Regioselective approach to phosphatidylinositol 3,5-bisphosphates: Syntheses of the native phospholipid and biotinylated short-chain derivative

Article abstract of DOI:10.1021/jo100393c

Figure presented A selective bis-silylation of 1D-O-TBDPS-myo-inositol leads to a 1,3,5-trisubstituted inositol, which can be advanced to the headgroup of phosphatidylinositol-3,5-bisphosphate [PI(3,5)P₂]. A mild, regioselective method for construction of the diacylglycerol moiety containing differing fatty acid chains, including the naturally occurring lipids, was developed. Their union in the synthesis of the cell-signaling molecule PI(3,5)P₂ containing the sn-1-stearoyl and sn-2-arachidonoyl groups is described. The methodology was also used to generate dioctanoyl-PI(3,5)P ₂ and a previously unreported biotin-PI(3,5)P₂ conjugate, which was coupled to neutravidin beads and used to pull down PI(3,5)P ₂-binding proteins from the cytosolic extract of adrenal neurosecretory cells. We report the specific pull-down of the PI(3,5)P ₂-binding protein svp1p, a known PI(3,5)P₂ effector involved in membrane trafficking.

Full text of DOI:10.1021/jo100393c

pubs.acs.org/joc
Regioselective Approach to Phosphatidylinositol 3,5-Bisphosphates:
Syntheses of the Native Phospholipid and Biotinylated
Short-Chain Derivative
Regan J. Anderson,† Shona L. Osborne,‡ Frederic A. Meunier,*,‡ and Gavin F. Painter*,†
†Carbohydrate Chemistry Team, Industrial Research Limited, PO Box 31-310, Lower Hutt, New Zealand,
and ‡Molecular Dynamics of Synaptic Function Laboratory, Queensland Brain Institute and School of
Biomedical Sciences, The University of Queensland, St. Lucia, 4072 Queensland, Australia
g.painter@irl.cri.nz; f.meunier@uq.edu.au
Received March 2, 2010
A selective bis-silylation of 1D-O-TBDPS-myo-inositol leads to a 1,3,5-trisubstituted inositol, which
can be advanced to the headgroup of phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2]. A mild,
regioselective method for construction of the diacylglycerol moiety containing differing fatty acid
chains, including the naturally occurring lipids, was developed. Their union in the synthesis of the
cell-signaling molecule PI(3,5)P2 containing the sn-1-stearoyl and sn-2-arachidonoyl groups is
described. The methodology was also used to generate dioctanoyl-PI(3,5)P2 and a previously
unreported biotin-PI(3,5)P2 conjugate, which was coupled to neutravidin beads and used to pull
down PI(3,5)P2-binding proteins from the cytosolic extract of adrenal neurosecretory cells. We report
the specific pull-down of the PI(3,5)P2-binding protein svp1p, a known PI(3,5)P2 effector involved in
membrane trafficking.
Introduction
3-, 4-, and 5-hydroxyls on the myo-inositol headgroup.
The intracellular concentrations and dynamic relationships
between the various PIP members are under tight control
from a series of kinases and phosphatases. The 3-phosphory-
lated PIPs PI(3,4)P2, PI(3,5)P2, and PI(3,4,5)P3 are intra-
cellular second messengers that are synthesized by the action
of PI-3-kinase, and in vivo, their half-life is relatively short as
the active signal is under tight control (see above). The
metabolic instability and relatively low concentrations of PIPs
(especially the 3-phosphorylated forms) are factors contribut-
ing to the inability to isolate useful amounts of these com-
poundsin pure formfor biologicalstudies. For thisreason, the
chemical synthesis of PIPs and derivatives remains an attrac-
tive endeavor. However, while the 3,4- and 3,4,5-headgroups
have received considerable attention, approaches to PI(3,5)P2
have been fewer due to its later discovery.7 Furthermore, there
Phosphatidylinositol phosphates (PIPs) are intracellular
amphiphilic molecules implicated in numerous cellular res-
ponses including growth,1 division,2 movement,3 differen-
tiation,4 neuro-exocytosis,5 and survival.6 There are a number
of forms that differ only in the phosphorylation state of the
(1) Lee, J.-H.; Overstreet, E.; Fitch, E.; Fleenor, S.; Fischer, J. A. Dev.
Biol. 2009, 331, 1–13.
(2) Liu, Y.; Boukhelifa, M.; Tribble, E.; Morin-Kensicki, E.; Uetrecht,
A.; Bear, J. E.; Bankaitis, V. A. Mol. Biol. Cell 2008, 19, 3080–3096.
(3) Waugh, C.; Sinclair, L.; Finlay, D.; Bayascas, J. R.; Cantrell, D. Mol.
Cell. Biol. 2009, 29, 5952–5962.
(4) Laketa, V.; Zarbakhsh, S.; Morbier, E.; Subramanian, D.; Dinkel, C.;
Brumbaugh, J.; Zimmermann, P.; Pepperkok, R.; Schultz, C. Chem. Biol.
2009, 16, 1190–1196.
(5) (a) Wen, P. J.; Osborne, S. L.; Morrow, I. C.; Parton, R. G.; Domin, J.;
Meunier, F. A. Mol. Biol. Cell 2008, 19, 5593–5603. (b) Osborne, S. L.;
Wallis, T. P.; Jimenez, J. L.; Gorman, J. J.; Meunier, F. A. Mol. Cell.
Proteomics 2007, 6, 1158–1169. (c) Meunier, F. A.; Osborne, S. L.;
Hammond, G. R.; Cooke, F. T.; Parker, P. J.; Domin, J.; Schiavo, G. Mol.
Biol. Cell 2005, 16, 4841–4851.
(7) (a) Cooke, F. T.; Dove, S. K.; McEwen, R. K.; Painter, G.; Holmes,
A. B.; Hall, M. N.; Michell, R. H.; Parker, P. J. Curr. Biol. 1998, 8, 1219–
1222. (b) Dove, S. K.; Cooke, F. T.; Douglas, M. R.; Sayers, L. G.; Parker,
P. J.; Michell, R. H. Nature 1997, 390, 187–192. Whiteford, C. C.; Brearley,
C. A.; Ulug, E. T. Biochem. J. 1997, 323, 597–601.
(6) Chow, C. Y.; Zhang, Y.; Dowling, J. J.; Jin, N.; Adamska, M.; Shiga,
K.; Szigeti, K.; Shy, M. E.; Li, J.; Zhang, X.; Lupski, J. R.; Weisman, L. S.;
Meisler, M. H. Nature 2007, 448, 68–72.
DOI: 10.1021/jo100393c
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Published on Web 05/05/2010
J. Org. Chem. 2010, 75, 3541–3551 3541
2010 American Chemical Society

Anderson et al.
JOCFeatured Article
are few syntheses of PIPs containing unsaturated fatty acids,
so new complementary methods are still needed.
Two chiral pool approaches have been reported, involving
Ferrier rearrangement of a glucose derivative18 or ring-closing
metathesis of a glucose-derived 1,7-diene followed by
dihydroxylation.19 Although lengthy sequences are some-
times required to access the 1,3,5-substitution pattern found
in PI(3,5)P2, several of the above approaches have utilized
tricyclic orthoesters of myo-inositol which provide rapid
differentiation of the 1-, 3-, and 5-hydroxyl groups from the
2-, 4-, and 6-positions.20
In this work, we present novel syntheses of the PI(3,5)P2
headgroup and mixed diacylglycerol moieties utilizing, re-
spectively, the regioselective bis-silylation of pentol 2 with
chlorotriethylsilane (TESCl) and the regioselective opening
of acetonide 12 with triethylsilyl trifluoromethanesulfonate
€
(TESOTf)/Hunig’s base. The novel methodologies were used
to prepare the natural sn-1-O-stearoyl-2-O-arachidonoyl
PI(3,5)P2, dioctanoyl PI(3,5)P2 and a biotin-tagged analogue
30. The biotin-tagged analogue was successfully employed in
an affinity pull-down strategy to isolate PI(3,5)P2 binding
proteins from the cytosolic extract of adrenal neurosecretory
cells.
The signaling properties of phosphatidylinositol 3,5-bis-
phosphate [PI(3,5)P2] were first characterized in yeast when
its concentration was shown to dramatically rise following
the application of hyperosmotic stress.7 In yeast, PI(3,5)P2 is
synthesized from PI(3)P by the PI(3)P 5-kinase, Fab1p.
Knockouts deficient in this enzyme exhibit vacuolar defects,
indicating the importance of PI(3)P 5-kinase activity, and
hence PI(3,5)P2, in membrane trafficking events. Other
work, utilizing siRNA, dominant negative constructs, and
chemical inhibitors, implies that PIKfyve (the murine ortho-
logue of Fab1p) is required for late endosome/lysosome
function8 and the full insulin stimulation of glucose trans-
port in adipose and muscle tissue.9 In contrast, PIKfyve
has also been implicated in the negative regulation of exo-
cytosis in neurosecretory cells.10 The details of this mecha-
nism are not currently known, but it is possible that PIKfyve
activity contributes to the negative regulation of exocytosis
by decreasing the levels of PI(3)P, as PI(3)P is known to
promote large dense core vesicle (LDCV) priming for
exocytosis.5a,c
To conduct further investigations into the role played by
PI(3,5)P2 in membrane trafficking, we sought a practical
route to this phosphoinositide and its derivatives. Entry into
the required D-enantiomeric series of PI(3,5)P2 has most
commonly involved the use of chiral auxiliaries, including
camphor acetals,11,12 camphanate esters,13,14 O-acetylman-
delate esters,15 and 5-oxo-2-tetrahydrofurancarboxylate
esters,16 attached to the myo-inositol core. More recently,
meso-triols have been desymmetrized by asymmetric phos-
phorylation, catalyzed by enantiodivergent pentapeptides.17
Results and Discussion
Synthesis of Inositol Headgroup. The synthetic method
developed by Bruzik’s group represents a direct entry into
useful homochiral starting materials for the synthesis of
PIPs.21 myo-Inositol is condensed with D-camphor dimethyl
acetal to give camphanylidene acetal 1 (Scheme 1) as the
major product, which is obtained in pure form after partial
hydrolysis and recrystallization without recourse to chro-
matography. The strategy is applicable on a large scale and
may be used to access all the naturally occurring members
of the PIP family.11 However, the approach is not equally
efficient in accessing all the different phosphorylation pat-
terns. In the synthesis of PI(3,5)P2, dibenzoate 3 was ob-
tained in only modest yield (∼ 40%) upon treatment of 2 with
benzoyl chloride (2 equiv) and proved difficult to separate
from the 3,4-isomer. In our hands, altering the temperature
and rate of addition did not improve the selectivity for the
3,5-isomer, so we decided to investigate alternative protect-
ing group strategies to access this substitution pattern. The
large silyl group at position 1 in compound 2 effectively
blocks reaction at the two neighboring hydroxyl groups, and
among the remaining three hydroxyl groups the 3-OH is
usually the most nucleophilic, as has been amply demon-
strated by various researchers.22 Reaction of pentol 2 with
a bulky group would initially form the 3-O-protected inter-
mediate, which should preferentially undergo further reac-
tion at the 5-OH to minimize steric interactions with the
groups at positions 1 and 3. We initially examined the
(8) de Lartigue, J.; Polson, H.; Feldman, M.; Shokat, K.; Tooze, S. A.;
ꢀ
Urbe, S.; Clague, M. J. Traffic 2009, 10, 883–893.
(9) Ikonomov, O. C.; Sbrissa, D.; Dondapati, R.; Shisheva, A. Exp. Cell
Res. 2007, 313, 2404–2416.
(10) Osborne, S. L.; Wen, P. J.; Boucheron, C.; Nguyen, H. N.; Hayakawa,
M.; Kaizawa, H.; Parker, P. J.; Vitale, N.; Meunier, F. A. J. Biol. Chem. 2008,
283, 2804–2813.
(11) Kubiak, R. J.; Bruzik, K. S. J. Org. Chem. 2003, 68, 960–968.
(12) Falck, J. R.; Krishna, M. U.; Katipally, K. R.; Capdevila, J. H.;
Ulug, E. T. Tetrahedron Lett. 2000, 41, 4271–4275.
