Synthesis of the pentasaccharide repeating unit of latosillan

Article abstract of DOI:10.1016/j.carres.2005.11.007

A pentasaccharide, β-d-Man-(1→2)-[β-d-GlcNAc-(1→4)]- α-l-Rha-(1→4)-α-l-Rha-(1→4)-α-l-Rha-1-OC ₈H₁₇, representing the repeating unit of latosillan, was convergently synthesized from the building blocks, ethyl 2,3-O-isopropylidene-1- t

Full text of DOI:10.1016/j.carres.2005.11.007

Carbohydrate
RESEARCH
Carbohydrate Research 341 (2006) 191–197
Synthesis of the pentasaccharide repeating unit of latosillan
Yuxia Hua,a Junjun Xiao,b Yingshen Huangb and Yuguo Dua,*
aState Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences,
Chinese Academy of Sciences, Beijing 100085, China
bDepartment of Cell Biology, School of Basic Medical Sciences, Peking University, Beijing 100083, China
Received 25 October 2005; received in revised form 9 November 2005; accepted 14 November 2005
Available online 5 December 2005
Abstract—A pentasaccharide, b-D-Man-(1!2)-[b-D-GlcNAc-(1!4)]-a-L-Rha-(1!4)-a-L-Rha-(1!4)-a-L-Rha-1-OC8H17, repre-
senting the repeating unit of latosillan, was convergently synthesized from the building blocks, ethyl 2,3-O-isopropylidene-1-thio-
a-L-rhamnopyranoside, 2-O-acetyl-3,4,6-tri-O-benzyl-b-D-glucopyranosyl trichloroacetimidate, and 3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-b-D-glucopyranosyl trichloroacetimidate under standard glycosylation conditions. The target pentasaccharide showed
acceptable differentiation-inducing activity on HL-60 cell lines at the dosages of 10–50 lg/mL.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Glycosylation; Latosillan; Cell differentiation; Oligosaccharides
1. Introduction
have isolated a polysaccharide (named as latosillan
later) from the culture filtrate of a bacterium, and a strong
differentiation inducer activity was observed when incu-
bated with M1 cells. The structure of latosillan was eluci-
dated, from a degradation study and NMR spectral
analysis, to be a heteroglycan composed of the repeating
units of a pentasaccharide,5 !2)-b-D-Man-(1!2)-[b-D-
GlcNAc-(1!4)]-a-L-Rha-(1!4)-a-L-Rha-(1!4)-a-L-
Rha-(1!, as shown in Figure 1.
The mouse myeloid leukemia cell line M1 was originally
established in vitro from a spontaneous leukemia SL
strain in the mouse.1 It has been shown that M1 cells
can be induced to differentiate into macrophages and
granulocytes when treated with proteinous factors (D-
factors) in conditioned media from various cells and in
various body fluids, and with chemicals such as gluco-
corticoid hormones, 1a,25-dihydroxyvitamin D, and
lipopolysaccharides.1–3 In the screening course for differ-
entiation inducer of M1 cells, Ando and his co-workers4
To have a better understanding of this immuno-
logically interesting observation and to compare the
bioactivities between natural polysaccharide and the
Figure 1. Structures of latosillan and compound 1.
*
Corresponding author. Tel.: +86 10 62914475; fax: +86 10 62923563; e-mail: duyuguo@mail.rcees.ac.cn
0008-6215/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.11.007

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Y. Hua et al. / Carbohydrate Research 341 (2006) 191–197
structural repeating unit, we launched a collaborative
project regarding the preparation and potential medical
application of latosillan-related analogues. Here, we
would like to report the synthesis and preliminary bio-
logical studies of a latosillan pentasaccharide derivative.
which provided a chemical shift of H-2 at 5.13 ppm
(J = 3.2, 1.4 Hz) in the H NMR spectrum, confirming
1
the structure of 8. Coupling of 8 and 2-O-acetyl-3,4,6-
tri-O-benzyl-b-D-glucopyranosyl trichloroacetimidate
(10)10 in the presence of a catalytic amount of TMSOTf
in CH2Cl2 at 0 ꢁC gave ethyl 2-O-acetyl-3,4,6-tri-O-benz-
yl-b-D-glucopyranosyl-(1!2)-3,4-O-(20,30-diethoxybu-
tane-20,30-diyl)-1-thio-a-L-rhamnopyranoside (11) in
2. Results and discussion
´
86% yield. Zemplen deacetylation of 11, followed by
Pentasaccharide 1 was prepared via a convergent Ô3+2Õ
strategy. The synthesis of disaccharide acceptor 7 is
described in Scheme 1. Ethyl 4-O-acetyl-2,3-O-isopropyl-
