Synthesis of conformationally locked L-iduronic acid derivatives: Direct evidence for a critical role of the skew-boat 2S0 conformer in the activation of antithrombin by heparin

Article abstract of DOI:10.1002/1521-3765(20011119)7:22<4821::AID-CHEM4821>3.0.CO;2-N

We have used organic synthesis to understand the role of L-iduronic acid conformational flexibility in the activation of antithrombin by heparin. Among known synthetic analogues of the genuine pentasaccharidic sequence representing the antithrombin bindin

Full text of DOI:10.1002/1521-3765(20011119)7:22<4821::AID-CHEM4821>3.0.CO;2-N

FULL PAPER
Synthesis of Conformationally Locked l-Iduronic Acid Derivatives:
2
Direct Evidence for a Critical Role of the Skew-Boat S0 Conformer in the
Activation of Antithrombin by Heparin
Sanjoy K. Das,[a] Jean-Maurice Mallet,[a] Jacques Esnault,[a] Pierre-Alexandre Driguez,[b]
Philippe Duchaussoy,[b] Philippe Sizun,[c] Jean-Pascal Herault,[b] Jean-Marc Herbert,[b]
Maurice Petitou,*[a, b] and Pierre Sinay*[a]
È
Abstract: We have used organic syn-
thesis to understand the role of l-
iduronic acid conformational flexibility
in the activation of antithrombin by
heparin. Among known synthetic ana-
logues of the genuine pentasaccharidic
sequence representing the antithrombin
binding site of heparin, we have selected
as a reference compound the methylated
anti-factor Xa pentasaccharide 1. As in
the genuine original fragment, the single
l-iduronic acid moiety of this molecule
exists in water solution as an equilibrium
between three conformers C4, C1 and
2S0. We have thus synthesized three
analogues of 1, in which the l-iduronic
acid unit is locked in one of these three
fixed conformations. A covalent two
atom bridge between carbon atoms two
and five of l-iduronic acid was first
iduronic acid moiety around the 1C4
chair form conformation, and the C4-
1
introduced to lock the pseudorotational
itinerary of the pyranoid ring around the
2S0 form. A key compound to achieve
this connection was the d-glucose deriv-
ative 5 in which the H-5 hydrogen atom
has been replaced by a vinyl group,
which is a progenitor of the carboxylic
acid. Selective manipulations of this
type pentasaccharide 43 was synthe-
sized. Finally the l-iduronic acid con-
taining disaccharide 58 which, due to the
presence of the methoxymethyl substitu-
ent at position five adopts a 4C1 con-
formation, was directly used to synthe-
size the 4C1-type pentasaccharide 61.
The locked pentasaccharide 23 showed
about the same activity as the reference
compound 1 in an antithrombin-medi-
ated anti-Xa assay, whereas the two
pentasaccharides 43 and 61 displayed
very low activity. These results clearly
establish the critical importance of the
2S0 conformation of l-iduronic acid in
the activation of antithrombin by hep-
arin.
2
molecule resulted in the S0-type penta-
saccharide 23. Starting from the d-glu-
cose derivative 28, a covalent two atom
bridge was now built up between carbon
atoms three and five to lock the l-
1
4
Keywords: antithrombin ´ carbohy-
drates ´ glycosylation ´ heparin ´
iduronic acid
complex glycosaminoglycans (GAGs): heparin, heparan sul-
fate, and dermatan sulfate. These remarkable polymers are
endowed with a fascinating array of biological functions[2]
which are still largely unexplained at the molecular level. It is
thus tempting to speculate that the presence of flexible l-
iduronic acid in GAGs boosts their biological responses.
The conformational behavior of the iduronate ring, obvi-
ously of intrinsic importance, has been the matter of a long
controversy[3] that has stimulated numerous conflicting stud-
ies. On the one hand, from the first NMR investigations, it has
been concluded that l-iduronic residues, whether sulfated, as
in heparin,[4] or unsulfated, as in dermatan sulfate,[5] adopt the
1C4 conformation. On the other hand, diffraction analysis of
crystalline fibers,[6] and periodate oxidation studies[7] of
Introduction
Conformational flexibility is a distinct feature of l-iduronic
acid,[1] a characteristic monosaccharide component of three
[a] Dr. M. Petitou, Prof. P. SinayÈ, Dr. S. K. Das,
Dr. J.-M. Mallet, Dr. J. Esnault
Â
Ecole Normale Superieure
Â
Â
Departement de Chimie Associe au CNRS
24, rue Lhomond; 75231 Paris cedex 05 (France)
Fax : (‡33) 1 44 32 33 90
E-mail: maurice.petitou@sanofi-synthelabo.com
pierre.sinay@ens.fr
[b] Dr. M. Petitou, Dr. P.-A. Driguez, Dr. P. Duchaussoy,
Dr. J.-P. Herault, Dr. J.-M. Herbert
Â
Departement Cardiovasculaire/Thrombose
4
Â
Sanofi-Synthelabo
dermatan sulfate, suggested the presence of the C1 confor-
195, route dꢀEspagne, 31036 Toulouse cedex (France)
Fax : (‡33) 5 61 16 22 86
mation. It has been finally suggested that several conformers,
coexisting in solution, could account for these opposing
experimental observations.[8] A further critical step was
accomplished after the chemical synthesis of the pentasac-
charide representing the antithrombin binding site of hep-
[c] Dr. P. Sizun
Â
DARA; Sanofi-Synthelabo
371 rue du Professeur Joseph Blayac
34184 Montpellier cedex (France)
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M. Petitou, P. SinayÈ et al.
FULL PAPER
arin,[9] which contains a single (but critical[10]) l-iduronic acid
residue. Indeed 1H NMR data on this synthetic compound was
best explained by taking into account the participation of the
very unusual 2S0 skew-boat conformer in addition to the above
locked in 1C4, 4C1, or 2S0 conformations.[18] The results,
presented here, about the ability of these compounds to
activate antithrombin with respect to blood coagulation
factor Xa inhibition, allow us to conclude that antithrombin-
bound l-iduronic acid adopts the 2S0 conformation and that a
conformational change from C4 to S0 is not required during
the activation process. Such an approach should be useful to
explore the role of l-iduronic acid conformation in other
biological systems where this monosaccharide is involved.
4
1
reported C1 and C4 to the conformational equilibrium of l-
iduronic acid.[11] Force-field studies and energy computation
further showed that the three conformers are almost equi-
1
2
2
energetic.[12] As a matter of fact, the S0 conformer is the
major contributor to the conformational equilibrium of l-
iduronic in the above pentasaccharide,[13] and numerous
studies have since considered the presence of the three above
conformers to describe this monosaccharide in glycosamino-
glycans and related compounds.[14]
Results and Discussion
The known[19] synthetic anti-factor Xa pentasaccharide 1
(Figure 1) was chosen as the reference compound. It strongly
binds to antithrombin, and the presence in the final structure
of methyl groups in place of hydroxyl groups, and of O-
sulfonates in place of N-sulfonates, simplifies the synthetic
route compared with that of the genuine heparin pentasac-
charidic sequence.
The fact that the above synthetic pentasaccharide displays
high affinity for antithrombin and activates its inhibitory
action against blood coagulation factor Xa, was for us an
incentive to investigate to what extent the unique conforma-
tional properties of l-iduronic acid translate in terms of
biological properties. Indeed, concerning the present penta-
saccharide and antithrombin, one could imagine that a precise
conformation was required, either in the early recognition
step, or to lock the protein in the active conformation reached
after induced-fit.[15] Alternatively, a conformational switch
from one conformer to another one might be necessary to
accomplish the transconformation known to occur during the
activation of the protein.
Synthesis of pentasaccharide 23 (2S0 conformer, Figure 1): A
literature search taught us that methyl 2,6-anhydro-3,4-di-O-
2
methyl-a-d-mannopyranoside prefers to exist as its S0 con-
Abstract in French: Nous avons employØ les mØthodes de la
To address these issues, we previously synthesized a
pentasaccharide containing a 3-deoxy-l-iduronic acid unit.[16]
As expected, the conformational equilibrium was shifted
Ã
synth›se organique afin de comprendre le role de la flexibilitØ
conformationnelle de lꢀacide l-iduronique dans lꢀactivation de
lꢀantithrombine par lꢀhØparine. Parmi les analogues synthØti-
ques connus de la sØquence pentasaccharidique dꢀorigine
reprØsentant le site de liaison de lꢀhØparine à lꢀantithrombine,
nous avons choisi le pentasaccharide mØthylØ anti-Xa 1 comme
composØ de rØfØrence. Tout comme dans le cas du site actif de
lꢀhØparine, la seule unitØ acide l-iduronique de cette molØcule
existe en solution aqueuse sous la forme dꢀun Øquilibre
1
towards C4. This compound displayed reduced affinity for
1
antithrombin, thus disqualifying this C4 conformer. In con-
1
trast, based on H NMR studies at various ionic strengths,
others proposed that the 1C4 conformation was adopted by the
pentasaccharide sequence when bound to antithrombin.[17]
To provide more direct evidence, we embarked on the
synthesis of pentasaccharides containing l-iduronic acid units
1
conformationnel C4 > 4C1 > 2S0. Nous avons donc synthØtisØ
trois analogues de cette molØcule de rØfØrence, dans lesquels
lꢀunitØ acide l-iduronique est verrouillØe sous une de ces trois
conformations. Lꢀintroduction dꢀun pont covalent à deux
atomes reliant les atomes de carbone deux et cinq de lꢀacide
l-iduronique fige lꢀitinØraire pseudorotationnel du cycle py-
Editorial Board Member:[‡] Pierre
Sinay: was born in 1938 in France
and graduated from Ecole Natio-
2
Â
nale Superieure des Industries
rannique autour de la forme S0. Un tel pont a ØtØ rØalisØ à
Chimiques, Nancy in 1961. In 1966
he received his PhD degree (with
Serge David). After postdoctoral
study with Roger Jeanloz at Havard
University, Cambridge (USA), he
became in 1969 an associate Profes-
sor then in 1972 a full Professor, at
partir de 5, un dØrivØ clØ du d-glucose, dans lequel lꢀatome
dꢀhydrog›ne H-5 a ØtØ remplacØ par un groupe vinyle,
prØcurseur de lꢀacide carboxylique. Des manipulations sØlecti-
ves de cette molØcule nous ont conduit au pentasaccharide 23
2
du type S0. En partant du dØrivØ 28 du d-glucose, un pont
covalent à deux atomes a maintenant ØtØ construit afin de figer
1
lꢀunitØ acide l-iduronique sous la conformation chaise C4, et
le pentasaccharide 43 du type 1C4 a ØtØ synthØtisØ. Finalement,
le disaccharide 58, dans lequel lꢀunitØ acide l-iduronique
adopte une conformation 4C1, a ØtØ directement utilisØ pour la
Â
Â
Â
the Universite dꢀOrleans, Orleans
(France). In 1986, he moved to Paris
Â
as a full Professor at Universite Pierre et Marie Curie, heading
4
a research group in the Chemistry Department at Ecole
Normale Superieure. Professor Sinay has been the co-author
of more than 250 papers. His studies are dealing with the
organic chemistry of carbohydrates.
synth›se du pentasacccharide 61 du type C1. Le pentasac-
Â
charide 23 prØsente une activitØ anti-Xa voisine de celle du
composØ de rØfØrence 1, alors que les deux pentasaccharides 43
et 61 sont pratiquement inactifs. Ces rØsultats montrent
clairement que la conformation 2S0 de lꢀacide l-iduronique
est critique pour lꢀactivation de lꢀantithrombine par lꢀhØparine.
‡
[ ] Members of the Editorial Board will be introduced to readers with their
first manuscript.
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l-Iduronic Acid Derivatives
4821±4834
raphy. Acid hydrolysis using
‡
IR-120 H resin at 808C, fol-
lowed by acetylation with acetic
anhydride in pyridine, gave the
tetracetate 5. The exclusive for-
mation of the b-anomer is as-
signed to the destabilizing 1,3-
diaxial interaction between O-1
and the C-5 vinyl group in the
a-anomer. The absolute config-
uration at C-5 in 5 was estab-
lished at this stage through
ROESY NMR experiments.
Thus, the observed NOE effects
between H-3 and the isolated
vinylic proton on the one hand,
and between H-4 and H-6a/H-
6b on the other hand confirmed
the assigned stereochemistry
(see Scheme 1). The large cou-
pling constants observed for the
ring protons (J1,2 ˆ 8.4, J2,3 ˆ 9.6,
J3,4 ˆ 10.1 Hz) clearly show that
this pyranosidic compound
adopts the 4C1 conformation,
and that only the 1,2-trans iso-
mer was formed.
The b-acetate 5 was ideally
suited for selective 1,2-trans
glycosylation of the known[24]
alcohol 6. Indeed they reacted
together to yield the disacchar-
ide 7 in 85% yield. As antici-
pated from the presence of a
participating group at position
2 of the glycosyl donor, only the
Figure 1. The known[19] biologically active pentasaccharide 1 was selected as the reference compound. In the
synthetic mimetic 23, the l-iduronate ring has been locked in the S0 conformation by connecting O-2 and C-5
2
b-d-anomer
7 was obtained
through a methylene bridge. A similar connection between O-3 and C-5 leads to an iduronate ring locked in the
1C4 conformation (pentasaccharide 43). Compound 56 was synthesized to demonstrate that introducing an ethyl
at C-5 of d-glucuronic acid does not affect the biological activity. The two disaccharide building blocks required
for the synthesis of the pentasaccharide 61 derive from the same intermediate. In 61, the iduronate ring adopts the
4C1 conformation. The letter code DEFGH is routinely used to designate the five monosaccharide units of the
antithrombin binding sequence in heparin.
(J1',2' ˆ 8.2 Hz). The disacchar-
ide 7 was finally deacetylated to
yield the triol 8.
The second part of the syn-
thesis consisted in the forma-
tion of the O-2/C-5 bridge. The
former,[20, 21] and it is on these grounds that we decided to
introduce a two-atom bridge between C-2 and C-5 of the l-
iduronic acid residue in 1 to freeze the l-iduronate ring in the
2S0 conformation.
To this end, we first had to replace the hydrogen atom at
C-5 of a gluco derivative by a substituent that should then be
converted into a carboxylate group. It was expected that the
reaction of vinyl magnesium bromide with the known[22]
5-keto-glucofuranose 2, controlled by chelation of the mag-
nesium by the ring oxygen atom, would lead to such a
derivative with high stereoselectivity.[23]
Thus a solution in tetrahydrofuran of the crude ketone 2,
resulting from Swern oxidation of 6-O-tert-butyldimethylsilyl-
1,2-O-isopropylidene-3-O-methyl-a-d-glucofuranose[22] was
treated with vinyl magnesium bromide to give alcohol 3 (see
Scheme 1), isolated in 70% yield after column chromatog-
most obvious option was to temporary protect the 4',6'-diol
system in 8, introduce a leaving group at position 2' (mesylate
10), and finally displace this group by attack by the O-6'
alcoholate. This approach has already met with success,[25] but
in our case led to frustation. The only isolated product was the
triol 8 resulting from the cleavage of the mesylate, a side
product already observed by others.[25] The monosaccharide
derivative 12 (Scheme 2), characterized by its NMR[26, 27] and
mass spectra, was also isolated in very small amounts. It
probably results from a Grob fragmentation of the diol 11.
Others[28] have also experienced difficulties in a similar
situation (displacement of a mesylate at position 2 of a
monosaccharide by a thiolate at position 6).
The second option (Scheme 3), starting from 9, was to first
invert the configuration at C-2', then to introduce a leaving
group at C-5' that can be next displaced by nucleophilic attack
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M. Petitou, P. SinayÈ et al.
FULL PAPER
was selectively introduced at
C-6' to give the tosylate 17.
The shorter route, that is direct
selective tosylation of the man-
no-triol (i.e., 16 where R ˆ H)
was impracticable due to the
formation of
a
significant
amount of the 2',6'-di-O-tosy-
lated compound. Various clas-
sical conditions were first inves-
tigated (sodium hydride, potas-
sium tert-butoxide either in
dimethylformamide or dime-
thylsulfoxide) to achieve the
intramolecular displacement of
the 6'-tosylate. The best result
was, however, obtained using
sodium hydroxide in ethanol.[30]
‡
Scheme 1. a) CH2CHMgBr, THF, 08C, 1 h, 70%; b) IR-120 H resin, H2O, 80 8C, 6 h; c) Ac2O, pyridine, RT, 16 h,
75% (two steps); d) 6, TMSOTf, CH2Cl2, 788C, 2 h, 85%; e) MeONa, MeOH, 08C then RT, 3 h;
f) (CH3O)2C(CH3)2, p-TsOH, acetone, RT, 16 h, 70% (two steps).
After heating at 708C for 3 h,
compound 18 could be isolated
in 70% yield after column chromatography. Ozonolysis,
followed by oxidation by sodium chlorite, and finally benzy-
lation by benzyl bromide in the presence of potassium
hydrogen carbonate, gave the disaccharide 19, ready for
addition of the remaining part of the pentasaccharide
molecule.
