Synthesis and biological action of novel 4-position-modified derivatives of D-myo-inositol 1,4,5-trisphosphate

Article abstract of DOI:10.1021/jo070611a

(Chemical Equation Presented) The design of a range of 4-position-modified D-myo-inositol 1,4,5-trisphosphate derivatives is described. The enantioselective synthesis of these compounds is reported, along with initial biological analysis, which indicates

Full text of DOI:10.1021/jo070611a

Synthesis and Biological Action of Novel 4-Position-Modified
Derivatives of D-myo-Inositol 1,4,5-Trisphosphate
Davide Bello,† Tashfeen Aslam,† Geert Bultynck,‡ Alexandra M. Z. Slawin,†
H. Llewelyn Roderick,§ Martin D. Bootman,§ and Stuart J. Conway*,†
EaStCHEM and School of Chemistry, Centre for Biomolecular Sciences, UniVersity of St Andrews,
North Haugh, St Andrews, Fife KY16 9ST, United Kingdom, Laboratory of Molecular Signalling,
Campus Gasthuisberg O/N1, Katholieke UniVersiteit LeuVen, Herestraat 49, Bus 802, B-3000 LeuVen,
Belgium, and Laboratory of Molecular Signalling, The Babraham Institute, Babraham,
Cambridge CB22 3AT, United Kingdom
sjc16@st-andrews.ac.uk
ReceiVed March 29, 2007
The design of a range of 4-position-modified D-myo-inositol 1,4,5-trisphosphate derivatives is described.
The enantioselective synthesis of these compounds is reported, along with initial biological analysis,
which indicates that these compounds do not act as D-myo-inositol 1,4,5-trisphosphate receptor agonists
or antagonists.
Introduction
behavior, memory, and learning.1 It is now clear that the InsP3
signaling cascade resides downstream of the receptors for most
important endogenous chemical transmitters, including acetyl-
choline, adrenaline, dopamine, glutamate, and serotonin, in
addition to some tyrosine kinase receptors.1 The biochemical
importance of InsP3 has prompted many syntheses of InsP3 and
numerous unnatural derivatives.2,3 These compounds have
proved vital in establishing the structural characteristics required
for agonist activity at InsP3Rs. Especially useful compounds
include the metabolically stable phosphorothioates2,3 the
adenophostins,4-11 photoactivated InsP3 derivatives,12,13 and
membrane-permeant InsP3 derivatives.12,14,15
It is well-established that D-myo-inositol 1,4,5-trisphosphate
(InsP3, 1; Figure 1) is a ubiquitous intracellular Ca2+-releasing
second messenger that mediates a wide range of cellular
functions.1 InsP3 is generated following G-protein activation,
as a result of extracellular receptor stimulation, which initiates
the intracellular PLC-mediated hydrolysis of phosphatidylinosi-
tol 4,5-bisphosphate [2, PtdIns(4,5)P2] to afford InsP3 and
diacylglycerol (DAG). Both of these compounds perform
important signaling roles within cells; DAG mediates its cellular
function through the activation of some isoforms of PKC. InsP3
is hydrophilic and diffuses through the cytosol to activate its
specific receptors (InsP3Rs), which are located predominantly
on the endoplasmic or sarcoplasmic reticulum membrane.
Activation of these InsP3-gated ion channels leads to the release
of intracellular Ca2+ into the cytosol and a plethora of
subsequent signaling events, including cell proliferation, muscle
contraction, apoptosis, and gene transcription. Ca2+ signaling
is also involved in fundamental processes such as fertilization,
(2) Conway, S. J.; Miller, G. J. Nat. Prod. Rep., in press.
(3) Potter, B. V. L.; Lampe, D. Angew. Chem., Int. Ed. Engl. 1995, 34,
1933.
(4) Morris, S. A.; Nerou, E. P.; Riley, A. M.; Potter, B. V. L.; Taylor,
C. W. Biochem. J. 2002, 367, 113.
(5) Correa, V.; Riley, A. M.; Shuto, S.; Horne, G.; Nerou, E. P.;
Marwood, R. D.; Potter, B. V. L.; Taylor, C. W. Mol. Pharmacol. 2001,
59, 1206.
(6) Marwood, R. D.; Correa, V.; Taylor, C. W.; Potter, B. V. L.,
Tetrahedron: Asymmetry 2000, 11, 397.
(7) Marwood, R. D.; Shuto, S.; Jenkins, D. J.; Potter, B. V. L. Chem.
Commun. 2000, 219.
(8) vanStraten, N. C. R.; vanderMarel, G. A.; vanBoom, J. H. Tetrahe-
dron 1997, 53, 6509.
* To whom correspondence should be addressed. Phone: +44 (0) 1334
463478. Fax: +44 (0) 1334 463808.
† University of St Andrews.
‡ Katholieke Universiteit Leuven.
§ The Babraham Institute.
(1) Berridge, M. J.; Bootman, M. D.; Roderick, H. L. Nat. ReV. Mol.
Cell Biol. 2003, 4, 517.
(9) vanStraten, N. C. R.; vanderMarel, G. A.; vanBoom, J. H. Tetrahe-
dron Lett. 1996, 37, 3599.
10.1021/jo070611a CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/22/2007
J. Org. Chem. 2007, 72, 5647-5659
5647

Bello et al.
FIGURE 1. Structures of InsP3 (1) and PtdIns(4,5)P2 (2).
In contrast, there are very few compounds that act as selective
antagonists of the InsP3Rs.3,16 2-Aminoethoxy diphenyl borate
(2-APB) is the most commonly used InsP3R antagonist, as it is
readily available and also membrane permeant.17 However,
2-APB is not selective for InsP3Rs and displays a number of
other actions including inhibition of SERCA pumps,18 inhibition
of store-operated Ca2+ entry,19,20 activation of TRPV1-3,21,22
inhibition of TRPC5,23 interactions with TRPM6 and TRPM7,
and possibly inhibition of Ca2+ ATPases.20 Xestospongin C, a
marine natural product that was isolated in 1984,24 has also been
reported to inhibit Ca2+ release from InsP3Rs.25,26 This com-
pound is potentially useful as, like 2-APB, it is membrane
permeant; however, xestospongin C does not selectively interact
with InsP3Rs and is known to inhibit SERCA pumps.25,27 More
recently, the structurally related xestospongin B has been
reported as a competitive inhibitor of InsP3-mediated Ca2+
signaling.28 Heparin and decavanadate also act as InsP3R
antagonists, but are not membrane permeant and thus not
suitable for use in many situations.3,29 Benzene-1,2,4-triol has
been reported to competitively block InsP3 binding to adrenal
cortex microsomes (IC50 ) 34 mM). The affinity of this
compound for the InsP3Rs is about 10000-fold less than that of
InsP3 and did not evoke Ca2+ release itself.3 Erneux and co-
workers have recently reported that another aromatic compound,
biphenyl 2,3′,4,5′,6-pentakisphosphate, inhibits InsP3-induced
Ca2+ release by blocking InsP3 binding to the InsP3Rs. However,
this compound is also known to act as an InsP3 5-phosphatase
inhibitor.30 The only InsP3R antagonist based on the inositol
architecture, to date, is the methylphosphonate reported by Dreef
and co-workers.31 Extensive pharmacological studies of this
molecule have not been published, as the production of the
required quantity of material presents a significant synthetic
challenge. In addition, the phosphate groups of this compound
would have to be modified for it to cross the cell membrane. It
is therefore clear that the development of selective, membrane-
permeant InsP3R antagonists is vital to enable further studies
of Ca2+ and InsP3 signaling.
The publication of the X-ray crystal structure of the InsP3-
binding domain of the mouse type 1 InsP3R provides a wealth
of information on the binding interaction of InsP3 with its
receptor.32 Only the ligand-bound structure was reported, which
is assumed to be in the “active” conformation. The apo form
of the binding domain would have provided further information
about the structure requirements for antagonist activity at InsP3-
Rs, as it can simplistically be assumed that an antagonist will
bind to the “inactive” form of the receptor. Nonetheless, we
have used this information to assist in the design of a range of
potential InsP3R antagonists.
From the X-ray crystal structure it can be seen that InsP3
binds in a cleft between the R-domain and the â-domain of the
InsP3-binding domain (Figure 2). Closer inspection reveals that
the 1-position phosphate group (P1) of InsP3 binds to residues
R568 and K569 and the 5-position phosphate (P5) binds to
residues R504, K508, R511, and Y567 on the R-domain.
Conversely, the 4-position phosphate (P4) binds predominantly
to residues on the â-domain, T266, T267, G268, and K569. In
addition, both P4 and P5 bind to R265 and R269 on the
â-domain (Figure 3).
Studies conducted by Mignery and Sudhof demonstrated that
InsP3 binding to the InsP3Rs causes a large conformation change
of the protein.33 Since the publication of the X-ray crystal
structure of the mouse type 1 InsP3R, it has been widely
postulated that this conformational change occurs as a result of
(10) Takahashi, M.; Tanzawa, K.; Takahashi, S. J. Biol. Chem. 1994,
269, 369.
(11) Takahashi, M.; Kagasaki, T.; Hosoya, T.; Takahashi, S. J. Antibiot.
1993, 46, 1643.
(12) Li, W. H.; Llopis, J.; Whitney, M.; Zlokarnik, G.; Tsien, R. Y.
Nature 1998, 392, 936.
(13) Walker, J. W.; Feeney, J.; Trentham, D. R. Biochemistry 1989, 28,
3272.
(14) Conway, S. J.; Thuring, J. W.; Andreu, S.; Kvinlaug, B. T.; Roderick,
H. L.; Bootman, M. D.; Holmes, A. B. Aust. J. Chem. 2006, 59, 887.
(15) Li, W. H.; Schultz, C.; Llopis, J.; Tsien, R. Y. Tetrahedron 1997,
53, 12017.
(16) Bultynck, G.; Sienaert, I.; Parys, J. B.; Callewaert, G.; De Smedt,
H.; Boens, N.; Dehaen, W.; Missiaen, L. Pfluegers Arch. 2003, 445, 629.
(17) Maruyama, T.; Kanaji, T.; Nakade, S.; Kanno, T.; Mikoshiba, K.
Jpn. J. Biochem. 1997, 122, 498.
(18) Bilmen, J. G.; Wootton, L. L.; Godfrey, R. E.; Smart, O. S.;
Michelangeli, F. Eur. J. Biochem. 2002, 269, 3678.
(19) Bootman, M. D.; Collins, T. J.; Mackenzie, L.; Roderick, H. L.;
Berridge, M. J.; Peppiatt, C. M. FASEB J. 2002, 16.
(20) Peppiatt, C. M.; Collins, T. J.; Mackenzie, L.; Conway, S. J.;
Holmes, A. B.; Bootman, M. D.; Berridge, M. J.; Seo, J. T.; Roderick, H.
L. Cell Calcium 2003, 34, 97.
(21) Hu, H. Z.; Gu, Q. H.; Wang, C. B.; Colton, C. K.; Tang, J. S.;
Kinoshita-Kawada, M.; Lee, L. Y.; Wood, J. D.; Zhu, M. X. J. Biol. Chem.
2004, 279, 35741.
(22) Chung, M. K.; Lee, H.; Mizuno, A.; Suzuki, M.; Caterina, M. J. J.
Neurosci. 2004, 24, 5177.
(23) Xu, S. Z.; Zeng, F. N.; Boulay, G.; Grimm, C.; Harteneck, C.; Beech,
D. J. Br. J. Pharmacol. 2005, 145, 405.
(24) Nakagawa, M.; Endo, M.; Tanaka, N.; Lee, G. P. Tetrahedron Lett.
1984, 25, 3227.
(25) De Smet, P.; Parys, J. B.; Callewaert, G.; Weidema, A. F.; Hill, E.;
De Smedt, H.; Erneux, C.; Sorrentino, V.; Missiaen, L. Cell Calcium 1999,
26, 9.
(29) Tones, M. A.; Bootman, M. D.; Higgins, B. F.; Lane, D. A.; Pay,
G. F.; Lindahl, U. FEBS Lett. 1989, 252, 105.
(30) Vandeput, F.; Combettes, L.; Mills, S. J.; Backers, K.; Wohlkonig,
A.; Parys, J. B.; De Smedt, H.; Missiaen, L.; Dupont, D.; Potter, B. V. L.;
Erneux, C. FASEB J. 2007, 21, 1481.
(26) Gafni, J.; Munsch, J. A.; Lam, T. H.; Catlin, M. C.; Costa, L. G.;
Molinski, T. F.; Pessah, I. N. Neuron 1997, 19, 723.
(27) Castonguay, A.; Robitaille, R. Cell Calcium 2002, 32, 39.
(28) Jaimovich, E.; Mattei, C.; Liberona, J. L.; Cardenas, C.; Estrada,
M.; Barbier, J.; Debitus, C.; Laurent, D.; Molgo, J. FEBS Lett. 2005, 579,
2051.
(31) Dreef, C. E.; Schiebler, W.; Vandermarel, G. A.; Vanboom, J. H.
Tetrahedron Lett. 1991, 32, 6021.
(32) Bosanac, I.; Alattia, J. R.; Mal, T. K.; Chan, J.; Talarico, S.; Tong,
F. K.; Tong, K. I.; Yoshikawa, F.; Furuichi, T.; Iwai, M.; Michikawa, T.;
Mikoshiba, K.; Ikura, M. Nature 2002, 420, 696.
(33) Mignery, G. A.; Sudhof, T. C. EMBO J. 1990, 9, 3893.
5648 J. Org. Chem., Vol. 72, No. 15, 2007

