Phosphite-based sialic acid donors in the synthesis of α(2→9) oligosialic acids

Article abstract of DOI:10.1016/j.tet.2009.04.022

The combination of a phosphite donor and an anomeric thiocresol-protected acceptor, both with a TFA protecting group at C-5 of the sialic acid, provides good α-selectivity and yield in sialylation. Although the convergent synthetic strategy of using a phosphite disialo-donor and a disialo-acceptor assembles tetra-sialic acid efficiently, overcoming the low α-selectivity of α-anomer and purifying it remain to be achieved. Furthermore, mono- and di-sialic acids were, respectively, conjugated on carrier protein, keyhole limpet hemocyanin. The enzymatic hydrolysis method is recommended for estimating the amount of sialic acid on a protein conjugate.

Full text of DOI:10.1016/j.tet.2009.04.022

Tetrahedron 65 (2009) 4714–4725
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Phosphite-based sialic acid donors in the synthesis of a(2/9) oligosialic acids
,b,
Chang-Ching Lin a, Avijit Kumar Adak a, Jia-Cherng Horng a, Chun-Cheng Lin a
*
a Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan
b Genomic Research Center, Academia Sinica, Taipei, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
The combination of a phosphite donor and an anomeric thiocresol-protected acceptor, both with a TFA
protecting group at C-5 of the sialic acid, provides good -selectivity and yield in sialylation. Although the
convergent synthetic strategy of using a phosphite disialo-donor and a disialo-acceptor assembles tetra-
sialic acid efficiently, overcoming the low -selectivity of -anomer and purifying it remain to be ach-
ieved. Furthermore, mono- and di-sialic acids were, respectively, conjugated on carrier protein, keyhole
limpet hemocyanin. The enzymatic hydrolysis method is recommended for estimating the amount of
sialic acid on a protein conjugate.
Received 19 February 2009
Received in revised form 3 April 2009
Accepted 7 April 2009
a
a
a
Available online 16 April 2009
Keywords:
Sialylation
Sialic acid
N-TFA
Ó 2009 Elsevier Ltd. All rights reserved.
Phosphite
Glycosylation
1. Introduction
cause the sialylation reaction commonly to proceed with low to
moderate yield of product, low a-selectivity, and the formation of
As a diverse, chiral pool of nine-carbon carboxylated saccha-
rides,1 sialic acids are commonly attached to the non-reducing ter-
minus of oligosaccharide chains in glycolipids or glycoproteins on
the surface of a cell.2 Since they are at the terminal positions of
glycan chains, sialic acids have very important roles in biological
events on the surface of the cell, serving as a mask for cell-surface
glycan receptors, ligands for proteins, and markers of some can-
an undesirable 2,3-elimination product. Additionally, the low re-
activity of hydroxyl groups at the C-8/C-9 of sialic acid in glyco-
sylation6 slows the synthesis of homo-oligosialic acids.
Recently, many attempts7 have been made to achieve high
a-
selectivity in sialylation. Most of these studies have focused on
changing the anomeric leaving group, installing an auxiliary group
at the C-1 or C-3 position, or modifying the amine protecting group
at the C-5 position of the sialyl donor.8 Although moderate to high
cers.1–3 Sialic acid-containing glycoconjugates exist in nature as
configuration sialosides. The linear homopolymer of sialic acids is
linked internally via contiguous -2–8, -2–9, or alternating -2–8
and -2–9 linkages, and functions as a virulence factor. These chains
a-
a-selectivities with variable yields have been obtained using halide,
a
a
a
sulfide, selenide, or oxygen substituent at the C-3 position9 or C-110
auxiliaries of the donors, additional steps are required to remove
these substituents at the end of the synthesis. However, the re-
placement of the acetyl group at the C-5 N-acetyl group of the sialyl
donor to a 2,2,2-trichloroethoxycarbonyl (NTroc),8c,d trifluoroacetyl
(NHTFA),8b azido (N3),8e,f Fmoc,8g phthalimide8h or diacetyl8a group
has been studied and demonstrated by the improved yield of
a
are found as capsular polysaccharides on the surface of bacterial
cells such as Escherichia coli K1 and Neisseria meningitidis groups B
and C, making them potential antigens for the development of an-
tibacterial vaccine.4 Polysaccharides that are adopted to prepare
vaccines are typically isolated from their natural sources, and so can
be heterogeneous and/or contaminated with other antigenic com-
ponents. Therefore, the development of synthetic polysaccharide
vaccines with high immunogenicity has attracted much attention.5
A challenging issue in synthesizing sialoconjugates is anomeric
product or
a-selectivity during sialylation. Recently, 1,5-lacta-
mized-sialyl acceptors have been developed and shown to increase
reactivity of the C-8 hydroxyl group in glycosylation.11 Despite the
potential of these sialyl donors, their use has been limited to the
a
-selectivity during the chemical sialylation of sialic acids. An
synthesis of
charide acceptors. Recently, Takahashi et al. adopted 5-N,4-O-car-
bonyl-protected sialyl donor to synthesize first (2,8) tetra-sialic
acid,12 and later
(2,9) tri-sialic acid via one-pot glycosylation and
polymer-assisted deprotection13 with exclusive
-selectivity in
sialylations. However, the -selectivity toward some primary hy-
droxyl acceptors remained low and is still a challenge.
a(2,8)-disialosides, and glycosylation with other sac-
electron-withdrawing carboxyl group at the anomeric center and
the lack of participating functionality at the C-3 of sialyl donor
a
a
a
a
* Corresponding author. Tel.: þ886 3 5753147; fax: þ886 3 5711082.
E-mail address: cclin66@mx.nthu.edu.tw (C.-C. Lin).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.04.022

C.-C. Lin et al. / Tetrahedron 65 (2009) 4714–4725
4715
HO
OH
CO2Me
STol
AcO
TFAHN
O
AcO
1. Sialylation
2. Acetylation
AcO
OAc
AcO
OAc
AcO
CO2Me
O
CO2Me
O
AcO
TFAHN
AcO
TFAHN
O
O
OAc
MeO2C
OAc
MeO2C
AcO
AcO
AcO
TFAHN
O
O
STol
OP(OBn)2
m
TFAHN
2
1
n
AcO
AcO
m = 0~3
n = m+1
1. Thiocresol deprotection
2. Phosphite formation
Scheme 1. Oligosialic acid synthesis through iterative sialylation.
Our previous study employed N-trifluoroacetyl-protected sialyl
phosphite as a general glycosyl donor in the synthesis of (2,9)-
oligosialic acids and has studied their -selectivity in sialylations
(Scheme 1).14
(2,9) Penta-sialic acid was synthesized using phos-
phite donor and iterative sialylations to elongate the sugar chain
from the non-reducing end to the reducing end. However, the
synthesis of homo-oligosaccharide using a convergent approach is
more practical and efficient. Thus, tetra-sialic acid can conceivably
be prepared by realization of the glycosylation of disaccharide
donor and acceptor, a [2þ2] coupling strategy. Herein, we describe
the synthesis of tetra-sialic acid by coupling disaccharide phosphite
donor with disaccharide acceptor. Also, mono- and di-sialic acids
are conjugated with keyhole limpet hemocyanin (KLH) to synthe-
acceptors.7–14 In the design of sialic acid donors and acceptors, many
efforts have been made to discover the best combination of the
protecting and leaving groups at the C-5 amino functional group
and the anomeric center, respectively. Based on our recent find-
ings,14 the strong electron-withdrawing nature of the C-5 NHTFA
a
a
a
group was expected to enhance the a-selectivityof sialic acid donors
and the primary C-9 hydroxyl group of sialic acid acceptors, as
shown in Figure 1. Additionally, the TFA group can be introduced
efficiently and removed easily under mild reaction conditions.
Although various leaving groups are known to increase the a-
selectivity of sialyl donors,15 per-O-acetyl-N-acetylneuraminic acid
methyl ester derivatives with either phosphites15d,e or aryl sulfi-
des15b,c at the anomeric center are mainly used in chemical sialy-
lation. Notably, the order of activation of donors can be tuned, not
only by the protecting groups, but also by a combination of acti-
vators and leaving groups on them.8g,14 The C-7, C-8, and C-9 hy-
droxyl groups on the exocyclic chain of sialyl acceptors and donors
can be selectively protected as the bulky tert-butyldimethylsilyl
(TBDMS)8g,14 group or as the electron-withdrawing chloroacetyl
size a(2,9) sialic acid-based vaccine. The conjugation efficiency is
examined by using the enzymatic or acidic hydrolysis of sialic acid
from carrier protein, followed by Warren’s method.
2. Results and discussion
(ClCH2CO)16 group. While the
a-phenylthiosialoside-based donors
2.1. Design of sialyl donors and acceptors
are relatively stable, the phosphite-based donors exhibit high ac-
tivity when a catalytic amount of promoter (typically trimethylsilyl
trifluoromethanesulfonate, TMSOTf) is used. Based on these
The outcomes of sialylations are well known to depend primarily
on the nature of the protecting groups on glycosyl donors and
AcO
AcO
OAc
HO
OTBDMS
O
OP(OBn)2
CO2Me
O
O
CO2Me
STol
OP(OBn)2
CO2Me
TBDMSO
TFAHN
O
AcO
RHN
O
TFAHN
AcO
TBDMSO
5
AcO
4
3a: R = Ac
3b: R = Troc
3c: R = TFA
HO
HO
OH
HO
OH
CO2Me
STol
CO2Me
HO
OH
CO2Me
AcO
O
O
TFAHN
O
TFAHN
AcO
O
STol
BzO
8
TFAHN
AcO
AcO
7
AcO
OR
6
OAc
O
RO
CO2Me
OP(OBn)2
CO2Me
AcO
TFAHN
AcO
O
OAc
O
TFAHN
OP(OBn)2
CO2Me
AcO
AcO
AcO
O
10
TFAHN
9: R = ClCH2CO
AcO
Figure 1. Structure of sialyl donors (3, 4, 9, and 10) and acceptors (5–8).

4716
C.-C. Lin et al. / Tetrahedron 65 (2009) 4714–4725
AcO
AcO
OAc
HO
OH
HO
OH
CO2Me
STol
CO2Me
STol
CO2H
OH
Ac2O, Pyr
ref. 8f
HO
RHN
HO
11: R = H
O
HO
O
TFAHN
O
AcHN
rt, 12h, 98%
AcO
HO
13
14
CF3CO2Me, Et3N
MeOH, 94%
12: R = TFA
AcO OAc
CO2Me
1H-tetrazole, CH3CN,
NBS, acetone/H2O
rt, 3 h, 86%
AcO
TFAHN
O
3c
OH
DDP, 1H-tetrazole
rt, 15 min, 97%
AcO
15
1) (CH3)2C(OMe)2,
acetone, CSA
rt, 12h
O
O
1) chloramine-T,
CO2Me
STol
CH3CN, rt. 4 h, 72%
AcO
TFAHN
O
12
4
2) DDP, 1H-tetrazole,
CH3CN, rt, 15 min, 97%
2) Ac2O, pyr. rt, 12 h
77% (two steps)
AcO
16
TFA, H2O, CH2Cl2,
0 °C, 10 min, 78%
6
Scheme 2. Synthesis of donors 3c and 4, and acceptor 6.
considerations, C-5 NHTFA-protected sialyl phosphites (3, 4, 9, and
10) were synthesized as building blocks for constructing the (2-9)-
linked oligosialic acids, while thiocresol was chosen as an anomeric
protecting group (Fig. 1).
(DDP) in CH3CN at room temperature in 97% yield. Selective 8,9-O-
isopropylidenation of 12 was followed by acetylation of secondary
hydroxyl groups to give thioglycoside 16 in 77% overall yield in two
steps. The aryl sulfide group without the deprotection of the aceto-
nide in 16 was selectively removed using chloramine-T.18 Notably, the
use of NBS led to the deprotection of thiocresol and isopropylidene.
The resulting free hydroxyl group at the anomeric center was con-
verted to phosphite 4 (97%) using DDP at room temperature in a very
short reaction period (w15 min). Meanwhile, the acid-catalyzed de-
O-isopropylidenation of 16 provided the desired acceptor 6 in 78%
yield.
a
2.2. Synthesis of donors and acceptors
As presented in Scheme 2, the synthesis of donors 3c14 and 4, and
acceptor 614 commenced from the key intermediate 12, which was
prepared by the acetylation of 11 obtained from Neu5Ac 13 by fol-
lowing reported procedures.8f The per-O-acetylation of 12 afforded 14
inalmost quantitative yield. Donor 3c wasthen efficientlyobtained by
the N-bromosuccinimide (NBS)-mediated removal of thiocresol-
protecting group17 to give anomeric alcohol 15 (86%) and then
phosphite was formed using dibenzyl diisopropylphosphoramidite
The synthesis ofacceptor 7 beganwith the known compound 1819
(Scheme 3). Saponification of 18 followed by N-trifluoroacetylation8c
with CF3CO2Me/Et3N/MeOH and then esterification using MeI in the
presence of Cs2CO3 gave TFA derivative 20.20 The desired acceptor 7
OR
RO
RO
RHN
OAc
AcO
AcO
AcHN
CO2R'
O
CO2Me
OAc
1) AcCl, HCl(g), 0 °C, 36h
O
O
2) allyl alcohol, silver salicylate,
rt, 2h, 88% (two steps)
RO
AcO
17
18: R = Ac, R' = Me
NaOH,
MeOH/H2O,
80 °C, 95%
19: R = R' = H
HO
OH
1) CF3CO2Me,
CO2Me
O
VO(OTf)2.2H2O,
DMP, CH3CN
92%
NEt3, MeOH
HO
TFAHN
O
2) Cs2CO3, MeI, DMF, rt,
86 % (two steps)
HO
20
O
O
CO2Me
O
TFA / H2O
CH2Cl2, 0 °C, 84%
7
RO
O
TFAHN
RO
21: R = H
Ac2O, Pyr
rt, 96%
22: R = Ac
Scheme 3. Synthesis of acceptor 7.

