Synthesis and preclinical evaluation of QS-21 variants leading to simplified vaccine adjuvants and mechanistic probes

Article abstract of DOI:10.1021/ja305121q

QS-21 is a potent immunostimulatory saponin that is currently under clinical investigation as an adjuvant in various vaccines to treat infectious diseases, cancers, and cognitive disorders. Herein, we report the design, synthesis, and preclinical evaluation of simplified QS-21 congeners to define key structural features that are critical for adjuvant activity. Truncation of the linear tetrasaccharide domain revealed that a trisaccharide variant is equipotent to QS-21, while the corresponding disaccharide and monosaccharide congeners are more toxic and less potent, respectively. Modification of the acyl chain domain in the trisaccharide series revealed that a terminal carboxylic acid is well-tolerated while a terminal amine results in reduced adjuvant activity. Acylation of the terminal amine can, in some cases, restore adjuvant activity and enables the synthesis of fluorescently labeled QS-21 variants. Cellular studies with these probes revealed that, contrary to conventional wisdom, the most highly adjuvant active of these fluorescently labeled saponins does not simply associate with the plasma membrane, but rather is internalized by dendritic cells.

Full text of DOI:10.1021/ja305121q

Article
pubs.acs.org/JACS
Synthesis and Preclinical Evaluation of QS-21 Variants Leading to
Simplified Vaccine Adjuvants and Mechanistic Probes
Eric K. Chea,† Alberto Fernandez-Tejada,‡ Payal Damani,§ Michelle M. Adams,‡ Jeffrey R. Gardner,‡
́
Philip O. Livingston,*,§ Govind Ragupathi,*,§ and David Y. Gin†,‡,¶
†Pharmacology Program, Weill Cornell Graduate School of Medical Sciences, ‡Molecular Pharmacology and Chemistry Program, and
§Melanoma and Sarcoma Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York,
New York 10065, United States
S
* Supporting Information
ABSTRACT: QS-21 is a potent immunostimulatory saponin
that is currently under clinical investigation as an adjuvant in
various vaccines to treat infectious diseases, cancers, and
cognitive disorders. Herein, we report the design, synthesis,
and preclinical evaluation of simplified QS-21 congeners to
define key structural features that are critical for adjuvant activity.
Truncation of the linear tetrasaccharide domain revealed that a
trisaccharide variant is equipotent to QS-21, while the
corresponding disaccharide and monosaccharide congeners are
more toxic and less potent, respectively. Modification of the acyl
chain domain in the trisaccharide series revealed that a terminal carboxylic acid is well-tolerated while a terminal amine results in
reduced adjuvant activity. Acylation of the terminal amine can, in some cases, restore adjuvant activity and enables the synthesis
of fluorescently labeled QS-21 variants. Cellular studies with these probes revealed that, contrary to conventional wisdom, the
most highly adjuvant active of these fluorescently labeled saponins does not simply associate with the plasma membrane, but
rather is internalized by dendritic cells.
combination of Alum and monophosphoryl lipid A)13 for the
Cervarix vaccine to immunize against human papillomavirus
(HPV). Thus, there remains a great need for novel adjuvants
for use in vaccine therapy.
INTRODUCTION
■
There is an ever increasing need to develop effective vaccines to
combat acute and chronic infections and diseases, in both
therapeutic and prophylactic settings. The traditional use of
live, attenuated pathogens as immunogens in vaccines has
Currently, one of the most promising adjuvants undergoing
clinical investigation is QS-21, a natural product extract
proven successful in addressing a host of maladies, yet there are
obtained from the bark of the Quillaja saponaria Molina tree,
many pathogens and diseases for which this strategy is
ineffective. As a consequence, many contemporary approaches
to vaccine development employ recombinant or synthetic
subunit vaccines, which offer improved safety and more precise
targeting. However, they are often characterized by poor
immunogenicity. As such, subunit antigens must be coadminis-
found in the desert regions of Chile, Bolivia, and Southern
Peru.30 This semipurified extract consists of a mixture of
triterpene saponins (Figure 1), whose principal constituents are
characterized by complex bis(desmosides) 1 (QS-21-apiose)
and 2 (QS-21-xylose).31,32 These immunoactive constituents of
tered with an adjuvant to enhance the immune response.1−8
QS-21 contain four distinct structural domains: the central
triterpene (quillaic acid), a branched trisaccharide at the C3
The discovery of novel adjuvants has emerged as a critical
frontline effort in the development of modern vaccine
formulations. The adjuvant component enables dose-sparing
of rare, expensive, and otherwise impotent antigens. An
position of the triterpene, a linear tetrasaccharide attached at
C28 of the triterpene, and a branched acyl chain linked to the
central fucose moiety of the linear tetrasaccharide.
