Synthesis of the repeating unit of O-specific polysaccharide isolated from the water-borne bacteria Aeromonas bestiarum 207

Article abstract of DOI:10.1016/j.carres.2017.11.010

Aeromonas bestiarum 207 is a bacterial pathogen with severe impact on aquaculture. In a recent study, the structure of OPS antigens from Aeromonas bestiarum was identified as pentasaccharide repeating units. Synthesis of the pentasaccharide repeating unit

Full text of DOI:10.1016/j.carres.2017.11.010

Accepted Manuscript
Synthesis of the repeating unit of O-specific polysaccharide isolated from the water-
borne bacteria Aeromonas bestiarum 207
Yiren Xu, Guanghui Zong, Shuhui Jin, Jianjun Zhang
PII:
S0008-6215(17)30780-2
10.1016/j.carres.2017.11.010
CAR 7488
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 17 October 2017
Revised Date: 15 November 2017
Accepted Date: 15 November 2017
Please cite this article as: Y. Xu, G. Zong, S. Jin, J. Zhang, Synthesis of the repeating unit of O-specific
polysaccharide isolated from the water-borne bacteria Aeromonas bestiarum 207, Carbohydrate
Research (2017), doi: 10.1016/j.carres.2017.11.010.
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ACCEPTED MANUSCRIPT
Synthesis of the repeating unit of O-specific polysaccharide
isolated from the water-borne bacteria Aeromonas bestiarum 207
HO
HO
HO
HO
O
OPMP
OPMP 1) Deprotection
2) Glycosylation
O
O
OPMP
O
BzO
O
O
BzO
BzO
O
O
HO
HO
OH
O
BzO
LevO
O
8
10
OH
O
OBz
AcHN
O
AcO
AcO
1) Deprotection
2) Glycosylation
O
O
OH
OH
OAc
NPhth
14
AcO
OH
Key intermidiate with
orthogonal protecting groups
OH
HO
2
5.2% overal yield through 11
linear steps from 10 and 14

ACCEPTED MANUSCRIPT
Synthesis of the repeating unit of O-specific polysaccharide isolated from
the water-borne bacteria Aeromonas bestiarum 207
Yiren Xu a, Guanghui Zonga,* Shuhui Jin a, Jianjun Zhang a,*
,
a
Department of Applied Chemistry, China Agricultural University, Beijing 100193, PR China.
Author. Tel/Fax: 86 10 62732219; E-mail: ryandota@sina.com (Y.X.);
*Corresponding author. Tel/Fax: 1 (479)575-5233;86 10 62732219; E-mail: zonggh@cau.edu.cn (G. Z.);
zhangjianjun@cau.edu.cn (J.Z.);

ACCEPTED MANUSCRIPT
Abstract
Aeromonas bestiarum 207 is a bacterial pathogen with severe impact on aquaculture. In a recent
study, the structure of OPS antigens from Aeromonas bestiarum was identified as pentasaccharide
repeating units. Synthesis of the pentasaccharide repeating unit and its derivative are reported. Stereo- and
regio-specific synthesis was achieved under Schmidt glycosylation conditions employing appropriately
protected L-rhamopyranosyl and D-glucopyranosylamine building blocks. The pentasaccharide synthesis
was achieved using a [3+2] strategy with an overall yield of 5.2% through 11 linear steps from the
monosaccharide building blocks 10 and 14.
Keywords: Aeromonas bestiarum; O-specific polysaccharide; pentasaccharide synthesis; aquaculture;
carbohydrate pesticide.
1. Introduction
Pathogenic bacteria from genus Aeromonas are widespread in various aquatic environments
and the intestinal tract of different animals.[1, 2] These bacteria are known as pathogens causing
a wide spectrum of diseases among reptiles, frogs, fish and some mammals (including
humans).[3] For example, Aeromonas sobria and Aeromonas caviae can cause wound infections,
meningitis, endocarditis, and osteomyelitis in humans[4-7]. Aeromonas punctata can cause red
leg disease in frogs[3]. Aeromona salmonicida and Aeromonas bestiarum can cause motile
aeromonad infection/motile aeromonad septicemia (MAI/MAS)[8, 9] and haemorrhagic
septicaemia[10] in fish. The putative virulence factors of genus Aeromonas include: exoproteins
(cytotoxins, entertoxins, haemolysins), proteases, the lipopolysaccharides (LPS) and other
cytoarchitecture on the cell surface layers. Among them, lipopolysaccharides (LPS) containing
O-specific polysaccharide (OPS) antigens have strong immunogenicity. In addition, LPS are
important in cell specific recognition[11, 12]. Identification of the virulent factor and molecular
mechanism behind the host-pathogen interaction, will be helpful to prevent and control such
outbreak of bacterial infections in aquaculture[1-7].The identification and synthesis of O-specific
polysaccharide (OPS) and their derivatives can shed more light on host-pathogen interaction
ways inhibit such interaction which can potentially lead to a safe carbohydrate based bio-
pesticide for application in aquaculture.

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→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→
2
1
β-D-GlupNAc
HO
OPMP
HO
HO
HO
O
O
OPMP
HO
O
O
HO
O
O
O
HO
O
O
HO
OH
O
O
OH
O
HO
HO
NHAc
AcHN
O
O
O
OH
OH
O
OH
OH
OH
OH
HO
OH
2
3
Scheme 1.The repeating unit of OPS from the water-borne bacteria A. bestiarum 207
Aeromonas bestiarum is a member of “Aeromonas hydrophila” complex, which belongs to
mesophilic and motile aeromonads group bacterial species[13]. Aeromonas bestiarum 207 was
isolated from diseased carp farmed in Poland and proved to be highly virulent for carp. High
infection and/or death rates related to this bacterium are an increasing problem in commercial
carp aquaculture[14].Recently, Anna Turska-Szewczuk and coworkers have isolated and
characterized a OPS antigens (scheme 1) from Aeromonas bestiarum 207 as a pentasaccharide
repeating unit[15],which consists of four α-linked L-Rhap in main chain with a terminal β-D-
GlcpNAc unit. As a part of our ongoing research project to develop carbohydrate based
pesticides from LPS O-antigens for controlling the genus Aeromonas, we studied the chemical
synthesis of a pentasaccharide analogue 2 (Scheme 1) related to this repeating unit, and its
tetrasaccharide derivative 3 with one L-Rhap missing in the reducing end of 2 (Scheme 1).
2. Results and Discussion
The retrosynthetic analysis of the target pentasaccharide 2 is as outlined in Scheme 2.
Synthesis of the pentasaccharide 2 could be achieved through a convergent strategy involving
[3+2] coupling of a trisaccharide acceptor 4 and a disaccharide donor 5. The trisaccharide
acceptor 4 could then be prepared by glycosylation of a α-(1→2)-linked disaccharide donor 6
and a rhamnosyl acceptor 7[16-20]. Coupling of the rhamnosyl acceptor 7 with another

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rhamnosyl donor 8 yield the disaccharide precursor for donor 6. Rhamnosyl acceptor 7 could
also be involved in the synthesis of the disaccharide donor 5 by coupling with a glucosyl donor
9[21], followed by a two-step sequence involving selective de-1-O-aetylation and
trichloroacetimidate formation.
HO
HO
HO
HO
O
OPMP
O
O
O
O
HO
O
OH
O
AcHN
O
OH
OH
O
OH
OH
HO
2
OCNHCCl3
NHAc
OBz
O
OBz
O
O
BzO
O
AcO
BzO
HO
BzO
AcO
BzO
O
O
O
O
4
OPMP
OAc
OAc
OAc
5
OPMP
O
OBz
O
BzO
OBz
O
BzO
LevO
BzO
OH
AcO
AcO
BzO
7
O
OCNHCCl3
NPhth
AcO
O
OCNHCCl3
OCNHCCl3
6
9
O
BzO
LevO
OBz
8
Scheme 2. Retrosynthetic analysis of the target pentasaccharide 2.

