Total synthesis of a tetra- and two pentasaccharide fragments of the O-specific polysaccharide of Shigella flexneri serotype 2a

Article abstract of DOI:10.1016/j.tet.2004.01.054

The synthesis of the methyl glycoside of the branched pentasaccharide biological repeating unit of the O-antigen of Shigella flexneri serotype 2a is described together with that of the methyl glycoside of the corresponding tetrasaccharide and frame-shifte

Full text of DOI:10.1016/j.tet.2004.01.054

Tetrahedron 60 (2004) 2475–2488
Total synthesis of a tetra- and two pentasaccharide fragments of
the O-specific polysaccharide of Shigella flexneri serotype 2aq
*
Laurence A. Mulard and Catherine Guerreiro
´
Unite de Chimie Organique, URA CNRS 2128, Institut Pasteur, 28 rue du Dr Roux, 75 724 Paris Cedex 15, France
Received 15 October 2003; revised 16 January 2004; accepted 20 January 2004
Abstract—The synthesis of the methyl glycoside of the branched pentasaccharide biological repeating unit of the O-antigen of Shigella
flexneri serotype 2a is described together with that of the methyl glycoside of the corresponding tetrasaccharide and frame-shifted linear
pentasaccharide. All the strategies disclosed herein involve a key disaccharide corresponding to the branching point and otherwise
appropriate monosaccharide building blocks activated as their trichloroacetimidate. Our data suggest partial lack of conformational
flexibility at the branched residue.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Allowing a better control of the various structural
parameters possibly involved in the immunogenicity of
glycoconjugate vaccines, oligosaccharide-protein conju-
gates were proposed as alternatives to polysaccharide–
protein conjugate vaccines against bacteria.24 Indeed, such
constructs were found immunogenic on several occasions,
including examples whereby the oligosaccharide portion
was made of one repeating unit only.5,18 We reasoned that
glycoconjugates incorporating chemically synthesized
oligosaccharides, appropriately selected for their ability to
mimic the native O-SP in terms of both antigenicity and
solution conformation, may offer an alternative to the
S. flexneri 2a O-SP-protein conjugates currently under
study. Our approach relies on a rational basis. Indeed, in
order to select the best oligosaccharide mimic, we have
undertaken the characterization of the antigenic determi-
nants of S. flexneri 2a O-SP recognized by serotype-specific
protective monoclonal antibodies. A panel of methyl
glycosides representative of fragments of S. flexneri 2a
O-SP was thus synthesized to be used as probes in the study
of antibody recognition.
Shigellosis, also known as bacillary dysentery, is a major
enteric disease which accounts for some 165 million annual
episodes, among which 1.1 million deaths, occurring mostly
in developing countries.12 Young children and immuno-
compromised individuals are the main victims. Some 15
years ago, vaccination was defined as a priority by the WHO
in its program on enteric diseases. However, there is still no
license vaccine against this bacterial infection although
intensive research is ongoing in the field.11 Shigellae are
Gram negative bacteria. As for other bacterial pathogens,
their lipopolysaccharide (LPS) is an important virulence
factor. It is also a major target of the host’s protective
immunity against infection.
Shigella flexneri 2a is the prevalent serotype in developing
countries, where it is responsible for the endemic form of
the disease.12 Based on the early hypothesis that a critical
level of serum IgG antibodies specific for the O-specific
polysaccharide (O-SP) moiety of the LPS was sufficient to
confer protection against homologous infections,26,27
several S. flexneri 2a O-SP-protein conjugates were
prepared. They were found safe and immunogenic in both
adults and children.2,22
The O-SP of S. flexneri 2a is a heteropolysaccharide defined
by the pentasaccharide repeating unit I.15,29 It features
a linear tetrasaccharide backbone, which is common to
all S. flexneri O-antigens and comprises a N-acetyl
q See Ref. 1.
Keywords: Carbohydrates; Glycosylation; Shigella flexneri; Lipopolysaccharide.
*
Corresponding author. Tel.: þ33-1-45-68-83-98; fax: þ33-1-45-68-84-04; e-mail address: lmulard@pasteur.fr
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.01.054

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L. A. Mulard, C. Guerreiro / Tetrahedron 60 (2004) 2475–2488
glucosamine and three rhamnose residues, together with an
a-D-glucopyranose residue branched at position 4 of one of
the rhamnoses. We have already reported on the synthesis of
the methyl glycosides of various fragments of the O-SP,
including the known EC disaccharide,6,16,19 the ECD19 and
B(E)C19 trisaccharides, the ECDA28 and AB(E)C9
tetrasaccharides, the B(E)CDA28 and DAB(E)C9 penta-
saccharides, the B00(E0)C0DAB(E)C octasaccharide3 and
more recently the D A0B0(E0)C0DAB(E)C decasaccharide.4
However, in order to complete the full set of frame-shifted
fragments of the repeating unit, the methyl glycosides of the
ECDAB, AB(E)CD pentasaccharides and that of the
B(E)CD tetrasaccharide, 1, 2 and 3, respectively, were
missing. Their synthesis is reported in the following.
C–D linkage was an appropriate disconnection site.3,4,28
Consequently, the synthesis of 1 was designed (Scheme 1)
based on the glycosylation of the known EC trichloro-
acetimidate donor 14,19 obtained in three steps (69%) from
the key diol 13,28 and the DAB trisaccharide acceptor 12.
The latter was obtained by the stepwise condensation of
known monosaccharide precursors, readily available by
selective protection, deprotection and activation sequences.
Thus, TMSOTf-catalysed condensation of the rhamno-
pyranoside acceptor 425 with the trichloroacetimidate
donor 57 in diethyl ether to give the fully protected
rhamnobioside 6,23 and subsequent de-O-acetylation gave
the AB disaccharide acceptor 725 in 91% overall yield,
which compares favourably with the previously described
preparation using the corresponding 1-O-acetyl donor.25
Analogously to previous work in a related series,4 the
known glucosaminyl trichloroacetimidate donor 9,8 was
chosen as the precursor to residue D. Conventional
glycosylation of 7 with 9 was best performed in acetonitrile
using tin trifluoromethanesulfonate (Sn(OTf)2) as the
catalyst17 to give the fully protected trisaccharide 10 in
2. Results and discussion
Analysis of the targets shows that all the glycosylation
reactions to set up involve 1,2-trans glycosidic linkages
except for that at the E–C junction which is 1,2-cis.
Consequently, the syntheses described herein rely on key
EC disaccharide building blocks as well as on appropriate
A, B and D monosaccharide synthons.
1
72% yield (extracted from the H NMR spectrum). When
TMSOTf was used instead of Sn(OTf)2, 10 was formed in
lower yield (52%) outlining the sensitivity of the tetra-
chlorophtaloyl group to these stronger conditions, as
previously noted.14 A three step process including heating
10 with ethylenediamine in dry ethanol,10 ensuing N-acetyl-
ation with acetic anhydride, and de-O-acetylation under
2.1. Synthesis of the linear ECDAB-OMe
pentasaccharide 1
´
Zemplen conditions, furnished the triol 11 (51% from 7). It
Earlier findings in the series have demonstrated that the
Scheme 1. (a) 6 from 5, 8 from 20, TMSOTf, Et2O, 235 8C!rt; (b) cat. MeONa, MeOH–CH2Cl2, rt; (c) from 7, Sn(OTf)2, CH3CN, rt; (d) (i)
˚
H2NCH2CH2NH2, EtOH, 60 8C, (ii) Ac2O, EtOH; (iii) MeONa, MeOH–CH2Cl2, rt; (e) Me2C(OMe)2, PTSA, acetone, rt; (f) see Ref. 19; (g) 4 A molecular
sieves, TfOH, CH2Cl2, 215 8C!rt; (h) 90% aq. TFA, 0 8C; (i) cat. MeONa, MeOH–CH2Cl2, rt; (j) H2, 10% Pd/C, EtOH–AcOH, rt.

L. A. Mulard, C. Guerreiro / Tetrahedron 60 (2004) 2475–2488
2477
was next protected at positions 4D and 6D by regioselective
introduction of an isopropylidene acetal upon reaction with
2,2-dimethoxypropane under acid-catalysis to give 12
(96%). The latter acetal-protecting group was selected
based on data previously obtained when synthesizing
shorter fragments in the series which had outlined the
interest of using 4,6-O-isopropylidene–glucosaminyl inter-
mediates instead of the more common benzylidene
analogues.19 Once the two key building blocks were made
available, their condensation was performed in dichloro-
methane in the presence of a catalytic amount of triflic acid
to give the fully protected pentasaccharide 15 (84%).
Conventional stepwise deprotection involving (i) acidic
hydrolysis of the isopropylidene acetal using 90% aq. TFA
to give diol 16 (95%), (ii) conversion of the latter into the
synthesis of 2. Indeed, most of our previous work in the
series relied on the use of the known 2-O-acetyl rhamno-
pyranosyl donor 5. In the reported syntheses,9 selective
de-O-acetylation at position 2B in the presence of a 2C-O-
benzoate was best performed by treatment with methanolic
HBF4·OEt2 for 5 days. Clearly, such de-O-acetylation
conditions are not compatible with the presence of an
isopropylidene acetal on the molecule. To overcome this
limitation, the corresponding 2-O-chloroacetyl rhamno-
pyranosyl trichloroacetimidate 20 was selected as an
alternate donor. In theory, the latter could also serve as an
appropriate precursor to residue A.
Regioselective conversion of diol 13 into its 2-O-benzoyl-
ated counterpart 21 was performed as described (Scheme
3).28 Treatment of the latter with chloroacetic anhydride and
pyridine gave the orthogonally protected 22 (95%), which
was smoothly de-O-allylated to yield the corresponding
hemiacetal 23 (91%) by a two-step process, involving (i)
iridium (I)-promoted isomerisation21 of the allyl glycoside
and (ii) subsequent hydrolysis in the presence of iodine.20
The selected trichloroacetimidate leaving group was
successfully introduced by treatment of 23 with
trichloroacetonitrile in the presence of 1,8-diazabicy-
clo[4.3.0]undec-7-ene (DBU), which resulted in the for-
mation of 19 (84%) together with the recovery of some
starting hemiacetal (14%) since partial hydrolysis during
column chromatography could not be avoided. TMSOTf-
mediated glycosylation of donor 19 and acceptor 18
furnished the fully protected ECD trisaccharide (24,
80%), which was readily converted to the required acceptor
25 upon selective deblocking of the chloroacetyl protecting
group with thiourea (97%). Following the two-step protocol
described above for the preparation of 19, the known allyl
rhamnopyranoside 27,33 bearing a 2-O-chloroacetyl protect-
ing group, was converted to the hemiacetal 28 (85%)
(Scheme 4). Next, treatment of the latter with trichloroace-
tonitrile and a slight amount of DBU gave at best donor 20
´
corresponding tetraol 17 under Zemplen conditions (86%),
and (iii) final hydrogenolysis of the benzyl protecting
groups, gave the linear pentasaccharide target 1 in 81%
yield.
2.2. Synthesis of the AB(E)CD pentasaccharide 2 and of
the B(E)CD tetrasaccharide 3
For reasons mentioned above, the glucosaminyl acceptor
18,19 protected at its 4 and 6 hydroxyl groups by an
isopropylidene acetal was the precursor of choice for
residue D (Scheme 2). In the past, introduction of residue
B at position 3C was performed on a 2C-O-benzoylated EC
acceptor resulting from the regioselective acidic hydrolysis
of the corresponding 2,3-orthoester intermediate.9,28 It
rapidly occurred to us that opening of the intermediate
phenyl orthoester was not compatible with the presence of
the 4D,6D-O-isopropylidene acetal. For that reason, the
trichloroacetimidate donor 19, suitably benzoylated at
position 2C and orthogonally protected by a chloroacetyl
group at position 3C was used as the EC building block
instead of the previously used 14. Protection at the 2-OH of
the rhamnosyl precursor to residue B was also crucial in the
Scheme 2. Retrosynthetic analysis of pentasaccharide 2.

