Synthesis of phosphotriester analogues of the phosphoinositides PtdIns(4,5)P2 and PtdIns(3,4,5)P3

Article abstract of DOI:10.1021/jo961226g

A synthetic route was developed for the preparation of novel O-(3-aminopropyl) tethered phosphotriester analogs (5) of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5,)P₂, or PIP₂) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃, or PIP₃) using the coupling reagent 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite. The phosphotriester ligand design introduced a reactive aminopropyl group at the polar lipid head of the ring-phosphorylated phosphoinositides, allowing a reporter moiety to be positioned at the surface of the bilayer and in the vicinity of the phosphorylated inositol. Such reporter groups may interact with membrane-proximal regions of PIP₂- and PIP₃-binding proteins recruited to membrane sites by electrostatic interactions between the phosphates of the phospholipid and basic regions of the proteins. Following a convergent strategy, phosphitylation of an optically-pure 1,2-O-diacyl-sn-glycerol with 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite was followed by coupling with protected inositol precursors to give adducts 8 in 80% to 95% yield. The 2-cyanoethyl phosphotriester was stable during the subsequent reaction steps and could be conveniently converted to the 3-aminopropyl group during the final hydrogenolysis of the benzyl protecting groups. Benzophenone-containing photoaffinity probes of the phosphotriester 11a and 11b were also synthesized. Alternatively, the versatile cyanoethyl group could be removed using diisopropylethylamine prior to hydrogenolysis, thereby furnishing the corresponding phosphodiesters, PIP₃ and PIP₂ (13a and 13b).

Full text of DOI:10.1021/jo961226g

8642
J . Org. Chem. 1996, 61, 8642-8647
Syn th esis of P h osp h otr iester An a logu es of th e P h osp h oin ositid es
P td In s(4,5)P 2 a n d P td In s(3,4,5)P 3
Qu-Ming Gu and Glenn D. Prestwich*,†
Department of Chemistry and Department of Biochemistry and Cell Biology,
State University of New York at Stony Brook, Stony Brook, New York 11794
Received J uly 1, 1996X
A synthetic route was developed for the preparation of novel O-(3-aminopropyl) tethered phospho-
triester analogs (5) of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5,)P2, or PIP2) and phos-
phatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3, or PIP3) using the coupling reagent 2-cyano-
ethyl N,N,N′,N′-tetraisopropylphosphorodiamidite. The phosphotriester ligand design introduced
a reactive aminopropyl group at the polar lipid head of the ring-phosphorylated phosphoinositides,
allowing a reporter moiety to be positioned at the surface of the bilayer and in the vicinity of the
phosphorylated inositol. Such reporter groups may interact with membrane-proximal regions of
PIP2- and PIP3-binding proteins recruited to membrane sites by electrostatic interactions between
the phosphates of the phospholipid and basic regions of the proteins. Following a convergent
strategy, phosphitylation of an optically-pure 1,2-O-diacyl-sn-glycerol with 2-cyanoethyl N,N,N′,N′-
tetraisopropylphosphorodiamidite was followed by coupling with protected inositol precursors to
give adducts 8 in 80% to 95% yield. The 2-cyanoethyl phosphotriester was stable during the
subsequent reaction steps and could be conveniently converted to the 3-aminopropyl group during
the final hydrogenolysis of the benzyl protecting groups. Benzophenone-containing photoaffinity
probes of the phosphotriester 11a and 11b were also synthesized. Alternatively, the versatile
cyanoethyl group could be removed using diisopropylethylamine prior to hydrogenolysis, thereby
furnishing the corresponding phosphodiesters, PIP3 and PIP2 (13a and 13b).
In tr od u ction
PtdIns(4,5,)P2 can be converted by agonist-stimulated,
receptor-mediated activation of phosphoinositide 3-kinase
(PI 3-K)10 to PtdIns(3,4,5,)P3, the key element in a new
intracellular signaling system (Scheme 1).11 Recent
efforts to understand the role of PI 3-K in cell signaling
have focused on possible targets for PIP3, in particular,
activation of Akt serine/threonine kinase,12,13 activation
of protein kinase C,14 and phosphorylation of pleckstrin
in platelets.15 For both PIP2 and PIP3, the question
remains as to what role the diacylglycerol moiety might
have in the interaction of these phosphoinositides with
hydrophobic regions of PIPn binding proteins. We report
herein the synthesis of novel triester analogues of PIPn
with pendant functionality for attaching reporter groups
that would enable investigation of protein-PIPn interac-
tions either in solution or at bilayer interfaces.
L-R-Phosphatidyl-D-myo-inositol 3,4,5-trisphosphate
(PtdIns(3,4,5)P3, or PIP3) (2) and L-R-phosphatidyl-D-myo-
inositol 4,5-bisphosphate (PtdIns(4,5,)P2, or PIP2) (1) play
a variety of essential roles in biological processes. PIP2
was first observed to be a substrate for phospholipase C
(Scheme 1), and the receptor-activated action of PLC on
PIP2 generated the second messengers Ins(1,4,5)P3 (3)
and 1,2-diacylglycerol (4).1-4 More recently, PIP2 has
been recognized to be a crucial element in the recruitment
of signaling proteins to membranes,5 as mediated by the
pleckstrin homology domains.6 Indeed, the recruitment
of the PLC δ1 isozyme is facilitated by the interaction of
its PH domain with 4,5-bisphosphate of PIP2.7 A three-
dimensional structure of the â-spectrin-Ins(1,4,5)P3 com-
plex has verified the importance of the 4,5-bisphosphate
interaction with hydrogen bonding and protonated basic
residues.8 In addition, PIP2 affects the organization of
the cytoskeleton by sequestering profilin, thereby pre-
venting the association of profilin with monomeric F-actin
and thus permitting the polymerization of actin.5,9
A number of published reports have described strate-
gies for the syntheses of diester and diether analogues
of PIP2 and PIP3.16-19 A typical synthetic strategy
(10) Stephens, L.; Cooke, F. T.; Walters, R.; J ackson, T.; Volinia,
S.; Gout, I.; Waterfield, M. D.; Hawkins, P. T. Curr. Biol. 1994, 4, 203-
214.
