Synthesis and biological activity of α-l-fucosyl ceramides, analogues of the potent agonist, α-d-galactosyl ceramide KRN7000

Article abstract of DOI:10.1016/j.bmcl.2010.04.079

Several l-fucoglycolipids are associated with diseases such as cancer, cystic fibrosis and rheumatoid arthritis. Activation of iNKT cells is known to lead to the production of cytokines that can help alleviate or exacerbate these conditions. α-Galactosyl ceramide (α-GalCer) is a known agonist of iNKT cells and it is believed that its fucosyl counterpart might have similar immunogenic properties. We herein report the synthesis of α-l-fucosyl ceramide derivatives and describe their biological evaluation. The key challenge in the synthesis of the target molecules involved the stereoselective synthesis of the α-glycosidic linkage. Of the methods examined, the per-TMS-protected glycosyl iodide donor was completely α-selective, and could be scaled up to provide gram quantities of the azide precursor 11, from which a range of N-acylated α-l-fucosyl ceramides were readily obtained and evaluated for ex vivo expansion of human iNKT cells.

Full text of DOI:10.1016/j.bmcl.2010.04.079

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3223–3226
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of
a-L-fucosyl ceramides, analogues
of the potent agonist, -D-galactosyl ceramide KRN7000
a
Natacha Veerapen a, Faye Reddington a, Gabriel Bricard b, Steven A. Porcelli b,c, Gurdyal S. Besra a,
*
a School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
b Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
c Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Several
arthritis. Activation of iNKT cells is known to lead to the production of cytokines that can help alleviate
or exacerbate these conditions. -Galactosyl ceramide ( -GalCer) is a known agonist of iNKT cells and
it is believed that its fucosyl counterpart might have similar immunogenic properties. We herein report
the synthesis of -fucosyl ceramide derivatives and describe their biological evaluation. The key chal-
lenge in the synthesis of the target molecules involved the stereoselective synthesis of the -glycosidic
linkage. Of the methods examined, the per-TMS-protected glycosyl iodide donor was completely
selective, and could be scaled up to provide gram quantities of the azide precursor 11, from which a
L-fucoglycolipids are associated with diseases such as cancer, cystic fibrosis and rheumatoid
Received 6 April 2010
Revised 15 April 2010
Accepted 16 April 2010
Available online 22 April 2010
a
a
a
-L
a
Keywords:
CD1d
iNKT
Antigen
Ceramide
Lipid
a
-
range of N-acylated
a
-
L
-fucosyl ceramides were readily obtained and evaluated for ex vivo expansion
of human iNKT cells.
Ó 2010 Elsevier Ltd. All rights reserved.
O
C15H31
Both
D
- and
L
-fucose (6-deoxy galactose) are widely found in
-fucose is predominantly found in the
nature.1 Of interest,
L
a-
NH
CH
configuration in the lipopolysaccharides (LPS) of Gram-negative
CH
OH
C
H
C CH2 CH3
bacteria and animal glycosphingolipids.1,2 With the exception
O
H
12
of the monohexylceramide,
a-L-fucosylceramide 1, initially iso-
O
lated from metastatic human adenocarcinoma,3 most fucosphin-
golipids are usually ceramide oligosaccharides.2,4 Many of these
fucolipids are antigenic5–7 and play a role in tumour cell biol-
OH
HO
OH
OH
1
ogy.8–10 Conversely, the synthetic glycolipid,
a-galactosyl cera-
OH
HO
HO
-GalCer)11 (2), is a strong agonist of iNKT cells when
O
C25H51
O
C25H51
mide (
a
O
O
bound to CD1d, triggering the release of diverse cytokines,
including both Th1 and Th2 cytokines.12–14 It is believed that
the release of Th1 cytokines may contribute to antitumour and
antimicrobial functions while that of Th2 cytokines may help
alleviate autoimmune diseases15–17 such as multiple sclerosis18
and arthritis.19
HO
NH OH
NH OH
HO
HO
O
O
C14H29
C14H29
OH
OH
2
3
a
-GalCer and its derivatives have proved to be and remain
invaluable tools in understanding the functioning of CD1d and
iNKT cells in a wide range of immune responses. Crystal structures
of the bound
xyl group at C-6 in the sugar head is not crucial for binding to the
T-cell receptor (TCR) as opposed to C-2, C-3 and C-4. Hence, the
-fucosylceramide 323 was shown to be a potent inducer of IFN-
a
-GalCer–CD1d complex20–22 showed that the hydro-
a
-
D
c
* Corresponding author. Tel.: +44 (0)121 4158125.
