A convergent, versatile route to two synthetic conjugate anti-toxin malaria vaccines

Article abstract of DOI:10.1039/b407323a

The synthesis of two glycosylphosphatidyl inositol (GPI) glycans that constitute the malaria toxin and promising anti-toxin vaccine constructs using a scalable route is described.

Full text of DOI:10.1039/b407323a

A convergent, versatile route to two synthetic conjugate anti-toxin
malaria vaccines
Peter H. Seeberger†,* Regina L. Soucy, Yong-Uk Kwon†, Daniel A. Snyder and Takuya Kanemitsu
Department of Chemistry: Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
E-mail: seeberger@org.chem.ethz.ch; Fax: +41) 1-633123; Tel: (+41) 1-6332103
Received (in Cambridge, UK) 14th May 2004, Accepted 1st June 2004
First published as an Advance Article on the web 9th July 2004
The synthesis of two glycosylphosphatidyl inositol (GPI)
glycans that constitute the malaria toxin and promising anti-
toxin vaccine constructs using a scalable route is described.
diester, we reasoned that presenting the antigen in the proper
orientation, as in 3a/3b, could result in better vaccination.
To obtain ready access to large quantities of 2a and 3a, we
devised a convergent, modular synthesis, involving a minimum of
late-stage modification, and using robust chemistry throughout.
Assembly proceeded via a key 4+2 glycosylation, which allowed
for the same tetrasaccharide building block to be used in generation
of both 2a and 3a. In addition to the two inositol-containing
disaccharides 12 and 13, three mannose synthons 4, 10 and 11 were
used (Scheme 1). After the completion of the hexamers, the
phosphate diester functions were installed using H-phosphonates
19 and 20 prior to global deprotection and conjugation (Scheme
2).
This synthesis built on previous efforts towards GPI structures by
our4,7 and other laboratories.8 Solution-phase methods are con-
siderably less rapid when compared to our automated assembly but
allow for ready scale-up, an important consideration in preparation
for preclinical and clinical trials.
The production of the key tetramannose trichloroacetimidate
building-block 7 started from C2-benzoyl mannose 4 (see Scheme
1). Glycosylation of 4 with 10, followed immediately by selective
removal of the 2-O-acetate in the presence of the benzoate ester
using acetyl chloride in methanol, provided disaccharide alcohol 5.
Repetition of this maneuver, first using donor 11 and then 10 again,
Malaria infects 5–10% of humanity, and kills up to three million
people each year, mostly children in Africa.1 Current malaria
treatments are often impractical in many endemic areas, and drug
resistance is a growing problem. At the same time, there is still no
effective malaria vaccine.2 Conjugate carbohydrate vaccines have
shown great utility as public health tools in preventing the infection
of children by Haemophilus influenzae type b and Streptococcus
pneumoniae.3 We have previously demonstrated the efficacy of
anti-toxin vaccination in a mouse model of malaria.4 Here, we
report the development of a general and practical synthesis strategy
for access to defined malaria toxin structures, and its application to
the synthesis of a second-generation vaccine.
The malaria parasite, Plasmodium falciparum, expresses a large
amount of glycosylphosphatidylinositol (GPI) in protein anchored
and free form on the cell surface.5 Mounting evidence suggests that
the proinflammatory-cytokine cascade triggered by this GPI is
responsible for much of malaria’s morbidity and mortality.6
Vaccination with synthetic GPI produces anti-GPI antibodies,
which neutralize this toxin and result in host survival.4 Based on the
GPI toxin 1, our initial vaccine candidate 2a was designed and
conjugated to a carrier protein (Fig. 1).4,7 However, as the native
toxin is linked to the cell membrane via an inositol phosphate
Fig. 1 Malarial GPI (1) and model vaccine constructs (2c and 3b).
Scheme 1 (a) 10, TMSOTf; (b) AcCl, MeOH; (c) 11, TMSOTf; (d)
Mg(OMe)2, MeOH; (e) 10, TMSOTf; (f) PdCl2, NaOAc, HOAc, H2O; (g)
Cl3CCN, DBU; (h) 12, TMSOTf; (i) 13, TMSOTf.
† New Address: Laboratory for Organic Chemistry, ETH Hönggerberg,
HCI F315, Wolfgang-Pauli-Str. 10, CH-8093 Zürich, Switzerland
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C h e m . C o m m u n . , 2 0 0 4 , 1 7 0 6 – 1 7 0 7
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

