Further evidence for the critical role of a non-chair conformation of L-iduronic acid in the activation of antithrombin

Article abstract of DOI:10.1002/1099-0690(200211)2002:21<3595::AID-EJOC3595>3.0.CO;2-F

L-iduronic acid, a conformationally flexible monosaccharide, imparts a remarkable protein adaptability to the glycosaminoglycans heparin, heparan sulfate, and dermatan sulfate. The pentasaccharide representing the antithrombin binding site of heparin contains one such L-iduronic acid residue, the conformation of which has been suspected for a long time to be a critical factor in the interaction with antithrombin. We have recently synthesized pentasaccharides containing an L-iduronic acid residue conformationally forced to exist within a restricted arc (²S₀ ? ^2,5B ? ⁵S₁) of the overall pseudorotational circle. We could thus demonstrate that the ²S₀ conformation is adopted upon binding to the protein. In the present work, we now describe the synthesis of a similar pentasaccharide containing a slightly more flexible L-iduronic acid unit with a three-atom bridge between C-2 and C5 of the hexopyranose ring. This pentasaccharide is a better activator of AT-III with respect to blood coagulation factor Xa inhibition. These results confirm that L-iduronic acid adopts an unusual non-chair conformation close to ²S₀ and clearly explains how the unique conformational behavior of L-iduronic acid is the key to heparin's interaction with AT-III. Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002.

Full text of DOI:10.1002/1099-0690(200211)2002:21<3595::AID-EJOC3595>3.0.CO;2-F

FULL PAPER
Further Evidence for the Critical Role of a Non-Chair Conformation of
L
-Iduronic Acid in the Activation of Antithrombin
[b]
[a]
[a]
[c]
´
Jose Kovensky, Jean-Maurice Mallet, Jacques Esnault, Pierre-Alexandre Driguez,
[d]
[c]
[c]
[c]
´
Philippe Sizun, Jean-Pascal Herault, Jean-Marc Herbert, Maurice Petitou,* and
[a]
¨
Pierre Sinay*
Dedicated to Professor Marc Julia on the occasion of his 80th birthday
Keywords: Antithrombin / Carbohydrates / Glycosylation / Heparin / Iduronic acid
L
-iduronic acid, a conformationally flexible monosaccharide,
sent work, we now describe the synthesis of a similar pentas-
accharide containing a slightly more flexible -iduronic acid
unit with a three-atom bridge between C-2 and C5 of the
hexopyranose ring. This pentasaccharide is a better activator
of AT-III with respect to blood coagulation factor Xa inhibi-
imparts a remarkable protein adaptability to the glycosamin-
oglycans heparin, heparan sulfate, and dermatan sulfate.
The pentasaccharide representing the antithrombin binding
L
site of heparin contains one such L-iduronic acid residue, the
conformation of which has been suspected for a long time to
be a critical factor in the interaction with antithrombin. We
tion. These results confirm that
usual non-chair conformation close to 2S0 and clearly ex-
plains how the unique conformational behavior of -iduronic
L-iduronic acid adopts an un-
have recently synthesized pentasaccharides containing an
L-
L
iduronic acid residue conformationally forced to exist within acid is the key to heparin’s interaction with AT-III.
a restricted arc (2S0 ǟǞ 2,5B Ǟ 5S1) of the overall pseudorota-
ǟ
tional circle. We could thus demonstrate that the 2S0 con-
( Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany,
formation is adopted upon binding to the protein. In the pre-
2002)
Introduction
chains having different lengths. Various modifications
within the basic disaccharide unit, including two possible
epimers of the uronic acid (-iduronic and -glucuronic),
sulfation at the N, 3-O, 6-O position of the glucosamine
and 2-O position of the uronic acid, generate a considerable
amount of diversity.[2] Understanding the influence of this
complex structure of HLGAGs on their function, repres-
ents one of the most exciting challenge for glycoscientists.
Thus far, the interaction with antithrombin, wherein a spe-
cific pentasaccharide sequence has been identified,[3,4] and
then chemical synthesis used to unambiguously prove the
specificity of the interaction,[5,6] constitute the only example
of a sequence designed for a specific interaction with a pro-
tein.
Heparin-
or
heparan-like
glycosaminoglycans
(HLGAGs) are complex polysaccharides that play import-
ant roles in several biological functions by binding to differ-
ent growth factors, morphogens, enzymes, and cytokines.[1]
The basic structure of HLGAGs is a disaccharide compris-
ing a uronic acid residue linked 1Ǟ4 to a glucosamine. It
is repeated many times to form heterogeneous mixtures of
[a]
´
´
Departement de Chimie, associe au CNRS, Ecole Normale
´
Superieure
24, Rue Lhomond; 75231 Paris cedex 05, France
Fax: (internat.) ϩ33-1/4432-33-97
E-mail: pierre.sinay@ens.fr
[b]
Research Member of the Consejo Nacional de Investigaciones
de la Republica Argentina (CONICET). Departamento de
It was first suspected,[7Ϫ9] then proved,[10,11] that in this
pentasaccharide the unusual[12,13] conformational proper-
ties of the single α--iduronate residue have a strong effect
on its interaction with antithrombin. Thus, whereas the -
iduronate ring can easily change its conformation between
4C1, 1C4 and 2S0, we recently showed[10] that the latter must
be adopted to efficiently bind to the protein antithrombin.
We could reach this conclusion by comparing the ability of
various conformationally constrained pentasaccharides to
´
´
´
Quımica Organica, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires,
Ciudad Universitaria, Pab. II, 3er. Piso, 1428 Buenos Aires,
Argentina
[c]
´
´
Departement Cardiovasculaire/Thrombose, Sanofi-Synthelabo,
195, Route dЈEspagne; 31036 Toulouse cedex, France
Fax: (internat.) ϩ 33-5/6116-2286
E-mail: maurice.petitou@sanofi-synthelabo.com
[d]
´
DARA, Sanofi-Synthelabo,
371, Rue du Professeur Joseph Blayac, 34184 Montpellier
cedex, France
Eur. J. Org. Chem. 2002, 3595Ϫ3603  2002 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim 1434Ϫ193X/02/1121Ϫ3595 $ 20.00ϩ.50/0
3595

M. Petitou, P. Sinay¨ et al.
FULL PAPER
activate antithrombin with respect to blood coagulation as an equilibrium between the three conformers C4 Ǟǟ 2S0
1
factor Xa inhibition.
Ǟ 4C1. The problem we addressed is how to confine the
ǟ
2
In this previous work, the iduronate residue was con- system so that only the S0 form can be formed. Inspection
formationally forced to exist for stringent geometrical of simple molecular models clearly shows that a covalent
reasons within the restricted section of the pseudorotational link between carbon atoms two and five would confine the
itinerary[14] of the pyranoid ring shown in Figure 1. For this itinerary to the section S0 ǟǞ 2,5B Ǟ 5S1 for geometrical
2
ǟ
purpose we used a two-atom bridge between C-2 and C-5 reasons (see Figure 1). More precisely, a one-atom bridge
2
to lock the ring in the S0 conformation. In the test for would probably force the pyranose ring to strictly exist as
factor Xa inhibition, the resulting pentasaccharide was its 2,5B conformation, whereas a longer spacer would allow
slightly less active than the reference compound. Molecular a restricted equilibration to take place, and thus facilitate
models’ examination as well as molecular modeling indic- the emergence of the desired 2S0 form wherein the substitu-
ated that introducing one more atom into the bridge might ents are in favorable equatorial and isoclinal orientations.
result in some more flexibility whilst maintaining the con- In order to select the most appropriate synthetic target, mo-
2
formation close to S0. Thus, in the present article we de- lecular modeling experiments were performed using the
scribe a similar pentasaccharide in which one more (car- SIBYL force field[17] as parameterized for carbohydrates.[18]
bon) atom has been introduced into the bridge. The com- These computations showed that in the case of -iduronic
2
pound thus obtained displays about the same ability as the acid, the best way to favor the S0 conformation would be
reference compound with respect to factor Xa inhibition. to use a three-atom bridge, which led us to select the pentas-
2
This result confirms that the S0 conformer of -iduronic accharide 2 as our synthetic target.
acid plays a key role during the process of antithrombin
The developed synthetic strategy is detailed in Schemes
activation by the pentasaccharides. Whether the other con-
1Ϫ3. According to previously developed chemistry,[10,11] the
formers play a role[15] in the many interactions where
key disaccharide 10 is well suited, after inversion of config-
HLGAGs are involved remains to be established.
