Synthesis of 3-O-arabinosylated (1→6)-β-D-galactan epitopes

Article abstract of DOI:10.1081/CAR-100104865

Two tetrameric arabinogalactans,β-D-galactopyranosyl-(1→6)-β-D-galactopyranosyl- (1→6)-[α-L-arabinofuranosyl-(1→3)]-D-galactopyranose (14) and α-L-arabinofuranosyl-(1→3)-β-D-galactopyranosyl-(1→6)-β-D-galactopyranosyl-(1→6)-D-galactopyranose (25), which are good candidates for CCRC-M7 epitope characterization, were synthesized efficiently using a convergent strategy. Migration of an acceptor acetyl group proved to be an obstacle to synthesis, but regioselective glycosylation or 4-O-benzyl protection of the acceptor circumvented this problem allowing efficient synthesis of the 1→6 linked target compounds.

Full text of DOI:10.1081/CAR-100104865

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SYNTHESIS OF 3-O-ARABINOSYLATED (1→6)-β-D-
GALACTAN EPITOPES
Qingfeng Pan a , Yuguo Du a , Fanzuo Kong* a , Jingqi Pan b & Mujian Lü b
a Research Center for Eco-Environmental Sciences, Academia Sinica , P.O.Box 2871,
Beijing, 100085, P. R. China
b College of Chemistry and Molecular Engineering, Peking University , Beijing, 100871, P.
R. China
Published online: 16 Aug 2006.
To cite this article: Qingfeng Pan , Yuguo Du , Fanzuo Kong* , Jingqi Pan & Mujian Lü (2001) SYNTHESIS OF 3-O-
ARABINOSYLATED (1→6)-β-D-GALACTAN EPITOPES, Journal of Carbohydrate Chemistry, 20:3-4, 297-306, DOI: 10.1081/
CAR-100104865
To link to this article: http://dx.doi.org/10.1081/CAR-100104865
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J. CARBOHYDRATE CHEMISTRY, 20(3&4), 297–306 (2001)
SYNTHESIS OF 3-O-ARABINOSYLATED
(1→6)-ꢀ-D-GALACTAN EPITOPES
Qingfeng Pan,1 Yuguo Du,1 Fanzuo Kong,1,* Jingqi Pan,2 and
Mujian Lü2
1Research Center for Eco-Environmental Sciences, Academia Sinica,
P.O.Box 2871, Beijing 100085, P. R. China
2College of Chemistry and Molecular Engineering, Peking University,
Beijing 100871, P. R. China
ABSTRACT
Two tetrameric arabinogalactans, ꢀ-D-galactopyranosyl-(1→6)-ꢀ-D-galac-
topyranosyl-(1→6)-[ꢁ-L-arabinofuranosyl-(1→3)]-D-galactopyranose (14)
and ꢁ-L-arabinofuranosyl-(1→3)-ꢀ-D-galactopyranosyl-(1→6)-ꢀ-D-galac-
topyranosyl-(1→6)-D-galactopyranose (25), which are good candidates for
CCRC-M7 epitope characterization, were synthesized efficiently using a con-
vergent strategy. Migration of an acceptor acetyl group proved to be an obsta-
cle to synthesis, but regioselective glycosylation or 4-O-benzyl protection of
the acceptor circumvented this problem allowing efficient synthesis of the
1→6 linked target compounds.
INTRODUCTION
As part of an ongoing research effort on arabinogalactan-protein (AGP) re-
lated epitopes, we have synthesized dodecyl ꢀ-D-galactopyranosyl-(1→6)-ꢀ-D-
galactopyranosyl-(1→6)-[ꢁ-L-arabinofuranosyl-(1→2)]-ꢀ-D-galactopyranoside.1
The synthesis of a 2-O-arabinofuranosylated tetrameric arabinogalactan modeled
on 1,2-anhydrosugars has been reported by van Boom’s group.2 The epitope of this
arabinogalactan recognized by the CCRC-M7 antibody consists of a ꢀ-(1→6)-
linked galactan containing at least three D-galactopyranosyl residues functional-
ized at one hydroxyl group with an ꢁ-(1→3)-linked L-arabinofuranosyl unit corre-
sponding to a proposal of Albersheim.3 Although structure and activity
297
Copyright © 2001 by Marcel Dekker, Inc.
www.dekker.com

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PAN ET AL.
