Versatile strategy for the divergent synthesis of linear oligosaccharide domain variants of Quillaja saponin vaccine adjuvants

Article abstract of DOI:10.1039/c5cc05244k

We describe a new, versatile synthetic approach to Quillaja saponin variants based on the natural product immunoadjuvant QS-21. This modular, divergent strategy provides efficient access to linear oligosaccharide domain variants with modified sugars and regiochemistries. This new synthetic approach opens the door to the rapid generation of diverse analogues to identify novel saponin adjuvants with improved synthetic accessibility.

Full text of DOI:10.1039/c5cc05244k

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Versatile strategy for the divergent synthesis of
linear oligosaccharide domain variants of Quillaja
saponin vaccine adjuvants†
Cite this:DOI: 10.1039/c5cc05244k
Received 25th June 2015,
Accepted 27th July 2015
Alberto Fernandez-Tejada,*a Derek S. Tan*ab and David Y. Gin‡ab
´
DOI: 10.1039/c5cc05244k
www.rsc.org/chemcomm
We describe a new, versatile synthetic approach to Quillaja saponin clinical research and, ultimately, large-scale deployment in vaccine
variants based on the natural product immunoadjuvant QS-21. This formulations. Thus, a key remaining challenge is to identify new
modular, divergent strategy provides efficient access to linear saponin variants that can be accessed via streamlined synthetic
oligosaccharide domain variants with modified sugars and regio- routes. This would be facilitated by the development of divergent
chemistries. This new synthetic approach opens the door to the synthetic approaches, in which a wide range of such scalable
rapid generation of diverse analogues to identify novel saponin saponin variants can be generated rapidly.
adjuvants with improved synthetic accessibility.
Herein, we report a new series of linear oligosaccharide domain
variants designed specifically for improved synthetic accessibility:
Adjuvants are critical components of modern subunit vaccines two terminal disaccharide variants, a dirhamnose variant 4 (SQS-1-
that induce enhanced immune responses to the coadminis- 0-10-18) and a lactose variant 5 (SQS-1-0-11-18), the latter synthe-
tered antigens.1 The saponin natural product QS-212 is a E2 : 1 sized in only 16 total steps, and a 2-galactosamine variant 6 (SQS-1-
mixture of apiose and xylose isomers at the terminal sugar3 of 0-12-18), which also features regiochemical modifications around
the linear oligosaccharide domain (Fig. 1). Despite being one of the bridging monosaccharide. To access the latter two saponin
the most potent4 and promising immunoadjuvants under variants efficiently, we developed a new, modular synthetic route
clinical investigation,5 QS-21 has several liabilities including involving initial attachment of the bridging monosaccharide
scarcity2 and heterogeneity6 from the natural source, chemical residue to the triterpene core followed by installation of various
instability,7 dose-limiting toxicity,5c and a poorly understood terminal disaccharides. In contrast to our previous convergent
mechanism of action8 that has impeded the rational design of approach,10–13 this new divergent route provides rapid access to
improved saponin adjuvants. To address these limitations, our diverse variants in the linear oligosaccharide domain of the
group previously completed the total syntheses of QS-21-Api (1) Quillaja saponins.
and QS-21-Xyl (2),9 and developed a semisynthetic approach10
Synthesis of our previously identified lead compound 3
that enabled systematic investigation of structural variations in required 23 total steps with 14 steps in the longest linear
each of the four domains of QS-21. Introduction of amide sequence (LLS).13,14 Notably, synthesis of the linear trisaccharide
linkages into and simplification of the acyl chain domain11 alone required 16 steps. Thus, we designed three new saponin
followed by extensive carbohydrate truncation12,13 provided variants with specific consideration to synthetic efficiency based
chemically stable, simplified saponins incorporating a trisac- on selection of readily-available carbohydrate building blocks. In
charide moiety in the linear oligosaccharide domain12 and dirhamnose variant 4, repetition of the rhamnose residue would
lacking the entire branched trisaccharide, exemplified by lead allow increased synthetic convergence. In lactose variant 5, use of
compound 3 (SQS-1-0-5-18).13 While these previous studies commercially available lactose as the terminal disaccharide
yielded potent, less toxic saponin variants in sufficient quantities would significantly reduce the overall step count. In the regioi-
for preclinical evaluation, larger amounts will be needed for someric 2-galactosamine variant 6, use of the readily available
2-azidogalactose residue would avoid the lengthy synthesis of the
a Chemical Biology Program, Memorial Sloan Kettering Cancer Center,
1 275 York Avenue, New York, NY 10065, USA. E-mail: alberto.fernandezt@gmail.com
b Tri-Institutional Research Program, Memorial Sloan Kettering Cancer Center, 1 275
York Avenue, New York, NY 10065, USA. E-mail: tand@mskcc.org
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5cc05244k
original 4-azidogalactose acceptor (cf. 3), while also changing the
regiochemical relationship of the appended acyl chain domain
and terminal disaccharide.
