Synthesis of a hexasaccharide fragment of group E streptococci polysaccharide and the tetrasaccharide repeating unit of E. coli O7:K98:H6

Article abstract of DOI:10.1016/j.carres.2004.03.027

Syntheses of a hexasaccharide, the dimer of the repeating unit of the group E streptococci polysaccharide, and a tetrasaccharide, the repeating unit of the E. coli O7:K98:H6, were achieved by constructing alternate α-L-(1→2) - and α-L-(1→3)-linked L-rhamnopyranose backbones and substituting with β-linked D-glucopyranose side chains for the former, and a D-glucopyranosyluronate branch for the latter, respectively, at O-2 of the L-rhamnose ring.

Full text of DOI:10.1016/j.carres.2004.03.027

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 1503–1510
Synthesis of a hexasaccharide fragment of group E streptococci
polysaccharide and the tetrasaccharide repeating unit
of E. coli O7:K98:H6
Youlin Zeng and Fanzuo Kong*
Research Center for Eco-Environmental Sciences, Academia Sinica, PO Box 2871, Beijing 100085, PR China
Received 9 February 2004; accepted 19 March 2004
Abstract—Syntheses of a hexasaccharide, the dimer of the repeating unit of the group E streptococci polysaccharide, and a
-(1 fi 3)-
D-gluco-
tetrasaccharide, the repeating unit of the E. coli O7:K98:H6, were achieved by constructing alternate a-
L
-(1 fi 2)- and a-
L
linked -rhamnopyranose backbones and substituting with b-linked -glucopyranose side chains for the former, and a
L
D
pyranosyluronate branch for the latter, respectively, at O-2 of the
Ó 2004 Elsevier Ltd. All rights reserved.
L
-rhamnose ring.
Keywords: Rhamnose oligosaccharides; Trichloroacetimidates
1. Introduction
linked b-(1 fi 2) or b-(1 fi 3) to an
L-rhamnose ring. The
syntheses of several structures corresponding to frag-
ments of the group A streptococci polysaccharides have
been reported.4–7 We present herein a facile synthesis of
two oligosaccharides consisting of (1 fi 2)- and (1 fi 3)-
The cell-wall polysaccharides of streptococci are
composed primarily of a peptidoglycan lattice and one
or more secondary polymers. These accessory wall
polymers can include teichoic or teichuronic acids as
well as neutral or acidic polysaccharides that contain
linked rhamnan backbone with a 2-O-D-Glcp side chain
(A) or a 2-O-
residues.
D
-GlcpA side chain (B) on the rhamnose
L
-rhamnose, such as group-specific polysaccharides of
group A streptococci1a;b and group E streptococci (A).2
Group A streptococci express a variety of both cell-
surface and extracellular virulence factors that are
responsible for a variety of human diseases, and are
defined by the presence of a cell-wall-associated poly-
saccharide composed of N-acetyl-D-glucosamine and L-
rhamnose. However, group E streptococci are defined
by the presence of a polysaccharide consisting of
→
L
L
2)-α- -Rhap-(1→3)-α- -Rhap-(1→
(A)
2
↑
1
β
D
- -Glcp
→
→
L
L
L
3)-α- -Rhap-(1→3)-α- -Rhap-(1→2)-α- -Rhap-(1
(B)
2
D
-glucose and
by a capsular polysaccharides (K98 antigen) composed
of -rhamnopyranose and -glucopyranosyluronate
(B).3 These polysaccharides consist of an alternating a-
(1 fi 2)- and -(1 fi 3)-linked -rhamnopyranose back-
bone with branching N-acetyl-
-glucopyranose residue or
L-rhamnose (A) and E. coli O7:K98:H6
↑
1
β
D
- -GlcpA
L
D
L
2. Results and discussion
D
-glucosamine residue, a
D
D-glucopyranosyluronate
Syntheses of the hexasaccharide consisting of two tri-
saccharide repeating units of the group E streptococci
polysaccharide and the tetrasaccharide repeating unit of
* Corresponding author. Tel.: +86-10-62936613; fax: +86-10-629235-
63; e-mail: fzkong@mail.rcees.ac.cn
0008-6215/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.03.027

1504
Y. Zeng, F. Kong / Carbohydrate Research 339 (2004) 1503–1510
the E. coli O7:K98:H6 are outlined in Scheme 1. A
strategy that first constructed the rhamnan backbone,
then attached the side chains was used. Thus, allyl 4-O-
yield (63.2%). In the present research, selective glyco-
sylation of 2 with 1 similarly gave the a-(1 fi 3)-linked
disaccharide 3 (40%) as the major product, together with
the a-(1 fi 2)-linked disaccharide 4 (20%) and a small
benzoyl-a-L-rhamnopyranoside (2) was used as the
starting material, since its 3-O-rhamnosylation with
perbenzoylated rhamnosyl donor, followed by acetyl-
ation, produced a key building block with a potential free
hydroxyl group at C-2. It has been reported8 that
amount of allyl 2,3,4-tri-O-benzoyl-a-
syl-(1 fi 2)-[2,3,4-tri-O-benzoyl-a- -rhamnopyranosyl-
(1 fi 3)]-4-O-benzoyl-a- -rhamnopyranoside. Since the
L-rhamnopyrano-
L
L
unreacted 2 (21%) was recoverable and the disaccharides
3 and 4 were readily separated on a gram scale, owing to
their relatively large difference in polarity, the selective
rhamnosylation of 2 is an acceptable procedure in
practical preparations. The regioselectivity was con-
firmed by benzoylation of 4, and the 1H NMR spectrum
condensation of the donor 2,3,4-tri-O-benzoyl-a-
rhamnopyranosyl trichloroacetimidate9 (1) with the
unprotected acceptor, allyl a- -rhamnopyranoside,
selectively gave allyl 2,3,4-tri-O-benzoyl-a- -rhamno-
L-
L
L
pyranosyl-(1 fi 3)-a-
L
-rhamnopyranoside in satisfactory
NH
OCCCl3
OAll
OAll
OAll
Me
BzO
Me
BzO
O
O
Me
BzO
O
Me
BzO
O
a
O
HO
O
+
O
OH
+
HO
Me
O
BzO
Me
BzO
OH
OBz
BzO
BzO
3 (40%)
BzO
1
2
OBz
OBz
4 (20%)
NH
OCCCl3
OAll
OH
Me
O
O
Me
O
O
O
BzO
Me
BzO
O
BzO
b
O
3
OAc
c
Me
OAc
O
Me
d
OAc
BzO
Me
BzO
O
BzO
BzO
BzO
OBz
OBz
BzO
OBz
5
6
7
OAll
OAll
OH
Me
O
Me
BzO
Me
O
BzO
Me
O
BzO
BzO
O
BzO
BzO
O
OH
O
8
c
Me
BzO
O
BzO
O
OAc
a
O
Me
BzO
O
OAc
Me
O
BzO
BzO
OBz
BzO
OBz
9
NH
OCCCl3
10
Me
O
BzO
OAll
OAll
BzO
O
Me
BzO
CAO
O
Me
O
d
e
b
Me
O
BzO
BzO
CAO
OH
O
OAc
OAc
Me
O
12
13
BzO
BzO
OBz
OAll
OAll
11
Me
BzO
Me
BzO
O
O
O
O
OAc
OH
OAll
Me
Me
BzO
O
O
11
BzO
Me
O
f
BzO
a
BzO
O
BzO
O
HO
14
Me
Me
BzO
O
O
OAc
BzO
O
O
OAc
OH
Me
BzO
Me
BzO
O
O
BzO
BzO
OBz
OBz
15
16
Scheme 1. Reagents and conditions: (a) TMSOTf, CH2Cl2, 0 °C to rt; (b) Ac2O–pyridine (dry), rt, 12 h; (c) PdCl2, MeOH–CH2Cl2, rt, 5 h; (d)
CCl3CN, CH2Cl2, DBU, rt; (e) thiourea in 1:4 EtOH–CH2Cl2, reflux, 16 h; (f) 7% CH3COCl–CH3OH–CH2Cl2, rt; (g) satd NH3–MeOH, rt, 2 weeks.