C.-C. Lin et al. / Tetrahedron 65 (2009) 4714–4725
4717
ClH2COCO
OCOCH2Cl
2.3. Evaluation of sialylation
CO2Me
STol
ClCH2COCl, pyr
AcO
O
With the sialyl phosphites 3–4 and 9, and acceptors 5–8 in hand,
we examined the sialylation reactions of these sialyl donors and
acceptors. The combination of TMSOTf as an activator and CH3CN as
solvent in the presence of molecular sieves (3 Å) at ꢀ40 ꢁC was used
6
TFAHN
dry CH2Cl2, - 10 °C, 90%
AcO
23
in the sialylation because it is known to improve a
-selectivity.23 The
1) NBS, acetone, H2O
rt, 86%
choice of an appropriate donor–acceptor combination was re-
sponsible for the obtaining of the predominantly -(2,9)-linked
disialosides 25–32 in 33–77% yields (Fig. 2 and Table 1).
9
23
a
2) DPP, 1H-tetrazole,
CH3CN, MS 3 °A, rt, 97%
Table 1
Sialylations of C-5 N-protected sialyl phosphites
1) VO(OTf)2.2H2O, DMP, CH3CN
2) BzCl, pyr, - 15 °C
3) TFA, H2O, CH2Cl2, - 10 °C,
65% (three steps)
PO
OP
PO
OP
8
12
CO2Me
OR
OP(OBn)2
CO2Me
Acceptor
TMSOTf (0.2 eq.),
CH3CN, 3 Å MS, -40 °C
AcO
RHN
AcO
RHN
O
O
AcO
AcO
Scheme 4. Synthesis of donor 9 and acceptor 8.
Entry
Donor
Acceptor
Product
Yield (%)
a/b
was easily obtained by selective protection of terminal diol using our
developed vanadyl triflate-catalyzed21 isopropylidenation followed
by O-acetylation with Ac2O/pyridine and then acid (aq CF3CO2H in
CH2Cl2)-catalyzed de-O-isopropylidenation.
1
2
3
4
5
6
7
8
9
3a
3b
3a
3c
3c
4
4
9
3c
5
5
6
6
7
6
7
6
8
24
25
26
27
28
29
30
31
32
83
33
55
77
70
68
64
75
72
d
1.5/1
4.7/1
a
The 8,9-O-dichloroacetyl-protected phosphite donor 9 was
synthesized from readily available acceptor 6 in three steps. Acyl-
ation of 6 with chloroacetyl chloride in CH2Cl2 followed by thio-
cresol deprotection (NBS, acetone/H2O) and subsequent phosphite
formation of the resulting anomeric hydroxyl group using DDP in
CH3CN gave the desired donor 9 in overall 80% yield (Scheme 4).
8,9-O-isopropylidenation of 12 followed by regioselective benzoy-
lation with benzoyl chloride in pyridine at ꢀ15 ꢁC and then de-O-
isopropylidenation (aq CF3CO2H in CH2Cl2) provided the acceptor
822 in 65% overall yield in three steps (Scheme 4).
6/1
3/1
1.3/1
2.5/1
a
According to Table 1, the bulky acceptor 524 coupled with donor
3b to give the desired dimer 25 in a modest yield with poor selec-
tivity (entry 2); only 2,3-elimination product 24 (entry 1) was
obtained when 3a was used as a donor. However, when less
AcO
OAc
AcO
AcO
AcO
OAc
CO2Me
O
AcO
O
CO2Me
O
TrocHN
OTBDMS
O
AcHN
CO2Me
STol
AcO
TBDMSO
AcHN
24
25
TBDMSO
AcO
AcO
OAc
CO2Me
O
AcO
OAc
AcO
RHN
O
OH
CO2Me
CO2Me
STol
AcO
AcO
AcO
O
O
TFAHN
O
OH
R/HN
CO2Me
O
AcO
AcO
TFAHN
O
26: R = Ac, R/ = TFA
27: R = R/ = TFA
28
AcO
O
O
O
O
CO2Me
CO2Me
AcO
AcO
O
O
TFAHN
O
OH
TFAHN
O
OH
CO2Me
O
CO2Me
STol
AcO
AcO
AcO
AcO
TFAHN
O
O
TFAHN
30
29
AcO
AcO
AcO
OAc
CO2Me
O
RO
AcO
OR
CO2Me
AcO
TFAHN
O
OH
O
CO2Me
STol
AcO
TFAHN
O
OH
CO2Me
STol
HO
AcO
O
TFAHN
AcO
O
BzO
TFAHN
AcO
31: R = ClCH2CO
32
Figure 2. Products of sialylations.

4718
C.-C. Lin et al. / Tetrahedron 65 (2009) 4714–4725
sterically hindered acceptor 6 was reacted with 3a, both the
a
-se-
2.4. Synthesis of oligosialic acid
lectivity and the product yield were markedly increased (entry 3).
These results suggest that the combination of the donor with the
electron-withdrawing group at the C-5 position and a less sterically
hindered acceptor can improve the a-selectivity and product yield.
Therefore, a TFA-protected, donor 3c was used to react with 6; TFA is
Although penta-sialic acid was successfully synthesized by
elongating the sugar chain from the non-reducing end to the re-
ducing end using monosaccharide acceptor,14 this approach gave
a low overall yield because of the consecutive linear synthetic
strategy. To assemble sugar chain efficiently, the [2þ2] convergent
strategy was adopted to synthesize tetra-sialoside 36. The synthesis
of tetra-sialoside depends on access to a suitable disialoside to
serve as a glycosyl donor and an acceptor. Therefore, disialyl
phosphite donor 10 and acceptor 33, as shown in Scheme 5, were
selected. Disialoside 10 was prepared by the same methods as de-
scribed for the synthesis of phosphite donor 3c, while disialyl ac-
ceptor 33 was obtained by the method used to prepare acceptor 6.
The optimized reaction conditions supported the synthesis of dis-
ialosides 10 and 33 in very high yields.
a more strongly electron-withdrawing protecting group. As expec-
ted, disialoside 27 was obtained with exclusive
yield (entry 4). Surprisingly, when the anomeric protecting group
STol of 6 was changed to an allyl group (7), the -selectivity of sia-
lylation with donor 3c was reduced (entry 5). Notably, when the
acetonide-protected donor 4 reacted with 6, the -selectivity also
declined (entry 6). Additionally, low -selectivity was observed
when 4 reacted with 7 (entry 7). Moreover, although the O-chloro-
acetyl-protecting sialyl donor exhibited good
-selectivity,16 the
a-anomer in 77%
a
a
a
a
8,9-O-dichloroacetyl-protected phosphite donor 9 reacted with
acceptor 6 to give disialoside 31 in 75% yield with relatively low
anomeric selectivity (entry 8). However, the sialylation of phosphite
donor 3c with thiosialoside 8 under the aforementioned conditions
Scheme 5 outlines a new route to tetra-sialoside 36 by a con-
vergent [2þ2] strategy from disialyl phosphite donor 10 and ac-
ceptor 33. As anticipated, upon O-sialylations under standard
conditions (with CH3CN as solvent and a catalytic amount of
TMSOTf as promoter at ꢀ40 ꢁC), the glycosylation proceeded with
resulted in 72% yield of disialoside 32 (entry 9) with exclusive
a-
anomer. These results show that the -selectivity was seriously af-
a
fected by the protecting group on the donor and the protecting
group at the anomeric center of the acceptor. The combination of
TFA as the C-5 protecting group, phosphite as the leaving group of
the donor, and STol as the anomeric protecting group of the acceptor
reasonable selectivity (
a
/
b
¼1.6/1), giving tetra-sialoside 36 in
moderate yield (58%). The newly generated glycosidic bond of the
tetra-sialoside 36 was identified by the analysis of 1H and 13C NMR
data of 36. Notably, however,
a and b anomers of 36 remained
ensured good
a-selectivity and yield in sialylation.
unseparable under many chromatographic conditions.
The configurations of the newly formed glycosidic bond of
disialosides were determined by 1H NMR spectroscopy from the
chemical shifts of H-3e of the non-reducing end sialic acid and
relevant empirical rules.15a,25 The chemical shifts of H-3e of
2.5. Conjugation of sialic acid with carrier protein
To synthesize an a(2–9) sialic acid-based vaccine, sialic acid was
a
-glycosides were more downfield than those of b-glycosides. In
conjugated with KLH and the conjugation efficiency was analyzed.
KLH is an extra-cellular respiratory protein that is widely recog-
nized as a hapten carrier and an immune stimulant. It has also been
found to be an effective immunogenic carrier protein for carbohy-
drate antigens.27 Sialic acid antigen was conjugated with KLH using
a bifunctional linker m-maleimidobenzoyl-N-hydroxysuccinimide
(MBS). As described by Scheme 6, the terminal olefin contained in
sialosides 37 was firstly transformed to thioacetate by the radical
addition of thiolacetic acid in the presence of AIBN27,28 to yield
quantitatively the desired thioacetates 38. The basic removal of the
acetates using sodium methoxide in methanol followed by
the case of compound 34 (Fig. 3(a)), only one isomer was obtained,
and the long-range JC-1,H-3ax coupling constant was measured to
verify the stereochemistry at the anomeric center. In a selective
proton decoupling 13C NMR experiment,26 the coupling pattern of
non-reducing C-1 gave a doublet signal with a 5.6 Hz coupling
constant. A similar method was applied to compound 28 to de-
termine the stereochemistry. As presented in Figure 3(b), two
doublet signals of C-1s with coupling constants 6.0 and 6.3 Hz,
respectively, were observed, verifying the assignment of two
a
-glycosidic bonds.
(b)
AcO
OAc
OAc
(a) AcO
CO2Me
O
CO2Me
O
AcO
TFAHN
O
AcO
TFAHN
O
OH
OAc
AcO
AcO
CO2Me
O
H3ax`
AcO
CO2Me
O
STol
H3ax`
AcO
O
28
AcO
TFAHN
34
TFAHN
AcO
H3ax
JC-1, H-3ax` = 5.6 Hz
JC-1, H-3ax` = 6.0 Hz
JC-1, H-3ax = 6.3 Hz
168.3 168.2 168.1 168.0 167.9 167.8 167.7 167.6 167.5 167.4 167.3
168.7 168.6 168.5 168.4 168.3 168.2 168.1 168.0 167.9 167.8
Figure 3. C-1 portion of selective proton-decoupled 13C NMR spectra of 28 and 34.

