Synthesis of a trisaccharide repeating unit of the O-antigen from Burkholderia anthina and its dimer

Article abstract of DOI:10.1016/j.carres.2016.03.023

A trisaccharide repeating unit of the O-antigen from Burkholderia anthina, α-L-Rha-(1→2)-α-L-Rha-(1→2)-β-D-Gal-O(CH₂)₃NH₂ (1), and its dimer, α-L-Rha-(1→2)-α-L-Rha-(1→2)-α-D-Gal-(1→3)-α-L-Rha-(1→2)-α-L-Rha-(1→2)-β-D-Gal-O(

Full text of DOI:10.1016/j.carres.2016.03.023

Accepted Manuscript
Title: Synthesis of a trisaccharide repeating unit of the o-antigen from
burkholderia anthina and its dimer
Author: Xueyun Geng, Lizhen Wang, Guofeng Gu, Zhongwu Guo
PII:
S0008-6215(16)30070-2
DOI:
Reference:
http://dx.doi.org/doi: 10.1016/j.carres.2016.03.023
CAR 7158
To appear in:
Carbohydrate Research
Received date: 15-2-2016
Revised date: 24-3-2016
Accepted date: 25-3-2016
Please cite this article as: Xueyun Geng, Lizhen Wang, Guofeng Gu, Zhongwu Guo, Synthesis
of a trisaccharide repeating unit of the o-antigen from burkholderia anthina and its dimer,
Carbohydrate Research (2016), http://dx.doi.org/doi: 10.1016/j.carres.2016.03.023.
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Synthesis of a Trisaccharide Repeating Unit of the O-Antigen from
Burkholderia anthina and Its Dimer
Xueyun Geng, Lizhen Wang, Guofeng Gu,* and Zhongwu Guo*
National Glycoengineering Research Center, Shandong Provincial Key Laboratory of
Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan 250010, PR China
Highlights:
 A trisaccharide repeating unit of the O-antigen from Burkholderia anthina and its
dimer-hexasaccharide were synthesized via a highly convergent and efficient assembly
strategy.
 The α-1,2-linked disaccharide was successfully achieved by the armed-disarmed
glycosylation strategy.
 The synthetic oligosaccharide fragments are useful antigen targets for the development of
O-antigen-based vaccines against B. anthina infectious disease.
Graphical Abstract
Abstract: A trisaccharide repeating unit of the O-antigen from Burkholderia anthina,
-L-Rha-(12)--L-Rha-(12)-β-D-Gal-O(CH2)3NH2
(1),
and
its
dimer,
-L-Rha-(12)--L-Rha-(12)--D-Gal-(13)--L-Rha-(12)--L-Rha-(12)-β-D-Gal-O(
CH2)3NH2 (2), were synthesized via a highly convergent and efficient assembly strategy.
Sequential glycosylation of galactosyl acceptor 6 with rhamnosyl thioglycoside 7, followed by
*Corresponding authors. Tel.: +86-531-88363612; fax: +86-531-88363602.
E-mail address: guofenggu@sdu.edu.cn (G. Gu), zwguo@sdu.edu.cn (Z. Guo)
Page 1 of 21

condensation of the resulting disaccharide acceptor 9 with rhamnosyl imidate donor 10, gave the
title molecule 1 after global deprotection. The title hexasaccharide 2 was assembled in a
convergent [2+2+2] manner, in which α-1,2-linked disaccharide 12 was initially obtained by the
coupling reaction of disarmed thiorhamnoside acceptor 15 with armed thiogalactoside donor 14.
Sequential glycosylation of disaccharide acceptor 9 with thioglycoside donors 12 and 13
afforded the target compound 2 after global deprotection.
Keywords: Burkholderia anthina; Lipopolysaccharide; O-Antigen; Carbohydrate vaccine;
Synthesis
1. Introduction
Lipopolysaccharides (LPSs) on the bacterial cell surface, which are the major components of
bacterial cell glycocalyx of Gram-negative bacteria, are important virulence factors for bacterial
pathogenicity.[1,2] O-Polysaccharides (OPSs), or O-antigens, are prominent part of LPSs exposed
on the bacterial cell surface, which have exhibited important roles in bacterial immunology, such
as protecting bacteria against host immune recognition, complement attack, and other related
responses.[2,3] Thereby, they are considered as the attractive candidates for the development of
novel protective and therapeutic carbohydrate-based antibacterial vaccines. [4]
The Burkholderia cepacia complex (Bcc), a group of phenotypically similar but genotypically
distinct Gram-negative bacteria,[5,6] is recognized as an important opportunistic human pathogen
that can cause fatal lung infections in cystic fibrosis patients, resulting in rapid and clinically
uncontrollable “cepacia syndrome” such as necrotizing pneumonia and septicemia that usually
lead to high mortality.[7-11] Therefore, the development of safe and effective therapeutic protocols
for prevention and treatment of Bcc infections are in urgent need. It has been clearly revealed
that the LPS O-antigens of Bcc were associated with bacterial invasion and virulence.[12,13] For
example, the O-antigen of B. cenocepacia K56-2 was conductive to inflammatory cytokine IL-1β
production.[12] Moreover, it also modulated phagocytosis of macrophages and interfered with
bacterial adhesion to bronchial epithelial cells.[13] Thus, the LPS O-antigens from Bcc bacteria
are useful antigen targets in developing carbohydrate-based vaccines against Bcc infection.[14,15]
Many O-antigens from Bcc have been structurally elucidated.[16] For example, Molinaro and
coworkers have recently isolated and identified the O-antigen from B. anthina strain LMG20983
Page 2 of 21

with the following trisaccharide repeat unit: -L-Rha-(1→2)--D-Rha-(12)--D-Gal-(13)[17]
(Figure 1). Subsequently, Vogel and coworkers reported the chemical synthesis of one form of
this trisaccharide repeating unit and its dimer in which the L-rhamnosyl residue was the reducing
end.[18,19] Using NMR technology and molecular dynamics (MD) simulation approaches, Silipo
and coworkers investigated the binding between these synthetic oligosaccharide ligands and the
lipopolysaccharide-specific monoclonal antibody (5D8).[19] Saturation transfer difference (STD)
NMR experiments revealed that the D-galactosyl residue might play an important role in the
intermolecular interactions because it gave rise to significant STD signals compared to that of the
rhamnosyl residues. In view of the important role played by galactosyl residue, we present herein
a chemical synthesis of the trisaccharide repeat unit of O-antigen from B. anthina and its dimer
1-2 containing terminal D-galactosyl residue (Figure 1). Moreover, these synthetic targets were
designed to contain a free amino group linked to their reducing end, which would enable their
regioselective conjugation with carrier molecules to generate glycoconjugate vaccines.
2. Results and Discussion
The synthesis of compound 1 is outlined in Scheme 1. First, 1-thio-β-D-galactopyranoside
(IPTG) 3 was converted into 4 in a good yield of 78% via tin complex-directed[20] regioselective
benzylation. Thereafter, the 4,6-O-positions in 4 were protected with α,α-dimethoxytoluene
using a catalytic amount of p-toluenesulfonic acid (p-TsOH) in CH3CN[21] to afford 5 in a 95%
yield. As previously reported,[22] the 2-O-unprotected thioglycoside 5 was directly used as a
glycosyl donor for the glycosylation of 3-azido-1-propanol[23] under the influence of
o
N-iodosuccinimide (NIS) and trimethylsilyl triflate (TMSOTf) at -40 C to generated β-linked
6[24] selectively in a high yield (80%). The coupling constant of H-1 signal (J1,2 = 7.8 Hz) at δ
1
4.30 ppm in the H NMR spectrum of 6 confirmed the β-configuration of the new glycosidic
bond. Glycosylation of 6 with thiorhamnoside 7[25] under the promotion of NIS and TMSOTf
furnished disaccharide 8 that was deacetylated with sodium methoxide in methanol to afford 9 as
a glycosyl acceptor in an overall yield of 82% for 2 steps. Then, 9 was glycosylated with
trichloroacetimidate 10[26] in the presence of TMSOTf in CH2Cl2 at 0 oC smoothly to produce the
fully protected trisaccharide 11 in an 85% yield. Finally, global deprotection of 11 in 2 steps,
including debenzoylation with sodium methoxide in methanol and debenzylation under
hydrogenation conditions in methanol and H2O (9:1) with 10% Pd/C as the catalyst, afforded
Page 3 of 21