idene-1-thio-a-L-rhamonopyranoside (3)6 was converted
into its octyl glycoside 4 under standard NIS/TMSOTf-
oxidation with DMSO/Ac2O,11 reduction with NaBH4,
and acetylation with Ac2O in pyridine, afforded ethyl
2-O-acetyl-3,4,6-tri-O-benzyl-b-D-mannopyranosyl-(1!2)-
3,4-O-(20,30-diethoxybutane-20,30-diyl)-1-thio-a-L-rham-
nopyranoside (14) in 62% yield for four steps. In
compound 11, the chemical shifts of H-10 and H-20
appear at 4.58 ppm (d, J = 8.4 Hz) and 5.07 ppm (t,
J = 8.4 Hz), while in 14, H-10 and H-40 appear at
5.10 ppm (J <1.0 Hz) and 5.76 ppm (d, J = 2.6 Hz),
respectively. This indicated a successful transformation
of the glucose derivative into the corresponding man-
nose derivative.12 Hydrolysis of 14 with aqueous 90%
TFA (!15), followed by regioselective 4-OH glycosyl-
ation with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-D-
glucopyranosyl trichloroacetimidate (16)13 (!17) and
acetylation with Ac2O in pyridine, delivered trisaccharide
donor ethyl 2-O-acetyl-3,4,6-tri-O-benzyl-b-D-manno-
pyranosyl-(1!2)-[3,4,6-tri-O-acetyl-2-deoxy-2-phthal-
imido-b-D-glucopyranosyl-(1!4)]-3-O-acetyl-1-thio-a-L-
rhamnopyranoside (18) in an overall yield of 55% for
three steps. The characteristic peaks corresponding to
´
catalyzed glycosylation conditions. Zemplen deacetyl-
ation7 of 4 with NaOMe in MeOH furnished octyl
2,3-O-isopropylidene-a-L-rhamonopyranoside (5) in a
yield of 86% for two steps. Glycosylation of 3 and 5 as
described in the preparation of 4 gave octyl 4-O-acetyl-
2,3-O-isopropylidene-a-L-rhamonopyranosyl-(1!4)-2,3-
O-isopropylidene-a-L-rhamonopyranoside (6), which was
further deacetylated with NaOMe in MeOH furnishing
disaccharide acceptor, octyl 2,3-O-isopropylidene-
a-L-rhamonopyranosyl-(1!4)-2,3-O-isopropylidene-a-L-
rhamnopyranoside (7) in 83% isolated yield for two steps.
It is noteworthy that the chemical shift of H-1II appears
at d 5.60 ppm (H-1I at d 4.95 ppm) in the 1H NMR spec-
trum of 7, a significant difference compared to a-(1!3)
linked rhamnopyranosyl disaccharide (around d 5.0 ppm).8
In our initial synthesis of trisaccharide donor, we
expected to establish a properly protected b-D-Glc-
NAc-(1!4)-a-L-Rha residue first, then attach the 2-O-
acetyl-3,4,6-tri-O-benzyl-b-D-glucopyranosyl residue to
the 2-OH of the above rhamnose unit, followed by
O-deacetylation–oxidation–reduction on C-2 of the glu-
cose unit to furnish a b-D-mannose containing trisaccha-
ride. However, the multistep reactions finally gave an
inseparable mixture having both b-D-GlcNAc-(1!4)-
[b-D-Man-(1!2)]-a-L-Rha and b-D-GlcNAc-(1!4)-[b-
D-Glc-(1!2)]-a-L-Rha in a ratio of about 4:1. We thus
modified our strategy towards the synthesis of trisaccha-
ride donor 18, as outlined in Scheme 2.
1
H-1III, H-4I, and H-3I in the H NMR spectrum of 18
that appear at 5.48 ppm (d, J = 8.3 Hz), 3.79 ppm (t,
J = 9.5 Hz), and 4.73 ppm (dd, J = 9.5, 3.1 Hz), respec-
tively, further confirmed the desired selectivity on C-4 in
the above glycosylation.
Condensation of trisaccharide donor 18 and disaccha-
ride acceptor 7 in CH2Cl2, using NIS/TMSOTf as co-
catalyst, stereoselectively gave pentasaccharide 19 in
good isolated yield. 2D NMR spectra of 19 clearly
showed 5 H-1s (H-1I: 4.94 ppm; H-1II: 5.52 ppm; H-
1III: 5.29 ppm; H-1IV: 4.36 ppm; H-1V: 5.47 ppm) and
5 C-1s (C-1I: 96.80 ppm; C-1II: 95.47 ppm; C-1III:
97.58 ppm; C-1IV: 100.04 ppm; C-1V: 97.37 ppm), which
are consistent with the desired structure. No b isomer
was isolated from this reaction. Acetal cleavage of com-
pound 19 was smoothly conducted with 90% aqueous
acetic acid under reflux, and the intermediate was subse-
Treatment of rhamnopyranosyl thioglycoside 2 with
butanedione, triethyl orthoformate, and TsOH in EtOH
furnished compound 8 with its trans-OHs blocked in
79% yield.9 To prove this regioselectivity, 8 was acetyl-
ated with acetic anhydride in pyridine generating 9,
Scheme 1. Synthesis of disaccharide acceptor 7. Reagents and conditions: (a) 2,2,-dimethoxypropane, acetone, TsOH, rt, 91%; (b) NIS, TMSOTf,
CH2Cl2, ꢀ20 ꢁC; (c) NaOMe, MeOH, rt, 86% for 5 from 3, 83% for 7 from 4.