(11) Stephens, L. R.; J ackson, T. R.; Hawkins, P. T. Biochim.
Biophys. Acta 1993, 1179, 27-75.
(12) Burgering, B. M.; Coffer, P. J . Nature 1995, 376, 599-602.
(13) Franke, T. F.; Yang, S.-I.; Chan, T. O.; Datta, K.; Kazlauskas,
A.; Morrison, D. K.; Kaplan, D. R.; Tsichlis, P. N. Cell 1995, 81,
1-20.
(14) Toker, A.; Meyer, M.; Reddy, K. K.; Falck, J . R.; Aneja, R.;
Aneja, S.; Parra, A.; Burns, D. J .; Ballas, L. M.; Cantley, L. C. J . Biol.
Chem. 1994, 269, 32358-32367.
(15) Toker, A.; Bachelot, C.; Chen, C. S.; Falck, J . R.; Hartwig, J .
H.; Cantley, L. C.; Kovacsovics, T. J . J . Biol. Chem. 1995, 270, 29525-
29531.
† Current address: Department of Medicinal Chemistry, 308 Skaggs
Hall, The University of Utah, Salt Lake City, Utah 84112. Phone:
801-585-9051. Fax: 801-585-9053. E-Mail: gprestwich@deans.pharm.
utah.edu.
X Abstract published in Advance ACS Abstracts, November 1, 1996.
(1) Berridge, M. J .; Irvine, R. F. Nature 1984, 312, 315-321.
(2) Berridge, M. J . Annu. Rev. Biochem. 1987, 56, 159-193.
(3) Berridge, M. J . Nature 1993, 361, 315-325.
(4) Nishizuka, Y. Science 1992, 258, 607-614.
(5) J anmey, P. Chem. Biol. 1995, 2, 61-65.
(6) Saraste, M.; Hyvo¨nen, M. Curr. Opin. Struct. Biol. 1995, 5, 403-
(16) Reddy, K. K.; Saady, M.; Falck, J . R.; Whited, G. J . Org. Chem.
1995, 60, 3385-3390.
408.
(7) Rebecchi, M.; Peterson, A.; McLaughlin, S. Biochemistry 1992,
31, 12742-12747.
(8) Hyvo¨nen, M.; Macias, M. J .; Nilges, M.; Oschkinat, H.; Saraste,
M.; Wilmanns, M. EMBO J . 1995, 14, 4676-4685.
(9) Lambrechts, A.; Vandamme, J .; Goethals, M.; Vandekerckhove,
J .; Ampe, C. Eur. J . Biochem. 1995, 230, 281-286.
(17) Watanabe, Y.; Hirofuji, H.; Ozaki, S. Tetrahedron Lett. 1994,
35, 123-124.
(18) Watanabe, Y.; Tomioka, M.; Ozaki, S. Tetrahedron 1995, 51,
8969-8976.
(19) Gou, D.-M.; Chen, C.-S. J . Chem. Soc., Chem. Commun. 1994,
2125-2126.
S0022-3263(96)01226-1 CCC: $12.00 © 1996 American Chemical Society

PIP2 and PIP3 Triester Synthesis
J . Org. Chem., Vol. 61, No. 24, 1996 8643
Sch em e 1. Meta bolic P a th w a ys for P td In s(4,5)P 2 in Eu k a r yotic Cells
involves incorporation of benzyl 1,2-diacylglyceryl (N,N-
diisopropylamino)phosphoramidite to a variety of pro-
tected inositols derived from different chiral synthons.
The bifunctional phosphitylating reagent benzyl N,N,N′,N′-
tetraisopropylphosphorodiamidite has been used most
frequently for tetrazole-mediated phosphitylation of 1,2-
diacylglycerol, an approach that was first effectively
employed20 in the synthesis of PIP2 analogues. The
required 1,2-diacylglyceryl (N,N-diisopropylamino)phos-
phoramidite could also be prepared from benzyl chloro
(N,N-diisopropylamino)phosphoramidite.21
Resu lts a n d Discu ssion
In order to gain further insight into the electrostatic
and hydrophobic interactions important in phospho-
inositide recognition and signal transduction, a strategy
has been developed that allows synthetic access to PIP3
and PIP2 analogues with an O-(3-aminopropyl) phosphate
linker group at polar lipid head. This “triester strategy”
provides a different spatial orientation for affinity probes
and reporter groups as compared to the modification of
one of the acyl chains of the 1,2-diacylglycerol moiety.21
Thus, the aminopropyl tethered triester PIP2 and PIP3
analogues should permit normal incorporation of the
diacylglycerol moiety in the phospholipid bilayer as well
as the presence of the unmodified 4,5-bisphosphate or
3,4,5-trisphosphate to recruit target proteins. The re-
porter group would reside at the interface of the lipid
bilayer and the aqueous environment, in an orientation
to potentially provide important information on PIP2
and PIP3 binding proteins. In this article, we present a
facile synthesis of these novel phosphotriesters (5) of PIP3
and PIP2. The synthetic strategy using 2-cyanoethyl
N,N,N′,N′-tetraisopropylphosphorodiamidite as a bifunc-
tional phosphitylating reagent also permits the prepara-
tion of several other phosphatidylinositol analogues.
The synthesis of the 1,2-O-diacyl 3-O-PMB-sn-glycerols
(PMB ) p-methoxybenzyl) was achieved from (+)-1,2-O-
isopropylidene-sn-glycerol as previously described.21,22
The selection of the PMB protecting group minimized 2-3
acyl migration. Thus, removal of the PMB group21 from
the sn-3-position of 1,2-O-diacyl 3-O-PMB-glycerols could
be achieved by either catalytic hydrogenolysis at 1 atm
or by oxidative cleavage using DDQ at 24 °C. Hydro-
genolysis of the PMB group using Pd/C (10%) was
achieved without detectable 2-3 acyl migration (98%
yield, recrystallized from EtOH). Pure 2-cyanoethyl
N,N,N′,N′-tetraisopropylphosphorodiamidite23 was con-
densed with 1,2-O-dipalmitoyl-sn-glycerol in the presence
of 1H-tetrazole to give 1,2-dipalmitoyl-sn-glyceryl 2-cya-
noethyl (N,N-diisopropylamino)phosphoramidite (6) in
90-95% yield after SiO2 chromatography.
