Synthetic studies of complex immunostimulants from Quillaja saponaria: Synthesis of the potent clinical immunoadjuvant QS-21Aapi

Article abstract of DOI:10.1021/ja062364i

QS-21 is one of the most promising new adjuvants for immune response potentiation and dose-sparing in vaccine therapy given its exceedingly high level of potency and its favorable toxicity profile. Melanoma, breast cancer, small cell lung cancer, prostate cancer, HIV-1, and malaria are among the numerous maladies targeted in more than 80 recent and ongoing vaccine therapy clinical trials involving QS-21 as a critical adjuvant component for immune response augmentation. QS-21 is a natural product immunostimulatory adjuvant, eliciting both T-cell- and antibody-mediated immune responses with microgram doses. Herein is reported the synthesis of QS-21 A_api in a highly modular strategy, applying novel glycosylation methodologies to a convergent construction of the potent saponin immunostimulant. The chemical synthesis of QS-21 offers unique opportunities to probe its mode of biological action through the preparation of otherwise unattainable nonnatural saponin analogues.

Full text of DOI:10.1021/ja062364i

Published on Web 08/22/2006
Synthetic Studies of Complex Immunostimulants from Quillaja
saponaria: Synthesis of the Potent Clinical Immunoadjuvant
QS-21Aapi
Yong-Jae Kim, Pengfei Wang, Mauricio Navarro-Villalobos, Bridget D. Rohde,
JohnMark Derryberry, and David Y. Gin*,†
Contribution from the Department of Chemistry, UniVersity of Illinois, Urbana, Illinois 61801,
and Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer
Center, 1275 York AVenue, New York, New York 10021
Received April 6, 2006; E-mail: gind@mskcc.org
Abstract: QS-21 is one of the most promising new adjuvants for immune response potentiation and dose-
sparing in vaccine therapy given its exceedingly high level of potency and its favorable toxicity profile.
Melanoma, breast cancer, small cell lung cancer, prostate cancer, HIV-1, and malaria are among the
numerous maladies targeted in more than 80 recent and ongoing vaccine therapy clinical trials involving
QS-21 as a critical adjuvant component for immune response augmentation. QS-21 is a natural product
immunostimulatory adjuvant, eliciting both T-cell- and antibody-mediated immune responses with microgram
doses. Herein is reported the synthesis of QS-21Aapi in a highly modular strategy, applying novel glycosylation
methodologies to a convergent construction of the potent saponin immunostimulant. The chemical synthesis
of QS-21 offers unique opportunities to probe its mode of biological action through the preparation of
otherwise unattainable nonnatural saponin analogues.
Introduction
17api/xyl (1 and 2), QS-18api/xyl (3 and 4), and QS-21Aapi/xyl
(5 and 6).3 Of these constituents, both QS-21Aapi (5) and QS-
The development of therapeutic vaccines for the treatment
of diseases has rapidly grown beyond the established strategy
of using lifeless or attenuated microorganisms to include the
use of macromolecules and/or small molecules to elicit immune
response. For example, the use of subunit vaccine antigens
encompasses selected disease-associated glycoproteins, recom-
binant proteins, synthetic peptides, and even nonimmunogenic
complex carbohydrates, which can be rendered immunogenic
when conjugated to an appropriate immunocarrier protein.
However, subunit antigen vaccines are inherently less im-
munogenic than those employing attenuated microorganisms.
Consequently many antigen formulations require co-administra-
tion with an adjuvant or immunostimulating complex (ISCOM),1
a substance that is itself not necessarily immunogenic but
functions in concert with the antigen to enhance/prolong immune
response.
It had been known for decades that semi-purified extracts of
the South American tree, Quillaja saponaria Molina, exhibit
remarkable adjuvant activity.2 In 1991, Kensil et al. reported
the purification, isolation, and partial characterization of selected
minor constituents of the bark extract from Quillaja saponaria.3
Among the more adjuvant-active components of these extracts
are several complex triterpene saponins (Chart 1), including QS-
21Axyl (6) have emerged as being among the most promising
new adjuvants for immune response potentiation and dose-
sparing in vaccine therapy, given their exceedingly high level
of potency and favorable toxicity profile.4 QS-21A (the 21st
fraction from RP-HPLC) was identified through extensive
chemical degradation and spectroscopic studies5 to be a mixture
of two principal isomeric triterpene glycoside saponins, QS-
21Aapi (5) and QS-21Axyl (6), each incorporating a central
quillaic acid triterpene core, flanked on either side by complex
oligosaccharides. The trisaccharide moiety attached to the C3-
position of quillaic acid is composed of D-glucuronic acid,
D-galactose, and D-xylose, while the tetrasaccharide substructure
attached at the C28 carboxylate of the triterpene comprises a
linear array of D-fucose, followed by L-rhamnose and D-xylose
linked to one of two isomeric sugars, D-apiose or D-xylose, for
QS-21Aapi (5) or QS-21Axyl (6), respectively. The remaining
component, a structurally elaborate L-arabinose-terminated fatty
acyl chain attached to the 4-position of the fucose residue,
completes the structural makeup of these amphiphilic triterpene
saponins.
Clinical trials employing QS-21A adjuvant have involved,
inter alia, formulations of ganglioside-keyhole limpet hemocy-
anin (KLH) conjugates for melanoma,6 globo-H oligosaccha-
† Address correspondence to: Memorial Sloan-Kettering Cancer Center.
(1) (a) Barr, I. G.; Mitchell, G. F. Immunol. Cell Biol. 1996, 74, 8. (b) Sjo¨lander,
A.; Cox, J. C. AdV. Drug DeliVery ReV. 1998, 34, 321.
(2) Dalsgaard, K. Arch. Gesamte Virusforsch. 1974, 44, 243.
(3) Kensil, C. R.; Patel, U.; Lennick, M.; Marciani, D. J. Immunol. 1991, 148,
431-437.
(4) Kensil, C. R. Crit. ReV. Ther. Drug Carrier Syst. 1996, 13, 1-55.
(5) (a) Jacobsen, N. E.; Fairbrother, W. J.; Kensil, C. R.; Lim, A.; Wheeler,
D. A.; Powell, M. F. Carbohydr. Res. 1996, 280, 1. (b) Zhu, X.; Yu, B.;
Hui, Y.; Higuchi, R.; Kusano, T.; Miyamoto, T. Tetrahedron Lett. 2000,
41, 717.
