Synthesis and NKT Cell Stimulating Properties of Fluorophore- and Biotin-Appended 6″-Amino-6″-deoxy-galactosylceramides

Article abstract of DOI:10.1021/ol025565+

(Matrix Presented) α-Galactosylceramides are potent stimulators of human T cells. Stimulation occurs through binding of the glycolipids by CD1d, Presentation to T cells, and formation of a CD1d-glycolipid-T cell receptor complex. To facilitate the elucida

Full text of DOI:10.1021/ol025565+

ORGANIC
LETTERS
2002
Vol. 4, No. 8
1267-1270
Synthesis and NKT Cell Stimulating
Properties of Fluorophore- and
Biotin-Appended
6′′-Amino-6′′-deoxy-galactosylceramides
Xiao-Ti Zhou,† Claire Forestier,‡ Randal D. Goff,† Chunhong Li,† Luc Teyton,§
,†
Albert Bendelac,‡ and Paul B. Savage*
Department of Chemistry and Biochemistry, Brigham Young UniVersity,
ProVo, Utah 84602, Department of Molecular Biology, Princeton UniVersity,
Princeton, New Jersey 08544, and Department of Immunology,
Scripps Research Institute, La Jolla, California 92037
paul_saVage@byu.edu
Received January 15, 2002
ABSTRACT
r-Galactosylceramides are potent stimulators of human T cells. Stimulation occurs through binding of the glycolipids by CD1d, presentation
to T cells, and formation of a CD1d−glycolipid−T cell receptor complex. To facilitate the elucidation of the structural features of glycolipids
necessary for T cell stimulation, r-galactosylceramides have been prepared with small molecules appended at the C6 position of the sugar.
The appended molecules do not significantly influence the abilities of the glycolipids to stimulate T cells.
Peptide antigen presentation via major histocompatability
complexes has long been recognized as a central element in
adaptive immune responses. Recently, a parallel pathway that
can elicit potent immune responses has begun to be
elucidated. This pathway involves the presentation of gly-
colipids by CD1 proteins and is believed to be responsible
for a portion of the innate immunity of mammals to bacteria.1
There are five members of the CD1 gene family, CD1a-e,
and these membrane-bound proteins have been characterized
by their abilities to present classes of glycolipids, including
those from bacterial membranes, to T cells. For example,
CD1b has been shown to selectively present glycolipids
found in the membranes of mycobacteria (e.g., Mycobacte-
rium tuberculosis) to T cells and stimulate proliferation.2
CD1d is another member of the gene family and has been
characterized by its ability to bind and present R-galacto-
sylceramides, including KRN70003 (1, in Figure 1), to natural
killer T cells (NKT cells).4 Glycosylceramides undergo
† Brigham Young University.
‡ Princeton University.
§ Scripps Research Institute.
(1) For recent reviews, see: (a) Porcelli, S. A.; Modlin, R. L. Annu.
ReV. Immunol. 1999, 17, 297. (b) Park, S.-H.; Bendelac, A. Nature 2000,
406, 788. (c) Moody, D. B.; Besra, G. S. Immunology 2001, 104, 243.
Figure 1. Structures of KRN7000 and 6-amino-6-deoxygalacto-
sylceramide 2.
10.1021/ol025565+ CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/20/2002

endocytosis by antigen-presenting cells, processing in which
oligosaccharides are truncated,5 loading into CD1d in the
endoplasmic reticulum, translocation to the cell surface, and
presentation to NKT cells. Complexes between glycolipid-
loaded CD1d proteins and T cell receptors form that can
lead to stimulation of the T cell.
Due in part to the complexity of the glycolipid presentation
pathway, little is known about the specific structural require-
ments for glycolipid binding by CD1d and T cell receptors.
The limited structure-activity studies that have been con-
ducted have used assays that do not distinguish between
CD1d binding and NKT cell receptor binding.3,4b,6,7 That is,
in a typical procedure, glycolipids are added to antigen-
presenting cells and T cells (of which NKT cells make up
only a small percentage), and T cell stimulation is gauged.
