The total synthesis of immunostimulant α-galactosylceramides from naturally configured α-galactoside raffinose

Article abstract of DOI:10.1021/ol201695n

The total synthesis of absolute anomeric confirmation α- galactosylceramide analogues from raffinose is described. Using the naturally occurring α-galactoside raffinose as the starting material, the easily maneuverable protocol without glycosylation react

Full text of DOI:10.1021/ol201695n

ORGANIC
LETTERS
2011
Vol. 13, No. 17
4530–4533
The Total Synthesis of Immunostimulant
r-Galactosylceramides from Naturally
Configured r-Galactoside Raffinose
Zhenxing Zhang,† Wei Zhao,† Bin Wang,† Chengfeng Xia,§ Wenpeng Zhang,*,§ and
Peng George Wang*,†,‡,§
College of Pharmacy, Nankai University, Tianjin 300071, China, State Key Laboratory
of Elemento-organic Chemistry, Nankai University, Tianjin 300071, China, and
Departments of Chemistry and Biochemistry, The Ohio State University, Columbus,
Ohio 43210, United States
zhang.472@osu.edu; pwang@nankai.edu.cn
Received June 24, 2011
ABSTRACT
The total synthesis of absolute anomeric confirmation R-galactosylceramide analogues from raffinose is described. Using the naturally occurring
R-galactoside raffinose as the starting material, the easily maneuverable protocol without glycosylation reactions ensured the critical R-linkage in
the product and simplified the synthetic procedures. The immunostimulatory activities of the new R-galactosylceramides were validated by both
in vitro and in vivo NKT cell stimulation assays.
R-Galactosylceramide is an agonist ligand for natural
killer T cells (NKT cells). It has been proven that R-
galactosylceramide is presented to the T cell receptor on
the NKT cell surface by the CD1d molecules of antigen-
presenting cells (APC). This stimulates the NKT immune
response, which results in the rapid release of the Th1 and
Th2 cytokineswithsignificant influences.1 KRN7000 isthe
most well-known molecule of the R-galactosylceramide
class, and its therapeutic effects have been well studied in
different disease models. Crystallographic analysis has
indicated that the mouse CD1d (mCD1d) has a deep,
narrow, and very hydrophobic ligand binding groove with
two pockets denoted as A0 and F0.2 Bioinformatic analysis
has also suggested that the two long alkyl chains of the
ligand are capable of being accommodated in the two
hydrophobic pockets. Removal of the hydroxyl from the
fatty acid chain has no significant effects on antigenicity.
However, the biological activity is sensitive to the config-
uration between the sugar and the lipid, with the β-
galactosylceramides only showing minimal or no activity.3
As the R-linkage is very critical for the biological func-
tions of the glycosphingolipids, many different methodol-
ogies have been developed to chemically synthesize this
critical structure. For example, the trichloroacetimidate
donorshavebeensuccessfully applied tothe preparation of
R-galactosylceramide.4 Thioalkyl activated donors have
also been used to prepare the R-galactosylceramide,
although the selectivity was not ideal.5 Glycosyl halides
are other documented donors for the preparation of R-
GalCer, with galactosyl fluoride, galactosyl bromide, and
galactosyl iodide all being applied as donors in moderate
yield and with good anomeric selectivity.6 AgClO4,7
† College of Pharmacy, Nankai University.
‡ State Key Laboratory of Elemento-organic Chemistry, Nankai
University.
BF3 Et2O,8 Bu4NBr,9 and phosphine oxide10 are reported
3
§ The Ohio State University.
to serve as promoters for glycosidation. Phosphate11 and
r
10.1021/ol201695n
Published on Web 08/04/2011
2011 American Chemical Society

