Synthesis of the trisaccharide portion of the immunologic adjuvant QS-21A via sulfonium-mediated oxidative and dehydrative glycosylation.

Article abstract of DOI:10.1021/ol015651u

[see structure]. The first synthesis of the trisaccharide fragment of the potent immunologic adjuvant QS-21A is reported. The key steps involve the application of sulfonium-mediated oxidative and dehydrative glycosidic couplings to construct the anomeric

Full text of DOI:10.1021/ol015651u

ORGANIC
LETTERS
2001
Vol. 3, No. 12
1801-1804
Synthesis of the Trisaccharide Portion
of the Immunologic Adjuvant QS-21A
via Sulfonium-Mediated Oxidative and
Dehydrative Glycosylation
Yong-Jae Kim and David Y. Gin*
Department of Chemistry, UniVersity of Illinois, Urbana, Illinois 61801
gin@scs.uiuc.edu
Received February 2, 2001
ABSTRACT
The first synthesis of the trisaccharide fragment of the potent immunologic adjuvant QS-21A is reported. The key steps involve the application
of sulfonium-mediated oxidative and dehydrative glycosidic couplings to construct the anomeric linkages in a short and convergent assembly
of the branched trisaccharide.
QS-21A (1) is a saponin adjuvant isolated from the bark of
the Quillaja saponaria Molina tree as a minor constituent
in a complex mixture of saponins.1,2 HPLC purification of 1
and assessment of its adjuvant activity revealed that it
enhances both humoral and cell-mediated immune responses
in a host of vaccine formulation assays. Among these
adjuvant enhanced vaccine studies are those involving
ganglioside subunit antigens against melanoma3 and prostate
cancer,4 TCR vaccines against T-cell lymphoma,5 protein/
peptide vaccine formulations against malaria,6 and recom-
binant gp120 and gp160 subunit antigens against HIV-1.7
QS-21A’s powerful immunostimulating properties, along
with its relative nontoxicity, has led to extensive clinical
development of this adjuvant. The complex oligosaccharide
architecture within 1 in combination with its exceedingly
potent adjuvant activity have engaged our efforts in the
synthesis of the triterpene saponin. Herein is reported the
(4) (a) Ragupathi, G.; Slovin, S. F.; Adluri, S.; Sames, D.; Kim, I. J.;
Kim, H. M.; Spassova, M.; Bornmann, W. G.; Lloyd, K. O.; Scher, H. I.;
Livingston, P. O.; Danishefsky, S. J. Angew. Chem., Int. Ed. Engl. 1999,
38, 563-566. (b) Slovin, S. F.; Ragupathi, G.; Adluri, S.; Ungers, G.; Terry,
K.; Kim, S.; Spassova, M.; Bornmann, W. G.; Fazzari, M.; Dantis, L.;
Olkiewicz, K.; Lloyd, K. O.; Livingston, P. O.; Danishefsky, S. J.; Scher,
H. I. Proc. Natl. Acad. Sci. 1999, 96, 5710-5715.
(1) Kensil, C. R.; Patel, U.; Lennick, M.; Marciani, D. J. Immunol. 1991,
146, 431-437.
(2) (a) Jacobsen, N. E.; Fairbrother, W. J.; Kensil, C. R.; Lim, A.;
Wheeler, D. A.; Powell, M. F. Carbohydr. Res. 1996, 280, 1-14. (b)
Saponins Used in Traditional and Modern Medicine; Waller, G. R.,
Yamasaki, K., Eds.; Advances in Experimental Medicine and Biology 404;
Plenum Press: New York, 1996; p 165.
(3) (a) Helling, F.; Shang, A.; Calves, M.; Zhang, S.; Ren, S.; Yu, R.
K.; Oettgen, H. F.; Livingston, P. O. Cancer Res. 1994, 54, 197-203. (b)
Helling, F.; Zhang, S.; Shang, A.; Adluri, S.; Calves, M.; Koganty, R.;
Longenecker, B. M.; Yao, T.-J.; Oettgen, H. F.; Livingston, P. O. Cancer
Res. 1995, 55, 2783-2788. (c) Ragupathi, G.; Meyers, M.; Adluri, S.;
Howard, L.; Musselli, C.; Livingston, P. O. Int. J. Cancer 2000, 85, 659-
666. (d) Brinckerhoff, L. H.; Thompson, L. W.; Slingluff, C. L. Curr. Opin.
Oncol. 2000, 12, 163-173. (e) Kim. S. K.; Ragupathi, G.; Cappello, S.;
Kagan, E.; Livingston, P. O. Vaccine 2001, 19, 530-537.
(5) Wong, C. P.; Okada, C. Y.; Levy, R. J. Immunol. 1999, 162, 2251-
2258.
(6) (a) Stoute, J. A.; Slaoui, M.; Heppner, D. G.; Momin, P.; Kester, K.
E.; Desmons, P.; Wellde, B. T.; Garcon, N.; Krzych, U.; Marchand, M.;
Ballou, W. R.; Cohen, J. D. New Engl. J. Med. 1997, 336, 86-91. (b)
Nardin, E. H.; Oliveira, G. A.; Calvo-Calle, J. M.; Castro, Z. R.;
Nussenzweig, R. S.; Schmeckpeper, B.; Hall, B. F.; Diggs, C.; Bodison,
S.; Edelman, R. J. Infect. Dis. 2000, 182, 1486-1496.
(7) (a) Newman, M. J.; Wu, J.-Y.; Gardner, B. H.; Anderson, C. A.;
Kensil, C. R.; Recchia, J.; Coughlin, R. T.; Powell, M. F. Vaccine 1997,
15, 1001-1007. (b) Sasaki, S.; Sumino, K.; Hamajima, K.; Fukushima, J.;
Ishii, N.; Kawamoto, S.; Mohri, H.; Kensil, C. R.; Okuda, K. J. Virol. 1998,
72, 4931-4939.
10.1021/ol015651u CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/11/2001

