that was free of unconverted 36 (checked
by 1H NMR spectra) as the starting materials, we still observed
O
HO
OH
HO
17
CMP-NeuNAc/CST-II
/MnCl2/Alkaline phosphotase
the formation of small amounts of
acids; this probably can be explained by a partial hydrolysis of
the CMP-NeuNAc at the binding site of CST-II.
a-(2?8)-linked oligosialic
OH
HO
CO2H
OMe
HO
O
O
AcHN
HO
Using pure 36 as a substrate, and under similar sialylation
conditions as 16; we isolated the disialoside 37 by size-exclusion
chromatography on Sephadex G-25 in 21.5% yield (Scheme 6),
together with some trisialoside which we could not isolate in
pure form.
HO
CO2H
O
HO
HO
O
OH
O
S
AcHN
HO
O
HO
HO
OH
In the 1H NMR spectra of 36 in D2O (see the Supplementary
data) as well as that of the formed disialoside 37, it was observed
that the unprotected anomeric center adopted almost exclusively
HO
%
(39
)
35
the b-configuration (36b/36
a
>23.5:1), as the 1H chemical shift of
Scheme 5.
a-(2?8)-Sialylations of substrates 17 with CMP-NeuNAc and CST-II.
H-3ax was found at 2.15 ppm. Combined with observations from
both Chen’s and our group, that CST-II tolerates significant struc-
tural variations at the anomeric center, this made us wonder if
the anomeric center of the monosialoside acceptor was actually
recognized at all by CST-II. If not, the binding domain of CST-II
for acceptor would be even smaller than a monosialoside.
We thus designed two b-sialosides 38 and 39 as a substrate to
probe the activities of CST-II (Scheme 7). Compound 38 was a thio-
glycoside prepared from 18 and had a large aglycone, while com-
pound 39 was a b-O-sialoside that had a small aglycone that was
prepared from 36 using the literature procedure.17 Our experi-
ments revealed that both b-sialosides 38 and 39 were not the sub-
significant difference in their ability to form hydrogen bonds be-
tween the O and S atoms, the above results further confirmed that
the anomeric glycosidic atom of the acceptor substrate did not in-
volve in key hydrogen bonds with the protein.
During the synthesis of 33 and 34, we observed the formation of
some by-products of similar molecular sizes, which created a sig-
nificant difficulty for us to isolate the desired oligosialosides in
pure form by size-exclusion chromatography. Analysis by mass
spectrometry indicated that these by-products were oligomers of
sialic acid, as their molecular weights correlated to analogs that
strates of CST-II as no a-(2?8)-sialylated products were formed in

1696
W. Li et al. / Carbohydrate Research 346 (2011) 1692–1704
OH
HO
CO 2H
OH
HO
O
HO
OH
O
AcHN
HO
HO
OH
a
HO
O
CO2H
O
AcHN
HO
CO2H
AcHN
HO
HO
37
36
(21.5%)
Scheme 6.
a-(2?8)-Sialylations of natural neuraminic acid (34). Reagents: (a) CMP-NeuNAc/CST-II/MnCI2/alkaline phosphatase.
OH
to the more stable b-anomer 37b as dictated by the anomeric
effect.
Combining all facts together, we see that CST-II is in fact very
tolerable toward structural variations (in term of size/shape) for
the group attached to the anomeric center, (see 1, 2, 13, 15, 16,
OAc
HO
SPhCl
AcO
SPhCl
b
b
a
O
no reaction
no reaction
O
CO2H
CO2Me
AcHN
AcHN
AcO
HO
HO
OAc
38
18
17, or 36
requires an
none of the b-sialosides (38 and 39) was accepted as a substrate.
This led us to conclude that the anomeric center of the -sialoside
a
as well as the previously reported GM3); but the enzyme
OH
OH
a
-sialoside for conducting -(2?8)-sialylation because
a
HO
OMe
HO
OH
c
O
O
CO2H
CO2H
a
AcHN
HO
AcHN
HO
was most probably located at the periphery of the binding site and,
that the aglycone was probably projecting into the bulk of water
(Fig. 2). This mode of binding explains the enzyme’s mechanics
HO
HO
39
36
very well: in all
product of the previous step as a substrate for the following step,
this is because CST-II only requires a small -sialoside for binding
a-(2?8)-sialylations, CST-II was able to use the
Scheme 7. Probing the CST-II’s activity using b-sialosides as a substrate. Reagents:
(a) NaOMe/MeOH then H2O; (b) CMP-NeuNAc/CST-II/MnCl2/alkaline phosphotase;
(c) (1) MeOH/HCl, (2) NaOH.
a
and such a key recognition element is found in every substrate/
product, that is why the enzyme is capable of repeating the cycle
many times to produce a-(2?8)-PSAs. We conjecture that similar
binding features could be true for many other enzymes of similar
catalytic properties in the nature.
OH
HO
CO2H
OH
HO
O
In conclusion, through a series of chemical mapping experi-
ments, we have provided convincing evidence that CST-II has a
small binding domain for the acceptor substrate, which is of
HO
OH
O
AcHN
HO
HO
OH
HO
O
CO2H
O
AcHN
HO
CO2H
AcHN
HO
the size of an
a-monosialoside. Furthermore, we demonstrated
HO
that functional groups introduced at the anomeric center could
be tolerated. These results revealed great potential of using
CST-II as a versatile tool to synthesize complex oligosaccharides
(not a substrate)
36β
37β
containing
a-(2?8)-oligosialyl fragments as well as their mimet-
OH
HO
HO
CO2H
ics that are difficult to obtain via conventional chemical synthe-
ses. Additionally, our studies also provided important guidance
for future design of inhibitors for CST-II using acceptor-based
approaches.
OH
O
HO
CO2H
O
AcHN
HO
HO
CO2H
a
O
OH
O
Ac HN
HO
OH
AcHN
HO
HO
HO
(a substrate)
36
α
37α
Scheme 8. Proposed mechanism for the formation of b-sialosides. Reagents: (a)
CMP-NeuNAc/CST-ll/MnCl2/alkaline phosphotase.
either case. These results suggested that although the glycosidic
atom is not involved in the key contact with the protein, the a-ano-
meric configuration that the sialoside adopted was a prerequisite
for enzymatic activities. The axial carboxylate could have engaged
in key interactions with the protein.
To explain why CST-II accepted 36 as a substrate, we could use
Scheme 8 to illustrate the process. In the aqueous solution of 36,
although the b-anomer 36b existed as the predominant form due
to anomeric effect, it was still in equilibrium with a very small
amount of
CST-II; after the first transfer of the NeuNAc residue to the 8-po-
sition, the product 37 was formed in the binding site; once
returning to solution, compound 37 quickly anomerized back
a-anomer 36a, which acted as the true substrate of
Figure 2. An illustration to show the estimated key binding domain of CST-II for a-
sialoside substrates and the variable group region (exposed to the bulk of water)
that could be tolerated by the enzyme.
a
a

W. Li et al. / Carbohydrate Research 346 (2011) 1692–1704
1697
H-3ax), 1.49–1.57 (m, 2H, OCHaCHbCH2), 1.19–1.40 (m, 18H,
9 ꢂ CH2 dodecyl), 0.91 (t, 3H, J = 7.1 Hz, CH3 dodecyl). 13C NMR
(From GHSQC, CD3OD, 100 MHz): dC 78.01 (C-8), 74.24 (C-6),
73.01 (C-60), 71.45 (C-80), 70.29 (C-7), 69.09 (C-70), 68.69 (C-4),
68.38 (C-40), 63.86 (OCHaHb), 63.29 (C-90), 62.06 (C-9), 52.84 (C-
5), 52.40 (C-50), 40.86 (C-3), 40.63 (C-30), 29.15–31.47 (8 ꢂ CH2
dodecyl), 25.64 (CH2 dodecyl), 22.09 (CH2 dodecyl), 21.48 (Ac),
21.02 (Ac), 12.93 (CH3 dodecyl). m/z (ESI-HRMS) calcd for
[C34H60N2O17ꢁ2H]2ꢁ 383.1868; found 383.1863.
3. Materials and methods
3.1. General methods
Optical rotations were determined in a 5 cm cell at 25 2 °C.
[a]
values are given in units of 10ꢁ1 deg cm2 gꢁ1. Analytical TLC
D
was performed on Silica Gel 60-F254 (Merck, Darmstadt) with
detection by quenching of fluorescence and/or by charring with
5% sulfuric acid in water or with a ceric ammonium molybdate
dip. All commercial reagents were used as supplied unless other-
wise stated. Column chromatography was performed on Silica
Gel 60 (Silicycle, Ontario). Molecular sieves are stored in an oven
at 100 °C and flame-dried under vacuum before use. Organic solu-
tions from extractions were dried with anhydrous Na2SO4 prior to
concentration under vacuum at <40 °C (bath). 1H NMR spectra
were recorded at 300 and 400 MHz on Bruker spectrometers. The
first order proton chemical shifts dH and dC are reported in d
(ppm) and referenced to either residual CHCl3 (dH 7.24, dC 77.0,
CDCl3) or residual CD2HOD (dH 3.30, dC 49.5, CD3OD). 1H and 13C
NMR spectra were assigned with the assistance of GCOSY, GHSQC.
Microanalyses and Electrospray Ionization (ESI) Mass Spectroscopy
were performed by the analytical services of the Department of
Chemistry, University of Calgary. For high resolution mass deter-
mination, spectra were obtained by voltage scan over a narrow
range at a resolution of approximately 10,000 and recorded by
the analytical services of the Department of Chemistry, University
of Alberta.
3.2.2. Dodecyl O-(5-acetamido-3,5-dideoxy-
galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-
dideoxy- -glycero- -galacto-non-2-ulopyranosylonic acid)-
D-glycero-a-D-
D
a-D
(2?8)-5-acetamido-3,5-dideoxy-D-glycero-a-D-galacto-non-
2-ulopyranosylonic acid (4)
1H NMR (CD3OD, 600 MHz): dY 4.29 (m, 1H, H-8 or H-80), 4.14–
4.24 (m, 2H, H-80 + H-9a0 or H-8 + H-9a), 4.09 (dd, 1H, J = 4.0,
12.1 Hz, H-9a or H-9a0), 3.83–3.98 (m, 4H, H-800 + H-7 + H-70 + H-
9a00), 3.54–3.84 (m, 13H, H-6 + H-60 + H-600 + H-5 + H-50 + H-
500 + H-4 + H-40 + H-400 + H-9b + H-9b0 + H-9b00 + OCHaHb),
3.47
(ddd, 1H, J = 6.9, 6.9, 9.2 Hz, OCHaHb), 3.43 (dd, 1H, J = 1.5,
9.1 Hz, H-700), 2.87 (dd, 1H, J = 4.5, 12.6 Hz, H-3eq000), 2.67–2.79
(m, 3H, H-3eq + H-3eq0 + H-3eq00), 2.08 (s, 3H, Ac), 2.05 (s, 3H,
Ac), 2.04 (s, 3H, Ac), 2.01 (s, 3H, Ac), 1.50–1.76 (m, 6H, H-
3ax + H-3ax0 + H-3ax00 + H-3ax000 + OCHaHbCH2), 1.23–1.40 (m,
18H, 9 ꢂ CH2 dodecyl), 0.90 (t, 3H, J = 6.8 Hz, CH3 dodecyl). 13C
NMR (CD3OD, 100 MHz, from HSQC): dC 78.80 (C-8 or C-80), 77.86
(C-80 or C-8), 74.09, 74.04, 73.06 (C-6 + H-60 + C-600), 71.82 (C-800),
70.32 (C-7 or C-70), 69.98 (C-70 or C-7), 68.99 (C-700), 68.39, 68.35,
68.09 (C-4 + C-40 + C-400), 64.02 (OCHaHb), 63.46 (C-900), 62.01
(C-9 or C-90, 61.67 (C-90 or C-9), 52.67, 52.59, 52.41 (C-5 +
C-50 + C-500), 41.13 (C-300), 40.69 (C-3 or C-30), 40.38 (C-30 or C-3),
22.16–31.81 (10 ꢂ CH2 dodecyl), 21.83 (ꢂ2) (2 ꢂ Ac), 21.19 (Ac),
3.2. Enzymatic reaction using a-sialoside (1) as a substrate
To a mixture of compound 1 (42 mg, 0.088 mmol) and crude
CMP-NeuNAc (ꢀ37%, 318 mg, 0.18 mmol, 2.0 equiv) was added a
solution of MnCl2 (0.5 M, 200 lL) and a solution of alkaline phos-
12.80
(CH3
dodecyl).
