The synthesis of membrane permeant derivatives of myo-inositol 1,4,5-trisphosphate

Article abstract of DOI:10.1071/CH06357

In order to enable the study of the intracellular second messenger d-myo-inositol 1,4,5-trisphosphate (InsP₃) and its receptors (InsP₃Rs), it has been desirable to develop protected derivatives of InsP₃ that are able to en

Full text of DOI:10.1071/CH06357

Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2006, 59, 887–893
The Synthesis of Membrane Permeant Derivatives
of myo-Inositol 1,4,5-Trisphosphate
Stuart J. Conway,A,B,G Jan W. Thuring,A Sylvain Andreu,A Brynn T. Kvinlaug,C
H. Llewelyn Roderick,C,D Martin D. Bootman,C and Andrew B. HolmesE,F,G
A Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK.
B School of Chemistry and Centre for Biomolecular Sciences, University of St Andrews,
St Andrews, Fife KY16 9ST, UK.
C Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK.
D Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, UK.
E School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne VIC 3010, Australia.
F Department of Chemistry, Imperial College London, London SW7 2AZ, UK.
G Corresponding authors. Email: sjc16@st-andrews.ac.uk; aholmes@unimelb.edu.au
In order to enable the study of the intracellular second messenger d-myo-inositol 1,4,5-trisphosphate (InsP3) and
its receptors (InsP3Rs), it has been desirable to develop protected derivatives of InsP3 that are able to enter the
cell, upon extracellular application. The subsequent removal of the lipophilic protecting groups, by intracellular
enzymes, releases InsP3 and leads to the activation of InsP3Rs. Two syntheses of d-myo-inositol 1,4,5-trisphosphate
hexakis(butyryloxymethyl) ester (d-InsP3/BM) and one of l-InsP3/BM are reported. It is demonstrated that extra-
cellular application of the d-enantiomer results in Ca2+ release, which is thought to occur via InsP3Rs. Application
of the l-enantiomer resulted in little Ca2+ release.
Manuscript received: 4 October 2006.
Final version: 28 October 2006.
Introduction
(AM) ester. This protecting group was first employed to
mask the carboxylate group of penicillins and hence improve
their bioavailability, with the AM group removed by esterase
enzymes once the drug had reached its target.[1,3] The AM
group shows improved lability within cells (compared to the
simple phosphate triesters) as the methylene linker removes
the ester moiety from the steric bulk of the protected com-
pound, allowing non-specific esterase enzymes to interact
with it. AM groups have also been used to mask phosphate
groups, the esterase enzymes hydrolyze the AM ester and
the spontaneous removal of formaldehyde reveals the free
phosphate (Fig. 1).
The intracellular study of phosphorylated small molecules
is complicated by the inability of these compounds to
passively diffuse across the cell membrane, a character-
istic that is conferred by their overall negative charge.
However, the delivery of multiply charged compounds,
across the lipophilic cell membrane, to an intracellular loca-
tion can be achieved by the masking of the charged moieties
with lipophilic groups.[1] To be useful, these groups must
allow the compound to diffuse across the cell membrane
before the lipophilic groups are removed by intracellu-
lar enzymes to reveal the biologically active compound.
Early work on membrane permeant phosphate derivatives
focused on the synthesis of simple phosphate triesters.[1,2]
However, it was found that the intracellular half-lives of
these compounds are very long, ranging from minutes or
hours to days. The intracellular stability of these groups
results from the lack of specific enzymes to hydrolyze
them and hence their intracellular cleavage is reliant on
uncatalyzed chemical hydrolysis rather than enzymatic
hydrolysis.[1]
Tsien and co-workers have applied this technology to the
synthesis of membrane permeant analogues of the intracel-
lular second messenger d-myo-inositol 1,4,5-trisphosphate
(InsP3).[4] The use of AM, propionyloxymethyl (PM), and
butyryloxymethyl (BM) moieties to protect the phosphate
groups of racemic InsP3 was reported (Fig. 2).[4] The
masked InsP3 diffused through the cell membrane and on
entering the cell, the protecting groups were removed by
esterase enzymes to give formaldehyde and InsP3, which
was demonstrated to evoke release of intracellular Ca2+
,
Lipophilic protecting groups that are stable outside of the
cell, but cleaved efficiently by intracellular enzymes, have
been used to furnish compounds that are of more use to bio-
logical investigators. One such group is the acetoxymethyl
presumably via activation of InsP3 receptors (InsP3Rs).[4]
Tsien has also reported a caged and membrane permeant ana-
logue of InsP3.[5] Schultz and co-workers have applied this
© CSIRO 2006
10.1071/CH06357
0004-9425/06/120887

888
S. J. Conway et al.
Cell
membrane
The racemic butyryloxymethyl ester of InsP3 (InsP3/
BM, 3)[4] and the caged membrane permeant InsP3
analogue[5] have proved to be of great use to those studying
the involvement of InsP3 in intracellular signalling.[5,9–13]
This led us to develop syntheses of both the d- and the
l-InsP3/BM for use as chemical probes of InsP3 signalling,
based on our previous investigations into the synthesis of
the phosphatidylinositol polyphosphates.[14–20] d-InsP3/BM
has proved to be invaluable in the study of InsP3 and Ca2+
signalling[12,13,21–27] and therefore we report the synthesis
of both enantiomers of InsP3/BM (3) and demonstrate the
differing Ca2+-releasing properties of each compound.
O
P
OϪ
H
H
O
P
OϪ
ϩ
O
RO
OϪ
RO
OϪ
Free phosphate
O
P
H
RO
O
O
O
O
Results and Discussion
O
R1
Synthesis of Compounds
In the first approach to the synthesis of d-InsP3/BM, (−)-3
(Scheme 1), the route utilized by Ozaki et al.[28] in the
synthesis of d-InsP3 was employed to furnish the opti-
cally pure alcohol (−)-4 {[α]2D0 = −14.6 (c 0.5, CHCl3)
[lit.,[28] [α]1D6 = −14.3 (CHCl3)]}. Benzylation of the axial
alcohol followed by Wilkinson’s catalyst [Rh(PPh3)3Cl]-
mediated isomerization of the allyl groups and subse-
quent methanolysis of the resulting enol ether afforded the
known triol (+)-6 {[α]2D0 = +12.3 (c 0.40, CHCl3) [lit.,[29]
[α]D = +12.4 (c 0.80, CHCl3)]} in good yield (81%).[28–30]
1H-Tetrazole-catalyzed phosphitylation of the triol (+)-6
with N,N-diisopropylamino bis-(2-cyanoethoxy)phosphine,
followed by P(iii) to P(v) oxidation using mCPBA, afforded
the fully protected InsP3 analogue (+)-7 (δP −2.5, −2.5,
and −2.7 ppm in CDCl3) in 86% yield. Treatment of the
cyanoethyl moieties with triethylamine furnished the pre-
sumed triethylammonium salt of InsP3, which reacted with
bromomethylbutyrate (12) to yield the fully benzylated
InsP3/BM (−)-8 (δP −4.6, −4.2, and −3.9 ppm). This reac-
tion was found to be capricious and only proceeded in poor to
moderate yield (29–55%). The poor yields for this reaction
reflects the fact that six eliminations and six substitutions
have to occur in order to furnish the desired product, and that
the triethylammonium salt of the phosphate group is likely
to be a poor nucleophile. Hydrogenolysis of the benzylated
InsP3/BM (−)-8 using palladium acetate and palladium tri-
fluoroacetate as catalysts afforded InsP3/BM (−)-3 (δP −4.1,
−3.8, and −3.6 ppm in MeOD) in good yield (88%). Palla-
dium acetate and palladium trifluoroacetate were employed
as Tsien has previously demonstrated that this reaction fails
when conducted with Pd on carbon as a catalyst.[4] The use
of neat acetic acid as the solvent during hydrogenolysis was
intended to maintain the product in a fully protonated state
and therefore minimize possible phosphate group migrations.
