Facile syntheses of the disaccharide repeating unit of the O-antigenic polysaccharide of Burkholderia pseudomallei strain 304b and its dimer and trimer

Article abstract of DOI:10.1016/j.carres.2011.09.001

A highly efficient strategy for the preparation of a disaccharide-repeating unit of the O-antigenic polysaccharide of Burkholderia pseudomallei strain 304b, and its dimer and trimer, has been developed through a regio- and stereoselective manner using p-m

Full text of DOI:10.1016/j.carres.2011.09.001

Carbohydrate Research 346 (2011) 2533–2539
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Facile syntheses of the disaccharide repeating unit of the O-antigenic
polysaccharide of Burkholderia pseudomallei strain 304b and its dimer
and trimer
⇑
Guanghui Zong, Xiangyan Cai, Xiaomei Liang, Jianjun Zhang , Daoquan Wang
Department of Applied Chemistry, China Agricultural University, Beijing 100193, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2011
Received in revised form 1 September 2011
Accepted 3 September 2011
Available online 9 September 2011
A highly efficient strategy for the preparation of a disaccharide-repeating unit of the O-antigenic polysac-
charide of Burkholderia pseudomallei strain 304b, and its dimer and trimer, has been developed through a
regio- and stereoselective manner using p-methoxylphenyl 2,4,6-tri-O-benzoyl-
a-D-glucopyranoside and
3-O-allyloxycarbonyl-2,4-di-O-benzoyl-6-deoxy- -talopyranosyl trichloroacetimidate as the key syn-
a
-L
thons. The target molecules were equipped with a p-methoxylphenyl handle at the reducing terminus
to allow for their further functionalization and attachment to a carrier protein.
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
Keywords:
Synthesis
D-Glucose
6-Deoxy-a-L-talose
Burkholderia pseudomallei
1. Introduction
infection, we have synthesized the monomer (2) and dimer (3) of
this repeating unit.12 Here, in order to compare the different effects
Lipopolysaccharides (LPSs), a glycoconjugate containing three
structural domains: lipid A, a core oligosaccharide, and the O-poly-
saccharides (O-PS),1 are ubiquitous components of the outer cell
membranes of Gram-negative bacteria, and are thought to be in-
volved in host-pathogen cross-talk. The O-PS, which are exposed to-
wards the external environment, play essential roles in this
course.2–4 The O-PS, normally contains multiple copies of repeating
units with typically 1–4 monosaccharide residues, is antigenic, and
a number of these glycans have been studied as vaccine candidates.5
Melioidosis, or Burkholderia pseudomallei infection, is an impor-
tant cause of mortality in Thailand and other parts of Southeast Asia
and Northern Australia.6 In Northeastern Thailand alone, a popula-
tion of 7 million people is at risk.7 In addition to its virulence, B.
pseudomallei is also intrinsically resistant to penicillin and gentami-
cin, the usual empirical treatment for suspected septicaemia in
many parts of the developing world.8 Since the small carbohydrates
can provoke the formation of antibodies,9–11 intensified efforts by
biochemists to detect B. pseudomallei identified unique molecules
on the surface of the bacteria. In B. pseudomallei strain 304b, a un-
ique disaccharide 1 (Fig. 1) was discovered8 containing glucose
and 6-deoxytalose. In our previous work, in a collaborative project
for an investigation of novel vaccines against B. pseudomallei
on pathogen detection caused by the difference in oligosaccharide
structure at the terminal position, we designed and synthesized an-
other three oligosaccharides: the monomer (4), dimer (5) and trimer
(6) (Fig. 2) of the frame shifted disaccharide repeating unit with glu-
cose at the terminal position. All the synthesized oligosaccharides
contain a terminal p-methoxylphenyl glycoside, which was neces-
sary for further functionalization and attachment to a carrier
protein.13
2. Results and discussion
The synthesis of the target oligosaccharides followed a conver-
gent approach. As shown in Scheme 1 and 1,2,4,6-tetra-O-acetyl-3-
O-allyl-
70%), was chosen as the starting material. It was converted into the
p-methoxyphenyl -glucoside (8) by treating 7 with p-methoxy-
D D-glucose (four steps,
-glucose (7),14 readily prepared from
a
-D
phenol and boron trifluoride diethyl etherate (70%). Deacetylation
of the compound 8, followed by perbenzoylation with 4 equiv of
benzoyl chloride, provided the p-methoxylphenyl 3-O-allyl-2,4,6-
tri-O-benzoyl-a-D-glucopyranoside (10) in 90% yield over two
steps. Selective cleavage of the 3-O-allyl group was then achieved
under the action of PdCl2 in a mixed solvent of MeOH/CH2Cl2 (2/1)
furnishing the key synthon 11, which is a ready acceptor for further
glycosylation reactions. We also acquired the X-ray crystal struc-
ture of 10 (Fig. 3); further confirming its structure.
⇑
Corresponding author. Tel.: +86 10 62731115; fax: +86 10 62732219.
E-mail address: zhangjianjun@cau.edu.cn (J. Zhang).
0008-6215/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.09.001

2534
G. Zong et al. / Carbohydrate Research 346 (2011) 2533–2539
HO
HO
O
O
HO
HO
HO
HO
O
O
O
HO
O
HO
OH
∗
O
OPMP
OPMP
O
2
OH
HO
∗
n
HO
HO
HO
O
O
1
HO
O
HO
OH
O
O
HO
HO
HO
O
3
HO
OH
Figure 1. O-Chain repeating unit of LPS from B. pseudomallei strain 304b (1) and the synthesized oligosaccharides 2 and 3.
HO
HO
HO
HO
HO
HO
O
O
HO
O
HO
O
HO
HO
HO
HO
HO
O
O
O
O
O
O
OH
OH
OH
HO
OPMP
OPMP
4
5
HO
HO
HO
HO
HO
HO
HO
O
HO
O
O
HO
O
O
O
O
HO
HO
O
O
O
O
OH
OH
HO
HO
OH
OPMP
6
Figure 2. Synthesized O-chain repeating unit of LPS from B. pseudomallei strain 304b.