m/z
(ESI-HRMS)
calcd
for
phatase (20 lL). A solution of crude CST-II (0.3 U/mL, 10 mL) was
[C45H77N3O25ꢁ2H]2ꢁ 528.7350; found 528.7346.
added and the mixture was gently tumbled overnight at room
temperature. Another batch of CST-II (0.3 U/mL, 10 mL), crude
CMP-NeuNAc (37%, 318 mg, 0.18 mmol, 2.0 equiv) and of alkaline
3.2.3. Dodecyl O-(5-acetamido-3,5-dideoxy-
D
-glycero-a-D-
galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-
dideoxy- -glycero- -galacto-non-2-ulopyranosylonic acid)-
(2?8)-(5-acetamido-3,5-dideoxy- -glycero- -galacto-non-
2-ulopyranosylonic acid)-(2?8)-5-acetamido-3,5-dideoxy-
glycero- -galacto-non-2-ulopyranosylonic acid (5)
phosphatase (20 lL) was added, and the reaction was continued
D
a-D
for another 24 h. The reaction mixture was diluted with EtOH
(50 mL) and concentrated under reduced vacuum. The residue
was suspended in H2O (5 mL), and the solution was recovered
by centrifugation. The solid was extracted two more times
(2 ꢂ 5 mL) as above, and the aqueous solutions were combined
and concentrated (ꢀ5 mL). The mixture was purified on Sephadex
G-25 using H2O as eluent. The fractions containing oligomers were
combined and concentrated. The residue was purified by HPLC on a
D
a-D
D-
a-D
1H NMR (CD3OD, 600 MHz): dY 4.29 (m, 1H, H-8 or H-80 or H-
800), 4.21–4.25 (m, 2H, H-8 or H-80 or H-800), 4.18 (dd, 1H, J = 3.7,
12.1 Hz, H-9a or H-9a0 or H-9a00), 4.07–4.13 (m, 2H, H-9a or H-
9a0 or H-9a00), 3.96 (ddd, 1H, J = 2.5, 7.1, 8.8 Hz, H-8000), 3.84–
3.92 (m, 4H, H-7 + H-70 + H-700 + H-9a000), 3.54 -3.82 (m, 17H, H-
6 + H-60 + H-600 + H-6000 + H-5 + H-50 + H-500 + H-5000 + H-9b + H-9b0 +
H-9b00 + H-9b000 + H-4 + H-40 + H-400 + H-4000 + OCHaHb), 3.46 (ddd,
1H, J = 6.9, 6.9, 9.1 Hz, OCHaHb), 3.41 (dd, 1H, J = 2.0, 9.0 Hz, H-
7000), 2.68–2.90 (m, 4H, H-3eq + H-3eq0 + H-3eq00 + H-3eq000), 2.08,
2.05, 2.04 2.01 (4 ꢂ s, 4 ꢂ 3H, 4 ꢂ NHAc), 1.47–1.76 (m, 6H, H-
3ax + H-3ax0 + H-3ax00 + H-3ax000 + OCHaHbCH2), 1.23 -1.40 (m,
18H, 9 ꢂ CH2 dodecyl), 0.90 (t, 3H, J = 6.8 Hz, CH3 dodecyl). 13C
NMR (CD3OD, 100 MHz, from HSQC): dC 80.11, 79.81, 79.45
(C-8 + C-80 + C-800), 76.10, 75.94, 75.50 (C-6 + C-60 + C-600), 74.69
(C-6000), 73.62 (C-8000), 71.73 (ꢂ2), 71.41 (C-7 + C-70 + C-700), 70.77
(C-7000), 69.81–70.09 (4C, C-4 + C-40 + C-400 + C-4000), 65.56 (OCH2),
65.12 (C-9000), 63.56, 63.47, 63.32 (C-9 + C-90 + C-900), 54.01–54.31
(C-5 + C-50 + C-500 + C-5000), 42.60 (C-3000), 42.31, 42.02, 41.77
(C-3 + C-30 + C-300), 33.10 (CH2 dodecyl), 30.81–31.13 (7 ꢂ CH2
dodecyl), 27.25 (CH2 dodecyl), 23.73 (CH2 dodecyl), 23.47 (Ac),
23.20 (ꢂ2, Ac ꢂ 2), 22.62 (Ac), 14.48 (CH3 dodecyl). m/z (ESI-
HRMS) calcd for [C56H94N4O33ꢁ3H]3ꢁ 449.1861; found:
449.1858.
reverse phase C18 column using
(0?100%) as eluent to afford sequentially
9 (3.8 mg, 1.7% yield), -(2?8)-heptasialoside 8 (2.9 mg, 1.5%
yield), -(2?8)-hexasialoside 7 (6.7 mg, 3.9% yield), -(2?8)-pent-
asialoside 6 (5.0 mg, 3.5% yield), -(2?8)-tetrasialoside 5 (8.2 mg,
6.9% yield), and -(2?8)-trisialoside 4 (6.6 mg, 7.1%), and
a
gradient of MeOH–H2O
a-(2?8)-octasialoside
a
a
a
a
a
a-
(2?8)-disialoside 3 (6.5 mg, 9.6% yield).
3.2.1. Dodecyl O-(5-acetamido-3,5-dideoxy-D-glycero-a-D-
galacto-non-2-ulopyranosylonic acid)-(2?8)-5-acetamido-3,5-
dideoxy- -glycero- -galacto-non-2-ulopyranosylonic acid (3)
D
a-D
1H NMR (400 MHz, CD3OD): dH 4.31 (m, 1H, H-8), 4.15 (dd, 1H,
J = 11.7, 3.8 Hz, H-9a), 3.94 (ddd, 1H, J = 3.9, 6.4, 9.1 Hz, H-80), 3.76–
3.90 (m, 4H, H-7 + H-9a0 + H-6 + H-5), 3.52–3.76 (m, 4H,
OCHaHb + H-50 + H-40 + H-9b), 3.51–3.62 (m, 3H, H-9b0 + H-4 + H-
60), 3.47 (m, 1H, OCHaHb), 3.44(dd, 1H, J = 1.8, 9.0 Hz, H-70), 2.84
(high order dd, 1H, J = 4.0, 12.3 Hz, H-3eq0), 2.72 (dd, 1H, J = 4.4,
12.1 Hz, H-3eq), 2.04 (s, 3H, Ac), 2.01 (s, 3H, Ac), 1.72 (high order
dd, 1H, J = 11.9, 11.9 Hz, H-3ax0), 1.57 (dd, 1H, J = 12.1, 12.1 Hz,

1698
W. Li et al. / Carbohydrate Research 346 (2011) 1692–1704
3.2.4. Dodecyl O-(5-acetamido-3,5-dideoxy-
D
-glycero-
a
-
D
-
m/z (ESI-HRMS) calcd for [C78H128N6O49ꢁ5H]5ꢁ 385.5469; found:
galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-
dideoxy- -glycero- -galacto-non-2-ulopyranosylonic acid)-
(2?8)-(5-acetamido-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-dideoxy-
glycero- -galacto-non-2-ulopyranosylonic acid)-(2?8)-5-
acetamido-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosylonic acid (6)
385.5467.
D
a-D
D
a
-
D
3.2.6. Dodecyl O-(5-acetamido-3,5-dideoxy-
galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-
dideoxy- -glycero- -galacto-non-2-ulopyranosylonic acid)-
(2?8)-(5-acetamido-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-dideoxy-
glycero-
acetamido-3,5-dideoxy-
ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-dideoxy-
glycero-
acetamido-3,5-dideoxy-
ulopyranosylonic acid (8)
1H NMR (CD3OD, 600 MHz): dY 3.99–4.32 (m, 12H, H-8 + H-
80 + H-800 + H-8000 + H-80000 + H-800000 + H-9a + H-9a0 +H-9a00 + H-9a000
H-9a0000 + H-9a00000), 3.95 (m, 1H, H-8000000), 3.54–3.93 (m, 37H, H-
7 + H-70 + H-700 + H-7000+ H-70000 + H-700000 + H-7000000 + H-9a000000 + H-
6 + H-60 + H-600 + H-6000 + H-60000 + H-600000 + H-6000000 + H-5 + H-50 + H-
500 + H-5000 + H-50000 H-500000 + H-5000000 + H-9b + H-9b0 + H-9b00 + H-
D-glycero-a-D-
D
-
a-
D
D
a-D
D
a
-D
D
a-D
D
D
-
-
1H NMR (CD3OD, 600 MHz): dY 4.06–4.30 (m, 8H, H-8 + H-
80 + H-800 + H-8000 + H-9a + H-9a0 +H-9a00 + H-9a000), 3.93 (ddd, 1H,
J = 2.4, 7.5, 9.0 Hz, H-80000), 3.83–3.91 (m, 7H, H-7 + H-70 +
H-700 + H-7000 + H-9a0000), 3.55–3.81 (m, 21H, H-6 + H-60 + H-600 +
H-6000 + H-60000 + H-5 + H-50 + H-500 + H-5000 + H-50000 + H-9b + H-
a-
D
-galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-
D-glycero-a-D-galacto-non-2-
a-
D-galacto-non-2-ulopyranosylonic acid)-(2?8)-5-
D
-glycero- -galacto-non-2-
a-D
9b0 + H-9b00 + H-9b000 + H-9b0000 + H-4 + H-40 + H-400 + H-4000 + H-40000
+
OCHaHb), 3.45 (m, 1H, OCHaHb), 3.42 (dd, 1H, J = 1.8, 9.3 Hz, H-7000),
2.86 (dd, 1H, J = 4.0, 12.5 Hz, H-3eq0000), 2.66–2.75 (m, 4H, H-
3eq + H-3eq0 + H-3eq00 + H-3eq000), 2.07 (s, 3H, Ac), 2.05 (s, 3H, Ac),
2.04 (s, 3H, Ac), 2.03 (s, 3H, Ac), 2.01 (s, 3H, Ac), 1.50–1.74 (m,
+
7H,
H-3ax + H-3ax0 + H-3ax00 + H-3ax000 + H-3ax0000 + OCHaHbCH2),
1.22–1.40 (m, 18H, 9 ꢂ CH2 dodecyl), 0.89 (t, 3H, J = 6.8 Hz, CH3
dodecyl). 13C NMR (CD3OD, 150 MHz, from HSQC): dC 80.09,
79.74 (ꢂ2), 79.25 (C-8 + C-80 + C-800 + C-8000), 76.01 (ꢂ2), 75.58
(ꢂ2), 74.69 (C-6 + C-60 + C-600 + C-6000), 73.84 (C-80000), 71.79, 71.60
(ꢂ3) (C-7 + C-70 + C-700 + C-7000), 70.61 (C-70000), 69.66–69.96 (5C, C-
4 + C-40 + C-400 + C-4000 + C-40000), 65.51 (OCHaHb), 65.05 (C-90000),
62.98–63.43 (4C, C-9 + C-90 + C-900 + C-9000), 53.95–54.29 (5C,
C-5 + C-50 + C-500 + C-5000 + C-50000), 42.65 (C-30000), 41.78–42.31 (4C,
C-3 + C-30 + C-300 + C-3000), 30.82–31.13 (8 ꢂ CH2 dodecyl), 27.39
(CH2 dodecyl), 23.80 (CH2 dodecyl), 23.50 (Ac), 23.30 (Ac), 23.23
(Ac), 23.07 (Ac), 22.71 (Ac), 14.53 (CH3 dodecyl). m/z (ESI-HRMS)
calcd for [C67H111N5O41ꢁ3H]3ꢁ 546.2168; found: 546.2165.