9
11906
J. AM. CHEM. SOC. 2006, 128, 11906-11915
10.1021/ja062364i CCC: $33.50 © 2006 American Chemical Society

Synthesis of QS-21
A R T I C L E S
Chart 1
Chart 2
ride-KLH conjugates for prostate7 and breast8 cancers, MUC1
peptide-KLH conjugates for breast cancer,9 and N-propion-
ylated polysialic acid-KLH conjugates for small-cell lung
cancer.10 In addition, several murine and human studies have
demonstrated promising adjuvant effects of QS-21A in vaccine
formulations with recombinant gp120 against HIV-111 and with
synthetic Plasmodium falciparum peptides against malaria.12
Both animal and human studies on the adjuvant activity of QS-
21A indicate that it elicits both Th1- and Th2-type cytokines
and amplifies both the T-cell- and B-cell-mediated immune
responses in microgram doses. Thus, QS-21A is an immuno-
stimulatory adjuvant, possibly functioning to facilitate the uptake
of the antigen into antigen-presenting cells.4
However, QS-21A is essentially nonrenewable in that it is
only present as a minor constituent in the bark of Quillaja
saponaria.3 The development of a chemical synthesis of QS-
21A would not only expand its availability but also offer
opportunities to access otherwise unattainable nonnatural sa-
ponin analogues to probe its mode of biological action. Reported
synthetic efforts directed at QS-21A are sparse, consisting of
the preparation of the fully acetylated trisaccharide and tet-
rasaccharide components.13 Herein is reported a detailed account
of the synthesis of QS-21Aapi (5).14
Results and Discussion
Construction of the triterpene saponin with maximum con-
vergence (Chart 2) relies on the acquisition of the triterpene
(7), the branched trisaccharide (8), the linear tetrasaccharide (10),
and fatty acyl chain (9) as the principal substructure quadrants.
With this strategy, the synthesis of the oligosaccharide portions
of the natural product through various glycosylation methods
appears to be, at least on the surface, a straightforward task in
that all of the glycosidic linkages are of the 1,2-trans config-
uration, and are thereby susceptible to traditional neighboring
group participation effects.15 However, the vast majority of
neighboring group participatory functionalities consist of acyl
protective groups such as esters, which have recently been
shown to be useful in the synthesis of the oligosaccharide
fragments of QS-21.13b Unfortunately, such intermediates are
unlikely to be directly viable in a synthesis of QS-21A without
extensive protective group exchange due to functional group
incompatibilities during late-stage protecting group (i.e., ester)
removal. Indeed, the reported hydrolytic lability of the acyl
(6) Ragupathi, G.; Livingston, P. O.; Hood, C.; Gathuru, J.; Krown, S. E.;
Chapman, P. B.; Wolchok, J. D.; Williams, L. J.; Oldfield, R. C.; Hwu,
W.-J. Clin. Cancer Res. 2003, 9, 5214.
(7) 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. U.S.A. 1999, 96, 5710.
(8) Gilewski, T.; Ragupathi, G.; Bhuta, S.; Williams, L. J.; Musselli, C.; Zhang,
X.-F.; Bencsath, K. P.; Panageas, K. S.; Chin, J.; Hudis, C. A.; Norton, L.;
Houghton, A. N.; Livingston, P. O.; Danishefsky, S. J. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 3270.
(9) Kim, S. K.; Ragupathi, G.; Cappello, S.; Kagan, E.; Livingston, P. O.
Vaccine 2001, 19, 530.
(10) Krug, L. M.; Ragupathi, G.; Ng, K. K.; Hood, C.; Jennings, H. J.; Guo, Z.;
Kris, M. G.; Miller, V.; Pizzo, B.; Tyson, L.; Baez, V.; Livingston P. O.
Clin. Cancer Res. 2004, 10, 916.
(11) (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.
(b) Evans, T. G.; McElrath, M. J.; Matthews, T.; Montefiori, D.; Weinhold,
K.; Wolff, M.; Keefer, M. C.; Kallas, E. G.; Corey, L.; Gorse, G. J.; Belshe,
R.; Graham, B. S.; Spearman, P. W.; Schwartz, D.; Mulligan, M. J.;
Goepfert, P.; Fast, P.; Berman, P.; Powell, M.; Francis, D. Vaccine 2001,
19, 2080.
(13) (a) Kim, Y.-J., Gin, D. Y. Org. Lett. 2001, 3, 1801-1804. (b) Zhu, X.;
Yu, B.; Hui, Y.; Schmidt, R. R. Eur. J. Org. Chem. 2004, 965.
(14) Wang, P.; Kim, Y.-J.; Navarro-Villalobos, M.; Rohde, B. D.; Gin, D. Y. J.
Am. Chem. Soc. 2005, 127, 3256-3257.
(15) Nukada, T.; Berces, A.; Zgierski, M. Z.; Whitfield, D. M. J. Am. Chem.
Soc. 1998, 120, 13291-13295.
(12) Carcaboso, A. M.; Herna´ndez, R. M.; Igartua, M.; Rosas, J. E.; Patarroyo,
M. E.; Pedraz, J. L. Vaccine 2004, 22, 1423.
9
J. AM. CHEM. SOC. VOL. 128, NO. 36, 2006 11907

A R T I C L E S
Scheme 1a
Kim et al.
Scheme 3 a
a Reagents and conditions: (a) MeI, Cs2CO3, DMF (68%).
Scheme 2 a
a Reagents and conditions: (a) Ph2SO, Tf2O, TBP; MeOH, Et3N; ROH,
ZnCl2.
Table 1. a
a Reagents and conditions: (a) NaOMe, MeOH (81%); (b) TBSCl,
imidazole, DMF (76%); (c) Ac2O, DMAP, CH2Cl2 (96%).
chain16 necessitates the selection of differentiated oligosaccha-
ride protective groups that can be removed without disrupting
this sensitive ester functionality in the final stages of the
synthesis.
Synthesis of the Branched Trisaccharide and Its Conjuga-
tion to Quillaic Acid. The quillaic acid subunit (11, Scheme
1) is a 30-carbon terpene of the ∆12-oleanane family. Semi-
purified bark of the Quillaja saponaria tree is commercially
available and has been analyzed to contain 25% of various
terpenes, with the major terpene constituent being the desired
quillaic acid. Quillaic acid has previously been isolated from
the Quillaja bark via acid hydrolysis followed by extraction
and recrystallization.17 Acid-mediated hydrolysis of the oli-
gosaccharide components from the crude commercial Quillaja
bark was performed in refluxing aqueous 1.0 N HCl followed
by continuous extraction with ether, providing gram quantities
of 11, following silica gel chromatography. The C28-carboxyl
group was then selectively protected as its methyl ester 12 (Cs2-
CO3, MeI, 68%) to provide a suitable model glycosyl acceptor
for coupling with a preformed trisaccharide fragment of QS-
21A.