The importance of QS-21 as an investigational adjuvant is
adjuvant also extends the immunotherapeutic benefits to poor
evident in its use in more than 100 clinical trials involving
responders, such as elderly or immunocompromised patients.
>6000 total patients.33 QS-21 has exhibited a remarkable ability
While numerous classes of adjuvants have been explored for
to augment clinically significant responses to vaccine antigens
targeting a wide landscape of diseases and degenerative
disorders (e.g., cancer, Alzheimer’s disease, malaria, HIV,
hepatitis). Despite these promising indications, further
vaccine therapies over the past several decades, Alum, a mixture
of aluminum salts (hydroxide, phosphate, sulfate),9−12 is still
the most popular and is present in approximately half of all
human vaccines in the U.S. First introduced for human use in
the 1930s, Alum was the only approved adjuvant in man for
more than 70 years. It was not until late 2009 that the FDA
approved GlaxoSmithKline’s AS04 adjuvant (a proprietary
Received: May 25, 2012
Published: August 6, 2012
© 2012 American Chemical Society
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Figure 1. Structure of the saponin adjuvant QS-21 and four key structural domains.
Figure 2. Semisynthetic approach to QS-21 analogues 5−7 with variations in the acyl chain domain (green).
commercial advancement of QS-21 is hampered by low
isolation yields, inconsistent composition, and lack of purity.
Moreover, the instability of QS-21, characterized by sponta-
neous hydrolysis of the acyl domain, limits its efficacy, enhances
its local reactogenicity, and complicates formulation and
storage protocols. Finally, these hurdles in advancing QS-21
are further exacerbated by the fact that the mechanism by which
it potentiates the immune response is unknown. In the absence
of insights on this front, there exists no rational path forward to
design novel saponin adjuvants with improved vaccine efficacy.
Our previous synthetic studies on QS-21 have permitted
access to each specific isomeric saponin within the
mixture,14−17 thereby obviating the problems associated with
inconsistent purity and composition. While the initial synthetic
route was rather lengthy, a significantly streamlined semi-
synthetic route was subsequently developed.18 This involved
isolation of the entire Quillaja prosapogenin half of the
adjuvant ([QPS]−OH, 3, Figure 2) from the commercial
semipurified Q. saponaria extract Quil-A, followed by
derivatization into a selectively protected form (protected
Quillaja prosapogenin, [PQPS]−OH, 4). Integration of this
technology into the synthesis of novel saponins offers a
convenient means by which to append distinct linear
tetrasaccharide domain and acyl chain domain variants for
evaluation.
acyl chains. This initial study revealed that significant structural
modification of the central fucosyl pyranose and of the acyl
chain of QS-21 causes little or no impairment of adjuvant
activity. Moreover, differing toxicity profiles were observed.
Although these initial findings highlighted the exciting
prospect of designing improved saponin adjuvants, these
three compounds offered a rather narrow insight into the
structure−activity profile of these adjuvants. For example, while
all three synthetic Quillaja saponins were competent
immunostimulators, the complex acyl chain in 5 required an
excessively long synthesis, the simplified acyl chain in 6
imparted unacceptable levels of toxicity, and the nonglycosy-
lated acyl chain in 7 limited its aqueous solubility.
Herein, we report efforts that have defined specific
substructures of the saponin adjuvant that are critical for
adjuvant activity. Multiple variations of both the acyl chain and
linear tetrasaccharide domains have been explored systemati-
cally, leading to the development of highly simplified structures
that show excellent immunostimulatory properties with
encouraging toxicity profiles. In addition, these efforts have
led to the development of a synthetic Quillaja saponin scaffold
that is amenable to attachment of fluorescent reporter groups
while still retaining adjuvant activity. The development of this
novel class of biochemical tools allows, for the first time, direct
investigations of QS-21-derived molecular probes to unravel the
cellular mechanisms of saponin immunostimulation.
Our initial work in this area involved the preparation and
evaluation of three non-natural analogues comprising acyl chain
domain variants 5−7 (Figure 2).19 These syntheses involved
modification of the fucose moiety within the linear
tetrasaccharide domain of the saponin to reduce hydrolytic
instability, allowing the attachment of three distinct lipophilic
RESULTS AND DISCUSSION
■
Synthesis and Evaluation of Initial Acyl Chain Domain
Variants. The nonoptimal chemical and biological properties
associated with the first three acyl chain domain variants 5−7
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Scheme 1. Synthesis of Acyl Chain Domain Variants 9 and
a
10
a
Reagents and conditions: (a) 5α-cholestan-3β-ol (11), COCl2,
PhMe; add to 8, 2,4,6-tri-t-butylpyridine, CH2Cl2, 21 °C, 58%; (b)
H2 (50 psi), Pd/C (Degussa), THF, EtOH, 21 °C; TFA, H2O, 0 °C,
58%; (c) HO2C(CH2)10CO2Bn (12), EtOCOCl, Et3N, THF, 0 °C;
add 8, 0 °C, 60%; (d) H2 (50 psi), Pd/C (Degussa), THF, EtOH, 21
°C; TFA/H2O (3:1 v/v), 0 °C, RP-HPLC, 89%.