+
9b000 + H-9b0000 + H-9b00000 + H-9b000000 + H-4 + H-40 + H-400 + H-4000 + H-
40000 + H-400000 + H-4000000 + OCHaHb), 3.47 (m, 1H, OCHaHb), 3.42 (over-
lapped, 1H, H-7000000), 2.88 (dd, 1H, J = 4.6, 12.4 Hz, H-3eq000000), 2.66–
2.79 (m, 6H, H-3eq + H-3eq0 + H-3eq00 + H-3eq000 + H-3eq0000 + H-
3eq00000), 2.07 (s, 3H, Ac), 2.06 (s, 3H, Ac), 2.05 (s, 3H, Ac), 2.04 (s,
6H, 2 ꢂ Ac), 2.03 (s, 3H, Ac), 2.01 (s, 3H, Ac), 1.76 (dd, 1H,
J = 11.9, 11.9 Hz, H-3ax000000), 1.51–1.73 (m, 8H, H-3ax + H-3ax0 + H-
3ax00 + H-3ax000 + H-3ax0000+ H-3ax00000 + OCHaHbCH2), 1.24 -1.39 (m,
18H, 9 ꢂ CH2 dodecyl), 0.89 (t, 3H, J = 6.8 Hz, CH3 dodecyl). 13C
NMR (CD3OD, 150 MHz, from HSQC): dC 78.43–79.89 (6C, C-8 + C-
80 + C-800 + C-8000 + C-80000 + C-800000), 75.48–75.97 (6C, C-6 + C-60 + C-
600 + C-6000 + C-60000 + C-600000), 74.89 (C-6000000), 73.78 (C-8000000), 71.59
(6C, C-7 + C-70 + C-700 + C-7000 + C-70000 + C-700000), 70.61 (C-7000000), 69.84
3.2.5. Dodecyl O-(5-acetamido-3,5-dideoxy-
galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-
dideoxy- -glycero- -galacto-non-2-ulopyranosylonic acid)-
(2?8)-(5-acetamido-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-dideoxy-
glycero- -galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-
acetamido-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosylonic acid)-(2?8)-5-acetamido-3,5-dideoxy-
D-glycero-a-D-
(7C,
(OCHaHb), 65.02 (C-9000000), 62.78–63.55 (6C, C-9 + C-90 + C-900 +
C-9000 + C-90000 + C-900000), C-5 + C-50 + C-500 +
54.14–54.30 (7C,
C-4 + C-40 + C-400 + C-4000 + C-40000 + C-400000 + C-4000000),
65.52
D
a-D
D
a-D
D
-
C-5000 + C-50000 + C-500000 + C-5000000), 42.82 (C-3000000), 41.75–42.34 (6C, C-
3 + C-30 + C-300 + C-3000 + C-30000 + C-300000), 33.21 (CH2 dodecyl),
30.74–31.24 (7 ꢂ CH2 dodecyl), 27.29 (CH2 dodecyl), 23.80 (CH2
dodecyl), 23.53 (Ac), 23.36 (2C, 2 ꢂAc), 23.26 (3C, 3 ꢂ Ac), 22.80
(Ac), 14.53 (CH3 dodecyl). m/z (ESI-HRMS) calcd for [C89H145N7-
O57ꢁ4H]4ꢁ 554.9582; found: 554.9583.
a-D
D
a-D
D
-
glycero-a-D-galacto-non-2-ulopyranosylonic acid (7)
1H NMR (CD3OD, 600 MHz): dY 4.02–4.31 (m, 10H, H-8 + H-
80 + H-800 + H-8000 + H-80000 + H-9a + H-9a0 +H-9a00 + H-9a000 + H-9a0000),
3.96 (ddd, 1H, J = 2.4, 7.1, 9.0 Hz, H-800000), 3.54–3.95 (m, 32H,
H-7 + H-70 + H-700 + H-7000+ H-70000 + H-700000+ H-9a00000 + H-6 + H-60 + H-
3.2.7. Dodecyl O-(5-acetamido-3,5-dideoxy-
galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-
dideoxy- -glycero- -galacto-non-2-ulopyranosylonic acid)-
(2?8)-(5-acetamido-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-dideoxy-
glycero-
acetamido-3,5-dideoxy-
ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-dideoxy-
glycero-
acetamido-3,5-dideoxy-
ulopyranosylonic acid)-(2?8)-5-acetamido-3,5-dideoxy-
glycero- -galacto-non-2-ulopyranosylonic acid (9)
D-glycero-a-D-
600 + H-6000 + H-60000 + H-600000 + H-5 + H-50 + H-500 + H-5000 + H-50000
+
D
a-D
H-500000 + H-9b + H-9b0 + H-9b00 + H-9b000 + H-9b0000 + H-9b00000 + H-4 + H-
40 + H-400 + H-4000 + H-40000 + H-400000 + OCHaHb), 3.46 (ddd, 1H, J = 6.9,
6.9, 9.4 Hz, OCHaHb), 3.40 (dd, 1H, J = 1.8, 9.0 Hz, H-700000), 2.89
(dd, 1H, J = 4.6, 12.2 Hz, H-3eq00000), 2.66–2.77 (m, 5H, H-3eq + H-
3eq0 + H-3eq00 + H-3eq000 + H-3eq0000), 2.08 (s, 3H, Ac), 2.06 (s, 3H,
Ac), 2.05 (s, 3H, Ac), 2.03 (s, 6H, 2 ꢂ Ac), 2.01 (s, 3H, Ac), 1.76
(dd, 1H, J = 11.9, 11.9 Hz, H-3ax00000), 1.51–1.72 (m, 7H, H-
D
a-D
D
D
-
-
a-
D
-galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-
D-glycero-a-D-galacto-non-2-
a-
D-galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-
D
-glycero- -galacto-non-2-
a-D
3ax + H-3ax0 + H-3ax00 + H-3ax000 + H-3ax0000 + OCHaHbCH2),
1.23–
D-
1.37 (m, 18H, 9 ꢂ CH2 dodecyl), 0.89 (t, 3H, J = 6.8 Hz, CH3 dode-
cyl). 13C NMR (CD3OD, 150 MHz, from HSQC): dC 80.11, 79.85,
79.75, 79.23, 78.62 (C-8 + C-80 + C-800 + C-8000 + C-80000), 75.64–
75.88 (5C, C-6 + C-60 + C-600 + C-6000 + C-60000), 74.80 (C-600000), 73.72
(C-800000), 71.64 (5C, C-7 + C-70 + C-700 + C-7000 + C-70000), 70.68 (C-
700000), 69.89 (6C, C-4 + C-40 + C-400 + C-4000 + C-40000 + C-400000), 65.52
(OCHaHb), 65.09 (C-900000), 62.85–63.58 (5C, C-9 + C-90 + C-900 + C-
9000 + C-90000), 54.10–54.55 (6C, C-5 + C-50 + C-500 + C-5000 + C-50000 + C-
500000), 42.86 (C-300000), 41.59–42.31 (5C, C-3 + C-30 + C-300 + C-
3000 + C-30000), 33.07 (CH2 dodecyl), 30.75–31.24 (7 ꢂ CH2 dodecyl),
27.22 (CH2 dodecyl), 23.77 (CH2 dodecyl), 23.53 (Ac), 23.31
(Ac), 23.20–23.22 (4C, 4 ꢂ Ac), 22.74 (Ac), 14.46 (CH3 dodecyl).
a-D
1H NMR (CD3OD, 400 MHz): dY 3.97–4.35 (m, 14H,
H-8 + H-80 + H-800 + H-8000 + H-80000 + H-800000 + H-8000000 + H-9a + H-9a0+H-
9a00 +H-9a000 + H-9a0000 + H-9a00000 + H-9a000000), 3.53–3.95 (m, 42H, H-7 +
H-70 + H-700 + H-7000+H-70000 + H-700000 + H-7000000 + H-70000000 + H-9a0000000 + H-
6 + H-60 + H-600 + H-6000 + H-60000 + H-600000 + H-6000000 + H-60000000 + H-5 + H-
50 + H-500 + H-5000 + H-50000
+
H-500000 + H-5000000 + H-50000000
+
H-9b + H-
9b0 + H-9b00 + H-9b000 + H-9b0000 + H-9b00000 + H-9b000000 + H-9b0000000 + H-
4 + H-40 + H-400 + H-4000 + H-40000 + H-400000 + H-4000000 + H-40000000 + OCHaHb),
3.48 (m, 1H, OCHaHb), 3.41 (overlapped, 1H, H-7000000), 2.90 (br dd, 1H,
H-3eq000000), 2.65–2.79 (m, 7H, H-3eq + H-3eq0 + H-3eq00 + H-
3eq000 + H-3eq0000 + H-3eq00000 + H-3eq000000), 2.07 (s, 3H, Ac), 2.065 (s,

W. Li et al. / Carbohydrate Research 346 (2011) 1692–1704
1699
3H, Ac), 2.05 (s, 3H, Ac), 2.046 (s, 3H, Ac), 2.04 (s, 3H, Ac), 2.035 (s, 3H,
Ac), 2.03 (s, 3H, Ac), 2.01 (s, 3H, Ac), 1.78 (br dd, 1H, H-3ax0000000), 1.50–
21.83 (ꢂ2, Ac ꢂ 2), 21.10 (Ac), 13.14 (CH3 dodecyl). m/z (ESI-
HRMS) calcd for [C45H77N3O24SꢁH]ꢁ 1074.4548; found:
1074.4548.
1.76 (m, 9H, H-3ax + H-3ax0 + H-3ax00 + H-3ax000 + H-3ax0000
+ H-
3ax00000+ H-3ax000000+ OCHaHbCH2), 1.23–1.38 (m, 18H, 9 ꢂ CH2 dode-
cyl), 0.89 (t, 3H, J = 6.8 Hz, CH3 dodecyl). m/z (ESI-HRMS) calcd for
[C100H162N8O65ꢁ6H]6ꢁ 418.1530; found: 418.1527.
3.3.3. Dodecyl O-(5-acetamido-3,5-dideoxy-
galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-
dideoxy- -glycero- -galacto-non-2-ulopyranosylonic acid)-
(2?8)-(5-acetamido-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosylonic acid)-(2?8)-5-acetamido-3,5-dideoxy-2-thio-
-glycero- -galacto-non-2-ulopyranosylonic acid (12)
1H NMR (CD3OD, 400 MHz): dY 4.14–4.41 (m, 6H, H-8 + H-
D-glycero-a-D-
D
a-D
3.3. Enzymatic reaction using
a-thiosialoside (2) as a substrate
D
a-D
To a mixture of thioglycoside 2 (30 mg, 0.061 mmol) and crude
CMP-NeuNAc (37%, 300 mg, ꢀ0.17 mmol, 2.8 equiv) was added a
D
a-D
solution of MnCl2 (0.5 M, 172
lL) and a solution of alkaline phos-
80 + H-800 + H-9a + H-9a0 + H-9a00), 3.84–4.00 (m, 5H, H-7 +
H-70 + H-700 + H-8000 + H-9a000), 3.54–3.84 (m, 16 H, H-5 + H-50 + H-
500 + H-5000 + H-6 + H-60 + H-600 + H-4 + H-40 + H-400 + H-4000 + H-9b +
H-9b0 + H-9b00 + H-9b000 + H-7000), 3.47 (dd, 1H, J = ꢀ1.0, 9.2 Hz, H-
phatase (30 lL). A solution of crude CST-II (0.3 U/mL, 10.5 U) was
added and the mixture was gently tumbled for 24 h. The reaction
mixture was worked up in a similar manner as 1, and the mixture
was purified by HPLC on a reverse phase C18 column using a gra-
dient of MeOH–H2O (0?100%) as eluent to afford tetrasialoside 12
(4.1 mg, 4.9% yield), trisialoside 11 (4.7 mg, 7.2%), and disialoside
10 (5.9 mg, 12.4% yield).