Y. Zeng, F. Kong / Carbohydrate Research 339 (2004) 1503–1510
1505
OAll
OAll
Me
BzO
O
O
Me
HO
O
O
O
BzO
BzO
BzO
O
O
Me
O
O
O
Me
HO
O
BzO
NH
OCCCl3
BzO
O
OBz
HO
BzO
17
O
OH
OH
Me
O
Me
HO
O
g
BzO
OBz
OBz
BzO
O
a
HO
O
O
OH
Me
O
O
Me
HO
O
BzO
O
OBz
O
BzO
OBz
HO
OH
OH
OH
OBz
OBz
BzO
HO
OH
18
19
OAll
Me
O
COOMe
O
BzO
AcO
AcO
BzO
O
O
NH
OCCCl3
Me
BzO
f
AcO
21
9
O
OH
Me
O
a
BzO
BzO
OBz
20
OAll
OAll
Me
HO
Me
O
O
BzO
Me
OH
BzO
O
O
Me
HO
O
O
BzO
g
O
O
O
O
O
Me
HO
Me
BzO
O
O
O
OH
OAc
OAc
BzO
OH
OH
OBz
MeOOC
+H4NOOC
OH
OH
OAc
22
23
Scheme 1 (continued)
of the product showed the same data as that reported in
the literature.10 Acetylation of 3 gave 5, whose H-2 at d
5.34 ppm with J1;2 ¼ 1:8 Hz and J2;3 ¼ 3:5 Hz, also veri-
fied the 3-O-glycosylation. The donor 7 was obtained
from disaccharide 5 by deallylation with PdCl2,11 and
then by trichloroacetimidate formation with trichloro-
acetonitrile.9 Coupling of 7 with allyl 3,4-di-O-benzoyl-
a-L
-rhamnopyranoside (8)12 gave trisaccharide 9 in high
yield (85%). Deallylation of 9, followed by trichloro-
acetimidate formation, furnished trisaccharide donor 11.
ment of the side chains was carried out by coupling of 16
with perbenzoylated glucosyl trichloroacetimidate 17,
giving hexasaccharide 18 (52%). Deacylation of the
protective hydroxyl groups with ammonia–saturated
methanol gave the unprotected hexasaccharide 19 (94%)
that was characterized by 13C NMR and 1H NMR
spectroscopy. The signals at d 5.25, 5.21, 5.09, 5.06 (4H,
J1;2 1.3 Hz, Rhap H-1), 4.84 (1H, J1;2 8.0 Hz, Glcp H-1),
4.80 (1H, J1;2 7.8 Hz, Glcp H-1), 103.9, 103.9 (2C, 2Glcp
C-1), 102.0, 100.7, 100.4, 97.4 (4C, 4Rhap C-1) corre-
spond with the designated structure.
2-O-Selective deacetylation of 9 gave the trisaccharide
acceptor 20 (80%). Condensation of 20 with 21 yielded
tetrasaccharide 22 (62%). Deacylation of 22 with
ammonia–saturated methanol gave the unprotected
tetrasaccharide 23 (95%) that showed characteristic
NMR signals at d 5.22, 5.04, 4.91 (3H, J1;2 1.3 Hz, Rhap
H-1), 4.63 (1H, J1;2 7.6 Hz, GlcpA H-1), 103.9 (GlcpA C-
1), 102.0, 100.7, and 96.9 (3C, 3Rhap C-1).
Allyl
anoside (12)12 was converted to allyl 2-O-acetyl-4-O-
benzoyl-a- -rhamnopyranoside (14) by acetylation and
4-O-benzoyl-3-O-chloroacetyl-a-
L-rhamnopyr-
L
dechloroacetylation. Compound 14 was also a key
component since it contains another potential hydroxyl
group at C-2 after its condensation with the trisaccharide
donor 11. The tetrasaccharide acceptor 16 with two free
hydroxyl groups at C-2 and C-200 was obtained by cou-
pling of 14 with 11, followed by 2-O-selective deacetyl-
ation by methanolysis.13a Due to the relatively large
steric hindrance at 2- and 200-OAc, 7% acetyl chloride in
1:2 methanol–dichloromethane (v/v) was used instead of
the usual 2–3% concentration of reagent.13b;c;d Attach-
In summary, we have presented herein a convergent
method that can be applied to the synthesis of (1 fi 2)-
and (1 fi 3)-linked rhamnans with b-linked GlcA and
Glc side chains at O-2 of the rhamnose residue.