C.-C. Lin et al. / Tetrahedron 65 (2009) 4714–4725
4719
27: R = H
Ac2O, Pyr,
rt, 93%
34: R = OAc
30
NBS,
acetone/H2O,
rt, 3h, 86%
1) Ac2O, Pyr, rt
2)TFA/H2O, CH2Cl2 , 0 °C,
78% (two steps)
AcO
AcO
OAc
AcO
CO2Me
O
TFAHN
O
OAc
CO2Me
OH
AcO
TFAHN
O
HO
OH
35
AcO
CO2Me
AcO
TFAHN
O
DDP, 1H-tetrazole,
CH3CN, rt, 15 min, 92%
O
OAc
AcO
AcO
CO2Me
O
AcO
TFAHN
O
10
33
TMSOTf (0.2 eq.)
CH3CN, 3Å MS,
- 40 °C, 58%
AcO
AcO
OAc
AcO
CO2Me
O
TFAHN
O
OAc
CO2Me
AcO
TFAHN
O
O
OH
CO2Me
O
AcO
AcO
O
TFAHN
OAc
CO2Me
O
AcO
36
AcO
O
TFAHN
AcO
Scheme 5. Synthesis of a-(2/9) linked tetra-sialoside 36.
saponification of the methyl ester and TFA amide yielded the fully
deprotected sialosides. Finally, per-O-acetylation and N-acetylation
with acetic anhydride and pyridine followed by global deacetyla-
tion under basic conditions (NaOMe in MeOH) gave the target
thiosialoglycosides 39a as a dimer and 39b as a monomer.
selectivity. Although the [2þ2] convergent strategy was an efficient
means of assembling tetra-sialic acid, the low -selectivity and puri-
fication of -anomer remain to be solved. The assay of the sialic acid
amounton synthetic vaccine byacidhydrolysis may beinterruptedby
the formation of Neu5Ac2,3ene. Enzymatic hydrolysis is recom-
mended to assay sialic acid on the carrier protein. The syntheses of
oligosialic acid-conjugated vaccines and the evaluation of their im-
munogenicities areunderwayandwillbedescribedinthenearfuture.
a
a
To covalently attach sialo-antigens to carrier protein, the lysine
residues of KLH were converted to maleimide by reacting with
MBS, as shown in Scheme 6. Conjugation to the carrier protein was
achieved through the Michael addition of the thiol nucleophile of
antigens 39, which was pretreated with NaBH4 to give the vaccine
constructs 40. Warren’s method29 was adopted to assay the
amounts of sialic acid on KLH. We used two methods to release free
sialic acid from carrier protein (see Experimental section for de-
tails). Firstly, the sialic acid was hydrolyzed by sulfuric acid and
then reacted with thiobarbituric acid to give a chromophoric
product, which was measured by UV. Alternatively, sialic acid was
hydrolyzed by the sialidase and the resulting mixture was then
assayed by Warren’s method. Surprisingly, the amount of sialic acid
obtained by sialidase hydrolysis was 10 times higher than that
obtained by acid hydrolysis. The amount of sialic acid estimated by
4. Experimental section
4.1. General methods
1H and 13C NMR spectra were recorded on Bruker AV-400 or
500 MHz. Assignment of 1H NMR spectra was achieved using 2D
methods (COSY). Chemical shifts were expressed in parts per mil-
lion using residual CHCl3 as reference. High-resolution mass spec-
tra were obtained by means of a Micromass (Autospec) mass
spectrometer. Analytical thin-layer chromatography (TLC) was
performed on precoated plates (Merck silica gel 60 F-254). Silica gel
60 (E. Merck Co.) was employed for all flash chromatographies. All
solvents are commercially available and dried by general methods.
Compounds 3a,14 3b,14 3c,14 6,14 8,22 12,14 16,14 18,19 19,20 21,21b
25,14 26,14 and 2714 have been previously prepared and reported in
the cited papers and their NMR spectra are in good agreement with
the literature data.
sialidase hydrolysis is 22.7
mg per 100
mg of vaccines 40a, while
acidic hydrolysis only gives 1.9
mg. This difference may arise be-
cause periodate oxidation of sialic acid in strong acidic solution
may lead to the elimination product Neu5Ac2,3ene, and thereby
reducing in the production of the final chromophoric compound.
3. Conclusion
4.1.1. Methyl (4-methylphenyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
The combination of phosphite donor and anomeric thiocresol-
protected acceptor, both with a TFA protecting group at the C-5 of
2-thio-5-trifluoroacetamido-
nonulopyranoside)onate (14)
D-glycero-a-D-galacto-2-
sialic acid, provided better
protecting group on both donor and acceptor strongly affected
a
-selectivity and yield in sialylation. The
To a solution of 11 (308 mg, 0.64 mmol) in pyridine (4 mL) was
added acetic anhydride (2 mL) at 0 ꢁC. The reaction was stirred for
a-

4720
C.-C. Lin et al. / Tetrahedron 65 (2009) 4714–4725
AcO
OAc
AcO
CO2Me
O
AcO
TFAHN
O
HSAc, AIBN
MeOH, reflux
OAc
MeO2C
O
AcO
TFAHN
O
n
37a: n = 0
37b: n = 1
AcO
AcO
OAc
CO2Me
AcO
TFAHN
O
1) NaOMe, MeOH
O
OAc
AcO
MeO2C
O
2) NaOH (0.5N)
AcO
TFAHN
3) Ac2O, Py
4) NaOMe, MeOH
38a: n = 0, 85%
38b: n = 1, 81%
O
n
SAc
AcO
HO
OH
CO2H
O
HO
AcHN
O
OH
HO
CO2H
HO
AcHN
O
O
n
S
HO
2
39a: n = 0, four steps, 60% (as dimer)
39b: n = 1, four steps, 51% (as monomer)
O
H
N
MBS
NH2
KLH
N
KLH
HO
O
O
39, NaBH4
OH
CO2H
O
KLH
HO
AcHN
O
O
N
N
H
O
OH
HO
CO2H
O
HO
AcHN
O
40a: n = 0
40b: n = 1
S
O
n
HO
Scheme 6. Functionalization of allyl glycosialosides en route to KLH conjugation.
12 h at room temperature and then concentrated in vacuo. The
residue was diluted with EtOAc and the resulting solution was
washed with 10% HCl, saturated NaHCO3, and saturated NaCl in
sequence. The organic phase was dried (MgSO4) and filtered, and
the filtrate was evaporated. The residue was purified by silica gel
column chromatography (EtOAc/hexane¼1/4) to afford 14 (301 mg,
EtOAc and the resulting solution was washed with H2O, saturated
NaHCO3, and saturated NaCl in sequence. The organic phase was
dried (MgSO4) and filtered, and the filtrate was evaporated. The
residue was purified by silica gel column chromatography (EtOAc/
hexane¼1/2) to afford 15 (165 mg, 86%): 1H NMR (400 MHz, CDCl3)
d
1.98 (s, 3H), 2.02 (s, 3H), 2.09 (s, 3H), 2.14 (s, 3H), 2.26 (m, 2H), 3.84
98%): 1H NMR (400 MHz, CDCl3)
d
1.97 (dd, J¼11.5,12.9 Hz,1H), 2.00
(s, 3H), 4.01 (dd, J¼8.7, 12.0 Hz, 1H), 4.15 (q, J¼10.2 Hz, 1H), 4.48 (d,
J¼10.2 Hz, 1H), 4.71 (d, J¼12.0 Hz, 1H), 5.22 (td, J¼5.2, 10.2 Hz, 1H),
5.31 (d, J¼8.7 Hz,1H), 5.36 (s,1H), 5.57 (s,1H), 7.84 (d, J¼10.0 Hz,1H);
(s, 3H), 2.04 (s, 3H), 2.05 (s, 3H), 2.14 (s, 3H), 2.36 (s, 3H), 2.84 (dd,
J¼4.7, 12.9 Hz, 1H), 3.6 (s, 3H), 3.84 (ddd, J¼9.6, 10.2, 10.2 Hz, 1H),
4.06 (dd, J¼1.7, 10.2 Hz, 1H), 4.23 (m, 1H), 4.42 (dd, J¼1.7, 11.5 Hz,
1H), 4.99 (ddd, J¼4.7, 10.2, 11.5 Hz, 1H), 5.24 (br, 2H), 6.42 (d,
J¼9.6 Hz, 1H), 7.14 (d, J¼8.0 Hz, 2H), 7.33 (d, J¼8.0 Hz, 2H); 13C NMR
13C NMR (100 MHz, CDCl3)
d 20.04, 20.09, 20.32, 20.34, 35.82, 49.32,
52.85, 67.84, 68.47, 70.67, 72.19, 94.58, 115.04, 157.35, 168.25, 168.25,
169.40, 170.08, 171.16, 172.38; HRMS (FAB) calcd for C20H27F3NO13
(MþH)þ: 546.1435, found: 546.1436.
(125 MHz, CDCl3)
d 20.50, 20.66, 20.87, 21.26, 37.81, 49.78, 52.72,
61.91, 67.59, 69.15, 73.91, 87.5, 115.37, 124.75, 129.66, 136.50, 140.39,
157.48, 167.76, 169.79, 170.58, 170.64, 171.01; HRMS (FAB) calcd for
C27H33F3NO12S (MþH)þ: 652.1676, found: 652.1664.
4.1.3. Dibenzyl [Methyl (4,7-di-O-acetyl-3,5-dideoxy-8,9-O-
isopropylidene-5-trifluoroacetamido-
D-glycero-a/b-D-galacto-
non-2-ulopyranosid)onate] phosphite (4)
4.1.2. Methyl (4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-
To a solution of 16 (121 mg, 0.2 mmol) in MeCN (2 mL) was added
chloramine-T (85 mg, 0.3 mmol). The reaction was stirred for 4 h at
room temperature. Then the solution was concentrated in vacuo. The
residuewasdilutedwithEtOAcandtheresultingsolutionwaswashed
with H2O, saturated NaHCO3, and saturated NaCl in sequence. The
organic phase was dried (MgSO4) and filtered, and the filtrate was
evaporated. The residue was purified by silica gel column
trifluoroacetamido-D-glycero-a-D-galacto-2-
nonulopyranosid)onate (15)
To a solution of 14 (230 mg, 0.35 mmol) in acetone (4.5 mL) and
water (0.4 mL) was added NBS (62 mg, 0.53 mmol). The reaction was
stirred for 30 min at room temperature. Then, the solution was
concentrated under reduced pressure. The residue was diluted with

