Chemical synthesis and molecular recognition of phosphatase-resistant analogues of phosphatidylinositol-3-phosphate

Article abstract of DOI:10.1021/ja0554716

The remodeling of phosphatidylinositol polyphosphates in cellular membranes by phosphatases and kinases orchestrates the signaling by these lipids in space and time. To provide chemical tools to study the changes in cell physiology mediated by these lipids, three new metabolically stabilized (ms) analogues of phosphatidylinositol-3-phosphate (Ptdlns(3)P) were synthesized. We describe herein the total asymmetric synthesis of 3-methylphosphonate, 3-(monofluoromethyl)phosphonate and 3-phosphorothioate analogues of Ptdlns(3)P. From differentially protected D-myo-inositol key intermediates, a versatile phosphoramidite reagent was employed in the synthesis of Ptdlns(3)P analogues with diacylglyceryl moieties containing dioleoyl, dipalmitoyl, and dibutyryl chains. In addition, we introduce a new phosphorylation reagent, (monofluoromethyl)phosphonyl chloride, which has general applications for the preparation of "pK_a-matched" monofluorophosphonates. These ms-Ptdlns(3)P analogues exhibited reduced binding activities with ¹⁵N-labeled FYVE and PX domains, as significant ¹H and ¹⁵N chemical shift changes in the FYVE domain were induced by titrating ms-Ptdlns(3)P analogues into membrane-mimetic dodecylphosphocholine micelles. In addition, the Ptdlns(3)P analogues with dioleoyl and dipalmitoyl chains were substrates for the 5-kinase enzyme PIKfyve; the corresponding phosphorylated ms-PI(3,5)P₂ products were detected by radio-TLC analysis.

Full text of DOI:10.1021/ja0554716

Published on Web 12/24/2005
Chemical Synthesis and Molecular Recognition of
Phosphatase-Resistant Analogues of
Phosphatidylinositol-3-phosphate
Yong Xu,† Stephanie A. Lee,‡ Tatiana G. Kutateladze,‡ Diego Sbrissa,§
Assia Shisheva,§ and Glenn D. Prestwich*,†
Contribution from the Department of Medicinal Chemistry, The UniVersity of Utah, 419 Wakara
Way, Suite 205, Salt Lake City, Utah 84108-1257, Department of Pharmacology, UniVersity of
Colorado Health Sciences Center, Aurora, Colorado 80045, and Department of Physiology,
Wayne State UniVersity School of Medicine, 540 East Canfield, Detroit, Michigan 48201
Received August 22, 2005; E-mail: gprestwich@pharm.utah.edu
Abstract: The remodeling of phosphatidylinositol polyphosphates in cellular membranes by phosphatases
and kinases orchestrates the signaling by these lipids in space and time. To provide chemical tools to
study the changes in cell physiology mediated by these lipids, three new metabolically stabilized (ms)
analogues of phosphatidylinositol-3-phosphate (PtdIns(3)P) were synthesized. We describe herein the total
asymmetric synthesis of 3-methylphosphonate, 3-(monofluoromethyl)phosphonate and 3-phosphorothioate
analogues of PtdIns(3)P. From differentially protected D-myo-inositol key intermediates, a versatile
phosphoramidite reagent was employed in the synthesis of PtdIns(3)P analogues with diacylglyceryl moieties
containing dioleoyl, dipalmitoyl, and dibutyryl chains. In addition, we introduce a new phosphorylation reagent,
(monofluoromethyl)phosphonyl chloride, which has general applications for the preparation of “pKa-matched”
monofluorophosphonates. These ms-PtdIns(3)P analogues exhibited reduced binding activities with 15N-
labeled FYVE and PX domains, as significant 1H and 15N chemical shift changes in the FYVE domain
were induced by titrating ms-PtdIns(3)P analogues into membrane-mimetic dodecylphosphocholine micelles.
In addition, the PtdIns(3)P analogues with dioleoyl and dipalmitoyl chains were substrates for the 5-kinase
enzyme PIKfyve; the corresponding phosphorylated ms-PI(3,5)P2 products were detected by radio-TLC
analysis.
Scheme 1. Metabolic Interconversions of PtdIns(3)P
Introduction
Phosphoinositide (PtdInsPn) signaling networks are dynami-
cally modulated by proteins with lipid recognition motifs as
well as kinase, phosphatase, and phospholipase enzymatic
activities. Lipid-protein interactions form the cornerstone for
many signaling pathways, and the new discipline of functional
lipidomics1 is defining many new targets for therapeutic
interaction.2 It is now accepted that 3-phosphorylated PtdInsPn
lipidssPtdIns(3)P, PtdIns(3,4)P2, PtdIns(3,5)P2, and PtdIns-
(3,4,5)P3sare intracellular signals in mammalian cells.3 These
signaling lipids have been implicated as activators of protein
kinase C isoforms, and are putative messengers in cellular signal
cascades pertinent to inflammation, cell proliferation, transfor-
mation, protein kinesis, and cytoskeletal assembly.4,5
PtdIns(3)P is produced by the action of phosphoinositide-3-
kinase (PI 3-K)3,6 on PtdIns (Scheme 1), and has cognate binding
† The University of Utah.
‡ University of Colorado Health Sciences Center.
proteins, kinase and phosphatases that are important in cell
physiology. The PI 3-K family of enzymes is an important
§ Wayne State University School of Medicine.
(1) Feng, L.; Ferguson, C. G.; Neilsen, P. O.; Chakravarty, L.; Rzepecki, P.;
Prestwich, G. D. In Functional Lipidomics; Feng, L., Prestwich, G. D.,
Eds.; Dekker-CRC Press: New York, 2005; pp 189-210.
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Driscoll, P. C.; Woscholski, R.; Parker, P. J.; Waterfield, M. D. Annu. ReV.
Biochem. 2001, 70, 535-602.
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J. AM. CHEM. SOC. 2006, 128, 885-897
885

A R T I C L E S
Xu et al.
Scheme 2. Synthesis of the Enantiomerically Pure D-myo-Inositol
Intermediate
therapeutic target for prevention of cancer, inflammation,
allergy, thrombosis, and autoimmune disorders.5,7-11 PtdIns-
(3)P is also a substrate for PIKfyve, a 5-kinase required for
endomembrane integrity and the formation of multivesicular
bodies.12 PtdIns(3)P binds FYVE domains,13,14 and is thus an
important player in phagocytosis15 and the biochemistry of
vesicular trafficking of proteins.16 PX domains also bind PtdIns-
(3)P with high affinity, and their spatiotemporal changes mediate
important aspects of cell respiration and physiology.17 The
3-phosphatase myotubularin (MTM) and MTM-related (MTMR)
proteins18 have been identified as important PtdIns(3)P phos-
phatases that contribute to lipid remodeling and are commonly
mutated in genetic diseases.19 Very recently, PtdIns(3)P phos-
phatase activity was observed for SapM, a Mycobacterium
tuberculosis enzyme required for bacterial viability and inhibi-
tion of host cell phagolysosome biogenesis.20 To gain deeper
insights into these biological pathways, selective reagents that
could block binding, inhibit enzyme activity, activate lipid-
protein mediate pathways, or act as alternative substrates would
be highly desirable. To date, 3-modified PtdIns(3)P analogues
with these properties have not been reported.
To create metabolically stabilized analogues of lysophospha-
tidic acid (LPA), we prepared two kinds of phosphatase-resistant
moietiess(fluoromethylene)phosphonates21,22 and phos-
phorothioates23swhich were found to be receptor subtype
specific agonists. These two functional groups have second pKa
values that are matched to the normal phosphomonoester pKa
value of 6.5.24 Phosphonate esters have been used extensively
to stabilize naturally occurring phosphates, replacing the bridg-
ing oxygen or one of the phosphate oxygens with a methylene
or methyl group. The resulting analogues were less susceptible
to either acidic or enzymatic cleavage. Methylphosphonates have
been studied as metabolically stable phosphate mimics, potential
enzyme inhibitors, and probes for the elucidation of biochemical
processes,25 but these analogues have significantly higher second
pKa values than the native phosphate group. Herein we describe
the asymmetric total synthesis of methylphosphonate, (fluoro-
methyl)phosphonate, and phosphorothioate analogues of PtdIns-
(3)P phosphatase-resistant analogues of PtdIns(3)P. The utility
of these analogues in cellular, molecular, and structural biology
will be described in due course.
Results and Discussion
Chemical Synthesis of Phosphatase-Resistant Analogues
of Phosphatidylinositol-3-phosphate. Several strategies have
been employed for total asymmetric synthesis of phospho-
inositides. One approach is the use of enantiopure natural
precursors, e.g., D-glucose,26-30 L-chiro-inositol derivatives,31
and quinic acid.32-34 Kinetic resolution or desymmetrization via
enantioselective enzymatic acylations and nonenzymatic phos-
phorylation of protected myo-inositol derivatives is another
common strategy.35-38 The separation of diastereomeric deriva-
tives of myo-inositol with chiral auxiliaries is also used in many
synthetic routes.39-41 In our early work, we developed the
synthesis of enantiomerically pure PtdInsPn and a variety of
derivatives from D-glucose via the Ferrier rearrangement.28,41
In this paper, we have employed the more atom-efficient
production of the enantiomeric inositol derivatives via the
resolution of myo-inositol by crystallization of diastereomeric
D-camphor ketals.40
Each of the phosphorylated phosphatidylinositols synthesized
in this work employed the simple and elegant protection scheme
developed by Bruzik.40 The inositol 1-position was silylated with
the TBDPS group, the phosphomonoester 3-position was
protected as a benzoate group, and all the remaining hydroxyl
groups were protected as methoxymethyl (MOM) ethers. In this
way, 1-O-(tert-butyldiphenylsilyl)-2,4,5,6-O-tetrakis(methoxym-
ethylene)-myo-inositol was synthesized from myo-inositol in six
steps (Scheme 2).40
The phosphonochloridates derived from simple phosphonic
acid alkyl esters are reliable and readily accessible reagents for
the phosphonylation of alcohols to produce the corresponding
phosphonates.42,43 O,O-Dimethyl methylphosphonyl chloride can
(7) Drees, B. E.; Mills, G. B.; Rommel, C.; Prestwich, G. D. Expert Opin.
Ther. Pat. 2004, 14, 703-732.
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24, 366-376.
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Phosphatase-Resistant Analogues of PtdIns(3)P
A R T I C L E S
Scheme 3. Synthesis of (Fluoromethyl)phosphonate Chloridea
vacuo eliminated the volatile reagents and left the desired
phosphonochloridate 9 as an oily yellow residue. The product
was then used without further purification, as attempts at vacuum
distillation resulted in decomposition.
Returning to the inositol ring intermediates, the secondary
alcohol 10 was phosphorylated with methyl (fluoromethyl)-
phosphonyl chloride in the presence of t-BuOK and gave the
protected 3-(fluoromethyl)phosphonate 11a in good yield (46-
48%) (Scheme 4). Using amine bases commonly employed for
this type of reaction, e.g., triethylamine and N-methylimidazole,
resulted in poorer yields. The silyl group in the 1-position was
removed with the neutral reagent TBAF-HOAc, and the
resulting alcohols 11 were treated with one of the three
diacylglyceryl phosphoramidite reagents 19a-19c in the pres-
ence of tetrazole followed by mild oxidation with n-Bu4NIO4
to give the fully protected PtdIns(3)P derivatives 14. Using the
mild oxidation reagent n-Bu4NIO4 avoided the undesired
epoxidation of the oleic acyl double bond.
a Reagents and conditions: (a) PCl5, benzene; (b) (CH2O)n, K2CO3,
CH3OH; (c) Tf2O, 2,6-lutidine, CH2Cl2; (d) TBAF, THF; (e) t-BuNH2;
Dowex resin; then (ClCO)2, DMF, CH2Cl2; (f) n-BuLi, THF; then
Selectfluor; (g) DAST, CH2Cl2.
To prepare the PtdIns(3)P diesters, different phosphoramidites
with preattached long, medium, and short diacyl chains were
prepared (Scheme 5). For evaluation in different biological and
biophysical systems, we prepared the dioleoyl, dipalmitoyl, and
dibutyryl reagents. Following our previously reported proce-
dures, the 3-O-PMB-sn-(2R)-glycerol was esterified with various
fatty acids using DCC/DMAP to give compounds in high
yields.26 Oxidative removal of the PMB protective group with
DDQ gave the corresponding 1,2-diacyl-sn-(2S)-glycerols in
good yields without significant acyl migration. Although the
1,2-diacyl-sn-(2S)-glycerol was reasonably stable, slow purifica-
tion on silica gel facilitated acyl chain migration. Therefore,
rapid flash chromatography was essential to obtain homogeneous
1,2-diacyl-sn-(2S)-glycerols without acyl migration. Finally,
coupling these alcohols with methyl N,N-diisopropylchloro-
phosphoramidite and cyanoethyl N,N-diisopropylchlorophos-
phoramidite gave phosphorylation reagents 19 and 20 in high
yields.
be readily prepared from the corresponding methylphosphonate
by chlorination with PCl5 at room temperature (rt).44
To prepare methyl (fluoromethyl)phosphonate, we first tried
N-F “electrophilic” fluorinating agents to fluorinate dimethyl
methylphosphonate 10. The commercially available fluorinating
agents N-fluoro-1,4-diazabicyclooctane bis(tetrafluoroborate) (F-
TEDA-BF4) and N-fluorobenzenesulfonimide (NFSI) were
chosen for investigation.45 A number of procedures for depro-
tonation of methylphosphonate, and its subsequent monoflu-
orination, were evaluated. The only positive result was obtained
using F-TEDA-BF4 in anhydrous THF solvent to fluorinate the
sodium enolate, but the product yields were too low (5%) to be
practical. Thus, as illustrated in Scheme 3, we developed a new
method to prepare methyl (fluoromethyl)phosphonic chloride
(9).