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OPMP
OPMP
OPMP
ref. 20
a
b
O
O
O
L-Rhamnose
BzO
BzO
LevO
BzO
HO
HO
OBz
11
OBz
OH
10
12
e
c
OPMP
OR
O
O
BzO
BzO
LevO
BzO
OH
OBz
7
13
R = H
d
8
R = CNHCCl3
AcO
AcO
AcO
ref. 22
O
O
f
AcO
OR
OAc
AcO
AcO
D-Glucosamine
NPhth
NPhth
14
15
9
R = H
R = CNHCCl3
g
Scheme 3. Synthesis of monosaccharide building blocks. Reagents and conditions: (a) triethyl orthobenzoate, p-
TsOH, CH2Cl2, rt, 4 h; then 80% aq AcOH, 20 min, 74%; (b) levulinic acid, DCC, DMAP, CH2Cl2, rt, 4 h, 93%; (c)
o
Ce(NH4)2(NO3)6, CH3CN/H2O (4:1 v/v), rt, 2 h, 76%; (d) CCl3CN, DBU, CH2Cl2, 0 C to rt, 2 h, 92%; (e) benzoyl
chloride, DMAP, Py, CH2Cl2, −25 oC, 4 h, 69%; (f) ethylenediamine/CH3COOH (1:1 v/v), THF, rt, 24 h, 72%; (g)
CCl3CN, DBU, CH2Cl2, 0 oC to rt, 2 h, 95%.
The synthesis commenced with preparation of
8. These two -rhamnosyl building blocks were obtained from the same intermediate 10[22],
which was synthesized through 6 steps from the commercially available -rhamnose following
L-rhamnosyl acceptor 7 and L-rhamnosyl donor
L
L
the literature reported procedure. Treatment of 10 with triethyl orthobenzoate in the presence of
p-TsOH formed an orthoester intermidiate, hydrolysis of which gave alcohol 11[22].Staglich
esterification of 11 with levulinic acid furnished compound 12. Donor 8 was then synthesized in
67% yield from 12 using a two-step sequence involving selective de-1-O-aetylation followed by
trichloroacetimidate formation. Starting from the commercially available
glucosyl donor 9[21] was synthesized over four steps
D
-glucosamine,
including the
ethylenediamine/CH3COOH[23, 24] involved selective removal of anomeric acetate followed by
reaction with trichloroacetonitrile and 1,8-diazobicyclo[5.4.0]undec-7-ene (DBU).

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Scheme 4. Synthesis of trisaccharide acceptor 4 and disaccharide donor 5. Reagents and conditions: (a) TMSOTf,
CH2Cl2, 4 Å molecular sieves, –10 oC, 8 h, 66% for 16, 75% for 18, 65% for 19; (b) Ce(NH4)2(NO3)6, CH3CN/H2O
(4:1 v/v), rt, 2 h, 68% for 17; 74% for 21; (c) CCl3CN, DBU, anhydrous CH2Cl2, 0 oC to rt, 2 h, 95% for 6, 90% for
o
5; (d) hydrazine acetate, DMF, rt, 4 h, 80%; (e) ethylenediamine, butanol, 85 C, 8 h; (f) Ac2O, Py, 24 h, 70% over
two steps.
With the monosaccharide building blocks 7, 8 and 9 in hand, we next focused on the
construction of the trisaccharide acceptor 4 and disaccharide donor 5 (Scheme 4). Glycosylation
of 7 with 8 promoted by TMSOTf afforded the corresponding α-(1→2)-linked disaccharide 16 in
65% yield. The cleavage of the 4-methoxyphenyl group in 16 with ceric ammonium nitrate
(CAN), followed by reacting the resulting hemiacetal 17 with trichloroacetonitrile in the
presence of DBU, afforded the disaccharide donor 6 in 67% yield over two steps. Further
glycosylation of disaccharide donor 6 with the same L-rhamnosyl acceptor 7 in CH2Cl2 generated
trisaccharide 18 smoothly in moderate yield (75%). Chemo-selective removal of Lev group on
C-3ʹʹ by treating 18 with hydrazine acetate in DMF[25], gave trisaccharide acceptor 4 in 80%
yield. Meanwhile, disaccharide donor 5 was synthesized following the route as shown in Scheme
4. The phthalimide (Phth) group at the C-2 position in glucosyl donor 9 lead the β-(1→2)-
linkage to form disaccharide 19 in the glycosylation with L-rhamnosyl acceptor 7. The 1,2-trans
1
configuration was confirmed by H NMR, in which the signal at 5.52 ppm showed a large
coupling (d, 1H, J 8.4 Hz) for H-1-GluNPhth. The Phth group was then removed together with
the other acetates when being treated with ethylenediamine in n-butanol [26]followed by per-
acetylation gave disaccharide 20 in 70% yield over two steps. Converting 20 to the

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corresponding disaccharide donor 5 was achieved in 63% yield over two steps following the
same strategy to prepare disaccharide donor 6.
Scheme 5. Synthesis of pentasaccharide 2. Reagents and conditions: (a) TMSOTf, CH2Cl2, 4 Å molecular sieves, –
10 oC, 8 h, 67%; (b) satd. NH3–MeOH, 14 days, 80%.
Finally, the construction of the target pentasaccharide 2 was achieved as shown in Scheme 5.
The Schmidt glycosylation of the trisaccharide acceptor 4 and the disaccharide donor 5 giving
the fully protected pentasaccharide 22. The successful glycosylation was supported by 13C NMR,
the signal at δ 97.6, 98.9, 99.3, 99.9 and 100.5 in which spectrum showed five anomeric carbons.
Global deprotection of 22 in satd. NH3–MeOH afforded the target pentasaccharide 2 in 80%
yield. High resolution mass data confirmed the accomplishment of the target compound.
Scheme 6. Synthesis of tetrasaccharide 3. Reagents and conditions: (a) hydrazine acetate, DMF, rt, 4 h, 79%; (b)
TMSOTf, CH2Cl2, 4 Å molecular sieves, –10 oC, 8 h, 73%; (c) satd. MeNH2–MeOH, 7 days, 80%.
During the synthesis of the target pentasaccharide 2, we were able to construct the one-
rhamnose missing analogue (tetrasaccharide 3) at the reducing end of 2 using the key
intermediate 16. Chemo-selective removal of Lev protecting group in 16 following the same
procedure to synthesis trisaccharide acceptor 4 giving disaccharide acceptor 23 in high yield.
Schmidt glycosylation of acceptor 23 with the disaccharide donor 5 afforded fully protected

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tetrasaccharide 24. Final complete deprotection of 24 following the same strategy to obtain 2
giving the tetrasaccharide 3 in high yield.
3. Conclusion
In summary, we have reported a convergent total synthesis of a pentasaccharide analogue 2
and its tetrasccharide derivative 3 related to the repeating unit of O-specific polysaccharide
isolated from water-borne bacteria Aeromonasbestiarum 207. The efficient synthesis features the
preparation of a key disaccharide intermediate 16 containing an orthogonal protecting group
(Lev), which could be easily converted to the disaccharide donor 6 or the disaccharide acceptor
23. This study will enrich the O-specific polysaccharide library for our carbohydrate pesticide
development project.
4. Experimental Procedures
4.1 Materials and methods
13
1H and C NMR spectra were recorded with Bruker AVANCE600 spectrometers (1H NMR-
300 MHz; 13C NMR-75 MHz) for solutions in CDCl3 or D2O. Internal references: TMS (δ 0.000
ppm for 1H), CDCl3 (δ 77.00 ppm for 13C). Data for 1H NMR are reported as follows: chemical
shift, integration, multiplicity (app = apparent, parobsc = partially obscure, ovrlp = overlapping,
s = singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, m= multiplet) and
coupling constants in Hertz. All 13C NMR spectra were recorded with complete proton
decoupling. High-resolution mass spectra (HRMS) were recorded with Bruker Daltonics Bio-
TOF-Q III (ESIMS). Thin layer chromatography (TLC) was performed on silica gel HF254
plates, detected by charring using 15% (v/v) H2SO4 in MeOH or by means of a UV detector. All
commercially available reagents were used without further purification, and purchased at
Sinopharm, Shanghai, China. All reactions were monitored by TLC or by iodine fuming
detection. Column chromatography was conducted on a silica gel plug (200-300 mesh) using a
mixture of ethyl actetate (EtOAc) and petroleum ether (bp 60-90 °C) as the eluent.
4.2 Synthetic procedures
4.2.1. p-Methoxyphenyl 3,4-di-O-benzoyl-α-L-rhamnopyranoside (7)

