Regioselective deacetylation and glycosylation in the synthesis of the sialyl lewis0 X tetrasaccharide, a key component of the recognition site of PSGL-1

Article abstract of DOI:10.1055/s-0030-1259054

A high-yielding regioselective deprotection of three out of five hydroxy groups of a Lewis X trisaccharide, which proceeds under very mild basic conditions, and a regio- and stereoselective sialylation reaction enable an efficient access to the sialyl Lew

Full text of DOI:10.1055/s-0030-1259054

LETTER
3023
Regioselective Deacetylation and Glycosylation in the Synthesis of the Sialyl
Lewis X Tetrasaccharide, a Key Component of the Recognition Site of PSGL-1
Regioselective
D
eacety
a
lationof
T
c
risaccharid
iej Pudelko,* Danuta Kowalczyk, Horst Kunz
Institut für Organische Chemie, Johannes-Gutenberg Universität Mainz, Duesbergweg 10–14, 55128 Mainz, Germany
Fax +49(6131)3922338; E-mail: pudelko@uni-mainz.de
Received 26 September 2010
.
Abstract: A high-yielding regioselective deprotection of three out
of five hydroxy groups of a Lewis X trisaccharide, which proceeds
under very mild basic conditions, and a regio- and stereoselective
sialylation reaction enable an efficient access to the sialyl Lewis X
tetrasaccharide.
OR2
R2O
COOR3
O
OR2
O
OR2
O
O
AcNH
R2O
O
O
O
OR2
OR2
R2O
AcHN
O
Key words: glycosylation, regioselective deacetylation, solid-
OR2
phase synthesis, glycopeptides, sialyl Lewis X
OR2
R2O
R2O
R2O
1 R1 = Fmoc-Thr-OH, R2 = Ac, R3 = Me
R2O
OR2
O
O
Murine P-selectin expressed by activated platelets and en-
dothelial cells exhibits the highest ligand affinity and
specificity of all selectins. It is thought to bind to the N-
terminus of murine PSGL-1, which has a different amino
acid sequence from the human PSGL-1.1 Recent studies
showed that for a sufficient binding of murine PSGL-1 to
P-selectin, the presence of sialyl Lewis X at Thr-17 and
the O-sulfation of Tyr-13 in glycopeptide 2 (Figure 1) are
crucial. Only then does the tethering and rolling of cells
expressing murine PSGL-1 to P-selectin occur.2 This sug-
gests that relatively short sulfated glycopeptides are well
suited for the inhibition of the P-selectin/PSGL-1 interac-
tion, which is an important step during the inflammatory
response and during the metastasis of tumor cells.
O
OR2
AcNH
R1
(13)(14)(15)
(17)
2 R1 = Ac-Phe-Glu-Asp-Pro-Asp-Tyr-Thr-Tyr-Asn-Thr-Asp-Pro-OH
–
–
R2 = R3 = H
OSO3 OSO3
Figure 1
In addition, the natural acetamido functionality in the glu-
cosamine unit should be re-established. This transforma-
tion is considered feasible when copper(II) salts are used
as mild Lewis acids7 for activating the oxazoline ring dur-
ing glycosylation.
The axial 4-OH group in 4 usually is of low reactivity, and
is not expected to compete with the primary hydroxy
group in position 6.
Herein, we report the regioselective de-O-acetylation of a
Lewis X trisaccharide 12 which is a crucial intermediate
in the preparation of the sialyl Lewis X tetrasaccharide 15
and of the oxazoline donor 5 derived from it. In our labo-
ratories, the chemical synthesis of different recognition
domains of PSGL-13 was previously developed, which
has afforded various glycopeptides and glycopeptide
mimics that served as selectin ligands containing sialyl
Lewis X.4 Some of them contained the sialyl Lewis X-
core2-threonine building block 1, which undoubtedly
constitutes the most demanding target in the synthesis of
the PSGL-1 glycopeptide 2. According to a novel ret-
rosynthetic analysis (Scheme 1), the hexasaccharide con-
struct 1 should be accessible from a partially deprotected
T antigen-threonine conjugate 4, which is a known inter-
mediate in the synthesis of tumor-associated antigens.5,6 It
is coupled to the sialyl Lewis X oxazoline derivative 5.
