Synthesis of QS-21-xylose: Establishment of the immunopotentiating activity of synthetic QS-21 adjuvant with a melanoma vaccine

Article abstract of DOI:10.1002/anie.200801885

(Chemical Equation Presented) A helping hand: The adjuvant QS-21-xylose (see structure; the xylose residue is shown in red) was obtained in pure form by chemical synthesis. When combined with its synthetic apiose isomer to furnish synthetic QS-21 (sQS-21), the saponin combination was shown to aid the production of antibodies in mice when injected with a melanoma vaccine. The preparation of sQS-21 provides access to pure, functional saponin adjuvants of defined composition.

Full text of DOI:10.1002/anie.200801885

Angewandte
Chemie
DOI: 10.1002/anie.200801885
Natural Products
Synthesis of QS-21-Xylose: Establishment of the Immunopotentiating
Activity of Synthetic QS-21 Adjuvant with a Melanoma Vaccine**
Kai Deng, Michelle M. Adams, Payal Damani, Philip O. Livingston, Govind Ragupathi,* and
David Y. Gin*
The success of antitumor and antiviral vaccines often requires
the use of an adjuvant, a substance that is itself not necessarily
immunogenic but significantly enhances the immune
response of a patient to a coadministered antigen.[1] The
adjuvant of choice in many recent immunotherapeutic
advances against cancer,[2] human immunodeficiency
virus,[3,4] and malaria[5,6] is the natural product saponin QS-
21.[7] Extensive studies in the development of conjugate
cancer vaccines demonstrated the superiority of QS-21 over
18 competing adjuvants in preclinical models.[8]
Isolated in minute quantities from the cortex of the
Quillaja saponaria (QS) tree, QS-21 is not a homogeneous
substance; rather, it is the 21st of 22 fractions in an early
reversed-phase (RP) HPLC trace of semipurified QS-bark
extracts.[7,9] This adjuvant-active fraction comprises at least
two principal constituents (Scheme 1), QS-21-Xyl (1) and QS-
21-Api (2), which differ in the terminal sugar residue within
the linear tetrasaccharide segment.[10,11] Although the multi-
component QS-21 fraction has fueled numerous previous and
ongoing vaccine clinical trials, the challenge to procure
samples of consistent composition from natural sources is
formidable. Indeed, high variability in saponin composition is
seen even among QS trees of similar age and local environ-
ment (e.g., soil, season).[12] Furthermore, recent metabolomic
analyses of semipurified QS-bark extracts revealed a complex
mixture of over 100 distinct saponins,[13] considerably more
than what the original 22-fraction RP HPLC profile might
imply.[7,9] Because of the variability and heterogeneity of QS
extracts, it is difficult to establish the adjuvant activity and
toxicity of the component saponins. The possibility that trace
quantities of additional saponins may be present in the QS-21
fraction impacts not only on efficacy and formulation aspects,
but also on regulatory hurdles in clinical vaccine development
in humans.
Scheme 1. Structures of the principal components of QS-21.
adjuvant-active samples of known composition. The recent
chemical synthesis of the isomer QS-21-Api (2) enabled
independent access to homogenous samples of this particular
saponin.[14] We now report the chemical synthesis of the
second principal isomer, QS-21-Xyl (1), and thereby verify its
structural constitution unambiguously. The synthesis of 1 has
enabled preclinical evaluation of the two synthetically
derived adjuvant isomers 1 and 2 (administered as a 35:65
mixture to mimic the composition of natural QS-21 isolated
from QS bark) by using the GD3-KLH conjugate melanoma
vaccine in mice.
The chemical synthesis of QS-21-Xyl (1) builds on the
foundation established in the synthesis of QS-21-Api (2).[14]
Given the many common structural elements of the two
saponins, several of the advanced synthetic intermediates in
the synthesis of 2 could be applied directly to the synthesis of
1. These intermediates include the selectively protected
trisaccharide–triterpene conjugate 3, the glycosylated pseu-
dodimeric normonoterpene acyl chain 4, and two selectively
protected monosaccharides, the xylopyranose 5 and the
fucopyranoside 6 (Scheme 2).
Efforts to further advance QS-21 in the clinic, as well as to
illuminate its unknown mechanism of action, require access to
[*]Dr. K. Deng, M. M. Adams, P. Damani, Prof. P. O. Livingston,
Dr. G. Ragupathi, Prof. D. Y. Gin
Memorial Sloan-Kettering Cancer Center
New York, NY 10065 (USA)
Fax: (+1)646-422-0416
E-mail: gind@mskcc.org
ragupatg@mskcc.org
[**]This research was supported by the NIH (GM58833), William H.
Goodwin & Alice Goodwin & The Commonwealth Foundation for
Cancer Research, and the Experimental Therapeutics Center of
MSKCC.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200801885.
