Synthesis of hybrid lipid probes: Derivatives of phosphatidylethanolamine-extended phosphatidylinositol 4,5-bisphosphate (Pea-PIP2)

Article abstract of DOI:10.1021/jo011185a

The total asymmetric synthesis of a novel hybrid lipid possessing a 2,3-diacylthreitol backbone, rather than a 1,2-diacylglycerol backbone, is described. The title compound, Pea-PIP₂, possesses a phosphatidylethanolamine (PE) headgroup at the 1-position and a phosphatidylinositol 4,5- bisphosphate (PtdIns(4,5)P₂) headgroup at the 4-position. Reporters (biotin, fluorophores, spin label) were covalently attached to the free amino group of the PE, such that these reporters were targeted to the lipid-water interface. The diacyl moieties allow incorporation of Pea-PIP₂ into a lipid bilayer, while the PtdIns(4,5)P₂ moiety in the aqueous layer was specifically recognized by PtdIns(4,5)P₂-specific binding proteins.

Full text of DOI:10.1021/jo011185a

Syn th esis of Hybr id Lip id P r obes: Der iva tives of
P h osp h a tid yleth a n ola m in e-Exten d ed P h osp h a tid ylin ositol
4,5-Bisp h osp h a te (P ea -P IP 2)
Piotr W. Rzepecki†,‡ and Glenn D. Prestwich*,†
Department of Medicinal Chemistry, The University of Utah, 419 Wakara Way, Suite 205,
Salt Lake City, Utah 84108, and Echelon Biosciences, Inc., 420 Chipeta Way, Suite 180,
Salt Lake City, Utah 84108
gprestwich@pharm.utah.edu
Received December 28, 2001
The total asymmetric synthesis of a novel hybrid lipid possessing a 2,3-diacylthreitol backbone,
rather than a 1,2-diacylglycerol backbone, is described. The title compound, Pea-PIP2, possesses a
phosphatidylethanolamine (PE) headgroup at the 1-position and a phosphatidylinositol 4,5-
bisphosphate (PtdIns(4,5)P2) headgroup at the 4-position. Reporters (biotin, fluorophores, spin label)
were covalently attached to the free amino group of the PE, such that these reporters were targeted
to the lipid-water interface. The diacyl moieties allow incorporation of Pea-PIP2 into a lipid bilayer,
while the PtdIns(4,5)P2 moiety in the aqueous layer was specifically recognized by PtdIns(4,5)P2-
specific binding proteins.
In tr od u ction
Phosphatidylinositol 4,5-bisphosphate, or PtdIns(4,5)-
P2, has been found to play a central role in a variety of
cellular functions, each utilizing different portions of the
total molecule.4,5 First, PtdIns(4,5)P2 is a substrate for
phospholipase C (PLC), a pleckstrin homology (PH)-
domain-containing enzyme whose three-dimensional struc-
ture has been determined.13 Hydrolysis of the phospho-
diester linkage releases the calcium-mobilizing second
messenger Ins(1,4,5)P3.14 Second, PtdIns(4,5)P2 itself
recruits PH-domain-containing proteins to membranes;15
PH domains occur in over 120 mammalian proteins, and
each exhibits distinctive PIPn-binding affinities and
selectivities.16-18 Among these, the best example is the
specific binding of PtdIns(4,5)P2 to the PH domain of the
δ1 isoform of PLC.19 Third, PtdIns(4,5)P2 binds to non-
PH-domain-containing proteins, e.g., gelsolin and profilin,
and regulates actin polymerization.5 PtdIns(4,5)P2 bind-
ing also modulates the GTPase activity of adenosine
ribosylation factor (ARF) and the phospholipase activity
of phospholipase D,20,21 recruits the human class II
phosphoinositide (PI) 3-kinase to membranes via its C2-γ
Phosphoinositides (PtdInsPns) are biosynthesized by
the interplay of kinases1 and phosphatases.2 These
charged lipids are minor components of cellular mem-
branes but are vital as second messengers for diverse
cellular functions.3,4 PtdInsPns are essential elements in
tyrosine kinase growth factor receptor and G-protein
receptor signaling pathways.5-7 Furthermore, these lipid
signals have important roles in membrane trafficking,8
including endocytosis, exocytosis, Golgi vesicle move-
ment, and protein trafficking,9,10 in cell adhesion and
migration,11 in remodeling of the actin cytoskeleton,12 and
in mitogenesis and oncogenesis.6,7 Activation of cellular
signaling pathways often results from production of one
of eight specific PtdInsPns in response to a stimuli, and
each PtdInsPn has a specific role for a given signaling
pathway in each cell-type.
† The University of Utah.
‡ Echelon Biosciences, Inc.
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10.1021/jo011185a CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/11/2002
5454
J . Org. Chem. 2002, 67, 5454-5460

Hybrid Phospholipid Synthesis
SCHEME 1. Syn th esis of Differ en tia lly F u n ction a lized 2,3-Dia cylth r eitol Ba ck bon ea
a
Reagents and conditions: (a) cyclopentanone, pTSA, toluene, reflux; (b) LiAlH4, THF; (c) NaH, PMBCl, DMF; (d) 1-H-tetrazole,
phosphoramidite 4, CH2Cl2; (e) 1 M HCl, THF; (f) C15H31COOH, DCC, DMAP, CH2Cl2; (g) DDQ, CH2Cl2/H2O
PX domain,22 and binds to the AP180 ENTH domain to
mediate endocytosis via clathrin-coated pits.23 The pro-
filin24 and gelsolin25 interactions with PtdIns(4,5)P2 have
been characterized by photoaffinity labeling. Finally,
PtdIns(4,5)P2 can be converted by PI 3-kinase26 to
PtdIns(3,4,5)P3, which interacts with many PH domains
and activates the specific protein kinase PDK1.3
Chemically modified derivatives of the natural
PtdInsPns27,28 have proven extremely valuable in isolat-
ing new PtdInsPn-binding proteins, identifying binding
sites, and unraveling the roles of specific molecular
species in cell signaling.28 These derivatives have in-
cluded photoaffinity analogues based on phosphotri-
esters29 and acyl modifications.27 In addition, fluores-
cently labeled analogues30 have been employed for
biophysical studies31,32 as well as investigations of cellular
uptake and subcellular localization.33 Biotinylated de-
rivatives have proven valuable as “bait” in fishing
through expression libraries for novel high-affinity
PtdInsPn-specific binding proteins.34 For each of these
derivatives, the modification has involved either the P-1
phosphate, thus placing the reporter moiety in potential
conflict with headgroup recognition, or the acyl chain,
thus altering the partitioning of the analogue into a lipid
bilayer environment.
