Synthesis of a tetrasaccharide related to the O-antigen from Azospirillum lipoferum SR65

Article abstract of DOI:10.1016/j.carres.2009.11.024

Concise synthesis of a tetrasaccharide repeating unit of the LPS isolated from Azospirillum lipoferum SR65 has been accomplished through suitable protecting group manipulations and stereoselective glycosylation starting from commercially available l-rhamn

Full text of DOI:10.1016/j.carres.2009.11.024

Carbohydrate Research 345 (2010) 432–436
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis of a tetrasaccharide related to the O-antigen from Azospirillum
lipoferum SR65
*
Prashant Ranjan Verma, Balaram Mukhopadhyay
Indian Institute of Science Education and Research-Kolkata (IISER-K), Mohanpur Campus, PO BCKV Campus Main Office, Mohanpur, Nadia, West Bengal, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 October 2009
Received in revised form 11 November 2009
Accepted 19 November 2009
Available online 5 December 2009
Concise synthesis of a tetrasaccharide repeating unit of the LPS isolated from Azospirillum lipoferum SR65
has been accomplished through suitable protecting group manipulations and stereoselective glycosyla-
tion starting from commercially available L-rhamnose and D-glucose. The target oligosaccharide in the
form of its p-methoxyphenyl glycoside is suitable for further glycoconjugate formation via selective
cleavage of the OMP glycoside. Plant growth-promoting bacteria (PGPB) of genus Azospirillum plays
important roles in the growth and development of plants. The interaction between the roots of the plants
and the microbes is governed by the cell surface carbohydrate polymers (CPS, LPS, etc.). The present
synthetic-based study elucidates aspects of plant-microbe interaction and future biofertiliser design.
Ó 2009 Elsevier Ltd. All rights reserved.
Dedicated to Professor Sanjib Bagchi, Indian
Institute of Science Education and Research-
Kolkata
Keywords:
Microbial LPS
Synthesis
Oligosaccharide
H2SO4-silica
Glycoconjugate
The Gram-negative nitrogen-fixing soil bacteria of genus Azo-
spirillum are known as plant growth-promoting bacteria (PGPB)
as they secrete many active substances that play vital roles in plant
growth and development.1 It is clear that these bacteria have a
high adaptation potential and therefore are promising for applica-
tion as biofertilizers. Cell surface carbohydrate polymers such as
EPS, CPS and LPS play important roles for the survival of the bacte-
ria in adverse environmental conditions as well as they regulate
the interaction with the roots of plants.1 Literature reports indicate
that the LPS of the Azospirillum outer membrane are involved in the
formation of bacterial association with the roots of cereals; for
example, mutants defective in LPS synthesis are worse adsorbers
to wheat roots2 and worse colonizers to maize roots3 compared
to their non-defective counterparts. Although the bacteria of the
genus Azospirillum have been used as model to study associative
plant-microbe relationship, only a few strains are studied so far.
To get a better understanding of these plant-microbe interactions,
synthetic studies on the LPSs will be useful. Recently, Fedonenko
et al.4 reported the structure of the LPS isolated from Azospirillum
lipoferum SR65. Herein we report the total synthesis of the tetra-
saccharide repeating unit (Fig. 1, 1) of the LPS in the form of its
p-methoxyphenyl glycoside. The choice of p-methoxyphenyl gly-
coside will enable us to conjugate the synthetic oligosaccharide
with suitable aglycon, when needed.
Synthesis of the tetrasaccharide (1) was started with the syn-
thesis of suitably protected
L-rhamnose and D-glucose synthons
followed by step-wise glycosylation and de-protection. Therefore,
known p-methoxyphenyl 2,3-O-isopropylidene-a-L-rhamnopyr-
anoside (2)5 was benzylated using BnBr in the presence of NaH6
to afford the corresponding benzylated derivative 3 in 87% yield.
Hydrolysis of the isopropylidene acetal using 80% AcOH at 80 °C7
followed by selective benzylation of the 2-OH position8 using
phase transfer catalyst furnished the required acceptor, p-
methoxyphenyl 2,4-di-O-benzyl-
a
-L
-rhamnopyranoside (5) in
OMP
Me
O
HO
O
OH
Me
HO
O
O
OH
Me
O
HO
HO
HO
HO
1
O
O
OH
OH
* Corresponding author. Tel.: +91 9748 261742; fax: +91 33 2587 3031.
E-mail address: sugarnet73@hotmail.com (B. Mukhopadhyay).
Figure 1. Structure of the target tetrasaccharide.
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.11.024

P. R. Verma, B. Mukhopadhyay / Carbohydrate Research 345 (2010) 432–436
433
OMP
OMP
OMP
OMP
Bu4NBr, CH2Cl2,
NaOH(10%)
Me
BnO
Me
HO
Me
BnO
80%AcOH
80 oC
Me
BnO
BnBr,NaH
DMF
O
O
O
O
BnBr
O
HO
O
HO
O
O
OBn
OH
5
4
2
3
STol
STol
STol
STol
Bu4NBr, CH2Cl2,
NaOH(10%)
Me
HO
Me
BnO
80%AcOH
80 ºC
Me
BnO
O
BnBr,NaH
DMF
O
Me
BnO
O
O
BnBr
O
O
HO
RO
O
O
OH
OBn
8
9
R = H
6
7
Ac2O, Py
10
R = Ac
Scheme 1. Synthesis of the monosaccharide acceptor 5 and donor 10.
