Catalyst-dependent syntheses of phosphatidylinositol-5-phosphate-DiC8 and its enantiomer

Article abstract of DOI:10.1016/j.tet.2008.03.037

Peptide-based catalysts have been applied to the enantioselective syntheses of the title compounds, with this being the first report of the synthesis of an ent-PI5P analogue. The key steps in the synthesis involve asymmetric phosphorylation catalysis. Add

Full text of DOI:10.1016/j.tet.2008.03.037

Tetrahedron 64 (2008) 7015–7020
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Catalyst-dependent syntheses of phosphatidylinositol-5-
phosphate–DiC8 and its enantiomer
*
Katherine J. Kayser-Bricker, Peter A. Jordan, Scott J. Miller
Department of Chemistry, Yale University, PO Box 208107, New Haven, CT 06520, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Peptide-based catalysts have been applied to the enantioselective syntheses of the title compounds, with
this being the first report of the synthesis of an ent-PI5P analogue. The key steps in the synthesis involve
asymmetric phosphorylation catalysis. Additional maneuvers were developed with a protecting groups
scheme that enabled efficient, streamlined syntheses of these important mediators of biochemical
events.
Received 31 January 2008
Received in revised form 10 March 2008
Accepted 12 March 2008
Available online 15 March 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Dedicated to Professor John F. Hartwig on
the occasion of his winning the Tetrahedron
Young Investigator Award
1. Introduction
dipalmitoyl-analogue of PI5P (PI5P–DiC16) has been previously
synthesized by Prestwich13 and Falck14 as the natural enantiomer.
Phosphatidylinositol (1) and its variously phosphorylated ana-
logues have been identified as tremendously important mediators
of biochemical processes.1 Among the monophosphates, phos-
phatidylinositol-5-phosphate (PtdIns-5P or ‘PI5P’, 2) has gained
significant attention (Fig. 1). Recent reports of cellular processes
regulated by this agent are emerging, and its role in biological
functions have only started to be appreciated. Among the examples,
PI5P has been associated as a regulator of chromatin modification
through its interaction with the tumor suppressor protein ING2.2 It
has also been implicated as a regulator of cell cycle progression,3 as
a modulator of gene expression,4 and as an activator of signal
transduction pathways.1,5 Its role in living systems reaches beyond
mammalian cells as it has been identified to play a role in the
osmotic-stress response in plants.6 Limited investigations of PI5P
have identified it as a critical regulator of various cellular functions,
however, further biochemical exploration is needed for our further
understanding of these processes.
Synthetic chemistry has played a defining role in developing the
chemical biology of PtdIns science.7 The synthetic achievements in
the field have yielded access to a number of important members of
this family of targets.8–11,14 The chiral pool has provided a common
starting place for many of the synthetic approaches that deliver
single-enantiomer compounds. The biomimetic approaches of
Prestwich in particular have afforded many members of this class,
as well as PI-based probes of PI-dependent biological events.12 The
Prestwich synthesized the inositol core via a Ferrier rearrangement
of methyl a- -glucopyranoside, while Falck relied on the dia-
stereomeric separation of a camphor ketal intermediate. In addi-
tion, Watanabe15 has synthesized the racemic form of PI5P–DiC16
directly from the meso-myo-inositol core.
D
Our own interest in these compounds stems from the synthetic
challenge in developing rapid, streamlined syntheses of these
molecules, and difficult-to-access analogues, such that high
precision biochemical studies may be undertaken. In particular, we
have reported enantioselective synthesis of phosphatidylinositol-
3-phosphate,16 phosphatidylinositol-3,5-bis(phosphate),17 several
deoxygenated analogues of these compounds, and in selected cases,
enantiomeric versions that may serve as biological probes. In each of
these syntheses, we capitalized on the power of desymmetrization
catalysis to enter a chosen enantiomeric series (Scheme 1).
