Introducing a C-interglycosidic bond in a biologically active pentasaccharide hardly affects its biological properties

Article abstract of DOI:10.1016/S0968-0896(98)00094-7

We describe here the synthesis and the biological activity of a 'C-pentasaccharide', a new analogue of the antithrombin III (AT III) binding region of heparin containing a methylene bridge in place of an interglycosidic oxygen atom. The affinity for AT III and the anti-factor Xa activity of this compound have been compared with that of the corresponding selected 'O-pentasaccharide'. Such a structural modification slightly decreased the affinity of this compound for AT III as well as its anti-factor Xa activity (880±40 anti-Xa units versus 1180±30 anti-Xa units for the C-pentasaccharide and the O-pentasaccharide, respectively). This compound therefore represents the first example of a new class of anti-factor Xa pentasaccharides containing a C-interglycosidic bond. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00094-7

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1509±1516
Introducing a C-Interglycosidic Bond in a Biologically Active
Pentasaccharide Hardly A€ects its Biological Properties
Maurice Petitou,a Jean-Pascal Herault,a Jean-Claude Lormeau,a
Arnim Helmboldt,b Jean-Maurice Mallet,b Pierre Sinay,b,*
and Jean-Marc Herberta
aHaemobiology Research Department, Sano® Recherche, 195 route d'Espagne, 31036 Toulouse Cedex, France
 Â
Ecole Normale Superieure, Departement de Chimie, URA CNRS 1686, 24 rue Lhomond, 75231 Paris Cedex 05, France
b
Received 18 December 1997; accepted 4 March 1998
AbstractÐWe describe here the synthesis and the biological activity of a `C-pentasaccharide', a new analogue of the
antithrombin III (AT III) binding region of heparin containing a methylene bridge in place of an interglycosidic oxygen
atom. The anity for AT III and the anti-factor Xa activity of this compound have been compared with that of the
corresponding selected `O-pentasaccharide'. Such a structural modi®cation slightly decreased the anity of this com-
pound for AT III as well as its anti-factor Xa activity (880‹40 anti-Xa units versus 1180‹30 anti-Xa units for the
C-pentasaccharide and the O-pentasaccharide, respectively). This compound therefore represents the ®rst example
of a new class of anti-factor Xa pentasaccharides containing a C-interglycosidic bond. # 1998 Published by Elsevier
Science Ltd. All rights reserved.
Introduction
the conformational properties of the molecule7 resulting
in enhanced or impaired biological activity. To docu-
ment this problem, the comparison of biologically active
compounds only di€ering by the presence of `C' versus
`O' interglycosidic bonds is therefore highly desirable.
Among the very restricted number of oligosaccharides
clinically investigated is the pentasaccharide that repro-
duces the exact sequence required in heparin for binding
and activation of antithrombin III (AT III).8 Several
analogues of this compound have been obtained which
are not susceptible to enzymatic degradation.9 However,
having such a series of biologically active pentasacchar-
ides at our disposal, we decided to investigate how a C-
disaccharide bond in one of them would in¯uence the
interaction with the target protein `receptor' anti-
thrombin III.10
Recent studies have demonstrated that oligosaccharides
are involved in a number of biological processes1 and
more and more constitute potential active substances
for drug development.2 One problem in this context is
the susceptibility of native structure to glycosidases.
Several structural modi®cations have thus been devel-
oped to increase the resistance of oligosaccharides to
enzymatic hydrolysis: introduction of S-interglycosidic
bonds,3 alkylation4 and acylation5 of noncritical hydro-
xyl functions. Various methods have also been reported
for the introduction of C-interglycosidic bonds, result-
ing in C-disaccharides.6 A critical feature is that the
biological activity must be present and if possible
increased in the analogue thus obtained, which requires
the active conformation being preserved. The under-
lying basic question is indeed to evaluate to what extend
the replacement of the oxygen atom by a methylene
groupÐwhich eradicates the exo anomeric e€ectÐa€ects
Results and Discussion
Total synthesis
Key words: Antithrombotics; heparin analogue; oligosacchar-
ide; C-pentasaccharide; antithrombin III.
*Corresponding author. Tel: +33 (0) 1 44 32 33 90; Fax: +33
(0) 1 44 32 33 97; E-mail: pierre.sinay@ens.fr
We selected 14 (Fig. 1) as the target pentasaccharide
because its structure was well adapted to the type of
chemistry we had previously developed for the synthesis
0968-0896/98/$Ðsee front matter # 1998 Published by Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00094-7

1510
M. Petitou et al./Bioorg. Med. Chem. 6 (1998) 1509±1516
Figure 1. Structure of the synthetic O- and C-pentasaccharides.
of the C-disaccharide building block precursor of
the DE part of the molecule.11 The preparation of
at position 4 of l-iduronic acid derivatives; only the a-
anomer was formed during the reaction. Conversion of
13 into 14 was achieved through hydrogenation, sapo-
ni®cation, and sulfation (65% overall yield for the three
steps).
the corresponding `O-pentasaccharide'
1 has been
described, and its biological properties thoroughly
investigated.12
The precursor of the unit F is the alcohol 5, which is
synthesized according to Scheme 1. The epoxide 213 was
opened in a trans diaxial manner with sodium benzylate
to give 3 which, after O-acetylation to 4, was hydrolysed
to provide the crystalline compound 5.
Biological properties in vitro
Anity for antithrombin III. AT III is the plasma
`receptor' of the synthetic pentasaccharides. It is there-
fore of prime importance to assess the in¯uence of the
structural modi®cations described above on the binding
anity for AT III. Interaction of the pentasaccharides
with AT III induces a change in ¯uorescence of AT III
which allows titration and determination of the anity
of the di€erent ligands.20 We used this method to
determine the anities of 1 and 14 for AT III (Table 1).
The substitution of a O-glycosidic bond by a C-glycosi-
dic bond resulted in a slight decrease in the anity for
AT III (Kd=1.9‹0.1 versus 2.8‹0.2 nM for 1 and 14,
respectively).
Compound 5 was then glycosylated with the previously
described10,11 derivative 6 to give the trisaccharide 7
(Scheme 2). The reaction was carried out in acetonitrile
to take advantage of the `nitrilium e€ect'.14 We thus
obtained in excellent yield (88%) a mixture containing 7
and its a-anomer 8 (b:a ratio 5.5:1).