1506
Y. Zeng, F. Kong / Carbohydrate Research 339 (2004) 1503–1510
3. Experimental
J3;4 9.9 Hz, H-30), 5.54 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:9 Hz, H-
40), 5.49 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:8 Hz, H-4), 5.39 (dd, 1H,
J1;2 1.7 Hz, J2;3 3.4 Hz, H-20), 5.53 (d, 1H, J 1.5 Hz, J
17.2 Hz, CH2–CH@CH2), 5.34 (dd, 1H, J1;2 1.8 Hz, J2;3
3.5 Hz, H-2), 5.25 (d, 1H, J 1.3 Hz, J 10.4 Hz, CH2–
CH@CH2), 5.16 (d, 1H, J1;2 1.7 Hz, H-10), 4.88 (d, 1H,
J1;2 1.6 Hz, H-1), 4.44 (dd, 1H, J2;3 3.5 Hz, J3;4 9.8 Hz, H-
3), 4.26–4.00 (m, 4H, H-50, H-5, 2CH2CH@CH2), 2.33
(s, 3H, CH3CO), 1.33 (d, 3H, J5;6 6.3 Hz, H-60), 1.30 (d,
3H, J5;6 6.3 Hz, H-6). Anal. Calcd for C45H44O14: C,
66.82; H, 5.48. Found: C, 66.69; H, 5.42.
3.1. General methods
Melting points were determined using a ‘Mel-Temp’
apparatus. Optical rotations were determined using a
Perkin–Elmer model 241-MC automatic polarimeter for
solutions in a 1-dm jacketed cell. 1H and 13C NMR
spectra were recorded with a Bruker ARX 400 spec-
1
13
trometer (400 MHz for H, 100 MHz for C) at 25 °C
for solutions in CDCl3 or D2O as indicated, and indi-
vidual resonances could not be identified with the specific
sugar residues. Chemical shifts are expressed in ppm
downfield from the Me4Si absorption. Mass spectra were
recorded on a VG Platform mass spectrometer in the
electrospray-ionization (ESI) mode. Thin-layer chro-
matography (TLC) was performed on silica gel HFwith
detection by charring with 30% (v/v) sulfuric acid in
MeOH or by UV detection. Column chromatography
was conducted by the elution of columns (8 · 100 mm,
16 · 240 mm, 18 · 300 mm, 35 · 400 mm) of silica gel
(100–200 mesh) with EtOAc–petroleum ether (bp 60–
90 °C) as the eluent. Analytical LC was performed with a
Gilson HPLC consisting of a pump (model 306), stain-
less-steel column packed with silica gel (Spherisorb SiO2,
10 300 mm or 4.6 · 250 mm), differential refractometer
(132-RI Detector), and a UV–vis detector (model 118).
EtOAc–petroleum ether (bp 60–90 °C) was used as the elu-
ent at a flow rate of 1–4 mL/min. Solutions were concen-
trated at a temperature <560 °C under reduced pressure.
3.3. 2,3,4-Tri-O-benzoyl-a-
2-O-acetyl-4-O-benzoyl-a-
acetimidate (7)
L
-rhamnopyranosyl-(1 fi 3)-
L-rhamnopyranosyl trichloro-
To a solution of compound 5 (3.4 g, 4.2 mmol) in MeOH
(100 mL) was added PdCl2 (50 mg, 0.28 mmol), and the
mixture was stirred at rt for 2 h, at the end of which time
TLC (3:1 petroleum ether–EtOAc) indicated that the
reaction was complete. The mixture was filtered, the
filter cake was washed with CH2Cl2, and the combined
filtrate and washings were concentrated. Purification by
column chromatography with 4:1 petroleum ether–
EtOAc as the eluent afforded compound 6. A mixture of
6, trichloroacetonitrile (2.1 mL, 10 mmol), and 1,8-
diazabicyclo[5.4.0]undecene (DBU, 0.20 mL, 1.6 mmol)
in dry CH2Cl2 (50 mL) was stirred for 3 h and then
concentrated. The residue was purified by flash chro-
matography with 4:1 petroleum ether–EtOAc as the
eluent to give 7 (3.1 g, 81% for two steps) as a white
25
1
3.2. Allyl 2,3,4-tri-O-benzoyl-a-
(1 fi 3)-2-O-acetyl-4-O-benzoyl-a-
pyranoside (5)
L
-rhamnopyranosyl-
-rhamno-
foam. ½aŠ +186.2 (c 1.0, CHCl3); H NMR (400 MHz,
D
L
CDCl3): d 8.77 (s, 1H, CNHCCl3), 8.08–7.21 (m, 20H,
Bz-H), 6.31 (d, 1H, J1;2 1.8 Hz, H-1), 5.67 (dd, 1H, J2;3
3.3 Hz, J3;4 10.1 Hz, H-30), 5.61–5.51 (m, 3H, H-20, H-4,
H-40), 5.36 (dd, 1H, J1;2 1.8 Hz, J2;3 3.5 Hz, H-2), 5.19 (d,
1H, J1;2 1.5 Hz, H-10), 4.51 (dd, 1H, J2;3 3.5 Hz, J3;4
9.9 Hz, H-3), 4.28 (ddd, 1H, J4;5 9.9 Hz, J5;6 6.2 Hz, H-
50), 4.17 (ddd, 1H, J4;5 9.9 Hz, J5;6 6.3 Hz, H-5), 2.38 (s,
3H, CH3CO), 1.35 (d, 3H, J5;6 6.2 Hz, H-60), 1.31 (d, 3H,
J5;6 6.3 Hz, H-6). Anal. Calcd for C44H40Cl3NO14: C,
57.87; H, 4.42. Found: C, 57.62; H, 4.39.
To a cooled solution (0 °C) of 1 (3.10 g, 5.0 mmol) and 2
(1.54 g, 5.0 mmol) in anhyd CH2Cl2 (50 mL) was added
TMSOTf (20 lL, 0.12 mmol). The mixture was stirred
for 2 h, during which time the temperature was gradu-
ally raised to ambient temperature. The mixture was
quenched with Et3N (four drops) and then evaporated
to give a residue that was purified by silica gel column
chromatography with 4:1 petroleum ether–EtOAc as the
eluent to give disaccharide 3 (1.53 g, 40%), disaccharide
4 (0.76 g, 20%), unreacted 2 (0.32 g, 21%), and a small
mount of trisaccharide. To a solution of 3 (1.46 g,
1.9 mmol) in pyridine (10 mL) was added Ac2O (5 mL).