The branched trisaccharide subunit of QS-21A (8, Chart 2)
is composed of a central D-glucuronate residue (GlcUA) and
two peripheral monosaccharides, D-galactopyranose (Gal) and
D-xylopyranose (Xyl). The presence of the C2-O-branched
substructure within the glucuronate fragment of the branched
trisaccharide reveals an opportunity to apply our sulfonium-
mediated oxidative glycosylation procedure18 with a glucuronal
substrate such as 15 (Scheme 2). The synthesis of the glucuronal
15 commenced with glucurono-6,3-lactone (13) according to
the four-step procedure of Nishimura19 providing the methyl
3,4-di-O-acetyl-D-glucuronal 14 in 72% overall yield. Installa-
tion of orthogonal protective groups was then accomplished by
methanolysis of the acetate esters within 14 followed by site-
selective sequential TBS protection of the C3-hydroxyl (TBSCl,
imidazole) and acetylation at the C4-hydroxyl (Ac2O, DMAP)
to provide the glucuronal 15 in 59% yield (three steps).
Access to glucuronal 15 permitted application of our oxidative
glycosylation employing triflic anhydride and diphenyl sulfoxide
entry
donor
solvent
yield (%)
(R:â)
1
2
3
22
23
23
CH2Cl2
PhMe/CH2Cl2 (3:1)
CHCl3
85 (24)
99 (25)
96 (25)
R only
3:2
1:3
a Reagents and conditions: (a) Ph2SO, Tf2O, TBP; add 20.
(Scheme 3).18 Initial model investigations with 2-propanol as a
glycosyl acceptor led to the efficient formation of the C2-
hydroxy-â-glucuronate 17 (85%). Good yields as well as
exclusive â-selectivities were also observed when allyl alcohol,
o-nitrobenzyl alcohol and p-methoxybenzyl alcohol were em-
ployed as acceptors, (i.e., 18-20), each of which could function
as a potentially useful anomeric protective group. It is worth
noting that 1H NMR studies conducted on the oxidative
glycosylation with glucuronal 15 revealed that the R-1,2-
anhydroglucuronate 16 was indeed formed in >90% as the
principal carbohydrate species prior to introduction of the
glycosyl acceptor. Finally, attempts at oxidative glycosylation
of quillaic acid methyl ester (12, HO-QA-Me) were less
successful, leading to a low yield of the GlcUA-O-QA-Me
conjugate 21 (31%), presumably a result of the increased steric
bulk associated with the neopentyl-like structure of this hydroxyl
glycosyl acceptor. As a consequence, the glucuronate 20,
incorporating the anomeric p-methoxybenzyl group, was ad-
vanced in the synthesis of an appropriate oligosaccharide
fragment suitable for glycosylation of the triterpene core.
With the availability of C2-hydroxyglucuronate 20, efforts
were directed toward the synthesis of the galactose â-(1f2)
glucuronate glycosidic linkage (Table 1) within the trisaccharide
subunit of QS-21. Activation of the 2,3,4,6-tetra-O-benzylga-
lactopyranose (22) with Ph2SO and Tf2O (entry 1),20 followed
by addition of glucuronate 20, provided the disaccharide 24
(85%), although with complete selectivity for the undesired
R-anomer. With the aim of effecting anomeric control through
neighboring-group participatory effects, 2,3,4,6-O-tetrabenzoyl-
D-galactopyranose (23) was employed as the donor. This proved
(16) Cleland, J. L.; Kensil, C. R.; Lim, A.; Jacobsen, N. E.; Basa, L.; Spellman,
M.; Wheeler, D. A.; Wu, J.-Y.; Powell, M. F. J. Pharm. Sci. 1996, 85, 22.
(17) Elliot, D. F.; Kon, G. A. R. J. Chem. Soc. 1939, 1130-1135.
(18) (a) Di Bussolo, V.; Kim, Y.-J.; Gin, D. Y. J. Am. Chem. Soc. 1998, 120,
13515-13516. (b) Honda, E.; Gin, D. Y. J. Am. Chem. Soc. 2002, 124,
7343-7352.
(20) (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.
(19) Nishimura, S.-I.; Nomura, S.; Yamada, K. Chem. Commun. 1998, 617.
9
11908 J. AM. CHEM. SOC. VOL. 128, NO. 36, 2006

Synthesis of QS-21
A R T I C L E S
Scheme 4 a
Table 2. a
entry
donor
acceptor
reagent
36 (%)
39 (%)
1
2
3
34
38
38
(Bu3SnO-QA-Me)
12
12
Ph2SO, Tf2O
TBSOTf
BF3‚OEt2
79 (R)
78 (R)
33 (â)
-
-
18 (R)
a Reagents and conditions: (a) CCl3CN, DBU, CH2Cl2 (96%); (b) 12 or
37, “reagents,” CH2Cl2.
respectively, in near quantitative yields. Although the â-ano-
meric selectivity with the use of 2,3,4-tri-O-benzyl-D-xylose (30)
to form 32 was lower than that with 2,3,4-tri-O-benzoyl-D-xylose
(31) to form 33, the isolated yield of the â-anomer of 32 versus
that of 33 were not significantly disparate. Given the likely
difficulties associated with multiple simultaneous saponification
events required for future protective group exchanges of the
ester functionalities in the synthesis, the trisaccharide 32â was
isolated and advanced to 34 for glycosylation of the triterpene
core.
a Reagents and conditions: (a) PhI(OBz)2, BF3‚OEt2, CH2Cl2 (83%); (b)
NH3, MeOH, THF (72%); (c) Ph2SO, Tf2O, TBP; add 20 (80%); (d) HF‚pyr,
THF (99%); (e) 31 or 32, Ph2SO, Tf2O, TBP; then 29; (f) TFA, H2O, CHCl3.
only moderately effective as sulfoxide-mediated dehydrative
glycosylation of 20 with 23 to form 25 (entries 2 and 3), in
either PhMe/CH2Cl2 (3:1) or CHCl3 as solvent, led to 3:2 and
1:3 (R:â) anomeric selectivities, respectively. The lack of high
â-selectivity in these glycosylation reactions is likely due to
competing anchimeric influences from the Lewis basic carbonyl
functionalities of both the C2-R-benzoate (favoring â-anomeric
selectivity) and the C4-â-benzoate (favoring R-anomeric selec-
tivity) within the donor 23.21
However, exclusive â-glycoside formation could be achieved
through a simple protective group modification of the galacto-
pyranose donor, such as 3,4,6-tetra-O-benzyl-2-O-benzoyl-D-
galactopyranose (27, Scheme 4), wherein the C4-hydroxyl is
capped with a nonparticipatory benzyl ether. This donor could
be easily accessed via our recently disclosed hypervalent iodine-
mediated 1,2-bis(acyloxylation)22 of tribenzyl galactal 26 em-
ploying PhI(OBz)2 and BF3‚OEt2 to provide benzoyl 3,4,6-tri-
O-benzyl-2-O-benzoyl-â-D-galactopyranoside. Subsequent depro-
tection of the resulting anomeric benzoate (NH3, MeOH) yielded
the desired galactose hemiacetal 27 in 60% yield over two steps,
whereas conventional methodologies would have required a
protracted multistep sequence to access 27. As expected, use
of the newly synthesized galactose hemiacetal donor 27 in the
glycosylation of the C2-hydroxy glucuronate acceptor 20
afforded exclusively the â-disaccharide 28 (80%).