provided fully-protected tetrasaccharide 6. Removal of the anome-
ric allyl group with PdCl2 in wet acetic acid, was followed by the
formation of glycosyl trichloroacetimidate 7 using Cl3CCN/DBU.
Demonstrating the scalability of this chemistry, central tetra-
saccharide 7 was produced readily on a 20 g scale. Coupling of 7
with 12 afforded hexamer 8 en route to 2a. The union of 7 and 13
provided 9 to be elaborated into 3a. It should be noted that the 2-O-
benzoate resulted in a significantly improved glycosylating agent
when compared to a tetrasaccharide containing the 2-O-benzyl
ether used previously: 85% yield as opposed to 39%.
The ester functions of hexamer 8 were first replaced with benzyl
ethers to fashion 14 (see Scheme 2). Elaboration of 14 as reported
previously furnished 15.7 Desilylation and phosphorylation using
H-phosphonate 19 provided fully-protected intermediate 16.
Global deprotection in one step using Pd(OH)2 was followed by
reaction of the primary amine with 2-iminothiolane,9 to generate
thiol 2b, ready for coupling to maleimide-activated BSA and
formation of model vaccine 2c.4
nate 19, yielding fully-protected 18. Global deprotection was
accomplished by the removal of ester groups using sodium
methoxide in methanol and subsequent Birch reaction using sodium
in ammonia to afford 3a, ready for conjugation to maleimide-
functionalized BSA, giving the new model vaccine 3b.
The products of these syntheses (2b and 3a) were attached to
BSA both as a model for attachment to the antigenic proteins
desired for vaccination, and to produce useful substrates for ELISA
tests for anti-GPI IGs in both naturally immune and vaccinated
individuals.10 Work is currently underway to determine, via rodent
trials, the best carrier protein and adjuvants for the vaccines. Also,
we are engaged in synthetic studies producing a variety of
substructures of the GPIs, to be used in determining the minimum
antigen structure necessary to produce good immune response. The
method presented here has 14 steps and provides 3a in 6.4% overall
yield.
In conclusion, we have demonstrated the development of a
practical synthesis of malarial GPI structures, and applied these
methods to the generation of conjugate anti-toxin malaria vaccines
from fully synthetic oligosaccharides, resulting in more efficient
access both to previously tested and second-generation vaccines.
This research was supported by GlaxoSmithKline (Scholar
Award to P.H.S.), the Alfred P. Sloan Foundation (Fellowship to
P.H.S.) and Merck & Co (Academic Development Award to
P.H.S). Y.-U. Kwon thanks Korea Science and Engineering
Foundation (KOSEF) for financial support (Postdoctoral Fellow-
ship).
Removal of the allyl ether from hexasaccharide 9 (see Scheme
2), using PdCl2 in wet acetic acid was followed by phosphorylation
with 20 to give 17. The TIPS ether was cleaved using Sc(OTf)3, and
the ethanolaminephosphate linker was installed using H-phospho-
Notes and references
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Scheme 2 (a) MeOH, NaOMe; (b) BnBr, NaH; (c)TsOH, MeOH; (d)
TBSCl, Im.; (e) Cl2PO2Me, Py.; (f) TBAF; (g) 1. 19, PivCl, pyridine; 2. I2;
(h) Pd(OH)2, H2; (i) Sc(OTf)3, H2O; (j) 1. 20, PivCl, pyridine; 2. I2; (k)
PdCl2, NaOAc, HOAc, H2O; (l) 1. 19, PivCl, pyridine; 2. I2; (m) 1. NaOMe,
MeOH; 2. Na, NH3.
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10 C. Evans, P. H. Seeberger and L. Schofield, unpublished results.
C h e m . C o m m u n . , 2 0 0 4 , 1 7 0 6 – 1 7 0 7
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