Synthesis of photoactivatable 1,2-O-diacyl-sn-glycerol derivatives of 1-L-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PtdInsP2) and 3,4,5-trisphosphate (PtdInsP3)

Article abstract of DOI:10.1021/jo960895r

Photoactivatable analogues of 1-L-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PtdIns(4,5)P₂ or PtdInsP₂) and the corresponding 3,4,5-trisphosphate (PtdIns(3,4,5)P₃ or PtdInsP₃) were prepared from the two chira

Full text of DOI:10.1021/jo960895r

J . Org. Chem. 1996, 61, 6305-6312
6305
Syn th esis of P h otoa ctiva ta ble 1,2-O-Dia cyl-sn -glycer ol Der iva tives
of 1-L-P h osp h a tid yl-D-m yo-in ositol 4,5-Bisp h osp h a te (P td In sP 2) a n d
3,4,5-Tr isp h osp h a te (P td In sP 3)
J ian Chen, Adam A. Profit, and Glenn D. Prestwich*,†
Department of Chemistry, University at Stony Brook, Stony Brook, New York 11794-3400
Received May 15, 1996X
Photoactivatable analogues of 1-L-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PtdIns(4,5)P2 or
PtdInsP2) and the corresponding 3,4,5-trisphosphate (PtdIns(3,4,5)P3 or PtdInsP3) were prepared
from the two chiral precursors, methyl R-D-glucopyranoside and 1,2-isopropylidene-sn-glycerol. Two
key synthetic transformations included the Ferrier rearrangement reaction to construct the optically-
pure inositol skeleton and the sequential acylation of the primary and secondary hydroxyl groups
on the glycerol derivatives. The sn-1-O-(6-aminohexanoyl) PtdInsP2 and PtdInsP3 derivatives were
further modified to contain benzophenone photophores in unlabeled and high specific activity
tritium-labeled forms.
In tr od u ction
PtdIns(3,4,5)P3, respectively. PI 3-K is a heterodimer
with a native molecular weight of 190 kDa and consists
of an 85 kDa regulatory subunit (p85) and a 110 kDa
catalytic subunit (p110).13 The p85 subunit has been
found to contain two SH2 domains and one SH3 domain,
while kinase activity has been demonstrated to reside
exclusively in the p110 subunit.14-16 PI 3-K is translo-
cated from the cytosol to the plasma membrane where it
is recruited to activated protein tyrosine kinase receptors
(PTKs) upon stimulation by various growth factors.12,17,18
This association is believed to occur through phospho-
tyrosine-SH2 domain interactions and serves to bring the
enzyme in proximity to its substrate(s), facilitating the
production of second messenger molecules.
Hydrolysis of PtdInsP2 by phospholipase C (PLC)
generates the now ubiquitous second messenger
1-4
Ins(1,4,5)P3
(Scheme 1). PtdInsP2 itself functions in
the recruitment of proteins to membranes5 via pleckstrin
homology (PH) domains.6 Indeed, the PH domain of PLC
allows membrane localization via the 4,5-bisphosphate
of PtdInsP2,7 while the catalytic domain cleaves the P-O
bond of an adjacent PtdInsP2 molecule. Three-dimen-
sional structures of the â-spectrin-Ins(1,4,5)P3 complex
and the PLC PH domain-complex have verified the
importance of the 4,5-bisphosphate interaction with
hydrogen bonding and protonated basic residues.8 In
addition, PtdInsP2 sequesters profilin to the inner surface
of the phospholipid bilayer, facilitating the polymeriza-
tion of actin.5,9 Release of profilin following PtdInsP2
hydrolysis suppresses further cytoskeletal reorganization.
In addition to its role as Ins(1,4,5)P3 precursor and
membrane protein anchor, 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 sys-
tem.11,12 PI 3-K catalyzes the transfer of the γ phosphate
of ATP to the D-3 hydroxyl of PtdIns, PtdIns(4)P, and
PtdIns(4,5)P2 to form PtdIns(3)P, PtdIns(3,4)P2, and
Recent studies have identified PtdIns(3,4,5)P3 as a
putative second messenger molecule acting independ-
ently of the classical inositol polyphosphate pathway.19-21
Although the molecular action of PtdIns(3,4,5)P3 is
unclear, several putative targets for this phosphoinositide
have been reported: the serine/threonine kinase Akt/
PKB,22,23 protein kinase C,24 and a mediator of the
(12) Stephens, L. R.; J ackson, T. R.; Hawkins, P. T. Biochim.
Biophys. Acta 1993, 1179, 27-75.
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Milfay, D.; Fried, V. A.; Williams, L. T. Cell 1991, 65, 75-82.
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R.; Panayotou, G.; Ruiz-Larrea, F.; Thompson, A.; Totty, N., F.; Hsuan,
J . J .; Courtneidge, S. A.; Parker, P. J .; Waterfield, M. D. Cell 1992,
70, 419-429.
† After Aug 1, 1996, address correspondence to: Department of
Medicinal Chemistry, The University of Utah, 308 Skaggs Hall, Salt
Lake City, UT 84112. Phone: (801) 581-7063. Fax: (801) 581-7087.
E-mail: gprestwich@deans.pharm.utah.edu.
X Abstract published in Advance ACS Abstracts, August 1, 1996.
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S0022-3263(96)00895-X CCC: $12.00 © 1996 American Chemical Society

6306 J . Org. Chem., Vol. 61, No. 18, 1996
Chen et al.
Sch em e 1. P h osp h a tid ylin ositol 4,5-Bisp h osp h a te (P td In sP 2) a s a Su bstr a te for P h osp h olip a se C a n d for
P h osp h oin ositid e 3-Kin a se
phosphorylation of platelet p47 (pleckstrin).25 In addi-
tion, a new 46-kDa protein, centaurin,26 has been purified
using a P-1-tethered Ins(1,3,4,5)P4 affinity column27 and
labeled by a photolabile Ins(1,3,4,5)P4 analogue28,29 and
appears to be a PtdInsP3 target.
In order to clarify the physiological role of the inositol
polyphosphate and phosphoinositide polyphosphates, we
have pursued a program of preparing P-1-tethered inosi-
tol polyphosphate derivatives.30 Scheme 2 illustrates the
parent ligands Ins(1,4,5)P3 and Ins(1,3,4,5)P4 and their
respective P-1-tethered photoaffinity reagents.28,31,32 The
selection of the benzophenone moiety optimized ease of
biochemical use, chemical stability, high specific activity
compatible with protein sequencing, and most impor-
tantly, nearly quantitative photocovalent modification of
the target proteins.33,34 For example, benzoyldihydro-
cinammoyl-IP3 (BZDC-IP3) has been employed to identify
the active site of the rat brain type I InsP3 receptor31 and
the PH domain of PLC δ1.35 We recognized that the lipid
group appended to the aminopropyl tether on the P-1
phosphate resembled the lipidic moiety provided by a
diacylglycerol. In several cases, proteins labeled by
[3H]BZDC-IP3 and [3H]BZDC-IP4 probes are in fact high
affinity targets for the PtdInsP2 and PtdInsP3 ligands,
respectively. Thus, we designed analogues that more
accurately mimic the PtdInsP2 and PtdInsP3 structures
by incorporating the photoactivatable BZDC amide moi-
ety in the backbone of the 1-O-acyl chain in unlabeled or
radiolabeled form.36 In this paper, we describe the
synthesis of PtdIns(4,5)P2 analogues 13a and 13b and
PtdIns(3,4,5)P3 analogues 13c and 13d pursuant to this
strategy.