(13) Painter, G. F.; Grove, S. J. A.; Gillbert, I. H.; Holmes, A. B.;
Raithby, P. R.; Hill, M. L.; Hawkins, P. T.; Stephens, L. R. J. Chem. Soc.,
Perkin Trans. 1 1999, 923–935.
(18) Peng, J.; Prestwich, G. D. Tetrahedron Lett. 1998, 39, 3965–3968.
(19) Nishikawa, A.; Saito, S.; Hashimoto, Y.; Koga, K.; Shirai, R.
Tetrahedron Lett. 2001, 42, 9195–9198.
(20) Lee, H. W.; Kishi, Y. J. Org. Chem. 1985, 50, 4402–4404.
(21) Bruzik, K. S.; Tsai, M.-D. J. Am. Chem. Soc. 1992, 114, 6361–6374.
ꢀ
Pietrusiewicz, K. M.; Salamonczyk, G. M.; Bruzik, K. S.; Wieczorek, W.
Tetrahedron 1992, 48, 5523–5542.
(14) Falck, J. R.; Krishna, U. M.; Capdevila, J. H. Bioorg. Med. Chem.
Lett. 2000, 10, 1711–1713.
(15) Han, F. S.; Hayashi, M.; Watanabe, Y. Chem. Lett. 2003, 32, 724–
725. Han, F. S.; Hayashi, M.; Watanabe, Y. Eur. J. Org. Chem. 2004, 558–
566.
(16) Riley, A. M.; Potter, B. V. L. Tetrahedron Lett. 1998, 39, 6769–6772.
(17) Xu, Y.; Sculimbrene, B. R.; Miller, S. J. J. Org. Chem. 2006, 71,
4919–4928.
(22) See, for example: Chung, S.-K.; Ryu, Y. Carbohydr. Res. 1994, 258,
145–167. Chung, S.-K.; Chang, Y.-T. Bioorg. Med. Chem. Lett. 1997, 7,
2715–2718. For a theoretical study concerning relative reactivities of inositol
hydroxyl groups, see: Kim, K. S.; Cho, S. J.; Oh, K. S.; Son, J. S.; Kim, J.;
Lee, J. Y.; Lee, S. J.; Lee, S.; Chang, Y.-T.; Chung, S.-K.; Ha, T.-K.; Lee,
B.-S.; Lee, I. J. Phys. Chem. A 1997, 101, 3776–3783.
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SCHEME 1. Bruzik’s Synthesis of 3,5-Dibenzoate 3
of acyl groups can occur at both the monoacyl- and diacyl-
glycerol stages (vide infra). The isopropylidene group of 1-O-
stearoyl-sn-glycerol 12 (Scheme 3) has previously been re-
moved by refluxing in MeOH with Amberlyst-Hþ resin.11
1
While no mention of any acyl migration was made, the H
NMR spectrum of the diol product indicated the presence of
some sn-2-O-acylated isomer (5-10%). Others have re-
ported similar results under these conditions.25 Also of
concern is the possible presence of 3-O-stearoyl-sn-glycerol
(the enantiomeric product), which would be expected to
form under conditions that promote acyl migration. Since
the equilibrium ratio of 1-monoacylglycerol to 2-monoacyl-
glycerol isomers is reported to be ∼9:1,26 the presence of 10%
of the 2-acylated isomer would indicate a near-equilibrium
mixture, suggesting that significant racemization may have
taken place. Gaffney and Reese24 have used a combination
of TFA, triethyl borate and trifluoroethanol to effect this
transformation, which relies on the formation of an inter-
mediate 2,3-borate ester to prevent acyl migration. We,
however, considered an alternative method for the opening
of terminal acetonides, described by Rychnovsky and co-
workers.27 Although this method has received little attention
since its initial report, we felt it would be well-suited to the
synthesis of mixed diacylglycerols. Thus, heating acetonide
reaction of 2 with a slight excess (1.2 equiv) of TESCl. As
expected, the 3-O-silylated inositol 4 was the major product,
isolated in 71% yield after column chromatography (Scheme 2).
Lesser amounts of 5-7 were also isolated, along with a trace
of starting material. Pleasingly, treatment of 2 with 2.5 equiv
of TESCl reproducibly led to the formation of the 3,5-bis-
silylated product 6 in 71% isolated yield, together with 14%
of the 3,4-isomer 7 which was readily separated by chroma-
tography on a multigram scale. The specific sites of silylation
in products 4-7 were deduced from 1H NMR coupling
patterns and 2D-COSY analysis. Of particular importance
to the regiochemical assignments were the well-known split-
ting patterns of myo-inositol ring-protons and cross-
couplings from these signals to the hydroxyl protons in the
2D-COSY spectra (see the Supporting Information).
Among the TES, triphenylsilyl, and tert-butyldimethylsi-
lyl protecting groups, TES was the most convenient in terms
of 3,5-selectivity, product purification, and subsequent facile
protection of the remaining hydroxyl groups. Although the
addition of a third silyl group to 6 or 7 is strongly retarded
due to the steric encumbrance around the remaining hydro-
xyl groups, exhaustive MOM protection of 6 could be readily
achieved, followed by desilylation with acid to give the 3,5-
diol 8 in excellent yield. Conversion of 8 to alcohol 9 followed
Bruzik’s reported procedure.11 In a similar manner, 4 was
transformed to alcohol 11, which has previously been used as
a precursor to PI(3)P.11 Incidentally, silylation of diol 8 with
TESCl was found to be completely selective for the 3-hydro-
xyl group to give compound 10, in which the 1-, 3-, and
5-positions are all differentiated. This compound could
provide a route into a series of PI(3,5)P2 analogues with
differing groups at positions 3 and 5.
€
12 with TESOTf in the presence of a slight excess of Hunig’s
base led to a labile 2-O-isopropenyl-3-O-triethylsilyl diether
13 which, without purification, was converted to the second-
ary alcohol 14 under the mild conditions reported by
Rychnovsky. Although a small amount (15%) of unreacted
acetonide 12 was recovered from the crude reaction mixture,
we observed no isomeric impurities in the crude mixture from
this reaction and alcohol 14 showed no propensity for either
silyl or acyl migration on chromatographic purification or in
subsequent derivatization. Thus, sn-1 T sn-2 T sn-3 acyl
migration which can occur during conventional acidic hy-
drolysis of the acetonide is completely avoided by this
procedure, and regioselective introduction of the second
fatty acid is ensured. Coupling with arachidonic acid was
followed by desilylation using catalytic FeCl3,28 which pro-
vided the desired diacylglycerol 16 in excellent yield and
purity. By comparison, removal of the bulkier TBDPS or
TBDMS groups often leads to extensive acyl migration
or requires reagents incompatible with unsaturated sub-
strates.29 The mild conditions used here for the removal of
the TES group, combined with the regioselective introduc-
tion of fatty acids at each position of the glycerol unit, should
engender further use of this protocol in di- and triacylglycer-
ol syntheses. Conversion to the phosphoramidite 17 was
achieved in the usual manner.
Synthesis of Mixed Diacylglycerol. Native PIPs generally
incorporate a mixed diacylglyceryl phosphate containing
unsaturation at the sn-2 position. The 2-O-arachidonyl-1-
O-stearoyl-sn-glycerol 16 is generally considered to be the most
abundant form of the diacylglycerol moiety in mammalian-
derived PIPs.23 We chose to modify the reported11,24 syn-
thesis of 16 to allow for selective functionalization of each
position on the glycerol and to minimize potential problems
of acyl migration under acidic or basic conditions. Migration
(25) Andrews, P. C.; Fraser, B. H.; Junk, P. C.; Massi, M.; Perlmutter, P.;
Thienthong, N.; Wijesundera, C. Tetrahedron 2008, 64, 9197–9202.
(26) Serdarevich, B. J. Am. Oil Chem. Soc. 1967, 44, 381–393.
(27) Rychnovsky, S. D.; Hoye, R. C. J. Am. Chem. Soc. 1994, 116, 1753–
1765. Rychnovsky, S. D.; Kim, J. Tetrahedron Lett. 1991, 32, 7219–7222.
(28) Yang, Y.-Q.; Cui, J.-R.; Zhu, L.-G.; Sun, Y.-P.; Wu, Y. Synlett 2006,
1260–1262. These authors suggest a single-electron-transfer mechanism may
operate in the FeCl3-catalyzed desilylation. In our case, the pH of the
reaction medium was determined to be 2-3 (using moistened pH paper),
so we cannot exclude the possibility that the FeCl3 serves only to provide a
trace amount of HCl which catalyses the TES-cleavage.
(23) Ikeda, M.; Yoshida, S.; Busto, R.; Santiso, M.; Ginsberg, M. D.
J. Neurochem. 1986, 47, 123–132. Vadnal, R. E.; Parthasarathy, R. Biochem.
Biophys. Res. Commun. 1989, 163, 995–1001. Pettitt, T. R.; Dove, S. K.;
Lubben, A.; Calaminus, S. D. J.; Wakelam, M. J. O. J. Lipid Res. 2006, 47,
1588–1596.
(29) For TBDPS deprotection, see: Greimel, P.; Ito, Y. Tetrahedron Lett.
2008, 49, 3562–3566. For TBDMS deprotection, see: Burgos, C. E.; Ayer.,
D. E.; Johnson, R. A. J. Org. Chem. 1987, 52, 4973–4977.
(24) Gaffney, P. R. J.; Reese, C. B. J. Chem. Soc., Perkin Trans. 1 2001,
192–205.
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Anderson et al.
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SCHEME 2. Synthesis of the Inositol Head Group 9
SCHEME 3. Synthesis of Phosphoramidite 17 Containing
Mixed Diacylglycerol
as a scavenger of adventitious HBr, the presence of which can
cause undesired side reactions.30 The 2-bromo-2-methylbu-
tane formed is simply removed under high vacuum. Finally,
treatment with ethanethiol31 removed any remaining MOM
groups to yield the target molecule PI(3,5)P2 (19). Synthetic
PI(3,5)P2 containing the naturally occurring lipid chains
has been described only once before in the literature;17 the
spectroscopic properties of our material compare well to the
published data.
We also chose to synthesize dioctanoyl PI(3,5)P2 (22) as its
increased water solubility, as compared with longer chain
derivatives, was expected to facilitate some biological studies.
Coupling of phosphoramidite 2032 with 9 gave fully protec-
ted dioctanoyl PI(3,5)P2 (21) which, upon deprotection,33
afforded 22 (Scheme 5) whose spectroscopic properties com-
pared well to previously reported data.14,17 The difference in
physical behavior between 19 and 22 was apparent from their
NMR spectra in D2O. Whereas 19 gave rather broad, poorly
defined peaks (1H and 31P) indicative of solution aggregation
behavior, the short-chain analogue 22 produced excellent 1H,
31P and 13C spectra, with both the lipid and inositol headgroup
regions of the H spectrum well resolved. Good H and 31P
spectra of 19 could be obtained from a dilute CD3OD solution.
Synthesis of Biotin-PI(3,5)P2 Affinity Probe. Our syn-
thesis of affinity probe 30 began with Cbz protection of
8-aminooctanoic acid, followed by esterification with
L-2,3-O-isopropylidene-sn-glycerol to afford acetonide 23
(Scheme 6). Opening of the acetonide and subsequent hydro-
lysis of the isopropenyl ether gave secondary alcohol 25
Completion of the Synthesis of PI(3,5)P2. Coupling of
phosphoramidite 17 (2.2 equiv) with alcohol 9, followed by
oxidation of the intermediate phosphite with tetrabutyl-
ammonium periodate,11 gave fully protected PI(3,5)P2 18 in
79% yield (Scheme 4). With slight modifications to Bruzik’s
deprotection protocol,11 debenzylation was accomplished
with bromotrimethylsilane (TMSBr) in the presence of
2-methylbut-2-ene. The latter was included in the reaction
1
1
(30) The side product(s) appeared in the 1H NMR as closely related minor
signals on the shoulders of some of the main signals. We did not attempt to
characterize them; however, Falck and co-workers have reported the migra-
tion of the sn-2-glycerylacyl group to an inositol hydroxyl group under acidic
conditions, and it is conceivable that, in our case, a similar process may be
promoted by HBr. See: Reddy, K. K.; Saady, M.; Falck, J. R. J. Org. Chem.