Nevertheless, it is well recognized that only the R anomers
(and not the â) of galactosylceramides cause stimulation, that
R-glucosylceramides are less active, and that the length of
the lipid chains can influence how T cells respond to the
compounds.7
Challenges in understanding the roles of glycolipid
structure in CD1d and NKT cell receptor binding have
included the inability to effectively observe trafficking of
glycolipids and difficulties in quantifying their association
with CD1d and NKT cell receptors. An attractive means of
overcoming these challenges is to label glycolipids with
fluorophores or other small molecules (e.g., biotin) that allow
observation of the compounds at low concentrations and/or
provide a means of quantifying the association with CD1d
and NKT cell receptors. R-Galactosylceramides have been
prepared with labels (a fluorophore8 or biotin9) at the end of
one of the lipid chains; however, CD1d binds lipid chains
within deep hydrophobic pockets.10 Consequently, addition
of the label on the lipid chains may interfere with the
association with CD1d.11
Modeling of the CD1d-1 complex suggested that the
hydroxyl groups at C4′′ and C6′′ on galactose are not
involved in complex formation.12 Consequently, these posi-
tions may be the best locations for attachment of fluorescent
tags or other small molecules. In addition, a derivative of 1
with a second sugar linked at the C6′′ position was shown
to not require processing for NKT cell stimulation,5 sug-
gesting that the CD1d-glycolipid-NKT cell receptor in-
teraction tolerates small molecules appended at C6′′.
By preparing 6-amino-6-deoxygalactosylceramide 2 (Fig-
ure 1), we have been able to attach a number of small
molecules to the glycolipid.13 The small molecules that have
been attached include dansyl, a prodan derivative, and biotin.
Examples of the labeled compounds have proven to have
NKT cell stimulating properties similar to the parent gly-
colipid (1), suggesting that in a manner similar to 1, the
compounds go through endocytosis, CD1d loading, presenta-
tion on the cell surface, and binding to T cell receptors
causing T cell stimulation.
The amine functionality was incorporated early onto the
carbohydrate as the azide (3,14 Scheme 1). The acetonides
Scheme 1. Preparation of
R-6-Amino-6-deoxygalactosylceramide 2a
(2) For leading references, see: (a) Moody, B. D.; Reinhold, B. B.; Guy,
M. R.; Beckman, E. M.; Frederique, D. E.; Furlong, S. T.; Ye, S.; Reinhold,
V. N.; Sieling, P. A.; Modlin, R. L.; Besra, G. S.; Porcelli, S. A. Science
1997, 278, 283. (b) Jackman, R. M.; Stenger, S.; Lee, A.; Moody, D. B.;
Rogers, R. A.; Niazi, K. R.; Sugita, M.; Modlin, R. L.; Peters, P. J.; Porcelli,
S. A. Immunity 1998, 8, 341.
(3) Morita, M.; Motoki, K.; Akimoto, K.; Natori, T.; Sukai, T.; Sawa,
E.; Yamaji, K.; Koezuka, Y.; Kobayashi, E.; Fukushima, H. J. Med. Chem.
1995, 38, 2176.
a Reagents: (a) AcCl, MeOH (86% yield); (b) BnBr, 18-crown-
6, NaH, THF (95% yield); (c) AcOH, H2SO4 (84% yield); (d)
HF‚pyridine, pyridine (78% yield); (e) MS 4Å, AgClO4, SnCl2,
THF (44%); (f) TBAF, THF (81% yield); (g) PPh3/H2O, THF
(quantitative yield); (h) NH3/Na, -78 °C (53% yield).
(4) For leading references, see: (a) Bendelac, A.; Rivera, M. N.; Park,
S.-H.; Roark, J. H. Annu. ReV. Immunol. 1997, 15, 535. (b) Kawano, T.;
Cui, J.; Koezuka, Y.; Toura, I.; Kaneko, Y.; Motoki, K.; Ueno, H.;
Nakagawa, R.; Sato, H.; Kondo, E.; Koseki, H.; Taniguchi, M. Science
1997, 278, 1626. (c) Burdin, N.; Brossay, L.; Koezuka, Y.; Smiley, S. T.;
Grusby, M. J.; Gui, M.; Taniguchi, M.; Hayakama, K.; Kronenberg, M. J.
Immunol. 1998, 161, 3271. (d) Park, S.-H.; Weiss, A.; Benlagha, K.; Kyin,
T.; Teyton, L.; Bendelac, A. J. Exp. Med. 2001, 193, 893.