Completion of the synthesis of the trisaccharide fragment
involved removal of the TBS group within 28 with HF‚pyridine,
unveiling alcohol 29 (99%) with no evidence of C4-acetyl
migration. Glycosylation of disaccharide 29 with 2,3,4-tri-O-
benzyl-D-xylose (30) afforded trisaccharide 32 (92%) with an
anomeric ratio of 1:2 (R:â). In an attempt to increase the
anomeric ratio to favor exclusively the â-xyloside, 2,3,4-tri-O-
benzoyl-D-xylose (31) was investigated as an alternate donor.
Indeed, with 31 and disaccharide acceptor 29, the corresponding
â-anomer of trisaccharide 33 was obtained exclusively (77%).
Subsequent removal of the anomeric PMB acetal in 32 and 33
(TFA) provided the trisaccharide hemiacetals 34 and 35,
Identification of feasible glycosylation conditions for the
coupling of the branched trisaccharide 34 with the triterpene
fragment turned out to be one of the most challenging tasks
during the synthesis of QS-21A. This difficulty arises from
pronounced steric hindrance in forming the glucuronate ano-
meric bond as this is proximal to a sterically demanding array
of both C2- and C3-carbohydrate appendages. Unfortunately,
several attempts at sulfoxide-mediated glycosylations with a
variety of selectively protected trisaccharide hemiacetals with
the triterpene 12 met with limited success. The most efficient
dehydrative glycosylation procedure arose from enhancement
of the nucleophilicity of the triterpene glycosyl acceptor in the
form of its 3-O-stannyl ether (37, entry 1, Table 2)23 to form
36 (79%), albeit with exclusive undesired R-anomeric selectiv-
ity. Moreover, extensive investigations into the use of other
classes of glycosyl donors such as anomeric phosphites,
fluorides, and sulfides were all unproductive.24 However, the
most promising method in this screen of glycosylation protocols
involved the use of the trichloroacetimidate donor, pioneered
by Schmidt.25 Thus, conversion of anomeric trisaccharide
hemiacetal 34 to the R-trichloroacetimidate donor 38 was
accomplished (CCl3CN, DBU) in 95% yield. The use of
TBSOTf (entry 2, Table 2) in the glycosylation of HO-QA-Me
(12) with imidate 38 proceeded with good efficiency to provide
glycoconjugate 36 (78%, comparable to entry 1), although not
surprisingly with the thermodynamically favored undesired
R-stereochemistry. On the other hand, the use of BF3‚OEt2
catalyst (entry 3, Table 2) provided a small but significant
quantity of the desired â-glycoside 36â (33% in the highest
yielding trial). Notably, the unproductive formation of the
glycosyl fluoride derivative 39 (g18%) during the course of
(23) David, S.; Hanessian, S. Tetrahedron 1985, 41, 643.
(24) Toshima, K.; Tatsuta, K. Chem. ReV. 1993, 93, 1503-1531.
(25) Schmidt, R. R.; Kinzy, W. AdV. Carbohydr. Chem. Biochem. 1994, 50,
21.
(21) Zinner, H.; Thielebeule, W. Chem. Ber. 1960, 93, 2791.
(22) Shi, L.; Kim, Y.-J.; Gin, D. Y. J. Am. Chem. Soc. 2001, 123, 6939-6940.
9
J. AM. CHEM. SOC. VOL. 128, NO. 36, 2006 11909

A R T I C L E S
Scheme 5 a
Kim et al.
Scheme 6 a
a Reagents and conditions: (a) TBSCl, imid, DMF (74%); (b) TIPSCl,
AgNO3, imid, DMF (48% â (and 40% R)); (c) K2CO3, MeOH (98%); (d)
allyl-OH, MeCOCl (77%, 3:1, R: â); (e) BnCl, NaH, 95 °C (45%); (f) KO-
t-Bu, DMSO, 100 °C; then HCl, acetone, reflux (71%); (g) Ac2O, Et3N,
CH2Cl2 (84%); (h) HBr, AcOH, CH2Cl2; (i) o-NO2-BnOH, Ag2O, CH2Cl2
(58%, 2 steps); (j) K2CO3, MeOH (90%); (k) Bu2SnO, BnBr, Bu4NBr,
PhCH3 (40%); (l) TBSCl, imid, DMF (96%); (m) Ac2O, DMAP, CH2Cl2
(84%); (n) p-TsOH, MeOH (72%).
a Reagents and conditions: (a) Ph2SO, Tf2O, TBP, CH2Cl2; 43 (55%);
(b) 41, Ph2SO, Tf2O, TBP, CH2Cl2; add 52 (88%); (c) TBAF, THF (98%);
(d) Ph2SO, Tf2O, TBP, CH2Cl2; add 51 (85%); (e) K2CO3, MeOH (91%).
with moderate selectivity (45%),28 and finally allyl acetal
removal (t-BuOK, then HCl) to provide 45 (71%). The initial
route to preparation of the differentially protected fucose moiety
51 (Scheme 5D) involved conversion of D-fucose (46) to tri-
O-acetylfucosyl bromide 47 (Ac2O, Et3N; HBr, AcOH), which
then served to glycosylate o-nitrobenzyl alcohol (49%, two
steps). The esters within the resulting fucoside were then
hydrolyzed (K2CO3, MeOH; 90%) to form triol 48,29 allowing
for monobenzylation of the C3-hydroxyl with moderate selectiv-
ity via its transient stannylene acetal derivative to generate 49.
Subsequent sequential C2-O-TBS protection and C4-O-acetyl-
ation, followed by acid hydrolysis of the TBS ether provided
o-nitrobenzyl 4-O-acetyl-3-O-benzyl-â-D-fucopyranoside (51).
While this initial attempt at securing a suitable differentially
protected D-fucose building block followed a somewhat sub-
optimal protracted sequence, it did serve us well in the
evaluation of initial model studies for the synthesis of a protected
this reaction could not be avoided in our hands, despite extensive
purification and drying of both the coupling substrates and the
acid catalyst. Nevertheless, the procedure in this last entry at
least indicated the feasibility of accessing the desired â-glyco-
conjugate, albeit with suboptimal efficiency. Therefore, probing
appropriate modifications of the trichloroacetimidate glycosyl-
ation protocol should lead to a viable means with which to
secure the trisaccharide-triterpene â-glycosidic linkage in QS-
21 (vide infra).
