Asymmetric Total Synthesis of Phosphatidylinositol 3-Phosphate and 4-Phosphate Derivatives

Article abstract of DOI:10.1021/jo980501r

New asymmetric syntheses of phosphatidylinositol 3-phosphate (PtdIns(3)P) and phosphatidylinositol 4-phosphate (PtdIns(4)P) derivatives are described. Key intermediates were used to prepare diacylglyceryl moieties with dibutyryl, dioctanoyl, and dihexadecanoyl chains. In addition, a modified route provided PtdIns(3)P and PtdIns(4)P triesters with P-1-linked aminopropyl groups for preparation of affinity probes. The synthesis of the inosityl precursor employed a dibutyltin oxide-mediated p-methoxybenzyl (PMB) etherification to give either the 2-PMB- or the 3-PMB-protected glucopyranosides. The Ferrier rearrangement was used to convert suitably protected glucose derivatives to enantiomerically pure, differentially protected D-myo-inositol key intermediates. A versatile phosphoramidite reagent was employed to allow synthesis of PtdInsP_n derivatives with diacylglyceryl moieties of different chain lengths.

Full text of DOI:10.1021/jo980501r

J . Org. Chem. 1998, 63, 6511-6522
6511
Asym m etr ic Tota l Syn th esis of P h osp h a tid ylin ositol 3-P h osp h a te
a n d 4-P h osp h a te Der iva tives
J ian Chen,† Li Feng,† and Glenn D. Prestwich*
Department of Medicinal Chemistry, The University of Utah, 30 South, 2000 East, Room 201,
Salt Lake City, Utah 84112-5820
Received March 17, 1998
New asymmetric syntheses of phosphatidylinositol 3-phosphate (PtdIns(3)P) and phosphatidyl-
inositol 4-phosphate (PtdIns(4)P) derivatives are described. Key intermediates were used to prepare
diacylglyceryl moieties with dibutyryl, dioctanoyl, and dihexadecanoyl chains. In addition, a
modified route provided PtdIns(3)P and PtdIns(4)P triesters with P-1-linked aminopropyl groups
for preparation of affinity probes. The synthesis of the inosityl precursor employed a dibutyltin
oxide-mediated p-methoxybenzyl (PMB) etherification to give either the 2-PMB- or the 3-PMB-
protected glucopyranosides. The Ferrier rearrangement was used to convert suitably protected
glucose derivatives to enantiomerically pure, differentially protected D-myo-inositol key intermedi-
ates. A versatile phosphoramidite reagent was employed to allow synthesis of PtdInsPn derivatives
with diacylglyceryl moieties of different chain lengths.
In tr od u ction
The phosphoinositide monophosphates PtdIns(3)P and
PtdIns(4)P and their protein targets have been less
extensively examined for their roles in cell signaling.
Studies have revealed that PtdIns(3)P, the product of the
PI 3-K VPS34, is required for proper targeting of newly
synthesized and endocytosed macromolecules in yeast.1
PtdIns(3)P and other D-3 phosphoinositides bind to
proteins that regulate vesicle coating, such as assembly
proteins AP-212 and AP-3 (also known as AP-180),13 and
proteins that mediate communication between the actin
cytoskeleton and membrane signaling proteins.14,15
PtdIns(3)P is also a substrate for a 5-kinase that produces
PtdIns(3,5)P2 in mammalian and yeast cells.16-18
PtdIns(4)P is a substrate for PI 5-kinases, which may
operate on substrate bound to phosphoinositide transport
proteins, for the biosynthesis of PtdIns(4,5)P2.19 Two
specific PtdIns(4)P 5-kinases are known that will also
phosphorylate D-3 phosphoinositides to give bis- and tris-
(phosphate) products.20,21 In addition, PtdIns(4)P shows
affinity for dynamin18 and modest inhibition of a PtdIns-
(3,4,5)P3 5-phosphatase.16
Phosphatidylinositol polyphosphates (PtdInsPns) are
key signaling molecules in cellular communication via
protein kinases in endo- and exocytosis and in vesicular
trafficking of proteins.1,2 The central molecule, L-R-
phosphatidyl-D-myo-inositol 4,5-bis(phosphate) (PtdIns-
(4,5)P2) serves four different cellular roles, each utilizing
different portions of the total molecule. First, PtdIns-
(4,5)P2 is a substrate for phospholipase C (PLC), releasing
the calcium-mobilizing second messenger Ins(1,4,5)P3.3
Second, PtdIns(4,5)P2 itself functions in the recruitment
of proteins to membranes via pleckstrin homology (PH)
domains.4 Third, PtdIns(4,5)P2 may bind to and regulate
cytoskeletal signaling proteins such as gelsolin and
profilin5 or it may regulate the GTPase function of ARF
or the phospholipase activity of PLD.6,7 Fourth, PtdIns-
(4,5)P2 can be converted by agonist-stimulated, receptor-
mediated activation of phosphoinositide 3-kinase (PI
3-K)8 to PtdIns(3,4,5)P3, an effector molecule in a new
intracellular signaling system.9 The PI 3-K pathway and
the corresponding D-3 PtdInsPn products10 have been
linked to mechanisms of oncogene transformation, cy-
toskeletal rearrangements, membrane association of
signaling proteins, and trafficking of proteins by coated
vesicles. Characteristics of the three families of PI 3-Ks
were recently reviewed.11
One constraint for the exploration of the biological roles
of PtdIns(3)P and PtdIns(4)P has been limited access to
synthetic materials with defined acyl chains; commer-
(11) Vanhaesebroeck, B.; Leevers, S.; Panayotou, G.; Waterfield, M.
TIBS 1997, 22, 267-272.
† These authors contributed equally to this research.
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1996, 271, 1533-1539.
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408.
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macol. Sci. 1996, 17, 182-185.
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Gu, Q.-M.; Cook, S.; Nozawa, Y.; Prestwich, G. D.; Frohman, M. A.;
Morris, A. J . J . Biol. Chem. 1997, 272, 3860-3868.
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S0022-3263(98)00501-5 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/01/1998

6512 J . Org. Chem., Vol. 63, No. 19, 1998
Chen et al.
Sch em e 1a
cially available materials from biological sources of
PtdIns have unavoidable heterogeneity in the glyceryl
acyl chains. Indeed, biologists have frequently resorted
to comparisons of binding or activation by symmetrical
dioctanoyl or dipalmitoyl PtdInsPn derivatives with
naturally occurring PtdInsPns, such as the sn-1-O-stearoyl-
2-O-arachidonyl PtdIns(4,5)P2, which is most abundant
in animal systems. To continue our efforts to prepare a
complete “matched set” of diacylglyceryl derivatives of
the PtdInsPns, which all derive their D-myo-inosityl
chirality retrosynthetically from R-D-glucose,22 we de-
scribe herein the preparation of PtdIns(3)P and PtdIns-
(4)P with short (dibutyryl), medium (dioctanoyl), and long
(dipalmitoyl) chains. We also describe the preparation
of the corresponding P-1-linked aminopropyl triesters23,24
and their use for the preparation of photoactivatable
reagents that probe the water-bilayer interface for
ligand-protein interactions.22
a
Reaction conditions: (a) Bu2SnO, n-Bu4NBr, or n-Bu4NI,
toluene, reflux for 2 h; then PMBCl, reflux 2 h to overnight.
Sch em e 2a
Resu lts a n d Discu ssion
The strategy for the synthesis of enantiomerically pure
PtdIns(3)P and PtdIns(4)P derivatives followed the route
developed to synthesize soluble inositol polyphosphates
(InsPn) and PtdInsPn congeners22-28 via the Ferrier
rearrangement.22,29 For these syntheses, methyl D-R-
glucopyranoside was chosen as the optically pure starting
material for the inositol moiety, and protecting groups
are installed to establish the desired regiochemistry for
the phosphate and hydroxyl groups. The key step
involves the unique protection of the glucosyl C-2 OH or
C-3 OH (corresponding to the C-3 OH or C-4 OH of
D-myo-inositol) relative to the other hydroxyl groups;
p-methoxybenzyl (PMB) was chosen for this purpose
because it could be easily removed by DDQ without
affecting other protecting groups.22 This protection
strategy was accomplished using dibutyltin oxide-
mediated30-34 selective PMB protection of the 2-OH of
methyl 4,6-O-(p-methoxybenzylidene)-D-R-glucopyrano-
side (1a ), giving the 2-PMB ether 2a a in ca. 45% isolated
yield (Scheme 1). Alternatively, the 3-PMB protected
glycopyranoside 2a b was obtained in 55% yield. The two
isomers were readily separable by chromatography on
silica gel. Use of either tetra-n-butylammonium bromide
or iodide was necessary for optimal etherification and for
a
Reaction conditions: (a) NaH, BnBr, DMF, rt, 2h.
shorter reaction time. Although better regioselectivity
has been reported using CH3CN instead of toluene as
solvent and by omission of the tetra-n-butylammonium
salt,28-31 lower yields and unacceptably long reaction
times were experienced for a modest increase in regiose-
lectivity. The 3-PMB ether 2a b was useful as the
precursor for synthesis of Ins(1,4,5)P327 and PtdIns(4,5)-
P2.23,25 Using the conditions described above, no 2,3-di-
PMB ether was detected. Similar yields and regiospec-
ificity were achieved from the commercially available
methyl 4,6-O-benzylidene-D-R-glucopyranoside (1b).
To confirm the structures of the two isomers, a short
chemical transformation was performed. Isomers 2a a
and 2a b were converted to benzyl ethers (Scheme 2).
(20) Rameh, L.; Tolias, K.; Duckworth, B.; Cantley, L. Nature 1997,
390, 192-196.
(21) Zhang, X. L.; Loijens, J . C.; Boronenkov, I. V.; Parker, G. J .;
Norris, F. A.; Chen, J .; Thum, O.; Prestwich, G. D.; Majerus, P. W.;
Anderson, R. A. J . Biol. Chem. 1997, 272, 17756-17761.
(22) Prestwich, G. D. Acc. Chem. Res. 1996, 29, 503-513.
(23) Gu, Q.-M.; Prestwich, G. D. J . Org. Chem. 1996, 61, 8642-
8647.
1
Comparison of the H NMR and melting points with the
known compounds27 allowed identification of the less
polar starting material as the 2-PMB ether 2a a and the
more polar isomer as the 3-PMB ether 2a b.
(24) Thum, O.; Chen, J .; Prestwich, G. D. Tetrahedron Lett. 1996,
37, 9017-9020.
As shown in Scheme 3, hydrolytic deprotection of the
benzylidene acetal in 2a a and 2bb under mild acidic
conditions unmasked the 4- and 6-hydroxyls (3a ,b ) in
high yield without affecting the PMB group. The primary
hydroxyl was selectively tritylated,28,35-37 the remaining
secondary hydroxyls were benzylated, and mild acidic
hydrolysis of the trityl group afforded alcohols 6a ,b.
Swern oxidation38 gave aldehydes 7a ,b, which were
(25) Chen, J .; Profit, A. A.; Prestwich, G. D. J . Org. Chem. 1996,
61, 6305-6312.
(26) Chen, J .; Dorma´n, G.; Prestwich, G. D. J . Org. Chem. 1996,
61, 393-397.
(27) Dorma´n, G.; Chen, J .; Prestwich, G. D. Tetrahedron Lett. 1995,
36, 8719-8722.
(28) Estevez, V. A.; Prestwich, G. D. J . Am. Chem. Soc. 1991, 113,
9885-9887.
(29) Ferrier, R.; Middleton, S. Chem. Rev. 1993, 93, 2779-2831.
(30) Qin, H.; Grindley, T. B. J . Carbohydr. Chem. 1996, 15, 95-
108.
(31) Riley, A. M.; J enkins, D. J .; Potter, B. V. L. J . Am. Chem. Soc.
1995, 117, 3300-3301.
(35) Chaudhary, S. K.; Hernandez, O. Tetrahedron Lett. 1979, 95.
(36) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis; J ohn Wiley & Sons: New York, 1991.
(37) Kocie´nski, P. J . Protecting Groups; Thieme: Stuttgart, Ger-
many, 1994.
(32) J enkins, D. J .; Potter, B. V. L. Carbohydr. Res. 1994, 265, 145.
(33) J enkins, D. J .; Dubreuil, D.; Potter, B. V. L. J . Chem. Soc.,
Perkin Trans. 1 1996, 1365-1372.
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(38) Mancuso, A. J .; Swern, P. Synthesis 1981, 165-185.

