Concise synthesis of the repeating units of the cell wall lipopolysaccharide of azospirillum brasilense SR80

Article abstract of DOI:10.1055/s-0033-1341256

Syntheses of trisaccharide and tetrasaccharide repeating units of the cell wall lipopolysaccharide of Azospirillum brasilense SR80 as their 4-methoxyphenyl (PMP) glycosides have been achieved following a sequential glycosylation strategy in good yield. All intermediate steps are high-yielding and the glycosylation steps are highly stereoselective. Georg Thieme Verlag Stuttgart New York.

Full text of DOI:10.1055/s-0033-1341256

PAPER
▌1947
paper
Concise Synthesis of the Repeating Units of the Cell Wall Lipopolysaccharide
of Azospirillum brasilense SR80
Lipopolysaccharide of Azospirillum brasilense SR80
Pintu Kumar Mandal,*a Debashis Dhara,b Anup Kumar Misrab
a
Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram Extension, Sitapur Road,
Lucknow-226 031, India
Fax +91(522)2623405; E-mail: pintuchem06@gmail.com
b
Bose Institute, Division of Molecular Medicine, P-1/12, C.I.T. Scheme VII-M, Kolkata-700054, India
Received: 08.11.2013; Accepted after revision: 31.03.2014
SR80 (Figure 1) presented by Sigida et al.7 have been re-
Abstract: Syntheses of trisaccharide and tetrasaccharide repeating
oriented without changing the sequence of the repeating
units of the cell wall lipopolysaccharide of Azospirillum brasilense
units to bring more complexity into the molecules. Since
the stereochemistry at the glycoside linkages and se-
SR80 as their 4-methoxyphenyl (PMP) glycosides have been
achieved following a sequential glycosylation strategy in good
yield. All intermediate steps are high-yielding and the glycosylation quence of the monosaccharide moieties were not changed,
steps are highly stereoselective.
the re-oriented structures can also be considered as the re-
peating units. In an ongoing program8 towards the synthe-
Key words: Azospirillum brasilense, tetrasaccharide, trisaccharide,
lipopolysaccharide, plant-growth-promoting bacteria
sis of bacterial polysaccharide fragments for their use in
the glycoconjugate preparation, chemical synthesis of a
trisaccharide and a tetrasaccharide repeating units of the
cell wall lipopolysaccharide of Azospirillum brasilense
SR80 in the form of their 4-methoxyphenyl (PMP) glyco-
sides have been carried out. The 4-methoxyphenyl group
acts as a temporary anomeric protecting group during the
synthesis of the oligosaccharide. Oxidative removal of the
4-methoxyphenyl group using ceric ammonium nitrate
can produce an oligosaccharide hemiacetal derivative,
which can be converted into its trichloroacetimidate de-
rivative for conjugation of the oligosaccharide moiety
with various aglycons to produce glycoconjugate deriva-
tives.
Among several plant-growth-promoting rhizobacteria
(PGPR), Azospirilla play a significant role in plant growth
in an asymbiotic manner by colonizing the rhizosphere of
several crop plants.1 Currently, PGPR are used as an alter-
native to chemical fertilizers to promote plant growth.2
Azospirillum species produce a number of surface-located
polysaccharides, such as lipopolysaccharides (LPSs), cap-
sular polysaccharides (CPSs), and exopolysaccharides,
that play a significant role in their interaction with plants.3
One well-studied species in this genus is Azospirillum
brasilense, which showed effectiveness in the biocontrol
of several plant diseases, such as crown gall disease,4 bac-
terial leaf and/or vascular diseases.5 It was also found to
promote disease resistance in rice crops.6 Recently, Sigida
et al. reported the presence of two repeating units in the
cell wall lipopolysaccharide of Azospirillum brasilense
SR80.7 It is not clear whether these two oligosaccharide
repeating units are linked together or not, but the existence
of more than one repeating unit in cell wall polysaccha-
rides of bacteria is common.
β-D-Xylp-(1→4)-[α-D-Rhap-(1→3)]-α-L-Fucp-(1→→3)-β-D-Galp-
(1→3)-β-D-GalNAcp-(1→3)-[α-L-Fucp-(1→2)]-α-D-Galp-(1→
Figure 1 Structure of the tri- and tetrasaccharide repeating units of
the cell wall lipopolysaccharide of Azospirillum brasilense SR80
The target tri- and tetrasaccharides 1 and 2 have been syn-
thesized in the form of their p-methoxyphenyl glycosides
using sequential glycosylation strategies by the stereose-
lective glycosylations of the suitably protected monosac-
charide intermediates 5, 6, 9, 13, 14, 17, and 20, which
were prepared from the reducing sugars following reac-
tion conditions reported in the literature (Scheme 1).
Since lipopolysaccharides are a major cell wall compo-
nent of Azospirillum species, it would be worthwhile to
study the role of lipopolysaccharides in plant–bacterial in-
teractions. However, isolation of significant amounts of
oligosaccharide with adequate purity from natural sources
is inconvenient. Chemical synthesis of the repeating units
of the cell wall lipopolysaccharides could provide the re-
quired quantity of pure oligosaccharides. The structures of
the trisaccharide and tetrasaccharide repeating units of the
cell wall lipopolysaccharide of Azospirillum brasilense
Transformation of L-fucose tetraacetate (3) into 4-meth-
oxyphenyl 2,3,4-tri-O-acetyl-α-L-fucopyranoside (4) was
carried out in 77% yield by treatment with 4-methoxy-
phenol and trimethylsilyl trifluoromethanesulfonate at 0
°C to room temperature. Compound 4 was transformed
into glycosyl acceptor 5 in 72% yield following a se-
quence of reactions consisting of saponification, 3,4-O-
isopropylidene ketal formation using 2,2-dimethoxypro-
pane in the presence of 4-toluenesulfonic acid,9 ben-
zylation of the remaining hydroxyl group using benzyl
SYNTHESIS 2014, 46, 1947–1953
Advanced online publication: 13.05.2014
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
DOI: 10.1055/s-0033-1341256; Art ID: ss-2013-n0733-op
© Georg Thieme Verlag Stuttgart · New York

1948
P. K. Mandal et al.
PAPER
OH
4-toluenesulfonic acid furnished 4-methoxyphenyl 4,6-O-
benzylidene-α-D-galactopyranoside (12) in 78% yield.
Treatment of compound 12 with benzoyl cyanide18 in
the presence of triethylamine in acetonitrile furnished
4-methoxyphenyl 3-O-benzoyl-4,6-O-benzylidene-α-D-
galactopyranoside (13) in 84% yield. Stereoselective con-
densation of compound 13 with thioglycoside donor 1419
in the presence of N-iodosuccinimide and trimethylsilyl
trifluoromethanesulfonate13 in dichloromethane–diethyl
ether (1:1) furnished disaccharide derivative 15 in 81%
yield together with a minor quantity (~5%) of its other iso-
mer, which was separated by column chromatography.
Stereoselective formation of compound 15 was supported
by its spectral analysis [signals at δ = 5.77 (d, J = 3.0 Hz,
H1A), 5.56 (s, PhCH), 4.98 (d, J = 3.5 Hz, H1B) and at δ =
100.5 (PhCH), 100.1 (C1B), 97.3 (C1A) in the 1H and 13C
NMR spectra respectively]. De-O-benzoylation of com-
pound 15 using sodium methoxide furnished disaccharide
acceptor 16 in 91% yield. Glycosylation of compound 16
with D-galactosamine derived thioglycoside 1720 in the
presence of N-iodosuccinimide and trimethylsilyl
trifluoromethanesulfonate13 furnished trisaccharide deriv-
ative 18 in 79% yield.