E-mail address: g.besra@bham.ac.uk (G.S. Besra).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.079

3224
N. Veerapen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3223–3226
in mice in vivo, while a slightly different derivative, synthesised by
Motoki et al.24 showed strong lymphocytic proliferation stimula-
tory effects in vitro in mice.
The phytosphingosine acceptor 729 was synthesised from
(2S,3S,4R)-2-azido-1,3,4-octadecanetriol30 as described before. Gi-
ven the previous success of a thiogalactoside in a similar glycosyl-
ation reaction with compound 728 we chose to use the thiofucosyl
donor 6, rather than compound 5. The thioglycoside 6 was thus ob-
While there are a few examples describing the synthesis of a-L-
fucosylceramide 4b25 and other derivatives in the literature,26,27
their biological activities are yet to be fully explored. As part of var-
ious ongoing studies, and because of the aforementioned reasons,
we have synthesised compounds 4a–4d for biological evaluation
and comparative studies.
tained from commercially available
L-fucose after standard proce-
dures, as previously reported.31
With both the acceptor and donor in hand, we then proceeded
to the critical glycosylation reaction. Interestingly, NIS/TfOH acti-
vation of 1 g of the thioglycoside 6 (Scheme 2) in anhydrous CH2Cl2
at À78 °C afforded the glycosylated compound 8, almost exclu-
C4H9
O
(CH2)8
sively as the
a-anomer (a:b ratio = 9:1), in 68% yield after 2 h.
NH OH
Our results show a definite improvement in selectivity from the
O
previously reported syntheses. Because the formation of the
a
-
O
(CH2)nCH3
NH OH
C14H29
C14H29
anomer is favoured by the anomeric effect, we rationalise that
the latter is a governing factor under our reaction conditions. Both
the lower temperature and the different reactivity of the acceptor 7
could potentially influence the stereoselectivity of the glycosyla-
tion reaction. These factors will have to be more thoroughly inves-
tigated in a later study. Subsequent Zemplen’s deprotection of the
benzoate protecting groups produced the azide intermediate in
quantitative yields. Tandem hydrogenation of the azido group
and hydrogenolysis of the benzyl ethers in methanol then pro-
duced the amine 9 as a white solid, which exhibited spectroscopic
data consistent with the literature.25
OH
O
OH
O
OH
OH
R
4a
OH
O
OH
OH
OH
O
C14H29
NH OH
4c n = 22, R = OH
4d n = 24, R = OH
4e n = 24, R = H
O
C14H29
OH
O
OH
In an attempt to improve the stereoselectivity and circumvent
the sometimes problematic removal of the benzyl ethers by
hydrogenolysis, we embarked on a different glycosylation route.
Recently, the use of glycosyl iodide donors has been revived by
Gervay-Hague’s group.32,33 They have demonstrated that glycosyl-
ation reactions employing glycosyl iodides and promoted by tetra-
butyl ammonium iodide (TBAI) are generally quite stereoselective
and fast. It has been hypothesized that TBAI catalyses the isomer-
OH
OH
4b
Fan et al.25 reported the synthesis of compound 4d, while
Okamoto et al.27 reported that of compound 4e. Both groups ob-
tained their target compounds as an a, b mixture (3:1 ratio) from
different glycosyl donors and acceptors (Scheme 1). On the other
hand, in the original reported synthesis of the fucosylceramide 1,
ization of the
to the formation of an
this strategy to the synthesis of
a
-glycosyl iodide to the b-anomer,34 thereby leading
-glycoside. They have successfully adapted
-GalCer35 and other
-fucosylgly-
only the
a-anomer was isolated when another ceramide acceptor
a
was used under different reaction conditions.
a
a
We recently reported the synthesis28 of
a
-GalCer and other
cosides.36 Furthermore, the replacement of the benzyl ethers with
trimethylsilylethers (TMS) made Gervay-Hague’s synthetic strat-
egy even more attractive. TMS protected sugars are easy to gener-
ate and their deprotection requires very mild acidic conditions,
compatible with the glycosidic linkage.
derivatives where we used N-iodosuccinimide (NIS) and triflic acid
(TfOH) as a promoter, and benzoate protecting groups on the
sphingosine base. Along with the nature of the protecting groups
on both the glycosyl acceptor and donor, the choice of promoter,
as well as temperature and reaction times can affect the stereose-
lectivity of the glycosylation reaction. In this study, we proposed to
investigate whether our glycosyl acceptor 7 and reaction condi-
SPh
OBz
C14H29
N3
HO
tions could offer a better
a
-selectivity in the case of L-fucose.