PtdIns(3)P and PtdIns(4)P Synthesis
J . Org. Chem., Vol. 63, No. 19, 1998 6513
Sch em e 3a
a
Reaction conditions: (a) 5% p-TsOH monohydrate (pH ∼ 2), MeOH, rt, 1 h; (b) TrCl, DMAP (cat.), Et3N, DMF, rt, overnight; (c) NaH,
BnBr, DMF, rt, 2 h; (d) 5% p-TsOH monohydrate or sulfuric acid (pH ∼ 2), MeOH, rt, 2 h; (e) oxalyl chloride, DMSO, CH2Cl2, -78 °C,
then Et3N, 1 h; (f) K2CO3, Ac2O, CH3CN, reflux, overnight; (g) Hg(OAc)2, acetone-water (3:2), 45 min, then saturated NaCl solution, rt,
overnight; (h) NaBH(OAc)3, CH3CN, HOAc, 45 min; (i) BOMCl, proton sponge, n-Bu4NBr, rt, 8 h, then, 35 °C, overnight, then 55 °C, 10
h to overnight; (j) 0.35 M NaOH, MeOH, reflux, 2 h; (k) DDQ, CH2Cl2-H2O (100:1, v/v), rt, 3 h; (l) (BnO)2PNPr2-i, 1H-tetrazole, rt, 30
min, then m-CPBA, -40 °C to room temperature, 60 min; (m) K2CO3, MeOH, 30 min.
Sch em e 4a
directly acetylated to form the precursors 8a ,b, respec-
tively. Ferrier rearrangement22,29 with Hg(OAc)2 gener-
ated the inositol skeletons 9a ,b. Highly stereoselective
reduction of the carbonyl group with triacetoxyborohy-
dride39 furnished the desired hydroxyl derivatives 10a ,b.
Careful reaction22 of the diols 10a and 10b with BOMCl
in the presence of proton sponge gave the bis-BOM
compounds 11a ,b with no detectable acyl migration.
DDQ removed the 4-PMB in 11b to give a free OH, which
was phosphorylated to triester 13b; hydrolysis of the
acetyl group then gave alcohol 14b, which was ready for
introduction of the phosphoglyceryl moiety.
a
Reaction conditions: (a) DDQ, CH2Cl2-H2O (100:1, v/v), rt, 2
h; (b) (BnO)2PNPr2-i, 1H-tetrazole, rt, 30 min, then m-CPBA, -40
°C to room temperature, 60 min; (c) 10% Pd/C, NaHCO3, t-BuOH-
H2O (6:1, v/v), H2, 50 psi, rt, 5 h; then Chelex (Na+ form) ion
exchange.
We required the soluble Ins(1,3)P2 for competitive
displacement studies in the study of protein-PtdInsPn
interactions. This ligand was obtained (Scheme 4) by
basic hydrolysis of the acetyl group of 11a , removal of
the PMB with DDQ, phosphorylation, and finally hydro-
genolysis at 50 psi with Pd/C to give the meso-myo-
inositol 1,3-bis(phosphate) 15, in sodium salt form.40
The differentially protected D-myo-inositol precursors
to PtdIns(3)P and PtdIns(4)P, 12a and 14b, respectively,
were employed for two sets of syntheses. In the first set
(Scheme 5), the dipalmitoyl glyceryl affinity probes
bearing the P-1-linked aminopropyl phosphotriester22-24
were prepared. Thus, coupling 12a or 14b with (cyano-
ethyl)phosphoramidite 16 gave 17a ,b, respectively. Re-
moval of PMB (from 17b) with DDQ gave phosphate 18
in 60% yield in two steps. If ceric ammonium nitrate41,42
was used for oxidative deprotection of the PMB group, a
slightly higher yield was obtained. Further phosphory-
lation furnished the fully protected phosphate 19 in 80%
yield. The cyanoethyl group could be reduced to an
aminopropyl group during hydrogenolysis of the benzyl
protecting groups.23 Thus, hydrogenation at 50 psi
removed all the protecting groups of 17b and 19 giving
PtdIns(3)P and PtdIns(4)P triesters 20a ,b, respectively,
as their sodium salts. Alternatively, initial removal of
(39) Evans, D. A.; Chopman, K. T.; Carreira, E. M. J . Am. Chem.
Soc. 1988, 110, 3560-3578.
(40) Billington, D. C.; Baker, R. J . Chem. Soc., Chem. Commun.
1987, 1011-1013.
(41) Wang, Y.; Babirad, S. A.; Kishi, Y. J . Org. Chem. 1992, 57, 468.
(42) J ohansson, R.; Samulsson, B. J . Chem. Soc., Perkin Trans. 1
1984, 2371-2374.

6514 J . Org. Chem., Vol. 63, No. 19, 1998
Chen et al.
Sch em e 5a
a
Reaction conditions: (a) 1H-tetrazole, rt, 30 min, then mCPBA, -40 °C to room temperature, 60 min; (b) DDQ, CH2Cl2-H2O (100:1,
v/v), rt, 2 h; (c) (BnO)2PNPr2-i, 1H-tetrazole, rt, 30 min, then m-CPBA, -40 °C to room temperature, 60 min; (d) 10% Pd/C, NaHCO3,
t-BuOH-H2O (6;1, v/v), H2, 50 psi, rt, 5 h; (e) i-Pr2NEt, MeOH, rt, overnight, then 10% Pd/C, NaHCO3, t-BuOH-H2O (6:1, v/v), H2, 50
psi, rt, 5 h.
glycerylphosphoramidite 2345 was prepared from reaction
of benzyl N,N,N,N-tetraisopropylphosphoramidite with
(S)-(+)-1,2-O-isopropylidene-sn-glycerol and showed chemi-
cal properties similar to those reported for similar
phosphoramidites.46 Reaction of inositol-protected 3-PMB
intermediate 12 with the protected glycerylphosphora-
midite 23 gave phosphate 24 in good yield. Removal of
PMB (DDQ) and further phosphorylation gave bis-
(phosphotriester) 26 in 60% yield. Hydrolysis of the
acetal gave diol 27,47 which could be readily acylated to
give a fully protected PtdIns(3)P precursor. As an
example, we coupled 27 with octanoic acid and butyric
acid in the presence of DCC and DMAP48,49 to give the
protected products 28a ,b, respectively, in high yields.
Hydrogenation gave PtdIns(3)P derivatives 29a ,b in
63% and 92% yields, respectively.
Sch em e 6a
Reaction conditions: (a) BZDC-NHS or [3H]-BZDC-NHS,
Et3N, DMF, rt, overnight; then DEAE chromatography.
a
the cyanoethyl group of 19 with i-Pr2NEt23 followed by
hydrogenation gave the desired PtdIns(3)P diester 21 in
77% yield.43
The 1H NMR of 21, the dipalmitoyl derivative of
PtdIns(3)P, illustrates the curious solubility of the Pt-
dInsP materials and the intrinsic difficulty in obtaining
The aminopropyl triesters were used in the preparation
of photoactivatable probes for the proteins recognizing
the inositol headgroup in the context of a lipid bilayer.22
Thus, reaction of the triester 20a with unlabeled p-
benzoyldihydrocinnamoyl N-hydroxysuccinimide ester
(BZDC-NHS) in DMF using Et3N as base gave BZDC-
triester-PtdIns(3)P (22a ) in ca. 50% yield.44 When high
specific activity [3H]BZDC-NHS was employed in a
mixed 0.25 M TEAB buffer-DMF solvent system, the
tritium-labeled 22a ′ was obtained in 8% radiochemical
yield (Scheme 6).
The long-chain diacyl derivatives of the phosphatidyli-
nositol mono- and bis(polyphosphate)s are insoluble in
water and form micelles and vesicles. However, the
dioctanoyl analogues of PtdInsP2 and PtdInsP3 regioiso-
mers are water soluble. It was unknown if the octanoyl
derivative of PtdInsP regioisomers would be water soluble.
To explore a variety of potential symmetrical diacylglyc-
eryl derivatives from a common PtdInsP precursor, we
prepared a versatile phosphoramidite (Scheme 7). Thus,
1
high-quality NMR spectroscopic data. In CDCl3, the H
NMR resonances of the acylglyceryl moiety are reason-
ably well resolved, while only broad, poorly resolved
resonances can be detected for the inositol phosphate
headgroup. In contrast, in D2O, the inositol phosphate
headgroup becomes better-resolved, while the resonances
for the fatty acyl chains and the glyceryl moiety become
severely broadened. Furthermore, 31P NMR in D2O
showed one very broad resonance of poor diagnostic
utility. Use of other solvents, e.g., DMSO-d6, makes
recovery of precious materials for biological studies not
feasible. Mixed solvents containing D2O, CD3OD, and
CDCl3 did not improve the situation. Changing cations
to ammonium or to the acidic form also simply made
spectra worse. In contrast, the dibutyryl and dioctanoyl
(45) Lemmen, P.; Buchweitz, K. M.; Stumpf, R. Chem. Phys. Lipids
1990, 53, 65-75.
(46) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 10441-10488.
(47) Heeb, N. V.; Nambiar, K. P. Tetrahedron Lett. 1993, 34, 6193-
6196.
(43) Bruzik, K. S.; Kubiak, R. J . Tetrahedron Lett. 1995, 36, 2415-
2418.
(44) Olszewski, J . D.; Dorma´n, G.; Elliott, J . T.; Hong, Y.; Ahern,
D. G.; Prestwich, G. D. Bioconjugate Chem. 1995, 6, 395-400.
(48) Martin, S. F.; Wong, Y.-L.; Wagman, A. J . Org. Chem. 1994,
59, 4821-4831.
(49) Vodovozova, E. L.; Molotkovsky, J . G. Tetrahedron Lett. 1994,
35, 1933-1936.

PtdIns(3)P and PtdIns(4)P Synthesis
J . Org. Chem., Vol. 63, No. 19, 1998 6515
Sch em e 7a
a
Reaction conditions: (a) 1H-tetrazole, rt, 30 min, then mCPBA, -40 °C to room temperature, 60 min; (b) DDQ, CH2Cl2-H2O (100:1,
v/v), rt, 2 h; (c) (BnO)2PNPr2-i, 1H-tetrazole, rt, 30 min, then m-CPBA, -40 °C to room temperature, 60 min; (d) 5% p-TsOH monohydrate
(pH ∼ 2), MeOH, rt, 2 h; (e) fatty acid, DMAP (cat.), DCC, CH2Cl2, rt, overnight; (f) 10% Pd/C, NaHCO3, t-BuOH-H2O (6:1, v/v), H2, 50
psi, rt, 5 h; then Chelex (Na+ form) ion exchange.
Sch em e 8a
Sch em e 9a
a
Reaction conditions: (a) Fatty acid, DMAP (cat.), DCC,
a
Reaction conditions: (a) 1H-tetrazole, rt, 30 min, then m-
CH2Cl2, rt, overnight; (b) DDQ, CH2Cl2-H2O (100:1, v/v), rt, 2 h;
CPBA, -40 °C to room temperature, 60 min; (b) 10% Pd/C,
NaHCO3, t-BuOH-H2O (6:1, v/v), H2, 50 psi, rt, 5 h; then Chelex
(Na+ form) ion exchange.
(c) (BnO)P(Cl)(NPr2-i), i-Pr2NEt, CH2Cl2, 0 °C, 2 h.
derivatives were water soluble and gave high-resolution
1H and 31P NMR spectra samples with a concentration
of 10 mg/mL. The narrow 31P lines (0.028-0.075 ppm
at half-height) for both dibutyryl and dioctanoyl PtdIns-
(3)P and PtdIns(4)P suggest monomeric and not micellar
forms at this concentration.
soluble Ins(1,3)P2 headgroup was synthesized from ra-
cemic myo-inositol derivatives.40,52 The synthesis re-
ported herein now allows preparation of diester reagents
or triester affinity probes22 for further examination of the
roles of these phosphoinositide monophosphates in cell
signaling. Applications of the [3H]BZDC-PtdIns(3)P and
-PtdIns(4)P photoaffinity labels for the identification of
new target proteins will be presented in due course.
Finally, with these syntheses and the synthesis of both
PtdIns(5)P and PtdIns(3,5)P2,53 biological researchers
may now select from a complete “matched set” of dibutryl,
dioctanoyl, or dipalmitoyl PtdInsPn derivatives with
either complete water solubility or complete bilayer
localization to determine the preferred number and
regiochemistry of phosphates required for optimal bio-
logical activity.
In the preparation of the series of PtdIns(4)P diesters,
an alternative strategy of using different phosphoramid-
ites with preattached long, medium, and short diacyl
chains was followed (Scheme 8). Following reported
procedures,23-25 the 3-O-PMB-sn-glycerol 30 was esteri-
fied with various fatty acids using DCC/DMAP to give
compounds 31a -c in high yields. Oxidative removal of
the PMB groups with DDQ gave the corresponding
alcohols 32a -c in good yields with no evidence of acyl
migration. Finally, coupling these alcohols with benzyl
N,N-isopropylchlorophosphoamidite gave reagents 33a -c
in high yields. Subsequent coupling of 4-phosphorylated
inosityl precursor 14b with 33a , 33b, or 33c followed by
mild oxidation gave the three protected PtdIns(4)P
precursors 34a -c in moderate to good yields (Scheme 9).
Hydrogenolysis provided PtdIns(4)P derivatives with
dipalmitoyl (35a ), dioctanoyl (35b), and dibutyryl (35c)
acyl chains. Both diester and triester with dipalmitoyl
glyceryl groups showed poor solubility in water and good
solubility in methanol-chloroform-water mixtures, while
the dibutyryl and dioctanoyl derivatives were water
soluble.
Exp er im en ta l Section
NMR spectra (1H, 13C, and 31P) were recorded in CDCl3 or
D2O on QE-300, AC-250, or AC-200 NMR spectrometers, and
shifts are reported relative to δ(TMS) ) 0 ppm. When
necessary, solvents and reagents were dried using standard
procedures. Elemental analysis was performed by M-H-W
Laboratories (Phoenix, AZ), and the mass spectra (low solution
and high resolution) were measured at The University of
California at Riverside (Riverside, CA) or at The University
of Utah (Salt Lake City, UT) using chemical ionization (CI,
+
with NH3 giving MNH4 peaks), EI, or FAB (BNA as matrix
Recently, a variety of synthetic approaches to PtdIn-
sPns have been summarized.22,50 Several recent ap-
proaches to PtdIns(3)P include preparation from D-1-O-
tert-butyldiphenylsilyl-myo-inositol43 or from enzymatically
resolved 1,2:5,6-di-O-cyclohexylidene-myo-inositol.51 The
giving MNa+ peaks). Chemicals from Aldrich Chemical Co.
(Milwaukee, WI) and Sigma Chemical Co. (St. Louis, MO) were
used without further purification. TLC (SiO2) plates for
inositol precursors were developed in ethyl acetate (EtOAc)
(52) Maryanoff, B. E.; Reitz, A. B.; Tutwiler, G. F.; Benkovic, S. J .;
Benkovic, P. A.; Pilkis, S. J . J . Am. Chem. Soc. 1984, 106, 7851.
(53) Peng, J .; Prestwich, G. D. Tetrahedron Lett. 1998, 39, 3965-
3968.
(50) Potter, B. V. L.; Lampe, D. Angew. Chem., Int. Ed. Engl. 1995,
34, 1933-1972.
(51) Wang, D.-S.; Chen, C.-S. J . Org. Chem. 1996, 61, 5905-5910.

