Synthesis and biological activity of α-glucosyl C24:0 and C20:2 ceramides

Article abstract of DOI:10.1016/j.bmcl.2010.05.010

α-Glucosyl ceramides 4 and 5 have been synthesised and evaluated for their ability to stimulate the activation and expansion of human iNKT cells. The key challenge in the synthesis of both target molecules was the stereoselective synthesis of the α-glycos

Full text of DOI:10.1016/j.bmcl.2010.05.010

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3475–3478
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of
a-glucosyl C24:0 and C20:2 ceramides
Peter J. Jervis a, Natacha Veerapen a, Gabriel Bricard c, Liam R. Cox b, Steven A. Porcelli c, Gurdyal S. Besra a,
*
a School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
b School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
c Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, 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:
a
-Glucosyl ceramides 4 and 5 have been synthesised and evaluated for their ability to stimulate the acti-
vation and expansion of human iNKT cells. The key challenge in the synthesis of both target molecules
was the stereoselective synthesis of the -glycosidic linkage. Of the methods examined, glycosylation
using per-TMS-protected glucosyl iodide 16 was completely -selective and provided gram quantities
of amine 11, from which -glucosyl ceramides 4 and 5 were obtained by N-acylation. -GlcCer 4, contain-
ing a C24 saturated acyl chain, stimulated a marked proliferation and expansion of human circulating
iNKT cells in short-term cultures. -GlcCer 5, which contains a C20 11,14-cis-diene acyl chain (C20:2),
induced extremely similar levels of iNKT cell activation and expansion.
Received 29 March 2010
Revised 4 May 2010
Accepted 5 May 2010
Available online 10 May 2010
a
a
a
a
Keywords:
CD1d
iNKT
Antigen
Ceramide
Lipid
a
Ó 2010 Elsevier Ltd. All rights reserved.
CD1d is a non-polymorphic glycoprotein expressed on the sur-
face of antigen-presenting cells (APCs). It is specifically associated
with presenting lipid antigens that activate the distinctive class of
T cells known as invariant Natural Killer T (iNKT) cells. iNKT cells
display characteristics of both T cells and NK cells and play a
crucial role in diverse immune responses and other pathologic con-
O
O
OH
OH
OH OH
HN
O
HN
O
n
OH
9
OH
4
HO
HO
HO
HO
O
O
13
13
OH
OH
ditions.1–4 When the synthetic glycolipid
a
-galactosyl ceramide
1 (KRN7000) (n = 24)
2 (n = 22)
Th1 and Th2 response
3
-GalCer),5 also known as KRN7000 (1, Fig. 1), is bound to CD1d
Th2-biased response
(a
and presented to T cell receptors (TCRs) on the surface of iNKT
cells, the latter are activated to release diverse cytokines, including
both Th1 and Th2 cytokines.6–8 Similar results are obtained with
the more readily obtained C24:0 analogue (2, Fig. 1).9,10 It is be-
lieved that the release of Th1 cytokines may contribute to antitu-
mour and antimicrobial functions, whilst the secretion of Th2
cytokines may help alleviate autoimmune diseases11–13 such as
multiple sclerosis14 and arthritis.15 The opposing effects induced
by Th1 and Th2 cytokines have complicated efforts to develop
KRN7000 as a therapeutic agent, since it induces high levels of both
types of cytokine and therefore may induce mixed and unpredict-
able biological effects.16 Switching the C26:0 acyl chain of
KRN7000 for a C20 11,14-cis-diene acyl chain modifies the out-
come of iNKT cell activation and potently induces a Th2-biased
cytokine response.9 This C20:2 analogue (3, Fig. 1) also exhibits less
stringent requirements for loading on to CD1d.10
Figure 1. -Galactosyl ceramides 1 (C26:0), 2 (C24:0) and 3 (C20:2).
a
and sphingosine structures of a-GalCer, there has been less analy-
sis of the effects of structural modifications of the carbohydrate
head group.17 Subtle changes in this part of the glycolipid are likely
to have significant effects on iNKT cell recognition since the mono-
saccharide group is exposed and makes direct contacts with the
TCR in complexes formed by the binding of
To this end, we now report the synthesis and preliminary biologi-
a
-GalCer to CD1d.18
cal activity of a-glucosyl ceramide analogues 4 and 5 (Fig. 2).
