Concise Chemical Synthesis of the Tetrasaccharide Repeating Unit of the O -Antigen from Bovine Mastitis Isolate Ecoli Serotype O174:H28

Article abstract of DOI:10.1055/s-0034-1380922

A concise strategy for the chemical synthesis of the tetrasaccharide repeating unit of the O-antigen from E. coli O174:H28 is reported. Synthesis of the target structure is accomplished by sequential glycosylation of rationally protected monosaccharide sy

Full text of DOI:10.1055/s-0034-1380922

SYNTHESIS
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
© Georg Thieme Verlag Stuttgart · New York
2015, 47, 3061–3066
paper
3061
A. Mitra, B. Mukhopadhyay
Paper
Synthesis
Concise Chemical Synthesis of the Tetrasaccharide Repeating Unit
of the O-Antigen from Bovine Mastitis Isolate E. coli Serotype
O174:H28
OH
Ankita Mitra
O
HO
HO
STol
Balaram Mukhopadhyay*
NHAc
OH
OH
Indian Institute of Science Education and Research Kolkata,
Mohanpur 741246, India
sugarnet73@hotmail.com
O
STol
HO
OH
O
OH
O
OH
OH
OH
O
HO
O
O
OPMP
O
HO
OH
O
HO
AcHN
O
OH
HO
HO
OH
NHAc
OH
O
HO
HO
OPMP
O
HO
HO
STol
NHAc
OH
Received: 13.02.2015
Accepted after revision: 30.04.2015
Published online: 03.07.2015
Bovine mastitis is an inflammation of the mammary
gland caused by a bacterial infection. This inflammation
greatly affects the production of milk and often results in
culling of the affected animals, thus, it has a high economic
impact. The initiation and the progression of the inflamma-
tion are strictly related to the invading pathogen; E. coli is
found to be the causative agent for acute mastitis as it
shows potent systemic inflammatory response immediately
after its entry to the teat canal. Although there is no report
of a specific virulence factor for mastitis-inducing E. coli, it
is evident that the ability of E. coli to multiply in the mam-
mary tissues and to release lipopolysaccharides is crucial in
the pathogenesis of mastitis. Recently, Holst et al.5 reported
the structure of the O-antigen repeating unit of the mastitis
causing E. coli strain O174:H28. Structural analysis of this
oligosaccharide revealed the presence of a glucuronic acid,
two glucosamines, and a galactose unit. Herein, we report
the total chemical synthesis of the tetrasaccharide repeat-
ing unit of the O-antigen from E. coli O174:H28 in the form
of its 4-methoxyphenyl (PMP) glycoside (Figure 1).
DOI: 10.1055/s-0034-1380922; Art ID: ss-2015-n0037-op
Abstract A concise strategy for the chemical synthesis of the tetrasac-
charide repeating unit of the O-antigen from E. coli O174:H28 is report-
ed. Synthesis of the target structure is accomplished by sequential gly-
cosylation of rationally protected monosaccharide synthons derived
from commercially available sugars. Glycosylation reactions were
achieved by the activation of the thioglycosides using N-iodosuccinim-
ide in the presence of H2SO4–silica. The carboxylic acid moiety of the
glucuronic acid unit was successfully installed by a late TEMPO-mediat-
ed oxidation of the primary hydroxy group. Phthalimido groups were
successfully used as the precursors of the required acetamido function-
alities for 1,2-trans-glycosylations.
Key words bacterial O-antigen, acetamido sugars, H2SO4–silica
The lipopolysaccharides (LPS) are important character-
istic components of Gram-negative bacteria. O-Polysaccha-
rides are the constituent of lipopolysaccharides responsible
for regulating various biological conditions of the organism.
O-Polysaccharides are made up of oligosaccharide repeating
units and remain exposed on the outer surface of the bacte-
rial cell wall; due to this exposure they play a pivotal role in
infections.1 Owing to the presence of different sugar resi-
dues, the O-antigens demonstrate a varied character and
act as the elicitor of innate immune responses. Literature
reports suggest wide scope with these bacterial O-antigens
as potential antimicrobial agents and vaccine targets.2–4
However, tedious isolation and purification processes block
the scope for accumulating adequate quantities of these im-
portant oligosaccharides from natural sources for detailed
biological studies and further use. Therefore, chemical syn-
thesis of these O-antigen repeating units becomes pertinent
to explore their vivid biological roles and potential as vac-
cine targets.
OH
O
OH
O
HO
HO
OH
O
HO
O
O
O
OPMP
HO
OH
AcHN
O
OH
O
HO
HO
NHAc
1
Figure 1 Structure of the target tetrasaccharide related to the repeat-
ing unit of the O-antigen from E. coli serotype O174:H28 in the form of
its PMP glycoside
Retrosynthetic analysis suggests that the tetrasaccha-
ride 1 can be disconnected into a tri- and a monosaccha-
ride. The trisaccharide may then be disconnected into a di-
and a monosaccharide and the disaccharide can be split
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, 3061–3066

3062
A. Mitra, B. Mukhopadhyay
Paper
Synthesis
into two monosaccharides (Scheme 1). All rationally pro-
tected monosaccharides may be obtained from commer-
cially available sugars using suitable protecting group ma-
nipulation strategies. As both the glucosamine units pres-
ent in the target structure are linked through 1,2-trans-
glycosidic bond, the corresponding phthalimido derivatives
were chosen as the precursors of the acetamido functional-
ity. 4-Methoxyphenyl glycoside at the reducing end was as-
sessed as suitable as it can be cleaved selectively from the
per-O-acetylated derivative of the target tetrasaccharide
and it allows further conjugation with the relevant aglycon
using trichloroacetimidate chemistry.