6516 J . Org. Chem., Vol. 63, No. 19, 1998
Chen et al.
and hexane mixtures and visualized by dipping in 5% phos-
phomolybdic acid in absolute ethanol followed by heating; for
acylglycerol derivatives, compounds were visualized by dipping
in anisaldehyde stains (ratio p-anisaldehyde:acetic acid:sul-
furic acid:ethanol 2.5:1:3.5:93, v/v/v/v) followed by heating.
Meth yl 2-O-(p-Meth oxyben zyl)-4,6-O-(p-m eth oxyben -
zylid en e)-r-D-glu cop yr a n osid e (2a a ). A mixture of methyl
4,6-O-(p-methoxybenzylidene)-R-D-glucopyranoside (1a , 5.21 g,
16.7 mmol), Bu2SnO (4.2 g, 16.9 mmol), and Bu4NI (0.6 g, 1.63
mmol) in 100 mL of toluene was refluxed for 2 h, then
p-methoxybenzyl chloride (PMBCl, 2.88 g, 18.3 mmol) was
added, and the mixture was stirred under reflux for 2 h, at
which point TLC showed no starting material. The mixture
was cooled, diluted with EtOAc, washed (H2O, brine), dried
(MgSO4), and concentrated in vacuo to a brown oil. Chroma-
tography on SiO2 (50% EtOAc-hexane) gave two products at
Rf ∼ 0.7. The first eluting product was the 2-PMB ether (2a a ,
3.30 g, yield 45%), and the second product was 3-PMB ether
(2a b, 3.8 g, 55% yield); the latter was less soluble in EtOAc-
hexane. No bis-PMB product was detected.
Met h yl 2-O-(p -Met h oxyb en zyl)-r-D-glu cop yr a n osid e
(3a ). A mixture of methyl 2-O-(p-methoxybenzyl)-4,6-O-(p-
methoxybenzylidene)-R-D-glucopyranoside (2aa, 4 g, 9.25 mmol)
and p-TsOH monohydrate (400 mg) in 100 mL of MeOH was
stirred for 1 h. TLC (Rf ∼ 0.5), ether) showed no starting
material. Then, 10 mL of 10% NaHCO3 solution was added
and the mixture was evaporated to dryness. The product was
extracted five times from the solid material with EtOAc and
then recrystallized from EtOAc-hexane to give 2.85 g of
pure methyl 2-O-(p-methoxybenzyl)-R-D-glucopyranoside 3a
(98%): 1H NMR (300 MHz, CDCl3) δ 7.22, 7.19, 6.82, 6.79 (4s,
4H, aromatic), 4.56 (AB type, J AB ) 11.4 Hz, 2H, CH2O), 4.48
(s, 1H), 3.85-3.70 (m, 2H), 3.72 (s, 3H, OMe), 3.61 (s, 1H),
3.49 (s, 2H), 3.37 (s, 2H), 3.25 (s, 3H, OMe), 2.52 (s, 1H), 2.01
(s, 1H); 13C NMR (75 MHz, CDCl3) δ 129.9, 129.7, 113.9, 97.9,
79.0, 77.2, 72.8, 72.7, 55.2 ppm. Anal. Calcd for C15H22O7:
C, 57.31; H, 7.06. Found: C, 57.47; H, 6.79.
Met h yl 3-O-(p -Met h oxyb en zyl)-r-D-glu cop yr a n osid e
(3b ). A mixture of methyl 3-O-(p-methoxybenzyl)-4,6-O-
benzylidene-R-D-glucopyranoside (2bb, 11.0 g, 27.3 mmol) and
p-TsOH monohydrate (930 mg) in 250 mL of MeOH was stirred
for 2 h. Then, 2 g of NaHCO3 was added, the NaHCO3 solid
was removed by filtration, and the filtrate was evaporated to
dryness. The crude product was extracted from the residue
with five portions of EtOAc, concentrated, and then purified
on SiO2 (75% EtOAc-hexane) to give 7.5 g of pure methyl 3-O-
Data for compound 2a a : mp 108-110 °C; 1H NMR (300
MHz, CDCl3) δ 7.36, 7.34, 7.26, 7.23 (4s, 4H, aromatic), 6.84-
6.81 (m, 4H, aromatic), 5.41 (s, 1H, CH), 4.61 (q, J ) 11.7 Hz,
2H), 4.50 (d, J ) 3.6 Hz, 1H), 4.15 (dd, J ) 4.5, 4.2 Hz, 1H),
4.05 (dt, J ) 9.2, 1.8 Hz, 1H), 3.74, 3.37 (2s, 6H, OMe), 3.43-
3.35 (m, 2H), 3.31 (s, 3H, OMe) ppm; 13C NMR (75 MHz,
CDCl3) δ 129.9, 129.8, 129.6, 127.6, 113.9, 113.6, 101.8, 98.7,
81.2, 79.1, 77.2, 73.0, 70.2, 68.9, 62.0, 55.4, 55.3 ppm. Anal.
Calcd for C23H28O8: C, 63.88; H, 6.53. Found: C, 63.64; H,
6.77.
Meth yl 3-O-(p-m eth oxyben zyl)-4,6-O-(p-m eth oxyben -
zylid en e)-r-D-glu cop yr a n osid e (2a b) was isolated from the
chromatography above: mp 163-165 °C; 1H NMR (300 MHz,
CDCl3) δ 7.40-7.20 (4s, 4H, aromatic), 6.84-6.81 (m, 4H,
aromatic), 5.51 (s, 1H, CH), 4.81 (q, J ) 11.7 Hz, 2H), 4.78 (d,
J ) 3.6 Hz, 1H), 4.30 (q, J ) 3.5 Hz, 1H), 3.74, 3.37 (2s, 6H,
OMe), 3.43-3.35 (m, 2H), 3.31 (s, 3H, OMe) ppm; 13C NMR
(75 MHz, CDCl3) δ 129.7, 129.0, 128.3, 126.0, 113.8, 101.3,
98.7, 82.0, 78.4, 77.2, 73.0, 70.2, 69.0, 62.6, 55.4, 55.3 ppm.
Anal. Calcd for C23H28O8: C, 63.88; H, 6.53. Found: C, 63.53;
H, 6.45.
Meth yl 2-O-(p-Meth oxyben zyl)-4,6-O-ben zylid en e-r-D-
glu cop yr a n osid e (2ba ). A mixture of methyl 4,6-O-ben-
zylidene-R-D-glucopyranoside (1b, 25 g, 88.6 mmol), and
Bu2SnO (22 g, 88.6 mmol), Bu4NBr (2.86 g, 8.86 mmol) in 300
mL of toluene was refluxed for 3 h, then PMBCl (16.7 g, 106.3
mmol) was added, and the mixture was stirred under reflux
overnight. After workup as above and SiO2 chromatography
with 50% EtOAc-hexane, two products were isolated. The
first eluting product (Rf ∼ 0.7) was identified as methyl 2-O-
(p-methoxybenzyl)-4,6-O-benzylidene-R-D-glucopyranoside (2-
PMB ether 2ba ) (15 g, yield 42%). The second eluting
compound was methyl 3-O-(p-methoxybenzyl)-4,6-O-benzylidene-
R-D-glucopyranoside (3-PMB ether 2bb) (20 g, 56% yield),
which was less soluble in EtOAc-hexane mixtures and easier
to recrystallize.
(p-methoxybenzyl)-R-D-glucopyranoside 3b (83%) with Rf
∼
0.21 (20% EtOAc-hexane): 1H NMR (200 MHz, CDCl3) δ 7.31,
7.28, 6.89, 6.85 (4s, 4H, PMB), 4.78 (J AB ) 11.2 Hz, 2H, CH2O),
4.72 (s, 1H), 3.78 (m, 5H), 3.57-3.54 (m, 4H), 3.40 (s, 3H,
OMe), 3.02 (s, 1H), 2.45 (s, 1H); 13C NMR (50 MHz, CDCl3) δ
159.1, 129.5, 113.8, 106.5, 104.5, 99.4, 82.0, 74.4, 72.4, 70.9,
69.7, 61.7, 55.1 ppm. MS(EI+): m/e 314 (M+), 150, 137, 121.
HRMS (EI+): calcd for C15H22O7 (M+), m/e 314.1366; found,
m/e 314.1349.
Meth yl 2-O-(p-Meth oxyben zyl)-6-O-tr ityl-r-D-glu cop y-
r a n osid e (4a ). A mixture of methyl 2-O-(p-methoxybenzyl)-
R-D-glucopyranoside (3a , 2.85 g, 9.10 mmol), trityl chloride
(2.79 g, 1.0 mmol), DMAP (40 mg), and triethylamine (3 mL)
in 100 mL of dry DMF was stirred overnight. TLC (Rf ∼ 0.3,
50% EtOAc-CH2Cl2) showed essentially complete conversion.
The mixture was then poured into water, extracted with
EtOAc, concentrated, and purified (SiO2) to give 4.79 g (94%)
of methyl 2-O-(p-methoxybenzyl)-6-O-trityl-R-D-glucopyrano-
side (4a ): 1H NMR (300 MHz, CDCl3) δ 7.45-7.10 (m, 17H,
aromatic), 6.79 (d, J ) 9.6 Hz, 2H, PMB), 4.61-4.51 (m, 3H),
3.80 (t, J ) 0.6 Hz, 1H), 3.73 (s, 3H, OMe), 3.64-3.59 (m, 2H),
3.43-3.20 (m, 3H), 3.31 (s, 3H, OMe), 3.00 (s, br, 1H, OH),
1.93 (s, 1H, OH) ppm; 13C NMR (75 MHz, CDCl3) δ: 143.8,
130.1, 129.6, 128.6, 128.0, 127.8, 127.0, 113.9, 97.6, 86.8, 78.8,
77.3, 73.1, 72.6, 71.7, 69.7, 64.0, 55.2, 55.1 ppm. MS (EI+):
m/e 556 (M+), 313, 243, 165, 121, 91. HRMS (EI+): calcd for
C
34H36O7 (M+), m/e 556.2461; found, m/e 556.2467.
Meth yl 3-O-(p-Meth oxyben zyl)-6-O-tr ityl-r-D-glu cop y-
r a n osid e (4b). A mixture of methyl 3-O-(p-methoxybenzyl)-
R-D-glucopyranoside (3b, 7.1 g, 22.4 mmol), trityl chloride (6.3
g, 22.5 mmol), DMAP (317 mg), and triethylamine (4.77 mL)
in 150 mL of dry DMF was stirred overnight. TLC showed
essentially complete conversion. The mixture was then poured
into water, extracted with EtOAc, washed (brine, water), dried
(MgSO4), and purified (SiO2) to give 8.2 g (65%) of methyl 3-O-
(p-methoxybenzyl)-6-O-trityl-R-D-glucopyranoside (4b): 1H NMR
(200 MHz, CDCl3) δ 7.52-7.21 (m, 17H, Ph + PMB), 6.88 (d,
2H, J ) 8.0 Hz, PMB), 4.91-4.72 (m, 3H), 3.79 (s, 3H, OMe),
3.76-3.57 (m, 4H), 3.47 (s, 3H, OMe), 3.41-3.37 (m, 2H), 2.46
(s, 1H, OH), 2.33 (d, 1H, J ) 6.0 Hz, OH); 13C NMR (50 MHz,
CDCl3) δ 159.5, 143.6, 130.5, 129.5, 128.5, 127.7, 126.9, 113.8,
105.5, 99.1, 86.7, 82.2, 74.5, 72.3, 71.3, 70.0 63.7, 55.1, 54.9
ppm. MS (EI+): m/e 555 (M - H+), 313, 243, 165, 137, 121.
HRMS (EI+): calcd for C34H36O7 (M+), m/e 556.2461; found,
m/e 556.2426.
Data for compound 2ba : 1H NMR (300 MHz, CDCl3) δ 7.60-
7.20 (m, 7H, aromatic), 6.90, 6.86 (2s, 2H, PMB), 5.50 (s, 1H,
CH), 4.63 (q, J ) 11.8 Hz, 2H), 4.55 (d, J ) 3.6 Hz, 1H), 4.25
(dd, J ) 4.5, 4.2 Hz, 1H), 4.10 (t, J ) 9.2 Hz, 1H), 3.76 (s, 3H,
OMe), 3.84-3.75 (m, 1H), 3.70 (q, J ) 10.1 Hz, 1H), 3.43-
3.35 (m, 2H), 3.31 (s, 3H, OMe) ppm; 13C NMR (75 MHz,
CDCl3) δ 130.0, 129.8, 129.2, 128.3, 126.4, 114.0, 101.9, 98.7,
81.3, 79.2, 73.0, 70.2, 69.0, 62.1, 55.4, 55.3 ppm. Anal. Calcd
for C22H26O7: C, 65.66; H, 6.51. Found: C, 65.40; H, 6.55.
Da t a for m et h yl 3-O-(p -m et h oxyb en zyl)-4,6-O-b en -
zylid en e-r-D-glu cop yr a n osid e (2bb): 1H NMR (300 MHz,
CDCl3) δ 7.60-7.30 (m, 7H, aromatic), 6.86, 6.82 (2s, 2H,
PMB), 5.56 (s, 1H, CH), 4.81 (q, J ) 11.7 Hz, 2H), 4.78 (d, J
) 3.6 Hz, 1H), 4.30 (q, J ) 3.5 Hz, 1H), 3.78 (s, 3H, OMe),
3.90-3.60 (m, 2H), 3.31 (s, 3H, OMe), 1.60 (br s, 1H, OH) ppm;
13C NMR (75 MHz, CDCl3) δ 129.7, 129.0, 128.3, 126.0, 113.8,
101.3, 98.7, 82.0, 78.4, 77.2, 73.0, 70.2, 69.0, 62.6, 55.4, 55.3
ppm. Anal. Calcd for C22H26O7: C, 65.66; H, 6.51. Found:
C, 65.46; H, 6.52.
Meth yl 3-O-(p-Meth oxyben zyl)-2,4-O-d iben zyl-6-O-tr i-
tyl-r-D-glu cop yr a n osid e (5b). A mixture of methyl 3-O-(p-
methoxybenzyl)-6-O-trityl-R-D-glucopyranoside (4b, 8.0 g, 14.3
mmol) and NaH (1.5 g, ∼60% oil, 37.5 mmol), and benzyl

