A concise synthesis of two isomeric pentasaccharides, the O repeat units from the lipopolysaccharides of P. syringae pv. porri NCPPB 3364T and NCPPB 3365

Article abstract of DOI:10.1016/j.carres.2003.09.014

A concise synthesis of two isomeric pentasaccharides, α-L-Rhap- (1→2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3) -[β-D-GlcpNAc-(1→2)]-α-L-Rhap (A) and α-L-Rhap-(1→2)- α-L-Rhap-(1→3)-[β-D-GlcpNAc-(1→2)]-α-L-Rhap- (1→3)-α-L-Rhap (B), the O repeats from the lipopolysacch

Full text of DOI:10.1016/j.carres.2003.09.014

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 43–49
A concise synthesis of two isomeric pentasaccharides, the O repeat
units from the lipopolysaccharides of P. syringae pv. porri
NCPPB 3364T and NCPPB 3365
Zuchao Ma, Jianjun Zhang and Fanzuo Kong*
Research Center for Eco-Environmental Sciences, Academia Sinica, Chinese Academy of Sciences, P.O. Box 2871,
Beijing 100085, China
Received 3 September 2003; accepted 10 September 2003
Abstract—A concise synthesis of two isomeric pentasaccharides, a-
GlcpNAc-(1 fi 2)]-a- -Rhap (A) and a- -Rhap-(1 fi 2)-a- -Rhap-(1 fi 3)-[b-
the O repeats from the lipopolysaccharides of Pseudonomonas syringae pv. porri NCPPB 3364T and 3365was achieved via assembly
of the building blocks, allyl 3,4-di-O-benzoyl-a- -rhamnopyranoside (1), 2,3,4-tri-O-benzoyl-a- -rhamnopyranosyl trichloroacet-
imidate (2), allyl 4-O-benzoyl-3-O-chloroacetyl-a- -rhamnopyranoside (6), 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b- -gluco-
pyranosyl trichloroacetimidate (7), and allyl 2,4-di-O-benzoyl-a- -rhamnopyranoside (10). Coupling of 1 with 2 followed by
L
-Rhap-(1 fi 2)-a-
L
-Rhap-(1 fi 3)-a-
L
-Rhap-(1 fi 3)-[b-
D-
L
L
L
D
-GlcpNAc-(1 fi 2)]-a-
L
-Rhap-(1 fi 3)-a-
L
-Rhap (B),
L
L
L
D
L
deallylation and trichloroacetimidate formation gave the disaccharide donor 5, while condensation of 6 with 7, followed by de-
chloroacetylation, offered the disaccharide acceptor 9. Then, 5 was coupled with 10 to obtain the trisaccharide 11, and subsequent
deallylation and trichloroacetimidate formation furnished the trisaccharide donor 13. Coupling of 9 with 13, followed by depro-
tection, afforded pentasaccharide 19, while condensation of 9 with 5, followed by deallylation and trichloroacetimidate formation,
gave the tetrasaccharide donor 16, whose coupling with 10 and subsequent deprotection yielded another pentasaccharide 22.
Ó 2003 Elsevier Ltd. All rights reserved.
Keywords: Oligosaccharide; Rhamnose; Glucosamine
1. Introduction
and in the site of attachment of GlcNAc, were found in
the two strains, O repeat A being the major in strain
NCPPB 3364T and B in strain NCPPB 3365:3
The lipopolysaccharides (LPS) of strains of Pseudono-
monas syringae, a phytopathogenic bacterium, was re-
ported as the causative agent of the bacterial blight of
leek (Allium porrum).1;2 The O polysaccharides (OPS)
obtained from the LPS of P. syringae pv. porri NCPPB
3364T and 3365possess multiple oligosaccharide O re-
peats, some of which are linear and composed of
→2)-α-
L
-Rhap-(1→2)-α-
L
-Rhap-(1→3)-α-
L
-Rhap-(1→3)-α-
L
-Rhap(1→ (A)
2
↑
1
β-
D
-GlcpNAc
→3)-α-
L
-Rhap-(1→2)-α-
L
-Rhap-(1→3)-α-
L
-Rhap-(1→3)-α-
L
-Rhap(1→ (B)
L
-rhamnose in the main chain and GlcNAc in the side
chains. Both branched O repeats, which differ in the
position of substitution of one of the rhamnose residues
2
↑
1
β-
D
-GlcpNAc
Synthetic samples of rhamnan structures with GlcNAc
side chains would be very valuable in research on plant
pathology and in the design of immunodiagnostic
* Corresponding author. Tel.: +86-10-62936613; fax: +86-10-629235-
63; e-mail: fzkong@mail.rcees.ac.cn
0008-6215/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2003.09.014

44
Z. Ma et al. / Carbohydrate Research 339 (2004) 43–49
reagents. The syntheses of several similar structures
occurring in the cell-wall polysaccharide with alternate
(1 fi 2)- and (1 fi 3)-linked rhamnan backbone and 3-O-
GlcNAc side chains on the fi2)-rhamnose residues have
been reported by PintoÕs group.4–7 A general and con-
vergent method for the synthesis of (1 fi 2)- and (1 fi 3)-
linked rhamnans with arbitrary sugar side chains on the
3-OH of the rhamnose residues has been reported by our
group.8 We present herein a facile synthesis of two iso-
meric pentasaccharides consisting of (1 fi 2)- and
(1 fi 3)-linked rhamnan backbone with 2-O-GlcNAc
side chain on the rhamnose residues.
was achieved. In terms of simplicity and efficiency, this
method can be used for construction of higher oligo-
saccharides with similar structures.
3. Experimental
3.1. General methods
Optical rotations were determined at 25 °C with a Per-
kin–Elmer Model 241-Mc automatic polarimeter. 1H
NMR and 13C NMR spectra were recorded with Bruker
ARX 400 spectrometers (400 MHz for 1H, 100 MHz
for 13C) at 25 °C for solutions in CDCl3 or D2O as
indicated. Mass spectra were recorded with a VG
PLATFORM mass spectrometer using the ESI mode.