Resu lts a n d Discu ssion
(25) 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.
(26) Hammonds-Odie, L. P.; J ackson, T. R.; Profit, A. A.; Blader, I.
J .; Turck, C.; Prestwich, G. D.; Theibert, A. B. J . Biol. Chem. 1996, in
press.
The convergent synthetic strategy (Schemes 3 and 4)
combined a simple but concise route to the mixed 1,2-O-
diacyl-sn-glycerols37 with differentially-protected inter-
mediates obtained via Ferrier rearrangements of glucose
derivatives.30 Thus, protection of (+)-1,2-O-isopropyl-
idene-sn-glycerol (1) was performed with p-methoxyl-
(27) Theibert, A. B.; Estevez, V. A.; Ferris, C. D.; Danoff, S. K.;
Barrow, R. K.; Prestwich, G. D.; Snyder, S. H. Proc. Natl. Acad. Sci.,
U.S.A. 1991, 88, 3165-3169.
(28) Estevez, V. A.; Prestwich, G. D. J . Am. Chem. Soc. 1991, 113,
9885-9887.
(33) Dorma´n, G.; Prestwich, G. D. Biochemistry 1994, 33, 5661-
5673.
(34) Prestwich, G. D.; Dorma´n, G.; Elliott, J . T.; Marecak, D. M.;
Chaudhary, A. Photochem. Photobiol. 1996, in press.
(35) Tall, E.; Dorma´n, G.; Garcia, P.; Shah, S.; Prestwich, G. D.;
Rebecchi, M. J . J . Biol. Chem. 1996, submitted.
(36) Olszewski, J . D.; Dorma´n G.; Elliott, J . T.; Hong, Y.; Ahern, D.
G.; Prestwich, G. D. Bioconjugate Chem. 1995, 6, 395-400.
(37) Martin, S. F.; J osey, J . A.; Wong, Y.-L.; Dean, D. W. J . Org.
Chem. 1994, 59, 4805-4820.
(29) Theibert, A. B.; Estevez, V. A.; Mourey, R. J .; Marecek, J . F.;
Barrow, R. K.; G. D. Prestwich; Snyder, S. H. J . Biol. Chem. 1992,
267, 9071-9079.
(30) Prestwich, G. D. Acc. Chem. Res. 1996, in press.
(31) Mourey, R. J .; Estevez, V. A.; Marecek, J . F.; Barrow, R. K.;
Prestwich, G. D.; Snyder, S. H. Biochemistry 1993, 32, 1719-1726.
(32) Prestwich, G. D.; Marecek, J . F.; Mourey, R. J .; Theibert, A.
B.; Ferris, C. D.; Danoff, S. K.; Snyder, S. H. J . Am. Chem. Soc. 1991,
113, 1822-1825.

PtdInsP2 and PtdInsP3 Photoaffinity Labels
J . Org. Chem., Vol. 61, No. 18, 1996 6307
Sch em e 2. Str u ctu r es of In ositol P olyp h osp h a tes (In sP n s), In sP n P h otop r obes, P h osp h oin ositid e
P olyp h osp h a tes P td In s(4,5)P 2 a n d P td In s(3,4,5)P 3, a n d P td In sP n P h otop r obes
Sch em e 3a
a
Reagents: (a) PMBCl/NaH, DMF, rt, 1 h; (b) 5% (wt) p-TsOH, MeOH, rt, 1 h; (c) CbzNHC5H10COOH, DMAP, DCC, CH2Cl2, 0 °C,
overnight; (d) stearic acid or oleic acid, DMAP, DCC, CH2Cl2, rt, overnight; (e) DDQ, wet CH2Cl2, rt, overnight; (f) (BnO)P(Cl)(NPr2-i),
i-Pr2NEt, CH2Cl2, 0 °C to rt, 2 h.
benzyl chloride (PMB-Cl)38 to give PMB ether 2, which
was transketalized (10 mol % of p-TsOH in MeOH) to
the 1,2-diol 3 in 83% isolated yield.39 The mixed 1,2-O-
diacylated glycerol was prepared by DCC/DMAP-medi-
ated sequential acylation.38 Thus, diol 3 was condensed
with 6-(N-Cbz-amino)hexanoic acid in the presence of
DCC and DMAP at 0 °C to give three products. The
desired 1-acylated compound 4 was the main product
obtained in 62% isolated yield; this regioisomer was
readily distinguished from the 2-acylated isomer by the
1H resonance of the methine proton (δ ) 5.2 ppm (m) for
the undesired 2-acyl material). Esterification of the
remaining secondary hydroxyl with either stearic acid
or oleic acid provided diacylglycerols 5a or 5b in 82% and
92% yield, respectively. The use of oleic acid at this step
set the stage for introduction of tritium into the fatty acyl
group during hydrogenolytic deprotection in the final
step. Removal of PMB with DDQ in wet CH2Cl2 (0.5%
water in volume) to give 1-[6-(N-Cbz-amino)hexanoyl]-
2-stearoylglycerol (6a and 6b) (in 96% and 79% yield)
occurred without any detectable migration of the stearoyl
group from the 2-position to the 3-position. Reaction of
diacylglycerol 6a with (benzyloxy)(N,N-diisopropyl-
amino)chlorophosphine28 gave phosphatidylating reagent
7a in good isolated yield. This reagent was found to be
unstable even when stored at -20 °C because of facile
air-oxidation detected by 31P NMR. Thus, the glyceryl
(38) Hebert, N.; Beck, A.; Lennox, R. B.; J ust, G. J . Org. Chem. 1992,
57, 1777-1783.
(39) Manley, P. W.; Tuffin, D. T.; Allanson, N. M.; Buckle, P. E.;
Lad, N.; Lai, S. M. F.; Lunt, D. O.; Porter, R. A.; Wade, P. J . J . Med.
Chem. 1987, 30, 1812-1818.

6308 J . Org. Chem., Vol. 61, No. 18, 1996
Chen et al.
Sch em e 4a
a
Reagents: (a) 1H-tetrazole, m-CPBA, -40 °C to rt; (b) DDQ, wet CH2Cl2; (c) (BnO)2PNPr2-i, 1H-tetrazole then m-CPBA; (d) 50 psi H2,
Pd/C, t-BuOH-H2O, NaHCO3; (e) BZDC-NHS or [3H]BZDC-NHS, DMF 0.25 M TEAB, rt, overnight.
phosphoramidite reagent was used immediately after
preparation for coupling to the protected inositol phos-
phate precursor.
overnight, followed by purification on DEAE-cellulose,
gave the cold BZDC derivative 13a in 70% yield. After
the reaction was scaled down to the microgram scale, the
[3H]BZDC derivative 13b was prepared similarly.
