A novel and efficient synthesis of a dimeric Le(x) oligosaccharide on polymeric support [9]

Full text of DOI:10.1021/ja001930l

10222
J. Am. Chem. Soc. 2000, 122, 10222-10223
A Novel and Efficient Synthesis of a Dimeric Lex
Oligosaccharide on Polymeric Support
Scheme 1. Concept of New Linker
Tong Zhu and Geert-Jan Boons*
Complex Carbohydrate Research Center
220 RiVerbend Road, Athens, Georgia 30602
ReceiVed June 1, 2000
Increased awareness of the biological importance of oligosac-
charides and glycoconjugates has stimulated the development of
efficient methods for the preparation of these compounds.
Improved chemical1 and enzymatic2 glycosylation procedures in
combination with convergent synthetic strategies make it possible
to execute multistep synthetic sequences that give well-defined
complex oligosaccharides in reasonable quantities. It is to be
expected that further improvements and eventually automation
will come from polymer-supported oligosaccharide synthesis.
Several relatively large oligosaccharides have been synthesized
on polymeric supports3 although most of these compounds were
relatively simple 1,2-trans linked linear homooligomers.
Herein, we report a highly efficient strategy for the polymer-
supported synthesis of the dimeric Lewis antigen Lewisx-Lewisx
(Lex-Lex).3b,d,4 Lewis antigens are an important family of tumor
associated antigens that offer promise for the development of
cancer vaccines for small cell lung, breast, prostate, lung, colon,
stomach, and ovary cancer.5 These branched compounds contain
both R- and â-glycosidic linkages attached to hindered hydroxyls
of a glucosamine moiety.
An important requirement for the synthesis of the target
compound is the selection of a set of temporary protecting groups
and a linker that are compatible with the acid-sensitive fucosidic
linkage. Furthermore, the linker and protecting groups should be
compatible with the base-sensitive amino protecting group
Trichloroethoxycarbonyl (Troc).6 Glycosyl donors protected with
this functionality are highly reactive and offer efficient neighbor-
ing group participation to give stereoselective formation of
â-glycosides. The Troc group also ensures high glycosyl accepting
properties of the C-3 hydroxyl of the glucosamine unit.4h
A strategy was adapted3f whereby a polymer-bound Lex
trisaccharide was prepared which could be converted into a
glycosyl acceptor by selective removal of a temporary protecting
group or into a soluble glycosyl donor by cleavage from the
polymeric support followed by activation of the anomeric center.
Coupling of the resulting glycosyl donor and acceptor followed
by cleavage from the solid support should give the target
hexasaccharide. This strategy requires a linker that attaches a
saccharide to a polymeric support via the anomeric center of the
reducing sugar.
To this end, a novel phenolic ester type linker was developed
(Scheme 1). The saccharide is attached to the polymer support
by glycosyaltion with the hydroxyl of the acyloxybenzyl linker.
Compared to well-established p-alkoxybenzyl glycosides, the
resulting p-acyloxybenzyl glycoside is significantly more stable
toward Lewis acidic conditions used in glycosylations. This bond,
however, is cleaved within minutes by treatment with hydrogen
peroxide/Et3N. After detachment, a stable p-hydroxyl benzyl
glycoside is obtained as a single anomer and this feature facilitates
purification. Oxidative removal of the p-hydroxyl benzyl moiety
with DDQ7 will give a lactol that can be easily converted into a
glycosyl donor (e.g. trichloroacetimidate).
The three saccharide building blocks 1,8 4, and 79 and linker
modified MPEG 2 were used for the assembly of the target
hexasaccharide. The temporary protecting groups 9-fluorenyl-
methoxycarbonyl (Fmoc) and diethylisopropylsilyl (DEIPS)10 of
these building blocks can be cleaved under very mild conditions
without affecting the linker and the Troc protecting group.
Methoxypoly(ethylene glycol) (MW 5000) MPEG was chosen
as the polymeric support to take advantage of its solubility in
many organic solvents. However, the workup procedure involves
precipitation of MPEG by addition of diethyl ether or tert-butyl
methyl ether. Thus, excess of reagents and other side products
can easily be removed by washing of the MPEG precipitate.11
Coupling of 1 with 2 in the presence of NIS/TMSOTf12 gave
formation of immobilized 3 (Scheme 2). Only 1.1 equiv of donor
1 was required to achieve complete conversion of the polymeric
acceptor. No self-condensation of 1 was observed which was
expected due to the much greater reactivity of the benzylic alcohol
of 2 compared to the C-4 hydroxyl of 1. The polymer-bound
monosaccharide 3, bearing a free C-4 hydroxyl, was immediately
used in a subsequent glycosylation without workup and purifica-
tion by precipitation. Thus, addition to the reaction mixture of
another amount of NIS/TMSOTf and galactosyl donor 4 gave,
after standard workup and purification by precipitation, im-
(1) (a) Toshima, K.; Tatsuta, K. Chem. ReV. 1993, 93, 1503. (b) Boons,
G.-J. Contemp. Org. Synth. 1996, 173. (c) Boons, G.-J. Tetrahedron 1996,
52, 1095.