Synthesis and Biological Action of InsP3 DeriVatiVes
FIGURE 2. A cartoon representation of the X-ray crystal structure of the mouse type 1 InsP3R reported by Bosanac and co-workers.32 It can be
seen that InsP3 binds between the R-domain (yellow) and â-domain (purple).
FIGURE 3. A cartoon representation of the X-ray crystal structure of the mouse type 1 InsP3R showing that P1 of InsP3 binds to residues R568
and K569 and P5 binds to residues R504, K508, R511, and Y567, which are all on the R-domain. Conversely, P4 binds predominantly to residues
on the â-domain, T266, T267, G268, and K569. In addition, both P4 and P5 bind to R265 and R269.
InsP3 binding between the R-domain and the â-domain, causing
the two domains to close together in a clam-shell-like manner.34
It is thought that this movement allows binding of Ca2+ to the
InsP3Rs and ultimately receptor activation. This method of
receptor activation is not uncommon and is present in the
subtype 1 metabotropic glutamate receptors, for example.35
If the mechanism of receptor activation is dependent on
closing of the clam shell, it can be suggested that compounds
that are able to bind to one domain but not the other will be
able to bind to the InsP3Rs but not activate them and hence
would act as competitive antagonists. As P1 and P5 bind
predominantly to the R-domain and P4 binds predominantly to
the â-domain, we decided to investigate the effect of altering
the P4 substituent to develop compounds that would still be
able to bind to the InsP3Rs through P1 and P5. However, the
lack of a polar 4-position substituent would prevent the
compounds from interacting with the â-domain, rendering them
unable to close the clam shell and hence not activate the
receptor. It was hypothesised that such compounds would act
as competitive antagonists of the InsP3Rs.
Using the above supposition, a range of 4-position-substituted
InsP3 derivatives was designed (3-6, Figure 4). We picked
uncharged substituents with approximately the same spatial
geometry as a phosphate group to eliminate the formation of
any salt bridges with the â-domain of the InsP3Rs. The
dimethylphosphinate 3 and the dibutylphosphinate 4 possess the
same geometry as a phosphate group and would allow us to
probe the steric bulk tolerated by the receptor. Furthermore,
the methanesulfonate 5 derivative would allow us to investigate
the effect of replacing the phosphorus atom with an ap-
proximately tetrahedral sulfur atom. In addition, a derivative
with an ethyl chain separating the 4-position phosphate group
from the inositol ring (6) was also designed. The synthesis and
biological evaluation of this range of compounds are described
below.
(34) Taylor, C. W.; da Fonseca, P. C. A.; Morris, E. P. Trends Biochem.
Sci. 2004, 29, 210.
(35) Kunishima, N.; Shimada, Y.; Tsuji, Y.; Sato, T.; Yamamoto, M.;
Kumasaka, T.; Nakanishi, S.; Jingami, H.; Morikawa, K. Nature 2000, 407,
971.
J. Org. Chem, Vol. 72, No. 15, 2007 5649

Bello et al.
readily methylated to afford the methyl ether 13 (Scheme 2)
before the O-allyl deprotection by Wilkinson’s catalyst-mediated
isomerization, followed by acid-catalyzed methanolysis of the
resultant enol ether.37 Treatment of the diol 14 with bis-
(benzyloxy)(N,N-diisopropylamino)phosphine and 1H-tetrazole,
followed by oxidation, afforded the protected phosphate 15.
Hydrogenolysis of the benzyl ethers was initially attempted
using palladium on carbon (10%) in ethanol. This afforded a
compound that displayed the expected mass ion upon mass
spectrometric analysis. However, the 1H and 31P NMR spectra
showed broad, unresolved peaks, and thus, the migration of the
phosphate groups could not be ruled out. Therefore, the
hydrogenolysis was repeated using palladium black as the
catalyst in the presence of sodium bicarbonate to facilitate the
formation of the sodium salt of the desired compound 16. It
has previously been demonstrated that the sodium salts of
phosphate groups are less susceptible to migration.36 Compound
FIGURE 4. Structures of the 4-position-modified InsP3 derivatives
3-6.
1
16 exhibited well-defined peaks in both its H and 31P NMR
Results and Discussion: Synthesis
spectra, indicating the presence of one compound. Other
analytical data were consistent with the assigned structure.
The synthetic route was designed to afford a key inositol
intermediate (7) that possesses orthogonal protection between
the 1- and 5-positions and the 4-position (Figure 5). It was
envisaged that elaboration of the key intermediate would enable
the introduction of several 4-position substituents at a late stage
of the synthesis. Following the route developed by Holmes, the
optically pure camphor acetal 8 (Scheme 1) [[R] 2D0 ) -11.9 (c
0.2 in CHCl3) [lit.36 [R] D22 ) -11.7 (c 1.3 in CHCl3)]] was
obtained in seven steps from myo-inositol.36-39 Treatment of 8
with sodium hydride followed by allyl bromide yielded the fully
protected inositol 9. Mild acid-catalyzed methanolysis of the
camphor acetal revealed the vicinal diol 10, which was
selectively benzylated at the 3-position by treatment with
dibutyltin oxide and benzyl bromide. The alcohol 11 displays
orthogonality between the 1- and 5-positions and the 4-position
and serves as a key intermediate in the synthesis of 4-position-
modified InsP3 derivatives.
To minimize the use of protecting groups, the installation of
the dimethylphosphinate prior to the phosphate groups was
attempted (Scheme 1). Treatment of the alcohol 11 with
(diisopropylamino)dimethylphosphine, followed by mCPBA
oxidation, resulted in the formation of the desired dimethylphos-
phinate 12. Subsequent attempts to remove the O-allyl protecting
groups were not successful despite numerous conditions being
investigated. Treatment with palladium on carbon (10%) and
toluenesulfonic acid monohydrate in methanol and water under
reflux conditions afforded a 21% yield of a product that appeared
to be a mixture of the desired compound and its regioisomer,
in which the phosphinate had migrated to the 5-position. An
alternative strategy, utilizing protection of the 4-position,
deprotection of the O-allyl groups, and subsequent phosphory-
lation, followed by unmasking of the 4-position, was therefore
investigated.
Having demonstrated the feasibility of the above approach,
it was necessary to introduce a protecting group that could be
cleaved once the dibenzyl phosphate groups had been installed.
There is precedent for a 4-methoxybenzyl (PMB) group being
removed in the presence of dibenzyl phosphate esters; hence,
this group was selected.37 PMB protection of the 4-position
hydroxyl group of alcohol 11 was achieved by reaction with
sodium hydride and 4-methoxybenzyl chloride to afford the
PMB ether 17 (Scheme 3). The conditions reported by Boons
and co-workers effected efficient deprotection of the allyl groups
of 17 to furnish the diol 18.40 The dibenzyl phosphate esters
were installed under standard conditions to yield 19 before the
PMB group was removed by oxidative cleavage, affording the
alcohol 20.
Compound 20 was a crystalline solid, and hence, a single-
crystal X-ray structure was obtained (Figure 6). The absolute
stereochemistry of compound 20 was confirmed unambiguously
using the anomalous dispersion effect [Flack parameter 0.03(4)].
With compound 20 in hand, a range of 4-position-substituted
InsP3 derivatives was synthesized (Scheme 4). The 4-position
substituents were chosen on the basis of their ability to mimic
the approximate shape and geometry of a phosphate group, but
with uncharged moieties. The first group installed was the
dimethylphosphinate. Initial attempts to install the dimeth-
ylphosphinate using phosphorus(III)-based reagents were unsuc-
cessful and seemed to promote migration of the existing
phosphate groups (not shown). Gratifyingly, dimethylphosphinic
chloride could be successfully employed to furnish the desired
compound 21 in good yield. Hydrogenolysis in the presence of
sodium bicarbonate afforded the sodium salt of the desired
compound 3. The dibutyl derivative 4 was synthesized in a
similar manner using dibutylphosphinic chloride to afford the
dibutylphosphinate 22. Hydrogenolysis in the presence of
sodium bicarbonate furnished the sodium salt of the dibu-
tylphosphinate 4. To investigate the effect of replacing phos-
phorus with sulfur, the methanesulfonate 23 was synthesized
by reaction of the alcohol 20 with methanesulfonyl chloride.
Hydrogenolysis under the conditions described above afforded
the sodium salt of 5. For completeness, the sodium salt of
D-myo-inositol 1,5-bisphosphate (24) was synthesized by hy-
To develop suitable conditions for this approach, methylation
of 11 was attempted as a model system. The alcohol 11 was
(36) Painter, G. F.; Grove, S. J. A.; Gilbert, 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.
(37) Lim, Z. Y.; Thuring, J. W.; Holmes, A. B.; Manifava, M.; Ktistakis,
N. T. J. Chem. Soc., Perkin Trans. 1 2002, 1067.
(38) Painter, G. F.; Thuring, J. W.; Lim, Z. Y.; Holmes, A. B.; Hawkins,
P. T.; Stephens, L. R. Chem. Commun. 2001, 645.
(39) Grove, S. J. A.; Gilbert, I. H.; Holmes, A. B.; Painter, G. F.; Hill,
M. L. Chem. Commun. 1997, 1633.
(40) Boons, G. J.; Burton, A.; Isles, S. Chem. Commun. 1996, 141.
5650 J. Org. Chem., Vol. 72, No. 15, 2007

Synthesis and Biological Action of InsP3 DeriVatiVes
FIGURE 5. Synthetic plan for the 4-position-modified InsP3 derivatives.
SCHEME 1. Synthesis of the Key Intermediate 11a
a TBAI ) tetrabutylammonium iodide.
SCHEME 2. Synthesis of the 4-Methyl-InsP3 Derivative 16
drogenolysis of 20 under standard conditions. All compounds
Results and Discussion: Biological Evaluation
were obtained as colorless lypholized powders.
Having established a robust synthetic route to the InsP3
derivatives 3-6, 16, and 24, we assessed the ability of these
compounds to act as competitive antagonists of InsP3R1. The
effect of the InsP3 derivatives on the InsP3-induced Ca2+ release
was investigated in permeabilized L15 by using a unidirectional
45Ca2+ flux assay. In this experiment, the nonmitochondrial
stores of permeabilized L15 cells are loaded to steady state with
45Ca2+, washed twice with nonlabeled efflux medium containing
thapsigargin (4 µM), and further incubated in a nonlabeled efflux
medium. Samples were collected every 2 min, and the loss of
Ca2+ from the internal stores under these conditions was plotted
as fractional Ca2+ loss (%/2 min) as a function of time.
Challenging the cells with InsP3R agonists leads to an increase
The synthesis of a compound that was structurally distinct
from the above series was also undertaken (Scheme 5). The
concept of inserting a linker between the inositol ring and the
phosphate group at the 4-position led to the design of compound
6. The synthesis of this compound commenced from the bisallyl
ether 11. Reaction of the 4-position hydroxyl group with
2-(allyloxy)ethyl bromide afforded the fully protected compound
25. Removal of the allyl ethers afforded the triol 26, with
phosphitylation and mCPBA oxidation affording the fully
perbenzylated phosphate ester 27. Hydrogenolysis of 27, using
the conditions described above, furnished the desired compound
6 as its sodium salt.
J. Org. Chem, Vol. 72, No. 15, 2007 5651