relationship studies have shown the importance of the arabinofuranose side chain
and its potential pharmacological activity,4 we do not yet have a complete struc-
tural knowledge of these AGP epitopes.5 We present here the assembly of two
tetrameric arabinogalactans, which represent the minimal AGP epitope units. We
believe these will prove extremely useful for antibody screening, immunogen de-
velopment and inhibitor construction.6
RESULTS AND DISCUSSION
2,3,5-Tri-O-benzoyl-ꢁ-L-arabinofuranosyl-(1→3)-6-O-triphenylmethyl-1,2-
O-ethylidene-ꢁ-D-galactopyranose (4) was prepared by regioselective coupling of
6-O-triphenylmethyl-1,2-O-ethylidene-ꢁ-D-galactopyranose (2) with O-(2,3,5-tri-
O-benzoyl-ꢁ-L-arabinofuranosyl) trichloroacetimidate (3) under standard glycosy-
lation conditions. Acetylation of 4 with acetic anhydride in pyridine gave 5 for
which the chemical shift of H-4 was found to be at ꢂ 5.64 ppm, thus confirming the
1→3 linkage in 4. Detritylation of 5 using ferric chloride hexahydrate in
dichloromethane7 was complicated by partial acetyl migration which occurred un-
der these conditions. Nevertheless, the desired 4-O-acetylated product 6 was ob-
tained in 48% yield. The reaction was followed by TLC. The acetyl migrated (6-
O-acetylated) byproduct 7 was found to be formed from the very beginning of the
reaction process and the amount of 7 continued to increase with reaction time. Gly-
cosylation of 6 with disaccharide donor 2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyra-
nosyl-(1→6)-2,3,4-tri-O-acetyl-ꢁ-D-galactopyranosyl trichloroacetimidate (10)1
in CH2Cl2 using TMSOTf as the catalyst furnished 2,3,4,6-tetra-O-acetyl-ꢀ-D-
galactopyranosyl-(1→6)-2,3,4-tri-O-acetyl-ꢀ-D-galactopyranosyl-(1→6)-[2,3,5-
tri-O-benzoyl-ꢁ-L-arabinofuranosyl-(1→3)]-4-O-acetyl-1,2-O-ethylidene-ꢁ-D-
galactopyranose (11) in 51% yield. Alternatively, removal of the triphenylmethyl
ether group of 4 gave the 4,6-diol 8 in high yield. Regioselective glycosylation of
8 with 10 provided the tetrasaccharide 12 in 87% yield. Acetylation of 12 resulted
in a product with the same NMR spectrum as 11 confirming the structural assign-
ment of 12. Trifluoroacetic acid promoted hydrolysis of the 1,2-O-ethylidene
group in 12 followed by acetylation with acetic anhydride in pyridine gave the fully
acylated tetrasaccharide 13. Treatment of 13 with a catalytic amount of NaOMe in
methanol provided the target compound 14 in good yield (70% from 12).
A similar synthetic strategy was also applied to the construction of the AG
derivative 25. Thus, 8 was acetylated, and then deacetalated using 90% tri-
fluroacetic acid to give 15. Acetylation of 15 with acetic anhydride in pyridine
(→ 16) followed by regioselective deacetylation of the anomeric carbon using ben-
zylamine (→ 17),8 and activation with trichloroacetonitrile using 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU) furnished 2,3,5-tri-O-benzoyl-ꢁ-L-arabinofura-
nosyl-(1→3)-2,4,6-tri-O-acetyl-ꢁ-D-galactopyranosyl trichloroacetimidate (18) in
good yield. In order to prevent undesired acetyl migration, 4-O-benzylation was
carried out on the acceptor. Therefore compound 191 was treated with methanolic
sodium methoxide (→20), regioselectively silylated with TBDPSCl in pyridine

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299
(→21) and benzylated with benzyl bromide and sodium hydride in DMF furnish-
ing 22 in 77.6% yield (from 19). Tetra-n-butylammonium fluoride (TBAF)-medi-
ated desilylation of 22 and condensation of the primary hydroxyl in 23 with imi-
date 18 led to the tetrameric derivative 24 in 85% yield. Treatment of 24 with 95%
TFA to remove the ethylidene group, acetylation with acetic anhydride in pyridine,
hydrogenolysis using H2 and Pd/C in MeOH/EtOAc, and finally deprotection by
Zemplén deacylation, provided the free tetrasaccharide 25 in a total yield of 49%
(from 24).
EXPERIMENTAL
General Methods. Optical rotations were determined at 25°C with a
Perkin-Elmer Model 241-Mc automatic polarimeter. 1H NMR, 13C NMR and 1H-
1H COSY spectra were recorded with ARX 400 spectrometers for solutions in
CDCl3, CD3OD and D2O. Chemical shifts are given in ppm downfield from inter-
nal Me4Si. Mass spectra were recorded with a VG PLATFORM mass spectrome-
ter using ESI technique to introduce the sample. Thin-layer chromatography (TLC)
was performed on silica gel HF254 with detection by charring with 30% (v/v)
H2SO4 in MeOH or in some cases by a UV detector. Column chromatography was
conducted by elution of a column (10ꢃ240 mm, 18ꢃ300 mm, 35ꢃ400 mm) of sil-
ica gel (100–200 mesh) with EtOAc - petroleum ether (60–90°C) as the eluent. So-
lutions were concentrated at ꢄ 60°C under diminished pressure. The (R and S) con-
figuration assignment for the ethylidene group in oligosaccharides was based on
Kochetkov’s report.9 Pure R or S isomers were obtained by column chromatogra-
phy. However, the isomers underwent isomerization during long term storage.
There was no difference in the reactivity for the two isomers.