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Scheme 2. Advanced intermediates prepared during the synthesis of
QS-21-Api (2) and employed in the current synthesis of QS-21-Xyl (1).
Bn=benzyl, TBS=tert-butyldimethylsilyl, TES=triethylsilyl,
TIPS=triisopropylsilyl.
Construction of the linear tetrasaccharide fragment of
QS-21-Xyl (1) commenced with 4-O-benzyl-2,3-isopropyli-
dene-l-rhamnopyranose (7; Scheme 3). The silylation of 7
with TIPSOTf afforded the corresponding a-silyl rhamnoside
(96%), which was subjected to hydrogenolysis of the 4-O-
benzyl ether to provide the rhamnoalcohol 8 (98%). This
alcohol served as a competent glycosyl acceptor in a chemo-
and
stereoselective
dehydrative
glycosylation
(Ph2SO·Tf2O)[15] with the xylopyranose diol 5 to afford the
b-disaccharide 9 (75%) as a single anomer. The remaining
hydroxy group in disaccharide 9 was then glycosylated with
the trichloroacetimidate 11[16] under BF3·OEt2 catalysis to
afford the b-trisaccharide 12 (77%). The final sugar residue of
the tetrasaccharide was introduced by conversion of the
anomeric silylacetal in trisaccharide 12 into its a-trichloroa-
cetimidate 13 in a two-step procedure through exposure first
to TBAF and then to trichloroacetonitrile and DBU (89%,
2 steps). This trisaccharide donor was then activated by
TMSOTf catalysis to effect a glycosylation of the selectively
protected fucopyranoside acceptor 6 and yield the linear
tetrasaccharide 14 (75%). Deacetylation of the oxygen atom
at C4 with DIBAL-H furnished the fully elaborated tetrasac-
charide fragment 15 (83%) for the late-stage convergent
assembly of the saponin target 1.
Scheme 3. Reagents and conditions: a) TIPSOTf, 2,6-lutidine, CH2Cl2,
0!238C, 96%; b) Pd-C, H2, MeOH, 238C, 98%; c) 5, Ph2SO, Tf2O,
TBP, CH2Cl2, ꢀ788C; then 8, ꢀ78!358C, 75%; d) CCl3CN, NaH,
CH2Cl2, 238C, 82% (a/b 4:1); e) BF3·OEt2, CH2Cl2, ꢀ358C, 77%;
f) TBAF, THF, 08C, >99%; g) CCl3CN, DBU, CH2Cl2, 08C, 90%; h) 6,
TMSOTf, Et2O, 08C, 75%; i) DIBAL-H, CH2Cl2, ꢀ788C, 83%.
DBU=1,8-diazabicyclo[5.4.0]undec-7-ene, DIBAL-H=diisobutylalumi-
num hydride, TBAF=tetrabutylammonium fluoride, TBP=tri-tert-butyl-
pyridine, Tf=trifluoromethanesulfonyl, TMS=trimethylsilyl.
functionality within the triterpene moiety. This sequence
resulted in the generation of QS-21-Xyl (1, 64% after RP
HPLC), which was found to be identical to the principal
component of 1 isolated from natural sources.[18]
The attachment of the normonoterpene-derived acyl
chain 4 to the tetrasaccharide 15 proceeded efficiently
under Yamaguchi mixed-anhydride conditions[17] to give 16
(89%). Subsequent desilylation (TBAF, 66%) was followed
by the formation of the a-anomeric trichloroacetimidate 17
(73%; Scheme 4). This complex intermediate served as an
efficient donor for b glycosylation of the triterpene–trisac-
charide conjugate 3 to afford the fully protected saponin
target 18 (80%). The final stage of the synthesis required
global deprotection, which was optimized through extensive
studies, by TFA-mediated hydrolysis of the silyl ethers and
isopropylidene ketal, followed by mild hydrogenolysis with
Pd-C (Degussa) of all benzyl protecting groups. Care was
taken in the final step to avoid reduction of the aldehyde
With QS-21-Xyl (1) and QS-21-Api (2) both accessible by
chemical synthesis, it was now possible to assess the adjuvant
activity of pure synthetic QS-21 devoid of any potential trace
quantities of other natural QS saponins. Adjuvant activity was
evaluated with the melanoma vaccine antigen GD3 conju-
gated to the immunocarrier protein keyhole limpet hemo-
cyanin (KLH; Scheme 5).
