Concise synthesis of clarhamnoside, a novel glycosphingolipid isolated from the marine sponge Agela clathrodes

Article abstract of DOI:10.1016/j.carres.2007.05.018

The first total synthesis of a novel α-galactoglycosphingolipid clarhamnoside has been achieved through a straightforward strategy. A thiogalactosyl donor with a benzylidene group at C-4 and C-6 and nonparticipating p-methoxybenzyl group at C-2 was succes

Full text of DOI:10.1016/j.carres.2007.05.018

Carbohydrate Research 342 (2007) 2003–2013
Concise synthesis of clarhamnoside, a novel
glycosphingolipid isolated from the marine sponge Agela clathrodes
Ning Ding, Chunxia Li, Yunpeng Liu, Zaihong Zhang and Yingxia Li*
Key Laboratory of Marine Drugs, The Ministration of Education of China, School of Pharmacy, Ocean University of China,
Qingdao 266003, China
Received 4 April 2007; received in revised form 8 May 2007; accepted 9 May 2007
Available online 18 May 2007
Abstract—The first total synthesis of a novel a-galactoglycosphingolipid clarhamnoside has been achieved through a straightfor-
ward strategy. A thiogalactosyl donor with a benzylidene group at C-4 and C-6 and nonparticipating p-methoxybenzyl group at
C-2 was successfully employed in the stereocontrolled syntheses of a-GalGSLs. The N-Phth-protected trifluoroacetimidate donor
for terminal disaccharide was successfully applied in constructing the [GalNAcb-(1!6)-Gal] glycosidic linkage.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: KRN7000; Clarhamnoside; Glycosphingolipids; Immunostimulatory; Sponge; Synthesis
1. Introduction
found to selectively induce IL-4, as opposed to IFNc,
and to offer protection in mice against experimental
autoimmune encephalomyelitis (EAE)4 and more
recently has been shown to offer protection against dia-
betes in NOD mice5 and against collagen-induced
arthritis.6 Agelagalastatin, isolated from the Western
Pacific marine sponge Agelas sp., displays significant
in vitro inhibitory activities against human cancer cell
growth.7
Besides its biological activities, spongal a-GalGSLs
appear to be a quite peculiar class of molecules in terms
of the structure of their carbohydrate moieties.8 Clar-
hamnoside, a novel a-GalGSL, which was recently iden-
tified by the Mangoni group from new specimens of A.
clathrodes,9 bears quite a unique structure containing
a-L-Rhap-(1!3)-b-D-GalpNAc-(1!6)-a-D-Galp-(1!2)-
a-D-Galp. It is one of the few natural a-GalGSLs glycos-
ylated at the inner galactose 2-OH and the only a-Gal-
GSL with an L-rhamnose unit in the sugar head.9 In
addition, the sequential two 1,2-cis-a-D-galactopyranos-
idic linkages [Gala-(100!20)-Gala-(10!1)-Cer] are also
an extraordinary and rare feature in nature (Fig. 1).
As part of our effort to understand the mechanism of
CD1-mediated T-cell activation, we are interested in
developing a facile approach to series of a-GalGSLs
with a glycosylated 20-OH or other substituting groups,
It is well established that sponges of the genus Agelas
and Axinella produce a-galactoglycosphingolipids (a-
GalGSLs), unique glycosphingolipids with an a-galact-
ose as the first sugar of the carbohydrate chain, unlike
the ubiquitous b-glycosidic bond from nearly all known
higher animals and plants.1 The scientific interests in a-
GalGSLs have recently increased on account of the role
they could play as therapeutic agents. a-GalGSLs are
potent ligands of the MHC class I-like CD1d protein,
which is present on the surface of the antigen-presenting
cells (APCs) and is capable of activating in vitro and
in vivo a specialized population of T cells, named natu-
ral killer T cells (NKT cells), which play an important
role in regulating innate and adaptive immunity during
infection, tumor growth, and autoimmune diseases.2 a-
Galactosyl ceramide (KRN7000), a potent analogue of
the natural agelasphins isolated from the marine sponge
Agelas mauritianus, is an important cerebroside exhibit-
ing immunostimulatory activity and antitumor proper-
ties.3 A truncated analogue of KRN7000, OCH, was
*
Corresponding author. Tel.: +86 532 82032150; fax: +86 532
82033054; e-mail: liyx417@ouc.edu.cn
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.05.018

2004
N. Ding et al. / Carbohydrate Research 342 (2007) 2003–2013
OH
NH OH
OH
gent synthesis thus calls for coupling of a tetrasaccha-
OH
C
19H39
O
HO
HO
ride donor with the aglycon. However, a-selective
glycosylation of the ceramide with galactosyl donors
has long been recognized as a difficult task,13 especially
for the inner galactose, which is (1!2) branched. The
potent steric effect between the bulky ceramide residue
and the tetrasaccharide donor requires attention in the
convergent synthesis.14,15 In addition, a general route
for preparation of a-GalGSLs with a free 20-OH group
were needed in our later work. For this purpose, a
straightforward strategy was described to complete the
synthetic work. The retrosynthetic analysis of cla-
rhamnoside is depicted in Scheme 1. a-Galactosyl lipid
7 with a free 2-OH group was anticipated as a key inter-
mediate for elaboration of the tetrasaccharide lipid via
sequential coupling with monosaccharide donor 10
and disaccharide donor 4. The stereocontrolled con-
struction of a-linked monosaccharide lipid 7 and disac-
charide lipid 3 turned out to be the crucial steps in our
synthesis.
O
O
OH
C13H27
O
O
O
HO
HO
O
OH
R'
OH
HO
HO
O
NHAc
HO
O
NH OH
O
OH
OH
R
HO
O
O
OH
Me
HO
KRN7000 R = C13H27, R' = C25H51
OCH R = C4H9, R' = C23H47
clarhamnoside
Figure 1. The structures of KRN7000, OCH, and clarhamnoside.
since structure–activity relationships showed that
substitution on 20-OH strongly affected the bioactivity
of these compounds.10 Although extensive syntheses of
KRN7000 and its analogues11 have been described,
there are only a few works reported on the syntheses
of C-20 modified a-GalGSLs10b,c,12 and spongal a-Gal-
For preparation of a-galactosyl ceramide (6 or 7), a
major effort was to find a suitable galactosyl donor,
which should be designed to obtain a high a:b ratio as
well as a satisfactory yield. Recently, galactosyl trichloro-
acetimidate 15 with a benzylidene group at C-4 and C-6
and nonparticipating benzyl groups at C-2 and C-3
has been successfully used in the stereocontrolled syn-
theses of a-GalGSLs16 (Scheme 2). This remarkable ste-
reoselectivity could be attributed to the cis-decalin ring
system with the equatorial phenyl group in 15 that hin-
ders the attack from the b-face.16 The result prompted
GSLs with
a
longer carbohydrate chain than
KRN7000.13 The unique structure and potent bioactivi-
ties make clarhamnoside a suitable goal for synthesis.
Herein, we describe the concise synthesis of clarhamno-
side using a-stereoselective glycosylations.
2. Results and discussion
Clarhamnoside consists of two synthetically distinct
parts, the ceramide and the tetrasaccharide. A conver-
OR
OAc
2V
OR
OR
RO
C19H39
O
C
19H39
O
O
1'
NH OR
OH
O
2'
5
NH
CCl3
1''
O
O
C13H27
O
2
OR
+
1
O
OR
6''
O
1'''
O
O
CF3
Me
RO
RO
O
RO
OR
RO
AcO
OAc
OAc
NHAc
11
12
NPh
NPhth
OR
RO
3'''
+
O
1IV
O
Ph
O
OR
O
O
O
O
OR
OMP
NPhth
OR
HO
1 R = H (clarhamnoside)
2 R = Ac
Me
Me
RO
RO
4
PhCH
+
N3 OBn
O
O
HO
C13H27
PhCH
BnO
O
O
O
1'
BnO
N3 OBn
OBn
8
OBn
+
O
1'
N3
OR
OBn
O
C
13H27
1''
O
O
OBn
1
C
13H27
O
PhCH
BnO
6 R = Bn
7 R = H
1
O
+
OBn
O
RO
RO
O
OR
O
X
OBn
RO
3
OR
STol
10
BnO
9 X = leaving groups
OBn
Scheme 1. Retrosynthetic analysis of clarhamnoside.

