Phosphorylation of unnatural phosphatidylinositols with phosphatidylinositol 3-kinase

Article abstract of DOI:10.1016/S0040-4020(00)00161-7

Phosphatidylinositol analogs (PI(C2)-PI(C18)) having a series of saturated fatty acid (C2-C18) at sn-2 position were synthesized and subjected to the phosphorylation reaction with phosphatidylinositol 3-kinase (PI 3- kinase). The reactivity of Pica with PI 3-kinase turned out to be comparable to that of natural PI, although PI(C18) was not phosphorylated under the same condition. The chirality of sn-2 center was not responsible for the phosphorylation reaction. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00161-7

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 2603±2614
Phosphorylation of Unnatural Phosphatidylinositols with
Phosphatidylinositol 3-Kinase
Naoko Morisaki,a Koji Morita,a Asuka Nishikawa,a Noriyuki Nakatsu,b Yasuhisa Fukui,b
Yuichi Hashimotoa and Ryuichi Shiraic,
*
aInstitute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
bGraduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
cResearch and Education Center for Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma-shi,
Nara 630-0101, Japan
Received 6 January 2000; accepted 21 February 2000
AbstractÐPhosphatidylinositol analogs (PIC2±PIC18) having a series of saturated fatty acid (C2±C18) at sn-2 position were synthesized and
subjected to the phosphorylation reaction with phosphatidylinositol 3-kinase (PI 3-kinase). The reactivity of PIC8 with PI 3-kinase turned out
to be comparable to that of natural PI, although PIC18 was not phosphorylated under the same condition. The chirality of sn-2 center was not
responsible for the phosphorylation reaction. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
regulation of cell proliferation, differentiation, apoptosis
and many other biological events are now becoming clear.5
Phosphatidylinositol 3-kinase (PI 3-kinase) is an enzyme
that catalyzes phosphorylation at D-3 position on the
inositol ring of polyphosphoinositides.1 The p110±p85
heterodimer type PI 3-kinase consists of catalytic subunit
(molecular weight 110 kDa) and regulatory adapter subunit
(85 kDa)2 is activated in vivo by platelet-derived growth
factor (PDGF), insulin and other extracellular stimulations.3
This enzyme phosphorylates phosphatidylinositol 4,5-
bisphosphate (PI 4,5-P2) to give phosphatidylinositol
3,4,5-trisphosphate (PIP3), which is then dephosphorylated
by PIP3 speci®c 5-phosphatase4 to produce phosphatidyl-
inositol 3,4-bisphosphate (PI 3,4-P2) (Scheme 1). PIP3 and
PI 3,4-P2 are considered to be the second messengers and/
or their precursors, and their roles and functions in the
In vitro, the PI 3-kinase phosphorylates phosphatidylinositol
(PI), phosphatidylinositol 4-phosphate (PI 4-P) and PI 4,5-
P2 to give phosphatidylinositol 3-phosphate (PI 3-P), PI 3,4-
P2 and PIP3, respectively.1b Natural PI (1), which mainly
contains stearoyl (COC17H35) at the sn-1 position and
arachidonoyl (COC19H31) at the sn-2 position of the diacyl-
glycerol (DAG) substructure, is an excellent substrate of the
PI 3-kinase.6 The syntheses of PI, PI phosphates and the
derivatives have been reported and several reports describe
the importance of the fatty acids in the DAG substructure.
However, systematic analysis focusing on their enzymatic
reactions has not been carried out yet.7 Recently, we found
that the saturated PI (2, sn-2 eicosanoate: COC19H39),
Scheme 1.
Keywords: carboxylic acids and derivatives; enzymes and enzyme reactions; inositols; phosphoric acids and derivatives.
* Corresponding author. Tel.: 181-743-72-6171; fax: 181-743-72-6179; e-mail: rshirai@ms.aist-nara.ac.jp
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00161-7

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N. Morisaki et al. / Tetrahedron 56 (2000) 2603±2614
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Figure 1.
prepared by hydrogenation of natural PI, was not phos-
phorylated by the PI 3-kinase. Synthetic distearate PI (3)
was also unchanged by the same reaction (Scheme 2).
Therefore, we designed a series of saturated PI to clarify
the contribution of the sn-2 fatty acid ester in this
phosphorylation reaction (Fig. 1). These unnatural PIs
[PIC2±PIC18] were prepared according to the syntheses of
PIP3 and its analogs.8 Synthesized unnatural PI analogs
were subjected to the enzymatic reaction with ATP, and
the products were analyzed by negative ion fast atom
bombardment mass spectrometry (FAB MS) using
optimized matrix.9±11 Phosphorylation of each unnatural
PI with [g-32P]-ATP was also monitored by autoradiography
of the phosphorylated products.12
PIs (PIC2, PIC4, PIC8, PIC12, PIC16, PIC18). To screen the pos-
sible substrate of the PI 3-kinase by negative ion FAB MS, a
mixture of seven unnatural PI analogs was also synthesized
by similar procedure utilizing the amidite 9 prepared from
an equimolar mixture of seven saturated carboxylic acids
with respective carbon numbers of 4, 6, 8, 10, 12, 14 and 16.
PI 3-kinase reaction of unnatural PIs
Synthesized unnatural PI analogs were mixed with phos-
phatidylserine (PS) and subjected to the PI 3-kinase
reaction.16 The procedures are described in Experimental.