In this paper, we describe the asymmetric total syn-
thesis of a new class of functionalized PtdInsPns, the Pea-
PIPs. Our strategy involves homologation of the 1,2-
diacylglycerol backbone to a 2,3-diacylthreitol back-
bone. These hybrid lipids thus possess a phosphatidyl-
ethanolamine (PE, or Pea) headgroup at the 1-position
and a PtdIns(4,5)P2 headgroup at the 4-position. The
reporter group, e.g., biotin, a fluorophore, or a spin label,
could then be covalently attached to the free PE amino
group. The reporter would thus be targeted to the lipid-
water interface at a site distant from the key PtdIns-
(4,5)P2 headgroup recognition features of the binding
protein. The diacyl moiety should permit insertion and
retention in a lipid bilayer to facilitate recruitment of
PtdIns(4,5)P2-specific binding proteins to a membrane
surface environment.
Resu lts a n d Discu ssion
(22) Song, X.; Xu, W.; Zhang, A. H.; Huang, G. Q.; Liang, X. S.;
Virbasius, J . V.; Czech, M. P.; Zhou, G. W. Biochemistry 2001, 40,
8940-8944.
Diethyl D-tartrate was chosen as the chiral precursor
for the extended glycerol backbone of the target hybrid
lipid. The absolute configuration of both stereogenic
centers at C-2 and C-3 is identical to the configuration
of glycerol sn-2 position in naturally occurring PtdInsPns
and in natural PE. Moreover, the C2 axis allowed the use
of a monoprotection step in the early stages of the
synthesis. As shown in Scheme 1, diethyl D-tartrate 1
was protected as a cyclopentylidene acetal, which was
found to be most readily removed after backbone func-
tionalization. Initially, an isopropylidene acetal was used,
but scale-up of the deprotection led to unsatisfactory
yields of the desired intermediate. Reduction of acetal 2
with lithium aluminum hydride afforded (2R,3R)-O-
cyclopentylidene threitol 3a , which was protected with
1 equiv of PMB-Cl to give the monobenzyl ether 3b. The
primary alcohol of 3b was converted to a Cbz-protected
PE headgroup by coupling to the phosphoramidite 4,
(23) Itoh, T.; Koshiba, S.; Kigawa, T.; Kikuchi, A.; Yokoyama, S.;
Takenawa, T. Science 2001, 291, 1047-1051.
(24) Chaudhary, A.; Chen, J .; Gu, Q.-M.; Witke, W.; Kwiatkowski,
D. J .; Prestwich, G. D. Chem. Biol. 1998, 5, 273-281.
(25) Feng, L.; Mejillano, M.; Yin, H. L.; Chen, J .; Prestwich, G. D.
Biochemistry 2001, 40, 904-913.
(26) Vanhaesebroeck, B.; Leevers, S. J .; Ahmadi, K.; Timms, J .;
Katso, R.; Driscoll, P. C.; Woscholski, R.; Parker, P. J .; Waterfield, M.
D. Annu. Rev. Biochem. 2001, 70, 535-602.
(27) Prestwich, G. D. Acc. Chem. Res. 1996, 29, 503-513.
(28) Prestwich, G. D.; Chaudhary, A.; Chen, J .; Feng, L.; Mehrotra,
B.; Peng, J . In Phosphoinositides: Chemistry, Biochemistry and
Biomedical Applications; Bruzik, K. S., Ed.; American Chemical
Society: Washington, DC, 1999; Vol. 818, pp 24-37.
(29) Gu, Q.-M.; Prestwich, G. D. J . Org. Chem. 1996, 61, 8642-
8647.
(30) Chen, J .; Profit, A. A.; Prestwich, G. D. J . Org. Chem. 1996,
61, 6305-6312.
(31) Glaser, M.; Wanaski, S.; Buser, C. A.; Boguslavsky, V.; Rash-
idzada, W.; Morris, A.; Rebecchi, M.; Scarlata, S. F.; Runnels, L. W.;
Prestwich, G. D.; Chen, J .; Aderem, A.; Ahn, J .; McLaughlin, S. J . Biol.
Chem. 1996, 271, 26187-26193.
(32) Tuominen, E. K. J .; Holopainen, J . M.; Chen, J .; Prestwich, G.
D.; Bachiller, P. R.; Kinnunen, P. K. J .; J anmey, P. A. Eur. J . Biochem.
1999, 263, 85-92.
(33) Ozaki, S.; DeWald, D. B.; Shope, J . C.; Chen, J .; Prestwich, G.
D. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 11286-11291.
(34) Rao, V. R.; Corradetti, M. N.; Yuan, J .; Chen, J .; Peng, J .;
Prestwich, G. D.; Yuan, J .; Brugge, J . S. J . Biol. Chem. 1999, 274,
37893-37900.
J . Org. Chem, Vol. 67, No. 16, 2002 5455

Rzepecki and Prestwich
SCHEME 2. Ba ck bon e P h osp h or yla tion a n d Syn th esis of P ea -P IP 2 Der iva tives 12a
a
Reagents and Conditions: (a) BnOP(NiPr2)2, 1-H-tetrazole, CH2Cl2; (b) 1-H-tetrazole, 4,5-HG, CH2Cl2; (c) H2 (60 psi), 10% Pd/C,
THF/H2O; (d) probe-NHS ester, 0.5 M TEAB, DMF
which after oxidation afforded the protected PE analogue
5. Acidic hydrolysis yielded diol 6 in 65% yield after silica
gel chromatography. Acylation with palmitic acid pro-
vided diester 7 and oxidative cleavage of the p-methoxy-
benzyl (PMB) ether with DDQ gave primary alcohol 8.