Three groups of five C57BL/6J mice (Table 1) received
three sequential vaccinations with GD3-KLH conjugate
(10 mg equivalent of GD3 in GD3-KLH conjugate). As a
negative control, the first group was vaccinated with no
adjuvant; the second group received 10 mg of synthetic QS-21
(sQS-21, a 65:35 mixture of QS-21-Api and QS-21-Xyl). As a
positive control, the third group was administered 100 mg of
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with GD3-KLH alone (without the adjuvant) failed to induce
any antibody response against GD3 (Table 1, column 1 and
Figure 1), and only low titers against KLH were observed
Table 1: Antibody responses in mice (C57BL/6J; female) after subcuta-
neous vaccination with GD3-KLH (10 mg) and various adjuvants.[a]
IgM titer against GD3
sQS-21 GPI-0100
adjuv.[b] (10 mg)[c,d] (100 mg)[e]
IgG titer against KLH
sQS-21 GPI-0100
adjuv.[b] (10 mg)[c,d] (100 mg)[e]
No
No
0
0
0
0
0
320
80
80
320
640
200
400
100
400
400
3200 204800
3200 204800
51200 819200
12800 819200
12800 819200
819200
204800
819200
819200
819200
[a]Serum was collected 7 days after the third injection. Antibody titers
(ELISA) for individual mice are reported. [b]Mouse group 1. [c]Mouse
group 2. [d]A 35:65 mixture of synthetic 1 and synthetic 2 was used.
[e]Mouse group 3.
Scheme 4. Reagents and conditions: a) 4, 2,4,6-C6H2Cl3COCl, Et3N,
PhMe; then 15, DMAP, 238C, 89%; b) TBAF, THF, 08C, 66% (16:
12%); c) CCl3CN, DBU, CH2Cl2, 08C, 73%; d) 3, BF3·OEt2, 4-Š molec-
ular sieves, CH2Cl2, ꢀ78!238C, 80%; e) TFA, H2O, 08C; f) H2, Pd-C,
MeOH, THF, 238C, 64% (2 steps). DMAP=4-dimethylaminopyridine,
TFA=trifluoroacetic acid.
Scheme 5. Structure of GD3-KLH conjugate.
GPI-0100, a semisynthetic adjuvant known to be active with
this antigen system, albeit with an approximately 10-fold
lower potency per dose.[19] Vaccines were administered
subcutaneously to each mouse in weeks 1, 2, and 3. Mice
were bled 7 days after the third vaccination, and the presence
of antibodies was examined by enzyme-linked immunosorb-
ent assays (ELISA) to determine antibody responses against
GD3 and KLH.[20] Serially diluted pre- and post-treatment
sera were added to GD3- and KLH-coated ELISA plates,
which were then washed. Specific antibody detection was
carried out by treatment with goat antimouse immunoglobu-
lin M (IgM) or immunoglobulin G (IgG) conjugated with
alkaline phosphatase, followed by measurement of the
absorbance of the sample at 405nm. The titer is defined as
the highest serum dilution at which an absorbance greater
than 0.1 OD405 higher than that of the preserum was observed.
No detectable anti-GD3 or anti-KLH antibodies were
present in prevaccination sera. As expected, mice immunized
Figure 1. Graphical representation of the results in Table 1. Ab=anti-
body.
(Table 1, column 4). Mice immunized with GD3-KLH con-
jugate plus either sQS-21 (10 mg) or GPI-0100 (100 mg)
produced antibodies against both GD3 and KLH. For mice
immunized with GD3-KLH plus sQS-21, the median anti-
GD3 IgM and anti-KLH IgG titers were 320 and 819200,
respectively (Table 1, columns 2 and 5). These titers were
comparable to the respective median values of 400 and
819200 (Table 1, columns 3 and 6) found with the positive
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Communications
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control GPI-0100. These data confirm the in vivo adjuvant
activity of sQS-21 against GD3-KLH at levels comparable to
that previously reported for GD3-KLH with natural QS-
21.[21,22]
In summary, we have described the chemical synthesis and
structural verification of QS-21-Xyl (1), a key isomeric
constituent of the clinical adjuvant QS-21. Moreover, the
establishment of the preclinical adjuvant activity of synthetic
QS-21 marks the necessary first step in the unambiguous
establishment of the bioactivity of the two principal constit-
uents of the natural product fraction, QS-21-Xyl (1) and QS-
21-Api (2). This study opens the door for the use of synthetic
QS saponins of defined molecular composition in imminent
clinical trials. Indeed, on the basis of these preclinical data, a
protocol for the use of synthetic QS-21 (sQS-21) for a phase-I
clinical trial with a GD3 and GD2 bivalent melanoma vaccine
has been approved by an institutional review board (IRB).[23]
Received: April 22, 2008
Published online: July 15, 2008
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Chem. Soc. Jpn. 1979, 52, 1989 – 1993.
[18] Natural QS-21-Xyl (1) was purified from commercial Quil A by
RP HPLC to approximately 70% purity.
[19] D. J. Marciani, J. B. Press, R. C. Reynolds, A. K. Pathak, V.
Pathak, L. E. Gundy, J. T. Farmer, M. S. Koratich, R. D. May,
Vaccine 2000, 18, 3141 – 3151.
Keywords: adjuvants · glycosylation · natural products ·
total synthesis · vaccines
.
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