Synthesis of two tetrasaccharides related to the O-Antigen from Azospirillum brasilense S17 and Azospirillum lipoferum SR65

Article abstract of DOI:10.1080/07328301003786789

Synthesis of two isomeric tetrasaccharides, β-D-Glup-(1→2)- α-L-Rhap-(1→3)-α-L-Rhap-(1→2)-α-L-Rhap (I) and β-D-Glup-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3) -α-L-Rhap (II), the repeating units from the lipopolysaccharides of the nitrogen-fixing bacterium Azosp

Full text of DOI:10.1080/07328301003786789

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Synthesis of Two Tetrasaccharides
Related to the O-Antigen from
Azospirillum brasilense S17 and
Azospirillum lipoferum SR65
Hanqing Zhao a , Huiqi Jia a , Hongxia Duan a , Jianjun Zhang a ,
Daoquan Wang a & Xiaomei Liang a
a Key Lab of Pesticide Chemistry and Application Technology,
Department of Applied Chemistry, China Agricultural University,
Beijing, 100193, China
Version of record first published: 02 Jun 2010.
To cite this article: Hanqing Zhao, Huiqi Jia, Hongxia Duan, Jianjun Zhang, Daoquan Wang & Xiaomei
Liang (2010): Synthesis of Two Tetrasaccharides Related to the O-Antigen from Azospirillum brasilense
S17 and Azospirillum lipoferum SR65, Journal of Carbohydrate Chemistry, 29:3, 103-117
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Journal of Carbohydrate Chemistry, 29:103–117, 2010
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Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328301003786789
Synthesis of Two
Tetrasaccharides Related
to the O-Antigen from
Azospirillum brasilense S17
and Azospirillum lipoferum
SR65
Hanqing Zhao, Huiqi Jia, Hongxia Duan, Jianjun Zhang,
Daoquan Wang, and Xiaomei Liang
Key Lab of Pesticide Chemistry and Application Technology, Department of Applied
Chemistry, China Agricultural University, Beijing 100193, China
Synthesis of two isomeric tetrasaccharides, β-D-Glup-(1→2)-α-L-Rhap-(1→3)-α-L-
Rhap-(1→2)-α-L-Rhap (I) and β-D-Glup-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)-α-L-
Rhap (II), the repeating units from the lipopolysaccharides of the nitrogen-fixing
bacterium Azospirillum brasilense S17 and Azospirillum lipoferum SR65, was
achieved via assembly of the building blocks 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl
trichloroacetimidate (2), p-methoxyphenyl 3,4-di-O-benzoyl-α-L-rhamnopyranoside (3),
3-O-allyloxycarbonyl-2,4-di-O-benzoyl-α-L-rhamnopyranosyl trichloroacetimidate (6),
2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl trichloroacetimidate (8), and p-methoxy
phenyl 2,4-di-O-benzoyl-α-L-rhamnopyranoside (14). Condensation of 3 with 6 or 8 pro-
vided the disaccharides 9 or 11, respectively. Deallyloxycarbonylation of 11 gave the
disaccharide aceptor 12, while removal of the p-methoxyphenyl group in 9 followed by
trichloroacetimidation of the anomeric hydroxyl group afforded the disaccharide donor
10. Meanwhile, disaccharide donor 16 and acceptor 18 were prepared from 6, 8, and 14
similarly. Finally, condensation of 10 with 12 or 16 with 18, followed by deprotection,
gave the target tetrasaccharides I or II, respectively.
Keywords Synthesis; D-Glucose; L-Rhamnose; Oligosaccharides; Nitrogen fixing
Received January 30, 2010; accepted March 17, 2010.
Address correspondence to Jianjun Zhang, Key Lab of Pesticide Chemistry and Appli-
cation Technology, Department of Applied Chemistry, China Agricultural University,
Beijing 100193, China. E-mail: zhangjianjun@cau.edu.cn
103

H. Zhao et al.
104
INTRODUCTION
Azospirilla species are gram-negative bacteria widely distributed in soils. They
colonize the rhizosphere and have a positive effect on plant growth and de-
velopment by excreting phytohormones, vitamins, and other biologically ac-
tive substances into the rhizosphere.[1] Very recently, it was reported that the
antigenic lipopolysaccharides from the nitrogen-fixing bacteria Azospirillum
brasilense S17 and Azospirillum lipoferum SR65 are made up of linear repeat-
ing units with (1→2)- and (1→3)-linked rhamnan backbone and D-glucose in
the side chains as shown in Figure 1A, B.[2,3]
The lipopolysaccharide (LPS) is the major antigen of the bacterial outer
membrane of the Azospirillum cell envelope. Together with other cell surface
carbohydrate polymers such as the exopolysaccharide (EPS) and the capsu-
lar polysaccharide (CPS), they play important roles for the survival of the
bacteria in adverse environmental conditions as well as regulate the inter-
action with the roots of plants.[1] The LPS is thought to play an important role
in the molecular mechanism of symbiotic infections, and the involvement of
the carbohydrate-rich molecules in establishing the interaction between the
nitrogen-fixing bacterium and the host has been reported.[4,5] It was also re-
vealed that the LPSs of the Azospirillum outer membrane play an important
Figure 1: Structure of the lipopolysaccharides of A. brasilense S17 (A), A. lipoferum SR65 (B),
and the synthesized tetrasaccharides I and II.

Synthesis of O-Antigen-Related Tetrasaccharides
105
role in the formation of bacterial association with the roots of cereals; for exam-
ple, mutants defective in LPS synthesis are worse colonizers to wheat root[6]
and worse absorbers to maize root[7] compared to their nondefective counter-
parts. These facts are of particular interest from the viewpoint of the biological
roles of carbohydrates. For a better understanding of the role the LPSs play in
the symbiotic infections of the bacterium with the host, considerable interest
has been paid to the synthesis of these antigenic repeating units.[8–10] Here we
report the efficient synthesis of the tetrasaccharide repeating units (Fig. 1A, B)
of the LPS from A. brasilense S17 and A. lipoferum SR65 in the form of their
p-methoxyphenyl glycosides.
RESULTS AND DISCUSSION
As shown in Scheme 1, synthesis of the tetrasaccharide I was commenced
with the synthesis of suitably protected L-rhamnose and D-glucose synthons
followed by stepwise glycosylation and deprotection. Therefore, known p-
methoxyphenyl 4-O-benzoyl-α-L-rhamnopyranoside (1)[11] was treated with
benzoyl chloride (or allyl chloroformate) in dichloromethane at –10◦C in the
Scheme 1: Synthesis of the target tetrasaccharide I. Reagents and conditions: (a) Benzoyl
chloride, CH2Cl2, pyridine, 92% for 3; 98% for 5; (b) AllocCl, Py, CH2Cl2, –10◦C, 93%; (c) 80%
MeCN, CAN, then Cl3CCN, DBU, CH2Cl2, 0◦C, 68% for 6 (two steps); 61% for 10 (two steps); (d)
TMSOTf, CH2Cl2, –10◦C to rt, 2 h, 81% for 7; 88% for 9; 89% for 11; 78% for 13; (e) MeOH-THF =
1:1, NaBH4, Pd[P(C6H5)3]4, 90%; (f) satd NH3–MeOH, rt, 96 h, 92%.

