Synthesis and structure verification of the vaccine adjuvant QS-7-Api. Synthetic access to homogeneous Quillaja saponaria immunostimulants

Article abstract of DOI:10.1021/ja801008m

QS-7-Api is an exceedingly potent immuno-adjuvant isolated from the bark of Quillaja saponaria. It is significantly less toxic than QS-21, a related saponin that is currently the favored adjuvant in anticancer and antiviral vaccine clinical trials. Tedious isolation/purification protocols and uncertainty in its structural constitution have hindered the clinical development of QS-7. A chemical synthesis of QS-7-Api is described, providing structural verification of the adjuvant. A novel semisynthetic sequence to QS-7-Api has also been established, greatly facilitating access to QS-7 for preclinical and clinical evaluation.Copyright

Full text of DOI:10.1021/ja801008m

Published on Web 04/15/2008
Synthesis and Structure Verification of the Vaccine Adjuvant QS-7-Api. Synthetic
Access to Homogeneous Quillaja saponaria Immunostimulants
Kai Deng, Michelle M. Adams, and David Y. Gin*
Memorial Sloan-Kettering Cancer Center, 1275 York AVenue, New York, New York 10065
Received February 8, 2008; E-mail: gind@mskcc.org
The clinical success of conjugate anticancer and antiviral
vaccines critically depends on the identification of, and access to,
novel potent adjuvants with attenuated toxicity. In this context,
specific fractions from extracts of the bark of Quillaja saponaria
(QS) have proven to be exceedingly powerful adjuvants in
immunotherapy. The QS-21 fraction,1–3 comprising isomeric forms
of a complex triterpene saponin, is currently the most promising
immuno-potentiator4 in several antitumor (melanoma, breast,
SCLC, prostate)5 and infectious-disease (HIV, malaria)6–9 vaccine
therapies. However, the tolerated dose of QS-21 in patients typically
does not exceed 100 µg, above which significant local erythema
and systemic flu-like symptoms arise. On the other hand, QS-7,
an additional QS extract fraction, was found not only to possess
Figure 1
significant stand-alone adjuvant activity,1,10 but also to induce
remarkable synergistic immune response augmentation11 when
coadministered with QS-21. Importantly, QS-7, unlike QS-21,
exhibited negligible toxicity in mice.1,10,11 That QS-7 has not been
advanced to the clinic likely stems from (1) a more difficult
purification protocol compared to that of QS-21 and (2) uncertainty
in its structural constitution, postulated to be QS-7-Api (1, Figure
1).10,11 We now report the synthesis and structural verification of
QS-7-Api (1).12 Furthermore, a novel semisynthetic sequence has
been developed, enabling procurement of homogeneous QS-7-Api
(1) for imminent preclinical evaluation.
The synthesis of the hexasaccharide fragment within QS-7-Api
(“R”, Figure 1) required initial preparation of the selectively
protected monosaccharides 2-4, 6, and 8 (Scheme 1). While the
xylo-, gluco-, and apio-derived monosaccharides 2-4 (Scheme 1A)
were obtained in multistep sequences by previously reported
procedures and modifications thereof,13,14 the novel sugars 6 and
8 were prepared from rhamnopyranose 5 and fucopyranoside 7,
respectively. Silylation of the selectively protected rhamnopyranose
5 (Scheme 1B) with TIPSOTf provided the R-TIPS glycoside
(96%), which subsequently underwent C4-O-debenzylation to
furnish the rhamnopyranoside 6 (98%). Synthesis of the fucosyl
residue within QS-7 commenced with selective C3-O-alkylation
of the allyl fucopyranoside 7 (Scheme 1C) with PMBCl (56%)
via its transient stannylene acetal. This allowed for sequential
selective silylation of the equatorial C2-OH (97%) and acetylation
of the axial C4-OH (>99%). Finally, oxidative removal of the PMB
ether with DDQ provided the selectively protected fucopyranoside
8 (86%).
Scheme 1a
a Reagents and conditions: (a) TIPSOTf, 2,6-lutidine, CH2Cl2, 0f23 °C,
96%; (b) H2, Pd-C, MeOH, 23 °C, 98%; (c) n-Bu2SnO, PhMe, reflux; CsF;
PMBCl, DMF, 23 °C, 56%; (d) TBSCl, imidazole, DMAP, CH2Cl2, 23 °C,
97%; (e) Ac2O, Et3N, DMAP, CH2Cl2, 23 °C, >99%; (f) DDQ, MeOH, H2O,
0f23 °C, 86%.
cosylation of rhamnopyranoside 6 with xylopyranose 2 to afford
the ꢀ-disaccharide 11 (75%), which directly underwent glyco-
sylation17 with the apiose-derived donor 4 to afford trisaccharide
12 (86%). The acetate esters in 12 were then exchanged for a
benzylidene acetal protective group (94%), followed by selective
acid hydrolysis of the rhamno-derived isopropylidene ketal to
afford the corresponding vicinal diol (71%). Selective alkylation
of the resulting axial rhamno-C2-OH with BnBr could then be
accomplished (84%), allowing for Schmidt glycosylation18 of
the C3-OH with the glucosyl imidate 3 to afford the tetrasac-
charide 13 (86%). Exchange of the benzoate ester for a TES