InsP3/BM (−)-3 was observed to be fairly unstable and broke
down if left at room temperature or in solvent for more than
a few hours.
HO
H
O
P
O
O
P
O
RO
O
O
R1
RO
O
O
O
R1
O
O
O
Esterase
O
O
R1
R1
Membrane permeant
analogue
Fig. 1. Acetoxymethyl-protected phosphates and derivatives can pas-
sively diffuse across the cell membrane. Hydrolysis by intracellu-
lar esterase enzymes reveals the biologically active compound and
formaldehyde. R1 = CH3 (AM), CH2CH3 (PM), (CH2)2CH3 (BM).
R
O
O
O
O
O
O
P
HO
HO
O
O
P
R
O
OH
O
O
O
R
O
O
O
O
O
O
R
P
O
O
O
O
O
O
R
R
InsP3/AM R ϭ CH3, 1
InsP3/PM R ϭ CH2CH3, 2
InsP3/BM R ϭ (CH2)2CH3, 3
Fig. 2. The membrane permeant analogues of rac-InsP3 reported
by Tsien and co-workers: rac-myo-inositol 1,4,5-trisphosphate hexa-
kis(acetoxymethyl) ester (InsP3/AM, 1); rac-myo-inositol 1,4,5-
trisphosphate hexakis(propionyloxymethyl) ester (InsP3/PM, 2);
rac-myo-inositol 1,4,5-trisphosphate hexakis(butyryloxymethyl) ester
(InsP3/BM, 3).[4]
The l-isomer (+)-3 was synthesized in an analogous
+14.6 (c 0.5, CHCl3) [lit.,[28] [α]D16 = +14.2 (CHCl3)]},
which was also furnished during the menthoxyacetyl-based
diastereomeric resolution. The yields obtained, shown in
approach to the synthesis of a membrane permeant derivative
of myo-inositol-1,3,4-trisphosphate,[6] membrane perme-
ant phosphatidylinositol polyphosphates,[7] and myo-inositol
3,4,5,6-tetrakisphosphate.[8]
manner using the optically antipodal alcohol (+)-4 {[α]D20
=

Synthesis of InsP3 Derivatives
889
HO
BnO
BnO
BnO
OAll
OAll
OH
OPMB
(f)
(a)
(b)
OBn
OAll
OBn
OAll
OBn
OH
OBn
OAll
BnO
BnO
BnO
BnO
OAll
OAll
OH
OH
(Ϫ)-9
(Ϫ)-4
(Ϫ)-5
(ϩ)-6
(c)
O
O
O(CH2)2CN
O
O
OBM
OBM
P
P
P
BnO
BnO
HO
BnO
O(CH2)2CN
O
OBM
OH
O
OBM
O
O
O
OBn
(e)
(d)
OBn
O
HO
BnO
O
P
O(CH2)2CN
O
P
P
OBM
OBM
OBM
OBM
O
P
O
O
O(CH2)2CN
O
O
O
NC(H2C)2O
P
P
OBM
OBM
OBM
OBM
O(CH2)2CN
(Ϫ)-3
(Ϫ)-8
(ϩ)-7
O
BM ϭ
O
Scheme 1. Synthesis of d-InsP3/BM (−)-3. Yields in parentheses refer to the l-isomer. Reagents and conditions: (a) NaH, BnBr, DMF,
0◦C → RT, 81% (85%). (b) (i) Wilkinson’s catalyst [Rh(PPh3)3Cl], Hünig’s base, EtOH, reflux; (ii) AcCl, MeOH, CH2Cl2, 75% (80%). (c) (i) N,N-
Diisopropylamino bis-(2-cyanoethoxy)phosphine, 1H-tetrazole, CH2Cl2; (ii) mCPBA, 86–93% (64%). (d) Et3N, CH2Cl2 then bromomethylbutyrate
and Hünig’s base, 29–48% (55%). (e) Pd(CF3CO2)2, Pd(CH3CO2)2, AcOH, H2 (1 atm), 15◦C, 88–91% (88%). (f) (i) Wilkinson’s catalyst
[Rh(PPh3)3Cl], Hünig’s base, EtOH, reflux; (ii) conc. HCl, MeOH, reflux, 66% yield over two steps.
O
O
O
O
O
(a)
(b)
OϪNϩBu4
O
Br
O
10
11
12
Scheme 2. Synthesis of bromomethylbutyrate (12). Reagents and conditions: (a) (i) 2 M NaOH
solution, tetrabutylammonium hydrogen sulfate, 56%; (ii) CH2Cl2 reflux, 2 days. (b) TMSBr, RT,
24 h, 48%.
parentheses (Scheme 1), were generally comparable with
those seen for the enantiomeric material.
of InsP3, which reacted with bromomethylbutyrate (12) to
yield the fully benzylated InsP3/BM (−)-8 (δP −4.7, −4.3,
and −4.0 ppm in CDCl3). Hydrogenolysis of the benzylated
InsP3/BM (−)-8 using palladium acetate and palladium tri-
fluoroacetate as catalysts afforded InsP3/BM (−)-3 (δP −4.1,
−3.8, and −3.6 ppm in MeOD).
