Synthesis of Lewis A trisaccharide analogues in which d-glucose and l-rhamnose replace d-galactose and l-fucose, respectively

Article abstract of DOI:10.1016/j.carres.2006.07.006

In our effort to design a safe anti-cancer vaccine based on the tumor associated carbohydrate antigen Le^aLe^x, we are studying the cross-reactivity between the Le^a natural trisaccharide antigen and analogues in which the l-

Full text of DOI:10.1016/j.carres.2006.07.006

Carbohydrate Research 341 (2006) 2426–2433
Note
Synthesis of Lewis A trisaccharide analogues in which D-glucose and
L-rhamnose replace D-galactose and L-fucose, respectively
Liang Liao and France-Isabelle Auzanneau*
Department of Chemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Received 12 June 2006; received in revised form 6 July 2006; accepted 12 July 2006
Available online 1 August 2006
Abstract—In our effort to design a safe anti-cancer vaccine based on the tumor associated carbohydrate antigen LeaLex, we are
studying the cross-reactivity between the Lea natural trisaccharide antigen and analogues in which the L-fucose, D-galactose,
and/or D-glucosamine residues are replaced by L-rhamnose or D-glucose, respectively. We describe here the chemical synthesis of
two such Lea trisaccharide analogues. In one trisaccharide, D-glucose replaces D-galactose and in the second analogue L-rhamnose
and D-glucose replace L-fucose and D-galactose, respectively. Introduction of the rhamnose and fucose moiety onto the poorly reac-
tive 4-OH group of the N-acetylglucosamine residue in a disaccharide acceptor was successful after bis-N-acetylation of the amine
group. These analogues will be used in competitive binding experiments with anti-Lea antibodies and their solution conformations
will be studied.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Synthetic carbohydrate-based anti-cancer vaccines; Cross-reactivity; Auto-immune reactions; LeaLex analogues; Lea analogues; GlcNAc-
containing acceptors
Synthetic anti-cancer vaccines based on carbohydrates
have been shown to hold great promise as immunother-
apeutics against cancer.1–3 In fact, candidates such as
Globo-H,4–6 Ley,7 and sTn-8,9 based vaccines are now
being investigated in clinical trials. Vaccination therapy
to treat cancer takes advantage of the abnormal glyco-
sylation10 on tumor cell surfaces. Indeed, since the early
reports by Hakomori and co-workers11,12 that large
quantities of fucose-containing blood group antigens
accumulated in human adenocarcinoma, tumor associ-
ated carbohydrate antigens (TACAs) have been exten-
sively studied and reviewed.10,13,14 The TACA LeaLex
(1) is a hexasaccharide that displays the Lea trisaccha-
ride linked to O-3 of the galactose residue of a reducing
end Lex trisaccharide.
immunization of mice with biopsied human squamous
lung carcinoma (SLC) cells.17 Interestingly, although
the mAb 43-9F also showed some cross-reactivity with
the Lea antigen, the hexasaccharide LeaLex was shown
to bind mAb 43-9F over 100 times more tightly than
the Lea antigen.18 Further studies by Hakomori and
co-workers19 using a synthetic glycosphingolipid dis-
playing LeaLex confirmed that the epitope recognized
by mAb 43-9F was indeed presented most efficiently
by this hexasaccharide. Moreover, it was also shown
that this epitope was largely associated with lung can-
cer17,20–22 and tumorigenicity of SLC cells,23,24 while it
was only found in normal tissues on a subset of seromu-
cous glands (trachea-bronchial tree) and on limited pop-
ulations of epithelial cells in the digestive and urogenital
systems.17 Therefore, LeaLex is an interesting target for
the development of anti-cancer vaccines.
Originally found in feces from preterm infants fed on
breast milk15 and later isolated from human milk,16
LeaLex was shown to bind strongly to a monoclonal
antibody (mAb) 43-9F that had been selected after
However, it is well known since Lemieux’s pioneering
work25 that the Lea trisaccharide may be converted to
an immunogenic determinant through its association
with an immunostimulant carrier protein. Indeed such
a Lea glycoconjugate has been used to raise an immune
*
Corresponding author. E-mail: fauzanne@uoguelph.ca
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.07.006

L. Liao, F.-I. Auzanneau / Carbohydrate Research 341 (2006) 2426–2433
2427
response in mice and the Abs obtained were shown, in
turn, to recognize Lea expressed at the surface of normal
human cells and tissues.26 Therefore, attempts to use
LeaLex for the development of anti-cancer vaccines
may lead to the production of anti-Lea antibodies that
could ultimately lead to auto-immune reactions that will
destroy normal tissues and cells expressing Lea. Thus,
our research program aims at discovering analogues of
LeaLex that could be used as anti-cancer vaccines, that
is, cross-react with the natural antigen, but would not
induce the production of anti-Lea antibodies. We postu-
late that replacing any of the L-fucose, D-galactose, or
N-acetyl D-glucosamine residues displayed by the nonre-
ducing end Lea trisaccharide by L-rhamnose or D-glu-
cose units, respectively, might abolish the secretion of
such anti-Lea antibodies while still allowing that of
anti-LeaLex specific antibodies such as mAb 43-9F.