Thus, known acceptor 4-methoxyphenyl 4,6-O-ben-
zylidene-3-O-benzyl-β-D-glucopyranoside (2)6 was glyco-
sylated with the known donor, p-tolyl 3,4,6-tri-O-acetyl-2-
deoxy-2-phthalimido-1-thio-β-D-glucopyranoside
(3)7
through activation of the thioglycoside using N-iodosuccin-
imide in the presence of H2SO4–silica8–12 to afford the disac-
charide 4 in 89% yield. Use of H2SO4–silica gel was beneficial
as the same glycosylation reaction with triflic acid or
trimethylsilyl triflate resulted in 73% and 78% yield of the
desired disaccharide, respectively. Moreover, the moisture-
tolerant solid H2SO4–silica is easier to handle compared to
fuming and moisture sensitive triflic acid and trimethylsilyl
triflate. Furthermore, regioselective opening of the ben-
zylidene using triethylsilane in the presence of boron tri-
OH
O
OH
O
HO
HO
OH
O
HO
O
O
O
OPMP
HO
OH
AcHN
O
OH
O
HO
HO
NHAc
1
OH
OH
O
HO
HO
OH
O
HO
O
O
O
OPMP
HO
PhthN
OH
O
OH
O
HO
HO
NPhth
OP
OP
O
O
PO
PO
HO
PO
OP
O
OPMP
PO
O
+
STol
PhthN
OP
O
O
OP
PO
PO
NPhth
commercially available
D-Gal
OP
OP
O
O
HO
PO
OPMP
+
PO
PO
STol
OP
O
PhthN
O
PO
PO
NPhth
commercially available
D-GlcNH2•HCl
OP
O
OP
O
PO
PO
PO
PO
STol
NPhth
OPMP
+
OH
P = protecting group
commercially available
D-Glc
commercially available
D-GlcNH2•HCl
Scheme 1 Retrosynthetic analysis for the target tetrasaccharide 1
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, 3061–3066

3063
A. Mitra, B. Mukhopadhyay
Paper
Synthesis
fluoride–diethyl ether complex13 gave the disaccharide ac-
ceptor 5 in 93% isolated yield. In a separate experiment, the
3-OH group of known p-tolyl 4,6-O-benzylidene-2-deoxy-
2-phthalimido-1-thio-β-D-glucopyranoside (6)14 was chlo-
roacetylated using chloroacetic anhydride in pyridine15 to
afford the donor 7 in 93% yield (Equation 1). Glycosylation
between disaccharide acceptor 5 and the donor 7 using N-
iodosuccinimide in conjunction with H2SO4–silica afforded
the trisaccharide 8 in 87% yield. The deprotection of the
chloroacetate group using thiourea16 in refluxing dichloro-
methane–methanol mixture resulted the trisaccharide ac-
ceptor 9 in 86% yield. Final glycosylation of the trisaccha-
ride acceptor 9 with the known donor 1017 furnished the
protected tetrasaccharide 11 in 85% yield. Now the ben-
zylidene group was hydrolyzed using 80% aqueous acetic
acid at 80 °C18 to afford the diol 12, which was used directly
in the following reactions. The phthalimido groups were re-
moved from 12 using ethylenediamine19 and the resulting
free amine groups were subsequently acetylated by acetic
anhydride in pyridine. The benzyl groups were hydrogeno-
lyzed using a 10% palladium on carbon cartridge in a
ThalesNano H-cube flow hydrogenation assembly. The pri-
mary hydroxy group of the resulting diol was selectively
oxidized by 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO)
together with (diacetoxyiodo)benzene as co-oxidant20 to af-
ford the desired uronic acid. It is worth noting that the 4-
methoxyphenyl group in the reducing end of the protected
tetrasaccharide was not affected by the TEMPO-mediated
oxidation. Finally, Zemplen de-O-acetylation of the acetates
using sodium methoxide in methanol21 furnished the target
tetrasaccharide 1 in 62% overall yield (Scheme 2).
