Synthesis of the pentasaccharide repeating unit of Escherichia coli O128 antigen

Article abstract of DOI:10.1016/j.carres.2010.07.025

A pentasaccharide, 4-methoxyphenyl 2-acetamido-2-deoxy-β-d- galactopyranosyl-(1→4)-α-d-galactopyranosyl-(1→3) -2-acetamido-2-deoxy-β-d-galactopyranosyl-(1→6)-[α-l- fucopyranosyl-(1→2)]-β-d-galactopyranoside (1), representing the repeating unit of Escherichia coli O128 antigen, was successfully prepared in 23% overall yield via a convergent '2+3' glycosylation strategy.

Full text of DOI:10.1016/j.carres.2010.07.025

Carbohydrate Research 345 (2010) 2272–2276
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis of the pentasaccharide repeating unit of Escherichia coli O128 antigen
Xun Lv a,b, Shuihong Cheng c, Guoha Wei a, Yuguo Du a,b,
⇑
a State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
b College of Chemistry and Chemical Engineering, Graduate University of Chinese Academy of Sciences, Beijing 100049, China
c CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 June 2010
Received in revised form 9 July 2010
Accepted 14 July 2010
Available online 21 July 2010
A
pentasaccharide, 4-methoxyphenyl 2-acetamido-2-deoxy-b-
D
-galactopyranosyl-(1?4)-
a-D-galacto-
pyranosyl-(1?3)-2-acetamido-2-deoxy-b- -galactopyranosyl-(1?6)-[
D
a
-
L
-fucopyranosyl-(1?2)]-b-D-
galactopyranoside (1), representing the repeating unit of Escherichia coli O128 antigen, was successfully
prepared in 23% overall yield via a convergent ‘2+3’ glycosylation strategy.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Glycosylation
Escherichia coli O128 antigen
Oligosaccharides
The O-antigens (O-specific polysaccharides) are the exposed part
of the lipopolysaccharides (LPS), the main outer-membrane compo-
nent of Gram-negative bacteria. Structural variations of O-antigens
contributeto thewidevarietyofantigenictypesofdifferentbacterial
species.1 O-antigens are furthermore recognized as important viru-
lence factors and have the potential to influence host–pathogen
interactions in many ways.2 During the investigation on the molec-
ular basis related to infantile diarrhea, E. coli O128 antigen has been
isolated and characterized from enteropathogenic E. coli strains, one
of the major species associated with diarrhea patients worldwide.3
The primary structure of the O-antigen repeating unit of E. coli
In our previous work, we found that using a b-(1?4)-linked
disaccharide donor might generate an -glycosidic product under
a
proper glycosylation conditions.9 Accordingly, E. coli O128 repeat-
ing pentasaccharide could be disconnected into a disaccharide do-
nor 2, in which the 2-OH was blocked with a non-neighboring
participation group such as benzyl, and a trisaccharide acceptor
3. Both 2 and 3 could be assembled from known or commercially
available monosaccharide building blocks 4–8 (Scheme 1).
In the preparation of disaccharide donor 2, we first tried the
coupling reaction between compounds 410 and 511 with the tradi-
tional method.12 Unfortunately, we observed a significant amount
of byproduct formed via thio-group transfer13–15 from 5 to 4. We
thus turned our attention to the ‘inverse procedure’16 under low
temperature (Scheme 2), in which donor 4 in CH2Cl2 was added
into a pre-cooled solution of acceptor 5 and the promoter, trimeth-
ylsilyl trifluoromethanesulfonate (TMSOTf), in CH2Cl2 at ꢀ15 °C. As
expected, synthesis of disaccharide 2 was successfully achieved in
O128 has been established4 as ?3)-b-
(1?3)-b- -GalNAc-(1?6)-[ -Fuc-(1?2)]-b-
saccharide (Fig. 1) in which [ -Fuc-(1?2)]-b-D
D
-GalNAc-(1?4)-
-Gal-(1?, a penta-
-Gal was proposed
a-D-Gal-
D
a-
L
D
a
-L
to be the immunodominant part. Due to frequently occurring antibi-
otic-resistant pathogens, there are stringent requirements in devel-
oping potent vaccines against infectious diseases. Vaccines
consisting of carbohydrates coupled to a carrier protein have been
found to be effective for the prevention of bacterial invasive dis-
eases.5,6 Thus, providing structurally defined glycoconjugates via
syntheticcarbohydrate antigens wouldbeone oftheattractive strat-
egies in healing infectious diseases.7,8 As a collaborative project, we
envisioned that the efficient synthesis of the pentasaccharide
repeating unit of the O-128 antigen would pave way for the develop-
ment of a well-structured glycoconjugate vaccine against infantile
diarrhea E. coli O128. We herein report the synthesis of the core
pentasaccharide using a convergent ‘2+3’ glycosylation strategy.