Figure 3. Preclinical evaluation of cholestanyl saponin 9 and
carboxyacyl saponin 10 in a two-component vaccine containing both
GD3−KLH and MUC1−KLH conjugates. Each adjuvant, plus a no-
adjuvant negative control, was evaluated by vaccination of a group of
five mice (C57BL/6J, female). Vaccinations were carried out by
weekly subcutaneous injection of GD3−KLH (5 μg), MUC1−KLH
(5.0 μg), and various saponin adjuvants (10 μg) for 3 weeks (days 0, 7,
and 14), followed by a booster at day 65. Postboost serological data at
day 72 are presented; *missing data points are from mice that did not
exhibit an antibody response to the antigen in question. Median values
of titers obtained are represented as a red horizontal bar. (a) Anti-KLH
titers (IgG), (b) Anti-MUC1 titers (IgG), and (c) Anti-GD3 titers
(IgM). (d) Toxicity assessment was done by tracking median weight
loss over the course of a week after the first vaccine injection.
prompted us to investigate two additional variants (Scheme 1).
The cholestanyl variant 9 was designed based on the known
propensity of QS-21 to associate with cholesterol,34 which has
led to hypotheses that saponin binding to membrane-associated
cholesterol may be a key enabling feature in its putative
mechanism of action.35 Indeed, the immunostimulatory
complex ISCOMATRIX20−22 comprises a proprietary mixture
of QS-21, cholesterol, and various phospholipids, and is CSL
Behring’s favored adjuvant product in clinical trials with
vaccines against influenza, hepatitis C virus (HCV), HPV,
and cancer.22 The carboxyacyl variant 10 was designed to
overcome the water solubility issues associated with the simple
dodecanoyl variant 7 through incorporation of a charged
carboxylate moiety to increase polarity of the otherwise
lipophilic acyl chain.
Governing criteria in both of these acyl chain domain
variants are simplicity and highly efficient synthesis. In this
regard, 5α-cholestan-3β-ol (11) is commercially available, and
dodecanedioic acid monobenzyl ester (12) is readily prepared
in one step from its commercially available diacid precursor.23
The preparation of these analogues began with the protected
Quillaja triterpene bis(desmoside) 8 (Scheme 1), previously
synthesized as a late stage intermediate in the preparation of the
first-generation analogues 5−7.19 Its amino functionality was
acylated with 5α-cholestan-3β-yl chloroformate, generated in
situ by treatment of 5α-cholestan-3β-ol with phosgene. The
resulting cholestanyl adduct was then exposed to global
deprotection conditions, via hydrogenolysis and acid hydrolysis,
to afford the cholestanyl saponin 9 (SQS-0-0-4-6). In a similar
manner, amine 8 was treated with the acyl chloride derivative of
dodecanedioic acid monobenzyl ester to afford the correspond-
ing fully protected saponin. Deprotection via hydrogenolysis
and acid hydrolysis afforded the carboxyacyl saponin 10 (SQS-
0-0-4-5).
Evaluation of these novel saponins for immunostimulatory
activity involved vaccination of mice with a multiple antigen
formulation that included both GD3−KLH and MUC1−KLH
conjugates. These constructs represent clinically relevant
oncology vaccine antigens (GD3: melanoma, sarcoma;
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Scheme 3. Synthesis of Disaccharide Donor 25 and
a
Monosaccharide Donor 26
a
Reagents and conditions: (a) Ph2SO, Tf2O, TBP, CH2Cl2, −78 °C →
21 °C, 82%; (b) TBAF, AcOH, THF, 0 °C → 21 °C, 88%; (c)
CCl3CN, DBU, CH2Cl2, 0 °C, 94%; (d) Ac2O, py, 21 °C, 97%; (e)
TBAF, AcOH, THF, 0 °C → 21 °C, 90%; (f) CCl3CN, DBU, CH2Cl2,
0 °C, 89%.
antigens were coadministered with the adjuvant of interest in a
vaccination protocol involving three subcutaneous injections at
days 0, 7, and 14, plus a booster at day 65. As the negative
control, mice were vaccinated with the GD3−KLH and
MUC1−KLH antigens without adjvant. As a positive control,
vaccinations were performed with naturally derived QS-21
(NQS-21), obtained by fractionating a mixture of saponins
from Q. saponaria,24 and also with synthetic QS-21 as its
reconstituted 65:35 mixture of apiose and xylose isomers (SQS-
21). Antibodies against each antigen in mice sera were detected
by ELISA (Figure 3a−c, see Supporting Information for
experimental details). As a standard initial assessment of overall
toxicity, the weight loss of the mice was monitored at 0, 24, 48,
and 72 h after the first vaccination (Figure 3d).