The second synthetic route (Scheme 1) is based on that
employed in the previously reported synthesis of phos-
phatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2].[19] The
synthesis of d-InsP3/BM (−)-3 commenced from the opti-
callypurealcohol(−)-9{[α]2D5 = −1.8(c1.6, CHCl3)[lit.,[19]
[α]2D0 = −0.6(c0.6, CHCl3)]}, whichwasobtainedfrommyo-
inositol orthoformate, as previously reported.[16,19] Wilkin-
son’s catalyst-mediated isomerization of the allyl group to
the corresponding enol ether, followed by acid catalyzed
methanolysis of the enol ether and the 1-position PMB
group, afforded the known triol (+)-6 {[α]2D0 = +12.1 (c 0.88,
CHCl3) [lit.,[29] [α]D = +12.4 (c 0.80, CHCl3)]}.[28–30] 1H-
Tetrazole-catalyzed phosphitylation of the triol (+)-6 with
N,N-diisopropylamino bis-(2-cyanoethoxy)phosphine, fol-
lowed by P(iii) to P(v) oxidation using mCPBA, afforded the
fully protected InsP3 analogue (+)-7 [δP −2.5 and −2.7 ppm
(2:1) in CDCl3]. Treatment of the cyanoethyl moieties with
triethylamine furnished the presumed triethylammonium salt
Methylene dibutyrate (11) was furnished heating the tetra-
butylammonium salt of butyric acid (10) in dichloromethane
under reflux, as described by Holmberg and Hansen
(Scheme 2).[31] The bromomethyl butyrate (12) was syn-
thesized as described by Tsien and co-workers by treating
methylene dibutyrate (11) with trimethylsilyl bromide to
yield the desired compound (12).[4,32] The compound was
purified by Kugelrohr distillation before each use, to ensure
optimum yield in the subsequent reaction.
Biological Evaluation of Compounds
Berridge and Irvine were the first to demonstrate that d-myo-
inositol1,4,5-trisphosphate(InsP3)releasecausesanincrease
in intracellular Ca2+ concentration.[33] InsP3 is a key second

890
S. J. Conway et al.
(a)
50 µM L-myo InsP3 ester
cells that did not respond to l-InsP3/BM (+)-3 did respond
to ATP, showing that InsP3Rs were present (Fig. 3a) and
that the cells were viable. Further studies indicate that the
100 µM ATP
products formed from BM-ester hydrolysis have no Ca2+
-
mobilizing ability. These results are as would be predicted
from the established pharmacology of d- and l-InsP3[37] and
therefore our data indicate that d-InsP3/BM (−)-3 exerts its
Ca2+-releasing actions via activation of InsP3Rs. This mate-
rial has proved also useful in studies conducted in a range of
biological systems.[12,13,21–27]
200
100
250 s
(b)
50 µM L-myo InsP3 ester
100 µM ATP
Conclusions
In conclusion we have synthesized both the d- and the
l-enantiomers of InsP3/BM, using two different approaches
for d-InsP3/BM (−)-3. It has been shown that the intra-
cellular Ca2+ releasing ability of InsP3/BM resides in the
d-enantiomer, while the l-enantiomer displays little biolog-
ical activity. This supports the theory that the membrane
permeant forms of InsP3 exert their Ca2+-mobilizing effects
through activation of InsP3Rs, rather than some non-specific
action on the cell. The d- and l-enantiomers of InsP3/BM are
likely to remain important tools for those studying the role
of InsP3Rs in Ca2+ signalling.
200
100
250 s
Fig. 3. d-InsP3/BM ester (−)-3, but not l-InsP3/BM ester mobilizes
calcium stores. Fura-2-loaded human embryonic kidney (HEK) cells
were incubated with the forms of InsP3 ester shown in the figures.
Adenosine triphosphate stimulates the production of InsP3 in HEK cells,
and was used in these experiments as a positive control. Please note that
the calcium signals observed during incubation with d-InsP3/BM (−)-3
reflected release of the ion from intracellular stores, since the compound
was applied in calcium-free medium. The traces in (a) and (b) are from
single experiments, and are representative of several trials. The trace
in (b) shows Ca2+ release from multiple cells on the same coverslip
and gives a representative picture of the effect of d-InsP3/BM (−)-3 on
Ca2+ release.
Experimental
1H NMR spectra were recorded on Bruker DPX-250 (250 MHz), DRX-
400 (400 MHz) and DRX-500 (500 MHz) instruments, using deuterated
chloroform as reference and internal deuterium lock. The chemical shift
data for each signal are given in units of δ in parts per million (ppm) rela-
tive to tetramethylsilane (TMS) where δTMS = 0. 13C NMR spectra were
recorded on Brucker DPX-250 (62.5 MHz), DRX-400 (100 MHz), and
DRX-500 (125 MHz) instruments using an internal deuterium lock and
proton decoupling. The chemical shift data for each signal are given
as δ in units of parts per million (ppm) relative to tetramethylsilane
where δTMS = 0. In some cases the multiplicity of each signal was
determined by an attached proton test experiment. 31P NMR spectra
were recorded on a Bruker DRX-400 (162 MHz) or Bruker DRX-500
(202 MHz) instrument with proton decoupling. The chemical shift data
for each signal are given as δ in units of ppm relative to external 85%
H3PO4.
messenger that is released as a result of cell-surface receptor
activation, triggering the phospholipase C (PLC)-mediated
hydrolysis of PtdIns(4,5)P2 to form diacylglycerol (DAG)
and InsP3. The lipophilic DAG remains in the plane of the
cell membrane and effects signal transduction by activation of
protein kinase C (PKC). InsP3, which is hydrophilic, diffuses
into the cytosol, and activates InsP3Rs to release Ca2+. Ca2+
is an almost universal intracellular messenger, controlling a
diverserangeofcellularprocesses, suchasgenetranscription,
muscle contraction, and cell proliferation.[34–36] The impor-
tance of Ca2+ signalling has led to prolific investigation in
this field, however, there are very few membrane permeant
small molecule InsP3R agonists or antagonists.
Infrared (IR) spectra were recorded on a Perkin–Elmer 1310 spec-
trometer.The sample was prepared as a solution in the indicated solvent.
Calibration was made relative to polystyrene at 1603 cm−1
.
Mass spectra were recorded at the EPSRC Mass Spectroscopy
Centre, University of Wales, Swansea, or at the Department of Chem-
istry, University of Cambridge. Microanalyses were carried out by the
staff of the University Chemical Laboratory, Cambridge, Analytical
Department.
Melting points were determined using Reichert Hotstage melting
point apparatus and are uncorrected. Kugelrohr bulb-to-bulb distilla-
tions were carried out using a Büchi GKR-51 machine. Boiling points
are the actual oven temperatures.
To investigate the Ca2+-releasing ability of d- and
l-InsP3/BM Fura-2-loaded human embryonic kidney (HEK)
cells were incubated with either d- or l-InsP3/BM (Fig. 3).
l-InsP3/BM (+)-3, which is ∼1000-fold less potent at
InsP3Rs than d-InsP3/BM (−)-3,[37] showed littleevidenceof
Ca2+-releasing ability (Fig. 3a). d-InsP3/BM (−)-3, however,
demonstrated Ca2+-releasing ability after an initial incuba-
tion period (Fig. 3b), which is presumably related to the
deprotection of the BM esters. This time delay is to be
expected, as all six esters have to be removed before the
compound will induce InsP3R activity. It should be noted that
Optical specific rotations were measured using a Perkin Elmer 241
polarimeter, in a cell of 1 dm path length. The concentration (c) is
expressed in g/100 cm3 (equivalent to g/0.1 dm3) and values are stated
in implied units of 10−1 deg cm2 g−1
.