Acetolysis of the 1,6-anhydro ring15 (Scheme 3) quanti-
tatively gave the expected a,b-acetates 9 (a:b 3:1) which
after selective removal of the anomeric O-acetyl group
by hydrazine acetate in DMF16 provided the hemiacetal
10 (90%, a:b 1:1). The latter was converted into the
imidate 11 using trichloroacetonitrile and DBU17 in
dichloromethane (94%, a:b 5:1). This trisaccharide 11
was then coupled to the alcohol 1218 to give the fully
protected pentasaccharide 13 in 80% yield from 11 (two
equivalents of 12 were used, and we did not try to
recover the excess). This previously reported 3+2 strat-
egy of coupling19 allowed us to take advantage of the
stereoselective a-coupling observed during glycosylation
Inhibition of factor Xa. Binding of the pentasaccharides
to AT III induces a conformational change of the pro-
tein20 which results in an accelerated inhibition of blood
coagulation factor Xa by AT III. The anti-factor Xa
activity was determined by an amidolytic method adap-
ted from Teien and Lie.21 Since the amount of AT III-
pentasaccharide complex depends on the anity of the
oligosaccharide for AT III, the anti-Xa activity of 1 and
14 followed the same ranking as for their anity for AT
III (Table 1).
Scheme 1. Synthesis of the alcohol 5. Reagents and conditions: (i) BnONa, BnOH, 110 ꢀC, 64%; (ii) Ac2O, Et3N, DMAP, CH2Cl2,
20 ꢀC; (iii) PTSA, MeOH, 20 ꢀC, 87% form 3.

M. Petitou et al./Bioorg. Med. Chem. 6 (1998) 1509±1516
1511
Scheme 2. Synthesis of the C-trisaccharide 8. Reagent and conditions: TMSOTf, CH3CN, 37 ꢀC, 88% 7:8 (5.5:1).
Scheme 3. Synthesis of the C-pentasaccharide 14. Reagents and conditions: (i) H2SO4, AcOH, Ac2O), 20 ꢀC, 100%; (ii)
NH3NH2OAc, DMF, 90%; (iii) CCl3CN, DBU, CH2Cl2, 94%; (iv) TMSOTf, CH2Cl2, 20 ꢀC, 79%; (v) 1. H2, Pd/C, CH2Cl2,
MeOH, 99%, 2. NaOH, H2O, MeOH, 0 ꢀC, 77%, 3. SO3. Et3N, DMF, 55 ꢀC, 85%.
factor Xa. Since we have recently shown22 that the DEF
trisaccharide part of the pentasaccharide molecules is
Conclusion
We conclude from the results of factor Xa inhibition
that substitution of an O-glycosidic by a C-glycosidic
bond does not change the ability to induce the con-
formational change in AT III resulting in active site
loop exposure and trapping of the target serine protease
responsible for the recognition of AT III, the active
conformation is preserved in the C-pentasaccharide.
The lower anity of the `C-glycoside' may be due to the
increased ¯exibility of the C-pentasaccharide leading to
a higher cost in conformational entropy when it binds to
its receptor protein.
Table 1. Biological properties of the pentasaccharides 1 and
14 in vitro
Experimental
General
Compd
Anity for AT III Anti-factor Xa activity
(Kd in nM)
(U/mg)
1 (O-glycoside)
14 (C-glycoside)
1.9‹0.1
2.8‹0.2
1180‹30
880‹40
All solvents and reagents were of the best commercially
available grade or were puri®ed and dried according to
standard procedures. Reactions were monitored by TLC
Values are arithmetic means‹SD (n=6).

1512
M. Petitou et al./Bioorg. Med. Chem. 6 (1998) 1509±1516
on silica gel 60 F254 (Merck) with detection by charring
with H2SO4. Column chromatography was performed
on silica gel 60 (E. Merck 63±200 mm). NMR spectra
were recorded with Bruker AM 100, AC 250, AM 400
or AM 500 instruments for solution in CDCl3 (internal
Me4Si) unless otherwise stated. Protons H-4ha and H-4hb
correspond respectively to the low ®eld and high ®eld
hydrogen of the methylene linkage. MS analyses were
performed on Nermag R10-10 instrument using chemi-
cal ionization (NH3) and detection of positive ions.
Melting points were determined in capillary tubes with a
Buchi 510 apparatus and are uncorrected. Optical rota-
tions were measured with a Perkin-Elmer Model 141
polarimeter at 23‹3 ꢀC. Elemental analyses were
carried out at the Service Central d'Analyses (C.N.R.S.,
Vernaison, France).
(m, 5H, Ph), 5.36 (br. s, 1H, H-1), 4.93 (br.s, 1H, H-3),
4.10 (dd, 1H, J=7.5Hz, H-6), 3.76 (dd, 1H, J=5.7 Hz,
7.5 Hz, H-60), 3.58 (br. m, 1H, H-4), 3.26 (br. m, 1H,
H-2), 2.08 (s, 3H, Ac); MS, FAB, positive mode: m/z
thioglycerol + NaCl, 317 (M+Na)+; thioglycerol +
KF, 333 (M+K)+; Anal. calcd for C15H18O6 (294.29):
C, 61.21 ; H, 6.16. Found : C, 61.33 ; H, 6.23.
O-(6-O-Acetyl-2,3,4-tri-O-methyl-ꢁ-D-glucopyranosyl-
methyl)-(1!4)-C-(benzyl 4-deoxy-2,3-di-O-methyl-ꢀ-D-
glucopyranosyluronate)-(1!4)-3-O-acetyl-1,6-anhydro-2-
O-benzyl-ꢀ-D-glucopyranose (7) and O-(6-O-Acetyl-
2,3,4-tri-O-methyl-ꢁ-D-glucopyranosylmethyl)-(1!4)-C-
(benzyl 4-deoxy-2,3-di-O-methyl-ꢁ-D-glucopyranosyl-
uronate)-(1!4)-3-O-acetyl-1,6-anhydro-2-O-benzyl-ꢀ-
D-glucopyranose (8). A 0.05 M solution of TMSOTf in
CH3CN (250 mL) was slowly added to a cooled solution
of 6 (179 mg, 255 mmol) and 5 (84 mg, 0.285 mmol) in
acetonitrile under argon at 37 ꢀC. After 1.5 h solid
KHCO3 (200 mg) was introduced and the temperature
was allowed to rise to room temperature. After ®ltration
and concentration, column chromatography of the resi-
due (CH2Cl2:EtOAc:acetone, 20:1:2) gave 7 (156 mg,
74%) and 8 (30 mg, 14%). For 7: [a]d 32ꢀ (c 1.0,
1,6-Anhydro-2-O-benzyl-4-O-(tetrahydropyran-2-yl)-ꢀ-
D-glucopyranose (3). A mixture of 1,6:2,3-dianhydro-4-
O-(tetrahydropyran-2-yl)-b-d-mannopyranose 2 (6.16 g,
27.05 mmol) and a solution of sodium benzylate in ben-
zyl alcohol (1 M, 135 mL) was heated at 110 ꢀC for 0.5 h.
