Synthesis of fluorescence-labeled sphingosine and sphingosine 1-phosphate; Effective tools for sphingosine and sphingosine 1-phosphate behavior

Article abstract of DOI:10.1016/S0960-894X(02)00999-X

A fluorescence-labeled sphingosine and sphingosine 1-phosphate have been successfully synthesized from the oxazolidinone methyl ester derived from glycidol via monoalkylation and the stereoselective reduction of the resulting ketone. The labeled sphingosine was converted into its phosphate by treatment with sphingosine kinase 1 (SPHK1) from mouse, and in platelets, and it was incorporated into the Chinese Hamster Ovarian (CHO) cells. In addition, MAPK was activated by NBD-Sph-1-P through Edg-1, Sph-1-P receptor.

Full text of DOI:10.1016/S0960-894X(02)00999-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 661–664
Synthesis of Fluorescence-Labeled Sphingosine and Sphingosine
1-Phosphate; Effective Tools for Sphingosine and Sphingosine
1-Phosphate Behavior
Toshikazu Hakogi,a Toshihiko Shigenari,a Shigeo Katsumura,a,*
Takamitsu Sano,b Takayuki Kohnob and Yasuyuki Igarashib,*
aSchool of Science and Technology, Kwansei Gakuin University, Gakuen, Sanda, Hyogo 669-1337, Japan
bGraduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido 060-0812, Japan
Received 20 September 2002;revised 18 November 2002;accepted 19 November 2002
Abstract—A fluorescence-labeled sphingosine and sphingosine 1-phosphate have been successfully synthesized from the oxazolidi-
none methyl ester derived from glycidol via monoalkylation and the stereoselective reduction of the resulting ketone. The labeled
sphingosine was converted into its phosphate by treatment with sphingosine kinase 1 (SPHK1) from mouse, and in platelets, and it
was incorporated into the Chinese Hamster Ovarian (CHO) cells. In addition, MAPK was activated by NBD-Sph-1-P through
Edg-1, Sph-1-P receptor.
# 2003 Elsevier Science Ltd. All rights reserved.
Sphingolipids are known as a lipid second messenger in
mammalian cells and cell membranes, and a great deal
of attention has been devoted to the studies of the bio-
logical process regulated by sphingolipids (Fig. 1).1
hydrolysis of the N-acyl group, and sphingosine
1-phosphate is generated from sphingosine by phos-
phorylation. We now report the synthesis of newly
designed fluorescence labeled analogues 1 and 2, in
which the fluorescent NBD group was situated in the
terminal of the sphingosine backbone. We also describe
their adaptability to sphingosine kinase and sphingosine
1-phosphate receptor as a substrate or a ligand (Fig. 2).
Now, it has been well accepted that the sphingolipids
play key roles in the cellular signal transmission path-
way. Among these sphingolipids, sphingosine 1-phos-
phate is regarded as a new remarkable phospholipid
based on the discovery of its biological receptor2 in
addition to its quite attractive biological activities.
Thus, sphingosine 1-phosphate stimulates DNA synth-
esis, cell division, cell growth,3 and platelet activation.4
Meanwhile, the fluorescence-labeled sphingolipids have
contributed in many areas as a tool to clarify the mode
of biological action of sphingolipids. Thus, nitrobenzo-
2-oxa-1,3-diazole(NBD)-labeled sphingomyelin and cer-
amide in which the fluorescent group is linked to the
N-acyl terminus, are now commercially available5 and
have been widely used.6 These compounds, however,
cannot be utilized in the research of the important sphin-
gosine and sphingosine-1-phosphate metabolism, because
sphingosine is biologically produced from ceramide by
We previously prepared both enantiomers of 4-methoxy-
carbonyloxazolidinone 7 as a chiral building block from
the enantiomerically pure glycidol, and succeeded in the
stereoselective syntheses of g-hydroxy-b-aminoalcohols,
sphingolipids including the short-chain sphingomyelin,
and azasugars by monoalkylation of the ester 7 fol-
lowed by highly syn- and anti-selective reduction of the
resulting ketones with L-selectride or diisobutylalumi-
num 2,6-di-t-butyl-4-methylphenoxide, respectively.7
These methods were then used for the synthesis of the
fluorescence labeled sphingolipids 1 and 2.