PtdIns(3)P and PtdIns(4)P Synthesis
J . Org. Chem., Vol. 63, No. 19, 1998 6517
bromide (5.76 g, 33.6 mmol) in 150 mL of dry DMF was stirred
for 2 h at room temperature. The reaction was quenched with
methanol, poured into 150 mL of water, extracted (3 volumes
of CH2Cl2), and purified on SiO2 (33% EtOAc-hexane) to give
10 g of pure 5b (95%), Rf ∼ 0.49 (20% EtOAc-hexane): 1H
NMR (200 MHz, CDCl3) δ 7.51-7.21 (m, 27H, Ph + PMB),
6.87 (d, J ) 8.8 Hz, 2H, PMB), 4.93-4.71 (m, 6H), 4.31 (d, J
) 10.6 Hz, 1H), 3.98 (t, J ) 9.2 Hz, 1H), 3.86-3.81 (m, 4H),
3.69-3.59 (m, 2H), 3.55-3.49 (m, 1H), 3.47 (s, 3H, OMe), 3.19
(dd, J ) 4.6, 10.0 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ: 159.0,
143.7, 138.2, 137.5, 130.7, 129.6, 128.6-127.3 (m), 126.7, 113.6,
108.5, 97.7, 86.1, 81.6, 80.0, 77.9, 75.5, 74.8, 73.2, 70.0, 62.4,
55.1, 54.8 ppm. MS (EI+): m/e 735 (M - H+), 645, 615, 493,
461, 243, 121, 91. HRMS (EI+): calcd for C48H48O7, m/e
736.3400; found, m/e 736.3364.
third, and the residue was diluted with water and extracted
with ether. The usual workup gave 2.2 g of enol acetate 8a
(85% in two steps), Rf ∼ 0.6, 50% EtOAc-hexane: 1H NMR
(250 MHz, CDCl3) δ 7.4-7.2 (m, 12H), 6.80, 6.76 (2s, 2H,
PMB), 4.90-4.60 (m, 8H), 4.00-3.90 (m, 2H), 3.80 (s, 3H,
OMe), 3.60-3.50 (m, 1H), 3.35 (s, 3H), 2.20 (s, 3H) ppm; 13C
NMR (63 MHz, CDCl3) δ 167.3, 159.2, 153.3, 138.0, 135.1,
130.8, 129.8, 128.5, 128.1, 123.0, 113.9, 113.8, 99.8, 81.1, 79.1,
75.4, 74.2, 73.7, 60.4, 56.2, 55.3 ppm. MS (CI+, NH3): m/e 552
(MNH4+), 432, 383, 325, 211, 137, 121, 108. HRMS (CI+):
calcd for
552.2597.
C
31H38NO8 (MNH4+), m/e 552.2597; found, m/e
Meth yl (Z)-2,4-O-Diben zyl-3-O-(p-m eth oxyben zyl)-6-O-
a cetyl-r-D-glu cos-5-en op yr a n osid e (8b). A solution of
(COCl)2 (1.29 mL, 14.78 mmol) in 50 mL of CH2Cl2 was cooled
to -78 °C, dry DMSO (2.1 mL, 29.6 mmol) was added
dropwise, the mixture was stirred at -78 °C for 10 min, and
a solution of alcohol 6b (2.8 g, 5.6 mmol) in 5 mL of CH2Cl2
was added over 10 min. The milky solution was stirred for
30 min, 6.6 mL of Et3N was added to give a clear solution,
and the mixture was warmed to room temperature and washed
(2 × 0.5 M KHSO4, water) to give crude aldehyde 7b. The
crude product was dissolved in 50 mL of dry CH3CN, and
anhydrous K2CO3 (5.25 g) was added. After the mixture was
stirred for 10 min, acetic anhydride (3.0 mL) was added and
the mixture was refluxed overnight under nitrogen. The
volume was reduced by rotary evaporation, and the residue
was diluted with water, extracted with ether, and purified on
SiO2 (33% EtOAc-hexane) to give 2.6 g of enol acetate 8b (94%
in two steps): 1H NMR (200 MHz, CDCl3) δ 7.39-7.17 (m, 12H,
Bn + PMB), 6.86 (d, 2H, J ) 8.7 Hz, PMB), 4.95-4.64 (m,
7H), 3.98-3.95 (m, 1H), 3.89-3.79 (m, 4H), 3.60-3.55 (m, 2H),
3.47 (s, 3H, OMe), 2.16 (s, 3H, Ac) ppm; 13C NMR (50 MHz,
CDCl3) δ 185.9, 159.0, 137.4, 134.8, 129.5-127.7 (m), 122.8,
113.7, 113.6, 108.5, 107.5, 105.5, 99.6, 80.6, 78.8, 75.2, 74.3,
73.5, 73.3, 72.6, 56.0, 55.0, 20.4 ppm. MS (EI+): m/e 534 (M+),
459, 443, 415, 383, 311, 301, 211, 91. HRMS (EI+): calcd for
Meth yl 2-O-(p-Meth oxyben zyl)-3,4-O-d iben zyl-r-D-glu -
cop yr a n osid e (6a ). A mixture of methyl 2-O-(p-methoxy-
benzyl)-6-O-trityl-R-D-glucopyranoside (4a , 4.79 g, 8.6 mmol)
and NaH (1.03 g, ∼60% oil, 25.8 mmol) and benzyl bromide
(4.4 g, 25.8 mmol) in 100 mL of dry DMF was stirred for 2 h.
TLC (Rf ∼ 0.9, 50% EtOAc-hexane) showed nearly complete
conversion. Then the mixture was poured into water and
extracted with EtOAc. The crude product 5a was dissolved
in 100 mL of MeOH containing p-TsOH monohydrate (40 mg,
to give pH ∼ 2), and the mixture was stirred for 1 h. TLC (Rf
∼ 0.45, 50% EtOAc-hexane) showed no starting material.
Then, 10 mL of 10% NaHCO3 solution was added and the
mixture was evaporated to dryness. The product was ex-
tracted from the solid material with five portions of EtOAc,
concentrated, and then purified on SiO2 using 50% EtOAc-
hexane as eluent to give 2.53 g of pure methyl 2-O-(p-
methoxybenzyl)-4,5-O-dibenzyl-R-D-glucopyranoside (6a ) (60%
in two steps): 1H NMR (300 MHz, CDCl3) δ 7.40-7.10 (m, 12H,
aromatic), 6.80 (d, J ) 8.4 Hz, 2H, PMB), 4.93 (d, J ) 11.1
Hz, 1H), 4.80 (AB type, J ) 10.8 Hz, 1H), 4.68 (d, J ) 12 Hz,
1H), 4.56 (t, J ) 13.2, Hz, 2H), 4.46 (d, J ) 3 Hz, 1H), 3.94 (t,
J ) 9.3 Hz, 1H), 3.75 (3H, OMe), 3.70-3.55 (m, 4H), 3.49-
3.40 (m, 2H), 3.20 (s, 3H, OMe), 1.65 (s, 1H, OH) ppm; 13C
NMR (75 MHz, CDCl3) δ 138.7, 138.1, 130.2, 129.7, 128.4-
127.3 (m), 113.8, 98.2, 81.9, 79.6, 77.4, 77.2, 75.7, 75.0, 73.0,
70.6, 61.8, 55.2, 55.1 ppm. MS (CI+, NH3): m/e 512 (MNH4+),
493, 461, 403, 373, 121, 108, 91. HRMS (CI+, NH3): calcd for
C
31H34O8 (M+), m/e 534.2254; found, m/e 534.2243.
1-O-Acet yl-3-O-(p -m et h oxyb en zyl)-4,5-O-d ib en zyl-2-
d eoxyl-2-oxo-m yo-in ositol (9a ). To a solution of enol acetate
8a (2.20 g, 4.12 mmol) in 100 mL of acetone-water (3:2) was
added mercuric acetate (13.20 g, 41.2 mmol); the solution was
stirred for 45 min, 57 mL of saturated NaCl solution was
added, and the mixture was stirred for 24 h. The acetone was
evaporated, and the residue was extracted (EtOAc), concen-
trated, and purified on SiO2 (30-50% EtOAc-hexane) to give
1.85 g of inositol 9a (86% yield, 90% pure): 1H NMR (250 MHz,
CDCl3) δ 7.40-7.10 (m, 12H, Bn + PMB), 6.86, 6.83 (2s, 2H,
PMB), 5.12 (bs, 1H), 4.86 (d, J ) 11.1 Hz, 2H), 4.75 (d, J )
10.8 Hz, 2H), 4.64 (q, J ) 11.4 Hz, 2H), 4.47 (d, J ) 11.1 Hz,
1H), 4.25 (s, 1H), 4.09 (m, 1H), 3.80-3.70 (m, 1H), 3.74 (s, 3H,
OMe), 2.17 (s, 3H, Ac) ppm; 13C NMR (75 MHz, CDCl3) δ 169.9,
159.9, 137.3, 129.8-128.1 (m), 114.1, 113.9, 83.5, 81.8, 78.5,
76.1, 74.9, 73.5, 73.1, 69.4, 55.3, 45.7 ppm. MS (CI+, NH3):
m/e 538 (MNH4+), 494, 461, 429, 418, 211, 181, 154, 137, 121,
108, 91. HRMS (CI+, NH3): calcd for C30H36NO8 (MNH4+), m/e
538.2441; found, m/e 538.2457.
C
29H38NO7 (MNH4+), m/e 512.2648; found, m/e 512.2632.
Meth yl 3-O-(p-Meth oxyben zyl)-2,4-O-d iben zyl-r-D-glu -
cop yr a n osid e (6b). Compound 5b in 100 mL of MeOH/
acetone containing 5% sulfuric acid (pH ∼ 2) was stirred for 1
h. Then the reaction was quenched by 10% NaHCO3 solution,
the mixture was evaporated to dryness, and the residue was
extracted with three volumes of EtOAc and the combined
organic phases were washed with 10% NaHCO3. Purification
on SiO2 (25% EtOAc-hexane) gave 2.8 g of glucopyranoside
6b (yield 42%), Rf ∼ 0.16 (33% EtOAc-hexane): 1H NMR (200
MHz, CDCl3) δ 7.38-7.27 (m, 12H, Bn + PMB), 6.86 (d, J )
8.6 Hz, 2H, PMB), 4.95-4.57 (m, 7H), 4.01 (t, J ) 9.1 Hz, 1H),
3.80 (s, 3H, OMe), 3.76-3.63 (m, 3H), 3.56-3.47 (m, 2H), 3.37
(s, 3H, OMe), 1.80 (s, 1H, OH); 13C NMR (50 MHz, CDCl3) δ
159.0, 137.9, 131.0, 129.5, 128.3-127.7 (m), 113.6, 98.0, 81.5,
79.9, 76.2, 75.3, 74.9, 73.2, 70.1, 61.6, 55.1, 55.0 ppm. MS
(EI+): m/e 493 (M - H+), 403, 373, 253, 175, 121, 91. HRMS
(EI+): calcd for C29H34O7 (M+), m/e 494.2305; found, m/e
494.2299.
Meth yl (Z)-3,4-O-Diben zyl-2-O-(p-m eth oxyben zyl)-6-O-
a cet yl-r-D-glu cos-5-en op yr a n osid e (8a ). A solution of
(COCl)2 (1.05 mL, 1.94 mmol) in 100 mL of CH2Cl2 was cooled
to -78 °C, dry DMSO (1.71 mL, 3.87 mmol) was added
dropwise, and the mixture was stirred 10 min at -78 °C. Next,
a solution of 6a (2.40 g, 4.86 mmol) in 5 mL of CH2Cl2 was
added over 10 min. The cloudy solution was stirred for 30 min,
5.4 mL of Et3N was added to produce a clear solution, and the
mixture was warmed to room temperature. The usual workup
gave oil 7a that was used directly in the next step. Thus, oil
7a was dissolved in 75 mL of dry CH3CN, anhydrous K2CO3
(4.13 g) was added, the mixture was stirred for 10 min, acetic
anhydride (2.7 mL) was added, and the mixture was refluxed
overnight under nitrogen. The volume was reduced to one-
1-O-Acet yl-4-O-(p -m et h oxyb en zyl)-3,5-O-d ib en zyl-2-
d eoxyl-2-oxo-m yo-in ositol (9b). To a solution of enol acetate
8b (3.0 g, 5.6 mmol) in 200 mL of acetone-water (3:2) was
added mercuric acetate (18.6 g, 58.3 mmol). The solution was
stirred for 45 min, and 100 mL of saturated NaCl solution was
added. The mixture was stirred for 24 h and concentrated in
vacuo, and the residue was extracted (3 volumes of CHCl3)
and purified by flash chromatography (33% EtOAc-hexane)
to give 1.7 g of protected inositol 9b (57% yield), Rf ∼ 0.36 (50%
EtOAc-hexane): 1H NMR (200 MHz, CDCl3) δ 7.41-7.23 (m,
12H, Bn + PMB), 6.89, 6.84 (d, J ) 8.6 Hz, 2H, PMB), 5.17
(d, J ) 2.4 Hz, 1H), 4.98-4.52 (m, 6H), 4.34 (m, 1H), 4.20-
4.04 (m, 2H), 3.87 (m, 1H), 3.82 (s, 3H, OMe), 2.73 (s, 1H, OH),
2.23 (s, 3H, Ac) ppm; 13C NMR (50 MHz, CDCl3) δ 197.7, 169.7,
159.1, 137.2, 130.2-127.7 (m), 113.6, 106.5, 105.5, 83.4, 81.3,
78.7, 75.7, 74.7, 73.4, 73.2, 69.1, 55.1, 20.3 ppm. MS (FAB):