Bello et al.
SCHEME 3. Synthesis of the Key Intermediate 20a
a CAN ) ammonium cerium nitrate.
Conclusion
We have described a robust synthesis of a range of novel
4-position-modified InsP3 derivatives. To the best of our
knowledge, these syntheses represent the first example of
dialkylphosphinate incorporation within an inositol phosphate-
based structure. Compound 6 is the first in a novel class of
compounds in which a linker is included between the inositol
ring and a phosphate (or other moiety).
Despite the compounds synthesized proving to be inactive
at InsP3Rs, a number of important conclusions concerning the
structure-activity relationship can be drawn from this work.
As expected, disruption of the 4,5-bisphosphate motif within
the InsP3 structure led to the agonist activity of these compounds
being abolished. However, it also seems removal of the
4-position phosphate group does not convey the ability of a
compound to bind to one domain of the InsP3Rs without causing
receptor activation. The most likely explanation for the lack of
InsP3 affinity displayed by these compounds is that the groups
installed at the 4-position were too lipophilic to enter the highly
charged InsP3-binding pocket of the InsP3Rs. We hypothesized
that the “clam shell” of the InsP3Rs would be mainly open in
the unactivated receptor, allowing compounds with 1- and
5-position phosphate groups to bind to the R-domain without
interacting with the â-domain. It seems that this hypothesis is
not correct, and this may suggest that the two domains of the
InsP3-binding domain do not fully open, even in the inactive
conformation of the receptor. In addition, our data may indicate
that the 4-position phosphate group is vital for receptor binding
and recognition, rather than activation; thus, any alteration of
this group removes the ability of the compound to bind to InsP3-
Rs. Future studies will seek to test these hypotheses.
FIGURE 6. A stick representation of the X-ray crystal structure of
compound 20. The absolute stereochemistry was confirmed unambigu-
ously using the anomalous dispersion effect [Flack parameter 0.03(4)].
Key: carbon, green; hydrogen, white; oxygen, red; phosphorus, orange.
in fractional loss (as observed with 0.2 µM InsP3). Furthermore,
we have used L15 cells for this analysis since these L fibroblasts
are a stable cell line overexpressing InsP3R1. They are therefore
a model system for the functional analysis of InsP3R1. Western
blot analysis of L15 cell lysates indicates a 3:1 ratio for InsP3-
R1/InsP3R3 (data not shown).
The compounds were first tested for their ability to evoke
Ca2+ release (i.e., behave as InsP3R agonists). None of the
compounds induced an increase in fractional loss when applied
for 2 min at a concentration of 20 µM (Figure 7), indicating
that these compounds do not act as InsP3R agonists.
The compounds were then tested for their ability to block
InsP3-evoked Ca2+ release (Figure 8). Experiments were
conducted in which the compounds were applied in 100-fold
excess (20 µM) at the same time as InsP3 (data not shown) and
also where the compounds were preincubated with the L15 cells
for 2 min prior to application of InsP3 (0.2 µM). In each case
it can be seen that the rise in Ca2+ concentration due to the
action of InsP3 was not abrogated by the presence of compounds
3-5, 16, and 24 (Figure 8). Compound 6 was evaluated in a
separate batch of experiments and also failed to prevent InsP3-
evoked Ca2+ release (data not shown). It can therefore be
concluded that these compounds are not acting as InsP3R
antagonists.
Experimental Section
For general experimental methods, please see the Supporting
Information.
(-)-1D-1,5-Bis-O-allyl-2,6-bis-O-benzyl-3-O-endo-4-O-exo-(L-
1′,7′,7′-trimethylbicyclo[2.2.1]hept-2′-ylidene)-myo-inositol (9).
(-)-1D-5-O-Allyl-2,6-bis-O-benzyl-3-O-endo-4-O-exo-(L-1′,7′,7′-
trimethylbicyclo[2.2.1]hept-2′-ylidene)-myo-inositol (8) (2.4 g, 4.5
mmol) was dissolved in dry tetrahydrofuran (20 mL) under an
atmosphere of nitrogen, the resulting mixture was cooled to 0 °C,
and sodium hydride (219 mg, 60% dispersion in mineral oil, 5.4
mmol) was added. The resulting mixture was allowed to warm to
rt and stirred for 1 h. The mixture was recooled to 0 °C, and
5652 J. Org. Chem., Vol. 72, No. 15, 2007

Synthesis and Biological Action of InsP3 DeriVatiVes
SCHEME 4. Synthesis of the 4-Position-Modified InsP3 Derivatives
SCHEME 5. Synthesis of the Trisphosphate 6
imidazole (catalytic amount) and tetra-n-butylammonium iodide
(catalytic amount) were added, followed by allyl bromide (653 mg,
472 µL, 5.4 mmol), which was added dropwise. The reaction
mixture was warmed to rt, then dry N,N-dimethylformamide (30
mL) was added, and the resulting mixture was stirred overnight.
The sodium hydride was quenched with water (2 mL), the solvent
removed under reduced pressure, and the residue reconstituted in
ethyl acetate (15 mL) and water (15 mL). The layers were separated,
and the aqueous layer was extracted with ethyl acetate (3 × 15
mL). The combined organic layers were washed with brine (10
mL), dried (magnesium sulfate), filtered, and concentrated under
reduced pressure to afford a yellow oil. Purification by silica gel
column chromatography, eluting with ethyl acetate and petroleum
ether (5:95), yielded 9 (2.4 g, 91%) as a colorless solid: Rf 0.45
(ethyl acetate/petroleum ether, 20:80); [R] 2D6 ) -23.0 (c 0.49 in
CHCl3); mp 55-57 °C (from ethyl acetate/petroleum ether); νmax
(KBr disk)/cm-1 3064.4 (w), 3025.2 (w), 2932.8 (s), 2868.3 (s),
1647.6 (w), 1453.9 (m), 1366.6 (m), 1309.5 (m), 1203.4 (m), 1092.7
(s), 1048.9 (s), 921.2 (s), 778.2 (w), 747.9 (s), 697.6 (s), 595.8
(w); H NMR (300 MHz, CDCl3) δ 7.40-7.25 (m, 10H), 6.06-
1
5.85 (m, 2H), 5.35 (ddt, J ) 17.1, 1.8, 1.5 Hz, 1H), 5.30 (ddt, J )
17.4, 1.8, 1.3 Hz, 1H), 5.19 (ddt, J ) 10.2, 1.8, 1.3 Hz, 2H), 4.93
(d, J ) 12.3 Hz, 1H), 4.88 (d, J ) 10.5 Hz, 1H), 4.86 (d, J ) 12.3
Hz, 1H), 4.84 (d, J ) 10.5 Hz, 1H), 4.40 (ddt, J ) 13.1, 5.4, 1.5
Hz, 1H), 4.27-4.20, (m, 2H), 4.09-4.02 (m, 3H), 3.85 (t, J ) 9.2
Hz, 1H), 3.51 (dd, J ) 9.5, 8.7 Hz, 1H), 3.42 (dd, J ) 9.7, 3.0 Hz,
1H), 3.22 (dd, J ) 9.7, 1.5 Hz, 1H), 2.16 (dt, J ) 13.6, 3.6 Hz,
1H), 2.02-1.93 (m, 1H), 1.77-1.71 (m, 2H), 1.48-1.36 (m, 2H),
1.29-1.19 (m, 1H), 1.05 (s, 3H), 0.91 (s, 3H), 0.88 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ 139.1, 138.5, 135.4, 134.9, 128.3, 128.2,
127.9, 127.55, 127.5, 120.4, 117.1, 116.4, 82.8, 81.2, 81.0, 77.2,
76.7, 76.5, 73.3, 71.7, 71.6, 70.8, 52.9, 48.2, 46.2, 45.0, 29.0, 26.8,
20.4, 20.2, 9.7; HRMS m/z (ES+) [found (M + Na)+ 597.3171,
J. Org. Chem, Vol. 72, No. 15, 2007 5653

Bello et al.
mg, 176 µL, 2.5 mmol) was added and the resulting solution stirred
for 4 h. The generated hydrochloric acid was quenched with
triethylamine (1 mL), the solvent removed under reduced pressure,
and the resulting yellow solid adsorbed onto silica and purified by
silica gel column chromatography, eluting with ethyl acetate and
petroleum ether (30:70), to yield 10 (1.6 g, 88%) as a colorless
solid: Rf 0.55 (ethyl acetate); [R] 26 -16.8 (c 0.64 in CHCl3); mp
D
119-120 °C (from ethyl acetate/petroleum ether); νmax (KBr disk)/
cm-1 3405.3 (s), 3066.5 (m), 3034.6 (m), 2910.9 (s), 2862.7 (s),
1647.7 (w), 1497.4 (m), 1455.7 (s), 1425.7 (s), 1354.7 (s), 1255.6
(w), 1160.8 (s), 1052.2 (s), 992.8 (s), 928.6 (s), 724.1 (s), 696.4
1
(s), 576.2 (w), 460.1 (w); H NMR (300 MHz, CDCl3) δ 7.43-
7.28 (m, 10H), 6.05-5.90 (m, 2H), 5.35 (ddt, J ) 17.1, 1.8, 1.5
Hz, 1H), 5.30 (ddt, J ) 17.2, 1.8, 1.5 Hz, 1H), 5.22 (ddt, J ) 10.5,
1.5, 1.3 Hz, 1H), 5.14 (ddt, J ) 10.2, 1.8, 1.3 Hz, 1H), 5.05 (d, J
) 11.8 Hz, 1H), 4.90 (d, J ) 10.5 Hz, 1H), 4.80 (d, J ) 10.5 Hz,
1H), 4.70 (d, J ) 11.8 Hz, 1H), 4.41 (ddt, J ) 12.3, 5.6, 1.5 Hz,
1H), 4.28 (ddt, J ) 12.3, 5.8, 1.3 Hz, 1H), 4.19-4.17, (m, 2H),
4.02 (t, J ) 2.3 Hz, 1H), 3.92 (t, J ) 9.7 Hz, 1H), 3.81 (t, J ) 9.5
Hz, 1H), 3.42-3.34 (m, 2H), 3.19 (t, J ) 9.2 Hz, 1H), 2.69 (br s,
2H); 13C NMR (75 MHz, CDCl3) δ 138.7, 138.67, 135.2, 134.8,
128.4, 128.3, 128.2, 127.8, 127.7, 127.6, 117.0, 116.9, 82.6, 81.4,
81.0, 77.2, 75.8, 74.8, 74.2, 73.8, 72.1, 71.9; HRMS m/z (ES+)
[found (M + Na)+ 463.2088, C26H32O6Na requires M+, 463.2097],
m/z (ES+) 463 ([M + Na]+, 100). Anal. Calcd for C26H32O6: C,
70.9; H, 7.3. Found: C, 70.6; H, 7.6.
FIGURE 7. Effect of compounds 3-6, 16, and 24 on 45Ca2+ release
from permeabilized L15 cells. It can be seen that none of the compounds
evoked Ca2+ release. The bold line, labeled “20 µM compound
application”, indicates the period of time in which a 20 µM solution
of each compound was applied to the cells.
(+)-1D-1,5-Bis-O-Allyl-2,3,6-tris-O-benzyl-myo-inositol (11).
10 (2.0 g, 4.5 mmol), di-n-butyltin oxide (1.2 g, 5.0 mmol), tetra-
n-butylammonium iodide (1.9 g, 4.5 mmol), and benzyl bromide
(3.7 g, 2.6 mL, 21.8 mmol) were dissolved in acetonitrile (80 mL)
under an atmosphere of nitrogen. The mixture was heated under
reflux for 24 h using a Soxhlet apparatus filled with 3 Å molecular
sieves to remove water generated in the reaction. The reaction
mixture was cooled to rt, and the solvent was removed under
reduced pressure. The residue was reconstituted in ethyl acetate
(20 mL) and water (20 mL), the layers were separated, and the
aqueous layer was extracted with ethyl acetate (3 × 20 mL). The
combined organic layers were washed with a saturated aqueous
solution of sodium hydrogen carbonate (20 mL), and the resulting
solid was removed by filtration through Celite. The filtrate was
washed with brine (20 mL), dried (magnesium sulfate), filtered,
and concentrated under reduced pressure to yield a yellow residue.
Purification by activated aluminum oxide column chromatography
(twice), eluting with ethyl acetate and petroleum ether (50:50),
yielded 11 (1.7 g, 71%) as a colorless solid: Rf 0.23 (ethyl acetate/
petroleum ether, 30:70); [R] 2D6 +2.8 (c 0.68 in CHCl3); mp 69-71
°C (from diethyl ether/petroleum ether); νmax (KBr disk)/cm-1
3530.9 (s), 3258.2 (s), 3064.2 (m), 3030.1 (m), 2893.6 (s), 2862.7
(s), 1648.1 (w), 1497.5 (m), 1454.3 (s), 1350.9 (s), 1210.4 (w),
1128.6 (s), 1069.3 (s), 1027.2 (s), 929.6 (m), 928.6 (s), 755.2 (w),
1
728.6 (s), 695.4 (s), 565.0 (w); H NMR (300 MHz, CDCl3) δ
7.44-7.25 (m, 15H), 6.06-5.87 (m, 2H), 5.33 (ddt, J ) 17.2, 1.8,
1.5 Hz, 1H), 5.30 (ddt, J ) 17.4, 1.8, 1.5 Hz, 1H), 5.20 (ddt, J )
10.5, 1.5, 1.3 Hz, 1H), 5.18 (ddt, J ) 10.2, 1.8, 1.5 Hz, 1H), 4.90
(d, J ) 12.0 Hz, 1H), 4.89 (d, J ) 10.5 Hz, 1H), 4.82-4.78 (m,
2H), 4.63 (d, J ) 11.8 Hz, 1H), 4.57 (d, J ) 11.8 Hz, 1H), 4.43-
4.30 (m, 2H), 4.16-4.09 (m, 3H), 4.05 (t, J ) 2.3 Hz, 1H), 4.00
(t, J ) 9.5 Hz, 1H), 3.29-3.18 (m, 3H), 2.55 (br s, 1H); 13C NMR
(75 MHz, CDCl3) δ 139.3, 138.4, 135.8, 135.3, 128.9, 128.8, 128.6,
128.3, 128.2, 128.1, 128.0, 127.8, 117.2, 83.3, 81.8, 81.3, 80.5,
76.3, 74.6, 74.4, 74.0, 73.1, 72.8, 72.2 (s); HRMS m/z (ES+) [found
(M + Na)+ 553.2563, C33H38O6Na requires M+, 553.2566], m/z
(ES+) 553 ([M + Na]+, 100). Anal. Calcd for C33H38O6: C, 74.7;
H, 7.2. Found: C, 74.5; H, 7.3.
FIGURE 8. Effect of compounds 3-5, 16, and 24 on InsP3-evoked
45Ca2+ release from permeabilized L15 cells. It can be seen that none
of the compounds prevented InsP3-evoked Ca2+ release. The bold line,
labeled “20 µM compound application”, indicates the period of time
in which a 20 µM solution of each compound was applied to the cells.
The bold dashed line, labeled “0.2 µM InsP3 application”, indicates
the period of time in which a 0.2 µM solution of InsP3 was applied to
the cells.
C36H46O6Na requires M+, 597.3192], m/z (ES+) 597 ([M + Na]+,
100). Anal. Calcd for C36H46O6: C, 75.2; H, 8.1. Found: C, 75.3;
H, 8.3.
(-)-1D-1,5-Bis-O-allyl-2,6-bis-O-benzyl-myo-inositol (10). 9
(2.4 g, 4.1 mmol) was dissolved in methanol and dichloromethane
(2:3, 50 mL) under an atmosphere of nitrogen. Acetyl chloride (194
(-)-1D-1,5-Bis-O-allyl-2,3,6-tris-O-benzyl-4-O-(dimethylphos-
phinyl)-myo-inositol (12). (Diisopropylamino)dimethylphosphine
(76 mg, 471 µmol) and 1H-tetrazole (0.43 M solution in acetonitrile,
1.1 mL, 471 µmol) were dissolved in dry dichloromethane (3 mL),
5654 J. Org. Chem., Vol. 72, No. 15, 2007