C.-C. Lin et al. / Tetrahedron 65 (2009) 4714–4725
4721
chromatography to afford hydroxyl compound (72%). To a solution of
the above hydroxyl compound (65 mg, 0.14 mmol) in MeCN (3.7 mL)
were added DDP (0.45 mL, 1.4 mmol) and 1H-tetrazole (0.1 g,
1.4 mmol) at ꢀ40 ꢁC under argon atmosphere. After being stirred for
5 min at ꢀ40 ꢁC, the solution was further stirred for 15 min at room
temperature. The solution was diluted with EtOAc, and the resulting
solution was washed with saturated aqueous NaHCO3. The organic
layer was dried (MgSO4) and concentrated. The residue was purified
by flashcolumnchromatography to give 4 (97%): some selected peaks
dried (MgSO4) and filtered, and the filtrate was concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (EtOAc/hexane¼1/1) to give 7 (85%): 1H NMR
(500 MHz, CDCl3)
d
2.02 (s, 3H), 2.04 (dd, J¼13.0, 11.9 Hz, 1H), 2.79
(dd, J¼5.0,13.0 Hz,1H), 3.0 (br, 2H), 3.49 (dd, J¼3.7,12.5 Hz,1H), 3.70
(dd, J¼2.5, 12.5 Hz, 1H), 3.87 (s, 3H), 3.90–3.96 (m, 2H), 4.11 (dd,
J¼2.0, 10.5 Hz, 1H), 4.22 (ddd, J¼9.9, 10.2, 10.5 Hz, 1H), 4.28 (dddd,
J¼1.6, 1.6, 5.6, 12.6 Hz, 1H), 4.97 (ddd, J¼5.0, 10.5, 11.9 Hz, 1H), 5.07
(dd, J¼2.0, 7.5 Hz, 1H), 5.19 (dddd, J¼1.6, 1.6, 1.6, 10.7 Hz, 1H), 5.28
(dddd, J¼1.6,1.6,1.6,17.2 Hz,1H), 5.86 (dddd, J¼5.6, 5.6,10.7,17.2 Hz,
of 4b d 1.29 (s, 3H), 1.32 (s, 3H), 1.93 (dd,
: 1H NMR (500 MHz, CDCl3)
J¼11.8, 13.0 Hz, 1H), 1.96 (s, 3H), 2.06 (s, 3H), 2.42 (dd, J¼4.9, 13.0 Hz,
1H), 3.50 (dd, J¼2.2, 10.5 Hz, 1H), 3.72 (s, 3H), 3.86 (dd, J¼7.8, 8.7 Hz,
1H), 3.94 (ddd, J¼10.2, 10.5, 10.5 Hz, 1H), 3.99 (dd, J¼6.5, 8.7 Hz, 1H),
4.10 (ddd, J¼3.3, 6.5, 7.8 Hz, 1H), 4.91 (ddd, J¼4.9, 10.5, 11.8 Hz, 1H),
4.95–5.07 (m, 5 H), 5.58 (d, J¼10.2 Hz, 1H), 7.24–7.44 (m, 10H).
1H), 7.76 (d, J¼9.9 Hz, 1H); 13C NMR (125 MHz, CD3OD)
d 20.83,
21.05, 37.63, 49.65, 53.79, 62.31, 65.87, 68.86, 69.34, 69.57, 72.06,
98.71, 115.80, 117.71, 133.40, 158.13, 169.89, 171.12, 171.72; HRMS
(FAB) calcd for C19H27F3NO11 (MþH)þ: 502.1536, found: 502.1534.
4.1.7. Methyl (4-methylphenyl 4,7-di-O-acetyl-8,9-di-O-
4.1.4. Methyl (2-allyl-3,5-dideoxy-5-trifluoroacetamido-
-galacto-2-nonulopyranosid)onate (20)
Compound 19 (2.56 g, 8.34 mmol) was dissolved in MeOH
D
-glycero-
chloroacetyl-3,5-dideoxy-2-thio-5-trifluoroacetamido-
-galacto-2-nonulopyranoside)onate (23)
To the solution of 6 in dry CH2Cl2 and pyridine was added
D-glycero-a-
a-D
D
(80 mL). Triethylamine (9.36 mL, 66.72 mmol) and methyl tri-
fluoroacetate (3.36 mL, 33.36 mmol) were added to the solution
dropwise at room temperature, respectively. The solution was
stirred for 1.5 h at room temperature. After the reaction was com-
pleted, the solution was evaporated and to the residue were added
DMF (80 mL), Cs2CO3 (2.7 g, 8.34 mmol), and MeI (2.6 mL,
41.7 mmol) sequentially. The reaction mixture was allowed to
proceed for 12 h at room temperature. After completion of the re-
action, the mixture was evaporated and the resulting residue was
purified by column chromatography (EtOAc/hexane¼1/1, with 15%
MeOH) to give product 20 (3.0 g, 86% for two steps): 1H NMR
chloroacetyl chloride at ꢀ10 ꢁC. The reaction was stirred for 3 h at
ꢀ10 ꢁC. Then the reaction was quenched with MeOH, and the re-
action mixture was diluted with dichloromethane (10 mL), and
then washed with saturated 10% HCl, NaHCO3, and saturated NaCl
in sequence. The organic phase was dried (MgSO4) and filtered, and
the filtrate was concentrated under reduced pressure. The residue
was purified by silica gel column chromatography to give 23 (90%):
1H NMR (400 MHz, CDCl3)
d
1.99 (s, 3H), 2.00 (dd, J¼11.6, 13.0 Hz,
1H), 2.14 (s, 3H), 2.35 (s, 3H), 2.82 (dd, J¼4.8, 13.0 Hz, 1H), 3.60 (s,
3H), 3.88–4.02 (m, 2H), 4.02 (d, J¼15.2 Hz, 1H), 4.03 (s, 2H), 4.10 (d,
J¼15.2 Hz, 1H), 4.26 (dd, J¼4.8, 12.4 Hz, 1H), 4.52 (dd, J¼2.0, 12.4 Hz,
1H), 4.91 (ddd, J¼4.8, 10.4, 11.6 Hz, 1H), 5.25 (dd, J¼7.6, 2.0 Hz, 1H),
5.29 (tdd, J¼7.6, 4.8, 2.0 Hz, 1H), 6.37 (br d, J¼9.2 Hz, 1H), 7.14 (d,
J¼8.0 Hz, 2H), 7.31 (d, J¼8.0 Hz, 2H); 13C NMR (125 MHz, CDCl3)
(400 MHz, CD3OD)
d
1.78 (dd, J¼12.4, 12.4 Hz, 1H), 2.69 (dd, J¼4.6,
12.4 Hz, 1H), 3.46 (dd, J¼1.4, 8.6 Hz, 1H), 3.63 (dd, J¼6.4, 11.9 Hz,
1H), 3.71 (ddd, J¼4.6, 10.0, 12.4 Hz, 1H), 3.81 (s, 3H), 3.82–3.84 (m,
2H), 3.87 (dd, J¼1.4, 10.5 Hz, 1H), 3.98 (dd, J¼10.0, 10.5 Hz, 1H), 4.00
(dddd, J¼1.6,1.6, 5.4,12.8 Hz,1H), 4.30 (dddd, J¼1.6,1.6, 5.4,12.8 Hz,
1H), 5.12 (dddd, J¼1.6,1.6,1.6,10.5 Hz,1H), 5.24 (dddd, J¼1.6,1.6,1.6,
17.2 Hz, 1H), 5.87 (dddd, J¼5.4, 5.4, 10.5, 17.2 Hz, 1H); 13C NMR
d
20.53, 20.61, 21.33, 37.95, 40.61, 40.82, 50.07, 52.96, 62.89, 66.76,
68.86, 70.77, 73.41, 87.45, 115.35, 124.41, 129.90, 136.48, 140.75,
157.91, 166.44, 167.02, 167.70, 169.94, 170.83; HRMS (FAB) calcd for
C27H31Cl2F3NO12S (MþH)þ: 720.0896, found: 720.0904.
(100 MHz, CD3OD)
d 41.83, 53.48, 54.02, 64.85, 66.43, 68.55, 70.27,
72.85, 73.95, 100.23, 117.12, 117.54, 135.56, 159.79, 170.92; HRMS
4.1.8. Dibenzyl [methyl (4,7-di-O-acetyl-8,9-O-chloroacetyl-3,5-
(FAB) calcd for C15H23F3NO9 (MþH)þ: 418.1325, found: 418.1317.
dideoxy-5-trifluoroacetamido-D-glycero-a/b-D-galacto-non-2-
ulopyranosid)onate] phosphite (9)
4.1.5. Methyl (2-allyl-4,7-di-O-acetyl-3,5-dideoxy-8,9-O-
isopropylidene-5-trifluoroacetamido-a-D-glycero-D-galacto-2-
nonulopyranosid)onate (22)
To a solution of 23 (0.38 mmol) in acetone (4.5 mL) and water
(0.4 mL) was added NBS (62.30 mg, 0.53 mmol). The reaction was
stirred for 30 min at room temperature. Then the solution was
concentrated in vacuo. The residue was diluted with EtOAc and
was washed with H2O, saturated NaHCO3, and saturated NaCl in
sequence. The organic phase was dried (MgSO4) and filtered, and
the filtrate was evaporated. The residue was purified by silica gel
column chromatography to afford hydroxyl compound (86%). To
a solution of the above hydroxyl compound (0.2 mmol) in MeCN
(3.7 mL) were added DDP (0.45 mL, 0.14 mmol) and 1H-tetrazole
(0.1 g, 1.4 mmol) at ꢀ40 ꢁC under argon atmosphere. After being
stirred for 5 min at ꢀ40 ꢁC, the solution was further stirred for
30 min at room temperature. The solution was diluted with EtOAc,
and washed with saturated aqueous NaHCO3. The organic layer was
dried (MgSO4) and concentrated. The residue was purified by flash
Compound 22 (96%) was obtained from 21 by the method de-
scribed in the synthesis of 14: 1H NMR (500 MHz, CDCl3)
d
1.99 (dd,
J¼12.0, 13.0 Hz, 1H), 2.03 (s, 3H), 2.16 (s, 3H), 2.72 (dd, J¼5.0,
13.0 Hz, 1H), 3.83 (s, 3H), 3.97–4.00 (m, 2H), 4.01 (dd, J¼6.5, 8.5 Hz,
1H), 4.07 (dd, J¼2.0, 9.5 Hz, 1H), 4.08 (dd, J¼6.5, 8.5 Hz, 1H), 4.26
(dddd, J¼1.5, 1.5, 5.5, 13.0 Hz, 1H), 4.38 (ddd, J¼4.0, 6.5, 6.5 Hz, 1H),
5.11 (ddd, J¼5.0,10.0,12.0 Hz,1H), 5.20 (dddd, J¼1.5,1.5,1.5,10.5 Hz,
1H), 5.29 (dddd, J¼1.5, 1.5, 1.5, 17.0 Hz, 1H), 5.37 (dd, J¼2.0, 4.0 Hz,
1H), 5.87 (dddd, J¼5.5, 5.5, 10.5, 17.0 Hz, 1H), 6.64 (d, J¼9.5 Hz, 1H);
13C NMR (125 MHz, CD3OD)
d 20.83, 21.00, 25.49, 26.54, 31.14, 37.67,
53.13, 53.79, 62.40, 65.70, 65.95, 68.92, 69.54, 72.08, 72.96, 99.07,
115.70, 117.67, 113.50, 158.00, 168.45, 170.46, 171.15; HRMS (FAB)
calcd for C22H31F3NO11 (MþH)þ: 542.1849, found: 542.1834.
column chromatography to give 9 (97%, ¼1/2): some selected
a/
b
peaks of 9 d 1.99 (s, 3H), 2.09 (s, 3H),
a
:
1H NMR (500 MHz, CDCl3)
4.1.6. Methyl (2-allyl-4,7-di-O-acetyl-3,5-dideoxy-5-
2.71 (dd, J¼4.8, 13.1 Hz, 1H), 3.69 (s, 3H), 4.20 (d, J¼15.2 Hz, 1H),
trifluoroacetamido-D-glycero-a-D-galacto-2-
4.32 (dd, J¼2.0, 10.6 Hz, 1H), 5.31 (dd, J¼2.0, 7.5 Hz, 1H), 6.95 (d,
nonulopyranosid)onate (7)
J¼9.9 Hz, 1H, NH). Some selected peaks of 9
b:
1H NMR (500 MHz,
1.97 (s, 3H), 2.09 (s, 3H), 2.46 (dd, J¼5.0, 13.2 Hz, 1H), 3.71
A mixture of trifluoroacetic acid (1.5 mL) and water (0.25 mL)
was added dropwise to a solution of 22 (0.87 mmol) in dichloro-
methane (15 mL) at 0 ꢁC. After being stirred for 10 min, the reaction
mixture was diluted with dichloromethane (10 mL), and then
washed with saturated NaHCO3 (30 mL). The organic phase was
CDCl3)
d
(s, 3H), 3.99 (s, 2H), 4.05 (d, J¼15.2 Hz, 1H), 4.12 (d, J¼15.2 Hz, 1H),
4.26 (dd, J¼7.4, 12.5 Hz, 1H), 4.70 (dd, J¼2.5, 12.5 Hz, 1H), 5.10 (dd,
J¼2.5, 4.1 Hz,1H), 5.15 (dd, J¼10.0,12.5 Hz,1H), 5.25 (ddd, J¼2.5, 4.1,
7.0 Hz, 1H), 5.66 (d, J¼10.0 Hz, 1H, NH).