(ClHCH2CO)2O
pyridine
CH2Cl2
O
O
Ph
Ph
O
O
O
O
STol
NPhth
STol
NPhth
O
HO
Cl
93%
O
6
7
Equation 1 Chloroacetylation of p-tolyl 4,6-O-benzylidene-2-deoxy-2-
phthalimido-1-thio-β-D-glucopyranoside
O
Ph
O
O
OPMP
OBn
O
BnO
O
Ph
O
O
OH
+
O
OPMP
HO
TES, BF3•Et2O
OPMP
NIS, H2SO4-silica
BnO
OAc
2
BnO
OAc
O
CH2Cl2, 0 °C
93%
CH2Cl2, 0 °C
89%
OAc
O
AcO
AcO
O
AcO
AcO
O
AcO
AcO
STol
NPhth
NPhth
NPhth
4
5
3
O
O
O
NIS, H2SO4-silica
CH2Cl2, 0 °C
87%
Ph
O
O
Ph
(ClCH2CO)2O
pyridine, CH2Cl2
O
O
STol
STol
HO
Cl
NPhth
NPhth
93%
AcO
O
7
6
OAc
O
OBn
O
O
OBn
O
O
Ph
O
Ph
OAc
O
O
O
AcO
AcO
STol
O
OPMP
O
RO
AcO
OPMP
O
BnO
BnO
OAc
O
PhthN
OAc
OAc
PhthN
O
10
O
O
O
AcO
AcO
NIS, H2SO4-silica
CH2Cl2, 0 °C
AcO
AcO
OAc
NPhth
NPhth
85%
thiourea, collidine
8 R = C(O)CH2Cl
9 R = H
11
CH2Cl2–MeOH, reflux
86%
80% AcOH
80 °C
89%
OH
OBn
O
OH
O
OH
O
HO
HO
O
OH
1. ethylenediamine
2. Ac2O, pyridine
HO
O
AcO
AcO
O
OAc
O
O
HO
O
OPMP
O
O
OPMP
BnO
HO
OH
PhthN
OAc
O
3. H2, Pd/C
4. PhI(OAc)2, TEMPO
5. NaOMe, MeOH
AcHN
O
OH
O
AcO
AcO
OAc
O
HO
HO
NPhth
62%
12
NHAc
1
Scheme 2 Synthesis of the target tetrasaccharide 1
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, 3061–3066

3064
A. Mitra, B. Mukhopadhyay
Paper
Synthesis
In conclusion, chemical synthesis of the tetrasaccharide
repeating unit of the O-antigen from E. coli O174:H28 is ac-
complished by sequential glycosylations of rationally pro-
tected monosaccharide synthons. Required glycosylations
were achieved by the activation thioglycosides with N-io-
dosuccinimide in conjunction with H2SO4–silica. Phthalimi-
do groups were successfully used as the precursors of the
desired acetamido functionalities in 1,2-trans-linked glyco-
sides. The required uronic acid moiety was installed by a
late-stage TEMPO/(diacetoxyiodo)benzene-mediated selec-
tive oxidation of the primary hydroxy group. Finally, the
presence of the 4-methoxyphenyl group at the reducing
end of the target tetrasaccharide would be cleaved selec-
tively using ammonium cerium(IV) nitrate (CAN) from the
per-O-acetylated derivative and this will allow further con-
jugation with a suitable aglycon to furnish desirable glyco-
conjugates for further biological study.
1H NMR (500 MHz, CDCl3): δ = 7.87–7.04 (m, 14 H, HAr), 6.98, 6.85 (2
d, J = 9.0 Hz, 4 H, C6H4OCH3), 5.87 (d, J1′,2′ = 8.5 Hz, 1 H, H1′), 5.75 (dd,
J2′,3′ = 9.5 Hz, J3′,4′ = 10.5 Hz, 1 H, H3′), 5.40 (s, 1 H, CHPh), 5.18 (dd, J3′,4′
10.5 Hz, J4′,5′ = 9.0 Hz, 1 H, H4′), 5.13 (d, J1,2 = 8.0 Hz, 1 H, H1), 4.49–
=
4.42 (ABq, JAB = 12.0 Hz, 2 H, CH2Ph), 4.39 (m, 1 H, H2′), 4.28 (dd, J5′,6a′
5.5 Hz, J6a′,6b′ = 11.0 Hz, 1 H, H6a′), 4.11 (m, 1 H, H6a), 3.86 (t, J1,2 = J2,3
=
=
8.0 Hz, 1 H, H2), 3.77 (s, 3 H, C6H4OCH3), 3.69 (m, 3 H, H3, H5′, H6b′),
3.61 (t, J3,4 = J4,5 = 9.0 Hz, 1 H, H4), 3.46 (dd, J5,6b = 2.5 Hz, J6a,6b = 12.0
Hz, 1 H, H6b), 3.40 (m, 1 H, H5), 2.01, 1.98, 1.83 (3 s, 9 H, 3 COCH3).
13C NMR (125 MHz, CDCl3): δ = 170.6, 170.1, 169.4 (3 COCH3), 155.4,
150.4, 148.1, 138.2, 136.9, 134.3 (2 C), 134.1, 131.7, 128.9, 128.1 (2 C),
128.0, 127.1 (2 C), 127.1, 125.9 (2 C), 123.7, 123.4, 118.3, 114.7 (CAr),
101.1 (CHPh), 100.5 (C1), 98.3 (C1′), 81.3, 80.8, 79.5, 74.6, 73.9, 71.9,
70.4, 68.4, 66.9, 65.6, 61.1 (C6), 55.7 (C6H4OCH3), 55.1 (C2′), 20.7, 20.6,
20.4 (3 COCH3).
HRMS: m/z [M + Na]+ calcd for C47H47O16NNa: 904.2793; found:
904.2790.
4-Methoxyphenyl 3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D-
glucopyranosyl-(1→2)-3,6-di-O-benzyl-β-D-glucopyranoside (5)
To a stirred solution of the disaccharide 4 (2.3 g, 2.7 mmol) and Et3SiH
(5.1 mL, 32 mmol) in anhyd CH2Cl2 (15 mL), BF3·OEt2 (670 μL, 5.4
mmol) was added at 0 °C and the reaction was stirred for 1 h at this
temperature (TLC, n-hexane–EtOAc, 3:2 showed complete conversion
of the starting material to a slower moving spot). The mixture was
washed successively with H2O (2 × 30 mL), sat. NaHCO3 (2 × 30 mL),
and brine (30 mL). The organic layer was separated, dried (Na2SO4),
filtered, and evaporated in vacuo. The residue was purified by flash
chromatography (n-hexane–EtOAc, 1:1) to afford pure 5 (2.2 g, 93%)
as a colorless foam; [α]D25 +80 (c 1.0, CHCl3).