HO
O
OH
HO
O
OH
HO
OPMP
O
O
O
A
C
A
D
O
HO
OH
O
O
NHAc
OH
HO
HO
C
OPMP
OH
O O
B
O
OH
B
HO
HO
OH
HO
2
O
OH
1
Ph
O
O
SEt
O
OPMP
OBn
OBn
O
OBn
O
OBn
BnO
O
BnO
SPh
BzO
BnO
HO
OH
OPMP
AcO
6
13
14
5
O
BzO
BzO
Ph
SEt
AcO OAc
O
O
OBz
O
9
SEt
AcO
O
OAc
SEt
NPhth
17
AcO
20
Scheme 1 Structure of the synthesized trisaccharide 1 and tetrasac-
charide 2 and their synthetic intermediates
bromide and sodium hydroxide,10 acidic hydrolysis of the
O-isopropylidene ring, and finally selective acetylation of
the 4-hydroxy group via 3,4-orthoester formation11 by the
reaction of triethyl orthoacetate and 4-toluenesulfonic
acid followed by hydrolysis of the orthoester. Stereoselec-
tive glycosylation of compound 5 with D-rhamnosyl
thioglycoside donor 612 promoted by the combination of
N-iodosuccinimide and trimethylsilyl trifluoromethane-
sulfonate13 furnished the required disaccharide derivative
7 in 89% yield. Formation of compound 7 was supported
by its spectral analysis [signals at δ = 5.33 (d, J = 3.5 Hz,
H1A), 5.12 (br s, H1B) and at δ = 96.9 (JC1,H1 = 169.0 Hz,
OAc
OAc
OPMP
OAc
O
a
O
OAc
OAc
AcO
AcO
4
3
b–f
OPMP
A
O
OBn
OPMP
OBn
6
O
O
RO
g
OH
BnO
BnO
B
AcO
O
OBn
5
C1A), 93.6 (JC1,H1 = 168.0 Hz, C1B) in the H and 13C
1
7 R = Ac
8 R = H
h
NMR spectra, respectively]. In compound 7, the JC1,H1
values (169.0 Hz and 168.0 Hz) for the anomeric carbons
confirmed the presence of two α-glycoside linkages.14 De-
O-acetylation of compound 7 using sodium methoxide
gave the disaccharide acceptor 8 in 92% yield, which was
stereoselectively coupled with D-xylosyl thioglycoside
derivative 915 in the presence of N-iodosuccinimide and
trimethylsilyl trifluoromethanesulfonate13 to furnish pro-
tected trisaccharide derivative 10 in 76% yield. The pres-
ence of signals at δ = 5.24 (d, J = 3.5 Hz, H1A), 5.01 (d,
J = 1.5 Hz, H1B), 4.77 (d, J = 7.5 Hz, H1C) in the 1H NMR
and at δ = 100.4 (C1C), 97.0 (C1A), 93.8 (C1B) in the 13C
NMR spectra confirmed its formation. Hydrogenolysis of
trisaccharide derivative 10 over Pearlman’s catalyst16 fol-
lowed by saponification using sodium methoxide fur-
nished trisaccharide 1 as its 4-methoxyphenyl glycoside
in 66% yield (2 steps) (Scheme 2). The presence of three
anomeric protons in the 1H NMR δ = 4.98 (d, J = 3.0 Hz,
H1A), 4.92 (br s, H1B), 4.29 (d, J = 7.5 Hz, H1C) and at
OPMP
OH
i
9
OPMP
OBn
A
O
A
O
O
HO
C
O
O O
HO
BzO
C
O O
BzO
j,k
OH
B
OBz
B
HO
HO
BnO
BnO
O
OH
O
OBn
1
10
Scheme 2 Reagents and conditions: (a) 4-methoxyphenol, TMS-
OTf, CH2Cl2, 0 °C to r.t., 5 h, 77%; (b) 0.1 M NaOMe, MeOH, r.t., 2
h; (c) Me2C(OMe)2, TsOH, DMF, r.t., 6 h; (d) BnBr, NaOH, TBAB,
THF, r.t., 5 h; (e) 80% aq AcOH, 80 °C, 1.5 h; (f) 1. MeC(OEt)3,
TsOH, DMF, r.t., 2 h; 2. H2O, r.t., 30 min, 72% (5 steps); (g) NIS,
TMSOTf, CH2Cl2, 4 Å MS, –25 °C, 1 h, 89%; (h) 0.1 M NaOMe,
MeOH, r.t., 3 h, 92%; (i) NIS, TMSOTf, CH2Cl2, 4 Å MS, –30 °C, 1
h, 76%; (j) H2, 20% Pd(OH)2/C, MeOH, r.t., 24 h; (k) 0.1 M NaOMe,
MeOH, r.t., 10 h, 66% (2 steps).
Formation of compound 18 was supported by its NMR
spectral analysis [signals at δ = 5.87 (d, J = 8.5 Hz, H1C),
5.56 (d, J = 3.0 Hz, H1A), 5.54 (s, PhCH), 5.43 (s, PhCH),
4.76 (d, J = 3.5 Hz, 1 H, H1B) in the 1H NMR and at δ =
100.7 (PhCH), 100.2 (C1B), 100.1 (PhCH), 97.0 (C1A),
96.2 (C1C) in the 13C NMR spectra]. The trisaccharide de-
rivative 18 was de-O-acetylated using sodium methoxide
to furnish trisaccharide acceptor 19 in 93% yield. Finally,
13
δ = 104.5 (C1C), 95.6 (C1B), 95.5 (C1A) in the C NMR
spectra confirmed the formation of compound 1 (Scheme
2). In another experiment, saponification of 4-methoxy-
phenyl 2,3,4,6-tetra-O-acetyl-α-D-galactopyranoside (11)17
using sodium methoxide followed by 4,6-O-benzylidene
acetal formation using benzaldehyde dimethyl acetal and
Synthesis 2014, 46, 1947–1953
© Georg Thieme Verlag Stuttgart · New York

PAPER
Lipopolysaccharide of Azospirillum brasilense SR80
1949
Ph
N-iodosuccinimide and trimethylsilyl trifluoromethane-
sulfonate13 mediated glycosylation of trisaccharide accep-
tor 19 with thioglycoside donor 2021 furnished the
tetrasaccharide derivative 21 in 83% yield. Spectroscopic
analysis of compound 21 confirmed its formation [signals
at δ = 5.71 (d, J = 8.0 Hz, H1C), 5.57 (d, J = 3.0 Hz, H1A),
5.56 (s, PhCH), 5.49 (s, PhCH), 5.04 (br s, H1B), 4.73 (d,
J = 8.0 Hz, H1D) and at δ = 106.9 (C1B), 101.6 (PhCH),
100.2 (2 C, C1D, PhCH), 97.0 (C1A), 96.6 (C1C) in the 1H
and 13C NMR spectra respectively]. Compound 21 was
subjected to a series of reactions involving (a) conversion
of the phthaloyl group to an acetamido group by treatment
with hydrazine hydrate followed by acetylation;22 (b) hy-
drogenolysis over palladium hydroxide on carbon;16 and
(c) de-O-acetylation using sodium methoxide to furnish
compound 2 in 60% yield. Spectroscopic analysis of com-
pound 2 unambiguously confirmed its formation [signals
at δ = 5.25 (d, J = 3.0 Hz, H1A), 4.97 (br s, H1B), 4.95 (d,
J = 7.0 Hz, H1D), 4.85 (d, J = 7.0 Hz, H1C) and at δ =
106.7 (C1C), 101.1 (C1D), 98.9 (C1B), 95.8 (C1A) in the 1H
and 13C NMR spectra, respectively] (Scheme 3).
Ph
O
O
OAc
O
AcO
AcO
O
O
O
14
a,b
O
A
RO
RO
d
O
AcO
OPMP
HO
OPMP
OPMP
12 R = H
11
B
O
OBn
c
13 R = Bz
OBn
BnO
e
15 R = Bz
16 R = H
Ph
Ph
O
O
O
O
17
O
O
O
A
C
20
h
O
RO
f
NPhth
OPMP
B
O
OBn
OBn
BnO
18 R = Ac
19 R = H
g
Ph
Ph
O
O
O
O
OAc
AcO
O
O
O
O
A
C
D
O
O
AcO
NPhth
OAc
OPMP
O
B
OBn
In summary, concise synthetic strategies have been devel-
oped for the synthesis of trisaccharide and tetrasaccharide
(1 and 2) repeating units of the cell wall lipopolysaccha-
ride of Azospirillum brasilense SR80 in the form of their
4-methoxyphenyl glycosides. The yields in the protecting
group manipulation steps and stereo outcome of the gly-
cosylation reactions were excellent.
OBn
BnO
21
OH
HO
O
OH
HO
OH
HO
O
O
O
A
C
D
O
HO
i,j,k
O
NHAc
OH
OPMP
OH
B
O
OH
HO
2
All reactions were monitored by TLC over silica gel coated TLC
plates; the plates were visualized by spraying with ceric sulfate so-
lution [2% Ce(SO4)2 in 5% H2SO4 in EtOH] then warming on a hot
plate. Silica gel 230–400 mesh was used for column chromatogra-
phy. 1H and 13C NMR, DEPT 135, 2D COSY, HSQC spectra were
recorded on Bruker Avance DRX 500 MHz spectrometers using
CDCl3–CCl4 and D2O as solvents and TMS as internal reference un-
less stated otherwise. ESI-MS were recorded on a Jeol spectrome-
ter. Elementary analysis was carried out on Carlo ERBA analyzer.
IR spectra were recorded on Shimadzu spectrophotometers. Optical
rotations were determined on an Autopol III polarimeter. Commer-
cially available grades of organic solvents of adequate purity were
used in all reactions.