Dirhamnose variant 4, was prepared using our original
convergent approach, via initial synthesis of the selectively
protected linear trisaccharide 12 from previously described
‡ Deceased March 22, 2011.
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Scheme 1 Synthesis of dirhamnose variant 4. Boc-6-Ahx-OH = N-Boc-
6-aminohexanoic acid; brsm = based on recovered starting material; MS =
molecular sieves; NHS = N-hydroxysuccinimide; PQA = protected quillaic
acid (13); TBP = 2,4,6-tri-tert-butylpyridine.
(17)13 gave dirhamnose variant 4 (SQS-1-0-10-18) in 22 total steps
(14 steps LLS).14
Next, we pursued synthesis of lactose variant 5. In initial
efforts using the original convergent approach, en bloc glycosy-
lation of the triterpene C28-carboxylic acid with a number of
pre-assembled lactosyl-4-azidogalactosyl donors was unsuccess-
ful under both Schmidt (the trisaccharide trichloroacetimidate
was unstable) and Ph2SO/Tf2O dehydrative conditions (the
glycosyl ester was formed with undesired a-selectivity). To over-
come this problem, we envisioned that the trisaccharide could
be installed in a modular fashion involving stepwise, b-selective
monoglycosylation of the triterpene with a 4-azidogalactose
residue and subsequent installation of the terminal disaccharide.
This new approach would also provide more efficient, divergent
access to analogues in the linear oligosaccharide domain than
our original convergent strategy. Thus, Schmidt glycosylation
of 13 with orthogonally protected imidate 18,12 followed by
careful deacetylation (NaOMe, MeOH) to avoid desilylation
of the triterpene, gave the glycosyl ester 19 with complete
Fig. 1 (a) Four structural domains of QS-21. (b) Structures of linear
oligosaccharide domain variants 3–6. (c) Previous convergent strategy
for synthesis of linear oligosaccharide variants 3 and 4. (d) New divergent
strategy for synthesis of linear oligosaccharide variants 5 and 6.
monosaccharide building blocks 7, 8, and 1011,12 using stereo- b-selectivity due to anchimeric assistance by the C2-acetate
selective dehydrative glycosylation (Ph2SO, Tf2O) reactions neighboring group (Scheme 2). We noted that this selectively
(Scheme 1).15 Notably, the rhamnosyl acceptor 8 was accessible protected triterpeneazidogalactose alcohol 19 is a versatile
in only two steps as an intermediate en route to rhamnosyl intermediate for modular diversification with a variety of
donor 7. Trichloroacetimidate donor 12 was then coupled with terminal disaccharides.