All solvents and reagents were dried prior to use according to stan-
dardized methods.22 Commercially purchased reagents were used
without further purification unless otherwise mentioned. Anhyd,
moisture-free CH2Cl2 was dried and distilled over P2O5. All reactions
were monitored by TLC on silica gel 60F254 with detection by fluores-
cence followed by charring after immersion in 10% H2SO4–EtOH. Flash
chromatography was performed with silica gel 230–400 mesh. Opti-
cal rotations were measured on the Na-line at r.t. 1H and 13C NMR
were recorded on Bruker 500 MHz spectrometer. In case of the tetra-
saccharide, 1H NMR values are denoted as H for the reducing end glu-
curonic acid unit, H′ for the glucosamine unit attached to the 4-posi-
tion of glucuronic acid moiety, H′′ for the galactose unit, and H′′′ for
the remaining glucosamine unit attached to the 2-position of glucu-
ronic acid moiety.
IR (neat): 2931, 2365, 1748, 1594, 1371, 1230, 1045 cm–1
.
1H NMR (500 MHz, CDCl3): δ = 7.60–7.01 (m, 14 H, HAr), 6.97–6.81 (2
d, J = 9.0 Hz, 4 H, C6H4OCH3), 5.91 (d, J1′,2′ = 8.5 Hz, 1 H, H1′), 5.74 (dd,
J2′,3′ = 9.0 Hz, J3′,4′ = 10.0 Hz, 1 H, H3′), 5.18 (dd, J3′,4′ = 10.0 Hz, J4′,5′ = 9.5
Hz, 1 H, H4′), 5.10 (d, J1,2 = 8.0 Hz, 1 H, H1), 4.50, 4.40 (ABq, JAB = 12.0
Hz, 2 H, CH2Ph), 4.48, 4.45 (ABq, JAB = 12.0 Hz, 2 H, CH2Ph), 4.35 (dd,
J1′,2′ = 8.5 Hz, J2′,3′ = 9.0 Hz, 1 H, H2′), 4.10 (t, J5,6a = J6a,6b = 7.0 Hz, 1 H,
H6a), 3.78 (dd, J1,2 = 8.0 Hz, J2,3 = 8.5 Hz, 1 H, H2), 3.77 (s, 3 H,
C6H4OCH3), 3.69 (m, 2 H, H5′, H6a′), 3.58 (m, 1 H, H6b′), 3.46 (m, 3 H,
H3, H4, H5), 3.34 (dd, J5,6a = J6a,6b = 7.0 Hz, 1 H, H6b), 1.99, 1.97, 1.82 (3
s, 9 H, 3 COCH3).
13C NMR (125 MHz, CDCl3): δ = 170.5, 170.1, 169.3 (3 COCH3), 155.1,
150.4, 138.4, 137.8, 134.1 (2 C), 131.1, 128.3 (3 C), 128.2 (3 C), 127.5
(3 C), 127.4, 127.0 (3 C), 123.4, 11.9 (2 C), 114.6 (2 C, CAr), 99.6 (C1),
97.9 (C1′), 83.4, 81.6, 74.6, 74.0, 73.5, 71.9, 70.9, 70.8, 69.7, 68.3, 60.9
(C6), 55.6 (C6H4OCH3), 55.1 (C2′), 20.6, 20.5, 20.3 (3 COCH3).
Preparation of H2SO4–Silica
To a slurry of silica gel 230–400 mesh (10 g) in anhyd Et2O (20 mL)
was added concd H2SO4 (1 mL) and the mixture was hand shaken for
couple of minutes. Then the solvents were evaporated in vacuo and
the residue was dried at 100 °C for 3 h. The dried material was kept in
an airtight bottle for further use.23
4-Methoxyphenyl 3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-D-
glucopyranosyl-(1→2)-4,6-O-benzylidene-3-O-benzyl-β-D-gluco-
pyranoside (4)
A mixture of the acceptor 26 (1.5 g, 3.2 mmol), donor 37 (2.3 g, 4.2
mmol), and activated molecular sieves 4 Å (2.0 g) in anhyd CH2Cl2 (25
mL) was stirred under N2 for 30 min. The mixture was cooled to 0 °C,
NIS (1.2 g, 5.5 mmol) was added followed by addition of H2SO4–silica
(100 mg) and the mixture was stirred at 0 °C for 15 min (TLC, n-hex-
ane–EtOAc, 3:2, showed complete consumption of the acceptor). The
mixture was filtered through a pad of Celite and the filtrate was suc-
cessively washed with aq Na2S2O3 (2 × 30 mL), sat. aq NaHCO3 (2 × 30
mL), and brine (30 mL). The organic layer was separated, dried
(Na2SO4), filtered, and concentrated in vacuo. The crude product thus
obtained was purified by flash chromatography (n-hexane–EtOAc,
1:1) to give 4 (2.3 g, 89%); [α]D25 +126 (c 1.1, CHCl3).
HRMS: m/z [M + Na]+ calcd for C47H49O16NNa: 906.2949; found:
906.2946.