H. Zhao et al.
106
presence of 4 equiv. of pyridine, and the C-3 hydroxyl group was selec-
tively blocked, giving acceptor 3 or compound 4 in 92% or 93%, respectively,
yield.[12,13] Low temperature and slow addition of the chlorides were nec-
essary for ensuring the regioselectivity. The regioselectivity of the process
1
was established by H NMR spectroscopy, and the characteristic C-3 proton
moved downfield upon acylation (δH-3 = 4.20 ppm in 1, δH-3 = 5.80 ppm in
3, and δH-3 = 5.50 ppm in 4). Benzoylation of 4 in pyridine with benzoyl
chloride provided p-methoxyphenyl 3-O-allyloxycarbonyl-2,4-di-O-benzoyl-α-
L-rhamnopyranoside (5) in 98% yield. Cleavage of the p-methoxyphenyl group
of 5 with ceric ammonium nitrate (CAN), followed by trichloroacetimida-
tion,[14] provided 3-O-allyloxycarbonyl-2,4-di-O-benzoyl-α-L-rhamnopyranosyl
trichloroacetimidate 6. At the beginning, we tried to synthesize the (1→2)-
linked glucose-containing disaccharide by the condensation of the 2,3,4,6-tetra-
O-acetyl-β-D-glucopyranosyl trichloroacetimidate 2[15] with acceptor 3. How-
ever, instead of getting the desired compound, (1→2)-linked orthoester 7 was
obtained as the main product. The formation of the orthoester was confirmed
1
from the H NMR spectrum, showing the characteristic signals at δ 1.64 for
CH3O3.[16] Later on, benzoylated glucose trichloroacetimidate 8[17] was used as
the glycosyl donor and the (1→2)-linked disaccharide 9 was obtained without
detecting the orthoester formation. Cleavage of the p-methoxyphenyl group of
9 with CAN followed by trichloroacetimidation with CCl3CN in the presence
of DBU or K2CO3 gave the disaccharide donor 10. At the same time, conden-
sation of the donor 6 with the acceptor 3 in the presence of catalytic TMSOTf
furnished the (1→2)-linked disaccharide 11. The allyloxycarbonyl group of 11
was successfully removed in MeOH-THF[18] in the presence of CH3COONH4,
Pd[P(C6H5)3]4, and NaBH4 within 5 min without affecting any of the ben-
zoyl groups, giving the desired disaccharide acceptor 12 in 90% yield. Con-
densation of the disaccharide acceptor 12 and donor 10 proceeded smoothly in
dichloromethane in the presence of TMSOTf, giving the tetrasaccharide 13 in
78% yields. Deacylation of 13 in ammonium-saturated methanol afforded the
1
target tetrasaccharide I. The structure of I was confirmed from its H NMR
and 13C NMR spectra and HSQC, showing the characteristic signals such as δ
5.45, 5.39, and 4.98 ppm for three H-1 (α) of rhamnose, and δ 4.56 ppm (J1,2
7.9 Hz) for H-1 (β) of glucose, and δ 98.1, 100.8, 101.9, and 104.2 ppm for the
anomeric C-1 signals.
=
Meanwhile, tetrasaccharide II was prepared in a similar way (Sch. 2). At
the beginning, the allyloxycarbonyl group of 5 was successfully removed in
MeOH-THF[18] with Pd[P(C6H5)3]4 to provide the monosaccharide acceptor 14
(95%), then condensation of donor 8 or 6 with C-3-OH acceptor 14 in the pres-
ence of TMSOTf gave the (1→3)-linked disaccharide 15 or 17, respectively. Re-
moval of the p-methoxyphenyl group of 15 followed by trichloroacetimidation
provided the disaccharide donor 16, and condensation of the 16 with acceptor
18, which was prepared from 17 through deallyloxycarbonylation, furnished

Synthesis of O-Antigen-Related Tetrasaccharides
107
Scheme 2: Synthesis of the target tetrasaccharide II. Reagents and conditions: (a) MeOH-
THF = 1:1, NaBH4, Pd[P(C6H5)3]4, 95% for 14; 90% for 18; (b) TMSOTf, CH2Cl2, –10◦C to rt, 2 h,
83% for 15; 86% for 17; 75% for 19; (c) 80% MeCN, CAN, then Cl3CCN, DBU, CH2Cl2, 0◦C, 68%
for two steps; (d) satd NH3–MeOH, rt, 96 h, 89%.
the tetrasaccharide 19 in 75% yield. Finally, deacylation of 19 in ammonium-
1
saturated methanol gave the target tetrasaccharide II. The H NMR and 13C
NMR spectra of II were in accordance with the recently reported data by
Prashant et al., and they synthesized this tetrasaccharide in different way.[9]
In summary, an efficient synthesis of p-methoxyphenyl β-D-gluco-
pyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-
L-rhamnopyranoside I and its isomer II were achieved through a [2 + 2] strat-
egy. Compared to Prashant’s synthesis of II, the procedure was more simple
owing to the use of only acyl groups in the syntheses. In terms of efficiency, the
method can be used for construction of higher oligosaccharides with similar
structures. The biological experiments of the synthetic tetrasaccharides are
currently under way in our research group and will be reported in due course.
EXPERIMENTAL
General Methods
Solvents were purified in the usual way. All commercially available
reagents were used as received. All reactions were monitored by TLC anal-
ysis and TLC was performed on silica gel HF with detection by charring with
30% (v/v) H2SO4 in CH3OH or by UV detection. Column chromatography was
conducted by elution of a column (8 × 100, 16 × 240, 18 × 300, 35 × 400mm) of
silica gel (200–300 mesh) with EtOAc-PE (b.p. 60–90◦C) as the eluent. Air- and
moisture-sensitive reactions were performed under dry N2 atmosphere. Opti-
cal rotations were recorded using a Perkin-Elmer 241 polarimeter. 1H and 13
NMR spectra were recorded with Varian XL-300 spectrometers in CDCl3 or
C