1995, 60, 3385–3390.
(31) A reviewer questioned whether, given the relatively weak Si-S bond
strength, EtSH is capable of solvolyzing the intermediate silyl phosphate
esters (which is required to provide the acidity necessary for the subsequent
MOM deprotection). We can only say that our experience in the deprotection
of compounds 18 and 29 and similar compounds (not reported here) has been
that solvolysis of the interemediate silyl phosphates with water or methanol
has not been necessary. EtSH has been, in our experience, adequate for this
purpose.
(32) Ali, S. M.; Ahmad, M. U.; Koslosky, P.; Kasireddy, K.; Krishna,
U. M.; Ahmad, I. Tetrahedron 2006, 62, 6990–6997.
(33) In contrast to the deprotection of compound 18, passage through
strongly acidic ion-exchange resin was sufficient to remove any MOM groups
remaining after TMSBr treatment of compound 21 (see the Experimental
Section).
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Anderson et al.
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SCHEME 4. Synthesis of Natural PI(3,5)P2 (19)
Identification of PI(3,5)P2-Binding Proteins from Bovine
Adrenal Chromaffin Cytosolic Fractions. PI(3,5)P2 is an
essential phospholipid in membrane-trafficking processes,
but the specific details of its involvement on a molecular level
remain unclear.35 The use of biotin-tagged affinity probes
can help to elucidate biochemical pathways by identifying
effectors of a given ligand. Isolation of bound effectors is
achieved using neutravidin-coated Sepharose beads, relying
on the tight biotin-neutravidin binding interaction. Bio-
tinylated PI(4,5)P2 has previously been used in this manner
to identify PI(4,5)P2-binding proteins from subcellular frac-
tions of bovine adrenal chromaffin cells.5b While many
PI(4,5)P2 binding proteins involved in a plethora of cellular
functions have been identified, very little is known about
potential PI(3,5)P2 effectors. We report here our initial
results using the biotinylated 3,5-phosphorylated isomer 30
and demonstrate its successful application in affinity purifi-
cation, combined with subcellular fractionation, to pull
down PI(3,5)P2 binding proteins from bovine adrenal chro-
maffin cytosolic extract. We have chosen to use bovine
adrenal chromaffin cells as a model system due to the
involvement of PI(3,5)P2 in constitutive membrane traffick-
ing and also regulated secretion in these cell types.10
SCHEME 5. Synthesis of Dioctanoyl PI(3,5)P2 (22)
Biotinylated PI(3,5)P2 was immobilized on beads and
incubated with bovine adrenal chromaffin cytosol, and
bound proteins were analyzed by SDS-PAGE and Western
blotting, using antibodies against svp1p (also known as
WIPI1, WIPI49, Atg18, and Aut10) and EEA1 (Figure 1A)
as a means of identification. Svp1p has previously been shown
to bind PI(3,5)P2 in yeast35 and mammalian cells36 and has
been implicated in the regulation of the endosomal pathway
and autophagy36,37 (the mechanism underpinning the degra-
dation of intracellular components). EEA1, on the other hand,
is a FYVE domain-containing protein that binds specifically to
PI(3)P38 and is used here to demonstrate the specificity of the
pull-down for PI(3,5)P2 binding proteins. As expected, svp1p
immunoreactivity is enriched in the PI(3,5)P2 lane compared
to the control pull-down, while EEA1 is not associated with
either control or PI(3,5)P2 beads. These two results demon-
strate the capacity of affinity probe 30 to specifically recruit
PI(3,5)P2 effectors. We further demonstrated that svp1p
binding to PI(3,5)P2 is sensitive to altered pH (Figure 1B).
Significantly higher binding is observed at pH 7.3 than pH 7.6.
A similar dependence on pH has previously been observed for
EEA1 binding to PI(3)P and these results may indicate that
binding of svp1p to membranes can be modulated by intracel-
lular pH, as demonstrated for EEA1.39
which was esterified with octanoic acid and desilylated to
furnish alcohol 27. The sequence 23 f 27 could be conve-
niently performed without purification of intermediates,
giving an improved yield of 51% over the four steps. The
fully elaborated compound 29 was obtained upon coupling
of phosphoramidite 28 with the inositol headgroup 9. A
stepwise deprotection of 29 in which the Cbz group is
removed last was employed as, otherwise, the free amino
group would buffer the acidity of the phosphates which is
required for MOM-deprotection.34 For the hydrogenolytic
removal of the Cbz group, we found the addition of dilute
aqueous ammonia to be beneficial with respect to reaction
time and product recovery from the catalyst. No purification
of the intermediates or the amine product was necessary, and
the latter was allowed to react with the NHS ester of biotin to
furnish the desired affinity probe 30. The synthesis of 30 has,
to our knowledge, not previously been reported in the
literature, and we include here its full characterization by
standard methods, including HPLC.
In summary, we present concise syntheses of native,
dioctanoyl, and biotin-tagged PI(3,5)P2 targets 19, 22, and
30 utilizing novel chemical methodologies. Key steps in the
(35) Dove, S. K.; Piper, R. C.; McEwen, R. K.; Yu, J. W.; King, M. C.;
Hughes, D. C.; Thuring, J.; Holmes, A. B.; Cooke, F. T.; Michell, R. H.;
Parker, P. J.; Lemmon, M. A. EMBO J. 2004, 23, 1922–1933.
(36) Jeffries, T. R.; Dove, S. K.; Michell, R. H.; Parker, P. J. Mol. Biol.
Cell 2004, 15, 2652–2663.
(37) Proikas-Cezanne, T.; Ruckerbauer, S.; Stierhof, Y.-D.; Berg, C.;
Nordheim, A. FEBS Lett. 2007, 581, 3396–3404.
(38) Patki, V.; Virbasius, J.; Lane, W. S.; Toh, B.-H.; Shpetner, H. S.;
Corvera, S. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 7326–7330. Stenmark, H.;
Aasland, R.; Toh, B.-H.; D’Arrigo, A. J. Biol. Chem. 1996, 271, 24048–
24054.
(39) Lee, S. A.; Eyeson, R.; Cheever, M. L.; Geng, J.; Verkhusha, V. V.;
Burd, C.; Overduin, M.; Kutateladze, T. G. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 13052–13057.
(34) While treatment of compound 29 with TMSBr/2-methyl-2-butene at
rt led to partial removal of the Cbz group, this could be kept to a minimum by
using a brief reaction time (35 min). Under more forcing conditions (40 °C, 19
h), products resulting from cleavage of the diacylglycerol moiety by Br-
attack at the sn-1 position were detected by MS and 1H NMR (∼10-15%).
The Cbz group was ∼90% deprotected in this experiment.
J. Org. Chem. Vol. 75, No. 11, 2010 3545

Anderson et al.
JOCFeatured Article
SCHEME 6. Synthesis of Biotin-PI(3,5)P2 Affinity Probe 30
synthetic protocol included regioselective silylation of pentol
2 with TESCl to give the 3,5-protected intermediate 6 in good
yield and a regioselective TESOTf-mediated acetonide open-
ing to access mixed diacylglycerols 16 and 27. The successful
application of the biotin-tagged derivative 30 was demon-
strated by its ability to specifically recruit a known PI(3,5)P2-
binding protein from a cytosolic fraction of adrenal neuro-
secretory cells. Further studies will utilize the affinity puri-
fication described here combined with mass spectrometry to
identify novel PI(3,5)P2 effectors.
NMR (500 MHz, CDCl3) δ 0.42-0.54 (m, 6 H), 0.86 (t, J = 7.9
Hz, 9 H), 1.11 (s, 9 H), 2.29 (d, J = 2.3 Hz, 1 H, 4-OH), 2.32 (s, 1
H, 2-OH), 2.36 (d, J = 2.6 Hz, 1 H, 6-OH), 2.66 (d, J = 2.0 Hz, 1
H, 5-OH), 3.17 (dd, J = 2.8, 9.2 Hz, 1 H, H-3), 3.22 (dt, J = 2.0,
9.3 Hz, 1 H, H-5), 3.54 (dd, J = 2.8, 9.3 Hz, 1 H, H-1), 3.57 (t,
J = 2.8 Hz, 1 H, H-2), 3.74 (dt, J = 2.3, 9.3 Hz, 1 H, H-4), 3.97 (dt,
J = 2.6, 9.3 Hz, 1 H, H-6), 7.37-7.41 (m, 4 H), 7.43-7.47 (m, 2
H), 7.72-7.76 (m, 4 H); 13C NMR (75 MHz, CDCl3) δ 4.7, 6.6,
19.4, 27.0, 72.8, 72.9, 73.0, 73.2, 73.9, 74.2, 127.8, 127.9, 130.0,
133.1, 133.4, 135.8; HRMS(ESI) calcd for C28H44NaO6Si2
[M þ Na]þ 555.2574, found 555.2562.
1-D-1-O-tert-Butyldiphenylsilyl-4-O-triethylsilyl-myo-inositol
(5): 1H NMR (500 MHz, CDCl3) δ 0.66 (q, J = 7.9 Hz, 6 H),
0.95 (t, J = 7.9 Hz, 9 H), 1.10 (s, 9 H), 2.18 (br, 1 H, 6-OH), 2.26
(d, J = 5.8 Hz, 1 H, 3-OH), 2.39 (br, 1 H, 5-OH), 2.44 (br, 1 H,
2-OH), 3.12 (t, J = 9.1 Hz, 1 H, H-5), 3.18 (ddd, J = 3.1, 5.8, 9.1
Hz, 1 H, H-3), 3.56 (dd, J = 2.9, 9.2 Hz, 1 H, H-1), 3.76 (t, J =
9.1 Hz, 1 H, H-4), 3.85-3.89 (m, 2 H, H-2 and H-6), 7.38-7.41
(m, 4 H), 7.43-7.47 (m, 2 H), 7.69-7.73 (m, 4 H); 13C NMR
(126 MHz, CDCl3) δ 5.2, 6.8, 19.4, 27.0, 72.0, 72.4, 73.4, 74.4,
74.57, 74.64, 127.9, 130.1, 130.2, 132.9, 133.2, 135.7, 135.8;
HRMS(ESI) calcd for C28H44NaO6Si2 [M þ Na]þ 555.2574,
found 555.2571.
Experimental Section
(i) Monosilylation of Pentol 2. Pentol 221 (590 mg, 1.41 mmol)
was dried by azeotropic distillation with pyridine under reduced
pressure and thoroughly concentrated under high vacuum. The
resultant foam was dissolved in pyridine (2.3 mL) and CH2Cl2
(1.0 mL) and stirred under argon at -78 °C while a solution of
TESCl (0.29 mL, 1.7 mmol) in CH2Cl2 (1.0 mL) was added over
15 min. After 3 h at -78 °C, the reaction was allowed to warm
to rt. The mixture was diluted with EtOAc, washed with water
and brine, and dried over MgSO4. Flash chromatography was
carried out on silica gel, using 10%, 12%, 20%, and 65%
EtOAc/petroleum ether as eluants, to give silyl ethers 6 (127
mg, 14%), 7 (20 mg, 2%), 5 (8 mg, 1%), and 4 (534 mg, 71%),
respectively. Compounds 5-7 were isolated as colorless gums,
while compound 4 was obtained as a white foam. (ii) Bis-
silylation of Pentol 2. A solution of 2 (6.18 g, 14.8 mmol, dried
using the above procedure) in CH2Cl2 (15 mL) and pyridine
(10 mL) was stirred under argon at -78 °C while a solution of
TESCl (4.52 g, 30.0 mmol) in CH2Cl2 (10 mL) was added over 15
min. The reaction was stirred for a further 1 h at -75 °C and then
allowed to warm to 0 °C over 3 h. Workup and purification, as
described above, gave compounds 6 (6.78 g, 71%), 7 (1.32 g,
14%), and 4 (0.62 g, 8%).
