Synthesis and plant growth regulation activity of α-d-ManpNAc- (1→2)-[α-l-Rhap-(1→3)-]α-l-Rhap-(1→4) -β-d-GlupNAc-(1→3)-α-l-Rhap, the repeating unit of O-antigen of Rhizobium trifolii 4s

Article abstract of DOI:10.1016/j.carres.2013.12.006

The synthesis of a pentasaccharide 2 containing acetamido-2-deoxy-d-glucose and acetamido-2-deoxy-d-mannose related to the cell wall polysaccharide of Rhizobium trifolii 4s has been achieved by a [2+3] approach from commercially available l-rhamnose, d-glucose, and d-glucosamine as the starting materials. The target molecule was equipped with a p-methoxylphenyl handle at the reducing terminus to allow for further glycoconjugate formation via selective cleavage of this group. The bioassay suggested that the synthetic pentasaccharide 2 can stimulate the growth of wheat coleoptile similarly to indole-3-acetic acid (IAA), and promote the wheat seedling development before winter by seed treatment at a concentration of 20 mg/L.

Full text of DOI:10.1016/j.carres.2013.12.006

Carbohydrate Research 388 (2014) 87–93
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and plant growth regulation activity of
a-D-ManpNAc-
(1?2)-[ -L-Rhap-(1?3)-] -L-Rhap-(1?4)-b-D-GlupNAc-(1?3)-a
a
a
-L-
Rhap, the repeating unit of O-antigen of Rhizobium trifolii 4s
b
b,
a
Guanghui Zong a, Xiaomei Liang a, Jianjun Zhang a, , Liusheng Duan , Weiming Tan , Daoquan Wang
⇑
⇑
a Department of Applied Chemistry, China Agricultural University, Beijing 100193, China
b Engineering Research Centre of Plant Growth Regulators, Ministry of Education, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a pentasaccharide 2 containing acetamido-2-deoxy-D-glucose and acetamido-2-deoxy-D-
Received 18 September 2013
Received in revised form 30 November 2013
Accepted 5 December 2013
Available online 12 December 2013
mannose related to the cell wall polysaccharide of Rhizobium trifolii 4s has been achieved by a [2+3]
approach from commercially available -rhamnose, -glucose, and -glucosamine as the starting
L
D
D
materials. The target molecule was equipped with a p-methoxylphenyl handle at the reducing terminus
to allow for further glycoconjugate formation via selective cleavage of this group. The bioassay suggested
that the synthetic pentasaccharide 2 can stimulate the growth of wheat coleoptile similarly to indole-3-
acetic acid (IAA), and promote the wheat seedling development before winter by seed treatment at a
concentration of 20 mg/L.
Keywords:
Synthesis
Oligosaccharide
Rhizobium trifolii 4s
Plant growth regulation activity
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
of
L-rhamnose, N-acetyl-D-glucosamine, and N-acetyl-D-mannosa-
mine in 3:1:1 molar proportion (Fig. 1, 1).10
Overuse of chemical fertilizer to maintain good harvest has
posed a threat to our environment and also the country’s food
security in the past decades. In fact, some plants may not need
so much chemical fertilizer, as they and their specific symbiotic
bacteria of genus Rhizobium can combine nitrogen gas with other
elements to form useful nitrogen compounds.1 The use of chemical
fertilizer will be greatly reduced if plants have a good use of biolog-
ical nitrogen fixation. The lipopolysaccharides (LPSs) of Rhizobium
are crucial to the bacteroid development and nodule occupancy.
Bacteria having defects in LPSs structure also have defects in nod-
ule invasion in plant symbiosis.2–4 Presenting at the distal part of
LPSs, the O-antigenic polysaccharides (OPSs) are in direct contact
with the environment, and are closely involved in the process of
exchanging signals between rhizobia and legumes.5 Mutants with
LPSs that lack their OPSs or have modified core components either
are defective in the formation of infection threads or have the nod-
ules unoccupied.6–9 With the aim of investigating the biological
roles of these OPSs, synthetic studies on the OPSs will be useful.
Wang et. al. reported that the structure of OPS of Rhizobium trifolii
4s was constituted of a pentasaccharide repeating unit composed
To obtain new oligosaccharide resources and discover novel
bioactive substances, we reported the first total synthesis of the
pentasaccharide repeating unit (Fig. 1, 2). Moreover, the biological
effect of the synthesized pentasaccharide on stimulating plant
growth has been determined.
2. Results and discussion
The design of the synthesis of the pentasaccharide 2 is outlined
in Scheme 1. Retrosynthetic analysis indicated that the pentasac-
charide
2 could be achieved through a convergent strategy
involving [2+3] glycosylation of a disaccharide acceptor 20 and a
trisaccharide donor 11. The trisaccharide 11 then could be
constructed from the a-(1?2)-linked disaccharide acceptor 8 and
the rhamnosyl donor 5, while the disaccharide acceptor 20 could
be built from the rhamnosyl acceptor 1311,12 and the glucosaminyl
donor 12.13
A
number of suitably functionalized monosaccharide
intermediates 4,14 5,15 6,11,12 12,13 and 1311,12 were prepared from
the commercially available reducing sugars, such as -rhamnose,
glucose, and
L
D-
D
-glucosamine, using the previously reported reaction
conditions. Compound 414 was prepared in 77% yield from
compound 316 using a two-step sequence involving selective
de-1-O-aetylation followed by trichloroacetimidate formation
(Scheme 2).
⇑
Corresponding authors. Tel.: +86 10 62731115; fax: +86 10 62732219.
E-mail addresses: zhangjianjun@cau.edu.cn (J. Zhang), tanwm@cau.edu.cn
(W. Tan).
http://dx.doi.org/10.1016/j.carres.2013.12.006
0008-6215/Ó 2013 Elsevier Ltd. All rights reserved.

88
G. Zong et al. / Carbohydrate Research 388 (2014) 87–93
OPMP
O
*
O
HO
O
O
HO
HO
O
A
HO
O
O
O
O
OH
OH
HO
HO
NHAc
O
B
NHAc
O
HO
C
HO
O
O
O
O
O
O
HO
OH
OH
OH
HO
OH
OH
OH
O
HO
OH
O
E
OH
O
*
D
NHAc
n
NHAc
1
2
Figure 1. O-Antigenic chain repeating unit of LPSs (1) from Rhizobium trifoki 4s and the synthesized oligosaccharide 2.
N3
N3
AcO
AcO
AcO
a
ref. 16
5 steps
O
O
D-glu
AcO
AcO
AcO
OCNHCCl3
OAc
3
4
Scheme 2. Synthesis of key synthon 4. Reagents and conditions: (a) BnNH2, THF, rt,
12 h; then CCl3CN, DBU, CH2Cl2, rt, 0.5 h, 77% over two steps for 4.
catalyst in the presence of 4 Å molecular sieves to afford the
disaccharide 7 in 86% yield. Deallyloxycarbonylation of 7 was suc-
cessfully achieved in MeOH–THF17 in the presence of CH3COONH4,
Pd[P(C6H5)3]4, and NaBH4, within 4 min without affecting any of
the other protecting groups, giving the desired acceptor 8 in 92%
yield. Formation of compound 8 was supported by its spectral
analysis [signals at 5.45 ppm (d, J = 1.4 Hz, H-1) and 5.06 ppm (d,
J = 1.4 Hz, H-10) in the 1H NMR, and 97.4 and 96.3 ppm (2ꢀ C-1)
in the 13C NMR spectra]. The coupling reaction between trichloro-
acetimidate 5 and acceptor 8 by using TMSOTf as the catalyst
smoothly yielded trisaccharide 9. The formation of compound 9
was confirmed by its 13C NMR spectrum [signals at 98.7, 96.3
and 95.8 ppm (3ꢀ C-1) corresponding to the three anomeric car-
bons]. Hydrogenolysis of compound 9 followed by N-acetylation
Figure 2. Partial HSQC spectra of the fully protected pentasaccharide 21.