2,3,5-Tri-O-benzoyl-ꢁ-L-arabinofuranosyl-(1→3)-6-O-triphenylmethyl-
1,2-O-ethylidene-ꢁ-D-galactopyranose (4). To a solution of 19 (3.4 g, 16.5
mmol) in pyridine (10 mL) was added TrCl (5.51 g, 19.8 mmol). The mixture was
stirred at rt overnight, then poured into cold water, extracted with CH2Cl2 (2 ꢃ 30
mL), and the organic layer was dried over Na2SO4, and concentrated. Column
chromatography (2/1 petroleum ether-ethyl acetate) of the residue gave 2 as a
syrup (R,S-mixture, 4.46 g, 61%). To a solution of 2 (2.11 g, 4.71 mmol) and 3
(2.806 g, 4.63 mmol) in anhydrous CH2Cl2 (25 mL) was added trimethylsilyl tri-
flate (30 ꢅL, 0.17 mmol) in the presence of 4 Å molecular sieves under a nitrogen
atmosphere at ꢆ40°C. The mixture was stirred at rt for 2 h, at the end of which time
TLC (2/1 petroleum ether-ethyl acetate) indicated that all starting material 3 was
consumed. The reaction mixture was neutralized with triethylamine, then filtered
and the filtrate was concentrated. Purification of the product by column chro-
matography (2:1 petroleum ether-ethyl acetate) gave syrupy 4 (R,S-mixture, 2.60
g, 63%); For R-isomer, [ꢁ]D25 ꢆ7° (c 2.0, CHCl3); 1H NMR (CDCl3, 400 MHz):
ꢂ 1.46 (d, 3 H, CH3CH), 3.00 (br t, 1 H, J 7.0 Hz, H-6a), 3.34 (dd, 1 H, J5,6a 5.8,
J
6a,6b 7.0 Hz, H-6b), 4.03–4.05 (m, 2 H, H-2, H-4), 4.30 (br t, 1 H, J 7.0 Hz, H-5),

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PAN ET AL.
4.73 (dd, 1 H, J 3.6, 11.6 Hz, H-5ꢇa), 4.86–4.92 (m, 2 H, H-4ꢇ, H-5ꢇb), 5.18 (q, 1
H, J 4.9 Hz, CH3CH), 5.45 (d, 1 H, J 3.6 Hz, H-1), 5.56 (s, 1 H, H-2ꢇ/H-1ꢇ), 5.58
(d, 1 H, H-3ꢇ), 5.60 (s, 1 H, H-1ꢇ/H-2ꢇ), 7.21–8.12 (m, 30 H, Ph).
Anal. Calcd for C53H48O13: C, 71.30; H, 5.38. Found C, 71.46; H, 5.19 (R,S
mixture).
2,3,5-Tri-O-benzoyl-ꢁ-L-arabinofuranosyl-(1→3)-4-O-acetyl-6-O-triph-
enylmethyl-1,2-O-ethylidene-ꢁ-D-galactopyranose (5). Compound 4 (2.54 g,
2.85 mmol) was acetylated with acetic anhydride (2 mL) in pyridine (5 mL) under
standard conditions to give 5 (2.53 g, 95%) as a syrup; For R-isomer, [ꢁ]D25 ꢈ3°
(c 0.6, CHCl3); 1H NMR (CDCl3, 400 MHz): ꢂ 1.46 (d, 3 H, CH3CH), 1.71 (s, 3
H, CH3CO), 3.00 (dd, 1 H, J5,6a 8.0, J6a,6b 9.0 Hz, H-6a), 3.35 (dd, 1 H, J5,6b 7.0 Hz,
H-6b), 4.03–4.05 (m, 2 H, H-2, H-3), 4.28–4.32 (m, 1 H, H-5), 4.73 (dd, 1 H, J2,3
11.3, J3,4 3.2 Hz, H-5ꢇa), 4.86–4.94 (m, 2 H, H-5ꢇb, H-4ꢇ), 5.18 (q, 1 H, J 4.8 Hz,
CH3CH), 5.45 (d, 1 H, J 3.8 Hz, H-1), 5.54 (s, 1 H, H-1ꢇ), 5.60 (d, 1 H, J 1.6 Hz,
H-3ꢇ), 5.61 (s, 1 H, H-2ꢇ), 5.64 (br s, 1 H, H-4), 7.22–8.12 (m, 30 H, Ph).
Anal. Calcd for C55H50O14: C, 70.66; H, 5.35. Found C, 70.72; H, 5.40 (R,S
mixture).
2,3,5-Tri-O-benzoyl-ꢁ-L-arabinofuranosyl-(1→3)-4-O-acetyl-1,2-O-
ethylidene-ꢁ-D-galactopyranose (6) and 2,3,5-tri-O-benzoyl-ꢁ-L-arabinofura-
nosyl-(1→3)-6-O-acetyl-1,2-O-ethylidene-ꢁ-D-galactopyranose (7). To a so-
lution of 5 (934 mg, 1.0 mmol) in CH2Cl2 (20 mL) was added ferric chloride
hexahydrate (670 mg, 2.5 mmol). The mixture was stirred at rt for 4 h, at the end of
which time TLC (2/1 petroleum ether-ethyl acetate) showed the reaction was com-
plete. The mixture was poured into cold water and extracted with dichloromethane.