2478
L. A. Mulard, C. Guerreiro / Tetrahedron 60 (2004) 2475–2488
Scheme 3. (a) see Ref. 28; (b) (ClAc)2O, Pyridine–CH2Cl2, 0 8C; (c) (i) (COD)Irþ(P(MePh2)2)PF62, THF; (ii) I2, THF/H2O, rt; (d) CCl3CN, DBU, CH2Cl2,
˚
0 8C; (e) 4 A molecular sieves, TMSOTf, CH2Cl2, 260 8C!rt; (f) thiourea, MeOH–pyridine, 65 8C.
in a yield of 73%. Although the isolated yield of 20 was not
better (72%), running the activation step in the presence of
K2CO3 instead of DBU resulted in a more reproducible
isolated yield of the activated donor. Glycosylation of the
ECD acceptor 25 and the B donor 20 was attempted under
various conditions of solvent and catalyst. Whatever the
conditions, hardly separable mixtures of compounds were
obtained, among which the yield of the target tetrasacchar-
ide reached 45–50%. Running the condensation in Et2O in
the presence of TMSOTf as the promoter were the best
conditions tested, although the expected tetrasaccharide 29
was often slightly contaminated with glycosylation inter-
mediates such as the silylated 26 or the orthoester 35, as
suggested from mass spectroscopy analysis and NMR data.
In fact, the nature of the latter was fully ascertained at the
next step in the synthesis. Indeed, full recovery of the
starting material was observed upon treatment of 35 with
thiourea (Fig. 1). On the contrary, treatment of a mixture of
the condensation products 29 and supposedly 26 under the
same conditions led to the expected tetrasaccharide acceptor
31 and the trisaccharide acceptor 25 (not described). The
bB-tetrasaccharide isomer could not be detected at this
stage, indicating that the corresponding chloroacetylated
bB-anomer was probably not part of the initial mixture.
Formation of the starting 25 during the dechloroacetylation
step was not unexpected, since loss of a trimethylsilyl group
under similar treatment was observed for a model
compound (not described). Although the fluoride analog
corresponding to donor 20 has been used successfully in a
prior report,33 the poor yield of 29 may be, in part,
associated to the sensitivity of the chloroacetyl group to the
glycosylation conditions. Thus, in order to investigate the
poor outcome of the condensation reaction, the donor
properties of the chloroacetylated 20 were compared to that
of the more common acetylated 5. When methyl rhamno-
pyranoside 4 was condensed with 20 as described for the
preparation of 6, the rhamnobioside 8 was isolated in 67%
yield. This result tends to suggest that indeed the acetylated
5 is a more powerful donor than 20.
Starting from 20 and 25, the isolated yield of the
tetrasaccharide acceptor 31 was 34%, which encouraged
us to reconsider the use of 5 as a precursor to residues B and
A in the synthesis of 2. Condensation of 5 and 25 in CH2Cl2
using TMSOTf as the promoter furnished the corresponding
tetrasaccharide 30 (72%). However, even though the yield
of 30 was better than that of 29, slight contamination by the
silylated side-product 26 was again apparent, outlining the
somewhat poor reactivity of the ECD acceptor. Subsequent
treatment of 30 with a 4 mM ethanolic solution of
guanidine13 resulted, as expected, in selective 2B-O-
deacetylation to give 31 in a satisfactory 83% yield,
which outlined the interest of the method. However,
previous experience in other closely related series has
shown that the selectivity of the method was highly
dependent on the nature of the substrate. Nevertheless, the
2-O-acetylated donor 5 was clearly preferred to the
chloroacetate analogue 20. Condensation of the tetra-
saccharide acceptor 31 and donor 5 in the presence of
TMSOTf gave the fully protected pentasaccharide 32 in a
yield of 52%. TFA-mediated hydrolysis of the isopropyl-
idene acetal followed by transesterification of the ester
groups and subsequent conventional hydrogenolysis of the
benzyl ethers finally gave the target pentasaccharide 2
(88%).
Figure 1.

L. A. Mulard, C. Guerreiro / Tetrahedron 60 (2004) 2475–2488
2479
Scheme 4. (a) (i) (COD)Irþ(P(MePh2)2)PF62, THF; (ii) I2, THF/H2O, rt; (b) CCl3CN, K2CO3, CH2Cl2, 0 8C; (c) 29 from 20, 30 from 5, TMSOTf, Et2O,
˚
260 8C!0 8C; (d) thiourea, MeOH–pyridine, 65 8C; (e) guanidine, EtOH–CH2Cl2, rt; (f) 4 A molecular sieves, TMSOTf, Et2O, 260 8C!rt; (g) 50% aq.
TFA, CH2Cl2, 0 8C; (h) 0.5% MeONa, MeOH, 55 8C; (i) 10% Pd/C, EtOH–AcOEt, 1 N aq. HCl, rt.
Alternatively, the fully protected tetrasaccharide 30 was
converted to the diol 36 by acidic removal of the
isopropylidene acetal (85%), and subsequently to the
corresponding tetraol 37 upon transesterification (83%).
Final hydrogenolysis of the benzyl groups furnished the
target tetrasaccharide 3 (71%) (Scheme 5).
tetra- and pentasaccharides were observed repeatedly. At
the protected and partially protected stage, major altered
signals are those tentatively assigned to C-3C and C-4C.
Besides, signals assigned to C-2D, C-3D as well as to C-1B
are significantly broader than expected. Loss of confor-
mational flexibility at the C ring is not totally unexpected
especially since the carbons involved are those correspond-
ing to the branching points. Of particular interest, however,
was the observation that residue D, the N-acetyl-glucosa-
minyl residue, was also partially constrained. Full confor-
mational freedom of residue D is recovered when the
B(E)CD and AB(E)CD oligosaccharides are in their free
form. However, this observation does not stand true for
residue C since characteristic broad signals for C-3C and C-
4C as well for C-1B and C-1E are still present in the 13C
NMR spectra of compounds 2 and 3, respectively.
Noteworthy, in the case of intermediates 33 and 36,
successful sodium methoxide-mediated transesterification
of the acyl groups required heating of the reaction
mixture.31 Isolation of the esters to be cleaved may best
explain the phenomenon. Indeed, the above-mentioned
procedure may be seen as an alternative to the use of
K2CO3 in dioxane/methanol30 or that of tBuOK in
methanol,32 which were found appropriate in related
cases. Steric hindrance may account for the poor outcome
of the condensation of the ECD acceptor 25 with the B
donors 20 and 5. Interestingly, 13C NMR data support this
hypothesis. Although no altered signals could be seen in the
13C NMR spectrum of the ECD acceptor 25 or in the 13C
NMR spectra of the fully protected precursor 24, significant
disturbance of several signals in the 13C NMR spectra of the
Overall, these observations suggest
a
somewhat
compact organisation at the branching point of the
B(E)CD structure. It is worth mentioning that none of
these disturbed signals are seen in the 13C NMR spectra of
the oligosaccharides corresponding to the linear ECDAB
fragment.
Scheme 5. (a) 50% aq. TFA, CH2Cl2, 0 8C; (b) 0.1% MeONa, MeOH, 55 8C; (c) 10% Pd/C, EtOH–AcOEt, 1 N aq. HCl, rt.

2480
L. A. Mulard, C. Guerreiro / Tetrahedron 60 (2004) 2475–2488
3. Conclusion
(200 mg) was added to a solution of alcohol25 4 (60 mg,
167 mmol) and trichloroacetimidate donor 20 (113 mg,
0.2 mmol) in dry Et2O (2 mL) and the solution was stirred at
rt for 30 min then cooled to 240 8C. TMSOTf (9 mL,
50 mmol) was added and the mixture was stirred for 1 h at
230 8C, then for 2 h while the bath temperature was coming
back to rt. TLC (solvent B, 4:1) showed the presence of a
major product less polar than 4. The mixture was neutralized
by addition of Et3N, and filtered on a pad of Celite.
Concentration of the filtrate and column chromatography of
the residue (solvent B, 4:1) gave 86 mg of 8 as a colourless
The synthesis of the methyl glycoside (2) of the repeating
unit I of the S. flexneri 2a O-SP, together with that of the
corresponding frame-shifted pentasaccharide 1 and tetra-
saccharide 3 were described. All the methyl glycosides of
the di- to pentasaccharides obtained by circular permutation
of the monosaccharide residues partaking in the linear
backbone of I, and comprising the EC portion, are now
available in the laboratory. Their binding to a set of
protective monoclonal IgG antibodies will be reported
elsewhere.
1
oil (67%). [a]D 213.6 (c 1.0); H NMR d 7.42–7.32 (m,
20H, Ph), 5.64 (dd, 1H, J1,2¼1.9 Hz, J2,3¼3.2 Hz, H-2A),
5.07 (d, 1H, H-1A), 4.98–4.93 (m, 2H, OCH2), 4.83–4.61
(m, 6H, OCH2), 4.64 (brs, 1H, H-1B), 4.18 (d, 1H,
J¼15.2 Hz, CH2Cl), 4.13 (d, 1H, OCH2Cl), 3.90 (dd, 1H,
J3,4¼9.3 Hz, H-3B), 3.89 (m, 1H, partially overlapped,
J5,6¼6.3 Hz, H-5A), 3.73 (dq, 1H, J4,5¼9.5 Hz, J5,6¼6.2 Hz,
H-5B), 3.48 (pt, 1H, J3,4¼9.4 Hz, H-4B), 3.45 (pt, 1H,
J3,4¼J4,5¼9.4 Hz, H-4A), 3.36 (s, 3H, OCH3), 1.37 (d, 3H,
H-6A), 1.35 (d, 3H, H-6B); 13C NMR d 165.5 (CvO),
137.4–126.4 (Ph), 100.2 (C-1A), 99.2 (C-1B), 80.4, 80.3,
80.2 (3C, C-4A, 4B, 3B), 77.9 (C-3A), 75.8, 75.7 (2C, OCH2),
74.8 (C-2B), 72.6, 72.5 (2C, OCH2), 71.2 (C-2A), 68.7
(C-5A), 68.2 (C-5B), 55.0 (OCH3), 41.4 (CH2Cl), 18.4 (2C,
C-6A, 6B). FABMS for C43H49ClNO10 (M, 760.3) m/z 783.3
[MþNa]þ. Anal. calcd for C43H49ClNO10: C, 67.84; H,
6.49%. Found: C, 68.03; H, 7.02.
4. Experimental
4.1. General methods
Melting points were determined in capillary tubes with an
electrothermal apparatus and are uncorrected. Optical
rotations were measured for CHCl3 solutions at 25 8C,
except where indicated otherwise, with a Perkin–Elmer
automatic polarimeter, Model 241 MC. TLC on precoated
slides of Silica Gel 60 F254 (Merck) was performed with
solvent mixtures of appropriately adjusted polarity
consisting of A, dichloromethane–methanol; B, cyclo-
hexane–ethyl acetate, C, cyclohexane–acetone, D,
water–acetonitrile, E, iso-propanol–ammonia–water; F,
0.01 M aq. TFA–acetonitrile. Detection was effected when
applicable, with UV light, and/or by charring with orcinol
(35 mM) in 4 N aq. H2SO4. Preparative chromatography
was performed by elution from columns of Silica Gel 60
(particle size 40–63 mm). RP-HPLC (215 nm) used a
4.1.2. Methyl (3,4-di-O-benzyl-a-L-rhamnopyranosyl)-
(1!2)-3,4-di-O-benzyl-a-L-rhamnopyranoside (7). Acti-
˚
vated powered 4 A molecular sieves was added to a solution
of alcohol 4 (322 mg, 0.90 mmol) and trichloroacetimidate
donor7 5 (573 mg, 1.08 mmol) in dry Et2O (9 mL) and the
solution was stirred at rt for 30 min then cooled to 235 8C.
TMSOTf (48 mL, 266 mmol) was added and the mixture
was stirred for 4 h, while the bath temperature was coming
back to rt. TLC (solvent B, 23:2) showed that only little
starting material remained and the mixture was neutralized
by addition of Et3N, and filtered on a pad of Celite.
Concentration of the filtrate and column chromatography of
the residue (solvent B, 9:1) gave 647 mg of slightly
contaminated 6. The later (626 mg) was dissolved in a
mixture of CH2Cl2 (2 mL) and MeOH (5 mL), and 1 M
methanolic sodium methoxide (300 mL) was added. The
mixture was stirred overnight, neutralized with Amberlite
IR 120 (Hþ), filtered and concentrated. Chromatography of
the residue (solvent G, 89:11) gave syrupy 7 (554 mg, 91%
from 4). Analytical data were as described.25
˚
Kromasil 5 mm C18 100 A 4.6£250 mm analytical column
(1 mL min21). The NMR spectra were recorded at 20 8C for
solution in CDCl3, unless stated otherwise, on a Brucker
1
Avance 400 spectrometer (400 MHz for H, 100 MHz for
13C). External references: for solutions in CDCl3, TMS
(0.00 ppm for both 1H and 13C); for solutions in D2O,
dioxane (67.4 ppm for 13C) and trimethylsilyl-3-propionic
1
acid sodium salt (0.00 ppm for H). Proton signal assign-
ments were made by first-order analysis of the spectra, as
well as analysis of two-dimensional 1H–1H correlation
maps (COSY) and selective TOCSY experiments. Of the
two magnetically non-equivalent geminal protons at C-6,
the one resonating at lower field is denoted H-6a and the one
at higher field is denoted H-6b. The 13C NMR assignments
were supported by two-dimensional 13C–1H correlation
maps (HETCOR). Interchangeable assignments are marked
with an asterisk in the listing of signal assignments. Sugar
residues in oligosaccharides are serially lettered according
to the lettering of the repeating unit of the O-SP and
identified by a subscript in the listing of signal assignments.
Low-resolution mass spectra were obtained by either
chemical ionisation (CIMS) using NH3 as the ionising gas,
by electrospray mass spectrometry (ESMS), or by fast atom
bombardment mass spectrometry (FABMS). HRMS were
obtained by Matrix Assisted Laser Desorption Ionisation
Mass Spectrometry (MALDIMS).
4.1.3. Methyl (3,4,6-tri-O-acetyl-2-deoxy-2-tetrachloro-
phtalimido-b-D-glucopyranosyl)-(1!2)-(3,4-di-O-ben-
zyl-a-L-rhamnopyranosyl)-(1!2)-3,4-di-O-benzyl-a-L-
rhamnopyranoside (10). A solution of disaccharide 7
(179 mg, 0.26 mmol) and trichloroacetimidate donor8
9
(436 mg, 0.60 mmol) in dry CH3CN (9 mL) was stirred at rt
˚
for 30 min in the presence of activated 4 A molecular sieves
(1.2 g). Tin(II) trifluoromethanesulfonate [Sn(OTf)2]
(75 mg, 180 mmol) was added and the mixture was stirred
at rt for 4 h, then neutralized with Et3N. Filtration on a pad
of Celite, concentration of the filtrate and column
chromatography of the residue (solvent B, 87:13) gave 10
(324 mg) as a slightly contaminated white foam (72% as
4.1.1. Methyl (3,4-di-O-benzyl-2-O-chloroacetyl-a-L-
rhamnopyranosyl)-(1!2)-3,4-di-O-benzyl-a-L-rhamno-
˚
pyranoside (8). Activated powered 4 A molecular sieves