78% yield over two steps. Subjected to the same benzylation-
hydrolysis of the isopropylidene acetal and selective benzylation
using phase transfer catalyst reaction sequence, known p-tolyl
charide acceptor 14 in 94% yield. Up to this stage, activation of
thioglycosides for the glycosylation reactions was satisfactory.
But when we tried to couple the known p-tolyl 2,3,4,6-tetra-O-
2,3-O-isopropylidene-1-thio-
a-
L-rhamnopyranoside (6) afforded
acetyl-1-thio-b-D-glucopyranoside with the trisaccharide acceptor
the protected derivative 9. Compound 9 upon acetylation using
14, it failed to afford the desired tetrasaccharide. Only the corre-
sponding hemiacetal of the donor and the unreacted trisaccharide
acceptor were recovered. Even the use of TMSOTf instead of H2SO4-
silica failed to produce the desired result. Anticipating the lesser
reactivity of thioglycoside donor, we used the known 2,3,4,6-tet-
Ac2O in pyridine9 furnished the required donor, p-tolyl 3-O-acet-
yl-2,4-di-O-benzyl-1-thio-a-L-rhamnopyranoside (10) in 94% yield
(Scheme 1).
Glycosylation between rhamnosyl acceptor 5 and donor 10 was
accomplished by using N-iodosuccinimide in conjunction with
H2SO4-silica10 to afford the disaccharide 11 in 87% yield. Use of
H2SO4-silica as the activator of NIS was found to be beneficial over
the use of traditional TfOH11 or TMSOTf12 as promoters. Glycosyl-
ation of acceptor 5 and donor 10 using TMSOTf and TfOH in con-
junction with NIS resulted in 79% and 76% yield, respectively.
The disaccharide 11 was reacted with NaOMe in MeOH to afford
the disaccharide acceptor 12 in 89% yield. It was further glycosyl-
ated with rhamnosyl donor 10 following the same glycosylation
strategy as mentioned above to furnish the trisaccharide 13 in
86% yield. NaOMe-catalyzed de-O-acetylation afforded the trisac-
ra-O-acetyl-a-D
-glucopyranosyl trichloroacetimidate13 and it gave
the desired tetrasaccharide 16 in 78% yield upon activation of tri-
chloroacetimidate by H2SO4-silica.14 The protected tetrasaccharide
subjected to catalytic hydrogenation using Pd–C followed by treat-
ment with NaOMe in MeOH afforded the target tetrasaccharide 1
(Scheme 2).
In conclusion, synthesis of the tetrasaccharide repeating unit of
the LPS isolated from A. lipoferum SR65 has been accomplished.
Since the protecting group manipulation strategies and glycosyla-
tion steps were selective and high-yielding, the present synthetic
strategy is capable of reasonably large-scale preparation. The syn-
AcO
AcO
AcO
OMP
O
OMP
Me
BnO
O
AcO
Me
BnO
O
CCl3
NH
O
O
OBn
15
10
NIS
O
Me
BnO
OBn
O
5
10
+
NIS
H2SO4-silica
Me
BnO
H2SO4-silica
O
H2SO4-silica
O
OBn
RO
Me
BnO
O
OBn
11 R = Ac
RO
OBn
NaOMe, MeOH
12
R = H
13
R = Ac
NaOMe, MeOH
14 R = H
OMP
O
OMP
Me
HO
Me
O
BnO
O
O
OH
OBn
Me
O
O
Me
O
O
HO
BnO
i. H2, Pd-C
O
O
OH
OBn
ii. NaOMe, MeOH
Me
Me
HO
BnO
HO
HO
AcO
AcO
AcO
O
O
O
O
OH
1
OBn
HO
16
OH
AcO
Scheme 2. Synthesis of the tetrasaccharide 1.

434
P. R. Verma, B. Mukhopadhyay / Carbohydrate Research 345 (2010) 432–436
thetic tetrasaccharide in the form of its p-methoxyphenyl glycoside
1.3. p-Tolyl 2,4-di-O-benzyl-3-O-acetyl-1-thio-a-L-
will be utilized for further biological experiments in due course.
