Synthesis of LeaLex oligosaccharide fragments and efficient one-step deprotection

Article abstract of DOI:10.1016/j.carres.2010.03.038

We describe here the synthesis of two oligosaccharide fragments of the tumor associated carbohydrate antigen Le^aLe^x. While the linear lacto-N-triose I: β-d-Galp-(1→4)-β-d-GlcNAcp-(1→3)-β-d-Galp-OMe is a known compound, this is the first reported preparation of the branched tetrasaccharide β-d-GlcNAcp-(1→3)-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-β-d-GlcNAcp-OMe. Our synthetic schemes involved using an N-trichloroacetylated trichloroacetimidate glucosaminyl donor activated with excess TMSOTf at 0 °C for glycosylation at O-3 of galactosyl residues and that of trichloroacetimidate galactosyl donors activated with excess BF₃·OEt₂ to glycosylate either O-3 or O-4 of glucosamine residues. The fucosylation at O-3 of the glucosamine acceptor was accomplished using a thiofucoside donor activated with copper(II) bromide and tetrabutylammonium bromide. Thus, syntheses of the protected tri- and tetrasaccharides were achieved easily and efficiently using known building blocks. Of particular interest, we also report that these protected oligosaccharides were submitted to dissolving metal conditions (Na-NH₃) to provide in one single step the corresponding deprotected compounds. Under these conditions all protecting groups (O-acyl, benzylidene, benzyl, and N-trichloroacetyl) were efficiently cleaved. The work-up procedure for such reactions usually involves quenching with excess methanol and then neutralization with acetic acid. In our work the neutralization was carried out using acetic anhydride rather than acetic acid to ensure N-acetylation of the glucosamine residue. Both fully deprotected compounds were then simply purified and desalted by gel permeation chromatography on a Biogel P2 column eluted with water.

Full text of DOI:10.1016/j.carres.2010.03.038

Carbohydrate Research 345 (2010) 1216–1221
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis of LeaLex oligosaccharide fragments and efficient
one-step deprotection
*
An Wang, France-Isabelle Auzanneau
Department of Chemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 March 2010
Received in revised form 25 March 2010
Accepted 28 March 2010
Available online 8 April 2010
We describe here the synthesis of two oligosaccharide fragments of the tumor associated carbohydrate
antigen LeaLex. While the linear lacto-N-triose I: b-
D
-Galp-(1?4)-b-
a known compound, this is the first reported preparation of the branched tetrasaccharide b-
(1?3)-b- -Galp-(1?4)-[ -Fucp-(1?3)]-b- -GlcNAcp-OMe. Our synthetic schemes involved using an
D
-GlcNAcp-(1?3)-b-
D
-Galp-OMe is
D
-GlcNAcp-
D
a-
L
D
N-trichloroacetylated trichloroacetimidate glucosaminyl donor activated with excess TMSOTf at 0 °C
for glycosylation at O-3 of galactosyl residues and that of trichloroacetimidate galactosyl donors activated
with excess BF3ꢀOEt2 to glycosylate either O-3 or O-4 of glucosamine residues. The fucosylation at O-3 of
the glucosamine acceptor was accomplished using a thiofucoside donor activated with copper(II) bro-
mide and tetrabutylammonium bromide. Thus, syntheses of the protected tri- and tetrasaccharides were
achieved easily and efficiently using known building blocks. Of particular interest, we also report that
these protected oligosaccharides were submitted to dissolving metal conditions (Na–NH3) to provide
in one single step the corresponding deprotected compounds. Under these conditions all protecting
groups (O-acyl, benzylidene, benzyl, and N-trichloroacetyl) were efficiently cleaved. The work-up proce-
dure for such reactions usually involves quenching with excess methanol and then neutralization with
acetic acid. In our work the neutralization was carried out using acetic anhydride rather than acetic acid
to ensure N-acetylation of the glucosamine residue. Both fully deprotected compounds were then simply
purified and desalted by gel permeation chromatography on a Biogel P2 column eluted with water.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Lacto-N-triose I
N-Acetylglucosamine
Oligosaccharide synthesis
Dissolving metal deprotections
The hexasaccharide LeaLex has been shown to be over-ex-
pressed at the surface of squamous carcinoma cells and as such
has been identified as a Tumor Associated Carbohydrate Antigen
(TACA).1 Our research efforts aim at discovering epitopes dis-
played by this antigen at the surface of tumor cells to eventually
develop an anti-cancer vaccine based on this hexasaccharide and
that would no-longer display the Lea trisaccharide also largely ex-
pressed on non-cancerous cells. In this context, we have recently
our knowledge, we are reporting here the first chemical synthesis
of tetrasaccharide 3. The synthetic approach that Matta and
co-workers followed for the preparation of trisaccharide 2 relied
on the use of an oxazoline glycosyl donor to form the b-
D-Glc-
NAcp-(1?3)- -Galp glycosidic bond. In our syntheses of tri- and
D
tetrasaccharides 2 and 3 we chose to use the more reactive N-tri-
chloroacetylated trichloroacetimidate glycosyl donor 4 introduced
by Blatter et al.6
described the synthesis of the trisaccharide fragment
a-
L-Fucp-
Thus, the known5 trisaccharide 2 was prepared in five steps
(Scheme 1) from the known monosaccharide building blocks 4,6
5,7 and 8.8 Under activation with 1 equiv of TMSOTf at 0 °C cou-
pling of acceptor 5 with donor 4 (2.5 equiv) was achieved in 70%
yield. The disaccharide 6 was then converted in two steps to the
acceptor 7. Applying conditions that were used previously in our
group for a similar disaccharide,2 the acetyl groups were first selec-
tively removed by methanolysis at room temperature using a 1 M
solution of HCl in MeOH (AcCl–MeOH). The resulting crude triol
was then selectively protected at positions 4 and 6 by reaction
with benzaldehyde dimethylacetal (3 equiv) under camphorsulfo-
nic acid catalysis in acetonitrile at 50 °C. Acceptor 7 was isolated
in an acceptable 56% yield over the two steps and was, in turn,
glycosylated with the galactosyl donor 8. Coupling of donor 8
(5 equiv) with acceptor 7 was carried out at room temperature in
(1?4)-b- -GlcpNAc-(1?3)-b- -Galp-OMe and established through
D
D
molecular mechanics and NMR experiments that it was highly
flexible. We describe here the synthesis of two additional frag-
ments of the LeaLex hexasaccharide: the so-called3 lacto-N-triose
I trisaccharide (2) and the branched fucosylated tetrasaccharide
fragment 3 (Chart 1).
