Synthetic carbohydrate derivatives as low sulfated heparin mimetics

Article abstract of DOI:10.1021/jo991152j

The total synthesis of a new family of heparin mimetics containing an hexadeca- (2), an octadeca- (3), and an eicosasaccharide (4) is described. All three oligosaccharides contain a pentasaccharidic antithrombin binding domain (DEFGH: van Boeckel, C. A. A.; Petitou, M. Angew. Chem., Int. Ed. Engl. 1993, 32, 1671-1690), extended at the nonreducing end by a thrombin binding domain composed of repeated 2,3-di-O-methyl-6-O-sodium sulfonato-α- D-glucosyl units. The targets were synthesized using a key dodecasaccharide imidate as glycosyl donor as well as di- and tetrasaccharide imidates, all derived from maltose. Condensation of these imidates with a tetrasaccharide precursor of the EFGH part of the antithrombin binding domain gave fully protected hexadeca-, octadeca-, and eicosasaccharide that were deprotected and sulfated to yield 2, 3, and 4. All three displayed antithrombin-mediated antifactor Xa and antithrombin (factor IIa) activity. The most active compound, the eicosasaccharide, showed activity similar to that of low molecular weight heparin. Significantly, unlike heparin and its derivatives, the present heparin mimetics do not interact with platelet factor 4, an interaction that can cause severe side effects in heparin-treated patients. Thus, this new family of compounds contains interesting drug candidates for the prevention and treatment of thrombosis.

Full text of DOI:10.1021/jo991152j

9512
J . Org. Chem. 1999, 64, 9512-9520
Syn th etic Ca r boh yd r a te Der iva tives a s Low Su lfa ted Hep a r in
Mim etics†
Pierre-A. Driguez, Isidore Lederman, J ean-M. Strassel, J ean-M. Herbert, and
Maurice Petitou*
Sanofi-Synthe´labo, 195 Route d’Espagne, 31036 Toulouse Cedex, France
Received J uly 20, 1999
The total synthesis of a new family of heparin mimetics containing an hexadeca- (2), an octadeca-
(3), and an eicosasaccharide (4) is described. All three oligosaccharides contain a pentasaccharidic
antithrombin binding domain (DEFGH: van Boeckel, C. A. A.; Petitou, M. Angew. Chem., Int. Ed.
Engl. 1993, 32, 1671-1690), extended at the nonreducing end by a thrombin binding domain
composed of repeated 2,3-di-O-methyl-6-O-sodium sulfonato-R-D-glucosyl units. The targets were
synthesized using a key dodecasaccharide imidate as glycosyl donor as well as di- and tetrasac-
charide imidates, all derived from maltose. Condensation of these imidates with a tetrasaccharide
precursor of the EFGH part of the antithrombin binding domain gave fully protected hexadeca-,
octadeca-, and eicosasaccharide that were deprotected and sulfated to yield 2, 3, and 4. All three
displayed antithrombin-mediated antifactor Xa and antithrombin (factor IIa) activity. The most
active compound, the eicosasaccharide, showed activity similar to that of low molecular weight
heparin. Significantly, unlike heparin and its derivatives, the present heparin mimetics do not
interact with platelet factor 4, an interaction that can cause severe side effects in heparin-treated
patients. Thus, this new family of compounds contains interesting drug candidates for the prevention
and treatment of thrombosis.
In tr od u ction
glucose residues alternatively R- and â-linked.7 The
antithrombotic activities of the compounds thus obtained
were similar to that of heparin, but also like heparin,
these compounds were neutralized by platelet factor 4
(PF4). PF4 is a platelet protein able to form a complex
with heparin that can induce thrombocytopenia, a side
reaction occurring in ca. 3% of heparin treated patients.
It has been stated that the interaction of heparin and
PF4 is related to two parameters: the size and the charge
density of the heparin fragments.11 Since the pentade-
casaccharide 1,7 which possesses the shortest size of an
oligosaccharide able to display dual anti-Xa and anti-
thrombin activities, still interacts with PF4, the only
possibility we had left to avoid this interaction was to
decrease the charge of the molecule. To this end, we first
synthesized compounds where the A-domain and the
T-domain were separated by a neutral domain composed
of either a flexible polyethylene spacer3 or by an array
of permethylated glucose residues that may be viewed
as a rigid spacer.8 However, in both cases, the prepara-
tion of this uncharged domain required numerous syn-
Heparin, a natural anionic polysaccharide, is used
worldwide for the treatment of thromboembolic disorders.
It is well established now that it displays antithrombotic
and anticoagulant activities mainly by inhibiting, via
antithrombin, two blood coagulation factors: factor Xa
and thrombin.1 Inhibition of factor Xa is due to a unique
pentasaccharide sequence (antithrombin binding domain;
A-domain, Chart 1) which has been chemically synthe-
sized2 and is currently being evaluated in clinical trials.
Molecular modeling and chemical synthesis3 suggested
that in order to inhibit thrombin, the pentasaccharide
must be extended at the nonreducing end by an anionic
domain (thrombin binding domain; T-domain) to consti-
tute a template required to form the thrombin-anti-
thrombin complex.4 In heparin, the T-domain is the so-
called “regular region”, i.e., the repetition of f4)-(2-O-
sulfonato-R-L-idopyranosyluronate)-(1f4)-(N-sulfonato-
6-O-sulfonato-R-D-glucosaminyl)-(1f disaccharide units.5
In our program devoted to the synthesis of new
antithrombotics, we synthesized heparin mimetics in
which, for easier synthesis, the structure of the T-domain
was dramatically simplified compared to that in native
heparin.6-10 Thus, in the most active series, it consisted
of repeated 3-O-methyl-2,6-di-O-sodium sulfonato-D-
(6) Petitou, M.; Duchaussoy, P.; Driguez, P. A.; J aurand, G.; He´rault,
J . P.; Lormeau, J . C.; van Boeckel, C. A. A.; Herbert, J . M. Angew.
Chem., Int. Ed. Engl. 1998, 37, 3009-3014.
(7) Petitou, M.; Duchaussoy, P.; Driguez, P. A.; He´rault, J . P.;
Lormeau, J . C.; Herbert, J . M. Bioorg. Med. Chem. Lett. 1999, 9, 1155-
1160.
(8) Petitou, M.; Driguez, P. A.; Duchaussoy, P.; He´rault, J . P.;
Lormeau, J . C.; Herbert, J . M. Bioorg. Med. Chem. Lett. 1999, 9, 1161-
1166.
(9) Duchaussoy, P.; J aurand, G.; Driguez, P. A.; Lederman, I.;
Gourvenec, F.; Strassel, J . M.; Sizun, P.; Petitou, M.; Herbert, J . M.
Carbohydr. Res. 1999, 317, 63-84.
† Dedicated to Professor Pierre Sinay¨ on the occasion of his 62th
birthday.
* To whom correspondence should be addressed. Tel: (33) 5 61 16
23 90. Fax: (33) 5 61 16 22 86. E-mail: maurice.petitou@sanofi.com.
(1) Heparin; Lane, D. A., Lindahl, U., Eds.; Arnold: London, 1989.
(2) van Boeckel, C. A. A.; Petitou, M. Angew. Chem., Int. Ed. Engl.
1993, 32, 1671-1690.
(3) Grootenhuis, P. D. J .; Westerduin, P.; Meuleman, D.; Petitou,
M.; van Boeckel, C. A. A. Nature Struct. Biol. 1995, 2, 736-739.
(4) Olson, S. T.; Bjo¨rk, I. Semin. Thromb. Hemostasis. 1994, 20,
373-409.
(10) Duchaussoy, P.; J aurand, G.; Driguez, P. A.; Lederman, I.;
Ceccato, M. L.; Gourvenec, F.; Strassel, J . M.; Sizun, P.; Petitou, M.;
Herbert, J . M. Carbohydr. Res. 1999, 317, 85-99.
(11) For a review: Warkentin, T. E.; Chong, B. H.; Greinacher, A.
Thromb. Haemost. 1998, 79, 1-7.
(5) Casu, B. Adv. Carbohydr. Chem. Biochem. 1985, 43, 51-134.
10.1021/jo991152j CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/02/1999

Carbohydrates as Low Sulfated Heparin Mimetics
J . Org. Chem., Vol. 64, No. 26, 1999 9513
Ch a r t 1
thetic steps, a matter of concern for the economic
feasibility of the synthesis.
uniformly sulfated T-domain, heparin mimetics devoid
of interaction with PF4 can be prepared in a very efficient
way.
In the approach reported here, we have investigated
the possibility of uniformly decreasing the charge density
along the molecule by using a low sulfated sequence
composed of a repetition of R-linked 2,3-di-O-methyl-6-
O-sodium sulfonato-D-glucose residues as the T-domain.
A high affinity pentasaccharide was used as A-domain.
Biochemical studies show that with only one sulfate on
each saccharide unit, the T-domain of these compounds
contains enough charges to attract thrombin, but not
enough to undergo neutralization by PF4. Thus, using a
Resu lts a n d Discu ssion
Syn th etic Str a tegy. As shown in Scheme 1, the three
target compounds, hexadecasaccharide 2, octadecasac-
charide 3, and eicosasaccharide 4, are composed of two
distinct domains: (i) an oligomer of R-linked 2,3-di-O-
methyl-6-O-sodium sulfonato-D-glucose as a T-domain,
and (ii) a pentasaccharide with high affinity for anti-
Sch em e 1. Retr osyn th etic An a lysis

9514 J . Org. Chem., Vol. 64, No. 26, 1999
Driguez et al.
Sch em e 2a
a
Key: (a) Ph3CCl, Et3N, DMAP, CH2Cl2; (b) MeI, NaH, DMF; (c) 60% aqueous AcOH; (d) N-acetylimidazole, (CH2Cl)2; (e) levulinic
acid, EDCI, DMAP, dioxane; (f) H2O, NIS, AgOTf, CH2Cl2/Et2O, then CCl3CN, Cs2CO3, CH2Cl2; (g) (CH3)3Si(CH2)2OH, NIS, AgOTf, CH2Cl2/
Et2O; (h) NH2NH2‚HOAc, toluene/EtOH.
thrombin as A-domain.2 We needed a T-domain with a
reduced number of sulfates, that could be obtained in a
small number of steps from an inexpensive starting
material. Because position six is an easy position to
differentiate, we decided to localize the single sulfate at
this position in the final compounds. As all the other
hydroxyls are methylated, we opted for an R-linked
oligomer because of the nonparticipating character of
O-methyl groups in glycosylation reactions.