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To a solution of 10 (5.00 g, 13.35 mmol) in CH2Cl2 (200 mL) was added pyridine (20 ml) and
DMAP (0.05 g, 0.41 mmol) at −25o C in N2 atmosphere. The reaction mixture was stirred for 30
min, and BzCl (1.53 mL, 13.30 mmol) in CH2Cl2 (25 ml) was added dropwise. The resulting
mixture was stirred for another 4 h, at which time TLC (petroleum ther-ethyl acetate 4:1)
indicated the completion of reaction. The reaction was then quenched with cooled H2O (200 mL),
and extracted with CH2Cl2 (3 × 150 mL). The organic phases were combined, dried over Na2SO4,
and concentrated. The residue was purified by flash column chromatography (petroleum
22
ether/ethyl acetate 8:1) to give 7 (4.48 g, 69%) as colorless syrup. [α]D −17.6° (c 1.0,
CHCl3).1H NMR (300 MHz, CDCl3): δ 7.99−7.94 (m, 4H, ArH), 7.53−7.47 (m, 2H, ArH),
7.39−7.34 (m, 4H, ArH), 7.12−7.06 (m, 2H, ArH), 6.89−6.85 (m, 2H, ArH), 5.79 (dd, 1H, J1=
3.5 Hz, J2= 10.0 Hz, H-3), 5.66 (t, 1H, J = 9.8 Hz, H-4), 5.51 (d, J = 1.7 Hz, H-1), 4.85 (dd, 1H,
J1 = 2.0 Hz, J2= 3.03 Hz, H-2), 4.28−4.18 (m, 1H, H-5), 3.79 (s, 3H, OCH3), 1.29 (d, 3H, J = 6.3
Hz, H-6); 13C NMR (75 MHz, CDCl3) : δ 165.4 (COPh), 165.3 (COPh), 154.8, 149.9, 132.9,
132.8, 129.4, 129.3, 129.0, 128.9, 128.1, 128.0, 117.25, 114.4, 98.1 (C-1), 72.1, 71.1, 69.2, 66.9,
55.3 (OCH3), 17.2 (C-6). ESI-HRMS [M + Na]+calculated for C27H26NaO8 512.1525 found
512.1521.
4.2.2. p-Methoxyphenyl 2,4-di-O-benzoyl-α-L-rhamnopyranoside (11)
To a solution of 10 (5.00 g, 13.35 mmol) in CH2Cl2 (100 mL) was added triethylorthobenzoate
(7.59 ml, 33.38 mmol) and p-TsOH (2.00 g, 0.12 mmol) at room temperature in N2 atmosphere.
The reaction mixture was stirred for 4 h, at which time TLC (petroleum ether-ethyl acetate 4:1)
indicated the completion of reaction, the reaction mixture was concentrated and added 80% aq.
AcOH 100 ml, the resulting mixture was stirred for another 1 h, atwhich time TLC (petroleum
ether-ethyl acetate 5:1) indicated the completion of reaction. The reaction was then quenched
withH2O (200 mL), and extracted with CH2Cl2 (3 ×75 mL). Theorganic phases were washed
with sated aq NaHCO3 (250 ml) and brine (2 × 200 ml), dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuo. The residue was purified by flash column chromatography
22
(petroleum ether/ethyl acetate 6:1) to give 11 (4.45 g, 74%) as colorless syrup. [α]D −14.6° (c
1
1.0, CHCl3). H NMR (300 MHz, CDCl3): δ 8.15−8.06 (m, 4H, ArH), 7.65−7.57 (m, 2H, ArH),
7.52−7.44 (m, 4H, ArH), 7.08−7.02 (m, 2H, ArH), 6.88−6.83 (m, 2H, ArH), 5.58−5.56 (m, 2H,
H-1, H-2), 5.34 (t, 1H, J = 9.9 Hz, H-4), 4.54−4.50 (m, 1H, H-3), 4.25−4.20 (m, 1H, H-5), 3.78
(s, 3H, OCH3), 2.56 (d, 1H, J = 6.8 Hz, OH), 1.31 (d, 3H, J = 6.2 Hz, H-6); 13C NMR (75 MHz,
CDCl3) : δ 166.7 (COPh), 165.6 (COPh), 154.9, 149.8, 133.2, 133.2, 129.6, 129.5, 129.1, 129.0,
128.3, 128.2, 117.4, 114.4, 96.2 (C-1), 75.2, 72.8, 68.6, 66.6, 55.3 (OCH3), 17.3 (C-6). ESI-
HRMS [M + Na]+calculated for C27H26NaO8 512.1525 found 512.1520.
4.2.3. p-Methoxyphenyl 2,4-di-O-benzoyl-3-O-levulinoyl-α-L-rhamnopyranoside (12)
To a solution of 11 (4.00 g, 8.36 mmol) in CH2Cl2 (100 mL) was added DCC (3.45 g, 16.72
mmol) and DMAP (0.05 g, 0.41 mmol) at 0o C in N2 atmosphere. The reaction mixture was
stirred for 10 min, and levulinic acid (1.46 g 12.54 mmol) in CH2Cl2 (20 ml) was added dropwise.
The resulting mixture was stirred for another 3 h, at which time TLC (petroleum ether-ethyl
acetate 1:1) indicated the completion of reaction. The reaction was then filtered through a pad of
celite, and the filtrate was concentrated. The residue was purified by flash column

