Synthesis and NMR study of a linear pentasaccharide fragment of the Shigella flexneri 5a O-specific polysaccharide

Article abstract of DOI:10.1016/S0040-4020(02)00148-5

A convergent chemical synthesis of the methyl glycoside of the linear epitope α-D-Glcp-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap- (1→3)-β-D-GlcNAcp-(1→2)-α-L-Rhap (EBCDA) corresponding to the ramification of the O-antigen of Shigella flexneri serotype 5a is described

Full text of DOI:10.1016/S0040-4020(02)00148-5

TETRAHEDRON
Pergamon
Tetrahedron 58 ,2002) 2593±2604
Synthesis and NMR study of a linear pentasaccharide fragment of
the Shigella ¯exneri 5a O-speci®c polysaccharideq
a,p
b
a,²
Laurence A. Mulard, Marie-Jeanne Clement, Fabienne Segat-Dioury and Muriel Delepierre
b
Â
a
Â
Unite de Chimie Organique, URA CNRS 2128, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France
b
    Â
Unite de Resonance Magnetique Nucleaire des Biomolecules, URA CNRS 2185, Institut Pasteur, 28 rue du Dr. Roux,
75724 Paris Cedex 15, France
Received 11 October 2001; revised 5 December 2001; accepted 4 February 2002
AbstractÐA convergent chemical synthesis of the methyl glycoside of the linear epitope a-d-Glcp-,1!3)-a-l-Rhap-,1!3)-a-l-Rhap-
,1!3)-b-d-GlcNAcp-,1!2)-a-l-Rhap ,EBCDA) corresponding to the rami®cation of the O-antigen of Shigella ¯exneri serotype 5a is
described. The strategy relies on the preparation of a key EB trichloroacetimidate donor and that of an appropriate CDA trisaccharide
acceptor. Trichloroacetimidate chemistry was used for the construction of all glycosidic linkages except that of DA, where a bromide donor
was preferred. In depth analysis of the pentasaccharide EBCDA 1H and 13C NMR spectra shows that its conformation approaches that of the
corresponding fragment in the native polysaccharide. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
on the model bacterium S. ¯exneri serotype 5a. The
approach focuses on the use of mimics of the antigenic
polysaccharide.5 Optimization of the mimics is supported,
in part, by a comparative study of their solution conforma-
tions and that of the native carbohydrate epitopes. Such a
study implies that ligands representative of the major struc-
tural features of the O-SP of S. ¯exneri serotype 5a be avail-
able in rather large amounts.
The use of polysaccharide±protein conjugates for the
prevention of infection by encapsulated bacteria is now
well-established.2 In the case of non-encapsulated bacteria,
such as Shigella, a Gram-negative enterobacterium that
can cause diarrhea and dysentery in humans, the bacterial
O-speci®c polysaccharide ,O-SP), a component of their
LPS, is both an essential virulence factor and a critical anti-
gen for host immunity. The O-SP of S. ¯exneri behaves as a
hapten, which acts as a T-dependent antigen when conju-
gated to an immunogenic protein carrier. In the case of
S. ¯exneri 2a and S. sonnei, such O-SP/protein conjugates
elicited high levels of anti-LPS IgG in healthy adults.3,4
The repeating unit of the O-SP of S. ¯exneri serotype 5a is
the branched pentasaccharide6,7 I, composed of a-linked
l-rhamnoses, b-linked 2-acetamido-2-deoxy-d-glucose, and
a-d-glucose branches. In spite of the large amount of work
on S. ¯exneri 5a reported previously in the literature,8,9 the
preparation of the required frame-shifted oligosaccharides
was undertaken.10±12 In this paper, we describe a convergent
synthesis of the linear pentasaccharide EBCDA prepared as
its methyl glycoside ,1) that has the natural anomeric
A program to develop chemically de®ned vaccines as a
possible alternative approach to the use of O-SP/protein
conjugates as human anti-bacterial vaccines is in progress
q See Ref. 1.
Keywords: bacterial oligosaccharides; Shigella ¯exneri; chemical glycosylation.
Corresponding author. Tel.: 133-1-40613677; fax: 133-1-45688404; e-mail: lmulard@pasteur.fr
Present address: Laboratoire de Chimie Organique±ESA CNRS 7084, CNAM, 292 Rue St-Martin, 75141 Paris Cedex 03, France.
p
²
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020,02)00148-5

2594
L. A. Mulard et al. / Tetrahedron 58 02002) 2593±2604
Scheme 1. Retrosynthetic approach to pentasaccharide 1.
con®guration at its reducing end. The NMR study of 1 using
a combination of one- and two-dimensional ,1D and 2D)
techniques is also presented with emphasis on its
characteristics in relation to the native O-SP.
can be blocked selectively at position 2, were selected as
suitable precursors to the intermediate 2. Trisaccharide 3
was readily accessible from the known methyl glycoside17,18
6, glucosaminyl bromide19 7 and trichloroacetimidate
donor12 8, which were chosen as appropriate precursors
for residues A, D, and C, respectively. Compound 8 features
easily removable protecting groups at positions 2 and 3,
allowing subsequent chain extension at position 3.
2. Results and discussion
The linear pentasaccharide 1 was prepared following a
block synthetic strategy ,Scheme 1), using the disaccharide
trichloroacetimidate 2 as the donor and the trisaccharide
acceptor 3 derived from a-l-Rhap-,1!3)-b-d-GlcNAcp-
,1!2)-a-l-Rhap-OMe. In designing the synthesis, attention
was paid to the fact that the construction of the EB moiety,
which involves a 1,2-cis glycosidic linkage, was the most
demanding. Based on previous experience in the methyl
glycoside series,10 the known trichloroacetimidate
donor13,14 4 and the allyl rhamnopyranoside15,16 5, which
2.1. Preparation of the disaccharide donor EB
Conventional regioselective opening of the intermediate
orthoester issued from diol 5 gave a 97:3 mixture of the
target 2-O-acetyl derivative20 9 and the corresponding
1
3-O-acetyl regioisomer, as seen by H NMR spectroscopy.
In order to prevent any potential migration of the acetyl
group to the vicinal hydroxyl group, the crude mixture
was used as such. Condensation of acceptor 9 with the
Scheme 2. Reagents and conditions: ,a) ,i) MeC,OMe)3 3 equiv., PTSA cat., CH3CN, rt, 1.5 h; ,ii) AcOH 80% aq., 08C, 30 min, 99%; ,b) 4 1.3 equiv.,
TMSOTf 0.05 equiv., Et2O, 2788C!rt, overnight; ,c) MeONa 1.5 equiv., MeOH, rt, 3 days, 57% for two steps; ,d) BzCl 1.5 equiv., Pyr, 708C, overnight,
95%; ,e) ,i) [Ir,COD){PCH3,C6H5)2}2]1PF62 0.09 equiv., THF, rt, overnight; ,ii) HgO 1.90 equiv., HgCl2 1.59 equiv., H2O/Me2CO, rt, 2.5 h, 86%; ,f) CCl3CN
10 equiv., DBU cat., CH2Cl2, 08C, 1h, 95%.

L. A. Mulard et al. / Tetrahedron 58 02002) 2593±2604
2595
Scheme 3. Reagents and conditions: ,a) see Ref. 10; ,b) Bu3SnH, AIBN, Toluene/DMA, 1008C; ,c) MeONa, MeOH/CH2Cl2; ,d) Ac2O, MeOH ,59% from 16);
,e) 7 1.38 equiv., AgOTf, DBMP, CH2Cl2, 2508C!rt, 89%; ,f) ,i) ,H2NCH2)2, EtOH, 708C; ,ii) Ac2O, EtOH ,83%); ,g) Me2C,OMe)2, PTSA, acetone ,87%
from 20).
trichloroacetimidate donor 4 was performed in diethyl ether
in the presence of a catalytic amount of trimethylsilyl
tri¯uoromethanesulfonate ,TMSOTf) to afford the conden-
sation products 1021 and 11 as an inseparable mixture.
Treatment of the anomeric mixture with an excess of metha-
nolic sodium methoxide afforded the monohydroxylated
disaccharide 12 ,57%) and contaminated b-anomer 13,
which could be separated at this stage ,Scheme 2).
The stereochemistry of the EC linkage in 12 and 13 was
to occur during TMSOTf catalyzed glycosylations.17 Next,
disaccharide 14 was converted to the hemiacetal 15 ,86%),
following a two-step selective deallylation procedure invol-
ving: ,i) isomerisation of the allyl ether into the correspond-
ing prop-1-enyl ether using a cationic iridium complex22
and ,ii) subsequent hydrolysis with mercury,II) chloride/
mercury,II) oxide.15,23 Finally, the EB disaccharide donor
2 was obtained in 95% yield as an anomeric mixture by
treatment of the hemiacetal 15 with trichloroacetonitrile
and a catalytic amount of DBU.
1
ascertained based on the JC-1,H-1 heteronuclear coupling
constants. Data for compound 12 were 168 and 170 Hz,
data for compound 13 were 159 and 169 Hz for residues E
and B, respectively. As observed in the methyl glycoside
series,10 position 2B is most probably sterically hindered due
to the presence of the 2,3,4,6-tetra-O-benzyl-a-d-gluco-
pyranose residue at O-3B, which renders the deacylation
step an unusually slow process. For this reason, benzoyl-
ation of 12 by treatment with benzoyl chloride in pyridine
was performed at 708C to give the fully protected 14 ,95%).
In that case, benzoylation was preferred to acetylation in
order to avoid potential acetylation of the acceptor, known
2.2. Preparation of the disaccharide acceptor DA
Two types of amine participating groups were considered in
the construction of the DA disaccharide ,Scheme 3), the
selectively removable N-trichloroacetyl group ,TCA, route
1) and the N-tetrachlorophtaloyl group ,TCP, route 2). In
route 1, the easily accessible disaccharide10 16 was the key
intermediate to the triol 21. Conversion of the N-trichloro-
acetyl group to the N-acetyl group was ®rst attempted
through tributyltin hydride-mediated reductive cleavage of
Scheme 4. Reagents and conditions: ,a) 8 1.55 equiv., TMSOTf 0.09 equiv., Et2O; ,b) MeONa, MeOH ,81% from 22); ,c) ,i) MeC,OMe)3, PTSA, CH3CN;
,ii) AcOH 80% aq.; ,d) 2 1.4 equiv., TMSOTf cat., Et2O ,67% from 24); ,e) TFA 50% aq. ,94%); ,f) MeONa, MeOH ,97%); ,g) H2, Pd/C, AcOH/EtOH ,67%).