With the monosaccharide building block 11 in hand, synthesis
of the target oligosaccharides was accomplished as shown in
Schemes 2 and 3. In our previous work, an efficient and large-scale
procedure for the preparation of p-methoxylphenyl 4-O-benzoyl-
groups, giving the desired acceptor 14 in 85% yield. Coupling of the
monosaccharide donor 15,17 which was obtained from
-rhamnose
in 10 steps with a total yield of 30%, with the glucosyl acceptor 11
afforded the expected (1?3)- -disaccharide 16 in 89% yield.
Cleavage of the 4-methoxyphenyl group of 16 with ceric ammo-
nium nitrate (CAN), followed by the reaction with trichloroacetoni-
trile and DBU,18 gave the trichloroacetimidate 17 in 75% yield over
two steps. Similar to the preparation of 13 and 16, condensation of
the disaccharide donor 17 with the disaccharide acceptor 14 fur-
nished the corresponding b-linked tetrasaccharide 18 in 77% yield.
The structure of 18 was confirmed from its 1H NMR and 13C NMR
spectra, and complete assignment of the 1H and 13C signals was
achieved by HSQC experiments (Fig. 4), showing the characteristic
L
a
6-deoxy-
Another key synthon 1215 could be easily obtained with this proce-
dure in 28% yield using -rhamnose as the starting material. Glyco-
a-
L-talopyranoside from
L
-rhamnose was developed.15
L
sylation between glucosyl acceptor 11 and 6-deoxytalosyl donor
12 was accomplished using TMSOTf as the catalyst in the presence
of 4 Å molecular sieves to afford the disaccharides 13 in 82% yield.
The configuration of the glycosyl bond formed in the product was
0
0
deduced from the corresponding coupling constants (J1 ,2 1.2 Hz).
Deallyloxycarbonylation of 13 was successfully achieved in
MeOH–THF16 in the presence of CH3COONH4, Pd[P(C6H5)3]4 and
NaBH4, within 4 min without affecting any of the other protecting
signals, such as d 5.73 (J 3.6 Hz, a-configuration), 4.90 (J 7.9 Hz, b-
configuration) for the two H-1’s of glucose, and d 5.40, 5.20 for the
two H-1’s of talose. Removal of the anomeric methoxylphenyl
group of 18 with CAN in 80% CH3CN–H2O, followed by trichloro-
acetimidate formation, provided tetrasaccharide donor 19 in 70%
yield over two steps.
OAc
ref . 17
O
AcO
AllylO
OAc
D-Glu
4 steps, 70%
OAc
a
Finally, condensation of the disaccharide acceptor 14 with the
tetrasaccharide donor 19, following the same glycosylation strat-
egy as mentioned above, gave the fully protected hexasaccharide
20 (65%) whose 1H NMR spectrum showed a signal at 4.65 ppm
(J 7.9 Hz), confirming the b glycosylation. The 13C NMR spectrum
of 20 showed six C-1 signals (d 99.9, 99.7, 99.7, 98.5, 98.1 and
95.7), and the complete assignment of the 1H and 13C signals of
20 was achieved by HSQC experiments (Fig. 5). Deacylation of
the disaccharide 16, the tetrasaccharide 18 and the hexasaccharide
20 with ammonia-saturated methanol afforded the target disac-
charide 4 (88%), tetrasaccharide 5 (84%) or hexasaccharide 6
(83%), respectively. The complete assignment of the 1H and 13C sig-
nals of 5 and 6 was achieved by HSQC experiments.
7
OBz
OR
O
O
d
BzO
HO
RO
AllylO
OBz
OPMP
OR
OPMP
11
8
9
R = Ac
R = H
10 R = Bz
b
c
Scheme 1. Synthesis of key synthon 11. Reagents and conditions: (a) 4-Methoxy-
phenol, BF3ꢀEt2O, CH2Cl2, 0–25 °C, 3 h; (b) MeOH–MeONa, rt, 1 h; (c) BzCl, Py,
CH2Cl2, ꢁ10 °C, 3 h, 90% for 10 over two steps; (d) PdCl2, 2:1 MeOH–CH2Cl2, rt,
overnight, 75% for 11.

G. Zong et al. / Carbohydrate Research 346 (2011) 2533–2539
2535
Figure 3. X-ray crystal structure of compound 10.
3. Conclusion
OBz
In conclusion, a convergent total synthesis of a disaccharide-
repeating unit of the O-antigenic polysaccharide of B. pseudomallei
strain 304b, and its dimer and also trimer, was achieved through a
regio- and stereoselective manner with suitably synthesized build-
ing blocks. The target molecules were functionalized with a
p-methoxylphenyl group at the reducing end of the glucoside,
which could be easily removed for subsequent attachment to a car-
rier protein. The described method can be used for construction of
higher oligosaccharides with similar structures. Immunological
investigations of the resultant disaccharide 4, tetrasaccharide 5
and hexasaccharide 6 are in progress and the results will be
reported in due course.
OCNHCCl3
11
O
BzO
O
O
ref. 17
OBz
O
OPMP
a
OBz
AllocO
OBz
11 steps, 28%
RO
OBz
OBz
13 R = Alloc
14 R = H
12
b
OBz
L-Rham
O
OCNHCCl3
11
BzO
O
O
OBz
OR
ref. 19
O
BzO
OBz
a
10 steps, 30%
BzO
OBz
OBz
OBz
4. Experimental procedures
4.1. General methods
15
16 R = PMP
c
17 R = CNHCCl3
14
a
Optical rotations were determined with a Perkin–Elmer model
241-MC automatic polarimeter for soln in a 1-dm, jacketed cell.
