A concise synthesis of the 6-O- and 6'-O-sulfated analogues of the sialyl Lewis X tetrasaccharide

Article abstract of DOI:10.1016/S0960-894X(00)00207-9

The octyl glycoside of the sialyl Lewis X tetrasaccharide and its 6-O-sulfated and 6'-O-sulfated analogues were chemically synthesized in a concise manner starting from readily accessible monosaccharide intermediates. The synthesis involved formation of an orthogonally protected tetrasaccharide intermediate from which all three materials were prepared. A selective catalytic hydrogenolysis of four O-benzyl ethers in presence of a 4,6-O-benzylidene group was the key step in the synthetic scheme. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00207-9

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1505±1509
A Concise Synthesis of the 6-O- and 60-O-Sulfated Analogues of
the Sialyl Lewis X Tetrasaccharide
Anup Kumar Misra,a Yili Ding,a John B. Lowea,b and Ole Hindsgaula,*
aThe Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
bHoward Hughes Medical Institute, University of Michigan Medical School, 1150 West Medical Center Drive,
Ann Arbor, MI 48109-0650, USA
Received 16 February 2000; accepted 28 March 2000
AbstractÐThe octyl glycoside of the sialyl Lewis X tetrasaccharide and its 6-O-sulfated and 60-O-sulfated analogues were
chemically synthesized in a concise manner starting from readily accessible monosaccharide intermediates. The synthesis involved
formation of an orthogonally protected tetrasaccharide intermediate from which all three materials were prepared. A selective
catalytic hydrogenolysis of four O-benzyl ethers in presence of a 4,6-O-benzylidene group was the key step in the synthetic scheme.
# 2000 Elsevier Science Ltd. All rights reserved.
During the in¯ammation process, the in®ltration of
leukocytes into tissues begins with carbohydrate medi-
ated endothelial cell adhesion involving E-, P- and L-
selectins.1 Selectins are a family of carbohydrate binding
membrane proteins, which play a vital role in leukocyte
homing, platelet binding and neutrophil extravasation.2
E-selectin appears on the vascular endothelial cells after
stimulation by cytokines during the preliminary stage of
in¯ammation and recruits neutrophils and monocytes to
in¯ammatory sites leading to the extravasation of leu-
kocytes.3 P-selectins are temporarily stored in alpha and
dense granules of platelets and Weibel±Palade bodies of
endothelial cells and rapidly distributed after activation
with thrombin, histamine, phorbol esters or the calcium
ionophores.4 L-selectin is the principal adhesion mole-
cule present on leukocytes involved in the interaction
with high endothelial venules (HEV) of peripheral lymph
nodes. GlyCAM-1 and CD34 are two mucin like O-
linked glycoproteins with sulfated, sialylated and fuco-
sylated oligosaccharide sequences which were found to
be the major ligands for L-selectin.5,6
including in¯ammation, reperfusion injury, metastasis,
angiogenesis, etc.7 It has been shown that the sialic acid
and fucose moieties are essential for binding.1c,d sLeX
has also been found on the surface of some tumor cells.8
It has been emphasized that L-selectin prefers 6-O-
sulfo-sLeX as a ligand over sLeX. Anti sLeX antibodies
that bind 6-O-sulfo-sLeX react with HEV in lymph
nodes inhibiting the binding of L-selectin to HEV.9,10
In order to study the function of sLeX and its sulfated
analogues and to evaluate their potential as carbohydrate
derived therapeutics, ecient and concise synthetic pro-
tocols are required. Several syntheses of these molecules
have been reported,11 and these require lengthy multistep
sequences. Chemoenzymatic synthesis shows great pro-
mise but requires access to a panel of glycosyltransferases
and sulfotransferases.
We report herein a concise chemical synthesis of the
octyl glycosides of sLeX (1), its 6-O-sulfate (2) and 60-O-
sulfate (3) from the readily accessible protected mono-
saccharide precursors 4±7 (Fig. 1). The key step in the
synthetic Scheme is the conversion of tetrasaccharide 12
to 16 which involves the selective hydrogenolysis of four
O-benzyl ethers in the presence of a 4,6-O-benzylidene
group.
The selectins recognize the sialyl Lewis X tetra-
saccharide (sLeX) determinant (the sequence in 1) which
is found as the terminal carbohydrate structure in both
glycolipids and glycoproteins, although these com-
pounds bind with low anity. The selectin-carbohydrate
interaction plays a crucial role in a number of events
Glycosylation of octyl 2-acetamido-4,6-O-benzylidine-2-
deoxy-b-d-glucopyranoside (4), prepared from N-acetyl-
d-glucosamine in four steps, and ethyl 2,3,4-tri-O-benzyl-
1-thio-b-l-fucopyranoside12 (5) using CuBr2-Bu4NBr13
*Corresponding author. Fax: +1-858-646-3193.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00207-9