The retrosynthetic analysis for the preparation of
hexadeca- to eicosamer (2-4) is depicted in Scheme 1.
The sulfated compounds derive from the corresponding
O-protected derivatives 29-31 themselves obtained by
reaction of the key dodecasaccharide imidate 22 with the
acceptors 24, 27, and 28. Among the various possible
strategies to synthesize imidate 22, we chose to add twice
a tetrasaccharide donor to the nonreducing end of a
tetrasaccharide acceptor. Both tetrasaccharides could, in
turn, be prepared from the same disaccharide (13).
Consequently, 22 is obtained in a reduced number of
steps compared to the strategy consisting of adding only
one disaccharide at the time. Imidate 13 can be prepared
from the known disaccharide 5.12
P r ep a r a tion of th e T-Dom a in . For obvious reasons,
we introduced the methyl groups at positions two and
three of the glucose units at an early stage of the
synthesis. To this end, the known ethyl (4,6-O-ben-
zylidene-R-D-glucopyranosyl)-(1f4)-1-thio-â-D-glucopyra-
noside (5)12 was considered as an appropriate starting
material. It was first treated with trityl chloride in the
presence of triethylamine and 4-(dimethylamino)pyridine
to give 6 (Scheme 2), and the remaining hydroxyls were
methylated to furnish 7. Hydrolysis of both the ben-
zylidene and trityl groups was carried out on crude 7
using 60% aqueous acetic acid at 80 °C. Saponification
was needed at this stage because some random acetyla-
tion of the free hydroxyls occurred during concentration
of the acetic acid. Pure 8 was obtained after column
chromatography (60% from 5). The two primary hy-
droxyls (sulfonated in the final compounds) were acety-
lated using N-acetylimidazole in refluxing dichloro-1,2-
ethane. Diacetate 9 was obtained in a moderate yield
(57%), together with triacetate 10 (9%) and the unex-
pected diacetate 11 (8%). The structure of the latter has
been unambiguously determined by mass spectroscopy
1
and H NMR (a shift of H-6a and H-6b from 3.70 to 4.32
The A-domain, also called DEFGH, is an analogue of
the antithrombin binding sequence of heparin, in which
the hydroxyls and N-sulfonates are replaced by methoxy
groups and O-sulfonates, respectively.2 The main advan-
tages of these modifications are simpler chemistry and
enhanced anti-factor Xa activity compared to the parent
pentasaccharide fragment contained in heparin. Accep-
tors 27 and 28 were obtained by adding disaccharide 13
or its tetrasaccharide analogue to tetrasaccharide 24,
followed by delevulinylation. Because the T-domain and
the D unit are composed of the same monomeric synthon,
the lacking D unit of the pentasaccharide is automatically
present as soon as 24 is elongated at the nonreducing
end. The synthesis of the EFGH tetrasaccharide 24 has
been previously described.13
ppm and 4.65 ppm was observed in the presence of
trichloroacetyl isocyanate). Another fraction containing
several unidentified byproducts was also obtained. The
remaining hydroxyl of 9 was protected (95% yield) with
levulinic acid using 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (EDCI) and catalytic amounts
of 4-(dimethylamino)pyridine to give 12.
The synthesis of dodecasaccharide imidate 22 required
the two tetrasaccharide building blocks 16 and 17, which
were prepared from 9 and 12. The choice of the trimeth-
ylsilyl ethyl group to block the anomeric position in 17
deserves some comment: we needed a protecting group
compatible with conditions of thioglycoside activation and
(13) Westerduin, P.; van Boeckel, C. A. A.; Basten, J . E. M.;
Broekhoven, M. A.; Lucas, H.; Rood, A.; van Der Heijden, H.; van
Amsterdam, R. G. M.; van Dinther, T. G.; Meuleman, D. G.; Visser,
A.; Vogel, G. M. T.; Damm, J . B. L.; Overklift, G. T. Bioorg. Med. Chem.
1994, 2, 1267-1280.
(12) Westman, J .; Nilsson, M. J . Carbohydr. Chem. 1995, 14, 949-
960.

Carbohydrates as Low Sulfated Heparin Mimetics
J . Org. Chem., Vol. 64, No. 26, 1999 9515
Sch em e 3a
Sch em e 4a
a
Key: (a) t-BDMSOTf, CH2Cl2/Et2O; (b) NIS, AgOTf, CH2Cl2/
Et2O.
levulinyl deprotection and that could be converted in a
high yield into imidate 22. In the present case, we might
have used the intermediates with the anomeric thioethyl
group as the glycosylating agent, which could have been
condensed on acceptors 24, 27, and 28. However, on some
occasions, we observed a more favorable R/â ratio with
2-O-methoxyglycosyl donor trichloroacetimidates com-
pared to the thioglycoside analogues. For this reason, a
trimethylsilyl ethyl ether was chosen to block the ano-
meric position of the reducing unit, and conversion to a
trichloroacetimidate was done at the dodecasaccharide
level (the decision to use a trimethylsilyl ethyl ether was
also motivated by other synthetic plans in which this
building block was required). Hydrolysis of thioglycoside
12 using N-iodosuccinimide (NIS) and silver trifluo-
romethane sulfonate (AgOTf) led to the free anomeric
hydroxyl, which was subsequently transformed into
trichloroacetimidate 13 using trichloroacetonitrile, in the
presence of cesium carbonate (89%, two steps). The
trimethylsilyl ethyl glycoside 14 was synthesized from
12 using the same activator as above, with an excess (2
equiv) of trimethylsilylethanol. Because the use of diethyl
ether generally enhances the thermodynamically more
stable axial glycoside,14 the coupling reaction was per-
formed in a 2:1 mixture of diethyl ether/dichloromethane.
Despite these precautions, an inseparable mixture of
anomers 14 was obtained (85%; 65:35 R/â), which was
quantitatively delevulinylated to 15 with hydrazine
acetate15 in 2:1 ethanol/toluene. At this stage, the R-gly-
coside (60%) could be partially separated from the other
anomer. Acceptor 9 and imidate 13 were coupled using
tert-butyldimethylsilyl trifluoromethanesulfonate (t-BDM-
SOTf)16 to yield tetrasaccharide 16 (71%; Scheme 3).
Similarly to the synthesis of 14, a 2:1 mixture of diethyl
ether/dichloromethane was used to minimize the forma-
tion of the undesired â-anomer. The new R-D linkage was
a
Key: (a) NH2NH2.HOAc, toluene/EtOH; (b) 16, NIS, AgOTf,
CH2Cl2/Et2O; (c) TFA/CH2Cl2, then CCl3CN, Cs2CO3, CH2Cl2.
tor 15 were coupled (NIS/AgOTf) to furnish tetrasaccha-
ride 17 in 69% yield. Again, H NMR analysis (δH-1 5.43
1
ppm, J 1,2 3.9 Hz) confirmed the R-D stereochemistry of
the new glycosidic bond. For the synthesis of both 16 and
17, no â-anomer could be isolated. Tetrasaccharide 17
was delevulinylated (Scheme 4) like 14 to give acceptor
18 (95%). The latter was coupled with thioglycoside 16
(NIS/AgOTf) to provide octasaccharide 19 (80%; δH-1
∼5.43 ppm, J 1,2 ≈ 3.8 Hz), and delevulinylation yielded
20 in 85% yield. The acceptor thus obtained was coupled
with 16 under the same conditions as above to give
dodecasaccharide 21 (71%). Although the major product
was the one expected (δH-1 ∼5.43 ppm, J 1,2 ≈ 3.8 Hz), for
the first time during the chain elongation, a small
amount of â-anomer (3%) was isolated and characterized
by high-field NMR (δH-1 4.26 ppm, J 1,2 ≈ 8 Hz). Activation
of 21 was realized first by deblocking17 the trimethysilyl
ether with the use of 2:1 trifluoroacetic acid/dichlo-
romethane, and then treatment as described for the
synthesis of 13 gave trichloroacetimidate 22 (86%, two
steps).
Elon ga tion of th e A-Dom a in . To use the dodecasac-
charide imidate 22 in the final step of the elongation
process, the three glycosyl acceptors 24, 27, and 28 were
required. Reaction (Scheme 5) of the tetrasaccharide 24
with the disaccharide imidate 13 gave 25 (60%; δH-1 5.50
ppm, J 1,2 ) 3.7 Hz), together with unreacted 24 (29%).
The hexasaccharide 27 was obtained (89%) after dele-
vulinylation. For the synthesis of 28, the tetrasaccharide
imidate 23 was synthesized first (77% yield from 17)
using the same route as for the synthesis of the imidate
22. Reaction of 23 with 24 gave 26 (69%; δH-1 5.50 ppm,
J 1,2 ) 3.7 Hz) together with unreacted 24 (14%). Finally,
removal of the levulinyl group gave 28 (83%).
1
unambiguously determined, using high-field H NMR, by
both the chemical shift (δ 5.39 ppm) and the coupling
constant (J 1,2 3.8 Hz) of the anomeric proton of the newly
synthesized glycosidic bond. Thioglycoside 12 and accep-
F in a l Cou p lin gs. The acceptors 24, 27, and 28 were
reacted (Scheme 6) with trichloroacetimidate 22 in a
(14) Wolff, G.; Ro¨hle, G. Angew. Chem., Int. Ed. Engl. 1974, 13, 157-
216.
(15) Slaghek, T. M.; Hyppo¨nen, T. K.; Ogawa, T.; Kamerling, J . P.;
Vliegenthart, J . F. G. Tetrahedron: Asymmetry 1994, 5, 2291-2301.