protected quillaic acid triterpene 13 ([PQA]-OH)13 under Schmidt
Initial attempts at glycosylation of triterpene–azidogalactose
conditions to afford, after benzene-selenol reduction of the alcohol 19 with a commercially available peracetylated lactosyl
azide,16 the b-glycosyl ester 14. The amine was treated with bromide under Koenigs–Knorr conditions were unproductive,
activated N-Boc-6-aminohexanoic acid (15), then subjected to with significant orthoester formation due to the steric hindrance
global deprotection via hydrogenolysis (H2, Pd/C) and acid hydro- at the azidogalactose C2 position. However, the corresponding
lysis (TFA/H2O) to provide the fully deprotected 6-aminocaproic perbenzoylated lactosyl bromide 20,17 prepared conveniently and
amide 16. Late-stage installation of the aryl iodide by acylation scalably in one step from commercially available lactose (cf. 7-step
with the N-hydroxysuccinimide (NHS) ester of 4-iodobenzoate synthesis for original rhamnose–xylose disaccharide in 3),
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triterpene–trisaccharide conjugate, whose azide group was reduced
(PhSeH) to give amine 26. Acylation with N-Boc-amino acid 15,
global deprotection to 6-aminocaproic amide 27, and final
installation of the aryl iodide moiety as above provided
2-galactosamine variant 6 (SQS-1-0-12-18) in 19 total steps
(13 steps LLS).14
In conclusion, we have demonstrated the efficient synthesis
of a series of novel Quillaja saponin variants and developed a
new, versatile synthetic route that provides modular, late-stage
access to diverse modifications in the linear oligosaccharide
domain of this class. Notably, the original 23-step route to the
lead compound 3 was progressively shortened in dirhamnose
variant 4 (22 steps), 2-galactosamine variant 6 (19 steps) and
lactose variant 5 (16 steps). In addition, this divergent synthesis
overcomes key limitations of our original convergent strategy,11–13
namely the demanding en bloc glycosylation of the triterpene with
the entire trisaccharide donor, which was unsuccessful en route
to lactose variant 5, and the need for pre-assembly of the entire
trisaccharide moiety, which limits rapid exploration of different
sugar residues in this domain. In contrast, this new divergent
approach benefits from efficient glycosylations with simpler mono-
saccharide donors to provide versatile triterpene–monosaccharide
intermediates for diversification with a variety of terminal dis-
accharides. Going forward, the efficiency and versatility of this
divergent route will facilitate the rapid, streamlined preparation of
a wide range of additional variants to identify novel saponin
adjuvants with improved synthetic accessibility and scalability that
will be necessary for future clinical advancement in vaccines.
This work is dedicated to the memory of our mentor and
colleague, Prof. David Y. Gin (1967–2011). We thank J. S. Lewis,
N. Pillarsetty, G. Ragupathi, and S. J. Danishefsky for helpful
discussions, and G. Sukenick, H. Liu, H. Fang, and S. Rusli
(MSKCC Analytical Core Facility) for expert mass spectral analyses.
This research was supported by the European Commission (Marie
Curie International Outgoing Fellowship to A.F.-T.), the U.S. NIH
(R01 AI085622 to D.Y.G. and J. S. Lewis, R01 GM058833 to D.Y.G.
and D.S.T., Cancer Center Support Grant P30 CA008748 to C. B.
Thompson), William and Alice Goodwin and the Commonwealth
Foundation for Cancer Research, and the Experimental
Therapeutics Center of MSKCC.
Scheme 2 Synthesis of lactose variant 5.
afforded the desired triterpene–trisaccharide in excellent yield
using a modified Koenigs–Knorr procedure with AgOTf as a
promoter and 2,4,6-tri-tert-butylpyridine as an acid scavenger.
Reduction of the azide gave amine 21, which underwent acylation
with N-Boc-amino acid 15. Global deprotection via hydrogenolysis
and acid hydrolysis with an additional Zemplen de-O-benzoylation
provided the fully deprotected 6-aminocaproic amide 22. Acylation
with NHS ester 17 yielded lactose variant 5 (SQS-1-0-11-18).
Notably, this high-yielding (Z80% per step) and streamlined
synthetic route required only 16 total steps (13 steps LLS),
compared to the previous 23-step synthesis of lead compound
3 (SQS-1-0-5-18).13,14
Finally, synthesis of the regioisomeric 2-galactosamine variant
6 started with protected 2-azidogalactosyl bromide 23, easily
obtained on multi-gram scale in three steps from commercially
available D-galactal18 (Scheme 3). Coupling of triterpene 13 with
glycosyl bromide 23 under optimized phase transfer conditions
(K2CO3, Bu4NBr, EtOAc, H2O, 45 1C)19 followed by careful deacetyla-
tion of the C6-hydroxyl gave the desired b-C28-galactosyl ester 24.
Glycosylation with disaccharide imidate 25 under TMSOTf catalysis
at À45 1C then proceeded with a-selectivity, providing the desired
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