The reaction was stopped by treatment with CHCl3±
CH3OH±c.HCl, and the crude CHCl3 extracts were
subjected to the following analyses.
Results
Analysis of the PI 3-kinase reaction products of
unnatural PI analogs by negative ion FAB MS: matrix
effect of PI, PI 4-P and PI 4,5-P2 in negative ion FAB MS
Synthesis of unnatural sn-2 fatty acid analogs of PI
Synthesis of a series of unnatural PI analogs with varied
chain length of saturated sn-2 fatty acid was carried out
by the procedure similar to our synthesis of PIP3 analogs.8c
The sn-1 fatty acid ester was ®xed as stearate in all
compounds. The synthesis of each PIcn (nˆ2±18) was
shown in Schemes 3 and 4. The diol 5 was prepared from
(S)-(1)-2,3-dimethyl-1,3-dioxolane-4-methanol (4) by
conventional method. The amidites with two different acyl
groups (9) were prepared from 5 according to the method of
Bannwarth et al.13 by utilizing a series of saturated fatty
acids.7e,8c Suitably protected homochiral myo-inositol (10)
was prepared8c from d-glucose by the method of Estevez
and Prestwich.7b Coupling of 10 and respective 9 followed
by oxidation with m-chloroperbenzoic acid (mCPBA) in one
pot gave fully protected PIs (11).8c,14 Oxidative removal of
the 4-methoxybenzyl groups with 2,3-dichloro-5,6-dicyano-
1,4-benzoquinone (DDQ) in wet CH2Cl2,15 and hydrogeno-
lysis of benzyloxymethyl ether with Pd black in 85%
Ef®cient detection of PI 3-P is indispensable because the
average yield of PI 3-P in the enzyme reaction of unnatural
PI was about 20±30%, and relatively large amount of
unreacted PI analogs were still contained in the CHCl3
extracts of the enzyme reaction mixture. To detect the
enzymatically produced PI 3-P, optimization of liquid
matrix is very important for ef®cient FAB MS analysis.17
Triethanolamine (TEA)11b,11d, diethanolamine (DEA)11e and
glycerol (GLY)11a have been used as matrices for negative
ion FAB MS analysis of PI and PI phosphates. Since PI 3-P
was commercially unavailable, PI 4-P was employed to
optimize matrices instead. Matrix effect was examined by
monitoring the intensity of the [M±H]2 ion peak with
matrices TEA, DEA, GLY and their mixtures, and results
are shown in Fig. 2.
By use of mixed matrix of TEA±GLY at a ratio of 3:1, the
maximum intensity of [M±H]2 ion peak of PI 4-P was two
and half times higher than with TEA alone. On the contrary,
7e
t-BuOH containing NaHCO3 gave a series of unnatural

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N. Morisaki et al. / Tetrahedron 56 (2000) 2603±2614
2607
Scheme 4.
Figure 2. Matrix effects of PI, PI 4-P and PI 4,5P2 in negative ion FAB MS. Intensities of [M±H]2 ion peaks were compared in matrices triethanolamine
(TEA), glycerol (GLY), diethanolamine (DEA) or their mixture.

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N. Morisaki et al. / Tetrahedron 56 (2000) 2603±2614
Figure 3. Negative ion FAB MS of a mixture of PIs and its PI 3-kinase reaction products (a) Spectrum of a mixture of synthetic unnatural PIs (PIC4, PIC6, PIC8
,
PIC10, PIC12, PIC14, PIC16). Triethanolamine (TEA) was used as the matrix. (b) Spectrum of PI 3-kinase reaction products derived from the mixture of synthetic
unnatural PIC4±C16. TEA±glycerol (3:1) was used as the matrix.
the intensity of [M±H]2 of PI was relatively low with TEA±
GLY(3:1), and high with TEA and DEA, respectively.
Therefore, it was expected that the relative intensity of
[M±H]2 ion of PI 4-P was higher than that of PI by analyz-
ing an equimolar mixture of PI 4-P and PI with TEA±
GLY(3:1). Assuming that the sensitivity of [M±H]2 ions
of PI 3-P and PI 4-P were obtained in similar fashion,
TEA±GLY (3:1) would be a desirable matrix for the analy-
sis of PI 3-P in an enzyme reaction mixture even though a
large amount of unreacted PI is contaminated.
Figure 4. Autoradiography of phosphorylation products of natural PI and
unnatural PIs with PI 3-kinase and [g-32P]-ATP.
Effects of matrices with PI and PI 4,5-P2 were also
examined. The intensity of [M±H]2 ion of PI 4,5-P2 was
Figure 5. Autoradiography of phosphorylation products of natural PI, unnatural PIC2 and PIC8 with PI 3-kinase and [g-32P]-ATP under competitive conditions.
(PS: phosphatidylserine).

N. Morisaki et al. / Tetrahedron 56 (2000) 2603±2614
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N. Morisaki et al. / Tetrahedron 56 (2000) 2603±2614
unstable in most matrices tested, however, relatively
constant and strong [M±H]2 ion was observed with
DEA.