The alkyl chain 6 as a backbone component was pre-
pared starting from 1,10-decanediol 3 as shown in
Scheme 1. Protection of the hydroxyl group as the THP
ether followed by the Swern oxidation gave aldehyde 4,
which was converted into the desired acetylene 6 by
utilizing Corey’s method in two steps;8 treatment with
*Corresponding author. Tel.: +81-7956-58314;fax: +81-7956-59077;
e-mail: katsumura@ksc.kwansei.ac.jp
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00999-X

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T. Hakogi et al. / Bioorg. Med. Chem. Lett. 13 (2003) 661–664
Figure 1. Sphingomyelin metabolism.
Figure 2. Designed NBD-labeled sphingolipids.
triphenylphosphine and carbon tetrabromide in the
presence of triethylamine9 followed by a reaction with 3
equivalents of n-butyllithium at À78 ꢀC produced the
desired acetylene 6 (Scheme 1).
The reaction of the lithium anion derived from 6 with
(S)-(À)-ester 7 at the temperature below À100 ꢀC in
THF selectively gave ketone 8 without any dialkylated
products (Scheme 2). The stereoselective reduction of
the obtained ketone was successfully achieved using
Scheme 2. Synthesis of sphingosine backbone. Reagents and condi-
tions;(i) 6, n-BuLi, THF, À100 ꢀC, 93%;(ii) diisobutylaluminum 2,6-
di-t-butyl-4-methylphenoxide, toluene, 0 ꢀC, 99%;(iii) Li, NH , THF,
3
diisobutylaluminum
2,6-di-tert-butyl-4-methylphen-
reflux, 96%;(iv) (a) KOH, EtOH, reflux;(b) Boc 2O, K2CO3, THF,
H2O, 0 ꢀC, 84% for two steps.
oxide10 to produce the desired anti aminoalcohol 9 in
99% yield, and the selectivity between the anti and syn
1
was confirmed by H NMR analysis to be 17:1. The
Introduction of the NBD group at the terminal position
was successfully realized as follows. The two hydroxy
groups of 11 were protected as tert-butyldimethylsilyl
ethers and then removal of the THP protecting group
desired acetylene 9 was then treated with Li in liquid
NH3 to produce oxazolidinone 10 in 96% yield resulting
from the E olefin preparation accompanied by removal
of the benzyl group. Hydrolysis of the oxazolidinone ring
of 10 with potassium hydroxide in ethanol under reflux
smoothly proceeded, and the subsequent protection of
the produced amine selectively afforded the desired 11 by
treatment with Boc2O in H2O and THF (50%) without
isolation of the intermediary primary amine, whose iso-
lation was difficult because of its strong polarity.
11
was completed by treatment with excess MgBr2 to
produce 13, whose terminal alcohol was activated as its
mesylate. The obtained mesylate was reacted with NaN3
in DMF to introduce an azide group. Reduction of the
azide with PPh3 in 10% aqueous THF at 60 ꢀC pro-
ceeded smoothly to afford the corresponding terminal
amine 15. A fluorescent NBD group was successfully
introduced by treatment with NBDCl in the presence of
Et3N, and the obtained compound was purified after
removal of the two silyl groups to furnish the Boc pro-
tected fluorescent sphingosine 16. Fortunately, this
fluorescent compound was stable enough in a bright
laboratory. Finally, deprotection of the amine with tri-
fluoroacetic acid gave the title compound 1,12 which was
1
purified by reverse phase HPLC. The H NMR spec-
trum of the triacetate derived from the compound 1 was
quite similar to those of natural sphingosine except for
the signals of the terminal group (Scheme 3).13
Scheme 1. Synthesis of acetylene. Reagents and conditions;(i) (a)
DHP, PPTS, CHCl3, 66%;(b) (COCl) 2, DMSO, Et3N, CH2Cl2,
À78 ꢀC, 99%;(ii) PPh 3, CBr4, CH2Cl2, 84%;(iii) n-BuLi, THF,
À78 ꢀC, quant.
Next, the synthesis of the labeled sphingosine 1-phos-
phate was accomplished according to the literature pro-
cedure.14 Thus, 16 was treated with carbon tetrabromide

T. Hakogi et al. / Bioorg. Med. Chem. Lett. 13 (2003) 661–664
663
The synthesized NBD-sphingosine was phosphorylated
by recombinant mouse sphingosine kinase 1 or sphin-
gosine kinase from human platelet lysates, yielding
NBD-sphingosine 1-phosphate under the conditions
applied to sphingosine phosphorylation.17 Thus,
NBD-sphingosine, like sphingosine, was recognized as a
substrate for sphingosine kinase. Next, we examined the
changes of intracellular distribution of NBD-sphingo-
sine and its metabolites in the Chinese Hamster Ovarian
(CHO) cells. In these experiments, cells were incubated
with 1 mM NBD-sphingosine at 37 ꢀC for varying times.