N. Ding et al. / Carbohydrate Research 342 (2007) 2003–2013
2005
PhCH
O
(i) Bu2SnO, BnBr, 78%
(ii) PhCH(OCH3)2, TsOH, 85%
HO OH
O
O
O
STol
STol
HO
BnO
OH
OH
13
17
NaH, PMBCl
PhCH
O
O
87%
PhCH
BnO
O
PhCH
BnO
O
BnO
O
O
O
O
STol
O
OR
STol
O
CCl3
OBn
15 R = Bn
19 R = PMB
OPMB
NH
14
18
BF3.Et2O
60% for 15
NIS/ AgOTf
62%
NIS/ AgOTf
67%
N3
OBn
C13H27
8
HO
PhCH
O
OBn
O
O
6
R = Bn
N3
OBn
BnO
20 R = PMB
7
DDQ
85%
OR
O
C13H27
R = H
OBn
(i) PPh3, THF-H 2O
(ii) C25H51COOH, HOBt, EDC, DIEA
78%
PhCH
O
C
25H51
O
O
Pd(OH)2-H2
O
KRN7000
NH
OBn
BnO
OBn
C
13H27
16
O
OBn
Scheme 2. Synthesis of KRN7000 and a-galactosyl ceramide 7.
us to turn our attention to the corresponding thiogalac-
tosyl donor 14 in forming a-GalGSLs. To the best of
our knowledge, thiogalactoside 14 has not been success-
fully employed as a donor in the stereocontrolled syn-
theses of a-GalGSLs. It has been used, however, as an
intermediate to trichloroacetimidate 15.16b,c Fortu-
nately, the glycosylation of azidosphingosine 812 with
14 was carried out successfully in the presence of NIS/
AgOTf to provide 6 exclusively and in a satisfactory
67% yield, while in our hands trichloroacetimidate 15
also delivered 6 exclusively in a clean reaction in 60%
yield when BF3Æetherate was chosen as a mild promoter.
Staudinger reduction17 of the azido group in 6 led to an
amine, which was then coupled with the hexacosanoic
acid in the presence of 1-hydroxybenzotriazole (HOBt),
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydro-
chloride (EDCÆHCl) to furnish 16. After global depro-
tection, KRN7000 was obtained.4a Thus the research
provides a more convenient donor than the most fre-
quently used trichloroacetimidates for a-stereoselective
preparation of KRN7000 analogues.
bonds, a PMB (p-methoxybenzyl) type ether should be
a good choice and simultaneously serve as a protecting
group at C-2 that can be selectively removed in the pres-
ence of benzyl groups. Therefore, thiolgalactosyl donor
18 with a PMB group at the 2-OH position was designed
and rapidly prepared from the known thiolglycoside 13
in three steps. To our delight, the glycosylation of donor
18 with acceptor 8 was carried out smoothly to afford a-
glycolipid 20 exclusively in a good yield (60%). Thus,
after selective removal of the PMB group in 20, the
key 20-OH free intermediate 7 for elaboration of disac-
charide lipid 3 or other derivatives was readily obtained.
It is worth noting that 3-O-benzyl-4,6-O-benzylidene-2-
p-methoxybenzyl trichloroacetimidate 19 was difficult to
prepare due to the lability of PMB ether during cleavage
of various of anomeric protective groups, such as thio-
anisole, p-methoxyphenol, and allyl by using N-bromo-
succinimide (NBS), ammonium cerium(IV) nitrate
(CAN), and PdCl2, respectively. Therefore, the applica-
tion of thiolgalactosyl donor 18 instead of the corre-
sponding trichloroacetimidate 19 provides a facile and
effective strategy to achieve the syntheses of a-GalGSLs
with a free 20-OH.
Since benzyl ethers have been excellent candidates as
nonparticipating groups at C-2 to form a-galactosidic

2006
N. Ding et al. / Carbohydrate Research 342 (2007) 2003–2013
Table 1. Introduce the second a-galactosidic unit to the 20-OH of
glycolipid 9
ride 25 as anomers (a:b 10:1, 64%), while donor 22 with
a remote participating benzoyl group19 showed an
improvement in a-selectivity from a:b 10:1 to a only
(71%). However, when the same conditions were applied
to the glycosylation of bulky substituted glycosyl accep-
tor 7, donor 22 afforded 27 in lower anomeric selectivity
(a:b 3:1, 76%). The decreased a-selectivity with acceptor
7 declared the great influence of the bulky azidosphingo-
sine residue. Fortunately, the a/b mixture of compound
27 was easily separated by column chromatography
(1:200 acetone–toluene). The reaction was also accom-
plished to give a-28 as the only identifiable anomer
(54%) when donor 23 was employed; however, the
manipulation of the benzylidene group to leave 600-OH
free was complex (in the presence of another benzyli-
dene). Thus after removing the benzoyl group in
600-OH of a-27 (86%), the crucial building block 29 was
obtained and used as a disaccharide acceptor for later
glycosylation (Scheme 3).
PhCH
OR1
O
PhCH
O
R2O
BnO
O
O
O
NIS/ AgOTf
O
O
O
+
STol
R3
BnO
BnO
OBn
OH
R3
O
21 R1 = R2 = Bn
22 R1 = Bz, R2 = Bn
23 R1, R2 = -PhCH-
OBn
24 R3 = L1
R3 = L2
R1O
R2O
7
BnO
L1:
O
25 R1 = R2 = Bn, R3 = L1
N3
26 R1 = Bz, R2 = Bn, R3 = L1
27 R1 = Bz, R2 = Bn, R3 = L2
28 R1, R2 = -PhCH-, R3 = L2
OBn
L2:
O
C13H27
OBn
Donor
Acceptor
Product
Yield (%)
a:ba
1
2
3
4
21
22
22
23
24
24
7
25
26
27
28
64
71
76
54
10:1
a
3:1
a
7
Trifluoroacetimidate20 4 was employed as the donor
for the glycosylation with 29, which had been synthe-
sized starting from L-rhamnosyl trichloroacetimidate
donor 11 and 2-N-phthalimido protected 12 from a
five-step procedure, and proved to be efficient in frag-
ment coupling for a b-glycosidic bond in our previous
work18 (Scheme 3). The coupling was carried out in
a The a:b ratio was determined by 1H NMR spectroscopy.
Toward the target molecule 1, the following effort was
to efficiently introduce the second a-galactosidic unit to
the 20-OH of glycolipid 7 (Table 1). We have examined
the glycosylation of two galactosyl donors (21 and 22)
with easily prepared model acceptor 24 in our previous
work.18 Promoted with NIS/AgOTf in 4:1 Et2O–
CH2Cl2, the glycosylation of 21 with 24 gave disaccha-
˚
the presence of TMSOTf and 4 A MS at ꢀ20 ꢁC, afford-
ing the desired b-glycoside 30 in 76% good yield.
Transformation of the N-phthalimido group into an
PhCH
O
CF3
NPh
O
O
RO
RO
O
O
O
NPhth
N3
OBn
BnO
O
C13H27
O
+
O
OBn
OR
O
OBn
RO
OR
Me
RO
BnO
4
α-27 R = OBz
29 R = OH
BnO
CH3ONa, CH3OH
86%
°
TMSOTf, -20 C
76%
OR3
PhCH
OR1
O
OR1
R2O
C19H39
O
O
O
NH OR2
O
O
(i) H2S, pyridine-H2O;
(ii) 5, HOBt, EDC.DIEA
N3
OBn
BnO
O
C13H27
O
OR2
C13H27
O
O
OR2
O
OBn
66%, 2 steps
OBn
O
R2O
O
BnO
NR
R3O
O
OR2
O
OBn
AcO
AcO
R3O
NHAc
O
O
OR3
OR3
OAc
OAc
O
O
Me
R3O
Me
AcO
32 R1 = -PhCH-, R2 = Bn, R3 = Ac
R1 = R2 = R3 = Ac
1 R1 = R2 = R3 = H, Clarhamnoside
ethylenediamide,
(i)
(i). Pd(OH)2-H2;
(ii) Ac2O, pyridine
30 R = Phth
31 R = HAc
°
CH3ONa,
CH3OH
n-BuOH, 90 C;
2
Ac O, pyridine,
(ii)
2
75%
72%, 2 steps
quant.