uration at C2Ј, to bridge C2Ј and C5Ј. It can be obtained
by condensation of 8 and the known[19] alcohol 9
(Scheme 1) The aldehyde 3, obtained as already de-
scribed,[20] reacted with vinylmagnesium bromide to give,
after treatment, a mixture of the two alcohols 4, which was
purified by chromatography (89%). Hydroboration followed
Figure 1. The selected section of the boat/skew-boat pseudorota-
tional itinerary of the pyranoid ring
by oxidation of the borane delivered the diols 5 (56%). Sub-
sequent selective silylation of the primary alcohol (85%),
Swern oxidation and addition of vinylmagnesium bromide
to the ketone delivered the tertiary alcohol 7 as a single
Results and Discussion
isomer (82%). The configurational assignment is based on
The synthetic biologically active pentasaccharide 1[16] the subsequent transformations. The face-selective addition
(Figure 2) already used in previous work was chosen as the of vinylmagnesium bromide to the ketone derived from 6 is
reference compound. The conformation of the iduronate explained by a chelation of the magnesium to the ring oxy-
unit of this molecule in aqueous solution is best represented gen. Acid hydrolysis of 7 followed by acetylation gave the
Figure 2. The known biologically active pentasaccharide 1 was selected as the reference compound; in the synthetic mimetic 22, the -
iduronate ring has been locked in the 2S0 conformation by connecting O-2 and C-5 through an ethylidene bridge; the letter code DEFGH
is routinely used to designate the five monosaccharide units of the antithrombin binding sequence in heparin
3596
Eur. J. Org. Chem. 2002, 3595Ϫ3603

A Non-Chair Conformation of L-Iduronic Acid
FULL PAPER
Scheme 1. a. CH2ϭCHMgBr, b. (i) BH3, (ii) H2O2, c. TBSCl, Et3N, CH2Cl2, d. (i) (CO)2Cl2, DMSO, Et3N, (ii) CH2ϭCHMgBr, e. (i)
Amberlite IR-120 (Hϩ), H2O, (ii) Ac2O, pyridine, f. TMSOTf, g. (i) MeONa, MeOH, (ii) (CH3O)2C(CH3)2, PTSA, h. (i) (CO)2Cl2,
DMSO, Et3N, (ii) LiEt3BH, i. Ac2O, pyridine, j. AcOH, k. TsCl; pyridine
desired β-acetate 8 (65%), ready for glycosylation with 9. which, after cyclisation (the isomeric tetrahydropyranyl
Selective 1,2-trans glycosylation[21] of the known alcohol 9 ethers were separated; both of them were used in the syn-
occurred in the presence of TMS triflate to give the ex- thesis of 18, which was obtained in different yields de-
pected disaccharide 10 in 80% yield.
pending on the isomer), was easily released to give the de-
The next step was the inversion of configuration at C-2Ј. sired C2ЈϪC5Ј bridged disaccharide 20, a locked protected
After deacetylation, protection of the 4Ј-7Ј diol system was equivalent of the GH part of the reference pentasaccharide 1.
achieved though the isopropylidene acetal 11. The config-
The known[22] trisaccharide imidate 21 already used for
uration at C-2 was then inverted by an oxidation-reduction the preparation of 1 was now condensed with the alcohol
(Swern-lithium triethylborohydride) sequence to give 12 20 to give the protected pentasaccharide 22 in modest (48%)
that was acetylated before acid hydrolysis to give the diol yield (Scheme 3). The transformation into the target pentas-
14 (70% from 10).
accharide 2 was achieved by a sequence of well-established
The key cyclisation could then be envisioned. In a first reactions.[23]
1
attempt we tried to avoid the protection of OH-4Ј. How-
The H NMR coupling constants of the locked -idu-
ever, when the tosyl derivative 15 was directly submitted ronic acid residue in G in the synthetic pentasaccharide 2
to cyclisation conditions (ethanol, sodium hydroxide), the (J1,2 ϭ 3.8, J2,3 ϭ 4.0, J3,4 ϭ 3.0 Hz) are somewhat different
product obtained, after ozonolysis and esterification, was from those calculated for a restrained ideal 2S0 form.[11]
the 4Ј-O cyclised compound 16 (Scheme 2). It was therefore This is especially true for the J3,4 value, which in any case
necessary to protect HO-4Ј as a tetrahydropyranyl ether is expected to change very rapidly with the dihedral angle.
Eur. J. Org. Chem. 2002, 3595Ϫ3603
3597

M. Petitou, P. Sinay¨ et al.
FULL PAPER
Scheme 2. a. NaOH, EtOH, b. (i) O3, Me2S, (ii) 2-methyl-2-butene, tBuOH, H2O, NaH2PO4, NaClO2, (iii) BnBr, Bu4NI, KHCO3, DMF,
c. DHP, CSA, d. NaOH, EtOH, e. MeOH, PTSA, f. (i) O3, Me2S, (ii) 2-methyl-2-butene, tBuOH, H2O, NaH2PO4, NaClO2, (iii) BnBr,
Bu4NI, KHCO3, DMF
Scheme 3. A. TMSOTf, b. (i) H2, Pd/C, (ii) NaOH, c. Et3N·SO3, DMF
The three conformers 2S0, 5S1 and 2,5B — or other interme- very close to that of the reference synthetic pentasaccharide
diate forms — are rather close in energy. Upon binding to 1 (1208 Ϯ 23 U/mg), and slightly better than that of the
2
AT-III, the S0 form can easily be adopted.
analogue pentasaccharide with a constrained -iduronic
Much to our delight the pentasaccharide 2 displays an acid having only one carbon atom to bridge C2 and C5
anti-factor Xa biological activity (1198 Ϯ 20 U/mg) that is (1073 Ϯ 61 U/mg).[10,11]
3598
Eur. J. Org. Chem. 2002, 3595Ϫ3603

A Non-Chair Conformation of L-Iduronic Acid
FULL PAPER
OH), 1.50 and 1.34 (two s, 6 H, Me). C11H18O5 (230.26): C 57.37,
H 7.88; found C 57.30, H 7.93.
Experimental Section
General Remarks: Melting points were determined with a Büchi
model 535 melting point apparatus and are uncorrected. Optical
rotations were measured at 20 Ϯ 2 °C with a PerkinϪElmer Model
241 digital polarimeter, using a 10 cm, 1 mL cell. Chemical Ionis-
ation Mass Spectra (CI-MS, ammonia) and Fast Atom Bombard-
ment Mass Spectra (FAB-MS) were obtained with a JMS-700 spec-
trometer. Elemental analyses were performed by Service de Mic-
6-Deoxy-1,2-O-isopropylidene-3-O-methyl-α-
(5a) and 6-Deoxy-1,2-O-isopropylidene-3-O-methyl-β-
D
-gluco-heptofuranose
-ido-heptofu-
L
ranose (5b): A 1  solution of BH3·THF complex in THF (19.6 mL,
19.6 mmol) was added to a solution of 4 (2.26 g, 9.81 mmol) in dry
THF (25 mL). The mixture was stirred at room temperature for
4 h, and then ethanol (10 mL) was slowly added, followed by 3 
NaOH (16.3 mL, 49.0 mmol) and 30% H2O2 (11.8 mL). After 2 h,
the mixture was poured into ice water and extracted three times
with dichloromethane. The organic layer was dried (MgSO4), fil-
tered and concentrated. Flash chromatography on silica gel (1:2,
cyclohexane/ethyl acetate) gave a mixture of 5a and 5b (1.43 g, 59%
yield) as a syrup.
´
roanalyse de lЈUniversite Pierre et Marie Curie, 4 Place Jussieu,
75005 Paris, France. 1H NMR spectra were recorded with a Bruker
AC 250 or a Bruker DRX 400 or a Bruker Avance 600 spectrometer
for solutions in CDCl3, CD3OD or D2O at ambient temperature.
Assignment were aided by COSY experiments. 13C NMR spectra
were recorded at 62.9 MHz with a Bruker AC 250, 100.6 MHz with
a Bruker DRX 400 or at 150.9 MHz with a Bruker DRX 600 spec-
trometer for solutions in CDCl3 adopting δ ϭ 77.00 ppm for the
central line of CDCl3. Assignments were aided by the J-mod tech-
nique and proton-carbon correlation. Reactions were monitored by
thin-layer chromatography (TLC) on a precoated plate of silica gel
60 F254 (layer thickness 0.2 mm; E. Merck, Darmstadt, Germany)
and detection was performed by charring with sulfuric acid. Flash
column chromatography was performed on silica gel 60 (230Ϫ400
mesh, E. Merck).