The cooled mixture was neutralized (Dowex 50 H+) ®l-
tered, and concentrated. The residue was puri®ed by
¯ash column chromatography (CH2Cl2/AcOEt 10:1 to
2:1) to give 3 as a white solid. (6.09 g, 64%). Isomer 1:
1
CHCl3); H NMR (400 MHz) d 7.37±7.24 (m, 10H, H
arom), 5.40 (s, 1H, H-1), 5.27 (s, 1H, H-3), 5.14 (s, 2H,
COOCH2Ph), 4.76 and 4.51 (d, 1H, J=12.2 Hz,
OCH2Ph), 4.68 (d,01H, J5,6b=5.5 Hz, H-5), 4.55 (m, 1H,
[a]d 69ꢀ (c 1.00; CH2Cl2); H NMR: d 7.42±7.25 (m, 5
1
H, Ph), 5.42 (s, 1 H, H-1), 4.52 (d, 1 H, J=5 Hz, H-5),
3.42 (dd, 1 H, J=5,6 Hz, H-4), 3.26 (d, 1 H, J=5.9 Hz,
H-2). Isomer 2: melting point 121 ꢀC; [a]d +29ꢀ (c 0.97;
CH2Cl2); 1H NMR d 7.39±7.28 (m, 5H, Ph), 5.44 (s, 1H,
H-1), 3.55 (dd, 1 H, J=1.3 Hz and 5.6 Hz, H-4), 3.26 (d,
1 H, J=4.5 Hz, H-2). MS, FAB, positive mode: m/z
thioglycerol + NaCl, 359 (M+Na)+; thioglycerol +
KF, 375 (M+K)+; Anal. calcd for C18H24O6 (336.37):
C, 64.27 ; H, 7.19. Found: C, 64.28 ; H, 7.34.
0
0
0
00
J1 ,2 =7.3 Hz, H-1 ), 4.39 (ddd, 10H0 , J4 ha,1 =11.7 Hz,
00 00
0
00
J1 ,2 =5.2 Hz, J4 hb,1 =2.8 Hz, H-1 ), 4.25±4.17 (m, 2H,
H-6a00, H-6b00), 3.96 (d, 1H, J6a,6b=7.3 Hz, H-6a), 3.89
0
0
0
(d, 1H, J4 ,5 =10.7 Hz, H-5 ), 3.75 (dd, 1H, H-6b), 3.66
(s, 1H, H-4), 3.59, 3.57, 3.52, 3.49 and 3.39 (s, 3H,
OCH3), 3.53±3.45 (m, 1H, H-50), 3.26±3.16 (m, 5H, H-2,
H-20, H-30, H-200, H-300), 3.01 (dd, 1H, J4 ,5 =9.5 Hz,
00 00
J3 ,4 =8.0 Hz, H-4 ), 2.25±2.17 (m, 1H, H-4 ), 2.06 and
00 00
0
00
0
0
2.05 (s, 3H, OCOCH3), 1.75 (ddd, 1H, J4 ,4 ha=2.3 Hz,
0
0
0
0
3-O-Acetyl-1,6-anhydro-2-O-benzyl-4-O-(tetrahydropyran-
2-yl)-ꢀ-D-glucopyranose (4).
H-40ha), 1.56 (ddd, 1H, J4 ha,4 hb=14.5 Hz, J4 ,4 hb
=8.0 Hz, H-40hb); 13C NMR (100.58 MHz) d 170.88,
169.30 and 168.53 (C=O), 137.71 and 135.02 (C arom),
128.47±127.58 (C arom), 102.67 (C-10), 100.64 (C-1),
84.53, 83.31, 82.75, 81.23, 79.58, 76.01, 75.82, 73.59,
73.21, 69.78 and 69.33 (C-2, C-3, C-4, C-5, C-20, C-30, C-
50, C-200, C-300, C-400, C-500), 71.01 (C-10, 70.96
(OCH2Ph), 67.11 (COOCH2Ph), 64.82 (C-6), 63.41 (C-
600), 60.29, 60.20, 60.16, 58.84 and 58.46, (OCH3), 38.40
(C-40), 23.66 (C methylene), 21.03 and 20.79 (COCH3).
MS, m/z 850 (M+NH4)+; Anal. calcd for C42H56O17
(832.894): C 60.57, H 6.78, found: C 60.72, H 7.03.
A solution of 3 (2 g,
6 mmol), triethylamine (5 mL, 35.6 mmol), 4-dimethyl-
aminopyridine (0.15 g, 1.2 mmol) and acetic anhydride
(2.8 mL, 29.8 mmol) in dichloromethane (20 mL) was
stirred overnight at room temperature, diluted with
dichloromethane, washed with 10% aq KHSO4, water
then satd aq NaHCO3 and water, dried (MgSO4) and
concentrated. The compound was used as such in the
next step.
3-O-Acetyl-1,6-anhydro-2-O-benzyl-ꢀ-D-glucopyranose
(5). A solution of crude 4 in a 0.25 M solution of p-
toluenesulfonic acid in methanol (24 mL) was stirred for
1 h at room temperature, diluted with dichloromethane,
washed with water, dried (MgSO4), and concentrated.
The residue was chromatographed on silica gel (cyclo-
hexane/AcOEt 1:1) to give 5 (1.53 g, 87% two steps). [a]d
82ꢀ (c 1.00; CH2Cl2), mp 79 ꢀC; 1H NMR: d 7.36±7.26
For 8: [a]d +21ꢀ (c 1.0, CHCl3); H NMR (400 MHz) d
7.40±7.25 (m, 10H, arom), 5.44 (s, 1H, H-1), 5.27 (d,
1H, J1 ,2 =3.5 Hz, H-10), 5.19 and 5.13 (d, 1H,
J=12.5 Hz, COOCH2Ph), 5.04 (m, 1H, H-3), 4.79 and
4.61 (d, 1H, J=12.0 Hz, OCH2Ph), 4.69 (d, 1H,
J5,6b=5.5 Hz, H-5), 4.44±4.38 (m, 1H, H-100), 4.34 (d,
1
0
0

M. Petitou et al./Bioorg. Med. Chem. 6 (1998) 1509±1516
1513
0
00
0
0
1H, J4 ,5 =9.5 Hz, H-5 ), 4.26±4.18 (m, 2H, H-6a , H-
6b00), 3.91 (d, 1H, J6a,6b=7.5 Hz, H-6a), 3.68 (dd, 1H,
H-6b), 3.57, 3.50, 3.48, 3.46 and 3.42 (s, 3H, OCH3),
3.57±3.51 (m, 2H, H-4, H-30), 3.47±3.42 (m, 1H, H-500),
128.66±127.68 (C arom), 103.65 (C-10), 89.09 (C-1),
85.23, 83.89, 82.72, 81.11, 79.54, 76.00, 75.98, 75.45,
70.96, 70.88, 70.70 and 69.89 (C-2, C-3, C-4, C-5, C-10,
C-20, C-30, C-50, C-200, C-300, C-400, C-500), 72.52
(OCH2Ph), 67.30 (COOCH2Ph), 63.33 (C-600), 61.69 (C-
6), 60.38, 60.27, 60.21, 59.32 and 58.54 (OCH3), 38.69
(C-40), 23.79 (C methylene), 20.97, 20.81 and 20.74
(OCOCH3). Selected data for b anomer: 103.53 (C-10),
93.62 (C-1). MS, m/z 952 (M+NH4)+; Anal. calcd for
C46H62O20 (934.984): C 59.09, H 6.68, found: C 59.04,
H 6.83.