For the PIP3 analogues, differentially protected inositol
7a ,24 an intermediate used in the synthesis of P-1-
modified D-myo-Ins(1,3,4,5)P4 via a Ferrier rearrange-
ment,25 was employed. For the PIP2 analogues, inter-
mediate 7b, a precursor for D-myo-Ins(1,4,5)P3 affinity
(22) Martin, S. F.; J osey, J . A.; Wong, Y.-L.; Dean, D. W. J . Org.
Chem. 1994, 59, 4805-4820.
(23) Bannwarth, W.; Trzeciak, A. Helv. Chim. Acta 1987, 70, 175-
186.
(24) Estevez, V. A.; Prestwich, G. D. J . Am. Chem. Soc. 1991, 113,
9885-9887.
(25) Ferrier, R. J .; Middleton, S. Chem. Rev. 1993, 93, 2779-2831.
(20) Dreef, C. E.; Elie, C. J . J .; Hoogerhout, P.; van der Marel, G.
A.; van Boom, J . H. Tetrahedron Lett. 1988, 29, 6513-6516.
(21) Chen, J .; Profit, A. A.; Prestwich, G. D. J . Org. Chem. 1996,
61, 6305-6312.

8644 J . Org. Chem., Vol. 61, No. 24, 1996
Gu and Prestwich
Sch em e 2a
a
(a) 1H-tetrazole, CH2Cl2, 24 °C, m-CPBA, -20 °C; (b) DDQ, CH2Cl2-H2O, 24 °C; (c) (i-Pr)2NP(OBn)2, 1H-tetrazole, 24 °C; m-CPBA;
(d) H2, Pd/C (10%), t-BuOH-H2O (6:1), NaHCO3, 50 psi, 24 °C; (e) BZDC-NHS, DMF, 0.2 M Et3NHCO3, pH 8.5, 24 °C.
probes26 and PIP2 analogues,21 was prepared. In each of
these precursors, the PMB ethers mask the inositol
hydroxyls destined for phosphorylation, while the benzyl
(Bn) and benzyloxymethyl (BOM) ethers mask hydroxyls
that will remain as hydroxyl groups in the final phos-
phoinositides. As shown in Scheme 2, coupling of pro-
tected inositol 7a with phosphoramidite 6 followed by the
low-temperature oxidation of the resulting phosphite
provided the fully-protected inositol phosphotriester 8a
in 82% yield. Only a single isomer was detected based
on TLC and 31P NMR analysis. Note that the inositol
moiety is optically-pure, arising from R-D-glucose, and the
1,2-diacylglycerol portion is also optically-pure. The
remaining stereogenic site is the phosphorus of the
triester, and in the absence of any asymmetric induction,
the product would consist of a diastereomeric mixture
differing only in the absolute stereochemistry at phos-
phorus. It is likely that neither technique could separate
these diastereomers, as has been observed for other
diastereomeric phosphoinositide intermediates.21
propylphosphoramidite followed by the m-CPBA oxida-
tion furnished 2,6-O-bis(benzyloxymethyl)-3,4,5-O-tris-
(dibenzylphosphoryl)-D-myo-inositol 1,2-O-diacyl-sn-glyceryl
2-cyanoethyl phosphate 10a in 73% yield. Under the
same reaction conditions, 10b was obtained in 98% yield.
Removal of Bn and BOM protecting groups as well as
conversion of the 2-cyanoethyl to 3-aminopropyl group
were accomplished simultaneously by catalytic hydroge-
nation (Pd/C, 50 psi) to give the 3-aminopropyl tethered
phosphotriesters of PIP3 (5a ) and PIP2 (5b) in 60% and
75% yields, respectively. Hydrogenation of the nitrile
group under neutral or acidic conditions was sufficiently
rapid that no remaining 2-cyanoethyl group was observed
based on the 1H NMR analysis. Reaction of the 3-(p-
benzoyldihydrocinnamoyl)-NHS reagent (BZDC-NHS)27
with 5a and 5b provided BZDC-derivatized photoaffinity
probes 11a and 11b in 91% and 85% yields, respectively.
Alternatively, the removal of 2-cyanoethyl groups from
the protected PIP3 and PIP2 derivatives 10a and 10b
could be accomplished by heating the compounds in a
1:40 mixture of diisopropylethylamine and acetonitrile
at 60 °C for 12 h, producing phosphodiesters 12a and 12b
in 85% and 90% yields, respectively (Scheme 3). Cleav-
age of 2-cyanoethyl group from the phosphate moiety via
a â-elimination28 could also be achieved by treatment
with other tertiary amines, e.g., triethylamine or tri-
Similarly, reaction of protected inositol 7b26 with 6 at
rt gave the protected inositol 8b in 93% yield. Removal
of the PMB protecting groups from the protected inositols
8 by the oxidation with DDQ in wet CH2Cl2 provided triol
9a and diol 9b, in 85% and 95% yields, respectively.
Subsequent phosphitylation using dibenzyl N,N-diiso-
(26) Dorma´n, G.; Chen, J .; Prestwich, G. D. Tetrahedron Lett. 1995,
36, 8719-8722.
(27) Olszewski, J . D.; Dorma´n, G.; Elliott, J . T.; Hong, Y.; Ahern,
D. G.; Prestwich, G. D. Bioconjugate Chem. 1995, 6, 395-400.

PIP2 and PIP3 Triester Synthesis
J . Org. Chem., Vol. 61, No. 24, 1996 8645
Sch em e 3a
a
(a) (i-Pr)2NEt, CH3CN, 60 °C; (b) H2, Pd/C (10%), t-BuOH-H2,O (6:1), NaHCO3, 50 psi, 24 °C.
0.20. 1H NMR (CDCl3, 300 MHz) δ 5.12-5.08 (m, 1H), 4.30-
methylamine. The cleavage rate was dramatically en-
hanced when a more polar organic solvent such as
methanol or ethanol was used instead of acetonitrile.