2596
L. A. Mulard et al. / Tetrahedron 58 02002) 2593±2604
Table 1. 1H NMR data for EBCDA-OMe ,1)
Residue
H-1H-2
H-3
H-4
H-5
H-6a
H-6b
a-l-Rhap ,A)a
a-l-Rhap ,B)
a-l-Rhap ,C)
b-d-GlcNAcp ,D)b
a-d-Glcp ,E)
4.82 ,0.32)
5.05 ,20.17)
4.84 ,20.04)
4.71, 20.07)
5.08 ,20.01)
3.98 ,20.16)
4.24 ,20.01)
3.87 ,0.00)
3.82 ,20.01)
3.56 ,20.04)
3.77 ,20.12)
3.89 ,20.12)
3.79 ,20.01)
3.60 ,20.06)
3.77 ,20.03)
3.29 ,0.10)
3.56 ,20.05)
3.51 ,20.05)
3.51,0.02)
3.62 ,20.08)
3.80 ,0.01)
4.00 ,0.01)
3.43 , 20.05)
3.96 ,20.04)
1.25 ,0.00)
1.30 ,20.04)
1.22 ,20.05)
3.90 ,20.04)
3.79 ,20.07)
3.73 ,20.03)
3.76 ,20.02)
3.44 ,0.02)
Chemicals shifts measured in ppm with an accuracy of ^0.01ppm are referenced to external DSS , dH 0.00). The numbers in parentheses represent the
difference in ppm between the chemical shifts of the protons in 1 and the chemical shifts of the respective protons in the O-SP of S. ¯exneri serotype 5a.37
The chemical shift of the aglyconic methyl is 3.37 ppm.
a
b
The chemical shift of the N-acetyl group is 2.05 ppm.
the chlorine atoms.24 Often yielding the N-chloroacetyl
disaccharide 17 together with the target 18, this approach
failed to give satisfactory results when applied to 16. That
the N-trichloroacetyl moiety was sensitive to treatment with
sodium methoxide10 or sodium hydroxide25 was reported
previously. Indeed, treatment of 16 with sodium methoxide
gave the aminotriol 19, which was next selectively N-acetyl-
ated into 21 ,59% for two steps). In route 2 to DA, the
glucosaminyl bromide 7 was preferred to the corresponding
trichloroacetimidate,26 which was found more dif®cult to
obtain. Condensation of the rhamnose acceptor 6 and
donor 7 was performed under base-de®cient conditions,27
using silver tri¯uoromethanesulfonate ,AgOTf) as the
promoter, to give the fully protected disaccharide 20
,89%). Treatment with ethylenediamine and subsequent
selective N-acetylation allowed the conversion of the
N-tetrachlorophthalimido disaccharide to the corresponding
N-acetamido triol 21 ,83%). Overall, the yield of 21
compares favorably with that described previously28 for an
analogous compound when using the corresponding
N-phthalimido glucosamine donor. Subsequent isopropyl-
idenation of 21 afforded the acceptor 22 ,87% from 20).
2.3. Assembly of the pentasaccharide EBCDA-OMe -1)
Condensation of 22 with the trichloroacetimidate donor 8 in
the presence of a catalytic amount of TMSOTf gave 23
,Scheme 4). Transesteri®cation gave the diol 24 ,81%
from 22) which was next regioselectively acetylated at
position 2C, by acidic hydrolysis of the intermediate ortho-
acetate, to give the CDA trisaccharide acceptor 3. TMSOTf
promoted glycosylation of this alcohol with the trichloro-
acetimidate donor 2 provided the fully protected penta-
saccharide 25 ,67% from 24). The a-anomery of residues
A, B, C and E in compound 25 was indicated by the 1JC-1,H-1
heteronuclear coupling constants, whose values were of
1
170, 172, 170, and 169 Hz, respectively. The JC-1,H-1
heteronuclear coupling constant for residue D was 161 Hz,
con®rming the b-anomery. Acetal cleavage of 25 gave diol
Â
26 ,94%), Zemplen deacylation of which afforded the
tetraol 27 ,97%), which was ®nally converted to the target
1 ,67%) by conventional debenzylation, using palladium on
charcoal as the catalyst.
2.4. NMR study of the pentasaccharide EBCDA-OMe -1)
in solution
Complete 1H NMR assignments of pentasaccharide
EBCDA-OMe ,1) ,Table 1) were obtained from 1D and
1
2D H NMR spectroscopy including DQF-COSY,29 and
1
TOCSY.30 The H chemical shifts were assigned to indi-
vidual rings based on straightforward analysis of charac-
teristic regions of the 1D proton spectrum ,Fig. 1), namely
the methyl groups and the anomeric proton region.31,32 13C
resonances were further assigned ,Table 2) from H±13C
1
heteronuclear NMR experiments such as gHSQC33 and
gHSQC-TOCSY.33,34 Quaternary carbons as well as
1
Figure 1. H NMR spectrum of the pentasaccharide EBCDA-OMe ,1) in
D2O at 388C at 500 MHz.
Table 2. 13C NMR data for EBCDA-OMe ,1)
1JC-1,H-1
a
Residue
C-1C-2
C-3
C-4
C-5
C-6
a-l-Rhap ,A)b
a-l-Rhap ,B)
a-l-Rhap ,C)
b-d-GlcNAcp ,D)c
a-d-Glcp ,E)
102.5 ,20.1)
104.6 ,1.3)
103.9 ,0.1)
104.7 ,0.1)
98.3 ,0.6)
81.0 ,20.2)
69.5 ,27.8)
73.2 ,20.2)
58.1 ,20.3)
74.0 ,20.1)
72.6 ,20.3)
78.2 ,1.3)
80.7 ,20.6)
84.1, 20.1)
75.6 ,20.3)
74.9 ,20.4)
73.0 ,20.4)
74.0 ,20.4)
71.1 ,20.4)
72.1, 20.5)
71.2 ,20.9)
71.8 ,20.1)
71.6 ,20.1)
78.4 ,20.3)
74.4 ,20.2)
19.1 ,20.1)
19.3 ,20.3)
19.1 ,20.3)
63.3 ,20.5)
63.0 ,20.5)
172.5
171.7
169.6
163.4
169.6
Chemicals shifts measured in ppm with an accuracy of ^0.2 ppm are referenced to external DSS ,dC 0.00). The numbers in parentheses represent the difference
in ppm between the chemical shifts of the protons in 1 and the chemical shifts of the respective protons in the O-SP of S. ¯exneri 5a.38
a
Anomeric 1JC-1,H-1 coupling constants is measured in Hz with a digital resolution of 0.5 Hz.
The chemical shift of the aglyconic methyl is 57.4 ppm.
The chemical shifts of the carboxyl and N-acetyl groups are 177.3 and 24.9 ppm, respectively.
b
c

L. A. Mulard et al. / Tetrahedron 58 02002) 2593±2604
2597
3
Table 3. J coupling constants measured for EBCDA-OMe ,1) ,n.d.: not
determined)
Residue
3J1,2 3J2,3 3J3,4 3J4,5 3J5,6a 3J5,6b 3J6a,6b
a-l-Rhap ,A)
a-l-Rhap ,B)
a-l-Rhap ,C)
1.3a 3.6a 9.7a 9.7a 6.3a
1.4a 3.2a 9.7a n.d. 6.3a
1.3a 3.2b 9.7b 9.7b 6.3a
±
±
±
±
±
±
b-d-GlcNAcp ,D) 8.5a 9.5b 9.7b 9.7b n.d.
6.4b
n.d.
12.3b
12.4b
a-d-Glcp ,E)
3.7a 9.7b 9.6b 9.2b n.d.
Directly measured on the 1D 1H NMR spectrum ,Hz^0.1Hz).
Measured on the DQF-COSY spectrum ,Hz^0.5 Hz).
a
b
Figure 2. The structure of EBCDA-OMe ,1) obtained by energy minimi-
zation with ROE-derived distances constraints. The methyl aglycone is
oriented towards the lower right.
glycosidic linkages H±C±O±C were then obtained from
gHMBC.33 The glycosidic linkages were con®rmed from
1H±1H dipolar interactions observed in the off-resonance
ROESY experiment.35 The vicinal coupling constants
,Table 3) of the ring protons in the monosaccharide units
within the pentasaccharide were found to be consistent with
observed earlier for other oligo- and polysaccharides
containing a 1!2 linkage.39,40 Interestingly, the chemical
shifts corresponding to residue E which is at the non-
reducing end of the linear 1 match with the corresponding
resonances in the O-SP, although E is a branched residue in
the latter. Residue A is involved in a 2,3-cis vicinal glyco-
sylation pattern, but it does not appear to interfere with the
conformational behavior of residue E. Comparison with
data corresponding to the branched synthetic trisaccharide
1
a C4 conformation for the l-rhamnopyranosyl rings and
a
4C1 conformation for the d-glucopyranosyl and the
N-acetyl-d-glucosaminyl units. The a and b con®gurations
1
were corroborated by heteronuclear one-bond JC-1,H-1
coupling constants ,Table 2) obtained from the gHMBC
experiment.36 Data in Tables 1and 2 show that the 1H and
13C chemical shifts of units A, B, C, D, and E in the
synthetic 1 are very close to those of the corresponding
residues in the native O-SP,37,38 although major differences
were observed for residues A and B. In the case of
the former, the observed divergences are likely due to the
presence of the anomeric O-methyl group and to the
external position of this residue in the pentasaccharide.
The substitution pattern may account for the differences in
the chemical shifts associated with residue B. Indeed, B is
monosubstituted in 1 whereas it is disubstituted at positions
2 and 3 in the O-SP. However, the large chemical shift
difference observed for C-2B can most likely be attributed
to a direct in¯uence of the upstream residue D present in the
O-SP but absent in the linear 1. This phenomenon has been
A,E)B-OMe41 ,Clement, unpublished data), shows that
Â
there is no direct in¯uence of the downstream units C or
D on the 1H and 13C signals of residue E. Consequently, it is
most probable that residue E is well-exposed to the solvent
in the O-SP.
The solution conformation of 1 was then investigated by
examination of NOEs determined from the off-resonance
ROESY experiment. Thus, based on the ROE-derived
distances ,Table 4), energy minimization using DISCOVER
resulted in an overview of the average structure of 1 ,Fig. 2).
Due to the high ¯exibility of such oligosaccharides, this
structure is provided as a representation that agrees with
NMR data, and which could be used as a good starting
point for further studies. Indeed, the only difference between
calculated and measured distances involves either long
distances or distances implying methyl groups.
Table 4. Inter-residue 1H±1H distances for EBCDA-OMe ,1)
a
Distance ,A) from
ROE intensity
a
Ê
Ê
Atom pairs
Distance ,A) from
minimized structure
with constraints
Taken as a whole, all the NMR results described herein
suggest a similar distribution of conformations in solution
for 1 and for the O-SP, which makes the former a good
candidate for studying the recognition pattern of mono-
clonal antibodies raised against the O-SP of S. ¯exneri
serotype 5a.
D-Ac/A-6b
E-1/B-2
E-1/B-3
E-5/B-3
E-5/B-4
B-1/C-2
B-1/C-3
B-1/D-Ac
B-2/C-3
B-6/C-2
B-6/D-Ac
C-1/D-3
C-1/D-Ac
C-2/D-Ac
D-1/A-1
D-1/A-2
D-2/A-14.73
D-5/A-2
D-Ac/A-15.80
D-Ac/A-4
6.13
2.46
2.51
3.06
3.74
3.44
2.24
5.05
3.74
4.413.88
5.48
2.27
4.53
4.66
3.26
2.27
6.50
2.44
2.66
3.00
3.82
3.18
2.15
5.00
3.84
3. Experimental
4.93
2.32
4.35
3.46
3.55
2.30
3.1. NMR experiments for compound 1
The solution concentration was 28 mM ,14 mg in 0.6 mL of
D2O). All NMR spectra were recorded at 388C on a Varian
Unity 500 spectrometer operating at a proton frequency of
500 MHz and a 13C frequency of 125 MHz and equipped
with a z-gradient triple resonance ,1H, 15N, 13C) probe.
Chemical shifts in ppm are referenced to external sodium
4,4-dimethyl-4-silapentane-1sulfonate ,DSS) , d 0). DQF-
COSY, TOCSY and off-resonance ROESY spectra were
4.05
6.01
3.18
4.57
3.53
4.68
a
The errors on distance values are ,10%.
Labeling: D-Ac, N-acetyl of residue D; A-6, H-6 of residue A.
b