Scheme 3. Synthesis of clarhamnoside.

N. Ding et al. / Carbohydrate Research 342 (2007) 2003–2013
2007
acetamido group was next accomplished using ethylene-
diamine, followed by acetylation to give 31 (72%).
Staudinger reduction of the azide utilizing triphenyl-
phosphine either in THF–water or pyridine–water
caused decomposition of substance 31. Another attempt
to use zinc powder with AcOH21 was shown to be innoc-
uous toward 31, but the reaction did not proceed effi-
ciently. Finally, the reduction reaction was carried out
smoothly with hydrogen sulfide (H2S) in pyridine–
water,22 giving the desired amine intermediate, which
was directly coupled with a-hydroxy acid 5 using
HOBt and EDCÆHCl as promoters to give the fully pro-
tected derivative 32 in 66% yield. a-Hydroxy acid 5 was
prepared based on the method published by Chen
et al.23
coated E. Merck Silica Gel 60 F254 plates. Flash column
chromatography was performed on silica gel (200–300
mesh, Qingdao, China). Optical rotations were deter-
mined with a Perkin–Elmer Model 241 MC polarimeter.
1H NMR and 13C NMR spectra were taken on a Jeol
JNM-ECP 600 spectrometer with tetramethylsilane
(Me4Si) as the internal standard, and chemical shifts
are recorded as d values. COSY, HMQC, and HMBC
NMR spectra were routinely used to definitively
1
assign the signals of H and 13C NMR spectra. Mass
spectra were recorded on a Global Q-TOF mass
spectrometer.
4.2. (2S,3S,4R)-1-O-(2,3-Di-O-benzyl-4,6-O-benzylidene-
a-D-galactopyranosyl)-2-azido-3,4-di-O-benzyl-octade-
cantriol (6)
Removal of the benzylidene and benzyl ether groups
in 32 by hydrogenolysis using palladium(II) hydroxide
(75%) in 3:1 EtOAc–MeOH proved successful,18 afford-
ing the desired tetrasaccharide intermediate, which was
followed by acetylation to give clarhamnoside perace-
Method A: To a solution of donor 14 (317.6 mg,
˚
0.57 mmol), acceptor 8 (250.0 mg, 0.48 mmol), and 4 A
MS (80 mg) in 1:5:1 CH2Cl2–Et2O–THF (21 mL) were
added NIS (173.6 mg, 0.77 mmol) and AgOTf
(12.3 mg, 0.05 mmol) at 0 ꢁC under Ar. The reaction
mixture was stirred at 0 ꢁC for 12 h and then concen-
trated. The residue was diluted with CH2Cl2 (20 mL),
washed with 10% Na2S2O3 (20 mL · 1) and brine
(20 mL · 1), dried over MgSO4, and concentrated under
reduced pressure. The residue was chromatographed on
a silica gel column (7:1 petroleum ether–EtOAc) to give
the desired product 6 (306.5 mg, 67.0%) as a colorless
oil: Rf 0.35 (7:1 petroleum ether–EtOAc).
1
tate 2. The diagnostic H and 13C NMR signals of 2
were shown to be identical to those provided in the liter-
ature.9 Removal of the acyl groups in 2 using the
reported procedure9 eventually furnished the target
1
glycolipid 1 (clarhamnoside). The H NMR spectrum
of clarhamnoside clearly shows the four anomeric pro-
tons (d 5.78, 5.63, 5.56, 5.27) and two NH signals (d
9.01, 8.59), which are in good agreement with those
listed in the literature.9 The 13C NMR signals are also
in good agreement with those listed in the literature,
except for the presence of an additional carbonyl signal
at 171.6 ppm (only one carbonyl signal d 175.3 was listed
in the literature).
Method B: A solution of trichloroacetimidate 15
(50.0 mg, 0.085 mmol) and phytosphingosine derivative
8 (40.0 mg, 0.076 mmol) in dry Et2O (3.5 mL) and dry
THF (0.5 mL) was added to freshly dried powdered
˚
4 A MS (20 mg) and cooled to ꢀ20 ꢁC. BF3ÆEt2O
3. Conclusions
(47%, 22.4 mg, 0.074 mmol) was added to the solution,
and the mixture was stirred at ꢀ20 ꢁC for 1 h. The
mixture was diluted with EtOAc (50 mL) and filtered
through Celite. The organic layer was washed with satd
aq NaHCO3 and brine, dried (MgSO4), and concen-
trated. The residue was purified by column chromato-
graphy on silica gel (7:1 petroleum ether–EtOAc) to
furnish 6 (48.4 mg, 60.0%) as a colorless oil: Rf 0.35
In conclusion, we have achieved the first total synthesis
of a novel a-GSL clarhamnoside in this paper by
employing a-galactosyl lipid 7 as the key intermediate.
Thiolgalactosyl donor 18, which has a benzylidene
group at C-4 and C-6 and a nonparticipating PMB
group at C-2, has been successfully used to achieve the
a-stereocontrolled synthesis of a-GalGSLs with 20-OH
groups free. The strategy described above provides a
facile and efficient approach to syntheses of a-GalGSLs
with glycosylated 20-OH groups or other substituents.
The syntheses of clarhamnoside analogues and their bio-
logical activities will be reported later.
22
(7:1 petroleum ether–EtOAc); ½aꢁD +50.1 (c 0.6,
CHCl3); 1H NMR (CDCl3): d 7.52–7.31 (25H, Ph),
5.45 (s, 1H, PhCH), 4.97 (d, 1H, J = 3.2 Hz, H-10),
4.87–4.48 (8H, 4PhCH2), 4.15 (d, 1H, J = 3.2 Hz, H-
40), 4.09 (dd, 1H, J = 10.1, 3.7 Hz, H-20), 4.08 (d,
1H, J = 12.8 Hz, H-60a), 4.02 (dd, 1H, J = 10.1,
3.2 Hz, H-30), 3.99 (d, 1H, J = 9.6 Hz, H-1a), 3.88 (d,
1H, J = 12.4 Hz, H-60b), 3.74–3.67 (m, 3H, H-1b, H-
3, H-4), 3.63 (m, 1H, H-2), 3.56 (s, 1H, H-50), 1.67
(m, 1H), 1.53 (m, 1H), 1.41 (m, 1H), 1.31–1.20 (m,
16H), 0.88 (t, 3H, J = 6.9 Hz, CH3); 13C NMR
(CDCl3): d 138.7, 138.3, 138.0, 137.8, 128.9, 128.5,
128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7,
4. Experimental
4.1. General
Solvents were purified in a conventional manner. Thin-
layer chromatography (TLC) was performed on pre-

2008
N. Ding et al. / Carbohydrate Research 342 (2007) 2003–2013
127.6, 127.5, 126.3, 101.0 (PhCH), 99.1 (C10), 79.4
(C2), 78.9, 75.7 (C30), 75.4 (C20), 74.6 (C40), 73.7,
73.5, 72.0, 69.3 (C60), 68.4 (C1), 62.9 (C50), 61.8,
31.9, 30.2, 30.0, 29.7, 29.3, 25.4, 22.7, 14.1; HRMS:
(m/z) [M+Na]+: calcd for C59H75N3O8Na+ 976.5452,
found 976.5428.
idue was purified by column chromatography (15:1
CHCl3–MeOH) to furnish a white solid (3.1 g, 77.7%).