Scheme 3 Reagents and conditions: (a) 0.1 M NaOMe, MeOH, r.t.,
3 h; (b) PhCH(OMe)2, TsOH, MeCN, r.t., 5 h, 78% (2 steps); (c) ben-
zoyl cyanide, Et3N, MeCN, r.t., 30 min, 84%; (d) NIS, TMSOTf, 4 Å
MS, CH2Cl2–Et2O (1:1), –25 °C, 30 min, 81%; (e) 0.1 M NaOMe,
MeOH, r.t., 3 h, 91%; (f) NIS, TMSOTf, 4 Å MS, CH2Cl2, –30 °C, 45
min, 79%; (g) 0.1 M NaOMe, MeOH, r.t., 30 min, 93%; (h) NIS,
TMSOTf, 4 Å MS, CH2Cl2, –10 °C, 1 h, 83%; (i) 1. NH2NH2·H2O,
EtOH, 80 °C, 8 h; 2. Ac2O, pyridine, r.t., 1 h; (j) H2, 20% Pd(OH)2/C,
MeOH, r.t., 24 h; (k) 0.1 M NaOMe, MeOH, r.t., 3 h, 60% (4 steps).
13C NMR (125 MHz, CDCl3): δ = 170.3 (COCH3), 170.2 (COCH3),
169.8 (COCH3), 155.2–114.7 (CAr), 95.7 (C1), 71.0 (C4), 67.9 (2 C,
C2, C3), 65.2 (C5), 55.5 (OCH3), 20.7 (COCH3), 20.6 (COCH3),
20.5 (COCH3), 15.9 (CCH3).
4-Methoxyphenyl 2,3,4-Tri-O-acetyl-α-L-fucopyranoside (4)
A solution of L-fucose tetraacetate (3, 5 g, 15.1 mmol), 4-methoxy-
phenol (2.2 g, 18.1 mmol), and 4 Å molecular sieves (2 g) in anhyd
CH2Cl2 (30 mL) was cooled to 0 °C. TMSOTf (2.9 mL, 16.5 mmol)
was added dropwise to the cooled mixture which was stirred at r.t.
for 5 h. The reaction was quenched with Et3N (1 mL) and filtered,
and the residue was washed with CH2Cl2 (50 mL). The filtrate was
concentrated under vacuum to give the crude product, which was
purified by column chromatography (silica gel, hexane–EtOAc,
3:1) to give pure 4 (4.6 g, 77%) as a colorless liquid; [α]D25 +23 (c
1.0, CHCl3).
MS (ESI): m/z = 419.0 [M + Na]+.
Anal. Calcd for C19H24O9 (396.14): C, 57.57; H, 6.10. Found: C,
57.44; H, 6.27.
4-Methoxyphenyl 4-O-Acetyl-2-O-benzyl-α-L-fucopyranoside
(5)
A solution of compound 4 (4 g, 10.1 mmol) in 0.1 M NaOMe in
MeOH (30 mL) was stirred at r.t. for 2 h. The mixture was neutral-
ized with Dowex 50W-X8 (H+) resin, filtered, and evaporated to
dryness. 2,2-Dimethoxypropane (1.9 mL, 15.2 mmol) and TsOH
(150 mg) were added to a solution of the de-O-acetylated product in
anhyd DMF (30 mL) and the mixture was stirred at r.t. for 6 h. To
the reaction mixture were added powdered NaOH (804 mg, 20.1
mmol), BnBr (1.4 mL, 12.0 mmol) and TBAB (100 mg, 0.31 mmol)
and the mixture was stirred at r.t. for 5 h. The mixture was poured
into H2O (200 mL) and extracted with EtOAc (100 mL). The organ-
ic layer was washed with H2O, dried (Na2SO4), and concentrated. A
IR (neat): 2925, 2339, 1750, 1663, 1595, 1440, 1373, 1222, 1048,
760 cm–1.
1H NMR (500 MHz, CDCl3): δ = 6.94 (d, J = 9.0 Hz, 2 H, HAr), 6.78
(d, J = 9.0 Hz, 2 H, HAr), 5.57 (d, J = 3.5 Hz, 1 H, H1), 5.51 (dd,
J = 10.5, 3.0 Hz, 1 H, H3), 5.32 (d, J = 2.5 Hz, 1 H, H4), 5.20 (dd,
J = 11.0, 3.5 Hz, 1 H, H2), 4.30–4.27 (m, 1 H, H5), 3.74 (s, 3 H,
OCH3), 2.17 (s, 3 H, COCH3), 2.05 (s, 3 H, COCH3), 1.99 (s, 3 H,
COCH3), 1.11 (d, J = 6.5 Hz, 3 H, CCH3).
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, 1947–1953

1950
P. K. Mandal et al.
PAPER
solution of the crude product in 80% aq AcOH (80 mL) was stirred
at 80 °C for 1.5 h. The solvents were removed under reduced pres-
sure and co-evaporated with toluene. Triethyl orthoacetate (2.3 mL,
20.0 mmol) and TsOH (100 mg) were added to a solution of the
crude product in anhyd DMF (20 mL), and the mixture was stirred
at r.t. for 2 h. H2O (100 mL) was added to the mixture and it was
stirred at r.t. for 30 min and extracted with EtOAc (100 mL). The
organic layer was dried (Na2SO4) and concentrated. The crude prod-
uct was purified by column chromatography (silica gel, hexane–
EtOAc, 5:1) to give pure 5 (2.9 g, 72% from 4) as a yellow oil;
[α]D25 +44 (c 1.0, CHCl3).
4-Methoxyphenyl (2,3,4-Tri-O-benzyl-α-D-rhamnopyranosyl)-
(1→3)-2-O-benzyl-α-L-fucopyranoside (8)
A solution of compound 7 (1.5 g, 1.8 mmol) in 0.1 M NaOMe in
MeOH (15 mL) was stirred at r.t. for 3 h. The mixture was neutral-
ized with Dowex 50W X8 (H+) resin, filtered, and concentrated to
give the crude product, which was passed through a short pad of sil-
ica gel (hexane–EtOAc, 1:1) to furnish pure 8 (1.3 g, 92%) as a yel-
low oil; [α]D25 –33 (c 1.0, CHCl3).
IR (neat): 3021, 2925, 1749, 1720, 1488, 1385, 1219, 1064, 757,
669 cm–1.
1H NMR (500 MHz, CDCl3): δ = 7.37–7.16 (m, 20 H, HAr), 6.96 (d,
J = 9.0 Hz, 2 H, HAr), 6.79 (d, J = 9.0 Hz, 2 H, HAr), 5.30 (d, J = 3.5
Hz, 1 H, H1A), 4.95 (d, J = 11.0 Hz, 1 H, PhCH2), 4.86 (d, J = 1.5
Hz, 1 H, H1B), 4.81 (d, J = 12.5 Hz, 1 H, PhCH2), 4.70–4.63 (m, 4
H, PhCH2), 4.60 (d, J = 12.0 Hz, 1 H, PhCH2), 4.48 (d, J = 11.5 Hz,
1 H, PhCH2), 4.22 (dd, J = 10.0, 3.0 Hz, 1 H, H3A), 4.06–4.01 (m,
2 H, H5A, H5B), 3.89 (dd, J = 9.0, 3.0 Hz, 1 H, H3B), 3.77 (s, 3 H,
OCH3), 3.74 (dd, J = 10.5, 3.5 Hz, 1 H, H2A), 3.70–3.68 (m, 2 H,
H2B, H4A), 3.62 (d, J = 10.0, 8.4 Hz, 1 H, H4B), 1.28 (d, J = 6.5 Hz,
3 H, CCH3), 1.22 (d, J = 6.5 Hz, 3 H, CCH3).
13C NMR (125 MHz, CDCl3): δ = 155.3–114.5 (CAr), 96.7 (C1A),
94.1 (C1B), 80.5 (C4B), 79.4 (C3B), 75.7 (C2B), 75.1 (PhCH2), 73.7
(C2A), 73.2 (PhCH2), 72.9 (PhCH2), 72.8 (C3A), 72.5 (PhCH2), 68.5
(C5B), 68.3 (C4A), 65.8 (C5A), 55.5 (OCH3), 18.1 (CCH3), 16.3
(CCH3).
IR (neat): 2925, 2339, 1750, 1663, 1595, 1440, 1373, 1222, 1048,
760 cm–1.
1H NMR (500 MHz, CDCl3): δ = 7.32–7.24 (m, 5 H, HAr), 6.96 (d,
J = 9.0 Hz, 2 H, HAr), 6.80 (d, J = 9.0 Hz, 2 H, HAr), 5.36 (d, J = 3.5
Hz, 1 H, H1), 5.27 (d, J = 2.5 Hz, 1 H, H4), 4.69–4.64 (AB q, 2 H,
PhCH2), 4.34–4.31 (m, 1 H, H3), 4.19–4.17 (m, 1 H, H5), 3.79 (dd,
J = 10.0, 3.5 Hz, 1 H, H2), 3.77 (s, 3 H, OCH3), 2.17 (s, 3 H,
COCH3), 1.10 (d, J = 6.5 Hz, 3 H, CCH3).
