Synthesis of a tetrasaccharide related to the repeating unit of the O-antigen from Escherichia coli K-12

Article abstract of DOI:10.1016/j.carres.2009.09.026

Synthesis of a tetrasaccharide related to the repeating unit of the O-antigen from Escherichia coli K-12 is reported in the form of its octyl glycoside. Syntheses of the 1,2-cis glycosidic linkages have been accomplished by using NIS in conjunction with H

Full text of DOI:10.1016/j.carres.2009.09.026

Carbohydrate Research 344 (2009) 2311–2316
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of a tetrasaccharide related to the repeating unit of the O-antigen
from Escherichia coli K-12
Bimalendu Roy a, Robert A. Field b, Balaram Mukhopadhyay a,
*
a Indian Institute of Science Education and Research-Kolkata (IISER-K), Mohanpur Campus, P.O. BCKV Campus Main Office, Mohanpur, Nadia, West Bengal, India
b Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2009
Received in revised form 19 September 2009
Accepted 22 September 2009
Available online 25 September 2009
Synthesis of a tetrasaccharide related to the repeating unit of the O-antigen from Escherichia coli K-12 is
reported in the form of its octyl glycoside. Syntheses of the 1,2-cis glycosidic linkages have been accom-
plished by using NIS in conjunction with H2SO4-silica, and it was found to be stereoselective and produc-
tive. The synthesized tetrasaccharide will be utilized as the substrate for galactofuranosyltransferase,
WbbI.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Bacterial oligosaccharide
1,2-Cis glycosylation
H2SO4-silica
1. Introduction
expression of the E. coli K-12 galactofuranosyltransferase, WbbI,17
and demonstrated that with monosaccharide acceptors (tagged
Protein–carbohydrate interactions are central to a wide variety
of microbial infection processes.1,2 As such, they present opportuni-
ties for microbe detection and therapeutic intervention, whether
through the action of novel small molecules or carbohydrate vac-
cines. A range of new analytical tools are beginning to pose as chal-
lenges such as those that are more tractable.3 Following on from our
work on cell surface carbohydrate recognition in relation to the
detection of bacteria4 and bacterial toxins,5–7 and the identification
of carbohydrate leads,8 targets9,10 and vaccine candidates11 to com-
bat human pathogens, we turned our attention to Escherichia coli
K-12 as a model organism. This strain of E. coli produces a galacto-
furanose-containing O-antigen12 and as such might prove useful in
investigating galactofuranose metabolism more generally. Whilst
higher organisms possess galactose in its 6-membered ring pyra-
nose form, numerous microbial pathogens produce essential mac-
romolecules containing the 5-membered ring galactofuranose.13
Hence the biochemistry of galactofuranose presents potential ther-
apeutic targets for diseases caused by, for instance, Mycobacterium
tuberculosis, Leishmania species and Trypanosma cruzi.14
as their octyl glycosides), this enzyme shows a remarkably relaxed
acceptor substrate specificity. This observation is not obviously in
accord with the need for the selective introduction of galactofura-
nose into the O-antigen repeat unit (Fig. 1). To address this point,
herein we report the chemical synthesis of a tetrasaccharide frag-
ment of the pentasaccharide repeat unit.
2. Results and discussion
Retrosynthetic analysis for the tetrasaccharide 1 suggested a
2+2 disconnection route where two disaccharides can be coupled
through the a-linked rhamnose residue. The most challenging part
of the synthesis was the installation of two 1,2-cis-linked glucose
units. Thus, the protecting group manipulations were chosen so
that 1,2-cis glycosylation becomes facile (Fig. 2).
Synthesis of a suitable armed glucoside donor was started with
the known ethyl 1-thio-b-D
-glucopyranoside (2).18 Reaction with
2,2-dimethoxypropane (DMP)19 afforded compound 3 which upon
benzylation using BnBr and NaH20 furnished the required donor,
In recent studies we have reported a practical chemoenzymatic
synthesis of UDP-galactofuranose15 and we have also investigated
UDP-galactofuranose formation from UDP-galactopyranose in
some detail.16 Further, we have reported the cloning and over-
ethyl 2,3-di-O-benzyl-4,6-O-isopropylidene-1-thio-b-
D-glucopy-
ranoside (4) in 82% overall yield. In another experiment, the known
octyl 2-acetamido-2-deoxy-6-O-trityl-a-D
-glucopyranoside (5)21
was benzoylated using benzoyl chloride in pyridine22 to afford com-
pound 6 in 81% yield. Hydrolysis of the trityl group using TFA23,24
furnished the acceptor, octyl 2-acetamido-3,4-di-O-benzoyl-2-
* Corresponding author.
E-mail addresses: rob.field@bbsrc.ac.uk (R.A. Field), sugarnet73@hotmail.com (B.