The oxazoline functionality should enable the facile stereo-
selective formation of the b-glycosidic bond between do-
nor 5 and acceptor 4.
The protected N-acetylglucosamine building block 9
equipped with the tert-butyldiphenylsilyl group8 at the
anomeric oxygen, which is stable under acidic and basic
conditions, was used as the starting material for the con-
struction of the sialyl Lewis X tetrasaccharide 15
(Scheme 2). Compound 9 was glycosylated with the thio-
fucoside donor 89 bearing acetyl protecting groups in po-
sitions 3 and 4 under adapted in situ anomerization
conditions10 and gave the disaccharide 10 in 58% yield. In
order to stereoselectively obtain the a-fucoside structure
the benzyl ether in position 2 had to be installed in the fu-
coside donor 8.
Acetyl protecting group at the a-fucoside moiety should
decrease the sensitivity of this glycosidic bond towards
acidic conditions as it is well established that O-benzyl
protecting groups on the fucose moiety are responsible for
the high acid-sensitivity of the fucoside bond.11 The new
protecting group strategy concerning the fucose portion
was different from the previous one.3 The intention was to
circumvent the exchange of protecting groups in order to
make the saccharide part suitable for the acidic conditions
SYNLETT 2010, No. 20, pp 3023–3026
x
x
.x
x
.2
0
1
0
Advanced online publication: 17.11.2010
DOI: 10.1055/s-0030-1259054; Art ID: G27410ST
© Georg Thieme Verlag Stuttgart · New York

3024
M. Pudelko et al.
LETTER
OAc
OAc
O
AcO
O
AcO
OAc
O
OH
HO
O
MeOOC
O
OAc
O
AcO
O
O
O
AcHN
AcO
O
AcO
NHFmoc
O
OAc
O
AcNH
AcNH
OAc
OAc
O
OAc
4
O
OAc
+
AcO
AcO
AcO
O
OAc
O
OAc
AcO
O
OAc
O
MeOOC
AcO
O
OAc
AcO
NHFmoc
OH
O
O
AcNH
O
OAc
1
AcHN
AcO
O
O
OAc
OAc
N
O
O
O
OAc
5
OAc
AcO
OAc
AcO
MeOOC
O
S
OEt
AcHN
AcO
OBn
O
AcO
AcO
OAc
S
6
SEt
AcO
Ph
O
O
CCl3
NH
O
AcNH
O
O
OBn
OTBDPS
HO
7
OAc
AcO
9
8
Scheme 1
of solid-phase glycopeptide synthesis. Trifluoromethane- yield. Acceptor 11 was then reacted with the galactosyl
sulfonic acid/triethylsilane-promoted regioselective ace- trichloroacetimidate 7 in a TMSOTf-promoted glycosyla-
tal ring-opening12 at –78 °C furnished the disaccharide 11 tion according to Schmidt,13 and gave the Lewis X trisac-
with a free 4-OH position at glucosamine unit in 81% charide 12 in 81% yield. In a crucial step the three O-
OBn
O
AcO
AcO
OTBDPS
SEt
O
OBn
O
Ph
OBn
O
AcO
O
O
CCl3
NH
O
HO
O
Ph
OTBDPS
O
OAc
O
O
O
AcO
O
7
AcNH
AcNH
OTBDPS
8
HO
O
OBn
10
AcNH
OBn
a)
b)
c)
9
OAc
OAc
AcO
AcO
11
OAc
AcO
COOMe
OBn
AcO
AcO
OBn
O
HO
HO
OBn
O
OBn
O
O
O
S
OEt
AcHN
AcO
O
O
OTBDPS
OTBDPS
O
AcO
O
HO
OAc
e)
S
AcNH
d)
AcNH
OBn
6
O
O
OBn
12
OAc
OAc
AcO
AcO
13