ether (91%, two steps) and conversion of the anomeric TIPS
group to its R-trichloroacetimidate counterpart 14 (92%, two
steps) secured a suitable donor for glycosylation of disaccharide
10. This was accomplished by treating the two components with
TMSOTf to afford hexasaccharide 15 (62%), whose fucosyl-
TIPS-acetal was then transformed to the R-trichloroacetimidate
16 (84%, two steps).
Convergent assembly of the branched hexasaccharide (Scheme
2) involved dehydrative glycosylation (Ph2SO•Tf2O)15 of fu-
copyranoside 8 with rhamnopyranose 5 (84%). The resulting
R-disaccharide 9 then underwent a series of protective group
exchanges, including TBS removal (95%), a novel Et2Zn/
Pd(PPh3)4-mediated anomeric deallylation (68%),16 and selective
anomeric silylation (75%) to afford the disaccharide 10 as a
suitable glycosyl acceptor. Its glycosyl donor coupling partner
was prepared by chemo- and stereoselective dehydrative gly-
Late stage construction of the full QS-7-Api skeleton involved
the elaborately protected triterpene-trisaccharide conjugate 17
(Scheme 3A), previously prepared in 18 steps from glucuronolac-
tone during the course of the synthesis of QS-21.13 This C28-
carboxylic acid glycosyl acceptor 17 responded well to glycosy-
lation with trichloroacetimidate glycosyl donor 16 (BF3 ·OEt2) to
9
5860 J. AM. CHEM. SOC. 2008, 130, 5860–5861
10.1021/ja801008m CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2a
The resulting product (71%) was found to be identical to naturally
derived QS-7-Api (1).19
This synthesis of 1 (Scheme 3A) from de novo construction of
all oligosaccharide fragments confirms the structure of QS-7-Api
and provides significantly more dependable access to homogeneous
samples of 1 than isolation from natural sources. This notwith-
standing, the synthesis of 1 can be further augmented. Quil-A (18,
Scheme 3B) is a commercially available semipurified extract from
Quillaja saponaria and contains variable quantities of >50 distinct
saponins,20 many of which incorporate the triterpene-trisaccharide
substructure within QS-7 (and QS-21). This monodesmoside
saponin 19 (Scheme 3B) can be isolated in semipure form via direct
base hydrolysis of the Quil-A mixture.21 Subsequent poly(silylation)
of 19 with excess TESOTf afforded the corresponding nonaki-
s(triethylsilyl ether) (257 mg from 1.15 g of 18), whose glucuronic
acid functionality could be selectively derivatized to the benzyl
ester 20 (CbzCl, 68%). This triterpene-trisaccharide conjugate,
obtained in only a three-step protocol from Quil-A (18), was an
effective acceptor in a C28-carboxylate glycosylation (80%) with
hexasaccharide 16 to provide, after global deprotection, QS-7-Api
(1) (77%). The evolution of the first synthesis of 1 to this
semisynthetic variant furnishes complex QS-saponin adjuvants (and
likely non-natural analogues) with markedly enhanced facility,
enabling heretofore untapped opportunities for novel adjuvant
discovery in antitumor and antiviral vaccine development.
Acknowledgment. This work is dedicated to Prof. E. J. Corey
on the occasion of his 80th birthday. This research was supported
by the NIH (Grant GM58833), Mr. William H. Goodwin, Mrs.
Alice Goodwin and The Commonwealth Foundation for Cancer
Research, and The Experimental Therapeutics Center of MSKCC.
a Reagents and conditions: (a) Ph2SO, Tf2O, TBP, CH2Cl2, -78f23 °C,
84%; (b) TBAF, THF, 0f23 °C, 95%; (c) Et2Zn, Pd(PPh3)4, Et2O, 23 °C;
68%; (d) TIPSCl, imidazole, DMAP, DMF, 23 °C, 75%; (e) Ph2SO, Tf2O,
TBP, CH2Cl2, -78f23 °C, 75%; (f) 4, TBSOTf, CH2Cl2, 0 °C, 86%; (g)
K2CO3, H2O, MeOH, 23 °C; (h) PhCH(OMe)2, p-TsOH, 23 °C, 94% (two
steps); (i) p-TsOH, H2O, MeOH, 23 °C, 71%; (j) BnBr, Bu4NBr, NaOH, H2O,
CH2Cl2, 23 °C, 84%; (k) 3, TMSOTf, Et2O, -45 °C, 86%; (l) DIBAL-H,
CH2Cl2, -78 °C, 92%; (m) TESOTf, 2,6-lutidine, CH2Cl2, 0f23 °C, 99%;
(n) TBAF, THF, 0 °C, >99%; (o) CCl3CN, DBU, CH2Cl2, 0 °C, 92%; (p)
TMSOTf, 4Å ms, CH2Cl2, -15 °C, 62%; (q) TBAF, THF, 0 °C; (r) CCl3CN,
DBU, CH2Cl2, 0f23 °C, 84% (two steps).
Supporting Information Available: Complete ref 7; experimental
details. This material is available free of charge via the Internet at
http://pubs.acs.org.
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a Reagents and conditions: (a) 16, BF3 ·OEt2, 4Å ms, CH2Cl2, -78f23
°C, 71%; (b) TFA, H2O, CH2Cl2, 0 °C; H2, Pd-C, EtOH, THF, 23 °C, 71%;
(c) KOH, EtOH, H2O, 80 °C; (d) TESOTf, Py, 40 °C; (e) CbzCl, Py, TBP,
CH2Cl2, 23 °C, 68%; (f) 16, BF3 ·OEt2, 4Å ms, CH2Cl2, -78f23 °C, 80%;
(g) H2, Pd-C, EtOH, THF, 23 °C; TFA, H2O, 0 °C, 77%.
afford fully protected QS-7-Api (71%), which underwent global
deprotection under carefully managed conditions (TFA; H2, Pd-C).
JA801008M
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