Synthesis of trisaccharide 11 was shown in Scheme 3. Glycosyl-
ation between rhamnosyl acceptor 6 and 2-acetamido-2-deoxy-
-mannosyl donor 4 was accomplished by using TMSOTf as the
a-
D
HO
O
HO
HO
HO
O
OPMP
O
HO
HO
O
O
O
O
O
NHAc
HO
HO
O
OH
OH
OH
AcHN
2
OCNHCCl3
O
BzO
OPMP
O
O
O
BzO
HO
Ac
O
O
Ac
BzO
O
Ac
Ac
O
Ac
Ac
OBz
BzO
NHAc
NHAc
11
20
OPMP
O
BzO
AllocO
OPMP
OCNHCCl3
OH
OCNHCCl3
NPhth
O
6
BzO
O
OPMP
+
BzO
O
HO
O
BzO
O
OBz
OCNHCCl3
BzO
AcO
AcO
5
Ac
Ac
HO
13
Ac
Ac
OBz
AcO
12
O
8
O
Ac
NHAc
Ac
4
N3
Scheme 1. A retrosynthetic strategy for the synthesis of the target pentasaccharide 2.

G. Zong et al. / Carbohydrate Research 388 (2014) 87–93
89
OCNHCCl3
+
20
11
ref. 15
O
O
BzO
BzO
OPMP
a
BzO
OBz
OPMP
O
O
AcO
O
5
BzO
O
BzO
O
L-Rha
OAc
BzO
RO
O
AcO
NHAc
BzO
BzO
BzO
OPMP
O
O
ref. 11 & 12
4
O
OAc
OAc
O
O
b
N3
2
AllocO
a
OAc
OH
O
7
R = Alloc
b
AcHN
8 R = H
OAc
OAc
6
OAc
21
a
5
OCNHCCl3
OPMP
Scheme 5. Synthesis of the target pentasaccharide 2. Reagents and conditions: (a)
TMSOTf, CH2Cl2, ꢂ10 °C to rt, 2 h, 69% for 21; (b) satd NH3–MeOH, rt, 96 h, 89% for 2.
O
O
BzO
O
BzO
O
O
d
O
O
O
BzO
BzO
OAc
OAc
O
AcHN
OAc
OAc
BzO
O
R
BzO
OBz
OBz
OAc
OAc
11
9
R = N3
c
10 R = NHAc
Scheme 3. Synthesis of trisaccharide donor 11. Reagents and conditions: (a)
TMSOTf, CH2Cl2, ꢂ10 °C to rt, 2 h, 86% for 7, 75% for 9; (b) CH3COONH4,
Pd[P(C6H5)3]4, NaBH4, MeOH–THF, ꢂ10 °C, 4 min, 92% for 8; (c) H2, Pd/C, 1 atm,
6 h; then Ac2O, Py, 25 °C, 12 h, 80% for 10; (d) 4:1 CH3CN–H2O, CAN, 30 °C, then
CCl3CN, DBU, CH2Cl2, rt, 0.5 h, 68% for two steps for 11.
produced compound 10 in 80% yield over two steps. The cleavage
of the 4-methoxyphenyl group in compound 10 with ceric ammo-
nium nitrate (CAN), followed by reacting with trichloroacetonitrile
in the presence of 1,8-diazobicyclo[5.4.0]undec-7-ene (DBU),18
afforded glycosyl trichloroacetimidate 11, which was then isolated
by flash chromatography on silica gel in 68% yield over two steps.
Having glycosyl donor 11 in hand, our attention was focused on
the construction of the disaccharide acceptor 20, as shown in
Scheme 4. The glycosylation of acceptor 1311,12 with trichloroace-
timidate 1213 having a C-2 acetyl ester to control b-anomeric selec-
tivity afforded the corresponding disaccharide 14 in 76% yield. 1H
NMR spectra confirmed its b-linkage formation [signals at
Figure 3. Partial HSQC spectra of the target pentasaccharide 2.
5.65 ppm (d, 1H, J 8.4 Hz, H-1-GluNAcp) in the 1H NMR spectra].
Compound 14 was then subjected to a series of transformations
involving (a) selective deacetylation with 2% CH3COCl–MeOH19
followed by isopropylidenation with 2,2-dimethyoxypropane in
dry DMF in the presence of catalytic TsOHꢁH2O, (b) removal of
N-phthalimido group using ethylenediamine and EtOH,20 followed
by N-acetylation, (c) deisopropylidenation in 70% AcOH and
OPMP
D-GluNH3Cl
O
selective benzoylation of the 6-OH of D-glucose moiety to furnish
BzO
OPMP
ref. 13
compound 20 in 23% overall yield over seven steps. The structure
of 20 was confirmed by its 1H and 13C NMR spectra, showing the
characteristic signals, such as 5.33 ppm (d, 1H, J 1.8 Hz, H-1-Rhap),
4.92 ppm (d, 1H, J 8.2 Hz, H-1-GluNAcp), and 100.8 (C-1), 96.3 ppm
(2ꢀ C-1).
HO
OBz
O
13
BzO
O
R1O
O
AcO
AcO
OBz
a
R O
1R2O
O
OCNHCCl3
NPhth
AcO
NPhth
14 R = R = Ac
b
c
15 R1 = R2 = H
Finally, the synthesis of the target pentasaccharide 2 was shown
in Scheme 5. The condensation of 11 with the acceptor 20 by
following the same glycosylation strategy as mentioned above
gave the fully protected pentasaccharide 21 in 69% yield. The pres-
ence of the five anomeric carbon signals in the 13C NMR spectra
confirmed its formation [100.8, 98.8, 98.3, 98.2, and 96.2 ppm].
The deacylation of the pentasaccharide 21 with ammonia-satu-
rated methanol afforded the target pentasaccharide 2 (89%). The
complete assignment of the 1H and 13C signals of 21 and 2 was
achieved by the HSQC experiments (Figs. 2 and 3).
16 R11 = is2opropylidene, R2 = H
12
d
OPMP
OPMP
O
O
AcO
R1O
f
R1O
R2O
O
O
O
O
O
OAc
OR1
O
AcO
R2O
NHAc
R3
19 R = R2 = H
17 R = R = H, R3 = NH2
g
e
20 R11 = Bz, R2 = H
18 R11 = R22 = Ac, R3 = NHAc
2.1. Effects on the growth of wheat plants
Scheme 4. Synthesis of disaccharide acceptor 20. Reagents and conditions: (a)
TMSOTf, CH2Cl2, ꢂ10 °C to rt, 2 h, 76% for 14; (b) AcCl, MeOH, rt, overnight, 90% for
15; (c) 2,2-dimethyoxypropane, DMF, rt, 2 h, 88% for 16; (d) 85% NH2NH2ꢁH2O,
EtOH, reflux, 3 h; (e) Ac2O, Py 25 °C,12 h, 79% for 18; (f) 70% AcOH, 70 °C, 1.5 h, 86 %
for 19; (g) BzCl, Py, –10 °C to rt, 3 h, 81% for 20.