O
+
OBn
OBz
OBn
OBn
OBn
N3
7
6
a
N3 OBn
C14H29
HO
OH
C14H29
O
N3 OBz
C14H29
OBn
a
OBn
O
O
O
OBn
OBn
OBz
O
OBn
OBn
OBn
OBn
OBn
OBn
OBn
α:β = 3:1
5
8
C13H27
N3
b, c
N3
C13H27
HO
SPh
NH2 OH
O
OBn
O
C14H29
OBn
O
O
OH
b
OBn
OBn
O
OH
OBn
OBn
OBn
OBn
OH
OH
6
α:β = 3:1
9
Scheme 1. (a) Me2S, 2-Cl-pyridine, Tf2O, CH2Cl2; (b) dimethyl(methylthio)sulfo-
nium triflate (DMTST), CH2Cl2, 0 °C-rt.
Scheme 2. (a) NIS/TfOH, CH2Cl2, À78 °C to À20 °C, 68%; (b) NaOMe/MeOH, 92%; (c)
H2, Pd, 76%.

N. Veerapen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3223–3226
3225
The per-O-trimethylsilyl-
a-
L-fucosylpyranosyl iodide 10 was
(4d) > C18:0 (OH) (4b) > C24:0 (4c). The
a-
L-fucosylceramide con-
generated by the reaction of the per-O-tetramethylsilyl-
a
-
L
-fucose
taining a C26:0 fatty acid (4d) was the most active of the fucosyl
series, and stimulated iNKT cell expansions in some donors that
approached those seen with the prototype iNKT cell agonist
with one equivalent of iodotrimethylsilane35 and then added to the
phytosphingosine acceptor 7 which was premixed with diisopro-
pylethylamine (DIPEA) and TBAI (Scheme 3). After 2 days at room
temperature, the solvent was evaporated and the TMS protecting
groups were removed by treatment with an acidic resin in MeOH.
KRN7000 (2). In contrast, the
a-L-fucosylceramide containing the
C20:2 fatty acid (4a) was found to lack detectable iNKT cell stimu-
lating activity in any of the donors tested (Fig. 1B). Representative
profiles obtained by flow cytometry of cultures from one normal
blood donor are shown in Figure 1A. This analysis was carried
out with PBMC from four separate donors (Fig. 1B). Although, dif-
ferences were observed for the levels of iNKT cell expansion be-
tween different donors, all donors responded significantly to two
Compound 11 was obtained as the
a-anomer exclusively in an
overall yield of 62%. The formation of the desired
a
-linkage was
confirmed by the H-1 and C-1 signals in 1H and 13C NMR. Methan-
olysis, followed by hydrogenation of the azide then afforded com-
pound 9.
Finally, N-acylation with the fully saturated fatty acids, tetraco-
sanoic acid (C24:0) and hexacosanoic acid (C26:0), was achieved
via reaction of the corresponding acid chloride with the free amine
9 in a 1:1 mixture of THF and saturated sodium acetate solution
(Scheme 3). Target compounds 4c and 4d were obtained as white
solids after concentration of the organic phase and purification of
the residue by flash chromatography. The spectroscopic data of
the final compounds were consistent with the literature.25 While
of the a-L-fucosylceramide analogues (4b and 4d).