1
5a: H NMR (250 MHz, CDCl3): δ ϭ 5.97 (d, J1,2 ϭ 3.9 Hz, 1 H,
H-1), 4.61 (d, 1 H, H-2), 4.20Ϫ4.05 (m, 2 H, H-4, H-5), 3.92Ϫ3.82
(m, 2 H, H-7a, H-7b), 3.90 (d, J3,4 ϭ 3.3 Hz, 1 H, H-3), 3.42 (s, 3
H, OMe), 3.10 (br. s, 1 H, OH), 1.95Ϫ1.75 (m, 2 H, H-6a, H-6b),
1.50 and 1.33 (two s, 6 H, Me) ppm.
1
5b: H NMR (250 MHz, CDCl3): δ ϭ 5.93 (d, J1,2 ϭ 3.9 Hz, 1 H,
H-1), 4.61 (d, 1 H, H-2), 4.20Ϫ4.05 (m, 2 H, H-4, H-5), 3.92Ϫ3.82
(m, 2 H, H-7a, H-7b), 3.78 (d, J3,4 ϭ 3.6 Hz, 1 H, H-3), 3.48 (s, 3
H, OMe), 3.10 (br. s, 1 H, OH), 1.95Ϫ1.75 (m, 2 H, H-6a, H-6b),
1.50 and 1.33 (two s, 6 H, Me). C10H20O6 (248.27): C 53.21, H
8.12; found C 53.21, H 8.30.
The biological activity of the pentasaccharide discussed in the pre-
sent study was determined as follows: Values are mean anti-factor
Xa units/mg (n ϭ 3). 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 ad-
apted from Teien and Lie.[24] For an accurate comparison, com-
7-O-tert-Butyldimethylsilyl-6-deoxy-1,2-O-isopropylidene-3-O-
methyl-α-D-gluco-heptofuranose (6a) and 6-Deoxy-1,2-O-isopropyli-
dene-3-O-methyl-β-L-ido-heptofuranose (6b): The diol 5 (1.19 g,
4.8 mmol) was dissolved in dry dichloromethane (12 mL) and then
tert-butyldimethylsilyl chloride (1.0 g, 6.6 mmol), triethylamine
(1.0 mL, 7.2 mmol) and dimethylaminopyridine (15 mg) were ad-
ded. The reaction mixture was stirred at room temperature. After
2 h it was diluted with dichloromethane and washed with water.
The organic layer was dried (MgSO4), concentrated and the residue
was purified by silica gel column chromatography (9:1, cyclohex-
ane/ethyl acetate) to give a mixture of 6a and 6b (1.47 g, 85% yield)
as a syrup.
1
pound concentrations were determined by H NMR spectroscopy
with reference to an internal standard.
6,7-Dideoxy-1,2-O-isopropylidene-3-O-methyl-α-
enofuranose (4a) and 6,7-Dideoxy-1,2-O-isopropylidene-3-O-methyl-
β- -ido-hept-6-enofuranose (4b): A solution of sodium metaperiod-
D-gluco-hept-6-
L
ate (8.13 g, 38 mmol) in methanol (150 mL) was added at 0 °C to
a solution of 1,2-O-isopropylidene-3-O-methyl-α--glucofuranose
(8.47 g, 36.2 mmol) in 150 mL of methanol,. After 1 h, the solvent
was removed, and the residue extracted with ethyl acetate. The
combined organic extracts were dried (MgSO4), concentrated and
the dry residue 3 (7.34 g) was used directly for the next step.
1
6a: H NMR (250 MHz, CDCl3): δ ϭ 5.89 (d, J1,2 ϭ 3.8 Hz, 1 H,
H-1), 4.52 (d, 1 H, H-2), 4.08Ϫ3.65 (m, 6 H, H-3, H-4, H-5, H-7a,
H-7b, OH), 3.32 (s, 3 H, OMe),1.98Ϫ1.60 (m, 2 H, H-6a, H-6b),
1.40 and 1.25 (two s, 6 H, Me), 0.80 (s, 9 H, Me3C), Ϫ0.02 (s, 6 H,
Me2Si) ppm.
1
6b: H NMR (250 MHz, CDCl3): δ ϭ 5.81 (d, J1,2 ϭ 3.8 Hz, 1 H,
Crude 3 (7.34 g) was dissolved in dry THF(200 mL) and a 1 
solution of vinylmagnesium bromide in THF (55 mL, 55.0 mmol)
was added at room temperature. The solution was then heated at
80 °C for 2 h. The reaction mixture was quenched with NH4Cl
and extracted with water. The organic layer was dried (MgSO4),
concentrated and the residue was purified by silica gel column
H-1), 4.50 (d, 1 H, H-2), 4.08Ϫ3.65 (m, 6 H, H-3, H-4, H-5, H-7a,
H-7b, OH), 3.40 (s, 3 H, OMe),1.98Ϫ1.60 (m, 2 H, H-6a, H-6b),
1.40 and 1.25 (two s, 6 H, Me), 0.80 (s, 9 H, Me3C), 0.00 (s, 6
H, Me2Si). C17H34O6Si (362.53): C 56.32, H 9.45; found C 56.26,
H 9.46.
chromatography (4:1, cyclohexane/ethyl acetate) to give a 1:1.2 7-O-tert-Butyldimethylsilyl-6-Deoxy-1,2-O-isopropylidene-3-O-
mixture of 4a and 4b (7.40 g, 89% from 1,2-O-isopropylidene-3-O-
methyl-α--glucofuranose) as a syrup.
methyl-5-C-vinyl-α-D-gluco-heptofuranose (7): Oxalyl chloride
(0.67 mL, 7.72 mmol) and dimethyl sulfoxide (1.10 mL,
15.44 mmol) were added to dry dichloromethane (2 mL) at Ϫ78 °C
4a: 1H NMR (250 MHz, CDCl3): δ ϭ 6.10Ϫ5.80 (m, 2 H, H-1, H-
6), 5.44 (dd, J7a,7b ϭ 1.8, J6,7a ϭ 17.2 Hz, 1 H, H-7a), 5.25 (dd, and stirred for 30 min. Then, a solution of compound 6 (1.4 g,
6,7b ϭ 10.6 Hz, 1 H, H-7b), 4.58 (d, J1,2 ϭ 4.0 Hz, 1 H, H-2), 4.50 3.86 mmol) in dichloromethane (6 mL) was added and stirred for
(m, 1 H, H-5), 4.20 (dd, J3,4 ϭ J4,5 ϭ 3.1 Hz, 1 H, H-4), 3.88 (d, 1 another 1 h. Triethylamine (3.23 mL, 23.16 mmol) was added and
J
H, H-3), 3.45 (s, 3 H, OMe), 2.95 (d, J5,OH ϭ 7.8 Hz, 1 H, OH),
1.50 and 1.34 (two s, 6 H, Me) ppm.
the mixture was kept at Ϫ60 °C for 5 min, then allowed to warm
slowly to room temperature. After 30 min the reaction mixture was
diluted with dichloromethane and washed with sat. aq NaCl, dried
4b: 1H NMR (250 MHz, CDCl3): δ ϭ 6.10Ϫ5.80 (m, 2 H, H-1, H-
6), 5.44 (dd, J7a,7b ϭ 1.8, J6,7a ϭ 17.2 Hz, 1 H, H-7a), 5.25 (dd, (MgSO4), and concentrated . The crude ketone was dissolved in
6,7b ϭ 10.6 Hz, 1 H, H-7b), 4.60 (d, J1,2 ϭ 4.0 Hz, 1 H, H-2), 4.50 dry THF (20 mL) and vinylmagnesium bromide (1  solution in
J
(m, 1 H, H-5), 4.04 (dd, J3,4 ϭ 3.4, J4,5 ϭ 6.7 Hz, 1 H, H-4), 3.70 THF, 5.8 mL, 5.79 mmol) was added at 0 °C. After 1 h the reaction
(d, 1 H, H-3), 3.40 (s, 3 H, OMe), 2.68 (d, J5,OH ϭ 1.65 Hz, 1 H, mixture was quenched with NH4Cl and extracted with ethyl acet-
Eur. J. Org. Chem. 2002, 3595Ϫ3603
3599