(5) 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.
were hydrolyzed with concomitant methylgalactoside forma-
tion,15 and benzyl ethers at C2, C3, and C4 were formed
(6) (a) Kobayashi, E.; Motoki, K.; Yamaguchi, Y.; Uchida, T.; Fuku-
shima, H.; Koezuka, Y. Bioorg. Med. Chem. 1996, 4, 615. (b) Uchimura,
A.; Shimizu, T.; Nakajima, M.; Ueno, H.; Motoki, K.; Fukushima, H.;
Natori, T.; Koezuka, Y. Bioorg. Med. Chem. 1997, 5, 1447. (c) Uchimura,
A.; Shimizu, T.; Morita, M.; Ueno, H.; Motoki, K.; Fukushima, H.; Natori,
T.; Koezuka, Y. Bioorg. Med. Chem. 1997, 5, 2245.
(7) Miyamoto, K.; Miyake, S.; Yamamura, T. Nature 2001, 413, 531.
(8) Sakai, T.; Ehara, H.; Koezuka, Y. Org. Lett. 1999, 1, 359.
(9) Sakai, T.; Naidenko, O. V.; Iijima, H.; Kronenberg, M.; Koezuka,
Y. J. Med. Chem. 1999, 42, 1836.
(11) Naidenko, O. V.; Maher, J. K.; Ernst, W. A.; Sukai, T.; Modlin, R.
L.; Kronenberg, M. J. Exp. Med. 1999, 190, 1069.
(12) Kamada, N.; Iijima, H.; Kimura, K.; Harada, M.; Shimizu, E.;
Motohashi, S.; Kawano, T.; Shinkai, H.; Nakayama, T.; Sakai, T.; Brossay,
L.; Kronenberg, M.; Taniguchi, M. Internat. Immunol. 2001, 13, 853.
(13) Fluorophores have been attached via amide bonds to C6 on other
biologically active galactosides. For example, see: Ueda, M.; Wada, Y.;
Yamamura, S. Tetrahedron Lett. 2001, 42, 3869.
(14) Corey, E. J.; Sameulsson, B.; Luzzio, F. A. J. Am. Chem. Soc. 1984,
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Org. Lett., Vol. 4, No. 8, 2002

giving 4. The methoxy group was then replaced by an
acetoxy group,16 followed by conversion to the anomeric
fluoride (5).17 Glycosyl bond formation18 with 619 gave 7.
The silyl protecting groups were removed20 followed by
reduction of the azide21 and removal of the benzyl groups
giving 2.22
Reaction of 2 with a variety of acid chlorides and
N-hydroxysuccinimidyl esters gave reasonable yields of the
corresponding amides. Glycosylceramides are notoriously
insoluble, and the influence of this insolubility in purification
may have decreased reaction yields. Nevertheless, using
dansyl chloride and a dansyl amide tethered to an N-
hydroxysuccinimidyl ester23 (9), we were able to prepare 8
and 10 (Scheme 2).
Figure 2. Structures of N-hydroxysuccinimidyl ester derivative
of prodan (11) and prodan- and biotin-labeled R-galactosylceramides
12 and 13, respectively.
Scheme 2. Preparation of Dansyl-Appended Glycolipids 8 and
10
Two other small molecules were appended onto 2. Prodan
is a fluorophore with a high quantum yield that responds to
the polarity of its environment via large changes in its
emission wavelength.24 It is probable that the environment
of a fluorophore appended to a galactosylceramide will
change upon complex formation with a protein. Conse-
quently, we anticipate being able to visualize the binding of
the appended glycolipid via fluorescence modulation. In
anticipation of attaching prodan to a glycolipid, compound
11 was prepared from 4-(6-methoxy-[2]naphthyl)-4-oxo-
butyric acid25 by nucleophilic displacement of the methoxy
group with lithium dimethylamide26 followed by reaction
with DCC and N-hydroxysuccinimide. Reaction of N-
hydroxysuccinimidyl ester 11 with 2 gave 12 in 46% yield.
Similarly, reaction of 2 with the N-hydroxysuccinimidyl ester
of biotin gave 13 in 52% yield.
a Reagents: (a) AcCl, MeOH (86% yield); (b) BnBr, 18-crown-
6, NaH, THF (95% yield); (c) AcOH, H2SO4 (84% yield); (d) HF‚
pyridine, pyridine (78% yield); (e) MS 4 Å, AgClO4, SnCl2, THF
(44% yield); (f) TBAF, THF (81% yield); (g) PPh3/H2O, THF
(quantitative yield); (h) NH3/Na, -78 °C (53% yield).
The abilities of compounds 8, 10, 12, and 13 to stimulate
NKT cells were gauged by measuring IL-2 production using
The constraints placed upon the carbohydrate portion of
glycosylceramides by CD1d and T cell receptors are not fully
understood, and it was not clear if the presence of a
fluorophore or similarly sized appendage would be tolerated.