Prior to embarking on the cumbersome synthesis of
such hexasaccharide analogues, it seemed reasonable
to first study the cross-reactivity of the natural Lea anti-
gen with trisaccharide analogues in which the individual
sugar residues have been substituted, as mentioned
above, by other monosaccharide units (D-Gal and/or
D-GlcNAc by D-Glu, and/or L-Fuc by L-Rha). These
analogues will be compared to the natural antigen not
only in terms of cross-reactivity with anti-Lea antibod-
ies, but also in terms of conformation using a combina-
tion of both NMR and molecular modeling
experiments. Only those analogues that do not bind to
anti-Lea antibodies even though they display a three-
dimensional conformation similar to the Lea trisaccha-
ride will be selected and further investigated as hexasac-
charides. In line with this approach, we have
reported27,28 the synthesis of a Lea analogue in which
the L-fucose moiety is replaced by L-rhamnose as well
as the synthesis of trisaccharide Lea. We report
here the synthesis of the Lea analogues 2 and 3. In ana-
logue 2 the D-galactose unit is replaced by a D-glucose
residue, while in analogue 3 both D-galactose and L-
fucose are substituted by D-glucose and L-rhamnose,
respectively.
The known29 monosaccharide glycosyl acceptor 4
(Scheme 1) was obtained according to the reported pro-
cedures. Coupling of acceptor 4 with peracetylated glu-
cosyl bromide 530 under Helferich conditions proceeded
uneventfully to afford disaccharide 6 in 74% yield.
Although the benzylidene acetal in 6 could be reduc-
tively opened to afford a disaccharide acceptor with a
free C-4 hydroxyl group, it is well known31,32 that the
4-OH group in N-acetylglucosamine derivatives is
poorly reactive toward glycosylation. In addition to ste-
ric hindrance at this position,31 it has been demonstrated
that the reactivity of this hydroxyl group was greatly
influenced by the presence of the N-acetyl group at C-
2 of the glucosamine residue.28,32 A similar observation
was made in sialic acid glycosyl acceptors in which the
reactivity of OH-8 is affected by the presence of the N-
acetyl group at C-5 of the sialic acid residue.33 Thus,
Demchenko and Boons33 established a synthetic strat-
egy for the glycosylation of such acceptors in which
the amino group of sialic acid was bis-acetylated prior
to glycosylation. This strategy was later applied success-
fully to achieve glycosylation of the 4-OH group of N-
acetylated glucosamine acceptors.28,32 Interestingly, di-
N,N-acetylated glucosamine derivatives have also
shown improved reactivity when used as glycosyl do-
nors.34 Applying this strategy to the synthesis of our tar-
get trisaccharides, disaccharide 6 was treated with acetyl
chloride in the presence of excess Hunig’s base and gave
¨
bis-N-acetylated disaccharide 7 in 89% yield. The benz-
ylidene acetal was then reductively opened in standard
conditions (NaBH3CN–HCl/diethyl ether) and the
desired di-N,N-acetate disaccharide glycosyl acceptor 8
was isolated in 62% yield (Scheme 1).
OH
OH
OH
OH
HO
O
OH
OH
O
OH
O
O
O
O
O
HO
O
O
OH
OH
O
NHAc
NHAc
O
OH
HO
OH
OH
HO
1
OH
OH
O
OH
O
HO
OH
O
OH
O
OH
O
OH
OH
O
O
O
HO
HO
HO
OMe
OMe
O
HO
O
NHAc
NHAc
OH
OH
3
2

2428
L. Liao, F.-I. Auzanneau / Carbohydrate Research 341 (2006) 2426–2433
OAc
AcO
AcO
O
Ph
O
O
Ph
O
O
O
a
AcO
AcO
O
+
O
O
OMe
HO
OMe
AcO
AcHN
AcO
AcHN
AcO
Br
6
4
5
OBn
OAc
Ph
O
O
O
AcO
AcO
O
O
N
O
b
HO
O
AcO
AcO
c
O
OMe
Ac
OMe
Ac
AcO
N
Ac
AcO
AcO
Ac
8
7
Scheme 1. Reagents and conditions: (a) Hg(CN)2, toluene/CH3NO2 (1:1), 40 ꢁC, 16 h, 74%; (b) AcCl, (i-Pr)2NEt, CH2Cl2, rt, 89%; (c) NaBH3CN,
HCl/Et2O, THF, 0 ꢁC, 62%.
BnO
BnO
OBn
O
OBn
O
OAc
SEt
OBn
O
O
O
O
AcO
AcO
OMe
Ac
OBn
BnO
N
Ac
AcO
9
a
11
8
OAc
O
AcO
b
AcO
NH
CCl3
O
OBn
OAc
O
O
O
AcO
AcO
O
O
OMe
Ac
AcO
N
Ac
AcO
OAc
OAc
10
12
Scheme 2. Reagents and conditions: (a) CH3OTf (5 equiv), Et2O, rt, 88%; (b) TESOTf (0.15 equiv), CH2Cl2, ꢀ78 ꢁC!rt, 86%.
