Expeditious Synthesis of a Tetrasaccharide Repeating Unit of the O-Antigen of Escherichia coli O163

Article abstract of DOI:10.1055/s-0035-1562606

The synthesis of the tetrasaccharide repeating unit of the O-antigen of Escherichia coli O163 as its p-methoxyphenyl (PMP) glycoside was achieved followed by sequential glycosylation strategy through thioglycoside activation using sulfuric acid immobilized on silica (H₂SO₄-silica) in conjunction with N-iodosuccinimide as a Bronsted acid catalyst. The application of one-pot reaction conditions for two glycosylations and in situ PMB-group removal reduced the number of reaction steps significantly. The l-QuipNAc building block was obtained from known carbohydrate l-rhamnose precursors. The stereoselective outcomes of all glycosylation reactions were found to be very good. A late-stage TEMPO-mediated oxidation was performed for the formation of required uronic acid moiety. An analogue of the target tetrasaccharide was also prepared by using one-pot glycosylation approach. Such synthetic oligosaccharides could later be effectively conjugated with an appropriate protein to furnish glycoconjugate derivatives for their use in immunochemical studies.

Full text of DOI:10.1055/s-0035-1562606

SYNLETT
0
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6
-
5
2
1
4
1
4
3
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-
2
0
9
6
© Georg Thieme Verlag Stuttgart · New York
2016, 27, A–F
letter
A
G. Karki et al.
Letter
Synlett
Expeditious Synthesis of a Tetrasaccharide Repeating Unit of the
O-Antigen of Escherichia coli O163
Geeta Karkia
OH
OH
Harikesh Kumara
Remya Rajana
Pintu Kumar Mandal*a,b
O
O
HO
HO
O
O
AcHN
NHAc
HO
HO
AcHN
NHAc
HO
HO
O
O
HO
HO
OH
HO
OH
O
O
HO
O
O
O
a Medicinal and Process Chemistry Division, CSIR-Central Drug
Research Institute, BS-10/1, Sector 10, Jankipuram extension,
Sitapur Road, Lucknow, 226 031, India
O
O
O
HO
HO
O
HO
OPMP
OPMP
HO
OH
OH
1
analogue 2
pintuchem06@gmail.com
b Academy of Scientific and Innovative Research, New Delhi,
11000, India
tetrasaccharide repeating unit of
Escherichia coli O163
Received: 04.05.2016
Accepted after revision: 08.07.2016
Published online: 05.08.2016
There is an increasing awareness that out of the several
pathogenic bacteria associated with sporadic cases of dis-
eases, as well as outbreaks of food-borne illnesses, E. coli is
the most predominant.6
DOI: 10.1055/s-0035-1562606; Art ID: st-2016-b0313-l
Abstract The synthesis of the tetrasaccharide repeating unit of the
O-antigen of Escherichia coli O163 as its p-methoxyphenyl (PMP) glyco-
side was achieved followed by sequential glycosylation strategy through
thioglycoside activation using sulfuric acid immobilized on silica
(H2SO4–silica) in conjunction with N-iodosuccinimide as a Brønsted acid
catalyst. The application of one-pot reaction conditions for two glyco-
sylations and in situ PMB-group removal reduced the number of reac-
tion steps significantly. The L-QuipNAc building block was obtained
from known carbohydrate L-rhamnose precursors. The stereoselective
outcomes of all glycosylation reactions were found to be very good. A
late-stage TEMPO-mediated oxidation was performed for the formation
of required uronic acid moiety. An analogue of the target tetrasaccha-
ride was also prepared by using one-pot glycosylation approach. Such
synthetic oligosaccharides could later be effectively conjugated with an
appropriate protein to furnish glycoconjugate derivatives for their use
in immunochemical studies.