L. A. Mulard, C. Guerreiro / Tetrahedron 60 (2004) 2475–2488
2481
estimated from the 1H NMR spectrum). An analytical
sample had [a]D þ23.3 (c 1.0); H NMR d 7.43–7.17 (m,
(C-6D), 58.9 (C-2D), 54.6 (OCH3), 22.3 (C(O)CH3), 17.9,
17.7 (2C, C-6A, 6B). FABMS for C49H61NO14 (M, 887.44)
m/z 910.1 [MþNa]þ. Anal. calcd for C49H61NO.14H2O: C,
64.96; H, 7.01; N, 1.55%. Found: C, 65.19; H, 6.83; N,
1.51%.
1
20H, Ph), 5.92 (d, 1H, J¼9.2, 10.5 Hz, H-3D), 5.24 (d, 1H,
J1,2¼8.4 Hz, H-1D), 5.14 (dd, 1H, J¼9.7, 9.4 Hz, H-4D),
5.00 (brs, 1H, H-1A), 4.79 (d, 1H, J¼10.8 Hz, OCH2), 4.65
(s, 2H, OCH2), 4.55 (d, 1H, J¼11.2 Hz, OCH2), 4.53 (brs,
1H, H-1B), 4.46–4.36 (m, 3H, H-2D, OCH2), 4.28 (d, 1H,
J¼12.4 Hz, OCH2), 4.26 (d, 1H, J¼10.6 Hz, OCH2), 4.06
(dd, 1H, J6a,6b¼12.5 Hz, J5,6a¼6.8 Hz, H-6aD), 3.91 (brs,
1H, H-2B), 3.85–3.69 (m, 5H, H-2A, 3B, 3A, 6bD, 5pA), 3.59
(dq, 1H, J4,5¼9.4 Hz, J5,6¼6.2 Hz, H-5pB), 3.40 (m, 1H,
H-5D), 3.27 (s, 3H, OCH3), 3.18 (m, 2H, H-4A, 4B), 2.03,
2.01, 1.94 (3s, 9H, C(O)CH3), 1.27, 1.25 (2d, 6H, H-6A, 6B);
13C NMR d 170.5, 170.4, 170.3, 163.8, 162.6 (5C, CvO),
140.3–128.0 (Ph), 101.1 (C-1A), 100.0 (C-1D), 99.8 (C-1B),
80.7 (2C, C-4A, 4B), 79.7 (C-2A), 78.9 (C-3B), 78.1 (C-3A),
76.2 (C-2B), 75.3, 75.2, 72.7, 71.4 (4C, OCH2), 71.3 (C-5D),
70.1 (C-3D), 68.5 (C-5Ap ), 68.4 (C-4D), 67.4 (C-5pB), 61.3
(C-6D), 55.4 (C-2D), 54.6 (OCH3), 20.7, 20.6 (3C,
C(O)CH3), 18.0, 17.7 (2C, C-6A, 6B). FABMS for
C61H63Cl4NO18 (M, 1237.3) m/z 1259.9 [MþNa]þ. Anal.
calcd for C61H63Cl4NO18·H2O: C, 58.24; H, 5.21; N, 1.11%.
Found: C, 58.21; H, 4.91; N, 1.01%.
4.1.5. Methyl (2-acetamido-2-deoxy-4,6-O-isopropyl-
idene-b-D-glucopyranosyl)-(1!2)-(3,4-di-O-benzyl-a-L-
rhamnopyranosyl)-(1!2)-3,4-di-O-benzyl-a-L-rhamno-
pyranoside
(12).
2,2-Dimethoxypropane
(4.9 mL,
39.8 mmol) and para-toluenesulfonic acid (18 mg,
95 mmol) were added to a solution of the triol 11
(964 mg, 1.09 mmol) in acetone (3 mL) and the mixture
was stirred at rt for 1 h. Et3N was added, and volatiles were
evaporated. Column chromatography of the residue (solvent
A, 99:1) gave the acceptor 12 as a white solid (969 mg,
96%) which could be crystallized from AcOEt/iPr2O; mp
164–165 8C [a]D 225.9 (c 1.0); 1H NMR d 7.45–7.31 (m,
20H, Ph), 6.98 (d, 1H, JNH,2¼2.4 Hz, NH), 6.37 (brs, 1H,
OH), 5.07 (d, 1H, J1,2¼1.9 Hz, H-1A), 4.90 (d, 1H,
J¼10.8 Hz, OCH2), 4.85 (d, 1H, J¼10.1 Hz, OCH2), 4.84
(d, 1H, J¼10.8 Hz, OCH2), 4.76 (d, 1H, OCH2), 4.69 (d, 1H,
OCH2), 4.68 (s, 2H, OCH2), 4.65 (d, 1H, OCH2), 4.61 (d,
1H, J1,2¼1.6 Hz, H-1B), 4.48 (d, 1H, J1,2¼8.3 Hz, H-1D),
4.09 (dd, 1H, H-2A), 4.01 (dd, 1H, J2,3¼3.2 Hz,
J3,4¼9.4 Hz, H-3A), 3.91 (dd, 1H, H-2B), 3.89–3.84 (m,
4.1.4. Methyl (2-acetamido-2-deoxy-b-D-glucopyrano-
syl)-(1!2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-
(1!2)-3,4-di-O-benzyl-a-L-rhamnopyranoside (11). A
solution of disaccharide 7 (179 mg, 0.26 mmol) and
trichloroacetimidate donor 9 (436 mg, 0.60 mmol) in dry
CH3CN (9 mL) was stirred at rt for 30 min in the presence of
0
0
0
0
2H, J5,6¼6.3 Hz, J4,5¼9.4 Hz, J2 ,3 ¼3.3 Hz, J3 ,4 ¼9.4 Hz,
H-5A, 3B), 3.68 (dq, partially overlapped, J5,6¼6.2 Hz,
J4,5¼9.5 Hz, H-5B), 3.66–3.58 (m, 5H, H-6aD, 6bD, 2D, 3D,
4D), 3.44 (pt, 1H, H-4A), 3.41 (pt, 1H, H-4B), 3.32 (s, 3H,
OCH3), 3.16 (m, 1H, H-5D), 1.60 (s, 3H, C(O)CH3), 1.54,
1.48 (2s, 6H, C(CH3)2), 1.35 (d, 6H, H-6A, 6B); 13C NMR d
173.9 (CvO), 138.8–128.0 (Ph), 103.7 (C-1D), 101.3
(C-1A), 100.3 (C(CH3)2), 100.2 (C-1B), 81.9 (C-4A), 80.8
(C-4B), 80.5 (C-3A), 79.7 (C-3B), 79.4 (C-2A), 76.2 (OCH2),
76.0 (C-2B), 75.6, 75.1 (2C, OCH2), 74.7 (C-4D), 74.4
(C-3D), 72.6 (OCH2), 68.6 (C-5A), 68.0, 67.9 (2C, C-5B,
5D), 62.2 (C-6D), 60.6 (C-2D), 55.1 (OCH3), 29.5 (C(CH3)2),
22.7 (C(O)CH3), 19.4 (C(CH3)2), 18.5, 18.2 (2C, C-6A, 6B).
FAB-MS for C52H65NO14 (M, 927.44) m/z 950.1 [MþNa]þ.
Anal. calcd for C52H65NO14: C, 67.30; H, 7.06; N, 1.51%.
Found: C, 67.12; H, 6.98; N, 1.44%.
˚
activated 4 A molecular sieves (1.2 g). Tin(II) trifluoro-
methanesulfonate [Sn(OTf)2] (75 mg, 180 mmol) was added
and the mixture was stirred at rt for 4 h, then neutralized
with Et3N. Filtration on a pad of Celite, concentration of the
filtrate and column chromatography of the residue (solvent
B, 87:13) gave 10 (324 mg) as a slightly contaminated
product. The latter was solubilized in dry ethanol (13 mL)
and diethylamine (200 mL, 3.0 mmol) was added and the
mixture was stirred overnight at 60 8C. The mixture was
cooled to rt and acetic anhydride (1.0 mL, 10.6 mmol) was
added and the mixture was stirred at this temperature for
2 h. The suspension was filtered and volatiles were
evaporated and co-evaporated repeatedly with toluene and
cyclohexane. The crude residue was taken up in a minimum
of CH2Cl2 and MeOH (10 mL). 1 N methanolic sodium
methoxide was added until the pH was 10 and the solution
was stirred overnight at rt, neutralized with IR 120 (Hþ),
filtered and concentrated. Chromatography of the residue
(solvent A, 24:1) gave foamy 11 (135 mg, 51% from 7).
4.1.6. Methyl (2,3,4,6-tetra-O-benzyl-a-D-glucopyrano-
syl)-(1!4)-(2,3-di-O-benzoyl-a-L-rhamnopyranosyl)-
(1!3)-(2-acetamido-2-deoxy-4,6-O-isopropylidene-b-D-
glucopyranosyl)-(1!2)-(3,4-di-O-benzyl-a-L-rhamno-
pyranosyl)-(1!2)-3,4-di-O-benzyl-a-L-rhamnopyrano-
˚
side (15). Activated powdered 4 A molecular sieves were
1
[a]D 215.0 (c 1.0); H NMR d 7.44–7.28 (m, 20H, Ph),
added to a solution of the trisaccharide acceptor 12 (202 mg,
0.22 mmol) and the disaccharide donor 1419 (263 mg,
0.25 mmol) in anhydrous CH2Cl2 (5 mL) and the suspen-
sion was stirred for 30 min at 215 8C. TfOH (7 mL,
34 mmol) was added and the mixture was stirred for 2 h
while the bath temperature was slowly coming back to
10 8C. TLC (solvent D, 49:1) showed that no 12 remained.
Et3N was added and after 30 min, the suspension was
filtered through a pad of Celite. Concentration of the filtrate
and chromatography of the residue (solvent B, 9:1!17:5)
gave the fully protected pentasaccharide 15 (330 mg, 84%)
as a white foam; [a]D þ63.3 (c 1.0); 1H NMR d 8.07–6.96
(m, 50H, Ph), 5.82 (d, 1H, JNH,2¼7.4 Hz, NH), 5.63 (dd, 1H,
J2,3¼3.5 Hz, J3,4¼9.5 Hz, H-3C), 5.43 (dd, 1H, J1,2¼1.6 Hz,
8.88 (brs, 1H, NHD), 5.28 (brs, 1H, H-1A), 4.93–4.61 (m,
8H, OCH2), 4.59 (s, 1H, J1,2¼1.3 Hz, H-1B), 4.41 (d, 1H,
J1,2¼8.3 Hz, H-1D), 4.06 (m, 2H, H-2A, 2B), 4.00 (dd, 1H,
J2,3¼3.3 Hz, J3,4¼9.4 Hz, H-3A), 3.86 (dd, 1H, J2,3¼2.9 Hz,
J3,4¼9.4 Hz, H-3B), 3.79 (dq, 1H, J4,5¼9.4 Hz, J5,6¼6.2 Hz,
H-5pA), 3.67 (m, 2H, H-5Bp , 6aD), 3.51 (m, 1H, H-2D), 3.49–
3.38 (m, 6H, H-6bD, 4D, 3D, 4B, 4A), 3.31 (s, 3H, OCH3),
3.29 (m, 1H, H-5D), 1.55 (s, 3H, C(O)CH3), 1.35 (d, 6H,
H-6A, 6B); 13C NMR d 173.6 (CvO), 138.5–127.6 (Ph),
103.2 (C-1D), 100.2 (C-1A), 99.9 (C-1B), 81.3, 80.7 (2C,
C-4A, 4B), 79.9 (2C, C-3A, 3B), 79.0 (C-2A), 77.2 (C-3D),
75.8 (C-5D), 75.7, 75.2, 74.6 (3C, OCH2), 73.4 (C-2B), 72.3
(OCH2), 71.8 (C-4D), 68.2, 67.7 (2C, C-5A, 5B), 62.5