3.54 (dd, 1H, J2 ,3 =8.5 Hz, H-20), 3.27 (dd, 1H,
0
0
00
00 00
00 00
J2 ,3 =9.0 Hz, J1 ,2 =5.5 Hz, H-2 ), 3.25 (s, 1H, H-2),
00 00 00
3.19 (t, 1H, J3 ,4 =8.8 Hz, H-300), 3.020 (dd, 1H,
00 00
J4 ,5 =9.8 Hz, H-4 ), 2.27±2.19 (m, 1H, H-4 ), 2.07 and
2.06 (s, 3H, OCOCH3), 1.81±1.67 (m, 2H, H-40ha, H-
40hb); 13C NMR (62.896 MHz) d 170.97, 170.10 and
169.66 (C=O), 137.68 and 135.14 (C arom), 128.56±
127.69 (C arom), 100.40 (C-1), 97.38 (C-10), 83.17,
81.40, 81.34, 80.79, 79.72, 75.86, 74.86, 74.33, 73.12,
71.65, 69.74 and 69.35 (C-2, C-3, C-4, C-5, C-20, C-30, C-
50, C-100, C-200, C-300, C-400, C-500), 71.35 (OCH2Ph), 67.15
(COOCH2Ph), 65.18 (C-6), 63.53 (C-600), 60.49, 60.32,
59.48, 58.54 and 58.33 (OCH3), 37.79 (C-40), 24.67 (C
methylene), 21.10 and 20.83 (COCH3). MS, m/z 850
(M+NH4)+
O-(6-O-Acetyl-2,3,4-tri-O-methyl-ꢁ-D-glucopyranosyl-
methyl)-(1!4)-C-(benzyl 4-deoxy-2,3-di-O-methyl-ꢀ-D-
glucopyranosyluronate)-(1!4)-3,6-di-O-acetyl-2-O-ben-
zyl-ꢁ,ꢀ-D-glucopyranose (10). Freshly prepared hydrazin-
ium acetate (33 mg, 0.358 mmol) was added to a solu-
tion of 9 (134 mg, 0.144 mmol) in DMF (10 mL). After
30 min at room temperature, saturated aqueous sodium
chloride (40 mL) was introduced and the product was
extracted by ethyl acetate. The organic solution was
dried (MgSO4) and concentrated. Flash chromato-
graphy of the residue (cyclohexane:ethyl acetate:acetone,
5:2:2) a€orded 10 (116 mg, 90%; a: b 1:1). 1H NMR
(400 MHz) d a anomer: 7.39±7.27 (m, 10H, H arom),
5.42 (t, 1H, J2,3=9.5 Hz, J3,4=9.5 Hz, H-3), 5.22 (d, 1H,
J1,2=3.5 Hz, H-1), 5.17 and 5.12 (d, 1H, J=12.5 Hz,
COOCH2Ph), 4.62 (s, 2H, OCH2Ph), 4.54 (dd, 1H,
J6a,6b=12.0 Hz, J5,6a=2.0 Hz, H-6a), 4.36 (ddd, 1H,
O-(6-O-Acetyl-2,3,4-tri-O-methyl-ꢁ-D-glucopyranosyl-
methyl)-(1!4)-C-(benzyl 4-deoxy-2,3-di-O-methyl-ꢀ-D-
glucopyranosyluronate)-(1!4)-1,3,6-tri-O-acetyl-2-O-
benzyl-ꢁ,ꢀ-D-glucopyranose (9). A 5% solution of concd
H2SO4 in acetic acid (80 mL) was added to a solution of
7 (120 mg, 0.144 mmol) in acetic anhydride (12 mL) at
20 ꢀC. After 15 min, CH2Cl2 (50 mL) and saturated aq
NaHCO3 (40 mL) were added. The aqueous phase was
extracted three times with CH2Cl2, the organic layers
were combined, dried (MgSO4) and concentrated. Flash
chromatography (cyclohexane:EtOAc:acetone, 3:1:1 )
gave 9 (134 mg, 100%; a,b 3:1 mixture). 1H NMR
(400 MHz) d: a anomer: 7.41±7.23 (m, 10H, arom), 6.30
(d, 1H, J1,2=3.5 Hz, H-1), 5.38 (t, 1H, J2,3=9.7 Hz,
J3,4=9.7 Hz, H-3), 5.16 and 5.11 (d, 1H, J=12.0 Hz,
COOCH2Ph), 4.62 and 4.49 (d, 1H, J=12.5 Hz,
J4 ha,1 =11.0 Hz, J1 ,2 =5.5 Hz, J4 hb,1 =2.8 Hz, H-100),
0
00
00 00
0
00
4.28±4.12 (m, 4H, H-5, H-6b, H-6a00, H-6b00), 4.14 (d,
1H, J1 ,2 =7.8 Hz, H-1 ), 3.72 (d, 1H, J4 ,5 =11.0 Hz, H-