The solid was dissolved in dry DMF (20 mL), then benz-
aldehyde dimethyl acetal (2.5 g, 16.3 mmol) and TsOH
(125.4 mg, 0.7 mmol) were added. After rotating at
30 ꢁC for 2 h and 50 ꢁC for 2.5 h under reduced pres-
sure, the mixture was neutralized by addition of Et3N
and then evaporated to dryness. Silica gel column chro-
matography of the crude product, using 3:1 petroleum
ether–EtOAc as eluent, furnished 17 (3.22 g, 85.3%) as
a foamy solid: Rf 0.33 (2:1 petroleum ether–EtOAc);
4.3. (2S,3S,4R)-1-O-(2,3-Di-O-benzyl-4,6-O-benzylidene-
a-D-galactopyranosyl)-2-hexacosanoylamino-3,4-di-O-
benzyl-octadecantriol (16)
22
½aꢁD +219.4 (c 0.5, CHCl3); 1H NMR (CDCl3): d
To a solution of compound 6 (130 mg, 0.136 mmol) in
pyridine (5 mL) and water (0.5 mL) cosolvent system
was added PPh3 (71.5 mg, 0.27 mmol). The reaction mix-
ture was heated at 50 ꢁC for 5 h and concentrated. To a
solution of the crude amino product and hexacosanoic
acid (64.7 mg, 0.163 mmol) in dried CH2Cl2 (10 mL)
were added HOBt (23.9 mg, 0.177 mmol), EDCÆHCl
(33.9 mg, 0.177 mmol), and DIPEA (26.4 mg,
0.204 mmol) at 0 ꢁC under Ar. The reaction mixture
was stirred at rt for 22 h, and then concentrated. The
residue was partitioned between CH2Cl2 and water. The
organic layer was separated, washed with brine, dried
over MgSO4, and concentrated. The residue was purified
by column chromatography on silica gel (7:1 petroleum
ether–EtOAc) to furnish 16 (138.7 mg, 78.1%) as a color-
7.58–7.05 (14H, Ph), 5.41 (s, 1H, PhCH), 4.73 (d, 1H,
J = 12.4 Hz, PhCHH), 4.70 (d, 1H, J = 12.4 Hz,
PhCHH), 4.46 (d, 1H, J = 9.5 Hz, H-1), 4.35 (dd, 1H,
J = 12.4, 1.4 Hz, H-6a), 4.12 (d, 1H, J = 2.9 Hz, H-4),
3.97 (dd, 1H, J = 12.4, 1.4 Hz, H-6b), 3.87 (dd, 1H,
J = 9.54, 9.48 Hz, H-2), 3.50 (dd, 1H, J = 9.5, 2.9 Hz,
H-3), 3.44 (s, 1H, H-5), 2.46 (br s, 1H, 2-OH), 2.33 (s,
3H, CH3); 13C NMR (CDCl3): d 138.4, 138.0, 137.8,
134.4, 129.7, 129.0, 128.4, 128.1, 127.9, 126.6, 126.5,
101.1 (PhCH), 87.1 (C1), 80.2 (C3), 73.3 (C4), 71.6
(PhCH2), 70.0 (C5), 69.4 (C6), 67.1 (C2), 21.1 (CH3);
HRMS: (m/z) [M+Na]+: calcd for C27H29O5S+,
465.1736, found 465.1738.
4.5. p-Methylphenyl 3-O-benzyl-4,6-O-benzylidene-2-O-
p-methoxybenzyl-1-thio-b-D-galactopyranoside (18)
less oil: Rf 0.32 (7:1 petroleum ether–EtOAc); ½aꢁ22 +48.2
D
1
(c 0.6, CHCl3); H NMR (CDCl3): d 7.52–7.26 (25H,
Ph), 5.80 (d, 1H, J = 8.3 Hz, NH), 5.45 (s, 1H, PhCH),
4.94 (d, 1H, J = 3.2 Hz, H-10), 4.86–4.73 (8H, 4PhCH2),
4.28 (m, 1H), 4.17 (d, 1H, J = 3.2 Hz, H-40), 4.10 (d, 1H,
J = 12.4 Hz), 4.06 (dd, 1H, J = 10.1, 3.2 Hz, H-20), 3.94–
3.90 (m, 3H), 3.80–3.74 (m, 2H), 3.57 (s, 1H, H-50), 3.53
(m, 1H), 1.88 (m, 2H), 1.63 (m, 2H), 1.50–1.4 (m), 1.31–
1.20 (m, 16H), 0.88 (t, 6H, J = 6.9 Hz, CH3); 13C NMR
(CDCl3): d 172.5, 138.6, 138.5, 137.4, 128.3, 127.9, 109.6,
105.9, 87.3, 83.7, 79.9, 76.1, 73.4, 72.2, 71.9, 71.8, 66.3,
65.3, 49.3, 36.8, 31.9, 29.8–25.3, 22.7, 14.1; HRMS:
(m/z) [M+Na]+: calcd for C85H127NO9Na+ 1328.9409,
found 1328.9412.
Compound 17 (2.5 g, 5.4 mmol) was dissolved in DMF
(15 mL). The reaction mixture was cooled to 0 ꢁC.
Sodium hydride (60%, 318.0 mg, 8.0 mmol) was added,
followed by p-methoxybenzyl chloride (1.52 g, 9.7 mmol).
The reaction was monitored by TLC and shown to be
complete after 2 h at 0 ꢁC. The mixture was diluted with
water (50 mL) and extracted with CH2Cl2 (50 mL · 3).
The organic extracts were combined, dried over MgSO4,
and concentrated. Silica gel column chromatography of
the crude product, using 3:1 petroleum ether–EtOAc as
eluent, furnished 18 (2.7 g, 86.9%) as a white solid; Rf
22
0.41 (2:1 petroleum ether–EtOAc); ½aꢁD ꢀ33.7 (c 0.4,
CHCl3); 1H NMR (CDCl3): d 7.61–6.86 (18H, Ph),
5.45 (s, 1H, PhCH), 4.70 (d, 1H, J = 12.4 Hz, PhCHH),
4.68 (d, 1H, J = 12.4 Hz, PhCHH), 4.65 (d, 1H,
J = 12.4 Hz, PhCHH), 4.61 (d, 1H, J = 12.4 Hz,
PhCHH) 4.53 (d, 1H, J = 9.7 Hz, H-1), 4.32 (dd, 1H,
J = 12.4, 1.4 Hz, H-6a), 4.10 (d, 1H, J = 2.9 Hz, H-4),
3.92 (dd, 1H, J = 12.4, 1.4 Hz, H-6b), 3.82 (dd, 1H,
J = 9.7, 9.1 Hz, H-2), 3.78 (s, 3H, OCH3), 3.58 (dd,
1H, J = 9.2, 3.2 Hz, H-3), 3.32 (s, 1H, H-5), 2.28 (s,
3H, CH3); 13C NMR (CDCl3): d 159.2, 138.1, 137.9,
137.5, 133.3, 129.8, 129.6, 128.3, 128.0, 127.7, 126.6,
113.7, 101.2 (PhCH), 86.6 (C1), 81.4, 75.1, 75.0, 73.6,
71.7, 69.7, 69.4, 55.2 (OCH3), 21.1; HRMS: (m/z)
[M+Na]+: calcd for C35H37O6S+, 585.2311, found
585.2322.
4.4. p-Methylphenyl 3-O-benzyl-4,6-O-benzylidene-1-
thio-D-galactopyranoside (17)
Compound 13 (3.0 g, 10.5 mmol) and Bu2SnO (3.9 g,
15.7 mmol) were stirred in MeOH (30 mL) and toluene
(30 mL) at reflux under argon for 4 h. The solvent was
evaporated under reduced pressure. The residue was
dissolved in toluene (60 mL), then TBAB (0.68 g,
2.1 mmol) and BnBr (2.7g, 15.7 mmol) were added.