Negative ion FAB MS of PI 3-kinase reaction products
Negative ion FAB MS spectrum of a mixture of synthetic PI
analogs [PIC4±PIC16] is shown in Fig. 3(a). The [M±H]2
ions of the respective PI analogs are indicated by the
peaks at m/z 669, 697, 725, 753, 781, 809 and 837. Although
the PI analogs were prepared starting from an equimolar
mixture of seven fatty acids, intensity of the [M±H]2 ions
of longer fatty acid analogs appeared lower. The enzymatic
phosphorylation of unnatural PI analogs with PI 3-kinase
was repeated several times to enrich PI 3-P and thus the
obtained reaction mixture was analyzed by negative ion
FAB MS with matrix of TEA±GLY (3:1). One of them is
shown in Fig. 3(b). The [M±H]2 ion peak of PIC4 3-P, PIC6
3-P, PIC8 3-P, PIC10 3-P is observed at m/z 749, 777, 805, and
833, respectively. These spectra indicated that PIC4, PIC6,
PIC8 and PIC10 were obviously phosphorylated by the PI
3-kinase giving PIC4 3-P±PIC10 3-P in good yield.18 As the
peak intensities of PI 3-P with longer fatty acids were very
low, the yield was shown quite low.
Figure 6. Autoradiography of phosphorylation products of natural PI,
hydrogenated PI, PIC18 and other unnatural PIs by PI 3-kinase and
[g-32P]-ATP.
of natural PI. Therefore, we turned our attention to whether
PI 3-kinase could differentiate the absolute stereochemistry
of sn-2 position. Synthesis of sn-2-epi PIC8 was performed
from R-(2)-2,2-dimethyl-1,3-dioxolane-4-methanol (13)19
according to the synthesis of unnatural PIs (Scheme 5).
The reactivity of PIC8 and sn-2-epi PIC8 was compared by
independent phosphorylation with the PI 3-kinase and
[g-32P]-ATP by the same procedure as those for unnatural
PIs, respectively. The reactions of natural PI (1), hydro-
genated PI (2), PIC18 (3), PIC4±16 mixture, and PIC4 were
also done for comparison. The crude reaction products
were developed on TLC plate and visualized by auto-
radiography, and the result was shown in Fig. 6. Interest-
ingly, phosphorylation of PIC8 and sn-2-epi PIC8 proceeded
equally to produce PIC8 3-P and sn-2-epi PIC8 3-P, indicating
that the PI 3-kinase did not differentiate the absolute stereo-
chemistry at sn-2 center.20
TLC and autoradiography analyses of PI 3-kinase
reaction products of unnatural PI analogs
The results of negative ion FAB MS described above were
also con®rmed by the following experiments. Each of the
six unnatural PI analogs [PIC2, PIC4, PIC8, PIC12, PIC16, PIC18
]
was subjected to the enzyme reaction with [g-32P]ATP, and
after development on TLC, the produced 32P-labeled PI 3-Ps
were detected by autoradiography. The results are shown in
Fig. 4. The spot of PIC2 3-P, PIC4 3-P, PIC8 3-P, PIC12 3-P was
clearly detected, respectively. On the other hand, the spot of
PIC16 3-P was very faint and that of PIC18 3-P was negligible.
These data demonstrated that PIC2, PIC4, PIC8 and PIC12
behaved as the substrate of PI 3-kinase (Fig. 4).
Discussion
Systematic studies on substrate speci®city of the enzymatic
reaction revealed an unknown structural factor of DAG
substructure. In the phosphorylation of PI by PI 3-kinase,
arachidonoate at sn-2 position of natural PI can be replaced
by short saturated fatty acyl group. Folded unsaturated
arachidonoate is expected to loosen the binding of PI to
lipid bilayer, which might be an essential factor for ef®cient
assembly of the enzyme and the substrate. PI with short fatty
acid at sn-2 also get ¯exibility and might be able to come
close to the enzyme like arachidonoate rather than e.g.
stearate which is tightly ®xed in the lipid bilayer.21 The
short saturated fatty acids such as octanoate might be
equivalent of the folded unsaturated arachidonoate and the
spherical size of the side chain at sn-2 might be crucial for
the PI 3-kinase activity. These results may be applied to the
design of arti®cial second messenger, PI 3-kinase inhibitors,
af®nity probes to ®nd the speci®c binding probes, and
photoaf®nity probes.8b,8d
Enzymatic reactivity of PIC2, PIC8, and natural PI
The reactivity of synthetic unnatural PI with PI 3-kinase was
evaluated by competitive phosphorylation with natural PI.
Equimolar mixtures of PIC2 and natural PI, and PIC8 and
natural PI were respectively incubated with the PI 3-kinase
for 5, 15 and 60 min in the presence of PS and [g-32P]-ATP.