Thin-layer chromatography (TLC) was performed on
silica gel HF254 plates with detection by charring
with 30% (v/v) H2SO4 in MeOH or in some cases by
a UV lamp. Column chromatography was conducted
by elution of a column (16 · 240 mm, 18 · 300 mm,
35 · 400 mm) of silica gel (100–200 mesh) with EtOAc–
petroleum ether (60–90 °C) as the eluent. Solutions were
concentrated at <60 °C under reduced pressure.
2. Results and discussion
As outlined in Scheme 1, condensation of allyl 3,4-di-O-
-rhamnopyranoside8 (1) with 2,3,4-tri-O-
-rhamnopyranosyl trichloroacetimidate8 (2)
benzoyl-a-
L
benzoyl-a-
L
in the presence of a catalytic amount of TMSOTf gave
allyl 2,3,4-tri-O-benzoyl-a- -rhamnopyranosyl-(1 fi 2)-
3,4-di-O-benzoyl-a- -rhamnopyranoside (3) in satisfac-
L
L
tory yield (84%). Deallylation of 3 with PdCl2 in meth-
anol,9 followed by trichloroacetimidate formation10 with
trichloroacetonitrile in the presence of DBU, produced
the disaccharide donor 5 (67% for two steps). Coupling
3.2. General procedure for the glycosylations
of allyl 4-O-benzoyl-3-O-chloroacetyl-a-
noside (6)8 with 3,4,6-tri-O-acetyl-2-deoxy-2-phthal-
imido-b- -glucopyranosyl trichloroacetimidate (7)
furnished disaccharide 8. Subsequent dechloroacetyl-
ation with thiourea gave a co-used disaccharide acceptor
9 (75%) for further couplings. Glycosylation of the
acceptor allyl 2,4-di-O-benzoyl-a-L
-rhamnopyranoside8
L-rhamnopyra-
A mixture of donor and acceptor was dried together
under high vacuum for 2 h, then dissolved in anhyd
CH2Cl2. TMSOTf (0.05equiv) was added dropwise at
)20 °C with nitrogen protection. The reaction mixture
was stirred for 3 h, during which time the temperature
was gradually raised to ambient temperature. Then the
mixture was neutralized with Et3N. Concentration of
the reaction mixture, followed by purification on a silica
gel column, gave the desired products.
D
(10) with donor 5 afforded trisaccharide 11 in high yield
(79%), which was activated by deallylation and trichloro-
acetimidate formation to furnish the trisaccharide donor
13 (72%, for two steps). Condensation of donor 5 and
acceptor 9 gave tetrasaccharide 14 in acceptable yield
(63%), and consecutive deallylation and trichloro-
acetimidate formation offered tetrasaccharide donor 16
(67%, for two steps). Condensation of the disaccharide
acceptor 9 with the trisaccharide donor 13 yielded pen-
tasaccharide 17 (60%), while coupling of the monosac-
charide acceptor 10 with the tetrasaccharide donor 16
furnished another pentasaccharide 20 (73%).
Hydrazinolysis to remove the phthalimido group from
17 or 20 was carried out in 10% hydrazine hydrate–EtOH
under reflux, and it was accompanied by reduction of the
allyl group and debenzoylation.8 Subsequent acetylation
of the resultant product with acetic anhydride in pyridine
readily gave acetylated pentasaccharide 18 or 21. Finally
deacetylation of 18 or 21 in ammonia–methanol gave the
target pentasaccharide 19 and 22.
3.3. Allyl 2,3,4-tri-O-benzoyl-a-
L-rhamnopyranosyl-
(1 fi 2)-3,4-di-O-benzoyl-a- -rhamnopyranoside (3)
L
As described in the general procedure, 1 (0.82 g,
1.98 mmol) and 2 (1.49 g, 2.40 mmol) were coupled, and
the product was purified by silica gel column chroma-
tography with 4:1 petroleum ether–EtOAc as the eluent
to give 3 (1.46 g, 84%) as a foamy solid: ½aŠD +80.2 (c 1.0,
1
CHCl3); H NMR (400 MHz, CDCl3): d 8.03–7.25(m,
25H, 5Ph), 6.02–5.85 (m, 2H, H-30, CH2–CH@CH2),
5.87 (dd, 1H, J1;2 1.5Hz, J2;3 3.3 Hz, H-20), 5.83 (dd, 1H,
J2;3 3.2 Hz, J3;4 10.0 Hz, H-3), 5.67 (dd, 1H, J3;4
¼
J4;5 ¼ 10:0 Hz, H-40), 5.66 (dd, 1H, J3;4 ¼ J4;5 ¼ 10:0 Hz,
H-4), 5.42–5.37 (m, 1H, CH2–CH@CHtrans), 5.30–5.27
(m, 1H, CH2–CH@CHcis), 5.15 (d, 1H, J1;2 1.5Hz, H-1 0),
5.06 (d, 1H, J1;2 1.5Hz, H-1), 4.37–4.29 (m, 2H, H-2,
CH2–CH@CH2), 4.18–4.12 (m, 2H, H-5, H-50), 1.41,
1.35(2d, 6H, J5;6 6.3 Hz, 2CH3). Anal. calcd for
C50H46O14: C, 68.96; H, 5.32. Found: C, 68.79; H, 5.29.
In summary, a concise synthesis of two branched
pentasaccharides, consisting of a-(1 fi 2)- and (1 fi 3)-
linked rhamnan backbone with 2-branched D-GlcpNAc

Z. Ma et al. / Carbohydrate Research 339 (2004) 43–49
45
Scheme 1. Reagents and conditions: (a) TMSOTf (0.01 equiv), CH2Cl2, )20–0 °C, 2–4 h; 84% for 3, 71% for 8, 79% for 11, 63% for 14, 60% for 17,
and 73% for 20, respectively; (b) PdCl2, MeOH, rt, 2 h; 78%, 83%, 80% for 4, 12, and 15, respectively; (c) Cl3CN, DBU, CH2Cl2, rt, 3 h; 86%, 86%,
84% for 5, 13, and 16, respectively; (d) thiourea in EtOH–CH2Cl2 (1:4), reflux, 16 h; 75%; (e) i: EtOH–10% hydrazine hydrate, reflux, 48 h; ii: Ac2O–
pyridine (dry), rt, 12 h; 83% for 18, 80% for 21; (f) satd NH3–MeOH, rt, 72 h; 89% for 19, 88% for 22.