1-D-3,5-O-Bis(triethylsilyl)-1-O-tert-butyldiphenylsilyl-myo-
1
inositol (6): [R]20 þ1.35 (c 2 g 100 mL-1, CHCl3); H NMR
D
(500 MHz, CDCl3) δ 0.39-0.51 (m, 6 H), 0.66 (q, J = 7.9 Hz,
6 H), 0.85 (t, J = 7.9 Hz, 9 H), 0.96 (t, J = 7.9 Hz, 9 H), 1.11 (s,
9 H), 2.09 (d, J = 2.1 Hz, 1 H, 4-OH), 2.23 (d, J = 2.3 Hz, 1 H,
6-OH), 2.28 (s, 1 H, 2-OH), 3.11 (dd, J = 2.8, 9.2 Hz, 1 H, H-3),
3.18 (t, J = 9.1 Hz, 1 H, H-5), 3.49-3.54 (m, 2 H, H-2 and H-1),
3.64 (dt, J = 2.1, 9.2 Hz, 1 H, H-4), 3.89 (dt, J = 2.3, 9.1 Hz,
1 H, H-6), 7.36-7.46 (m, 6 H), 7.74-7.77 (m, 4 H); 13C NMR
(75 MHz, CDCl3) δ 4.8, 5.3, 6.7, 6.9, 19.5, 27.0, 72.8, 73.1, 73.3,
73.4, 74.2, 76.0, 127.7, 129.8, 129.9, 133.3, 133.8, 135.8, 136.0;
HRMS(ESI) calcd for C34H58NaO6Si3 [M þ Na]þ 669.3439,
found 669.3428.
1-D-1-O-tert-Butyldiphenylsilyl-3-O-triethylsilyl-myo-inositol
1-D-3,4-O-Bis(triethylsilyl)-1-O-tert-butyldiphenylsilyl-myo-
(4): mp 55-57 °C; [R]20D -10.4 (c 2 g 100 mL-1, CHCl3); 1H
inositol (7): [R]20 -13.2 (c 2 g 100 mL-1, CHCl3); H NMR
1
D
3546 J. Org. Chem. Vol. 75, No. 11, 2010

Anderson et al.
JOCFeatured Article
127.6, 127.8, 129.6, 129.9, 133.1, 134.4, 136.0, 136.4; HRMS-
(ESI) calcd for C40H70NaO9Si3 [M þ Na]þ 801.4225, found
801.4221.
1-D-1-O-tert-Butyldiphenylsilyl-2,4,6-O-tris(methoxymethylene)-
myo-inositol (8).11 The crude MOM-protected product obtained
above (3.3 g) was dissolved in 3:3:1 THF/HOAc/water (28 mL)
and heated at 40 °C for 48 h. After concentration of the solvents
under reduced pressure, the crude product was purified by flash
chromatography on silica gel, using 40-45% EtOAc/petroleum
ether as eluant, to afford diol 8 as a white crystalline solid (1.90 g,
90% over two steps): mp 116-117 °C; [R]20 þ77.8 (c 2 g 100
D
mL-1, CHCl3); 1H NMR (500 MHz, CDCl3) δ 1.07 (s, 9 H), 3.19
(ddd, J = 2.5, 7.0, 9.7 Hz, 1 H), 3.25 (m, 1 H), 3.33 (s, 3 H), 3.41 (s,
3 H), 3.43 (s, 3 H), 3.57-3.61 (m, 2 H), 3.70-3.76 (m, 3 H), 4.32 (d,
J = 1.2 Hz, 1 H), 4.34 (d, J = 6.8 Hz, 1 H), 4.53 (d, J = 6.5 Hz,
1 H), 4.62 (d, J = 6.8 Hz, 1 H), 4.76 (d, J = 6.5 Hz, 1 H), 4.80 (d,
J = 6.6 Hz, 1 H), 4.84 (d, J = 6.6 Hz, 1 H), 7.36-7.45 (m, 6 H),
7.68-7.71 (m, 4 H); 13C NMR (126 MHz, CDCl3) δ 19.3, 27.0,
55.7, 55.8, 56.0, 70.4, 72.6, 73.5, 81.91, 81.93, 85.0, 98.1, 98.60,
98.63, 127.6, 127.7, 129.7, 130.0, 133.6, 133.7, 135.8, 135.9; HRMS-
(ESI) calcd for C28H42NaO9Si [M þ Na]þ 573.2496, found
573.2491.
1-D-2,4,6-O-Tris(methoxymethylene)-myo-inositol 3,5-Bis(dibenzyl
phosphate) (9).Alcohol 9was obtained from diol 8intwosteps(90%)
according to the literature procedure.11 1H and 13C NMR data were
in good agreement with those previously reported. Additional spec-
troscopic data: [R]20D -8.4 (c 2.4 g 100 mL-1, CDCl3); 31P NMR
(121 MHz, CDCl3) δ -1.3; HRMS(ESI) calcd for C40H50NaO15P2
[M þ Na]þ 855.2523, found 855.2529.
FIGURE 1. (A) Bovine adrenal chromaffin cytosol extract was
incubated with biotin-PI(3,5)P2 conjugated to Neutravidin Plus
beads [PtdIns(3,5)P2] or beads alone (control). Beads were washed
extensively, and associated proteins were analyzed by SDS-PAGE
and Western blotting together with an untreated portion of the
cytosolic extract (0.25 T). Antibodies against svp1p and EEA1 were
used to identify these proteins. (B) Biotin-PI(3,5)P2 pull-downs
were performed at pH 7.3 or 7.6 and associated proteins analyzed by
SDS-PAGE and Western blotting.
(300 MHz, CDCl3) δ 0.47 (q, J = 8.0 Hz, 6 H), 0.63 (q, J = 8.0
Hz, 6 H), 0.87 (t, J = 8.0 Hz, 9 H), 0.94 (t, J = 8.0 Hz, 9 H), 1.11
(s, 9 H), 2.33 (d, J = 1.5 Hz, 1 H, 2-OH), 2.42 (d, J = 3.0 Hz, 1 H,
6-OH), 2.45 (d, J = 3.1 Hz, 1 H, 5-OH), 3.16 (dt, J = 3.1, 8.7 Hz,
1 H, H-5), 3.25 (dd, J = 2.8, 8.5 Hz, 1 H, H-3), 3.57 (dd, J = 2.8,
8.8 Hz, 1 H, H-1), 3.62 (m, 1 H, H-2), 3.79 (t, J = 8.5 Hz, 1 H,
H-4), 3.94 (dt, J = 3.0, 8.8 Hz, 1 H, H-6), 7.35-7.47 (m, 6 H),
7.70-7.77 (m, 4 H); 13C NMR (75 MHz, CDCl3) δ 4.9, 5.2, 6.8,
6.9, 19.4, 27.0, 72.4, 72.9, 73.7, 74.2, 74.4, 74.5, 127.7, 127.8,
129.89, 129.93, 133.2, 133.5, 135.8, 135.9; HRMS(ESI) calcd for
C34H58NaO6Si3 [M þ Na]þ 669.3439, found 669.3422.
1D-1-O-tert-Butyldiphenylsilyl-3-O-triethylsilyl-2,4,6-O-tris-
(methoxymethylene)-myo-inositol (10). A solution of diol 8
(1.86 g, 3.38 mmol) in dry pyridine (3.4 mL) and CH2Cl2 (3.4
mL) was stirred at -90 °C while a solution of TESCl (0.67 mL,
4.0 mmol) in CH2Cl2 (1.5 mL) was added over 2 min. After
being stirred at -78 °C for 3 h, the mixture was allowed to warm
to rt overnight. The reaction was diluted with EtOAc, washed
with water and brine, and dried over MgSO4. Purification by
flash chromatography on silica gel, using 15-20% EtOAc/
petroleum ether as eluant, gave 10 as a colorless gum (1.79 g,
80%): [R]20D þ10.7 (c 2.5 g 100 mL-1, CHCl3); 1H NMR (300
MHz, CDCl3) δ 0.44 (q, J = 7.9 Hz, 6 H), 0.86 (t, J = 7.9 Hz, 9
H), 1.08 (s, 9 H), 3.20-3.25 (m, 2 H), 3.34 (s, 3 H), 3.39 (s, 3 H),
3.41 (m, 1 H), 3.43 (s, 3 H), 3.66 (t, J = 9.2 Hz, 1 H), 3.73 (dd,
J = 2.0, 9.6 Hz, 1 H), 3.84 (t, J = 9.1 Hz, 1 H), 4.24 (d, J = 1.0
Hz, 1 H, exch), 4.51 (d, J = 6.5 Hz, 1 H), 4.66 (d, J = 6.1 Hz,
1 H), 4.69-4.73 (m, 2 H), 4.75-4.78 (m, 2 H), 7.33-7.46 (m,
6 H), 7.68-7.76 (m, 4 H); 1H NMR (300 MHz, d6-acetone) δ
0.49 (q, J = 7.9 Hz, 6 H), 0.88 (t, J = 7.9 Hz, 9 H), 1.10 (s, 9 H),
3.26 (m, 1 H), 3.33 (s, 3 H), 3.37 (s, 3 H), 3.38 (s, 3 H), 3.47 (dd,
J = 2.2, 9.4 Hz, 1 H), 3.51 (t, J = 2.2 Hz, 1 H), 3.64 (t, J =
9.4 Hz, 1 H), 3.81 (t, J = 9.4 Hz, 1 H), 3.97 (dd, J = 2.2, 9.4 Hz,
1 H), 4.15 (d, J = 2.2 Hz, 0.7 H, exch), 4.62 (d, J = 6.4 Hz, 1 H),
4.69 (d, J = 6.0 Hz, 1 H), 4.72-4.80 (m, 4 H), 7.40-7.51 (m,
6 H), 7.75-7.83 (m, 4 H); 13C NMR (75 MHz, CDCl3) δ 4.7,
6.7, 19.3, 27.1, 55.7, 55.8, 72.4, 73.1, 73.3, 79.6, 81.5, 83.3, 97.5,
98.3, 98.5, 127.5, 127.7, 129.6, 129.8, 133.5, 133.9, 136.0, 136.1;
HRMS(ESI) calcd for C34H56NaO9Si2 [M þ Na]þ 687.3361,
found 687.3340.
1-D-3,5-O-Bis(triethylsilyl)-1-O-tert-butyldiphenylsilyl-2,4,6-
O-tris(methoxymethylene)-myo-inositol. To a solution of triol 6
(2.48 g, 3.83 mmol, previously dried by azeotropic distillation
with toluene under reduced pressure) in DMF (9 mL) was
added N,N-diisopropylethylamine (7.0 mL, 40 mmol) followed
by MOMCl solution40 (∼6 M in MeOAc, 4.3 mL, 26 mmol).
The reaction vessel was sealed and heated at 65 °C for 64 h.
After being cooled to rt, the mixture was partitioned between
petroleum ether and water, and the organic phase was washed
with 0.1 M aq HCl, water, and brine and dried over MgSO4 to
give the crude MOM-protected product (3.3 g). A comparable
sample was purified by flash chromatography on silica gel,
using 8% EtOAc/petroleum ether as eluant, to afford the title
compound as a colorless gum: [R]20 þ18.6 (c 2 g 100 mL-1
,
D
1
CHCl3); H NMR (500 MHz, CDCl3) δ 0.29-0.38 (m, 6 H),
0.72 (q, J = 7.9 Hz, 6 H), 0.82 (t, J = 7.9 Hz, 9 H), 1.00 (t, J =
7.9 Hz, 9 H), 1.09 (s, 9 H), 3.10 (dd, J = 2.2, 9.5 Hz, 1 H), 3.17
(t, J = 2.2 Hz, 1 H), 3.30 (t, J = 9.2 Hz, 1 H), 3.34 (s, 3 H), 3.39
(s, 3 H), 3.47 (s, 3 H), 3.62 (t, J = 9.4 Hz, 1 H), 3.74 (dd, J = 2.2,
9.8 Hz, 1 H), 3.90 (t, J = 9.5 Hz, 9 H), 4.60 (d, J = 6.0 Hz, 1 H),
4.64 (d, J = 6.0 Hz, 1 H), 4.72 (d, J = 5.8 Hz, 1 H), 4.74 (d, J =
5.8 Hz, 1 H), 4.87 (d, J = 5.7 Hz, 1 H), 4.94 (d, J = 5.7 Hz, 1 H),
7.35-7.40 (m, 4 H), 7.42-7.46 (m, 2 H), 7.72 (m, 2 H), 7.86 (m, 2
H); 13C NMR (126 MHz, CDCl3) δ 4.7, 5.2, 6.8, 7.1, 19.2, 27.3,
55.7, 56.5, 56.9, 73.0, 73.9, 75.0, 78.7, 79.0, 79.8, 97.7, 98.4, 99.0,
1D-1-O-tert-Butyldiphenylsilyl-2,4,5,6-O-tetrakis(methoxymethy-
lene)-3-O-triethylsilyl-myo-inositol. To a solution of tetrol 4 (687
mg, 1.29 mmol) in DMF (3 mL) in a pressure vessel was added
N,N-diisopropylethylamine (2.2 mL, 13 mmol) followed by MOMCl
solution40 (∼6 M in MeOAc, 1.8 mL, 11 mmol). The reaction vessel
was sealed and heated at 60 °C for 4 days. After being cooled to rt,
the mixture was partitioned between petroleum ether and water,
and the organic phase was washed with 0.1 M aq HCl, water and
brine and dried over MgSO4 to give the crude MOM-protected
product (996 mg). A comparable sample was purified by flash
(40) Amato, J. S.; Karady, S.; Sletzinger, M.; Weinstock, L. M. Synthesis
1979, 970.