As shown in Scheme 1, catalysts 3 and 4 have emerged as
workhorses in our laboratory for the rapid syntheses of compounds
in the PtdIns family. Initially, we targeted enantioselective
synthesis of inositol-1-phosphate, and its enantiomer inositol-
3-phosphate. As shown in Scheme 1, rapid syntheses were indeed
possible as a function of the two catalysts we discovered for these
purposes.18,19 Herein, we report the application of chiral catalysts 3
and 4 to the enantioselective syntheses of PI5P as well as ent-PI5P.
Catalyst-dependent syntheses of these particular phosphatidyl-
inositol-monophosphates have not yet been reported, and thus
provide a new entry into each enantiomeric series. Efficient
synthetic routes to these compounds could allow for further bio-
chemical investigation of these important cellular messengers in an
effort to elucidate their biological roles.
* Corresponding author. Tel.: þ1 203 432 9885; fax: þ1 203 436 4900.
E-mail address: scott.miller@yale.edu (S.J. Miller).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.03.037

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K.J. Kayser-Bricker et al. / Tetrahedron 64 (2008) 7015–7020
OH
O
OH
HO
HO
O
P
O
O
P
O
HO
HO
HO
O
HO
HO
O
P
O
O
O
O
O
O
R1
R1
HO
HO
HO
HO
O
O
O
R2
R2
Phosphatidylinositol-5-phosphate
Phosphatidylinositol
(2)
(1)
OH
OH
O
O
O
P
OH
O
OH
OH
ent-Phosphatidylinositol-5-phosphate
(ent-2)
P
R1
O
O
O
OH
OH
O
O
R2
Figure 1. Phosphatidylinositol and its 5-phosphorylated variants.
t-BuO
H
N
OH
OH
N
Me
P
O
OH
OH
O
H
HO
N
O
O
O
3
1
5
N
O
HO
N
N
H
BOC
O
OH
NH
I-3P
2 mol % 3
Ph
O
OBn
t-Bu
O
MeO
P
O
OH
PhO
PhO
3
1
5
>98% ee
O
P
BnO
OBn
3
HO
OPh
OPh
OBn
OH
Cl
BnO
HO
ent-5
OH
1
Et3N, PhCH3
0 °C
5
BnO
OBn
O
P
NHTrt
HO
O
OPh
OPh
OBn
3
1
O
N
5
H
N
BnO
Ph
HN
OH
N
Me
OH
O
N
OH
N
N
O
O
O
5
H
N
HO
HO
O
P
OH
3
1
O
OH
H
5
BOC
O
NH
Me
Scheme 1.
O
O
2 mol % 4
OH
I-1P
t-Bu
OMe
2. Results and discussion
the synthesis of each single-enantiomer PI5P compound (Scheme
1). As previously reported, it was necessary to exchange the phenyl
phosphate ester groups to benzyl phosphate ester groups to
provide an efficient final deprotection of the desired compounds.
Thus the two remaining hydroxyl groups of 5 were protected as the
silyl ethers followed by transesterification and subsequent depro-
tection to provide intermediate 7 (Scheme 2).