0
0
0
0
0
50), 3.65±3.57 (m, 1H, H-4), 3.55, 3.51, 3.50, 3.49 and
3.39 (s, 3H, OCH3), 3.52±3.47 (m, 1H, H-2), 3.44±3.37
00
(m, 1H, H-500), 3.22 (dd, 1H, J2 ,3 =9.0 Hz, H-2 ), 3.14
00 00
00
00 00
0
0
(t, 1H, J3 ,4 =9.0 Hz, H-3 ), 3.09 (t, 1H, J2 ,3 =9.5 Hz,
J3 ,4 9.5 Hz, H-30), 3.02±2.96 (m, 2H, H-20, H-400),
2.16±2.07 (m, 1H, H-40), 2.09 (s, 6H, OCOCH3), 2.08
(s, 3H, OCOCH3), 1.70±1.61 (m, 1H, H-40ha), 1.54±1.57
0
0
OCH2Ph), 4.53±4.47 (m, 1H, H-6a), 4.35 (ddd, 1H,
00
0
00
00 00
0
00
J4 ha,1 =11.5 Hz, J1 ,2 =5.5 Hz, J4 hb,1 =2.8 Hz, H-1 ),
4.24 (dd, 1H, J6a,6b=12.8 Hz, J5,6b=4.3 Hz, H-6b),
4.22±4.19 (m, 2H, H-6a00, H-6b00), 4.13 (d, 1H,
(m, 1H, J4 ha,4 hb=15.5 Hz, H-40hb). Selected data for
anomer: 5.16 and 5.11 (d, 1H, J=12.5 Hz,
COOCH2Ph), 4.84 and 5.66 (d, 1H, J=12.0 Hz,
0
0
J1 ,2 =8.0 Hz, H-10), 3.98 (ddd, 1H, J4,5=10.0 Hz,
b
0
0
0
0
0
J5,6a=2.0 Hz, H-5), 3.70 (d, 1H, J4 ,5 =11.0 Hz, H-5 ),
3.65 (t, 1H, H-4), 3.55 (dd, 1H, H-2), 3.54, 3.51, 3.50,
3.49 and 3.39 (s, 3H, OCH3), 3.43±3.39 (m, 1H, H-500),
OCH2Ph), 4.78 (d, 01H, J1,2=7.8 Hz, H-1), 3.71 (d, 1H,
0
0
J4 ,5 =11.0 Hz, H-5 ), 3.31 (dd, 1H, J2,3=9.5 Hz, H-2);
13C NMR (100.58 MHz) d mixture of anomers: 170.93,
170.47, 170.43, 170.18, 168.44 and 168.39 (C=O),
138.04, 137.41, 134.71 and 134.68 (C quart arom),
3.21 (dd, 1H, J2 ,3 =9.0 Hz, H-200), 3.13 (t, 1H,
00 00
J3 ,4 =9.0 Hz, H-0300), 3.08 (dd, 1H, J2 ,3 =9.5 Hz,
00 00
0
0 0
00
0
J3 ,4 =9.5 Hz, H-3 ), 3.01±2.96 (m, 2H, H-2 , H-4 ),
0
2.16, 2.09, 2.07 and 1.92 (s, 3H, OCOCH3), 2.15±2.08
0
128.54±127.47 (C arom), 103.50 and 103.45 (C-10
a
(m, 1H, H-40), 1.65 (ddd, 1H, J4 ,4 ha=2.0 Hz, H-40ha),
and b), 97.10 (C-1 b), 90.53 (C-1 a), 85.05, 85.00,
83.76, 82.63, 81.00, 79.70, 79.49, 76.33, 76.21, 75.88,
75.82, 73.02, 72.86, 70.87, 70.81, 69.78 and 68.46,
(C-2, C-3, C-4, C-5, C-20, C-30, C-50, C-100, C-200, C-300,
C-400, C-500), 73.60, 72.51 and 67.17 (OCH2Ph,
COOCH2Ph a and b), 63.30 (C-60 a and b), 62.18 and
62.04 (C-6 a and b), 60.22, 60.18, 60.11, 59.09 and 58.40
(OCH3), 38.60 and 38.55 (C-40 a and b), 23.69 (C
0
1.49 (ddd, J4 ha,4 hb=15.5 Hz, J4 ,4 hb=8.0 Hz, H-40hb).
Selected data for b anomer: 5.62 (d, 1H, J1,2=8.3 Hz,
H-1), 5.22 (t, 1H, J2,3=9.4 Hz, J3,4=9.4 Hz, H-3), 4.68
0
0
0
0
and 4.60 (d, 1H, J=12.0 Hz, OCH2Ph), 4.12 (d, 1H,
0
J1 ,2 =7.8 Hz, H-1 ), 3.74 (m, 1H, H-5); 13C NMR
(100.58 MHz) d a anomer: 170.92, 170.30, 170.14,
169.17 and 168.45 (C=O), 137.35 and 134.74 (C arom),
0
0

1514
M. Petitou et al./Bioorg. Med. Chem. 6 (1998) 1509±1516
methylene a and b), 20.77, 20.75, 20.72 and 20.63
(OCOCH3). MS, m/z 910 (M+NH4)+; Anal. calcd. for
C44H60O19 (892.947): C 59.18, H 6.77, found: C 59.15,
H 6.73.