Lacto-N-triose I was first isolated following partial hydrolysis of
the human milk tetrasaccharide lacto-N-tetraose3 and was later
purified by Morgan and co-workers from a partial acid-hydrolysate
of blood group substances.4 While the synthesis of trisaccharide 2
has, in turn, been reported by Matta and co-workers,5 to the best of
* Corresponding author. Tel: +1 519 824 4120x53809; fax: +1 519 766 1499.
E-mail address: fauzanne@uoguelph.ca (F.-I. Auzanneau).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.03.038

A. Wang, F.-I. Auzanneau / Carbohydrate Research 345 (2010) 1216–1221
1217
HO
OH
OH
O
OH
OH
OH
HO OH
O
HO
O
OH
HO
HO
OH
HO
HO
OH
O
O
O
O
HO
O
O
O
O
OMe
O
O
O
O
O
OH
NHAc
OH
NHAc
OH
NHAc
OH
O
2
OH
OH
HO
HO
1
OH
OH
HO
O
OH
O
O
O
O
HO
OMe
O
OH
NHAc
NHAc
O
OH
OH
3
HO
Chart 1.
dichloromethane under promotion by 2 equiv of BF3ꢀOEt2. Flash
column chromatography followed by centrifugal chromatography
of the impure fractions gave the protected trisaccharide 9 in 64%
yield.
55% yield after gel permeation chromatography (2 ꢂ Biogel P2,
100 cm ꢂ 1 cm, water). The NMR data recorded for trisaccharide
2 were in total agreement with the data reported for lacto-N-tri-
ose I (Scheme 2).5
In our quest for an efficient one-step deprotection procedure,
we investigated the removal of all protecting groups from trisac-
charide 9 using sodium–liquid ammonia in THF at ꢁ78 °C (Birch
reduction conditions). Indeed, expanding on the work by Seeber-
ger and co-workers,9 we have reported that Birch reduction con-
ditions led not only to the easy removal of O-benzyl and O-acyl
groups but also to the concurrent reduction of 6-chlorohexyl gly-
cosides to the corresponding hexyl glycosides.10 Thus, we postu-
lated that under these conditions the O-acyl groups would be
removed, the benzylidene group reductively cleaved, and that
the N-trichloroacetyl group would be either reduced to the cor-
responding acetamido or removed to give the corresponding free
amine. Because it is more likely that, under such basic condi-
tions, the N-trichloroacetyl group would be removed rather than
de-chlorinated, the work-up procedure was modified to ensure
N-acetylation of the resulting free amine. Thus, after quenching
of the reaction with methanol and evaporation of the liquid
ammonia at room temperature, the resulting mixture was trea-
ted with an excess acetic anhydride (1.3 equiv to the sodium
added) rather than neutralized with acetic acid. After concentra-
tion, the deprotected trisaccharide 2 did not require silica gel
chromatography and was obtained pure and free of salts in
The trisaccharide 3 was prepared in six steps from the donor
1010 and acceptor 11,11 which we have reported previously and
from the known donors 46 and 17.12 The coupling of acceptor 11
with trichloroacetimidate donor 10 was carried out under condi-
tions that we have established for the efficient glycosylation at
the poorly reactive O-4 position of N-acetylated glucosamine gly-
cosyl acceptors.11,13 Thus, donor 10 (5 equiv) was activated with
2 equiv of BF3ꢀOEt2 and was allowed to react with acceptor 11 for
1 h at 40 °C. Upon flash chromatography (EtOAc and hexanes,
8:2) the desired disaccharide 12 was obtained in 60% yield (esti-
mated by 1H NMR) contaminated with unreacted acceptor 11 in
a 3:1 ratio. Centrifugal chromatography of this mixture using a
CHCl3–MeOH (20:1) eluent provided the pure desired disaccharide
12 in 54% yield.
This mediocre yield was in sharp contrast with the ꢃ90% yield
that we have reported for the glycosylation of acceptor 11 with
a
-trichloroacetimidate peracetylated glucopyranose under the
same coupling conditions.11 The observation that some acceptor re-
mained unreacted suggests that galactosylation at O-4 of acceptor
11 is more difficult to achieve than its glucosylation. Indeed, other
galactosylations at O-4 of N-acetylglucosamine acceptors that we
have recently described10 using the same coupling conditions also
OR2 BzO
OBz
OAc
BzO
HO
OBz
O
O
R2O
R1O
O
O
AcO
AcO
TMSOTf
0 ºC
+
OMe
OMe
O
OBz
TCAHN
TCAHN
OBz
O
CCl3
6 R1, R2 = Ac
7 R1= H, R2 = PhC<
1. HCl/MeOH
2. PhCH(OMe)2, H+
NH
5
4
AcO OAc
BzO
O
OBz
Ph
O
.
AcO
AcO
OAc
BF3 OEt2
rt
O
O
O
Na/NH3(l)
7 +
O
O
O
OMe
2
AcO
AcO
OBz
TCAHN
O
CCl3
AcO
8
9
NH
Scheme 1. Synthesis of trisaccharide 2.

1218
A. Wang, F.-I. Auzanneau / Carbohydrate Research 345 (2010) 1216–1221
OAc
O
AcO
ClAcO
OBn
OAc
AcO
R2O
OBn
.
O
O
HO
ClAcO
BF3 OEt2
40 oC
+
O
O
OMe
R1O
AcO
OMe
AcHN
O
NH
CCl3
AcO
AcHN
11
10
12 R1, R2 = ClAc
13 R1 = ClAc , R2 = H
14 R1 , R2 = H
(H2N)2CS
OAc
OAc
AcO
BnO
O
O
TMSOTf
0 oC
AcO
O
O
13 +
4
O
AcO
RO
OMe
AcO
TCAHN
(H2N)2CS
AcHN
15 R = ClAc
16 R = H
OAc
OAc
AcO
BnO
O
O
O
SEt
O
CuBr2
O
AcO
AcO
O
Na/NH3(l)
16 +
BnO
OBn
O
OMe
3
Bu4NBr, rt
AcO
OBn
OBn
17
AcHN
TCAHN
O
OBn
BnO
18
Scheme 2. Synthesis of tetrasaccharide 3.
led to maximum yields around ꢃ70%. Observing that the chloroace-
tyl group at O-30 of disaccharide 12 was less sterically hindered
than that at O-3, we reasoned that it should be possible to
selectively remove it using controlled reaction conditions to give
acceptor 13. Indeed, best yields of acceptor 13 were obtained
applying conditions established by Naruto et al.,14 that is, treating
disaccharide 12 with thiourea (1.3 equiv) and sodium bicarbonate
(20 mg/mL) in ethanol at 70 °C (4 h). Under these conditions, the
desired acceptor 13 was isolated in 65% yield while the diol 14
was obtained in 13% yield.