3.4. 2,3,4-Tri-O-benzoyl-a-
3,4-di-O-benzoyl-a-
acetimidate (5)
L
-rhamnopyranosyl-(1 fi 2)-
L-rhamnopyranosyl trichloro-
and the resultant residue was purified by flash chroma-
tography (3:1 petroleum ether–EtOAc) to give 4
(864 mg, 78%) as a white foam. A mixture of 4 (851 mg,
1.02 mmol), trichloroacetonitrile (204 lL, 2.04 mmol)
and 1,8-diazabicyclo[5.4.0]-undecene (DBU) (55 lL) in
dry CH2Cl2 (10 mL) was stirred under nitrogen for 3 h
and then concentrated. The residue was purified by flash
chromatography (4:1 petroleum ether–EtOAc) to give 5
To a solution of 3 (1.16 g, 1.33 mmol) in anhyd MeOH
(13 mL) was added PdCl2 (30 mg). After stirring the
mixture at rt for 2 h, TLC (2:1 petroleum ether–EtOAc)
indicated that the reaction was complete. The mixture
was filtered, the solution was concentrated to dryness,
(856 mg, 86%) as a foamy solid: ½aŠ +160.5( c 1.0,
D

46
Z. Ma et al. / Carbohydrate Research 339 (2004) 43–49
1
CHCl3); H NMR (400 MHz, CDCl3): d 8.75(s, 1H,
CNHCCl3), 8.04–7.26 (m, 25H, 5Ph), 6.50 (d, 1H, J1;2
1.7 Hz, H-1), 5.97 (dd, 1H, J2;3 3.4 Hz, J3;4 10.1 Hz,
(m, 2H, H-50, H-60a), 4.02–3.85 (m, 5H, H-2, H-3, H-5,
CH2–CH@CH2), 2.12, 2.04, 1.90 (3s, 3H, 3CH3CO), 1.17
(d, 3H, J5;6 6.2 Hz, H-6). Anal. Calcd for C36H39NO15: C,
59.58; H, 5.42. Found: C, 59.48; H, 5.40.
H-30), 5.87–5.84 (m, 2H, H-20, H-3), 5.78 (dd, 1H, J3;4
¼
J4;5 ¼ 9:8 Hz, H-40), 5.70 (dd, 1H, J3;4 ¼ J4;5 ¼ 10:0 Hz,
H-4), 5.22 (dd, 1H, J1;2 1.3 Hz, H-10), 4.58 (m, 1H, H-2),
4.43–4.34 (m, 2H, H-5, H-50), 1.46 (d, 3H, J5;6 6.2 Hz,
H-6), 1.40 (d, 3H, J5;6 6.2 Hz, H-6). Anal. Calcd for
C49H42Cl3NO14: C, 60.35; H, 4.34. Found: C, 60.08;
H, 4.46.
3.7. Allyl 2,3,4-tri-O-benzoyl-a-L-rhamnopyranosyl-
(1 fi 2)-3,4-di-O-benzoyl-a-
(1 fi 3)-2,4-di-O-benzoyl-a-
L
-rhamnopyranosyl-
-rhamnopyranoside (11)
L
Donor 5 (355 mg, 0.36 mmol) was coupled with acceptor
10 (150 mg, 0.36 mmol) as described in the general pro-
cedure, and the product was purified by chromato-
graphy with 3:1 petroleum ether–EtOAc as the eluent to
3.5. Allyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-
b-
D
-glucopyranosyl-(1 fi 2)-4-O-benzoyl-3-O-
-rhamnopyranoside (8)
chloroacetyl-a-
L
give 11 (352 mg, 79%) as a foamy solid: ½aŠ +140.5
D
1
(c 1.0, CHCl3); H NMR (400 MHz, CDCl3): d 8.24–
Donor 7 (878 mg, 1.5mmol) was coupled with acceptor
6 (486 mg, 1.26 mmol) as described in the general pro-
cedure, and the product was purified by chromato-
graphy with 3:1 petroleum ether–EtOAc as the eluent to
7.24 (m, 35H, 7Ph), 6.03–5.96 (m, 1H, CH2–CH@CH2),
5.81 (dd, 1H, J2;3 3.4 Hz, J3;4 10.2 Hz, H-300), 5.63 (dd,
1H, J1;2 1.7 Hz, J2;3 3.4 Hz, H-200), 5.57–5.47 (m, 5H, H-2,
H-30, H-4, H-40, H-400), 5.38 (m, 1H, CH2–CH@CHtrans),
5.30 (m, 1H, CH2–CH@CHcis), 5.24 (d, 1H, J1;2 1.7 Hz,
H-100), 5.08 (d, 1H, J1;2 1.6 Hz, H-10), 4.66 (d, 1H, J1;2
1.6 Hz, H-1), 4.49 (dd, 010 H, J2;3 3.6 Hz, J3;4 9.7 Hz, H-3),
4.30–4.25(m, 1H, H-5 ), 4.14–4.08 (m, 4H, H-5, H-50,
CH2–CH@CH2), 3.97 (dd, 1H, J1;2 1.6 Hz, J2;3 2.8 Hz,
H-20), 1.33, 1.26, 1.08 (3d, 9H, J5;6 6.2 Hz, Rhap H-6);
13C NMR (100 MHz, CDCl3), d 166.4, 166.0, 165.7,
165.5, 165.4, 165.3, 165.0 (PhCO), 133.7–128.4 (PhCO,
–CH2–CH@CH2), 118.2 (–CH2–CH@CH2), 100.7, 99.2,
96.8 (C-1), 76.0, 75.3, 73.8, 72.6, 71.7, 70.7, 70.5, 69.8,
68.8, 68.2, 67.8, 67.6, 66.9 (C-2–5, –CH2–CH@CH2),
17.8–17.6 (Rhap C-6). Anal. Calcd for C70H64O20:
C, 68.62; H, 5.26. Found: C, 68.83; H, 5.37.