J. Org. Chem. Vol. 75, No. 11, 2010 3547

Anderson et al.
JOCFeatured Article
chromatography on silica gel, using 15% EtOAc/petroleum ether as
(630 mg, 2.07 mmol), and DMAP (50 mg, 0.41 mmol). After being
stirred at rt for 2.5 h, the reaction was quenched by the addition of
HOAc (50 μL) followed by water (20 μL). The mixture was diluted
with petroleum ether, filtered, and concentrated to give an oil. Flash
chromatography on silica gel, using 1.5-2% acetone/petroleum
ether as eluant, followed by a second column using 5-7% Et2O/
petroleum ether as eluant, gave the protected diacylgycerol 15
(1.178 g, 82%): [R]20D þ8.1 (c 4.0 g 100 mL-1, CHCl3); 1H NMR
(500 MHz, CDCl3) δ 0.60 (q, J = 8.0 Hz, 6 H), 0.87-0.91 (m,
6 H), 0.95 (t, J = 8.0 Hz, 9 H), 1.26-1.39 (m, 34 H), 1.61 (pentet,
J = 7.3 Hz, 2 H), 1.70 (pentet, J = 7.5 Hz, 2 H), 2.06 (q, J =
7.1 Hz, 2 H), 2.12 (q, J = 6.9 Hz, 2 H), 2.28-2.34 (m, 4 H),
2.80-2.85 (m, 6 H), 3.69-3.75 (m, 2 H), 4.16 (dd, J = 6.2, 11.8
Hz, 1 H), 4.35 (dd, J = 3.7, 11.8 Hz, 1 H), 5.07 (m, 1 H), 5.31-
5.43 (m, 8 H); 13C NMR (126 MHz, CDCl3) δ 4.3, 6.6, 14.0, 14.1,
22.6, 22.7, 24.8, 24.9, 25.6, 25.7, 26.5, 27.2, 29.1, 29.30, 29.32, 29.4,
29.5, 29.6, 29.65, 29.70, 31.5, 31.9, 33.7, 34.2, 61.2, 62.4, 71.9,
127.6, 127.9, 128.1, 128.3, 128.6, 128.9, 130.5, 172.8, 173.4;
HRMS(ESI) calcd for C47H86NaO5Si [M þ Na]þ 781.6142, found
781.6154.
eluant, to afford the title compound as a colorless gum: [R]20
D
þ10.9 (c 1.8 g 100 mL-1, CDCl3); 1H NMR (500 MHz, CDCl3)
δ 0.28-0.39 (m, 6 H), 0.82 (t, J = 8.0 Hz, 9 H), 1.08 (s, 9 H), 3.16
(dd, J = 2.1, 9.5 Hz, 1 H), 3.19 (t, J = 2.1 Hz, 1 H), 3.29-3.32
(m, 4 H), 3.42 (s, 3 H), 3.48 (s, 3 H), 3.49 (s, 3 H), 3.74-3.79 (m,
2H),4.01(t,J= 9.6 Hz, 1 H), 4.57 (d, J= 6.0 Hz, 1 H), 4.62 (d, J=
6.0 Hz, 1 H), 4.72 (d, J = 6.1 Hz, 1 H), 4.79 (d, J = 6.1 Hz, 1 H),
4.83 (d, J = 6.0 Hz, 1 H), 4.91 (d, J = 6.0 Hz, 1 H), 4.94 (d, J = 6.0
Hz, 1 H), 5.00 (d, J = 6.0 Hz, 1 H), 7.35-7.46 (m, 6 H), 7.71 (m, 2
H), 7.79 (m, 2 H); 13C NMR (126 MHz, CDCl3) δ 4.7, 6.8, 19.3,
27.3, 55.7, 56.5, 56.6, 56.7, 73.2, 74.1, 78.6, 79.0, 79.2, 79.7, 97.7,
98.66, 98.69, 99.3, 127.7, 127.8, 129.7, 129.9, 133.1, 134.1,
136.0, 136.2; HRMS(ESI) calcd for C36H60NaO10Si2 [M þ
Na]þ 731.3623, found 731.3622.
1D-1-O-tert-Butyldiphenylsilyl-2,4,5,6-O-tetrakis(methoxymethy-
lene)-myo-inositol (11).11 The crude MOM-protected product ob-
tained above (996 mg) was dissolved in 3:3:1 THF/HOAc/water
(8.4 mL) and stirred at 40 °C for 24 h. The volatiles were
concentrated under reduced pressure, and the residue was coeva-
porated with toluene twice to remove residual HOAc. Purification
by flash chromatography on silica gel, using 20% acetone/petro-
leum ether as eluant, gave 11 as a colorless gum (521 mg, 68% over
two steps): [R]20D þ52 (c 2.0 g 100 mL-1, CHCl3); 1H NMR (500
MHz, CDCl3) 1.09 (s, 9 H), 3.05 (ddd, J = 2.3, 7.1, 9.4 Hz, 1 H),
3.21 (t, J = 2.3 Hz, 1 H), 3.34-3.38 (m, 4 H), 3.39 (s, 3 H), 3.44 (s, 3
H), 3.45 (s, 3 H), 3.60 (t, J = 9.4 Hz, 1 H), 3.81 (dd, J = 2.3, 9.5 Hz,
1H),3.85(d,J= 7.1 Hz, 1 H), 3.98 (t, J= 9.5 Hz, 1 H), 4.30 (d, J=
6.5 Hz, 1 H), 4.60 (d, J = 6.5 Hz, 1 H), 4.74-4.78 (m, 3 H), 4.82 (d,
J = 6.3 Hz, 1 H), 4.92 (d, J = 6.3 Hz, 1 H), 4.99 (d, J = 6.2 Hz,
1 H), 7.36-7.40 (m, 4 H), 7.43-7.46 (m, 2 H), 7.70 (m, 2 H), 7.73
(m, 2H);13C NMR (126 MHz, CDCl3) δ19.2, 27.1, 55.8, 55.9, 56.3,
56.5, 70.6, 73.9, 78.9, 79.1, 81.2, 81.4, 98.3, 98.36, 98.44, 99.0, 127.77,
127.83, 129.9, 130.1, 132.9, 134.0, 135.9, 136.0; HRMS(ESI) calcd
for C30H46NaO10Si [M þ Na]þ 617.2758, found 617.2756.
2-O-Arachidonoyl-1-O-stearoyl-sn-glycerol (16).24 The protec-
ted diacylglycerol 15 (1.09 g, 1.43 mmol) was stirred at rt with
a solution of FeCl3 in 3:1 reagent-grade MeOH/CH2Cl2 (5 mM,
14 mL). The starting material fully dissolved after 25 min,
and after 8.5 h the solution was partitioned between Et2O and
water. The organic phase was washed with brine and dried over
MgSO4. Flash chromatography on silica gel, using 25-35%
Et2O/petroleum ether (containing 0.05% HOAc) as eluant, gave
the desired product 16 as an oil (836 mg, 90%): [R]20D þ2.6 (c 4.0
g 100 mL-1, pyridine), -0.45 (c 3.42 g 100 mL-1, toluene) [lit.24
[R]20D -0.4 (c 3.42 g 100 mL-1, toluene)]; 1H NMR (500 MHz,
CDCl3) δ 0.87-0.90 (m, 6 H), 1.26-1.39 (m, 34 H), 1.61 (pentet,
J = 7.4 Hz, 2 H), 1.72 (pentet, J = 7.5 Hz, 2 H), 2.00 (t, J = 8.0
Hz, 1 H), 2.06 (m, 2 H), 2.13 (m, 2 H), 2.32 (t, J = 7.4 Hz, 2 H),
2.37 (t, J = 7.5 Hz, 2 H), 2.80-2.85 (m, 6 H), 3.69-3.77 (m,
2 H), 4.24 (dd, J = 5.6, 11.9 Hz, 1 H), 4.32 (dd, J = 4.6, 11.9 Hz,
1 H), 5.08 (m, 1 H), 5.31-5.44 (m, 8 H); 13C NMR (126 MHz,
CDCl3) δ 14.0, 14.1, 22.6, 22.7, 24.8, 24.9, 25.62, 25.64, 26.5,
27.2, 29.1, 29.26, 29.32, 29.35, 29.5, 29.6, 29.65, 29.69, 31.5, 31.9,
33.7, 34.1, 61.6, 62.0, 72.2, 127.5, 127.8, 128.1, 128.3, 128.6,
128.8, 129.0, 130.5, 173.1, 173.8; HRMS(ESI) calcd for
C41H72NaO5 [M þ Na]þ 667.5277, found 667.5271.
1-O-Stearoyl-3-O-triethylsilyl-sn-glycerol (14). To a solution
of acetonide 1224 (1.02 g, 2.56 mmol) in dry DCE (10 mL) was
added N,N-diisopropylethylamine (0.90 mL, 5.2 mmol) fol-
lowed by TESOTf (0.70 mL, 3.1 mmol). The mixture was stirred
at reflux for 12 h, after which time further TESOTf (0.30 mL, 1.3
mmol) was added and heating was continued for an additional
22 h. After being cooled to rt, the reaction was diluted with
petroleum ether, washed with 0.1 M aq HCl, water, and brine,
and dried over MgSO4 to afford the intermediate 2-O-isopro-
penyl-1-O-stearoyl-3-O-triethylsilyl-sn-glycerol (13), together
with ∼15% unreacted starting material. This material was
dissolved in THF (21 mL), and 10% aq NaHCO3 (9 mL) was
added, followed by iodine (956 mg, 3.77 mmol). The mixture
was stirred vigorously at rt for 80 min, at which time TLC
analysis indicated that the isopropenyl ether had been comple-
tely consumed. After being quenched with saturated aq
Na2S2O3, the product was extracted with Et2O, washed with
brine, and dried over MgSO4. Flash chromatography on silica
gel, using 3-5% acetone/petroleum ether as eluant, gave the
pure secondary alcohol 14 as a colorless low-melting solid (890
mg, 74%, two steps): [R]20D þ1.1 (c 4.0 g 100 mL-1, CHCl3); 1H
NMR (500 MHz, CDCl3) δ 0.62 (q, J = 8.0 Hz, 6 H), 0.88 (t, J =
6.9 Hz, 3 H), 0.96 (t, J = 8.0 Hz, 9 H), 1.25-1.33 (m, 28 H), 1.62
(pentet, J = 7.5 Hz, 2 H), 2.33 (t, J = 7.5 Hz, 2 H), 2.53 (d, J =
5.2 Hz, 1 H), 3.60 (dd, J = 5.8, 10.1 Hz, 1 H), 3.67 (dd, J = 4.6,
10.1 Hz, 1 H), 3.87 (m, 1 H), 4.12 (dd, J = 4.9, 11.5 Hz, 1 H), 4.15
(dd, J = 6.0, 11.5 Hz, 1 H); 13C NMR (126 MHz, CDCl3) δ 4.3,
6.7, 14.1, 22.7, 25.0, 29.2, 29.3, 29.4, 29.5, 29.6, 29.65, 29.67,
29.69, 31.9, 34.2, 63.4, 65.0, 70.1, 173.9; HRMS(ESI) calcd for
C27H56NaO4Si [M þ Na]þ 495.3846, found 495.3846.