HO
OH
O
O
OH
O
OH
AcHN
OH
HO
O
HO
O
O
O
HO
AcHN
HO
O
O
O
OH
OH
OH
⇑
Corresponding author. Tel./fax: +86 10 62849126.
E-mail address: duyuguo@rcees.ac.cn (Y. Du).
Figure 1. Structure of E. coli O128 antigen.
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.07.025

X. Lv et al. / Carbohydrate Research 345 (2010) 2272–2276
2273
OAcOAc
HO
HO
OH
O
OAc
OAc
O
O
OH
OBn
O
OBn
O
AcO
TrocHN
O
O
OH
O
AcO
SCHMe2
BnO
TrocHN
OH
HO
AcHN
BnO
SCHMe2
OC(NH)CCl3
OBn
HO
OBn
O
2
5
4
HO
O
O
O
Ph
HO
AcHN
O
O
O
O
O
OMP
HO
O
OMP
O
OH
O
HO
HO
1
OH
O
HO
SEt
OBn
O
O
O
O
TrocHN
O
SPh
NHTroc
OMP
OH
O
OBn
O
O
O
OH
OH
HO
6
7
8
O
O
Scheme 1. Retrosynthetic analysis of E. coli O128 pentasaccharide.
OAc
OAc
O
OAc
OAc
O
OH
OBn
O
SCHMe2
OBn
O
Inverse Procedure
AcO
OBn
O
+
AcO
TrocHN
BnO
SCHMe2
BnO
TrocHN
OC(NH)CCl3
OBn
4
5
2
Scheme 2. Synthesis of disaccharide donor 2.
Ph
0 °C, generating 4-methoxyphenyl 6-O-tert-butyldimethylsilyl-
3,4-O-isopropylidene-b- -galactopyranoside (11). Condensation of
ethyl 2-O-benzyl-3,4-O-isopropylidene-b-
-fucopyranoside (8)18
D
O
OH
HO
HO
O
L
PhCHO, TsOH
O
O
and 11 in the presence of N-iodosuccinimide (NIS) and TMSOTf at
0 °C in CH2Cl2 gave disaccharide 12 (81%). Desilylation of 12 with
tetrabutylammonium fluoride (TBAF) in THF yielded the disaccha-
ride acceptor 13, which was further coupled with 10, under the same
reaction conditions as described in the preparation of 12, to afford
trisaccharide 14. Deacetylation of 14 with NaOMe in MeOH
furnished trisaccharide acceptor 3 in a yield of 70% over three steps.
In order to improve the efficiency in making trisaccharide deriv-
ative 14, we also investigated one-pot glycosylation as shown in
Scheme 4. Taking advantage of different reactivity between the pri-
mary and secondary alcohols of acceptor 7, donor 10 was first
added to a pre-cooled (ꢀ42 °C) solution of 7 in CH2Cl2 in the pres-
ence of co-catalysts NIS–TMSOTf. An additional amount of 10 was
added on occasion to push the reaction to completion. When
acceptor 7 was completely consumed, the second glycosyl donor
8 was added to the same reaction system at ꢀ15 °C, together with
another portion of NIS–TMSOTf. Compound 14 was eventually ob-
tained in 28–43% isolated yields. However, this method provided
unreliable yield, and the purification step was also tedious. We
thus prepared 14 step-by-step as shown in Scheme 3.
SPh
SPh
RO
CH(OEt)3, CH3CN, 83%
NHTroc
NHTroc
6
9 R = H
10 R = Ac
Ac2O, Pyr, 96%
OH
OTBS
O
O
TBSCl, Pyr
73%
O
O
OMP
OMP
O
O
OH
OH
7
11
O
OR
O
OMP
O
NIS, TMSOTf
O
8
11
DCM, 0 oC, 81%
O
OBn
Ph
O
O
O
III
OMP
O
O
O
O
O
II
12 R = TBS
13 R = H
TBAF, THF
95%
RO
O
OBn
TrocHN
O
O
NIS, TMSOTf
DCM, -42 oC, 79%
I
With both disaccharide donor and trisaccharide acceptor in
hands, we next explored the condensation of 2 and 3 catalyzed by
NIS and TMSOTf in anhyd CH2Cl2 at ꢀ20 °C (Scheme 5). As we ex-
O
NaOMe
MeOH
95%
O
14 R = Ac
3 R = H
pected, pentasaccharide 15 was formed with the
a-glycoside as
Scheme 3. Synthesis of trisaccharide acceptor 3.
the major product in 84% isolated yield. Based on COSY spectra of
15, the chemical shifts corresponding to the new glycosidic bond
showed at 5.25 ppm (1H NMR, H-1IV, J = 3.0 Hz) and 95.45 ppm
an acceptable isolated yield of 73%, in addition to its 6% of the
isomer.