give 8 (714 mg, 71%) as a foamy solid: ½aŠ )5.6 (c 1.0,
D
1
CHCl3); H NMR (400 MHz, CDCl3): d 7.88–7.27 (m,
9H, Ph, Pth), 5.94–5.83 (m, 2H, H-30, CH2–CH@CH2),
5.42–5.37 (m, 2H, H-10, H-3), 5.34–5.29 (m, 1H, CH2–
CH@CHtrans), 5.23–5.20 (m, 1H, CH2–CH@CHcis), 5.16
(dd, 1H, J3;4 ¼ J4;5 ¼ 9:5Hz, H-4), 4.95 (d, 1H, J1;2
2.0 Hz, H-1), 4.90 (dd, 1H, J3;4 ¼ J4;5 ¼ 10:0 Hz, H-40),
4.45(dd, 1H, J1;2 8.1 Hz, J2;3 10.8 Hz, H-20), 4.29 (dd,
1H, J5;6b 4.9 Hz, J6a;6b 12.3 Hz, H-60b), 4.22–4.17 (m, 2H,
H-5, H-60a), 4.08 (dd, 1H, J1;2 2.0 Hz, J2;3 2.7 Hz, H-2),
4.06–4.07 (m, 1H, H-50), 3.88, 3.52 (ABq, 2H, J 14.9 Hz,
CH2ClCO), 3.88–3.83 (m, 2H, CH2–CH@CH2), 2.12,
2.04, 1.89 (3s, 3H, 3CH3CO), 1.14 (d, 3H, J5;6 6.2 Hz,
H-6). Anal. Calcd for C38H40ClNO16: C, 56.90; H, 5.03.
Found: C, 57.12; H, 5.09.
3.8. 2,3,4-Tri-O-benzoyl-a-
L
-rhamnopyranosyl-(1 fi 2)-
3.6. Allyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-
L
3,4-di-O-benzoyl-a-
L
-rhamnopyranosyl-(1 fi 3)-
-rhamnopyranosyl
b-
rhamnopyranoside (9)
D
-glucopyranosyl-(1 fi 2)-4-O-benzoyl-a-
-
2,4-di-O-benzoyl-a-
L
trichloroacetimidate (13)
To a solution of 8 (695g, 0.87 mmol) in EtOH (5mL)–
CH2Cl2 (20 mL) was added thiourea (0.33 g), and the
mixture was refluxed for 16 h, at the end of which time
TLC (1:1 petroleum ether–EtOAc) indicated that the
reaction was complete. The mixture was concentrated.
The residue was passed through a silica gel column with
2:1 petroleum ether–EtOAc as the eluent to give 9
Compound 11 (340 mg, 0.8 mmol) was deallylated and
subsequently trichloroacetimidated under the same
conditions as those used for the preparation of 5 from 3,
giving 13 (265mg, 72% for two steps) as a foamy solid:
½aŠ +126.3 (c 1.0, CHCl3); 1H NMR (400 MHz,
D
CDCl3): d 8.83 (s, 1H, CNHCCl3), 6.38 (d, 1H, J1;2
1.7 Hz, H-1), 5.82 (dd, 1H, J2;3 3.4 Hz, J3;4 ¼ 10:1 Hz,
H-300), 5.76 (dd, 1H, J1;2 1.8 Hz, J2;3 3.4 Hz, H-200), 5.66–
5.48 (m, 5H, H-2, H-30, H-4, H-40, H-400), 5.26 (d, 1H,
J1;2 1.8 Hz, H-100), 4.75(d, 1H, J1;2 1.6 Hz, H-10), 4.59
(dd, 1H, J2;3 3.4 Hz, J3;4 9.7 Hz, H-3), 4.31–4.26 (m, 1H,
H-500), 4.22–4.18 (m, 1H, H-50), 4.07–3.99 (m, 2H, H-20,
H-5), 1.37, 1.26, 0.96 (3d, 9H, J5;6 6.2 Hz, Rhap H-6).
Anal. Calcd for C69H60Cl3NO20: C, 62.33; H, 4.55.
Found: C, 62.56; H, 4.64.
(473 mg, 75%) as a foamy solid: ½aŠ )36.2 (c 1.0,
D
1
CHCl3); H NMR (400 MHz, CDCl3): d 7.84–7.27 (m,
9H, Ph, Pth), 5.91–5.86 (m, 2H, H-30, CH2–CH@CH2),
5.56 (d, 1H, J1;2 8.5Hz, H-1 0), 5.34–5.29 (m, 1H, CH2–
CH@CHtrans), 5.32–5.15 (m, 3H, H-4, CH2–CH@CHcis),
4.95(d, 1H, J1;2 1.1 Hz, H-1), 4.65(dd, 1H, J3;4 ¼ J4;5
¼
9:7 Hz, H-40), 4.46 (dd, 1H, J1;2 8.5Hz, J2;3 10.7 Hz, H-20),
4.31 (dd, 1H, J5;6b 4.9 Hz, J6a;6b 12.2 Hz, H-60b), 4.21–4.15

Z. Ma et al. / Carbohydrate Research 339 (2004) 43–49
47
3.9. Allyl 2,3,4-tri-O-benzoyl-a-
(1 fi 2)-3,4-di-O-benzoyl-a- -rhamnopyranosyl-
(1 fi 3)-[3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-
b- -glucopyranosyl-(1 fi 2)]-4-O-benzoyl-a-
rhamnopyranoside (14)
L
-rhamnopyranosyl-
(3s, 3H, 3CH3CO), 1.40, 1.04, 0.70 (d, 9H, J5;6 6.2 Hz,
Rhap H-6). Anal. Calcd for C82H75Cl3N2O28: C, 59.95;
H, 4.60. Found: C, 59.77; H, 4.49.