Analytical thin layer chromatography (TLC) was carried out on pre-
coated 0.25 mm thick Merck 60 F254 silica gel plates. Visualization
was by absorption of UV light, thermal development after treatment
with basic potassium permanganate solution or an ethanolic solution
of phosphomolybdic acid. Flash chromatography was carried out using
Merck Kieselgel 60 (230–400 mesh) under a pressure of compressed air
(typically 0.2–0.3 bar).

Synthesis of InsP3 Derivatives
891
Dry THF was distilled from potassium in a recycling still using
benzophenone ketyl as an indicator. Reagents were purified and dried
where necessary by standard techniques. Ether refers to diethyl ether and
n-hexane is referred to as hexane. Where appropriate and if not stated
non-aqueous reactions were carried out under an atmosphere of argon. In
vacuo refers to the removal of volatile solvents using a rotary evaporator.
Room temperature (RT) refers to ambient laboratory temperature.
(+)-1D-2,3,6-Tris-O-benzyl-myo-inositol (+)-6 (Method 2)
(−)-5-O-Allyl-2,3,6-tris-O-benzyl-1-O-(4-methoxybenzyl)-myo-inosi-
tol (−)-9 (300 mg, 0.47 mmol),[19] Wilkinson’s catalyst (131.3 mg,
0.14 mmol) and Hünig’s base (61.2 mg, 82.4 µL, 0.47 mmol) were
dissolved in ethanol (15 mL) and heated under reflux. As the start-
ing material and product have the same TLC Rf value the reaction
progress was monitored by 1H NMR spectroscopy. The resonance for
the alkene CH proton is observed at δH ∼5.9 ppm in the starting mate-
rial and δH ∼6.3 ppm in the enol ether. The reaction was seen to
be complete by 1H NMR analysis after 1.5 h. The reaction mixture
was filtered through Celite and then concentrated under reduced pres-
sure. The resulting oil was suspended in water (30 mL) and extracted
with ethyl acetate (4 × 20 mL). The combined organic fractions were
washed with saturated brine (20 mL), dried (MgSO4), filtered, and
concentrated under reduced pressure. This afforded crude 1-O-(4-
methoxybenzyl)-5-O-propenyl-2,3,6-tris-O-benzyl-myo-inositol as an
oil which was used, without purification. (−)-1-O-(4-Methoxybenzyl)-
5-O-propenyl-2,3,6-tris-O-benzyl-myo-inositol (assumed 300 mg) was
dissolved in methanol (20 mL) and concentrated HCl (1 mL) was added.
The solution was heated under reflux in air for 2 h, by which time the
reaction was adjudged to be complete by TLC analysis. The solution
was cooled and solid NaHCO3 (840 mg, 10 mmol, 1 equiv wrt HCl)
was added to neutralize the HCl. The resulting mixture was filtered and
the methanol removed from the filtrate under reduced pressure. Purifi-
cation using silica gel column chromatography eluted with ethyl acetate
and hexane (2:3) furnished (−)-2,3,6-tris-O-benzyl-myo-inositol (+)-6
(147.8 mg, 66% yield) as an off-white solid. Rf 0.62 (ethyl acetate).
mp 118–119◦C (from ethyl acetate) [lit.,[28] mp 117–119◦C; lit.,[29] mp
117–119◦C; lit.,[30] mp 122–123◦C]. [α]D25 = +12.1 (c 0.88, CHCl3)
[lit.,[28] [α]1D6 = +15.5 (CHCl3); lit.,[29] [α]D = +12.4 (c 0.80, CHCl3);
lit.,[30] [α]D = +10.3(c1.73, CHCl3)]. δH (500 MHz;CDCl3)7.41–7.30
(−)-1D-1,4,5-Tris-O-allyl-2,3,6-tris-O-benzyl-myo-inositol (−)-5
Into a suspension of sodium hydride (21.0 mg, 60% dispersion in min-
eral oil, 0.524 mmol) in DMF (2 mL) at 0◦C was cannulated a solution
of the alcohol (−)-4 (210 mg, 0.437 mmol) in DMF (4 mL). The sus-
pension was stirred at 0◦C for 15 min before benzyl bromide (89.6 mg,
62.4 µL, 0.524 mmol) was added dropwise and the suspension stirred
for 16 h at RT. The reaction was quenched by the addition of methanol
(0.2 mL). The volatile components were removed in vacuo. The residue
was partitioned between diethyl ether (10 mL) and water (5 mL) and
the organic layer was separated. The aqueous layer was extracted with
diethyl ether (10 mL) and the combined organic extracts washed with
brine (5 mL), dried (MgSO4), filtered, and the solvent removed in vacuo.
Silica gel column chromatography of the residue, eluting with hexane
and Et2O (6:1 then 2:1), gave the title compound (−)-5 (202 mg, 81%)
as a colourless oil. Rf 0.57 (Et2O/hexane 3:1). [α]2D0 = −3.5 (c 1.12,
CHCl3) [lit.,[38] [α]D30 = −2.5 (c 1.6, CHCl3)]. δH (250 MHz; CDCl3)
7.42–7.23 (15H, m, Ph), 6.04–5.85 (3H, m, CH2CH=CH2), 5.32–
5.12 (6H, m, CH2CH=CH2), 4.86 (2H, s, CH2-Ph), 4.82 (2H, ABq,
2J 10.3, CH2-Ph), 4.63 (2H, ABq, 2J 11.9, CH2-Ph), 4.36–4.31 (4H, m,
CH2CH=CH2), 4.09–4.07 (2H, m, CH2CH=CH2), 3.99–3.97 (1H, m,
inositol ring H), 3.92 (1H, apparent t, J 9.6, inositol ring H), 3.87 (1H,
t, J 9.6, inositol ring H), 3.28–3.17 (3H, m, 3 × inositol ring H). These
data are in agreement with the literature values.[38]
ꢀ
ꢀ
(15H, m, Ph), 4.96 (1H, d, JAB 11.4, CHAHBPh), 4.90 (1H, d, JA B 11.4,
(+)-1L-1,4,5-Tris-O-allyl-2,3,6-tris-O-benzyl-myo-inositol (+)-5
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ ꢀꢀ
CHA HB Ph), 4.86 (1H, d, JA B 11.4, CHA HB Ph), 4.74 (1H, d, JA B
ꢀꢀ
ꢀꢀ
11.7, CHA HB Ph), 4.71 (1H, d, JAB 11.4, CHAHBPh), 4.61 (1H, d,
(+)-1l-1,4,5-Tris-O-allyl-2,3,6-tris-O-benzyl-myo-inositol (+)-5 was
prepared in a manner similar to that described for its antipode, from
(+)-4.Yield 295 mg (73%). [α]2D0 = +3.0 (c 1.05, CHCl3).[39] All other
spectroscopic and analytical data were identical to that reported for the
enantiomeric material, (−)-5.