a
Ph
O
As depicted in Scheme 3, treatment of phenyl 2-deoxy-2-(2,2,2-
OMP
O
O
O
trichloroethoxycarbonylamino)-1-thio-b-
D
-galactopyranoside (6)17
O
OH
O
O
with PhCHO, trimethyl orthoformate [CH(OEt)3], and TsOH in aceto-
nitrile gave phenyl 4,6-O-benzylidene-2-deoxy-2-(2,2,2-trichloro-
ethoxycarbonylamino)-1-thio-b-D-galactopyranoside (9), which
was then acetylated with Ac2O in pyridine to afford 10 in 79% yield
8
+
AcO
10
O
+
O
O
OMP
OBn
O
TrocHN
O
NIS
NIS
OH
7
TMSOTf
CH2Cl2
TMSOTf
CH2Cl2
O
O
14
-42 o
C
-15 o
C
over two steps. To differentiate the 2-OH from the 6-OH of
4-methoxyphenyl 3,4-O-isopropylidene-b-
D-galactopyranoside (7),
commercially available 7 was treated with TBSCl in pyridine at
Scheme 4. One-pot synthesis of trisaccharide 14.

2274
X. Lv et al. / Carbohydrate Research 345 (2010) 2272–2276
OAc
tored by TLC until compound 5 disappeared. The mixture was neu-
tralized with Et3N, and filtered, and the filtrate was concentrated.
Column chromatography (4:1 petroleum ether–EtOAc) of the resi-
OAc
OAc
Ph
O
OBn
O
O
O
III
II
OMP
O
O
duegave compound2 (709 mg, 73%)as a syrup: ½a D25
ꢁ
+86(c 1, CHCl3);
O
TrocHN
OBn
V
O
O
1H NMR (500 MHz, CDCl3): d 7.26–7.42 (m, 15H, Ph), 5.62 (d, 1H, J
5.2 Hz, NH), 5.26 (d, 1H, J 2.4 Hz, H-40), 4.92 (d, 1H, J 8.8 Hz, H-1),
4.88–4.91 (m, 2H), 4.79 (d, 1H, J 7.6 Hz, H-10), 4.49–4.69 (m, 6H),
4.40 (d, 1H, J 11.5 Hz), 4.00–4.11 (m, 3H), 4.92–4.94 (m, 1H), 3.56–
3.77 (m, 6H), 3.22–3.24 (m, 1H, SCH), 2.14, 1.99, 1.93 (3s, 9H, 3Ac),
0.85–0.88 (m, 6H, CH(CH3)2). 13C NMR (125 MHz, CDCl3): d 170.3,
170.1, 170.0, 154.1, 138.1, 137.1, 129.0, 128.7, 128.4, 128.2, 127.9,
127.7, 127.6, 101.9, 95.7, 84.7, 83.5, 78.9, 77.3, 77.0, 76.9, 76.7,
76.0, 75.7, 74.4, 74.3, 73.5, 71.8, 70.8, 69.2, 66.5, 61.1, 52.6, 35.3,
23.9, 23.8, 22.6, 20.6, 20.5. Anal. Calcd for C45H54Cl3NO14S: C,
55.64; H, 5.60. Found: C, 55.49; H, 5.50.
NIS, TMSOTf
BOnO
O
O
IV
2
3
OBn
I
+
15
TrocHN
O
CH2Cl2, -20 o
84%
C
(1) i) 80% AcOH; ii) Ac O, Pyr
(2) i) Zn, AcOH; ii) Ac2O, Pyr
O
O
(3) i) Pd/C, H2, MeOH; ii) Ac2O,
Pyr, 71 % in three steps
OR
OR
OR
O
O
OR
III
O
RO
O
O
OR
V
II
AcHN
OR
OR
O
OMP
ROO
AcHN
IV
O
OR
O
OR
I
16 R = Ac
1 R = H
1.3. Phenyl 3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-b-D-galactopyranoside (10)
NaOMe, MeOH, 92%
RO
A mixture of 6 (4.5 g, 10.0 mmol), PhCHO (1.2 mL, 12.0 mmol),
CH(OEt)3 (2.0 mL, 12.0 mmol), and TsOH (190 mg, 1.0 mmol) in
CH3CN (50 mL) was stirred at 45 °C for 5 h, at the end of which time
TLC indicated that most of compound 6 was consumed. The reaction
mixture was neutralized with Et3N and concentrated under dimin-
ished pressure, and the residue was purified by column chromatog-
raphy (3:1 petroleum ether–EtOAc) to give 9 as a foamy solid (4.47 g,
83%). A solution of 9 (4.27 g, 7.98 mmol) in Pyr (15 mL) and Ac2O
(8 mL) was stirred at rt for 6 h, then concentrated. Column chroma-
tography (4:1 petroleum ether–EtOAc) of the residue gave 10
Scheme 5. Synthesis of pentasaccharide 1.