L
D
L-
3.11. Allyl 2,3,4-tri-O-benzoyl-a-
(1 fi 2)-3,4-di-O-benzoyl-a- -rhamnopyranosyl-(1 fi 3)-
2,4-di-O-benzoyl-a- -rhamnopyranosyl-(1 fi 3)-[3,4,6-tri-
O-acetyl-2-deoxy-2-phthalimido-b- -glucopyranosyl-
(1 fi 2)]-4-O-benzoyl-a- -rhamnopyranoside (17)
L-rhamnopyranosyl-
L
Compound 5 (472 mg, 0.48 mmol) and 9 (293 mg,
0.37 mmol) were coupled under the same conditions as
described in the general procedure. Purification of the
product by chromatography with 1:1 petroleum ether–
EtOAc as the eluent afforded 14 (392 g, 63%) as a foamy
L
D
L
As described in the general procedure, 9 (164 mg,
0.23 mmol) and 13 (250 mg, 0.19 mmol) were coupled,
and the product was purified by silica gel column
chromatography with 1:1 petroleum ether–EtOAc as the
1
solid: ½aŠ +76.4 (c 1.0, CHCl3); H NMR (400 MHz,
D
CDCl3): d 8.09–7.17 (m, 34H, 6Ph, Pth), 6.14 (dd, 1H,
J2;3 10.7 Hz, J3;4 9.4 Hz, Glcp H-30), 6.01–5.86 (m, 4H,
Glcp H-1, 2Rhap H-3, CH2–CH@CH2), 5.82 (dd, 1H,
eluent to give 17 (214 g, 60%) as a foamy solid: ½aŠ
D
J1;2 1.3 Hz, J2;3 3.3 Hz, Rhap H-200), 5.58 (dd, 1H, J3;4
J4;5 ¼ 10:0 Hz, Rhap H-40), 5.49 (dd, 1H, J3;4 ¼ J4;5
¼
+64.6 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
8.25–7.21 (m, 44H, 8Ph, Pth), 6.02 (dd, 1H, J2;3 10.6 Hz,
J3;4 9.1 Hz, Glcp H-30), 5.95–5.87 (m, 4H, Glcp H-10,
Rhap H-3000, H-300, CH2–CH@CH2), 5.77 (dd, 1H, J1;2
1.7 Hz, J2;3 3.2 Hz, Rhap H-200), 5.58–5.44 (m, 4H), 5.32–
5.27 (m, 1H, CH2–CH@CHtrans), 5.21–5.18 (m, 1H,
CH2–CH@CHcis), 5.10 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:4 Hz,
Rhap H-4), 5.08 (d, 1H, J1;2 1.3 Hz, Rhap H-1), 5.01 (dd,
1H, J1;2 1.8 Hz, J2;3 3.2 Hz, Rhap H-20), 4.44 (dd, 1H, J1;2
8.3 Hz, J2;3 10.6 Hz, Glcp H-30), 4.36–3.69 (m, 12H, Glcp
H-5, H-6, Rhap 2H-2, H-3, 4H-5, CH2–CH@CH2), 1.98,
1.94, 1.28 (3s, 3H, 3CH3CO), 1.38, 1.21, 1.07, 0.74
(d, 12H, J5;6 6.2 Hz, Rhap H-6); 13C NMR (100 MHz,
CDCl3): d 170.5, 169.5, 169.4 (CH3CO), 165.6, 165.3,
165.2, 165.1, 165.0, 164.9, 164.8, 163.7 (PthCO, PhCO),
133.7–128.0 (Ph, –CH2–CH@CH2), 117.3 (–CH2–
CH@CH2), 100.3, 100.2, 98.8, 98.4, 97.6 (C-1), 75.7,
75.4, 73.5, 72.4, 72.2, 71.8, 71.6, 71.0, 70.4, 69.9, 69.6,
69.5, 68.2, 67.8, 67.7, 67.4, 67.2, 62.1, 54.9 (C-2–6,
–CH2–CH@CH2), 20.8, 20.6, 20.5( CH3CO), 17.9, 17.7,
17.6, 17.5(Rha p C-6). Anal. Calcd for C103H97NO34:
C, 65.36; H, 5.17. Found: C, 65.55; H, 5.20.
¼
9:9 Hz, Rhap H-4), 5.36–5.32 (m, 1H, CH2–
CH@CHtrans), 5.28 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:7 Hz, Rhap
H-4), 5.25–5.22 (dq, 1H, CH2–CH@CHcis), 5.04 (d, 1H,
J1;2 1.3 Hz, Rhap H-1), 4.95(d, 2H, J1;2 1.4 Hz, 2Rhap
H-1), 4.87 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:4 Hz, Glcp H-40),
4.51–3.71 (m, 12H, Glcp H-2, H-5, H-6, Rhap 2H-2,
H-3, 3H-5, CH2–CH@CH2), 2.11, 2.10, 1.90 (3s, 3H,
3CH3CO), 1.48, 1.01, 0.77 (d, 9H, J5;6 6.2 Hz, Rhap
H-6); 13C NMR (100 MHz, CDCl3): d 170.8, 170.0,
169.8 (CH3CO), 167.9 (PthCO), 165.9, 165.7, 165.3,
165.2, 165.0, 164.5 (PhCO), 134.1–128.2 (Ph, –CH2–
CH@CH2), 117.1 (–CH2–CH@CH2), 99.6, 99.2, 98.4,
98.3 (C-1), 76.6, 74.2, 72.8, 72.6, 71.9, 70.5, 70.4, 69.8,
69.6, 68.5, 68.3, 68.1, 67.5, 67.3, 62.2, 61.7, 54.9, 52.2
(C-2–6, –CH2–CH@CH2), 20.8–20.7 (CH3CO), 17.8,
17.6 17.5(Rha p C-6). Anal. Calcd for C83H79NO28:
C, 64.80; H, 5.18. Found: C, 65.05; H, 5.23.