Mukhopadhyay).
deoxy-a-D-glucopyranoside (7). Glycosylation between the donor
4 and the acceptor 7 was accomplished using NIS in conjunction
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.09.026

2312
B. Roy et al. / Carbohydrate Research 344 (2009) 2311–2316
HO
HO
HO
O
HO
HO
O
HO
O
HO
O
HO
HO
O
O
O
AcNH
O
HO
O
Me
HO
OH
O
AcNH
O
Me
OC8H17
OAc
HO
HO
HO
HO
O
OAc
O
HO
HO
HO
HO
O
1
HO
HO
O
O
Repeating unit
OH
HO
Figure 1. Structure of the repeating unit of the O-antigen from E. coli K-12 and the synthetic target.
required acceptor, p-methoxyphenyl 2-O-acetyl-4-O-benzyl-
rhamnopyranoside (13) in 86% yield. Subsequently, acceptor 13
was coupled with donor using NIS in conjunction with
H2SO4-silica27–30 to afford the disaccharide, p-methoxyphenyl 2,3-
di-O-benzyl-4,6-O-isopropylidene- -glucopyranosyl-(1?3)-
2-O-acetyl-4-O-benzyl- -rhamnopyranoside (14) in 65% isolated
a-L-
HO
O
HO
HO
4
HO
O
O
HO
O
a
-D
O
a-L
AcNH
yield. It is worth noting that having evaluated HClO4-silica31 and
H2SO4-silica as activators of NIS, we found the latter to be more prac-
tical. In this case also, the desired 1,2-cis glycoside was isolated as
the major product as confirmed by the H-10 signal at d 5.10 (J
3.3 Hz) in the 1H NMR spectrum. At this stage, CAN-mediated oxida-
tive cleavage of the p-methoxyphenyl glycoside was required. From
our previous experience it was evident that the isopropylidene ace-
tal is not compatible with the CAN-mediated reaction. Thus, the iso-
propylidene derivative 14 was hydrolysed using 80% AcOH at 80 °C
and then the CAN-mediated32 oxidative cleavage was accomplished.
Reinstallation of the isopropylidene acetal followed by reaction with
trichloroacetonitrile in the presence of DBU33 afforded the required
Me
OC8H17
HO
O
OAc
HO
HO
HO
O
HO
PO
PO
PO
O
SEt
Me
PO
O
PO
O
+
O
O
OAc
HO
PO
HO
PO
PO
AcNH
O
OC8H17
PO
disaccharide donor, 2,3-di-O-benzyl-4,6-O-isopropylidene-
a-D-glu-
HO
PO
PO
PO
PO
copyranosyl-(1?3)-2-O-acetyl-4-O-benzyl- -rhamnopyranosyl
a
-L
O
O
SEt
+
trichloroacetimidate (18). Due to the stability concern associated
with reactive trichloroacetimidate donors, compound 18 was uti-
lized for further reaction without characterization (Scheme 2).
Relying on the higher reactivity of the 3-OH compared to the 4-
OH position,8 diol 9 was allowed to react with the trichloroacetimi-
date donor 18 in the presence of H2SO4-silica to afford the protected
PO
PO
AcNH
OC8H17
Commercially available
D-Glc and D-GlcNAc
OMP
PO
PO
PO
O
Me
O
SEt
+
PO
tetrasaccharide, octyl 2,3-di-O-benzyl-4,6-O-isopropylidene-
glucopyranosyl-(1?3)-4-O-benzyl-2-O-acetyl- -rhamnopyrano-
syl-(1?3)-2-acetamido-2-deoxy-6-O-(2,3-di-O-benzyl-4,6-O-iso-
propylidene- -glucopyranosyl)- -glucopyranoside (19) in 78%
a-D-
HO
PO
a-L
OAc
a-
D
a-D
Commercially available
L-Rha and D-Glc
yield. It was then treated with 80% AcOH at 80 °C to hydrolyse the
isopropylidene acetals followed by Pd(OH)2–C catalysed hydrogen-
olysis34 for the removal of the benzyl groups to furnish the target
Figure 2. Retrosynthetic analysis for the tetrasaccharide 1.
tetrasaccharide, octyl
rhamnopyranosyl-(1?3)-2-acetamido-2-deoxy-6-O-(
anosyl)- -glucopyranoside (1) in 85% overall yield (Scheme 3).
a
-
D
-glucopyranosyl-(1?3)-2-O-acetyl-
a-L-
a-D-glucopyr-
a-D
with H2SO4-silica at ꢀ60 °C. Due to the low temperature, the reac-
tion took longer time (ꢁ12 h) for completion but to our satisfaction,
only the 1,2-cis-linked disaccharide (8), as confirmed by 1H NMR
from the H-10 signal at d 4.78 (J 2.4 Hz), was formed in 67% yield.
Compound 8 was then treated with NaOMe in MeOH to afford the
disaccharide diol 9 in 83% yield (Scheme 1).
3. Conclusions
In conclusion, we have developed a concise convergent synthe-
sis of a tetrasaccharide related to the pentasaccharide repeating
unit of E. coli K-12. In particular, H2SO4-silica in conjunction with
NIS was found to be an effective catalyst for maximizing the yield
of the key 1,2-cis glycosylation reactions using thioglycoside donor
building blocks. The target tetrasaccharide, 1, is currently under
further investigation as a substrate for E. coli K-12 galactofurano-
syltransferase WbbI; full details will be reported in due course.