The reaction mixture was refluxed for 3 h. The solvent
was evaporated under reduced pressure, and the residue
was diluted with CH2Cl2 (100 mL) and washed with
water (50 mL · 2). The organic extracts were combined,
dried over MgSO4, and concentrated. The resulting res-

N. Ding et al. / Carbohydrate Research 342 (2007) 2003–2013
2009
4.6. (2S,3S,4R)-1-O-(3-O-Benzyl-4,6-O-benzylidene-2-O-
p-methoxybenzyl-a-D-galactopyranosyl)-2-azido-3,4-di-
O-benzyl-octadecantriol (20)
1.8 Hz, H-60a), 3.89 (dd, 1H, J = 12.4, 1.4 Hz, H-60b),
3.75 (dd, 1H, J = 10.1, 3.2 Hz, H-30), 3.73 (m, 2H, H-
1b, H-2), 3.67 (dd, 1H, J = 5.0, 4.1 Hz, H-3), 3.62 (m,
1H, H-4), 3.55 (s, 1H, H-50), 1.67 (m, 1H), 1.55 (m,
1H), 1.41 (m, 1H), 1.31–1.22 (m, 16H), 0.88 (t, 3H,
J = 6.9 Hz, CH3); 13C NMR (CDCl3): d 138.32,
138.26, 137.8, 137.7, 128.9, 128.5, 128.4, 128.1, 128.0,
127.8, 127.7, 126.2, 100.9 (PhCH), 100.1 (C10), 79.3
(C4), 79.2 (C3), 76.3 (C30), 73.9, 73.7 (C40), 72.1, 71.6,
69.3 (C60), 69.1 (C1), 68.0 (C20), 63.2 (C50), 62.0 (C2),
31.9, 30.0, 29.7, 29.6, 29.3, 25.3, 22.7, 14.1; HRMS:
(m/z) [M+Na]+: calcd for C52H69N3O8Na+ 886.4982,
found 886.4998.
To a mixture of glycosyl donor 18 (1.37 g, 2.4 mmol),
˚
acceptor 8 (1.05 g, 2.0 mmol) and 4 A MS (200 mg) in
1:5:1 CH2Cl2–Et2O–THF (63 mL) were added NIS
(723.2 mg, 3.2 mmol) and AgOTf (51.4 mg, 0.2 mmol)
at ꢀ20 ꢁC under Ar. The reaction mixture was stirred
for 30 min at ꢀ20 ꢁC and then warmed to 0 ꢁC. After
stirring for 12 h at 0 ꢁC the reaction mixture was concen-
trated. The residue was diluted with CH2Cl2 (50 mL)
and washed with 10% Na2S2O3 (50 mL · 1) and brine
(50 mL · 1), dried over MgSO4, and concentrated under
reduced pressure. The residue was chromatographed on
a silica gel column (7:1 petroleum ether–EtOAc) to give
the desired product 20 as a colorless oil (1.22 g, 61.9%):
4.8. (2S,3S,4R)-1-O-[6-O-Benzoyl-2,3,4-tri-O-benzyl-a/
b-D-galactopyranosyl-(1!2)-3-O-benzyl-4,6-O-benzyl-
idene-a-D-galactopyranosyl]-2-azido-3,4-di-O-benzyl-
octadecantriol (27)
22
Rf 0.67 (3:1 petroleum ether–EtOAc); ½aꢁD +44.3 (c 0.2,
CHCl3); 1H NMR (CDCl3): d 7.52–6.76 (24H, Ph), 5.45
(s, 1H, PhCH), 4.93 (d, 1H, J = 3.7 Hz, H-10), 4.82–4.48
(8H, 4PhCH2), 4.15 (d, 1H, J = 3.2 Hz, H-40), 4.07 (dd,
1H, J = 10.1, 3.7 Hz, H-20), 4.06 (dd, 1H, J = 12.4,
1.4 Hz, H-60a), 4.00 (m, 2H, H-30, H-1a), 3.88 (dd, 1H,
J = 12.4, 1.4 Hz, H-60b), 3.74 (s, 3H, OCH3), 3.74–
3.67 (m, 3H), 3.62 (m, 1H), 3.56 (s, 1H, H-50), 1.66
(m, 1H), 1.53 (m, 1H), 1.41 (m, 1H), 1.30–1.21 (m,
16H), 0.88 (t, 3H, J = 6.9 Hz, CH3); 13C NMR (CDCl3):
d 159.1, 138.8, 138.4, 138.0, 137.8, 130.8, 129.4, 128.9,
128.4, 128.3, 128.1, 127.9, 127.8, 127.7, 127.6, 127.5,
126.3, 113.6, 101.0 (PhCH), 99.2 (C10), 79.4, 78.9, 75.8
(C30), 75.0 (C20), 74.7 (C40), 73.8, 73.2, 72.1, 69.3
(C60), 68.4 (C1), 63.0 (C50), 61.8, 55.2 (OCH3), 31.9,
30.0, 29.7, 29.4, 25.4, 22.7, 14.1; HRMS: (m/z)
[M+Na]+: calcd for C60H77N3O9Na+ 1006.5558, found
1006.5594.
To
a
mixture of glycosyl donor 22 (52.8 mg,
˚
0.08 mmol), acceptor 7 (63.4 mg, 0.096 mmol) and 4 A
MS (20 mg) in 1:5 CH2Cl2–Et2O (3 mL) were added
NIS (28.9 mg, 0.128 mmol) and AgOTf (2.0 mg,
0.008 mmol) at ꢀ20 ꢁC under Ar. The reaction mixture
was stirred for 30 min at ꢀ20 ꢁC and then warmed to
0 ꢁC. After stirring for 12 h at 0 ꢁC, the reaction mix-
ture was concentrated. The residue was diluted with
CH2Cl2 (20 mL) and washed with 10% Na2S2O3
(20 mL · 1) and brine (20 mL · 1), dried over MgSO4,
and concentrated under reduced pressure. The residue
was chromatographed on a silica gel column (7:1 petro-
leum ether–EtOAc) to give the desired product 27
(84.9 mg, 75.8%) as a colorless oil: Rf 0.40 (7:1 petro-
1
leum ether–EtOAc); Representative H NMR (CDCl3)
signals: d 5.38 (s, 1H, PhCH-a), 5.35 (s, 0.33H,
PhCH-b), 5.23 (d, 0.33H, J = 3.7 Hz, H-10-b), 5.17 (s,
1H, H-100-a), 5.12 (d, 0.33H, J = 11.5 Hz, H-100-b)
5.10 (d, 1H, J = 3.7 Hz, H-10-a); 13C NMR (CDCl3):
d 165.9, 138.8–137.7, 133.1, 132.6, 129.8–126.3, 101.1,
99.9, 98.5, 97.3, 79.4–79.0, 74.9–68.9, 63.6, 62.9, 62.2,
31.9, 29.9–29.4, 25.2, 22.7, 14.1. a/b Mixtures of com-
pound 27 were easily separated by column chromato-
graphy on a silica gel column (250:1 toluene–acetone,
300–400 mesh) to give the desired a-27: Rf 0.10 (100:1
4.7. (2S,3S,4R)-1-O-(3-O-Benzyl-4,6-O-benzylidene-a-D-
galactopyranosyl)-2-azido-3,4-di-O-benzyl-octadecantriol
(7)
A solution of 20 (550.0 mg, 0.56 mmol) in CH2Cl2
(15 mL) was treated with 1 mL of water and DDQ
(190.4 mg, 0.84 mmol) and stirred at 0 ꢁC for 3 h. The
reaction mixture was poured into satd aq NaHCO3
(50 mL) and extracted with CH2Cl2 (50 mL · 2). The
organic extract was washed with satd aq NaHCO3
(50 mL · 2) and water, dried over MgSO4, and concen-
trated under reduced pressure. The residue was chro-
matographed on a silica gel column (5:1 petroleum
ether–EtOAc) to give the desired product 7 as a colorless
oil (410.1 mg, 84.8%): Rf 0.58 (3:1 petroleum ether–
22
toluene–acetone,); ½aꢁD ꢀ2.0 (c 0.68, CHCl3); 1H
NMR (CDCl3): d 7.86–7.12 (35H, Ph), 5.38 (s, 1H,
PhCH), 5.17 (s, 1H, H-100), 5.11 (d, 1H, J = 3.6 Hz,
H-10), 4.95 (d, 1H, J = 11.0 Hz, CHHPh), 4.82–4.47
(m, 13H, CHHPh), 4.40 (t, 1H, J = 6.4 Hz, H-500),
4.30 (dd, 1H, J = 10.1, 3.2 Hz, H-20), 4.24 (m, 2H, H-
600a, H-600b), 4.09 (s, 2H, H-200, H-300), 4.01 (dd, 1H,
J = 10.1, 3.2 Hz, H-1a), 3.98 (d, 1H, J = 12.4 Hz, H-
60a), 3.93 (dd, 1H, J = 10.1, 3.2 Hz, H-30), 3.87 (s,
1H, H-400), 3.80 (m, 1H, H-2), 3.77 (d, 1H,
J = 12.4 Hz, H-60b), 3.59 (m, 1H, H-4), 3.54 (dd, 1H,
J = 9.7, 9.6 Hz, H-1b), 3.48 (t, 1H, J = 5.0 Hz, H-3),
22
1
EtOAc); ½aꢁD +67.5 (c 0.4, CHCl3); H NMR (CDCl3):
d 7.52–7.25 (20H, Ph), 5.42 (s, 1H, PhCH), 5.03 (d, 1H,
J = 3.7 Hz, H-10), 4.76–4.53 (6H, 3PhCH2), 4.21 (dd,
1H, J = 10.1, 3.7 Hz, H-20), 4.15 (d, 1H, J = 3.2 Hz,
H-40), 4.11 (m, 1H, H-1a), 4.09 (dd, 1H, J = 12.4,

2010
N. Ding et al. / Carbohydrate Research 342 (2007) 2003–2013
3.46 (s, 1H, H-50), 1.61–1.53 (m, 4H), 1.30–1.20 (m,
the CH3ONa, the mixture was then diluted with
CH3OH, and the ion-exchange resin was filtered off.