Y. Hua et al. / Carbohydrate Research 341 (2006) 191–197
193
Scheme 2. Synthesis of pentasaccharide 1. Reagents and conditions: (a) triethyl orthoformate, butanedione, TsOH, EtOH, rt, 79%; (b) Ac2O, Py; (c)
TMSOTf, CH2Cl2, 0 ꢁC, 86% for 11, 65% for 17 at ꢀ60 ꢁC; (d) NaOMe, MeOH, rt, 83%; (e) Ac2O, DMSO, rt; NaBH4, 1:1 CH2Cl2–MeOH, 0 ꢁC; (f)
Ac2O, Py, 75% for 14 (from 12), 100% for 18; (g) 90% TFA aqueous solution, rt, 85%; (h) NIS/TMSOTf, ꢀ20 ꢁC, 80%; (i) 90% AcOH aq, reflux; 20%
Pd(OH)2/C, H2; Ac2O, Py; (j) NH3, 4:1 MeOH–CH2Cl2, 6 days; (k) Ac2O, Py; NaOMe, MeOH, 59% based on 19.
quently debenzylated with H2 and Pd(OH)2/C. The
above residue was further treated with NH3-saturated
MeOH for 6 days to deprotect acetyl and phthalyl pro-
tecting groups. Global N,O-acetylation with acetic
anhydride in pyridine, followed by O-deacetylation with
NaOMe in MeOH, furnished pentasaccharide derivative
1 as a white foam in 59% yield from 19.
The differentiation-inducing activity of pentasaccha-
ride 1 in the HL-60 cell line was preliminarily studied
according to the published method.4,14 The results are
summarized in Table 1. Our experiments indicate that
compound 1 can induce NBT reduction and is a dose-
dependent differentiation inducer for HL-60 cells. At a
dosage of 50 lg/mL, compound 1 showed the same
inducing activity as the commonly used positive control
(ATRA).
In conclusion, we have prepared a pentasaccharide
derivative representing the natural latosillan repeating
unit. In this convergent synthesis, a b-D-mannose unit
was introduced by oxidation–reduction of a b-D-glucos-
yl 2-OH group using Ac2O/DMSO–NaBH4 conditions.
L-Rhamnosyl thioglycosides were used as donors and
provided good stereo outcomes in generating the a-gly-
cosidic bond in NIS/TMSOTf-catalyzed glycosylations.
The compound prepared showed acceptable differentia-
tion-inducing activity on HL-60 cell lines at the dosages
of 10–50 lg/mL. These results should be valuable in cell
differentiation-related SAR studies. Synthesis and bioac-
tivity studies regarding this pentasaccharide linear oligo-
mer and dendrimer are currently under investigation in
our group.
Table 1. Compound 1 induced differentiation activities of HL-60 cells
Concentrations
NBT reduction (A560 nm/106cells)a
DEME
ATRA (lmol/L)
0
1
1
0.093 0.025b
0.191 0.006c
0.095 0.003b,c
Compound 1 (lg/mL) 10 0.130 0.003b,c
20 0.167 0.005b,c
3. Experimental
3.1. General
50 0.212 0.003b,c
a Data are presented as means SD from three separate experiments;
q-values are calculated using one-factor analysis of variance with
one-way ANOVA.
b q* <0.01 compared with the negative control.
c q* <0.01 compared with the positive control.
Optical rotations were determined at 25 ꢁC with a Per-
kin–Elmer Model 241-Mc automatic polarimeter, and

194
Y. Hua et al. / Carbohydrate Research 341 (2006) 191–197
[a]D-values are in units of 10ꢀ1 deg cm2 gꢀ1. H NMR,
13C NMR and 1H–1H, 1H–13C COSY spectra were
recorded with a Bruker ARX 400 spectrometer for solu-
tions in CDCl3 or CD3OD. Chemical shifts are given
in parts per million downfield from internal Me4Si.
Mass spectra were measured using a MALDITOF-MS
with a-cyano-4-hydroxycinnamic acid (CCA) as matrix.
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 UV detector.
Column chromatography was conducted by elution of a
column of silica gel (100–200 mesh) with EtOAc–petro-
leum ether (60–90 ꢁC) as the eluent. Solutions were con-
centrated at <60 ꢁC under reduced pressure.
red under these conditions for 30 min, quenched by
Et3N, and concentrated. The residue was purified by
silica gel column chromatography (5:1 petroleum
1
25
ether–EtOAc) to give 6 as a syrup (2.45 g, 90%): ½aꢁD
ꢀ67 (c 1, CHCl3); 1H NMR (400 MHz, CDCl3) d
5.63 (s, 1H, H-10), 4.95 (s, 1H, H-1), 4.88 (dd, 1H, J
10.1, 7.9 Hz, H-40), 4.21 (dd, 1H, J 7.1, 5.6 Hz, H-3),
4.19 (d, 1H, J 5.6 Hz, H-2), 4.17 (dd, 1H, J 7.9,
5.8 Hz, H-30), 4.10 (d, 1H, J 5.8 Hz, H-20), 3.76–3.60
(m, 2H, H-5, H-50), 3.67, 3.41 (2dt, 2H, J 9.6, 6.6 Hz,
OCH2), 3.59 (dd, 1H, J 9.9, 7.1 Hz, H-4), 2.10 (s,
3H, CH3CO), 1.60–1.58 (m, 2H, OCH2CH2), 1.55,
1.53, 1.36, 1.33 (4s, 4 · 3H, 2(CH3)2C), 1.30–1.26
(m, 10H, 5CH2), 1.21, 1.14 (d, 2 · 3H, J 6.3 Hz, H-6,
H-60), 0.89 (t, 3H, J 7.0 Hz, CH3). Anal. Calcd for
C28H48O10: C, 61.74; H, 8.88. Found: C, 62.02; H,
8.96.