O
O
OH
OH
HN
HN
O
O
9
OH
HO
HO
HO
22
OH
4
HO
Although extensive studies have examined the impact on the
iNKT cell-stimulating activities of modifications to the fatty acyl
HO
HO
O
O
13
13
OH
OH
4
5
* Corresponding author. Tel.: +44 (0) 121 4158125.
E-mail address: g.besra@bham.ac.uk (G.S. Besra).
Figure 2. Target a-glucosyl ceramides 4 (C24) and 5 (C20:2).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.010

3476
P. J. Jervis et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3475–3478
OBn
O
Since targets 4 and 5 differ only in their acyl chain substitution,
OBn
O
we elected to pursue a synthetic strategy that would allow the
introduction of this point of diversity in the final step. We therefore
examined several routes to amine 11 from which both glucosyl
ceramide targets would then be accessed through chemoselective
acylation of the amino residue. The key challenge in a synthesis
a or b
BnO
BnO
BnO
BnO
N3 OBn
OBn
BnO
X
O
BnO
13
12 (X = OH, α:β mixture)
13 (X = β-F)
14
of amine 11 is to form the glycosidic linkage with high a-selectiv-
Scheme 2. Reagents: (a) From 12: CBr4, PPh3, CH2Cl2, then 8, nBu4NBr, tetramethyl
urea, 67% ( :b ratio: 1:1); (b) from 13: 8, SnCl4, AgIO4, THF, 41% ( :b ratio: 5:1).
ity. To this end, we first opted to employ a stereospecific glucosy-
lation method developed by Bols (Scheme 1).19 This method
involves the use of a silyl tether to attach the acceptor temporarily
to the 2-position of the glucosyl donor prior to the key glycosyla-
tion step. Glycosylation proceeds with 1,2-syn specificity, owing
to the formation of a five-membered silylacetal intermediate,
which in the case of glucosyl donors, ensures the formation of
a
a
variety of alcohol acceptors.26 The reaction conditions for this gly-
cosylation are also extremely mild and the silyl protecting groups
are easily removed using an acid work-up. We reasoned that the
use of phytosphingosine acceptor 17,27 in which the internal
1,2-diol is protected as an acetal, would deliver the completely
O-deprotected glucoside 18 upon acid work-up. To this end,
1,2,3,4,6-penta-O-trimethylsilyl glucose 15, which is commercially
available or can be readily synthesised on large scale by treating
the
steps from
a
-glycoside product. Thioglucoside 7, synthesised in three
D
-glucal 6,20 was reacted with a fivefold excess of
dichlorodimethylsilane. This reaction afforded a silyl chloride
intermediate, which, after removal of the excess dichlorosilane re-
agent under reduced pressure, reacted with known alcohol 821 to
form mixed silyl acetal 9, our glucosylation precursor, in modest
yield. Treatment of silyl acetal 9 with N-iodosuccinimide (NIS) fur-
nished the desired glucoside 10 as a single diastereoisomer, albeit
in modest yield. Hydrogenolysis of the benzyl groups and reduc-
tion of the azide in 10 using Pd(OH)2 as the catalyst,22 provided
our acylation precursor, amine 11 in 57% yield (Scheme 1).
Although this synthetic approach allowed a completely stereo-
selective route to our target amine 11, a number of steps in the se-
quence suffered from poor yields, which hindered access to
significant quantities of material. We therefore examined other
glycosylation methods. Kobayashi has described a stereoselective
glucose with
a mixture of TMSCl and hexamethyldisilazane
(HMDS) in pyridine,28 was converted to glycosyl iodide 16 by treat-
ment with TMSI in CH2Cl2 (Scheme 3). Adding a solution of crude
16 to a solution of alcohol 17, nBu4NI, Hünig’s base and 4 Å molec-
ular sieves in CH2Cl2 successfully effected glycosylation. Treating
the initially formed glycoside product with p-toluenesulfonic acid
(pTSA) in methanol provided the fully O-deprotected glycoside
18 as a single anomer. Although the yield for this three-step pro-
cess was a modest 45%, we now had very rapid access to our target
molecules. A final Staudinger reduction of azide 18 delivered our
requisite amine 11 in quantitative yield (Scheme 3).29 This reaction
sequence is short and scalable and proved to be particularly
effective for accessing multigram quantities of amine 11. The final
a
-glycosylation using a galactosyl bromide generated in situ from
2,3,4,6-tetra-O-benzyl galactose.23 Unfortunately, we found that
glycosylation using the corresponding glucosyl bromide derived
from 12 afforded significant amounts of the unwanted b-anomer
(Scheme 2). The use of perbenzylated glucosyl fluoride 1324 also
OTMS
O
OTMS
O
a
b
TMSO
TMSO
TMSO
TMSO
provided a mixture of a- and b-glycosides 14, which proved diffi-
cult to separate (Scheme 2).