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A. K. Misra et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1505±1509
Figure 1. Key features of the synthetic approach to the sLeX analogues 1±3.
a€orded the a(1!3) linked disaccharide (8) in 88%
yield. Regioselective reductive ring opening14 of the
benzylidene acetal in 8 using NaBH3CN, furnished the
disaccharide acceptor (9) in 79% yield (Scheme 1).
Doublets at d 4.88 (J1,2=9.0 Hz; GlcNAc H-1) and at d
proton and carbon signals in the 1H and 13C NMR
spectra (d 5.43 (s, PhCH), 5.05 (d, J=8.1 Hz, GlcNAc H-
1), 4.94 (d, J=2.6 Hz, Fuc H-1), 4.60 (d, J=7.8 Hz, Gal
H-1), 2.76 (dd, J=12.9 Hz, 4.5 Hz, H-3e000); 13C NMR: d
101.5, 99.5, 99.2, 97.8, 97.5) con®rmed the structure of
12. Several others sialyl donors and various promoters17
were examined but the yields were similar.
1
4.61 (J1,2=3.6 Hz; Fuc H-1) in the H NMR spectrum
con®rmed the stereoselective glycosylations. The b-d-
Gal-trichloroacetimidate (6) proved e€ective as donor
.
with BF3 Et2O as promoter (Et2O:CH2Cl2, 2:1). On
The key step in the use of the single intermediate 12 to
furnish both the 6-O and 60-O-sulfo-sLeX was the con-
trolled removal of the O-benzyl groups in 12 employing
catalytic hydrogenation using Pearlman's catalyst18
(20% Pd(OH)2-C) (Scheme 2). In order to remove ben-
zyl ethers in presence of the benzylidene acetal several
trials for selective hydrogenolysis of the benzyl ethers
were carried out. Using 10% Pd-C in 2-propanol, 20%
Pd(OH)2-C in cyclohexene or 10% Pd-C and ammonium
formate in re¯uxing methanol as the indirect hydrogen
sources gave only low yields of product. Use of Pd-C in
ethanol and H2 resulted in the slow complete removal of
the benzyl ethers and benzylidene acetal. Use of acetic
acid:ethanol (1:2) as the hydrogenation solvent resulted
deacetylation (NaOMe/MeOH), trisaccharide tetraol 10
was obtained in 77% yield. Careful benzylidenation of
10 using benzaldehyde dimethylacetal and p-TsOH a€or-
ded the trisaccharide acceptor 11 in 74% yield (longer
reaction times lead to defucosylation). The observed che-
mical shifts and coupling constants (d 5.07 (d, J1,2=8.1
Hz, GlcNAc H-1), 4.93 (d, J1,2=2.7 Hz, Fuc H-1), 4.41 (d,
1
J1,2=7.8 Hz, Gal H-1)) in the H NMR spectra unam-
biguously established the expected stereochemistry.
Sialylation of the trisaccharide diol acceptor 11 using
the sialyl donor 715 and NIS/TfOH as promoter16
a€orded tetrasaccharide 12 in 46% yield. Characteristic