In the same way, when reacted with N-hydroxy-
succinimidoyl 6-[(7-nitro-2-oxa-1,3-diazobenz-4-yl)amino]-
hexanoate (NBD aminohexanoic NHS ester), a fluores-
cent PtdInsP2-NBD (14) was obtained. This fluorescent
analogue has been used to demonstrate the effect of a
highly basic peptide in sequestering PtdInsP2 into two-
dimensional lateral domains in phospholipid vesicles.41
Construction of 2,6-bis-O-(benzyloxymethyl)-3-O-ben-
zyl-4,5-bis-O-(4-methoxybenzyl)-myo-inositol (8a ) from
methyl R-D-glucose was accomplished as described for the
synthesis of a P-1-tethered Ins(1,4,5)P3 photoaffinity
label.40 Coupling of intermediate 8a with phospha-
tidylating reagent 7a in the presence of 1H-tetrazole
followed by m-CPBA oxidation gave the protected phos-
phoinositide precursor 9a in 87% yield. Removal of two
PMB groups from compound 9a with DDQ in wet CH2Cl2
gave diol 10a that was further phosphorylated to give
the PtdInsP2 analogue precursor 11a . Deprotection of
the benzyl and BOM groups was carried out at an initial
pressure of 50 psi of H2 in 2-methyl-2-propanol-H2O
containing NaHCO3 using 5% Pd/C as catalyst at rt for
4 h. After removal of the catalyst and concentration in
vacuo, an amorphous solid was obtained. Purification
by Chelex chromatography afforded sodium salt 12a in
78% yield. This solid was partially dissolved in water to
yield a cloudy solution.
The synthetic strategy for the 1-L-phosphatidyl-D-myo-
inositol 3,4,5-trisphosphate analogues parallels that of
our PtdInsP2 compounds, except that the PtdInsP3 prod-
Reaction of the aminohexanoyl PtdInsP2 analogue 12
with N-hydroxysuccinimidyl benzoyldihydrocinnamic acid
ester (BZDC-NHS ester)36 in 1:1 (v/v) DMF-0.25 M
triethylammonium bicarbonate (TEAB) buffer at rt for
(41) Glaser, M.; Wanaski, S.; Buser, C. A.; Boguslavsky, V.; Rash-
idzada, W.; Morris, A.; Rebecchi, M.; Scarlata, S.; Runnels, L. W.;
Prestwich, G. D.; Chen, J .; Aderem, A.; Ahn, J .; McLaughlin, S. J . Biol.
Chem. 1996, in press.
(40) Dorma´n, G.; Chen, J .; Prestwich, G. D. Tetrahedron Lett. 1995,
36, 8719-8722.

PtdInsP2 and PtdInsP3 Photoaffinity Labels
J . Org. Chem., Vol. 61, No. 18, 1996 6309
reagents were dried using standard procedures. FAB HRMS
were performed at the University of California at Riverside,
using NBA matrix to obtain MNa+ as formula peak.
uct requires an inositol intermediate (obtained via a
Ferrier rearrangement) equipped with PMB ethers pro-
tecting the C3, -4, and -5 hydroxyl groups (8b).28 Phos-
phitylation of 8b with glycerol phosphoramidite 7b in the
presence of 1H-tetrazole followed by oxidation with
m-CPBA furnished the protected phosphatidylinositol 9b
in 88% yield. Cleavage of the PMB groups with DDQ
afforded triol 10b in 61% yield, and phosphorylation was
achieved using dibenzyl N,N-diisopropylphosphoramidite
followed by oxidation to give compound 11b (83% yield).
Removal of all benzyl-derived protecting groups was
accomplished by catalytic hydrogenation on 10% Pd/C in
2-methyl-2-propanol/H2O with seven equivalents of NaH-
CO3, giving the PtdInsP3 analogue 12b as a hexasodium
salt. As with the PtdInsP2 analogue, condensation of the
primary amine with BZDC-NHS ester afforded the ben-
zophenone-based photoaffinity probe in both radiolabeled
and nonlabeled forms (13c and 13d ).
Earlier syntheses of a number of phosphatidylinositol
polyphosphates have been reported, and these synthetic
compounds have been employed to verify specific biologi-
cal roles for these ligands. Falck and co-workers de-
scribed the synthesis of the sn-1,2-distearoyl analogue
of PtdInsP3 using (-)-quinic acid as the chiral starting
material for the construction of D-myo-inositol skeleton.42-44
Gou and Chen have completed an analogous synthesis
from enzymatically-resolved 1-D-dicyclohexylidene-myo-
inositol,45 while Bruzik and Kubiak started from the
camphor ketal of myo-inositol.46 Watanabe et al. reported
concise routes to the racemic form of PtdIns(3,4,5)P3 and
recently the optically pure form from the enzymatically
resolved 1-D-1,2-cyclohexylidene-myo-inositol.47,48
The syntheses of modified PtdInsP2 and PtdInsP3
analogues described herein offer three unique features.
First, the Ferrier rearrangement49 of an inexpensive R-D-
glucose derivative provides the enantiomerically-pure
inositol framework. Second, the selective esterification
of the sn-1 position with an amino-functionalized ester
allows introduction of a variety of reporter groups. Third,
the syntheses described herein represent the first high
specific activity, tritium-labeled photoactivatable ana-
logues of the phosphoinositide polyphosphates available
for exploration of lipid binding regions of PtdInsP2 and
PtdInsP3 biochemical targets. These photoactivatable
acyl-modified analogues, as well as a novel series of
phosphotriester head group-modified analogues50 of Pt-
dInsP2 and PtdInsP3, have been evaluated in biochemical
experiments and appear to be readily recognized as
PtdInsP2 or PtdInsP3 mimics. These results will be
reported elsewhere in due course.
3-(4-Meth oxyben zyl)-sn -glycer ol (3). To a solution of 1,2-
O-isopropylidene-sn-glycerol (1, 1000 mg, 7.6 mmol) in 100 mL
of dry DMF was added NaH (455 mg, 60%, 11.4 mmol),
followed by addition of p-methoxybenzyl chloride (1,188 mg,
9.1 mmol). The mixture was stirred at rt for 1 h (TLC, 2 Rf )
0.82, 33% ethyl acetate/hexane). Excess NaH was destroyed
by dropwise addition of MeOH, and CH2Cl2 was added to
extract. The CH2Cl2 layer was washed with brine and dried
over Na2SO4. After concentration, an oil was obtained and
redissolved in 20 mL of MeOH containing p-toluenesulfonic
acid monohydrate (70 mg, 0.05 equiv, 0.37 mmol). The
mixture was stirred at rt for 1 h (TLC, 3 Rf ) 0.24, 75% ethyl
acetate/hexane). Next, NaHCO3 (100 mg) was added, and the
solvent was evaporated. The residue was purified by flash
chromatography on silica gel (“purified on SiO2”) with 75%
ethyl acetate/hexane to give 1.322 g of 3-(4-methoxybenzyl)-
sn-glycerol (3) (83% yield from 1): mp 40-42 °C (lit.6 mp 40-
41 °C). 1H NMR (CDCl3, 250 MHz) δ: 7.25, 7.22, 6.89, 6.85
(4s, 4H, phenyl), 4.46 (s, 2H, PMBCH2O), 3.90-3.75 (s, 1H,
H-2), 3.80 (s, 3H, MeO), 3.64-3.45 (m, 4H, H-1 and H-3), 2.86
(s and br, 1H, OH), 2.42 (s and br, 1H, OH) ppm. 13C NMR
(CDCl3, 63 MHz) δ: 129.46, 113.91, 73.24, 71.48, 70.64, 64.01,
55.29 ppm.