trisaccharide 1 in a good 75% overall yield, after purification by size-exclusion chromatography
on a Bio-Gel P-2 column. The 1H, 13C NMR and MS data of the product were in full agreement
with the desired structure of 1.
For the synthesis of compound 2, the dimer of the above repeating unit, the least
straightforward step was constructing the α-galactopyranosyl linkage. Accordingly, our plan was
to accomplish the α-galactopyranosylation first, and the whole molecule could be assembled via
a convergent [2+2+2] strategy with 9, 12 and 13 as the key disaccharide blocks (Scheme 2).
Consequently, the α-1,2-linked disaccharide 12 could be prepared from glycosylation of
disarmed thiorhamnoside 15 with armed thiogalactoside 14,[27] whereas 13 could be obtained
from the coupling reaction of rhamnosyl acceptor 16[25] with trichloroacetimidate 10.
As shown in Scheme 3, treatment of 5 with para-methoxybenzyl chloride and sodium hydride
o
in DMF at 0 C afforded thiogalactoside 14 (87%) smoothly, which was used to successfully
glycosylate the disarmed thiorhamnoside 15 under the promotion of NIS and AgOTf in Et2O[28]
at -30 oC to furnish the desired disaccharide 12 in a 73% yield. The α-linkage in 12 was assigned
based on the coupling constant of the galactosyl H-1' signal (J1',2' = 3.6 Hz) at δ 5.27 ppm of its
1H NMR spectrum. On the other hand, glycosylation of 16 with trichloroacetimidate 10 in the
o
presence of a catalytic amount of TMSOTf in anhydrous CH2Cl2 at 0 C afforded disaccharide
13 in an excellent yield (85%).
With disaccharides 9, 12, and 13 in hand, compound 2 was assembled in a [2+2+2] manner
(Scheme 4). Glycosylation of 9 with 12 at -78 oC in CH2Cl2 using NIS and AgOTf as promoters
gave tetrasaccharide 17, which was then treated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ)[29] in CH2Cl2 and H2O (v/v 14:1) to selectively cleavage the PMB group at the galactosyl
residue 2-O-postison, furnishing tetrasaccharide 18 in a 57% overall yield. Condensation
reaction between 18 and 13 in CH2Cl2 was promoted with NIS and AgOTf at -78 oC to afford the
fully protected hexasaccharide 19 in a high yield (85%). Subsequently, the target compound 2
was obtained upon global deprotection in two steps, as described for 1, in a 90% overall yield.
1
1
The JCH coupling constants of all rhamnosyl residues in the H-coupled gHSQC spectrum of 2
were over 172 Hz, which confirmed the α-configuration of all rhamnosidic bonds. The synthetic
Page 4 of 21

1
target and all of the synthetic intermediates involved were fully characterized with 1D, 2D H
and 13C NMR spectrometry and MS.
In conclusion, a highly efficient and convergent strategy was developed for the synthesis of
the trisaccharide repeating unit of B. anthina O-antigen and its dimer, in which the D-galactosyl
residue is at the reducing end. The trisaccharide was assembled via stepwise glycosylation using
monosaccharyl building blocks 6, 7 and 8. The dimer-hexasaccharide was prepared via [2+2+2]
glycosylation reaction, in which the α-1,2-linked disaccharide 12 was successfully achieved by
the armed-disarmed glycosylation strategy. Moreover, there was a 3-aminopropanyl linker at the
reducing end of both target molecules 1 and 2, which would enable them convenient attachment
to functionalized biomolecules, such as immunological carrier molecules, for the exploration of
B. anthina immunology and the development of O-antigen-based vaccines against B. anthina.
3. Experimental Section
o
3.1 General methods: Optical rotations were determined at 25 C with a Rudolph Autopol I
1
automatic polarimeter. H and 13C NMR spectra were recorded with an Agilent 600 MHz
spectrometer for solutions in CDCl3 or D2O. Chemical shifts (δ) are given in ppm downfield
from internal Me4Si or with the DHO signal as reference when D2O was used as the solvent.
Positive-mode electrospray ionization (ESI) high-resolution mass spectroscopy (HRMS) was
recorded on a JEOL JMS-DX-303HF spectrometer. MALDI-TOF mass spectra were recorded on
an AXIMA Confidence spectrometer using 2,5-dihydroxybenzoic acid (DHB) as matrix. Thin
layer chromatography (TLC) was performed on silica gel HF254 plates, detected by charring
using 30% (v/v) H2SO4 in MeOH or by means of a UV detector. Silica gel column
chromatography was conducted with mixtures of ethyl acetate and petroleum ether (b. p. 60–90
o
oC), hexane, or toluene as the eluents. Solution concentration was performed at <60 C under
diminished pressure.
3.2 Isopropyl 3-O-Benzyl-1-thio-β-D-galactopyranoside (4)
A mixture of 3 (IPTG, 3.0 g, 12.6 mmol) and Bu2SnO (4.72 g, 18.9 mmol) in dry MeOH and
toluene (v/v 1:1, 60 mL) was refluxed for 4 h. After the solvent was removed under reduced
pressure, the resulting residue was dissolved in toluene (60 mL), and TBAB (0.81 g, 2.52 mmol)
Page 5 of 21

o
and BnBr (2.3 mL, 18.9 mmol) were added. The mixture was stirred at 70 C for 5 h, and then
concentrated under reduced pressure. The above residue was diluted with CH2Cl2 (100 mL) and
washed with water (250 mL). The organic phase was dried over Na2SO4, and then concentrated.
Purification of the residue by column chromatography (CHCl3/MeOH 15:1) yielded 4 (3.2 g,
78%) as a white solid. []D25 -7o (c 0.2, CHCl3); H NMR (600 MHz, CDCl3): δ 7.41-7.27 (m,
1
5H, Ph), 4.78 (d, 1H, J = 11.8 Hz, CH2Ph), 4.76 (d, 1H, J = 11.8 Hz, CH2Ph), 4.37 (d, 1H, J =
9.6 Hz, H-1), 4.04 (br s, 1H, H-4), 3.94 (ddd, 1H, J = 11.4, 6.8, 4.2 Hz, H-6a), 3.82-3.73 (m, 2H,
H-2, H-6b), 3.52 (m, 1H, H-5), 3.45 (dd, 1H, J = 9.0, 3.6 Hz, H-3), 3.28-3.16 (m, 1H, -SCH-),
2.63 (s, 1H, -OH), 2.49 (s, 1H, -OH), 2.20 (m, 1H, -OH), 1.34 (d, 3H, J = 6.6 Hz, -SCH(CH3)2),
13
1.33 (d, 3H, J = 6.6 Hz, -SCH(CH3)2); C NMR (150 MHz, CDCl3): δ 137.62, 128.62, 128.12,
127.92, 86.09, 81.14, 78.27, 72.20, 69.63, 67.43, 62.70, 35.56, 24.20, 24.09; MALDI-TOF MS:
calcd for (C16H24O5S+Na+) m/z, 351.12; found, 351.74.
3.3 Isopropyl 3-O-Benzyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (5)
To a solution of 4 (3.0 g, 9.14mmol) in dry CH3CN (20 mL) was added benzaldehyde
dimethyl acetal (1.9 mL, 13.72 mmol) and TsOH (120 mg, 0.7 mmol) at rt. The reaction mixture
was stirred at 50 oC for 5 h, at which time TLC indicated the completion of reaction. The mixture
was neutralized with Et3N and concentrated. The residue was purified by flush silica gel column
chromatography with petroleum ether and ethyl acetate (3:1) as the eluents to afford 5 (3.61 g,
95%) as a white foamy solid. []D25 -4o (c 0.5, CHCl3); 1H NMR (600 MHz, CDCl3): δ 7.52-7.48
(m, 2H, Ph), 7.41-7.38 (m, 2H, Ph), 7.37-7.26 (m, 6H, Ph), 5.44 (s, 1H, CHPh), 4.77 (d, 1H, J =
12.0 Hz, CH2Ph), 4.74 (d, 1H, J = 12.0 Hz, CH2Ph), 4.41 (d, 1H, J = 9.6 Hz, H-1), 4.27 (dd, 1H,
J = 12.6, 1.2 Hz, H-6a), 4.17 (d, 1H, J = 3.6 Hz, H-4), 4.01 (td, 1H, J = 9.6, 1.2 Hz, H-2), 3.96
(dd, 1H, J = 12.6, 1.8 Hz, H-6b), 3.50 (dd, 1H, J = 9.6, 3.6 Hz, H-3), 3.36 (s, 1H, H-5), 3.32-3.23
(m, 1H, -SCH-), 2.68 (d, 1H, J = 1.2 Hz, -OH), 1.37 (d, 3H, J = 6.6 Hz, -SCH(CH3)2), 1.32 (d,
13
3H, J = 6.6 Hz, -SCH(CH3)2); C NMR (150 MHz, CDCl3): δ 138.13, 137.86, 128.94, 128.77,
128.44, 128.12, 127.87, 127.83, 126.42, 125.89, 101.19, 85.48, 80.34, 73.51, 71.48, 70.00, 69.44,
68.46, 34.57, 24.49, 23.99; MALDI-TOF MS: calcd for (C23H28O5S+Na+) m/z, 439.15; found,
439.77.
3.4 3-Azidopropyl 3-O-Benzyl-4,6-O-benzylidene-β-D-galactopyranoside (6)
Page 6 of 21