A. K. Misra et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1505±1509
1507
.
in some cleavage of sialic acid and defucosylation.
Finally, using 20% Pd(OH)2-C (1:1 by wt) in MeOH
with a reaction time for 5 h at room temperature furn-
ished the debenzylated product in 77% yield with the
benzylidene acetal intact which was con®rmed by its
proton signal d 5.43 (s, PhCH) in the 1H NMR spectrum.
This reaction condition has been applied for several
other selective hydrogenolysis of Lewis X trisaccharide
derivative and for some other disaccharides. In every
case, a satisfactory yields and reproducibility was
achieved. Prolonged (36 h) hydrogenation of 12 under
the same reaction conditions gave tetrasaccharide hep-
taol 13 (87%), which on saponi®cation gave 1 in 68%
yield. Selective silylation19 (TBDMS) of the primary
hydroxyl group in 14 followed by conventional acetyla-
tion of the secondary hydroxyl groups a€orded the
orthogonally protected key tetrasaccharide intermediate
15 in 70% yield (Scheme 2).
which on sulfation (SO3 Pyr) gave the 6-O-sulfate 17 in
73% yield. Removal of the benzylidene acetal through
catalytic hydrogenolysis using Pearlman's catalyst fol-
lowed by saponi®cation gave 2 in 61% yield (Scheme 3).
Extended hydrogenolysis of 15 gave the 40, 60-diol 18 in
74% yield. Selective sulfation of the 60-hydroxyl group
of 18 followed by saponi®cation of 19 then a€orded the
60-O-sulfate 3 in 64% yield (Scheme 4). Selected 1H
Tetrasaccharide 15 was treated with excess HF-Pyr20 in
THF to produce the 6-OH derivative 16 in 68% yield,
Scheme 2. (a) H2, Pd(OH)2-C (20%), MeOH, rt, 5 h (77%); (b)
TBDMS-Cl, imidazole, DMF, rt, 2 h; (c) Ac2O, pyridine, rt, 12 h
(70% in two steps).
Scheme 1. (a) CuBr2, Bu4NBr, 1,2-DCE:DMF (5:1), MS-4 A, rt, 24 h
(88%); (b) NaBH3CN, HCl-Et2O, MS-3 A, THF, 0±5ꢀC, 3 h (79%); (c) (i)
6, BF3 Et2O:CH2Cl2 (2:1), 10 ꢀC±rt,3h; (ii)solidNaHCO3,1MMeONa,
.
ꢀ
.
MeOH (77%); (d) PhCH(OMe)2, p-TsOH, CH3CN, rt, 2 h (74%); (e) NIS,
TfOH, CH3CN:CH2Cl2 (5:1), MS-3 A, 20 ꢀC, 16 h (43%); (f) H2,
Pd(OH)2-C (20%), MeOH, rt, 36 h (87%); (g) 0.1 M MeONa, MeOH, rt,
12 h, then H2O was added and stirred at rt for 12 h (68%).
Scheme 3. (a) HF-pyridine, THF, 0±5 C, 4 h (68%); (b) SO3 Pyr
complex, pyridine, 0 ꢀC±rt, 7 h, then Dowex 50W X8 (Na+) (73%); (c)
H2, Pd(OH)2-C (20%), MeOH, rt, 32 h (72%); (d) 0.1 M MeONa,
MeOH, rt, 12 h, then H2O was added and stirred at rt for 12 h (61%).

1508
A. K. Misra et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1505±1509
5. (a) Rosen, S. D. Semin, Immunol. 1993, 5, 237. (b) Imai, Y.;
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Scheme 4. (a) H2, Pd(OH)2-C (20%), MeOH, rt, 48 h (74%); (b)
ꢀ
SO3 Pyr complex, Pyridine, 0 10 ꢀC, 7 h, then Dowex 50W X8
(Na+) (70%); (c) 0.1 M MeONa, MeOH, rt, 12 h, then H2O was
added and stirred at rt for 12 h (64%).
.
NMR and MS data for key compounds are presented
below.21 Compounds 1±3 are under evaluation as selectin
inhibitors and as sulfotransferase acceptors.
13. Sato, S.; Mori, M.; Ito, Y.; Ogawa, T.. Carbohydr. Res.
155, C6-C10..
14. Garegg, P. J.; Hultberg, H.; Oscarson, S. J. Chem. Soc.,
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In conclusion, the 6- and 60-O-sulfated analogues of
sLeX were synthesized in a concise and practical way.
16. (a) Konradsson, P.; Udodong, U. E.; Fraser-Reid, B. Tet-
rahedron Lett. 1990, 31, 4313. (b) Veenemann, G. H.; van-
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1331.
Acknowledgements
The authors would like to thank Dr. O. P. Srivastava,
Alberta Research Council for providing conditions for
the synthesis of 9. This work was supported by National
Institutes of Health Grant PO1 CA 71932.
17. (a) Martin, T. J.; Brescello, R.; Toepfer, A.; Schmidt, R. R.
Glycoconjugate J. 1993, 10, 16. (b) Marra, A.; Sinay, P. Car-
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18. Pearlman, W. M. Tetrahedron Lett. 1967, 1663.
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References and Notes
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Soc. 1985, 107, 5556.
0
0
21. Partial NMR and MS data: 8: d 5.03 (d, J1 ,2 =3.6 Hz, 1H,
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Watson, S. R.; Fennie, C.; Lasky, L. A. Nature 1991, 349, 164.
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H-10), 4.88 (d, J1,2=9.0 Hz, 1H, H-1), 1.60 (s, 3H, NHAc),
0.78 (d, J=6.6 Hz, 3H, H-60). 11: d 5.55 (s, 1H, PhCH), 5.07
0
0
0
(d, J1,2=8.1 Hz, 1H, H-1), 4.93 (bd, J1 ,2 =2.7 Hz, 1H, H-1 ),
00
00 00
4.41 (d, J1 ,2 =7.8 Hz, 1H, H-1 ), 1.58 (s, 3H, NHAc), 1.02
(d, J=6.6 Hz, 3H, H-60). 12: d 5.43 (s, 1H, PhCH), 5.05 (d,
0
0
J1,2=8.1 Hz, 1H, H-1), 4.94 (d, J1 , 2 =2.1 Hz, 1H, H-1 ), 4.59
0
00
00 00
(d, J1 ,2 =7.8 Hz, 1H, H-1 ), 3.63 (s, 3H, COOMe), 2.76 (dd,
J=12.9 Hz, 4.5 Hz, H-3e000), 1.56±2.12 (6s, 18H, 4 OAc, and 2
NHAc), 1.01 (d, J=6.6 Hz, 3H, H-60), 16: d 5.42 (m, 1H, H-
8000), 5.31 (s, 1H, PhCH), 5.12 (d, J1 ,2 =3.0 Hz, 1H, H-1 ),
0
0
0
00 00
4.73 (d, J1,2=8.1 Hz, 1H, H-1), 4.44 (d, J1 ,2 =7.5 Hz, 1H, H-
100), 3.56 (s, 3H, COOMe), 2.72 (dd, 1H, H-3e000), 1.85±2.12
(10s, 30H, 8 OAc and 2 NHAc), 0.88 (s, 9H, CMe3), 0.71 (d,
J=6.6 Hz, 3H, H-60), 0.06, 0.07 (2s, 3H each, 2 Me). 17 : d
0
0
5.35 (s, 1H, PhCH), 5.21 (d, J1 ,2 =3.0 Hz, 1H, H-1 ), 4.73 (d,
0
4. Johnston, G. I.; Kurosky, A.; McEver, R. P. J. Biol. Chem.
1989, 264, 1816.
00
00 00
J1,2=8.1 Hz, 1H, H-1), 4.53 (d, J1 ,2 =8.1 Hz, 1H, H-1 ),

