Thio-isoglobotrihexosylceramide, an agonist for activating invariant natural killer T cells

Article abstract of DOI:10.1021/ol062199b

Thio-isoglobotrihexosylceramide (S-iGb3) might be resistant to α-galactosidases in antigen-presenting cells and have a longer retaining time in the lysosome before being loaded to CD1d. The biological assay showed that S-iGb3 demonstrates a much higher in

Full text of DOI:10.1021/ol062199b

ORGANIC
LETTERS
2006
Vol. 8, No. 24
5493-5496
Thio-isoglobotrihexosylceramide, an
Agonist for Activating Invariant Natural
Killer T Cells
Chengfeng Xia,† Dapeng Zhou,*,‡ Chengwen Liu,‡ Yanyan Lou,‡ Qingjia Yao,†
Wenpeng Zhang,† and Peng George Wang*,†
Departments of Biochemistry and Chemistry, The Ohio State UniVersity, Columbus,
Ohio 43210, and the Department of Melanoma Medical Oncology, UniVersity of Texas
M.D. Anderson Cancer Center, 7455 Fannin Street #SCR2.3150,
Houston, Texas 77054
pgwang@chemistry.ohio-state.edu; dzhou@mdanderson.org
Received September 5, 2006
ABSTRACT
Thio-isoglobotrihexosylceramide (S-iGb3) might be resistant to
r-galactosidases in antigen-presenting cells and have a longer retaining time
in the lysosome before being loaded to CD1d. The biological assay showed that S-iGb3 demonstrates a much higher increase as a stimulatory
ligand toward invariant natural killer T (iNKT) cells as compared to iGb3.
The well-known CD4+ and CD8+ T cells of the immune sys-
tem typically recognize specific peptide antigens presented
by major histocompatibility complex (MHC) class I or MHC
class II molecules.1 However, a unique subset of T cells called
invariant natural killer T (iNKT) cells recognize glycolipid
antigens presented by the MHC class I-like molecule called
CD1d. iNKT cells represent a distinct population of T cells
that express conserved Râ T cell receptors (TCR) and natural
killer (NK) receptors.2,3 Mouse (m) VR14 and human (h)
VR24 NKT cells regulate a number of critical biological
conditions in vivo, including malignancy, infection, and
autoimmune diseases, through the rapid secretion of T helper
1 (Th1) and Th2 cytokines and chemokines (Figure 1).4,5
It was found that R-galactosylceramides can stimulate
iNKT cells to produce both interferon (IFN)-γ and interleukin
(IL)-4 cytokines.6 Recently, the first natural glycosphingo-
lipid ligand, isoglobotrihexosylceramide (iGb3), was found
to stimulate both human VR24 NKT cells and mouse VR14
iNKT cells.7 In the lysosome, â-hexosaminidases remove the
terminal GalNAc of iGb4 to produce iGb3 (Figure 2). Subse-
quently, iGb3 is degraded to â-lactosylceramide (â-LacCer)
by the removal of the terminal Gal with R-galactosidase.
(4) Smyth, M. J.; Crowe, N. Y.; Hayakawa, Y.; Takeda, K.; Yagita, H.;
Godfrey, D. I. Curr. Opin. Immunol. 2002, 14, 165.
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Sagiv, Y.; Hudspeth, K.; Wu, Y.-P.; Yamashita, T.; Teneberg, S.; Wang,
D.; Proia Richard, L.; Levery Steven, B.; Savage Paul, B.; Teyton, L.;
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† The Ohio State University.
‡ University of Texas M.D. Anderson Cancer Center.
(1) (a) Krummel, M. F.; Davis, M. M. Curr. Opin. Immunol. 2002, 14,
66. (b) Bromley, S. K.; Burack, W. R.; Johnson, K. G.; Somersalo, K.;
Sims, T. N.; Sumen, C.; Davis, M. M.; Shaw, A. S.; Allen, P. M.; Dustin,
M. L. Annu. ReV. Immunol. 2001, 19, 375. (c) Gao, G. F.; Jakobsen, B. K.
Immunol. Today 2000, 21, 630.
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2005, 25, 183.
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10.1021/ol062199b CCC: $33.50
© 2006 American Chemical Society
Published on Web 10/31/2006