Glycosylation of acceptor 8 with the known35 b-thio-
ethyl per-benzylated fucosyl donor 9 went smoothly un-
der CH3OTf catalysis using diethyl ether as a solvent to
assist in the formation of the a-anomer (Scheme 2). In-
deed trisaccharide 11 was obtained as the single product
and in an excellent 88% yield. Glycosylation of disaccha-
ride 8 with the known36 trichloroacetimidate donor 10
(Scheme 2) was catalyzed by TESOTf and afforded the
desired trisaccharide 12 in excellent 86% yield. The
a-(1!4) linkage of the newly introduced L-rhamnose
unit was unambiguously confirmed by the one bond
coupling constant measured for the rhamnose anomeric
1
center JC1–H1 (173 Hz).37
Trisaccharides 11 and 12 were then submitted to Zem-
´
plen de-acetylation to remove the O-acetyl groups as well
as one of the N-acetyl groups, affording polyols 13 and
14 in 78% and 84% yields, respectively. Finally, hydrog-
enolysis of 13 and 14 over 10% Pd/C gave the desired Lea
trisaccharide analogues 2 and 3 in good yields. Future
study will use 2 and 3 as soluble antigens to test their
BnO
OBn
OBn
OH
HO
HO
O
OBn
O
OMe
NHAc
OBn
O
OMe
NHAc
O
OH
OH
O
O
O
O
HO
HO
HO
HO
O
O
OH
HO
14
13

L. Liao, F.-I. Auzanneau / Carbohydrate Research 341 (2006) 2426–2433
2429
cross-reactivity to anti-Lea antibodies. Conformational
analysis of these analogues with a combination of
NMR and molecular modeling is also underway.
and brine (100 mL). The aqueous phases were re-ex-
tracted with CH2Cl2 (50 mL) and the combined organic
layers were dried (Na2SO4) and concentrated. Chroma-
tography (98:2, CH2Cl2/CH3OH) of the dry residue
1
gave compound 6 (75 mg, 74%) as a colorless glass. H
1. Experimental
NMR (400 MHz, CDCl3): d 7.58–7.35 (m, 5H, Ar),
5.83 (d, 1H, J = 6.6 Hz, NH), 5.53 (s, 1H, CHPh),
5.12–5.09 (m, 2H, H-1, H-30), 5.03 (br t, 1H,
J = 9.7 Hz, H-40), 4.95 (dd, 1H, J = 9.2, 8.1 Hz, H-20),
4.77 (d, 1H, J = 8.0 Hz, H-10), 4.71 (br t, 1H,
J = 9.5 Hz, H-3), 4.34 (dd, J = 4.9, 10.5 Hz, H-6a),
4.08–3.92 (m, 2H, H-6a0, H-6b0), 3.79 (dd, 1H,
J = 10.3, 10.2 Hz, H-6b), 3.68 (br t, 1H, J = 9.2 Hz,
H-4), 3.57 (m, 1H, H-5), 3.51 (s, 3H, OCH3), 3.37 (m,
1H, H-50), 3.04 (m, 1H, H-2), 2.02–1.92 (5br s, 5 · 3H,
N-acetyl and O-acetyl); 13C NMR (100.6 MHz, CDCl3):
d 167.9, 166.8, 166.1 (C@O), 138.5, 138.0, 130.2–128.0
(Ar), 101.6 (CHPh), 100.3 (C-1), 99.3 (C-10), 80.2 (C-
4), 76.4 (C-3), 72.9 (C-30), 71.7, 71.7 (C-20 and C-50),
68.8 (C-60), 67.9 (C-40), 65.8 (C-5), 62.3 (C-6), 58.5 (C-
50), 57.3 (OCH3), 20.7, 20.6 (O- and N-COCH3);
ESIMS: m/z calcd for C30H40NO15 [M+H]+: 654.2398.
Found: 654.2426.
1.1. General methods
1H NMR (400.13 and 600.13 MHz) and 13C NMR
(75.5, 100.6, and 150.9 MHz) spectra were recorded in
CDCl3 (internal standard, for 1H residual CHCl3
d
7.24; for 13C: CDCl3 d 77.0), CD3OD (internal standard,
for 1H residual CH3OD d 3.30; for 13C: CD3OD d 49.0),
or D2O [external standard 3-(trimethylsilyl)-propionic
acid-d4, sodium salt (TSP) for 1H d 0.00, for 13C d
0.00]. H NMR and 13C NMR chemical shifts are re-
1
ported in parts per million (ppm). Coupling constants
(J) are reported in hertz (Hz). Chemical shifts and cou-
pling constants were obtained from a first-order analysis
of one-dimensional spectra. Assignments of proton and
carbon resonances were based on two-dimensional
1H–1H and 13C–1H correlation experiments. Multiplici-
ties are abbreviated as follows: singlet (s), doublet (d),
triplet (t), quartet (q), multiplet (m), and broadened
(br). All NMR spectra were recorded at 300 K. Analyt-
ical thin-layer chromatography (TLC) was performed
using silica gel 60 F254 precoated plates (250 lm) with
a fluorescent indicator, visualized under UV and charred
with 10% sulfuric acid in ethanol. Flash chromatogra-
phy was performed using silica gel 60 (230–400 mesh)
from EM science. All reactions were carried out under
nitrogen atmosphere with anhyd solvents freshly dis-
tilled according to standard procedures. Commercially
available methyl triflate and TESOTf were freshly
distilled under reduced pressure prior to their use in gly-
cosylation reactions. All other reagents were purchased
from commercial suppliers and used without further
purification.