ACCEPTED MANUSCRIPT
chromatography (petroleum ether/ethyl acetate 1:1) to give 3 (4.44 g, 93%) as colorless syrup.
22
[α]D 37.1° (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): δ 8.15−8.01 (m, 4H, ArH), 7.65−7.43
(m, 6H, ArH), 7.09−7.04 (m, 2H, ArH), 6.87−6.82 (m, 2H, ArH), 5.81 (dd, 1H, J1 = 3.4 Hz, J2 =
10.1 Hz, H-3), 5.71 (dd, 1H, J1 = 1.9 Hz, J2 = 3.4 Hz, H-2), 5.58− 5.51 (m, 2H, H-1, H-4),
4.29−4.20 (m, 1H, H-5), 3.77 (s, 3H, OCH3), 2.64−2.34 (m, 4H, COCH2CH2COCH3), 1.93 (s,
3H, COCH2CH2COCH3), 1.31 (d, J = 6.2 Hz, H-6); 13C NMR (75 MHz, CDCl3) :δ 205.4
(CH2COCH3), 171.3 (C(O)OCH2CH2), 165.4 (COPh), 165.2 (COPh), 154.9, 149.7, 133.2, 133.0,
129.6, 129.5, 128.9, 128.3, 128.1, 117.3, 114.3, 96.1 (C-1), 71.3, 70.1, 69.0, 67.0, 55.3 (OCH3),
37.5, 29.0, 27.6, 17.3 (C-6). ESI-HRMS [M + Na]+ calculated for C32H32NaO10 599.1898 found
599.1895.
4.2.4. 2,4-di-O-benzoyl-3-O-levulinoyl-α-L-trichloroacetimidate (8)
To a solution of 12 (4.00 g, 6.93 mmol) in CH3CN:H2O (4:1 v/v) 75 ml was added
Ce(NH4)2(NO3)6 (15.20 g, 27.75 mmol) at room temperature. The resulting mixture was stirred
for another 2 h, at which time TLC (petroleum ether-ethyl acetate 4:1) indicated the completion
of reaction. The reaction was then quenched with cooled H2O (200 mL), and extracted with
CH2Cl2 (3 × 150 mL). The organic phases were combined, dried over Na2SO4, and concentrated.
The residue was purified by flash column chromatography (petroleum ether/ethyl acetate 3:1) to
give 13 (2.30 g, 76%). Subsequently dissolving 13 in CH2Cl2 (50 ml) and added
trichloroacetonitrile (1.23 ml, 12.2 mmol) and DBU (0.05mL) in N2 atmosphere. The reaction
mixture was stirred for 2 h under this condition, and then concentrated. The residue was
subjected to flash column chromatography (petroleum ether/ethyl acetate 4:1) to give 8 (2.88 g
92%) as colorless syrup. [α]D22 16.6° (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): δ 8.80 (s, 1H,
OCNHCCl3), 7.98−7.88 (m, 4H, ArH), 7.53−7.46 (m, 2H, ArH), 7.40−7.32 (m, 4H, ArH), 6.32
(d, 1H, J = 1.7 Hz, H-1), 5.76 (dd, 1H, J1 = 3.4 Hz, J2 = 10.2 Hz, H-3), 5.68−5.59 (m, 2H, H-2,
H-4), 4.37−4.28 (m, 1H, H-5), 2.73 (s, 4H, OCOCH2CH2COCH3), 2.14 (s, 3H,
OCOCH2CH2COCH3), 1.37 (d, 3H, J = 6.2 Hz, H-6); 13C NMR (75 MHz, CDCl3) :δ 205.7
(COCH3), 171.4C(O)OCH2CH2, 165.6COPh, 165.4COPh, 159.9 (OCNHCH3), 133.4, 133.2,
129.7, 129.7, 129.0, 129.0, 128.4, 128.3, 94.7(C-1), 90.7, 70.8, 69.6, 69.5, 68.6, 37.7, 29.6, 27.8,
17.6 (C-6). ESI-HRMS [M + Na]+ calculated for C27H26NNaO9 636.0576 found 636.0572.
4.2.5. 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-D-glucopyranoside (15)
To a solution of ethylene diamine : CH3COOH (0.85 ml : 0.85 ml v/v) in THF (250 ml) at 0oC
and compound 14 (5.00 g, 10.47 mmol) in THF (50 ml) was added dropwise. The resulting
mixture was stirred for 24 h at room temperature, at which time TLC (petroleum ether-ethyl
acetate 3:1) indicated the completion of reaction. The reaction was then quenched with 1M HCl
(200 mL), and extracted with CH2Cl2 (3 × 100 mL). The organic phases were washed with sated
aq. NaHCO3 (200 ml), and brine (2 × 200 ml), dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The residue was purified by flash column chromatography (petroleum
22
ether/ethyl acetate 4:1) to give 15 (3.43 g, 72%) as colorless syrup. [α]D 16.7° (c 1.0, CHCl3).
1H NMR (300 MHz, CDCl3): δ 7.90−7.82 (m, 2H, ArH), 7.78−7.71 (m, 2H, ArH), 5.84 (dd, 1H,
J1 = 9.1 Hz, J2 = 10.7 Hz, H-3), 5.63 (t, 1H, J = 7.2 Hz, H-4), 5.17 (dd, 1H, J1 = 9.2 Hz, J2 = 10.1
Hz, H-2), 4.32−4.16 (m, 3H, H-1, H-6), 3.95−3.90 (m, 1H, H-5), 3.48 (d, 1H, J = 6.4 Hz, OH),

ACCEPTED MANUSCRIPT
1.86, 2.03, 2.10 (3s, 9H, OCOCH3); 13C NMR (75 MHz, CDCl3) : δ 170.4 (COCH3), 169.7
(COCH3), 169.1 (COCH3), 134.0, 131.0, 123.3, 92.3 (C-1), 71.7, 70.1, 68.6, 61.7, 55.7 (C-6),
20.4 (COCH3), 20.2 (COCH3), 20.0 (COCH3). ESI-HRMS [M + Na]+ calculated for
C20H21NNaO10 458.1063 found 458.1058.
4.2.6. 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-D-trichloroacetimidate (9)
To a solution of 15 (3.00 g, 6.89 mmol) in CH2Cl2 (50 mL) at 0oC with in N2 atmosphere was
added trichloroacetonitrile (1.72 ml, 17.2mmol) and DBU (0.05 mL). The reaction mixture was
stirred for 2 h under this condition, and then concentrated. The residue was subjected to flash
column chromatography (petroleum ether/ethyl acetate 4:1) to give 15 (3.75 g 95%) which was
immediately used in the synthesis of 19.
4.2.7. p-Methoxyphenyl 2,4-di-O-benzoyl-3-O-levulinoyl-α-L-rhamnopyranosyl-(1→2)-3,4-di-O-
benzoyl-α- -rhamnopyranoside (16)
L
A mixture of 8 (2.50 g, 4.18 mmol), 7 (2.00 g, 4.18 mmol) and 4 Å MS (0.5 g) in anhydrous
CH2Cl2 (70 mL) was stirred at rt for 30 min in N2 atmosphere, before it was cooled to 0o C, then
TMSOTf (10 uL) was added. The mixture was stirred at same temperature for 8 h, and then
neutralized with Et3N. The mixture was diluted with CH2Cl2 (10 mL), filtered through a pad of
celite, and the filtrate was concentrated. The residue was purified by flash column
chromatography (petroleum ether/ethyl acetate 4:1) to give 16 (2.53 g, 66%) as colorless syrup.
[α]D22 11.9° (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): δ 8.08–7.95 (m, 8H, ArH), 7.62− 7.56
(m, 2H, ArH), 7.52−7.29 (m, 10H, ArH), 7.12−7.08 (m, 2H, ArH), 6.89−6.86 (m, 2H, ArH), 5.96
(dd, 1H, J1 = 3.5 Hz, J2 = 10.0 Hz, H-3), 5.76−5.73 (m, 2H, H-2, H-3), 5.68 (t, 1H, J = 9.8 Hz,
H-4), 5.55 (d, 1H, J = 1.6 Hz, H-1), 5.48 (t, 1H, J = 9.9 Hz, H-4), 5.14 (s, 1H, H-1), 4.47 (m, 1H,
H-2), 4.28−4.22 (m, 2H, H-5, H-5), 3.79 (s, 3H, OCH3), 2.59−2.38 (m, 4H,
OCOCH2CH2COCH3), 1.94 (s, 3H, OCOCH2CH2COCH3), 1.36 (d, 3H, J = 6.3 Hz, H-6), 1.30 (d,
13
3H, J = 6.3 Hz, H-6); C NMR (75 MHz, CDCl3) : δ 205.4 (COCH3), 171.0 (C(O)OCH2CH2),
165.5 (COPh), 165.4 (COPh), 165.1 (COPh), 164.7 (COPh), 154.9, 149.8, 133.0, 132.8, 132.7,
129.8, 129.6, 129.5, 129.4, 129.1, 128.9, 128.6, 128.1, 128.1, 127.9, 117.3, 114.3, 99.1 (C-1),
97.4 (C-1), 75.9, 71.5, 71.3, 70.6, 69.0, 68.8, 67.3, 67.2, 55.3 (OCH3), 37.5, 29.0, 27.7, 17.3 (C-
6), 17.2 (C-6). ESI-HRMS [M+NH4]+ calculated for C52H54NO16 948.3437, found 948.3431.
4.2.8. 2,4-di-O-benzoyl-3-O-levulinoyl-α-
trichloroacetimidate (6)
L-rhamnopyranosyl-(1→2)-3,4-di-O-benzoyl-α-L-
To a solution of 16 (1.50 g, 1.61 mmol) in CH3CN : H2O (4:1 v/v) 25 ml was added
Ce(NH4)2(NO3)6 (3.70 g, 6.45 mmol) at room temperature. The resulting mixture was stirred for
another 2 h, at which time TLC (petroleum ether-ethyl acetate 5:1) indicated the completion of
reaction. The reaction was then quenched with cooled H2O (50 mL), and extracted with CH2Cl2
(3 ×25 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The
residue was purified by flash column chromatography (petroleum ether/ethyl acetate 5:1) to give
17 (0.93 g 68%). Subsequently dissolving 17 in CH2Cl2 20 ml and added trichloroacetonitrile
(0.28 mL, 2.82 mmol) and DBU (0.02 mL). The reaction mixture was stirred for 2 h under this
condition, and then concentrated. The residue was subjected to flash column chromatography