The organic phase was washed with saturated NaHCO3, then dried over Na2SO4 and
concentrated. Column chromatography (2/1 petroleum ether-ethyl acetate) of the
residue gave 6 (304 mg, 48%), 7 (101 mg, 16%), and recovered starting material 5
(300 mg). For S-isomer of 6, [ꢁ]D25 ꢈ18° (c 2.0, CHCl3); 1H NMR (CDCl3, 400
MHz): ꢂ 1.38 (d, 3 H, J 4.8 Hz, CH3CH), 2.00 (s, 3 H, CH3CO), 3.45 (dd, 1 H, J5,6a
4.4, J6a,6b 9 Hz, H-6a), 3.69 (dd, 1 H, J5,6b 6.0, J6a,6b 9.0 Hz, H-6b), 4.05–4.15 (m, 2
H, H-2, H-5), 4.38 (dd, 1 H, J 4.8, 7.6 Hz, H-3), 4.63–4.71 (m, 2 H, H-4, H-5ꢇa),
4.81–4.85 (m, 1 H, H-5ꢇb), 5.45 (d, 1 H, J 2.8 Hz, H-4), 5.49 (q, 1 H, CH3CH),
5.50–5.65 (m, 4 H, H-1, H-1ꢇ, 2ꢇ, 3ꢇ), 7.26–8.08 (m, 15 H, Ph).
Anal. Calcd for C36H36O14: C, 62.43; H, 5.20. Found: C, 62.31; H, 5.33.
For compound 7 (R:S ꢉ 1:2); 1H NMR (CDCl3, 400 MHz): ꢂ 1.36 (d, 2 H,
CH3CH), 1.45 (d, 1 H, CH3CH), 1.99 (s, 1 H, CH3CO), 2.07 (s, 2 H, CH3CO),
4.02–4.16 (m, 4 H, H-2,5,6a,6b), 4.30–4.39 (m, 2 H, H-3, H-4), 4.68–4.83 (m, 3 H,
H-4ꢇ, H-5ꢇa, H-5ꢇb), 5.14 (q, 0.33 H, H-1(S)), 5.60–5.68 (m, 3.67 H, H-1ꢇ,2ꢇ,3ꢇ, H-
1 (R)), 7.25–8.11 (m, 15 H, Ph). ESMS Calcd for C36H36O14 (692.2), Found ESI-
MS(ꢈ) 710.4 (MꢈNH4) ꢈ.
2,3,5-Tri-O-benzoyl-ꢁ-L-arabinofuranosyl-(1→3)-1,2-O-ethylidene-ꢁ-D-
galacto-pyranose (8). To a solution of 4 (2.54 g, 2.85 mmol) in CH2Cl2 (50 mL)
was added ferric chloride hexahydrate (1.68 g, 6.3 mmol) as described in the prepa-

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ration of 6 to give 8 as a syrup (R,S-mixture, 1.518 g, 82%); 1H NMR (CDCl3, 400
MHz): ꢂ 1.34 (d, 1.5 H, CH3CH), 1.45 (d, 1.5 H, CH3CH), 3.80–4.21 (m, 5.5 H, H-
2, H-4, H-5, H-6a, H-6b and H-3 (R or S)), 4.40 (br t, 0.5 H, J 6.2 Hz, H-3 (S or R)),
4.65–4.81 (m, 3 H, H-4ꢇ, H-5ꢇa, H-5ꢇb), 5.14 (q, 0.5 H, J 4.8 Hz, CH3CH (S)), 5.48
(q, 0.5 H, J 4.8 Hz, CH3CH (R)), 5.60–5.67 (m, 3 H, H-1ꢇ, H-2ꢇ, H-3ꢇ), 7.25–8.07
(m, 15 H, Ph). ESMS Calcd for: C34H34O13 (650.2). Found ESI-MS(ꢈ) 673.3
(MꢈNa) ꢈ.
2,3,4,6-Tetra-O-acetyl-ꢀ-D-galactopyranosyl-(1→6)-2,3,4-tri-O-acetyl-
ꢀ-D-galacto-pyranosyl-(1→6)-[2,3,5-tri-O-benzoyl-ꢁ-L-arabinofuranosyl-
(1→3)]-4-O-acetyl-1,2-O-ethylidene-ꢁ-D-galactopyranose (11). To a solution
of 6 (240 mg, 0.35 mmol) and 10 (271 mg, 0.35 mmol) in anhydrous CH2Cl2 (10
mL) was added trimethylsilyl triflate (11 ꢅL, 0.06 mmol) under a nitrogen atmo-
sphere at ꢆ15°C. The mixture was stirred under this condition for 2 h, at the end
of which time TLC (1/1 petroleum ether-ethyl acetate) indicated that the starting
material 10 was completely consumed. The reaction mixture was neutralized with
triethylamine, then filtered and the filtrate was concentrated. Purification of the
product by column chromatography (3:2 petroleum ether-ethyl acetate) gave 11 as
25
1
a syrup (234 mg, 51%); For S-isomer, [ꢁ]D ꢈ8° (c 0.8, CHCl3); H NMR
(CDCl3, 400 MHz): ꢂ 1.36 (d, 3 H, J 4.6 Hz, CH3CH), 1.93, 1.99, 2.01, 2.07, 2.11,
2.13 (6 s, 24 H, 8 CH3CO), 3.69–3.89 (m, 6 H), 4.01–4.15 (m, 4 H), 4.37 (m, 1 H),
4.54 (d, 1 H, J 9.0 Hz, H-1B), 4.58 (d, 1 H, J 8.5 Hz, H-1C), 4.68–4.71 (m, 2 H, H-
4D, H-5aD), 4.90 (dd, 1 H, J 4.2, 8.5 Hz, H-5bD), 4.85–5.00 (m, 4 H), 5.37 (br s, 2
H, H-4B, H-4C), 5.45 (q, 1 H, CH3CH), 5.53–5.60 (m, 5 H, H-4A,H-1D,2D,3D, H-
1A), 7.26–8.10 (m, 15 H, Ph). ESMS Calcd for C62H70O31 (1310); Found ESI-
MS(ꢈ) 1333 (MꢈNa) ꢈ.