People ingesting food contaminated with Shiga toxin-
producing E. coli (STEC) may get infected with watery diar-
rhoea, abdominal pain, fever, vomiting, and in severe cases
hemorrhagic colitis. It is also characterized by hemolytic-
uremic syndrome, the symptoms of which include acute
kidney failure, microangiopathic hemolytic anemia, and
thrombocytopenia. These multidrug-resistant bacterial
strains are a major threat to the mankind, especially chil-
dren, and is an issue of serious concern.4,7
The O-antigen is an important component of the lipo-
polysaccharide unit of the bacteria. Till date, 174 types of
O-antigenic strains of E.coli have been reported. The reason
for this huge diversification is the genetic variation be-
tween galF and gnd genes on the chromosome of E.coli that
codes for O-antigen.8,9 Based on the types of pathogenic in-
fections they cause, they are categorized as: a) entero-
pathogenic E. coli (EPEC), b) enterohemorrhagic E. coli
(EHEC), c) enterotoxigenic E. coli (ETEC), d) enteroaggrega-
tive E. coli (EAEC), e) enteroinvasive E. coli (EIEC), etc.10,11
Out of the various strains so far identified, which are as-
sociated with each clinical symptom, E. coli 0163 strains
had been found to cause results that shows a high rate of
acute renal failure in children and renal and nonrenal se-
quelae in survivors. These strains are also identified as STEC
strain and cause diarrhoea, gastrointestinal infections. E.
coli O163 isolated from children suffering from hemolytic-
uremic syndrome has been found to be transmitted
through sheep and swine feces.12
Keywords polysaccharide, Escherichia coli, glycosylation, antigen, ste-
reoselectivity
Escherichia coli (E. coli), immensely diversified gram-
negative rod-shaped, anaerobic bacterial species facultative
in nature resides in the lower intestine of humans and are
generally harmless. They contribute a major part to the mi-
croflora within the gut and make themselves available for
suppressing the growth and harmful effects of other micro-
organisms.1,2 They are also found to be helpful in synthesiz-
ing an appreciable amount of vitamins within the body, for
example, the synthesis of vitamin K2 (menaquinone).3 Al-
though some of its bacterial strains are beneficial to the hu-
man hosts, a large diversity of its pathogenic forms are also
found in nature.4 The pathogenic forms are either food-
borne or water-borne pathogens.5
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–F

B
G. Karki et al.
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The structure of the O-antigenic polysaccharide present
in the cell wall of E. coli 0163 strain has been reported by
A. V. Perepelov et al.13 which is composed of D-glucuronic
acid (GlcA), 2-acetamido-2,6-dideoxy-L-glucose (QuiNAc),
D-mannose (Man), D-glucasamine (GlcNAc, Figure 1).
In addition, we therefore decided to synthesize the tar-
get tetrasaccharide 1 and its analogue 2 moiety corre-
sponding to the repeating unit of the cell wall O-specific
polysaccharide of E. coli O163 strain using a one-pot syn-
thetic strategy.
The strategy for the synthesis of the tetrasaccharide as
its p-methoxyphenyl (PMP) glycoside 1 and its analogue 2
involves sequential glycosylations of suitably functional-
ized monosaccharide intermediates (Scheme 1). The selec-
tion of the p-methoxyphenyl (PMP) glycoside as the final
reducing-end glycoside could provide the option handle for
further glycoconjugate formation after oxidative removal of
the PMP group16 through trichloroacetimidate chemistry.
As per the requirement, a set of suitably functionalized
monosaccharide intermediates 3, 4,20 5,21 and 6 were pre-
pared in good yield from the commercially available reduc-
ing sugars using the reaction conditions available in the lit-
erature (Scheme 2).
→3)-α-L-QuipNAc-(1→3)-α-D-GlcpNAc-(1→2)-β-D-Manp-(1→4)-β-D-GlcpA-(1→
Figure 1 Structure of the repeating unit of the O-antigen of Escherichia
coli O163
Due to the emerging resistance of the bacterial cells to-
wards the various antibacterial agents presently available
in market, the synthesis of potent vaccines is the only hope
left for the treatment of these bacterial infections which
when left untreated may even be fatal. Vaccines are biologi-
cal preparations that provide active acquired immunity to a
particular disease. Since cell wall O-antigen contributes to
the virulence factor of the bacterial cells, its biological stud-
ies is of prime importance.14,15 Since the antigen plays an
important role in the initial stages of infection, vaccines
with O-antigens linked to a conjugate (protein, lipid) seems
to be pertinent. In the recent past, a number of reports ap-
peared in the literature for the synthesis and evaluation of
glycoconjugate vaccines against bacterial infections.16–19
However, due to the difficulty in isolation of these lipopoly-
saccharides from bacterial cell walls in sufficient amount
and purity followed by problems related with handling of
live bacterial strains and tedious isolation procedures, de-
velopment of appropriate synthetic strategies for the syn-
thesis of these lipopolysaccharides has become a prime
requisite. This will help in synthesizing the antigens in ap-
preciable amounts both for the biological studies and vac-
cine synthesis.