(
13C NMR, C-1IV), respectively. Deacetalation of 15 with 80% AcOH at
85 °C, followed by removal of Troc with zinc in AcOH, debenzylation
with 10% Pd/C under H2 pressure, and acetylation of the residue with
Ac2O in pyridine, generated fully acetylated pentasaccharide 16 in
71% yield from 15.19 In the COSY spectra of 16, a broad singlet at
5.13 ppm (J <3.0 Hz) and the peak at 94.6 ppm represent H-1IV and
C-1IV, respectively, further confirming its structural assignment.
Saponification of 16 with NaOMe in MeOH quantitatively furnished
the desired pentasaccharide 1.
In summary, we have developed a practical route for the syn-
thesis of the pentasaccharide repeating unit of E. coli O128 antigen
via a facile a-bond formation. This successful example supports the
argument that remote controls affect the stereoselectivity in the
construction of an oligosaccharide, leading to a new glycosyl bond
with an opposite configuration to that of the established bond in
either the donor or acceptor.20 Application of this pentasaccharide
in vaccine development is currently under exploration.
(4.42 g, 96%) as an amorphous solid: ½a D25
ꢁ
ꢀ53 (c 1, CHCl3); 1H
NMR (400 MHz, CDCl3): d 7.25–7.66 (m, 10H, Ph), 5.55 (s, 1H, PhCH),
5.31 (d, 1H, J 10.2 Hz, H-3), 5.11 (d, 1H, J 8.0 Hz, NH), 5.07 (d, 1H, J
10.0 Hz, H-1), 4.73–4.75 (m, 2H, CH2CCl3), 4.37 (d, 1H, J 12.3 Hz, H-
6a), 4.35 (s, 1H, H-4), 4.03 (d, 1H, J 12.3 Hz, H-6b), 3.90–3.93 (m,
1H, H-2), 3.60 (s, 1H, H-5), 2.03 (s, 3H, Ac). Anal. Calcd for
C24H24Cl3NO7S: C, 49.97; H, 4.19. Found: C, 49.81; H, 4.25.
1.4. 4-Methoxyphenyl 6-O-tert-butyldimethylsilyl-3,4-O-
isopropylidene-b- -galactopyranoside (11)
D
1. Experimental
To a commercially available compound 7 (3.27 g, 10.0 mmol) in
pyridine (25 mL) was added TBSCl (1.7 g, 12.0 mmol) at 0 °C. After
stirring under these conditions for 3 h, the reaction was quenched
by MeOH, and the mixture was concentrated. The residue was di-
luted with dichloromethane (50 mL), washed with satd aq NaHCO3,
and the organic phase was dried over Na2SO4 and concentrated. The
residue was purified by column chromatography (4:1 petroleum
1.1. General methods
Optical rotations were determined at 25 °C with a Perkin–Elmer
Model 241-Mc automatic polarimeter. 1H NMR, 13C NMR, and COSY
spectra were recorded with Bruker ARX 500 (or 400) spectrometers
for solutions in CDCl3 or D2O. Chemical shifts are given in ppm
downfield from internal Me4Si. MALDI-TOF mass spectra (MALDI-
ether–EtOAc) to give 11 (3.21 g, 73%) as a syrup: ½a D25
ꢀ28 (c 1,
ꢁ
CHCl3); 1H NMR (400 MHz, CDCl3): d 7.00, 6.79 (2d, 2 ꢂ 2H, J
8.0 Hz, CH3OC6H4O), 4.63 (d, 1H, J 8.2 Hz, H-1), 4.22 (d, 1H, J 5.4 Hz,
H-4), 4.13 (t, 1H, J 6.6 Hz, H-3), 4.88–4.90 (m, 3H, H-2, H-6a, H-6b),
3.80 (t, 1H, J 7.7 Hz, H-5), 3.75 (s, 3H, OCH3), 2.76 (br s, 1H, OH),
1.55, 1.35 (2s, 6H, 2CH3), 0.89, 0.06, 0.05 (3s, 15H, 5CH3). Anal. Calcd
for C22H36O7Si: C, 59.97; H, 8.24. Found: C, 60.21; H, 8.17.
TOFMS) were measured using
a-cyano-4-hydroxycinnamic acid
(CCA) as the matrix. Thin-layer chromatography (TLC) was per-
formed on silica gel HF254 with detection by charring with 30%
(v/v) H2SO4 in MeOH or in some cases by UV detection. Column
chromatography was conducted by elution of a silica gel column
(100–200 mesh) with EtOAc–petroleum ether (60–90 °C) as the
eluent, or a column of Bio-Gel P2 with water as the eluent. Solu-
tions were concentrated at <50 °C under reduced pressure.