4722
C.-C. Lin et al. / Tetrahedron 65 (2009) 4714–4725
4.1.9. Methyl [2-allyl 4,7-di-O-acetyl-3,5-dideoxy-9-O-(methyl-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido- -glycero-
-galacto-2-nonulopyranosylonate)-5-trifluoroacetamido-
glycero- -galacto-2-nonulopyranosid]onate (28)
A mixture of donor 3c (0.074 mmol), acceptor 7 (0.049 mmol),
and activated molecular sieves (3 Å, 120 mg) in MeCN (3.0 mL) was
stirred for 15 min under argon at room temperature. The mixture
4.1.11. Methyl [2-allyl-4,7-di-O-acetyl-3,5-dideoxy-9-O-(methyl-5-
trifluoroacetamido-4,7-di-O-acetyl-3,5-dideoxy-8,9-O-
D
a-D
D-
isopropylidene-
trifluoroacetamido-
nonulopyranosid]onate (30)
Compound 30 (64%,
¼1.3/1) was obtained from donor 4 and
acceptor 7 by the method described for the synthesis of 28: 30
1H
NMR (500 MHz, CDCl3)
1.34 (s, 3H), 1.35 (s, 3H), 1.98 (dd, J¼12.5,
D
-glycero-
a
-D-galacto-2-nonulopyranosylonate)-5-
a/
b-
D
D-glycero-a/b-D
-galacto-2-
a/b
a
was cooled to ꢀ40 ꢁC for 15 min and TMSOTf (1.47
m
mol) was
d
added. The reaction mixture was stirred until TLC analysis indicated
that reaction was completed. The residue was concentrated in
vacuo and purified by silica gel column chromatography to afford
13.0 Hz, 1H), 2.06 (dd, J¼12.5, 13.0 Hz, 1H), 2.70 (dd, J¼5.0, 13.0, Hz,
1H), 2.78 (dd, J¼5.0, 13.0 Hz, 1H), 3.58 (dd, J¼3.4, 10.5 Hz, 1H), 3.77
(dd, J¼6.1, 10.5 Hz, 1H), 3.85 (s, 3H), 3.87 (s, 3H), 3.91 (ddd, J¼9.7,
10.4, 10.4, Hz, 1H), 3.94–4.00 (m, 1H), 4.02 (ddd, J¼8.0, 10.4, 10.4 Hz,
1H), 4.05–4.12 (m, 4H), 4.25–4.30 (m, 2H), 4.35 (ddd, J¼4.3, 4.3,
6.5 Hz, 1H), 4.96 (ddd, J¼5.0, 10.4, 12.5 Hz, 1H), 5.05 (br d, J¼8.8 Hz,
1H), 5.15 (ddd, J¼5.0, 10.4, 12.5 Hz, 1H), 5.21 (br d, J¼10.8 Hz, 1H),
5.26 (dd, J¼1.6, 4.3, 1H), 5.35 (dddd, J¼1.7, 1.7, 1.7, 17.5 Hz, 1H), 5.87
(dddd, J¼5.4, 5.4, 10.8, 17.5 Hz, 1H), 6.34 (d, J¼9.7 Hz, 1H), 6.54 (d,
28 (70%,
a/b
¼6/1): 28
a d 1.99–2.04 (m,
1H NMR (500 MHz, CDCl3)
2H), 2.01 (s, 3H), 2.02 (s, 3H), 2.03 (s, 3H), 2.14 (s, 9H), 2.63 (dd,
J¼4.5, 13.0 Hz, 1H), 2.74 (dd, J¼4.5, 13.0 Hz, 1H), 3.55 (dd, J¼3.5,
10.5 Hz, 1H), 3.81 (s, 3H), 3.82 (dd, J¼6.5, 10.5 Hz, 1H), 3.85 (s, 3H),
3.98 (dd, J¼5.5, 13.0 Hz, 1H), 3.99 (ddd, J¼10.0, 10.0, 10.0 Hz, 1H),
4.07–4.11 (m, 1H), 4.08 (ddd, J¼10.0, 10.0, 10.0 Hz, 1H), 4.14 (dd,
J¼6.5, 12.5 Hz, 1H), 4.22 (dd, J¼1.5, 10.0 Hz, 1H), 4.24 (br d,
J¼10.0 Hz, 1H), 4.27 (dd, J¼5.5, 13.0 Hz, 1H), 4.36 (dd, J¼2.5,
12.5 Hz, 1H), 4.99–5.03 (m, 3H), 5.19 (dd, J¼1.5, 10.5 Hz, 1H), 5.27
(dd, J¼1.5, 9.0 Hz, 1H), 5.28 (dd, J¼1.5, 17.0 Hz, 1H), 5.38 (ddd,
J¼3.5, 6.5, 9.0 Hz, 1H), 5.86 (ddd, J¼5.5, 10.5, 17.0 Hz, 1H), 6.51 (d,
J¼10.0 Hz, 1H), 7.01 (d, J¼10.0 Hz, 1H); 13C NMR (125 MHz, CDCl3)
J¼8.0 Hz, 1H); 13C NMR (125 MHz, CDCl3)
d 20.78, 20.80, 20.96,
20.97, 25.46, 26.62, 31.14, 37.59, 50.25, 50.88, 53.19, 53.74, 65.88,
65.97, 68.76, 68.79, 69.12, 69.20, 70.47, 72.11, 72.85, 75.50, 98.86,
99.21, 109.00, 115.47, 115.63, 117.82, 133.32, 157.68, 157.99, 166.97,
168.28, 170.16, 170.54, 170.55, 170.96; HRMS (FAB) calcd for
C38H50F6N2NaO21 (MþNa)þ: 1007.2708, found: 1007.2690.
d
20.79, 20.98, 21.28, 37.13, 37.50, 50.16, 50.38, 53.25, 53.44, 62.57,
65.76, 65.95, 67.42, 68.13, 68.51, 68.79, 68.90, 68.95, 69.28, 69.33,
72.09, 72.45, 98.81, 98.99, 115.65, 115.76, 117.81, 133.39, 157.83,
168.04, 169.08, 170.12, 170.13, 170.43, 170.86, 171.11, 171.40; HRMS
(FAB) calcd for C39H50F6N2NaO23 (MþNa)þ: 1051.2606, found:
1051.2628.
4.1.12. Methyl [4-methylphenyl-4,7-di-O-acetyl-3,5-dideoxy-2-
thio-9-O-(methyl-4,7-di-O-acetyl-8,9-O-chloroacetyl-3,5-dideoxy-
5-trifluoroacetamido-D-glycero-a/b-D-galacto-2-
nonulopyranosylonate)-5-trifluoroacetamido-D-glycero-a-D-
galacto-2-nonulopyranosid]onate (31)
Compound 31 (75%,
acceptor 6 by the method described for the synthesis of 28: 31
NMR (400 MHz, CDCl3) 1.98–2.00 (m,1H),1.99 (s, 3H),1.99–2.01 (m,
a
/
b
¼2.5/1) was obtained from donor 9 and
4.1.10. Methyl [4-methylphenyl-4,7-di-O-acetyl-3,5-dideoxy-2-
thio-9-O-(methyl-4,7-di-O-acetyl-3,5-dideoxy-8,9-O-
a
1H
d
isopropylidene-5-trifluoroacetamido-
nonulopyranosylonate)-5-trifluoroacetamido-
galacto-2-nonulopyranosid]onate (29)
D
-glycero-
a
/
b
-
D
-galacto-2-
1H), 2.00 (s, 3H), 2.12 (s, 3H), 2.14 (s, 3H), 2.36 (s, 3H), 2.61 (dd, J¼4.7,
13.0 Hz,1H), 2.86 (dd, J¼4.9,13.0 Hz,1H), 3.39 (dd, J¼3.0,10.5 Hz,1H),
3.69 (s, 3H), 3.69–3.72 (m, 1H), 3.80 (s, 3H), 3.91 (dd, J¼1.9, 10.5 Hz,
1H), 3.94–4.25 (m, 7H), 4.15 (d, J¼15.2 Hz,1H), 4.26 (d, J¼15.2 Hz, 1H),
4.43 (ddd, J¼2.4, 4.8, 12.5 Hz, 1H), 4.90–4.98 (m, 2H), 5.03 (dd, J¼1.7,
8.3 Hz, 1H), 5.26 (dd, J¼1.7, 8.3 Hz, 1H), 5.44 (ddd, J¼2.5, 8.3, 13.5 Hz,
1H), 6.46 (d, J¼9.8 Hz, 1H, NH), 6.67 (t, J¼9.6 Hz, 1H, NH), 7.16 (d,
J¼7.9 Hz, 2H), 7.38 (d, J¼7.9 Hz, 2H); 13C NMR (100 MHz, CDCl3)
D-glycero-
a-D-
Compound 29 (68%,
acceptor 6 by the method described in the synthesis of 28: 29
NMR (400 MHz, CDCl3)
1.34 (s, 3H), 1.36 (s, 3H), 1.97 (dd, J¼12.0,
a
/
b
¼3/1) was obtained from donor 4 and
a
1H
d
13.0 Hz, 1H), 2.02 (s, 6H), 2.06 (dd, J¼11.5, 13.0 Hz, 1H), 2.14 (s,
3H), 2.16 (s, 3H), 2.39 (s, 3H), 2.70 (dd, J¼5.0, 13.0 Hz, 1H), 2.87
(dd, J¼5.0, 13.0 Hz, 1H), 3.53 (dd, J¼3.4, 10.5 Hz, 1H), 3.70 (dd,
J¼6.2, 10.5 Hz, 1H), 3.71 (s, 3H), 3.85 (s, 3H), 3.90 (dd, J¼2.0,
10.5 Hz, 1H), 3.93–4.00 (m, 2H), 4.01–4.10 (m, 4H), 4.35 (ddd,
J¼4.3, 4.3, 6.4 Hz, 1H), 4.95 (ddd, J¼5.0, 10.5, 11.5 Hz, 1H), 5.01 (dd,
J¼2.0, 8.0 Hz, 1H), 5.14 (ddd, J¼5.0, 10.2, 12.0 Hz, 1H), 5.30 (dd,
J¼2.0, 4.3 Hz, 1H), 6.72 (d, J¼9.2 Hz, 1H), 6.84 (d, J¼10.0 Hz, 1H),
7.17 (d, J¼7.6 Hz, 2H), 7.39 (d, J¼8.0 Hz, 2H); 13C NMR (100 MHz,
d
20.54, 20.55, 20.56, 20.81, 21.40, 37.20, 37.28, 40.59, 40.60, 41.04,
49.72,50.11, 53.18,53.33,63.56,65.27, 66.70,68.13,68.70,69.26,69.81,
71.66, 74.09, 86.71, 98.63,115.35,115.43,124.36,129.86,136.69,141.01,
157.54, 157.60, 166.86, 167.22, 168.07, 169.05, 169.78, 170.05, 170.60,
170.92; 31
b
1H NMR (500 MHz, CDCl3)
d
1.84 (dd, J¼11.8,13.2 Hz,1H),
1.99 (s, 3H), 2.00 (s, 3H), 2.06 (dd, J¼11.0.13.1 Hz,1H), 2.13 (s, 3H), 2.17
(s, 3H), 2.36 (s, 3H), 2.47 (dd, J¼4.9, 13.2 Hz, 1H), 2.90 (dd, J¼4.8,
13.1 Hz,1H), 3.51(d, J¼9.9 Hz,1H), 3.62 (dd, J¼2.3, 9.9 Hz,1H), 3.71 (s,
3H), 3.74–3.76 (m,1H), 3.75 (s, 3H), 3.97–4.21 (m, 8H), 4.48 (dd, J¼2.3,
10.6 Hz, 1H), 4.67 (ddd, J¼2.3, 12.8, 14.8 Hz, 1H), 4.87 (tdd, J¼2.5, 4.8,
11.0 Hz,1H), 5.01 (ddd, J¼4.9,11.8,16.0 Hz,1H), 5.26 (dd, J¼1.1, 4.5 Hz,
1H), 5.31–5.40 (m, 2H), 7.16 (d, J¼8.0 Hz, 2H), 7.36 (d, J¼8.0 Hz, 2H);
CDCl3)
d 20.77, 20.81, 20.91, 20.95, 21.57, 31.12, 37.47, 49.58, 49.68,
49.75, 53.67, 53.74, 53.83, 69.12, 69.22, 69.68, 72.18, 72.77, 74.18,
74.33, 115.40, 124.50, 124.57, 130.04, 130.07, 136.82, 141.26, 141.31,
157.91, 158.20, 169.36, 169.61, 170.14, 170.40, 171.74, 172.19; HRMS
(FAB) calcd for C42H52F6N2NaO20S (MþNa)þ: 1073.2636, found:
1073.2634. 29
b
1H NMR (400 MHz, CDCl3)
d
1.97 (s, 3H), 1.98 (s,
13C NMR (100 MHz, CDCl3)
d 20.53, 20.57, 21.05, 21.35, 21.37, 37.30,
3H), 1.99–2.05 (m, 4H), 2.08 (dd, J¼11.2, 12.8 Hz, 1H), 2.10 (s, 3H),
2.16 (s, 3H), 2.19 (s, 3H), 2.35 (s, 3H), 2.37 (dd, J¼5.2, 14.0 Hz, 1H),
2.89 (dd, J¼4.4, 12.8 Hz, 1H), 3.34 (dd, J¼9.2, 12.0 Hz, 1H), 3.43–
3.45 (m, 1H), 3.46 (s, 3H), 3.62–3.66 (m, 2H), 3.76 (dd, J¼2.0,
10.0 Hz, 1H), 3.81 (s, 3H), 4.05–4.16 (m, 4H), 4.63 (ddd, J¼4.4, 11.2,
11.2 Hz, 1H), 4.80 (d, J¼10.0 Hz, 1H), 4.89 (ddd, J¼5.2, 11.2, 10.4 Hz,
1H), 5.42 (dd, J¼2.0, 8.8 Hz, 1H), 7.09 (d, J¼10 Hz, 1H), 7.14 (d,
J¼8.0 Hz, 2H), 7.36 (d, J¼8.0 Hz, 2H); 13C NMR (100 MHz, CDCl3)
40.61, 40.93, 48.31, 49.31, 52.89, 53.52, 53.57, 60.39, 63.82, 64.29,
67.23, 68.41, 68.93, 69.30, 70.04, 71.80, 74.88, 86.75, 97.88, 115.42,
115.43, 124.17, 129.91, 136.62, 141.21, 157.68, 158.06, 166.62, 166.85,
167.40, 169.56, 169.82, 170.39, 170.94, 171.85; HRMS (FAB) calcd for
C43H51Cl2F6N2O22S (MþH)þ: 1163.1935, found: 1163.1954.
4.1.13. Methyl [4-methylphenyl 4-O-benzoyl-3,5-dideoxy-2-thio-9-
O-(methyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-
trifluoroacetamido-D-glycero-a-D-galacto-2-
d
20.60, 20.68, 20.78, 21.49, 21.51, 31.51, 31.12, 37.68, 48.14, 48.81,
52.96, 54.32, 60.61, 64.08, 68.57, 68.83, 69.45, 69.73, 87.06,
98.56, 115.72, 115.88, 167.52, 170.11, 170.43, 170.73, 172.81; HRMS
(FAB) calcd for C42H52F6N2NaO20S (MþNa)þ: 1073.2636, found:
1073.2627.
nonulopyranosylonate)-5-trifluoroacetamido-D-glycero-a-D-
galacto-2-nonulopyranosid]onate (32)
Compound 32 (72%, only
a) was obtained from donor 3c and
acceptor 8 by the method described for the synthesis of 28: 1H NMR