2598
L. A. Mulard et al. / Tetrahedron 58 02002) 2593±2604
recorded using a spectral width of 2.2 kHz in both dimen-
sions, a 908 pulse of 4.3 ms, 2K ,F1)£2K ,F2) points data
sets, zero-®lled to 4K in F1dimension and using 16, 8 and
32 scans per increment, respectively. The TOCSY experi-
ment was acquired using a mixing time of 80 ms. The
off-resonance ROESY experiment was recorded with a
11.6 kHz effective spin lock ®eld generated by a series of
308 pulses during 400 ms. To avoid Hartmann±Hahn
artifacts, the offset of the spin lock carrier was shifted by
approximately 8 kHz from the center of the spectrum in
order to create an angle of 54.78 between the effective
spin lock axis and the static magnetic ®eld. These 1H
NMR experiments were processed using shifted sine-bells
windows in both dimensions. Gradient-enhanced HSQC,
gHSQC-TOCSY and gHMBC experiments were performed
,A) dichloromethane±methanol, ,B) cyclohexane±ethyl
acetate, ,C) toluene±acetone, ,D) toluene±ethyl acetate,
,E) iso-propanol±ammonia±water, ,F) water±acetonitrile.
Detection was effected when applicable, with UV light,
and/or by charring with orcinol ,35 mM) in aq. H2SO4
,4N). Preparative chromatography was performed by
elution from columns of Silica Gel 60 ,particle size
0.040±0.063 mm). For all compounds excepts the target 1,
the NMR spectra were recorded at 258C for solutions in
CDCl3, on a Bruker AC 300P spectrometer ,300 MHz for
1H, 75 MHz for 13C). External references: for solutions in
1
CDCl3, TMS ,0.00 ppm for both H and 13C); for solutions
in D2O, dioxane ,67.4 ppm for 13C) and trimethylsilyl-3
propionic acid sodium salt ,0.00 ppm for 1H). Proton-signal
assignments were made by ®rst-order analysis of the
1
1
with spectral width of 2.2 and 24 kHz in the H and 13C
spectra, as well as analysis of 2D H±1H correlation maps
1
dimensions, respectively, with H and 13C 908 pulse width
,COSY) and selective TOCSY experiments. Of the two
magnetically non-equivalent geminal protons at C-6, the
one resonating at lower ®eld is denoted H-6a and the one
at higher ®eld is denoted H-6b. The 13C NMR assignments
were supported by 2D 13C±1H correlation maps ,HETCOR).
Interchangeable assignments are marked with an asterisk in
the listing of signal assignments. Sugar residues in oligo-
saccharides are serially lettered according to the lettering of
the repeating unit of the O-SP and identi®ed by a subscript
in the listing of signal assignments. Low-resolution chemi-
cal ionization mass spectra ,CIMS) were obtained using
NH3 as the ionizing gas. Fast atom bombardment mass spec-
tra ,FABMS) were recorded in the positive-ion mode using
dithioerythritol/dithio-l-threitol ,4:1, MB) as the matrix, in
the presence of NaI, and Xenon as the gas. Before use,
AgOTf was dried at 133 Pa/508C for 2 h. Anhydrous
CH2Cl2, sold on molecular sieves was used as such. Et2O
and THF were distilled over sodium/benzophenone. CH3CN
suitable for DNA synthesis and kept on Trap-Pack molecu-
lar sieves bags was used as such. Solutions in organic
solvents were dried by passing through phase separator
®lters.
of 4.3 and 15.9 ms, respectively, 16 scans per increment and
a recycle delay of 1.5 s. Zero-®lling was set to 4K in F1
dimension and 2K in F2 dimension. For gHSQC
1
experiment, a delay corresponding to a H±13C one-bond
coupling constant value of 150 Hz was used. The gHSQC-
TOCSY experiment was recorded with a mixing time of
150 ms. The gHMBC experiment was performed with a
delay of 60 ms for evolution of long-range couplings. For
these heteronuclear experiments, shifted Gaussian windows
were applied in both dimensions. All 2D data, except
gHMBC were collected in the phase sensitive mode
3
using the States±Haberkorn method.42 The JH,H coupling
constants were obtained from 1D spectrum with a digital
resolution of 0.1Hz/point, or from the DQF-COSY
experiment with a digital resolution of 0.5 Hz/point. The
1JC-1,H-1 coupling constants were measured from the
gHMBC spectrum with a digital resolution of 0.5 Hz/point.
Inter-proton distances from cross-peak volumes. The cross-
peak volumes from off-resonance ROESY were measured
with the VNMR software. The intra-residue distance of
Ê
2.52 A between H-1and H-2 protons of the a-rhamnose
unit B was used as the reference for distance calibration.
The proton±proton distances were calculated using the
usual 1/r6 NOE/distance relationship.43
3.3.1. Allyl -2,3,4,6-tetra-O-benzyl-a-d-glucopyranosyl)-
-1!3)-4-O-benzyl-a-l-rhamnopyranoside 12 and allyl
-2,3,4,6-tetra-O-benzyl-b-d-glucopyranosyl)--1!3)-4-O-
benzyl-a-l-rhamnopyranoside 13. Allyl 4-O-benzyl-a-l-
rhamnopyranoside16 ,5, 1.47 g, 5.0 mmol) was dissolved in
MeCN ,2.5 mL) containing methyl orthoacetate ,1.85 mL,
14.5 mmol). p-Toluenesulphonic acid ,13 mg) was added,
the mixture was stirred for 1.5 h at rt TLC ,solvent D, 9:1)
showed that no starting material remained. The reaction
mixture was cooled to 08C, and 80% aq. AcOH ,2.5 mL)
was added. After 30 min at rt, TLC ,solvent D, 9:1) showed
that the intermediate orthoester had turned into a more polar
product. CH2Cl2 was added, and the organic phase was
washed with ice-water and satd aq. NaCl, dried and evapo-
3.2. Energy minimization
Energy minimization was performed on Silicon Graphics
Octane workstation running under the IRIX 6.5 operating
system using MSI INSIGHT II/DISCOVER software
package. The computations were performed using
AMBER force®eld with Homans extensions for the ano-
meric atoms,44 with a distance-dependent dielectric constant
of 4.0 and 5000 steps of energy minimization. Twenty inter-
residual distance constraints were used.
1
rated to dryness to give a yellow syrup. H NMR showed
that the product 9 was identical to that described20 and that it
was only slightly contaminated by the regiosiomer acetyl-
ated at position 3 ,2-OAc/3-OAc, 97:3).
3.3. General methods
Melting points were determined in capillary tubes with an
electrothermal apparatus and are uncorrected. Optical rota-
tions were measured for CHCl3 solutions at 258C, except
where indicated otherwise, with a Perkin±Elmer automatic
polarimeter, Model 241MC. TLC on precoated slides of
Silica Gel 60 F254 ,Merck) was performed with solvent
mixtures of appropriately adjusted polarity consisting of
TMSOTf ,45 mL, 0.23 mmol) was added to a solution of the
crude 9 and donor13,14 4 ,4.5 g, 6.57 mmol) in Et2O ,50 mL),
at 2788C. The reaction mixture was stirred overnight while
slowly coming back to rt. TLC ,solvent B, 3:1) showed the
complete disappearance of 5. Et3N ,1mL) was added and