M. Petitou, P. Sinay¨ et al.
FULL PAPER
ate. The organic layer was dried (MgSO4), concentrated and the
(0.5 mL) and p-toluenesulfonic acid (catalytic) were added. The re-
residue was purified by silica gel column chromatography (9:1, action mixture was stirred at room temperature overnight. The
cyclohexane/ethyl acetate) to give the desired compound 7 (1.27 g, solvent was removed, the residue was partitioned between water
82% yield) as a syrup. [α]2D0 ϭ Ϫ52 (c ϭ 0.95, CHCl3). 1H NMR
(250 MHz, CDCl3): δ ϭ 5.95 (d, J1,2 ϭ 3.7 Hz, 1 H, H-1), 5.87 (dd,
J8,9a ϭ 17.2, J8,9b ϭ 10.6 Hz, 1 H, H-8), 5.45 (dd, J9a,9b ϭ 2.0 Hz,
and chloroform. The organic layer was dried (MgSO4), and concen-
trated. To prevent decomposition, the crude isopropylidene derivat-
ive was used directly for the next step. A small (20 mg) quantity
1 H, H-9a), 5.22 (dd, 1 H, H-9b), 4.53 (d, 1 H, H-2), 4.24 (s, 1 H, was chromatographed (3:1 cyclohexane/ethyl acetate) for character-
OH), 4.02 (d, J3,4 ϭ 3.2 Hz, 1 H, H-4), 3.85 (d, 1 H, H-3), ization of 11 (syrup). [α]2D0 ϭ 0.0° (c ϭ 1.5, CHCl3). 1H NMR
3.90Ϫ3.69 (m, 2 H, H-7a, H-7b), 3.40 (s, 3 H, OMe), 2.05Ϫ1.85 (250 MHz, CDCl3): δ ϭ 7.46Ϫ7.20 (m, 15 H, arom.), 6.05 (dd,
(m, 2 H, H-6a, H-6b), 1.49 and 1.31 (two s, 6 H, Me), 0.89 (s, 9 H,
Me3C), 0.05 (s, 6 H, Me2Si). C19H36O6Si (388.57): C 58.72, H 9.34;
found C 58.81, H 9.38.
J8Ј,9Јa ϭ 18.0, J8Ј,9Јb ϭ 11.2 Hz, 1 H, H-8Ј), 5.56 (dd, J9Јa,9Јb ϭ
1.6 Hz, 1 H, H-9Јa), 5.12 (dd, 1 H, H-9Јb), 5.05 and 4.82 (two d,
2 H, CH2Ph), 4.77 (d, 1 H, CHPh), 4.65 (d, J1Ј,2Ј ϭ 7.9 Hz, 1 H,
H-1Ј), 4.62Ϫ4.55 (m, 4 H, H-1, CH2Ph), 4.00Ϫ3.25 (m, 10 H, H-
2, H-3, H-4, H-5, H-6a, H-6b, H-2Ј, H-4Ј, H-7Јa, H-7Јb), 3.60 (s,
3 H, OMe), 3.35 (s, 3 H, OMe), 3.08 (dd, J2Ј,3Ј ϭ J3Ј,4Ј ϭ 9.6 Hz,
1 H, H-3Ј), 2.60 (d, J2Ј,OH ϭ 2.0 Hz, 1 H, OH), 1.80Ϫ1.00 (m, 2
H, H-6Јa, H-6Јb), 1.40 and 1.32 (two s, 6 H, Me). CI-MS: m/z ϭ
738 [M ϩ NH4ϩ]. C41H52O11 (720.84): C 68.31, H 7.27; found C
68.24, H 7.41.
1,2,4,7-Tetra-O-acetyl-6-deoxy-3-O-methyl-5-C-vinyl-β-D-gluco-
heptopyranose (8): Compound 7 (1.20 g, 3.09 mmol) was dissolved
in water (17 mL), Amberlite IR-120 (Hϩ form, 0.35 g) was added,
and the mixture was heated at 80 °C for 16 h. The resin was then
filtered off and the filtrate concentrated. The residue was dissolved
in pyridine (7 mL) and acetic anhydride (6 mL) was added. After
stirring overnight at room temperature, the excess acetic anhydride
was quenched by methanol and solvents were removed on a rotary
evaporator. The residue was extracted between water and dichloro-
methane. The organic layer was then dried (MgSO4) and concen-
trated. Flash chromatography on silica gel (4:1, cyclohexane/ethyl
acetate) yielded 8 (0.805 g, 65%) as a white solid, m.p. 136 °C.
Methyl 2,3,6-Tri-O-benzyl-4-O-(2-O-acetyl-6-deoxy-3-O-methyl-5-
C-vinyl-β-D-manno-hepto-pyranosyl)-α-D-glucopyranoside (14): Ox-
alyl chloride (0.11 mL, 1.24 mmol) and dry DMSO (0.18 mL,
2.48 mmol) were stirred in dry dichloromethane (2 mL) at Ϫ78 °C
for 30 min. Compound 11 (450 mg, 0.62 mmol) in dry dichlorome-
thane (2 mL) was added to the solution and the mixture stirred for
another 45 min. Triethylamine (0.52 mL, 3.72 mmol) was added at
Ϫ60 °C, and the mixture was kept for 5 min at this temperature,
then allowed to warm slowly to room temperature. After dilution
with dichloromethane, the organic layer was washed with sat. aq
NaCl, dried (MgSO4), concentrated and the residue was used dir-
ectly for the next reaction without further purification. The crude
ketone was dissolved in dry THF (7 mL) and lithium triethyl-
borohydride (1  solution in THF, 1.24 mL, 1.24 mmol) was added
at Ϫ 78 °C. The reaction mixture was allowed to stir at room tem-
perature for 1 h and then 5% sodium hydroxide (1 mL) and hydro-
gen peroxide (0.5 mL) were added. The solvent was removed and
the residue was partitioned between water and ethyl acetate. The
organic layer was dried (MgSO4), concentrated and the residue
containing crude 12 was acetylated directly with pyridine (3 mL)
and acetic anhydride (0.5 mL). After stirring overnight at room
temperature, the excess pyridine and acetic anhydride were removed
on a rotary evaporator and the residue 13 was used directly for
isopropylidene deprotection with 80% acetic acid (5 mL) at 60 °C
for 2 h. After solvent evaporation, the residue was purified by silica
gel column chromatography (3:2, cyclohexane/ethyl acetate) to
[α]2D0 ϭ Ϫ75 (c ϭ 1.0, CHCl3). H NMR (250 MHz, CDCl3): δ ϭ
1
5.82 (dd, J8,9a ϭ 17.8, J8,9b ϭ 10.7 Hz, 1 H, H-8), 5.80 (d, J1,2
8.2 Hz, 1 H, H-1), 5.67 (dd, J9a,9b ϭ 1.8 Hz, 1 H, H-9a), 5.47 (dd,
1 H, H-9b), 5.02 (d, J3,4 ϭ 10.1 Hz, 1 H, H-4), 5.01 (dd, J2,3
ϭ
ϭ
9.4 Hz, 1 H, H-2), 4.05 (t, J6,7 ϭ 7.3 Hz, 2 H, H-7a, H-7b), 3.41
(t, 1 H, H-3), 3.40 (s, 3 H, OMe), 2.08, 2.02, 1.99 and 1.93 (four s,
12 H, Ac), 1.84 (dd, J6a,6b ϭ 15.0 Hz, 1 H, H-6a), 1.72 (dd, 1 H,
H-6b). C18H26O10 (402.39): C 53.72, H 6.51; found C 53.86, H 6.66.