NBD-sphingosine was rapidly incorporated into cells,
and most of intracellular NBD-fluorescence was incor-
porated into cytoplasm in 5-min incubation, and to the
Golgi apparatus in 15-min incubation (Fig. 3A). We
examined the metabolic changes of the incorporated
NBD-sphingosine in CHO cells using thin-layer chro-
matography
(Butanol/Acetic
acid/H2O=3:1:1).
NBD-sphingosine was converted to NBD-sphingosine
1-phosphate, NBD-ceramide and NBD-sphingomyelin
(Fig. 3B). The pattern of metabolic changes of
3
NBD-sphingosine was similar to that of H-sphingosine
(data not shown). Based on these experiments, we con-
cluded that NBD-sphingosine could be a useful tool to
visualize the sphingosine dynamics in the cells under the
fluorescent microscope.
Scheme 3. Synthesis of the NBD-labeled sphingosine. Reagents and
conditions;(i) TBSCl, imid., DMF;(ii) MgBr 2, Et2O, 82% for two
steps;(iii) (a) MsCl, Et 3N, THF;(b) NaN 3, DMF, 50 ꢀC, 89% for two
steps;(iv) PPh , 10% aq THF, 60 ꢀC;(v) (a) NBDCl, Et N, THF;(b)
In addition, we examined whether MAPK was activated
by NBD-Sph-1-P according to the method described
previously.18 Briefly, Edg-1 expressing CHO cells
(8Â105) were cultured in serum free Ham F-12 medium
for 24 h followed by incubation in the absence or pre-
sence of 500 ng/mL pertusis toxin (PTx) for 6 h. Then,
the cells were stimulated with 100 nM Sph-1-P of 1 nM
NBD-Sph-1-P for 5 min. Total cell lysates were pre-
pared using lysis buffer (50 mM Tris–HCl (pH 7.5), 500
mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1%
Triton X-100, 10 mM MgCl2, protease inhibitors cock-
tail19 (complete EDTA free) and 5 mM EDTA). These
lysates were resuspended in Laemmli sample buffer20
followed by boiling for 3 min. Then these samples were
subjected to SDS-PAGE, and analyzed by Western
3
3
2N-HCl, MeOH, 82% for three steps;(vi) TFA, CH 2Cl2, 33%.
and trimethylphosphite in pyridine at À10 ꢀC to furnish
the dimethyl phosphonic ester 17. When this reaction
was carried out at 0 ꢀC, the secondly hydroxyl group of
16 was also phosphorylated. Finally, deprotection of the
amino group and hydrolysis of the methyl ester were
achieved by treatment with bromotrimethylsilane in
acetonitrile to give sphingosine 1-phosphate 2,15 which
1
was purified by reversed phase HPLC. The H NMR
spectrum of the obtained compound was quite similar
to those of the natural sphingosine 1-phosphate except
for the signals of the terminal group (Scheme 4).16 Thus,
the fluorescent NBD labeled sphingosine 1 and sphin-
gosine 1-phosphate 2 were stereoselectively synthesized.
Scheme 4. Synthesis of the NBD-labeled sphingosine 1-phosphate.
Reagents and conditions;(i) CBr 4, P(OMe)3, pyr. À10 ꢀC, 82%;(ii)
TMSBr, CH3CN, 71%.
Figure 3. Incorporation into the CHO cells.

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T. Hakogi et al. / Bioorg. Med. Chem. Lett. 13 (2003) 661–664
3. Olivera, A.;Spiegel, S. Nature 1993, 365, 557.
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(c=0.86, CH3OH);IR (KBr disk): 3281, 3081, 2928, 1678,
1620, 1589, 1300, 1202, 1138 cmÀ1 1H NMR (CD3OD, 400
;
MHz) d: 8.51 (d, J=8.78 Hz, 1H), 6.33 (d, J=9.02 Hz, 1H),
5.83 (dtd, J=0.98, 6.83, 15.13 Hz, 1H), 5.46 (tdd, J=1.47,
7.08, 15.37 Hz, 1H), 4.27 (t, J=5.85 Hz, 1H), 3.79 (dd,
J=4.15, 11.71 Hz, 1H), 3.66 (dd, J=8.30, 11.71 Hz, 1H),
3.42–3.60 (m, 2H), 3.20 (td, J=4.39, 8.29 Hz, 1H), 2.08 (m,
2H), 1.77 (qd, J=7.32, 7.32 Hz, 2H), 1.27–1.51 (m, 14H); 13C
NMR (CD3OD, 100 MHz) d: 146.7, 145.8, 145.5, 138.5, 136.5,
128.5, 122.8, 99.6, 71.0, 59.5, 58.5, 44.8, 33.3, 30.6, 30.5, 30.35,
30.30, 30.1, 29.3, 28.0;FAB HRMS m/z calcd for C20H32N5O5
(M++H ) 422.2403, found 422.2409.