Synthesis of the disaccharide donor was started with the known
p-methoxyphenyl
2,3-O-isopropylidene-a-L-rhamnopyranoside
(10).25 Benzylation of the free –OH group with BnBr in the presence
of NaH followed by the hydrolysis of the isopropylidene acetal
afforded the diol 12. Further, selective acetylation of the 2-OH
through orthoester formation and rearrangement26 furnished the

B. Roy et al. / Carbohydrate Research 344 (2009) 2311–2316
2313
OH
O
O
2,2-DMP, CSA
HO
HO
O
SEt
O
SEt
HO
OH
OH
2
3 R=H
BnBr, NaH
4 R=Bn
O
O
O
OTr
O
OR1
O
O
NaOMe
M
O
4
BnO
O
BnO
BzCl, Py R2O
HO
HO
NIS,
H2SO4-silica
BnO
BnO
R2O
O
O
O
AcNH
AcNH
BzO
BzO
O
OC8H17
OC8H17
BzO
BzO
5
AcNH
6 R1=Tr, R2=Bz
7 R1=H,R2=Bz
AcNH
TFA, CH2Cl2
OC8H17
OC8H17
9
8
Scheme 1. Synthesis of the disaccharide diol 9.
OMP
OMP
OMP
OMP
Me
O
Me
BnO
O
º
Me
RO
80% AcOH, 80 C
4
NIS
º
80% AcOH, 80 C
O
BnO
Me
O
BnO
O
O
OAc
O
OAc
BnO
H2SO4-silica
BnO
O
R2O
OR1
BnO
O
BnO
HO
HO
O
O
O
i. Trimethyl
12 R1=R2=H
10 R=H
15
14
orthoacetate, CSA
BnBr, NaH
11 R=Bn
13 R1=Ac, R2=H
ii. 1N HCl
NH
CCl3
OH
OH
O
Me
O
Me
BnO
O
BnO
CCl3CN
DBU
Me
BnO
2,2-DMP
CSA
O
CAN
O
O
OAc
BnO
OAc
BnO
O
O
BnO
O
OAc
18
BnO
O
BnO
HO
O
BnO
O
HO
O
O
17
16
Scheme 2. Synthesis of the trichloroacetimidate donor 18.
OH
O
O
O
HO
HO
O
BnO
BnO
BnO
O
O
O
O
i. 80% AcOH
H2SO4-silica
HO
HO
O
9 + 18
O
ii. Pd(OH)2-C, MeOH
AcNH
AcNH
Me
HO
OC8H17
O
Me
OC8H17
O
BnO
O
O
OAc
OAc
HO
BnO
HO
HO
BnO
O
19
O
OH
1
O
O
Scheme 3. Synthesis of the target tetrasaccharide 1.
matography was performed with silica gel 60–120. Optical rotation
was measured at the sodium D-line at 25 °C. 1H and 13C NMR spec-
tra were recorded on a spectrometer (Bruker). In the case of the
tetrasaccharide 1H NMR values are denoted as H for reducing
end glucosamine moiety, H0 for rhamnose, H00 for the non-reducing
end glucose unit attached to rhamnose moiety and H000 for the non-
reducing end glucose unit attached to glucosamine moiety. For 13C
NMR assignment similar primes are used.
4. Experimental section
4.1. General methods
All reactions were monitored by TLC on Silica Gel 60-F254 with
detection by fluorescence and/or by charring following immersion
in 10% ethanolic solution of sulfuric acid. An orcinol dip prepared
by the careful addition of sulfuric acid (20 mL) to an ice-cold solu-
tion of 3,5-dihydroxytoluene (360 mg) in ethanol (250 mL) and
water (10 mL) was used to detect the deprotected compound by
charring. All reagents and solvents were dried prior to use accord-
ing to the standard methods.35 Commercial reagents were used
without further purification unless otherwise stated. Flash chro-
4.2. Preparation of H2SO4-silica
To a slurry of silica gel (10 g, 200–400 mesh) in dry diethyl ether
(50 mL) was added commercially available concd H2SO4 (1 mL) and

2314
B. Roy et al. / Carbohydrate Research 344 (2009) 2311–2316
the slurry was shaken for 5 min. The solvent was evaporated under
reduced pressure resulting in free flowing H2SO4-silica which was
dried at 110 °C for 3 h and was used for the reactions.
(NHCOCH3), 167.0, (COC6H5), 165.9 (COC6H5), 133.4, 133.3, 129.9,
129.8, 129.0, 128.8, 128.4 (ArC), 97.3 (C-1), 71.7, 70.4, 69.4, 68.4,
61.2, (C-6), 52.3 (C-2), 31.8, 29.4, 29.3, 26.2, 23.0 (NHCOCH3), 22.6,
14.1 (CH3). HRMS calcd for C30H39NO8Na (M+Na)+: 564.2573; found
m/z 564.2570.