L. A. Mulard et al. / Tetrahedron 58 02002) 2593±2604
2599
1
volatiles were evaporated. Chromatography of the residue
,solvent E, 24:1) gave the condensation products as a
contaminated syrupy mixture of a- ,10)21 and b- ,11)
isomers ,5.82 g). The syrup was dissolved in CH2Cl2 and
MeOH ,8:11) to which 1 M MeONa ,5 mL) was added, and
the mixture was kept for 5 days at rt under N2. The mixture
was neutralized by addition of resin IR-120 ,H1), and
®ltered. Evaporation of the ®ltrate gave a syrup which was
puri®ed by chromatography ,solvent D, 47:3) to give 12
,2.31g, 57%) as a colorless oil, and 13 ,1.19 g) contami-
nated by a 2,3,4,6-tetra-O-benzyl-d-glucopyranose deriva-
tive as the second eluting product. Compound 12 had
[a]Dˆ1438 ,c 1.0); NMR: H, d 8.08±7.05 ,m, 30H, Ph),
5.89 ,m, 1H, CHvCH2), 5.63 ,dd, 1H, J1,2ˆ2.3 Hz, J2,3ˆ
2.5 Hz, H-2B), 5.29 ,m, 1H, CHvCH2), 5.25 ,d, 1H, J1,2ˆ
3.6 Hz, H-1E), 5.19 ,m, 1H, CHvCH2), 4.97 ,d, 1H, Jˆ
10.2 Hz, OCH2), 4.93 ,d, 1H, J1,2ˆ1.5 Hz, H-1B), 4.86±
4.35 ,m, 9H, OCH2), 4.36 ,dd, 1H, H-3B), 4.16 ,m, 1H,
OCH2), 4.06±4.00 ,m, 3H, H-3E, 5E, OCH2), 3.87 ,dq, 1H,
J4,5ˆ9.4 Hz, H-5B), 3.70 ,m, 2H, H-4B, 4E), 3.64±3.52 ,m,
3H, H-6aE, 2E, 6bE), and 1.43 ,d, 3H, J5,6ˆ6.2 Hz, H-6B);
13C, d 166.1 ,CvO), 138.8±127.5 ,Ph, CHvCH2), 117.5
,CHvCH2), 96.6 ,C-1B), 92.7 ,C-1E), 82.0 ,C-3E), 79.9
p
p
,C-4E ), 79.0 ,C-2E), 77.5 ,C-4B ), 76.2, 75.4, 74.9, 73.3,
,4C, OCH2), 72.6 ,C-3B), 72.2 ,OCH2), 70.2 ,C-5E), 68.6
,C-2B), 68.1,2C, C-6 E, OCH2), 68.0 ,C-5B), and 18.0
,C-6B); CIMS for C57H60O11 ,M, 920.4) m/z 943.4 ,[M1
Na]1). Anal. Calcd for C57H60O11: C, 74.33; H, 6.57%.
Found: C, 74.18; H, 6.71%.
1
[a]Dˆ1328 ,c 1.0); NMR: H, d 7.57±7.13 ,m, 25H, Ph),
5.92 ,m, 1H, CHvCH2), 5.30 ,m, 1H, CHvCH2), 5.22 ,m,
1H, CHvCH2), 4.94 ,m, 4H, H-1E, 1B, OCH2), 4.87±4.48
,m, 7H, OCH2), 4.31,d, 1H, Jˆ12.0 Hz, OCH2), 4.19 ,m,
1H, OCH2), 4.11±3.94 ,m, 5H, H-3E, 3B, 5E, 2B, OCH2),
3.80 ,m, 1H, J4,5ˆ9.6 Hz, H-5B), 3.75 ,dd, 1H, J3,4ˆ
9.2 Hz, J4,5ˆ9.8 Hz, H-4E), 3.62 ,dd, 1H, J1,2ˆ3.6 Hz,
J2,3ˆ9.6 Hz, H-2E), 3.52 ,t, 1H, J3,4ˆ9.4 Hz, H-4B), 3.48
,dd, 1H, J5,6aˆ2.3 Hz, H-6aE), 3.44 ,bs, 1H, OH-2), 3.36
,dd, 1H, J6a,6bˆ10.6 Hz, H-6bE), and 1.40 ,d, 3H, J5,6ˆ
6.2 Hz, H-6B); 13C, d 134.0±127.7 ,Ph, CHvCH2), 117.5
3.3.3.
-2,3,4,6-Tetra-O-benzyl-a-d-glucopyranosyl)-
-1!3)-2-O-benzoyl-4-O-benzyl-a/b-l-rhamnopyranose
15. A solution of 1,5-cyclooctadiene-bis,methyldiphenyl-
phosphine)iridium hexa¯uorophosphate ,150 mg, 0.18
mmol, Ir,COD){PCH3,C6H5)2]2PF62) in anhydrous THF
,20 mL) was degassed, and the catalyst was activated by
passing a stream of hydrogen until the solution had turned
yellow ,ca. 3 min). The solution was degassed again, and a
degassed solution of compound 14 ,21.0 g, 22.8 mmol) in
anhydrous THF ,100 mL) was added. The reaction was
stirred overnight under Ar. TLC ,solvent D, 23:2) showed
that only little starting material remained, and the mixture
was concentrated to dryness. Mercuric oxide ,9.0 g,
41.9 mmol) and mercuric chloride ,10 g, 37.2 mmol) were
added to a solution of the residue in a mixture of acetone and
water ,1.5 L, 9:1). The suspension, protected from light, was
stirred at rt for 1.5 h, and acetone was evaporated. The
resulting suspension was taken up in CH2Cl2, washed
twice with 50% KI, water and satd aq NaCl, dried and
concentrated. Puri®cation of the crude material was effected
by silica gel chromatography ,solvent B, 4:1) to furnish
the hemiacetal 15 ,17.2 g, 86%) as a mixture of a and b
anomers. NMR ,a-anomer): 1H, d 8.08±7.07 ,m, 30H, Ph),
5.65 ,dd, 1H, J1,2ˆJ2,3ˆ2.6 Hz, H-2B), 5.29 ,bs, 1H, H-1B),
5.28 ,d, 1H, J1,2ˆ3.6 Hz, H-1E), 4.97 ,d, 1H, Jˆ10.2 Hz,
OCH2), 4.86±4.35 ,m, 10H, H-3B, OCH2), 4.06±4.00 ,m,
3H, H-3E, 5E, 5B), 3.75±3.65 ,m, 2H, H-4B, 4E), 3.64±3.52
,m, 3H, H-6aB, 2B, 6bB), 3.18 ,bs, 1H, OH-1B), and 1.41 ,d,
3H, J5,6ˆ6.2 Hz, H-6B); 13C, d 166.1 ,CvO), 138.7±127.3
,CHvCH2), 98.2 ,C-1B, JC,Hˆ170 Hz), 93.9 ,C-1E, JC,H
ˆ
168 Hz), 82.4 ,C-3E), 79.3 ,C-4B), 78.9 ,C-2E), 77.7 ,C-4E),
76.6 ,C-3B), 75.6, 74.9, 74.3, 73.4 ,5C, OCH2), 70.7 ,C-5E),
67.8 ,2C, C-6E, OCH2), 67.4 ,C-2B), 67.2 ,C-5B), and 18.0
,C-6B). FABMS for C50H56O10 ,M, 816.39) m/z 839.5
,[M1Na]1). Anal. Calcd for C50H56O10: C, 73.51; H,
6.91%. Found: C, 73.42; H, 7.04%.
Analytical data for compound 13 were [a]Dˆ2128 ,c 1.0);
NMR: 1H, d 7.35±7.13 ,m, 25H, Ph), 5.90 ,m, 1H,
CHvCH2), 5.33±5.18 ,m, 2H, CHvCH2), 4.99±4.47 ,m,
12H, H-1E, 1B, OCH2), 4.18 ,m, 1H, OCH2), 4.13 ,m, 2H,
H-3B, 2B), 3.98 ,m, 1H, OCH2), 3.80 ,dq, 1H, J4,5ˆ9.4 Hz,
H-5B), 3.74±3.55 ,m, 6H, H-6aE, 3E, 6bE, 4E, 4B, 2E), 3.53
,m, 1H, H-5E), 3.25 ,bs, 1H, OH), and 1.34 ,d, 3H, J5,6ˆ
6.2 Hz, H-6B); 13C, d 138.4±127.5 ,Ph, CHvCH2), 117.5
,CHvCH2), 102.6 ,C-1E, JC,Hˆ159 Hz), 98.6 ,C-1B, JC,H
p
77.7 ,C-4E ), 75.6, 75.0, 74.7 ,4C, OCH2), 74.4 ,C-5E), 73.6
ˆ
p
169 Hz), 84.8 ,C-3E), 82.0 ,C-2E), 80.9 ,C-3B), 80.0 ,C-4B ),
,OCH2), 69.9 ,C-2B), 68.8 ,C-6E), 67.4 ,OCH2), 52.6 ,C-5B),
and 18.0 ,C-6B). FABMS for C50H56O10 ,M, 816.39) m/z
839.4 ,[M1Na]1). Anal. Calcd for C50H56O10: C, 73.51;
H, 6.91%. Found: C, 73.41; H, 6.89%.
p
p
3.3.2. Allyl -2,3,4,6-tetra-O-benzyl-a-d-glucopyranosyl)-
-1!3)-2-O-benzoyl-4-O-benzyl-a-l-rhamnopyranoside
14. A solution of disaccharide 12 ,1.85 g, 2.27 mmol) in
anhydrous pyridine ,5 mL) containing benzoyl chloride
,390 mL, 3.44 mmol) was stirred overnight at 708C. TLC
,solvent D, 23:2) indicated that the reaction was essentially
quantitative. Methanol ,5 mL) was added to the cooled
mixture, and the solution was stirred at rt for a further
time of 1h. Volatiles were evaporated and the residue was
taken up in CH2Cl2. The organic phase was washed succes-
sively with 5% aq. HCl, water, and satd aq NaCl. Concen-
tration of the organic phase followed by chromatography of
the residue ,solvent B, 9:1) gave pure 14 ,1.86 g, 89%) as a
colorless oil, together with a slightly contaminated fraction
,180 mg, 8.6%) which could be used as such in the next step
,the estimated yield of pure 14 is 95%). Compound 14 had
,Ph), 92.5 ,C-1B ), 92.2 ,C-1E ), 82.0 ,C-3E), 79.8 ,C-4B),
79.0 ,C-2E), 77.6 ,C-4E), 76.1, 75.4, 74.9, 73.2, 72.2
,5C, OCH2), 72.0 ,C-3B), 70.2 ,C-5E), 68.8 ,C-2B), 68.3
,C-6E), 68.0 ,C-5B), and 18.2 ,C-6B); CIMS for C54H56O11
,M, 880.4) m/z 903.4 ,[M1Na]1). Anal. Calcd for
C54H56O11´0.5H2O: C, 72.88; H, 6.46%. Found: C, 72.82;
H, 6.60%.
3.3.4.
-2,3,4,6-Tetra-O-benzyl-a-d-glucopyranosyl)-
-1!3)-2-O-benzoyl-4-O-benzyl-a/b-l-rhamnopyranosyl
trichloroacetimidate 2. Trichloroacetonitrile ,1.2 mL,
12.0 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene ,DBU,
18 mL) were added to a solution of the hemiacetal 15
,1.06 g, 1.20 mmol) in anhydrous CH2Cl2 ,600 mL). The
solution was kept for 1h at rt, at which time TLC ,solvent
B containing Et3N 2½, 3:1) showed that the reaction was