The results of the growth regulation activity tests given in
Tables 1 and 2 clearly show that the synthetic pentasaccharide
2
promoted the elongation of wheat coleoptile and also

90
G. Zong et al. / Carbohydrate Research 388 (2014) 87–93
Table 1
Plant growth promotion activity data of synthetic pentasaccharide for wheat coleoptile
Concentration (mg/L)
b-Indoleacetic acid, IAA
Coleoptile length (mm) Promotion rate (%)
Synthetic pentasaccharide 2
Coleoptile length (mm) Promotion rate (%)
0
6.47 0.24
7.16 0.31
7.81 0.35
8.43 0.38
/
6.47 0.24
6.95 0.32
8.03 0.38
8.66 0.41
/
0.01
0.1
1.0
+27.9
+54.3
+79.4
+19.4
+63.2
+88.7
Table 2
Wheat seedling growth morphological characters on 49th day after planting, using the synthetic pentasaccharide 2 for seed treatment
Treatments
Control (tap water)
Synthetic pentasaccharide 2, 20 mg/L
Expansion leaves number of the stem
Till number
Till number with no less than 3 leaves
Total root number
6.0 0.3
4.9 0.3
1.5 0.1
11.2 0.7
7.3 0.4
6.2 0.4
2.5 0.2
13.1 0.6
stimulated the growth of wheat seedlings. For wheat coleoptile
growth (Table 1), the promotion effects of 0.1 to 1.0 mg/L syn-
thetic pentasaccharide exceeded the control compound IAA.
When used for the wheat seed treatment (Table 2), the number
of the expansion leaves on the main stem, the till number, the till
number with no less than 3 leaves and the total root number of
each plant were all higher than those of the control seedlings,
indicating that the stimulation effects of the synthetic pentasac-
charide were evident.
4.2. Synthesis
4.2.1. p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-
mannopyranosyl-(1?2)-3-O-allyloxycarbonyl-4-O-benzoyl-a-L-
rhamnopyranoside (7)
Compound 6 (3.6 g, 7.9 mmol), 4 (4.1 g, 8.6 mmol), and 4 Å
molecular sieves (4 g) were dried together under high vacuum
for 2 h, then dissolved in anhydrous, redistilled CH2Cl2 (100 mL).
a-D-
TMSOTf (54
l
L, 0.3 mmol) was added dropwise at ꢂ10 °C under
an N2 atmosphere. The reaction mixture was stirred for 0.5 h, dur-
ing which time the mixture was allowed to gradually warm to
ambient temperature. TLC (3:6:1 petroleum ether–toluene–EtOAc)
indicated that the reaction was complete. Then the reaction mix-
ture was neutralized with triethylamine and filtrated, and the fil-
trate was concentrated. Purification of the residue by column
chromatography (4:6:1 petroleum ether–toluene–EtOAc) gave 7
3. Conclusion
In conclusion, a pentasaccharide analog (2), related to the
repeating unit of the O-antigenic polysaccharide of Rhizobium
trifoki 4s, has been synthesized using a [2+3] block glycosylation
strategy for the first time. All intermediates were obtained in rela-
tively high yield. Also, we presented the first report regarding the
plant growth activity of this O-antigenic polysaccharide. By our
bioassay studies, we found the synthetic pentasaccharide could
promote wheat coleoptile growth at low concentration (<1 mg/L),
and had the similar growth promotion effect compared to the
known plant harmony IAA. Moreover, we demonstrated that the
synthetic pentasaccharide 2 could be used as a wheat seed treat-
ment agent to gain flourishing seedlings before winter.
(5.2 g, 86%) as a white solid. ½a D25
ꢃ
þ 55:9ꢄ (c 0.3 CHCl3). 1H NMR
(300 MHz, CDCl3): d 8.04–8.02 (m, 2H, Bz-H), 7.57 (m, 1H, Bz-H),
7.47–7.42 (m, 2H, Bz-H), 7.06–7.03 (m, 2H, Ar-H), 6.87–6.84 (m,
0
2H, Ar-H), 5.69 (m, 1H, CH2@CHCH2OCO), 5.53 (dd, 1H, J2 ,3
3.4 Hz, J3 ,4 10.1 Hz, H-30), 5.46–5.37 (m, 4H, H-10, H-3, H-4, H-40),
5.23–5.06 (m, 2H, CH2@CHCH2OCO), 4.96 (d, 1H, J1,2 1.5 Hz, H-1),
0
0
0
0
4.52–4.49 (m, 2H, CH2@CHCH2OCO), 4.36 (dd, 1H, J1 ,2 2.0 Hz,
J2 ,3 3.3 Hz, H-20), 4.32–4.23 (m, 2H, H-5, H-50), 4.22 (dd, 1H, J1,2
1.7 Hz, J2,3 3.2 Hz, H-2), 4.17–4.02 (m, 2H, 2ꢀ H-60), 2.12, 2.09,
2.07 (3s, 9H, 3ꢀ CH3CO), 1.28 (d, 1H, J 6.3 Hz, H-6). 13C NMR
(75 MHz, CDCl3): d 170.7, 169.7, 169.6 (3ꢀ COCH3), 165.4 (COPh),
165.4, 154.2, 130.8, 119.2 (CH2@CHCH2OCO), 97.6, 96.2 (2ꢀ C-1),
55.6 (C6H4OCH3), 20.6(2), 20.4 (3ꢀ COCH3), 17.5 (C-6). ESI-MS m/
z calcd for C36H41N3NaO16 (M+Na)+ 794.2. Found: 793.9. Anal.
Calcd for C36H41N3O16: C, 56.03; H, 5.35. Found: C, 56.22; H, 5.31.
0
0
4. Experimental procedures
4.1. General methods
Optical rotations were determined with
a Perkin–Elmer
model 241-MC automatic polarimeter for solutions in a 1-dm,
jacketed cell. 1H and 13C NMR spectra were recorded with Bru-
ker DPX300 and Bruker AVANCE600 spectrometers in CDCl3 or
D2O solutions. Internal references: TMS (d 0.000 ppm for 1H),
CDCl3 (d 77.00 ppm for 13C), HOD (d 4.700 for 1H). Elemental
analysis was performed on a Yanaco CHN Corder MF-3 auto-
matic elemental analyzer. High-resolution mass spectra (HRMS)
were acquired by the Peking University, and electrospray-ioni-
zation mass spectra (ESIMS) were acquired by the China
Agricultural University. Thin-layer chromatography (TLC) was
performed on silica gel HF with detection by charring with
30% (v/v) H2SO4 in MeOH or by UV. Column chromatography
was conducted by elution of a column of silica gel (200–300
mesh) with EtOAc/petroleum ether (bp 60–90 °C) as the eluent.
Solutions were concentrated at temperature <60 °C under
diminished pressure.