The trisaccharide imidate 20[31] reacted with the alcohol 19
to give the pentasaccharide 21 (67%, Scheme 4). The
observed coupling constant for H-1'' (J1'',2'' ˆ 3.6 Hz) clearly
proved the a-anomeric configuration of the newly established
interglycosidic bond. Pentasaccharide 21 was then submitted
to catalytic hydrogenation followed by saponification, and
1H NMR analysis was used at this stage to check the complete
removal of all protective groups in alcohol 22. The hydroxyl
groups thus liberated were sulfated in dimethylformamide at
558C using triethylamine/sulfur trioxide complex. The struc-
ture and purity of the final pentasaccharide 23, isolated by gel
Scheme 2. Basic treatment of the mesylate 11 gave the triol 8 and a small
amount of 12.
by O-2'. In this strategy it must be noted that the configuration
inverting step could have been omitted starting from a
compound having the d-manno instead of the d-gluco
configuration. However, in this case the synthesis of the b-
d-mannoside, a classical challenge in carbohydrate chemis-
try,[29] might have been problematic or at least less straightfor-
ward. Swern oxidation of 9 followed by reduction of the crude
ketone 13 by lithium triethylborohydride uneventfully gave
the expected manno disaccharide 14 (70%). The latter was
temporarily acetylated at position 2', then the isopropylidene
group was hydrolysed to give compound 16 and a tosyl group
1
permeation chromatography, were ascertained by H NMR
analysis and mass spectrometry.
1H NMR spectroscopy was also used to investigate the
conformation of the l-iduronate ring in pentasaccharide 23.
Comparison of the coupling
constants (J1,2 ˆ 1.3, J2,3 ˆ 1.4,
4
J3,4 ˆ 2.7, J2,4 ˆ 0.5 Hz) with
those reported by Köll et al.
for methyl 2,6-anhydro-d-
mannopyranoside derivatives,
which give
a
2S0 conforma-
tion[32] (J1,2 ˆ 1.2 ± 1.4, J2,3
ˆ
4
1.2 ± 2.4, J3,4 ˆ 3.0 ± 3.4, J2,4
ˆ
0.7 Hz), confirm that our deriv-
ative adopts the same confor-
mation. In another approach,
using the methodology report-
[33]
Â
ed by Perez et al., from the
same observed coupling con-
stants we could compute dihe-
dral angles (O5-C1-C2-C3
Scheme 3. a) (COCl)2, DMSO, CH2Cl2, 788C, 45 min; b) LiEt3BH, THF, 788C then RT, 1 h, 70%, two steps;
c) Ac2O, pyridine, RT, 3 h; d) AcOH, 608C, 2 h, 70%, two steps; e) TsCl, pyridine, RT, 3 h, 80%; f) NaOH,
EtOH, 708C, 3 h, 70%; g) O3, CH2Cl2, 788C, Me2S; then 2-methyl-2-butene, tBuOH, H2O, NaH2PO4, NaClO2,
RT, 16 h; then BnBr, Bu4NI, KHCO3, DMF, RT, 5 h, 80% three steps.
55.68, C1-C2-C3-C4
62.28,
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l-Iduronic Acid Derivatives
4821±4834
that led from 18 to 19. Finally,
coupling of the trisaccharide
imidate 20 onto the alcohol 40
proceeded in 83% yield to give
the pentasaccharide 41. The a
anomeric configuration of the
new interglycosidic bond was
proven by 1H NMR spectrosco-
py (J1'',2'' ˆ 3.0 Hz). Finally de-
protection and sulfation as pre-
viously described gave the de-
sired pentasaccharide 43.
1H NMR study demonstrates
that the locked iduronic acid
ring stands in the 1C4 conforma-
tion. The coupling constants
observed (J1,2 ˆ 0, J2,3 ˆ 3.7,
Scheme 4. a) 19, TBDMS-OTf, CH2Cl2, Et2O, 208C, 30 min, 67%; b) H2, Pd/C, AcOH, 408C, 12 h; then
NaOH, H2O, 55 8C, 3 h, 86% (two steps); c) Et3N ´ SO3, DMF, 558C, 18.5 h, 85%.
C2-C3-C4-C5 7.38) and compare them with angles computed
for a true S0 conformer (O5-C1-C2-C3 37.98, C1-C2-C3-C4
J3,4 ˆ 5.5 Hz) are very close to the expected ones (J1,2 ꢀ1.8 ±
2.0, J2,3 ꢀ2.6, J3,4 ꢀ2.6 ± 3.0 Hz), and they are in excellent
agreement with those already reported[18] for another deriv-
2
638, C2-C3-C4-C5 21.78). The good agreement also proves
that in the pentasaccharide 23
the locked l-iduronate ring
adopts a conformation close to
2S0.
Synthesis of pentasaccharide 43
(1C4 conformer, Figure 1): We
also wished to obtain a true
analogue of pentasaccharide 1
where the l-iduronic acid unit
would give the 1C4 conforma-
tion.[34] The sequence of reac-
tions that led to disaccharide 9
was repeated (Scheme 5) from
compound 25, obtained by sily-
lation of the known 3-O-allyl-
1,2-O-isopropylidene-a-d-glu-
cofuranose (24),[35] to give di-
saccharide 31. The alcohol 31
was methylated to give the di-
saccharide 32 and the allyl pro-
tecting group was selectively
removed following the classical
isomerisation ± hydrolysis path-
way. The resulting disaccharide
33 was acetylated; acid hydrol-
ysis of the isopropylidene group
gave the 4,6-diol 35, which was
selectively tosylated at position
6' to furnish the intermediate
36, ready for ring closure. The
latter was achieved using the
Scheme 5. a) TBDMSCl, imidazole, CH2Cl2, RT, 4 h, 95%; b) (COCl)2, DMSO, CH2Cl2, 788C, 1 h; then
CH2CHMgBr, THF, 08C, 1 h, 89% two steps; c) IR-120 H resin, H2O, 80 8C, 8 h; d) Ac2O, pyridine, RT, 16 h,
conditions previously described
for the transformation 17 ! 18
to give the locked 37 in excel-
lent yield (85%). It was then
converted into the desired l-
iduronic acid derivative 40 us-
ing similar conditions as those
‡
52%, two steps; e) 6, TMSOTf, CH2Cl2, 788C to RT, 3 h, 78%; f) MeONa, MeOH, 08C then RT, 6 h;
g) (CH3O)2C(CH3)2, p-TsOH, acetone, RT, 16 h, 74% (two steps); h) MeI, NaH, DMF, 08C then RT, 4 h, 92%;
i) tBuOK, DMSO, 808C, 1 h; then HgO, HgCl2, acetone, H2O, RT, 1 h, 65%; j) Ac2O, pyridine, CH2Cl2, RT, 3 h;
k) AcOH, H2O, 70 8C, 1 h, 71% two steps; l) TsCl, CH2Cl2, RT, 6 h, 75%; m) NaOH, EtOH, 708C, 2 h, 85%;
n) O3, CH2Cl2, 788C, Me2S; o) 2-methyl-2-butene, tBuOH, H2O, NaH2PO4, NaClO2, RT, 5 h; p) BnBr, Bu4NI,
KHCO3, DMF, RT, 5 h, 73% three steps; q) 20, TMSOTf, CH2Cl2, 208C, 1 h, 83%; r) H2, Pd/C, AcOH, 508C,
12 h; then NaOH, H2O, MeOH, RT, 12 h, 88% (two steps); s) Et3N:SO3, DMF, 558C, 20 h, 94%.
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M. Petitou, P. SinayÈ et al.
FULL PAPER
ative of l-iduronic acid similarly locked in the 1C4 conforma-
was selectively oxidized at C-6 using TEMPO/hypochlorite[36]
to give the corresponding glucuronic acid, isolated as its
benzyl ester 47. Glycosylation of the alcohol 47 with the
thioethyl glycoside 48,[37] in the presence of N-iodosuccini-
mide and triflic acid,[38] delivered a 5:1 mixture of the a- and b-
linked trisaccharides in 87% yield, easily separated by column
chromatography. The trisaccharide 49 was then submitted to a
series of classical transformations.[39]Acetolysis by a mixture
acetic anhydride/acetic acid/sulfuric acid led to replacement
of the anomeric methoxy group as well as the benzyl ethers at
positions 3, 6, and 6'' by acetyl groups. Trisaccharide 51 was
then obtained after selective removal of the anomeric acetate
using hydrazine acetate in N,N-dimethylformamide. Reaction
of 51 with trichloroacetonitrile in the presence of DBU[40]
gave the imidate 52 which was immediately engaged in the
glycosylation of the alcohol 53 to selectively provide the
pentasaccharide 54. The a anomeric configuration of the new
glycosidic bond was confirmed by 1H NMR spectroscopy
(J1'',2'' ˆ 3.6 Hz). Hydrogenation of the benzyl groups was
followed by saponification of the esters to give the inter-
mediate polyol 55, and finally sulfation gave the desired
pentasaccharide 56.
tion (J1,2 ˆ 0, J3,4 ˆ 4.0 Hz). The long range couplings (4J1,3
ˆ
4
0.5; J2,4 ˆ 0.5 Hz) confirm the chair conformation.
Converging synthesis of biologically active pentasaccharides:
It is worth noting that the synthetic monosaccharide units that
contain a tertiary carbon at position 5 are remarkably
versatile intermediates that can be oxidized into uronic acid
derivatives having either the d-gluco or the l-ido configu-
rations. The former are obtained after oxidation of the
primary alcohol function, while the second derive from
ozonolysis of the double bond followed by oxidation of the
resulting aldehyde (see previously described preparations of
compounds 19 and 40). Since the biologically active penta-
saccharides we are dealing with in this work contain both
types of uronic acids, it was interesting to design a converging
synthesis of these compounds where the versatility mentioned
above is exploited. There is, however, a prerequisite: that the
ethyl group present at C-5 of glucuronic acid does not impair
the interaction between the pentasaccharide and antithrom-
bin. To test this we decided to synthesize (Scheme 6) the
pentasaccharide 56, prepared from the fully protected inter-
mediate 54. The EF part of 54 (see Figure 1 for the code
DEFGH) can be obtained from the disaccharide 9 which was
methylated at 2' to give compound 44. Acid hydrolysis
followed by selective hydrogenation of the double bond in
the presence of PtO2 in ethyl acetate yielded the diol 46 which
Synthesis of pentasaccharide 61 (4C1 conformer, Figure 1):
Finally, considering that the non-constrained monosacchar-
ides units bearing a hydrocarbon chain instead of a hydrogen
4
atom at C-5 adopted the C1 conformation, we decided, on
these premises, to explore the
conformation of an iduronic
acid moiety bearing a methox-
ymethyl substituent at C-5, with
a view to synthesize a pentasac-
charide in which the iduronate
4
ring would adopt the C1 con-
formation. The synthesis of
such a derivative from 45 was
straightforward
(Scheme 7).
Thus, selective methylation at
position 6' of the disaccharide
was first carried out with meth-
yl iodide, using the stannyli-
dene procedure[41] (65%).
Then, ozonolysis of the double
bond, followed by oxidation of
the aldehyde and finally ester-
ification, gave disaccharide 58
(77% from 57). The pentasac-
charide 59 was prepared by
condensation of 58 with the
imidate 52. 1H NMR analysis
proved the a anomeric config-
uration of the new glycosidic
bond (J1'',2'' ˆ 3.8 Hz). Hydroge-
nation followed by saponifica-
tion gave in 87% yield the
polyol 60, which was finally
sulfated to provide 61 in 90%
Scheme 6. a) MeI, NaH, THF, 08C then RT, 1 h, 92%; b) AcOH, H2O, 70 8C, 3 h, 75% (two steps); c) H2, PtO2,
AcOEt, RT, 10 min, 100%; d) NaCl, NaHCO3, NaOCl, H2O, TEMPO, CH2Cl2, KBr, Bu4NCl, NaHCO3, 08C,
30 min; then BnBr, Bu4NCl, NaHCO3, RT, 45 min, 75% (two steps); e) 48, NIS, TfOH, CH2Cl2, Et2O, 408C,
30 min, 72%; f) H2SO4, AcOH, Ac2O, 0 8C, 2 h, 73%; g) NH2NH2 ´ HOAc, DMF, RT, 1 h, 90%; h) DBU, CCl3CN,
CH2Cl2, RT, 30 min, 87%; i) 53, TMSOTf, CH2Cl2, 208C, 30 min, 76%; j) H2, Pd/C, AcOH, 508C, 12 h; then
NaOH, H2O, MeOH, RT, 12 h, 87% (two steps); k) Et3N ´ SO3, DMF, 558C, 20.5 h, 90%.
1
yield. H NMR investigation of
the uronic acid unit G in 61
4826
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0947-6539/01/0722-4826 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 22

l-Iduronic Acid Derivatives
4821±4834
conformation is not the active
2
one, and that either the S0 is
essential or that the conforma-
tional flexibility of l-iduronic
1
2
acid (switch from C4 to S0) is
required during antithrombin
activation. The present data
rule out the second hypothesis,
and unambiguously establish
that l-iduronic acid adopts the
2S0 conformation in antithrom-
bin bound synthetic pentasac-
charides. It is also highly prob-
able that the single l-iduronic
acid unit contained in the an-
tithrombin binding site of hep-
arin itself also adopts this con-
formation when heparin binds
to the protein.
Scheme 7. a) Bu2SnO, toluene, reflux; then MeI, DMF, 508C, 6 h, 65%; b) O3, CH2Cl2, 788C, Me2S; then
2-methyl-2-butene, tBuOH, H2O, NaH2PO4, NaClO2, RT, 16 h; then BnBr, Bu4NI, KHCO3, DMF, RT, 6 h, 77%
(three steps); c) 52, TMSOTf, CH2Cl2, 208C, 0.5 h, 71%; d) H2, Pd/C, AcOH, 508C, 12 h; then NaOH, H2O,
MeOH, RT, 12 h, 87% (two steps); e) Et3N ´ SO3, DMF, 558C, 20.5 h, 90%.
clearly demonstrated that it adopts a conformation very close
to C1 (J1,2 ˆ 7.6, J2,3 ˆ 8.5, J3,4 ˆ 9.5 Hz).
The synthesis of such conformationally locked monosac-
charide units should prove to be very useful in the study of
other biological effects where carbohydrate conformation
might play a predominant role.
4
l-Iduronic acid conformation and interaction with antithrom-
bin: The ultimate goal of the present synthetic work was to
investigate the role of l-iduronic acid conformation in the
interaction of these heparin mimetics with antithrombin. The
biological activities of the four pentasaccharides described
here (23, 43, 56, and 61) were determined and compared to the
activity of the reference compound 1. The results (Table 1)
Experimental Section
General procedures: All compounds were homogeneous by TLC analysis
and had spectral properties consistent with their assigned structures.
Melting points were determined in capillary tubes in a Mettler or a Büchi
510 apparatus, and are uncorrected. Optical rotations were measured with a
Perkin ± Elmer Model 241 digital polarimeter at 22 Æ 38C. Compound
purity was checked by TLC on aluminum supported silica gel 60 F254 (E.
Merck) with detection by charring with a 5% ethanolic sulfuric acid
solution. Unless otherwise stated, column chromatography were performed
on silica gel 60, 40 ± 63 (flash) or 63 ± 200 mm (E. Merck). 1H NMR spectra
were recorded with Bruker AC200, AM250, AC300, AM400 or AM500
instruments. Before analysis in D2O, samples were passed through a Chelex
(Bio-Rad) ion exchange column. Chemical shifts are relative to external
TMS (CDCl3) or to external TSP (D2O). MS analyses were performed on a
Nermag R 10-10 or a ZAB-2E instrument (Fisons). Elemental analyses
Table 1. Biological activity of the pentasaccharides discussed in the
present study. Values are mean anti-factor Xa (n ˆ 3).
Compound
1
anti Xa [Umg
]
1
23
43
56
61
1208 Æ 63
1073 Æ 61
43 Æ 3
1345 Æ 65
115 Æ 3
Â
were performed by Service dꢀAnalyse de Universite Pierre et Marie Curie
or using a Fisons elemental analyzer.
Human factor Xa (71 nkat per vial), antithrombin, and S-2222 substrate
(Bz-Ile-Glu-Gly-Arg-pNA) were from Chromogenix (Mölndal, Sweden).