2482
L. A. Mulard, C. Guerreiro / Tetrahedron 60 (2004) 2475–2488
H-2C), 5.09 (brs, 1H, H-1A), 5.02 (d, 1H, J1,2¼3.4 Hz,
H-1E), 4.99 (d, 1H, J1,2¼8.3 Hz, H-1D), 4.95 (d, 1H,
J1,2¼1.1 Hz, H-1C), 4.94–4.63 (m, 13H, OCH2), 4.63 (s,
1H, H-1B), 4.37 (d, 1H, J¼11.0 Hz, OCH2), 4.29 (dq, 1H,
J4,5¼9.5 Hz, J5,6¼6.2 Hz, H-5C), 4.25 (d, 1H, J¼9.5 Hz,
OCH2), 4.23 (pt, 1H, J3,4¼J4,5¼9.5 Hz, H-3D), 4.01 (m, 1H,
H-2A), 3.97–3.86 (m, 5H, H-3A, 2B, 3E, 4C, OCH2), 3.82 (m,
1H, H-3B, 5A), 3.71–3.57 (m, 6H, H-5D, 4E, 5B, 4D, 6aD,
6bD), 3.54–3.41 (m, 3H, H-2E, 4A, 2D) 3.38–3.31 (m, 2H,
H-4B, 6aE), 3.31 (s, 3H, OCH3), 3.17 (m, 1H, H-5D), 3.08 (d,
1H, J6a,6b¼10.1 Hz, H-6bE), 1.84 (s, 3H, C(O)CH3), 1.46 (s,
3H, C(CH3)2), 1.45 (d, 3H, J5,6¼5.9 Hz, H-6C), 1.35 (m, 6H,
J5,6¼5.9 Hz, H-6A, C(CH3)2), 1.31 (d, 3H, J5,6¼6.2 Hz,
H-6B); 13C NMR d 171.7, 165.9, 165.8 (3C, CvO), 138.9–
127.9 (Ph), 102.3 (C-1D, J¼167 Hz), 101.5 (C-1A,
J¼170 Hz), 100.3 (C-1B, J¼170 Hz), 99.8 (C(CH3)2),
99.6 (C-1E, J¼172 Hz), 98.2 (C-1C, J¼172 Hz), 82.0
(C-3E), 81.2, 80.9, 80.7 (3C, C-4A, 4B, 2E), 80.0, 79.7,
79.3 (3C, C-3B, 3A, 4C), 78.1, 77.8, 77.4 (3C, C-2A, 4E, 3D),
75.9, 75.8, 75.6 (3C, OCH2), 75.5 (C-2B), 75.0, 74.4, 73.7
(3C, OCH2), 73.2 (2C, C-4D, OCH2), 72.2 (OCH2), 71.7,
71.6 (3C, C-2C, 3C, 5E), 68.8 (C-5B), 68.0 (C-6E), 68.0 (2C,
C-5A, 5B), 67.6 (C-5D), 62.5 (C-6D), 58.9 (C-2D), 55.0
(OCH3), 29.5 (C(CH3)2), 23.8 (C(O)CH3), 19.8 (C(CH3)2),
18.6 (C-6C), 18.5 (C-6A), 18.3 (C-6B). FAB-MS for
C106H117NO25 (M, 1803.79) m/z 1826.4 [MþH]þ. Anal.
calcd for C106H117NO25·H2O: C, 69.83; H, 6.58; N, 0.77%.
Found: C, 69.86; H, 6.33; N, 0.71%.
71.3 (C-3C), 71.1 (C-2C), 69.4 (C-5C), 68.8 (C-5A), 68.3
(C-5B), 68.1 (C-6E), 63.0 (C-6D), 57.6 (C-2D), 55.0 (OCH3),
23.8 (C(O)CH3), 18.8 (C-6C), 18.6, 18.5 (2C, C-6A, 6B).
FAB-MS for C103H113NO25 (M, 1763.76) m/z 1786.2
[MþH]þ. Anal. calcd for C103H113NO25·2H2O: C, 68.69;
H, 6.55; N, 0.78%. Found: C, 68.74; H, 6.45; N, 0.65%.
4.1.8. Methyl (2,3,4,6-tetra-O-benzyl-a-D-glucopyrano-
syl)-(1!4)-a-L-rhamnopyranosyl-(1!3)-(2-acetamido-
2-deoxy-b-D-glucopyranosyl)-(1!2)-(3,4-di-O-benzyl-a-
L-rhamnopyranosyl)-(1!2)-3,4-di-O-benzyl-a-L-rham-
nopyranoside (17). 1 M Methanolic sodium methoxide was
added to a solution of 16 (277 mg, 157 mmol) in a 1:1
mixture of CH2Cl2 and MeOH (6 mL) until the pH was 10.
The mixture was stirred overnight at rt and neutralized with
Amberlite IR-120 (Hþ). The crude material was chromato-
graphed (solvent A, 49:1) to give 17 (211 mg, 86%) as a
white foam; [a]D þ23.8 (c 1.0); 1H NMR d 7.33–7.16 (m,
40H, Ph), 5.34 (d, 1H, JNH,2¼7.6 Hz, NH), 5.18 (brs, 1H,
H-1A), 4.79 (d, partially overlapped, 1H, H-1E), 4.67 (brs,
1H, H-1C), 4.50 (d, partially overlapped, 1H, H-1D), 4.49
(brs, 1H, H-1B), 4.88–4.33 (m, 16H, OCH2), 3.98–3.81 (m,
6H, H-2A, 2B, 5E, 3A, 3E, 5pB), 3.77–3.70 (m, 3H, H-3B, 2C,
5pC), 3.65 (dq, 1H, J4,5¼9.4 Hz, J5,6¼6.2 Hz, H-5pA), 3.62–
3.51 (m, 4H, H-2D, 6aD, 6aE, 6bE), 3.48–3.27 (m, 7H, H-2E,
4E, 3D, 4A, 4B, 3C, 4C), 3.23–3.12 (m, 6H, H-4D, 6bD, 5D,
OCH3), 2.76 (brs, 1H, OH), 1.72 (brs, 3H, OH), 1.65 (s, 3H,
NHAc), 1.32, 1.25 (2d, 9H, H-6C, 6B, 6A); 13C NMR d 170.6
(CvO), 138.5–128.0 (Ph), 103.0 (C-1D), 101.8 (C-1C),
100.7 (C-1A), 100.4 (C-1B), 99.6 (C-1E), 87.3 (C-3D), 85.0
(C-4pC), 82.0 (C-3E), 81.2, 80.7, 80.5, 80.2, 79.7, 78.1, 77.9
(7C, C-2B, 3A, 3B, 4A, 4B, 2E, 4E), 76.2 (C-5D), 76.1, 75.9,
75.6, 75.4, 74.0, 73.9, 73.6 (7C, OCH2), 73.0 (C-2A), 72.8
(OCH2), 71.7, 71.2, 71.1, 69.8 (4C, C-4D, 5E, 2C, 3C), 68.8,
68.2 (3C, C-5A, 5B, 5C), 63.1 (C-6D), 55.6 (C-2D), 55.0
(OCH3), 23.7 (C(O)CH3), 18.6, 18.3, 18.1 (3C, C-6A, 6B,
6C). FAB-MS for C89H105NO23 (M, 1555.71) m/z 1578.2
[MþH]þ. Anal. calcd for C89H105NO23: C, 68.66; H, 6.80;
N, 0.90%. Found: C, 68.41; H, 6.78; N, 0.61%.
4.1.7. Methyl (2,3,4,6-tetra-O-benzyl-a-D-glucopyrano-
syl)-(1!4)-(2,3-di-O-benzoyl-a-L-rhamnopyranosyl)-
(1!3)-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-
(1!2)-(3,4-di-O-benzyl-a-L-rhamnopyranosyl)-(1!2)-
3,4-di-O-benzyl-a-L-rhamnopyranoside (16). Aq. TFA
(750 mL) was added at 0 8C to a solution of the fully
protected 15 (588 mg, 326 mmol) in CH2Cl2 (6.7 mL) and
the mixture was stirred at this temperature for 1 h TLC
(solvent B, 1.5:1) showed that no 15 remained. Volatiles
were evaporated by repeated addition of toluene. Chroma-
tography of the residue (solvent B, 4:1!1:1) gave 16
1
(544 mg, 95%) as a white foam; [a]D þ58.8 (c 1.0); H
4.1.9. Methyl a-D-glucopyranosyl-(1!4)-a-L-rhamno-
pyranosyl-(1!3)-2-acetamido-2-deoxy-b-D-glucopyra-
nosyl-(1!2)-a-L-rhamnopyranosyl-(1!2)-a-L-rhamno-
pyranoside (1). The benzylated tetrasaccharide 17 (352 mg,
226 mmol) was dissolved in a mixture of ethanol (14 mL)
and AcOH (1 mL), treated with 10% Pd–C catalyst
(200 mg), and the suspension was stirred for 5 days at rt.
TLC (solvent A, 1:1) showed that the starting material had
been transformed into a more polar product. The suspension
was filtered on a pad of Celite. The filtrate was concentrated
and co-evaporated repeatedly with cyclohexane. Reverse
phase chromatography of the residue (solvent D,
100:0!49:1), followed by freeze-drying, gave the target
tetrasaccharide 1 as an amorphous powder (153 mg, 81%).
RP-HPLC gave a single product eluting at rt: 15.21 min
(solvent F, 1:0!80:20 over 20 min); [a]D 23.2 (c 1.0,
NMR d 8.06–7.06 (m, 50H, Ph), 5.82 (d, 1H, JNH,2¼7.1 Hz,
NH), 5.65 (dd, 1H, J2,3¼3.8 Hz, J3,4¼9.0 Hz, H-3C), 5.53
(m, 1H, H-2C), 5.34 (brs, 1H, H-1A), 5.04 (d, 1H,
J1,2¼8.3 Hz, H-1D), 5.00 (m, 2H, H-1C, 1E), 4.97–4.63
(m, 13H, OCH2), 4.48 (brs, 1H, H-1B), 4.40 (d, 1H,
J¼8.4 Hz, OCH2), 4.29 (d, 1H, J¼8.0 Hz, OCH2), 4.28–
4.21 (m, 2H, H-3D, 5C), 4.10 (m, 1H, H-2B), 4.04 (m, 1H,
H-2A), 3.99 (d, 1H, OCH2), 3.95–3.89 (m, 3H, H-3A, 3E,
4C), 3.87 (dd, 1H, J2,3¼2.7 Hz, J3,4¼9.7 Hz, H-3B), 3.81–
3.64 (m, 5H, H-5E, 5A, 6aD, 4E, 5B), 3.54 (dd, 1H,
J1,2¼3.2 Hz, J2,3¼9.7 Hz, H-2E), 3.51 (pt, 1H, J3,4
J4,5¼9.5 Hz, H-4A), 3.45–3.37 (m, 4H, H-4B, 4D, 6aE,
2D), 3.33 (m, 5H, H-5D, 6bD, OCH3), 3.12 (d, 1H, J6a,6b
¼
¼
10.6 Hz, H-6bE), 2.28 (brs, 1H, OH),1.97 (brs, 1H, OH),
1.84 (s, 3H, C(O)CH3), 1.54 (d, 3H, J5,6¼6.1 Hz, H-6C),
1.37 (m, 6H, H-6B, 6A); 13C NMR d 171.5, 165.8, 165.6 (3C,
CvO), 138.8–127.9 (Ph), 101.6 (C-1D), 100.8 (C-1A),
100.5 (C-1B), 100.1 (C-1pE), 99.9 (C-1Cp ), 84.9 (C-3D), 82.1
(C-3E), 80.9, 80.7, 80.6, 80.5 (4C, C-4B, 3B, 4A, 2E), 79.7
(C-4C), 79.3 (C-3A), 77.8 (2C, C-2A, 4E), 76.0, 75.9 (2C,
OCH2), 75.8 (C-5D), 75.6, 75.1, 74.6, 73.7, 73.1 (5C,
OCH2), 72.8 (C-2B), 72.6 (OCH2), 71.8 (C-5E), 71.6 (C-4D),
1
methanol); H NMR (D2O) d 5.08 (d, 1H, J1,2¼1.2 Hz,
H-1A), 4.97 (d, 1H, J1,2¼3.9 Hz, H-1E), 4.79 (d, 1H,
J1,2¼1.3 Hz, H-1C), 4.69 (m, 2H, H-1B, 1D), 4.07 (dd, 1H,
J2,3¼3.3 Hz, H-2A), 4.02 (dq, 1H, J4,5¼9.3 Hz, J5,6¼6.2 Hz,
H-5C), 3.93 (m, 1H, H-5E), 3.86 (m, 2H, H-2B, 3A), 3.82–
3.73 (m, 7H, H-3C, 2D, 6aE, 6bE, 3B, 2C, 6aD), 3.70–3.59 (m,
4H, H-5A, 3E, 6bD, 5B), 3.56 (pt, 1H, J3,4¼J4,5¼9.4 Hz,

