Synthesis of diglycosylceramides and evaluation of their iNKT cell stimulatory properties

Article abstract of DOI:10.1016/j.bmcl.2007.12.067

Stimulation of iNKT cells is highly dependent on the structures of the glycolipids presented by CD1d. Furthermore, antigen processing and CD1d loading in lysosomes play central roles in controlling the stimulatory properties of glycolipid antigens. Previo

Full text of DOI:10.1016/j.bmcl.2007.12.067

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 3052–3055
Synthesis of diglycosylceramides and evaluation
of their iNKT cell stimulatory properties
Yang Liu,a Shenglou Deng,a Li Bai,b Stefan Freigang,c Jochen Mattner,b
Luc Teyton,c Albert Bendelacb and Paul B. Savagea,*
aDepartment of Chemistry and Biochemistry, C100 BNSN, Brigham Young University, Provo, UT 84602, United States
bHoward Hughes Medical Institute, Department of Pathology, 929 East 7th Street, University of Chicago,
Chicago, IL 60637, United States
cImmunology Department, 10550 North Torrey Pines Road, Scripps Research Institute, La Jolla, CA 92037, United States
Received 23 October 2007; revised 15 December 2007; accepted 17 December 2007
Available online 1 January 2008
Abstract—Stimulation of iNKT cells is highly dependent on the structures of the glycolipids presented by CD1d. Furthermore, anti-
gen processing and CD1d loading in lysosomes play central roles in controlling the stimulatory properties of glycolipid antigens.
Previously, we determined that substitution at C600 on a-galactosylceramides did not significantly impact stimulatory properties;
however, it was not known if substitution at this position influenced lysosomal processing of oligoglycosylceramides. We have pre-
pared a series of mono- and di-galactosylceramides to observe the impact of C600 substitution on glycosidase truncation of these
glycolipids. We found that substitution did not significantly impact glycosidase activity or loading into CD1d.
Ó 2007 Elsevier Ltd. All rights reserved.
Natural killer T (NKT) cells are a subset of T cells and
have been characterized as regulatory T cells.1 The most
prevalent and best studied subset of NKT cells expresses
an invariant T cell Va14/24 (mouse/human) receptor
and is referred to as invariant NKT cells (iNKT) cells.
Through this invariant T cell receptor, iNKT cells recog-
nize bacterial and endogenous glycolipid antigens pre-
sented by CD1d, an antigen presentation protein
related to major histocompatibility complex proteins.
The hallmark response of iNKT cells is the rapid pro-
duction and release of large quantities of cytokines,
including proinflammatory cytokines (e.g., IFN-c, IL-
2) and immuno-modulatory cytokines (e.g., IL-4). These
cytokines play critical roles in inducing a series of events
leading to the activation of innate and adaptive immune
cells. The most well-studied ligand for iNKT cells is a
glycolipid, KRN7000 (Fig. 1), developed through anti-
tumor structure–activity studies of glycolipids from the
marine sponge Agelas mauritianus.2 In further structure
activity studies using cytokine release from NKT cells as
an end point, we have determined that shortening the
acyl chain in KRN7000 to eight carbons (from 26) yields
a compound (1 in Fig. 1) that loads effectively into
CD1d without dependence on lipid-transfer proteins
that are required for loading most glycolipids into
CD1d.3,4 We have also found that replacement of the
hydroxyl group at C600 (see Fig. 1 for glycosylceramide
numbering) with an acetylamide gives a compound, 2,
that is a more potent stimulator of NKT cells than
KRN7000 (both in vitro and in vivo).5
An important aspect of glycolipid presentation by CD1d
and recognition by iNKT cells is lysosomal processing of
glycolipids. Lysosomes in antigen-presenting cells con-
tain an array of glycosidases that can truncate oligogly-
cosylceramides to monoglycosylceramides. Work by
Prigozy et al.6 demonstrated that diglycosylceramides
with 1–2 or 1–3 glycosidic linkages (e.g., 3 in Fig. 1) re-
quire lysosomal processing and truncation to KRN7000
for presentation by CD1d and stimulation of iNKT cells.
CD1d is cycled between lysosomes, where it is loaded
with glycolipids, and the cell surface, where it presents
bound glycolipids to iNKT cells. Deletion of the peptide
sequence required for this cycling yields ‘tail-deleted’
CD1d (tdCD1d), which does not effectively cycle between
the cell surface and lysosomes.7 Consequently, glycolip-
ids that are processed in the lysosome are not loaded into
Keywords: Glycosylation; Glycosphingolipid synthesis; Stimulatory
activity; IL-2 release; NKT cells.
*
Corresponding author. Tel.: +1 8014224020; fax: +1 8014220153;
e-mail: pbs@byu.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.067