Synthesis and Biological Action of InsP3 DeriVatiVes
the resulting mixture was cooled to -78 °C, and 11 (100 mg, 188
µmol) dissolved in dry dichloromethane (2 mL) was added by
cannula. The resulting mixture was allowed to warm to rt and stirred
overnight. 31P NMR analysis indicated the complete conversion of
(diisopropylamino)dimethylphosphine in the intermediate phos-
phinite (δP 130.0). The mixture was recooled to -78 °C, 3-chlo-
roperoxybenzoic acid (60% (w/w), 112 mg, 471 µmol) was added,
and the resulting mixture was warmed to rt and stirred for 30 min.
The 3-chloroperoxybenzoic acid was quenched with a 10% aqueous
solution of sodium hydrogen sulfite (5 mL), the layers were
separated, and the aqueous layer was extracted with dichlo-
romethane (3 × 5 mL). The combined organic layers were washed
with a 10% aqueous solution of sodium hydrogen bicarbonate (5
mL) and brine (5 mL), dried (magnesium sulfate), filtered, and
concentrated under reduced pressure. Purification by silica gel
column chromatography, eluting with methanol and ethyl acetate
(2:98), yielded 12 (107 mg, 94%) as a colorless solid. Further
purification was achieved by crystallization from diethyl ether/
dichloromethane/petroleum ether: Rf 0.38 (ethyl acetate); [R] 26
-1.9 (c 0.27 in CHCl3); mp 122-124 °C (from diethyl etheDr/
dichloromethane/petroleum ether); νmax (KBr disk)/cm-1 3064.3 (w),
3031.7 (w), 2823.2 (s), 2851.5 (s), 1454.5 (m), 1302.9 (m), 1216.5
(s), 1130.6 (m), 1096.4 (s), 1050.2 (s), 935.2 (s), 866.9 (m), 736.2
(s), 698.9 (w); 1H NMR (300 MHz, CDCl3) δ 7.41-7.28 (m, 15H),
6.04-5.83 (m, 2H), 5.33-5.25 (m, 2H), 5.18 (ddt, J ) 10.5, 1.8,
1.5 Hz, 1H), 5.15 (ddt, J ) 10.2, 1.5, 1.3 Hz, 1H), 4.88-4.74 (m,
4H), 4.66-4.54 (m, 3H), 4.39 (ddt, J ) 12.3, 5.6, 1.5 Hz, 1H),
4.27 (ddt, J ) 12.3, 5.6, 1.5 Hz, 1H), 4.13-4.05 (m, 2H), 4.00-
3.94 (m, 2H), 3.33-3.29 (m, 2H), 3.24 (dd, J ) 9.8, 2.1 Hz, 1H),
1.50 (d, J ) 14.1 Hz, 3H), 1.49 (d, J ) 14.1 Hz, 3H); 13C NMR
(75 MHz, CDCl3) δ 139.1, 139.0, 138.0, 135.3, 135.1, 128.9, 128.9,
128.8, 128.6, 128.4, 128.3, 128.2, 128.1, 127.9, 117.3, 117.1, 81.9
(d, J ) 2.8 Hz), 81.7, 80.7, 79.4 (d, J ) 2.2 Hz), 76.6, (d, J ) 8.3
Hz), 76.3, 74.7, 74.6, 73.9, 73.0, 72.1, 17.0 (d, J ) 94.0 Hz), 16.9
(d, J ) 94.0 Hz); 31P NMR (121 MHz, CDCl3) δ 54.8; HRMS m/z
(ES+) [found (M + Na)+ 629.2643, C35H43O7NaP requires M+,
629.2644], m/z (ES+) 629 ([M + Na]+, 100). Anal. Calcd for
C35H43O7P: C, 69.3; H, 7.1. Found: C, 69.3; H, 7.2.
61.8; HRMS m/z (ES+) [found (M + Na)+ 567.2716, C34H40O6Na
requires M+, 567.2723], m/z (ES+) 567 ([M + Na]+, 100). Anal.
Calcd for C34H40O6: C, 75.0; H, 7.4. Found: C, 75.2; H, 7.4.
(+)-1D-2,3,6-Tris-O-benzyl-4-O-methyl-myo-inositol (14). 13
(80 mg, 147 µmol), Wilkinson’s catalyst (41 mg, 44 µmol), and
Hunig’s base (38 mg, 51 µL, 294 µmol) were suspended in ethanol
(8 mL), and the resulting mixture was heated under reflux for 3 h.
The mixture was then cooled to 0 °C and filtered through Celite
and the filtrate concentrated under reduced pressure. The resulting
red residue was dissolved in methanol and dichloromethane (2:3,
8 mL), acetyl chloride (7 mg, 6 µL, 88 µmol) was added, and the
mixture was stirred for 2 h. The generated hydrochloric acid was
quenched with triethylamine (1 mL), the solvent was removed under
reduced pressure, the residue was reconstituted in ethyl acetate (5
mL) and water (5 mL), the layers were separated, and the aqueous
layer was extracted with ethyl acetate (3 × 5 mL). The combined
organic layers were washed with a saturated aqueous solution of
sodium hydrogen carbonate (5 mL) and brine (5 mL), dried
(magnesium sulfate), filtered, and concentrated under reduced
pressure. Purification by silica gel column chromatography (twice),
eluting with ethyl acetate and petroleum ether (30:70), yielded 14
(54 mg, 79%) as a colorless solid: Rf 0.5 (ethyl acetate/petroleum
ether, 50:50); mp 80-81 °C (from ethyl acetate/petroleum ether);
[R] D25 +2.1 (c 0.45 in CHCl3); νmax (KBr disk)/cm-1 3474.9 (s),
3032.1 (w), 2914.8 (m), 1719.3 (w), 1605.0 (w), 1496.9 (m), 1454.8
(m), 1357.7 (m), 1206.2 (w), 1119.6 (s), 1070.8 (s), 1027.4 (s),
934.3 (w), 869.9 (w), 727.0 (s), 696.2 (s), 572.0 (w), 518.1 (w);
1H NMR (300 MHz, CDCl3) δ 7.41-7.29 (m, 15H), 4.99 (d, J )
11.5 Hz, 1H), 4.90 (d, J ) 11.5 Hz, 1H), 4.82 (d, J ) 11.5 Hz,
1H), 4.71 (d, J ) 11.5 Hz, 1H), 4.67 (s, 2H), 4.03 (t, J ) 2.3 Hz,
1H), 3.71-3.59 (m, 5H), 3.47 (dd, J ) 9.5, 2.8 Hz, 1H), 3.44 (t,
J ) 9.0 Hz, 1H), 3.36 (dd, J ) 9.7, 2.3 Hz, 1H), 2.30 (br s, 2H);
13C NMR (75 MHz, CDCl3) δ 138.7, 138.6, 138.2, 128.6, 128.5,
128.4, 128.1, 127.8, 127.7, 127.7, 127.7, 127.5, 82.9, 81.7, 80.8,
77.2, 75.0, 74.9, 74.7, 72.6, 72.2, 61.4; HRMS m/z (ES+) [found
(M + Na)+ 487.2088, C28H32O6Na requires M+, 487.2097], m/z
(ES+) 487 ([M + Na]+, 100). Anal. Calcd for C28H32O6: C, 72.4;
H, 6.9. Found: C, 72.5; H, 6.9.
(-)-1D-1,5-Bis-O-allyl-2,3,6-tris-O-benzyl-4-O-methyl-myo-
inositol (13). 11 (150 mg, 283 µmol) was dissolved in dry
tetrahydrofuran (8 mL) under an atmosphere of nitrogen, the
solution was cooled to 0 °C, and sodium hydride (13 mg, 60%
dispersion in mineral oil, 311 µmol) was added. The mixture was
allowed to warm to rt and stirred for 2 h, then it was recooled to
0 °C, and methyl iodide (44 mg, 19 µL, 311 µmol) was added.
The mixture was warmed to rt and stirred overnight. The sodium
hydride was quenched with water (1 mL), the solvent removed
under reduced pressure, and the residue reconstituted in ethyl acetate
(10 mL) and water (10 mL). The layers were separated, and the
aqueous layer was extracted with ethyl acetate (3 × 10 mL). The
combined organic layers were washed with brine (10 mL), dried
(magnesium sulfate), filtered, and concentrated under reduced
pressure. Purification by silica gel column chromatography, eluting
with ethyl acetate and petroleum ether (10:90), yielded 13 (184
mg, 92%) as a colorless waxy solid: Rf 0.70 (ethyl acetate/
petroleum ether, 30:70); mp 35-36 °C (from ethyl acetate/
petroleum ether); [R] D26 -4.05 (c 0.41 in CHCl3); νmax (KBr disk)/
cm-1 3064.6 (w), 3030.5 (w), 2925.6 (m), 1647.5 (w), 1496.9 (m),
1454.8 (m), 1357.4 (m), 1207.7 (w), 1132.9 (s), 1088.2 (s), 1028.3
(+)-1D-2,3,6-Tris-O-benzyl-4-O-methyl-myo-inositol 1,5-Bis-
(dibenzyl phosphate) (15). Bis(benzyloxy)(N,N-diisopropylamino)-
phosphine (353 mg, 1.0 mmol) was stirred with 1H-tetrazole (0.43
M solution in acetonitrile, 2.4 mL, 1.0 mmol) for 30 min under an
atmosphere of nitrogen. 14 (95 mg, 204 µmol) dissolved in dry
dichloromethane (8 mL) was added by cannula and the resulting
solution stirred overnight. The solution was cooled to -78 °C, and
3-chloroperoxybenzoic acid (176 mg, 1.0 mmol) was added. The
resulting mixture was allowed to warm to rt and stirred for 30 min.
The 3-chloroperoxybenzoic acid was quenched with a 10% aqueous
solution of sodium hydrogen sulfite (5 mL). The layers were
separated, and the aqueous layer was extracted with dichlo-
romethane (3 × 10 mL). The combined organic layers were washed
with a saturated aqueous solution of sodium hydrogen carbonate
(5 mL) and brine (5 mL), dried (magnesium sulfate), filtered, and
concentrated under reduced pressure. Purification by silica gel
column chromatography, eluting with ethyl acetate and petroleum
ether (30:70, then 40:60, then 50:50), yielded 15 (132 mg, 66%)
as a colorless gum: Rf 0.37 (ethyl acetate/petroleum ether, 50:50);
[R] D25 +7.6 (c 0.2 in CHCl3); νmax (thin film)/cm-1 3064.4 (w),
3033.3 (w), 2933.0 (m), 1497.5 (m), 1455.5 (s), 1379.8 (m), 1269.5
(s), 1214.3 (m), 1124.9 (m), 1091.8 (s), 1013.9 (s), 881.1 (m), 800.0
1
(m), 995.6 (w), 924.5 (m), 734.9 (m), 697.2 (m); H NMR (300
1
MHz, CDCl3) δ 7.36-7.16 (m, 15H), 5.98-5.76 (m, 2H), 5.26-
5.18 (m, 2H), 5.11-5.06 (m, 2H), 4.79 (s, 2H), 4.78 (d, J ) 10.5
Hz, 1H), 4.70 (d, J ) 10.5 Hz, 1H), 4.61 (d, J ) 11.8 Hz, 1H)
4.51 (d, J ) 11.8 Hz, 1H), 4.25 (dt, J ) 5.6, 1.3 Hz, 2H), 4.02-
3.98 (m, 2H), 3.90 (t, J ) 2.3 Hz, 1H), 3.85 (t, J ) 9.4 Hz, 1H),
3.64 (t, J ) 9.7 Hz, 1H), 3.58 (s, 3H), 3.17-3.09 (m, 3H); 13C
NMR (75 MHz, CDCl3) δ 139.4, 139.3, 139.1, 135.9, 135.4, 128.8,
128.76, 128.7, 128.5, 128.2, 128.01, 128.0, 127.8, 127.7, 117.1,
116.9, 84.0, 83.9, 81.9, 81.1, 80.9, 76.3, 75.0, 74.8, 74.4, 73.2, 72.8,
(w), 736.5 (s), 696.6 (s); H NMR (300 MHz, CDCl3) δ 7.31-
7.00 (m, 35H), 4.96-4.62 (m, 12H), 4.48 (s, 2H), 4.35-4.24 (m,
2H), 4.18-4.12 (m, 1H), 3.98 (t, J ) 9.4 Hz, 1H), 3.68 (t, J ) 9.4
Hz, 1H), 3.45 (s, 3H), 3.25 (dd, J ) 9.7, 2.3 Hz, 1H); 13C NMR
(75 MHz, CDCl3) δ 139.0, 138.6, 138.3, 136.6 (d, J ) 7.8 Hz),
136.4 (d, J ) 7.8 Hz), 136.1 (d, J ) 1.7 Hz), 136.0 (d, J ) 1.7
Hz), 129.0, 128.95, 128.9, 128.86, 128.8, 128.7, 128.6, 128.3, 128.2,
128.1, 128.02, 128.0, 127.93, 127.9, 127.8, 127.7, 81.4 (d, J ) 1.7
Hz), 80.6 (dd, J ) 6.9, 1.6 Hz), 80.3, 78.7 (dd, J ) 7.7, 4.5 Hz),
J. Org. Chem, Vol. 72, No. 15, 2007 5655