ꢀꢀ ꢀꢀ
ꢀꢀ
ꢀꢀ
JA B 11.7, CHA HB Ph), 4.10 (1H, apparent t, J 2.6, inositol ring H),
4.03 (1H, td, J 9.4, 1.9, inositol ring H), 3.70 (1H, apparent t, J 9.3,
inositol ring H), 3.58–3.54 (1H, m, inositol ring H), 3.50 (1H, td, J 9.1,
2.1, inositol ring H), 3.32 (1H, dd, J 9.7, 2.4, inositol ring H), 2.60 (2H,
apparent dd, J 8.5, 2.1, 2 × OH), 2.35 (1H, d, J 6.6, OH). These data are
in agreement with the literature values.[28–30]
(+)-1D-2,3,6-Tris-O-benzyl-myo-inositol (+)-6 (Method 1)
To a solution of the allyl ether (−)-5 (198 mg, 0.347 mmol) and
Hünig’s base (40.3 mg, 54 µL, 0.312 mmol) in ethanol–toluene–water
(7:3:1, 11 mL) was added Wilkinson’s catalyst [(Ph3P)3RhCl] (144 mg,
0.156 mmol) and the solution heated under reflux for 2 h, then cooled
to RT. The mixture was diluted with ethyl acetate (5 mL) and insolu-
ble material was removed by filtration through Hyflo, under vacuum.
To the filtrate was added water (10 mL) and ethyl acetate (15 mL).
The layers were separated and the aqueous phase was extracted with
ethyl acetate (10 mL). The combined organic layers were washed with
brine (10 mL), dried (MgSO4), filtered, and evaporated in vacuo. The
residue was dissolved in MeOH (7 mL) and dichloromethane (14 mL)
and acetyl chloride (20.4 mg, 18.5 µL, 0.260 mmol) was added. The
solutionwasstirredatRTfor70 min. Et3N(79 mg, 109 µL, 0.781 mmol)
was added and the mixture was concentrated in vacuo. Silica gel col-
umn chromatography eluting with hexane and ethyl acetate (2:1 then
1:1) afforded the triol (+)-6 (117 mg, 75%) as an off-white solid. Rf
0.19 (Et2O/hexanes 1:1). mp 119–120◦C (from ethyl acetate) [lit.,[28]
mp 117–119◦C; lit.,[29] mp 117–119◦C; lit.,[30] mp 122–123◦C; lit.,[38]
117–119◦C]. [α]2D0 = +12.3 (c 0.40, CHCl3) [lit.,[28] [α]1D6 = +15.5
(CHCl3); lit.,[29] [α]D = +12.4 (c 0.80, CHCl3); lit.,[30] [α]D = +10.3
(c 1.73, CHCl3); lit.,[38] [α]D16 = +15.5 (c 1.0, CHCl3)]. δH (250 MHz;
CDCl3) 7.37–7.33 (15H, m, Ph), 4.83 (2H, ABq, 2J 11.6), 4.87 (2H,
ABq, 2J 11.8), 4.64 (2H, ABq, 2J 11.7), 4.09 (1H, apparent t, J 2.5,
inositol ring H), 4.02 (1H, dt, J 9.6, 1.9, inositol ring H), 3.69 (1H,
apparent t, J 9.2 inositol ring H), 3.44–3.58 (2H, m, 2 × inositol ring
H), 3.30 (1H, dd, 1H, J 9.7, 2.4, inositol ring H), 2.61–2.58 (2H, m, OH),
2.33 (1H, d, J 6.5, OH). These data are in agreement with the literature
values.[28–30,38]
(+)-1D-2,3,6-Tris-O-benzyl-myo-inositol 1,4,5-Trisphosphate
Hexakis-(2-cyanoethyl) Ester (+)-7
N,N-Diisopropylamino
bis-(2-cyanoethoxy)phosphine
(633 mg,
2.33 mmol) and 1H-tetrazole (196.2 mg, 2.8 mmol) were dissolved in
dry dichloromethane (4 mL) and stirred for 1 h at RT. (+)-2,3,6-Tris-
O-benzyl-myo-inositol (+)-6 (140 mg, 0.31 mmol) was dissolved in dry
dichloromethane (3 mL) and cannulated into the stirred phosphite solu-
tion and washed in with dry dichloromethane (3 mL). The resulting
solution was stirred for 45 h when a new product and no starting material
were observed by TLC analysis. The solution was cooled to −78◦C and
solid mCPBA (483 mg, 2.8 mmol) was added.The resulting mixture was
stirred for 1 h at −78◦C then overnight at RT. Formation of a more polar
spot than previously observed was seen by TLC analysis. The organic
layer was washed with a saturated aqueous solution of NaHSO3 (10 mL),
a saturated aqueous solution of NaHCO3 (20 mL) then water (20 mL).
The combined aqueous layers were washed with dichloromethane
(3 × 30 mL). The combined organic layers were washed with satu-
rated brine (20 mL), dried (MgSO4), filtered, and concentrated in
vacuo. Purification using silica gel column chromatography eluted with
ethyl acetate then ethyl acetate and methanol (90:10) afforded pure
(+)-1D-2,3,6-tris-O-benzyl-myo-inositol 1,4,5-trisphosphate hexakis-
(2-cyanoethyl) ester ( +)-7 (291 mg, 93% yield) as a clear colourless
resin. Rf 0.1 (ethyl acetate). [α][D26] = +3.7 (c 0.6, CHCl3). νmax
(CHCl3)/cm−1 2969 (w) (CH), 2937 (w) (CH), 2255 (w) (CN), 1717
(w), 1498 (w) (Ph), 1471 (w) (Ph), 1456 (w) (Ph), 1279 (s) (P=O), 1028
(s), 752 (m) (5 adjacent Ph), 700 (m) (5 adjacent Ph). δH (500 MHz;
CDCl3) 7.46–7.42 (15H, m, Ph), 4.97–4.75 (6H, m, Ph-CH2),

892
S. J. Conway et al.
4.58–3.96 (17H, m, 6 × PO-CH2 and 5 × inositol ring H), 3.63 (1H, dd,
J 9.9, 2.0, 3-H), 2.70–2.21 (12H, m, CH2-CN). δC (125 MHz; CDCl3)
137.9, 137.8, 137.1, 128.6, 128.5, 128.4, 128.2, 128.0, 127.8, 127.8,
127.7, 126.7, 117.0 (CN), 116.8 (CN), 116.6 (CN), 116.5 (CN), 116.4
(CN), 116.3 (CN), 79.3, 78.2–78.1 (2C, m, 2 × inositol ring C), 77.6–
77.5 (2C, m, 2 × inositol ring C), 75.6, 74.8 (inositol ring C), 74.8,
72.5, 63.0 (1C, d, JCP 5.5, PO-CH2), 62.8 (1C, d, JCP 5.5, PO-CH2),
62.5–62.4 (2C, m, PO-CH2), 62.3–62.2 (2C, m, PO-CH2), 19.6 (4C,
m, PO-CH2CH2), 19.1 (1C, d, JCP 7.8, PO-CH2CH2), 19.0 (1C, d, JCP
8.1, PO-CH2CH2). δP (202 MHz; CDCl3) −2.7, −2.5 (1:2). m/z (ES+,
NH3) 1031.4 [100, (M + Na)+], 1026.5 [35, (M + NH4)+], 1009.4 [50,
(M + H)+], 956.4 (10), 829.3 (20), 739.2 (20), 310.2 (15), 235 (15),
181.1 (60), 143.7 (60), 90.8 (70). The appropriate data are in agreement
with literature values for the racemic compound.[4]
(c 1.9, CHCl3).All other spectroscopic and analytical data were identical
to that reported for the enantiomeric material, (−)-8.