(2) Gijsen, H. J. M.; Lei, Q.; Fitz, W.; Wong, C. H. Chem. ReV. 1996, 96,
443.
(3) (a) Verduyn, R.; van der Klein, P. A. M.; Douwes, M.; van der Marel,
G. A.; van Boom, J. H. Recl. TraV. Chim. Pays-Bas 1993, 112, 464. (b)
Randolph, J. T.; McClure, K. F.; Danishefsky, S. J. J. Am. Chem. Soc. 1995,
117, 5712. (c) Shimizu, H.; Ito, Y.; Kanie, O.; Ogawa, T. Bioorg. Med. Chem.
Lett. 1996, 6, 2841. (d) Danishefsky, S. J.; Bilodeau, M. T. Angew. Chem.,
Int. Ed. 1996, 35, 1380. (e) Rademann, J.; Schmidt, R. R. J. Org. Chem.
1997, 62, 3650. (f) Nicolaou, K. C.; Winssinger, N.; Pastor, J.; DeRoose, F.
J. Am. Chem. Soc. 1997, 119, 449. (g) Nicolaou, K. C.; Watanabe, N.; Li, J.;
Pastor, J.; Winssinger, N. Angew. Chem., Int. Ed. 1998, 37, 1559. (h) Ito, Y.;
Manabe, S. Curr. Chem. Bio. 1998, 2, 701 and references therein. (i) Andrade,
R. B.; Plante, O. J.; Melean, L. G.; Seeberger, P. H. Org. Lett. 1999, 1, 1811.
(j) Osborn, H. M. I., Khan, T. H. Tetrahedron 1999, 55, 1807.
(4) Several elegant chemical syntheses of Lewis antigens have been
reported. For examples, see: (a) Sato, S.; Ito, Y.; Ogawa, T. Tetrahedron
Lett. 1988, 29, 5267. (b) Nicolaou, K. C.; Hummel, C. W.; Iwabuchi, Y. J.
Am. Chem. Soc. 1992, 114, 3126. (c) Toepfer, A.; Schmidt, R. R. Tetrahedron
Lett. 1992, 33, 5161. (d) Windmuller, R.; Schmidt, R. R. Tetrahedron Lett.
1994, 35, 7927. (e) Yan, L.; Kahne, D. J. Am. Chem. Soc. 1996, 118, 9239
and references therein. (f) Deshpande, P. P.; Kim, H. M.; Zatorski, A.; Park,
T.; Ragupathi, G.; Livingston, P. O.; Live, D.; Danishefsky, S. J. J. Am. Chem.
Soc. 1998, 120, 1600. (g) Kanemitsu, T.; Kanie, O.; Wong, C. H. Angew.
Chem., Int. Ed. 1998, 37, 3415. (h) Ellervik, U.; Magnusson, G. J. Org. Chem.
1998, 63, 9314.
(7) Jobron, L.; Hindsgaul, O. J. Am. Chem. Soc. 1999, 121, 5835.
(8) Zhu, T.; Boons, G.-J. Tetrahedron: Asymmetry 2000, 11, 199.
(9) Depre´, D.; Du¨ffels, A.; Green, L. G.; Lenz, R.; Ley, S. V.; Wong, C.
H. Chem. Eur. J. 1999, 5, 3326
(10) Toshima, K.; Mukaiyama, S.; Kinoshita, M.; Tatsuta, K. Tetrahedron
Lett. 1989, 30, 6413.
(11) (a) Douglas, S. P.; Whitfield, D. M.; Krepinsky, J. J. J. Am. Chem.
Soc. 1991, 113, 5095. (b) Douglas, S. P.; Whitfield, D. M.; Krepinsky, J. J.
J. Am. Chem. Soc. 1995, 117, 2116.
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1990, 31, 4313. (b) Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H.
Tetrahedron Lett. 1990, 31, 1331.
(5) Hakomori, S. Acta Anat. 1998, 161, 79.
(6) (a) Windholz, T. B.; Johnston, D. B. R. Tetrahedron Lett. 1967, 2555.
(b) Just, G.; Grozinger, K. Synthesis 1976, 457. (c) Zhang, Z.; Ollmann, E.