To start with, known diol glucoside 722 (prepared from
commercial D-glucose23) was allowed to react with TBDPSCl
and imidazole in dry DMF to give 4-O-unprotected gluco-
side 3 in 88% yield. The synthesis of the building block 6
was carried out with efficient approaches towards starting
from commercially available L-rhamnose monohydrate.
The commercially available reducing sugars L-rhamnose
(8) was transformed into the rhamnal 9 in 72% yield in
three steps in the following sequence of reactions: per-
acetylation with acetic anhydride in the presence of per-
chloric acid; bromination using HBr/AcOH; and finally re-
ductive elimination using NaH2PO4 and zinc dust.24,25
Azidonitration26,27 of 9 with sodium azide in the pres-
ence of ceric ammonium nitrate (CAN) in MeCN at –15 °C
followed by acetolysis25 with acetic acid, Ac2O, and NaOAc
OH
O
OH
HO
C
O
O
HO
C
O
AcHN
O
AcHN
D
NHAc
HO
O
D
NHAc
OH
HO
HO
O
O
HO
OH
HO
O
O
HO
HO
HO
O
HO
HO
O
B
O
HO
A
OPMP
O
B
O
OPMP
A
HO
OH
OH
1
analogue 2
tetrasaccharide repeating unit of
Escherichia coli O163
OTBDPS
Ph
O
O
O
O
HO
BnO
OBn
SEt
BnO
OPMP
OAc
TBDPS = t-BuPh2Si
PMP = p-MeOC6H4
PMB = p-MeOC6H4CH2
3
4
O
6
Ph
PMBO
O
STol
N3
O
O
AcO
N3
STol
AcO
5
Scheme 1 Structure of the synthesized tetrasaccharide 1 and its analogue 2 as their p-methoxyphenyl (PMP) glycosides and their synthetic intermedi-
ates
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–F

C
G. Karki et al.
Letter
Synlett
yield. The disaccharide 11 was then deacetylated under
Zemplén conditions34 to furnish the disaccharide alcohol 12
in 93% yield. Oxidation of the C-2 hydroxyl group of com-
pound 12 using Dess–Martin periodinane35 followed by ste-
reoselective reduction of the resulted ketone using sodium
borohydride36 furnished disaccharide acceptor 13 in a yield
of 74% (Scheme 3). Formation of the β-D-mannoside con-
taining disaccharide moiety 13 with exact stereochemistry
was prepared exclusively from the β-D-glucoside containing
disaccharide derivative 12 by epimerization at C-2, and it
was unambiguously confirmed by NMR spectroscopy.
OH
OTBDPS
O
O
a
HO
BnO
HO
OPMP
OPMP
BnO
OBn
OBn
3
7
OH
O
O
b, c, d
e
HO
AcO
HO
AcO
OH
8
9
STol
N3
OAc
N3
f
O
O
AcO
AcO
AcO
AcO
In order to synthesize the target tetrasaccharide deriva-
tive 14, a three-step, one-pot-sequence starting from disac-
charide acceptor 13 was employed (Scheme 4). Thus, accep-
tor 13 and 3-O-PMB-protected 2-azido-glucoside donor 5
was made to react in the presence of NIS and H2SO4–silica
at a low temperature, providing the expected trisaccharide
intermediate. After complete consumption of the 3-O-PMB-
protected 2-azido-glucoside donor 5, which was indicated
by TLC, by raising the reaction temperature of the reaction
vessel, the 3-O-PMB group was removed in the same pot37
and produced the desired trisaccharide acceptor. The reac-
tion mixture was once again cooled, and a mixture of 2-azi-
do-2,6-dideoxy-L-glucopyranoside donor 6 and another
6
10
Scheme 2 Reagents and conditions: (a) TBDPSCl, imidazole, DMF, r.t., 6
h, 88%; (b) Ac2O, HClO4 (cat.), 0 °C, 10 min; (c) HBr/AcOH, CH2Cl2, 4 h;
(d) Zn, NaH2PO4, EtOAc–H2O, 30 min, (9: 72%, from L-rhamnose); (e)
NaN3, CAN, MeCN, –15 °C, 3 h, then NaOAc, AcOH, Ac2O (cat.), 100 °C,
1 h, 77%; (f) p-thiocresol, BF3·OEt2, CH2Cl2, r.t., 4 h, 85%.
at reflux for one hour gave the reducing deoxy glucoside 10
in 77% yield (Scheme 2). Compound 10 was further subject-
ed to treatment with p-thiocresol in the presence boron tri-
fluoridediethyl etherate, and the glycosylating donor 6 was
synthesized in 85% yield.