7000),
2.68–2.96
(m,
6H,
H-3eq + H-3eq0 + H-3eq00 + H-
3eq000 + SCHaHb), 2.09 (s, 3H, NHAc), 2.07 (s, 3H, NHAc), 2.05 (s,
3H, NHAc), 2.01 (s, 3H, NHAc), 1.50–1.83 (m, 6H, H-3ax + H-
3ax0 + H-3ax00 + H-3ax000 + SCHaHbCH2), 1.22–1.43 (m, 18H, 9 ꢂ CH2
dodecyl), 0.90 (t, 3H, J = 6.8 Hz, CH3 dodecyl). 13C NMR (CD3OD,
100 MHz, from HSQC): dC 77.80 (ꢂ2), 77.57 (C-8 or C-80 or C-800),
75.50, 73.73 (ꢂ2) (C-6 or C-60 or C-600), 73.14 (C-6000), 71.49 (C-
8000), 69.78, 69.48, 68.97 (C-7 or C-70 or C-700), 68.95 (C-7000), 68.89,
68.72, 68.60, 68.30 (C-4 or C-40 or C-400 or C-4000), 62.99 (C-9000),
62.28, 61.52, 61.40 (C-9 or C-90 or C-900), 52.67, 52.55, 52.49,
52.43 (C-5 or C-50 or C-500 or C-5000), 41.28, 41.15, 40.69 (2ꢂ) (C-3
or C-30 or C-300 or C-3000), 22.16–28.94 (11 ꢂ CH2 dodecyl), 22.10
(Ac), 21.79 (Ac), 21.76 (Ac), 21.06 (Ac), 13.08 (CH3 dodecyl). m/z
(ESI-HRMS) calcd for [C56H94N4O32Sꢁ2H]2ꢁ 682.2713; found:
682.2710.
3.3.1. Dodecyl O-(5-acetamido-3,5-dideoxy-D-glycero-a-D-
galacto-non-2-ulopyranosylonic acid)-(2?8)-5-acetamido-3,5-
dideoxy-2-thio-
acid (10)
D-glycero-a-D-galacto-non-2-ulopyranosylonic
1H NMR (CD3OD, 400 MHz): dY 4.30 (m, 1H, H-8), 4.16 (dd, 1H,
J = 4.4, 11.7 Hz, H-9a), 3.95 (ddd, 1H, J = 2.4, 6.0, 8.8 Hz, H-80), 3.88
(dd, 1H, J = 1.6, 9.2 Hz, H-7), 3.86 (dd, 1H, J = 2.4, 11.2 Hz, H-9a0),
3.82 (dd, 1H, J = 10.0, 10.0 Hz, H-5), 3.65–3.75 (m, 4H, H-6 + H-
40 + H-50 + H-9b), 3.51–3.63 (m, 3H, H-4 + H-9b0 + H-60), 3.45 (dd,
1H, J = 1.6, 9.2 Hz, H-70), 2.70–2.87 (m, 4H, H-3eq + H-
3eq0 + SCHaCHb + SCHaHb), 2.04 (s, 3H, Ac), 2.01 (s, 3H, Ac), 1.73
(high order dd, 1H, J = 11.0, 11.0 Hz, H-3ax0), 1.65 (dd, 1H,
J = 11.6, 11.6 Hz, H-3ax), 1.52–1.60 (m, 2H, 1 ꢂ CH2 dodecyl),
1.24–1.43 (m, 18H, 9 ꢂ CH2 dodecyl), 0.90 (t, 3H, J = 6.8 Hz, CH3
dodecyl). 13C NMR (From HSQC experiment, CD3OD, 100 MHz): dC
79.25 (C-8), 77.09 (C-6), 74.33 (C-60), 72.64 (C-80), 71.71 (C-7),
70.41 (C-70), 70.38 (C-4), 69.67 (C-40), 64.64 (C-90), 63.57 (C-9),
53.89 (C-5), 53.73 (C-50), 43.08 (C-3), 41.74 (C-30), 30.18 (CH2 dode-
cyl)), 29.88 (SCH2), 28.88–31.66 (9 ꢂ CH2 dodecyl), 23.55 (CH2
dodecyl), 23.26 (Ac), 22.72 (Ac), 14.35 (CH3 dodecyl). m/z (ESI-
HRMS) calcd for [C34H60N2O16S–H]ꢁ 783.3580; found: 783.3573.
3.4. 4-Chlorophenyl O-(5-acetamido-3,5-dideoxy-
galacto-non-2-ulopyranosylonic acid)–(2?8)-(5-acetamido-
3,5-dideoxy-2-thio- -glycero- -galacto-non-2-
D-glycero-a-D-
D
a-D
ulopyranosylonic acid)-(2?6)-S-1,6-dithio-b-D-
galactopyranoside (14)
To a mixture containing compound 13 (10 mg, 0.016 mmol) and
crude CMP-NeuNAc (64 mg, 37%, ꢀ0.29 mmol) was added an aque-
ous solution of MnCl2 (0.5 M, 63 lL), a solution of alkaline phos-
phate (94 lL), and a solution of the crude CST-II (6.5 mL). The
mixture was gently tumbled for three days at room temperature.
After filtration, the mixture was concentrated. The residue was
purified by gel filtration chromatography on a LH-20 column using
MeOH as the eluent to afford the desired trisaccharide 14 (5.0 mg,
47.8% yield). A small amount of starting material 13 (3.0 mg) was
3.3.2. Dodecyl O-(5-acetamido-3,5-dideoxy-
galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-
dideoxy- -glycero- -galacto-non-2-ulopyranosylonic acid)-
(2?8)-5-acetamido-3,5-dideoxy-2-thio-D-glycero-a-D-galacto-
non-2-ulopyranosylonic acid (11)
D-glycero-a-D-
D
a-D
recovered. Rf: 0.25 (isopropanol–H2O–NH3ꢃH2O 7:1:1); ½a D25
ꢄ
+10.5
1H NMR (CD3OD, 400 MHz): dY 4.35 (m, 1H, H-8 or H-80), 4.10–
4.24 (m, 3H, H-80 or H-8 + H-9a + H-9a0), 3.96–3.84 (m, 4H, H-
800 + H-7 + H-70 + H-9a00), 3.55–3.84 (m, 12H, H-5 + H-50 + H-
500 + H-6 + H-60 + H-600 + H-9b +H-9b0 + H-9b00 + H-4 + H-40 + H-400),
3.47 (dd, 1H, J = ꢀ1.0, 9.2 Hz, H-700), 2.64–2.93 (m, 5H + H-
3eq + H-30eq + H-300eq + SCHaHb + SCHaHb), 2.04 (s, 6H, 2 ꢂ Ac),
2.01 (s, 3H, Ac), 1.61–1.82 (m, 3H, H-3ax + H-3ax0 + H-3ax00),
1.50–1.61 (m, 2H, SCHaHbCH2), 1.14–1.43 (m, 18H, 9 ꢂ CH2 dode-
cyl), 0.90 (t, 3H, J = 6.8 Hz, CH3 dodecyl). 13C NMR (CD3OD,
100 MHz, from HSQC): dC 77.95 (C-8 or C-80), 77.47 (C-80 or C-8),
75.60 (C-6, or C-60), 73.87 (C-60 or C-6), 72.94 (C-600), 71.61 (C-
800), 70.13 (C-7 or C-70), 69.43 (C-70 or C-7), 68.82 (C-700), 68.78
(C-400), 68.19 (ꢂ2, C-4 + C-40), 63.29 (C-900), 61.92 (C-9 or C-90),
61.59 (C-90 or C-9), 52.63 (ꢂ2, C-5 or C-50 or C-500), 52.54 (C-5 or
C-50 or C-500), 41.56 (C-3 or C-30 or C-300), 41.08 (C-3 or C-30 or C-
300), 40.07 (C-3 or C-30 or C-300), 22.26–31.77 (11 ꢂ CH2 dodecyl),
(c 0.2, MeOH); 1H NMR (D2O, 400 MHz): dY 7.45 (d, 2H, J = 8.4 Hz,
PhCl), 7.35 (d, 2H, J = 8.4 Hz, PhCl), 4.70 (d, overlapped with HDO,
1H, H-1), 4.03–4.12 (m, 2H, H-80 + H-9a0), 3.92 (dd, 1H, J = 3.2,
ꢀ1.0 Hz, H-4), 3.64–3.86 (m, 6H, H-8a00 + H-9a00 + H-50 + H-500 + H-
70 + H-5), 3.44–3.64 (m, 9H, H-60 + H-600 + H-9b0 + H-9b00 + H-
700 + H-40 + H-400 + H-3 + H-2), 2.91 (dd, 1H, J = 13.4, 8.9 Hz, H-6a),
2.83 (dd, 1H, J = 4.5, 13.7 Hz, H-6b), 2.64–2.70 (m, 2H, H-
3eq0 + H-3eq00), 1.98 (s, 3H, NHAc), 1.95 (s, 3H, NHAc), 1.65 (dd,
1H, J = 12.4, 12.4 Hz, H-3ax0 or H-3ax00), 1.62 (dd, 1H, J = 12.4,
13.7 Hz, H-3ax00 or H-3ax0); 13C NMR (D2O, 100 MHz, from HSQC):
dC 131.93, 128.87, 87.40 (C-1), 78.49 (C-80), 77.76 (C-5), 75.24,
73.56, 72.43, 71.37, 69.69, 69.13, 68.34, 68.29, 62.51 (C-900), 62.12
(C-90), 52.09 (C-50 or C-500), 51.47 (C-500 or C-50), 40.44 (C-30 or C-
300), 40.27(C-300 or C-30), 29.40 (C-6), 21.99 (ꢂ2, NHAc). m/z (ESI-
HRMS) calcd for [C34H49N2O20S2Clꢁ2H]2ꢁ 451.0932; found:
451.0931.

1700
W. Li et al. / Carbohydrate Research 346 (2011) 1692–1704
3.5. Methyl (11-azidoundecyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosid)onate (20) and Methyl (11-azidoundecyl 5-
for 30 min and concentrated under vacuum. The residue was dis-
solved in a 1:1 H2O–MeOH mixture (10 mL) and the saponifica-
tion was continued at rt overnight. The pH of the solution was
neutralized with AC2O and the solvent was removed under re-
duced pressure. The residue was finally purified by silica gel col-
umn chromatography using a mixture of CH2Cl2–MeOH–H2O
(80:20:2) as eluent to afford the desired 15 as a white solid after
lyophilization (230 mg, 64% yield). Rf: 0.2 (CH2Cl2–MeOH–H2O
D
a-D
acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-b-D-
galacto-non-2-ulopyranosid)onate (21)
Thioglycoside 18 (870 mg, 1.41 mmol) and 11-azido-1-undeca-
nol 19 (600 mg, 4.7 mmol) were dissolved in a mixture of anhy-
drous CH2Cl2 (5.0 mL) and CH3CN (2.0 mL) under an
atomosphere of argon; 4 Å molecular sieves (300 mg) were added
and the mixture was stirred at room temperature for 1.5 h. The
temperature was cooled to ꢁ50 °C and N-iodosuccinimide
(633 mg, 2.8 mmol) was added. After stirring for 10 min, trifluoro-
70:30:3). [a]
+17.45 (c 1.05, MeOH). 1H NMR (D2O, 400 MHz):
D
dH 3.93–3.60 (m, 8H, H-8, H-9a, H-5, H-4, H-9b, OCHaHb, H-7,
H-6), 3.46 (dd, 1H, J = 6.8, 6.8, 9.0 Hz, OCHaHb), 3.30 (t, 2H,
J = 6.9 Hz, CH2N3), 2.76 (dd, 1H, J = 12.2, 4.4 Hz, H-3eq), 2.05 (s,
3H, Ac), 1.72–1.50 (m, 5H, H-3ax, OCH2CH2, CH2CH2N3), 1.44–
1.23 (m, 14H, 7 ꢂ CH2 chain). 13C NMR (D2O, 100 MHz): dC
178.07, 174.99 (CO), 100.64 (C-2), 72.53 (C-6), 71.55 (C-8), 68.40
(C-7), 67.88(C-4), 64.87 (OCHaHb), 62.19 (C-9), 51.99 (C-5),
51.26 (CH2N3), 40.51 (C-3), 29.41, 29.23 (ꢂ2), 29.09, 28.87,
28.47, 26.44 (ꢂ2), 25.58 (9 ꢂ CH2 chain), 22.07 (Ac). HRMS m/z
calcd for C22H39N4O9 (M-H)ꢁ: 503.2723; found: 503.2719.
methanesulfonic acid (40 lL) was added dropwise and the reac-
tion was slowly warmed to rt for another 2 h. TLC showed that
all the starting material had been consumed. Et3N (1.0 mL) was
added and mixture was diluted with EtOAc (20 mL). After filtra-
tion, the organic solution was washed with a 1:1 mixture of 10%
Na2S2O3 and satd NaHCO3, dried, and evaporated. The residue
was purified by column chromatography on silica gel using a mix-
ture of acetone–CH2Cl2 (1:5) as eluent to afford sequentially the
pure b-sialoside (21, 94.8 mg, yield: 12%) and the desired pure
3.7. (Methyl S-5-acetamido-3,5-dideoxy-2-thio-D-glycero-a-D-
galacto-non-2-ulopyranosyl)onic acid (16)
a-sialoside (20, 506 mg, yield 64%).