The reaction mixtures were developed on TLC, and the
radioactivity of the resulting PI 3-P was visualized by auto-
radiography (Fig. 5). The blank phosphorylation reaction
was also carried out with PS, natural PI and PS, PIC2 and
PS, and PIC8 and PS, respectively. From the equimolar
mixture of natural PI and PIC8, equivalent amounts of PI
3-P and PIC8 3-P were produced. This result demonstrated
that the reactivity of PIC8 was comparable to that of
natural PI. As the amount of PIC2 3-P was smaller than
that of natural PI 3-P, PIC2 is slightly less reactive than
natural PI.6
Experimental
Materials
Comparison of the enzymatic reactivity of PIC8 and sn-2
epimer of PIC8
Bovine liver PI ammonium salt and bovine brain PI 4-P
ammonium salt, and bovine brain PI 4,5-P2 ammonium
As shown in Fig. 5, the reactivity of PIC8 was similar to that

N. Morisaki et al. / Tetrahedron 56 (2000) 2603±2614
2611
salt for FAB MS analyses were purchased from Sigma
Chemical Co. and Boehringer Mannheim Corp., respec-
tively, and bovine brain PI and bovine brain PS for
enzymatic reaction were from Avanti. TEA, GLY and
DEA were purchased from Tokyo Kasei Kogyo Co., Ltd.
and Wako Pure Chemical Industries, Ltd., respectively.
by developing with methanol±1% potassium oxalate (1:1)
and activated at 808C for 3 h. After samples were
spotted, the TLC plate was developed with a solvent system
of H2O±acetic acid±methanol±acetone±chloroform (7:12:
13:15:40)4.
Synthesis of unnatural PIs
Enzymatic reaction with PI 3-kinase16
Typical representative synthesis of PIC8 and sn-2-epi PIC8
are described. 1H NMR and 31P NMR spectra were
measured on JEOL Alpha-500 NMR spectrometer at 500
and 202.35 MHz, respectively. Chemical shifts of 1H
NMR were recorded in d unit relative to internal tetra-
methylsilane (TMS) (dˆ0) in CDCl3, and relative to DHO
(dˆ4.65 ppm) in D2O. Chemical shifts of 31P NMR were
relative to external potassium phosphate (dˆ0). IR spectra
were recorded on a JASCO A-102 instrument. FAB MS and
high resolution FAB MS (HRFAB MS) were measured on a
JEOL JMS-HX110 instrument. mNBA or TEA±GLY (3:1)
was used as the matrix. Elementary analyses were
performed by the Microanalytical Laboratory, Faculty of
Pharmaceutical Sciences, The University of Tokyo. Optical
rotations were recorded on a JASCO DIP 1000 digital
polarimeter.
The PI 3-kinase was puri®ed from Sf9 cells co-expressing
GST-p85 and p110, the two subunits of the heterodimer.22
The enzymatic reaction was carried out as follows: A solu-
tion of unnatural PI analog (0.2 mg) and PS (10 mg) in
dimethylsulfoxide (0.5 ml) was dispersed into 50 ml of
buffer A containing 20 mM Tris/HCl buffer (pH 7.5),
100 mM NaCl and 0.5 mM EGTA to form micelles. The
reaction was started by the addition of the PI 3-kinase,
and [g-32P]-ATP (10 mM, 5 mCi) and MgCl2 (®nal 5 mM)
dissolved in the buffer A. After incubation of the mixture for
1 h at 258C, the reaction was stopped by the addition of
100 ml of CHCl3±CH3OH±c.HCl (100:200:10). The solu-
tion was stirred with a Vortex mixer, and the CHCl3 solution
containing unnatural PI and the reaction product was
separated by centrifugation. The aqueous solution was
re-extracted with 200 ml of CHCl3, and the CHCl3 solutions
were combined. The solvent of the solution was removed in
vacuo. The extracts were prepared by TLC, and the reaction
products were detected by autoradiography. Samples for
FAB MS analyses were obtained by the reaction of a
mixture of seven unnatural PI analogs (PIC4±PIC16, 20±
30 mg) and PS (200±300 mg), and with unlabeled ATP.
The yield of PI 3-kinase was 20±30%.
1-O-Stearoyl-3-O-benzyl-sn-glycerol (6). 3-O-Benzyl-sn-
glycerol (5, 4 g, 22.0 mmol), prepared from 4 and
dehydrated by the benzene azeotrope, was dissolved in
dry pyridine (100 ml) and cooled to 08C. To this solution,
stearoyl chloride (6.5 g, 21.5 mmol in dry CH2Cl2) was
added and stirred overnight at room temperature. The
reaction mixture was poured into AcOEt, and the whole
was washed with saturated CuSO4 and with brine. After
being dried over anhydrous Na2SO4, the solvent was
removed to give the crude ester 6, which was puri®ed by
silica gel column chromatography (n-hexane: AcOEtˆ6:1)
to give 6 (5.9 g, 13.2 mmol, 60% yield) as colorless crystals.
1H NMR (CDCl3): d 0.98 (3H, dt, Jˆ1.0, 6.5 Hz, CH3), 1.26
(30H, brm), 2.33 (2H, t, Jˆ7.0 Hz, COCH2), 3.50 (1H, ddd,
Jˆ1.0, 6.0, 10.0 Hz), 3.56 (1H, ddd, Jˆ1.0, 4.5, 10.0 Hz),
4.03 (1H, m, H-2), 4.14 (1H, ddd, Jˆ1.0, 6.0, 12.0 Hz), 4.21
(1H, ddd, Jˆ1.0, 4.5, 12.0 Hz), 4.56 (2H, s, benzyl), 7.26±
7.38 (5H, m, phenyl); IR (CHCl3): 2920, 2850, 1730, 1455,
1360±1380, 1240, 1170, 1090±1110 cm21; FAB MS
(mNBA): m/z 449 (M1H); [a]Dˆ12.0 (c 0.42, CHCl3);
Anal. Calcd for C28H48O4: C, 74.95; H, 10.78. Found: C,
75.23; H, 10.56.