Dimethyl hydroxymethylphosphonate (6) was prepared in
quantitative yield by the reaction of dimethyl phosphite with
paraformaldehdye and anhydrous potassium carbonate in metha-
nol.46 Direct fluorination of 6 with DAST at -78 °C gave a
low yield (5%). In contrast, activation of the alcohol 6 as triflate
7 (with 2,6-lutidine as the base), followed by nucleophilic
displacement with tetrabutylammonium fluoride (TBAF) in
THF, proved to be a safe and simple route to the protected
(monofluoromethyl)phosphonate 8.47 Direct chlorination of
dialkyl phosphonate with oxalyl chloride48 was unsuccessful.
However, a two-step protocol was successful. Thus, aminolysis
of 8 with tert-butylamine afforded the desired phosphonic acid
monomethyl ester tert-butylamine salt in quantitative yield.43
The tert-butylamine salt was then converted to the free mono-
methyl phosphonic acid by passage through a Dowex acidic
resin. The monomethyl phosphonate was then treated with oxalyl
chloride and catalytic DMF in CH2Cl2 initially at 0°C, followed
by stirring for 2 h at rt.49 Analysis of a reaction aliquot by 31P
NMR demonstrated complete conversion. Concentration in
The removal of protective groups from the phosphate and
hydroxyl of the fully protected inositol lipid intermediates was
performed as follows. First, (TMS)Br was used to deprotect
the phosphate methyl esters.40 The fully protected derivatives
were dissolved in (TMS)Br and CH2Cl2 (1:1, v/v) under strictly
anhydrous conditions, and the resulting solution was stirred at
rt for 1 h. After concentration in vacuo, the residue was dissolved
in a methanol-water mixture (95:5, v/v) and the resulting
solution then stirred for an additional 30 min to hydrolyze the
silyl phosphate esters. After complete drying of the reaction
mixture in vacuo, ethanethiol was added to remove all MOM
groups and provide the final products in >98% purity.40
The reported methods of preparation of phosphorothioates
vary considerably. Some methods that are suitable for specific
compounds utilize conditions that would preclude application
to the synthesis of derivatives bearing more labile functional
groups. For example, methyl and ethyl esters have often been
used as protecting groups; deprotection is then accomplished
with (TMS)I or (TMS)Br. However, this approach fails to give
practical yields with phosphonothioate or phosphorothioate
derivatives.50 Deprotection of the dibenzyl esters of phos-
(44) Esalthazor, M.; Flores, R. A. J. Org. Chem. 1980, 45, 529-531.
(45) Lal, G. S.; Pez, G. P.; Syvret, R. G. Chem. ReV. 1996, 96, 1737-1755.
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177, 1137-1145.
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1056.
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P. A.; Phillips, B. W.; Moore, W.; Smith, A. B. I. J. Am. Chem. Soc. 1997,
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Chem. 2000, 8, 507-513.
9
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A R T I C L E S
Xu et al.
Scheme 4. Synthesis of 3-(Fluoromethyl)phosphonate Analogues of PtdIns(3)Pa
a Reagents and conditions: (a) 4 or 9, t-BuOK, CH2Cl2; (b) TBAF‚3H2O, HOAc, THF; (c) 19a-19c, 1H-tetrazole, THF/CH3CN; then n-Bu4NIO4, CH3CN;
(d) (TMS)Br, CH2Cl2; then CH3OH/H2O; then EtSH.
Scheme 5. Synthesis of Glyceryl Phosphoramiditesa
The reaction did not occur in common glassware, which was
damaged in the process. The reaction rate could not be increased
without also accelerating side reactions. Finally, TASF (tris-
(dimethylamino)sulfonium difluorotrimethylsilicate) was also
explored for this deprotection,50,53 but TASF was unable to
cleave the secondary alcohol silyl ether 21.
Three phosphoramidite reagents, 20a-20c, were prepared by
the reaction of 2-cyanoethyl N,N-diisopropylchlorophosphor-
amidite with diacylglycerol preattached with different acyl
chains using N,N-diisopropylethylamine (DIPEA) as the base.54
The air-sensitive phosphoramidites were purified by rapid flash
chromatography using a basic solvent system. The phosphor-
a Reagents and conditions: (a) methyl N,N-diisopropylchlorophosphora-
midite, DIPEA, CH2Cl2; (b) 2-cyanoethyl N,N-diisopropylchlorophosphora-
midite, DIPEA, CH2Cl2.
amidites were then condensed with secondary alcohol 22 in the
presence of 1H-tetrazole to yield the phosphoramidite inter-
mediates.
Oxidation of phosphoramidite triester proved to be problem-
atic. The commonly used oxidation reagents, including MCPBA,
n-Bu4NIO4, and H2O2, oxidized both the phosphoramidite
triester and the 3-position phosphorothioate, even at low
temperatures (-78 to -20 °C). Indeed, the phosphorothioate
is readily oxidized to the corresponding phosphate when exposed
to standard oxidation reagents. Therefore, a mild oxidation
reagent was required to selectively oxidize the phosphoramidite
triester without overoxidation of the phosphorothioate. Recently,
t-BuOOSi(CH3)3 and t-BuOOH have been utilized to selectively
oxidize phosphoramidite triesters.55,56 Using t-BuOOH gave
complete oxidation of the phosphoramidite triester to phosphate
without overoxidation of the phosphorothioate. The CE groups
on the phosphorothioate were removed by using triethylamine
(TEA) plus bis((trifluoromethyl)silyl)acetamide (BTFSA) in
anhydrous acetonitrile.52 BTFSA was added to prevent the
phosphorothioate anion from undergoing realkylation. The
cleavage of the O-silyl derivatives was achieved by aqueous
hydrolysis at neutral pH to give the MOM ether-protected
intermediate. The MOM groups were removed using ethanethiol-
BF3 at rt to give the final products.57
phonothioic acids has been achieved with sodium in liquid
ammonia,51 but we sought milder reaction conditions. We turned
to the cyanoethyl ester (CE), which is widely used for the
synthesis of phosphonothioate or phosphorothioate acids. The
CE group can be deprotected under mild basic conditions.23,52
Primary, secondary, and tertiary amine, e.g., triethylamine and
tert-butylamine, will readily remove the CE protection from
phosphates and phosphorothioates at rt.
Alcohol 10 was then phosphorylated employing phosphor-
amidite methodology (Scheme 6). The resulting phosphoramidite
triester was oxidized with elemental sulfur to yield the corre-
sponding phosphorothioate triester. TBAF was frequently used
to deprotect the TBDPS protective group. However, we found
TBAF not only removed the TBDPS ether, but simultaneously
removed the CE group, despite the fact that the TBAF reagent
had been neutralized with 1 equiv of acetic acid. Apparently
the basicity of TBAF was sufficient to cleave the CE phosphate
linkage. Thus, to selectively deprotect the TBDPS group, we
selected the HF- pyridine complex, which in Py-THF solution
selectively removed the TBDPS ether. This deprotection reaction
was very slow, and required three weeks to reach completion.
Importantly, the reaction was conducted in a Teflon container.
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Phosphatase-Resistant Analogues of PtdIns(3)P
A R T I C L E S
Scheme 6. Synthesis of 3-Phosphorothioate Analogues of PtdIns(3)Pa
a Reagents and conditions: (a) bis(2-cyanoethyl) diisopropylphosphorodiamidite, 1H-tetrazole, CH3CN; then S8, CS2/Py; (b) HF‚Py-Py, THF; (c) 20a-
20c, 1H-tetrazole, THF/CH3CN; t-BuOOH, CH3CN; (d) TEA, TFBSA, CH3CN; then NH4OAc/H2O; then EtSH, BF3‚Et2O.
Consistent with the analytical TLC behavior of these mol-
ecules, each of the final amphiphilic ms-PtdIns(3)P derivatives
could be purified by conventional chromatography using a
CHCl3-MeOH-NH4OH solvent system on silica gel.58 The 1H
NMR of the PtdIns(3)P analogues illustrated the curious
solubility of these compounds and the intrinsic difficulty in
obtaining high-quality NMR spectroscopic data.27 Only broad,
15N HSQC spectra of the proteins, although the pattern of
resonance perturbations remained essentially unchanged (Figure
1C,D and data not shown).
PIKfyve Uses ms-PtdIns(3)P Analogues as Substrates.
PtdIns(3,5)P2 and PtdIns(5)P could be produced in vitro by two
mammalian enzymes: type I PIP kinase60 and PIKfyve.61
Genetic and biochemical evidence has recently accumulated to
implicate PIKfyve as the principal enzyme responsible for their
biosynthesis in the cellular context.62 We now provide evidence
for specific and high-affinity interactions between the PIKfyve
and metabolically stabilized analogues of PtdIns(3)P in vitro.
PIKfyve activity was assayed as described previously by the
[γ-32P]ATP-dependent phosphorylation of PtdIns. It was found
that each of the three PtdIns(3)P analogues having dioleoyl and
dipalmitoyl chains were substrates for the kinase PIKfyve
(Scheme 7), as phosphorylated products were detected by radio-
TLC analysis (Figure 2). Since (monofluoromethyl)phosphonate
and phosphorothioate are less polar than the phosphate group,
the resulting [5-32P]ms-PtdIns(3,5)P2 analogues migrated faster
than the unmodified [5-32P]PtdIns(3,5)P2 (Figure 2). Thus, the
stabilized phosphorothioate and phosphonate groups at the
3-position of these PtdIns(3)P analogues are recognized by the
nonclassical FYVE motif within the PIKfyve catalytic domain.63
Conclusions. In summary, we have developed a general
approach to the synthesis of methylphosphonate, (mono-
fluoromethyl)phosphonate, and phosphorothioate analogues of
PtdIns(3)P. This strategy also is applicable to the synthesis of
analogues of all other PtdInsPn and InsPn compounds. In
addition, our method enables synthesis of both saturated and
unsaturated acyl analogues of PtdInsPn. These PI(3)P analogues
exhibited reduced binding activities with 15N-labeled FYVE and
1
poorly resolved H NMR resonances could be detected in the
single-solvent system CDCl3 or CD3OD. In contrast, in CDCl3-
1
CD3OD (3:2, v/v), the H NMR resonances for the headgroup
and acyl chains were well resolved.
Binding to FYVE and PX Domains. In cellular membranes,
PtdIns(3)P is specifically recognized by a number of protein
binding partners including FYVE and PX domains.14,59 To test
whether the PtdIns(3)P analogues are able to bind the physi-
ological targets, we investigated interactions of human EEA1
FYVE and yeast Vam7 PX domains by NMR spectroscopy.
1
Significant H and 15N chemical shift changes in the FYVE
domain were induced by titrating C16-PtdIns(3)P-CH2F (16b)
embedded in membrane-mimetic dodecylphosphocholine (DPC)
micelles (Figure 1A). These perturbations paralleled chemical
shift changes seen in the FYVE domain as C4-PtdIns(3)P-
enriched DPC micelles were titrated in (Figure 1B). Thus, the
PtdIns(3)P analogue and unmodified lipid are accommodated
by the same binding pocket consisting of four Arg and two His
residues of the FYVE domain.59 However, the resonance
perturbations observed during PtdIns(3)P-CH2F titration were
smaller in magnitude, indicating weaker binding. These results
reveal the importance of two negatively charged oxygen atoms
of the 3-phosphate group of PtdIns(3)P and their hydrogen-
bonding potential for the strong anchoring of the inositol ring
by the basic residues of the FYVE domain. Similarly, when
the PX domain was treated with PtdIns(3)P-CH2F 16b(c), which
was prebound to the mixed diheptanoylphosphatidylcholine
(DHPC)/CHAPS micelles, the observed chemical shift changes
mirrored those seen upon binding of unmodified PtdIns(3)P.
Addition of PtdIns(3)P(S) 24b(c) and PtdIns(3)P-CH3 15b(c)
lipids or titrating the soluble C4 forms of PtdIns(3)P analogues
resulted in considerably smaller chemical shift changes in 1H-
1
PX domains as significant H and 15N chemical shift changes
in the FYVE domain were induced by titrating ms-PtdIns(3)P
in membrane-mimetic DPC micelles. In addition, these PtdIns-
(3)P analogues with dioleoyl and dipalmitoyl chains were
recognized by PIKfyve, as phosphorylated ms-PtdIns(3,5)P2
(60) Tollas, K. F.; Rameh, L. E.; Ishihara, H.; Shibashi, Y.; Chen, J.; Prestwich,
G. D.; Cantley, L. C.; Carpenter, C. L. J. Biol. Chem. 1998, 273, 18040-
18046.
(61) Sbrissa, D.; Ikonomov, O. C.; Shisheva, A. J. Biol. Chem. 1999, 274,
21589-21597.
(62) Shisheva, A. Cell Biol. Int. 2001, 25, 1201-1206.
(63) Sbrissa, D.; Ikonomov, O. C.; Shisheva, A. J. Biol. Chem. 2002, 277, 6073-
6079.