Compounds 12a and 12b could be either purified on silica
gel or directly subjected to catalytic hydrogenolysis after
removal of the solvents in vacuo. Thus, Pd/C-catalyzed
hydrogenolysis of 12a and 12b provided corresponding
phosphodiesters, dipalmitoyl PIP3 (13a ), and dipalmitoyl
PIP2 (13b) in overall yields of 60% and 70% from 10a
and 10b, respectively.
4.22 (m, 2H), 3.72 (t, 2H, J ) 6.0 Hz), 2.30 (q, 4H), 1.60-1.50
(m, 4H), 1.38-1.20 (m, 48H), 0.80 (t, 3H, J ) 7.2 Hz). FAB
MS: 551 (M+ - OH, 24.3); 569 (MH+, 2.1). Anal. Calcd for
C35H68O5: C, 73.88; H, 12.05. Found: C, 74.02; H, 11.81.
2-Cya n oet h yl N,N,N′,N′-Tet r a isop r op ylp h osp h or od i-
a m id ite. To a solution of PCl3 (13.7 g, 8.55 mL, 0.1 mol) and
dry pyridine (8.1 mL, 0.1 mol) in 20 mL of dry Et2O under
nitrogen at -78 °C was added dropwise over 1 h 3-hydroxy-
propanenitrile (7.1 g, 6.8 mL, 0.1 mol) in 40 mL of dry Et2O.
The mixture was warmed to rt and stirred for an additional 1
h at 24 °C. Precipitates were removed by filtration under
nitrogen and washed twice with dry Et2O (2 × 40 mL). The
solvent was evaporated in vacuo, and the oily residue was
dried for 2 h at 20 Torr. Next, a solution of this oily residue
(7.4 g, 43 mmol) in 30 mL of dry Et2O (20 mL) under nitrogen
was treated with (i-Pr)2NH (53.6 mL, 0.38 mol) over 10 min
at -20 °C. The mixture was stirred at 24 °C for 1.5 h, and
precipitated solids were removed by filtration under nitrogen
and washed twice with Et2O (2 × 20 mL). After evaporation
of solvent, the oily residue was distilled over a 10-cm Vigreux
column to give 8.6 g of the phosphoramidite reagent (70%
yield), bp 115 °C/0.6 Torr.
1,2-O-Dip a lm itoyl-sn -glycer yl 2-Cya n oeth yl (N,N-Di-
isop r op yla m in o)p h osp h or a m id ite (6). To a mixture of the
1,2-O-dipalmitoyl-sn-glycerol (150 mg, 0.26 mmol) and 1H-
tetrazole (36.4 mg, 0.52 mmol) in dry CH2Cl2 (1 mL) under
nitrogen was added a solution of the phosphoamidite (157 mg,
0.52 mmol) in CH2Cl2 (1 mL). The mixture was stirred at 24
°C for 40 min, concentrated in vacuo, and purified on SiO2
(EtOAc/hexane/Et3N, 1:5:0.1) to give 190 mg of product (94%
yield) as a colorless oil. TLC (SiO2) EtOAc/hexane (1:2), Rf ∼
0.9. 1H NMR (CDCl3, 300 MHz) δ 5.20 (br, s, 1H), 4.85-4.20
(m, 4H), 3.80-3.70 (t, 2H, J ) 1.5 Hz), 3.65-3.60 (m, 2H),
3.14 (m, 2H), 2.65 (t, 2H, J ) 1.5 Hz), 2.35-2.20 (m, 4H), 1.60-
1.50 (m, 4H), 1.30-1.20 (m, 60H), 0.88-0.82 (t, 6H).
Con clu sion
Novel affinity probes based on phosphotriester ana-
logues of PIP3 and PIP2 offer the opportunity to examine
PIPn-protein interactions at membrane surfaces. In
preparing these affinity probes, we found that the use of
2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamid-
ite provided a general approach to the synthesis of novel
3-aminopropyl phosphotriesters of phosphatidylinositides
in excellent overall yield. In particular, we have dem-
onstrated that this methodology can be applied to the
synthesis of two aminopropyl tethered phosphatidyl-
inositides as well as to other analogues of PIP2 and PIP3.
Preparation and biological function of specific affinity
probes derived from these aminopropyl and aminoacyl
derivatives will be reported elsewhere.29 In addition, the
synthetic approach described herein is extremely versa-
tile, allowing access to the synthesis of additional PIPn
and phosphatidic acid derivatives that will be reported
in due course.29a
Exp er im en ta l Section
2,6-O-Bis(ben zyloxym eth yl)-3,4,5-O-tr is(p-m eth oxyben -
zyl)-D-m yo-in osit ol 1-O-(1,2-O-Dip a lm it oyl)-sn -glycer yl
2-Cya n oeth yl P h osp h a te (8a ). To a mixture of 6 (171 mg,
0.22 mmol) and 1H-tetrazole (15.4 mg, 0.22 mmol) in dry CH2-
Cl2 (1 mL) was added a solution of 2,6-O-bis(benzyloxymethyl)-
3,4,5-O-tris(p-methoxybenzyl)-D-myo-inositol 7a (79 mg, 0.1
mmol) in CH2Cl2 (1 mL). After the mixture was stirred at 24
°C for 1 h under nitrogen, TLC analysis showed that the
reaction was complete (Rf of the coupled product: 0.80; SiO2;
EtOAc/hexane 1:1). The reaction mixture was then cooled to
-40 °C, and a solution of m-CPBA (43 mg, 0.25 mmol) in CH2-
Cl2 was added. After stirring at 24 °C for an additional 1 h,
the mixture was diluted with CH2Cl2 (40 mL), washed once
with 10% aqueous NaHCO3 solution (20 mL) and brine (20
mL), dried over MgSO4, concentrated in vacuo, and purified
on SiO2 (EtOAc/hexane, 1:1) to give 121 mg of product 8a (82%
yield) as a colorless oil. TLC (SiO2) EtOAc/hexane (1:1), Rf ∼
0.55. 1H NMR (CDCl3, 300 MHz) δ 7.29-7.10 (m, 22H), 6.80-
6.75 (m, 6H), 5.20 (br, s, 1H), 4.95-4.50 (m, 10H), 4.40-4.05
(m, 4H), 3.80 (s, 9H), 3.75 (t, 2H), 2.70 (t, 2H), 2.30-2.20 (m,
4H), 1.60-1.50 (m, 4H), 1.30-1.20 (m, 48H), 0.88-0.82 (t, 6H).
