Synthesis of the potent immunostimulatory adjuvant QS-21A

Article abstract of DOI:10.1021/ja0422007

QS-21A 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 clinical trials involving QS-21A as a critical component for immune response augmentation in microgram doses. Herein is reported the first synthesis and structure verification of QS-21A_api, applying novel glycosylation methodologies in the convergent modular construction of this rare and potent natural product immunostimulant. Copyright

Full text of DOI:10.1021/ja0422007

Published on Web 02/18/2005
Synthesis of the Potent Immunostimulatory Adjuvant QS-21A
Pengfei Wang, Yong-Jae Kim, Mauricio Navarro-Villalobos, Bridget D. Rohde, and David Y. Gin*
Department of Chemistry, UniVersity of Illinois, Urbana, Illinois 61801
Received December 27, 2004; E-mail: gin@scs.uiuc.edu
Scheme 1a
It has been known for decades that semi-purified extracts from
the bark of the South American tree, Quillaja saponaria Molina,
exhibit remarkable immunoadjuvant activity. The most active
components of these extracts, designated QS-21A, were identified1
to be a mixture of two principal isomeric triterpene glycoside
saponins, QS-21Aapi (1) and QS-21Axyl (2), each incorporating a
quillaic acid triterpene core, flanked on either side by complex
oligosaccharides and a stereochemically rich glycosylated fatty acyl
chain.2 The potency of QS-21A and its favorable toxicity profile
in more than 80 recent and ongoing vaccine clinical trials3
(melanoma, breast cancer, small cell lung cancer, prostate cancer,
HIV-1, malaria) have established it as one of the most promising
new adjuvants for immune response potentiation and dose-sparing.
We report the first synthesis and unambiguous structural determi-
nation4 of QS-21Aapi (1).5
a Reagents and conditions: (a) (Z)-butene, nBuLi, KO-t-Bu, -78 °C to
-45 °C to -78 °C, (+)-Ipc2BOMe, BF3‚OEt2, -78 °C, then 3, -78 °C to
23 °C, NaOH, 23 °C, 89%, >99:1 dr, 98:2 er; (b) BnBr, NaHMDS, 0 °C
to 23 °C, 91%; (c) TBAF, 23 °C, 94%; (d) DMSO, (COCl)2, Et3N, -78 °C
to -45 °C, 95%; (e) 5, LDA, -78 °C to 0 °C, aldehyde 4, MgBr2, -115
°C, 4:1 dr; (f) NaOMe, 23 °C, 89% (two steps); (g) TBSCl, imidazole, 23
°C, 98%; (h) H2, 10% Pd/C, 23 °C, 92%; (i) 2,3,5-tri-O-TBS-L-arabino-
furanose, Ph2SO, Tf2O, -78 °C to -45 °C, then 7, -78 °C to 23 °C, 72%;
(j) Ba(OH)2‚8H2O, 23 °C, 77%; (k) 2,4,6-C6H2Cl3COCl, Et3N, 23 °C, then
7, DMAP, >99%; (l) Ba(OH)2‚8H2O, 23 °C, 83%.
The preparation of the linear tetrasaccharide fragment of QS-
21Aapi (Scheme 2) employs a novel chemoselective application of
our dehydrative glycosylation8 in which a 1,3-diol glycosyl donor
is used. 2,4-Di-O-benzyl-D-xylopyranose (10)11 is activated with
excess Ph2SO and Tf2O, followed by the introduction of triisopro-
pylsilyl 2,3-di-O-isopropylidene-â-L-rhamnopyranose (11)11 to af-
ford the 1f4-â-linked disaccharide 14 (66%) as a single anomer.
The resulting disaccharide 14 is immediately used as the glycosyl
acceptor in a TESOTf-catalyzed glycosylation12 with acetyl 2,3-
Enantioselective synthesis of the fatty acyl chain within QS-
21A (Scheme 1) begins with the conversion of 3-(O-TBS)-
propionaldehyde (3) to the â-benzyloxyaldehyde 4 involving the
sequential operations of: (1) asymmetric diastereoselective croty-
lation6 (89%, >99:1 dr, 98:2 er), (2) benzylation of the resulting
hydroxyl group, (3) removal of the primary TBS ether, and (4)
Swern oxidation of the resulting primary alcohol (81%, three steps).
Diastereoselective aldol reaction of the aldehyde 4 with the enolate
derived from (R)-2-acetoxy-1,1,2-triphenylethanol (5)7 affords the
corresponding â-hydroxy ester (89%, 4:1 dr), which then undergoes
ester methanolysis and TBS protection of the â-hydroxy group to
form 6. Simultaneous benzyl ether hydrogenolysis and alkene
hydrogenation provides the δ-hydroxy ester 7 (80%, four steps from
4). Dehydrative glycosylation8 of 7 with 2,3,5-tri-O-TBS-L-ara-
binofuranose9 (Ph2SO, Tf2O) proceeds to afford the R-glycocon-
jugate (72%), which then provides the carboxylic acid 8 after ester
saponification with Ba(OH)2‚8H2O (77%). The carboxylic acid 8
is then activated as its mixed 2,4,6-trichlorobenzoyl anhydride10
which then engages in quantitative acylation of the δ-hydroxyl
group in the previously synthesized hydroxy-ester intermediate 7.
Subsequent base-mediated hydrolysis of the methyl ester with Ba-
(OH)2‚8H2O is then accomplished to yield 9 (83%, two steps).
di-O-acetyl-5-O-benzyl-D-apiofuranose (12)13 to provide the trisac-