To a mixture of 5 (1.0 g, 2.4 mmol), 3-azido-1-propanol (364 mg, 3.6 mmol), and 4 Å MS
(1.0 g) in anhydrous CH2Cl2 (15 mL) were added NIS (648 mg, 2.88 mmol) and AgOTf (10.2
mg, 0.04 mmol) at -20 oC under a N2 atmosphere. The mixture was stirred under these conditions
for 30 min, and then neutralized with Et3N and concentrated. The residue was purified by
column chromatography (petroleum ether/ethyl acetate 8:1) to give 6 (847 mg, 80%) as syrup.
[]D25 6o (c 0.2, CHCl3); 1H NMR (600 MHz, CDCl3): δ 7.53-7.49 (m, 2H, Ph), 7.40-7.37 (m, 2H,
Ph), 7.37-7.26 (m, 6H, Ph), 5.45 (s, 1H, CHPh), 4.74 (d, 1H, J = 12.0 Hz, CH2Ph), 4.72 (d, 1H, J
= 12.0 Hz, CH2Ph), 4.30 (d, 1H, J = 7.8 Hz, H-1), 4.29 (dd, 1H, J = 12.6, 1.2 Hz, H-6a), 4.12 (d,
1H, J = 3.0 Hz, H-4), 4.04-3.96 (m, 3H, H-2, H-6b, -OCH2-), 3.66-3.60 (m, 1H, -OCH2-), 3.48
(dd, 1H, J = 9.6, 3.6 Hz, H-3), 3.47-3.40 (m, 2H,-CH2N3), 3.36 (br s, 1H, H-5), 2.46 (d, 1H, J =
13
1.8 Hz, -OH), 1.96-1.85 (m, 2H, -OCH2CH2CH2N3); C NMR (150 MHz, CDCl3): δ 137.97,
137.67, 128.91, 128.46, 128.11, 127.89, 127.84, 126.31, 103.00, 101.07, 79.14, 72.98, 71.48,
+
69.91, 69.25, 66.67, 66.48, 48.42, 29.09; ESI-HRMS: calcd for (C16H24O5S+NH4 ) m/z,
459.2244; found, 459.2233.
3.5
3-Azidopropyl
2-O-Acetyl-3,4-di-O-benzyl--L-rhamnopyranosyl-(12)-3-O-benzyl-4,6-O-benzylidene-β-
D-galactopyranoside (8)
To a solution of 6 (300 mg, 0.678 mmol) and 7 (401 mg, 0.816 mmol) in anhydrous CH2Cl2
(10 mL) was added NIS (184 mg, 0.813 mmol) and AgOTf (15 mg, 0.038 mmol) at -78 oC under
a N2 atmosphere. The reaction mixture was stirred for 2 h under these conditions, when TLC
indicated the disappearance of 6. The mixture was neutralized with Et3N and concentrated. The
residue was purified by flash column chromatography (petroleum ether/ethyl acetate 3:1) to yield
8 (468 mg, 85%) as a white foamy solid. []D25 4o (c 0.2, CHCl3); 1H NMR (600 MHz, CDCl3): δ
7.61-7.18 (m, 20H, Ph), 5.52 (dd, 1H, J = 3.6, 1.8 Hz, H-2), 5.46 (s, 1H, CHPh), 5.29 (d,1H, J =
1.8 Hz, H-1), 4.92 (d, 1H, J = 10.8 Hz, CH2Ph), 4.73 (d, 1H, J = 10.8 Hz, CH2Ph), 4.69 (d, 1H, J
= 12.0 Hz, CH2Ph), 4.66 (d, 1H, J = 12.0 Hz, CH2Ph), 4.63 (d, 1H, J = 10.8 Hz, CH2Ph), 4.51 (d,
1H, J = 10.8 Hz, CH2Ph), 4.30 (dd, 1H, J = 12.0, 1.2 Hz, H-6a), 4.29 (d, 1H, J = 7.8 Hz, H-1),
4.19-4.13 (m, 1H, H-5), 4.13-4.09 (br d, 1H, J = 3.6 Hz, H-4), 4.03 (t, 1H, J = 9.6 Hz, H-2), 4.02
(dd, 1H, J = 12.0, 1.2 Hz, H-6b), 4.00-3.94 (m, 1H, -OCH2-), 3.89 (dd, 1H, J = 9.6, 3.6 Hz, H-3),
3.56 (dd, 1H, J = 9.6, 3.6 Hz, H-3), 3.55-3.50 (m, 1H, -OCH2-), 3.42 (t, 1H, J = 9.6 Hz, H-4),
Page 7 of 21