The anti-factor Xa activity was determined, in buffer, by an amidolytic
method adapted from Teien and Lie.[42] For an accurate comparison,
compound concentrations were determined by 1H NMR with reference to
an internal standard.
indicate that the the pentasaccharide 23 containing an
iduronic acid moiety in the S0 conformation is able to bind
2
to antithrombin, and thereby to strongly reinforce its ability to
inhibit the blood coagulation proteinase factor Xa. In con-
trast, pentasaccharides 43 and 61 which contain the unit G
locked in the 1C4 and 4C1 conformation, respectively, only very
slightly potentiate this inhibition. The high activity found for
pentasaccharide 56 shows that the dramatically decreased
activity of pentasaccharide 61 only results from a conforma-
tional feature of unit G, and not from converging introduction
of an ethyl group on residue E. These results are in agreement
with previous work showing that, in antithrombin binding
pentasaccharides, a shift in the conformational equilibrium
6-O-tert-Butyldimethylsilyl-1,2-O-isopropylidene-3-O-methyl-5-C-vinyl-
a-d-glucofuranose (3): A solution of oxalyl chloride (3.2 mL, 36.8 mmol)
and DMSO (5.2 mL, 73.4 mmol) in dry dichloromethane (40 mL) was
stirred at 788C for 30 min. 6-O-tert-Butyldimethylsilyl-1,2-O-isopropyli-
dene-3-O-methyl-a-d-glucofuranose (6.4 g, 18.4 mmol) was then added
and stirring was prolonged for 1 h. Triethylamine (15.3 mL, 110 mmol) was
then added, and after 30 min the reaction mixture was diluted with
dichloromethane, washed with water, dried (MgSO4), and concentrated to
give compound 2 which was used directly for the next reaction. A 1m
solution of vinyl magnesium bromide in THF (28 mL, 28 mmol) was added
to a cooled (08C) solution of the crude ketone 2 in dry THF (100 mL).
After 1 h the reaction mixture was quenched with aq. NH4Cl, the organic
layer was dried (MgSO4), concentrated, and the residue was purified by
column chromatography (ethyl acetate/cyclohexane 1:9) to give compound
1
toward the C4 conformation resulted in a reduced biological
activity.[16] They are also in agreement with those reported by
Sakairi et al.[18] using a pentasaccharide where the l-iduronic
1
acid moiety had been similarly locked in the C4 conforma-
3 (4.8 g, 70%) as a syrup. [a]D
(250 MHz, CDCl3): d ˆ 5.95 (m, 1H, H-7), 5.89 (d, J1,2 ˆ 3.8 Hz, 1H, H-1),
ˆ
40 (c ˆ 1.3 in chloroform); 1H NMR
tion. These authors were able to conclude that the 1C4
Chem. Eur. J. 2001, 7, No. 22
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0947-6539/01/0722-4827 $ 17.50+.50/0
4827

M. Petitou, P. SinayÈ et al.
FULL PAPER
5.40 (dd, J8a,8b ˆ 1.8, J7,8a ˆ 17.3 Hz, 1H, H-8a), 5.14 (dd, J7,8b ˆ 10.8 Hz, 1H,
H-8b), 4.50 (d, 1H, H-2), 4.20 (d, J3,4 ˆ 3.1 Hz, 1H, H-4), 3.85 (d, 1H, H-3),
3.81 (brs, 1H, OH), 3.60, 3.50 (2d, J6a,6b ˆ 9.6 Hz, 2H, H-6a, H-6b), 3.38 (s,
3H, OMe), 1.42, 1.22 (2s, 6H, 2Me), 0.85 (s, 9H, tBu), 0.00 (s, 6H, 2Me);
elemental analysis calcd (%) for C18H34O6Si: C 57.72, H 9.15; found: C
57.77, H 9.23.
Ketone 13 was dissolved in dry THF (15 mL) and 1m lithium triethylboro-
hydride in THF (4 mL, 4.0 mmol) was added at 788C. After 1 h stirring at
room temperature, 5% sodium hydroxide (2 mL) and hydrogen peroxide
(1 mL) were added, the solvent was evaporated and the residue partitioned
between water and ethyl acetate. The organic layer was dried (MgSO4),
concentrated, and column chromatography (ethyl acetate/cyclohexane 2:1)
gave pure disaccharide 14 (1.0 g, 70%). [a]D
ˆ
11 (c ˆ 0.5 in chloroform);
1,2,4,6-Tetra-O-acetyl-3-O-methyl-5-C-vinyl-b-d-glucopyranose (5): IR-
120 H resin (1 g) was added to a solution of ketone 3 (3.5 g, 9.4 mmol)
1H NMR (200 MHz, CDCl3): d ˆ 7.40 ± 7.10 (m, 15H, aromatic), 6.05 (dd,
J7',8'a ˆ 11.2, J7',8'b ˆ 18.0 Hz, 1H, H-7'), 5.42 (d, 1H, H-8'a), 5.20 (d, 1H,
H-8'b), 4.92, 4.80 (2d, J ˆ 11.1 Hz, 2H, CH2Ph), 4.80 (s, 1H, H-1'), 4.70, 4.60
(2d, J ˆ 12.2 Hz, 2H, CH2Ph), 4.60, 4.40 (2d, J ˆ 12.1 Hz, 2H, CH2Ph), 4.52
(d, J1,2 ˆ 3.5 Hz, 1H, H-1), 4.10 (d, J3',4' ˆ 10.2 Hz, 1H, H-4'), 3.90 ± 3.40 (m,
‡
in water (50 mL). After 6 h of heating at 808C, the resin was filtered off
and, after concentration, acetic anhydride (12 mL) and pyridine (13 mL)
were added. After 16 h of stirring, methanol was added and, after
concentration, the residue was dissolved in dichloromethane, washed with
water, dried (MgSO4), and concentrated. Column chromatography (ethyl
acetate/cyclohexane 3:2) gave compound 5 (2.7 g, 75%) as a solid. M.p.
8H, H-2, 4, 5, 6a, 6b, 2', 6'a, 6'b), 3.30 (s, 6H, 2OMe), 3.25 (t, J2,3 ˆ J3,4
ˆ
10.0 Hz, 1H, H-3), 3.05 (dd, J2',3' ˆ 3.4 Hz, 1H, H-3'), 2.53 (brs, 1H, OH),
‡
‡
1.40, 1.35 (2s, 6H, 2CH3); CI-MS: 707 [M‡H] , 724 [M‡NH4] .
Methyl 2,3,6-tri-O-benzyl-4-O-(2-O-acetyl-3-O-methyl-5-C-vinyl-b-d-
508C; [a]D
ˆ
84 (c ˆ 1.6 in chloroform); 1H NMR (250 MHz, CDCl3): d ˆ
5.70 (m, 3H, olefinic, H-1), 5.45 (dd, J ˆ 3.1 Hz, 8.9 Hz, 1H, olefinic), 5.15
(d, J3,4 ˆ 10.1 Hz, 1H, H-4), 4.95 (dd, J1,2 ˆ 8.4, J2,3 ˆ 9.6 Hz, 1H, H-2), 3.95,
3.52 (2d, J6a,6b ˆ 12.5 Hz, 2H, H-6a, H-6b), 3.30 (t, 1H, H-3), 3.24 (s, 3H,
mannopyranosyl)-a-d-glucopyranoside (16): Acetic anhydride (0.3 mL)
was added to a solution of disaccharide 14 (940 mg, 1.3 mmol) in pyridine
(3 mL). After 3 h of stirring at room temperature, concentration under
reduced pressure gave crude disaccharide 15 that was dissolved in 80%
acetic acid (5 mL). After 2 h at 608C, evaporation to dryness gave a residue
that was purified by column chromatography (ethyl acetate/cyclohexane
‡
‡
OMe), 1.90 (4s, 12H, 4OAc); CI-MS: 389 [M‡H] , 406 [M‡NH4] ;
elemental analysis calcd (%) for C17H24O10: C 52.47, H 6.19; found: C 52.51,
H 6.19.
Methyl 2,3,6-tri-O-benzyl-4-O-(2,4,6-tri-O-acetyl-3-O-methyl-5-C-vinyl-b-
4:1) to give diol 16 as a solid (660 mg, 70%). M.p. 538C; [a]D
ˆ
10 (c ˆ 0.8
in chloroform); 1H NMR (200 MHz, CDCl3): d ˆ 7.30 (m, 15H, aromatic),
5.85 (dd, J7',8'a ˆ 11.1, J7',8'b ˆ 17.9 Hz, 1H, H-7'), 5.45 (dd, J8'a,8'b ˆ 1.5 Hz, 1H,
H-8'a), 5.20 (dd, 1H, H-8'b), 5.19 (d, J2',3' ˆ 3.1 Hz, 1H, H-2'), 4.99, 4.75 (2d,
J ˆ 11.5 Hz, 2H, CH2Ph), 4.79 (s, 1H, H-1'), 4.75, 4.60 (2d, J ˆ 11.5 Hz, 2H,
CH2Ph), 4.60, 4.40 (2d, J ˆ 11.9 Hz, 2H, CH2Ph), 4.55 (d, J1,2 ˆ 3.5 Hz, 1H,
H-1), 3.95 (d, J3',4' ˆ 10.2 Hz, 1H, H-4'), 3.82 ± 3.00 (m, 7H, H-2, 3, 4, 5, 6a,
6b, 6'b), 3.30, 3.23 (2s, 6H, 2OMe), 3.15 (t, J2,3 ˆ J3,4 ˆ 10.0 Hz, 1H, H-3),
d-glucopyranosyl)-a-d-glucopyranoside (7):
A mixture of 5 (1.6 g,
4.1 mmol) and methyl 2,3,6-tri-O-benzyl-a-d-glucopyranoside (6; 2.1 g,
4.5 mmol) in dry dichloromethane (50 mL) containing molecular sieves
(4.0 g) was stirred at room temperature for 1 h. After cooling to 788C,
TMSOTf (0.95 mL, 5.2 mmol) was added and the temperature was allowed
to slowly raise to room temperature. After 2 h, the reaction was quenched
with triethylamine. After filtration (Celite), the solution was washed with
water, dried (MgSO4), concentrated, and the residue was purified by
column chromatography (ethyl acetate/cyclohexane 4:1) to give disacchar-
‡
‡
2.92 (dd, 1H, H-3'), 2.00 (s, 3H, OAc); CI-MS: 709 [M‡H] , 726 [M‡H4] .
Methyl 2,3,6-tri-O-benzyl-4-O-(2-O-acetyl-3-O-methyl-6-O-tosyl-5-C-vi-
nyl-b-d-mannopyranosyl)-a-d-glucopyranoside (17): Tosyl chloride
(240 mg, 1.3 mmol) was added to a solution of diol 16 (600 mg, 0.9 mmol)
in pyridine (3 mL). After 3 h of stirring, evaporation to dryness gave a
residue that was dissolved in chloroform and washed with water. The
organic layer was dried (MgSO4), and after concentration in vacuo, the
residue was purified by a silica gel column chromatography (ethyl acetate/
ide 7 (2.77 g, 85%) as a solid. M.p. 478C; [a]D
ˆ
36 (c ˆ 0.5 in chloro-
form); 1H NMR (200 MHz, CDCl3): d ˆ 7.40 (m, 15H, aromatic), 6.00 (dd,
J7',8'a ˆ 10.7, J7',8'b ˆ 17.8 Hz, 1H, H-7'), 5.75 (dd, J8'a,8'b <1 Hz, 1H, H-8'a),
5.45 (dd, 1H, H-8'b), 5.40 (d, J3',4' ˆ 10.5 Hz, 1H, H-4'), 5.30, 4.90 (2d, 2H,
CH2Ph), 5.09 (dd, J1',2' ˆ 8.2, J2',3' ˆ 9.4 Hz, 1H, H-2'), 4.85, 4.70 (2d, 2H,
CH2Ph), 4.80 (d, 1H, H-1'), 4.80, 4.58 (2d, 2H, CH2Ph), 4.70 (d, J1,2
ˆ
3.7 Hz, 1H, H-1), 4.20 (d, J6'a,6'b ˆ 12.3 Hz, 1H, H-6'a), 3.90 ± 3.40 (m, 8H,
H-2, 3, 4, 5, 6a, 6b, 3', 6'b), 3.50 (s, 6H, 2OMe), 2.20, 2.10, 2.00 (3s, 9H,
3OAc); elemental analysis calcd (%) for C43H52O14: C 65.14, H 6.61; found:
C 65.09, H 6.70.
cyclohexane 1:1) to give disaccharide 17 (297 mg, 80%) as a syrup. [a]D
ˆ
26 (c ˆ 0.8 in chloroform); 1H NMR (250 MHz, CDCl3): d ˆ 7.70 ± 7.10
(m, 19H, aromatic), 5.79 (dd, J7',8'a ˆ 10.9, J7',8'b ˆ 17.9 Hz, 1H, H-7'), 5.50
(dd, J8'a,8'b ˆ 1.3 Hz, 1H, H-8'a), 5.22 (dd, 1H, H-8'b), 5.13 (dd, J1',2' ˆ 1.1,
J2',3' ˆ 3.0 Hz, 1H, H-2'), 5.01, 4.65 (2d, J ˆ 11.9 Hz, 2H, CH2Ph), 4.73 (d,
1H, H-1'), 4.68, 4.62 (2d, J ˆ 11.9 Hz, 2H, CH2Ph), 4.59, 4.40 (2d, J ˆ
12.2 Hz, 2H, CH2Ph), 4.49 (d, J1,2 ˆ 3.4 Hz, 1H, H-1), 4.07 (dd, J3',4' ˆ 10.2,
J4',OH ˆ 3.6 Hz, 1H, H-4'), 4.04 (d, J6'a,6'b ˆ 11.0 Hz, 1H, H-6'a), 3.77 ± 3.39
(m, 7H, H-2, 3, 4, 5, 6a, 6b, 6'b), 3.27, 3.20 (2s, 6H, 2OMe), 2.89 (dd, 1H,
H-3'), 2.54 (d, 1H, OH), 2.32 (s, 3H, Me), 2.10 (s, 3H, OAc).
Methyl 2,3,6-O-tri-O-benzyl-4-O-(4,6-O-isopropylidene-3-O-methyl-5-C-
vinyl-b-d-glucopyranosyl)-a-d-glucopyranoside (9): A catalytic amount of
sodium was added at 08C to a solution of disaccharide 7 (2.7 g, 3.4 mmol) in
methanol (40 mL). After 3 h of stirring at room temperature, the mixture
was acidified with acetic acid, and the solvents were evaporated. The
residue (crude disaccharide 8) was dissolved in dry acetone (40 mL) and
2,2-dimethoxypropane (2 mL), then a catalytic amount of p-toluenesul-
fonic acid was added, and the reaction mixture was stirred at room
temperature overnight. After concentration, the residue was partitioned
between water and chloroform. The organic layer was dried (MgSO4),
concentrated and column chromatography (ethyl acetate/cyclohexane 1:1)
Methyl
2,3,6-tri-O-benzyl-4-O-(2,6-anhydro-3-O-methyl-5-C-vinyl-b-d-
mannopyranosyl)-a-d-glucopyranoside (18):
hydroxide solution (5 mL) was added to
(550 mg, 0.6 mmol) in ethanol (3 mL) and the mixture was heated at
708C for 3 h. After neutralisation with Amberlite IR-120 H resin,
A
a
0.1m ethanolic sodium
solution of tosylate 17
‡
of the residue gave disaccharide 9 as a solid (1.7 g, 70%). M.p. 558C; [a]D
ˆ
concentration and column chromatography (ethyl acetate/cyclohexane
1:1) gave disaccharide 18 (292 mg, 70%) as a syrup. [a]D ˆ ‡13 (c ˆ 0.5 in
chloroform); 1H NMR (250 MHz, CDCl3): d ˆ 7.40 ± 7.00 (m, 15H, aro-
matic), 5.47 (dd, J7',8'a ˆ 10.4 Hz, 1H, H-7'), 5.33 (dd, J8'a,8'b ˆ 2.0 Hz, 1H,
H-8'a), 5.20 (dd, 1H, H-8'b), 5.05 (brs, 1H, H-1'), 4.89 (AB system, 2H,
CH2Ph), 4.70, 4.55 (2d, J ˆ 12.2 Hz, 2H, CH2Ph), 4.55, 4.42 (2d, J ˆ
12.1 Hz, 2H, CH2Ph), 4.55 (d, J1,2 ˆ 3.8 Hz, 1H, H-1), 4.00 (m, 1H, H-3,
virtual coupling), 3.81, 3.60 (2d, J6'a,6'b ˆ 9.9 Hz, 2H, H-6'a, H-6'b), 3.74 (m,
4H, H-2', 4, 6a, 6b), 3.60 (m, 2H, H-4', 5), 3.47 (dd, J2,3 ˆ 9.6 Hz, 1H, H-2),
3.30, 3.25 (2s, 6H, 2OMe), 2.97 (t, J2',3' ˆ J3',4' ˆ 1 Hz, 1H, H-3'); CI-MS: 666
‡13 (c ˆ 0.8 in chloroform); 1H NMR (250 MHz, CDCl3): d ˆ 7.5 (m, 15H,
aromatic), 6.10 (dd, J7',8'a ˆ 18.1, J7',8'b ˆ 11.3 Hz, 1H, H-7'), 5.6 (dd, J8'a,8'b
ˆ
2.0 Hz, 1H, H-8'a), 5.25 (dd, 1H, H-8'b), 4.90 (d, J1',2' ˆ 7.6 Hz, 1H, H-1'),
4.65 (d, J1,2 ˆ 3.5 Hz, 1H, H-1), 5.10 ± 4.64 (m, 6H, 3CH2Ph), 4.08 ± 3.28 (m,
11H, H-2, 3, 4, 5, 6a, 6b, 2', 3', 4', 6'a, 6'b), 3.65, 3.44 (2s, 6H, 2OMe), 2.75
‡
(d, JOH,2 ˆ 2.7 Hz, 1H, OH), 1.58, 1.68 (2s, 6H, 2Me); CI-MS: 707 [M‡H] ,
‡
724 [M‡NH4] ; elemental analysis calcd (%) for C40H50O11: C 67.97, H 7.13;
found: C 67.87, H 7.16.