rhamnopyranoside (10)
To a cold (0 °C) solution of p-tolyl 2,3-O-isopropylidene-1-thio-
-L-rhamnopyranoside (6) (3.0 g, 9.7 mmol) in dry DMF (25 mL),
1. Experimental
a
NaH in mineral oil (700 mg, 14.6 mmol) was added followed by
BnBr (1.5 mL, 12.6 mmol). After complete addition, the mixture
was allowed to stir at room temperature for 2 h when TLC (n-hex-
ane–EtOAc; 3:1) showed complete conversion of the starting mate-
rial to a faster moving spot. Excess NaH was neutralized by the
careful addition of MeOH and the mixture was diluted with H2O
(30 mL). Stirring was continued for another 30 min. Then it was ex-
tracted with Et2O (2 ꢀ 25 mL). The combined Et2O layer was
washed with water (2 ꢀ 25 mL), organic layer was separated, dried
(Na2SO4) and evaporated in vacuo. The crude mixture thus ob-
tained was purified by flash chromatography using n-hexane–
EtOAc (5:1) to afford pure p-tolyl 4-O-benzyl-2,3-O-isopropyli-
1.1. General methods
All reagents and solvents were dried prior to use according to
standard methods.15 Commercial reagents were used without fur-
ther purification unless otherwise stated. Analytical TLC was per-
formed on Silica Gel 60-F254 (Merck or Whatman) with detection
by fluorescence and/or by charring following immersion in a 10%
ethanolic solution of sulfuric acid. An orcinol dip, prepared by
the careful addition of concentrated sulfuric acid (20 cm3) to an
ice-cold solution of 3,5-dihydroxytoluene (360 mg) in EtOH
(150 cm3) and water (10 cm3), was used to detect deprotected
compounds by charring. Flash chromatography was performed
with silica gel 230–400 mesh (Qualigens, India). Optical rotations
were measured at the sodium D-line at ambient temperature with
a Perkin Elmer 141 polarimeter. 1H NMR and 13C NMR spectra were
recorded on a Bruker Avance spectrometer at 500 and 125 MHz,
respectively, using Me4Si or CH3OH as internal standards, as
appropriate.
dene-1-thio-a-L-rhamnopyranoside (7) (3.4 g, 89%) as a colourless
gel. Compound 7 (3.0 g, 7.5 mmol) was then dissolved in 80% aq
AcOH (25 mL) and the solution was stirred at 80 °C for 2 h. The sol-
vents were evaporated in vacuo to afford the diol 8 which was dis-
solved in CH2Cl2 (30 mL). To it Bu4NBr (2.4 g, 7.5 mmol), 10% aq
NaOH (10 mL) and BnBr (1.1 mL, 9.0 mmol) were added and the
mixture was stirred at room temperature for 36 h when TLC
showed complete conversion of the starting diol to a faster moving
spot. The mixture was diluted with CH2Cl2 (10 mL) and washed
with H2O (3 ꢀ 30 mL). Organic layer was separated, dried and
evaporated in vacuo. The crude material thus obtained was puri-
fied by flash chromatography using n-hexane–EtOAc (3:1) to afford
1.2. p-Methoxyphenyl 2,4-di-O-benzyl-
a-L-rhamnopyranoside (5)
A
solution of p-methoxyphenyl 2,3-O-isopropylidene-a-L-
rhamnopyranoside (2) (3.0 g, 9.7 mmol) in dry DMF (25 mL) was
cooled to 0 °C, NaH in 50% mineral oil (930 mg, 19.4 mmol) was
added followed by BnBr (1.5 mL, 12.6 mmol) and the mixture
was allowed to stir at room temperature for 2 h when TLC (n-hex-
ane–EtOAc; 3:1) showed complete conversion of the starting
material to a faster moving spot. Excess NaH was neutralized by
the careful addition of MeOH and the mixture was diluted with
H2O (30 mL). Stirring was continued for another 30 min. Then it
was extracted with Et2O (2 ꢀ 25 mL). The combined Et2O layer
was washed with water (2 ꢀ 25 mL), organic layer was separated,
dried (Na2SO4) and evaporated in vacuo. The crude mixture thus
obtained was purified by flash chromatography using n-hexane–
EtOAc (5:1) to afford pure p-methoxyphenyl 4-O-benzyl-2,3-O-
p-tolyl 2,4-di-O-benzyl-1-thio-a-L-rhamnopyranoside (9) (2.6 g,
76% over two steps) as a colourless syrup. Compound 9 (2.5 g,
5.5 mmol) was dissolved in dry pyridine (15 mL) followed by
Ac2O (5 mL) and the solution was stirred at room temperature
for 2 h. Solvents were evaporated and co-evaporated with toluene
to remove residual pyridine. The crude material was purified by
flash chromatography using n-hexane–EtOAc (4:1) as an eluent
to give p-tolyl 2,4-di-O-benzyl-3-O-acetyl-1-thio-
a
-
L
-rhamnopyr-
+101 (c 1.0,
anoside (10) (2.6 g, 94%) as a colourless gel. ½a D25
ꢁ
CHCl3). 1H NMR (500 MHz, CDCl3) d: 7.34–7.09 (m, 14H, ArH),
5.39 (d, 1H, J1,2 1.5 Hz, H-1), 5.18 (dd, 1H, J2,3 3.0 Hz, J3,4 8.5 Hz,
H-3), 4.74–4.46 (3d, 4H, J 12.0 Hz, 2 ꢀ CH2Ph), 4.22 (m, 1H, H-5),
4.09 (dd, 1H, J1,2 1.5 Hz, J2,3 3.0 Hz, H-2), 3.69 (t, 1H, J3,4, J4,5
8.5 Hz, H-4), 2.31 (s, 3H, SC6H4CH3), 1.95 (s, 3H, COCH3), 1.34 (d,
3H, J5,6 6.0 Hz, C–CH3). 13C NMR (125 MHz, CDCl3) d: 170.1
(COCH3), 138.2, 137.6, 132.2(2), 130.5, 129.8(2), 128.4(5),
127.9(2), 127.8, 127.7, 127.6(2) (ArC), 85.6 (C-1), 79.2, 74.9, 73.6,
72.4, 72.1, 69.0, 21.1 (S-Ph-CH3), 20.9 (COCH3), 17.9 (C–CH3). HRMS
calcd for C29H32O5SNa (M+Na)+: 515.1868, found: 515.1864.