13C NMR (125 MHz, CDCl3): δ = 170.8 (COCH3), 155.1–114.6
(CAr), 96.4 (C1), 76.5 (C2), 72.8 (C3), 72.7 (PhCH2), 67.9 (C4),
65.5 (C5), 55.5 (OCH3), 20.8 (COCH3), 16.2 (CCH3).
MS (ESI): m/z = 425.0 [M + Na]+.
Anal. Calcd for C22H26O7 (402.17): C, 65.66; H, 6.51. Found: C,
65.50; H, 6.68.
MS (ESI): m/z = 799.1 [M + Na]+.
4-Methoxyphenyl (2,3,4-Tri-O-benzyl-α-D-rhamnopyranosyl)-
(1→3)-4-O-acetyl-2-O-benzyl-α-L-fucopyranoside (7)
Anal. Calcd for C47H52O10 (776.35): C, 72.66; H, 6.75. Found: C,
72.48; H, 6.90.
To a solution of compound 5 (1.5 g, 3.7 mmol) and thioglycoside 6
(2.3 g, 4.4 mmol) in CH2Cl2 (20 mL) was added 4 Å molecular
sieves (2 g) and the mixture was stirred at r.t. under argon for 30
min. The mixture was cooled to –25 °C and NIS (1.2 g, 5.3 mmol)
and TMSOTf (25 μL) were added. The mixture was stirred at this
temperature for 1 h, and then it was filtered through Celite and the
Celite was washed with CH2Cl2 (50 mL). The organic layer was
washed successively with 5% Na2S2O3 (100 mL), sat. NaHCO3
(100 mL), and H2O (100 mL), dried (Na2SO4), and evaporated to
dryness. The crude mass was purified by column chromatography
(silica gel, hexane–EtOAc, 5:1) to furnish pure 7 (2.7 g, 89%) as a
yellow oil; [α]D25 +55 (c 1.0, CHCl3).
4-Methoxyphenyl (2,3,4-Tri-O-benzoyl-β-D-xylopyranosyl)-
(1→4)-[(2,3,4-tri-O-benzyl-α-D-rhamnopyranosyl)-(1→3)]-2-
O-benzyl-α-L-fucopyranoside (10)
To a solution of compound 8 (1 g, 1.3 mmol) and thioglycoside 9
(780 mg, 1.5 mmol) in anhyd CH2Cl2 (20 mL) was added 4 Å mo-
lecular sieves (1 g) and the mixture was stirred at r.t. under argon
for 30 min. The mixture was cooled to –30 °C, and NIS (370 mg,
1.7 mmol) and TMSOTf (10 μL) were added. The mixture was
stirred at this temperature for 1 h, and then it was filtered through
Celite and the Celite was washed with CH2Cl2 (100 mL). The organ-
ic layer was washed successively with 5% Na2S2O3 (100 mL), sat.
NaHCO3 (100 mL), and H2O (100 mL), dried (Na2SO4), and evap-
orated to dryness. The crude mass was purified by column chroma-
tography (silica gel, hexane–EtOAc, 4:1) to furnish pure 10 (1.2 g,
76%) as a colorless syrup; [α]D25 +62 (c 1.0, CHCl3).
IR (neat): 2924, 2856, 2363, 1654, 1649, 1515, 1460, 1218, 1072,
759, 671 cm–1.
1H NMR (500 MHz, CDCl3): δ = 7.49–7.13 (m, 20 H, HAr), 6.96 (d,
J = 9.0 Hz, 2 H, HAr), 6.80 (d, J = 9.0 Hz, 2 H, HAr), 5.33 (d, J = 3.5
Hz, 1 H, H1A), 5.32 (d, J = 2.5 Hz, 1 H, H4A), 5.12 (br s, 1 H, H1B),
4.97 (d, J = 11.0 Hz, 1 H, PhCH2), 4.80 (d, J = 12.5 Hz, 1 H,
PhCH2), 4.68–4.62 (m, 3 H, PhCH2), 4.50 (br s, 2 H, PhCH2), 4.45
(d, J = 11.5 Hz, 1 H, PhCH2), 4.38 (dd, J = 10.0, 3.0 Hz, 1 H, H3A),
4.20–4.18 (m, 1 H, H5A), 4.04–4.01 (m, 1 H, H5B), 3.80 (dd, J = 9.5,
3.0 Hz, 1 H, H3B), 3.77 (s, 3 H, OCH3), 3.70 (dd, J = 10.0, 3.5 Hz,
1 H, H2A), 3.61 (dd, J = 10.0, 8.5 Hz, 1 H, H4B), 3.57 (br s, 1 H,
H2B), 2.10 (s, 3 H, COCH3), 1.29 (d, J = 6.5 Hz, 3 H, CCH3), 1.12
(d, J = 6.5 Hz, 3 H, CCH3).
13C NMR (125 MHz, CDCl3): δ = 170.4 (COCH3), 155.0–114.5
(CAr), 96.9 (JC1,H1 = 169.0 Hz, C1A), 93.6 (JC1,H1 = 168.0 Hz, C1B),
80.5 (C4B), 79.1 (C3B), 75.1 (PhCH2), 74.6 (C2B), 74.4 (C2A), 73.3
(PhCH2), 71.9 (PhCH2), 71.7 (PhCH2), 70.3 (C3A), 69.6 (C5B), 68.3
(C4A), 65.0 (C5A), 55.5 (OCH3), 20.8 (COCH3), 18.9 (CCH3), 16.2
(CCH3).
IR (neat): 2923, 2372, 2142, 1661, 1591, 1055, 611 cm–1.
1H NMR (500 MHz, CDCl3): δ = 8.14–8.00 (m, 6 H, HAr), 7.54–
7.03 (m, 29 H, HAr), 6.95 (d, J = 9.0 Hz, 2 H, HAr), 6.79 (d, J = 9.0
Hz, 2 H, HAr), 5.50–5.49 (m, 1 H, H3C), 5.35 (t, J = 8.0 Hz each, 1
H, H2C), 5.24 (d, J = 3.5 Hz, 1 H, H1A), 5.15–5.12 (m, 1 H, H4C),
5.01 (d, J = 1.5 Hz, 1 H, H1B), 4.98 (d, J = 11.5 Hz, 1 H, PhCH2),
4.78 (d, J = 10.5 Hz, 2 H, PhCH2), 4.77 (d, J = 7.5 Hz, 1 H, H1C),
4.69 (d, J = 11.0 Hz, 1 H, PhCH2), 4.63 (d, J = 12.0 Hz, 1 H,
PhCH2), 4.55 (d, J = 11.0 Hz, 1 H, PhCH2), 4.50 (d, J = 12.0 Hz, 1
H, PhCH2), 4.39 (d, J = 12.0 Hz, 1 H, PhCH2), 4.22 (dd, J = 10.5,
3.0 Hz, 1 H, H3A), 4.12–4.04 (m, 3 H, H2B, H5A, H5aC), 3.91 (dd,
J = 9.0, 3.0 Hz, 1 H, H3B), 3.84–3.79 (m, 3 H, H2A, H4A, H5B), 3.77
(s, 3 H, OCH3), 3.65 (d, J = 10.0, 8.4 Hz, 1 H, H4B), 3.25–3.23 (m,
1 H, H5bC), 1.31 (d, J = 6.5 Hz, 3 H, CCH3), 1.21 (d, J = 6.5 Hz, 3
H, CCH3).
13C NMR (125 MHz, CDCl3): δ = 165.3 (COPh), 165.2 (COPh),
165.0 (COPh), 155.4–114.5 (CAr), 100.4 (C1C), 97.0 (C1A), 93.8
(C1B), 80.8 (C4B), 79.4 (C3B), 78.7 (C2B), 75.1 (PhCH2), 75.0
(C2A), 73.5 (C4A), 72.9 (PhCH2), 72.5 (PhCH2), 72.3 (PhCH2), 70.8
(C3A), 69.0 (C2C), 68.9 (C3C), 68.6 (C5B), 67.6 (C4C), 66.7 (C5A),
59.5 (C5C), 55.5 (OCH3), 18.0 (CCH3), 16.8 (CCH3).
MS (ESI): m/z = 841.2 [M + Na]+.
Anal. Calcd for C49H54O11 (818.37): C, 71.86; H, 6.65. Found: C,
71.70; H, 6.82.
MS (ESI): m/z = 1243.3 [M + Na]+.
Synthesis 2014, 46, 1947–1953
© Georg Thieme Verlag Stuttgart · New York

PAPER
Lipopolysaccharide of Azospirillum brasilense SR80
1951
Anal. Calcd for C73H72O17 (1220.48): C, 71.79; H, 5.94. Found: C,
71.64; H, 6.15.
IR (neat): 3428, 2831, 1725, 1552, 1370, 1276, 1013, 1045, 822,
756 cm–1.