Methyl 2,3,6-tri-O-benzyl-4-O-(4,6-O-isopropylidene-3-O-methyl-5-C-vi-
nyl-b-d-mannopyranosyl)-a-d-glucopyranoside (14): A mixture of oxalyl
chloride (0.35 mL, 4.0 mmol) and dry DMSO (0.57 mL, 8.0 mmol) in
dichloromethane (10 mL) was stirred at 788C for 30 min. Compound 9
(1.4 g, 2.0 mmol) in dry dichloromethane (10 mL) was then added to the
solution and stirred for 45 min. Dry triethylamine (1.7 mL, 12.0 mmol) was
added, the solution was diluted with dichloromethane, and washed with
water. The organic layer was dried (MgSO4), concentrated, and the residue
13 was directly used for the next reaction.
‡
[M‡NH4] .
Methyl 2,3,6-tri-O-benzyl-4-O-(2,6-anhydro-5-C-benzyloxycarbonyl-3-O-
methyl-b-d-mannopyranosyl)-a-d-glucopyranoside (19): Ozone was bub-
bled into a stirred, cooled ( 788C), solution of disaccharide 18 (260 mg,
0.4 mmol) in dichloromethane (20 mL) until the color turned to pale blue.
Dimethylsulfide was added and, after washing with water, the organic layer
was dried (MgSO4), and concentrated. The residue was dissolved in tert-
butanol (16 mL), and 2-methyl-2-butene (5 mL) then water (16 mL) were
4828
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0947-6539/01/0722-4828 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 22

l-Iduronic Acid Derivatives
4821±4834
added, followed by NaH2PO4 (700 mg) and NaClO2 (700 mg). The
suspension was vigorously stirred at room temperature overnight and
partitioned between water and ethyl acetate. The organic layer was dried
(MgSO4), and concentrated. The residue was dissolved in dry DMF
(25 mL), then tetrabutylammonium iodide (0.7 g, 2.0 mmol), potassium
hydrogencarbonate (0.25 g, 2.5 mmol) and benzyl bromide (0.250 mL,
2.1 mmol) were introduced. After 5 h, the product was extracted with
diethyl ether. The organic layer was washed with water, dried (MgSO4),
concentrated, and the residue was purified by silica gel column chroma-
tography (ethyl acetate/cyclohexane 2:1) to yield the benzyl ester
derivative 19 as a syrup (236 mg, 80%). [a]D ˆ ‡34 (c ˆ 0.93 in dichloro-
methane); 1H NMR (200 MHz, CDCl3): d ˆ 7.40 ± 7.10 (m, 20H, aromatic),
5.10 (brs, 1H, H-1'), 5.05 (ABq, 2H, CH2Ph), 4.90 (s, 2H, CH2Ph), 4.70,
4.60 (2d, J ˆ 12.1 Hz, 2H, CH2Ph), 4.60 (d, J1,2 ˆ 3.5 Hz, 1H, H-1), 4.60,
4.40 (2d, J ˆ 12.1 Hz, 2H, CH2Ph), 4.05, 3.90 (2d, J6'a,6'b ˆ 9.8 Hz, 2H,
H-6'a,6'b), 4.05 ± 3.90 (m, 2H, H-3, 4'), 3.8 ± 3.5 (m, 4H, H-4, 5, 6a, 6b), 3.65
(s, 1H, H-2'), 3.45 (dd, J1,2 ˆ 3.5, J2,3 ˆ 9.5 Hz, 1H, H-2), 3.35, 3.30 (2s, 6H,
2OMe), 2.92 (brs, 1H, H-3'), 2.30 (brs, 1H, OH); ESI-MS positive mode:
H-2GlcIII, J2,3 ˆ 9.5 Hz, H-2 GlcI), 4.29 (dd, J6a,6b ˆ 11.3 Hz, 2H, H-6a GlcV,
H-6b GlcIII), 4.24 (d, 1H, H-6a ManII), 4.20 (ddd, J5,6a ˆ 3.8, J5,6b ˆ 2.2 Hz,
1H, H-5 GlcI), 4.17 (d, 1H, H-4 ManII), 4.12 (dd, J6a,6b ˆ 11.2 Hz, 1H, H-6b
GlcV), 4.09 (d, J6a,6b ˆ 10.2 Hz, 1H, H-6b ManII), 4.08 (ddd, J5,6a ˆ 1.8, J5,6b
ˆ
2.0 Hz, 1H, H-5 GlcIII), 4.01 (dd, 2H, J4,5 ˆ 9.7 Hz, H-4 GlcIII, J4,5 ˆ 10.0 Hz,
H-4 GlcI), 3.90 (dd, J4,5 ˆ 9.6 Hz, 1H, H-4 GlcUAIV), 3.88 (dt, J5,6a ˆ 1.6,
J5,6b ˆ 1.6 Hz, 1H, H-5 GlcV), 3.74 (dd, J3,4 ˆ 2.7 Hz, 1H, H-3 ManII), 3.72 (d,
1H, H-5 GlcIV), 3.55 (dd, J3,4 ˆ 9.5 Hz, 1H, H-3 GlcV), 3.53 (dd, J3,4
ˆ
9.1 Hz, 1H, H-3 GlcUAIV), 3.34 (dd, J4,5 ˆ 9.9 Hz, 1H, H-4 GlcV), 3.31
(dd, J2,3 ˆ 9.9 Hz, 1H, H-2 GlcV), 3.26 (dd, J2,3 ˆ 9.4 Hz, 1H, H-2 GlcUAIV);
ESI-MS, negative mode: monoisotopic mass: 1727.19; calcd: 1725.89;
found: 1725.46 Æ 0.19.
3-O-Allyl-6-O-tert-butyldimethylsilyl-1,2-O-isopropylidene-a-d-glucofur-
anose (24): 3-O-Allyl-1,2-O-isopropylidene-a-d-glucofuranose (7.5 g,
28.8 mmol) was dissolved in dry dichloromethane (100 mL), then
TBDMSCl (4.75 g, 31.7 mmol) and imidazole (3.9 g, 57.7 mmol) were
added. The reaction mixture was stirred at RT for 4 h, then diluted with
dichloromethane, washed with water, the organic layer was dried (MgSO4),
concentrated and the residue purified by column chromatography (ethyl
‡
‡
m/z: 779 [M‡Na] , 795 [M‡K] ; elemental analysis calcd (%) for
C43H48O12: C 68.24, H 6.39; found: C 68.35, H 6.80.
acetate/cyclohexane 1:9) to give the desired product 24 (10.3 g, 95%) as a
1
syrup. [a]D
ˆ
31 (c ˆ 1 in chloroform); H NMR (200 MHz, CDCl3): d ˆ
Pentasaccharide 21: A solution of tert-butyldimethylsilyl trifluorometha-
nesulfonate (TBDMS-OTf) in dichloromethane (0.156m, 100 mL) was
added, at 208C to a stirred mixture of imidate 20 (81 mg, 78.2 mmol),
alcohol 19 (65 mg, 86 mmol), and finely grounded 4 Š molecular sieves
(70 mg) in dichloromethane/diethyl ether 1:2 (2.4 mL). After 30 min of
stirring, the reaction was complete (TLC: toluene/ethyl acetate 7:3), and
solid NaHCO3 was added until neutral pH. After filtration and concen-
tration, the residue was first purified using a Sephadex LH 20 gel column
(dichloromethane/ethanol 1:1), and then over silica gel (ethyl acetate/
cyclohexane 1:1) to yield pentasaccharide 21 (86 mg, 67%). [a]D ˆ ‡66
(c ˆ 1.0 in dichloromethane); 1H NMR (500 MHz, CDCl3): d ˆ 5.50 (d,
J1,2 ˆ 3.7 Hz, 1H, H-1 GlcV), 5.41 (dd, 1H, H-3 GlcIII), 5.18 (brs, J1,2 ꢀ1 Hz,
1H, H-1 ManII), 4.98 (d, J1,2 ˆ 3.6 Hz, 1H, H-1 GlcIII), 4.58 (d, J1,2 ˆ 3.6 Hz,
1H, H-1 GlcI), 4.58 (dd, 1H, H-6a GlcIII), 4.28 (dd, 1H, H-6a GlcV), 4.22
(dd, 1H, H-6b GlcV), 4.19 (dd, 1H, H-6b GlcIII), 4.14 (d, J1,2 ꢀ9 Hz, 1H, H-1
GlcUAIV), 4.12 (d, 1H, H-4 ManII), 4.10 (d, 1H, H-6a ManII), 4.01 (dd, 1H,
H-3 GlcI), 3.98 (d, 1H, H-6b ManII), 3.94 (ddd, 1H, H-5 GlcIII), 3.92 (dd,
1H, H-4 GlcUAIV), 3.83 (m, 3H, H-2 GlcUAIV, H-5 GlcIV, H-6a GlcI), 3.80
(ddd, 1H, H-5 GlcI), 3.78 (dd, 1H, H-4 GlcI), 3.63 (dd, 1H, H-4 GlcIII), 3.61
(dd, 1H, H-6b GlcI), 3.51 (dd, 1H, H-2 GlcI), 3.45 (dd, 1H, H-2 GlcIII), 3.43
(ddd, 1H, H-5 GlcV), 3.37 (dd, 1H, H-3 GlcV), 3.31 (dd, 1H, H-3 GlcUAIV),
3.12 (dd, 1H, H-3 ManII), 3.11 (dd, 1H, H-2 GlcV), 3.03 (dd, 1H, H-4 GlcV),
2.93 (dd, 1H, H-2 GlcUAIV); ESI-MS, positive mode: monoisotopic mass:
1632.65; calcd: 1633.75; found: 1633.77 Æ 0.10; elemental analysis calcd
(%) for C86H104O31: C 63.23, H 6.42; found: C 63.01, H 6.69.
5.80 (d, J1,2 ˆ 3.6 Hz, 1H, H-1), 5.80 (m, 1H, CH allylic), 4.45 (d, 1H, H-2),
4.0 (d, J3,4 ˆ 2.9 Hz, 1H, H-4), 3.95 (d, 1H, H-3), 3.90 (m, 1H, H-5), 3.75
(dd, J5,6b ˆ 3.6, J6a,6b ˆ 10.1 Hz, 1H, H-6b), 3.65 (dd, J5,6a ˆ 5.0 Hz, 1H,
H-6a), 2.60 (d, J5,OH ˆ 6.5 Hz, 1H, OH), 1.40, 1.20 (2s, 6H, 2Me), 0.80 (s,
9H, tBu), 0.0 (s, 6H, 2Me).
3-O-Allyl-6-O-tert-butyldimethylsilyl-1,2-O-isopropylidene-5-C-vinyl-a-d-
glucofuranose (26): Oxalyl chloride (4.6 mL, 53.5 mmol) and DMSO
(7.6 mL, 107 mmol) were added in dry dichloromethane (40 mL) at 788C
and stirred for 30 min. A solution of compound 24 (10.0 g, 26.7 mmol) in
dichloromethane (50 mL) was slowly added and stirred for 1 h. Triethyl-
amine (22.3 mL, 160.4 mmol) was added and after 30 min of stirring, the
reaction mixture was diluted with dichloromethane, washed with water,
dried (MgSO4), and concentrated to give the ketone which was used as such
for the next reaction. The crude ketone 25 was dissolved in dry THF
(80 mL), and
a 1m solution of vinylmagnesium bromide (40.3 mL,
40.3 mmol) in THF was added at 08C. After 1 h the reaction mixture was
quenched with sat. NH4Cl and extracted with ethyl acetate. The organic
layer was dried (MgSO4), concentrated and the residue was purified by
silica gel column chromatography (ethyl acetate/cyclohexane 2:8) to give
compound 26 (9.9 g, 89%) as a syrup. [a]D
ˆ
28 (c ˆ 1 in chloroform);
1H NMR (200 MHz, CDCl3): d ˆ 6.00 (m, 1H, H-7), 5.90 (d, J1,2 ˆ 3.93 Hz,
1H, H-1), 5.80 (m, 1H, -CH allylic), 5.40 (dd, J8a,8b ˆ 1.8, J7,8b ˆ 17.4 Hz, 1H,
H-8b), 5.30 ± 5.20 (m, 2H, CH2 allylic), 5.14 (dd, J7,8a ˆ 10.7 Hz, 1H, H-8a),
4.50 (d, 1H, H-2), 4.20 (d, J3,4 ˆ 3.04 Hz, 1H, H-4), 3.90 ± 4.10 (m, 2H, CH2
allylic), 4.0 (d, 1H, H-3), 3.81 (brs, 1H, OH), 3.50 (2d, J6a,6b ˆ 9.6 Hz, 2H,
H-6a, H-6b), 1.42, 1.22 (2s, 6H, 2Me), 0.85 (s, 9H, tBu), 0.00 (s, 6H, 2Me);
elemental analysis calcd (%) for C20H34O6Si: C 59.97, H 9.05; found: C
60.00, H 9.05.
Pentasaccharide 22: A solution of pentasaccharide 21 (49 mg, 30.0 mmol) in
AcOH (3 mL) was stirred under H2 (35 bar) at 408C for 12 h in the
presence of 5% Pd/C (73 mg). The mixture was then filtered (Celite),
concentrated, and codistilled with water (4 Â 5 mL). The residue was
dissolved in aq. 1m NaOH (3 mL), and heated at 558C for 3 h. The solution
was cooled then loaded on top of a Sephadex G25F column (170 mL)
equilibrated in water. The fractions containing the compound were
1,2,4,6-Tetra-O-acetyl-3-O-allyl-5-C-vinyl-b-d-glucopyranose (28): A mix-
‡
ture of compound 26 (8.0 g, 20 mmol) and IR-120 H resin (2.5 g) in water
(200 mL) was heated at 808C. After 8 h of stirring, the resin was filtered off
and the filtrate concentrated to give the crude product 27. The latter was
acetylated using acetic anhydride (40 mL) and pyridine (80 mL). After 12 h
of stirring, the excess of acetic anhydride was quenched by methanol and
solvents were concentrated to yield the crude residue which was partitioned
between ethyl acetate and water. The organic layer was dried (MgSO4),
concentrated, and the residue purified by column chromatography (ethyl
‡
collected, passed through a Dowex H resin column, and concentrated to
give pentasaccharide 22 (25 mg, 86% from pentasaccharide 21). [a]D
ˆ
‡105 (c ˆ 1.0 in water); ESI-MS, negative mode: monoisotopic mass:
966.34; calcd: 966.89; found: 966.5.
2S0 Pentasaccharide 23:
A solution of pentasaccharide 22 (20 mg,
20.7 mmol) and Et3N ´ SO3 (164 mg, 0.90 mmol) in DMF (2 mL) was heated
at 558C with protection from light for 18 h. After cooling to room
temperature, the solution was diluted with aq. 0.2m NaCl, and layered on
top of a Sephadex G25F gel column (170 mL) equilibrated in aq. 0.2m
NaCl. The fractions containing the pentasaccharide were pooled and the
compound was desalted using a Sephadex G25F gel column (170 mL)
equilibrated in water. After freeze-drying, compound 23 (30.5 mg, 85%)
was obtained. [a]D ˆ ‡49 (c ˆ 0.63 in water); 1H NMR (500 MHz, D2O):
d ˆ 5.51 (d, J1,2 ˆ 3.8 Hz, 1H, H-1 GlcIII), 5.50 (brs, J1,2 ˆ 1.3, J1,6b ˆ 0.5 Hz,
1H, H-1 ManII), 5.47 (d, J1,2 ˆ 3.7 Hz, 1H, H-1 GlcV), 5.17 (d, J1,2 ˆ 3.6 Hz,
1H, H-1 GlcI), 4.83 (dd, J3,4 ˆ 9.7 Hz, 1H, H-3 GlcI), 4.66 (d, J1,2 ˆ 7.8 Hz,
1H, H-1 GlcUAIV), 4.52 (dd, 1H, H-6a GlcI), 4.51 (dd, J3,4 ˆ 9.7 Hz, 1H,
H-3 GlcIII), 4.41 (dd, J2,3 ˆ 1.4, J2,4 ˆ 0.5 Hz, 2H, H-2 ManII, H-6a GlcIII),
4.40 (dd, J6a,6b ˆ 11.3 Hz, 1H, H-6b GlcI), 4.36 (dd, 2H, J2,3 ˆ 9.7 Hz,
acetate/cyclohexane 1:1) to furnish compound 28 (4.3 g, 52%) as a syrup.