2-O-Arachidonoyl-1-O-stearoyl-sn-glycero-3-(benzyl diisopropyl-
phosphoramidite) (17).17 Diacylglycerol 16 (798 mg, 1.24 mmol)
and 1H-tetrazole (88 mg, 1.26 mmol), dried by azeotropic distilla-
tion of toluene on a rotary evaporator, were suspended in dry
CH2Cl2 (12 mL) and stirred in an ice bath under Ar while benzyl N,
N,N0,N0-tetraisopropylphosphorodiamidite (540 mg, 1.60 mmol)
was quickly added. After 10 min, the ice bath was removed, and
stirring was continued at rt for 45 min. The mixture was concen-
trated under reduced pressure, suspended in petroleum ether, and
immediately purified by rapid flash chromatography on Et3N-
neutralized silica gel, using 10:90:3 EtOAc/petroleum ether/Et3N
as eluant, to afford the phosphoramidite 17 (1:1 mixture of dias-
tereomers) as a colorless oil (929 mg, 85%). The oil was stored
under argon at -20 °C until ready for use: 1H NMR (500 MHz,
CDCl3) δ 0.87-0.90 (m, 6 H), 1.17-1.19 (m, 12 H), 1.25-1.39 (m,
34 H), 1.57-1.63(m, 2H), 1.66-1.72 (m, 2 H), 2.03-2.08(m, 2H),
2.08-2.12 (m, 2 H), 2.27-2.33 (m, 4 H), 2.79-2.85 (m, 6 H),
3.60-3.67 (m, 2 H), 3.69-3.81 (m, 2 H), 4.15-4.19 (m, 1 H), 4.32-
4.37 (m, 1 H), 4.63-4.68 (m, 1 H), 4.71-4.76 (m, 1 H), 5.17-5.21
(m, 1 H), 5.31-5.43 (m, 8 H) 7.24-7.27 (m, 1 H), 7.31-7.35 (m, 4
H); 13C NMR (126 MHz, CDCl3) δ 14.05, 14.09, 22.6, 22.7,
24.5-24.59 (2 ꢀ d), 24.63, 24.68, 24.8, 24.9, 25.62, 25.63, 25.65,
26.6, 27.2, 29.2, 29.30, 29.32, 29.36, 29.5, 29.64, 29.66, 29.67, 29.70,
31.5, 31.9, 33.8, 34.1, 43.1-43.2 (2 ꢀ d), 61.5-61.8 (2 ꢀ d), 62.46,
62.51, 65.3-65.5 (2 ꢀ d), 70.9-71.0 (2 ꢀ d), 127.0, 127.3, 127.6,
127.9, 128.2, 128.3, 128.6, 128.87, 128.91, 130.5, 139.3-139.4 (2 ꢀ d),
2-O-Arachidonoyl-1-O-stearoyl-3-O-triethylsilyl-sn-glycerol (15).
To a solution of alcohol 14 (890 mg, 1.88 mmol) in dry CH2Cl2
(10 mL) were added DCC (564 mg, 2.73 mmol), arachidonic acid
3548 J. Org. Chem. Vol. 75, No. 11, 2010

Anderson et al.
JOCFeatured Article
172.7, 173.4; 31P NMR (202 MHz, CDCl3) δ 148.9, 149.1;
HRMS(ESI) calcd for C54H92NNaO6P [M þ Na]þ 904.6560,
found 904.6554.
phosphite intermediate was oxidized with m-CPBA (1.0 mmol,
prepared by dissolving 350 mg of 50-55% m-CPBA in CHCl3,
drying over MgSO4, filtering, and concentrating to ∼4 mL). After
being stirred at rt overnight, the solution was diluted with EtOAc,
washed with 10% aq Na2S2O3, 10% aq NaHCO3, and brine, and
dried over MgSO4. Flash chromatography on silica gel, using
20-30% acetone/petroleum ether as eluant, afforded the fully
protected dioctanoyl PI(3,5)P2 21 (mixture of diastereomers) as a
1D-1-O-(2-O-Arachidonoyl-1-O-stearoyl-sn-glycero-3-O-benzyl-
phospho)-2,4,6-O-tris(methoxymethylene)-myo-inositol 3,5-Bis-
(dibenzyl phosphate) (18). A mixture of inositol 9 (160 mg,
0.192 mmol) and 1H-tetrazole (41 mg, 0.59 mmol), dried by
azeotropic distillation of toluene under reduced pressure, was
stirred in dry CH2Cl2 (1 mL) at rt while neat phosphoramidite 17
(373 mg, 0.423 mmol) was added from a syringe over 1 h. The
reaction was stirred at rt for a further 17 h and then cooled to
-25 °C, and the phosphite intermediate was oxidized with a
solution of Bu4NIO4 (220 mg, 0.508 mmol) in CH2Cl2 (1.5 mL).
After being warmed to rt over 1.5 h, the reaction was quenched
with 10% aq Na2S2O3 and diluted with EtOAc. The organic
phase was washed with water and brine and dried over MgSO4.
Purification by flash chromatography on silica gel, using 20%-
25% acetone/petroleum ether as eluant, gave the fully protected
PI(3,5)P2 18 (mixture of diastereomers) as a colorless gum (247
mg, 79%): 1H NMR (500 MHz, CDCl3) δ 0.87-0.90 (m, 6 H),
1.25-1.40 (m, 34 H), 1.53-1.60 (m, 2 H), 1.62-1.70 (m, 2 H),
2.03-2.10 (m, 4 H), 2.23-2.32 (m, 4 H), 2.77-2.84 (m, 6 H), 3.31
(s, 1.5 H), 3.317 (s, 1.5 H), 3.319 (s, 1.5 H), 3.34 (s, 1.5 H), 3.35 (s,
1.5 H), 3.37 (s, 1.5 H), 4.02-4.30 (m, 9 H), 4.50 (m, 1 H),
4.67-4.78 (m, 6 H), 5.00-5.09 (m, 9 H), 5.12 (m, 1 H), 5.17 (m,
1 H), 5.30-5.42 (8 H), 7.27-7.38 (m, 25 H); 13C NMR(126 MHz,
CDCl3) δ 14.0, 14.1, 22.6, 22.7, 24.7, 24.8, 25.59, 25.61, 25.64,
26.5, 27.2, 29.1, 29.29, 29.30, 29.34, 29.5, 29.64, 29.67, 29.69, 31.5,
31.9, 33.49, 33.51, 34.0, 55.9, 56.58, 56.64, 56.65, 61.6, 65.53,
65.57, 65.74, 65.79, 69.38, 69.42, 69.50, 69.52, 69.55, 69.56, 69.62,
69.67, 69.85, 69.90, 75.45, 75.50, 75.64, 75.67, 75.82, 75.86, 75.95,
75.98, 76.02, 76.06, 79.7, 79,75, 79.79, 98.08, 98.10, 98.48, 98.53,
127.5, 127.8, 127.95, 127.96, 127.98, 128.01, 128.03, 128.1, 128.3,
128.5, 128.55, 128.60, 128.7, 128.8, 128.90, 128.91, 130.5, 135.5,
135.6, 135.65, 135.69, 135.71, 135.75, 135.77, 135.88, 135.93,
172.48, 172.51, 173.1; 31P NMR (202 MHz, CDCl3) δ -1.42,
-1.38, -1.373, -1.366, -1.18, -1.17; HRMS(ESI) calcd for
C88H127NaO22P3 [M þ Na]þ 1651.7930, found 1651.7916.
L-r-Phosphatidyl-D-myo-inositol 3,5-Bisphosphate (19).17 Un-
der an atmosphere of dry argon, TMSBr (∼0.5 mL) was distilled
from calcium hydride directly into an ice-cooled solution of the
fully protected PI(3,5)P2 18 (31 mg, 0.019 mmol) in 2-methylbut-
2-ene (0.5 mL). The solution was protected from light and stirred
at rt for 1.5 h and then, without exposure to air, concentrated
under high vacuum overnight. The solid was dissolved in freshly
distilled EtSH (0.5 mL) at rt. After 20 h, the solution was con-
centrated under reduced pressure. The resulting solid was dis-
solved in dilute aq NH3 (pH 8.5), and theproduct was lyophilized.
Conversion to the sodium salt by ion-exchange (Na form) gave
PI(3,5)P2 (19) as a white powder (17.7 mg, 84%): 1H NMR (500
MHz, CD3OD, ammonium salt) δ 0.89-0.92 (m, 6 H), 1.29-1.39
(m, 34 H), 1.59 (m, 2 H), 1.68 (m, 2 H), 2.07 (q, J = 6.9 Hz, 2 H),
2.13 (q, J = 6.0 Hz, 2 H), 2.30 (t, J = 7.4 Hz, 2 H), 2.36 (m, 2 H),
2.81-2.86 (m, 6 H), 3.91-4.15 (m, 7 H), 4.21 (dd, J = 6.8, 12.1
Hz, 1 H), 4.37 (t, J =2.4Hz, 1H), 4.48 (dd, J = 2.9, 12.1Hz, 1 H),
5.25 (m, 1 H), 5.33-5.39 (m, 8 H); 31P NMR (202 MHz, CD3OD,
ammonium salt) δ -0.1, 1.3, 2.1; HRMS(ESI) calcd for C47H84-
O19P3 [M - H]- 1045.4820, found 1045.4828.
1
colorless gum (404 mg, 84%): H NMR (500 MHz, CDCl3) δ
0.85-0.89 (m, 6 H), 1.26, (m, 16 H), 1.57 (m, 4 H), 2.22-2.29 (m, 4
H), 3.31 (s, 1.5 H), 3.318 (s, 1.5 H), 3.319 (s, 1.5 H), 3.34 (s, 1.5 H),
3.35 (s, 1.5 H), 3.37 (s, 1.5 H), 4.03-4.30 (m, 9 H), 4.50 (m, 1 H),
4.67-4.78 (m, 6 H), 5.01-5.14 (m, 10 H), 5.17 (m, 1 H), 7.28-7.38
(m, 25 H); 13C NMR (75 MHz, CDCl3) δ 14.0, 22.6, 24.8, 28.9,
29.00, 29.04, 31.6, 33.9, 34.1, 55.9, 56.56, 56.62, 61.7, 65.5-65.8 (m,
4 peaks), 69.3-69.7 (m, 7 peaks), 69.8, 69.9, 75.3-76.1 (m, 12
peaks), 79.6-79.8 (m, 6 peaks), 98.06, 98.09, 98.46, 98.50, 127.95,
128.02, 128.47, 128.53, 128.6, 128.7, 135.5, 135.56, 135.59, 135.65,
135.74, 135.8, 135.9, 172.7, 173.1; 31P NMR (121 MHz, CDCl3) δ
-1.43 (0.5 P), -1.40 (1.5 P), -1.2 (1 P); HRMS(ESI) calcd for
C66H91NaO22P3 [M þ Na]þ 1351.5113, found 1351.5083.
Dioctanoyl L-r-Phosphatidyl-D-myo-inositol 3,5-Bisphosphate
(22).17 Under an atmosphere of dry argon, TMSBr (∼1 mL) was
distilled directly into a solution of the fully protected dioctanoyl
PI(3,5)P2 21 (50 mg, 0.038 mmol) in dry pyridine (0.2 mL)
cooled to -78 °C. The reaction was stirred at rt for 50 min and
then, without exposure to air, concentrated under high vacuum
for 16 h. The residue was dissolved in 8 mM aq NH4OAc
(50 mL) and lyophilized to give a white solid (47 mg). The solid
was dissolved in CHCl3-MeOH and passed through Amberlite
IR-120-Hþ resin to remove pyridinium salts. After concentra-
tion under reduced pressure, the product was dissolved in water,
passed through Amberlite IR-120-Naþ, and finally lyophilized
to give dioctanoyl PI(3,5)P2 22 (sodium salt) as a white powder
(29 mg, 97%): 1H NMR (300 MHz, D2O) δ 0.94 (m, 6 H), 1.36,
(m, 16 H), 1.68 (m, 4 H), 2.49 (m, 4 H), 3.94-4.05 (m, 3 H),
4.08-4.19 (m, 4 H), 4.36 (dd, J = 7.1, 12.3 Hz, 1 H), 4.50-4.56
(m, 2 H), 5.38 (m, 1 H); 13C NMR (75 MHz, D2O) δ 14.1, 22.7,
25.0, 25.1, 28.8, 28.87, 28.91, 31.7, 34.5, 34.6, 63.6, 64.5 (d, J =
5.5 Hz), 70.7, 71.3-71.6 (m, g 6 peaks), 75.5 (d, J = 5.6 Hz),
76.0 (d, J = 5.8 Hz), 79.5 (d, J = 6.1 Hz), 176.7, 177.1; 31P NMR
(121 MHz, D2O) δ -0.6, 0.0, 0.9; HRMS(ESI) calcd for
C25H48O19P3 [M - H]- 745.2003, found 745.2003.