L. A. Mulard, C. Guerreiro / Tetrahedron 60 (2004) 2475–2488
2483
H-3D), 3.49 (dd, 1H, J2,3¼9.6 Hz, H-2E), 3.46–3.38 (m, 5H,
H-4C, 4B, 4D, 5D, 4E), 3.32 (s, 3H, OCH3), 3.24 (pt, 1H,
J3,4¼J4,5¼9.6 Hz, H-4A), 2.00 (s, 3H, C(O)CH3), 1.25 (d,
3H, partially overlapped, H-6C), 1.23 (d, 3H, partially
overlapped, H-6B), 1.18 (d, 3H, J5,6¼6.2 Hz, H-6A); 13C
NMR (D2O) d 175.0 (CvO), 102.3 (C-1D, J¼162 Hz),
101.5 (C-1C, J¼170 Hz), 101.3 (C-1A, J¼173 Hz), 100.0
(C-1E, J¼170 Hz), 99.9 (C-1B, J¼172 Hz), 81.9 (C-3D),
81.4 (C-4C), 79.2 (C-2A), 79.0 (C-2B), 76.2, 73.1, 72.6, 72.2,
72.0, 71.4, 70.4, 70.0, 69.8, 69.7, 69.6, 69.3, 68.9, 68.7
(14C, 3A, 4A, 5A, 3B, 4B, 5B, 2C, 3C, 4D, 5D, 2E, 3E, 4E, 5E),
68.4 (C-5C), 60.5 (2C, C-6D, 6E), 56.0 (C-2D), 55.3 (OCH3),
22.6 (C(O)CH3), 17.0 (3C, C-6A, 6B, 6C). HRMS (MALDI)
calcd for C27H47NO19 þNa: 858.3214. Found: 858.3206.
in CH2Cl2 (11 mL) and freshly activated K2CO3 (1.1 g,
8.0 mmol) was added. The suspension was cooled to 0 8C,
and trichloroacetonitrile (1.0 mL) was added. The mixture
was stirred vigorously at rt for 5 h. The suspension was
filtered on a pad of Celite, and concentrated. The crude
material was purified by flash chromatography (solvent B,
9:1þ0.1% Et3N) to give 20 as a white foam (840 mg, 72%,
a/b: 9:1 from the 1H NMR spectrum). 1H NMR (a-anomer)
d 8.71 (s, 1H, NH), 7.40–7.30 (m, 10H, Ph), 6.24 (d, 1H,
J1,2¼1.8 Hz, H-1), 5.57 (dd, 1H, H-2), 4.94 (d, 1H,
J¼10.8 Hz, OCH2), 4.76 (d, 1H, J¼11.2 Hz, OCH2), 4.67
(d, 1H, OCH2), 4.62 (d, 1H, OCH2), 4.22 (s, 2H, CH2Cl),
4.04 (dd, 1H, J2,3¼3.2 Hz, H-3), 3.99 (dq, 1H, J4,5¼9.6 Hz,
H-5), 3.53 (pt, 1H, H-4), 1.37 (d, 3H, J5,6¼6.2 Hz, H-6); 13
C
NMR (a-anomer) d 166.9 (CvO), 160.4 (CvNH), 138.4–
128.3 (Ph), 95.2 (C-1), 91.1 (CCl3), 79.5 (C-4), 77.6 (C-3),
76.1, 72.9 (2C, OCH2), 71.2 (C-5), 69.8 (C-2), 41.1
(CH2Cl), 18.3 (C-6).
4.1.10. 3,4-Di-O-benzyl-2-O-chloroacetyl-a/b-L-rham-
nopyranose (28). 1,5-Cyclooctadiene-bis(methyldiphenyl-
phosphine)iridium hexafluorophosphate (Ir(I), 25 mg) was
dissolved in dry THF (5 mL) and the resulting red solution
was degassed in an argon stream. Hydrogen was then
bubbled through the solution, causing the colour to change
to yellow. The solution was then degassed again in an argon
stream. A solution of rhamnopyranoside33 27 (3.28 g,
7.12 mmol) in THF (30 mL) was degassed and added. The
mixture was stirred overnight at rt, and a solution of iodine
(3.6 g, 14.2 mmol) in a mixture of THF (70 mL) and water
(20 mL) was added. The mixture was stirred at rt for 1 h,
then concentrated. The residue was taken up in CH2Cl2 and
washed twice with 5% aq. NaHSO4. The organic phase was
dried and concentrated. The residue was purified by column
chromatography (solvent B, 9:1) to give 28 (2.53 g, 85%).
1H NMR d 7.40–7.28 (m, 10H, Ph), 5.57 (brd, 0.2H, H-2b),
5.45 (dd, 0.8H, J1,2¼2.0 Hz, H-2a), 5.13 (brd, 0.8H, H-1a),
4.92 (d, 1H, J¼10.9 Hz, OCH2a, OCH2b), 4.79 (d, 0.2H,
J¼11.2 Hz, OCH2b), 4.74 (d, 1H, J¼11.2 Hz, OCH2a,
H-1b), 4.65 (d, 0.8H, OCH2a), 4.64 (d, 0.2H, OCH2b), 4.58
(d, 0.8H, OCH2a), 4.54 (d, 0.2H, OCH2b), 4.30 (d, 0.2H,
J¼15.1 Hz, CH2Clb), 4.26 (d, 0.2H, CH2Clb), 4.20 (s,
1.6H, CH2Cla), 4.08 (dd, 0.8H, J2,3¼3.3 Hz, J3,4¼9.6 Hz,
H-3a), 4.04 (dq, 0.8H, J4,5¼9.5 Hz, H-5a), 3.66 (dd, 0.2H,
J2,3¼3.2 Hz, J3,4¼8.7 Hz, H-3b), 3.44 (pt, 2H, H-4a, 5b,
OH-1a, 1b), 3.38 (pt, 0.2H, J4,5¼9.5 Hz, H-4b), 1.37 (d,
0.6H, J5,6¼5.7 Hz, H-6b), 1.34 (d, 2.4H, J5,6¼6.2 Hz,
H-6a); 13C NMR d 167.8 (CvOb), 167.4 (CvOa), 138.6–
128.2 (Ph), 93.0 (C-1b), 92.4 (C-1a), 80.3 (C-4a), 80.2
(C-3b), 79.6 (C-4b), 77.8(C-3a), 75.9 (OCH2b), 75.8
(OCH2a), 72.5 (OCH2a), 72.3 (0.4C, C-5b, OCH2b), 71.9
(C-2-b), 71.7 (C-2a), 68.2(C-5a), 41.3 (CH2Cla, CH2Clb),
18.3 (C-6a, 6b); FAB-MS for C22H25ClO6 (M, 420.5) m/z
443.1 [MþNa]þ. Anal. calcd for C22H25ClO6: C, 62.78; H,
5.94%. Found: C, 62.92; H, 6.11%.
4.1.12. Allyl (2,3,4,6-tetra-O-benzyl-a-D-glucopyrano-
syl)-(1!4)-2-O-benzoyl-3-O-chloroacetyl-a-L-rhamno-
pyranoside (22). To a solution of the known 2128 (7.10 g,
8.55 mmol) in a mixture of CH2Cl2 (40 mL) and pyridine
(5 mL) at 0 8C was added chloroacetic anhydride (3.65 g,
21.3 mmol), and the mixture was stirred at this temperature
for 2 h. TLC (solvent C, 9:1) showed the complete
disappearance of the starting material. MeOH (10 mL)
was added, and after 30 min, volatiles were evaporated.
Column chromatography (solvent B, 1:0!4:1) of the crude
yellow oil afforded 22 as a colourless foam (7.34 g, 95%).
1
[a]D þ47.5 (c 1.0); H NMR d 8.12–7.13 (m, 25H, Ph),
5.95 (m, 1H, CHv), 5.50–5.42 (m, 2H, J2,3¼3.6 Hz, H-2C,
3C), 5.37 (m, 1H, vCH2), 5.28 (m, 1H, vCH2), 4.96 (d, 1H,
J¼11.0 Hz, OCH2), 4.93 (d, 1H, J1,2¼1.5 Hz, H-1C), 4.90
(d, 1H, J1,2¼3.3 Hz, H-1E), 4.87–4.81 (m, 3H, OCH2), 4.67
(d, 1H, J¼12.1 Hz, OCH2), 4.64 (d, 1H, J¼12.8 Hz, OCH2),
4.47 (d, 1H, J¼10.8 Hz, OCH2), 4.43 (d, 1H, J¼12.0 Hz,
OCH2), 4.25 (m, 2H, OCH2), 4.09 (d, 1H, J¼15.5 Hz,
CH2Cl), 3.99–3.93 (m, 3H, CH2Cl, H-5C, 3C), 3.84 (m, 1H,
H-5E), 3.78–3.74 (m, 2H, H-6aE, 4E), 3.70 (pt, 1H,
J4,5¼J3,4¼9.3 Hz, H-4C), 3.58–3.54 (m, 2H, H-6bE, 2E),
1.50 (d, 3H, J5,6¼6.2 Hz, H-6C); 13C NMR d 167.0, 166.0
(2C, CvO), 139.1–128.0 (Ph, All), 118.5 (All), 99.5
(C-1E), 96.8 (C-1C), 81.9 (C-3E), 81.0 (C-2E), 79.7 (C-4C),
77.7 (C-4E), 76.0, 75.4, 74.1, 73.8 (4C, OCH2), 73.5 (C-3C),
71.8 (C-5E), 70.9 (C-2C), 68.8 (OCH2), 68.1 (C-6E), 67.7
(C-5C), 41.5 (CH2Cl), 18.6 (C-6C); FAB-MS for C52H55O12
(M, 906.5) m/z 929.3 [MþNa]þ. Anal. calcd for
C52H55ClO12: C, 68.83; H, 6.11%. Found: C, 68.74; H,
6.19%.
4.1.13. (2,3,4,6-Tetra-O-benzyl-a-D-glucopyranosyl)-
(1!4)-2-O-benzoyl-3-O-chloroacetyl-a/b-L-rhamno-
pyranose (23). A solution of 22 (7.21 g, 7.95 mmol) in THF
(80 mL) containing activated iridium complex (60 mg) was
treated as described for the preparation of 28. The mixture
was stirred at rt for 3 h, at which point a solution of iodine
(4.0 g, 15.7 mmol) in a mixture of THF (90 mL) and water
(24 mL) was added. The mixture was stirred at rt for 30 min,
then concentrated. The residue was taken up in CH2Cl2 and
washed twice with 5% aq. NaHSO4, then with brine. The
organic phase was dried and concentrated. The residue was
purified by column chromatography (solvent B, 4:1) to give
4.1.11. 3,4-Di-O-benzyl-2-O-chloroacetyl-a/b-L-rham-
nopyranosyl trichloroacetimidate (20). (a) The hemi-
acetal 28 (700 mg, 1.66 mmol) was dissolved in CH2Cl2
(6 mL) and the solution was cooled to 0 8C. Trichloroace-
tonitrile (1.7 mL) and DBU (26 mL) were added. The
mixture was stirred at rt for 2 h. Toluene was added, and
co-evaporated twice from the residue. The crude material
was purified by flash chromatography (solvent B 4:1þ0.1%
Et3N) to give 20 as a white foam (687 mg, 73%, a/b: 4:1).
(b) The hemiacetal 28 (858 mg, 2.04 mmol) was dissolved