Figure 4. Truncated linear tetrasaccharide domain variants.
a
Scheme 2. Synthesis of Trisaccharide Donor 22
The resulting antibody titers indicate that both acyl chain
domain variants, cholestanyl saponin 9 (SQS-0-0-4-6) and
carboxyacyl saponin 10 (SQS-0-0-4-5), induce anti-GD3 titers
comparable to those of NQS-21 and SQS-21 (Figure 3c);
however, stark differences were observed in comparisons of
antibody levels generated against the MUC1 peptide and KLH
protein antigens (Figure 3a,b). Cholestanyl saponin 9 was
essentially inactive as an adjuvant in mice vaccinated with either
of these antigens. In contrast, mice vaccinated in the presence
of carboxyacyl saponin 10 exhibited anti-MUC1 and anti-KLH
titers comparable to those of both positive controls (NQS-21,
SQS-21). Interestingly, both synthetic saponins 9 and 10
exhibited noticeably lower toxicity, as judged by weight loss, in
comparison to NQS-21 and SQS-21 (Figure 3d). On the basis
of its superior immunostimulatory performance, carboxyacyl
saponin 10 was selected as a lead for further structural
modifications.
a
Reagents and conditions: (a) Ph2SO, Tf2O, TBP, CH2Cl2, −60 °C →
0 °C, 71%; (b) Et2NH, Pd(OAc)2, PPh3, CH2Cl2, MeOH, 21 °C,
>99% (9:1, α:β); (c) Ph2SO, Tf2O, TBP, CH2Cl2, −72 °C → 0 °C,
74%; (d) TBAF, AcOH, THF, 0 °C; (d) Cl3CCN, DBU, CH2Cl2, 3
°C → 21 °C, 82% (2 steps).
Synthesis and Evaluation of Truncated Linear
Tetrasaccharide Domain Variants. Having identified in
carboxyacyl saponin 10 an acyl chain domain that offers both
ease of synthesis and potent immunostimulating effects, we
next focused our efforts on modification of the linear
tetrasaccharide domain of QS-21. Although the synthetic
route for the preparation of the linear tetrasaccharide in 10 is
highly streamlined, the synthesis of each of the selectively
protected monosaccharide building blocks requires 3−7 steps,
with preparation and installation of the terminal apiose sugar
being particularly lengthy. Thus, we prepared systematically
truncated versions of this linear tetrasaccharide in analogues
MUC1: prostate, breast), conjugated to the immunological
carrier KLH, and serve as useful models for comparing adjuvant
performance. Monitoring antibody responses to both the
antigen and the carrier protein provides a useful assessment
of adjuvant performance in antigens of diverse immunogenicity,
ranging from a poorly immunogenic glycolipid (GD3), to a
moderately immunogenic nonglycosylated peptide (MUC1),
and to a highly immunogenic protein (KLH). Groups of five
mice (C57BL/6J, female, 6−8 weeks of age) were vaccinated
with GD3−KLH and MUC1−KLH at a 5 μg antigen dose. The
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a
Scheme 4. Synthesis of Truncated Linear Tetrasaccharide Domain Variants 13−15
a
Reagents and conditions: (a) BF3·OEt2, 4 Å M.S., CH2Cl2, −40 °C → 21 °C, 73%; (b) PhSeH, Et3N, 40 °C, 87%; (c) HO2C(CH2)10CO2Bn (12),
EtOCOCl, Et3N, THF, add to 28, 0 °C, 75%; (d) H2 (50 psi), Pd/C (Degussa), THF, EtOH, 21 °C; (e) TFA, H2O, 0 °C, RP-HPLC, 92% (2
steps); (f) BF3·OEt2, 4 Å M.S., CH2Cl2, −78 °C → 21 °C, 41%; (g) PhSeH, Et3N, 36 °C, 92%; (h) HO2C(CH2)10CO2Bn (12), EtOCOCl, Et3N,
THF, add to 30, 0 °C, 81%; (i) H2 (50 psi), Pd/C (Degussa), THF, EtOH, 21 °C; (j) TFA, H2O, 0 °C, RP-HPLC, 70% (2 steps); (k) BF3·OEt2, 4 Å
M.S., CH2Cl2, −78 °C → 21 °C, 45%; (l) PhSeH, Et3N, 36 °C, 90%; (m) HO2C(CH2)10CO2Bn (12), EtOCOCl, Et3N, THF, add to 32, 0 °C, 72%;
(n) H2 (50 psi), Pd/C (Degussa), THF, EtOH, 21 °C; (o) TFA, H2O, 0 °C; NaOMe, MeOH, H2O, 0 °C, RP-HPLC, 71% (3 steps).