Instrumentation and sample preparation for FAB MS
analysis
Negative ion FAB mass spectra were recorded on a JEOL
JMS-HX110 double-focusing mass spectrometer of EBE
arrangement with a JMS-DA7000 data system. Ion
acceleration voltage was 10 kV, and the fast-atom xenon
gas was accelerated at a voltage of 6 kV.
Sample solutions for experiments of matrix effects shown in
Fig. 2 were prepared by mixing 1 mg sample and 0.5 ml of
matrix on FAB MS target. Negative ion FAB mass spectra
were obtained by means of a 2.8 s scan from m/z 810 to 940
for PI, 2.7 s scan from m/z 890 to 1020 for PI 4-P, and 2.6 s
scan from m/z 970 to 1100 for PI 4,5-P2. Data were collected
every 10 s, and seven values from the start to 1 min of the
analysis were averaged.
1-O-Stearoyl-2-O-octanoyl-3-O-benzyl-sn-glycerol (7c).
Compound 6 (600 mg, 1.34 mmol), dehydrated by the
benzene azeotrope, and catalytic amount of N,N-dimethyl-
aminopyridine (DMAP) were dissolved in dry pyridine
(16 ml), and cooled to 08C. To this solution, octanoyl
chloride (256 ml, 1.5 mmol, in 10 ml of dry CH2Cl2) was
added dropwise, and the solution was warmed to room
temperature. The reaction mixture was poured in AcOEt,
and the solution was washed successively with saturated
CuSO4, saturated NaHCO3, and brine, then dried over
anhydrous Na2SO4. The solvent was removed, and the
ester 7c was puri®ed by silica gel column chromatography
(n-hexane: AcOEtˆ25: 1) to give 7c (685 mg, 1.19 mmol,
89% yield) as colorless oil. 1H NMR (CDCl3): d 0.87 (3H, t,
Jˆ7.0 Hz, CH3), 0.88 (3H, t, Jˆ7.0 Hz, CH3), 1.25 (36H,
For the analysis of unnatural PI analogs, TEA was used as
the matrix, and TEA±GLY (3:1) was used for the PI
3-kinase reaction products of the unnatural PI analogs.
Prior to the FAB MS analysis, the CHCl3 extracts of
the enzyme reaction products were re-treated with
CHCl3±CH3OH±1 N HCl (10:5:1), and the CHCl3 layer
containing free acid form PIs and PI 3-Ps were analyzed.
TLC preparation
TLC plate (Silica Gel 60 plastic sheet) was pre-treated

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N. Morisaki et al. / Tetrahedron 56 (2000) 2603±2614
brm), 1.54 (4H, m), 2.20±2.26 (4H, m, COCH2), 3.73 (2H,
m, sn-H-3), 4.24 (1H, dd, Jˆ5.5, 12.0 Hz, sn-H-1), 4.32
(1H, dd, Jˆ4.5, 12.0 Hz, sn-H-1), 4.51 (1H, d, Jˆ12.0 Hz,
benzyl), 4.55 (1H, d, Jˆ12.0 Hz, benzyl), 5.08 (1H, m,
sn-H-2), 7.25±7.45 (5H, m, phenyl); IR (CHCl3, cm21):
2920, 2850, 2250, 1720±1740, 1455, 1370, 1250±1270,
1160, 1100, 900; FAB MS (mNBA): m/z 575 (M1H);
[a]Dˆ16.7 (c 0.76, CHCl3).
Compound 11c (425 mg, 0.30 mmol) was dissolved in a
mixture of CH2Cl2 (4.5 ml) and water (0.5 ml), and was
treated with 95% DDQ (355 mg, 1.49 mmol). After
90 min of stirring at room temperature, the reaction mixture
was poured in AcOEt, and the solution was washed with
saturated NaHCO3 and with brine, then dried over
anhydrous Na2SO4. The solvent was removed, and the
product 12c was puri®ed by silica gel column chromato-
graphy (CHCl3:MeOHˆ20:1) to give 12c (122 mg,
1
1-O-Stearoyl-2-O-octanoyl-sn-glycerol (8c). Compound
7c (680 mg, 1.2 mmol) was dissolved in 40 ml of AcOEt,
and hydrogenated with Pd±C (380 mg) under H2 overnight
at room temperature. The catalyst was removed by ®ltration,
the solvent was removed in vacuo, and the residue was
chromatographed on silica gel (n-hexane:AcOEtˆ5:1) to
give 8c (486 mg, 1.0 mmol, 84% yield). 1H NMR
(CDCl3): d 0.87 (3H, t, Jˆ7.0 Hz), 0.88 (3H, t, Jˆ
7.0 Hz), 1.25 (36H, brm), 1.54 (4H, m), 2.20±2.26 (4H,
m, COCH2), 3.73 (2H, m), 4.24 (1H, dd, Jˆ5.5, 12.0 Hz),
4.32 (1H, dd, Jˆ4.5, 12.0 Hz), 5.08 (1H, m); IR (CHCl3,
cm21): 2920, 2850, 2250, 1730, 1460, 1380, 1260, 1160,
1100, 900; FAB MS (mNBA): m/z 485 (M1H);
[a]Dˆ23.1 (c 1.0, CHCl3).