(16) Tabeur, C.; Machetto, F.; Mallet, J . M.; Duchaussoy, P.; Petitou,
M.; Sinay¨, P. Carbohydr. Res. 1996, 281, 253-276.
(17) J ansson, K.; Ahlfors, S.; Fredj, T.; Kihlberg, J .; Magnusson, G.;
Dahmen, J .; Noori, G.; Stenvall, K. J . Org. Chem. 1988, 53, 5629-
5647.

9516 J . Org. Chem., Vol. 64, No. 26, 1999
Driguez et al.
Sch em e 5a
a
Key: (a) t-BDMSOTf, CH2Cl2/Et2O; (b) NH2NH2‚HOAc, toluene/EtOH.
Sch em e 6a
a
Key: (a) t-BDMSOTf, CH2Cl2/Et2O; (b) 5% Pd/C, H2, AcOH; (c) aqueous NaOH, MeOH; (d) Et3N‚SO3, DMF.
Ta ble 1. Com p a r ison of th e Biologica l Activity of
heparin in inhibiting thrombin. However, 4 displayed
activities comparable to that of low molecular weight
heparin. Particularly noteworthy is that none of the
synthetic compounds 2-4 were neutralized by PF4 even
at high protein concentrations (100 µg/mL, data not
shown), indicating that this new family of synthetic
compounds might constitute valuable alternatives to
currently used standard heparin and low molecular
weight heparin. Pharmacological evaluation of com-
pounds 2-4 is currently being investigated.
Oligosa cch a r id es 2-4 w ith th a t of Hep a r in a n d Low
Molecu la r Weigh t Hep a r in
low
mol wt
compd
2
3
4
heparin heparin
no. of saccharide units
molecular wt
KD antithrombin (nM)
factor Xa inhibition
(units/mg)
16
18
20
∼10-50 ∼6-30
5030 5614 6199 ∼15 000 ∼4500
3.3
6
18
25
25
350 260 210 180
170
thrombin inhibition-
antithrombin-mediated
(IC50, ng/mL)
490 360 88
3.3
27
Exp er im en ta l Section
Gen er a l Meth od s. All compounds were homogeneous by
TLC analysis and had spectral properties consistent with their
assigned structures. Melting points are uncorrected. Optical
rotations were measured at room temperature (22 ( 3 °C).
Compound purity was checked by TLC on silica gel 60 F254
(E. Merck) with detection by charring with sulfuric acid.
Unless otherwise stated, column chromatography was per-
mixture of diethyl ether and dichloromethane, using
t-BDMSOTf as the activator to provide, respectively,
hexadecasaccharide 29 (44%), octadecasaccharide 30
(48%), and eicosasaccharide 31 (51%). In each case, gel
permeation chromatography followed by silica gel chro-
matography allowed easy purification of the product. As
in the synthesis of 25 and 26, some unreacted acceptor
was isolated (45%, 39%, and 23%, respectively). Several
byproducts were formed during the three coupling reac-
tions, including likely some â-anomers. These complex
mixtures were not characterized. Catalytic hydrogenation
of the fully protected oligosaccharides 29-31, followed
by saponification of acyl groups with aqueous sodium
hydroxide in methanol, and finally sulfonation of the
hydroxyls using triethylamine sulfur trioxide complex
yielded 2 (82% over the three steps), 3 (87%), and 4 (77%).
Biologica l Assa ys. The in vitro activities of com-
pounds 2-4 were determined (Table 1) and compared to
that of heparin and low molecular weight heparin. It
appeared that for 2-4, the affinity for antithrombin as
well as inhibition of factor Xa are in the same range.
None of the synthetic compounds was as potent as
1
formed on silica gel 60, 40-63, or 63-200 µm (E. Merck). H
NMR spectra were recorded on Bruker AC 200, AM 250, AC
300, or AM 500 instruments, in CDCl3 or D2O. Before analysis
in D2O, samples were passed through a Chelex (Bio-Rad) ion-
exchange column and lyophilized three times from D2O.
Chemical shifts are relative to external TMS (CDCl3) or
external TSP (D2O). For NMR assignment purposes, the carbo-
hydrate residues for products larger than monosaccharides
have been designed starting from the reducing end (unit I).
Cr u d e Eth yl (4,6-O-Ben zylid en e-r-D-glu cop yr a n osyl)-
(1f4)-6-O-tr ityl-1-th io-â-D-glu cop yr a n osid e (6). To a sus-
pension of 512 (50.0 g, 0.105 mol) in CH2Cl2 (620 mL) were
added, under argon, triethylamine (35 mL, 0.252 mol), trityl
chloride (29.3 g, 0.105 mol), and DMAP (1.28 g, 10 mmol). The
mixture was refluxed, and after 2 h, the same quantity of trityl
chloride was added. After an additional 2.5 h of reflux, starting
material was no longer visible by TLC. After being cooled to
room temperature, the solution was diluted with CH2Cl2,
successively washed with cold 10% aqueous KHSO4, H2O, and

Carbohydrates as Low Sulfated Heparin Mimetics
J . Org. Chem., Vol. 64, No. 26, 1999 9517
brine, dried (Na2SO4), and concentrated. Filtration over silica
gel (2:1 then 1:1 toluene/acetone) gave crude 6, which was
sufficiently pure for use in the next step. An analytical sample
was chromatographed: [R]D +53° (c 0.74, CH2Cl2); 1H NMR
(CD2Cl2) δ 7.52-7.25 (m, 20H, 4Ph), 5.42 (s, CHPh), 4.97 (d,
J ) 3.5 Hz, H-1 GlcII), 4.40 (d, J ) 9.6 Hz, H-1 GlcI), 3.82 (t,
J ) 9.3 Hz, H-3 GlcII), 3.70, 3.68 (m, 2H, H-3 GlcI, H-4 GlcI),
3.60 (dd, J ≈ 2.0, 11.0 Hz, H-6a GlcI), 3.55 (td, J ) 5.2, 9.7,
9.7 Hz, H-5 GlcII), 3.49-3.45 (m, 3H, H-2 GlcI, H-2 GlcII, H-5
GlcI), 3.38 (dd, J ) 10.5 Hz, H-6a GlcII), 3.33 (dd, H-6b GlcII),
3.30-3.27 (m, 2H, H-4 GlcII, H-6b GlcI), 2.90-2.77 (m, 2 H,
SCH2CH3), 1.40-1.37 (t, 3H, SCH2CH3); mass spectrum (ESI)
m/z 715 [(M - H)-]. Anal. Calcd for C40H44O10S: C, 67.02; H,
6.19; S, 4.47. Found: C, 66.83; H, 6.19; S, 4.19.
Cr u d e Eth yl (4,6-O-Ben zylid en e-2,3-d i-O-m eth yl-r-D-
glu cop yr a n osyl)-(1f4)-2,3-d i-O-m eth yl-6-O-tr ityl-1-th io-
â-D-glu cop yr a n osid e (7). MeI (34 mL, 0.536 mol) was added
dropwise under argon to a solution of the preceding compound
(64.1 g) in DMF (600 mL), and after the mixture was cooled
in a water/ice mixture, NaH (13.5 g, 0.536 mol) was added
portionwise. The suspension was stirred for 2 h at room
temperature and then cooled to 0 °C, MeOH (35 mL) was
added dropwise, and after 2 h of stirring, the mixture was
diluted with EtOAc (500 mL) and H2O (600 mL). The aqueous
phase was extracted with EtOAc, and the combined organic
phases were washed with H2O, dried (Na2SO4), and concen-
trated to give a residue that was considered pure enough for
the next step. An analytical sample was purified on silica gel
(7:3 cyclohexane/acetone): [R]D +45° (c 0.83, CH2Cl2); 1H NMR
(CD2Cl2) δ 7.52-7.19 (m, 20H, 4Ph), 5.51 (d, J ) 3.3 Hz, H-1
GlcII), 5.43 (s, CHPh), 4.45 (d, J ) 9.8 Hz, H-1 GlcI), 3.60, 3.59,
3.51, 3.49 (4s, 12H, 4OMe), 2.86 (q, 2H, J ) 7.5 Hz, SCH2-
CH3), 1.40 (t, 3H, SCH2CH3); mass spectrum (ESI) m/z 795
[(M + Na)+]. Anal. Calcd for C44H52O10S: C, 68.37; H, 6.78; S,
4.15. Found: C, 68.28; H, 6.98; S, 4.09.
1.30 (t, 3H, J ) 7.5 Hz, SCH2CH3); mass spectrum (ESI) m/z
591 [(M + Na)+]. Anal. Calcd for C24H40O13S: C, 50.69; H, 7.09;
S, 5.64. Found: C, 50.85; H, 7.29; S, 5.45. 11: [R]D + 36° (c
1.0, CH2Cl2); 1H NMR (CDCl3) δ 5.61 (d, J ) 3.9 Hz, H-1 GlcII),
4.35 (d, J ) 9.7 Hz, H-1 GlcI), 3.74, 3.62, 3.59, 3.57 (4s, 12H,
4OMe), 2.08, 2.06 (2s, 6H, 2Ac), 1.30 (t, 3H, J ) 7.5 Hz,
SCH2CH3), 1.96 (t, J ) 7.0 Hz, OH); mass spectrum (ESI) m/z
549 [(M + Na)+]. Anal. Calcd for C22H38O12S: C, 50.17; H, 7.27;
S, 6.09. Found: C, 50.28; H, 7.45; S, 5.89.