3.10. 2,3,4-Tri-O-benzoyl-a-
(1 fi 2)-3,4-di-O-benzoyl-a-
(1 fi 3)-[3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-
b- -glucopyranosyl-(1 fi 2)]-4-O-benzoyl-a-
rhamnopyranosyl trichloroacetimidate (16)
L
-rhamnopyranosyl-
L
-rhamnopyranosyl-
D
L
-
3.12. Propyl 2,3,4-tri-O-acetyl-a-
(1 fi 2)-3,4-di-O-acetyl-a- -rhamnopyranosyl-(1 fi 3)-
2,4-di-O-acetyl-a- -rhamnopyranosyl-(1 fi 3)-[3,4,6-tri-
O-acetyl-2-acetamido-2-deoxy-b- -glucopyranosyl-
(1 fi 2)]-4-O-acetyl-a- -rhamnopyranoside (18)
L-rhamnopyranosyl-
L
L
Compound 14 (305mg, 0.8 mmol) was deallylated and
subsequently trichloroacetimidated under the same
conditions as those used for the preparation of 5 from 3,
giving 16 (218 mg, 67% for two steps) as a foamy solid:
D
L
Pentasaccharide 17 (166 mg, 0.09 mmol) was dissolved in
EtOH (20 mL) to which was added 100% hydrazine
hydrate (4 mL), and the solution was refluxed for 48 h.
The solution was then concentrated, and the residue was
co-evaporated several times with toluene. The residue
was dissolved in pyridine (5mL) to which was added
Ac2O (3 mL). The solution was stirred for 12 h at rt and
then evaporated to dryness. Purification of the residue
by column chromatography (EtOAc) gave 18 (95mg,
½aŠ +84.6 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
D
d 8.73 (s, 1H, CNHCCl3), 8.02–7.25(m, 34H, 6 Ph, Pth),
6.37 (d, 1H, Rhap H-1), 6.08 (dd, 1H, J2;3 10.7 Hz, J3;4
9.0 Hz, Glcp H-30), 6.02 (d, 1H, J1;2 8.4 Hz, Glcp H-10),
5.87–5.79 (m, 3H, Rhap H-200, 2H-3), 5.56 (dd, 1H,
J3;4 ¼ J4;5 ¼ 9:9 Hz, Rhap H-400), 5.45 (dd, 1H, J3;4
¼
J4;5 ¼ 9:9 Hz, Rhap H-40), 5.29 (dd, 1H, J3;4 ¼ J4;5
¼
9:6 Hz, Rhap H-4), 5.05 (d, 1H, J1;2 1.3 Hz, Rhap H-100),
5.00 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:8 Hz, Glcp H-4), 4.95(d, 2H,
J1;2 1.3 Hz, 2Rhap H-10), 4.51–3.93 (m, 10H, Glcp H-20,
H-5, Rhap 2H-2, H-3, 3H-5, Glcp H-6), 2.07, 2.06, 1.87
83% for two steps) as a foamy solid: ½aŠ )21.5( c 1.0,
D
CHCl3); 1H NMR (400 MHz, CDCl3): d 6.14 (d, 1H, J1;2
8.0 Hz, NHAc), 5.56 (dd, 1H, J2;3 ¼ J3;4 ¼ 9:4 Hz, Glcp

48
Z. Ma et al. / Carbohydrate Research 339 (2004) 43–49
1
H-30), 5.34 (dd, 1H, J2;3 3.5Hz, J3;4 10.0 Hz, Rhap H-3000),
5.30–5.28 (m, 2H, Rhap H-2000, H-300), 5.22 (d, 1H, J1;2
1.6 Hz, Rhap H-1000), 5.11–4.92 (m, 7H), 4.84 (d, 1H, J1;2
2.5Hz, Rha p H-1000), 4.80 (d, 1H, J1;2 1.6 Hz, Rhap H-10),
4.74 (d, 1H, J1;2 1.8 Hz, Rhap H-1), 4.31 (dd, 1H, J2;3
3.4 Hz, J3;4 8.5Hz, Rha p H-3), 4.25(dd, 1H, J5;6b 4.9 Hz,
J6a;6b 12.2 Hz, Glcp H-6b), 4.11 (dd, 1H, J5;6a 2.5Hz,
J6a;6b 12.2 Hz, Glcp H-6a), 4.05–3.37 (m, 9H), 2.18, 2.17,
2.16, 2.15, 2.13, 2.07, 2.06, 2.04, 2.03, 2.00, 1.99, 1.98
(12s, 36H, 12CH3CO), 1.59 (m, 2H, OCH2CH2CH3),
1.27, 1.23, 1.16, 1.15(d, 12H, J5;6 6.2 Hz, Rhap H-6),
0.92 (t, 3H, J 7.4 Hz, OCH2CH2CH3); 13C NMR
(100 MHz, CDCl3), d 171.7, 171.3, 170.7, 170.6, 170.5,
+104.6 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3):
8.13–7.25(m, 44H, 8 Ph, Pth), 6.02–5.90 (m, 2H, Glcp
H-300, CH2–CH@CH2), 5.81 (dd, 1H, J2;3 3.6 Hz, J2;3
10.2 Hz, Rhap H-3000), 5.78–5.73 (m, 2H, Rhap H-2000,
H-300), 5.65 (d, 1H, J1;2 8.4 Hz, Glcp H-100), 5.49 (dd, 1H,