4.3. Ethyl 2,3-di-O-benzyl-4,6-O-isopropylidene-1-thio-b-D-
glucopyranoside (4)
4.5. Octyl 2,3-di-O-benzyl-4,6-O-isopropylidene-
glucopyranosyl-(1?6)-2-acetamido-3,4-di-O-benzoyl-2-deoxy-
-glucopyranoside (8)
a-D-
To a suspension of ethyl 1-thio-b-D-glucopyranoside 2 (3 g,
9.8 mmol) in dry acetone (30 mL) was added DMP (2.4 mL,
19.6 mmol) followed by CSA (15 mg) and the mixture was allowed
to stir at rt for 4 h when the solution became clear and TLC (n-hex-
ane–EtOAc; 1:1) showed a complete conversion of the starting
material to a faster moving spot. The solution was neutralized with
Et3N and the solvents were evaporated in vacuo. The semi-solid
mass thus obtained was purified by flash chromatography using
a gradient of n-hexane–EtOAc (2:1 to 1:1) as eluent to afford pure
isopropylidene derivative 3 (3.2 g, 90%). To a cold solution (ice-
bath) of purified isopropylidene derivative in dry DMF (40 mL)
was added NaH (950 mg, 20 mmol) and the solution was allowed
to stir for 15 min with slow warming up to rt. Then BnBr (1.5 mL,
12.8 mmol) was added to the reaction mixture and allowed to stir
at rt for 30 min. When TLC (n-hexane–EtOAc; 4:1) showed a com-
plete conversion of the starting material to a faster moving spot,
the excess NaH was quenched by using MeOH (3 mL) and the sol-
vents were evaporated in vacuo. The solution was diluted with
Et2O (20 mL) and washed with H2O (2 ꢂ 50 mL) and brine
(50 mL). The organic layer was collected, dried (Na2SO4) and puri-
fied by flash chromatography after evaporation to afford pure com-
a-D
A mixture of acceptor 7 (1.5 g, 2.8 mmol), donor 4 (1.8 g,
3.6 mmol) and activated MS 4 Å (2 g) in dry CH2Cl2 (25 mL) was
stirred under nitrogen atmosphere for 1 h. After cooling in a meth-
anol bath at ꢀ80 °C, NIS (920 mg, 4.1 mmol) was added followed
by H2SO4-silica (40 mg) and the mixture was stirred for 12 h when
TLC (n-hexane–EtOAc, 2:1) showed a complete conversion of the
acceptor. The mixture was allowed to warm up to rt, filtered
through CeliteÒ and the filtrate was diluted with CH2Cl2 (30 mL)
and was washed successively with aq Na2S2O3 (2 ꢂ 50 mL), aq
NaHCO3 (2 ꢂ 50 mL) and brine (50 mL). The organic layer was sep-
arated, dried (Na2SO4) and evaporated to a syrup. The crude prod-
uct was purified by flash chromatography using n-hexane–EtOAc
(4:1) to afford pure disaccharide 8 (1.1 g, 67%) as a colourless foam.
[a]
D +87 (c 0.9, CHCl3). 1H NMR (CDCl3, 300 MHz) d: 7.95–7.20 (m,
20H, ArH), 5.86 (d, 1H, J 6.3 Hz, NH), 5.69 (t, 1H, J 9.9 Hz, H-3), 5.42
(t, 1H, J 9.6 Hz, H-4), 4.96 (d, 1H, J 2.7, H-1), 4.88–4.81 (2d, 4H, J
11.4 Hz, 2 ꢂ CH2C6H4), 4.78 (d, 1H, J 2.4 Hz, H-10), 4.54 (dt, J
2.7 Hz, H-2), 4.34 (m, 1H, H-50), 3.98 (m, 1H, H-6a), 3.95 (m, 3H,
H-5, OCH2), 3.86–3.80 (m, 2H, H-40, H-30), 3.78 (t, 1H, H-20), 3.77–
3.73 (m, 2H, H-6a0, H-6b0), 3.64–3.51 (dd, 1H, H-6b), 1.86 (s, 3H,
NHCOCH3), 1.45, 1.39 (2s, 6H, isopropylidene-CH3), 1.32 (m, 12H,
6ꢂCH2), 0.89 (t, 3H, J 6.6, CH3). 13C NMR (CDCl3, 75 MHz) d:
177.4 (NHCOCH3), 167.0 (COC6H5), 165.2 (COC6H5), 139.1, 138.3,
133.3 (2), 129.7 (4), 128.9, 128.3 (4), 128.2 (2), 128.1 (2), 127.8
(2), 127.7 (2), 127.4, 127.3 (2), 99.1 (isopropylidene C), 98.8 (C-
1), 96.9 (C-10), 79.0, 78.9, 74.9, 74.6, 73.4, 71.9, 69.5, 69.1, 68.5,
67.3, 63.3, 62.4 (C-60), 62.0 (C-6), 31.7, 29.6–29.0 (6), 28.0, 26.1,
23.0, 2.6, 19.1, 14.01 (C–CH3). HRMS calcd for C53H65NO13Na
(M+Na)+: 946.4354; found m/z 946.4352.
pound 4 (4.9 g, 82%). [
a]
D
+109 (c 1.1, CHCl3). 1H NMR (CDCl3,
500 MHz) d: 7.38–7.25 (m, 10H, ArH), 4.88–4.83 (2d, 2H,
J
11.3 Hz, CH2Ph), 4.79–4.74 (2d, 2H, J 11.3 Hz, CH2Ph), 4.5 (d, 1H, J
9.8 Hz, H-1), 3.92 (dd, 1H, J 5.4 Hz, H-4), 3.76 (m, 2H, H-6), 3.64
(t, 1H, J 8.5 Hz, H-3), 3.41 (t, 1H, J 8.5 Hz, H-2), 3.27 (m, 1H, H-5),
2.77 (m, 2H, S–CH2), 1.49, 1.42 (2s, 6H, isopropylidene-CH3), 1.29
(t, 3H, J 7.4 Hz, S–CH2–CH3). 13C NMR (CDCl3, 125 MHz) d: 142.3,
141.4, 128.4, 128.2, 128.2, 127.8 (ArC), 99.1 (isopropylidene-C),
85.6 (C-1), 83.1, 81.2, 75.8, 74.8, 74.3, 72.8, 71.1, 62.1, 44.8, 29.6,
29.1, 25.0, 19.9, 19.1 (isopropylidene-CH3), 15.0 (CH3). HRMS calcd
for C25H32O5SNa (M+Na)+: 467.1868; found m/z 467.1865.