Scheme 2 illustrates the installation of two different
phosphorylated headgroups on the 2,3-diacylthreitol
backbone. Thus, reaction of alcohol 8 with benzyloxybis-
(N,N-diisopropylamino)phosphine yielded a homologated
PE-like phosphoramidite reagent 9, which was coupled
with the protected myo-inositol 4,5-bisphosphate head-
group obtained as previously described,29 to give the fully
protected Pea-PIP2 precursor 10. Global debenzylation
of 10 was accomplished by hydrogenolysis to give the free
phosphate monoesters and phosphodiesters in the hybrid
lipid Pea-PIP2 (11). Reaction of the free amino group of
11 with four N-hydroxysuccinimidyl (NHS) esters35 af-
forded the corresponding biotinylated derivative 12a , the
fluorescent N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)
and 6-carboxyfluorescein derivatives 12b and 12c, and
the spin-labeled 3-carboxy-2,2,5,5-tetramethyl-1-pyrroli-
dinyloxy (PROXYL) derivative 12d . Biological results are
described below for biotinylated derivative 12a and
fluorescent analogue 12c. Use of the spin-labeled deriva-
tive 12d to probe interfacial protein-lipid interactions
in liposomes will be described elsewhere, in analogy to
the use of acyl spin-labeled probes to characterize the
MARCKS peptide-PtdIns(4,5)P2 interaction in lipo-
somes.36
Two preliminary biological assays were conducted to
establish the utility of this reagent in a biochemical
context. First, we established biochemical relevance by
demonstrating that the Pea-PIP2 12 derivatives could
bind to the PtdIns(4,5)P2-selective PH domain of the PLC
δ1 isoform. We employed a bioluminescence assay (Alpha-
Screen)37 in which the biotinylated lipid 12a was bound
to a streptavidin-coated donor bead and the GST-tagged
PLC δ1-PH domain was attached to an anti-GST-coated
acceptor bead. A luminescent signal quantitatively re-
ported the interaction between the biotinylated lipid and
the binding protein. Thus, in the absence of a lipid or a
specific binding protein, no signal was seen. A biotinyl-
ated PtdIns(4,5)P2 analogue27 exhibited the benchmark
binding interaction, and the Pea-PIP2 analogue 12a
showed approximately 30% of the full PtdIns(4,5)P2
response. As expected, the biotinylated InsP6 derivative38
did not bind to the GST-PLC δ1-PH construct (Figure 1).
Next, competitive binding assays were conducted (data
not shown) in which the GST-PLC δ1-PH protein was
preincubated for 30 min with 10 nM to 10 fM of unlabeled
di-C4 PtdIns(4)P, PtdIns(3,4,5)P3, and PtdIns(4,5)P2 prior
to addition of the other reagents, using biotinylated Pea-
PIP2 12a as the probe lipid. Over 100-fold selectivity was
observed for displacement of the GST-PLC δ1-PH from
binding to 12a by the di-C4 PtdIns(4,5)P2, relative to the
other lipids.
Second, preliminary data were obtained with im-
mobilized lipids.18 Eight different quantities (from 200
(35) Olszewski, J . D.; Dorman, G.; Elliott, J . T.; Hong, Y.; Ahern,
D. G.; Prestwich, G. D. Bioconjugate Chem. 1995, 6, 395-400.
(36) Rauch, M.; Ferguson, C.; Prestwich, G. D.; Cafiso, D. J . Biol.
Chem. 2002, in press.
(37) Beaudet, L.; Bedard, J .; Breton, B.; Mercuri, R.; Budarf, M.
Genome Res. 2001, 11, 600-608.
(38) Chaudhary, A.; Mehrotra, B.; Prestwich, G. D. Biotechniques
1997, 23, 427-30.
5456 J . Org. Chem., Vol. 67, No. 16, 2002

Hybrid Phospholipid Synthesis
SCHEME 3. Str u ctu r es of Dip a lm itoyl-P td In s(4,5)P 2 a n d F ou r P ea -P IP 2 P r obes (12a -d ) P r ep a r ed for
Bioch em ica l a n d Biologica l Stu d ies
to 2 pmol) of PE, PtdIns(4,5)P2, and biotinylated Pea-
PIP2 12a were spotted onto nitrocellulose, and the
binding of an anti-PtdIns(4,5)P2 IgM monoclonal antibody
(Echelon) and the GST-PLC δ1-PH construct were exam-
ined. Neither protein recognized the PE control lipid, but
both proteins showed dose-dependent recognition of the
immobilized lipids. The antibody recognition was more
robust than that of the GST-PLC δ1-PH protein. Full
details for all biochemical assays will be published in due
course.
Exp er im en ta l Section
Gen er a l. Reactions requiring anhydrous conditions were
carried out in oven-dried glassware (2 h, 120 °C). Dry solvents
were prepared using standard procedures. Concentration in
vacuo refers to the use of a rotary evaporator for solvent
removal, and purification on SiO2 refers to flash chromatog-
raphy on silica gel. Reactions were monitored by TLC (SiO2)
on Whatman glass plates using a mixture of Ce(SO4)2, MoO3,
and H2SO4 in water for visualization. NMR spectra were
recorded on Varian INOVA instrument in chloroform-d or D2O
with TMS as internal reference. Coupling constants (J ) are
reported in Hz. IR spectra were recorded on J ASCO FT/IR-
420 apparatus (film on NaCl disk). Elemental analyses were
performed by M-H-W Laboratories (Phoenix, AZ), and mass
spectra were measured at The University of California at
Riverside (Riverside, CA) using matrix-assisted laser desorp-
tion ionization (MALDI).