3.2. Octyl 2,3-O-isopropylidene-a-L-rhamnopyranoside
(5)
To a solution of compound 3 (5.80 g, 20.00 mmol) and
3.4. Octyl 2,3-O-isopropylidene-a-L-rhamnopyranosyl-
(1!4)-2,3-O-isopropylidene-a-L-rhamnopyranoside (7)
1-octanol (2.90 mL, 18.26 mmol) in dry CH2Cl2
˚
(50 mL) was added 4 A molecular sieves (3 g). The mix-
ture was stirred at ꢀ20 ꢁC for 20 min under an N2 atmo-
sphere, then N-iodosuccinimide (7.35 g, 30.00 mmol)
and TMSOTf (100 lL, 0.55 mmol) were added. The
mixture was stirred under these conditions for 30 min,
quenched by Et3N, diluted with CH2Cl2 (100 mL), and
washed with water (2 · 20 mL). The organic phase was
dried over Na2SO4 and concentrated under diminished
pressure to give crude product, which was subsequently
dissolved in MeOH (100 mL). To this mixture was
added NaOMe (1.0 M, kept pH at 9–10) at rt, stirred
under these conditions for 3 h, then neutralized with
Amberlite IR-120 (H+). The mixture was filtered, and
the filtrate was concentrated. The residue was purified
by silica gel column chromatography (4:1 petroleum
ether–EtOAc) to give 5 as a white foam (5.44 g, 86%):
½aꢁD ꢀ49 (c 1.2, CHCl3); H NMR (400 MHz, CDCl3)
d 4.98 (s, 1H, H-1), 4.19 (dd, 1H, J 3.5, 1.7 Hz, H-2),
4.14 (dd, 1H, J 10.0, 3.5 Hz, H-3), 3.76–3.85 (m, 2H,
H-4, H-5), 3.66, 3.42 (2dt, 2H, J 6.5, 9.7 Hz, OCH2),
1.59–1.57 (m, 2H, OCH2CH2), 1.55, 1.34 (2s, 6H,
(CH3)2C), 1.29–1.26 (m, 10H, 5CH2), 1.16 (d, 3H, J
6.3 Hz, H-6), 0.89 (t, 3H, J 7.0 Hz, CH3). Anal. Calcd
for C17H32O5: C, 64.53; H, 10.19. Found: C, 64.28; H,
10.25.
Removal of the acetyl group from compound 6 (2.40 g,
4.41 mmol), as described in the preparation of 5, gave 7
25
as a white foam (2.03 g, 92%): ½aꢁD ꢀ6 (c 1, CHCl3); 1H
NMR (400 MHz, CDCl3) d 5.60 (s, 1H, H-10), 4.95 (s,
1H, H-1), 4.23 (dd, 1H, J 7.0, 5.6 Hz, H-3), 4.19 (d,
1H, J 5.6 Hz, H-2), 4.10 (d, 1H, J 5.7 Hz, H-20), 4.01
(dd, 1H, J 7.5, 5.7 Hz, H-30), 3.69–3.56 (m, 4H, H-4,
H-40, H-5, H-50), 3.42–3.39 (m, 2H, OCH2), 1.55, 1.53,
1.36, 1.33 (4s, 4 · 3H, (CH3)2C), 1.60–1.58 (m, 2H,
CH2), 1.32–1.28 (m, 10H, 5CH2), 1.27, 1.25 (2d,
2 · 3H, J 6.3 Hz, H-6, H-60), 0.89 (t, 3H, J 7.7 Hz,
CH3). Anal. Calcd for C26H46O9: C, 62.13; H, 9.22.
Found: C, 61.89; H, 9.14.
3.5. Ethyl 3,4-O-(20,30-diethoxybutane-20,30-diyl)-1-thio-
a-L-rhamnopyranoside (8)
25
1
To a solution of compound 2 (4.20 g, 20.17 mmol) in
EtOH (50 mL) was added triethyl orthoformate
(21.3 mL, 161 mmol), butanedione (5.3 mL, 60.39
mmol), and TsOH (pH 3) at rt. The mixture was stirred
under these conditions for 1 h, quenched by Et3N, and
concentrated. The residue was purified by silica gel col-
umn chromatography (6:1 petroleum ether–EtOAc) to
give foamy 8 (5.60 g, 79%). To confirm the structure,
compound 8 (30 mg) was acetylated with Ac2O (1 mL)
in pyridine (2 mL) affording 9 as a syrup, quantitatively:
3.3. Octyl 4-O-acetyl-2,3-O-isopropylidene-a-L-rhamno-
pyranosyl-(1!4)-2,3-O-isopropylidene-a-L-rhamno-
pyranoside (6)
25
1
½aꢁD ꢀ5 (c 1, CHCl3); H NMR (400 MHz, CDCl3) d
5.18 (d, 1H, J 1.4 Hz, H-1), 5.13 (dd, 1H, J 3.2,
1.4 Hz, H-2), 4.15 (m, 1H, H-5), 4.07 (dd, 1H, J 10.1,
3.2 Hz, H-3), 3.73 (t, 1H, J 10.1 Hz, H-4), 3.51–3.49
(m, 2 · 2H, 2OCH2), 2.64–2.62 (m, 2H, SCH2), 2.13 (s,
3H, CH3CO), 1.26–1.20 (m, 18H, H-6 and 5CH3). For
8: Anal. Calcd for C16H30O6S: C, 54.83; H, 8.63. Found:
C, 55.01; H, 8.72.