In summary, in the current study we have developed an
efficient method for the synthesis of a series of biologically active
a-L
-fucosylceramides.40 The second method, employing the per-
O-trimethylsilyl-
superior with a better
the glycosylation reaction. Given the marked difference in the ste-
reochemistry of the -fucosyl head group of these glycolipids com-
pared to -galactosyl group of strong iNKT cell activators like
a-
L-fucosylpyranosyl iodide 10, proved to be
a
-selectivity and reasonably good yield in
a
compound 4a was obtained by heating amine
9
with the
a
N-hydroxysuccinimide activated ester of C20:2 fatty acid in a mix-
ture of pyridine: water (9:1) at 50 °C overnight, compound 4b was
obtained via dicyclohexylcarbodiimide (DCC) activated coupling
using procedures described previously.37,38
KRN7000, it is surprising that the compounds in the current series
show such substantial activity. This would seem to further rein-
force the notion that the TCR of iNKT cells, in spite of its relative
limited variability, is nevertheless able to interact efficiently with
a broad range of structurally diverse ligands. It is noteworthy that
To assess the biological activity of the
a-L-fucosylceramides and
compare these to -GalCer (KRN7000, 2), we assessed the ability of
a
each compound to induce the expansion of iNKT cells in samples of
human peripheral blood mononuclear cells (PBMC) during an
eight-day in vitro culture.39 The results showed that both the per-
centages and absolute numbers of iNKT cells in cultures were in-
creased by stimulation with
a-
L-fucosylceramides with C26:0
I
O
7
OTMS
+
OTMS
OTMS
a, b
10
N3 OBz
O
C14H29
OBz
O
HO
OH
11
HO
c,d
9
e
O
(CH2)nCH3
NH OH
C14H29
O
Figure 1. Ex vivo expansion of human iNKT cells by a-L-fucosylceramides. PBMC from
OH
four different donors were stimulated with the indicated glycolipids at a concen-
tration of 250 nM in the presence of low levels of exogenous IL-2 and IL-7. At day 8,
cultures were harvested and analysed by flow cytometry using monoclonal
O
OH
OH
antibodies specific for CD3 and for the invariant TCR
a chain expressed by iNKT
OH
cells (6B11). (A) Dot plots showing relative levels of CD3+ 6B11+ iNKT cells are
shown for one representative donor. Numbers in upper right quadrant indicate
percentages of total lymphocytes that are iNKT cells. (B) Absolute numbers of iNKT
cells in the cultures were determined by flow cytometry using fluorescent counting
beads, and the values of iNKT cell fold expansion were determined by dividing by
the input number of iNKT cells.
4c n = 22, R = OH
4d n = 24, R = OH
Scheme 3. (a) TBAI, DIPEA, CH2Cl2, rt; (b) Dowex 50WX8-200, MeOH, rt, 62% over
two steps; (c) NaOMe/MeOH, quantitative; (d) H2, Pd, MeOH, 80%; (e) C25H51COCl or
C23H47COC1, THF, NaOAc, 78–80%.

3226
N. Veerapen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3223–3226
22. Borg, N. A.; Wun, K. S.; Kjer-Nielsen, L.; Wilce, M. C. J.; Pellicci, D. G.; Koh, R.;
the C20:2 N-acyl variant of
detectable iNKT cell stimulating activity, given that the
a
-fucosylceramide (4a) showed no
Besra, G. S.; Bharadwaj, M.; Godfrey, D. I.; McCluskey, J.; Rossjohn, J. Nature
2007, 448, 44.
a
-galacto-
syl version of this compound is an extremely potent iNKT cell ago-
nist.41 This suggests that modification of the carbohydrate moiety
can significantly alter the influence of the lipid moiety of the ligand
on CD1d presentation and iNKT cell responses. Although the mech-
anism for this remains unclear, it is an important consideration for
synthetic strategies that seek to combine biologically active alter-
ations of the carbohydrate and lipid moieties of iNKT cell ligands.
23. Tashiro, T.; Nakagawa, R.; Inoue, S.; Shiozaki, M.; Watarai, H.; Taniguchi, M.;
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C.-H.; Fang, J.-M.; Lin, C.-C. Tetrahedron 2005, 61, 1855.
26. Yoshino, T.; Watanabe, K.; Hakomori, S.-I. Biochemistry 1982, 21, 928.
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39. Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-hypaque
centrifugation of leucocyte concentrates obtained from healthy volunteer
blood donors. For ex vivo expansion of human iNKT cells, 4 million PBMC were
cultured in wells of 24-well tissue culture plates in RPMI-1640 medium with
10% foetal calf serum and recombinant IL-2 (60 IU/mL) and IL-7 (5 ng/mL)
(Peprotech). Glycolipids were solubilised in 100% DMSO and added directly to
Acknowledgements
G.S.B. acknowledges support in the form of a Personal Research
Chair from Mr. James Badrick, Royal Society Wolfson Research
Merit Award, as a former Lister Institute-Jenner Research Fellow,
the Medical Council and The Wellcome Trust (084923/B/08/7).
S.A.P. and G.B. were supported by NIH/NIAID grant AI45889. Core
resources that facilitated flow cytometry were supported by the
Einstein Center for AIDS Research(AI 051519) and the Einstein Can-
cer Center (CA 13330). The NMR spectrometers used in this re-
search were funded in part through Birmingham Science City
with support from Advantage West Midlands (AWM) and part-
funded by the European Regional Development Fund (ERDF).