31P NMR (CDCl3, 101 MHz) δ 0.03. FAB MS: 1487 (MNa+,
0.8). Anal. Calcd for C84H122NO18P: C, 68.86; H, 8.40; N, 0.96.
Found: C, 68.82; H, 8.21; N, 1.04.
General procedures were described previously.21
1,2-O-Dip a lm itoyl-sn -glycer ol. To a solution of 3-O-(p-
methoxybenzyl)-sn-glycerol21 (0.89 g, 4.2 mmol), DMAP (0.24
g, 2 mmol), and palmitic acid (2.18 g, 8.5 mmol) in dry CH2Cl2
(50 mL) was added a solution of DCC (1.85 g, 9 mmol) in 40
mL of dry CH2Cl2 (20 mL). The resulting mixture was stirred
at 24 °C for 12 h. The mixture was filtered through Celite,
concentrated in vacuo, and purified on SiO2 (EtOAc/hexane,
1:3) to give 2.54 g of product (88% yield). TLC (SiO2) EtOAc/
hexane (1:2), Rf ∼ 0.8.
A mixture of the PMB ether (2.0 g, 2.9 mmol) and 5% Pd/C
(250 mg) in 50 mL of EtOH/AcOH (19:1) was shaken under
H2 at atmosphere for 3 h. The catalyst was removed by
filtration on Celite (1 g), concentrated in vacuo, and purified
on SiO2 (EtOAc/hexane, 1:1) to give 1.48 g of product (98%
yield) as a white solid. TLC (SiO2) EtOAc/hexane (1:2), Rf ∼
(28) Beld, A.; Claesen, C. C. A.; Roersma, E. S.; Schippers, W. J .
M.; Keizer, L. M.; Tesser, G. I. Recl. Trav. Chim. Pays-Bas 1984, 103,
196-202.
(29) (a) Prestwich, G. D. Acc. Chem. Res. 1996, 29, 503-513. (b)
Prestwich, G. D.; Dorma´n, G.; Elliott, J. T.; Marecak, D. M.; Chaudhary,
A. Photochem. Photobiol. 1996, in press.

8646 J . Org. Chem., Vol. 61, No. 24, 1996
Gu and Prestwich
2,6-O-Bis(ben zyloxym eth yl)-3-O-(ben zyloxy)-4,5-O-bis-
(p-m eth oxyben zyl)-D-myo-in ositol 1-O-(1,2-O-Dipalm itoyl)-
sn -glycer yl 2-Cya n oeth yl P h osp h a te (8b). To a mixture
of 6 (64 mg, 0.083 mmol) and 1H-tetrazole (7 mg, 0.10 mmol)
in dry CH2Cl2 (1 mL) was added a solution of 2,6-O-bis-
(benzyloxymethyl)-3-O-(benzyloxy)-4,5-O-bis(p-methoxybenzyl)-
D-myo-inositol 7b (60 mg, 0.075 mmol) in CH2Cl2 (1 mL). After
stirring at 24 °C for 30 min under nitrogen, the reaction
mixture was cooled to -40 °C and a solution of m-CPBA (21
mg, 0.12 mmol) in 2 mL of CH2Cl2 was added. After 20 min,
the mixture was diluted with CH2Cl2 (20 mL), washed with
10% aqueous NaHCO3 solution (20 mL) and brine (20 mL),
dried over MgSO4, concentrated in vacuo, and purified on SiO2
(EtOAc/hexane, 1:2) to give 101 mg of product 8b (93% yield)
as a colorless syrup. TLC (SiO2) EtOAc/hexane (1:2), Rf ∼ 0.25.
1H NMR (CDCl3, 300 MHz) δ 7.45-7.20 (m, 23H), 6.85-6.75
(m, 4H), 5.15-5.12 (m, 1H), 4.85-4.50 (m, 11H), 4.42-4.20
(m, 6H), 3.79 (s, 6H), 3.75-3.60 (t, 2H), 2.45 (t, 2H), 2.35-
2.25 (m, 4H), 1.55-1.50 (m, 4H), 1.30-1.10 (m, 48H), 0.88-
0.75 (t, 6H, J ) 7.5 Hz). 31P NMR (CDCl3, 101 MHz) δ 0.03.
FAB MS: 1457 (MNa+, 2.4). Anal. Calcd for C83H120NO17P:
C, 69.46; H, 8.43; N, 0.98. Found: C, 69.10; H, 8.80; N, 1.22.
2,6-O-Bis(ben zyloxym eth yl)-D-m yo-in ositol 1-O-(1,2-O-
Dip a lm itoyl)-sn -glycer yl 2-Cya n oeth yl P h osp h a te (9a ).
A mixture of 8a (100 mg, 0.068 mmol) and DDQ (45.5 mg, 0.21
mmol) in 50 mL of CH2Cl2-H2O (100:0.5) was stirred at 24
°C for 3 h. The resulting solution was diluted with 50 mL of
CH2Cl2 and washed with NaHCO3 (10%) (2 × 30 mL) and
brine (40 mL), dried over MgSO4, and concentrated in vacuo.
The residue was purified on SiO2 (EtOAc/hexane, 5:1) to give
64 mg of product 9a (85% yield) as a white solid:. TLC (SiO2)
EtOAc/hexane (5:1), Rf ∼ 0.20. 1H NMR (CDCl3, 300 MHz) δ
7.30-7.21 (m, 10H), 5.20 (br s, 1H), 4.90-4.50 (m, 8H),
4.45-4.05 (m, 4H), 3.75 (t, 2H), 3.55 (m, 2H), 2.65 (t, 2H),
2.30-2.25 (m, 4H), 1.66-1.45 (m, 4H), 1.35-1.10 (m, 48H),
0.80 (t, 6H). 31P NMR (CDCl3, 101 MHz) δ 0.03. FAB MS:
1127 (MNa+, 7.7). Anal. Calcd for C60H98NO15P: C, 65.24;
H, 8.95; N, 1.27. Found: C, 65.12; H, 8.98; N, 1.26.