3.41-3.28 (m, 3H, H-5, -CH2N3), 2.12 (s, 3H, -COCH3), 1.89-1.80 (m, 2H, -OCH2CH2CH2N3),
1.30 (d, 3H, J = 6.0 Hz, H-6); 13C NMR (150 MHz, CDCl3): δ 170.22 (-COCH3), 138.77, 138.23,
137.81, 137.67, 128.89, 128.43, 128.33, 128.26, 128.13, 128.07, 128.02, 127.88, 127.79, 127.60,
127.52, 126.29, 101.82 (C-1), 100.96 (CHPh), 98.20 (C-1), 80.39 (C-3), 80.11 (C-4), 78.23
(C-3), 75.24 (CH2Ph), 73.08 (C-2), 72.64 (C-4), 71.62 (CH2Ph), 71.08 (CH2Ph), 69.24 (C-6),
68.68 (C-2), 67.63 (C-5), 66.42 (C-5), 66.17 (-OCH2CH2-), 48.43 (-CH2N3), 29.19
+
(-OCH2CH2CH2N3), 21.10 (-COCH3), 17.85 (C-6); ESI-HRMS: calcd for (C45H51O3S11+NH4 )
m/z, 827.3867 found, 827.3883.
3.6
3-Azidopropyl
3,4-di-O-Benzyl--D-rhamnopyranosyl-(12)-3-O-benzyl-4,6-O-benzylidene-β-D-galactop
yranoside (9)
To a solution of 8 (468 mg, 0.58 mmol) in CH2Cl2 and MeOH (v/v 1:1, 10 mL) was added
NaOMe (0.5 M) in MeOH (1 mL) at rt. After the reaction mixture was stirred for 2 h, it was
neutralized with Amberlite IR-120 (H+), filtered, and concentrated. Purification of the residue by
flash column chromatography with hexane and ethyl acetate (7:3) as the eluents gave 3 (430 mg,
97%) as a white foamy solid. []D25 -7o (c 0.2, CHCl3); 1H NMR (600 MHz, CDCl3): δ 7.69-7.15
(m, 20H, Ph), 5.46 (s, 1H, CHPh), 5.33 (s, 1H, H-1), 4.89 (d, 1H, J = 11.4 Hz, CH2Ph), 4.70 (d,
1H, J = 12.0 Hz, CH2Ph), 4.66 (d, 1H, J = 11.4 Hz, CH2Ph), 4.65-4.60 (m, 3H, CH2Ph),
4.35-4.25 (m, 2H, H-1, H-6a), 4.17-4.07 (m, 2H, H-4, H-5), 4.05-3.96 (m, 4H, H-2, H-2', H-6b,
-OCH2-), 3.80 (dd, 1H, J = 9.6, 3.0 Hz, H-3), 3.60-3.51 (m, 2H, H-3, -OCH2-,), 3.46 (t, 1H, J =
9.6 Hz, H-4'), 3.45-3.34 (m, 2H, -CH2N3), 3.32 (br s, 1H, H-5), 2.37 (d, 1H, J = 1.8 Hz, -OH),
13
1.91-1.81 (m, 2H, -OCH2CH2CH2N3), 1.30 (d, 3H, J = 6.0 Hz, H-6); C NMR (150 MHz,
CDCl3): δ 138.65, 138.17, 137.96, 137.69, 128.95, 128.47, 128.46, 128.32, 128.17, 127.89,
127.87, 127.80, 127.72, 127.61, 126.38, 102.12 (C-1), 101.10 (CHPh), 99.74 (C-1), 80.34 (C-3),
80.11 (C-4), 80.09 (C-3), 75.25 (CH2Ph), 73.67 (C-2), 72.73 (C-4), 71.76 (CH2Ph), 71.06
(CH2Ph), 69.24 (C-6), 68.37 (C-2), 67.44 (C-5), 66.40 (C-5), 66.19 (-OCH2-), 48.43 (-CH2N3),
+
29.22 (-OCH2CH2CH2N3), 17.88 (C-6); ESI-HRMS: calcd for (C43H49O3S10+NH4 ) m/z,
785.3762; found, 785.3768.
Page 8 of 21

3.7
3-Azidopropyl
2,3,4-tri-O-Benzoyl--L-rhamnopyranosyl-(12)-3,4-di-O-benzyl--L-rhamnopyranosyl-(
12)-3-O-benzyl-4,6-O-benzylidene-β-D-galactopyranoside (11)
To a solution of 9 (50 mg, 0.065 mmol) and 10 (68 mg, 0.097 mmol) in anhydrous CH2Cl2
o
(10 mL) was added TMSOTf (7 L, 0.038 mmol) at -78 C under a N2 atmosphere. After the
reaction mixture was stirred for 2 h, it was neutralized with Et3N and concentrated. The residue
was purified by flash column chromatography (petroleum ether/ethyl acetate 3:1) to yield 11 (68
mg, 85%) as a white foamy solid. []D25 124o (c 0.6, CHCl3); H NMR (600 MHz, CDCl3): δ
1
8.08 (d, 2H, J = 7.8 Hz, Ph), 7.97 (d, 2H, J = 7.8 Hz, Ph), 7.82 (d, 2H, J = 7.8 Hz, Ph), 7.61 (t,
1H, J = 7.8 Hz, Ph), 7.55-7.46 (m, 5H, Ph), 7.43-7.30 (m, 13H, Ph), 7.29-7.22 (m, 5H, Ph),
7.20-7.13 (m, 3H, Ph), 7.11 (t, 2H, J = 7.8 Hz, Ph), 5.87 (dd, 1H, J = 3.0, 1.8 Hz, H-2), 5.85 (dd,
1H, J = 9.6, 3.0 Hz, H-3), 5.60 (t, 1H, J = 9.6 Hz, H-4), 5.40 (s, 1H, CHPh), 5.38 (d, 1H, J =
1.8 Hz, H-1), 5.18 (d, 1H, J = 1.8 Hz, H-1), 4.95 (d, 1H, J = 11.4 Hz, CH2Ph), 4.74 (d, 1H, J =
11.4 Hz, CH2Ph), 4.71 (d, 1H, J = 12.0 Hz, CH2Ph), 4.68 (d, 1H, J = 13.2 Hz, CH2Ph), 4.65 (d,
1H, J = 12.0 Hz, CH2Ph), 4.64 (d, 1H, J = 13.2 Hz, CH2Ph), 4.29 (d, 1H, J = 7.8 Hz, H-1),
4.28-4.23 (m, 2H, H-6a, H-5), 4.18-4.11 (m, 1H, H-5'), 4.10 (t, 1H, J = 2.4 Hz, H-2'), 4.07-4.03
(m, 2H, H-2, H-4), 4.01-3.96 (m, 2H, H-6b, -OCH2-), 3.87 (dd, 1H, J = 9.6, 3.0 Hz, H-3'), 3.68 (t,
1H, J = 9.5 Hz, H-4'), 3.58-3.53 (m, 1H,-OCH2-), 3.51 (dd, 1H, J = 9.6, 3.6 Hz, H-3), 3.44-3.33
(m, 2H, -CH2N3), 3.30 (s, 1H, H-5), 1.90-1.84 (m, 2H, -CH2CH2CH2N3), 1.37 (d, 3H, J = 6.0 Hz,
13
H-6), 1.19 (d, 3H, J = 6.6 Hz, H-6); C NMR (150 MHz, CDCl3): δ 165.84, 165.35, 165.17,
138.80, 138.52, 137.91, 137.66, 133.27, 133.22, 132.90, 129.91, 129.80, 129.66, 129.41, 129.28,
129.02, 128.90, 128.49, 128.45, 128.37, 128.26, 128.19, 128.12, 128.10, 127.83, 127.64, 127.47,
127.32, 126.31, 101.98 (C-1), 100.97 (CHPh), 99.54 (C-1), 99.38 (C-1), 80.23 (C-4), 80.17
(C-3), 79.81 (C-3), 76.14 (C-2), 75.38 (CH2Ph), 73.56 (C-2), 72.68 (C-4), 72.24(CH2Ph), 71.90
(C-4), 70.91(CH2Ph), 70.64 (C-2), 69.99 (C-3), 69.20 (C-6), 68.17 (C-5), 67.02 (C-5),
66.36 (C-5), 66.20 (-OCH2-), 48.45 (-CH2N3), 29.20 (-CH2CH2CH2N3), 17.89 (C-6), 17.53
+
(C-6); ESI-HRMS: calcd for (C70H71N3O17+NH4 ) m/z, 1243.5122; found, 1243.5146.
3.8
3-Aminopropyl
-L-Rhamnopyranosyl-(12)--L-rhamnopyranosyl-(12)-β-D-galactopyranoside (1)
Page 9 of 21