1H and 13C NMR spectra were recorded with Bruker DPX300 and
Bruker AVANCE600 spectrometers in CDCl3 or D2O solns. Internal
references: TMS (d 0.000 ppm for 1H), CDCl3 (d 77.00 ppm for
13C), HOD (d 4.700 for 1H). Elemental analysis was performed on
a Yanaco CHN Corder MF-3 automatic elemental analyzer. High-
resolution mass spectra (HRMS) was performed by the Peking
University, and electrospray-ionization mass spectra (ESIMS) was
performed by the China Agricultural University. Thin-layer chro-
matography (TLC) was performed on silica gel HF with detection
by charring with 30% (v/v) H2SO4 in MeOH or by UV detection.
Column chromatography was conducted by elution of a column
of silica gel (200–300 mesh) with EtOAc/petroleum ether (bp
60–90 °C) as the eluent. Solns were concd at a temperature
<60 °C under diminished pressure.
OBz
OBz
BzO
BzO
O
BzO
BzO
O
BzO
O
O
O
O
O
OBz
OR
OBz
BzO
BzO
18 R = PMP
c
19
R = CNHCCl3
d
d
16
4
18
5
Scheme 2. Synthesis of the target disaccharide 4 and tetrasaccharide 5. Reagents
and conditions: (a) TMSOTf, CH2Cl2, ꢁ10 °C to rt, 2 h, 82% for 13, 89% for 16, 77% for
18; (b) CH3COONH4, Pd[P(C6H5)3]4, NaBH4, MeOH–THF, ꢁ10 °C, 4 min, 85% for 14;
(c) 4:1 CH3CN–H2O, CAN, 30 °C, then CCl3CN, DBU, CH2Cl2, rt, 0.5 h, 75% over two
steps for 17, 70% over two steps for 19; (d) satd NH3–MeOH, rt, 96 h, 88% for 4, 84%
for 5.

2536
G. Zong et al. / Carbohydrate Research 346 (2011) 2533–2539
14
19
+
a
OBz
OBz
OBz
BzO
BzO
O
O
BzO
BzO
BzO
O
BzO
BzO
O
O
O
O
O
O
O
O
OBz
OPMP
OBz
OBz
BzO
BzO
BzO
20
b
6
Scheme 3. Synthesis of the target hexasaccharide 6. Reagents and conditions: (a) TMSOTf, CH2Cl2, ꢁ10 °C to rt, 2 h, 65% for 20; (b) satd NH3–MeOH, rt, 96 h, 83% for 6.
Figure 4. Partial HSQC spectra of the fully protected hexasaccharide 18.
Figure 5. Partial HSQC spectra of the fully protected tetrasaccharide 20.

G. Zong et al. / Carbohydrate Research 346 (2011) 2533–2539
2537
4.2. p-Methoxylphenyl 3-O-allyl-2,4,6-tri-O-benzoyl-
glucopyranoside (10)
a
-D
-
white foam. ½a 2D5
ꢃ
+5.9 (c 1.0 CHCl3). 1H NMR (300 MHz, CDCl3): d
8.14–7.11 (m, 25H, Bz-H), 7.02–6.99 (m, 2H, Ar-H), 6.71–6.68 (m,
2H, Ar-H), 5.85 (m, 1H, CH2@CHCH2OCO), 5.78 (d, 1H, J1,2 3.7 Hz,
Compound 819 (9.0 g, 20 mmol) was dissolved in 150 mL abso-
lute MeOH, 200 mg MeONa was added to the reaction mixture and
stirred at rt for 3 h. Afterwards, neutralization of the reaction mix-
ture with acidic ion exchange resin [Amberlite IR-120 (H+), Alfa
Aesar] was conducted and after filtration, the organic phase was
concentrated in vacuo, and then co-evaporated with toluene, pro-
viding compound 9 as a syrup. Then to a cold (0 °C) solution of
the syrup in dry pyridine (100 mL) was added benzoyl chloride
(2.8 mL, 24 mmol). The reaction mixture was slowly raised to room
temperature and stirred for 2 h, at the end of which time TLC (3:1
petroleum ether–EtOAc) indicated that the reaction was complete.
The reaction mixture was diluted with CH2Cl2 (100 ml), washed
with 1 M HCl (50 mL), water (2 ꢂ 50 mL) and dried (Na2SO4). The
solvent was evaporated under vacuum and the residue was puri-
fied by column chromatography (petroleum ether–EtOAc 4:1) to
afford compound 10 (11.5 g, 90%) over two steps as a white solid.
H-1), 5.67 (t, 1H, J3,4, J4,5 9.5 Hz, H-4), 5.53 (d, 1H, J1 ,2 1.2 Hz, H-
10), 5.44 (dd, 1H, J1,2 3.6 Hz, J2,3 9.8 Hz, H-2), 5.36–5.19 (m, 4H, H-
0
0
20, H-40, CH2@CHCH2OCO), 5.16 (t, 1H, J2 ,3 , J3 ,4 3.9 Hz, H-30), 4.87
0
0
0
0
(t, 1H, J2,3
, J3,4 9.5 Hz, H-3), 4.65–4.48 (m, 4H, H-5, H-6,
CH2@CHCH2OCO), 4.42 (m, 1H, H-6), 4.04 (m, 1H, H-50), 3.70 (s,
3H, C6H4OCH3), 0.74 (d, 3H, J 6.5 Hz, C-CH3). ESI-MS m/z calcd for
C
C
58H52O18Na (M+Na)+ 1059.3. Found: 1059.3. Anal. Calcd for
58H52O18: C, 67.18; H, 5.05. Found: C, 67.43; H, 5.20.