(58) Auger, K. R.; Carpenterg, C. L.; Cantley, L. C.; Varticovski, L. J. Biol.
Chem. 1989, 264, 20181-20184.
(59) Kutateladze, T.; Overduin, M. Science 2001, 291, 1793-1796.
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Xu et al.
1
Figure 1. PtdIns(3)P analogues are recognized by the FYVE and PX domains. Superimposed H-15N HSQC NMR spectra of the (A, B, upper left and
right) 0.2 mM EEA1 FYVE domain and (C, D, lower left and right) 0.2 mM Vam7 PX domain collected before and after addition of (A) 4 mM C16-PtdIns-
(3)P-CH2F (16b) and 250 mM d38-DPC, (B) 1 mM C4-PtdIns(3)P and 250 mM d38-DPC, (C) 2 mM C4-PtdIns(3)P-CH2F (16c) in 100 mM DHPC and 33
mM CHAPS, and (D) 2 mM C4-PtdIns(3)P in 100 mM DHPC and 33 mM CHAPS. Directions of the chemical shift changes are indicated by arrows.
Scheme 7. Phosphorylation of PtdIns(3)P Analogues by PIKfyve
Experimental Section
products were detected by radio-TLC analysis. The metaboli-
cally stabilized analogues of PtdIns(3)P reported herein provide
new tools for the elucidation of the roles of these phospho-
inositide monophosphates in cell signaling. Specific applications
of these analogues in cell and molecular biology will be
presented in due course.
General Procedures. Chemicals were purchased from Aldrich and
Acros Chemical Corp. and used without prior purification. Solvents
were reagent-grade and distilled before use: CH2Cl2 was distilled from
CaH2, and THF was distilled from sodium wire. TLC used precoated
silica gel aluminum sheets (EM Science silica gel 60F254). Flash
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Phosphatase-Resistant Analogues of PtdIns(3)P
A R T I C L E S
Figure 2. Radio-TLC shows that PtdIns(3)P analogs are recognized and phosphorylated by PIKfyve. PIKfyve activity was determined in anti-PIKfyve
immunoprecipitates with the indicated substrates and [γ-32P]ATP. Refer to the Experimental Section for details. IP, immunoprecipitates; P, preimmune
serum; I, immune anti-PIKfyve-serum.
chromatography (FC) employed Whatman 230-400 mesh ASTM silica
gel. NMR spectra were recorded on a Varian INOVA 400 at 400 MHz
(1H), 101 MHz (13C), 162 MHz (31P), and 376 MHz (19F) at 25 °C.
Chemical shifts are reported in parts per million with TMS as the
internal standard (δ ) 0.00). The internal standard for 31P NMR is
85% H3PO4 (δ ) 0.00), and that for 19F NMR is CFCl3 (δ ) 0.00).
Low- and high-resolution mass spectra were obtained on HP5971A
MSD and Finnigan MAT95 double-focusing mass spectrometer (MS)
instruments, respectively. Dibutyryl- and dipalmitoyl-PtdIns(3)P were
obtained from Echelon Biosciences (Salt Lake City, UT).
wise. The resulting mixture was allowed to warm to 0 °C over a period
of 1.5 h, whereupon the dark brown solution was diluted with ether
(300 mL). The precipitates formed were removed by filtration. The
ethereal solution was successively washed with water, 1 N HCl, and
brine and then dried over Na2SO4. After concentration, a yellow oil
was obtained, which was used in the next step without further
purification.
Dimethyl (Fluoromethyl)phosphonate (8). A solution of the triflate
7 (5.08 g, 0.015 mol) in THF (22 mL) was cooled to 0 °C before 20
mL (0.02 mol) of a 1 M solution of tetrabutylammonium fluoride in
THF was added dropwise. The solution was stirred at 0 °C for 1 h.
Solvents were then removed, and CH2Cl2 (35 mL) was added. The
organic layer was washed (H2O, 3 × 8 mL), dried (MgSO4), and
evaporated to a crude oil. This was purified by FC, using EtOAc-
hexane (1:1) as the eluent, to give 8 as a pale yellow oil (1.67 g, 67%).
1H NMR (CDCl3): δ 4.68 (dd, J ) 46.8, 4.8 Hz, 2H), 3.81 (d, J )
10.8 Hz, 6 H). 31P NMR (CDCl3): δ 19.83 (d, J ) 63.0 Hz). 19F NMR
(CDCl3): δ -250.74 (dt, J ) 61.7, 47.0 Hz). MS (CI): m/z 143.0
(M+ + 1, 100.00). HRMS (CI) (m/z) (M+ + 1): found, 143.0262; calcd
for C3H9FO3P, 143.0267.
Methyl Methylphosphonyl Chloride (4). To 12.87 g (0.104 mol)
of dimethyl methylphosphonate in 30 mL of anhydrous benzene was
added 21.6 g (0.104 mol) of PCl5 at 0 °C. The solution was stirred
vigorously for 1 h at 0 °C. The solvent was removed under vacuum.
Vacuum distillation (80 °C, 22 mmHg) gave 12.1 g (0.094 mol, 94%)
1
of homogeneous product. H NMR (CDCl3): δ 3.72 (d, J ) 6.0 Hz,
2H), 3.57 (d, J ) 10.5 Hz, 6H). 13C NMR (CDCl3): δ 56.40 (s), 54.78
(s), 53.14 (s), 53.07 (s). 31P NMR (CDCl3): δ 28.18 (s). MS (CI): m/z
129.0 (M+ + 1, 8.32). HRMS (CI) (m/z) (M+ + 1): found, 128.9872;
calcd for C2H6ClO2P, 128.9871.
(Dimethoxyphosphinyl)methyl Triflates (7). To a stirred solution
of dimethoxy (hydroxymethyl)phosphonate (30.6 mmol) and 2,6-
lutidine (37.6 mmol) in anhydrous CH2Cl2 (50 mL) at -50 °C under
N2 was added trifluoromethanesulfonic anhydride (35.5 mmol) drop-
Methyl (Fluoromethyl)phosphonate Chloride (9). 8 (0.221 g, 0.592
mmol) was dissolved in tert-butylamine (8 mL, 76 mmol) and the
resulting solution was heated at reflux overnight. The reaction was
concentrated and gave 0.256 g of product as a white salt (quantitative
9
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A R T I C L E S
Xu et al.
yield). Methyl (fluoromethyl)phosphonate tert-butylamine salt (0.290
g, 0.670 mmol) was dissolved in CHCl3 and the resulting solution
treated with cation-exchange resin (Dowex 50W-X8 (H+ form), 200-
400 mesh). The Dowex resin was removed by filtration, and the filtrate
was concentrated in vacuo to give 0.241 g (quantitative yield) of the
oily phosphonate. Next, oxalyl chloride (0.132 g, 1.04 mmol) was added
dropwise to a solution of the methyl (fluoromethyl)phosphonic acid
(0.240 g, 0.668 mmol) and DMF (2.5 µL, 0.033 mmol) dissolved in
CH2Cl2 at 0 °C. The solution was stirred at 0 °C for 20 min, then
warmed to rt, and stirred for 1.5 h. The reaction was concentrated,
dissolved in toluene (2 mL), and then reconcentrated in vacuo to remove
the volatile reagents. This gave phosphonochloridate 9 as a yellow oil,
which was then used immediately for reaction with the 3-hydroxy-
1D O-(1,2-Di-O-oleoyl-sn-(2S)-glycerol-3-O-methylphospho)-3-
(methyl methylphosphonate)-2,4,5,6-O-tetrakis(methoxymethylene)-
myo-inositol (13a). N,N-Diisopropyl-O-methyl-O-(di-(2S)-oleoyl-sn-
glycerol)phosphonamidite (0.187 g, 0.135 mmol) was added under an
argon atmosphere to a solution of 12a (110 mg, 0.089 mmol) and 1H-
tetrazole (26 mg, 0.374 mmol) in 4 mL of dry CH2Cl2/THF (1:1, v/v).
After the resulting solution was stirred for 20 h at rt, oxidation was
performed with (n-C4H9)4NIO4 (78 mg, 0.180 mmol) at -20 °C for 1
h. The reaction mixture was warmed to rt for an additional 30 min,
and after aqueous workup, the crude product was purified by FC on
silica gel (n-hexane/acetone, 3:1, v/v) to give 77 mg of a homogeneous
1
colorless oil (0.068 mmol, 76%). H NMR (CDCl3): δ 5.32 (m, 2H),
5.21 (m, 1H), 4.84-4.71 (m, 8H), 4.32-4.09 (m, 8H), 3.96-3.89 (m,
2H), 3.77-3.72 (m, 6H), 3.41-3.33 (m, 12H), 2.29 (m, 4H), 1.96 (m,
8H), 1.55 (m, 4H), 1.52 (dd, J ) 17.6, 6.8 Hz, 3H), 1.21 (m, 42H),
0.84 (t, J ) 7.2 Hz, 6H). 13C NMR (CDCl3): δ 173.15 (s), 172.73 (s),
129.91 (s), 129.66 (s), 98.91 (s), 98.54 (s), 98.45 (s), 98.37 (s), 98.03
(s), 97.85 (s), 79.13 (d, J ) 13.78 Hz), 74.24 (dd, J ) 31.48, 6.13 Hz),
69.37 (s), 65.80 (s), 65.52 (s), 61.61 (s), 56.78 (s), 56.65 (s), 56.56 (s),
56.51 (s), 55.76 (s), 54.81 (s), 53.75 (s), 52.18 (dd, J ) 35.30, 6.13
Hz), 34.08 (s), 33.96 (s), 31.88 (s), 29.65 (s), 29.63 (s), 29.61 (s), 29.45
(s), 29.31 (s), 29.25 (s), 29.22 (s), 29.09 (s), 29.06 (s), 24.78 (s), 22.64
(s), 14.07 (s), 11.03 (dd, J ) 148.83, 12.27 Hz). 31P NMR (CDCl3):
δ 34.60 (s), 33.49 (s), 0.74 (s), 0.52 (d, J ) 7.61 Hz). MS (CI): m/z
1145.7 (M+ + 1, 100.00). HRMS (CI) (m/z) (M+ + 1): found,
1145.6897; calcd for C56H107O19P2, 1145.6882.
1
containing protected inositol derivatives. H NMR (CDCl3): δ 4.62
(dd, J ) 46.8, 4.4 Hz, 2H), 3.74 (d, J ) 11.2 Hz, 3 H). 13C NMR
(CDCl3): δ 75.72 (dd, J ) 180.2, 168.7 Hz), 53.14 (d, J ) 6.1 Hz).
31P NMR (CDCl3): δ 19.80 (d, J ) 63.0 Hz). 19F NMR(CDCl3): δ
-250.98 (dt, J ) 62.8, 47.0 Hz). MS (CI): m/z 147.0 (M+ + 1, 100.00).
HRMS (CI) (m/z) (M+ + 1): found, 146.9759; calcd for C2H6ClFO2P,
146.9778.
1D 1-O-(tert-Butyldiphenylsilyl)-3-(methyl methylphosphonate)-
2,4,5,6-O-tetrakis(methoxymethylene)-myo-inositol (11a). t-BuOK
(18 mg, 0.163 mmol, 1.4 equiv) was added to a stirred solution (69
mg, 0.116 mmol) of 10 and methyl methylphosphonate chloride (18
mg, 0.139 mmol, 1.2 equiv) in CH2Cl2 (1 mL) at 0 °C, then the resulting
solution was stirred for 2 h at rt, and the reaction was complete. A
saturated aqueous solution of NH4Cl (1 mL) was added, the resulting
solution was stirred for 10 min, the aqueous phase was extracted with
CH2Cl2 (3 × 5 mL), the organic solution was dried with anhydrous
Na2SO4, and the solvent was evaporated under reduced pressure. The
crude product was purified by chromatography (n-hexane-acetone, 2:1,
1D O-(1,2-Di-O-oleoyl-sn-(2S)-glycerol-3-phospho)-3-(methylphos-
phonate)-myo-inositol (15a). The phosphate 13a (22 mg, 0.019 mmol)
in a 5 mL flask was dried in vacuo, and then anhydrous (TMS)Br (0.2
mL) and CH2Cl2 (0.2 mL) were added into the flask. The solution was
stirred at rt for 30 min. (TMS)Br and volatile products were evaporated
under high vacuum for 6 h. The residue was dissolved in MeOH-
H2O (95:5, 1.0 mL) and the resulting solution stirred for 30 min at rt.
The solution was thoroughly concentrated for an additional 3 h under
high vacuum. Ethanethiol (1 mL) was added, and the solution was kept
at rt for 1 h and then concentrated to yield the crude product.
Chromatography on silica gel (CHCl3-CH3OH-NH4OH (2.0 M), 65:
25:3, v/v/v) provided 16 mg of pure product 15a (0.017 mmol, 89%).