4.2.2. p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-
a-D-
mannopyranosyl-(1?2)-4-O-benzoyl- -rhamnopyranoside (8)
a-L
To a cooled (ꢂ5 °C) solution of compound 7 (4.0 g, 5.2 mmol) in
1:1 MeOH–THF (100 mL) in 500 mL flask was added CH3COONH4
(4.0 g, 52 mmol). With vigorous stirring, NaBH4 (47 mg, 1.3 mmol),
Pd[P(C6H5)3]4 (240 mg, 0.21 mmol), and NaBH4 (234 mg, 6.5 mmol)
were added in 3 portions immediately one after another. 1 min
after the addition of the second portion of NaBH4, TLC (2:1 petro-
leum ether–EtOAc) indicated that the reaction was complete. The
reaction mixture was concentrated under diminished pressure,
the residue was dissolved in CH2Cl2 (150 mL) and washed with
water (50 mL), then the organic phase was dried over Na2SO4. Evap-
oration and purification by flash column chromatography (petro-
leum ether–EtOAc 3:1) afforded compound 8 as a white foam

G. Zong et al. / Carbohydrate Research 388 (2014) 87–93
91
(3.3 g, 92%). ½a 2D5
ꢃ
þ 2:4ꢄ (c 0.6 CHCl3). 1H NMR (300 MHz, CDCl3): d
1184.4. Found: 1184.5. HRMS for C61H64NO22 (M+H)+ 1162.3920.
Found: 1162.3897.
8.07–8.05 (m, 2H, Bz-H), 7.58 (m, 1H, Bz-H), 7.48–7.43 (m, 2H,
Bz-H), 7.05–7.01 (m, 2H, Ar-H), 6.87–6.83 (m, 2H, Ar-H), 5.48 (dd,
1H, J2 ,3 3.8 Hz, J3 ,4 9.4 Hz, H-30), 5.45 (d, 1H, J1,2 1.4 Hz, H-1),
4.2.5. 3,4,6-Tri-O-acetyl-2-acetamido-2-deoxy-
a-D-
0
0
0
0
5.34 (t, 1H, J3 ,4 , J4 ,5 9.4 Hz, H-40), 5.13 (t, 1H, J3,4, J4,5 9.7 Hz, H-4),
mannopyranosyl-(1?2)-[2,3,4-tri-O-benzoyl-a-L-
0
0
0
0
5.06 (d, 1H, J1 ,2 1.4 Hz, H-10), 4.39 (m, 1H, H-3), 4.33–4.26 (m, 2H,
H-5, H-50), 4.24 (dd, 1H, J1,2 1.7 Hz, J2,3 3.4 Hz, H-2), 4.19 (dd, 1H,
rhamnopyranosyl-(1?3)-]4-O-benzoyl-a-L-rhamnopyranosyl
0
0
trichloroacetimidate (11)
J1 ,2 2.0 Hz, J2 ,3 3.8 Hz, H-20), 4.15–4.08 (m, 2H, 2ꢀ H-60), 3.10 (d,
1H, J 8.8 Hz, OH), 2.12, 2.09, 2.05 (3s, 9H, 3ꢀ CH3CO), 1.27 (d, 1H,
J 6.2 Hz, H-6). 13C NMR (75 MHz, CDCl3): d 170.7, 169.9, 169.5 (3ꢀ
COCH3), 166.9 (COPh), 155.2, 150.2, 117.6(2), 114.7(2) (C6H4OCH3),
97.4, 96.3 (2ꢀ C-1), 55.6 (C6H4OCH3), 20.6(2), 20.5 (3ꢀ COCH3), 17.5
(C-6). ESI-MS m/z calcd for C32H37N3NaO14 (M+Na)+ 710.2. Found:
709.9. Anal. Calcd for C32H37N3O14: C, 55.89; H, 5.42; N, 6.11. Found:
C, 55.70; H, 5.36; N, 6.23.
To a solution of compound 10 (1.1 g, 0.95 mmol) in acetonitrile
(16 mL) and water (4 mL) was added ceric ammonium nitrate
(CAN) (2.1 g, 3.8 mmol). The mixture was stirred for 20 min at
30 °C, at the end of which time TLC (1:1 petroleum ether–EtOAc)
indicated that the reaction was complete. The solvent was evapo-
rated under diminished pressure at 50 °C to give a residue, which
was diluted with CH2Cl2, and washed with water. The organic phase
was dried and concentrated. Purification by silica gel chromatogra-
phy with 2:1 petroleum ether–EtOAc as the eluent afforded 3,4,
0
0
0
0
4.2.3. p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-
mannopyranosyl-(1?2)-[2,3,4-tri-O-benzoyl- -rhamnopyran-
osyl-(1?3)-]4-O-benzoyl- -rhamnopyranoside (9)
Compound 8 (2.9 g, 4.2 mmol) and 5 (2.9 g, 4.7 mmol) were cou-
pled in the presence of catalytic TMSOTf (54 L, 0.3 mmol) under
a
-
D
-
6-tri-O-acetyl-2-acetamido-2-deoxy-
[2,3,4-tri-O-benzoyl- -rhamnopyranosyl-(1?3)-]4-O-benzoyl-
-rhamnopyranoside. mixture of this compound,
trichloroacetonitrile (0.5 mL, 5 mmol), and 1,8-diazabicy-
clo[5.4.0]undecene (DBU) (0.05 mL, 0.4 mmol) in dry CH2Cl2
(30 mL) was stirred for 0.5 h and then concentrated. The residue
was purified by chromatography with 2:1 petroleum ether–EtOAc
a-D-mannopyranosyl-(1?2)-
a
-L
a-L
a-
L
a
-
L
A
l
the same conditions as described above for the coupling of 6 with
4. Purification by silica gel chromatography with 4:4:1 petroleum
ether–toluene–EtOAc as the eluent gave trisaccharide 9 (3.6 g,
as the eluent to give 11 (0.75 g, 68%) as a white foam. ½a D25
ꢃ
þ 98:3ꢄ
75%) as a foamy solid. ½a D25
ꢃ
þ 101:8ꢄ (c 0.5 CHCl3). 1H NMR
(c 0.6 CHCl3). 1H NMR (300 MHz, CDCl3): d 8.72 (s, 1H, CNHCCl3),
8.04–7.17 (m, 20H, Bz-H), 6.41 (d, 1H, J1,2 1.6 Hz, H-1), 5.85 (d, 1H,
J 8.2 Hz, NH), 5.75–5.45 (m, 6H, H-1, H-2, 2ꢀ H-3, 2ꢀ H-4), 5.33 (t,
1H, J 9.9 Hz, H-4), 5.23 (br, 1H, H-1), 4.80 (m, 1H, H-2-Manp), 4.70
(m, 1H, H-3), 4.57–4.53 (m, 2H, H-2, H-5), 4.50–4.00 (m, 4H, 2ꢀ
H-5, 2ꢀ H-6), 2.15 (s, 3H, CH3COO), 2.10 (s, 3H, CH3CONH), 2.04(2)
(s, 6H, 2ꢀ CH3COO), 1.35 (d, 1H, J 6.5 Hz, H-6-Rhap), 1.33 (d, 1H, J
(300 MHz, CDCl3): d 8.04–7.17 (m, 20H, Bz-H), 7.08–7.04 (m, 2H,
Ar-H), 6.88–6.84 (m, 2H, Ar-H), 5.62–5.60 (m, 2H, 2ꢀ H-3), 5.60–
5.49 (m, 5H, H-1, H-2, 3ꢀ H-4), 5.34 (br, 1H, H-1), 5.16 (br, 1H, H-
1), 4.75–4.55 (m, 2H, 2ꢀ H-2), 4.50–4.00 (m, 6H, H-3, 3ꢀ H-5, 2ꢀ
H-6), 3.80 (s, 1H, C6H4OCH3), 2.11, 2.10, 2.06 (3s, 9H, 3ꢀ CH3CO),
1.42 (d, 1H, J 6.2 Hz, H-6), 1.29 (d, 1H, J 6.0 Hz, H-6). 13C NMR
(75 MHz, CDCl3): d 170.4, 170.0, 170.5 (3ꢀ COCH3), 165.9, 165.2,
164.9, 164.5 (4ꢀ COPh), 155.4, 150.2, 117.8(2), 114.7(2) (C6H4OCH3),
98.7, 96.3, 95.8 (3ꢀ C-1), 55.6 (C6H4OCH3), 20.6(2), 20.4 (3ꢀ COCH3),
17.7, 17.6 (2ꢀ C-6). ESI-MS m/z calcd for C59H59N3NaO21 (M+Na)+
1168.3. Found: 1168.1. Anal. Calcd for C59H59N3O21: C, 61.83; H,
5.19; N, 3.67. Found: C, 62.11; H, 5.20; N, 3.78.