With all the required building blocks in hand, 4-O-un-
protected glucoside 3 was glycosylated with thioglycoside
derivative 4 in the presence of N-iodosuccinimide (NIS) in
conjunction with sulfuric acid immobilized on silica
(H2SO4–silica)28–33 to afford the disaccharide 11 in 87%
OH
OTBDPS
Ph
O
O
O
O
B
O
BnO
OPMP
A
BnO
OBn
13
OTBDPS
Ph
PMBO
O
Ph
O
O
O
O
O
O
HO
BnO
SEt
BnO
OPMP
+
N3
STol
OAc
OBn
two glycosylations and
removal of PMB group
in one pot
5
a
3
4
a
STol
O
N3
AcO
OTBDPS
O
Ph
O
AcO
O
OAc
O
B
O
6
BnO
OPMP
A
BnO
OBn
Ph
O
O
O
11
O
C
N3
b
O
D
N3
AcO
AcO
O
OTBDPS
OTBDPS
O
Ph
O
Ph
O
O
O
O
O
O
O
B
B
O
BnO
BnO
OPMP
A
OPMP
A
BnO
BnO
OH
OBn
OBn
14
12
b, c, e, f
b, c, d, e, f
c, d
1
2
OH
O
OTBDPS
O
Ph
O
O
B
O
BnO
Scheme 4 Reagents and conditions: (a) 5, NIS, H2SO4–silica, CH2Cl2–
Et2O (1:2), MS 4 Å, –20 °C, 30 min; then 10 °C, 30 min; then 6, NIS, –20
°C, 30 min, 61%; (b) MeCOSH, pyridine, r.t., 10 h; (c) Bu4NF–THF, AcOH,
0 °C to r.t., 6 h; (d) TEMPO, NaBr, NaOCl, TBAB, NaClO2, 2-methylbut-
2-ene, NaH2PO4, NaHCO3, CH2Cl2, 0 °C to r.t., 5 h; (e) H2, 10% Pd(OH)2/C,
MeOH, r.t., 24 h; (f) 0.1 M NaOMe, MeOH, r.t., 1 h (overall yield for 1
54% and for 2 62%).
OPMP
A
BnO
OBn
13
Scheme 3 Reagents and conditions: (a) NIS, H2SO4–silica, CH2Cl2, MS 4
Å, –20 °C, 30 min, 87%; (b) NaOMe, MeOH, r.t., 1 h, 93%; (c) Dess–
Martin periodinane, CH2Cl2, r.t., 3 h; (d) NaBH4, MeOH, r.t., 10 h, 74%
over two steps.
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–F

D
G. Karki et al.
Letter
Synlett
fresh portion of NIS were added to the reaction mixture.
The tetrasaccharide derivative 1438 was obtained in 61%
overall yield using a three-step, one-pot-sequence strategy
and the formation of compound 1438 was unambiguously
confirmed by a combination of 2D-NMR techniques includ-
ing 1H–1H and 1H–13C experiments.
Generation of a minor amount of other isomeric glyco-
sylation products was also observed, which were removed
from compound 14 using flash column chromatography.
Carrying out three reactions in a one-pot setup significant-
ly reduced the number of steps in this process which would
improve ease in synthesis of the target compound. A one-
pot multistep strategy required less bench time and afford-
ed superior yields, avoiding the workup and isolation pro-
cedures required in the intermediate steps.
References and Notes
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Immun. 1990, 58, 2710.
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2004, 2, 123.
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Zimmerhackl, L. B. J. Infect. Dis. 2002, 186, 493.
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J. J. Med. Microbiol. 1988, 25, 237.
Finally, the tetraaccharide derivative 14 was subjected
to a series of functional-group transformations, which
comprised of: a) treatment with thioacetic acid39 to convert
azido group into acetamido group, b) fluoride ion mediated
desilylation40, c) TEMPO-mediated oxidation of the primary
hydroxyl group,41 d) removal of benzyl ethers and ben-
zylideneacetal under catalytic transfer hydrogenation con-
ditions using hydrogen and 10% Pd/C,42 and finally, e)
saponfication using sodium methoxide34 to furnish the de-
sired tetrasaccharide p-methoxyphenyl (PMP) glycoside 143
in 54% overall yield. The NMR spectrum of compound 143
unambiguously supported its structure. The analogue 244 of
the desired tetrasaccharide p-methoxyphenyl (PMP) glyco-
side 1 was also synthesized from compound 14 with the
same experimental protocol that was used for compound 1
skipping the TEMPO-mediated oxidation of the primary hy-
droxyl group (Scheme 4).