H. Zhao et al.
108
1
D2O solutions. Internal references: TMS (δ 0.000 ppm for H), CDCl3 (δ 77.00
1
1
ppm for 13C), HOD (δ 4.700 for H). H NMR and 13C NMR signals of some
compounds were assigned with the aid of COSY and HSQC. Elemental anal-
ysis was performed on a Yanaco CHN Corder MF-3 automatic elemental ana-
lyzer. Mass spectra were recorded with a VG PLATFORM mass spectrometer
using the electronspray ionization (ESI) mode. Solutions were concentrated at
a temperature less than 60◦C under diminished pressure.
p-Methoxyphenyl 3,4-di-O-benzoyl-6-deoxy-α-L-
rhamnopyranoside (3)
Benzoyl chloride (0.55 mL, 4.80 mmol) in dry dichloromethane (1.7 mL)
was added dropwise to the solution of compound 1[11] (1.7 g, 4.5 mmol) and dry
pyridine (1.8 mL) in dry dichloromethane (10 mL) over 30 min under nitrogen
atmosphere, which was cooled in an ice-salt bath. The reaction mixture was
slowly raised to rt and stirred for 12 h, at the end of which time TLC (2:1
petroleum ether-EtOAc) indicated that the reaction was complete. The reaction
mixture was diluted with CH2Cl2 (100 mL), washed with ice water and 1 M
HCl, and dried (Na2SO4). The solution was concentrated, and the residue was
subjected to column chromatography (4:1 petroleum ether-EtOAc) to give the
desired product 3 (2.0 g, 92%) as a foamy solid. Rf = 0.23 (3:1 petroleum ether-
25
1
EtOAc); [α]D –42.1 (c 1.0, CHCl3); H NMR (CDCl3): δ 7.99–7.32 (m, 10 H,
Bz-H), 7.11–6.85 (m, 4 H, MeOC6H4), 5.80 (dd, J = 2.9, 10.0 Hz, 1 H, H-3),
5.68 (dd, J = 10.0, 9.9 Hz, 1 H, H-4), 5.51 (d, J = 1.8 Hz, 1 H, H-1), 4.49 (dd,
J = 1.8, 2.9 Hz, 1 H, H-2), 4.28–4.19 (m, 1 H, H-5), 3.78 (s, 3 H, OCH3), 2.72 (s,
1 H, OH), 1.29 (d, J = 6.3 Hz, 3 H, H-6); Anal. Calcd for C27H26O8: C, 67.77; H,
5.48. Found: C, 67.81; H, 5.50.
p-Methoxyphenyl 3-O-allyloxycarbonyl-4-O-benzoyl-α-L-
rhamnopyranoside (4)
Compound 1[11] (3.7 g, 10 mmol) was dissolved in dry dichloromethane (40
mL) containing pyridine (8.1 mL, 100 mmol); then under N2 atmosphere, al-
lyl chloroformate (1.2 mL, 11 mmol) in anhydrous dichloromethane (10 mL)
was added dropwise to the solution over 30 min at 0◦C. The reaction mixture
was slowly raised to rt and stirred for 2 h, at the end of which time TLC (3:1
petroleum ether-EtOAc) indicated that the reaction was complete. The reaction
mixture was diluted with dichloromethane (100 mL), washed with water and
1 M HCl, and dried (Na2SO4). The solution was concentrated, and purification
of the residue by column chromatography on silica gel (3:1 petroleum ether-
EtOAc) gave compound 4 (4.2 g, 93%) as a syrup. Rf = 0.4 (3:1 petroleum ether-
25
1
EtOAc); [α]D –50.3 (c 1.0, CHCl3); H NMR (CDCl3): δ 8.05–7.41 (m, 5 H,

Synthesis of O-Antigen-Related Tetrasaccharides
109
Bz-H), 7.07–6.83 (m, 4 H, MeOC6H4), 5.76–5.67 (m, 1 H, CH2 CH-CH2OCO),
5.54–5.47 (m, 3 H, H-1, H-3, H-4), 5.21–5.04 (m, 2 H, CH2 CH-CH2OCO),
4.51–4.48 (m, 2 H, CH2 = CH-CH2OCO), 4.38 (dd, J = 0.5, 2.7 Hz, 1 H, H-2),
4.16–4.11 (m, 1 H, H-5), 3.77 (s, 3 H, OCH3), 2.85 (s, 1 H, OH), 1.25 (d, J = 6.3
Hz, 3 H, H-6); Anal. Calcd for C24H26O9: C, 62.88; H, 5.72. Found: C, 62.71; H,
5.89.
p-Methoxyphenyl 3-O-allyloxycarbonyl-2,4-di-O-
benzoyl-α-L-rhamnopyranoside (5)
Compound 4 (4.0 g, 8.7 mmol) was benzoylated under the same conditions
as that used for the preparation of 3 from 1,[11] giving 5 (4.8 g, 98%) as a foamy
25
1
solid. Rf = 0.7 (3:1 petroleum ether-EtOAc); [α]D +36.8 (c 0.5, CHCl3); H
NMR (CDCl3): δ 8.14–7.43 (m, 10 H, Bz-H), 7.09–6.84 (m, 4 H, MeOC6H4),
5.79–5.58 (m, 5 H), 5.18–5.02 (m, 2 H, CH2 CH-CH2OCO), 4.51–4.49 (m, 2
H, CH2 CH-CH2OCO), 4.26–4.21 (m, 1 H, H-5), 3.77 (s, 3 H, OCH3), 1.29 (d,
J = 6.3 Hz, 3 H, H-6). Anal. Calcd for C31H30O10: C, 66.18; H, 5.38. Found: C,
66.03; H, 5.77.
3-O-Allyloxycarbonyl-2,4-di-O-benzoyl-α-L-rhamnopyranosyl
trichloroacetimidate (6)
To a solution of 5 (3.0 g, 5.3 mmol) in 80% MeCN (100 mL) was added
ceric ammonium nitrate (11.7 g, 21.3 mmol). The mixture was stirred for 20
min at 35◦C, at the end of which time TLC (3:1 petroleum ether-EtOAc) indi-
cated that the reaction was complete. The solvents were evaporated in vacuo at
50◦C to give a residue, which was dissolved in CH2Cl2, and washed with water.
The organic phase was dried (Na2SO4) and concentrated. Purification by silica
gel chromatography with 3:1 petroleum ether-EtOAc as the eluent afforded a
foamy residue. The residue was dried under high vacuum for 2 h, then was
dissolved in dry CH2Cl2 (50 mL) and trichloroacetonitrile (2 mL, 19.4 mmol)
and 1,8-diazabicyclo[5.4.0] undecene (DBU) (0.2 mL, 20 mmol) were added.
The mixture was aged under the nitrogen atmosphere until completion (TLC,
3:1 petroleum ether-EtOAc). Concentration of the reaction mixture and purifi-
cation of the residue by column chromatography (4:1 petroleum ether-EtOAc)
gave 6 (2.2 g, 68%) as a white foamy solid. Rf = 0.65 (3:1 petroleum ether-
25
1
EtOAc); [α]D +98.2 (c 0.5, CHCl3); H NMR (CDCl3): δ 8.81 (s, 1 H, C NH),
8.14–7.44 (m, 10 H, Bz-H), 6.45 (d, J = 1.8 Hz, 1 H, H-1), 5.82 (dd, J = 1.8, 3.2
Hz, 1 H, H-2), 5.72–5.51 (m, 3 H, CH2 CH-CH2OCO, H-3, H-4), 5.18–5.03 (m,
2 H, CH2 CH-CH2OCO), 4.51–4.49 (m, 2 H, CH2 CH-CH2OCO), 4.35–4.26
(m, 1 H, H-5), 1.39 (d, J = 6.3 Hz, 3 H, H-6). Anal. Calcd for C26H24Cl3NO9: C,
51.97; H, 4.03; N, 2.33. Found: C, 52.30; H, 3.91; N, 2.59.