Y. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3052–3055
3053
OH
OH
HO
HO
C2'
C6"
O
O
O
O
HO
HO
HN
OH
HN
OH
OH
OH
O
O
C2
OH
OH
1
KRN7000
O
NH
OH
HO
HO
HO
O
O
O
O
HO
HN
OH
HN
OH
HO
O
HO
O
O
HO
HO
OH
OH
O
2
3
OH
Figure 1. Structure of glycolipids KRN7000, 1, 2, and 3.
CD1d, and tdCD1d is useful for observing dependence
on lysosomal glycolipid processing.6,7
pected. That is, 6 should lose stimulatory properties with
tdCD1d.
The rationale for preparing glycolipids substituted at
C600 was to use groups appended at this position to mon-
itor trafficking, processing, and presentation of glycolip-
ids.8 We have found that fluorophores appended at C600
do not significantly alter stimulation of iNKT cells.9
However, we have not determined if C600 substitution af-
fects lysosomal processing. To this end, we prepared a
series of diglycosylceramides including one with C600
substitution. For this series, we used the C8 acyl chain
in 1 to remove possible influences of lipid-transfer pro-
teins10,11 and thereby only focus on activities of lyso-
somal glycosidases.
The synthesis of disaccharide 4 is described in Scheme 1.
A Schmidt coupling12 of 7 and 8 gave the corresponding
disaccharide, and the anomeric hydroxyl group was re-
vealed after deacylation with the mono-acetate salt of
ethylene diamine. The disaccharide was coupled with
the appropriately protected ceramide using Gin’s meth-
od,13 and deprotection gave 4.
The preparation of C600 modified galactosylceramide 5 is
detailed in Scheme 2. The synthesis borrows from work
with fluorophore labeled galactosylceramides.9 Cou-
pling of C6 azido galactose 12 with ceramide gave 13.
Dissolving metal deprotection also caused reduction of
the azide to the amine, and subsequent acylation gave 5.
The glycolipids prepared to determine the effects of C600
substitution on lysosomal processing are shown in Fig-
ure 2. Comparison of iNKT cell stimulation of 1 and
4 with wild-type CD1d and tdCD1d provides a measure
of the influences of lysosomal processing on these glyco-
lipids. Glycolipid 1 would be expected to load into
CD1d well and stimulate iNKT cells comparably with
wild-type and tdCD1d. However, the requirement for
processing would make 4 less stimulatory with tdCD1d.
If the substitution at C600 does not significantly influence
lysosomal trafficking, then the same trend would be ex-
For the preparation of 6 (Scheme 3), C6 azido acceptor
15 was used, and a pathway similar to that used for the
synthesis of 4 was followed, except that deprotection
was achieved using dissolving metal conditions. The
acyl group was installed as described in the preparation
of 5.
The iNKT cell stimulatory properties of glycolipids 1, 4,
5, and 6 were determined using iNKT cell hybridoma
O
O
OH
NH
NH
HO
HO
HO
O
HO
HO
O
O
O
O
O
HO
HN
OH
HN
OH
HN
OH
O
HO
O
O
HO
O
O
HO
HO
HO
HO
HO
OH
OH
OH
O
O
4
6
5
OH
OH
Figure 2. Structures of diglycosyl and C600 appended glycolipids used in the study of lysosomal processing of C600 substituted glycolipids.
OAc
OAc
O
AcO
AcO
AcO
AcO
O
C7H15
OAc
OAc
OBn
O
O
C7H15
OAc
BnO
BnO
O
AcO
AcO
NH
CCl3
HN
HN
OH
c
a,b
d
O
O
O
4
HO
BnO
BnO
O
O
C14H29
C14H29
OBn
HO
OAc
BnO
BnO
BnO
BnO
OAc
10
O
O
OAc
7
8
9
11
OBn
OBn
Scheme 1. Preparation of diglycosylceramide 4. Reagents (yields in parentheses) (a) TMSOTf, CH2Cl2 (40%); (b) elthylene diamine, AcOH, THF
(75%); (c) diphenylsulfoxide, Tf2O, tri-t-butylpyrimidine, CH2Cl2 (71%); (d) H2, Pd/C, THF, methanol; MeONa, methanol (56%).