(−)-1D-myo-Inositol 1,4,5-Trisphosphate Hexakis(butyryloxymethyl)
Ester (−)-3 (D-InsP3/BM)
(−)-1d-2,3,6-Tris-O-benzyl-myo-inositol 1,4,5-trisphosphate hexakis
(butyryloxymethyl) ester (25.8 mg, 20.0 µmol) (−)-8 was dis-
solved in glacial acetic acid (2 mL) under argon. Palladium
bis(trifluoromethylacetate) (19.3 mg, 58.1 µmol) and palladium diac-
etate (38.6 mg, 171.9 µmol) were added and the mixture was stirred at
10◦C for 2.5 h under an atmosphere of hydrogen. The suspension was
filtered through Hyflo, which was then washed with glacial acetic. The
resulting solution was diluted with Milli-Q purified water (1 mL) and
lyophilized to afford ( −)-1D-myo-inositol 1,4,5-trisphosphate hexakis
(butyryloxymethyl) ester ( −)-3 (17.8 mg, 91% yield) as a colourless
solid. [α]2D5 = −10.8 (c 0.46, MeOH). νmax (neat film)/cm−1 3727 (w)
(OH), 2925 (s) (CH), 2855 (m) (O-CH2-O), 1733 (s) (CO), 1465 (m),
1380 (m), 1262 (m) (P=O), 997 (s). δH (400 MHz; MeOD) 5.71–5.64
(12H, m, O-CH2-O), 4.62 (1H, q, J 9.4, inositol ring H), 4.38–4.24 (2H,
m, 2H, inositol ring H), 4.18 (1H, apparent t, J 2.5, inositol ring H),
4.02 (1H, apparent t, J 9.5, inositol ring H), 3.68 (1H, dd, J 9.7, 2.5,
inositol ring H), 2.42–2.37 (12H, m, C(O)CH2), 1.65 (12H, hx, J 7.4,
C(O)CH2-CH2), 0.96 (18H, t, J 7.4, CH3). δP (162 MHz; MeOD) −4.1,
−3.8, −3.6. m/z (ES+) 1043.8 [20, (M + Na)+], 960.4 (60), 471.7 (80),
417.0 (65), 116.6 (50), 81.3 (70), 74.1 (100). The appropriate data are
in agreement with literature values for the racemic compound.[4]
(−)-1L-2,3,6-Tris-O-benzyl-myo-inositol 1,4,5-Trisphosphate
Hexakis-(2-cyanoethyl) Ester (−)-7
(−)-1l-2,3,6-Tris-O-benzyl-myo-inositol 1,4,5-trisphosphate hexakis-
(2-cyanoethyl) ester (−)-7 was prepared in a manner similar to
that described for its antipode, from (−)-6. Yield 246 mg (92%).
[α]2D0 = −4.7 (c 1.6, CHCl3).All other spectroscopic and analytical data
were identical to that reported for the enantiomeric material, (+)-7.
(−)-1D-2,3,6-Tris-O-benzyl-myo-inositol 1,4,5-trisphosphate
Hexakis(butyryloxymethyl) Ester (−)-8
(+)-1d-2,3,6-Tris-O-benzyl-myo-inositol 1,4,5-trisphosphate hexakis-
(2-cyanoethyl) ester (+)-7 (47.5 mg, 0.05 mmol) was dissolved in
dry dichloromethane (1 mL) under argon and triethylamine (47.6 mg,
65.4 µL, 0.47 mmol, 10 equiv) was added. After stirring for 16 h at
RT the solvent was removed in vacuo. Freshly distilled bromomethyl
butyrate (255 mg, 1.41 mmol) was added to the residue and mixture
was placed under an atmosphere of argon. Dry acetonitrile (2 mL) fol-
lowed by Hünig’s base (182.6 mg, 246.0 µL, 1.41 mmol) were added
and the resulting solution was stirred for 48 h. A reaction was adjudged
to have occurred by TLC analysis. The solvent was removed in vacuo
and purification by silica gel column chromatography eluted with ethyl
acetate and hexane (40:60 then 50:50) furnished (−)-1D-2,3,6-tris-
O-benzyl-myo-inositol 1,4,5-trisphosphate hexakis(butyryloxymethyl)
ester ( −)-8 (29.4 mg, 48% yield) as a clear colourless oil. Rf 0.14
(ethyl acetate/hexane, 50:50). [α]2D0 = −3.9 (c 1.6, CHCl3). νmax (neat
film)/cm−1 2964 (m) (CH), 2932 (m) (CH), 2877 (m) (O-CH2-O), 1763
(s) (CO), 1458 (m) (Ph), 1416 (m) (Ph), 1364 (m), 1283 (m) (P-O-aryl),
1262 (m) (P=O), 1151 (s) (C–O stretch ether), 1099 (m) (P-O-alkyl),
963 (s). δH (500 MHz; CDCl3) 7.41–7.23 (15H, m, Ph), 5.65–5.58 (4H,
m, O-CH2-O), 5.51–5.38 (7H, m, O-CH2-O), 5.31–5.27 (1H, m, O-
CH2-O), 4.89–4.79 (3H, m, CH2-Ph and 1 × inositol ring H), 4.74–4.71
(2H, m, CH2-Ph), 4.66 (1H, d, JAB 11.5, CHAHB-Ph), 4.62 (1H, d, JAB
11.5, CHAHB-Ph), 4.41 (1H, apparent q, J 9.3, inositol ring H), 4.33
(1H, t, J 2.3, inositol ring H), 4.30–4.26 (1H, m, inositol ring H), 4.03
(1H, t, J 9.6, inositol ring H), 3.47 (1H, dd, J 9.9, 2.2, inositol ring H),
2.31–2.22 (12H, m, O=C-CH2), 1.65–1.55 (12H, m, O=C-CH2-CH2),
0.94–0.82 (18H, m, CH3). δC (125 MHz; CDCl3) 171.8, 171.8, 171.7,
137.9, 137.7, 137.0, 128.8, 128.5, 128.3, 128.2, 127.9, 127.8, 127.7,
127.7, 127.5, 82.9 (2C, m, O-CH2-O), 82.6–82.5 (3C, m, O-CH2-O),
82.4 (1C, d, JCP 4.5, O-CH2-O), 79.2, 78.3 (1C, d, JCP 5.5, inositol ring
C), 78.2–78.1 (1C, m, inositol ring C), 77.4–77.3 (2C, m, 2 × inositol
ring C), 75.3, 75.1, 74.9 (inositol ring C), 72.6, 35.6–35.5 (6C, m, O=C-
CH2), 17.9, 13.5–13.4 (6C, m, O=C-CH2-CH2-CH2). δP (202 MHz;
CDCl3) −4.7, −4.3, −4.0. m/z (ES+, NH3) 1313.6 [60, (M + Na)+],
1308.6 [100, (M + NH4)+], 932.5 (100), 860.3 (70), 739.5 (85), 663.4
(70), 491.2 (100), 42.1 (100).