J3;4 ¼ J4;5 ¼ 9:8 Hz, Rhap H-4000), 5.46 (dd, 1H,
J3;4 ¼ J4;5 ¼ 10:2 Hz, Rhap H-400), 5.41 (dd, 1H,
J3;4 ¼ J4;5 ¼ 9:9 Hz, Rhap H-40), 5.40–5.25 (m, 2H, Rhap
H-20, CH2–CH@CHtrans), 5.28–5.25 (m, 1H, CH2–
CH@CHcis), 5.07 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:5Hz, Rha p
H-4), 5.04 (d, 1H, J1;2 1.3 Hz, Rhap H-1000), 5.02 (d, 1H,
J1;2 1.5Hz, Rha p H-100), 4.85(d, 1H, J1;2 1.3 Hz, Rhap
H-10), 4.79 (d, 1H, J1;2 1.2 Hz, Rhap H-1), 4.68 (dd, 1H,
J2;3 ¼ J3;4 ¼ 9:8 Hz, Glcp H-400), 4.39 (dd, 1H, J2;3
3.4 Hz, J3;4 9.8 Hz, Rhap H-3), 4.29 (dd, 1H, J1;2 8.4 Hz,
J2;3 10.8 Hz, Glcp H-200), 4.26–3.53 (m, 12H), 2.06, 2.03,
1.83 (3s, 3H, 3CH3CO), 1.28, 1.26, 0.82, 0.69 (d, 12H,
J5;6 6.2 Hz, Rhap H-6); 13C NMR (100 MHz, CDCl3),
d 170.5, 169.6, 169.5 (CH3CO), 167.5(Pth CO), 165.8,
165.6, 165.4, 165.1, 165.0, 164.9, 164.8, 164.0 (PhCO),
133.9–128.0 (Ph, –CH2–CH@CH2), 117.8 (–CH2–
CH@CH2), 100.9, 98.8, 98.7, 98.0, 96.2 (C-1), 76.2, 76.1,
73.6, 72.9, 72.7, 72.3, 71.6, 71.1, 70.2, 70.1, 70.0, 69.3,
68.9, 68.4, 68.1, 67.7, 67.1, 66.4, 61.4, 54.4 (C-2–6,
–CH2–CH@CH2), 20.6, 20.5, 20.3 (CH3CO), 17.4, 17.3,
17.2, 17.0 (Rhap C-6). Anal. Calcd for C103H97NO34:
C, 65.36; H, 5.17. Found: C, 65.63; H, 5.13.
170.4, 170.2, 170.1, 170.0, 169.8, 169.7, 169.5(CH CO),
3
99.5, 99.4, 99.3, 99.1, 99.0 (C-1), 75.7, 74.1, 73.0, 72.6,
71.8, 71.5, 71.2, 70.1, 70.9, 70.3, 69.9, 69.4, 68.8, 67.3,
67.7, 67.0, 66.9, 62.2, 55.8 (C-2–6, OCH2CH2CH3), 24.9,
23.1, 22.7, 21.9, 20.9, 20.8, 20.8, 20.7, 20.7, 20.6, 20.6,
20.6 (CH3CO), 17.6, 17.3, 17.2, 17.2 (Rhap C-6), 10.5
(OCH2CH2CH3). Anal. Calcd for C57H83NO33: C, 52.25;
H, 6.38. Found: C, 52.04; H, 6.50.
3.13. Propyl a-
rhamnopyranosyl-(1 fi 3)-a-
(1 fi 3)-[2-acetamido-2-deoxy-b-
(1 fi 2)]-a- -rhamnopyranoside (19)
L
-rhamnopyranosyl-(1 fi 2)-a-
-rhamnopyranosyl-
-glucopyranosyl-
L-
L
D
L
Pentasaccharide 18 (84 mg, 0.06 mmol) was dissolved in
satd NH3–MeOH (20 mL). After 96 h at rt, the reaction
mixture was concentrated, and the residue was purified
by chromatography on Sephadex LH-20 (MeOH) to
3.15. Propyl 2,3,4-tri-O-acetyl-a-
(1 fi 2)-3,4-di-O-acetyl-a- -rhamnopyranosyl-(1 fi 3)-
[3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-b- -glucopyr-
anosyl-(1 fi 2)]-4-O-acetyl-a- -rhamnopyranosyl-
(1 fi 3)-2,4-di-O-acetyl-a- -rhamnopyranoside (21)
L-rhamnopyranosyl-
L
D
L
afford 19 (48 mg, 89%) as a foamy solid: ½aŠ )17.6
D
L
1
(c 1.0, H2O); H NMR (400 MHz, D2O): d 5.21 (s, 1H,
Rhap H-1000), 5.00 (s, 1H, Rhap H-100), 4.98 (d, 1H, J1;2
1.5Hz, Rha p H-10), 4.95(s, 1H, Rha p H-1), 4.58 (d, 1H,
J1;2 8.3 Hz, Glcp H-10), 1.99 (s, 3H, CH3CONH), 1.60
(m, 2H, OCH2CH2CH3), 1.33–1.27 (m, 12H, Rhap H-6),
0.92 (t, 3H, J 7.4 Hz, OCH2CH2CH3); 13C NMR
(100 MHz, D2O): d 173.0 (CH3CONH), 102.9, 102.1,
101.8, 100.5, 98.6 (C-1), 55.5 (OCH2CH2CH3), 16.5,
16.4, 16.3, 16.2 (Rhap C-6), 9.6 (OCH2CH2CH3). Anal.
Calcd for C35H61NO22: C, 49.58; H, 7.25. Found: C,
49.69; H, 7.24.
Pentasaccharide 20 (164 mg, 0.09 mmol) was dissolved in
EtOH (20 mL) to which was added 100% hydrazine
hydrate (4 mL), and the solution was refluxed for 48 h.