3054
Y. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3052–3055
O
NH2
N3
N3
HO
HO
BnO
BnO
O
C7H15
OAc
BnO
BnO
O
O
a
C7H15
OH
O
b
HN
c
C7H15
OAc
O
5
HN
HN
HO
F
HO
BnO
C14H29
BnO
O
O
C14H29
C14H29
OAc
10
12
OH
14
OAc
13
Scheme 2. Preparation of C600 N-acyl amino substituted galactosylceramide 5. Reagents (yields in parentheses) (a) AgClO4, SnCl2 CH2Cl2 (54%); (b)
Na°, NH3 (40%); (c) AcOH, HOBt, DCC (90%).
N3
N3
N3
AcO
AcO
AcO
AcO
AcO
AcO
O
N3
OBn
O
O
O
BnO
BnO
O
O
O
O
C7H15
OAc
AcO
AcO
NH
CCl3
OH
HN
a
d,e
c
b
O
6
BnO
BnO
BnO
O
O
OAc
C14H29
OBn
HO
OAc
15
BnO
BnO
BnO
BnO
BnO
BnO
O
O
O
OAc
7
16
17
18
OBn
OBn
OBn
Scheme 3. Preparation of C600 N-acyl amino substituted di-galactosylceramide 6. Reagents (yields in parentheses) (a) TMSOTf, CH2Cl2 (37%); (b)
ethylene diamine, AcOH, THF (64%); (c) diphenylsulfoxide, Tf2O, tri-t-butylpyrimidine, CH2Cl2 (53%); (d) Na/NH3, (27%); (e) AcOH, DCC, HOBt
(22%).
and dendritic cells as antigen-presenting cells. Dendritic
cells generate a well-established lysosome and effectively
truncate many oligoglycosylceramides to stimulatory
compounds. As shown in Figure 3A, all four of the gly-
colipids stimulated cytokine release. The monoglycosyl-
ceramides are not dependent on processing and can load
into CD1d directly, and as expected they proved to be
more potent than the diglycosylceramides. Notably,
C600 did not play a significant role in stimulatory proper-
ties. To further establish the dependency of the diglyco-
sylceramides for lysosomal processing, dendritic cells
with tdCD1d were used as antigen-presenting cells. Be-
cause tdCD1d does not effectively sample the lipids in
lysosomes, it was expected that stimulation with 4 and
6 would be decreased with tdCD1d. As shown in Figure
3B, this trend was observed, while the monoglycosyl-
ceramides were less dependent on CD1d sampling of
lysosomes.
tution at C600 indicate that substitution at this position
does not play a significant role in stimulatory properties
of these glycolipids or their abilities to act as substrates
for lysosomal glycosidases. Small changes in glycosyl-
ceramide structure can change iNKT cell stimulatory
properties dramatically: a-galactosylceramides (e.g.,
KRN7000 and 1) are potent stimulators while b-galacto-
sylceramide is only very weakly active or inactive;
a-glucosylceramide is much less potent than a-galactosyl-
ceramide. However, the C600 position appears to be well
suited for manipulation without significantly impacting
glycolipid processing and presentation. We have shown
that fluorophores of modest size (e.g., a danyl group)
can be appended at C600 without significantly altering
NKT cell responses,9 and the acetamide at C600 in 6 does
not interfere with processing. It is anticipated that larger
groups, such as fluorophores of modest size, appended at
this position with no impact processing; however, this has
not yet been demonstrated. Furthermore, it is not yet well
understood how substitution at this position affects gly-
colipid trafficking and lipid-transfer protein interactions,
Comparisons of the iNKT cell stimulatory properties of
mono- and diglycosylceramides with and without substi-
Figure 3. Comparison of stimulatory activity on mouse Va14 iNKT hybridoma DN3.2.D3 by murine bone marrow-derived dendritic cells in the
presence of different concentrations of glycosylceramides 1, 4, 5, and 6. Indicated are the mean release amounts of IL-2 (pg/mL) in cell culture
supernatants determined by ELISA. (A) Wild-type CD1d. (B) Comparison of stimulation with wild-type CD1d and tdCD1d.