1-[6-(N-Cbz-a m in o)h exa n oyl]-3-(4-m eth oxyben zyl)-sn -
glycer ol (4). To a solution of the 3-(4-methoxybenzyl)-sn-
glycerol (3, 772 mg, 3.64 mmol) and 6-[(carbobenzyloxy)amino]-
hexanoic acid (927 mg, 3.5 mmol) in 40 mL of dry CH2Cl2 at 0
°C was added dropwise a solution of DCC (865 mg, 4.2 mmol)
and DMAP (512 mg, 4.2 mmol) in 10 mL of dry CH2Cl2. The
solution was stirred at 0 °C for 16 h and concentrated. The
residue was redissolved in 20 mL of dry ethyl acetate, filtered,
concentrated in vacuo, and purified on SiO2 (25% ethyl acetate
in CH2Cl2, Rf ) 0.7; the Rf of the other isomer is 0.65, and the
Rf of the diester is 0.95) giving 1.041 g (62% yield) of glycerol
derivative 4. 1H NMR (CDCl3, 250 MHz) δ: 7.40-7.30 (m,
5H, Ph), 7.25, 7.21, 6.89, 6.85 (4s, 4H, PMB), 5.08 (s, 2H, Bn),
4.84 (s and br, 1H, NH), 4.47 (s, 2H, PMBCH2), 4.20-4.09 (m,
2H, H-1 and H-2), 4.08-3.97 (m, 1H, H-1), 3.79 (s, 3H, MeO),
3.53-3.40 (m, 2H, H-3), 3.27 (q, J ) 6.5 Hz, CH2N), 2.61 (d, J
) 4.5 Hz, 1H, OH), 2.31 (t, J ) 7.2 Hz, 2H, CH2CO), 1.70-
1.30 (m, 6H) ppm. 13C NMR (CDCl3, 63 MHz) δ: 173.64,
159.36, 129.74, 129.46, 128.52, 128.12, 113.87, 73.15, 70.51,
68.85, 66.62, 65.48, 55.29, 40.77, 33.94, 29.57, 26.08, 24.44
ppm. HRMS: calcd for C25H34NO7(MH+) 460.2335, found
460.2344.
1-O-[6-(N-Cb z-a m in o)h exa n oyl]-2-O-st ea r oyl-3-O-(4-
m eth oxyben zyl)-sn -glycer ol (5a ). A solution of DCC (453
mg, 2.2 mmol) and DMAP (112 mg, 1 mmol) in 5 mL of CH2Cl2
was added to a solution of glycerol 4 (841 mg, 1.83 mmol) and
stearic acid (624 mg, 2.2 mmol) in 10 mL of dry CH2Cl2.
Stirring was continued overnight at rt. Similar workup as
above and purification on SiO2 (20% ethyl acetate/hexane, Rf
) 0.55) gave 1.225 g (92% yield) of product 5a . 1H NMR
(CDCl3, 250 MHz) δ: 7.40-7.30 (m, 5H, Ph), 7.25, 7.21, 6.89,
6.85 (4s, 4H, PMB), 5.25-5.17 (m, 1H, H-2), 5.08 (s, 2H, Bn),
4.84 (s and br, 1H, NH), 4.45 (AB, J AB ) 11.8 Hz, 2H,
PMBCH2), 4.32 (dd, J ) 3.7 Hz, 11.8 Hz, 1H, H-1), 4.12 (dd, J
) 11.8 Hz, 3.7 Hz, 1H, H-1), 3.78 (s, 3H, MeO), 3.53 (d, J )
5.1 Hz, 2H, H-3), 3.17 (q, J ) 6.5 Hz, CH2N), 2.28 (2t, J ) 7.2,
8.9 Hz, 4H, CH2CO), 1.70-1.10 (m, 36H), 0.88 (t, J ) 6.2 Hz,
3H CH3) ppm. 13C NMR (CDCl3, 63 MHz) δ: 173.16, 173.10,
159.31, 156.39, 136.65, 129.74, 129.32, 128.51, 128.08, 113.82,
72.94, 70.00, 67.87, 66.56, 62.84, 55.26, 40.82, 33.94, 29.51,
29.30, 29.09, 26.15, 24.96, 24.41, 22.72, 14.14 ppm. HRMS:
calcd for C43H67NNaO8(MNa+) 748.4764, found 748.4746.
1-[6-(N-Cb z-Am in o)h exa n oyl]-2-st ea r oyl-sn -glycer ol
(6a ). To a solution of glyceryl ether 5 (1.152 mg, 1.59 mmol)
in 50 mL of wet CH2Cl2 was added DDQ (750 mg, 3.30 mmol).
The mixture was stirred at rt overnight, diluted with CH2Cl2,
washed with 10% NaHCO3, dried over Na2SO4, concentrated,
and purified on SiO2 (30% ethyl acetate/hexane, Rf ) 0.55) to
give compound 6a (930 mg, 96% yield). 1H NMR (CDCl3, 250
MHz) δ: 7.35-7.25 (m, 5H, Ph), 5.15-5.00 (m, 3H, H-2 and
Exp er im en ta l Section
1H, 13C, and 31P NMR spectra were recorded in CDCl3 or
D2O on a QE-300 or AC-250 NMR spectrometer and reported
relative to δ(TMS) ) 0 ppm. When necessary, solvents and
(42) Falck, J . R.; Abdali, A. BioMed. Chem. Lett. 1993, 3, 717-720.
(43) Reddy, K. K.; Falck, J . R.; Capdevila, J . Tetrahedron Lett. 1993,
34, 7869-7872.
(44) Reddy, K. K.; Saady, M.; Falck, J . R.; Whited, G. J . Org. Chem.
1995, 60, 3385-3390.
(45) Gou, D.-M.; Chen, C.-S. J . Chem. Soc., Chem. Commun. 1994,
2125-2126.
(46) Bruzik, K. S.; Kubiak, R. J . Tetrahedron Lett. 1995, 36, 2415-
2418.
(47) Watanabe, Y.; Hirofuji, H.; Ozaki, S. Tetrahedron Lett. 1994,
35, 123-124.
(48) Watanabe, Y.; Tomioka, M.; Ozaki, S. Tetrahedron 1995, 51,
8969-8976.
(49) Ferrier, R. J .; Middleton, S. Chem. Rev. 1993, 93, 2779-2831.
(50) Gu, Q.-M.; Prestwich, G. D. J . Org. Chem. 1996, submitted.

6310 J . Org. Chem., Vol. 61, No. 18, 1996
Chen et al.
BnCH2), 4.86 (s and br, 1H, NH), 4.32 (dd, J ) 3.7 Hz, 11.8
Hz, 1H, H-1), 4.12 (dd, J ) 11.8, 3.7, 1H, H-1), 3.69 (dd, J )
5.6, J ) 5.7 Hz, 2H, H-3), 3.17 (q, J ) 6.5 Hz, CH2N), 2.41-
2.30 (m, 4H, CH2CO), 1.70-1.10 (m, 36H), 0.88 (t, J ) 6.2 Hz,
3H CH3) ppm. 13C NMR (CDCl3, 63 MHz) δ: 173.43, 156.45,
136.56, 128.52, 128.10, 72.02, 66.63, 62.17, 61.40, 40.82, 33.94,
29.51, 29.30, 29.09, 26.15, 24.96, 24.41, 22.72, 14.14 ppm.
HRMS: calcd for C35H60NO7(MH+) 606.4370, found 606.4384.