6518 J . Org. Chem., Vol. 63, No. 19, 1998
Chen et al.
m/e 519 (M - H+) 429, 399, 386, 273, 211, 121. HRMS (FAB):
113.5, 95.7, 95.0, 83.1, 81.0, 80.0, 76.3, 75.6, 75.4, 72.8, 72.6,
69.4, 69.1, 55.0, 20.7 ppm. HRMS (FAB): calcd for C46H50O10
(M - H+), m/e 761.3326; found, m/e 761.3355.
calcd for
519.2020.
C
30H34O8 (M - H+), m/e 519.2019; found, m/e
1-O-Acetyl-3-O-(p-m eth oxyben zyl)-4,5-O-d iben zyl-m yo-
in ositol (10a ). To a solution of inosose 9a (1.85 g, 3.56 mmol)
in 60 mL of dry acetonitrile was added NaBH(OAc)3 (7.55 g,
35.6 mmol) and 19 mL of glacial HOAc. The mixture was
stirred for 45 min at room temperature, and the excess NaBH-
(OAc)3 was destroyed by dropwise addition of 0.5 M NaHSO4.
The mixture was extracted with EtOAc, washed (0.5 M
NaHSO4, saturated Na2HPO4), dried (Na2SO4), and concen-
trated. The residue was recrystallized from EtOAc/H to yield
1.5 g of 10a (81%), Rf ∼ 0.21 (67% EtOAc-hexane): 1H NMR
(300 MHz, CDCl3) δ 7.40-7.10 (m, 12H, aromatic), 6.78 (m,
2H, PMB), 5.20-4.37 (m, 7H), 4.20 (m, 1H), 4.10-3.41 (m, 6H),
3.74 (s, 3H, OMe), 2.10 (s, 3H, Ac) ppm; 13C NMR (63 MHz,
CDCl3) δ 172.3, 158.7, 130.0, 129.6, 128.1, 128.0, 113.8, 113.6,
82.6, 80.6, 80.1, 75.5, 72.9, 67.1, 55.8 ppm. MS (CI+, NH3):
m/e 540 (MNH4+), 431, 401, 330, 311, 295, 277, 205, 181, 137,
121, 108, 91. HRMS: calcd for C30H38NO8 (MNH4+), m/e
540.2597; found, m/e 540.2596.
1-O-Acetyl-4-O-(p-m eth oxyben zyl)-3,5-O-d iben zyl-m yo-
in ositol (10b). To a solution of inosose 9b (970 mg, 1.8 mmol)
in 50 mL of dry acetonitrile was added NaBH(OAc)3 (4.0 g,
18.8 mmol) and 8.8 mL of glacial HOAc. The mixture was
stirred for 45 min at room temperature, and the excess NaBH-
(OAc)3 was destroyed by dropwise addition of 0.5 M NaHSO4.
The mixture was extracted (EtOAc), washed (0.5 M NaHSO4),
dried (Na2SO4), and purified on deactivated SiO2 to give 800
mg of compound 10b (83% yield): 1H NMR (200 MHz, CDCl3)
δ 7.34-7.22 (m, 12H, Bn + PMB), 6.85 (d, J ) 8.6 Hz, 2H,
PMB), 4.99-4.70 (m, 7H), 4.28 (t, J ) 2.6 Hz, 1H), 4.10 (t, J
) 9.4 Hz, 1H), 3.80 (s, 3H, OMe), 3.55 (dd, J ) 2.6, 9.4 Hz,
1H), 3.36 (t, J ) 9.4 Hz, 1H), 2.42 (s, 1H, OH), 2.22 (s, 1H,
OH), 2.16 (s, 3H, Ac) ppm; 13C NMR (50 MHz, CDCl3) δ 170.8,
129.4-127.7 (m), 113.5, 108.5, 106.5, 83.0, 80.5, 80.0, 76.3,
75.5, 73.1, 73.0, 70.1, 68.0, 55.1, 21.1 ppm. HRMS (FAB):
calcd for C30H34O8 (M+), m/e 522.2254; found, m/e 522.2267.
3-O-(p -Met h oxyb en zyl)-4,5-O-d ib en zyl-2,6-O-b is(b en -
zoxym eth yl)-m yo-in ositol (12a ). A mixture of protected
inositol 10a (1.50 g, 2.87 mmol), proton sponge (1.5 g), n-Bu4-
NBr (100 mg), and benzoxyl chloromethyl ether (BOMCl, 2
mL) in 40 mL of dry acetonitrile was stirred overnight at room
temperature, then at 35 °C for 10 h, and then at 55 °C
overnight. TLC (product Rf ∼ 0.57, 33% EtOAc-hexane)
showed no starting material. The mixture was concentrated
3-fold, diluted with water, and extracted with EtOAc, and the
extracts were washed (water), dried (MgSO4), and concentrated
to give oil 11a . The crude material 11a was dissolved in 50
mL of MeOH, 0.35 M NaOH (20 mL) was added, and the
mixture was refluxed 2 h. The usual workup gave 1.52 g of
protected inositol 12a (76%): 1H NMR (300 MHz, CDCl3) δ
7.40-7.10 (m, 22H), 6.90-6.70 (m, 2H, PMB), 5.00-4.50 (m,
15H), 4.20-4.10 (m, 1H), 3.96 (t, J ) 9 Hz, 1H), 3.88-3.80
(m, 2H), 3.76 (s, 3H, OMe), 3.43 (m, 2H) ppm; 13C NMR (75
MHz, CDCl3) δ 138.7, 130.1-127.0 (m), 113.8, 113.7, 96.6, 95.9,
83.0, 82.8, 81.7, 79.6, 75.8, 72.3, 71.1, 70.1, 69.8, 65.3, 55.3
ppm. MS (FAB): m/e 743 (MNa+), 599, 491, 371, 227, 211,
181, 176, 121. HRMS: calcd for C44H48NaO9 (MNa+), m/e
743.3196; found, m/e 743.3192.
1-O-Acet yl-3,5-O-d ib en zyl-2,6-O-b is(b en zoxym et h yl)-
m yo-in ositol (12b). To the intermediate 11b (660 mg, 0.866
mmol) in 30 mL of wet CH2Cl2 was added DDQ (231 mg, 1.01
mmol). The mixture was stirred at room temperature for 3 h,
diluted to 60 mL CH2Cl2, washed (2 × 10% aqueous NaHCO3,
brine, water), dried (MgSO4), concentrated, and purified on
SiO2 (25% EtOAc-hexane) to give 4-hydroxy compound 12b
(500 mg, 89%) as a colorless oil, Rf ∼ 0.3 (25% EtOAc-
hexane): 1H NMR (200 MHz, CDCl3) δ 7.37-7.26 (m, 20H,
Bn), 5.03-4.52 (m, 13H), 4.41 (t, J ) 2.4 Hz, 1H), 4.23 (t, J )
8.8 Hz, 1H), 4.14 (t, J ) 9.6 Hz, 1H), 3.49-3.35 (m, 2H), 2.52
(s, 1H, OH), 1.85 (s, 3H, Ac); 13C NMR (50 MHz, CDCl3) δ
170.3, 138.3, 137.6, 137.2, 128.4-127.3 (m), 107.5, 105.5, 95.7,
94.7, 82.8, 79.1, 75.1, 72.7, 72.1, 71.7, 69.5, 69.1, 20.7 ppm.
HRMS (FAB): calcd for C38H42O9 (MH+): m/e 643.2907; found,
m/e 643.2926.
4,5-O-Dib en zyl-2,6-O-b is(b en zoxym et h yl)-m yo-in osi-
tol (13a ). A mixture of protected inositol 12a (40 mg, 0.056
mmol) and DDQ (50 mg) in 10 mL of CH2Cl2-H2O (100/1 v/v)
was stirred at room temperature for 2 h. The usual workup
gave 22 mg (67% yield) of diol 13a (Rf ∼ 0.36, 1.5:1 EtOAc-
hexane); 1H NMR (300 MHz, CDCl3) δ 7.50-7.00 (m, 22H,
aromatic), 5.00-4.50 (m, 12H), 4.25-4.00 (m, 1H), 3.85-3.30
(6H), 3.00 (br, s, 1H) ppm; 13C NMR (75 MHz, CDCl3) δ 138.6-
137.4 (m), 129.0-128.0 (m), 82.4, 77.2, 75.5, 75.4, 71.8, 71.0,
70.3, 70.1, 64.8 ppm. MS (FAB): m/e 623 (MNa+).
1-O-Acet yl-3,5-O-d ib en zyl-4-O-(d ib en zylp h osp h or yl)-
2,6-O-bis(ben zoxym eth yl)-m yo-in ositol (13b). To 4-alcohol
12b (100 mg, 0.159 mmol) and 1H-tetrazole (134 mg, 1.91
mmol) in 10 mL of dry CH2Cl2 was added a solution of dibenzyl
N,N-diisopropylphosphoramidite (132 mg, 0.38 mmol). The
solution was stirred at room temperature under nitrogen for
1 h and cooled to -40 °C, and a solution of m-CPBA (83 mg,
0.65 mmol) was added. The mixture was stirred for 1 h at
room temperature, diluted with 50 mL of CH2Cl2, washed (10%
aqueous NaHCO3, brine), dried (MgSO4), concentrated, and
purified on SiO2 (50% EtOAc-hexane) to give 100 mg (71%)
of product 13b as a colorless oil, Rf ∼ 0.72 (50% EtOAc-
hexane): 1H NMR (200 MHz, CDCl3) δ 7.34-7.04 (m, 30H,
Bn), 4.98-4.59 (m, 17H), 4.49 (J ) 12.0 Hz, 1H), 4.37 (m, 1H),
4.27 (t, J ) 12.0 Hz, 1H), 3.60-3.50 (m, 2H), 1.81 (s, 3H); 13
C
NMR (50 MHz, CDCl3) δ 170.5, 138.1, 137.7, 137.1, 128.4-
127.4 (m), 106.7, 105.7, 104.7, 96.0, 95.0, 81.8, 81.7, 79.6, 79.5,
78.0, 75.0, 72.4, 72.3, 72.0, 69.7, 69.3, 69.2, 69.1, 69.0, 20.7
ppm; 31P NMR (81 MHz, CDCl3) δ -0.56 ppm. HRMS (FAB):
calcd for C52H55O12P (M+), m/e 902.3431; found, m/e 902.3480.
Tet r a b en zyl
1,3-(4,5-O-d ib en zyl-2,6-O-b is(b en zoxy-
m eth yl)-m yo-in osityl) Bis(p h osp h a te) (14a ). A solution of
diol 13a (20 mg, 0.038 mmol) in 2 mL of CH2Cl2 and
1H-tetrazole (10 mg, 0.14 mmol) was stirred at room temper-
ature, while a solution of dibenzyl N,N-diisopropylphosphora-
midite (40 mg, 0.12 mmol) in 1 mL of CH2Cl2 was added. The
mixture was stirred at room temperature for 1 h and cooled
to -40 °C, m-CPBA (20 mg) was added, and the reaction was
stirred for 30 min at 0 °C and then 30 min at room temper-
ature. The mixture was diluted with CH2Cl2, washed (10%
aqueous NaHCO3), dried (Na2SO4), concentrated, and chro-
matographed on SiO2 using 50% EtOAc-hexane to give 40 mg
(87%) of protected bis(phosphate) 14a as a viscous colorless
oil, Rf ∼ 0.3: 1H NMR (300 MHz, CDCl3) δ 7.4-7.2 (m, 40H,
phenyl), 5.0-4.4 (m, 22H), 4.4-4.1 (m, 2H), 4.0 (m, 1H), 3.4
(m, 1H) ppm; 13C NMR (63 MHz, CDCl3) δ 138.1, 137.8, 135.7,
130-127 (m), 96.1, 75-74 (m), 76-69 (m) ppm; 31P NMR (101
MHz, CDCl3) δ 0.33, 0.09 (1:1) ppm.
1,3-m yo-In osityl Bis(p h osp h a te) Tetr a sod iu m Sa lt (15).
A solution of bis(phosphate) 14a (140 mg, 0.125 mmol),
NaHCO3 (42 mg, 0.5 mmol), and Pd/C (10%, 50 mg) in
t-BuOH-H2O (6:1 v/v, 35 mL) was shaken at 50 psi initial
pressure for 5 h. The catalyst was filtered off and washed with
2 × 5 mL of EtOH, 2 × 5 mL of EtOH-H2O (1:1 v/v), and 2 ×
5 mL of water. Removal of solvent gave 50 mg of solid bis-
(phosphate) 15: 1H NMR (200 MHz, D2O) δ 4.27 (t, J ) 2.8
1-O-Acetyl-3,5-O-d iben zyl-4-O-(p-m eth oxyben zyl)-2,6-
O-bis(ben zoxym eth yl)-m yo-in ositol (11b). A mixture of
inositol 10b (800 mg, 1.5 mmol), proton sponge (830 mg), n-Bu4-
NBr (25 mg), and BOMCl (0.86 mL) in 40 mL of dry acetoni-
trile was stirred overnight at room temperature, at 35 °C for
10 h, and then 55 °C overnight. The mixture was concentrated
3-fold, diluted with water, and extracted with EtOAc, and the
extracts were washed (water), dried (MgSO4), concentrated,
and purified on SiO2 to give intermediate 11b as a colorless
oil in 57% yield: 1H NMR (CDCl3, 200 MHz) δ 7.36-7.18 (m,
22H, Bn + PMB), 6.81 (d, J ) 8.7 Hz, 2H, PMB), 5.04-4.70
(m, 14H), 4.56 (t, J ) 9.6 Hz, 1H), 4.41-4.39 (m, 1H), 4.27 (t,
J ) 10.0 Hz, 1H), 4.06 (t, J ) 9.5 Hz, 1H), 3.80 (s, 3H, OMe),
3.59-3.48 (m, 2H), 1.85 (s, 3H, Ac) ppm; 13C NMR (50 MHz,
CDCl3) δ 170.3, 159.0, 138.3, 137.7, 137.5, 129.5-127.2 (m),