2484
L. A. Mulard, C. Guerreiro / Tetrahedron 60 (2004) 2475–2488
23 (6.7 g, 97%) as a slightly yellow foam. 1H NMR d 8.10–
7.09 (m, 25H, Ph), 5.47 (dd, 1H, J2,3¼3.5 Hz, J3,4¼9.3 Hz,
H-3C), 5.41 (brs, 1H, H-2C), 5.03 (brs, 1H, H-1C), 4.94 (d,
1H, J¼10.9 Hz, OCH2), 4.87 (d, 1H, J1,2¼3.4 Hz, H-1E),
4.85 (d, 1H, OCH2), 4.80 (m, 2H, OCH2), 4.64 (m, 2H,
OCH2), 4.45 (d, 1H, J¼10.7 Hz, OCH2), 4.41 (d, 1H,
J¼12.1 Hz, OCH2), 4.16 (dq, 1H, J4,5¼9.3 Hz, H-5C), 4.09
(d, 1H, J¼15.6 Hz, CH2Cl), 3.96 (d, 1H, CH2Cl), 3.93 (pt,
1H, H-3E), 3.83 (m, 1H, H-5E), 3.77–3.68 (m, 2H, H-4E,
6aE), 3.65 (pt, 1H, H-4C), 3.54 (m, 2H, H-6bE, 2E), 1.48 (d,
3H, J5,6¼6.2 Hz, H-6C); 13C NMR d 167.0, 166.0 (2C,
CvO), 139.1–127.9 (Ph), 99.5 (C-1E), 92.3 (C-1C), 81.9
(C-3E), 81.0 (C-2E), 79.9 (C-4C), 77.6 (C-4E), 76.0, 75.6,
74.2, 74.1 (4C, OCH2), 72.1 (C-3C), 71.7 (C-4E), 71.1
(C-2C), 68.0 (C-6E), 67.5 (C-5C), 41.5 (CH2Cl), 18.9 (C-6C);
FAB-MS for C49H51ClO12 (M, 866.3) m/z 889.3 [MþNa]þ.
Anal. calcd for C49H51ClO12: C, 67.85; H, 5.93%. Found: C,
67.72; H, 6.00%.
(solvent B, 85:15!1:1) to give 24 (1.64 g, 80%) as a
white powder. [a]D þ55.1 (c 1.0); 1H NMR d 8.06–6.93 (m,
25H, Ph), 6.18 (d, 1H, JNH,2¼7.3 Hz, NHD), 5.40 (dd, 1H,
J2,3¼3.5 Hz, H-3C), 5.38 (brs, 1H, H-2C), 4.98 (d, 1H,
J1,2¼8.3 Hz, H-1D), 4.94 (brs, 1H, H-1C), 4.94 (d, 1H,
OCH2), 4.93 (d, 1H, J1,2¼3.4 Hz, H-1E), 4.83 (d, 2H,
J¼10.7 Hz, OCH2), 4.81 (d, 1H, J¼10.6 Hz, OCH2), 4.67
(d, 1H, J¼11.7 Hz, OCH2), 4.62 (d, 1H, J¼11.4 Hz, OCH2),
4.47 (m, 3H, H-3D, OCH2), 4.22 (dq, 1H, J4,5¼9.4 Hz,
J5,6¼6.2 Hz, H-5C), 4.10 (d, 1H, J¼15.5 Hz, CH2Cl), 3.96
(m, 2H, H-6aD, CH2Cl), 3.91 (pt, 1H, H-3E), 3.82 (m, 2H,
H-5E, 6bD), 3.72 (m, 3H, H-6aE, 4E, 4C), 3.62 (pt, 1H,
J3,4¼J4,5¼9.4 Hz, H-4D), 3.55 (m, 2H, H-6bE, 2E), 3.51 (s,
3H, OCH3), 3.41 (m, 1H, H-5D), 3.15 (m, 1H, H-2D), 2.04
(s, 3H, C(O)CH3), 1.51 (s, 3H, C(CH3)2), 1.42 (m, 6H, H-6C,
C(CH3)2), 1.51 (d, 3H, J5,6¼6.2 Hz, H-6C); 13C NMR d
171.8, 167.3, 166.1 (3C, CvO), 139.0–128.0 (Ph), 101.1
(C-1D, JCH,164 Hz), 99.9 (C(CH3)2), 99.4 (C-1E,
JCH.165 Hz), 98.2 (C-1C, JCH¼172 Hz), 81.8 (C-3E),
80.9 (C-2E), 79.0 (C-4Cp ), 77.7 (C-4Ep), 76.7 (C-3D), 75.9,
75.3, 74.2, 73.9 (4C, OCH2), 73.7 (C-4D), 73.4 (C-3C), 71.9
(C-5E), 71.2 (C-2C), 68.2 (C-6E), 67.8 (C-5C), 67.4 (C-5D),
62.7 (C-6D), 59.6 (C-2D), 57.6 (OCH3), 41.5 (CH2Cl), 29.5
(C(CH3)2), 27.3 (C(O)CH3), 19.7 (C(CH3)2), 18.6 (C-6C);
FAB-MS for C61H70ClNO17 (M, 1123.4) m/z 1146.5
[MþNa]þ. Anal. calcd for C61H70ClNO17: C, 65.15; H,
6.27; N, 1.25%. Found: C, 65.13; H, 6.23; N, 1.22%.
4.1.14. (2,3,4,6-Tetra-O-benzyl-a-D-glucopyranosyl)-
(1!4)-2-O-benzoyl-3-O-chloroacetyl-a-L-rhamno-
pyranosyl trichloroacetimidate (19). Trichloroacetonitrile
(1.1 mL, 10.9 mmol) and DBU (17 mL) were added to a
solution of the hemiacetal 23 (950 mg, 1.09 mmol) in dry
CH2Cl2 (8 mL), and the mixture was stirred at 0 8C for 1.5 h.
Toluene was added, and volatiles were evaporated. The
residue was purified by flash chromatography (solvent B,
3:2 containing 0.1% Et3N) to give 19 (930 mg, 84%) as a
colourless foam. Further elution gave some remaining
starting material 23 (136 mg, 14%). [a]D þ39.3 (c 1.0); 1H
NMR d 8.76 (s, 1H, NH), 8.12–7.17 (m, 25H, Ph), 6.34 (d,
1H, J1,2¼1.5 Hz, H-1C), 5.67 (dd, 1H, H-2C), 5.54 (dd, 1H,
J2,3¼3.4 Hz, J3,4¼8.8 Hz, H-3C), 4.98 (d, 1H, OCH2), 4.88
(d, 1H, J1,2¼3.4 Hz, H-1E), 4.84 (d, 1H, J¼11.1 Hz, OCH2),
4.82 (d, 1H, J¼11.2 Hz, OCH2), 4.65 (d, 1H, OCH2), 4.62
(d, 1H, OCH2), 4.44 (d, 1H, J¼11.4 Hz, OCH2), 4.41 (d, 1H,
J¼11.8 Hz, OCH2), 4.14 (dq, 1H, J4,5¼9.5 Hz, H-5C), 4.11
(d, 1H, J¼15.5 Hz, CH2Cl), 3.98 (d, 1H, CH2Cl), 3.94 (pt,
1H, H-3E), 3.83–3.71 (m, 4H, H-5E, 6aE, 4E, 4C), 3.56–3.51
(m, 2H, H-6bE, 2E), 1.51 (d, 3H, J5,6¼6.2 Hz, H-6C); 13C
NMR d 167.1, 165.7, 160.6 (3C, Cv), 139.0–127.9 (Ph),
99.9 (C-1E), 95.2 (C-1C), 82.1 (C-3E), 80.9 (C-2E), 79.0
(C-4C), 77.6 (C-4E), 76.0, 75.6, 74.2, 73.8 (4C, OCH2), 73.0
(C-3C), 71.9 (C-5E), 70.7 (C-5C), 69.2 (C-2C), 68.0 (C-6E),
67.7 (C-5C), 41.4 (CH2Cl), 18.6 (C-6C). Anal. calcd for
C51H51Cl4NO12: C, 60.54; H, 5.08; N, 1.38%. Found: C,
60.49; H, 5.01; N, 1.34%.
4.1.16. Methyl (2,3,4,6-tetra-O-benzyl-a-D-glucopyrano-
syl)-(1!4)-(2-O-benzoyl-a-L-rhamnopyranosyl)-(1!3)-
2-acetamido-2-deoxy-4,6-O-isopropylidene-b-D-gluco-
pyranoside (25). To a solution of the fully protected 24
(1.40 g, 1.25 mmol) in a mixture of methanol (18 mL) and
pyridine (18 mL) was added thiourea (951 mg, 12.5 mmol).
The mixture was stirred at 65 8C for 5 h, at which time TLC
(solvent C, 4:1) showed that no starting material remained.
Evaporation of the volatiles and co-evaporation of
petroleum ether from the residue resulted in a crude solid,
which was taken up in a minimum of methanol. A large
excess of CH2Cl2 was added and the mixture was left to
stand at 0 8C for 1 h. The precipitate was filtrated on a pad of
Celite and the filtrate was concentrated. Column chroma-
tography of the residue (solvent C, 4:1) gave the
trisaccharide acceptor 25 (1.28 g, 97%) as a white powder.
1
[a]D þ33.5 (c 1.0); H NMR d 8.10–6.96 (m, 25H, Ph),
6.09 (d, 1H, JNH,2¼7.9 Hz, NHD), 5.26 (dd, 1H,
J1,2¼1.6 Hz, J2,3¼3.4 Hz, H-2C), 4.97 (m, 3H, H-1C, 1E,
OCH2), 4.86 (m, 3H, H-1D, OCH2), 4.81 (d, 1H, OCH2),
4.72 (d, 1H, OCH2), 4.58 (d, 1H, J¼12.2 Hz, OCH2), 4.51
(d, 1H, J¼10.9 Hz, OCH2), 4.48 (d, 1H, J¼12.2 Hz, OCH2),
4.23 (pt, 1H, J2,3¼J3,4¼9.4 Hz, H-3D), 4.18–4.10 (m, 2H,
H-5C, 5E), 4.06–3.95 (m, 3H, H-3C, 3E, 6aD), 3.80 (pt, 1H,
J5,6b¼J6a,6b¼10.4 Hz, H-6bD), 3.66 (m, 2H, H-6aE, 6bE),
3.62 (dd, 1H, J2,3¼9.8 Hz, J1,2¼4.1 Hz, H-2E), 3.59 (pt, 1H,
J3,4¼J4,5¼8.9 Hz, H-4E), 3.55 (pt, 1H, J3,4¼J4,5¼9.2 Hz,
H-4D), 3.51 (pt, 1H, J3,4¼J4,5¼9.3 Hz, H-4C), 3.49 (s, 3H,
OCH3), 2.22 (s, 3H, C(O)CH3), 1.90 (brs, 1H, OH), 1.49 (s,
3H, CMe2), 1.43 (s, 3H, CMe2), 1.40 (s, 3H, J5,6¼6.2 Hz,
H-6C); 13C NMR d 171.8, 166.6 (2C, CvO), 138.9–128.1
(Ph), 101.6 (C-1D), 99.8 (C(CH3)2), 98.6 (C-1pE), 98.3
(C-1pC), 85.4 (C-4C), 82.0 (C-3E), 80.4 (C-2E), 78.2 (C-4E),
77.1 (C-3D), 75.9, 75.5, 74.2, 73.9 (4C, OCH2), 73.6 (C-4pD),
73.5 (C-2pC), 71.7 (C-5E), 69.0 (C-6E), 68.3 (C-3C), 67.5
4.1.15. Methyl (2,3,4,6-tetra-O-benzyl-a-D-glucopyrano-
syl)-(1!4)-(2-O-benzoyl-3-O-chloroacetyl-a-L-rhamno-
pyranosyl)-(1!3)-2-acetamido-2-deoxy-4,6-O-iso-
propylidene-b-D-glucopyranoside (24). The acceptor19 18
(500 mg, 1.82 mmol) was dissolved in CH2Cl2 (5.5 mL) and
˚
4 A molecular sieves (300 mg) were added. The mixture
was cooled to 260 8C and stirred for 15 min. TMSOTf
(35 mL, mmol) and a solution of the disaccharide donor 19
(2.39 g, 2.36 mmol) in CH2Cl2 (7.5 mL) were added. The
mixture was stirred for 45 min while the cooling bath was
coming back to rt, and for more 3 h at rt. The mixture was
then heated at 65 8C for 1 h 30 min. Et3N was added and the
mixture was stirred at rt for 20 min, then diluted with
CH2Cl2 and filtered through a pad of Celite. The filtrate was
concentrated and purified by column chromatography