The mixture was stirred for 2 h at rt, at the end of which
time TLC (3:1 petroleum ether–EtOAc) indicted that the
reaction was complete. The mixture was dried and co-
vaporized with toluene (5 mL) three times. The residue
was purified by chromatography with 4:1 petroleum
ether–EtOAc as the eluent to give disaccharide 5 (1.41 g,
3.4. Allyl 2,3,4-tri-O-benzoyl-a-
(1 fi 3)-2-O-acetyl-4-O-benzoyl-a-
(1 fi 2)-3,4-di-O-benzoyl-a- -rhamnopyranoside (9)
L
-rhamnopyranosyl-
L
-rhamnopyranosyl-
L
Donor 7 (3.0 g, 3.28 mmol) and acceptor 8 (1.18 g,
2.86 mmol) were coupled in the presence of a catalytic
amount of TMSOTf (20 lL, 0.11 mmol) under the same
conditions as described for the coupling of 2 with 1.
Purification by chromatography with 3:1 petroleum
ether–EtOAc as the eluent gave triasaccharide 9 (2.83 g,
25
25
1
1
92%) as a foamy solid. ½aŠ +141.9 (c 1.0, CHCl3); H
85%) as a foamy solid. ½aŠ +129.5 (c 1.0, CHCl3); H
D
D
NMR (400 MHz, CDCl3): d 8.10–7.18 (m, 20H, Bz-H),
5.95 (m, 1H, CH2CH@CH2), 5.65 (dd, 1H, J2;3 3.4 Hz,
NMR (400 MHz, CDCl3): d 8.20–7.15 (m, 30H, Bz-H),
6.02–5.89 (m, 3H, H-200, H-300, CH2CH@CH2), 5.85 (dd,

Y. Zeng, F. Kong / Carbohydrate Research 339 (2004) 1503–1510
1507
1H, J2;3 3.3 Hz, J3;4 10.0 Hz, H-3), 5.76 (dd, 1H,
mixture was dried and co-vaporized with toluene (10 mL)
three times. The residue was purified by chromatography
with 4:1 petroleum ether–EtOAc as the eluent to give 13
J3;4 ¼ J4;5 ¼ 10:0 Hz, H-400), 5.63 (dd, 1H, J3;4 ¼ J4;5
¼
9:9 Hz, H-40), 5.61 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:9 Hz, H-4),
5.48 (d, 1H, J1;2 1.7 Hz, H-100), 5.45 (d, 1H, J1;2 1.7 Hz,
H-10), 5.34 (d, 1H, J 1.4 Hz, J 17.2 Hz, CH2–CH@CH2),
5.27 (dd, 1H, J1;2 1.7 Hz,J2;3 3.5 Hz, H-20), 5.26 (d, 1H, J
1.4 Hz, J 10.4 Hz, CH2–CH@CH2), 5.09 (d, 1H, J1;2
1.8 Hz, H-1), 4.47–4.00 (m, 7H, H-2, H-30, H-500, H-50,
H-5, 2CH2CH@CH2), 2.04 (s, 3H, CH3CO), 1.39 (d,
3H, J5;6 6.2 Hz, H-600), 1.38 (d, 3H, J5;6 6.2 Hz, H-60), 1.30
(d, 3H, J5;6 6.2 Hz, H-6). Anal. Calcd for C65H62O20: C,
67.12; H, 5.37. Found: C, 66.89; H, 5.46.
25
(3.88 g, 91%) as a foamy solid. ½aŠ +152.9 (c 1.0,
D
1
CHCl3); H NMR (400 MHz, CDCl3): d 8.02–7.43 (m,
5H, Bz-H), 5.95 (m, 1H, CH2CH@CH2), 5.65 (dd, 1H,
J2;3 3.5 Hz, J3;4 10.1 Hz, H-3), 5.38 (dd, 1H, J3;4
¼
J4;5 ¼ 10:1 Hz, H-4), 5.36 (d, 1H, J 1.5 Hz, J 17.2 Hz,
CH2–CH@CH2), 5.33 (dd, 1H, J1;2 1.7 Hz, J2;3 3.5 Hz, H-
2), 5.53 (d, 1H, J 1.5 Hz, J 10.4 Hz, CH2–CH@CH2),
5.16 (d, 1H, J1;2 1.7 Hz, H-1), 4.26–4.00 (m, 3H, H-5,
2CH2CH@CH2), 3.89, 3.88 (ABq, 2H, J 18.2 Hz,
ClCH2CO), 2.18 (s, 3H, CH3CO), 1.28 (d, 3H, J5;6
6.2 Hz, H-6). Anal. Calcd for C20H23ClO8: C, 56.28; H,
5.43. Found: C, 56.11; H, 5.38.
3.5. 2,3,4-Tri-O-benzoyl-a-
L
-rhamnopyranosyl-(1 fi 3)-2-
-rhamnopyranosyl-(1 fi 2)-3,4-
L-rhamnopyranosyl trichloroacetimidate
O-acetyl-4-O-benzoyl-a-
L
di-O-benzoyl-a-
(11)
3.7. Allyl 2-O-acetyl-4-O-benzoyl-a-L-rhamno-
pyranoside (14)
To a solution of 9 (2.8 g, 2.4 mmol) in MeOH (100 mL)
was added PdCl2 (30 mg, 0.17 mmol), and the mixture
was stirred at rt for 2 h, at the end of which time TLC (3:1
petroleum ether–EtOAc) indicated that the reaction was
complete. The mixture was filtered, the filter cake was
washed with CH2Cl2, and the combined filtrate and
washings were concentrated. Purification by column
chromatography with 3:1 petroleum ether–EtOAc as the
eluent afforded compound 10. A mixture of 10, trichlo-
roacetonitrile (1.1 mL, 5 mmol), and 1,8-diazabicy-
clo[5.4.0]undecene (DBU) (0.10 mL, 0.8 mmol) in dry
CH2Cl2 (50 mL) was stirred for 3 h and then concen-
trated. The residue was purified by chromatography with
Thiourea (1.6 g, 20.9 mmol) was added to a solution
(100 mL) of 13 (1.63 g, 3.81 mmol) in MeOH and CH2Cl2
(v/v, 1:2), then the reaction mixture was refluxed for 17 h, at
the end of which time TLC (3:1 petroleum ether–EtOAc)