1.5. 4-Methoxyphenyl 2-O-benzyl-3,4-O-isopropylidene-
fucopyranosyl-(1?2)-6-O-tert-butyldimethylsilyl-3,4-O-
a-L-
1.2. Isopropyl 3,4,6-tri-O-acetyl-4,6-O-benzylidene-2-deoxy-2-
isopropylidene-b-D-galactopyranoside (12)
(2,2,2-trichloroethoxycarbonylamino)-b-
D-galactopyranosyl-
(1?4)-2,3,6-tri-O-benzyl-1-thio-b- -galactopyranoside (2)
D
To a mixture of compounds 8 (406 mg, 1.2 mmol) and 11
(480 mg, 1.09 mmol) in anhyd CH2Cl2 (10 mL) were added NIS
(405 mg, 1.8 mmol) and TMSOTf (22 lL, 0.12 mmol) under an N2
atmosphere at 0 °C. The mixture was stirred under these conditions
for 30 min, at the end of which time TLC (3:1 petroleum ether–
EtOAc) indicated that all the starting materials were consumed.
The reaction mixture was neutralized with Et3N, diluted with CH2Cl2
To a mixture of 5 (508 mg, 1.0 mmol) and 4 Å molecular sieves in
anhyd CH2Cl2 (5 mL) was added TMSOTf (22 L, 0.12 mmol) at
ꢀ42 °C. The mixture was stirred under these conditions for 10 min
with N2 protection, and then a solution of 4 (750 mg, 1.2 mmol) in
CH2Cl2 (10 mL) was added drop-by-drop. The reaction was moni-
l

X. Lv et al. / Carbohydrate Research 345 (2010) 2272–2276
2275
(10 mL), and washed with aq Na2S2O3. The organic layer was dried
over Na2SO4 and concentrated. Column chromatography (6:1 petro-
leum ether–EtOAc) of the residue gave disaccharide 12 (633 mg,
and neutralized with Dowex-50 (H+) ion-exchange resin. The mix-
ture was filtered, the filtrate was concentrated, and the resulting
residue was purified by silica gel column chromatography (3:1
petroleum ether–EtOAc) to afford compound 3 (98 mg, 95%) as
81%) as a foamy solid: ½a D25
ꢁ
ꢀ81 (c 1, CHCl3); 1H NMR (400 MHz,
CDCl3):d7.28–7.42(m, 5H, Ph), 6.92, 6.80(2d, 2 ꢂ 2H, J9.1 Hz, CH3O-
C6H4O), 5.49 (d, 1H, J 3.6 Hz, H-1), 4.82 (d, 1H, J 8.2 Hz, H-10), 4.78 (dd,
2H, PhCH2), 4.58–4.60 (m, 1H), 4.34 (t, 1H, J 6.6 Hz, H-30), 4.20–4.22
(m, 2H), 4.00–4.02 (m, 2H), 3.86–3.89 (m, 3H), 3.76 (s, 3H, CH3OPh),
3.53 (dd, 1H, J 3.6, 8.0 Hz, H-2), 1.57 (s, 3H, H-6), 1.33–1.56 (2s, 12H,
4CH3), 0.92 (s, 9H, C(CH3)3), 0.07 (2s, 6H, 2CH3 of TBS). Anal. Calcd for
an amorphous solid: ½a D25
ꢁ
ꢀ51 (c 1, CHCl3); 1H NMR (500 MHz,
CDCl3): d 7.25–7.50 (m, 10H, Ph), 6.97, 6.89 (2d, 2 ꢂ 2H, 9.0 Hz,
CH3OC6H4O), 5.54 (s, 1H, CHPh), 5.45 (d, 1H, J 3.5 Hz, H-1I), 4.91
(d, 1H, J 8.0 Hz, H-1II), 4.78 (dd, 2H, PhCH2), 4.67–4.69 (m, 2H),
4.24–4.35 (m, 3H), 3.93–4.12 (m, 9H), 3.75 (s, 3H, CH3OPh), 3.51–
3.53 (m, 1H), 3.41 (s, 1H), 1.56, 1.38, 1.36, 1.34, 1.33 (5s, 15H, H-
6I, 4CH3). 13C NMR (125 MHz, CDCl3): d 155.5, 154.9, 150.8,
138.1, 137.3, 129.2, 128.3, 128.2, 127.9, 127.6, 126.3, 118.1,
114.9, 110.6, 108.7, 101.3, 100.8, 99.6, 95.5, 79.8, 77.3, 77.0, 76.7,
76.2, 75.9, 75.7, 75.6, 74.9, 74.5, 73.9, 73.6, 71.8, 70.9, 69.0, 68.2,
66.5, 62.8, 55.6, 55.3, 28.2, 27.9, 26.5, 26.4, 16.3. Anal. Calcd for
C38H56O11Si: C, 63.66; H, 7.87. Found: C, 63.49; H, 7.96.
1.6. 4-Methoxyphenyl 2-O-benzyl-3,4-O-isopropylidene-
a-L-
fucopyranosyl-(1?3)-3,4-O-isopropylidene-b-D-
galactopyranoside (13)
C48H58Cl3NO17: C, 56.12; H, 5.69. Found: C, 56.34; H, 5.58.