(2R,3R)-1,4-Dioxa -sp ir o[4.4]n on a n e-2,3-d ica r b oxylic
Acid Dieth yl Ester 2. Diethyl D-tartrate 1 (1.004 g, 4.87
mmol), cyclopentanone (2.2 mL, 24.35 mmol, 5 equiv), and
p-toluenesulfonic acid (93 mg, 0.49 mmol, 0.1 equiv) were
dissolved in toluene (75 mL) and stirred under reflux for 36 h
with azeotropic removal of water using a Dean-Stark trap.
Upon completion, the reaction mixture was cooled to room
temperature and neutralized with solid NaHCO3. Solid salts
were removed by filtration, the filtrate was concentrated in
vacuo, and the crude product was purified on SiO2 (hexane/
acetone 4:1 containing 10% v/v Et3N) to give 1.166 g (4.28
mmol, 88%) of acetal 2 (enantiomer39) as a colorless oil, [R]D
) +30.5° (0.36 g/dL).1H NMR (400 MHz, CDCl3) δ 4.73 (s, 2H),
F IGURE 1. Binding of biotinylated Pea-PIP2 12a and control
biotinylated inositide lipids to GST-PLC δ1-PH (“LRP”).
(39) Tsuzuki, Y. Bull. Chem. Soc. J pn. 1937, 12, 487-492.
J . Org. Chem, Vol. 67, No. 16, 2002 5457

Rzepecki and Prestwich
4.28 (q, 4H, J ) 7.2), 1.94-2.04 (m, 2H), 1.80-1.90 (m, 2H),
1.65-1.76 (m, 4H), 1.32 (t, 6H, J ) 7.2). 13C NMR (100 MHz,
CDCl3) δ 169.67, 123.34, 77.08, 61.85, 36.66, 23.50, 14.15. IR
2980, 1756, 1337, 1119, 1115, 1023, 460, 453. Anal. Calcd for
(5 mL) was added via cannula, and the mixture was stirred
for 2 h (until all starting material was consumed). After cooling
to -40 °C, mCPBA (1.360 g, 6 mmol, 3 equiv, 60%) in CH2Cl2
(5 mL) was added, and the reaction mixture was stirred for 5
min. The cold bath was then removed and stirring was
continued for an additional 1 h. The reaction was diluted with
CH2Cl2, poured into saturated NaHCO3, and after 20 min of
vigorous stirring extracted 3× with CH2Cl2. Combined organic
phases were dried (MgSO4) and concentrated in vacuo, and
the crude product was purified on SiO2 (hexane/acetone 3:2)
to give 1.033 g (1.57 mmol, 99%) of compound 5 as a colorless
oil, [R]D ) +3.7° (4.83 g/dL). 1H NMR (400 MHz, CDCl3) δ
7.28-7.38 (m, 10H), 7.18-7.24 (m, 2H), 6.82-6.88 (m, 2H),
5.44-5.54 (m, 1H), 5.00-5.12 (m, 4H), 4.42-4.51 (m, 2H),
3.99-4.17 (m, 4H), 3.89-3.99 (m, 2H), 3.76 (s, 3H), 3.56 (dd,
1H, J 1 ) 4.8, J 2 ) 10.0), 3.43-3.50 (m, 1H), 3.33-3.41 (m, 2H),
1.70-1.84 (m, 4H), 1.54-1.70 (m, 4H). 13C NMR (100 MHz,
CDCl3) δ 159.30, 156.34, 136.47, 135.65, 135.58, 129.92, 129.79,
129.35, 128.68, 128.63, 128.48, 128.35, 128.07, 128.03, 119.95,
113.80, 77.24, 76.00, 73.18, 69.95, 69.53, 67.46, 66.94, 66.71,
55.21, 53.47, 41.30, 37.26, 37.20, 23.49, 23.42. 31P NMR (162
MHz, CDCl3) δ 2.23. IR 3316, 2956, 1722, 1514, 1250, 1023.
Anal. Calcd for C34H42NO10P: C, 62.28; H, 6.46; N, 2.14.
Found: C, 62.38; H, 6.29; N, 2.18.
C
13H20O6: C, 57.34; H, 7.40. Found: C, 57.34; H, 7.21.
