Synthesis of a monophosphoryl lipid A derivative and its conjugation to a modified form of a tumor-associated carbohydrate antigen GM3

Article abstract of DOI:10.1039/b907351e

An efficient synthesis of a derivative of monophosphoryl lipid A suitable for coupling to various structures for the construction of glycoconjugate vaccines and its conjugation with an N-modified form of the tumor-associated antigen GM3 is presented.

Full text of DOI:10.1039/b907351e

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Synthesis of a monophosphoryl lipid A derivative and its conjugation
to a modified form of a tumor-associated carbohydrate antigen GM3w
Qianli Wang, Jie Xue and Zhongwu Guo*
Received (in College Park, MD, USA) 14th April 2009, Accepted 10th August 2009
First published as an Advance Article on the web 27th August 2009
DOI: 10.1039/b907351e
An efficient synthesis of a derivative of monophosphoryl lipid A
suitable for coupling to various structures for the construction of
glycoconjugate vaccines and its conjugation with an N-modified
form of the tumor-associated antigen GM3 is presented.
To probe MPLA for the development of effective conjugate
vaccines, we prepared a monophosphoryl form, 3 (Scheme 1),
of Neisseria meningitidis lipid A analogue.16 SAR studies
showed that modification of the reducing end of the MPLA
had little influence on its immunological activity.10 The
syntheses of lipid A and MPLA have been reported17–23
(incomplete), but 3 is designed to have a functionality that
will facilitate its coupling to other structures. For example, the
azido group of 3 will enable the coupling of 3 to other
molecules by click chemistry. The azido group can also be
readily reduced to form a primary amine to facilitate the
introduction of other functionalities, and therefore MPLA
coupling to various structures by other methods.
Lipid A (1) is the hydrophobic domain of lipopolysaccharides
(LPS) of the outer membranes of Gram-negative bacteria.1
Its structure consists of a b-1,6-linked di-glucosamine with
1,40-di-O-phosphorylation, and 2,20-N- and 3,30-O-acylation
(Fig. 1). Lipid A of different bacterial origins can vary
significantly in terms of the number and structure of the fatty
acids.1
Lipid A is a strong immunostimulator. It functions through
Toll-like receptor 4 (TLR4), the activation of which triggers a
cascade of immunological responses, including the production
of various cytokines and chemokines, such as tumor necrosis
factor-a, interleukin-1b (IL-1b), IL-6 and interferon-b
(IFN-b).2 Therefore, lipid A is an important target for the
development of useful immunomodulators,3,4 such as vaccine
adjuvants, and for the development of new conjugate
vaccines.5 However, a serious problem associated with lipid
A is that it is pro-inflammatory and can cause fatal septic
shock.6 Recent structure–activity relationship (SAR) studies
have disclosed that both the fatty acid structure and the
phosphorylation degree of lipid A can affect its activity and
toxicity.7–10 Most notably, it was found that monophosphoryl
lipid A (MPLA 2), which has only one phosphate group linked
to the 40-OH, had a drastically reduced toxicity but retained
immunostimulatory activity.11 MPLA has been proved to be
clinically safe.12
Our synthesis of 3 is outlined in Scheme 1. Monosaccharide
blocks 4 and 5 were synthesized from commercially available
glucosamine by following a series of established transformations.
The glycosylation reaction between 4 and 5 was achieved with
N-iodosuccinimide (NIS) and silver trifluoromethanesulfonate
(AgOTf) as promoters. This reaction was unexpectedly slow,
took about 2 d at room temperature to complete but
was stereoselective and afforded the desired b-anomer 6
0
0
(J1 ,2 = 8.8 Hz) in a 54% isolated yield. Next, the phthalyl
and acetyl groups in 6 were simultaneously removed by
treatment with hydrazine in refluxing ethanol to give 7.
Selective acylation of the free amino groups of 7 by
(R)-3-dodecanoyl-tetradecanoic acid (8), using 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (EDC) as the conden-
sation reagent, went smoothly to furnish 9 in a yield of 80%.
Acylation of both the C-3 and C-30 hydroxyl groups of 9 by
lauric acid was slow and required a higher temperature
(45 1C), but the reaction was clean and produced 10 in an
excellent yield. Thereafter, regioselective opening of the
benzylidene acetal ring of 10 was accomplished using sodium
cyanoborohydride (NaCNBH3) and HCl in dry diethyl ether
to produce 11, which has a free hydroxyl group on C-40. The
regiochemistry was confirmed via an acetylation experiment.
Finally, 11 was phosphorylated in a two-step-one-pot manner
to afford synthetic target 3.
Meanwhile, carbohydrate-based vaccines for cancer and
various infectious diseases are currently a hot topic.13 However,
a crucial issue related to carbohydrate antigens is that they are
poorly immunogenic; so to form effective vaccines, they have to
be covalently linked to an immunologically-active carrier.14 In
this regard, MPLA could be of great potential. First, owing to its
bacterial origins, MPLA could be a ‘‘danger signal’’ to the
immune system to significantly enhance the immunogenicity of
a carbohydrate antigen once it is covalently coupled, as observed
by Boons et al.15 with a different conjugate. Second, as noted
above, MPLA is an immunostimulator and adjuvant; this could
further help to stimulate the immune system.
To demonstrate that 3 can be useful for the development
of conjugate vaccines, we then examined the coupling of 3 to
Department of Chemistry, Wayne State University, 5101 Cass Avenue,
Detroit, MI 48202, USA. E-mail: zwguo@chem.wayne.edu;
Fax: +1 313-577-8822; Tel: +1 313-577-2557
w Electronic supplementary information (ESI) available: Experimental
procedures and data, and NMR and MS spectra of the intermediates
and final products. See DOI: 10.1039/b907351e
Fig. 1 Generic structures of lipid A (1) and MPLA (2).
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transformed into active ester 14 to facilitate the coupling to 15.
The reaction between 14 and 15 afforded the desired
MPLA–NPhAcGM3 conjugate, 16, in a good yield.
In summary, a highly convergent and efficient method has
been established for the synthesis of an MPLA derivative, 3,
derived from monosaccharides 4 and 5 in 6 separate steps and
a 17% overall yield. This MPLA derivative is suitable for
coupling to various structures. As a demonstration, 3 was
effectively coupled with a modified tumor-associated antigen
to afford 16, which was readily deprotected via Pd-catalyzed
hydrogenolysis. The immunological properties of conjugate 16
and its deprotected product as cancer vaccines are now under
investigation in our laboratory.
The authors thank NIH (CA95142) for their financial
support of this work. They also thank Dr B. Shay and
Dr L. Hryhorczuk for HRMS measurements, and Dr B. Ksebati
for NMR measurements.
Notes and references
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Scheme 1 The synthesis of target molecule 3.
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Scheme 2 The synthesis of MPLA–NPhAcGM3 conjugate 16.
N-phenylacetyl GM3 (NPhAcGM3), a neoantigen investi-
gated in our laboratory for cancer immunotherapy.24 As
outlined in Scheme 2, after the azido group of 3 was reduced
with Zn under acidic conditions, the resultant free amine was
acylated with succinic anhydride to form 13, which was then
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Chem. Commun., 2009, 5536–5537 | 5537