Bello et al.
78.4 (d, J ) 5.5 Hz), 76.4, 75.5, 75.1, 73.3, 69.9 (d, J ) 5.6 Hz),
69.7 (d, J ) 5.4 Hz), 69.5 (d, J ) 5.3 Hz), 69.4 (d, J ) 5.2 Hz),
61.5; 31P NMR (121 MHz, CDCl3) δ 0.15, -0.56; HRMS m/z (ES+)
[found (M + Na)+ 1007.3288, C56H58O12NaP2 requires M+,
1007.3301], m/z (ES+) 1007 ([M + Na]+, 100). Anal. Calcd for
C56H58O12P2: C, 68.3; H, 5.9. Found: C, 68.25; H, 5.8.
added, and the resulting mixture was stirred for 10 min at rt. The
mixture was then cannulated onto a solution of 17 (40 mg, 61 µmol)
in dry tetrahydrofuran (0.5 mL) under an atmosphere of nitrogen
and the resulting mixture heated under reflux for 6 h. The mixture
was cooled to rt and the solvent removed under reduced pressure
to give a dark red residue. 1H NMR analysis indicated that the allyl
groups had completely isomerized. The residue was suspended in
ethanol, the resulting mixture filtered through Celite, and the solvent
removed under reduced pressure. The resulting residue was
dissolved in a solution of methanol and dichloromethane (2:3, 1
mL) under an atmosphere of nitrogen, acetyl chloride (3 mg, 3 µL,
37 µmol) was added, and the resulting mixture was stirred for 3 h.
The generated hydrochloric acid was quenched with triethylamine
(50 µL), the solvent removed under reduced pressure, and the
residue adsorbed onto silica gel and purified by column chroma-
tography, eluting with ethyl acetate and petroleum ether (20:80),
to yield 18 (31 mg, 89%) as a colorless gum: Rf 0.54 (ethyl acetate/
petroleum ether, 50:50); [R] 2D5 -6.7 (c 0.56 in CHCl3); νmax (thin
film)/cm-1 3555.0 (m), 3449.2 (m), 3055.3 (m), 2924.8 (s), 1612.8
(m), 1586.1 (w), 1514.1 (s), 1455.0 (s), 1364.3 (m), 1265.7 (s),
1250.2 (m), 1113.2 (m), 1069.2 (s), 1028.0 (m), 933.9 (w), 822.7
(-)-1D-4-O-Methyl-myo-inositol 1,5-Bisphosphate (Sodium
Salt) (16). 15 (11 mg, 11 µmol) was dissolved in tert-butyl alcohol
and water (6:1, 3.5 mL). Sodium hydrogen carbonate (4 mg, 43
µmol) and palladium black (23 mg, 213 µmol) were added, and
the flask was flushed three times with a balloon of hydrogen and
then stirred for 4 h at rt under an atmosphere of hydrogen. The
organic layer was removed by filtration, the dark residue washed
with water (3 mL), and the collected aqueous layer lyophilized to
yield 16 as a colorless solid (4 mg, 82%): [R] 2D2 -4.6 (c 0.2 in
H2O); νmax (KBr disk)/cm-1 3423.1 (s), 1686.1 (s), 1650.3 (w),
1384.5 (s), 1205.6 (w), 1133.8 (s), 1085.3 (s), 1029.4 (s), 973.2
(s), 917.5 (w), 804.9 (m), 724.8 (m), 595.8 (w), 551.6 (m); 1H NMR
(300 MHz, D2O) δ 4.10 (br s, 1H), 3.74-3.65 (m, 3H), 3.45-3.39
(m, 4H), 3.23 (dd, J ) 10.2, 8.5 Hz, 1H); 13C NMR (75 MHz,
D2O) δ 81.3 (d, J ) 5.5 Hz), 78.0 (dd, J ) 6.1, 1.1 Hz), 74.6 (d,
J ) 5.5 Hz), 72.2 (d, J ) 6.1 Hz), 70.9 (d, J ) 1.1 Hz), 69.6 (d,
J ) 1.7 Hz), 60.2; 31P NMR (121 MHz, D2O) δ 3.56, 2.99; HRMS
m/z (ES-) [found M- 374.9855, C7H14O12NaP2 requires M-,
374.9858], m/z 375 ([C7H14NaO12P2], 70), 352 ([C7H15O12P2]-,
100), 273 ([C7H14O9P], 10).
1
(w), 737.3 (s), 701.9 (s); H NMR (300 MHz, CDCl3) δ 7.29-
7.18 (m, 17H), 6.79 (d, J ) 8.7 Hz, 2H), 4.92 (d, J ) 11.5 Hz,
1H), 4.84 (d, J ) 11.0 Hz, 1H), 4.83 (d, J ) 11.4 Hz, 1H), 4.73-
4.65 (m, 2H), 4.62 (s, 2H), 4.61 (d, J ) 11.0 Hz, 1H), 3.98 (t, J )
2.6 Hz, 1H), 3.81 (J ) 9.2 Hz, 1H), 3.73 (s, 3H), 3.61 (d, J ) 9.5
Hz, 1H), 3.46-3.34 (m, 3H), 2.39 (d, J ) 2.0 Hz, 1H), 2.22 (d, J
) 6.4 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 159.7, 139.2, 139.1,
138.6, 131.2, 130.2, 129.0, 128.9, 128.8, 128.5, 128.2, 128.2, 128.1,
128.0, 114.4, 82.1, 81.4, 81.3, 77.9, 77.5, 77.1, 75.4, 73.1, 72.6,
55.7; HRMS m/z (ES+) [found (M + Na)+ 593.2504, C35H38O7Na
requires M+, 593.2515], m/z (ES+) 593 ([M + Na]+, 100). Anal.
Calcd for C35H38O7: C, 73.66; H, 6.7. Found: C, 73.25; H, 6.75.
(+)-1D-1,5-Bis-O-allyl-2,3,6-tris-O-benzyl-4-O-(4-methoxyben-
zyl)-myo-inositol (17). 11 (2.3 g, 4.3 mmol) was dissolved in dry
N,N-dimethylformamide (80 mL) under an atmosphere of nitrogen,
the solution was cooled to 0 °C, and sodium hydride (191 mg, 60%
dispersion in mineral oil, 4.8 mmol) was added. The resulting
mixture was allowed to warm to rt and stirred for 1 h, then it was
recooled to 0 °C, and tetra-n-butylammonium iodide (80 mg, 216
µmol) and 4-methoxybenzyl chloride (747 mg, 647 µL, 4.8 mmol)
were added. The mixture was allowed to warm to rt and stirred
overnight. The sodium hydride was quenched with water (3 mL),
the solvent removed under reduced pressure, and the residue
reconstituted in ethyl acetate (20 mL) and water (20 mL). The layers
were separated, and the aqueous layer was extracted with ethyl
acetate (3 × 10 mL). The combined organic layers were washed
with brine (20 mL), dried (magnesium sulfate), filtered, and
concentrated under reduced pressure to give a pale yellow oil.
Purification by silica gel column chromatography, eluting with ethyl
acetate and petroleum ether (10:90), yielded 17 (2.7 g, 95%) as a
colorless solid: Rf 0.6 (ethyl acetate/petroleum ether, 30:70);
[R]2D5 +6.4 (c 0.6 in CHCl3); mp 58-59 °C (from ethyl acetate/
petroleum ether); νmax (KBr disk)/cm-1 3058.8 (w), 3031.8 (w),
2921.3 (m), 1725.6 (w), 1613.9 (m), 1514.1 (s), 1454.3 (m), 1359.1
(m), 1302.1 (w), 1250.3 (s), 1172.6 (w), 1074.4 (s), 1035.6 (s),
(+)-1D-2,3,6-Tris-O-benzyl-4-O-(4-methoxybenzyl)-myo-inosi-
tol 1,5-Bis(dibenzyl phosphate) (19). Bis(benzyloxy)(N,N-diiso-
propylamino)phosphine (3.0 g, 8.8 mmol) was stirred with 1H-
tetrazole (613 mg, 8.8 mmol) for 10 min under an atmosphere of
nitrogen at rt. 18 (1.0 g, 1.8 mmol) dissolved in dry dichloromethane
(20 mL) was added by cannula and the resulting mixture stirred
overnight. The mixture was cooled to -78 °C, and 3-chloroper-
oxybenzoic acid (1.5 g, 8.8 mmol) was added. The resulting mixture
was allowed to warm to rt and stirred for 30 min. The 3-chlorop-
eroxybenzoic acid was quenched with a 10% aqueous solution of
sodium hydrogen sulfite (20 mL). The layers were separated, and
the aqueous layer was extracted with dichloromethane (3 × 10 mL).
The combined organic layers were washed with a saturated aqueous
solution of sodium hydrogen carbonate (10 mL) and brine (10 mL),
dried (magnesium sulfate), filtered, and concentrated under reduced
pressure. Purification by silica gel column chromatography, eluting
with ethyl acetate and petroleum ether (30:70, then 50:50), yielded
19 (1.4 g, 75%) as a colorless gum: Rf 0.39 (ethyl acetate/petroleum,
50:50); [R] 2D5 +7.5 (c 0.3 in CHCl3); νmax (thin film)/cm-1 3064.2
(m), 3033.0 (m), 2934.8 (m), 1612.8 (m), 1586.4 (w), 1514.3 (s),
1497.6 (m), 1455.6 (s), 1364.6 (m), 1250.1 (s), 1214.6 (m), 1073.8
(w), 1012.2 (s), 880.7 (m), 823.0 (w), 737.3 (s), 696.6 (s); 1H NMR
(300 MHz, CDCl3) δ 7.30-6.93 (m, 37H), 6.67 (d, J ) 8.7 Hz,
2H), 4.87-4.62 (m, 14H), 4.48 (d, J ) 11.5 Hz, 1H), 4.43 (d, J )
11.5 Hz, 1H), 4.34 (dd, J ) 18.2, 9.0 Hz, 1H), 4.26 (t, J ) 2.3 Hz,
1H), 4.18-4.12 (m, 1H), 4.03-3.93 (m, 2H), 3.67 (s, 3H), 3.31
(dd, J ) 9.7, 2.3 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 159.3,
139.0, 138.6, 138.2, 136.5 (d, J ) 4.8 Hz), 136.4 (d, J ) 4.8 Hz),
136.1 (d, J ) 2.3 Hz), 136.0 (d, J ) 1.8 Hz), 131.0, 129.8, 129.0,
128.9, 128.8, 128.8, 128.7, 128.5, 128.2, 128.1, 128.1, 128.0, 127.9,
127.7, 113.9, 80.9 (dd, J ) 7.0, 1.5 Hz), 80.4, 79.1 (d, J ) 2.8
Hz), 78.8 (dd, J ) 7.5, 3.3 Hz), 78.5 (d, J ) 5.9 Hz), 76.3, 75.5,
75.1, 75.0, 73.1, 69.9 (d, J ) 5.7 Hz), 69.7 (d, J ) 5.5 Hz), 69.6
(d, J ) 4.9 Hz), 55.6; 31P NMR (121 MHz, CDCl3) δ -0.22, -0.61;
HRMS m/z (ES+) [found (M + Na)+ 1113.3711, C63H64O13NaP2
1
917.3 (m), 821.8 (m), 744.7 (s), 697.4 (s), 605.7 (w); H NMR
(300 MHz, CDCl3) δ 7.37-7.16 (m, 17H), 6.76 (d, J ) 8.7 Hz,
2H), 5.99-5.77 (m, 2H), 5.26-5.19 (m, 2H), 5.12-5.07 (m, 2H),
4.81-4.77 (m, 3H), 4.73 (d, J ) 10.2 Hz, 1H), 4.71 (d, J ) 10.5
Hz, 1H), 4.67 (d, J ) 10.2 Hz, 1H), 4.61 (d, J ) 11.8 Hz, 1H),
4.53 (d, J ) 11.8 Hz, 1H), 4.28 (dt, J ) 5.6, 1.5 Hz, 2H), 4.03-
3.99 (m, 2H) 3.96-3.86 (m, 3H), 3.72 (s, 3H), 3.24-3.18 (m, 2H),
3.12 (dd, J ) 10.0, 2.3 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ
159.6, 139.5, 139.4, 139.0, 135.9, 135.4, 131.5, 130.3, 128.83,
128.8, 128.7, 128.6, 128.3, 128.03, 128.0, 127.8, 117.1, 117.0,
114.2, 83.8, 82.1, 81.8, 81.3, 81.0, 76.4, 76.1, 75.1, 74.8, 74.5, 73.3,
72.1, 55.7; HRMS m/z (ES+) [found (M + Na)+ 673.3165,
C41H46O7Na requires M+, 673.3141], m/z (ES+) 673 ([M + Na]+,
100). Anal. Calcd for C41H46O7: C, 75.7; H, 7.1. Found: C, 75.7;
H, 7.4.
(-)-1D-2,3,6-Tris-O-benzyl-4-O-(4-methoxybenzyl)-myo-inosi-
tol (18). Wilkinson’s catalyst (22 mg, 24 µmol) was dissolved in
dry tetrahydrofuran (0.5 mL) under an atmosphere of nitrogen,
n-butyllithium (1.6 M solution in hexanes, 23 µL, 36 µmol) was
5656 J. Org. Chem., Vol. 72, No. 15, 2007