Methyl
methyl-5-C-vinyl-β-
2,3,6-Tri-O-benzyl-4-O-(2,4,7-tri-O-acetyl-6-deoxy-3-O-
-gluco-heptopyranosyl)-α- -glucopyranoside
D
D
(10): Compound 8 (395 mg, 0.98 mmol) and methyl 2,3,6-tri-O-
benzyl-α--glucopyranoside (9, 500 mg, 1.08 mmol) were dissolved
in dry dichloromethane (12 mL) and the reaction mixture was
stirred at room temperature for 1 h in the presence of molecular
sieves (1.0 g) and then cooled to Ϫ78 °C . Trimethylsilyl trifluoro-
methanesulfonate (0.23 mL, 1.24 mmol) was added and the mixture
was slowly allowed to reach room temperature. After 2 h, the reac-
tion mixture was quenched with N,N-diisopropyl-N-ethylamine, fil-
tered through celite, and concentrated. Silica gel column chromato-
graphy (4:1, cyclohexane/ethyl acetate) gave the desired compound
1
10 (628.3 mg, 80%) as a syrup. [α]2D0 ϭ Ϫ14 (c ϭ 1.0, CHCl3). H
yield the diol 14 as a colourless oil (342.7 mg, 70% from 10). [α]2D0
Ϫ28 (c ϭ 1.07, CHCl3). 1H NMR (250 MHz, CDCl3): δ ϭ
7.50Ϫ7.20 (m, 15 H, arom.), 5.90 (dd, J8Ј,9Јa ϭ 18.0, J8Ј,9Јb
ϭ
NMR (250 MHz, CDCl3): δ ϭ 7.42Ϫ7.20 (m, 15 H, arom.), 5.82
(dd, J8Ј,9Јa ϭ 18.0, J8Ј,9Јb ϭ 11.2 Hz, 1 H, H-8Ј), 5.45 (dd, J9Јa,9Јb
Ͻ 1 Hz, 1 H, H-9Јa), 5.25 (dd, 1 H, H-9Јb), 5.05Ϫ4.90 (m, 2 H,
ϭ
11.2 Hz, 1 H, H-8Ј), 5.52 (dd, J9Јa,9Јb ϭ 1.5 Hz, 1 H, H-9Јa), 5.30
(m, 2 H, H-2Ј, H-9Јb), 5.10 (d, 1 H, CHPh), 4.90 (d, J1Ј,2Ј ϭ 1.0 Hz,
1 H, H-1Ј), 4.84Ϫ4.47 (m, 6 H, H-1, CHPh), 3.95Ϫ3.45 (m, 9 H,
H-2, H-3, H-4, H-5, H-6a, H-6b, H-4Ј, H-7Јa, H-7Јb), 3.35 (s, 3 H,
OMe), 3.31 (s, 3 H, OMe), 3.01 (dd, J2Ј,3Ј ϭ 3.0, J3Ј,4Ј ϭ 10.1 Hz,
1 H, H-3Ј), 2.90Ϫ2.70 (broad s, 2 H, OH), 2.02 (s, 3 H, Ac),
1.95Ϫ1.60 (m, 2 H, H-6Јa, H-6Јb). CI-MS: m/z ϭ 740 [M ϩ NH4ϩ].
C40H50O12 (722.82): C 66.46, H 6.97; found C 65.90, H 7.33.
H-2Ј, H-4Ј), 4.86 and 4.75 (two d, 2 H, CH2Ph), 4.84 (d, J1Ј,2Ј
ϭ
8.3 Hz, 1 H, H-1Ј), 4.82 and 4.64 (two d, 2 H, CH2Ph), 4.70 and
4.50 (two d, 2 H, CH2Ph), 4.55 (d, J1,2 ϭ 3.7 Hz, 1 H, H-1),
4.20Ϫ3.25 (m, 9 H, H-2, H-3, H-4, H-5, H-6a, H-6b, H-3Ј, H-7Јa,
H-7Јb), 3.36 and 3.34 (two s, 6 H, OMe), 2.14, 1.94 and 1.93 (three
s, 9 H, Ac), 1.80Ϫ1.60 (m, 2 H, H-6Јa, H-6Јb). C44H54O14 (806.89):
C 65.49, H 6.75; found C 65.60, H 6.88.
Methyl 2,3,6-Tri-O-benzyl-4-O-(4,7-O-isopropylidene-6-deoxy-3-O-
methyl-5-C-vinyl-β-
D
-gluco-heptopyranosyl)-α-
D
-glucopyranoside
Methyl 2,3,6-Tri-O-benzyl-4-O-(2-O-acetyl-6-deoxy-3-O-methyl-7-
(11): Compound 10 (530 mg, 0.66 mmol) was dissolved in meth-
anol (40 mL), sodium (catalytic) was added at 0 °C and the re-
sulting mixture was allowed to stir at room temperature for 8 h.
The solvent was removed on a rotary evaporator, the residue was
redissolved in dry acetone (20 mL) and then 2,2-dimethoxypropane
O-tosyl-5-C-vinyl-β-D-manno-heptopyranosyl)-α-D-glucopyranoside
(15): The diol 14 (317.4 mg, 0.44 mmol) was dissolved in dry
dichloromethane (10 mL) and tosyl chloride (150 mg, 0.79 mmol),
triethylamine (0.14 mL, 1.0 mmol) and dimethylaminopyridine
(10 mg) were added. The reaction mixture was stirred at room tem-
3600
Eur. J. Org. Chem. 2002, 3595Ϫ3603

A Non-Chair Conformation of L-Iduronic Acid
perature overnight and then it was diluted with dichloromethane (0.32 mL, 3.45 mmol) was then added dropwise. The reaction mix-
FULL PAPER
and washed with water. The organic layer was dried (MgSO4), con-
centrated and the residue was purified by silica gel column chroma-
tography (5:2, cyclohexane/ethyl acetate) to give 15 (349.1 mg, 91%)
ture was stirred for 20 min and neutralised with K2CO3, filtered,
and the solvent was evaporated to dryness. Silica gel column chro-
matography (5:2, cyclohexane/ethyl acetate) gave the two diastereo-
mers 17a (845.0 mg) and 17b (186 mg) with a total yield of 93%.
1
as a syrup. [α]2D0 ϭ Ϫ20 (c ϭ 1.32, CHCl3). H NMR (250 MHz,
CDCl3): δ ϭ 7.70Ϫ7.20 (m, 19 H, arom.), 5.85 (dd, J8Ј,9Јa ϭ 18.0, 17a: Syrup. [α]2D0 ϭ Ϫ3 (c ϭ 0.7, CHCl3). 1H NMR (400 MHz,
J8Ј,9Јb ϭ 11.2 Hz, 1 H, H-8Ј), 5.40 (dd, J9Јa,9Јb ϭ 1.5 Hz, 1 H, H- CDCl3): δ ϭ 7.70Ϫ7.20 (m, 19 H, arom.), 5.85 (dd, J8Ј,9Јa ϭ 18.0,
9Јa), 5.20 (m, 2 H, H-2Ј, H-9Јb), 5.00 (d, 1 H, CHPh), 4.82 (d, J8Ј,9Јb ϭ 11.2 Hz, 1 H, H-8Ј), 5.40 (dd, J9Јa,9Јb ϭ 0.8 Hz, 1 H, H-
J1Ј,2Ј ϭ 1.2 Hz, 1 H, H-1Ј), 4.80Ϫ4.55 (m, 5 H, H-1, CHPh), 4.50
(d, 1 H, CHPh), 4.05Ϫ3.50 (m, 9 H, H-2, H-3, H-4, H-5, H-6a, H- 1 H, J8Ј,9Јb ϭ 11.2 Hz, H-9Јb), 5.05 (d, 1 H, CHPh), 4.85 (d, J1Ј,2Ј
6b, H-4Ј, H-7Јa, H-7Јb), 3.37 (s, 3 H, OMe), 3.25 (s, 3 H, OMe), 1.0 Hz, 1 H, H-1Ј), 4.80Ϫ4.65 (m, 6 H, H-1, CHPh, CH), 4.52 (d,
2.90 (dd, J2Ј,3Ј ϭ 3.0, J3Ј,4Ј ϭ 10.1 Hz, 1 H, H-3Ј), 2.40 (s, 3 H, 1 H, CHPh), 4.20Ϫ4.05 (m, 2 H, CH2), 3.90Ϫ3.45 (m, 9 H, H-2,
9Јa), 5.25 (dd, J1Ј,2Ј ϭ 0.7, J2Ј,3Ј ϭ 2.8 Hz, 1 H, H-2Ј), 5.18 (br. d,
ϭ
CH3Ph), 2.35 (d, J4Ј,OH ϭ 2.2 Hz, 1 H, OH), 2.10 (s, 3 H, Ac), H-3, H-4, H-5, H-6a, H-6b, H-4Ј, H-7Јa, H-7Јb), 3.37 (s, 3 H,
1.90Ϫ1.60 (m, 2 H, H-6Јa, H-6Јb). CI-MS: m/z ϭ 894 [M ϩ NH4ϩ].
C47H56O14S (877.01): C 64.36, H 6.44; found C 64.26, H 6.57.