1.3. Methyl 2-(N-acetylacetamido)-3-O-(2,3,4,6-tetra-O-
acetyl-b-D-glucopyranosyl)-6-O-benzyl-2-deoxy-b-D-
glucopyranoside (7)
Compound
6 (67 mg, 0.1 mmol), acetyl chloride
(374 lL, 5 mmol, 50 equiv), and N,N-diisopropylethyl-
amine (180 lL, 1 mmol, 10 equiv) were mixed together
˚
in CH2Cl2 (7 mL) containing 4 A molecular sieves
(670 mg). The reaction mixture was stirred at rt over-
night, then diluted with CH2Cl2 (50 mL) and washed
sequentially with satd aq NaHCO3 (30 mL) and brine
(30 mL). The aqueous phases were re-extracted with
CH2Cl2 (30 mL) and the combined organic layers were
dried (Na2SO4) and concentrated. Chromatography
(30:70!40:60, EtOAc/hexanes) gave 7 as a colorless
glass (69 mg, 89%). 1H NMR (400 MHz, CDCl3): d
7.51–7.45 (m, 5H, Ar), 5.56 (s, 1H, CHPh), 5.11 (d,
1H, J = 7.7 Hz, H-1), 5.06–5.03 (m, 2H, H-30, H-40),
4.95 (br t, 1H, J = 8.2 Hz, H-2), 4.75 (dd, 1H, J = 9.4,
8.5 Hz, H-3), 4.56 (d, 1H, J = 8.0 Hz, H-10), 4.35 (dd,
1H, J = 10.4, 4.9 Hz, H-6a0), 3.97 (dd, 1H, J = 12.5,
3.5 Hz, H-6a), 3.80 (t, 1H, J = 10.3 Hz, H-6b0), 3.76
(br s, 1H, H-6b), 3.74–3.66 (m, 2H, H-2, H-5), 3.59
(m, 1H, H-5), 3.46 (s, 3H, OCH3), 3.28 (m, 1H, H-50),
2.49–1.93 (6s, 6 · 3H, N-acetyl and O-acetyl); 13C
NMR (100.6 MHz, CDCl3): d 170.1 (C@O), 129.4–
126.0 (Ar), 101.6 (CHPh), 100.3, 100.3 (C-1, C-10),
81.3 (C-4), 76.7 (C-3), 73.1 (C-30 or C-40), 71.5, 71.5
(C-2, C-5), 68.9 (C-60), 67.7 (C-30 or C-40), 65.4 (C-5),
63.3 (C-2), 61.1 (C-6), 57.5 (OCH3), 25.2-20.5 (O- and
N-COCH3); ESIMS: m/z calcd for C32H42NO16
[M+H]+: 696.2504. Found: 696.2535.
1.2. Methyl 2-acetamido-3-O-(2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl)-4,6-O-benzylidene-2-deoxy-b-D-gluco-
pyranoside (6)
The known29 glycosyl acceptor 4 (50 mg, 0.15 mmol)
and Hg(CN)2 (88 mg, 0.46 mmol) were mixed in a mix-
ture of anhyd toluene (2.5 mL) and anhyd CH3NO2
˚
(2.5 mL) containing activated 3 A molecular sieves
(500 mg). This mixture was stirred at rt for 1 h and
heated to 40 ꢁC. 2,3,4,6-Tetra-O-acetyl-D-glucopyrano-
syl bromide30 5 (95.5 mg, 0.23 mmol) was added and
the mixture was stirred at 40 ꢁC for 16 h. The mixture
was diluted with CH2Cl2 (100 mL) and filtered through
Celiteꢂ. The solids were washed with CH2Cl2
(10 mL · 3) and the combined filtrate and washings were
washed sequentially with satd aq NaHCO3 (100 mL)

2430
L. Liao, F.-I. Auzanneau / Carbohydrate Research 341 (2006) 2426–2433
1.4. Methyl 2-(N-acetylacetamido)-3-O-(2,3,4,6-tetra-O-
acetyl-b-D-galactopyranosyl)-6-O-benzyl-2-deoxy-b-D-
glucopyranoside (8)
and concentrated. Chromatography (3:7, EtOAc/hex-
anes) gave trisaccharide 11 as a colorless glass (70 mg,
88%). 1H NMR (400 MHz, CDCl3): d 7.35–7.19 (m,
20H, Ar), 5.15 (d, 1H, J = 3.7 Hz, H-100), 5.01–4.98
(m, 2H, H-30, OCH2Ph), 4.92 (br t, 2H, J = 8.5 Hz, H-
20, H-40), 4.86–4.79 (m, 4H, H-1, H-3, OCH2Ph), 4.72–
4.58 (m, 4H, H-500, OCH2Ph), 4.50–4.38 (m, 4H, H-10,
H-6a0, OCH2Ph), 4.12 (d, 1H, J = 7.0, 2.7 Hz, H-200),
3.99 (dd, 1H, J = 12.4, 2.0 Hz, H-6b0), 3.96–3.90 (m,
3H, H-4, H-6a, H-300), 3.77 (br s, 1H, H-400), 3.68–3.54
(m, 4H, H-6b, H-2, H-20, H-50), 3.49 (m, 1H, H-5),
3.41 (s, 3H, OCH3), 2.50, 2.26 (2br s, 2 · 3H, N-acetyl),
2.02, 1.99, 1.97, 1.94 (4s, 4 · 3H, O-acetyl), 1.29 (d, 3H,
J = 6.5 Hz, H-600); 13C NMR (75.5 MHz, CDCl3): d
170.4, 170.0, 169.5, 169.1 (C@O), 138.8, 138.7, 138.2,
128.4–127.0 (Ar), 99.6 (C-10), 99.4 (C-1), 97.6 (C-100),
80.0 (C-300 or C-4), 78.4 (C-400), 77.5 (C-200), 75.4 (C-3),
75.0 (C-2 or C-20 or C-50), 74.9, 74.6, 73.2, 72.7
(OCH2Ph), 74.4 (C-4 or C-300), 73.6 (C-30), 72.1 (C-2
or C-20 or C-50), 71.4 (C-20 or C-40), 68.2 (C-20 or C-
40), 67.6 (OCH2Ph), 66.6 (C-500), 64.5 (C-2 or C-20 or
C-50), 61.9 (OCH2Ph), 57.0 (OCH3), 20.6 (O- and N-
COCH3), 16.9 (C-600); ESIMS: m/z calcd for
C59H71N2O20 [M+H]+: 1131.4913. Found: 1136.4845.