To a solution of 11 (50 mg, 0.041mmol) in MeOH (5 mL) was added NaOMe (1.0 M) in
MeOH dropwise until the pH value reached 10. The mixture was stirred at rt for 2 h, and then
neutralized with Amberlite IR 120 (H+). The solution was filtered and concentrated. The residue
was purified by flash column chromatography (petroleum ether/ethyl acetate 1:1). This product
was dissolved in MeOH and H2O (v/v 1:9, 2 mL), to which was added 10% Pd/C (5 mg). The
suspension was stirred under a H2 atmosphere at rt for 12 h. The solid materials were filtrated off,
and the solution was concentrated. The residue was purified by size exclusion chromatography
on a Bio-Gel P-2 column with distilled water as the eluent, which was followed by lyophilization
1
to give 1 (16.3 mg, 75% for 2 steps) as a white solid. []D25 -97o (c 0.1, MeOH); H NMR (600
MHz, D2O): δ 5.06 (s, 1H, H-1), 4.78 (s, 1H, H-1), 4.32 (d, 1H, J = 7.8 Hz, H-1), 3.89 (dd, 1H,
J = 3.0, 1.8 Hz, H-2), 3.85 (br s, 1H, H-2), 3.80 (m, 1H, -OCH2-), 3.76 (m, 1H, H-5), 3.71 (d,
1H, J = 3.0 Hz, H-4), 3.70-3.63 (m, 2H, H-3, -OCH2-), 3.63-3.56 (m, 4H, H-3, H-3, H-6a,b),
3.56-3.47 (m, H-5, H-5), 3.39 (dd, 1H, J = 9.0, 7.8 Hz, H-2), 3.31 (t, 1H, J = 9.6 Hz, H-4), 3.26
(t, 1H, J = 9.6 Hz, H-4), 2.97-2.85 (m, 2H, -CH2NH2), 1.85-1.76 (m, 2H, -OCH2CH2CH2NH2),
13
1.11 (d, 3H, J = 6.0 Hz, H-6), 1.09 (d, 3H, J = 6.0 Hz, H-6); C NMR (150 MHz, CDCl3): δ
102.06 (C-1), 101.44 (C-1), 99.44 (C-1), 77.87 (C-2), 76.71 (C-2), 74.94 (C-5), 73.35 (C-3),
71.85 (2C, C-4, C-4), 69.89 (C-3), 69.86 (C-2), 69.81 (C-3), 69.01 (C-5), 68.87 (C-4),
68.79 (C-5), 67.30 (-OCH2-), 60.85 (C-6), 37.12 (-CH2NH2), 27.41 (-OCH2CH2CH2NH2), 16.54
(C-6), 16.50 (C-6); ESI-HRMS: calcd for (C21H39NO14+H+) m/z, 530.2443; found, 530.2441.
3.9
Isopropyl
3-O-benzyl-4,6-O-benzylidene-2-O-para-methoxybenzyl-β-D-galactopyranoside (14)
To an ice-bath cooled solution of 5 (2.5 g, 6.0 mmol) in dry DMF (15 mL) was added NaH
o
(318 mg, 60% in kerosene, 8.0 mmol) and PMBCl (1.32 mL, 9.7 mmol) at 0 C. The mixture
o
was stirred at 0 C for 1 h, diluted with EtOAc (100 mL), and washed successively with cooled
water, 1 M aq. HCl, and brine. The organic phase was dried over Na2SO4, and concentrated. The
residue was purified by flash column chromatography (petroleum ether/ethyl acetate 3:1) to
1
afford 7 (2.8 g, 87%) as a white foamy solid. []D25 89o (c 0.5, CHCl3); H NMR (600 MHz,
CDCl3): δ 7.54 (d, 2H, J = 7.8 Hz, Ph), 7.41-7.26 (m, 10H, Ph), 6.85 (d, 2H, J = 9.0 Hz, Ph),
5.46 (s, 1H, CHPh), 4.81 (d, 1H, J = 10.2 Hz, CH2Ph), 4.77 (d, 1H, J = 12.0 Hz, CH2Ph), 4.74 (d,
Page 10 of 21

1H, J = 12.0 Hz, CH2Ph), 4.73 (d, 1H, J = 10.2 Hz, CH2Ph), 4.47 (d, 1H, J = 9.6 Hz, H-1), 4.28
(dd, 1H, J = 12.0, 1.2 Hz, H-6a), 4.13 (d, 1H, J = 3.0 Hz, H-4), 3.95 (dd, 1H, J = 12.0, 1.2 Hz,
H-6b), 3.84 (t, 1H, J= 9.6 Hz, H-2), 3.79 (s, 3H, -OCH3), 3.57 (dd, 1H, J = 9.0, 3.6 Hz, H-3),
3.32 (s, 1H, H-5), 3.31-3.25 (m, 1H, -SCH-), 1.37 (dd, 3H, J = 6.6 Hz, -SCH(CH3)2), 1.33 (dd,
13
3H, J = 6.8 Hz, -SCH(CH3)2); C NMR (150 MHz, CDCl3): δ 159.23, 138.35, 137.88, 130.66,
130.01, 128.97, 128.36, 128.14, 127.73, 127.70, 126.57, 113.69, 101.48, 84.39, 81.11, 75.41,
+
74.05, 71.82, 69.69, 69.42, 55.28, 34.62, 24.37, 23.81; ESI-HRMS: calcd for (C31H36O6S+NH4 )
m/z, 554.2576; found, 554.2571.
3.10
p-Tolyl
3-O-Benzyl-4,6-O-benzylidene-2-O-para-methoxybenzyl--D-galactopyranosyl-(13)-2-O-
benzoyl-4-O-benzyl--L-rhamnopyranoside (12)
To a solution of 14 (254 mg, 0.047 mmol), 16 (200 mg, 0.43 mmol), and 4 Å MS (600 mg) in
anhydrous Et2O (15 mL) was added NIS (117 mg, 0.52 mmol) and AgOTf (5.2 mg, 0.02mmol) at
o
-30 C under a N2 atmosphere. The reaction mixture was stirred for 30 min, at which time TLC
indicated the disappearance of 14. The reaction mixture was neutralized with Et3N, filtered and
concentrated. The residue was purified by flash column chromatography (petroleum ether/ethyl
1
acetate 3:1) to yield 12 (290 mg, 73%) as a white foamy solid. []D25 28o (c 0.45, CHCl3); H
NMR (600 MHz, CDCl3): δ 8.04 (d, 2H, J = 7.8 Hz, Ph), 7.58 (t, 1H, J = 7.8 Hz, Ph), 7.50-7.40
(m, 4H, Ph), 7.40-7.22 (m, 13H, Ph), 7.21-7.15 (m, 2H, Ph), 7.12 (d, 2H, J = 7.8 Hz, Ph)), 7.02
(d, 2H, J = 8.4 Hz, Ph), 6.64 (d, 2H, J = 8.4 Hz, Ph), 5.83 (dd, 1H, J = 3.0, 1.2 Hz, H-2), 5.49 (d,
1H, J = 1.2 Hz, H-1), 5.34 (s, 1H, CHPh), 5.27 (d, 1H, J = 3.6 Hz, H-1), 4.77 (d, 1H, J = 11.4
Hz, CH2Ph), 4.71 (d, 1H, J = 12.0 Hz, CH2Ph), 4.67 (d, 1H, J = 11.4 Hz, CH2Ph), 4.65 (d, 1H, J
= 12.0 Hz, CH2Ph), 4.50 (d, 1H, J = 11.4 Hz, CH2Ph), 4.47 (d, 1H, J = 11.4 Hz, CH2Ph),
4.39-4.31 (m, 1H, H-5), 4.27 (dd, 1H, J = 9.6, 3.0 Hz, H-3), 4.12 (br d, 1H, J = 12.6, 1.2 H,
H-6a), 4.07 (dd, 1H, J = 10.2, 3.6 Hz, H-2), 3.92 (dd, 1H, J = 10.2, 3.6 Hz, H-3), 3.82 (br d, 1H,
J = 3.0 Hz, H-4), 3.76 (s, 3H, -OCH3), 3.71 (t, 1H, J = 9.6 Hz, H-4), 3.66 (br s, 1H, H-5), 3.57
13
(br d, 1H, J = 12.6 Hz, H-6b), 2.33 (s, 3H, -SPhCH3), 1.42 (d, 3H, J = 6.0 Hz, H-6); C NMR
(150 MHz, CDCl3): δ 165.79, 158.79, 138.77, 138.04, 137.91, 137.86, 133.15, 132.35, 130.47,
130.09, 129.98, 129.91, 129.88, 129.23, 129.04, 128.88, 128.47, 128.35, 128.27, 128.23, 128.10,
127.83, 127.78, 127.57, 127.37, 126.40, 125.30, 113.41, 101.05 (CHPh), 93.94 (C-1), 86.50
Page 11 of 21