2,3,4,6-Tetra-O-acetyl-ꢀ-D-galactopyranosyl-(1→6)-2,3,4-tri-O-acetyl-
ꢀ-D-galacto-pyranosyl-(1→6)-[2,3,5-tri-O-benzoyl-ꢁ-L-arabinofuranosyl-
(1→3)]-1,2-O-ethylidene-ꢁ-D-galactopyranose (12). To a solution of 8 (680
mg, 1.05 mmol) and 10 (871 mg, 1.11 mmol) in anhydrous CH2Cl2 (15 mL) was
added trimethylsilyl triflate (20 ꢅL, 0.11 mmol) in the presence of 4 Å molecular
sieves under a nitrogen atmosphere at ꢆ40°C. The mixture was stirred under this
condition for 2 h, at the end of which time TLC (1/1 petroleum ether-ethyl acetate)
indicated that the starting material 10 was completely consumed. The reaction mix-
ture was neutralized with triethylamine, then filtered and the filtrate was concen-
trated. Purification of the product by column chromatography (3:2 petroleum
ether-ethyl acetate) gave 12 as a syrup (1.158 g, 87%); For S-isomer, [ꢁ]D25 ꢈ22°
(c 4.8, CHCl3); 1H NMR (CDCl3, 400 MHz): ꢂ 1.36 (d, 3 H, J 4.6 Hz, CH3CH),
1.97, 1.98, 2.04, 2.07, 2.11, 2.13, 2.14 (7 s, 21 H, 7 CH3CO), 3.66–3.90 (m, 6 H),
4.01–4.15 (m, 5 H), 4.32 (br t, 1 H, J 6.6 Hz), 4.49 (d, 1 H, J 9.1 Hz, H-1B), 4.58
(d, 1 H, J 8.5 Hz, H-1C), 4.68–4.71 (m, 2 H, H-4D, H-5aD), 4.90 (dd, 1 H, J 3.6, 9.2
Hz, H-5bD), 4.85–5.00 (m, 4 H), 5.37 (br s, 2 H, H-4B, H-4C), 5.45 (q, 1 H,
CH3CH), 5.53–5.60 (m, 4 H, H-1D,2D,3D, H-1A), 7.26–8.10 (m, 15 H, Ph). ESMS
Calcd for C60H68O30 (1268.4); Found ESI-MS(ꢈ) 1291.2 (MꢈNa)ꢈ.
Anal. Calcd for C60H68O30: C, 56.78; H, 5.36. Found : C, 56.59; H, 5.44.

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ꢀ-D-Galactopyranosyl-(1→6)-ꢀ-D-galactopyranosyl-(1→6)-[ꢁ-L-arabi-
nofuranosyl-(1→3)]-D-galactopyranose (14). Compound 12 (340 mg, 0.27
mmol) was dissolved in aqueous 90% TFA (5 mL) and the mixture was stirred at
rt. The reaction was monitored by TLC (2/3 petroleum ether-ethyl acetate) until all
of the starting material was consumed (about 40 min). The mixture was diluted
with toluene and then concentrated to dryness. After acetylation of the residue with
acetic anhydride (2 mL) in pyridine (5 mL), the solution was co-evaporated with
toluene. Purification of the products on column chromatography (2/3 petroleum
ether-ethyl acetate) gave 13 as a syrup which was subsequently treated with
NaOMe in MeOH (keep pH at 9) at rt for 24 h, then neutralized with Dowex-50
(Hꢈ) resin and, concentrated. The product was purified by Sephadex LH-20 col-
umn chromatography using methanol as the eluent to afford 14 (120 mg, 70% from
12); [ꢁ]D25 ꢆ48° (c 0.9, H2O); 13C NMR (D2O, 100 MHz): ꢂ 62.3 (C-6C), 63.1 (C-
5D), 69.4, 69.5, 69.9, 71.5, 71.8, 72.6, 73.4, 73.8, 73.9, 74.0, 74.5, 74.8, 76.6, 78.2,
79.2, 82.1, 85.8, 103.0, 104.5, 104.7 (C-1A/C-1B/C-1C), 111.2 (C-1D). ESMS
Calcd for C23H40O20 (636.2). Found ESI-MS(ꢈ): 659.1 (MꢈNa) ꢈ.
Anal. Calcd for C23H40O20: C, 43.40; H, 6.29. Found: C, 43.25; H, 6.40.