Compound 7 (50 mg, 72 lmol), sodium cyanoboro-
hydride (37 mg, 590 lmol, 8 equiv), and powdered acti-
˚
vated molecular sieves 4 A (100 mg) were mixed together
in anhyd THF (2 mL). A 2 M solution of HCl in Et2O
(697 lL) was added slowly at 0 ꢁC and stirring was
maintained at 0 ꢁC for an additional 30 min, once the
addition was complete (ꢁ30 min). The reaction mixture
was then diluted with CH2Cl2 (50 mL), washed sequen-
tially with 0.5 M HCl (30 mL), satd aq NaHCO3
(30 mL), and brine (30 mL). The aqueous phases were
re-extracted with CH2Cl2 (30 mL) and the combined
organic layers were dried (Na2SO4) and concentrated.
Chromatography (50:50, EtOAc/hexanes) gave acceptor
8 as a colorless glass (31 mg, 62%). 1H NMR (400 MHz,
CDCl3): d 7.32–7.27 (m, 5H, Ar), 5.15 (br t, 1H,
J = 9.5 Hz, H-20), 5.04–4.97 (m, 3H, H-10, H-30, H-40),
4.63 (s, 2H, OCH2Ph), 4.45 (d, 1H, J = 8.0 Hz, H-1),
4.45 (dd, 1H, J = 10.4, 7.0 Hz, H-3), 4.24–4.11 (m, 2H,
H-6a0, H-6b0), 4.05 (s, 1H, OH), 3.88 (dd, 1H,
J = 11.0, 1.6 Hz, H-6a), 3.82 (m, 1H, H-50), 3.71 (dd,
1H, J = 10.1, 5.1 Hz, H-6b), 3.61 (dd, 1H, J = 10.2,
7.8 Hz, H-2), 3.54 (m, 2H, H-4, H-5), 3.46 (s, 3H,
OCH3), 2.49–1.99 (6s, 6 · 3H, N-acetyl and O-acetyl);
13C NMR (100.6 MHz, CDCl3): d 175.2, 174.7, 170.5,
170.0, 169.9, 169.3 (C@O), 138.2, 128.3, 127.6 (Ar),
100.9 (C-1), 99.6 (C-10), 83.8 (C-3), 75.0 (C-4 or C-5),
73.5 (OCH2Ph), 72.8 (C-20), 71.9 (C-50), 71.2 (C-30 or
C-40), 70.3 (C-40 or C-50), 69.2 (C-6), 68.2 (C-30 or C-
40), 62.8 (C-2), 61.9 (C-60), 57.1 (OCH3), 28.4, 25.4,
20.6, 20.4, 20.4 (O- and N-COCH3); ESIMS: m/z calcd
for C32H47N2O16 [M+H]+: 698.2660. Found: 698.2662.
1.6. Methyl 2-(N-acetylacetamido)-3-O-(2,3,4,6-tetra-O-
acetyl-b-D-glucopyranosyl)-4-O-(2,3,4-tri-O-acetyl-a-L-
rhamnopyranosyl)-6-O-benzyl-2-deoxy-b-D-glucopyranos-
ide (12)
Compound 8 (50 mg, 72 lmol), peracetylated a-L-
rhamnopyranosyl trichloroacetimidate36 10 (155 mg,
0.36 mmol, 5 equiv), and powdered activated molecular
˚
sieves 4 A (100 mg) were mixed in anhyd CH2Cl2
(1.25 mL) and the mixture was stirred at rt for 3 h.