6.3 Hz, H-6-Rhap). ESI-MS m/z calcd for
C56H57Cl3N2NaO21
(M+Na)+ 1221.2. Found: 1221.3. Anal. Calcd for C56H57Cl3N2O21: C,
56.03; H, 4.79; N, 2.33. Found: C, 56.24; H, 4.58; N, 2.08.
4.2.6. p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-phthalimido-2-
deoxy-b-
rhamnopyranoside (14)
Compound 13 (2.4 g, 5.0 mmol) and 12 (3.2 g, 5.5 mmol) were
coupled in the presence of catalytic TMSOTf (54 L, 0.3 mmol) under
D-glucopyranosyl-(1?3)-2,4-di-O-benzoyl-a-L-
4.2.4. p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-acetamido-2-deo-
xy-
a
-
D
-mannopyranosyl-(1?2)-[2,3,4-tri-O-benzoyl-
a
-
L
-rha-
l
mnopyranosyl-(1?3)-]4-O-benzoyl-
a
-
L
-rhamnopyranoside (10)
the same conditions as described above for the coupling of 6 with 4.
Purification by silica gel chromatography with 2:1 petroleum ether–
EtOAc as the eluent gave disaccharide 14 (3.4 g, 76%) as a foamy so-
A mixture of compound 9 (1.5 g, 1.3 mmol) and Pd/C (0.25 g) in
methanol (100 mL) was stirred under 1 atm of H2 at rt for 4 h. TLC
(2:1 petroleum ether–EtOAc) indicated that the reaction was com-
plete. The reaction mixture was filtered and the filtrate was con-
centrated under diminished pressure, and the residue was used
for next step directly without purification. A solution of the residue
in pyridine (10 mL) and Ac2O (3 mL) was stirred at rt for 12 h, and
TLC (1:1 petroleum ether–EtOAc) indicated that the reaction was
complete. The reaction mixture was concentrated, and then the
residue was purified by flash column chromatography on a silica
gel column (2:1 petroleum ether–EtOAc) to give trisaccharide 10
lid. ½a 2D5
ꢃ
þ 9:6ꢄ (c 0.5 CHCl3). 1H NMR (300 MHz, CDCl3): d 8.13–7.16
(m, 14H, Bz-H, Phth-H), 7.06–7.03 (m, 2H, Ar-H), 5.67 (m, 1H, H-2),
5.65 (d, 1H, J 8.4 Hz, H-10), 5.64 (t, 1H, J2 ,3 , J3 ,4 10.6 Hz, H-30), 5.56
(d, 1H, J 1.6 Hz, H-1), 5.43 (t, 1H, J 9.8 Hz, H-4), 5.03 (t, 1H, J 9.9 Hz,
0
0
0
0
H-40), 5.49 (dd, 1H, J2,3 3.7 Hz, J3,4 9.7 Hz, H-3), 4.26 (dd, 1H, J1 ,2
0
0
8.4 Hz, J2 ,3 10.7 Hz, H-20), 4.20–4.11 (m, 2H, 2ꢀ H-60), 4.04, 3.89
(2 m, 2H, 2ꢀ H-5), 3.78 (s, 1H, C6H4OCH3), 1.96, 1.88, 1.72 (3s, 9H,
3ꢀ CH3CO), 1.09 (d, 1H, J 6.2 Hz, H-6). 13C NMR (75 MHz, CDCl3): d
170.6, 169.9, 169.2, 165.9, 164.7 (5ꢀ CO), 155.2, 150.0, 117.7(2),
114.6(2) (C6H4OCH3), 98.5, 96.3 (2ꢀ C-1), 72.3, 71.9, 71.5, 70.4,
68.5, 66.8, 61.7, 55.5 (C6H4OCH3), 54.5, 20.4(2), 20.2 (3ꢀ CH3CO),
17.4 (C-6-Rhap). ESI-MS m/z calcd for C47H45NNaO17 (M+Na)+
918.2. Found: 918.1. Anal. Calcd for C47H45NO17: C, 63.01; H, 5.06;
N, 1.56. Found: C, 62.79; H, 5.21; N, 1.40.
0
0
(1.2 g, 80%) as a white foamy solid. ½a D25
ꢃ
þ 81:6ꢄ (c 0.7 CHCl3). 1H
NMR (300 MHz, CDCl3): d 8.04–7.17 (m, 20H, Bz-H), 7.08–7.05
(m, 2H, Ar-H), 6.88–6.84 (m, 2H, Ar-H), 5.91 (d, 1H, J 8.2 Hz, NH),
5.68 (dd, 1H, J2,3 3.1 Hz, J3,4 10.2 Hz, H-3), 5.62-5.48 (m, 5H, H-1,
H-2-Rhap, H-3, 2ꢀ H-4), 5.35–5.25 (m, 2H, H-1, H-4), 5.18 (br,
1H, H-1), 4.77 (m, 1H, H-2-Manp), 4.67–4.38 (m, 3H, H-2, H-3, H-
5), 4.38–4.03 (m, 4H, H-5, 2ꢀ H-6), 2.14, 2.03, 2.01 (3s, 9H, 3ꢀ CH3-
COO), 2.08 (s, 3H, CH3CONH), 1.41 (d, 1H, J 5.9 Hz, H-6-Rhap), 1.29
(d, 1H, J 6.0 Hz, H-6-Rhap). 13C NMR (75 MHz, CDCl3): d 170.4,
170.2, 169.5 (3ꢀ COCH3), 165.8, 165.2, 165.0, 164.5 (4ꢀ COPh),
155.2, 150.0, 117.6(2), 114.7(2) (C6H4OCH3), 98.8, 98.1, 96.0 (3ꢀ
C-1), 55.6 (C6H4OCH3), 23.2 (NHCOCH3), 20.7(2), 20.6 (3ꢀ COCH3),
17.7, 17.6 (2ꢀ C-6). ESI-MS m/z calcd for C61H63NNaO22 (M+Na)+
4.2.7. p-Methoxyphenyl 2-phthalimido-2-deoxy-b-
D-glucopyr-
anosyl-(1?3)-2,4-di-O-benzoyl- -rhamnopyranoside (15)
a-L
To a solution of compound 14 (1.8 g, 2.0 mmol) in anhyd MeOH
(40 mL) was added AcCl (0.40 mL). The reaction mixture was stir-
red at rt overnight until which time TLC (1:2 petroleum ether–
EtOAc) showed that the starting material had disappeared. The
solution was neutralized with Et3N, then concentrated to dryness.