0.115 mmol, 66% yield for 2 steps) as colorless oil. H
NMR (CDCl3): d 0.87 (3H, t, Jˆ7.0 Hz), 0.88 (3H, t,
Jˆ7.0 Hz), 1.25 (36H, brm), 1.57 (4H, m), 2.24±2.30
(4H, m, COCH2), 3.36±3.43 (2H, m), 3.69 (1H, m), 3.80
(1H, m), 4.05±4.31 (7H, m), 4.53±5.09 (10H, m, benzyl),
5.15 (1H, m), 7.26±7.37 (15H, m, phenyl); IR (CHCl3,
cm21): 3400, 2920, 2850, 2250, 1735, 1710, 1455, 1360,
1260, 1150, 1100, 1000, 890; FAB MS (mNBA) m/z 1080
(M1Na); HRFAB MS. Calcd for C58H89O15PNa (M1Na):
1079.5837, Found: 1079.5885.
PIC8
Compound 12c (35 mg, 0.033 mmol) was dissolved in
14 ml of 85% t-BuOH in water, and Pd black (50 mg) and
NaHCO3 (3 mg, 0.035 mmol) were added to the solution,
and the mixture was shaken under H2 (4.0 kgf/cm2) for 7 h.
The mixture was ®ltered, the catalyst was washed with
EtOH and water, and the ®ltrate was concentrated in
vacuo and lyophilized to give PIC8 (25 mg, 0.033 mmol,
2,6-Di-O-benzyloxymethyl-3,4,5-tri-O-p-methoxybenzyl-
d-myo-inositol (1-O-stearoyl-2-O-octanoyl-sn-glycerol)
benzylphosphate (11c). Benzyl-N,N,N0,N0-tetraisopropyl-
phosphoramide8c,13 (650 mg, 2.0 mmol) and 1H-tetrazole
(140 mg, 2.0 mmol) were dissolved in dry CH2Cl2
(20 ml). To this solution, compound 8c (460 mg,
0.95 mmol) in dry CH2Cl2 (10 ml) was added dropwise,
and stirred at room temperature. After the reaction was
completed, the solvent was removed with a rotary evapora-
tor. The residue was chromatographed on a silica gel
column with n-hexane:AcOEt:Et3Nˆ25:4:1). The oily
product 9c (651 mg, 95% yield) was used as such in subse-
quent experiments. 2,6-Di-O-benzyloxymethyl-3,4,5-tri-O-
p-methoxybenzyl-d-myo-inositol (10, 234 mg, 0.3 mmol),
prepared by Prestwich's procedure,7b was dehydrated by
the benzene azeotrope. Compound 9c (651 mg,
0.90 mmol) and 1H-tetrazole (120 mg, 1.7 mmol) were
added to 10 and the whole was dehydrated with a rotary
pump for 1 h. The mixture was dissolved in dry CH2Cl2
(5 ml), and the solution was stirred for 1 h at room tempera-
ture. A small amount of water was added, and the whole was
stirred for 10 min. Then, 70% mCPBA (300 mg, 1.74 mmol)
was added at 2788C, and the reaction mixture was allowed
to warm to room temperature. The whole was poured in
AcOEt, and successively washed with 10% Na2SO3, satu-
rated NaHCO3 and brine, then dried over anhydrous
Na2SO4. The solvent was removed, and the product 11c
was puri®ed by silica gel column chromatography (n-hexa-
ne:AcOEtˆ2.5:1) (735 mg, containing a small amount of
1
quant.). H NMR (D2O): d 0.60 (3H, t, Jˆ7.0 Hz, CH3),
0.78 (3H, t, Jˆ7.0 Hz, CH3), 1.10±1.20 (40H, brm), 1.40±
1.55 (4H, m), 2.20 (4H, m, COCH2), 3.20±4.35 (10H, m),
5.20 (1H, br); 31P NMR (D2O):(d 20.43; negative ion FAB
MS (TEA±GLYˆ3:1): m/z 725 (M±H).
1-O-Benzyl-3-O-stearoyl-sn-glycerol (15). 1-O-Benzyl-
sn-glycerol (14, 967 mg, 4.37 mmol), prepared from 13
and dehydrated by the benzene azeotrope, was dissolved
in 16 ml of dry pyridine and cooled to 08C. To this solution,
stearoyl chloride (1.33 g, 4.38 mmol in dry CH2Cl2) was
added and stirred for 2 h at room temperature. The reaction
mixture was poured into AcOEt, and the whole was washed
with saturated CuSO4 and with brine. After being dried over
anhydrous Na2SO4, the solvent was removed to give the
crude ester 15, which was puri®ed by silica gel column
chromatography (n-hexane:AcOEtˆ9:1) to give 15
(1.06 g, 2.37 mmol, 54% yield) as colorless crystal. 1H
NMR (CDCl3): d 0.98 (3H, dt, Jˆ1.0, 6.5 Hz, CH3), 1.26
(30H, brm), 2.33 (2H, t, Jˆ7.0 Hz, COCH2), 3.50 (1H, ddd,
Jˆ1.0, 6.0, 10.0 Hz), 3.56 (1H, ddd, Jˆ1.0, 4.5, 10.0 Hz),
4.03 (1H, m), 4.14 (1H, ddd, Jˆ1.0, 6.0, 12.0 Hz), 4.21 (1H,
ddd, Jˆ1.0, 4.5, 12.0 Hz), 4.56 (2H, s, benzyl), 7.26±7.38
(5H, m, phenyl); IR (CHCl3, cm21): 2920, 2850, 1730,
1455, 1360±1380, 1240, 1170, 1090±1110; FAB MS
(mNBA): m/z 449 (M1H); [a]dˆ21.9 (c 0.94, CHCl3);
Anal. Calcd for C28H48O4: C, 74.95; H, 10.78. Found: C,
74.72; H, 10.64.