We therefore turned our attention to the use of glucosyl
iodides,25 specifically per-TMS-protected glucosyl iodide 16, as
an alternative donor. Du et al. have shown that the corresponding
TMSO
TMSO
I
OTMS
16
15
OH
N3
O
galactosyl iodide provides excellent levels of
a-selectivity with a
O
HO
HO
HO
N3 OH
OH
13
HO
OH
O
OBn
O
ref 20
13
O
O
HO
HO
BnO
BnO
(3 steps)
18
SEt
acceptor 17
OH
D-glucal
6
7
c
O
OH
OH
a,b
HN
O
O
HO
HO
HO
HO
22
OH
d
OBn
O
N3 OBn
NH2 OH
OH
HO
HO
HO
O
O
BnO
BnO
SEt
N3 OBn
13
13
13
OBn
OH
O
O
11
4
Si
acceptor 8
13
OBn
9
e
O
OH
c
HN
OBn
O
OH
O
9
OH
HO
HO
4
O
BnO
BnO
HO
HO
d
HO
O
N3 OBn
OBn
NH2 OH
OH
HO
HO
13
O
O
OH
13
13
5
10
11
Scheme 3. Reagents: (a) TMSI, CH2Cl2; (b) 17, nBu4NI, iPr2NEt, 4 Å molecular sieves,
CH2Cl2; then pTSA, MeOH, 45% from 15; (c) PMe3, wet THF, quant.; (d) tetracosanoyl
chloride, THF/8 M NaOAc, 68%; (e) 11,14-eicosadienoyl chloride, THF/8 M NaOAc,
66%.
Scheme 1. Reagents: (a) Me2SiCl2, pyridine, toluene; (b) acceptor 8, pyridine,
toluene, 38% over two steps; (c) NIS, MeNO2, 47%; (d) H2, Pd(OH)2, CHCl3/MeOH
(1:1), 57%.

P. J. Jervis et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3475–3478
3477
acylation reactions were accomplished by adding either tetracosa-
noyl chloride or 11,14-eicosadienoyl chloride (formed from the
corresponding carboxylic acids using oxalyl chloride) in THF to
amine 11 in a vigorously stirred biphasic mixture of THF and 8 M
NaOAc solution. Both reactions provided the desired amide prod-
ucts 4 and 5 in good yields (Scheme 3).30,31
The strong biological activity of the
consistent with findings from the initial study that described the
a
-GlcCer compounds was
reactivity of CD1d-restricted iNKT cells to synthetic glycosylcera-
mides.5 This showed an
a-GlcCer with a C26 saturated acyl group
to be stimulatory for mouse iNKT cells, with a level of activity only
slightly less than that of KRN7000 (1). Our analysis confirms the
To assess the biological activity of the
and 5 and compare these to KRN7000 1 and the
a
-glucosyl ceramides 4
activity of
It is also notable that we observed human iNKT cell activation
and expansion for an -GlcCer with a shorter acyl chain containing
unsaturations (5). Previous work with analogues of -GalCer con-
taining C20:2 or other unsaturated fatty acyl groups revealed a
marked tendency for these to bias iNKT cell-dependent cytokine
responses in mice to give preferential secretion of Th2 cytokines
such as IL-4 and IL-13.9 This Th2 cytokine bias has been associated
with therapeutic benefits in a variety of mouse models of autoim-
mune and inflammatory diseases, indicating potential therapeutic
applications for such glycolipids in human diseases.17 It will thus
a-GlcCer compounds as ligands for human iNKT cells.