H. Zhao et al.
110
p-Methoxyphenyl 3ꢀ,4ꢀ,6ꢀ-tri-O-acetyl-α-D-glucopyranose-
1ꢀ,2ꢀ-(3,4-di-O-benzoyl-α-L- rhamnopyranoside 2-yl)
Orthoacetate (7)
Compound 3 (0.56 g, 1.2 mmol) and 2[15] (0.62 g, 1.3 mmol) and 4 A molec-
ular sieves (1.0 g) were dried together under high vacuum for 2 h, then dis-
solved in anhydrous redistilled CH2Cl2 (50 mL). TMSOTf (18 uL, 0.10 mmol)
was added dropwise at –10◦C with nitrogen protection. The reaction mixture
was allowed to rise to rt and was stirred for 2 h, and then was quenched with
Et3N (2 drops). Filtration of the reaction mixture and concentration of the fil-
trate, followed by purification of the residue by column chromatography (5:1
petroleum ether-EtOAc), provided the orthoester 7 (0.87 g, 81%). Rf = 0.30
˚
25
1
(3:1 petroleum ether-EtOAc). [α]D +5.26 (c 1.0, CHCl3), H NMR (CDCl3): δ
7.98–7.34 (m, 10 H, Bz-H), 7.11–6.86 (m, 4 H, MeOC6H4), 5.71 (d, J = 4.9 Hz,
1 H, H-1ꢁ), 5.69 (dd, J = 3.2, 9.5 Hz, 1 H, H-3), 5.60 (dd, J = 9.5, 10.2 Hz, 1 H,
H-4), 5.38 (d, J = 1.8 Hz, 1 H, H-1), 4.95 (dd, J = 2.8, 3.0 Hz, 1 H, H-3ꢁ), 4.81
(dd, J = 2.8, 9.3 Hz, 1 H, H-4ꢁ), 4.51 (dd, J = 1.8, 3.2 Hz, 1 H, H-2), 4.39 (dd,
J = 4.9, 3.0 Hz, 1 H, H-2ꢁ), 4.12–4.10 (m, 3 H), 3.83–3.75 (m, 4 H), 2.05–2.04
(m, 9 H, 3 × CH3CO), 1.64 (s, 3 H, CH3CO3), 1.30 (d, J = 6.2 Hz, 3 H, H-6).
13C NMR (CDCl3): δ 170.4, 169.3, 168.6, 165.9, 165.5, (5 C O), 155.1, 150.0,
133.2, 133.1, 129.5, 129.3, 129.2, 129.2, 128.3, 128.3, 121.9, 117.7, 114.6, 98.2
(C-1), 97.0 (C-1), 77.2, 73.3, 71.1, 71.1, 70.3, 69.7, 67.6, 67.3, 67.1, 62.9, 55.5
(OCH3), 21.5 (CH3CO3), 20.6, 20.6, 20.5 (3 CH3CO), 17.4 (C-6). Anal. Calcd for
C41H44O17: C, 60.89; H, 5.48. Found: C, 60.95; H, 5.21.
p-Methoxyphenyl 2,3,4,6-tetra-O-benzoyl-β-D-
glucopyranose-(1→2)-3,4-di-O-benzoyl -α-L-
rhamnopyranoside (9)
Compound 3 (1.0 g, 2.1 mmol) and 8[17] (1.8 g, 2.4 mmol) were coupled
under the same conditions as that used for the preparation of 7 from 3 and 2,
giving 9 (1.9 g, 88%) as a foamy solid. Rf = 0.17 (3:1 petroleum ether-EtOAc);
25
1
[α]D +0.65 (c 0.5, CHCl3); H NMR (CDCl3): δ 7.97–7.08 (m, 30 H, Bz-H),
7.06–7.78 (m, 4 H, MeOC6H4), 5.86–5.51 (m, 6 H), 5.0 (d, J = 7.7 Hz, 1 H,
H-1ꢁ), 4.61–4.38 (m, 3 H), 4.41 (dd, J = 5.6, 12.2 Hz, 1 H, H-3), 4.14–4.01 (m,
2 H, H-5, H-5ꢁ), 3.77 (s, 3 H, OCH3), 1.24 (d, J = 6.2 Hz, 3 H, H-6); 13C NMR
(CDCl3): δ 166.0, 165.9, 165.7, 165.1, 165.0, 164.9 (6 C O), 154.9, 149.9, 133.4,
133.2, 133.0, 132.9, 132.8, 130.0, 129.8, 129.7, 129.6, 129.6, 129.5, 129.4, 129.2,
128.8, 128.7, 128.6, 128.4, 128.3, 128.2, 128.1, 128.0, 117.3, 114.5, 101.9 (C-1),
97.6 (C-1), 76.4, 72.5, 72.3, 72.0, 71.6, 71.3, 69.3, 67.3, 62.6, 55.5 (OCH3), 17.5
(C-6). Anal. Calcd for C61H52O17: C, 69.31; H, 4.96. Found: C, 69.19; H, 4.90.

Synthesis of O-Antigen-Related Tetrasaccharides
111
2,3,4,6-Tetra-O-benzoyl-β-D-glucopyranose-(1→2)-3,
4-di-O-benzoyl-α-L-rhamnopyranosyl
trichloroacetimidate (10)
Compound 9 (1.7 g, 1.6 mmol) was trichloroacetimidated under the same
conditions as that used for the preparation of 6 from 5, giving 10 (1.1 g, 61%)
25
as a foamy solid. Rf = 0.20 (3:1 petroleum ether-EtOAc); [α]D +14.7 (c 0.5,
CHCl3); 1H NMR (CDCl3): δ 8.5 (s, 1 H, C NH), 8.01–7.00 (m, 30 H, Bz-H),
6.59 (d, J = 1.8 Hz, 1 H, H-1), 5.86–5.58 (m, 5 H), 5.01 (d, J = 7.7 Hz, 1 H,
H-1ꢁ), 4.62–4.48 (m, 3 H), 4.33–4.07 (m, 2 H), 1.33 (d, J = 6.2 Hz, 3 H, H-6).
Anal. Calcd for C56H46Cl3NO16: C, 61.41; H, 4.23; N, 1.28. Found: C, 61.53; H,
4.54; N, 1.65.
p-Methoxyphenyl 3-O-allyloxycarbonyl-2,4-di-O-benzoyl-α-L-
rhamnopyranosyl-(1→2) -3,4-di-O-benzoyl-α-L-
rhamnopyranoside (11)
Compound 3 (0.47 g, 0.97 mmol) and 6 (0.64 g, 1.1 mmol) were coupled
under the same conditions as that used for the preparation of 7 from 3 and 2,
giving 11 (0.79 g, 89%) as a foamy solid. Rf = 0.31 (3:1 petroleum ether-EtOAc);
25
1
[α]D +57.5 (c 0.5, CHCl3); H NMR (CDCl3): δ 8.10–7.34 (m, 20 H, Bz-H),
7.13–6.87 (m, 4 H, MeOC6H4), 5.98 (dd, J = 3.3, 10.0 Hz, 1 H, H-3), 5.82–5.50
(m, 6 H), 5.20–5.02 (m, 3 H), 4.54–4.51 (m, 2 H, CH2 CH-CH2), 4.48 (dd,
J = 2.0, 3.3 Hz, 1 H, H-2), 4.28–4.23 (m, 2 H), 3.79 (s, 3 H, OCH3), 1.37 (d,
J = 6.3 Hz, 3 H, H-6), 1.31 (d, J = 6.3 Hz, 3 H, H-6); 13C NMR (CDCl3): δ 165.7,
165.6, 165.1, 163.6, 155.2, 153.9, 150.1, 133.4, 133.4, 133.2, 133.2, 131.1, 130.0,
129.9, 129.7, 129.3, 129.1, 129.0, 128.9, 128.4, 128.3, 118.8, 117.6, 114.7, 99.2
(C-1), 97.7 (C-1), 72.8, 71.8, 71.5, 70.9, 70.1, 68.8, 67.7, 67.5, 55.6 (OCH3), 17.6
(C-6), 17.5 (C-6). Anal. Calcd for C51H48O16: C, 66.80; H, 5.28. Found: C, 66.86;
H, 5.13.
p-Methoxyphenyl 2,4-di-O-benzoyl-α-L-rhamnopyranosyl-
(1→2)-3,4-di-O-benzoyl-α-L-rhamnopyranoside (12)
To a cooled (–5◦C) solution of 11 (0.60 g, 0.69 mmol) and CH3COONH4
(0.50 g, 6.9 mmol) in 1:1 MeOH-THF (50 mL) in a 150-mL flask were added
NaBH4 (0.02 g, 0.46 mmol), Pd[P(C6H5)3]4 (0.03 g, 0.02 mmol), and NaBH4
(0.06 mg, 1.5 mmol) in three portions immediately one after another. The mix-
ture was vigorously stirred until TLC (3:1 petroleum ether-EtOAc) indicated
completion of the reaction. The reaction mixture was concentrated under vac-
uum, the residue was dissolved in CH2Cl2 (100 mL) and washed with water
(20 mL), and then the organic phase was dried over Na2SO4. Evaporation and
purification by flash column chromatography (4:1 petroleum ether-EtOAc) af-
forded compound 12 as a foamy solid (0.52 g, 90%). Rf = 0.24 (3:1 petroleum