2600
L. A. Mulard et al. / Tetrahedron 58 02002) 2593±2604
over. Volatiles were evaporated and the residue was ¯ash-
chromatographed ,solvent B containing Et3N 5½) to give 2
,1.17 g, 95%) as a sticky foam. Compound 2 was isolated as
a mixture of a and b anomers. NMR ,a anomer): 1H, d 8.71
,s, 1H, NH), 8.09±7.06 ,m, 30H, Ph), 6.35 ,d, 1H,
J1,2ˆ1.7 Hz, H-1B), 5.82 ,dd, 1H, J2,3ˆ2.5 Hz, H-2B), 5.20
,d, 1H, H-1E), 5.01±4.36 ,m, 10H, OCH2), 4.93 ,dd, 1H,
J3,4ˆ9.6 Hz, H-3B), 4.06±4.00 ,m, 3H, H-5B, 3E, 5E), 3.79
,pt, 1H, J4,5ˆ9.6 Hz, H-4B), 3.74 ,m, 1H, J3,4ˆ9.4 Hz,
H-4E), 3.66 ,dd, 1H, J5,6ˆ2.6 Hz, J6a,6bˆ10.9 Hz, H-6aE),
3.60 ,dd, 1H, J1,2ˆ3.3 Hz, J2,3ˆ9.6 Hz, H-2E), 3.48 ,d,
1H, H-6bE), and 1.45 ,d, 3H, J5,6ˆ6.2 Hz, H-6B); 13C, d
165.7 ,CvO), 160.2 ,CvNH), 138.6±127.4 ,Ph), 95.0
J2,3ˆ9.6 Hz, H-3D), 4.82 ,d, 1H, Jˆ10.6 Hz, OCH2), 4.75
,d, 1H, Jˆ10.7 Hz, OCH2), 4.71,bs, 1H, H-1 A), 4.61,d, 2H,
OCH2), 4.53 ,d, 1H, J1,2ˆ8.5 Hz, H-1D), 4.22 ,dd, 1H, J5,6a
ˆ
4.4 Hz, J6a,6bˆ12.2 Hz, H-6aD), 4.13 ,bd, 1H, H-6bD), 4.01
,bq, 1H, H-2D), 3.90 ,bs, 1H, H-2A), 3.86 ,bd, 1H, J3,4ˆ
9.6 Hz, H-3A), 3.63 ,m, 2H, H-5D, 5A), 3.37 ,pt, 1H,
J4,5ˆ9.7 Hz, H-4A), 3.33 ,s, 3H, OCH3), 2.08, 2.03, 2.00
,3s, 9H, C,O)CH3), 1.62 ,s, 3H, NC,O)CH3), and 1.32 ,d,
3H, J5,6ˆ5.9 Hz, H-6A); 13C, d 170.8, 170.7, 169.9, 169.3
,4C, CvO), 138.3±127.7 ,Ph), 103.1 ,C-1D), 99.8 ,C-1A),
80.9 ,C-4A), 80.1,C-3 A), 78.1,C-2 A), 75.5, 73.6 ,2C,
OCH2), 73.3 ,C-3D), 72.0 ,C-5D), 68.3 ,C-4D), 67.6
,C-5A), 62.0 ,C-6D), 54.6 ,OCH3), 54.1,C-2 ), 23.1, 20.8,
D
,C-1B), 92.9 ,C-1E), 90.8 ,CCl3), 82.0 ,C-3E), 79.1,C-4 ),
B
20.7, 20.6 ,4C, C,O)CH3), and 17.9 ,C-6A); ESMS for
C35H45NO13 ,M, 687.29) m/z 688.3 ,[M1H]1). Anal.
Calcd for C35H45NO13´H2O: C, 60.34; H, 6.51; N, 2.01%.
Found: C, 60.49; H, 6.71; N, 1.94%.
78.8 ,C-2E), 77.4 ,C-4E), 76.4, 75.5, 75.0, 73.4, ,4C, OCH2),
72.3 ,C-3B), 72.2 ,OCH2), 70.9 ,C-5B), 70.4 ,C-5E), 68.1
,C-6E), 66.9 ,C-2B), and 18.2 ,C-6B). Anal. Calcd for
C56H56Cl3NO11: C, 65.59; H, 5.50; N, 1.37%. Found: C,
65.49; H, 5.68; N, 1.28%.
3.3.6. Methyl -3,4,6-tri-O-acetyl-2-deoxy-2-N-tetrachloro-
phthalimido-b-d-glucopyranosyl)--1!2)-3,4-di-O-benzyl-
a-l-rhamnopyranoside 20. AgOTf ,16.0 g, 62.2 mmol)
was added in one portion to a solution of donor 7 ,28.1g,
44.2 mmol), alcohol 6 ,11.47 g, 32.0 mmol) and 2,6-di-tert-
butyl-4-methyl pyridine ,11.04 g, 53.8 mmol) in CH2Cl2
,600 mL) stirred at 2508C. Stirring was continued over-
night, at which time the bath temperature had reached
208C. TLC ,solvent D, 17:3) showed the complete dis-
appearance of 6. The mixture was ®ltered through a bed
of Celite. The ®ltrate was washed with a 1:1 mixture of
5% aq. NaHCO3 and 5% aq. Na2S2O3, then water and satd
aq. NaCl. The organic phase was dried and concentrated.
The crude product was chromatographed ,solvent D, 9:1) to
give pure disaccharide 20 ,12.17 g, 89%) as a crystalline
material. Compound 20 had, mp 154±1558C ,from
3.3.5. Methyl -3,4,6-tri-O-acetyl-2-chloroacetamido-2-
deoxy-b-d-glucopyranosyl)--1!2)-3,4-di-O-benzyl-a-l-
rhamnopyranoside 17 and methyl -2-acetamido-3,4,6-
tri-O-acetyl-2-deoxy-b-d-glucopyranosyl)--1!2)-3,4-di-
O-benzyl-a-l-rhamnopyranoside 18. Compound 1610
,4.86 g, 6.15 mmol) in a mixture of toluene ,175 mL) and
N,N-dimethylacetamide ,60 mL) was stirred for 20 min
under a ¯ow of dry Ar. Tributyltin hydride ,7.3 mL,
27.1mmol) and a,a0-azobisisobutyronitrile ,AIBN, 196
mg)) were added and the reaction mixture was heated at
1008C for 45 min under a ¯ow of Ar. TLC ,solvent C,
4:1; solvent B, 1:1) showed that no starting material
remained and that one more major polar product had been
formed. The reaction mixture was concentrated into an oily
residue which was triturated in petroleum ether to give a
white solid. Chromatography of the latter ,solvent C, 22:3)
gave the slightly contaminated chloroacetamide 17 ,2.06 g,
1
AcOEt); [a]Dˆ168 ,c 1.0); NMR: H, d 7.61±7.10 ,m,
10H, Ph), 5.97 ,dd, 1H, J2,3ˆ9.1Hz, J3,4ˆ10.5 Hz, H-3D),
5.28 ,d, 1H, J1,2ˆ8.4 Hz, H-1D), 5.17 ,dd, 1H, J4,5ˆ9.4 Hz,
H-4D), 4.68 ,bs, 1H, H-1A), 4.44 ,dd, 1H, H-2D), 4.40±4.21
1
41% yield from the H NMR spectrum) as the ®rst eluting
product and the target 18 as the second eluting product
,m, 5H, OCH2, H-6aD), 4.15 ,dd, 1H, J5,6ˆ2.2 Hz, J6a,6bˆ
1
,1.95 g, 30% from the H NMR spectrum).
12.1 Hz, H-6bD), 3.83 ,m, 1H, H-5D), 3.64 ,bs, 1H, H-2A),
3.61,dd, 1H, J2,3ˆ2.9 Hz, H-3A), 3.54 ,dq, 1H, J4,5ˆ9.4 Hz,
H-5A), 3.26 ,s, 3H, OCH3), 3.11 ,m, 1H, J3,4ˆ9.4 Hz, H-4A),
2.11, 2.04, 1.93 ,3s, 9H, C,O)CH3), and 1.25 ,d, 3H, J5,6ˆ
6.2 Hz, H-6A); 13C, d 170.7, 170.5, 169.5 ,CvO), 138.0±
127.2 ,Ph), 99.9 ,C-1D), 99.6 ,C-1A), 80.6 ,C-4A), 78.8 ,2C,
C-3A, 2A), 75.1, 72.4 ,2C, OCH2), 71.5 ,C-5D), 70.0 ,C-3D),
68.8 ,C-4D), 67.7 ,C-5A), 61.9 ,C-6D), 55.4 ,C-2D), 54.6
,OCH3), 20.8, 20.7 ,3C, C,O)CH3), and 17.8 ,C-6A).
FABMS for C41H41Cl4NO14 ,M, 913.57) m/z 936.2
,[M1Na]1). Anal. Calcd for C41H41Cl4NO14: C, 53.90; H,
4.52; N, 1.53%. Found: C, 53.75; H, 4.56; N, 1.46%.
An analytical sample of 17, isolated as a white solid, had
[a]Dˆ08 ,c 1.0); NMR: 1H, d 7.39±7.29 ,m, 10H, Ph), 6.55
,d, 1H, JNH,2ˆ8.2 Hz, NH), 5.06 ,dd, 1H, J2,3ˆ10.4 Hz,
J3,4ˆ9.4 Hz, H-3D), 5.05 ,t, 1H, J4,5ˆ9.7 Hz, H-4D), 4.87
,d, 1H, Jˆ10.8 Hz, OCH2), 4.72 ,d, 1H, OCH2), 4.71,d,
1H, H-1D), 4.69 ,bs, 1H, H-1A), 4.61,d, 1H, OCH ), 4.60 ,d,
2
1H, OCH2), 4.23 ,dd, 1H, J5,6aˆ4.7 Hz, J6a,6bˆ12.2 Hz,
H-6aD), 4.12 ,dd, 1H, J5,6bˆ2.6 Hz, H-6bD), 3.95±3.82 ,m,
3H, H-2D, 2A, 3A), 3.83 ,d, 1H, CH2Cl), 3.76 ,d, 1H,
Jˆ13.0 Hz, CH2Cl), 3.65 ,dq, 1H, H-5A), 3.59 ,m, 1H,
H-5D), 3.37 ,t, 1H, J3,4ˆJ4,5ˆ9.3 Hz, H-4A), 3.32 ,s, 3H,
OCH3), 2.08, 2.03, 2.02 ,3s, 9H, C,O)CH3), and 1.31 ,d,
3H, J5,6ˆ6.3 Hz, H-6A); 13C, d 170.6, 170.5, 169.4, 169.2
,4C, CvO), 138.4±127.7 ,Ph), 101.8 ,C-1D), 99.9 ,C-1A),
80.7 ,C-4A), 80.0 ,C-3A), 77.3 ,C-2A), 75.4, 73.3 ,2C,
OCH2), 72.1,C-3 D), 71.8 ,C-5D), 68.5 ,C-4D), 67.7
,C-5A), 62.0 ,C-6D), 55.1,C-2 D), 54.6 ,OCH3), 42.4
,CH2Cl), 20.8, 20.7, 20.6 ,3C, C,O)CH3), and 17.9 ,C-6A).
3.3.7. Methyl -2-acetamido-2-deoxy-b-d-glucopyranosyl)-
-1!2)-3,4-di-O-benzyl-a-l-rhamnopyranoside 21. ,a)
Ethylenediamine ,8.77 mL, 131 mmol) was added to a
suspension of the fully protected disaccharide 20 ,12.0 g,
13.1 mmol) in absolute ethanol ,300 mL), and the reaction
mixture was heated at 708C for 9 h. TLC ,solvent A, 9:1)
showed that no starting material remained and that two more
polar products had been formed. The reaction mixture was
cooled to 08C, acetic anhydride ,50 mL, 503 mmol) was
slowly added, and stirring went on for 2 h at rt. TLC ,solvent
A, 9:1) showed that the more polar compound had been
An analytical sample of 18 had [a]Dˆ238 ,c 1.0); NMR:
1H, d 7.42±7.35 ,m, 10H, Ph), 5.48 ,d, 1H, JNH,2ˆ8.6 Hz,
NH), 5.06 ,pt, 1H, J3,4ˆJ4,5ˆ9.6 Hz, H-4D), 4.97 ,pt, 1H,

