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H.-S. Lim et al. / Bioorg. Med. Chem. Lett. 13 (2003) 237–240
only EDG-1. It indicates that the C3 hydroxyl group of
S1P as well as the C1 phosphate group and the C2
ammonium moiety are crucial for its binding to EDG
receptors.
Biophy. Res. Commun. 1999, 264, 743. (d) Wang, F.; Brocklyn,
J. R. V.; Hobson, J. P.; Movafagh, S.; Zukowska-Grojec, Z.;
Milstien, S.; Spiegel, S. J. Biol. Chem. 1999, 274, 35343.
4. Parrill, A. L.; Wang, D.; Bautista, D. L.; Brocklyn, J. R. V.;
Lorincz, Z.; Fischer, D. J.; Baker, D. L.; Liliom, K.; Spiegel,
S.; Tigyi, G. J. Biol. Chem. 2000, 275, 39379.
In summary, we have synthesized all four stereoisomers
of S1P and dihydro S1P, and their analogues which are
modified at the C3 position and evaluated their binding
affinity to EDG receptors for the first time. On the basis
of our experimental and computational data,13 it might
be concluded that (1) the d-erythro configuration of S1P
is important for a high affinity binding to EDGs, (2) the
phosphate group of S1P is essential for ligand recogni-
tion of EDGs, (3) besides the C1 phosphate group and
the C2 ammonium moiety of S1P, the presence and
configuration of the C3 hydroxyl group of S1P appears
to be very important for specific binding to EDGs.
These insights and findings will substantially contribute
to developing potent and selective agonist and antago-
nist for EDG receptors. Further works along these lines
are currently in progress.
5. Lee, J. M.; Lim, H. S.; Chung, S. K. Tetrahedron: Asym-
metry 2002, 13, 343.
6. (a) Szulc, Z. M.; Hannun, Y. A.; Bielawska, A. Tetrahedron
Lett. 2000, 41, 7821. (b) Boumendjel, A.; Miller, S. P. F. J.
Lipid Res. 1994, 35, 2305. (c) Kratzer, B.; Schmidt, R. R. Tet-
rahedron Lett. 1993, 34, 1761.
7. Compounds 1a and 7a were identical with the reported
data with respect to TLC, NMR (1H, 13C and 31P) and FAB-
MS.6b,c
8. Compound 5a: [a]D20 À2.61ꢀ (c 0.34, CHCl3), mp 79–81 ꢀC
(lit.6a [a]D24 À4.0ꢀ (c 1.0, CHCl3), mp 82–83 ꢀC).
9. Liang, X.; Andersch, J.; Bols, M. J. Chem. Soc., Perkin
Trans. 1 2001, 2136.
10. Murata, N.; Sato, K.; Kon, J.; Tomura, H.; Okajima, F.
Anal. Biochem. 2000, 282, 115.
11. Assay protocol: [3H]-S1P was purchased from American
Radiolabeled Chemicals, Inc. CHO cells (CHO/Edg-1, CHO/
Edg-3, CHO/Edg-5, and CHO/Mock)14 in confluent six mul-
tiplates were washed twice with the ice-cold binding buffer
consisting of 20mM Tris–HCl (pH 7.5), 10mM NaCl, 15
mM NaF, and 0.4% (w/v) BSA, and then incubated with the
same buffer containing 2 nM [3H]-S1P (about 40,000 dpm per
well) and 100 nM of S1P or S1P analogues in a final volume of
0.5 mL. The plates were kept on ice for 90 min, and the cells
were washed twice with the same ice-cold binding buffer to
remove unbounded ligand. The cells were solubilized with the
solubilizing solution composed of 0.1% SDS, 0.4% NaOH,
and 2% Na2CO3, and the radioactivity was measured by a
liquid scintillation counter after the addition of scintillation
cocktail solution. The relative binding affinity of each analo-
gues to each EDG receptors (EDG-1,-3 and-5) were presented
as percentage to S1P.
Acknowledgements
We thank Mr. Kwangseok Ko at C&C Research
Laboratories for the homology modeling of EDG-1
receptor and docking studies with our compounds.
Financial support from the Ministry of Education/BSRI
Fund is gratefully acknowledged.
References and Notes
1. (a) Lee, M. J.; Brocklyn, J. R. V.; Thangada, S.; Liu, C. H.;
Hand, A. R.; Menzeleev, R.; Spiegel, S.; Hla, T. Science 1998,
279, 1552. (b) Brocklyn, J. R. V.; Lee, M. J.; Menzeleev, R.;
Olivera, A.; Edsall, L.; Cuvillier, O.; Thomas, D. M.; Coop-
man, P. J. P.; Thangada, S.; Liu, C. H.; Hla, T.; Spiegel, S.
J. Cell Biol. 1998, 142, 229. (c) Hla, T.; Lee, M. J.; Ancellin,
N.; Liu, C. H.; Thangada, S.; Thompson, B. D.; Kluk, M.
Biochem. Pharmacol. 1999, 58, 201. (d) Spiegel, S.; Melstien, S.
FEBS Lett. 2000, 476, 55.
2. (a) Ancellin, N.; Hla, T. J. Biol. Chem. 1999, 274, 18997. (b)
Pyne, S.; Pyne, N. J. Biochem. J. 2000, 349, 385. (c) Spiegel, S.;
Milstein, S. Biochim. Biophys. Acta 2000, 1484, 107. (d) Pyne,
S.; Pyne, N. Pharmacol. Therapeutics 2000, 88, 115.
3. (a) Lee, M. J.; Thangada, S.; Claffey, K. P.; Ancellin, N.;
Liu, C. H.; Kluk, M.; Volpi, M.; Sha’afi, R. I.; Hla, T. Cell
1999, 99, 301. (b) Levade, T.; Auge, N.; Veldman, R. J.;
Cuvillier, O.; Negre-Salvayre, A.; Salvayre, R. Circ. Res. 2001,
89, 957. (c) Lee, O. H.; Kim, Y. M.; Lee, Y. M.; Moon, E. J.;
Lee, D. J.; Kim, J. H.; Kim, K. W.; Kwon, Y. K. Biochem.
12. (a) Brocklyn, J. R. V.; Tu, Z.; Edsall, L. C.; Schmidt,
R. R.; Spiegel, S. J. Biol. Chem. 1999, 274, 4626. (b) Tamama,
K.; Kon, J.; Sato, K.; Tomura, H.; Kuwabara, A.; Kimura,
T.; Kanda, T.; Ohta, H.; Ui, M.; Kobayashi, I.; Okajima, F.
Biochem. J. 2001, 353, 139.
13. The homology model of EDG-1 was developed using
Modeller4 (Laboratory of Molecular Biophysics in Rock-
efeller University, New York, USA) and Insight II program
(Accelrys, San Diego, CA, USA) on a Silicon Graphics Octane
workstation (1.2GB RAM, IRIX 6.5). The EDG-1 sequence
was obtained from GenBankTM (AAF43420) and the bovine
rhodopsin model composed of 7 transmembrane helices (PDB
code 1boj) was used as a template structure for homology
modeling. The results of docking studies with S1P isomers and
analogues will be reported in due course.
14. CHO cells used in our bioassays were provided by Dr.
Yoshiko Banno (Gifu University, Japan) and Dr. Yoh
Takuwa (Kanazawa University, Japan), and we wish to thank
them.