1H NMR (CDCl3/CD3OD, 3:2, v/v): δ 5.32 (m, 2H), 5.21 (m, 1H),
3.93 (dd, J ) 12.0, 3.6 Hz, 1H), 3.82 (m, 2H), 3.73-3.60 (m, 5H),
3.30 (m, 2H), 2.33 (m, 4H), 1.96 (m, 8H), 1.55 (m, 4H), 1.52 (dd, J )
17.6, 6.8 Hz, 3H), 1.21 (m, 42H), 0.84 (t, J ) 7.2 Hz, 6H). 13C NMR
(CDCl3/CD3OD, 3:2, v/v): δ 173.28 (s), 172.89 (s), 129.25 (s), 128.98
(s), 97.13 (s), 73.70 (s), 70.87 (s), 70.46 (s), 69.98 (s), 69.33 (s), 64.39
(s), 61.60 (s), 54.88 (s), 42.53 (s), 42.46 (s), 33.30 (s), 31.24 (s), 29.26
(s), 29.06 (s), 28.83 (s), 28.64 (s), 28.60 (s), 28.56 (s), 28.53 (s), 28.45
(s), 28.43 (s), 28.39 (s), 26.46 (s), 24.17 (s), 21.96 (s), 13.08 (s), 10.62
(d, J ) 140.80 Hz). 31P NMR (CDCl3/CD3OD, 3:2, v/v): δ 32.56 (s),
32.40 (s), -0.51 (s). MS (ESI): m/z 963.53 (M+ + Na, C46H86NaO15P2,
100.0). HRMS (MALDI) (m/z) (M+ + Na): found, 963.5312; calcd
for C46H86NaO15P2, 963.5340.
1
v/v) to afford a colorless liquid (37 mg, 0.054 mmol, 46%). H NMR
(CDCl3): δ 7.72-7.67 (m, 4H), 7.41-7.36 (m, 6H), 4.98 (d, J ) 4.4
Hz, 1H), 4.91 (d, J ) 4.4 Hz, 1H), 4.79 (d, J ) 4.4 Hz, 1H), 4.75 (m,
3H), 4.57 (d, J ) 4.8 Hz, 1H), 4.26 (d, J ) 4.8 Hz, 1H), 3.92-3.85
(m, 4H), 3.86 (d, J ) 6.0 Hz, 3H), 3.42-3.32 (m, 12H), 3.20 (d, J )
23.0 Hz, 2H), 1.34 (d, J ) 17.6 Hz, 3H), 1.06 (s, 9H). 13C NMR
(CDCl3): δ 135.93 (s), 135.81 (s), 133.80 (s), 133.77 (s), 132.51 (s),
132.48 (s), 129.95 (s), 129.86 (s), 129.80 (s), 128.01 (s), 127.89 (s),
127.71 (s), 127.69 (s), 98.93 (s), 98.35 (s), 98.30 (s), 98.22 (s), 97.46
(d, J ) 14.58 Hz), 82.04 (d, J ) 8.45 Hz), 81.28 (d, J ) 5.43 Hz),
78.88 (d, J ) 34.49 Hz), 74.84 (s), 70.49 (s), 56.05 (dd, J ) 62.05,
24.54 Hz), 27.07 (s). 31P NMR (CDCl3): δ 33.89 (s), 33.04 (s). MS
(CI): m/z 687.1 (M+ + 1, 79.67), 655.1 (M+ - OCH3, 100.00). HRMS
(CI) (m/z) (M+ + 1): found, 687.2962; calcd for C32H52O12PSi,
687.2966.
1D 3-(Methyl methylphosphonate)-2,4,5,6-O-tetrakis(methoxy-
methylene)-myo-inositol (12a). A solution of 11a (24 mg, 0.035 mmol)
in THF (1 mL) was treated with tetrabutylammonium fluoride trihydrate
(16 mg, 0.049 mmol) at rt. After the solution was stirred for 18 h, the
reaction was complete (monitored by TLC). The solvent was evaporated
under reduced pressure, and the crude product was purified by passage
through a short silica gel column (n-hexane-acetone, 3:1, v/v) to afford
12 mg of a colorless liquid (0.027 mmol, 77%). 1H NMR (CDCl3): δ
4.79-4.64 (m, 8H), 4.17 (t, J ) 10.4 Hz, 1H), 4.10 (d, J ) 20.4 Hz,
1H), 3.98 (dd, J ) 11.6, 4.4 Hz, 1H), 3.89 (dd, J ) 21.2, 9.6 Hz, 1H),
3.69 (dd, J ) 20.0, 9.2 Hz, 1H), 3.58 (td, J ) 9.2, 1.2 Hz, 1H), 3.42
(m, 1H), 3.41-3.34 (m, 15H), 1.46 (dd, J ) 18.0, 3.6 Hz, 3H). 13C
NMR (CDCl3): δ 98.52 (s), 98.35 (s), 98.31 (s), 98.26 (s), 98.17 (s),
98.11 (s), 97.97 (s), 82.93 (s), 79.11 (d, J ) 16.09 Hz), 78.41 (d, J )
6.94 Hz), 76.50 (d, J ) 6.13 Hz), 74.82 (dd, J ) 29.06, 6.84 Hz),
70.40 (d, J ) 5.33 Hz), 52.03 (dd, J ) 40.63, 6.13 Hz), 10.97 (dd, J
) 146.52, 23.73 Hz). 31P NMR (CDCl3): δ 34.60 (s), 33.36 (s). MS
(CI): m/z 449.1 (M+ + 1, 100.00). HRMS (CI) (m/z) (M+ + 1): found,
449.1779; calcd for C16H34O12P, 449.1788.
1D O-(1,2-Di-O-palmitoyl-sn-(2S)-glycerol-3-O-methylphospho)-
3-(methyl methylphosphonate)-2,4,5,6-O-tetrakis(methoxymethyl-
ene)-myo-inositol (13b). N,N-Diisopropyl-O-methyl-O-(di-(2S)-palmitoyl-
sn-glycerol)phosphonamidite (91 mg, 0.125 mmol) was added under
an argon atmosphere to a solution of 12a (37 mg, 0.083 mmol) and
1H-tetrazole (25 mg, 0.349 mmol) in 4 mL of dry CH2Cl2-THF (1:1,
v/v). After the resulting solution was stirred for 20 h at rt, oxidation
was performed with (n-C4H9)4NIO4 (69 mg, 0.160 mmol) at -20 °C
for 1 h. The reaction mixture was warmed to rt for an additional 30
min, and after aqueous workup, the crude product was chromatographed
on silica gel (n-hexane-acetone, 3:1, v/v) to give 67 mg of pure 13b
1
as a colorless oil (0.061 mmol, 73%). H NMR (CDCl3): δ 5.21 (m,
1H), 4.84-4.71 (m, 8H), 4.34-4.07 (m, 8H), 3.96-3.89 (m, 2H), 3.77-
3.72 (m, 6H), 3.41-3.33 (m, 12H), 2.29 (m, 4H), 1.55 (m, 4H), 1.52
(dd, J ) 17.6, 6.8 Hz, 3H), 1.21 (m, 48H), 0.84 (t, J ) 7.2 Hz, 6H).
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Phosphatase-Resistant Analogues of PtdIns(3)P
A R T I C L E S
13C NMR (CDCl3): δ 173.19 (s), 172.80 (s), 98.90 (s), 98.53 (s), 98.43
(s), 98.35 (s), 98.03 (s), 97.83 (s), 79.13 (d, J ) 13.78 Hz), 77.21 (s),
76.90 (s), 76.35 (s), 74.24 (dd, J ) 31.48, 6.13 Hz), 69.44 (s), 69.35
(s), 65.75 (s), 65.57 (s), 61.60 (s), 56.77 (s), 56.64 (s), 56.55 (s), 56.50
(s), 55.75 (s), 53.75 (s), 52.18 (dd, J ) 35.30, 6.13 Hz), 34.08 (s),
33.96 (s), 31.88 (s), 29.65 (s), 29.63 (s), 29.61 (s), 29.45 (s), 29.31 (s),
29.25 (s), 29.22 (s), 29.09 (s), 29.06 (s), 24.78 (s), 22.64 (s), 14.07 (s),
11.03 (dd, J ) 148.83, 12.27 Hz). 31P NMR (CDCl3): δ 34.60 (d, J )
8.74 Hz), 33.51 (d, J ) 6.64 Hz), 0.74 (s), 0.53 (d, J ) 8.74 Hz). MS
(CI): m/z 1093.7 (M+ + 1, 4.58). HRMS (CI) (m/z) (M+ + 1): found,
1093.6583; calcd for C52H103O19P2, 1093.6569.
8 mg of pure product 15c (0.016 mmol, 94%). 1H NMR (CDCl3/CD3-
OD, 3:2, v/v): δ 5.27 (m, 1H), 4.74 (s, 1H), 4.36 (dd, J ) 12.0, 3.2
Hz, 1H), 4.28 (m, 1H), 4.18-4.06 (m, 5H), 3.78 (m, 2H), 3.23 (t, J )
9.2 Hz, 1H), 2.28 (m, 4H), 1.58 (m, 7H), 0.99 (m, 6H). 13C NMR
(CDCl3/CD3OD, 3:2, v/v): δ 174.32 (s), 173.94 (s), 98.25 (s), 78.74
(m), 78.36 (m), 76.19 (m), 74.75 (s), 71.99 (s), 71.57 (s), 71.08 (s),
70.32 (s), 65.56 (s), 62.60 (s), 56.07 (s), 36.42 (s), 36.29 (s), 18.72 (s),
13.73 (d, J ) 3.84 Hz). 31P NMR (CDCl3/CD3OD, 3:2, v/v): δ 36.57
(s), 36.42 (s), 3.47 (s), 3.31 (s). MS (ESI): m/z 575.13 (M+ + Na,
C18H34NaO15P2). HRMS (MALDI) (m/z) (M+ + Na): found, 575.1296;
calcd for C18H34NaO15P2, 575.1271.
1D O-(1,2-Di-O-palmitoyl-sn-(2S)-glycerol-3-phospho)-3-(meth-
ylphosphonate)-myo-inositol (15b). The phosphate 13b (29 mg, 0.027
mmol) in a 5 mL flask was thoroughly dried in vacuo, and then
anhydrous (TMS)Br (0.2 mL) and CH2Cl2 (0.2 mL) were added. The
solution was stirred at rt for 30 min. (TMS)Br and volatile products
were evaporated under high vacuum for 6 h. The residue was dissolved
in MeOH-H2O (95:5, 1.0 mL) and the resulting solution stirred for
30 min at rt. The solution was thoroughly concentrated for an additional
3 h under high vacuum. Ethanethiol (1 mL) was added, and the solution
was kept at rt for 1 h and then concentrated to yield the crude product.
Chromatography on silica gel (CHCl3-CH3OH-NH4OH (2.0 M), 65:
25:3, v/v/v) provided 22 mg of pure product 15b (0.025 mmol, 92%).
1H NMR (CDCl3-CD3OD, 3:2, v/v): δ 5.27 (m, 1H), 3.93 (dd, J )
12.0, 3.6 Hz, 1H), 3.82 (m, 2H), 3.73-3.60 (m, 6H), 3.30 (m, 2H),
2.33 (m, 4H), 1.62 (m, 4H), 1.56 (dd, J ) 17.6, 6.8 Hz, 3H), 1.27 (m,
48H), 0.84 (t, J ) 7.2 Hz, 6H). 13C NMR (CDCl3/CD3OD, 3:2, v/v):
δ 173.17 (s), 172.75 (s), 98.20 (s), 73.80 (s), 70.97 (s), 70.55 (s), 70.02
(s), 69.74 (s), 64.52 (s), 61.81 (s), 54.97 (s), 33.45 (s), 33.42 (s), 31.36
(s), 29.32 (s), 29.12 (s), 28.85 (s), 28.64 (s), 28.70 (s), 14.01 (s), 10.58
(d, J ) 138.20 Hz). 31P NMR (CDCl3/CD3OD, 3:2, v/v): δ 32.45 (s),
32.30 (s), 0.74 (s), -0.55 (s). MS (ESI): m/z 911.50 (M+ + Na, C42H82-
NaO15P2, 100.00). HRMS (MALDI) (m/z) (M+ + Na): found,
911.5045; calcd for C42H82NaO15P2, 911.5027.
1D 1-O-(tert-Butyldiphenylsilyl)-3-(methyl (fluoromethyl)phos-
phonate)-2,4,5,6-O-tetrakis(methoxymethylene)-myo-inositol (11b).
t-BuOK (230 mg, 2.31 mmol, 1.4 equiv) was added to a stirred solution
of 980 mg (1.65 mmol) of 10 and methyl (fluoromethyl)phosphonate
chloride (1.99 mmol, 1.2 equiv) in CH2Cl2 (10 mL) at 0 °C; stirring
was continued for 2 h at rt to complete the reaction. A saturated aqueous
solution of NH4Cl (1 mL) was added, the resulting solution was stirred
for 10 min, and the aqueous phase was extracted with CH2Cl2 (3 × 5
mL). The combined organic phases were dried with anhydrous Na2-
SO4, and the solvent was removed in vacuo. The crude product was
purified by chromatography (n-hexane-acetone, 3:1, v/v) to afford 552
mg of 11b as a colorless liquid (0.784 mmol, 48%). 1H NMR
(CDCl3): δ 7.70-7.65 (m, 4H), 7.42-7.34 (m, 6H), 4.97 (d, J ) 2.0
Hz, 1H), 4.96 (d, J ) 2.4 Hz, 1H), 4.90 (d, J ) 6.0 Hz, 1H), 4.76 (m,
3H), 4.62 (m, 1H), 4.50 (m, 2H), 4.46 (m, 1H), 3.98-3.73 (m, 5H),
3.52 (m, 2H), 3.43-3.23 (m, 14H), 1.05 (s, 9H). 13C NMR (CDCl3):
δ 135.99 (s), 135.85 (s), 133.79 (s), 133.76 (s), 132.48 (s), 130.08 (s),
129.97 (s), 129.91 (s), 129.89 (s), 128.10 (s), 127.99 (s), 127.81 (s),
127.77 (s), 99.14 (s), 99.09 (s), 98.86 (s), 98.67 (s), 98.38 (s), 97.78
(s), 97.55 (s), 78.85 (m), 78.04 (s), 77.76 (s), 76.42 (m), 75.38 (s),
75.31 (s), 75.23 (s), 73.54 (d, J ) 7.68 Hz), 56.68 (d, J ) 3.84 Hz),
56.63 (s), 56.43 (s), 53.10 (d, J ) 6.16 Hz), 52.46 (d, J ) 6.97 Hz),
27.13 (s), 27.10 (s), 19.11 (s), 19.08 (s). 31P NMR (CDCl3): δ 19.57
(d, J ) 61.88 Hz), 18.91 (d, J ) 64.15 Hz). 19F NMR (CDCl3): δ
-249.64 (m). MS (ESI): m/z 705.3 (M+ + 1, 100.00). HRMS
(MALDI): (m/z) (M+ + Na): found, 727.2685; calcd for C32H50FNaO12-
PSi, 727.2691.