Data for 20: Rf: 0.45 (acetone–CH2Cl2 1:4). [
a
]
ꢁ3.77 (c 3.9,
Method A: To a solution of compound 22 (1.0 g, 1.8 mmol) in dry
MeOH (20 mL), was added t-BuOK (0.19 mg, 1.8 mmol) at ꢁ40 °C.
The reaction mixture was stirred for 5 min, and concentrated un-
der vacuum. The residue was dissolved in dry DMF (15 mL), and
MeI (0.38 mL, 5.4 mmol) was added. After stirring for 6 h at rt,
the mixture was concentrated under high vacuum to give a syrup.
Dry MeOH (10 mL) was added, and a solution of MeONa in MeOH
(1.5 M, 0.5 mL) was added. After stirring for 1 h, the solvent was re-
moved under reduced pressure to give crude 23, which was redis-
solved in the mixture of MeOH and water (1:1, 10 mL). After
stirring for 2 h, the solution was concentrated under reduced pres-
sure. The residue was purified by chromatography on silica gel
using CH2Cl2–MeOH–H2O (80:20:2) as eluent to afford target prod-
uct 16 as a white solid after lyophilization (0.21 g, 35% yield over
four steps).
D
CHCl3). 1H NMR (CDCl3, 400 MHz): dH 5.38 (ddd, 1H, J = 8.1, 5.5,
2.6 Hz, H-8), 5.32 (dd, 1H, J = 8.2, 1.8 Hz, H-7), 5.28 (d, 1H,
J = 9.6 Hz, NHAc), 4.86–4.78 (m, 1H, H-4), 4.30 (dd, 1H, J = 12.4,
2.6 Hz, H-9a), 4.13–4.01 (m, 3H, H-9b, H-5, H-6), 3.78 (s, 3H,
OMe), 3.73 (ddd, 1H, J = 9.3, 6.4, 6.4 Hz, OCHaHb), 3.24 (t, 2H,
J = 7.0 Hz, CH2N3), 3.19 (ddd, 1H, J = 9.3, 6.6 Hz, OCHaHb), 2.57
(dd, 1H, J = 12.8, 4.6 Hz, H-3eq), 2.13 (s, 3H, Ac), 2.12 (s, 3H, Ac),
2.03 (s, 3H,Ac), 2.01 (s, 3H, Ac), 1.93 (dd, 1H, J = 12.6, 12.6 Hz,
H-3ax), 1.87 (s, 3H,Ac), 1.62–1.46 (m, 4H, OCH2CH2, CH2CH2N3),
1.39–1.21 (m, 14H, 7 ꢂ CH2 chain). 13C NMR (CDCl3, 100 MHz):
dC 171.02, 170.63, 170.20, 170.12, 170.05, 168.55 (6 ꢂ CO), 98.72
(C-2), 72.44 (C-6), 69.20 (C-4), 68.74 (C-8), 67.37 (C-7), 65.07
(OCHaHb), 62.34 (C-9), 52.60 (OMe), 51.46 (CH2N3), 49.40 (C-5),
38.10 (C-3), 29.56, 29.50, 29.46, 29.28, 29.13, 28.82, 26.70, 25.84
(9 ꢂ CH2 chain), 23.18, 21.10, 20.85, 20.82, 20.77 (5 ꢂ Ac). HRMS
Method B: To a mixture of MeI (0.114 mL, 1.82 mmol) and
compound 22 (0.500 g, 0.91 mmol) was added anhydrous DMF
(3.5 mL) under argon, and the reaction mixture was stirred until
the solid had dissolved. Et2NH (0.47 mL, 4.55 mmol) was added
dropwise and the reaction mixture was stirred at rt for 12 h.
The reaction was diluted with EtOAc (15 mL) and then washed
with H2O (3 ꢂ 10 mL) and brine (2 ꢂ 10 mL). The organic phase
was dried over anhydrous Na2SO4, filtered, and concentrated in
vacuo. The crude residue was purified by column chromatogra-
phy on silica gel using a gradient of EtOAc–toluene (20?30%)
as the eluent to afford 24 (320 mg, 67%). Rf: 0.28 (toluene–ace-
m/z calcd for
C
31H50N4O13Na (M+Na)+: 709.3267; found:
709.3266.
Data for 21: Rf: 0.48 (acetone–CH2Cl2 1:4), [a]
ꢁ5.98 (c 0.7,
D
CHCl3). 1H NMR (CDCl3, 400 MHz): dH 5.39 (dd, 1H, J = 3.8, 2.3 Hz,
H-7), 5.27 (d, 1H, J = 10.2 Hz, NHAc), 5.26 (ddd, 1H, J = 15.5, 9.7,
5.1 Hz, H-4), 5.18 (ddd, 1H, J = 7.7, 3.7, 2.5 Hz, H-8), 4.79 (dd, 1H,
J = 12.4, 2.5 Hz, H-9a), 4.17–4.06 (m, 2H, H-9b, H-5), 3.92 (dd, 1H,
J = 10.5, 2.3 Hz, H-6), 3.79 (s, 1H, OMe), 3.45 (ddd, 1H, J = 9.3, 6.5,
6.5 Hz, OCHaHb), 3.31 (t, 2H, J = 7.0 Hz, CH2N3), 3.26 (ddd, 1H,
J = 9.3, 6.9, 6.9 Hz, OCHaHb), 2.45 (dd, 1H, J = 12.9, 5.0 Hz, H-3eq),
2.14 (s, 3H, Ac), 2.07 (s, 3H, Ac), 2.03 (s, 3H, Ac), 2.01 (s, 3H, Ac),
1.88 (s, 3H, Ac), 1.86 (dd, 1H, J = 12.6, 11.4 Hz, H-3ax), 1.64–1.50
(m, 4H, OCH2CH2, CH2CH2N3), 1.41–1.23 (m, 14H, 7 ꢂ CH2 chain).
13C NMR (CDCl3, 100 MHz): dC 171.08, 170.68, 170.52, 170.20,
170.18, 167.62 (CO), 98.48 (C-2), 72.15 (C-8), 71.71 (C-6), 68.99
(C-4), 68.46 (C-7), 64.23 (OCHaHb), 62.40 (C-9), 52.62 (OMe),
51.47 (CH2N3), 49.43 (C-5), 37.47 (C-3), 29.59, 29.47 (ꢂ2), 29.43,
29.41, 29.12, 28.82, 26.70, 26.06 (9 ꢂ CH2 chain), 23.18, 21.03,
20.89 (ꢂ2), 20.79 (5 ꢂ Ac). HRMS m/z calcd for C31H50N4O13Na
(M+Na)+: 709.3267; found: 709.3265.
tone 3:2) [
a
]
D
+29.7 (c 0.6, CHCl3). 1H NMR (CDCl3, 400 MHz):
dY 5.37 (ddd, 1H, J = 2.8, 5.2, 8.4 Hz, H-8), 5.31 (dd, 1H, J = 2.1,
8.4 Hz, H-7), 5.17 (br b, 1H, J = 10.0 Hz, NH), 4.86 (ddd, 1H,
J = 4.8, 11.4, 11.6 Hz, H-4), 4.30 (dd, 1H, J = 2.6, 12.4 Hz, H-9a),
4.08 (dd, 1H, J = 5.2, 12.4 Hz, H-9b), 4.03 (ddd, 1H, J = 10.4,
10.4, 10.4 Hz, H-5), 3.82–3.87 (m, 1H, H-6), 3.78 (s, 3H, OMe),
2.70 (dd, 1H, J = 4.7, 12.7 Hz, H-3eq), 2.14 (Ac), 2.11 (Ac), 2.09
(SMe), 2.01 (Ac), 2.01 (Ac), 1.85 (Ac). 13C NMR (CDCl3,
100 MHz): dC 170.92 (CO), 170.64 (CO), 170.17 (CO), 170.10
(CO), 170.00 (CO), 167.89 (CO), 82.90 (C-2), 74.02 (C-6), 69.72
(C-4), 68.55 (C-8), 67.32 (C-7), 62.19 (C-9), 52.91 (OMe), 49.40
(C-5), 37.81 (C-3), 23.19 (Ac), 21.16, 20.83, 20.75 (4 ꢂ Ac),
12.01 (SMe).
3.6. (11-Azidoundecyl 5-acetamido-3,5-dideoxy-D-glycero-a-D-
galacto-non-2-ulopyranosid)onic acid (15)
A portion of 24 (0.154 g, 0.296 mmol) was dissolved in anhy-
drous MeOH (3.5 mL) and t-BuOK (0.066 g, 0.592 mmol) was added
until pH ꢀ9. The reaction mixture was stirred at rt for 90 min, and
evaporated under reduced pressure. The residue was re-dissolved
The fully protected
dissolved in anhydrous MeOH (10 mL), and a solution of 1.5 M
NaOMe in MeOH (400 L) was added. The mixture was stirred
a-sialoside 20 (500 mg, 0.89 mmol) was
l

W. Li et al. / Carbohydrate Research 346 (2011) 1692–1704
1701
in 3.5 mL of MeOH (3.5 mL) and H2O (1 mL), and the reaction mix-
3.9. Methyl S-(5-acetamido-3,5-dideoxy-
non-2-ulopyranosylonic acid)-(2?6)-(6-thio-b-
galactopyranosyl)-(1?4)-O-b- -glucopyranoside (17)
D-glycero-a-D-galacto-
ture was stirred at 50 °C overnight. The reaction mixture was
cooled to rt and neutralized with one drop of Ac2O. After concen-
tration under high vacuum, the residue was purified by reverse
phase column chromatography on C18 silica gel using a gradient
of MeOH–H2O (10?80%) as the eluent. The compound was then
lyophilized to afford 16 a white solid (66 mg, 82%). Rf: 0.35
D-
D
Compound 27 (52 mg, 0.05 mmol) was dissolved in anhydrous
MeOH (2.0 mL) and a solution of NaOMe in anhydrous MeOH
(1.5 M, 0.4 mL) was added under argon; the reaction mixture was
stirred at rt for 8 h. After concentration under reduced pressure,
the residue was redissolved in a 1:1 mixture of MeOH–H2O
(4.0 mL) and the stirring was continued overnight. Several drops
of Ac2O was added to neutralize the reaction and the mixture was
concentrated to afford a residue which was purified by HPLC on a
reverse-phase C18 silica gel column using H2O (100%) as eluent to
afford compound 17 (25 mg, 80% yield) which was lyophilized. Rf:
(CH2Cl2–MeOH–H2O 70:30:3). [a]
+24.4 (c 1.75, MeOH). 1H NMR
D
(D2O, 400 MHz): dH 3.82–3.74 (m, 2H, H-8, H-9a), 3.73 (dd, 1H,
J = 10.2, 10.2 Hz, H-5), 3.59 (ddd, 1H, J = 4.8, 10.3, 11.2 Hz, H-4),
3.56–3.45 (m, 3H, H-9b, H-6, H-7), 2.70 (dd, 1H, J = 12.7, 4.7 Hz,
H-3eq), 2.04 (s, 3H, SMe), 1.94 (s, 3H, Ac), 1.67 (dd, 1H, J = 12.8,
12.8 Hz, H-3ax). 13C NMR (D2O, 100 MHz): dC 175.04, 174.27
(CO), 85.13 (C-2), 74.58 (C-6), 71.85 (C-8), 68.62 (C-4), 68.22 (C-
7), 62.56 (C-9), 51.80 (C-5), 40.64 (C-3), 22.01(Ac), 11.5 (SMe). m/
0.3 (CH2Cl2–MeOH–H2O–HOAc 65:35:5:0.1); [
a
]
+34.5 (c 0.1,
D
z
(ESI-HRMS) calcd for [C12H21NO8SꢁH]ꢁ 338.0915; found:
MeOH). 1H NMR (D2O, 400 MHz): dH 4.33 (d, 1H, J = 7.5 Hz, H-10),
4.31 (d, 1H, J = 8.0 Hz, H-1), 3.92 (dd, 1H, J = 3.5, ꢀ1.0 Hz, H-40),
3.89 (dd, 1H, J = 2.0, 12.4 Hz, H-6a), 3.78 (dd, 1H, J = 2.4 11.9 Hz,
H-9a00), 3.75–3.75 (m, 4H, H-500 + H-6b + H-800 + H-50), 3.45–3.63
338.0909.