4-Tolyl 4,6-O-Benzylidine-3-O-chloroacetyl-2-deoxy-2-phthalimi-
do-1-thio-β-D-glucopyranoside (7)
To a solution of p-tolyl 4,6-O-benzylidine-2-deoxy-2-phthalimido-1-
thio-β-D-glucopyranoside (6,14 2.0 g, 4 mmol) in anhyd CH2Cl2 (20
mL) and pyridine (1.3 mL, 4 mmol), chloroacetic anhydride (1.3 g, 7.7
mmol) was added at –5 °C. The solution was stirred at –5 °C for 45
min (TLC, n-hexane–EtOAc, 2:1, showed complete conversion of the
starting material to a faster moving spot). The solvents were evapo-
rated in vacuo, coevaporated with toluene to remove residual pyri-
IR (neat): 2945, 2368, 1691, 1590, 1280, 1094, 1012, 706 cm–1
.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, 3061–3066

3065
A. Mitra, B. Mukhopadhyay
Paper
Synthesis
dine, and dried under vacuum. The crude product thus obtained was
purified by flash chromatography (n-hexane–EtOAc, 3:1) to afford
pure 7 (2.1 g, 93%) as a white solid; [α]D25 +108 (c 1.1, CHCl3).
4-Methoxyphenyl 4,6-O-Benzylidene-2-deoxy-2-phthalimido-β-D-
glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-O-(2,4,6-tri-O-acetyl-2-
deoxy-2-phthalimido-β-D-glucopyranosyl)-β-D-glucopyranoside
(9)
IR (neat): 2358, 1596, 1371, 1228, 1061, 698 cm–1
.
A mixture of the trisaccharide 8 (2.3 g, 1.8 mmol), thiourea (650 mg,
8.6 mmol), and 2,4,6-collidine (1.1 mL, 8.6 mmol) in CH2Cl2–MeOH
(2:3, 20 mL) was refluxed for 12 h. The solvents were evaporated in
vacuo and the residue was dissolved in CH2Cl2 (30 mL) and washed
with water (2 × 30 mL). The organic layer was separated, dried
(Na2SO4), and evaporated. The crude product thus obtained was puri-
fied by flash chromatography (n-hexane–EtOAc, 1:1) to afford 9 (2.0
g, 86%) as a colourless gel; [α]D25 +132 (c 1.0, CHCl3).
1H NMR (500 MHz, CDCl3): δ = 7.88–7.35 (m, 9 H, HAr), 7.29, 7.09 (2 d,
J = 8.0 Hz, 4 H, C6H4CH3), 5.98 (t, J2,3 = J3,4 = 9.5 Hz, 1 H, H3), 5.77 (d,
J1,2 = 10.5 Hz, 1 H, H1), 5.53 (s, 1 H, CHPh), 4.43 (m, 2 H, H2, H4), 3.88
(s, 2 H, COCH2Cl), 3.80 (m, 3 H, H5, H6a, H6b), 2.29 (s, 3 H, SC6H4CH3).
13C NMR (125 MHz, CDCl3): δ = 167.7, 167.1 (2 phthalimido C=O),
166.6 (COCH2Cl), 138.7, 136.6, 134.4, 134.3, 133.6 (2 C), 131.4, 131.0,
129.7 (2 C), 129.1, 128.2 (2 C), 126.9, 126.1 (2 C), 123.7, 123.6 (CAr),
101.5 (CHPh), 83.9 (C1), 78.6, 72.3, 70.3, 68.4, 53.9 (C2), 40.2 (COCH2Cl),
21.1 (SC6H4CH3).
IR (neat): 2928, 2363, 1747, 1591, 1378, 1231, 1045 cm–1
.
1H NMR (500 MHz, CDCl3): δ = 7.81–7.04 (m, 23 H, HAr), 6.88, 6.75 (2
d, J = 9.0 Hz, 4 H, C6H4OCH3), 5.82 (d, J1′,2′ = 8.0 Hz, 1 H, H1′), 5.69 (dd,
J2′,3′ = 10.5 Hz, J3′,4′ = 10.0 Hz, 1 H, H3′), 5.35 (d, J1′′,2′′ = 9.0 Hz, 1 H, H1′′),
HRMS: m/z [M + Na]+ calcd for C30H26O7ClSNNa: 602.1016; found:
602.1012.
5.27 (s, 1 H, CHPh), 5.13 (t, J3′,4′ = J4′,5′ = 10.0 Hz, 1 H, H4′), 4.98 (d, J1,2
7.5 Hz, 1 H, H1), 4.63, 4.56 (ABq, JAB = 12.0 Hz, 2 H, CH2Ph), 4.38 (dd,
J4,5 = 8.5 Hz, J5,6a = J5,6b = 9.0 Hz, 1 H, H-5), 4.28 (dd, J1′,2′ = 8.0 Hz, J2′,3′
10.5 Hz, 1 H, H2′), 4.26, 4.18 (2 d, J = 12.0 Hz, 2 H, CH2Ph), 4.01 (m, 2
H, H2′′, H6a′′), 3.89 (t, J5,6a = J6a,6b = 9.0 Hz, 1 H, H6a), 3.82 (dd, J1,2 = 7.5
Hz, J2,3 = 8.5 Hz, 1 H, H2), 3.75 (s, 3 H, C6H4OCH3), 3.61 (m, 2 H, H5′,
H6a′), 3.54 (t, J2,3 = J3,4 = 8.5 Hz, 1 H, H3), 3.42 (dd, J3′′,4′′ = J4′′,5′′ = 9.0 Hz,
1 H, H4′′), 3.28 (m, 3 H, H3′′, H6b′, H6b′′), 3.22 (t, J5,6b = J6a,6b = 9.0 Hz, 1
H, H6b), 3.11 (t, J3,4 = J4,5 = 8.5 Hz, 1 H, H4), 2.95 (m, 1 H, H5′′), 1.99,
1.96, 1.78 (3 s, 9 H, 3 COCH3).