L. A. Mulard et al. / Tetrahedron 58 02002) 2593±2604
2601
fully transformed into the less polar one. Volatiles were
eliminated by repeated coevaporations with cyclohexane.
The residue was re¯uxed in EtOAc and ®ltered on a pad
of Celite. More salts crystallized out of an EtOAc/MeOH
solution. Chromatography of the mother liquor ,solvent A,
residue was re¯uxed in EtOAc and ®ltered on a pad of
Celite. 2,2-Dimethoxypropane ,30 mL) and p-toluene-
sulfonic acid ,200 mg) was added to a suspension of the
resulting crude 21 in acetone ,20 mL), and the mixture
was stirred at rt overnight. TLC ,solvent A, 23:2) showed
that no starting material remained. Et3N was added, and
volatiles were evaporated. Chromatography of the residue
,solvent A, 99:1) gave monohydroxylated 22 as a white
foam ,6.88 g, 87%); [a]Dˆ2278 ,c 1.0); NMR: 1H, d
7.42±7.27 ,m, 10H, Ph), 6.98 ,d, 1H, J2,NHˆ2.0 Hz, NH),
4.84±4.64 ,m, 4H, OCH2), 4.68 ,bs, 1H, H-1A), 4.42 ,d, 1H,
J1,2ˆ8.3 Hz, H-1D), 3.96±3.91,m, 3H, H-2 A, 3A, 6aD), 3.80
,t, 1H, J6a,6bˆ10.4 Hz, H-6bD), 3.69 ,dq, 1H, J4,5ˆ9.4 Hz,
H-5A), 3.64±3.50 ,m, 3H, H-2D, 3D, 4D), 3.38 ,pt, 1H, J3,4ˆ
9.1Hz, H-4 A), 3.35 ,s, 3H, OCH3), 3.23 ,m, 1H, H-5D), 1.57
,s, 3H, C,O)CH3), 1.54, 1.47 ,2s, 6H, C,CH3)2), and 1.34 ,d,
3H, J5,6ˆ6.2 Hz, H-6A); 13C, d 173.6 ,CvO), 138.0±127.5
,Ph), 103.5 ,C-1D), 99.9 ,C,CH3)2), 99.7 ,C-1A), 81.4
9:1) gave the triol 21 ,6.11 g, 83%); [a]Dˆ2138 ,c 1.0);
1
NMR: H, d 7.42±7.27 ,m, 10H, Ph), 7.05 ,d, 1H, J2,NH
2.7 Hz, NH), 4.84±4.65 ,m, 4H, OCH2), 4.73 ,d, 1H,
J1,2ˆ1.0 Hz, H-1A), 4.42 ,d, 1H, J1,2ˆ8.1Hz, H-1 D),
3.96±3.86 ,m, 3H, H-2A, 3A, 6aD), 3.82 ,m, 1H, H-6bD),
ˆ
3.70 ,dq, 1H, J4,5ˆ9.4 Hz, H-5A), 3.60±3.51,m, 2H, H-2 ,
D
5D), 3.43±3.34 ,m, 6H, H-4A, 3D, 4D, OCH3), 2.50 ,t, 1H,
JOH,6ˆ6.2 Hz, OH-6D), 1.58 ,s, 3H, C,O)CH3), and 1.34 ,d,
3H, J5,6ˆ6.2 Hz, H-6A); 13C, d 173.5 ,CvO), 138.0±127.4
p
,Ph), 103.2 ,C-1D), 99.7 ,C-1A), 81.2 ,C-4A), 80.4 ,C-3A ),
p
78.8 ,C-2A), 77.1,C-3 p), 75.6 ,OCH2), 75.4 ,C-4D ), 74.4
D
,OCH2), 71.4 ,C-5D), 67.4 ,C-5A), 62.3 ,C-6D), 58.5 ,C-2D),
54.6 ,OCH3), 22.3 ,C,O)CH3), and 17.7 ,C-6A). FABMS for
C29H39NO10 ,M, 561.26) m/z 584.3 [,M1 Na]1). Anal. Calcd
for C29H39NO10´1.5H2O: C, 59.17; H, 7.19; N, 2.38%. Found:
C, 58.95; H, 7.08; N, 2.34%.
p
p
,C-4A), 80.6 ,C-3A ), 78.8 ,C-2A ), 75.7, 74.7 ,2C, OCH2),
74.4 ,C-3D ), 74.0 ,C-4D ), 67.6 ,C-5D), 67.5 ,C-5A), 61.9
p
p
,C-6D), 60.1,C-2 D), 54.6 ,OCH3), 29.1,C,CH ) ), 22.4
3 2
,C,O)CH3), 19.0 ,C,CH3)2), and 17.8 ,C-6A); ESMS for
C32H43NO10 ,M, 601.29) m/z 602.4 ,[M1H]1). Anal.
Calcd for C32H43NO10: C, 63.88; H, 7.20; N, 2.33%.
Found: C, 63.72; H, 7.35; N, 2.28%.
,b) 1N sodium methoxide ,15 mL, 15 mmol) was added to a
solution of the fully protected disaccharide 16 ,273 mg,
345 mmol) in CH2Cl2 ,1mL), and the reaction mixture
was stirred at rt for 17 h. TLC ,solvent A, 9:1) showed
that no starting material remained and that two more polar
products had been formed. No further evolution was
observed if stirring was pursued. Resin IR 120 ,H1) was
added until the pH was 7±8. The resin was ®ltered and
washed with methanol. Concentration of the ®ltrate, and
chromatography of the residue ,solvent A, 9:1) gave methyl
,2-amino-2-deoxy-b-d-glucopyranosyl)-,1!2)-3,4-di-O-
benzyl-a-l-rhamnopyranoside ,19, 162 mg), which was
dissolved in methanol ,7 mL). Acetic anhydride ,700 mL,
7.4 mmol) was slowly added, and stirring went on for 1.25 h
at rt. TLC ,solvent A, 9:1) showed that the aminotriol inter-
mediate had been fully transformed into 21. Volatiles were
eliminated by repeated coevaporations with cyclohexane
and toluene. Chromatography of the residue ,solvent A,
19:1) gave the triol 21 ,107 mg, 59%).
3.3.9. Methyl -4-O-benzyl-2,3-di-O-acetyl-a-l-rhamno-
pyranosyl)--1!3)--2-acetamido-2-deoxy-4,6-O-isopropyl-
idene-b-d-glucopyranosyl)--1!2)-3,4-di-O-benzyl-a-l-
rhamnopyranoside 23. TMSOTf ,190 mL, 100 mmol) was
added to a solution of disaccharide 22 ,689 mg, 1.14 mmol)
and donor 8 ,880 mg, 1.62 mmol) in Et2O ,10 mL) at
2788C. The mixture was stirred overnight while the bath
temperature slowly came back to rt. TLC ,solvent C, 4:1)
showed that no acceptor remained, Et3N was added and
volatiles were evaporated. Chromatography of the residue
,solvent D, 22:3) provided the fully protected trisaccharide
23 ,971mg) as a slightly contaminated white foam.
1
Available data for 23 are NMR: H, d 7.37±7.27 ,m, 15H,
Ph), 5.86 ,d, 1H, JNH,2ˆ7.5 Hz, NH), 5.27 ,dd, 1H, J2,3ˆ
3.3 Hz, J3,4ˆ9.8 Hz, H-3C), 5.10 ,dd, 1H, J1,2ˆ1.6 Hz,
H-2C), 4.87 ,d, 1H, Jˆ10.8 Hz, OCH2), 4.85 ,d, 1H, J1,2ˆ
8.4 Hz, H-1D), 4.73±4.59 ,m, 7H, H-1A, 1C, OCH2), 4.11
,dq, 1H, J4,5ˆ9.7 Hz, H-5C), 4.03 ,t, 1H, J2,3ˆJ3,4ˆ9.5 Hz,
H-3D), 3.92±3.73 ,m, 4H, H-2A, 3A, 6aD, 6bD), 3.63 ,dq, 1H,
J4,5ˆ9.4 Hz, H-5A), 3.59 ,pt, 1H, J4,5ˆ9.4 Hz, H-4D), 3.52±
3.42 ,m, 2H, H-4C, 2D), 3.39 ,pt, 1H, H-4A), 3.29 ,m, 4H,
H-5D, OCH3), 2.12, 1.97, 1.78 ,3s, 9H, C,O)CH3), 1.48, 1.40
,2s, 6H, C,CH3)2), 1.33 ,d, 3H, J5,6ˆ6.2 Hz, H-6A), and 1.28
,d, 3H, J5,6ˆ6.2 Hz, H-6C); 13C, d 171.3, 170.1, 170.0 ,3C,
1
Compound 19 had NMR ,CD3OD): H, d 7.40±7.27 ,m,
10H, Ph), 4.87 ,d, 1H, OCH2), 4.85 ,d, 1H, J1,2ˆ1.7 Hz,
H-1A), 4.74 ,d, 1H, Jˆ11.6 Hz, OCH2), 4.66 ,d, 1H, Jˆ
11.0 Hz, OCH2), 4.63 ,d, 1H, OCH2), 4.49 ,d, 1H, J1,2ˆ
8.0 Hz, H-1D), 4.12 ,dd, 1H, H-2A), 3.86 ,bd, 1H, J6a,6b
ˆ
11.4 Hz, H-6aD), 3.82 ,dd, 1H, J2,3ˆ3.2 Hz, H-3A), 3.67 ,m,
1H, H-6bD), 3.61,dq, 1H, H-5 A), 3.51,pt, 1H, J3,4ˆ J4,5ˆ
9.4 Hz, H-4A), 3.35±3.26 ,m, 6H, H-3D, 4D, 5D, OCH3), 2.63
,m, 1H, H-2D), and 1.27 ,d, 3H, J5,6ˆ6.1Hz, H-6 A); 13C, d
139.8±128.6 ,Ph), 105.6 ,C-1D), 101.5 ,C-1A), 81.3 ,C-4A),
80.5 ,C-3A), 78.1,C-4 D), 77.7 ,C-2A), 77.2 ,C-3D), 76.1,
p
C,O)), 138.4±127.5 ,Ph), 102.0 ,C-1D), 100.1 ,C-1A ), 99.5
p
,C,CH3)2), 97.9 ,C-1C ), 80.8 ,C-4A), 79.5 ,C-3A), 78.8
,C-4C), 77.3 ,C-3D), 77.2 ,C-2A), 75.4, 74.7, 72.8 ,3C,
OCH2), 72.7 ,C-4D), 71.3 ,C-3C), 71.0 ,C-2C), 67.6 ,2C,
C-5A, 5C), 67.3 ,C-5D), 62.2 ,C-6D), 58.0 ,C-2D), 54.5
,OCH3), 29.2 ,CCH3), 23.4, 21.5, 20.9 ,3C, C,O)CH3),
19.3 ,CCH3), and 17.9 ,2C, C-6A, 6C); CIMS for
C49H63NO16 ,M, 921.41) m/z 922.0 ,[M1H]2).
73.1,2C, OCH ), 71.5 ,C-5D), 68.8 ,C-5A), 62.5 ,C-6D),
2
58.7 ,C-2D), 55.2 ,OCH3), and 18.2 ,C-6A); ESMS for
C27H37NO9 ,M, 519.60) m/z 520.3 ,[M1H]2).
3.3.8. Methyl -2-acetamido-2-deoxy-4,6-O-isopropyli-
dene-b-d-glucopyranosyl)--1!2)-3,4-di-O-benzyl-a-l-
rhamnopyranoside 22. A suspension of the fully protected
disaccharide 20 ,12.0 g, 13.1 mmol) in absolute ethanol
,300 mL) was treated with ethylenediamine ,8.77 mL,
131 mmol) as described for the preparation of 21. The
3.3.10. Methyl -4-O-benzyl-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-
pyranoside 24. A mixture of disaccharide 22 ,1.45 g,