3.8. Methyl S-[methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-2-thio-
D
-glycero-
a
-D
-galacto-non-2-
(m,
11H,
H-400 + H-30 + H-4 + H-3 + H-9b00 + H-5 + H-600 + H-
ulopyranosylonate]-(2?6)-(2,3-di-O-acetyl-6-thio-b-
galactopyranosyl)-(1?4)-O-2,3,6-tri-O-acetyl-b-D-
glucopyranoside (27)
D
-
700 + OMe), 3.41 (dd, 1H, J = 7.7, 10.1 Hz, H-20), 3.25 (high order dd,
1H, J = 8.1, 9.2 Hz, H-2), 2.87 (high order dd, 1H, J = 7.0, 14.6 Hz,
H-6a0), 2.83 (high order dd, 1H, J = 6.6, 14.6 Hz, H-6a0), 2.71 (dd,
1H, J = 4.6, 12.8 Hz, H-3eq00), 1.93 (s, 3H, NHAc), 1.68 (dd, 1H,
J = 11.5, 12.6 Hz, H-3ax00); 13C NMR (D2O, 100 MHz): dC 175.01
(CO), 174.01 (CO), 103.00 (C-1), 102.95 (C-10), 85.84 (C-200), 78.91
(C-4), 74.87 (C-600), 74.75, 74.32, 74.11, 72.75, 72.46 (C-2), 72.22
(C-50), 70.70 (C-20), 68.99 (C-40), 68.49 (C-400), 68.04 (C-700), 62.58
(C-900), 60.14 (C-6), 57.17 (OMe), 51.63 (C-500), 40.76, 21.98. HRMS
m/z calcd for [C24H41NO18SꢁH]ꢁ 662.1972; found: 662.1973.
Compound 25 (176 mg, 0.312 mmol) was dissolved in a mix-
ture of anhydrous CH2Cl2 (2.5 mL) and pyridine (0.13 mL,
1.56 mmol, 5.0 equiv) under argon. After cooling the solution to
ꢁ25 °C, trifloromethanesulfonic anhydride (0.063 mL, 0.37 mmol,
1.2 equiv) was added dropwise over approximately 1 min. After
stirring for 1 h, the reaction mixture was diluted with more
CH2Cl2 (50 mL) and washed successively with cold 1.0 M HCl,
cold satd NaHCO3 solution, and ice-cold water. The organic layer
was dried over Na2SO4, filtered, and concentrated at 18 °C under
high vacuum. The crude residue (26) was combined with 22
(257 mg, 0.47 mmol, 1.5 equiv), and the mixture was dissolved
in dry DMF (2.0 mL) under argon. After cooling to 0 °C, Et2NH
(0.32 mL, 3.1 mmol, 10 equiv) was added and the solution was
stirred at rt for 4 h. The solution was concentrated under reduced
pressure, the residue was purified by column chromatography on
silica gel using MeOH–CH2Cl2 (2:98) as eluent to afford 27
(85 mg, 26% yield) as an amorphous solid. Rf: 0.3 (MeOH–CH2Cl2
3.10. Enzymatic reaction using a-sialoside (15) as a substrate
To a mixture containing glycoside 15 (35 mg, 0.069 mmol) and
crude CMP-NeuAc (ꢀ35%, 902 mg, 6.8 equiv) was added a solution
of MnCl2(0.5 M, 125
(25
lL) and a solution of alkaline phosphatase
L). A solution of crude CST-II (8 mL) was added and the mix-
l
ture was gently tumbled for 24 h at rt. The reaction mixture was
worked up in a similar manner as 1, and purified by HPLC on a re-
verse phase C18 column using a gradient of MeOH–H2O (0?100%)
as eluent to afford sequentially
2.2%), -(2?8)-pentasialoside 31 (5.2 mg, 4.5%),
aloside 30 (5.0 mg, 5.3%), -(2?8)-trisialoside 29 (5.8 mg, 7.7%),
and -(2?8)-disialoside 28 (6.6 mg, 12%).
a
-(2?8)-hexasialoside 32 (3.0 mg,
3:97). [a]
+14.2 (c 2.5, CHCl3). 1H NMR (CDCl3, 400 MHz): dH
a
a-(2?8)-tetrasi-
D
5.33 (dd, 1H, J = 1.9, 9.5 Hz, H-700), 5.24 (ddd, 1H, J = 3.2, 4.5,
9.5 Hz, H-800), 5.17 (d, 1H, J = 9.9 Hz, NHAc), 5.17 (dd, 1H,
J = 9.5, 9.5 Hz, H-3), 5.12 (dd, 1H, J = 7.9, 9.9 Hz, H-20), 4.86–
4.97 (m, 3H, H-2 + H-30 + H-400), 4.60 (d, 1H, J = 7.9 Hz, H-10),
4.50 (dd, 1H, J = 2.2, 11.8 Hz, H-6a), 4.38 (d, 1H, J = 7.9 Hz, H-1),
4.29 (dd, 1H, J = 2.9, 12.7 Hz, H-9a00), 4.21 (overlapped, 1H, H-
40), 4.17 (dd, 1H, J = 6.6, 11.8 Hz, H-6b), 4.12 (dd, 1H, J = 4.1,
12.4 Hz, H-9b00), 3.96 (ddd, 1H, J = 10.2, 10.2, 10.2 Hz, H-500),
3.79–3.89 (m, 5H, CO2Me + H-4 + H-600), 3.60–3.69 (m, 2H, H-
5 + H-50), 3.48 (s, 3H, OMe), 2.89 (dd, 1H, J = 7.3, 14.3 Hz, H-
6a0), 2.81, dd, 1H, J = 7.0, 14.3 Hz, H-6b0), 2.76 (dd, 1H, J = 4.5,
12.7 Hz, H-3eq00), 2.55 (d, 1H, J = 4.8 Hz, OH-40), 2.19 (s, 3H, Ac),
2.14 (s, 3H, Ac), 2.10 (s, 3H, Ac), 2.07 (s, 3H, Ac), 2.05 (s, 6H,
2 ꢂ Ac), 2.046 (s, 3H, Ac), 2.04 (s, 6H, 2 ꢂ Ac), 1.98 (dd, 1H,
J = 12.1, 12.1 Hz, H-3ax00), 1.89 (s, 3H, Ac); 13C NMR (CDCl3,
100 MHz, from HSQC): dC 101.28 (C-1), 100.17 (C-10), 75.10 (C-
4), 73.88 (C-600), 73.28 (C-5 or C-50), 72.97, 72.37 (C-3), 71.81
(C-400), 69.14 (C-20), 68.69, 67.86 (C-800), 67.33 (C-40), 66.74 (C-
700), 62.11 (C-6), 61.93 (C-900), 56.72 (OMe), 52.83 (CO2Me),
49.43 (C-500), 38.13 (C-300), 37.89 (C-300), 29.18 (C-60), 22.76 (Ac),
21.17 (ꢂ2, 2 ꢂ Ac), 20.62 (ꢂ4, 4 ꢂ Ac), 20.59 (ꢂ2, 2 ꢂ Ac), 20.52
(Ac). HRMS m/z calcd for [C43H61NO27S+Na]+ 1078.3044; found:
1078.3049.
a
a
3.10.1. 11-Azidoundecyl O-(5-acetamido-3,5-dideoxy-
D-glycero-
a-D-galacto-non-2-ulopyranosylonic acid)-(2?8)-5-acetamido-
3,5-dideoxy-
D-glycero-
a-
D-galacto-non-2-ulopyranosylonic acid
(28)
Rf: 0.40 (i-PrOH–H2O–NH4OH 6:1:1); [a]
D
+1.96 (c 0.3, MeOH).
1H NMR (CD3OD, 400 MHz,) dH 4.43–4.36 (m, 1H, H-8), 4.23 (dd,
1H, J = 11.8, 3.8 Hz, H-9a), 4.10–4.02 (ddd, 1H, J = 2.4, 6.4, 8.8 Hz,
H-80), 4.03–3.94 (m, 2H, H-9a0 + H-7), 3.91–3.86 (m, 2H, H-50 + H-
60), 3.86–3.76 (m, 4H, H-5 + H-4 + OCHaHb + H-9b), 3.75–3.63 (m,
3H, H-40 + H-9b0 + H-6), 3.63–3.52 (m, 2H, OCHaHb + H-70), 3.41
(t, 2H, J = 6.8 Hz, CH2N3), 2.95 (dd, 1H, J = 12.2, 8.3 Hz, H-3eq),
2.82 (dd, 1H, J = 12.2, 4.6 Hz, H-3eq0), 2.17 (s, 3H, Ac), 2.13 (s, 3H,
Ac), 1.86–1.76 (m, 2H, H-3ax + H-3ax0), 1.74–1.60 (m, 4H,
OCH2CH2 + CH2CH2N3), 1.54–1.37 (m, 14H, 7 ꢂ CH2 chain). 13C
NMR (CD3OD, 100 MHz) dC 175.54, 175.16, 174.95, 174.68 (CO),
102.70, 101.91 (C-2), 79.53 (C-8), 74.61(C-6), 73.14 (C-60), 72.02
(C-80), 70.75 (C-60), 70.19 (C-7, C-70), 69.84 (C-4), 68.69 (C-40),
65.49 (OCHaHb), 65.00 (C-90), 63.81 (C-9), 54.58 (C-5), 54.10 (C-
50), 52.63 (CH2N3), 42.48 (C-3, C-30), 31.24, 30.85 (ꢂ2), 30.78
(ꢂ2), 30.42, 30.07, 27.98, 27.38, 27.17 (9ꢂCH2 chain), 23.27 (Ac),

1702
W. Li et al. / Carbohydrate Research 346 (2011) 1692–1704
22.74 (Ac). m/z (ESI-HRMS) calcd for [C33H57N5O17ꢁ2H]2ꢁ
800 + H-8000 + H-9a + H-9a0 + H-9a00 + H-9a000), 4.00–3.54 (m, 26H),
3.52–3.39 (m, 5H), 3.27 (t, 2H, J = 6.9 Hz, CH2N3), 2.94–2.89 (m,
1H, H-3eq), 2.78–2.69 (m, 4H, 4 ꢂ H-3eq), 2.09–2.07 (m, 9H, Ac),
2.04 (s, 3H, Ac), 2.02 (s, 3H, Ac), 1.82–1.61 (m, 5H, 5 ꢂ H-3ax),
1.61–1.48 (m, 4H, OCH2CH2, CH2CH2N3), 1.43–1.23 (m, 14H,
7 ꢂ CH2 chain). 13C NMR (CD3OD, 100 MHz, from HSQC): dC
78.04, 77.17, 77.00, 75.44, 75.02, 74.65, 74.28, 74.10, 73.80,
73.42, 71.77, 69.70, 69.08, 68.90, 68.78, 68.11, 67.92, 67.11,
63.54, 61.78, 61.55, 61.42, 52.74, 52.50, 50.61, 50.20, 40.78,
29.36, 28.80, 21.90, 21.27, 21.19 (Ac). HRMS m/z calcd for
[C66H108N8O41ꢁ4H]4ꢁ: 416.15802; found: 416.15782; calcd for
[C66H108N8O41ꢁ3H]3ꢁ: 555.2131; found: 555.2126.