1
impurity). H NMR (CDCl3): d 0.87 (3H, t, Jˆ7.0 Hz),
0.88 (3H, t, Jˆ7.0 Hz), 1.25 (36H, brm), 1.54 (4H, m),
2.20±2.26 (4H, m, COCH2), 3.37±3.43 (2H, m), 3.78 (9H,
s, PMB±OMe), 3.93±4.24 (6H, m), 4.43±5.10 (18H, m),
5.14 (1H, m), 6.78±7.32 (27H, m, phenyl); IR (CHCl3,
cm21): 2920, 2850, 1740, 1615, 1515, 1460, 1360, 1200±
1240, 1160, 1020; FAB MS (mNBA): m/z 1440 (M1Na).
1-O-Benzyl-2-O-octanoyl-3-O-stearoyl-sn-glycerol (16).
Compound 15 (500 mg, 1.12 mmol) dehydrated by the
benzene azeotrope and catalytic amount of DMAP were
dissolved in dry pyridine (12 ml) and cooled to 08C. To
this solution, octanoyl chloride (350 ml, 2.05 mmol in
2,6-Di-O-benzyloxymethyl-d-myo-inositol (1-O-stearoyl-
2-O-octanoyl-sn-glycerol)
benzylphosphate
(12c).

N. Morisaki et al. / Tetrahedron 56 (2000) 2603±2614
2613
1
6 ml of dry CH2Cl2) was added with stirring, and the solu-
tion was warmed to room temperature. The reaction mixture
was poured in AcOEt, and the solution was successively
washed with saturated CuSO4 solution, saturated NaHCO3
solution, and brine, then dried over anhydrous Na2SO4. The
solvent was removed, and the ester 16 was puri®ed by silica
gel column chromatography (n-hexane:AcOEtˆ25:1) to
give 16 (550 mg, 0.96 mmol, 86% yield) as colorless oil.
1H NMR (CDCl3): d 0.87 (3H, t, Jˆ7.0 Hz, CH3), 0.88 (3H,
t, Jˆ7.0 Hz, CH3), 1.25 (40H, brm), 1.55±1.65 (4H, m),
2.27 (2H, t, Jˆ7.5 Hz, COCH2), 2.32 (2H, t, Jˆ7.5 Hz,
COCH2), 3.58 (2H, dd, Jˆ1.5, 5.0 Hz), 4.18 (1H, dd,
Jˆ6.5, 12.0 Hz), 4.34 (1H, dd, Jˆ5.0, 12.0 Hz), 4.51 (1H,
d, Jˆ12.0 Hz, benzyl), 4.55 (1H, d, Jˆ12.0 Hz, benzyl),
5.31 (1H, m), 7.25±7.45 (5H, m, phenyl); IR (CHCl3,
cm21): 2920, 2850, 2250, 1720±1740, 1455, 1370, 1250±
1270, 1160, 1100, 900; FAB MS (mNBA): m/z 575 (M1H);
[a]Dˆ26.9 (c 1.6, CHCl3).
graphy to give 19 (460 mg, 0.32 mmol, 90% yield). H
NMR (CDCl3): d 0.87 (3H, t, Jˆ7.0 Hz), 0.88 (3H, t,
Jˆ7.0 Hz), 1.25 (36H, brm), 1.54 (4H, m), 2.20±2.26
(4H, m, COCH2), 3.37±3.43 (2H, m), 3.78 (9H, s, PMB±
OMe), 3.93±4.24 (6H, m), 4.43±5.10 (18H, m), 5.14 (1H,
m), 6.78±7.32 (27H, m, phenyl); IR (CHCl3, cm21): 2920,
2850, 1740, 1615, 1515, 1460, 1360, 1200±1240, 1160,
1020; FAB MS (mNBA): m/z 1440 (M1Na).
2,6-Di-O-benzyloxymethyl-d-myo-inositol (2-O-octanoyl-
3-O-stearoyl-sn-glycerol)
benzylphosphate
(20).
Compound 19 (380 mg, 0.27 mmol) was dissolved in a
mixture of CH2Cl2 (4.5 ml) and water (0.5 ml), and was
treated with 95% DDQ (300 mg, 1.26 mmol). After 2 h of
stirring at room temperature, the reaction mixture was
poured in AcOEt, and the solution was washed with satu-
rated NaHCO3 and with brine, then dried over anhydrous
Na2SO4. The solvent was removed, and the product 20 was
puri®ed by silica gel column chromatography (n-hexane:
AcOEtˆ1:2) to give 20 (220 mg, 0.21 mmol, 77% yield)
as colorless oil. 1H NMR (CDCl3): d 0.87 (3H, t,
Jˆ7.0 Hz), 0.88 (3H, t, Jˆ7.0 Hz), 1.25 (36H, brm), 1.57
(2H, m), 2.24±2.30 (4H, m, COCH2), 3.36±3.43 (2H, m),
3.69 (1H, m), 3.80 (1H, m), 4.05±4.31 (7H, m), 4.53±5.09
(9H, m), 5.15 (1H, m), 7.26±7.37 (15H, m, phenyl); IR
(CHCl3, cm21): 3400, 2920, 2850, 2250, 1735, 1710,
1455, 1360, 1260, 1150, 1100, 1000, 890; FAB MS
(mNBA): m/z 1080 (M1Na); HRFAB MS. Calcd for
C58H89O15PNa (M1Na). 1079.5837, Found: 1079.5867.