(10) Beutin, L.; Aleksic, S.; Zimmermann, S.; Gleier, K. Med. Microbiol.
Immunol. 1994, 183, 13.
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Caprioli, A.; Karch, H. J. Med. Microbiol. 1995, 42, 404.
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Shashkov, A. S.; Wang, L.; Knirel, Y. A. Carbohydr. Res. 2015, 404,
34.
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Herrera, L.; Izquierdo, M.; Villar, A.; Valdés, Y.; Cosme, K.; Deler,
M. L.; Montane, M.; Garcia, E.; Ramos, A.; Aguilar, A.; Medina, E.;
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Carmenates, A.; Costa, L.; Cardoso, F.; Campa, C.; Diaz, M.; Diaz,
R. Science 2004, 305, 522.
In summary, oligosaccharide unit corresponding to the
O-antigen of E. coli O163 as its sodium salt and p-methoxy-
phenyl glycoside was synthesized, applying three-step,
one-pot sequence reaction conditions for two stereoselec-
tive glycosylation reactions and removal of the PMB group
in situ. TEMPO-mediated phase-transfer oxidation was per-
formed efficiently at a late stage of the total synthesis. An
analogue of the concerned tetrasaccharide was also synthe-
sized in very good yield using the same glycosylation ap-
proach. During the synthetic process, H2SO4 immobilized
on silica was used as a thiophilic glycosylation activator in
all stereoselective glycosylation reactions. The choice of the
p-methoxyphenyl group as reducing end glycoside in all
cases offers further glycoconjugate formation for future bi-
ological evaluation of the synthesized oligosaccharides.
(17) Roy, R. Drug Discovery Today: Technol. 2004, 1, 327.
(18) (a) Werz, D. B.; Seeberger, P. H. Angew. Chem. Int. Ed. 2005, 44,
6315. (b) Tamborrini, M.; Werz, D. B.; Frey, J.; Pluschke, G.;
Seeberger, P. H. Angew. Chem. Int. Ed. 2006, 45, 6581.
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Kovác, P.; Wade, W. F. Infect. Immun. 2004, 72, 4090.
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Acknowledgment
Author gratefully acknowledges financial support by Department of
Science and Technology (DST), India under Fast Track Proposal
Scheme for Young Scientists (SB/FT/CS-127/2012) and SAIF Division
of CSIR-CDRI for providing the spectroscopic and analytical data.
CDRI communication no 9272.
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© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–F

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2311.
5D), 55.7 (OCH3), 26.8 [SiC(CH3)3], 20.7 (COCH3), 20.6 (COCH3),
19.4 [SiC(CH3)3], 16.1 (CH3). HRMS (ESI-TOF): m/z calcd for
C
86H94N6O21Si [M + Na]: 1597.6241; found: 1597.6259.
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(43) p-Methoxyphenyl (2-Acetamido-2,6-dideoxy-α-L-glucopyra-
nosyl)-(1→3)-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-
(1→2)-(β-D-mannopyranosyl)-(1→4)-β-D-glucopyranosy-
luronic Acid (1)
A solution of compound 14 (400 mg, 0.26 mmol) and thioacetic
acid (0.2 mL) in pyridine (15 mL) was stirred at room tempera-
ture for 10 h. The solvent was removed under reduced pressure,
and the crude product was passed through a short pad of SiO2.