E t h yl (6-O-Acet yl-4-O-levu lin yl-2,3-d i-O-m et h yl-r-D-
glu cop yr a n osyl)-(1f4)-6-O-a cetyl-2,3-d i-O-m eth yl-1-th io-
â-D-glu cop yr a n osid e (12). To a solution of diacetate 9 (19.4
g, 36.7 mmol) in dioxane (400 mL) were added, under argon,
levulinic acid (7.53 mL, 73.5 mmol), EDCI (14.1 g, 73.5 mmol),
and DMAP (900 mg, 7.35 mmol). The mixture was stirred for
3.5 h, diluted with CH2Cl2, washed with H2O, 10% aqueous
KHSO4, H2O, 2% aqueous NaHCO3, and H2O, dried (Na2SO4),
and concentrated. The residue was flash chromatographed
(97:3 and then 79:21 CH2Cl2/acetone) to give 12 (21.8 g, 95%):
[R]D +40° (c 0.72, CH2Cl2); 1H NMR (CDCl3) δ 5.56 (d, J ) 3.9
Hz, H-1 GlcII), 4.35 (d, J ) 9.8 Hz, H-1 GlcI), 3.64, 3.60, 3.58,
3.55 (4s, 12H, 4OMe), 2.76-2.71 (m, 4H, O(CdO)CH2CH2(Cd
O)CH3); 2.19, 2.08, 2.07 (3s, 9H, 2Ac and O(CdO)CH2CH2(Cd
O)CH3), 1.31 (t, 3H, J ) 7.4 Hz, SCH2CH3); mass spectrum
(ESI) m/z 647 [(M + Na)+]. Anal. Calcd for C27H44O14S: C,
51.91; H, 7.10; S, 5.13. Found: C, 51.88; H, 7.05; S, 4.96.
(6-O-Acetyl-4-O-levu lin yl-2,3-d i-O-m eth yl-r-D-glu cop y-
r a n osyl)-(1f4)-6-O-a cet yl-2,3-d i-O-m et h yl-1-t r ich lor o-
a cetim id oyl-D-glu cop yr a n ose (13). To a solution of thiogly-
coside 12 (9.53 g, 15.3 mmol) in 1:1 CH2Cl2/Et2O (180 mL) were
added H2O (1.4 mL, 76.3 mmol), NIS (6.84 g, 30.5 mmol), and
AgOTf (0.51 g, 1.98 mmol). After 15 min, saturated aqueous
NaHCO3 (5 mL) was added, and the reaction mixture was
diluted with CH2Cl2, washed with H2O, M Na2S2O3, 2%
aqueous NaHCO3, and H2O. The organic layer was dried (Na2-
SO4) and concentrated, and the residue was purified over silica
gel (2:1 then 1:0 EtOAc/cyclohexane) to give a solid that was
engaged in the next step without characterization. A solution
of the preceding compound (7.88 g, 13.59 mmol) in CH2Cl2 (120
mL) was treated, under argon, with Cs2CO3 (7.08 g, 21.74
mmol) and CCl3CN (6.81 mL, 67.93 mmol). After 40 min, the
mixture was filtered, concentrated, and flash chromatographed
(85:15 toluene/acetone) to give imidate 13 (9.16 g, 89%): [R]D
+ 118° (c 1.00, CH2Cl2); 1H NMR (CDCl3) δ 8.66, 8.65 (2s, 1H,
R and â N:H), 6.52 (d, J ) 3.6 Hz, H-1R GlcI), 5.70 (d, J ) 7.5
Hz, H-1â GlcI), 5.58 (d, J ) 3.7 Hz, H-1 GlcII), 2.78-2.57 (m,
4H, O(CdO)CH2CH2(CdO)CH3), 2.18, 2.07, 2.06 (3s, 9H, 2Ac
and O(CdO)CH2CH2(CdO)CH3); mass spectrum (ESI) m/z 746
[(M + Na)+]. Anal. Calcd for C27H40Cl3NO15‚0.5H2O: C, 44.18;
H, 5.63; N, 1.98. Found: C, 44.14; H, 5.61; N, 1.97.
Eth yl (2,3-Di-O-m eth yl-r-D-glu cop yr a n osyl)-(1f4)-2,3-
d i-O-m eth yl-1-th io-â-D-glu cop yr a n osid e (8). A suspension
of crude 7 (67.4 g) in 60% aqueous AcOH (470 mL) was heated
at 80 °C for 2 h. The reaction mixture was cooled, filtered,
and concentrated. The residue was treated with MeONa (940
mg) in MeOH (200 mL) for 1 h. The solution was then
neutralized with Dowex 50 WX4 (H+) resin, and after filtration,
and concentration, column chromatography (3:2 toluene/
acetone) gave 8 (27.9 g, 60% from 5): [R]D + 26° (c 1.07, CH2-
1
Cl2); H NMR (CDCl3) δ 5.62 (d, J ) 3.9 Hz, H-1 GlcII), 4.35
(d, J ) 9.8 Hz, H-1 GlcI), 3.64, 3.64, 3.59, 3.58 (4s, 12H, 4OMe),
1.29 (t, 3H, J ) 7.4 Hz, SCH2CH3); mass spectrum (ESI) m/z
465 [(M + Na)+]. Anal. Calcd for C18H34O10S‚ H2O: C, 46.94;
H, 7.87; S, 6.96. Found: C, 47.19; H, 7.72; S, 6.70.
Eth yl (6-O-Acetyl-2,3-d i-O-m eth yl-r-D-glu cop yr a n osyl)-
(1f4)-6-O-a cetyl-2,3-d i-O-m eth yl-1-th io-â-D-glu cop yr a n o-
sid e (9), Eth yl (4,6-Di-O-a cetyl-2,3-d i-O-m eth yl-r-D-glu -
cop yr a n osyl)-(1f4)-6-O-a cetyl-2,3-d i-O-m eth yl-1-th io-â-
D-glu cop yr a n osid e (10), a n d Eth yl (4,6-Di-O-a cetyl-2,3-
d i-O-m eth yl-r-D-glu cop yr a n osyl)-(1f4)-2,3-d i-O-m eth yl-
1-th io-â-D-glu cop yr a n osid e (11). Triol 8 (5.86 g, 13.2 mmol)
was refluxed overnight with N-acetylimidazole (3.21 g, 29.1
mmol) in ClCH2CH2Cl (120 mL). Another portion of N-
acetylimidazole (440 mg, 3.96 mmol) was added, and stirring
was continued an additional 4 h. The reaction mixture was
then allowed to reach room temperature, and MeOH (2 mL)
was added. After 1 h of stirring, the mixture was diluted with
CH2Cl2, successively washed with cold M HCl, H2O, saturated
aqueous NaHCO3, and H2O, dried (Na2SO4), and concentrated.
Column chromatography (3.5:1 toluene/acetone) of the residue
yielded diacetate 9 (3.97 g, 57%), triacetate 10 (652 mg, 9%),
and diacetate 11 (539 mg, 8%). 9: [R]D +33° (c 1.90, CH2Cl2);
1H NMR (CDCl3) δ 5.51 (d, J ) 3.9 Hz, H-1 GlcII), 4.34 (d, J )
9.8 Hz, H-1 GlcI), 3.64, 3.63, 3.59, 3.56 (4s, 12H, 4OMe), 2.11,
2.06 (2s, 6H, 2Ac), 1.31 (t, 3H, J ) 7.4 Hz, SCH2CH3); mass
spectrum (ESI) m/z 549 [(M + Na)+]. Anal. Calcd for
2-(Tr im et h ylsilyl)et h yl (6-O-Acet yl-4-O-levu lin yl-2,3-
d i-O-m eth yl-r-D-glu cop yr a n osyl)-(1f4)-6-O-a cetyl-2,3-d i-
O-m eth yl-D-glu cop yr a n osid e (14). A mixture of thioglyco-
side 12 (10.6 g, 16.9 mmol), 2-(trimethylsilyl)ethanol (4.8 mL,
33.9 mmol), and powdered molecular sieves (5.07 g, 4 Å) in
1:2 CH2Cl2/Et2O (105 mL) was stirred under argon for 1 h at
23 °C with protection from light, cooled to 0 °C, and treated
with NIS (11.4 g, 50.82 mmol) and AgOTf (565 mg, 2.20 mmol).
The reaction was quenched after 10 min with solid NaHCO3
(250 mg), and after filtration (Celite), the reaction mixture was
diluted with CH2Cl2 and successively washed with M Na2S2O3,
H2O, 2% aqueous NaHCO3, and H2O. The organic phase was
dried (Na2SO4) and concentrated, and the residue obtained was
purified by chromatography (1:1 acetone/CH2Cl2) to give com-
pound 14 (9.80 g, 85%) as an inseparable mixture of anomers
(R/â 2:1): 1H NMR (CDCl3) δ 5.58 (d, J ) 3.9 Hz, H-1 GlcII),
4.94 (d, J ) 3.5 Hz, H-1R GlcI), 4.26 (d, J ) 7.7 Hz, H-1â GlcI),
2.76-2.56 (m, 4H, O(CdO)CH2CH2(CdO)CH3), 2.17, 2.08, 2.05
(3s, 9H, 2Ac and O(CdO)CH2CH2(CdO)CH3), 1.18-0.88 (m,
2H, OCH2CH2Si(CH3)3), 0.02 (s, 9H, OCH2CH2Si(CH3)3); mass
spectrum (ESI) m/z 703 [(M + Na)+]. Anal. Calcd for C30H52O15-
Si: C, 52.92; H, 7.69. Found: C, 53.29; H, 7.75.