OAc
AcO
RO
O
OBn
MeOOC
O
OBn
O
O
O
O
OTBDPS
AcHN
AcO
14 R = H
RO
AcNH
OAc
f)
O
OBn
OAc
15 R = Ac
AcO
Scheme 2 Reagents and conditions: (a) 8 (1.1 equiv), CuBr2 (1.37 equiv), NBu4Br (1.37 equiv), 4Å MS, DMF–CH2Cl2 (1:1), r.t., 2 d, 58%
yield; (b) TfOH (2.9 equiv), Et3SiH (1.5 equiv), 4Å MS, –78 °C, 4.5 h, CH2Cl2, 81% yield; (c) 7 (1.8 equiv), TMSOTf (0.23 equiv), CH2Cl2,
– 50 °C slowly to r.t., 17 h, 81% yield; (d) cat. NaOMe–MeOH, r.t., pH 7.5, 94% yield; (e) 6 (2.32 equiv), AgOTf (2.33 equiv), MeSBr (2.33
equiv), 4Å MS, MeCN–CH2Cl2 (5:1), –50 °C → –28 °C, 17 h, 76% yield; (f) Ac2O–Py, DMAP (0.9 equiv), r.t., 90% yield.
Synlett 2010, No. 20, 3023–3026 © Thieme Stuttgart · New York

LETTER
Regioselective Deacetylation of Trisaccharide
3025
acetyl groups of the galactose unit of Lewis X trisaccha-
ride 12 were selectively cleaved by very mild transesteri-
fication with a freshly prepared solution of catalytic
sodium methoxide in methanol. Interestingly, when the
pH value did not exceed 7.5 (wet pH indicator), it was
possible to obtain 13 as a single regioisomer in 94% yield,
with unaffected O-acetyl groups on the fucose moiety af-
ter neutralization with the acidic ion-exchanger Am-
berlyst 15. The NMR spectrum of 13 displays the typical
downfield shifts of protons H3 and H4 on the fucose moi-
ety that bear O-acetyl protecting groups. However, when
the same reaction was performed at pH 8.5–9.0, the com-
pletely deacetylated Lewis X trisaccharide was obtained
after 20 hours at room temperature. We presume that the
methoxide ion in catalytic amounts initially attacks the
acetyl group on the most readily accessible 3-position of
the galactose unit. Subsequently, intramolecular acetyl
transfer occurs which leads to the removal of the O-acetyl
groups from the adjacent 2-OH and 4-OH positions in the
galactose. It appears that this acetyl migration does not ap-
ply to the two O-acetyl groups on the fucose moiety.
References and Notes
(1) (a) Moore, K. L.; Stults, N. L.; Diaz, S.; Smith, D. F.;
Cummings, R. D.; Varki, A.; McEver, R. P. J. Cell Biol.
1992, 118, 445. (b) Sperandio, M. FEBS J. 2006, 273, 4377.
(c) Carlow, D. A.; Gossens, K.; Naus, S.; Veerman, K. M.;
Seo, W.; Ziltener, H. J. Immunol. Rev. 2009, 230, 75.
(2) Xia, L. J.; Ramachandran, V.; McDaniel, J. M.; Nguyen, K.
N.; Cummings, R. D.; McEver, R. P. Blood 2003, 101, 552.
(3) (a) Baumann, K.; Kowalczyk, D.; Kunz, H. Angew. Chem.
Int. Ed. 2008, 47, 3445. (b) Baumann, K.; Kowalczyk, D.;
Gutjahr, T.; Pieczyk, M.; Jones, C.; Wild, M. K.; Vestweber,
D.; Kunz, H. Angew. Chem. Int. Ed. 2009, 48, 3174.
(4) Pudelko, M.; Bull, J.; Kunz, H. ChemBioChem 2010, 11,
904.