396.6802; found: 396.6803.
3.10.2. 11-Azidoundecyl O-(5-acetamido-3,5-dideoxy-
-galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-
acetamido-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosylonic acid)-(2?8)-5-acetamido-3,5-dideoxy-
glycero- -galacto-non-2-ulopyranosylonic acid (29)
Rf: 0.25 (i-PrOH–H2O–NH4OH 6:1:1); [ D +0.69 (c 0.57, MeOH).
D-glycero-
a-D
D
a-D
D
-
a-D
a
]
1H NMR (CD3OD, 400 MHz) dH 4.36–4.30 (m, 1H, H-8), 4.29–4.19 (m,
2H, H-80 + H-9a0), 4.14 (dd, 1H, J = 11.8, 4.1 Hz, H-9a), 3.98–3.84 (m,
4H, H-7 + H-70 + H-800 + H-9a00), 3.83–3.57 (m, 12H, H-6 + H-60 + H-
600 + H-4 + H-400 + H-400 + H-700 + H-50 + H-500 + H-9b + H-9b0 + H-
9b00 + OCHaHb), 3.53–3.44 (m, 2H, H-5 + OCHaHb), 3.28 (t, 2H,
J = 6.8 Hz, CH2N3), 2.89 (dd, J = 1H, 12.6, 4.8 Hz, H-3eq), 2.72 (dd,
2H, J = 12.1, 4.4 Hz, H-3eq0 + H-3eq00), 2.05 (s, 3H, Ac), 2.05 (s, 3H,
Ac), 2.02 (s, 3H, Ac), 1.69 (dd, 2H, J = 12.5, 12.2 Hz, H-3ax + H-
3ax0), 1.63–1.49 (m, 5H, H-3ax00 + OCH2CH2 + CH2CH2N3), 1.43–
1.25 (m, 14H, 7 ꢂ CH2 chain).13C DEPT135 NMR (CD3OD, 100 MHz)
dC 78.14 (C-8), 77.79 (C-80), 74.29, 74.07, 73.03, 71.77, 70.04,
69.43, 68.87, 68.76, 68.52, 68.19 (C-4, C-40, C-400, C-6, C-60, C-600, C-
7, C-70, C-700, C-800), 63.97 (OCHaHb), 63.19 (C-900), 62.07 (C-90),
61.69 (C-9), 53.02 (C-5), 52.72 (C-50), 52.50 (C-500), 51.07 (CH2N3),
41.30 (C-3), 40.72 (C-30), 40.56 (C-300), 29.69, 29.30(ꢂ2), 29.23(ꢂ2),
28.86, 28.50, 26.42, 25.81 (9 ꢂ CH2 chain), 21.83 (Ac), 21.72 (Ac),
21.20 (Ac). HRMS m/z calcd for C44H71N6O25 (M-3H)3ꢁ:361.1495;
found: 361.1495.
3.10.5. 11-Azidoundecyl O-(5-acetamido-3,5-dideoxy-
-galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-
acetamido-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-dideoxy-
glycero-
acetamido-3,5-dideoxy-
ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-dideoxy-
glycero-
acetamido-3,5-dideoxy-
ulopyranosylonic acid (32)
Rf: 0.10 (i-PrOH–H2O–NH4OH 6:1:1); [
D-glycero-
a-D
D
a-D
D
-
-
a
-
D
-galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-
D-glycero-a-D-galacto-non-2-
D
a-
D
-galacto-non-2-ulopyranosylonic acid)-(2?8)-5-
D-glycero- -galacto-non-2-
a-D
a]
D ꢁ4.81 (c 0.21, MeOH).
1H NMR (600 MHz, CD3OD) dH 4.35–4.25 (m, 2H, 2 ꢂ H-8), 4.25–
4.11 (m, 3H, 3 ꢂ H-9a), 4.01–3.55 (m, 26H), 3.54–3.44 (m, 3H),
3.27 (t, 2H, J = 6.9 Hz, CH2N3), 2.95–2.89 (m, 1H, H-3eq), 2.83–
2.67 (m, 5H, 5 ꢂ H-3eq), 2.11–1.99 (m, 18H, 6 ꢂ Ac), 1.73–1.63
(m, 5H, 5 ꢂ H-3ax), 1.62–1.50 (m, 5H, H-3ax, OCH2CH2, CH2CH2N3),
1.42–1.25 (m, 14H, 7 ꢂ CH2 chain). 13C NMR (CD3OD, 100 MHz,
from HSQC): dC 79.44, 75.78, 75.74, 75.59, 74.81, 73.32, 71.45,
71.16, 70.77, 70.57, 70.49, 69.07, 69.92, 69.87 (C-8, C-6, C-4, C-7,
C-2), 65.44 (OCHaHb), 64.71, 63.44, 63.21, 62.93 (C-9), 54.14 (C-
5), 52.54 (CH2N3), 42.54, 42.49, 42.39, 42.19, 42.00 (C-3), 31.11,
30.74, 29.89, 27.60, 27.37 (9 ꢂ CH2 chain), 23.32, 23.13, 22.64
(Ac). HRMS m/z calcd for [C77H125N9O49ꢁ5H]5ꢁ: 390.94405; found:
3.10.3. 11-Azidoundecyl O-(5-acetamido-3,5-dideoxy-
-galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-
acetamido-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-dideoxy-
glycero- -galacto-non-2-ulopyranosylonic acid)-(2?8)-5-
acetamido-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosylonic acid (30)
Rf: 0.20 (i-PrOH–H2O–NH4OH 6:1:1); [a]D +0.88 (c 0.45, MeOH).
D-glycero-
a-D
D
a-D
D
-
a-D
D
a-D
1H NMR (CD3OD, 600 MHz) dH 4.41–4.37 (m, 1H, H-8), 4.34–4.27 (m,
2H, H-80, H-9a), 4.17 (m, 2H, H-9a0 + H-9b0), 3.99–3.92 (m, 3H, H-
8000 + H-800 + H-9a00), 3.92–3.86 (m, 3H, H-9a000 + H-7 + H-70), 3.83–
3.56 (m, 17H, H-700 + H-4 + H-40 + H-400 + H-4000 + H-6 + H-60 + H-
600 + H-6000 + H-5 + H-50 + H-500 + H-5000 + OCHaHb + H-9b + H-9b00 +
H-9b000), 3.52–3.45 (m, 2H, OCHaHb + H-7000), 3.28 (t, 2H, J = 6.8 Hz,
CH2N3), 2.95 (dd, 1H, J = 12.8, 4.2 Hz, H-3eq), 2.80–2.70 (m, 3H, H-
3eq0 + H-3eq00 + H-3eq000), 2.08 (s, 3H, Ac), 2.07 (s, 3H, Ac), 2.05 (s,
3H, Ac), 2.02 (s, 3H, Ac), 1.76 (dd, 1H, J = 11.8, 12.0 Hz, H-3ax),
1.70–1.62 (m, 3H, H-3ax0 + H-3ax00 + H-3ax000), 1.62–1.51 (m, 4H,
OCH2CH2 + CH2CH2N3), 1.41–1.27 (m, 14H, 7 ꢂ CH2 chain). 13C
NMR (CD3OD, 100 MHz, from HSQC): dC 79.75, 79.37 (C-8, C-80),
75.74 (ꢂ2), 75.46, 74.56, 73.23, 71.01, 70.60, 70.50, 70.43, 69.93,
69.77, 69.66 (C-4, C-40, C-400, C-4000, C-6, C-60, C-600, C-6000, C-7, C-70,
C-700, C-7000, C-800, C-8000), 65.27 (OCHaHb), 64.68 (C-9000), 63.51 (C-
900), 63.26 (C-90), 62.91 (C-9), 54.27 (ꢂ4) (C-5, C-50, C-500, C-5000),
52.49 (CH2N3), 42.70 (C-3), 42.43, 42.25 (ꢂ2) (C-30, C-300, C-3000),
31.2–27.20 (9 ꢂ CH2 chain), 23.26 (ꢂ2), 22.78, 22.40 (Ac). HRMS
m/z calcd for [C55H91N7O33ꢁ4H]4ꢁ:343.3842; found: 343.3844.
390.94382; calcd for [C77H125N9O49ꢁ4H]4ꢁ
: 488.9319; found:
488.9324.
3.11. Enzymatic reaction using a-sialoside (16) as a substrate
To a mixture containing thioglycoside 16 (20 mg, 0.059 mmol)
and crude CMP-NeuAc (ꢀ37%, 420 mg, 4.0 equiv) was added a
solution of MnCl2 (0.5 M, 100 lL) and a solution of alkaline phos-
phatase (15 lL). A solution of crude CST-II (10 mL, 0.3 U/mL) was
added and the mixture was gently tumbled for 24 h at rt. The reac-
tion mixture was filtered off and concentrated in vacuo. The resi-
due was re-suspended in MeOH (20 mL), and the supernatant
was removed by centrifugation; after concentration of the solution,
the residue was purified by silica gel column using a gradient of
IPA–H2O–NH4OH (9:1:1?5:2:1) as eluent to afford sequentially
a-(2?8)-disialoside 33 (6.3 mg, 17% yield) and a-(2?8)-trisialo-
side 34 (5.5 mg, 10.1%).
3.11.1. Methyl O-(5-acetamido-3,5-dideoxy-D-glycero-a-D-
galacto-non-2-ulopyranosylonic acid)-(2?8)-5-acetamido-3,5-
3.10.4. 11-Azidoundecyl O-(5-acetamido-3,5-dideoxy-
-galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-
acetamido-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-dideoxy-
glycero- -galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-
acetamido-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosylonic acid)-(2?8)-5-acetamido-3,5-dideoxy-
glycero- -galacto-non-2-ulopyranosylonic acid (31)
Rf: 0.15 (i-PrOH–H2O–NH4OH 6:1:1); [
D ꢁ0.84 (c 0.71, MeOH).
1H NMR (400 MHz, CD3OD) dH 4.39–4.10 (m, 8H, H-8 + H-80 + H-
D
-glycero-
dideoxy-2-thio-
acid (33)
D
-glycero-
a
-
D
-galacto-non-2-ulopyranosylonic
a-D
D
a
-
D
Rf: 0.50 (i-PrOH–H2O–NH4OH 7:2:1). [a]D +21.1 (c 0.17, MeOH).
D
-
1H NMR (CD3OD, 400 MHz): dY 4.30 (m, 1H, H-8), 4.14 (dd, 1H,
J = 4.1, 11.8 Hz, H-9a), 3.91 (ddd, 1H, J = 2.5, 6.5, 9.0 Hz, H-80),
3.80–3.87 (m, 2H, H-7 and H-9a0), 3.78 (dd, 1H, J =10.0, 10.0 Hz,
H-5), 3.65–3.75 (m, 4H, H-6+ H-40 + H-50 + H-9b), 3.61 (ddd, 1H, J
=3.9, 10.5, 10.5 Hz, H-4), 3.53–3.60 (m, 2H, H-9b0 + H-60), 3.42
(dd, 1H, J =1.8, 9.0 Hz, H-70), 2.83 (dd, 1H, J = 4.2, 12.3 Hz, H-3eq),
2.80 (dd, 1H, J = 4.4, 12.2 Hz, H-3eq0) 2.17 (SMe), 2.02, 2.00 (6H,
a
-D
D
a-D
D
-
a-D
a]

W. Li et al. / Carbohydrate Research 346 (2011) 1692–1704
1703
2 ꢂ Ac), 1.70 (higher order dd, 1H, J = 9.1, 9.1 Hz, H-3ax), 1.63
(higher order dd, 1H, J = 11.0, 11.0 Hz, H-3ax0). 13C NMR assigned
through HSQC (CD3OD, 100 MHz) d 79.01 (C-8), 77.17 (C-6),
74.31 (C-60), 72.88 (C-80), 71.85 (C-7), 70.27 (C-70), 70.11 (C-4),
69.63 (C-40), 64.59 (C-90), 63.43 (C-9), 53.93 (C-5), 53.84 (C-50),
42.66 (C-3), 42.22 (C-30), 23.07 (Ac), 22.57 (Ac), 13.17 (SMe). m/z
(ESI-HRMS) calcd for [C23H36N2O16Sꢁ2H]2ꢁ 314.0898; found:
314.0898.