1
[a]D
ˆ
43 (c ˆ 1 in chloroform); H NMR (250 MHz, CDCl3): d ˆ 5.90 ±
5.61 (m, 3H, H-7, allylic proton), 5.80 (d, J1,2 ˆ 8.4 Hz, 1H, H-1), 5.58 (dd,
J7,8a ˆ 8.5 Hz, 1H, H-8a), 5.30 (d, J3,4 ˆ 10.1 Hz, 1H, H-4), 5.10 (dd, J2,3
ˆ
9.8 Hz, 1H, H-2), 5.10 (m, 1H, allylic), 4.50 (d, J6a,6b ˆ 12.5 Hz, 1H, H-6b),
4.0 (dd, J ˆ 1.4 Hz, 4.4 Hz, 1H, allylic CH2), 3.62 (d, 1H, H-6a), 3.50 (t,
J3,4 ˆ J2,3 ˆ 9.8 Hz, 1H, H-3), 2.70 (4s, 12H, 4OAc); elemental analysis
calcd (%) for C19H26O10: C 55.06, H 6.32; found: C 55.13, H 6.33.
Methyl 2,3,6-tri-O-benzyl-4-O-(2,4,6-tri-O-acetyl-3-O-allyl-5-C-vinyl-b-d-
glucopyranosyl)-a-d-glucopyranoside (29): A solution of compound 28
(3.2 g, 8.0 mmol) and alcohol 6 (4.45 g, 9.6 mmol) in dichloromethane
(75 mL) was stirred at RT in the presence of powdered molecular sieves
(8.0 g) for 1 h. Then TMSOTf (1.75 mL, 9.6 mmol) was added at 788C
Chem. Eur. J. 2001, 7, No. 22
ꢁ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0722-4829 $ 17.50+.50/0
4829

M. Petitou, P. SinayÈ et al.
FULL PAPER
and slowly allowed to reach room temperature under stirring. After 3 h, the
reaction mixture was quenched with triethylamine, filtered (Celite), and
the filtrate was washed with water. The organic layer was dried (MgSO4),
concentrated, and the residue was purified by column chromatography
(ethyl acetate/cyclohexane 3:7) to furnish compound 29 (5.1 g, 78%) as a
The residue was dissolved in dichloromethane and washed successively
with aq. KI, water, and brine. The organic layer was dried (MgSO4),
concentrated, and the residue was purified by column chromatography
(ethyl acetate/cyclohexane 2:3) to provide disaccharide 33 as a syrup
(1.83 g, 65%). [a]D
ˆ
2 (c ˆ 1.6 in chloroform); 1H NMR (400 MHz,
white solid. M.p. 1248C; [a]D
ˆ
45 (c ˆ 1.1 in chloroform); 1H NMR
CDCl3): d ˆ 6.05 (dd, J7',8'a ˆ 11.3, J7',8'b ˆ 18.0 Hz, 1H, H-7'), 5.52 (dd,
J8'a,8'b ˆ 0.9 Hz, 1H, H-8'b), 5.15 (dd, 1H, H-8'a), 5.05, 4.86 (2d, J ˆ 11.1 Hz,
2H, CH2Ph), 4.82 (d, J1',2' ˆ 8.2 Hz, 1H, H-1'), 4.85, 4.67 (2d, J ˆ 12.2 Hz,
2H, CH2Ph), 4.65 (d, J1,2 ˆ 3.8 Hz, 1H, H-1), 4.56 (ABq, 2H, CH2Ph),
3.97 ± 3.85 (m, 3H, H-6b,4,3), 3.72 (ddd, J4,5 ˆ 8.9, J5,6b ˆ 1.7, J5,6a ˆ 3.1 Hz,
1H, H-5), 3.70 (t, J2',3' ˆ J3',4' ˆ 10.2 Hz, 1H, H-3'), 3.69 (d, J6a,6b ˆ 12.0 Hz,
1H, H-6a), 3.60 (dd, J2,3 ˆ 8.9 Hz, 1H, H-2), 3.62 (d, 1H, H-4'), 3.60, 3.42
(2s, 6H, 2OMe), 3.52 (d, J6'a,6'b ˆ 10.2 Hz, 1H, H-6'b), 3.40 (d, 1H, H-6'a),
3.05 (dd, 1H, H-2'); elemental analysis calcd (%) for C40H51O11: C 67.87, H
7.26; found: C 67.75, H 7.30.
(400 MHz, CDCl3): d ˆ 5.90 (dd, J7',8'b ˆ 11.0, J7',8'a ˆ 17.8 Hz, 1H, H-7'), 5.80
(m, 1H, allylic), 5.70 (dd, J8'a,8'b ˆ 0.6 Hz, 1H, H-8'b), 5.37 (d, J3',4' ˆ 9.6 Hz,
1H, H-4'), 5.35 (dd, 1H, H-8'a), 5.20 (m, 2H, allylic), 5.02 (dd, J1',2' ˆ 8.2,
J2',3' ˆ 9.5 Hz, 1H, H-2'), 4.86 (d, 1H, H-1'), 5.21, 4.72 (2d, J ˆ 12.6 Hz, 2H,
CH2Ph), 4.72, 4.61 (2d, J ˆ 12.4 Hz, 2H, CH2Ph), 4.71, 4.49 (2d, J ˆ
12.0 Hz, 2H, CH2Ph), 4.60 (d, J1,2 ˆ 4.1 Hz, 1H, H-1), 4.1 (d, J6'a,6'b
ˆ
12.3 Hz, 1H, H-6'b), 4.05 (m, 2H, allylic CH2), 3.87 (t, J2,3 ˆ J3,4 ˆ 9.0 Hz,
1H, H-3), 3.82 (t, 1H, H-4), 3.75 (dd, J6a,6b ˆ 11.7, J5,6b ˆ 3.7 Hz, 1H, H-6b),
3.60 (ddd, J5,6a ˆ 1.7, J4,5 ˆ 9.0 Hz, 1H, H-5), 3.56 (dd, 1H, H-6a), 3.52 (dd,
1H, H-2), 3.46 (d, 1H, H-6'a), 3.45 (t, 1H, H-3'), 3.40 (s, 3H, OMe), 2.10,
2.0, 1.90 (3s, 9H, 3OAc); elemental analysis calcd (%) for C45H54O14: C
66.00, H 6.67; found: C 66.03, H 6.59.
Methyl 2,3,6-tri-O-benzyl-4-O-(3-O-acetyl-2-O-methyl-5-C-vinyl-b-d-glu-
copyranosyl)-a-d-glucopyranoside (35): Acetic anhydride (0.7 mL) and
pyridine (1.6 mL) were added to a solution of compound 33 (1.4 g,
1.98 mmol) in dichloromethane (20 mL). The reaction mixture was stirred
at RT for 3 h. Excess of the reagent was quenched with MeOH and
concentration furnished the crude disaccharide 34 which was used as such
for the next reaction. A solution of compound 34 in a mixture of 60% acetic
acid in water (20 mL) was added and heated at 708C for 1 h. Concentration
and co-concentration with toluene gave a crude product that was purified
by column chromatography (ethyl acetate/cyclohexane 7:3) to furnish
Methyl 2,3,6-O-tri-O-benzyl-4-O-(3-O-allyl-4,6-O-isopropylidene-5-C-vi-
nyl-b-d-glucopyranosyl)-a-d-glucopyranoside (31): Catalytic amount so-
dium was added at 08C to a solution of compound 29 (5.0 g, 6.11 mmol) in
methanol (100 mL). After 6 h of stirring at RT, the solvent was concen-
trated. The residue (crude disaccharide 30) was dissolved in dry acetone
(100 mL), and 2,2'-dimethoxypropane (6 mL) then p-TsOH (catalytic) was
added. The reaction mixture was allowed to stir at RT overnight. The
solvent was evaporated and the residue was partitioned between water and
chloroform. The organic layer was dried (MgSO4), concentrated and the
residue was purified by column chromatography (ethyl acetate/cyclo-
disaccharide 35 (1.0 g, 71%) as a solid. M.p. 1238C; [a]D
ˆ
12 (c ˆ 1.53 in
chloroform); 1H NMR (400 MHz, CDCl3): d ˆ 5.87 (dd, J7',8'a ˆ 11.2, J7',8'b
ˆ
17.0 Hz, 1H, H-7'), 5.45 (dd, J8'a,8'b ˆ 1.1 Hz, 1H, H-8'b), 5.12 (dd, 1H,
H-8'a), 5.05, 4.92 (2d, J ˆ 12.0 Hz, 2H, CH2Ph), 4.93 (t, J2',3' ˆ 10.1 Hz, 1H,
H-3'), 4.85, 4.70 (2d, J ˆ 12.2 Hz, 2H, CH2Ph), 4.80 (d, J1',2' ˆ 7.9 Hz, 1H,
H-1'), 4.65 (d, J1,2 ˆ 3.7 Hz, H1H, -1), 4.57 (ABq, 2H, CH2Ph), 3.92 ± 3.85
(m, 4H, H-4', 6b, 4, 3), 3.72 (ddd, J5,6a ˆ 1.7, J5,6b ˆ 3.7, J4,5 ˆ 8.9 Hz, 1H,
H-5), 3.67 (dd, J6a,6b ˆ 10.8 Hz, 1H, H-6b), 3.60 (dd, J2,3 ˆ 9.5 Hz, 1H, H-2),
3.50, 3.40 (2s, 6H, 2OMe), 3.37 (m, 1H, H-6'b), 3.15 (d, J ˆ 11.8 Hz, 1H,
H-6'a), 3.05 (dd, 1H, H-2'), 2.20 (s, 3H, OAc); elemental analysis calcd (%)
for C39H48O12: C 66.09, H 6.83; found: C 66.02, H 6.84.
hexane 3:7) to furnish disaccharide 31 (3.3 g, 74%) as a syrup. [a]D
ˆ
16
(c ˆ 1 in chloroform); 1H NMR (400 MHz, CDCl3): d ˆ 6.02 (dd, J7',8'a
11.3, J7',8'b ˆ 18.0 Hz, 1H, H-7'), 5.95 (m, 1H, allylic), 5.50 (dd, J8'a,8'b
ˆ
ˆ
1.3 Hz, 1H, H-8'b), 5.20 ± 5.35 (m, 2H, allylic), 5.19 (dd, 1H, H-8'a), 5.05,
4.91 (2d, J ˆ 11.3 Hz, 2H, CH2Ph), 4.87 (d, J1',2' ˆ 7.4 Hz, 1H, H-1'), 4.82,
4.65 (2d, J ˆ 12.2 Hz, 2H, CH2Ph), 4.60 (ABq, 2H, CH2Ph), 4.40 ± 4.20 (m,
2H, allylic), 4.02 (dd, J6a,6b ˆ 11.0, J5,6b ˆ 3.0 Hz, 1H, H-6b), 3.89 ± 3.99 (m,
2H, H-3, H-4), 3.78 (ddd, J5,6a ˆ 2.0 Hz, 1H, H-5), 3.72 (d, J3',4' ˆ 9.8 Hz, 1H,
H-4'), 3.67 (dd, 1H, H-6a), 3.57 (d, J6'a,6'b ˆ 10.1 Hz, 1H, H-6'b), 3.56 (dd,
J1,2 ˆ 3.2, J2,3 ˆ 10.1 Hz, 1H, H-2), 3.46 (m, 2H, H-2', H-3'), 3.37 (s, 3H,
OMe), 3.35 (d, 1H, H-6'a); elemental analysis calcd (%) for C42H52O11: C
68.83, H. 7.15; found: C 68.90, H 7.11.
Methyl 2,3,6-tri-O-benzyl-4-O-(3-O-acetyl-2-O-methyl-6-O-tosyl-5-C-vi-
nyl-b-d-glucopyranosyl)-a-d-glucopyranoside
(36):
Tosyl
chloride
(0.145 g, 0.745 mmol) and triethylamine (0.2 mL) were added to a solution
of compound 35 (0.48 g, 0.68 mmol) in dichloromethane (10 mL), and the
reaction mixture was stirred at RT for 6 h. The organic layer was then
washed with water, dried (MgSO4), and concentrated. The residue was
purified by column chromatography (ethyl acetate/cyclohexane 3:7) to
Methyl 2,3,6-tri-O-benzyl-4-O-(3-O-allyl-4,6-O-isopropylidene-2-O-meth-
yl-5-C-vinyl-b-d-glucopyranosyl)-a-d-glucopyranoside (32): NaH (0.26 g,
5.4 mmol, 60% dispersion in paraffin oil), and methyl iodide (0.42 mL,
6.75 mmol) were added to a solution of disaccharide 31 (3.3 g, 4.5 mmol) in
DMF (50 mL) at 08C. The reaction mixture was stirred at room temper-
ature for 4 h. Excess of NaH was quenched with methanol, solvents were
evaporated, and the crude residue was partitioned between ethyl acetate
and water. The organic layer was separated, dried (MgSO4), and
concentrated. Purification by column chromatography (ethyl acetate/
cyclohexane 3:2) furnished disaccharide 32 (3.1 g, 92%) as a syrup.
[a]D ˆ ‡5 (c ˆ 1.5 in chloroform); 1H NMR (400 MHz, CDCl3): d ˆ 6.05
(dd, J7',8'a ˆ 11.3, J7',8'b ˆ 18.0 Hz, 1H, H-7'), 5.52 (dd, J8'a,8'b ˆ 0.9 Hz, 1H,
H-8'b), 5.15 (dd, 1H, H-8'a), 5.05, 4.86 (2d, J ˆ 11.1 Hz, 2H, CH2Ph), 4.82
(d, J1',2' ˆ 8.2 Hz, 1H, H-1'), 4.85, 4.62 (2d, J ˆ 12.2 Hz, 2H, CH2Ph), 4.65 (d,
J1,2 ˆ 3.8 Hz, 1H, H-1), 4.56 (ABq, 2H, CH2Ph), 3.97 ± 3.85 (m, 3H,
H-6b,4,3), 3.72 (ddd, J4,5 ˆ 8.9, J5,6b ˆ 1.7, J5,6a ˆ 3.1 Hz, 1H, H-5), 3.69 (dd,
J6a,6b ˆ 12.0 Hz, 1H, H-6a), 3.60 (dd, J2,3 ˆ 8.9 Hz, 1H, H-2), 3.62 (d, 1H,
H-4'), 3.60 (s, 3H, OMe), 3.52 (d, J6'a,6'b ˆ 10.2 Hz, 1H, H-6'a), 3.42 (s, 3H,
OMe), 3.40 (d, 1H, H-6'b), 3.05 (d, 1H, H-2'); elemental analysis calcd (%)
for C43H54O11: C 69.15, H 7.29; found: C 69.26, H 7.22.
furnish disaccharide 36 (0.438 g, 75%) as a syrup. [a]D
ˆ
10 (c ˆ 0.85 in
chloroform); 1H NMR (400 MHz, CDCl3): d ˆ 5.90 (dd, J7',8'a ˆ 11.1, J7',8'b
ˆ
17.9 Hz, 1H, H-7'), 5.52 (dd, J8'a,8'b ˆ 0.6 Hz, 1H, H-8'b), 5.12 (dd, 1H,
H-8'a), 5.01 (t, J2',3' ˆ J3',4' ˆ 9.9 Hz, 1H, H-3'), 5.20, 4.80 (2d, J ˆ 11.8 Hz,
2H, CH2Ph), 4.75 (d, J1',2' ˆ 8.1 Hz, 1H, H-1'), 4.75, 4.60 (2d, J ˆ 12.0 Hz,
2H, CH2Ph), 4.52 (ABq, 2H, CH2Ph), 4.09 (d, J6'a,6'b ˆ 11.4 Hz, 1H, H-6'b),
4.02 (dd, J4',OH ˆ 5.5 Hz, 1H, H-4'), 3.93 (dd, J6a,6b ˆ 10.7, J5,6b ˆ 2.7 Hz, 1H,
H-6b), 3.87 (t, J3,4 ˆ 9.7 Hz, 1H, H-3), 3.82 (t, 1H, H-4), 3.69 (ddd, J5,6a ˆ 1.7,
J4,5 ˆ 8.8 Hz, 1H, H-5), 3.65 (dd, 1H, H-6a), 3.53 (dd, 1H, H-2), 3.52 (d, 1H,
H-6'a), 3.47, 3.39 (2s, 6H, 2OMe), 3.15 (dd, 1H, H-2'), 3.05 (d, 1H, OH).