OMe), 3.25 (s, 3 H, OMe), 3.05 (dd, J2Ј,3Ј ϭ 3.0, J3Ј,4Ј ϭ 10.0 Hz,
1 H, H-3Ј), 2.40 (s, 3 H, CH3Ph), 2.09 (s, 3 H, Ac), 2.07Ϫ1.95 (m,
2 H, CH2), 1.90Ϫ1.60 (m, 2 H, H-6Јa, H-6Јb), 1.58Ϫ1.45 (m, 2 H,
CH2) ppm. C52H64O15S (961.1): C 64.98, H 6.71; found C 64.77,
H 6.74.
Methyl 2,3,6-Tri-O-benzyl-4-O-(4-O-5-C-ethylidene-α-
L-gulopyran-
uronate)-α- -glucopyranoside (16): Compound 15 (275.0 mg,
D
1
17b: Syrup. [α]2D0 ϭ Ϫ43 (c ϭ 1.03, CHCl3). H NMR (400 MHz,
0.31 mmol) was dissolved in ethanol (40 mL) and 0.1  ethanolic
sodium hydroxide solution (3.2 mL) was added. The reaction mix-
ture was heated at 70 °C for 2 h and then quenched with IR-120
resin (Hϩ form) and filtered through celite. The residue, after con-
centration, was purified by column chromatography (2:1, cyclohex-
ane/ethyl acetate) to give 194.5 mg of a syrup, which was redis-
solved in dichloromethane (25 mL) and cooled to Ϫ78 °C. Ozone
was bubbled through the reaction mixture until the solution ob-
tained a blue coloration (2 min). The reaction mixture was then
purged with oxygen to remove the excess ozone. Dimethylsulfide
was added and the reaction mixture allowed to warm to room tem-
perature over a period of 1 h. After dilution with dichloromethane,
the solution was washed with water. The organic layer was dried
(MgSO4), concentrated, and the next reaction was performed with-
out further purification. The crude aldehyde was dissolved in tert-
butyl alcohol (10 mL) and 2-methyl-2-butene (5 mL) and water
(10 mL) were added. NaH2PO4 (500 mg) and NaClO2 (500 mg)
were added to the mixture. The suspension was vigorously stirred
at room temperature overnight and then partitioned between water
and ethyl acetate. The organic layer was dried (MgSO4) and con-
centrated to a syrup. The crude acid was dissolved in dry DMF
(18 mL) and tetrabutylammonium iodide (0.5 g, 1.45 mmol), potas-
sium bicarbonate (0.18 g, 1.8 mmol) and benzyl bromide (0.18 mL,
1.5 mmol) were added. After stirring overnight at room temper-
ature, the reaction mixture was partitioned between water and di-
ethyl ether. The diethyl ether layer was dried (MgSO4), concen-
trated and the residue was purified by silica gel column chromato-
graphy (2:1, cyclohexane/ethyl acetate). The benzyl ester derivative
(16) was obtained as a white foam (167.7 mg, 74%). [α]2D0 ϭ Ϫ1.3
(c ϭ 0.9, CHCl3). 1H NMR (400 MHz, CDCl3): δ ϭ 7.50Ϫ7.20
(m, 20 H, arom.), 5.22 (d, J1Ј,2Ј ϭ 2.4 Hz, 1 H, H-1Ј), 5.19 and 5.10
(two d, 2 H, CH2Ph), 4.96 (ABq, 2 H, CH2Ph), 4.80 and 4.65 (two
d, 2 H, CH2Ph), 4.64 and 4.52 (two d, 2 H, CH2Ph), 4.62 (d, J1,2 ϭ
3.6 Hz, 1 H, H-1), 4.16 (d, J6a,6b ϭ 10.0 Hz, 1 H, H-6a), 3.97Ϫ3.84
(m, 5 H, H-3, H-5, H-2Ј, H-7Јa, H-7Јb), 3.82Ϫ3.75 (m, 2 H, H-4,
OH), 3.72 (dd, J5,6b ϭ 5.2 Hz, 1 H, H-6b), 3.62Ϫ3.53 (m, 2 H, H-
CDCl3): δ ϭ 7.70Ϫ7.20 (m, 19 H, arom.), 5.85 (dd, J8Ј,9Јa ϭ 18.0,
J8Ј,9Јb ϭ 11.2 Hz, 1 H, H-8Ј), 5.38 (dd, J9Јa,9Јb ϭ 1.0 Hz, 1 H, H-
9Јa), 5.28 (dd, J1Ј,2Ј ϭ 1.0, J2Ј,3Ј ϭ 3.2 Hz, 1 H, H-2Ј), 5.18 (br. d,
1 H, J8Ј,9Јb ϭ 11.8 Hz, H-9Јb), 5.05 (d, 1 H, CHPh), 4.85 (d, J1Ј,2Ј
ϭ
1.0 Hz, 1 H, H-1Ј), 4.84Ϫ4.60 (m, 6 H, H-1, CHPh, CH), 4.52 (d,
1 H, CHPh), 4.06Ϫ3.95 (m, 2 H, CH2), 3.87Ϫ3.48 (m, 9 H, H-2,
H-3, H-4, H-5, H-6a, H-6b, H-4Ј, H-7Јa, H-7Јb), 3.37 (s, 3 H,
OMe), 3.32 (s, 3 H, OMe), 3.05 (dd, J2Ј,3Ј ϭ 3.1, J3Ј,4Ј ϭ 10.0 Hz,
1 H, H-3Ј), 2.40 (s, 3 H, CH3Ph), 2.09 (s, 3 H, Ac), 1.90Ϫ1.45 (m,
6 H, H-6Јa, H-6Јb, CH2) ppm. C52H64O15S (961.1): C 64.98, H
6.71; found C 64.80, H 6.87.
Methyl 2,3,6-Tri-O-benzyl-4-O-(2,7-anhydro-6-deoxy-3-O-methyl-4-
O-tetrahydropyranyl-5-C-vinyl-β-D-manno-heptopyranosyl)-α-D-
glucopyranoside (18): A solution of 17a (840 mg, 1.13 mmol) in eth-
anol (80 mL) was heated to 80 °C and 0.1  NaOH in ethanol
(27 mL) was added. After 72 h the starting material had been tot-
ally consumed, and the mixture was concentrated and extracted
with dichloromethane. The organic layer was washed with satd. aq.
NaCl, dried over MgSO4, filtered, and concentrated to a syrup.
Flash chromatography on silica gel (65:35, toluene/ethyl acetate)
gave 18a (335 mg, 51% yield). The diastereomer 17b (145 mg,
0.19 mmol) was submitted to the above cyclisation conditions to
give 18b (85 mg, 75% yield).
18a: Syrup. [α]2D0 ϭ ϩ45 (c ϭ 1.2, CHCl3). 1H NMR (400 MHz,
CDCl3): δ ϭ 7.70Ϫ7.20 (m, 15 H, arom.), 5.94 (dd, J8Ј,9Јa ϭ 17.5,
J8Ј,9Јb ϭ 11.0 Hz, 1 H, H-8Ј), 5.37 (dd, J9Јa,9Јb ϭ 1.3 Hz, 1 H, H-
9Јa), 5.12 (d, J1Ј,2Ј ϭ 3.0 Hz, 1 H, H-1Ј), 5.07 (dd, J8Ј,9Јb ϭ 11.0 Hz,
1 H, H-9Јb), 5.06Ϫ4.90 (m, 2 H, CH2Ph), 4.83Ϫ4.51 (m, 6 H, H-
1, CHPh, CH), 4.25 (dd, J1Ј,2Ј ϭ 3.0, J2Ј,3Ј ϭ 4.2 Hz, 1 H, H-2Ј),
4.19Ϫ4.10 (m, 2 H, CH, H-3), 3.95Ϫ3.70 (m, 7 H, H-2, H-4, H-
6a, H-6b, H-7Јa, H-7Јb, CH), 3.62Ϫ3.50 (m, 2 H, H-4Ј, H-5), 3.41
(s, 3 H, OMe), 3.19 (s, 3 H, OMe), 3.15 (dd, J2Ј,3Ј ϭ 4.2, J3Ј,4Ј
ϭ
2.2 Hz, 1 H, H-3Ј), 2.18Ϫ2.07 (m, 2 H, CH, H-6Јa), 1.93Ϫ1.50 (m,
6 H, CH2, H-6Јb) ppm. CI-MS: m/z ϭ 764 [M ϩ NH4ϩ].
C43H54O11 (746.9): C 69.14, H 7.29; found C 68.99, H 7.45.