4.4. Octyl 2-acetamido-3,4-di-O-benzoyl-2-deoxy-
a
-D
-
4.6. Octyl 2,3-di-O-benzyl-4,6-O-isopropylidene-
a-D
-
glucopyranoside (7)
glucopyranosyl-(1?6)-2-acetamido-2-deoxy-
a
-D
-
glucopyranoside (9)
To a cold solution (ice-bath) of compound 5 (3 g, 5.21 mmol) in
dry pyridine (30 mL) was added BzCl (1.80 mL, 15.6 mmol) and the
solution was allowed to stir for 4 h with slow warming up to rt. Then
MeOH (3 mL) was added to quench the reaction and the solvents
were evaporated in vacuo. The resulting semi-solid mass was dis-
solved in CH2Cl2 (50 mL) and was washed successively with ice-cold
1 M HCl (2 ꢂ 50 mL), aq NaHCO3 (2 ꢂ 50 mL) and brine (50 mL). The
organic layer was collected, dried (Na2SO4) and concentrated under
reduced pressure. The residue was dissolved in CH2Cl2 (30 mL) fol-
lowed by the addition of 90% aq TFA (10 mL) and was allowed to stir
at rt till TLC showed a completeconversion of the starting material to
a slower running spot (1 h). The solution was diluted with CH2Cl2
(20 mL) and was washed successively with H2O (2 ꢂ 50 mL), aq
NaHCO3 (2 ꢂ 50 mL) and brine (50 mL). The organic layer was col-
lected, dried (Na2SO4) and was purified by flash chromatography
after concentration under reduced pressure to afford pure 7 (2.6 g,
To a solution of the disaccharide 8 (1.1 g, 1.5 mmol) in dry
MeOH (25 mL), NaOMe (25 mg) was added and the solution was
stirred at rt for 2 h. After completion of the reaction, the solution
was neutralized with DOWEX 50W H+ resin, filtered and evapo-
rated to a semi-solid mass. The crude product was purified by flash
chromatography using n-hexane–EtOAc (2:1) to afford pure disac-
charide 9 (850 mg, 83%) as white amorphous powder. [
a]
D
+97
(c 1.0, CHCl3). 1H NMR (CDCl3 + CCl4, 300 MHz) d: 7.39–7.28 (m,
10H, ArH), 5.99 (d, 1H, NH), 4.85 (d, 1H, J 2.7 Hz, H-1), 4.83–4.76
(2d, 4H, J 11.3 Hz, 2 ꢂ CH2C6H5), 4.73 (d, 1H, J 2.7 Hz, H-10), 4.69
(d, 1H, J 2.5 Hz, H-2), 4.08 (m, 1H, H-50), 3.92 (dd, 1H, H-6a), 3.89
(m, 3H, H-5, OCH2), 3.89 (d, 1H, J 9.6 Hz, 3.8 (m, 2H, H-30, H-40),
3.64 (m, 4H), 3.55 (m, 2H, H-3, H-4), 3.4 (m, H-5), 2.05 (s, 3H,
COCH3), 1.48, 1.42 (2s, 6H, 2 ꢂ isopropylidene CH3), 1.28 (m, 12H,
6 ꢂ CH2), 0.89 (t, 3H, J 6.4 Hz, C–CH3). 13C NMR (CDCl3, 75 MHz)
d: 171.4 (NHCOCH3), 138.9, 137.9, 128.4, 128.2, 128.1, 128.0,
127.8, 127.8, 127.7, 127.4 (ArC), 99.3 (isopropylidene C), 98.53
(C-1), 96.95 (C-10), 79.0, 78.8, 74.9, 74.7, 73.6, 73.2, 73.1, 69.5,
68.6, 68.0, 63.3 (C-60), 62.4 (C-6), 33.7, 29.9, 29.62, 29.3, 29.2,
29.1, 29.1 (Octyl-CH2), 23.2, 22.5 (isopropylidene CH3), 19.1
(CH3), 14.0 (C–CH3). HRMS calcd for C39H57NO11Na (M+Na)+:
738.3829; found m/z 738.3826.