The resin was washed thoroughly, and the filtrate was
concentrated. The residue was chromatographed on a
silica gel column (3:1 petroleum ether–EtOAc) to give
the desired product a-29 (216.1 mg, 85.7%) as a foamy
large band, alkyl chains), 0.88 (t, 3H, J = 6.8 Hz,
CH3); HRMS: (m/z) [M+Na]+: calcd for C86H101
-
N3O14Na+ 1422.7184, found 1422.7176.
22
4.9. (2S,3S,4R)-1-O-[2,3-Di-O-benzyl-4,6-O-benzylidene-
a-D-galactopyranosyl-(1!2)-3-O-benzyl-4,6-O-benzyl-
idene-a-D-galactopyranosyl]-2-azido-3,4-di-O-benzyl-
octadecantriol (28)
solid: Rf 0.35 (4:1 petroleum ether–EtOAc); ½aꢁD +82.4
1
(c 0.2, CHCl3); H NMR (CDCl3): d 7.57–7.26 (35H,
Ph), 5.46 (s, 1H, PhCH), 5.12 (s, 2H, H-10, H-100),
4.97–4.53 (12H, 6PhCH2), 4.34 (dd, 1H, J = 10.1,
3.2 Hz, H-20), 4.17 (d, 1H, J = 3.2 Hz, H-40), 4.12–4.01
(m, 6H), 3.87 (m, 1H), 3.83 (d, 1H, J = 12.4 Hz, H-
600b), 3.78 (s, 1H, H-400), 3.66 (m, 2H), 3.59 (s, 1H, H-
5 ), 3.56 (m, 2H), 3.34 (dd, 1H, J = 11.0, 5.0 Hz, H-
600b), 1.68 (m, 1H), 1.59 (m, 1H), 1.35–1.28 (m, large
band, alkyl chains), 0.94 (t, 3H, J = 6.8 Hz, –CH3);
13C NMR (CDCl3): d 138.8, 138.7, 138.4, 138.3, 138.2,
137.7, 128.8 128.3–127.4, 126.1, 100.7, 98.2, 96.8, 79.2,
78.9, 76.4, 75.3, 74.6, 74.3, 73.9, 73.3, 73.0, 72.9, 72.7,
72.0, 71.4, 70.6, 69.2, 69.1, 62.8, 62.4, 62.1, 31.8, 29.9,
29.7-29.6, 29.3, 25.1, 22.6, 17.9, 14.1; HRMS: (m/z)
[MꢀN2+Na]+: calcd for C79H97NO13Na+ 1290.6858,
found 1290.6856.
To
a
mixture of glycosyl donor 23 (44.3 mg,
0.08 mmol), acceptor 7 (63.4 mg, 0.096 mmol) and
˚
4 A MS (20 mg) in 1:5 CH2Cl2–Et2O (3 mL) were
added NIS (28.9 mg, 0.128 mmol) and AgOTf
(2.0 mg, 0.008 mmol) at ꢀ20 ꢁC under Ar. The reaction
mixture was stirred for 30 min at ꢀ20 ꢁC and then
warmed to 0 ꢁC. After stirring for 12 h at 0 ꢁC, the
reaction mixture was concentrated. The residue was
diluted with CH2Cl2 (20 mL) and washed with 10%
Na2S2O3 (20 mL · 1) and brine (20 mL · 1), dried over
MgSO4, and concentrated under reduced pressure. The
residue was chromatographed on a silica gel column
(7:1 petroleum ether–EtOAc) to give the desired prod-
uct 28 (56.3 mg, 54.4%) as a colorless oil: Rf 0.68 (3:1
petroleum ether–EtOAc); 1H NMR (CDCl3): d 7.85–
7.00 (35H, Ph), 5.46 (s, 1H, PhCH), 5.34 (s, 1H,
PhCH), 5.12 (s, 1H, H-100), 5.07 (d, 1H, J = 3.3 Hz,
H-10), 4.81-4.47 (5 PhCH2), 4.29 (dd, 1H, J = 10.1,
3.2 Hz, H-20), 4.18 (d, 1H, J = 2.8 Hz, H-40), 4.05 (s,
2H, H-200, H-300), 4.01 (dd, 1H, J = 10.1, 3.7 Hz, H-
1a), 3.99 (d, 1H, J = 12.4 Hz, H-60a), 3.95 (dd, 1H,
J = 10.6, 3.2 Hz, H-30), 3.91 (d, 1H, J = 12.8 Hz, H-
600a), 3.90 (s, 2H, H-400, H-500), 3.82 (m, 2H, H-60b,
H-2), 3.58 (m, 1H, H-4, H-1b), 3.56 (s, 1H, H-50),
3.51 (d, 1H, J = 12.4 Hz, H-600b), 3.48 (dd, 1H,
J = 5.5, 4.6 Hz, H-3), 1.68–1.20 (m, large band, alkyl
chains), 0.88 (t, 3H, J = 6.9 Hz, CH3); 13C NMR
(CDCl3): d 138.9, 138.5, 138.3, 138.0, 137.7, 132.0,
128.9, 128.7, 128.4–127.4, 126.4, 126.3, 114.3, 100.9
(2C), 98.3, 97.4, 79.3, 79.2, 76.0, 75.6, 74.7, 74.5,
73.9, 73.5, 73.3, 72.9, 72.1, 71.8, 71.1, 69.3, 69.2,
62.9, 62.7, 62.2, 55.6, 31.9, 29.9–29.4, 25.2, 22.7, 14.1;
HRMS: (m/z) [M+Na]+: calcd for C79H95N3O13Na+
1316.6765, found 1316.6767.
5.1. (2S,3S,4R)-1-O-[2,3,4-Tri-O-acetyl-a-L-rhamnopyr-
anosyl-(1!3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-b-
D-galactopyranosyl-(1!6)-2,3,4-tri-O-benzyl-a-D-galac-
topyranosyl-(1!2)-3-O-benzyl-4,6-O-benzylidene-a-D-
galactopyranosyl]-2-azido-3,4-di-O-benzyl-octadecantriol
(30)
To
a mixture of a-29 (216.1 mg, 0.17 mmol), 6
˚
(167.4 mg, 0.20 mmol), and 4 A molecular sieves
(50 mg) in dried CH2Cl2 (15 mL) were added TMSOTf
(3.7 mg, 0.017 mmol) at ꢀ20 ꢁC under Ar protection.