With late stage intermediate 7 in hand, our synthetic plan could
either first install the glycerol functionality and then protect the C3
hydroxyl or vice versa. In the examination of the Mitsunobu reaction,
it wasintriguingtorecount thereactionsof 7 anddeoxy-analogue 10,
using identical conditions.16 Both analogues underwent mono-
hydrolysis under basic conditions to deliver the corresponding
phosphoric acids20 followed by reaction with 8 employing Mitsu-
nobu-type conditions to obtain phosphatidylinositol derivatives 9
and 11 (Scheme 3).16 The yields for these two processes are re-
markably different, withthedeoxy-compoundproducedin89%yield
compared to 40% for 9. Further, the purification of 9 was also difficult
as a result of poor separation of the DEAD byproduct that is produced
in the reaction. The requirement for multiple chromatographic
Peptide-based asymmetric catalysis provided direct routes to
desymmetrized phosphoinositols 5 and ent-5 and we envisioned
manipulating these enantiomers to synthesize each PI5P and
ent-PI5P, with dioctanoyl side chains, in an optically pure fashion
(Fig. 2). The synthesis of these phosphatidylinositide derivatives
bears inherent challenges due to the decreased reactivity toward
phosphorylation of the C5 hydroxyl, in comparison to the C3
hydroxyl. Thus, retrosynthetically the C5 phosphate could be pre-
pared from protected derivative 6, which could be obtained
through a selective protection (PG) of the C3 hydroxyl (Fig. 2). The
glycerol fragment could be installed through the previously
developed reaction for these systems of the corresponding
phosphoric acid of 7 and alcohol 8 under Mitsunobu-type condi-
tions.16 Inositol derivative 7 can be readily derived from 5 through
a previously developed three step sequence to convert the phenyl
phosphate ester groups to benzyl phosphate ester groups.16
Large-scale production of both 5 and ent-5, utilizing selective
catalysts 4 and 3, respectively, provided a suitable starting point for

K.J. Kayser-Bricker et al. / Tetrahedron 64 (2008) 7015–7020
7017
O
O
O
P
OH
O
P
OH
OBn
HO
HO
O
PGO
BnO
O
O
O
C7H15
O
O
C7H15
3
OBn
OBn
3
1
5
1
5
O
C7H15
O
C7H15
OH
O
O
PI5P-DiC8
O
O
OH
P
6
HO OH
O
OBn
OBn
O
O P
O
HO
HO
O P
OPh
OPh
OBn
OBn
+
HO
O
C7H15
3
3
1
1
OBn
OBn
O
C7H15
5
5
BnO
BnO
O
OH
5
OH
7
8
Figure 2. Retrosynthetic analysis for an enantiomeric series of PI5P–DiC8.
procedures could have contributed to the decreased isolated yield of
the Mitsunobu product. Based on this evidence, we suspected that
the relatively solvent exposed C3 hydroxyl may be impeding the
reaction. Thus, masking this functionality could lead to increased
yield of the desired product. As a result, our synthetic strategy
evolved in the direction of protecting the C3 hydroxyl followed by
subsequent installation of the glycerol fragment.
Two issues guided the choice of protecting group for the C3
hydroxyl. First, it was desirable to have one set of global
deprotection conditions as the final synthetic step. Therefore the
protecting group to be employed should be removable under
previously developed hydrogenolysis conditions. Second, the pro-
tection needed to be selective for the C3 hydroxyl over the C5
hydroxyl. Initially, we attempted protection of the alcohol as the
benzyl ether. We turned our attention toward Dudley’s benzylation
conditions, which uses MgO as a mild base.21 These conditions
were chosen with the thought that they should be sufficiently mild
in order to preclude phosphate migration. The use of strong bases,
such as NaH in the presence of benzyl bromide, was avoided as it
was expected to cause racemization of 7 through a migration event.
The reaction utilizing Dudley’s reagent was sluggish with the use of
toluene at 80 ꢀC and provided only trace amounts of the desired
product after 12 h. Dudley reports the reaction to be more efficient
with the use of a,a,a-trifluorotoluene as a solvent, and thus we
examined these conditions. These conditions proved to be more
reactive, however, even at 80 ꢀC for 3 days with 4 equiv of both the
benzylating reagent and MgO, the desired product was obtained in
only 18% yield in a 1:2:1 mixture of benzylation products (3,5-OBn/
3-OBn/5-OBn). The poor selectivity and yield of the reaction was
consistent with our previous experience of low site-selectivity for
alkylation reactions when comparing the reactivity of the C1 or C3
hydroxyl with the more hindered C5 hydroxyl.