1.14 mmol) in dry CH2Cl2 (2 mL) was added a solution of
dibenzyl N,N-diisopropylphosphoramidite (79 mg, 0.23 mmol)
in CH2Cl2 (2 mL). After stirring at 24 °C for 1 h, the reaction
mixture was cooled to -40 °C, and a solution of m-CPBA (60
mg, 0.35 mmol) in CH2Cl2 (2 mL) was added. After an
additional 1 h, the mixture was diluted with CH2Cl2 (20 mL),
washed once with 10% aqueous NaHCO3 solution (20 mL) and
brine (20 mL), dried over Na2SO4, concentrated in vacuo, and
purified on SiO2 (EtOAc/hexane, 1:2) to give 92 mg of product
10b (98% yield) as a colorless syrup. TLC (SiO2) EtOAc/
hexane (1:2), Rf ∼ 0.35. 1H NMR (CDCl3, 300 MHz) δ 7.45-
7.05 (m, 35H), 5.20-4.75 (m, 16H), 4.65-3.75 (m, 8H), 3.50-
3.45 (t, 2H), 2.42 (t, 2H), 2.25-2.10 (m, 4H), 1.60-1.50 (m,
4H), 1.35-1.10 (m, 48H), 0.88-0.85 (t, 6H, J ) 7.0 Hz). 31P
NMR (CDCl3, 101 MHz) δ 0.045, -0.20, -0.65. FAB MS: 1737
(MNa+, 6.6). Anal. Calcd for C95H130NO21P3: C, 66.52; H, 7.64;
N, 0.82. Found: C, 66.82; H, 7.66; N, 1.04.
3,4,5-Tr i-O-p h osp h or yl-D-m yo-in osit ol 1-O-(1,2-O-Di-
p a lm itoyl)-sn -glycer yl 3-Am in op r op yl P h osp h a te So-
d iu m Sa lt (5a ). A mixture of 10a (60 mg, 0.032 mmol),
NaHCO3 (16 mg, 0.19 mmol), and Pd/C (10%) (120 mg) in 20
mL of t-BuOH/H2O (6:1) was shaken under H2 (51 psi) for 21
h. The catalyst was removed by filtration over 1 g of Celite,
and the filter was washed thoroughly with water (20 mL) and
EtOH (20 mL). The combined filtrates were lyophilized to give
30 mg of product 5a (80% yield) as a solid, which was positive
to ninhydrin. TLC (SiO2) CH2Cl2/MeOH/concd NH4OH (5:4:
1), Rf ∼ 0.70. 1H NMR (D2O, 300 MHz) δ 5.20-5.00 (m, 1H),
4.40-4.10 (m, 4H), 4.06-3.60 (m, 2H), 3.15 (m, 2H), 2.20-
2.10 (m, 4H), 1.78 (m, 2H), 1.50 (m, 4H), 1.30-1.10 (m, 48H),
0.88-0.75 (t, 6H). 31P NMR (D2O, 101 MHz) δ 8.1, 7.8, 5.1,
1.1. Mass (MALDI-TOF) 1106 (M+ - 1); 1128 (MNa+ - 1);
calcd: 1106.
4,5-Di-O-p h osp h or yl-D-m yo-in ositol 1-O-(1,2-O-Dip a lm -
itoyl)-sn -glycer yl 3-Am in op r op yl P h osp h a te Sod iu m Sa lt
(5b). A mixture of 10b (72 mg, 0.045 mmol), NaHCO3 (15 mg,
0.18 mmol), and Pd/C (10%) (100 mg) in 20 mL of t-BuOH/
H2O (6:1) was shaken under H2 (51 psi) for 22 h. The catalyst
was removed by filtration over 1 g of Celite, and the filter was
washed thoroughly with water (20 mL) and EtOH (20 mL).
The combined filtrates were lyophilized to give 35 mg of crude
product 5b (72% yield) as a solid, which was positive to
ninhydrin. TLC (SiO2) CH2Cl2/MeOH/concd NH4OH (5:4:1),
Rf ∼ 0.80. 1H NMR (D2O, 300 MHz) δ 5.15-5.00 (s, 1H), 4.40-
4.15 (m, 4H), 4.00-3.90 (m, 2H), 3.20 (m, 2H), 2.15-2.05 (m,
4H), 1.78 (m, 2H), 1.60-1.45 (m, 4H), 1.40-1.05 (m, 48H),
0.85-0.70 (t, 6H). 31P NMR (D2O, 101 MHz) δ 7.01, 6.60, 0.8.
Mass (MALDI-TOF), 1027 (M+ - 1); 1049 (MNa+ - 1), calcd:
1027.
2,6-O-Bis(ben zyloxym eth yl)-3-O-(ben zyloxy)-D-myo-in os-
it ol 1-O-(1,2-O-Dip a lm it oyl)-sn -glycer yl 2-Cya n oet h yl
P h osp h a te (9b). A mixture of 8b (90 mg, 0.062 mmol) and
DDQ (32.5 mg, 0.15 mmol) in 6 mL of CH2Cl2-H2O (100:0.5)
was stirred at 24 °C for 3 h. The resulting solution was diluted
with 50 mL of CH2Cl2 and washed with NaHCO3 (10%) (2 ×
30 mL) and brine (40 mL), dried over MgSO4, and concentrated
in vacuo. The residue was purified on SiO2 (EtOAc/hexane,
5:1) to give 71 mg of product 9b (95% yield) as a white solid.