indicated that the reaction was complete. The mixture
was concentrated to 1/5 of the original volume, diluted
with CH2Cl2, and washed with satd aq NaHCO3 and
water, and the organic phase was dried and concen-
trated. Purification by silica gel chromatography with 3:1
petroleum ether–EtOAc as the eluent afforded 14 (1.16 g,
25
1
87 %). ½aŠ +154.1 (c 1.0, CHCl3); H NMR (400 MHz,
D
CDCl3): d 8.04–7.43 (m, 5H, Bz-H), 5.89 (m, 1H,
CH2CH@CH2), 5.31 (d, 1H, J 1.5 Hz, J 17.2 Hz, CH2–
CH@CH2), 5.22 (d, 1H, J 1.5 Hz, J 10.4 Hz, CH2–
CH@CH2), 5.13–5.06 (m, 2H, H-2, H-4), 4.86 (d, 1H, J1;2
1.7 Hz, H-1), 4.22–3.94 (m, 4H, H-3, H-5,
CH2CH@CH2), 2.16 (s, 3H, CH3CO), 1.24 (d, 3H, J5;6
6.2 Hz, H-6). Anal. Calcd for C18H22O7: C, 61.71; H,
6.33. Found: C, 61.42; H, 6.39.
4:1 petroleum ether–EtOAc as the eluent to give 11
25
(2.4 g, 79% for two steps) as a white foam. ½aŠ +93.2 (c
D
1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d 8.76 (s, 1H,
CNHCCl3), 8.10–7.22 (m, 30H, Bz-H), 6.43 (d, 1H, J1;2
1.8 Hz, H-1), 5.78 (dd, 1H, J2;3 3.2 Hz, J3;4 10.0 Hz, H-300),
5.69 (dd, 1H, J3;4 ¼ J4;5 ¼ 10:0 Hz, H-400), 5.67 (dd, 1H,
J2;3 3.4 Hz, J3;4 10.2 Hz, H-3), 5.59 (dd, 1H, J3;4 ¼ J4;5
¼
9:8 Hz, H-40), 5.57 (dd, 1H, J1;2 1.4 Hz, J2;3 3.2 Hz, H-2),
5.52 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:8 Hz, H-4), 5.38 (dd, 1H, J1;2
1.5 Hz, J2;3 3.2 Hz, H-20), 5.24 (d, 1H, J1;2 1.4 Hz, H-100),
5.03 (d, 1H, J1;2 1.5 Hz, H-10), 4.58 (dd, 1H, J2;3 3.4 Hz,
J3;4 9.8 Hz, H-30), 4.51 (dd, 1H, J1;2 1.8 Hz, J2;3 3.2 Hz,
H-2), 4.39–4.30 (m, 2H, H-500, H-50), 4.18 (ddd, 1H, J4;5
10.0 Hz, J5;6 6.2 Hz, H-5), 2.24 (s, 3H, CH3CO), 1.41 (d,
3H, J5;6 6.2 Hz, H-600), 1.39 (d, 3H, J5;6 6.2 Hz, H-60), 1.33
(d, 3H, J5;6 6.2 Hz, H-6). Anal. Calcd for C64H58Cl3NO20:
C, 60.65; H, 4.61. Found: C, 60.41; H, 4.74.
3.8. Allyl 2,3,4-tri-O-benzoyl-a-
(1 fi 3)-2-O-acetyl-4-O-benzoyl-a-
(1 fi 2)-3,4-di-O-benzoyl-a- -rhamnopyranosyl-(1 fi 3)-
2-O-acetyl-4-O-benzoyl-a- -rhamnopyranoside (15)
L
-rhamnopyranosyl-
L
-rhamnopyranosyl-
L
L
Compound 11 (1.52 g, 1.2 mmol) and 14 (351 mg,
1.0 mmol) were coupled in the presence of catalytic
TMSOTf (20 lL, 0.11 mmol) under the same conditions
as described for the coupling of 2 with 1. Purification by
silica gel chromatography with 3:1 petroleum ether–
EtOAc as the eluent gave tetrasaccharide 15 (1.31 g,
3.6. Allyl 2-O-acetyl-4-O-benzoyl-3-O-chloroacetyl-a-L-
rhamnopyranoside (13)
25
D
1
90%) as a foamy solid. ½aŠ +208.2 (c 1.0, CHCl3); H
NMR (400 MHz, CDCl3): d 8.07–7.15 (m, 35H, Bz-H),
5.93 (m, 1H, CH2CH@CH2), 5.65–5.52 (m, 3H, 2H-3,
H-4), 5.49 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:7 Hz, H-4), 5.42–5.29
(m, 6H, 3H-2, CH2–CH@CH2, 2H-4), 5.26 (d, 1H, J
1.4 Hz, J 10.4 Hz, CH2–CH@CH2), 5.18 (d, 1H, J1;2
To a solution of 12 (3.84 g, 10 mmol) in pyridine (30 mL)
was added Ac2O (10 mL). The mixture was stirred for 2 h
at rt, at the end of which time TLC (4:1 petroleum ether–
EtOAc) indicted that the reaction was complete. The

1508
Y. Zeng, F. Kong / Carbohydrate Research 339 (2004) 1503–1510
1.4 Hz, H-1), 5.12 (d, 1H, J1;2 1.5 Hz, H-1), 4.86 (d, 1H,
J1;2 1.5 Hz, H-1), 4.54 (d, 1H, J1;2 1.3 Hz, H-1), 4.46 (dd,
1H, J2;3 3.4 Hz, J3;4 9.8 Hz, H-3), 4.40 (dd, 1H, J2;3
3.5 Hz, J3;4 9.7 Hz, H-3), 4.35–3.78 (m, 7H, H-2, 4H-5,
CH2CH@CH2), 2.33 (s, 3H, CH3CO), 2.04 (s, 3H,
CH3CO), 1.36 (d, 3H, J5;6 6.2 Hz, H-6), 1.29 (d, 3H, J5;6
6.2 Hz, H-6), 1.26 (d, 3H, J5;6 6.2 Hz, H-6), 0.92 (d, 3H,
J5;6 6.2 Hz, H-6). 13C NMR (100 MHz, CDCl3): d 170.6,
169.8 (2C, 2CH3CO), 165.7, 165.6, 165.5, 165.4, 165.1,
165.1, 164.6 (7C, 7PhCO), 133.4–128.6 (PhCO, –CH2–
CH@CH2), 117.9 (1C, –CH2–CH@CH2), 100.0, 99.3,
98.5, 96.6 (4C, 4C-1), 73.8, 73.7, 73.4, 72.5, 71.6, 71.4,
71.3, 70.8, 70.4, 70.3, 69.2, 68.4, 67.6, 67.5, 67.4, 66.7 (C-
2–5, –CH2–CH@CH2), 20.9, 20.8 (2C, 2CH3CO), 17.5,
17.4, 17.4, 17.1 (4C, 4C-6). Anal. Calcd for C80H78O26:
C, 66.02; H, 5.40. Found: C, 65.93; H, 5.52.
conditions as described for the coupling of 2 with 1.