(2R,3R)-(3-H yd r oxym et h yl-1,4-d ioxa -sp ir o[4.4]n on -2-
yl)-m eth a n ol 3a . A solution of diethyl ester 2 (1165 mg, 4.28
mmol, 1 equiv) in dry THF was transferred via cannula to a
suspension of LiAlH4 (244 mg, 6.42 mmol, 1.5 equiv) in dry
THF that had been precooled in a brine/ice bath. The reaction
mixture was stirred at room temperature for 24 h, and then
saturated aqueous potassium sodium tartrate was added
dropwise to decompose excess hydride reagent. The mixture
was stirred for an additional 24 h and then extracted with
three portions of CH2Cl2. The combined organic phases were
dried over MgSO4 and concentrated in vacuo, and the crude
product was purified on SiO2 (hexane/acetone 3:2 containing
10% v/v Et3N) to give 805 mg (4.27 mmol, 99%) of the diol 3a
as a colorless oil, [R]D ) +6.7° (4.08 g/dL).1H NMR (400 MHz,
CDCl3) δ 3.85-3.95 (m, 2H), 3.60-3.80 (m, 4H), 1.73-1.85 (m,
4H), 1.60-1.73 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 119.36,
78.40, 62.46, 37.32, 23.44. IR 3390, 2956, 2875, 1434, 1335,
1204, 1112, 1041, 973. Anal. Calcd for C9H16O4: C, 57.43; H,
8.57. Found: C, 57.48; H, 8.52.
(2R,3R)-[3-(4′-Met h oxy-b en zyloxym et h yl)-1,4-d ioxa -
sp ir o[4.4]n on -2-yl]-m eth a n ol 3b. To a suspension of NaH
(175 mg, 4.38 mmol, 1 equiv) in dry DMF was added alcohol
3a (824 mg, 4.38 mmol, 1 equiv) in dry DMF via cannula. The
mixture was cooled to 0 °C, and then 4-methoxybenzyl chloride
(0.65 mL, 4.82 mmol, 1.1 equiv) was added dropwise over 20
min. The ice bath was removed, and the mixture was stirred
at room temperature for 18 h. Traces of NaH were decomposed
by slow addition of water. The mixture was extracted 3× with
CH2Cl2, dried (MgSO4), and concentrated in vacuo. The crude
product was purified on SiO2 (hexane/acetone 4:1 containing
10% v/v Et3N) to give 879 mg (2.85 mmol, 65%) of compound
3b as a colorless oil, [R]D ) -7.7° (3.82 g/dL). 1H NMR (400
MHz, CDCl3) δ 7.20-7.30 (m, 2H), 6.80-6.95 (m, 2H), 4.45-
4.55 (m, 2H), 3.92-4.00 (m, 1H), 3.82-3.89 (m, 1H), 3.79 (s,
3H), 3.60-3.76 (m, 3H), 3.49 (dd, 1H, J 1 ) 5.6, J 2 ) 9.6), 2.45
(bs, 1H), 1.73-1.89 (m, 4H), 1.58-1.73 (m, 4H). 13C NMR (100
MHz, CDCl3) δ 159.29, 129.70, 129.40, 128.52, 119.31, 113.82,
79.56, 76.58, 73.21, 70.14, 62.63, 55.19, 37.26, 37.20, 23.52,
23.40. IR 3466, 2955, 2872, 1612, 1514, 1248, 1101, 1035. Anal.
Calcd for C17H24O5: C, 66.21; H, 7.84. Found: C, 66.27, H,
7.76.
[2-(Ben zyloxy-d iisop r op yla m in o-p h osp h a n yloxy)-eth -
yl]-ca r ba m ic Acid Ben zyl Ester 4.40 To a solution of
benzyloxybis(N,N-diisopropylamino)phosphine41 (1.538 g, 4.54
mmol, 1.5 equiv) and 1-H-tetrazole (106 mg, 1.51 mmol, 0.5
equiv) in dry CH2Cl2 was added a solution of (2-hydroxyethyl)-
carbamic acid benzyl ester (591 mg, 3.03 mmol) in CH2Cl2. The
mixture was stirred at room temperature for 3 h and concen-
trated in vacuo, and the crude product was purified on SiO2
(hexane/acetone/Et3N 6:4:1) to give 886 mg (2.05 mmol, 68%)
of compound 4 as a colorless oil. 1H NMR (400 MHz, CDCl3) δ
7.20-7.40 (m, 10H), 5.21 (bs, 1H), 5.08 (s, 2H), 4.72 (dd, 1H,
J 1 ) 8.2, J 2 ) 12.4), 4.63 (dd, 1H, J 1 ) 8.2, J 2 ) 12.4), 3.64-
3.79 (m, 2H), 3.56-3.70 (m, 2H), 3.39 (m, 2H), 1.18 (d, 12H, J
) 7.2). 31P NMR (162 MHz, CDCl3) δ 149.1.
(2-{Ben zyloxy-[(2R,3R)-2,3-d ih yd r oxy-4-(4′-m et h oxy-
ben zyloxy)-bu toxy]-ph osph or yloxy}-eth yl)-car bam ic Acid
Ben zyl Ester 6. To a solution of 5 (2.161 g, 3.3 mmol) in dry
THF (100 mL) was added 100 mL of 1 M HCl. The mixture
was stirred at room temperature for 3 h, and then saturated
aqueous NaHCO3 was added. The mixture was transferred to
a separatory funnel and extracted with CH2Cl2. The crude
product was purified on SiO2 (toluene/acetone 1:4) to give 1.267
g (2.15 mmol, 65%) of the diol 6 as a colorless oil, [R]D ) 0.0°
(2.14 g/dL). 1H NMR (400 MHz, CDCl3) δ 7.25-7-35 (m, 10H),
7.17-7.22 (m, 2H), 6.81-6.86 (m, 2H), 5.64-5.75 (m, 1H),
4.95-5.10 (m, 4H), 4.35-4.46 (m, 2H), 3.95-4.12 (m, 4H),
3.78-3.86 (m, 1H), 3.65-3.78 (m, 1H), 3.75 (s, 3H), 3.45-3.55
(m, 2H), 3.30-3.40 (m, 2H). 13C NMR (100 MHz, CDCl3) δ
159.31, 156.51, 136.45, 135.50, 129.77, 129.42, 128.73, 128.64,
128.48, 128.06, 113.83, 73.14, 71.24, 70.50, 69.65, 69.25, 68.90,
66.95, 66.73, 55.22, 41.24. 31P NMR (162 MHz, CDCl3) δ 0.52.
IR 3349, 2954, 1719, 1612, 1513, 1456, 1248, 1023, 821, 739,
698. Anal. Calcd for C29H36NO10P: C, 59.08; H, 6.15; N, 2.38;
O, 27.14; P, 5.25. Found: C, 58.96; H, 6.27; N, 2.44; P, 5.44.
Hexa d eca n oic Acid {(2R,3R)-3-[Ben zyloxy-(2-ben zyl-
oxycar bon ylam in o-eth oxy)-ph osph or yloxy]-2-h exadecan -
oyloxy-1-(4′-m eth oxy-ben zyloxym eth yl)}-p r op yl Ester 7.
DCC (1.377 g, 6.67 mmol, 3 equiv) and DMAP (299 mg, 2.45
mmol, 1.1 equiv) were added in one portion to a solution of 6
(1.311 g, 2.22 mmol, 1 equiv) and hexadecanoic acid (1.901 g,
6.67 mmol, 3 equiv) in dry CH2Cl2. After stirring for 18 h, the
reaction mixture was concentrated in vacuo and purified on
SiO2 (hexane/acetone 4:1) to give 1.68 g (1.58 mmol, 71%) of
product 7 as a waxy solid, melting range 40-60 °C, [R]D
)
-31.8° (0.93 g/dL). 1H NMR (400 MHz, CDCl3) δ 7.26-7.40
(m, 10H), 7.18-7.24 (m, 2H), 6.82-6.88 (m, 2H), 5.08 (s, 2H),
4.95-5.06 (m, 2H), 4.32-4.46 (m, 2H), 3.88-4.24 (m, 4H), 3.76
(s, 3H), 3.43-3.60 (m, 2H), 3.34-3.43 (m, 2H), 2.21-2.32 (m,
4H), 1.50-1.64 (m, 4H), 1.25 (bs, 48H), 0.88 (t, 6H, J ) 7.0).