H. Zhao et al.
112
25
1
ether-EtOAc); [α]D +14.7 (c 0.5, CHCl3); H NMR (CDCl3): δ 8.13–7.36 (m,
20 H, Bz-H), 7.13–6.87 (m, 4 H, MeOC6H4), 5.97 (dd, J = 3.2, 10.0 Hz, 1 H,
H-3), 5.69–5.57 (m, 3 H), 5.31 (dd, J = 9.8, 9.4 Hz, 1 H, H-4), 5.18 (d, J = 1.5
Hz, 1 H, H-1), 4.47–4.45 (m, 2 H), 4.30–4.21 (m, 2 H), 3.79 (s, 3 H, OCH3),
2.47 (d, J = 7.1 Hz, 1 H, OH), 1.35 (d, J = 6.3 Hz, 3 H, H-6), 1.31 (d, J = 6.3
Hz, 3 H, H-6); 13C NMR (CDCl3): δ 166.8, 165.7, 165.5, 165.4 (4 C O), 155.2,
150.1, 133.4, 133.3, 133.2, 133.1, 129.9, 129.8, 129.8, 129.7, 129.6, 129.6, 129.3,
129.2, 128.9, 128.4, 128.2, 117.6, 114.7, 99.5 (C-1), 97.8 (C-1), 76.7, 75.0, 72.8,
71.8, 70.7, 68.7, 67.5, 67.2, 55.5 (OCH3), 17.7 (C-6), 17.5 (C-6). Anal. Calcd for
C47H44O14: C, 67.78; H, 5.33. Found: C, 67.91; H, 5.38.
p-Methoxyphenyl 2,3,4,6-tetra-O-benzoyl-β-D-glucopyranose-
(1→2)-3,4-di-O-benzoyl-α-L-rhamnopyranosyl-(1→3)-2,
4-di-O-benzoyl-α-L-rhamnopyranosyl-(1→2)-3,
4-di-O-benzoyl-α-L-rhamnopyranoside (13)
Compound 12 (0.50 g, 0.60 mmol) and 10 (0.90 g, 0.80 mmol) were coupled
under the same conditions as that used for the preparation of 7 from 3 and
2, giving 13 (0.83 g, 78%) as a foamy solid. Rf = 0.31 (2:1 petroleum ether-
EtOAc); [α]D +73.7 (c 0.5, CHCl3), 1H NMR (CDCl3): δ 8.12–7.26 (m, 50 H,
25
Bz-H), 7.12–6.85 (m, 4 H, MeOC6H4), 5.97 (dd, J = 3.2, 10.1 Hz, 1 H, H-3),
5.74 (dd, J = 1.8, 3.2 Hz, 1 H, H-2ꢁ), 5.70 (dd, J = 9.8, 10.1 Hz, 1 H, H-4), 5.63
(dd, J = 9.7, 9.9 Hz, 1 H), 5.61 (d, J = 1.8 Hz, 1 H, H-1), 5.56 (dd, J = 9.5,
9.7 Hz, 1 H), 5.48–5.40 (m, 3 H), 5.33 (d, J = 1.8 Hz, 1 H, H-1ꢁ), 5.30 (dd, J =
9.6, 9.9 Hz, 1 H, H-4ꢁꢁ), 5.26 (d, J = 1.7 Hz, 1 H, H-1ꢁꢁ), 4.77 (d, J = 7.6 Hz,
1 H, H-1ꢁꢁꢁ), 4.67 (dd, J = 3.2, 9.4 Hz, 1 H, H-3ꢁ), 4.50 (dd, J = 1.8, 3.2 Hz, 1
H, H-2), 4.33–3.97 (m, 6 H), 3.79 (s, 3 H, OCH3), 3.51–3.45 (m, 1 H), 1.32 (d, J
= 6.2 Hz, 3 H, H-6), 1.30 (d, J = 6.2 Hz, 3 H, H-6), 1.04 (d, J = 6.2 Hz, 3 H,
H-6); 13C NMR (CDCl3): δ 165.8, 165.7, 165.5, 165.5, 165.3, 165.0, 165.0, 165.0,
164.8, 164.9 (10 C O), 155.2, 150.3, 133.2, 133.2, 133.1, 133.0, 132.9, 132.7,
132.7, 130.0, 129.9, 129.8, 129.7, 129.7, 129.6, 129.5, 129.3, 129.1, 129.1, 128.9,
128.9, 128.6, 128.5, 128.4, 128.3, 128.1, 128.1, 117.7, 114.8, 100.9 (C-1), 100.4
(C-1), 99.5 (C-1), 97.9 (C-1), 76.7, 75.5, 74.6, 73.3, 72.7, 72.1, 72.0, 72.0, 71.8,
71.6, 71.4, 71.0, 69.7, 67.8, 67.7, 67.6, 62.8, 55.7 (OCH3), 17.7 (C-6), 17.3 (C-6).
Anal. Calcd for C101H88O29: C, 68.70; H, 5.02. Found: C, 68.88; H, 5.10.
p-Methoxyphenyl β-D-glucopyranose-(1→2)-α-L-
rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-
(1→2)-α-L-rhamnopyranoside (I)
Tetrasaccharide 13 (400 mg, 0.23 mmol) was dissolved in satd NH3-MeOH
(30 mL). After 96 h at rt, the reaction mixture was concentrated, and the