Therefore, we used a tether between the fluorophore and the
carbohydrate in 10 to determine how a flexible tether would
influence CD1d and T cell receptor binding.
(16) Davis, N. J.; Flitsch, S. L. J. Chem. Soc., Perkin Trans. 1 1994,
359.
(17) Hayashi, M.; Hashimoto, S.; Noyori, R. Chem. Lett. 1984, 1747.
(18) Hashimoto, S.; Hayashi, M.; Noyori, R. Tetrahedron Lett. 1984,
25, 1379.
(19) (2S,3S,4R)-3,4-Bis-tert-butyldimethylsisyloxy-2-hexaosanoylamino-
4-octadecanol was prepared by coupling of phytosphingosine (Avanti Polar
Lipids) with hexacosanoic acid (DIC, HOBT) followed by the protection/
deprotection scheme from ref 20.
(20) Takikawa, H.; Muto, S.; Mori, K. Tetrahedron 1998, 54, 3141.
(21) Vaultier, M.; Knouzi, N.; Carrie´, R. Tetrahedron Lett. 1983, 24,
763.
(22) Sakai, T.; Ueno, H.; Natori, T.; Uchimura, A.; Motoki, K.; Koezuka,
Y. J. Med. Chem. 1998, 41, 650.
Figure 3. NKT cell stimulatory activity of fluorophore-appended
6-amino-6-deoxygalactosylceramides 8, 10, 12. 1 ((); 8 (b); 10
(2); 12 (9).
(23) Wang, F.; Schwabacher, A. W. J. Org. Chem. 1999, 64, 8922.
Org. Lett., Vol. 4, No. 8, 2002
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an immobilized CD1d assay.27 Briefly, soluble, biotinylated
CD1d was loaded onto precoated avidin plates, and the plates
were pulsed with incrementally varied concentrations of
glycolipids. After being washed, the plates were treated with
a CD1d-restricted VR24 NKT cell hybridoma and IL-2
release was measured using ELISA.
Compounds 8, 10, 12, and 13 were highly stimulatory.
Although 8 and 12 appear to be slightly less efficient in the
results from the experiment shown in Figure 3, no significant
differences among the compounds were found in repeated
experiments (at least three experiments for each compound).
In a separate series of experiments (e.g., Figure 4), 13 was
slightly but reproducibly more efficient in stimulating NKT
cells than 1. Similar results were observed using CD1d-
transfected rat basophilic leukemia cells for antigen presenta-
tion to NKT cell hybridomas.
Interestingly, attachment of a dansyl group directly at C6′′
(as in 8) or through a tether did not cause a significant loss
of stimulating properties. Similarly, alteration of the ap-
pended group (i.e., dansyl vs prodan vs biotin) did not greatly
affect the abilities of glycolipids to stimulate NKT cells.
Because the glycolipids are appended in the carbohydrate
portion of the molecules, it was anticipated that the labels
would not interfere with association of the glycolipids with
CD1d. Gratifyingly, we found that modification of the
glycolipids at C6′′ with small molecules allowed retention
of NKT cell stimulating properties. Attachment of fluoro-
phores and biotin to R-galactosylceramides is expected to
facilitate elucidation of the structural features of glycolipids
Figure 4. NKT cell stimulatory activity of biotin-appended
6-amino-6-deoxygalactosylceramide 13. 1 ((); 13 (9).
necessary for NKT cell stimulation. Fluorescence and surface
plasmon resonance studies are underway to characterize the
association of appended glycolipids with CD1d, and results
will be reported in due course.
Acknowledgment. Generous financial support from the
NSF (CAREER) (P.B.S.) and the Human Frontier Science
Program (C.F.) is gratefully acknowledged.
(24) Weber, G.; Farris, F. J. Biochemistry 1979, 18, 3075.
(25) Khan, M. S. Y.; Siddiqui, A. A. Indian J. Chem., Sect. B 2000, 39,
614.
(26) Weber, G.; Farris, F. J. Biochemistry 1979, 18, 3075.
(27) Benlagha, K.; Weiss, A.; Beavis, A.; Teyton, L.; Bendelac, A. J.
Exp. Med. 2000, 191, 1895.
Supporting Information Available: Experimental details
for the syntheses of 8, 10, 12, and 13. This material is
available free of charge via the Internet at http://pubs.acs.org.
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