Synthesis of the Tetrasaccharide Fragment of QS-21Aapi
.
The linear tetrasaccharide component of QS-21A is composed
of the four constituent monosaccharides, L-rhamnose, D-xylose,
D-apiose, and D-fucose. Each of the selectively protected forms
of these monosaccharides was prepared according to standard
carbohydrate functional group interconversion processes (Scheme
5). The selectively protected apiose derivative 41 (Scheme 5A)
was synthesized from 2,3-di-O-isopropylidene-D-apiose (40)26
via selective silylation of the C4′-hydroxyl (74%). Triisopro-
pylsilyl 2,3-di-O-isopropylidene-â-L-rhamnopyranoside (43,
Scheme 5B) was accessed from 4-O-acetyl-2,3-di-O-isopropyl-
idene-â-D-rhamnopyranose (42)27 via anomeric silylation (TIP-
SCl, imidazole) followed by acetate hydrolysis (K2CO3, MeOH).
Preparation of 2,4-di-O-benzyl-D-xylose (45, Scheme 5C) was
achieved via a short sequence involving anomeric acetal
exchange of D-xylose (44) with allyl alcohol (77%), one-pot
dibenzylation of the C2- and C4-hydroxyl groups (BnCl, NaH)
form of QS-21Aapi
.
The assembly of the linear tetrasaccharide fragment of QS-
21Aapi commenced with a novel chemoselective application of
our sulfoxide-mediated dehydrative glycosylation20 in which a
1,3-diol glycosyl donor is employed in the coupling (Scheme
6). In this sequence, 2,4-di-O-benzyl-D-xylopyranose (45) was
activated with excess Ph2SO (5.6 equiv) and Tf2O (2.8 equiv),
followed by the introduction of triisopropylsilyl 2,3-di-O-
isopropylidene-â-L-rhamnopyranose (43) to afford the 1f4-â-
linked disaccharide 52 (55%) as a single constitutional and
(28) Fukase, K.; Hase, S.; Ikenaka, T.; Kusumoto, S. Bull. Chem. Soc. Jpn.
1992, 65, 436.
(29) Nicolaou, K. C.; Hummel, C. W.; Nakada, M.; Shibayama, K.; Pitsinos,
E. N.; Saimoto, H.; Mizuno, Y.; Baldenium, K.-U.; Smith, A. L. J. Am.
Chem. Soc. 1993, 115, 7625-7635.
(26) Ho, P.-T. Can. J. Chem. 1979, 57, 381.
(27) Nguyen, H. M.; Poole, J. L.; Gin, D. Y. Angew. Chem., Int. Ed. 2001, 40,
414.
9
11910 J. AM. CHEM. SOC. VOL. 128, NO. 36, 2006

Synthesis of QS-21
A R T I C L E S
Scheme 7 a
BINAP-RuBr2 catalyst31 at elevated pressure (750 psi) provided
the desired (R)-enantiomer 59 in near quantitative yield and
excellent enantioselectivity (>98:2 er). The resulting alcohol
59 was protected as its TBS ether (>99%), which underwent
ester reduction (DIBAL-H) to afford aldehyde 60 (93%).
Diastereoselective Brown crotylation32 of aldehyde 60 with (Z)-
crotyl(diisopinocamphenyl)borane provided a mixture of the
diastereomers 61 and 62 (83%) in a diastereomeric ratio of 1:2
favoring the desired (S,S,S)-diastereomer 62.33 Despite the poor
diastereoselectivity in this crotylation due to a stereochemical
“mismatch” in this pair of reagent substrates, the major isomer
62 was separated and advanced in the synthesis of the acyl chain.
At this point, it was prudent to verify that the homoallylic
alcohol 62 indeed possessed the resident stereocenters whose
absolute configurations conformed to that of the natural product.
This involved its derivatization to the lactone 63, which had
been previously synthesized and verified as containing the
correct absolute configuration (S,S,S) of the QS-21 acyl chain.
Thus, hydrogenation/hydrogenolysis of 62 (H2, Pd/C) provided
the corresponding saturated diol (73%), which was then
subjected to TPAP oxidation (85%) and TBS removal (51%)
to provide lactone 63. Comparison of data of this compound to
the reported data of the naturally derived lactone revealed
1
identical H NMR resonances and comparable optical rotation
values.5b
With access to the desired diastereomer of the homoallylic
alcohol 62, efforts were directed at glycosylation of its hydroxyl
group with the arabinofuranose 64. Following extensive inves-
tigations, the best result obtained involved sulfoxide-mediated
dehydrative glycosylation (Ph2SO, Tf2O) performed in a 1:1
(v:v) solvent mixture of PhMe and CH2Cl2, providing the desired
R-anomer 65 in 60% yield along with the undesired â-anomer
in 30% yield. Completion of the synthesis of the dimeric acyl
chain was then accomplished with a series of operations
including: (1) simultaneous alkene hydrogenation and benzyl
ether hydrogenolysis (94%); (2) oxidation of the primary alcohol
to the carboxylic acid 66 with RuCl3 and NaIO4 (88%); (3)
esterification of the homoallylic alcohol 62 with the activated
Yamaguchi anhydride34 derivative of 66 (96%); and (4)
sequential hydrogenation/hydrogenolysis of the alkene/benzyl
ether (73%) and alcohol oxidation to provide the carboxylic
acid 67 (96%).
a Reagents and conditions: (a) NaH, THF; n-BuLi; BOMCl (70%); (b)
(R)-BINAP‚RuBr2‚Et3N (cat.), H2, MeOH (>99%); (c) TBSCl, imid, DMF
(>99%); (d) DIBAL-H, PhMe, -78 °C (93%); (e) (+)-(ipc)2B(OMe),
Z-MeCHdCHCH2Li (83%, 1:2, 61:62); (f) H2, Pd/C, MeOH (73%); (g)
TPAP, NMO, CH2Cl2 (85%); (h) TBAF, THF (51%); (i) 64, Ph2SO, Tf2O,
TBP, PhMe/CH2Cl2 (1:1); 62 (90%, 2:1, R: â); (j) H2, Pd/C, MeOH (94%);
(k) RuCl3‚H2O, NaIO4, H2O, MeCN (88%); (l) 2,4,6-Cl3C6H2COCl, Et3N,
DMAP, CH2Cl2; add 62 (96%); (m) H2, Pd/C, MeOH (73%); (n)
RuCl3‚H2O, NaIO4, H2O, MeCN (96%).
stereoisomer.30 The resulting disaccharide 52, with its free
hydroxyl group on the xylose ring, was immediately used as
the nucleophilic glycosyl acceptor in a direct glycosylation with
apiose 41, providing the trisaccharide 53 (88%, R only).