C.-C. Lin et al. / Tetrahedron 65 (2009) 4714–4725
4723
(500 MHz, CDCl3)
d
1.96 (dd, J¼11.9, 12.6 Hz, 1H), 2.02 (s, 3H), 2.04
4.1.16. Methyl [4,7,8-tri-O-acetyl-3,5-dideoxy-9-O-(methyl-5-
trifluoroacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy- -glycero-
-galacto-non-2-ulopyranosylonate)-5-trifluoroacetamido-
glycero- -galacto-non-2-ulopyranosid]onate (35)
Compound 35 (93%) was obtained from 34 by the method de-
scribed for the synthesis of 15: 1H NMR (400 MHz, CDCl3)
1.86
(s, 3H), 2.13 (s, 3H), 2.14 (s, 3H), 2.23 (dd, J¼11.3, 12.3 Hz, 1H), 2.37
(s, 3H), 2.68 (dd, J¼4.8, 12.6 Hz, 1H), 3.04 (dd, J¼4.9, 12.3 Hz, 1H),
3.58 (d, J¼7.7 Hz, 1H), 3.64 (s, 3H), 3.71 (d, J¼7.7 Hz, 1H), 3.76 (d,
J¼10.3 Hz, 1H), 3.81 (s, 3H), 3.90–3.96 (m, 2H), 3.99 (q, J¼10.2 Hz,
1H), 4.13 (dd, J¼6.1, 12.5 Hz, 1H), 4.27 (dd, J¼2.0, 10.2 Hz, 1H), 4.38
(q, J¼10.3 Hz, 1H), 4.42 (dd, J¼3.0, 12.5 Hz, 1H), 5.07 (ddd, J¼4.8,
10.2, 11.9 Hz, H), 5.22 (ddd, J¼4.9, 10.3, 11.3 Hz, 1H), 5.31 (dd, J¼2.0,
7.0 Hz, 1H), 5.37 (ddd, J¼3.0, 6.1, 7.1 Hz, 1H), 6.86 (d, J¼10.2 Hz, 1H,
NH), 7.17 (d, J¼7.0 Hz, 2H), 7.38 (d, J¼10.3 Hz, 1H, NH), 7.39 (d,
J¼7.0 Hz, 2H), 7.46 (t, J¼7.5 Hz, 2H), 7.61 (t, J¼7.5 Hz, 1H), 7.99 (d,
D
a-
D
D-
b-D
d
(dd, J¼12.4, 13.2 Hz, 1H), 1.92 (br, 1H), 1.99 (s, 3H), 2.00 (s, 3H), 2.02
(s, 3H), 2.11 (s, 3H), 2.14 (s, 3H), 2.15 (s, 3H), 2.18 (s, 3H), 2.24 (dd,
J¼12.4, 13.2 Hz, 1H), 2.34 (dd, J¼4.8, 13.2 Hz, 1H), 2.43 (dd, J¼5.2,
13.2 Hz, 1H), 3.57 (dd, J¼6.8, 10.8 Hz, 1H), 3.81 (s, 3H), 3.82 (dd,
J¼2.0, 6.8 Hz, 1H), 3.86 (m, 1H), 3.88 (s, 3H), 4.06–4.20 (m, 3H),
4.57–4.61 (m, 2H), 5.22–5.30 (m, 4H), 5.33 (br, 1H), 5.39 (br, 1H),
7.35 (d, J¼9.6 Hz, 1H), 7.79 (d, J¼10.0 Hz, 1H); 13C NMR (125 MHz,
J¼7.5 Hz, 2H); 13C NMR (125 MHz, CDCl3)
d 20.50, 20.54, 20.62,
21.02, 21.35, 37.18, 37.25, 50.13, 50.80, 52.93, 53.90, 62.26, 66.14,
67.32, 68.29, 68.36, 69.41, 69.97, 70.44, 71.78, 75.09, 86.54, 98.63,
115.38, 115.41, 124.51, 128.57, 128.63, 129.80, 129.82, 133.90, 136.59,
140.86, 157.55, 158.49, 166.96, 168.02, 168.89, 170.16, 170.71, 170.95,
170.97; HRMS (FAB) calcd for C43H51Cl2F6N2O22SNa (MþNa)þ:
1137.2585, found: 1137.2582.
CDCl3) d 14.40, 20.78, 20.83, 20.85, 21.07, 21.09, 21.26, 36.37, 36.47,
49.39, 50.21, 53.08, 53.57, 60.62, 62.34, 63.33, 67.94, 68.78, 68.82,
68.89, 70.58, 71.61, 95.46, 98.56, 115.81, 115.87, 157.76, 158.11,
166.43, 169.89, 170.08, 170.59, 170.71, 170.92, 171.05; HRMS
(FAB) calcd for C38H48F6N2NaO24 (MþNa)þ: 1053.2399, found:
1053.2406.
4.1.14. Methyl [2-allyl-4,7,8-tri-O-acetyl-3,5-dideoxy-9-O-(methyl-
4,7-di-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-
D
-glycero-a-D
-
4.1.17. Dibenzyl {methyl [4,7,8-tri-O-acetyl-3,5-dideoxy-9-O-
galacto-2-nonulopyranosylonate)-5-trifluoroacetamido-
D
-glycero-
(methyl-5-trifluoroacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
glycero- -galacto-non-2-ulopyranosylonate)-5-
trifluoroacetamido- -glycero- -galacto-non-2-
ulopyranosid]onate} phosphite (10)
D-
a-D
-galacto-2-nonulopyranosid]onate (33)
a-D
Compound 33 (84%) was obtained from 30
a
by the methods
D
a/b-D
described in the synthesis of 14 and 7: 1H NMR (400 MHz,
CDCl3)
d
1.96 (t, J¼12.8 Hz, 2H), 2.03 (s, 3H), 2.04 (s, 3H), 2.13 (s,
To a solution of 35 (0.14 mmol) in MeCN (3.7 mL) were added
DDP (0.45 mL, 1.4 mmol) and 1H-tetrazole (0.1 g, 1.4 mmol) at
ꢀ40 ꢁC under argon atmosphere. After being stirred for 5 min at
ꢀ40 ꢁC, the solution was further stirred for 15 min at room tem-
perature. The solution was diluted with EtOAc, and washed with
saturated aqueous NaHCO3. The organic layer was dried (MgSO4)
and concentrated. The residue was purified by flash column chro-
3H), 2.17 (s, 6H), 2.69 (dd, J¼4.4, 12.8 Hz, 1H), 2.72 (dd, J¼5.2,
12.8 Hz, 1H), 3.50 (br, 1H), 3.62 (dd, J¼2.8, 10.8 Hz, 1H), 3.70 (br,
1H), 3.81 (s, 3H), 3.84–3.91 (m, 3H), 3.88 (s, 3H), 3.93 (q,
J¼10.0 Hz, 1H), 4.02 (br d, J¼10.0 Hz, 1H), 4.15 (ddd, J¼10.0, 10.0,
10.0 Hz, 1H), 4.25–4.30 (m, 1H), 4.26 (dd, J¼2.0, 10.0 Hz, 1H), 4.95
(ddd, J¼5.2, 10.0, 12.8 Hz, 1H), 5.00 (br d, J¼8.0 Hz, 1H), 5.04
(ddd, J¼4.4, 10.0, 12.8 Hz, 1H), 5.19 (dddd, J¼1.6, 1.6, 1.6, 10.8 Hz,
1H), 5.28 (dd, J¼2.0, 9.2 Hz, 1H), 5.29 (dddd, J¼1.6, 1.6, 1.6,
17.2 Hz, 1H), 5.38 (ddd, J¼3.2, 4.8, 8.0 Hz, 1H), 5.86 (dddd, J¼5.6,
5.6, 10.8, 17.2 Hz, 1H), 6.57 (d, J¼10.0 Hz, 1H), 7.04 (d, J¼10.0 Hz,
matography to give 10 (92%): some selected peaks of 10
(500 MHz, CDCl3)
1.85 (dd, J¼12.7, 13.1 Hz, 1H), 1.86 (dd, J¼12.7,
b
: 1H NMR
d
13.1 Hz, 1H), 1.97 (s, 3H), 1.98 (s, 3H), 2.01 (s, 3H), 2.03 (s, 3H), 2.07
(s, 3H), 2.11 (s, 3H), 2.12 (s, 3H), 2.46 (dd, J¼4.9, 13.1 Hz, 1H), 2.55
(dd, J¼4.3, 13.1 Hz, 1H), 3.70 (s, 3H), 3.76 (s, 3H), 3.94 (ddd, J¼10.2,
10.5, 10.5 Hz,1H), 3.96 (ddd, J¼9.7,10.5,10.5 Hz,1H), 4.14 (dd, J¼2.2,
10.6 Hz, 1H), 4.20 (dd, J¼2.2, 10.6 Hz, 1H), 4.25 (dd, J¼2.6, 12.5 Hz,
1H), 4.87 (ddd, J¼4.3, 10.5, 12.7 Hz, 1H), 4.91 (d, J¼7.8 Hz, 1H), 4.93
(d, J¼8.0 Hz, 1H), 4.95 (d, J¼7.8 Hz, 1H), 5.08 (ddd, J¼4.9, 10.5,
13.1 Hz, 1H), 5.25 (dd, J¼2.2, 8.4 Hz, 1H), 5.32 (ddd, J¼2.6, 5.5,
8.4 Hz, 1H), 6.08 (d, J¼9.7 Hz, 1H), 6.48 (d, J¼10.2 Hz, 1H), 7.14–7.37
(m, 10H).
1H); 13C NMR (125 MHz, CDCl3)
d 20.78, 20.79, 20.94, 20.99,
21.32, 37.20, 38.16, 49.95, 50.64, 53.05, 53.91, 62.59, 63.36, 66.16,
67.54, 68.44, 68.64, 68.99, 69.01, 69.55, 71.46, 72.27, 98.46, 98.60,
115.63, 117.62, 133.62, 157.88, 168.34, 169.20, 170.13, 170.36,
170.96, 171.16, 171.52; HRMS (FAB) calcd for C37H48F6N2NaO22
(MþNa)þ: 1009.2501, found: 1009.2520.
4.1.15. Methyl [4-methylphenyl 4,7,8-tri-O-acetyl-3,5-dideoxy-2-
thio-9-O-(methyl-5-trifluoroacetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-
trifluoroacetamido-
ulopyranosid]onate (34)
Compound 34 (93%) was obtained from 27b by the method
described for the synthesis of 14: 1H NMR (400 MHz, CDCl3)
1.93
D
-glycero-
a
-
D
-galacto-non-2-ulopyranosylonate)-5-
4.1.18. Tetramer (36)
D
-glycero- -galacto-non-2-
a-D
Compound 36 (58%) was obtained from donor 10 and acceptor
33 by the method described for the synthesis of 28: some selected
peaks of 36: 13C NMR (125 MHz, CDCl3)
d 20.51, 20.67, 20.77, 20.96,
d
21.02, 29.57, 35.59, 36.25, 36.39, 36.58, 36.89, 37.31, 37.49, 37.89,
48.78, 49.10, 49.32, 49.54, 49.61, 49.79, 49.92, 50.03, 50.24, 50.30,
52.75, 52.89, 53.02, 53.17, 53.44, 53.84, 62.08, 62.46, 63.03, 63.15,
63.40, 63.52, 63.65, 63.82, 65.24, 65.88, 66.93, 67.23, 67.41, 67.66,
67.87, 68.26, 68.39, 68.53, 68.76, 69.17, 69.57, 69.92, 71.17, 71.26,
71.39, 71.65, 71.83, 72.02, 72.13, 72.47, 98.33, 98.38, 98.48, 98.57,
112.01, 114.30, 116.60, 117.32, 118.89, 133.34, 133.39, 157.13, 157.43,
157.73, 158.2, 166.60, 167.76, 167.86, 168.08, 168.13, 168.41, 168.97,
169.29, 169.67, 169.81, 169.89, 170.06, 170.17, 170.51, 170.68,
170.74, 170.83, 171.09, 171.30, 171.46; HRMS (FAB) calcd for
C75H94N4F12NaO45 (MþNa)þ: 2021.4896, found: 2021.4882.
(t, J¼12.8 Hz, 1H), 1.95 (t, J¼12.8 Hz, 1H), 2.00 (s, 3H), 2.03 (s, 3H),
2.05 (s, 3H), 2.11 (s, 3H), 2.12 (s, 3H), 2.15 (s, 3H), 2.17 (s, 3H), 2.39
(s, 3H), 2.62 (dd, J¼4.6, 12.8 Hz, 1H), 2.80 (dd, J¼4.6, 12.8 Hz, 1H),
3.62 (s, 3H), 3.77 (dd, J¼2.6, 11.4 Hz, 1H), 3.80 (s, 3H), 3.88 (dt,
J¼9.7, 10.7 Hz, 1H), 3.95 (dd, J¼6.3, 11.4 Hz, 1H), 4.02 (dt, J¼9.7,
10.7 Hz, 1H), 4.09–4.15 (m, 2H), 4.22 (dd, J¼2.2, 10.7 Hz, 1H), 4.33
(dd, J¼2.6, 12.4 Hz, 1H), 4.94–5.01 (m, 2H), 5.16 (dd, J¼2.2, 6.3 Hz,
1H), 5.25 (ddd, J¼2.6, 6.3, 6.3 Hz, 1H), 5.32 (dd, J¼2.2, 8.1 Hz, 1H),
5.38 (ddd, J¼2.6, 8.1, 10.5 Hz, 1H), 6.65 (d, J¼9.7 Hz, 1H), 6.96 (d,
J¼9.7 Hz,1H), 7.17 (d, J¼8.0 Hz, 2H), 7.45 (d, J¼8.0 Hz, 2H); 13C NMR
(125 MHz, CDCl3)
d 20.78, 21.20, 21.29, 21.55, 37.58, 37.99, 50.14,
50.20, 52.99, 53.27, 62.52, 63.19, 67.39, 67.98, 68.70, 68.86, 69.32,
70.73, 71.87, 73.52, 116.18, 117.15, 125.23, 129.87, 136.59, 140.52,
157.55, 157.66, 168.09, 170.02, 170.14, 170.69, 170.81, 171.20, 171.25;
HRMS (FAB) calcd for C45H55F6N2O23S (MþH)þ: 1137.2821, found:
1137.2826.
4.1.19. Methyl(2-allyl-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-5-
trifluoroacetamido-a-D-glycero-D-galacto-2-
nonulopyranosid)onate (37a)
Compound 37a (95%) was obtained from 20 by the method
described for the synthesis of 14: 1H NMR (400 MHz, CDCl3)