synthesis of the trisaccharide component of the natural
product, employing sulfonium-mediated glycosidic bond
formation.
In this approach, two key challenges are addressed. First,
it was envisioned that an oxidative glycosylation reaction
performed with the glucuronate-glycal 3 would generate a
C(2)-hydroxy glucuronate ester, which in turn would im-
mediately function as the carbohydrate acceptor for a
subsequent glycosidic coupling with the galactopyranose
donor 4. However, reports on the direct conversion of
glucuronate glycal donors to the corresponding C(2)-hydroxy
glycoside have been scarce;10 thus, we were eager to
determine whether our sulfonium-mediated oxidative gly-
cosylation protocol could effect this transformation directly
on a glucuronal substrate such as 3, thereby expanding the
scope of this new method in the context of the synthesis of
2. Second, the installation of both the galactopyranose and
xylopyranose moieties at neighboring C(2)- and C(3)-
positions on the glucuronate ester component would provide
a notable challenge for the dehydrative glycosylation method
in sterically congested environments.
The first step to establish the feasibility of oxidative
glycosylation in the synthesis of 2 is the preparation of the
appropriate selectively protected glucuronate-glycal donor.
Methyl 3,4-di-O-acetylglucuronate-D-glycal (6, Scheme 2)
Scheme 2a
We have recently developed a series of new glycosylation
methods involving the reagent combination of diphenyl
sulfoxide and triflic anhydride. These methods include
dehydrative glycosylation involving direct glycosidic bond
formation from a hemiacetal donor8 and oxidative glycosy-
lation for the generation of C(2)-hydroxy glycosides from
glycal donors.9 Since the trisaccharide portion of QS-21A
(2, Scheme 1) is composed of a C(2)-branched oligosaccha-
a NaOMe, MeOH, 81%; (b) TBSCl, imidazole, DMF, 76%; (c)
Ac2O, DMAP, CH2Cl2, 95%.
was readily obtained from commercially available glucu-
ronolactone.11 For the synthesis of trisaccharide 2, it was
imperative to construct a glucuronate glycal donor 3 that
incorporates orthogonal protective groups at C(3) and at C(4),
as this would provide a 2-hyroxyglucuronate substrate with
chemically distinct functionalities at positions 1, 2, 3, and 4
upon oxidative glycosylation. Thus, saponification of the
acetate esters within 6 (Scheme 2), followed by selective
C(3)-O-silylation (TBSCl, imidazole, 76%) and re-acetylation
of the C(4)-hydroxyl afforded 7 (95%) as an appropriate
glycal donor.
Scheme 1
The feasibility of the sulfonium-mediated oxidative cou-
pling procedure on glucuronate glycal donors was first
established by oxidative glycosylation of 2-propanol with
7, which proceeded with high efficiency (10, Figure 1). In
this reaction, treatment of a solution of glucuronal 7,
(tBu)3pyr (3 equiv), and Ph2SO (3 equiv) in CH2Cl2 with
Tf2O (1.5 equiv) led to complete activation of the glycal
donor at -78 °C. Following the sequential addition of
methanol (1 equiv), triethylamine (3 equiv), 2-propanol (10
equiv), and ZnCl2 (2 equiv), the 2-hydroxy glycoside 10 was
isolated in 85% yield with complete diastereoselectivity. The
formation of 10 is likely a result of the generation of an
intermediate 1,2-anhydroglucuronate ester 9 arising from
ride, these new sulfonium-mediated glycosylation methods
would likely be particularly efficacious in the efficient
construction of these glycosidic bonds. Consequently, the
selectively protected glucuronate glycal 3, the galacto-
pyranose 4, and the xylopyranose 5 would serve as the
principal carbohydrate coupling partners in the strategy
(Scheme 1).
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Org. Lett., Vol. 3, No. 12, 2001