molecular sieves at 20 ꢀC. After 15 min solid KHCO3
(500 mg) was introduced and the temperature was
allowed to rise to room temperature. After ®ltration and
concentration, column chromatography of the residue
(CH2Cl2:EtOAc:acetone, 20:1:2) followed by gel ®ltra-
tion through a Sephadex LH-20 column (CH2Cl2/
MeOH) gave 13 (96 mg, 79%). [a]d +15ꢀ (c 0.24,
CHCl3); 1H NMR (500 MHz): 7.38±7.22 (m, 30H, 00H
[O-(6-O-Acetyl-2,3,4-tri-O-methyl-ꢁ-D-glucopyranosyl-
methyl)-(1!4)-C-(benzyl 4-deoxy-2,3-di-O-methyl-ꢀ-D-
glucopyranosyluronate)-(1!4)-3,6-di-O-acetyl-2-O-ben-
zyl-ꢁ,ꢀ-D-glucopyranosyl] trichloroacetimidate (11). Tri-
chloroacetonitrile (0.65 mL, 6.5 mmol) and DBU (15 mL,
0.1 mmol) were added to a solution of 10 (117 mg,
0.131 mmol) in CH2Cl2 (10 mL). After 20 min at room
temperature, the solution was concentrated. Flash
chromatography (cyclohexane:ethyl acetate, 13:10 +
00 00
00 00
arom), 5.40 (t, 1H, J2 ,3 =9.7 Hz, J3 ,4 =9.7 Hz, H-3 ),
0
0
5.31 (d, 1H, J1 ,2 =6.8 Hz, H-1 ), 5.26 and 4.97 (d, 1H,
0
00 00
Jgem=12.2 Hz, OCH2Ph), 5.20 (d, 1H, J1 ,2 =3.5 Hz,
H-100), 5.14 and 5.10 (d, 1H, Jgem=12.2 Hz, OCH2Ph),
4.89 and 4.77 (d, 1H, Jgem=11.0 Hz, OCH2Ph), 4.75
and 4.58 (d, 1H, Jgem=12.4 Hz, OCH2Ph), 4.69 and
4.53 (d, 1H, Jgem=12.3 Hz, OCH2Ph), 4.64 and 4.51 (d,
1H, Jgem=12.1 Hz, OCH2Ph), 4.56 (d, 01H, J1,2=3.6 Hz,
1
1% Et3N) gave 11 (128 mg, 94%; a:b : 5:1). H NMR
(400 MHz, C6D6) d a anomer: 8.51 (s, 1H, N-H), 7.45±
7.05 (m, 10H, arom), 6.73 (d, 1H, J1,2=3.5 Hz, H-1),
6.06 (t, 1H, J2,3=9.7 Hz, J3,4 9.7 Hz, H-3), 5.12 and 5.01
(d, 1H, J=12.0 Hz, COOCH2Ph), 4.79±4.74 (m, 1H, H-
0
0
H-1), 4.46 (d, 1H, J4 ,5 =6.2 Hz, H-5 ), 4.43 (dd, 1H,
00
00
J6a ,6b =12.0 Hz, J5 ,6a =1.7 Hz, H-6 a), 4.36 (ddd, 1H,
00
00
00
000ha,10000
0000,20000
000hb,10000
=2.6 Hz, H-
J4
=11.4 Hz, J1
=5.5 Hz, J4
6a), 4.61 (dd, 1H, J6a ,6b =11.7 Hz, J5 ,6a =2.0 Hz, H-
6a00), 4.54±4.49 (m, 3H, H-5, H-6b, OCH2Ph), 4.48 (ddd,
10000), 4.25±4.18 (m, 3H, H-6b00,H-6a0000, H-6b0000), 4.06±
00
00
00
00
4.02 (m, 1H, H-500), 4.06 (d, 1H, J1
=7.9 Hz, H-1000),
3.85 (dd, 1H, J3 ,4 =8.5 Hz, H-4 ), 3.84±3.78 (m, 2H, H-
000,2000
0
0
00
00 00
0
00
0
0
1H, J4 ha,1 =11.6 Hz, J1 ,2 =5.5 Hz, J4 hb,1 =2.5 Hz, H-
00
100), 4.37 (dd, 1H, J5 ,6b =5.5 Hz, H-6b )0, 4.34 (2d, 2H,
3, H-4), 3.76±3.65 (m, 4H, H-5, H-6a, H-6b, H-30), 3.67
00
00
000
0
J=12.0 Hz, OCH2Ph, J1 ,2 =7.5 Hz, H-1 ), 3.84 (t, 1H,
0
00 00
000,5000
(d, 1H, J4
=10.0 Hz, H-5 ), 3.55 (t, 1H, J4 ,5
0
=9.7 Hz, H-400), 3.54, 3.48, 3.47, 3.46, 3.45, 3.39, 3.36
0
J4,5=9.7 Hz, H-4), 3.79 (d, 1H, J4 ,5 =11.0 Hz, H-5 ),
00 00
3.67 (ddd, 1H, J4 ,5 =9.5 Hz, H-5 ), 3.59 (dd, 1H, H-2),
3.50, 3.39, 3.33, 3.32 and 3.15 (s, 3H, OCH3), 3.29 (t,
0
00
and 3.18 (s, 3H, OCH3), 3.48±3.37 (m, 3H, H-2, H-200,
H-50000), 3.21 (dd, 1H, J2
(t, 1H, J3
=8.7 Hz, H-20000), 3.12
0000,30000
00
1H, J2 ,3 =9.5 Hz, J3 ,4 =9.5 Hz, H-3 ), 3.22 (dd, 1H,
H-200), 3.09±2.97 (m, 3H, H-20, H-30, H-400), 2.45±2.36
(m, 1H, H-40), 2.05, 1.94 and 1.71 (s, 3H, OCOCH3),
=8.7 Hz, H-30000), 3.02 (dd, 1H,
=10.3 Hz, J2
=8.7 Hz, H-40000), 2.93 (dd, 1H, J2 ,3 =8.1 Hz, H-
00 00
00 00
0000,40000
8.3 Hz, H-3000), 2.99 (t, 1H,
000,3000
000,4000
J3
J4
0000,50000
0
0
1.77 (ddd, 1H, J4 ,4 ha=1.8 Hz, H-40ha), 1.52 (ddd, 1H,
20), 2.85 (dd, 1H, H-2000), 2.12±2.07 (m, 1H, H-4000), 2.09,
0
0
J4 ha,4 hb=15.5 Hz, J4 ,4 hb=8.0 Hz, H-40hb). Selected
data for b anomer: 8.62 (s, 1H, N-H), 6.04 (d, 1H,
J1,2=7.5 Hz, H-1), 5.58 (t, 1H, J2,3=9.0 Hz, H-3), 4.87
and 4.67 (d, 1H, J=12.0 Hz, OCH2Ph); 13C NMR
(100.58 MHz, C6D6) d a anomer: 170.99, 170.44, 170.10
and 169.53 (C=O), 163.41 (CCl3), 161.93 (C=NH),
138.73 and 135.98 (C arom), 129.73±128.14 (C arom),
104.32 (C-10), 94.38 (C-1), 86.05, 84.86, 84.25, 82.48,
81.04, 77.47, 77.15, 77.11, 77.05, 72.60, 72.17 and 71.13
(C-2, C-3, C-4, C-5, C-20, C-30, C-50, C-100, C-200, C-300,
C-400, C-500), 73.18 (OCH2Ph), 68.10 (COOCH2Ph),
64.30 and 62.87 (C-6, C-600), 60.80, 60.75, 60.71, 59.66
and 58.73 (OCH3), 39.91 (C-40), 24.40 (C methylene),
21.45, 21.16 and 20.84 (OCOCH3). MS, m/z 1055
(M+NH4)+, 892 (M-OCNHCCl3+NH3)+.
1.95 and 1.90 (s, 3H, OCOCH3), 1.64 (ddd, 1H,
=1.8 Hz, H-4000ha), 1.57
0
0
0
0
000ha,4000
000,4ha000
J4
(ddd, 1H, J4
hb=15.3 Hz, J4
000,4hb000
=8.1 Hz, H-4000hb). MS, m/z 1650
(M+NH4)+; Anal. calcd for C87H108O30 (1633.792): C
63.96, H 6.66, Found: C 63.65, H 6.52.