a
-galactosyl cera-
mide analogues 2 (C24:0) and 3 (C20:2), we assessed the ability of
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.32 The results showed that both the per-
centages and absolute numbers of iNKT cells in the cultures were
markedly increased to similar levels by stimulation with both of
a
a
the
a-GlcCer analogues 4 and 5 (Fig. 3). The level of iNKT cell
expansion, at least with a relatively high concentration of the gly-
colipids (250 nM), was comparable for both of the N-acyl variants
of
a-GlcCer and very similar to levels obtained with the related
be important to determine whether compound 5 or other a-GlcCer
a
-GalCer analogues (2 (C24:0) and 3 (C20:2)) and with the proto-
analogues bearing an unsaturated acyl chain also show an ability
to induce Th2-biased cytokine responses, which is a focus for fu-
ture studies.
typical iNKT cell activator KRN7000 (1 (C26:0)). Representative
profiles obtained by flow cytometry of cultures from one normal
blood donor are shown in Figure 3A. This analysis was carried
out with PBMC from four separate donors (Fig. 3B). Although differ-
ences were observed for the levels of iNKT cell expansion between
different donors, all donors responded well to the two
analogues. In all cases, these responses were similar to those gen-
erated by the analogous -GalCer compounds.
In summary, we have developed an efficient route to a-glucosyl
ceramides that provided two biologically active ligands 4 and 5 for
stimulation of human iNKT cell responses. Of the range of glycosyl-
ation methods that were investigated for accessing the target
molecules with high levels of stereoselectivity, the use of per-
TMS-protected glucosyl iodide 16 as the donor is the most attrac-
a-GlcCer
a
tive, reacting with acceptor 17 to provide a single
a-glycoside
product. This glycosylation reaction is also scalable and with an
acidic work-up effecting global deprotection, followed by Stau-
dinger reduction of the azide, allows rapid access to advanced
intermediate 11, which can now be used to provide a broad range
of
a-GlcCer compounds with different acyl chains. Compounds
produced using this approach will assist in expanding the current
understanding of the structure–activity relationships for glycolipid
activators of iNKT cells, which is of central importance to the fur-
ther development of this class of compounds as clinically useful
immunomodulators.
Acknowledgements
G.S.B. acknowledges support from a Personal Research Chair
from Mr. James Bardrick, a Royal Society Wolfson Research Merit
Award, a former Lister Institute-Jenner Research Fellowship, the
Medical Research Council and The Wellcome Trust (084923/B/08/
Z). S.A.P. and G.B. were supported by NIH/NIAIDGrant AI45889. Core
resources that facilitated flow cytometry were supported by the
Einstein Center for AIDS Research (AI 051519) and the Einstein
Cancer Center (CA 13330). The NMR spectrometers used in this
research were funded in part through Birmingham Science
City: Innovative Uses for Advanced Materials in the Modern World
(West Midlands Centre for Advanced Materials Project 2), with
support from Advantage West Midlands (AWM) and part-funded
by the European Regional Development Fund (ERDF).
Supplementary data
Figure 3. Ex vivo expansion of human iNKT cells by
a-GlcCer and a-GalCer
analogues. Peripheral blood mononuclear cells (PBMC) from four different donors
were stimulated with the indicated glycolipids at a concentration 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 antibodies specific for CD3 and
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.010.
for the invariant TCRa chain expressed by iNKT cells (6B11). (A) Dot plots showing
References and notes
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.
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a
-GlcCer 4: Mp 151–152 °C; Rf = 0.21 (10% MeOH in CHCl3); ½a D20
ꢀ
+47.2
(c 0.50, CHCl3/MeOH (1:1)); m
max(film)/cmꢁ1 3336 br s (OH, NH2), 2917 s, 2850
s, 1623 m (C@O), 1463 m, 1350 s, 1143 s, 1071 m, 1033 m, 798 s, 718 m; dH
(500 MHz, CDCl3/CD3OD (2:1)) 0.88 (6H, t, J = 6.7, 2ꢂ terminal CH3), 1.20–1.44
(64H, stack, alkyl chain), 1.51–1.69 (4H, stack, alkyl chain), 2.19 (2H, app. t,
J = 7.8, CH2CONH), 3.31–3.37 (1H, m, 4-H), 3.45 (1H, dd, J = 9.8, 3.8, 2-H), 3.52–
3.59 (3H, stack, 5-H, 30-H, 40-H), 3.62 (1H, app. t, J = 9.5, 3-H), 3.65 (2H, stack, 6-
Ha, 10-Ha), 3.79 (1H, dd, J = 12.0, 2.5, 6-Hb), 3.86 (1H, dd, J = 10.5, 4.5, 10-Hb),
4.17 (1H, app. q, J = 4.5, 20-H), 4.85 (1H, d, J = 3.5, 1-H), CONH resonance not
observed; dC (125 MHz, CHCl3/CD3OD (3:1)) 13.5 (CH3, 2ꢂ terminal CH3), [22.2,
25.5, 28.9, 29.0, 29.2, 29.27, 29.30, 29.4, 31.5, 32.0, 36.0 (CH2, alkyl chain, some
overlapping resonances)], 50.1 (CH, C-20), 61.2 (CH2, C-6), 66.8 (CH2, C-10), 69.9
(CH, C-4), [71.6 (CH), 71.6 (CH, C-2), overlapping resonances], 71.8 (CH), 73.5
(CH, C-3), 74.2 (CH, C-30), 98.9 (CH, anomeric-C), 174.2 (quat. C, CONH); m/z
(TOF ES+) 852.8 ([M+Na]+, 100%); HRMS m/z (TOF ES+) 852.6912. C48H95NO9Na
requires 852.6905.