The mixture was stirred under these conditions for 1 h,
then neutralized with Et3N. The solid was then filtered
off, and the filtrate was concentrated under vacuum to
give a yellow syrup, which was purified by column chro-
matography (3:1 petroleum ether–EtOAc) to give com-
pound 30 (240.0 mg, 75.9%) as a foamy solid: Rf 0.45
(3:2 petroleum ether–EtOAc); ½aꢁ2D2 +45.8 (c 0.1, CHCl3);
1H NMR (CDCl3): d 7.70–7.25 (39H, Phth, Ph), 5.34 (d,
1H, J = 3.2 Hz, H-4000), 5.29 (s, 1H, PhCH), 5.03 (d, 1H,
J = 8.2 Hz, H-1000), 5.01 (dd, 1H, J = 7.7, 3.2 Hz), 4.99
(d, 1H, J = 3.2 Hz, Gal H-1), 4.96 (d, 1H, J = 3.2 Hz,
Gal H-1), 4.92 (dd, 1H, J = 10.1, 9.6 Hz, H-4IV), 4.92
(d, 1H, J = 4.3 Hz), 4.75 (d, 1H, J = 11.0 Hz, PhCHH),
4.70 (d, 1H, J = 11.9 Hz, PhCHH), 4.63 (m, 4H), 4.60
(br s, 1H, H-1IV), 4.56 (m, 2H), 4.52–4.45 (m, 4H),
4.38 (m, 2H), 4.16–4.11 (m, 2H), 4.07 (dd, 1H,
J = 11.5, 6.0 Hz), 4.04 (dd, 1H, J = 9.7, 9.6 Hz), 3.96–
3.86 (m, 7H), 3.82 (br s, 1H), 3.77 (dd, 1H, J = 10.1,
3.2 Hz), 3.71 (m, 2H), 3.59 (t, 1H, J = 6.4 Hz), 3.54
(m, 2H), 3.47 (t, 1H, J = 9.6 Hz), 3.41 (m, 2H), 2.18
5. (2S,3S,4R)-1-O-[2,3,4-Tri-O-benzyl-a-D-galactopyr-
anosyl-(1!2)-3-O-benzyl-4,6-O-benzylidene-a-D-galacto-
pyranosyl]-2-azido-3,4-di-O-benzyl-octadecantriol (a-29)
Compound a-27 (272.4 mg, 0.20 mmol) was dissolved in
dry CH3OH (2 mL) and dry CH2Cl2 (2 mL). CH3ONa
(99%, 57 mg) was added, and the mixture was stirred
for 20 h. After almost all starting material was con-
sumed, Amberlyst 15 (H+-form) was added to neutralize

N. Ding et al. / Carbohydrate Research 342 (2007) 2003–2013
2011
(s, 3H, CH3CO), 2.01 (s, 3H, CH3CO), 1.97 (s, 3H,
CH3CO), 1.86 (s, 3H, CH3CO), 1.83 (s, 3H, CH3CO),
1.60 (m, 1H), 1.50 (m, 1H), 1.31–1.20 (broad band, alkyl
chain protons), 1.17 (d, 3H, J = 6.4 Hz, CH3-6IV), 0.88
(t, 3H, J = 6.8 Hz, CH3); 13C NMR (CDCl3): d 170.5,
170.4, 170.1, 169.4, 169.3, 169.0, 167.3, 139.1, 138.9,
138.8, 138.7, 138.3, 137.8, 137.7, 134.0, 133.8, 131.4,
131.0, 128.9, 128.4-127.2, 126.3, 123.7, 123.1, 101.1
(PhCH), 98.6, 98.5, 98.1, 97.7, 79.2, 79.1, 78.7, 76.1,
75.0, 74.65, 74.56, 74.3, 74.2, 73.5, 73.4, 73.2, 72.0,
71.9, 71.8, 71.0, 70.6, 69.9, 69.3, 69.1, 68.6, 68.2, 68.1,
67.3, 65.2, 62.8, 62.1, 61.8, 53.0, 31.9, 29.8-29.6, 29.3,
25.2, 22.7, 20.8, 20.7, 20.6, 20.5, 17.3, 14.1; HRMS:
17.3, 14.1; HRMS: (m/z) [M+H]+: calcd for
þ
C
109H131N4O28 1855.8923, found 1856.1011.
5.3. (2S,3S,4R)-1-O-[2,3,4-Tri-O-acetyl-a-L-rhamnopyr-
anosyl-(1!3)-4,6-di-O-acetyl-2-deoxy-2-acetamido-b-D-
galactopyranosyl-(1!6)-2,3,4-tri-O-benzyl-a-D-galacto-
pyranosyl-(1!2)-3-O-benzyl-4,6-O-benzylidene-a-D-
galactopyranosyl]-2-[N-(R)-2-acetoxydocosanoylamino]-
3,4-di-O-benzyl-octadecantriol (32)
Compound 31 (80.0 mg, 0.043 mmol) was dissolved in
pyridine (3 mL) and H2O (3 mL). H2S, generated from
FeS and 3 M H2SO4, was bubbled in over a period of
1 h. The reaction was kept sealed overnight. The solvent
was removed in vacuo. The resulting residue was dis-
solved in CH2Cl2 (2 mL), and 5 (17.5 mg, 0.044 mmol),
HOBt (6.5 mg, 0.048 mmol), EDCÆHCl (9.2 mg,
0.048 mmol), DIPEA (7.6 mg, 0.060 mmol) were added.
The reaction mixture was stirred under argon overnight.
Solvent was removed in vacuo, and the resulting residue
was purified by column chromatography (3:1 petroleum
ether–EtOAc) to give compound 32 (62.9 mg, 66.0%) as
a colorless oil: Rf 0.51 (1:1 petroleum ether–EtOAc);
(m/z) [M+H]+: calcd for C109H131N4O
1943.8950,
þ
28
found 1943.8872.
