Ready access to sialylated oligosaccharide donors

Article abstract of DOI:10.1021/ol990406k

(figure presented) Numerous glycoconjugates contain the disaccharide Neu5Acα(2→S)DGalp. An efficient way to incorporate this disaccharide into synthetic glycoconjugates is to develop a disaccharide building block. This communication reports a chemoenzymatic route to such a building block which requires as few as four steps. Some examples using more chemical steps are also presented, which increase the flexibility. These disaccharide donors were used to prepare synthetic trisaccharides.

Full text of DOI:10.1021/ol990406k

ORGANIC
LETTERS
2000
Vol. 2, No. 6
751-753
Ready Access to Sialylated
Oligosaccharide Donors
Seema Mehta, Michel Gilbert, Warren W. Wakarchuk, and Dennis M. Whitfield*
National Research Council, Ottawa, Ontario, Canada
dennis.whitfield@nrc.ca
Received December 20, 1999
ABSTRACT
Numerous glycoconjugates contain the disaccharide Neu5Acr(2f3)DGalp. An efficient way to incorporate this disaccharide into synthetic
glycoconjugates is to develop a disaccharide building block. This communication reports a chemoenzymatic route to such a building block
which requires as few as four steps. Some examples using more chemical steps are also presented, which increase the flexibility. These
disaccharide donors were used to prepare synthetic trisaccharides.
Pathogenic bacteria such as Campylobacter jejuni, Neisseria
meningitidis, and Group B Streptococcus have sialylated
oligosaccharides as integral parts of their outer surface. It is
widely believed that these sialic acid residues mimic host
cell surface oligosaccharides. This leads to reduced immu-
nogenicity and, hence, evolutionary advantages for these
pathogens.1 As part of our Institute’s program to develop
protective vaccines against these organisms, we have initiated
a program to synthesize some of these sialylated oligosac-
charides.2 Since we wish to make a large number of structural
variants and derivatives, we envisaged a building block
approach. This communication reports a highly efficient
chemoenzymatic route to Neu5AcR(2f3)DGalp disaccharide
donors.
improved the efficiency,4 but such procedures still require
too many synthetic steps and too many chromatographic
separations.
In our synthesis, arylthio glycosides of galactose and
lactose have been enzymatically sialylated to afford glycoside
donors with minimal chemical steps and chromatography.
Our new method involves a two-step preparation of an
arylthio glycoside donor which requires one simple chro-
matographic separation (cf. 2b and 3b in Scheme 1). This is
followed by enzymatic glycosylation using sialic acid, CTP,
and glycosides as substrates and CMP-Neu5Ac synthetase
and sialyl transferase as catalysts5 to produce the Neu5AcR-
(2f3)DGalpâ(1f)SAr disaccharides in >80% isolated yield
after gel filtration or reverse phase purification; cf. 4a and
4b. We have scaled these reactions from 10 mg to 100 mg
followed by 1 g with no diminution in yield.
Previously, we have accessed such donors by a chemical
method involving a three-step preparation of a sialic acid
glycosyl donor and a six-step synthesis of a DGalp acceptor.
These monosaccharides are coupled in 60% yield, and three
additional steps of functional group manipulations are
required to make a disaccharide donor. The whole process
requires nine chromatographic separations. A number of
groups have employed similar strategies in their syntheses
of sialylated oligosaccharides.3 Some recent syntheses have
(3) (a) Pozsgay, V.; Jennings, H. J.; Kasper, D. L. J. Carbohydr. Chem.
1987, 6, 41. (b) Paulsen, H.; Tietz, H. Carbohydr. Res. 1985, 144, 205. (c)
Murase, T.; Kameyama, A.; Kartha, K. P. R.; Ishida, H.; Kiso, M.;
Hasegawa, A. J. Carbohydr. Chem. 1989, 8, 265.
(4) (a) Schwarz, J. B.; Kuduk, S. D.; Chen, X. T.; Sames, D.; Glunz, P.
W.; Danishefsky, S. J. J. Am. Chem. Soc. 1999, 121, 2662. (b) Demchenko,
A. V.; Boons, G.-J. Tetrahedron. Lett. 1998, 39, 3065.
(5) The transferases and the synthetase have been cloned and expressed
in the NRC laboratories; for experimental details see: (a) Gilbert, M.;
Brisson, J.-R.; Karwaski, M.-F.; Michniewicz, J.; Cunningham, A.-M.; Wu,
Y.; Young, N. M.; Wakarchuk, W. W. J. Biol. Chem. 2000, 275, 3896. (b)
Gilbert, M.; Watson, D. C.; Wakarchuk, W. W. Biotechnol. Lett. 1997, 19,
417.
(1) Jennings, H. J. Curr. Top. Microbiol. Immunol. 1990, 150, 97.
(2) Eichler, E.; Jennings, H. J.; Whitfield, D. M. J. Carbohydr. Chem.
1997, 16, 385.
10.1021/ol990406k CCC: $19.00 Published 2000 by the American Chemical Society
Published on Web 02/16/2000