4.5. p-Methoxylphenyl 2,4-di-O-benzoyl-6-deoxy-a-L-talopyran
osyl-(1?3)-2,4,6-tri-O-benzoyl- -glucopyranoside (14)
a-D
To a cooled (ꢁ5 °C) solution of compound 13 (2.1 g, 2.0 mmol)
in 1:1 MeOH–THF (60 mL) in 250 mL flask was added CH3COONH4
(1.6 g, 20 mmol). With vigorous stirring, NaBH4 (18 mg, 0.5 mmol),
Pd[P(C6H5)3]4 (92 mg, 0.08 mmol), and then additional NaBH4
(93 mg, 2.5 mmol) was added in 3 portions immediately one after
another. One min after the addition of the second portion of NaBH4,
TLC (2:1 petroleum ether–EtOAc) indicated that the reaction was
complete. The reaction mixture was concentrated under dimin-
ished pressure, the residue was dissolved in CH2Cl2 (150 mL) and
washed with water (50 mL), then the organic phase was dried over
Na2SO4. Evaporation and purification by flash column chromatog-
raphy (petroleum ether–EtOAc 3:1) afforded compound 14 as a
½
a 2D5
ꢃ
+89.6 (c 1.0 CHCl3); 1H NMR (300 MHz, CDCl3): d8.13–7.36 (m,
15H, Bz-H), 7.03–7.00 (m, 2H, Ar-H), 6.70–6.67 (m, 2H, Ar-H), 5.76
(d, 1H, J1,2 3.7 Hz, H-1), 5.67 (m, 1H, CH2@CHCH2), 5.53 (t, 1H, J3,4
,
J4,5 9.3 Hz, H-4), 5.29 (dd, 1H, J1,2 3.7 Hz, J2,3 9.9 Hz, H-2), 5.14–4.94
(m, 2H, CH2@CHCH2), 4.56–4.37 (m, 4H, H-3, H-5, CH2@CHCH2),
4.22–4.16 (m, 1H, H-6a), 4.13–4.07 (m, 1H, H-6b), 3.70 (s, 3H,
C6H4OCH3). ESI-MS m/z calcd for C37H34O10Na (M+Na)+ 661.2.
Found: 661.2. Anal. Calcd for C37H34O10: C, 69.58; H, 5.37. Found:
C, 69.47; H, 5.31.
foamy solid (1.65 g, 85%). ½a D25
ꢃ
+20.4 (c 1.0 CHCl3). 1H NMR
(300 MHz, CDCl3): d 8.13–7.17 (m, 25H, Bz-H), 7.03–7.00 (m, 2H,
Ar-H), 6.72–6.69 (m, 2H, Ar-H), 5.76 (d, 1H, J1,2 3.6 Hz, H-1), 5.65
(t, 1H, J3,4, J4,5 9.5 Hz, H-4), 5.53 (s, 1H, H-10), 5.45 (dd, 1H, J1,2
3.6 Hz, J2,3 9.8 Hz, H-2), 5.09–5.07 (m, 2H, H-20, H-40), 4.86 (t, 1H,
J2,3, J3,4 9.5 Hz, H-3), 4.58–4.51 (m, 2H, H-5, H-6), 4.42 (m, 1H, H-
6), 4.24 (m, 1H, H-30), 3.95 (m, 1H, H-50), 3.70 (s, 3H, C6H4OCH3),
2.67 (d, 1H, J 5.8 Hz, OH), 0.74 (d, 3H, J 6.5 Hz, C-CH3). 13C NMR
(75 MHz, CDCl3): d 167.0, 166.1, 166.0, 165.5, 165.1 (5 ꢂ COPh),
155.5, 150.3, 118.3(2), 114.6(2) (C6H4OCH3), 99.6, 95.7 (2 ꢂ C-1),
75.5, 73.9, 71.7, 70.4, 69.6, 68.5, 66.0, 65.3, 63.0, 55.5 (C6H4OCH3),
16.0 (C-CH3). ESI-MS m/z calcd for C54H48O16Na (M+Na)+ 975.3.
Found: 975.3. Anal. Calcd for C54H48O16: C, 68.06; H, 5.08. Found:
C, 67.88; H, 5.05.
4.3. p-Methoxylphenyl 2,4,6-tri-O-benzoyl-a-D-glucopyranoside
(11)
To a solution of 10 (9.2 g, 14.4 mmol) in MeOH (100 mL) and
CH2Cl2 (50 mL) was added PdCl2 (0.20 g, 1.2 mmol), and the mix-
ture was stirred at rt overnight, at the end of which time TLC
(3:1 petroleum ether–EtOAc) indicated that the reaction was com-
plete. The mixture was filtered, the filter cake was washed with
CH2Cl2 and the combined filtrate and washings were concentrated.
Purification by column chromatography with 3:1 petroleum ether–
EtOAc as the eluent afforded compound 11 (6.5 g, 75%) as a white
foam. ½a 2D5
ꢃ
+102.1 (c 1.0 CHCl3). 1H NMR (300 MHz, CDCl3): d 8.12–
7.38 (m, 15H, Bz-H), 7.05–7.03 (m, 2H, Ar-H), 6.73–6.70 (m, 2H, Ar-
H), 5.74 (d, 1H, J1,2 3.7 Hz, H-1), 5.44 (t, 1H, J3,4, J4,5 9.4 Hz, H-4),
5.26 (dd, 1H, J1,2 3.7 Hz, J2,3 9.9 Hz, H-2), 4.70–4.62 (m, 1H, H-5),
4.60–4.50 (m, 2H, H-3, H-6a), 4.47–4.40 (m, 1H, H-6b), 3.71 (s,
3H, C6H4OCH3), 2.73 (d, 1H, J 5.3 Hz, OH). ESI-MS m/z calcd for
4.6. p-Methoxylphenyl 2,3,4-tri-O-benzoyl-6-deoxy-
a-L-talopyr
anosyl-(1?3)-2,4,6-tri-O-benzoyl- -glucopyranoside (16)
a-D
Compound 11 (1.79 g, 3.0 mmol) and 15 (2.3 g, 3.7 mmol) were
coupled in the presence of catalytic TMSOTf (54 L, 0.3 mmol) un-
C
C
34H30O10Na (M+Na)+ 621.2. Found: 621.2. Anal. Calcd for
37H34O10: C, 68.22; H, 5.05. Found: C, 68.31; H, 5.23.