2, H-4Ј), 3.48 (s, 3 H, OMe), 3.41 (s, 3 H, OMe), 2.87 (d, J2Ј,3Ј
ϭ
1
18b: Syrup. [α]2D0 ϭ Ϫ27 (c ϭ 2.40, CHCl3). H NMR (400 MHz,
5.1 Hz, 1 H, H-3Ј), 2.10 (m, 1 H, H-6Јa), 1.95 (m, 1 H, H-6Јb). CI-
MS: m/z ϭ 788 [M ϩ NH4ϩ]. C44H50O12 (770.86): C 68.55, H 6.54;
found C 68.52, H 6.60.
CDCl3): δ ϭ 7.70Ϫ7.20 (m, 15 H, arom.), 5.85 (dd, J8Ј,9Јa ϭ 17.5,
J8Ј,9Јb ϭ 11.0 Hz, 1 H, H-8Ј), 5.35 (dd, J9Јa,9Јb ϭ 1.2 Hz, 1 H, H-
9Јa), 5.17 (d, J1Ј,2Ј ϭ 3.0 Hz, 1 H, H-1Ј), 5.09 (dd, J8Ј,9Јb ϭ 11.0 Hz,
1 H, H-9Јb), 5.06 and 4.95 (two d, 2 H, CH2Ph), 4.81 (d, 1 H,
Methyl 2,3,6-Tri-O-benzyl-4-O-(2-O-acetyl-6-deoxy-3-O-methyl-4-
O-tetrahydropyranyl-7-O-tosyl-5-C-vinyl-β-
D
-manno-hepto- CHPh), 4.68Ϫ4.58 (m, 5 H, H-1, CHPh, CH), 4.22 (dd, J1Ј,2Ј
ϭ
pyranosyl)-α- -glucopyranoside (17): Camphorsulfonic acid (14 mg)
D
3.0, J2Ј,3Ј ϭ 4.2 Hz, 1 H, H-2Ј), 4.19Ϫ4.10 (m, 2 H, CH, H-3),
was added to a solution of 15 (1.01 g, 1.15 mmol) in dry dichloro-
methane (30 mL), and the mixture cooled to 0 °C. Dihydropyran
3.93Ϫ3.72 (m, 7 H, H-2, H-4, H-6a, H-6b, H-7Јa, H-7Јb, CH),
3.60Ϫ3.50 (m, 2 H, H-4Ј, H-5), 3.42 (s, 3 H, OMe), 3.37 (dd,
Eur. J. Org. Chem. 2002, 3595Ϫ3603
3601

M. Petitou, P. Sinay¨ et al.
FULL PAPER
J2Ј,3Ј ϭ 4.2, J3Ј,4Ј ϭ 2.4 Hz, 1 H, H-3Ј), 3.30 (s, 3 H, OMe),
purified using a Sephadex LH 20 gel column (1:1, dichlorome-
2.16Ϫ2.06 (m, 2 H, CH, H-6Јa), 1.93Ϫ1.50 (m, 6 H, CH2, H-6Јb). thane/ethanol), and then over silica gel (3:2, ethyl acetate/cyclohex-
CI-MS: m/z ϭ 764 [M ϩ NH4ϩ]. C43H54O11 (746.9): C 69.14, H ane) to yield pentasaccharide 22 (28.2 mg, 48%). [α]2D0 ϭ ϩ70 (c ϭ
1
7.29; found C 69.11, H 7.45.
1.0, dichloromethane). H NMR (500 MHz, CDCl3): δ ϭ 5.47 (d,
J1,2 ϭ 3.8 Hz, 1 H, H-1 GlcV), 5.34 (dd, 1 H, H-3 GlcIII), 5.09 (d,
J1,2 ϭ 3.5 Hz, 1 H, H-1 ManII), 4.98 (d, J1,2 ϭ 3.7 Hz, 1 H, H-1
GlcIII), 4.56 (d, J1,2 ϭ 3.7 Hz, 1 H, H-1 GlcI), 4.50 (dd, 1 H, H-6a
GlcIII), 4.24 (dd, 1 H, H-6a GlcV), 4.25 (dd, 1 H, H-6b GlcIII), 4.18
(dd, 1 H, H-6b GlcV), 4.12 (d, J1,2 ഠ 7.9 Hz, 1 H, H-1 GlcUAIV),
4.11 (d, J3,4 ϭ 2.9 Hz, 1 H, H-4 ManII), 4.08 (dd, J2,3 ϭ 4.0 Hz, 1
H, H-2 ManII), 3.98 (dd, 1 H, H-3 GlcI), 3.96 (ddd, 1 H, H-5
GlcIII), 3.89 (dd, 1 H, H-4 GlcUAIV), 3.83 (m, 3 H, H-2 GlcUAIV,
H-5 GlcIV, H-6a GlcI), 3.77 (ddd, 1 H, H-5 GlcI), 3.73 (m, 3 H, H-
4 GlcI, H-7a ManII, H-7b ManII), 3.62 (dd, 1 H, H-4 GlcIII), 3.64
(dd, 1 H, H-6b GlcI), 3.47 (dd, 1 H, H-2 GlcIII), 3.46 (dd, 1 H, H-
2 GlcI), 3.40 (ddd, 1 H, H-5 GlcV), 3.37 (dd, 1 H, H-3 GlcV), 3.32
(dd, 1 H, H-3 GlcUAIV), 3.07 (dd, 1 H, H-2 GlcV), 3.03 (dd, 1 H,
H-3 ManII), 3.00 (dd, 1 H, H-4 GlcV), 2.93 (dd, 1 H, H-2
GlcUAIV), 2.19 (m, 2 H, H-6a ManII, H-6b ManII) ppm. ESI-MS,
positive mode: monoisotopic mass ϭ 1647.42; chemical mass ϭ
Methyl 2,3,6-Tri-O-benzyl-4-O-(2,7-anhydro-6-deoxy-3-O-methyl-5-
C-vinyl-β-D-manno-heptopyranosyl)-α-D-glucopyranoside (19): p-
Toluenesulfonic acid (15 mg) was added to a solution of 18a
(294 mg, 0.39 mmol) in methanol (5 mL). After 15 min at room
temperature, triethylamine was added and the mixture was concen-
trated to a syrup. Flash chromatography on silica gel (45:55, tolu-
ene/ethyl acetate) gave 19 (220 mg, 84% yield). [α]2D0 ϭ ϩ15 (c ϭ
0.87, CHCl3). 1H NMR (400 MHz, CDCl3): δ ϭ 7.70Ϫ7.20 (m, 15
H, arom.), 5.67 (dd, J8Ј,9Јa ϭ 17.3, J8Ј,9Јb ϭ 10.7 Hz, 1 H, H-8Ј),
5.48 (dd, J9Јa,9Јb ϭ 1.7 Hz, 1 H, H-9Јa), 5.23 (dd, J8Ј,9Јb ϭ 10.7,
J9Јa,9Јb ϭ 1.7 Hz, 1 H, H-9Јb), 5.10 (d, J1Ј,2Ј ϭ 2.9 Hz, 1 H, H-1Ј),
5.00 (br. s, 2 H, CH2Ph), 4.79 (d, 1 H, CHPh), 4.72Ϫ4.54 (m, 4 H,
CH2Ph, H-1), 4.20Ϫ3.90 (m, 2 H, H-3, H-2Ј), 3.93Ϫ3.76 (m, 7 H,
H-4, H-5, H-6a, H-6b, H-4Ј, H-7Јa, H-7Јb), 3.75 (dd, J1,2 ϭ 3.5,
J2,3 ϭ 9.0 Hz, 1 H, H-2), 3.42 (s, 3 H, OMe), 3.41 (s, 3 H, OMe),
3.15 (dd, J2Ј,3Ј ϭ 4.4, J3Ј,4Ј ϭ 2.0 Hz, 1 H, H-3Ј), 2.15 (m, 1 H, H-
6Јa), 1.93 (d, J4Ј,OH ϭ 3.8 Hz, 1 H, OH), 1.84 (pseudo dt, 1 H,
J6Ј,7Јa ϭ J6Ј,7Јb ϭ 3.8, J6Јa,6Јb ϭ 15 Hz, H-6Јb) ppm.
1647.80; experimental mass
ϭ 1647.30 Ϯ 0.27. C87H106O31
(1647.8): C 63.42, H 6.48; found C 63.40, H 6.69.