2602
L. A. Mulard et al. / Tetrahedron 58 02002) 2593±2604
mmol) and donor12 8 ,1.80 g, 2.41 mmol) in anhydrous Et2O
was treated with TMSOTf as described above for the
preparation of 23. The crude mixture was roughly puri®ed
by chromatography yielding contaminated 23 ,2.19 g). 1N
methanolic sodium methoxide was added dropwise to a
solution of the latter ,2.04 g) in methanol ,20 mL) until
pH 10 was reached. The mixture was stirred at rt for 7 h
when TLC ,solvent C, 4:1) showed that no starting material
remained. Neutralization with resin IR 120 ,H1) followed
by column chromatography ,solvent A, 49:1) of the crude
material gave diol 24 ,1.53 g, 81%) as a white foam;
3.73 ,C-3C), 67.7 ,C-5D), 67.6 ,C-5A), 67.4 ,C-5C), 62.2 ,C-
6D), 56.9 ,C-2D), 54.5 ,OCH3), 29.1,CCH ), 23.2, 21.1
,C,O)CH3), 19.1 ,CCH3), 17.9 ,C-6C), and 17.8 ,C-6A).
3
Crude 3 ,990 mg, 1.07 mmol) and donor 2 ,1.54 g,
1.5 mmol) were dissolved in anhydrous Et2O ,10 mL) and
the solution was cooled to 2788C. TMSOTf ,20 mL,
100 mmol) was added, and the reaction mixture was stirred
overnight while the bath temperature was slowly coming
back to rt. TLC ,solvent A, 97:3) showed that only little
acceptor remained, Et3N was added and volatiles were
evaporated. Chromatography ,solvent A, 12:1) provided
the fully protected pentasaccharide 25 ,1.28 g, 67%) as a
white foam; [a]Dˆ1248 ,c 1.0); NMR: 1H, d 8.13±7.05 ,m,
45H, Ph), 5.70 ,bs, 1H, H-2B), 5.60 ,d, 1H, JNH,2ˆ7.7 Hz,
NH), 5.21,d, 1H, J1,2ˆ3.5 Hz, H-1E), 5.20 ,bs, 1H, H-1B),
4.99 ,bd, 1H, J1,2ˆ1.7 Hz, H-2C), 4.98±4.36 ,m, 15H,
OCH2), 4.82 ,d, overlapped, 1H, H-1D), 4.69 ,bs, 1H,
H-1C), 4.66 ,bs, 1H, H-1A), 4.34 ,dd, 1H, J2,3ˆ3.0 Hz,
J1,2ˆ9.5 Hz, H-3B), 4.24 ,d, 1H, Jˆ12.1 Hz, OCH2), 4.13
,dd, 1H, J2,3ˆ3.2 Hz, J3,4ˆ9.5 Hz, H-3C), 4.06±3.85 ,m,
8H, H-3E, 5E, 5C, 3D, 6aD, 2A, 5B, 3A), 3.75±3.41,m, 10H,
H-6bD, 4E, 4B, 5A, 6aE, 2E, 4D, 6bE, 4C, 2D), 3.38 ,pt, 1H,
J3,4ˆJ4,5ˆ9.3 Hz, H-4A), 3.30 ,s, 3H, OCH3), 3.25 ,m, 1H,
H-5D), 2.09, 1.82 ,2s, 6H, C,O)CH3), 1.46 ,s, 3H, C,CH3)2),
1.41 ,d, 3H, H-6B), 1.40 ,s, 3H, C,CH3)2), 1.32 ,d, 3H,
J5,6ˆ6.2 Hz, H-6A), and 1.20 ,d, 3H, J5,6ˆ6.2 Hz, H-6C);
13C, d 171.1, 170.3, 165.8 ,3C, CvO), 138.7±127.3 ,Ph),
102.2 ,C-1D, JC,Hˆ161 Hz), 100.1 ,C-1A, JC,Hˆ170 Hz),
99.4 ,C,CH3)2), 99.3 ,C-1B, JC,Hˆ172 Hz), 97.8 ,C-1C,
JC,Hˆ170 Hz), 92.3 ,C-1E, JC,Hˆ169 Hz), 81.9 ,C-3E),
1
[a]Dˆ2288 ,c 1.0); NMR: H, d 7.36±7.21,m, 15H, Ph),
6.29 ,bd, 1H, JNH,2ˆ6.6 Hz, NH), 4.86 ,d, 2H, Jˆ11.8 Hz,
OCH2), 4.75 ,d, 1H, J1,2ˆ1.0 Hz, H-1A), 4.71,bs, 1H, H-1 C),
4.70±4.56 ,m, 5H, H-1D, OCH2), 4.07 ,dq, 1H, J4,5ˆ9.6 Hz,
H-5C), 3.97±3.71,m, 7H, H-2 C, 3D, 3C, 2A, 3A, 6aD, 6bD), 3.70
,m, 1H, H-2D), 3.66 ,m, 1H, H-5A), 3.58 ,dd, 1H, H-4D), 3.38
,m, 2H, H-4A, 4C), 3.32 ,s, 3H, OCH3), 3.27 ,m, 2H, H-5D,
OH), 2.92 ,d, 1H, Jˆ4.7 Hz, OH), 2.06 ,s, 3H, C,O)CH3),
1.39, 1.38 ,2s, 6H, C,CH3)2), 1.31 ,d, 3H, J5,6ˆ6.2 Hz, H-6A),
and 1.27 ,d, 3H, J5,6ˆ6.2 Hz, H-6C); 13C, d 170.9 ,CvO),
139.1±127.3 ,Ph), 103.1 ,C-1D), 100.5 ,C-1C), 99.7 ,C-1A),
99.4 ,C,CH3)2), 80.9 ,C-4A), 80.6 ,C-4C), 79.4 ,C-3A), 77.9
,C-2A), 75.9 ,C-3D), 75.4, 75.6, 73.1,3C, OCH 2), 72.3
,C-4D), 72.0 ,C-3C), 71.1 ,C-2C), 67.9 ,C-5A), 67.5 ,C-5D),
67.4 ,C-5C), 61.8 ,C-6D), 57.5 ,C-2D), 54.7 ,OCH3), 29.6
,CCH3), 23.1,NHAc), 18.9 ,CCH 3), and 17.8, 17.6 ,2C,
C-6C, 6A); CIMS for C45H59NO14 ,M, 837.39) m/z838.4
,[M1H]2). Anal. Calcd for C45H59NO14: C, 64.50; H, 7.1 0;
N, 1.67%. Found: C, 64.50; H, 7.17; N, 1.61%.
p
3.3.11. Methyl -2,3,4,6-tetra-O-benzyl-a-d-glucopyrano-
syl)--1!3)--2-O-benzoyl-4-O-benzyl-a-l-rhamnopyrano-
syl)--1!3)--2-O-acetyl-4-O-benzyl-a-l-rhamnopyrano-
syl)--1!3)--2-acetamido-2-deoxy-4,6-O-isopropylidene-
b-d-glucopyranosyl)--1!2)-3,4-di-O-benzyl-a-l-rhamno-
pyranoside 25. p-Toluenesulphonic acid ,10 mg) was
added to a suspension of diol 24 ,950 mg, 1.07 mmol) in
MeCN ,8 mL) containing methyl orthoacetate ,550 mL,
4.3 mmol). The mixture was stirred for 45 min at rt. TLC
,solvent A, 19:1) showed that no starting material remained.
The reaction mixture was cooled to 08C, and 80% aq. AcOH
,1.5 mL) was added. After 30 min at rt, TLC ,solvent A,
19:1) showed that the intermediate orthoester had turned
into a more polar product. CH2Cl2 was added, and the
organic phase was washed with ice-water and satd aq.
NaCl, dried and evaporated to dryness to give 3 quantita-
80.8 ,C-4A), 79.8 ,C-4C), 79.6 ,C-4E ), 79.5 ,C-3A), 79.0
p
,C-2E), 77.9 ,C-3C), 77.8 ,C-3D), 77.5 ,C-4B ), 77.3
,C-2A), 76.2, 75.4, 75.3, 74.9, 73.1, 72.8 ,7C, OCH2), 72.7
,C-2C), 72.5 ,C-4D), 72.2 ,C-3B), 72.1,OCH 2), 70.1,C-5 E),
68.9 ,C-5B), 68.4 ,C-2B), 68.3 ,C-6E), 67.6 ,C-5A ), 67.5
p
p
,C-5C ), 67.4 ,C-5D), 62.2 ,C-6D), 57.8 ,C-2D), 54.5
,OCH3), 29.0 ,CCH3), 23.2, 21.0 ,C,O)CH3), 19.0
,CCH3), 17.8 ,C-6B), 17.7 ,C-6A), and 17.6 ,C-6C).
FABMS for C101H115NO25 ,M, 1741.78) m/z 1764.8
,[M1Na]1). Anal. Calcd for C101H115NO25: C, 69.60; H,
6.65; N, 0.80%. Found: C, 69.48; H, 6.81; N, 0.96%.
3.3.12. Methyl -2,3,4,6-tetra-O-benzyl-a-d-glucopyrano-
syl)--1!3)--2-O-benzoyl-4-O-benzyl-a-l-rhamnopyrano-
syl)--1!3)--2-O-acetyl-4-O-benzyl-a-l-rhamnopyranosyl)-
-1!3)--2-acetamido-2-deoxy-b-d-glucopyranosyl)--1!2)-
3,4-di-O-benzyl-a-l-rhamnopyranoside 26. 50% aq. TFA
,12 mL) was added, at 08C, to a solution of pentasaccharide
25 ,1.19 g, 683 mmol) in CH2Cl2 ,20 mL), and the biphasic
mixture was stirred vigorously at rt for 1h. Repeated
coevaporation with toluene and chromatography of the
residue ,solvent A, 193:7) provided diol 26 ,1.09 g, 94%)
as a white foam; [a]Dˆ1298 ,c 1.0); NMR: 1H, d 8.00±7.06
1
tively, as a white foam; NMR: H, d 7.40±7.27 ,m, 15H,
Ph), 5.74 ,bd, 1H, JNH,2ˆ7.7 Hz, NH), 4.90 ,dd, 1H, J1,2ˆ
1.5 Hz, J2,3ˆ3.3 Hz, H-2C), 4.86 ,d, 2H, Jˆ11.3 Hz, OCH2),
4.74±4.57 ,m, 5H, H-1D, OCH2), 4.65 ,bs, 2H, H-1A, 1C),
4.08 ,dd, 1H, J3,4ˆ9.5 Hz, H-3C), 3.99 ,dq, 1H, J4,5ˆ9.6 Hz,
H-5C), 3.92±3.84 ,m, 4H, H-2A, 3A, 6aD, 6bD), 3.70 ,m, 1H,
H-2D), 3.65 ,m, 1H, H-5A), 3.61,pt, 1H, J2,3ˆJ3,4ˆ8.7 Hz,
H-3D), 3.55 ,dd, 1H, J4,5ˆ8.9 Hz, H-4D), 3.36 ,pt, 1H,
H-4A), 3.35 ,pt, 1H, J3,4ˆJ4,5ˆ9.5 Hz, H-4C), 3.30 ,s, 3H,
OCH3), 3.22 ,m, 1H, H-5D), 2.13, 1.83 ,2s, 6H, C,O)CH3),
1.47, 1.41 ,2s, 6H, C,CH3)2), 1.32 ,d, 3H, J5,6ˆ6.2 Hz,
H-6A), and 1.27 ,d, 3H, J5,6ˆ6.2 Hz, H-6C); 13C, d 171.6,
170.6 ,2C, CvO), 138.7±127.6 ,Ph), 102.8 ,C-1D), 99.9
,m, 45H, Ph), 5.69 ,bs, 1H, H-2B), 5.62 ,d, 1H, JNH,2
ˆ
7.3 Hz, NH), 5.20 ,bs, 2H, H-1B, 1E), 5.00 ,bs, 1H, H-2C),
4.97±4.34 ,m, 18H, OCH2, H-1D, 1A, 1C), 4.31,dd, 1H,
J2,3ˆ2.9 Hz, J1,2ˆ9.4 Hz, H-3B), 4.22 ,d, 1H, Jˆ12.0 Hz,
OCH2), 4.15 ,dd, 1H, J2,3ˆ3.2 Hz, J3,4ˆ9.4 Hz, H-3C),
4.01,m, 2H, H-3 , 5E), 3.94±3.73 ,m, 6H, H-5C, 2A, 6aD,
E
p
p
,C-1A ), 99.5 ,C,CH3)2), 98.3 ,C-1C ), 81.3 ,C-4C), 80.9
,C-4A), 79.7 ,C-3A), 79.2 ,C-3D), 77.6 ,C-2A), 75.4, 74.8
,2C, OCH2), 73.6 ,C-2C), 73.2 ,OCH2), 72.4 ,C-4D), 69.5
3A, 5B, 6bD), 3.67 ,dd, 2H, H-4E, 4B), 3.59±3.40 ,m, 8H,
H-5A, 6aE, 3D, 2E, 2D, 4C, 6bE, 4D), 3.37 ,pt, 1H, J3,4ˆ
J4,5ˆ9.4 Hz, H-4A), 3.30 ,s, 3H, OCH3), 3.28 ,m, 1H,