l
der the same conditions as described above for the coupling of 11
with 12. Purification by silica gel chromatography with 6:3:1
petroleum ether–toluene–EtOAc as the eluent gave disaccharide
4.4. p-Methoxylphenyl 3-O-allyloxycarbonyl-2,4-di-O-benzoyl-
6-deoxy- -talopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-
glucopyranoside (13)
a
-L
a
-D
-
16 (2.8 g, 89%) as a foamy solid. ½a D25
ꢃ
+8.8 (c 1.0 CHCl3). 1H NMR
(300 MHz, CDCl3): d 8.17–7.13 (m, 30H, Bz-H), 7.03–7.00 (m, 2H,
Ar-H), 6.72–6.69 (m, 2H, Ar-H), 5.79 (d, 1H, J1,2 3.6 Hz, H-1), 5.71
0
0
Compound 11 (3.3 g, 5.5 mmol), 12 (4.0 g, 6.6 mmol) and 4 Å
molecular sieves (4 g) were dried together under high vacuum for
2 h, then dissolved in anhydrous, redistilled CH2Cl2 (100 mL).
(t, 1H, J3,4, J4,5 9.5 Hz, H-4), 5.57 (s, 1H, H-10), 5.56 (t, 1H, J2 ,3
,
J3 ,4 3.9 Hz, H-30), 5.47 (dd, 1H, J1,2 3.6 Hz, J2,3 9.8 Hz, H-2), 5.35
(m, 1H, H-20), 5.31 (m, 1H, H-40), 4.91 (t, 1H, J2,3, J3,4 9.5 Hz, H-3),
4.61–4.53 (m, 2H, H-5, H-6), 4.43 (m, 1H, H-6), 4.14 (m, 1H, H-
50), 3.70 (s, 3H, C6H4OCH3), 0.76 (d, 3H, J 6.5 Hz, C-CH3). 13C NMR
(75 MHz, CDCl3): d 166.1(2), 165.4, 165.1, 165.0, 164.8 (6 ꢂ COPh),
155.5, 150.3, 118.3(2), 114.7(2) (C6H4OCH3), 100.0, 95.7 (2 ꢂ C-1),
76.1, 73.9, 69.6, 69.2, 68.5, 67.7, 66.3, 65.9, 63.0, 55.5 (C6H4OCH3),
15.9 (C-CH3). ESI-MS m/z calcd for C61H52O17Na (M+Na)+ 1079.3.
Found: 1079.3. Anal. Calcd for C61H52O16: C, 69.31; H, 4.96. Found:
C, 69.48; H, 4.70.
0
0
TMSOTf (54
lL, 0.3 mmol) was added dropwise at ꢁ10 °C under
an N2 atmosphere. The reaction mixture was stirred for 0.5 h, during
which time the mixture was allowed to gradually warm to ambient
temperature. TLC (4:2:1 petroleum ether–toluene–EtOAc) indicated
that the reaction was complete. Then the reaction mixture was neu-
tralized with triethylamine and filtrated, and the filtrate was con-
centrated. Purification of the residue by column chromatography
(6:3:1 petroleum ether–toluene–EtOAc) gave 13 (4.7 g, 82%) as a

2538
G. Zong et al. / Carbohydrate Research 346 (2011) 2533–2539
4.7. 2,3,4-Tri-O-benzoyl-6-deoxy-
a
-
L
-talopyranosyl-(1?3)-
½
a 2D5
ꢃ
ꢁ60.0 (c 1.0 CHCl3). 1H NMR (300 MHz, CDCl3): d 8.06–6.68
2,4,6-tri-O-benzoyl-
a-
D-glucopyranosyl trichloroacetimidate
(m, 59H, Ar-H), 5.73 (d, 1H, J1,2 3.6 Hz, H-1), 5.56 (t, 1H, J3,4, J4,5
9.6 Hz, H-4), 5.41–5.38 (m, 2H, H-10, H-30), 5.36 (t, 1H, J3,4, J4,5
9.8 Hz, H-400), 5.30 (dd, 1H, J1,2 3.6 Hz, J2,3 9.8 Hz, H-2), 5.22–5.16
(m, 5H, H-1000, H-20, H-40, H-3000, H-4000), 5.08 (m, 1H, H-2000), 4.90 (d,
1H, J1,2 7.9 Hz, H-100), 4.80 (t, 1H, J2,3, J3,4 9.5 Hz, H-3), 4.51–4.26
(m, 7H, H-5, 4 ꢂ H-6, H-200, H-300), 4.00–3.85 (m, 3H, 3 ꢂ H-5),
3.70 (s, 3H, C6H4OCH3), 0.68, 0.64 (2d, 6H, J 6.4 Hz, 2 ꢂ C-CH3).
13C NMR (75 MHz, D2O): d 166.2, 166.1, 166.0(3), 165.7, 165.2,
164.8, 164.7, 164.6, 163.8 (11 ꢂ COPh), 155.4, 150.4, 118.4(2),
114.6(2) (C6H4OCH3), 100.0, 99.7, 97.8, 95.8 (4 ꢂ C-1), 78.7, 73.6,
73.2, 71.9, 70.2, 69.8, 69.1, 69.0, 68.3, 67.6, 67.5, 66.1, 66.0, 65.7,
63.1, 62.9, 55.5 (C6H4OCH3), 15.8, 15.7 (2 ꢂ C-CH3). ESI-MS m/z
calcd for C108H92O31Na (M+Na)+ 1907.6. Found: 1907.6. Anal. Calcd
for C108H92O31: C, 68.78; H, 4.92. Found: C, 68.93; H, 4.75.