Table 1. Dehydrative Glycosylation of 13
sylation of 13 with 15 in chloroform led to increased
efficiency in the coupling reaction, affording the disaccharide
17 in 96% yield with 1:4 (R:â) selectivity (entry 4). However,
the use of 3,4,6-tri-O-benzyl-2-O-benzoyl-D-galactopyranose
(16) as the donor provided the highest selectivity to afford
the â-disaccharide 19 in 80% yield.
The final steps in the synthesis of 2 involved attachment
of the xylopyranose component via a second dehydrative
coupling. Deprotection of the C(3)-O-TBS ether in 19
(Scheme 3) was effected under mild conditions (HF‚Pyridine,
0 f 23 °C) in order to avoid undesired migration of the
C(4)-acetyl protective group. Dehydrative glycosylation of
Figure 1. Oxidative glycosylation with glycal 7.
oxygen atom transfer from the sulfoxide reagent to the glycal
enol ether functionality.12 It is worth noting that in this
1
reaction, H NMR analysis of the reaction mixture im-
mediately following the addition of MeOH and Et3N allowed
for the detection of the 1,2-anhydropyranoside 9 as the
principle species in solution, which is consistent with the
proposed mechanism of oxygen transfer in this oxidative
glycosylation.13 In addition, the use of other nucleophilic
acceptors such as allyl alcohol, o-nitrobenzyl alcohol, and
p-methoxybenzyl alcohol in this transformation also led to
good yields of the corresponding 2-hydroxyglycosides 11,
12, and 13, all possessing convenient orthogonal protective
groups at the anomeric center. Of these, the p-methoxybenzyl
C(2)-hydroxyglycoside 13 was found to be the most ap-
propriate substrate for the synthesis of the trisaccharide
fragment of 1.
Scheme 3a
Access to the selectively protected methyl 2-hydroxyglu-
curonate 13 allowed for direct attachment of the galactose
fragment via sulfonium-mediated dehydrative glycosylation
with a suitable galactopyranose donor. Glycosylation of
13 with 2,3,4,6-tetra-O-benzyl-D-galactopyranose (14)14 re-
sulted in a high yielding coupling, although the undesired
R-galactopyranoside 16 was formed exclusively (Table 1,
entry 1). In the hopes of capitalizing on neighboring group
participatory effects to produce the â-galacto-linkage, 2,3,4,6-
tetra-O-benzoyl-D-galactopyranose (15)15 was employed as
the donor (entries 2-4). As expected, â-selectivity increased
with this acceptor substrate; moreover, the trend of increased
â-selectivity in the presence of more polar solvents was
observed in this glycosylation reaction. Dehydrative glyco-
(8) (a) Garcia, B. A.; Poole, J. L.; Gin, D. Y. J. Am. Chem. Soc. 1997,
119, 7597-7598. (b) Garcia, B. A.; Gin, D. Y. J. Am. Chem. Soc. 2000,
122, 4269-4279.
(9) Di Bussolo, V.; Kim, Y.-J.; Gin, D. Y. J. Am. Chem. Soc. 1998,
120, 13515-13516.
a (a) HF‚Pyr, THF, pyr, 85%; (b) Ph2SO, Tf2O, (t-Bu)3pyr, 1:3
v/v CH2Cl2/PhMe, 77% (â); (c) CF3CO2H, H2O, CHCl3, >99%.
Org. Lett., Vol. 3, No. 12, 2001
1803