ACCEPTED MANUSCRIPT
(petroleum ether/ethyl acetate 7:1) to give 6 (980 mg 95%) which was immediately used in the
synthesis of 18.
4.2.9. p-Methoxyphenyl 2,4-di-O-benzoyl-3-O-levulinoyl-α-
L
-rhamnopyranosyl-(1→2)-2,4-di-
-rhamnopyranoside (18)
O-benzoyl-α- -rhamnopyranosyl-(1→2)-3,4-di-O-benzoyl-α-
L
L
A mixture of 7 (572 mg, 1.20 mmol), 6 (980 mg, 1.01 mmol) and 4 Å MS (0.4 g) in anhydrous
CH2Cl2 (30 mL) was stirred at room temperature for 30 min in N2 atmosphere, before it was
cooled to −10o C, then TMSOTf (10 uL) was added. The mixture was stirred at same temperature
for 8 h, and then neutralized with Et3N. The mixture was diluted with CH2Cl2 (20 mL), filtered
through a pad of celite, and the filtrate was concentrated. The residue was purified by flash
column chromatography (petroleum ether/ethyl acetate 4:1) to give 18 (890 mg, 75%) as
22
colorless syrup. [α]D 37.9° (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): δ 8.02−7.94 (m, 12H,
ArH), 7.63−7.53 (m, 2H, ArH), 7.51−7.30 (m, 14H, ArH), 7.24−7.17 (m, 2H, ArH), 7.17−7.10
(m, 2H, ArH), 6.92−6.82 (m, 2H, ArH), 5.90−5.80 (m, 2H, H-3, H-3), 5.71−5.57 (m, 4H, H-2,
H-3, H-4, H-4), 5.60 (s, 1H, H-1), 5.32 (t, 1H, J = 9.7 Hz, H-4), 5.17 (d, 1H, J = 1.4 Hz, H-1),
4.79 (s, 1H, H-1), 4.63−4.51 (m, 1H, H-2), 4.40−4.33 (m, 1H, H-2), 4.32−4.23 (m, 2H, H-5, H-5),
4.01−3.99 (m, 1H, H-5), 3.79 (s, 3H, OCH3), 2.60−2.34 (2m, 4H, OCOCH2CH2CH3), 1.92 (s, 3H,
COCH3), 1.38 (d, 3H, J = 6.3 Hz, H-6), 1.34 (d, 3H, J = 6.3 Hz, H-6), 0.97 (d, 3H, J = 6.26 Hz,
H-6); 13C NMR (75 MHz, CDCl3) : δ 205.4 (COCH3), 170.9 (C(O)OCH2CH2), 165.6 (COPh),
165.4 (COPh), 165.1 (COPh), 165.1 (COPh), 164.6 (COPh), 154.9, 149.9, 133.0, 133.0, 132.9,
132.8, 132.7, 129.6, 129.5, 129.4, 129.3, 129.1, 129.0, 128.9, 128.8, 128.7, 128.1, 128.1, 128.0,
128.0, 117.3, 114.4, 100.3 (C-1), 99.0(C-1), 97.7 (C-1), 71.4, 71.2, 70.2, 69.8, 68.8, 67.4, 67.1,
55.3 (OCH3), 37.4, 29.0, 27.6, 17.3 (C-6), 17.3 (C-6), 16.8 (C-6). ESI-HRMS [M+NH4]+
calculated for C71H69NO22 1302.4540 found 1302.4531.
4.3.0. p-Methoxyphenyl 2,4-di-O-benzoyl-α-
L
-rhamnopyranosyl-(1→2)-2,4-di-O-benzoyl-α-
L-
rhamnopyranosyl-(1→2)-3,4-di-O-benzoyl-α-
L
-rhamnopyranoside (4)
To a solution of 16 (800 mg, 0.62 mmol) in DMF (25 mL) was added hydrazine acetate (243 mg,
3.1 mmol) at room temperature in N2 atmosphere. The reaction mixture was stirred for 4 h, at
which time TLC (petroleum ether/ethyl acetate 2:1) indicated the completion of reaction. The
reaction was then quenched with sat. aq. NH4Cl (50 mL) and then extracted with
dichloromethane (100 mL). The organic phase was washed with sat. aq. NaHCO3 (2 x 50 mL)
and water (50 mL). After the solution was dried over Na2SO4, the solvent was removed under
reduced pressure and the residue was purified by flash chromatography (petroleum ether/ethyl
22
1
acetate 3:1) to give 4 (589 mg, 80%) as colorless syrup. [α]D 38.4° (c 1.0, CHCl3). H NMR
(300 MHz, CDCl3): δ8.09−7.93 (m, 12H, ArH), 7.62−7.52 (m, 2H, ArH), 7.62−7.32 (m, 14H,
ArH), 7.21−7.04 (m, 4H, ArH), 6.91−6.86 (m, 2H, ArH), 5.89−5.79 (m, 2H, H-3, H-3), 5.68 (t,
1H, J = 9.7 Hz, H-4), 5.56 (s, 1H, H-1), 5.59−5.53 (t, 1H, J = 9.8 Hz, H-4), 5.43 (dd, 1H, J1 = 1.6
Hz, J2 = 3.3 Hz, H-2), 5.16 (s, 1H, H-1), 5.16−5.09 (t, 1H, J = 11.7 Hz, H-4), 4.78 (d, 1H, J = 1.3
Hz, H-1), 4.56 (dd, 1H, J1 = 2.1 Hz, J2 = 3.0 Hz, H-2), 4.36−4.32 (m, 2H, H-2, H-3), 4.30−4.22
(m, 2H, H-5, H-5), 4.06−3.96 (m, 1H, H-5), 3.80 (s, 3H, OCH3), 2.34 (d, 1H, J = 7.3 Hz, OH),
13
1.37 (d, 3H, J = 6.2 Hz, H-6), 1.32 (d, 3H, J = 6.2 Hz, H-6), 0.97 (d, 3H, J = 6.2 Hz, H-6); C
NMR (75 MHz, CDCl3) : δ 166.6 (COPh), 165.6 (COPh), 165.4 (COPh), 165.2 (COPh), 165.1
(COPh), 165.0 (COPh), 154.9, 149.9, 133.0, 132.9, 132.8, 132.8, 132.7, 129.5, 129.5, 129.3,