1D O-(1,2-Di-O-butanoyl-sn-(2S)-glycerol-3-O-methylphospho)-
3-(methyl methylphosphonate)-2,4,5,6-O-tetrakis(methoxymethyl-
ene)-myo-inositol (13c). To a solution of alcohol 12a (17 mg, 0.038
mmol) in dry THF (0.5 mL) were added N,N-diisopropyl-O-(methyl)-
O-di-O-butanoyl-sn-(2S)-glycerol)phosphonamidite (25 mg, 24 µL,
0.125 mmol) and 1H-tetrazole (26 mg, 0.374 mmol). The mixture was
stirred at rt for 16 h. Then oxidation was performed with (n-C4H9)4-
NIO4 (76 mg, 0.174 mmol) at -20 °C for 1 h. The reaction mixture
was warmed to rt for an additional 30 min. The solution was diluted
with methylene chloride (20 mL) and washed with 10% sodium
bisulfite. The organic layer was concentrated, and the residue was
chromatographed on silica gel (n-hexane-acetone, 1:1, v/v) to give
1D 3-(Methyl (fluoromethyl)phosphonate)-2,4,5,6-O-tetrakis-
(methoxymethylene)-myo-inositol (12b). A solution of 11b (40 mg,
0.057 mmol) in THF (1 mL) was treated with tetrabutylammonium
fluoride trihydrate (36 mg, 0.114 mmol) and acetic acid (7 µL, 0.114
mmol) at rt. After the resulting solution was stirred for 12 h, TLC
indicated that the reaction was complete; solvent was evaporated in
vacuo, and the crude product was purified by passage through a short
silica column (n-hexane-acetone, 1:1, v/v) to afford 15 mg of 12b as
1
1
25 mg of pure 13c as a colorless oil (0.033 mmol, 87%). H NMR
a colorless liquid (0.032 mmol, 56%). H NMR (CDCl3): δ 4.86-
(CDCl3): δ 5.23 (m, 1H), 4.84-4.74 (m, 8H), 4.34-4.07 (m, 8H),
3.96-3.89 (m, 2H), 3.77-3.72 (m, 6H), 3.41-3.33 (m, 12H), 2.31
(m, 4H), 1.62 (m, 4H), 1.52 (dd, J ) 17.6, 6.8 Hz, 3H), 0.93 (t, J )
7.2 Hz, 3H), 0.90 (t, J ) 7.2 Hz, 3H). 13C NMR (CDCl3): δ 173.03
(s), 172.90 (s), 98.90 (s), 98.45 (s), 97.98 (s), 97.85 (s), 98.03 (s), 79.20
(m), 74.28 (d, J ) 31.48 Hz), 69.36 (s), 65.65 (d, J ) 23.03 Hz), 61.57
(s), 56.79 (s), 56.57 (s), 55.78 (s), 54.59 (s), 52.20 (dd, J ) 35.30,
6.13 Hz), 35.93 (s), 35.83 (s), 18.26 (s), 13.57 (s), 11.03 (dd, J )
148.83, 12.27 Hz). 31P NMR (CDCl3): δ 34.65 (s), 33.54 (s), 0.75 (s),
0.52 (d, J ) 7.61 Hz). MS (ESI): m/z 757.41 (M+ + 1, 100.00). HRMS
(MALDI) (m/z) (M+ + Na): found, 779.2627; calcd for C28H54NaO19P2,
779.2632.
4.68 (m, 10H), 4.32 (m, 1H), 4.17 (m, 1H), 3.98 (m, 1H), 3.87 (dd, J
) 10.8, 3.2 Hz, 3H), 3.65 (t, J ) 9.6 Hz, 1H), 3.41-3.34 (m, 12H),
3.20 (m, 2 H). 13C NMR (CDCl3): δ 98.73 (s), 98.66 (s), 98.48 (s),
98.38 (s), 98.18 (s), 83.40 (s), 83.00 (s), 78.72 (d, J ) 90.09 Hz), 78.33
(d, J ) 90.09 Hz), 76.10 (d, J ) 10.00 Hz), 70.43 (d, J ) 6.16 Hz),
58.68 (s), 56.65 (s), 56.52 (s), 56.37 (s), 56.12 (s), 55.91 (s), 55.85 (s),
53.52 (s), 53.11 (s). 31P NMR (CDCl3): δ 34.60 (d, J ) 62.05 Hz),
33.36 (d, J ) 65.12 Hz). 19F NMR (CDCl3): δ -249.36 (dt, J ) 60.19,
45.88 Hz), -250.08 (dt, J ) 62.78, 45.88 Hz). MS (CI): m/z 467.2
(M+ + 1, 100.00). HRMS (MALDI) (m/z) (M+ + Na): found,
489.1540; calcd for C16H32FNaO12P, 489.1508.
1D O-(1,2-Di-O-oleoyl-sn-(2S)-glycerol-3-O-methylphospho)-3-
(methyl (fluoromethyl)phosphonate)-2,4,5,6-O-tetrakis(methoxy-
methylene)-myo-inositol (14a). N,N-Diisopropyl-O-methyl-O-(di-(2S)-
oleoyl-sn-glycerol)phosphoramidite (77 mg, 0.093 mmol) was added
under an argon atmosphere to a solution of 12b (29 mg, 0.062 mmol)
and 1H-tetrazole (0.73 mL, 3 wt %, 0.249 mmol) in 4 mL of dry CH3-
CN-THF (1:1, v/v). After the resulting solution was stirred for 20 h
at rt, oxidation was performed with (n-C4H9)4NIO4 (40 mg, 0.093 mmol)
1D O-(1,2-Di-O-butanoyl-sn-(2S)-glycerol-3-phospho)-3-(meth-
ylphosphonate)-myo-inositol (15c). The phosphate 13c (12 mg, 0.016
mmol) was dried and reacted with (TMS)Br and the resulting compound
then hydrolyzed in aqueous MeOH as described above for 15a.
Ethanethiol (1 mL) was added, and the solution was kept at rt for 1 h
and then concentrated to yield the crude product. Chromatography on
silica gel (CHCl3-CH3OH-NH4OH (2.0 M), 65:25:3, v/v/v) provided
9
J. AM. CHEM. SOC. VOL. 128, NO. 3, 2006 893

A R T I C L E S
Xu et al.
at -20 °C for 1 h. The reaction mixture was warmed to rt for an
additional 30 min, and after aqueous workup the crude product was
purified by FC (n-hexane-acetone, 2:1, v/v) to give 60 mg of pure
product 14a as colorless oil (0.052 mmol, 83%). 1H NMR (CDCl3): δ
5.30 (m, 4H), 5.21 (m, 1H), 4.84-4.67 (m, 10H), 4.32-4.09 (m, 8H),
3.93 (m, 2H), 3.86 (d, J ) 11.2 Hz, 3H), 3.78 (dd, J ) 10.8, 3.2 Hz,
3H), 3.41 (m, 12H), 2.29 (m, 4H), 1.96 (m, 8H), 1.57 (m, 4H), 1.26
(m, 42H), 0.84 (t, J ) 7.2 Hz, 6H). 13C NMR (CDCl3): δ 173.16 (s),
172.78 (s), 129.96 (s), 129.64 (s), 98.86 (s), 98.70 (s), 98.42 (s), 98.18
(s), 97.93 (s), 78.94 (d, J ) 6.16 Hz), 76.39 (m), 75.40 (d, J ) 22.32
Hz), 75.30 (d, J ) 22.42 Hz), 69.34 (m), 65.82 (s), 65.51 (s), 61.56
(s), 56.73 (s), 56.56 (s), 55.83 (s), 55.56 (s), 54.80 (d, J ) 6.16 Hz),
54.60 (d, J ) 8.08 Hz), 53.60 (d, J ) 6.06 Hz), 53.25 (d, J ) 6.16
Hz), 34.04 (s), 33.92 (s), 31.85 (s), 29.70 (s), 29.67 (s), 29.47 (s), 29.26
(s), 29.17 (s), 29.15 (s), 29.08 (s), 29.05 (s), 29.02 (s), 27.16 (s), 27.12
(s), 24.75 (s), 22.63 (s), 14.06 (s). 31P NMR (CDCl3): δ 19.53 (d, J )
63.02 Hz), 19.13 (d, J ) 67.39 Hz), 0.57 (s). 19F NMR (CDCl3): δ
-249.23 (m), -250.08 (m). MS (ESI): m/z 1163.7 (M+ + 1, 100.00).
HRMS (MALDI) (m/z) (M+ + Na): found, 1185.6608; calcd for
C56H105FNaO19P2, 1185.6607.
(MALDI) (m/z) (M+ + Na): found, 1133.6325; calcd for C52H101
-
FNaO19P2, 1133.6294.
1D O-(1,2-Di-O-palmitoyl-sn-(2S)-glycerol-3-phospho)-3-((fluoro-
methyl)phosphonate)-myo-inositol (16b). The phosphate 14b (35 mg,
0.032 mmol) was dried and treated with (TMS)Br, the resulting ethers
were hydrolyzed in aqueous MeOH, and the crude product was dried
as described above for 15a. Ethanethiol (1 mL) was added, and the
solution was kept at rt for 1 h and concentrated to yield the crude
product. FC on silica gel (CHCl3-CH3OH-NH4OH (2.0 M), 65:25:3,
1
v/v/v) provided 21 mg of 16b (0.022 mmol, 67%). H NMR (CDCl3/
CD3OD, 3:2, v/v): δ 5.21 (m, 1H), 4.78 (m, 1H), 4.73 (m, 1H), 4.66
(m, 1H), 4.14 (m, 4H), 3.77 (m, 2H), 3.38 (m, 2H), 2.28 (m, 4H), 1.56
(m, 4H), 1.24 (m, 48H), 0.83 (t, J ) 7.2 Hz, 6H). 13C NMR (CDCl3/
CD3OD, 3:2, v/v): δ 174.26 (s), 173.87 (s), 98.10 (s), 71.70 (m), 71.24
(m), 70.12 (s), 70.04 (s), 65.37 (m), 62.43 (m), 56.01 (s), 34.36 (s),
30.03 (s), 29.80 (s), 29.60 (s), 29.58 (s), 29.42 (m), 27.47 (s), 27.44
(s), 25.12 (s), 22.9 (s), 14.22 (s). 31P NMR (CDCl3/CD3OD, 3:2, v/v):
δ 21.25 (d, J ) 63.02 Hz), 21.10 (d, J ) 67.39 Hz), 3.61 (s), 3.36 (s).
19F NMR (CDCl3/CD3OD, 3:2, v/v): δ -245.96 (m). MS (ESI): m/z
959.63 (M+ + NH4, C42H91FN3O15P2, 100.00). HRMS (MALDI) (m/z)
(M+ + Na): found, 958.5884; calcd for C42H91FN3O15P2, 958.5909.