13−15 (SQS-0-0-5-5, SQS-0-0-6-5, and SQS-0-0-9-5) (Figure
4).
pyranoside 20, involving C2-O-acetylation, carefully monitored
desilylation to avoid unwanted acyl transfer, and trichloroace-
timidate formation.
We first sought to assemble the selectively protected linear
trisaccharide 22 (Scheme 2), in which the terminal apiose sugar
is omitted. As shown, commencing with allyl 2,3-di-O-
isopropylidinerhamnopyranoside (17), the C4-hydroxyl group
was glycosylated with tri-O-benzylxylopyranose (16) under
dehydrative coupling conditions with Ph2SO and Tf2O.36,37
The condensation proceeded with β-anomeric selectivity to
afford the disaccharide 18 in 71% yield. Selective removal of the
allyl acetal in 18 proceeded in quantitative yield to provide
disaccharide 19, which served as a competent glycosyl donor in
a second dehydrative glycosylation with the previously prepared
4-deoxy-4-azidogalactopyranoside acceptor 20,19 to furnish
trisaccharide 21 with complete α-selectivity. Subsequent
conversion of the silyl acetal in 21 to a latent leaving group
was performed in a two-step procedure, involving desilylation
followed by treatment with CCl3CN in the presence of DBU to
give the α-trichloroacetimidate 22.
The two other truncated carbohydrate fragments, 25 and 26,
were prepared in an analogous fashion from similar substrates
(Scheme 3). Thus, the C2-hydroxyl group of the 4-deoxy-4-
azidogalactopyranoside acceptor 20 underwent dehydrative
glycosylation with 4-O-benzyl-2,3-di-O-isopropylidenerhamno-
pyranoside (23) to afford disaccharide 24, allowing for
subsequent conversion of its anomeric functionality from the
TIPS acetal to the trichloroacetimidate 25. Finally, preparation
of monosaccharide 26 was accomplished through straightfor-
ward functional group modifications of 4-deoxy-4-azidogalacto-
The protected carbohydrate donors 22, 25, and 26 were then
advanced to saponin analogues 13, 14, and 15, respectively,
having truncated linear tetrasaccharide domains (Scheme 4).
These late stage assembly steps are exemplified by the synthesis
of the saponin 13 (Scheme 4a), in which the previously
prepared selectively protected Quillaja prosapogenin 4
([PQPS]−OH)18 is glycosylated at its C28-carboxylic acid by
the trisaccharide 22 under Schmidt coupling conditions to
afford the glycosyl ester 27 with complete β-anomeric
selectivity. Reduction of the azide 27 to its corresponding
amine 28 was accomplished with PhSeH under basic
conditions. On the basis of the promising activity of the
carboxyacyl saponin 10 above (Figure 3), the amino group of
28 was also acylated with dodecanedioic acid monobenzyl ester
12 to install the acyl chain domain. Subsequent global
deprotection via hydrogenolysis and acid hydrolysis provided
the carboxyacyl trisaccharide 13 as the single-sugar truncation
analogue of the immunoactive carboxyacyl tetrasaccharide
saponin 10. Following similar synthetic protocols, the
progressively carbohydrate-truncated saponins carboxyacyl
disaccharide 14 (Scheme 4b) and carboxyacyl monosaccharide
15 (Scheme 4c) were prepared from their corresponding
protected saccharides 25 and 26, respectively.
Evaluation of these three truncated saponin analogues for
immunoadjuvant activity involved vaccination of mice with a
three-component vaccine that contained the GD3−KLH and
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a
Scheme 5. Synthesis of Acyl Chain Domain Variants 33−35
a
Reagents and conditions: (a) Boc2O, CH2Cl2, 21 °C; (b) H2 (50 psi),
Pd/C (Degussa), THF, EtOH, 21 °C; (c) TFA, H2O, 0 °C, RP-
HPLC, 59% (3 steps); (d) HO2C(CH2)5NHBoc (36), EtOCOCl,
Et3N, THF, add to 28, 0 °C; (e) H2 (50 psi), Pd/C (Degussa), THF,
EtOH, 21 °C; (f) TFA, H2O, 0 °C, RP-HPLC, 63% (3 steps); (g)
FITC (37), Et3N, DMF, 21 °C, RP-HPLC, 75%.
Figure 5. Preclinical evaluation of carboxyacyl trisaccharide saponin
13, carboxyacyl disaccharide 14, and carboxyacyl monosaccharide 15
with GD3−KLH, MUC1−KLH, and OVA three-component vaccine.