The solution was then concentrated, and the residue was
co-evaporated several times with toluene. The residue
was taken up in pyridine (5mL) to which was added
Ac2O (3 mL). The solution was stirred for 12 h at rt and
then evaporated to dryness. Purification of the residue
by column chromatography (EtOAc) gave 21 (91 mg,
80% for two steps) as a foamy solid: ½aŠ )36.6 (c 1.0,
D
CHCl3); 1H NMR (400 MHz, CDCl3): d 6.46 (d, 1H, J1;2
9.8 Hz, NHAc), 5.30 (dd, 1H, J2;3 3.5Hz, J3;4 10.8 Hz,
Rhap H-3), 5.29 (d, 1H, J1;2 1.4 Hz, Rhap H-1000), 5.22
(dd, 1H, J2;3 3.0 Hz, J3;4 10.5Hz, Rha p H-3), 5.16 (dd,
1H, J1;2 1.4 Hz, J2;3 3.5Hz, Rha p H-2), 5.09–5.01
(m, 5H), 4.93 (d, 1H, J1;2 1.6 Hz, Rhap H-100), 4.89
(d, 1H, J1;2 8.5Hz, Glc p H-100), 4.77 (d, 1H, J1;2 1.2 Hz,
Rhap H-10), 4.69 (d, 1H, J1;2 1.3 Hz, Rhap H-1), 2.17,
2.15, 2.15, 2.10, 2.09, 2.09, 2.07, 2.06, 2.05, 2.02, 2.02,
1.97 (12s, 36H, 12CH3CO), 1.60 (m, 2H, OCH2-
CH2CH3), 1.32, 1.21, 1.19, 1.14 (d, 12H, J5;6 6.2 Hz,
Rhap H-6), 0.94 (t, 3H, J 7.4 Hz, OCH2CH2CH3);
3.14. Allyl 2,3,4-tri-O-benzoyl-a-
(1 fi 2)-3,4-di-O-benzoyl-a- -rhamnopyranosyl-(1 fi 3)-
[3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b- -glucopyr-
anosyl-(1 fi 2)]-4-O-benzoyl-a- -rhamnopyranosyl-
(1 fi 3)-2,4-di-O-benzoyl-a- -rhamnopyranoside (20)
L-rhamnopyranosyl-
L
D
L
L
Donor 16 (206 mg, 0.13 mmol) was coupled with ac-
ceptor 10 (103 mg, 0.25mmol) as described in the gen-
eral procedure, and the product was purified by
chromatography with 1:1 petroleum ether–EtOAc as the
eluent to give 20 (173 mg, 73%) as a foamy solid: ½aŠ
D

Z. Ma et al. / Carbohydrate Research 339 (2004) 43–49
49
13C NMR (100 MHz, CDCl3), d 171.4, 171.0, 170.7,
170.4, 170.3, 170.1, 170.0, 170.0, 169.9, 169.8, 169.4,
169.3 (CH3CO), 101.7, 99.9, 99.7, 99.4, 97.3 (C-1), 75.2,
74.9, 73.6, 73.4, 72.5, 72.4, 71.9, 71.6, 71.4, 70.8, 69.8,
69.8, 69.6, 69.0, 68.6, 67.8, 67.4, 67.3, 66.2, 62.2, 55.9
(C-2–6, OCH2CH2CH3), 17.8, 17.3, 17.2, 17.1 (Rhap
C-6), 10.6 (OCH2CH2CH3). Anal. Calcd for C57H83-
NO33: C, 52.25; H, 6.38. Found: C, 52.14; H, 6.43.
(Rhap C-6), 9.7 (OCH2CH2CH3). Anal. Calcd for
C35H61NO22: C, 49.58; H, 7.25. Found: C, 49.72; H,
7.28.
Acknowledgements
This work was supported by The National Natural
Science Foundation of China (Projects 39970864 and
30070815).
3.16. Propyl a-
rhamnopyranosyl-(1 fi 3)-[2-acetamido-2-deoxy-
b- -glucopyranosyl-(1 fi 2)]-a- -rhamnopyranosyl-
(1 fi 3)-a- -rhamnopyranoside (22)
L
-rhamnopyranosyl-(1 fi 2)-a-
L-
References
D
L
1. Keoike, S. T.; Barak, J. D.; Gilbertson, R. L. Plant Dis.
1999, 83, 165–170.
2. Samson, R.; Shafik, H.; Benjama, A.; Gardan, L. Phyto-
pathology 1998, 88, 844–850.
3. Zdorovenko, E. L.; Zatonskii, G. V.; Kocharova, N. A.;
Shashkov, A. S.; Knirel, Y. A.; Ovod, V. V. Eur. J.
Biochem. 2003, 270, 20–27.
4. Reimer, K. B.; Harris, S. L.; Varma, V.; Pinto, B. M.
Carbohydr. Res. 1992, 228, 399–414.
5. Marino-Albernas, J.; Harris, S. L.; Varma, V.; Pinto, B.
M. Carbohydr. Res. 1993, 245, 245–257.
6. Auzanneau, F.-I.; Forooghian, F.; Pinto, B. M. Carbo-
hydr. Res. 1996, 291, 21–24.
7. Hoog, C.; Rotondo, A.; Johnston, B. D.; Pinto, B. M.
Carbohydr. Res. 2002, 337, 2023–2036.
8. Zhang, J.; Kong, F. Tetrahedron 2003, 59, 1429–1441.
9. Ogawa, T.; Yamamoto, H. Carbohydr. Res. 1985, 137, 79.
10. Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem.
Biochem. 1994, 50, 21–125.
L
Pentasaccharide 21 (88 mg, 0.07 mmol) was dissolved in
satd NH3–MeOH (20 mL). After 96 h at rt, the reaction
mixture was concentrated, and the residue was purified
by chromatography on Sephadex LH-20 (MeOH) to
afford 22 (50 mg, 88%) as a foamy solid: ½aŠ )42.1
D
(c 1.0, H2O); 1H NMR (D2O): d 5.25 (s, 1H, Rhap
H-1000), 5.24 (s, 1H, Rhap H-100), 5.06 (s, 1H, Rhap H-10),
4.98 (s, 1H, Rhap H-1), 4.55 (d, 1H, J1;2 8.5Hz, Glc p
H-100), 1.99 (s, 3H, CH3CONH), 1.61 (m, 2H,
OCH2CH2CH3), 1.33–1.24 (m, 12H, Rhap H-6), 0.92
(t, 3H, J 7.4 Hz, OCH2CH2CH3); 13C NMR (100 MHz,
D2O): d 173.2 (CH3CONH), 102.7, 101.9, 101.1, 100.3,
99.3 (C-1), 55.5 (OCH2CH2CH3), 16.5, 16.4, 16.3, 16.2