isopropylidene-a-L-rhamnopyranoside (3) (3.4 g, 87%) as a colour-
less syrup. Compound 3 (3.0 g, 7.5 mmol) was then dissolved in
80% aq AcOH (25 mL) and the solution was stirred at 80 °C for
2 h. The solvents were evaporated in vacuo to afford the diol 4
which was dissolved in CH2Cl2 (30 mL). To it Bu4NBr (2.4 g,
7.5 mmol), 10% aq NaOH (10 mL) and BnBr (1.2 mL, 9.8 mmol)
were added and the mixture was stirred at room temperature
for 36 h when TLC showed complete conversion of the starting
diol to a faster moving spot. The mixture was diluted with CH2Cl2
(10 mL) and washed with H2O (3 ꢀ 30 mL). Organic layer was sep-
arated, dried and evaporated in vacuo. The crude material thus
obtained was purified by flash chromatography using n-hexane–
1.4. p-Methoxyphenyl 3-O-acetyl-2,4-di-O-benzyl-a-L-rhamno-
pyranosyl-(1?3)-2,4-di-O-benzyl- -rhamnopyranoside (11)
a-L
EtOAc (3:1) to afford p-methoxyphenyl 2,4-di-O-benzyl-a-L-
To a solution of acceptor 5 (1.2 g, 2.7 mmol) and0donor 10 (1.7 g,
3.5 mmol) in dry CH2Cl2 (25 mL) was added MS 4 AÅ (2.0 g) and the
mixture was stirred under nitrogen for 30 min. Then NIS (1.0 g,
4.6 mmol) was added followed by H2SO4-silica (50 mg) and the
mixture was allowed to stir at room temperature for 45 min when
TLC (n-hexane–EtOAc; 3:1) showed complete consumption of the
acceptor. The mixture was filtered through a pad of Celite, washed
with CH2Cl2 (10 mL) and the combined filtrate was washed succes-
sively with Na2S2O3 (2 ꢀ 30 mL), saturated NaHCO3 (2 ꢀ 30 mL)
and brine (30 mL). The organic layer was separated, dried (Na2SO4)
and evaporated in vacuo. The crude material thus obtained was
purified by flash chromatography using n-hexane–EtOAc (4:1) as
an eluent to afford pure disaccharide 11 (1.9 g, 87%) as white foam.
rhamnopyranoside (5) (2.6 g, 78% over two steps) as a colourless
oil. ½a 2D5
ꢁ
+112 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) d: 7.38–
7.25 (m, 10H, ArH), 6.92, 6.81 (2d, 4H, O–C6H4–OCH3), 5.41 (d,
1H, J1,2 1.5 Hz, H-1), 4.90, 4.79, 4.68, 4.65 (4d, AB system, 4H, J
11.0 Hz, 2 ꢀ CH2Ph), 4.13 (dt, 1H, J2,3 4.0 Hz, J3,4 9.0 Hz, J3,OH
9.0 Hz, H-3), 3.91 (dd, 1H, J1,2 1.5 Hz, J2,3 4.0 Hz, H-2), 3.80 (m,
1H, H-5), 3.77 (s, 3H, C6H4OCH3), 3.40 (t, 1H, J3,4, J4,5 9.0 Hz, H-
4), 2.34 (d, 1H, J3,OH 9.0 Hz, OH), 1.29 (d, 3H, J5,6 6.0 Hz, C–CH3).
13C NMR (125 MHz, CDCl3) d: 154.9, 150.4, 138.5, 137.6,
128.7(2), 128.4(2), 128.2, 128.0(2), 127.9(2), 127.8, 117.6(2),
114.6(2) (ArC), 96.1 (C-1), 82.2, 78.5, 75.1, 73.2, 71.5, 67.9, 55.7
(OC6H4OCH3), 18.1 (C–CH3). HRMS calcd for C27H30O6Na
(M+Na)+: 473.1940, found: 473.1936.
