The synthesis and biological characterization of a ceramide library

Article abstract of DOI:10.1021/ja017576o

A facile synthesis of a combinatorial ceramide library and their activities in the NF-κB pathway and in apoptosis induction/prevention were demonstrated. A novel NF-κB activating molecule was discovered among ceramide containing β-galactose, and the structural requirements of ceramides for apoptosis induction was elucidated. Copyright

Full text of DOI:10.1021/ja017576o

Published on Web 02/05/2002
The Synthesis and Biological Characterization of a Ceramide Library
Young-Tae Chang,*,§ Jaehwa Choi, Sheng Ding, Eva E. Prieschl,† Thomas Baumruker,†
Jae-Mok Lee,‡ Sung-Kee Chung,*,‡ and Peter G. Schultz*
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road,
San Diego, California 92037, Department of Immunology, NoVartis Research Institute, Vienna, Austria,
and Department of Chemistry, DiVision of Molecular and Life Sciences,
Pohang UniVersity of Science and Technology, Pohang 790-784, Korea
Received November 20, 2001
Fatty acids and their metabolites play key roles as second
messengers in cellular signal transduction and as hormones that
regulate an enormous array of physiological processes. For example,
ceramides are sphingosine-based signaling molecules that regulate
cell cycle arrest, proliferation, differentiation, and apoptosis.1
Although many chemical libraries have been synthesized and
screened for biologically active molecules over the past decade,
combinatorial libraries of fatty acids, their derivatives, and analogues
have not yet been extensively explored. Herein we report the
synthesis of a combinatorial ceramide library, and present prelimi-
nary results obtained in cell-based screens of apoptosis and signal
transduction that suggest libraries of this sort will be a rich source
of biologically active synthetic molecules.
To synthesize the ceramide library, the core structure was
derivatized in solution with a series of solid-phase reagents. A
number of reaction conditions were screened for the formation of
ceramides using sphingosine and solid-phase activated esters as acyl
donor with various resins, solvents, and activated ester groups. Use
of a nitrophenol ester on polystyrene in tetrahydrofuran was found
to yield a very pure product after a simple filtration step, without
a trace of sphingosine or byproducts containing O-acylation
(Scheme 1).2 With these optimized conditions, a library of 528
compounds was generated using 16 sphingosine-like core structures
(Figure 1, Cores) and 33 acyl groups (2-50 in Figure 1). Four
stereoisomers of sphingosine, DES, DTS, LES, and LTS, were
synthesized by a slightly modified literature procedure.3
Scheme 1. Representative Synthetic Scheme for Natural
Ceramide
Activated esters on solid support were prepared using either the
acyl halide in pyridine or the diisopropylcarbodiimide-activated
acid. Completion of the reaction was confirmed by negative
ninhydrin staining and thin-layer chromatography with phospho-
molybdic acid staining. About 100 synthetic compounds among
the 528 in the library were selected randomly and characterized by
high-resolution mass spectrometry (MALDI-FTMS) and 1H NMR
to confirm their identity and purity.
Figure 1. Structures of cores and tails of the ceramides.
apoptosis. Several cytokines, including tumor necrosis factor-R
(TNF-R) and interleukin-1â (IL-1â), are known to induce NF-κB
activation, concomitant with ceramide accumulation in various cell
types.4 However, it is not clear whether ceramide plays a direct
role in NF-κB activation.5 Thus it was of interest to assay the
agonistic effects of the library in an NF-κB reporter gene (luciferase)
assay using C6 glioma cells.6 When 10 µM compound was added
to the cells, a marked direct activation of NF-κB was only observed
for the PC ceramides with a â-galactose headgroup: PC9 was the
most potent (>8-fold increase of NF-κB activity). None of the
The biological activities of members of this library were tested
in two cell-based assays: NF-κB signaling and the induction of
§ Present address: Department of Chemistry, New York University.
† Novartis Research Instiute.
‡ Pohang University of Science and Technology.
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10.1021/ja017576o CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Apoptosis inducing effect of ceramide libraries. The Z axis is the reciprocal of IC50 (µM).
natural ceramides (DES) or their cell membrane permeable deriva-
tives showed any effects in this assay. It is noteworthy that direct
activation was sensitive to the sugar stereochemistry, as analogues
containing â-glucose (GP1, 5, 7, 9, 12, and 32) proved to be inactive
in this system. This strict stereospecificity was not observed in a
related cellular screen involving TNF-R transcription in a murine
mast cell line.7 While R-galactose-containing ceramides have
already been demonstrated to activate the immune system,8 this is
the first example of such effects with â-sugar-containing ceramides.
We also screened the library molecules in a apoptosis screen
with U937 leukemic cells, in which it is known that exogenous
ceramide promotes apoptosis, and endogenous ceramide generation
plays an essential role in activation of apoptosis under stress
conditions (Figure 2).9 In general, the most active members of the
library had IC50 values in the micromolar range with 12ES10 as
the most potent compound (IC50 4 µM). The data revealed several
important structural elements of ceramides that are required to
induce apoptosis. The activity vs carbon length profile has a
Gaussian shape with an optimum around a sum of carbon chain
length (SCCL) of 18 [the most potent compounds from each core
structure with their IC50 and SCCL were 10ES10 (IC50 12 µM,
SCCL ) 16), 12ES10 (IC50 4 µM, SCCL ) 18), 15ES6 (IC50 20
µM, SCCL ) 17), DES6 (IC50 12 µM, SCCL ) 20), and 20ES4
(IC50 50 µM, SCCL ) 20)]. The stereochemistry of the headgroup
is not critical as the four streoisomers, DES, DTS, LES, and LTS,
showed similar activities throughout. However, a C4-C5 double
bond in the ceramide core structure does appear to be important
for apoptotic activity, as both erythro (DED, ED) and threo (TD)
dihydro-ceramides were generally inactive. This result contrasts with
a previous study that indicated threo isomers are active whereas
erythro isomers are not.10 Activity is restored when a bulky tail is
introduced with a preference for the D-configuration in ED (see 32
and 45). This result suggests that a hydrophobic moiety may be
required near the headgroup of the ceramide for biological activity.
Finally, compounds based on a short chain core, i.e., NE, were not
active with short carbon tails, but activity was restored with long
unsaturated tails (see 46, 47, and 49), consistent with the reported
trend.11 This study suggests that the apoptotic activity of these
ceramide analogues depends on specific structural features; how-
ever, the exact mechanism of action is unclear at present.
In conclusion, we have developed a facile synthetic pathway to
generate a library of ceramides and have demonstrated that members
of this library have novel biological activities that may ultimately
provide unique insights into cellular processes or possibly serve as
leads for the development of therapeutic agents.
Acknowledgment. The work at Pohang University was sup-
ported by the Korea Science & Engineering Foundation (981-0302-
008-2, 985-0302-002-2).
Supporting Information Available: The full name for the ab-
breviation used, experimental procedures, and characterization data;
NF-κB activation data for PC; and IC50 values of all available
compounds in the U937 cell assay and the murine mast cell data
(PDF). This material is available free of charge via the Internet at
http://pubs.acs.org.
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