8-(Benzyloxycarbonylamino)octanoic Acid.41 To a solution of
8-aminooctanoic acid (1.00 g, 6.28 mmol) in 2 N aq NaOH (3.4
mL) were added benzyl chloroformate (0.97 mL, 6.8 mmol)
and 2 N aq NaOH (3.4 mL) simultaneously over 1 min, with
vigorous stirring. Additional water (10 mL) was added, and
after 20 min, the solution was washed with Et2O. The aqueous
layer was acidified to pH 1-2 with 1 M aq HCl, and the resulting
white precipitate was extracted with EtOAc. The organic ex-
tracts were dried with brine and MgSO4 and concentrated under
reduced pressure to give the title compound as a white solid
(1.75 g, 95%): mp = 63-64 °C (lit.42 mp 63-64 °C [EtOAc/
petroleum ether]).
1-O-[8-(Benzyloxycarbonylamino)octanoyl]-2,3-O,O0-isopro-
pylidene-sn-glycerol (23). To a solution of Cbz-protected 8-ami-
nooctanoic acid (1.75 g, 5.96 mmol) in dry CH2Cl2 (12 mL) were
added DCC (1.23 g, 5.96 mmol), L-2,3-O-isopropylidene-sn-
glycerol (0.73 g, 5.52 mmol), and DMAP (0.22 g, 1.8 mmol).
After being stirred for 2 h at rt, the reaction mixture was
concentrated under reduced pressure and the residue was taken
up in Et2O. The dicyclohexyl urea was removed by filtration,
1D-1-O-(1,2-O-Dioctanoyl-sn-glycero-3-O-benzylphospho)-2,4,6-
O-tris(methoxymethylene)-myo-inositol 3,5-bis(dibenzyl phosphate)
(21). A mixture of inositol 9 (301 mg, 0.36 mmol) and 1H-tetrazole
(75 mg, 1.1 mmol), dried by azeotropic distillation with toluene
under reduced pressure, was stirred in dry CH2Cl2 (3.5 mL) at 0 °C
while neat phosphoramidite 2032 (376 mg, 0.65 mmol) was added
from a syringe. After the mixture was warmed to rt and stirred for
15 h, TLC analysis indicated some starting material remained, and
the reaction was treated with a further portion of 20 (62 mg, 0.11
mmol). After 3 h, the mixture was cooled to -25 °C, and the
ꢀ
ꢀ
(41) D’Aleo, A.; Pozzo, J.-L.; Heuze, K.; Vogtle, F.; Fages, F. Tetrahe-
€
dron 2007, 63, 7482–7488.
(42) Okuno, Y.; Horita, K.; Yonemitsu, O. Chem. Pharm. Bull. 1983, 31,
737–740.
J. Org. Chem. Vol. 75, No. 11, 2010 3549

Anderson et al.
JOCFeatured Article
and the filtrate was purified by flash chromatography on silica
gel, using 25-30% EtOAc/petroleum ether as eluant, to give
acetonide 23 as a white solid (2.035 g, 90% from isopropylidene
petroleum ether as eluant, gave diacylglycerol 27 as a gum (237
mg, 99%). The four-stepsequencefrom23 f27 couldalsobe con-
ducted without chromatographic purification of intermediates,
glycerol): mp 38-39 °C; [R]20 þ1.5 (c 2.0 g 100 mL-1
,
and withthe replacement of EDCIfor DCC, to give the product in
D
chloroform); 1H NMR (500 MHz, CDCl3) δ 1.31 (m, 6 H),
1.36 (s, 3 H), 1.43 (s, 3 H), 1.49 (m, 2 H), 1.62 (m, 2 H), 2.33 (t,
J = 7.5 Hz, 2 H), 3.18 (m, 2 H), 3.73 (dd, J = 6.2, 8.5 Hz, 1 H),
4.07 (dd, J = 6.5, 8.5 Hz, 1 H), 4.09 (dd, J = 5.9, 11.5 Hz, 1 H),
4.15 (dd, J = 4.7, 11.5 Hz, 1 H), 4.31 (m, 1 H), 4.52 (br, 0.15 H),
4.74 (br, 0.85 H), 5.09 (s, 2 H), 7.29-7.36 (m, 5 H); 13C NMR
(126 MHz, CDCl3) 24.7, 25.4, 26.5, 26.7, 28.8, 28.9, 29.9, 34.0,
41.0, 64.5, 66.3, 66.6, 73.7, 109.8, 128.0, 128.1, 128.5, 136.7,
156.4, 173.5; HRMS(ESI) calcd for C22H33NNaO6 [M þ Na]þ
430.2206, found 430.2204.
51% overall yield: [R]20 -3.2 (c 4.0 g 100 mL-1, CDCl3); H
1
D
NMR (500 MHz, CDCl3) δ 0.88 (t, J = 7.0 Hz, 3 H), 1.26-
1.32 (m, 14 H), 1.49 (m, 2 H), 1.58-1.65 (m, 4 H), 2.27 (t,
J = 6.2 Hz, 1 H), 2.31 (t, J = 7.5 Hz, 2 H), 2.34 (t, J = 7.5 Hz,
2 H), 3.18 (m, 2 H), 3.71-3.73 (m, 2 H), 4.22 (dd, J = 5.7, 11.9 Hz,
1 H), 4.32 (dd, J = 4.4, 11.9 Hz, 1 H), 4.64 (br, 0.15 H), 4.79 (br,
0.85 H), 5.06-5.10 (m, 3 H), 7.29-7.35 (m, 5 H); 13C NMR
(75 MHz, CDCl3) δ 14.0, 22.5, 24.7, 24.9, 26.4, 28.8, 28.9, 29.0,
29.8, 31.6, 34.0, 34.3, 41.0, 61.5, 62.1, 66.6, 72.1, 128.0, 128.5,
136.6, 156.4, 173.4, 173.6; HRMS(ESI) calcd for C27H43NNaO7
[M þ Na]þ 516.2937, found 516.2933.
1-O-[8-(Benzyloxycarbonylamino)octanoyl]-3-O-triethylsilyl-
sn-glycerol (25). To a solution of acetonide 23 (700 mg, 1.72
mmol) in DCE (7 mL) was added N,N-diisopropylethylamine
(1.2 mL, 6.9 mmol), followed by TESOTf (1.2 mL, 5.3 mmol),
and the mixture was stirred at 90 °C for 6 h. After being cooled
to rt, the mixture was diluted with petroleum ether, washed with
0.1 M aq HCl, water, and brine, and dried over MgSO4. The
resulting oil was dissolved in CH2Cl2 (20 mL) and stirred
vigorously with 1:1 HOAc/water (20 mL) at rt, following the
progress of the reaction by TLC (25% EtOAc/petroleum ether,
product Rf 0.15). At approximately 75% conversion, the reac-
tion mixture was extracted with petroleum ether (ꢀ3), and the
extracts were dried with brine and MgSO4. Flash chromatog-
raphy on silica gel, using 25-30% EtOAc/petroleum ether as
1-O-[8-(Benzyloxycarbonylamino)octanoyl]-2-O-octanoyl-sn-
glycero-3-(benzyl diisopropylphosphoramidite) (28). To an ice-
cooled solution of diacylglycerol 27 (237 mg, 0.48 mmol, dried
by azeotropic distillation of toluene on a rotary evaporator) in
CH2Cl2 (2.5 mL) were added benzyl N,N,N0,N0-tetraisopropyl-
phosphorodiamidite (214 mg, 0.63 mmol) and 1H-tetrazole (33
mg, 0.47 mmol). After 10 min, the ice bath was removed, and
stirring was continued at room temperature for 100 min. The
mixture was concentrated under reduced pressure and immedi-
ately purified by rapid flash chromatography on Et3N-neutra-
lized silica gel, using 10:88:2 to 15:83:2 EtOAc/petroleum ether/
Et3N as eluant, to afford the phosphoramidite 28 (1:1 mixture of
1
diastereomers) as a gum (307 mg, 87%): H NMR (500 MHz,
CDCl3) δ 0.87 (t, J = 6.9 Hz, 3 H), 1.17-1.19 (m, 12 H),
1.26-1.30 (m, 14 H), 1.48 (m, 2 H), 1.56-1.63 (m, 4 H), 2.26-
2.30 (m, 4 H), 3.17 (m, 2 H), 3.59-3.67 (m, 2 H), 3.68-3.81 (m,
2 H), 4.14-4.19 (m, 1 H), 4.31-4.37 (m, 1 H), 4.50 (br, 0.15 H),
4.63-4.75 (m, 2.85 H), 5.09 (s, 2 H), 5.18 (m, 1 H), 7.23-7.27 (m,
1 H), 7.28-7.35 (m, 9 H); 13C NMR (126 MHz, CDCl3) δ 14.0,
22.6, 24.5-24.57 (2 ꢀ d), 24.60, 24.66, 24.73, 24.9, 26.5, 28.86,
28.90, 28.95, 29.03, 29.9, 31.6, 34.0, 34.3, 41.0, 43.0-43.2 (2 ꢀ
d), 61.5-61.8 (2 ꢀ d), 62.5, 62.6, 65.3-65.5 (2 ꢀ d), 66.5, 70.8-
70.9 (2 ꢀ d), 126.9, 127.3, 128.0, 128.1, 128.2, 128.5, 136.7,
139.3-139.4 (2 ꢀ d), 156.4, 173.0, 173.2; 31P NMR (202 MHz,
CDCl3) δ 148.9, 149.1; HRMS(ESI) calcd for C40H63N2NaO8P
[M þ Na]þ 753.4220, found 753.4224.
eluant, gave the secondary alcohol 25 as a gum (296 mg, 36%):
1
[R]20 þ1.9 (c 5.0 g 100 mL-1, CDCl3); H NMR (500 MHz,
D
CDCl3) δ 0.62 (q, J = 8.0 Hz, 6 H), 0.96 (t, J = 8.0 Hz, 9 H),
1.31 (m, 6 H), 1.49 (m, 2 H), 1.62 (m, 2 H), 2.33 (t, J = 7.5 Hz, 2
H), 2.60 (d, J = 5.2 Hz, 1 H), 3.17 (m, 2 H), 3.60 (dd, J = 5.7,
10.1 Hz, 1 H), 3.67 (dd, J = 4.7, 10.1 Hz, 1 H), 3.87 (m, 1 H),
4.11 (dd, J = 6.1, 11.5 Hz, 1 H), 4.15 (dd, J = 4.7, 11.5 Hz, 1 H),
4.58 (br, 0.15 H), 4.77 (br, 0.85 H), 5.09 (s, 2 H), 7.28-7.35 (m, 5
H); 13C NMR (126 MHz, CDCl3) δ 4.3, 6.6, 24.8, 26.4, 28.8,
28.9, 29.9, 34.1, 41.0, 63.4, 65.1, 66.5, 70.0, 128.01, 128.05,
128.5, 136.7, 156.4, 173.8; HRMS(ESI) calcd for C25H43-
NNaO6Si [M þ Na]þ 504.2757, found 504.2756.