Disaccharide acceptor 13 was, in turn, glycosylated with the
N-trichloroacetylated trichloroacetimidate donor 4. This coupling
gave best results when it was carried out in dichloromethane
using a large excess of TMSOTf (2 equiv) at 0 °C for 1 h. Under
these conditions, trisaccharide 15 was isolated in 74% yield after
two chromatographic purification steps (CHCl3–MeOH, 20:1; then
EtOAc–hexanes, 9:1). It is worth mentioning that lower concentra-
tions of TMSOTf, as well as using 2 equiv of BF3ꢀOEt2 at 40 °C, did
not lead to acceptable yields of the desired trisaccharide but to the
rapid formation of the corresponding known6 oxazoline, which
was then unreactive as a glycosyl donor under these conditions.
Removal of the O-3 chloroacetate from trisaccharide 15 was
accomplished easily using thiourea in a pyridine–ethanol mix-
ture at 65 °C and gave trisaccharide acceptor 16 in 76%. Finally,
coupling of acceptor 16 with fucosyl donor 17 proceeded well
under activation with copper(II) bromide and tertabutylammoni-
um bromide at room temperature and the desired protected tet-
ammonia the mixture was treated with acetic anhydride to neu-
tralize the sodium methoxide formed and to acetylate the C-2000
free amine. The desired deprotected tetrasaccharide 3 was then
purified and desalted by passing twice on a gel permeation col-
umn (Biogel P2) eluted with water and was isolated in 72% yield.
In conclusion, syntheses of the protected tri- and tetrasaccha-
rides were achieved easily and efficiently via the assembly of
known building blocks. These protected oligosaccharides were
submitted to dissolving metal conditions (Na–NH3) and provided
in one single step after N-acetylating work-up the corresponding
deprotected compounds. Both fully deprotected compounds were
then simply purified and desalted by gel permeation chromatogra-
phy on a Biogel P2 column eluted with water and will be used in
molecular modeling and binding experiments.
1. Experimental
1.1. General methods
1H (600.13, 400.13 or 300.13 MHz) and 13C NMR (150, 100 or
75 MHz) spectra were recorded at 300 K for solution in CDCl3
(internal standard, for 1H residual CHCl3 d 7.24; for 13C CDCl3
d
77.0) or D2O [external standard 3-(trimethylsilyl)-propionic acid-
d4, sodium salt (TSP) for 1H d 0.00, for 13C d 0.00]. 1H NMR and 13
C
NMR chemical shifts are reported in parts per million (ppm). Cou-
pling constants (J) are reported in hertz (Hz). Chemical shifts and
coupling constants were obtained from a first-order analysis of
one-dimensional spectra. Assignments of proton and carbon reso-
nances were based on two dimensional 1H–1H and 13C–1H correla-
tion experiments. Multiplicities are abbreviated as follows: singlet
(s), doublet (d), triplet (t), quartet (q), multiplet (m), and broadened
(br). Analytical thin-layer chromatography (TLC) was performed
rasaccharide was isolated in 76% yield. The
a configuration of the
newly formed fucosidic bond was confirmed by 1H NMR using
the coupling constant measured between H-1 and H-2 of the
fucosyl residue (3.7 Hz). The tetrasaccharide was, in turn, sub-
mitted to dissolving metal conditions [NaꢁNH3(l)] as described
above for the one-step deprotection of trisaccharide 9. Expecting
that these conditions would lead to the removal of all protecting
groups including that of the trichloroacetamido, the reaction was
quenched with methanol and after evaporation of the excess
using Silica Gel 60 F254 precoated plates (250 lm) with a fluores-
cent indicator, visualized under UV, and charred with 10% sulfuric
acid in ethanol. Compounds were purified by flash chromatography

A. Wang, F.-I. Auzanneau / Carbohydrate Research 345 (2010) 1216–1221
1219
with Silica Gel 60 (230–400 mesh) unless otherwise stated. Sol-
62.76 (C-6), 60.04 (C-20), 56.98 (OCH3). HRESIMS calcd for C43H44
Cl3N2O14 [M+NH4]+: 917.1858, found: 917.1873.
-
vents were distilled and dried according to standard procedures,15
and organic solutions were dried over Na2SO4 and concentrated be-