TLC (SiO2) EtOAc/hexane (5:1), Rf ∼ 0.40. 1H NMR (CDCl3,
300 MHz) δ 7.35-7.20 (m, 15H), 5.15 (br, s, 1H), 5.12-4.55
(m, 8H), 4.44-3.85 (m, 8H), 2.66 (t, 2H), 2.25 (m, 4H), 1.55-
3,4,5-Tr i-O-p h osp h or yl-D-m yo-in osit ol 1-O-(1,2-O-Di-
palm itoyl)-sn -glycer yl (3-BZDC-am ido)pr opyl P h osph ate
Sod iu m Sa lt (11a ). PIP3 derivative 5a (10 mg, 9 µmol) was
suspended in a solution of 0.2 M Et3NHCO3 buffer, pH 8.8 (0.8
mL), and DMF (0.8 mL) and stirred at 24 °C for 3 h until a
clear solution was obtained. To the resulting solution was
added BZDC-NHS27 (5 mg, 0.014 mmol) in DMF (0.2 mL),
and the mixture was stirred for 16 h at 24 °C. Solvents were
evaporated in vacuo, and the residue was washed with acetone
(4 × 1 mL) to remove most of the unreacted or hydrolyzed
BZDC derivatives. The residue was then dissolved in 1 mL
of 0.1 M Et3NHCO3 buffer, pH 8.8, and loaded onto a DEAE-
cellulose column (3 cm × 0.5 cm i.d.) preequilibrated with same
buffer. The column was eluted using a linear gradient of
Et3NHCO3 buffer (pH 8.8) from 0.1 M to 1.2 M, and the product
was eluted out at a concentration between 1.0 to 1.2 M. The
fractions were collected and concentrated to give 11 mg of
product 11a (91% yield) as a solid. 1H NMR (D2O, 300 MHz)
δ 7.72-7.35 (m, 9H), 4.40-3.50 (m, 10H), 3.15-3.00 (m, 4H),
2.50-2.40 (m, 4H), 2.20-2.10 (m, 4H), 1.80-1.70 (m, 2H),
1.45 (m, 4H), 1.30-1.10 (m, 48H), 0.82 (t, 6H, J ) 8.0 Hz). 31
P
NMR (CDCl3, 101 MHz) δ -0.58. FAB MS: 1217 (MNa+, 4.4).
2,6-O-Bis(ben zyloxym eth yl)-3,4,5-O-tr is(d iben zylp h os-
p h or yl)-D-m yo-in ositol 1-O-(1,2-O-d ip a lm itoyl)-sn -glyc-
er yl 2-Cya n oeth yl P h osp h a te (10a ). To a mixture of 9a
(64 mg, 0.057 mmol) and 1H-tetrazole (40 mg, 0.57 mmol) in
dry CH2Cl2 (1 mL) was added a solution of dibenzyl N,N-
diisopropylphosphoramidite (117 mg, 0.34 mmol) in CH2Cl2 (2
mL). After stirring at 24 °C for 1 h, the reaction mixture was
cooled to -40 °C and a solution of m-CPBA (60 mg, 0.35 mmol)
in CH2Cl2 (2 mL) was added. After an additional 1 h, the
mixture was diluted with CH2Cl2 (20 mL), washed once with
10% aqueous NaHCO3 solution (20 mL) and brine (20 mL),
dried over Na2SO4, concentrated in vacuo, and purified on SiO2
(EtOAc/hexane, 1:3) to give 79 mg of product 10a (73% yield)
as a colorless syrup. TLC (SiO2) EtOAc/hexane (2:1), Rf ∼ 0.32.
1H NMR (CDCl3, 300 MHz) δ 7.45-7.05 (m, 40H), 5.15-4.60
(m, 22H), 4.55-4.05 (m, 6H), 2.42 (t, 2H), 2.25-2.10 (m, 4H),
1.60-1.50 (m, 4H), 1.40-1.20 (48H), 0.88-0.85 (t, 6H). 31P
NMR (CDCl3, 101 MHz) δ 0.15, -0.03, -0.47, -1.06. FAB
MS: 1907 (MNa+, 4.7). Anal. Calcd for C102H137NO24P4: C,
64.97; H, 7.33; N, 0.74. Found: C, 64.82; H, 7.36; N, 0.92.
2,6-O-Bis(ben zyloxym eth yl)-3-O-(ben zyloxy)-4,5-O-bis-
(d iben zylp h osp h or yl)-D-m yo-in ositol 1-O-(1,2-O-Dip a lm i-
toyl)-sn -glycer yl 2-Cya n oeth yl P h osp h a te (10b). To a
mixture of 9b (70 mg, 0.058 mmol) and 1H-tetrazole (80 mg,
1.50-1.45 (m, 4H), 1.30-1.10 (m, 48H), 0.88-0.82 (t, 6H). 31
P
NMR (D2O, 101 MHz) δ 7.2, 5.6, 5.1, 1.9. Mass (MALDI-TOF),
1342 (M+ - 1); 1364 (M+ + Na - 1); 1386 (M+ + 2Na - 1);
1408 (M+ + 3Na - 1); calcd: 1342.
4,5-Di-O-p h osp h or yl-D-m yo-in ositol 1-O-(1,2-O-Dip a lm -
itoyl)-sn -glycer yl (3-BZDC-a m id o)p r op yl P h osp h a te So-
d iu m Sa lt (11b). PIP2 analogue 5b (5 mg, 4.9 µmol) was

PIP2 and PIP3 Triester Synthesis
J . Org. Chem., Vol. 61, No. 24, 1996 8647
suspended in a solution of 0.2 M Et3NHCO3 buffer, pH 8.8 (0.6
mL), and DMF (0.6 mL), and stirred at 24 °C for 1 h. To the
resulting solution was added BZDC-NHS27 (2.6 mg, 7.4 µmol)
in DMF (0.2 mL), and the mixture was stirred for 16 h at 24
°C. Solvents were evaporated in vacuo, and the residue was
washed with acetone (4 × 1 mL) to remove most of soluble
BZDC derivatives. The residue was then dissolved in 1 mL
of 0.1 M Et3NHCO3 buffer, pH 8.8, and loaded onto a DEAE-
cellulose column (3 cm × 0.5 cm i.d.) preequilibrated with the
same buffer. The column was eluted with a gradient of Et3-
NHCO3 buffer, pH 8.8, from 0.1 to 1.2 M concentration. The
product was eluted at a concentration between 0.9 to 1.0 M.