(17)
To a solution of compound 16 (2.5 g, 2.4 mmol) in acetonitrile
(80 mL) and water (20 mL) was added ceric ammonium nitrate
(CAN) (5.2 g, 9.6 mmol). The mixture was stirred for 20 min at
30 °C, at the end of which time TLC (2:1 petroleum ether–EtOAc)
indicated that the reaction was complete. The solvent was evapo-
rated under diminished pressure at 50 °C to give a residue, which
was diluted with CH2Cl2 and washed with water. The organic
phase was dried and concentrated. Purification by silica gel chro-
matography with 3:1 petroleum ether–EtOAc as the eluent
afforded
2,3,4-tri-O-benzoyl-6-deoxy-
a-L-talopyranosyl-(1?3)-
2,4,6-tri-O-benzoyl-
a-D-glucopyranose. A mixture of this com-
pound, trichloroacetonitrile (2 mL, 20 mmol), and 1,8-diazabicyclo
[5.4.0] undecene (DBU) (0.2 mL, 1.6 mmol) in dry CH2Cl2 (50 mL)
was stirred for 0.5 h and then concentrated. The residue was puri-
fied by chromatography with 4:1 petroleum ether–EtOAc as the
4.10. p-Methoxylphenyl 6-deoxy-
a
L
-
L
-talopyranosyl-(1?3)-b-
D
-
glucopyranosyl-(1?3)-6-deoxy-
a-
-talopyranosyl-(1?3)-
a-
D-
glucopyranoside (5)
eluent to give 17 (1.95 g, 75%) as a white foam. ½a D25
ꢃ
+19.2 (c 1.0
CHCl3). 1H NMR (300 MHz, CDCl3): d 8.60 (s, 1H, CNHCCl3), 8.17–
Compound 18 (0.25 g, 0.13 mmol) was dissolved in satd NH3–
MeOH (40 mL). After 1 week at rt, the reaction mixture was con-
centrated, and the residue was purified by chromatography on
Sephadex LH-20 (MeOH) to afford 5 (82 mg, 84%) as a foamy solid.
7.10 (m, 30H, Bz-H), 5.76 (d, 1H, J1,2 3.6 Hz, H-1), 5.79 (t, 1H, J3,4
,
J4,5 9.8 Hz, H-4), 5.62 (dd, 1H, J1,2 3.7 Hz, J2,3 9.7 Hz, H-2), 5.54 (t,
1H, J2 ,3 , J3 ,4 3.9 Hz, H-30), 5.50 (s, 1H, H-10), 5.32–5.29 (m, 2H,
H-20, H-40), 4.77 (t, 1H, J2,3, J3,4 9.6 Hz, H-3), 4.65 (dd, 1H, J 2.5 Hz,
12.2 Hz, H-6), 4.56 (m, 1H, H-5), 4.43 (dd, 1H, J 4.5 Hz, 12.2 Hz,
H-6), 4.11 (m, 1H, H-50), 0.74 (d, 3H, J 6.5 Hz, C-CH3). ESI-MS m/z
calcd for C56H46Cl3NO16Na (M+Na)+ 1116.2. Found: 1116.2. Anal.
Calcd for C56H46Cl3NO16: C, 61.41; H, 4.23; N, 1.28. Found: C,
61.56; H, 4.37; N, 1.43.
0
0
0
0
½
a 2D5 +35.6 (c 0.5 CHCl3). 1H NMR (300 MHz, CDCl3): d 6.99, 6.82
ꢃ
(2d, 4H, J 9.0 Hz, C6H4OCH3), 5.34 (d, 1H, J1,2 3.4 Hz, H-1), 5.15 (s,
2H, H-10, H-1000), 4.55 (d, 1H, J010 3.4 Hz, H-100), 4.27–4.17 (m, 2H,
00
,2
2 ꢂ H-5), 4.05 (m, 1H, H-2000), 3.99 (t, 1H, J2 ,3 , J3 ,4 3.0 Hz, H-30),
3.88 (t, 1H, J2,3, J3,4 9.4 Hz, H-3), 3.85–3.82 (m, 2H, H-40, H-4000),
3.80–3.77 (m, 2H), 3.72–3.62 (m, 9H), 3.51 (m, 1H), 3.45–3.38
(m, 2H), 3.38–3.33 (m, 2H), 1.14, 1.10 (2d, 6H, J 6.6 Hz, 2 ꢂ C-
CH3). 13C NMR (75 MHz, D2O): d 154.6, 150.4, 118.8(2), 115.1(2)
(C6H4OCH3), 101.6, 101.5 (2 ꢂ C-1-Talp), 101.1 (C-100), 98.2 (C-1),
81.9, 79.9, 75.9, 73.8, 73.7, 72.5, 72.2, 71.7, 70.0, 69.9, 69.6, 68.0,
67.9, 67.5(2), 65.6, 60.7, 60.3, 55.8 (C6H4OCH3), 15.5, 15.4 (2 ꢂ C-
CH3). HRMS calcd for C31H48O20Na (M+Na)+ 763.2637. Found:
763.2624.