Y. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3052–3055
3055
3. Goff, R. D.; Gao, Y.; Mattner, J.; Zhou, D. P.; Yin, N.;
Cantu, C.; Teyton, L.; Bendelac, A.; Savage, P. B. J. Am.
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and studies are ongoing to determine impacts on these
interactions.
4. Zajonc, D. M.; Cantu, C.; Mattner, J.; Zhou, D. P.;
Savage, P. B.; Bendelac, A.; Wilson, I. A.; Teyton, L. Nat.
Immunol. 2005, 6, 810.
Acknowledgments
5. Liu, Y.; Goff, R. D.; Zhou, D.; Mattner, J.; Sullivan, B. A.;
Khurana, A.; Cantu, C.; Altman, J. D.; Teyton, L.;Bendelac,
A.; Savage, P. B. J. Immunol. Methods 2006, 312, 34.
6. Prigozy, T. I.; Naidenko, O.; Qasba, P.; Elewaut, D.;
Brossay, L.; Khurana, A.; Natori, T.; Koezuka, Y.;
Kulkarni, A.; Kronenberg, M. Science 2001, 291, 664.
7. Chiu, Y.-H.; Park, S.-H.; Benlagha, K.; Forestier, C.;
Jayawardena-Wolf, J.; Savage, P. B.; Teyton, L.; Bende-
lac, A. Nat. Immunol. 2002, 3, 55.
8. Sagiv, Y.; Hudspeth, K.; Mattner, J.; Schrantz, N.; Stern,
R. K.; Zhou, D.; Savage, P. B.; Teyton, L.; Bendelac, A. J.
Immunol. 2006, 177, 26.
9. Zhou, X. T.; Forestier, C.; Goff, R. D.; Li, C. H.; Teyton,
L.; Bendelac, A.; Savage, P. B. Org. Lett. 2002, 4, 1267.
10. Zhou, D.; Cantu, C. C.; Sagiv, Y.; Kulkarni, A. B.; Qi, X.;
Morales, C.; Grabowski, G. A.; Benlagha, K.; Savage, P.
B.; Bendelac, A.; Teyton, L. Science 2004, 303, 523.
11. Schrantz, N.; Sagiv, Y.; Liu, Y.; Savage, P. B.; Bendelac,
A.; Teyton, L. J. Exp. Med. 2007, 204, 841.
We gratefully acknowledge generous financial support
from the National Institutes of Health (NIAID
AI053725). J.M. is a CRI Fellow and was supported
by a grant from LRI. S.F. is a Swiss National Science
Foundation/SSMBS Fellow.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2007.
12.067.
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