charide 15 (51%). The acetate protecting groups are exchanged for
the benzylidene acetal, and subsequent removal of the anomeric
TIPS group on the rhamnose residue affords the trisaccharide
hemiacetal 16 (95%, three steps). Dehydrative glycosylation8 of
triisopropyl 4-O-acetyl-3-O-benzyl-â-D-fucopyranose (13)11 with the
trisaccharide 16 provides the fully protected tetrasaccharide 17
(54%). Site-selective installation of the glycosylated acyl chain is
then effected by sequential acetate methanolysis in 17 followed by
esterification with the mixed 2,4,6-trichlorobenzoyl anhydride10 of
9 (90%). Removal of the anomeric TIPS group in 18 with TBAF
provides 19 (81%), whose hemiacetal group is then converted to
its R-trichloroacetimidate 20 (56%, plus 40% recovered 19).
Construction of the triterpene-trisaccharide fragment (Scheme
3) commences with quillaic acid (24), isolated from commercially
available mixtures of natural Quillaja sapogenins.14 Allylation of
the carboxylate in 24 provides the triterpene 25 (70%), which is
glycosylated with the branched trisaccharide fragment 225a in one
of the more challenging couplings in the synthesis. Following the
screening of several glycosylation methods to stereoselectively
couple derivatives of 22 with 25, one promising protocol involved
the coupling of anomeric R-trichloroacetimidate 23, derived from
9
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J. AM. CHEM. SOC. 2005, 127, 3256-3257
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C O M M U N I C A T I O N S
Scheme 2a
the glycosyl fluoride 21 also ensued (18%). However, this problem
is avoided by using (C6F5)3B (3 mol %)16 as a glycosylation catalyst
to afford the glycoconjugate 26 (59%, R: â 1:7, plus 15% of 22
and 21% of 25) with no evidence of unwanted glycosyl fluoride
21.
Being mindful of the hydrolytic instability of the fatty acyl chain
in QS-21A,1 exchange of the ester protecting groups in 26 to
alternate groups that would be readily removable in the projected
final steps of the synthesis include: (1) base-mediated ester group
hydrolysis, (2) benzylation of the glucuronic acid carboxylate group,
(3) protection of the remaining hydroxyl groups as the TES ethers,
and (4) removal of the allyl ester in the triterpene (75%, four steps)
to provide 27. The final convergent step involves the glycosylation
of 27 with the acylated tetrasaccharide 20 (BF3‚OEt2)15 to afford
fully protected QS-21Aapi 28 (70%). Finally, mild acid hydrolysis
of the isopropylidene ketal and of the silicon ethers is accomplished
with TFA/H2O (4:1 v:v), without compromising the glycosidic
linkages nor the ester linkages on the acyl chain. Subsequent
hydrogenolysis of all benzylic protecting groups (H2, Pd/C) occurs
efficiently without reduction of the trisubstituted alkene, providing
synthetic QS-21Aapi (1, 75%).17
With the completion of the first synthesis of QS-21Aapi (1), its
structure has been verified, and availability of this powerful clinical
immunostimulant has been expanded to synthetic sources. Genera-
tion of analogues of 1 is underway to probe its mechanism of
immunostimulatory activity, which has yet to be ascertained.1b
a Reagents and conditions: (a) 10, Ph2SO, Tf2O, -78 °C, then 11, -78
°C to 23 °C, 66%; (b) 12, TESOTf, 0 °C, 51%; (c) K2CO3, 23 °C; (d)
C6H5CH(OMe)2, pTsOH, 23 °C; (e) TBAF, 23 °C, 95% (three steps); (f)
Ph2SO, Tf2O,-78 °C, then 13, -78 °C to 23 °C, 54%; (g) K2CO3, 40 °C,
>99%; (h) 9, 2,4,6-C6H2Cl3COCl, Et3N, 23 °C, then 17, DMAP, 90%; (i)
TBAF, 0 °C, 81%; (j) CCl3CN, DBU, 0 °C, 56% (40% recovered 19).
Acknowledgment. This research was supported by the NIH
(GM58833).
Scheme 3a
Supporting Information Available: Complete refs 3a,b; experi-
mental procedures and spectroscopic data for synthetic intermediates.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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a Reagents and conditions: (a) CCl3CN, DBU, 0 °C, 95%; (b) Cs2CO3,
allylBr, 0 °C, 70%; (c) (B(C6F5)3), 23 °C, 59%, (R: â 1:7), (plus 15% 22
and 21% 25); (d) NaOH, 23 °C, then Cs2CO3, H2O, 58 °C; (e) KHCO3,
BnBr, 23 °C, 92% (two steps); (f) TESOTf, 2,6-lutidine, 23 °C; (g) HCO2H,
Pd(OAc)2), Et3N, PPh3, 23 °C, 81% (two steps); (h) BF3‚OEt2, 20, -78
°C, 70%; (i) TFA, H2O, 0 °C; (j) 150 psi H2, Pd/C, 23 °C, 75% (two steps).
22 (95%), with 25 and BF3‚OEt2 catalysis.15 While some of the
â-glycoconjugate 26 is formed (33%), competitive formation of
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