1558
P. Ettmayer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1555–1558
5. Brodesser, S.; Sawatzki, P.; Kolter, T. Eur. J. Org. Chem.
2003, 2021.
6. Hakogi, T.; Shigenari, T.; Katsumura, S.; Sano, T.;
Kohno, T.; Igarashi, Y. Bioorg. Med. Chem. Lett. 2003,
13, 661.
(dd, J=5.4, 6.8 Hz; 1H), 4.19 (t, J=5.3 Hz; 1H), 3.69/
3.57 (AB-system, J=11.6 Hz, 4.0, 8.4 Hz; 2H), 3.11 (m;
2H), 2.80 (s; 6H), 2.73 (t, J=6.9 Hz; 2H), 2.00 (q, J=6.9
Hz, 2H), 1.33–1.22 (m; 2H), 1.22–0.90 (m; 14H). Electro-
spray MS: 506.3 (M+H)+, C27H43N3O4S, calcd 505.3.
Data for the NBD labeled sphingosine 2d: [a]2D0 À3.8
7. Kozikowski, A. P.; Ding, Q.; Spiegel, S. Tetrahedron Lett.
1996, 37, 3279.
1
(c=1.0, CH3OH); H NMR (CD3OD, 400 MHz) d: 8.43
8. Garner, P.; Park, J. M.; Malecki, E. J. Org. Chem. 1988,
53, 4395.
9. Characteristic NMR data for the Mosher esters. 6a,b
(n=3): 1H NMR (DMSO-d6, 500 MHz, 330 K) d: 3.43 (s;
3H, 6a-OCH3), 3.54 (s, 3H; 6b-OCH3). 19F NMR
(d, J=9 Hz; 1H), 6.25 (d, J=9 Hz; 1H), 5.74 (dt, J=15, 7
Hz; 1H), 5.36 (dd, J=15, 7 Hz; 1H), 4.18 (dd, J=6, 7 Hz;
1H); 3.69 (dd, 4, 12 Hz; 1H); 3.56 (dd, 8, 12 Hz; 1H); 3.44
(bs; 2H), 3.12–3.03 (m; 1H), 2.00–1.95 (m; 2H), 1.72–1.64
(m; 2H); 1.40–1.25 (m; 14H). Electrospray MS: 436.2
(M+H)+, C21H33N5O5, calcd 435.2.
(DMSO-d6, 377 MHz, 299K)
À0.192 (s; 6b-CF3).
d
À0.194 (s; 6a-CF3),
12. The phosphorylation reactions were performed essentially
as described in ref 4. Briefly, the cytoplasmic fraction of
recombinant HEK-293 cells overexpressing human
SPHK-1or-2 was incubated at 30 ꢀC in total volumes of
100 mL with SP derivatives (20 mM; added from stock
10. Data for the pyrene labeled sphingosine 1d: [a]2D0 À4.2
(c=1.0, CH3OH); 1H NMR (CDCl3, 400 MHz) d: 9.16 (d,
J=9 Hz; 1H), 8.51 (d, J=9 Hz; 1H), 8.15–7.80 (m; 7H),
5.78 (bs; 1H), 5.42 (bs; 1H), 4.56 (bs; 1H); 4.41 (t, J=9
Hz; 2H); 3.97–3.60 (m; 2H), 3.50 (bs; 3H), 1.96 (bs; 2H),
1.84–1.77 (m; 2H), 1.50–1.20 (m; 14H). Electrospray MS:
502.3 (M+H)+, C32H39NO4, calcd 501.3.
solutions in DMSO), 1mM of ATP, and
2
mCi
[g-32P]ATP in 50 mM Hepes buffer (pH 7.4) containing 15
mM MgCl2, 0.005% Triton X-100, 10 mM KCl, 10 mM
NaF and 1.5 mM semicarbazide. Following incubations
for different time points up to 2 h, lipids were extracted
and separated by thin-layer chromotography (TLC) plates
(Merck). Radiolabeled SPP derivatives were visualized
and quantified using a Molecular Dynamics Storm Phos-
phorImager (Sunnyvale, CA). The rate of phosphoryl-
ation was calculated and is reported for the SP derivatives
as value relative to the rate for sphingosine (for which the
rate was 41and 25 nmol/min/mg with SPHK-1and -2,
respectively).
11. Data for the pyrene labeled sphingosine 2a: [a]2D0 À2.4
(c=1.0, CH3OH); 1H NMR (CDCl3+DMSO-d6,
400 MHz) d: 8.55 (d, J=9 Hz; 1H), 8.28–8.04 (m; 8H),
7.60 (bs; 1H), 5.80 (dt, J=7, 15 Hz; 1H), 5.45 (dd, J=6,
15 Hz; 1H), 4.42 (bs; 1H); 3.83–3.75 (m; 2H), 3.57 (dt,
J=7, 6 Hz; 2H), 3.20 (bs; 1H), 2.04 (dt, J=7, 6 Hz; 2H),
1.77–1.70 (m; 2H), 1.52–1.26 (m; 14H). Electrospray MS:
501.3 (M+H)+, C32H40N2O3, calcd 500.3. Data for the
pyrene labeled sphingosine 2b: [a]2D0 À2.2 (c=1.0, CH3OH);
(CD3OD) d: 8.94 (d, J=9.4 Hz; 1H), 8.56 (d, J=8.2 Hz;
1H), 8.60–8.00 (m; 7H), 5.72 (dt, J=6.6, 15.3 Hz; 1H),
5.34 (dd, J=6.8, 15.3 Hz; 1H), 4.18 (t; J=5.6 Hz; 1H),
3.69/3.56 (AB-System, J=4.0, 11.6, 8.3 Hz; 2H), 3.08 (m;
1H), 2.72 (t, J=6.7 Hz; 2H), 2.93 (m; 2H), 1.23–1.07 (m;
4H), 1.07–0.55 (m; 12H). Electrospray MS: 537.3
(M+H)+, C31H40N2O4S, calcd 536.3. Data for the dan-
syl labeled sphingosine 2c: [a]2D0 À1.8 (c=2, CH3OH);
(CD3OD) d: 8.46 (d, J=8.5 Hz; 1H), 8.27 (d, J=8.7 Hz;
1H), 8.11 (d, J=7.4 Hz; 1H), 7.51–7.46 (m; 2H), 7.19 (d,
J=7.5 Hz; 1H), 5.75 (dt, J=6.4 Hz, 16.2 Hz; 1H), 4.30
13. Thin-layer chromatography was performed on silica plates,
using either butanol/acetic acid/ water 3:1:1 or CHCl3/
MeOH/H2O/NH4OH (28w/w%) 200:150:29:1 as mobile
phase. In these systems sphingosine and 1d co-migrate.
Identity of the metabolites with pyrene-labeled SPP and
SM was established by co-migration with 1d-phosphate
(prepared with recombinant SPHK-1) and commercially
available tritium-labeled SPP and SM as standards.
14. Yang, L.; Yatomi, Y.; Miura, Y.; Satoh, K.; Ozaki, Y.
British Journal of Haematology 1999, 107, 282.