13C NMR (125 MHz, CDCl3): δ = 170.5, 170.1, 169.4 (3 COCH3), 154.9,
150.1, 138.9, 137.8, 136.8, 134.2, 133.9, 131.3, 130.8, 129.1, 128.2 (3
C), 128.1 (3 C), 128.0 (3 C), 127.6 (3 C), 127.4 (2 C), 126.6, 126.2, 125.7
(2 C), 123.3 (2 C), 116.9 (2 C), 114.5 (2 C) (CAr), 101.6 (CHPh), 99.5
(C1), 97.8 (C1′′), 97.7 (C1′), 82.3, 81.7, 81.5, 75.4, 74.3, 74.1, 72.7, 71.7,
70.9, 68.3, 68.3, 68.1, 67.6, 65.7, 60.9, 57.1, 55.6 (C6H4OCH3), 54.9,
20.6, 20.5, 20.3 (3 COCH3).
=
4-Methoxyphenyl 4,6-O-Benzylidine-3-O-chloroacetyl-2-deoxy-2-
phthalimido-β-D-glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-O-
(2,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-
β-D-glucopyranoside (8)
=
A mixture of disaccharide acceptor 5 (2.1 g, 2.4 mmol), donor 7 (2.1 g,
3.6 mmol), and activated molecular sieves 4 Å (2 g) in anhyd CH2Cl2
(20 mL) was stirred under N2 for 30 min. The mixture was cooled to 0
°C, NIS (895 mg, 4.0 mmol) was added followed by H2SO4–silica (100
mg) and the mixture was stirred at this temperature. Within 5 min
TLC (n-hexane–EtOAc, 3:2) showed complete consumption of the di-
saccharide acceptor. The mixture was filtered through a pad of Celite
and the filtrate was successively washed with aq Na2S2O3 (2 × 30 mL),
sat. aq NaHCO3 (2 × 30 mL), and brine (30 mL).The organic layer was
separated, dried (Na2SO4), filtered, and evaporated in vacuo. The
crude product thus obtained was purified by flash chromatography
25
(n-hexane–EtOAc, 1:1) to give 8 (2.7 g, 87%) as a colorless foam; [α]D
+98 (c 1.0, CHCl3).
HRMS: m/z [M + Na]+ calcd for C68H66O22N2Na: 1285.4005; found:
1285.4001.
IR (neat): 2930, 2368, 1751, 1588, 1368, 1233, 1048, 695 cm–1
.
1H NMR (500 MHz, CDCl3): δ = 7.80–7.04 (m, 23 H, HAr), 6.89, 6.75 (2
d, J = 9.0 Hz, 4 H, C6H4OCH3), 5.83 (d, J1′,2′ = 8.5 Hz, 1 H, H1′), 5.71 (dd,
4-Methoxyphenyl 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-
(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyra-
nosyl-(1→4)-3,6-di-O-benzyl-2-O-(2,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-β-D-glucopyranosyl)-β-D-glucopyranoside (11)
J2′,3′ = 9.5 Hz, J3′,4′ = 10.0 Hz, 1 H, H3′), 5.68 (dd, J2′′,3′′ = 10.5 Hz, J3′′,4′′
=
10.0 Hz, 1 H, H3′′), 5.52 (d, J1′′,2′′ = 8.5 Hz, 1 H, H1′′), 5.23 (s, 1 H, CHPh),
5.13 (t, J3′,4′ = J4′,5′ = 10.0 Hz, 1 H, H4′), 4.99 (d, J1,2 = 8.0 Hz, 1 H, H1),
4.60 (s, 2 H, CH2Ph), 4.29 (dd, J1′,2′ = 8.5 Hz, J2′,3′ = 9.5 Hz, 1 H, H2′), 4.25,
4.14 (ABq, JAB = 12.0 Hz, 2 H, CH2Ph), 4.07 (dd, J1′′,2′′ = 8.5 Hz, J2′′,3′′ = 10.5
Hz, 1 H, H2′′), 4.01 (dd, J5′′,6a′′ = 3.0 Hz, J6a′′,6b′′ = 12.5 Hz, 1 H, H6a′′), 3.89
