6′-Derivatised α-GalCer analogues capable of inducing strong CD1d-mediated Th1-biased NKT cell responses in mice

Article abstract of DOI:10.1021/ja8064182

α-Galactosyl ceramide (α-GalCer, also known as KRN 7000) is known as the prototypical antigen for invariant natural killer T (NKT) cells. Stimulation of NKT cells by CD1d-mediated α-GalCer presentation leads to rapid release of Th1 and Th2 cytokines. Since Th1 and Th2 cytokines antagonize each other′s effects, α-GalCer analogues that induce a biased Th1/Th2 response are highly awaited. With the exception of a C-glycoside (α-C-GalCer), most of the known α-GalCer analogues able to induce polarized cytokine responses are characterized by modifications of the phytosphingosine or fatty acyl chains, expected to alter the affinity for CD1d. Herein we describe the synthesis of 6′-modified α-GalCer analogues with an intact phytoceramide moiety that are capable to skew the cytokine release profile to Th1, while maintaining strong antigenic activity. These analogues are characterized by the presence of an aromatic moiety that is connected via an amide or an urea linkage to C′-6 of the galactopyranose ring. Copyright

Full text of DOI:10.1021/ja8064182

Published on Web 11/14/2008
6′-Derivatised r-GalCer Analogues Capable of Inducing Strong
CD1d-Mediated Th1-Biased NKT Cell Responses in Mice
Matthias Trappeniers, Katrien Van Beneden, Tine Decruy, Ulrik Hillaert, Bruno Linclau, Dirk Elewaut,
and Serge Van Calenbergh*
Laboratory for Medicinal Chemistry (FFW), Ghent UniVersity, Harelbekestraat 72, B-9000 Gent, Belgium,
Laboratory for Molecular Immunology and Inflammation, Ghent UniVersity Hospital, Ghent UniVersity, De
Pintelaan 185, 9000 Ghent, Belgium, and School of Chemistry, UniVersity of Southampton,
Highfield, Southampton SO17 1BJ, U.K.
Received August 13, 2008; E-mail: serge.vancalenbergh@ugent.be
Chart 1. Structures of Selected R-Galactosyl Ceramides
During the past years the use of glycolipids as immunostimulating
agents has become increasingly important.1,2 When presented by
the major histocompatibility complex (MHC) class I-like molecule
CD1d, certain glycolipids are recognized by the semi-invariant T
cell receptors (TCR) of natural killer T (NKT) cells.3 The
prototypical antigen for NKT cells is R-galactosyl ceramide (R-
GalCer; 1).4 Upon recognition of the CD1d-R-GalCer bimolecular
complex by their TCR, NKT cells are activated, resulting in the
rapid release of T helper 1 (Th1) and T helper 2 (Th2) cytokines.5
As both Th1 and Th2 cytokines influence the outcome of
different immune responses, disruption of the carefully controlled
Th1/Th2 balance can lead to disease induction and progression.
While certain autoimmune diseases are characteristic of hypore-
sponsiveness to Th2 and overactivation of pathogenic Th1 cells,
the opposite is true for many types of cancer that have a
predominant Th2 response. Hence, R-GalCer analogues that induce
a biased Th1/Th2 response are highly awaited.
Attempts to selectively control the rapid secretion of cytokines
by NKT cells have led to the development of several R-GalCer
analogues with interesting immunomodulatory properties (Chart 1).
With the synthesis of OCH (2), a direct relationship has been shown
between the shortening of lipid tail lengths and biasing of the
cytokine release profile toward a Th2 response.6 Porcelli reported
that substituting the N-acyl chain of R-GalCer with shorter,
unsaturated fatty acids modifies the outcome of VR14i NKT cell
activation.7 Analogues containing multiple cis-double bonds in the
acyl chain (e.g., 5) potently induced a Th2-biased cytokine response.
Conversely, Wong et al. have found that introduction of terminal
aromatic groups into the fatty acyl tail (as in 6) biases the profile
toward a Th1 response.8
the previously proposed structures,11,12 R-GalCer protrudes mini-
mally from the CD1d cleft with only the galactosyl headgroup
exposed for recognition by the NKT TCR and interacting solely
with its CDR1R and CDR3R loops. The galactose ring is sand-
wiched between Trp-153 of CD1d and the aliphatic moiety of Arg-
95R. The 2′-OH, 3′-OH, and 4′-OH of the galactose ring form
hydrogen bonds with amino acid residues of the invariant TCR
R-chain. This mode of binding is consistent with the specificity
the NKT TCR exhibits for R-GalCer versus closely related
analogues modified at the sugar part.14