Purification by a chromatography with 3:1 petroleum
ether–EtOAc as the eluent gave hexasaccharide 18
25
D
(331 mg, 52%) as a foamy solid. ½aŠ +100.2 (c 1.0,
1
CHCl3); H NMR (400 MHz, CDCl3): d 8.24–7.11 (m,
75H, Bz-H), 6.22 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:7 Hz, H-4),
6.14 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:6 Hz, H-4), 5.95 (dd, 1H,
J2;3 ¼ J3;4 ¼ 9:7 Hz, H-3), 5.87–5.16 (m, 14H, 1H-1, 3H-
2, 3H-3, 4H-4, CH2–CH@CH2, CH2–CH@CH2), 4.97
(d, 1H, J1;2 1.5 Hz, H-1), 4.94 (d, 1H, J1;2 1.5 Hz, H-1),
4.94 (d, 1H, J1;2 1.5 Hz, H-1), 4.69–3.54 (m, 17H, 3H-2,
2H-3, 6H-5, 4H-6, CH2CH@CH2), 1.52 (d, 3H, J5;6
6.2 Hz, H-6), 1.35 (d, 3H, J5;6 6.2 Hz, H-6), 1.26 (d, 3H,
J5;6 6.2 Hz, H-6), 0.99 (d, 3H, J5;6 6.2 Hz, H-6). 13C
NMR (100 MHz, CDCl3): d 166.1, 165.9, 165.9, 165.6,
165.5, 165.5, 165.5, 165.5, 165.4, 165.2, 165.2, 165.0,
164.9, 164.8, 164.4 (15C, 15PhCO), 133.76–128.1
(PhCO, –CH2–CH@CH2), 117.1 (1C, –CH2–CH@CH2),
101.0 (JC-1;H-1 162.1 Hz, b-C-1), 100.4 (JC-1;H-1 167.6 Hz,
b-C-1), 99.2 (JC-1;H-1 177.6 Hz, a-C-1), 98.4 (JC-1;H-1
172.3 Hz, a-C-1), 98.3 (JC-1;H-1 172.3 Hz, a-C-1), 98.3
(JC-1;H-1 172.3 Hz, a-C-1), 77.4, 76.2, 74.1, 73.8, 73.7,
73.6, 72.9, 72.2, 72.1, 72.0, 71.9, 71.6, 70.6, 70.1, 69.7,
69.3, 69.1, 68.4, 68.3, 68.1, 67.3, 62.7, 62.4, 60.4 (27C, C-
2–5, C-6, –CH2–CH@CH2), 17.8, 17.7, 17.2, 16.6 (4C,
4C-6). Anal. Calcd for C144H126O42: C, 68.40; H, 5.02.
Found: C, 68.20; H, 4.93.
3.9. Allyl 2,3,4-tri-O-benzoyl-a-
(1 fi 3)-4-O-benzoyl-a- -rhamnopyranosyl-(1 fi 2)-3,4-
di-O-benzoyl-a- -rhamnopyranosyl-(1 fi 3)-4-O-benzoyl-
a- -rhamnopyranoside (16)
L-rhamnopyranosyl-
L
L
L
To a solution of 15 (1.1 mg, 0.74 mmol) in mixed sol-
vents of MeOH (33 mL) and CH2Cl2 (67 mL) was added
AcCl (7 mL). The mixture was stirred at rt until the TLC
(2:1 petroleum ether–EtOAc) suggested that the reaction
was complete. The mixture was dried, and purified by
silica gel chromatography with 2:1 petroleum ether–
EtOAc as the eluent to give 16 (612 mg, 62%) as a foamy
3.11. Allyl a-
anosyl-(1 fi 2)]-a-
rhamnopyranosyl-(1 fi 3)-[b-
(1 fi 2)]-a- -rhamnopyranoside (19)
L
-rhamnopyranosyl-(1 fi 3)-[b-
-rhamnopyranosyl-(1 fi 2)-a-
-glucopyranosyl-
D-glucopyr-
25
1
solid. ½aŠ +155.7 (c 1.0, CHCl3); H NMR (400 MHz,
L
L-
D
CDCl3): d 8.07–7.19 (m, 35H, Bz-H), 5.93 (m, 1H,
CH2CH@CH2), 5.81–5.17 (m, 10H, 1H-1, 1H-2, 2H-3,
4H-4, CH2–CH@CH2), 5.14 (d, 1H, J1;2 1.5 Hz, H-1),
4.96 (d, 1H, J1;2 1.5 Hz, H-1), 4.70 (d, 1H, J1;2 1.4 Hz, H-
1), 4.36–3.78 (m, 11H, 3H-2, 2H-3, 4H-5,
CH2CH@CH2), 1.36 (d, 3H, J5;6 6.2 Hz, H-6), 1.29 (d,
3H, J5;6 6.2 Hz, H-6), 1.26 (d, 3H, J5;6 6.2 Hz, H-6), 0.99
(d, 3H, J5;6 6.2 Hz, H-6).13C NMR (100 MHz, CDCl3): d
165.7, 165.7, 165.7, 165.5, 165.2, 165.1, 164.6 (7C,
7PhCO), 133.6–128.1 (PhCO, –CH2–CH@CH2), 117.7
(1C, –CH2–CH@CH2), 100.1, 100.4, 98.8, 98.4 (4C, 4C-
1), 75.8, 73.3, 72.9, 71.8, 71.5, 71.3, 71.0, 70.4, 69.4, 68.3,
67.6, 67.5, 67.4, 66.6, 60.4 (C-2–5, –CH2–CH@CH2),
17.6, 17.5, 17.5, 17.3 (4C, 4C-6). Anal. Calcd for
C76H74O24: C, 66.56; H, 5.44. Found: C, 66.37; H, 5.37.