(t, J5,6a = J6a,6b = 9.0 Hz, 1 H, H6a), 3.82 (m, 3 H, H2, COCH2Cl), 3.77 (s, 3
H, C6H4OCH3), 3.62 (m, 1 H, H5′), 3.55 (m, 2 H, H6a′, H6b), 3.41 (m, 2
H, H3, H4′′), 3.26 (m, 3 H, H5′′, H6b′, H6b′′), 3.13 (t, J3,4 = J4,5 = 10.0 Hz,
1 H, H4), 3.05 (m, 1 H, H5), 1.98, 1.95, 1.78 (3 s, 9 H, 3 COCH3).
13C NMR (125 MHz, CDCl3): δ = 171.1, 170.5, 169.3 (3 COCH3), 166.6
(COCH2Cl), 138.9, 137.8, 136.7, 134.3, 133.9, 131.3, 130.8, 129.1, 128.2
(3 C), 128.1 (3 C), 128.0 (2 C), 127.6, 127.4 (3 C), 127.1, 126.6, 126.2 (3
C), 125.5 (2 C), 123.6, 123.3 (2 C), 116.8 (2 C), 114.6 (2 C), 101.4
(CHPh), 99.4 (C1), 97.7 (C1′, C1′′), 82.4, 81.8, 78.5, 75.6, 74.3, 74.1,
72.7, 71.8, 71.4, 70.9, 68.3, 68.0, 67.4, 65.7, 60.8, 60.4, 55.7
(C6H4OCH3), 55.6 (C2′), 40.2 (COCH2Cl), 20.6, 20.5, 20.3 (3 COCH3).
A mixture of trisaccharide acceptor 9 (2 g, 1.6 mmol), known donor
1017 (935 mg, 2.1 mmol), and activated molecular sieves 4 Å (2.0 g) in
anhyd CH2Cl2 (20 mL) was stirred under N2 for 30 min. The mixture
was cooled to 10 °C in an ice–water bath, NIS (603 mg, 2.7 mmol) was
added followed by H2SO4–silica (100 mg), and the mixture was stirred
in the ice–water bath for a further 10 min (TLC, n-hexane–EtOAc, 1:1,
showed complete consumption of the acceptor). The mixture was fil-
tered through Celite and the filtrate was washed successively with aq
Na2S2O3 (2 × 30 mL), sat. aq NaHCO3 (2 × 30 mL), and brine (30 mL).
The organic layer was separated, dried (Na2SO4), filtered, and evapo-
rated in vacuo. The crude product was purified by flash chromatogra-
phy (n-hexane–EtOAc, 1:1) to afford pure 11 (2.1 g, 85%) as a white
amorphous solid; [α]D25 +86 (c 0.9, CHCl3).
IR (KBr): 1748, 1721, 1632, 1603, 1365, 1218, 1039 cm–1
1H NMR (500 MHz, CDCl3): δ = 7.85–6.73 (m, 27 H, HAr), 5.80 (d, J1′′′,2′′′
8.5 Hz, 1 H, H1′′′), 5.70 (dd, J2′′′,3′′′ = 10.5 Hz, J3′′′,4′′′ = 9.0 Hz, 1 H, H3′′′),
5.27 (s, 1 H, CHPh), 5.23 (d, J1′,2′ = 8.5 Hz, 1 H, H1′), 5.15 (t, J3′′′,4′′′ = J4′′′,5′′′
.
HRMS: m/z [M + Na]+ calcd for C70H67O23ClN2Na: 1361.3721; found:
1361.3717.
=
=
9.0 Hz, 1 H, H4′′′), 5.13 (br s, 1 H, H4′′), 4.95 (d, J1,2 = 7.5 Hz, 1 H, H1),
4.88 (dd, J1′′,2′′ = 8.0 Hz, J2′′,3′′ = 10.5 Hz, 1 H, H2′′), 4.68 (dd, J2′′,3′′ = 10.5
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, 3061–3066

3066
A. Mitra, B. Mukhopadhyay
Paper
Synthesis
Hz, J3′′,4′′ = 3.5 Hz, 1 H, H3′′), 4.61, 4.52 (ABq, JAB = 12.0 Hz, 2 H, CH2Ph),
4.53 (dd, J2′,3′ = 10.5 Hz, J3′,4′ = 9.0 Hz, 1 H, H3′), 4.45 (d, J1′′,2′′ = 8.0 Hz, 1
H, H1′′), 4.30, 4.20 (ABq, JAB = 12.0 Hz, 2 H, CH2Ph), 4.27 (dd, J1′′′,2′′′ = 8.5
Hz, J2′′′,3′′′ = 10.5 Hz, 1 H, H2′′′), 4.07 (dd, J1′,2′ = 8.5 Hz, J2′,3′ = 10.5 Hz, 1
H, H2′), 4.01–3.95 (m, 2 H, H6a, H6a′), 3.88 (t, J3,4 = J4,5 = 9.0 Hz, 1 H,
H4), 3.82 (dd, J1,2 = 7.5 Hz, J2,3 = 9.0 Hz, 1 H, H2), 3.79–3.74 (m, 2 H,
H5′′, H6b), 3.74 (s, 3 H, C6H4OCH3), 3.68 (dd, J5′,6b′ = 5.0 Hz, J6a′,6b′ = 10.5
Hz, 1 H, H6b′), 3.62 (m, 1 H, H5′′′), 3.51 (t, J2,3 = J3,4 = 9.0 Hz, 1 H, H3),
3.44 (t, J3′,4′ = J4′,5′ = 9.0 Hz, 1 H, H4′), 3.38 (m, 2 H, H5, H6a′′), 3.31 (dd,
J5′′,6b′′ = 2.0 Hz, J6a′′,6b′′ = 12.0 Hz, 1 H, H6b′′), 3.22 (m, 1 H, H6a′′′), 3.08
(m, 1 H, H6b′′′), 3.02 (m, 1 H, H5′), 2.05, 2.04, 1.99, 1.96, 1.90, 1.82,
1.78 (7 s, 21 H, 7 COCH3).