3.13. Enzymatic reaction using N-acetylneuraminic acid (36) as
a substrate
To a mixture of compound 36 (18 mg, 0.058 mmol) and crude
CMP-NANA (ꢀ29%, 295 mg, 0.13 mmol, 2.2 equiv) was added a
solution of MnCl2 (0.5 M, 80 lL) and a solution of alkaline phos-
phatase (25 lL). A solution of crude CST-II (5.0 mL, 0.3 U/mL) was
added and the mixture was gently tumbled for 4 days. The reaction
mixture was concentrated in vacuo and re-suspended in MeOH
(20 mL). The supernatant was removed by centrifugation, and the
solvent was concentrated. The residue was purified by gel-filtra-
tion chromatography on G-15 using H2O as eluent to afford the
disialoside 37 (7.5 mg, 21.5%). Rf: 0.30 (i-PrOH–H2O–NH4OH
3.11.2. Methyl O-(5-acetamido-3,5-dideoxy-
galacto-non-2-ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-
dideoxy- -glycero- -galacto-non-2-ulopyranosylonic acid)-
D-glycero-a-D-
D
a-D
(2?8)-5-acetamido-3,5-dideoxy-2-thio-
D
-glycero-
a-D-galacto-
non-2-ulopyranosylonic acid (34)
6:2:1). ½a 2D5
ꢄ
ꢁ4.0 (c 0.35, MeOH). 1H NMR (400 MHz, D2O) dH
Rf: 0.38 (i-PrOH–H2O–NH4OH 7:2:1). [
a
]
D
+8.9 (c 0.18, MeOH).
4.00–3.88 (m, 3H, H-4, H-8 H-9a), 3.88–3.71 (m, 6H), 3.69–3.47
(m, 5H), 2.72 (dd, 1H, J = 12.3, 4.6 Hz, H-3eq), 2.17 (dd, 1H,
J = 13.1, 5.0 Hz, H-3eq), 2.02 (s, 1H, Ac), 1.98 (s, 1H, Ac), 1.70 (dd,
1H, J = 12.2, 12.32 Hz, H-3ax), 1.68 (dd, 1H, J = 12.2, 12.3 Hz, H-
3ax0). 13C DEPT NMR (100 MHz, D2O) dC 75.29 (C-8), 72.87, 72.13,
70.37, 68.46, 68.09, 67.63, 67.18 (C-6, C-60, C-80, C-4, C-40, C-2, C-
20, C-7, C-70), 62.65 (C-9), 60.98 (C-90), 52.42 (C-5), 51.74 (C-50),
41.05 (C-3), 39.31 (C-30), 22.22 (Ac), 22.08 (Ac).
1H NMR (CD3OD, 400 MHz): dY 4.35 (m, 1H, H-8 or H-80), 4.22
(m, 2H, H-80 or H-8 + H-9a or H-9a0), 4.14 (dd, 1H, J = 5.4,
11.4 Hz, H-9a or H-9a0), 3.81–3.95 (m, 4H, H-800+ H-7 +H-70 + H-
9a00), 3.58–3.81 (m, 12H, H-5 + H-50 + H-500 + H-6 + H-60 + H-
600 + H-9b + H-9b0 + H-9b00 + H-4 + H-40 + H-400), 3.48 (dd, 1H, J =
1.2, 9.0 Hz, H-700), 2.91 (dd, 1H,, J = 4.0, 12.6 Hz, H-3eq or H-3eq0
or H-3eq00), 2.81 (dd, 1H, J = 4.5, 12.2 Hz, H-3eq or H-3eq0 or H-
3eq00), 2.68 (dd, 1H, J = 4.7, 10.7 Hz H-3eq or H-3eq or H-3eq00),
2.19 (s, 3H, SMe), 2.04 (Ac), 2.03 (Ac), 2.01 (Ac), 1.77–2.61 (m,
3H, H-3ax + H-3ax0 + H-3ax00). 13C NMR assigned through HSQC
(CD3OD, 100 MHz) dC 78.89 (C-80 or C-8), 78.46 (C-8 or C-80),
76.94, 75.06, 74.22, 72.88, 70.82, 70.33, 70.09, 69.91, 69.78, 69.36
(C-6, C-60, C-600, C-7, C-70, C-700, C-4, C-40, C-400, C-800), 64.45 (C-900),
63.11 (C-9 or C-90), 62.63 (C-9 or C-90), 53.83 (C-5 or C-50), 53.79
(C-5 or C-50), 53.62 (C-500), 42.47 (C-3 or C-30 or C-300), 42.42 (C-3
or C-30 or C-300), 41.29 (C-3 or C-30 or C-300), 22.89 (Ac), 22.39
(Ac), 22.56 (Ac) 12.32 (SMe). m/z (ESI-HRMS) calcd for
[C34H55N3O24Sꢁ3H]3ꢁ 306.0893; found: 306.0891.
3.14. (p-Chlorophenyl 5-acetamido-3,5-dideoxy-2-thio-
D-
glycero- -galacto-non-2-ulopyranosid)onic acid (38)
a-D
Thioglycoside 18 (1.0 g, 1.62 mmol) was dissolved in anhydrous
MeOH (12 mL) and a solution of NaOMe in MeOH (0.5 M, 8.0 mL)
was added. The reaction was stirred at room temperature for 4 h.
The solvent was removed under reduced pressure. The residue
was re-dissolved in a 1:1 mixture of MeOH–H2O (18 mL), and the
solution was heated to 50 °C for 18 h. The reaction mixture was
cooled to room temperature and neutralized with Ac2O to pH ꢀ8.
After concentration under high vacuum, the residue was purified
by reverse phase column chromatography on C18 silica gel using
a gradient of MeOH–H2O (0?50%) as the eluent to afford com-
pound 38 as a white solid after lyophilization (592 mg, 84% yield).
3.12. Methyl O-(5-acetamido-3,5-dideoxy-
non-2-ulopyranosylonic acid)-(2?8)-(5-acetamido-3,5-
dideoxy-2-thio- -glycero- -galacto-non-2-ulopyranosylonic
-galactopyranosyl)-(1?4)-b-
D-glycero-a-D-galacto-
D
a-D
acid)-(2?6)-(6-thio-b-
D
D-
Rf: 0.14 (i-PrOH–H2O–NH4OH 70:30:3). ½a D25
ꢄ
ꢁ66.8 (c 0.2, H2O). 1H
glucopyranoside (35)
NMR (400 MHz, D2O) dH 7.41 (d, 2H, J = 8.6 Hz, PhCl), 7.30 (d, 2H,
J = 8.6 Hz, PhCl), 4.31 (dd, 1H, J = 10.5, ꢀ1 Hz, H-6), 4.06 (ddd, 1H,
J = 4.7, 10.1, 11.6 Hz, H-4), 3.82 (dd, 1H, J = 10.2, 10.2 Hz, H-5),
3.71 (dd, 1H, J = 11.6, 2.6 Hz, H-9a), 3.66 (ddd, 1H, J = 2.6, 5.5,
9.1 Hz, H-8), 3.56 (dd, 1H, J = 5.4, 11.6 Hz, H-9b), 3.48 (d, 1H,
J = 9.0, ꢀ1 Hz, H-7), 2.59 (dd, 1H, J = 13.7, 4.8 Hz, H-3e), 1.99 (s,
3H, Ac), 1.89 (dd, 1H, J = 13.7, 11.8 Hz, H-3a). 13C NMR (100 MHz,
D2O) dC 174.75 (CO), 173.93 (CO), 135.51 (Ar), 134.56 (Ar),
129.44 (Ar), 128.97 (Ar), 91.44 (C-2), 71.64 (C-6), 70.06 (C-8),
68.60 (C-7), 67.33 (C-4), 63.12 (C-9), 52.32 (C-5), 40.88 (C-3),
22.15 (Ac). m/z (ESI-HRMS) calcd for [C17H22NO8SClꢁH]ꢁ
434.0682; found: 434.0682.
To a mixture containing compound 17 (16 mg, 0.024 mmol),
was added crude CMP-NANA (77 mg, 0.096 mmol), an aqueous
solution of MnCl2 (0.5 M, 80
lL), a solution of alkaline phosphate
(61 L), and a solution of the crude
l
a
-(2?8)-sialyltransferase
(7.0 mL, 3.0 U/mL). The mixture was gently tumbled at room tem-
perature for three days. The reaction mixture was filtered off and
concentrated. The residue was purified by gel filtration chromatog-
raphy on a LH-20 column using MeOH as the eluent to afford the
desired tetrasaccharide 35 (9.0 mg, 39% yield). Rf: 0.2 (i-PrOH–
H2O–NH4OH 7:2:1); [
400 MHz): dH 4.36 (d, 1H, J = 7.6 Hz, H-10), 4.32 (d, 1H, J = 7.9 Hz,
H-1), 4.04–4.11 (m, 2H, H-800 + H-9a00), 3.64–3.87 (m, 9H, H-8000
a
]
+26.7 (c 0.1, MeOH). 1H NMR (D2O,
D
+
H-9a000 + H-40 + H-500 + H-5000 + H-6b + H-600 + H-700 + H-50), 3.44–3.64
(m, 13H, H-9b00 + H-9b000 + H-400 + H-4000 + H-30 + H-3 + H-4 + H-5 +
H-6000 + H-7000 + OMe), 3.41 (dd, 1H, J = 7.9, 9.5 Hz, H-20), 3.26 (high
order m, 1H, H-2), 2.85–2.96 (m, 2H, H-6a0 + H-6b0), 2.68 (dd, 1H,
J = 4.8, 12.4 Hz, H-3eq000), 2.66 (dd, 1H, J = 4.4, 12.5 Hz, H-3eq00),
1.98 (s, 3H, NHAc), 1.95 (s, 3H, NHAc), 1.65 (dd, 1H, J = 11.8,
12.4 Hz, H-3ax000), 1.64 (dd, 1H, J = 11.8, 11.8 Hz, H-3ax00); 13C
NMR (D2O, 100 MHz, from HSQC): dC 102.87 (C-1), 102.64 (C-10),
78.76 (C-4), 78.35 (C-800), 74.71, 73.88, 73.83, 72.69, 72.59, 71.21,
70.57, 69.88, 69.14, 68.91, 68.32, 68.18, 62.43 (C-9000), 61.42 (C-
900), 60.09 (C-6), 57.06 (OMe), 52.00 (C-500 or C-5000), 51.72 (C-5000
or C-500), 40.59 (C-300 or C-3000), 40.54 (C-3000 or C-300), 29.28 (C-60),
22.25 (NHAc), 21.97 (NHAc). m/z (ESI-HRMS) calcd for
[C35H58N2O26Sꢁ2H]2ꢁ 476.1427; found: 476.1428.
Acknowledgments
We thank Dr. W. Wakarchuk and Dr. M. Gilbert from the
Institute for Biological Sciences, NRCC, for providing clones of
the enzymes and Professor D. Bundle and Dr. A. Morales-Izquier-
do from the Department of Chemistry, University of Alberta, for
acquiring mass spectra. The financial support from the Natural
Sciences and Engineering Research Council of Canada is
acknowledged.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.05.009.

1704
W. Li et al. / Carbohydrate Research 346 (2011) 1692–1704
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