78%) as white amorphous powder. [
a]
+123 (c 1.0, CHCl3). 1H
D
NMR (CDCl3 + CCl4, 300 MHz) d: 7.94–7.28 (m, 10 H, ArH), 5.95 (d,
1H, J 9.3 Hz, NH), 5.69 (t, 1H, J 9.9 Hz, H-3), 5.45 (t, 1H, J 9.6 Hz, H-
3), 4.98 (d, 1H, J 3.3, H-1), 4.54 (dt, 1H, J 2.7 Hz, H-2), 3.97 (m, 1H,
H-6a), 3.94 (m, 3H, H-5, OCH2), 3.50 (dd, 1H, J 6.6 Hz, 2.7 Hz, H-6b),
2.80 (br s, 1H, OH), 1.86 (s, 3H, NHCOCH3), 1.3 (m, 12H, 6 ꢂ CH2),
0.90 (d, 3H, J 6.6 Hz, CH3). 13C NMR (CDCl3 + CCl4, 75 MHz) d: 169.7

B. Roy et al. / Carbohydrate Research 344 (2009) 2311–2316
2315
4.7. p-Methoxyphenyl 2-O-acetyl-4-O-benzyl-
rhamnopyranoside (13)
a
-
L
-
The solvents were evaporated in vacuo and co-evaporated with tol-
uene. The crude product was purified by flash chromatography
using n-hexane–EtOAc (2:1) to afford pure disaccharide diol 15
To a solution of p-methoxyphenyl 4-O-benzyl-
a
-L-rhamnopyr-
(1.0 g, 95%) as a colourless foam. [a]
+82 (c 1.1, CHCl3). 1H NMR
D
anoside 12 (2.5 g, 6.9 mmol) in dry CH3CN (25 mL), trimethyl ort-
(CDCl3, 300 MHz) d: 7.42–7.28 (m, 15H, ArH), 6.97, 6.82 (2d, J
7.8 Hz, C6H4OMe), 5.55 (dd, 1H, J 1.5 Hz, J 1.8 Hz, H-2), 5.34 (d,
1H, J 1.5 Hz, H-1), 5.20 (d, 1H, J 3.3 Hz, H-10), 5.05–4.70 (6d, 6H, J
11.4 Hz, 3 ꢂ CH2C6H5), 4.33 (dd, 1H, J 3.3 Hz, 8.4 Hz), 3.91 (dd,
1H, J 1.5 Hz, 9.0 Hz), 3.87 (m, 2H), 3.78 (s, 3H, C6H4OCH3), 3.68–
3.61 (m, 4H), 3.55 (dd, 1H, J 3.3 Hz, 9.6 Hz), 2.10 (br s, 1H, OH),
1.99 (s, 3H, COCH3), 1.70 (br s, 1H, OH), 1.34 (d, 3H, J 6.3 Hz, C–
CH3). 13C NMR (CDCl3, 75 MHz) d: 170.5 (COCH3), 155.1, 150.0,
138.6, 138.0, 137.7, 128.5 (2), 128.4 (2), 128.3, 128.2 (2), 127.9
(2), 127.8 (2), 127.6 (2), 117.8 (2), 114.6 (2), 96.6 (C-10), 93.0 (C-
1), 96.6, 93.0, 81.0, 79.4, 79.2, 75.7, 75.0, 72.8, 72.2, 71.0, 70.3,
68.5, 67.8, 62.0 (C-60), 55.6 (C6H4OCH3), 20.7 (COCH3), 17.9 (C–
CH3). HRMS calcd for C42H48O12Na (M+Na)+: 767.3043; found m/z
767.3040.
hoacetate (900 L, 10.4 mmol) was added followed by CSA
l
(20 mg) and the mixture was allowed to stir at rt until a complete
conversion of the starting material was evident by TLC (5:1 n-hex-
ane–EtOAc). After 45 min, the solution was neutralized with Et3N
and was evaporated in vacuo. The resulting syrupy mass was dis-
solved in CH2Cl2 (30 mL) and washed successively with 1 N HCl
(3 ꢂ 50 mL) to rearrange the orthoester to the corresponding 2-
O-acetate, followed by aq NaHCO3 (2 ꢂ 50 mL) and brine (50 mL).
The organic layer was separated, dried (Na2SO4) and evaporated
to a syrup. The crude product was purified by flash chromatogra-
phy (4:1 n-hexane–EtOAc) to give pure monosaccharide acceptor
13 (2.3 g, 86%) as a colourless liquid. [a]
+77 (c 1.2, CHCl3). 1H
D
NMR (CDCl3 + CCl4, 300 MHz) d: 7.3–7.2 (m, 5H, C6H5), 6.97, 6.81
(2d, 4H, ArH), 5.32 (s,1H, H-2), 5.27 (d, 1H, J 1.8 Hz, H-1), 4.89,
4.75 (2d, J 11.4 Hz, OCH2C6H5), 4.31 (dd, 1H, J 3.6, Hz, H-3), 3.91
(m, 1H, H-5), 3.43 (t, 1H, J 9.3 Hz, H-4), 2.2 (s, 3H, COCH3), 1.32
(d, 3H, J 6.3 Hz, CH3). 13C NMR (CDCl3 + CCl4, 75 MHz) d: 170.4
(COCH3), 154.9, 150.0, 138.3, 128.3 (2), 127.7 (2), 117.6 (2), 114.5
(2) (ArC), 96.3 (C-1), 81.3, 75.0, 72.0, 69.91, 68.0, 55.3. 20.9
(COCH3), 17.9 (CH3). HRMS calcd for C23H30O7Na (M+Na)+:
441.1889; found m/z 441.1885.