C
22H38O12S: C, 50.17; H, 7.27; S, 6.09. Found: C, 50.15; H,
1
7.49; S, 5.89. 10: [R]D +42° (c 1.0, CH2Cl2); H NMR (CDCl3)
δ 5.55 (d, J ) 3.9 Hz, H-1 GlcII), 4.33 (d, J ) 9.7 Hz, H-1 GlcII),
3.63, 3.58, 3.57, 3.52 (4s, 12H, 4OMe), 2.08, 2.06 (2s, 9H, 3Ac),
2-(Tr im eth ylsilyl)eth yl (6-O-Acetyl-2,3-d i-O-m eth yl-r-
D-glu cop yr a n osyl)-(1f4)-6-O-a cet yl-2,3-d i-O-m et h yl-D-
glu cop yr a n osid e (15). Hydrazine acetate (12.7 g, 138.0

9518 J . Org. Chem., Vol. 64, No. 26, 1999
Driguez et al.
mmol) was added, under argon, to a solution of compound 14
(9.41 g, 13.8 mmol) in 1:2 toluene/EtOH (300 mL). After 15
min of stirring, the reaction mixture was concentrated, diluted
with CH2Cl2, washed with H2O, 2% aqueous NaHCO3, and
H2O, dried (Na2SO4), and concentrated. The residue was
chromatographed (3:2 acetone/toluene) and afforded pure 15R
(4.81 g, 60%), together with a R/â mixture (3.06 g, 37%). 15R:
were treated for 10 min under the conditions described for 17
with NIS (2.38 g, 10.58 mmol) and AgOTf (118 mg, 0.461
mmol) in 1:2 CH2Cl2/Et2O (60 mL) in the presence of molecular
sieves (1.77 g, 4 Å). After workup and chromatography (7:2
then 2:1 CH2Cl2/acetone), pure 19 (5.71 g, 80%) was obtained:
1
[R]D +161° (c 0.65, CH2Cl2); H NMR (CDCl3) δ 5.54 (d, J )
3.8 Hz, H-1 GlcVIII), 5.47-5.40 (m, 6H, H-1 GlcII-VII), 4.95 (d,
J ) 3.7 Hz, H-1 GlcI), 2.84-2.51 (m, 4H, O(CdO)CH2CH2(Cd
O)CH3), 2.17, 2.13, 2.12, 2.11, 2.11, 2.08, 2.05 (7s, 27H, 8Ac
and O(CdO)CH2CH2(CdO)CH3), 1.18-0.97 (m, 2H, OCH2CH2-
Si(CH3)3), 0.03 (s, 9H, OCH2CH2Si(CH3)3); mass spectrum
(ESI) m/z 2112 [(M + K)+]. Anal. Calcd for C90H148O51Si: C,
52.12; H, 7.19. Found: C, 51.98; H, 7.25.
1
[R]D +132° (c 0.61, CH2Cl2); H NMR (CDCl3) δ 5.55 (d, J )
3.8 Hz, H-1 GlcII), 4.95 (d, J ) 3.6 Hz, H-1 GlcI), 2.10, 2.08
(2s, 6H, 2Ac), 1.16-0.89 (m, 2H, OCH2CH2Si(CH3)3), 0.02 (s,
9H, OCH2CH2Si(CH3)3; mass spectrum (ESI) m/z 621 [(M +
K)+]. Anal. Calcd for C25H46O13Si: C, 51.53; H, 7.96. Found:
C, 51.37; H, 8.06.
E t h yl (6-O-Acet yl-4-O-levu lin yl-2,3-d i-O-m et h yl-r-D-
glu cop yr a n osyl)-(1f4)-[(6-O-a cetyl-2,3-d i-O-m eth yl-r-D-
glu copyr an osyl)-(1f4)]2-6-O-acetyl-2,3-di-O-m eth yl-1-th io-
â-D-glu cop yr a n osid e (16). A solution of imidate 13 (1.10 g,
1.52 mmol) and acceptor 9 (806 mg, 1.38 mmol) was stirred
under argon for 1 h in 1:2 CH2Cl2/Et2O (22 mL) in the presence
of finely grounded molecular sieves (1.10 g, 4 Å). After the
solution was cooled to -20 °C, a solution of M tert-butyldim-
ethysilyl trifluoromethanesulfonate (t-BDMSOTf) in CH2Cl2
(0.77 mL) was added, and the mixture was stirred for 30 min,
diluted with CH2Cl2, and filtered (Celite). The organic phase
was then washed with 2% aqueous NaHCO3 and H2O, dried
(Na2SO4), concentrated, and purified by chromatography (2.5:1
then 3:1 EtOAc/cyclohexane) to yield pure 16 (1.12 g, 71%):
[R]D +95° (c 1.00, CH2Cl2); 1H NMR (CDCl3) δ 5.55 (d, J ) 3.9
Hz, H-1 GlcIV), 5.39, 5.37 (2d, J ) 3.8, 3.9 Hz, H-1 GlcII, H-1
GlcIII), 4.34 (d, J ) 9.7 Hz, H-1 GlcI), 2.84-2.51 (m, 6H, SCH2-
CH3, O(CdO)CH2CH2(CdO)CH3), 2.17, 2.10, 2.09, 2.08, 2.04
(5s, 15H, 4Ac and O(CdO)CH2CH2(CdO)CH3), 1.30 (t, 3H, J
) 7.4 Hz, SCH2CH3); mass spectrum (ESI) m/z 1111 [(M +
Na)+]. Anal. Calcd for C47H76O26S: C, 51.83; H, 7.03; S, 2.94.
Found: C, 51.66; H, 7.02; S, 2.94.
2-(Tr im et h ylsilyl)et h yl (6-O-Acet yl-4-O-levu lin yl-2,3-
d i-O-m eth yl-r-D-glu cop yr a n osyl)-(1f4)-[(6-O-a cetyl-2,3-
d i-O-m eth yl-r-D-glu cop yr a n osyl)-(1f4)-]2-6-O-a cetyl-2,3-
d i-O-m eth yl-r-D-glu cop yr a n osid e (17). Thioglycoside 12
(4.21 g, 6.74 mmol) and glycosyl acceptor 15 (3.57 g, 6.13 mmol)
were stirred in the dark for 45 min in 1:2 CH2Cl2/Et2O (105
mL), in the presence of powdered molecular sieves (3.57 g, 4
Å). NIS (4.53 g, 20.2 mmol) and AgOTf (225 mg, 0.13 equiv)
were then added under argon atmosphere at 0 °C. After 10
min of stirring, the reaction mixture was filtered (Celite),
diluted with CH2Cl2, washed with M Na2S2O3, H2O, 2%
aqueous NaHCO3, and H2O, dried (Na2SO4), and concentrated.
Purification of the residue by chromatography (3:1 then 9:1
acetone/cyclohexane) afforded 17 (4.81 g, 69%): [R]D +143° (c
0.56, CH2Cl2); 1H NMR (CDCl3) δ 5.57 (d, J ) 3.9 Hz, H-1
GlcIV), 5.44, 5.41 (2d, J ) 3.8 Hz, H-1 GlcIII, H-1 GlcII), 4.96
(d, J ) 3.6 Hz, H-1 GlcI), 2.78-2.58 (m, 4H, O(CdO)CH2CH2-
(CdO)CH3), 2.18, 2.12, 2.12, 2.09, 2.06 (5s, 15H, 4Ac and O(Cd
O)CH2CH2(CdO)CH3), 1.21-0.97 (m, 2H, OCH2CH2Si(CH3)3),
0.03 (s, 9H, OCH2CH2Si(CH3)3); mass spectrum (ESI) m/z 1167
[(M + Na)+]. Anal. Calcd for C50H84O27Si: C, 52.44; H, 7.39.
Found: C, 52.29; H, 7.46.
2-(Tr im eth ylsilyl)eth yl [(6-O-Acetyl-2,3-d i-O-m eth yl-r-
D-glu cop yr a n osyl)-(1f4)]7-6-O-a cetyl-2,3-d i-O-m eth yl-r-
D-glu cop yr a n osid e (20). Compound 19 (3.00 g, 1.45 mmol)
was treated like 14 to give 20 (2.43 g, 85%): [R]D +167° (c
0.57, CH2Cl2); 1H NMR (CDCl3) δ 5.47-5.40 (m, 7H, H-1
GlcII-VIII), 4.95 (d, J ) 3.7, H-1 GlcI), 2.80 (d, J ) 4.4 Hz, OH),
2.13, 2.11, 2.10, 2.08, 2.07 (5s, 24H, 8Ac), 1.18-0.97 (m, 2H,
OCH2CH2Si(CH3)3), 0.03 (s, 9H, OCH2CH2Si(CH3)3); mass
spectrum (ESI) m/z 2014 [(M + K)+]. Anal. Calcd for C85H142O49-
Si: C, 51.66; H, 7.24. Found: C, 51.32; H, 7.26.
2-(Tr im et h ylsilyl)et h yl (6-O-Acet yl-4-O-levu lin yl-2,3-
d i-O-m eth yl-r-D-glu cop yr a n osyl)-(1f4)-[(6-O-a cetyl-2,3-
d i-O-m eth yl-r-D-glu cop yr a n osyl)-(1f4)]10-6-O-a cetyl-2,3-
d i-O-m eth yl-r-D-glu cop yr a n osid e (21r). Compounds 16
(1.35 g, 1.24 mmol) and 20 (2.38 g, 1.20 mmol) were treated
for 10 min under the conditions described for the synthesis of
17, with NIS (834 mg, 3.72 mmol) and AgOTf (41 mg, 0.160
mmol) in 1:2 CH2Cl2/Et2O (19 mL) in the presence of molecular
sieves (620 mg, 4 Å). Treatment and column chromatography
yielded 21R (2.56 g, 71%) together with 21â (116 mg, 3%).
1
21r: [R]D +166° (c 0.88, CH2Cl2); H NMR (CDCl3) δ 5.54 (d,
J ) 3.8 Hz, H-1 GlcXII), 5.47-5.40 (m, 10H, H-1 GlcII-XI), 4.95
(d, J ) 3.7 Hz, H-1 GlcI), 2.81-2.51 (m, 4H, O(CdO)CH2CH2-
(CdO)CH3), 2.17, 2.13, 2.12, 2.11, 2.11, 2.08, 2.05 (7s, 39H,
12Ac and O(CdO)CH2CH2(CdO)CH3), 1.17-0.96 (m, 2H,
OCH2CH2Si(CH3)3), 0.03 (s, 9 H, OCH2CH2Si(CH3)3); mass
spectrum (ESI) m/z 1525 [(M + 2Na)2+/2]. Anal. Calcd for
C
130H212O75Si: C, 51.99; H, 7.12. Found: C, 51.63; H, 7.12. 21â:
1
[R]D +151° (c 0.93, CH2Cl2); H NMR (CDCl3) δ 5.56 (d, J )
3.8 Hz, H-1 GlcXII), 5.47-5.40 (m, 9H, H-1 GlcII-VIII,X,XI), 4.95
(d, J ) 3.7 Hz, H-1 GlcI), 4.26 (d, J ∼ 8 Hz, H-1 GlcIX), 2.81-
2.51 (m, 4H, O(CdO)CH2CH2(CdO)CH3), 2.17, 2.13, 2.11, 2.10,
2.09, 2.09, 2.05 (7s, 39H, 12Ac and O(CdO)CH2CH2(CdO)-
CH3), 1.17-0.96 (m, 2H, OCH2CH2Si(CH3)3), 0.03 (s, 9 H,
OCH2CH2Si(CH3)3); mass spectrum (ESI) m/z 1525 [(M +
2Na)2+/2].