Synthesis of O-Antigen-Related Tetrasaccharides
113
residue was puried by chromatography on Sephadex LH-20 (MeOH) to afford
I (153 mg, 92%) as a foamy solid. [α]D +23.0 (c 0.5, water); 1H NMR (D2O): δ
25
7.07–6.93 (m, 4 H, MeOC6H4), 5.45 (s, 1 H, H-1), 5.39 (s, 1 H, H-1), 4.98 (s, 1
H, H-1), 4.56 (d, J = 7.9 Hz, 1 H, H-1ꢁꢁꢁ), 4.15–4.03 (m, 3 H), 3.93–3.66 (m, 11
H), 3.55–3.29 (m, 7 H), 1.28 (d, J = 6.2 Hz, H-6), 1.22 (d, J = 6.1 Hz, H-6), 1.22
(d, J = 6.1 Hz, H-6); 13C NMR (D2O): δ 154.6, 149.3, 118.9, 118.9, 115.0, 115.0
(C6H4), 104.2 (C-1), 101.9 (C-1), 100.8 (C-1), 98.1 (C-1), 80.1, 78.3, 77.8, 75.6,
75.3, 73.1, 72.0, 72.0, 71.2, 69.7, 69.7, 69.6, 69.4, 69.1, 69.1, 68.8, 60.3, 55.7
(OCH3), 16.5 (C-6), 16.5 (C-6). ESIHRMS: m/z calcd for C31H48O19Na[M+Na+]:
747.2687; C31H48O19K[M+K+]: 763.2427. Found: m/z 747.2673; 763.2413.
p-Methoxyphenyl 2,4-di-O-benzoyl-α-L-rhamnopyranoside (14)
Compound 5 (2.8 g, 4.9 mmol) was deallyloxycarbonylated under the same
conditions as that used for the preparation of 12 from 11, giving 14 (2.2 g,
25
95%) as a foamy solid. Rf = 0.23 (3:1 petroleum ether-EtOAc); [α]D –17.5 (c
1
1.0, CHCl3); H NMR (CDCl3): δ 8.15–7.44 (m, 10 H, Bz-H), 7.07–6.83 (m, 4
H, MeOC6H4), 5.59–5.56 (m, 2 H, H-1, H-2), 5.35 (dd, J = 9.8, 9.9 Hz, 1 H,
H-4), 4.52 (dd, J = 3.3, 9.9 Hz, 1 H, H-3), 4.27–4.20 (m, 1 H, H-5), 3.78 (s, 3
H, OCH3), 2.57 (s, 1 H, OH), 1.31 (d, J = 6.3 Hz, 3 H, H-6). Anal. Calcd for
C27H26O8: C, 67.77; H, 5.48. Found: C, 67.67; H, 5.30.
p-Methoxyphenyl 2,3,4,6-tetra-O-benzoyl-β-D-
glucopyranose-(1→3)-2,4-di-O-benzoyl -α-L-
rhamnopyranoside (15)
Compound 14 (0.60 g, 1.3 mmol) and 8[17] (1.1 g, 1.4 mmol) were coupled
under the same conditions as that used for the preparation of 7 from 3 and 2,
giving 15 (1.1 g, 83%) as a foamy solid. Rf = 0.15 (3:1 petroleum ether-EtOAc);
25
1
[α]D +12.9 (c 0.5, CHCl3); H NMR (CDCl3): δ 8.08–7.17 (m, 30 H, Bz-H),
7.00–6.80 (m, 4 H, MeOC6H4), 5.78–5.46 (m, 6 H), 5.1 (d, J = 7.8 Hz, 1 H, H-1ꢁ),
4.63–4.39 (m, 3 H), 4.42–4.10 (m, 2 H), 3.78 (s, 3 H, OCH3), 1.16 (d, J = 6.2 Hz,
1 H, H-6); 13C NMR (CDCl3): δ 166.0, 165.9, 165.6, 165.0, 164.9, 164.4 (6 C O),
155.2, 150.0, 133.2, 133.0, 133.0, 132.9, 132.7, 132.5, 129.9, 129.8, 129.6, 129.6,
129.5, 129.4, 129.3, 129.1, 128.8, 128.7, 128.6, 128.4, 128.4, 128.3, 128.2, 128.2,
128.1, 128.0, 127.9, 117.9, 114.5, 101.5, (C-1), 96.4 (C-1), 75.9, 72.8, 72.5, 72.1,
72.0, 71.8, 69.1, 67.0, 62.7, 55.5 (OCH3), 17.5 (C-6). Anal. Calcd for C61H52O17:
C, 69.31; H, 4.96. Found: C, 69.52; H, 4.81.