Subsequent selective anomeric desilylation of 53 (TBAF)
furnished the trisaccharide hemiacetal 54 (98%), which served
to glycosylate the C2-hydroxyl of fucose 51 (Ph2SO, Tf2O) to
provide the fully protected linear tetrasaccharide 55 (85%). With
de-acylation of the fucosyl-C4-hydroxyl group in 55, the
tetrasaccharide 56 was produced (91%), ready to be coupled to
the acyl side chain.
Synthesis of the Acyl Chain of QS-21. The asymmetric
synthesis of the dimeric fatty acyl chain of QS-21A began
(Scheme 7) with treatment of commercially available isobutyl-
acetoacetate (57) with sodium hydride followed by n-BuLi to
provide the corresponding dianion, which immediately was
exposed to BOMCl to provide the â-ketoester 58 (70%).
Asymmetric reduction of the ketone in 58 with Noyori’s (R)-
Synthesis of Protected QS-21Aapi. With the availability of
all fully protected quadrants of QS-21Aapi, efforts focused on
their late-stage convergent assembly. The initial task involved
the preparation of the trisaccharide-triterpene substructure
following a modified protocol for trichloroacetimidate glyco-
sylation. It was clear from our earlier model studies for
trichloroacetimidate glycosylation of protected quillaic acid (see
Table 2, entry 2) that the key issue in maximizing the coupling
yield with trisaccharide 38 was to minimize the unwanted
formation of glycosyl fluoride 39. To circumvent this problem
of glycosyl fluoride formation, tris(pentafluorophenyl)borane
(31) Noyori, R.; Ohkuma, T.; Kitamura, M.; Takaya, H.; Sayo, N.; Kumobayashi,
H.; Akutagawa, S. J. Am. Chem. Soc. 1987, 109, 5856-5858.
(32) Brown, H. C.; Bhat, K. S. J. Am. Chem. Soc. 1986, 108, 5919-5931.
(33) (a) White, J. D.; Hong, J.; Robarge, L. A. Tetrahedron Lett. 1999, 40,
1463. (b) Blakemore, P. R.; Browder, C. C.; Hong, J.; Lincoln, C. M.;
Nagornyy, P. A.; Robarge, L. A.; Wardrop, D. J.; White, J. D. J. Org.
Chem. 2005, 70, 5449-5460.
(30) The specific reason for the high â-selectivity in the glycosylation of 43 in
the absence of the C2-participatory group in 45 is unclear, although it is
likely that this outcome is highly substrate dependent.
(34) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull. Chem.
Soc. Jpn. 1979, 52, 1989.
9
J. AM. CHEM. SOC. VOL. 128, NO. 36, 2006 11911

A R T I C L E S
Scheme 8 a
Kim et al.
with the R-trichloroacetimidate 38 employing 3 mol % of tris-
(pentafluorophenyl)borane ((C6F5)3B) as the catalyst. Under
these conditions, the desired glycoconjugate 69 was produced
in 59% (R: â, 1:7) (15% recovered 34) with no evidence of
formation of unwanted glycosyl fluoride that plagued previous
glycosylations employing BF3‚OEt2 as catalyst. Subsequent
exchange of the ester protecting groups, initially required to
secure anomeric selectivity in the preceding coupling reactions,
to groups which are labile to either mild acid or hydrogenolysis
in the final deprotection steps in the synthesis was then
performed. These interconversions include: (1) sequential
treatment of 69 with sodium hydroxide (NaOH) and Cs2CO3
for ester group hydrolysis; (2) protection of the glucuronic acid
carboxylate group as the benzyl ester using benzyl bromide
(BnBr) and KHCO3 (92%, two steps); and (3) removal of the
allyl ester on C28 in the triterpene (70%) to afford triol-acid
70.
In assembling the tetrasaccharide-acyl chain hemisphere of
the immunostimulant (Scheme 9), acylation of the fucose C4-
hydroxyl on the linear tetrasaccharide 56 was effected by way
of the Yamaguchi anhydride derivative of 67 to form 71 (75%).
Exchange of the o-nitrobenzyl acetal to the anomeric trichlo-
roacetimidate was achieved in moderate yield (50%, two steps).
Trichloroacetimidate 71 and the trisaccharide-triterpene con-
jugate 70 were then subjected to BF3‚OEt2 activation, resulting
in the formation of the â-glycosyl ester 72 (72%), which could
be separated from minor side products derived from multiple
glycosylations of the hydroxyl groups in 70. During the initial
stages of planning the synthesis, care was taken to include only
acid- and hydrogenolysis-labile protective groups in the final
stages of the synthesis to facilitate global deprotection without
basic hydrolysis of the noted labile of ester functionality of the
acyl chain. While this particular goal was attained, critical
problematic deprotection issues surfaced with the attempted
acid-mediated removal of the isopropylidene acetal on the apiose
a Reagents and conditions: (a) CCl3CN, DBU, CH2Cl2 (95%); (b)
Cs2CO3, allyl-Br, DMF (70%); (c) (B(C6F5)3), CH2Cl2 (59%, 1:7, R: â; plus
15% 34 and 21% 68); (d) NaOH, 1,4-dioxane; then Cs2CO3, H2O, MeOH;
(e) KHCO3, BnBr, DMF (92%, 2 steps); (e) HCO2H, Pd(OAc)2), Et3N,
PPh3, 1,4-dioxane (70%).
(B(C6F5)3)35 was selected as a potentially useful catalyst in this
context. B(C6F5)3, lacking the reactive B-F bond, has been used
as a catalyst in a variety of transformations, including Mu-
kaiyama aldol,36 Sakurai-Hosomi allylation,37 and epoxide
rearrangement,38 exhibiting a reactivity profile similar to that
of BF3‚OEt2.
Thus, the suitably protected quillaic acid C28-O-allyl deriva-
tive 68 (Scheme 8) was prepared by allylation of the cesium
carboxylate of 11, and 68 was then subjected to glycosylation
Scheme 9 a
a Reagents and conditions: (a) 67, 2,4,6-Cl3C6H2COCl, Et3N, DMAP, CH2Cl2; add 56 (75%); (b) hν (350 nm), THF; (c) CCl3CN, DBU, CH2Cl2 (50%,
2 steps); (d) 70, BF3‚OEt2, CH2Cl2 (72%).