low 40 °C, under reduced pressure. High-resolution electrospray
ionization mass spectra (HR-ESI-MS) were recorded by the analyt-
ical services of the McMaster Regional Center for Mass Spectrome-
try, Hamilton, Ontario.
1.4. Methyl 3-O-[3-O-(2,3,4,6-tetra-O-acetyl-b-
osyl)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-b-
glucopyranosyl]-2,4,6-tri-O-benzoyl-b- -galactopyranoside (9)
D-galactopyran-
D
-
D
BF3ꢀOEt2 (44
lL, 0.36 mmol, 2.0 equiv) was added at rt to a
1.2. Methyl 3-O-(3,4,6-tri-O-acetyl-2-deoxy-2-trichloroacetami-
solution stirred under N2 of disaccharide acceptor 7 (161 mg,
0.179 mmol) and donor 8 (439 mg, 0.891 mmol, 5.0 equiv) in an-
hyd CH2Cl2 (8 mL). The reaction mixture was stirred for 30 min
do-b-D-glucopyranosyl)2,4,6-tri-O-benzoyl-b-D-galactopyranoside
(6)
and the reaction quenched by the addition of Et3N (50 lL). The sol-
Acceptor 5 (500 mg, 0.99 mmol) and donor 4 (1.47 g, 2.47 mmol,
2.5 equiv) were dissolved in anhyd CH2Cl2 (30 mL) under N2 and the
solution was cooled down to 0 °C. Freshly distilled TMSOTf (180 L,
0.99 mmol, 1.0 equiv) was added, the mixture was stirred at 0 °C for
1 h and the reaction was quenched by the addition of Et3N (138 L,
vent was evaporated and flash chromatography of the residue
(EtOAc–hexanes, 3:7) followed by centrifugal chromatography
(EtOAc–hexanes, 3:7) of the impure fractions gave pure trisaccha-
l
ride 9 (140 mg, 64%) as a yellowish amorphous glass. [a] +21(c
D
l
1.0, CHCl3). 1H NMR (400 MHz, CDCl3): d 8.09–8.15, 7.98–8.06,
7.52–7.63, 7.39–7.50, 7.30–7.38 (20H, Ar), 6.83 (d, 1H, J = 6.8 Hz,
NH), 5.81 (d, 1H, J = 2.8 Hz, H-4), 5.54 (dd, 1H, J = 7.8, 9.9 Hz, H-
2), 5.44 (s, 1H, PhCH), 5.30 (d, 1H, J = 8.1 Hz, H-10), 5.21 (dd, 1H,
J = 0.6, 3.3 Hz, H-400), 5.50 (dd, 1H, J = 8.0, 10.4 Hz, H-200), 4.81
(dd, 1H, J = 3.4, 10.4 Hz, H-300), 4.59 (d, 1H, J = 8.0 Hz, H-100), 4.53
(d, 1H, J = 7.7 Hz, H-1), 4.46–4.58 (m, 3H, H-6a, H-6b, H-30),
4.21–4.32 (m, 2H, H-3, H-6a0), 4.09–4.14 (m, 1H, H-5), 3.97 (dd,
1H, J = 6.7, 11.3 Hz, H-6a00), 3.82 (dd, 1H, J = 6.8, 11.2 Hz, H-6b00),
3.53–3.63 (m, 3H, H-40, H-6b0, H-500), 3.42–3.51 (m, 4H, H-50,
OCH3), 3.10–3.17 (m, 1H, H-20), 2.04, 1.90, 1.89, 1.70 (4s, 12H,
0.99 mmol). The solvent was evaporated and flash chromatography
of the residue (EtOAc–hexanes, 2:3) gave the pure disaccharide 6
(660 mg, 70%) as a yellowish foam. [
a]
+25 (c 1.0, CHCl3). 1H
D
NMR (400 MHz, CDCl3): d 7.96–8.11, 7.35–7.60 (15H, Ar), 6.59 (d,
1H, J = 8.1 Hz, NH), 5.81 (br d, 1H, J = 3.4 Hz, H-4), 5.55 (dd, 1H,
J = 7.7, 9.8 Hz, H-2), 5.29 (dd, 1H, J = 9.2, 10.6 Hz, H-30), 5.01 (d,
1H, J = 8.2 Hz, H-10), 4.96 (t, 1H, J = 9.6 Hz, H-40), 4.54 (d, 1H,
J = 7.7 Hz, H-1), 4.41–4.53 (m, 2H, H-6a, H-6b), 4.24 (dd, 1H,
J = 3.4, 9.8 Hz, H-3), 4.19 (dd, 1H, J = 2.4, 12.3 Hz, H-6a0), 4.03–4.16
(m, 2H, H-5, H-6b0), 3.53–3.68 (m, 2H, H-20, H-50), 3.45 (OCH3),
1.95, 1.94, 1.86 (3s, 9H, CH3CO ꢂ 3). 13C NMR (100 MHz, CDCl3): d
170.69, 170.22, 169.25, 166.16, 165.52, 165.07, 161.56 (C@O),
133.43, 133.22, 133.13, 130.07, 129.87, 129.64, 129.61, 129.46,
129.37, 128.53, 128.42, 128.34 (Ar), 102.10 (C-1), 99.26 (C-10),
91.64 (CCl3), 76.53 (C-3), 71.87, 71.67, 71.45 (C-2, C-5, C-50), 70.55
(C-30), 69.85 (C-4), 68.24 (C-40), 62.95 (C-6), 61.34 (C-60), 56.79
(OCH3), 56.49 (C-20), 20.57, 20.49, 20.37 (CH3CO ꢂ 3). HRESIMS
calcd for C42H46Cl3N2O17 [M+NH4]+: 955.1862, found:955.1829.
CH3CO ꢂ 4). 13C NMR (100 MHz, CDCl3):
d 170.21, 170.11,
170.00, 169.25, 166.14, 165.56, 165.10, 161.83 (C@O), 136.86,
133.50, 133.36, 133.24, 130.05, 129.94, 129.69, 129.62, 129.59,
129.40, 129.29, 128.57, 128.49, 128.42, 128.26, 126.21 (Ar),
102.33 (C-1), 101.36 (PhCH), 98.79 (C-100), 98.36 (C-10), 91.72
(CCl3), 78.24 (C-40), 76.46 (C-3), 74.83 (C-30), 71.50 (C-5), 71.19
(C-2), 70.90 (C-300), 70.52 (C-500), 69.93 (C-4), 68.77 (C-200), 68.31
(C-60), 66.79 (C-400), 66.05 (C-50), 62.70 (C-6), 61.15 (C-600), 59.48
(C-20), 56.98 (OCH3), 20.57, 20.53, 20.46 (CH3CO ꢂ 4). HRESIMS
calcd for C57H62Cl3N2O23 [M+NH4]+: 1247.2809, found: 1247.2798.