To the solution of the N-acetylated product in THF (10 mL) was
added Bu4NF in THF (5 mL), and the reaction mixture was
stirred at room temperature for 6 h. The solvents were removed,
and the crude mass was dissolved in CH2Cl2 (40 mL). The
organic layer was washed with sat. NaHCO3 and water, dried,
and concentrated. To a solution of the crude product in CH2Cl2
(20 mL) and H2O (3.5 mL) were added aqueous solution of NaBr
(1 mL, 1 M), aqueous solution of TBAB (2 mL; 1 M), TEMPO (80
mg, 0.4 mmol), sat. aqueous solution of NaHCO3 (8 mL), and 4%
NaOCl aq (10 mL) in succession, and the reaction mixture was
stirred at 0–5 °C for 2 h. The reaction mixture was neutralized
with 1 N HCl aq solution followed by addition of t-BuOH (25
mL), 2-methyl-but-2-ene (30 mL, 2 M solution in THF), aqueous
solution of NaClO2 (1 g in 5 mL), and aqueous solution of
NaH2PO4 (1 g in 5 mL). The resultant mixture was allowed to stir
at room temperature for 5 h and then diluted with sat. aqueous
solution of NaH2PO4 and extracted with CH2Cl2 (3 × 50 mL). The
combined organic layer was washed with water, dried (Na2SO4),
and concentrated to dryness to give the oxidized product. To a
solution of the oxidized product in MeOH (10 mL) was added
10% Pd/C (100 mg) and it was allowed to stir at room tempera-
ture for 24 h under a positive pressure of hydrogen. The reac-
tion mixture was filtered through a Celite® bed and the filtering
bed was washed with MeOH (50 mL). The combined solution
was concentrated under reduced pressure to give the crude
product, which was dissolved in 0.1 M NaOMe in MeOH (20 mL),
and the solution was stirred at room temperature for 1 h. The
reaction mixture was neutralized with Dowex 50W-X8 (H+)
resin, filtered, and concentrated to give compound 1, which was
passed through a column of Sephadex LH-20 (25% MeOH–H2O)
to furnish pure compound 1 (121 mg, 54%) as white powder;
[α]D25 –28 (c 1.0, H2O). IR (neat): 3419, 2910, 1598, 1327, 1142,
988, 679 cm–1. 1H NMR (400 MHz, D2O): δ = 7.06 (d, J = 8.1 Hz, 2
H, ArH), 6.92 (d, J = 8.1 Hz, 2 H, ArH), 5.18 (br s, 1 H, H-1C), 5.04
(d, J = 7.6 Hz, 1 H, H-1A), 4.83 (br s, 1 H, H-1D), 4.69 (br s, 1 H, H-
1B), 4.59–4.57 (m, 1 H, H-2D), 4.22–4.20 (m, 1 H, H-2C), 4.11–
4.09 (m, 3 H, H-5A, H-2B, H-5C), 3.95–3.90 (m, 4 H, H-4B, H-5D, H-
(38) p-Methoxyphenyl (3,4-di-O-Acetyl 2-azido-2,6-dideoxy-α-L-
glucopyranosyl)-(1→3)-(2-azido-4,6-O-benzylidene-2-
deoxy-α-D-glucopyranosyl)-(1→2)-(3-O-benzyl-4,6-O-ben-
zylidene-β-D-mannopyranosyl)-(1→4)-6-O-tert-butyldiphe-
nylsilyl-2,3-di-O-benzyl-β-D-glucopyranoside (14)
To a solution of compound 13 (1.5 g, 1.50 mmol) and compound
5 (730 mg, 1.65 mmol) in anhydrous CH2Cl2–Et2O (1:2 v/v, 30
mL) MS 4 Å (3 g) was added and cooled to –20 °C under argon.
To the cooled reaction mixture, NIS (410 mg, 1.82 mmol) and
H2SO4–SiO2 (100 mg) were added, and it was allowed to stir at
the same temperature for 30 min. After consumption of the
starting materials (TLC; hexane–EtOAc, 3:1), the temperature of
the reaction mixture was raised to 10 °C and stirred for 30 min.