To a solution of compounds 3 (1.60 g, 5.5 mmol) and 5
(1.58 g, 5.0 mmol) in dry CH2Cl2 (20 mL) was added
˚
4 A molecular sieves (3 g). The mixture was stirred at
ꢀ20 ꢁC for 20 min under an N2 atmosphere, then N-
iodosuccinimide (2.02 g, 8.25 mmol) and TMSOTf
(50 lL, 0.28 mmol) were added. The mixture was stir-

Y. Hua et al. / Carbohydrate Research 341 (2006) 191–197
195
3.6. Ethyl 2-O-acetyl-3,4,6-tri-O-benzyl-b-D-glucopyr-
anosyl-(1!2)-3,4-O-(20,30-diethoxybutane-20,30-diyl)-1-
thio-a-L-rhamnopyranoside (11)
portion. The reaction mixture was stirred at rt for 6 h,
then diluted with CH2C12, and the organic phase was
successively washed with water, aq NaHCO3, and brine.
The organic solvent was evaporated in vacuum, and the
crude residue was acetylated with Ac2O (5 mL) in dry
pyridine (10 mL) at rt for 12 h, then concentrated to
dryness with the help of toluene. The residue was purified
by silica gel column chromatography (5:1 petroleum
ether–EtOAc) to yield 14 (1.20 g, 75% for three steps)
To a solution of compound 8 (1.76 g, 5.02 mmol) and
compound 10 (3.84 g, 6.02 mmol) in dry CH2Cl2
˚
(30 mL) was added 4 A molecular sieves (3 g) at 0 ꢁC
under an N2 atmosphere. The mixture was stirred under
these conditions for 20 min, then TMSOTf (21 lL,
0.12 mmol) was added. The reaction mixture was stirred
under these conditions for 1 h, quenched by Et3N, and
concentrated. The residue was purified by silica gel col-
umn chromatography (4:1 petroleum ether–EtOAc) to
25
as a white foam: ½aꢁD ꢀ52 (c 2, CHCl3); 1H NMR
(400 MHz, CDCl3) d 7.40–7.28 (m, 15H, PhH), 5.76
(br d, 1H, J 2.6 Hz, H-20), 5.32 (d, 1H, J 1.1 Hz, H-1),
5.10 (br s, 1H, H-10), 4.85, 4.80, 4.66, 4.58, 4.50, 4.47
(6d, 6H, J 10.2 Hz, PhCH2), 4.16–4.14 (m, 1H, H-40),
4.10–4.07 (m, 1H, H-5), 4.01 (dd, 1H, J 10.2, 2.6 Hz,
H-30), 3.86 (dd, 1H, J 3.0, 1.1 Hz, H-2), 3.69–3.64 (m,
5H, H-4, H-3, H-40, H-50, H-6a0), 3.49–3.44 (m, 5H, H-
6b0, OCH2), 2.57–2.54 (m, 2H, SCH2), 2.15 (s, 3H,
CH3CO), 1.23–1.11 (m, 18H, H-6, 5CH3). Anal. Calcd
for C45H60O12S: C, 65.51; H, 7.33. Found: C, 65.77; H,
7.29.