As previously mentioned, these two positions have shown
remarkable differences in reactivity for the phosphorylation re-
action.18 Therefore, we envisioned protecting the C3 hydroxyl with
a benzyloxycarbonyl, which should provide a more selective reaction
as it proceeds through a tetrahedral intermediate, similar to the
phosphorylation reaction. In addition, this group is removable under
O
P
O
P
OBn
O
P
OBn
OBn
OBn
TBSO
BnO
O
TBSO
BnO
O
HO
O
OPh
OBn
OPh
a
b
c
OPh
OPh
OBn
3
1
5
3
1
3
1
5
5
OBn
OBn
BnO
OTBS
OTBS
OH
13
12
5
O
O
P
O
P
O
P
OBn
OBn
OBn
OBn
CbzO
BnO
O
CbzO
BnO
O
HO
O
OBn
O
O
C7H15
OBn
d
e,f
OBn
OBn
3
1
3
3
OBn
OBn
1
5
1
O
C7H15
5
5
BnO
OBn
O
OH
OH
OH
15
7
14
O
O
O
O
P
ONa
O
P
OBn
OH
OBn
O
O
C7H15
HO
HO
O
CbzO
BnO
O
O
C7H15
g
h
3
1
5
3
1
5
O
C7H15
O
C7H15
OH
O
OBn
O
O
PI5P-DiC8
O
O
O
P
BnO OBn
16
P
NaO ONa
Scheme 2. Reagents and conditions: (a) TBSCl, imidazole, DMF, 89%; (b) NaH, BnOH, THF, 84%; (c) HF–pyridine, THF, 86%; (d) CbzCl, Et3N, cat. DMAP, CH2Cl2, 75%; (e) LiBr, acetone,
reflux; then DOWEX 50ꢁ2-200; (f) DEAD, Ph3P, 8, THF, 74%, two steps; (g) 4,5-dicyanoimidazole, dibenzyl diisopropylphosphoramidite, CH2Cl2; then 30% H2O2/H2O, 85%; (h)
Pd(OH)2/C, H2, t-BuOH/H2O (5:1), Chelex 100 (sodium form), 61%.

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K.J. Kayser-Bricker et al. / Tetrahedron 64 (2008) 7015–7020
O
hydroxyl of the inositol ring. The route employed in our synthesis
O
P
OBn
OBn
OBn
O
was highly efficient and provided both enantiomers in their opti-
cally pure form, thus providing facile access to either a PI5P, or
an ent-PI5P analogue (ent-PI5P–DiC8), from a common starting
material. PI5P is a newly emerging member of the phospholipid
family and its synthesis is essential toward the identification of its
role in cellular processes. These syntheses provide rapid, efficient
access to these molecules thus enabling the further exploration and
identification of novel cellular functions of not only the natural
enantiomer, but also the unnatural enantiomer, which could play
alternate cellular roles.
HO
O P
HO
O
O
C7H15
O
OBn
a, b
40%
3
1
5
O
O
C7H15
OBn
BnO
OBn
BnO
OBn
O
O
OH
7
OH
9
O
O
O
P
OBn
OBn
OBn
O
O P
O
O
C7H15
OBn
OBn
OBn
a, b
89%
3
1
5
C7H15
BnO
BnO
OBn
OH
10
OH
4. Experimental
4.1. General
11
Scheme 3. Reagents and conditions: (a) LiBr, acetone, reflux; then DOWEX 50ꢁ2-200;
(b) DEAD, Ph3P, 8, THF.
Proton NMR spectra were recorded on 400 or 500 MHz
spectrometer. Proton chemical shifts are reported in parts per
million (d) relative to internal tetramethylsilane (TMS, d 0.0 ppm) or
with the solvent reference relative to TMS employed as the internal
standard (CDCl3, d 7.26 ppm; CD3OD, d 3.31 ppm; D2O, d 4.79 ppm).
Data are reported as follows: chemical shift (multiplicity [singlet
(s), doublet (d), triplet (t), quartet (q), and multiplet (m)], coupling
constants [Hz], integration). Carbon NMR spectra were recorded on
400 or 500 MHz spectrometer with complete proton decoupling.