References and notes
culture media to achieve
a final concentration of 250 nM. Control wells
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received an amount of DMSO vehicle identical to that added with the
glycolipids (0.00125%). Cultures were incubated for 8 days at 37 °C in a 5%
CO2 humidified incubator. Cultures were harvested and the cells stained with
fluorochrome labelled monoclonal antibodies specific for CD3 and the iNKT cell
TCR (6B11, BD Biosciences). Samples were also stained with propidium iodide
to exclude dead cells, and a known number of Caltag fluorescent counting
beads (Invitrogen) were added to the samples to allow quantitation of absolute
cell numbers. The total number of iNKT cells (CD3+, 6B11+, PI negative
lymphocytes) was calculated by normalising according to counting beads and
the fold expansion values were calculated based on the initial number of iNKT
cells. Data were collected on a FACSCalibur flow cytometer using CellQuest
software (BD Biosciences).
40. Selected data for new compounds. Compound 8: 1H NMR (CDCl3): d 7.92–8.04
(10H, m, Ar-H), 7.10–7.64 (15H, m, Ar-H), 5.42–5.50 (2H, m, H-3Cer, H-4Cer),
5.02 (1H, d, J1,2 = 3.6 Hz, H-1), 4.72–4.84 (6H, m, 3 Â CH2Ph), 3.98 (1H, dd,
J2,3 = 11.5 Hz, H-2), 3.75–3.90 (3H, m, H-4, H-3, H-1aCer), 3.55 (1H, m, H-5), 3.68
(1H, dd, J1a,1b = 10.4, J1b,2 = 8.6 Hz, H-1bCer), 1.88 (2H, m, H-5aCer, H-5bCer), 1.27
(3H, d, J5,6 = 6.4 Hz, H-6), 1.24 (24H, m, CH2), 0.89 (3H, t, CH3); 13C NMR
(75 MHz, CDCl3): d 99.5 (C-1); HRMS calcd for C59H73N3O9 [M+Na]+: 990.5245,
found 990.5248. Compound 9: 1H NMR (CD3OD): d 4.73 (1H, d, J1,2 = 3.7 Hz,
H-1), 3.91–3.96 (2H, m, H-3, H-4), 3.68–3.72 (1H, dd, J1a,1b = 9.9, J1b,2 = 3.9 Hz,
H-1aCer), 3.61–3.68 (2H, m, H-4, H-3Cer, H-4Cer), 3.52–3.55 (1H, dd, J2,3 = 6.0 Hz,
H-2), 3.43–3.49 (1H, m, H-5), 3.39–3.42 (1H, dd, J1b,2 = 8.6 Hz, H-1bCer), 3.35
(1H, m, H-2Cer), 1.89 (2H, m, H-5aCer, H-5bCer), 1.28 (3H, d, J5,6 = 6.4 Hz, H-6),
1.24 (24H, m, CH2), 0.89 (3H, t, CH3); 13C NMR (75 MHz, CD3OD): d 102.5 (C-1);
HRMS calcd for C24H49NO7 [M+Na]+: 486.3403, found 486.3309. Compound 11:
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Immnunol. 2003, 21, 483.
1H NMR (CDCl3):
d 7.36–7.86 (10H, m, Ar-H), 5.60 (1H, dd, J3,4 = 4.8,
J3,2 = 6.8 Hz, H-3Cer), 5.51 (1H, ddd, J4,5a = 4.2, J4,5b = 8.5 Hz, H-4Cer), 4.82 (1H,
d, J1,2 = 2.8 Hz, H-1), 3.88–4.00 (3H, m, H-2, H-4, H-5), 3.62–3.80 (3H, m, H-3,
H-2Cer, H-1aCer), 3.71 (1H, dd, J1a,1b = 10.4, J1b,2 = 6.5 Hz, H-1bCer), 1.76–1.91 (2H,
m, H-5aCer, H-5bCer), 1.10–1.43 (24 H, m, CH2), 1.07 (3H, d, J5,6 = 6.6 Hz, H-6),
0.85 (3H, t, CH3); 13C NMR (75 MHz, CDCl3): d 100.9 (C-1); HRMS calcd for
C38H55N3O10 [M+Na]+: 720.3836, found 720.3811. Compound 4a: HRMS calcd
for C44H83NO8 [M+Na]+: 776.6013, found 776.6011. Compound 4b: HRMS calcd
for C42H83NO9 [M+Na]+: 768.5962, found 768.5941. Compound 4c: HRMS calcd
for C48H95NO8 [M+Na]+: 836.6956, found 836.6953. Compound 4d: HRMS calcd
for C50H99NO8 [M+Na]+: 864.7268, found 864.7240.
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