Enzymatic Preparation of Sphingosine 1-Phosphate

Article abstract of DOI:10.1039/a804770g

Sphingosine 1-phosphate was prepared from sphingosine by two steps involving conversion of sphingosine to phosphatidylsphingosine catalyzed by phospholipase D and enzymatic hydrolysis of the intermediate to a diacyglycerol and sphingosine 1-phosphate with phospholipase C.

Full text of DOI:10.1039/a804770g

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774 J. CHEM. RESEARCH (S), 1999
J. Chem. Research (S),
1999, 774±775$
Enzymatic Preparation of Sphingosine 1-Phosphate$
Eiji Morigaki, Yoshie Miura, Kyoya Takahata, Mikiro Tada,
Shuhei Nakajima and Naomichi Baba*
Department of Bioresource Chemistry, Faculty of Agriculture, Okayama University, Tsushimanka,
Okayama, 700-8530, Japan
Sphingosine 1-phosphate was prepared from sphingosine by two steps involving conversion of sphingosine to
phosphatidylsphingosine catalyzed by phospholipase D and enzymatic hydrolysis of the intermediate to a diacyglycerol
and sphingosine 1-phosphate with phospholipase C.
In the sphingolipid metabolic cycle, sphingosine 1-phosphate
(Sph 1-P) is produced from sphingosine (Sph) by sphingo-
sine kinase and converted back to Sph catalyzed by sphingo-
sine 1-phosphate phosphatase. Sph 1-P has been identi®ed
as a second messenger for various biochemical events.
Zhang et al. suggested1 that, in their study using Swiss 3T3
®broblasts, Sph 1-P was involved in calcium release and
the regulation of cell growth induced by sphingosine. By the
same research group,2 it was also demonstrated using the
Swiss 3T3 cells that Sph 1-P induced a rapid increase in
phosphatidic acid level which might be correlated with its
e€ect on DNA synthesis in addition to calcium immobiliz-
ation. This group also reported very recently3 that Sph 1-P
activated G protein-coupled orphan receptor EDG-1, over
expression of which induced exaggerated cell±cell aggrega-
tion, enhanced expression of cadherins and formation of
well-developed adherens junctions. Thus, Sph 1-P has been
implicated as an important second messenger in prolifer-
ation and survival of cells. However, its receptors on the
cell surface have not been identi®ed yet. To promote studies
of this ®eld it is essential to supply Sph 1-P in sucient
and Schmidt.4 Their method is based on the use of azido-
sphingosine in the phosphite amide method. This method
seems to be the most ecient and versatile so far. However,
it still requires several steps. The present report describes a
very simple and convenient two-step enzymatic preparation
of Sph 1-P using phospholinase D (PLD) and phospho-
lipase C (PLC) via phosphatidylsphingosine (PSph) as an
intermediate (Scheme 1). This strategy is basically the same
as that reported by D'Arrigo et al.5 In the present case,
however, a problem arose in the PLC-catalyzed phosphate
ester cleavage step.
PSph could be prepared very easily from phosphatidyl-
choline (PC) 1 and commercially available sphingosine (Sph)
2 by phospholipase D-catalyzed transphosphatidylation. In
fact, as described in the experimental section, commercially
available PLD from Streptomyces sp. catalyzed the reaction
to a€ord PSph. Here, PC 1 bearing a linoleoyl group at the
sn-2 position was employed to utilize the ole®nic protons as
a marker for structural analyses by NMR. As a key and
last step, the PSph 3 was submitted to PLC-catalyzed phos-
phate ester cleavage. The enzymic reaction was conducted
using PSph and PLC from Streptomyces chromofuscus in
the presence of sodium deoxycholate, calcium chloride
and lecitin in tris±HCl, pH 7.5. However, no reaction was
observed. As another candidate, PLC from Bacillus cereus
quantities.
A one step synthesis from sphingosine by
utilizing sphingosine kinase seems to be impracticable since
the enzyme is not readily accessible. The most recent report
on the chemical synthesis of Sph 1-P was done by Kratzer
Scheme 1 Enzymic synthesis of sphingosine 1-phosphate
was examined under the same conditions. This enzyme is
known to be inactive to phosphate cleavage at the sphingo-
myelin site suggesting that such a cleavage occurs at the
phosphatidyl site of 36 which is marked with a curved arrow
in the reaction scheme. Here, the addition of lecitin broad-
*To receive any correspondence (e-mail: babanaom@news2.cc.
okayama-u.ac.jp).
$This is a Short Paper as de®ned in the Instructions for Authors,
Section 5.0 [see J. Chem. Research (S), 1998, Issue 1]; there is there-
fore no corresponding material in J. Chem. Research (M).