L. A. Mulard, C. Guerreiro / Tetrahedron 60 (2004) 2475–2488
2485
(C-5D), 66.9 (C-5C), 62.7 (C-6D), 58.9 (C-2D), 57.5 (OCH3),
29.5 (C(CH3)2), 24.0 (C(O)CH3), 19.7 (C(CH3)2), 18.2
(C-6C); FAB-MS for C59H69NO16 (M, 1047.5) m/z 1070.4
[MþNa]þ. Anal. calcd for C70H76O16: C, 67.61; H, 6.64; N,
1.34%. Found: C, 67.46; H, 6.78; N, 1.24%.
J2,3¼4.0 Hz, H-2B), 4.65–4.47 (m, 4H, OCH2), 4.44–4.32
(m, 4H, H-5E, 3D, 3C, OCH2), 4.15 (m, 1H, H-5C), 4.05
(pt, 1H, J2,3¼J3,4¼9.5 Hz, H-3E), 4.03 (pt, 1H,
J3,4¼J4,5¼9.4 Hz, H-4C), 3.94 (dd, 1H, J5,6a¼5.3 Hz,
J6a,6b¼10.7 Hz, H-6aD), 3.83–3.75 (m, 4H, H-6aE, 6bD,
CH2Cl), 3.74–3.70 (m, 3H, H-4E, 6E, 3B), 3.65 (dd, 1H,
J1,2¼3.4 Hz, J2,3¼9.4 Hz, H-2E), 3.48 (pt, 2H, H-4B, 4D),
3.46 (s, 3H, OCH3), 3.38 (m, 1H, H-5D), 3.22 (dq, 1H,
J4,5¼9.5 Hz, J5,6¼6.2 Hz, H-5B), 2.88 (m, 1H, H-2D), 1.90
(s, 3H, C(O)CH3), 1.42 (s, 3H, C(CH3)2), 1.36 (s, 6H,
C(CH3)2, H-6C), 1.30 (d, 3H, J5,6¼6.3 Hz, H-6B); 13C NMR
d 171.8, 166.4 (2C, CvO), 139.1–122.5 (Ph), 101.0 (C-1D,
JCH¼165 Hz), 99.7 (C(CH3)2), 98.3 (C-1C, JCH¼172 Hz),
97.8 (brs, C-1E, JCH¼170 Hz), 97.5 (C-1B, JCH¼176 Hz),
82.2 (C-3E), 80.7 (C-2E), 79.3 (brs, C-4B), 78.8 (C-3B), 78.1
(brs, C-4E), 77.3 (C-2B), 76.2 (brs, C-3C), 75.8, 75.6, 74.9,
74.6, 73.9 (6C, C-4C, OCH2), 73.5 (2C, C-4D, 2C), 71.4
(OCH2), 71.0 (C-3D), 70.7 (2C, C-5E, 5B), 69.0 (C-5C), 68.8
(C-6E), 67.2 (C-5D), 62.5 (C-6D), 60.0 (C-2D), 57.6 (OCH3),
46.9 (CH2Cl), 29.5 (C(CH3)2), 23.9 (C(O)CH3), 19.7
(C(CH3)2), 19.0 (C-6B), 18.4 (C-6C); FAB-MS for
C81H92NClO21 (M, 1449.5) m/z 1472.7 [MþNa]þ. Anal.
calcd for C81H92NClO21·H2O: C, 66.23; H, 6.34; N, 0.96%.
Found: C, 66.11; H, 6.62; N, 0.85%.
4.1.17. Methyl (3,4-di-O-benzyl-2-O-chloroacetyl-a-L-
rhamnopyranosyl)-(1!3)-[2,3,4,6-tetra-O-benzyl-a-D-
glucopyranosyl-(1!4)]-(2-O-benzoyl-a-L-rhamnopyra-
nosyl)-(1!3)-2-acetamido-2-deoxy-4,6-O-isopropyl-
idene-b-D-glucopyranoside (29). (a) The trisaccharide
acceptor 25 (615 mg, 0.58 mmol) was dissolved in Et2O
(10 mL) and the solution was cooled to 260 8C. TMSOTf
(32 mL) and donor 20 (497 mg, 0.88 mmol) in Et2O (12 mL)
were added, and the mixture was stirred for 1 h while the
bath was slowly coming back to 220 8C. The mixture was
stirred for 4 h at this temperature, then at 0 8C overnight.
More 20 (50 mg, 88 mmol) was added, and the mixture was
stirred at rt for 3 h more at 0 8C. Et3N was added, and the
mixture was concentrated. Column chromatography of the
residue (solvent B, 9:1!1:1) gave the orthoester 35 (44 mg,
5%) then the fully protected 29 (445 mg, 52%) contami-
nated with the trimethylsilyl side product 26 (29/26: 9:1)
together with a mixture of 29 and 35 (65 mg, 8%), and the
starting 25 (27 mg, 4%). An analytical sample of compound
29 had [a]D þ17.9 (c 1.0); 1H NMR d 8.07–7.12 (m, 35H,
Ph), 5.96 (d, 1H, JNH,2¼7.9 Hz, NH), 5.82 (m, 1H, H-2B),
5.33 (dd, 1H, J1,2¼1.8 Hz, J2,3¼3.2 Hz, H-2C), 5.07 (d, 1H,
J1,2¼3.2 Hz, H-1E), 5.05 (d, 1H, J1,2¼1.7 Hz, H-1B), 4.98
(d, 1H, OCH2), 4.97 (brs, 1H, H-1C), 4.91–4.78 (m, 5H,
H-1D, OCH2), 4.64 (d, 1H, J¼11.6 Hz, OCH2), 4.60–4.45
(m, 5H, OCH2), 4.36 (d, 1H, J¼11.9 Hz, OCH2), 4.26 (pt,
1H, J2,3¼J3,4¼9.5 Hz, H-3D), 4.17 (dd, 1H, J2,3¼3.4 Hz,
H-3C), 4.16 (d, 1H, J¼15.1 Hz, CH2Cl), 4.11 (d, 1H,
CH2Cl), 4.10 (dq, 1H, J4,5¼9.1 Hz, J5,6¼6.3 Hz, H-5C),
4.06 (m, 1H, H-5E), 4.00 (pt, 1H, J3,4¼J2,3¼9.4 Hz, H-3E),
3.97 (dd, 1H, J5,6a¼5.3 Hz, J6a,6b¼10.8 Hz, H-6aD), 3.89
(m, 1H, H-6aE), 3.88–3.68 (m, 4H, H-6bE, 6bD, 4C, 3B),
3.67 (m, 1H, H-5B), 3.58 (pt, 1H, J3,4¼J4,5¼9.4 Hz, H-4D),
3.52 (dd, 1H, J1,2¼3.3 Hz, J2,3¼9.8 Hz, H-2E), 3.49 (s, 3H,
OCH3), 3.39 (m, 1H, H-5D), 3.30 (m, 2H, H-2D, 4B), 2.12 (s,
3H, C(O)CH3), 1.52 (s, 3H, C(CH3)2), 1.42 (s, 3H,
C(CH3)2), 1.33, 0.96 (2d, 6H, J5,6¼6.2 Hz, H-6B, 6C); 13C
NMR d 171.9, 167.0, 166.3 (3C, CvO), 138.8–128.0 (Ph),
101.4 (C-1D, JCH¼164 Hz), 99.9 (C(CH3)2), 99.3 (C-1C,
JCH¼168 Hz), 98.3 (C-1E, JCH¼168 Hz), 97.9 (C-1B,
JCH¼171 Hz), 82.1 (C-3E), 81.8 (C-2E), 80.4 (brs, C-3B),
80.0 (C-4C), 78.8 (brs, C-4pE), 78.3 (C-4Bp ), 77.7 (C-3pC), 76.9
(C-3D), 75.9, 75.5, 75.3, 74.3 (4C, OCH2), 73.4 (C-4D), 73.2
(OCH2), 72.7 (C-2B), 72.1 (C-5E), 69.1 (C-5C), 67.7 (C-5pD),
67.6 (C-5pB), 62.7 (C-6D), 59.1 (C-2D), 57.5 (OCH3), 41.4
(CH2Cl), 29.5 (C(CH3)2), 24.0 (C(O)CH3), 19.7 (C(CH3)2),
18.8, 18.2 (2C, C-6B, 6C); FAB-MS for C81H92NClO21 (M,
1449.5) m/z 1472.7 [MþNa]þ. Anal. calcd for
C81H92NClO21: C, 67.05; H, 6.39; N, 0.97%. Found: C,
66.21; H, 6.46; N, 1.01%.
4.1.18. Methyl (2-O-acetyl-3,4-di-O-benzyl-a-L-rhamno-
pyranosyl)-(1!3)-[2,3,4,6-tetra-O-benzyl-a-D-gluco-
pyranosyl-(1!4)]-(2-O-benzoyl-a-L-rhamnopyranosyl)-
(1!3)-2-acetamido-2-deoxy-4,6-O-isopropylidene-b-D-
glucopyranoside (30). The trisaccharide acceptor 25
(500 mg, 0.47 mmol) was dissolved in CH2Cl2 (5 mL) and
the solution was cooled to 240 8C. TMSOTf (21 mL) and
donor 5 (328 mg, 0.62 mmol) were added and the mixture
was left under stirring while the bath was slowly coming
back to rt. After 5 h, more 5 (50 mg, 94 mmol) was added
and the mixture was stirred at rt for 1 h more. Et3N was
added and the mixture was concentrated. Column chroma-
tography of the residue (solvent B, 4:1!1:1) gave the fully
protected 30 (484 mg, 72%) slightly contaminated with the
corresponding trimethylsilyl side product 26. The 30/26
ratio was estimated to be 85:15 from the 1H NMR spectrum.
Eluting next was some residual starting 25 (45 mg, 9%).
Thus, based on the consumed acceptor, the estimated yield
of contaminated 30 was 79%. An analytical sample of 30
had [a]D þ15.9 (c 0.8); 1H NMR d 8.09–7.14 (m, 35H, Ph),
6.04 (brs, 1H, NHD), 5.76 (m, 1H, H-2B), 5.37 (dd, 1H,
J1,2¼1.9 Hz, J2,3¼2.8 Hz, H-2C), 5.11 (d, 1H, J1,2¼3.1 Hz,
H-1E), 5.06 (d, 1H, H-1B), 4.96 (brs, 1H, H-1C), 5.02–4.82
(m, 7H, H-1D, OCH2), 4.69–4.37 (m, 6H, OCH2), 4.28 (pt,
1H, J2,3¼J3,4¼9.5 Hz, H-3D), 4.15 (dd, 1H, J2,3¼3.3 Hz,
J3,4¼9.4 Hz, H-3C), 4.13–3.93 (m, 5H, H-5E, 6aE, 3E, 5C,
6aD), 3.87–3.76 (m, 5H, H-4E, 6bE, 3B, 4C, 6bD), 3.68 (dq,
1H, J4,5¼9.5 Hz, H-5B), 3.57 (pt, 1H, J3,4¼J4,5¼9.4 Hz,
H-4D), 3.54 (dd, 1H, J2,3¼3.2 Hz, H-2E), 3.48 (s, 3H,
OCH3), 3.40 (m, 1H, H-5D), 3.34 (pt, 1H, J3,4¼9.7 Hz,
H-4B), 3.27 (m, 1H, H-2D), 2.18, 2.13 (2s, 6H, C(O)CH3),
1.51, 1.42 (2s, 6H, C(CH3)2), 1.33 (d, 3H, J5,6¼6.2 Hz,
H-6C), 0.98 (d, 3H, J5,6¼6.2 Hz, H-6B); 13C NMR d 171.9,
170.5, 166.3 (3C, CvO), 139.3–127.7 (Ph), 101.3 (C-1D),
99.9 (C(CH3)2), 99.6 (C-1B), 98.4 (C-1E), 98.0 (C-1C), 82.1
(C-3E), 81.8 (C-2E), 80.3 (2C, C-3C, 4B), 78.7 (brs, C-4C),
78.2 (C-3pB), 77.7 (C-4Ep), 76.9 (brs, C-3D), 75.9, 75.4, 75.3,
74.3 (4C, OCH2), 73.4 (C-4D), 73.3 (OCH2), 72.7 (C-2C),
Compound 35 had [a]D þ26.7 (c 0.8); 1H NMR d 8.07–7.15
(m, 35H, Ph), 5.47 (d, 1H, JNH,2¼7.4 Hz, NHD), 5.45 (brs,
1H, H-2C), 5.42 (d, 1H, J1,2¼2.3 Hz, H-1B), 5.24 (d, 1H,
J1,2¼3.4 Hz, H-1E), 4.94 (d, 1H, J1,2¼8.2 Hz, H-1D), 4.91–
4.82 (m, 7H, H-1C, OCH2), 4.80 (d, 1H, J¼11.0 Hz, OCH2),
4.75 (d, 1H, J¼11.6 Hz, OCH2), 4.68 (dd, 1H, J1,2¼2.4 Hz,

2486
L. A. Mulard, C. Guerreiro / Tetrahedron 60 (2004) 2475–2488
72.1 (C-5E), 70.9 (OCH2), 69.0 (3C, C-2B, 5B, 6E), 67.8
(C-5C), 67.6 (C-5D), 62.7 (C-6D), 59.2 (C-2D), 57.5 (OCH3),
29.5 (C(CH3)2), 24.0, 21.6 (2C, C(O)CH3), 19.7 (C(CH3)2),
18.9 (C-6C), 18.2 (C-6B). FAB-MS for C81H93NO21 (M,
1415) m/z 1438.6 [MþNa]þ.
(10 mL), and the mixture was stirred at 260 8C for 30 min.
The donor 5 (234 mg, 0.44 mmol) in CH2Cl2 (7 mL) was
added, and the mixture was stirred for 1 h while the bath
temperature was reaching rt. After a further 1 h at this
temperature, more 5 (50 mg, 94 mmol) was added, and the
mixture was stirred for 1 h before Et3N was added. Filtration
through a pad of Celite and evaporation of the volatiles gave
a residue which was column chromatographed twice
(solvent B, 4:1; then solvent A, 17:3) to give 32 (262 mg,
4.1.19. Methyl (3,4-di-O-benzyl-a-L-rhamnopyranosyl)-
(1!3)-[2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-
(1!4)]-(2-O-benzoyl-a-L-rhamnopyranosyl)-(1!3)-2-
acetamido-2-deoxy-4,6-O-isopropylidene-b-D-gluco-
pyranoside (31). (a) Thiourea (22 mg, 0.29 mmol) was
added to the chloroacetylated 29 (83 mg, 57 mmol) in
MeOH/pyridine (1:1, 2.8 mL), and the mixture was heated
overnight at 65 8C. Volatiles were evaporated, and the solid
residue thus obtained was taken up in the minimum of
MeOH. CH2Cl2 was added, and the suspension was left
standing at 0 8C for 1 h. The precipitate was filtered on a pad
of Celite, and the filtrate was concentrated. Column
chromatography of the residue (solvent B, 9:1!1:1) gave
the tetrasaccharide acceptor 31 (74 mg, 94%).
1
52%) as a white powder. [a]D þ25.9 (c 1.0); H NMR d
8.07–7.13 (m, 45H, Ph), 6.03 (brs, 1H, NHD), 5.59 (brs, 1H,
H-2A), 5.35 (brs, 1H, H-2C), 5.16 (brs, 1H, H-1E), 5.13 (brs,
1H, H-1A), 5.06 (brs, 1H, H-1B), 5.02–4.97 (m, 4H, H-1D,
1C, OCH2), 4.91–4.50 (m, 12H, OCH2), 4.44–4.32 (m, 4H,
H-2B, 3D, OCH2), 4.20–3.96 (m, 7H, H-5E, 5A, 3C, 3E, 6aD,
5C, 3A), 3.87–3.68 (m, 6H, H-4E, 6aE, 6bE, 6bD, 4C, 3B),
3.64–3.47 (m, 7H, H-5B, 4D, 2E, 4A, OCH3), 3.42 (m, 1H,
H-5D), 3.34 (pt, 1H, J3,4¼J4,5¼9.3 Hz, H-4B), 3.17 (m, 1H,
H-2D), 2.13 (s, 3H, C(O)CH3), 1.49 (s, 3H, C(CH3)2), 1.43
(s, 6H, C(CH3)2, H-6C), 1.33 (d, 3H, J5,6¼6.1 Hz, H-6A),
1.01 (d, 3H, J5,6¼5.8 Hz, H-6B); 13C NMR d 171.9, 170.3,
166.3 (3C, CvO), 139.2–127.6 (Ph), 101.5 (brs, C-1B,
JCH¼171 Hz), 101.2 (C-1D, JCH¼163 Hz), 99.8 (C(CH3)2),
99.7 (C-1A, JCH¼171 Hz), 97.9 (2C, C-1E, 1C, JCH¼172,
169 Hz), 82.4 (C-3E), 82.1 (C-2E), 80.5 (C-4A), 80.2 (brs,
C-3C), 80.1 (C-4B), 79.4, 78.1, 78.0 (4C, C-3B, 4E, 3A, 4C),
76.6 (brs, C-3D), 75.9, 75.8, 75.4 (3C, OCH2), 74.8 (2C,
C-2B, OCH2), 73.5 (C-4D), 73.4 (OCH2), 73.2 (C-2C), 72.1
(OCH2), 71.8 (C-5A), 71.2 (OCH2), 69.4 (C-2A), 69.2
(C-5B), 68.9 (C-6E), 68.7 (C-5C), 67.8 (C-5E), 67.5 (C-5D),
62.7 (C-6D), 59.6 (brs, C-2D), 57.6 (OCH3), 29.5 (C(CH3)2),
24.0, 21.4 (2C, C(O)CH3), 19.7 (C(CH3)2), 19.1 (C-6A),
18.8 (C-6C), 18.2 (C-6B); FAB-MS for C101H115NO25 (M,
1741.7) m/z 1765.9 [MþNa]þ. Anal. calcd for
C101H115NO25: C, 69.60; H, 6.65; N, 0.80%. Found: C,
69.56; H, 6.75; N, 0.73%.
(b) The monoacetylated 30 (52 mg, 37 mmol) was dissolved
in a mixture of EtOH (10 mL) and CH2Cl2 (100 mL). A
freshly prepared 0.4 M ethanolic solution of guanidine
(92 mL, 37 mmol) was added and the mixture was stirred at
rt overnight. Volatiles were evaporated, and the residue
taken up in CH2Cl2 was washed with water. The organic
phase was dried and concentrated. Column chromatography
of the crude product gave 31 (42 mg, 83%) as a glassy solid.
Compound 31 had [a]D þ27.3 (c 1.0); 1H NMR d 8.24–6.88
(m, 35H, Ph), 5.90 (brs, 1H, NHD), 5.29 (brs, 1H, H-2C),
5.14 (d, 1H, J1,2¼3.0 Hz, H-1E), 5.06 (d, 1H, J1,2¼1.6 Hz,
H-1B), 5.00–4.95 (m, 3H, H-1D, 1C, OCH2), 4.88–4.46 (m,
9H, OCH2), 4.31 (pt, 1H, J2,3¼J3,4¼9.4 Hz, H-3D), 4.24
(brs, 1H, H-2B), 4.14–3.08 (m, 3H, H-3C, 5C, 5E), 4.02 (pt,
1H, J2,3¼J3,4¼9.3 Hz, H-3E), 3.97 (dd, 1H, J5,6a¼5.2 Hz,
J6a,6b¼10.7 Hz, H-6aD), 3.80 (m, 2H, H-4C, 6bD), 3.71 (m,
2H, H-6aE, 6bE), 3.66 (pt, 1H, J4,5¼9.5 Hz, H-4E), 3.61–
3.55 (m, 4H, H-3B, 2E, 5B, 4D), 3.50 (s, 3H, OCH3), 3.42–
3.36 (m, 2H, H-5D, 4B), 3.20 (m, 1H, H-2D), 2.85 (brs, 1H,
OH), 2.10 (s, 3H, C(O)CH3), 1.51, 1.41 (2s, 6H, C(CH3)2),
1.33 (d, 3H, J5,6¼6.2 Hz, H-6C), 1.15 (s, 3H, J5,6¼6.2 Hz,
H-6B); 13C NMR d 171.7, 166.3 (2C, CvO), 139.0–127.8
(Ph), 103.1 (C-1B), 101.2 (C-1D), 99.8 (C(CH3)2), 98.2, 98.1
(2C, C-1E, 1C), 82.0 (C-3E), 81.5 (C-3pB), 80.6 (C-4B), 79.4
(C-2pE), 79.1 (2C, C-4C, 3C), 78.2 (C-4B), 76.8 (C-3D), 76.0,
75.5, 74.5, 74.2 (4C, OCH2), 73.9 (C-2C), 73.7 (OCH2), 73.5
(C-4D), 72.1 (OCH2), 71.6 (C-5E), 69.0 (C-6E), 68.7 (2C,
C-2B, 5B), 67.9 (C-5C), 67.5 (C-5D), 62.7 (C-6D), 59.4
(C-2D), 57.5 (OCH3), 29.5 (C(CH3)2), 24.0 (C(O)CH3), 19.7
(C(CH3)2), 19.0 (C-6C), 18.3 (C-6B); FAB-MS for
C79H91NO20 (M, 1373) m/z 1396.5 [MþNa]þ. Anal. calcd
for C79H91NO200.5H2O: C, 68.56; H, 6.65; N, 1.01%.
Found: C, 68.53; H, 6.71; N, 1.01%.
4.1.21. Methyl a-L-rhamnopyranosyl-(1!2)-a-L-rham-
nopyranosyl-(1!3)-[a-D-glucopyranosyl-(1!4)]-a-L-
rhamnopyranosyl-(1!3)-2-acetamido-2-deoxy-b-D-
glucopyranoside (2). 50% aq. TFA (1 mL) was added at
0 8C to a solution of the fully protected pentasaccharide 32
(155 mg, 89 mmol) dissolved in CH2Cl2 (4 mL) After 1 h at
this temperature, volatiles were evaporated. The residue,
containing diol 33, was taken up in 0.5% methanolic sodium
methoxide (8 mL) and the mixture was heated overnight at
55 8C. Neutralisation with Dowex X8 (Hþ), evaporation of
the volatiles and column chromatography of the residue
gave 34 (121 mg, 98%). Compound 34 (111 mg, 81 mmol)
was dissolved in a mixture of ethanol (13 mL) and ethyl
acetate (2.6 mL) containing 1 N aq. HCl (130 mL).
Palladium on charcoal (130 mg) was added, and the
suspension was stirred under a hydrogen atmosphere for
2 h. Filtration of the catalyst and reverse phase chromato-
graphy gave the target pentasaccharide (60 mg, 88%) as a
slightly yellow foam. RP-HPLC purification followed by
freeze-drying gave pure 2 (36 mg). Compound 2 had rt:
9.63 min (solvent F, 100:0!80:20 over 20 min); [a]D
218.6 (c 1.0, methanol); 1H NMR d 5.13 (d, 1H,
J1,2¼3.7 Hz, H-1E), 4.98 (brs, 1H, H-1B), 4.90 (d, 1H,
J1,2¼1.4 Hz, H-1A), 4.72 (d, 1H, J1,2¼1.4 Hz, H-1C), 4.39
(d, 1H, J1,2¼8.6 Hz, H-1D), 4.09 (dq, 1H, J4,5¼9.2 Hz,
H-5C), 4.00 (m, 2H, H-2B, 2A), 3.94–3.79 (m, 7H, H-5E, 2C,
4.1.20. Methyl (2-O-acetyl-3,4-di-O-benzyl-a-L-rhamno-
pyranosyl)-(1!2)-(3,4-di-O-benzyl-a-L-rhamnopyrano-
syl)-(1!3)-[2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-
(1!4)]-(2-O-benzoyl-a-L-rhamnopyranosyl)-(1!3)-2-
acetamido-2-deoxy-4,6-O-isopropylidene-b-D-gluco-
˚
pyranoside (32). Activated 4 A molecular sieves and
TMSOTf (16 mL) were added to a solution of the
tetrasaccharide acceptor 31 (406 mg, 0.29 mmol) in Et2O