A. K. Misra et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1505±1509
1509
0
3.75 (s, 3H, COOMe), 2.75 (dd, 1H, H-3e000), 1.84±2.16 (10s,
977.9397. 2: d 5.03 (d, J1 ,2 =3.9 Hz, 1H, H-1 ), 4.55 (J1,2=7.8
0
0
30H, 8 OAc and 2 NHAc), 0.62 (d, J=6.6 Hz, 3H, H-60). 20: d
Hz, 1H, H-1), 4.48 (d, J =7.8 Hz, 1H, H-10, 2.67 (dd, 1H, H-
00,200
0
0
0
5.20 (d, J1 ,2 =2.7 Hz, 1H, H-1 ),004.75 (d, J1,2=8.1 Hz, 1H, H-
3e000), 1.93, 1.96 (2s, 6H, 2NHAc), 1.73 (t, 1H, H-3a000), 1.11 (d,
00 00
1), 4.45 (d, J1 ,2 =8.1 Hz, 1H, H-1 ), 3.76 (s, 3H, COOMe), 2.80
J=6.6 Hz, 3H, H-60); 13C NMR: d 101.8, 101.6, 100.2, 99.2;
(dd, 1H, H-3e000), 1.84±2.11 (10s, 30H, 8 OAc and 2 NHAc), 1.15
HRMS: calcd For C39H66O26N2Na2S (M+Na+) 1079.9862;
(d, J=6.6 Hz, 3H, H-60), 0.87 (s, 9H, CMe3),00.03 and 0.05 (2s,
found 1079.9855. 3: d 5.05 (d, J1 ,2 =3.9 Hz, 1H, H-1 ), 4.50
(d, J1, 2=7.8 Hz, 1H, H-1), 4.47 (d, J1 ,2 =8.1 Hz, 1H, H-1 ),
2.73 (dd, 1H, H-3e000), 1.98, 1.99 (2s, 6H, 2NHAc), 1.76 (t, 1H,
H-3a000), 1.14 (d, J=6.6 Hz, 3H, H-60); 13C NMR: d 102.2,
101.5, 100.3, 99.2; HRMS: calcd for C39H66O26N2Na2S
(M+Na+) 1079.9862; found 1079.9858.
0
0
0
00
0
0
00 00
6H, 2Me). 1: d 5.05 (d, J1 ,2 =3.9 Hz, 1H, H-1 ), 4.48 (2d, J=7.5
Hz, 2H, H-1 and H-100), 4.04 (dd, 1H, H-300), 2.72 (dd, 1H, H-
3e000), 1.98, 1.99 (2s, 6H, 2 NHAc), 1.76 (t, 1H, H-3a000), 1.12 (d,
J=6.6 Hz, 3H, H-60); 13C NMR: d 102.2, 101.5, 100.2, 99.2.
HRMS: calcd for C39H67O23N2Na (M+Na+) 977.9402; found