1.3. Methyl 3-O-(4,6-O-benzylidene-2-deoxy-2-trichloroaceta-
mido -b-D-glucopyranosyl)2,4,6-tri-O-benzoyl-b-D-galactopyran-
oside (7)
1.5. Methyl 3-O-[2-deoxy-2-acetamido-3-O-(b-
D-galactopyran-
AcCl (1 mL) was added to a solution of disaccharide 6 (444 mg,
0.473 mmol) in anhyd MeOH (12.5 mL) and the reaction mixture
was stirred under N2 at rt for 12 h. NaHCO3was added to the reac-
tion mixture and once gas evolution had ceased the solids were fil-
tered off and rinsed with MeOH. The combined filtrate and
washings were concentrated and the residue was dried under high
osyl)-b- -glucopyranosyl]-b- -galactopyranoside (2)
D
D
Trisaccharide 9 (20 mg, 0.016 mmol) was dissolved in THF
(5 mL) and liquid ammonia (20 mL) was condensed into the reac-
tion flask at ꢁ78 °C. Sodium (83 mg, 3.6 mmol) was added into
the reaction mixture which turned deep blue. After 50 min at
ꢁ78 °C, the reaction was quenched by the addition of MeOH
(5 mL) and the ammonia was allowed to evaporate at rt. Ac2O
vacuum. Benzaldehyde dimethyl acetal (213 lL, 3.0 equiv) and CSA
(80 mg) were added to a suspension of the crude triol in anhyd
MeCN (8 mL) stirred under N2 at 50 °C. The reaction was allowed
to proceed for 30 min at 50 °C and was quenched by the addition
(445lL, 4.7 mmol) was added to the remaining solution, the solvent
was evaporated and the residue was submitted to gel permeation
chromatography on a Biogel P2 column eluted with water. The de-
sired compound was obtained contaminated with residual acetate
salts and submitted again to chromatography on Biogel P2 (water)
to give the known5 trisaccharide 2 (5 mg, 55%), which was isolated
of Et3N (50
phy of the residue (EtOAc–hexanes, 1:4) gave pure benzylidene ace-
tal 7 (238 mg, 56% over two steps) as a yellowish glass. [ D +13 (c
lL). Solvents were evaporated and flash chromatogra-
a]
0.5, CHCl3). 1H NMR (400 MHz, CDCl3): d 7.98–8.15, 7.30–7.62 (20H,
Ar), 6.76 (d, 1H, J = 6.4 Hz, NH), 5.81 (d, 1H, J = 2.9 Hz, H-4), 5.59 (dd,
1H, J = 7.8, 9.8 Hz, H-2), 5.44 (s, 1H, PhCH), 5.13 (d, 1H, J = 8.1 Hz, H-
10), 4.56 (d, 1H, J = 7.8 Hz, H-1), 4.46–4.53 (m, 2H, H-6a, H-6b), 4.30
(dd, 1H, J = 4.3, 10.4 Hz, H-6a0), 4.20–4.27 (m, 2H, H-3, H-30), 4.13 (t,
1H, J = 6.2 Hz, H-5), 3.53–3.62 (m, 1H, H-6b0), 3.49 (s, 3H, OCH3),
3.34–3.45 (m, 2H, H-40, H-50), 3.16–3.23 (m, 1H, H-20).13C NMR
as a white amorphous powder upon freeze-drying. [a] +9 (c 0.3,
D
1
MeOH). H NMR (600 MHz, D2O): d 4.72 (d, 1H, J = 8.4 Hz, H-100),
4.42 (d, 1H, J = 7.7 Hz, H-10), 4.28 (d, 1H, J = 8.0 Hz, H-1), 4.12 (d,
1H, J = 3.2 Hz, H-4), 3.85–3.90 (m, 3H, H-20, H-6a0, H-400), 3.65–3.82
(m, 9H, H-3, H-5, H-6a, H-6b, H-30, H-6b0, H-500, H-6a00, H-6b00), 3.62
(dd, 1H, J = 3.4, 9.9 Hz, H-300), 3.44–3.57 (m, 7H, H-2, H-40, H-50,
H-200, OCH3), 2.0 (s, 3H, CH3CO). 13C NMR (150 MHz, D2O): d
174.97 (C@O), 103.83 (C-10), 103.44 (C-100), 102.38 (C-1), 82.18,
82.05 (C-3, C-30), 75.23, 75.15, 74.67 (C-5, C-50, C-500), 72.41 (C-300),
70.63 (C-200), 69.69 (C-2), 68.47, 68.41, 68.30 (C-4, C-40, C-400),
60.98, 60.86, 60.45 (C-6, C-60, C-600), 57.15 (OCH3), 54.71 (C-20),
22.19 (CH3CO).
(100 MHz, CDCl3):
d 166.20, 165.63, 165.34, 162.41 (C@O),
136.82, 133.52, 133.42, 133.25, 130.11, 129.95, 129.73, 129.66,
129.47, 129.41, 129.30, 128.61, 128.56, 128.44, 128.38, 128.32,
126.25, 126.21 (Ar), 102.20 (C-1), 101.77 (PhCH), 98.93 (C-10),
91.80 (CCl3), 81.11 (C-40), 76.53 (C-3), 71.60 (C-2, C-5), 71.67,
71.45 (C-50), 69.83 (C-4), 68.89 (C-30), 68.28 (C-60), 66.13 (C-50),

1220
A. Wang, F.-I. Auzanneau / Carbohydrate Research 345 (2010) 1216–1221
1.6. Methyl 2-acetamido-4-O-(2,4,6-tri-O-acetyl-3-O-
chloroacetyl-b- -galactopyranosyl)-6-O-benzyl-3-O-
chloroacetyl-2-deoxy-b- -glucopyranoside (12)
PhCHH), 4.56 (d, 1H, J = 8.2 Hz, H-1), 4.50 (d, 1H, J = 12.1 Hz,
PhCHH), 4.43 (d, 1H, J = 8.1 Hz, H-10), 3.97–4.17 (m, 2H, H-6a0, H-
6b0), 3.78-3.91 (m, 2H, H-4, H-50), 3.63–3.77 (m, 3H, H-6a, H-6b,
H-30), 3.55–3.63 (m, 1H, H-3), 3.46 (s, 3H, OCH3), 3.41–3.52 (m,
2H, H-2, H-5), 2.13, 2.03, 1.97 (3s, 12H, CH3CO ꢂ 4). 13C NMR
D
D
BF3ꢀEt2O (220
lL, 1.75 mmol, 2.0 equiv) was added to a solution
stirred at 40 °C of acceptor 11 (350 mg, 0.87 mmol) and donor 10
(2.3 g, 4.4 mmol, 5.0 equiv) in anhyd CH2Cl2 (20 mL). The reaction
mixture was stirred at 40 °C for 1 h and the reaction quenched
(100 MHz, CDCl3): d 170.75, 170.59 (C@O), 138.10, 128.40,
127.72, 127.63 (Ar), 101.12 (C-1, C-10), 81.27 (C-3), 73.98 (C-5),
73.53 (PhCH2), 72.31 (C-20), 71.81 (C-4), 71.42 (C-50), 70.93 (C-30),
69.49 (C-40), 6.13 (C-6), 61.88 (C-60), 56.56, 56.41 (OCH3, C-2),
23.48, 20.87, 20.68, 20.52 (CH3CO ꢂ 4). HRESIMS calcd for
C28H40NO14 [M+H]+: 614.2449, found: 614.2424.