2,3,5-Tri-O-benzoyl-ꢁ-L-arabinofuranosyl-(1→3)-4,6-di-O-acetyl-1,2-O-
ethylidene-ꢁ-D-galactopyranose (9). Compound 8 (1.345 g, 2.07 mmol) was
acetylated with acetic anhydride (3 mL) in pyridine (5 mL) under standard condi-
tions to give 9 (1.49 g, 98%) as a syrup; For R-isomer, [ꢁ]D25 ꢈ3° (c 0.5, CHCl3);
1H NMR (CDCl3, 400 MHz): ꢂ 1.42 (d, 3 H, J 4.8 Hz, CH3CH), 1.96, 2.05 (2 s, 6
H, 2 CH3CO), 4.08–4.15 (m, 3 H, H-2, 2 H-6), 4.26–4.30 (m, 1 H, H-5), 4.34 (dd,
1 H, J2,3 7.1, J3,4 4.5 Hz, H-3), 4.68–4.76 (m, 2 H, H-4ꢇ, H-5ꢇa), 4.89 (dd, 1 H, J4ꢇ,5ꢇb
2.7, J5ꢇa,5ꢇb 11.6 Hz, H-5ꢇb), 5.56 (d, 1 H, J1,2 4.7 Hz, H-1), 5.57 (s, 1 H, H-1ꢇ/H-
2ꢇ), 5.61–5.63 (d, 2 H, J3ꢇ,4ꢇ 4.6 Hz, H-3ꢇ, H-2ꢇ/H-1ꢇ), 7.27–8.11 (m, 15 H, Ph).
Anal. Calcd for C38H38O15: C, 62.12; H, 5.18. Found: C, 62.31; H, 5.15.
2,3,5-Tri-O-benzoyl-ꢁ-L-arabinofuranosyl-(1→3)-2,4,6-tri-O-acetyl-ꢁ-
D-galactopyra-nosyl 2, 2, 2-trichloroacetimidate (18). Compound 9 (2.2 g, 3
mmol) was dissolved in aqueous 90% trifluoroacetic acid (10 mL). The solution
was stirred at rt for 3 h and then co-evaporated with toluene to dryness under re-
duced pressure. The syrup generated above was fully acetylated with acetic anhy-
dride (4 mL) in pyridine (8 mL) for 4 h at room temperature, then co-evaporated
with toluene (2 ꢃ 20 mL) to dryness. Column chromatography using 2/1 petroleum
ether-ethyl acetate as the eluent gave syrupy 16 quantitatively. To a solution of 16
in THF (20 mL) was added benzylamine (3.4 mL, 31 mmol) under ice-water bath
conditions. The solution was stirred at rt for 10 h, at the end of which time TLC
(1/1 petroleum ether-ethyl acetate) indicated that the reaction was complete. The
mixture was poured into cold water and extracted with CH2Cl2. The organic phase
was washed with 5% HCl (2 ꢃ 100 mL) and water (100 mL), then dried over
Na2SO4 and concentrated. Column chromatography (1/1 petroleum ether-ethyl ac-
etate) of the residue gave 17 as a syrup (1.69 g, 75%). To a solution of 17 (2.47 g,
3.3 mmol) in anhydrous CH2Cl2 (20 mL) were added trichloroacetonitrile (1.5 mL,

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303
15 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene(1,5-5) (DBU, 0.2 mL), and the
mixture was stirred at room temperature for 2 h. TLC (2/1 petroleum ether-ethyl
acetate) indicated that the reaction was complete. The mixture was concentrated
and purified on a silica gel column using 2:1 petroleum ether-ethyl acetate as elu-
ent to give 18 as a syrup (2.20 g, 74.8%); [ꢁ]D25 ꢆ40° (c 1.0, CHCl3); 1H NMR
(CDCl3, 400MHz): ꢂ 1.90, 2.00, 2.12 (3 s, 9 H, 3 CH3CO), 4.04 (dd, 1 H, J5,6a 6.8,
J
6a,6b 10.1 Hz, H-6a), 4.10–4.20 (m, 2 H, H-3, H-6b), 4.32–4.42 (m, 1 H, H-5), 4.76
(dd, 1 H, J 3.6, J 11.9 Hz, H-5ꢇa), 4.80–4.90 (m, 2 H, H-4, H-5ꢇb), 4.90 (dd, 1 H,
2,3 12.0 Hz, H-2), 5.40, 5.42 (2 s, 2 H, H-1ꢇ, H-2ꢇ), 5.58 (d, 1 H, J 4.2 Hz, H-3ꢇ),
J
5.64 (d, 1 H, J 2.2 Hz, H-4), 6.60 (d, 1 H, J1,2 3.1 Hz, H-1), 7.23–8.14 (m, 15 H,
Ph), 8.65 (s,1 H,NH).
Anal. Calcd for C40H38Cl3NO16: C, 53.66; H, 4.25. Found: C, 53.52; H, 4.41.