5.2. (2S,3S,4R)-1-O-[2,3,4-Tri-O-acetyl-a-L-rhamnopyr-
anosyl-(1!3)-4,6-di-O-acetyl-2-deoxy-2-acetamido-b-D-
galactopyranosyl-(1!6)-2,3,4-tri-O-benzyl-a-D-galacto-
pyranosyl-(1!2)-3-O-benzyl-4,6-O-benzylidene-a-D-
galactopyranosyl]-2-azido-3,4-di-O-benzyl-octadecantriol
(31)
22
1
A solution of 30 (271.0 mg, 0.14 mmol) in ethylenedi-
amide (4 mL) and n-BuOH (8 mL) was heated at 90 ꢁC
for 6 h. The reaction mixture was cooled and concen-
trated under diminished pressure. The residue was then
dissolved in pyridine (3 mL) and Ac2O (3 mL). The
mixture was stirred at rt for 12 h, MeOH (5 mL) was
added, and the mixture was then concentrated to give
a residue that was purified by column chromatography
(3:2 petroleum ether–EtOAc) to give compound 31
(185.6 mg, 71.7%) as a foamy solid: Rf 0.80 (1:1 petro-
½aꢁD +0.3 (c 0.3, CHCl3); H NMR (CDCl3): d 7.48–
7.15 (35H, Ph), 6.64 (d, 1H, J = 8.7 Hz, NH), 5.41 (s,
1H, PhCH), 5.30 (d, 1H, J = 6.8 Hz, NH), 5.22 (d,
1H, J = 3.2 Hz), 5.10 (dd, 1H, J = 10.1, 3.7 Hz), 5.06
(dd, 1H, J = 3.2, 1.4 Hz), 5.03 (d, 1H, J = 10.1,
9.6 Hz), 5.00 (d, 1H, J = 3.7 Hz, sugar H-1), 4.97 (dd,
1H, J = 7.8, 4.6 Hz), 4.96 (d, 1H, J = 4.1 Hz, sugar H-
1), 4.89 (d, 1H, J = 11.0 Hz), 4.79 (d, 1H,
J = 12.8 Hz), 4.75 (d, 1H, J = 8.2 Hz, H-1000), 4.73 (d,
1H, J = 11.9 Hz), 4.70–4.50 (m, 11H), 4.67 (m, 1H,
sugar H-1), 4.40 (m, 1H, H-2V), 4.21 (dd, 1H, J = 6.8,
6.4 Hz), 4.17 (dd, 1H, J = 10.5, 3.2 Hz), 4.14–4.10 (m,
4H), 4.08 (d, 1H, J = 12.8 Hz, H-6a0), 4.04 (dd, 1H,
J = 11.5, 5.0 Hz), 4.02 (dd, 1H, J = 10.1, 3.2 Hz),
3.99–3.92 (m, 4H), 3.88–3.84 (m, 4H), 3.82 (d, 1H,
J = 11.9 Hz, H-6b0), 3.69 (dd, 1H, J = 6.9, 6.4 Hz),
3.62 (dd, 1H, J = 4.6, 4.1 Hz), 3.54 (m, 1H), 3.51 (s,
1H, H-50), 3.47 (dd, 1H, J = 10.1, 6.8 Hz), 2.92 (m,
1H), 2.15 (s, 3H, CH3CO), 2.10 (s, 3H, CH3CO), 2.06
(s, 3H, CH3CO), 2.02 (s, 3H, CH3CO), 2.00 (s, 3H,
CH3CO), 1.97 (s, 3H, CH3CO), 1.76 (m, 1H), 1.57 (s,
3H, CH3CONH), 1.40 (m, 1H), 1.30–1.21 (broad band,
alkyl chain protons), 1.18 (d, 3H, J = 6.4 Hz, CH3-6IV),
0.88 (t, 6H, J = 6.8 Hz, CH3); 13C NMR (CDCl3): d
202.9, 172.03,171.21, 170.49, 170.46, 170.28, 170.18,
170.12, 170.04, 139.1, 138.61, 138.58, 138.46, 138.39,
138.20, 137.9, 129.1, 128.6, 128.41, 128.38, 128.32,
128.20, 127.99, 127.96, 127.77, 127.74, 127.70, 127,67,
127.56, 127.30, 127.21, 126.1, 100.7, 99.5, 99.3, 98.3,
95.9, 80.3, 79.5, 78.9, 75.8, 75.0, 74.6, 74.3, 73.3, 73.1,
72.9, 72.6, 72.1, 72.0, 70.8, 70.1, 69.9, 69.4, 69.3, 69.0,
68.8, 67.1, 63.1, 62.0, 60.4, 55.4, 50.6, 32.1, 31.9, 30.4,
29.8, 29.7, 29.6, 29.4, 25.8, 25.1, 23.2, 22.7, 21.1, 20.9,
22
leum ether–EtOAc); ½aꢁD +53.3 (c 0.2, CHCl3); 1H
NMR (CDCl3): d 7.51–7.26 (35H, Ph), 5.45 (d, 1H,
J = 6.8 Hz, NH), 5.41 (s, 1H, PhCH), 5.22 (d, 1H,
J = 3.2 Hz), 5.12–5.10 (m, 3H), 5.07 (m, 2H, sugar
H-1, sugar H-1), 5.04 (d, 1H, J = 9.6 Hz, H-4IV), 4.94
(d, 1H, J = 2.3 Hz), 4.92 (d, 1H, J = 4.3 Hz), 4.80–
4.58 (m, 11H), 3.52–4.47 (m, 3H), 4.21–4.17 (m, 2H),
4.06–4.03 (m, 4H), 3.98–3.91 (m, 5H), 3.83 (dd, 1H,
J = 10.1, 5.0 Hz), 3.82 (m, 1H), 3.79–3.76 (m, 2H),
3.71 (dd, 1H, J = 6.9, 6.4 Hz), 3.57 (m, 1H), 3.52–
3.48 (m, 3H), 3.45 (t, 1H, J = 5.0 Hz), 2.85 (m, 1H),
2.11 (s, 3H, CH3CO), 2.08 (s, 3H, CH3CO), 2.06 (s,
3H, CH3CO), 2.00 (s, 3H, CH3CO), 1.98 (s, 3H,
CH3CO), 1.28–1.23 (broad band, alkyl chain protons),
1.18 (d, 3H, J = 6.4 Hz, CH3-6IV), 0.88 (t, 3H,
J = 6.9 Hz, CH3); 13C NMR (CDCl3): d 170.5, 170.2,
170.03, 170.00, 139.1, 138.8, 138.7, 138.6, 138.3,
137.8, 137.7, 129.1, 128.6–128.2, 127.9, 127.7, 127.6,
127.4, 127.2, 126.1, 100.7 (PhCH), 99.4, 98.3, 98.2,
97.5, 79.3, 79.0, 76.3, 75.1, 75.0, 74.5, 73.9, 73.5,
73.2, 73.0, 72.6, 72.5, 72.0, 70.7, 70.1, 69.9, 69.7,
69.2, 68.9, 68.8, 67.1, 63.0, 62.3, 62.0, 55.8,
31.9, 29.9-29.7, 29.4, 25.2, 23.3, 22.7, 20.9, 20.8, 20.7,

2012
N. Ding et al. / Carbohydrate Research 342 (2007) 2003–2013
20.8, 20.71, 20.68, 17.3, 14.21, 14.15; HRMS: (m/z)
Supplementary data
[M+H]+: calcd for C127H176N2O30 2232.2208, found
þ
2232.2200.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2007.05.018.
5.4. Clarhamnoside peracetate 2
References
To a solution of 32 (50.0 mg, 0.036 mmol) in MeOH
(1 mL) and EtOAc (3 mL) was added Pd(OH)2/C (75%
wt% Pd dry basis on carbon, 25.0 mg), and the mixture
was hydrogenated at rt for 12 h. The suspension was fil-
tered through a Celite pad, and the filter cake was rinsed
with 5:1 CHCl3–MeOH (10 mL). The combined filtrate
and washings were concentrated in vacuo. The residue
was then dissolved in pyridine (1 mL) and Ac2O
(1 mL). After stirring at rt overnight, the excess Ac2O
was decomposed by adding MeOH. The mixture was
concentrated in vacuo, and the resulting residue was
purified by column chromatography (3:1 petroleum
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22
leum ether–EtOAc); ½aꢁD +38 (c 0.43, CHCl3); the diag-
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(m/z) [M+H]+: calcd for C94H153N2O
1918.0132,
þ
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found 1918.0101.
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gave a foamy solid (6.90 mg, quant): Rf 0.32 (4:1
22
CHCl3–MeOH); ½aꢁ +22 (c 0.2, CHCl3); 1H NMR
D
(pyridine-d5): d 9.04 (d, 1H, J = 9.2 Hz, NH), 8.59 (d,
1H, J = 9.6 Hz, NH), 5.78 (s, 1H, H-1IV), 5.65 (dd,
1H, J = 3.2 Hz, H-10), 5.56 (dd, 1H, J = 2.8 Hz, H-100),
5.26 (d, 1H, J = 7.8 Hz, H-1000), 5.25–4.25 (m, over-
lapped), 3.96 (m, 1H, H-5000), 2.24–1.62 (m), 1.57 (d,
3H, J = 5.9 Hz, H-6IV), 1.40–1.20 (m, large band, alkyl
chains), 0.89 (m, 6H, CH3); 13C NMR (pyridine-d5): d
175.2 (C@O), 171.6 (C@O), 104.2, 102.4, 98.8, 98.0,
79.7, 76.6, 75.8, 75.4, 73.8, 72.9, 72.8, 72.7, 72.3, 71.1–
70.1, 69.6, 69.2, 68.4, 62.3, 62.0, 57.3, 53.4, 50.9, 35.4,
33.6, 32.0, 30.3–29.5, 26.9, 26.6, 25.8, 23.2, 22.8, 19.1,
18.4, 14.2; HRMS (MALDI-TOF): (m/z) [M+Na]+:
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