After the formation of a new spot which was confirmed by TLC
(hexane–EtOAc, 3:1), again the reaction mixture was cooled to –
20 °C. To the cooled reaction mixture, a solution of compound 6
(689 mg, 1.81 mmol) in CH2Cl2–Et2O (1:2 v/v, 5 mL) and NIS
(448 mg, 1.99 mmol) were added, and the reaction mixture was
then again stirred at –20 °C for another 30 min. The reaction
mixture was filtered through a Celite bed, and the filtering bed
was washed with CH2Cl2 (100 mL). The combined organic layer
was successively washed with 5% Na2S2O3, sat. NaHCO3 and
water, dried (Na2SO4), and concentrated. The crude product was
purified over SiO2 using hexane–EtOAc (4:1) as the eluent to
25
give pure compound 14 (1.4 g, 61%); colorless oil; [α]D +18 (c
1.0, CHCl3). IR (neat): 3089, 2866, 1849, 1732, 1435, 1236, 1022,
988, 698 cm–1. 1H NMR (400 MHz, CDCl3): δ = 7.66–7.64 (m, 4 H,
ArH), 7.52–7.50 (m, 2 H, ArH), 7.40–7.20 (m, 26 H, ArH), 7.15–
7.11 (m, 1 H, ArH), 7.01–6.97 (m, 2 H, ArH), 6.96 (d, J = 9.0 Hz, 2
H, ArH), 6.80 (d, J = 9.0 Hz, 2 H, ArH), 5.62 (s, 1 H, PhCH), 5.55 (s,
1 H, PhCH), 5.32 (dd, J = 3.8, 9.8 Hz, 1 H, H-3D), 5.25 (br s, 1 H, H-
1C), 5.10 (d, J = 10.4 Hz, 1 H, PhCH2), 5.02 (d, J = 11.0 Hz, 1 H,
PhCH2), 4.89 (d, J = 7.8 Hz, 1 H, H-1A), 4.87 (br s, 1 H, H-1B),
4.86–4.83 (m, 2 H, H-4D, PhCH2),4.82 (d, J = 11.7 Hz, 1 H, PhCH2),
4.77 (d, J = 10.8 Hz, 1 H, PhCH2), 4.65 (d, J =11.8 Hz, 1 H, PhCH2),
4.64–4.59 (m, 1 H, H-2C), 4.56 (br s, 1 H, H-1D), 4.27–4.22 (m, 3
H, H-3A, H-3C, H-6aB), 4.15–4.13 (m, 1 H, H-2D), 4.09–4.05 (m, 3
H, H-2B, H-3B, H-6bB), 4.01–3.96 (m, 2 H, H-6aA, H-4C), 3.94–3.88
(m, 2 H, H-5C, H-5D), 3.83–3.76 (m, 3 H, H-2A, H-6bA, H-6aC), 3.78
(s, 3 H, OCH3), 3.72–3.67 (m, 2 H, H-4B, H-6bC), 3.52–3.49 (m, 1
H, H-5A), 3.41–3.78 (m, 1 H, H-4A), 3.28–3.22 (m, 1 H, H-5B),
2.04 (s, 3 H, COCH3), 1.92 (s, 3 H, COCH3), 1.06 [s, 9 H, SiC(CH3)3],
0.35 (d, J = 6.1 Hz, 3 H, CH3). 13C NMR (100 MHz, CDCl3): δ =
169.9 (2 C, COCH3), 155.4–114.6 (ArC), 103.2 (C-1A), 102.2
(PhCH), 101.5 (PhCH), 99.7 (2 C, C-1B, C-1C), 95.7 (C-1D), 83.5 (C-
2A), 82.0 (C-4B), 79.1 (C-3B), 78.7 (C-5A), 77.4 (C-4C), 76.8 (C-3A),
76.4 (PhCH2), 75.7 (C-4A), 75.1 (PhCH2), 74.6 (C-2B), 73.8 (C-3C),
73.6 (PhCH2), 70.9 (2 C, C-3D, C-4D), 68.7 (C-6B), 68.5 (C-6C), 67.2
(C-5B), 66.3 (C-5C), 63.8 (C-2C), 62.2 (C-6A), 61.6 (C-2D), 61.4 (C-
6
aB, H-6aC), 3.88–3.82 (m, 3 H, H-4A, H-6bB, H-3C), 3.76 (s, 3 H,
OCH3), 3.72–3.61 (m, 6 H, H-2A, H-3A, H-3B, H-4C, H-6bC, H-3D),
3.42–3.33 (m, 2 H, H-5B, H-4D), 2.02 (s, 6 H, NHCOCH3), 1.25 (d, J
= 6.0 Hz, 3 H, CH3). 13C NMR (100 MHz, D2O, CD3OD internal
standard at δ = 49.5 ppm): δ = 175.5(2 C, C-6A, NHCOCH3), 174.9
(NHCOCH3), 155.5–115.6 (ArC), 101.6 (C-1A), 100.7 (C-1B), 100.4
(C-1C), 95.9 (C-1D), 81.1 (C-4A), 77.5 (C-5B), 76.3 (C-2B), 74.3 (2 C,
C-5A, C 3C), 74.1 (C-4D), 73.3 (C-3B), 72.9 (2 C, C-5C, C-3D), 70.0 (C-
3A), 69.5 (C-5D), 69.2 (C-4B), 67.2 (C-4C), 65.2 (C-2A), 61.4 (C-6C),
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–F

F
G. Karki et al.
Letter
Synlett
60.7 (C-6B), 56.4 (OCH3), 53.3 (C-2C), 49.0 (C-2D), 22.6 (NHCO-
CH3), 22.3 (NHCOCH3), 17.2 (CH3). HRMS (ESI-TOF): m/z calcd
for C35H51N2O22 [M + Na]: 874.2831; found: 874.2843.