20 with Ph2SO and Tf2O was best performed with 2,3,4-tri-
O-benzoylxylopyranose (21), affording the trisaccharide 22
with complete â-selectivity at the newly formed xylosyl
anomeric bond. Finally, selective removal of the anomeric
p-methoxybenzyl protective group within 22 was accom-
plished with trifluoroacetic acid, affording the selectively
protected trisaccharide donor 23 (77%), a versatile advanced
carbohydrate intermediate for the synthesis of 1.
glycosylation reaction with glucuronal donors, one can
directly access a variety of â-glycosides of 2-hydroxyglu-
curonate esters. This, in combination with the dehydrative
glycosylation protocol, allows for an exceedingly short
synthetic sequence for the preparation of the branched
trisaccharide 23 as a key carbohydrate fragment in the
synthesis of QS-21A.
In summary, the synthesis of the trisaccharide portion of
the immunologic adjuvant QS-21A is reported. By establish-
ing the feasibility of the sulfonium-mediated oxidative
Acknowledgment. This research was supported by the
National Institutes of Health (GM-58833), Glaxo Wellcome,
Inc., and the Alfred P. Sloan Foundation. D.Y.G. is a Cottrell
Scholar of Research Corporation. Y.J.K. thanks Lubrizol and
Pharmacia for predoctoral fellowships.
(10) Recently a few 2-hydroxythioglycosides of methyl glucoronate were
prepared employing dimethyldioxirane as the glycal oxidant. See: Ichikawa,
S.; Shuto, S.; Matsuda, A. J. Am. Chem. Soc. 1999, 121, 10270-10280.
(11) Nishimura, S.-I.; Nomura, S.; Yamada, K. Chem. Commun. 1998,
617-618.
(12) Previous investigations on the sulfonium-mediated oxidative gly-
cosylation using protected glucal donors have established, through 18O-
labeling studies, that the oxygen atom from the excess sulfoxide reagent is
stereoselectively transferred to the C(2)-position of the donor (see ref 9). It
is likely that the transformation of 7 f 10 proceeds in like manner.
(13) Formation of the intermediate anhydropyranoside occurs only after
introduction of the nucleophilic acceptor. We postulate that the first
equivalent of the acceptor serves to generate the anhydropyranoside via
expulsion of diphenylsulfide from an activated C(2)-diphenylsulfonium-
C(1)-oxosulfonium glucoronate intermediate (see ref 9). Following the
addition of the Lewis acid, epoxide opening occurs with the excess acceptor
to afford the 2-hydroxyglycoside product.
Supporting Information Available: Experimental details
and spectral/analytical data for the glycoside products. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL015651U
(14) Bieg, T.; Szeja, W. Carbohydr. Res. 1990, c10-c11.
(15) Fiandor, J.; Garcia-Lopez, M. T.; de las Heras, F. G.; Mendez-
Castrillon, P. P. Synthesis 1985, 1121-1123.
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Org. Lett., Vol. 3, No. 12, 2001