Carbon chemical shifts are reported in parts per million (d) relative
to TMS with the respective solvent resonance as the internal
standard (CDCl3, d 77.0 ppm) except for D2O in which case a drop of
methanol was added as internal standard (CH3OH, d 49.5 ppm).22
Phosphorous NMR spectra were recorded on 400 (162 MHz) or 500
(202 MHz) spectrometer with complete proton decoupling. Phos-
phorous chemical shifts are reported in parts per million (d) relative
to a 85% H3PO4 external standard. NMR data were collected at
25 ꢀC, unless otherwise indicated. Analytical thin-layer chroma-
tography (TLC) was performed using Silica Gel 60 Å F254 precoated
plates (0.25 mm thickness). TLC Rf values are reported. Visualiza-
tion was accomplished by irradiation with an UV lamp and/or
staining with KMnO4 or ceric ammonium molybdenate (CAM)
solutions. Flash column chromatography was performed using
Silica Gel 60 Å (32–63 mm).23 Optical rotations were recorded at
20 ꢀC at the sodium D line (path length 1.0 cm). High resolution
mass spectra were obtained at institutional providers. The method
of ionization is given in parentheses.
the global hydrogenolysis deprotection conditions. As predicted, the
3-OCbz-inositol derivative 14 was delivered in good yield with no
detection of the 5-OCbz regioisomer. This reaction can be catalyzed
by either N-methyl-imidazole (NMI), or 4-dimethylaminopyridine
(DMAP), and shows a strong dependence on a 1.67:1 molar ratio
between benzyl chloroformate and triethylamine. Protected
derivative 14 was then subjected to monodebenzylation conditions
and subsequent Mitsunobu reaction with glycerol fragment 8 to
provide 15 as a mixture of diastereomers at the phosphorus atom.
The Mitsunobu reaction with this derivative proceeded in 74% yield,
which was a significant increase from the unprotected C3 hydroxyl
derivative, which gave only 40% of the desired product under iden-
tical conditions. The Cbz protection may have further contributed to
the yield of the reaction, as 15 was more easily separated from the
many components of the reaction when compared to adduct 9.
From 15, the free hydroxyl at C5 of the myo-inositol core was
subjected to coupling with dibenzyl diisopropylphosphoramidite
under standard conditions to install the phosphate and provide
fully protected PI5P–DiC8, 16 in excellent yield. Deprotection of 16
employing previously optimized hydrogenolysis conditions with
Pearlman’s catalyst (palladium hydroxide on carbon) and Chelex
100 sodium form resin under a hydrogen atmosphere in a water/
tert-butanol mixture proceeded well to provide PI5P–DiC8 as the
sodium salt. On occasion, an unidentified impurity was present in
the sample, manifested by a large singlet at 1.91 ppm in the 1H
NMR. The presence of this contaminant at this stage has sporadi-
cally been a problem in previous synthesis of other phosphoinosi-
tide and phosphatidylinositide derivatives. However, exhaustive
washing of Pearlman’s catalyst prior to use, typically allows the
global deprotection to proceed to deliver a pure product. Moreover,
size exclusion chromatography proved effective for isolation of
analytically pure PI5P–DiC8. In preparative runs, the deprotection
sequence may be carried out, with passage of the product through
a size exclusion column, to allow for the isolation of analytically
pure PI5P–DiC8 in 61% yield, from the fully protected derivative. As
a testimony to the robustness of this streamlined synthetic
sequence, the entire synthetic procedure was repeated, starting
from ent-5, to provide the unnatural enantiomer, ent-PI5P–DiC8,
with comparable yields at every step.
Analytical and preparative reverse phase and normal phase HPLC
wereperformedemployingasinglewavelengthUVdetector(214 nm
or 254 nm). Measurements of enantiomeric excess were carried out
via analytical normal phase HPLC equipped with a diode array
detector (214 nm and 254 nm) and employing a ChiralcelÒ OD col-
umnat aflow rate of0.5 mL/min. Allreactionswerecarried out under
an argon or nitrogen atmosphere employing oven- and flame-dried
glassware. All solvents were distilled from appropriate drying agents
prior to use. Compounds 7 and ent-7 were synthesized from peptide
catalyzed phosphorylation and transesterification reactions.16,17
4.2. Specific procedures
4.2.1. Compound 14
3. Conclusion
To a stirred solution of 7 (190 mg, 0.267 mmol) in CH2Cl2
(0.534 mL) was added 4-(dimethylamino)pyridine (DMAP) (33 mg,
0.267 mmol) followed by triethylamine (0.112 mL, 0.801 mmol).