Pentasaccharide 23: A solution of pentasaccharide 22 (17.8 mg,
10.8 µmol) in dichloromethane (0.9 mL) and tert-butyl alcohol
(0.6 mL) was stirred under H2 (35 bar) at 40 °C for 20 h in the
presence of 5% Pd/C (40 mg). The mixture was then filtered (Cel-
ite), concentrated, and codistilled with water (4 ϫ 5 mL). The res-
idue was dissolved in methanol (0.3 mL) and aq. 5  NaOH
(1.17 mL), and stirred at room temp. for 21 h. The solution was
neutralized with Dowex 50WX4 (Hϩ) and then loaded on top of
a Sephadex G25F column (170 mL) equilibrated in water. The frac-
tions containing the compound were collected, passed through a
Dowex Hϩ resin column, and concentrated to give pentasaccharide
23 (5.5 mg, [α]2D0 ϭ ϩ96 (c ϭ 0.44, water), which was directly sul-
fated without further characterisation.
Methyl 2,3,6-Tri-O-benzyl-4-O-(2,7-anhydro-5-C-benzyloxycarbon-
yl-3-O-methyl-β-D-manno-heptopyranosyl)-α-D-glucopyranoside
(20): Ozone was bubbled through a stirred, cooled (Ϫ78 °C), solu-
tion of disaccharide 19 (100 mg, 0.15 mmol) in dichloromethane
(30 mL) until the color turned pale blue. Dimethylsulfide was ad-
ded and, after washing with water, the organic layer was dried
(MgSO4), and concentrated. The residue was dissolved in tert-butyl
alcohol (5.5 mL), and 2-methyl-2-butene (2.75 mL) then water
(5.5 mL) were added, followed by NaH2PO4 (274 mg) and NaClO2
(274 mg). The suspension was stirred vigorously at room temper-
ature overnight and then partitioned between water and ethyl acet-
ate. The organic layer was dried (MgSO4), and concentrated. The
residue was dissolved in dry DMF (10 mL), then tetrabutylammo-
nium iodide (275 mg), potassium hydrogencarbonate (100 mg) and
benzyl bromide (0.1 mL) were introduced. After stirring overnight,
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 chromatography (70:30, hexanes/
acetone) to yield the benzyl ester derivative 20 as a syrup (84 mg,
72%). [α]2D0 ϭ ϩ40 (c ϭ 1.06, dichloromethane). 1H NMR
(200 MHz, CDCl3): δ ϭ 7.40Ϫ7.10 (m, 20 H, aromatic), 5.08 (d,
J1Ј,2Ј ϭ 3.2 Hz, 1 H, H-1Ј), 5.05 (ABq, 2 H, CH2Ph), 4.90 (s, 2 H,
Pentasaccharide 2: A solution of pentasaccharide 23 (5.5 mg, 5.61
µmol) and Et3N·SO3 (142.4 mg, 0.78 mmol) in DMF (2 mL) was
heated at 55 °C in the dark for 18 h. After cooling to room temper-
ature, the solution was diluted with aq. 0.2  NaCl, and layered
on top of a Sephadex G25F gel column (170 mL) equilibrated in
aq. 0.2  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 2 (1.7 mg; accidental loss of material) was obtained.
[α]2D0 ϭ ϩ62 (c ϭ 0.13, water). 1H NMR (500 MHz, D2O): δ ϭ
5.54 (d, J1,2 ϭ 3.7 Hz, 1 H, H-1 GlcIII), 5.49 (d, J1,2 ϭ 3.7 Hz, 1
H, H-1 GlcV), 5.42 (d, J1,2 ϭ 3.8 Hz, 1 H, H-1 ManII), 5.19 (d,
J1,2 ϭ 3.7 Hz, 1 H, H-1 GlcI), 4.85 (dd, J2,3 ϭ J3,4 ϭ 9.7 Hz, 1 H,
H-3 GlcI), 4.80 (dd, J2,3 ϭ 4.0 Hz, 2 H, H-2 ManII), 4.68 (d, J1,2 ϭ
7.9 Hz, 1 H, H-1 GlcUAIV), 4.50 (dd, 1 H, H-6a GlcI), 4.50 (dd,
J2,3 ϭ J3,4 ϭ 9.7 Hz, 1 H, H-3 GlcIII), 4.43 (m, 1 H, H-6a GlcIII),
4.40 (dd, 1 H, H-2 GlcI), 4.38 (dd, J5,6b ϭ 2.2 Hz, J6a,6b ϭ 11.3 Hz,
1 H, H-6b GlcI), 4.36 (dd, 2 H, H-2 GlcIII), 4.34 (dd, J5,6 ϭ 2 Hz,
CH2Ph), 4.70, 4.60 (2d, 2 H, J ϭ 12.1 Hz, CH2Ph), 4.61 (d, J1,2
ϭ
3.7 Hz, 1 H, H-1), 4.60, 4.40 (2d, 2 H, J ϭ 12.1 Hz, CH2Ph),
4.07Ϫ3.90 (m, 3 H, H-3, H-2Ј, H-4Ј), 3.85Ϫ3.50 (m, 7 H, H-2, H-
4, H-5, H-6a, H-6b, H-7Јa, H-7Јb), 3.35, 3.30 (2s, 6 H, 2OMe), 3.04
(dd, J2Ј,3Ј ϭ 4.0, J3Ј,4Ј ϭ 2.4 Hz, 1 H, H-3Ј), 2.38 (d, J4,OH ϭ 6.7 Hz,
1 H, OH), 2.27Ϫ2.04 (m, 2 H, H-6Јa, H-6Јb) ppm. ESI-MS positive
mode: m/z ϭ 793 [M ϩ Na]ϩ, 809 [M ϩ K]ϩ. C44H50O12 (770.9):
C 68.55, H 6.53; found C 68.57, H 6.54.
J6a,6b ϭ 11.3 Hz, 2 H, H-6a GlcV, H-6b GlcIII), 4.25 (ddd, J5,6a
3.8, 1 H, H-5 GlcI), 4.27 (d, J3,4 ϭ 3.0 Hz, 1 H, H-4 ManII), 4.15
ϭ
Pentasaccharide 22: A solution of tert-butyldimethylsilyl trifluoro-
methanesulfonate (TBDMSOTf) in dichloromethane (0.1 , 64 µL) (dd, J5,6b ϭ 2 Hz, J6a,6b ϭ 11.2 Hz, 1 H, H-6b GlcV), 4.02 (ddd,
was added, at Ϫ20 °C, to a stirred mixture of imidate 21 (44.4 mg,
J5,6a ϭ 1.8, J5,6b ϭ 2.0 Hz, 1 H, H-5 GlcIII), 4.01 (dd, 2 H, H-4
42.7 µmol), alcohol 20 (27.6 mg, 35.8 µmol), and finely ground 4 GlcIII, J4,5 ϭ 10.0 Hz, H-4 GlcI), 3.92 (dd, J3,4 ϭ J4,5 ϭ 9.6 Hz, 1
˚
A molecular sieves (67 mg) in dichloromethane/diethyl ether (1:2;
H, H-4 GlcUAIV), 3.96Ϫ3.80 (m, 3 H, H-5 GlcV, H-7a, H-7b
1.9 mL). After 30 min of stirring, the reaction was complete (TLC
toluene/ethyl acetate, 7:3), and solid NaHCO3 was added until
neutral. After filtration and concentration, the residue was first
ManII), 3.75 (d, 1 H, H-5 GlcUAIV), 3.72 (dd, 1 H, H-3 ManII),
3.58 (dd, J2,3 ϭ J3,4 ϭ 9.5 Hz, 1 H, H-3 GlcV), 3.56 (dd, J3,4
ϭ
9.1 Hz, 1 H, H-3 GlcUAIV), 3.36 (dd, J4,5 ϭ 9.9 Hz, 1 H, H-4
3602
Eur. J. Org. Chem. 2002, 3595Ϫ3603

A Non-Chair Conformation of L-Iduronic Acid
FULL PAPER
GlcV), 3.33 (dd, J2,3 ϭ 9.9 Hz, 1 H, H-2 GlcV), 3.30 (dd, J2,3
ϭ
´
soy, P. Sizun, J.-P. Herault, J.-M. Herbert, M. Petitou, P. Sinay¨,
Chem. Eur. J. 2001, 4821Ϫ4833.
9.4 Hz, 1 H, H-2 GlcUAIV), 2.53Ϫ2.45 (m, 2 H, H-6a, H-6b
ManII). ESI-MS for C39H55Na9O49S7, negative mode: monoiso-
topic mass ϭ 1739.34; chemical mass ϭ 1739.20; experimental
mass ϭ 1739.60 Ϯ 0.75.
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3603