PtdIns(3)P and PtdIns(4)P Synthesis
J . Org. Chem., Vol. 63, No. 19, 1998 6519
Hz, 1H), 4.0-3.80 (m, 2H), 3.72 (t, J ) 9.2 Hz, 2H), 3.35 (t, J
) 9.2 Hz, 1H) ppm; 31P NMR (81 MHz, D2O) δ 6.70, 5.0 (1:1)
ppm.
Cya n oet h yl 1,2-O-Dip a lm it oyl-sn -glycer yl 1-(4,5-O-
d iben zyl-2,6-O-bis(ben zoxym eth yl)-m yo-in osityl) P h os-
p h a te (18). A mixture of PMB ether 17a (800 mg, ∼90% pure,
0.556 mmol) and DDQ (150 mg, 0.661 mmol) was stirred in
50 mL of CH2Cl2-H2O (100:1, v:v) at room temperature for 2
h. The mixture was washed (10% aqueous NaHCO3), dried
(MgSO4), and purified on SiO2 using 67% EtOAc-hexane to
give 430 mg of phosphate 18 (60% yield from 12a ) as an oil,
Rf ∼ 0.34: 1H NMR (250 MHz, CDCl3) δ 7.35-7.20 (m, 20H,
aromatic), 5.30-5.10 (m, 1H), 5.05-4.40 (m, 12H), 4.35-4.15
(m, 10H), 3.80-3.30 (m, 2H), 3.00 (br, 1H, OH), 2.52 (J ) 6.1
Hz, CH2CN), 2.28 (t, J ) 7.2 Hz, 4H, COCH2), 1.70-1.50 (m,
4H), 1.30-1.10 (m, 48H), 0.90 (t, J ) 4.8 Hz, 6H, 2 × CH3)
ppm; 31P NMR (81 MHz, CDCl3) δ -0.63, -0.81 (1:1) ppm.
Cyan oeth yl 1,2-O-Dipalm itoyl-sn -glycer yl 1-(3-O-Diben -
zylp h osp h or yl-4,5-O-d iben zyl-2,6-O-bis(ben zoxym eth yl)-
m yo-in osityl) P h osp h a te (19). A solution of hydroxy com-
pound 18 (430 mg, 0.335 mmol) in 5 mL of CH2Cl2 and 1H-
tetrazole (95 mg, 1.36 mmol) was stirred at room temperature,
while a solution of dibenzyl N,N-diisopropylphosphoramidite
(230 mg, 0.67 mmol) in 1 mL of CH2Cl2 was added. The
mixture was stirred at room temperature for 1 h and cooled
to -40 °C, m-CPBA (115 mg) was added, and the reaction was
stirred for 30 min at 0 °C and then 30 min at room temper-
ature. The mixture was diluted with CH2Cl2 and washed (10%
NaHCO3, water), dried (Na2SO4), concentrated, and chromato-
graphed on SiO2 using 50% EtOAc-hexane to give 414 mg
(80%) of compound 19 (Rf ∼ 0.38) as a viscous colorless oil:
1H NMR (300 MHz, CDCl3) δ 7.4-7.2 (m, 40H, phenyl), 5.0-
4.4 (m, 22H), 4.4-4.1 (m, 2H), 4.0 (m, 1H), 3.4 (m, 1H) ppm;
31P NMR (81 MHz, CDCl3) δ 0.33, 0.09 (1:1) ppm. MS (FAB):
m/e 1567 (MNa+), 808, 553, 552, 550, 313, 281, 267, 221, 207,
147, 136, 133. HRMS (FAB): calcd for C88H123NNaO18P2
(MNa+), m/e 1566.8113; found, m/e 1566.8160.
Cyan oeth yl 1,2-O-Dipalm itoyl-sn -glycer yl 1-[3-(p-Meth -
oxyben zyl-4,5-O-diben zyl)-2,6-O-bis(ben zoxym eth yl)-myo-
in osityl] P h osp h a te (17a ). To a mixture of protected inositol
12a (400 mg, 0.556 mmol) and 1H-tetrazole (155 mg, 2.21
mmol) in 5 mL of dry CH2Cl2 was added phosphoramidite 16
(840 mg, 1.09 mmol) in 2 mL of CH2Cl2, and the mixture was
stirred at room temperature for 30 min. The mixture was
cooled to -40 °C, and m-CPBA (250 mg, 60-85%) was added
and stirred as it warmed to room temperature. The mixture
was diluted to 20 mL with CH2Cl2, washed (10% NaHCO3,
water), dried (Na2SO4), concentrated, and chromatographed
on SiO2 using 50% EtOAc-hexane to give 760 mg (97%) of
protected phosphoinositide 17a (Rf ∼ 0.3) as a viscous, colorless
oil: 1H NMR (250 MHz, CDCl3) δ 7.35-7.20 (m, 22H, aro-
matic), 6.80-6.60 (m, 2H, PMB), 5.30-5.10 (m, 1H), 5.05-
4.40 (m, 14H), 4.35-4.15 (m, 10H), 3.78 (s, 3H, OMe), 3.80-
3.30 (m, 2H), 2.52 (J ) 6.1 Hz, CH2CN), 2.28 (t, J ) 7.2 Hz,
4H, COCH2), 1.70-1.50 (m, 4H), 1.30-1.10 (m, 48H), 0.90 (t,
J ) 4.8 Hz, 6H, 2 × CH3) ppm; 13C NMR (63 MHz, CDCl3) δ
171.1, 133.4, 129.4-127.6 (m), 113.8, 95.5, 87.5, 82.8, 81.4,
75.7, 72.6, 70.3, 69.8, 69.7, 61.5, 55.2, 34.1, 34.0, 29.7-29.1
(m), 24.8, 22.7, 22.5, 14.3 ppm; 31P NMR (81 MHz, CDCl3) δ
-0.44, -0.63 (1:1) ppm.
3,5-O-Diben zyl-4-O-d iben zylp h osp h or yl-2,6-O-bis(ben -
zoxym eth yl)-m yo-in ositol (14b). To a solution of acetate
13b (200 mg, 0.221 mmol) in 20 mL of methanol was added
K2CO3 (61.3 mg, 0.443 mmol), and the mixture was stirred at
room temperature for 45 min until TLC showed that the
reaction was complete. The solvent was evaporated, and the
residue was dissolved in water and extracted with 3 × with
EtOAc. The combined organics were washed (water, brine),
and dried (MgSO4), and the crude product was purified on SiO2
with 4% EtOAc-CH2Cl2 to give 130 mg of 14b (68%) (Rf ∼ 0.48,
50% EtOAc-hexane): 1H NMR (200 MHz, CDCl3) δ 7.38-7.08
(m, 30H, aromatic), 5.02-4.52 (m, 17H), 4.26 (s, 1H), 3.94-
3.84 (m, 2H), 3.56-3.45 (m, 2H) ppm; 13C NMR (50 MHz,
CDCl3) δ 139.2-136.8 (m), 128.3-127.1 (m), 96.4, 95.5, 82.8,
81.1, 79.7, 79.5, 74.8, 74.7, 71.7, 70.8, 70.0, 69.5, 69.0, 68.9,
66.8 ppm; 31P NMR (81 MHz, CDCl3) δ -0.56 ppm. HRMS
(FAB): calcd for C50H54O11P (MH+), m/e 861.3404; found, m/e
861.3438.
Cya n oeth yl 1,2-O-Dip a lm itoyl-sn -glycer yl 3,5-O-d iben -
zyl-4-O-d iben zylph osph or yl-2,6-O-d iben zoxym eth yl-myo-
in ositol P h osp h a te (17b). To a mixture of inositol 14b (43
mg, 0.050 mmol) and 14 mg (0.20 mmol) of 1H-tetrazole in
dry CH2Cl2 (10 mL) was slowly added cyanoethyl 1,2-O-
dipalmitoyl-sn-glyceryl N,N-diisopropylphosphoramidite (16)
(150 mg, 0.20 mmol) in CH2Cl2 (1 mL), and the mixture was
stirred for 1 h at room temperature. The reaction was cooled
to -40 °C, and m-CPBA (43 mg, 0.25 mmol) was added and
stirred as it warmed to room temperature for another 1 h.
Workup as above gave phosphate 17b (28 mg, 36% yield) as a
colorless oil that slowly solidified: 1H NMR (200 MHz, CDCl3)
δ 7.39-7.00 (m, 30H, Bn), 5.31-5.13 (m, 1H), 5.00-4.48 (m,
17H), 4.26-4.09 (m, 9H), 3.67-3.48 (m, 2H), 2.51-2.45 (t, J
) 6.2 Hz, 2H), 2.28 (t, J ) 7.0 Hz, 2H, CH2CO), 2.27 (t, J )
7.2 Hz, 2H, CH2CO), 1.80-1.50 (m, 4H), 1.25 (m, 48H), 0.95-
0.91 (t, J ) 6.2 Hz, 6H, CH3) ppm; 13C NMR (50 MHz, CDCl3)
δ 173.2, 172.8, 138.0, 137.7, 137.6, 137.3, 136.1, 136.0, 135.8,
128.3-127.3 (m), 116.5, 96.5, 95.3, 81.3, 79.5, 79.3, 75.0, 73.1,
72.4, 70.3, 69.6, 69.1-68.0 (m), 67.9, 66.0, 65.9, 62.3, 62.3, 61.5,
34.1, 33.9, 31.9, 29.7-29.1 (m), 25.5, 24.8, 22.7, 19.5, 19.4, 14.1
ppm; 31P NMR (81 MHz, CDCl3) δ -0.47 (1 P), -1.09 (1 P)
ppm. MS (FAB): m/e 1567 (MNa+), 551, 313, 181. HRMS
(FAB): calcd for C88H123NNaO18P2 (MNa+), m/e 1566.8113;
found, m/e 1566.8078.
3-Am in op r op yl 1,2-O-Dip a lm it oyl-sn -glycer yl 1-(3-O-
P h osp h or yl-m yo-in osityl) P h osp h a te (20a ). A solution of
fully protected precursor 19 (170 mg, 0.11 mmol), NaHCO3 (9
mg, 0.10 mmol), and Pd/C (10%, 100 mg) in t-BuOH-H2O (6:
1, v:v, 35 mL) was added, and the mixture was shaken at 50
psi initial pressure for 5 h. The catalyst was filtered off and
washed with 3 × 5 mL of EtOH, 5 mL of EtOH-H2O (v/v 1:1),
and 1 × 5 mL of water. Removal of solvent gave 52 mg (63%
yield) of solid PtdIns(3)P triester 20a : 1H NMR (200 MHz,
D2O) δ 4.30-3.20 (m, 13H), 2.70 (m, 2H), 2.30-2.20 (m, 4H,
COCH2), 1.7-1.4 (m, 6H), 1.30-1.00 (m, 48H), 0.90-0.70 (m,
6H) ppm; 31P NMR (81 MHz, CDCl3) δ 8.8 (s and sharp), 1.9
(br) (∼ 1:1) ppm.
3-Am in opr opyl 1,2-O-Dipalm itoyl-sn -glycer yl 4-O-P h os-
p h or yl-D-m yo-in ositol P h osp h a te Sod iu m Sa lt (20b). A
mixture of cyanoethyl ester 17b (43 mg, 0.028 mmol), sodium
bicarbonate (2.4 mg, 0.028 mmol), and Pd/C (10%) (100 mg)
in 14 mL of t-BuOH/water (6:1) was shaken under hydrogen
(50 psi) for 10 h and filtered, the catalyst was washed with a
CHCl3-CH3OH-H2O mixture, and the filtrate was concen-
trated under N2 to give PtdIns(4)P triester 20b: 1H NMR (200
MHz, D2O) δ 5.24 (s, 1H), 5.00-2.60 (br), 2.40-2.14 (m, 4H),
1.77-1.50 (m, 4H), 1.47-1.04 (m, 48H), 0.95-0.84 (m, 6H);
31P NMR (81 MHz, CDCl3) δ 0.09 (s), 2.6 (s) ppm.
1,2-O-Dip a lm itoyl-sn -glycer yl 1-[3-O-P h osp h or yl-m yo-
in osityl] P h osp h a te (21). A solution of precursor 19 (130
mg, 0.086 mmol) and i-Pr2NEt (200 µL) in 2 mL of MeOH was
stirred overnight (TLC showed no remaining starting material)
and evaporated to dryness. The residue was dissolved in
t-BuOH-H2O (6:1 v/v, 35 mL) and NaHCO3 (10 mg, 0.11
mmol), Pd/C (10%, 100 mg) was added, and the mixture was
shaken at 50 psi initial pressure for 5 h. The catalyst was
filtered off and washed with 5 × 5 mL of EtOH, 3 × 5 mL of
EtOH-H2O (v/v 1:1), and 2 × 5 mL of water. Removal of
solvent gave 62 mg (77% yield) of solid PtdIns(3)P di-C16
-
The low yields (36-40%) in the coupling reactions to give
17b and 34a below do not reflect a fundamental limitation in
the reaction. Instead, low recoveries simply reflect difficulties
in workup, primarily due to (a) loss of material at foamy
aqueous/organic interfaces and (b) irreversible adsorption to
silica gel during purification.
diester 21: 1H NMR (200 MHz, CDCl3) δ 5.30-3.50 (very broad
and poorly resolved peaks, 11H), 2.30-2.10 (m, 4H), 1.70-
1.30 (m, 52H), 0.90-0.80 (m, 6H) ppm; 31P NMR (81 MHz,
CDCl3) δ 3.8, 0.1 (∼1:1) ppm.
In the processing of this and other hydrogenolytic depro-
tection reactions of these detergent-like molecules, an esti-