Benzyl chloroformate (0.188 mL, 1.337 mmol) was then added
quickly. The reaction solution was bubbled and a precipitate
formed. The reaction mixture stirred at room temperature under
a nitrogen atmosphere for 12 h. The reaction was then diluted with
CH2Cl2 (50 mL) and the organic layer was washed with saturated
NaHCO3 (50 mL) and brine (50 mL). The organic layer was dried
In summary, we have completed the total syntheses of both the
natural and unnatural enantiomers of PI5P with dioctanoyl side
chains on the glycerol fragment. Following the catalytic desym-
metrization of a central myo-inositol derivative, the key synthetic
steps employed are the Mitsunobu coupling reaction and the use of
a benzyloxycarbonyl protecting group. The use of the Cbz group
provided selective protection of the C3 hydroxyl over the C5

K.J. Kayser-Bricker et al. / Tetrahedron 64 (2008) 7015–7020
7019
(Na2SO4), filtered, and concentrated in vacuo to afford a yellow oil.
This crude material was then purified by column chromatography
(0–45% ethyl acetate/hexanes) to afford 14 as a clear thick oil
(170 mg, 75%). 1H NMR (CDCl3, 500 MHz) d 7.28–7.12 (m, 30H), 5.04
(ABq, J¼12.1 Hz, 2H), 4.93–4.86 (m, 4H), 4.70 (ABq, J¼11.2 Hz, 2H),
4.64–4.56 (m, 5H), 4.23–4.20 (m, 2H), 3.83 (td, J¼9.5 and 2.3 Hz,
2H), 3.47 (td, J¼9.2 and 2.3 Hz, 1H), 2.37 (d, J¼2.4 Hz, 1H); 13C NMR
(CDCl3, 500 MHz) d 154.4, 138.3, 138.2, 138.1, 135.6, 135.6, 135.1,
128.6, 128.6, 128.5, 128.5, 128.5, 128.4, 128.3, 128.2, 127.9, 127.8,
127.8, 127.7, 127.7, 127.5, 79.6, 79.6, 78.9, 77.8, 77.8, 75.3, 75.2, 75.2,
74.4, 74.4, 69.8, 69.5, 69.5, 69.4, 69.3; 31P NMR (CDCl3, 202 Hz)
d ꢂ1.7; IR (film, cmꢂ1) 3387, 3089, 3064, 3032, 2946, 2880, 1744,
1495, 1450, 1262, 1013; TLC Rf 0.41 (40% ethyl acetate/hexanes);
exact mass calcd for [C49H49O11P]þ requires m/z 845.3085, found
845.3064 (ESIþ); [a]D þ26.4 (c 1.0, CHCl3).
1.23–1.15 (m, 16H), 0.82–0.77 (m, 6H); 13C NMR (CDCl3, 500 MHz)
d 173.0,172.7,172.6,154.2,154.2,138.0,138.0,137.9,137.8,135.9,135.9,
135.8,135.8,135.4,135.4,135.3,135.3,134.9,134.9,128.6,128.6,128.3,
128.3, 128.2, 128.1, 128.1, 127.9, 127.8, 127.8, 127.7, 127.7, 127.6, 127.6,
127.5, 127.5, 127.4, 127.3, 79.9, 79.8, 78.2, 78.2, 78.2, 78.1, 77.6, 77.5,
77.5, 77.5, 77.2, 76.6, 76.5, 76.3, 76.2, 75.4, 75.3, 74.7, 74.7, 69.9, 69.9,
69.8, 69.8, 69.6, 69.6, 69.3, 69.3, 69.2, 69.2, 69.2, 69.1, 65.8, 65.7, 65.6,
65.5, 61.4, 61.4, 34.0, 34.0, 33.9, 31.6, 29.0, 29.0, 28.9, 24.8, 24.7, 22.6,
14.0; 31P NMR (CDCl3, 202 Hz) d ꢂ1.6, ꢂ1.9, ꢂ2.0; IR (film, cmꢂ1); TLC
Rf 0.37 (30% ethyl acetate/hexanes); exact mass calcd for
[C75H91O18P2]þ requires m/z 1341.5675, found 1341.5661 (ESIþ); [a]D
þ8.8 (c 1.0, CHCl3).