4.10. Octyl 2,3-di-O-benzyl-4,6-O-isopropylidene-
glucopyranosyl-(1?3)-2-O-acetyl-4-O-benzyl-
rhamnopyranosyl-(1?3)-2-acetamido-2-deoxy-6-O-(2,3-di-O-
a-D-
a-L-
benzyl-4,6-O-isopropylidene-a-D-glucopyranosyl)-a-D-
glucopyranoside (19)
To a solution of disaccharide 15 (1.0 g, 1.3 mmol) in CH3CN–H2O
(9:1, 20 mL) was added CAN (1.5 g, 2.7 mmol) and the mixture was
stirred at rt for 45 min. The solvents were evaporated off and the res-
idue was dissolved in CH2Cl2 (30 mL) and was washed with H2O
(2 ꢂ 50 mL); the organic layer was separated, dried (Na2SO4) and
was concentrated to a syrup. The syrupy 16 (850 mg, 1.3 mmol) thus
4.8. p-Methoxyphenyl 2,3-di-O-benzyl-4,6-O-isopropylidene-
-glucopyranosyl-(1?3)-2-O-acetyl-4-O-benzyl-
rhamnopyranoside (14)
a-
D
a-L-
A mixture of acceptor 13 (1.5 g, 3.7 mmol), donor 4 (2.8 g,
5.6 mmol) and activated MS 4 Å (2 g) in dry CH2Cl2 (25 mL) was
stirred under nitrogen atmosphere for 1 h. After cooling in a meth-
anol bath at ꢀ80 °C, NIS (1.6 g, 7.3 mmol) was added followed by
H2SO4-silica (40 mg) and the mixture was stirred for 12 h when
TLC (n-hexane–EtOAc, 3:1) showed a complete conversion of the
acceptor. The mixture was allowed to warm up to rt, was filtered
through CeliteÒ and the filtrate was diluted with CH2Cl2 (30 mL)
and was washed successively with aq Na2S2O3 (2 ꢂ 50 mL), aq
NaHCO3 (2 ꢂ 50 mL) and brine (50 mL). The organic layer was sep-
arated, dried (Na2SO4) and was concentrated to a syrup. The crude
product was purified by flash chromatography using n-hexane–
EtOAc (5:1) to afford pure disaccharide 14 (1.2 g, 65%) as a colour-
obtained was dissolved in dry acetone (10 mL) and DMP (320 lL,
2.6 mmol) was added followed by CSA (15 mg) and the mixture
was allowed to stir at rt for 1 h when the solution became clear
and TLC (n-hexane–EtOAc; 1:1) showed a complete conversion of
the starting material to a faster moving compound. The solution
was neutralized with Et3N and the solvents were evaporated off.
Compound 17 thus obtained as a semi-solid mass was re-dissolved
in dry CH2Cl2 (20 mL), CCl3CN (640
lL, 6.60 mmol) was added fol-
lowed by DBU (220 L, 1.5 mmol) and the mixture was stirred at rt
l
for 1 h. The solvents were then evaporated off and the residue was
charged directly to a flash column and was eluted with n-hexane–
EtOAc (2:1) to afford pure trichloroacetimidate donor 18 (910 mg,
85%). Next, a mixture of 18 (910 mg, 1.1 mmol), acceptor 9
(950 mg, 1.3 mmol) and MS 4 Å (1 g) in dry CH2Cl2 (15 mL) was stir-
red under nitrogen atmosphere for 1 h. H2SO4-silica (15 mg) was
added and the mixture was stirred at rt for 3 h. The mixture, after
neutralization withEt3N, wasfiltered throughCeliteÒ andthefiltrate
was concentrated and the residue was purified by flash chromatog-
raphy (2:1 n-hexane–EtOAc) to afford pure tetrasaccharide 19 (1.0 g,
less foam. [a]
D +112 (c 1.1, CHCl3). 1H NMR (CDCl3 + CCl4, 500 MHz)
d: 7.36–7.25 (m, 15H, ArH), 6.94, 6.80 (2d, 4H, J 7.8 Hz, C6H4OMe),
5.49 (dd, 1H, J 1.5 Hz, 1.8 Hz, H-2), 5.31 (d, 1H, J 1.5 Hz, H-1), 5.10
(d, 1H, J 3.3 Hz, H-10), 5.02–4.71 (6d, 6H, J 11.4 Hz, 3 ꢂ CH2C6H5),
4.34 (dd, 1H, J 3.3 Hz, 8.4 Hz), 3.98 (dd, 1H, J 5.4 Hz, H-4), 3.82
(m, 2H), 3.75 (s, 3H, C6H4OCH3), 3.69–3.61 (m, 4H), 3.46 (dd, 1H,
J 3.3 Hz, 9.6 Hz), 1.93 (s, 3H, COCH3), 1.47, 1.35 (2s, 6H, 2 ꢂ isopro-
pylidene CH3), 1.31 (d, 3H, J 6.3 Hz, C–CH3). 13C NMR (CDCl3 + CCl4,
125 MHz) d: 170.4 (COCH3), 155.1, 150.0, 138.3, 138.0, 137.7,
137.7, 128.4 (2), 128.2, 128.1 (2), 127.8 (2), 127.8 (2), 127.7 (2),
117.8 (2), 114.5 (2), 99.2 (isopropylidene C), 96.6 (C-10), 94.0 (C-
1), 79.5, 78.7, 78.6, 76.1, 75.0, 74.7, 73.6, 72.3, 68.5, 67.6, 63.7,
62.5 (C-60), 55.5 (COCH3), 29.6, 29.0, 20.7, 19.2, 17.8 (C–CH3). HRMS
calcd for C45H52O12Na (M+Na)+: 807.3356; found m/z 807.3353.