1H NMR (500 MHz, CDCl3): δ = 8.12–8.10 (m, 2 H, HAr), 7.55–
7.33 (m, 8 H, HAr), 7.06 (d, J = 9.0 Hz, 2 H, HAr), 6.81 (d, J = 9.0
Hz, 2 H, HAr), 5.67 (d, J = 3.5 Hz, 1 H, H1), 5.62 (dd, J = 10.5, 3.5
Hz, 1 H, H3), 5.52 (s, 1 H, PhCH), 4.52 (d, J = 3.0 Hz, 1 H, H4),
4.49 (dd, J = 10.0, 3.0 Hz, 1 H, H2), 4.26 (d, J = 12.5 Hz, 1 H, H6a),
4.00 (d, J = 12.5 Hz, 1 H, H6b), 3.89 (br s, 1 H, H5), 3.76 (s, 3 H,
OCH3).
13C NMR (125 MHz, CDCl3): δ = 166.8 (COPh), 155.3–114.7 (CAr),
100.6 (PhCH), 98.8 (C1), 74.4 (C2), 72.1 (C4), 69.1 (C6), 66.9
(C3), 63.3 (C5), 55.6 (OCH3).
4-Methoxyphenyl (β-D-Xylopyranosyl)-(1→4)-[(α-D-rhamno-
pyranosyl)-(1→3)]-α-L-fucopyranoside (1)
20% Pd(OH)2/C (100 mg) was added to a soln of compound 10 (800
mg, 0.66 mmol) in MeOH (20 mL) and the mixture was stirred at
r.t. for 24 h under a positive pressure of H2. The mixture was filtered
through Celite and concentrated to dryness. A solution of the hydro-
genated product in 0.1 M NaOMe in MeOH (10 mL) was stirred at
r.t. for 10 h. The mixture was neutralized with Dowex 50W-X8 (H+)
resin, filtered, and evaporated to dryness. The crude product was
passed through a Sephadex LH20 column (MeOH–H2O, 3:1) to
give pure 1 (240 mg, 66%) as a powder; [α]D25 +22 (c 1.0, H2O).
MS (ESI): m/z = 501.0 [M + Na]+.
IR (KBr): 3445, 2361, 1670, 1649, 1541, 1462, 1026, 767 cm–1.
Anal. Calcd for C27H26O8 (478.16): C, 67.77; H, 5.48. Found: C,
67.60; H, 5.63.
1H NMR (500 MHz, D2O): δ = 7.01 (d, J = 9.0 Hz, 2 H, HAr), 6.89
(d, J = 9.0 Hz, 2 H, HAr), 4.98 (d, J = 3.0 Hz, 1 H, H1A), 4.92 (br s,
1 H, H1B), 4.29 (d, J = 7.5 Hz, 1 H, H1C), 4.11 (br s, 1 H, H4A),
4.08–4.03 (m, 1 H, H5A), 3.89–3.70 (m, 5 H, H2A, H2B, H3A, H3B,
H5B), 3.56–3.49 (m, 2 H, H4C, H5aC), 3.34–3.24 (m, 3 H, H2C, H3C,
H4B), 3.25 (s, 3 H, OCH3), 3.18–3.14 (m, 1 H, H5bC), 1.21 (d,
J = 6.5 Hz, 3 H, CCH3), 1.17 (d, J = 6.5 Hz, 3 H, CCH3).
13C NMR (125 MHz, D2O): δ = 155.4–115.7 (CAr), 104.5 (C1C),
95.6 (C1B), 95.5 (C1A), 75.6 (2 C, C2C, C4A,), 73.5 (2 C, C3C, C4C),
72.1 (C3A), 70.2 (2 C, C2B, C4B), 69.4 (C3B), 68.6 (C5B), 66.6
(C2A), 66.4 (C5A), 64.9 (C5C), 55.1 (OCH3), 16.7 (CCH3), 15.1
(CCH3).
4-Methoxyphenyl (2,3,4-Tri-O-benzyl-α-L-fucopyranosyl)-
(1→2)-3-O-benzoyl-4,6-O-benzylidene-α-D-galactopyranoside
(15)
To a solution of compound 13 (2 g, 4.2 mmol) and compound 14
(2.4 g, 5.02 mmol) in anhyd CH2Cl2–Et2O (1:1, 20 mL) was added
4 Å molecular sieves (1 g) and the mixture was stirred at r.t. for 30
min under argon and cooled to –25 °C. NIS (510 mg, 2.27 mmol)
and TMSOTf (15 μL) were added to the cooled mixture and it was
stirred at this temperature for 30 min. The mixture was diluted with
CH2Cl2 (50 mL) and filtered through Celite and the Celite was
washed with CH2Cl2 (100 mL). The organic layer was washed suc-
cessively with 5% aq Na2S2O3, sat. NaHCO3, and H2O, dried
(Na2SO4), and concentrated under reduced pressure. The crude
product was purified by column chromatography (silica gel, hex-
ane–EtOAc, 6:1) to afford pure 15 (3 g, 81%) as a colorless oil;
[α]D25 +45 (c 1.0, CHCl3).
MS (ESI): m/z = 571.1 [M + Na]+.
Anal. Calcd for C24H36O14 (548.21): C, 52.55; H, 6.62. Found: C,
52.37; H, 6.78.
4-Methoxyphenyl 4,6-O-Benzylidene-α-D-galactopyranoside
(12)
IR (neat): 3418, 2917, 2857, 1702, 1623, 1585, 1443, 1374, 1316,
1275, 1115, 1069, 1028, 786, 712 cm–1.
A solution of compound 11 (5 g, 11.0 mmol) in 0.1 M NaOMe (25
mL) was stirred at r.t. for 3 h. The mixture was neutralized with
Dowex 50W X8 (H+) resin, filtered, and concentrated to give the de-
O-acetylated product quantitatively. To a solution of the de-O-acet-
ylated product in MeCN (20 mL) was added PhCH(OMe)2 (2.5 mL,
16.5 mmol) followed by TsOH (150 mg) and mixture was stirred at
r.t. for 5 h. The reaction was quenched with Et3N (2 mL) and the sol-
vents were removed under reduced pressure. The crude product was
passed through a short pad of silica gel (hexane–EtOAc, 1:1) to give
pure 12 (3.2 g, 78%) as a white foam; [α]D25 +33 (c 1.0, CHCl3).
1H NMR (500 MHz, CDCl3): δ = 8.11–8.09 (m, 2 H, HAr), 7.54–
7.22 (m, 18 H, HAr), 7.11–7.05 (m, 5 H, HAr), 7.03 (d, J = 9.0 Hz, 2
H, HAr), 6.79 (d, J = 9.0 Hz, 2 H, HAr), 5.80 (dd, J = 10.5, 3.5 Hz, 1
H, H3A), 5.77 (d, J = 3.0 Hz, 1 H, H1A), 5.56 (s, 1 H, PhCH), 4.98
(d, J = 3.5 Hz, 1 H, H1B), 4.87 (d, J = 11.5 Hz, 1 H, PhCH2), 4.71
(d, J = 12.0 Hz, 1 H, PhCH2), 4.66 (d, J = 3.0 Hz, 1 H, H4A), 4.59
(d, J = 11.5 Hz, 1 H, PhCH2), 4.53 (d, J = 12.0 Hz, 1 H, PhCH2),
4.45 (dd, J = 10.5, 3.0 Hz, 1 H, H2A), 4.38 (AB q, 2 H, PhCH2),
4.27–4.25 (m, 1 H, H6aA), 4.07–4.05 (m, 1 H, H6bA), 3.92 (br s, 1 H,
H5A), 3.89 (dd, J = 10.5, 3.5 Hz, 1 H, H2B), 3.85 (dd, J = 10.0, 2.5
Hz, 1 H, H3B), 3.81–3.78 (m, 1 H, H5B), 3.76 (s, 3 H, OCH3), 3.46
(br s, 1 H, H4B), 0.67 (d, J = 6.5 Hz, 3 H, CCH3).
13C NMR (125 MHz, CDCl3): δ = 166.3 (COPh), 154.8–114.7 (CAr),
100.5 (PhCH), 100.1 (C1B), 97.3 (C1A), 78.7 (C4B), 77.9 (C3B),
76.2 (C2B), 74.6 (PhCH2), 74.5 (C2A), 74.3 (C4A), 73.2 (PhCH2),
72.5 (PhCH2), 70.3 (C3A), 69.2 (C6A), 67.2 (C5B), 62.9 (C5A), 55.5
(OCH3), 16.3 (CCH3).
IR (neat): 3408, 2911, 1815, 1562, 1359, 1286, 1113, 1035, 822,
788 cm–1.