(5) (a) Dziadek, S.; Kowalczyk, D.; Kunz, H. Angew. Chem. Int.
Ed. 2005, 44, 7624. (b) Brocke, C.; Kunz, H. Synlett 2003,
2052.
(6) Brocke, C.; Kunz, H. Synthesis 2004, 525.
(7) Wittmann, V.; Lennartz, D. Eur. J. Org. Chem. 2002, 1363.
(8) Hanessian, S.; Lavallee, P. Can. J. Chem. 1975, 53, 2975.
(9) (a) Windmüller, R.; Schmidt, R. R. Tetrahedron Lett. 1994,
35, 7927. (b) Zhu, T.; Boons, G.-J. J. Am. Chem. Soc. 2000,
122, 10222.
(10) Sato, S.; Mori, M.; Ito, Y.; Ogawa, T. Carbohydr. Res. 1986,
155, C6.
(11) Kunz, H.; Unverzagt, C. Angew. Chem., Int. Ed. Engl. 1988,
27, 1697.
(12) Sakagami, M.; Hamana, H. Tetrahedron Lett. 2000, 41,
5547.
(13) Schmidt, R. R.; Michel, J. Angew. Chem., Int. Ed. Engl.
1980, 19, 731.
(14) Marra, A.; Sinaÿ, P. Carbohydr. Res. 1990, 195, 303.
(15) (a) Liebe, B.; Kunz, H. Angew. Chem., Int. Ed. Engl. 1997,
36, 618. (b) Filser, C.; Kowalczyk, D.; Jones, C.; Wild, M.
K.; Ipe, U.; Vestweber, D.; Kunz, H. Angew. Chem. Int. Ed.
2007, 46, 2108.
Taking advantage of this highly regioselective deacetyla-
tion of 12, the reaction of 13 with the sialyl donor 614 ac-
cording to previously published methodology6,15 using
AgOTf/Me–S–Br promoting system stereo- and regiose-
lectively gave the tetrasaccharide 14 in 76% yield, thereby
exploiting the nitrile effect.16 Acetylation of the 2-OH and
4-OH groups of the galactose unit gave the sialyl Lewis X
15 in 90% yield. It is worth noting that the anomeric
TBDPS group remained unaffected during all these con-
versions towards 15 (Scheme 2).
In conclusion, we have efficiently synthesized the sialyl
Lewis X tetrasaccharide 15 using an altered protective
group strategy concerning the glucosamine acceptor 9 and
the fucose donor 8. The key step in this synthesis is the re-
gioselective deacetylation of three out of five hydroxy
groups of a Lewis X trisaccharide 12 using very mild
transesterification protocol,17 in which a strict control of
the pH was a pivotal feature. It is also worth noting, that
the glycosylation of typically unreactive 4-hydroxy group
of the disaccharide unit 11 with the galactosyl donor 7
proceeds with high yield. Further work towards the tet-
rasaccharide oxazoline 5 and the hexasaccharide 1 repre-
senting the building block for solid-phase synthesis of
glycopeptide 2 is currently in progress.
(16) (a) Schmidt, R. R.; Rücker, E. Tetrahedron Lett. 1980, 21,
1421. (b) Ratcliffe, A. J.; Fraser-Reid, B. J. Chem. Soc.,
Perkin Trans. 1 1990, 747.
(17) Typical Procedure for Regioselective O-Deacetylation of
the Trisaccharide 12: Trisaccharide 12 (2.11 g, 1.69 mmol)
was dissolved in MeOH (150 mL) and freshly prepared
NaOMe solution in MeOH was added carefully until pH 7.5
was obtained. Reaction was performed at r.t. and monitored
by TLC every 30 min. After completion of the reaction (4 h
40 min), the mixture was neutralized with Amberlyst 15 to
pH 6–6.5. The resin was removed by filtration, washed with
MeOH, and the filtrate was concentrated under vacuum to
give the crude 13 (1.78 g, 94% yield) as a white amorphous
material. Trisaccharide 13 can be additionally purified on
silica gel with EtOAc as eluent (Rf 0.35 in EtOAc).