reaction mixture was neutralized with Dowex 50W-X8 (H+)
resin, filtered, and concentrated to give compound 1, which was
passed through a column of Sephadex LH-20 (25% MeOH–H2O)
to furnish pure compound 1 (142 mg, 62%) as white pow-
(44) p-Methoxyphenyl (2-Acetamido-2,6-dideoxy-α-L-glucopyra-
nosyl)-(1→3)-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-
(1→2)-(β-D-mannopyranosyl)-(1→4)-β-D-glucopyranoside
(2)
25
der;[α]D –12 (c 1.0, H2O). IR (neat): 3445, 2936, 1546, 1377,
1030, 988, 669 cm–1. 1H (400 MHz, D2O): δ = 7.12 (d, J = 8.1 Hz, 2
H, ArH), 6.98 (d, J = 8.1 Hz, 2 H, ArH), 5.20 (br s, 1 H, H-1C), 5.03
(d, J = 7.4 Hz, 1 H, H-1A), 4.87 (br s, 1 H, H-1D), 4.81 (br s, 1 H, H-
1B), 4.63–4.62 (m, 1 H, H-2D), 4.26–4.25 (m, 1 H, H-2C), 4.19 (br
s, 1 H, H-2B), 4.15–4.13 (m, 2 H, H-5A, H-5C), 3.99–3.90 (m, 6 H,
H-4B, H-5D, H-6abA, H-6aB, H-6aC), 3.82 (s, 3 H, OCH3), 3.79–3.71
(m, 7 H, H-2A, H-4A, H-3B, H-3C, H-4C, H-6bB, H-6bC), 3.70–3.58
(m, 2 H, H-3A, H-3D), 3.51–3.48 (m, 1 H, H-5B), 3.38–3.36 (m, 1
H, H-4D), 2.07 (s, 3 H, NHCOCH3), 2.06 (s, 3 H, NHCOCH3), 1.31
(d, J = 6.0 Hz, 3 H, CH3). 13C NMR (100 MHz, D2O, CH3OD internal
standard at δ = 49.5 ppm): δ = 175.2 (NHCOCH3), 174.6 (NHCO-
CH3), 155.3–115.6 (ArC), 101.6 (C-1A), 100.6 (2 C, C-1B, C-1C),
95.9 (C-1D), 79.9 (C-4A), 77.5 (C-2B), 76.8 (C-5B), 75.4 (C-3D), 74.7
(C-3C), 74.1 (C-3B), 73.4 (C-5C), 73.0 (C-5A), 72.6 (C-3A), 72.3 (C-
4D), 69.5 (C-5D), 69.2 (C-4B), 67.3 (C-4C), 65.2 (C-2A), 61.5 (C-6C),
60.8 (C-6B), 60.6 (C-6A), 56.4 (OCH3), 53.3 (C-2C), 49.1 (C-2D),
22.6 (NHCOCH3), 22.3 (NHCOCH3), 17.2 (CH3). HRMS (ESI-TOF):
m/z calcd for C35H54N2O21 [M + Na]: 861.3219; found: 861.3158.
A solution of compound 14 (600 mg, 0.39 mmol) and thioacetic
acid (0.2 mL) in pyridine (20 mL) was stirred at room tempera-
ture for 10 h. The solvent was removed under reduced pressure,
and the crude product was passed through a short pad of SiO2.
To the solution of the N-acetylated product in THF (10 mL) was
added Bu4NF in THF (5 mL), and the reaction mixture was
stirred at room temperature for 6 h. The solvents were removed,
and the crude product was passed through a short pad of SiO2.
To a solution of the desilylated product in MeOH (10 mL) was
added 10% Pd/C (150 mg), and it was allowed to stir at room
temperature for 24 h under a positive pressure of hydrogen. The
reaction mixture was filtered through a Celite® bed, and the fil-
tering bed was washed with MeOH (60 mL). The combined solu-
tion was concentrated under reduced pressure to give the crude
product, which was dissolved in 0.1 M MeONa in MeOH (30 mL),
and the solution was stirred at room temperature for 1 h. The
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–F