(Ben zyloxy)3-[1-[6-(N-Cbz-am in o)h exan oyl]-2-stear oyl-
sn -glycer yl](N,N-diisopr opylam in o)ph osph in e (7a). Glyc-
erol derivative 6a (240 mg, 0.04 mmol) was dissolved in 10
mL of CH2Cl2, and diisopropylethylamine (103 mg, 0.04 mmol)
was added. The solution was cooled to 0 °C under N2, and a
solution of (benzyloxy)(N,N-diisopropylamino)chlorophosphine
(110 mg, 0.04 mmol) in 2 mL of CH2Cl2 was added. The
mixture was stirred at 0 °C for 10 min and at rt for 75 min,
diluted with CH2Cl2, washed with 10% aqueous NaHCO3, dried
(Na2SO4), concentrated, and purified on SiO2 (ethyl acetate/
hexane/triethylamine (20:10:1) Rf ) 0.96) to give 300 mg (90%
yield) of product 7a as a colorless oil. 1H NMR (CDCl3, 250
MHz) δ: 7.34-7.24 (m, 10H, phenyl), 5.15-5.00 (m, 5H, H-2
and BnCH2), 4.77-3.34 (m, 15H), 3.17 (q, J ) 6.5 Hz, CH2N),
2.41-2.30 (m, 4H, CH2CO), 1.70-1.10 (m, 48H), 0.88 (t, J )
6.2 Hz, 3H, CH3) ppm. 13C NMR (CDCl3, 63 MHz) δ: 173.07,
156.45, 141.96, 128.52, 128.28, 128.11, 127.31, 126.95, 70.79,
66.60, 62.17, 61.87, 43.15, 42.95, 40.82, 33.94, 29.51, 29.30,
29.09, 26.15, 24.96, 24.41, 22.72, 14.17 ppm. 31P NMR (101
MHz, CDCl3) δ: 150.07, 149.94 (1:1) ppm.
Ben zyl 3-[1-[6-(N-Cbz-a m in o)h exa n oyl]-2-stea r oyl-sn -
glycer yl] 1-[2,6-Bis-O-[(b en zyloxy)m et h yl]-3-O-b en zyl-
m yo-in osityl] P h osp h a te (9a ). A mixture of protected 8a
(150 mg, 0.20 mmol) in 8 mL of CH2Cl2 and 1H-tetrazole (56
mg, 0.8 mmol) was stirred at rt, while a solution of glyceryl
phosphoramidite 7a (202 mg, 0.24 mmol) in 2 mL of CH2Cl2
was added in one portion. The mixture was stirred at rt for 1
h and then cooled to -40 °C, and m-CPBA (70 mg, 0.4 mmol)
was added while stirring continued at this temperature for 5
min. Next, the mixture was stirred at 0 °C for 30 min and rt
for 30 min. The mixture was diluted to 50 mL with CH2Cl2,
washed with 10% aqueous Na2SO3, 10% NaHCO3, and water,
dried (MgSO4), and concentrated. The residue was purified
by chromatography on silica gel using 30% ethyl acetate/
hexane (Rf ) 0.50) to give 160 mg (87% yield based on
consumed starting material) of compound 9a as a colorless
syrup. Unreacted inositol 8a (58 mg, 39% yield) was recovered.
1H NMR (CDCl3, 250 MHz) δ: 7.40-7.15 (m, 29H, phenyl and
PMB), 6.83, 6.81, 6.79, 6.77 (4s, 4H, PMB), 5.20-4.45 (m, 20H),
4.30-3.90 (m, 6H), 3.77 (s, 6H, MeO), 3.55-3.30 (m, 2H), 3.16
(q, J ) 6.5 Hz, 2H, CH2N), 2.36-2.16 (m, 4H, CH2CO), 1.70-
1.20 (m, 36H), 0.87 (t, J ) 6.2 Hz, 3H, CH3) ppm. 13C NMR
(CDCl3, 63 MHz) δ: 173.04, 159.67, 137.83, 129.67, 129.13,
128.58, 1128.37, 128.23, 127.97, 127.86, 127.71, 127.59, 127.45,
127.37, 113.73, 96.04, 70.79, 66.60, 62.17, 61.87, 43.15, 42.95,
40.82, 33.94, 29.51, 29.30, 29.09, 26.15, 24.96, 24.41, 22.72,
14.17 ppm. 31P NMR (250 MHz, CDCl3) δ: 9.74, 0.27 (1:1)
ppm. (The minor impurity 31P NMR was readily removed after
the next step.) HRMS: calcd for C87H114NNaO19P(MNa+)
1530.7620, found 1530.7703.
δ: 0.15, 0.04 (near 1:1) ppm. HRMS: calcd for C71H98-
NNaO17P(MNa+) 1290.6470, found 1290.6399.
2,6-Bis-O-[(b en zyloxy)m et h yl]-3-O-b en zyl-4,5-b is-O-
(dibenzylphosphonyl)-D-m yo-in osityl 1-O-[1-[6′-(N-Cbz-a m i-
n o)h exa n oyl]-2-stea r oyl-sn -glycer yl Ben zyl P h osp h a te
(11a ). A mixture of diol 10a (90 mg, 0.076 mmol) in 8 mL of
CH2Cl2 and 1H-tetrazole (43 mg, 0.61 mmol) was stirred at
rt, while a solution of dibenzyl N,N-diisopropylphosphoramid-
ite (105 mg, 0.305 mmol) in 2 mL of CH2Cl2 was added in one
portion. The mixture was stirred at rt for 30 min, and then
m-CPBA (65 mg, 0.38 mmol) was added to effect oxidation as
described for compound 9a . Similar workup gave 109 mg of
fully-protected PtdInsP2
using 30% ethyl acetate/hexane (Rf
11a by chromatography on silica gel
) 0.21). 1H NMR (CDCl3,
250 MHz) δ: 7.40-7.15 (m, 45H, phenyl), 5.20-4.45 (m, 27H),
4.30-3.90 (m, 6H), 3.50-3.44 (m, 1H), 3.16 (q, J ) 6.5 Hz,
2H, CH2
N), 2.36-2.16 (m, 4H, CH2CO), 1.70-1.20 (m, 36H),
0.87 (t, J ) 6.2 Hz, 3H, CH3) ppm. 13C NMR (CDCl3, 63 MHz)
δ: 172.86, 171.77, 144.50, 133.20, 130.10-127.50 (m), 96.53,
95.46, 76.54, 73.65, 73.13, 72.14, 70.49, 69.55, 69.30, 69.17,
66.60, 65.32, 61.61, 58.11, 40.82, 34.08, 33.70, 31.94, 29.72,
29.52, 29.39, 29.33, 29.10, 26.15, 24.96, 24.41, 22.72, 14.14
ppm. 31P NMR (101 MHz, CDCl3) δ: 0.09, -0.03, -0.39 ppm
(near 1:3:2, corresponding to two diastereoisomers). HRMS:
calcd for C99H124NNaO23P3 1810.7675, found 1810.7672.