Interestingly, the Gal 6′-OH is the only sugar alcohol not involved
in H-bond formation, suggesting the possibility of introducing
modifications at that position. In this respect, we were drawn to a
report from Savage et al. who attached small fluorophores at that
position, resulting in modified R-GalCers that retained the capacity
to stimulate NKT cells.15 As it was envisioned that extra interactions
might be established between CD1d and a 6′-OH modified
R-GalCer, we decided to investigate such analogues.
The synthetic strategy was similar to that used by Savage;15
however, we chose to protect the secondary hydroxyl groups of
the phytoceramide aglycon as benzyl ethers to facilitate the final
deprotection (Scheme 1). Furthermore, to avoid solubility problems,
the final debenzylation step was performed after modification of
the 6′-amino group. The synthesis of 6′-azido-6′-deoxygalactosyl-
ceramide 9 started from the D-ribo-phytosphingosine building block
8.16 Mukaiyama glycosidation17 involving 715 as the donor afforded
the desired R-glycoside 9 in 46% yield. Reaction of the crude amine
10, obtained by Staudinger reduction of 9, with the appropriate
acid and EDC as the coupling reagent, followed by hydrogenolysis
of the benzyl groups, gave the desired amide analogues 11-14.
Treatment of 10 with the appropriate isocyanates afforded, after
final deprotection, the 6′-ureido-6′-deoxygalactosylceramide ana-
logues 15-19.
A proof-of-principle that R-GalCer analogues capable of skewing
the cytokine release profile may translate in an improved thera-
peutical outcome was established with R-C-GalCer (3). Character-
ized by a marked Th1 response, this compound exhibits markedly
improved activity against melanoma metastases and malaria
compared with R-GalCer.9 Bittman recently reported a truncated
nonisosteric C-glycoside, termed R-1C-GalCer (4), which produced
less IFN-γ than R-GalCer but showed higher IFN-γ/IL-4 and IFN-
γ/IL-13 ratios, typical for a Th1-immune response.10
In 2005, the crystal structures of human11 and mouse12 CD1d
complexed with R-GalCer or a short-chain variant were elucidated,
revealing that the galactose ring is well ordered and extends above
the surface of a lipid-binding groove. Different intermolecular
hydrogen bonds are assumed to anchor R-GalCer in a proper
orientation for recognition by the TCR of NKT cells.
All analogues were tested by measuring the serum cytokine levels
after injection into C57Bl/6 mice. Based on their ability to induce
significantly reduced IL-4 production and comparable levels of
IFN-γ compared to R-GalCer (Figure 1), 12-19 were identified
as Th1-skewing compounds.
Recently, Borg et al. reported the structure of a human NKT
TCR in complex with CD1d bound to R-GalCer.13 Consistent with
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16468 J. AM. CHEM. SOC. 2008, 130, 16468–16469
10.1021/ja8064182 CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1. Synthesis of NKT Cell Agonists 11-19a
resulting in the cytokine polarization. It is interesting to observe
that, at least in the amide series, electron withdrawing substituents
on the aryl ring tend to induce the most promising Th1 cytokine
profile. Interestingly, in the proximity of the 6′-position hCD1d
structurally differs from mCD1d in that it contains a Trp-153 instead
of Gly-155.19 Hence, π-π interaction with the electron rich indole
ring could lead to additional effect on the cytokine polarization.20
In vitro assays to investigate the effect of our analogues using
human antigen presenting cells are in progress.
Acknowledgment. M.T. is an aspirant, and K.V.B. a postdoc-
toral researcher of the Fund for Scientific Research-Flanders
(F.W.O.-Vlaanderen). Financial support by FWO and Cancer
Research Technology is gratefully acknowledged.
Supporting Information Available: Experimental procedures for
the preparation of 9-19 and for the in ViVo stimulation with R-GalCer-
analogues. This material is available free of charge via the Internet at
http://pubs.acs.org.
a Reagents (yields in parentheses): (a) SnCl2, AgClO4, THF, 4 Å MS,
-10 °C to rt, 2 h (46%); (b) (i) PMe3, THF, rt, 4 h; (ii) NaOH 1 M, rt, 2 h
(quant.); (c) R-COOH, EDC, DMF, 4 h, rt; (d) R-NCO, DMF, 0 °C to rt,
2 h; (e) H2, Pd black, CHCl3/EtOH 1/3, 5 h (33-65% over 2 steps).
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