D
L
Compound 18 (301 mg, 0.12 mmol) was dissolved in satd
ammonia–MeOH (10 mL). After 2 weeks at rt, the
reaction mixture was concentrated, and the residue was
purified on a BioGel P2 column with MeOH–water as
the eluent to afford 19 (105 mg, 93%) as an amorphous
25
1
solid. ½aŠ À153.5 (c 1.0, H2O); H NMR (400 MHz,
D
D2O): d 5.93 (m, 1H, OCH2CH@CH2), 5.33 (dd, 1H, J
1.4 Hz, J 17.2 Hz, CH2–CH@CH2), 5.28 (dd, 1H, J
1.4 Hz, J 10.2 Hz, CH2–CH@CH2), 5.25 (d, 1H, J1;2
1.3 Hz, H-1), 5.21 (d, 1H, J1;2 1.3 Hz, H-1), 5.09 (d, 1H,
J1;2 1.3 Hz, H-1), 5.06 (d, 1H, J1;2 1.3 Hz, H-1), 4.84 (d,
1H, J1;2 8.0 Hz, H-1), 4.80 (d, 1H, J1;2 7.8 Hz, H-1), 4.58–
3.31 (m, 30H, H-2–5, 4H-6, CH2CH@CH2), 1.32 (d, 3H,
J5;6 5.6 Hz, H-6), 1.30 (d, 3H, J5;6 6.0 Hz, H-6), 1.28 (d,
3H, J5;6 5.8 Hz, H-6), 1.26 (d, 3H, J5;6 6.2 Hz, H-6). 13C
NMR (100 MHz, D2O): d 134.0 (1C, –CH2–CH@CH2),
118.1 (1C, –CH2–CH@C2), 103.9, 103.9, 102.0, 100.7,
100.4, 97.4 (6C, 6C-1), 78.9, 77.6, 76.3, 75.9, 75.5, 75.3,
73.0, 72.1, 71.9, 71.8, 71.6, 70.4, 69.9, 69.8, 69.7, 69.2,
68.9, 68.5, 67.9, 60.3 (27C, C-2–5, 2C-6, –CH2–
CH@CH2), 16.5, 16.4, 16.3, 16.2 (4C, 4C-6). Anal.
Calcd for C39H66O27: C, 48.44; H, 6.88. Found: C, 48.29;
H, 6.90.
3.10. Allyl 2,3,4-tri-O-benzoyl-a-
(1 fi 3)-[2,3,4,6-tetra-O-benzoyl-b-
(1 fi 2)]-4-O-benzoyl-a- -rhamnopyranosyl-(1 fi 2)-3,4-
di-O-benzoyl-a- -rhamnopyranosyl-(1 fi 3)-[2,3,4,6-tet-
ra-O-benzoyl-b- -glucopyranosyl-(1 fi 2)]-4-O-benzoyl-
a- -rhamnopyranoside (18)
L
-rhamnopyranosyl-
D
-glucopyranosyl-
L
L
D
L
Compound 16 (345 mg, 0.25 mmol) and 17 (555 mg,
0.75 mmol) were coupled in the presence of a catalytic
amount of TMSOTf (10 lL, 0.06 mmol) under the same

Y. Zeng, F. Kong / Carbohydrate Research 339 (2004) 1503–1510
1509
3.12. Allyl 2,3,4-tri-O-benzoyl-a-
(1 fi 3)-4-O-benzoyl-a- -rhamnopyranosyl-(1 fi 2)-3,4-
di-O-benzoyl-a- -rhamnopyranoside (20)
L
-rhamnopyranosyl-
J3;4 10.0 Hz, H-3), 4.38–4.04 (m, 8H, 2H-2, H-3, 3H-5,
2CH2CH@CH2), 3.83 (d, 1H, J5;6 10.0 Hz, H-5), 3.46 (s,
3H, COOCH3), 2.32 (s, 3H, CH3CO), 2.04 (s, 3H,
CH3CO), 2.01 (s, 3H, CH3CO),1.50 (d, 3H, J5;6 6.3 Hz,
H-6), 1.34 (d, 3H, 6.2 Hz, H-6), 1.23 (d, 3H, J5;6 6.2 Hz,
H-6). 13C NMR (100 MHz, CDCl3): d 170.0, 169.9,
169.4, 166.8 (4C, 3CH3CO, COOCH3), 165.8, 165.4,
165.3, 165.3, 164.9, 164.7 (6C, 6PhCO), 133.4–128.1
(PhCO, –CH2–CH@CH2), 117.9 (1C, –CH2–CH@CH2),
101.1, 100.9, 98.5, 97.8, (4C, JC1–H1 174.4, 165.0, 171.2,
170.2 Hz, 4C-1), 76.8, 74.7, 73.1, 72.0, 71.9, 71.8, 71.7,
71.3, 70.9, 70.5, 69.4, 68.9, 68.3, 68.1, 67.9, 66.8, 52.5
(17C, C-2–5, –CH2–CH@CH2), 29.6 (C, COOCH3),
22.6, 20.6, 20.5 (3C, 3CH3CO), 17.7, 17.5, 17.5 (3C, 3C-
6). Anal. Calcd for C76H76O28: C, 63.50; H, 5.33. Found:
C, 63.31; H, 5.28.