Spectroscopic data for 13: [a]24D –26.6 (c = 2.00, MeOH). 1H
NMR (COSY, CD3OD): d = 7.66–7.72 (4 H, m, Har), 7.19–
7.39 (21 H, m, Har), 5.30–5.34 (2 H, m, H1-Fuc {5.33}, H3-
Fuc {5.32}), 5.22 (1 H, d, H4-Fuc, J3,4 = 2.6 Hz), 5.04–5.06
(1 H, m, H5-Fuc), 4.29–4.80 (8 H, m, CH2Bn-2a {4.78},
CH2-Bn-1a {4.66}, H1-GlcNAc {4.60}, CH2Bn-1b {4.58},
CH2Bn-2b {4.45}, CH2Bn-3a {4.44}, H1-Gal {4.42},
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett. Included are
experimental procedures and spectroscopic characterization data
for compounds 11, 12, 14, and 15.
CH2Bn-3b {4.30}), 4.06 (1 H, t*, H4-GlcNAc, J3,4 @ J4,5
@
9.3 Hz), 3.92–3.96 (1 H, m, H3-GlcNAc), 3.77–3.86 (5 H,
m, H6a,b-Gal {3.85}, H6a-GlcNAc {3.84}, H2-Fuc {3.82},
H4-Gal {3.78}), 3.40–3.47 (3 H, m, H2-Gal {3.46}, H5-Gal
{3.43}, H6b-GlcNAc {3.42}), 3.30–3.35 (2 H, m, H3-Gal
{3.33}, H2-GlcNAc {3.31}), 3.07–3.10 (1 H, m, H5-
GlcNAc), 2.03, 1.94, 1.80 (9 H, 3 × s, MeAc), 1.05 (9 H, s,
Me-t-Bu), 1.04 [3 H, d, Me(6)-Fuc, J5,6 = 6.5 Hz]. 13C NMR
(BB, HSQC, 100.6 MHz, CD3OD): d = 173.18, 172.48,
Acknowledgment
M.P. is grateful for the stipend provided by the Alexander von
Humboldt Foundation. Support from the Deutsche Forschungs-
gemeinschaft is gratefully acknowledged.
Synlett 2010, No. 20, 3023–3026 © Thieme Stuttgart · New York

3026
M. Pudelko et al.
LETTER
Fuc), 73.39 (CH2Bn-2), 72.86 (C2-Gal), 71.25 (C3-Fuc),
70.34 (C6-Gal), 69.77 (C4-Gal), 68.87 (C6-GlcNAc), 65.53
(C5-Fuc), 48.80 (C2-GlcNAc), 27.41 (Me-t-Bu), 23.69,
20.99, 19.98 (MeAc), 16.33 [Me(6)-Fuc]. MS (ESI): m/z
[M + Na]+ calcd for C61H75NO17SiNa: 1144.47; found:
1144.48. HRMS (ESI–TOF): m/z [M + Na]+ calcd for
C61H75NO17SiNa: 1144.4702; found: 1144.4690.
171.56 (C=O), 140.18, 139.78 (Cipso-Ar), 137.17, 137.09
(Co,m,p-Ar), 134.78, 134.18, 130.97 (Cipso-Ar), 130.79,
129.35, 129.18, 129.06, 128.71, 128.65, 128.49, 128.43
(Co,m,p-Ar), 103.41 (C1-Gal), 98.04 (C1-Fuc), 97.77 (C1-
GlcNAc), 76.68 (C5-GlcNAc), 75.45 (C3-GlcNAc), 75.05
(C5-Gal), 75.02 (C2-Fuc), 74.82 (C3-Gal), 74.75 (C4-
GlcNAc), 74.44 (CH2Bn-1), 74.29 (CH2Bn-3), 73.56 (C4-
Synlett 2010, No. 20, 3023–3026 © Thieme Stuttgart · New York

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