2-O-Octanoyl-3-O-stearoyl-sn-glycerol (17). Compound
16 (520 mg, 0.91 mmol) was dissolved in 35 ml of
AcOEt, and hydrogenated with Pd±C (290 mg) under H2
overnight at room temperature. The catalyst was removed
by ®ltration, the solvent was removed in vacuo, and the
residue was chromatographed on silica gel (n-hexane:
AcOEtˆ5:1) to give 17, colorless oil, 395 mg (0.82 mmol,
1
90% yield): H NMR (CDCl3): d 0.88 (3H, t, Jˆ7.0 Hz,
CH3), 0.88 (3H, 3H, t, Jˆ7.0 Hz, CH3), 1.25 (36H, brm),
1.63 (4H, m), 2.32 (2H, t, Jˆ7.5 Hz, COCH2), 2.35 (2H, t,
Jˆ7.5 Hz, COCH2), 3.73 (2H, m), 4.24 (1H, dd, Jˆ5.5,
12.0 Hz), 4.32 (1H, dd, Jˆ4.5, 12.0 Hz), 5.08 (1H, m); IR
(CHCl3, cm21): 2920, 2850, 2250, 1730, 1460, 1380, 1260,
1160, 1100, 900; FAB MS (mNBA): m/z 485 (M1H);
[a]dˆ13.3 (c 0.75, CHCl3); Anal. Calcd for C29H56O5: C,
71.85; H, 11.64. Found: C, 72.05; H, 11.51.
sn-2-epi PIC8
Compound 20 (29 mg, 0.027 mmol) was dissolved in 14 ml
of 85% t-BuOH in water, and Pd black (40 mg) and
NaHCO3 (2.3 mg, 0.027 mmol) were added to the solution,
and the mixture was shaken under H2 (4.0 kgf/cm2) for 7 h.
The mixture was ®ltered, the catalyst was washed with
EtOH and water, and the ®ltrate was concentrated in
vacuo and lyophilized to give sn-2-epi PIC8, (17.5 mg,
2,6-Di-O-benzyloxymethyl-3,4,5-tri-O-p-methoxybenzyl-
d-myo-inositol (2-O-octanoyl-3-O-stearoyl-sn-glycerol)
benzylphosphate (19). Benzyl-N,N,N0,N0-tetraisopropyl-
phosphoramide8c,13 (650 mg, 2.0 mmol) and 1H-tetrazole
(140 mg, 2.0 mmol) were dissolved in dry CH2Cl2
(20 ml). To this solution, compound 17 (372 mg,
0.77 mmol) in dry CH2Cl2 (10 ml) was added dropwise,
and stirred at room temperature. After the reaction was
completed, the solvent was removed with a rotary evapora-
tor. The residue was chromatographed on a silica gel
column with n-hexane: AcOEt:Et3Nˆ25:4:1). The oily
product 18 (385 mg, 69% yield) was used as such in sub-
sequent experiments. Compound 10 (280 mg, 0.36 mmol),
prepared by Prestwich's procedure,7b was dehydrated by the
benzene azeotrope. Compound 18 (385 mg, 0.53 mmol) and
1H-tetrazole (100 mg, 1.43 mmol) were added, and the
whole was dehydrated with a rotary pump for 1 h. The
mixture was dissolved in dry CH2Cl2 (5 ml), and the solu-
tion was stirred for 1 h at room temperature. A small amount
of water was added, and the whole was stirred for 10 min.
Then, the mixture was cooled to 2788C, and 70% mCPBA
(350 mg, 2.03 mmol) was added, and the reaction mixture
was allowed to warm to room temperature. The whole was
poured in AcOEt, and washed successively with 10%
Na2SO3, saturated NaHCO3 and brine, then dried over
anhydrous Na2SO4. The solvent was removed, and the
product 19 was puri®ed by silica gel column chromato-
1
0.023 mmol, 85% yield): H NMR (D2O): d 0.60 (3H, br,
CH3), 0.78 (3H, br, CH3), 1.10±1.20 (40H, brm), 1.40±1.55
(4H, m), 2.20 (4H, m, COCH2), 3.20±4.35 (10H, m), 5.20
(1H, br); 31P NMR (D2O):(d 20.43; negative ion FAB MS
(TEA±GLYˆ3:1): m/z 725 (M±H).
Acknowledgements
We are grateful to Dr Kazuo Furihata, Division of Agricul-
ture and Agricultural Life Sciences, The University of
Tokyo, for the measurement of 31P NMR. This work was
supported in part by The Naito Foundation, The Mochida
Foundation for Medical and Pharmaceutical Research, and
Grant-in-Aid for Scienti®c Research from The Ministry of
Education, Science, Sports and Culture, Japan.
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