View Article Online
J. CHEM. RESEARCH (S), 1999 775
‡
4.12±4.36 [m, 4H, POCH2CH(OCOR)CH2OCO, POCH2CHCN3 ],
4.47 [m, 1H, CH(NH3 )CHOH], 5.20 (m, 1H, CHOCO), 5.34
ens the speci®city of the enzyme. Unexpectedly however,
only a trace of Sph 1-P product was detected on TLC by
molybdenum-blue coloration for phosphorus and ninhydrin-
violet coloration for the amino group. Very recently,
Kamata et al. found7 that, in the phospholipase
C-catalyzed hydrolysis of PC, calcium ion was not essential
and it rather retarded the reaction. Thus, we used the same
PLC from Bacillus cereus as described above for cleavage
of the phosphate ester bond of 3 without Ca2‡ under the
reaction conditions described in the experimental section.
TLC analysis indeed showed the formation of Sph 1-P.
After puri®cation by silica gel preparative TLC, the isolated
yield was 30%. Although, the yields are unsatisfactory and
should be improved further by alteration of enzyme source
and/or reaction conditions, the present synthetic route with
only two steps may be usable to obtain Sph 1-P in a
preparative scale in a much shorter time.
‡
(m, 4H, ole®n protons in the linoleoyl group), 5.42 [m, 1H,
.
.
CH(OH)CH CH)], 5.83 [m, 1H, CH(OH)CH CH)]. Ion spray MS:
‡
m/z 982.8 (M ‡ H) . Calc. for C57H104NO9P, 981.8.
Preparation of Sphingosine 1-phosphate.ÐTo a solution of PSph 3
(4 mg, 4 ꢀmol) in diethyl ether (80 ꢀl) was added phosphate bu€er
(0.48 ml, pH 7.0, 1/15 M), phospholipase C from Bacillus cereus
(500 units, Sigma) and a trace of butylated hydroxytoluene, and
the solution was stirred at 30 8C for 4 h in a nitrogen atmosphere
in the dark. The reaction mixture was then made slightly acidic with
0.1 M HCl and the product was extracted with chloroform ten times.
The solvent was evaporated under reduced pressure and the residue
was chromatographed on preparative TLC a€ording the title com-
±
±
pound. Yield 30%. TLC (silica gel G, CHCl3 CH3OH H2O,
50:40:10), Rf ˆ 0.36. ꢁH 0.88 (br, 3H, CH3), 1.27 (m, 22H,
±
±
.
11 ÂCH2), 2.08 [m, 2H, C C CH2 (CH2)11CH3], 3.60±4.42 [m, 4H,
‡
±
±
±
±
P O CH2 CH(NH3 ) CH(OH)], 5.44±5.53 [m, 1H, CH(OH)
CH CH], 5.70±5.81 [m, 1H, CH(OH) CH CH]. This NMR spec-
±
±
.
.
trum coincided with that reported.8 Ion spray MS: m/z 380.3
‡
(M ‡ H) . Calc. for C18H38NO5P, 379.2.
Experimental
1H NMR spectra (dH) were recorded on a Varian VXR 200 or
500 spectrophotometer. Ion spray mass spectra were taken with an
API III triple quadrupole mass spectrometer (PE-Sciex) equipped
with an ion spray interface.
Received, 23rd June 1998; Accepted, 3rd August 1998
Paper E/8/04770G
References
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a solution of
1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (13.1 mg, 17 ꢀmol)
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(0.1 M), excess of sphingosine (25 mg, 84 ꢀmol), phospholipase D
from Streptomyces sp. (ca. 50 units) and a trace of butylated
hydroxytoluene (an antioxidant), and the solution was stirred
at 30 8C for 4 h in a nitrogen atmosphere in the dark. Ethylene-
diaminetetraacetic acid (EDTA) (0.52 ml, 0.1 M) was added to
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32ÂCH2), 1.58 (m, 4H, 2ÂOCOCH2CH2), 2.04 (m, 6H,
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.
3ÂC CCH2CH2), 2.28 (m, 4H, 2ÂOCOCH2), 2.76 (m, 2H,
‡
.
C CCH2C C), 3.72 (m, 1H, CH2CHNH3 ), 3.96 (m, 2H, CH2OP),
.