Figure 3. Structure of S-iGb3.
We reasoned that S-iGb3 could be synthesized from thio-
trisaccharide donor 2 and a ceramide acceptor. The thio-
trisaccharide would be prepared by the direct glycosylation
of 3′-thio-lactoside derivative 3 with a galactosyl donor
(Figure 4).
Figure 1. Activation of iNKT cells by R-GalCer/APC.
iGb3, but not iGb4 or â-LacCer, can be presented as an ago-
nist ligand toward iNKT cell lines by plate-bound recom-
binant CD1d molecules. Hexb-/- cells can present iGb3 but
fail to present iGb4, which directly indicates that processing
of iGb4 into iGb3 is necessary for iNKT cell recognition.7
Figure 4. Retrosynthetic analysis.
Figure 2. Metabolism of iGb4 in human cells.
Because the iGb3 is a natural “self” ligand, it is critical
that the expression of this ligand should be tightly regulated.
iGb3 can be hydrolyzed to â-LacCer to lose its agonist
activity. The dynamics of the iGb3/CD1d complex that is
presented to iNKT cells remains a puzzle.
First, we explored an efficient way to replace 3′-OH by a
thio group, leaving the stereochemistry intact (Scheme 1).
We hypothesized that an R-galactosidase-resistant iGb3
analogue might be a valuable tool for studying the turnover
of iGb3 in antigen-presenting cells. However, we first had
to determine whether changes in the carbohydrate linkage
preserve the agonist activity of iGb3. It is well-known that
thioglycosides are resistant to glycosidase.8 Conformational
studies of S-linked oligosaccharides (thio-oligosaccharides)
have suggested that the flexibility about the anomeric linkage
is increased but that they still adopt a conformational space
very similar to their O-linked counterparts.9 Therefore, we
hypothesize that the iNKT cell receptor should recognize
the conformation of an iGb3 analogue with the distal galac-
tose linked to LacCer through a thioglycoside. Herein, we
report an efficient synthesis of S-iGb3 (Figure 3) and its bio-
assay, which showed that S-iGb3 can dramatically increase
its stimulatory ability to iNKT cells compared to iGb3.
Scheme 1. Synthesis of a Thio-lactose Moiety
(8) Albrecht, B.; Puetz, U.; Schwarzmann, G. Carbohydr. Res. 1995,
276, 289.
(9) (a) Geyer, A.; Hummel, G.; Eisele, T.; Stefan, R.; Schjmidt, R. R.
Chem.-Eur. J. 1996, 2, 981. (b) Aguilera, B.; Jimenez-Barbero, J.;
Fernandez-Mayoralas, A. Carbohydr. Res. 1998, 308, 19. (c) Weimar, T.;
Kreis, U. C.; Andrews, J. S.; Pinto, B. M. Carbohydr. Res. 1999, 315, 222.
Compound 6 was prepared from the known compound 5,10
which was generated from allyl â-D-lactose 410 by selectively
dibutyltinoxide-promoted alkylation with p-methoxylbenzyl
5494
Org. Lett., Vol. 8, No. 24, 2006

(PMB) chloride,11 in two steps including pivaloylation and
PMB removal using DDQ. To get the original stereochem-
istry of the thio group, the inversion of 3′-OH stereochemistry
was required before the second reversal in the introduction
of thio. Therefore, the equatorial 3′-OH of 6 was converted
to an axial configuration by treatment with triflic anhydride
followed by KNO2. Compound 7 was triflated again, and
3′-O-triflate was substituted with AcSNBu4 to give S-acetate
8 in a SN2 manner reforming the original stereochemistry.12
However, the yield was very low (<10%) and varied in every
trial. We assumed that the C-4′ hydroxyl derivative was
formed because of the neighboring participation of 4′-O-
ester in compound 9 via the cyclic intermediate 10 (Scheme
1).13
Scheme 3. Synthesis of a Galactosyl Donor
deprotection stage. Our initial attempts toward the synthesis
of the per-PMB protected galactoside donor gave a poor yield
in the allyl group removal stage because some of the PMB
groups were not stable. So, we switched and employed acetyl
groups to protect the C-3, C-4, and C-6 hydroxyl groups,
and only the C-2 hydroxyl group was protected with a PMB
group for R-selectivity in glycosylation. The commercially
available allyl â-D-galactose 17 was treated with dimethox-
ypropane (DMP) and a catalytic amount of p-TsOH to give
18.14 Then, the C-2 hydroxyl group was protected with PMB.
Removal of the ketal in acidic conditions and direct acetyl-
ation of the resulting triol afforded compound 20. The allyl
group was removed by Pd(PPh3)4 in the presence of ZnCl2
and Et3SiH15 followed by treatment with CCl3CN and DBU
to afford the donor 21.16
To prevent this neighboring group participation, an acetal
group was used to protect the 4′-OH instead of an ester. The
benzylidene was selected as the protecting group to protect
the C-4′ and C-6′ hydroxyl groups.13 Compound 11 was
prepared by 4,6-benzylidene protection of lactoside 5 and
subsequent benzoylation (Scheme 2). Following the same
Scheme 2. Improved Synthetic Protocol of a Thio Acceptor
The glycosylation of thio 16 with donor 21 using TMSOTf
as a catalyst gave S-trisaccharide 22 in good yield (Scheme
4). The allyl group was removed and converted to Schmidt’s-
type donor 23 following procedures similar to those in the
preparation of donor 21. The protected glycolipid 25 was
generated by glycosylation of donor 23 with azido-sphin-
gosine 24.6a,17 The employment of an azido instead of a
ceramide was to avoid the low reactivity resulting from
H-bonding of the amido group. The azide was reduced with
PPh3 in the presence of trace amounts of water. The resulting
amine was directly amidated with cerotic acid without further
purification to give the protected S-iGb3 27. The benzylidene
and two PMB groups were removed by 10% TFA in DCM.
Finally, all the acyl groups were removed with NaOMe in
methanol to afford the S-iGb3.
The bioassay using mouse (C57BL6) bone marrow derived
dendritic cells showed that the S-iGb3, similar to iGb3,
stimulates iNKT cell lines at a concentration above 10 ng/
mL. A 10-fold increase was observed for S-iGb3 compared
to iGb3 (Figure 5). The results indicated that the conforma-
tion change of S-iGb3 does not abolish the iNKT cell
recognition. We cannot conclusively explain our observation
that S-iGb3 is a stronger agonist ligand by using bone marrow
derived dendritic cells as antigen-presenting cells. The most
procedure as that in the synthesis of compound 8, the
thioester 15 was obtained in high yield. The acetyl was
selectively removed by treatment with hydrazine acetate in
degassed DMF to afford the thio-lactose derivative 16.
The preparation of the galactosyl donor was outlined in
Scheme 3. The benzyl group could not be used as a
protecting group because of the incompatibility with the
double bond in the lipid part at hydrogenation in the
(10) Kononov, L. O.; Kornilov, A. V.; Sherman, A. A.; Zyryanov, E.
V.; Zatonsky, G. V.; Shashkov, A. S.; Nifant’ev, N. E. Bioorg. Khim. 1998,
24, 608.
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Chem. 1999, 18, 1009.
(14) Pozsgay, V.; Jennings, H. J. Carbohydr. Res. 1988, 179, 61.
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306, 81.
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Chem., Int. Ed. 2004, 43, 613.
(13) Liakatos, A.; Kiefel, M. J.; Von Itzstein, M. Org. Lett. 2003, 5,
4365.
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Org. Lett., Vol. 8, No. 24, 2006
5495