Methyl C-(6-O-sulfo-2,3,4-tri-O-methyl-ꢁ-D-glucopyrano-
sylmethyl)-(1! 4)-O-(4-deoxy-2,3-di-O-methyl-ꢀ-D-
glucopyranosyluronic acid)-(1!4)-O-(2,3,6-tri-O-sulfo-
ꢁ-D-glucopyranosyl)-(1!4)-O-(2,3-di-O-methyl-ꢁ-L-ido-
pyranosyluronic acid)-(1!4)-2,3,6-tri-O-sulfo-ꢁ-D-gluco-
pyranoside (14). Hydrogenolysis of benzyl ethers and
benzyl esters. A solution of 13 (70 mg, 0.043 mmol), in
CH2Cl2:MeOH (1:1, 2 mL), was stirred for 5 h at room
temperature under hydrogen atmosphere (5 bar) in the
presence of 10% Pd/C (40 mg). After ®ltration and
concentration, the residue (46.3 mg, 99%) was used in
the next step.
Methyl C-(6-O-acetyl-2,3,4-tri-O-methyl-ꢁ-D-glucopyr-
anosylmethyl)-(1!4)-O-(benzyl 4-deoxy-2,3-di-O-methyl-
ꢀ-D-glucopyranosyluronate)-(1!4)-O-(3,6-di-O-acetyl-
2-O-benzyl-ꢁ-D-glucopyranosyl)-(1!4)-O-(benzyl 2,3-di-
O-methyl-ꢁ-L-idopyranosyl uronate)-(1!4)-2,3,6-tri-O-
benzyl-ꢁ-D-glucopyranoside (13). A 0.05 M solution of
TMSOTf in CH2Cl2 (0.35 mL) was slowly added under
argon to a stirred solution of 11 (77 mg, 0.074 mmol)
and 12 (118 mg, 0.155 mmol) in CH2Cl2, containing 4 A
Saponi®cation of the esters. NaOH (4 M aq) was added
(0.5 M ®nal concentration) at 0 ꢀC to a solution of the
residue in MeOH (2 mL). After 2 h at 0 ꢀC water was
introduced, followed by Dowex 50 H+ resin, until pH
1±2. After ®ltration and concentration, the residue was
passed through a Sephadex G-25 column (1.6Â115 cm)

M. Petitou et al./Bioorg. Med. Chem. 6 (1998) 1509±1516
1515
eluted with water to give pure fully deprotected com-
pound (33 mg, 77%). At this stage, complete removal of
protective groups was checked by high ®eld H NMR.
1993, 3, 97. (d) Feizi, T. Curr. Opin. Struct. Biol. 1993, 3, 701.
(e) Boren, T.; Falk, P.; Roth, K. A.; Larson, G.; Normak, S.
Science 1993, 262, 1892. (f) Varki, A. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 7390.
1
2. (a) Petitou M. A. C. S. Symp. Series 1994, 560, 19. (b)
Musser, J. F.; Fugedi, P.; Anderson, M. B.; Rao, N.; Peto, C.;
Tyrrell, D.; Holme, K.; Tressler, R. Drugs News Perspect. 1996,
9, 133. (c) Zopf, D.; Roth, S. Lancet 1996, 347, 1017.
Sulfation. Et3N±sulfur trioxide complex (5 mmol/mmol
of hydroxyl function) was added to a solution of the
deprotected compound (29.8 mg, 0.030 mmol) in DMF
(3 mL). After one day at 55 ꢀC with protection from
light, the solution was layered on top of a Sephadex G-
25 column (1.6Â115 cm) eluted with 0.2 M NaCl. The
fractions containing the product were concentrated, and
desalted using the same column, equilibrated in water.
Lyophilization provided 14 (45.2 mg, 85%). [a]d +45ꢀ
(c 0.99, H2O). 1H NMR (500 MHz, D2O) d 5.41 (d, 1H,
J1,2=3.7 Hz, H-1 F-unit), 5.15 (d, 1H, J1,2=3.6 Hz, H-1
H-unit), 5.07 (d, 1H, J1,2=2.8 Hz, H-1 G-unit), 4.60 (d,
1H, J1,2=7.9 Hz, H-1 E-unit), 4.43 (m, 1H, J1,2=3.7 Hz,
H-1 D-unit), 3.63, 3.60, 3.57, 3.56, 3.52, 3.47, 3.46, (s,
24H, 8 OMe), 2.00 (H-4, E-unit), 1.84 (ddd, Hha
CHaHb), 1.58 (ddd, Hhb CHhaHhb). MS: m/z 840
[(M-2Na)2-/2].
3. Driguez, H. Top. Curr. Chem. 1997, 187, 85.
4. (a) Jaurand, G.; Basten, J.; Lederman, I.; van Boeckel, C. A.
A.; Petitou, M. Bioorg. Med. Chem. Lett. 1992, 2, 897. (b)
Basten, J.; Jaurand, G.; Olde-Hanter, B.; Petitou, M.; van
Boeckel, C. A. A. Bioorg. Med. Chem. Lett. 1992, 2, 901. (c)
Basten, J.; Jaurand, G.; Olde-Hanter, B.; Duchaussoy, P.;
Petitou, M.; van Boeckel, C. A. A. Bioorg. Med. Chem. Lett.
1992, 2, 905.
5. Petitou, M.; Coudert, C.; Level, M.; Lormeau, J.-C.; Zuber,
M.; Simenel, C.; Fournier, J.-P.; Choay, J. Carbohydr. Res.
1992, 236, 107.
6. Representative selection of stereoselective syntheses of C-
disaccharides: (a) Rouzaud, D.; Sinay, P. J. Chem. Soc., Chem.
Commun. 1983, 1353. (b) Vauzeilles, B.; Cravo, D.; Mallet, J.-
M.; Sinay, P. Synlett 1993, 522. (c) Xin, Y.-C.; Mallet, J.-M.;
Sinay, P. J. Chem. Soc., Chem. Commun. 1993, 864. (d) Martin,
O. R.; Lai, W. J. Org. Chem. 1993, 58, 176. (e) Mallet, A.;
Mallet, J.-M.; Sinay, P. Tetrahedron: Asymm. 1994, 5, 2593. (f)
Chenede, A.; Perrin, E.; Rekaõ, E. D.; Sinay, P. Synlett 1994,
420. (g) Mazeas, D.; Skrydstrup, T.; Doumeix, O.; Beau, J.-M.