L. A. Mulard, C. Guerreiro / Tetrahedron 60 (2004) 2475–2488
2487
3C, 6aE, 6aD, 2D, 3A), 3.76–3.65 (m, 7H, H-4C, 3E, 6bE, 6bD,
5A, 5B, 3B), 3.52 (pt, 1H, J3,4¼8.8 Hz, H-3D), 3.49–3.33 (m,
9H, H-4D, 2E, 4A, 4B, 5D, 4E, OCH3), 1.98 (s, 3H, C(O)CH3),
1.27 (d, 3H, J5,6¼6.3 Hz, H-6C), 1.24, 1.23 (d, 6H, H-6A,
6B); 13C NMR d 172.3 (CvO), 100.7 (C-1A, JCH¼171 Hz),
99.6 (2C, C-1D, 1B, JCH¼163, 170 Hz), 99.2 (C-1C,
JCH¼170 Hz), 95.7 (brs, C-1E, JCH¼170 Hz), 82.0 (C-3D),
79.1 (C-2B), 79.4 (brs, C-3C), 76.4 (C-5pD), 75.4 (brs, C-4C),
73.0 (C-3E), 72.4 (2C, C-4A, 4B), 72.2 (C-5E), 71.7 (C-2E),
71.1 (C-2C), 70.4, 70.1, 70.0 (4C, C-2A, 3A, 3B, 4E), 69.7,
69.6, 69.3 (3C, C-5A, 5B, 5C), 68.8 (C-4D), 61.2, 61.0 (2C,
C-6D, 6E), 57.4 (OCH3), 55.4 (C-2D), 22.6 (C(O)CH3), 18.2
(C-6C), 17.2, 17.0 (2C, C-6A, 6B); HRMS (MALDI) calcd
for C33H57NO23 þNa: 858.3219. Found: 858.3089.
a single product. Filtration of the catalyst and reverse phase
chromatography, followed by RP-HPLC purification and
freeze-drying gave pure 3 (19 mg, 71%). Rt: 9.35 min
(solvent F, 100:0!80:20 over 20 min); [a]D þ12.5 (c 0.8,
methanol); 1H NMR d 5.09 (d, 1H, J1,2¼3.7 Hz, H-1E), 4.89
(brs, 1H, H-1B), 4.71 (d, 1H, J1,2¼1.1 Hz, H-1C), 4.39 (d,
1H, J1,2¼8.6 Hz, H-1D), 4.08 (dq, 1H, J4,5¼9.3 Hz, H-5C),
3.96 (dd, 1H, J1,2¼1.4 Hz, J2,3¼3.2 Hz, H-2B), 3.88–3.80
(m, 4H, H-2C, 3C, 6aE, 6bE, 5D), 3.77–3.62 (m, 6H, H-6aD,
6bD, 3B, 5B, 2D, 4C), 3.59 (pt, 1H, J3,4¼J4,5¼9.4 Hz, H-3E),
3.50 (pt, 1H, J3,4¼J4,5¼9.4 Hz, H-3E), 3.50 (pt, 1H,
J3,4¼J4,5¼8.7 Hz, H-3D), 3.47–3.34 (m, 8H, H-2E, 4E, 4B,
4D, 5E, OCH3), 1.98 (s, 3H, C(O)CH3), 1.27 (d, 3H,
J5,6¼6.3 Hz, H-6C), 1.21 (d, 3H, J5,6¼6.3 Hz, H-6B); 13C
NMR d 174.5 (CvO), 103.2 (brs, C-1B, JCH¼172 Hz),
101.8 (C-1D, JCH¼160 Hz), 101.5 (C-1C, JCH¼170 Hz),
98.0 (C-1E, JCH¼170 Hz), 82.2 (C-3D), 79.1 (brs, C-3C),
76.6 (brs, C-4C), 76.4 (C-4pB), 72.9 (C-3E), 72.3, 72.2 (2C,
C-4D, 5D), 71.87 (C-2E), 71.1 (brs, C-2C), 70.6 (2C, C-2B,
3B), 69.7, 69.6 (2C, C-5E, 5B), 69.2, 68.9 (2C, C-6D, 6E),
57.4 (OCH3), 55.4 (C-2D), 22.6 (C(O)CH3), 18.0 (C-6C),
17.0 (C-6B). HRMS (MALDI) calcd for C27H47NO19 þNa:
712.2635. Found: 712.2635.
4.1.22. Methyl (2-O-acetyl-3,4-di-O-benzyl-a-L-rhamno-
pyranosyl)-(1!3)-[2,3,4,6-tetra-O-benzyl-a-D-gluco-
pyranosyl-(1!4)]-(2-O-benzoyl-a-L-rhamnopyranosyl)-
(1!3)-2-acetamido-2-deoxy-b-D-glucopyranoside (36).
50% aq. TFA (400 mL) was added to a solution of the
fully protected tetrasaccharide 30 (57 mg, 40 mmol) in
CH2Cl2 (1 mL) at 0 8C, and the mixture was stirred
overnight at this temperature. Volatiles were evaporated
and the residue was purified by column chromatography
(solvent B, 1:1) to give diol 36 (47 mg, 85%). [a]D þ19.5 (c
1
0.9); H NMR d 8.10–7.16 (m, 35H, Ph), 5.80 (d, 1H,
Acknowledgements
J¼8.8 Hz, NHD), 5.66 (m, 1H, H-2B), 5.39 (pt, 1H,
J1,2¼2.8 Hz, H-2C), 5.01 (m, 2H, H-1B, 1E), 4.96 (m, 2H,
H-1C, OCH2), 4.90–4.81 (m, 5H, H-1D, OCH2), 4.66–4.41
(m, 7H, OCH2), 4.18 (dd, 1H, J2,3¼2.9 Hz, J3,4¼7.4 Hz,
H-3C), 4.10 (pt, 1H, H-3D), 4.08–3.95 (m, 5H, H-5E, 3E,
5C), 3.89–3.64 (m, 8H, H-6aD, 6bD, 6aE, 6bE, 3B, 4C, 4E,
5B), 3.54–3.49 (m, 5H, H-2E, 4D, OCH3), 3.45 (m, 1H,
H-5D), 3.33 (pt, 1H, J3,4¼J4,5¼9.4 Hz, H-4B), 3.27 (m, 1H,
H-2D), 2.26 (brs, 1H, OH), 2.17 (s, 6H, C(O)CH3), 1.99 (brs,
1H, OH), 1.39 (d, 3H, J5,6¼6.2 Hz, H-6C), 0.95 (d, 3H,
J5,6¼6.1 Hz, H-6B); 13C NMR d 171.5, 170.4, 166.1 (3C,
CvO), 139.1–127.8 (Ph), 100.9 (C-1D), 99.7 (2C, C-1pB,
1C), 99.2 (brs, C-1E), 85.0 (C-3D), 82.1 (C-3E), 81.3 (brs,
C-3E), 80.1 (C-4B), 78.0, 77.8 (4C, C-3C, 4C, 3B, 4E), 76.0
(OCH2), 75.6 (C-5D), 75.3, 75.2, 74.4, 73.4 (4C, OCH2),
72.3 (C-2C), 72.1 (C-5Cp ), 71.3 (C-4D), 71.2 (OCH2), 69.2
(C-5B), 69.0 (C-5E, 2B), 68.4 (C-6E), 63.2 (C-6D), 57.4 (2C,
C-2D, OCH3), 23.9, 21.0 (2C, C(O)CH3), 19.1 (C-6C), 18.0
(C-6B). FAB-MS for C78H89NO21 (M, 1375.6) m/z 1398.6
[MþNa]þ. Anal. calcd for C78H89NO21: C, 68.06; H, 6.52;
N, 1.02%. Found: C, 68.10; H, 6.62; N, 0.98%.
The authors thank the Institut Pasteur (Contract PTR 99) for
financial support. The authors are grateful to J. Ughetto-
´
Monfrin (Unite de Chimie Organique, Institut Pasteur) for
recording all the NMR spectra.
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