1-O-[8-(Benzyloxycarbonylamino)octanoyl]-2-O-octanoyl-3-
O-triethylsilyl-sn-glycerol (26). To a solution of alcohol 25 (296
mg, 0.61 mmol) in CH2Cl2 (6 mL) were added DCC (154 mg,
0.75 mmol), octanoic acid (99 mg, 0.69 mmol), and DMAP (20
mg, 0.16 mmol). After being stirred for 5.5 h at rt, the reaction
mixture was concentrated under reduced pressure and purified
by flash chromatography on silica gel, using 8-10% acetone/
petroleum ether as eluant, to give the diacylated product 26 as a
1-D-1-O-[1-O-[8-(Benzyloxycarbonylamino)octanoyl]-2-O-
octanoyl-sn-glycero-3-O-benzylphospho]-2,4,6-O-tris(methoxymethy-
lene)-myo-inositol 3,5-Bis(dibenzyl phosphate) (29). To an ice-
cooled suspension of inositol 9 (99 mg, 0.12 mmol) and 1H-
tetrazole (15 mg, 0.21 mmol) in CH2Cl2 (0.5 mL) was added neat
phosphoramidite 28 (96 mg, 0.13 mmol). After being stirred
at rt for 2 h, the reaction mixture was cooled to -75 °C and the
phosphite intermediate was oxidized with m-CPBA (0.18 mmol,
prepared by dissolving 62 mg of 50% m-CPBA in CH2Cl2, drying
over MgSO4, filtering and concentrating to ∼2 mL). After being
warmed to rt, the solution was diluted with EtOAc, washed with
10% aq Na2S2O3 (ꢀ3), 10% aq NaHCO3 (ꢀ3), and brine, and
dried over MgSO4. Flash chromatography on silica gel, using
70-80% EtOAc/petroleum ether as eluant, afforded fully pro-
tected PI(3,5)P2-NH(Cbz) 29 (mixture of diastereomers) as a
colorless gum (134 mg, 76%): 1H NMR (500 MHz, CDCl3) δ 0.86
(t, J = 7.0 Hz, 3 H), 1.23-1.30 (m, 14 H), 1.47 (m, 2 H), 1.52-1.60
(m, 4 H), 2.22-2.29 (m, 4 H), 3.16 (m, 2 H), 3.306 (s, 1.5 H), 3.313
(s, 1.5 H), 3.32 (s, 1.5 H), 3.34 (s, 1.5 H), 3.35 (s, 1.5 H), 3.37 (s, 1.5
H), 4.03-4.29 (m, 9 H), 4.50 (s, 1 H), 4.60 (br, 0.2 H), 4.67-4.77
(m, 6 H), 4.82 (br, 0.8 H), 5.00-5.06 (m, 8 H), 5.07-5.13 (m, 4 H),
5.17 (m, 1 H), 7.26-7.38 (m, 30 H); 13C NMR (126 MHz, CDCl3)
δ 14.0, 22.5, 24.6, 24.7, 26.5, 28.8, 28.85, 28.88, 29.0, 29.8, 31.6,
33.8, 34.01, 34.03, 41.0, 55.9, 56.5, 56.6, 61.6, 65.50, 65.54, 65.71,
65.75, 66.5, 69.29, 69.32, 69.36, 69.40, 69.45, 69.47, 69.49, 69.51,
69.54, 69.57, 69.59, 69.61, 69.79, 69.84, 75.4-75.5 (m), 75.59,
75.62, 75.77, 75.81, 75.89, 75.94, 75.96, 76.02, 79.65, 79.70, 79.76,
colorless oil (298 mg, 80%): [R]20 þ10.5 (c 3.5 g 100 mL-1
,
D
CDCl3); 1H NMR (500 MHz, CDCl3) δ 0.59 (q, J = 8.0 Hz, 6
H), 0.88 (t, J = 7.0 Hz, 3 H), 0.95 (t, J = 8.0 Hz, 9 H),
1.26-1.32 (m, 14 H), 1.49 (m, 2 H), 1.57-1.64 (m, 4 H),
2.28-2.32 (m, 4 H), 3.18 (m, 2 H), 3.71 (dd, J = 5.6, 10.8
Hz, 1 H), 3.73 (dd, J = 5.1, 10.8 Hz, 1 H), 4.16 (dd, J = 6.4, 11.8
Hz, 1 H), 4.35 (dd, J = 3.7, 11.8 Hz, 1 H), 4.51 (br, 0.15 H), 4.74
(br, 0.85 H), 5.05-5.09 (m, 3 H), 7.29-7.36 (m, 5 H); 13C NMR
(126 MHz, CDCl3) δ 4.3, 6.6, 14.0, 22.6, 24.7, 24.9, 26.5, 28.87,
28.90, 28.96, 29.02, 29.9, 31.6, 34.0, 34.3, 41.0, 61.3, 62.5, 66.6,
71.7, 128.0, 128.1, 128.5, 136.7, 156.4, 173.1, 173.3; HRMS-
(ESI) calcd for C33H57NNaO7Si [M þ Na]þ 630.3802, found
630.3805.
1-O-[8-(Benzyloxycarbonylamino)octanoyl]-2-O-octanoyl-sn-
glycerol (27). To silyl ether 26 (293 mg, 0.48 mmol) was added a
solution of FeCl3 in 3:1 reagent-grade MeOH/CH2Cl2 (4.9 mL,
5 mM). After 90 min at 17 °C, the solution was partitioned
between Et2O and water, and the organic phase was washed with
brine and dried over MgSO4. Flash chromatography on silica
gel (deactivated with 5% v/w water), using 30-40% EtOAc/
3550 J. Org. Chem. Vol. 75, No. 11, 2010

Anderson et al.
JOCFeatured Article
98.0, 98.1, 98.4, 98.5, 127.89, 127.91, 127.92, 127.96, 127.98, 128.03,
128.4, 128.50, 128.54, 128.6, 135.5, 135.55, 135.61, 135.65, 135.66,
135.70, 135.72, 135.8, 135.9, 136.7, 156.3, 172.69, 172.71, 172.96,
172.98; 31P NMR (202 MHz, CDCl3) δ -1.45, -1.41, -1.40,
-1.39, -1.19, -1.18; HRMS(ESI) calcd for C74H98NNaO24P3
[M þ Na]þ 1500.5589, found 1500.5591.
1 H), 4.69 (dd, J = 5.0, 7.9 Hz, 1 H), 5.39 (m, 1 H); 13C NMR
(75 MHz, D2O) δ 14.1, 22.7, 24.9, 25.1, 25.9, 26.6, 28.4, 28.5, 28.7,
28.8, 28.85, 28.89, 31.6, 34.5, 34.7, 36.2, 39.9, 40.3, 56.1, 60.9, 62.7,
63.6, 64.4 (d, J = 5.5 Hz), 70.8, 71.5, 71.6, 71.8 (m), 75.2 (d, J =
5.7 Hz), 76.2 (d, J = 5.9 Hz), 79.0 (m), 176.8, 177.09, 177.13; 31
P
NMR (202 MHz, D2O) δ -0.6, 1.0, 2.6; HRMS(ESI) calcd for
C35H63N3O21P3S [M - H]- 986.2888, found 986.2874. HPLC
analysis was carried out on a Kinetex 2.6 μm C18 column at 40 °C
with a flow rate of 0.5 mL/min, eluting with a linear gradient of
10:80:10 to 60:30:10 MeCN/water/100 mM aq NH4OAc over
20 min. Detection was by electrospray MS in the negative ion
mode. Compound 30 had a retention time of 8.0 min and a purity
of 95%. See the Supporting Information for trace.
Biotinylated L-r-Phosphatidyl-D-myo-inositol 3,5-Bisphosphate
(30). Under anhydrous conditions, TMSBr (∼1 mL) was distilled
from CaH2 directly into an ice-cooled flask containing fully
protected PI(3,5)P2-NH(Cbz) 29 (34.7 mg, 0.023 mmol) and
2-methyl-2-butene(0.3 mL), andthe resulting solutionwas stirred
at rt for 35 min. Without exposure to air, the solution was
concentrated under reduced pressure and dried under high
vacuum for 3.5 h. After the vacuum was replaced with Ar, the
gummy product was dissolved in freshly distilled ethanethiol (1.5
mL) and stirred at 25 °C for 6 h before the solution was
concentrated to a white solid. 1H NMR spectroscopy at this
stage indicated the complete removal of all MOM groups
(absence of CH3O signal at 3.49 ppm, solvent: D2O). After
dissolution in aq NH3 (∼8 mM, 20 mL) and subsequent
lyophilization, the solid was stirred in 7:5 THF/0.5 M aq
NH3 (3 mL) with 20% Pd(OH)2/C (20 mg) under H2 (balloon
pressure) at rt for 1 h. The mixture was filtered through a glass
fiber filter paper, washing the catalyst thoroughly with MeOH,
and the filtrate concentrated under redþuced pressure to afford
the primary amine product. The NH4 counterions were ex-
changed for Et3NHþ counterions by dissolution in 1 M triethyl-
ammonium bicarbonate (TEAB) buffer (pH 8.5) and subse-
quent lyophilization.
Pull-down Experiments. Chromaffin cytosol5b was diluted in
pull-down buffer (20 mM Hepes-NaOH, pH 7.3, 150 mM
NaCl, 0.5% Nonidet P40) containing Complete protease inhi-
bitor mixture to a final concentration of 2 mg/mL and incubated
for 30 min on ice before preclearing by centrifugation for 15 min
at 13000 rpm at 4 °C. The cytosolic extract was incubated for 3 h
at 4 °C with either 1 nmol of biotinylated PI(3,5)P2 (30) pre-
bound to UltraLink Plus NeutrAvidin beads or beads alone.
Beads were washed extensively in pull-down buffer and then
resuspended in Laemmli sample buffer containing β-mercapto-
ethanol. For pH binding studies, chromaffin cytosol was pre-
pared with the pH indicated (Figure 1B), and beads were washed
in the respective pH pull-down buffers. Proteins associated with
the pull-downs were analyzed by Western blotting with anti-
bodies raised against svp1p (gift from Peter Parker (Cancer
Research UK)).
To a solution of the resulting solid in 1 M TEAB (pH ∼7.7, 1.0
mL) was added a solution of biotin-NHS ester (11.3 mg, 0.033
mmol) in DMF (0.9 mL). After 1 h at rt, the solution (whose
pH was now 9.3) was diluted with water and lyophilized.
The product was purified by flash chromatography on silica gel,
using 9:7:2 CHCl3/MeOH/water as eluant, followed by reversed-
phase flash chromatography on C18 silica gel, using 20-35%
MeOH/water as eluant, to furnish the title compound 30. Con-
version tothe sodiumsaltbyion exchange (Naform), followed by
lyophilization, gave a fluffy white solid (14.6 mg, 59%): 1H NMR
(500 MHz, D2O) δ 0.94 (t, J = 6.9 Hz, 3 H), 1.34-1.41 (m, 14 H),
1.48 (m, 2 H), 1.58 (m, 2 H), 1.65-1.84 (m, 8 H), 2.32 (t, J = 7.1
Hz, 2 H), 2.47 (m, 2 H), 2.51 (t, J = 7.3 Hz, 2 H), 2.87 (d, J = 13.1
Hz, 1 H), 3.07 (dd, J = 5.0, 13.1 Hz, 1 H), 3.25 (m, 2 H), 3.39(m, 1
H), 3.94-4.00 (m, 3 H), 4.09-4.20 (m, 4 H), 4.37 (dd, J = 7.2,
12.3 Hz, 1 H), 4.48-4.51 (m, 2 H), 4.54 (dd, J = 2.6, 12.3 Hz,
Acknowledgment. We thank the New Zealand Founda-
tion for Research and Technology for financial support
(C08X0701 and NZFRST fellowship, IRLX0502, to R.J.A.).
This work was also supported by the NHMRC grants(631568
to F.A.M. and 631418 to S.L.O.) and NHMRC SRF fellow-
ship to F.A.M. We express our gratitude to P. Parker (Cancer
Research UK) for providing the anti-svp1p antibody and
helpful discussions.
Supporting Information Available: General experimental
details, 1H, 13C, and 31P NMR spectra for all new compounds
reported in the manuscript, COSY spectra for compounds 4-7,
and HPLC trace for compound 30. This material is available
free of charge via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 75, No. 11, 2010 3551