1D O-(1,2-Di-O-oleoyl-sn-(2S)-glycerol-3-phospho)-3-((fluoro-
methyl)phosphonate)-myo-inositol (16a). The phosphate 14a (32 mg,
0.028 mmol) was dried and reacted with (TMS)Br, the resulting
compound then hydrolyzed in aqueous MeOH, and the crude product
dried as described above for 15a. Ethanethiol (1 mL) was added, and
the solution was kept at rt for 1 h and concentrated to yield the crude
product. Chromatography on silica gel (CHCl3-CH3OH-NH4OH (2.0
M), 65:25:3, v/v/v) provided 20 mg of pure product 16a (0.021 mmol,
1D O-(1,2-Di-O-butanoyl-sn-(2S)-glycerol-3-O-methylphospho)-
3-(methyl (fluoromethyl)phosphonate)-2,4,5,6-O-tetrakis(methoxy-
methylene)-myo-inositol (14c). To a solution of alcohol 12b (12 mg,
0.026 mmol) in dry THF (0.5 mL) were added N,N-diisopropyl-O-
(methyl-O-di-butanoyl-sn-(2S)-glycerol)phosphoramidite (145 mg, 0.039
mmol) and 1H-tetrazole (0.31 mL, 3 wt %, 0.104 mmol). The mixture
was stirred at rt for 16 h. Then oxidation was performed with (n-C4H9)4-
NIO4 (17 mg, 0.039 mmol) at -20 °C for 1 h. The reaction mixture
was warmed to rt for an additional 30 min. The solution was diluted
with methylene chloride (20 mL) and washed with 10% sodium
bisulfite. The organic layer was concentrated, and the residue was
purified by FC (n-hexane-acetone, 2:1, v/v) to give 14 mg of pure
1
75%). H NMR (CDCl3/CD3OD, 3:2, v/v): δ 5.29 (m, 4H), 5.21 (m,
1H), 4.78 (m, 1H), 4.73 (m, 1H), 4.66 (m, 1H), 4.14 (m, 4H), 3.77 (m,
2H), 3.38 (m, 2H), 2.28 (m, 4H), 1.96 (m, 8H), 1.56 (m, 4H), 1.24 (m,
42H), 0.83 (t, J ) 7.2 Hz, 6H). 13C NMR (CDCl3/CD3OD, 3:2, v/v):
δ 174.26 (s), 173.87 (s), 130.27 (s), 129.97 (s), 98.10 (s), 71.70 (m),
71.24 (m), 70.12 (s), 70.04 (s), 65.37 (m), 62.43 (m), 56.01 (s), 34.36
(s), 30.03 (s), 29.80 (s), 29.60 (s), 29.58 (s), 29.42 (m), 27.47 (s), 27.44
(s), 25.12 (s), 22.9 (s), 14.22 (s). 31P NMR (CDCl3/CD3OD, 3:2, v/v):
δ 21.25 (d, J ) 63.02 Hz), 21.10 (d, J ) 67.39 Hz), 3.61 (s), 3.36 (s).
19F NMR (CDCl3/CD3OD, 3:2, v/v): δ -245.96 (m). MS (ESI): m/z
1015.6 (M+ + Na, C46H91FN2NaO15P2). HRMS (MALDI) (m/z) (M+
+ Na): found, 1015.5803; calcd for C46H91FN2NaO15P2, 1015.5776.
1
14c as a colorless oil (0.018 mmol, 70%). H NMR (CDCl3): δ 5.23
(m, 1H), 4.85-4.69 (m, 10H), 4.34-4.07 (m, 8H), 3.94 (t, J ) 10.0
Hz, 2H), 3.87 (t, J ) 9.6 Hz, 3H), 3.97 (dd, J ) 5.6, 4.4 Hz, 3H),
3.42-3.37 (m, 12H), 2.29 (m, 4H), 1.62 (m, 4H), 0.93 (t, J ) 7.2 Hz,
3H), 0.90 (t, J ) 7.2 Hz, 3H). 13C NMR (CDCl3): δ 173.06 (s), 172.90
(s), 98.89 (s), 98.73 (s), 98.46 (s), 97.96 (s), 79.01 (s), 76.44 (s), 75.54
(s), 69.34 (s), 65.85 (s), 61.56 (s), 56.81 (s), 56.59 (s), 55.87 (s), 35.94
(s), 35.83 (s), 18.28 (s), 13.60 (s), 13.54(s). 31P NMR (CDCl3): δ 19.55
(dd, J ) 60.75, 3.24 Hz), 19.11 (dd, J ) 63.99, 5.34 Hz), 0.74 (s),
0.68 (s), 0.59 (s), 0.56 (s). 19F NMR (CDCl3): δ -249.26 (m), -250.01
(m). MS (CI): m/z 775.46 (M+ + 1, 100.00). HRMS (CI) (m/z) (M+
+ 1): found, 775.2751; calcd for C28H54FO19P2, 775.2719.
1D O-(1,2-Di-O-palmitoyl-sn-(2S)-glycerol-3-O-methylphospho)-
3-(methyl (fluoromethyl)phosphonate)-2,4,5,6-O-tetrakis(methoxy-
methylene)-myo-inositol (14b). N,N-Diisopropyl-O-methyl-O-(di-(2S)-
palmitoyl-sn-glycerol)phosphoramidite (90 mg, 0.123 mmol) was added
under an argon atmosphere to a solution of 12b (38 mg, 0.082 mmol)
and 1H-tetrazole (1.0 mL, 3 wt %, 0.320 mmol) in 4 mL of dry CH3-
CN-THF (1:1, v/v). After the resulting solution was stirrred for 20 h
at rt, oxidation was performed with (n-C4H9)4NIO4 (60 mg, 0.123 mmol)
at -20 °C for 1 h. The reaction mixture was warmed to rt for an
additional 30 min, and after aqueous workup the crude product was
purified by FC (n-hexane-acetone, 2:1, v/v) to give 49 mg of pure
1D O-(1,2-Di-O-butanoyl-sn-(2S)-glycerol-3-phospho)-3-((fluo-
romethyl)phosphonate)-myo-inositol (16c). The phosphate 14c (17
mg, 0.022 mmol) was dried and treated with (TMS)Br, the silyl ethers
were hydrolyzed in aqueous MeOH, and the crude product was dried
as described above for 16a. Ethanethiol (1 mL) was added, the solution
was kept at rt for 1 h and concentrated, and the crude product was
chromatographed on silica gel (CHCl3-CH3OH-NH4OH (2.0 M),
65:25:3, v/v/v) to give pure product as a white powder (9 mg, 0.015
1
14b as a colorless oil (49 mg, 0.044 mmol, 54%). H NMR (CDCl3):
δ 5.22 (m, 1H), 4.85-4.69 (m, 10H), 4.34-4.10 (m, 8H), 3.95 (t, J )
9.6 Hz, 2H), 3.87 (d, J ) 10.8 Hz, 1H), 3.79 (d, J ) 11.2 Hz, 1H),
3.43-3.39 (m, 12H), 2.29 (m, 4H), 1.58 (m, 8H), 1.23 (m, 48H), 0.86
(t, J ) 7.2 Hz, 6H). 13C NMR (CDCl3): δ 173.19 (s), 172.80 (s), 98.90
(s), 98.53 (s), 98.43 (s), 98.35 (s), 98.03 (s), 97.83 (s), 79.13 (d, J )
13.78 Hz), 77.21 (s), 76.90 (s), 76.35 (s), 74.24 (dd, J ) 31.48, 6.13
Hz), 69.44 (s), 69.35 (s), 65.75 (s), 65.57 (s), 61.60 (s), 56.77 (s), 56.64
(s), 56.55 (s), 56.50 (s), 55.75 (s), 53.75 (s), 52.18 (dd, J ) 35.30,
6.13 Hz), 34.08 (s), 33.96 (s), 31.88 (s), 29.65 (s), 29.63 (s), 29.61 (s),
29.45 (s), 29.31 (s), 29.25 (s), 29.22 (s), 29.09 (s), 29.06 (s), 24.78 (s),
22.64 (s), 14.07 (s), 11.03 (dd, J ) 148.83, 12.27 Hz). 31P NMR
(CDCl3): δ 19.53 (d, J ) 61.88 Hz), 0.74 (s). 19F NMR (CDCl3): δ
-249.09 (m). MS (ESI): m/z 1133.6 (M+ + 1, 100.00). HRMS
1
mmol, 68%). H NMR (CDCl3/CD3OD, 3:2, v/v): δ 5.18 (m, 1H),
4.77 (m, 1H), 4.66 (dd, J ) 12.0, 5.2 Hz, 1H), 4.43-4.15 (m, 2H),
4.12-4.07 (m, 4H), 3.95 (m, 1H), 3.74 (m, 1H), 3.15 (m, 2H), 2.23
(m, 4H), 1.56 (m, 4H), 0.86 (m, 6H). 13C NMR (CDCl3): δ 174.46
(s), 174.07 (s), 130.44 (s), 130.16 (s), 74.70 (d, J ) 9.16 Hz), 71.51
(s), 71.15 (s), 70.36 (d, J ) 7.64 Hz), 65.61 (s), 62.77 (s), 34.61 (s),
34.49 (s), 32.40 (s), 30.23 (s), 30.00 (s), 29.81 (s), 29.77 (s), 29.72 (s),
29.70 (s), 29.61 (s), 29.55 (s), 27.65 (s), 25.34 (s), 23.13 (s), 14.32 (s).
31P NMR (CDCl3/CD3OD, 3:2, v/v): δ 21.49 (s), 21.09 (s), 3.35 (s),
3.32 (s). 19F NMR (CDCl3/CD3OD, 3:2, v/v): δ -246.28 (m). MS
(ESI): m/z 571.26 (M+ + 1, 18.00). HRMS (MALDI) (m/z) (M+
+
Na): found, 593.1199; calcd for C18H33FNaO15P2, 593.1176.
9
894 J. AM. CHEM. SOC. VOL. 128, NO. 3, 2006

Phosphatase-Resistant Analogues of PtdIns(3)P
A R T I C L E S
N,N-Diisopropyl-O-(cyanoethyl)-O-(dioleoyl-sn-(2S)-glycerol)-
phosphonamidite (20a). A solution of 53 mg of 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite (0.226 mmol) and 35 mg (47 µL,
0.271 mmol) of DIPEA in 3 mL of anhydrous CH2Cl2 was cooled to
0 °C under nitrogen. To this solution was added 140 mg (0.226 mmol)
of 1,2-dioleoyl-sn-(2S)-glycerol with vigorous stirring. After being
stirred at 0 °C for 1 h and at rt for 3 h under nitrogen, the solution was
separated from the white precipitate. FC on silica gel (hexanes-
EtOAc-TEA, 100:10:1, v/v/v) gave 96 mg of 20a (0.118 mmol, 52%)
2 h, sulfur (100 mg) and CS2/pyridine (1.0 mL, 1:1, v/v) were added.
After being stirred at rt for 2 h, the reaction mixture was filtered, and
the filtrate was washed with brine, dried over Na2SO4, and concentrated.
FC (EtOAc-hexane, 1:3, v/v) gave 68 mg of 21 as a colorless oil (0.085
mmol, 91%). 1H NMR (CDCl3): δ 7.71-7.65 (m, 4H), 7.44-7.34 (m,
6H), 4.97 (d, J ) 6.4 Hz, 1H), 4.92 (d, J ) 6.0 Hz, 1H), 4.85 (d, J )
6.4 Hz, 1H), 4.78 (d, J ) 7.2 Hz, 1H), 4.73 (d, J ) 6.4 Hz, 1H), 4.53
(d, J ) 6.8 Hz, 1H), 4.43 (d, J ) 6.8 Hz, 1H), 4.22-3.86 (m, 8H),
3.44-3.34 (m, 12H), 3.23 (s, 3H), 2.67-2.58 (m, 4H), 1.06 (s, 9H).
13C NMR (CDCl3): δ 136.00 (s), 135.89 (s),133.88 (s), 132.60 (s),
129.89 (s), 128.16 (s), 127.82 (s), 116.39 (s), 99.19 (s), 98.86 (s), 98.45
(s), 97.71 (s), 78.91 (s), 78.76 (s), 78.16 (d, J ) 5.45 Hz), 77.88 (s),
77.24 (d, J ) 6.16 Hz), 73.52 (s), 62.37 (d, J ) 3.84 Hz), 61.98 (d, J
) 3.13 Hz), 56.74 (s), 56.72 (s), 55.59 (s), 27.15 (s), 19.22 (s), 19.17
(s), 19.14 (s), 19.07 (s), 18.98 (s). 31P NMR (CDCl3): δ 68.69 (s). MS
(CI): m/z 797.3 (M+ + 1, 43.15). HRMS (CI) (m/z) (M+): found,
796.2845; calcd for C36H53N2O12PSSi, 796.2826.
1
as a colorless oil. H NMR (CDCl3): δ 5.32 (m, 4H), 5.17 (m, 1H),
4.32 (m, 1H), 4.13 (m, 1H), 3.85-3.52 (m, 6H), 2.61 (t, J ) 6.8 Hz,
2H), 2.28 (m, 4H), 1.98 (m, 8H), 1.58 (m, 4H), 1.26 (m, 42H), 1.14
(m, 12H), 0.86 (t, J ) 7.2 Hz, 6H). 13C NMR (CDCl3): δ 173.35 (s),
172.96 (s), 129.99 (s), 129.69 (s), 62.36 (s), 61.77 (s), 61.62 (s), 58.57
(s), 58.48 (s), 58.30 (s), 43.20 (s), 43.08 (s), 34.28 (s), 34.08 (s), 33.36
(s), 31.88 (s), 29.75 (s), 29.70 (s), 29.51 (s), 29.31 (s), 29.19 (s), 29.12
(s), 28.96 (s), 27.20 (s), 27.16 (s), 24.89 (s), 24.86 (s), 24.60 (s), 24.53
(s), 22.66 (s), 20.38 (s), 20.31 (s), 14.10 (s). 31P NMR (CDCl3): δ
150.61 (s), 150.46 (s). MS (CI): m/z 821.6 (M+ + 1, C48H90N2O6P,
41.22). HRMS (CI) (m/z) (M+ + 1): found, 821.6562; calcd for
C48H90N2O6P, 821.6537.