½
a 2D5 +128 (c 1.1, CHCl3). 1H NMR (400 MHz, CDCl3) d: 7.43–7.14 (m,
ꢁ

P. R. Verma, B. Mukhopadhyay / Carbohydrate Research 345 (2010) 432–436
435
20H, ArH), 6.93, 6.80 (2d, 4H, C6H4OCH3), 5.37 (s, 1H, H-1), 5.33
1.7. p-Methoxyphenyl 2,4-di-O-benzyl-
(1?3)-2,4-di-O-benzyl- -rhamnopyranosyl-(1?3)-2,4-di-O-
benzyl- -rhamnopyranoside (14)
a-L-rhamnopyranosyl-
(dd, 1H, J2 ,3 2.8 Hz, J3 ,4 9.6 Hz, H-30), 5.20 (s, 1H, H-10), 4.85–
4.53 (m, 6H, 3 ꢀ CH2Ph), 4.32, 4.19 (2d, J 12.4 Hz, 2H, CH2Ph),
4.29 (dd, 1H, J2,3 2.8 Hz, J3,4 9.6 Hz, H-3), 3.90 (bd, 2H, J 2.8 Hz, H-
2, H-20), 3.83 (m, 2H, H-5, H-50), 3.76 (s, 3H, C6H4OCH3), 3.70 (t,
a-L
0
0
0
0
a-L
Compound 13 (1.5 g, 1.3 mmol) was de-O-acetylated by fol-
lowing the same procedure as described above for the prepara-
tion of compound 12 to afford the trisaccharide acceptor 14
1H, J3,4, J4,5 9.6 Hz, H-4), 3.62 (t, 1H, J3 ,4 , J4 ,5 9.6 Hz, H-40), 1.95
(s, 3H, COCH3), 1.29, 1.27 (2d, 3H, J 6.0 Hz, 2 ꢀ C–CH3). 13C NMR
(100 MHz, CDCl3) d: 170.2 (COCH3), 154.8, 150.3, 138.5, 138.4,
137.9, 137.8, 128.5(2), 128.4(2), 128.3(2), 128.2(2), 127.8(2),
127.7(2), 127.6(2), 127.5(2), 127.4(2), 127.0(2), 117.5(2), 114.5(2)
(ArC), 99.6 (C-10), 96.7 (C-1), 80.7, 79.0, 77.9, 77.6, 74.7, 74.6,
73.5, 72.9, 72.6, 68.9, 68.4, 55.6 (C6H4OCH3), 21.1 (COCH3), 18.1,
17.9 (2 ꢀ C–CH3). HRMS calcd for C49H54O11Na (M+Na)+:
841.3564, found: 841.3562.
0
0
0
0
(1.4 g, 94%) as white foam. ½a D25
ꢁ
+78 (c 1.1, CHCl3). 1H NMR
(500 MHz, CDCl3) d: 7.37–7.04 (m, 30H, ArH), 6.86, 6.71 (2d,
4H, C6H4OCH3), 5.32 (s, 1H, H-1), 5.14 (s, 1H, H-10), 5.10 (s,
1H, H-100), 4.79–4.51 (m, 8H, 4 ꢀ CH2Ph), 4.34 (s, 2H, CH2Ph),
4.22, 4.13 (2d, J 12.4 Hz, 2H, CH2Ph), 4.21 (dd, 1H, J2,3 2.4 Hz,
J3,4 9.6 Hz, H-3), 4.01 (dd, 1H, J2 ,3 2.0 Hz, J3 ,4 9.6 Hz, H-30),
3.89 (m, 3H, H-2, H-200, H-300), 3.80–3.72 (m, 4H, H-20, H-5, H-
50, H-500), 3.66 (s, 3H, C6H4OCH3), 3.59 (m, 2H, H-4, H-40), 3.22
0
0
0
0
(t, 1H, J3 ,4 , J4 ,5 9.0 Hz, H-400), 2.32 (br s, 1H, OH), 1.18, 1.16,
1.11 (3d, 9H, J 6.0 Hz, 3 ꢀ C–CH3). 13C NMR (125 MHz, CDCl3)
d: 154.9, 150.4, 138.8, 138.7, 138.3, 138.1, 138.0, 137.8,
128.5(3), 128.4(3), 128.3(3), 128.3(3), 128.2(3), 127.8(3),
127.6(4), 127.5(4), 127.4, 127.2(2), 126.7, 117.6(2), 114.6(2)
(ArC), 99.8 (C-10), 99.0 (C-100), 96.6 (C-1), 82.2, 81.0, 80.6, 79.2,
78.7, 78.2, 74.9, 74.7, 74.6, 72.9, 72.5, 72.4, 71.6, 68.1(2), 67.8,
55.7 (C6H4OCH3), 18.2, 18.1, 18.0 (3 ꢀ C–CH3). HRMS calcd for
C67H74O14Na (M+Na)+: 1125.4976, found: 1125.4973.
00 00
00 00
1.5. p-Methoxyphenyl 2,4-di-O-benzyl-a-L-rhamnopyranosyl-
(1?3)-2,4-di-O-benzyl- -rhamnopyranoside (12)
a
-L
To a solution of compound 11 (1.8 g, 2.2 mmol) in dry MeOH
(10 mL) was added NaOMe in MeOH (0.5 M, 1 mL) and the solution
was allowed to stir at room temperature for 2 h. Then the solution
was neutralized with DOWEX 50W H+ resin and filtered. The sol-
vents were evaporated in vacuo and the residue was purified by
flash chromatography using n-hexane–EtOAc (3:1) to afford pure
compound 12 (1.5 g, 89%) as white foam. ½a D25
ꢁ
+112 (c 1.0, CHCl3).