(+)-1L-myo-Inositol 1,4,5-Trisphosphate hexakis(butyryloxymethyl)
Ester (+)-3 (L-InsP3/BM)
(+)-1L-myo-Inositol 1,4,5-trisphosphate hexakis(butyryloxy-methyl)
ester ( +)-3 was prepared in a manner similar to that described for its
antipode, from (+)-8.Yield 26 mg (88%). [α][D20] = +8.3 (c 1.4, CHCl3).
All other spectroscopic and analytical data were identical to that reported
for the enantiomeric material, (−)-3.
Methylene Dibutyrate 11
Butyric acid (5.3 g, 5.5 mL, 60.2 mmol) was added to a 2 M solution
of sodium hydroxide (4.8 g, in 60 mL of water) and stirred for 30 min
under air. Tetrabutylammoniumhydrogen sulfate (20.4 g, 60.1 mmol)
was added, heat was evolved and the resulting solution was stirred
for 30 min. The aqueous solution was extracted with dichloromethane
(4 × 100 mL). The combined organic fractions were dried (MgSO4),
filtered, and heated under reflux for 2 days. The dichloromethane was
removed by distillation and the resulting oil was transferred to a smaller
vessel. This oil was purified by vacuum distillation, the fraction boiling
at 43◦C under 0.3 mmHg of pressure was pure methylene dibutyrate 11
(3.15 g, 55.7% yield) as a clear colourless oil. δH (250 MHz; CDCl3)
5.75 (2H, s, OCH2O), 2.34 (4H, t, J 7.4, CH2C=O), 1.67 (4H, sx, J 7.4,
CH3CH2), 0.95 (6H, t, J 7.4, CH3CH2). These data are in agreement
with the literature values.[31]
Bromomethyl Butyrate 12
Methylene dibutyrate 11 (1.02 g, 5.4 mmol, 1 equiv), trimethylsilyl
bromide (1.08 g, 0.93 mL, 7.1 mmol, 1.3 equiv) and zinc(ii) bromide
(61 mg, 0.27 mmol, 0.05 equiv) were stirred together overnight. The
reaction was adjudged to be incomplete by 1H NMR analysis, there-
fore trimethylsilyl bromide (0.54 g, 0.47 mL, 3.6 mmol, 0.7 equiv) was
added and the resulting solution was stirred for a further 24 h when the
reaction was adjudged to be complete by 1H NMR analysis. The solu-
tion was diluted with ether (20 mL) and a 1 M aqueous HCl solution
was added (10 mL) and the mixture was stirred for 10 min. The aqueous
phase was removed and the organic fraction was stirred with a satu-
rated solution of Na2CO3 (20 mL) for 30 min. The organic fraction was
washed with water (10 mL) and a saturated brine solution (10 mL), dried
(MgSO4), filtered, and the ether was removed under reduced pressure
at RT. The resulting oil was adjudged to consist mainly of the required
product contaminated with ∼10% of methylene butyrate. The oil was
further purified by Kugelrohr distillation, the fraction which boiled at
(+)-1L-2,3,6-Tris-O-benzyl-myo-inositol 1,4,5-Trisphosphate
Hexakis(butyryloxymethyl) Ester (+)-8
(+)-1L-2,3,6-Tris-O-benzyl-myo-inositol 1,4,5-trisphosphate hexakis
(butyryloxymethyl) ester ( +)-8 was prepared in a manner similar to that
described for its antipode, from (−)-7.Yield 76 mg (55%). [α]2D0 = +3.5

Synthesis of InsP3 Derivatives
893
90◦C under 5 mmHg of pressure was pure bromomethyl butyrate 12
(472.2 mg, 48.2% yield) as a clear colourless oil. δH (250 MHz; CDCl3)
5.81 (2H, s, CH2Br), 2.36 (2H, t, J 7.4, CH2C=O), 1.69 (2H, sx, J 7.4,
CH3CH2), 0.97 (3H, t, J 7.4, CH3CH2).[32]
[14] S. J. A. Grove, I. H. Gilbert, A. B. Holmes, G. F. Painter,
M. L. Hill, Chem. Commun. 1997, 1633. doi:10.1039/A703044D
[15] S. J. A. Grove, A. B. Holmes, G. F. Painter, P. T. Hawkins,
L. R. Stephens, Chem. Commun. 1997, 1635. doi:10.1039/
A703045B
[16] G. F. Painter, S. J. A. Grove, I. H. Gilbert, A. B. Holmes,
P. R. Raithby, M. L. Hill, P. T. Hawkins, L. R. Stephens,
J. Chem. Soc., PerkinTrans. 1 1999, 923. doi:10.1039/A900278B
[17] M. Manifava, J. Thuring, Z. Y. Lim, L. Packman, A. B. Holmes,
N. T. Ktistakis, J. Biol. Chem. 2001, 276, 8987. doi:10.1074/
JBC.M010308200
[18] G. F. Painter, J.W.Thuring, Z.Y. Lim,A. B. Holmes, P.T. Hawkins,
L. R. Stephens, Chem. Commun. 2001, 645. doi:10.1039/
B101320N
Cell Culture
Human embryonic kidney cells (HEK-293) were grown at 37◦C in
Dulbecco’s modified Eagle’s medium supplemented with 10% heat-
inactivated fetal bovine serum, 100 U mL−1 penicillin, 100 µg mL−1
streptomycin, and 2 mM l-glutamine in a humidified 95% air, 5% CO2
incubator. All experimental procedures were carried out at room tem-
perature (20–22◦C). Prior to imaging, the culture medium was replaced
with an extracellular medium (EM) containing (mM): NaCl, 121; KCl,
5.4; MgCl2, 0.8; CaCl2, 1.8; NaHCO3, 6; d-glucose, 5.5; Hepes, 25;
pH 7.3.
[19] Z. Y. Lim, J. W. Thuring, A. B. Holmes, M. Manifava,
N. T. Ktistakis, J. Chem. Soc., Perkin Trans. 1 2002, 1067.
doi:10.1039/B200585A
[20] M. K. Johns, S. J. Conway, A. B. Holmes, Z. Y. Lim, C. Meyer,
Abstr. Pap. Am. Chem. Soc. 2004, 227, 515.
Acknowledgments
[21] S. C. Tovey, P. de Smet, P. Lipp, D. Thomas, K. W. Young,
L. Missiaen, H. De Smedt, J. B. Parys, M. J. Berridge,
J. Thuring, A. Holmes, M. D. Bootman, J. Cell Sci. 2001, 114,
3979.