1H NMR (500 MHz, CDCl3): δ = 7.50–7.36 (m, 5 H, HAr), 7.03 (d,
J = 9.0 Hz, 2 H, HAr), 6.82 (d, J = 9.0 Hz, 2 H, HAr), 5.62 (d, J = 3.0
Hz, 1 H, H1), 5.56 (s, 1 H, PhCH), 4.32 (d, J = 3.0 Hz, 1 H, H4),
4.23 (dd, J = 12.5, 2.5 Hz, 1 H, H6a), 4.11–4.03 (m, 3 H, H3, H2,
H6b), 3.84–3.83 (m, 1 H, H5), 3.79 (s, 3 H, OCH3).
13C NMR (125 MHz, CDCl3): δ = 155.5–114.7 (CAr), 101.3 (PhCH),
98.4 (C1), 75.7 (C3), 69.9 (C4), 69.7 (C5), 69.2 (C2), 63.5 (C6),
55.6 (OCH3).
MS (ESI): m/z = 917.2 [M + Na]+.
Anal. Calcd for C54H54O12 (894.36): C, 72.47; H, 6.08. Found: C,
72.32; H, 6.25.
MS (ESI): m/z = 397.1 [M + Na]+.
Anal. Calcd for C20H22O7 (374.13): C, 64.16; H, 5.92. Found: C,
64.03; H, 6.03.
4-Methoxyphenyl (2,3,4-Tri-O-benzyl-α-L-fucopyranosyl)-
(1→2)-4,6-O-benzylidene-α-D-galactopyranoside (16)
A solution of compound 15 (2 g, 2.2 mmol) in 0.1 M NaOMe in
MeOH (25 mL) was stirred at r.t. for 3 h. The mixture was neutral-
ized with Dowex 50W X8 (H+) resin, filtered, and concentrated to
give the crude product, which was passed through a short pad of sil-
ica gel (hexane–EtOAc, 1:1) to furnish pure 16 (1.6 g, 91%) as a
yellow oil; [α]D25 +35 (c 1.0, CHCl3).
4-Methoxyphenyl 3-O-Benzoyl-4,6-O-benzylidene-α-D-galacto-
pyranoside (13)
To a solution of compound 12 (3 g, 8.0 mmol) and benzoyl cyanide
(1.4 g, 10.4 mmol) in anhyd MeCN (40 mL) was added Et3N (0.5
mL) and the mixture was stirred for 30 min at r.t.. The mixture was
quenched with MeOH (1 mL) and the solvents were removed under
reduced pressure. The crude product was purified by column chro-
matography (silica gel; hexane–EtOAc, 4:1) to furnish pure 13 (3.2
g, 84%) as a yellow oil; [α]D25 +52 (c 1.0, CHCl3).
IR (neat): 3409, 2927, 2747, 1734, 1555, 1238, 1113, 1102, 1059,
998, 782, 698 cm–1.
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, 1947–1953

1952
P. K. Mandal et al.
PAPER
1H NMR (500 MHz, CDCl3): δ = 7.62–7.59 (m, 2 H, HAr), 7.41–
7.23 (m, 18 H, HAr), 7.01 (d, J = 9.0 Hz, 2 H, HAr), 6.81 (d, J = 9.0
Hz, 2 H, HAr), 5.69 (d, J = 3.0 Hz, 1 H, H1A), 5.59 (s, 1 H, PhCH),
4.95 (d, J = 3.5 Hz, 1 H, H1B), 4.94 (d, J = 12.0 Hz, 1 H, PhCH2),
4.84 (d, J = 11.5 Hz, 2 H, PhCH2), 4.77–4.69 (m, 2 H, PhCH2), 4.62
(d, J = 11.5 Hz, 1 H, PhCH2), 4.38–4.36 (m, 2 H, H3A, H4A), 4.23–
4.20 (m, 1 H, H6aA), 4.06–4.03 (m, 3 H, H6bA, H2A, H2B), 3.98 (dd,
J = 10.0, 3.5 Hz, 1 H, H3B), 3.85–3.83 (m, 1 H, H5B), 3.78–3.75 (m,
1 H, H5A), 3.77 (s, 3 H, OCH3), 3.58 (br s, 1 H, H4B), 0.77 (d,
J = 6.5 Hz, 3 H, CCH3).
13C NMR (125 MHz, CDCl3): δ = 154.8–114.7 (CAr), 100.2 (2 C,
PhCH, C1B), 97.7 (C1A), 79.6 (C3B), 78.7 (C4B), 77.4 (C2B), 76.6
(C2A), 75.7 (C4A), 77.8 (PhCH2), 73.9 (PhCH2), 73.0 (PhCH2), 69.4
(C6A), 67.7 (C3A), 67.1 (C5B), 63.3 (C5A), 55.6 (OCH3), 16.5
(CCH3).
a short column of silica gel (hexane–EtOAc, 2:1) to give pure 19
(900 mg, 93%) as a colorless syrup; [α]D25 +29 (c 1.0, CHCl3).
IR (neat): 3029, 2210, 1849, 1628, 1415, 1322, 1042, 988, 756, 667
cm–1.
1H NMR (500 MHz, CDCl3): δ = 7.67–7.16 (m, 29 H, HAr), 6.99 (d,
J = 9.0 Hz, 2 H, HAr), 6.77 (d, J = 9.0 Hz, 2 H, HAr), 5.86 (d, J = 8.0
Hz, 1 H, H1C), 5.58 (d, J = 3.0 Hz, 1 H, H1A), 5.51 (s, 1 H, PhCH),
5.45 (s, 1 H, PhCH), 4.93 (d, J = 3.0 Hz, 1 H, H1B), 4.88–4.81 (m,
4 H, PhCH2), 4.72–4.69 (m, 2 H, H3A, PhCH2), 4.54 (d, J = 12.0 Hz,
1 H, PhCH2), 4.39 (d, J = 3.0 Hz, 1 H, H4A), 4.37–4.35 (m, 1 H,
H2C), 4.33–4.30 (m, 1 H, H6aA), 4.24–4.21 (m, 1 H, H3C), 4.14–4.12
(m, 1 H, H6bA), 4.06 (dd, J = 10.5, 3.5 Hz, 1 H, H2A), 4.02 (dd,
J = 10.0, 3.0 Hz, 1 H, H2B), 3.97–3.91 (m, 3 H, H3B, H4C, H6aC),
3.84–3.64 (m, 3 H, H5B, H5C, H6bC), 3.76 (s, 3 H, OCH3), 3.51 (br
s, 1 H, H4B), 3.12 (br s, 1 H, H5A), 0.68 (d, J = 6.5 Hz, 3 H, CCH3).
13C NMR (125 MHz, CDCl3): δ = 167.8, 167.2 (2 C, Phth), 154.6–
114.6 (CAr), 101.3 (PhCH), 100.1 (2 C, C1B, PhCH), 97.1 (C1A),
96.2 (C1C), 79.3 (C3B), 77.3 (C4B), 76.9 (C2A), 76.7 (C2B), 76.5
(C4A),), 75.2 (C4C), 74.7 (PhCH2), 74.4 (PhCH2), 72.4 (PhCH2),
69.2 (C6A), 69.1 (C3A), 68.9 (C6C), 67.5 (C3C), 66.8 (C5B), 66.2
(C5A), 63.2 (C5C), 55.9 (C2C), 55.5 (OCH3), 16.4 (CCH3).
MS (ESI): m/z = 813.1 [M + Na]+.
Anal. Calcd for C47H50O11 (790.34): C, 71.38; H, 6.37. Found: C,
71.20; H, 6.56.
4-Methoxyphenyl (3-O-Acetyl-4,6-O-benzylidene-2-deoxy-2-
phthalimido-β-D-galactopyranosyl)-(1→3)-[(2,3,4-tri-O-ben-
zyl-α-L-fucopyranosyl)-(1→2)]-4,6-O-benzylidene-α-D-galacto-
pyranoside (18)
MS (ESI): m/z = 1192.3 [M + Na]+.
To a solution of compound 16 (1 g, 1.26 mmol) and compound 17
(730 mg, 1.51 mmol) in anhyd CH2Cl2 (10 mL) was added 4 Å mo-
lecular sieves (1 g) and the mixture was stirred at –30 °C for 30 min
under argon. To the cooled mixture was added NIS (375 mg, 1.66
mmol) followed by TMSOTf (10 μL), and it was stirred at this tem-
perature for 45 min. The mixture was poured into aq 5% Na2S2O3
and extracted with CH2Cl2 (50 mL). The organic layer was washed
with sat. NaHCO3 and H2O, dried (Na2SO4), and concentrated. The
crude product was purified by column chromatography (silica gel,
hexane–EtOAc, 7:1) to give pure 18 (1.2 g, 79%) as a yellow oil;
[α]D25 +76 (c 1.0, CHCl3).
Anal. Calcd for C68H67NO17 (1169.44): C, 69.79; H, 5.77. Found: C,
69.62; H, 5.90.