13C NMR (125 MHz, CDCl3): δ = 170.6, 170.2, 170.1, 170.0 (2 C), 169.4,
168.7 (7 COCH3), 155.0, 150.6, 138.8, 137.9 (2 C), 134.2, 133.9 (2 C),
130.8 (2 C), 129.3, 128.3 (3 C), 128.1 (3 C), 128.0 (3 C), 127.5, 127.4 (3
C), 126.6, 126.2, 126.0 (2 C), 125.8 (2 C), 123.4 (2 C), 117.0 (2 C), 114.5
(2 C) (CAr), 101.3 (CHPh), 100.1 (C1′′), 99.6 (C1), 97.7 (C1′′′), 97.6 (C1′),
82.1, 81.3, 80.7, 75.3, 74.1 (3 C), 72.7, 71.0, 70.9, 70.1, 69.2, 68.4, 68.2,
67.7, 66.5, 65.8, 60.9, 60.7, 60.4, 55.7 (2 C), 55.6 (C6H4OCH3), 54.9,
20.7, 20.6 (2 C), 20.5, 20.4, 20.3, 20.0 (7 COCH3).
13C NMR (125 MHz, D2O): δ = 175.0 (COOH), 174.5, 174.1 (2 NHCOCH3),
155.2, 149.6, 119.8 (2 C), 115.0 (2 C) (CAr), 100.5 (C1′′), 100.3 (C1),
97.6 (C1′), 97.3 (C1′′′), 81.5, 76.0, 75.2, 73.4, 72.0, 71.6, 70.7, 70.5, 70.2,
69.8, 69.6, 69.5, 62.5, 62.1, 61.0, 60.7, 60.6, 59.7, 55.7, 55.4, 55.1, 21.9,
22.0 (2 NHCOCH3).
HRMS: m/z [M + Na]+ calcd for C35H52O23N2Na: 891.2859; found:
891.2856.
Acknowledgment
A.M. is thankful to the Indian Institute of Science Education and Re-
search (IISER) Kolkata for Junior Research Fellowship. The work is
supported by the Science and Engineering Research Board (SERB)
SB/S1/OC-48/2013 to BM.
Supporting Information
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0034-1380922.
S
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HRMS: m/z [M + Na]+ calcd for C82H84O31N2Na: 1615.4956; found:
1615.4953.
Reference
4-Methoxyphenyl β-D-Galactopyranosyl-(1→3)-2-acetamido-2-de-
oxy-β-D-glucopyranosyl-(1→4)-2-O-(2-acetamido-2-deoxy-β-D-
glucopyranosyl)-β-D-glucopyranosiduronic Acid (1)
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A mixture of the tetrasaccharide 11 (1.0 g, 0.6 mmol) in 80% aq AcOH
(20 mL) was stirred at 80 °C for 2 h. After evaporation of the solvents
in vacuo and co-evaporation with toluene to remove traces of AcOH,
the colorless gum was dissolved in BuOH (15 mL). Ethylenediamine
(2.0 mL) was added and the solution was stirred at 110 °C for 12 h.
The solvents were evaporated and the residue was dissolved in dry
pyridine (10 mL), Ac2O (10 mL) was added and the solution was
stirred at r.t. for 2 h. The solution was evaporated in vacuo, co-evapo-
rated with toluene, and the residue thus obtained was dissolved in
MeOH (50 mL) and passed through a 10% Pd/C cartridge in a Thale-
sNano flow hydrogenation assembly under continuous flow of H2 at
atmospheric pressure. The hydrogenolysis of the benzyl groups was
complete after 3 such cycles as evident from mass spectroscopy. Sol-
vents were evaporated in vacuo and the resulting diol was dissolved
in CH2Cl2–H2O (1.5:1; 20 mL). TEMPO (30 mg, 0.2 mmol) was added
followed by (diacetoxyiodo)benzene (480 mg, 1.6 mmol) and the mix-
ture was stirred at 5 °C for 7 h. Aq sat. Na2S2O3 (5 mL) was added to
stop the reaction and the mixture was diluted with CH2Cl2 (20 mL)
and washed successively with brine (2 × 30 mL). The organic layer was
separated, dried (Na2SO4), filtered, and evaporated to a syrup. The res-
idue was dissolved in MeOH (10 mL) and 0.5 M NaOMe in MeOH (1
mL) was added and the solution was stirred at r.t. for 12 h. The solu-
tion was neutralized by DOWEX 50W H+ resin, filtered, and evaporat-
ed in vacuo to afford the final tetrasaccharide 1 (275 mg, 62%) as a
white amorphous solid; [α]D25 +102 (c 0.6, MeOH).
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IR (KBr): 2350, 1683, 1194, 782 cm–1
.
Partial 1H NMR (500 MHz, D2O): δ = 7.11, 6.98 (2 d, J = 9.0 Hz, 4 H,
OC6H4OMe), 5.93 (d, J1′,2′ = 7.5 Hz, 1 H, H1′), 5.91 (d, J1′′′,2′′′ = 7.5 Hz, 1 H,
H1′′′), 4.77 (d, J1,2 = 8.0 Hz, 1 H, H1), 4.75 (d, J1′′,2′′ = 7.5 Hz, 1 H, H1′′),
3.82 (s, 3 H, OC6H4OCH3), 2.01, 2.00 (2 s, 6 H, 2 NHCOCH3).
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© Georg Thieme Verlag Stuttgart · New York — Synthesis 2015, 47, 3061–3066