6-O-tert-Butyldiphenylsilyl-2,3,4-tri-O-benzyl-ꢀ-D-galactopyranosyl-
(1→6)-1,2:3,4-di-O-isopropylidene-ꢁ-D-galactopyranose (22). Compound 19
(4.5 g, 7.56 mmol) was treated with NaOMe in MeOH (keep pH at 9) at rt for 24
h, then neutralized with Dowex-50 (Hꢈ) resin and concentrated. The crude prod-
uct was dissolved in pyridine (20 mL) and tert-butyldiphenylsilyl chloride (2.5 g,
9 mmol) and 4-dimethylaminopyridine (30 mg) were added to the solution. The
mixture was stirred at rt for 16 h, then concentrated under reduced pressure. Flash
column chromatography (2/3 petroleum ether-ethyl acetate) of the residue gave
crude 21 as a syrup. To a cooled solution of 21 in DMF (12 mL) was added NaH
(2.17 g, 50%, 45 mmol) and BnBr (2.8 mL, 24.4 mmol). The mixture was stirred
at rt for 6 h, at the end of which time TLC (3/1 petroleum ether-ethyl acetate) in-
dicated that the reaction was complete. The mixture was poured into cold water and
extracted with CH2Cl2. The organic phase was washed with water, then dried over
Na2SO4 and concentrated. Column chromatography (4/1 petroleum ether-ethyl ac-
etate) of the residue gave 22 as a syrup (5.29 g, 77.6% from 19). [ꢁ]D25 ꢆ56° (c
1
1.1, CHCl3); H NMR (CDCl3, 400MHz): ꢂ 1.04 (s, 9 H, C(CH3)3), 1.27, 1.29,
1.42, 1.46 (4 s, 12 H, 2 (CH3)2C), 3.36 (dd, 1 H, J5,6a 7.8, J6a,6b 9.2 Hz, H-6a), 3.50
(dd, 1 H, J5ꢇ,6ꢇa 2.8, J6ꢇa,6ꢇb 11.4 Hz, H-6ꢇa), 3.62 (dd, 1 H, J5ꢇ,6ꢇb 7.6 Hz, H-6ꢇb),
3.70–3.85 (m, 2 H, H-5ꢇ, H-6b), 3.92 (d, 1 H, J 2.7 Hz, H-4ꢇ), 4.01–4.10 (m, 2 H,
H-3ꢇ, H-5), 4.15 (dd, 1 H, J2,3 2.4, J3,4 7.9 Hz, H-3), 4.28 (dd, 1 H, J1,2 5.0, J2,3 2.4
Hz, H-2), 4.34 (d, 1 H, J1,2 7.7 Hz, H-1ꢇ), 4.53 (dd, 1 H, J3,4 7.9, J4,5 2.4 Hz, H-4),
4.61 (d, 1 H, J 11.3 Hz, PhCH2), 4.73 (d, 2 H, J 10.7 Hz, 2 PhCH2), 4.83, 4.98, 5.03
(3 d, 3 H, J 11.3, 10.7, 10.7 Hz, 3 PhCH2), 5.55 (d, 1 H, J1,2 5.0 Hz, H-1), 7.20–7.62
(m, 25 H, Ph).
Anal. Calcd for C55H66O11: C, 73.17; H, 7.32. Found: C, 73.24; H, 7.20.
2,3,4-Tri-O-benzyl-ꢀ-D-galactopyranosyl-(1→6)-1,2:3,4-di-O-isopropy-
lidene-ꢁ-D-galactopyranose (23). To a solution of 22 (840 mg, 0.93 mmol) in
THF (20 mL) was added tetrabutylammonium fluoride trihydrate (300 mg, 0.95
mmol). The mixture was stirred at rt for 5 h, at the end of which time TLC (2/1
petroleum ether-ethyl acetate) showed the reaction was complete. The mixture was
concentrated under reduced pressure. Column chromatography (2/1 petroleum

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PAN ET AL.
ether-ethyl acetate) of the residue gave 23 as a syrup (574 mg, 89%). [ꢁ]D25 ꢆ6°
(c 2.1, CHCl3); 1H NMR (CDCl3, 400MHz): ꢂ 1.31, 1.32, 1.43, 1.50 (4 s, 12 H, 2
(CH3)2C), 3.36 (dd, 1 H, J6ꢇa,6ꢇb 5.2, J5ꢇ,6ꢇb 6.8 Hz, H-6ꢇa), 3.42 (dd, 1 H, J5ꢇ, 6ꢇb 11.1
Hz, H-6ꢇb), 3.51 (dd, 1 H, J5,6a 2.9, J6a,6b 9.7 Hz, H-6a), 3.71–3.78 (m, 3 H, H-
4ꢇ,5ꢇ,H-6b), 3.82 (dd, 1 H, J1ꢇ,2ꢇ 7.7 Hz, J2ꢇ,3ꢇ 10.0 Hz, H-2ꢇ), 4.03–4.05 (m, 1 H, H-
5), 4.10 (dd, 1 H, J2ꢇ,3ꢇ 10, J3ꢇ,4ꢇ 4.7 Hz, H-3ꢇ), 4.28 (dd, 1 H, J2,3 2.0 Hz, H-2), 4.41
(d, 1 H, J1ꢇ,2ꢇ 7.7 Hz, H-1ꢇ), 4.60 (dd, 1 H, J3,4 7.9, J4,5 2.4 Hz, H-4), 4.65, 4.72,
4.75, 4.83, 4.93, 5.03 (6 d, 6 H, J 12.0, 11.7, 11.0 Hz, 3 PhCH2), 5.55 (d, 1 H, J1,2
5.0 Hz, H-1), 7.25–7.45 (m, 15 H, Ph).
Anal. Calcd for C39H48O11: C, 67.63; H, 6.94. Found: C, 67.55; H, 7.08.