4.2.4. PI5P–DiC8
Compound 16 (0.095 g, 0.071 mmol) was dissolved in t-BuOH/
H2O (5:1) (6 mL) and Chelex (Na form) resin was added till a thick
slurry. Palladium hydroxide on carbon (0.190 g) was added and the
chamber evacuated. The solution was placed under 1 atm of H2 and
stirred for 18 h. The reaction mixture was filtered over a Celite pad
and the Celite was washed with ethanol (20 mL), ethanol/water
(1:1) (15 mL), and H2O (15 mL). The filtrate was filtered through
a 0.22 mm filter and concentrated under reduced pressure (no
heating). The resulting solid was dissolved in a minimal amount of
water and run through a Sephadex G-10 size exclusion column
eluting with water. Fractions containing pure product were com-
bined and lyophilized. The solid was dissolved in a minimal amount
of waterand run through a Chelex (Na form) ion exchange column to
fully form the desired sodium salt. Fractions containing the desired
product were combined and lyophilized to yield the sodium salt of
PI5P–DiC8 as a white fluffy solid (0.032 g, 61% yield). 1H NMR (D2O,
500 MHz) d 5.32 (q, J¼7.7 Hz, 1H), 4.43 (dd, J¼12.2 and 1.5 Hz, 1H),
4.27 (dd, J¼12.1 and 8.2 Hz,1H), 4.22 (t, J¼2.6 Hz,1H), 4.08–4.03 (m,
3H), 3.93–3.85 (m, 2H), 3.80 (t, J¼9.3 Hz, 1H), 3.62 (dd, J¼10.0 and
2.4 Hz,1H), 2.48–2.30 (m, 4H),1.64–1.58 (m, 4H),1.37–1.25 (m,16H),
0.89–0.85 (m, 6H); 13C NMR (D2O, 500 MHz) d 175.8,175.7, 79.7, 79.7,
76.6, 76.5, 72.5, 71.7, 71.5, 71.5, 71.2, 63.9, 34.8, 34.8, 32.4, 29.7, 29.6,
29.6, 29.5, 25.5, 25.4, 23.2, 23.2, 14.5, 14.4; 31P NMR (D2O, 202 Hz)
d 4.3, ꢂ0.6; exact mass calcd for [C25H49O16P2]þ requires m/z
667.2496, found 667.2476 (ESIþ); [a]D þ2.75 (c 2.0, H2O, pH 9).