9
11912 J. AM. CHEM. SOC. VOL. 128, NO. 36, 2006

Synthesis of QS-21
A R T I C L E S
Scheme 10 a
Scheme 11a
a Reagents and conditions: (a) allyl-OH, AcCl (88%); (b) Bu2SnO, PhMe;
CsF, BnBr, DMF (67%); (c) TBSCl, imid, DMF (94%); (d) Ac2O, Et3N,
DMAP, CH2Cl2 (99%); (e) Pd(PPh3)4, Et2Zn, THF; (f) TBAF, THF (59%,
2 steps); (g) TIPSCl, imid, DMAP, DMF (49%).
fragment. Despite earlier model studies which suggested
otherwise, this ketal could not be removed without acid
hydrolysis of specific glycosidic linkages within the saponin
construct. Indeed, after extensive experimentation on the depro-
tection of 72, only a trace quantity of QS-21Aapi among a variety
of glycoconjugate fragments could be detected by LC-MS
under various hydrogenolysis-acid hydrolysis conditions.
Preparation of a New Acylated Tetrasaccharide Frag-
ment: Synthesis of QS-21Aapi. The unsuccessful attempts at
global deprotection of 72, coupled with some suboptimal
stereoselective transformations involved in the preparation of
the acyl chain, led us to overhaul the synthesis of the entire
acylated tetrasaccharide fragment of the natural product. One
of the key bottlenecks in acquiring adequate quantities of
forefront synthetic intermediates involved the rather protracted
preparation of the selectively protected D-fucose moiety within
QS-21A (Scheme 5D, vide supra). Attempts to streamline this
process involved a preparation of 75 (Scheme 10) commencing
with the formation of allyl fucopyranoside (88%) from D-fucose
(46). Regioselective benzylation (CsF, BnBr) of the intermediate
allyl fucoside via its stannylene acetal intermediate afforded the
diol 73 (67%), whose remaining equatorial hydroxyl group was
silylated (TBSCl, imidazole) as the TBS ether (94%). Following
acetylation of the axial C4-hydroxyl (99%), anomeric allyl acetal
removal (Et2Zn, P(PPh3)4) and subsequent desilylation (TBAF)
afforded the corresponding 1,2-diol (59%, two steps), which
could be resilylated at the anomeric position to afford the
â-anomeric TIPS fucoside 75 (49% plus 30% 1,2-di-O-TIPS
derivative available for recycling).
a Reagents and conditions: (a) 76, TESOTf, CH2Cl2 (51%); (b) K2CO3,
MeOH; (c) C6H5CH(OMe)2, p-TsOH; (d) TBAF, THF (95%, 3 steps); (e)
Ph2SO, Tf2O, TBP, CH2Cl2; then 75 (54%).
Scheme 12 a
With 75 available in sufficient quantity, preparation of a new
suitably protected tetrasaccharide fragment that would obviate
the problematic late-stage hydrolysis of the apiose-2,3-di-O-
isopropylidene was initiated. Thus, the previously prepared
xylose-rhamnose disaccharide 52 (Scheme 6, vide supra), with
its free hydroxyl group on the xylose ring, was employed
(Scheme 11) as the nucleophilic glycosyl acceptor in a TESOTf-
catalyzed glycosylation39 with acetyl 2,3-di-O-acetyl-5-O-ben-
zylapiofuranose (76) to provide the â-apiose anomer of the fully
protected trisaccharide (51%). Being mindful of the base-
hydrolytic instability of QS-21A, the acetate protecting groups,
which controlled anomeric selectivity in the glycosylation, were
exchanged for the benzylidene acetal (K2CO3, MeOH; then
a Reagents and conditions: (a) (+)-(ipc)2B(OMe), Z-MeCHdCHCH2Li
(89%, >99:1 dr, 98:2 er); (b) BnBr, NaHMDS, THF, DMF (91%); (c)
TBAF, THF (94%); (d) DMSO, (COCl)2, Et3N, CH2Cl2 (95%); (e) 82, LDA,
THF; aldehyde 81, MgBr2, -115 °C, (4:1 dr); (f) NaOMe, MeOH (89%, 2
steps); (g) TBSCl, imid, DMF (82%); (h) H2, 10% Pd/C, MeOH (92%); (i)
64, Ph2SO, Tf2O, TBP, CH2Cl2; then 84 (72%); (j) Ba(OH)2‚8H2O, MeOH
(77%); (k) 2,4,6-C6H2Cl3COCl, Et3N, PhMe; then 84, DMAP (>99%); (l)
Ba(OH)2‚8H2O, MeOH (83%).
C6H5CH(OMe)2, p-TsOH). Subsequent removal of the anomeric
TIPS group (TBAF) on the rhamnose residue afforded the
trisaccharide hemiacetal 77 in 95% over three steps.40 Dehy-
(35) Ishihara, K.; Yamamoto, H. Eur. J. Org. Chem. 1999, 527.
(36) Ishihara, K.; Hanaki, H.; Yamamoto, H. Synlett 1993, 577.
(37) Ishihara, K.; Hanaki, H.; Funahashi, M.; Miyata, M.; Yamamoto, H. Bull.
Chem. Soc. Jpn. 1995, 68, 1721-1730.
(38) Ishihara, K.; Hanaki, H.; Yamamoto, H. Synlett 1995, 721-722.
(39) Roush, W. R.; Bennett, C. E.; Roberts, S. E. J. Org. Chem. 2001, 66, 6389-
6393.
(40) The strategy of using 2,3-di-O-benzylidine-5-O-benzylapiofuranose instead
of 76 as a the glycosyl donor for direct coupling with 52 was less attractive,
given that such a donor required its preparation from 76 in a low-yielding
sequence. Given that the conversion of 52 + 76 f f 77 is nearly
quantitative over three steps, the alternate approach would have been
considerably less efficient.
9
J. AM. CHEM. SOC. VOL. 128, NO. 36, 2006 11913

A R T I C L E S
Scheme 13 a
Kim et al.
a Reagents and conditions: (a) K2CO3, MeOH; (b) 67, 2,4,6-C6H2Cl3COCl, Et3N, PhMe; 85, DMAP (90%, 2 steps); (c) TBAF, THF (81%); (d) CCl3CN,
DBU, CH2Cl2 (56% R, plus 40% recovered 87); (e) NaOH, 1,4-dioxane; then Cs2CO3, H2O, MeOH; (f) KHCO3, BnBr, DMF (92%, 2 steps); (g) TESOTf,
2,6-lutidine, CH2Cl2; (h) HCO2H, Pd(OAc)2, Et3N, PPh3, 1,4-dioxane (81%, 2 steps); (i) BF3‚OEt2, CH2Cl2 (70%); (j) TFA, H2O, CH2Cl2, 0 °C; (k) 150 psi
H2, Pd/C, THF, MeOH (75%, 2 steps).
drative glycosylation (Ph2SO, Tf2O) of triisopropyl 4-O-acetyl-
3-O-benzyl-â-D-fucopyranose (75) with the trisaccharide hemi-
acetal 77 provided the fully protected tetrasaccharide fragment
78 (54%).
ester (4:1 dr), whose chiral auxiliary was removed by metha-
nolysis to provide 83 (89%, two steps). The methyl ester 83
was then subjected to a series of functional group interconver-
sions, including: (1) protection of the â-hydroxyl group as its
TBS ether and (2) reduction with H2 (Pd/C) to effect simulta-
neous benzyl ether hydrogenolysis and alkene hydrogenation,
providing the δ-hydroxy ester 84 in 75% over two steps.