Angew. Chem. Int. Ed. Engl. 1994, 33, 1383. (h) Ferritto, R.;
Vogel, P. Tetrahedron: Asymm. 1994, 5, 2077. (i) Dietrich, H.;
Schmidt, R. R. Liebigs Ann. Chem. 1994, 975. (j) Armstrong,
R. W.; Sutherlin, D. P. Tetrahedron Lett. 1994, 35, 7743. (k)
Wei, A.; Haudrechy, A.; Audin, C.; Jun, H.-S.; Haudrechy-
Bretel, N.; Kishi, Y. J. Org. Chem. 1995, 60, 2160. (l) Sutherlin,
D. P.; Armstrong, R. W. J. Am. Chem. Soc. 1996, 118, 9802.
(m) Sinay, P. Pure Appl. Chem. 1997, 69, 459.
Anity for AT III. Fluorescence measurements were
performed using a Perkin±Elmer LS-50 type spectro-
¯uorimeter (excitation l=280 nm, emission l=338 nm)
equipped with a thermostated sample compartment, at
37 ꢀC, under continuous stirring. Oligosaccharides were
added into the sample containing 2 mL of 0.01 M Tris±
HCl bu€er, pH 7.5, 0.15 M NaCl and 5±60 nM human
AT-III. The ratio and the concentrations of AT III-oligo-
saccharide complexes were calculated considering a 1:1
reaction stoichiometry, and dissociation constants (KD)
were determined by Scatchard analysis using the RS/1
computer program (BBN Software Product Corpora-
tion, Cambridge, MA, USA).
7. (a) Wang, Y.; Goekjian, P. G.; Ryckmann, D. M.; Miller,
W. H.; Babirad, S. A.; Kishi, Y. J. Org. Chem. 1992, 57, 482.
(b) Espinosa, J.-F.; Martõn-Pastor, M.; Asensio, J. L.; Dietrich,
H.; Martõn-Lomas, M.; Schmidt, R. R.; Jimenez-Barbero, J.
Tetrahedron Lett. 1995, 36, 6329. (c) Berthault, P.; Birliradis,
N.; Rubinstenn, G.; Sinay, P.; Desvaux, H. J. Biomol. NMR
1996, 8, 23. (d) Rubinstenn, G.; Sinay, P.; Berthault, P. J. Phys.
Chem. A 1997, 101, 2536.
Anti-factor Xa activity. Human factor Xa (2.4 nkat/mL)
was incubated for 2 min with human AT III (0.17 U/
mL) at 37 ꢀC in the presence of various concentrations
of oligosaccharides in Tris±maleate 20 mM bu€er pH
7.4, NaCl 150 mM. To measure the residual factor Xa,
S-2222 substrate (dissolved in 50 mM Tris±HCl bu€er,
pH 8.4, NaCl 175 mM, EDTA 2 7.5 mM) was added
(0.25 mM ®nal). The reaction was stopped 2 min later by
addition of 50% aqueous acetic acid and the absorbance
at 405 nm was read. The percentage of inhibition was
then calculated [inhibition %=100 ((OD blank-OD
sample)/OD blank], and the activity of the compounds
determined by comparison with a calibrated standard,
using Excel 4.0 Software (Microsoft, Redmond, USA).
8. (a) Choay, J.; Petitou, M.; Lormeau, J.-C.; Sinay, P.; Casu,
B.; Gatti, G. Biochem. Biophys. Res. Commun. 1983, 116, 492.
(b) Sinay, P.; Jacquinet, J.-C.; Petitou, M.; Duchaussoy, P.;
Lederman, I.; Choay, J.; Torri, G. Carbohydr. Res. 1984, 132,
C5.
9. (a) Petitou, M.; van Boeckel, C. A. A. In Progress in the
Chemistry of Organic Natural Products; W. Herz, W.; Wein, Eds;
Springer-Verlag, New York 1992; p 143. (b) van Boeckel, C. A.
A.; Petitou, M. Angew. Chem., Int. Ed. Engl. 1993, 32, 1671.
10. A preliminary communication on this work has been pub-
lished: Helmboldt, A.; Petitou, M.; Mallet, J.-M.; Herault, J.-
P.; Lormeau, J.-C.; Driguez, P.-A.; Herbert, J.-M.; Sinay, P.
Bioorg. Med. Chem. Lett. 1997, 7, 1507.
References and Notes
1. For selected references, see (a) Perez, M.; Sindal, A. K.;
Hakomori, S.-I.; Paulson, J. C. Science 1990, 250, 1130. (b)
Lasky, L. A. Science 1992, 258, 964. (c) Varki, A. Glycobiology
11. Helmboldt, A.; Mallet, J.-M. Petitou, M.; Sinay, P. Bull.
Soc. Chim. Fr. 1997, 134, 1057.

1516
M. Petitou et al./Bioorg. Med. Chem. 6 (1998) 1509±1516
12. 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.
C.; Mallet, J.-M.; Michon, V.; Sinay, P. Carbohydr. Res. 1993,
246, 23.
15. (a) Paulsen, H.; Paul, M. Carbohydr. Res. 1984, 135, 71. (b)
Paulsen, H.; Heume, M.; Gyorgydeak, M.; Lebuhn, R. Carbo-
hydr. Res. 1985, 144, 57.
13. Paulsen, H.; Stenzel, W. Chem. Ber. 1978, 111, 2348.
16. Excoer, G.; Gagnaire, D.; Utille, J.-P. Carbohydr. Res.
1975, 39, 368.
14. (a) Pougny, J.-R.; Sinay, P. Tetrahedron Lett. 1976, 4073.
(b) Schmidt, R. R.; Rucker, E. Tetrahedron Lett. 1980, 1421.
(c) Pavia, A. A.; Ung-Chhun, S. N.; Durand, J.-L. J. Org.
Chem. 1981, 46, 3158. (d) Schmidt, R. R.; Michel, J. J. Carbo-
hydr. Chem. 1985, 4, 141. (e) Klemer, A.; Kohla, M. J. Carbo-
hydr. Chem. 1988, 7, 785. (f) Murase, T.; Kameyama, K.;
Kartha, P. R.; Ishida, H.; Kiso, M.; Hasegawa, A. J. Carbo-
hydr. Chem. 1989, 8, 265. (g) Marra, A.; Sinay, P. Carbohydr.
Res. 1990, 195, 303. (h) Marra, A.; Mallet, J.-M.; Amatore,
C.; Sinay, P. Synlett 1990, 572. (i) Ratcli€e, A. J.; Fraser-Reid,
B. J. Chem. Soc. Perkin Trans. 1 1990, 747. (j) Gordon, D.
M.; Danishefsky, S. J. J. Org. Chem. 1991, 56, 3713. (k)
Marra, A.; Gau€eny, F.; Sinay, P. Tetrahedron 1991, 47, 5149.
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17. Sakai, K.; Nakahara, Y.; Ogawa, T. Tetrahedron Lett.
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18. 12 was prepared as reported in ref 11 for the corresponding
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