The reaction mixture was cooled down to ꢀ78 ꢁC and
a freshly prepared 0.37 M solution of TESOTf in anhyd
CH2Cl2 (30 lL, 10.8 lmol, 0.15 equiv) was added. The
reaction was then allowed to reach rt slowly (over 2 h)
and was quenched with NEt3 (10 lL). The reaction mix-
ture was diluted with CH2Cl2 (50 mL) and filtered
through Celiteꢂ. The solids were washed with CH2Cl2
(10 mL · 3) and the combined filtrate and washings were
washed sequentially with satd aq NaHCO3 (50 mL) and
brine (50 mL). The aqueous phases were re-extracted
with CH2Cl2 (30 mL) and the combined organic layers
were dried (Na2SO4) and concentrated. Chromatogra-
phy (40:60!50:50, EtOAc/hexane) gave trisaccharide
12 as a colorless glass (57 mg, 82%). 1H NMR
(400 MHz, CDCl3): d 7.32–7.25 (m, 5H, Ar), 5.28 (dd,
1H, J = 10.6, 3.8 Hz, H-300), 5.21 (m, 1H, H-200), 5.14
(br t, 1H, J = 10.0 Hz, H-400), 5.07 (br t, 1H,
J = 9.3 Hz, H-30), 5.01 (br s, 1H, H-100), 4.97–4.90 (m,
3H, H-1, H-20, H-40), 4.84 (br t, 1H, J = 10.0 Hz, H-
3), 4.59 (m, 1H, H-500), 4.54 (2d, 2H, J = 12.2 Hz,
OCH2Ph), 4.38 (d, 1H, J = 8.0 Hz, H-10), 4.26 (d, 2H,
1.5. Methyl 2-(N-acetylacetamido)-3-O-(2,3,4,6-tetra-O-
acetyl-b-D-glucopyranosyl)-6-O-benzyl-4-O-(2,3,4-tri-O-
benzyl-a-L-fucopyranosyl)-2-deoxy-b-D-glucopyranoside
(11)
Glycosyl acceptor 8 (50 mg, 72 lmol) and glycosyl do-
nor ethyl 2,3,4-tri-O-benzyl-1-thio-b-L-fucopyranoside35
9 (107.5 mg, 216 lmol, 3 equiv) were mixed together in
anhyd Et2O (2.5 mL) containing powdered, activated
˚
4 A molecular sieves (250 mg). The mixture was stirred
at rt for 3 h, CH3OTf (42.5 lL, 0.36 mmol, 5 equiv)
was added and the reaction was allowed to proceed at
rt for 18 h and was then quenched by the addition of
NEt3 (100 lL). The reaction mixture was diluted with
CH2Cl2 (50 mL) and filtered through Celiteꢂ. The solids
were washed with CH2Cl2 (10 mL · 3) and the com-
bined filtrate and washings were washed sequentially
with satd aq NaHCO3 (50 mL) and brine (50 mL). The
aqueous phases were re-extracted with CH2Cl2 (30 mL)
and the combined organic layers were dried (Na2SO4)

L. Liao, F.-I. Auzanneau / Carbohydrate Research 341 (2006) 2426–2433
2431
J = 5.3 Hz, H-6a0, H-6b0), 3.93 (br t, 1H, J = 9.4 Hz, H-
4), 3.74 (br s, 2H, H-6a, H-6b), 3.67–3.58 (m, 2H, H-50,
H-2), 3.49 (m, 1H, H-5), 3.43 (s, 3H, OCH3), 2.49, 2.34,
2.18, 2.14, 2.10, 2.03, 2.02, 1.99, 1.96 (9s, 9 · 3H, N-acet-
yl and O-acetyl), 1.30 (d, 3H, J = 6.3 Hz, H-600); 13C
NMR (100.6 MHz, CDCl3): d 170.4, 169.1 (C@O),
128.2, 127.5 (Ar), 99.5 (C-1), 99.2 (C-10), 96.0 (C-100,
1JC–H = 173 Hz), 74.4 (C-5), 73.8 (C-3), 73.6 (C-30),
73.1 (C-4), 73.0 (OCH2Ph), 72.2 (C-2 or C-50), 71.6
(C-20 or C-40), 71.3 (C-400), 70.3 (C-200), 69.5 (C-20 or
C-40), 68.9 (C-300), 67.9 (C-6), 66.1 (C-500), 64.2 (C-2 or
C-50), 62.7 (C-60), 57.1 (OCH3), 20.9, 20.7, 20.5 (O-
and N-COCH3), 17.2 (C-600); ESIMS: m/z calcd for
C44H63NO23 [M+Na]+: 992.3418. Found: 992.3376.
diluted with CH3OH, filtered, and concentrated. Chro-
matography with CHCl3/CH3OH (1:1) gave 2 that was
lyophilized from H2O after filtration on glass wool
and pure amorphous powder (15 mg, 75%) was ob-
tained. [a]D ꢀ73 (c 0.4, H2O); 1H NMR (600 MHz,
D2O): d 4.94 (d, 1H, J = 3.9 Hz, H-100), 4.70 (m, 1H,
H-500, overlap with solvent signal), 4.47 (d, 1H,
J = 7.8 Hz, H-10), 4.37 (d, 1H, J = 8.5 Hz, H-1), 3.97
(br t, 1H, J = 9.8 Hz, H-3), 3.92 (dd, 1H, J = 12.4,
2.2 Hz, H-6a), 3.87 (dd, 1H, J = 12.2, 2.0 Hz, H-6a0),
3.80 (m, 3H, H-2, H-6b, H-300), 3.73 (dd, 1H, J = 10.5,
4.0 Hz, H-200), 3.69 (br d, 1H, J = 2.8 Hz, H-400), 3.65
(br t, 1H, J = 9.4 Hz, H-4), 3.48 (m, 2H, H-5, H-6b0),
3.42 (s, 3H, OCH3), 3.38 (br t, 1H, J = 9.2 Hz, H-30),
3.31 (m, 2H, H-20, H-40), 2.06 (s, 3H, N-acetyl), 1.10
(d, 3H, J = 6.7 Hz, H-600); 13C NMR (150.9 MHz,
D2O): d 174.6 (C@O), 102.4 (C-10), 101.8 (C-1), 98.1
(C-100), 76.6 (C-3), 76.1 (C-50), 75.5 (C-5), 75.4 (C-30),
73.1 (C-20 or C-40), 72.9 (C-4), 72.0 (C-400), 70.5 (C-20
or C-40), 69.2 (C-300), 67.8 (C-200), 61.8 (C-60), 59.8 (C-
6), 57.1 (OCH3), 55.6 (C-2), 22.3 (N-COCH3), 15.5 (C-
600); ESIMS: m/z calcd for C21H38NO15 [M+H]+:
544.2241. Found: 544.2259.
1.7. Methyl 2-acetamido-6-O-benzyl-4-O-(2,3,4-tri-O-
benzyl-a-L-fucopyranosyl)-2-deoxy-3-O-(b-D-gluco-
pyranosyl)-b-D-glucopyranoside (13)
Compound 11 (63 mg, 57 lmol) was dissolved in
CH3OH (2.5 mL) and sodium (1 mg) was added. The
mixture was stirred at rt for 30 min and neutralized with
Dowexꢂ 50WX8-100 H+ resin. The resin was filtered off,
rinsed with CH3OH and the combined filtrate and wash-
ings were concentrated. Column chromatography (9:1,
CHCl3/CH3OH) gave 13 as a pure, colorless glass
1.9. Methyl 2-acetamido-6-O-benzyl-2-deoxy-3-O-(b-D-
galactopyranosyl)-4-O-(a-L-rhamnopyranosyl)-b-D-
glucopyranoside (14)
1
(40 mg, 78%). H NMR (400 MHz, CD3OD): d 7.49–
7.15 (m, 20H, Ar), 4.99 (d, 1H, J = 3.6 Hz, H-100),
4.95–4.82 (m, 2H, 2 · OCHPh), 4.72 (m, 1H, H-500),
4.75–4.57 (m, 4H, 4 · OCHPh), 4.46–4.38 (m, 3H, H-
10, 2 · OCHPh), 4.30 (d, 1H, J = 7.8 Hz, H-1), 3.98
(dd, 1H, J = 13.3, 2.6 Hz, H-200), 3.96–3.81 (m, 7H, H-
2, H-3, H-4, H-6a, H-6a0, H300, H-400), 3.67 (m, 1H, H-
6b), 3.58 (m, 1H, H-6b0), 3.46 (m, 1H, H-5), 3.45 (s,
3H, OCH3), 3.28 (m, 2H, H-30, H-50), 3.15 (m, 2H, H-
20, H-40), 1.94 (s, 3H, N-acetyl), 1.16 (d, 3H,
J = 6.5 Hz, H-600); 13C NMR (100.6 MHz, CD3OD): d
173.9 (C@O), 140.2, 139.4, 129.8–128.6 (Ar), 104.4 (C-
10), 103.2 (C-1), 98.5 (C-100), 81.1 (C-200), 79.5 (C-2 or
C-3 or C-4 or C-300 or C-400), 78.5 0(0C-30 or C-50), 78.5
(C2 or C-3 or C-4 or C-300 or C-4 ), 77.8 (C-30 or C-
50), 77.3 (C-2 or C-3 or C-4 or C-300 or C-400), 76.4 (C-
5), 76.3, 75.8, 74.5, 73.4 (4 · OCH2Ph), 74.9 (C-20 or
C-40), 74.2 (C-2 or C-3 or C-4 or C-300 or C-400), 74.2
(C-20 or C-40), 73.4 (C-6), 71.9 (C-500), 69.1 (C-60), 67.9
(OCH3), 67.9 (C-2 or C-3 or C-4 or C-300 or C-400),
23.1 (N-COCH3), 16.7 (C-600); ESIMS: m/z calcd for
C49H62NO15 [M+H]+: 904.4119. Found: 904.4123.
Compound 12 (47 mg, 0.049 mmol) was dissolved in
CH3OH (2.5 mL) and sodium (1 mg) was added. The
mixture was stirred at rt for 30 min and neutralized with
Dowexꢂ 50WX8-100 H+ resin. The resin was filtered,
rinsed with CH3OH and the combined filtrate and wash-
ings were concentrated. Column chromatography (9:1,
CHCl3/CH3OH) gave 14 as a pure, colorless glass
1
(26 mg, 84%). H NMR (400 MHz, CD3OD): d 7.48–
7.25 (m, 5H, Ar), 4.95 (br d, 1H, J = 1.4 Hz, H-100),
4.59 (br s, 2H, OCH2Ph), 4.45 (d, 1H, J = 7.6 Hz, H-
10), 4.44 (m, 1H, H-500), 4.38 (d, 1H, J = 8.2 Hz, H-1),
3.95–3.68 (m, 9H, H-2, H-3, H-4, H-6a, H-6b, H-40,
H-6a0, H-6b0, H-200), 3.48–3.42 (m, 5H, H-30, H-50, and
OCH3), 3.35–3.27 (m, 2H, H-5, H-400), 3.17 (m, 2H, H-
20, H-300), 1.95 (s, 3H, N-acetyl), 1.25 (d, 3H,
J = 6.3 Hz, H-600); 13C NMR (100.6 MHz, CD3OD): d
174.0 (C@O), 139.4, 129.4, 129.0, 128.9, 128.7 (Ar),
104.7 (C-10), 103.0 (C-1), 101.3 (C-100), 79.0 (C-2 or C-
3 or C-4 or C-40 or C-200), 78.0 (C-20 or C-300), 78.0 (C-
5 or C-400), 76.5 (C-50 or C-30), 74.9 (C-2 or C-3 or
C-4 or C-40 or C-200), 74.8 (C-20 or C-300), 74.5
(OCH2Ph), 74.1 (C-5 or C-400), 72.4 (C-2 or C-3 or C-4
or C-40 or C-200), 72.1 (C-2 or C-3 or C-4 or C-40 or
C-200), 70.5 (C-5 or C-30), 69.7 (C-60), 69.6 (C-500),
62.0 (C-6), 57.4 (C-2 or C-3 or C-4 or C-40 or C-200),
56.9 (OCH3), 23.2 (N-COCH3), 18.0 (C-600); CIMS:
m/z calcd for C28H44NO15 [M+H]+: 634.2711. Found:
634.2727.