(C-1), 79.91(C-4), 75.33 (CH2Ph), 75.13 (C-3), 74.67 (C-4), 74.31 (C-2), 72.75 (C-3), 72.49
(CH2Ph), 71.81 (CH2Ph), 70.22 (C-2), 69.20 (C-5), 69.17 (C-6), 62.53 (C-5), 55.18 (-OCH3),
+
21.14 (-SPhCH3), 18.05 (C-6); ESI-HRMS: calcd for (C55H56O11S+NH4 ) m/z, 942.3887; found,
942.3902.
3.11
p-Tolyl
2,3,4-tri-O-Benzoyl--L-rhamnopyranosyl-(12)-3,4-di-O-benzyl--L-rhamnopyranosyide
(13)
To a solution of 16 (184 mg, 0.41mmol) and 10 (280 mg, 0.45mmol) in anhydrous CH2Cl2
(10 mL) was added TMSOTf (15 L, 0.08 mmol) at -78 oC under a N2 atmosphere. The reaction
mixture was stirred under these conditions for 2 h, when TLC indicated the disappearance of 10.
The mixture was neutralized with Et3N and then concentrated. The residue was purified by flash
column chromatography (petroleum ether/ethyl acetate 6:1) to yield 13 (317 mg, 85%) as a white
1
foamy solid. []D25 22o (c 0.5, CHCl3); H NMR (600 MHz, CDCl3): δ 8.09 (d, 2H, J = 7.2 Hz,
Ph), 7.97 (d, 2H, J = 7.2 Hz, Ph), 7.82 (d, 2H, J = 7.2 Hz, Ph), 7.63 (t, 1H, J = 7.2 Hz, Ph),
7.52-7.48 (m, 3H, Ph), 7.43 (t, 1H, J = 7.2 Hz, Ph), 7.40-7.32 (m, 10H, Ph), 7.30 (t, 1H, J = 7.2
Hz, Ph), 7.28-7.21 (m, 4H, Ph), 7.17 (t, 1H, J = 7.2 Hz, Ph), 7.12 (d, 2H, J = 7.8 Hz, Ph),
5.88-5.83 (m, 2H, H-2,3), 5.63 (t, 1H, J = 9.6 Hz, H-4), 5.44 (d, 1H, J = 1.2 Hz, H-1), 5.16 (s,
1H, H-1), 4.99 (d, 1H, J = 11.0 Hz, CH2Ph), 4.79 (d, 1H, J = 12.0 Hz, CH2Ph), 4.75 (d, 1H, J =
11.0 Hz, CH2Ph), 4.70 (d, 1H, J = 12.0 Hz, CH2Ph), 4.29-4.16 (m, 3H, H-2,5, H-5), 3.92 (dd, 1H,
J = 9.6, 3.0 Hz, H-3), 3.74 (dd, 1H, J = 9.6 Hz, H-4), 2.32 (s, 3H, -SPhCH3), 1.41 (d, 3H, J = 6.0
Hz, H-6), 1.25 (d, 3H, J = 6.0 Hz, H-6); 13C NMR (150 MHz, CDCl3): δ 165.78, 165.44, 165.26,
138.45, 138.02, 137.83, 133.39, 133.29, 133.03, 132.23, 130.48, 129.91, 129.77, 129.67, 129.50,
129.28, 129.22, 128.54, 128.47, 128.40, 128.39, 128.25, 128.21, 127.85, 127.72, 127.68, 99.59
(C-1), 87.94 (C-1), 80.20 (C-4), 79.83 (C-3), 78.04 (C-2), 75.60 (CH2Ph), 72.67 (CH2Ph), 71.77
(C-4), 70.60 (C-2), 69.90 (C-3), 69.54 (C-5), 67.21 (C-5), 21.11 (-SPhCH3), 17.88 (C-6),
+
17.56 (C-6); ESI-HRMS: calcd for (C54H52SO11+NH4 ) m/z, 926.3574; found, 926.3584.
3.12
3-Azidopropyl
3-O-Benzyl-4,6-O-benzylidene-2-O-para-methoxybenzyl--D-galactopyranosyl-(13)-2-O-
Page 12 of 21

benzoyl-4-O-benzyl--L-rhamnopyranosyl-(12)-3,4-di-O-benzyl--L-rhamnopyranosyl-(
12)-3-O-benzyl-4,6-O-benzylidene-β-D-galactopyranoside (17)
To a solution of 9 (75 mg, 0.098 mmol), 12 (100 mg, 0.1082 mmol), and 4Å MS (300 mg) in
anhydrous CH2Cl2 (4 mL) were added NIS (30 mg, 0.14 mmol) and AgOTf (13 mg, 0.05mmol)
o
at -78 C under a N2 atmosphere. After the reaction mixture was stirred under these conditions
for 2 h, it was neutralized with Et3N, filtered, and concentrated. The residue was purified by flash
column chromatography (petroleum ether/ethyl acetate 3:1) to yield 17 (133 mg, 85%) as a white
foamy solid. []D25 124o (c 0.6, CHCl3); 1H NMR (600 MHz, CDCl3): δ 8.05 (d, 2H, J = 7.2 Hz,
Ph), 7.58 (t, 1H, J = 7.2 Hz, Ph), 7.55-7.50 (m, 2H, Ph), 7.47-7.41 (m, 4H, Ph), 7.41-7.10 (m,
31H, Ph), 7.02 (d, 2H, J = 8.4 Hz, Ph), 6.60 (d, 2H, J = 8.4 Hz, Ph), 5.77 (dd, 1H, J = 3.0, 1.8 Hz,
H-2), 5.37 (s, 1H, CHPh), 5.35 (d, 1H, J = 3.6 Hz, H-1), 5.30 (s, 1H, CHPh), 5.28 (d, 1H, J =
1.8 Hz, H-1), 5.15 (d, 1H, J = 1.8 Hz, H-1), 4.92 (d, 1H, J = 10.8 Hz, CH2Ph), 4.66 (m, 9H,
CH2Ph), 4.51 (d, 1H, J = 11.4 Hz, CH2Ph), 4.42 (d, 1H, J = 11.4 Hz, CH2Ph), 4.32 (dd, 1H, J =
10.2, 3.0 Hz, H-3), 4.26 (br d, 1H, J = 11.4 Hz, H-6a), 4.25 (d, 1H, J = 7.8 Hz, H-1), 4.12-4.06
(m, 3H, H-6a, H-2, H-5), 4.03 (dd, 1H, J = 10.2, 3.6 Hz, H-2), 4.01 (dd, 1H, J = 9.6, 7.8 Hz,
H-2), 3.99-3.92 (m, 4H, H-4, H-5, H-6b, -OCH2-), 3.88 (dd, 1H, J = 10.2, 3.6 Hz, H-3), 3.82
(dd,1H, J = 9.6, 3.0 Hz, H-3), 3.75 (d, 1H, J = 3.0 Hz, H-4), 3.72 (s, 3H, -OCH3), 3.65 (br s,
1H, H-5), 3.60-3.49 (m, 4H, H-4, H-4, H-6b, -OCH2-), 3.47 (dd, 1H, J = 9.6, 3.6 Hz, H-3),
3.41-3.30 (m, 2H, -CH2N3), 3.26 (br s, 1H, H-5), 1.88-1.80 (m, 2H, -OCH2CH2CH2N3), 1.28 (d,
13
3H, J = 6.0 Hz, H-6), 1.24 (d, 3H, J = 6.6 Hz, H-6); C NMR (150 MHz, CDCl3): δ 165.43,
158.67, 138.86, 138.84, 138.63, 138.27, 138.01, 137.93, 137.69, 132.93, 130.64, 130.15, 130.03,
129.18, 129.03, 128.92, 128.78, 128.46, 128.33, 128.25, 128.23, 128.21, 128.14, 128.03, 127.94,
127.84, 127.72, 127.70, 127.57, 127.46, 127.41, 127.31, 127.24, 127.15, 126.41, 126.37, 113.31,
102.00 (C-1), 101.04 (CHPh), 100.96 (CHPh), 99.51 (C-1), 99.21 (C-1), 93.20 (C-1), 80.45
(C-4), 80.06 (C-3), 79.74 (2C, C-4 and C-3), 75.39 (CH2Ph), 75.08 (C-3), 75.07 (CH2Ph),
74.88 (C-2), 74.61 (C-4), 74.43 (C-2), 73.41 (C-2), 72.86 (C-4), 72.26 (CH2Ph), 72.08
(CH2Ph), 71.62 (2C, C-3 and CH2Ph), 71.09 (CH2Ph), 69.30 (C-6), 69.19 (C-6), 68.31 (C-2),
68.12 (C-5), 68.00 (C-5), 66.31 (C-5), 66.17 (-OCH2CH2-), 62.21 (C-5), 55.14 (-OCH3),
48.43 (-CH2N3), 29.16 (-OCH2CH2CH2N3), 18.10 (C-6), 17.92 (C-6); ESI-HRMS: calcd for
+
(C91H97N3O21+NH4 ) m/z, 1585.6958; found, 1585.6962.
Page 13 of 21