Methyl 2,3,6-tri-O-benzyl-4-O-(3-O-5-C-methylidene-2-O-methyl-5-C-vi-
nyl-a-l-idopyranosyl)-a-d-glucopyranoside (37): A 0.1n ethanolic sodium
hydroxide solution (10 mL) of compound 36 (0.4 g, 0.464 mmol) was heated
‡
at 70 ± 808C for 2 h. The reaction mixture was neutralized with IR H resin
and filtered. The filtrate was concentrated and purified by column
chromatography (ethyl acetate/cyclohexane 8:2) to furnish disaccharide
37 (0.255 g, 85%) as a syrup. [a]D ˆ ‡6 (c ˆ 1 in chloroform); 1H NMR
(400 MHz, CDCl3): d ˆ 5.70 (dd, J7',8'b ˆ 10.8, J7',8'b ˆ 17.2 Hz, 1H, H-7'), 5.50
(dd, J8'a,8'b ˆ 1.5 Hz, 1H, H-8'b), 5.40 (s, 1H, H-1'), 5.27 (dd, 1H, H-8'a),
5.02, 4.92 (2d, J ˆ 10.7 Hz, 2H, CH2Ph), 4.80, 4.65 (2d, J ˆ 12.1 Hz, 2H,
CH2Ph), 4.67, 4.55 (2d, J ˆ 11.9 Hz, 2H, CH2Ph), 4.65 (d, J1,2 ˆ 3.5 Hz, 1H,
H-1), 4.45 (d, J6'a,6'b ˆ 9.7 Hz, 1H, H-6'b), 4.29 (t, J2',3' ˆ J3',4' ˆ 4.9 Hz, 1H,
H-3'), 4.07 (t, J2,3 ˆ J3,4 ˆ 9.8 Hz, 1H, H-3), 4.02 (t, 1H, H-4'), 3.92 (dd,
J6a,6b ˆ 10.7, J5,6b ˆ 2.8 Hz, 1H, H-6b), 3.85 (t, 1H, H-4), 3.75 (ddd, J4,5 ˆ 9.8,
J5,6a ˆ 1.8 Hz, 1H, H-5), 3.72 (d, 1H, H-6'a), 3.69 (dd, 1H, H-6a), 3.6 (dd,
1H, H-2), 3.58 (d, 1H, H-2'), 3.40 (s, 3H, OMe), 3.30 (s, 3H, OMe); CI-MS:
Methyl 2,3,6-tri-O-benzyl-4-O-(4,6-O-isopropylidene-2-O-methyl-5-C-vi-
nyl-b-d-glucopyranosyl)-a-d-glucopyranoside (33): A mixture of com-
pound 32 (3.0 g, 4.01 mmol) and tBuOK (3.6 g, 32.1 mmol) in DMSO
(30 mL) was heated at 808C for 1 h. After cooling to 08C, sat. aq. NH4Cl
was added and the reaction mixture was stirred for 15 min, and extracted
repeatedly with dichloromethane. The organic layer was dried (MgSO4)
and concentrated to obtain the crude prop-1'-enyl ether, which was used as
such for the next reaction. To a mixture of this compound, HgO (1.74 g,
8.03 mmol), acetone (50 mL), and water (8 mL), was added dropwise a
solution of HgCl2 (1.09 g, 4.01 mmol) in acetone (5 mL). The reaction
mixture was stirred at RT for 1 h, then filtered (Celite), and concentrated.
‡
m/z:666 [M‡NH3] .
4830
ꢁ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0722-4830 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 22

l-Iduronic Acid Derivatives
4821±4834
Methyl 2,3,6-tri-O-benzyl-4-O-(benzyl 2-O-methyl-3-O-5-C-methylidene-
a-l-idopyranosyluronate)-a-d-glucopyranoside (40): A solution of com-
pound 37 (0.2 g, 0.308 mmol) in dichloromethane (30 mL) was stirred at
788C, then ozone was bubbled until the solution turned pale blue. Excess
of ozone was quenched with dimethylsulphide and was kept at RT for 1 h.
The reaction mixture was partitioned between dichloromethane and water.
The organic layer was dried (MgSO4), and concentrated to furnish the
crude aldehyde 38 which was used as such for the next reaction. The latter
was dissolved in a mixture of 2-methyl-2-butene/water/tert-butanol (1:2:2,
25 mL), then sodium dihydrogenphosphate (500 mg) and sodium chlorite
(500 mg) were added, and the mixture was stirred at RT for 5 h. After
dilution with ethyl acetate, the organic layer was washed with water, dried
(MgSO4) and concentrated to obtain the crude acid (39). The latter was
dissolved in DMF (10 mL) and potassium hydrogen carbonate (0.2 g) then
benzyl bromide (0.2 mL) were added, and the reaction mixture was allowed
to stir at room temperature for 5 h. After dilution with diethyl ether, the
organic layer was washed (water), dried (MgSO4), and concentrated to
obtain a residue which was purified by column chromatography (ethyl
acetate/cyclohexane 3:2) to furnish compound 40 (0.17 g, 73%) as a syrup.
[a]D ˆ ‡18 (c ˆ 0.75 in chloroform); 1H NMR (200 MHz, CDCl3): d ˆ 5.40
(s, 1H, H-1'), 5.15 (s, 2H, CH2Ph), 4.90, 4.50 (2d, J ˆ 10.6 Hz, 2H, CH2Ph),
4.78, 4.63 (2d, J ˆ 12.1 Hz, 2H, CH2Ph), 4.65 (d, J1,2 ˆ 3.7 Hz, 1H, H-1),
4.69, 4.55 (2d, J ˆ 12.0 Hz, 2H, CH2Ph), 4.60 (d, J6'a,6'b ˆ 9.4 Hz, 1H, H-6'b),
4.49 (dd, JOH,4' ˆ 10.5, J3',4' ˆ 5.5 Hz, 1H, H-4'), 4.29 (t, J2',3' ˆ J3',4' ˆ 4.5 Hz,
1H, H-3'), 4.10 (d, 1H, H-6'a), 4.05 (t, J3,4 ˆ J2,3 ˆ 9.2 Hz, 1H, H-3), 4.01 (d,
NaCl. The fractions containing the pentasaccharide were pooled together
and the compound was desalted using a Sephadex G25F gel column
(190 mL) equilibrated in water. After freeze-drying, compound 43
(43.7 mg, 94%) was obtained. [a]D ˆ ‡61 (c ˆ 1 in water); 1H NMR
(500 MHz, D2O): d ˆ 5.46 (d, J1,2 ˆ 3.8 Hz, 1H, H-1 GlcV), 5.39 (brs, J1,2 ˆ 0,
J1,3 ˆ 0.5, J1,6b ˆ 0.5 Hz, 1H, H-1 IdoUAII), 5.33 (d, J1,2 ˆ 3.6 Hz, 1H, H-1
GlcIII), 5.16 (d, J1,2 ˆ 3.6 Hz, 1H, H-1 GlcI), 4.79 (dd, J3,4 ˆ 9.1 Hz, 1H, H-3
GlcI), 4.66 (dd, J3,4 ˆ 5.5, 4J3,6b ˆ 0 ꢀ 0.5 Hz, 1H, H-3 IdoUAII), 4.65 (d, J1,2
ˆ
7.8 Hz, 1H, H-1 GlcUAIV), 4.62 (dd, J3,4 ˆ 9.6 Hz, 1H, H-3 GlcIII), 4.57 (d,
1H, H-4 IdoUAII), 4.48 (d, 1H, H-6a IdoUAII), 4.45 (dd, 1H, H-6a GlcI),
4.40 (dd, 1H, H-6a GlcIII), 4.36 (dd, J2,3 ˆ 9.2 Hz, 1H, H-2 GlcI), 4.34 (dd,
J6a,6b ˆ 10.8 Hz, 1H, H-6b GlcI), 4.30 (dd, J6a,6b ˆ 11.3 Hz, 1H, H-6b GlcIII),
4.28 (dd, 1H, H-6a GlcV), 4.27 (dd, J2,3 ˆ 10.1 Hz, 1H, H-2 GlcIII), 4.14 (d,
J6a,6b ˆ 10.0 Hz, 1H, H-6b IdoUAII), 4.11 (dd, J6a,6b ˆ 11.2 Hz, 1H, H-6b
GlcV), 4.10 (ddd, J5,6a ˆ 3.7, J5,6b ˆ 2.2 Hz, 1H, H-5 GlcI), 4.07 (ddd, J5,6a
ˆ
1.8, J5,6b ˆ 1.2 Hz, 1H, H-5 GlcIII), 3.98 (dd, J4,5 ˆ 9.8 Hz, 1H, H-4 GlcIII),
3.88 (dd, J4,5 ˆ 9.7 Hz, 1H, H-4 GlcUAIV), 3.87 (dt, J5,6a ˆ 1.8, J5,6b ˆ 1.8 Hz,
1H, H-5 GlcV; dd, J4,5 ˆ 9.7 Hz, 1H, H-4 GlcI), 3.71 (d, 1H, H-5 GlcIV), 3.67
(dd, J2,3 ˆ 3.7, J2,4 ˆ 0.5 Hz, 1H, H-2 IdoUAII), 3.54 (dd, J3,4 ˆ 9.7 Hz, 1H,
H-3 GlcV), 3.51 (dd, J3,4 ˆ 9.1 Hz, 1H, H-3 GlcUAIV), 3.33 (dd, J4,5
ˆ
10.1 Hz, 1H, H-4 GlcV), 3.30 (dd, J2,3 ˆ 10.0 Hz, 1H, H-2 GlcV), 3.25 (dd,
J2,3 ˆ 9.4 Hz, 1H, H-2 GlcUAIV). ESI-MS: negative mode: monoisotopic
mass: 1724; calcd: 1725.17; found: 1725.13.
Methyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-methyl-5-C-vinyl-b-d-glucopyra-
nosyl)-a-d-glucopyranoside (45): Methyl iodide (0.28 mL, 4.5 mmol) and
NaH (60% dispersion in oil, 0.18 g, 4.5 mmol) were added at 08C to a
solution of compound 9 (1.6 g, 2.3 mmol) in dry THF (20 mL), and the
reaction mixture was stirred at room temperature for 1 h. After addition of
MeOH, the solvents were removed and the residue was partitioned
between water and ethyl acetate. The organic layer was dried (MgSO4) and
concentrated to give crude disaccharide 44 that was used without
purification. Acetic acid (80% in water; 5 mL) was added, and the solution
was heated at 708C for 3 h. After evaporation the residue was purified by
column chromatography (ethyl acetate/cyclohexane 4:1) to give disacchar-
1H, OH), 3.85 (t, J4,5 ˆ 9.2 Hz, 1H, H-4), 3.85 (dd, J6a,6b ˆ 10.8, J5,6b
ˆ
3.0 Hz, 1H, H-6b), 3.75 (ddd, J5,6a ˆ 1.9 Hz, 1H, H-5), 3.65 (dd, 1H,
H-6a), 3.62 (dd, 1H, H-2), 3.51 (d, 1H, H-2'), 3.40 (s, 3H, OMe), 3.20 (s,
3H, OMe); elemental analysis calcd (%) for C43H48O12: C 68.24, H 6.39;
found: C 68.29, H 6.79.
Pentasaccharide 41: A mixture of imidate 20 (81 mg, 81 mmol), compound
40 (61 mg, 80 mmol), powdered molecular sieves (150 mg) in anhydrous
dichloromethane (5 mL) was stirred at 208C for 30 min. Then TMSOTf
(3 mL) was added and stirring was continued for 1 h. The reaction mixture
was quenched with triethylamine, stirred 30 min, and filtered (Celite). The
filtrate was partitioned between water and dichloromethane. The organic
layer was dried (MgSO4) and concentrated to furnish a residue that was
purified by column chromatography (ethyl acetate/cyclohexane 7:3) to
furnish pentasaccharide 41 (107 mg, 83%) as a syrup. [a]D ˆ ‡80 (c ˆ 1 in
dichloromethane); 1H NMR (500 MHz, CDCl3): d ˆ 5.50 (d, J1,2 ˆ 3.7 Hz,
1H, H-1 GlcV), 5.42 (dd, 1H, H-3 GlcIII), 5.29 (d, J1,2 ꢀ1 Hz, 1H, H-1
IdoUAII), 4.92 (d, J1,2 ˆ 3.0 Hz, 1H, H-1 GlcIII), 4.57 (d, J1,2 ˆ 3.6 Hz, 1H,
H-1 GlcI), 4.54 (dd, 1H, H-6a GlcIII), 4.41 (d, 1H, H-4 IdoUAII), 4.36 (dd,
1H, H-3 IdoUAII), 4.27 (dd, 1H, H-6a GlcV), 4.13 (d, J1,2 ꢀ8 Hz, 1H, H-1
GlcUAIV), 4.21 (dd, 1H, H-6b GlcV), 4.19 (dd, 1H, H-6b GlcIII), 4.10 (d, 1H,
H-6a IdoUAII), 4.03 (ddd, 1H, H-5 GlcIII), 3.92 (dd, 1H, H-3 GlcI), 3.91 (dd,
1H, H-4 GlcUAIV), 3.82 (d, 1H, H-5 GlcIV), 3.79 (d, 1H, H-6b IdoUAII),
3.78 (ddd, 1H, H-5 GlcI), 3.73 (dd, 1H, H-4 GlcI), 3.62 (dd, 1H, H-4 GlcIII),
3.60 (m, 2H, H-6a, H-6b GlcI), 3.47 (dd, 1H, H-2 GlcI), 3.43 (ddd, 1H, H-5
GlcV), 3.42 (dd, 1H, H-2 GlcIII), 3.36 (dd, 1H, H-2 GlcUAIV), 3.34 (dd, 1H,
H-3 GlcV), 3.30 (dd, 1H, H-3 GlcUAIV), 3.10 (dd, 1H, H-2 GlcV), 3.03 (dd,
1H, H-4 GlcV), 2.92 (dd, 1H, H-2 GlcUAIV); FAB-MS, positive mode: m/z:
ide 45 (1.16 g, 75%) as
a
solid. M.p. 408C; [a]D
ˆ
17 (c ˆ 1.3 in
chloroform); 1H NMR (200 MHz, CDCl3): d ˆ 7.40 ± 7.10 (m, 15H, aro-
matic), 5.85 (dd, J8a,8b <1 Hz, 1H, H-8b GlcII), 5.80 (dd, J7,8a ˆ 11.1, J7,8b
ˆ
19.9 Hz, 1H, H-7 GlcII), 5.05 (dd, 1H, H-8a GlcII), 4.95, 4.80 (2d, J ˆ
11.7 Hz, 2H, CH2Ph), 4.73, 4.60 (2d, J ˆ 11.7 Hz, 2H, CH2Ph), 4.61 (d,
J1,2 ˆ 9.2 Hz, 1H, H-1 GlcII), 4.55 (d, J1,2 ˆ 3.7 Hz, 1H, H-1 GlcI), 4.50 (s,
2H, CH2Ph), 3.90 ± 3.45 (m, 7H, H-2 GlcI, H-3 GlcI, H-4 GlcI, H-5 GlcI,
H-6a GlcI, H-6b GlcI, H-4 GlcII), 3.50, 3.42, 3.30 (3s, 12H, 3OMe), 3.28 (d,
J6a,6b ˆ 11.8 Hz, 1H, H-6b GlcII), 3.05 (d, 1H, H-6a GlcI), 3.00 (t, J2,3 ˆ J3,4
ˆ
9.5 Hz, 1H, H-3 GlcII), 2.85 (t, 1H, H-2 GlcII); CI-MS: m/z: 688 [M‡NH4] ;
elemental analysis calcd (%) for C38H48O11: C 67.04, H 7.11; found: C 67.13,
H 7.08.
‡
Methyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-methyl-5-C-ethyl-b-d-glucopyra-
nosyl)-a-d-glucopyranoside (46): A solution of disaccharide 45 (700 mg,
1.03 mmol) in ethyl acetate (50 mL) was stirred for 10 min under hydrogen
atmosphere (1.4 bar) in the presence of PtO2, filtered through Celite, and
concentrated to give compound 46 (700 mg, quantitative). [a]D
ˆ
2 (c ˆ
1.4 in chloroform); 1H NMR (250 MHz, CDCl3): d ˆ 7.62 ± 7.40 (m, 15H,
aromatic), 5.20 (d, J ˆ 11.7 Hz, 1H, CH2Ph), 5.01 (d, J ˆ 11.7 Hz, 1H,
CH2Ph), 5.00 (d, J ˆ 12 Hz, 1H, CH2Ph), 4.87 ± 4.78 (m, J1,2 ˆ 3.6 Hz, 4H,
H-1 GlcI, CH2Ph), 4.70 (d, J1,2 ˆ 7.9 Hz, 1H, H-1 GlcII), 4.43 (d, J ˆ 12 Hz,
1H, CH2Ph), 4.18 (dd, J5,6a ˆ 2.8, J6a,6b ˆ 10.8 Hz, 1H, H-6a GlcI), 4.04 ± 4.00
(m, 2H), 3.93 ± 3.45 (m, 7H), 3.83, 3.71, 3.58 (3s, 3H, 3OMe), 3.32 (dd,
J3,4 ˆ J2,3 ˆ 9 Hz, 1H, H-3 GlcII), 3.08 (dd, 1H, H-2 GlcII), 2.82 (brs, 1H,
OH-4 GlcII), 1.64 (m, 2H, CH2CH3), 0.95 (t, 3H, CH2CH3); CI-MS: 683
‡
thioglycerol‡NaCl: 1655.8 [M‡Na] ; m/z: thioglycerol‡KF: 1671.6
‡
[M‡K] ; elemental analysis calcd (%) for C86H104O31: C 63.23, H 6.42;
found: C 63.19, H 6.63.