(6-O-Acetyl-4-O-levu lin yl-2,3-d i-O-m eth yl-r-D-glu cop y-
r a n osyl)-(1f4)-[(6-O-a cetyl-2,3-d i-O-m eth yl-r-D-glu cop y-
r a n osyl)-(1f4)]10-6-O-a cetyl-2,3-d i-O-m eth yl-1-tr ich lor o-
a cetim id oyl-D-glu cop yr a n ose (22). A solution of 21R (400
mg, 0.133 mmol) in 2:1 TFA/CH2Cl2 (2 mL) was stirred for 1.5
h and then diluted with 2:1 toluene/n-PrOAc (12 mL), concen-
trated, and co-concentrated with toluene. Purification of the
residue (4:3 acetone/cyclohexane) gave a solid (364 mg) that
was dissolved in CH2Cl2 (2.5 mL). Cs2CO3 (65 mg, 0.200 mmol)
and CCl3CN (63 mL, 0.620 mmol) were added to the mixture,
which was stirred for 2.5 h and then filtered (Celite), concen-
trated, and purified on silica gel (50:50:0.1 cyclohexane/
acetone/Et3N) to give imidate 22 (348 mg, 86%): [R]D +185°
(c 0.91, CH2Cl2); 1H NMR (CD2Cl2) δ 8.61, 8.58 (2s, 1H, R and
â N:H), 6.35 (d, J ) 3.7 Hz, H-1R GlcI), 5.59 (d, J ) 7.5 Hz,
H-1â GlcI), 5.38 (d, J ) 3.8 Hz, H-1 GlcXII), 5.32-5.25 (m, 10H,
H-1 GlcII-XI), 2.64-2.40 (m, 4H, O(CdO)CH2CH2(CdO)CH3),
2.02, 1.96, 1.95, 1.94, 1.93, 1.89 (6s, 39H, 12Ac and O(CdO)-
CH2CH2(CdO)CH3); mass spectrum (ESI) m/z 1546 [(M +
2Na)2+/2]. Anal. Calcd for C127H200Cl3NO25: C, 50.06; H, 6.61;
N, 0.46. Found: C, 49.93; H, 6.52; N, 0.42.
2-(Tr im eth ylsilyl)eth yl [(6-O-Acetyl-2,3-d i-O-m eth yl-r-
D-glu cop yr a n osyl)-(1f4)]3-6-O-a cetyl-2,3-d i-O-m eth yl-r-
D-glu cop yr a n osid e (18). Compound 17 (4.71 g, 4.11 mmol)
was reacted like 14 with hydrazine acetate (3.79 g, 41.1 mmol)
in 1:2 toluene/EtOH (90 mL) for 2 h. After workup, column
chromatography (3:2 cyclohexane/acetone) yielded 18 (4.11 g,
1
95%): [R]D +154° (c 0.63, CH2Cl2); H NMR (CDCl3) δ 5.46,
5.46, 5.41 (2d, 3H, J ) 3.9 Hz, H-1 GlcII-IV), 4.95 (d, J ) 3.5
Hz, H-1 GlcI), 2.81 (d, J ) 4.4 Hz, OH), 2.11, 2.09, 2.08 (3s,
12H, 4Ac), 1.19-0.97 (m, 2H, OCH2CH2Si(CH3)3), 0.03 (s, 9H,
OCH2CH2Si(CH3)3); mass spectrum (ESI) m/z 1085 [(M + K)+].
Anal. Calcd for C45H78O25Si: C, 51.61; H, 7.51. Found: C,
51.39; H, 7.54.
2-(Tr im et h ylsilyl)et h yl (6-O-Acet yl-4-O-levu lin yl-2,3-
d i-O-m eth yl-r-D-glu cop yr a n osyl)-(1f4)-[(6-O-a cetyl-2,3-
d i-O-m eth yl-r-D-glu cop yr a n osyl)-(1f4)]6-6-O-a cetyl-2,3-
d i-O-m eth yl-r-D-glu cop yr a n osid e (19). Thioglycoside 16
(3.86 g, 3.54 mmol) and glycosyl acceptor 18 (3.60 g, 3.44 mmol)
(6-O-Acetyl-4-O-levu lin yl-2,3-d i-O-m eth yl-r-D-glu cop y-
r a n osyl)-[(1f4)-(6-O-a cetyl-2,3-d i-O-m eth yl-r-D-glu cop y-
r a n osyl)]2-(1f4)-6-O-a cetyl-2,3-d i-O-m eth yl-1-tr ich lor o-
a cetim id oyl-D-glu cop yr a n ose (23). A solution of 17 (200 mg,
0.174 mmol) in 2:1 TFA/CH2Cl2 (2.5 mL) was stirred for 3 h,

Carbohydrates as Low Sulfated Heparin Mimetics
J . Org. Chem., Vol. 64, No. 26, 1999 9519
2:1 toluene/n-PrOAc (16 mL) was added, and the mixture was
concentrated and co-concentrated with toluene. Column chro-
matography (3:2 toluene/acetone) of the residue yielded a solid
(178 mg), which was dissolved in CH2Cl2 (5 mL) and was
treated under argon with Cs2CO3 (140 mg, 0.40 mmol) and
CCl3CN (75 µL, 0.749 mmol) for 2 h. The reaction mixture was
then filtered, concentrated, and purified over silica gel (3:2
toluene/acetone) to yield 23 (230 mg, 77%): [R]D +112° (c 0.99,
CH2Cl2); 1H NMR (CDCl3) δ 8.66-8.64 (2s, 1 H, R and â N:H),
6.51 (d, J ) 3.6 Hz, H-1R GlcI), 5.71 (d, J ) 7.5 Hz, H-1â GlcI),
2.90-2.52 (m, 4 H, O(CdO)CH2CH2(CdO)CH3, 2.17, 2.11, 2.11,
2.09, 2.05 (5s, 4Ac and O(CdO)CH2CH2(CdO)CH3); mass
spectrum (ESI) m/z 1226 [(M + K)+].
Meth yl (6-O-Acetyl-4-O-levu lin yl-2,3-d i-O-m eth yl-r-D-
glu cop yr a n osyl)-(1f4)-(6-O-a cet yl-2,3-d i-O-m et h yl-r-D-
glu cop yr a n osyl)-(1f4)-(ben zyl 2,3-d i-O-m eth yl-â-D-glu -
cop yr a n osylu r on a te)-(1f4)-(3,6-d i-O-a cetyl-2-O-ben zyl-
r-D-glu cop yr a n osyl)-(1f4)-(b en zyl 2,3-d i-O-m et h yl-r-L-
id o p y r a n o s y lu r o n a t e )-(1f4)-2,3,6-t r i-O -b e n zy l-r-D -
glu cop yr a n osid e (25). Imidate 13 (175 mg, 0.242 mmol) and
acceptor 24 (306 mg, 0.220 mmol) were treated in 1:2 CH2Cl2/
Et2O (6 mL) under the same conditions described for the
synthesis of 16, with molecular sieves (170 mg, 4 Å) and
t-BDMSOTf (26 µL, 0.5 equiv), for 40 min. Neutralization was
realized by adding solid NaHCO3, the mixture was filtered,
diluted with CH2Cl2, and processed as for 16. Column chro-
matography (7:3 cyclohexane/acetone) yielded unreacted 24 (88
mg, 29%) and 25 (260 mg, 60%): [R]D +76° (c 0.97, CH2Cl2);
1H NMR (CDCl3) δ 7.44-7.21 (m, 30H, 6Ph), 5.54 (d, J ) 3.8
Hz, H-1 GlcVI), 5.50 (d, J ) 3.7 Hz, H-1 GlcV), 5.29 (d, J ) 6.6
Hz, H-1 GlcII), 5.18 (d, J ) 3.5 Hz, H-1 GlcIII), 4.56 (d, J ) 3.7
Hz, H-1 GlcI), 4.08 (d, J ) 7.7 Hz, H-1 IdoUAII), 2.80-2.60
(m, 4H, O(CdO)CH2CH2(CdO)CH3), 2.17, 2.13, 2.05, 2.00, 1.88
(5s, 15H, 4Ac and O(CdO)CH2CH2(CdO)CH3); mass spectrum
(ESI) m/z 1974 [(M + Na)+]. Anal. Calcd for C100H126O39: C,
61.53; H, 6.51. Found: C, 61.28; H, 6.59.
1876 [(M + Na)+]. Anal. Calcd for C95H120O37: C, 61.54; H,
6.52. Found: C, 61.29; H, 6.51.
Meth yl [(6-O-Acetyl-2,3-d i-O-m eth yl-r-D-glu cop yr a n o-
syl)-(1f4)]4-(ben zyl 2,3-d i-O-m eth yl-â-D-glu cop yr a n osyl-
u r on a te)-(1f4)-(3,6-d i-O-a cetyl-2-O-ben zyl-r-D-glu cop y-
r a n osyl)-(1f4)-(ben zyl 2,3-d i-O-m eth yl-r-L-id op yr a n osyl-
u r on a t e)-(1f4)-2,3,6-t r i -O-b en zyl-r-D-glu cop yr a n osid e
(28). Octasaccharide 26 (120 mg, 50 µmol) was reacted like
14 with hydrazine acetate (45 mg, 0.496 mmol) in 2:1 EtOH/
toluene (22 mL) for 1.5 h. The reaction mixture was concen-
trated, diluted in CH2Cl2, washed with 10% aqueous KHSO4,
H2O, saturated aqueous NaHCO3, and brine, dried (Na2SO4),
and concentrated. The residue was finally purified over silica
gel (3:1 toluene/acetone) to give 28 (95 mg, 83%): [R]D +80° (c
0.62, CH2Cl2); 1H NMR (CDCl3) δ 7.42-7.12 (m, 30H, 6Ph),
5.50, 5.46, 5.43, 5.40 (4d, J ) 3.9, 3.9, 3.7, 3.7 Hz, H-1 GlcV-VIII),
2.14, 2.10, 2.09, 2.08, 2.00, 1.88 (6s, 18H, 6Ac); mass spectrum
(ESI) m/z 2357 [(M + K)+]. Anal. Calcd for C115H152O49: C,
59.57; H, 6.60. Found: C, 59.49; H, 6.61.