The fractions were collected and dried to give 5.3 mg of product
11b (85% yield) as a solid. TLC (SiO2) CH2Cl2/MeOH/concd
NH4OH (5:4:1), Rf ∼ 0.65. 1H NMR (D2O, 300 MHz) δ 7.82-
7.30 (m, 9H), 5.20 (m, 1H), 4.40-4.10 (m, 4H), 4.06-3.60 (m,
7H), 3.12 (m, 2H), 2.20-2.05 (m, 4H), 1.75 (m, 2H), 1.50-1.35
(m, 4H), 1.30-1.10 (m, 48H), 0.88-0.80 (t, 6H, J ) 7.0 Hz).
31P NMR (D2O, 101 MHz) δ 8.1, 7.2, 3.1. Mass (MALDI-TOF),
1263 (M+ - 1); calcd: 1263.
for 12 h. Solvents were evaporated under reduced pressure,
and the residue was then purified on SiO2 (MeOH/CH2Cl2, 1:1)
to give 48 mg of product 12b (93% yield) as a colorless syrup.
TLC (SiO2) MeOH/CH2Cl2 (1:1), Rf ∼ 0.60. 1H NMR (CDCl3,
300 MHz) δ 7.40-7.10 (m, 35H), 5.18 (m, 1H), 5.02-4.75 (m,
15H), 4.70-3.75 (m, 10H), 3.55-3.40 (m, 3H), 2.90 (m, 2H),
1.60-1.50 (m, 4H), 1.38-1.12 (m, 60H), 0.95-0.85 (t, 6H, J )
7.0 Hz). 31P NMR (CDCl3, 101 MHz) δ 0.26, -0.24, -0.25.
3,4,5-Tr i-O-p h osp h or yl-D-m yo-in osit ol 1-O-(1,2-O-d i-
p a lm itoyl)-sn -glycer yl P h osp h a te Sod iu m Sa lt (13a ). A
mixture of 12a (148 mg, 0.082 mmol), NaHCO3 (12.6 mg, 0.15
mmol), and Pd/C (10%) (80 mg) in 15 mL of t-BuOH/H2O (6:1)
was shaken under H2 (51 psi) for 6 h. Catalyst was removed
by filtration over 1 g of Celite, and the filter was washed with
0.2 M NH4OH (2 × 20 mL). The combined filtrates were
lyophilized to provide 84 mg of product 13a (87% yield) as a
white solid. 1H NMR (D2O, 300 MHz) δ 5.18-5.00 (m, 1H),
4.56-4.00 (m, 8H), 3.63-3.40 (m, 2H), 2.46-2.27 (m, 4H),
1.96-1.62 (m, 4H), 1.58-1.02 (m, 48H), 0.93 (t, 6H). 31P NMR
(D2O, 101 MHz) δ 8.45, 7.52, 6.38, 3.66. Mass (MALDI-TOF),
1049 (M+ - 1); calcd: 1049.
4,5-Di-O-p h osp h or yl-D-m yo-in ositol 1-O-(1,2-O-Dip a lm -
itoyl)-sn -glycer yl P h osp h a te Sod iu m Sa lt (13b). A mix-
ture of 12b (47 mg, 0.030 mmol), NaHCO3 (12.6 mg, 0.15
mmol), and Pd/C (10%) (80 mg) in 15 mL of t-BuOH/H2O (6:1)
was shaken under H2 (51 psi) for 6 h. Catalyst was removed
by filtration over 1 g of Celite, and the filter was washed with
0.2 M NH4OH (2 × 20 mL). The combined filtrates were
lyophilized to give 25 mg of product 13b (83% yield) as a white
solid. 1H NMR (D2O, 300 MHz) δ 5.20-5.00 (m, 1H), 4.40-
4.10 (m, 4H), 4.06-3.60 (m, 6H), 2.30-2.10 (m, 4H), 1.85-
1.75 (m, 4H), 1.55-1.05 (m, 48H), 0.85 (t, 6H). 31P NMR (D2O,
101 MHz) δ 8.9, 8.1, 4.5. Mass (MALDI-TOF), 970 (M+ - 1);
992 (MNa+ - 1); calcd: 970.
2,6-O-Bis(ben zyloxym eth yl)-3,4,5-O-tr is(d iben zylp h os-
p h or yl)-D-m yo-in ositol 1-O-(1,2-O-Dip a lm itoyl)-sn -glyc-
er yl Diisop r op yleth yla m m on iu m P h osp h a te (12a ).
A
mixture of 10a (150 mg, 0.080 mmol) and diisopropyleth-
ylamine (100 µL) in dry CH3CN (4 mL) was heated to 60 °C
with stirring for 12 h. Solvents were evaporated under
reduced pressure, and the residue was then purified on SiO2
(MeOH/CH2Cl2, 1:1) to give 148 mg of product 12a (97% yield)
as a colorless syrup. TLC (SiO2) MeOH/CH2Cl2 (1:1), Rf ∼ 0.25.
1H NMR (CDCl3, 300 MHz) δ 7.30-7.15 (m, 40H), 5.21 (m,
1H), 5.04-4.72 (m, 21H), 4.70-3.70 (m, 8H), 3.50-3.47 (m,
3H), 2.92-2.90 (m, 2H), 1.60-1.50 (m, 4H), 1.38-1.12 (m,
60H), 0.95-0.85 (t, 6H, J ) 7.0 Hz). 31P NMR (CDCl3, 101
MHz) δ 0.33, 0.21, -0.45, -1.59. FAB MS: 1855 (MNa+, 5.7).
2,6-O-Bis(ben zyloxym eth yl)-3-O-(ben zyloxy)-4,5-O-bis-
(d iben zylp h osp h or yl)-D-m yo-in ositol 1-O-(1,2-O-Dip a lm i-
toyl)-sn -glycer yl Diisop r op yleth yla m m on iu m P h osp h a te
(12b). A mixture of 10b (52 mg, 0.033 mmol) and diisopro-
pylethylamine (60 µL) in CH3OH (4 mL) was stirred at 24 °C
Ack n ow led gm en t. We thank the NIH for grant NS
29632 for support of this work.
J O961226G