Synthesis and Biological Action of InsP3 DeriVatiVes
requires M+, 1113.3720], m/z (ES+) 1113 ([M + Na]+, 100). Anal.
(+)-1D-4-O-(Dimethylphosphinyl)-myo-inositol 1,5-Bisphos-
phate (Sodium Salt) (3). 21 (71 mg, 68 µmol) was dissolved in
tert-butyl alcohol and water (6:1, 12 mL), sodium hydrogen
carbonate (23 mg, 271 µmol) and palladium black (145 mg, 1.4
mmol) were added, and the flask was flushed three times with a
balloon of hydrogen and then stirred for 7 h at rt under an
atmosphere of hydrogen. The organic layer was removed by
filtration, the dark residue washed with water (4 × 3 mL), and the
collected aqueous layer lyophilized to yield 3 as a colorless solid
(32 mg, 93%); [R] D22 +0.81 (c 0.6 in H2O); νmax (KBr disk)/cm-1
3423.3 (s), 2198.8 (m), 1655.3 (w), 1309.1 (w), 1188.8 (s), 1116.1
(s), 1053.1 (s), 950.1 (s), 920.3 (m), 883.9 (m), 811.2 (w), 721.7
Calcd for C63H64O13P2: C, 69.35; H, 5.9. Found: C, 69.7; H, 5.8.
(+)-1D-2,3,6-Tris-O-benzyl-myo-inositol 1,5-Bis(dibenzyl phos-
phate) (20). 19 (327 mg, 0.3 mmol) was dissolved in acetonitrile
and water (4:1, 5 mL), and ceric ammonium nitrate (987 mg, 1.8
mmol) was added at rt. The resulting orange solution was stirred
for 2 h. The solvent was removed under reduced pressure, the
residue was reconstituted in ethyl acetate (5 mL) and water (5 mL),
the layers were separated, and the aqueous layer was extracted with
ethyl acetate (3 × 5 mL). The combined organic layers were washed
with a saturated aqueous solution of sodium hydrogen carbonate
(10 mL), dried (magnesium sulfate), filtered, and concentrated under
reduced pressure to give an orange residue. Silica gel column
chromatography, eluting with ethyl acetate and petroleum ether (50:
50), followed by crystallization from diethyl ether/dichloromethane/
petroleum ether, yielded 20 (232 mg, 73%) as a colorless solid: Rf
0.24 (ethyl acetate/petroleum, 50:50); [R] 2D5 +1.6 (c 0.6 in
CHCl3); mp 125-126 °C; νmax (thin film)/cm-1 3397.3 (s), 3064.6
(m), 3030.5 (m), 2938.6 (m), 2890.7 (m), 1497.5 (m), 1455.5 (s),
1367.4 (m), 1269.4 (s), 1240.1 (s), 1216.2 (m), 1162.8 (m), 1129.1
(m), 1068.7 (s), 1013.4 (s), 888.8 (m), 737.0 (s), 695.3 (s), 589.3
1
(w), 513.8 (m); H NMR (300 MHz, D2O) δ 4.29-4.15 (m, 2H),
3.97-3.77 (m, 3H), 3.63 (dd, J ) 9.7, 2.8 Hz, 1H), 1.58 (d, J )
11.0 Hz, 3H), 1.53 (d, J ) 11.0 Hz, 3H); 13C NMR (75 MHz,
D2O) δ 76.8 (dd, J ) 7.7, 6.3 Hz), 76.1-75.9 (m), 74.4 (d, J )
5.5 Hz), 72.2 (d, J ) 7.2 Hz), 70.8, 69.2, 15.3 (d, J ) 95.0 Hz),
15.1 (d, J ) 95.0 Hz); 31P NMR (121 MHz, D2O) δ 67.1, 1.63,
1.41; HRMS m/z (MALDI, matrix 3AQ, internal calculation on
glucose sulfate and ATP) [found (C8H18O13P3)- 414.9939, C8H18O13P3
requires M-, 414.9960], m/z (MALDI, matrix 3AQ, external
calculation on glucose sulfate and ATP) 415 [(C8H18O13P3)-].
1
(w), 554.7 (w), 502.2 (m); H NMR (300 MHz, CDCl3) δ 7.37-
7.12 (m, 35H), 5.05-4.70 (m, 12H), 4.62 (d, J ) 11.8 Hz, 1H),
4.57 (d, J ) 11.8 Hz, 1H), 4.30 (t, J ) 2.3 Hz, 1H), 4.25-4.17
(m, 3H), 4.08-3.98 (m, 1H), 3.87 (br s, 1H), 3.25 (dd, J ) 9.2,
2.0 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 138.6, 138.0, 137.8,
135.8-135.6 (m) 128.6, 128.52, 128.5, 128.46, 128.4, 128.25,
128.2, 127.8, 127.7, 127.6, 127.4, 82.4 (dd, J ) 6.1, 1.9 Hz), 79.1,
78.3-78.0 (m), 76.0, 75.2, 75.1, 72.9, 72.0, 69.6 (d, J ) 5.2 Hz),
69.5 (d, J ) 5.8 Hz), 69.3 (d, J ) 5.5 Hz); 31P NMR (121 MHz,
CDCl3): δ 1.34, -0.49; HRMS m/z (ES+) [found (M + Na)+
993.3147, C55H56O12NaP2 requires M+, 993.3145], m/z (ES+) 993
([M + Na]+, 100). Anal. Calcd for C55H56O12P2: C, 68.0; H, 5.8.
Found: C, 68.1; H, 5.65.
(+)-1D-2,3,6-Tris-O-benzyl-4-O-(di-n-butylphosphinyl)-myo-
inositol 1,5-Bis(dibenzyl phosphate) (22). 20 (100 mg, 103 µmol)
was dissolved in dry N,N-dimethylformamide (4 mL) under an
atmosphere of nitrogen and the mixture cooled to -42 °C.
4-(Dimethylamino)pyridine (catalytic amount) was added, followed
by dry triethylamine (52 mg, 72 µL, 515 µmol) and di-n-
butylphosphinyl chloride (82 mg, 79 µL, 416 µmol). The mixture
was allowed to warm to rt and stirred overnight. The di-n-
butylphosphinic chloride was quenched with water (0.5 mL), the
solvent was removed under reduced pressure, and the residue was
reconstituted in ethyl acetate (2 mL) and water (2 mL). The layers
were separated, and the aqueous layer was extracted with ethyl
acetate (3 × 5 mL). The combined organic layers were dried
(magnesium sulfate), filtered, and concentrated under reduced
pressure. Purification by silica gel column chromatography, eluting
with ethyl acetate and petroleum ether (60:40), gave 22 as a
colorless solid which was crystallized from ethyl acetate and
petroleum ether (76 mg, 65%): Rf 0.25 (ethyl acetate/petroleum
ether, 60:40); [R] 2D5 +0.5 (c 0.27 in CHCl3); mp 104-105 °C
(from ethyl acetate/petroleum ether); νmax (KBr disc)/cm-1 3064.4
(m), 3030.8 (m), 2930.0 (m), 1457.3 (w), 1381.8 (w), 1261.3 (m),
1211.2 (w), 1160.8 (w), 1127.3 (w), 1037.8 (s), 1015.5 (s), 881.1
(+)-1D-2,3,6-Tris-O-benzyl-4-O-(dimethylphosphinyl)-myo-
inositol 1,5-Bis(dibenzyl phosphate) (21). 20 (40 mg, 41 µmol)
was dissolved in dry N,N-dimethylformamide (1 mL) under an
atmosphere of nitrogen. 2,6-Lutidine (22 mg, 24 µL, 206 µmol)
was added, and the resulting mixture was cooled to -42 °C.
Dimethylphosphinic chloride (19 mg, 165 µmol) dissolved in dry
N,N-dimethylformamide (0.5 mL) was added by cannula. The
resulting mixture was allowed to warm to rt and stirred for 22 h.
The solvent was removed under reduced pressure and the residue
adsorbed onto silica gel and purified by silica gel column chro-
matography, eluting with methanol and ethyl acetate (1:99) (three
times), to give 21 (33 mg, 76%) as a colorless solid. Further
purification was achieved by crystallization from ethyl acetate and
1
(w), 867.1 (w), 135.7 (m), 695.9 (s), 593.0 (w); H NMR (300
MHz, CDCl3) δ 7.28-6.91 (m, 35H), 5.11 (dd, J ) 11.8, 6.1 Hz,
1H), 4.92-4.57 (m, 12H), 4.45 (d, J ) 11.5 Hz, 1H), 4.35-4.30
(m, 3H), 4.23-4.16 (m, 1H), 4.02 (t, J ) 9.5 Hz, 1H), 3.30 (dd, J
) 9.7, 1.8 Hz, 1H), 1.73-0.91 (m, 12H) 0.71 (t, J ) 7.2 Hz, 3H),
0.65 (t, J ) 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 138.3,
138.2, 137.8, 136.2 (d, J ) 8.1 Hz), 135.9 (d, J ) 6.6 Hz), 135.6
(d, J ) 7.5 Hz), 135.5 (d, J ) 6.9 Hz), 128.6, 128.5, 128.4, 128.31,
128.3, 128.13, 128.1, 127.84, 127.8, 127.7, 127.64, 127.6, 127.3,
127.2, 79.6 (d, J ) 6.1 Hz), 78.2-77.9 (m), 75.3, 74.72, 74.7, 73.2,
72.0, 69.5 (d, J ) 4.2 Hz), 69.3 (d, J ) 6.7 Hz), 69.2 (d, J ) 5.4
Hz), 28.7 (d, J ) 30.9 Hz), 27.5 (d, J ) 33.3 Hz), 24.5 (d, J ) 2.5
Hz), 24.4 (d, J ) 3.6 Hz), 24.1-23.8 (m), 13.7, 13.6; 31P NMR
(121 MHz, CDCl3) δ 62.1, -0.37, -0.60; HRMS m/z (ES+) [found
[C63H73O13NaP3]+ 1153.4181, C63H73O13NaP3 requires M+, 1153.-
4162], m/z (ES+) 1153 ([M + Na]+, 100).
(-)-1D-4-O-(Di-n-butylphosphinyl)-myo-inositol 1,5-Bispho-
sphate (Sodium Salt) (4). 22 (79 mg, 70 µmol) was dissolved in
tert-butyl alcohol and water (5:1, 12 mL), sodium hydrogen
carbonate (24 mg, 280 µmol) and palladium black (149 mg, 1.4
mmol) were added, and the flask was flushed three times with a
balloon of hydrogen and then stirred for 8 h at rt under an
atmosphere of hydrogen. The organic layer was removed by
filtration, the dark residue washed with water (3 × 5 mL), and the
petroleum ether: Rf 0.52 (methanol/ethyl acetate, 5:95); [R] 22
D
+6.2 (c 0.85 in CHCl3); mp 105-106 °C (from ethyl acetate/
petroleum ether); νmax (KBr disk)/cm-1 3058.8 (m), 3033.4 (m),
2924.4 (m), 2879.5 (m), 1498.2 (m), 1455.4 (m), 1381.0 (w), 1262.8
(s), 1215.8 (s), 1124.5 (w), 1017.2 (s), 939.9 (w), 872.2 (m), 736.4
1
(s), 695.9 (s), 594.5 (w), 507.5 (w); H NMR (300 MHz, CDCl3)
δ 7.32-6.95 (m, 35H), 5.05 (dd, J ) 11.8, 6.1 Hz, 1H), 4.92-
4.59 (m, 12H), 4.44 (d, J ) 11.3 Hz, 1H), 4.38-4.32 (m, 2H),
4.28 (t, J ) 2.6 Hz, 1H), 4.21-4.15 (m, 1H), 4.01 (t, J ) 9.5 Hz,
1H), 3.29 (dd, J ) 10.0, 2.0 Hz, 1H), 1.40 (d, J ) 14.0 Hz, 3H),
1.26 (d, J ) 14.0 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 138.24,
138.2, 136.9, 136.0 (d, J ) 7.1 Hz), 135.9 (d, J ) 5.8 Hz) 135.6-
135.5 (m), 128.6, 128.5, 128.5, 128.4, 128.3, 128.2, 128.1, 128.06,
127.9, 127.8, 127.7, 127.6, 127.4, 127.2, 79.4-79.3 (m), 78.1-
77.9 (m), 75.3, 74.9, 74.8, 73.3-73.2 (m), 72.2, 69.6 (d, J ) 6.2
Hz), 69.5 (d, J ) 5.5 Hz), 69.3 (d, J ) 4.9 Hz), 17.6 (d, J ) 69.7
Hz), 16.3 (d, J ) 73.5 Hz); 31P NMR (121 MHz, CDCl3) δ 57.3,
-0.17, -0.54; HRMS m/z (ES+) [found (M + Na)+ 1069.3218,
C57H61O13NaP3 requires M+, 1069.3223], m/z (ES+) 1069 ([M +
Na]+, 100). Anal. Calcd for C57H61O13P3: C, 65.4; H, 5.9. Found:
C, 65.05; H, 5.6.
J. Org. Chem, Vol. 72, No. 15, 2007 5657