Meth yl (6-O-Acetyl-4-O-levu lin yl-2,3-d i-O-m eth yl-r-D-
glu cop yr a n osyl)-(1f4)-[(6-O-a cetyl-2,3-d i-O-m eth yl-r-D-
glu cop yr a n osyl)-(1f4)]11-(b en zyl 2,3-d i-O-m et h yl-â-D-
glu copyr an osylu r on ate)-(1f4)-(3,6-di-O-acetyl-2-O-ben zyl-
r-D-glu cop yr a n osyl)-(1f4)-(b en zyl 2,3-d i-O-m et h yl-r-L-
id o p y r a n o s y lu r o n a t e )-(1f4)-2,3,6-t r i-O -b e n zy l-r-D -
glu cop yr a n osid e (29). Imidate 22 (75 mg, 24.6 µmol) and
acceptor 24 (33 mg, 23.9 µmol) were reacted in the presence
of powdered molecular sieves (18 mg, 4 Å) in 1:2 CH2Cl2/Et2O
(0.75 mL) with a M solution of t-BDMSOTf (12.3 µL) in CH2-
Cl2, as described for the synthesis of 16. After 1 h of stirring,
the same amount of t-BDMSOTf was added, stirring was
continued for 10 min, and the reaction mixture was neutralized
with NaHCO3. After filtration (Celite), the mixture was diluted
with CH2Cl2, washed with 2% aqueous NaHCO3 and H2O,
dried (Na2SO4), and filtered. The residue was first purified over
a Toyopearl HW40 gel column using 1:1 CH2Cl2/EtOH as an
eluant to remove unreacted 24 (15 mg, 45%) and then using
silica gel to yield 29 (45 mg, 44%): [R]D +135° (c 0.53, CH2-
Cl2); 1H NMR (CDCl3) δ 5.53 (d, J ) 3.8 Hz, H-1 GlcXVI), 5.50-
5.40 (m, 11H, H-1 GlcV-XV), 5.28 (d, J ) 6.6 Hz, H-1 IdoUAII),
5.17 (d, J ) 3.5 Hz, H-1 GlcIII), 4.58 (d, J ) 3.6 Hz, H-1 GlcI),
4.11 (d, J ) 7.9 Hz, H-1 GlcUAIV), 2.83-2.52 (m, 4H, O(Cd
O)CH2CH2(CdO)CH3), 2.17, 2.15, 2.12, 2.11, 2.08, 2.05, 1.99,
1.87 (8s, 45H, 14Ac and O(CdO)CH2CH2(CdO)CH3); mass
spectrum (ESI) m/z 4274 [(M + 2K)2+]/2. Anal. Calcd for
Meth yl (6-O-Acetyl-4-O-levu lin yl-2,3-d i-O-m eth yl-r-D-
glu cop yr a n osyl)-[(1f4)-(6-O-a cetyl-2,3-d i-O-m eth yl-r-D-
glu cop yr a n osyl)]3-(1f4)-(ben zyl 2,3-d i-O-m eth yl-â-D-glu -
cop yr a n osylu r on a te)-(1f4)-(3,6-d i-O-a cetyl-2-O-ben zyl-
r-D-glu cop yr a n osyl)-(1f4)-(b en zyl 2,3-d i-O-m et h yl-r-L-
id o p y r a n o s y lu r o n a t e )-(1f4)-2,3,6-t r i-O -b e n zy l-r-D -
glu cop yr a n osid e (26). A solution of 23 (73 mg, 0.061 mmol)
and acceptor 24 (82 mg, 59 µmol) was treated for 30 min in
2:1 Et2O/CH2Cl2 (1 mL) as for the synthesis of 16. After
workup, the compound was first purified using a Sephadex
LH-20 chromatography column (1:1 CH2Cl2/EtOH) to remove
unreacted 24 (20 mg, 14%) and then over a silica gel column
C
200H286O99: C, 56.20; H, 6.74. Found: C, 55.81; H, 6.58.
Meth yl (6-O-Acetyl-4-O-levu lin yl-2,3-d i-O-m eth yl-r-D-
glu cop yr a n osyl)-[(1f4)-(6-O-a cetyl-2,3-d i-O-m eth yl-r-D-
glu cop yr a n osyl)]13-(1f4)-(b en zyl 2,3-d i-O-m et h yl-â-D-
glu copyr an osylu r on ate)-(1f4)-(3,6-di-O-acetyl-2-O-ben zyl-
r-D-glu cop yr a n osyl)-(1f4)-(b en zyl 2,3-d i-O-m et h yl-r-L-
id o p y r a n o s y lu r o n a t e )-(1f4)-2,3,6-t r i-O -b e n zy l-r-D -
glu cop yr a n osid e (30). A solution of 22 (144 mg, 47.3 µmol)
and 27 (85 mg, 45.8 µmol) was reacted in 1:2 CH2Cl2/Et2O (2.5
mL) in the presence of molecular sieves (41 mg, 4 Å), with a
M solution of t-BDMSOTf (23.6 µL) in CH2Cl2 under the
conditions described for 16. After 10 min of stirring, the
reaction mixture was filtered (Celite), diluted with CH2Cl2,
washed with 2% aqueous NaHCO3 and H2O, dried (Na2SO4),
filtered, and concentrated. The residue was first purified over
a Toyopearl HW50 gel column (1:1 CH2Cl2/EtOH) to separate
unreacted 27 (33 mg, 39%) and then by silica gel chromatog-
raphy (3:2 toluene/acetone) to yield pure octadecasaccharide
30 (103 mg, 48%): [R]D +143° (c 0.57, CH2Cl2); 1H NMR
(CDCl3) δ 7.34-7.25 (m, 30H, 6Ph), 5.54 (d, J ) 3.8 Hz, H-1
GlcXVIII), 5.51-5.40 (m, 13H, H-1 GlcV-XVII), 5.30 (d, J ) 6.6
Hz, H-1 IdoUAII), 5.17 (d, J ) 3.5 Hz, H-1 GlcIII), 4.56 (d, J )
3.6 Hz, H-1 GlcI), 4.09 (d, J ) 7.9 Hz, H-1 GlcUAIV), 2.82-
2.51 (m, 4H, O(CdO)CH2CH2(CdO)CH3), 2.17, 2.16, 2.13, 2.11,
2.08, 2.05, 2.00, 1.88, 1.61 (9s, 51H, 16Ac and O(CdO)CH2-
1
to give pure 26 (98 mg, 69%): [R]D +95° (c 1.01, CH2Cl2); H
NMR (CDCl3) δ 7.43-7.20 (m, 30H, 6Ph), 5.55 (d, J ) 3.9 Hz,
H-1 GlcVIII), 5.50 (d, J ) 3.9 Hz, H-1 GlcV), 5.44, 5.38 (2d, J )
3.7, 3.9 Hz, H-1 GlcVI, H-1 GlcVII), 5.29 (d, J ) 6.8 Hz, H-1
IdoUAII), 5.17 (d, J ) 3.5 Hz, H-1 GlcIII), 4.56 (d, J ) 3.7 Hz,
H-1 GlcI), 4.10 (d, J ) 7.9 Hz, H-1 GlcUAIV), 2.81-2.50 (m,
4H, O(CdO)CH2CH2(CdO)CH3), 2.17, 2.15, 2.11, 2.09, 2.05,
2.00 (7s, 21H, 6Ac and O(CdO)CH2CH2(CdO)CH3); mass
spectrum (ESI) m/z 2456 [(M + K)+]. Anal. Calcd for
C
120H158O51: C, 59.63; H, 6.59. Found: C, 59.23; H, 6.58.
Meth yl [(6-O-Acetyl-2,3-d i-O-m eth yl-r-D-glu cop yr a n o-
syl)-(1f4)]2-(ben zyl 2,3-d i-O-m eth yl-â-D-glu cop yr a n osyl-
u r on a te)-(1f4)-(3,6-d i-O-a cetyl-2-O-ben zyl-r-D-glu cop y-
r a n osyl)-(1f4)-(ben zyl 2,3-d i-O-m eth yl-r-L-id op yr a n osyl-
u r on a te)-(1f4)-2,3,6-tr i-O -ben zyl-r-D -glu cop yr a n osid e
(27). A solution of hexasaccharide 25 (149 mg, 76 µmol) in 1:2
toluene/EtOH (21 mL) was treated like 14 with hydrazine
acetate (70 mg, 0.76 mmol), for 4 h. The reaction mixture was
then concentrated, diluted in CH2Cl2, washed with 10%
aqueous KHSO4, H2O, saturated aqueous NaHCO3, and brine,
dried (Na2SO4), and concentrated. Chromatography (7:3 cy-
clohexane/acetone) of the residue gave 27 (126 mg, 89%): [R]D
+94° (c 0.77, CH2Cl2); 1H NMR (CDCl3) δ 7.42-7.23 (m, 30H,
6Ph), 5.50, 5.48 (2d, J ) 3.7 Hz, H-1 GlcV, H-1 GlcVI), 5.30 (d,
J ) 6.8 Hz, H-1 IdoUAII), 5.17 (d, J ) 3.5 Hz, H-1 GlcIII), 4.57
(d, J ) 3.6 Hz, H-1 GlcI), 4.08 (d, J ) 8.1 Hz, H-1 GlcUAIV),
2.12, 2.09, 2.00, 1.87 (4s, 12H, 4Ac); mass spectrum (ESI) m/z
CH2(CdO)CH3); mass spectrum (ESI) m/z 2408 [(M + 2K)2+
/
2]. Anal. Calcd for C220H318O111: C, 55.76; H, 6.76. Found: C,
55.29; H, 6.76.