L. A. Mulard et al. / Tetrahedron 58 02002) 2593±2604
2603
H-5D), 2.10 ,s, 3H, OC,O)CH3), 2.07 ,m, 1H, OH-6D), 1.78
,s, 3H, NC,O)CH3), 1.36 ,d, 3H, H-6B), 1.31 ,d, 3H,
J5,6ˆ6.2 Hz, H-6A), and 1.27 ,d, 3H, J5,6ˆ6.2 Hz, H-6C);
13C, d 170.5, 170.2, 165.8 ,3C, CvO), 138.7±127.4 ,Ph),
pentasaccharide 1 ,284 mg, 67%) as a white powder;
1
[a]Dˆ08 ,c 1.0, water). The H and 13C NMR data are
reported in Tables 1±3. FABMS for C33H57NO23 ,M,
835.3) m/z 858.4 ,[M1Na]1). Anal. Calcd for
C33H57NO23±3H2O: C, 44.54; H, 7.14; N, 1.57%. Found:
C, 44.45; H, 7.03; N, 1.58%.
p
p
102.0 ,C-1D), 100.0 ,C-1A ), 99.6 ,C-1C ), 99.4 ,C-1B), 92.4
,C-1E), 86.2 ,C-3D), 81.9 ,C-3E), 80.8 ,C-4A), 79.8 ,C-3A),
p
p
79.5 ,2C, C-4B , 4C), 79.0 ,C-2E), 77.5 ,C-4E ), 77.2 ,C-2A),
76.9 ,C-3C), 76.0, 75.6, 75.5 ,3C, OCH2), 75.4 ,2C, OCH2,
C-5D), 74.9, 73.2, 72.9 ,3C, OCH2), 72.2 ,2C, OCH2, C-3B),
72.1,C-2 C), 70.3 ,C-4D), 70.1,C-5 E), 69.1,C-5 C), 69.0
,C-5B), 68.3 ,C-6E), 68.2 ,C-2B), 67.6 ,C-5A), 62.6 ,C-6D),
55.6 ,C-2D), 54.6 ,OCH3), 23.3, 21.1 ,2C, C,O)CH3), and
18.0, 17.9 ,3C, C-6B, 6A, 6C); CIMS for C98H111NO25 ,M,
1701.74) m/z 1719.8 ,[M1NH4]1). Anal. Calcd for
Acknowledgements
Â
È
We are grateful to Joel Ughetto-Monfrin ,Unite de Chimie
Organique, Institut Pasteur) for recording all the 300 MHz
NMR spectra. We thank the MENRT ,Program Recherche
Fondamentale en Microbiologie et Maladies Infectieuses et
Parasitaires), the DGA ,contract 99 34 029) and CNRS
,program PCV) for supporting this work ®nancially.
.
C98H111NO25 H2O: C, 68.40; H, 6.62; N, 0.81%. Found: C,
68.20; H, 6.67; N, 1.15%.
3.3.13. Methyl -2,3,4,6-tetra-O-benzyl-a-d-glucopyrano-
syl)--1!3)--4-O-benzyl-a-l-rhamnopyranosyl)--1!3)-
-4-O-benzyl-a-l-rhamnopyranosyl)--1!3)--2-acetamido-
2-deoxy-b-d-glucopyranosyl)--1!2)-3,4-di-O-benzyl-a-
l-rhamnopyranoside 27. 1N methanolic sodium methox-
ide was added dropwise to a solution of diol 26 ,1.09 g,
640 mmol) in methanol ,5 mL) until pH 10 was reached.
The mixture was stirred at rt overnight, and neutralized
with resin IR 120 ,H1). The crude material was puri®ed
by chromatography ,solvent A, 49:1) to give the tetraol 27
,963 mg, 97%) as a white foam; [a]Dˆ1208 ,c 1.0); NMR:
1H, d 7.36±7.08 ,m, 40H, Ph), 5.62 ,d, 1H, JNH,2ˆ7.0 Hz,
NH), 5.13 ,bs, 1H, H-1B), 4.96±4.19 ,m, 16H, OCH2), 4.85
,d, overlapped, 1H, H-1E), 4.68 ,m, overlapped, 3H, H-1D,
1A, 1C), 4.07±3.62 ,m, 14H, H-3E, 3C, 3B, 5C, 5E, 2A, 2B, 2C,
5B, 3A, 6aD, 6bD, 4E, 5A), 3.60±3.29 ,m, 13H, H-2D, 2E, 4B,
4D, 3D, 6aE, 6bE, 4C, 4A, 5D, OCH3), 2.50 ,d, 1H, Jˆ2.4 Hz,
OH), 2.12 ,m, 3H, OH), 1.72 ,s, 3H, C,O)CH3), 1.39 ,d, 3H,
J5,6ˆ6.1Hz, H-6 B), 1.33 ,d, 3H, H-6A), and 1.30 ,d, 3H,
H-6C); 13C, d 170.3 ,CvO), 138.4±127.5 ,Ph), 102.3
References
1. Part 6 of the series Synthesis of ligands related to the
O-speci®c polysaccharides of S. ¯exneri serotypes 2a and
5a. For part 5 see: Costachel, C.; Sansonetti, P. J.; Mulard,
L. A. J. Carbohydr. Chem. 2000, 19, 1131±1150.
2. Lindberg, A. A. Vaccine 1999, 17, S28±S36.
3. Taylor, D. N.; Trofa, A. C.; Sadoff, J.; Chu, C.; Bryla, D.;
Shiloach, J.; Cohen, D.; Ashkenazi, S.; Lerman, Y.; Egan,
W.; Schneerson, R.; Robbins, J. B. Infect. Immun. 1993, 61,
3678±3687.
4. Passwell, J. H.; Harlev, E.; Ashkenazi, S.; Chu, C.; Miron, D.;
Ramon, R.; Farzan, N.; Shiloach, J.; Bryla, D. A.; Majadly, F.;
Roberson, R.; Robbins, J. B.; Schneerson, R. Infect. Immun.
2001, 69, 1351±1357.
5. Phalipon, A.; Folgori, A.; Arondel, J.; Sgamarella, G.;
Fortugno, P.; Cortese, R.; Sansonetti, P. J.; Felici, F. Eur. J.
Immunol. 1997, 27, 2620±2625.
p
p
,C-1D), 101.5 ,C-1A ), 100.3 ,C-1B), 100.0 ,C-1C ), 94.2
,C-1E), 86.3 ,C-3D), 82.3 ,C-3E), 80.9 ,C-4A), 79.9 ,C-3A),
79.7 ,C-4C), 79.1,C-4 B), 78.8 ,C-2E), 77.9 ,C-3C), 77.7
,C-4E), 77.4 ,C-2A), 76.7 ,C-3B), 75.7, 75.6 ,2C, OCH2),
75.5 ,2C, OCH2, C-5D), 75.0, 74.5, 74.4, 73.4, 73.1,5C,
OCH2), 70.8 ,C-5E), 70.5 ,C-2C), 70.4 ,C-4D), 69.3 ,C-5C),
68.0 ,C-5B), 67.9 ,C-6E), 67.6 ,2C, C-5A, 2B), 62.8 ,C-6D),
55.4 ,C-2D), 54.6 ,OCH3), 23.4 ,C,O)CH3), and 18.2, 18.0
,3C, C-6A, 6B, 6C). FABMS for C89H105NO23 ,M, 1555.71)
m/z 1578.5 ,[M1Na]1). Anal. Calcd for C89H105NO23: C,
68.66; H, 6.80; N, 0.90%. Found: C, 68.51; H, 6.90; N,
0.93%.
6. Simmons, D. A. R. Bacteriol. Rev. 1971, 35, 117±148.
7. Lindberg, A. A.; Karnell, A.; Weintraub, A. Rev. Infect. Dis.
1991, 13, S279±S284.
8. Kochetkov, N. K.; Byramova, N. E.; Tsvetkov, Y. E.;
Backinovsky, L. V. Tetrahedron 1985, 41, 3363±3375.
9. Bakinovskii, L. V.; Gomtsyan, A. R.; Bairamova, N. E.;
Kochetkov, N. K.; Yankina, N. F. Bioorg. Khim. 1985, 11,
1562±1571.
10. Mulard, L. A.; Ughetto-Monfrin, J. J. Carbohydr. Chem.
1999, 18, 721±753.
11. Mulard, L. A.; Ughetto-Monfrin, J. J. Carbohydr. Chem.
2000, 19, 193±220.
12. Mulard, L. A.; Ughetto-Monfrin, J. J. Carbohydr. Chem.
2000, 19, 503±526.
3.3.14. Methyl a-d-glucopyranosyl--1!3)-a-l-rhamno-
pyranosyl--1!3)-a-l-rhamnopyranosyl--1!3)--2-aceta-
mido-2-deoxy-b-d-glucopyranosyl)--1!2)-a-l-rhamno-
pyranoside 1. The benzylated pentasaccharide 27 ,411 mg,
264 mmol) was dissolved in a 9:1methanol/acetic acid
mixture ,20 mL), treated with 10% Pd±C catalyst
,200 mg), and the suspension was stirred at rt for 4 days,
under a hydrogen atmosphere. At this time, TLC ,solvent E,
7:1:2) showed that the starting material had been trans-
formed into a more major polar product. The suspension
was ®ltered on a bed of Celite, and the ®ltrate was concen-
trated. Reverse phase chromatography ,solvent F, gradient)
of the residue, followed by lyophilization, gave the target
13. Schmidt, R. R.; Michel, J. Tetrahedron Lett. 1984, 25, 821±
824.
14. Schmidt, R. R.; Michel, J.; Roos, M. Liebigs Ann. Chem. 1984,
1343±1357.
15. Gigg, R.; Payne, S.; Conant, R. J. Carbohydr. Chem. 1983, 2,
207±223.
16. Pinto, B. M.; Morissette, D. G.; Bundle, D. R. J. Chem. Soc.,
Perkin Trans. 1 1987, 9±14.
17. Pozsgay, V.; Brisson, J.-R.; Jennings, H. J. Can. J. Chem.
1987, 65, 2764±2769.
18. Maddali, U. B.; Ray, A. K.; Roy, N. Carbohydr. Res. 1990,
208, 59±66.

2604
L. A. Mulard et al. / Tetrahedron 58 02002) 2593±2604
19. Debenham, J. S.; Madsen, R.; Roberts, C.; Fraser-Reid, B.
J. Am. Chem. Soc. 1995, 117, 3302±3303.
31. Pozsgay, V.; Jennings, H. J. J. Org. Chem. 1988, 53, 4042±
4052.
32. Jones, C. Adv. Carbohydr. Anal. 1991, 1, 145±194.
33. Willer, W.; Leibfritz, D.; Kerssebaum, R.; Bermel, W. Magn.
Reson. Chem. 1993, 31, 287±292.
20. Pinto, B. M.; Reimer, K. B.; Tixidre, A. Carbohydr. Res. 1991,
210, 199±219.
21. Zhang, K. Q.; Li, S. C.; Mao, J. M.; Chen, H. M.; Cai, M. S.
Chem. J. Chin. Univ. 1997, 18, 1469±1473.
34. Delay, C.; Gavin, J. A.; Aumelas, A.; Bonnet, P. A.;
Roumestand, C. Carbohydr. Res. 1997, 302, 67±78.
35. Desvaux, H.; Berthault, P.; Birlirakis, N.; Goldman, M.
J. Magn. Reson. 1994, 108, 219±229.
22. Oltvoort, J. J.; van Boeckel, C. A. A.; der Koning, J. H.; van
Boom, J. Synthesis 1981, 305±308.
23. Gigg, R.; Warren, C. D. J. Chem. Soc. C 1968, 1903.
24. Blatter, G.; Beau, J.-M.; Jacquinet, J.-C. Carbohydr. Res.
1994, 260, 189±202.
36. Bock, K.; Pedersen, C. J. Chem. Soc., Perkin Trans. 2 1974,
293±297.
37. Jansson, P. E.; Kenne, L.; Wehler, T. Carbohydr. Res. 1987,
166, 271±282.
25. Toth, A.; Medgyes, A.; Bajza, I.; Liptak, A.; Batta, G.;
Â
Kontrohr, T.; Peterffy, K.; Pozsgay, V. Bioorg. Med. Chem.
Lett. 2000, 10, 19±21.
38. Jansson, P. E.; Kenne, L.; Wehler, T. Carbohydr. Res. 1988,
179, 359±368.
26. Castro-Palomino, J. C.; Schmidt, R. R. Tetrahedron Lett.
1995, 36, 5343±5346.
39. Shashkov, A. S.; Vinogradov, E. V.; Knirel, Y. A.; Nifant'ev,
N. E.; Kochetkov, N. K.; Dabrowski, J.; Kholodkova, E. V.;
Stanislavsky, E. S. Carbohydr. Res. 1993, 241, 177±188.
40. Pozsgay, V.; Coxon, B. Carbohydr. Res. 1994, 257, 189±215.
41. Nifant'ev, N. E.; Lipkind, G. M.; Shashkov, A. S.; Kochetkov,
N. K. Carbohydr. Res. 1992, 223, 109±128.
42. States, D. J.; Haberkorn, R. A.; Ruben, D. J. J. Magn. Reson.
1982, 48, 286±292.
27. Garegg, P. J.; Norberg, T. Acta Chem. Scand. Ser. B 1979, 33,
116±118.
28. Bundle, D. R.; Josephson, S. Can. J. Chem. 1979, 57, 662±
668.
29. Rance, M.; Sorensen, O. W.; Bodenhausen, G.; Wagner, G.;
Ernst, R. R.; WuÈthrich, K. Biochem. Biophys. Res. Commun.
1983, 117, 479±485.
43. Baleja, J.; Moult, J.; Sykes, B. D. J. Magn. Reson. 1990, 87,
375±384.
44. Homans, S. W. Biochemistry 1990, 29, 9110±9118.
30. Griesinger, C.; Otting, G.; WuÈtrich, K.; Ernst, R. R. J. Am.
Chem. Soc. 1988, 110, 7870±7872.