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a
-GlcCer 5: Mp 83–85 °C; Rf = 0.23 (10% MeOH in CHCl3); ½a D20
ꢀ
+41.3 (c
0.50, CHCl3/MeOH (1:1)); m
max(film)/cmꢁ1 3300 br s (OH), 3009 m, 2922 s,
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2851 s, 1697 m (C@O), 1633 s (C@C), 1540 m, 1466 m, 1378 w, 1282 w, 1215
w, 1044 m, 760 m; dH (400 MHz, CDCl3/CD3OD (2:1)) 0.86 (3H, t, J = 6.6,
terminal CH3), 0.87 (3H, t, J = 7.0, terminal CH3), 1.17–1.43 (42H, stack, alkyl
chain), 1.47–1.64 (4H, stack, alkyl chain), 2.00–2.08 (4H, stack,
CH2CH@CHCH2CH@CHCH2), 2.19 (2H, app. t, J = 7.6, CH2CONH), 2.75 (2H,
app. t, J = 6.4, CH@CHCH2CH@CH), 3.31–3.38 (1H, m, 4-H), 3.44 (1H, dd, J = 9.8,
3.7, 2-H), 3.51–3.58 (2H, stack, 5-H, 40-H), 3.58–3.73 (4H, stack, 10-Ha, 30-H, 3-H,
6-Ha), 3.78 (1H, dd, J = 11.8, 2.6, 6-Hb), 3.84 (1H, dd, J = 10.6, 4.2, 10-Hb), 4.11–
4.18 (1H, m, 20-H), 4.84 (1H, d, J = 3.7, 1-H), 5.25–5.40 (4H, stack, 2ꢂ CH@CH),
CONH resonance not observed; dC (100 MHz, CDCl3) [15.2, 15.3 (CH3, 2ꢂ
terminal CH3)], [23.9, 24.0, 26.9, 27.2, 28.5, 28.6, 30.76, 30.81, 30.9, 31.1, 32.9,
33.3, 33.4, 37.7 (CH2, alkyl chain, some resonance overlap)], 51.7 (CH, C-20),
62.8 (CH2, C-6), 68.6 (CH2, C-10), 71.5 (CH, C-2), 73.26 (CH), 73.32 (CH), 73.6
(CH), 75.1 (CH, C-3), 75.6 (CH, C-30), 100.6 (CH, anomeric-C), 129.26 (CH,
CH@CH), 129.30 (CH, CH@CH), 131.38 (CH, CH@CH), 131.45 (CH, CH@CH),
175.8 (quat. C, CONH); m/z (TOF ES+) 792.7 ([M+Na]+, 100%); HRMS m/z (TOF
ES+) 792.5963. C44H83NO9Na requires 792.5966.
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32. Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll–Hypaque
centrifugation of leukocyte 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
22. Lu, X.; Bittman, R. Tetrahedron Lett. 2005, 46, 3165.
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, eBiosciences). 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
28. Toubiana, R.; Das, B. C.; Defaye, J.; Mompon, B.; Toubiana, M. J. Carbohydr. Res.
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iNKT cells. Data were collected on a FACSCalibur flow cytometer using
CellQuest software (BD Biosciences).