0
0
0
0
4.8. p-Methoxylphenyl 6-deoxy-a-L-talopyranosyl-(1?3)-a-D-
glucopyranoside (4)
Compound 16 (0.15 g, 0.14 mmol) was dissolved in satd
NH3–MeOH (40 mL). After 120 h at rt, the reaction mixture was
concentrated, and the residue was purified by chromatography
on Sephadex LH-20 (MeOH) to afford 4 (0.054 g, 88%) as a white
foamy solid. ½a 2D5
ꢃ
ꢁ46.5 (c 0.5 H2O). 1H NMR (300 MHz, D2O):
4.11. 2,3,4-Tri-O-benzoyl-6-deoxy-
2,4,6-tri-O-benzoyl-b- -glucopyranosyl-(1?3)-2,4-di-O-
benzoyl-6-deoxy- -talopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-
-glucopyranosyl trichloroacetimidate (19)
a-L-talopyranosyl-(1?3)-
d 6.95 (dd, 4H, Ar-H), 5.37 (d, 1H, J1,2 3.6 Hz, H-1), 5.17 (s, 1H,
H-10), 4.26 (m, 1H, H-50), 3.90 (t, 1H, J2,3, J3,4 9.5 Hz, H-3), 3.89
D
a-L
(m, 1H, H-20), 3.85 (t, 1H, J2 ,3 , J3 ,4 3.3 Hz, H-30), 3.75–3.66 (m,
8H, H-2, H-40, H-5, 2 ꢂ H-6, C6H4OCH3), 3.44 (t, 1H, J3,4, J4,5
9.6 Hz, H-4), 1.16 (d, 3H, J 6.6 Hz, C-CH3). 13C NMR (75 MHz,
D2O): d 154.6, 150.3, 118.7(2), 115.0(2) (C6H4OCH3), 101.8, 98.2
(2 ꢂ C-1), 79.9, 72.5, 72.2, 71.6, 69.9, 67.8, 67.5, 65.6, 60.3, 55.8
(C6H4OCH3), 15.5 (C-CH3). HRMS calcd for C19H28O11Na (M+Na)+
455.1529. Found: 455.1512.
a-D
0
0
0
0
To a solution of compound 18 (1.0 g, 0.53 mmol) in acetonitrile
(40 mL), and water (10 mL) was added CAN (1.2 g, 2.2 mmol). The
mixture was stirred for 20 min at 30 °C, at the end of which time
TLC (petroleum ether–EtOAc 1:1) indicated that the reaction was
complete. The solvent was evaporated under diminished pressure
to give a residue, which was dissolved in CH2Cl2, and washed with
water. The organic phase was dried (Na2SO4) and concd. Purifica-
tion by silica gel chromatography with petroleum ether–EtOAc
4.9. p-Methoxylphenyl 2,3,4-tri-O-benzoyl-6-deoxy-
ranosyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyranosyl-(1?3)-
2,4-di-O-benzoyl-6-deoxy- -talopyranosyl-(1?3)-2,4,6-tri-O-
benzoyl- -glucopyranoside (18)
a-L-talopy
D
a-
L
3:1 as the eluent afforded 2,3,4-tri-O-benzoyl-6-deoxy-
pyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyranosyl-(1?3)-
2, 4 - d i - O-benzoyl-6-deoxy- -talopyranosyl-(1?3)-2,4,6-tri-O-
benzoyl- -glucopyranose as a slight yellow foamy solid. The foa-
a-L-talo-
a-
D
D
a-L
To a cooled (ꢁ10 °C) solution of 14 (0.85 g, 0.89 mmol) and 17
(1.07 g, 0.98 mmol) in anhydrous, redistilled CH2Cl2 (40 mL) was
added 4 Å molecular sieves (1.5 g) and the mixture was stirred un-
a-D
my solid was dried under high vacuum for 2 h, then dissolved in
anhyd CH2Cl2 (30 mL) and trichloroacetonitrile (0.5 mL, 5 mmol)
and DBU (0.03 mL, 0.3 mmol) were added successively under an
N2 atmosphere. The mixture was stirred for 0.5 h and then concd.
The residue was purified by chromatography with petroleum
der an N2 atmosphere for 30 min. Then TMSOTf (27 lL, 0.15 mmol)
was added to the mixture. The reaction mixture was stirred at
ꢁ10 °C for 0.5 h, during which time the mixture was allowed to
gradually warm to ambient temperature. TLC (petroleum ether–
EtOAc 3:1) indicated that the reaction was complete. Then the
reaction mixture was quenched with Et3N (2 drops) and filtered.
The filtrate was evaporated in vacuo to give a residue, which was
purified by silica gel column chromatography (petroleum ether–
EtOAc 3:1) to give tetrasaccharide 18 (1.3 g, 77%) a white foam.
ether–EtOAc 4:1 to give 19 (0.71 g, 70%) as a foamy solid. ½a D25
ꢃ
ꢁ40.0 (c 0.7 CHCl3). 1H NMR (300 MHz, CDCl3): d 8.57 (s, 1H,
CNHCCl3), 8.09–6.86 (m, 55H, Ar-H), 6.73 (d, 1H, J1,2 3.6 Hz, H-1),
5.65 (t, 1H, J3,4, J4,5 9.7 Hz, H-4), 5.41 (dd, 1H, J1,2 3.7 Hz, J2,3
9.7 Hz, H-2), 5.39 (t, 1H, J2 ,3 , J3 ,4 3.8 Hz, H-30), 5.36–5.33 (m, 2H,
H-10, H-400), 5.22–5.19 (m, 2H, H-20, H-40), 5.17 (d, 1H, J1,2 1.2 Hz,
0
0
0
0

G. Zong et al. / Carbohydrate Research 346 (2011) 2533–2539
2539
H-1000), 5.15 (m, 1H, H-3000), 5.07 (m, 1H, H-2000), 4.89 (d, 1H, J1,2
7.8 Hz, H-100), 4.67 (t, 1H, J2,3, J3,4 9.5 Hz, H-3), 4.59–4.78 (m, 2H,
H-5, H-6), 4.44–4.25 (m, 5H, H-200, H-300, 3 ꢂ H-6), 4.00–3.88 (m,
3H, 3 ꢂ H-5), 3.70 (s, 3H, C6H4OCH3), 0.68, 0.64 (2d, 6H, J 6.4 Hz,
2 ꢂ C-CH3). ESI-MS m/z calcd for C103H86Cl3NO30Na (M+Na)+
1944.4. Found: 1944.8. Anal. Calcd for C103H86Cl3NO30: C, 64.29;
H, 4.51; N, 0.73. Found: C,64.44; H, 4.68; N, 0.79.