by the addition of Et3N (250 lL). The solvent was evaporated and
flash chromatography of the residue (EtOAc–hexanes, 4:1) gave a
mixture of disaccharide 12 and unreacted acceptor 11 (484 mg,
12:11 = 3:1 by NMR integration, 60%). Further purification by cen-
trifugal chromatography (CHCl3–MeOH, 20:1) gave the pure disac-
1.8. Methyl 2-acetamido-3-O-[2,4,6-tri-O-acetyl-4-O-(3,4,6-tri-O
charide 12 as a white amorphous solid (362 mg, 54%). [
a
]
ꢁ8 (c
-acetyl-2-deoxy-2-trichloroacetamido-b-D-glucopyranosyl)-b-D-
galactopyranosyl]-6-O-benzyl-3-O-chloroacetyl-2-deoxy-b-D-
glucopyranoside (15)
D
1.0, CHCl3). 1H NMR (400 MHz, CDCl3): d 7.25–7.40 (m, 5H, Ar),
5.79 (d, 1H, J = 9.3 Hz, NH), 5.23 (d, 1H, J = 3.3 Hz, H-40), 5.10 (dd,
1H, J = 8.9, 10.3 Hz, H-3), 4.95 (dd, 1H, J = 8.0, 10.3 Hz, H-20), 4.80
(dd, 1H, J = 3.5, 10.4 Hz, H-30), 4.74 (d, 1H, J = 12.1 Hz, PhCH2),
4.34–4.45 (m, 3H, H-1, H-10, PhCH2), 4.00–4.13 (m, 4H, H-6a0, H-
6b0, ClCH2CO), 3.88–4.00 (m, 4H, H-2, H-4, ClCH2CO), 3.66–3.75
(m, 2H, H-6a, H-6b), 3.55–3.62 (m, 1H, H-50), 3.40–3.50 (m, 4H,
H-5, OCH3), 2.10, 2.05, 1.93, 1.92 (4s, 12H, CH3CO ꢂ 4). 13C NMR
Freshly distilled TMSOTf (26
lL, 0.14 mmol, 2.0 equiv) was
added to solution of disaccharide acceptor 13 (50 mg,
a
0.072 mmol) and donor 4 (216 mg, 0.363 mmol, 5.0 equiv) stirred
at 0 °C under N2 in anhyd CH2Cl2 (3.5 mL). The reaction mixture
was stirred at 0 °C for 1 h, the reaction quenched by the addition
(100 MHz, CDCl3):
d
170.39, 170.34, 170.27, 168.85, 167.38,
of Et3N (20 lL, 0.14 mmol) and the solvent evaporated. Flash chro-
166.55 (C@O), 137.59, 128.63, 128.18, 128.13 (Ar), 101.79 (C-1),
99.97 (C-10), 74.60, 74.36 (C-3, C-4, C-5), 73.64 (PhCH2), 72.52
(C-30), 70.41 (C-50), 68.74 (C-20), 67.12 (C-6), 66.60 (C-40), 60.86
(C-60), 56.63 (OCH3), 53.33 (C-2), 40.80, 40.33 (ClCH2CO ꢂ 2),
23.30, 20.64, 20.60 (CH3CO ꢂ 4). HRESIMS calcd for C32H42Cl2NO16
[M+H]+: 766.1881, found: 766.1821.
matography (CHCl3–MeOH, 20:1) of the residue followed by fur-
ther chromatography (EtOAc–hexanes, 9:1) of the fractions
containing the desired product gave pure trisaccharide 15
(60 mg, 74%) as a white amorphous solid. [a] +11 (c 0.7, CHCl3).
D
1H NMR (400 MHz, CDCl3): d 7.26–7.40 (m, 5H, Ar), 6.90 (d, 1H,
J = 8.2 Hz, NH), 5.88 (d, 1H, J = 9.4 Hz, NH), 5.39 (dd, 1H, J = 9.2,
10.7 Hz, H-300), 5.29 (d, 1H, J = 3.3 Hz, H-40), 5.51–5.11 (m, 2H, H-
3, H-400), 4.90 (dd, 1H, J = 8.1, 10.1 Hz, H-20), 4.88 (d, 1H,
J = 8.0 Hz, H-100), 4.72 (d, 1H, J = 12.1 Hz, PhCHH), 4.46 (d, 1H,
J = 12.1 Hz, PhCHH), 4.34 (d, 1H, J = 7.7 Hz, H-1), 4.29 (d, 1H,
J = 8.0 Hz, H-10), 4.28–4.34 (m, 1H, H-6a00), 3.94–4.16 (m, 6H, H-2,
H-6a0, H-6b0, H-6b00, ClCH2CO), 3.89 (t, 1H, J = 8.6 Hz, H-4), 3.65-
3.78 (m, 4H, H-6a, H-6b, H-30, H-500), 3.54–3.65 (m, 2H, H-50,
H-200), 3.40–3.53 (m, 4H, H-5, OCH3), 2.08, 2.07, 2.00, 1.97, 1.96,
1.94 (6s, 21H, CH3CO ꢂ 7). 13C NMR (100 MHz, CDCl3): d 170.72,
170.65, 170.53, 170.45, 169.95, 169.37, 168.99, 167.49, 161.79
(C@O), 137.89, 128.58, 128.02, 127.97 (Ar), 101.88 (C-1), 100.24
(C-10), 99.27 (C-100), 92.14 (CCl3), 75.84 (C-30), 74.61, 74.46, 74.09
(C-3, C-4, C-5), 73.66 (PhCH2), 72.04 (C-500), 71.15 (C-50), 70.76
(C-20), 70.48 (C-300), 68.76 (C-40), 68.37 (C-400), 67.48 (C-6), 61.96
(C-60), 61.14 (C-600), 56.80, 56.67 (C-200, OCH3), 52.85 (C-2), 40.83
(ClCH2CO), 23.31, 21.09, 20.77, 20.73, 20.62, 20.57 (CH3CO ꢂ 7).
HRESIMS calcd for C44H57Cl4N2O23 [M+H]+:1121.2106, found:
1121.2057.
1.7. Methyl 2-acetamido-4-O-(2,4,6-tri-O-acetyl-b-
anosyl)-6-O-benzyl-3-O-chloroacetyl-2-deoxy-b- -glucopyrano-
side (13) and methyl2-acetamido-4-O-(2,4,6-tri-O-acetyl-b- -ga-
lactopyranosyl)-6-O-benzyl-2-deoxy-b- -glucopyranoside (14)
D-galactopyr-
D
D
D
Thiourea (47 mg, 0.617 mmol, 1.3 equiv) and NaHCO3 (260 mg,
20 mg/mL) were added to a stirred solution of disaccharide 12
(365 mg, 0.476 mmol) in EtOH (13 mL). The reaction mixture was
stirred at 70 °C for 4 h, solids were filtered off, rinsed with EtOH
and the combined filtrate and washing were concentrated. Flash
chromatography of the residue (CHCl3–MeOH, 20:1) gave the de-
sired acceptor 13 isolated as a white amorphous solid (213 mg,
65%) followed by diol 14 also obtained as a white amorphous solid
(37 mg, 13%).