4724
C.-C. Lin et al. / Tetrahedron 65 (2009) 4714–4725
d
1.98 (dd, J¼12.3, 12.9 Hz, 1H), 2.03 (s, 3H), 2.05 (s, 3H), 2.16 (s,
4.1.22. Methyl[2-(3-acetylsulfanyl-propoxy)-4,7,8-tri-O-acetyl-3,5-
dideoxy-9-O-(methyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-
3H), 2.18 (s, 3H), 2.69 (dd, J¼4.7, 12.9 Hz, 1H), 3.81 (s, 3H), 3.89
(dddd, J¼1.4, 1.4, 5.2, 12.8 Hz, 1H), 3.99 (ddd, J¼10.4, 10.4,
10.4 Hz, 1H), 4.14 (dd, J¼5.4, 12.6 Hz, 1H), 4.27–4.29 (m, 2H),
4.32 (dd, J¼2.6, 12.6 Hz, 1H), 5.01 (ddd, J¼4.7, 10.4, 12.3 Hz, 1H),
5.18 (dddd, J¼1.4, 1.4, 1.4, 10.5 Hz, 1H), 5.29 (dddd, J¼1.4, 1.4, 1.4,
17.2 Hz, 1H), 5.30 (dd, J¼2.2, 8.1 Hz, 1H), 5.42 (ddd, J¼2.6, 5.4,
8.1 Hz, 1H), 5.86 (dddd, J¼5.2, 5.2, 10.5, 17.2, Hz, 1H), 6.36 (d,
trifluoroacetamido-
5-trifluoroacetamido-
nonulopyranosid]onate (38b)
D
-glycero- -galacto-2-nonlopyranosylonate)-
a
-
D
D-glycero-a/b-D-galacto-2-
Compound 38b (81%) was obtained from 28 by the methods
described in the synthesis of 14 and 38a: 1H NMR (400 MHz, CDCl3)
d
1.79–1.85 (m, 2H), 1.91 (t, J¼12.1 Hz, 1H), 1.93 (t, J¼12.1 Hz, 1H),
J¼10.4 Hz, 1H); 13C NMR (100 MHz, CD3OD)
d
20.77, 20.89, 21.32,
2.02 (s, 6H), 2.05 (s, 3H), 2.13 (s, 3H), 2.14 (s, 3H), 2.15 (s, 3H), 2.17 (s,
3H), 2.33 (s, 3H), 2.62 (dd, J¼4.7, 12.1 Hz, 1H), 2.63 (dd, J¼4.7,
12.1 Hz, 1H), 2.94 (ddd, J¼7.4, 7.4, 13.7 Hz, 1H), 2.97 (ddd, 6.4, 7.8,
13.7 Hz, 1H), 3.32 (ddd, J¼5.0, 7.4, 9.7 Hz, 1H), 3.58 (dd, J¼2.8,
11.2 Hz, 1H), 3.81(s, 3H), 3.83 (s, 3H), 3.88 (ddd, J¼7.4, 7.8, 9.7 Hz,
1H), 3.90 (dd, J¼6.4, 11.2 Hz, 1H), 3.97 (q, J¼10.5 Hz, 1H), 4.00 (q,
J¼10.5 Hz, 1H), 4.14 (dd, J¼5.2, 12.1 Hz, 1H), 4.19 (dd, J¼2.0, 10.5 Hz,
1H), 4.32 (dd, J¼2.0, 10.5 Hz, 1H), 4.35 (dd, J¼2.2, 12.1 Hz, 1H), 4.97
(ddd, J¼4.7, 10.5, 12.1 Hz, 1H), 5.00 (ddd, J¼4.7, 10.5, 12.1 Hz, 1H),
5.20 (dd, J¼2.0, 8.0 Hz, 1H), 5.32 (dd, 2.0, 7.7 Hz, 1H), 5.33–5.39 (m,
2H), 6.59 (d, J¼10.5 Hz, 1H), 6.81 (d, J¼10.5 Hz, 1H); 13C NMR
38.22, 50.57, 53.03, 62.34, 66.29, 67.34, 68.50, 68.68, 71.83,
75.66, 98.68, 117.67, 117.54, 159.70, 168.32, 170.19, 170.39, 170.87,
171.06; HRMS (FAB) calcd for C23H30F3NNaO13 (MþNa)þ:
608.1567, found: 608.1575.
4.1.20. Methyl[2-(3-acetylsulfanyl-propoxy)-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-5-trifluoroacetamido-D-glycero-a-D-galacto-2-
nonulopyranosid]onate (38a)
HSAc (0.39 mL, 5.1 mmol) and AIBN (14 mg, 0.085 mmol) were
added to a solution of compound 37a (200 mg, 0.34 mmol) in dry
MeOH (15 mL). The mixture was then degassed for three times
and then stirred at 75 ꢁC for 24 h. The mixture was concentrated
in vacuo and then purified by flash silica gel chromatography
(EtOAc/hexane¼1/3) to yield product 38a (191.2 mg, 85%): 1H
(100 MHz, CDCl3)
d 20.81, 20.87, 21.27, 21.33, 25.99, 29.72, 30.83,
37.65, 38.16, 50.30, 50.50, 62.54, 63.55, 63.75, 67.45, 67.57, 68.54,
68.66, 68.89, 69.15, 71.47, 72.07, 98.51, 98.83, 115.62, 115.72, 157.79,
157.84, 167.97, 168.58, 169.96, 170.13, 170.25, 170.83, 170.96, 171.08,
171.24, 195.98; HRMS (FAB) calcd for C43H56F6N2NaO25S (MþNa)þ:
1169.2695, found: 1169.2688.
NMR (400 MHz, CDCl3)
d
1.78–1.83 (m, 2H), 1.89 (dd, J¼12.4,
12.9 Hz, 1H), 1.97 (s, 3H), 1.99 (s, 3H), 2.09 (s, 3H), 2.10 (s, 3H), 2.27
(s, 3H), 2.57 (dd, J¼4.7, 12.9 Hz, 1H), 2.82–2.94 (m, 2H), 3.21–3.28
(m, 1H), 3.76 (s, 3H), 3.77 (m, 1H), 3.97 (q, J¼10.0 Hz, 1H), 4.09
(dd, J¼6.0, 12.4 Hz, 1H), 4.25 (dd, J¼2.0, 10.0 Hz, 1H), 4.31 (dd,
J¼2.4, 12.4 Hz, 1H), 4.93 (ddd, J¼4.7, 10.0, 12.4 Hz, 1H), 5.26 (dd,
J¼2.0, 7.5 Hz, 1H), 5.32 (ddd, J¼2.4, 6.0, 7.5 Hz, 1H), 7.00 (d,
4.1.23. 2-(3-Mercapto-propoxy)-5-acetamido-3,5-dideoxy-9-O-(5-
acetamido-3,5-dideoxy-
nonulopyranosidonic acid)-
nonulopyranosidonic acid (39b)
D
-glycero-
a
-
D
-galacto-2-
-galacto-2-
D
-glycero-a-D
J¼10.0 Hz, 1H); 13C NMR (100 MHz, CDCl3)
d
20.47, 20.54, 20.61,
Compound 39b (51%) was obtained as a monomer from 38b by
20.98, 25.61, 29.37, 30.50, 37.78, 49.94, 52.73, 62.16, 63.33, 67.16,
68.47, 69.14, 71.71, 98.62, 168.10, 169.86, 170.37, 170.63, 170.98,
195.77; HRMS (FAB) calcd for C25H35F3NO14S (MþH)þ: 662.1730,
found: 662.1721.
the method described in the synthesis of 39a: 1H NMR (500 MHz,
D2O)
d
1.70 (t, J¼12.5 Hz, 1H), 1.88 (t, J¼12.5 Hz, 1H), 1.99–2.03 (m,
2H), 2.08 (s, 3H), 2.09 (s, 3H), 2.79 (dd, J¼5.0,12.5 Hz,1H), 2.84–2.91
(m, 3H), 3.57–3.63 (m, 1H), 3.67–3.77 (m, 5H), 3.78–4.08 (m, 10H);
13C NMR (125 MHz, CD3OD)
d 23.12, 23.17, 29.77, 35.41, 40.52, 41.59,
4.1.21. 3-(5-Acetamido-3,5-dideoxy-
nonulopyranosidonic acid)-oxypropyldisulfide (39a)
D
-glycero-
a
-
D
-galacto-2-
53.04, 53.05, 63.41, 63.79, 64.13, 67.90, 68.37, 69.13, 69.55, 71.34,
71.74, 72.63, 73.55, 101.52, 101.74, 171.14, 172.87, 174.97, 176.14;
HRMS (FAB) calcd for C25H42N2NaO17S (MþNa)þ: 697.2102, found:
697.2091.
To a stirred solution of compound 38a (0.11 mmol) in dry
MeOH (1.5 mL) was added NaOMe (catalytic amount). After being
stirred at room temperature for 2 h, the solution was neutralized
with Amberlite IR-120 (Hþ) resin, filtered, and concentrated to
dryness. To the residue was then added 0.5 N NaOH (1 mL) and
allowed to stir at room temperature for 30 min. The solution was
neutralized with Dowex 50Wꢂ8 (Hþ) resin, filtered, and con-
centrated. To a solution of the residue in pyridine (2 mL) was
added acetic anhydride (1 mL) at 0 ꢁC. The reaction mixture was
stirred for 12 h at room temperature and then concentrated in
vacuo. The residue left was diluted with EtOAc and was washed
with 10% HCl, saturated NaHCO3, and saturated NaCl sequen-
tially. The organic phase was dried (MgSO4) and filtered, and the
filtrate was evaporated. The residue was purified by silica gel
column chromatography to afford peracetyl compound. To
a stirred solution of peracetyl compound in dry MeOH (1.5 mL)
was added NaOMe (catalytic amount). After being stirred at
room temperature for 2 h, the solution was neutralized with
Amberlite IR-120 (Hþ) resin, filtered, and concentrated to dry-
ness. The dried residue was purified by P2 biogel to give dimer
4.2. Conjugation of sialic acid with carrier protein (KLH)
The solution of KLH (5 mg) in 0.01 M PBS buffer (pH¼7) was put
inside a dialysis membrane, and then the membrane was put inside
a 4 L 0.01 M PBS buffer and stirred for more than 4 h. The process
was repeated three times. After that, KLH was taken out and dis-
solved in 70 mL m-maleimidobenzoyl-N-hydroxysulfosuccinimide
ester (MBS)/DMF. The solution was stirred for 30 min at room
temperature. PD-10 column was applied to purify the maleimide-
linked KLH, and 0.05 M PBS buffer (pH¼6) was used as the eluent.
All tubes containing protein, which was indicated by Coomassie
Brilliant Blue, were combined together. To the solution of disulfide-
linked sialic acid 39 (5 mg) in PBS (0.05 M, 1 mL) was added NaBH4
(10 equiv). The solution was stirred for 20 min and then added to
the combined protein solution. The mixture was adjusted to pH 7.3
and stirred for 3 h. Then, the mixture was dialyzed and lyophilized
to obtain sialic acid-linked KLH.
product 39a (60%): 1H NMR (500 MHz, CD3OD)
d 1.60 (t,
J¼12.0 Hz, 1H), 1.89–1.94 (m, 2H), 2.03 (s, 3H), 2.73–2.76 (m, 2H),
2.77 (dd, J¼4.5, 12.0 Hz, 1H), 3.54 (dd, J¼6.5, 16.0 Hz, 2H), 3.59 (br
d, J¼9.5 Hz, 1H), 3.64–3.71 (m, 2H), 3.82–3.89 (m, 4H); 13C NMR
4.3. Determination of sialic acid amount on sialic acid-linked
KLH by Warren’s method
(125 MHz, CD3OD)
d
22.98, 30.40, 35.92, 42.16, 53.76, 63.93,
Four kinds of solution were prepared. Solution A: a solution of
periodic acid (0.025 M) in diluted sulfuric acid (0.063 M, pH 1.2).
Solution B: 2% solution of sodium arsenite in diluted hydrochloric
acid (0.5 M). Solution C: thiobarbituric acid (0.1 M) adjusted to pH
63.99, 69.44, 69.88, 73.01, 74.49, 101.85, 171.22, 176.07; HRMS
(FAB) calcd for C28H48N2NaO18S2 (MþNa)þ: 787.2241, found:
787.2236.