4,5-Di-O-p h osp h or yl-D-m yo-in osityl 1-O-[1-(6′-Am in o-
h exa n oyl)-2-st ea r oyl-sn -glycer yl] P h osp h a t e Tet r a -
sod iu m Sa lt (12a ). A mixture of protected PtdInsP2 11a (130
mg, 0.074 mmol), 5% Pd/C (190 mg), and NaHCO3 (43 mg, 0.51
mmol) in 55 mL of t-BuOH-H2O (6:1) was shaken under H2
(50 psi) at rt for 4 h. The catalyst was filtered through Celite,
and the Celite pad was washed with 10 mL of ethyl acetate,
10 mL of EtOH, 10 mL of EtOH-H2O, and 10 mL of H2O. The
combined filtrate was concentrated in vacuo giving a solid,
which made a cloudy solution when dissolved in water. This
solution was adsorbed to a Chelex column (Na+ form), and the
phosphoinositide product was eluted with water. Concentra-
tion in vacuo gave an amorphous solid of compound 12a (56
mg, 78% yield). 1H NMR (D2O, 250 MHz) δ: 5.20-5.00 (m,
1H), 4.30-3.50 (m, 10H), 2.90 (m, 2H, CH2N), 2.36-2.16 (m,
4H, CH2CO), 1.70-1.20 (m, 36H), 0.87 (m, 3H, CH3) ppm. 31P
NMR (D2O, 101 MHz) δ: 8.70, 8.60, 3.29 (near 1:1:1) ppm.
4,5-Di-O-p h osp h or yl-D-m yo-in osityl 1-O-[1-[6′-[(p-Ben -
zoyld ih yd r ocin n a m oyl)a m in o]h exa n oyl]-2-st ea r oyl-sn -
glycer yl] P h osp h a te Tetr a k is(tr ieth yla m m on iu m ) Sa lt
(P td In sP 2-BZDC, 13a ). PtdInsP2 analogue 12a (8 mg, 8.3
µmol) was dissolved in 1.2 mL of 0.25 M TEAB buffer, and a
solution of N-hydroxysuccinimidyl p-benzoyldihydrocinnamic
acid ester (BZDC-NHS ester) (6 mg, 17.1 µmol) in 400 µL of
DMF was added. The mixture was stirred at rt overnight and
concentrated in vacuo, and the residue was redissolved in 1
mL of water and reconcentrated to remove the remaining
triethylammonium salt. Acetone was added, the precipitate
formed was centrifuged, and the acetone was decanted. The
solid was washed with acetone and centrifuged five times, until
the acetone solution showed no UV-active materials. The solid,
which was found to be free of N-hydroxysuccinimide and
p-benzoylhydrocinnamic acid, was then dissolved in 1 mL of
water and applied to a DEAE cellulose column (55 × 10 mm,
HCO3- form). The column was eluted with two 3-mL portions
of water, and then with 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, and 1.0 M
TEAB (3 mL for each concentration), and finally with three
3-mL portions of 1.28 M TEAB. The product eluted in 1.0 and
1.28 M buffer; fractions were concentrated and analyzed by
TLC developed in CHCl3:MeOH:10 N NH4OH ) 6:4:1. The
fractions were lyophilized to give 8 mg (80% yield) of PtdInsP2-
BZDC (13a ). 1H NMR (250 MHz, D2O) δ: 7.70-7.55 (m, 5H),
7.40 (t, 2H), 7.26 (t, 2H), 5.2 (m, 1H), 4.8-3.4 (m, 10H), 3.06
(t, J ) 6.5 Hz, 2H), 2.90 (t, J ) 7.5 Hz, 2H), 2.45 (t, J ) 7.5
Hz, 2H), 2.1 (m, 4H), 1.4-1.0 (m, 36H), 0.8(br s, 3H) ppm. 31P
NMR δ: 4.97 (s), 4.37 (s), 3.71 (s) (1:1:1) ppm. MALDI-TOF
MS: 1131 (M- + Na - 1).
Ben zyl 1-[6′-(N-Cb z-a m in o)h exa n oyl]-2-st ea r oyl-sn -
glycer yl 1-O-[2,6-Bis-O-[(ben zyloxy)m eth yl]-3-O-ben zyl-
D-m yo-in osityl] P h osp h a te (10a ). A mixture of the pro-
tected coupled phosphoinositide 9a (160 mg, 0.113 mmol) and
DDQ (100 mg, 0.44 mmol) in 10 mL of wet CH2Cl2 was stirred
at rt for 4 h. TLC showed the reaction was complete. (Rf (9a )
) 0.50; Rf (10a ) ) 0.22, 50% ethyl acetate/hexane). Usual
workup followed by purification of SiO2 (50% ethyl acetate/
hexane) gave 100 mg of pure compound 10a (75% yield). 1H
NMR (CDCl3, 250 MHz) δ: 7.35-7.15 (m, 25H, phenyl), 5.20-
4.45 (m, 18H), 4.30-3.90 (m, 8H), 3.55-3.30 (m, 2H), 3.16 (q,
J ) 6.5 Hz, 2H, CH2N), 2.36-2.16 (m, 4H, CH2CO), 1.70-
1.20 (m, 36H), 0.87 (t, J ) 6.2 Hz, 3H, CH3) ppm. 13C NMR
(CDCl3, 63 MHz) δ: 172.83, 170.46, 144.50, 137.76, 128.71-
127.57 (m), 121.27, 96.55, 95.31, 76.54, 73.65, 73.13, 72.14,
70.49, 69.55, 69.30, 69.17, 66.60, 65.32, 61.61, 58.11, 40.82,
34.08, 33.70, 31.94, 29.72, 29.52, 29.39, 29.33, 29.10, 26.15,
24.96, 24.41, 22.72, 14.17 ppm. 31P NMR (101 MHz, CDCl3)
4,5-Di-O-p h osp h or yl-D-m yo-in osityl 1-O-[1-[6′-[(p-Ben -
zoyld it r it iocin n a m oyl)-a m in o]h exa n oyl]-2-st ea r oyl-sn -
glycer yl] P h osp h a te Tetr a k is(tr ieth yla m m on iu m ) sa lt
([3H]P td In sP 2-BZDC, 13b). Aminohexanoyl derivative 12a

PtdInsP2 and PtdInsP3 Photoaffinity Labels
J . Org. Chem., Vol. 61, No. 18, 1996 6311
(0.25 mg/1 mL of 0.25 M TEAB buffer stock solution, 100 µL)
was added to 30 µL of DMF containing [3H]BZDC-NHS ester
(2 mCi) and stirred overnight at rt. The solvents were
evaporated in vacuo, and the residue was evaporated with 100
µL of water to remove the remaining triethylammonium salt.
The residue was dissolved in 1 mL of water and applied to
(107.3 mg, 1.53 mmol) was coevaporated three times with 1
mL of dry benzene (azeotropic removal of water) and dried
under vacuum for approximately 3 h. The dry mixture was
placed under an atmosphere of N2, and (benzyloxy)3-[1-[6-
[(carbobenzyloxy)amino]hexanoyl]-2-oleoyl-sn-glyceroxy](N,N-
diisopropylamino)phosphine (556 mg, 0.661 mmol) dissolved
in 4 mL of dry CH2Cl2 was added. The reaction was stirred
for 1 h at rt, the mixture was cooled to -40 °C, and m-CPBA
(203 mg of 57-85% pure material, a minimum of 0.809 mmol)
was added. Oxidation was allowed to proceed at -40 °C for 5
min and at 0 °C for 30 min. The mixture was diluted with 80
mL of CH2Cl2 and extracted with 2 × 30 mL 10% Na2SO3
followed by 2 × 30 mL 10% NaHCO3. The organic phase was
dried over MgSO4, filtered, concentrated to a yellowish oil, and
purified on SiO2 with 33% ethyl acetate/hexane (Rf ) 0.25) to
yield the desired product 9b (297 mg, 88%) as a colorless oil.