92
G. Zong et al. / Carbohydrate Research 388 (2014) 87–93
The residue was passed through a short silica gel column (1:1
TLC (1:4 petroleum ether–EtOAc) indicated completion of the reac-
tion. The mixture was concentrated under diminished pressure and
then co-evaporated with toluene for two times. The residue was
passed through a short silica gel column with 1:3 petroleum
ether–EtOAc as the eluent to give 19 (0.50 g, 86%) as a white solid.
petroleum ether–EtOAc) to give 15 (1.4 g, 90%) as a white solid.
½
a 2D5
ꢃ
ꢂ 17:0ꢄ (c 0.9 CHCl3). 1H NMR (300 MHz, CDCl3): d 8.11–7.10
(m, 14H, Bz-H, Phth-H), 7.06–7.03 (m, 2H, Ar-H), 6.85–6.82 (m,
2H, Ar-H), 5.87 (dd, 1H, J1,2 1.8 Hz, J2,33.5 Hz, H-2), 5.46 (d, 1H, J
8.2 Hz, H-10), 5.42 (d, 1H, J 1.6 Hz, H-1), 5.38 (t, 1H, J 9.7 Hz, H-4),
4.42 (dd, 1H, J2,3 3.7 Hz, J3,4 9.7 Hz, H-3), 4.11–4.01 (m, 2H, 2ꢀ H-
5), 3.92–3.82 (m, 2H, H-30, OH), 3.77 (s, 1H, C6H4OCH3), 3.61 (m,
1H, H-20), 3.46 (m, 1H, H-40), 3.38–3.30 (m, 2H, 2ꢀ H-60), 3.24 (t,
1H, J 7.5 Hz, OH), 2.98 (d, 1H, J 5.8 Hz, OH), 1.09 (d, 1H, J 6.2 Hz,
H-6). ESI-MS m/z calcd for C41H39NNaO14 (M+Na)+ 792.2. Found:
792.0. Anal. Calcd for C41H39NO14: C, 63.97; H, 5.11; N, 1.82. Found:
C, 64.28; H, 5.30; N, 1.87.
½
a 2D5
ꢃ
ꢂ 40:2ꢄ (c 0.4 CHCl3). 1H NMR (300 MHz, CDCl3): d 6.98 (d, 2H,
J 9.1 Hz, Ar-H), 6.82 (d, 2H, J 9.1 Hz, Ar-H), 6.12 (d, 1H, J 8.6 Hz,
NHAc), 5.55 (dd, 1H, J1,2 1.8 Hz, J2,3 3.4 Hz, H-2-Rhap), 5.27 (d,
0
0
0
0
1H, J 1.5 Hz, H-1-Rhap), 5.20 (t, 1H, J2 ,3 , J3 ,4 9.3 Hz, H-3-Glup),
5.08 (t, 1H, J3,4, J4,5 9.7 Hz, H-4-Rhap), 4.91 (d, 1H, J 8.2 Hz, H-1-
Glup), 4.19 (dd, 1H, J2,3 3.5 Hz, J3,4 9.7 Hz, H-3-Rhap), 3.94–3.89
(m, 3H, 2ꢀ H-6-Glup, H-2-Glup), 3.77 (s, 3H, C6H4OCH3), 3.66–
3.63 (m, 3H, 2ꢀ OH, H-4-Glup), 3.51 (m, 1H, H-5), 3.33 (m, 1H,
H-5), 2.18, 2.10, 2.08, 1.94 (4s, 12H, 4ꢀ CH3CO), 1.16 (d, 3H, J
6.2 Hz, H-6-Rhap). 13C NMR (75 MHz, CDCl3): d 171.9, 171.3,
170.4, 169.8 (4ꢀ MeCO), 155.2, 149.7, 117.7(2), 114.6(2) (C6H4-
OCH3), 101.1, 96.4 (2ꢀ C-1), 75.8, 75.1, 72.0, 71.2, 68.6, 68.5,
66.8, 55.6 (C6H4OCH3), 55.0, 23.2, 21.1, 20.9(2) (4ꢀ CH3CO), 17.4
(C-6-Rhap). ESI-MS m/z calcd for C27H37NNaO14 (M+Na)+ 622.2.
Found: 622.2. Anal. Calcd for C27H37NO14: C, 54.09; H, 6.22; N,
2.34. Found: C, 54.13; H, 6.06; N, 2.17.
4.2.8. p-Methoxyphenyl 4,6-O-isopropylidene-2-phthalimido-2-
deoxy-b-
D-glucopyranosyl-(1?3)-2,4-di-O-benzoyl-a-L-rhamn-
opyranoside (16)
To a solution of compound 15 (1.3 g, 1.7 mmol) in anhyd DMF
(20 mL) was added TsOHꢁH2O (6.4 mg, 0.034 mmol) and 2-
methoxypropene (0.31 mL, 2.5 mmol) under a N2 atmosphere. The
mixture was stirred at rt for 3 h, at the end of which time TLC (1:1
petroleum ether–EtOAc) indicated that the reaction was complete,
then concentrated, and co-evaporated with toluene for two times
to give a residue that was purified by column chromatography
(2:1 petroleum ether–EtOAc) to give 16 (1.2 g, 88%) as a white foam.
4.2.11. p-Methoxyphenyl 3-O-acetyl-4-O-benzoyl-2-acetamido-
2-deoxy-b-
rhamnopyranoside (20)
To a solution of compound 19 (0.38 g, 0.63 mmol) in pyridine
(5 mL) was added dropwise solution of benzoyl chloride
D-glucopyranosyl-(1?3)-2,4-di-O-acetyl-a-L-
½
a 2D5
ꢃ
ꢂ 25:0ꢄ (c 0.6 CHCl3). 1H NMR (300 MHz, CDCl3): d 8.14–7.21
(m, 14H, Bz-H, Phth-H), 7.06–7.03 (m, 2H, Ar-H), 6.85–6.82 (m,
2H, Ar-H), 5.59 (dd, 1H, J1,2 1.9 Hz, J2,3 3.6 Hz, H-2-Rhap), 5.53 (d,
1H, J 1.8 Hz, H-1-Rhap), 5.45 (d, 1H, J 8.2 Hz, H-1-Glup), 5.40 (t,
1H, J 9.8 Hz, H-4-Rhap), 4.48 (dd, 1H, J2,3 3.6 Hz, J3,4 9.7 Hz, H-3-
Rhap), 4.29 (m, 1H, H-2-Glup), 4.12–3.91 (m, 3H, H-3-Glup, H-4-
Glup, H-5), 3.77 (s, 1H, C6H4OCH3), 3.59 (m, 1H, H-5), 3.47–3.42
(m, 2H, 2ꢀ H-6-Glup), 2.14 (d, 1H, J 3.6 Hz, OH), 1.40, 1.37 (2s, 6H,
Me2C), 1.08 (d, 1H, J 6.3 Hz, H-6). ESI-MS m/z calcd for C44H43NNaO14
(M+Na)+ 832.2. Found: 831.9. Anal. Calcd for C44H43NO14: C, 65.26;
H, 5.35; N, 1.73. Found: C, 65.41; H, 5.14; N, 1.55.
a
(0.081 mL, 0.70 mmol) in pyridine (3 mL) over 30 min at –10 °C.