4.2.2. Compound 15
To a stirred solution of 14 (0.185 g, 0.219 mmol) in 14 mL of
acetone (reagent grade) was added LiBr (0.035 g, 0.408 mmol), and
the reaction mixture was refluxed for 10 h. It was then cooled to
room temperature and concentrated. The residue was purified by
silica gel chromatography eluting with 50% EtOAc/hexanes to 15%
CH3OH/CH2Cl2 to give the crude product as a lithium salt. The salt
was dissolved in a minimal amount of CH3OH and run through a Hþ
DOWEX 50ꢁ2-200 ion exchange column. Fractions containing the
desired product were combined and concentrated to give the crude
phosphoric product. The crude residue was then dissolved in THF
(1.1 mL), and diacylglycerol 8 (0.151 g, 0.437 mmol) and triphenyl-
phosphine (0.115 g, 0.437 mmol) were added and the reaction was
cooled to 0 ꢀC. DEAD (68 mL, 0.437 mmol) was then added and the
reaction mixture was stirred at 0 ꢀC under N2 for 48 h. The mixture
was then concentrated under reduced pressure and purified by
silica gel chromatography eluting with 0–40% EtOAc/hexanes to
yield 15 as a colorless thick oil (0.165 g, 74%, over two steps). 1H
NMR (CDCl3, 400 MHz) d 7.29–7.15 (m, 25H), 5.07–4.88 (m, 5H),
4.76–4.57 (m, 7H), 4.28–3.81 (m, 8H), 3.50 (m, 1H), 2.48 (s, 1H),
2.17–2.10 (m, 4H), 1.48–1.46 (m, 4H), 1.18–1.15 (m, 16H), 0.79–0.77
(m, 6H); 13C NMR (CDCl3, 400 MHz) d 173.0, 173.0, 172.6, 154.3,
154.3, 138.2, 138.2, 138.1, 138.0, 137.9, 135.4, 135.4, 135.4, 135.3,
135.0, 135.0, 128.7, 128.5, 128.3, 128.2, 128.2, 128.2, 128.0, 127.9,
127.8, 127.8, 127.7, 127.7, 127.6, 127.5, 127.5, 79.5, 79.4, 79.4, 78.9,
78.9, 77.9, 77.9, 77.8, 77.8, 77.8, 77.2, 76.8, 75.3, 75.2, 75.1, 74.4, 69.8,
69.7, 69.6, 69.5, 69.5, 69.2, 69.1, 65.7, 65.6, 65.4, 65.4, 61.5, 61.4, 34.0,
34.0, 33.9, 31.6, 29.0, 28.9, 28.8, 24.7, 24.7, 22.5, 14.0; 31P NMR
(CDCl3, 162 Hz) d ꢂ1.7, ꢂ1.8; IR (film, cmꢂ1) 3026, 2951, 2925, 2849,
1738, 1456, 1260, 1158, 1113, 1022; TLC Rf 0.21 (30% ethyl acetate/
hexanes); exact mass calcd for [C61H78O15P]þ requires m/z
1081.5073, found 1081.5035 (ESIþ); [a]D þ11.6 (c 1.0, CHCl3).
4.2.5. ent-PI5P–DiC8
Synthesis and spectral data were identical to PI5P–DiC8. [a]D
ꢂ8.75 (c 2.0, H2O, pH 8).24
Acknowledgements
We thank the National Institutes of General Medical Sciences of
the National Institutes of Health for support (GM-068649).
4.2.3. Protected PI5P–DiC8 (16)
To a stirred solution of 15 (0.100 mg, 0.092 mmol) in CH2Cl2
(18.5 mL) was added dibenzyl diisopropylphosphoramidite (311 mL,
0.92 mmol) followed by 4,5-dicyanoimidazole (0.131 g,1.109 mmol).
The reaction was stirred at room temperature under a nitrogen
atmosphere for 15 h. The reaction was then cooled to 0 ꢀC and 30%
H2O2 (8.6 mL) was added. The reaction was stirred at 0 ꢀC for 1 h at
which time the reaction was quenched with saturated Na2SO3
(w70 mL) until no peroxides were detected via starch paper. The
reaction was then extracted with DCM (3ꢁ50 mL) and the organic
layers were combined, dried (Na2SO4), filtered, and concentrated
under reduced pressure. The resulting residue was then purified by
silica gel column chromatography (0–38% ethyl acetate/hexanes)
and a second column (70% diethyl ether/hexanes) to afford pure 16 as
a thick oil (0.105 g, 85%). 1H NMR (CDCl3, 500 MHz) d 7.315–7.32 (m,
31H), 6.98–6.94 (m, 4H), 4.98 (s, 2H), 4.94–4.61 (m, 14H), 4.45–4.15
(m, 3H), 4.04–3.72 (m, 6H), 2.17–2.09 (m, 4H), 1.51–1.44 (m, 4H),
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