1H NMR (CDCl3, 250 MHz) δ: 7.33-7.17 (m, 26H), 6.82-6.77
(m, 6H), 5.33-5.31 (m, 1H), 5.29 (s, 2H), 5.14-4.44 (m, 18H),
4.26-3.91 (m, 8H), 3.77 (s, 9H), 3.48-3.35 (m, 2H), 3.20-3.15
(q, 2H), 2.8 (br s, 1H), 2.26-2.17 (m, 4H), 2.04-1.10 (m, 4H),
1.60-1.27 (m, 28H), 0.90-0.85 (t, J ) 6.1 Hz, 3H) ppm. 31P
NMR (101 MHz, CDCl3) δ: 0.23 ppm. HRMS: calcd for
C88H114NO20NaP 1558.7570, found 1558.7660.
Ben zyl 3-[1-[6′-(N-Cb z-a m in o)h exa n oyl]-2-oleoyl-sn -
glycer oxy] 1-[2,6-Bis-O-(ben zyloxym eth yl)-m yo-in osityl]
P h osp h a te (10b). A mixture of coupled derivatives 9b 148
mg, 0.0964 mmol) and DDQ (140 mg, 0.617 mmol) in 15 mL
of wet CH2Cl2 was stirred for 5 h at rt. Next, the mixture
was diluted with 85 mL CH2Cl2 and extracted 2 × 35 mL of
10% NaHCO3, followed by 35 mL of saturated brine. The
organic phase was dried over MgSO4, filtered, concentrated
to an oily residue, and purified on SiO2 with ethyl acetate (Rf
) 0.42) to give triol 10b (68.9 mg, 61%) as a colorless oil. 1H
NMR (CDCl3, 250 MHz) δ: 7.45-7.30 (s, 20H), 5.41-5.30 (m,
2H), 5.15-4.51 (m, 13H), 4.28-4.04 (m, 8H), 3.82-3.69 (br s,
1H), 3.46-3.31 (m, 3H), 3.22-3.11 (q, J ) 6.1 Hz, 2H), 2.89
(br s, 1H), 2.72 (br s, 1H), 2.29-2.27 (t, J ) 6.8 Hz, 4H), 2.04-
1.94 (m, 4H), 1.61-1.22 (m, 28), 0.89-0.84 (t, J ) 6.7 Hz, 3H)
ppm. 31P NMR (101 MHz, CDCl3) δ: -0.09, -0.3 (near 1:1)
ppm. HRMS: calcd for C64H90NO17NaP 1198.5844, found
1198.5774.
2,6-Bis-O-[(b e n zyloxy)m e t h yl]-3,4,5-t r is-O-(d ib e n -
zylp h osp h on yl)-D-m yo-in osityl 1-O-[1-[6′-(N-Cbz-a m in o)-
h exa n oyl]-2-oleoyl-sn -glycer yl Ben zyl P h osp h a te (11b).
Triol 10b (63.9 mg, 0.054 mmol) along with 1H-tetrazole (52.3
mg, 0.747 mmol) was coevaporated twice with 1 mL of dry
benzene and dried under vacuum for approximately 2 h. Bis-
(benzyloxy) N,N-diisopropylphosphoramidite (122 mg, 0.354
mmol) dissolved in 2 mL of CH2Cl2 was then added, and the
reaction was allowed to proceed for 2.5 h. The mixture was
cooled to -40 °C, and m-CPBA (142 mg of 57-85% pure
material, a minimum of 0.469 mmol) was added. Oxidation
proceeded at -40 °C for 5 min, at 0 °C for 30 min, and at rt
for an additional 30 min. The mixture was diluted with 120
mL of CH2Cl2 and extracted twice with 40 mL of 10% Na2SO3,
followed by two extractions with 50 mL of 10% NaHCO3. The
organic phase was dried over MgSO4, filtered, concentrated
to a cloudy oil, and purified on SiO2 with 50% ethyl acetate/
hexane (Rf ) 0.14) to give 88 mg (83% yield) of the compound
11b as a colorless oil. 1H NMR (250 MHz, CDCl3) δ: 7.31-
7.18 (m, 50H), 5.06-3.99 (m, 35H), 3.12 (m, 2H), 2.91 (m, 2H),
2.19 (m, 4H), 1.85-1.25 (m, 32H), 0.87-0.79 (t, 3H) ppm. 31P
NMR (101 MHz, CDCl3) δ: 0.334, 0.142, -0.390 (1:2:1) ppm.
3,4,5-Tr is-O-p h osp h or yl-D-m yo-in osityl 1-O-[1-(6′-Am i-
n oh exa n oyl)-2-st er oyl-sn -glycer yl] P h osp h a t e H exa -
sod iu m Sa lt (12b). A mixture of fully-protected PtdInsP3
derivative 11b (71.3 mg, 0.037 mmol), NaHCO3 (28.2 mg, 0.336
mmol), and 233 mg of 10% Pd/C catalyst in 30 mL of t-BuOH/
H2O 6:1 was shaken under an atmosphere of H2 (50 psi) in a
Parr apparatus for 24 h. The catalyst was removed by
filtration through a bed of Celite and washed with 10 mL of
t-BuOH, 2 × 10 mL of t-BuOH/H2O 1:1, and 4 × 10 mL of
H2O. The filtrate was concentrated in vacuo and lyophilized,
leaving the PtdInsP3 derivative 12b as a white solid. 1H NMR
(250 MHz, D2O) δ: 5.35 (m, 1H), 4.51-3.97 (m, 10H), 3.02 (m,
2H), 2.72 (m, 4H), 1.70-1.30 (m, 36H), 0.89 (m, 3H) ppm. 31P
-
DEAE cellulose column (15 × 4 mm, HCO3 form). The
column was eluted with two 1-mL portions of water, and then
with 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, and 1.0 M TEAB (1 mL for each
concentration), and finally with three 1-mL portions of 1.28
M TEAB. A 1-µL aliquot of each fraction was analyzed by
liquid scintillation counting, the 1.0 M and 1.28 M fractions
contained 120 µCi of 13b (6% radiochemical yield).51
4,5-Di-O-p h osp h or yl-D-m yo-in osityl 1-O-[1-[6′-[[6-[(7-n i-
tr o-2-oxa -1,3-d ia zoloben z-4-yl)a m in o]h exa n oyl]a m in o]-
h exa n oyl]-2-stea r oyl-sn -glycer yl] P h osp h a te Tetr a k is-
(tr ieth yla m m on iu m ) Sa lt (P td In sP 2-NBD, 14). Amino-
hexanoyl derivative 12a (7 mg, 7.3 µmol) was dissolved in 1200
µL of 0.25 M TEAB buffer, and a solution of N-hydroxy-
succinimidyl
6-[7-nitro-2-oxa-1,3-diazolobenz-4-yl)amino]-
hexanoate (NBD-aminocaproic NHS ester, 5 mg, 12.8 µmol)
in 400 µL of DMF was added. The mixture was stirred at rt
overnight and concentrated in vacuo, and the residue was
redissolved in 1 mL of water and reconcentrated to remove
the remaining triethylammonium salt. Acetone was added,
the precipitate formed was centrifuged, and the acetone was
decanted. The solid was washed with acetone and centrifuged
five times, until the acetone solution showed no fluorescent-
active (NBD-aminocaproic acid) or UV-active (N-hydrosuccin-
imide) materials. The solid, now free of N-hydroxysuccinimide
and NBD-aminocaproic acid by TLC was dissolved in 1 mL of
water and applied to a 55 mm × 10 mm column of DEAE
cellulose (HCO3- form). The column was eluted with two 3-mL
portions of water, and then 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, and 1.0
M TEAB (3 mL for each concentration), and three 3-mL
portions of 1.28 M TEAB. The product was in the 1.0 and 1.28
M TEAB fractions that were yellow and showed fluorescence.