Scheme 1
Scheme 3
to yield trisaccharide 9 in 90% and 86% yields, respectively
(see Scheme 3). In a more demanding application the
polymer-bound diol 10 has been glycosylated with donor 7
to yield 11. The polymer is the soluble monomethyl ether
of poly(ethylene glycol) (MeO(CH2CH2O)nOH, MPEG),9 and
dioxyxylene (p-OCH2C6H4CH2O-, DOX) is the linker.10
Routine cleavage of 11 from the polymer using Sc(OTf)3/
Ac2O afforded the trisaccharide 12 in 36% overall yield (see
Scheme 4).11 This trisaccharide is a synthon for the Neu5AcR-
A simple acetylation produces the lactones 5a and 5b in
75% and 90% yields with the Neu5Ac carboxyl esterified
with DGalp O-2 (see Scheme 2).6 The lactone either can be
Scheme 2
Scheme 4
used directly as a glycosyl donor or can be opened to the
methyl ester 67 and the DGalp O-2 acetylated to give 7 in
71% yield from 5a. It is readily apparent that other esters
and other acyl groups could be prepared (see below). These
arylthio glycosides can be used directly as glycosyl donors.
In certain cases other activating groups may be desirable and
thioglycosides can be converted into bromides, fluorides, or
trichloroacetimidates as required.8 This procedure is suf-
ficiently simple that these donors could be produced com-
mercially, thereby allowing for the synthesis of bacterial
antigens and other sialylated oligosaccharides.
(2f3)DGalpâ(1f4)DGlcNAcpâ(1f) grouping found on
many glycoconjugates.
In an extension of our idea, the phenylthio glycoside of
lactose (DGalpâ(1f4)DGlcp, 13) has been enzymatically
sialylated to afford the trisaccharide 14 (see Scheme 5). Since
glycosylations with O-2 acetylated lactose donors12 are
(9) (a) Jiang, L.; Hartley, R. C.; Chan, T. H. Chem. Commun. 1996,
2193. (b) Wang, Z. G.; Douglas, S. P.; Krepinsky, J. J. Tetrahedron Lett.
1996, 39, 6985. (c) Ito, Y.; Kanie, O.; Ogawa, T. Angew. Chem., Int. Ed.
Engl. 1996, 35, 2510. (d) Ito, Y.; Ogawa, T. J. Am. Chem. Soc. 1997, 119,
5562. (e) Dreef-Tromp, C. M.; Willems, H. A. M.; Westerduin, P.; van
Veelen, P.; van Boeckel, C. A. A. Biorg. Med. Chem. Lett. 1997, 7, 1175.
(f) Eggenweiler, H. M.; Bayer, E. InnoVation Perspect. Solid-Phase Synth.
Comb. Libr. Collect. 1996, 4, 363.
As proof of principle, disaccharides 5a and 5b have been
used as donors to glycosylate the O-6 of DGlcp acceptor 8
(6) Yu, R. K.; Koerner, T. A. W.; Ando, S.; Yohe, H. C.; Prestegard, J.
H. J. Biochem. 1985, 98, 1367.
(7) Hayashi, M.; Tanaka, M.; Itoh, M.; Miyauchi, H. J. Org. Chem. 1996,
61, 2938.
(8) Garegg, P. J. AdV. Carbohydr. Chem. Biochem. 1997, 52, 179.
(10) Douglas, S. P.; Whitfield, D. M.; Krepinsky, J. J. J. Am. Chem.
Soc. 1995, 117, 2116.
(11) Mehta, S.; Whitfield, D. M. Tetrahedron Lett. 1998, 39, 5907.
752
Org. Lett., Vol. 2, No. 6, 2000

functional group manipulations.14 This synthesis is a good
precedent for our work, but our procedure is significantly
simpler. Benzoylation of 14 produced the lactone between
DGalp O-2 and the Neu5Ac carboxyl 15 as with acetylation
of the Neu5AcR(2f3)DGalp disaccharides. The highly
hindered Neu5Ac O-7 was not benzoylated under these
conditions. Subsequent esterification with methanol 16
followed by N- and O-acetylation15 led to the peracylated
trisaccharide donor 17 in 51% overall yield. Preliminary
experiments have shown that di-N-acetylated Neu5Ac con-
taining oligosaccharide donors give higher glycosylation
yields than mono-N-acetylated donors.16 The regiochemistry
of the acyl groups in 15 and 17 was confirmed by 13C-1H
HMBC correlation experiments. Thus, a synthon for GM3
and derivatives has been efficiently prepared. Extrapolation
of this procedure to larger oligosaccharides related to
common gangliosides is in progress.
Scheme 5
Acknowledgment. We thank E. Eichler for measuring
optical rotations, M. F. Karwaski for technical assistance,
K. Chan for measuring FAB MS spectra, D. Krajcarski for
measuring the MALDI MS spectra, and J. R. Brisson for
advice with the NMR spectra. This is NRC paper #42420.
frequently complicated by ortho ester formation and acyl
transfer, the trisaccharide 14 was benzoylated to give 15 in
44% yield.13 A previous communication used a 2-O pivaloyl
lactose derivative as glycosyl acceptor for enzymatic gly-
cosylation and subsequently successfully used this to prepare
synthetic GM3 glycolipid by chemical glycosylation and
Supporting Information Available: Full synthetic pro-
cedures for the oligosaccharides prepared in this communica-
tion, including spectral characterization. This material is
available free of charge via the Internet at http://pubs.acs.org.
(12) (a) Kunz, H.; Harreus, A. Liebigs Ann. Chem. 1982, 41. (b) Sato,
S.; Nunomura, T.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1988, 29, 4097. (c)
Sato, S.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1988, 29, 5267. (d) Nunomura,
S.; Ogawa, T. Tetrahedron Lett. 1988, 29, 5681.
(13) Nukada, T.; Be´rces, A.; Whitfield, D. M. J. Org. Chem. 1999, 64,
9030.
OL990406K
(14) Ito, Y.; Paulson, J. C. J. Am. Chem. Soc. 1993, 115, 1603.
(15) Demchenko, A. V.; Boons, G.-J. Chem. Eur. J. 1999, 5, 1278.
(16) Eichler, E.; Whitfield, D. M. Unpublished observations.
Org. Lett., Vol. 2, No. 6, 2000
753