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10.1021/ja001930l CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/27/2000

Communications to the Editor
J. Am. Chem. Soc., Vol. 122, No. 41, 2000 10223
Scheme 2.a Synthesis of Lex-Lex Hexasaccharide 14
buffered tetrabutylammonium fluoride (TBAF) to remove the
DEIPS group to give the trisaccharide acceptor 9. Under these
conditions a TBDMS group could not be removed and the use of
other desilylating reagents resulted in concomitant cleavage of
the fucoside or linker. The protected Lex trisaccharide of pool B
was released from the polymeric support by treatment with
hydrogen peroxide in the presence of Et3N in DCM to give, after
silica gel column chromatography, trisaccharide 10 in an overall
yield of 35% (based on initial loading of polymer). Oxidative
removal of the anomeric p-hydroxy-benzyl moiety of 10 with
DDQ gave within minutes lactol 11. Next, 11 was converted into
trichloracetimidate 12 by treatment with trichloroacetonitrile in
the presence of a catalytic amount of DBU (R/â, 1/1, overall yield
75%).13 Next, trisaccharide donor 12 (1.5 equiv) was coupled with
polymer-bound trisaccharide acceptor 9 in the presence of
TMSOTf.13b The reaction was repeated to achieve complete
consumption of immobilized acceptor and quantitative formation
of 13. Precipitation of 13 with diethyl ether did not pose any
problems and the NMR spectrum indicated a pure polymer-bound
compound. Finally, the protected Lex-Lex hexasaccharde 14 was
obtained in overall yield of 20% (based on loading) by cleavage
of 13 from the polymeric support using standard conditions.
Compound 14 was conveniently converted into methyl glycoside
17 by oxidative removal of the anomeric p-hydroxybenzyl
followed by conversion of the resulting lactol 15 into the
trichloroacetimidate 16 which was glycosylated with methanol
in the presence of TMSOTf. Deprotection of 17 was easily
accomplished by a five-step procedure. The DEIPS group was
removed by treatment with buffered TBAF. Next, the N-Troc was
cleaved by treatment with activated Zn in acetic acid and the
resulting amino functionality was acetylated with acetic anhydride.
Finally, target hexasaccharide 18 was obtained by removal of the
benzyl ethers by catalytic hydrogenation over Pd followed by
deacylation with NaOMe in methanol. The NMR and mass
spectroscopy data of 18 were in agreement with the proposed
structure.
In conclusion, we have described a highly efficient synthesis
of Lex-Lex hexasaccharide on MPEG. This compound, which is
branched and contains both R- and â-glycosidic linkage, is one
of the most complex saccharides ever synthesized on polymeric
support. A key feature of the approach is a novel phenolic ester
linker that is compatible with the hydroxyl protecting groups Fmoc
and DEIPS and the amino protecting group Troc. This set of
protecting groups and linker can be used for the synthesis of acid-
sensitive oligosaccharides that contain fucosides and amino sugars.
Cleavage of the linker gives an oligosaccharide that can easily
be converted into a glycosyl donor for further synthesis. A one-
pot multistep glycosylation sequence was employed to minimize
the number of protecting group manipulations.
a Reagents and conditions: (i) NIS, TMSOTf, MS 4 Å, 0 °C, DCM;
(ii) Et3N, DCM; (iii) TBAF, HOAc, THF; (iv) H2O2, Et3N, THF; (v)
DDQ, DCM, H2O; (vi) CCl3CN, DBU, DCM; (vii) TMSOTf, MS 4 Å;
(viii) MeOH, TMSOTf, DCM, MS 3 Å; (ix) a. TBAF, HOAc, THF; b.
Zn, AcOH then Ac2O, Py; c. Pd(OAc)2, H2, EtOH, AcOH; d. NaOMe,
MeOH.
mobilized disaccharide 5. The NMR spectrum indicated clean
formation of 5 and no production of truncated or incomplete
glycosylated structures. To the best of our knowledge, this
glycosylation sequence represents the first example of a one-pot
multistep glycosylation sequence performed on polymeric support.
Next, the Fmoc of 5 was removed by the treatment with
nonnucleophilic base Et3N in DCM to give 6. No cleavage of
disaccharide from the polymer support was observed. The revealed
hydroxyl of 6 was glycosylated with fucosyl donor 7 in the
presence of NIS/TMSOTf to give trisaccharide 8. The NMR
spectrum indicated the formation of only one anomer. It is
noteworthy that only a very small amount of trisaccharide was
formed when the amino function was protected as phthaloyl (Phth)
instead of Troc. It is well-known that the reactivity of a hydroxyl
adjacent to Phth is reduced due to steric hindrance.4h
Acknowledgment. We thank the office of the vice president of
research of the University of Georgia, Athens for financial support.
Supporting Information Available: Schemes for preparation of
compounds 2 and 4, 500 MHz 1H NMR spectra of 4, 10, 14, and 18, 1H
NMR and 13C NMR spectral data of 10, 14, and 18, general procedure
for NIS/TMSOTf mediated glycosylation on MPEG, general procedure
for the cleavage of product from MPEG, and procedures for the
deprotection of 14 to give 18 (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
JA001930L
The polymer-bound trisaccharide 8 was split into two pools,
A and B (ratio, A/B ) 1/4). Pool A was treated with acetic acid
(13) (a) Schmidt, R. R. Angew. Chem., Int. Ed. 1986, 25, 212. (b) Manzoni,
L.; Lay, L.; Schmidt, R. R. J. Carbohydr. Chem. 1998, 739.