Each adjuvant, plus a no-adjuvant negative control, was evaluated by
vaccination of a group of five mice (C57BL/6J, female). Vaccinations
were carried out by weekly subcutaneous injection of GD3−KLH (5
μg), MUC1−KLH (2.5 μg), OVA (20 μg), and various saponin
adjuvants (20 μg) for 3 weeks (days 0, 7, and 14), followed by a
booster at day 65. Postboost serological data at day 72 are presented;
*missing data points are from mice that did not exhibit an antibody
response to the antigen in question; **missing data points are from
dead mice. Median values of titers obtained are represented as a red
horizontal bar. (a) Anti-KLH titers (IgG), (b) anti-OVA titers (IgG),
(c) anti-MUC1 titers (IgG), and (d) anti-GD3 titers (IgM). (e)
Toxicity assessment was done by tracking median weight loss over the
course of a week after the first vaccine injection.
after vaccination with disaccharide 14, while only one mouse
died when treated with trisaccharide 13 or monosaccharide 15.
In addition, while monosaccharide 15 was the least toxic among
the saponins tested, as assessed by weight loss, it was also the
least efficacious. Thus, taking mortality, toxicity, and efficacy
into consideration, the carboxyacyl trisaccharide saponin 13
was deemed superior to the disaccharide 14 and the
monosaccharide 15. Overall, trisaccharide 13 also exhibits
comparable efficacy to SQS-21, but is more readily accessible
(26 vs 76 synthetic steps for QS-21-apiose (1)).
Synthesis and Evaluation of Functionalized Acyl
Chain Domain Variants. The above results reveal the
importance of incorporating at least the trisaccharide moiety
of the linear tetrasaccharide domain to maintain optimal
adjuvant activity. They also present an attractive path forward
for accessing additional simplified adjuvants, given that the
significant efforts required for the synthesis and installation of
the selectively protected apiose sugar can be circumvented.
Thus, the simplified amino trisaccharide intermediate 28 was
subsequently used as the workhorse scaffold for the
investigation of an additional property of the acyl chain
domain, ionic charge. Initial studies with the 12-carboxy-
dodecanoyl chain in both carboxyacyl tetrasaccharide 10 and
carboxyacyl trisaccharide 13 had signaled that adjuvant activity
can be maintained with the introduction of a carboxylate anion
(at physiological pH).
MUC1−KLH conjugates as well as ovalbumin (OVA), the
main protein in egg white, which is known to be a reliable
immunogen that induces both antibody and T-cell responses in
immunized mice. The resulting antibody responses to GD3
were similar in the mice vaccinated with carboxyacyl
trisaccharide 13, carboxyacyl disaccharide 14, carboxyacyl
monosaccharide 15, and the positive controls, NQS-21 and
SQS-21 (Figure 5d). However, antibody titers against KLH,
OVA, and MUC1 peptide decreased with each subsequent
saccharide truncation (Figure 5a−c). Although trisaccharide 13
induces greater weight loss (Figure 5d), two of five mice died
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5). Acylation of the amino group in 28 with (Boc)2O provided
the corresponding carbamate. The fully protected intermediate
was then subjected to the standard hydrogenolysis/hydrolysis
deprotection protocol to provide amino trisaccharide 33 (SQS-
0-0-5-10), which lacks the lipophilic acyl chain domain entirely.
Preparation of a second amine-containing variant involved
acylation of the amino group in 28 with 6-((t-butoxycarbonyl)-
amino)hexanoic acid (36) followed by hydrogenolyis/hydrol-
ysis deprotection, providing the extended lipophilic aminoacyl
trisaccharide 34 (SQS-0-0-5-11).
We recognized that the amine-containing saponin 34 also
opened the door to late-stage, chemoselective elaboration with
other moieties, such as fluorescent labels that might be useful in
studying saponin subcellular localization. Along these lines,
treatment of aminoacyl trisaccharide 34 with fluorescein
isothiocyanate (FITC) (37) afforded the fluoresceinyl amino-
acyl trisaccharide 35 (SQS-0-0-5-12).
Immunological evaluation of this new group of saponins was
accomplished through vaccination of mice with the MUC1−
KLH conjugate. Antibody responses to KLH and MUC1 in
mice vaccinated with carboxyacyl trisaccharide saponin 13
(SQS-0-0-5-5) were again comparable to both NQS-21 and
SQS-21 (Figure 6a,b). In contrast, mice vaccinated with either
amino trisaccharide 33 (SQS-0-0-5-10) or aminoacyl trisac-
charide 34 (SQS-0-0-5-11) generated antibody titers that were
similar to no-adjuvant treatment negative control. This suggests
that the positive charge imparted by the ammonium group in
the acyl chain domain attenuates saponin adjuvant activity. The
toxicity (weight loss) of the amine-containing saponins 33 and
34 was also decreased compared to NQS-21, SQS-21, and
carboxyacyl saponin 13 (Figure 6c).