3.13
3-Azidopropyl
3-O-Benzyl-4,6-O-benzylidene--D-galactopyranosyl-(13)-2-O-benzoyl-4-O-benzyl--L-r
hamnopyranosyl-(12)-3,4-di-O-benzyl--L-rhamnopyranosyl-(12)-3-O-benzyl-4,6-O-be
nzylidene-β-D-galactopyranoside (18)
To solution of 17 (100 mg, 0.069 mmol) in CH2Cl2 and H2O (v/v 14:1, 15 mL) was added
DDQ (19 mg, 0.083 mmol) at rt. The reaction mixture was stirred for 2 h, at which time TLC
indicated the complete consumption of 17. The reaction mixture was diluted with CH2Cl2 (50
mL), washed with 10% aq. NaHSO3 (30 mL). The organic phase was dried over Na2SO4, and
concentrated. The residue was subjected to silica gel column chromatograph using toluene and
ethyl acetate (3:1) as the eluents to give 18 (67 mg, 67%) as a white foamy solid. []D25 61o (c
0.3, CHCl3); 1H NMR (600 MHz, CDCl3): δ 8.09 (d, 2H, J = 7.8 Hz, Ph), 7.64 (t, 1H, J = 7.8 Hz,
Ph), 8.11-8.07 (m, 4H, Ph), 7.47-7.43 (m, 2H, Ph), 7.41-7.05 (m, 31H, Ph), 5.67 (dd, 1H, J = 3.0,
1.2 Hz, H-2), 5.37 (s, 1H, CHPh), 5.36 (d, 1H, J = 3.6 Hz, H-1), 5.28 (d, 1H, J = 1.8 Hz, H-1),
5.21 (s, 1H, CHPh), 5.06 (d, 1H, J = 1.2 Hz, H-1), 4.89 (d, 1H, J = 10.8 Hz, CH2Ph), 4.76-4.57
(m, 8H, CH2Ph), 4.42 (d, 1H, J = 11.4 Hz, CH2Ph), 4.32 (dd, 1H, J = 9.6, 3.0 Hz, H-3), 4.26 (dd,
1H, J = 12.6, 1.2 Hz, H-6a), 4.25 (d, 1H, J = 7.8 Hz, H-1), 4.22 (ddd, 1H, J = 10.2, 6.6, 3.6 Hz,
H-2), 4.12-4.06 (m, 2H, H-2, H-5), 4.04-3.93 (m, 6H, H-2, H-4, H-5, H-6b, H-6a, -OCH2-),
3.81 (dd, 1H, J = 9.6, 3.0 Hz, H-3), 3.68 (d, 1H, J = 3.6 Hz, H-4), 3.60 (dd, 1H, J = 10.2, 3.6
Hz, H-3), 3.56-3.51 (m, 2H, H-5, -OCH2-), 3.51-3.45 (m, 2H, H-3, H-4), 3.42 (t, 1H, J = 9.6
Hz, H-4), 3.40-3.31 (m, 3H, H-6b, -CH2N3), 3.27 (s, 1H, H-5), 2.23 (d, 1H, J = 6.6 Hz, -OH),
1.89-1.80 (m, 2H, -OCH2CH2CH2N3), 1.29 (d, 3H, J = 6.0 Hz, H-6), 1.25 (d, 3H, J = 6.0 Hz,
13
H-6); C NMR (150 MHz, CDCl3): δ 166.26, 138.75, 138.65, 138.45, 138.01, 137.85, 137.67,
133.42, 130.02, 129.75, 129.02, 128.92, 128.77, 128.47, 128.38, 128.25, 128.13, 128.04, 127.91,
127.86, 127.69, 127.55, 127.45, 127.30, 127.17, 126.98, 126.35, 126.28, 125.28, 102.00 (C-1),
101.02 (CHPh), 100.88 (CHPh), 99.52 (C-1), 99.34 (C-1), 95.74 (C-1), 80.34 (C-4), 80.06
(C-3), 79.94 (C-4), 79.88 (C-3), 75.68 (C-3), 75.37 (CH2Ph), 75.08 (2C, C-2 and CH2Ph),
74.10 (C-4), 73.48 (C-2), 73.00 (C-3), 72.82 (C-4), 72.22 (CH2Ph), 71.38 (CH2Ph), 71.05
(CH2Ph), 69.19 (C-6), 69.16 (C-6), 69.10 (C-2), 68.46 (C-5), 68.05 (C-5), 67.85 (C-2),
66.32 (C-5), 66.18 (-OCH2-), 62.58 (C-5), 48.42 (-CH2N3), 29.16 (-OCH2CH2CH2N3), 18.10
Page 14 of 21