78%) as a white foam. [
a
]
D
+91 (c 0.8, CHCl3). 1H NMR (CDCl3,
300 MHz) d: 7.39–7.28 (m, 25H, ArH), 5.67 (d, 1H, J 8.4 Hz, NH),
5.31 (br s, 1H, H-20), 5.07 (br s, 1H, H-10), 4.99 (br s, 2H, H-100, H-
1000), 4.84 (br s, 1H, H-1), 4.79–4.56 (m, 10H, 5 ꢂ CH2C6H5), 2.01 (s,
3H, OCOCH3), 1.90 (s, 3H, NHCOCH3), 1.55, 1.47, 1.46, 1.41 (4s,
12H, 4 ꢂ isopropylidene CH3), 1.86 (m, 2H, O–CH2–CH2–C5H10
–
CH3), 1.33 (m, 10H, O–CH2–CH2–C5H10–CH3), 0.86 (m, 6H, C–CH3,
O–CH2–CH2–C5H10–CH3). 13C NMR (CDCl3, 75 MHz) d: 170.7
(NHCOCH3), 170.2 (COCH3), 138.9, 138.3, 128.4, 128.3, 128.28,
128.2, 127.9, 127.8, 127.6, 127.5, 127.4, 127.3(ArC), 99.2(2)(2 ꢂ iso-
propylidene C), 99.0 (C-1), 98.3 (C-1000), 96.7 (C-100), 93.6 (C-10), 80.3,
79.6, 79.1, 79.0, 78.9, 78.7, 78.7, 76.0, 75.0, 74.9, 74.8, 74.7, 73.5, 73.1,
71.9, 70.1, 68.9, 68.2, 67.9, 67.9, 67.4, 65.9, 63.6, 63.4, 62.6(C-6), 53.8,
31.8, 29.6, 29.3, 29.2, 29.0, 26.1, 23.3, 22.6, 20.7, 19.2, 17.7, 14.0 (C–
CH3). HRMS calcd for C77H101NO21Na (M+Na)+: 1398.6764; found m/
z 1398.6761.
4.9. p-Methoxyphenyl 2,3-di-O-benzyl-a-D-glucopyranosyl-
(1?3)-2-O-acetyl-4-O-benzyl- -rhamnopyranoside (15)
a-L
To a solution of the disaccharide 14 (1.2 g, 1.5 mmol) in AcOH
(16 mL), H2O (4 mL) was added and the suspension was stirred at
80 °C for 3 h when TLC (1:2 n-hexane–EtOAc) showed a complete
conversion of the starting material to a slower running component.

2316
B. Roy et al. / Carbohydrate Research 344 (2009) 2311–2316
4.11. Octyl a-D-glucopyranosyl-(1?3)-2-O-acetyl-a-L-
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To a solution of tetrasaccharide 19 (1.0 g, 0.7 mmol) in AcOH
(16 mL), H2O (4 mL) was added and the suspension was stirred at
80 °C for 3 h when TLC (1:2; n-hexane–EtOAc) showed a complete
conversion of the starting material to a slower running component.
The solvents were evaporated off in vacuo and were co-evaporated
with toluene. To the resulting syrupy mass (740 mg, 0.6 mmol) dis-
solved in CH3OH was added 20% Pd(OH)2/C (500 mg) and the reac-
tion mixture was allowed to stir at rt under a positive pressure of
hydrogen for 24 h. The reaction mixture was filtered through a Cel-
iteÒ bed and the filtrate was concentrated to dryness to afford pure
target tetrasaccharide 1 (432 mg, 85%) as a white foam. [a]D +78 (c
0.7, H2O). 1H NMR (D2O, 400 MHz) d: 5.59 (br s, 1H, H-20), 5.22 (br
s, 2H, H-100, H-1000), 5.09 (br s, 1H, H-1), 2.09 (s 3H, COCH3), 1.98 (s,
3H, NHCOCH3), 1.84 (m, 2H, O–CH2–CH2–C5H10–CH3), 1.54 (m,
10H, O–CH2–CH2–C5H10–CH3), 1.12 (m, 6H, octyl-CH3, Rha-CH3).
NMR (D2O, 75 MHz) d: 174.0 (COCH3), 173.1 (NHCOCH3), 99.1 (C-
1), 97.7 (C-1000), 96.7 (C-100), 94.5 (C-10), 77.26, 72.8, 72.2, 71.5,
71.0, 70.3, 69.9, 69.6, 69.3, 69.0, 68.1, 60.3 (C-6), 54.0, 31.3, 28.,
28.5, 25.4, 22.1, 22.1, 21.9, 20.3, 16.5 (C–CH3), 13.6 (2 ꢂ C–CH3).
HRMS calcd for C36H63NO21Na (M+Na): 868.3790; found m/z
868.3787.
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Supplementary data
Supplementary data (copies of 1H and 13C NMR spectra of all
new compounds) associated with this article can be found, in the
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