2,3,5-Tri-O-benzoyl-ꢁ-L-arabinofuranosyl-(1→3)-2,4,6-tri-O-acetyl-ꢀ-
D-(1→ 6)-2,3,4-tri-O-benzyl-ꢀ-D-galactopyranosyl-(1→6)-1,2:3,4-di-O-iso-
propylidene-ꢁ-D-galactopyranose (24). A solution of 18 (190 mg, 0.21 mmol)
and 23 (140 mg, 0.2 mmol) in anhydrous dichloromethane (5 mL) was cooled to
0°C, then to this solution was added TMSOTf (12 ꢅL, 0.04 mmol) under nitrogen
protection. The mixture was stirred at rt for 1 h, at the end of which time TLC (1/1
petroleum ether-ethyl acetate) showed that the reaction was complete. The reaction
mixture was neutralized with triethylamine (0.01 mL), then concentrated. Column
chromatography (1:1 petroleum ether-ethyl acetate) of the residue gave 24 as a
syrup (233 mg, 81%). [ꢁ]D25 ꢆ8° (c 3.2, CHCl3); 1H NMR (CDCl3, 400MHz): ꢂ
1.13, 1.24, 1.26, 1.47 (4 s, 12 H, CH3), 1.87, 2.02, 2.14 (3 s, 9 H, CH3CO),
3.46–3.50 (m, 2 H, H-5B), H-6A), 3.61 (dd, 1 H, J 7.4, 9.3 Hz, H-6bA), 3.72–3.73
(br d, 2 H, 2 H-6B), 3.78–3.86 (m, 4 H, H-3C, H-2B, 2 H-6C), 4.00–4.16 (m, 4 H,
H-3B, H-4B, H-5A), H-5C), 4.18 (dd, 1 H, J 1.7, 8.0 Hz, H-3A), 4.24 (dd, 1 H, J 5.0
Hz, H-2A), 4.37 (d, 1 H, J 7.7Hz, H-1B), 4.55 (dd, 1 H, J 2.4, 8.0 Hz, H-4A), 4.58
(d, 1 H, J 8.1 Hz, H-1C), 4.65 (d, 1 H, J 11.5 Hz, one proton of PhCH2), 4.69–4.75
(m, 3 H, H-5D, PhCH2), 4.79–4.82 (m, 2 H, H-4D and one proton of PhCH2),
4.89–4.94 (m, 2 H, one proton of PhCH2 and H-5D), 5.04 (d, 1 H, J 11.0 Hz, one
proton of PhCH2), 5.25 (dd, 1 H, J 9.8, 8.1 Hz, H-2C), 5.30 (s, 1 H, H-1D), 5.36 (s,
1 H, H-2D), 5.46 (d, 1 H, J 3.1 Hz, H-4C), 5.50 (d, 1 H, J 5.0 Hz, H-1A), 5.57 (d, 1
H, J 5.2 Hz, H-3D), 7.26–8.10 (m, 30 H, Ph).
Anal. Calcd for C77H84O26: C, 64.89; H, 5.90. Found: C, 64.78; H, 5.83.
ꢁ-L-Arabinofuranosyl-(1→3)-ꢀ-D-galactopyranosyl-(1→6)-ꢀ-D-galac-
topyranosyl-(1→6)-D-galactopyranose (25). A solution of 24 (198 mg, 0.14
mmol) was dissolved in CH2Cl2 (2 mL) and the mixture was treated with 95% TFA
(4 mL) for 2 h and then co-evaporated with toluene to dryness under reduced pres-
sure. The syrup generated above was fully acetylated with acetic anhydride (1.5
mL) in pyridine (4 mL) for 5 h at rt, then co-evaporated with toluene (3 ꢃ 7 mL)
to dryness. The resulting colorless oil was dissolved in MeOH/EtOAc/H2O
(3/1/0.05, v/v/v, 10 mL) and Pd/C (10%, 200 mg) was added. Hydrogen was bub-
bled into the mixture at rt for 24 h, during which time solvents were added occa-
sionally and then filtered. The filtrate was concentrated and the residue was dis-
solved in MeOH (10 mL). NaOMe (0.5 M, keep pH at 9) was added and the

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PAN ET AL.
mixture was stirred at room temperature overnight. After neutralization with Am-
berlite IR 120 (Hꢈ), the mixture was filtered and the filtrate was concentrated un-
der reduced pressure. The crude product was purified by Sephadex LH-20 column
chromatography using MeOH as the eluent to afford 25 (43 mg, 49% from 24).
[ꢁ]D25 ꢆ7° (c 0.3, H2O); 13C NMR (D2O, 100 MHz): ꢂ 63.0 (C-6C), 63.6 (C-5D),
69.8, 69.9, 71.1, 71.5, 71.8, 72.7, 73.5, 73.8, 73.9, 74.2, 74.6, 75.2, 76.9, 79.2, 80.5,
83.2, 85.9, 104.1, 105.5, 105.7 (C-1A/C-1B/C-1C), 111.6 (C-1D). ESIMS calcd for
C23H40O20 (636). Found ESI-MS(-): 635 (M-H) ꢈ.
ACKNOWLEDGMENTS
This work was supported by NNSF of China (Projects 29972053, 39970179)
and CAS KIP-RCEES9904, KJ952J1510.
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Received January 11, 2001
Accepted March 22, 2001

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