Notably, upon masking the free amine of 34 by acylation
with FITC, efficacy was restored in the resulting fluoresceinyl
aminoacyl trisaccharide 35 (SQS-0-0-5-12) to levels compara-
ble to both positive controls NQS-21 and SQS-21 (Figure
6a,b). Importantly, in addition to this potent adjuvant activity,
35 exhibited lower toxicity (weight loss) compared to NQS-21,
SQS-21, and carboxyacyl saponin 13 (Figure 6c). To the best of
our knowledge, while other fluorescently labeled saponins have
Figure 6. Preclinical evaluation of carboxyacyl trisaccharide saponin
13, amino trisaccharide saponin 33, aminoacyl trisaccharide saponin
34, and fluoresceinyl aminoacyl trisaccharide saponin 35 with MUC1−
KLH vaccine. Each adjuvant, plus a no-adjuvant negative control, was
evaluated by vaccination of a group of five mice (C57BL/6J, female).
Vaccinations were carried out by weekly subcutaneous injection of
MUC1−KLH (2.5 μg) and various saponin adjuvants (10 μg) for 3
weeks (days 0, 7, and 14), followed by a booster at day 65. Postboost
serological data at day 72 are presented. Median values of titers
obtained are represented as a red horizontal bar. (a) Anti-KLH titers
(IgG) and (b) anti-MUC1 titers (IgG). (c) Toxicity assessment was
done by tracking median weight loss over the course of a week after
the first vaccine injection.
To explore whether a cationic moiety would impart the same
beneficial properties, two amine-containing saponins 33 and 34
were prepared from selectively protected precursor 28 (Scheme
a
Scheme 6. Synthesis of Fluorescently Labeled Variants 38 and 39
a
Reagents and conditions: (a) 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one, Et3N, DMF, triethylammonium 2-(2-(3,6,8-trisulfonato-
pyren-1-yloxy)acetamido)acetate (40), 21 °C, RP-HPLC, 36%; (b) BODIPY 581/591 C3 succinimidyl ester (41), Et3N, DMF, 21 °C, RP-HPLC,
28%.
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Figure 8. Subcellular localization of fluorescently labeled saponins 35,
38, and 39 in immature dendritic cells. Immature dendritic cells were
treated with (a) 1 μM fluoresceinlabeled saponin 35 (SQS-0-0-5-12),
(b) 1 μM BODIPY-labeled saponin 39 (SQS-0-0-5-17), or (c) 1 μM
Cascade Blue-labeled saponin 38 (SQS-0-0-5-15) for 1 h then
visualized by confocal microscopy. As controls, immature dendritic
cells were treated with glycine methyl ester labeled with each
corresponding fluorophore (d−f).
C3 succinimidyl ester (41) afforded BODIPY-labeled saponin
39 (SQS-0-0-5-17).
Preclinical evaluation of these fluorescently labeled saponins
revealed that, although median antibody titers to KLH from
mice treated with Cascade Blue-labeled saponin 38 were
comparable to SQS-21, antibody responses to both OVA and
MUC1 were attenuated (Figure 7a−c). Since Cascade Blue
imparts polyanionic character to the saponin, the attenuation in
antibody response to OVA and MUC1 suggests that saponin
adjuvant activity is highly sensitive to charge. In mice treated
with BODIPY-labeled saponin 39, antibody titers generated to
KLH, OVA, and MUC1 were all lower compared to NQS-21
and SQS-21. Although introducing either of these two
fluorophores attenuates adjuvant activity, both fluorescently
labeled saponins were also less toxic compared to the positive
controls (Figure 7d).
Subcellular Localization of Fluorescently Labeled
Saponins. Currently, there are only indirect means to
investigate the mechanism of action of immunopotentiating
saponins. Theories for the saponin mode of action include
perturbation of cell membrane components to increase immune
cell maturation as well as increased delivery of antigens into
cells.38,39 Although there is limited knowledge on which cell
types are involved in saponin-mediated immunostimulation, we
elected to investigate dendritic cells, as there is mounting
evidence of their importance in adjuvant activity across a panel
of various adjuvants, including MF59,27 α-galactosylceramide,28
and MPL-A.29
Figure 7. Preclinical evaluation of Cascade Blue-labeled saponin 38
and BODIPY-labeled saponin 39 with OVA and MUC1−KLH two-
component vaccine. Each adjuvant, plus a no-adjuvant negative
control, was evaluated by vaccination of a group of five mice (C57BL/
6J, female). Vaccinations took the form of weekly subcutaneous
injection of OVA (20 μg), MUC1-KLH (2.5 μg), and various saponin
adjuvants (20 μg) for 3 weeks (days 0, 7, and 14), followed by a
booster at day 65. Postboost serological data at day 72 are presented.