A solution of 12 (700 mg, 0.98 mmol) and TBAF (523 mg,
2.0 mmol) in THF (15 mL) was stirred at 0 °C for 1 h, then for a fur-
ther 4 h at rt until the starting material was consumed. The reac-
tion mixture was diluted with EtOAc (10 mL), then washed with
aq NH4Cl and water, respectively. The water phase was extracted
with EtOAc (3 ꢂ 3 mL), and the combined organic phase was
washed with water and brine, dried, filtered, and concentrated to
dryness. The residue was purified by silica gel column chromatog-
raphy (3:1 petroleum ether–EtOAc) to give 13 (560 mg, 95%) as a
1.9. 4-Methoxyphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-b- -galactopyranosyl-(1?4)-
2,3,6-tri-O-benzyl- -galactopyranosyl-(1?3)-4,6-O-benzyl
idene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-b-
galactopyranosyl-(1?6)-[2-O-benzyl-3,4-O-isopropylidene-
-fucopyranosyl-(1?2)]-3,4-O-isopropylidene-b-
galactopyranoside (15)
D
a-D
D-
a-
L
D-
foamy solid: ½a 2D5
ꢁ
ꢀ70 (c 1, CHCl3); 1H NMR (400 MHz, CDCl3): d
To
0.2 mmol) in anhyd CH2Cl2 (10 mL) were added NIS (81 mg,
0.36 mmol) and TMSOTf (4.0 L, 0.022 mmol) under an N2 atmo-
a mixture of 2 (233 mg, 0.24 mmol) and 3 (205 mg,
7.28–7.42 (m, 5H, Ph), 6.89, 6.82 (2d, 2 ꢂ 2H, J 9.1 Hz, CH3OC6H4O),
5.47 (d, 1H, J 3.6 Hz, H-1), 4.89 (d, 1H, J 8.2 Hz, H-10), 4.75 (dd, 2H,
PhCH2), 4.54–4.56 (m, 1H), 4.38 (t, 1H, J 6.1 Hz, H-30), 4.24 (dd, 1H, J
5.6, 8.0 Hz, H-20), 4.24 (dd, 1H, J 1.6, 5.6 Hz, H-4), 4.00–4.02 (m,
4H), 3.92 (br s, 1H, OH), 3.84 (s, 3H, CH3OPh), 3.53 (dd, 1H, J 3.6,
8.0 Hz, H-2), 2.17 (s, 3H, CH3), 1.34–1.38 (m, 12H, H-6 and 3CH3).
Anal. Calcd for C32H42O11: C, 63.77; H, 7.02. Found: C, 63.60; H,
7.11.
l
sphere at ꢀ20 °C. The mixture was stirred under these conditions
for 1.5 h, quenched by the addition of Et3N, diluted with CH2Cl2
(20 mL), and washed with aq Na2S2O3. The organic layer was dried
over Na2SO4 and concentrated. The residue was purified by silica
gel column chromatography (1.5:1 petroleum ether–EtOAc) to give
compound 15 (322 mg, 84%) as a white foamy solid: ½a D25
ꢁ
+20 (c 2,
CHCl3); 1H NMR (500 MHz, CDCl3): d 7.02–7.50 (m, 25H, Ph), 6.86–
6.89 (m, 4H, CH3OC6H4O), 5.52 (s, 1H, PhCH), 5.48 (d, 1H, J 3.2 Hz,
H-1I), 5.43 (d, 1H, J 7.4 Hz, NH), 5.25 (d, 1H, J 3.0 Hz, H-1IV), 5.12 (s,
1H, H-4V), 4.88 (d, 2H, J 11.0 Hz, PhCH2), 4.78–4.81 (m, 3H), 4.67 (d,
1H, J 12.2 Hz, PhCH2), 4.46–4.58 (m, 8H), 4.33 (t, 1H, J 6.3 Hz, H-3II),
3.90–4.29 (m, 21H), 3.64–3.73 (m, 6H), 3.62–3.64 (m, 1H), 3.54 (dd,
1H, J 3.3, 8.1 Hz, H-5I), 3.14 (s, 1H), 2.14, 2.03, 1.91 (3s, 9H, 3Ac),
1.59 (s, 3H), 1.37 (2s, 12H). 13C NMR (125 MHz, CDCl3): d 176.9,
173.2, 172.9, 170.0, 155.0, 154.4, 153.8, 151.4, 138.2, 137.9,
137.6, 128.9, 128.6, 128.2, 127.9, 127.6, 126.4, 117.5, 114.8,
110.4, 108.7, 102.0, 101.2, 101.1, 99.4,95.4, 80.0, 78.2, 77.3, 77.0,
76.7, 76.5, 76.3, 76.0, 75.7, 74.6, 74.1, 73.8, 72.5, 71.8, 71.2, 71.0,
70.4, 69.2, 68.8, 68.5, 66.6, 66.3, 62.8, 62.1, 61.4, 60.4, 55.6, 52.5,
39.3, 37.4, 37.1, 35.9, 34.0, 33.7, 33.4, 32.7, 31.9, 30.0, 29.7, 29.3,
29.1, 28.2, 28.0, 27.4, 27.2, 26.5, 24.8, 24.4, 22.7, 20.7, 19.7, 19.2,
18.3, 16.3, 14.4, 14.1. Anal. Calcd for C90H104Cl6N2O31: C, 56.23;
H, 5.45. Found: C, 56.02; H, 5.53.