1.7.1. Analytical data for 13
[a
]
D
ꢁ4 (c 1.2, CHCl3). 1H NMR (400 MHz, CDCl3): d 7.26–7.37
(m, 5H, Ar), 5.97 (d, 1H, J = 9.3 Hz, NH), 5.18 (d, 1H, J = 3.4 Hz,
H-40), 5.10 (dd, 1H, J = 9.0, 10.0 Hz, H-3), 4.67–4.77 (m, 2H, H-20,
PhCHH), 4.45 (d, 1H, J = 12.1 Hz, PhCHH), 4.37 (d, 1H, J = 8.2 Hz,
H-1), 4.35 (d, 1H, J = 8.0 Hz, H-10), 3.95–4.33 (m, 5H, H-2, H-6a0,
H-6b0, ClCH2CO), 3.90 (t, 1H, J = 9.2 Hz, H-4), 3.70–3.78 (m, 2H,
H-6a, H-6b), 3.53–3.62 (m, 1H, H-30, H-50), 3.41–3.51 (m, 4H, H-5,
OCH3), 2.80–2.88 (br, 1H, OH), 2.11, 2.06, 2.01, 1.92 (4s, 12H,
CH3CO ꢂ 4). 13C NMR (100 MHz, CDCl3): d 170.82, 170.77, 170.44,
167.51 (C@O), 137.76, 128.50, 127.92, 127.86 (Ar), 101.85 (C-1),
100.02 (C-10), 74.66, 74.55, 74.41 (C-3, C-4, C-5), 73.52 (PhCH2),
72.73 (C-20), 70.99, 70.83 (C-30, C-50), 69.50 (C-40), 67.31 (C-6),
61.66 (C-60), 56.59 (OCH3), 53.29 (C-2), 40.89 (ClCH2CO), 23.23,
20.93, 20.70, 20.69 (CH3CO ꢂ 4). HRESIMS calcd for C30H41ClNO15
[M+H]+: 690.2165, found: 690.2175.
1.9. Methyl 2-acetamido-4-O-[2,4,6-tri-O-acetyl-3-O-(3,4,6-tri-
O-acetyl-2-deoxy-2-trichloroacetamido-b-
D-glucopyranosyl)-b-
D-galactopyranosyl]-6-O-benzyl-2-deoxy-b-D-glucopyranoside (16)
Thiourea (120 mg, 1.58 mmol, 9 equiv) was added to a solution
of trisaccharide 15 (200 mg, 0.178 mmol) in a mixture of pyridine
and EtOH (2:1, 10 mL). The reaction mixture was warmed up to
65 °C and stirred for 12 h. The solvent was evaporated and the res-
idue was co-concentrated with toluene (10 mL ꢂ 2), dissolved in
CH2Cl2 (20 mL) and washed successively with 8% HCl (10 mL), satd
aq NaHCO3 (10 mL) and brine (10 mL). The aqueous phases were
re-extracted with CH2Cl2 and the combined organic phases were
dried and concentrated. Flash chromatography of the residue
(CHCl3–MeOH, 20:1?9:1) gave pure trisaccharide acceptor 16
1.7.2. Analytical data for 14
(142 mg, 76%) as a white amorphous solid. [a] +21 (c 0.9, CHCl3).
D
[a
]
D
+12 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3): d 7.26–7.40
1H NMR (400 MHz, CDCl3): d 7.26–7.40 (m, 5H, Ar), 7.03 (d, 1H,
J = 8.3 Hz, NH), 5.65 (d, 1H, J = 8.2 Hz, NH), 5.30–5.38 (m, 2H,
H-40, H-300), 5.00–5.14 (m, 2H, H-20, H-400), 4.89 (d, 1H, J = 8.0 Hz,
(m, 5H, Ar), 5.97 (d, 1H, J = 8.3 Hz, NH), 5.23 (d, 1H, J = 3.4 Hz, H-
40), 4.92 (dd, 1H, J = 8.1, 9.9 Hz, H-20), 4.67 (d, 1H, J = 12.1 Hz,

A. Wang, F.-I. Auzanneau / Carbohydrate Research 345 (2010) 1216–1221
1221
H-100), 4.67 (d, 1H, J = 12.2 Hz, PhCHH), 4.44–4.54 (m, 2H, H-1,
1.11. Methyl 2-acetamido-2-deoxy-3-O-
[3-O -(2-deoxy-2-acetamido-b-
pyranosyl]-b- -glucopyranoside (3)
a
-l-fucopyranosyl-4-O-
PhCHH), 4.38 (d, 1H, J = 8.1 Hz, H-10), 4.27–4.34 (m, 1H, H-6a00),
4.05–4.20 (m, 3H, H-6a0, H-6b00, OH), 3.94–4.04 (m, 1H, H-6b0),
3.77–3.88 (m, 3H, H-3, H-30, H-50), 3.51–3.74 (m, 6H, H-2, H-4,
H-6a, H-6b, H-200, H-500), 3.40–3.50 (m, 4H, H-5, OCH3), 2.10, 2.08,
2.03, 2.00, 1.98, 1.97 (6s, 21H, CH3CO ꢂ 7). 13C NMR (100 MHz,
CDCl3): d 170.71, 170.58, 169.96, 169.30, 169.13, 161.81 (C@O),
138.25, 128.40, 127.67 (Ar), 101.30 (C-1, C-10), 99.37 (C-100),
92.12 (CCl3), 81.15 (C-4), 75.69 (C-30), 74.12 (C-5), 73.58 (PhCH2),
72.10, 71.58 (C-3, C-50, C-500), 70.73 (C-300), 70.32 (C-20), 68.56
(C-40), 68.19 (C-400), 68.09 (C-6), 62.18 (C-60), 61.06 (C-600), 56.62,
56.54 (C-200, OCH3), 55.97 (C-2), 23.58, 21.08, 20.77, 20.66, 20.58,
20.54 (CH3CO ꢂ 7). HRESIMS calcd for C42H56Cl3N2O22 [M+H]+:
1045.2390, found: 1045.2360.
D-glucopyranosyl)-b-D-galacto-
D
Tetrasaccharide 18 (42 mg, 0.027 mmol) was deprotected as de-
scribed above Section 1.5 for the preparation of trisaccharide 2.
Purification by gel permeation chromatography (2 ꢂ Biogel P2,
eluted with water) gave pure tetrasaccharide 3 (16 mg, 72%) which
was obtained as an amorphous white powder upon freeze-drying.