4-Methoxyphenyl (2,3,4,6-Tetra-O-acetyl-β-D-galactopyrano-
syl)-(1→3)-(4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-
galactopyranosyl)-(1→3)-[(2,3,4-tri-O-benzyl-α-L-fucopyrano-
syl)-(1→2)]-4,6-O-benzylidene-α-D-galactopyranoside (21)
To a solution of compound 19 (800 mg, 0.68 mmol) and compound
20 (320 mg, 0.82 mmol) in anhyd CH2Cl2 (10 mL) was added 4 Å
molecular sieves (500 mg) and the mixture was stirred under argon
at r.t. for 30 min. The mixture was cooled to –10 °C, NIS (200 mg,
0.89 mmol) and TMSOTf (5 μL) were added, and it was stirred at
this temperature for 1 h. The mixture was filtered through Celite and
the Celite was washed with CH2Cl2 (50 mL). The combined organic
layers were washed successively with 5% aq Na2S2O3, sat.
NaHCO3, and H2O, dried (Na2SO4), and concentrated. The crude
product was purified by column chromatography (silica gel,
hexane–EtOAc, 5:1) to give pure 21 (850 mg, 83%) as a yellow oil;
[α]D25 +79 (c 1.0, CHCl3).
IR (neat): 3418, 2911, 2769, 2211, 1664, 1532, 1498, 1411, 1379,
1255, 1198, 1063, 982, 661 cm–1.
1H NMR (500 MHz, CDCl3): δ = 7.55–7.14 (m, 29 H, HAr), 6.96 (d,
J = 9.0 Hz, 2 H, HAr), 6.75 (d, J = 9.0 Hz, 2 H, HAr), 5.87 (d, J = 8.5
Hz, 1 H, H1C), 5.65 (dd, J = 11.5, 3.5 Hz, 1 H, H3C), 5.56 (d, J = 3.0
Hz, 1 H, H1A), 5.54 (s, 1 H, PhCH), 5.43 (s, 1 H, PhCH), 4.89–4.83
(m, 3 H, PhCH2, H3A), 4.79–4.72 (m, 3 H, PhCH2), 4.76 (d, J = 3.5
Hz, 1 H, H1B), 4.69 (dd, J = 11.5, 8.0 Hz, 1 H, H2C), 4.54 (d,
J = 11.0 Hz, 1 H, PhCH2), 4.40 (br s, 1 H, H4A), 4.26–4.23 (m, 1 H,
H6aA), 4.13–4.11 (m, 1 H, H6bA), 4.05 (d, J = 3.0 Hz, 1 H, H4C),
4.01 (dd, J = 10.0, 3.0 Hz, 1 H, H2B), 3.97–3.94 (m, 2 H, H2A,
H6aC), 3.88–3.86 (m, 1 H, H3B), 3.75 (s, 3 H, OCH3), 3.73–3.66 (m,
3 H, H5B, H5C, H6bC), 3.50 (br s, 1 H, H4B), 2.92 (br s, 1 H, H5A),
1.92 (s, 3 H, COCH3), 0.61 (d, J = 6.5 Hz, 3 H, CCH3).
13C NMR (125 MHz, CDCl3): δ = 170.1 (COCH3), 167.9, 167.3 (2
C, Phth), 154.6–114.6 (CAr), 100.7 (PhCH), 100.2 (C1B), 100.1
(PhCH), 97.0 (C1A), 96.2 (C1C), 79.1 (C3B), 77.3 (C4B), 76.9 (C2A),
76.6 (C2B), 76.5 (C4A), 74.7 (PhCH2), 74.2 (PhCH2), 72.9 (C4C),
72.2 (PhCH2), 69.1 (C6A), 69.0 (C6C), 68.9 (C3A), 68.7 (C3C), 66.8
(C5B), 66.0 (C5A), 63.3 (C5C), 55.5 (OCH3), 52.4 (C2C), 20.8
(COCH3), 16.3 (CCH3).
IR (neat): 3459, 3021, 2927, 2847, 1734, 1555, 1323, 1238, 1133,
1102, 1079, 782, 698 cm–1.
1H NMR (500 MHz, CDCl3): δ = 7.62–7.11 (m, 29 H, HAr), 6.95 (d,
J = 9.0 Hz, 2 H, HAr), 6.76 (d, J = 9.0 Hz, 2 H, HAr), 5.71 (d, J = 8.0
Hz, 1 H, H1C), 5.57 (d, J = 3.0 Hz, 1 H, H1A), 5.56 (s, 1 H, PhCH),
5.49 (s, 1 H, PhCH), 5.30–5.28 (m, 1 H, H4D), 5.04 (br s, 1 H, H1B),
4.88–4.81 (m, 5 H, PhCH2), 4.78–4.74 (m, 2 H, H2D, H3A), 4.73 (d,
J = 8.0 Hz, 1 H, H1D), 4.68 (d, J = 3.0 Hz, 1 H, H4A), 4.64–4.62 (m,
2 H, H2C, H3D), 4.54 (d, J = 11.5 Hz, 1 H, PhCH2), 4.40 (br s, 1 H,
H4C), 4.29–4.26 (m, 1 H, H6aA), 4.24–4.14 (m, 3 H, H3C, H6abD),
4.13–4.10 (m, 1 H, H6bA), 4.02–4.00 (m, 2 H, H2B, H5D), 3.97–3.91
(m, 2 H, H3B, H6aC), 3.85–3.83 (m, 1 H, H2A), 3.80–3.78 (m, 1 H,
H6bC), 3.74 (s, 3 H, OCH3), 3.71–3.64 (m, 2 H, H5B, H5C), 3.51 (br
s, 1 H, H4B), 2.92 (br s, 1 H, H5A), 2.07 (s, 3 H, COCH3), 1.92 (s, 3
H, COCH3), 1.88 (s, 3 H, COCH3), 1.84 (s, 3 H, COCH3), 0.58 (d,
J = 6.5 Hz, 3 H, CCH3).
13C NMR (125 MHz, CDCl3): δ = 169.9 (COCH3), 169.8 (COCH3),
168.9 (COCH3), 168.8 (COCH3), 167.9, 167.3 (2 C, Phth), 154.6–
114.6 (CAr), 106.9 (C1B), 101.6 (PhCH), 100.2 (2 C, C1D, PhCH),
97.0 (C1A), 96.6 (C1C), 81.4 (C3D), 80.2 (C3C), 79.3 (C3B), 77.2
(C4B), 76.9 (2 C, C2A, C2B), 76.5 (C5D), 75.8 (C4C), 75.3 (C4A),
74.7 (PhCH2), 74.5 (C3A), 74.2 (PhCH2), 72.2 (PhCH2), 69.5 (C4D),
69.4 (C6A), 68.9 (C6C), 68.4 (C2D), 66.8 (C5B), 66.1 (C5A), 63.3
(C5C), 62.6 (C6D), 55.6 (OCH3), 53.3 (C2C), 20.8 (COCH3), 20.6
(COCH3), 20.5 (COCH3), 20.4 (COCH3), 16.2 (CCH3).
MS (ESI): m/z = 1234.4 [M + Na]+.
Anal. Calcd for C70H69NO18 (1211.45): C, 69.35; H, 5.74. Found: C,
69.22; H, 5.90.
4-Methoxyphenyl (4,6-O-Benzylidene-2-deoxy-2-phthalimido-
β-D-galactopyranosyl)-(1→3)-[(2,3,4-tri-O-benzyl-α-L-fucopy-
ranosyl)-(1→2)]-4,6-O-benzylidene-α-D-galactopyranoside (19)
A solution of compound 18 (1 g, 0.83 mmol) in 0.1 M NaOMe in
MeOH (30 mL) was stirred at r.t. for 30 min and neutralized with
Dowex 50W X8 (H+) resin. The mixture was filtered and evaporat-
ed to dryness to give the crude product, which was passed through
Synthesis 2014, 46, 1947–1953
© Georg Thieme Verlag Stuttgart · New York

PAPER
Lipopolysaccharide of Azospirillum brasilense SR80
1953
MS (ESI): m/z = 1522.3 [M + Na]+.
J. Chem. Pharm. Res. 2013, 5, 46. (c) Rajasekar, S.; Elango,
R. Curr. Botany 2011, 2, 27. (d) Bloemberg, G. V.;
Lugtenberg, B. J. J. Curr. Opin. Plant Biol. 2001, 4, 343.
(3) (a) Jofre, E.; Lagares, A.; Mori, G. FEMS Microbiol. Lett.
2004, 231, 267. (b) Burdman, S.; Okon, Y.; Jurkevitch, E.
Crit. Rev. Microbiol. 2000, 26, 91. (c) Egorenkova, I. V.;
Konnova, S. A.; Sachuk, V. N.; Ignatov, V. V. Plant Soil
2001, 231, 275.