C.-C. Lin et al. / Tetrahedron 65 (2009) 4714–4725
4725
Molecular Immunology of Complex Carbohydrates 2; Kluwer–Plenum: New York,
NY, 2001; (c) Halcomb, R. L.; Cappell, M. D. J. Carbohydr. Chem. 2002, 21, 723–
768; (d) Ress, D. K.; Linhardt, R. J. Curr. Org. Synth. 2004, 1, 31–46; (e) De Meo, C.;
Priyadarshani, U. Carbohydr. Res. 2008, 343, 1540–1552.
8. (a) Demchenko, A. V.; Boons, G. J. Chem.dEur. J. 1999, 5, 1278–1283; (b) De Meo,
C.; Demchenko, A. V.; Boons, G. J. J. Org. Chem. 2001, 66, 5490–5498; (c) Ren, C.
T.; Chen, C.-S.; Wu, S.-H. J. Org. Chem. 2002, 67, 1376–1379; (d) Ando, H.; Koike,
Y.; Ishida, H.; Kiso, M. Tetrahedron Lett. 2003, 44, 6883–6886; (e) Lu, K.-C.;
Tseng, S.-Y.; Lin, C.-C. Carbohydr. Res. 2002, 337, 755–760; (f) Yu, C.-S.; Niikura,
K.; Lin, C.-C.; Wong, C.-H. Angew. Chem., Int. Ed. 2001, 40, 2900–2903; (g) Ta-
naka, H.; Adachi, M.; Takahashi, T. Chem.dEur. J. 2005, 11, 849–862; (h) Tanaka,
K.; Goi, T.; Fukase, K. Synlett 2005, 2958–2962.
9.0 with NaOH. Solution D: 1-butanol containing 5% hydrochloric
acid (12 M). To the solution of sialic acid-linked KLH (10 g, 200 g,
300 g, and 400 g) in 500 L water was added 250 L of solution
A. The resulting mixture was heated in a water bath at 37 ꢁC for
30 min. Then, 200 L of solution B was added to it at room tem-
perature for 2 min. To this, solution C (2 mL) was added, and the
mixture was heated in boiling water bath for 15 min. After that, the
solution was cooled to 0 ꢁC and extracted by solution D (3 mL).
The solution was centrifuged at 1000 rpm for 5 min. The upper
clear layer was separated and measured by UV.
m
m
m
m
m
m
m
9. (a) Okamoto, K.; Kondo, T.; Goto, T. Tetrahedron 1987, 43, 5909–5918; (b) Er-
´
cegovec, T.; Magnusson, G. J. Org. Chem. 1995, 60, 3378–3384; (c) Ito, Y.; Ogawa,
Alternatively, the sialic acid was removed from the sialic acid-
linked KLH by sialidase. In this method, the above A solution was
changed to sialidase (Arthrobacter ureafaciens, 8 mU) solution and
the reaction time was extended to 12 h at 37 ꢁC.
T. Tetrahedron 1990, 46, 89–102; (d) Martichonok, V.; Whitesides, G. M. J. Am.
´
Chem. Soc. 1996, 118, 8187–8191; (e) Ercegovec, T.; Magnusson, G. J. Org. Chem.
1996, 61, 179–184; (f) Castro-Palomino, J. C.; Tsvetkov, Y. E.; Schmidt, R. R. J. Am.
Chem. Soc. 1998, 120, 5434–5440; (g) Hossain, N.; Magnusson, G. Tetrahedron
Lett. 1999, 40, 2217–2220; (h) Okamoto, K.; Kondo, T.; Goto, T. Tetrahedron 1987,
43, 5919–5928.
Acknowledgements
10. (a) Takahashi, T.; Tsukamoto, H.; Yamada, H. Tetrahedron Lett. 1997, 38, 8223–
8226; (b) Danishefsky, S. J.; DeNinno, M. P.; Chen, S. J. Am. Chem. Soc. 1988, 110,
3929–3940; (c) Haberman, J. M.; Gin, D. Y. Org. Lett. 2001, 3, 1665–1668; (d) Ye,
X.-S.; Huang, X.; Wong, C.-H. Chem. Commun. 2001, 974–975; (e) Haberman, J.
M.; Gin, D. Y. Org. Lett. 2003, 5, 2539–2541; (f) Ishiwata, A.; Ito, Y. Synlett 2003,
1339–1343.
The authors would like to thank the National Science Council of
Taiwan (NSC97-2752-B-007-002-PAE), National Tsing Hua Univer-
sity, and Academia Sinica, Taiwan for financially supporting this
research.
11. Ando, H.; Koike, Y.; Koizumi, S.; Ishida, H.; Kiso, M. Angew. Chem., Int. Ed. 2005,
44, 6759–6763.
12. Tanaka, H.; Nishiura, Y.; Takahashi, T. J. Am. Chem. Soc. 2006, 128, 7124–7125.
13. Tanaka, H.; Tateno, Y.; Nishiura, Y.; Takahashi, T. Org. Lett. 2008, 10, 5597–5600.
14. Lin, C.-C.; Huang, K.-T.; Lin, C.-C. Org. Lett. 2005, 7, 4169–4172.
15. (a) Kanie, O.; Kiso, M.; Hasegawa, A. J. Carbohydr. Chem. 1988, 7, 501–506; (b)
Marra, A.; Sinay, P. Carbohydr. Res. 1989, 187, 35–42; (c) Roy, R.; Andersson, F. O.;
Letellier, M. Tetrahedron Lett. 1992, 33, 6053–6056; (d) Martin, T. J.; Schmidt, R.
R. Tetrahedron Lett. 1992, 33, 6123–6126; (e) Kondo, H.; Icjikawa, Y.; Wong, C.-H.
J. Am. Chem. Soc. 1992, 411, 8748–8750; (f) Cai, S.; Yu, B. Org. Lett. 2003, 5, 3827–
3830; (g) Liu, Y.; Ruan, X.; Li, X.; Li, Y. J. Org. Chem. 2008, 73, 4287–4290.
16. Tsvetkov, Y. E.; Nifantiev, N. E. Synlett 2005, 1375–1380.
17. Lin, C.-C.; Hsu, T.-S.; Lu, K.-C.; Huang, I.-T. J. Chin. Chem. Soc. 2000, 47, 921–928.
18. Misra, A. K.; Agnihotri, G. Carbohydr. Res. 2004, 339, 885–890.
19. Eby, R.; Schuerch, C. Carbohydr. Res. 1982, 102, 131–138.
20. Fan, G.-T.; Lee, C.-C.; Lin, C.-C.; Fang, J.-M. J. Org. Chem. 2002, 67, 7565–7568.
21. (a) Chen, C.-T.; Weng, S.-S.; Kao, J.-Q.; Lin, C.-C.; Jan, M.-D. Org. Lett. 2005, 7,
3343–3346; (b) Lin, C.-C.; Jan, M.-D.; Weng, S.-S.; Lin, C.-C.; Chen, C.-T. Carbo-
hydr. Res. 2006, 341, 1948–1953.
Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2009.04.022.
References and notes
1. (a) Schauer, R.; Kamerling, J. P. In Glycoproteins II; Montreuil, J., Vliegenthart, J.
F. G., Schachter, H., Eds.; Elsevier: Amsterdam, 1997; pp 243–402; (b) Angata, T.;
Varki, A. Chem. Rev. 2002, 102, 439–469.
2. (a) Bomsel, M.; Alfsen, A. Nat. Rev. Mol. Cell Biol. 2003, 4, 57–68; (b) Allende, M.
L.; Proia, R. Curr. Opin. Struct. Biol. 2002, 12, 587–592; (c) Crocker, P. R. Curr.
Opin. Struct. Biol. 2002, 12, 609–615; (d) Biology of the Sialic Acids; Rosenberg, A.,
Ed.; Plenum: New York, NY, 1995.
3. (a) Kiefel, M. J.; von Itzstein, M. Chem. Rev. 2002, 102, 471–490; (b) Inoue, Y.;
Inoue, S. Pure Appl. Chem. 1999, 71, 789–800.
4. Jennings, H. J. In Carbohydrate-based Drug Discovery; Wong, C. H., Ed.; Wiley-
VCH: Weinheim, 2003; pp 357–380.
22. Yan, M.-C.; Chen, Y.-N.; Wu, H.-T.; Lin, C.-C.; Chen, C.-T.; Lin, C.-C. J. Org. Chem.
2007, 72, 299–302.
23. Hasegawa, A.; Nagahama, T.; Ohki, H.; Hotta, K.; Ishida, H.; Kiso, M. J. Carbohydr.
Chem. 1991, 10, 493–498.
´
5. (a) Verez-Bencome, V.; Fernandez-Santana, V.; Hardy, E.; Toledo, M. E.; Ro-
24. For a representative synthesis, see: Chen, M.-Y.; Patkar, L.-N.; Lu, K.-C.; Lee,
S.-Y.; Lin, C.-C. Tetrahedron Lett. 2004, 45, 635–639.
drı´guez, M. C.; Heynngnezz, L.; Rodriguez, A.; Baly, A.; Herrera, L.; Izquierdo,
´
M.; Villar, A.; Valdes, Y.; Cosme, K.; Deler, M. L.; Montane, M.; Garcia, E.; Ramos,
25. (a) Hasegawa, A.; Ohki, H.; Nagahama, T.; Ishida, H.; Kiso, M. Carbohydr. Res.
1991, 212, 277–281; (b) Okamoto, K.; Kondo, T.; Goto, T. Bull. Chem. Soc. Jpn.
1987, 60, 637–643; (c) Van der Vleugel, D. J. M.; Van Heeswijk, W. A. R.; Vlie-
genthart, J. F. G. Carbohydr. Res. 1982, 102, 121–130; (d) Dabrowski, U.; Friebolin,
H.; Brossmer, R.; Supp, M. Tetrahedron Lett. 1979, 48, 4637–4640.
26. Hori, H.; Nakajima, T.; Nishida, Y.; Ohrui, H.; Meguro, H. Tetrahedron Lett. 1988,
29, 6317–6320.
27. Danishefsky, S. J.; Allen, J. R. Angew. Chem., Int. Ed. 2000, 39, 837–863.
28. Roy, R.; Baek, M.-G.; Rittenhouse-Olson, K. J. Am. Chem. Soc. 2001, 123, 1809–
1816.
A.; Aguilar, A.; Medina, E.; Yoran˜o, G.; Sosa, I.; Hernandez, I.; Martı´nez, R.;
Muzachio, A.; Carmenates, A.; Costa, L.; Cardoso, F.; Campa, C.; Diaz, M.; Roy, R.
Science 2004, 305, 522–525; (b) Phalipon, A.; Costachel, C.; Grandjean, C.;
Thuizat, A.; Guerreiro, C.; Tanguy, M.; Nato, F.; Vulliez-Le Normand, B.; Belot, F.;
Wright, K. J. Immunol. 2006, 176, 1686–1694; (c) Pozsgay, V.; Kubler-Kielb, J.;
Schneerson, R.; Robbins, J. B. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 14478–14482;
(d) Xin, H.; Dziadek, S.; Bundle, D. R.; Cutler, J. E. Proc. Natl. Acad. Sci. U.S.A. 2008,
105, 13526–13531.
6. Castro-Palomino, J. C.; Tsvetkov, Y. E.; Schneider, R.; Schmidt, R. R. Tetrahedron
Lett. 1997, 38, 6837–6840.
29. (a) Warren, L. J. Biol. Chem. 1959, 234, 1971–1975; (b) Romero, E. L.; Pardo, M. F.;
Porro, S.; Alonso, S. J. Biochem. Biophys. Methods 1997, 35, 129–134.
7. For recent reviews of glycosidations of sialic acid, see: (a) Boons, G. J.; Dem-
chenko, A. V. Chem. Rev. 2000, 100, 4539–4565; (b) Lin, C. H.; Lin, C. C. The