25
give 11 (3.56 g, 86%) as a white foam: ½aꢁD ꢀ29 (c 1.5,
1
CHCl3); H NMR (400 MHz, CDCl3) d 7.40–7.28 (m,
15H, PhH), 5.42 (d, 1H, J 1.1 Hz, H-1), 5.07 (t, 1H, J
8.4 Hz, H-20), 4.80, 4.77, 4.68, 4.56 (4d, 4H, J 10.2 Hz,
2PhCH2), 4.58 (dd, 1H, J 8.4 Hz, H-10), 4.55 (s, 2H,
PhCH2), 4.05–4.03 (m, 1H, H-5), 3.93 (dd, 1H, J 10.2,
3.0 Hz, H-3), 3.86 (dd, 1H, J 3.0, 1.1 Hz, H-2), 3.69–
3.64 (m, 5H, H-4, H-30, H-40, H-50, H-6a0), 3.48–3.44
(m, 5H, H-6b0, 2OCH2CH3), 2.55–3.53 (m, 2H,
SCH2CH3), 2.15 (s, 3H, CH3CO), 1.23–1.11 (m, 18H,
H-6, 5CH3). Anal. Calcd for C45H60O12S: C, 65.51; H,
7.33. Found: C, 65.82; H, 7.24.
3.9. Ethyl 2-O-acetyl-3,4,6-tri-O-benzyl-b-D-manno-
pyranosyl-(1!2)-1-thio-a-L-rhamnopyranoside (15)
Compound 14 (1.15 g, 1.39 mmol) was dissolved in 90%
aqueous TFA (8 mL), stirred at rt for 30 min, and co-
evaporated with toluene to dryness under diminished
pressure. The residue was purified by silica gel column
chromatography (2:1 petroleum ether–EtOAc) to give
3.7. Ethyl 3,4,6-tri-O-benzyl-b-D-glucopyranosyl-(1!2)-
3,4-O-(20,30-diethoxybutane-20,30-diyl)-1-thio-a-L-
rhamnopyranoside (12)
25
Removal of the acetyl group from compound 11 (3.20 g,
3.88 mmol) as described in the preparation of 5 gave 12 as
15 (810 mg, 85%) as a colorless syrup: ½aꢁD ꢀ35 (c 1.5,
CHCl3). To confirm the structure, compound 15
(50 mg, 0.07 mmol) was acetylated with Ac2O (0.5 mL)
in pyridine (1 mL) affording ethyl 2-O-acetyl-3,4,6-tri-
O-benzyl-b-D-mannopyranosyl-(1!2)-3,4-di-O-acetyl-
25
1
a white foam (2.52 g, 83%): ½aꢁD ꢀ17 (c 2, CHCl3); H
NMR (400 MHz, CDCl3) d 7.45–7.28 (m, 15H, PhH),
5.34 (s, 1H, H-1), 5.07, 4.86, 4.79, 4.56, 4.53, 4.50 (6d,
6H, J 10.2 Hz, PhCH2), 4.42 (d, 1H, J 7.3 Hz, H-10),
4.14–4.12 (m, 1H, H-5), 4.01 (dd, 1H, J 10.0, 2.9 Hz, H-
3), 3.98 (d, 1H, J 2.9 Hz, H-2), 3.66–3.61 (m, 4H, H-20,
H-30, H-40, H-50), 3.53–3.48 (m, 6H, H-60, OCH2CH3),
2.62–2.59 (m, 2H, SCH2CH3), 1.23–1.11 (m, 18H, H-6,
CH3). MALDITOF-MS: calcd for C43H58O11S, m/z
782; found: m/z 805.3 (M+Na)+. Anal. Calcd for
C43H58O11S: C, 65.96; H, 7.47. Found: C, 66.25; H, 7.41.
25
1-thio-a-L-rhamnopyranoside, quantitatively: ½aꢁD ꢀ13
1
(c 1, CHCl3); H NMR (400 MHz, CDCl3) d 7.40–7.28
(m, 15H, PhH), 5.67 (br d, 1H, J 2.6 Hz, H-20), 5.41
(d, 1H, J 1.4 Hz, H-1), 5.10 (dd, 1H, J 10.0, 3.1 Hz,
H-3), 5.05 (dd, 1H, J 10.0, 9.4 Hz, H-4), 4.85, 4.80,
4.66, 4.58, 4.50, 4.47 (6d, 6 · 1H, J 10.2 Hz, PhCH2),
4.61 (br s, 1H, H-10), 4.26–4.24 (m, 1H, H-6a0), 4.14–
4.11 (m, 1H, H-5), 3.74–3.67 (m, 3H, H-2, H-40, H-
6b0), 3.64 (dd, 1H, J 10.2, 2.6 Hz, H-30), 3.44–3.42 (m,
1H, H-50), 2.56–2.53 (m, 2H, SCH2), 2.21, 2.06, 2.01
(s, 3 · 3H, 3CH3CO), 1.23–1.11 (m, 6H, H-6,
SCH2CH3). Anal. Calcd for C37H46O10S (compound
15): C, 65.08; H, 6.79. Found: C, 64.80; H, 6.86.
3.8. Ethyl 2-O-acetyl-3,4,6-tri-O-benzyl-b-D-manno-
pyranosyl-(1!2)-3,4-O-(20,30-diethoxybutane-20,30-diyl)-
1-thio-a-L-rhamnopyranoside (14)
A solution of compound 12 (1.51 g, 1.93 mmol) in 1,2
Ac2O–DMSO (15 mL) was kept at room temperature
until all starting materials were consumed based on
TLC monitoring. The mixture was diluted with CH2Cl2
(90 mL) and washed with water (3 · 40 mL). The organic