13C NMR (100 MHz, CDCl3) δ 172.90, 172.77, 159.35, 129.41,
128.73, 128.65, 128.47, 128.08, 128.01, 113.80, 72.97, 70.04,
69.97, 69.74, 69.66, 69.62, 67.45, 67.02, 66.71, 65.65, 55.18,
41.30, 34.15, 31.94, 29.72, 29.67, 29.51, 29.38, 29.31, 29.15,
24.92, 24.86, 22.70, 14.12. 31P NMR (162 MHz, CDCl3) δ 0.26,
0.14. IR 2924, 2853, 1741, 1612, 1514, 1456, 1249, 1154, 1112,
1035, 736, 697. Anal. Calcd for C61H96NO12P: C, 68.70; H, 9.07;
N, 1.31. Found: C, 68.87; H, 8.81; N, 1.32.
(2-{Ben zyloxy-[(2R,3R)-3-(4′-m eth oxyben zyloxym eth yl)-
1,4-d ioxa -sp ir o[4.4]n on -2-ylm et h oxy]-p h osp h or yloxy}-
eth yl)-ca r ba m ic Acid Ben zyl Ester 5. A solution of the
monoprotected alcohol 3b (486 mg, 1.58 mmol, 1 equiv) and
tetrazole (331 mg, 473 mmol, 3 equiv) in dry CH2Cl2 (5 mL)
was stirred under N2 for 5 min at room temperature. Phos-
phoramidite 4 (886 mg, 2.05 mmol, 1.3 equiv) in dry CH2Cl2
Hexa d eca n oic Acid {(2R,3R)-3-[Ben zyloxy-(2-ben zyl-
oxycar bon ylam in o-eth oxy)-ph osph or yloxy]-2-h exadecan -
oyloxy-1-h yd r oxym eth yl}-p r op yl Ester 8. To a solution of
7 (234 mg, 0.23 mmol, 1 equiv) in CH2Cl2 (23 mL) was added
water (0.23 mL) followed by DDQ (103 mg, 0.46 mmol, 2 equiv).
(40) Campbell, A. S.; Fraser-Reid, B. Bioorg. Med. Chem. 1994, 2,
1209-1219.
(41) Buijsman, R. C.; Basten, J . E. M.; Dreef-Tromp, C. M.; van der
Marel, G. A.; van Boeckel, C. A. A.; van Boom, J . H. Bioorg. Med. Chem.
1999, 7, 1881-1890.
5458 J . Org. Chem., Vol. 67, No. 16, 2002

Hybrid Phospholipid Synthesis
When TLC indicated that the reaction was complete, the
mixture was transferred to a separatory funnel and washed
with 5% Na2SO3 and saturated NaHCO3 (2×). The aqueous
phases were back-extracted once with CH2Cl2, the combined
organic phases were dried (MgSO4) and concentrated in vacuo,
and product was purified on SiO2 (hexane/acetone 4:1) to give
125 mg (0.13 mmol, 60%) of product 8 as a waxy solid, melting
range 55-65 °C, [R]D ) -39.4° (1.09 g/dL). 1H NMR (400 MHz,
CDCl3) δ 7.20-7.40 (m, 10H), 5.0-5.16 (m, 6H), 3.92-4.23 (m,
4H), 3.53-3.72 (m, 2H), 3.30-3.45 (m, 2H), 2.80-2.94 (m, 1H),
2.27-2.33 (m, 4H), 1.54-1.64 (m, 4H), 1.25 (bs, 48H), 0.88 (t,
6H, J ) 7.0). 13C NMR (100 MHz, CDCl3) δ 173.44, 173.16,
156.46, 136.45, 135.46, 128.81, 128.70, 128.51, 128.13, 128.06,
71.64, 70.05, 69.81, 67.08, 66.80, 65.79, 60.70, 41.30, 34.15,
31.94, 29.73, 29.68, 29.52, 29.38, 29.31, 29.16, 24.90, 22.70,
14.13. 31P NMR (162 MHz, CDCl3) δ 1.68, 1.61. IR 2917, 2850,
1739, 1467, 1263, 1017. Anal. Calcd for C53H88NO11P: C, 67.27;
H, 9.37; N, 1.48. Found: C, 67.32; H, 9.49; N, 1.50.
1-[(2R,3R)-4-(2-Am in oeth oxyp h osp h or yloxy)-2,3-d i-O-
p a lm it oylb u t oxyp h osp h or yloxy]-4,5-m yo-b isp h osp h a t e
11. To a solution of compound 10 (193.4 mg, 0.091 mmol) in a
mixture of THF/water (4:1, v/v, 50 mL) was added 10%
palladium on charcoal (387 mg). The mixture was shaken for
18 h at room temperature under 60 psi of H2. The catalyst
was removed by filtration, and solvent was removed in vacuo.