ACCEPTED MANUSCRIPT
129.1, 129.0, 128.9, 128.7, 128.1, 128.0, 128.0, 117.3, 114.4, 100.5 (C-1), 98.9 (C-1), 97.7 (C-1),
76.5, 74.7, 72.4, 71.4, 71.3, 71.2, 70.1, 68.3, 67.4, 67.1, 66.7, 55.3 (OCH3), 17.4 (C-6), 17.3 (C-
6), 16.9 (C-6). ESI-HRMS [M+NH4]+ calculated for C67H66NO20 1204.4178 found 1204.4174.
4.3.1. p- Methoxyphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→2)
-3,4-di-O-benzoyl-α- -rhamnopyranoside (19)
L
A mixture of 10 (1.80 g, 3.47 mmol), 9 (2.00 g, 3.45 mmol) and 4 Å MS (0.5 g) in anhydrous
CH2Cl2 (40 mL) was stirred at rt for 30 min in N2 atmosphere, before it was cooled to −10o C,
then TMSOTf (10 uL) was added. The mixture was stirred at same temperature for 8 h, and then
neutralized with Et3N. The mixture was diluted with CH2Cl2 (10 mL), filtered through a pad of
celite, and the filtrate was concentrated. The residue was purified by flash column
chromatography (petroleum ether/ethyl acetate 4:1) to give 19 (2.00 g 65%) as colorless syrup.
[α]D22 −15.5° (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): δ 7.80−7.77 (m, 2H, ArH), 7.54−7.51
(m, 4H, ArH), 7.45−7.28 (m, 6H, ArH), 7.11−7.04 (m, 4H, ArH), 6.87−6.82 (m, 2H, ArH), 5.71
(s, 1H, H-1-Rhamp), 5.70 (t, 1H, J = 10.5 Hz, H-3-Glcp), 5.52 (d, 1H, J = 8.4 Hz, H-1-Glcp),
5.49 (dd, 1H, J1 = 3.3 Hz, J2 = 10.2 Hz, H-3-Rhamp), 5.33 (t, 1H, J = 9.9 Hz, H-4-Rhamp), 5.08
(t, 1H, J = 10.1 Hz, H-4-Glcp), 4.61 (m, 1H, H-2-Rhamp), 4.48 (dd, 1H, J1 = 8.4 Hz, J2 = 10.5
Hz, H-2-Glcp), 4.19 (dd, 1H, J1 = 6.1 Hz, J2 = 12.2 Hz, H-6-Glcp), 4.11−4.06 (m, 2H, H-5, H-6),
3.83−3.78 (m, 1H, H-5), 3.77 (s, 3H, OCH3), 1.80, 1.84, 1.98 (3s, 9H, OCOCH3), 1.20 (d, 3H, J
= 6.2 Hz, H-6); 13C NMR (75 MHz, CDCl3) :δ 170.2 (COCH3), 169.6 (COCH3), 169.0 (COCH3),
167.0 (Phth), 165.9 (COPh), 164.7 (COPh), 154.7, 149.9, 133.5, 132.6, 132.4, 130.8, 129.1,
129.1, 128.7, 127.9, 123.1, 117.1, 114.3, 98.9 (C-1), 97.6 (C-1), 71.9, 70.6, 70.1, 68.7, 66.8, 61.8,
55.3, 54.2 (OCH3), 20.2 (COCH3), 20.0 (COCH3), 17.2 (C-6). ESI-HRMS [M+NH4]+ calculated
for C47H49N2O17 913.3026 found 913.3024.
4.3.2. p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-acetamido-β-D-glucopyranosyl-(1→2)-
3,4-di-O-acetyl-α- -rhamnopyranose (20)
L
Ethylene diamine (1.49 ml, 22.3 mmol) was added to a suspension of compound 19 (2.00 g, 2.23
mmol) in n-butyl alcohol (100 mL). The reaction mixture was stirred for 8 h at 85o C. The
solvent was removed under reduced pressure and repeatedly co-distilled with toluene (3 x 150
mL) and ethanol (2 x 100 mL). The resulting oil was dissolved in pyridine (50 mL), treated with
acetic anhydride (10 mL) and stirred for 24 h at room temperature. The reaction was poured into
ice-water (200 mL) and then extracted with dichloromethane (200 mL). The organic phase was
washed with 0.1 M HCl (150 mL), sat. aq. NaHCO3 (2 x 100 mL) and water (100 mL). After the
solution was dried over Na2SO4, the solvent was removed under reduced pressure and the residue
was purified by flash chromatography (petroleum ether/ethyl acetate 1:2) to give 20 (1.60 g 70%)
as colorless syrup. [α]D22 −7.7° (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): δ 6.98−6.95 (m, 2H,
ArH), 6.82−6.74 (m, 2H, ArH), 5.87 (d, 1H, J = 7.6 Hz, H-1-Glcp), 5.65 (dd, 1H, J1 = 9.3 Hz, J2
= 10.5 Hz, H-3-Glcp), 5.43 (d, 1H, J = 1.7 Hz, H-1-Rhamp), 5.39 (dd, 1H, J1 = 3.1 Hz, J2 = 10.2
Hz, H-3-Rhamp), 5.10−5.03 (m, 2H, H-2-Glcp, H-4), 4.95 (t, 1h, J = 9.9 Hz, H-4), 4.22−4.16 (m,
2H, H-2-Rhamp, H-6-Glcp), 4.03−3.98 (dd, 1H, J1 = 2.1 Hz, J2 = 12.1 Hz, H-6-Glcp), 3.99−3.89
(m, 1H, H-5), 3.73 (s, 3H, OCH3), 3.50−3.41 (m, 1H, H-5), 1.88, 1.97, 1.98, 2.00, 2.09 (5s, 18H,
OCOCH3), 1.14 (d, 3H, J = 6.2 Hz, H-6); 13C NMR (75 MHz, CDCl3) :δ 170.5 (COCH3), 170.2
(COCH3), 170.1 (COCH3), 170.0 (COCH3), 169.5 (COCH3), 169.2(COCH3), 154.6, 149.6, 133.8,

ACCEPTED MANUSCRIPT
131.5, 123.0, 117.0, 114.3, 100.1 (C-1), 97.4 (C-1), 75.7, 71.3, 70.8, 70.5, 68.8, 66.6, 61.8, 55.7,
55.2, 22.9 (COCH3), 22.3 (COCH3), 20.4 (COCH3), 20.3 (COCH3), 20.2 (COCH3), 20.1
(COCH3), 17.0 (C-6). ESI-HRMS [M+NH4]+ calculated for C31H45N2O16 701.2764 found
701.2758.
4.3.3. 3,4,6-tri-O-acetyl-2-deoxy-2-acetamido-β-
trichloroacetimidate (5)
D-glucopyranosyl-(1→2)-3,4-di-O-acetyl-L-
To a solution of 20 (1.50 g, 2.18 mmol) in CH3CN : H2O (4:1 v/v) 25 ml was added
Ce(NH4)2(NO3)6 (4.90 g, 8.75 mmol) at room temperature. The resulting mixture was stirred for
another 2 h, at which time TLC (petroleum ether-ethyl acetate 4:1) indicated the completion of
reaction. The reaction was then quenched with cooled H2O (50 mL), and extracted with CH2Cl2
(3 ×25 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The
residue was purified by flash column chromatography (petroleum ether/ethyl acetate 1:3) to give
21 (0.90 g, 70%). Subsequently dissolving 21 in CH2Cl2 30 ml and added DBU (0.1 ml) with N2
protection. The reaction mixture was stirred for 12 h under this condition, and then filtered
through a pad of celite, and the filtrate was concentrated. The residue was subjected to flash
column chromatography (petroleum ether/ethyl acetate 1:4) to give 5 (2.85 g 90%) as colorless
22
1
syrup. [α]D −14.3° (c 1.0, CHCl3). H NMR (300 MHz, CDCl3): δ 8.66 (s, 1H, OCNHCCl3),
6.27 (d, 1H, J = 2.0 Hz, H-1-Rhamp), 5.83 (d, 1H, J = 7.4 Hz, H-1-Glcp), 5.67 (dd, 1H, J1 = 9.2
Hz, J2 = 10.5 Hz, H-3-Glcp), 5.25 (dd, 1H, J1 = 3.0 Hz, J2 = 10.3 Hz, H-3-Rhamp), 5.16−5.09 (m,
2H, H-4-Rhamp, H-4-Glcp), 5.00 (t, 1H, J = 9.8 Hz, H-2-Glcp), 4.23 (m, 2H, H-2-Rhamp, H-6-
Glcp), 4.15-4.05 (m, 1H, H-6-Glcp), 3.74−3.68 (m, 1H, H-5), 3.46−3.37 (m, 1H, H-5), 2.08, 2.03,
13
2.03, 2.02, 1.99, 1.98 (6s, 18H, OCOCH3), 1.22 (d, 3H, J = 6.2 Hz, H-6-Rhamp); C NMR (75
MHz, CDCl3) :δ 170.4 (COCH3), 170.3 (COCH3), 170.0 (COCH3), 169.9 (COCH3), 169.5
(COCH3), 169.1 (COCH3), 159.7 (OCNHCH3), 99.9 (C-1), 96.0 (C-1), 90.5 (OCNHCCl3), 74.2,
71.3, 70.8, 70.2, 70.1, 68.9, 68.7, 61.7, 56.0 (C-2), 22.9 (COCH3), 20.3 (COCH3), 20.3 (COCH3),
20.2 (COCH3), 17.0 (C-6). ESI-HRMS [M+NH4]+ calculated for C26H39N3O15 738.1449 found
738.1442.
4.3.4. p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-acetamido-β-D-glucopyranosyl-(1→2)-3,4-
di-O-acetyl-α- -rhamnopyranosyl-(1→3)-2,4-di-O-benzoyl-α-
L
L
-rhamnopyranosyl-(1→2)-3,4-di
-O-benzoyl-α- -rhamnopyranosyl-(1→2)-3,4-di-O-benzoyl-α-L- rhamnopyranose (22)
L
A mixture of 5 (304 mg, 0.43 mmol), 4 (500 mg, 0.42 mmol) and 4 Å MS (0.3 g) in anhydrous
CH2Cl2 (30 mL) was stirred at rt for 30 min in N2 atmosphere, before it was cooled to −10o C,
then TMSOTf (10 uL) was added.The mixture wasstirred at same temperature for 8 h, and then
neutralized with Et3N. The mixture was diluted with CH2Cl2 (10 mL), filtered through a pad of
celite, and the filtrate was concentrated. The residue was purified by flash column
chromatography (petroleum ether/ethyl acetate 1:4) to give 22 (447 mg 67%) as colorless syrup.
[α]D22 28.4° (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): δ 8.07−7.91 (m, 12H, ArH), 7.62−7.32
(m, 15H, ArH),7.20−7.06 (m, 5H, ArH), 6.89−6.86 (m, 2H, ArH), 5.88−5.84 (dd, 1H, J1 = 3.0
Hz, J2 =10.2 Hz, H-3), 5.82−5.78 (dd, 1H, J1 = 3.0 Hz, J2 =9.8 Hz, H-3), 5.70−5.64 (m, 2H, H-3,
H-4), 5.60−5.43 (m, 3H, H-4, H-4, H-4), 5.57 (s, 1H, H-1-Rhamp),5.29 (t, 1H, J = 9.8 Hz, H-4),
5.14 (s, 1H, H-1-Rhamp), 5.08−5.04 (dd, 1H, J1 = 2.1 Hz, J2 = 9.5 Hz, H-2), 5.01 (s, 1H, H-1-
Rhamp), 4.89 (t, 1H, J = 9.7 Hz, H-3), 4.80 (s, 1H, H-1-Rhamp), 4.77−4.71 (m, 1H, H-2), 4.72