layer was dried over MgSO4 and evaporated. To the
above-mentioned crude residue in 1:1 CH2Cl2–MeOH
(20 mL) at 0 ꢁC was added NaBH4 (700 mg) in one
3.10. Ethyl 2-O-acetyl-3,4,6-tri-O-benzyl-b-D-manno-
pyranosyl-(1!2)-[3,4,6-tri-O-acetyl-2-deoxy-2-phthal-
imido-b-D-glucopyranosyl-(1!4)]-3-O-acetyl-1-thio-a-L-
rhamnopyranoside (18)
To a solution of compounds 15 (500 mg, 0.73 mmol) and
16 (465 mg, 0.80 mmol) in dry CH2Cl2 (10 mL) was

196
Y. Hua et al. / Carbohydrate Research 341 (2006) 191–197
˚
added 4 A molecular sieves (1 g) at ꢀ60 ꢁC under an N2
atmosphere. The mixture was stirred under these condi-
tions for 20 min, and then TMSOTf (10 lL, 0.06 mmol)
was added and stirred for another 30 min, quenched by
Et3N. The mixture was filtered and the filtrate was con-
centrated. The residue was purified by silica gel column
chromatography (3:1 petroleum ether–EtOAc) to give 17
as a white foam, which was acetylated with Ac2O (1 mL)
in pyridine (3 mL) to furnish 18 (543 mg, 65% for two
H-4II, H-5IV, OCH), 2.20, 2.12, 2.10, 2.03, 1.85 (5s,
5 · 3H, 5 CH3CO), 1.56, 1.42, 1.34, 1.19 (4s, 4 · 3H,
2(CH3)2C), 1.61–1.57 (m, 2H, OCH2CH2), 1.30–1.27
(m, 10H, 5CH2), 1.20 (d, 6H, J 6.3 Hz, H-6I, H-6III),
1.14 (d, 3H, J 6.3 Hz, H-6II), 0.89 (t, 3H, J 7.0 Hz,
CH3). 13C NMR (100 MHz, CDCl3): d 170.7, 170.4,
170.2, 170.1, 169.5, 138.3, 138.1, 137.8, 137.4, 134.2,
129.0, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8,
127.7, 127.6, 127.5, 127.4, 109.3, 109.0, 100.0, 97.6,
97.4, 96.8, 95.4, 79.8, 78.5, 78.0, 77.5, 77.3, 77.0, 76.7,
75.8, 75.1, 74.6, 74.0, 73.6, 73.1, 71.4, 71.3, 70.6, 69.1,
69.0, 67.8, 67.6, 67.3, 64.5, 63.8, 61.6, 54.8, 31.8, 29.3,
29.2, 29.1, 27.9, 27.8, 26.3, 26.1, 22.6, 21.4, 21.2, 21.0,
20.7, 20.6, 20.6, 18.0, 17.7, 17.6, 14.0. MALDITOF-
MS: calcd for C83H107NO29, m/z 1581; found: m/z
1604.8 (M+Na)+, 1620.8 (M+K)+. Anal. Calcd for C83-
25
steps) as a syrup: ½aꢁD ꢀ42 (c 1, CHCl3); 1H NMR
(400 MHz, CDCl3) d 7.90–7.18 (m, 19H, PhH), 5.83
(dd, 1H, J 10.8, 9.0 Hz, H-3III), 5.67 (br d, 1H, J
3.0 Hz, H-2II), 5.48 (d, 1H, J 8.3 Hz, H-1III), 5.28 (d,
1H, J 1.4 Hz, H-1I), 5.20 (dd, 1H, J 10.0, 9.0 Hz, H-
4III), 4.87, 4.77, 4.57, 4.55, 4.53, 4.49 (6d, 6H, J
10.2 Hz, 3PhCH2), 4.73 (dd, 1H, J 9.5, 3.1 Hz, H-3I),
4.40 (dd, 1H, J 12.3, 3.7 Hz, H-6aIII), 4.37 (br s, 1H,
H-1II), 4.30 (dd, 1H, J 12.3, 3.7 Hz, H-6bIII), 4.23 (dd,
1H, J 10.8, 8.3 Hz, H-2III), 4.03 (dd, 1H, J 1.4, 3.1 Hz,
H-2I), 3.98 (dt, 1H, H-5III), 3.90–3.85 (m, 1H, H-5I),
3.79 (t, 1H, J 9.5 Hz, H-4I), 3.73 (t, 1H, J 9.5 Hz, H-
4II), 3.70 (d, 2H, J 3.6 Hz, H-6II), 3.60 (dd, 1H, J 9.5,
3.0 Hz, H-3II), 3.39–3.31 (dt, 1H, H-5II), 2.51–2.46 (m,
2H, SCH2), 2.21, 2.15, 2.06, 2.01, 1.88 (5s, 5 · 3H,
5CH3CO), 1.24 (d, 3H, J 6.3 Hz, H-6I), 1.15 (t, 3H, J
7.3 Hz, CH3). Anal. Calcd for C59H67NO20S: C, 62.04;
H, 5.91. Found: C, 62.31; H, 5.80.
H
107NO29: C, 62.99; H, 7.81. Found: C, 63.28; H, 7.72.
3.12. Octyl 2-acetamido-2-deoxy-b-D-glucopyranosyl-
(1!4)-[b-D-mannopyranosyl-(1!2)]-a-L-rhamno-
pyranoyl-(1!4)-a-L-rhamnopyranosyl-(1!4)-a-L-
rhamnopyranoside (1)
Compound 19 (410 mg, 0.26 mmol) was dissolved in 90%
aq acetic acid (8 mL) and stirred under reflux for 30 min.