H. Zhao et al.
114
2,3,4,6-Tetra–O-benzoyl-β-D-glucopyranose-(1→3)-2,
4-di-O-benzoyl-α-L-rhamnopyranosyl
trichloroacetimidate (16)
Compound 15 (1.0 g, 1.0 mmol) was trichloroacetimidated under the same
conditions as that used for the preparation of 6 from 5, giving 16 (0.7 g, 68%)
25
as a foamy solid. Rf = 0.17 (3:1 petroleum ether-EtOAc). [α]D +20.0 (c 0.5,
1
CHCl3); H NMR (CDCl3): δ 8.8 (s, 1 H, C NH), 8.09–7.10 (m, 30 H, Bz-H),
6.45 (d, J = 2.0 Hz, 1 H, H-1), 5.80–5.41 (m, 5 H), 5.1 (d, J = 7.8 Hz, 1 H, H-1ꢁ),
4.53–4.37 (m, 3 H), 4.20–4.10 (m, 2 H), 1.22 (d, J = 6.2 Hz, 3 H, H-6). Anal.
Calcd for C56H46Cl3NO16: C, 61.41; H, 4.23; N, 1.28. Found: C, 61.27; H, 4.22;
N, 1.54.
p-Methoxyphenyl 3-O-allyloxycarbonyl-2,4-di-O-benzoyl-
α-L-rhamnopyranosyl-(1→3) -2,4-di-O-benzoyl-α
-L-rhamnopyranoside (17)
Compound 14 (0.40 g, 0.80 mmol) and 6 (0.60 g, 0.90 mmol) were coupled
under the same conditions as that used for the preparation of 7 from 3 and
2, giving 17 (0.7 g, 86%) as a foamy solid. Rf = 0.31 (3:1 petroleum ether-
25
1
EtOAc). [α]D +28.3 (c 0.5, CHCl3); H NMR (CDCl3): δ 8.10–7.38 (m, 20 H,
Bz-H), 7.07–6.84 (m, 4 H, MeOC6H4), 5.68–5.54 (m, 4 H), 5.37–5.22 (m, 4 H),
5.08–4.95 (m, 2 H), 4.66 (dd, J = 3.5, 9.8 Hz, 1 H, H-3), 4.35–4.33 (m, 2 H),
4.23–4.05 (m, 2 H), 3.78 (s, 3 H, OCH3), 1.33 (d, J = 6.2 Hz, 3 H, H-6), 1.14 (d,
J = 6.3 Hz, 3 H, H-6); 13C NMR (CDCl3): δ 166.0, 165.8, 165.3, 165.1, 155.3,
153.5, 150.0, 133.6, 133.5, 133.3, 133.2, 133.2, 131.1, 130.0, 130.0, 129.9, 129.9,
129.8, 129.7, 129.6, 129.4, 129.4, 129.3, 129.3, 129.2, 129.1, 128.7, 128.6, 128.5,
128.5, 128.4, 128.4, 128.3, 128.1, 118.6, 117.7, 117.7, 114.7, 99.2 (C-1), 96.4 (C-
1), 72.9, 72.5, 72.1, 71.4, 70.4, 68.5, 67.6, 67.4, 55.6 (OCH3), 17.7 (C-6), 17.3
(C-6). Anal. Calcd for C51H48O16: C, 66.80; H, 5.28. Found: C, 66.63; H, 5.49.
p-Methoxyphenyl 2,4-di-O-benzoyl-α-L-rhamnopyranosyl-
(1→3)-2,4-di-O-benzoyl -α-L-rhamnopyranoside (18)
Compound 17 (0.60 g, 0.70 mmol) was deallyloxycarbonylated under the
same conditions as that used for the preparation of 12 from 11, giving 18 (0.50
25
g, 90%) as a foamy solid. Rf = 0.24 (3:1 petroleum ether-EtOAc); [α]D +28.9
1
(c 0.5, CHCl3); H NMR (CDCl3): δ 8.24–7.38 (m, 20 H, Bz-H), 7.08–6.84 (m,
4 H, MeOC6H4), 5.70–5.58 (m, 3 H), 5.27 (d, J = 1.5 Hz, 1 H, H-1), 5.1 (dd, J
= 9.7, 9.7 Hz, 1 H, H-4), 5.0 (dd, J = 1.5, 3.3 Hz, 1 H, H-2), 4.6 (dd, J = 3.4,
9.8 Hz, 1 H, H-3), 4.25–3.98 (m, 3 H), 3.78 (s, 3 H, OCH3), 2.27 (d, J = 5.6 Hz,
1 H, OH), 1.33 (d, J = 6.2 Hz, 3 H, H-6), 1.15 (d, J = 6.2 Hz, 3 H, H-6); 13C

Synthesis of O-Antigen-Related Tetrasaccharides
115
NMR (CDCl3): δ 166.6, 165.9, 165.8, 165.4 (4 C O), 155.2, 149.9, 133.5, 133.3,
133.2, 129.9, 129.8, 129.6, 129.3, 129.2, 129.1, 128.7, 128.5, 128.3, 128.2, 117.6,
114.6, 99.3 (C-1), 96.3 (C-1), 76.3, 75.0, 72.9, 72.9, 72.2, 68.4, 67.3, 67.0, 55.5
(OCH3), 17.7 (C-6), 17.3 (C-6). Anal. Calcd for C47H44O14: C, 67.78; H, 5.33.
Found: C, 67.72; H, 5.14.
2,3,4,6-Tetra-O-benzoyl-β-D-glucopyranose-(1→3)-2,
4-di-O-benzoyl-α-L-rhamnopyranosyl-(1→3)-2,4-di-O-
benzoyl-α-L-rhamnopyranosyl-(1→3)-2,4-di-O-benzoyl-
α-L-rhamnopyranoside (19)
Compound 18 (0.37 g, 0.44 mmol) and 16 (0.53 g, 0.48 mmol) were coupled
under the same conditions as that used for the preparation of 7 from 3 and
2, giving 19 (0.58 mg, 75%) as a foamy solid. Rf = 0.29 (2:1 petroleum ether-
25
1
EtOAc); [α]D +208.8 (c 0.5, CHCl3); H NMR (CDCl3): δ 8.04–7.08 (m, 50 H,
Bz-H), 7.07–6.83 (m, 4 H, MeOC6H4), 5.71 (dd, J = 1.8, 3.4 Hz, 1 H, H-2), 5.61
(dd, J = 9.8, 9.8 Hz, 1 H, H-4), 5.60 (dd, J = 1.8 Hz, 1 H, H-1), 5.51 (dd, J =
9.7, 9.7 Hz, 1 H), 5.44 (dd, J = 7.8, 9.7 Hz, 1 H), 5.34 (dd, J = 9.7, 9.7 Hz, 1 H),
5.28–5.22 (m, 2 H, H-1ꢁ, H-4ꢁ), 5.19 (dd, J = 9.8, 9.9 Hz, 1 H, H-4ꢁꢁ), 5.13 (dd, J =
1.8, 3.2 Hz, 1 H, H-2ꢁ), 5.02 (dd, J = 1.9, 3.2 Hz, 1 H, H-2ꢁꢁ), 4.68 (d, J = 1.9 Hz,
1 H, H-1ꢁꢁ), 4.63 (dd, J = 3.4, 9.7 Hz, 1 H, H-3), 4.36 (d, J = 7.8 Hz, 1 H, H-1ꢁꢁꢁ),
4.23–4.00 (m, 6 H), 3.81–3.76 (m, 4 H), 3.61–3.56 (m, 1 H), 1.32 (d, J = 6.2 Hz, 3
H, H-6), 1.11 (d, J = 6.2 Hz, 3 H, H-6), 0.52 (d, J = 6.2 Hz, 3 H, H-6); 13C NMR
(CDCl3): δ 166.0, 165.9, 165.9, 165.8, 165.7, 165.3, 165.2, 164.8, 164.5, 164.3
(10 C O), 155.3, 150.1, 133.7, 133.3, 133.2, 133.0, 132.5, 130.0, 129.7, 129.7,
129.6, 129.6, 129.5, 129.5, 129.4, 129.3, 129.4, 129.2, 129.0, 128.8, 128.8, 128.7,
128.5, 128.4, 128.3, 128.1, 128.1, 128.0, 127.9, 127.9, 117.7, 114.7, 101.1 (C-1),
98.9 (C-1), 98.6 (C-1), 96.4 (C-1), 77.2, 77.2, 76.8, 76.8, 72.9, 72.8, 72.4, 72.2,
71.9, 71.7, 71.3, 70.8, 68.3, 67.6, 67.4, 67.1, 61.3, 55.6 (OCH3), 17.7 (C-6), 17.3
(C-6), 16.7 (C-6). Anal. Calcd for C101H88O29: C, 68.70; H, 5.02. Found: C, 68.90;
H, 4.81.
p-Methoxyphenyl β-D-glucopyranose-(1→3)-α-L-
rhamnopyranosyl-(1→3)-α-L- rhamnopyranosyl-(1→3)-
α-L-rhamnopyranoside (II)
Compound 19 (400 mg, 0.23 mmol) was deblocked under the same condi-
tions as that used for the preparation of I from 13, giving II (146 mg, 89%) as
25
1
a foamy solid. [α]D +64.3 (c 0.5, water), H NMR (D2O): δ 7.06–6.90 (m, 4 H,
MeOC6H4), 5.35 (s, 1 H, H-1), 5.01 (s, 2 H, H-1ꢁ, H-1ꢁꢁ), 4.65 (d, J = 7.8 Hz, 1 H,
H-1ꢁꢁꢁ), 4.27 (bs, 1 H, H-2), 4.18 (bs, 1 H, H-2ꢁ), 4.13 (bs, 1 H, H-2ꢁꢁ), 3.98–3.95
(m, 2 H), 3.90–3.78 (m, 5 H), 3.74 (s, 3 H, OCH3), 3.67–3.51 (m, 4 H), 3.48–3.28