ACCEPTED MANUSCRIPT
(d, 1H, J = 8.0 Hz, H-1-Glcp), 4.55 (m, 1H, H-2), 4.48−4.43 (dd, 1H, J1 = 2.9 Hz, J2 =9.7 Hz, H-
3), 4.55 (m, 1H, H-2), 4.31−4.22 (m, 2H, H-6), 3.95−3.79 (m, 2H, H-5), 3.79 (s, 3H, OCH3),
3.59 (m, 2H, H-2, H-5), 3.38−3.30 (m, 2H, H-5),1.89, 1.93, 1,95, 1.96, 1.98, 2.00 (6s, 18H,
OCOCH3), 1.37 (d, 3H, J = 6.2 Hz, H-6), 1.31 (d, 3H, J = 6.2 Hz, H-6), 1.03 (d, 3H, J = 6.2 Hz,
13
H-6), 0.89 (d, 3H, J = 6.2 Hz, H-6); C NMR (75 MHz, CDCl3) : δ 170.3, 170.2, 169.9, 169.7,
169.2, 165.7, 165.2, 164.9, 154.9, 132.93, 132.8, 129.6, 129.5, 129.4, 129.3, 129.2, 129.2, 129.1,
129.0, 128.7, 128.2, 128.1, 128.0, 128.0, 117.4, 114.7, 100.5 (C-1-Rhamp), 99.9 (C-1-Rhamp),
99.3 (C-1-Glcp), 98.9 (C-1-Rhamp), 97.6 (C-1-Rhamp), 76.5 (C-2, C-2), 74.8 (C-3, C-4), 73.4
(C-3, C-4, C-4), 72.8 (C-4, C-4, C-3), 71.5 (C-3), 71.2 (C-5), 70.9 (C-5), 70.4 (C-5), 70.2 (C-5),
68.6 (C-2), 67.4 (C-2), 67.1(C-3), 67.0 (C-6), 61.4 (C-5), 55.5 (C-2), 55.3 (OCH3), 22.8, 20.4,
20.3, 20.2, 17.3 (C-6, C-6), 16.9 (C-6), 16.7 (C-6). ESI-HRMS [M+NH4]+ calculated for
C91H99N2O34 1763.6079 found 1763.6075.
4.3.5. p-Methoxyphenyl 2-deoxy-2-acetamido-β-
D
-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-
-rhamnopyranose (2)
(1→3)-α- -rhamnopyranosyl-(1→2)-α- -rhamnopyranosyl-(1→2)-α-L
L
L
To a solution of 22 (400 mg, 0.23 mmol) in MeOH (20 mL) was added satd. MeOH-NH3
(MeOH solution) 40 ml. The resulting mixture was stirred for 14 days and the solvent was
22
removed under reduced pressure to give 2 (168 mg 80%) as yellow syrup. [α]D 26.3°(c 1.0,
MeOH). 1H NMR (300 MHz, D2O): δ 6.96−6.93 (m, 2H, ArH), 6.84−6.81 (m, 2H, ArH), 5.34 (s,
1H, H-1-Rhamp), 5.15 (s, 1H, H-1-Rhamp), 5.06 (s, 1H, H-1-Rhamp), 4.86 (s, 1H, H-1-Rhamp),
4.57 (d, 1H, J = 8.4 Hz, H-1-Glcp), 4.04−3.99 (m, 5H), 3.83−3.57 (m, 15H, OCH3), 3.48−3.20
(m, 10H), 1.94 (s, 3H, ONHCOCH3), 1.19−1.13 (m, 12H, H-6); 13C NMR (75 MHz, D2O) : δ
174.5, 154.5, 149.3, 118.6, 114.9, 102.5 (C-1-Glcp), 101.6 (C-1-Rhamp), 100.7 (C-1-Rhamp),
100.6 (C-1-Rhamp), 98.0 (C-1-Rhamp), 78.9, 78.1, 77.9, 75.5 , 73.3, 72.0, 71.8, 69.7, 69.6, 69.5,
69.3, 69.1, 68.9, 60.3, 55.6, 55.5, 22.0, 16.5 (C-6, C-6), 16.4 (C-6). ESI-HRMS [M +
H]+calculated for C39H62NO23 912.3707 found 912.3700.
4.3.6. p-Methoxyphenyl 2,4-di-O-benzoyl -α-L-rhamnopyranosyl-(1→2)-3,4-di-O-benzoyl-α-L
-rhamnopyranose (23)
To a solution of 16 (2.00 g, 2.15 mmol) in DMF (100 mL) was added hydrazine acetate (0.80 g,
10.75 mmol) and at room temperature in N2 atmosphere. The reaction mixture was stirred for 4 h,
at which time TLC (petroleum ether/ethyl acetate 2:1) indicated the completion of reaction. The
reaction was then quenched with sat. aq. NH4Cl (200 mL) and then extracted with
dichloromethane (200 mL). The organic phase was washed with sat. aq. NaHCO3 (2 x 100 mL)
and water (100 mL). After the solution was dried over Na2SO4, the solvent was removed under
reduced pressure and the residue was purified by flash chromatography (petroleum ether/ethyl
22
1
acetate 3:1) to give 23 (1.38 g 79%) as colorless syrup. [α]D 18.8° (c 1.0, CHCl3). H NMR
(300 MHz, CDCl3): δ 8.13−7.97 (m, 8H, ArH), 7.64−7.33 (m, 12H, ArH), 7.13−7.09 (m, 2H,
ArH), 6.91−6.87 (m, 2H, ArH), 5.98 (dd, 1H, J1 = 3.2 Hz, J2 = 10.0 Hz, H-2), 5.70−5.64 (m, 2H,
H-3, H-4), 5.58 (d, 1H, J = 1.7 Hz, H-1), 5.32 (t, 1H, J = 9.8 Hz, H-4), 5.19 (s, 1H, H-1),
4.53−4.46 (m, 2H, H-2, H-3), 4.31−4.24 (m, 2H, H-5, H-5), 3.79 (s, 3H, OCH3), 2.52 (d, 1H, J =
13
7.4 Hz, OH), 1.36 (d, 3H, J = 6.3 Hz, H-6), 1.31 (d, 3H, J = 6.3 Hz, H-6); C NMR (75 MHz,