1H NMR (400 MHz, CDCl3) d: 7.40–7.15 (m, 20H, ArH), 6.94, 6.81
(2d, 4H, C6H4OCH3), 5.37 (s, 1H, H-1), 5.26 (s, 1H, H-10), 4.93 (d,
1H, J 11.2 Hz, CH2Ph), 4.83–4.73 (m, 4H, 2 ꢀ CH2Ph), 4.65 (d, 1H, J
11.2 Hz, CH2Ph), 4.35, 4.13 (2d, J 11.6 Hz, 2H, CH2Ph), 4.31 (dd,
1H, J2,3 2.5 Hz, J3,4 9.6 Hz, H-3), 4.02 (m, 1H, H-30), 3.89 (br s, 1H,
H-2), 3.86 (m, 2H, H-5, H-50), 3.77 (s, 3H, C6H4OCH3), 3.75 (br s,
1.8. p-Methoxyphenyl 2,3,4,6-tetra-O-acetyl-b-
glucopyranosyl-(1?3)-2,4-di-O-benzyl- -rhamnopyranosyl-
(1?3)-2,4-di-O-benzyl- -rhamnopyranosyl-(1?3)-2,4-di-O-
benzyl- -rhamnopyranoside (16)
D-
a-L
a-L
a-L
A mixture of compound 14 (1.0 g, 0.9 mmol), compound 15
0
1H, H-20), 3.69 (t, 1H, J3,4, J4,5 9.6 Hz, H-4), 3.62 (t, 1H, J3 ,4 , J4 ,5
(890 mg, 1.8 mmol) and MS 4 ÅA (1.0 g) in dry CH2Cl2 (15 mL)
was stirred under nitrogen atmosphere for 30 min. H2SO4-silica
(20 mg) was added and the mixture was allowed to stir at room
temperature for 45 min when the TLC (n-hexane–EtOAc; 3:1)
showed complete conversion of the starting material. The mix-
ture was filtered through a pad of Celite and washed with
CH2Cl2 (10 mL). The combined filtrate was washed successively
with aq satd NaHCO3 (2 ꢀ 20 mL) and brine (20 mL). The organic
layer was separated, dried (Na2SO4), filtered through cotton plug
and evaporated in vacuo. The crude mixture thus obtained was
purified by flash chromatography using n-hexane–EtOAc (3:1)
0
0
0
0
9.3 Hz, H-40), 2.30 (d, 1H, J 9.2 Hz, OH), 1.30, 1.27 (2d, 3H, J
6.0 Hz, 2 ꢀ C–CH3). 13C NMR (100 MHz, CDCl3) d: 154.8, 150.3,
138.7, 138.4, 137.8, 137.7, 128.5(2), 128.4(2), 128.3(2), 128.2(2),
127.7(8), 127.6(2), 126.8(2), 117.6(2), 114.6(2) (ArC), 98.8 (C-10),
96.7 (C-1), 82.2, 80.9, 79.1, 77.7, 74.8, 74.7, 72.9, 72.5, 71.6, 69.0,
67.9, 55.6 (C6H4OCH3), 18.1, 18.0 (2 ꢀ C–CH3). HRMS calcd for
C47H52O10Na (M+Na)+: 799.3458, found: 799.3455.
1.6. p-Methoxyphenyl 3-O-acetyl-2,4-di-O-benzyl-
pyranosyl-(1?3)-2,4-di-O-benzyl- -rhamnopyranosyl-(1?3)-
2,4-di-O-benzyl- -rhamnopyranoside (13)
a-L-rhamno-
a-L
a
-L
as an eluent to afford pure tetrasaccharide 16 (1.0 g, 78%). ½a D25
ꢁ
+118 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) d: 7.40–7.21 (m,
30H, ArH), 6.93, 6.80 (2d, 4H, C6H4OCH3), 5.37 (d, 1H, J1,2
Glycosylation between disaccharide acceptor 12 (1.4 g,
1.8 mmol) and donor 10 (1.1 g, 2.3 mmol) was accomplished by
following the same reaction protocol as described above for the
preparation of disaccharide 11. After purification, the trisaccharide
1.5 Hz, H-1), 5.22 (br s, 1H, H-10), 5.14 (t, 1H, J3
, J4
000,4000
000,5000
9.5 Hz, H-4000), 5.10 (dd, 1H, J1
7.5 Hz, J2
9.5 Hz, H-2000),
000,2000
000,3000
5.07 (br s, 1H, H-100), 5.03 (t, 1H, J2 , J3
9.5 Hz, H-3000),
000,3000
000,4000
13 (1.8 g, 86%) was obtained as a colourless gel. ½a D25
ꢁ
+83 (c 1.0,
4.83 (d, 1H, J1
7.5 Hz, H-1000), 4.84–4.70 (m, 6H, 3ꢀCH2Ph),
000,2000
CHCl3). 1H NMR (400 MHz, CDCl3) d: 7.44–7.11 (m, 30H, ArH),
6.93, 6.80 (2d, 4H, C6H4OCH3), 5.38 (s, 1H, H-1), 5.28 (m, 1H, H-
300), 5.22 (s, 1H, H-10), 5.14 (s, 1H, H-100), 4.80–4.57 (m, 8H,
4 ꢀ CH2Ph), 4.44 (s, 2H, CH2Ph), 4.31, 4.15 (2d, J 12.4 Hz, 2H,