6520 J . Org. Chem., Vol. 63, No. 19, 1998
Chen et al.
mated equimolar (according to the number of negative phos-
phate charges in the molecule) amount of NaHCO3 was
generally added during hydrogenation to suppress acyl migra-
tion. When traces of Na+ salts remain, the 31P NMR peaks
become complex as the different counterions result in different
chemical shifts. Thus, the hydrogenolysis reaction mixture
was first passed through a Dowex 50WX8-200 ion-exchange
resin and concentrated to dryness to remove excess HCO3-. It
was then passed through a Chelex column to exchange the
cation back to Na+. When NaHCO3 was not used, this extra
step was unnecessary.
3-((p -Ben zoyld ih yd r ocin n a m yl)a m in o)p r op yl 1,2-O-
Dip a lm itoyl-sn -glycer yl 1-(3-O-P h osp h or yl-m yo-in osityl)
P h osp h a te (22a ). PtdIns(3)P aminopropyl triester 20a (5 mg,
0.0056 mmol) was suspended in 2.5 mL of anhydrous DMF,
and N-hydroxysuccinimido p-benzoyldihydrocinnamate ester
(BZDC-NHS ester, 3.6 mg, 0.010 mmol) was added, followed
by 0.2 mL of dry Et3N. The suspension was stirred overnight
and concentrated in vacuo, and the residue was evaporated
with 2 mL of water. The residue was centrifuged with acetone
several times until the acetone showed no remaining UV-
absorbing material. Then the solid was dissolved in 1 mL of
water and applied to a 50 × 6 mm column of DEAE cellulose
(OH- form). The column was washed with 2 × 1 mL of water
and then eluted with 2 × 1 mL of 0.1 M TEAB, 2 × 1 mL of
0.2 M TEAB, 2 × 1 mL of 0.3 M TEAB, and 1 mL of 0.4 M
TEAB buffer. The product eluted in 0.2 M buffer. Evaporation
of these fractions in vacuo followed by evaporating with several
small volumes of methanol gave the triethylamine salt of
BZDC-PtdIns(3)P triester 22a . After ion-exchange using a
column packed with Bio-Rad Chelex 100 resin (Na form), the
sodium salt of 22a (2.5 mg, 40% yield) was obtained as a
glass: 1H NMR (250 MHz, D2O) δ 7.86-7.75 (m, 5H), 7.50 (t,
2H), 7.36 (t, 2H), 4.30-3.20 (m, 13H), 3.10 (m, 2H), 2.90 (t, J
) 7.5 Hz, 2H), 2.45 (t, J ) 7.5 Hz, 2H), 2.30-2.20 (m, 4H,
COCH2), 1.7-1.4 (m, 6H), 1.30-1.00 (m, 48H), 0.90-0.70 (m,
6H) ppm; 31P NMR (81 MHz, CDCl3) δ 8.8 (s), 1.9 (s) (∼1:1)
ppm.
3-((p-Ben zoyld itr itiocin n a m yl)a m in o)p r op yl 1,2-O-Di-
p a lm it oyl-sn -glycer yl 1-[3-O-P h osp h or yl-m yo-in osit yl]
P h osph ate (22a′). [3H2]-N-Hydroxysuccinimido p-benzoyldihy-
drocinnamate ester ([3H]BZDC-NHS ester, 2 mCi, 40 Ci/
mmol) in EtOAc was gently concentrated with dry N2, and then
3-aminopropyl triester 20a (800 µg/mL of DMF stock solution,
15 µL) was added followed by 10 mL of dry triethylamine. The
suspension was stirred overnight. The solvents were evapo-
rated in vacuo, the residue was evaporated with 1 mL of water,
and the residue was dissolved in 1 mL of water and then
applied to 50 × 6 mm column of DEAE cellulose (OH- form).
The column was washed with 2 × 1 mL of water and then
eluted with 2 × 1 mL of 0.1 M TEAB, 2 × 1 mL of 0.2 M TEAB,
2 × 1 mL of 0.3 M TEAB, and 1 mL of 0.4 M TEAB buffer.
The product 22a ′ eluted in 0.1 M buffer. Radioactive fractions
were pooled to give ca. 150 µCi of purified 22a ′, specific activity
42.5 Ci/mmol (radiochemical yield, 7.5%).
3-((p -Ben zoyld ih yd r ocin n a m yl)a m in o)p r op yl 1,2-O-
Dip a lm itoyl-sn -glycer yl 1-(4-O-p h osp h or yl-m yo-in osityl)
P h osp h a te (22b). To aminopropyl triester 20b (5 mg, 0.0056
mmol) in 2 mL of DMF (distilled after incubation with P2O5
for 5 days) was added BZDC-NHS ester (3.6 mg, 0.010 mmol),
and the mixture was stirred for 30 min. Dry Et3N (0.2 mL)
was then added, and the suspension was stirred overnight.
DMF was removed under a stream of N2, and the residue was
washed with acetone (5 × 1 mL) to give 22b as a white powder
(2.3 mg, 37% yield), which was homogeneous by TLC: 1H NMR
(250 MHz, D2O) δ 7.83-7.71 (m, 5H), 7.64-7.54 (m, 2 H),
7.45-7.38 (m, 2H), 4.29-3.40 (m, 13H), 3.23-3.09 (m, 2H),
2.75-2.70 (m, 4H), 2.03-1.96 (m, 4H), 1.67-1.49 (m, 4H),
1.31-1.20 (m, 48H), 0.96-0.79 (m, 6H) ppm; 31P NMR (81 Hz,
D2O) 4.89 (br) ppm.
h and then washed (10% NaHCO3), dried (MgSO4), and
purified on SiO2 using EtOAc-hexane-Et3N (100:300:10, v/v/
v), to give 732 mg of phosphoramidite 23 (84%), Rf ∼ 0.95: 1H
NMR (200 MHz, CDCl3) δ 7.40-7.20 (m, 5H, aromatic), 4.89
(d, J ) 8.4 Hz, CH2O), 4.80-4.50 (m, 1H), 4.30-4.10 (m, 2H),
4.01, 3.99 (2t, J ) 8.2 Hz, 2H), 3.80-3.50 (m, 2H), 1.41, 1.35
(2s, 6H, isopropylidene) ppm; 31P NMR (81 MHz, CDCl3) δ
148.8, 148.7 (∼1:1) ppm.
Ben zyl 1,2-O-Diisop r op ylid en e-sn -glycer yl 1-[3-(p -
Meth oxyben zyl)-4,5-O-d iben zyl-2,6-O-d iben zoxym eth yl-
m yo-in osityl] P h osp h a te (24). To a mixture of precursor
12a (200 mg, 0.28 mmol) and 1H-tetrazole (80 mg, 1.1 mmol)
in 5 mL of dry CH2Cl2 was added phosphoramidite 23 (200
mg, 1.09 mmol) in 2 mL of CH2Cl2, and the mixture was stirred
at room temperature for 30 min. The mixture was cooled to
-40 °C, and m-CPBA (250 mg, 60-85%) was added and stirred
as the reaction warmed to room temperature. The mixture
was diluted to 20 mL with CH2Cl2, washed (10% NaHCO3,
water), dried (Na2SO4), concentrated, and chromatographed
on SiO2 using 50% EtOAc-hexane to give 240 mg (97%) of
compound 24 as a viscous colorless oil (Rf ∼ 0.4): 1H NMR
(200 MHz, CDCl3) δ 7.40-7.20 (m, 27H, aromatic), 6.80 (m,
2H, PMB), 5.20-4.50 (m, 14H), 4.30-3.90 (m, 8H), 3.78 (s,
3H, OMe), 3.70-3.40 (m, 6H), 1.37, 1.31 (2s, 6H, isopropy-
lidene) ppm; 31P NMR (81 MHz, CDCl3) δ:-0.19, -0.34 (∼1:1)
ppm. MS (FAB): m/e 1027 (MNa+), 669, 386, 370, 328, 211,
181, 121. HRMS (FAB): calcd for C57H65NaO14P (MNa+), m/e
1027.4010; found, m/e 1027.4031.
Ben zyl 1,2-O-Diisop r op ylid en e-sn -glycer yl 1-[4,5-O-
Diben zyl-2,6-O-bis(ben zoxym eth yl)-m yo-in osityl] P h os-
p h a te (25). A mixture of phosphate 24 (230 mg, 0.23 mmol)
and DDQ (80 mg, 0.35 mmol) in 20 mL of CH2Cl2-H2O (100/1
v/v) was stirred at room temperature for 2 h. The mixture
was washed (10% NaHCO3), dried (MgSO4), and purified on
SiO2 using 50% EtOAc-hexane to give 140 mg (69% yield) of
phosphate 25 as an oil (Rf ∼ 0.20): 1H NMR (200 MHz, CDCl3)
δ 7.40-7.15 (m, 25H, aromatic), 5.20-4.50 (m, 12H), 4.30-
3.90 (m, 7H), 3.80-3.40 (m, 6H), 3.30 (br., 1H, OH), 1.39, 1.32
(2s, 6H, isopropylidene) ppm; 31P NMR (81 MHz, CDCl3) δ
-0.45, -0.56 (∼1:1) ppm. MS (FAB): m/e 907 (MNa+), 855
(MH+), 777, 669, 657, 579, 393, 271, 182, 181, 165, 154, 136,
115. HRMS: calcd for C49H57NaO13P (MNa+), m/e 907.3435;
found, m/e 907.3476.
Ben zyl sn -glycer yl 1-(3-O-Diben zylp h osp h or yl-4,5-O-
diben zyl-2,6-O-diben zoxym eth yl-myo-in osityl) P h osph ate
(27). A solution of protected phosphoinositide 25 (136 mg,
0.154 mmol) in 2 mL of CH2Cl2 and 1H-tetrazole (43 mg, 0.61
mmol) was stirred at room temperature, while a solution of
dibenzyl N,N-diisopropylphosphoramidite (110 mg, 0.32 mmol)
in 1 mL of CH2Cl2 was added. The mixture was stirred at
room temperature for 1 h and cooled to -40 °C, and m-CPBA
(20 mg) was added. The reaction was stirred for 30 min at 0
°C and then 30 min at room temperature, and the mixture
was diluted with CH2Cl2, washed (10% NaHCO3, water), dried
(Na2SO4), and concentrated to give a crude residue 26 (33%
EtOAc-hexane, Rf ∼ 0.5). This residue was dissolved in
MeOH, 10 mg of p-TsOH monohydrate was added, and the
mixture was stirred at room temperature for 1 h. The acid
was neutralized (0.5 mL of 10% aqueous NaHCO3), and the
solvent was evaporated. Purification on SiO2 eluting with 67%
EtOAc-hexane gave 100 mg (Rf ∼ 0.12) of compound 27 (59%
for two steps) as an oil: 1H NMR (200 MHz, CDCl3) δ 7.40-
7.10 (m, 35H, aromatic), 5.20-4.50 (m, 18H), 4.45-3.50 (m,
11H), 2.0 (br, 2H, OH) ppm; 31P NMR (81 MHz, CDCl3) δ 0.98,
-0.64, 0.75, -1.09 (∼1:1:1:1) ppm.
Ben zyl 1,2-O-Dia cyl-sn -glycer yl 1-(3-O-Diben zylp h os-
ph or yl-4,5-O-diben zyl-2,6-O-bis(ben zoxym eth yl)-myo-in os-
ityl) P h osp h a te (28). A solution of diol 27 (1 equiv) and fatty
acid (octanoic acid or butyric acid, 3 equiv), DCC (3 equiv),
and DMAP (0.05-0.1 equiv) in 5 mL of dry CH2Cl2 was stirred
overnight. The solid was removed by filtration, washed with
ether, and concentrated, the residue was dissolved in 50%
EtOAc-hexane, and the precipitate was removed by filtration
and washed with the same solvent. Concentration of the
Ben zyl 1,2-O-Diisop r op ylid en e-sn -glycer yl N,N-Diiso-
p r op ylp h osp h or a m id ite (23). A mixture of BnOP(NPr2-i)2
(800 mg, 2.36 mmol), 1,2-O-diisopropylidene-sn-glycerol (312
mg, 2.36 mmol), and diisopropylammonium tetrazole (200 mg)
in 10 mL of dry CH2Cl2 was stirred at room temperature for 1