Figure 4. MAPK activation by Sph-1-P and NBD-Sph-1-P.
blotting using an anti-MAPK antibody19 and an anti-
phospho MAPK antibody.19 These results clearly
showed that NBD-Sph-1-P as well as Sph-1-P induced
MAPK activation through the Edg-1 in a PTx sensitive
manner. Based upon these results, we concluded that
NBD-Sph-1-P is recognized as a ligand by Sph-1-P
receptor, Edg-1 (Fig. 4)
In conclusion, the NBD labeled sphingosine 1 and
sphingosine 1-phosphate 2 were synthesized from (À)-4-
methoxycarbonyloxazolidinone. The synthesized 1 was
recognized as a substrate by sphingosine kinase, and
was converted into the corresponding 1-phosphate 2. In
addition, 2 was recognized as a ligand by Edg-1, Sph-1-
P receptor. Thus, 1 and 2 would be useful tools to
visualize the sphingosine and sphingosine 1-phosphate
dynamics, respectively.
13. Herold, P. Helv. Chim. Acta 1988, 71, 354.
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15. Data for fluorescence-labeled sphingosine 1-phosphate 2;
[a]1D9.0 À9.88 (c=0.52, CH3OH);IR (KBr disk): 3401, 2928,
1588, 1530, 1300, 1044 cmÀ1; 1H NMR (CD3OD, 400 MHz) d:
8.51 (d, J=8.78 Hz, 1H), 6.34 (d, J=9.03 Hz, 1H), 5.88 (dtd,
J=0.98, 6.83, 15.36 Hz, 1H), 5.48 (tdd, J=1.22, 6.83, 15.37
Hz, 1H), 4.34 (dd, J=5.37, 5.61 Hz, 1H), 4.24 (ddd, J=3.66,
6.10, 11.46 Hz, 1H), 4.12 (ddd, J=6.59, 8.29, 11.47 Hz, 1H),
3.47–3.59 (m, 2H), 3.45 (td, J=3.91, 9.02 Hz, 1H), 2.09 (td,
J=6.83, 6.83 Hz, 2H), 1.78 (tt, J=7.32, 7.32 Hz, 2H),
1.28–1.50 (m, 12H); 13C NMR (CD3OD, 100MHz) d: 146.7,
145.7, 145.5, 138.6, 137.0, 128.0, 122.6, 99.7, 70.6, 64.3
(JC-P=3.31 Hz), 56.9 (JC-P=7.44 Hz), 44.8, 33.3, 30.5, 30.4,
30.33, 30.28, 30.0, 29.3, 28.0.
Acknowledgements
We thank Daiso Co., Ltd., for providing the enantio-
merically pure glycidol. This research was supported by
a Grant-in-Aid for Scientific Research from the Minis-
try of Education, Science and Culture, Japan. T.H. is
grateful to the JSPS for a Research Fellowship for
Young Scientists.
16. Li, S.;Wilson, W. K.;Schroepfer, G. J., Jr. J. Lipid Res.
1999, 40, 117.
References and Notes
17. Olivera, A.;Kohama, T.;Tu, Z.;Milstien, S.;Spiegel, S.
J. Biol. Chem. 1998, 273, 12576.
1. For recent reviews on signal transduction mediated by
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K. M. Biochem. 2001, 40, 4893. (b) Kolter, T.;Sandhoff, K.
Angew. Chem. Int. Ed. 1999, 38, 1532, and references cited
therein.
2. Lee, M.-J.;Van Brocklyn, J. R.;Thangada, S.;Liu, C. H.;
Hand, A. R.;Menzeleev, R.;Spiegel, S.;Hla, T. Science 1998,
279, 1552.
18. Kohno, T.;Wada, A.;Igarashi, Y. FASEB J. 2002, 16, 983.
19. The following substances were purchased;protease inhi-
bitors cocktail: Roche Molecular Biochemicals, Mannheim,
Germany;anti-MAPK antibody: Cell signaling, Beverly, MA;
anti-phospho MAPK antibody: Santa Cruz Biotechnology,
Santa Cruz, CA.
20. Laemmli, U. K. Nature 1970, 227, 680.