CH2Ph), 4.27 (dd, 1H, J2,3 2.4 Hz, J3,4 9.6 Hz, H-3), 4.21 (dd, 1H,
4.65, 4.57 (2d, 2H, J 11.5 Hz, CH2Ph), 4.52 (m, 2H, CH2Ph),
4.39–4. 31 (m, 3H, H-6a000, CH2Ph), 4.26 (dd, 1H, J2,3 3.5 Hz, J3,4
10.0 Hz, H-3), 4.15 (dd, 1H, J2 ,3 3.0 Hz, J3 ,4 9.0 Hz, H-30), 4.11–
4.07 (m, 2H, H-300, H-6b000), 3.90–3.78 (m, 6H, H-2, H-20, H-200,
H-5, H-50, H-500), 3.76 (s, 3H, C6H4OCH3), 3.66 (t, 1H, J3,4, J4,5
9.5 Hz, H-4), 3.55 (t, 2H, J 9.5 Hz, H-40, H-400), 3.47 (m, 1H, H-
5000), 2.01, 1.99, 1.83, 1.81 (4s, 12H, 4 ꢀ COCH3), 1.26, 1.23, 1.17
(3d, 9H, J 6.0 Hz, 3 ꢀ C–CH3). 13C NMR (125 MHz, CDCl3) d:
170.4, 170.3, 169.4, 169.3 (4 ꢀ COCH3), 154.9, 150.3, 138.6,
138.5, 138.4, 138.3, 138.1, 137.9, 128.6(2), 128.5(5), 128.4(3),
128.3, 128.2(3), 127.9(3), 127.8(3), 127.7(3), 127.6, 127.5(2),
127.4(2), 127.1, 126.9, 117.6(2), 114.6(2) (ArC), 101.4 (C-1000),
100.4 (C-10), 99.4 (C-100), 96.6 (C-1), 80.7, 80.6, 80.2, 80.0, 79.0,
78.7, 78.2, 78.0, 77.6, 77.3, 74.9, 74.8, 73.4, 73.1, 72.9, 72.2,
71.8(2), 69.2, 68.9, 68.7, 68.5, 61.7, 55.7 (C6H4OCH3), 20.6(2),
20.5, 20.4 (4 ꢀ COCH3), 18.1, 18.0, 17.9 (3 ꢀ C–CH3). HRMS calcd
for C81H92O23Na (M+Na)+: 1455.5927, found: 1455.5923.
0
0
0
0
J2 ,3 2.0 Hz, J3 ,4 9.6 Hz, H-30), 3.92 (br s, 2H, H-2, H-200), 3.83–3.78
0
0
0
0
(m, 4H, H-20, H-5, H-50, H-500), 3.76 (s, 3H, C6H4OCH3), 3.65 (t, 1H,
J3,4, J4,5 9.6 Hz, H-4), 3.62 (t, 1H, J3 ,4 , J4 ,5 9.2 Hz, H-40), 3.60 (m,
1H, H-400), 2.01 (s, 3H, COCH3), 1.26, 1.21, 1.17 (3d, 9H, J 6.0 Hz,
3 ꢀ C–CH3). 13C NMR (100 MHz, CDCl3) d: 170.1 (COCH3), 154.8,
150.3, 138.8, 138.5, 138.2, 138.0, 137.9, 137.8, 128.5(3), 128.4(3),
128.3(4), 128.2, 127.7(3), 127.6(2), 127.5(2), 127.4(3), 127.3(3),
127.1(3), 126.8(3), 117.5(2), 114.5(2) (ArC), 99.6(2) (C-10, C-100),
96.5 (C-1), 80.6, 80.5, 79.0, 78.5, 78.3, 78.0, 74.9, 74.6, 73.5, 72.8,
72.6, 72.4, 68.8, 68.4, 55.6 (C6H4OCH3), 21.0 (COCH3), 18.1, 17.9,
17.8 (3 ꢀ C–CH3). HRMS calcd for C69H76O15Na (M+Na)+:
1167.5082, found: 1167.5080.
0
0
0
0

436
P. R. Verma, B. Mukhopadhyay / Carbohydrate Research 345 (2010) 432–436
1.9. p-Methoxyphenyl b-
D
-glucopyranosyl-(1?3)-
a-L
-rhamno-
DST, New Delhi, India through SERC Fast-Track Grant SR/FTP/CS-
110/2005.
pyranosyl-(1?3)-
a-L-rhamnopyranosyl-(1?3)-a-L-
rhamnopyranoside (1)
References
A dilute solution of compound 16 (800 mg, 0.6 mmol) in MeOH
(80 mL) was passed through the flow hydrogenation assembly fit-
ted with a Pd–C cartridge at room temperature and 2 atm pressure
of hydrogen. After completion, as evident from TLC (n-hexane–
EtOAc; 1:1), the solvents were evaporated in vacuo and the residue
was re-dissolved in dry MeOH (10 mL). NaOMe (1 mL, 0.5 M in
MeOH) was added and the solution was stirred overnight at room
temperature. It was neutralized with DOWEX 50W H+ resin, fil-
tered and evaporated in vacuo to afford the pure target tetrasac-
charide 1 (330 mg, 82% over two steps) as amorphous white
1. Steenhoudt, O.; Vanderleyden, J. FEMS Microbiol. Rev. 2000, 24, 487–506.
2. Fedonenko, Y. P.; Egorenkova, I. V.; Konnova, S. A.; Ignatov, V. V. Microbiology
2001, 70, 329–334.