acetate12 were also exploited for the preparation of R-
GalCer. However, these glycosylation reactions produced
mostly a mixture of R/β isomers which were very difficult
to separate.
There is always suspicion about the anomeric purity of
synthetic galactosylceramide as it is difficult toseparate the
R isomers from the β isomers, especially when they are
present in trace amounts in the glycosylation reactions.
Until now, there is no chemical glycosylation reaction in
this area with 100% R/β selectivity. Time-consuming
separation procedures are required to separate the isomers
from the above synthetic methodologies, which lowers the
reaction efficiency and prevents the methods from being
extended to large scale synthesis. To overcome the diffi-
culties and flaws of current synthetic methodologies, we
chose to begin our synthesis of R-galactosyl lipids from the
naturally configured R-galactoside, raffinose. By avoiding
glycosylation reactions, a new and easily scaled synthesis
of several KRN7000 analogues was developed via high
yield reactions that could be easily adapted to industrial
scale synthesis.
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By comparing the KRN7000 structure and raffinose, we
designed a series of new potential NKT cell ligands as
shown in Scheme 1 (compounds 1, 2, 3, 4, 5).
Scheme 1. Structures of KRN7000, Raffinose, and the Proposed
R-Galactosylipids
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From raffinose 6, the fully benzylated derivative 7 was
prepared in 80% yield by treatment with benzyl bromide
and sodium hydride in DMF (Scheme 2). Partially benzy-
lated sugar 8 was accessed, using a described procedure,13
by selective cleavage of the β-fructofuranosidic linkage in 7
under acidic conditions in 90% yield. A subsequent reduc-
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as the reductant which, among many other reductants
screened, was found to be the best in terms of safety and
reaction yield (Scheme 2).
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reactive sites for introducing the lipid chains. The easiest
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Org. Lett., Vol. 13, No. 17, 2011
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Scheme 2. Preparation of Intermediate Diol 9
Scheme 4. Synthesis of the New R-Galactosylceramides
the two ꢀOH groups with a 1-bromoalkane and NaH in
DMF. Via this method, and following deprotection, a
series of R-galactosyllipids 3, 4, 5 (Scheme 1) was obtained.
Unfortunately, they had no stimulatory effect on our
studied cells. So, we decided to produce a molecule that
was more like the known KRN7000, and the amide moiety
was needed.
Without purification of the reduced diol 9, monopivalo-
ate 10 was synthesized in 97% yield by regioselective
pivaloylation (PivCl/pyridine) at the primary ꢀOH group.
Scheme 3. Synthesis of Azide 11
The synthesis of azide 11 was achieved in 92% yield by
treating alcohol 10 with DPPA in the presence of Ph3P and
DEAD in toluene (Scheme 3). Deacylation via base-cata-
lyzed methanolysis and alkylation with 1-bromododecane
in the presence of NaH in DMF afforded ether 13 in 77%
yield (Scheme 4).
Figure 1. IL-2 cytokine releasing profile from stimulated NKT
hybrodoma cells. (A) Hybridoma DN3A4-1.2. (B) Hybridoma
N38-2H4.
Reduction of the azide 13 was achieved with sodium
borohydride in the presence of a catalytic amount of nickel
chloride, affording the corresponding amine. Treatment of
this amine with active ester in the presence of Et3N gave
amide 14 and 15 both in 95% overall yield (2 steps).
Finally, global debenzylation via catalytic hydrogenation
over Pd/C gave the final R-galactosylceramide 1 and 2 in
70% and 63% yield respectively (Scheme 4).
For the in vitro hybridoma assay, as Figure 1 shows,
among all of the new analogues synthesized, compound 1
caused significant stimulation of the two hybridoma cell
lines, with comparable strength to KRN7000. All of the
other four analogues did not show any stimulation in the
hybridoma assay.
In the case of the in vivo mouse splenocytes culture assay,
as Figure 2 shows, compound 1 showed significant stimu-
lation of both cytokines, especially the Th2 cytokine. The
activity of compound 1 is quite impressive among all of the
The NKT stimulation activities of the new compounds
were evaluated in both an in vitro hybridoma assay and in
vivo mouse splenocytes stimulation assay.
4532
Org. Lett., Vol. 13, No. 17, 2011

configuration via a new and simplified procedure. The
biological results also reveal that replacement of the acyl
chain with a short carbon chain (compounds 3, 4, 5)
eliminates the analogues’ immunogenicity, probably due
to a lack of affinity or a lack of flexibility when the
compounds bind to CD1d. At the same time, compound
1 maintained a high immunogenicity by keeping its long
acyl chain for binding affinity (compound 2 does not have
a long enough acyl chain). The introduction of an ꢀOꢀ
group in the sphingosine chain does not significantly
change the immunogenicity of the compound.
In summary, a new kind of R-galactosylceramide has
been successfully synthesized by an easy method from
raffinose. This group of novel R-galactosylceramides has
been shown to be immunostimulatory for NKT cells by
biological evaluation. In our synthesis, the naturally oc-
curring R-galactoside, raffinose, was applied as the starting
material to ensure the emergence of the R-linkage in the
product and to simplify the synthetic route. This method
alsoprovides anefficient methodologyfor producing other
galactosylceramides if the protocol is extended to other
available saccharides and varied lipid chains. It will also
prove useful for the facile generation of glycolipid libraries
for novel NKT cell ligand discovery.
Figure 2. IFN-γ (A) and IL-4 (B) releasing profile from glyco-
lipids stimulated mouse splenocytes.
Acknowledgment. This work was supported by the Na-
tional Basic Research Program of China (973 Program:
No.2007CB914403), NSFC 20902050, and NIH (R21
A1083513 to P.G.W.).
known KRN7000 analogues, as it has comparable cyto-
kine stimulation efficiency.
Based on these results, it can be concluded that the
analogue from thenewlydevelopedsyntheticmethodology
still maintains the immunogenicity against NKT cells. The
synthetic strategy achieved its target of producing a func-
tional KRN7000 analogue with the correct absolute
Supporting Information Available. Experimental pro-
cedures for the preparation of all new compounds and the
detail experimental procedures of the biological activity.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Org. Lett., Vol. 13, No. 17, 2011
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