Median values of titers obtained are represented as a red horizontal
bar. (a) Anti-KLH titers (IgG), (b) anti-MUC1 titers (IgG), and (c)
anti-GD3 titers (IgM). (d) Toxicity assessment was done by tracking
median weight loss over the course of a week after the first vaccine
injection.
been reported,25 the fluorescein-labeled saponin 35 represents
the first example of a fluorescent saponin with confirmed
adjuvant activity.
Synthesis and Evaluation of Fluorescently Labeled
Saponin Probes. Building upon the promising adjuvant
activity and toxicity profile of the fluorescein-labeled saponin 35
above, we next investigated two additional fluorescently labeled
saponins (Scheme 6) to provide probes with alternative
excitation and emission wavelengths and increased stability at
acidic pH that may be encountered in the intracellular
environment. Chemoselective acylation of the aminoacyl
trisaccharide scaffold 34 with Cascade Blue derivative 4026
afforded the corresponding Cascade Blue-labeled saponin 38
(SQS-0-0-5-15). Analogous acylation with BODIPY 581/591
Immature dendritic cells were treated with the fluorescently
labeled saponins 35, 38, and 39, and imaged via confocal
microscopy. Interestingly, the immunopotentiating fluorescein-
labeled saponin 35 (SQS-0-0-5-12) (Figure 8a) localized to a
discrete structure within the cell, distinguishing itself from the
other two probes 38 (SQS-0-0-15) and 39 (SQS-0-0-5-17),
which are poor immunopotentiators. BODIPY-labeled saponin
39 (Figure 8b) localized to the plasma membrane, while
Cascade Blue-labeled saponin 38 (Figure 8c) did not associate
with dendritic cells at all. These findings suggest that, in order
for a saponin to be an efficacious adjuvant, it is necessary for it
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to be internalized into dendritic cells. This mechanism is shared
by Alum, wherein internalization results in activation of the
NALP-3 inflammasome.11 The low efficacy observed in
Cascade Blue-labeled saponin 38 may result from its inability
to engage the cell membrane due to the repulsion between its
three sulfonate anions with the phospholipid anions that adorn
the plasma membrane. The importance of the saponin scaffold
in the trafficking of these fluorescently labeled saponins within
dendritic cells was established by comparison to the trafficking
of the corresponding fluorescently labeled glycine methyl esters
(Figure 8d−f; see Supporting Information for synthesis and
characterization).
To determine whether the fluorescein-labeled saponin 35
remains intact intracellularly, peripheral blood mononuclear
cells were treated with 35 and the cellular lysate was analyzed
by RP-HPLC. A major peak (tr = 18.1 min) corresponding to
35 was observed (Supporting Information Figure S1),
supporting the supposition that the immunoactive fluores-
cein-labeled saponin 35 survives as an intact molecule within
the cells.
ASSOCIATED CONTENT
* Supporting Information
Experimental details and analytical data for isolable synthetic
intermediates. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
livings43@gmail.com; ragupatg@mskcc.org.
■
Notes
The authors declare the following competing financial
interest(s): J. R. G., P. O. L., G. R., and D. Y. G. are founders
of and have financial interests in Adjuvance Technologies, Inc.
¶Deceased March 22, 2011.
ACKNOWLEDGMENTS
■
This work is dedicated to the memory of our friend, mentor,
and colleague, Prof. David Y. Gin. This work was supported by
the NIH (R01 GM058833, R01 AI085622), and Experimental
Therapeutics Center of MSKCC. We thank Prof. Samuel J.
Danishefsky (MSKCC) and Prof. Derek S. Tan (MSKCC) for
helpful discussions and assistance with the preparation of the
manuscript, and Dr. George Sukenick, Dr. Hui Liu, Hui Fang,
and Dr. Sylvi Rusli (MSKCC Analytical Core Facility) for
expert mass spectral analyses. A.F.-T. thanks the Ministerio de
CONCLUSION
■
The synthesis and preclinical evaluation of the tailored QS-21
derived saponins reported herein have defined key structural
features that are critical for adjuvant activity and have enabled
the development of the first generation of chemical probes to
study saponin mechanisms of action. Through systematic
truncation of the linear tetrasaccharide domain, it was
discovered that an optimal balance of synthetic accessibility,
toxicity, and efficacy is achieved with the corresponding
trisaccharide variant 13 (SQS-0-0-5-5) (Figure 4). As a result,
synthetic challenges associated with the 40-step synthesis of the
initially identified carboxyacyl tetrasaccharide 10 (SQS-0-0-4-5)
have been circumvented with the identification of this
significantly simplified analogue 13, which is accessed in only
26 steps. This will allow future investigations of other domains
of QS-21, such as the branched trisaccharide and triterpene
core, to be carried out more efficiently.
́ ́
Educacion of Spain and Comision Fulbright for postdoctoral
fellowship support.
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