Bello et al.
collected aqueous layer lyophilized to yield 4 as a colorless solid
(39 mg, 95%): [R] D25 -1.43 (c 0.5 in H2O); νmax (KBr disk)/cm-1
3428.6 (s), 2959.6 (s), 2930.0 (s), 2868.3 (s), 1650.3 (m), 1457.3,
(w), 1376.2 (w), 1236.4 (w), 1114.6 (s), 972.2 (s), 900.7 (w), 800.0
(w), 724.5 (w), 576.2 (w), 537.1 (w); 1H NMR (300 MHz, D2O) δ
4.28-4.25 (m, 1H), 4.20 (t, J ) 9.2 Hz, 1H), 3.86-3.72 (m, 3H),
3.63 (dd, J ) 9.7, 2.8 Hz, 1H), 1.97-1.77 (m, 4H), 1.51-1.22 (m,
8H), 0.82-0.76 (m, 6H); 13C NMR (75 MHz, D2O) δ 76.4 (dd, J
) 8.3, 6.6 Hz), 76.2-76.1 (m), 74.3 (d, J ) 5.5 Hz), 72.2 (d, J )
7.7 Hz), 70.9, 69.4, 23.6-23.1 (m), 13.0 (d, J ) 1.1 Hz), 12.8 (d,
J ) 1.1 Hz); 31P NMR (121 MHz, D2O) δ 70.3, 3.9, 3.0; HRMS
m/z (ES+) [found [C14H29O13Na3P3]+ 567.0522, C14H29O13Na3P3
requires M+, 567.0514], m/z (ES+) 567 ([C14H29O13Na3P3]+, 100).
alcohol and water (5:1, 10 mL), sodium hydrogen carbonate (32
mg, 377 µmol) and palladium black (201 mg, 1.9 mmol) were
added, and the flask was flushed three times with a balloon of
hydrogen and then stirred for 8 h at rt under an atmosphere of
hydrogen. The organic layer was removed by filtration, the dark
residue washed with water (3 × 5 mL), and the collected aqueous
layer lyophilized to yield 24 as a colorless solid (37 mg, 92%):
[R] 2D5 +5.7 (c 0.53 in H2O) [lit.41 [R2D5 +6.0 (c 0.5 in H2O)]; νmax
(KBr disk)/cm-1 3423.5 (s), 2930.0 (m), 1655.1 (m), 1560.7 (w),
1376.3 (w), 1094.1 (s), 968.2 (s), 897.9 (w), 808.6 (m), 718.9 (m),
1
568.0 (m); H NMR (300 MHz, D2O) δ 4.18 (t, J ) 2.8 Hz, 1H),
3.87-3.81 (m, 1H), 3.75-3.64 (m, 3H), 3.53 (dd, J ) 9.5, 2.8 Hz,
1H); 13C NMR (75 MHz, D2O) δ 78.3 (d, J ) 5.6 Hz), 74.6
(d, J ) 5.1 Hz), 72.6 (d, J ) 1.5 Hz), 71.9 (t, J ) 5.0 Hz), 71.6,
71.0; 31P NMR (121 MHz, D2O) δ 5.4, 4.5; m/z (ES-) 405
([C6H10Na3O12P2]-, 15), 383 ([C6H11Na2O12P2]-, 20), 361 ([C6H12-
NaO12P2]-, 50), 339 ([C6H13O12P2]-, 45), 281 ([C6H11NaO9P]-, 70),
259 ([C6H12O9P]-, 100). These data are in good agreement with
the literature values.41
(+)-1D-2,3,6-Tris-O-benzyl-4-O-(methylsulfonyl)-myo-inosi-
tol 1,5-Bis(dibenzyl phosphate) (23). 20 (70 mg, 72 µmol) was
dissolved in dry dichloromethane (4 mL) under an atmosphere of
nitrogen and the mixture cooled to -78 °C. 4-(Dimethylamino)-
pyridine (catalytic amount) was added, followed by dry triethy-
lamine (36 mg, 50 µL, 360 µmol) and methanesulfonyl chloride
(33 mg, 22 µL, 288 µmol). The mixture was allowed to warm to
rt and stirred for 2 days. The methanesulfonyl chloride was
quenched with a saturated aqueous solution of sodium hydrogen
carbonate (2 mL). The layers were separated, and the aqueous layer
was extracted with dichloromethane (3 × 5 mL). The combined
organic layers were dried (magnesium sulfate), filtered, and
concentrated under reduced pressure. Purification by silica gel
column chromatography, eluting with ethyl acetate and petroleum
ether (50:50, then 70:30), gave 23 (42 mg, 56%) as a colorless
gum: Rf 0.25 (ethyl acetate/petroleum ether, 50:50); [R] 25 +1.5
(c 0.94 in CHCl3); νmax (thin film)/cm-1 3064.4 (s), 303D3.2 (s),
2931.9 (s), 1956.3 (w), 1884.7 (w), 1813.1 (w), 1726.8 (m), 1606.2
(w), 1497.6 (s), 1455.6 (s), 1355.1 (s), 1272.3 (s), 1214.7 (m),
1176.6 (m), 1124.5 (w), 1099.3 (w), 1014.2 (m), 880.9 (m), 847.7
(-)-1D-1,5-Bis-O-allyl-4-O-[2-(allyloxy)ethyl]-2,3,6-tris-O-ben-
zyl-myo-inositol (25). 11 (170 mg, 320 µmol) was dissolved in
dry N,N-dimethylformamide (5 mL) under an atmosphere of
nitrogen. The resulting solution was cooled to 0 °C and sodium
hydride (60% (w/w), 15 mg, 384 µmol) added with stirring. The
mixture was allowed to warm to rt and stirred for 2 h, it was then
recooled to 0 °C, and tetra-n-butylammonium iodide (catalytic
amount) and 2-(allyloxy)ethyl bromide (48 µL, 63 mg, 384 µmol)
were added. The resulting mixture was allowed to warm to rt and
stirred overnight. The sodium hydride was quenched with water
(0.5 mL), the solvent removed under reduced pressure, and the
residue reconstituted with ethyl acetate (10 mL) and water (10 mL).
The layers were separated, and the aqueous layer was extracted
with ethyl acetate (3 × 5 mL). The combined organic layers were
washed with brine (10 mL), dried (magnesium sulfate), filtered,
and concentrated under reduced pressure. Purification by silica gel
column chromatography, eluting with ethyl acetate and petroleum
ether (10:90), gave 25 (158 mg, 80%) as a colorless oil: Rf 0.6
(ethyl acetate/petroleum ether, 30:70); [R] 2D5 -7.8 (c 0.51 in
CHCl3); νmax (thin film)/cm-1 3064.0 (m), 3031.6 (m), 2984.1 (s),
2869.3 (s), 1647.2 (w), 1496.9 (w), 1454.9 (m), 1421.2 (w), 1266.0
(s), 1208.6 (w), 1131.9 (m), 1086.6 (s), 1028.1 (m), 996.0 (w),
1
(w), 736.2 (s), 696.5 (s), 599.6 (m); H NMR (300 MHz, CDCl3)
δ 7.39-7.00 (m, 35H), 5.14-4.41 (m, 16H), 4.37 (t, J ) 2.3, 1H),
4.26-4.17 (m, 1H), 4.09 (t, J ) 9.5 Hz, 1H), 3.40 (dd, J ) 10.2,
2.0 Hz, 1H), 2.91 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 138.1,
138.0, 136.6, 135.9 (d, J ) 7.7 Hz), 135.7 (d, J ) 6.8 Hz) 135.5
(d, J ) 7.0 Hz), 128.6, 128.4, 128.34, 128.3, 128.2, 128.1, 128.0,
128.0, 127.9, 127.9, 127.8, 127.3, 79.9 (d, J ) 4.5 Hz), 78.1 (dd,
J ) 8.1, 1.5 Hz), 77.7-77.6 (m), 77.1, 75.3, 74.9, 74.8, 72.6, 69.9
(d, J ) 5.5 Hz), 69.6 (d, J ) 5.6 Hz), 69.5 (d, J ) 5.4 Hz), 39.3;
31P NMR (121 MHz, CDCl3) δ -0.22, -0.53; m/z (ES+) 1071
([M + Na]+, 100). Anal. Calcd for C56H58O14P2S: C, 64.1; H, 5.6.
Found: C, 64.0; H, 5.3.
1
926.4 (m), 737.3 (s), 699.3 (m); H NMR (300 MHz, CDCl3) δ
7.43-7.29 (m, 15H), 6.06-5.83 (m, 3H), 5.32-5.12 (m, 6H), 4.86
(s, 2H), 4.85 (d, J ) 10.2 Hz, 1H), 4.78 (d, J ) 10.2 Hz, 1H), 4.72
(d, J ) 11.8 Hz, 1H), 4.59 (d, J ) 11.8 Hz, 1H), 4.43-4.28 (m,
2H), 4.10-4.06 (m, 2H), 4.02-3.89 (m, 6H), 3.82 (t, J ) 9.5 Hz,
1H), 3.61 (t, J ) 4.9 Hz, 2H), 3.29 (d, J ) 9.2 Hz, 1H), 3.27 (dd,
J ) 9.7, 5.9 Hz, 1H), 3.18 (dd, J ) 9.7, 2.3 Hz, 1H); 13C NMR
(75 MHz, CDCl3) δ 139.05, 139.0, 138.7, 135.6, 135.0, 134.9,
128.3, 128.3, 128.1, 127.8, 127.6, 127.5, 127.3, 116.8, 116.6, 116.4,
83.1, 82.5, 81.6, 80.53, 80.5, 75.9, 74.6, 74.5, 74.0, 72.9, 72.7, 72.0,
71.7, 69.9; HRMS m/z (ES+) [found (M + Na)+ 637.3140,
C38H46O7Na requires M+, 637.3141], m/z (ES+) 637 ([M + Na]+,
100).
(+)-1D-4-O-(Methylsulfonyl)-myo-inositol 1,5-Bisphosphate
(Sodium Salt) (5). 23 (73 mg, 70 µmol) was dissolved in tert-
butyl alcohol and water (10:1, 11 mL), sodium hydrogen carbonate
(24 mg, 280 µmol) and palladium black (149 mg, 1.4 mmol) were
added, and the flask was flushed three times with a balloon of
hydrogen and then stirred for 8 h at rt under an atmosphere of
hydrogen. The organic layer was removed by filtration, the dark
residue washed with water (3 × 5 mL), and the collected aqueous
layer lyophilized to yield 5 as a colorless solid (32 mg, 91%):
[R] D22 +1.64 (c 0.3 in H2O); νmax (KBr disk)/cm-1 3448.9 (s),
2969.2 (s), 2924.4 (s), 1655.1 (m), 1340.9 (m), 1158.0 (s), 1107.8
(s), 973.7 (s), 942.6 (w), 869.9 (w), 802.8 (w), 724.5 (w), 598.6
(-)-1D-4-O-(2-Hydroxyethyl)-2,3,6-tris-O-benzyl-myo-inosi-
tol (26). Wilkinson’s catalyst (53 mg, 49 µmol) was dissolved in
dry tetrahydrofuran (1.0 mL) under an atmosphere of nitrogen,
n-butyllithium (1.6 M solution in hexanes, 308 µL, 207 µmol) was
added, and the resulting mixture was stirred for 10 min at rt. The
mixture was then cannulated onto a solution of 25 (303 mg, 493
µmol) in dry tetrahydrofuran (0.5 mL) under an atmosphere of
nitrogen and the resulting mixture heated under reflux for 6 h. The
mixture was cooled to rt and the solvent removed under reduced
1
(w), 539.9 (w); H NMR (300 MHz, D2O) δ 4.60 (t, J ) 9.5 Hz,
1H), 4.33 (m, 1H), 4.00-3.91 (m, 1H), 3.82-2.80 (m, 2H), 3.75
(dd, J ) 10.2, 3.0 Hz, 1H), 3.25 (s, 3H); 13C NMR (75 MHz, D2O)
δ 83.8 (d, J ) 6.6 Hz), 75.0 (d, J ) 5.5 Hz), 74.2 (d, J ) 5.5 Hz),
72.3 (d, J ) 7.7 Hz), 70.8, 68.3, 38.9; 31P NMR (121 MHz, D2O)
δ 4.7, 4.1; HRMS m/z (ES+) [found [C7H13O14Na4P2S]+ 506.9090,
C7H13O14Na4P2S requires M+, 506.9092], m/z (ES-); 439 ([C7H14O14-
NaP2S]-, 5), 417 ([C7H15O14P2S]-, 15), 365 (20), 343 (100), 321
(40), 303 (15), 208 (15).
1
pressure to give a dark red residue. H NMR analysis indicated
that the allyl groups had completely isomerized. The residue was
dissolved in a mixture of methanol and dichloromethane (2:3, 5
(+)-1D-myo-Inositol 1,5-Bisphosphate (Sodium Salt) (24). 20
(100 mg, 103 µmol) (92 mg, 94 µmol) was dissolved in tert-butyl
(41) Westerduin, P.; Willems, H. A. M.; Vanboeckel, C. A. A.
Tetrahedron Lett. 1990, 31, 6915.
5658 J. Org. Chem., Vol. 72, No. 15, 2007