1D 3-(Bis(cyanoethyl) phosphothionate)-2,4,5,6-O-tetrakis(meth-
oxymethylene)-myo-inositol (22). A solution of 35 mg (0.035 mmol)
of 21 in THF (1 mL) was added along with anhydrous pyridine (0.4
mL) and hydrogen fluoride-pyridine complex (70%, 0.2 mL) at rt to
a Teflon container. After the resulting solution was stirred for 3 weeks,
the reaction was complete as determined by TLC; the reaction was
then diluted with ethyl acetate (30 mL) and washed with 10% sodium
bicarbonate (8 mL × 2). The organic layer was washed with brine,
dried over Na2SO4, and concentrated. FC (acetone-hexane, 1:3, v/v)
N,N-Diisopropyl-O-(cyanoethyl)-O-(dipalmitoyl-sn-(2S)-glycerol)-
phosphonamidite (20b). A solution of 38 mg (0.162 mmol) of
2-cyanoethyl N,N-diisopropylchlorophosphoramidite and 25 mg (34 µL,
0.194 mmol) of DIPEA in 3 mL of anhydrous CH2Cl2 was cooled to
0 °C under nitrogen. To this solution was added 92 mg (0.162 mmol)
of 1,2-dipalmitoyl-sn-(2S)-glycerol with vigorous stirring. After being
stirred at 0 °C for 1 h and at rt for 3 h under nitrogen, the solution was
separated from the white precipitate. FC on silica gel (hexanes-
EtOAc-TEA, 100:10:1, v/v/v) gave 67 mg of 20b (0.087 mmol, 54%)
1
gave 21 mg of 22 as a colorless liquid (0.038 mmol, 86%). H NMR
(CDCl3): δ 4.85-4.71 (m, 8H), 4.34 (m, 5H), 4.20 (s, 1H), 4.10 (s,
1H), 4.01 (t, J ) 10.0 Hz, 1H), 3.64 (t, J ) 10.0 Hz, 1H), 3.44 (m,
14H), 2.75 (m, 4H). 13C NMR (CDCl3): δ 115.48 (s), 108.75 (s), 97.73
(s), 97.50 (s), 97.14 (s), 82.50 (s), 78.21 (d, J ) 1.62 Hz), 77.34 (d, J
) 6.87 Hz), 75.57 (d, J ) 6.97 Hz), 69.43 (s), 61.72 (d, J ) 3.84 Hz),
61.47 (d, J ) 3.94 Hz), 55.76 (s), 55.39 (s), 55.12 (s), 54.99 (s), 18.48
(s), 18.45 (s), 18.39 (s), 18.37 (s). 31P NMR (CDCl3): δ 68.12 (s). MS
(CI): m/z 559.2 (M+ + 1, 17.87). HRMS (CI) (m/z) (M+): found,
558.1686; calcd for C20H35N2O12PS, 558.1648.
1
as a white solid. H NMR (CDCl3): δ 5.16 (m, 1H), 4.31 (m, 1H),
4.15 (m, 1H), 3.85-3.54 (m, 6H), 2.60 (t, J ) 6.8 Hz, 2H), 2.29 (m,
4H), 1.59 (m, 4H), 1.26 (m, 48H), 1.26 (m, 42H), 1.14 (m, 12H), 0.85
(t, J ) 7.2 Hz, 6H). 13C NMR (CDCl3): δ 173.36 (s), 172.97 (s), 117.50
(s), 70.56 (d, J ) 3.84 Hz) 62.34 (s), 61.76 (s), 61.61 (s), 61.44 (s),
58.56 (s), 58.48 (s), 58.38 (s), 58.29 (s), 43.19 (s), 43.06 (s), 34.29 (s),
34.10 (s), 33.35 (s), 31.90 (s), 29.67 (s), 29.63 (s), 29.61 (s), 29.47 (s),
29.34 (s), 29.28 (s), 29.12 (s), 29.09 (s), 24.90 (s), 24.87 (s), 24.59 (s),
24.52 (s), 22.66 (s), 20.37 (s), 20.29 (s), 14.09 (s). 31P NMR (CDCl3):
δ 150.60 (s), 150.45 (s). MS (CI): m/z 769.6 (M+ + 1, 22.44). HRMS
(CI) (m/z) (M+): found, 768.6141; calcd for C44H85N2O6P, 768.6145.
1D O-(1,2-Di-O-oleoyl-sn-(2S)-glycerol-3-O-(cyanoethyl)phospho)-
3-(bis(cyanoethyl) phosphothionate)-2,4,5,6-O-tetrakis(methoxy-
methylene)-myo-inositol (23a). To a solution of 12 mg of alcohol 22
(0.022 mmol) in dry THF (0.5 mL) were added N,N-diisopropyl-O-
(cyanoethyl)-O-(dioleoyl-sn-(2S)-glycerol)phosphonamidite (25 mg,
0.030 mmol) and 1H-tetrazole (6 mg, 0.26 mL, 0.088 mmol). The
mixture was stirred at rt for 16 h. Oxidation was then performed with
t-BuOOH (9.9 mg, 11 µL, 0.110 mmol) at rt for 1 h. The solution was
diluted with CH2Cl2 (20 mL) and washed with 10% sodium bisulfite.
The organic layer was concentrated and the residue purified by FC
(acetone-hexane, 1:3, v/v) to give 25 mg of 23a as a colorless oil
(0.019 mmol, 88%). 1H NMR (CDCl3): δ 5.35 (m, 4H), 5.22 (m, 1H),
4.83-4.74 (m, 8H), 4.42-4.19 (m, 12H), 4.10 (m, 2H), 3.42 (m, 12H),
2.78 (m, 6H), 2.29 (m, 4H), 1.99 (m, 8H), 1.57 (m, 4H), 1.28 (m, 42H),
0.85 (t, J ) 7.2 Hz, 6H). 13C NMR (CDCl3): δ 173.23 (s), 172.84 (s),
172.81 (s), 130.00 (s), 129.67 (s), 116.63 (s), 116.49 (s), 98.91 (s),
98.93 (s), 98.55 (s), 98.49 (s), 98.01 (s), 97.96 (s), 79.17 (s), 76.40
(m), 75.70 (d, J ) 19.29 Hz), 69.28 (d, J ) 5.35 Hz), 66.09 (d, J )
5.35 Hz), 65.98 (d, J ) 5.45 Hz), 62.89 (d, J ) 3.84 Hz), 62.70 (m),
62.30 (d, J ) 5.35 Hz), 62.24 (d, J ) 4.55 Hz), 61.60 (s), 56.64 (s),
56.62 (s), 56.59 (s), 56.03 (s), 34.10 (s), 33.94 (s), 31.87 (s), 29.73 (s),
29.70 (s), 29.50 (s), 29.29 (s), 29.21 (s), 29.19 (s), 29.12 (s), 29.08 (s),
29.06 (s), 27.19 (s), 27.16 (s), 24.79 (s), 22.65 (s), 19.69 (s), 19.62 (s),
19.44 (s), 19.36 (s), 14.09 (s). 31P NMR (CDCl3): δ 67.86 (s), 67.70
(s), -0.84 (s), -1.25 (s). MS (CI): m/z 1294.3 (M+, 40.74), 1262.2
N,N-Diisopropyl-O-(cyanoethyl)-O-(dibutanoyl-sn-(2S)-glycerol)-
phosphonamidite (20c). A solution of 220 mg (0.931 mmol) of
2-cyanoethyl N,N-diisopropylchlorophosphoramidite and 144 mg (200
µL, 1.117 mmol) of DIPEA in 5 mL of anhydrous CH2Cl2 was cooled
to 0 °C under nitrogen. To this solution was added 1,2-dibutanoyl-sn-
(2S)-glycerol (216 mg, 0.931 mmol) with vigorous stirring. After being
stirred at 0 °C for 1 h and at rt for 3 h under nitrogen, the solution was
separated from the white precipitate. FC on silica gel (hexane-EtOAc-
TEA, 100:10:1, v/v/v) gave 285 mg of pure 20c (0.661 mmol, 71%)
1
as a colorless oil. H NMR (CDCl3): δ 5.07 (m, 1H), 4.22 (m, 1H),
4.05 (m, 1H), 3.76-3.47 (m, 6H), 2.54 (t, J ) 6.4 Hz, 2H), 2.19 (m,
4H), 1.53 (m, 4H), 1.06 (m, 12H), 0.84 (t, J ) 7.2 Hz, 3H), 0.81 (t, J
) 7.2 Hz, 3H). 13C NMR (CDCl3): δ 172.78 (s), 172.43 (s), 117.32
(s), 70.34 (dd, J ) 7.68, 3.84 Hz), 62.01 (d, J ) 1.52 Hz), 61.51 (s),
61.35 (s), 61.21 (s), 58.28 (d, J ) 7.68 Hz), 58.09 (d, J ) 8.48 Hz),
42.90 (s), 42.77 (s), 35.82 (s), 35.62 (s), 24.28 (s), 24.22 (s), 20.08 (s),
20.02 (s), 18.07 (s), 18.04 (s), 13.31 (s), 13.27 (s). 31P NMR (CDCl3):
δ 150.38 (s), 150.27 (s). MS (CI): m/z 433.3 (M+ + 1, 100.00). HRMS
(CI): (m/z) (M+): found, 432.2408; calcd for C20H37N2O6P, 432.2389.
1D 1-O-(tert-Butyldiphenylsilyl)-3-(bis(cyanoethyl) phosphothion-
ate)-2,4,5,6-O-tetrakis(methoxymethylene)-myo-inositol (21). Bis(2-
cyanoethyl) diisopropylphosphorodiamidite (28 mg, 0.104 mmol) was
added under an argon atmosphere to a solution of 10 (56 mg, 0.094
mmol) and 1H-tetrazole (0.48 mL, 3 wt % in CH3CN, 0.207 mmol) in
1 mL of dry CH3CN. After the resulting solution was stirred at rt for
-
(M+ OCH4, 100.00). HRMS (CI) (m/z) (M+): found, 1293.6830;
calcd for C62H109N3O19P2S, 1293.6851.
1D O-(1,2-Di-O-oleoyl-sn-(2S)-glycerol-3-phospho)-3-(phospho-
thionate)-myo-inositol (24a). To a solution of 21 mg of compound
23a (0.016 mmol) in CH3CN (1.0 mL) under N2 was added triethyl-
9
J. AM. CHEM. SOC. VOL. 128, NO. 3, 2006 895

A R T I C L E S
Xu et al.
amine (0.5 mL) followed by the addition of bis((trifluoromethyl)silyl)-
acetamide (0.50 mL). After 24 h, the reaction mixture was concentrated,
and the residue was dissolved in 30 mL of 8 mM ammonium acetate
(pH 7.1). The water phase was lyophilized, and a white powder was
obtained. The anhydrous white powder was dissolved in ethanethiol
(1 mL) and the resulting solution treated with boron trifluoride diethyl
etherate (13 µL). After 3 h, the reaction was stopped by adding dry
triethylamine (20 µL). The thiol was removed by evaporation, and the
semisolid residue was dissolved in ammonium acetate buffer. FC
(CHCl3-CH3OH-NH4OH (2.0 M) 9:7:2, v/v/v) provided 15 mg of
(M+ + Na, 17.00). HRMS (MALDI) (m/z) (M+ + Na+): found,
980.5410; calcd for C41H89N3NaO15P2S, 980.5387.
1D O-(1,2-Di-O-butanoyl-sn-(2S)-glycerol-3-O-(cyanoethyl)phos-
pho)-3-(bis(cyanoethyl) phosphothionate)-2,4,5,6-O-tetrakis(methoxy-
methylene)-myo-inositol (23c). To a solution of alcohol 22 (35 mg,
0.063 mmol) in dry THF (0.5 mL) were added N,N-diisopropyl-O-
(cyanoethyl)-O-(dibutanoyl-sn-(2S)-glycerol)phosphonamidite (38 mg,
0.088 mmol) and 1H-tetrazole (0.74 mL, 3 wt %, 0.252 mmol). The
mixture was stirred at rt for 16 h. Then oxidation was performed with
t-BuOOH (33 µL, 0.300 mmol) at rt for 1 h. The solution was diluted
with CH2Cl2 (20 mL) and washed with 10% sodium bisulfite. The
organic layer was concentrated, and the residue was chromatographed
(acetone-hexane, 1:3, v/v) on silica gel to give pure product as a
1
pure 24a (0.015 mmol, 93%). H NMR (CDCl3/CD3OD, 3:2, v/v): δ
5.32 (m, 4H), 5.24 (m, 1H), 4.20 (m, 3H), 4.04-3.95 (m, 5H), 3.77
(m, 2H), 3.20 (m, 2H), 2.29 (m, 4H), 2.00 (m, 8H), 1.57 (m, 4H), 1.27
(m, 42H), 0.86 (t, J ) 7.2 Hz, 6H). 13C NMR (CDCl3/CD3OD, 3:2,
v/v): δ 175.15 (s), 174.88 (s), 130.71 (s), 130.43 (s), 76.82 (s), 75.73
(s), 75.13 (s), 72.63 (s), 72.27 (s), 71.61 (s), 71.42 (s), 64.53 (s), 63.86
(s), 34.95 (s), 34.83 (s), 32.60 (s), 30.50 (s), 30.47 (s), 30.44 (s), 30.19
(s), 30.06 (s), 30.00 (s), 29.97 (s), 29.90 (s), 29.87 (s), 29.82 (s), 27.91
(s), 27.88 (s), 25.67 (s), 25.57 (s), 23.35 (s), 14.58 (s). 31P NMR (CDCl3/
CD3OD, 3:2, v/v): δ 48.96 (s), 0.74 (s). MS (ESI): m/z 981.64 (M+ -
NH4+, 100.00). HRMS (MALDI) (m/z) (M+ + 2NH4+ + H+): found,
1046.6530; calcd for C45H102NO15P2S, 1046.6568.