Meth yl (6-O-Acetyl-4-O-levu lin yl-2,3-d i-O-m eth yl-r-D-
glu cop yr a n osyl)-[(1f4)-(6-O-a cetyl-2,3-d i-O-m eth yl-r-D-

9520 J . Org. Chem., Vol. 64, No. 26, 1999
Driguez et al.
glu cop yr a n osyl)]15-(1f4)-(b en zyl 2,3-d i-O-m et h yl-â-D-
glu copyr an osylu r on ate)-(1f4)-(3,6-di-O-acetyl-2-O-ben zyl-
r-D-glu cop yr a n osyl)-(1f4)-(b en zyl 2,3-d i-O-m et h yl-r-L-
id o p y r a n o s y lu r o n a t e )-(1f4)-2,3,6-t r i-O -b e n zy l-r-D -
glu cop yr a n osid e (31). Imidate 22 (84 mg, 27 µmol) was
reacted with acceptor 28 (62 mg, 27 µmol) in 1:3 CH2Cl2/Et2O
(4 mL) under the conditions described for 16, using powdered
molecular sieves (85 mg, 4 Å) and a M solution of t-BDMSOTf
(14 µL) in CH2Cl2. After 1 h of stirring, the reaction mixture
was diluted with CH2Cl2, filtered (Celite), washed with satu-
rated aqueous NaHCO3, dried (Na2SO4), filtered, and concen-
trated. The residue was first purified using a Toyopearl HW40
gel column to remove unreacted 28 (14 mg, 23%) and then on
silica gel (1:1 acetone/cyclohexane) to yield pure 31 (71 mg,
fon a t o-r-D-glu cop yr a n osyl)]13-(1f4)-(sod iu m 2,3-d i-O-
m et h yl-â-D-glu cop yr a n osylu r on a t e)-(1f4)-(2,3,6-t r i-O-
sodiu m su lfon ato-r-D-glu copyr an osyl)-(1f4)-(sodiu m 2,3-
d i-O-m eth yl-r-L-id op yr a n osylu r on a te)-(1f4)-2,3,6-tr i-O-
sod iu m su lfon a to-r-D-glu cop yr a n osid e (3). Compound 30
(71 mg, 15 µmol) was hydrogenolyzed in AcOH (3 mL) with
5% Pd/C (140 mg, 40 bar) for 3.5 h at 50 °C. After filtration
(Celite) and co-distillation with toluene and then with MeOH,
the residue (62 mg) was dissolved in MeOH (6.5 mL), and 5
M aqueous NaOH (0.256 mL) was added at 0 °C. After 2 h of
stirring, the reaction mixture was acidified (pH 3) with H+
Dowex AG 50 WX4, concentrated, and loaded on a Toyopearl
HW40 gel column (H2O). The fractions were pooled and dried.
The residue (42 mg) thus obtained was dissolved in DMF (3
mL), and Et3N/SO3 complex (111 mg, 0.613 mmol) was added.
The reaction mixture was stirred in the dark at 55 °C for 21
h and then cooled and processed as for 2. Lyophilization
1
51%): [R]D +136° (c 0.95, CH2Cl2); H NMR (CDCl3) δ 7.42-
7.18 (m, 30H, 6Ph), 5.54 (d, J ) 3.8 Hz, H-1 GlcXX), 5.51-5.40
(m, 15H, H-1 GlcV-XIX), 5.30 (d, J ) 6.8 Hz, H-1 IdoUAII), 5.17
(d, J ) 3.5 Hz, H-1 GlcIII), 4.56 (d, J ) 3.7 Hz, H-1 GlcI), 4.09
(d, J ) 7.9 Hz, H-1 GlcUAIV), 2.85-2.53 (m, 4H, O(CdO)-
CH2CH2(CdO)CH3), 2.17, 2.16, 2.13, 2.11, 2.08, 2.05, 2.00, 1.88
(8s, 57H, 18Ac and O(CdO)CH2CH2(CdO)CH3); mass spec-
1
yielded 3 (29 mg, 87% from 30): [R]D +124° (c 1.25, H2O); H
NMR (D2O) δ 5.71-5.67 (m, 13H, H-1 GlcVI-XVIII), 5.48 (d, J )
3.5 Hz, H-1 GlcV), 5.42 (d, J ) 3.3 Hz, H-1 GlcIII), 5.17 (d, J )
3.7 Hz, H-1 GlcI), 5.11 (br s, H-1 IdoUAII), 4.68 (d, J ) 7.5 Hz,
H-1 GlcUAIV); mass spectrum (ESI) m/z 779 [(M - 7Na)7-]/7;
CE 97% (tR ) 15.16 min.).
trum (ESI) m/z 2641 [(M + 2K)2+/2]. Anal. Calcd for C240H350
O123‚4H2O: C, 54.64; H, 6.84. Found: C, 54.51; H, 6.79.
-
Meth yl (2,3-Di-O-m eth yl-4,6-d i-O-sod iu m su lfon a to-r-
D-glu copyr an osyl)-[(1f4)-(2,3-di-O-m eth yl-6-O-sodiu m su l-
fon a t o-r-D-glu cop yr a n osyl)]11-(1f4)-(sod iu m 2,3-d i-O-
m et h yl-â-D-glu cop yr a n osylu r on a t e)-(1f4)-(2,3,6-t r i-O-
sodiu m su lfon ato-r-D-glu copyr an osyl)-(1f4)-(sodiu m 2,3-
d i-O-m eth yl-r-L-id op yr a n osylu r on a te)-(1f4)-2,3,6-tr i-O-
sod iu m su lfon a to-r-D-glu cop yr a n osid e (2). A solution of
29 (31 mg, 7.25 µmol) in AcOH (3 mL) was stirred for 4 h at
50 °C in the presence of 5% Pd/C (62 mg, 40 bar). The mixture
was filtered (Celite), concentrated, and codistilled with H2O.
To a solution of the residue (27 mg) in MeOH (3 mL) was
added, at 0 °C, 5 M aqueous NaOH (0.311 mL). After 4.5 h of
stirring, the reaction mixture was diluted with H2O and loaded
over a Sephadex G25 F gel column (H2O). The residue (20 mg)
obtained after concentration was acidified through a H+ Dowex
AG50WX4 (3 mL) column and concentrated. Et3N/SO3 complex
(113 mg, 0.624 mmol) was added to a solution of the compound
obtained in the preceding step in DMF (3 mL), and the mixture
was heated at 55 °C for 20 h with protection from light. After
cooling, the reaction mixture was diluted with 0.2 M aqueous
NaCl (2 mL) and layered on top of a Sephadex G25 F gel
column (0.2 M aqueous NaCl). The fractions were pooled,
concentrated, and desalted on the same gel filtration column,
equilibrated with H2O. Lyophilization gave 2 (35 mg, 82% from
29): [R]D +119° (c 0.56, H2O); 1H NMR (D2O) δ 5.71-5.67 (m,
11H, H-1 GlcVI-XVI), 5.48 (d, J ) 3.5 Hz, H-1 GlcV), 5.43 (d, J
) 3.3 Hz, H-1 GlcIII), 5.16 (d, J ) 3.7 Hz, H-1 GlcI), 5.11 (br s,
H-1 IdoUAII), 4.68 (d, J ) 7.5 Hz, H-1 GlcUAIV); mass spectrum
(ESI) m/z 815 [(M - 6Na)6-]/6; CE 94% (tR ) 15.07 min).
Meth yl (2,3-Di-O-m eth yl-4,6-d i-O-sod iu m su lfon a to-r-
D-glu copyr an osyl)-[(1f4)-(2,3-di-O-m eth yl-6-O-sodiu m su l-
Meth yl (2,3-Di-O-m eth yl-4,6-d i-O-sod iu m su lfon a to-r-
D-glu copyr an osyl)-[(1f4)-(2,3-di-O-m eth yl-6-O-sodiu m su l-
fon a t o-r-D-glu cop yr a n osyl)]15-(1f4)-(sod iu m 2,3-d i-O-
m et h yl-â-D-glu cop yr a n osylu r on a t e)-(1f4)-(2,3,6-t r i-O-
sodiu m su lfon ato-r-D-glu copyr an osyl)-(1f4)-(sodiu m 2,3-
d i-O-m eth yl-r-L-id op yr a n osylu r on a te)-(1f4)-(2,3,6-tr i-O-
sod iu m su lfon a to-r-D-glu cop yr a n osid e (4). Compound 31
(55 mg, 10.5 µmol) was hydrogenolyzed in AcOH (3 mL) in
the presence of Pd/C 5% (110 mg) with H2 (40 bar) during 4 h.
Charcoal was filtered off (Celite), volatiles were removed under
vacuum, and co-distillation with H2O was realized. The residue
(48 mg) was dissolved in MeOH (5 mL), and 5 M aqueous
NaOH (0.193 mL) was slowly added. After 3 h of stirring, the
reaction mixture was processed as described for 29. Finally,
the residue (34 mg) was dissolved in DMF (4 mL), reacted with
Et3N/SO3 (186 mg, 1.03 mmol), and purified as for 2. Freeze-
drying gave 4 (50 mg, 77%): [R]D +107° (c 0.52, H2O); 1H NMR
(D2O) δ 5.71-5.67 (m, 15H, H-1 GlcVI-XX), 5.48 (d, J ) 3.5 Hz,
H-1 GlcV), 5.43 (d, J ) 3.3 Hz, H-1 GlcIII), 5.17 (d, J ) 3.7 Hz,
H-1 GlcI), 5.10 (br s, H-1 IdoUAII), 4.68 (d, J ) 7.5 Hz, H-1
GlcUAIV); mass spectrum (ESI) m/z 596 [(M - 10Na)10-/10];
CE 88% (15.27 min).
Ack n ow led gm en t. This work is part of a collabora-
tive project between N.V. Organon (The Netherlands)
and Sanofi (France) on antithrombotic oligosaccharides.
The authors also wish to thank Dr. P. Sizun (high-field
NMR experiments) and Dr. J .P. He´rault (biological
assays).
J O991152J