3. Jofre, E.; Lagares, A.; Mori, G. FEMS Microbiol. Lett. 2004, 231, 267–275.
4. Fedonenko, Y. P.; Zdorovenko, E. L.; Konnova, S. A.; Kachala, V. V.; Ignatov, V. V.
Carbohydr. Res. 2008, 343, 2841–2844.
5. (a) Werz, D. B.; Seeberger, P. H. Angew. Chem., Int. Ed. 2005, 44, 6315–6318; (b)
Sarkar, K.; Mukherjee, I.; Roy, N. J. Carbohydr. Chem. 2003, 22, 95–108.
6. Thollas, B.; Biosset, C. Synlett 2007, 1736–1738.
7. Mukhopadhyay, B.; Field, R. A. Carbohydr. Res. 2006, 341, 1697–1701.
8. (a) Garegg, P. J. Acta Chem. Scand. 1963, 17, 1343–1348; (b) Garegg, P. J.;
Iversen, T.; Oscarson, S. Carbohydr. Res. 1976, 50, C12.
9. Hudson, C. S.; Dale, J. K. J. Am. Chem. Soc. 1915, 37, 1264–1270.
10. 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 concentrated H2SO4
(1 mL) and the slurry 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 used for the reactions. For the use of H2SO4-silica in various
carbohydrate reactions see: (a) Rajput, V. K.; Roy, B.; Mukhopadhyay, B.
Tetrahedron Lett. 2006, 47, 6987–6991; (b) Rajput, V. K.; Mukhopadhyay, B.
Tetrahedron Lett. 2006, 47, 5939–5941; (c) Mukhopadhyay, B. Tetrahedron Lett.
2006, 47, 4337–4341; (d) Roy, B.; Mukhopadhyay, B. Tetrahedron Lett. 2007, 48,
3783–3787; (e) Dasgupta, S.; Pramanik, K.; Mukhopadhyay, B. Tetrahedron
2007, 63, 12310–12316; (f) Mandal, S.; Mukhopadhyay, B. Tetrahedron 2007,
63, 11363–11370; (g) Dasgupta, S.; Mukhopadhyay, B. Eur. J. Org. Chem. 2008,
34, 5770–5777; (h) Roy, B.; Pramanik, K.; Mukhopadhyay, B. Glycoconjugate J.
2008, 25, 157–166.
powder. ½a 2D5
ꢁ
+62 (c 0.8, H2O). 1H NMR (500 MHz, D2O) d: 7.02,
6.90 (2d, 4H, J 8.5 Hz, C6H4OCH3), 5.33 (s, 1H, H-1), 4.99 (s, 1H,
H-10), 4.98 (s, 1H, H-100), 4.62 (d, 1H, J1
8.0 Hz, H-1000), 4.24 (br
000,2000
s, 1H, H-2), 4.15 (br s, 1H, H-20), 4.09 (br s, 1H, H-200), 3.93 (m,
2H, H-3, H-30), 3.85–3.78 (m, 5H, H-4, H-40, H-400, H-5000, H-6a000),
000,6b000
000,6b000
5.0 Hz, J6a
3.72 (s, 3H, C6H4OCH3), 3.64 (dd, 1H, J5
12.0 Hz, H-6b000), 3.56–3.51 (m, 2H, H-5, H-50), 3.48 (t, 1H, J3
,
000,4000
9.0 Hz, H-4000), 3.42 (t, 1H, J2 , J3
9.0 Hz, H-3000), 3.37 (m,
9.0 Hz, H-2000), 1.22,
000,3000
000,5000
000,3000 000,4000
J4
1H, H-500), 3.31 (dd, 1H, J1
8.0 Hz, J2
000,2000
1.18, 1.15 (3d, 9H, J 6.0 Hz, 3ꢀC-CH3). 13C NMR (125 MHz, D2O)
d: 154.7, 149.3, 118.8(2), 115.1(2) (ArC), 103.7 (C-1000), 102.3 (C-
10), 102.1 (C-100), 99.0 (C-1), 79.9, 78.3, 77.9, 75.7, 75.5, 73.3,
71.3(2), 71.0, 69.9, 69.8, 69.7, 69.6, 69.5, 69.3, 69.0, 60.5, 55.7
(C6H4OCH3), 16.6, 16.5, 16.4 (3 ꢀ C–CH3). HRMS calcd for
C31H48O19Na (M+Na)+: 747.2688, found: 747.2686.
11. Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H. Tetrahedron Lett. 1990, 31,
1331–1334.
12. Terada, T.; Ishida, H.; Kiso, M.; Hasegawa, A. J. Carbohydr. Chem. 1995, 14, 751–
768.
13. Kerékgyártó, J.; Szurmai, Z.; Liptak, A. Carbohydr. Res. 1993, 245, 65–80.
14. Rajput, V. K.; Mukhopadhyay, B. J. Org. Chem. 2008, 73, 6924–6927.
15. Perrin, D. D.; Amarego, W. L.; Perrin, D. R. Purification of Laboratory Chemicals;
Pergamon: London, 1996.
Acknowledgements
P. R. V. is thankful to Indian Institute of Science Education and
Research-Kolkata (IISER-K) for fellowship. The work is funded by