Scheme 4. Synthesis of S-iGb3
likely explanation is that the thioglycoside is resistant to
to apo-E, which was recently identified as a necessary
component for lipid antigen uptake by antigen-presenting
cells.19
R-galactosidases in the antigen-presenting cells, thus S-iGb3
has a longer retaining time in the lysosome before being
loaded to CD1d. We have assumed that the degradation of
iGb3 requires the physical interaction of glycolipids with
saposins, and iGb3/CD1d will be inaccessible to glycosidase
cleavage.18
However, we cannot exclude the possibility that the
conformational change of S-iGb3 creates a conformation that
binds to TCR differentially from iGb3. It is also possible
that S-iGb3 and iGb3 prefer a different binding orientation
In summary, we successfully constructed 3′-thio-lactose
via a repeated triflation-stereochemistry inversion sequence
by using benzylidene as a protecting group on the C-4′ and
C-6′ hydroxyl groups of lactose to eliminate neighboring
group participation. Glycosylation of the 3′-thiolactose with
a suitable galactosyl donor afforded the thio-trisaccharide.
After introduction of the ceramide moiety, S-iGb3 was
synthesized in 18 steps with 6.5% overall yield. The bioassay
proved that S-iGb3 had significantly higher activity in
stimulation of iNKT cells. S-iGb3 is a valuable tool for
mechanistic studies on iGb3 processing in antigen-presenting
cells. Further bioassays are ongoing in vivo and in vitro to
ascertain the detailed mechanism.
Acknowledgment. This project was supported by The
Ohio State University to Peng George Wang and the M.D.
Anderson Cancer Center to Dapeng Zhou.
Supporting Information Available: Full experimental
details as well as spectral data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL062199B
Figure 5. S-iGb3 and iGb3 dissolved in DMSO at 1.0 mg/mL.
They were further diluted in a cell culture medium at indicated
concentrations and pulsed to 100 000 mouse (C57BL6) bone
marrow derived dendritic cells overnight, after the dendritic cells
were washed with a culture medium and mixed with 50 000
DN32.D3 hybridoma cells. They were cocultured for 24 h, and the
released IL2 was measured by CTLL assay. Data are representative
of three independent experiments.
(18) (a) Zhou, D.; Cantu, C.; Sagiv, Y., III; Schrantz, N.; Kulkarni, A.
B.; Qi, X.; Mahuran, D. J.; Morales, C. R.; Grabowski, G. A.; Benlagha,
K.; Savage, P. B.; Bendelac, A.; Teyton, L. Science 2004, 303, 523. (b)
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