[22] F. W. Johenning, M. Zochowski, S. J. Conway, A. B. Holmes,
P. Koulen, B. E. Ehrlich, J. Neurosci. 2002, 22, 5344.
[23] F. W. Johenning, M. Zochowski, J. Thuring, A. Holmes,
P. Koulen, B. E. Ehrlich, Biophys. J. 2002, 82, 838.
[24] C. M. Peppiatt, T. J. Collins, L. Mackenzie, S. J. Conway,
A. B. Holmes, M. D. Bootman, M. J. Berridge, J. T. Seo,
H. L. Roderick, Cell Calcium 2003, 34, 97. doi:10.1016/S0143-
4160(03)00026-5
The authors gratefully acknowledge funding from the
Biotechnology and Biological Sciences Research Council
and thank the Engineering and Physical Sciences Research
Council Mass Spectrometry service, Swansea. S.J.C. thanks
Hughes Hall, Cambridge, for a Research Fellowship. B.T.K.
thanks the NewtonTrust for funding. H.L.R. thanks the Royal
Society for a Research Fellowship. A.B.H. acknowledges
generous support from the Australian Research Council, the
Commonwealth Scientific and Industrial Research Organisa-
tion, the Victorian Endowment for Science, Knowledge and
Innovation, and the University of Melbourne.
[25] S. C. Tovey, T. A. Goraya, C. W. Taylor, Br. J. Pharmacol. 2003,
138, 81. doi:10.1038/SJ.BJP.0705011
[26] R. Chen, I. Valencia, F. Zhong, K. S. McColl, H. L. Roderick,
M. D. Bootman, M. J. Berridge, S. J. Conway, A. B. Holmes,
G. A. Mignery, P. Velez, C. W. Distelhorst, J. Cell Biol. 2004,
166, 193. doi:10.1083/JCB.200309146
References
[1] C. Schultz, Bioorg. Med. Chem. 2003, 11, 885. doi:10.1016/
S0968-0896(02)00552-7
[27] N. N. Kasri, A. M. Holmes, G. Bultynck, J. B. Parys,
M. D. Bootman, K. Rietdorf, L. Missiaen, F. McDonald,
H. De Smedt, S. J. Conway, A. B. Holmes, M. J. Berridge,
H. L. Roderick, EMBO J. 2004, 23, 312. doi:10.1038/
SJ.EMBOJ.7600037
[28] S. Ozaki, Y. Watanabe, T. Ogasawara, Y. Kondo, N. Shiotani,
H. Nishii, T. Matsuki, Tetrahedron Lett. 1986, 27, 3157. doi:
10.1016/S0040-4039(00)84742-5
[29] K. Sato, S. Sakuma, S. Muramatsu, M. Bokura, Chem. Lett. (Jpn.)
1991, 20, 1473. doi:10.1246/CL.1991.1473
[30] D. L. J. Clive, X. He, M. H. D. Postema, M. J. Mashimbye, J. Org.
Chem. 1999, 64, 4397. doi:10.1021/JO990086I
[31] K. Holmberg, B. Hansen, Tetrahedron Lett. 1975, 16, 2303. doi:
10.1016/S0040-4039(00)75108-2
[32] G. Grynkiewicz, R. Y. Tsien, Pol. J. Chem. 1987, 61, 443.
[33] H. Streb, R. F. Irvine, M. J. Berridge, I. Schulz, Nature 1983, 306,
67. doi:10.1038/306067A0
[34] M. J. Berridge, R. F. Irvine, Nature 1989, 341, 197. doi:10.1038/
341197A0
[35] M. J. Berridge, P. Lipp, M. D. Bootman, Nat. Rev. Mol. Cell Biol.
2000, 1, 11. doi:10.1038/35036035
[2] J. Engels, E. J. Schlaeger, J. Med. Chem. 1977, 20, 907. doi:
10.1021/JM00217A008
[3] A. B. A. Jansen, T. J. Russell, J. Chem. Soc. 1965, 2127. doi:
10.1039/JR9650002127
[4] W. H. Li, C. Schultz, J. Llopis, R.Y. Tsien, Tetrahedron 1997, 53,
12017. doi:10.1016/S0040-4020(97)00714-X
[5] W. H. Li, J. Llopis, M. Whitney, G. Zlokarnik, R.Y. Tsien, Nature
1998, 392, 936. doi:10.1038/31965
[6] M. T. Rudolf, A. E. Traynor-Kaplan, C. Schultz, Bioorg. Med.
Chem. Lett. 1998, 8, 1857. doi:10.1016/S0960-894X(98)00322-9
[7] C. Dinkel, M. Moody, A. Traynor-Kaplan, C. Schultz,
Angew. Chem. Int. Ed. 2001, 40, 3004. doi:10.1002/1521-3773
(20010817)40:16<3004::AID-ANIE3004>3.0.CO;2-O
[8] M. T. Rudolf, C. Dinkel, A. E. Traynor-Kaplan, C. Schultz,
Bioorg. Med. Chem. 2003, 11, 3315. doi:10.1016/S0968-
0896(03)00188-3
[9] D. Thomas, P. Lipp, S. C. Tovey, M. J. Berridge, W. H. Li,
R.Y. Tsien, M. D. Bootman, Curr. Biol. 2000, 10, 8. doi:10.1016/
S0960-9822(99)00258-4
[10] T. J. Collins, P. Lipp, M. J. Berridge, W. H. Li, M. D. Bootman,
Biochem. J. 2000, 347, 593. doi:10.1042/0264-6021:3470593
[11] P. Lipp, M. Laine, S. C. Tovey, K. M. Burrell, M. J. Berridge,
W. H. Li, M. D. Bootman, Curr. Biol. 2000, 10, 939. doi:10.1016/
S0960-9822(00)00624-2
[36] M. J. Berridge, M. D. Bootman, H. L. Roderick, Nat. Rev. Mol.
Cell Biol. 2003, 4, 517. doi:10.1038/NRM1155
[37] B. V. L. Potter, D. Lampe, Angew. Chem. Int. Ed. Engl. 1995, 34,
1933. doi:10.1002/ANIE.199519331
[12] L. Mackenzie, M. D. Bootman, M. Laine, M. J. Berridge,
J. Thuring, A. Holmes, W. H. Li, P. Lipp, J. Physiol. 2002, 541,
395. doi:10.1113/JPHYSIOL.2001.013411
[13] D. S. K. Samways, W. H. Li, S. J. Conway, A. B. Holmes,
M. D. Bootman, G. Henderson, Biochem. J. 2003, 375, 713.
doi:10.1042/BJ20030508
[38] S. Ozaki, Y. Kondo, N. Shiotani, T. Ogasawara, Y. Watanabe,
J. Chem. Soc., Perkin Trans.
1 1992, 729. doi:10.1039/
P19920000729
[39] S. Ozaki,Y. Watanabe,A.Awaya,Y. Ishizuka, US Patent 4952717
1990.