The fractions were lyophilized to give 7 mg (78% yield) of
yellow PtdInsP2-NBD 14. 1H NMR (250 MHz, D2O) δ: 8.43
(m, 1H, very weak), 61.5 (m, 1H, very weak), 5.20 (m, 1H),
4.4-3.8 (m, 10H), 3.1-3.0 (m, 4H), 2.1 (m, 6H), 1.4-1.0 (m,
42H), 0.8 (br s, 3H) ppm. 31P NMR δ: 4.80 (s), 4.17 (s), 3.34
(s) (1:1:1) ppm. MALDI-TOF MS: found 1151, required for
free acid 1148 (M- - 1).
(Ben zyloxy) 3-[1-[6-(N-Cbz-a m in o)h exa n oyl]-2-oleoyl-
sn -glycer oxy](N,N-diisopr opylam in o)ph osph in e (7b). 1-[6-
[(Carbobenzyloxy)amino]hexanoyl]-2-oleoyl-sn-glycerol (600 mg,
0.993 mmol) was dissolved in 10 mL of dry CH2Cl2, and
diisopropylethylamine (346 µL, 1.99 mmol) was added. The
mixture was cooled to 0 °C under an atmosphere of N2 and
(benzyloxy)(N,N-diisopropylamino)chlorophosphine (326 mg,
1.19 mmol) dissolved in 15 mL CH2Cl2 added dropwise over
30 min. The mixture was kept at 0 °C for an additional 15
min. Next, the cooling bath was removed and the reaction
allowed to proceed at rt for 2 h. The mixture was evaporated
to dryness, and the crude product was purified on SiO2 with
33% ethyl acetate/hexane containing 5% triethylamine (Rf )
0.84). The purified product 7b (721 mg, 86% yield) was
obtained as a colorless oil. 1H NMR (CDCl3, 250 MHz) δ:
7.45-7.25 (m, 10H), 5.40-5.28 (m, 2H), 5.20-5.14 (m, 1H),
5.08 (s, 2H), 4.77-4.58 (m, 3H), 4.39-4.30 (m, 1H), 4.19-4.11
(m, 1H), 3.83-3.55 (m, 4H), 3.18-3.15 (q, J ) 6.1 Hz, 2H),
2.32-2.26 (t, J ) 7.4 Hz, 4H), 2.01-1.98 (q, J ) 5.5 Hz, 4H),
1.67-1.14 (m, 6H), 1.28-1.26 (m, 22H), 1.19 -1.16 (2d, 12H),
0.89-0.84 (t, J ) 6.3 Hz, 3H) ppm. 31P NMR (101 MHz,
CDCl3) δ: 150.18, 150.07 (near 1:1) ppm.
Ben zyl 3-[1-[6-(N-Cb z-a m in o)h exa n oyl]-2-oleoyl-sn -
glycer oxy] 1-[2,6-Bis-O-(ben zyloxym eth yl)-3,4,5-tr is-O-(p-
m eth oxyben zyl)-m yo-in osityl) P h osp h a te (9b). A mixture
of protected inositol 8b (171 mg, 0.219 mmol) and 1H-tetrazole
(51) Radiochemical yields for carrier-free materials, particularly the
detergent-like phosphoinositides, are invariably lower than chemical
yields. Since reactions are performed at higher dilution and with 100-
to 1,000-fold less mass, adsorptive losses and incomplete conversion
are common occurrences.

6312 J . Org. Chem., Vol. 61, No. 18, 1996
Chen et al.
NMR (101 MHz, D2O) δ: 8.25, 7.02, 5.21, 3.65 ppm. MALDI-
TOF MS: 952.4 (M - 1), calcd 952.8.
mg/1 mL of stock solution in H2O, 0.056 mg, 52 nmol) was
added to 33 µL of 0.38 M TEAB buffer (final TEAB concentra-
tion, 0.25 M). The resulting phospholipid solution was added
to 70 µL of DMF containing [3H]BZDC-NHS ester (2 mCi, 58
nmol) and the mixture stirred overnight at rt. The solvent
was evaporated in vacuo, and residual DMF and buffer salts
were removed by the addition and evaporation of 100 µL of
H2O. The residue was dissolved in 1 mL of H2O and applied
to a Pasteur pipet column of DEAE cellulose. The column was
washed twice with 1-mL portions of H2O and eluted with a
linear gradient of TEAB buffer (0.1-1.3 M). One microliter
aliquots from each fraction were subjected to liquid scintilla-
tion counting, and the radioligand 13d was determined to be
present in the 1.0-1.3 M TEAB fractions (2.6% radiochemical
yield).
3,4,5-Tr is-O-p h osp h or yl-D-m yo-in osityl 1-O-[1-[6′-[(p-
ben zoyld ih yd r ocin n a m oyl)a m in o]h exa n oyl]-2-stea r oyl-
sn -glycer yl] P h osph ate Hexakis(tr ieth ylam m on iu m ) Salt
(13c). A suspension of aminohexanoyl PtdInsP3 analogue 12b
(4.88 mg, 4.51 µmol) in 200 µL of 0.25 M TEAB buffer was
stirred at rt for 3 h until a clear solution was obtained. BZDC-
NHS ester (5.05 mg, 14.4 µmol) dissolved in 200 µL of DMF
was added and the resulting cloudy solution stirred at rt
overnight. The mixture was evaporated to dryness, and
residual buffer salts were removed by the addition and
evaporation twice with 0.5 mL of MeOH. The residue was
washed by centrifugation with four portions of acetone and
dried, leaving 5.85 mg of photolabel 13c as a white solid. 1H
NMR (D2O, 250 MHz) δ: 7.81-7.50 (m, 9H), 4.45-3.86 (m,
10H), 3.06-3.00 (t, J ) 7.5 Hz, 2H), 2.61-2.55 (t, J ) 7.5 Hz,
2H), 2.47-2.14 (m, 6H), 1.87-1.23 (m, 36H), 0.89 (m, 3H) ppm.
MALDI-TOF MS: 1189.4 (M - 1), calcd 1188.1 (free acid).
3,4,5-Tr is-O-p h osp h or yl-D-m yo-in osityl 1-O-[1-[6′-[(p-
ben zoyld itr itiu m cin n a m oyl)a m in o]ca p r oyl]-2-stea r oyl-
sn -glycer yl] P h osph ate Hexakis(tr ieth ylam m on iu m ) Salt
(13d ). Aminohexanoyl PtdInsP3 analogue 12b (17 µL of a 3.3
Ack n ow led gm en t. We thank the NIH (Grant No.
NS 29632) for financial support and Dr. David G. Ahern
(Dupont-New England Nuclear) for providing [3H]-
BZDC-NHS ester.
J O960895R