H. Zhao et al.
116
(m, 4 H); 13C NMR (D2O): δ 154.5, 149.2, 118.6, 118.6, 114.9, 114.9 (C6H4),
103.4 (C-1), 102.0 (C-1), 101.8 (C-1), 98.8 (C-1), 79.7, 78.1, 77.6, 75.5, 75.3, 73.1,
71.1, 71.1, 70.8, 69.7, 69.6, 69.5, 69.3, 69.3, 69.0, 68.8, 60.4, 55.6 (OCH3), 16.4
(C-6), 16.4 (C-6), 16.4 (C-6). ESIHRMS: m/z calcd for C31H48O19Na[M+Na+]:
747.2687. Found: m/z 747.2674.
ACKNOWLEDGEMENTS
This work was supported by the Chinese Universities Scientific Fund (No.
2009–1-43), the National Basic Research Program of China (2010CB126105),
and NSFC (No. 20902108) of China.
REFERENCES
1. Steenhoudt, O.; Vanderleyden, J. Azospirillum, a free-living nitrogen-fixing bac-
terium closely associated with grasses: genetic, biochemical and ecological aspects.
FEMS Microbiol. Rev. 2000, FEMS Microbiol. Rev., 487–506.
2. Fedonenko, Y.P.; Konnova, O.N.; Zdorovenko, E.L.; Konnova, S.A.; Zatonsky, G.V.;
Shashkov, A.S.; Ignatov, V.V.; Knirel, Y.A. Structural analysis of the O-polysaccharide
from the lipopolysaccharide of Azospirillum brasilense S17. Carbohydr. Res. 2008, 343,
810–816.
3. Fedonenko, Y.P.; Zdorovenko, E.L.; Konnova, S.A.; Kachala, V.V.; Ignatov, V.V.
Structural analysis of the O-antigen of the lipopolysaccharide from Azospirillum
lipoferum SR65. Carbohydr. Res. 2008, 343, 2841–2844.
4. Lima, E.; Boddey, R.M.; Do¨bereiner, J. Quantification of biological nitrogen fixa-
tion associated with sugar cane using a nitrogen-15 aided nitrogen balance. Soil Biol.
Biochem. 1987, 19, 165–170.
5. Battisti, L.; Lara, J.C.; Leigh, J.A. Specific oligosaccharide form of the Rhizobium
meliloti exopolysaccharide promotes nodule invasion in alfalfa. Proc. Natl. Acad. Sci. U.
S. A. 1992, 89, 5625–5629.
6. Fedonenko, Y.P.; Egorenkova, I.V.; Konnova, S.A.; Ignatov, V.V. Involvement of the
lipopolysaccharides of Azospirilla in the interaction with wheat seedling roots. Mi-
crobiology (Moscow, Russian Federation) (Translation of Mikrobiologiya) 2001, 70(3),
329–334.
7. Jofre, E.; Lagares, A.; Mori, G. Disruption of dTDP-rhamnose biosynthesis mod-
ifies lipopolysaccharide core, exopolysaccharide production, and root colonization in
Azospirillum brasilense. FEMS Microbiol. Lett. 2004, 231, 267–275.
8. Zhang, J.J.; Kong, F. Z. Synthesis of β-D-glcp-(1→2)-[β-D-ribf-(1→3)-]α-L-rhap-
(1→3)-α-L-rhap-(1→2)-α-L-rhap, the repeating unit of the lipopolysaccharide of Ace-
tobacter diazotrophicus PAL 5. J. Carbohydr. Chem. 2002, 21(6), 579–589.
9. Prashant, R.V.; Balaram, M. Synthesis of a tetrasaccharide related to the O-
antigen from Azospirillum lipoferum SR65. Carbohydr. Res. 2010, 345, 432–436.
10. Pandey, S.; Ghosh, S.; Misra, A.K. Synthesis of a trisaccharide and a tetrasaccha-
ride from the cell-wall lipopolysaccharides of Azospirillum brasilense S17. Synthesis
2009, 15, 2584–2590.

Synthesis of O-Antigen-Related Tetrasaccharides
117
11. Sarkar, K.; Mukherjee, I.; Roy, N. Synthesis of the trisaccharide repeating unit of
the O-antigen related to the enterohemorrhagic Escherichia coli type O26:H. J. Carbo-
hydr. Chem. 2003, 22, 95–107.
12. Zhang, J.J.; Kong, F.Z. A general method for the synthesis of oligosaccharides con-
sisting of α-(1→2)- and α-(1→3)-linked rhamnan backbones and GlcNAc side chains.
Tetrahedron 2003, 59, 1429–1441.
13. Zhang, J.J.; Yan, S.Q.; Liang, X.M.; Wu, J.P.; Wang, D.Q.; Kong, F.Z. Practical
preparation of 2-azido-2-deoxy-β-D-mannopyranosyl carbonates and their application
in the synthesis of oligosaccharides. Carbohydr. Res. 2007, 342, 2810–2817.
14. Schmidt, R.R.; Kinzy, W. Anomeric-oxygen activation for glycoside synthesis: the
trichloroacetimidate method. Adv. Carbohydr. Chem. Biochem. 1994, 50, 21–123.
15. Lee, H.Y.; Kwon, J.T.; Koh, M.; Cho, M.H.; Park, S.B. Enhanced efficacy of 7-
hydroxy-3-methoxycadalene via glycosylation in in vivo xenograft study. Bioorg. Med.
Chem. Lett. 2007, 17, 6335–6339.
16. Wang, W.; Kong, F.Z. Highly regio- and stereoselective synthesis of mannose-
containing oligosaccharides with acetobromo sugars as the donors and partially pro-
tected mannose derivatives as the acceptors via sugar orthoester intermediates. Angew.
Chem. Int. Ed. 1999, 38, 1247–1250.
17. Zhao, W.; Yang, G.B.; Kong, F.Z. Synthesis of two heptasaccharide analogues of the
lentinan repeating unit. Carbohydr. Res. 2003, 338, 2813–2823.
18. Zong, G.H.; Yan, S.Q.; Liang, X.M.; Zhang, J.J.; Wang, D.Q.; Kong, F.Z. Highly
efficient removal of allyloxycarbonyl (Alloc) function provides a practical orthogonal
protective strategy for carbohydrates. Chin. Chem. Lett. 2009, 20, 127–130.