The crude product was redissolved in water and stirred for 3
h with Dowex 50X-100 resin (Na+ form). The resin was
removed by filtration, and the filtrate was lyophilized to give
77.3 mg (0.062 mmol, 62%) as the sodium salt. The dried crude
product was used for coupling with activated (NHS) esters as
described below. 1H NMR (400 MHz, D2O) δ 5.15-5.30 (m, 2H),
4.10-4.30 (m, 2H), 3.85-4.10 (m, 7H), 3.75-3.85 (m, 1H), 3.6-
3.7 (m, 1H), 3.4-3.5 (m, 1H), 3.18 (bs, 2H), 2.1-2.5 (m, 4H),
1.4-1.6 (bs, 4H), 1.18 (bs, 48H), 0.76 (bs, 6H). 31P NMR (162
MHz, D2O) δ 2.36, 1.80, 1.09, 0.56 (ratio 1:1:1:1). MS MALDI
(free acid) 1146 (M + Na), 950 (M + 3Na - C15H31CO), 928
(M + 2Na - C15H31CO), 906 (M + Na - C15H31CO), 884 (M -
Hexadecan oic Acid {(2R,3R)-1-[Ben zyloxy-(2-ben zyloxy-
carbonylamino-ethoxy)-phosphoryloxymethyl]-3-(benzyloxy-
d iisop r op yla m in o-p h osp h a n yloxy)-2-h exa d eca n oyloxy}-
p r op yl E st er 9. To a solution of benzyloxybis(N,N-diiso-
propylamino)phosphine (1.164 g, 3.44 mmol, 1.5 equiv) and
tetrazole (80 mg, 1.15 mmol, 0.5 equiv) in dry CH2Cl2 was
added a solution of ester 8 (2.169 g, 2.30 mmol, 1 equiv) in
CH2Cl2 via cannula. After 2 h, the reaction was concentrated
in vacuo, and the residue was purified on SiO2 (ethyl acetate/
toluene 4:1 containing 5% Et3N) to give 2.190 g (1.85 mmol,
81%) of phosphoramidite 9. 1H NMR (400 MHz, CDCl3) δ 7.20-
7.40 (m, 10H), 5.0-5.16 (m, 6H), 3.92-4.23 (m, 4H), 3.53-
3.72 (m, 2H), 3.30-3.45 (m, 2H), 2.80-2.94 (m, 1H), 2.27-
2.33 (m, 4H), 1.54-1.64 (m, 4H), 1.25 (bs, 48H), 0.88 (t, 6H, J
) 7.0). 13C NMR (100 MHz, CDCl3) δ 172.91, 172.75, 139.18,
135.58, 135.52, 128.74, 128.67, 128.49, 128.31, 128.26, 128.09,
128.02, 127.36, 127.29, 126.96, 126.93, 70.59, 69.72, 67.01,
66.72, 65.70, 65.37, 65.20, 61.69, 43.10, 34.18, 31.95, 29.73,
29.68, 29.52, 29.38, 29.33, 29.17, 24.88, 24.58, 22.70, 14.13.
31P NMR (162 MHz, CDCl3) δ 150.18, 149.79, 0.32, 0.22.
Hexa d eca n oic Acid 1-[Ben zyloxy-(2-ben zyloxyca r bon -
ylam in o-eth oxy)-ph osph or yloxym eth yl]-2-h exadecan oyl-
oxy-3-[1-b en zyloxy-p h osp h or yloxy-3-b en zyloxy-2,6-bis-
(ben zyloxym eth oxy)-4,5-bis-(bis-ben zyloxy-p h osp h or yl-
oxy)-D-m yo-in ositol]-p r op yl Ester 10. To a solution of 4,5-
headgroup (4,5-HG, 250 mg, 0.24 mmol, 1 equiv) and tetrazole
(51 mg, 0.73 mmol, 3 equiv) in dry CH2Cl2 was added a solution
of phosphoramidite 9 (345 mg, 0.29 mmol, 1.2 equiv) in CH2Cl2.
The mixture was stirred at room temperature for about 3 h
and cooled to -40 °C, and mCPBA (210 mg, 0.73 mmol, 3
equiv, 60%) was added. After 15 min, the cold bath was
removed, and stirring was continued for an additional hour.
The reaction mixture was diluted with CH2Cl2 and poured into
a solution of 5% Na2SO3 and saturated NaHCO3. The mixture
was extracted 3× with CH2Cl2, and the combined organic
fractions were dried over MgSO4, concentrated in vacuo, and
purified on SiO2 (hexane/acetone 3:2) to give 306 mg (0.14
mmol, 59%) of product 10 as a colorless oil, [R]D ) -14.43°
(2.47 g/dL). 1H NMR (400 MHz, CDCl3) δ 7.00-7.35 (m, 50H),
5.55-5.95 (m, 1H), 5.15-5.35 (m, 2H), 4.40-5.15 (m, 27H),
4.20-4.40 (m, 2H), 3.90-4.20 (m, 6H), 3.45-3.60 (m, 1H),
3.25-3.45 (m, 2H), 2.10-2.30 (m, 4H), 1.40-1.60 (m, 4H),
1.05-1.35 (m, 48H), 0.88 (t, 6H, J ) 7.0). 13C NMR (100 MHz,
CDCl3) δ 172.59, 138.09, 137.71, 137.25, 136.87, 136.56, 136.23,
136.17, 136.10, 136.06, 135.99, 135.90, 135.82, 135.51, 135.54,
128.65, 128.44, 128.34, 128.26, 128.02, 127.84, 127.75, 127.42,
96.64, 95.46, 78.99, 77.61, 77.34, 76.93, 76.60, 74.89, 72.79,
72.02, 70.45, 69.90, 69.70, 69.44, 69.12, 66.97, 66.62, 65.20,
41.29, 33.97, 31.92, 29.70, 29.67, 29.66, 29.51, 29.35, 29.29,
29.13, 24.78, 22.68, 14.12. 31P NMR (162 MHz, CDCl3) δ 0.35,
-0.15, -0.56 (ratio 1:2:1). IR 2924, 2853, 1743, 1455, 1273,
1022. Anal. Calcd for C117H153NO27P4: C, 65.99; H, 7.24; N,
0.66; P, 5.82. Found: C, 65.75; H, 7.24; N, 0.73; P, 6.12.
C
15H31CO).
Gen er a l P r oced u r e for Cou p lin g w ith NHS Ester s. To
a solution of compound 11 (∼10 µmol, 1 equiv) in 0.5 M TEAB
(0.5 mL, pH 7.5) was added a solution of appropriate NHS
ester (∼12 µmol, 1.2 equiv) (three of which were obtained from
Molecular Probes, Inc.) in 0.5 mL of DMF. PROXYL-SE was
prepared as described.36 The mixture was stirred at room
temperature for 18 h, and solvents were then removed in
vacuo. The residue was washed 4× with acetone and then
purified on DEAE-cellulose column with a step gradient (0 to
2 M) of triethylammonium bicarbonate (TEAB). The desired
fractions were pooled, lyophilized, converted by ion exchange
into a sodium salt, and lyophilized again.