(4) (a) Weller, D. M. Annu. Rev. Phytopathol. 1988, 26, 379.
(b) Guo, Y. B.; Li, J.; Li, L.; Chen, F.; Wu, W.; Wang, J.;
Wang, H. Appl. Environ. Microbiol. 2009, 75, 6792.
(c) Bashan, Y.; de-Bashan, L. E. Adv. Agron. 2010, 108, 77.
(5) (a) Bashan, Y.; de-Bashan, L. E. Appl. Environ. Microbiol.
2002, 68, 2637. (b) Bashan, Y.; de-Bashan, L. E. Eur. J.
Plant Pathol. 2002, 108, 821. (c) Romero, A. M.; Correa, O.;
Moccia, S.; Rivas, J. G. J. Appl. Microbiol. 2003, 95, 832.
(6) (a) Figueiredo, M. V. B.; Seldin, L.; de Araujo, F. F.; de
Lima Ramos Mariano, R. In Plant Growth and Health
Promoting Bacteria; Maheshwari, D. K., Ed.; Springer:
Berlin, 2011, 21–43. (b) Ptacek, S. D.; Gysegom, P.;
Srinivasan, M.; Vanderleyden, J. Appl. Environ. Microbiol.
2005, 71, 1803.
Anal. Calcd for C82H85NO26 (1499.54): C, 65.63; H, 5.71. Found: C,
65.46; H, 5.90.
4-Methoxyphenyl (β-D-Galactopyranosyl)-(1→3)-(2-acetami-
do-2-deoxy-β-D-galactopyranosyl)-(1→3)-[(α-L-fucopyrano-
syl)-(1→2)]-α-D-galactopyranoside (2)
To a solution of compound 21 (700 mg, 0.47 mmol) in EtOH (20
mL) was added hydrazine hydrate (1 mL) and the mixture was
stirred at 80 °C for 8 h. The solvents were removed under reduced
pressure and a solution of the crude mass in pyridine (5 mL) and
Ac2O (5 mL) was kept at r.t. for 1 h. The solvents were evaporated
and co-evaporated with toluene under reduced pressure. 20%
Pd(OH)2/C (100 mg) was added to a solution of the acetylated prod-
uct in MeOH (20 mL) and the mixture was stirred at r.t. for 24 h un-
der a positive pressure of H2. The mixture was filtered through
Celite, the Celite was washed with MeOH (20 mL) and the com-
bined filtrate was concentrated under reduced pressure. A solution
of the hydrogenolyzed product in 0.1 M NaOMe in MeOH (10 mL)
was stirred at r.t. for 3 h. The mixture was neutralized with Dowex
50W-X8 (H+) resin, filtered, and concentrated. The crude product
was passed through a Sephadex LH-20 column (MeOH–H2O, 3:1)
to give pure 2 (250 mg, 60%) as a white powder; [α]D25 +53 (c 1.0,
H2O).
(7) Sigida, E. N.; Fedonenko, Y. P.; Zdorovenko, E. L.;
Konnova, S. A.; Shashkov, A. S.; Ignatov, V. V.; Knirel, Y.
A. Carbohydr. Res. 2013, 371, 40.
IR (KBr): 3456, 2935, 1622, 1356, 1236, 1165, 1067, 697 cm–1.
1H NMR (500 MHz, D2O): δ = 7.03 (d, J = 9.0 Hz, 2 H, HAr), 6.87
(d, J = 9.0 Hz, 2 H, HAr), 5.25 (d, J = 3.0 Hz, 1 H, H1A), 4.97 (br s,
1 H, H1B), 4.95 (d, J = 7.0 Hz, 1 H, H1D), 4.85 (d, J = 7.0 Hz, 1 H,
H1C), 4.43–4.39 (m, 1 H, H5B), 4.20–4.11 (m, 2 H, H4D, H5A),
3.89–3.71 (m, 4 H, H2C, H4A, H5D, H6aD), 3.67–3.53 (m, 7 H, H2A,
H2D, H3A, H3D, H4C, H6aC, H6bD), 3.65 (s, 3 H, OCH3), 3.51–3.33
(m, 4 H, H5C, H6abA, H6bC), 3.31–3.21 (m, 2 H, H2B, H4B), 3.09–
2.97 (m, 2 H, H3B, H3C), 1.97 (s, 3 H, COCH3), 1.13 (d, J = 6.5 Hz,
3 H, CCH3).
13C NMR (125 MHz, D2O): δ = 174.8 (COCH3), 155.5–115.9 (CAr),
106.7 (C1C), 101.1 (C1D), 98.9 (C1B), 95.8 (C1A), 82.6 (C3A), 80.9
(C4C), 76.5 (C3D), 75.6 (C5C), 72.8 (C5D), 72.2 (C2A), 71.6 (2 C,
C4B, C5A), 70.7 (C3B), 70.5 (C4A), 70.2 (C3C), 69.2 (C2B), 68.9
(C2D), 68.2 (C5B), 67.4 (C4D), 66.2 (C6A), 62.7 (C6C), 60.5 (C6D),
55.0 (OCH3), 54.9 (C2C), 21.9 (COCH3), 16.5 (CCH3).
(8) (a) Mandal, P. K.; Misra, A. K. Bioorg. Chem. 2010, 38, 56.
(b) Mandal, P. K.; Misra, A. K. Glycoconjugate J. 2008, 25,
713. (c) Mandal, P. K.; Misra, A. K. Tetrahedron 2008, 64,
8685. (d) Agnihotri, G.; Mandal, P. K.; Misra, A. K.
Tetrahedron 2007, 63, 7240. (e) Mandal, P. K.; Misra, A. K.
Synthesis 2007, 2660. (f) Mandal, P. K.; Branson, T. R.;
Hayes, E. D.; Ross, J. F.; Gavín, J. A.; Daranas, A. H.;
Turnbull, W. B. Angew. Chem. Int. Ed. 2012, 51, 5143.
(9) Liptak, A.; Imre, J.; Nanasi, P. Carbohydr. Res. 1981, 92,
154.
(10) Madhusudan, S. K.; Agnihotri, G.; Negi, D. S.; Misra, A. K.
Carbohydr. Res. 2005, 340, 1373.
(11) Bouchra, M.; Calinaud, P.; Gelas, J. Carbohydr. Res. 1995,
267, 227.
(12) Dinkelaar, J.; de Jong, A. R.; Van Meer, R.; Somers, M.;
Lodder, G.; Overkleeft, H. S.; Codee, J. D. C.; Van der
Marel, G. A. J. Org. Chem. 2009, 74, 4982.
(13) (a) Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H.
Tetrahedron Lett. 1990, 31, 1331. (b) Konradsson, P.;
Udodong, U. E.; Fraser-Reid, B. Tetrahedron Lett. 1990, 31,
4313.
MS (ESI): m/z = 820.3 [M + Na]+.
Anal. Calcd for C33H21NO21 (797.30): C, 49.68; H, 6.44. Found: C,
49.55; H, 6.65.
Acknowledgment
(14) Bock, K.; Lundt, I.; Pedersen, C. Tetrahedron Lett. 1973, 14,
1037.
(15) Paulsen, H.; Brenken, M. Liebigs Ann. Chem. 1988, 649.
(16) Pearlman, W. M. Tetrahedron Lett. 1967, 8, 1663.
(17) Ichikawa, Y.; Matsukawa, Y.; Tamura, M.; Ohara, F.; Isobe,
M.; Kotasuki, H. Chem. Asian J. 2006, 1, 717.
D. D. thanks CSIR, New Delhi for providing a Junior Research Fel-
lowship. This work was supported by CDRI, India. CDRI Commu-
nication No: 8698.
(18) Jacquinet, J.-C. Carbohydr. Res. 2004, 339, 349.
(19) Lönn, H. Carbohydr. Res. 1985, 139, 105.
(20) Kumar, R.; Maulik, P. R.; Misra, A. K. Glycoconjugate J.
2008, 25, 511.
(21) Vic, G.; Hasting, J. J.; Howarth, O. W.; Crout, D. H. G.
Tetrahedron: Asymmetry 1996, 7, 709.
References
(1) (a) Bekri, M. A.; Desair, J.; Proost, P.; Leeuwen, M. S.-V.;
Vanderleyden, J.; Vande Broek, A. J. Bacteriol. 1999, 182,
2440. (b) Okon, Y. Trends Biotechnol. 1985, 3, 223.
(c) Steenhoudt, O.; Vanderleyden, J. FEMS Microbiol. Rev.
2000, 24, 487.
(22) Kanie, O.; Crawley, S. C.; Palcic, M. M.; Hindsgaul, O.
Carbohydr. Res. 1993, 243, 139.
(2) (a) Das, A. J.; Kumar, M.; Kumar, R. Res. J. Agriculture
Forestry Sci. 2013, 1, 21. (b) Kannahi, M.; Kowsalya, M.
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, 1947–1953