PtdIns(3)P and PtdIns(4)P Synthesis
J . Org. Chem., Vol. 63, No. 19, 1998 6521
filtrates gave an oil that was purified on SiO2 using 50%
EtOAc-hexane to give the phosphoinositides 28a or 28b with
Rf ∼ 0.2.
0.91 (2t, J ) 7.3 Hz, 6H, CH3) ppm; 13C NMR (63 MHz, CDCl3)
δ: 173.2, 172.9, 159.3, 129.8, 129.3, 113.8, 87.7, 72.9, 70.0, 67.9,
62.7, 55.2, 36.2, 35.9, 18.4, 18.3, 13.6 ppm. MS (CI+): m/e 352
(M+) 281, 215, 145, 136, 121, 71. HRMS (CI+): calcd for
C19H28O6, m/e 352.1886; found, m/e 352.1881. Anal. Calcd for
Da ta for ben zyl 1,2-O-d iocta n oyl-sn -glycer yl 1-(3-O-
d iben zylp h osp h or yl-4,5-O-d iben zyl-2,6-O-bis(ben zoxym -
eth yl)-m yo-in osityl) p h osp h a te (28a ): 40 mg yield; 1H NMR
(200 MHz, CDCl3) δ 7.35-7.10 (m, 35H, aromatic), 5.15-4.00
(m, 27H), 3.60-3.40 (m, 2H), 2.2 (m, 4H, COCH2), 1.70-1.20
(m, 20H), 0.9-0.7 (m, 6H, 2 × CH3) ppm; 31P NMR (81 MHz,
CDCl3) δ -0.48, -0.53, -0.58, -0.64 (∼1:1:1:1) ppm. MS
(FAB): m/e 1380 (MNa+), 327, 255, 181, 127. HRMS (FAB):
calcd for C76H94NaO18P2 (MNa+), m/e 1379.5813; found, m/e
1379.5759.
C
19H28O6: C, 64.77; H, 7.95. Found: C, 65.00, H, 7.88.
Gen er a l P r oced u r e for Dep r otection of P MB Eth er s.
A mixture of a PMB ether 31 (1 equiv) in 20 mL of wet CH2-
Cl2 (CH2Cl2-H2O, 100:1 v/v) and DDQ (1.2 equiv) was stirred
at room temperature for 3 h. It was then washed with 10%
aqueous NaHCO3 and brine, dried over MgSO4, and concen-
trated. Purification on SiO2 gel using 95% ether-CH2Cl2
mixtures gave the corresponding 1,2-O-diacyl-sn-glycerol 32.
Da ta for 1,2-O-d iocta n oyl-sn -glycer ol (32b): obtained in
Da ta for ben zyl 1,2-O-d ibu tyr oyl-sn -glycer yl 1-(3-O-
d iben zylp h osp h or yl-4,5-O-d iben zyl-2,6-O-bis(ben zoxym -
eth yl)-m yo-in osityl) p h osp h a te (28b): 50 mg yield; 1H NMR
(200 MHz, CDCl3) δ 7.40-7.10 (m, 35H, aromatic), 5.20-4.50
(m, 20H), 4.40-3.90 (m, 7H), 3.60-3.40 (m, 2H), 2.30-2.20
(m, 4H, COCH2), 1.70-1.50 (m, 4H), 1.00-0.80 (m, 6H, 2 ×
CH3) ppm; 31P NMR (81 MHz, CDCl3) δ -0.22, -0.45, -0.53
(∼1:1:2) ppm. MS (FAB): m/e 1267 (MNa+), 215, 181.
HRMS: calcd for C68H78NaO18P2 (MNa+), m/e 1267.4561;
found, m/e 1267.4610.
1,2-O-Diocta n oyl-sn -glycer yl 1-(3-O-P h osp h or yl-m yo-
in osityl) P h osp h a te (29a ). To a solution of fully protected
phosphate 28a (50 mg, 0.037 mmol), t-BuOH-H2O (6:1 v/v,
35 mL) and NaHCO3 (9 mg, 0.10 mmol) was added Pd/C (10%,
50 mg), and the mixture was shaken at 50 psi initial pressure
for 5 h. The catalyst was filtered off and washed with 5 mL
of EtOH, 5 mL of EtOH-H2O (v/v 1:1), and 3 × 5 mL of water.
Removal of solvent gave 17 mg (63% yield) of solid PtdIns(3)P
di-C8-ester 29a : 1H NMR (200 MHz, D2O) δ 5.35-5.15 (m, 1H),
4.45-4.20 (m, 2H), 4.10-3.60 (m, 7H), 3.35 (t, J ) 9.2 Hz, 1H),
2.38, 2.34 (2t, J ) 7.2 Hz, 4H, COCH2), 1.7-1.2 (m, 20H), 0.82
(t, J ) 6.2 Hz, 6H) ppm; 31P NMR (81 MHz, D2O) δ 7.04, 2.65
ppm.
1,2-O-Dib u t yr yl-sn -glycer yl-1-(3-O-p h osp h or yl-m yo-
in osityl) P h osp h a te (29b). A solution of protected phos-
phoinositide 28b (50 mg, 0.040 mmol), NaHCO3 (10 mg, 0.11
mmol), and Pd/C (10%, 50 mg) in t-BuOH-H2O (6:1 v/v, 35
mL) was shaken at 50 psi initial pressure for 5 h. The catalyst
was filtered off and washed with 5 mL of EtOH, 5 mL of
EtOH-H2O (1:1 v/v), and 3 × 5 mL of water. Removal of
solvent gave 22 mg (92%) of solid PtdIns(3)P di-C4-ester 29b:
1H NMR (200 MHz, D2O) δ 5.35-5.25 (m, 1H), 4.50-4.20 (m,
2H), 4.10-3.37 (m, 7H), 3.35 (t, J ) 9.2 Hz, 1H), 2.37, 2.34
(2t, J ) 7.2 Hz, 4H), 1.70-1.50 (m, 4H), 0.88, 0.86 (2t, J ) 6.2
Hz, 6H, 2 × CH3) ppm; 31P NMR (81 MHz, D2O) δ 7.43, 2.65
(∼1:1) ppm.
1
80% yield; H NMR (200 MHz, CDCl3) δ 5.07 (quint, J ) 5.6
Hz, 1H), 4.30-4.08 (m, 2H), 3.70 (d, J ) 5.0 Hz, 2H), 3.37-
2.27 (m, 4H), 1.66-1.54 (m, 4H), 1.28-1.20 (m, 16H), 0.89-
0.83 (m, 6H) ppm. MS (CI+): m/e 362 (MNH4+), 345, 327,
269, 226, 127, 98. HRMS (CI+): calcd for C19H40NO5 (MNH4+),
m/e 362.2906; found, m/e 362.2909.
Da ta for 1,2-O-d ibu tyr oyl-sn -glycer ol (32c) (Rf ∼ 0.35,
33% EtOAc-hexane): obtained in72% yield; 1H NMR (200
MHz, CDCl3) δ 5.09 (quint., J ) 5.0 Hz, 1H), 4.29, 4.26 (2q, J
) 11.9 Hz, 2H), 3.72 (d, J ) 4.9 Hz, 2H), 2.33, 2.30 (2t, J )
7.4 Hz, 4H, COCH2), 1.70-1.53 (m, 4H), 0.95, 0.94 (2t, J )
7.2 Hz, 6H, CH3) ppm. MS (CI+): 250 (MNH4+), 233 (MH+),
215, 145, 88, 71. HRMS (CI+): calcd for C11H24NO5 (MNH4+),
m/e 250.1654; found, m/e 250.1661.
Gen er a l P r oced u r e for P r ep a r a tion of P h osp h or a -
m id ites 33. A mixture of 1,2-O-diacyl-sn-glycerol and benzyl
N,N-diisopropylchlorophosphoramidite and N,N-diisopropyl-
ethylamine was stirred at 0 °C for 2 h. It was washed with
10% aqueous NaHCO3 solution and dried over MgSO4. Puri-
fication on SiO2 eluting with EtOAc-hexane-Et3N (from 10:
50:3 to 10:30:3, v/v/v) gave the phosphoramidites 33. Spec-
troscopic data are shown; however, analytical data on these
unstable materials were not obtained. These materials were
employed directly for the coupling reactions to give adducts
34.
Da ta for ben zyl 1,2-O-d iocta n oyl-sn -glycer ol N,N-d i-
isop r op ylp h osp h or a m id ite (33b): 1H NMR (200 MHz,
CDCl3) δ 7.38-7.24 (m, 5H, Bn), 5.25-5.12 (m, 1H), 4.70 (t, J
) 10.0 Hz, 2H), 4.40-4.31 (m, 1H), 4.23-4.10 (m, 1H), 3.81-
3.53 (m, 4H), 2.30 (t, J ) 7.4 Hz, 4H), 1.67-1.56 (m, 4H), 1.27-
1.17 (m, 28H), 0.92-0.85 (m, 6H) ppm; 31P NMR (81 MHz,
CDCl3) δ 149.6 (s), 149.4 (s) (1:1) ppm.
Da ta for ben zyl 1,2-O-d ibu tyr yl-sn -glycer ol N,N-d iiso-
p r op ylp h osp h or a m id ite (33c): 1H NMR (200 MHz, CDCl3)
δ 7.40-7.20 (m, 5H, Bn), 5.20-5.10 (m, 1H), 4.72 (d, J ) 7.4
Hz, 2H), 4.40-4.20 (m, 2H), 3.80-3.50 (m, 4H), 2.30 (t, J )
7.2 Hz, 4H, COCH2), 1.80-1.60 (m, 4H), 1.30 (m, 12H), 0.94
(2t, J ) 7.4 Hz, 6H) ppm; 31P NMR (81 MHz, CDCl3) δ 149.5
(s), 149.4 (s) (1:1) ppm.
Gen er a l P r oced u r e for th e DCC/DMAP -P r om oted Es-
ter ifica tion . A mixture of a PMB-protected glycerol 30 (1
equiv), DMAP (0.05-0.1 equiv), fatty acid (2.5 equiv), and DCC
(2.5 equiv) in dry CH2Cl2 (10 mL) was stirred at room
temperature overnight. The mixture was filtered and concen-
trated. The residue was then dissolved in EtOAc, and the
precipitate was filtered again and washed with EtOAc. The
filtrate was concentrated and purified on SiO2 column eluting
with EtOAc-hexane mixtures to give the corresponding 1,2-
O-diacyl-3-O-(p-methoxybenzyl)-sn-glycerol 31.
Da ta for 1,2-O-d iocta n oyl-3-O-(p-m eth oxyben zyl)-sn -
glycer ol (31b): obtained in 95% yield; 1H NMR (200 MHz,
CDCl3) δ 7.24, 7.20, 6.88, 6.83 (4s, 4H, PMB), 5.28-5.17 (m,
1H), 4.46-4.45 (m, 2H), 4.31 (dd, J ) 4.0, 12.0 Hz, 1H), 4.15
(dd, J ) 6.4 Hz, 11.8 Hz, 1H), 3.79 (s, 3H, OMe), 3.54 (d, J )
5.2 Hz, 2H), 2.30, 2.26 (2t, J ) 7.8 Hz, 4H), 1.60-1.57 (m,
4H), 1.48-1.05 (m, 16H), 0.86 (t, J ) 6.6 Hz, 6H, CH3) ppm.
MS (EI+): m/e 464 (M+) 352, 327, 226, 201, 135, 121. HRMS
(EI+): calcd for C27H44O6 (M+), m/e 464.3139; found, m/e
464.3120.
Ben zyl 1,2-O-Dip a lm itoyl-sn -glycer yl 1-[3,5-O-Diben -
zyl-2,6-O-d iben zoxym eth yl-4-O-(d iben zylp h osp h or yl)-D-
m yo-in ositol] P h osp h a te (34a ). To a mixture of alcohol 14b
(40 mg, 0.047 mmol) and 1H-tetrazole (24 mg, 0.34 mmol) in
CH2Cl2 (10 mL) was added
a solution of benzyl 1,2-O-
dipalmitoyl-sn-glyceryl N,N-diisopropylphosphoramidite (33a ,
190 mg) in 3 mL of dry CH2Cl2. Workup as in the preparation
of 13b gave phosphate 34a (25 mg, 40% yield) as a colorless
oil: 1H NMR (200 MHz, CDCl3) δ 7.38-6.99 (m, 35H, aro-
matic), 5.19-4.45 (m, 20H), 4.26-3.99 (m, 7H), 3.53-3.43 (m,
2H), 2.27-2.16 (m, 4H, CH2CO), 1.64-1.44 (m, 4H), 1.25 (m,
48H), 0.88 (t, 6H, J ) 6.6 Hz, 6H, CH3) ppm; 13C NMR (50
MHz, CDCl3) δ 137.9-135.8 (m), 128.4-127.0 (m), 107.5,
106.4, 105.5, 71.8, 69.9-66.7 (m), 61.3, 33.9-33.7 (m), 31.7,
29.5-28.9 (m), 24.6, 22.5, 13.9 ppm; 31P NMR (81 MHz, CDCl3)
δ -0.22, -0.50 (∼1:1) ppm. MS (FAB): m/e 1604 (MNa+), 551,
313, 181. HRMS (FAB): calcd for C92H126NaO18P2 (MNa+), m/e
1603.8317; found, m/e 1603.8382.
Da t a for 1,2-O-d ib u t yr yl-3-O-(p-m et h oxyb en zyl)-sn -
glycer ol (31c): obtained in 90% yield; 1H NMR (300 MHz,
CDCl3) δ 7.23, 7.19, 6.86, 6.84 (4s, 4H, PMB), 5.25-5.17 (m,
1H), 4.44 (AB, J ) 11.7 Hz, 2H, OCH2), 4.20 (d, AB, J ) 3.8
Hz, 2H), 3.77 (s, 3H, OMe), 3.53 (d, J ) 5.2 Hz, 2H), 2.26,
2.24 (2t, J ) 7.4 Hz, 4H, COCH2), 1.70-1.53 (m, 4H), 0.92,
Ben zyl 1,2-O-d iocta n oyl-sn -glycer yl 1-[3,5-O-d iben zyl-
2,6-O-b is(b en zoxym et h yl)-4-O-(d ib en zylp h osp h or yl)-D-
m yo-in ositol] p h osp h a te (34b) was synthesized as described
above: 1H NMR (200 MHz, CDCl3) δ 7.39-7.01 (m, 35H, Bn),

6522 J . Org. Chem., Vol. 63, No. 19, 1998
Chen et al.
5.15-4.45 (m, 20 H), 4.25-3.96 (m, 7H), 3.49-3.44 (m, 2H),
2.35-2.17 (m, 4H, CH2CO), 1.70-1.45 (m, 4H), 1.26-1.24 (m,
16H), 0.88-0.84 (m, 6H, CH3); 13C NMR (50 MHz, CDCl3) δ
172.9, 172.5, 137.6-137.0 (m), 128.5-127.0 (m), 110.5, 109.5,
107.5, 106.5, 104.5, 102.5, 69.9-69.0 (m), 61.3, 33.8, 33.7, 31.4,
28.8-28.7 (m), 24.6, 22.4, 13.8 ppm; 31P NMR (81 MHz, CDCl3)
δ -0.20, -0.24 (combined as 1P), -0.53 (1P) ppm. MS
(FAB): m/e 1380 (MNa+), 551, 327, 255, 181. HRMS (FAB):
calcd for C76H94NaO18P2 (MNa+), m/e 1379.5813; found, m/e
1379.5795.
1.00 (m, 48H), 0.90-0.84 (m, 6H); 31P NMR (81 MHz, D2O) δ
7.67 (sharp), 2.92 (br) ppm.
1,2-O-Diocta n oyl-sn -glycer yl 1-(4-O-P h osp h or yl-D-m yo-
in osit ol) P h osp h a t e Sod iu m Sa lt (35b ). Preparation as
above from protected phosphoinositide 34b gave final product
35b (80% yield) as the sodium salt: 1H NMR (200 MHz, D2O)
δ 5.31-5.22 (m, 1H), 4.41 (dd, J ) 4.0, 13.4 Hz, 1H), 4.28-
3.87 (m, 5 H), 3.77 (t, J ) 9.2 Hz, 1H), 3.58 (dd, J ) 2.6, 9.6
Hz, 1H), 3.41 (t, J ) 9.0 Hz, 1H), 3.21 (J ) 7.4 Hz, 1H), 2.43-
2.31 (m, 4H), 1.66-1.47 (m, 4H), 1.29-1.16 (m, 16H), 0.84-
0.78 (m, 6H, CH3) ppm; 31P NMR (81 MHz, D2O) δ 7.66 (s,
1P), 2.76 (s, 1P) ppm.
1,2-O-Dibu tyr yl-sn -glycer yl 1-(4-O-P h osp h or yl-D-m yo-
in ositol) P h osp h a te Sod iu m Sa lt (35c). Preparation as
above from protected phosphoinositide 34c gave final product
35b (85% yield) as the sodium salt: 1H NMR (200 MHz, D2O)
δ 5.40-5.26 (m, 1H), 4.37 (dd, J ) 3.2, 12.0 Hz, 1H), 4.30-
4.17 (m, 2H), 4.12-4.03 (m, 3H), 3.98-3.88 (m, 1H), 3.79 (t, J
) 9.6 Hz, 1H), 3.60 (dd, J ) 2.6, 9.8 Hz, 1H), 3.43 (t, J ) 9.3
Hz, 1H), 2.41-2.31 (m, 4H), 1.69-1.47 (m, 4H), 0.89, 0.87 (2t,
J ) 7.4 Hz, 6H, CH3) ppm; 31P NMR (81 MHz, D2O) δ 7.81 (s,
1P), 2.73 (s, 1P) ppm.
Ben zyl 1,2-O-d ibu ta n oyl-sn -glycer yl 1-[3,5-O-d iben zyl-
2,6-O-bis(ben zyloxym eth yl)-4-O-(d iben zylp h osp h or yl)-D-
m yo-in ositol] p h osp h a te (34c) was synthesized as described
above: 1H NMR (200 MHz, CDCl3) δ 7.38-7.00 (m, 35H, Bn),
5.20-4.51 (m, 20H), 4.27-4.00 (m, 7H), 3.53-3.43 (m, 2H),
2.27-2.16 (m, 4H, CH2CO), 1.66-1.53 (m, 4H), 0.95-0.85 (m,
6 H, CH3) ppm; 13C NMR (50 MHz, CDCl3) δ 172.7, 172.4,
137.9, 137.6, 137.5, 137.0, 128.5-127.1 (m), 112.5, 109.5, 108.5,
107.5, 106.5, 105.5, 104.5, 99.5, 69.9-66.9 (m), 61.3, 35.7, 35.6,
18.1, 13.4, 13.3 ppm; 31P NMR (81 MHz, CDCl3) δ -0.19, -0.25
(combined as 1P), -0.53 (1P) ppm. HRMS (FAB): calcd for
C
68H78O18P2 (MH+), m/e 1245.4742; found, m/e 1245.4836.
1,2-O-Dip a lm it oyl-sn -glycer yl 1-(4-O-P h osp h or yl-D-
m yo-in ositol) P h osp h a te Sod iu m Sa lt (35a ). A mixture
of protected phosphoinositide 34a (36 mg, 0.023 mmol),
NaHCO3 (6 mg, 0.072 mmol), and Pd/C (10%) (100 mg) in 12
mL of t-BuOH/water (6:1) was shaken under hydrogen (50 psi)
for 14 h. The mixture was filtered, washed with a mixed
solution of chloroform, methanol, and water, the solvents were
evaporated, and the remaining product 35a (17 mg, 84% yield)
was lyophilized: 1H NMR (200 MHz, CDCl3) δ 5.20-3.70 (br),
3.20-2.50 (br), 2.20-1.90 (m, 4H), 1.55-1.35 (m, 4H), 1.35-
Ack n ow led gm en t. We thank the NIH (Grant NS
29632) for financial support, Dr. D. G. Ahern (NEN Life
Science Products, Inc., Boston, MA) for providing [3H]-
BZDC-NHS, and our many phosphoinoisitide collabo-
rators for suggesting specific biologically useful
derivatives.
J O980501R