1.8. Methyl 2-acetamido-2-deoxy-b-D-glucopyranosyl-
(1!3)-[a-L-fucopyranosyl-(1!4)]-b-D-glucopyranoside
(2)
Trisaccharide 13 (33 mg, 37 lmol) was dissolved in
CH3OH (5 mL) and 10% Pd/C (50 mg) was added.
The mixture was stirred under H2 for 24 h and then

2432
L. Liao, F.-I. Auzanneau / Carbohydrate Research 341 (2006) 2426–2433
Dantis, L.; Olkiewicz, K.; Llloyd, K. O.; Linvingston, P.
O.; Danishefsky, S. J.; Scher, H. I. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 5710–5715.
1.10. Methyl 2-acetamido-b-D-glucopyranosyl-(1!3)-
[a-L-rhamnopyranosyl-(1!4)]-2-deoxy-b-D-glucopyrano-
side (3)
5. Ragupathi, G.; Slovin, S. F.; Alduri, A.; Sames, D.;
Kim, I. J.; Kim, H. M.; Spassova, M.; Bornmann, W.
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Danishefsky, S. J. Angew. Chem., Intl. Ed. 1999, 38,
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6. Gilewski, T.; Ragupathi, G.; Bhuta, S.; Williams, L. J.;
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Trisaccharide 14 (19 mg, 30 lmol) was dissolved in
CH3OH (5 mL) and 10% Pd/C (50 mg) was added.
The mixture was stirred under H2 for 24 h. The reaction
mixture was diluted with CH3OH, filtered, and concen-
trated. Column chromatography with CHCl3/CH3OH
(1:1) gave 3 that was lyophilized from H2O after filtra-
tion on glass wool and obtained pure as an amorphous
powder (12 mg, 74%). [a]D ꢀ122 (c 0.4, H2O); 1H NMR
(600 MHz, D2O): d 4.87 (br s, 1H, H-100), 4.43 (d, 1H,
J = 7.8 Hz, H-10), 4.38 (d, 1H, J = 8.4 Hz, H-1), 4.30
(m, 1H, H-500), 3.93 (br t, 1H, J = 9.6 Hz, H-3), 3.91
(dd, 1H, J = 3.0, 1.4 Hz, H-200), 3.84 (br d, 2H,
J = 12.1 Hz, H-6a, H-6a0), 3.81 (br t, 1H, J = 9.6 Hz,
H-2), 3.72 (m, 2H, H-4, H-6b0), 3.69 (m, 1H, H-40),
3.67 (m, 1H, H-6b), 3.47 (m, 1H, H-5), 3.42 (s, 3H,
OCH3), 3.37 (m, 2H, H-30, H-400), 3.28 (m, 2H, H-50,
H-300), 3.16 (dd, 1H, J = 8.9, 8.1 Hz, H-20), 1.96 (s,
3H, N-acetyl), 1.17 (d, 3H, J = 6.3 Hz, H-600); 13C
NMR (150.9 MHz, D2O): d 174.6 (C@O), 102.5 (C-10),
101.8 (C-1), 100.2 (C-100), 76.4 (C-3), 76.1 (C-50 or C-
300), 75.5 (C-30 or C-400), 75.2 (C-5), 73.9 (C-4), 73.1 (C-
20), 71.9 (C-30 or C-400), 70.2 (C-200), 70.1 (C-40), 69.7
(C-300 or C-50), 68.9 (C-500), 61.1 (C-60), 60.1 (C-6), 57.2
(OCH3), 55.6 (C-2), 22.4 (N-COCH3), 16.8 (C-600);
ESIMS: m/z calcd for C21H38NO15 [M+H]+: 544.2241.
Found: 544.2259.
13. Hakomori, S.-I.; Zhang, Y. Chem. Biol. 1997, 4, 97–
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`
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Acknowledgments
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Eriksson, H.; Zopf, D.; Olsson, L.; Lundblad, A. Cancer
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We thank the Research Corporation and the National
Science and Engineering Research Council of Canada
for financial support of this work. L.L. thanks the
Ontario Government for a graduate scholarship.
20. Battifora, H.; Sørenson, H. R.; Mehta, P.; Ahn, C.;
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Supplementary data
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Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2006.07.006.
23. Pettijohn, D. E.; Pfenninger, O.; Brown, J.; Duke, R.;
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