[
a
]
ꢁ18 (c 0.5, MeOH). 1H NMR (600 MHz, D2O): d 5.07 (d, 1H,
D
J = 4.0 Hz, H-10), 4.77–4.83 (m, 1H, H-50), 4.66 (d, 1H, J = 8.4 Hz, H-
1000), 4.45 (d, 1H, J = 8.2 Hz, H-1), 4.41 (d, 1H, J = 7.9 Hz, H-100), 4.07
(d, 1H, J = 3.0 Hz, H-400), 3.96–4.01 (m, 1H, H-6a), 3.79–3.92 (m,
6H, H-2, H-3, H-6b, H-30, H-3000, H-6a000), 3.62–3.78 (m, 7H, H-20,
H-40, H-300, H-6a00, H-6b00, H-2000, H-6b000), 3.39-3.61 (m, 9H, H-4, H-
5, H-200, H-500, H-4000, H-5000, OCH3), 2.01, 2.00 (2s, 6H, CH3CO ꢂ 2),
1.13 (d, 3H, J = 6.6 Hz, H-60). 13C NMR (150 MHz, D2O): d 174.89,
174.42 (C@O), 102.77 (C-1000), 101.79, 101.72 (C-1, C-100), 98.74
(C-10), 81.50 (C-300), 75.60, 75.33, 74.98, 74.43, 73.49 (C-3, C-4,
C-5, C-4000, C-5000), 73.14 (C-3000), 71.83 (C-40), 70.51 (C-500), 69.68
(C-200), 69.18 (C-30), 68.24 (C-400), 67.61 (C-20), 66.69 (C-50), 61.42
(C-600), 60.45 (C-6000), 59.74 (C-6), 57.11 (OCH3), 55.61 (C-2, C-2000),
22.19 and 22.12 (CH3CO ꢂ 2), 15.24 (C-60). HRESIMS calcd for
C29H50N2NaO20 [M+Na]+: 769.2855, found:769.2845.
1.10. Methyl 2-acetamido-4-O-[2,4,6-tri-O-acetyl-3-O-(3,4,6-tri-
O-acetyl-2-deoxy-2-trichloroacetamido-b-
D
-glucopyranosyl)-b-
D
-galactopyranosyl]-6-O-benzyl-3-O-(2,3,4-tri-O-benzyl-
a-L-
fucopyranosyl)-2-deoxy-b-D-glucopyranoside (18)
Trisaccharide acceptor 16 (34 mg, 0.032 mmol) and fucosyl
donor 17 (47 mg, 0.097 mmol, 3.0 equiv) were dissolved in a mix-
ture of CH2Cl2 and DMF (1:1, 2 mL) containing activated pow-
dered MS 4 Å (100 mg). The mixture was stirred at rt for
30 min, Cu(II) Br2 (22 mg, 0.098 mmol, 3.0 equiv) followed by n-
Bu4NBr (33 mg, 0.099 mmol, 3.1 equiv) were added and the reac-
tion was allowed to proceed under stirring for 20 h at rt. The sol-
ids were filtered off on CeliteÒ and washed with CH2Cl2 (5 mL).
The combined filtrate and washing were diluted with CH2Cl2
(50 mL) and washed successively with brine (15 mL) and satd
aq NaHCO3(15 mL ꢂ 6). The aqueous phases were re-extracted
with CH2Cl2 (50 mL) and the combined organic phases were dried
and concentrated. Flash chromatography of the residue (EtOAc–
hexanes, 1:1 to CHCl3–MeOH, 20:1) gave pure tetrasaccharide
Acknowledgments
The authors are grateful for financial support by NSERC, the
Canada foundation for Innovation, and the Ontario Innovation Trust.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2010.03.038.
18 (34 mg, 73%) as a colorless oil. [
a
]
D
ꢁ18 (c 0.9, CHCl3). 1H
References
NMR (400 MHz, CDCl3): d 7.24–7.44 (m, 20H, Ar), 6.78 (d, 1H,
J = 8.3 Hz, NH), 6.68 (d, 1H, J = 8.2 Hz, NH), 5.36–5.41 (m, 2H, H-
400, H-3000), 5.17 (d, 1H, J = 3.7 Hz, H-10), 5.12 (t, 1H, J = 9.6 Hz, H-
4000), 5.02 (dd, 1H, J = 7.9, 9.9 Hz, H-200), 4.97 (d, 1H, J = 11.8 Hz,
PhCH2), 4.90 (d, 1H, J = 8.1 Hz, H-1000), 4.70–4.83 (m, 4H, PhCH2),
4.65–4.70 (m, 2H, H-1, PhCH2), 4.58 (d, 1H, J = 12.0 Hz, PhCH2),
4.40–4.45 (m, 2H, H-100, PhCH2), 4.38 (dd, 1H, J = 2.4, 12.3 Hz,
H-6a000), 4.16 (dd, 1H, J = 4.0, 12.3 Hz, H-6b000), 4.09–4.14 (m, 3H,
H-20, H-50, H-6a00), 4.06 (t, 1H, J = 5.6 Hz, H-4), 4.01 (dd, 1H,
J = 6.4, 11.4 Hz, H-6b00), 3.89–3.93 (m, 2H, H-3, H-6a), 3.86 (dd,
1H, J = 2.6, 10.1 Hz, H-30), 3.75–3.83 (m, 3H, H-2, H-6b, H-300),
3.63–3.73 (m, 4H, H-5, H-500, H-2000, H-5000), 3.60–3.62 (m, 1H, H-
40), 3.33 (s, 3H, OCH3), 2.13, 2.06, 2.05, 2.03, 2.02, 1.87 (6s, 21H,
CH3CO ꢂ 7), 1.13 (d, 3H, J = 6.5 Hz, H-60). 13C NMR (150 MHz,
CDCl3): d 170.63, 170.57, 170.33, 170.23, 169.62, 169.42, 169.28,
161.74 (C@O), 138.86, 138.82, 138.49, 138.07, 128.43, 128.34,
128.30, 128.27, 128.16, 127.77, 127.65, 127.58, 127.56, 127.44,
127.33, 127.25, 125.48 (Ar), 100.76 (C-1), 99.43, 99.39 (C-100,
C-1000), 96.01 (C-10), 92.02 (CCl3), 79.45 (C-30), 77.14 (C-40), 76.25
(C-20), 75.05 (C-300), 74.46 (PhCH2), 73.94 (C-5), 73.49 (C-3),
73.34, 72.84, 72.71 (PhCH2 ꢂ 3), 72.05 (C-4, C-5000), 71.05 (C-500),
70.77 (C-200), 70.54 (C-3000), 69.06 (C-6), 68.53 (C-400), 68.20
(C-4000), 66.67 (C-50), 61.22 (C-600), 61.14 (C-6000), 56.59 (C-2000),
56.39 (OCH3), 23.11, 21.12, 20.73, 20.69, 20.61, 20.62, 20.49
(CH3CO ꢂ 7), 16.57 (C-60). HRESIMS calcd for C69H84Cl3N2O26
[M+H]+: 1461.4378, found: 1461.4384.
1. (a) Pettijohn, D. E.; Stranahan, P. L.; Due, C.; Rønne, E.; Sørenson, H. R.; Olsson,
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