+
(C-6), 17.82 (C-6); ESI-HRMS: calcd for (C83H89N3O20+NH4 ) m/z, 1465.6383; found,
1465.6401.
3.14
3-Azidopropyl
2,3,4-tri-O-Benzoyl--L-rhamnopyranosyl-(12)-3,4-di-O-benzyl--L-rhamnopyranosyl-(
12)-3-O-benzyl-4,6-O-benzylidene--D-galactopyranosyl-(13)-2-O-benzoyl-4-O-benzyl-
-L-rhamnopyranosyl-(12)-3,4-di-O-benzyl--L-rhamnopyranosyl-(12)-3-O-benzyl-4,6
-O-benzylidene-β-D-galactopyranoside (19)
To a solution of 18 (60 mg, 0.041 mmol) and 13 (56 mg, 0.062 mmol) in anhydrous CH2Cl2
o
(4 mL) were added NIS(17 mg, 0.074 mmol) and AgOTf (13 mg, 0.05mmol) at -78 C under a
N2 atmosphere. The reaction mixture was stirred for 2 h, neutralized with Et3N, and concentrated.
The residue was purified by flash column chromatography (toluene/ethyl acetate 6:1) to yield 19
(78 mg, 85%) as a white foamy solid. []D25 46o (c 0.2, CHCl3); 1H NMR (600 MHz, CDCl3): δ
8.12 (d, 2H, J = 8.4 Hz, Ph), 8.00 (d, 2H, J = 8.4 Hz, Ph), 7.93 (d, 2H, J = 8.4 Hz, Ph), 7.76 (d,
2H, J = 8.4 Hz, Ph), 7.60-7.00 (m, 57H, Ph), 5.80-5.75 (m, 2H, H-2,4), 5.71 (t, 1H, J = 2.4 Hz,
H-2), 5.48 (t, 1H, J = 9.6 Hz, H-3), 5.41 (d, 1H, J = 3.0 Hz, H-1), 5.35 (s, 1H, CHPh), 5.27
(d, 1H, J = 1.2 Hz, H-1), 5.26 (d, 1H, J = 1.2 Hz, H-1), 5.20 (s, 1H, CHPh), 5.06 (d, 1H, J =
1.8 Hz, H-1), 4.97 (br s, 1H, H-1), 4.91 (d, 1H, J = 10.8 Hz, CH2Ph), 4.84 (d, 1H, J = 11.4
Hz, CH2Ph), 4.76 (d, 1H, J = 11.4 Hz, CH2Ph), 4.74-4.59 (m, 7H, CH2Ph), 4.58 (d, 1H, J = 13.2
Hz, CH2Ph), 4.45 (d, 1H, J = 13.2 Hz, CH2Ph), 4.42 (dd, 1H, J = 9.6, 3.0 Hz, H-2), 4.35 (dd,
1H, J = 9.6, 2.4 Hz, H-3), 4.25 (dd, 1H, J = 12.0, 1.2 Hz, H-6a), 4.23 (d, 1H, J = 7.8 Hz, H-1),
4.18 (m, 1H, H-5), 4.12 (br d, 1H, J = 12.0 Hz, H-6a), 4.09-3.99 (m, 5H, H-2,5, H-2,5,
CH2Ph), 3.98 (dd, 1H, J = 9.6, 7.8 Hz, H-2), 3.96-3.90 (m, 6H, H-3, H-5, H-4,6b, CH2Ph,
-OCH2-), 3.80 (br s, 1H, H-5), 3.78 (dd, 1H, J = 9.6, 3.0 Hz, H-3), 3.66 (d, 1H, J = 3.0 Hz,
H-4), 3.61 (t, 1H, J = 9.6 Hz, H-4), 3.59 (t, 1H, J = 9.6 Hz, H-4), 3.57 (br d, 1H, J = 12.0 Hz,
H-6b), 3.51 (m, 1H, -OCH2-), 3.50 (t, 1H, J = 9.6 Hz, H-4), 3.45 (dd, 1H, J = 9.6, 3.0 Hz,
H-3), 3.43 (dd, 1H, J = 9.6, 3.0 Hz, H-3), 3.40-3.28 (m, 2H, -CH2N3), 3.25 (s, 1H, H-5),
1.86-1.79 (m, 2H, -OCH2CH2CH2N3), 1.39 (d, 3H, J = 6.0 Hz, H-6), 1.27 (d, 3H, J = 6.0 Hz,
13
H-6), 1.23 (d, 3H, J = 6.0 Hz, H-6), 1.06 (d, 3H, J = 6.0 Hz, H-6); C NMR (150 MHz,
CDCl3): δ 165.84, 165.22, 164.99, 164.65, 139.07, 138.86, 138.70, 138.63, 138.56, 138.18,
Page 15 of 21

137.98, 137.81, 137.68, 133.16, 132.87, 132.81, 130.69, 129.87, 129.80, 129.64, 129.43, 129.28,
128.93, 128.70, 128.44, 128.41, 128.38, 128.33, 128.28, 128.25, 128.23, 128.19, 128.14, 128.08,
128.02, 127.94, 127.84, 127.70, 127.62, 127.44, 127.40, 127.38, 127.23, 127.21, 127.04, 126.97,
126.90, 126.81, 126.38, 126.21, 101.99 (C-1), 101.09 (CHPh), 100.78 (C-1), 100.62 (CHPh),
99.49 (C-1), 99.21 (2C, C-1, C-1), 94.21 (C-1), 80.41 (C-4), 80.33 (C-3), 80.08 (C-4),
79.77 (C-3), 79.70 (C-4), 79.51 (C-3), 76.46 (C-3), 76.33 (C-2), 75.38 (CH2Ph), 75.31
(CH2Ph), 75.05 (CH2Ph), 74.94 (C-2), 73.65 (C-4), 73.46 (C-2), 72.92 (C-4), 72.18 (C-2),
71.91 (2C, C-4, CH2Ph), 71.74 (CH2Ph), 71.16 (CH2Ph), 70.99 (C-3), 70.85 (CH2Ph), 70.46
(C-2), 69.95 (C-3), 69.31 (C-6), 69.18 (C-6), 68.66 (C-5), 68.60 (C-5), 67.97 (C-5),
67.82 (C-2), 66.87 (C-5), 66.29 (C-5), 66.19 (-OCH2-), 62.02 (C-5), 48.42 (-CH2N3), 29.15
(-OCH2CH2CH2N3), 18.18 (C-6), 18.02 (2C, C-6, C-6), 17.34 (C-6); ESI-HRMS: calcd
+
for (C130H133N3O31+2NH4 ) m/z, 1134.4822; found, 1134.4834.
3.15
3-Aminopropyl
-L-Rhamnopyranosyl-(12)--L-rhamnopyranosyl-(12)--D-galactopyranosyl-(13)-
-L-rhamnopyranosyl-(12)--L-rhamnopyranosyl-(12)-β-D-galactopyranoside (2)
To a solution of 19 (30 mg, 0.0134 mmol) in MeOH (3 mL) was added NaOMe (1.0 M) in
MeOH dropwise until the pH value reached 10. The mixture was stirred at rt for 5 h, and then
neutralized with Amberlite IR 120 (H+). The solution was filtered and concentrated. The residue
was purified by flash column chromatography (petroleum ether/ethyl acetate 4:1). The product
was dissolved in MeOH and H2O (v/v 1:9, 2 mL), and then 10% Pd/C (5 mg) was added. The
suspension was stirred under a H2 atmosphere at rt for 24 h. The solid materials were filtrated off,
and the solution was concentrated. The residue was purified by size exclusion chromatography
on a Bio-Gel P-2 column with distilled water as the eluent, which was followed by lyophilization
1
to give 2 (11 mg, 90% for 2 steps) as a white solid. []D25 -10o (c 0.1, MeOH); H NMR (600
MHz, D2O): δ 5.09 (s, 1H, H-1), 5.03 (s, 1H, H-1), 5.01 (d, 1H, J = 2.4 Hz, H-1), 4.80 (br s, 2H,
2H-1), 4.33 (d, 1H, J = 7.8 Hz, H-1), 4.11 (s, 1H, H-2), 4.03 (t, 1H, J = 6.0 Hz), 3.94-3.84 (m,
5H), 3.84-3.65 (m, 8H), 3.64-3.47 (m, 10H), 3.46-3.37 (m, 2H), 3.36-3.23 (m, 3H), 2.99-2.89 (m,
13
2H, -CH2NH2), 1.87-1.77 (m, 2H, -OCH2CH2CH2NH2), 1.16-1.08 (m, 43H, 4H-6); C NMR
(150 MHz, D2O): δ 102.07 (2C, 2C-1), 101.46 (C-1), 100.24 (C-1), 99.42 (C-1), 94.15 (C-1),
Page 16 of 21

78.22, 77.75, 76.80, 74.96, 74.28, 74.03, 73.34, 71.85, 71.84, 71.77, 70.66, 70.10, 69.90, 69.87,
69.79, 69.74, 69.38, 69.24, 69.11, 69.08, 68.98, 68.87, 68.80, 67.26 (-OCH2-), 66.23, 60.86 (C-6),
60.69 (C-6), 37.12 (-CH2NH2), 27.06 (-OCH2CH2CH2NH2), 16.65 (C-6), 16.61 (C-6), 16.57
(C-6), 16.49 (C-6); ESI HRMS: calcd for (C39H69NO27+H+) m/z, 984.4135; found, 984.4135.
Acknowledgement
This work was supported by research grants from the National Major Scientific and
Technological Special Project for Significant New Drugs Development (No.
2012ZX09502001-005), the Major State Basic Research Development Program of China (973
Program) (No. 2012CB822102), and Shandong Provincial National Science Foundation (No.
ZR2015BM020).
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Supplementary data
1
Supplementary data including H NMR, 13C NMR, and ESI-HRMS spectra of all final
products and intermediates are available from the corresponding authors.
Page 18 of 21

Figure 1. Structures of the O-antigen from B. anthina strain LMG20983 and the synthetic targets
1 and 2.
Scheme 1. Synthesis of target molecule 1
Page 19 of 21

Scheme 2. Retro-synthetic analysis of target molecule 2
Scheme 3. Synthesis of key disaccharide blocks 12 and 13
Page 20 of 21

Scheme 4. Synthesis of target molecule 2
Page 21 of 21