1
colorless oil (49 mg, 0.054 mmol, 86%). H NMR (CDCl3): δ 5.26
(m, 1H), 4.83-4.74 (m, 9H), 4.40 (d, J ) 2.4 Hz, 1H), 4.35-4.12 (m,
12H), 3.79 (m, 2H), 3.45-3.38 (m, 12H), 2.76 (m, 6H), 2.31 (m, 4H),
1.67 (m, 4H), 0.95 (t, J ) 7.2 Hz, 3H), 0.91 (t, J ) 7.2 Hz, 3H). 13C
NMR (CDCl3): δ 173.07 (s), 172.68 (s), 116.63 (s), 116.50 (s), 98.91
(s), 98.89 (s), 98.48 (s), 98.00 (s), 97.94 (s), 79.16 (m), 76.41 (m),
75.69 (d, J ) 19.91 Hz), 69.27 (d, J ) 6.13 Hz), 66.14 (dd, J ) 12.97,
7.64 Hz), 62.90 (d, J ) 3.82 Hz), 62.70 (s), 62.38 (d, J ) 4.63 Hz),
62.22 (d, J ) 4.20 Hz), 61.53 (s), 56.84 (s), 56.61 (s), 56.02 (s), 35.94
(s), 35.80 (s), 19.68 9s), 19.61 (s), 19.43 (s), 19.35 (s), 18.27 (s), 13.58
(s), 13.54 (s). 31P NMR (CDCl3): δ 67.83 (s), 67.70 (s), -0.90 (s),
-1.30 (s). MS (ESI): m/z 906.41 (M+ + 1, 30.00). HRMS (MALDI)
(m/z) (M+ + Na + NH4+): found, 946.2996; calcd for C34H61N4-
NaO19P2S, 946.3024.
1D O-(1,2-Di-O-pamitoyl-sn-(2S)-glycerol-3-O-(cyanoethyl)phos-
pho)-3-(bis(cyanoethyl) phosphothionate)-2,4,5,6-O-tetrakis(methoxy-
methylene)-myo-inositol (23b). To a solution of alcohol 22 (53 mg,
0.095 mmol) in dry THF (0.5 mL) were added N,N-diisopropyl-O-
(cyanoethyl)-O-(dipalmitoyl-sn-(2S)-glycerol)phosphonamidite (102 mg,
0.133 mmol) and 1H-tetrazole (1.12 mL, 3 wt %, 0.380 mmol). The
mixture was stirred at rt for 16 h. Then oxidation was performed with
t-BuOOH (60 µL, 0.380 mmol) at rt for 1 h. The solution was diluted
with methylene chloride (20 mL) and washed with 10% sodium
bisulfite. The organic layer was concentrated, and the residue was
purified by FC (acetone-hexane, 1:3, v/v) to give 95 mg of pure
1D-O-(1,2-Di-O-butanoyl-sn-(2S)-glycerol-3-phospho)-3-(phos-
phothionate)-myo-inositol (24c). To a solution of 20 mg of compound
23a (0.022 mmol) in CH3CN (1.0 mL) under N2 was added triethy-
lamine (0.5 mL) followed by the addition of bis((trifluoromethyl)silyl)-
acetamide (0.50 mL). After 24 h, the reaction mixture was concentrated,
and the residue was dissolved in 30 mL of 8 mM ammonium acetate
(pH 7.1). The water phase was lyophilized, and white powder was
obtained. The anhydrous white powder was dissolved in ethanethiol
(1 mL) and the resulting solution treated with boron trifluoride diethyl
etherate (11 µL). After 3 h, the reaction was stopped by adding dry
triethylamine (20 µL). The thiol was removed by evaporation, and the
semisolid residue was dissolved in ammonium acetate buffer. FC
(CHCl3-CH3OH-NH4OH (2.0 M) 9:7:2, v/v/v) provided 11 mg of
1
product (0.077 mmol, 81%) as a colorless oil. H NMR (CDCl3): δ
5.24 (m, 1H), 4.83-4.72 (m, 7H), 4.39 (m, 1H), 4.31-4.13 (m, 12H),
3.93 (m, 2H), 3.44 (m, 2H), 3.40 (m, 12H), 2.77 (m, 6H), 2.28 (m,
4H), 1.56 (m, 4H), 1.21 (m, 48H), 0.84 (t, J ) 7.2 Hz, 6H). 13C NMR
(CDCl3): δ 173.23 (s), 172.84 (s), 116.61 (s), 116.48 (s), 98.87 (s),
98.51 (s), 98.45 (s), 97.97 (s), 97.92 (s), 79.14 (s), 76.34 (m), 75.77
(s), 75.56 (s), 69.29 (s), 66.11 (m), 62.71 (d, J ) 3.84 Hz), 62.20 (d,
J ) 12.32 Hz,), 61.60 (s), 58.31 (s), 56.80 (s), 56.55 (s), 55.99 (s),
34.09 (s), 33.94 (s), 31.86 (s), 29.64 (s), 29.60 (s), 29.45 (s), 29.30 (s),
29.26 (s), 29.08 (s), 29.06 (s), 24.78 (s), 22.63 (s), 19.66 (s), 19.58 (s),
19.40 (s), 19.32 (s), 14.06 (s). 31P NMR (CDCl3): δ 67.84 (s), 67.68
(s), -0.88 (s), -1.28 (s). MS (ESI): m/z 1242.8 (M+ + 1, 18.00).
HRMS (MALDI) (m/z) (M+ + Na): found, 1264.6463; calcd for
C58H105N3NaO19P2S, 1264.6436.
1
pure 24c (0.018 mmol, 80%). H NMR (CDCl3/CD3OD, 3:2, v/v): δ
5.26 (m, 1H), 3.93 (m, 2H), 3.82 (m, 2H), 3.73-3.60 (m, 5H), 3.30
(m, 2H), 2.31 (m, 4H), 1.67 (m, 4H), 0.95 (t, J ) 7.2 Hz, 3H), 0.91 (t,
J ) 7.2 Hz, 3H). 31P NMR (CDCl3/CD3OD, 3:2, v/v): δ 48.96 (s),
0.74 (s). MS (ESI): m/z 624.27 (M+ + 3, 21.00), 667.28 (M+ + 2 +
2Na+, 100.00). HRMS (MALDI) (m/z) (M+ + Na+): found, 644.1652;
calcd for C17H41N3O15P2S, 644.1631.
Protein Expression and Purification. DNA fragments encoding
residues 1325-1410 of human EEA1 FYVE and residues 2-122 of
yeast Vam7 PX were cloned in pGEX-KG and pGEX-2T vectors
(Amersham). The 15N-labeled proteins were expressed in Escherichia
coli BL21 (DE3) pLysS and BL21 Codon Plus RP strains in minimal
media supplemented with 15NH4Cl (Cambridge Isotope). Bacteria were
harvested by centrifugation after induction with IPTG (0.5 mM) and
lysed with a French press. The glutathione S-transferase (GST) fusion
FYVE and PX were purified on a Glutathione Sepharose 4B column
(Amersham). The GST tag was cleaved with thrombin (Sigma). The
proteins were further purified by FPLC and concentrated in Milli-
pore concentrators (Millipore). The buffers were exchanged into
20 mM d11-Tris (FYVE) or 50 mM potassium phosphate (PX), pH
6.8, 100-200 mM KCl, 1-20 mM perdeuterated dithiothreitol,
50 µM 4-amidinophenylmethanesulfonyl fluoride, 1 mM NaN3, and
1D O-(1,2-Di-O-palmitoyl-sn-(2S)-glycerol-3-phospho)-3-(phos-
phothionate)-myo-inositol (24b). To a solution of 35 mg of compound
23b (0.028 mmol) in CH3CN (1.0 mL) under N2 was added triethyl-
amine (0.5 mL) followed by the addition of bis((trifluoromethyl)silyl)-
acetamide (0.50 mL). After 24 h, the reaction mixture was concentrated,
and the residue was dissolved in 30 mL of 8 mM ammonium acetate
(pH 7.1). The water phase was lyophilized, and a white powder was
obtained. The anhydrous white powder was dissolved in ethanethiol
(2 mL) and the resulting solution treated with boron trifluoride diethyl
etherate (14 µL). After 3 h, the reaction was stopped by adding dry
triethylamine (30 µL). The thiol was removed by evaporation, and the
semisolid residue was dissolved in ammonium acetate buffer. FC
(CHCl3-CH3OH-NH4OH (2.0 M) 9:7:2, v/v/v) provided 22 mg of
1
pure 24b (0.022 mmol, 79%). H NMR (CDCl3/CD3OD, 3:2, v/v): δ
2
7% H2O.
5.27 (m, 1H), 3.93 (dd, J ) 12.0, 3.6 Hz, 1H), 3.82 (m, 2H), 3.73-
3.60 (m, 5H), 3.30 (m, 2H), 2.33 (m, 4H), 1.62 (m, 4H), 1.56 (dd, J )
17.6, 6.8 Hz, 3H), 1.27 (m, 48H), 0.84 (t, J ) 7.2 Hz, 6H). 31P NMR
(CDCl3/CD3OD, 3:2, v/v): δ 48.96 (s), 0.74 (s). MS (ESI): m/z 981.64
NMR Spectroscopy and Titration of PtdIns(3)P Analogues. NMR
spectra were recorded at 25 °C on a Varian INOVA 500 MHz
spectrometer. The 1H-15N heteronuclear single-quantum coherence
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896 J. AM. CHEM. SOC. VOL. 128, NO. 3, 2006

Phosphatase-Resistant Analogues of PtdIns(3)P
A R T I C L E S
(HSQC) spectra of 0.2-0.3 mM uniformly 15N-labeled FYVE and PX
domains were collected while dibutanoyl (C4)- or dipalmitoyl (C16)-
PtdIns(3)P analogues (Echelon Biosciences) (up to 4 mM) embedded
in micelles consisting of d38-DPC (250 mM) (Cambridge Isotopes) or
DHPC/CHAPS (100 mM/17 mM) (Avanti/Anatrace) were added
stepwise.
N HCl and extraction with 160 µL of 1:1 (v/v) CHCl3-CH3OH. The
lower chloroform layer containing the 32P-labeled lipid product was
collected by centrifugation for 30 s at 5000 rpm in a microfuge and
quickly rinsed twice with 100 µL of 1:1 (v/v) 1 N HCl-CH3OH before
50 µL was spotted onto a silica gel glass TLC plate (20 × 20 cm ×
0.25 mm layer thickness, Merck) and developed up to the top (4-5 h)
at room temperature with 65:35 (v/v) 1-propanol-2 M acetic acid.
Following exposure with an X-omat autoradiography film for the
appropriate length of time the radiolabeled spots were scraped into glass
scintillation vials and deacylated by methylaminolysis at 54 °C for 50
min by the standard protocol.61 Recovery of the aqueously soluble
deacylation products was then analyzed by HPLC on a Whatman 5
µm Partisphere SAX (H2PO4-) column as formerly described61 and the
32P detected with a Radiomatic 525TR online flow scintillation analyzer
and FLO-ONE radiochromatography software (Packard Instrument Co.,
Downers Grove, IL) by Cerenkov emission in the low-energy tritium
channel.
Phosphorylation of PtdIns(3)P Analogues by PIKfyve. Phos-
phatase-resistant dipalmitoyl and dioleoyl analogues of PtdIns(3)P and
di-C16-PtdIns(3)P (Echelon Biosciences, Inc.) were separately prepared
as 0.5 mM aqueous stocks and stored at -80 °C. Just before use as
substrates in the phosphorylation reaction with PIKfyve immunopre-
cipitates, a 60 µL aliquot of PtdIns(3)P, the stabilized analogues, or
soybean PtdIns (Avanti Polar Lipids, Inc.) was evaporated down to
dryness with absolute ethanol (2 × 200 µL) under a stream of dry N2.
Lipids were then reconstituted in an equal volume of lipid buffer (20
mM, pH 7.5, HEPES, 1 mM EDTA) by sonication (2 × 30 s) in a
bath sonicator at room temperature. A 10 µL aliquot of each 0.5 mM
phosphoinositide reconstituted in lipid buffer was then phosphorylated
with immunoprecipitates of PIKfyve immune (R7069) or preimmune
sera derived from PC-12 cell lysates prepared in RIPA buffer (50 mM
Tris-HCl, pH 8.0, 150 mM NaCl, 1% w/v Nonidet P-40, 0.5% w/v
sodium deoxycholate) plus protease inhibitors (1 mM phenylmethyl-
sulfonyl fluoride, 5 µg/mL leupeptin, 5 µg/mL aprotinin, 1 µg/mL
pepstatin, and 1 mM benzamidine) and immunoadsorbed onto 10 µL
of packed Protein A Sepharose beads, as previously described.61 The
reaction in 50 µL of assay buffer (50 mM, pH 7.5, Tris-HCl/2.5 mM
MnCl2/2.5 mM MgCl2) containing 10 µCi of [γ-32P]ATP (50 µM) was
incubated for 15 min at 37 °C before being stopped with 200 µL of 1
Acknowledgment. We thank the NIH (Grant NS29632), the
American Cancer Society (Grant RSG0513601) (T.G.K.), and
the American Diabetes Association and NIH (Grant DK58058)
(A.S.) for financial support of this work.
Supporting Information Available: Experimental procedures
and characterization for all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
JA0554716
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