The reaction mixture was slowly raised to rt and stirred for a fur-
ther 2 h, at the end of which time TLC (1:4 petroleum ether–EtOAc)
indicated that the reaction was complete. The reaction mixture
was concentrated, and purification of the residue by column chro-
matography on silica gel (1:2 petroleum ether–EtOAc) gave 20
(0.36 g, 81%) as a white foam. ½a D25
ꢃ
ꢂ 52:1ꢄ (c 0.8 CHCl3). 1H NMR
(300 MHz, CDCl3): d 8.08–8.05 (m, 2H, Bz-H), 7.51 (m, 1H, Bz-H),
7.38–7.33 (m, 2H, Bz-H), 6.89 (d, 2H, J 9.1 Hz, Ar-H), 6.78 (d, 2H, J
9.1 Hz, Ar-H), 5.87 (d, 1H, J 8.5 Hz, NHAc), 5.46 (dd, 1H, J1,2
2.0 Hz, J2,3 3.4 Hz, H-2-Rhap), 5.33 (d, 1H, J 1.8 Hz, H-1-Rhap),
4.2.9. p-Methoxyphenyl 3-O-acetyl-4,6-O-isopropylidene-2-
0
0
0
0
acetamido-2-deoxy-b-
-rhamnopyranoside (18)
A solution of compound 16 (1.1 g, 1.4 mmol) in 85% NH2NH2ꢁH2O
D
-glucopyranosyl-(1?3)-2,4-di-O-acetyl-
5.28 (t, 1H, J2 ,3 , J3 ,4 9.3 Hz, H-3-Glup), 5.11 (t, 1H, J3,4, J4,5 9.7 Hz,
H-4-Rhap), 4.92 (d, 1H, J 8.2 Hz, H-1-Glup), 4.67 (dd, 1H, J 4.5 Hz,
12.2 Hz, H-6-Glup), 4.56 (dd, 1H, J 1.9 Hz, 12.2 Hz, H-6-Glup),
4.27 (dd, 1H, J2,3 3.5 Hz, J3,4 9.7 Hz, H-3-Rhap), 3.90 (m, 1H, H-2-
Glup), 3.77 (s, 3H, C6H4OCH3), 3.70–3.54 (m, 4H, 2ꢀ H-5, OH, H-
4-Glup), 2.12, 2.10, 2.08, 1.95 (4s, 12H, 4ꢀ CH3CO), 1.16 (d, 3H, J
6.2 Hz, H-6-Rhap). 13C NMR (75 MHz, CDCl3): d 171.7, 170.4,
170.2, 169.8 (4ꢀ CH3CO), 167.0 (C6H5CO), 155.1, 149.8, 117.7(2),
114.6(2) (C6H4OCH3), 100.8, 96.3 (2ꢀ C-1), 75.0, 74.6, 74.0, 72.2,
71.4, 68.9, 66.7, 63.6, 55.5 (C6H4OCH3), 55.1, 23.1, 20.9(2), 20.8,
(4ꢀ CH3CO), 17.3 (C-6-Rhap). ESI-MS m/z calcd for C34H41NNaO15
(M+Na)+ 726.2. Found: 726.3. Anal. Calcd for C34H41NO15: C,
58.03; H, 5.87; N, 1.99. Found: C, 58.30; H, 5.65; N, 2.07.
a-L
(4 mL) and EtOH (36 mL) was heated at 90 °C for 48 h. The reaction
mixture was cooled and concentrated and co-evaporated with tolu-
ene for two times to give 17 as a syrup. The syrup was then dissolved
in pyridine (10 mL) and Ac2O (3 mL). The mixture was stirred at rt
for 12 h at the end of which time TLC (1:2 petroleum ether–EtOAc)
indicates that the reaction was complete. The solvents were concen-
trated and then co-evaporated with toluene for two times to give a
residue that was purified by column chromatography (1:1 petro-
leum ether–EtOAc) to give compound 18 (0.69 g, 79%) as a white
foam. ½a 2D5
ꢃ
ꢂ 3:8ꢄ (c 0.3 CHCl3). 1H NMR (300 MHz, CDCl3): d 6.98–
6.93 (m, 2H, Ar-H), 6.84–6.80 (m, 2H, Ar-H), 5.72 (m, 1H, NHAc),
5.32–5.30 (m, 2H, H-1-Rhap, H-2-Rhap), 5.14 (t, 1H, J3,4, J4,5 9.6 Hz,
4.2.12. p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-acetamido-2-
0
0
0
0
H-4-Rhap), 5.10 (t, 1H, J2 ,3 , J3 ,4 9.8 Hz, H-3-Glup), 4.71 (d, 1H, J
8.2 Hz, H-1-Glup), 4.22 (dd, 1H, J2,3 2.8 Hz, J3,4 9.6 Hz, H-3-Rhap),
3.37-3.77 (m, 7H, H-2-Glup, 2ꢀ H-6-Glup, H-5, C6H4OCH3), 3.71 (t,
deoxy-
nopyranosyl-(1?3)-]4-O-benzoyl-
3-O-acetyl-4-O-benzoyl-2-acetamido-2-deoxy-b-
yl-(1?3)-2,4-di-O-acetyl- -rhamnopyranoside (21)
Compound 21 (220 mg, 69%) was prepared by coupling of 20
(190 mg, 0.27 mmol) with 11 (390 mg, 0.33 mmol) under the same
conditions as described for the synthesis of 7 by coupling of 4 with
a
-
D
-mannopyranosyl-(1?2)-[2,3,4-tri-O-benzoyl-
-rhamnopyranosyl-(1?4)-
-glucopyranos
a-L-rham-
a-L
D
0
0
0
0
1H, J3 ,4 , J4 ,5 9.6 Hz, H-4-Glup), 2.16, 2.10, 2.07, 1.95 (4s, 12H, 4ꢀ
a-L
CH3CO), 1.47, 1.38 (2s, 6H, Me2C). ESI-MS m/z calcd for C30H41-
NNaO14 (M+Na)+ 662.2. Found: 661.9. Anal. Calcd for C30H41NO14
C, 56.33; H, 6.46; N, 2.19. Found: C, 56.14; H, 6.28; N, 2.17.