L
L
To a solution of 9 (512 mg, 0.44 mmol) in mixed solvents
of MeOH (33 mL) and CH2Cl2 (67 mL) was added AcCl
(5 mL). The mixture was stirred at rt until the TLC (2:1
petroleum ether–EtOAc) indicated that the reaction was
complete. The mixture was dried and purified by chro-
matography with 2:1 petroleum ether–EtOAc as the
25
eluent to give 20 (395 mg, 80%) as a foamy solid. ½aŠ
D
1
+127.5 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): d
8.07–7.23 (m, 30H, Bz-H), 5.97 (m, 1H, OCH2-
CH@CH2), 5.76 (dd, 1H, J2;3 3.5 Hz, J3;4 9.9 Hz, H-300),
5.69 (dd, 1H, J2;3 3.2 Hz, J3;4 9.9 Hz, H-3), 5.63 (dd, 1H,
J3;4 ¼ J4;5 ¼ 9:9 Hz, H-400), 5.58 (dd, 1H, J3;4 ¼ J4;5
¼
9:8 Hz, H-40), 5.49 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:9 Hz, H-4),
5.44 (dd, 1H, J1;2 1.3 Hz, J2;3 3.5 Hz, H-200), 5.37 (dd,
1H,J 1.4 Hz, J 17.2 Hz, CH2–CH@CH2), 5.29 (d, 1H,
J1;2 1.3 Hz, H-100), 5.27 (dd, 1H, J 1.4 Hz, J 10.4 Hz,
CH2–CH@CH2), 5.03 (d, 1H, J1;2 1.3 Hz, H-10), 5.02 (d,
1H, J1;2 1.3 Hz, H-1), 4.45 (dd, 1H, J2;3 3.2 Hz, J3;4
9.8 Hz, H-30), 4.35 (dd, 1H, J1;2 1.3 Hz, J2;3 3.2 Hz, H-2),
4.35–4.26 (m, 4H, 2H-20, H-500, 1CH2CH@CH2), 4.15–
4.07 (m, 3H, H-50, H-5, 1CH2CH@CH2), 1.39 (d, 3H,
J5;6 6.3 Hz, H-600), 1.34 (d, 3H, J5;6 6.2 Hz, H-60), 1.26 (d,
3H, J5;6 6.2 Hz, H-6). 13C NMR (100 MHz, CDCl3): d
165.7, 165.6, 165.5, 165.5, 165.1, 164.6 (6C, 6PhCO),
133.4–128.1 (PhCO, –CH2–CH@CH2), 117.8 (1C, –
CH2–CH@CH2), 101.5, 98.9, 97.7 (3C, 3C-1), 77.2, 76.0,
76.5, 72.9, 71.8, 71.7, 71.1, 70.4, 69.3, 68.3, 67.6, 67.5,
66.8 (13C, C-2–5, –CH2–CH@CH2), 17.6, 17.5, 17.4
(3C, 3C-6). Anal. Calcd for C63H60O19: C, 67.49; H,
5.39. Found: C, 67.42; H, 5.46.
3.14. Allyl a-
anosyluronic acid-(1 fi 2)]-a-
a- -rhamnopyranoside, ammonium salt (23)
L
-rhamnopyranosyl-(1 fi 3)-[b-
D-glucopyr-
-rhamnopyranosyl-(1 fi 2)-
L
L
Compound 22 (142 mg, 0.10 mmol) was dissolved in a
satd ammonia–MeOH (10 mL). After 2 weeks at rt, the
reaction mixture was concentrated, and the residue was
purified on a BioGel P2 column with MeOH–water as
the eluent to afford 23 (64 mg, 95%) as an amorphous
25
D
1
solid. ½aŠ +180.3 (c 1.0, H2O); H NMR (400 MHz,
D2O): d 5.94 (m, 1H, OCH2CH@CH2), 5.34 (dd, 1H, J
1.4, J 17.2 Hz, CH2–CH@CH2), 5.29 (dd, 1H, J 1.4 Hz,
J 10.2 Hz, CH2–CH@CH2), 5.22 (d, 1H, J1;2 1.3 Hz, H-
1), 5.04 (d, 1H, J1;2 1.3 Hz, H-1), 4.91 (d, 1H, J1;2 1.3 Hz,
H-1), 4.63 (d, 1H, J1;2 7.6 Hz, H-1), 4.23–3.37 (m, 18H,
H-2–5, 2CH2CH@CH2), 1. 32 (d, 3H, J5;6 6.3 Hz, H-6),
1.29 (d, 3H, J5;6 6.2 Hz, H-6), 1.26 (d, 3H, J5;6 6.2 Hz, H-
6). 13C NMR (100 MHz, D2O): d 172.9 (C, COONH4þ),
134.2 (1C, –CH2–CH@CH2), 118.4 (1C, –CH2–
CH@CH2), 103.9, 102.0, 100.7, 96.9 (4C, 4C-1), 79.2,
78.2, 76.2, 74.7, 74.6, 74.5, 72.5, 71.9, 71.6, 71.1, 69.8,
69.7, 69.1, 68.6, 67.9 (18C, C-2–5, C-6, –CH2–
CH@CH2), 16.5, 16.3, 16.1 (3C, 3C-6). MALDI-TOF
MS: Calcd for the ammonium salt of 23, C27 H47 NO19:
689.6 [M]. Found: 689.3; 694.3 (M)NH4þ+Naþ).
3.13. Allyl 2,3,4-tri-O-benzoyl-a-
(1 fi 3)-[methyl 2,3,4-tri-O-acetyl-b-
uronate-(1 fi 2)]-4-O-benzoyl-a- -rhamnopyranosyl-
(1 fi 2)-3,4-di-O-benzoyl-a- -rhamnopyranoside (22)
L
-rhamnopyranosyl-
D
-glucopyranosyl-
L
L
Donor 21 (109 mg, 0.23 mmol) and acceptor 20 (214 mg,
0.19 mmol) were coupled in the presence of a catalytic
amount of TMSOTf (3 lL, 0.02 mmol) under the same
conditions as described for the coupling of 2 with 1.
Purification by a chromatography with 2:1 petroleum
Acknowledgements
ether–EtOAc as the eluent gave tetrasaccharide 22
25
D
(169 mg, 62%) as a foamy solid. ½aŠ +134.6 (c 1.0,
This work was supported by The Chinese Academy of
Sciences (KZCX3-J-08) and by The National Natural
Science Foundation of China (Projects 30070185 and
39970864).
1
CHCl3); H NMR (400 MHz, CDCl3): d 8.08–7.23 (m,
30H, Bz-H), 5.95 (m, 1H, OCH2CH@CH2), 5.81 (dd,
1H, J2;3 3.2 Hz, J3;4 10.0 Hz, H-3), 5.67 (dd, 1H, J2;3
3.2 Hz, J3;4 10.0 Hz, H-3), 5.64 (dd, 1H, J3;4 ¼ J4;5
¼
10.0 Hz, H-4), 5.57 (dd, 1H, J3;4 ¼ J4;5 ¼ 10:0 Hz, H-4),
5.45–5.23 (m, 7H, H-1, H-2, H-3, 2H-4, 2CH2–
CH@CH2), 5.21 (d, 1H, J1;2 1.3 Hz, H-1), 5.15 (d, 1H,
J1;2 8.0 Hz, J2;3 9.6 Hz, H-2), 5.06 (d, 1H, J1;2 8.0 Hz, H-
1), 5.02 (d, 1H, J1;2 1.3 Hz, H-1), 4.55 (d, 1H, J2;3 3.2 Hz,
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