Dehydrative glycosylation (Ph2SO, Tf2O) of the hydroxyl group
in 84 with 2,3,5-tri-O-TBS-L-arabinofuranose (64) afforded the
corresponding R-glycoconjugate (72%), which then provided
the carboxylic acid 66 after saponification of its methyl ester
(77%) with Ba(OH)2‚8H2O. The corresponding carboxylic acid
in 66 was then activated as the mixed 2,4,6-trichlorobenzoyl
anhydride, which engaged in quantitative acylation of the
previously synthesized hydroxy-ester 84. Subsequent hydrolysis
of the methyl ester with Ba(OH)2‚8H2O, taking care to avoid
unwanted hydrolysis of the internal ester group, was ac-
complished to yield the fully intact glycosylated fatty acyl chain
subunit 67 (83%, two steps).
The synthesis of the acyl chain of QS-21A was also
overhauled in light of the “mismatched” diastereoselective
crotylation reaction in our initial synthesis (Scheme 7) of this
fragment. The modified sequence (Scheme 12) began with
asymmetric diastereoselective crotylation32 of 3-O-TBS-propi-
onaldehyde (79) with (+)-(ipc)2B(OMe) and (Z)-MeCHd
CHCH2Li to afford the homoallylic alcohol 80 as a single
diastereomer (89%, >99:1 dr, 98:2 er). Protection of the
hydroxyl group as the benzyl ether, followed by sequential
desilylation (TBAF) and Swern oxidation (DMSO, (COCl)2)
of the resulting primary alcohol provided the â-alkoxy aldehyde
81 in 81% yield over three steps.
Diastereoselective aldol reaction of the aldehyde 81 with the
enolate derived from (R)-2-acetoxy-1,1,2-triphenylethanol (82,
plus 2.4 equiv LDA)41 afforded the corresponding â-hydroxy
The final convergent stages of the synthesis involved initial
(41) Braun, M.; Waldmuller, D. Synthesis 1989, 856.
acetate saponification in tetrasaccharide 79 to form 85, followed
9
11914 J. AM. CHEM. SOC. VOL. 128, NO. 36, 2006

Synthesis of QS-21
A R T I C L E S
by esterification (90%, two steps) of the fucose C4-hydroxyl
in 85 with the mixed 2,4,6-trichlorobenzoyl anhydride of 67,
generated in situ from its treatment with 2,4,6-trichlorobenzoyl
chloride (2,4,6-C6H2Cl3COCl). Removal of the anomeric TIPS
group in 86 (TBAF) provided the hemiacetal 87 (81%), whose
anomeric hydroxyl was converted (CCl3CN, DBU) to the
corresponding R-trichloroacetimidate 88 (56%, plus 40% re-
covered hemiacetal 87) to serve as the glycosyl donor in
triterpenoid glycosylation.
Generation of the corresponding trisaccharide-triterpene
glycosyl acceptor for coupling with 88 also incorporated minor
modifications relative to the earlier synthetic route to 72
(Scheme 9, vide supra). To avoid the potential for multiple
glycosylations onto a partially protected glycosyl acceptor such
as 70 (Scheme 8, vide supra), a fully protected version of the
trisaccharide-triterpene-C28-carboxylic acid (89, Scheme 13)
was prepared from 69, involving the steps of (1) treatment of
69 with sodium hydroxide (NaOH) and Cs2CO3 for ester group
hydrolysis; (2) protection of the glucuronic acid carboxylate
group as the benzyl ester using benzyl bromide (BnBr) and
KHCO3; (3) protection of the remaining hydroxyl groups as
the TES ethers; and (4) removal of the allyl ester on C28 in the
triterpene, all of which proceeded in 75% yield over the four
steps to afford 89.
The final coupling in the synthesis involves the glycosylation
of the trisaccharide-triterpene 70 with 89, employing BF3‚OEt2
catalyst to afford the fully protected QS-21Aapi intermediate 90
(70%). The remaining critical operations to complete the
synthesis relied on the proper early selection of protecting
groups. Thus, mild acid hydrolysis of the rhamnose isopropyl-
idene ketal and all of the silicon (TBS and TES) ethers was
accomplished with a 4:1 (v:v) mixture of trifluoroacetic acid
(TFA) and water, without compromising the glycosidic linkages
or the sensitive ester linkages on the acyl chain of the natural
product. Subsequent hydrogenolysis of all benzylic protecting
groups with H2 and Pd/C occurred efficiently without reduction
of the trisubstituted alkene in the triterpene, providing synthetic
QS-21Aapi (5) in 75% yield.42
Conclusion
With the completion of the first synthesis of QS-21Aapi (5),
the initial reported structure of the adjuvant has been verified,
and availability of this therapeutically important immunostimu-
lant has been expanded to synthetic sources. The potency of
QS-21A and its favorable toxicity profile over a broad spectrum
of vaccine formulations have established it as one of the most
promising new adjuvants for immune response potentiation and
dose-sparing. Although reasonable hypotheses highlight the
potential of QS-21A to influence cell membrane permeability
or to induce cytokine response in local tissue as its means of
immunostimulation,4 the mechanism of action of QS-21A has
yet to be ascertained. The highly modular synthetic approach
to 5 should allow for facile generation of designed structural
analogues for determination of its putative minimal pharma-
cophore requirements as well as investigations into its mech-
anism of immunostimulatory activity.
Acknowledgment. This research was supported by the NIH-
NIGMS (GM58833), the Alfred P. Sloan Foundation, and
Research Corporation. Y.J.K. thanks Lubrizol and Pharmacia
(Pfizer) for predoctoral fellowships. We thank Ms. Michelle
Adams for proofreading of this manuscript.
Supporting Information Available: Experimental details for
the preparation and analytical characterization of synthetic
intermediates. This information is available free of charge via
the Internet at http://pubs.acs.org.
JA062364I
(42) RP-HPLC, 1H NMR, and MS data of synthetic 5 were identical to those of
natural 5, obtained by RP-HPLC of Quil A (Accurate Chemical & Scientific
Corp.), a Quillaja extract known to contain traces of 5.
9
J. AM. CHEM. SOC. VOL. 128, NO. 36, 2006 11915