ACCEPTED MANUSCRIPT
CDCl3) : δ 166.6 (COCH3), 165.5 (COCH3), 165.3 (COCH3), 165.2 (COCH3), 153.9, 149.9,
133.1, 133.0, 132.9, 132.8, 129.6, 129.5, 129.5, 129.5, 129.4, 129.1, 129.0, 129.0, 128.2, 128.2,
128.1, 128.0, 117.3, 114.4, 99.2 (C-1), 97.5 (C-1), 76.5, 74.8, 72.5, 71.5, 70.5, 68.5, 67.2, 67.0,
55.3 (OCH3), 17.4 (C-6), 17.3 (C-6). ESI-HRMS [M+NH4]+ calculated for C47H48NO14 850.3069
found 850.3064.
4.3.7. p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-acetamido-β-
di-O-acetyl-α- -rhamnopyranosyl-(1→3)-2,4-di-O-benzoyl-α- -rhamnopyranosyl-(1→2)-3,4-
di-O-benzoyl-α- -rhamnopyranose (24)
D-glucopyranosyl-(1→2)-3,4-
L
L
L
After a mixture of 5 (430 mg, 0.60 mmol), 23 (500 mg, 0.60 mmol) and 4 Å MS (0.2 g) in
anhydrous CH2Cl2 (20 mL) was stirred at rt for 30 min in N2 atmosphere, before it was cooled to
0o C, then TMSOTf (10 uL) was added. The mixture was stirred at same temperature for 8 h, and
then neutralized with Et3N. The mixture was diluted with CH2Cl2 (10 mL), filtered through a pad
of celite, and the filtrate was concentrated. The residue was purified by flash column
chromatography (petroleum ether/ethyl acetate 1:2) to give 24 (586 mg 73%) as colorless syrup.
22
[α]D 29.7° (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): δ 8.09−8.05 (m, 4H, ArH), 8.00−7.94
(m, 4H, ArH), 7.63−7.30 (m, 12H, ArH), 7.10−7.15 (m, 2H, ArH), 6.89−6.84 (m, 2H, ArH),
5.97-5.93 (dd, 1H, J1 = 3.2 Hz, J2 = 10.0 Hz, H-3), 5.67-5.60 (m, 3H, H-4, H-4, H-4), 5.55 (d,
1H, J = 1.7 Hz, H-1), 5.51−5.43 (m, 2H, H-2, H-3), 5.18 (d, 1H, J = 1.7 Hz, H-1), 5.12−5.08 (dd,
1H, J1 = 2.9 Hz, J2 = 10.0 Hz, H-2), 5.07 (d, 1H, J = 1.7 Hz, H-1), 4.92 (t, 1H, J = 9.8 Hz, H-3),
4.76−4.73 (d, 1H, J = 8.7 Hz, H-1), 4.79−4.73 (t, 1H, J = 10.1 Hz, H-4), 4.59−4.55 (dd, 1H, J1 =
3.4 Hz, J2 = 9.7 Hz, H-3), 4.47−4.45 (dd, J1 = 2.0 Hz, J2 = 3.1 Hz, H-2), 4.26−4.14 (m, 2H, H-6),
3.94−3.89 (m, 1H, H-5), 3.84−3.82 (dd, 1H, J1 = 2.0 Hz, J2 = 3.1 Hz, H-2), 3.78 (s, 3H, OCH3),
3.65−3.61 (m, 1H, H-5), 3.40−3.31 (m, 2H, H-5), 2.04, 1.99, 1.97, 1.96, 1.94, 1.91 (6s, 18H,
OCOCH3), 1.33 (d, 3H, J = 6.3 Hz, H-6), 1.25 (d, 3H, J = 6.3 Hz, H-6), 1.08 (d, 3H, J = 6.3 Hz,
H-6); 13C NMR (75 MHz, CDCl3) : δ 170.3 (COCH3), 170.2 (COCH3), 170.0 (COCH3), 169.7
(COCH3), 165.4 (COCH3), 165.1 (COCH3), 154.9, 149.9, 133.1, 132.9, 132.8, 129.6, 129.5,
129.5, 129.4, 129.1, 128.7, 128.2, 128.1, 128.0, 117.3, 114.3, 100.0 (C-1-Rhamp), 99.3 (C-1-
Glcp), 99.0 (C-1-Rhamp), 97.5 (C-1-Rhamp), 76.8 (C-2), 74.9 (C-3), 73.4 (C-4, C-5), 72.9 (C-4),
71.5 (C-3), 71.2 (C-3), 70.9 (C-2), 70.6 (C-2), 70.4 (C-3), 68.6 (C-4), 67.4 (C-4), 67.2 (C-6),
67.1 (C-5), 61.4 (C-5), 55.6 (C-2, C-5), 55.3 (OCH3), 22.9, 20.5, 20.4, 20.3, 17.4 (C-6, C-6),
16.7 (C-6). ESI-HRMS [M+NH4]+ calculated for C71H81N2O28 1409.4970 found 1409.4972.
4.3.8. p-Methoxyphenyl 2-deoxy-2-acetamido-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl
-(1→3)-α- -rhamnopyranosyl-(1→2)-α- -rhamnopyranose (3)
L
L
To a solution of 24 (500 mg, 0.36 mmol) in MeOH (20 mL) was added 30% MeNH2 (MeOH
solution) 30 ml. The resulting mixture was stirred for 7 days and the solvent was removed under
reduced pressure to give 3 (220 mg, 80%) as yellow syrup. [α]D22 29.7° (c 1.0, MeOH).1H NMR
(300 MHz, D2O): δ 6.97−6.83 (2m, 4H, ArH), 5.39 (s,1H, H-1-Rhamp), 5.18 (s,1H, H-1-Rhamp),
4.91 (s,1H, H-1-Rhamp), 4.59 (d, 1H, J = 8.1 Hz, H-1-Glcp), 4.10−3.96 (m, 5H), 3.85−3.57 (m,

ACCEPTED MANUSCRIPT
9H), 3.69 (s, 3H, OCH3), 3.49−3.24 (m, 8H) , 1.97 (s, 3H, OCOCH3), 1.15−1.12 (m, 9H, H-6).
13C NMR (75 MHz, D2O) : δ 174.6, 154.6, 149.3, 118.7, 114.9, 102.5 (C-1-Glcp), 101.8 (C-1-
Rhamp), 100.7 (C-1-Rhamp), 98.0 (C-1-Rhamp), 78.9, 78.3, 77.8, 75.5, 73.3, 72.0, 71.9, 71.1,
69.6, 69.5, 69.4, 69.4, 69.1, 68.9, 60.3, 55.6, 55.5, 24.3, 22.1, 16.5, 16.4. ESI-HRMS [M +
H]+calculated for C33H52NO19 766.3128 found 766.3121.
Acknowledgments
This work was partially supported by the NSFC (21772230) and the National S&T Pillar
Program (2015BAK45B01) of China.
Supplementary Data
Supplementary data associated with this article can be found, in the online version, at http://~~.
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Highlights:
1. we have reported a convergent total synthesis of a pentasaccharide analogue
and its tetrasccharide derivative
2. Stereo- and regio-specific synthesis was achieved in Schmidt glycosylation
employing appropriately protected
building blocks.
L-rhamopyranosyl and D-glucopyranosylamine
3. The efficient synthesis features the preparation of a key disaccharide
intermediate containing an orthogonal protecting group (Lev), which could be
easily converted to the disaccharide donor or the disaccharide acceptor