:
6. ½a 2D5
ꢃ
þ 50:3ꢄ (c 0.5 CHCl3). 1H NMR (300 MHz, CDCl3): d 8.07–
4.2.10. p-Methoxyphenyl 3-O-acetyl-2-acetamido-2-deoxy-b-
glucopyranosyl-(1?3)-2,4-di-O-acetyl-a-L-rhamnopyranoside
D
-
8.03 (m, 4H, Bz-H), 7.91 (d, 2H, J 7.4 Hz, Bz-H), 7.81 (d, 2H, J
7.3 Hz, Bz-H), 7.69 (d, 2H, J 7.6 Hz, Bz-H), 7.55–7.27 (m, 14H, Bz-
H), 7.19 (t, 2H, J 7.7 Hz, Bz-H), 6.91 (d, 2H, J 9.1 Hz, Ar-H), 6.78
(d, 2H, J 9.1 Hz, Ar-H), 5.90–5.85 (m, 2H, 2ꢀ NHAc), 5.64 (dd, 1H,
J2,3 3.0 Hz, J3,4 10.2 Hz, H-3-RhapD), 5.60 (t, 1H, J2,3, J3,4 9.4 Hz, H-
(19)
Compound 18 (0.62 g, 0.97 mmol) was dissolved in 70% AcOH
(20 mL) and stirred at 75 °C for 1.5 h, at the end of which time

G. Zong et al. / Carbohydrate Research 388 (2014) 87–93
93
PRð%Þ ¼ ðLtreated ꢂ 4:0Þ=ðLcontrol ꢂ 4:0Þ ꢀ 100
where L is the average length of coleoptile section.
3-Glup), 5.55–5.39 (m, 5H, H-2-RhapA, H-2-RhapD, H-3-Manp,
H-4-RhapC, H-4-RhapD), 5.34 (d, 1H, J 1.6 Hz, H-1-Manp), 5.29 (t,
1H, J3,4, J4,5 9.6 Hz, H-4-Manp), 5.21 (s, 1H, H-1-Rhap), 5.14 (t, 1H,
J3,4, J4,5 9.6 Hz, H-4-RhapA), 5.08 (d, 1H, J 8.2 Hz, H-1-Glup), 5.05
(s, 1H, H-1-Rhap), 4.92 (s, 1H, H-1-Rhap), 4.90–4.70 (m, 3H, H-2-
Manp, H-2-RhapC, H-3-RhapC), 4.60–4.25 (m, 6H, H-3-RhapA, 2ꢀ
H-6-Manp, 3ꢀ H-5), 4.17 (m, 1H, H-2-Glup), 4.10–3.80 (m, 3H, H-
4-Glup, H-5-Glup, H-6-Glup), 3.80–3.70 (m, 4H, H-6-Glup, C6H4-
OCH3), 3.63 (m, 1H, H-5), 2.17–2.04 (m, 21H, 7ꢀ CH3CO), 1.94 (s,
3H, CH3CO), 1.37 (d, 3H, J 6.1 Hz, H-6-Rhap), 1.23 (d, 3H, J 5.7 Hz,
H-6-Rhap), 1.16 (d, 3H, J 6.2 Hz, H-6-Rhap). 13C NMR (75 MHz,
CDCl3): d 171.1, 170.3, 170.2(3), 170.1, 169.7(2) (8ꢀ CH3CO),
166.1, 165.8, 165.2, 164.9, 164.5 (5ꢀ C6H5CO), 155.1, 149.8,
117.7(2), 114.5(2) (C6H4OCH3), 100.8 (C-1-Glup), 98.8, 98.3, 98.2
(3ꢀ C-1-Rhap), 96.2 (C-1-Manp), 75.2, 73.5, 73.2, 73.2, 72.7, 72.2,
72.1, 71.6, 71.3, 70.3, 69.3, 69.2, 69.0, 68.6, 67.7, 66.8, 65.0, 62.5,
62.1, 55.6, 55.5 (C6H4OCH3), 50.7, 23.3, 23.1, 21.1, 20.9(2),
20.7(2), 20.6 8ꢀ CH3CO), 17.7, 17.4, 17.2 (5ꢀ C-6-Rhap). ESI-MS
m/z calcd for C88H96KN2O35 (M+K)+ 1779.5. Found: 1779.8. HRMS
for C88H97N2O235 (M+H)+ 1741.5872. Found: 1741.5921.
4.4. Field experiment
The experiment was conducted in the China Agricultural Uni-
versity Shangzhuang Experiment Station (Haidian, Beijing) in
2011, using Triticum aestivum L. (cv. ‘Jingdong 8’). The wheat seeds
were immersed with 20 mg/L pentasaccharide aqueous solution
for 8 h, and tap water as control. The soaked seeds were grown
in the fertile loam soil on September 28. Before the wintering stage
(November 15), the expansion leaves number of the main stem, till
number, till number with no less than 3 leaves, and total root num-
ber of each plant were investigated. Each treatment was replicated
4 times, and the experiment was carried out as a completely ran-
domized block design and each treatment had 50 plants.
Acknowledgments
This work was supported by the National Basic Research Pro-
gram of China (Nos. 2011AA10A206, 2010CB126105, 2011BAE06
B02-01 and 2012BAK25B03-01), the NSFC (20902108 and
21172257) of China, and the Chinese Universities Scientific Fund
(2011JS030).
4.2.13. p-Methoxyphenyl 2-acetamido-2-deoxy-
anosyl-(1?2)-[ -rhamnopyranosyl-(1?3)-]
anosyl-(1?4)-2-acetamido-2-deoxy-b- -glucopyranosyl-(1?3)-
-rhamnopyranoside (2)
Pentasaccharide 21 (0.16 g, 0.066 mmol) was dissolved in satd
a
-
D
-mannopyr-
a
-
L
a-L-rhamnopyr-
D
a-L
Supplementary data
NH3–MeOH (40 mL). After 1 week at rt, the reaction mixture was
concentrated, and the residue was purified by chromatography
on Sephadex LH-20 (MeOH) to afford 2 (79 mg, 89%) as a foamy so-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2013.12.
006.
lid. ½a 2D5
ꢃ
ꢂ 39:5ꢄ (c 0.3 CHCl3). 1H NMR (300 MHz, MeOD): d 6.95 (d,
2H, J 8.4 Hz, Ar-H), 6.83 (d, 2H, J 8.2 Hz, Ar-H), 5.28 (s, 1H, H-1, H-1-
Manp), 5.04 (s, 1H, H-1, H-1-Rhap), 4.97 (s, 1H, H-1, H-1-Rhap),
4.85 (s, 1H, H-1, H-1-Rhap), 4.61 (d, 1H, J 8.0 Hz, H-1-Glup),
4.26–4.24 (m, 2H), 4.00–3.82 (m, 6H), 3.80–3.51 (m, 16H), 3.50–
3.30 (m, 3H), 1.95, 1.94 (2s, 6H, 2ꢀ COCH3), 1.25–1.15 (m, 6H,
2ꢀ H-6), 1.11 (d, 1H, J 5.9 Hz, H-6). 13C NMR (75 MHz, MeOD): d
174.8, 174.7 (2ꢀ COCH3), 154.6, 149.3, 118.7(2), 115.1(2) (C6H4-
OCH3), 102.8 (C-1-Glup), 101.7, 98.8, 97.2, 97.0 (4ꢀ C-1), 80.1,
77.5, 75.0, 74.8, 74.6, 72.8, 72.3, 72.1, 71.9, 70.7, 70.2, 69.9, 69.8,
69.7, 69.6, 69.0, 68.7, 66.4, 60.4, 60.2, 56.1, 55.8, 52.8 (C6H4OCH3),
22.2, 21.9 (2ꢀ COCH3), 16.7, 16.6, 16.4 (3ꢀ C-6). HRMS for
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