1.7. 4-Methoxyphenyl 2-O-benzyl-3,4-O-isopropylidene-
fucopyranosyl-(1?2)-[3-O-acetyl-4,6-O-benzylidene-2-deoxy-
2-(2,2,2-trichloroethoxycarbonylamino)-b- -galactopyranosyl-
(1?6)]-3,4-O-isopropylidene-b- -galactopyranoside (14)
a-L-
D
D
To a mixture of compounds 10 (750 mg, 1.3 mmol) and 13
(602 mg, 1.0 mmol) in anhyd CH2Cl2 (12 mL) were added NIS
(450 mg, 2.0 mmol) and TMSOTf (24 lL, 0.13 mmol) under an N2
atmosphere at ꢀ42 °C. The mixture was stirred under these condi-
tions for 1.5 h, quenched by Et3N, diluted with CH2Cl2, and washed
with aq Na2S2O3. The organic layer was dried over Na2SO4 and con-
centrated. The residue was purified by silica gel column chroma-
tography (2:1 petroleum ether–EtOAc) to give compound 14
(845 mg, 79%) as a white foamy solid: ½a D25
ꢁ
ꢀ27 (c 2, CHCl3); 1H
NMR (400 MHz, CDCl3): d 7.24–7.52 (m, 10H, Ph), 6.96, 6.89 (2d,
2 ꢂ 2H, J 9.1 Hz, CH3OC6H4O), 5.51 (s, 1H, CHPh), 5.45 (d, 1H, J
3.6 Hz, H-1I), 4.92 (d, 1H, J 8.4 Hz, H-1II), 4.85–4.88 (m, 1H), 4.78
(dd, 2H, PhCH2), 4.50 (d, 1H, J 10.0 Hz, H-1III), 4.52–4.55 (m, 3H),
4.22–4.35 (m, 3H), 3.93–4.10 (m, 4H), 3.75 (s, 3H, CH3OPh), 3.51–
3.53 (m, 1H), 3.41 (s, 1H), 2.06 (s, 3H, Ac), 1.56, 1.38, 1.36, 1.34,
1.33 (5s, 15H, H-6I, 4CH3). Anal. Calcd for C50H60Cll3NO18: C,
56.16; H, 5.66. Found: C, 56.32; H, 5.41.
1.10. 4-Methoxyphenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
b-
osyl-(1?3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-b-
pyranosyl-(1?6)-[2,3,4-tri-O-acetyl- -fucopyranosyl-
(1?2)]-3,4-di-O-acetyl-b- -galactopyranoside (16)
D
-galactopyranosyl-(1?4)-2,3,6-tri-O-acetyl-
a-
D-galactopyran
D
-galacto
a
-L
D
1.8. 4-Methoxyphenyl 2-O-benzyl-3,4-O-isopropylidene-
fucopyranosyl-(1?2)-[4,6-O-benzylidene-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-b- -galactopyranosyl-(1?6)]-
3,4-O-isopropylidene-b- -galactopyranoside (3)
a
-
L
-
Compound 15 (401 mg, 0.21 mmol) in 80% aq AcOH (15 mL)
was stirred at 85 °C for 35 min, at the end of which time the mix-
ture was concentrated, and the residue was treated with Ac2O
(1 mL) in pyridine (2 mL) at rt for 4 h. The mixture was concen-
trated, and the residue was purified by flash silica gel column chro-
matography. The white foamy product thus generated was then
dissolved in HOAc (12 mL), and zinc (nano-size activated powder,
99.9%, 800 mg) was added. After stirring for 6 h at 30 °C, TLC
D
D
NaOMe (1 M) was added to a solution of compound 14 (107 mg,
0.1 mmol) in MeOH (3 mL) at 0 °C until the pH of the solution
reached 9–10. The reaction mixture was then stirred at rt for 4 h

2276
X. Lv et al. / Carbohydrate Research 345 (2010) 2272–2276
(20:1 EtOAc–MeOH) showed that the starting material was com-
pletely consumed. The reaction mixture was passed through a
short silica gel column (eluent: ethyl acetate), the combined eluent
was concentrated, and the residue was again acetylated with Ac2O
in pyridine. The acetylated product was then dissolved in MeOH
containing Pd/C (10% Pd, 50 mg), and H2 was bubbled into the mix-
ture at rt for about 12 h. The reaction mixture was filtered, and the
filtrate was concentrated to dryness. Treatment of the residue with
Ac2O in pyridine as described above gave a syrup that was sub-
jected to silica gel column chromatography (1:1 petroleum
5.27 (s, 1H, H-1I), 5.19 (d, 1H, J 7.5 Hz, H-1II), 5.06 (d, 1H, J 4.0 Hz,
H-1IV), 4.59 (d, 1H, J 8.5 Hz, H-1III), 4.53 (d, 1H, J 8.5 Hz, H-1V),
3.58–4.21 (m, 33H), 2.05, 1.75 (2s, 6H, 2Ac), 1.11 (s, 3H, H-6I). 13C
NMR (125 MHz, D2O): d 175.8, 174.9, 155.2, 151.5, 118.6, 118.1,
116.0, 103.7, 101.8, 100.7, 100.0, 96.3, 78.0, 77.5, 76.7, 75.6, 75.5,
74.5, 74.0, 72.5, 71.6, 71.3, 70.2, 69.9, 69.6, 69.2, 69.1, 69.0, 68.5,
67.7, 64.8, 61.8, 61.7, 60.8, 56.6, 53.3, 51.3, 23.1, 22.7, 20.8, 16.0.