Biotin Der iva tive 12a . Reaction of 11 (9.4 mg, 7.5 µmol)
with 6-[(biotinyoyl)amino]hexanoic acid, succinimidyl ester
(Biotin-X, SE) (4.4 mg, 9.7 µmol) yielded 6.4 mg (4 µmol, 53%)
1
of 12a . H NMR (400 MHz, D2O) δ 5.20-5.40 (m, 4H), 4.50-
4.60 (m, 1H), 4.30-4.45 (m, 1H), 3.70-4.45 (m, 9H), 3.60-
3.70 (m, 1H), 3.30-3.45 (m, 1H), 3.05-3.20 (m, 4H), 2.65-
2.95 (m, 2H), 2.00-2.50 (m, 8H), 1.40-1.80 (m, 12H), 0.90-
1.40 (m, 52H), 0.70-0.90 (m, 6H). 31P NMR (162 MHz, D2O) δ
3.22, 2.32, 1.34, 0.41 (ratio 1:1:1:1). MS MALDI (free acid) 1528
(M + 3Na), 1506 (M + 2Na), 1484 (M + Na), 1462 (M - H),
1223 (M - H - C15H31CO), 1122 (M - H - biotin). HR MALDI
C60H113N4O26P4S [M - H]- calcd 1461.60388, found 1461.60491.
NBD Der iva tive 12b. Reaction of 11 (9.9 mg, 7.9 µmol)
with 6-NBD-aminohexanoic acid, succinimidyl ester (NBD-X,
SE) (4.0 mg, 10.2 µmol) afforded 7.7 mg (5 µmol, 64%) of 12b.
1H NMR (400 MHz, D2O) δ 8.10-8.30 (m, 1H), 6.00-6.20 (m,
1H), 5.10-5.30 (m, 2H), 3.65-4.20 (m, 12H), 3.20-3.65 (m,
5H), 2.95-3.25 (m, 4H), 2.20-2.40 (m, 2H), 2.15-2.25 (m, 4H),
1.65-1.80 (m, 2H), 1.50-1.65 (m, 2H), 1.30-1.50 (m, 4H),
0.80-1.30 (m, 50H), 0.50-0.80 (m, 6H). 31P NMR (162 MHz,
D2O) δ 4.03, 2.92, 1.37, 0.47 (ratio 1:1:1:1). MS MALDI (free
acid) 1161 (M - C15H31CO).
F lu or escein Der iva tive 12c. Reaction of 11 (9.5 mg, 7.6
µmol) with 6-carboxyfluorescein, succinimidyl ester (6-FAM-
SE) (4.7 mg, 9.8 µmol) yielded 9.1 mg (5.6 µmol, 74%) of 12c.
1H NMR (400 MHz, D2O) δ 7.30-7.50 (m, 8H), 5.20-5.40 (m,
2H), 3.10-4.30 (m, 14H), 2.20-2.40 (m, 4H), 1.40-1.60 (m,
4H), 0.90-1.40 (m, 48H), 0.60-0.90 (m, 6H). 31P NMR (162
MHz, D2O) δ 4.94, 4.33, 1.21, 0.65 (ratio 1:1:1:1). MS MALDI
(free acid) 1243 (M - C15H31CO), 1123 (M - fluorescein), 884
(M - C15H31CO - fluorescein), 841 (M - C15H31CO - fluores-
cein - aminoethyl).
P ROXYL Der iva tive 12d . Reaction of 11 (9.8 mg, 7.8 µmol)
with PROXYL-SE (2.9 mg, 10.1 µmol) afforded 6.1 mg (4.3
µmol, 55%) of 12d . MS MALDI (free acid) 1292 (M-), 1123 (M
- proxyl), 1053 (M - C15H31CO), 884 (M - C15H31CO - proxyl).
HR MALDI C53H103N2O25P4 [M] calcd 1291.57950, found
1291.57679.
Bin d in g Assa ys w it h 12a a n d Ot h er Biot in yla t ed
In ositid es. A competitive homogeneous assay (AlphaScreen,
J . Org. Chem, Vol. 67, No. 16, 2002 5459

Rzepecki and Prestwich
Packard Biosciences) was employed. First, 0.2 pmol/well GST-
PLC δ1-PH domain protein was added to a white 384-well
polystyrene plate, and then 0.4 pmol/well of biotinylated
inositide, 5 µL of streptavidin donor beads, and 5 µL of anti-
GST acceptor beads (25 µL final volume) were added. The
biotinylated inositides tested were biotinylated Pea-PIP2 (12a ),
biotinylated PtdIns(4,5)P2, and biotinylated InsP6. The con-
tents of the wells were mixed, covered to shield from light,
and incubated at room temperature for 2 h before being read
on a Packard Fusion plate reader. For competition assays (data
not shown), different concentrations (from 10 nM to 10 fM) of
unlabeled di-C4 PtdIns(4)P, PtdIns(3,4,5)P3, and PtdIns(4,5)P2
were preincubated with the GST-PLC δ1-PH for 30 min prior
to addition of the other reagents. All biotinylated and short
chain inositides were obtained from Echelon Biosciences, Inc.
Ack n ow led gm en t. We thank the NIH for funding
to G.D.P. (NS 29236 and GM 57705) for this project.
The PtdIns(4,5)P2 headgroup intermediate and other
biotinylated lipids were provided by Dr. C. G. Ferguson
(Echelon Biosciences, Inc.). We also thank Dr. P. O.
Neilsen, Ms. A. Branch, and Ms. H.A. Hudson (all
Echelon) for assistance with protein reagents and with
binding assays.
J O011185A
5460 J . Org. Chem., Vol. 67, No. 16, 2002