Synthesis and Biological Action of InsP3 DeriVatiVes
mL) under an atmosphere of nitrogen, acetyl chloride (23 mg, 21
µL, 296 µmol) was added, and the resulting solution was stirred
for 2 h. The generated hydrochloric acid was quenched with
triethylamine (0.2 mL), the solvent removed under reduced pressure,
and the residue adsorbed onto silica gel and purified using silica
gel column chromatography, eluting with ethyl acetate and petro-
leum ether (60:40), to give 26 (195 mg, 80%) as a colorless solid.
A very pure sample was obtained by crystallization from ethyl
acetate and petroleum ether: Rf 0.46 (ethyl acetate/petroleum ether,
60:40); [R] 2D5 -0.45 (c 1.1 in CHCl3); mp 92-93 °C (from ethyl
acetate/petroleum ether); νmax (KBr disk)/cm-1 3398.6 (s), 3064.4
(w), 3025.2 (w), 2911.9 (m), 2873.9 (m), 1496.9 (w), 1454.7 (m),
1364.5 (m), 1249.1 (w), 1208.9 (w), 1131.7 (s), 1085.6 (s), 2068.5
(s), 1023.2 (s), 928.7 (w), 723.3 (s), 969.8 (s), 607.0 (w), 539.9
(w); 1H NMR (300 MHz, CDCl3) δ 7.37-7.29 (m, 15H), 5.00 (d,
J ) 11.8 Hz, 1H), 4.89 (1H, d, JA′B′ ) 11.3 Hz, OCHA′HB′), 4.80
(1H, d, JA′B′ ) 11.3 Hz, OCHA′HB′), 4.70 (1H, d, JA′B′ ) 11.8 Hz,
OCHAHB), 4.69 (2H, s, OCHA′′HB′′), 4.05-3.46 (m, 9H), 3.37 (1H,
dd, J 9.7, 2.3, inositol ring), 3.30 (1H, br s, OH), 3.06 (1H, br s,
OH), 2.26 (1H, d, J 7,4, OH); 13C NMR (75 MHz, CDCl3) δ 138.5,
138.49, 137.8, 128.6, 128.55, 128.5, 128.1, 127.95, 127.93, 127.9,
127.8, 127.7, 82.1, 81.5, 80.6, 77.2, 75.0, 74.91, 74.9, 74.7, 72.8,
72.3, 62.2; HRMS m/z (ES+) [found (M + Na)+ 517.2192,
C29H34O7Na requires M+, 517.2202], m/z (ES+) 517 ([M + Na]+,
100). Anal. Calcd for C29H34O7: C, 70.4; H, 6.9. Found: C, 70.4;
H, 6.8.
filtration, the dark residue washed with water (3 × 5 mL), and the
collected aqueous layer lyophilized to yield 6 as a colorless solid
(40 mg, 89%): [R] D25 -2.95 (c 0.44 in H2O); νmax (KBr disk)/
cm-1 3290.0 (s), 2963.6 (m), 2930.0 (m), 1655.1 (s), 1639.2 (s),
1
1093.2 (s), 978.3 (s), 802.8 (w), 721.7 (w), 550.5 (m); H NMR
(300 MHz, D2O) δ 4.30 (br s, 1H), 4.11-4.06 (m, 1H), 3.81-3.69
(m, 6H), 3.58-3.45 (m, 2H); 13C NMR (75 MHz, D2O) δ 81.3 (d,
J ) 6.0 Hz), 78.4 (d, J ) 5.4 Hz), 74.8 (d, J ) 5.7 Hz), 73.2 (d,
J ) 7.3 Hz), 72.8 (d, J ) 7.0 Hz), 70.9, 69.9, 64.2 (d, J ) 4.5 Hz);
31P NMR (121 MHz, D2O) δ 5.2, 4.9, 4.1; HRMS m/z (ES+) [found
(M + H)+ 596.8883, C8H14O16Na6P3 requires M+, 596.8881], m/z
(ES+), 597 ([M + H]+, 50), 289 (100).
45Ca2+ Flux Assay. L15 cells were obtained by stable exogenous
expression of InsP3R1 in L fibroblasts.42,43 The cells were cultured
in Dulbecco’s modified Eagle’s medium supplemented with 10%
fetal calf serum, 3.8 mM L-glutamine, 0.9% (v/v) nonessential
amino acids, 85 IU/mL penicillin, 85 µg/mL streptomycin, and 20
mM HEPES, pH 7.4.
45Ca2+ fluxes were performed on saponin-permeabilized cells.
The cells were seeded in 12-well clusters (Costar, Cambridge, MA)
at a density of approximately 4 × 104 cm-2. Experiments were
carried out on confluent monolayers of cells at the seventh day
after plating. The cells were permeabilized by incubating them for
10 min with a solution containing 120 mM KCl, 30 mM imidazole
hydrochloride, pH 6.8, 2 mM MgCl2, 1 mM ATP, 1 mM EGTA,
and 20 µg/mL saponin at 25 °C. The nonmitochondrial Ca2+ stores
were loaded for 45 min at 25 °C in 120 mM KCl, 30 mM imidazole
hydrochloride, pH 6.8, 5 mM MgCl2, 5 mM ATP, 0.44 mM EGTA,
10 mM NaN3, and 150 nM free 45Ca2+ (28 µCi/mL). The cells
were then washed twice with 1 mL of efflux medium containing
120 mM KCl, 30 mM imidazole hydrochloride, pH 6.8, 1 mM
EGTA, and 10 µM thapsigargin (TG). The efflux medium was
replaced every 2 min, and the efflux was performed at 25 °C. The
additions of InsP3 and the 4-position-modified compounds are
indicated in the figures. At the end of the experiment, the 45Ca2+
remaining in the stores was released by incubation with 1 mL of a
2% sodium dodecyl sulfate solution for 30 min. Ca2+ release is
plotted as the fractional loss (i.e., the amount of Ca2+ released in
2 min divided by the total store Ca2+ content at that time). The
latter value was calculated by summing in retrograde order the
amount of tracer remaining in the cells at the end of the efflux and
the amounts of tracer collected during the successive time intervals.
(+)-1D-4-O-[2-[(Dibenzylphosphoryl)oxy]ethyl]-2,3,6-tris-O-
benzyl-myo-inositol 1,5-Bis(dibenzyl phosphate) (27). Bis(ben-
zyloxy)(N,N-diisopropylamino)phosphine (1.00 g, 2.9 mmol) was
stirred with 1H-tetrazole (0.43 M solution in acetonitrile, 6.9 mL,
2.9 mmol) for 30 min under an atmosphere of nitrogen. 26 (195
mg, 394 µmol) dissolved in dry dichloromethane (8 mL) was added
by cannula and the resulting mixture stirred overnight. The mixture
was cooled to -78 °C, and 3-chloroperoxybenzoic acid (510 mg,
2.9 mmol) was added. The resulting mixture was allowed to warm
to rt and stirred for 30 min. The 3-chloroperoxybenzoic acid was
quenched with a 10% aqueous solution of sodium hydrogen sulfite
(10 mL). The layers were separated, and the aqueous layer was
extracted with dichloromethane (3 × 10 mL). The combined organic
layers were washed with a saturated aqueous solution of sodium
hydrogen carbonate (10 mL), dried (magnesium sulfate), filtered,
and concentrated under reduced pressure. Purification by silica gel
column chromatography, eluting with ethyl acetate and petroleum
ether (40:60, then 60:40), gave 27 (232 mg, 46%) as a colorless
oil: Rf 0.54 (ethyl acetate/petroleum ether, 80:20); [R] 25 +9.7 (c
D
0.88 in CHCl3); νmax (thin film)/cm-1 3064.3 (w), 3033.2 (w),
2948.6 (m), 2885.2 (m), 1497.5 (m), 1455.6 (s), 1273.7 (s), 1214.7
(m), 1012.1 (s), 920.3 (w), 881.7 (m), 736.5 (s), 696.4 (s); 1H NMR
(300 MHz, CDCl3) δ 7.28-6.97 (m, 45H), 4.98-4.50 (s, 14H),
4.89-4.59 (m, 19H), 4.46 (d, J ) 11.8 Hz, 1H), 4.37 (d, J ) 11.8
Hz, 1H), 4.15 (t, J ) 2.0 Hz, 1H), 4.09-3.84 (m, 5H), 3.74 (t, J
) 9.5 Hz, 2H), 3.09 (dd, J ) 9.7, 2.0 Hz, 1H); 13C NMR (75 MHz,
CDCl3) δ 138.5, 138.1, 137.7, 136.0-135.5 (m), 128.52, 128.5,
128.4, 128.3, 128.24, 128.2, 128.1, 127.84, 127.8, 127.7, 127.6,
127.5, 127.4, 127.3, 80.4 (d, J ) 6.6 Hz), 79.9 (d, J ) 1.7 Hz),
79.3, 78.1 (dd, J ) 11.4, 4.0 Hz), 77.9 (d, J ) 5.9 Hz), 76.0, 75.1,
74.6, 72.7, 71.6 (d, J ) 7.8 Hz), 69.4 (d, J ) 5.9 Hz), 69.3-69.2
(m) 69.0 (d, J ) 5.7 Hz), 66.9 (d, J ) 6.1 Hz); 31P NMR (121
MHz, CDCl3) δ 0.38, -0.35, -0.61; m/z (ES+) 1297 ([M + Na]+,
100). Anal. Calcd for C71H73O16P3: C, 66.9; H, 5.8. Found: C,
66.7; H, 5.8.
Acknowledgment. This work was funded by the Biotech-
nology and Biological Sciences Research Council (BBSRC;
U.K.) (Grant Number BB/C515255/1) and the University of St
Andrews. We are grateful to the technical staff of the University
of St Andrews for providing mass spectrometry and elemental
analysis data. We are grateful to Tomas Luyten for technical
assistance. H.L.R. thanks the Royal Society for a Research
Fellowship.
Supporting Information Available: General experimental
methods, 1H, 13C, and, where appropriate, 31P NMR spectra for all
reported compounds, raw data from the 45Ca2+ flux assay, and CIF
file for the X-ray crystal structure of compound 20. This material
is available free of charge via the Internet at http://pubs.ac.org.
(-)-1D-4-O-[2-(Phosphoryloxy)ethyl]-myo-inositol 1,5-Bispho-
sphate (Sodium Salt) (6). 27 (97 mg, 76 µmol, 1.0) was dissolved
in tert-butyl alcohol and water (5:1, 12 mL), sodium hydrogen
carbonate (38 mg, 455 µmol) and palladium black (162 mg, 1.5
mmol) were added, and the flask was flushed three times with a
balloon of hydrogen and then stirred for 8 h at rt under an
atmosphere of hydrogen. The organic layer was removed by
JO070611A
(42) Mackrill, J. J.; Wilcox, R. A.; Miyawaki, A.; Mikoshiba, K.;
Nahorski, S. R.; Challiss, R. A. J. Biochem. J. 1996, 318, 871.
(43) Miyawaki, A.; Furuichi, T.; Maeda, N.; Mikoshiba, K. Neuron 1990,
5, 11.
J. Org. Chem, Vol. 72, No. 15, 2007 5659