Pentasaccharide 42: A solution of pentasaccharide 41 (50 mg, 30.6 mmol) in
acetic acid (2 mL) was stirred under H2 in the presence of 5% Pd/C
(100 mg, 20 bar) for 12 h at 508C. The mixture was then filtered (Celite),
concentrated, and codistilled with water (5 Â 2 mL). The residue was
dissolved in methanol (1.38 mL), and aq. 5n NaOH (1.10 mL) was added
(final concentration: 1m). After 12 h of stirring, the solution was loaded on
top of a Sephadex G25F column (190 mL) equilibrated with water. The
fractions containing the compound were collected, passed through a
‡
‡
[M‡H] , 700 [M‡NH4] ; elemental analysis calcd (%) for C38H50O11: C
66.84, H 7.38; found: C 66.74, H 7.43.
Methyl 2,3,6-tri-O-benzyl-4-O-(benzyl 2,3-di-O-methyl-5-C-ethyl-b-d-glu-
copyranuronate)-a-d-glucopyranoside (47): A mixture of sat. aq. NaCl
(1.38 mL), sat. aq. NaHCO3 (0.72 mL) and aq. NaOCl (1.3m, 1.72 mL) was
added dropwise, at 08C, to a mixture of compound 46 (480 mg, 0.70 mmol),
dichloromethane (2 mL), 2,2,6,5-tetramethylpiperidinoxy radical (1.5 mg,
0.1 equiv), KBr (7.3 mg), Bu4NCl (9.6 mg), and sat. aq. NaHCO3 (1.3 mL).
The resulting mixture was stirred for 30 min at 08C. Bu4NCl (96 mg),
NaHCO3 (57 mg) and benzyl bromide (0.8 mL) were then introduced and
stirring was continued at RT for 45 min. After dilution with water and
dichloromethane the organic layer was dried (MgSO4) and concentrated.
‡
Dowex H resin column, and concentrated to give pentasaccharide 42
(26 mg, 88% from 41). FAB-MS: positive mode: m/z: thioglycerol‡NaCl:
‡
989 [M‡Na] .
1C4 Pentasaccharide 43:
A solution of pentasaccharide 42 (26 mg,
26.8 mmol) and Et3N ´ SO3 (170 mg, 0.94 mmol) in DMF (2.8 mL) was
heated at 558C with protection from light for 20 h. After cooling to room
temperature, the solution was diluted with aq. 0.2m NaCl, and layered on
top of a Sephadex G25F gel column (190 mL) equilibrated in aq. 0.2m
Chem. Eur. J. 2001, 7, No. 22
ꢁ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0722-4831 $ 17.50+.50/0
4831

M. Petitou, P. SinayÈ et al.
FULL PAPER
Column chromatography of the residue (hexane/ethyl acetate 65:35) gave
trisaccharide 51 (256 mg, 90%). [a]D ˆ ‡33 (c ˆ 1.0 in chloroform);
1H NMR (400 MHz, CDCl3): d ˆ 5.45 (dd, J2,3 ˆ J3,4 ˆ 9.4 Hz, H-3a GlcI),
5.21 (dd, J2,3 ˆ J3,4 ˆ 9.4 Hz, H-3bGlcI), 5.28 ± 5.22 (m, CH2Ph, H-1a GlcI,
H-1 GlcIII), 5.05 (d, J ˆ 12.5 Hz, CH2Ph), 5.03 (d, J ˆ 12.4 Hz, CH2Ph), 4.87
(d, J ˆ 11.8 Hz, CH2Ph), 4.81 (dd, J1,2 ˆ 7.5, J1,OH ˆ 4.4 Hz, H-1bGlcI), 4.70,
4.60, 4.33, 4.24 (4dd, H-6a a/b, H-6b a/b GlcI), 4.50, 4.47 (2d, J3,4 ˆ 8.0 Hz,
H-4 GlcUAII), 4.18 (ddd, H-5bGlcI), 4.07, 4.05 (2d, J1,2 ˆ 7.5 Hz, H-1
GlcUAII), 3.86 (brd, OHbGlcI), 3.62, 3.53, 3.50, 3.49, 3.48, 3.46 (6s, 18H,
6MeO), 2.11, 2.09, 2.08, 1.90, 1.85 (5s, AcO a and b), 1.04 ± 0.95 (m,
CH2CH3); elemental analysis calcd (%) for C45H62O20: C 58.56, H 6.77;
found: C 58.55, H 6.79.
disaccharide 47 (415 mg, 75%). [a]D
ˆ
7 (c ˆ 0.7 in chloroform); 1H NMR
(400 MHz, CDCl3): d ˆ 7.40 ± 7.20 (m, 20H, aromatic), 5.19 (d, J ˆ 11.7 Hz,
1H, CH2Ph), 4.93 (s, 2H, CH2Ph), 4.79 (d, J ˆ 12 Hz, 1H, CH2Ph), 4.75 (d,
J ˆ 11.8 Hz, 1H, CH2Ph), 4.68 ± 4.61 (m, 4H, H-1 GlcI, H-1 GlcUAII,
CH2Ph), 4.55 (d, J ˆ 12 Hz, 1H, CH2Ph), 4.03 (dd, J5,6a ˆ 2.8, J6a,6b
ˆ
10.9 Hz, 1H, H-6a GlcI), 3.95 (dd, J4,5 ˆ J3,4 ˆ 9.0 Hz, 1H, H-4 GlcI), 3.91
(dd, J2,3 ˆ 9.0 Hz, 1H, H-3 GlcI), 3.82 (dd, J4,OH ˆ 2.2, J3,4 ˆ 9.3 Hz, 1H, H-4
GlcUAII), 3.74 (ddd, J5,6b ˆ 1.7 Hz, 1H, H-5 GlcI), 3.66 (s, 3H, OMe), 3.65
(dd, 1H, H-6b GlcI), 3.56 (dd, J1,2 ˆ 3.7 Hz, 1H, H-2 GlcI), 3.53 (s, 3H,
OMe), 3.39 (s, 3H, OMe), 3.20 (dd, J2,3 ˆ J3,4 ˆ 8.9 Hz, 1H, H-3 GlcUAII),
3.04 (dd, J1,2 ˆ 7.5 Hz, 1H, H-2 GlcUAII), 2.99 (d, J ˆ 1 Hz, OH), 2.02 (dq,
J ˆ 7.2, J ˆ 15.3 Hz, 1H, CH2), 1.57 (dq, 1H, CH2), 0.80 (t, 3H, Me); CI-MS:
(6-O-Acetyl-2,3,4-tri-O-methyl-a-d-glucopyranosyl)-(1 ! 4)-(benzyl 5-C-
ethyl-2,3-di-O-methyl-b-d-glucopyranosyluronate)-(1 ! 4)-1-trichloroace-
timidoyl-3,6-di-O-acetyl-2-O-benzyl-a,b-d-glucopyranose (52): A mixture
of compound 51 (75 mg, 0.091 mmol), trichloroacetonitrile (0.456 mL,
4.55 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (10 mL, 0.07 mmol) in
dichloromethane (5 mL) was stirred at room temperature for 30 min. After
concentration, column chromatography (hexane/acetone 65:35) gave
compound 52 (76 mg, 87%). This compound was directly used in the next
reaction.
‡
804 [M‡NH4] ; elemental analysis calcd (%) for C45H54O12: C 68.68, H
6.91; found: C 68.67, H 6.99.
Methyl (6-O-benzyl-2,3,4-tri-O-methyl-a-d-glucopyranosyl)-(1 ! 4)-(ben-
zyl 5-C-ethyl-2,3-di-O-methyl-b-d-glucopyranuronate)-(1 ! 4)-2,3,6-tri-O-
benzyl a-d-glucopyranoside (49): A mixture of ethyl 6-O-benzyl-2,3,4-tri-
O-methyl-1-thio-b-d-glucopyranoside (48; 292 mg, 0.82 mmol), disacchar-
ide 47 (323 mg, 0.41 mmol), and finely grounded 4 Š molecular sieves
(700 mg) in dichloromethane/diethyl ether (3:2, 10 mL) was stirred under
argon for 30 min at room temperature. N-iodosuccinimide (424 mg,
1.89 mmol) was added and, after cooling to 408C, trifluoromethanesul-
fonic acid (25 mL, 0.28 mmol) was introduced. After stirring for 30 min, the
reaction was complete (TLC: toluene/diethyl ether 1:1). After neutraliza-
tion by addition of triethylamine, the mixture was filtered (Celite), the
solution was diluted with dichloromethane, washed with aq. Na2S2O3,
water, dried (MgSO4) and concentrated. Column chromatography (ethyl
acetate/hexane 3:7) gave trisaccharide 49 (319 mg, 72%) and the b anomer
(67 mg, 15%). 49: [a]D ˆ ‡33 (c ˆ 1.0 in chloroform); 1H NMR (400 MHz,
CDCl3): d ˆ 7.40 ± 7.20 (m, 25H, aromatic), 5.27 (d, J1,2 ˆ 3.7 Hz, 1H, H-1
GlcIII), 5.15 (d, J ˆ 11.8 Hz, 1H, CH2Ph), 4.97 (d, J ˆ 12.5 Hz, 1H, CH2Ph),
4.64 ± 4.57 (m, 6H, CH2Ph, H-1 GlcI, H-1 GlcUAII), 4.46 (d, J ˆ 12.4 Hz,
1H, CH2Ph), 4.44 (d, J ˆ 12.2 Hz, 1H, CH2Ph), 4.05 (d, J ˆ 8.2 Hz, 1H, H-4
GlcUAII), 4.00 (dd, J6a,6b ˆ 11.1, J5,6a ˆ 3.3 Hz, 1H, H-6a GlcI), 3.92 ± 3.84
(m, 2H, H-3,4 GlcI), 3.75 ± 3.32 (m, 10H), 3.64, 3.59, 3.53, 3.52, 3.48, 3.39
Pentasaccharide 54: To a mixture of imidate 52 (75 mg, 0.07 mmol),
disaccharide 53 (90 mg, 0.105 mmol), and finely grounded 4 Š molecular
sieves (200 mg) in dichloromethane (2.5 mL) was added, at 208C, a
dichloromethane solution of trimethylsilyl trifluoromethanesulfonate
(0.05m, 0.4 mL). After 30 min under stirring, triethylamine was introduced
until neutralisation. After filtration and concentration, the residue was
purified first using a Sephadex LH 20 gel column (dichloromethane/
ethanol 1:1), then over silica gel (hexane/acetone 75:20, then 70:30) to yield
pentasaccharide 54 (90 mg, 76%). [a]D ˆ ‡53 (c ˆ 1 in dichloromethane);
1H NMR (500 MHz, CDCl3): d ˆ 5.37 (dd, 1H, H-3 GlcIII), 5.30 (d, J1,2
ˆ
6.9 Hz, 1H, H-1 IdoUAII), 5.20 (d, J1,2 ˆ 3.6 Hz, 1H, H-1 GlcV), 5.19 (d,
J1,2 ˆ 3.6 Hz, 1H, H-1 GlcIII), 4.56 (d, J1,2 ˆ 3.7 Hz, 1H, H-1 GlcI), 4.46 (dd,
1H, H-6a GlcIII), 4.43 (dd, 1H, H-4 IdoUAII), 4.30 (d, J1,2 ˆ 8.0 Hz, 1H, H-1
GlcUAIV), 4.25 (m, 2H, H-6a GlcV, H-6b GlcIII), 4.22 (dd, 1H, H-6b GlcV),
3.99 (m, 2H, H-4 GlcUAIV, H-5 GlcIII), 3.86 (dd, 1H, H-2 GlcI), 3.82 (dd,
1H, H-4 GlcI), 3.81 (dd, 1H, H-3 GlcI), 3.75 (dd, 1H, H-6a GlcI), 3.69 (m,
2H, H-5, H-6b GlcI), 3.66 (dd, 1H, H-3 IdoUAII), 3.59 (ddd, 1H, H-5 GlcV),
3.54 (dd, 1H, H-4 GlcIII), 3.44 (dd, 1H, H-2 GlcIII), 3.35 (dd, 1H, H-3 GlcV),
3.32 (dd, 1H, H-3 GlcUAIV), 3.06 (m, 2H, H-2, H-4 GlcV), 2.92 (dd, 1H,
H-2 IdoUAII), 2.88 (dd, 1H, H-2 GlcUAIV); FAB-MS, positive mode:
monoisotopic mass: 1662.70; calcd: 1663.82; found: 1662.9; rlemental
analysis calcd (%) for C88H110O31: C 63.52, H 6.66; found: C 64.41, H 7.02.
(6s, 18H, 6MeO), 3.16 (dd, J2,3 ˆ 9.7 Hz, 1H, H-2 GlcIII), 3.11 (dd, J1,2
ˆ
J2,3 ˆ 8 Hz, 1H, H-2 GlcUAII), 2.12 (dq, J ˆ 7.2, J ˆ 15.3 Hz, 1H, CH2CH3),
‡
1.62 (dq, 1H, CH2CH3), 0.95 (t, 3H, CH2CH3); CI-MS: 1098 [M‡NH4] ;
elemental analysis calcd (%) for C61H76O17: C 67.78, H 7.08; found: C 67.80,
H 7.15.
(6-O-Acetyl-2,3,4-tri-O-methyl-a-d-glucopyranosyl)-(1 ! 4)-(benzyl 5-C-
ethyl-2,3-di-O-methyl-b-d-glucopyranosyluronate)-(1 ! 4)-1,3,6-tri-O-
acetyl-2-O-benzyl-a,b-d-glucopyranose (50): A 5% solution of sulfuric
Pentasaccharide 55: A solution of pentasaccharide 54 (42 mg, 0.025 mmol)
in acetic acid (2 mL) was stirred under H2 in the presence of 5% Pd/C
(84 mg, 20 bar) for 12 h at 508C. The mixture was then filtered (Celite),
concentrated, and codistilled with water (5 Â 5 mL). The residue was
dissolved in methanol (0.9 mL) and aq. NaOH was added (final concen-
tration: 1m). After 12 h of stirring, the solution was loaded on top of a
Sephadex G25F column (170 mL) equilibrated with water. The fractions
acid in acetic acid (0.272 mL) was added at 08C to
a solution of
trisaccharide 49 (494 mg, 0.453 mmol) in acetic anhydride (40 mL). After
stirring for 2 h at 08C the mixture was poured dropwise into an aq.
saturated solution of NaHCO3. The product was extracted with dichloro-
methane, washed with water, dried (MgSO4) and concentrated. Column
chromatography (hexane/acetone 7:3) gave trisaccharide 50 (a/b 6:1;
322 mg, 73%). [a]D ˆ ‡33 (c ˆ 1.0 in chloroform); 50 a: 1H NMR
‡
containing the compound were collected, passed through a Dowex H resin
column, and concentrated to give pentasaccharide 55 (22.0 mg, 87% from
pentasaccharide 54).
(400 MHz, CDCl3): d ˆ 7.40 ± 7.25 (m, 10H, aromatic), 6.33 (d, J1,2
ˆ
3.7 Hz, 1H, H-1 GlcI), 5.46 (dd, J2,3 ˆ J3,4 ˆ 9.7 Hz, 1H, H-3 GlcI), 5.26 (d,
J ˆ 12.5 Hz, 1H, CH2Ph), 5.23 (d, J1,2 ˆ 3.7 Hz, 1H, H-1 GlcIII), 5.06 (d, J ˆ
2S0 Pentasaccharide 56:
A solution of pentasaccharide 55 (22.0 mg,
22.0 mmol) and Et3N ´ SO3 (139.6 mg, 0.77 mmol) in DMF (2.0 mL) was
heated at 558C with protection from light for 20 h. After cooling to room
temperature, the solution was diluted with aq. 0.2m NaCl, and layered on
top of a Sephadex G25F gel column (650 mL) equilibrated in aq. 0.2m
NaCl. The fractions containing the pentasaccharide were pooled together
and the compound was desalted using a Sephadex G25F gel column
(650 mL) equilibrated in water. After freeze-drying, pentasaccharide 56
(35.0 mg, 90%) was obtained: [a]D ˆ ‡43 (c ˆ 1 in water); 1H NMR
12.5 Hz, 1H, CH2Ph), 4.65 (d, J ˆ 12.3 Hz, 1H, CH2Ph), 4.54 (dd, J6a,6b
ˆ
12.2, J5,6a ˆ 2.7Hz, 1H, H-6a GlcI), 4.52 (d, J ˆ 12.5 Hz, 1H, CH2Ph), 4.48
(d, J3,4 ˆ 8.0 Hz, 1H, H-4 GlcUAII), 4.33 (dd, J5,6b ˆ 4.1 Hz, 1H, H-6b GlcI),
4.29 ± 4.26 (m, 2H, H-6a, 6b GlcIII), 4.05 (d, J1,2 ˆ 7.5 Hz, 1H, H-1 GlcUAII),
4.02 (m, 1H, H-5 GlcI), 3.75 ± 3.38 (m, 5H), 3.63, 3.54, 3.51, 3.49 (4s, 15H,
5MeO), 3.15 ± 3.05 (m, 3H), 2.18, 2.11, 2.10, 1.91 (4s, 12H, 4Ac), 2.10 (m,
1H, CH2CH3), 1.82 (m, 1H, CH2CH3), 1.02 (t, 3H, CH2CH3); selected data
for 50 b: d ˆ 5.65 (d, J1,2 ˆ 8.0 Hz, 1H, H-1 b GlcI); CI-MS: 982 [M‡NH4] ;
‡
(500 MHz, D2O): d ˆ 5.42 (d, J1,2 ˆ 3.8 Hz, 1H, H-1 GlcV), 5.40 (d, J1,2
3.7 Hz, 1H, H-1 GlcIII), 5.14 (d, J1,2 ˆ 3.7 Hz, 1H, H-1 GlcI), 5.06 (d, J1,2
ˆ
ˆ
elemental analysis calcd (%) for C47H64O21: C 58.49, H 6.68; found: C 58.58,
H 6.68.