MALDI-TOFMS calcd for C41H64N2O26: 1000.4 [M]+; found, 1023.7
[M+Na]+. Anal. Calcd for C41H64N2O26: C, 49.20; H, 6.44. Found: C,
49.01; H, 6.52.
ether–EtOAc) to afford 16 (230 mg, 71%) as a foamy solid: ½a D25
ꢁ
+17 (c 2, CHCl3); 1H NMR (500 MHz, CDCl3): d 6.97, 6.89 (2d,
2 ꢂ 2H, J 9.0 Hz, CH3OC6H4O), 5.99 (d, 1H, J 7.5 Hz), 5.80 (dd, 1H,
J 3.5, 13.5 Hz), 5.69 (d, 1H, J 7.5 Hz), 5.46 (d, 1H, J 3.5 Hz), 5.42
(dd, 1H, J 3.5, 11.0 Hz), 5.38 (d, 1H, J 3.5 Hz), 5.36 (d, 1H, J
4.0 Hz), 5.31 (dd, 1H, J 3.0, 11.0 Hz), 5.30 (d, 1H, J 4.0 Hz), 5.25
(d, 1H, J 3.0 Hz), 5.01–5.14 (m, 7H), 4.56–4.58 (m, 1H), 4.38–4.41
(m, 2H), 4.18–4.21 (m, 2H), 4.03–4.15 (m, 7H), 3.98 (t, 1H, J
8.5 Hz), 3.90 (t, 1H, J 7.0 Hz), 3.83–3.86 (m, 1H), 3.72–3.80 (m,
5H), 3.42–3.47 (m, 2H), 1.88–2.16 (m, 45H, 15Ac), 1.89 (s, 3H).
13C NMR (125 MHz, CDCl3): d 171.2, 171.0, 170.6, 170.5, 170.4,
170.3, 170.2, 170.1, 170.0, 169.8, 155.5, 150.4, 117.6, 114.9, 99.6,
99.2, 99.1, 95.8, 94.6, 77.3, 77.0, 76.7, 73.8, 73.6, 72.4, 71.7, 71.5,
71.1, 70.7, 69.7, 68.3, 68.2, 67.4, 67.3, 66.7, 66.4, 66.2, 65.3, 64.7,
63.0, 61.7, 61.3, 55.7, 53.3, 23.3, 23.1, 21.0, 20.7, 20.6, 20.5, 15.8,
14.1. MALDI-TOFMS calcd for C67H90N2O39: 1456.5 [M]+; found,
1569.7 [M+Na]+. Anal. Calcd for C67H90N2O39: C, 52.00; H, 5.86.
Found: C, 52.17; H, 5.96.
Acknowledgments
This work was supported by NNSF projects of China(20621703,
20872172, 20732001) and Project 2009ZX09501-011.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2010.07.025.
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1.11. 4-Methoxyphenyl 2-acetamido-2-deoxy-b-
osyl-(1?4)- -galactopyranosyl-(1?3)-2-acetamido-2-deoxy-
b- -galactopyranosyl-(1?6)-[ -fucopyranosyl-(1?2)]-b-
galactopyranoside (1)
D-galactopyran-
a-D
D
a-
L
D-
To a solution of 16 (232 mg, 0.15 mmol) in MeOH (10 mL) was
added 1 M NaOMe until the pH of the solution reached 9–10. The
reaction mixture was allowed to stir at rt for 10 h, then neutralized
with Dowex-50 (H+) ion exchange resin. The mixture was filtered,
the filtrate was concentrated, and the resulting residue was purified
by a Bio-Gel P2 column using H2O as eluent. The desired fractions
were combined and freeze dried to afford compound 1 (138 mg,
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92%) as an amorphous solid: ½a D25
ꢁ
+5 (c 0.4, H2O); 1H NMR
(500 MHz, D2O): d 7.08, 6.99 (2d, 2 ꢂ 2H, J 9.0 Hz, CH3OC6H4O),