(m, 3H, 3 ꢂ H-1-Talp), 4.57, 4.55 (2d, 2H, J 7.8 Hz, H-1-Glup), 4.27–
4.18 (m, 3H, 3 ꢂ H-5-Talp), 4.05–4.97 (m, 4H), 3.88 (t, 1H, J2,3, J3,4
9.8 Hz, H-3), 3.85–3.78 (m, 6H), 3.72–3.59 (m, 10H), 3.56–3.35
(m, 10H), 1.14 (d, 1H, J 6.4 Hz, C-CH3), 1.12 (d, 1H, J 6.2 Hz, C-
CH3), 1.10 (d, 1H, J 6.4 Hz, C-CH3). 13C NMR (75 MHz, D2O): d
154.7, 150.3, 118.8(2), 115.1(2) (C6H4OCH3), 101.6, 101.5, 101.4
(3 ꢂ C-1-Talp), 101.1, 101.1, 98.2 (3 ꢂ C-1-Glup), 55.9 (C6H4OCH3),
15.5(2), 15.4 (3 ꢂ C-CH3). HRMS for C43H68O29Na (M+Na)+
1071.3744. Found: 1071.3719.
4.12. p-Methoxylphenyl 2,3,4-tri-O-benzoyl-6-deoxy-
talopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyranosyl-
(1?3)-2,4-di-O-benzoyl-6-deoxy- -talopyranosyl-(1?3)-
2,4,6-tri-O-benzoyl-b- -glucopyranosyl-(1?3)-2,4-di-O-
benzoyl-6-deoxy- -talopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-
-glucopyranoside (20)
a-L-
D
a
-L
Acknowledgements
D
a-L
This work was supported by NSFC of China (20902108), the
National Basic Research Program of China (2010CB126105), the
Chinese Universities Scientific Fund (2011JS030) and Scholarship
Award for Excellent Doctoral Student granted by Ministry of
Education of China.
a-D
Glycosylation between disaccharide acceptor 14 (0.14 g,
0.15 mmol) and tetrasaccharide donor 19 (0.30 g, 0.16 mmol)
was accomplished by following the same procedure as described
above for the preparation of tetrasaccharide 18. After purification,
hexasaccharide 20 (0.26 g, 65%) was afforded as a white foamy so-
Supplementary data
lid. ½a 2D5
ꢃ
ꢁ59.1 (c 1.0 CHCl3). 1H NMR (300 MHz, CDCl3): d8.03–6.68
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.09.001.
(m, 84H, Ar-H), 5.72 (d, 1H, J1,2 3.6 Hz, H-1), 5.56 (t, 1H, J3,4, J4,5
0
0
0
0
9.6 Hz, H-4), 5.42 (s, 1H, H-1-Talp), 5.37 (t, 1H, J2 ,3 , J3 ,4 9.5 Hz,
H-3-Glup), 5.35–5.27 (m, 3H, H-2, 2 ꢂ H-4-Glup), 5.22–5.09 (m,
7H, 2 ꢂ H-1-Talp, 2 ꢂ H-3-Talp, 3 ꢂ H-4-Talp), 5.08–5.06 (m, 2H,
2 ꢂ H-2-Talp), 4.90 (m, 1H, H-2-Talp), 4.86 (d, 1H, J1,2 7.9 Hz, H-
1-Glup), 4.80 (t, 1H, J2,3, J3,4 9.4 Hz, H-3), 4.65 (d, 1H, J1,2 7.9 Hz,
H-1-Glup), 4.52–4.08 (m, 11H, 2 ꢂ H-2-Glup, 2 ꢂ H-3-Glup,
6 ꢂ H-6-Glup, H-5), 3.97–3.93 (m, 2H, 2 ꢂ H-5), 3.85–3.73 (m,
2H, 2 ꢂ H-5), 3.70 (s, 3H, C6H4OCH3), 3.47 (m,1H, H-5), 0.67, 0.62,
0.58 (3d, 9H, J 6.4 Hz, 3 ꢂ C-CH3). 13C NMR (75 MHz, D2O): d
166.1(2), 166.0(2), 165.9(4), 165.6, 165.2, 164.9, 164.7, 164.6,
164.5, 164.4, 163.8 (16 ꢂ COPh), 155.4, 150.4, 118.4(2), 114.6(2)
(C6H4OCH3), 99.9, 99.7(2), 98.5, 98.1, 95.7 (6 ꢂ C-1), 55.5
(C6H4OCH3), 15.7, 15.5, 14.1 (3 ꢂ C-CH3). ESI-MS m/z calcd for
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C
155H132O45Na (M+Na)+ 2735.8. Found: 2735.9. Anal. Calcd for
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4.13. p-Methoxylphenyl 6-deoxy-
a
-
L
-talopyranosyl-(1?3)-b-
-talopyranosyl-(1?3)-b-
-talopyranosyl-(1?3)-a-D-
D-
glucopyranosyl-(1?3)-6-deoxy-
glucopyranosyl-(1?3)-6-deoxy-
glucopyranoside (6)
a
-
L
D-
a-
L
Hexasaccharide 20 (0.18 g, 0.066 mmol) was dissolved in satd
NH3–MeOH (40 mL). After 1 week at rt, the reaction mixture was
concentrated, and the residue was purified by chromatography
on Sephadex LH-20 (MeOH) to afford 6 (57 mg, 83%) as a foamy so-
16. Zong, G. H.; Yan, S. Q.; Liang, X. M.; Zhang, J. J.; Wang, D. Q.; Kong, F. Z. Chin.
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lid. ½a 2D5
ꢃ
+67.0 (c 0.5 CHCl3). 1H NMR (300 MHz, CDCl3): d 7.02, 6.85
(2d, 4H, J 9.0 Hz, C6H4OCH3), 5.36 (d, 1H, J1,2 3.5 Hz, H-1), 5.16–5.14