2.8 Hz, 1H, H-1 IdoUAII), 4.76 (d, 1H, H-5 IdoUAII), 4.67 (d, J1,2 ˆ 8.1 Hz,
(6-O-Acetyl-2,3,4-tri-O-methyl-a-d-glucopyranosyl)-(1 ! 4)-(benzyl 5-C-
ethyl-2,3-di-O-methyl-b-d-glucopyranosyluronate)-(1 ! 4)-3,6-di-O-ace-
tyl-2-O-benzyl-a,b-d-glucopyranose (51): Hydrazine acetate (73 mg,
0.8 mmol) was added to a solution of compound 50 (300 mg, 0.310 mmol)
in DMF (22 mL). After stirring for 1 h at room temperature ethyl acetate
was added. The solution was washed with water, dried (MgSO4) and
concentrated. Column chromatography (hexane/acetone 65:35) gave
1H, H-1 GlcUAIV), 4.66 (dd, J3,4 ˆ 9.7 Hz, 1H, H-3 GlcI), 4.58 (dd, J5,6b
ˆ
2.1 Hz, 1H, H-6a GlcIII), 4.51 (dd, J3,4 ˆ 9.4 Hz, 1H, H-3 GlcIII), 4.35 (dd,
J2,3 ˆ 9.8 Hz, 1H, H-2 GlcI), 4.38 (dd, J5,6b ˆ 4.9 Hz, 1H, H-6a GlcI), 4.29
(dd, J2,3 ˆ 10.0 Hz, 1H, H-2 GlcIII), 4.27 (dd, J6a,6b ˆ 11.2 Hz, 1H, H-6b
GlcIII), 4.26 (m, J5,6b ˆ 1.9 Hz, H-6a GlcV, J6a,6b ˆ 11.4 Hz, 2H, H-6b GlcI),
4.20 (ddd, J5,6a ˆ 1.4 Hz, 1H, H-5 GlcIII), 4.19 (dd, J4,5 ˆ 2.6 Hz, 1H, H-4
IdoUAII), 4.13 (dd, J6a,6b ˆ 11.2 Hz, 1H, H-6b GlcV), 4.09 (ddd, J5,6a
ˆ
4832
ꢁ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0722-4832 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 22

l-Iduronic Acid Derivatives
4821±4834
2.2 Hz, 1H, H-5 GlcI), 4.02 (ddd, J5,6a ˆ 1.6 Hz, 1H, H-5 GlcV), 3.97 (d, 1H,
H-4 GlcUAIV), 3.95 (dd, J4,5 ˆ 9.2 Hz, 1H, H-4 GlcI), 3.80 (dd, J4,5 ˆ 9.9 Hz,
3.07 (m, 2H, H-2, H-4 GlcV), 3.02 (dd, H-2 GlcUAIV), 2.96 (dd, H-2
IdoUAII); FAB-MS, positive mode: monoisotopic mass: 1706.73; calcd:
1707.89; found: 1707.6; elemental analysis calcd (%) for C90H114O32: C
63.29, H 6.73; found: C 63.25, H 6.76.
1H, H-4 GlcIII), 3.77 (dd, J3,4 ˆ 3.5 Hz, 1H, H-3 IdoUAII), 3.60 (dd, J3,4
ˆ
9.2 Hz, 1H, H-3 GlcUAIV), 3.52 (dd, J3,4 ˆ 9.6 Hz, 1H, H-3 GlcV), 3.49 (dd,
J2,3 ˆ 5.3 Hz, 1H, H-2 IdoUAII), 3.30 (dd, J4,5 ˆ 9.9 Hz, 1H, H-4 GlcV), 3.27
(dd, J2,3 ˆ 10.0 Hz, 1H, H-2 GlcV), 3.26 (dd, J2,3 ˆ 9.3 Hz, 1H, H-2
GlcUAIV); ESI-MS, negative mode: monoisotopic mass: 1753.9, average
mass: 1755.2, found: 1755.3Æ0.1.
Methyl 2,3,6-tri-O-benzyl-4-O-(2,3,6-tri-O-methyl-5-C-vinyl-b-d-glucopyr-
anosyl)-a-d-glucopyranoside (57): Dibutyltin oxide (0.44 g, 1.76 mmol) was
added to a solution of disaccharide 45 (1.0 g, 1.47 mmol) in toluene
(15 mL). After heating under reflux conditions with azeotropic removal of
water for 3 h, toluene was removed and the residue was taken in dry DMF
(10 mL). Methyl iodide (0.11 mL, 1.76 mmol) was added to the solution and
after stirring at 508C for 6 h, the reaction mixture was partitioned between
water and diethyl ether. The organic layer was dried (MgSO4), concen-
trated, and the residue was purified by column chromatography (ethyl
acetate/cyclohexane 1:1) to give disaccharide 57 (0.663 g, 65%) as a syrup.
Pentasaccharide 60: A solution of pentasaccharide 59 (37 mg, 21.7 mmol) in
acetic acid (2 mL) was stirred under H2 in the presence of 5% Pd/C (74 mg,
20 bar) for 12 h at 508C. The mixture was then filtered (Celite),
concentrated, and codistilled with water (5 Â 5 mL). The residue was
dissolved in methanol (0.8 mL), and aq. NaOH was added (final concen-
tration: 1m). After 12 h of stirring, the solution was loaded on top of a
Sephadex G25F column (170 mL) equilibrated with water. The fractions
‡
containing the compound were collected, passed through a Dowex H resin
column, and concentrated to give pentasaccharide 60 (19.5 mg, 87% from
pentasaccharide 59): ESI-MS, negative mode: monoisotopic mass: 1040.41;
calcd: 1041.41; found: 1041.0.
4C1 Pentasaccharide 61: A solution of compound 60 (19.5 mg, 18.7 mmol)
and Et3N ´ SO3 (94 mg, 0.52 mmol) in DMF (1.4 mL) was heated at 558C
with protection from light for 20 h. After cooling to room temperature, the
solution was diluted with aq. 0.2m NaCl, and layered on top of a Sephadex
G25F gel column (170 mL) equilibrated in aq. 0.2m NaCl. The fractions
containing the pentasaccharide were pooled together and the compound
was desalted using a Sephadex G25F gel column (170 mL) equilibrated in
water. After freeze-drying, pentasaccharide 61 (30.5 mg, 90%) was
obtained. [a]D ˆ ‡41 (c ˆ 0.9 in water); 1H NMR (500 MHz, D2O): d ˆ
5.53 (d, J1,2 ˆ 3.4 Hz, 1H, H-1 GlcIII), 5.42 (d, J1,2 ˆ 3.9 Hz, 1H, H-1 GlcV),
5.15 (d, J1,2 ˆ 3.7 Hz, 1H, H-1 GlcI), 4.93 (d, J1,2 ˆ 7.8 Hz, 1H, H-1 IdoUAII),
4.67 (dd, J3,4 ˆ 8.3 Hz, 1H, H-3 GlcIII), 4.66 (d, J1,2 ˆ 8.1 Hz, 1H, H-1
GlcUAIV), 4.61 (dd, J3,4 ˆ 8.7 Hz, 1H, H-3 GlcI), 4.58 (dd, J5,6b ˆ 1.9 Hz, 1H,
H-6a GlcIII), 4.55 (dd, J5,6b ˆ 8.3 Hz, 1H, H-6a GlcI), 4.43 (dd, J2,3 ˆ 9.3 Hz,
[a]D
ˆ
13 (c ˆ 0.6 in chloroform); 1H NMR (250 MHz, CDCl3): d ˆ 7.25
(m, 15H, aromatic), 5.93 (dd, J7,8b ˆ 17.9, J7,8b ˆ 11.1 Hz, 1H, H-7 GlcII), 5.45
(dd, J8a,8b <1 Hz, 1H, H-8b GlcII), 5.10 (dd, 1H, H-8a GlcII), 5.07, 4.70 (2d,
J ˆ 11.6 Hz, 2H, CH2Ph), 4.70, 4.56 (2d, J ˆ 11.6 Hz, 2H, CH2Ph), 4.58 (d,
J1,2 ˆ 7.2 Hz, 1H, H-1 GlcII), 4.55 (d, J1,2 ˆ 3.9 Hz, 1H, H-1 GlcI), 4.48 (s, 2H,
CH2Ph), 3.85 (dd, J5,6b ˆ 7.8, J6a,6b ˆ 10.9 Hz, 1H, H-6b GlcI), 3.80 ± 3.60 (m,
4H, H-3 GlcI, H-4 GlcI, H-5 GlcI, H-6a GlcI), 3.62 (d, J3,4 ˆ 9.5 Hz, 1H, H-4
GlcII), 3.58 (s, 3H, OMe), 3.45, 3.30, 3.08 (3s, 9H, 3OMe), 3.48 (dd, J2,3
ˆ
9.2 Hz, 1H, H-2 GlcI), 3.12 ± 2.91 (m, 4H, H-2 GlcII, H-3 GlcII, H-6a GlcII,
‡
H-6b GlcII); CI-MS: 712 [M‡NH4 ].
Methyl 2,3,6-tri-O-benzyl-4-O-(benzyl 2,3-di-O-methyl-5-C-methoxymeth-
yl-a-l-idopyranosyluronate)-a-d-glucopyranoside (58): Ozone was bub-
bled into a cooled ( 788C) and stirred solution of disaccharide 57 (0.60 g,
0.87 mmol) in dichloromethane (12 mL) until the solution turned pale blue
(about 1 min). Dimethylsulfide was then introduced, the reaction mixture
was washed with water, and the organic layer was dried (MgSO4) and
concentrated. tert-Butanol (36 mL), 2-methyl-2-butene (14 mL) and water
(36 mL) were added to the crude aldehyde thus obtained, followed by
NaH2PO4 (1.5 g) and NaClO2 (1.5 g). After vigorous stirring for one night
at room temperature the mixture was partitioned between water and ethyl
acetate, the organic layer was dried (MgSO4), concentrated, and the crude
acid was used as such in the next step. It was dissolved in dry DMF (60 mL),
and tetrabutylammonium iodide (1.7 g, 4.58 mmol), potassium bicarbonate
(0.5 g, 5.44 mmol) and benzyl bromide (0.54 mL, 4.58 mmol) were added.
After stirring at room temperature for 6 h, the product was extracted with
diethyl ether, dried (MgSO4), concentrated, and purified by column
chromatography (ethyl acetate/cyclohexane 2:1). The ester 58 was thus
1H, H-2 GlcI), 4.33 (dd, J2,3 ˆ 8.7 Hz, 1H, H-2 GlcIII), 4.27 (m, 2H, J5,6b
ˆ
1.9 Hz, H-6a GlcV, J6a,6b ˆ 11.2 Hz, H-6b GlcIII), 4.23 (dd, J6a,6b ˆ 11.0 Hz,
1H, H-6b GlcI), 4.15 (ddd, J5,6a ˆ 1.9 Hz, 1H, H-5 GlcIII), 4.14 (dd, J6a,6b
ˆ
11.2 Hz, 1H, H-6b GlcV), 4.13 (ddd, J5,6a ˆ 2.2 Hz, 1H, H-5 GlcI), 4.03 (dd,
J3,4 ˆ 9.5 Hz, 1H, H-3 IdoUAII), 4.02 (ddd, J5,6a ˆ 1.9 Hz, 1H, H-5 GlcV),
3.98 (m, 2H, H-4 IdoUAII, H-4 GlcUAIV), 3.86 (dd, J4,5 ˆ 9.4 Hz, 1H, H-4
GlcIII), 3.83 (dd, J4,5 ˆ 9.5 Hz, 1H, H-4 GlcI), 3.60 (dd, J3,4 ˆ 9.2 Hz, 1H, H-3
GlcUAIV), 3.53 (dd, J3,4 ˆ 9.4 Hz, H1H, -3 GlcV), 3.36 (dd, J4,5 ˆ 10.0 Hz,
1H, H-4 GlcV), 3.28 (m, J2,3 ˆ 9.3 Hz, 2H, H-2 GlcUAIV, J2,3 ˆ 9.9 Hz, H-2
GlcV), 3.19 (dd, J2,3 ˆ 9.0 Hz, 1H, H-2 IdoUAII); ESI-MS (negative mode):
monoisotopic mass: 1797.95; calcd: 1799.30; found: 1799.0.
Acknowledgement
This work is part of a collaboration between N.V. Organon (Oss, The
Netherlands), and Sanofi Recherche on antithrombotic oligosaccharides.
We thank Isidore Lederman for technical assistance, and the Toulouse Staff
of the ªService dꢀAnalyse de la Recherche Amontº (C. Picard, Head) for
elemental analyses (M. Maftouh, S. Albugues), NMR analyses (C. Ponthus,
D. Albene, M. Rival), and MS analyses (F. Uzabiaga, V. Videau).
obtained (0.534 g, 77%) as a syrup. [a]D
ˆ
17 (c ˆ 0.3 in chloroform);
1H NMR (400 MHz, CDCl3): d ˆ 7.30 (m, 20H, aromatic), 5.08, 4.81 (2d,
J ˆ 12.3 Hz, 2H, CH2Ph), 5.02, 4.77 (2d, J ˆ 11.6 Hz, 2H, CH2Ph), 4.77, 4.63
(2d, J ˆ 11.6 Hz, 2H, CH2Ph), 4.71 (d, J1,2 ˆ 8.1 Hz, 1H, H-1 IdoUAII), 4.64
(d, J1,2 ˆ 3.2 Hz, 1H, H-1 GlcI), 4.61, 4.57 (2d, J ˆ 11.8 Hz, 2H, CH2Ph),
3.90 ± 3.70 (m, 6H, H-3 GlcI, H-4 GlcI, H-5 GlcI, H-6a GlcI, H-6b GlcI, H-4
IdoUAII), 3.68, 3.54, 3.42, 3.10 (4s, 12H, 4OMe), 3.50 ± 3.40 (m, 4H, H-2
GlcI, H-3 IdoUAII, H-6a IdoUAII, H-6b IdoUAII), 3.00 (t, J2,3 ˆ 8.1 Hz, 1H,
‡
H-2 IdoUAII); CI-MS: 820 [M‡NH4] .
[1] B. Casu, M. Petitou, A. Provasoli, P. SinayÈ, Trends Biochem. Sci. 1988,
13, 221 ± 225.
Pentasaccharide 59: A solution of trimethylsilyl trifluoromethanesulfonate
(0.05m, 0.65 mL) in dichloromethane (0.65 mL) was added at 208C to a
mixture of imidate 52 (120 mg, 0.112 mmol), disaccharide 58 (140 mg,
0.168 mmol), and finely grounded 4 Š molecular sieves (300 mg) in
dichloromethane (5 mL). After 30 min under stirring, triethylamine was
added until neutralisation. After filtration and concentration, the residue
was purified first using a Sephadex LH 20 gel column (dichloromethane/
ethanol 1:1), then over silica gel (hexane/acetone 65:35) to yield compound
59 (136 mg, 71%). [a]D ˆ ‡63 (c ˆ 0.88 in dichloromethane); 1H NMR
(500 MHz, CDCl3): d ˆ 5.43 (dd, H-3 GlcIII), 5.25 (d, J1,2 ˆ 3.8 Hz, H-1
GlcIII), 5.19 (d, J1,2 ˆ 3.8 Hz, H-1 GlcV), 5.07 (d, J1,2 ˆ 7.9 Hz, H-1 IdoUAII),
4.59 (d, J1,2 ˆ 3.8 Hz, H-1 GlcI), 4.49 (dd, H-6a GlcIII), 4.37 (d, J1,2 ˆ 7.6 Hz,
H-1 GlcUAIV), 4.24 (m, 2H, H-6a, H-6b GlcV), 4.22 (dd, H-6b GlcIII), 4.05
(d, H-4 IdoUAII), 4.03 (d, H-4 GlcUAIV), 3.97 (ddd, H-5 GlcIII), 3.86 (ddd,
H-5 GlcI), 3.79 (m, 2H, H-3, H-6a GlcI), 3.71 (dd, H-6b GlcI), 3.69 (dd, H-3
IdoUAII), 3.60 (ddd, H-5 GlcV), 3.55 (dd, H-4 GlcIII), 3.43 (m, 2H, H-2 GlcI,
GlcIII), 3.42 (dd, H-4 GlcI), 3.37 (dd, H-3 GlcV), 3.36 (dd, H-3 GlcUAIV),
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1
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Received: February 15, 2001 [F3078]
4834
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0947-6539/01/0722-4834 $ 17.50+.50/0
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