At the end of this time, TLC indicated all starting mate-
rial was consumed. The mixture was co-evaporated with
toluene under diminished pressure to give a syrup, which
was subjected to hydrogenation with H2 under a flow
rate of 100 mL/min in the presence of 20% Pd(OH)2 on
charcoal (209 mg, 0.14 mmol) in 1:1 MeOH–EtOAc
(20 mL) for 70 h. The reaction mixture was filtered, the
filtrate was concentrated, and the syrup was treated with
Ac2O (2 mL) in pyridine (4 mL) for 4 h at rt. After
co-evaporation with toluene, the residue (about 320 mg)
was dissolved into NH3-saturated 4:1 MeOH–CH2Cl2
(50 mL) and stirred at rt for 6 days, then concentrated
under diminished pressure. The residue was dissolved
in H2O (1 mL) and passed through a Sephadex LH-20
column with H2O as eluent yielding foamy intermediate
(about 175 mg) after freeze drying. Acetylation of this
intermediate as described in the preparation of 14, fol-
lowed by purification on a silica gel column (3:2 petro-
leum ether–EtOAc) gave an amorphous solid. To a
solution of the above solid in MeOH was added NaOMe
(1.0 M, kept at pH 9–10) at rt. The reaction mixture was
stirred for 3 h, then neutralized with Amberlite IR-120
(H+), and filtered. The filtrate was concentrated, and
the residue was purified on a Sephadex LH-20 column
with H2O as eluent to finish compound 1 (143 mg, 59%
3.11. Octyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-
D-glucopyranosyl-(1!4)-[2-O-acetyl-3,4,6-tri-O-benzyl-
b-D-mannopyranosyl-(1!2)]-3-O-acetyl-a-L-rhamno-
pyranoyl-(1!4)-4-O-acetyl-2,3-O-isopropylidene-a-L-
rhamnopyranosyl-(1!4)-2,3-O-isopropylidene-a-L-
rhamnopyranoside (19)
Coupling of disaccharide 7 (200 mg, 0.40 mmol) and tri-
saccharide 18 (490 mg, 0.43 mmol) was carried out as
described in the preparation of 6. The crude product
was purified on a silica gel column (2:1 petroleum
ether–EtOAc) to yield 19 as a foamy solid (504 mg,
25
80%): ½aꢁD ꢀ100 (c 1, CHCl3); 1H NMR (400 MHz,
CDCl3) d 7.78–7.18 (m, 19H, PhH), 5.84 (dd, 1H, J
10.8, 9.0 Hz, H-3V), 5.67 (br d, 1H, J 3.2 Hz, H-2IV),
5.52 (s, 1H, H-1II), 5.48 (d, 1H, J 8.4 Hz, H-1V), 5.28
(d, 1H, J 1.6 Hz, H-1III), 5.20 (dd, 1H, J 10.0, 9.0 Hz,
H-4V), 4.93 (s, 1H, H-1I), 4.87, 4.80, 4.64, 4.57, 4.53,
4.48 (6d, 6 · 1H, J 10.8 Hz, 3 PhCH2), 4.78 (dd, 1H, J
9.6, 3.2 Hz, H-3III), 4.41 (dd, 1H, J 12.4, 3.0 Hz, H-
6aV), 4.37 (br s, 1H, H-1IV), 4.30 (dd, 1H, J 12.4,
3.0 Hz, H-6bV), 4.23 (dd, 1H, J 10.8, 8.4 Hz, H-2V),
4.16 (dd, 1H, J 7.2, 5.6 Hz, H-3II), 4.06 (d, 1H, J
5.6 Hz, H-2II), 4.00–3.93 (m, 4H, H-2III, H-3I, H-5V,
H-6aIV), 3.81 (t, 3H, J 9.6 Hz, H-4III), 3.79 (t, 3H, J
10.0 Hz, H-4IV), 3.75 (d, 1H, J 5.6 Hz, H-2I), 3.70 (dd,
1H, J 1.2, 11.2 Hz, H-6bIV), 3.68–3.50 (m, 6H, H-4I,
H-5I, H-5II, H-5III, H-3IV, OCH), 3.37–3.33 (m, 3H,
25
from 19) as a white foam after freeze drying: ½aꢁD ꢀ32
1
(c 1, H2O); Selected H NMR (400 MHz, CD3OD) d
5.46 (d, 1H, J 1.2 Hz, H-1II), 5.14 (d, 1H, J 1.6 Hz, H-
1III), 4.76 (d, 1H, J 8.3 Hz, H-1V), 4.62 (br s, 2H, H-1I,
H-1IV), 4.03 (dd, 1H, J 1.6, 3.4 Hz, H-2III), 4.01 (d, 1H,
J 2.7 Hz, H-2IV);13C NMR (100 MHz, CD3OD): d

Y. Hua et al. / Carbohydrate Research 341 (2006) 191–197
197
174.4, 103.7, 103.3, 102.9, 102.3, 101.4, 81.7, 81.3, 81.2,
References
80.9, 78.7, 77.8, 76.7, 75.2, 73.3, 73.2, 72.9, 72.7, 72.2,
72.0, 68.8, 68.5, 68.2, 63.1, 62.9, 58.2, 33.0, 30.5, 30.4,
27.3, 23.7, 23.0, 18.7, 18.4, 18.1, 14.5. MALDITOF-
MS: calcd for C40H71NO23, m/z 933; found: m/z 956.1
(M+Na)+, 972.1 (M+K)+. Anal. Calcd for C40H71-
NO23: C, 51.44; H, 7.66. Found: C, 51.19; H, 7.79.
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3.13. Bioassays of compound 1
M1 cells (3 · 105/well) were cultured in a suspension of
DulbccoÕs Modified Eagle Medium (DMEM) supple-
mented with 10% newborn calf serum in a humidified
atmosphere at 37 ꢁC containing 5% CO2. Compound 1
was applied at concentrations of 10, 20, and 50 lg/mL
according to the dosage applied for natural latosillan
in the literature,4 while positive control all-trans-retinoic
acid (ATRA) was used at 1 lmol/L. DMEM medium
was used as a negative control. At the end of 3 days of
incubation, the cells were harvested by centrifugation,
and then suspended in nitroblue tetrazolium (NBT)
solution (100 lL, 4.0 mg/mL), and 12-O-tetradecanoyl-
phorbol-13-acetate (TPA, 100 lL, 2.0 lg/mL) was
added. The cell suspension was incubated at 37 ꢁC for
20 min, and 1 N HCl (200 lL) was added at 4 ꢁC to ter-
minate the reaction. After centrifugation, DMSO
(600 lL) was added to the cell pellets, and the amount
of formazan formed in this process was measured at
560 nm with a microplate reader.
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Acknowledgments
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This work was supported by NNSF of China (Projects
20372081, 30330690).